Monsanto Company CR240

- 13U1 1 of 374 Petition for Determination of Nonregulated Status for Corn Rootworm Protected and Glyphosate Tolerant MON 87411 Maize

The undersigned submits this petition under 7 CFR § 340.6 to request that the Administrator make a determination that the article should not be regulated under

7 CFR p art 340

October 4, 2013 (Revised December 18, 2013)

OECD Unique Identifier: MON

- 87411

- 9

Monsanto Petition Number: CR240

- 13U1

USDA

- APHIS Petition Number 13

- 290

- 01p

Submitted by:

John M. Cordts, M.S., M.B.A Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167 Phone: (314) 694

- 4831

Fax: (314) 694

- 3080

E

- mail: John.M.Cordts @monsanto.com

A. Ahmad , Ph.D., P.M. B achman , Ph.D., B.A. Comstoc k , B.S., Prepared by:

J.M. Cordts , M.S., M.B.A., S. Dubelman, Ph.D., T.H. Klusmeyer, Ph.D., D.K. Kovalic, Ph.D., C.A. Lawrence , Ph.D., J.S. Petrick , Ph.D.

, Q. Tian, M.D., Ph.D.

Contributors and/or Principal Investigators:

K. Adu

- tutu, Ph.D., S. Arackal, M.S.

, D. Anstrom, M.S., A. Beyene

, Ph.D., J. Bynum , Ph.D., S.M. Carleton

, Ph.D., D.B. Carson , Ph.D.

, M. Chen , Ph.D.

, K. Crowley, Ph.D., S. Drury, B.A., J. Fischer, Ph.D., R. Hileman, Ph.D., K. Howard, Ph.D., T. Lee, Ph.D., S.L. Levine , Ph.D., G. Mueller, M.A., M. Paradise, M.A., E. Parks, Ph.D., B. Sammons, Ph.D. A. Silvanovich , Ph.D., Z. Song , M.S., J. Tan, Ph.D., J. Uffman, B.S., W. Urqhart

, Ph.D., J.M. Ward , Ph.D.,

B.J. Warner , M.S., J. Warren, B.S. , F. Zapata, M.S.Monsanto Company CR240

- 13U1 2 of 374 RELEASE OF INFORMATION

Monsanto is submitting the information in this petition for review by the USDA as part of the regulatory process. Monsanto understands that the USDA complies with the provisions of the Freedom of Information Act (FOIA). In the event the USDA receives a FOIA request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of the information in this petition, Monsanto expects that, in advance of the release o f the document(s), USDA will provide Monsanto with a copy of the material proposed to be released and the opportunity to object to the release of any information based on appropriate legal grounds, e.g., responsiveness, confidentiality, and/or competitive

concerns. Monsanto understands that this information may be made available to the public in a reading room and upon individual request as part of a public comment period. Monsanto also understands that when deemed complete, a copy of the petition may be posted to the USDA

- APHIS BRS website or other U.S. government websites (e.g., www.regulations.gov ). Except in accordance with the foregoing, Monsanto does not authorize the release, publication or other distribution of this information without

Monsan to's prior notice and consent.

© 2013 Monsanto Company. All Rights Reserved. This document is protected under national and international copyright law and treaties. This document and any accompanying material are for use only by the regulatory

auth ority to which it has been submitted by Monsanto Company and its affiliates, collectively “Monsanto Company

, ” and only in support of actions requested by Monsanto Company. Any other use, copying, or transmission, including internet posting, of this

docume nt and the materials described in or accompanying this document, without prior consent of Monsanto Company, is strictly prohibited; except that Monsanto Company hereby grants such consent to the regulatory authority where required under applicable

law or r egulation. The intellectual property, information and materials described in or accompanying this document are owned by Monsanto Company, which has filed for or been granted patents on those materials. By submitting this document and any accompanying mat erials, Monsanto Company does not grant any party or entity any right or license to the information, material or intellectual property described or contained in

this submission.CERTIFICATIO N

Th e undersigne d certifie s that

, t o th e bes t knowledg e an d behe f

o f th e undersigned

, thi s petitio n include s al l informatio n an d view s o n whic h t o bas e a determination

, an d tha t i t

include s al l relevan t

dat a an d informatio n know n t o th e petitione r tha t ar e unfavorabl e t o
th e petition

.

Joh n M

. Cordts

, M.S.

, M.B.A

. Regulator y Affair s Manage r
Monsant o Compan y
80 0 Nort h Lindberg h Blvd.

, Mai l Sto p C3S D

St

. Louis

, M O 6316 7

Tel : (314)-694-483 1

Fax : (314)-694-308 0

Monsant o Compan y CR240-13U 1 3 of37 4Monsanto Company CR240

- 13U1 4 of 374 EXECUTIVE SUMMARY

The Animal and Plant Health Inspection Service (APHIS) of the United States ( U.S. ) Department of Agriculture (USDA) has responsibility under the Plant Protection Act (Title IV Pub. L. 106

- 224, 114 Stat. 438, 7 U.S.C. § 7701

- 7772) to prevent the

introductio n and dissemination of plant pests into the U

. S. APHIS regulation

7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and no longer shou ld be regulated. If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction

of the article.

Monsanto Company is submitting this request to APHIS for a determinatio n of nonregulated status for the new biotechnology

- derived maize product, MON 87411 , any progeny derived from crosses between MON 87411 and conventional maize , and any progeny derived from crosses of MON 87411 with biotechnology

- derived maize that

ha ve pre viously been granted nonregulated status under 7 CFR p art 340.

Monsanto Company has developed biotechnology

- derived maize , MON 87411, that

confers protection against corn rootworm (CRW) ( Diabrotica spp .) and tolerance to the herbicide glyphosate . MON 87411 contains a suppression cassette that expresses an inverted repeat sequence de signed to match the sequence of western corn rootworm (WCR; Diabrotica virgifera virgifera ) . The expression of the suppress ion cassette results

in the formation of a double

- stranded RNA (dsRNA) transcript containing a 240 bp fragment of the WCR Snf7 gene ( DvSnf7 ) . Upon consumption, the plant

- produced

ds RNA in MON 87411 is recognized by the CRW’s RNA interference (RNAi)

machin ery resulting in down

- regulation of the targeted DvSnf7 gene leading to CRW

mortalit y . MON 87411 also contains a cry3Bb1 gene that produces a modified Bacillus thuringiensis (subsp. kumamotoensis ) Cry3Bb1 protein to protect against CRW larval feeding

. In addition, MON 87411 contains the cp4 epsps gene from Agrobacterium sp

. strain CP4 that encodes for the 5

- enolpyruvylshikimate

- 3

- phosphate synthase (EPSPS) protein, which confers tolerance to glyphosate, the active ingredient in Roundup Product Description

®

MON 87411 builds upon the current Bt protein

- based mode

- of

- action (MOA) for CRW

control by the addition of a new RNA

- mediated MO A that offers enhanced control of target insect pests and prolong ed durability of existing Bt technologies designed to

c ontrol CRW

. MON 87411 will provide benefits to growers similar to those obtained by use of existing CRW

- protected maize hybrids, which include reduced need for

insecticides and associated improvements in worker safety , increased yield protection,

and wate r conservati on

. MON 87411 is also glyphosate tolerant and will continue to agricultural herbi cides.

® Roundup and Roundup Ready are registered trademarks of Monsanto Technology LLCMonsanto Company CR240

- 13U1 5 of 374 provide benefits associated with conservation tillage methods, including reduced soil erosion, reduced fuel and labor costs, improved air quality and conservation of soil

moisture.

MON 87411 will not be offered for commercial use as a stand

- alone product, but will be combined, through traditional breeding methods, with other deregulated biotechnology

derived traits to provide protection against both above

- ground and below

- ground maize

pe sts as well as tolerance to multiple herbicides. These next generation combined

- trait maize products will offer broader grower choice, improve d production efficienc y
, increase d pest control d urability, a nd enhanced grower profit potential s

.

The data and information presented in this petition demonstrate that MON 87411 is agronomically, phenotypically, and composi tionally comparable to commercially

cultivated maize . Moreover, the data and information presented herein demonstrate that

MON 87411 is not expected to pose an increased plant pest risk, including weediness, compared to commercially cultivated maize . The food, feed, and environmental safety of

MON 87411 was confirmed based on multiple, well

- established lines of evidence: Data and Infor mation Presented Confirms the Lack of Plant Pest Potential and the Food and Feed Safety of MON 87411 Compared to Conventional Maize

**•** The CP4 EPSPS protein in MON 87411 is identical to the CP4 EPSPS protein pr esent in several other commercially available crops that have been reviewed by USDA and previously deregulated ( e.g ., Roundup Ready varieties of soybean, maize , cotton, sugarbeet, canola, and alfalfa). The safety and mode

- of

- action of CP4 EPSPS proteins

is well documented and is the subject of numerous publications

. Similarly, the safety of the Cry3Bb1 protein has been previously assessed in two other corn rootworm

protected products (MON 863 and MON 88017) that have been grown on tens of

millions of acres in t he U.S. since their introduction. The mode

- of

- action of Bt proteins has also been extensively studied and is well

- documented in numerous publications.

**•** The RNA

- based suppression of the Snf7 gene in western corn rootworm that results from expression of the DvSnf7 suppression cassette in MON 87411 is mediated by

dsRNA molecules. Double

- stranded RNAs are commonly used by eukaryotes, including plants, for endogenous gene suppression and as described in this petition, pose no novel risks from a feed/food an d environment perspective. Nucleic acids, as the components of RNA, have a long history of safe consumption and are considered GRAS by the U.S. FDA.

**•** A compositional assessment supports the conclusion that MON 87411 grain and forage are equivalent to grai n and forage of conventional maize

.

**•** Evaluation of the agronomic and phenotypic characteristics of MON 87411

, using

current maize cultivation and management practices

, leads to the conclu sion that deregulation of MON 87411 would not have an effect on maize agronomic practices.Monsanto Company CR240

- 13U1 6 of 374 Maize is grown extensively throughout the world, and is the largest cultivated crop followed by wheat ( Triticum sp

. ) and rice ( Oryza sativa L

. ) in total global production. In

the U.S., maize is grown in al most all states and is the largest crop grown in terms of acreage planted and net value. Maize has been studied extensively, and the domestication of maize can be traced back to approximately 10,000 years ago in southern

Mexico. Maize is a Familiar Crop Lacking Weedy Characteristics

Maize is not listed as a weed in the major literature references on weeds , nor is it present on the lists of noxious weed species published by the federal government (7 CFR Part 360). In addition, maize has been grown throughout the world without any rep ort that it

is a serious weed. Maize is poorly suited to survive without human assistance and is not capable of surviving as a weed due to past selection in the domestication of maize

. During domestication of maize , traits often associated with weediness , such as seed dormancy, a dispersal mechanism, or the ability to establish reproducing populations

outside of cultivation

, have not been selected. Similarly, the history of hybrid breeding in the U.S. does not indicate there are any changes in the charac teristics of maize that would change the weediness profile of the crop. Although maize seed can overwinter into a rotation with soybeans and other crops , mechanical and chemical measures are routinely

used to control maize volunteers. Some populations of wild annual and perennial species

tha t could hybridize with MON 87411 are known to exist in the U.S., however key

d ifferences in several factors such as flowering time, geographical separation, and development timings make natural cro sses in the U.S. highly unlikely.

Based on seed availability and the appropriate fit for various studies, c onventional control materials were developed for use as comparator s in safety assessment studies

. The conventional control materials included the original transformation line (LH244) and two hybrid conventional control lines ( hybrid s MPA640 B and/ or NL6169 ) , both of which

ha ve similar genetic background s to the hybrid MON 87411 test material (LH244 is one parent of each of the control hybrids)

. Both MPA640B (LH244 × LH287) and NL6169 ( LH244 × HCL645 ) w ere used as controls in molecular characterization studies. NL6169

was used as the conventional control in compositional analysis studies while MPA640B

was used as the conventional control in phenotypic, agronomic and environmental

interactions assessment s . Where appropriate, commercial reference maize hybrids were used to establish a range of variability or responses representative of commercial maize (reference hybrids) in the U.S. Conventional Maize MPA640B and NL6169 are Appropriate Comparator s for

MON 87411

MON 874 11 was developed through Agrobacterium

- mediated transformation of maize immature embryos from line LH 244 utilizing plasmid vector PV

- Z MIR10871

. PV

- ZMIR10871 contains one transfer DNA ( T

- DNA ) that is delineated by Left and Right Molecular Characterization Verified the Integrity and Stability of the Inserted DNA

in MON 87411Monsanto Company CR240

- 13U1 7 of 374 Border regions. The T

- DNA contains the DvSnf7 suppression cassette, the cry3Bb1 expression cassette , and the cp4 epsps expression cassette. The DvSnf7 suppression cassette is regulated by the e35S promoter from the 35S RNA of cauliflower mosaic virus (CaMV), the heat shock protein 70 ( Hsp70 ) intron from Zea mays , and the 3' untranslated sequence of the E9 gene from Pisum sativum . The cry3Bb1 expression cassette is regulated by the pIIG promoter from Zea mays , the chlorophyll a/b binding protein (CAB) leader from Triticum aestivum , the Ract1 intron from Oryza sativa , and the heat shock protein 17 ( Hsp17 ) 3 ′ untranslated region from Triticum aestivum

. The cp4 epsps expression cassette is regulated by the T ubA promoter from Oryza sativa , the TubA leader

from Oryza sativa , the TubA intron from Oryza sativa , the CTP2 chloroplast

- targeting sequence from Arabidopsis thaliana , and the TubA 3 ′ untranslated region from Oryza

sativa

.

Characterization of the DNA insert in MON 87411 was conducted using a combination of sequencing, PCR, and bioinformatics. The results of this characterization demonstrate

that MON 87411 contains one copy of the intended T

- DNA containing the DvSnf7 suppression cassette and the cry3Bb1 and cp4 epsps expression cassettes that is stably integrated at a single locus and is inherited according to Mendelian principles over multiple gen erations. These conclusions are based on several lines of evidence:

**•** Molecular characterization of MON 87411 by Next Generation Sequencing and Junction Sequence Analysis (NGS/JSA) demonstrated that DNA from

PV

- ZMIR10871 DNA was integrated at a single locu s in MON 87411.

**•** Directed sequencing (locus

- specific PCR, DNA sequencing and analyses) was performed on MON 87411

, which determined the complete sequence of the single

PV

- ZMIR10871 T

- DNA insert , the adjacent flanking DNA

, and the 5' and 3' insert

to

- flank j unctions. This confirmed that the sequence and organization of the T

- DNA insert is identical to the corresponding region in PV

- ZMIR10871. The sequencing analysis, along with the NGS/JSA result showing that MON 87411 contains only a single DNA insert with no unintended fragments, also confirms that no vector backbone or other unintended plasmid sequences are present in MON 87411. Furthermore, the genomic organization at the insertion site was asse ssed by comparing the sequence s flanking the T

- DNA insert in MON 87411 to the sequence

of the insertion site in conventional maize

. This analysis also assessed potential

rearrangements at the insertion site in MON 87411 upon T

- DNA integration.

**•** Generational stability analysis by NGS/JSA demonstrated that the single T

- DNA

insert in MON 87411 has been maintained through five breeding generations, thereby confirming the stability of the intend ed T

- DNA in MON 87411.

**•** Segregation analyses showed expected heritability that, along with NGS/JSA, demonstrated stability of the T

- DNA insert across multiple generations.

Taken together, the characterization of the genetic modification in MON 87411

demonst rates that a single copy of the intended T

- DNA was stably integrated at a singleMonsanto Company CR240

- 13U1 8 of 374 locus of the maize genome and that no plasmid backbone sequences are present in

MON 87411.

A multistep approach was used to characterize and assess the safety of the CP4 EPSPS and Cry3Bb1 protein s expressed in MON 87411 . The expression level s of the

CP4 EPSPS and Cry3Bb1 protein s in selected tissues of MON 87411 w ere determined and exposure to humans and animals through diet was evaluated

. In addition , the donor organism s for the CP4 EPSPS and Cry3Bb1 protein coding sequence s
, Agrobacterium sp

. strain CP4 and Bacillus thuringiensis ssp kumamotoensis

, are ubiquitous in the

environment and are not commonly known for human or animal pathogenicity or allergenicity. Bioinformatics analysis determined that the CP4 EPSPS and Cry3Bb1 proteins l ack structural similarity to known allergens or protein toxins. As ha s been previously shown in safety assessments of other Roundup Ready and Cry3Bb1

- containing crops , t he CP4 EPSPS and Cry3Bb1 proteins are rapidly digested in simulated digestive fluids and demonstrate no acute oral toxicity in mice at the levels tested. Hence, the consumption of the CP4 EPSPS and Cry3Bb1 proteins from MON 87411 or its progeny poses no meaningful risk to human and animal health or an increased plant pest risk

. Data Confirms CP4 EPSPS and Cry3Bb1 Protein Safety

DvSnf7 RNA from MON 87411 is a dsRNA that upon consumption by w estern corn

rootworm causes gene suppression of the targeted DvSnf7 gene

. In MON 87411, the predominant RNA transcript produced from the suppression cassette was identified as being 968 nucleotides (nt) in length. Because of the extremely low expression of DvSnf7

RNA in MON 87411, it was necessary to produce RNA through in vitro transcription methods in order to obtain sufficient quantities of DvSnf7_968 RNA for subsequent safety studies

. T he molecular characteristics of the MON 87411 DvSnf7 RNA were determined and equivalence between MON 87411 DvSnf7 RNA and in vitro

- produced DvSnf7_968 RNA was demonstrated. This equivalence justifies the use of the

in vitro

- produced DvSnf7_968 RNA as a test substance in studies assessing the specificity and potential impact of DvSnf7 RNA on non

- target organisms

. Tissue specific e xpression studies demonstrated that MON 87411 DvSnf7 RNA was expressed

at mean levels ranging fro m 0.091 × 10 - 3 μg/g fw ( in grain) to 14.4 × 10 - 3 μg/g fw ( in over season leaf at growth stage V14

- R1). Anticipated human dietary exposure to
DvSnf7 RNA is also very low ( ≤ 0.4 ng/kg body weight per day) relative to estimated

total daily RNA intake

. Based on the ubiquitous nature of RNAi suppressio n utilizing

dsRNA in a wide variety of consumed plant species , demonstration of the specificity of DvSnf7 suppression in CRW, the long history of safe consumption of RNA from a range

of sources , and the apparent lack of toxicity or allergenicity of dietary RNA; the DvSnf7

RNA i suppression sequence used in MON 87411 poses no observed or theoretical risks to humans or animals. Therefore, the consumption of the DvSnf7 RNA from

MON 87411 or its progeny is considered safe for humans and animals and poses no increased plant pest risk

. Data Confirms DvSnf7 RNA S afetyMonsanto Company CR240

- 13U1 9 of 374 Compositional analysis was conducted on grain and forage of MON 87411

, a conventional control and 20 different commercial reference hybrids grown at eight representative sites in a 2011/2012 field production in Argentina. Production in the U.S.

c orn belt and Argentina maize

- growing regions occurs at relatively similar latitudes with an approximate 6 month offset. The averag e growing season temperatures and precipitation are comparable and as a result, maize hybrids developed in the U.S. are often used directly by farmers in the southern growing regions of Argentina. As such, compositional analyses from maize grown in Argent ina are appropriate for a comparative safety assessment and study results are relevant to the use of this maize grown in the U.S. MON 87411 is Compositionally Equivalent to Conventional Maize

The compositional analysis, based on the OECD co nsensus document for maize, included measurement of nutrients, anti

- nutrients and secondary metabolites in conventional commercial reference hybrids to provide data on the natural variability of each compositional component analyzed. A total of 78 components were assayed (nine in forage and 69 in grain). Of th e 78 components assayed , 18 had more than 50% of observations that were below the assay limit of quantitation and were therefore excluded from statistical analy sis. Of the 60 remaining components statistically assessed, only 12 components (protein, histidine, tyrosine, oleic acid, neutral detergent fiber, copper, iron, manganese, zinc, niacin, vitamin B1 in grain

, and ash in forage) showed a statistically significant difference between MON 87411 and the conventional control. For these 12 components, the mean difference in component values between MON 87411 and the

conventional control , however, was less than the natural variation found with in the

conventional control and reference hy brid values

. Additionally, MON 87411 mean component values were within the tolerance int erval s of the reference hybrids , the values

for maize observed in the literature, and/or the I nternational L ife S ciences I nstitute C rop Composition Database (ILSI

- CCDB) values. These data indicated that the compositional components with statistically significant differences were not meaningful from a food and feed safety or nutritional perspective.

These results support the overall conclusion that MON 87411 was not a major contributor to variation in component levels in maize grain and forage

, and conf irmed the compositional equivalence of grain and forage from MON 87411 to co nventional maize

. These results support the overall food and feed safety and lack of plant pest risk of

MON 87411.

Plant pest potential of a biotechnology

- derived crop is assessed from the basis of familiarity that the USDA recognizes as an important underlying concept in risk assessment. The concept of familiarity is based on the fact that the biot echnology

derived plant is developed from a conventional plant hybrid or variety whose biological properties and plant pest potential are well known. Familiarity considers the biology of the plant, the introduced trait, the receiving environment, and the interactions among these factors. This provides a basis for comparative risk assessment between a MON 87411 Does Not Change Maize Plant Pest Potentia l or Environm ental

InteractionsMonsanto Company CR240

- 13U1 10 of 37 4 biotechnology

- derived plant and the conventional control. Thus, the phenotypic, agronomic, and environmental interaction assessment of MON 87411 included a genetically sim i lar conventional control as a comparator. This evaluation used a weight of evidence approach and considered statistical differences between MON 87411 and the conventional control with respect to reproducibility, magnitude, and directionali ty

. Comparison to a range of commercial reference hybrids grown concurrently established the range of natural variability for maize , and provided a context from which to further evaluate any observed statistical differences. Cha racteristics assessed incl uded: seed dormancy and germination, pollen morphology , and plant phenotypic observations and environmental interaction evaluations conducted in the field. The phenotypic, agronomic, and environmental interaction assessment demonstrated that MON 87411 is comparable to the conventional control. Thus, MON 87411 is not expected to have increased weediness or plant pest risk compared to conventional maize

.

Seed dormancy and germination characterization indicated that MON 87411 seed had dormancy and germination characteristics similar to seed of the conventional control . In particular, the lack of hard seed, a well recognized seed characteristic associated w ith weediness, supports a conclusion of no increased weediness of MON 87411 compared to

the conventional control . For pollen characteristic assessments, there were no statistically significant differences (α=0.05) detected between MON 87411 and the

conven tional control for pollen viability and diameter, and no visual differences in general pollen morphology were observed.

The field evaluation of phenotypic, agronomic, and environmental characteristics also supports the conclusion that MON 87411 is not lik ely to have increased weediness or plant pest potential compared to conventional maize. Evaluations were conducted at nine replicated field sites across the U.S. corn belt . These assessments included 13 plant growth and development characteristics, as we ll as observations for plant responses to abiotic stressors and plant

- disease and plant

- arthropod interactions. The observed phenotypic characteristics were comparable between MON 87411 and the conventional control. Across sites, data show no statistical ly significant differences between

MON 87411 and the conventional control for any of the assessed characteristics, including early stand count, days to 50% pollen shed and silking, stay green, ear height, plant height, dropped ears, stalk and root lodging, final stand count, grain moisture, test weight, and yield. Thus, the phenotypic characteristics of MON 87411 were not altered in terms of pest/weed potential compared to the conventional control

.

In an assessment of abiotic stress response and disease da mage, no differences were

observed between MON 87411 and the conventional control for any of the 100 comparisons for the assessed abiotic stressors or for any of the 119 comparisons for the

assessed diseases among all observations across the sites . In an assessment of arthropod

related damage, no differences were detected between MON 87411and the conventional control for any of the 102 comparisons for the assessed arthropods. Additionally, no statistically significant differences were detected across sites between MON 87411 and the conventional control for quantitative evaluations of corn earworm or European corn borer damage. The lack of differences in plant response to abiotic stress, disease damage, and arthropod

- related damage support the conclusion th at the introduc ed traits inMonsanto Company CR240

- 13U1 11 of 374 MON 87411 are not expected to pose an increased plant pest/weed potential compared to

the conventional control

.

In an assessment of arthropod abundance collected using sticky trap s , no statistically significant differences wer e detected between MON 87411 and the conventional control plots for 104 out of 108 comparisons among the collections at the four sites where these evaluations were made. The mean arthropod abundance values from MON 87411 were within the respective range o f reference hybrids for one of the four detected differences. For the remaining three differences, the mean abundance values for MON 87411 were outside of the reference range; however, these differences were not consistent across

collection times or sites. These results are not indicative of a consistent response

associated with the traits and are not considered biologically meaningful in terms of increased pest/weed potential of MON 87411 compared to the conventional control

.

In an assessment of arthropod abundance from visual counts , no statistically significant

differences were detected between MON 87411 and the conventional control for 60 out of 61 comparisons among the collections at the four sites where these evaluations were

made. For the single detected difference, the mean ab undance value for MON 87411 was

outside of the reference range; however, this difference was not consistent across

collections. Thus, this difference was not indicative of a consistent response as sociated

with the tr aits and is not considered biologically meaningful in terms of increased pest/weed potential of MON 87411 compared to the conventional control

.

Separate studies demonstrated the efficacy of MON 87411 against two different CRW species and low root feeding d amage ratings to MON 87411 hybrids in CRW

- infested

fields.

In summary, the phenotypic, agronomic, and environmental interaction data were

evaluated to characterize MON 87411

, and to assess whether the traits introdu ced in

MON 87411 alter the plant pest potential compared to conventional maize . The evaluation, using a weight of evidence approach, considered the reproducibility, magnitude, and direction of detected differences between MON 87411 and the

conventional control , and compar ison to the range of the commercial reference hybrids

. Results from the phenotypic, agronomic, and environmental interactions assessment

indicate that MON 87411 does not possess enhanced weed iness characteristics, increased susceptibility or tolerance to specific abiotic stress ors , diseases, or arthropods, or characteristics that would confer a plant pest risk compared to conventional maize

.

An evaluation of the impacts of MON 87411 on non

- target organisms (NTOs) is a component of the plant pest risk assessment. The NTO assessment has taken into consideration a number of characteristics of the expressed products in MON 87411 to evaluate potential hazards t o NTOs, including threatened and endangered species and organisms beneficial to agriculture. Characteristics evaluated included MOA, spectrum of insecticidal activity and exposure levels to the CP4 EPSPS and Cry3Bb1 proteins and MON 87411 Will Not Negatively Affect Non

- target Organism s Including Those Beneficial to AgricultureMonsanto Company CR240

- 13U1 12 of 374 DvSnf7 RNA. Both the CP4 EPSPS and Cry3Bb1 proteins have been assessed in multiple products by USDA

- APHIS , U.S. FDA, and U.S. EPA in past years. Cry3Bb1 protein is produced in both MON 863 and MON 88017 that were granted non

- regulated status and comparable levels of this protein are produced in MON 87411. Additionally

, starting in 1994 with Monsanto’s 40

- 3

- 2 soybean, a number of Roundup Ready crops (canola, maize, sugarbeet, cotton, alfalfa) containing CP4 EPSPS protein s have been

g ranted non

- regulated status by USDA

- APHIS

. Afte r both extensive testing and

widescale commercial cultivation, i n n o instance have adverse impacts to NTOs been associated with exposure to Cry3Bb1 or CP4 EPSPS proteins from these biotechnology

derived crop s

.

As noted previously, t h e suppression cassette in MON 87411 contains the CaMV e 35S promoter, maize hsp70 intron, the two DvSnf7 p sequences (240 nt each) separated by a 150 bp intervening sequence, and a pea E9 3’ untranslated region. When the suppression

cassette is transcribed

, the predominant RNA transcript expressed is 968 nt in length and forms a hairpin loop

, thereby allowing the formation of the 240 bp DvSnf7 ds RNA. When consumed by CRW, th is 240 bp DvSnf7 dsRNA activates the RNAi process leading to suppression of the targeted CRW Snf7 gen e.

To address potential impacts to NTOs, s pecific laboratory bioassay studies using DvSnf7_240 dsRNA , the active insecticidal product in MON 87411, were conducted on a variety of NTOs including several Coleopteran, two Hymenopteran, one Hemipteran and

fo ur Lepidopteran species. No impacts to survival, growth, or development of these species were noted when fed extremely high doses ( relative to level s present in

MON 87411 ) of DvSnf7_240 dsRNA over multi

- day bioassay periods.

Additional NTO assessments w ere conducted on a battery of organisms based on recommendations p ublished by the U.S. EPA. Organisms tested included earthworm, honeybee, parasitic wasp, ladybird beetle, carabid beetle and the insidious flower bug. In these studies, test concentrations were based on the measured DvSnf7 RNA expression in the tissue types that the NTO would most likely be exposed to in the environment. Based on U.S. EPA recommendations, a targeted margin of exposure (MOE) of gr eater than 10

- times the maximum expected environmental concentration (MEEC) was used to establish

test concentrations. MOEs that exceed 10 are considered as indicative of minimal risk in

worst

- case laboratory assays by U.S. EPA. In all cases where MOEs c ould be calculated, they were >10

- fold the predicted exposure level for these species, indicating that DvSnf7 RNA is not likely to have effects on terrestrial beneficial invertebrate species at field exposure levels.

Additional assessments for potential exposure of aquatic organisms and threatened and endangered species to DvSnf7 RNA contained in MON 87411 conclude that due to the lack of proximity of these organisms to maize cultivation, lack of relevant exposure because of feeding ecology and the restri cted activity of the DvSnf7 RNA, that

cultivation of MON 87 4 11 will have no e ffect on these species or their habitats

.Monsanto Company CR240

- 13U1 13 of 374 An assessment of current maize agronomic practices was conducted to determine whether

the cultivation of MON 87411 has the potential to impact current maize agronomic practices. Maize fields are typically h ighly managed areas that are dedicated to grain and/or forage production. Deregulation of MON 87411 is Not Expected to Have Effects on Maize Agronomic

Practices

MON 87411 was developed to provide two effective MOA s against the target corn

rootworm pests of maize in the U.S. c orn b elt as well as glyphosate tolerance in a single product. As tolerance to glyphosate and protection from corn rootworm complex pests are present in ma ny currently available maize hybrids that have been widely grown in the

U.S. since 2003 , the introduction of MON 87411 is expected to have no impact on

cu rrent agronomic or management practices for maize. As phenotypic evaluations,

evaluations of stress r esponses, and pest/disease susceptibility showed no difference

between MON 87411 and reference hybrids (other than protection from CRW larval feeding), n o changes are anticipated in crop rotations, tillage practices, planting practices, fertility managemen t, weed and disease management, and volunteer management from

the introduction of MON 87411.

MON 87411 is similar to conventional maize in its agronomic, phenotypic, environmental, and compositional characteristics and has naturally occurring levels of

p rotection against pests (other than CRW) and diseases co mparable to and typical of

conventional commercial maize hybrids . Ba sed on this assessment, the introduction of

MON 87411 is not expected to result in changes or impacts to current maize agronomic practices.

Based on the data and information presented in this petition, it is concluded that

MON 87411 is not expected to be a plant pest. Therefore, Monsanto Company requests a

determination from USDA

- APHIS that MON 87411 and any progeny derived from

crosses between MON 87411 and conventional maize or deregulated biotechnology

derived maize be granted nonregulated status under 7 CFR p art 340. ConclusionMonsanto Company CR240

- 13U1 14 of 374 TABLE OF CONTENTS

REL EASE OF INFORMATION

................................ ................................ ...................... 2

CERTIFICATION

................................ ................................ ................................ ............. 3

EXECUTIVE SUMMARY

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

TABLE OF CONTENTS

................................ ................................ ................................ 14

LIST OF TABLES

................................ ................................ ................................ ........... 20

LIST OF FIGURES

................................ ................................ ................................ ......... 23

ABBREVIATIONS AND DEFINITIONS

................................ ................................ ..... 25

I. RATIONALE FOR THE DEVELOPMENT OF MON 87411

............................... 28

I.A. Basis for the Request for a Determination of Nonregulated Status under

7 CFR § 340.6

................................ ................................ ................................ ............... 28

I.B. Rationale for the Development of Insect

- Protected and Glyphosate

Tolerant Maize MON 87411

................................ ................................ ......................... 28

I.B.1 Benefits of Insect

- Protection and Herbicide Tolerance Traits

...................... 29

I.B.2. Introduction of Insect

- Protection Traits in the U.S.

................................ .... 30

I.B.3. Development of CRW

- Protected and Glyphosate Tolerant Maize

MON 87411

................................ ................................ ................................ ........... 30

I.C. RNA Interference ( RNAi)

................................ ................................ ..................... 31

I.C.1. Applications of RNAi in Plants

................................ ................................ .. 31

I.C.2. Applications of RNAi in Insects

................................ ................................ . 32

I.D. Modes

- of

- Action of the Inserted Genetic Components

................................ ........ 32

I.D.1. Mode

- of

- Action of the RNAi Component of MON 87411

........................ 32

I.D.2. Modes

- of

- Action of the CP4 EPSPS a nd Cry3Bb1 Proteins

...................... 35

I.E. Product Efficacy

................................ ................................ ................................ .... 35

I.F. Submissions to Other Regulatory Agencies

................................ .......................... 36

I.F.1. Submission to FDA

................................ ................................ ..................... 36

I.F.2. Submission to EPA

................................ ................................ ...................... 36

I.F.3. Submissions to Foreign Government Agencies

................................ .......... 37

II. THE BIOLOGY OF MAIZE

................................ ................................ .................... 38

II.A. Maize as a Crop

................................ ................................ ................................ ... 38

II.B. Characteristics of the Recipient Plant

................................ ................................ .. 39

II.C. Maize as a Test System in Product Saf ety Assessment

................................ ....... 39

III. DESCRIPTION OF THE GENETIC MODIFICATION

................................ .... 41

III.A. The Plasmid Vector PV

- ZMIR10871

................................ ................................ . 41

III.B. Description of the Transformation System

................................ ......................... 41Monsanto Company CR240

- 13U1 15 of 374 III.C. The cry3Bb1 Coding Sequence and the Cry3Bb1 Protein

................................ .. 45

III.D. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein

........................... 45

III.E. DvSnf7 p sequence

................................ ................................ ............................... 45

III.F. Regulatory Sequences

................................ ................................ ......................... 45

III.G. T

- DNA Borders

................................ ................................ ................................ .. 46

III.H. Genetic Elements Outside of the T

- DNA Borders

................................ ............. 46

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION

..................... 52

IV.A. Determining the Number of DNA inserts in MON 8741 1

................................ . 61

IV.A.1. Next Generation Sequencing (NGS) for MON 87411 and

Conventional Control Genomic DNA

................................ ................................ ... 62

IV.A.2 Characterization of insert number in MON 87411 using Bioinformatic Analysis

................................ ................................ .......................... 64

IV.B. Organization and Sequence of the Insert and Adjacent Flanking DNA in

MON 87411

................................ ................................ ................................ ................... 68

IV.C. Sequencing of the MON 87411 Insertion Site

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

IV.D. Determination of Insert Stability over Multiple Generations of

MON 87411

................................ ................................ ................................ ................... 73

IV.D.1 Determination of the Insert Number

................................ ......................... 73

IV.E. Inheritance of the Genetic Insert in MON 87411

................................ ............... 77

IV.F. Characterization of the Genetic Modification Summary and Conclusion

.......... 81

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE Cry3Bb1 and CP4 EPSPS PROTEINS PRODUCED IN MON 87411

....................... 82

V.A. Identity and Function of the Cry3Bb1 and CP4 EPSPS Proteins from

MON 87411

................................ ................................ ................................ ................... 82

V.A.1. Identity and Function of the Cry3Bb1 Protein from MON 87411

............. 82

V.A.2. Identity and Function of the CP4 EPSPS Protein from MON 87411

........ 83

V.B. Characterization and Equivalence of Cry3Bb1 and CP4 EPSPS Proteins

from MON 87411

................................ ................................ ................................ .......... 84

V.C. Expres sion Levels of Cry3Bb1 and CP4 EPSPS Proteins in MON 87411

......... 84

V.C.1. Expression Levels of Cry3Bb1 Protein

................................ ..................... 85

V.C.2. Expression Levels of CP4 EPSPS Protein

................................ ................ 87

V.D. Assessment of Potential Allergenicity of the Cry3Bb1 and CP4 EPSPS

Proteins

................................ ................................ ................................ .......................... 90

V.D.1. Assessment of Potential Allergenicity of the Cry3Bb1 Protein

................ 90

V.D.2. Assessment of Potential Allergenicity of the CP4 EPSPS Protein

........... 90

V.E. Safety Assessment Summary of Cry3Bb1 and CP4 EPSPS Proteins in

MON 87411

................................ ................................ ................................ ................... 91Monsanto Company CR240

- 13U1 16 of 374 V.E.1. Cry3Bb1 Donor Organism, History of Safe Use, and Specificity

............. 91

V.E.2 CP4 EPSPS Donor Organism, History of Safe Use, and Specificity

......... 92

V.E.3. Cry3Bb1 and CP4 EPSPS Proteins in MON 87411 are Not Homologous to Known Allergens or Toxins

................................ ......................... 93

V.E.4. Cry3Bb1 and CP4 EPSPS Proteins in MON 87411 are La bile in in

vitro Digestion Assays

................................ ................................ ........................... 93

V.E.5. Cry3Bb1 and CP4 EPSPS Proteins in MON 87411 are Not Acutely Toxic

................................ ................................ ................................ ......... 93

V.E.6. Human and Animal Exposure to the Cry3Bb1 and CP4 EPSPS

Proteins

................................ ................................ ................................ .................. 93

V.E.7. CP4 EPSPS Activity

................................ ................................ .................. 95

V.E.8. Cry3Bb1 Activity

................................ ................................ ...................... 95

V.E.9. Non

- Target Assessment for CP4 EPSPS Protein

................................ ....... 95

V.E.10. Non

- Target Assessment for Cry3Bb1 Protein

................................ .......... 95

V.F. Cry3Bb1 and CP4 EPSPS Proteins Characterization and Safety

Conclusion

................................ ................................ ................................ ..................... 97

VI. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE

DvSnf7 RNA PRODUCED IN MON 87411

................................ ................................ .. 98

VI.A. History of Safe Use of RNA

- mediated Gene Suppression in Plants

.................. 98

VI.B. Characterization and Equivalence of DvSnf7 RNA from MON 87411

........... 10 0

VI.C. Expression Levels of DvSnf7 RNA in MON 87411

................................ ........ 101

VI.D. Human and Animal Exposure to the DvSnf7 RNA

................................ ......... 104

VI.E. Laboratory Tests to Characterize the Spectrum of Activity of DvSnf7

RNA

................................ ................................ ................................ ............................. 105

VI.E.1. RNAi in Ins ects

................................ ................................ ...................... 105

VI.E.2. Test Substances Used to Assess the Activity Spectrum of DvSnf7

RNA

................................ ................................ ................................ ..................... 105

VI.E.3. Results from Activity Spectrum Bioassays for DvSnf7 RNA

............... 106

VI.E.4. Non

- Target Organism Assessment for DvSnf7

................................ ...... 109

VI.F. Characterization and Safety Conclusions

................................ ......................... 112

VII. COMPOSITIONAL ASSESSMENT OF MON 87411

................................ ...... 113

VII.A. Compositional Equivalence of MON 87411 Grain and Forage to

Conventional Maize

................................ ................................ ................................ .... 114

VII.A.1. Nutrient Levels in Maize Grain

................................ ............................ 116

VII.A.2. Anti

- Nutrient Levels i n Maize Grain

................................ ................... 121

VII.A.3. Secondary Metabolites Levels in Maize Grain

................................ .... 121

VII.A.4. Nutrient Levels in Maize Forage

................................ .......................... 122Monsanto Company CR240

- 13U1 17 of 374 VII.B. Compositional Assessment of MON 87411 Conclusion

................................ 139

VIII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

................................ ................................ ............... 140

VIII.A. Characteristics Measured for Assessment

................................ ..................... 140

VIII.B. Interpretation of Phenotypic and Environmental Interaction Data

................ 144

VIII.B.1. Interpretation of Detected Differences Criteria

................................ ... 145

VIII.B.2. Interpretation of Environmental Interactions Data

.............................. 146

VIII.C. Comparative Assessments of the Phenotypic, Agronomic, and

Environmental Interaction Characteristics of MON 87411

................................ ........ 147

VIII.C.1. Seed Dormancy and Germination Characteristics

............................... 147

VIII.C.2. Field Phenotypic, Agronomic, and Environmental Interactions

Characteristics

................................ ................................ ................................ ...... 150

VIII.C.3. Pollen Characteristics

................................ ................................ .......... 161

VIII.D. Conclusions for Phenotypic, Agronomic, and Environmental

Interactions Evaluation

................................ ................................ ................................ 162

IX. U.S. AGRONOMIC PRACTICES

................................ ................................ ........ 164

IX.A. Introduction

................................ ................................ ................................ ...... 164

IX.B. Overview of U.S. Maize Production

................................ ................................ 164

IX.B.1. Maize Production

................................ ................................ ................... 164

IX.C. Production Management Considerations

................................ .......................... 166

IX.D. Management of Insect Pests

................................ ................................ ............. 166

IX.D.1. Insect Resistance Management

................................ .............................. 170

IX.E. Management of Diseases and Other Pests

................................ ........................ 171

IX.F. Weed Management

................................ ................................ ........................... 171

IX.F.1. Weed Resistance Management

................................ ............................... 173

IX.G. Crop Rotation Practices in Maize

................................ ................................ ..... 173

IX.H. Maize Volunteer Management

................................ ................................ ......... 174

IX.I. Stewardship of MON 87411

................................ ................................ .............. 174

IX.J. Impact of the Introduction of MON 87411 on Agricultural Practices

.............. 175

X. PLANT PEST ASSESSMENT

................................ ................................ ................ 176

X.A. Characterist ics of the Genetic Insert and Expressed Products

.......................... 176

X.A.1. Genetic Insert

................................ ................................ .......................... 176

X.B. Mode

- of

- Action

................................ ................................ ................................ . 177

X.C. Expr ession and Characterization of Gene Products

................................ ........... 177

X.C.1. Protein Safety and Expression Levels

................................ ..................... 177Monsanto Company CR240

- 13U1 18 of 374 X.C.2. RNA Safety and Expression Levels

................................ ........................ 178

X.D. Compositional Characteristics

................................ ................................ ........... 178

X.E. Phenotypic, Agronomic, and Environmental Interaction Characteristics

.......... 179

X.F. Weediness Potential of MON 87411

................................ ................................ .. 179

X.F.1. Seed Dormancy and Germination

................................ ............................ 180

X.F.2. Plant Growth and Development

................................ ............................... 180

X.F.3. Pollen Morphology and Viability

................................ ............................ 181

X.G. Impact to Non

- Target Organisms, Including Those Beneficial to Agriculture

................................ ................................ ................................ ................... 181

X.G.1. Impact on Threatened and Endangered Species

................................ ...... 181

X.H. Environmental Fate of CRW Products Expressing DvSnf7_968

...................... 182

X.I. Potential for Pollen Mediated Gene Flow and Introgression

.............................. 183

X.I.1. Hybridization with Cultivated Maize

................................ ....................... 183

X.I.2. Hybridization with Wild Annual Species of Zea mays subsp.

mexicana

................................ ................................ ................................ .............. 192

X.I.3. Hybridization with the Wild Perennial Species of Subgenus Tripsacum

................................ ................................ ................................ ............ 192

X.J. Transfer of Genetic Information to Species wit h which Maize Cannot Interbreed (Horizontal Gene Flow)

................................ ................................ ............. 192

X.K. Potential Impact on Maize Agronomic Practices

................................ .............. 193

X.L. Conventional Breeding with Other Biotechnology

- derived or

Conventional Maize

................................ ................................ ................................ .... 193

X.M. Summary of Plant Pest Assessments

................................ ................................ 194

XI. ADVERSE CONSEQUENCES OF INTRODUCTION

................................ ..... 196

REFERENCES

................................ ................................ ................................ ............... 197

APPENDICES

................................ ................................ ................................ ................ 213

Appendix A: USDA Notifications and Permits

................................ .......................... 214

Appendix B: Materials, Methods, and Supplementary Results for Molecular Analyses of MON 87411

................................ ................................ ................................ 218

Appendix C: Materials, Methods and Results for Characterization of Cry3Bb1 and CP4 EPSPS Proteins Produced in MON 87411

................................ .. 250

Appendix D: Materials and Methods Used for the Analysis of the Levels of Cry3Bb1 and CP4 EPSPS Proteins in MON 87411

................................ .................... 280

Appendix E: Materials, Methods and Results for Characterization of the

DvSnf7 RNA Produced in MON 87411

................................ ................................ ........ 285

Appendix F: Materials and Methods Used for the Analysis of Expression

Levels of DvSnf7 RNA in MON 87411

................................ ................................ ......... 294Monsanto Company CR240

- 13U1 19 of 374 Appendix G: Materials, Methods, and Individual Site Results for Compositional Analysis of MON 87411 Maize Grain and Forage

............................ 300

Appendix H: Materials, Methods, and Individual Site Results for Seed Dormancy and Germination Assessment of MON 87411

................................ .......... 316

Appendix I: Materials, Methods, and Individual Site Results from Phenotypic, Agronomic, and Environmental Interaction Assessment of

MON 87411 under Field Conditions

................................ ................................ ............ 323

Appendix J: Materials and Methods for Pollen Morphology and Viability

Assessment

................................ ................................ ................................ ...................... 358

Appendix K: Summary of Non

- target Organism Studies

................................ ......... 362

Appendix L: Next

- Generati on Sequencing and Junction Sequence Analysis for the Characterization of DNA Inserted into Crop Plants

................................ ...... 368Monsanto Company CR240

- 13U1 20 of 374 LIST OF TABLES

Table III

- 1. Summary of Genetic Elements in PV

- ZMIR10871

................................ . 47

Table IV

- 1. Summary of Genetic Elements in MON 87411

................................ ........ 59

Table IV

- 2. Sequencing (NGS) Conducted for MON 87411 and Control

Genomic DNA

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

Table IV

- 3. Summary of NGS Data for the Conventional Control DNA Sample Spiked with PV

- ZMIR10871 DNA

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

Table IV

- 4. Unique Junction Sequence Class Results

................................ ................. 65

Table IV

- 5. Junction Sequence Classes Detected

................................ ......................... 73

Table IV

- 6. Segregation of the T

- DNA During the Development of

MON 87411 1:1 Segregation

................................ ................................ ........................... 80

Table IV

- 7. Segregation of the T

- DNA During the Development of

MON 87411 1:2:1 Segregation

................................ ................................ ........................ 80

Table V

- 1. Summary of Cry3Bb1 Protein Levels in Tissues from

MON 87411 Grown in 2011

– 2012 Argentina Field Trials

................................ ......... 86

Table V

- 2. Summary of CP4 EPSPS Protein Levels in Tissues from

MON 87411 Grown in 2011

– 2012 Argentina Field Trials

................................ ......... 88

Table VI

- 1. Summary of DvSnf7 RNA Levels in Maize Tissues Collected

from MON 87411 Produced in Argentina Field Trials during 2011

- 2012

............... 1 02

Table VI

- 2. Susceptibility to DvSnf7_240 dsRNA in laboratory bioassays

.............. 108

Table VI

- 3. Maximum expected environmental concentrations (MEECs), no observed effect concentrations (NOECs) from non

- target organism studies and estimated marg ins of exposure (MOEs) for DvSnf7 RNA

................................ .. 111

Table VII

- 1. Summary of Maize Grain Protein and Amino Acids for

MON 87411, Conventional Co ntrol, and Reference Hybrids

................................ .... 123

Table VII

- 2. Summary of Maize Grain Fat and Fatty Acids for MON 87411, Conventional Control, and R eference Hybrids

................................ ........................... 127

Table VII

- 3. Summary of Maize Grain Carbohydrates by Calculation and

Fiber for MON 87411, Conventional Control , and Reference Hybrids

.................... 129

Table VII

- 4. Summary of Maize Grain Ash and Minerals for MON 87411, Conventional Control, and Reference Hybrids

................................ ........................... 130

Table VII

- 5. Summary of Maize Grain Vitamins for MON 87411, Conventional Control, and Reference Hybrids

................................ ........................... 132

Table VII

- 6. Summary of Maize Grain Anti

- nutrients and Secondary

Metabolites for MON 87411, Conventional Control, and Reference Hybrids

......... 134

Table VII

- 7. Summary of Maize Forage Proximates, Fiber and Minerals for

MON 87411, Conventional Control, and Reference Hybrids

................................ .... 135Monsanto Company CR240

- 13U1 21 of 374 Table VII

- 8. Literature and ILSI Database Ranges for Components in Maize Forage and Grain

................................ ................................ ................................ ........... 137

Table VIII

- 1. Phenotypic, Agronomic, and Environmental Interaction Characteristics Evaluated in U.S. Field Trials and Laboratory Studies

.................. 142

Table VIII

- 2. Germination Characteristics of MON 87411 and the

Conventional Control

................................ ................................ ................................ .... 149

Table VIII

- 3. Field Phenotypic Evaluation Sites for MON 87411 during 2012

...... 152

Table VIII

- 4. Combined

- Site Comparison of MON 87411 to Conventional Control for Phenotypic and Agronomic Characteristics During 2012

..................... 153

Table VIII

- 5. Summary of Qualitative Environmental Interactions Assessments during 2012

................................ ................................ ............................... 156

Ta ble VIII

- 6 Combined

- Site Comparison of Pest Damage to MON 87411 Compared to the Conventional Control during 2012

................................ ................. 159

Table VIII

- 7. Summary of Arthropod Abundance Assessments and Detected Differences during 2012

................................ ................................ ................................ . 160

Table VIII

- 8. Pollen Characteristics of MON 8 7411 Compared to the Conventional Control during 2012

................................ ................................ ............... 162

Table IX

- 1. Maize Production in the U.S., 2007

- 2012

................................ ............... 165

Table IX

- 2. Insect Pests in Maize in the North Central States and Typical Time of Damage

................................ ................................ ................................ ............. 167

Table IX

- 3. Insecticide Applications in Maize in 2010 in the U.S.

........................... 168

Table IX

- 4. Troublesome Weeds in Maize

................................ ................................ . 172

Table X

- 1. Summary of Published Literature on Maize Cross Pollination

............. 185

Table A

- 1. USDA Notifications and Permits Approved for MON 87411 and

Status of Trials Planted under These N otifications

................................ .................... 215

Table C

- 1. N

- Terminal Sequence of the MON 87411

- produced Cry3Bb1

.............. 252

Table C

- 2. Summary of the Tryptic Masses Identified for the

MON 87411

- Produced Cry3Bb1 Using MALDI

- TOF MS

................................ ........ 254

Table C

- 3. Comparison of Immunoreactive Signal Between MON 87411

- and E coli

- produced Cry3Bb1 Proteins

................................ ................................ ....... 258

Table C

- 4. Molecular Weight Comparison Between the MON 87411

- and

E. coli

- produced Cry3Bb1 Proteins

................................ ................................ .............. 258

Table C

- 5. Cry3Bb1 Functional Activity Assay

................................ ......................... 264

Table C

- 6. N

- Terminal Sequence of the MON 87411

- produced CP4EPSPS

.......... 267

Table C

- 7. Summary of the Tryptic Masses Identified for the

MON 87411

- produced CP4 EPSPS Using MA LDI

- TOF MS

................................ .... 269

Table C

- 8. Comparison of Immunoreactive Signal Between MON 87411

- and E coli

- produced CP4 EPSPS Protein

................................ ................................ ... 273Monsanto Company CR240

- 13U1 22 of 374 Table C

- 9. Molecular Weight Comparison Between the MON 87411

- and

E. coli

- produced CP4 EPSPS Proteins

................................ ................................ ......... 274

Table C

- 10. CP4 EPSPS Functional Activity Assay

................................ .................. 278

Table D

- 1. Cry3Bb1 Protein Extraction Methods 1 for Tissue Samples

................... 281

Table D

- 2. CP4 EPSPS Protein Ex traction Methods 1 for Tissue Samples

.............. 281

Table G

- 1. Conventional Commercial Reference Maize Hybrids

............................ 300

Table G

- 2. Re

- expression Formulas for Statistical Analysis of Composition

Data

................................ ................................ ................................ ................................ . 310

Table H

- 1. Starting Seed of MON 87411, Conventional Control and Commercial Maize Reference Hybrids Used in Dormancy Assessment

................... 319

Table H

- 2. Germination and Dormancy Characteristics of MON 87411 and the Conventional Control Seed (Selfed F2 Grain) Produced at each of the

Three Field Sites

................................ ................................ ................................ ............. 320

Table I

- 1. Starting Seed for Phenotypic, Agronomic, and Environmental

Interaction Assessment

................................ ................................ ................................ .. 325

Table I

- 2. Field and Planting Information

................................ ................................ . 326

Table I

- 3. Data Missing or Excluded from Analysis

................................ .................. 334

Table I

- 4. Individual Site Phenotypic Comparison of MON 87411 C ompared

to the Conventional Control

................................ ................................ .......................... 336

Table I

- 5. Qualitative Assessment: Abiotic Stressor Evaluations Using a Categorical Scale fo r MON 87411 and the Conventional Control

............................ 341

Table I

- 6. Qualitative Assessment: Disease Damage Evaluations Using a Categorical Scal e for MON 87411 and the Conventional Control

............................ 342

Table I

- 7. Qualitative Assessment: Arthropod Damage Evaluations Using a Categorical Scale for MON 87411 and the Conventional Control

............................ 343

Table I

- 8. Individual

- Site Analysis: Quantitative Assessment of Co rn Earworm and European Corn Borer Damage to MON 87411 Compared to

the Conventional Control

................................ ................................ .............................. 344

Table I

- 9. Individual

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411 Compared to the Conventional Control

....... 345

Table I

- 10. Individual

- Site Analysis: Arthropod Abundance in Visual Counts

from MON 87411 Compared to the Conventional Control

................................ ....... 351

Table J

- 1. Starting Se ed for Pollen Morphology and Viability Assessment

............. 359Monsanto Company CR240

- 13U1 23 of 374 LIST OF FIGURES

Figure I

- 1. Diagram of MON 87411 dsRNA Oral Delivery to Suppress DvSnf7 Expression via the RNAi Pathway in CRW

................................ ..................... 33

Figure I

- 2. Model depicting e ndosomal

- autophagy pathway involved in intracellular sorting and degradation of receptors along with other macromolecules in a normal cell (left) and a DvSnf7 deficient cell (right).

............... 34

Figure III

- 1. Circular Map of PV

- ZMIR10871

................................ ........................... 43

Figure III

- 2. Schematic of th e Development of MON 87411

................................ ...... 44

Figure III

- 3. Deduced Amino Acid Sequence of the Cry3Bb1 Protein

...................... 51

Figure III

- 4. Deduced Amino Acid Sequence of the CTP2 Targeting Sequence and CP4 EPSPS Protein

................................ ................................ ................. 51

Figure IV

- 1. Molecular Characterization using Sequencing and

Bioinformatics

................................ ................................ ................................ .................. 53

Figure IV

- 2. Junctions and Junction Sequences

................................ .......................... 54

Figure IV

- 3. Two Unique Junction Sequence Classes are Produced by the Insertion of a Single Plasmid Region

................................ ................................ ............. 54

Figure IV

- 4. Breeding History of MON 87411

................................ ............................. 57

Figure IV

- 5. Schematic Representation of the Insert and Flanking Sequences

in MON 87411

................................ ................................ ................................ .................. 58

Figure IV

- 6. Junction Sequences Detected by NGS/JSA

................................ ............ 66

Figure IV

- 7. Analysis of Overlapping PCR Products Across the MON 87411

Insert

................................ ................................ ................................ ................................ . 69

Figure IV

- 8. PCR Amplificatio n of the MON 87411 Insertion Site

........................... 72

Figure IV

- 9. Junction sequences detected by JSA. Junction Sequence Class A Alignment (All Ge nerations Tested)

................................ ................................ ........... 75

Figure IV

- 10. Junction sequences detected by JSA. Junction Sequence Class B Alignment (All Generations Teste d)

................................ ................................ ........... 76

Figure IV

- 11. Breeding Path for Generating Segregation Data for

MON 87411

................................ ................................ ................................ ....................... 79

Figure VIII

- 1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods

................................ ................................ ................................ . 145

Figure IX

- 1. Planted Maize Acres by County in the U.S. in 2012

............................ 165

Figure IX

- 2. Maize acres in the U.S. planted to hybrids containing Bt proteins for corn rootworm protection

................................ ................................ ........ 170

Figure C

- 1. MALDI

- TOF MS Coverage Map of the MON 87411

- produced Cry3Bb1

................................ ................................ ................................ .......................... 255

Figure C

- 2. Western Blot Analysis of MO N 87411

- and E. coli

- produced Cry3Bb1 Proteins

................................ ................................ ................................ ........... 257Monsanto Company CR240

- 13U1 24 of 374 Figure C

- 3. Molecular Weight and Purity Analysis of the

MON 87411

- produced Cry3Bb1 Protein

................................ ................................ ..... 259

Figure C

- 4. Glycosylation Analysis of the MON 87411

- produced Cry3Bb1

Protein

................................ ................................ ................................ ............................. 262

Figure C

- 5. MALDI

- TOF MS Coverage Map of the MON 87411

- Produced

CP4 EPSPS Protein

................................ ................................ ................................ ........ 270

Figure C

- 6. Western Blot Analysis of MON 87411

- and E. coli

- Produced

CP4 EPSPS Proteins

................................ ................................ ................................ ...... 272

Figure C

- 7. Molecular Weight and Purity Analysis of the

MON 87411

- produced CP4EPSPS Protein

................................ ................................ . 274

Figure C

- 8. Glycosylation Analysis of the MON 87411

- produced CP4 EPSPS

Protein

................................ ................................ ................................ ............................. 276

Figure E

- 1. PCR Amplification of MON 87411 DvSnf7 cDNA

................................ 289

Figure E

- 2. Sequence Alignment between MO N 87411 DvSnf7 cDNA and DvSnf7_968 cDNA

................................ ................................ ................................ ......... 290

Figure E

- 3. Northern Blot Analysis to Confirm the Equivalence between the

dsRNA in DvSn f7_968 RNA and MON 87411 DvSnf7 RNA

................................ ..... 292

Figure J

- 1. General Morphology of Pollen from MON 87411, the Conventional Control, and the Reference Hybrids under 200X Magnification

...... 360

Figure L

- 1. Sequencing and Sequence Selection

................................ ......................... 370

Figure L

- 2. Junctions and Junction Sequences

................................ .......................... 371

Figure L

- 3. Two Unique Junction Sequence Classes are Produced by the Insertion of a Single Plasmid Region

................................ ................................ ........... 372Monsanto Company CR240

- 13U1 25 of 374 ABBREV IATIONS AND DEFINITIONS 1

~ approximately

ADF acid detergent fiber

ANOVA a nalysis of variance

AOSA Association of Official Seed Analysts

APHIS Animal and Plant Health Inspection Service

APS analytical protein standards bp base pairs

BSA bovine serum albumin

Bt Bacillus thuringiensis

bu/A bushels per acre bw body weight

cDNA complementary deoxyribonucleic acid

CEW corn earworm CFR Code of Federal Regulations

CHT ceramic hydroxyapatite

CP4 EPSPS 5

- e nolpyruvylshikimate

- 3

- phosphate synthase protein from Agrobacterium tumefaciens strain CP4

CRW corn rootworm

CTAB h exadecyltrimethylammonium bromide

CV coefficient of variation

DAP days after planting

dATP deoxyadenosine triphosphate

DDI daily dietary intake

DEEM

- F C I D Dietary Exposure Evaluation Model

- Food Commodity Intake Database

DHB 2,5

- dihydroxybenzoic acid

dNTP deoxyribonucleotide

dsRNA double stranded RNA

DTT dithiothreitol

DvSnf7 Snf7 gene from Diabrotica virgifera virgifera encoding the SNF7 subunit

of the ESCRT

- III complex

DvSnf7 RNA RNA expressed from the suppression cassette that contains an inverted repeat sequence de sig ned to match the western corn rootworm (WCR; Diabrotica virgifera virgifera ) DvSnf7 gene
DvSnf7_240 t he active insecticidal RNA in MON 87411
DvSnf7_968 an in vitro transcribed DvSnf7 single stranded RNA

DvSnf7 p partial coding sequence of the Snf7 gene from Diabrotica virgifera virgifera encoding the S nf 7 subunit of the ESCRT

- III complex

dw dry weight

DWCF dry weight conversion factor

ECB European corn borer

1 Alred, G.J., C.T. Brusaw, and W.E. Oliu. 2003. Handbook of Technical Writing, 7th edn., pp. 2

- 7. Bedford/St. Martin's, Boston, MA.Monsanto Company CR240

- 13U1 26 of 374 EDV extended diapause variant

E. coli Escherichia coli

ELISA enzyme

- linked immunosorbent assay

EPA Environmental Protection Agency

ESCRT Endosomal Sorting Complex Required for Transport

EUP experimental use permit

ETS Excellence Through Stewardship

FA fatty aci d

FDA U.S. Food and Drug Administration

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FMOC fluorenylmethyl chloroformate

FS E farm scale evaluation

fw fresh weight

GC gas chromatography

Gb gigabases

ha hectare

HPLC high

- performance liquid chromatography

HRP horseradish peroxidase

HT herbicide tolerance

ILSI CCDB International Life Sciences Institute

- Crop Composition Database

IPM integrated pest management

IRM insect resistance management

JSC junction sequence class

kDa kilodalton kg/hl k ilograms per hectoliter

LOD limit of detection LOQ limit of quantitation

MEEC maximum expected environmental concentration

MESA 4

- Morpholinepranesulfonic acid

- e thylenediaminetetraacetic acid

- sodium acetate

MFI median fluorescence intensity Mg/ha megagrams/hectare

miRNA micro RNA

MMT million metric tons

MOA mode

- of

- action

MOE margin of exposure

MVB multi

- vesicular bodies

n number of samples

NCR northern corn rootworm

NDF neutral detergent fiber

NFDM nonfat dry milk

NGS/JSA Next Generation Sequencing/Junction Sequence Analysis

NHANE S National Health and Nutrition Examination Survey

NOAEL no observable adverse effect level

NOEC no observable effect concentration

nt nucleotide

NTO n on

- target organismMonsanto Company CR240

- 13U1 27 of 374 OECD Organi s ation for Economic Co

- operation and Development

OM organic matter

OPA o

- phthalaldehyde

OSL over season leaf

OSR over season root

OSWP over season whole plant

PBST phosphate buffered saline containing 0.05% (v/v) Tween

PCR polymerase chain reaction

PIP plant incorporated protectant Poly(A) multiple adenosine monophosphates

PPA P lant P rotection A ct

PTH

- AA phenylthiohydantoin

- amino acid QC

- negative quality control QC+ positive quality control

RDR root damage rating

RH relative humidity

RISC RNA

- induced silencing complexes

RNA ribonucleic acid

RNAi RNA interference

RNase r ibonuclease

RT room temperature

SAP Scientific Advisory Panel

SBV soybean variant

SCR s outhern c orn r ootworm

SD standard deviation

SDS sodium dodecyl sulfate

S.E. standard error

SGF simulated gastric fluid

SIF simulated intestinal fluid

siRNA small interfering RNA

sp. s pecies

TDF total dietary fiber

T

- DNA transfer DNA

TFA trifluoroacetic acid

TSSP tissue

- specific site pool

TTC threshold of toxicological concern

Tz t etrazolium

USDA United States Department of Agriculture

UTR untranslated region

UV u ltraviolet

v/v volume to volume

WCR western corn rootwormMonsanto Company CR240

- 13U1 28 of 374 I. RATIONALE FOR THE DEVELOPMENT OF MON 87411

I.A. Basis for the Request for a Determination of Nonregulated Status under

7 CFR § 340. 6

The Animal and Plant Health Inspection Service (APHIS) of the United States ( U.S. ) Department of Agriculture (USDA) has responsibility, under the Plant Protection Act ( Title IV Pub. L. 106

- 224, 114 Stat. 438, 7 U.S.C. § 7701

- 7772)

, to prevent the introduction and dissemination of plant pests into the U.S. APHIS regulation

7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and no longer should be regulated. If APHIS determines that the regulated article does not

presen t a plant pest risk, the petition is granted, thereby allowing unrestricted introduction

of the article.

Monsanto Company is submitting this request to APHIS for a determination of nonregulated status for the new biotechnology

- derived maize product, MON 87411 , any progeny derived from crosses between MON 87411 and conventional maize , and any progeny derived from crosses of MON 87411 with biotechnology

- derived maize that

ha ve previously been granted nonregulated status under 7 CFR Part 340.

I.B. Rational e for the Development of Insect

- Protected and Glyphosate Tolerant

Maize MON 87411

Maize ( Zea mays L.) is the largest crop grown in the U.S. in terms of acreage planted and net value. In 2012, maize was planted on over 97 million acr es and grain harvested from

87.4 million acres ( USDA

- NASS 2013a )

. Average yields in the previous fiv e years ranged from 147 bushels per acre (bu/ A ) (2011) to 165 bu/ A (2009) and were valued between $46.7 billion (2009) and $76.9 billion (2011) ( USDA

- NASS 2013d ) ; however, in 2012, a widespread drought in the U.S. resulted in an average yield of only 123 bu/ A

,

w ith a total production of about 10.8 billion bushels ( USDA

- NASS 2013a )

, v alued at approximately $77 billion ( USDA

- NASS 2013c )

.

In 2012, approximately 85 million a c res in the U.S. (or 88 % of the total U.S. maize acreage) w ere planted with biotechnology

- derived maize hybrids, and approximately 64 million acres (or 67 % of the total maize acreage) were planted with maize hybrids containing insect icidal crystal (Cry) proteins derived from Bacillus thuringiensis ( Bt ) ( USDA

- ERS 2013 )

. Of those 64 million acres, over 50 million acres were planted with

combined

- trait hybrids containing Bt and herbicide tolerance (HT) traits ( USDA

- ERS

2013 )

. Since the early to mid

- 2000’s, many of these combined

- trait hybrids have contained multiple Bt genes with multiple modes

- of

- action (MOA) for robust and durable efficacy against a broad range of above

- ground lepidopteran and below

- ground coleopteran maize pests. Es timates are that approximately 50 million acres of corn rootworm ( CRW )

- protected hybrids were planted in the U.S. in 2011 ( Marra, et al. 2012 )

.Monsanto Company CR240

- 13U1 29 of 374 I.B.1 Benefits of Insect

- Protection and Herbicide Tolerance Traits

T he introduction of insect

- protected and herbicide tolerant (HT) biotechnology

- derived

maize hy brids has been valuable to growers for two primary reasons: 1) they permit the

in

- crop application of broad

- spectrum agricultural herbicides for effective weed control

, which promotes the adoption of conservation tillage practices ; and 2) they provide highly effective targeted pest control to manage some of a grower’s most damaging maize pests.

The value of HT maize hybrids to growers has been demonstrated by the significant growth in acres planted to HT maize. In 2000, just 7% o f maize acres were planted with hybrids containing a trait conferring herbicide tolerance

, while that percentage had increased to 73% by 2012 ( USDA

- ERS 2013 )

. Competition for light, nutrients, and moisture resources by weeds can lead to proportional and significant reductions in crop yield ( Knake, et al. 1990 ) . Numerous studies have shown that weed control early in the growing season is necessary to reduce yield los ses in corn.

I nsect

- protected maize hybrids have also delivered significant value to growers as demonstrated by significant growth in insect

- protected maize acres planted. In 2000, 19% of planted maize acres had insect

- protection traits

, while that percentage had increased to 67% by 2012 ( USDA

- ERS 2013 )

. Included in that total are insect

- protected products for above

- ground lepidopteran control, below

- ground coleopteran control and hybrids containing traits for control o f both types of pests

.

Of the several insect species that can cause damage to maize plants, the most damaging in major U.S. maize growing regions are larvae of the CRW complex 2

Chandler, et al. 2008 ( Diabrotica s p p., Coleoptera : Chrysomelidae) ( )

. The corn rootworm complex

includes Diabrotica species that are significant pests of maize including western CRW ( D. virgifera virgifera ), northern CRW ( D. barber i ), and southern CRW ( D. undecimpunctata howardi ). These insect larvae damage maize by feeding on the roots, reducing the ability of the pl ant to absorb water and nutrients from the soil, and causing harvesting difficulties because of plant lodging ( Riedell 1990 ; Spike and Tollefson 1991 )

. C RW has long been described as the “billion

- dollar pest” complex, based on costs associated with the application of soil insecticides and crop losses from pest damage ( Mitchell, et al. 2004 )

. As the adoption of CRW

- protect ion traits has increased from its

first plantings i n 2003 to approximately 50 million acres in 2011 ( Marra et al. 2012 ) , the

use of these traits has led to the decreased use of conventional insectici des for CRW control by more than 75% ( U.S. EPA 2011 )

.

2 The corn rootworm complex includes Diabrotica species that are significant pests of maize including western CRW ( D. virgifera virg ifera ), northern CRW ( D. barber i ), and southern CRW ( D. undecimpunctata howardi ).Monsanto Company CR240

- 13U1 30 of 374 I.B.2. Introduction of Insect

- Protect ion T raits in the U.S.

Biotechnology traits providing protection against CRW were first introduced in the

United States in 2003 when YieldGard ®

Marra et al. 2012 Rootworm (MON 863 that expresses Cry3Bb1 protein) maize hybrids were commercialized. This trait provided a highly effective solution for CRW control. Shortly after YieldGard ® Rootworm was launched, other products providing CRW protection from Dow AgroSc iences (H erculex ® /DAS 59122 that expresses Cry34/35Ab1 proteins) and Syngenta ( Agrisure ® /MIR 604 that expresses a modified Cry3A protein (mCry3A) ) were introduced ( ) . Following the development and introduction of YieldGard ® Rootworm maize hybrids, Monsanto

introduced YieldGard VT Rootworm/RR2 ® (MON 88017 that expresses Cry3Bb 1 and CP4 EPSPS proteins ) with genes for both CRW protection and Roundup ® herbicide tolerance in a single product. That introduction was followed shortly thereafter by

combined

- trait hybrids containing Bt proteins for both above

- ground lepidopteran and

below

- ground coleopteran (CRW) pest control, as well as herbicide tolerance (YieldGard VT Triple ®

Bates, et al. 2005 , Genuity ® VT Triple PRO ®

, and Genuity ® Smartstax ® ). Monsanto’s YieldGard VT Triple (MON 88017 × MON 810) contains single Bt proteins to control targeted lepidopteran (Cry1Ab) and CRW (Cry3Bb1) maize pests whil e Monsanto’s Genuity VT Triple PRO (MON 88017 × MON 89034) provides two Bt proteins with two

MOA s (Cry1A.105 and Cry2Ab2) for lepidopteran pests and a single Bt protein (Cry3Bb1) for CRW control

. The joint introduction of SmartStax maize hybrids by Monsanto and Dow AgroSciences introduced the first maize hybrids with six Bt proteins (Cry1A.105, Cry2Ab2, Cry1F, Cry34/35Ab1, and Cry3Bb1), three effective MOA against the primary lepidopteran pests of maize in the U.S. corn belt, and two effec tive MOA against the primary CRW pests of maize in the U.S. corn belt

. These two MOA provided in MON 87411 ( from Cry3Bb1 protein and DvSnf7 RNA) can be expected to improve the durability of CRW

- protection traits and extend the useful lifetime of these

pro ducts ( ; Roush 1998 ) . All of these noted CRW

- protected product s have provided highly effective control of CRW across wide growin g regions in the U.S.

I.B.3. Development of CRW

- Protected and Glyphosate Tolerant Maize MON 87411

In its continuing efforts to provide highly effective, durable control of CRW for its

customers, Monsanto Company has developed biotechnology

- derived maize MON 87411

that confers protection against CRW ( Diabrotica spp .) and tolerance to the herbicide glyphosate

. MON 87411 builds upon current Bt protein

- based CRW control technology by introducing a new MOA based on RNA

- mediated gene suppression (RNAi) that o ffers increased control of target insect pests and will prolong the durability of existing CRW

controlling Bt technologies.

® YieldGard and YieldGard VT Rootworm/RR2 are registered trademarks of Monsanto Technology LLC. Herculex is a registered trademark of Dow AgroSciences LLC. Agrisure is a registered trademark of Syngenta Participations AG.

® YieldGard VT Triple, Genuity VT T riple Pro, Genuity SmartStax, and Roundup are registered trademarks of Monsanto Technology LLC.Monsanto Company CR240

- 13U1 31 of 374 MON 87411 contains a suppression cassette that expresses an inverted repeat sequence

de signed to match the sequence in western cor n rootworm (WCR; Diabrotica virgifera virgifera ) . The expression of the suppression cassette results in the formation of a

double

- stranded RNA (dsRNA) transcript containing a 240 bp fragment of the WCR Snf7 gene ( DvSnf7 ) . Upon consumption, the plant

- produced ds RNA in MON 87411 is recognized by the CRW’s RNA interference (RNAi) machinery ( Hammond 2005 ; Ketting and Plasterk 2004 ; Tomari and Zamore 2005 ) resulting in the down

- regulation of the targeted DvSnf7 gene leading to CRW mortalit y ( Bolognesi, et al. 2012 )

. MON 87411 also produces a modified Bacillus thuringiensis (subsp. kumamotoensis ) Cry3Bb1 protein to protect against CRW larval feeding

. In addition, MON 87411

contains the cp4 epsps gene from Agrobacterium sp . strain CP4 that encodes for the

5

- enolpyruvylshikimate

- 3

- phosphate synthase (EPSPS) protein, which confers tolerance to glyphosate, the active ingredient in Roundup agricultural herbicides.

MON 87411 wi ll provide benefits to growers similar to those obtained by use of existing

CRW

- protected maize hybrids, which includ es reduced use of insecticides, increased yield protection, water conservation, and increased worker safety ( Rice 2004 )

. MON 87411 is also glyphosate tolerant and will continue to provide benefits associated with conservation tillage methods, including reduced soil erosion, reduced fuel and labor costs, improved air quality and conservation of soil moisture ( CTIC 2011 ; Hurley, et al.

2009 ; Towery and Werblow 2010 )

.

MON 87411 will not be offered for commercial use as a stand

- alo ne product, but will be combined, through traditional breeding methods, with other deregulated biotechnology

derived traits to provide protection against both above

- ground and below

- ground maize pests as well as tolerance to multiple herbicides. These nex t generation combined

- trait maize products will offer the ability to maximize grower choice, improve production efficiency, increase pest control durability, and improve grower profit potentials.

I.C. RNA Interference (RNAi)

I.C.1. Applications of RNA i i n Plants

Naturally occurring RNA

- mediated gene suppression (RNAi) in plants ha s been previously documented and include s selection for soybean seed coat color ( Tuteja, et al.

2004 ) and maize stalk color ( Della Vedova, et al. 2005 ) . In both of these instances, production of chalcone synthase was suppressed leading to significantly decreased pigmentation in soybean seed coats and maize stalks, respectively

. In addition, a low glutelin rice variety has been studied and has been determined to res ult from production of a dsRNA and concomitant suppression of glutelin genes ( Kusaba, et al. 2003 ) . RNA

me diated gene suppression has also been used in a number of biotechnology

- derived food

crops that have previously been deregulated by USDA or other regulatory authorities

includin g virus resistant papaya, squash, potato, common bean, and plum as well as a delayed ripening tomato and a soybean with altered oil composition ( Parrott, et al. 2010 )

. S afety assessments have been conducted ( Parrott et al. 2010 ; Petrick, et al. 2013 ) and global r egulatory approvals have been obtained for products employing RNAi gene suppression

.Monsanto Company CR240

- 13U1 32 of 374 I.C.2. Applications of RNAi in Insects

RNAi can also achieve gene silencing in susceptible insects following ingestion of

dsRNAs ( Baum, et al. 2007a ; Terenius, et al. 2011 ; Whyard, et al. 2009 ) . Insect control products can be developed utilizing RNAi sequence

- specific gene silencing to suppress genes critical for insect survival. Because of this sequence

- specific gene silencing, these products have the potential to selectively target a narrow group of closely related pest species and gr eatly reduce the likelihood of adverse effects on non

- target organisms (NTOs), including those beneficial to agriculture. The spectrum of activity for DvSnf7 dsRNA has been shown to be narrow and activity is only evident in a subset of beetles

within the Galerucinae subfamily of Chrysomelidae within the Order Coleoptera ( Bachman, et al. 2013 ) , as described in more detail in Section VI.E below

.

I. D . Mode s

- of

- Action of the Inserted Genetic Components

I. D .1. Mode

- of

- Action of t he RNAi Component of MON 87411

MON 87411 contains a DvSnf7 suppression cassette that expresses an inverted repeat sequence de signed to match the sequence in WCR and thereby utilizes the RNAi pathway to control CRW ( Diabrotica s p p.)

. The expression of the suppression cassette results in the formation of a dsRNA transcript containing a 240 bp fragment of the WCR Snf7 gene (DvSnf7 ) . Upon consumption of MON 87411 by WCR

, DvSnf7 ds RNA is recognized by the pest’s RNAi machinery

, resulting in the down

- regulation of the targeted DvSnf7 gene leading to W CR mortality ( Bolognesi et al. 2012 ) (Figure I

- 1)

.Monsanto Company CR240

- 13U1 33 of 374 Figure I

- 1

. Diagram of MON 87411 dsRNA Oral Deliver y to Suppress DvSnf7 Expression via the RNAi Pathway in CRW

The RNAi pathway is a natural process in eukaryotic organisms for the regulation of endogenous gene expression ( Dykxhoorn, et al. 2003 ; Parrott et al. 2010 )

. T he dsRNA molecule that activates the mechanism is first processed by a class of RNase III enzymes called Dicers into small interfering RNAs (siRNAs , ~21

- 2 5 nucleotides ) ( Hammond

2005 ; Siomi and Siomi 2009 ; Zamore, et al. 2000 ) . The resulting siRNA molecules are then incorporated into multiprotein RNA

- induced silencing complexes (RISC), which

facilitate complementary sequence recognition a nd mRNA cleavage that leads to specific suppression of the target mRNA ( Hammond 2005 ; Tomari and Zamore 2005 )

. I n the

case of CRW that consume MON 87411 , the DvSnf7 gene in CRW is suppressed

.

DvSnf7 was selected as the target mRNA in CRW d ue to its vital cellul ar function that can be suppressed at relatively low concentrations when targeted by dsRNA ( Baum et al.

2007a )

. Snf7 is a class E vacuolar sorting protein and belongs to the Endosomal Sorting Complex Required for Transport ( ESCRT )

– III complex, which has been shown to be

i nvolved in sorting of transmembrane proteins enroute to lysosomal degradation through

the endosomal

- autophagic pathway in a number of organisms ( Kim, et al. 2011 ; Lee and

Gao 2008 ; Rusten, et al. 2008 ; Teis, et al. 2008 ; Vaccari, et al. 2009 ) (Figure I

- 2)

. ESCRT

- III components play critical roles in distinct steps of this pathway ( Henne, et al.

2011 ; Roxrud, et al. 2010 )

. Data h ave shown that suppression of DvSnf7 in WCR leadsMonsanto Company CR240

- 13U1 34 of 374 to accumulation of ubiquitinated proteins 3

Ramaseshadri, et al. 2013 destined for lysosomal degradation ( ) . Sorting of t ransmembrane proteins is critical to regulate signal transduction in cells and as such, suppression of this sorting through the ESCRT

III complex impairs cell homeostasis and functionin g, leading to cellular death and CRW mortality. We have also shown systemic spread of the RNAi effect to tissues distal to the

WCR midgut ( Bolognesi et al. 2012 )

. Similar lethal pheno types resulting from

knockdown of Snf7 have been shown in Drosophila ( Sweeney, et al. 2006 ) and C. elegans ( Michelet, et al. 2010 )

.

Figure I

- 2 . Model depicting endosomal

- autophagy pathway involved in intracellular sorting and degradation of receptors along with other macromolecules in a normal cell (left) and a DvSnf7 deficient cell (right).

In the normal cell, internalization and ubiquitinat ion of cargo proteins (1 ) de

- ubiquitination of

cargo proteins (2), biogenesis of multi

- vesicular bodies ( MVB ) (3), formation of autophagosomes engulfing macromolecules (4), and fusion of late endosomes, autophagosome and lysosomes into autolysosomes for degradation of cargo proteins and macromolecules (5) are depicted. In the

3 Ubiquitination is a post

- translational modification of a protein in which one or more ubiquitin molecules are added to the protein ( Pickart 2001 )

. Ubiquitins are small regulatory proteins found in all eukaryotic cells and their addition to a protein often leads to degradation of that protein. This process of ubiquitination and protein degradation allows the cell to modulate the co ncentration of essential proteins within that cell.Monsanto Company CR240

- 13U1 35 of 374 DvSnf7 deficient cell, the impairment of de

- ubiquitination, accumulation of autophagosomes, and failure of fusion of endosomes, autophagosomes and lysosomes and autolysosomal activit y are highlighted ( Ramaseshadri et al. 2013 )

.

I nduction of RNAi

- mediated gene suppression in insects via an oral route of exposure requires efficient uptake of dsRNA s by m idgut cells followed by suppression of the target mRNA leading to significant effects on growth, development and survival. In the case of

WCR , only the relatively l ong dsRNA ( e.g ., DvSnf7 240

- mer ) is taken up by the insect and significant biological activ ity was only observed with dsRNA sequences ≥ 60 bp ( Bolognesi et al. 2012 )

. Finally, s everal key points have been identified in demonstrat ing efficacy of MON 87411 against WCR: 1) oral delivery/uptake of dsRNA into WCR gut

cells , 2) suppression of the targeted DvSnf7 mRNA expression followed by suppression

of the production of the DvSnf7 protein , 3) systemic spread of suppression o f DvSnf7 expression beyond WCR midgut tissues, and 4) growth inhibition and WCR mor tality ( Bolognesi et al. 2012 )

.

I. D .2 . Modes

- of

- Acti on of the CP4 EPSPS and Cry3Bb1 Proteins

MON 87411 con tains the identical CP4 EPSPS protein that is expressed in MON 88017

maize ( USDA

- APHIS Petition No. 04

- 125

- 01p) and numerous other Roundup Ready crops (maize, cotton, soybean, canola, alfalfa, sugar beet)

. The CP4 EPSPS protein is structurally similar and functionally identical to endogenous plant EPSPS enzymes, but has a much reduced affinity for glyphosate relat ive to endogenous plant EPSPS ( Padgette, et al. 1996 )

. In MON 87411, as in other Roundup Ready plants, aromatic

amino acid s and other metabolites necessary for plant growth and developme nt are produced by the continued action of the CP4 EPSPS enzyme in the presence of glyphosate ( Padgette et al. 1996 )

.

MON 87411 also contains an expression cassette that codes for the same Cry3Bb1 protein as the expression cassette that is present in MON 88017 maize ( USDA

- APHIS

Petition No. 04

- 125

- 01p) that was granted non

- regulated status by USDA

- APHIS in 2006 ( USDA

- APHIS 2013 )

. The amino acid sequence deduced from the Cry3Bb1 expression

cassettes of MON 87411 and MON 88017 is also 99.8% identical to the deduced amino acid sequence for Cry3Bb1 protein in MON 863 (USDA

- APHIS Petition No. 01

- 137

- 01p) that was granted non

- regulated status by USDA

- APHIS in 2002 ( USDA

- APHIS

2013 )

. The use of Bt

- containing crops in U.S. agriculture has been widespread and the

mode

- of

- action ( i.e

., solubilization of C ry protein, processing to the active form, binding to midgut receptors and insertion of the toxin in to cellular membranes) and specificity of

Bt proteins has been studied extensively and is well understood ( Gill, et al. 1992 ; Whalon and Wingerd 2003 )

.

I

. E

. Product Efficacy

Monsanto conducted field trials in 2011 and 2012 to assess the efficacy of MON 87411 in reducing root damage caused by CRW larvae . In both years, MON 87411 was compared to genetically similar control hybrids (one parent of each hybrid was LH244)

which also contained biotechnolo gy

- derived MON 89034 expressing two Cry proteinsMonsanto Company CR240

- 13U1 36 of 374 ( Cry1A.105 and Cry2Ab2 ) for protection against above

- ground lepidopteran pests. Cry1A.105 and Cry2Ab2 are not active against Coleopteran insects and th erefore do not imp act r oot feeding by CRW larvae. Data were collected from replicated blocks at nine

trial locations in 2011 and five locations in 2012 from maize production regions in Iowa, Illinois, and Indiana.

In both years, when plants reached the V2 growth stage , five pl ants per plot were infested

with corn rootworm eggs at a rate of over 3200 eggs per plant. At the V10 growth stage,

these five plants were dug and the roots were washed and evaluated for feeding damage.

The evaluation s w ere based on a root damage rating (RDR) ( Oleson, et al. 2005 ) scale of 0 to 3, where 0 is no root damage detected and a 3 is where all three below

- ground nodes are completely missing or totally damaged

.

In 2011 trials, the average RDR across all 9 locations for control hybrids not containing Cry3Bb1 or DvSnf7 dsRNA was 1.5. Damage ratings across these locations ranged from

0.9 to 2. 4. These ratings are indicative of the relatively high rootworm pressure overall. The average RDR for MON 87411 hybrids was 0.13, demonstrating significant efficacy against larval CRW feeding. In 2012 trials, the average RDR across the 5 l ocations for

c ontrol hybrids was 1.06, confirming significant rootworm pressure. Averaged RDR’s

across these sites for MON 87411 hybrids in these trials was 0.07, again demonstrating significant efficacy against larval CRW feeding.

I. F . Submissions to Other Regulato ry Agencies

Under the Coordinated Framework for Regulation of Biotechnology (CFR) ( USDA

APHIS 1986 )

, the responsibility for regulatory oversight of biotechnology

- derived crops

falls primarily on three U.S. agencies : U.S. Food and Drug Administration ( FDA )

, the United States Department of Agriculture ( USDA )

, and in the case of plant incorporated

protectan ts (PIPs) , the Environmental Protection Agency (EPA) . Deregulation of

MON 87411 by USDA constitutes only one component of the overall regulatory oversight and review of this product. As a practical matter, MON 87411 cannot be

released and marketed until FDA , EPA, and USDA have completed their reviews and assessments under their respective jurisdictions.

I. F .1. Submission to FDA

MON 87411 falls within the scope of the 1992 FDA policy statement concerning regulation of products derived from new plant varieties, including those developed through biotechnology ( U.S. FDA 1992 ) . In comp liance with this policy, Monsanto

submitted a food/feed safety and nutritional assessment summary document to FDA in

November 2013

.

I. F .2. Submission to EPA

Substances that are pesticides, as defined under the Federal Inse cticide, Fungicide and Rodenticide Act (FIFRA) [7 U.S.C. §136(u)], are subject to regulation by the EPA. Pesticides produced in planta , referred to as PIPs, are also subject to regulation by the

EPA under FIFRA.Monsanto Company CR240

- 13U1 37 of 374 Pursuant to §408(d) of the Federal Food Drug and Cosmetic Act [21 U.S.C. 346 a(d)]

, Monsanto Company petitioned EPA for an exemption from the requirement of a

tolerance for the PIP Bacillus thuringiensis Cry3Bb1 protein in or on all food and feed

commoditi es of field corn, sweet corn

, and popcorn and the genetic material necessary for its production in these products in 1997 . On March 31, 2004 , the EPA established a

permanent exemption from the requirement of a tolerance for the PIP Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for its production in food and feed commodities of field corn, sweet corn and popcorn (40 CFR § 180.1214 ). Additionally, and applicable to MON 87411, is EPA’s establishment of an exemption from the requir ement of a tolerance for residues of nucleic acids that are part of a plant

incorporated protectant (40 CFR § 174.475).

On May 4, 2012, Monsanto filed an experimental use permit (EUP) application for

MON 87411 and the genetic material necessary for its pr oduction with the U.S. EPA to

facilitate MON 87411 field testing and safety evaluations. EPA granted the EUP ( 524

- EUP

- 104 ) on March 1, 2013

. Monsanto will make an application to the EPA for a Breeding R egistration for MON 87411 and the genetic material (PV

- ZMIR10 87 1) necessary for its production in maize in the near future. Additionally, Monsanto will make the appropriate Section 3 registration application(s) when final decisions about specific stacked maize products (breeding stacks) are made.

I .F .3. Submissions to Foreign Government Agencies

Consistent with our commitments to the Biotechnology Industry Organization’s Excellence Through Stewardship ® (ETS) Program 4

® Excellence Through Stewardship is a registered trademark of Excellence Through Stewardship, Washington, DC. , Monsanto intends to obtain the appropriate approvals from all key maize import markets with functioning regulatory systems prior to commercial planting of MON 87411. As appropriate, notifications will be made to countries that import significant quantities of maize and maize products and do not have formal regulatory review processes for bi otechnology

- derived crops.

4 http://www.excellencethroughstew ardship.org/

.Monsanto Company CR240

- 13U1 38 of 374 II. THE BIOLOGY OF MAIZE

The Organisation for Economic Co

- Operation and Development (OECD) Consensus Document on the biology of maize ( OECD 2003 ) provide s key information on:

- general description of maize biology, including taxonomy and morphology and use of maize as a crop plant

- agronomic practices in maize cultivation

- geographic centers of origin

- reproductive biology

- cultivated maize as a volunteer weed

- inter

- species/genus introgression into relatives and interactions with other organisms

- a summary of the ecology of maize

Additional information on the biology and uses of maize can also be found on the Australian Government Department of Health and Agein g (Office of the Gene Technology Regulator) web site ( OGTR 2008 ) , and in the USDA

- ARS GRIN database ( USDA

- ARS 2013 )

.

To support the evaluation of the plant pest potential of MON 87411 relativ e to conventional maize, additional information regarding several aspects of maize biology can be found elsewhere in this petition. This includes: agronomic practices for maize in Section IX; volunteer management of maize in Section IX. H ; and inter

- speci es/genus introgression potential in Section X. I

.

II.A. Maize as a Crop

Maize is grown in nearly all areas of the world and is the largest cultivated crop in the world followed by wheat ( Triticum sp.) and rice ( Oryza sativa L.) in total global metric

ton production . In 2012, maize was planted globally on ~174 million hectares (ha) with a

total grain production of an estimated 8 54 million metric tons ( MMT ) ( USDA

- FAS

2013 ) . The top five production regions in 2012 were: USA (27 4 MMT), China (208 MMT), Brazil (7 3 MMT), EU

- 27 (5 5 MMT), and Argentina (27 MMT) ( USDA

- FAS

2013 ) . In the U.S., maize is grown in almost every state and in 2012, its production value

of over $77 billion was the highest of any crop ( USDA

- NASS 2013c )

.

In industrialize d countries maize has two major uses: (1) as animal feed in the form of grain, forage or silage; and (2) as a raw material for wet

- or dry

- milled processed products such as high fructose maize syrup, oil, starch, glucose, dextrose and ethanol. By

- products of the wet

- and dry

- mill processes are also used as animal feed. These processed products are used as ingredients in many industrial applications and in human food products. Most maize produced in industrialized countries is used as animal feed or

for i ndustrial purposes, but maize remains an important food staple in many developing regions, especially sub

- Saharan Africa and Central America, where it is frequently the mainstay of human diets ( Morris 1998 )

.

Maize is a very familiar plant that has been rigorously studied due to its use as a staple food/feed and the economic opportunity it brings to growers. The domestication of maizeMonsanto Company CR240

- 13U1 39 of 374 likely occurred in so uthern Mexico between 7,000 and 10,000 years ago ( Goodman

1988 )

. While the putative progenitor species of maize have not been recovered, it is likely that teosinte played an important role in co ntributing to the genetic background of maize. Although grown extensively throughout the world, maize is not considered a persistent weed or a plant that is difficult to control. Maize, as we know it today, cannot

survive in the wild because the female i nflorescence (the ear) is covered by a husk thereby restricting seed dispersal , it has no seed dormancy, and is a poor competitor in an unmanaged ecosystem

. The transformation from a wild, weedy species to one dependent

on humans for its survival most lik ely evolved over a long period of time through plant breeding by the indigenous inhabitants of the Western Hemisphere. Today, virtually all the maize grown in the U.S. is a hybrid, a production practice that started in the 1930’s ( Wych 1988 ) . Maize hybrids are developed and used based on the posi tive yield increases and plant vigor associated with heterosis, also known as hybrid vigor.

Conventional plant breeding results in desirable characteristics in a plant through the unique combination of genes already present in the plant. However, there is a limit to genetic diversity with conventional plant breeding. Biotechnology, as an additional tool to conventional breeding, offers access to greater genetic diversity than conventional breeding alone, resulting in expression of highly desirable traits that are profitable to

growers.

II. B

. Characteristics of the Recipient Plant

The transformation for MON 87411 was conducted with inbred maize line LH24 4 , a patented maize line assigned to Holden’s Foundation Seeds LLC in 2001 ( U.S. patent #6,252,148 )

. LH244 is a medium season yellow dent maize line with a Stiff Stalk background that is best adapted to the central regions of the U.S. c orn b elt . LH244 was initiated from a single cross of LH197 × LH199 followed by a backcross to LH197

. The

F 2 combination ( ( LH19 7 × LH197) × LH199) was then selfed and used in the development of LH244.

Following transformation of immature LH244 embryos , a single transformed plant was

selected and self

- crossed to increase seed supplies. A homozygous inbred line was develope d though further self

- crossing and selection and was then used to produce other lines which were used for product testing, safety assessment studies, and commercial production.

I I

. C

. Maize as a Test System in Product Safety Assessment

Based on seed availability and appropriate fit for various studies, h ybrid maize line s MPA640B (LH244 × LH287) and NL6169 (LH244 × HCL645) w ere used as near isogenic, conventional control s for this submission (hereafter referred to as conventional

control s )

. As noted, o ne parent of each of these control maize lines is LH244 , the inbred

from which MON 87411 i s derived , while the other parents (LH287 and HCL645) are

other maize inbreds

. As such, b oth of these maize lines constitute relevant comparators

for MON 87411. In addition, other commercial maize hybrids (hereafter referr ed to as reference hybrids) were used to establish ranges of natural variability representative of

commercial maize hybrids

. R eference hybrids used at each field trial location were

selected based on their availability and agronomic fit for the respective geographicMonsanto Company CR240

- 13U1 40 of 374 region s

. Both MPA640B and NL6169 were used in molecular characterization studies. NL6169 was used as the conventional control in compositional analysis while MPA640B was used in pheno typic, agronomic and environmental interactions assessments. Where appropriate, reference hybrids were used to establish a range of variability or responses representative of commercial maize in the U.S.

In developing the data to support this petition, ap propriate MON 87411 test materials were generated for the molecular characterization (Sections III and IV), protein characterization and expression analysis (Section V), RNA characterization and expression (Section VI), composition al analysis (Section VI I ), and phenotypic, agronomic and environmental interactions assessment (Section VII I ). The full m olecular

characterization studies (NGS/JSA) were conducted with the R 4 generation (Figure IV

- 4).

I nitiation of commercial breeding efforts w as conducted with the R 5 generation (Figure

IV

- 4 ) . Protein and RNA characterization and expression analysis, composition analysis, and phenotypic, agronomic and environmental interactions assessment were conducted

with various MON 87411 breeding generations as noted in th e Breeding Tree (Figure IV

- 4)

.Monsanto Company CR240

- 13U1 41 of 374 I II

. DESCRIPTION OF THE GENETIC MODIFICATION

MON 87411 was developed through Agrobacterium tumefaciens

- mediated transformation of maize immature embryos from line LH244 utilizing PV

- ZMIR10871.

This section describes the plasmid vector, the donor gene, and the regulatory elements used in the development of MON 87411 as well as the deduced amino acid sequence of the Cry3Bb1 protein and CP4 EPSPS protein produced in MON 87411. In this section, transfer DNA (T

- DNA) refer s to DNA that is transferred to the plant during transformation. An expression ( or suppression ) cassette is comprised of sequences to be transcribed and the regulatory elements necessary for the expression of those sequences.

I II

. A

. The Plasmid Vector PV

- ZMIR10871

PV

- ZMIR10871 was used in the transformation of maize to produce MON 87411 and its plasmid map is shown in Figure III

- 1. The elements included in this plasmid vector are

described in Table III

- 1. PV

- ZMIR10871 is approximately 16.5 kb and contai ns one
T

- DNA that is delineated by Left and Right Border regions. The T

- DNA contains the

DvSnf7 suppression cassette, the cry3Bb1 expression cassette, and the cp4 epsps expression cassette. The DvSnf7 suppression cassette is regulated by the e35S promote r from the 35S RNA of cauliflower mosaic virus (CaMV), the heat shock protein 70 ( Hsp70 ) intron from Zea mays , and the 3' untranslated sequence of the E9 gene from

Pisum sativum . The cry3Bb1 expression cassette is regulated by the pIIG promoter from

Zea m ays , the chlorophyll a/b binding protein (CAB) leader from Triticum aestivum , the

Ract1 intron from Oryza sativa , and the heat shock protein 17 ( Hsp17 ) 3 ′ untranslated region from Triticum aestivum

. The cp4 epsps expression cassette is regulated by the

T

ubA promoter from Oryza sativa , the TubA leader from Oryza sativa , the TubA intron

from Oryza sativa , the CTP2 targeting sequence from Arabidopsis thaliana , and the TubA

3 ′ untranslated region from Oryza sativa

.

The backbone region of PV

- ZMIR10871, locate d outside of the T

- DNA, contains two origins of replication for maintenance of the plasmid vector in bacteria ( ori V

, ori

- pBR322 ), a bacterial selectable marker gene ( aadA ), and a coding sequence for repressor of primer (ROP) protein for maintenance of pla smid vector copy number in

Escherichia coli ( E. coli ) . A description of the genetic elements and their prefixes ( e.g. , B

- , P

- , L

- , I

- , TS

- , CS

- , T

- , and OR

- ) in PV

- ZMIR10871 is provided in Table III

- 1.

III.B. Description of the Transformation System

MON 87411 was developed through Agrobacterium

- mediated transformation of immature maize embryos based on the method described by Sidorov and Duncan ( 2009 )

, utilizing PV

- ZMIR10871. Immature embryos were excised from a post

- pollinated maize

ear of LH244. After co

- culturing the excised immature embryos with Agrobacterium carrying the plasmid vector, the immature embryos were placed on selection medium

contai ning glyphosate and carbenicillin disodium salt in order to inhibit the growth of untransformed plant cells and excess Agrobacterium . Once transformed callus developed, the callus was placed on media conducive to shoot and root development. The rooted pl ants ( R 0 ) with normal phenotypic characteristics were selected and transferred to soil for growth and further assessment. As demonstrated in this petition,Monsanto Company CR240

- 13U1 42 of 374 the use of disarmed Agrobacterium tumefaciens strain ABI, a designated plant pest, as the

transform ation vector has not imparted plant pest characteristics to MON 87411.

The R 0 plants generated through the transformation process described above had already been exposed to glyphosate in the selection medium and demonstrated glyphosate

tolerance. The R 0 plants self

- pollinated to produce R 1 seed and R 1 plants were evaluated for the presence of the T

- DNA via quantitative polymerase chain reaction (PCR) analysis.

The R 1 plants homozygous for the T

- DNA were selected for further development and their progenies were subjected to further molecular and phenotypic assessments. As is typical of a commercial event production and selection process , hundreds of different

transformation events (regenerants) were generated in the lab oratory using

PV

- ZMIR10871. After many months of careful selection and evaluation of these

hundreds of events in the lab oratory , greenhouse and field

, MON 87411 was selected as the lead event based on superior agronomic, phenotypic

, and molecular characteristics.

Studies on MON 87411 wer e initiated to further characterize the genetic insertion and the expressed products , and to establish the food, feed, and environmental safety relative to

commercial maize. The major steps involved in the development of MON 87411 are depicted in Figure II I

- 2.Monsanto Company CR240

- 13U1 43 of 374 Figure III

- 1. Circular Map of PV

- ZMIR10871 A circular map of the plasmid vector PV

- ZMIR10871 used to develop MON 87411 is shown.

PV

- ZMIR10871 contains a single T

- DNA. Genetic elements are shown on the exterior of the

map. P Superscript in Dv S nf7 indicates partial sequence.Monsanto Company CR240

- 13U1 44 of 374 Figure III

- 2. Schematic of the Development of MON 87411 Transformed LH244 (a maize line for more efficient transformation) immature embryos with PV

- ZMIR10871 in Agrobacterium tumefaciens

Selected transformants and generated rooted shoots from the

transformed callus tissue s

Identified MON 87411 as lead candidate and further evaluated its

progeny in laboratory and field assessments for insert integrity, glyphosate tolerance, efficacy against CRW larval damage and superior phenotypic characteristics Assembled Agrobacterium binary plasmid vector PV

- ZMIR10871 and

transferred to Agrobacterium tumefaciens strain ABI

Evaluated the transformed plants for the presence of the T

- DNA and selected homozygous plants by quantitative poly merase chain reaction (PCR) analyse s

Evaluate d plants for insert integrity by Southern blot analysisMonsanto Company CR240

- 13U1 45 of 374 III.C. The cry3Bb1 Coding Sequence and the Cry3Bb1 Protein

The cry3Bb1 expression cassette encodes a 74.5 kDa Cry3Bb1 protein consisting of a single polypeptide of 653 amino acids (Figure III

- 3)

. The cry3Bb1 coding sequence is the codon optimized coding sequence from Bacillus thuringiensis that encodes the Cry3Bb1 protein ( English, et al. 2000 ) . The presence of Cry3Bb1 protein provides protection from corn rootworm feeding

.

III.D. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein

The cp4 epsps expression cassette, encodes a 47.6 kDa CP4 EPSPS protein consisting of a single polypeptide of 455 amino acids (Figure III

- 4) ( Padgette et al. 1996 )

. The cp4 epsps coding sequence is th e codon optimized coding sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding CP4 EPSPS ( Barry, et al. 2001 ; Padgette et

al. 1996 ) . The CP4 EPSPS protein is sim ilar and functionally identical to endogenous plant EPSPS enzymes, but has a much reduced affinity for glyphosate, the active ingredient in Roundup agricultural herbicides, relative to endogenous plant EPSPS ( Barry

et al. 2001 ; Padgette et al. 1996 )

. The presence of this protein renders the plant tolerant to Roundup.

II I.E. DvSnf7 p sequence

The DvSnf7 p sequence is the partial coding sequence of the Snf7 gene from Diabrotica virgifera virgifera ( Baum et al. 2007a ; Baum, et al. 2007b ) encoding the S NF 7 subunit of

the ESCRT

- III complex ( Babst, et al. 2002 )

. The DvSnf7 suppression cassette contains

two 240 bp DvSnf7 p sequences in an inverted orientation. The re is an intervening

sequence of 1 50 nucleotides between the two DvSnf7 p sequences (noted on Tables III

- 1

and IV

- 1) . W hen the suppression cassette is transcribed

, the RNA expressed forms a hairpin loop thereby allowing the formation of double stranded DvSnf7 RNA. The

DvSnf7 p sequence s in the suppression cassette produce a 240 bp dsRNA that upon transcription triggers the RNAi mechanism.

III.F. Regulatory Sequences

The cry3Bb1 coding sequence in MON 87411 is under the regulation of the pIIG promoter, the chlorophyll a/b binding protein (CAB) leader, the Ract1 intron, and the

h eat shock protein 17 ( Hsp17) 3 ′ untranslated region. The pIIG promoter, which directs transcription in plant cells, is from the pIIG gene family encoding the physical impedance induced protein from Zea mays ( Huang, et al. 1998 )

. The CAB leader is the 5' untranslated region from the chlorophyll a/b

- binding (CAB) protein of Triticum aestivum and is involved in regulating gene expression ( Lamppa, et al. 1985 ) . The Ract1 intron is

the intron from the act1 gene from Oryza sativa ( McElroy, et al. 1990 ) . The Hsp17 3 ′

non

- translated region is the 3 ′ untranslated region from the heat shock protein, Hsp17, of

Triticum aestivum ( McElwain and Spiker 1989 ) that directs polyadenylation of the

mRNA.

The cp4 epsps coding sequence in MON 87411 is under the regulation of the TubA promoter, the TubA leader, the TubA intron, the CTP2 targeting sequence, and the TubA

3 ′ untranslated region. The TubA promoter, which directs transcription in plant cells, isMonsanto Company CR240

- 13U1 46 of 374 from the OsTubA gene family from Oryza sativa (rice) encoding α

- tubulin ( Jeon, et al.

2000 ) . The TubA intron is the intron from the OsTubA gene family from Oryza sativa (rice) encoding α

- tubulin ( Jeon et al. 2000 ) . The chloroplast transit peptide CTP2 directs transport of the CP4 EPSPS protein to the chloroplast in MON 87411 and is derived from

CTP2 target sequence of the Arabidopsis thaliana ShkG gene ( Herrmann 1995 ; Klee, et

al. 1987 ) . The TubA 3 ′ non

- translated region is the 3 ′ untranslated region from the

OsTubA gene family from Oryza sativa (rice) encoding α

- tubulin ( Jeon et al. 2000 ) that directs polyadenylation of mRNA.

The DvSnf7 p sequence in MON 87411 is under the regulation of the e35S promoter, the heat shock protein 70 ( Hsp70) intron, and the E9 3 ′ untranslated region. The e35S promoter, which directs transcription in plant cells, contains the duplicated enhancer region ( Kay, et al. 1987 ) from the cauliflower mosaic virus (CaMV) 35S RNA promoter ( Odell, et al. 1985 )

. As demonstrated in this petition, the use of the CaMV 35S promoter containing the duplicated enhancer region, derived from a designated plant pest, has not imparted plant pest characteristics to MON 87411

. The hsp70 intron is the first intron from the maize heat shock protein 70 gene ( Brown and Santino 1997 ; Rochester, et al.

1986 )

. The E9 3 ′ non

- translated region is the 3 ′ untranslated region from the rbcS gene of

Pisum sativum (pea) encoding the small subunit of ribulose bisphosphate carboxylase protein ( Coruzzi, et al. 1984 ) that directs polyadenylation of the mRNA.

III.G. T

- DNA Borders

PV

- ZMIR10871 contains Right Border and Left Border regions (Figure III

- 1 and

Table III

- 1) that were derived from Agrobacterium tumefaciens plasmids. The border regions each contain a nick site that is the site of DNA exchange during transformation ( Barker, et al. 1983 ; Depicker, et al. 1982 ; Zambryski, et al. 1982 ) . The border regions separate the T

- DNA from the plasmid backbone region and are involved in the efficient

transfer of T

- DNA into the maize genome. As demonstrated in this petition, the use of Right Border and Left Border regions derived from Agrobacterium tumefaciens , a designated plant pest, has not imparted plant pest characteristics to MON 87411.

III.H. Genetic Elements Outside of the T

- DNA Bor ders

Genetic elements that exist outside of the T

- DNA borders are those that are essential for

the maintenance or selection of PV

- ZMIR10871 in bacteria. The origin of replication

ori V is required for the maintenance of the plasmid in Agrobacterium and is derived from the broad host plasmid RK2 ( Stalker, et al. 1981 ) . The origi n of replication

ori

- pBR322 is required for the maintenance of the plasmid in E. coli and is derived from the plasmid vector pBR322 ( Sutcliffe 1979 ) . Coding sequence rop is the coding sequence of the repressor of primer (ROP) protein and is necessary for the maintenance of plasmid copy number in E. coli ( Giza and Huang 1989 )

. The selectable marker aadA is a bacterial pro moter and coding sequence for an enzyme from transposon Tn 7 that confers spectinomycin and streptomycin resistance ( Fling, et al. 1985 ) in E. coli and Agrobacterium during molecular cloning. Because these elements are outside the border regions, they are not expected to be transferred into th e maize genome. The absence of the backbone and other unintended plasmid sequence in MON 87411 has been confirmed by sequencing and bioinformatic analyses (see Section IV.A).Monsanto Company CR240

- 13U1 47 of 374 Table III

- 1. Summary of Genetic Elements in PV

- ZMIR10871

Genetic Element Loca tion in

Plasmid

Vector Function (Reference)

T

- DNA

B 1

- Left Border Region 1

- 442 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T

- DNA ( Barker et al. 1983 ) Intervening Sequence 443

- 485 Sequence used in DNA cloning

T 2

- E9 486

- 1118 3 ′ UTR of the rbcS gene family from

Pisum sativum (pea) encoding the small subunit of ribulose bisphosphate carboxylase protein ( Coruzzi et al. 1984 ) that directs polyadenylation of the mRNA Intervening Sequence 1119

- 1147 Sequence used in DNA cloning

DvSnf7 p 1148

- 1387 Partial coding sequence of the Snf7 gene designed to match that from Diabrotica virgifera virgifera ( Baum et al. 2007a ; Baum et al. 2007b ) encoding the SNF7

subunit of the ESCRT

- III complex ( Babst

et al. 2002 ) that forms part of the suppression cassette Intervening Sequence 1388

- 1537 Sequence used in DNA cloning

DvSnf7 p 1538

- 1777 Partial coding sequence of the Snf7 gene designed to match that from Diabrotica virgifera virgifera ( Baum et al. 2007a ; Baum et al. 2007b ) encoding the SNF7

subunit of the ESCRT

- III complex ( Babst

et al. 2002 ) that forms part of the suppression cassette Intervening Sequence 1778

- 1813 Sequence used in DNA cloning

I 3

- Hsp70 1814

- 2617 Intron and flanking exon sequence of the hsp70 gene from Zea mays (maize) encoding the heat shock protein 70 (HSP70) ( Rochester et al. 1986 ) that is involved in regulating gene expre ssion ( Brown and Sant ino 1997 )

P 4

- e35S 2618

- 3238 Promoter from the 35S RNA of cauliflower mosaic virus (CaMV) ( Odell

et al. 1985 ) containing the duplic ated enhancer region ( Kay et al. 1987 ) that directs transcription in plant cells Intervening Sequence 3239

- 3264 Sequence used in DNA cloningMonsanto Company CR240

- 13U1 48 of 374 Table III

- 1 (continued) . Summary of Genetic Elements in PV

- ZMIR10871

Genetic Element Location in

Plasmid

Vector Function (Reference)

P

-

pIIG 3265

- 4213 Promoter sequence of the pIIG gene encoding the physical impedance induced protein from Zea mays ( Huang et al. 1998 ) (maize) that directs transcription in plant

cells Intervening Sequence 4214

- 4219 Sequence used in DNA cloning

L 5

- Cab 4220

- 4280 5' UTR leader sequence from chlorophyll

a/b

- binding (CAB) protein of Triticum

aestivum (wheat) that is involved in regulating gene expression ( Lamppa et al.

1985 ) Intervening Sequence 4281

- 4296 Sequence used in DNA cloning

I

- Ract1 4297

- 4776 Intron and flanking UTR sequence of the

act1 gene from Oryza sativa (rice) encoding rice Actin 1 protein is involved in regulating gene expression ( McElroy et al.

1990 ) Intervening Sequence 4777

- 4785 Sequence used in DNA cloning

CS 6

- cry3Bb1 4786

- 6747 Codon optimized coding sequence from Cry3Bb1 protein of Bacillus thuringiensis that provides insect resistance ( English et

al . 2000 ) Intervening Sequence 6748

- 6766 Sequence used in DNA cloning

T

- Hsp17 6767

- 6976 3' UTR sequence from a heat shock protein, Hsp17, of Triticum aestivum (wheat) ( McElwain and Spiker 1989 ) that directs polyadenylation of the mRNA Intervening Sequence 6977

- 7024 Sequence used in DNA cloning

P

- TubA 7025

- 9205 Promoter , 5 ′UTR leader and intron

sequence s of the OsTubA gene family from Oryza sativa (rice) encoding α

- tubulin ( Jeon et al. 2000 ) that directs transcription in plant cells Intervening Sequence 9206

- 9209 Sequence used in DNA cloningMonsanto Company CR240

- 13U1 49 of 374 Table III

- 1 (continued) . Summary of Genetic Element s in PV

- ZMIR10871

Genetic Element Location in

Plasmid

Vector Function (Reference)

TS 7

- CTP2 9210

- 9437 Targeting sequence of the ShkG g ene from Arabidopsis thaliana encoding the EPSPS transit peptide region that directs transport of the protein to the chloroplast ( Herrmann

1995 ; Klee et al. 1987 )

CS

- cp4 epsps 9438

- 10805 Codon optimized coding sequence of the

aroA gene from Agrobacterium sp. strain CP4 encoding the native CP4 EPSPS protein that provides herbicide tolerance ( Barry et al. 2001 ; Padgette et al. 1996 ) Intervening Sequence 10806

- 10812 Sequence used in DNA cloning

T

- TubA 10813

- 11394 3' UTR sequence of the OsTubA gene family from Oryza sativa (rice) encoding

α

- tubulin ( Jeon et al. 2000 ) that directs polyadenylation of mRNA Intervening Sequence 11395

- 11412 Sequence used in DNA cloning

B

- Right Border Region 11413

- 11743 DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T

- DNA ( Depicker et al. 1982 ; Zambryski et al.

1982 )

V ector Backbone Intervening Sequence 11744

- 11879 Sequence used in DNA cloning

aadA 11880

- 12768 Bacterial promoter, coding sequence, and 3' UTR for an aminoglycoside

- modifying enzyme, 3''(9)

- O

- nucleotidyltransferase from the transposon Tn 7 ( Fling et al. 1985 ) that confers spectinomycin and

stre ptomycin resistance Intervening Sequence 12769

- 13298 Sequence used in DNA cloning

OR 8

- ori

- pBR322 13299

- 13887 Origin of replication from plasmid pBR322 for maintenance of plasmid in E. coli ( Sutcliffe 1979 ) Intervening Sequence 13888

- 14314 Sequence used in DNA cloning

CS

- rop 14315

- 14506 Coding sequence for repressor of primer protein from the ColE1 plasmid for maintenance of plasmid copy number in E.

coli ( Giza and Huang 1989 ) Intervening Sequence 14507

- 16014 Sequence used in DNA cloningMonsanto Company CR240

- 13U1 50 of 374 Table III

- 1 (continued) . Summary of Genetic Elements in PV

- ZMIR10871

Genetic Element Location in

Plasmid

Vector Function (Refere nce)

OR

- ori V 16015

- 16411 Origin of replication from the broad host range plasmid RK2 for maintenance of plasmid in Agrobacterium ( Stalker et al.

1981 ) Intervening Sequence 16412

- 16497 Sequence used in DNA cloning

1 B, Border

2 T, T ranscription Termination Sequence

3 I, Intron

4 P, Promoter

5 L, Leader

6 CS, Coding Sequence

7 TS, Targeting Sequence

8 OR, Origin of Replication

p Superscript in DvSnf7 indicates the partial sequence. Within the DvSnf7 cassette, bases 1148

- 1387 are reverse complement to bases 1538

- 1777

.Monsanto Company CR240

- 13U1 51 of 374 1 MANPNNRSEH DTIKVTPNSE LQTNHNQYPL ADNPNSTLEE LNYKEFLRMT EDSSTEVLDN
61 STVKDAVGTG ISVVGQILGV VGVPFAGALT SFYQSFLNTI WPSDADPWKA FMAQVEVLID
121 KKIEEYAKSK ALAELQGLQN NFEDYVNALN SWKKTPLSLR SKRSQDRIRE LFSQAESHFR
181 NSMPSFAVSK FEVLFLPTYA QAANTHLLLL KDAQVFGEEW GYSSEDVAEF YRRQLKLTQQ

241 YTDHCVNWYN VGLNGLRGST YDAWVKFNRF RREMTLTVLD LIVLFPFYDI RLYSKGVKTE
301 LTRDIFTDPI FLLTTLQKYG PTFLSIENSI RKPHLFDYLQ GIEFHTRLRP GYFGKDSFNY
361 WSGNYVETRP SIGSSKTITS PFYGDKSTEP VQKLSFDGQK VYRTIANTDV AAWPNGKVYL
421 GVTKVDFSQ Y DDQKNETSTQ TYDSKRNNGH VSAQDSIDQL PPETTDEPLE KAYSHQLNYA
481 ECFLMQDRRG TIPFFTWTHR SVDFFNTIDA EKITQLPVVK AYALSSGASI IEGPGFTGGN
541 LLFLKESSNS IAKFKVTLNS AALLQRYRVR IRYASTTNLR LFVQNSNNDF LVIYINKTMN
601 KDDDLTYQTF DLATTNSNMG FSGDKNELII GAESFVSNEK IYIDKIEFIP VQL

Figure III

- 3. Deduced Amino Acid Sequence of the Cry3Bb1 Protein The amino acid sequence of the Cry3Bb1 protein was deduced from the full

- length coding nucleotide sequence present in PV

- ZMIR10871.

1

61 MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG LKKSGMTLIG

SELRPLKVMS SVSTA C
121 TRITGLLEGE DVINTGKAMQ AMGARIRKEG DTWIIDGVGN GGLLAPEAPL DFGNAATGCR MLHG ASSRPATARK SSGLSGTVRI PGDKSISHRS FMFGGLASGE

181 LTMGLVGVYD FDSTFIGDAS LTKRPMGRVL NPLREMGVQV KSEDGDRLPV TLRGPKTPTP
241 ITYRVPMASA QVKSAVLLA G LNTPGITTVI EPIMTRDHTE KMLQGFGANL TVETDADGVR
301 TIRLEGRGKL TGQVIDVPGD PSSTAFPLVA ALLVPGSDVT ILNVLMNPTR TGLILTLQEM

361 GADIEVINPR LAGGEDVADL RVRSSTLKGV TVPEDRAPSM IDEYPILAVA AAFAEGATVM

421 NGLEELRVKE SDRLSAVANG LKLNGVDCDE GETSLVVRGR PDGKGLGNAS GAAVATHLDH

481 RIAMSFLVMG LVSENPVTVD DATMIATSFP EFMDLMAGLG AKIELSDTKA A

Figure III

- 4 . Deduced Amino Acid Sequence of the CTP2 Targeting Sequence and

CP4 EPSPS Protein The transit peptide CTP2 for the CP4 EPSPS protein is underlined. Accumulation of the

CP4 EPSPS protein is targeted to the chloroplasts using cleavable CTP2, the transit peptide of the Arabidopsis thaliana EPSPS protein. The amino acid sequence of the CP4 EPSPS protein was

deduced from the full

- length coding nucleotide sequence present in PV

- ZMIR108 71.Monsanto Company CR240

- 13U1 52 of 374 I V. CHARACTERIZATION OF THE GENETIC MODIFICATION

Characterization of the DNA insert in MON 87411 was conducted using a combination of sequencing, PCR, and bioinformatics. The results of this characterization demonstrate

that MON 87411 contains one copy of the intended transfer DNA (T

- DNA) containing the DvSnf7 suppression cassette and the cry3Bb1 and cp4 epsps expression cassettes that is stably integrated at a single locus and is inherited according to Mendelian principles over multipl e generations. These conclusions are based on several lines of evidence:

**•** Molecular characterization of MON 87411 by Next Generation Sequencing and Junction Sequence Analysis (NGS/JSA) demonstrated that MON 87411 contained a single DNA insert. These whol e

- genome sequence analys e s provide d a comprehensive assessment of MON 87411 to determine the presence of sequences

derived from PV

- ZMIR10871 ( DuBose, et al. 2013 ; Kovalic, et al. 2012 )

, demonstrated that MON 87411 contained a single DNA insert

.

**•** D i rected sequencing (locus

- specific PCR, DNA sequencing and analyses) of

MON 8 7411 w as used to determine the complete sequence of the single DNA insert

from PV

- ZMIR10871, the adjacent flanking DNA, and the 5' and 3' insert

- to

- flank junctions. This analysis confirmed that the sequence and organization of the DNA is identical to the corresponding region in the PV

- ZMIR10871 T

- DNA. The sequencing analysis, along wit h the NGS/JSA result showing that MON 87411 contains only a single DNA insert with no unintended fragments, also confirms that no vector backbone or other unintended plasmid sequences are present in

MON 87411. Furthermore, the genomic organization at the insertion site was assessed by comparing the sequences flanking the T

- DNA insert in MON 87411 to the sequence of the insertion site in conventional maize. This analysis determined that no major DNA rearrangement o ccurred at the insertion site in MON 87411 upon DNA integration.

**•** Generational stability analysis by NGS/JSA demonstrated that the single

PV

- ZMIR10871 T

- DNA insert in MON 87411 has been maintained through five breeding generations, thereby confirming the stability of the T

- DNA in MON 87411.

**•** Segregation analysis corroborates the insert stability demonstrated by NGS/JSA and independently establishes the nature of the T

- DNA as a single chrom osomal locus.

Taken together, the characterization of the genetic modification in MON 87411 demonstrates that a single copy of the intended T

- DNA was stably integrated at a single locus of the maize genome and that no plasmid backbone sequences are presen t in

MON 87411.

A schematic representation of the NGS/JSA methodology and the basis of the characterization using NGS/JSA and PCR sequencing are illustrated in Figure IV

- 1

below. These techniques and their value in DNA characterization in crop plants are further described in Appendi ces B and L.Monsanto Company CR240

- 13U1 53 of 374 Step 1 : Next generation sequencing ( NGS ) of genomic DNA samples

. A collection of 100

- mer sequences are generated which comprehensively cover the test and control sample genomes

.

Step 2 : Selection of all 100

- mers containing sequence similar to that of the transformation plasmid

Step 3 : Junction Sequence Analysis

Bioinformatics ( JSA ) to find and characterize all

selected 100

- mer sequences defining transgenic

insertions

Step 4 : Directed sequencing across the insertion

from 5 ' flank to 3 ' flank 1 ) Insert number determined from junction sequence pairs ( in this case of a single insert one pair is expected )

2 ) Exact sequence of insert ( s ) determined

.

3 ) Organization

, intactness and copy number of

genetic elements demonstrated

4 ) Demonstrates no backbone sequence has been

incorporated

Step 5 : Directed sequencing across wild type

insertion site 5 ) Integrity and organization of the insertion site ( s ) Experimental Stage Resultant Molecular Characterization

Figure IV

- 1. Molecular Characterization using Sequencing and Bioinformatics

Genomic DNA from MON 87411 and the conventional control was sequenced using NGS technology that produces a set of short, randomly distributed sequence reads (each approximately 100 bp long) that comprehensively cover the genomes (Step 1). Utilizing these genomic sequences, bioinformatics search tools were used to select all sequence reads (100

- mers) that were significantly similar to the transformation plasmid (Step 2) and Junction Sequence Analysis (JSA) bioinformatics was used to determine the insert number (Step 3). Overlapping PCR products are produced which span any in sert(s) and their wild type loci (Step 4 and Step 5, respectively). These PCR products are sequenced to provide a detailed characterization of the insertion site(s).

The NGS/JSA method characterized the genomic DNA from MON 87411 and the

conventional control using short (~100 bp) randomly distributed sequence fragments (sequencing reads) generated in sufficient number to ensure comprehensive coverage of the sample genomes. Bioinformatics analysis was then used to select sequencing reads

that contained sequences similar to the transformation plasmid, and these were analysed

to determine the number of DNA inserts. NGS/JSA was run on all MON 87411 samples

and the conventional controls ; results of NGS/JSA are shown in Section IV.A and IV.D

below.

The numb er of DNA inserts was determined by analyzing sequences for novel junctions. The junctions of the DNA insert and the flanking DNA are unique for each insertion ; an example is shown in Figure IV

- 2 below ( Kovalic et al. 2012 ) . Therefore, insertion sites can be recognized by analyzing for sequence reads containing such junctions. NGS/JSA

Directed SequencingMonsanto Company CR240

- 13U1 54 of 374 Figure IV

- 2. Junctions and J unction Sequences Depicted above are five example junction sequences formatted and labeled to indicate the plasmid/flanking DNA portions of the sequences and with the junction point indicated (plasmid DNA is shown in bold, underlined text and flank DNA is shown in plain text). Junctions are detected by examining the NGS data for sequences having portions of plasmid sequences that span less than the full read. Detected junctions are typically characteristic of plasmid insertions in the genome.

Each inser tion will produce two unique junction sequence classes characteristic of the genomic locus, one at the 5' end of the insert (illustrated in see Figure IV

- 3 below , and named junction sequence class A, or JSC

- A, in this case) and one at the 3' end of the

ins ert , named junction sequence class B, or JSC

- B, in this case ( Kovalic et al. 2012 )

.

Figure IV

- 3. Two Unique Junction Sequence Classes are Produced by the Insertion of a Single Plasmid Region A schematic representation of a single DNA insertion within the genome showing the inserted DNA, the 5' and 3' flanks (depicted as areas bounded by dotted lines), and the two distinct regions spanning the junctions between inserted DNA and flanking DNA (shaded boxes). The group of

~100

- mer sequences in which each read contains sequences from both the DNA insert and the adjacent flanking DNA at a given junction is call ed a Junction Sequence Class. In this example, two distinct junction sequence classes (in this case: Class A at the 5' end and Class B at the 3' end) are represented.

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACG

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACGTGT

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACGTGTCG

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACGTGTCGGG

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACGTGTCGGGAT

Flanking DNA Plasmid DNA Junction



## DNA insert 5’ Flank 3’ Flank Insert Junction Regions

Junction Sequences: Class A

Junction Sequences: Class BMonsanto Company CR240

- 13U1 55 of 374 By evaluating the number of unique junction classes detected, the number of insertion

si tes of the plasmid sequence can be determined. A longer description of the molecular

methods used to characterize MON 87411 can be found in Appendi ces B and L

. For a single insert two junction sequence classes are expected, one each originating from eith er end of the insert, both containing portions of plasmid DNA insert and flanking sequence.

In the case of MON 87411, two unique junction sequence classes, both containing portions of T

- DNA and flanking sequence, were detected indicating MON 87411

contain s a single DNA insert (results are described in Section IV.A, methods and supplementary data are presented in Appendix B). The identity of the DNA insert ( i.e.

, T

- DNA or backbone) is determined by direct sequencing described below.

The NGS/JSA strategy to determine insert number of the integrated plasmid DNA was designed to ensure that all transgenic segments would be identified. The depth of coverage (the average number of times each base of the genome is independently sequenced) was ≥ 75 × for each sampl e genome. It has previously been demonstrated that

≥ 75 × coverage of the soybean genome is adequate to provide comprehensive coverage

and ensure detection of inserted DNA ( Kovalic et al. 2012 ) and similarly ≥ 75 × coverage provides comprehensive coverage of the maize genome ( Clarke and Carbon 1976 ) . The level of sensitivity of this method was demonstrated by detection of a positive control spiked at 1/10 th copy

- per

- genome equivalent.

Directed sequencing (locus

- specific PCR and DNA sequencing analyses, Figure IV

- 1, step 4) complements the NGS/JSA analyses. As indicated above, NGS/JSA results

determined that MON 87411 contains a single DNA insertion si te. Sequencing of the insert and flanking genomic DNA determined the complete sequence of the insert and flanks; it determined that the sequence and organization of the DNA insert is identical to

those in the T

- DNA of PV

- ZMIR10871, and that each genetic e lement (except for the border regions) in the insert is intact, and also that no vector backbone, or other unintended plasmid sequences were inserted in MON 87411. Furthermore, the genomic organization at the insertion site was assessed by comparing the i nsert and MON 87411 flanking sequence to the sequence of the insertion site in conventional maize. This assessment indicated that the integration site in the MON 87411 genome included a

118 bp deletion of genomic DNA but is otherwise identical to the nati ve sequence. Results are described in Section IV.B and Section IV.C; methods are presented in Appendix B.

The stability of the T

- DNA present in MON 87411 across multiple generations was evaluated by NGS/JSA analyses. Genomic DNA from five generations of MON 87411 (Figure IV

- 4) was assayed for all unique junction sequence classes as described above. This information was used to determine the number and identity of insertion sites. For a single insert, two junction sequence classes are expected; each one originates from either end of the insert, both containing portions of DNA insert and flanking sequence. In the

case of MON 87411, two identical junction sequence classes were detected in all the generations tested, confirming that the single insert is sta bly inherited over multiple generations.

Segregation analysis of the T

- DNA was conducted to determine the inheritance and stability of the insert in MON 87411. Results from this analysis demonstrate inheritance according to Mendelian principles and the st ability of the insert is as expected acrossMonsanto Company CR240

- 13U1 56 of 374 multiple generations (Figure IV

- 11, Table IV

- 4

, and Table IV

- 5). The segregation analysis corroborates the insert stability demonstrated by NGS/JSA and independently establishes the genetic behavior of the T

- DNA as a single chromosomal locus.

The results of these analyses of MON 87411 demonstrate that a single copy of the

intended T

- DNA derived from PV

- ZMIR10871 was inserted at a single locus of the

MON 87411 genome, that the sequence and organization of the T

- DN A insert is identical to the corresponding region in PV

- ZMIR10871 and that no additional genetic elements, including backbone sequences, were detected in MON 87411. Generational stability analysis demonstrated that the single insert in MON 87411 was maint ained through five generations of the breeding history, thereby confirming the stability of T

- DNA in

MON 87411. In addition, results from segregation analyses confirmed the genetic

behavio r of the T

- DNA as a single chromosomal locus.Monsanto Company CR240

- 13U1 57 of 374 Figure IV

- 4. Breeding History of MON 87411

R 0 corresponds to the transformed plant, F # is the filial generation, R # is the regenerant and subsequent generations,  designates self

- pollination.

1 Generation used for full molecular characterization

. 2 Generations used to confirm insert stability

. 3 Generation used for commercial development of MON 87411

. 4 Generation used for c ompositional analysis and RNA expression studies

. 5 Generation used for agronomic/phenotypic studi es LH244 R 1

LH244 R 5 2,3 LH244 R 4 1,2 LH244 R 3 LH244 R 2 

  

R 4 F 1 (LH244 × HCL645) 2,4 LH244 R 0



LH244 R 6 2 R 5 F 1 (LH244 × LH287) 2,5

 × HCL645

× LH287Monsanto Company CR240

- 13U1 58 of 374 Figure IV

- 5. Schematic Representation of the Insert and Flanking Sequences in MON 87411 Linear map showing DNA derived from the T

- DNA of PV

- ZMIR10871 integrated in to MON 87411. Right

- angled arrows indicate the ends of the integrated T

- DNA and the beginning of the flanking sequence. Identified on the map are genetic elements within the insert. This schematic diagram is drawn to scale ; the exact coordinates of every element is shown in Table IV

- 1.

r1 Superscript in Left and Right Border Regions indicate that the sequence in MON 87411 was truncated compared to the sequences in PV

- ZMIR10871.

P Superscript in DvSnf7 indicates the partial sequence

.Monsanto Company CR240

- 13U1 59 of 374 Table IV

- 1. Summary of Genetic Elements in MON 87411

Genetic Element 1 Location in Sequence 2 Function (Reference)

5 ′ Flank 1

- 1460 Sequence flanking the 5 ′ end of the insert

B 3

- Left Border Region r1 1461

- 1723 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the

T

- DNA ( Barker et al. 1983 ) Intervening Sequence 1724

- 1766 Sequence used in DNA cloning

T 4

- E9 1767

- 2399 3' UTR sequence from Pisum sativum rbcS gene family encoding the small subunit of ribulose bisphosphate carboxylase protein ( Coruzzi et al. 1984 ) that directs polyadenylation of the mRNA Intervening Sequence 2400

- 2428 Sequence used in DNA cloning

DvSnf7 P 2429

- 2668 Partial coding sequence of the Snf7 gene designed to match that from Diabrotica virgifera virgifera ( Baum et al. 2007a ; Baum

et al. 2007b ) encoding the Snf7 subunit of

the ESCR T

- III complex ( Babst et al. 2002 )

that form s part of the suppression cassette Intervening Sequence 2669

- 2818 Sequence used in DNA cloning

DvSnf7 P 2819

- 3058 Partial coding sequence of the Snf7 gene designed to match that from Diabrotica virgifera virgifera ( Baum et al. 2007a ; Baum et al. 2007b ) encoding the Snf7

subunit of the ESCR T

- III complex ( Babst et

al. 2002 ) that forms part of the suppression

cassette Intervening Sequence 3059

- 3094 Sequence used in DNA cloning

I 5

- Hsp70 3095

- 3898 Intron and flanking exon sequence of the hsp70 gene from Zea mays (maize) encoding the heat shock protein 70 (HSP70) ( Rochester et al. 1986 ) is involved in regulating gene expression ( Brown and

Santino 1997 )

P 6

- e35S 3899

- 4519 Promoter from the 35S RNA of cauliflower mosaic virus (CaMV) ( Odell et al. 1985 ) containing the duplicated enhancer region ( Kay et al. 1987 ) that directs transcription in plant cells Intervening Sequence 4520

- 4545 Sequence used in DNA cloning

P

-

pIIG 4546

- 5494 Promoter sequence from the physical impedance induced protein of Zea mays (maize) ( Huang et al. 1998 ) that directs transcription in plant cellsMonsanto Company CR240

- 13U1 60 of 374 Table IV

- 1 (continued). Summary of Genetic Elements in MON 87411

Genetic Element 1 Location in Sequence 2 Function (Reference)

Intervening Sequence 5495

- 5500 Sequence used in DNA cloning

L 7

- Cab 5501

- 5561 5' UTR leader sequence from chlorophyll a/b

binding (CAB) protein of Triticum aestivum (wheat) that is involved in regulating gene expression ( Lamppa et al. 1985 ) Intervening Sequence 5562

- 5577 Sequence used in DNA cloning

I

- Ract1 5578

- 6057 Intron and flanking UTR sequence of the

act1 gene from Oryza sativa (rice) encoding rice Actin 1 protein is involved in regulating gene expression ( McElroy et al. 1990 ) Intervening Sequence 60 58

- 6066 Sequence used in DNA cloning

CS 8

- cry3Bb1 6067

- 8028 Codon optimized coding sequence for Cry3Bb1 protein of Bacillus thuringiensis that provides insect resistance ( English et al.

2000 ) Intervening Sequence 8029

- 8047 Sequence used in DNA cloning

T

- Hsp17 8048

- 8257 3' UTR sequence from a heat shock protein,

H SP 17, of Triticum aestivum (wheat) ( McElwain and Spiker 1989 ) that directs polyadenylation of the mRNA Intervening Sequence 8258

- 8305 Sequence used in DNA cloning

P

- TubA 8306

- 10486 Promoter, 5 ′ UTR leader and intron sequences

of the OsTubA gene family from Oryza sativa (rice) encoding α

- tubulin ( Jeon et al. 2000 ) that directs transcription in plant cells Intervening Sequence 10487

- 10490 Sequence used in DNA cloning

TS 9

- CTP2 10491

- 10718 Targeting sequence of the ShkG gene from Arabidopsis thaliana encoding the EPSPS transit peptide region that directs transport of the protein to the chloroplast ( Herrmann

1995 ; Klee et al. 1987 )

CS

- cp4 epsps 10719

- 12086 Coding sequence of the ar oA gene from Agrobacterium sp. strain CP4 encoding the native CP4 EPSPS protein that provides

herbicide tolerance ( Barry et al. 2001 ; Padgette et al. 1996 ) Intervening Sequence 12087

- 12093 Sequence used in DNA cloning

T

- TubA 12094

- 12675 3' UTR sequence of the OsTubA gene from Oryza sativa (rice) encoding α

- tubulin ( Jeon

et al. 2000 ) that directs polyadenylation of

mRNAMonsanto Company CR240

- 13U1 61 of 374 Table IV

- 1 (continued). Summary of Genetic Elements in MON 87411

Genetic Element 1 Location in Sequence 2 Function (Reference)

Intervening Sequence 12676

- 12693 Sequence used in DNA cloning

B

- Right Border Region r1 12694

- 12708 DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T

- DNA ( Depicker et al. 1982 ; Zambryski et al.

1982 )

3 ′ Flank 12709

- 14511 Sequence flanking the 3 ′ end of the insert

1 Although flanking sequences and intervening sequence are not functional genetic elements, they comprise a portion of the sequence.

2 Numbering refers to the sequence of the insert in MON 87411 and adjacent DNA.

3 B, Border

4 T, Transcription Termination Sequence

5 I, Intron

6 P, Promoter

7 L, Leader

8 CS, Coding Sequence

9 TS, Targeting Sequence

r1 Superscript in Left and Right Border Regions indicate that the sequence in MON 87411 was truncated compared to the sequences in PV

- ZMIR10871

P Superscript in DvSnf7 indicates the partial sequence. Within the DvSnf7 cassette, bases 2429

- 2668 are reverse complement to bases 2819

- 3058.

IV.A

. Determining the Number of DNA inserts in MON 87411

The number of insertion sites of PV

- ZMIR10871 DNA in the MON 87411 was assessed by performing NGS/JSA on MON 87411 genomic DNA. A single genomic DNA insertion is expected to produce two junction sequen ce classes and any additional integration sites would produce additional junction sequence classes. A plasmid map of

PV

- ZMIR10871 is shown in Figure III

- 1. A schematic representation of the insert and flanking sequences in MON 87411 is shown in Figure IV

- 5. The generations studied are depicted in the breeding history diagram shown in Figure IV

- 4. The NGS conducted for all samples and its adequate depth of coverage in each case is summari z ed in

Section IV.A.1, Section IV.A.2 and Table IV

- 2. The sensitiv ity of the method is

demonstrated in Section IV.A.1 with data shown in Table IV

- 3. The JSA analysis of the

R 4 generation is shown in Section IV.A.2 with data presented in Figure IV

- 6 and supplemental data shown in Appendix B; the other generations that we re used in the generational stability analysis are shown in Figure IV

- 4 with the results of JSA analysis described in Section IV.D, with JSA results shown in Figure IV

- 9, Figure IV

- 10 and supplemental data included in Appendix B. For full details on mater ials and methods see Appendix B.Monsanto Company CR240

- 13U1 62 of 374 IV.A.1. Next Generation Sequencing (NGS) for MON 87411 and Conventional

Control Genomic DNA

Genomic DNA from five generations of MON 87411 and the conventional controls (inbred LH244 and hybrids NL6169 and MPA640B ) were i solated and prepared for sequencing according to the manufacturer’s protocol (Illumina, TruSeq library protocol. For material and method details see Appendix B). These genomic DNA libraries were used to generate short (~100 bp) randomly distributed seque nce fragments (sequencing reads) in sufficient numbers to ensure comprehensive coverage of the maize genome (see Figure IV

- 1, Step 1).

To confirm sufficient sequence coverage in all generations of MON 87411 and the conventional controls, the 100

- mer sequen ce reads from all samples were analyzed to determine the effective depth of coverage ( i.e. , the average number of times any base of the genome is expected to be independently sequenced) by mapping all reads to a known single

- copy endogenous gene (Pyruvate decarboxylase ( pdc3 ), GenBank accession.version: AF370006.2). The analysis showed that pdc3 was covered by

100

- mers at >107 × for each sample (Table IV

- 2). It has previously been demonstrated

that ≥ 75 × coverage of the soybean genome is adequate to provide comprehensive coverage and ensure detection of inserted DNA ( Kovalic et al. 2012 ) and similarly ≥ 75 ×

cover age provides comprehensive coverage of the maize genome ( Clarke and Carbon

1976 ) . A summary of NGS and effective depth of coverage are shown in Table IV

- 2.

In order to confirm the method’s ability to detect any sequences derived from the

PV

- ZMIR10871 transformation plasmid, a sample of conventio nal control maize DNA spiked with PV

- ZMIR10871 DNA at 1 and 1/10 th genome equivalent was analyzed by NGS and bioinformatics. At 1 genome equivalent, 100% nucleotide identity was

observed over 100% of PV

- ZMIR10871 (Table IV

- 3). This result demonstrates th at all nucleotides of the transformation plasmid are observed by the sequencing and bioinformatic assessments performed. Also, observed coverage was adequate ( Clarke

and Carbon 1976 ) at a level of at most 1/10 th genomic equivalent (99.64% coverage at 100% identity for the 1/10 th genome equivalent spiked control sample, Table IV

- 3) and, hence, a detection level of at least 1/10 th genome equivalent was achieved for the plasmid DNA sequence assessment.Monsanto Company CR240

- 13U1 63 of 374 Table IV

- 2. Sequencing (NGS) Conducted for MON 87411 and Control Genomic

DNA

Sample T otal Nucleotides (Gb) Effective Median Depth of Coverage (x

- fold)

LH244 346.9 126x

LH244 × HCL645 309.5 107x

LH244 × LH287 342.3 118x

R 4 334.9 125x

R 4 F 1 338.9 113x

R 5 F 1 346.6 126x

R 5 352.3 140x

R 6 363.9 134x
For each sample the raw data produced are presented in terms of total nucleotide number. Effective depth of coverage is determined by mapping and alignment of all raw data to a well known single copy locus ( pdc3 : pyruvate decarboxylase) within the maize g enome

. The median effective depth s of coverage are shown for all samples.

Table IV

- 3. Summary of NGS Data for the Conventional Control DNA Sample Spiked with PV

- ZMIR10871 DNA

1 Extent of coverage is calculated as the percent of all PV

- ZMIR10871 bases observed in the sequencing of the spike

in samples:

extent of coverage = number of spike in bases detected

total length ( bp ) of spike in plasmid × 100

2 Percent identity of coverage is calculated as the percent of all PV

- ZMIR10871 bases observed in the sequencing of the spike

- in samples:

Percent identity of coverage = number of identical bases ( spike in vs

. plasmid sequence ) detected

total length ( bp ) of spike in plasmid detected × 100 1/10 th copy Spike 1 copy Spike

Extent of coverage 1 of PV

- ZMIR10871 99.64% 100%

Percent identity of coverage 2 of PV

- ZMIR10871 100% 100%Monsanto Company CR240

- 13U1 64 of 374 IV.A.2 Characterization of insert number in MON 87411 using Bioinformatic Analysis

The number of insertion sites of DNA from PV

- ZMIR10871 in the MON 87411 was assessed by performing NGS/JSA on MON 87411 genomic DNA using the R 4 generation . A single genomic DNA insertion is expected to produce two junction sequence classes and any additional integration sites would produce additional juncti on sequence classes.

IV.A.2.1 Selection of Sequence Reads Containing Sequence of the PV

- ZMIR10871

PV

- ZMIR10871 was transformed into the parental line LH244 to produce MON 87411. Consequently, any DNA inserted into MON 87411 will consist of sequences that are similar to the PV

- ZMIR10871 DNA sequence. To fully characterize the DNA from

PV

- ZMIR10871 inserted in MON 87411, it is sufficient to completely analyze only the sequence reads that have similarity to the transformation plasmid (Figure IV

- 1, Step 2). In order to analyze the sequence data for insert number, all sequences that have significant sequence similarity to PV

- ZMIR10871 were selected from the full sequencing

datasets ( Kovalic et al. 2012 ) . Due to the depth of sequence coverage demonstrated with this methodology (see Section IV.A.1), on average, any area of the genome will be covered by more than 10 7 of the 100

- mer sequences; this ensures that sequences from

PV

- ZMIR10871 inserted into the genome will be detected by the analysis.

Using established criteria (which are described in the materials and methods, Appendix B), reads similar to the transformat ion plasmid were selected from

MON 87411 and the conventional control sequence datasets and were then used as input data for bioinformatic junction sequence analysis.

IV.A.2.2 Determination of the Insert Number

The NGS/JSA method described above used the entire PV

- ZMIR10871 plasmid as a query to determine the DNA insertion site number. Any DNA inserts, regardless of

whether the sequence was from backbone or T

- DNA, can be detected by junction

sequences. Therefore

, unlike the traditional Southern blot analysis that separately hybridizes T

- DNA or backbone probes, in NGA/JSA the determination of the T

- DNA insert number and of the absence of backbone or unintended sequences are simply represented by the determination o f the overall insert number in the genome followed by determination of the exact identity of any DNA insert using directed sequencing and sequence analysis.

By evaluating the number of unique junction classes, the number of DNA insertion sites

can be dete rmined (Figure IV

- 1, Step 3). For a single insert, at a single genomic locus, a single pair of junction sequences classes, each one originating from either end of the insert, is expected. If MON 87411 contains a single T

- DNA insert two junction sequence classes (JSCs) each containing portions of T

- DNA sequence and flanking sequence will

be detected.Monsanto Company CR240

- 13U1 65 of 374 To determine the insert number in MON 87411, the selected sequence reads described above were analyzed using JSA ( Kovalic et al. 2012 ) . JSA uses bioinformatic analysis to find and classify partially matched reads characteristic of the ends of insertions. The

numb er of resultant unique JSCs were determined by this analysis and are shown in the

table below.

Table IV

- 4. Unique Junction Sequence Class Results

Sample JSCs Detected

MON 87411 2

LH244 0

Detailed sequence information of the junction sequences detected by JSA is shown in Figure IV

- 6 (Panel B) and Figure B

- 1 in Appendix B. The location and orientation of the junction sequences relative to the T

- DNA insert determined for MON 87411 (as

described in Section IV.B) is shown in Figure IV

- 6, Panel A. As shown in the figure, there are two junction sequence classes identified in MON 87411. Junction Sequence Class A and Class B (JSC

- A and JSC

- B) both contain the T

- DNA border sequence joined to flanking sequence, indicating that they represent the sequenc es at the junctions of the

intended T

- DNA insert and flanking sequence.

The presence of two, and only two, junction sequence classes (joining T

- DNA border and flanking sequences) indicate this single pair of JSCs likely arises from the insertion of the

int ended PV

- ZMIR10871 T

- DNA at a single locus in the genome of MON 87411. JSC

A represents the junction of the T

- DNA Left Border sequence to the 5' flank and JSC

- B represents the junction of the T

- DNA Right Border sequence to the 3' flank. As shown by exact and complete alignment of the JSCs to the full flank/insert sequence (described in Section IV.B and shown in Figure IV

- 6, Panel B) both of these JSCs originate from the

same locus of the MON 87411 genome and are linked by contiguous, known and expected DN A that makes up the single insert.

Based on this comprehensive NGS/JSA study it is concluded that MON 87411 contains one DNA inserted into a single locus, as shown in Figure IV

- 5. The identity of the DNA insert was determined by the sequencing and analysi s of overlapping PCR products from

this locus as described below in Section IV.B. Additionally, the lack of detectable junction sequences attributable to plasmid backbone sequences leads to the conclusion that no backbone sequences from PV

- ZMIR10871 are p resent in MON 87411.Monsanto Company CR240

- 13U1 66 of 374 Panel A. Figure IV

- 6. Junction Sequences Detected by NGS/JSA Panel A: Linear map of MON 87411 illustrating the relationship of the detected junction sequences to the insert locus. The individual junction sequences detected by JSA are illustrated as stacked bars; each detected junction sequence read is shown trimmed to include only 30 bases of plasmid sequence. The scale of the identified junction sequences relative to the insert map is depicted by the br aces.

r1 Superscript in Left and Right Border Regions indicate that the sequence in MON 87411 was truncated compar ed to the sequences in

PV

- ZMIR10871.

P Superscript in DvSnf7 indicates the partial sequence

.Monsanto Company CR240

- 13U1 67 of 374 **JSC**

**- A Alignment:** 

**JSC_A: 1**  **AATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCATT 60**  **||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||**
**Directed Seq.: 1554 AATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCATT**  **1495**

**JSC_A: 61 GCTGATCCATGTAGATTTCCCGGACATGAAGCCA**  **^ CTTAACTATTCATTAGTGTTTTGCCT**  **|||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||| 120** 

**Directed Seq.: 1494 GCTGATCCATGTAGATTTCCCGGACATGAAGCCA**  **^ CTTAACTATTCATTAGTGTTTTGCCT**  **1435**

**JSC_A: 121 TTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC**  **|||||||||||||||||||||||||||||||||||||||||||| 164** 

**Directed Seq.: 1434 TTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC**  **1391**

**JSC**

**- B Alignment:** 

**JSC**

**- B:**  **1 GGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACACCA**  **60 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||** 
**Directed Seq.: 12616 GGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACACCA**  **12675**

**JSC**

**- B:**  **61 TCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT**  **^ AGAGAATCACAAACCTCTAGATGTATT**  **||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||| 120** 

**Directed Seq.: 12676 TCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT**  **^ AGAGAATCACAAACCTCTAGATGTATT**  **12735**

**JSC**

**- B:**  **121 AATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT**  **||||||||||||||||||||||||||||||||||||||||||| 163** 

**Directed Seq.: 12736 AATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT**  **12778**

Panel B. Figure IV

- 6 ( c ontinued). Junction Sequences Detected by NGS/JSA Panel B: Full consensus sequence for junction sequence Classes A and B (JSC

- A and JSC

- B) showing exact alignment to the independently determined in planta sequence at the insert locus (labeled “Directed Seq.”). The numbers flanking the sequence text represent the base pair numbering of the JSA consensus sequence or the insert sequence

, res pectively. Double underlined text indicates plasmid DNA sequence, single underlined text indicates plant genome sequence, and the carat character “^” indicates the junction point

between the MON 87411 insert and the flank.Monsanto Company CR240

- 13U1 68 of 374 IV. B

. Organization and Sequence of the Insert and Adjacent Flanking DNA in

MON 87411

The organization of the elements within the DNA insert and the adjacent genomic DNA was assessed using directed DNA sequence analysis (refer to Figure IV

- 1, Step 4). PCR primers were designed to amplify eight overlapping regions of the MON 87411 genomic DNA that span the entire length of the insert (Figure IV

- 7). The amplified PCR products were subjected to DNA sequen cing analyses. The results of this analysis confirm that the

MON 87411 insert is 11,248 bp and that each genetic element in the insert is intact, with the exception of the R ight and L eft border regions. The border regions both contain

small terminal dele tions with the remainder of the inserted border regions being identical to the sequence in PV

- ZMIR10871. The sequence and organization of the insert was also shown to be identical to the corresponding T

- DNA of PV

- ZMIR10871, confirming that a single copy o f T

- DNA was inserted as intended. This analysis also shows that only

T

- DNA elements (described in Table IV

- 1) were present and no PV

- ZMIR10871

backbone sequences were present in MON 87411.Monsanto Company CR240

- 13U1 69 of 374 Figure IV

- 7. Analysis of Overlapping PCR Products Across the MON 87411 Insert PCR was performed on both conventional control genomic DNA and genomic DNA of the R 4 generation of MON 87411 using eight pairs of primers to generate overlapping PCR fragments from MON 87411 for sequencing analysis. To verify the production of PCR products,

5 μl of each of the PCR reactions was loaded on the gel, except where noted below. The expected product size for each amplicon is provided in the illustratio n.

r1 Superscript in Left and Right Border Regions indicate that the sequence in MON 87411 was truncated compared to the sequences in PV

- ZMIR10871.

P Superscript in DvSnf7 indicates the partial sequence

. Product B: ~ 1.1 kb Product A: ~ 2.1 kb

Product C: ~ 2.1 kb Product D: ~ 2.2 kb

Product E: ~ 3.2 kb Product F: ~ 2.1 kb

Product G: ~ 2.5 kb Product H: ~ 2.8 kb 4.0

2.0

1.0 40

3.0

1.6 1 2 3 4 5 6 7 8 Product A Product B

9 10 11 12 13 14 15 16 17 Product C Product D

19 20 21 22 23 24 25 26 18 Product E Product F

2.0 1.6

1.0 4.0 3.0 40 27 28 29 30 31 32 33 34 Product G Product HMonsanto Company CR240

- 13U1 70 of 374 Lane designations are as follows:

Lane Sample Lane Sample

1 1 k b DNA Extension Ladder 18 1 k b DNA Extension Ladder

2 Conventional Control LH244 19 Conventional Control LH244

3 MON 87411 20 MON 87411

4 No template control 21 PV

- ZMIR10871

5 Conventional Control LH244 22 No template control

6 MON 87411 23 Conventional Control LH244

7 PV

- ZMIR10871 (2 μl) 24 MON 87411

8 No template control 25 PV

- ZMIR10871

9 Conventional Control LH244 26 No template control

10 MON 87411 27 Conventional Control LH244

11 PV

- ZMIR10871 (2 μl) 28 MON 87411

12 No template control 29 PV

- ZMIR10871

13 Conventional Control LH244 30 No template control

14 MON 87411 (10 μl) 31 Conventional Control LH244

15 PV

- ZMIR10871 32 MON 87411

16 No template control 33 No template control

17 1 k b DNA Extension Ladder 34 1 k b DNA Extension Ladder Figure IV

- 7 (continued). Analysis of Overlapping PCR Products Across the MON 87411 Insert Arrows next to the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained from the 1 k b DNA Extension Ladder (Invitrogen, Grand Island, NY) on the ethidium bromide stained gel.Monsanto Company CR240

- 13U1 71 of 374 IV.C. Sequencing of the MON 87411 Insertion Site

PCR and sequence analysis were performed on genomic DNA extracted from the conventional control to examine the insertion site in conventional maize (refer to Figure IV

- 1, Step 5). The PCR was performed with one primer specific to the genomic DNA sequence flanking the 5' end of the MON 87411 insert paired with a second prime r specific to the genomic DNA sequence flanking the 3' end of the insert (Figure IV

- 8). A sequence comparison between the PCR product generated from the conventional control and the sequence generated from the 5' and 3' flanking sequences of MON 87411

ind icates there was a 118 base pair deletion that occurred during integration of the

T

- DNA, with the remainder of the flanks in MON 87411 being identical to the conventional control. Such changes are common during plant transformation and these changes presu mably resulted from double

- stranded break repair mechanisms in the plant during the Agrobacterium

- mediated transformation process ( Salomon and Puch ta 1998 )

.Monsanto Company CR240

- 13U1 72 of 374 Figure IV

- 8. PCR Amplification of the MON 87411 Insertion Site PCR Analysis was performed to evaluate the MON 87411 insertion site. PCR was performed on conventional control DNA using Primer A, specific to the 5 ′ flanking sequence, and Primer B, specific to the 3 ′ flanking sequence of the MON 87411 insert. The DNA g enerated from the conventional control PCR was used for sequencing analysis. This illustration depicts the

MON 87411 insertion site in the conventional control (upper panel) and a schematic of the

MON 87411 insert (lower panel). Approximately 5 μl of eac h of the PCR reactions was loaded on the gel. Lane designations are as follows:

Lane Sample

1 1 k b DNA Extension Ladder

2 Conventional Control LH244

3 No template DNA control

4 1 k b DNA Extension Ladder

Arrows on the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained

from the 1 k b DNA Extension Ladder (Invitrogen, Grand Island, NY) on the ethidium bromide stained gel.

r1 Superscript in Left and Right Border Regions indicate th at the sequence in MON 87411 was truncated compared to the sequences in PV

- ZMIR10871.

P Superscript in DvSnf7 indicates the partial sequence

. 5.0
4.0 40

3.0

2.0

1.6

1.0

0.5 4 3 2 1

1.6 3.0

2.0 5.0
4.0 40

1.0

0.5

Primer A Primer B

Primer A Primer BMonsanto Company CR240

- 13U1 73 of 374 IV.D. Determination of Insert Stability over Multiple Generations of MON 87411

In order to demonstrate the stabil ity of the T

- DNA present in MON 87411 through multiple generations, NGS/JSA analysis was performed using DNA obtained from five breeding generations of MON 87411. The breeding history of MON 87411 is presented in Figure IV

- 4, and the specific generations tested are indicated in the figure legend. The

MON 87411 (R 4 ) generation was used for the molecular characterization analyses

discussed in Sections IV.A

- IV.C and shown in Figure IV

- 6 and Figure B

- 1 in Appendix B. To assess stability, four additional gene rations were evaluated by NGS/JSA analysis as previously described in Section IV.A, and compared to the fully characterized

MON 87411 (R 4 ) generation. The conventional controls used for the generational stability analysis included LH244, which has a genet ic background similar to the

MON 87411 (R 4 ), MON 87411 (R 5 ) and MON 87411 (R 6 ) generations and represents the original transformation line. The conventional control hybrid LH244 × HCL645 has a genetic background similar to the MON 87411 R 4 F 1 hybrid. In a ddition, the conventional control hybrid LH244 × LH287 , has a genetic background similar to the

MON 87411 R 5 F 1 hybrid. Genomic DNA isolated from each of the selected generations

of MON 87411 and conventional controls were used for NGS/JSA analysis. The r esults are shown in Figure IV

- 9, Figure IV

- 10 and Figure B

- 1 in Appendix B.

IV.D.1 Determination of the Insert Number

To determine the insert number in the MON 87411 generations, the sequences selected as described in Section IV.A.2.1 were analyzed using JSA ( Kovalic et al. 2012 ) , where the number of resultant JSCs containing PV

- ZMIR10871 DNA sequence determined by this analysis is shown in the table below.

Table IV

- 5. Junction Sequence Classes Detected

Sample JSC s Detected

MON 87411 (R 4 ) 2

MON 87411 (R 4 F 1 ) 2

MON 87411 (R 5 ) 2

MON 87411 (R 5 F 1 ) 2

MON 87411 (R 6 ) 2

LH244 0

LH244 × HCL645 0

LH244 × LH287 0Monsanto Company CR240

- 13U1 74 of 374 Figure IV

- 9 and Figure IV

- 10

, below, identif y the presence of two, and only two, identical junction sequence classes in each of the five assessed MON 87411 generations (R 4 , R 5 , R 6 , R 4 F 1 and R 5 F 1 ) as expected for a stably maintained single insert. This single identical pair of JSCs is observed due to the insertion of PV

- ZMIR10871 T

- DNA at a single locus in the genome of MON 87411. The consistency of these JSC data across all generations tested demonstrates that this single locus is stably maintained throughout the

MON 87411 breeding process.

These re sults, therefore, demonstrate that the MON 87411 single locus of integration has been maintained through several generations of the breeding of MON 87411; thereby confirming the stability of the insert. Based on this comprehensive sequence data and

bioinf ormatic analysis (NGS/JSA), it is concluded that MON 87411 contains a single and

stable T

- DNA insertion.Monsanto Company CR240

- 13U1 75 of 374 R 4 - AATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT Directed Seq. GAATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R 6 GAATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R 4 F 1 - AATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R 5 GAATTGAAAAAAAA TTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R 5 F 1 GAATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R 4 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA ^ CTTAACTATTCATTAGTGTTTTGCC Directed Seq. TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA ^ CTTAACTATTCATTAGTGTTTTGCC

R 6 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA ^ CTTAACTATTCATTAGTGTTTTGCC

R 4 F 1 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA ^ CTTAACTATTCATTAGTGTTTTGCC

R 5 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA ^ CTTAACTATTCATTAGTGTTTTGCC

R 5 F 1 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA ^ CTTAACTATTCATTAGTGTTTTGCC

R 4 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC Directed Seq. TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC

R 6 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC

R 4 F 1 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

R 5 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

----
R 5 F 1 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

---

Figure IV

- 9. Junction sequences detected by JSA. Junction Sequence Class A Alignment (All Generations Tested) Full consensus sequence for JSC

- A showing exact alignment to the independently determined in planta locus specific sequence (labeled “Directed Seq.” in the figure); individual consensus sequences for each of the five generations are labeled according to th eir generation (R 4 , R 5 , R 6 , R 4 F 1 and R 5 F 1 ). Double underlined text indicates plasmid DNA sequence, single underlined text indicates plant genome sequence, and the carat character “^” indicates the junction point between the MON 87411 insert and the flank. The asterisk character “*” indicates identical nucleotide in every sequence at that position in the alignment. Dash characters indicate positions past the end of the consensus sequence for a particular generation.Monsanto Company CR240

- 13U1 76 of 374 R 5 F 1 AGCGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA Directed Seq. AGCGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R 5 -- CGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R 4 F 1 - GCGGCTAGA GCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R 4 --- GGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R 6 AGCGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R 5 F 1 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT ^ AGAGAATCACAAACCTCTAGATGT Directed Seq. CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT ^ AGAGAATCACAAACCTCTAGATGT

R 5 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT ^ AGAGAATCACAAACCTCTAGATGT

R 4 F 1 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT ^ AGAGAATCACAAACCTCTAGATGT

R 4 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT ^ AGAGAATCACAAACCTCTAGATGT

R 6 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT ^ AGAGAATCACAAACCTCTAGATGT

R 5 F 1 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT

---- Directed Seq. ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R 5 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R 4 F 1 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R 4 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R 6 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

Figure IV

- 10. Junction sequences detected by JSA. Junction Sequence Class B Alignment (All Generations Tested) Full consensus sequence for JSC

- B showing exact alignment to the independently determined in planta locus specific sequence (labeled “Directed Seq.” in the figure) ; individual consensus sequenc es for each of the five generations are labeled according to their generation (R 4 , R 5 , R 6 , R 4 F 1 and R 5 F 1 ). Double underlined text indicates plasmid DNA sequence, single underlined text indicates plant genome sequence, and the carat character “^” indicates the junction point between the MON 87411 insert and the flank. The asterisk character “*” indicates identical nucleotide in every sequence at that position in the alignment. Dash characters indicate positions past the end of the consensus sequence for a particular generation.Monsanto Company CR240

- 13U1 77 of 374 IV.E. Inheritance of the Genetic Insert in MON 87411

The MON 87411 T

- DNA resides at a single locus within the maize genome and therefore

should be inherited according to Mendelian principles of inheritance. During development of MON 87411, phenotypic and genotypic segregation data were recorded to assess the inheritance and stability of the MON 87411 T

- DNA using Chi

- square (χ 2 ) analysis over severa l generations. The χ 2 analysis is based on comparing the observed segregation ratio to the expected segregation ratio according to Mendelian principles.

The MON 87411 breeding path for generating segregation data is described in Figure IV

- 11. The transfo rmed R 0 plant was self

- pollinated to generate R 1 seed . An individual homozygous positive plant was identified in the R1 segregating population via

a Real

- Time TaqMan ****

The homozygous positive R 1 plant was self

- pollinated to give rise to R 2 seed. The R 2 plants were self

- pollinated to produce R 3 seed. The R 3 plants were self

- pollinated to produce R 4 seed. Homozygous positive R 4 plants were crossed via traditional breeding techniques to a recurrent parent (HCL645) that does not contain the DvSnf7 p

, cp4 epsps ,

or c ry3Bb1 coding sequences to produce hemizygous R 4 F 1 seed. The R 4 F 1 plants were crossed with the recurrent parent to produce BC 1 F 1 seed. The BC 1 F 1 generation was tested for the presence of the T

- DNA by End

- Point TaqMan PCR to select for hemizygous MON 87411 plants. BC 1 F 1 plants hemizygous for MON 87411 T

- DNA were crossed with the recurrent parent to produce the BC 2 F 1 plants. BC 2 F 1 plants hemizygous for MON 87411 T

- DNA were self

- pollinated to produce the BC 2 F 2 plants.

BC 2 F 1 plants hemizygous for MON 87411 T

- DNA were crossed with the recurrent parent to produce the BC 3 F 1 plants. PCR assay.

The inheritance of the MON 87411 T

- DNA was assessed in the BC 2 F 1 , BC 2 F 2 , and

BC3F 1 generations. At the BC 2 F 1 and BC 3 F 1 generations, the MON 87411 T

- DNA wa s predicted to segregate at a 1:1 ratio (hemizygous positive : homozygous negative) according to Mendelian inheritance principles. At the BC 2 F 2 generation, the MON 87411

T

- DNA was predicted to segregate at a 1:2:1 ratio (homozygous positive: hemizygous

pos itive: homozygous negative) according to Mendelian inheritance principles.

A Pearson’s Chi

- square (χ 2 ) analysis was used to compare the observed segregation ratios

of the MON 87411 T

- DNA to the expected ratios. The Chi

- square (χ 2 ) analysis used the

stat istical program R Version 2.15.2 (2012

- 10

- 26).

The Chi

- square was calculated as:

χ 2 = ∑ [( | o

– e | ) 2 / e]

**** TaqMan is a registered trademark of Roche Molecular Systems, Inc.Monsanto Company CR240

- 13U1 78 of 374 where o = observed frequency of the genotype or phenotype and e = expected frequency of the genotype or phenotype. The level of statistical significance was predetermined to be 5% (α = 0.05).

The results of the χ 2 analysis of the MON 87411 segregating progeny are presented in

Table IV

- 6 and Table IV

- 7 . The χ 2 values in the BC 2 F 1 and BC 3 F 1 generations indicated no statistically significant difference between the observed and expected 1:1 segregation ratio (hemizygous positive: homozygous negative) of the MON 87 411 T

- DNA. The χ 2

value for the BC 2 F 2 generation indicated no statistically significant difference between the observed and expected 1:2:1 segregation ratio (homozygous positive: hemizygous positive: homozygous negative) of MON 87411 T

- DNA. These results support the

conclusion that the MON 87411 T

- DNA resides at a single locus within the maize genome and is inherited according to Mendelian principles. These results are also consistent with the molecular characterization data indicating that MON 87411 con tains a single intact copy of the T

- DNA inserted at a single locus in the maize genome.Monsanto Company CR240

- 13U1 79 of 374 Figure IV

- 11. Breeding Path for Generating Segregation Data for MON 87411

*Chi

- square analysis was conducted on segregation data from the BC 2 F 1 , BC 2 F 2 , and BC 3 F 1 generations (bolded text).

TI: T rait I ntegration: Replacement of gene tic background of MON 87411 by recurrent background except inserted gene. RP: Recurring parent.
 =Self

- Pollinated LH244 R 1

Breeding path

continued LH244 R 4 LH244 R 3 LH244 R 2 

  Transformed LH244 R 0 Plant



R 4 F 1 (LH244 x HCL645)

TI:BC 1 F 1

TI:BC 2 F 1 × RP

× RP × RP

TI:BC 3 F 1 x RP (Expected segregation 1:1)
(Hemizygous positive: Homozygous negative)

(Expected segregation 1:1) ( Hemizygous positive: Homozygous negative) 

TI:BC 2 F 2 (Expected segregation 1:2:1)

(Homozygous positive: Hemizygous positive: Homozygous negative)Monsanto Company CR240

- 13U1 80 of 374 Table IV

- 6 . Segregation of the T

- DNA During the Development of MON 87411 1:1 Segregation

1:1 Segregation

Generation Total Plants Observed #

Plants

Positive Observed #

Plants Expected #

Plants
Hemizygous

(Positive) Expected #

Plants
Homozygous

χ 2 Probability 2 Negative Negative

BC 2 F 1 1 351 172 179 175.50 175.50 0.14 0.709

BC 3 F 1 1 223 104 119 111.50 111.50 1.01 0.315

1 Segregation was evaluated using an End

- Point TaqMan analysis for the MON 87411 insert.

2 Chi

- square analysis was performed to analyze the segregation ratios (p ≤ 0.05).

Table IV

- 7 . Segregation of the T

- DNA During the Development of MON 87411 1:2:1 Segregation

1:2:1 Segregation

Generation Total

Plants Observed #

Plants
Homozygous

Positive Observed #

Plants
Hemizygous Observed #

Plants
Homozygous

Negative Expected #

Plants
Homozygous

Positive Expected #

Plants
Hemizygous Expected #

Plants
Homozygous

Negative χ 2 Probability 2

BC 2 F 2 1 623 152 314 157 155.75 311.50 155.75 0.12 0.942

1 Segregation was evaluated using Real

- Time TaqMan analysis for the MON 87411 insert

. 2 Chi

- square analysis was performed to analyze the segregation ratios (p ≤ 0.05).Monsanto Company CR240

- 13U1 81 of 374 IV.F. Characterization of the Genetic Modification Summary and Conclusion

Molecular characterization of MON 87411 by NGS/JSA and directed sequencing demonstrated that a single copy of the intended transfer DNA (T

- DNA) containing the DvSnf7 suppression cassette and the cry3Bb1 and cp4 epsps expression cassettes from

PV

- ZMIR10871 was integrated into the maize genome at a single locus. These analyses

also showed no PV

- ZMIR10871 backbone DNA had been inserted.

Directed sequence analyses performed on MON 87411 confirmed the organization and

intactness of the full T

- DNA and all expected elements within the insert, with the exception of incomplete Right and Left Border sequences that do not affect the functionality of the DvSnf7 suppression or cry3Bb1 and cp4 epsps expression cassettes. Analysis of the T

- DNA insertion site in maize show s the flanks in MON 87411 are identical to the conventional control, excepting a 118 bp deletion of genomic DNA at the

insertion si te in MON 87411

. This deletion is not expected to affect food or feed safety.

Generational stability analysis by NGS/JSA demonstrated that the T

- DNA in

MON 87411 was maintained through five breeding generations, thereby confirming the stability of the ins ert. Results from segregation analyses show heritability and stability of the insert occurred as expected across multiple generations, which corroborates the molecular insert stability analysis and establishes the presence of the T

- DNA in

MON 87411 at a s ingle chromosomal locus.Monsanto Company CR240

- 13U1 82 of 374 V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE Cry3Bb1

and CP4 EPSPS PROTEIN S PRODUCED IN MON 87411

Characterization of the introduced protein(s) in a biotechnology

- derived crop is important to establishing food, feed, and env ironmental safety. As described in Section IV,

MON 87411 contains cry3Bb1 and cp4 epsps expression cassette s that, when transcribed and translated, result in the expression of the Cry3Bb1 and CP4 EPSPS protein s

. The characterization and safety assessment for the DvSnf7 s uppression cassette is described

in Section VI.

This section summarizes: 1) the identity and function of the Cry3Bb1 and CP4 EPSPS proteins produced in MON 87411 ; 2) assessment of equivalenc e between the plant

- produced and E. coli

- produced proteins; 3) the level of the Cry3Bb1 and

CP4 EPSPS proteins in plant tissues from MON 87411 ; 4) assessment of the potential allergenicity of the Cry3Bb1 and CP4 EPSPS proteins produced in MON 87411 ; and 5) the food and feed safety assessment of the Cry3Bb1 and CP4 EPSPS proteins produced in MON 87411 . The data are consistent with prior safety assessments of these two proteins and support a conclusion that the proteins produced in MON 87411 are safe

for hum an or animal consumption and safe for the environment based on several lines of

evidence summarized below.

V.A. Identity and Function of the Cry3Bb1 and CP4 EPSPS Protein s from

MON 87411

V.A.1. Identity and Function of the C ry3Bb1 Protein from MON 87411

Cry3Bb1 protein originates from Bacillus thuringiensis ( Bt ), a ubiquitous gram

- positive soil bacterium that accumulates crystal pro teins during sporulation. Cry3Bb1 protein is a

member of the 3 D

- Cry family of insecticidal proteins ( Crickmore 2012 )

. Pro teins within

the 3 D

- Cry proteins are subdivided into different groups base d on the high specificity they have for their target categor y of insects . Because of their narrow spectrum of activity, they lack an impact on broader insect populations or other or ganisms. For example, Cry3 proteins have insecticidal activity specifically against coleopteran insects

, while Cry1A proteins have insecticidal activity specifically aga inst lepidopteran insects ( Höfte and Whiteley 1989 )

.

The generalized MOA for Cry proteins was described by English and Slatin ( 1992 ) . It includes ingestion of the crystals by insects and solubilization of the crystals in the insect midgut, followed by activation through proteolytic processing of the soluble Cry protein by digestive enzymes in the midguts. The activ ated protein then binds to specific receptors on the surface of the midgut epithelium of target insects and inserts into the membrane, leading to pore formation and generalized disruption of the transmembrane gradients and, therefore, cell integrity. Whil e alternate mechanisms have also been proposed, a review of the available data has recently been published and the authors concluded that the original model, pore formation, is the most valid model for Cry protein

mode of action ( Vachon, et al. 2012 )

.Monsanto Company CR240

- 13U1 83 of 374 Cry3Bb1 protein in MON 87411 is a protein con sisting of a single polypeptide of 65 2 amino acids. Like other Cry proteins, it is synthesized as a prototoxin and is likely cleaved by digestive enzymes in the midgut of target organisms to an approximately

60 kDa activated protein ( Bravo, et al. 2 007 )

. Cry3Bb1 is also expressed in commercially available YieldGard VT Rootworm/RR2 (MON 88017) maize and

SmartStax ® maize . The amino acid sequence deduced from the Cry3Bb1 expression cassette present in YieldGard VT Rootworm/RR2 is i dentical to that deduced from the Cry3Bb1 expression cassette present in MON 87411. A related Cry3Bb1 protein, which has over 99% amino acid identity to the Cry3Bb1 in YieldGard VT Rootworm/RR2 and

MON 87411, is expr essed in YieldGard Rootworm maize (MON 863)

. E ach of these products were previously reviewed by USDA

- APHIS and found to not have any unique plant pest risks relative to conventional maize and were subsequently deregulated

.

V.A.2. Identity and Function of the C P4 EPSPS Protein from MON 87411

The enzyme, 5

- enolpyruvylshikimate

- 3

- phosphate synthase (EPSPS), catalyzes one of the enzymatic steps of the shikimic acid pathway, and is the target for the broad spectrum herbicide glyphosate ( Haslam 1993 ; Herrmann and Weaver 1999 ; Kishore, et al. 1988 ; Steinrücken and Amrhein 1980 ) . The shikimic acid pathway and EPSPS enzymes are ubiquitous to plants and microorganisms, but absent in mammals, fish, birds, reptiles, and

insects ( Alibhai and Stallings 2001 ) . EPSPS proteins have been isolated from both pla nt and microbial sources and their properties have been extensively studied ( Harrison, et al.

1996 ; Haslam 1993 ; Schönbrunn, et al. 2001 ; Steinrücken and Amrhein 1984 ) . The plant and microbial enzymes are mono

- functional with a molecular weight of 44

- 51 kDa ( Franz, et al. 1997 ; Kishore et al. 1988 ) . EPSPS enzymes catalyze the transfer of the enolpyruvyl group from phosphoenolpyru vate (PEP) to the 5

- hydroxyl of

shikimate

- 3

- phosphate (S3P), thereby yielding inorganic phosphate and

5

- enolpyruvylshikimate

- 3

- phosphate (EPSP) ( Alibhai and Stallings 2001 ) . Shikimic acid

is a substr ate for the biosynthesis of the aromatic amino acids (phenylalanine, tryptophan and tyrosine) and other aromatic molecules necessary for plant growth.

The EPSPS transgene in MON 87411 is derived from Agrobacterium sp. strain CP4 ( cp4 epsps ). The cp4 eps ps coding sequence encodes a n EPSPS protein consisting of a single polypeptide of 455 amino acids ( Padgette et al. 1996 ) . The CP4 EPSPS protein is similar and functionally identical to en dogenous plant EPSPS enzymes, but has a much reduced affinity for glyphosate, the active ingredient in Roundup agricultural herbicides, relative to endogenous plant EPSPS ( Padgette et al. 199 6 ) . In conventional plants, including weeds, glyphosate blocks the biosynthesis of EPSP, thereby depriving plants of

essential amino acids ( Haslam 1993 ; Steinrücken and Amrhein 1980 ) . In Roundup Ready plants, which are tolerant to Roundup agricultural herbicides, requirement s for aromatic amino acids and other metabolites are met by the continued action of the

CP4 EPSPS enzyme in the presence of glyphosate ( Padgette et al. 1996 ) . The

CP4 EPSPS protein expres sed in MON 87411 is identical to the CP4 EPSPS protein expressed in Roundup Ready products across several crops
, including soybeans

, corn
, canola, cotton, sugar beet, and alfalfa.Monsanto Company CR240

- 13U1 84 of 374 V.B. Characterization and Equivalence of Cry3Bb1 and CP4 EPSPS Proteins f rom MON 87411

The safety assessment of crops derived through biotechnology includes characterization of the physicochemical and functional properties of the protein(s) produced from the inserted DNA, and confirmation of the safety of the protein(s). For s afety data generated using proteins produced from a heterologous source ( e.g ., E. coli

- produced protein) to be applied to plant

- produced protein(s), the equivalence of the plant and E. coli

- produced proteins must be assessed. The physicochemical and funct ional characteristics of the

MON 87411

- produced CP4 EPSPS and MON 87411

- produced Cry3Bb1 proteins were determined and each was shown to be equivalent to its respective E. coli

- produced protein. A summary of the analytical results for each protein are show n below and the details of the materials, methods, and results are described in Appendix C.

The Cry3Bb1 and CP4 EPSPS protein s purified from grain of MON 87411 w ere characterized and the equivalence of the physicochemical characteristics between the

MON 87411

- produced and the E. coli

- produced proteins was established using a panel of analytical tests: 1) N

- terminal sequence analys i s of the MON 87411

- produced protein s established identity; 2) MALDI

- TOF MS analys i s yielded peptide masses consistent with the expected peptide masses from the theoretical trypsin digest of the

MON 87411

- produced sequence s ; 3) MON 87411

- produced Cry3Bb1 and CP4 EPSPS protein s w ere detected on western blot s probed with their respective protein

- specific

a ntibodies and the immunoreactive properties of the MON 87411

- produced and

E. coli

- produced proteins were shown to be equivalent; 4) the apparent molecular weight s of the MON 87411

- produced and E. coli

- produced proteins, assessed by SDS

- PAGE, were shown to be equivalent; 5) MON 87411

- produced and E. coli

- produced

MON 87411 proteins were both determined to be non

- glycosylated; and 6) functional (biological) activity of the MON 87411

- produced and E. coli

- produced proteins were demonstrated to be equivalent for both Cry3Bb1 and CP4 EPSPS

.

Taken together, these data provide a detailed characterization of the

MON 87411

- produced Cry3Bb1 and CP4 EPSPS proteins and establish their respective equivalence to E. coli

- produced Cry3Bb1 and CP4 EPSPS proteins. This equivalence justifies the use of previously conducted protein studies using E. coli

- produced Cry3Bb1

and CP4 EPSPS proteins to establish the safety of the Cry3Bb1 and CP4 EPSPS proteins expressed i n MON 87411, summarized in section V.E.

V.C. Expression Levels of Cry3Bb1 and CP4 EPSPS Protein s in MON 87411

Cry3Bb1 and CP4 EPSPS protein levels in various tissues of MON 87411 relevant to the risk assessment were determined by a validated enzyme

- linked immunosorbent assa y (ELISA). Tissues of MON 87411 were collected from four replicate plots planted in a randomized complete block field des ign during the 2011

- 2012 growing season from the

followi ng five field sites in Argentina : Pergamino, Buenos Aires (Site Code BAFO); Hunter, Buenos Aires (Site Code BAHT); Pergamino, Buenos Aires (Site Code BAPE); Sarasa, Buenos Aires (Site Code BASS) and Salto, Buenos Aires (Site Code BATC). The field sites were representative of maize

- producing regions suitable for commercial maize production. Maize production in the U.S. c orn belt and Argentina growing regionsMonsanto Company CR240

- 13U1 85 of 374 occurs at relatively similar latitudes with an approximate 6 month offset ( Schnepf, et al.

2001 ) . The average growing season temperatures and precipitation are comparable ( Schnepf et al. 2001 ) and , as a result, maize hybrids developed in the U.S. are often used directly by farmers in the southern growing regions of Argentina. As such, protein expression analyses from maize grown at these sites are appropriate for a comparative

assessment

. Nineteen total tissue samples were collected from each replicated plot at each site, many over several time points/growth stages, throughout the season. Samples

included o ver season leaf (OSL1 through OSL4), over season root (O SR1 through

OSR 4) , over season whole plant (OSWP1 through OSWP4), stover, senescent root, forage root, forage, grain , pollen and silk

. MON 87411 plots were treated with glyphosate to generate samples under c onditions of the intended use (0.95 lbs active ingredient/ hectare ) of the product.

V.C.1. Expression Levels of Cry3Bb1 Protein

Cry3Bb1 protein levels were determined in 19 tissue types. The ELISA results obtained for each sample were averaged across the five sites and are summarized in Table V

- 1.

T he details of the materials and met hods are described in Appendix D . The individual Cry3Bb1 protein levels in MON 8 7411 across all samples analyzed from all sites ranged

from 3.0 to 460 μg/g dw. The mean Cry3Bb1 protein level among all tissue type s was highest in OSWP1 at 340 μg/g dw and lowest in grain at 4.0 μg/g dw.Monsanto Company CR240

- 13U1 86 of 374 Table V

- 1. Summary of Cry3Bb1 Protein Levels in Tissues from MON 87411

Grown in 2011

– 2012 Argentina Field Trials

Tissue 1 Development Stage 2 Days After Planting (DAP) 3 Cry3Bb1

Mean (SD) Range (μg /g fw ) 4 Cry3Bb1 Mean (SD) Range (μg/g dw ) 5 LOQ/LOD (μg/g fw ) 6

OSL1 V3

- V4 21

- 22 45 (9.0 ) 270 (65) 0.035/0.006

31

– 64 160

- 39 0

OSL2 V 6

- V 8 35

- 44 40 (7.8) 210 (40) 0.035/0.006

26

– 56 120

– 270

OSL3 V10

- V1 3 50

- 55 40 (7.9) 170 (35) 0.035/0.006

21

– 52 92

– 220

OSL4 V 14

- R1 59

- 78 56 (19) 220 (63) 0.035/0.006

31

– 89 130

– 340

OSR1 V3

- V4 21

- 22 25 (4.6) 180 (43) 0.035/0.028

16

– 32 130

– 280

OSR2 V 6

- V 8 35

- 44 16 (4.0) 120 (24) 0.035/0.028

9.4

– 25 67

– 170

OSR3 V10

- V1 3 50

- 55 15 (4.0) 84 (21) 0.035/0.028

9.6

– 24 54

– 130

OSR4 V 14

- R1 59

- 78 14 (3.3) 75 (19) 0.035/0.028

9.0

– 21 43

– 120

OSWP1 V3

- V4 21

- 22 44 (4.9) 340 (49) 0.035/0.008

33

– 53 250

– 460

OSWP2 V 6

- V 8 35

- 44 30 (5.3) 190 (30) 0.035/0.008

21

– 40 130

– 270

OSWP3 V10

- V1 3 50

- 55 20 (6.8) 140 (39) 0.035/0.008

9.2

– 33 59

– 210

OSWP4 V 14

- R1 59

- 78 20 (4.8) 120 (28) 0.035/0.008

12

– 29 71

– 170

Stover R6 136

- 155 10 (6.2) 21 (13) 0.035/0.008

1.9

– 19 4.7

– 44

Senescent Root R6 136

- 155 4.8 (3.1) 19 (13) 0.035/0.028

0.76

– 12 3.0

– 50Monsanto Company CR240

- 13U1 87 of 374 Table V

- 1. (continued) Summary of Cry3Bb1 Protein Levels in Tissues from MON 87411

Grown in 2011

– 2012 Argentina Field Trials

Tissue 1 Development Stage 2 Days After Planting (DAP) 3 Cry3Bb1 Mean (SD) Range (μg/g fw ) 4 Cry3Bb1 Mean (SD) Range (μg/g dw ) 5 LOQ/LOD (μg/g fw ) 6

Forage Root R5 101

- 111 7.9 (3.5) 36 (16) 0.035/0.028

2.6

– 15 13

– 66

Forage R5 101

- 111 12 (4.9) 39 (17) 0.035/0.008

5.5

– 23 18

– 75

Grain R 6 139

- 154 3.5 (0.45) 4.0 (0.56) 0.035/0.007

2.7

– 4.4 3.1

– 5.1

Pollen VT

- R1 65

- 80 29 (3.0) 36 (4.0) 0.035/0.018

23

– 34 30

– 42

Silk R1 65

- 81 16 (3.8) 160 (37) 0.035/0.010

8.5

– 23 89

– 220

1 OSL= over season leaf ; OSR= over season root ; OSWP= over season whole plant

2 T he crop development stage each tissue was collected.

3 The number of day s after planting that each tissue was collected.

4 Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of

protein per gram (g) of tissue on a fresh weight basis (fw). The means, SD, and ranges (minimum and maximum values) were calc ulated for each tissue across all sites (n=20).

5 Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of

protein per gram (g) of t issue on a dry weight basis (dw ). The dry weight values were cal culated by

div iding the μg/g fw by the dry weight conversion factor obtained from moisture analysis data.

6 LOQ=limit of quantitation; LOD=limit of detection.

V.C.2

. Expression Levels of CP4 EPSPS Protein

CP4 EPSPS protein levels were determined in all 19 tissue types . The ELISA results

obtained for each sample were averaged across the five sites and are summarized in Table

V

- 2. The details of the materials and methods are described in Appendix D

. The

individual CP4 EPSPS protein levels in MON 87411 across all sampl es analyzed from all

sites ranged from less than the limit of quantitation (<LOQ) to 76 μg/g dw. The mean

CP4 EPSPS protein level among all tissue type s was highest in OSWP1 at 63 μg/g dw and lowest in grain at 1.9 μg/g dw

.Monsanto Company CR240

- 13U1 88 of 374 Table V

- 2. Summary of CP4 EPSPS Protein Levels in Tissues from MO N 87411

Grown in 2011

– 2012 Argentina F ield Trials

Tissue 1 Development Stage 2 Days After Planting (DAP) 3 CP4 EPSPS Mean (SD) Range (μg/g fw ) 4 CP4 EPSPS Mean (SD) Range (μg/g dw ) 5 LOQ/LOD (μg/g fw ) 6

OSL1 V3

- V4 21

- 22 7.1 (0.83) 42 (5.9) 0.137/0.071

5.8

– 9.0 33

– 55

OSL2 V 6

- V 8 35

- 44 7.0 (0.64) 36 (3.1) 0.137/0.071

6.0

– 7.9 29

– 39

OSL3 V10

- V1 3 50

- 55 7.4 (0.78) 32 (3.8) 0.137/0.071

6.4

– 8.9 27

– 42

OSL4 V 14

- R1 59

- 78 7.8 (0.85) 31 (3.5) 0.137/0.071

6.6

– 9.5 24

– 37

OSR1 V3

- V4 21

- 22 6.5 (0.86) 48 (6.6) 0.068/0.033

4.4

– 8.0 38

– 63

OSR2 V 6

- V 8 35

- 44 5.2 (1.0) 37 (7.0) 0.068/0.033

3.8

– 7.1 23

– 48

OSR3 V10

- V1 3 50

- 55 5.6 (0.84) 31 (4.7) 0.068/0.033

4.0

– 7.1 24

– 37

OSR4 V 14

- R1 59

- 78 5.7 (0.80) 30 (4.8) 0.068/0.033

4.2

– 7.1 20

– 38

OSWP1 V3

- V4 21

- 22 8.1 (0.90) 63 (6.7 ) 0.137/0.070

6.6

– 9.8 54

– 76

OSWP2 V 6

- V 8 35

- 44 5.6 (0.94) 36 (5.8) 0.137/0.070

3.4

– 7.4 21

– 46

OSWP3 V10

- V1 3 50

- 55 4.6 (1.1) 33 (6.2) 0.137/0.070

2.3

– 6.6 21

– 45

OSWP4 V 14

- R1 59

- 78 4.3 (0.87) 25 (5.0) 0.137/0.070

2.9

– 5.5 17

– 32

Stover R6 136

- 155 1.0 (0.60) 2.2 (1.2) 0.137/0.070

0.30

– 2.1 0.59

– 4.9

Senescent Root R6 136

- 155 1.4 (0.69) 5.4 (2.9) 0.068/0.033

0.49

– 2. 6 1.8

– 11Monsanto Company CR240

- 13U1 89 of 374 Table V

- 2. (continued ) Summary of CP4 EPSPS Protein Levels in Tissues from

MON 87411 Grown in 2011

– 2012 Argentina Field Trials

Tissue 1 Development Stage 2 Days After Planting (DAP) 3 CP4 EPSPS Mean (SD) Range (μg/g fw ) 4 CP4 EPSPS Mean (SD) Range (μg/g dw ) 5 LOQ/LOD (μg/g fw ) 6

Forage Root R 5 101

- 111 2.2 (0.81) 10 (3.7) 0.068/0.033

1.1

– 4.1 5.1

– 19

Forage R5 101

- 111 2.4 (0.71) 8.0 (2.3) 0.137/0.070

1.5

– 3.8 5.2

– 13

Grain R 6 139

- 154 1.7 (0.27) 1.9 (0.31) 0.228/0.152

1.4

– 2.7 1.6

– 3.1

Pollen VT

- R1 65

- 80 15 (1.9) 19 (2.8) 0.137 /0.099

12

– 19 16

– 24

Silk R1 65

- 81 4.0 (0.69) 40 (5.0) 0.137/0.121

3.1

– 5.1 32

– 49

1 OSL= over season leaf ; OSR= over season root ; OSWP= over season whole plant

2 The crop development stage each tissue was collected.

3 The number of day s after planting that each tissue was collected.

4 Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of

protein per gram (g) of tissue on a fresh weight basis (fw). The means, SD, and ranges (minimum and maximum values) were calculated for each tissue across all sites (n=20, except for stover where n=19 due to one sample expressing <LOQ).

5 Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of

protein per gram (g) of tissue on a dry weight basis (dw). The dry weight values were calculated by dividing the μg/g fw by the dry weight conversion factor obtained from moisture analysis data.

6 LOQ=limit of quantitat ion; LOD=limit of detection

.

.Monsanto Company CR240

- 13U1 90 of 374 V.D. Assessment of Potential Allergenicity of the Cry3Bb1 and CP4 EPSPS

Protein s

The allergenic potential of an introduced protein is assessed by comparing the physiochemical characteristics of the introduced protein to physiochemical characteristics of known allergens ( Codex Alimentarius 2009 )

. Using a weight of evidence approach, a protein is not likely to be associated with allergenicity if: 1) the protein is from a non

allergenic source; 2) the protein represents a small portion of the total plant protein; 3) the protein does not share structural similarities to known allergens b ased on the amino acid sequence; and 4) the protein does not show resistance to pepsin digestion. The Cry3Bb1 and CP4 EPSPS proteins have been assessed for their potential allergenicity according to these safety assessment guidelines.

V.D. 1. Assessment of Potential Allergenicity of the Cry3Bb1 Protein

The Cry3Bb1 protein has been assessed for its potential allergenicity according to the

Codex safety assessment guidelines described above, and conclusions were as follows

.

1) The Cry3Bb1 protein originate s fro m B t

, an organism that has not been reported to be a source of known allergens.

2) The Cry3Bb1 protein represents no more than 0.004 % of the total protein in the grain of MON 87411

.

3) Bioinformatics analyses demonstrated that the Cry3Bb1 protein does not shar e amino acid sequence similarities with known allergens and, therefore, is highly unlikely to contain immunologically cross

- reactive allergenic epitopes.

4) Finally, in vitro digestive fate experiments conducted with the Cry3Bb1 protein demonstrate that the protein is rapidly digested by pepsin in a simulated gastric fluid (SGF) assay

.

Taken together, these data support the conclusion that the Cry3Bb1 protein does no t pose a significant allergenic risk to humans or animals.

V.D. 2. Assessment of Potential Allergenicity of the CP4 EPSPS Protein

The CP4 EPSPS protein has been assessed for its potential allergenicity according to the

Codex safety assessment guidelines described above, and conclusions are as follows

.

1) The CP4 EPSPS pr otein originate s from Agrobacterium sp

. strain CP4

, an organism that has not been reported to be a source of known allergens.

2) The CP4 EPSPS protein represents no more than 0.002 % of the total protein in

the seed of MON 87411

.

3) Bioinformatics analyses demo nstrated that the CP4 EPSPS protein does not share amino acid sequence similarities with known allergens and, therefore, is highly unlikely to contain immunologically cross

- reactive allergenic epitopes.Monsanto Company CR240

- 13U1 91 of 374 4) Finally, in vitro digestive fate experiments conducted with the CP4 EPSPS protein demonstrate that the protein is rapidly digested by pepsin in a SGF assay.

Taken together, these data support the conclusion that the CP4 EPSPS protein does no t pose a significant allergen ic risk to humans or animals.

V.E. Safety Assessment Summary of Cry3Bb1 and CP4 EPSPS Protein s in

MON 87411

A comprehensive set of factors have been considered and assessed in the safety

assessment of the Cry3Bb1 and CP4 EPSPS protein s in food and feed o r the

environment . The results are summarized below along with the conclusions reached

from each assessment.

V.E.1. Cry3Bb1 Donor Organism, History of Safe Use, and Specificity

V.E.1

. 1 The cry3Bb1 Donor Organism is Safe

The donor organism for cry3Bb1

, Bacillus thuringiensis, has been used commercially in

the United States since 1958 to produce microbial

- derived products with insecticidal activity. The extremely low mammalian toxicity of Bt

- based insecticide products has been demonstrated in numerous safety studies, and there are no confirmed cases of allergic reactions to Cry proteins in applicators of microbial

- derived Bt products during

5 0 years of use.

Applications of sporulated Bt have a long history of safe use for pest control in agriculture, especially in organic farming ( Cannon 1993 ; IPCS 1999 ; U.S. EPA 1988 )

. Microbial pesticides containing Bt Cry3 proteins have been used for more than 30 years and subjected to extensive toxicity testing showing no adverse effects to human health ( Baum, et al. 1999 ; Betz, et al. 2000 ; McClintock, et al. 1995 ; Mendelsohn, et al. 2003 ; U.S. EPA 2001b ; 2005 )

.

V.E. 1. 2. Cry3Bb1 Protein has a History of Safe Use

A history of safe use of Cry3Bb1 protein has been established ( Cannon 1993 ; IPCS 1999 ; U.S. EPA 1988 ) . Microbial pesticides containing Bt Cry proteins have been used for

more than 50 years and subjected to extensive toxicity testing showing no adv erse effects

to human health ( Baum et al. 1999 ; Betz et al. 2000 ; McClintock et al. 1995 ; Mendelsohn

et al. 2003 ; U.S. EPA 2001b ; 2005 )

. Cry3Bb1, specifically, is one of the active ingredients in the microbial pesticide mixture Raven ® Oil Flowable Bioinsecticide that

was commercialized in the United States to control Colorado Potato Beetle ( Baum et al.

1999 )

. Cry3Bb1 is also expressed in commercially available YieldGard VT

Roo tworm/RR2 (MON 88017) maize and SmartStax ® maize. The amino acid sequence deduced from the Cry3Bb1 expression cassette present in YieldGard VT Rootworm/RR2 is identical to that deduced from the Cry3Bb1 expression cassette present in MON 87411.

A related Cry3Bb1 protein, which has over 99% amino acid identity to the Cry3Bb1 in

YieldGard VT Rootworm/RR2 and MON 87411, is expressed in YieldGard Rootworm

maize (MON 863)

. The Un ited States EPA has approved commercial use of theMonsanto Company CR240

- 13U1 92 of 374 Cry3Bb1 as expressed in maize and has established an exemption from the requirement of a tolerance for residues of the Cry3Bb1 protein and the genetic material for its production in maize ( U.S. EPA 2004a )

. The history of large scal e cultivation of these Cry3Bb1

- expressing crops without any indication of harmful impact on the environment,

non

- target o rganisms , or mammals provides additional evidence for the safety of the Cry3Bb1 protein. Taken together, these data demonstrate that the Cry3Bb1 protein has a history of safe use and does not pose any unexpected effects to human and animal health

or the e nvironment

.

V.E. 1 .3. Cry3Bb1 Protein Demo n strates Specificity

Cry3 Bb1 protein, like other Cry3 proteins, has insecticidal activity specifically against coleopteran insects ( Höfte and Whiteley 1989 ) and is covered in more detail in Section

X. C.1

.

V.E.2 CP4 EPSPS Donor Organism, History of Safe Use, and Specificity

V.E. 2

. 1 The cp4 epsps Donor Organism is Safe

The donor organism for cp4 epsps , Agrobacterium sp. strain CP4

, is not known for human or animal pathogenicity, and is not commonly allergenic ( FAO

- WHO 1991 )

. The history of safe use of Agrobacterium sp. strain CP4 has been previously reviewed as a part of the safety assessment of th i s donor organism for USDA

- APHIS deregulations, as

well as completed Monsanto consultations with the FDA regarding Roundup Ready

varieties of soybean (199 5 and 2007 ), canola (1995 , 2002, and 2012 ), maize (199 8 and 2000) , sugar beet (1998 and 2004 ), alfalfa (2004), and cotton ( 1995 and 2005).

V.E. 2. 2. CP4 EPSPS Protein has a History of Safe Use

The CP4 EP SPS protein present in MON 87411 is similar to EPSPS proteins consumed in a variety of food and feed sources. CP4 EPSPS protein is homologous to EPSPS proteins naturally present in plants, including food and feed crops ( e.g., soybean and maize) and fungal and microbial food sources such as baker’s yeast ( Saccharomyces

cerevisiae ), all of which have a history of safe consumption ( Harrison et al. 1996 ; Padgette et al. 1996 ) . The similarity of the CP4 EPSPS protein to EPSPS proteins i n a variety of foods and feeds supports extensive safe consumption of the family of EPSPS proteins and the lack of human or animal health concerns. The ubiquitous presence of homologous EPSPS enzymes in crops and common microorganisms establishes that

EPS PS proteins, and their enzymatic activity, pose no hazards to human s, animal s, or the

environment

. In addition, the CP4 EPSPS protein in MON 87411 is identical to the

CP4 EPSPS protein in numerous other Roundup Ready varieties of soybean, maize, canola, sugar beet, cotton and alfalfa. Further, the U .S. EPA has established an exemption from the requirement of a tolerance for residues of CP4 EPSPS protein and the genetic material necessary for its production in all plan ts ( U.S. EPA 1996 )

.Monsanto Company CR240

- 13U1 93 of 374 V.E .2 .3. CP4 EPSPS Catalyzes a Specific Enzyme R eaction

CP4 EPSPS, like other EPSPS enzymes, functions in the shikimate pathway that is integral to aromatic amino acid biosynthesis in plants and microorganisms ( Levin and Sprinson 1964 ; Steinrücken and Amrhein 1980 ) . Therefore, EPSPS and its activity are found widely in food and feed derived from plant and microbial sources. Genes for numerous EPSPS proteins have been cloned ( Padgette et al. 19 96 ) and the catalytic domains of this group of proteins are conserved. Bacterial EPSPS proteins have been well characterized with respect to their three dimensional X

- ray crystal structures ( Stallings, et al. 1991 ) and detailed kinetic and chemical mechanisms ( Anderson and

Johnson 1990 )

.

V.E.3. Cry3Bb1 and CP4 EPSPS Proteins i n MON 87411 are Not Homologous to Known Allergens or Toxins

Bioinformatics analyses were performed to assess the potential for allerge nicity

, toxicity, or biological activity of Cry3Bb1 and CP4 EPSPS . The analys e s demonstrated that neither protein shares amino acid sequence similarities with known allergens, gliadins, glutenins, or protein toxins that could have adverse effects to human or anim al health

.

V.E.4. Cry3Bb1 and CP4 EPSPS Proteins in MON 87411 are Labile in in vitro Digestion Assays

As has been d escribed in previous regulatory submissions for a number of Roundup Ready crops a nd MON 88017 (APHIS Petition No. 04

- 125

- 01) , b oth the Cry3B b1 and

CP4 EPSPS proteins are readily digestible in SGF ( USDA

- APHIS 2013 ) . Digestion in simulated intestinal fluid (SIF) was also assessed. CP4 EPSPS was readily digestible in SIF. As expected, Cry3Bb1 subjected to SIF alone was processed to a trypsin

- resistant

core of ~66 kDa which w as stable throughout the SIF digestion period. This result is consistent with observations for this and other Cry proteins subjected to SIF digestion. Rapid degradation of the Cry3Bb1 and CP4 EPSPS proteins in SGF makes it highly unlikely that either pro tein would be absorbed in a form other than as component
nutritional amino acids in the small intestine or have any adverse effects on human or

animal health

.

V.E. 5

. Cry3Bb1 and CP4 EPSPS Proteins in MON 87411 are Not Acutely Toxic

Acute oral toxicology studies were conducted with Cry3Bb1 and CP4 EPSPS proteins individually . Results indicate that neither Cry3Bb1 nor CP4 EPSPS caused any adverse effects in mice, with No Observable Adverse Effect Levels (NOAELs) for CP4 EPSPS at
572 mg/kg ( Harrison et al. 1 996 ) and for Cry3Bb1 at 1930 mg/kg bw ( FSANZ 2006 ) , the

hi ghest doses tested

. As described below, the highest dose levels tested are many fold higher than levels of Cry3Bb1 or CP4 EPSPS that are present in tissues of MON 87411.

V.E. 6. Human and Animal Exposure to the Cry3Bb1 and CP4 EPSPS Protein s

A common app roach used to assess potential health risks for potentially toxic materials is to calculate a Margin of Exposure (MOE) between the lowest NOAEL from anMonsanto Company CR240

- 13U1 94 of 374 appropriate animal toxicity study and an estimate of human exposure. Since no evidence

of mammalian tox icity has been reported for Cry3Bb1 or CP4 EPSPS, dietary risk assessments would normally not be considered necessary. Nevertheless, a dietary risk assessment was still conducted for these proteins in order to provide further assurances of safety by calcu lating a MOE between the NOAELs for the Cry3Bb1 and CP4 EPSPS proteins in mouse acute oral toxicity studies (Section V.E.5) and 95 th percentile consumption estimates of acute dietary exposure determined using the Dietary Exposure

Evaluation Model

- Food Co mmodity Intake Database (DEEM

- FCID) ( U.S. EPA 2013 )

. DEEM

- FCID utilizes food consumption data from the National Health and Nutrition Examination Survey (NHANES) conducted in 2003

- 2008 ( Included: vegetable maize, popcorn, and field maize (flour, meal, bran and starch) )

. Based on l evels of expressed protein on a fresh weight basis ( **μ** g/g) discussed above (Sections V.C.1 and V.C.2), 95 th
percentile exposures to Cry3Bb1 and CP4 EPSPS for the general U.S. population were estimated to be 7.2 and 3.5 μg/kg body weight (bw) , respectively. For non

- nursing infants in the U.S., the most highly exposed sub

- populati on, 95 th percentile exposures to Cry3Bb1 and CP4 EPSPS were estimated to be 17.1 and 8.3 μg/kg, respectively. For the Cry3Bb1 protein, MOEs for acute dietary intake were estimated to be 270,000 ( 2.7 **×** 10 5 )

and 110,000 ( 1.1 **×** 10 5 ) for the general population and non

- nursing infants, respectively.

For the CP4 EPSPS protein, MOEs for acute dietary intake were estimated to be 160,000
( 1.6 **×** 10 5 ) and 69,000 ( 6.9 **×** 10 4 ) for the general population and non

- nursing infants, respectively. A ctual MOEs will likely be much higher because: 1) t he exposure estimates utilized are conservative (95 th percentile, assume 100% MON 87411) and 2) as described

in section V.E. 4 , Cry3Bb1 and CP4 EPSPS are rapidly digested, further minimizing exposures. The se very large MOEs 5

There are a number of steps in the processing of maize to make food ingredients

, including hig h temperature treatments, hydrolyses, soaking in slightly acidic water, and drying that can denature a protein

. Changes in temperature, pH, and physical disruptions associated with food processing and cooking/preparation generally lead to loss of protein structure and functionality ( indicate that there is no meaningful risk to human health from dietary exposure to the Cry3Bb1 or CP4 EPSPS proteins produced by

MON 87411.

Hammond and Jez 2011 ) . Like other proteins, the Cry3Bb1 and CP4 EPSPS proteins in MON 87411 are expected to be similarly susceptible to denaturation when exposed to high temperatures, pH extremes, and digestive environments encountered during processing and cooking of foods containing

MON 87411. Thus, there are likely to be significantly lower exposures to the functionally active forms of these proteins through consumption of MON 87411 than the

levels estimated above.

The potential Cry3Bb1 and CP4 EPSPS protein exposure to animals from consumption of

MON 87411 in feeds was evaluated by calculating an estimate of daily dietary intake (DDI). The highest percentage of Cry3Bb1 and CP4 EPSPS proteins (g/ kg bw ) per total

5 These MOEs reflect that a human would have to eat thousands of kilograms of maize in a short time period to achieve exposures t o the expressed proteins in MON 87411 that we re not toxic to mice, which would be a physical impossibility

.Monsanto Company CR240

- 13U1 95 of 374 protein consumed was in the lactating dairy cow, 0.0 14 % (g/g) and 0.00 34 % (g/g) of the total dietary protein intake (0.000 876 g Cry3Bb1/kg bw divided by 6.2 g dietary protein,

and 0.000 210 g CP4 EPSPS/kg bw divided by 6.2 g dietary protein which is the total dietary protein int ake for the cow), respectively. The chicken and pig percentages of the Cry3Bb1 and CP4 EPSPS proteins consumed as part of the daily protein intake are much

less than for the lactating dairy cow. In the worst case sc enario, per kg b ody weight

, poultry, swine and lactating dairy cattle would be consuming 0.02% ( g/g ) and 0.004% ( g/g ) o r less of their total protein as Cry3Bb1 and CP4 EPSPS proteins , respectively,

from MON 87411 maize

.

In summary, there is no significant risk to human and animal health associated with dietary exposure to the Cry3Bb1 and CP4 EPSPS proteins in food and feed products

derived from MON 87411.

V.E.7. CP4 EPSPS Activity

The CP4 EPSPS protein produced in MON 87411 is similar to native EPSPS proteins that are ubiquitous in plant and microbial tissues in the environment and is not known to be toxic to other organisms ( ILSI

- CERA 2010 ; 2011 ; USDA

- APHIS 2013 )

.

V.E.8 . Cry3Bb1 Activity

The spectrum of activity of the modified Cry3Bb1 protein has previously been reviewed by USDA

- APHIS in submissions for both MON 863 and MON 88017 ( USDA

- APHIS

2013 ) and U.S. EPA ( U.S. EPA 2010 )

. Significant insecticidal activity has only been seen in the family Chrysomelidae within the Order Coleoptera ( U.S. EPA 2010 )

. The Cry3Bb1 genes present in MON 863 and MON 88017 code for Cry3Bb1 proteins that are

> 99.8% identical and which have been shown to be functionally equivalent in insect bioassays with two species sensitive to the Cry3Bb1 protein ( U.S. EPA 2010 )

. The Cry3Bb1 gene present in MON 87411 codes for Cry3Bb1 protein that is identical to that

in MON 88017

. Consequently, the Cry3Bb1 in MON 87411 is predicted to have equivalent functional activity and spectrum of activity as in those two previously registered and deregulated maize products

.

V

. E.9 . Non

- Target Assessment for CP4 EPSPS Protein

The USDA

- APHIS has previously determined that the gene imparting glyph osate
toleran ce
, cp4 epsps

, and the CP4 EPSPS protein that it encodes, poses no significant risk

to non

- target organisms due to its long history of use and no known toxicity to non

- target organisms ( Harrison et al. 1996 ; ILSI

- CERA 2010 ; USDA

- APHIS 2013 )

.

V

. E.10 . Non

- Target Assessment for Cry3Bb1 Protein

USDA

- APHIS and EPA have conducted plant pest and environmental assessments of

MON 863 and MON 88017 that produce the Cry3Bb1 protein. The Cry3Bb1 gene present in MON 87411 has the same coding sequence as the Cry3Bb1 gene pr esent in

MON 88017 and the tissue expression of the Cry3Bb1 p rotein is comparable to the previously registered maize products MON 88017 and MON 863. For MON 863, aMonsanto Company CR240

- 13U1 96 of 374 series of NTOs were evaluated to a ssess for potential negative impacts o f the Cry3Bb1 protein ( U.S. EPA 2010 ) . Exposure concentrations for the MON 863 studies was based

upon ≥10X the maximum observed Cry3Bb1 expression in tissue and no unacceptable negative impacts were observed in the NTO testing battery. Because the maximum Cry3Bb1 protein levels are higher in MON 863 compared to MON 87411 pr evious studies performed for MON 863 are sufficient to inform and provide a protective NTO

assessment for MON 87411. In the environmental assessment for MON 863 ( USDA

APHIS 2013 ) (USDA

- APHIS Petition No. 01

- 137

- 01p), USDA

- APHIS considered the potential impact of the Cry3Bb1 protein on NTOs, inc luding beneficial organisms and threatened or endangered specie s conclud ing :

“It [MON 863] has no potential to have a greater damaging, harmful, or toxic effect on organisms beneficial to agriculture than does other

cultivated corn. In addition to our finding of no plant pest risk, there will be no effect on threatened or endangered species resulting from a determination of non

- regulated status for MON 863 corn and its progeny.” ( USDA 2002 )

Additionally, USDA

- APHIS concluded in their assessment of MON 8 8017 that;

“The Cry3Bb1 protein expressed in MON 88017 corn has activity only against select beetle (Order Coleoptera) species within the family Chrysomelidae, namely CRW [corn rootworm] and Colorado potato

beetle

. F ield observations, composition al analyses, and data on the safety of the engineered EPSPS and Cry3Bb1 proteins all indicate that

MON 88017 should not have greater potential than other cultivated corn to damage or harm organisms beneficial to agriculture. In addition to our finding of no plant pest risk, there will be no effect on the threatened or endangered species resulting from a determination of non

- regulated status for MON 88017 and its progeny.” ( USDA 2005 )

The EPA also conducted an extensive envi ronmental hazard asse ssment of Cry3Bb1

- producing maize based on studies conducted with similar C ry3Bb1 proteins on representative species of bird, fish, and terrestrial non

- target insects , including endangered species

, to support the registration of MON 86 3 and MON 88017 concluding:

“ Due to a demonstrated lack of toxicity and/or exposure, no effects from Cry3Bb1 protein are anticipated for any non

- target species, including federally listed threatened and endangered (“listed”) lepidopteran and coleopteran species and their designated cr itical habitats.” ( U.S. EPA

2010 )Monsanto Company CR240

- 13U1 97 of 374 V. F

. Cry3Bb1 and CP4 EPSPS Protei ns Characterization and Safety Conclusi on

The data and information provided in this section address the questions important to the

food

, feed and environmental safety of the Cry3Bb1 and CP4 EPSPS protein s in

MON 87411

, including their potential allergenicity and toxicity. To summarize, the phy sicochemical characteristics of the Cry3Bb1 and the CP4 EPSPS protein s from

MON 87411 were determined and shown to be equivalent to those of their

E. coli

- produced counterparts

. Given t he very large MOEs for Cry3Bb1 and CP4 EPSPS protein s for humans and the minimal exposure to animals

, both support a conclusion of

no significant risk to human or animal health associated with dietary exposure to these proteins in food and feed products derived from MON 87411 . An assessment of the allergenic potential of t he protein s supports the conclusion that the Cry3Bb1 and

CP4 EPSPS protein do not pose a significant allergenic risk to humans or animals. The donor organisms for the Cry3Bb1 and CP4 EPSPS coding sequences, Bacillus thuringiensis and Agrobacterium sp. str ain CP4, respectively, are ubiquitous in the environment and not commonly known for human or animal pathogenicity, or allergenicity. The Cry3Bb1 and CP4 EPSPS protein s lack structural similarity to allergens, toxins or other proteins known to have adverse effects on mammals. The Cry3Bb1 and CP4 EPSPS proteins are rapidly digested in simulated d igestive fluid and

demonstrate no oral toxicity in mice at the level tested. Based on the above information, the consumption of the Cry3Bb1 and CP4 EPSPS protein s from MON 87411 or its

progeny is considered safe for humans and animals.

The protein safety data presented herein support the conclusion that food and feed products containing MON 87411 or derived from MON 87411 are as safe as maize currently on the mar ket for human and animal consumption. Given the identical nature of these proteins to proteins contained in other products that have been registered by EPA and deregulated by USDA

- APHIS, as well as previous safety assessments, the proteins

contained in MO N 87411 are also considered as safe for the environment as other maize products.Monsanto Company CR240

- 13U1 98 of 374 VI. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE DvSnf7

RNA PRODUCED IN MON 87411

VI.A. History of Safe Use of RNA

- mediated Gene Suppression in Plants

RNA

- mediated gene suppression (RNAi) is a naturally occurring, ubiquitous process in eukaryotes, including plants and animals consumed as food and feed. Endogen ous RNA

mediated gene modulation is responsible for certain characteristics of conventional crops ( Della Vedova et al. 2005 ; Kusaba et al. 2003 ; Tuteja et al. 2004 ) and has also been utilized in some biotechnology

- derived crops approved for cultivation and use as food

and feed ( Ivashuta, et al. 2009 ; Parrott et al. 2010 ; Petrick et al. 2013 ) . Therefore, there is a history of safe consumption of the RNA molecules mediating gene suppression in plants , including those with homology to genes in humans and other animals ( Ivashuta et

al. 2009 ; Jensen, et al. 2013 )

. Additionally , there is no evidence to suggest that dietary consumption of nucleic acids is associated with toxicity ( Petrick et al. 2013 ; U.S. FDA

1992 ) and U.S. EPA has an established tolerance exemption for nucleic acids that are part of PIP products ( U.S. EPA 2001a ) . FDA recognizes that all food allergens are proteins ( U.S. FDA 1992 ; 2001 ) and there is also no evidence of allergenicity of dietary RNA in the peer reviewed scientific literature. Th is lack of toxicity or allergenicity f or ingested

RNA also extend s to RNA molecules associated with dsRNA

- mediated gene regulation. Therefore an extensive history of safe consumption for dietary RNAs, including dsRNAs, has been established, as reviewed ( Petrick et al. 2013 )

. The reason for this history of safe consumption of dietary RNAs is that e xtensive sequence

- independent physiological and

biochemical ba rriers are known to exist in humans and other animals that limit the potential for uptake or activity of ingested nucleic acids ( Juliano, et al. 2009 ; O'Neill, et

al. 2011 ; Petrick et al. 2013 )

.

A recent publication reported that ingest ion of large doses of a particular small ( micro ) RNA (miRNA) from rice led to some absorption of the miRN A, detection of the miRNA

in serum and liver

, and an apparent impact on a target protein and plasma LDL in mice ( Zhang, et al. 2012a ) . The authors suggest that a “cross

- kingdom” effect

– a plant gene product ( mi R168a) regulating animal gene expression

– may be a common phenomenon; and that miRNAs in food may regulate specific genes in animals based upon sequen ce identity between plant miRNAs and mammalian genes. A second publication ( Heinemann, et al. 2013 ) , a review article relying almost exclusively on the Zhang ( 201 2a) study, suggests that the current safe ty/risk assessment approach is not sufficient

for RNA

- based biotechnology

- derived products. As stated in the preceding paragraph, there are no safety concerns related to the consumption of RNA and RNAi in plants and

therefore the current safety assessment approach for dsRNA

- containing products, including MON 87411, is a ppropriate for assessing product safety. The following evidence supports this conclusion :

**•** Humans regularly consume plants that contain small RNAs. Recent research demonstrates that many existing plant RNAs share sequences with human genes ( Ivashuta et al. 2009 )

. This work has also been followed up by studies demonstrating that not only small RNAs but also long dsRNAs in plants share sequence identity to human transcripts ( Jensen et al. 2013 ) . Further, humans regularly consume animal

- derived foods that are likely to contain more animalMonsanto Company CR240

- 13U1 99 of 374 miRNAs that have 100% identity to human genes than plant miRNAs . Despite this routine ingestion of plant and animal small RNAs, no impacts on human health have been reported

**•** A follow up study to Zhang et al. ( 2012a ) invo lving Monsanto scientists ( Zhang,

et al. 2012b ) revealed confoundin g factors which likely explain, in part, the unexpected findings in the 201 2 study ( Zhang et al. 2012a )

. In this follow up study

, plant miRNAs (including miR168a) were shown to be over

- represented relative to their dietary abundance in some public animal small RNA datasets

. This indicates that their apparent presence in mouse tissues likely resulted

, at least

i n part, from cross

- contamination during the sequencing procedure , thus call ing into question the potential for significant uptake of ingested plant miRNAs ( Zhang et al. 2012b )

.

**•** Differences in diet composition, rather than cross kingdom gene regulation by plant miRNAs, were likely responsible for alterations in plasma LDL cholesterol when Zhang et al., ( 2012a ) fed an all

- rice diet to mice ( Petrick et al. 2013 ) . These results are consistent with the known challenges to oral delivery of nucleic acids ( O'Neill et al. 2011 )

.

**•** In a recent study, Monsanto and miRagen Therapeutics scientists collaborated on a rice feeding study in mice to evaluate cla ims of dietary miRNA uptake and physiological impact ( Di ckinson, et al. 2013 )

. In this study, miR168a uptake was not reproduced, LDLRAP1 protein levels were unaffected, and LDL was only modulated with a high rice diet and not with a nutritionally balanced rice diet. These results support the conclusion that previously reported ob servations ( Zhang

et al. 2012a ) were due to variability in gene expression and protein expression

data and n utritional differences in animal diets, rather than dietary exposure to

miR168a.

**•** A recent report ( Snow, et al. 2013 ) notes the presence of endogenous miRNAs at substantial levels in diets of humans, mice and honey bees. The authors provide empirical data to demonstrate that despite consumption of miRNAs, horizontal delivery via oral ingestion from a typical diet is neither frequent nor prevalent across these consuming organisms

.

**•** A recent report ( Witwer, et al. 2013 ) conducted a feeding study in nonhuman primates with an miRNA rich food source and based on their results, concluded that, “there is little evidence for presence of these plant miRNA in nonhuman primat e blood prior to or following dietary intake of a plant miRNA

- rich

substance.”

**•** Numerous barriers to the systemic and cellular uptake of exogenous nucleic acids exist (salivary enzymes, stomach and intestinal acids and enzymes, nucleases in serum, etc.) ( Akhtar 2009 ; Haupenthal, et al. 2006 ; Jai n 2008 ; O'Neill et al. 2011 ; Petrick et al. 2013 )

.

**•** Other authors, organizations, and regulatory bodies have looked specifically at biotechnology

- derived RNA

- based products and support current risk assessment approaches ( FSANZ 2013 ; ILSI

- CERA 2011 ; Parrott et al. 2010 )

.

**•** Data presente d below in this petition regarding specificity and lack of adverse impacts on NTOs (Section VI.E.) supports the safety of th e consumption of

MON 87411 and the expressed construct

- derived RNA molecules contained

within it.Monsanto Company CR240

- 13U1 100 of 374 Finally, these numerous point s re garding the ubiquitous nature of RNA in foods

, the known barriers to systemic and cellular uptake of exogenous nucleic acids, the known

sequence similarity between some plant and human RNAs, and empirical data demonstrating the lack of adverse impacts on non

- target organisms lead us to conclude

that unique risk issues related to expression of DvSnf7 RNA in MON 87411 do not exist

and the current risk/ safety assessment approach used by USDA

- APHIS is a pprop riate for assessing this product

.

V I

. B

. Characteriz ation and Equivalence of DvSnf7 RNA from MON 87411

Despite the long history of exposure to and consumption of nucleic acids, a

characterization and safety assessment of the DvSnf7 RNA produced in MON 87411 was conducted

. The safety assessment of the DvSnf 7 transcript require d large amounts of

DvSnf7 RNA as the test substance. Because of the extremely low expression of DvSnf7

RNA in MON 87411 (Table VI

- 1), i t was necessary to produce RNA through in vitro transcription method s in order to obtain sufficient quantities of DvSnf7 RNA (DvSnf7_968 RNA 6

Regarding the overall potential for toxicity and allergenicity, RNA is different from proteins. While certain rare proteins are capabl e of demonstrating toxicity (usually occurring acutely) ( ) for subsequent safety studies . In order to demonstrate that the

in vitro

- produced RNA wa s the appropriate test material for the safety assessment studies, the molecular characteristics of the RNA produced in MON 87411 (MON 87411 DvSnf7 RNA) were determined and were shown to be equivalent to th ose of in vitro

produced RNA, DvSnf7_968 RNA. A s ummary of the results is shown below and the details of the materials, methods, and results are described in Appendix E

.

Pariza and Johnson 2001 ; Sjoblad, et al. 1992 ) , there is no evidence that ingested dsRNA is capable of eliciting toxicity in humans regardless of sequence ( Petrick et al. 2013 ) . Additionally, all known food allergens are proteins ( U.S. FDA

1992 ; 2001 ) and there is no evidence to suggest that ingested RNA can be allergenic. Therefore, experimental endpoints intended to address toxici ty and/or allergenicity of proteins are not necessary or useful for evaluating the food and feed safety of ingested

RNA.

The characterization and equivalence between MON 87411 DvSnf7 RNA and DvSnf7_968 RNA was established by two molecular analyses: 1) DNA sequencing of

the reverse transcribed RNA determined that the sequence of the MON 87411 DvSnf7

and DvSnf7_968 RNAs was identical, including the sequence within the 240 bp inverted repeat regions ; and 2) RNase I f 7

6 DvSnf7_968 is the predominant RNA transcript produced from the DvSnf7 suppression cassette and, in

addition to the DvSnf7 p sequences, contains portions of the CaMV 35S promoter, maize hsp70 i ntron, and

pea E9 3’ untranslated region from PV

- ZMIR10871. digestion followed by northern blot analys is detected the 240 bp dsRNA in MON 87411 DvSnf7 RNA and demonstrated its equivalence to the

in vitro produced DvSnf7 _968 RNA.

7 Endonuclease RNase I f specifically digests single stranded RNA, leaving dsRNA intactMonsanto Company CR240

- 13U1 101 of 374 Taken together, these data provide a detailed characterization of MON 87411 DvSnf7

RNA and establish its equivalence to the in vitro

- produced DvSnf7_968 RNA

. The equivalence justifies the use of the DvSnf7_968 RNA as a test subtance in the safety

assessment studies (Section VI.E. 4 .) ( e.g ., studies on earthworms, honey bees, Lady beetle, environmental fate of DvSnf7 RNA, etc.) of MON 87411.

VI. C

. Expression Levels of DvSnf7 RNA in MON 87411

D vSnf7 RNA levels in various tissues of MON 87411 were determined by a validated QuantiGene ® Plex 2.0 Assay. Plant material from MON 87411 w as collected in 2011

- 2012 from the same replicated field sites in Argentina (BAFO, BAHT, BAPE, BASS, and BATC) using the same 19 tissue types as described for protein expression analysis (Section V.C.).

DvSnf7 RNA levels were determined in all 19 tissue types. The results obtained fo r each sample by the QuantiGene ® Plex 2.0 Assay were averaged across the five sites and are

summarized in Table VI

- 1 . The details of the materials and methods are described in Appendix F . The individual DvSnf7 RNA levels in MON 87411 across all samples

a nalyzed from all sites ranged from lower than the limit of detection (<LOD) to

213 × 10 - 3 **μ** g/g dw. The mean DvSnf7 RNA level for each tissue type was highest in

OSWP1 at 84.8 × 10 - 3 μg/ g dw and lowest in grain at 0.104 × 10 - 3 μg/g dw. Samples with an exp ression level <LOD or lower than the limit of quantitation (<LOQ) were not

included in the mean determinations.Monsanto Company CR240

- 13U1 102 of 374 Table VI

- 1. Summary of DvSnf7 RNA Levels in Maize Tissues Collected from

MON 87411 Produced in Argentina Field Trials during 2011

- 2012

Tissue Type 1 Development Stage 2 Mean (SD) Range (μg/g fw) 3 Mean (SD) Range (μg/g dw) 4 LOD/ LOQ 5
(μg/g fw)

OSL1 V3

- V4 12.6 × 10
- 3 73.9 × 10
- 3 0.129 × 10
- 3 / (2.11 × 10
- 3 ) (14.5 × 10
- 3 ) 0.566 × 10
- 3

8.53 × 10
- 3

- 16.6 × 10
- 3 43.3 × 10
- 3

- 103 × 10
- 3

OSL2 V6

- V8 13.2 × 10
- 3 67.3 × 10
- 3 0.114 × 10
- 3 / (4.08 × 10
- 3 ) (19.4 × 10
- 3 ) 0.502 × 10
- 3

7.77 × 10
- 3

- 20.1 × 10
- 3 37.1 × 10
- 3

- 98.9 × 10
- 3

OSL3 V10

- V13 10.3 × 10
- 3 44.6 × 10
- 3 0.106 × 10
- 3 / (1.89 × 10
- 3 ) (8.51 × 10
- 3 ) 0.468 × 10
- 3

6.19 × 10
- 3

- 12.8 × 10
- 3 27.5 × 10
- 3

- 58.8 × 10
- 3

OSL4 V14

- R1 14.4 × 10
- 3 56.9 × 10
- 3 0.110 × 10
- 3 / (6.71 × 10
- 3 ) (28.5 × 10
- 3 ) 0.482 × 10
- 3

5.40 × 10
- 3

- 33.8 × 10
- 3 22.1 × 10
- 3

- 153 × 10
- 3

OSR1 V3

- V4 3.15 × 10
- 3 23.9 × 10
- 3 0.029 × 10
- 3 / (1.79 × 10
- 3 ) (15.1 × 10
- 3 ) 0.128 × 10
- 3

1.74 × 10
- 3

- 8.00 × 10
- 3 12.5 × 10
- 3

- 67.0 × 10
- 3

OSR2 V6

- V8 2.32 × 10
- 3 16.3 × 10
- 3 0.021 × 10
- 3 / (0.758 × 10
- 3 ) (4.84 × 10
- 3 ) 0.093 × 10
- 3

0.928 × 10
- 3

- 3.76 × 10
- 3 6.62 × 10
- 3

- 25.7 × 10
- 3

OSR3 V10

- V13 1.81 × 10
- 3 10.2 × 10
- 3 0.020 × 10
- 3 / (0.749 × 10
- 3 ) (4.77 × 10
- 3 ) 0.088 × 10
- 3

0.942 × 10
- 3

- 4.00 × 10
- 3 5.13 × 10
- 3

- 24.3 × 10
- 3

OSR4 V14

- R1 1.28 × 10
- 3 6.84 × 10
- 3 0.015 × 10
- 3 / (0.471 × 10
- 3 ) (2.67 × 10
- 3 ) 0.067 × 10
- 3

0.530 × 10
- 3

- 2.40 × 10
- 3 2.66 × 10
- 3

- 13.0 × 10
- 3

OSWP1 V3

- V4 10

. 5 × 10
- 3 84 .8 × 10
- 3 0

. 078 × 10
- 3 / ( 4 .25 × 10
- 3 ) ( 43 .8 × 10
- 3 ) 0

. 345 × 10
- 3

6 .78 × 10
- 3

- 23 .1 × 10
- 3 51

. 1 × 10
- 3

- 213 × 10
- 3

OSWP2 V6

- V8 8 .54 × 10
- 3 55 .1 × 10
- 3 0

. 054 × 10
- 3 / ( 3 .54 × 10
- 3 ) ( 23 .1 × 10
- 3 ) 0

. 239 × 10
- 3

5 .01 × 10
- 3

- 16

. 0 × 10
- 3 33 .0 × 10
- 3

- 106 × 10
- 3

OSWP3 V10

- V13 3 .53 × 10
- 3 25 .5 × 10
- 3 0

. 027 × 10
- 3 / ( 1 .17 × 10
- 3 ) ( 9 .53 × 10
- 3 ) 0

. 119 × 10
- 3

2 .03 × 10
- 3

- 5 .89 × 10
- 3 13 .0 × 10
- 3

- 45 .9 × 10
- 3Monsanto Company CR240

- 13U1 103 of 374 Table VI

- 1. (continued). Summary of DvSnf7 RNA Levels in Maize Tissues

Collected from MON 87411 Produced in Argentina Field Trials during 2011

- 2012

Tissue Type 1 Development Stage 2 Mean (SD) Range (μg/g fw) 3 ,6 Mean (SD) Range (μg/g dw) 4 LOD/ LOQ 5
(μg/g fw)

OSWP4 V14

- R1 3 .16 × 10
- 3 18 .5 × 10
- 3 0

. 028 × 10
- 3 / ( 1 .03 × 10
- 3 ) ( 6 .27 × 10
- 3 ) 0

. 123 × 10
- 3

1 .89 × 10
- 3

- 5 .37 × 10
- 3 10 .3 × 10
- 3

- 32 .2 × 10
- 3

Forage

Root R5 0.536 × 10
- 3 2.37 × 10
- 3 0.013 × 10
- 3 / (0.295 × 10
- 3 ) (1.29 × 10
- 3 ) 0.059 × 10
- 3

0.086 × 10
- 3

- 1.07 × 10
- 3 0.425 × 10
- 3

- 4.61 × 10
- 3

Forage R5 1.28 × 10
- 3 4.26 × 10
- 3 0.036 × 10
- 3 / (0.361 × 10
- 3 ) (1.26 × 10
- 3 ) 0.157 × 10
- 3

0.601 × 10
- 3

- 2.31 × 10
- 3 2.00 × 10
- 3

- 7.72 × 10
- 3

Senescent

Root R6 0.353 × 10
- 3 1.39 × 10
- 3 0.015 × 10
- 3 / (0.203 × 10
- 3 ) (0.815 × 10
- 3 ) 0.065 × 10
- 3

0.127 × 10
- 3

- 0.947 × 10
- 3 0.478 × 10
- 3

- 3.68 × 10
- 3

Stover R6 0.310 × 10
- 3 0.677 × 10
- 3 0.047 × 10
- 3 / (0.077 × 10
- 3 ) (0.201 × 10
- 3 ) 0.207 × 10
- 3

0.190 × 10
- 3

- 0.449 × 10
- 3 0.401 × 10
- 3

- 1.04 × 10
- 3

Pollen VT

- R1 0.103 × 10
- 3 0.134 × 10
- 3 0.013 × 10
- 3 / (0.069 × 10
- 3 ) (0.090 × 10
- 3 ) 0.057 × 10
- 3

0.056 × 10
- 3

- 0.224 × 10
- 3 0.073 × 10
- 3

- 0.292 × 10
- 3

Silk R1 0.530 × 10
- 3 5.42 × 10
- 3 0.004 × 10
- 3 / (0.190 × 10
- 3 ) (2.05 × 10
- 3 ) 0.019 × 10
- 3

0.215 × 10
- 3

- 0.893 × 10
- 3 1.99 × 10
- 3

- 9.03 × 10
- 3

Grain R6 0.091 × 10
- 3 0.104 × 10
- 3 0.008 × 10
- 3 / (0.028 × 10
- 3 ) (0.033 × 10
- 3 ) 0.036 × 10
- 3

0.049 × 10
- 3

- 0.153 × 10
- 3 0.056 × 10
- 3

- 0.175 × 10
- 3

1 OSL=over season leaf, OSR= over season root, and OSWP= over season whole plant

2 The crop development stages at which each tissue was collected.

3 The DvSnf7 RNA levels are calculated as microgram (μg) of DvSnf7 RNA ( ≥ 200 nt) per gram (g) of

tissue on a fresh weight (fw) basis. The sample means, SDs, and ranges (minimum and maximum values) were calculated for each tissue type across all 5 sites (n=20, except for senescent root n=19, stover n=16, pollen n=5, and grain n=18 due to expressions from two p ollen samples <LOD and from the rest of the samples for senescent root, stover, pollen, and grain <LOQ)

4 The DvSnf7 RNA levels are calculated as microgram (μg) of DvSnf7 RNA per gram of tissue on a dry weight (dw) b asis. The sample means, SDs, and range s (minimum and maximum values) were calculated for each tissue type across all 5 sites (n=20, except for senescent root n=19, stover n=16, pollen n=5, and grain n=18 due to expressions from two pollen samples <LOD and from the rest of the samples for

senes cent root, stover, pollen, and grain <LOQ).

5 LOD=limit of detection; LOQ=limit of quantitation.

6 Converting μg/g to parts per billion (ppb) shows DvSnf7 RNA expression ranging from 0.091 ppb in grain to 14.4 ppb in over season leaf at these growth stages

.Monsanto Company CR240

- 13U1 104 of 374 V I. D

. Human and Animal Exposure to the DvSnf7 RNA

The potential dietary intake of DvSnf7 RNA from the consumption of food derived from

M ON 87411 can be estimated by multiplying maize consumption (total of all maize fractions consumed) by the level of DvSnf7 RNA in MON 87411. For the purposes of this assessment, the concentration of the DvSn f 7 RNA in the included maize fractions ( Determined using DEEM

- FCID 8

Total nucleic acid consumption i n the diet for DNA an d RNA (based on purine and pyrimidine intake) is estimated to be 1

- 2 grams per day ( as in Section V.E.6.; flour, meal, bran, starch, sweet corn and popcorn) was assumed to be equal to the mean expression level in whole

MON 87411 grain. Based on DEEM

- FCID exposure estimates and l evels of expressed DvSnf7 RNA in grain from MON 8 7411 on a fresh weight basis ( **μ** g/g) discussed above (Table VI

- 1 )

, 95 th percentile exposure estimates for the general U.S. population and for

non

- nursing infants (the most highly exposed sub

- population) were estimated to be 0.2

and 0.4 n g/kg body weight per day , respectively. These exp osure estimates are likely to

be overestimates because they assume that there is no loss of DvSnf7 RNA during

storage, processing and/or cooking of the grain or food.

Suchner, et al. 2000 ) which would equate to a maximal intake of 0.2 g/kg/day (2 g in a 10 kg non

- nursing infant; assuming all of the intake is RNA). In comparison, anticipated exposures to DvSnf7 RNA from consumption of MON 87411 are extremely low ( ≤ 0 .4 ng/kg/day) relative to this estimate of total daily RNA intake (a 500 m illion fold difference between daily consumption of DvSnf7 RNA from MON 87411 and total RNA)

. Estimated DvSnf7 exposure values are more than 3,300 fold lower than the 1.5 μg/kg/day intake value defined as the threshold of toxicological concern (TTC) for compounds not triggering structural alerts for toxicity ( Kroes, et al. 2005 ) . The TTC concept establishes an exposure level for chemicals (with or without toxicity data) below which there would be negligible risk to human health. Furthermore, it is noteworthy to point out that the TTC value reflects chronic (mean per capita) intake values and the exposures herein are conservative, as they reflect acute (short

- term, 95th percentile) intake values. Based on

this info rmation, the exposure assessment described above , rather than a dietary risk

assessment is considered mo st appropriate for evaluation of the DvSnf7 RNA and the determination of safety is based on the weight

- of

- the

- evidence for RNA safety ( e.g. , general RNA history of safe use, very low exposure to the specific DvSnf7 RNA , and the lack of oral toxicity to nucleic acids ).

The potential DvSnf7 RNA exposure to animals from consumption of MON 87411 in feeds was evaluated by calculating an estimate of daily dietary intake. The highest dietary exposure of DvSnf7 RNA is 81.0 ng/kg bw /day in the lactating dairy cow. The dietary exposure of DvSnf7 RNA for broiler chicken and finishing pig is

8 D ietary E xposure E valuation M odel

– F ood C ommodity I ntake D atabase. This is a software

- based exposure analysis system for performing chronic and acute dietary exposure assessments developed by the

U.S. EPA. DEEM

- FCID can be used to estimate dietary intake of any component of food or water.Monsanto Company CR240

- 13U1 105 of 374 10.5 ng/kg bw /day and 4.6 ng/kg bw /day, respectively. Therefore, there is minimal dietary exposure of animals to DvSnf7 RNA from MON 87411.

Negligible exposures and lack of oral toxicity of RNA in higher organisms indicate s that there would be no significant risk to human and animal health associated with

c onsumption of DvSnf7 RNA in food and feed products derived from MON 87411.

VI.E

. Laboratory Tests to Characterize the Spectrum of Activity of DvSnf7 RNA

VI.E.1. RNAi in Insects

In plants and nematodes, exogenous dsRNAs that enter the cell are amplified via RNA

dependent RNA polymerases (RdRPs) to produce endogenous dsRNAs that supplement the RNAi pathway and prolong the RNAi effect ( Miller, et al. 2012 ) . However, insects

have been shown to lack RdRPs ( Miller et al. 2012 ) . The ability of WCR and Tribolium

castaneum to produce dose

- dependent responses with RNAi is consistent with the absence of an endogenous amplification mechanism ( Bolognesi et al. 2012 ; Miller et al.

2012 ) . The lack of an endogenous amplification mechanism in insects suggests that exposure to dsRNA in higher trophic levels, via ingested prey species, will be limited because a mechanism for bioamplific ation is not evident. Other factors can also influence the efficiency of RNAi in insects, including concentration, potency and efficacy against the target, sequence and length, persistence of gene silencing and the insect life

stage ( Baum et al. 2007a ; Huvenne and Smagghe 2010 ; Whyard et al. 2009 ) . In general, long dsRNAs that incorporate a high degree of sequence match to mRNAs in the target insect have great er potential for efficacy as a result of the number of siRNAs that can be produced ( Baum et al. 2007a ) . Another mechanism that can affect RNAi efficiency in insects, and potentially limit environmental exposure, is the length of the dsRNA. Bolognesi et al. ( 2012 ) and Miller et al., ( 2012 ) recently demonstrated that a ds RNA must be of sufficient length (e.g. ≥ 60 bp) to result in efficacy against WCR and T. castaneum

, respectively.

VI.E. 2

. Test Substances Used to Assess the Activity Spectrum of DvSnf7 RNA

As noted earlier, MON 87411 maize, expresses a 968 nucleotide (nt ) DvSnf7 RNA primary transcript. The DvSnf7 target sequence or “active” region is a 240 bp stretch of dsRNA included within the larger 968 nt RNA molecule. The DvSnf7 240 bp dsRNA was confirmed to be equipotent (in terms of mortality to WCR and southern corn rootworm ( SCR

, Diabrotica undecimpunctata howardi ) ) to the DvSnf7 968 nt RNA in diet bioassays with both the WCR and SCR, justifying the use of the 240 bp dsRNA. The DvSnf7 240 bp dsRNA, herein referred to as DvSnf7_240 dsRNA, was synthesized in

vitro for use in initial insect diet bioassays to characterize the spectrum of activity (Table

VI

- 2 ). Definitive studies used for the quantitative ecological risk assessment utilized an

in vitro synthesized 968 nt DvSnf7 (known as DvSnf7_968 RNA) as ex pressed in

MON 87411 maize and are described below (Table VI

- 3 ).Monsanto Company CR240

- 13U1 106 of 374 VI.E. 3

. Results from Activity Spectrum Bioassays for DvSnf7 RNA

An assessment of the spectrum of activity for a pesticide is designed to characterize activity against a range of insect tax a that includes the target organism as well as non

target organisms ( Raybould 2006 ; Romeis, et al. 2013 ; U.S. EPA 2007 )

. Efficacy of the

DvSnf7 dsRNA against the target organism WCR, and the closely related SCR, was

characterized with larvae in 12

- day continuous

- feeding diet

- incorporation bioas says to
characterize the concentration

- effect relationship and to estimate the LC 50 9

Bachman et al. 2013 value for DvSnf7_240 dsRNA ( Table VI

- 2 ) ( ; Bolognesi et al. 2012 ) . Both

WCR and SCR demonstrated comparable concentration

- dependent effect s with mean 12

- day LC 50 values of 4.4 ng DvSnf7_240 dsRNA/m l diet and 1 .2 ng DvSnf7_240

dsRNA/m l diet , respectively (Table VI

- 2 )

. The relatively small difference in sensitivities between SCR and WCR species to DvSnf7_240 dsRNA may be related to each species’ feeding physiology, how well they perform on an artificial diet in the laboratory

bi oassays and SCR typically having higher growth rates in laboratory settings than WCR. Results from feeding DvSnf7_240 dsRNA to these two species demonstrates efficacy against target pests in these assays.

Characterization of the spectrum of insecticidal activity, MOA

, as well as an understanding of environmental exposure levels and pathways provides important information that can narrow the scope of NTO testing for an ecological risk assessment ( Romeis, et al. 2008 ; Romeis et al. 2013 )

. NTO testing is typically performed in a sequential scheme ( e.g. Tier 1, Tier 2, etc.) and builds upon characterization of the activity spectrum . Results from the NTO battery compl e ments specificity data by evaluating organisms that may be phylogenetically related and/or provide important

functional roles ( e.g. , detritivores, predators, parasitoids, pollinators) in relevant and

reliable laboratory studies. In 2011, a tripartite group (government, industry and academia) evaluate d the environmental risk assessment approach for genetically engineered plants ( e.g. Bt

- expressing plants) and concluded that the current ecological risk assessment framework and effects tests for NTOs are applicable to plants expressing

an RNAi trait ( ILSI

- CERA 2011 )

.

The spectrum of activity for DvSnf7 RNA was characterized by selecting and testing insects based upon their taxonomic relatedness to the WCR (Table VI

- 2 ) ( Bachman et al.

2013 )

. In total, 14 insect species were tested, representing 10 families and 4 orders. Representative insects from the following orders were tested: Hemiptera ( Orius

insidiosus ), Hymenoptera ( Nasonia vitripennis and Pediobius foveolatus ), Lepidoptera ( Spodoptera frugiperda, Helicoverpa zea

, Ostrinia nubilalis , an d Bombyx mori ) and Coleoptera (WCR, SCR, Leptinotarsa decemlineata, Tribolium castaneum, Coleomegilla maculata, Epilachna varivestis, and Poecilus chalcites)

. A representative insect from the Order Diptera was not examined because a previous study on a Dipteran species ( Drosophila spp.) had shown it to be in sensitive to dietary dsRNA without the use of a

9 The LC 50 value is the measure of toxicity of a substance ( L ethal C oncentration), that when fed to a test organism over a given time period, results in death of 50% of those organisms.Monsanto Company CR240

- 13U1 107 of 374 transfection agent ( Whyard et al. 2009 )

. Given that the target species are in the Order Coleoptera, a strong focus was placed on this order and i n total, 7 species across four

families in th is Order were tested with the goal to more fully characterize th e range of activity of DvSnf7 dsRNA within the Order Coleoptera.

Diet bioassays were conducted utilizing in vitro synthesized DvSnf7_240 dsRNA

, at test concentrations ranging from 500 to 5000 ng DvSnf7_240 dsRNA/m l diet and exceed DvSnf7 dsRNA expression v alues in planta ( Section VI. B

. ). Bioassays for activity spectrum studies were designed to: 1) provide continuous exposure of the DvSnf7_240 dsRNA to each test species

, and 2) provide a sufficient duration of exposure to evaluate the potential effects of D vSnf7_240 dsRNA on growth, development and survival. At exposure concentrations that will greatly exceed field exposure levels, no negative impacts attributable to DvSnf7_240 were detected against the Chrysomelidae species, Leptinotarsa decemlineata (phylogenetically the closest to the target species), or in representative species that are even less phylogenetically related from the other three families (Coccinellidae, Tenebrionidae, and Carabidae) tested in the order Coleoptera. Of the Coleoptera te sted, two species L

. decemlineata and Tribolium castaneum ha ve, however, been show n to be sensitive to ingested dsRNA when fed their conspecific (species

- specific) dsRNA ( Bachman et al. 2013 ; Baum et al. 2007a ; Whyard et al. 2009 )

. Additionally, no negative impacts were detected in representative species from the orders Hemiptera, Hymenoptera, or Lepidoptera. Results from these bioassays demonstrate that the spectrum of activity of DvSnf7_240 dsRNA is restricted to the subfamily Galerucinae in the family Chrysomelidae within the order Coleoptera (Table VI

- 2 ).Monsanto Company CR240

- 13U1 108 of 374 Table VI

- 2

. Susceptibility to DvSnf7_24 0 dsRNA in laboratory bioassays

Order Family Subfamily Species Common Name Bioassay

Duration (days) Endpoints 1 LC 50 2 or No

Observed Effect

Concentration 3
(ng/m l or g diet)

Coleoptera Chrysomelidae Galerucinae Diabrotica virgifera virgifera Western corn rootworm 12 S 4.4 2

Chrysomelidae Galerucinae Diabrotica undecimpunctata howardi Southern corn rootworm 12 S 1.2 2

Chrysomelidae Chrysomelinae Leptinotarsa decemlineata Colorado potato beetle 12 S, G 5,000 3

Tenebrionidae Tenebrioninae Tribolium castaneum Red flour beetle 30 S, G 5,000 3

Coccinellidae Coccinellinae Coleomegilla maculata Pink

- spotted lady beetle 24 S, G, D 3,000 3

Coccinellidae Epilachninae Epilachna varivestis Mexican bean beetle 28 S, G, D 3,000 3

Carabidae Harpalinae Poecilus chalcites Carabid beetle 35 S, G, D 5,000 3

Hemiptera Anthocoridae Anthocorinae Orius insidiosus Insidious Flower bug 9 S, D 5,000 3

Hymenoptera Eulophidae Entedoninae Pediobius foveolatus Eulophid wasp 21 S 3,000 3

Pteromalidae Pteromalinae Nasonia vitripennis Jewel wasp 20 S 5,000 3

Lepidoptera Noctuidae Noctuinae Spodoptera frugiperda Fall armyworm 8 S, G 500 3

Noctuidae Heliothinae Helicoverpa zea Corn earworm 12 S, G 5,000 3

Crambidae Pyraustinae Ostrinia nubilalis European corn borer 12 S, G 5,000 3

Bombycidae Bombycinae Bombyx mori Silkworm 14 S, G 5,000 3

1 S: Survival; G: Growth; D: Development

2 LC 50 survival d ata reported in ( Bachman et al. 2013 )

3 No observed effect concentration (NOEC) is equal to the maximum concentration tested in these bioassays.Monsanto Company CR240

- 13U1 109 of 374 VI.E. 4 . Non

- Target Organism Assessment for DvSnf7

E valuation of the potential risks to NTOs is a n important component of APHIS’s plant pest risk assessment of a biotechnology

- derived crop. Assessment of the potential risks

to NTO s associated with the introduction of a biotechnology

- derived crop producing an insecticidal trait is based on the characteristics of the crop and the introduced trait

. Since risk is a function of hazard and exposure, it is critical to determine the potential hazard and exposure scenarios that are most likely and that require eval uation through experimental studies. Selection of the test organisms and test material are important

decisions that are based on the characteristics of the trait and the product ( Romeis et al.

2008 ) . In the U.S., regulatory guidelines for NTO testing and risk assessment of insect

protected crops have been developed by the EPA and testing is conducted according to a

tier

- based system ( U.S. EPA 2010 ) . Additionally , the EPA has convened several Scientific Advisory Panel (SAP) meetings to make recommendations and provide guidance for NTO testing and risk assessment for agricultural products produced by methods of biotechnology ( U.S. EPA 2001c ; 2002 ; 2004b ; 2010 )

.

B ased on the resul ts from the activity spectrum (Section VI.E. 3 .) and known expression

of DvSnf7 RNA in MON 87411 (Section VI.B.), an evaluation of the potential toxicity to

selected NTOs at field exposure levels was conducted. The hazard assessment included toxicity test ing against a soil decomposer [ earthworm ( Eisenia andrei )], and f ive beneficial insect species [honeybee ( Apis mellifera )

, parasitic wasp ( Pediobius foveolatus ), ladybird beetle ( Coleomegilla maculata )

, carabid beetle ( Poecilus chalcites ),

and Insidious Flower bug ( Orius insidiosus )] (Appendix K). All studies were conducted using in vitro produced DvSnf7_968 RNA as the test substance. With the exception of the earthworm study, all studies utilizing the in vitro

- produced test substance included a diet analysis using a sensitive insect ( Diabrotica undecimpunctata howardi; Southern

corn rootworm , SCR ) to confirm that the DvSnf7_968 RNA contained in the test substance diet was biologically active and had the expected level of biological activity. Additionally, where appropriate based upon the diet matrix, the homogeneity of the test material and stability (of the DvSnf7_968 RNA test substance) over the period of storage

was also confirmed. The no observed effect concentrations ( NOECs ) determined for

each of the tests used in the NTO risk assessment for MON 87411 are summarized in

Tab le VI.3

.

Exposure information was developed to determine the maximum expected environmental concentration ( M EEC ) for the DvSnf7 RNA produced in MON 87411 to NTOs

. T est
concentrations were based on the measured DvSnf7 RNA expression in the tissue(s)

type s that the NTO would most likely be exposed to in the environment. A targeted MOE of greater than 10

- times the M EEC was used to set test concentrations ( U.S. EPA

2010 )

. The NTO studies were performed at a nominal concentration of 1,000 ng

DvSnf7 _968 RNA /ml or g diet. In addition to this concentration providing an adequate MOE, it also was high enough t o allow for the assessment of the functional activity of the

RNA in 12

- day dose confirmation diet bioassays with SCR. For predatory beetles that consume herbivorous prey and have an indirect exposure to maize expressed DvSnf7 RNA, t he maximum expression v alue from the leaf development stage with the highest expression (V14

- R1) was used to represent worst

- case scenario to calculate the MOE forMonsanto Company CR240

- 13U1 110 of 374 these species. The most ecologically relevant route of exposure for soil

- dwelling organisms, such earthworms, was considered to be from decomposing late season plant tissue that enters the soil environment. Consequently, for soil

- dwelling NTOs, the

MEECs were based on the level of estimated DvSnf7 RNA in senescent maize roots

.

For the non

- target organism studies, MO Es were calculated based on the ratio of the

NOECs to the M EECs

. MOEs calculated for each species were >10

- fold of the predicted exposure level for NTOs ( Table VI

- 3 ). MOEs that exceed 10 are considered as indicative

of minimal risk in worst

- case laboratory assays by ( U.S. EPA 2010 ) . Therefore, as with the previously assessed Cry3Bb1 prote in, DvSnf7 RNA is not likely to produce adverse

effects on terrestrial beneficial invertebrate species at field exposure levels

. This conclusion is in agreement with prior published literature which reported that DvSnf7 activity is very specific and is re stricted to the Galerucinae subfamily within the Chrysomelidae family in the Order Coleoptera ( Bachman et al. 2013 )

.

The August 2002 EPA SAP report ( U.S. EPA 2002 ) recommended that non

- target testing should be focused on species exposed to the cro p being evaluated ( i.e. , for MON 87411

beneficial organisms found in maize fields). Though aquatic habitats may be located near agricultural areas, the EPA concluded that exposure of aquatic organisms to biotech crops

is limited temporally and spatially and that the potential exposure of aquatic organisms is

therefore low to negligible ( U.S. EPA 2010 )

. S ince there is no meaningful ecologically

relevant exposure to aquatic organisms from maize

, other than through purposeful feeding of processed maize products , e ffects tests on aquatic species were not conducted

for MON 87411 . A dditionally, based upon the narrow spectrum of activity for DvSnf7 RNA the likelihood of adverse effects to aquatic organisms from DvSnf7 is extremely

low ( Bachman et al. 2013 )

. Furthermore, s uccessful RNA i in organisms such as fish, reptiles, and birds has only been achieved with cell lines and/or embryos and has required the use of transfection agents, direct injection, electroporation, or other invasive RNA delivery techniques ( Schyth 2008 ; Sifuentes

- Romero, et al. 2011 ; Ubuka, et al. 2012 )

. Based on minimal exposure and the narrow spectrum of activity of DvSnf7, the

likelihood of negative impacts to non

- target aquatic organisms from cultivation of

MON 87411 is concluded to be extremely low.Monsanto Company CR240

- 13U1 111 of 374 Table VI

- 3

. Maximum expected environmental concentrations (MEEC s) , no observed effect concentrations (NOECs) from non

target organism studies and estimated margins of exposure (MOEs) for DvSnf7 RNA

Test organism Order MEEC 1 NOEC MOE 2

Coleomegilla maculata Coleoptera 0.224 ng/g fw pollen 1000 ng/g ≥4464

Poecilus chalcites Coleoptera 33.8 ng/g fw leaf 3 1000 ng/g ≥30

Apis mellifera larvae Hymenoptera 0.000448 ng 4 11.3 ng/larvae 5 ≥25,223

Apis mellifera adult Hymenoptera 0.224 ng/g fw pollen 1000 ng/g ≥4464

Pediobius foveolatus Hymenoptera 0.224 ng/g fw pollen 1000 ng/g ≥4464

Orius insidiosus Hemiptera 0.224 ng/g fw pollen 1000 ng/g ≥4464

Eisenia andrei Haplotaxida 3.68 ng/g dw senescent root 5000 ng/g dry soil ≥1359

1 M aximum expression levels determined from MON 87411

. 2 MOE values were calculated based on the ratio of the NOEC to M EEC. The MOE was determined based on the maximum expression level of the DvSnf7 RNA

in the tissue from MON 87411 deemed most relevant to the NTO exposure.

3 The maximum expression value from the leaf development stage with the highest expression (V14

- R1) was used to represent worst

- case

- scenario for a predator consuming an herbivorous prey.

4 MEEC based upon mean amount of DvSnf7 RNA expressed in 2 mg of MON 87411 pollen (fw). The average consumption of pollen by honey bee larvae is 2 mg during development ( Babendreier, et al. 2004 ) . The MEEC was calculated as follows: (2 mg pollen × (0.224 ng DvSnf7 RNA/1000 **μ** g)).

5 The NOEC represents the concentration of the test solution used for dosing individual larval cells. A s ingle dose of 10 **μ** l of 1000 ng/g solution was added to each larval cell for a total mass of 11.3 ng DvSnf7/cell. The concentration of 1000 ng/g DvSnf7

_ 968 RNA in the diet solution is calculated based on the density of the 30% sucrose/water (w/v) solution of 1.1270 g/ml

.Monsanto Company CR240

- 13U1 112 of 374 VI. F . Characterization and Safety Conclusion s

DvSnf7 RNA from MON 87411 is a dsRNA that upon consumption b y CRW species causes gene suppression of the targeted DvSnf7 gene. The molecular characteristics of

the MON 87411 DvSnf7 RNA were determined and equivalence between MON 87411

DvSnf7 RNA and in vitro

- produced DvSnf7_968 RNAs was demonstrated. This equivalence justifie s the use of the in vitro

- produced DvSnf7_968 RNA as a test substance in the RNA safety studies (described in more detail in Section VI.E. 4

. )

. Expression studies using a Quantigene Plex 2.0 assay demonstrated that MON 87411 DvSnf7 was expressed at very l ow mean levels ranging from 0.104 × 10 - 3 μg/g dw to

84.8 × 10 - 3 μg/g dw, representing a low percentage of the total RNA. Anticipated dietary exposures to DvSnf7 in humans are very low ( ≤ 0.4 ng/kg b w per day). Anticipated dietary exposures to DvSnf7 in a nimals are also very low, the highest anticipated levels being in la ctating dairy cows (81 ng/kg bw per day).

Additionally , activity spectrum testing on 14 different species showed that activity of

DvSnf7 RNA is restricted to the subfamily Galerucinae in the family Chr y somelidae within the order Coleoptera

. Specific testing on a battery of NTO s (honeybee, earthworm, ladybird beetle , and others) further support s a conclusion that negative impacts resulting from exposure to DvSnf7 RNA from MON 87411 are unlikely. Based on the ubiquitous nature of the RNA

- based suppression mechanism utilizing dsRNA,

demonstration of specificity of DvSnf7 suppression in CRW, lack of impact to NTOs, the history of safe consumption of RNA, and the apparent lack of toxicity or allergenicity of dietary RNA; the RNA

- mediated gene suppression used in MON 87411 poses no observed or theoretical risks to humans or animals. Therefore, the consumption of the

DvSnf7 RNA from MON 87411 or its progeny is considered safe for humans and

animals

.

The safety data presented herein support the conclusion that food and feed products containing MON 87411 or derived from MON 87411 are as safe as maize products currently on the market for human and anima l consumption and the environment

.Monsanto Company CR240

- 13U1 113 of 374 VI I

. COMPOSITIONAL ASSESSMENT OF MON 87411

Safety assessments of biotechnology

- derived crops follow the comparative safety

assessment process ( Codex Alimentarius 2009 ) in which the composition of grain and/or other raw agricultural commodities of the biotechnology

- derived crop are compared to the appropriate conventional control that has a history of safe use. Compositio nal assessments are performed using the principles and analytes outlined in the OECD consensus document for maize composition ( OECD 2002b )

.

A recent review of compositional assessments conducted according to O ECD guidelines, which encompassed a total of seven biotechnology

- derived crop varieties, nine countries and eleven growing seasons concluded that incorporation of biotechnology

- derived agronomic traits has had little impact on natural variation in crop com position. Most compositional variation is attributable to growing region, agronomic practices, and genetic background ( Harrigan, et al. 2010 ) . Numerous scientific publications have further documented the extensive variability in the concentrations of crop nutrients, anti

nutrients, and secondary metabolites that reflect the influence of environmental and genetic factors as well as extensive conventional breeding efforts to improve nutrition, agronomics, and yield ( Harrigan et al. 2010 ; Harrigan, et al. 2009 ; Ridley, et al. 2011 ; Zhou, et al. 2011 )

.

Compositional equivalence between biotechnology

- derived and conventional crops supports an “equal or increased assurance of the safety of foods derived from genetically modified plants” ( OECD 2002a )

. OECD consensus documents on compositional considerations for new crop varieties emphasize quantitative measurements of essential

nutrients and known anti

- nutrients. These quantitative measurements effectively discern any compositional changes that imply potential nutritional or safety ( e.g., anti

- nutritional) concerns. Levels of the components in grain and/or other raw agricultural commodities of the biotechnology

- derived crop product are compared to: 1) corresponding levels in a conventional comparator , a genetically similar conventional line, grown concurrently under similar field conditions, and 2) natural ranges generated from an evaluation of commercial reference hybrids grown concurrently and from data published in the

scientific literature. The c omparison to data published in the literature places any potential differences between the assessed crop and its comparator in the context of the

well

- documented variation in the concentrations of crop nutrients, anti

- nutrients, and secondary metabolites.

This section provides analyses of concentrations of key nutrients, anti

- nutrients, and secondary metabolites o f MON 87411 compared to that of a conventional control maize hybrid grown and harvested under similar conditions, as appropriate. In addition, conventional commercial reference maize hybrids (hereafter refered to as reference hybrids) were included in the composition analyses to provide additional information on the range of natural variability for each component. The production of materials for compositional analyses used a sufficient variety of field trial sites, robust field designs (randomized complete block design with four blocks), and sensitive analytical methods that allow accurate assessments of compositional characteristics over a range of

environmental conditions under which MON 87411 is expected to be grown.Monsanto Company CR240

- 13U1 114 of 374 The information provided in this section addresses relevant factors in Codex Plant Guidelines, Section 4, paragraphs 44 and 45 for compositional analyses ( Codex

Alimentarius 2009 )

.

VI I .A. Compositional Equivalence of MON 87411 Grain and Forage to
Conventional Maize

Grain and forage samples were collected from MON 87411, the conventional control, and a total of 20 different reference hybrids grown in Argentina during a 2011/2012 field production. The reference hybrids were included in the composition analyses to prov ide data on the natural variability for each component. The field production was conducted at eight sites (Appendix G ). The field sites were planted in a randomized complete block design with four blocks per site. MON 87411, conventional control, and re ference hybrids were grown under normal agronomic field conditions for their respective geographic regions, in areas that were typical for maize production in Argentina. Production in the U.S. corn belt and Argentina maize

- growing regions occurs at relati vely similar latitudes with an approximate 6 month offset ( Schnepf et al. 2001 ) . The average growing season temperatures and preci pitation are comparable ( Schnepf et al. 2001 ) and maize hybrids developed in the U.S. are often used directly by farmers in Argenti na. As such, compositional analyses from maize grown in Argentina are approriate for a comparative safety assessment. MON 87411 plots were treated with glyphosate ( 0.95 lb s a.i./ha ) to generate samples under conditions of the intended use of the product.

The evaluation of MON 87411 followed considerations relevant to the compositional quality of maize as defined by the OECD consensus document ( OECD 2002b ) . Grain samples were analyzed for levels of nutrients including proximates, carbohydrates by calculation, fiber, amino acids, fatty acids, minerals, and vitamins. The anti

- nutrients analyzed in grain included phytic acid and raffinose. Secondary metabolites analyzed in grain included furfural, ferulic acid, and p

- coumaric acid. Forage samples were analyzed for levels of proximates, carbohydrates by calculation, fiber, and minerals. In total, 78 different components were assayed (nine in forage and 69 in grain).

Of those 78 components, 14 fatty acids (cap rylic, capric, lauric, myristic, myristoleic, pentadecanoic, pentadecenoic, palmitoleic, heptadecanoic, heptadecenoic, gamma linolenic, eicosadienoic, eicosatrienoic, and arachidonic acids), sodium, and furfural had

more than 50% of observations below the assay limit of quantitiation (LOQ) and were excluded from statistical analysis. Moisture on two components (grain and forage) was measured for conversion of components to dry weight, but was not statistically analyzed. Therefore, 60 components were stati stically analyzed.

The statistical comparison of MON 87411 and the conventional control was based on compositional data combined across all field sites (the combined

- site analysis). Statistically significant differences were identified at the 5% level ( α = 0.05). The compositional data from the reference hybrids were combined across all field sites to calculate a 99% tolerance interval for each component to estimate the natural variability of each component in maize.Monsanto Company CR240

- 13U1 115 of 374 A statistically significan t differ ence between MON 87411 and the conventional control does not imply biological relevance from a food and feed safety perspective. Therefore, statistically significant differences observed in the combined

- site analysis between

MON 87411 and the conventional control were evaluated further to determine whether the detected difference indicated a biologically relevant compositional change or supported a conclusion of compositional equivalence, as follows:

1) Determination of the mean difference between MON 87411 a nd the conventional control that was used in steps two and three , below

. For protein and amino acids only 10

2) Assessment of the relative impact of MON 87411 compared to variation within the conventional control germplasm grown across multiple sites ( i.e ., variation due to environmental influence). This assessment compares the mean difference between

MON 87411 and the conventional control to the range of values for the conventional

control (maximum value minus the minimum value) derived from the combined

- site analysis. When a mean difference is less than ranges seen due to natural environmental variation within even a single, closely related germplasm, the

difference i s typically not a food or feed safety concern ; and , the relative magnitude of the difference (percent change relative to the control) between MON 87411 and the conventional control was determined to allow an assessment of the difference in amino acids in relation to a difference in protein;

3) Assessment of the relative impact of MON 87411 compared to natural variation due to multiple sources ( e.g. , environmental and germplasm influences). This assessment

c ompar e s the mean difference between MO N 87411 and the conventional control to

variation in conventional maize as estimated by in

- study reference hybrid values and assessing whether the mean value of MON 87411 was within the 99% tolerance interval, the literature values, and /or the ILSI Crop Co mposition Database values (ILSI

- CCDB) ( ILSI 2011 )

.

These evaluations of natural variation within the context of the conventional control and conventional maize references are important as crop composition is known to be influenced by environment and germplasm. Although use d as the first step in the comparative assessment process, detection of statistically significant differences between

MON 87411 and conventional control mean values does not imply a meaningful contribution by MON 87411 to compositional variability. Only i f mean differences

between MON 87411 and the conventional control are large relative to natural variation inherent to conventional maize would further assessments be required to establish

10 Since total amino acids measured in a grain analysis are predominantly derived from hydrolysis of protein, changes in protein levels will likely result in corresponding changes in amino acids levels. However, the mean difference for individual amino acid levels will be less than the mean difference for protein because each amino acid constitutes only a part of the protein. For this reason, the relative magnitudes of difference (percent change relative to the control) for amino acids and protein were determined to allow an assessment of the difference in amino acids in relation to a difference in prot ein. When the relative magnitudes of difference for amino acids were related to the relative magnitude of difference for protein, then steps 2 and 3 are not discussed for amino acids.Monsanto Company CR240

- 13U1 116 of 374 whether the change in composition would have an impact from a food a nd feed safety perspective. The steps reviewed in this assessment therefore describe whether the

differences between MON 87411 and the conventional control are meaningful from a food/feed perspective or whether they support a conclusion of compositional e quivalence.

The compositional analysis provided a comprehensive comparative assessment of the levels of key nutrients, anti

- nutrients, and secondary metabolites in maize grain and forage of MON 87411 and the conventional control. Of the 60 components stat istically assessed there were no statistically significant differences in 48 components. Only 12 components (protein, histidine, tyrosine, oleic acid, neutral detergent fiber, copper, iron, manganese, zinc, niacin, vitamin B1 in grain and ash in forage) s howed a significant

difference between MON 87411 and the conventional control in the combined site analysis. For these 12 components, the mean difference s in component values between

MON 87411 and the conventional control were less than the observed range of the

conventional control values (Step 2 above) and the reference hybrid values (Step 3 above) (Tables VII

- 1,

- 3,

- 4,

- 5, and

- 7 below) . The MON 87411 mean component values of the 12 components that were statistically different were within the 99%

tole rance intervals, the values observed in the literature, and/or the ILSI

- CCDB values (Step 3 above). These results support the overall conclusion that MON 87411 was not a major contributor to variation in component levels in maize grain and forage and

conf irmed the equivalence of MON 87411 to the conventional control in levels of all measured components. A detailed description of the assessment of statistically significant

differences observed between MON 87411 and the conventional control is provided in

th e following section. These data confirmed that the components with observed significant differences were not compositionally meaningful from a food and feed safety perspective.

VI I .A. 1 . Nutrient Levels in Maize Grain

Grain samples were analyzed for levels of nutrients including ash, protein, total fat, carbohydrates by calculation, fiber (three components), amino acids (18 components), fatty acids (22 components), minerals (nine components), and vitamins (seven

compone nts). Moisture was measured for conversion of components from fresh to dry weight, but was not statistically analyzed.

VII.A.1.1. Proteins and Amino Acids

Maize grain is typically composed of approximately 10% protein and the levels of protein

and asso ciated amino acids can vary depending on local growing conditions ( Harrigan et

al. 2009 ; Ridley et al. 2011 ; Zhou et al. 2011 ) . This naturally occurring variability is important in assessing the biological relevance of statistically s ignificant differences in composition.

A statistically significant difference (p<0.05) between MON 87411 and the conventional control was observed for protein (Table VII

- 1). The mean protein value was 10.71% dw

for MON 87411 and 10.28% dw for the conven tional control, a mean difference of

0.43% dw . This difference was evaluated in the context of the range of the conventional control values, 4.54% dw , calculated from the minimum (8.06% dw ) and maximumMonsanto Company CR240

- 13U1 117 of 374 (12.60% dw ) protein values. The mean difference in p rotein values between MON 87411 and the conventional control was less than the range of the conventional control values, indicating that MON 87411 does not impact levels more than natural variation within the conventional control grown at multiple location s. The mean difference in protein values

between MON 87411 and the conventional control was also less than the variation seen in the reference hybrid values (ranged 7.21 to 14.59% dw , a magnitude of 7.38% dw ),

and the MON 87411 mean value for protein was within the 99% tolerance interval, the values observed in the published literature and/or the ILSI

- CCDB values (Table VII

- 8).

Since total amino acids measured in a seed analysis are predominantly derived from hydrolysis of protein, differences in amino a cid levels between MON 87411 and the

conventional control were assessed relative to the difference in protein levels. The relative magnitude of the difference in mean protein values for MON 87411 and the conventional control, when expressed as a percent of the conventional control, was 4.19% (Table VII

- 1). Cor respondingly, relative magnitudes of difference for the 18

amino acids measured were ≤4.77%. These differences were significant for two of the

amino acids (histidine, tyrosine) (Table VII

- 1), and reflected small relative magnit udes of

differences between MON 87411 and the conventional control, as would be expected

based on the small relative magnitude of difference in protein

.

The data demonstrated that MON 87411 was not a major contributor to variation in protein and amino acid levels in maize grain and c onfirmed the compositional equivalence of MON 87411 to the conventional control in levels of these components. Also, the mean values of these components were within the 99% tolerance intervals, the values observed in the literature, and/or the ILSI

- CCDB v alues. These data confirmed that the significant differences (p<0.05) in mean values of protein and two amino acids were not compositionally meaningful from a food and feed safety perspective.

VII.A. 1.2. Total Fat and Fatty Acids

Maize grain is typically composed of approximately 4% fat and the levels of total fat and fatty acid levels can vary depending on local growing conditions ( Harrigan et al. 2009 ; Ridley et al. 2011 ; Zhou et al. 2011 ) . This naturally occ urring variability is important in assessing the biological relevance of statistically significant differences in composition.

No statistically significant difference was observed for total fat (Table VII

- 2). There were also no statistically significant differences for seven of the eight fatty acids (FA) assessed. One statistically significant difference (p<0.05) was observed for oleic acid (Table VII

- 2). The mean oleic acid value was 21.89% total FA for MON 87411 and

21.70% total FA for the convention al control, a difference of 0.19% total FA. This

observed statistically significant difference was evaluated in the context of the range of the conventional control values, 2.67% total FA, calculated from the minimum (20.81% total FA) and maximum (23.49% total FA) oleic acid values. The mean difference in

oleic acid values between MON 87411 and the conventional control was less than the range of the conventional control values, indicating that MON 87411 does not impact

levels more than natural variation w ithin the conventional control grown at multiple

locations. The mean difference in oleic acid values between MON 87411 and the conventional control was also less than the variation seen in the reference hybrid valuesMonsanto Company CR240

- 13U1 118 of 374 (ranged from 20.52 to 42.88% total FA, a magnitude of 22.36% total FA), and the

MON 87411 mean value was within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values (Table VII

- 8).

The data demonstrated that MON 87411 was not a major contributor to va riation in total fat and fatty acid levels in maize grain and confirmed the compositional equivalence of

MON 87411 to the conventional control in levels of these components. Also, the mean values of these components were within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values. These data confirmed that the significant

difference in the mean value of oleic acid was not compositionally meaningful from a food and feed safety perspective.

VII.A. 1.3. Carbohydrates by Calculation and Fiber

In addition to protein and fat, major biomass components assessed in maize grain included carbohydrates by c alculation and fiber [acid detergent fiber (ADF), neutral detergent fiber (NDF), and total dietary fiber (TDF)]. Maize grain is typically composed of approximately 85% carbohydrates by calculation, which includes fiber (ADF, NDF, and TDF), and the levels of these components can vary widely depending on local growing conditions ( Harrigan et al. 2009 ; Ridley et al. 2011 ; Zhou et al. 2011 ) . This naturally occurring variability is important in assessing the biological relevance of

statist ically significant differences in composition.

No statistically significant differences were observed for carbohydrates by calculation, ADF, or TDF (Table VII

- 3). A statistically significant difference (p<0.05) was observed

for NDF. The mean NDF value was 8.26% dw for MON 87411 and 8.74% dw for the conventional control, a mean difference of

- 0.48% dw . This difference was evaluated in the context of the range of the conventional control values, 2.94% dw , calculated from the minimum (7.36% dw ) and maximu m (10.30% dw ) NDF values. The mean difference

in NDF values between MON 87411 and the conventional control was less than the range of the conventional control values, indicating that MON 87411 does not impact levels

more than natural variation within the conventional control grown at multiple locations.

The mean difference in NDF values was also less than the variation seen in the reference hybrid values (ranged from 7.41 to 14.80% dw , a magnitude of 7.39% dw ), and the

MON 87411 mean value for NDF was wit hin the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values (Table VII

- 8).

The data demonstrated that MON 87411 was not a major contributor to variation in carbohydrates by calculation and fiber levels in maize grai n and confirmed the compositional equivalence of MON 87411 to the conventional control in levels of these components. Also, the mean values of these components were within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values. These data confirmed that the significant difference in the mean value of NDF was not compositionally meaningful from a food and feed safety perspective.Monsanto Company CR240

- 13U1 119 of 374 VII.A .1.4 Ash and Minerals

Ash and minerals were also assessed in maize grain. Mineral components (calcium, copper, iron, magnesium, manganese, phosphorus, potassium and zinc), constituents of ash, are also discussed in this section. The levels of these components can vary widely depending on local growing conditions ( Harrigan et al. 2009 ; Ridley et al. 2011 ; Zhou et

al. 2011 ) . This naturally occurring variability is important in assessing the biological relevance of statistically significant differences in co mposition.

Ash levels were not statistically significantly different between MON 87411 and the conventional control (Table VII

- 4). There were also no statistically significant differences observed for four of the eight minerals assessed, including calci um, magnesium, phosphorus, or potassium. Statistically significant differences (p<0.05) were

observed between MON 87411 and the conventional control for copper, iron, manganese, and zinc (Table VII

- 4).

For copper, the mean value was 1.33 mg/kg dw for MO N 87411 and 1.41 mg/kg dw for the conventional control, a difference of

- 0.077 mg/kg dw . This difference was evaluated in the context of the range of the conventional control values, 0.65 mg/kg dw , calculated from the minimum (1.13 mg/kg dw ) and maximum ( 1.78 mg/kg dw ) copper values. The mean difference in copper values between MON 87411 and the conventional control was less than the range of the conventional control values, indicating that MON 87411 does not impact levels more than natural variation with in the conventional control grown at multiple locations. The mean difference in copper values was also less than the variation seen in the reference hybrid values (ranged 1.10 to 3.23 mg/kg dw , a magnitude of 2.13 mg/kg dw ), and the MON 87411 mean value w as within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values (Table VII

- 8).

For iron, the mean value was 16.84 mg/kg dw for MON 87411 and 16.33 mg/kg dw for the conventional control, a difference of 0.51 mg/kg dw . The mean difference in iron

values between MON 87411 and the conventional control was less than the range of the conventional control values (4.67 mg/kg dw ; 13.72 to 18.39 mg/kg dw ), indicating that

MON 87411 does not impact levels more than natural v ariation within the conventional control grown at multiple locations. The mean difference in iron values was also less than the variation seen in the reference hybrid values (ranged 13.80 to 24.48 mg/kg dw , a magnitude of 10.68 mg/kg dw ), and the MON 8741 1 mean value was within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values (Table VII

- 8).

For manganese, the mean value was 6.16 mg/kg dw for MON 87411 and 5.99 mg/kg dw for the conventional control, a difference of 0.17 mg/kg dw . The mean difference in manganese values between MON 87411 and the conventional control was less than the range of the conventional control values (2.68 mg/kg dw ; 4.63 to 7.32 mg/kg dw ),

indicat ing that MON 87411 does not impact levels more than natural variation within the conventional control grown at multiple locations. The mean difference in manganese values was also less than the variation seen in the reference hybrid values (ranged 4.93 to 10.42 mg/kg dw , a magnitude of 5.49 mg/kg dw ), and the MON 87411 mean value wasMonsanto Company CR240

- 13U1 120 of 374 within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

CCDB values (Table VII

- 8).

For zinc, the mean value was 21.44 mg/kg dw for MON 8741 1 and 20.93 mg/kg dw for the conventional control, a difference of 0.50 mg/kg dw . The mean difference in zinc

values between MON 87411 and the conventional control was less than the range of the conventional control values (6.45 mg/kg dw ; 17.76 to 24.21 m g/kg dw ), indicating that

MON 87411 does not impact levels more than natural variation within the conventional control grown at multiple locations. The mean difference in zinc values was also less than the variation seen in the reference hybrid values (ra nged 16.40 to 33.92 mg/kg dw , a magnitude of 17.52 mg/kg dw ), and the MON 87411 mean value was within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values (Table VII

- 8).

The data demonstrated that MON 87411 was n ot a major contributor to variation in ash and mineral levels in maize grain and confirmed the compositional equivalence of

MON 87411 to the conventional control in levels of these components. Also, the mean values of these components were within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values. These data confirmed that the significant differences in mean values of these several minerals were not compositionally meaningful from a food and feed safety perspect ive.

VII.A. 1.5 Vitamins

Maize grain contains both water

- soluble vitamins (folic acid, niacin, B1, B2, and B6) and

fat

- soluble vitamins [vitamins A (β

- carotene) and E]. The levels of these components can vary widely depending on local growing conditio ns ( Harrigan et al. 2009 ; Ridley et al.

2011 ; Safawo, et al. 2010 ) . This naturally occurring variability is important in assess ing the biological relevance of statistically significant differences in composition.

No statistically significant differences were observed for folic acid, vitamin A, vitamin B2, vitamin B6, and vitamin E (Table VII

- 5). Statistically significant differ ences (p<0.05) were observed between MON 87411 and the conventional control for niacin and

vitamin B1.

For niacin, the mean value was 17.33 mg/kg dw for MON 87411 and 18.78 mg/kg dw for the conventional control, a difference of

- 1.45 mg/kg dw . This diff erence was evaluated in the context of the range of the conventional control values, 20.69 mg/kg dw , calculated from the minimum (13.73 mg/kg dw ) and maximum (34.41 mg/kg dw ) values. The mean

difference in niacin values between MON 87411 and the conventio nal control was less than the range of the conventional control values, indicating that MON 87411 does not impact levels more than natural variation within the conventional control grown at multiple locations. The mean difference in niacin values was also less than the variation seen in the reference hybrid values (ranged 14.90 to 38.07 mg/kg dw , a magnitude of 23.17 mg/kg dw ), and the MON 87411 mean value was within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB va lues (Table VII

8).Monsanto Company CR240

- 13U1 121 of 374 For vitamin B1, the mean value was 3.44 mg/kg dw for MON 87411 and 3.56 mg/kg dw for the conventional control, a difference of

- 0.12 mg/kg dw . The mean difference in

vitamin B1 values between MON 87411 and the conventional control wa s less than the range of the conventional control values (0.85 mg/kg dw ; 3.10 to 3.94 mg/kg dw ), indicating that MON 87411 does not impact levels more than natural variation within the conventional control grown at multiple locations. The mean difference in vitamin B1 values was also less than the variation seen in the reference hybrid values (ranged 2.79 to 4.96 mg/kg dw , a magnitude of 2.17 mg/kg dw ), and the MON 87411 mean value was within the 99% tolerance interval, the values observed in the literatur e, and/or the ILSI

CCDB values (Table VII

- 8).

The data demonstrated that MON 87411 was not a major contributor to variation in vitamin levels in maize grain and confirmed the compositional equivalence of

MON 87411 to the conventional control in levels of these components. Also, the mean values of these components were within the 99% tolerance interval, the values observed in the literature, and/or the ILSI

- CCDB values. These data confirmed that the significant

differences in mean values of these vitamin s were not compositionally meaningful from a food and feed safety perspective.

VI I .A. 2 . Anti

- Nutrient Levels in Maize Grain

Anti

- nutrients assessed include d phytic acid and raffinose. Phytic acid, the major storage form of phosphorus in maize grain, is considered an anti

- nutrient due to its mineral

chelating properties and the sequestration of phosphorus in phytic acid, reducing phosphorus bioavailabity. Ra ffinose is a low molecular weight non

- digestible carbohydrate that is considered to be an anti

- nutrient due to the enteric gas production and resulting flatulence caused by its consumption ( Liener 2000 ) . The levels of these components can vary widely depending on local growing conditions ( Harrigan et al.

2009 ; Ridley et al. 2011 ) . This naturally occurring variability is important in assessing the biological relevance of statistically significant differences in composition.

No statistically significant differences w ere observed for phytic acid and raffinose (Table VII

- 6). The data demonstrated that MON 87411 was not a major contributor to variation in phytic acid and raffinose levels in maize grain and confirmed the compositional equivalence of MON 87411 to the conv entional control in levels of these

components.

VI I .A. 3 . Secondary Metabolites Levels in Maize Grain

Secondary metabolites measured in MON 87411 grain included furfural, ferulic acid, and

p

- coumaric acid according to the OECD consensus document ( OECD 2002b ) . Furfural was not detected in the grain of MON 87411, the conventional control, or reference hybrids. Ferulic acid and p

- coumaric acid are derived from phenylalanine and tyrosine ( Buchanan, et al. 2000 ) and serve as precursors for a large group of phenylpropanoid compounds and fiber. The levels of these seco ndary metabolites can vary widely depending on local growing conditions ( Harrigan et al. 2009 ; Ridley et al. 2011 )

. This naturally occurring variability is important in assessing the biological relevance of statistically significant differences in composition.Monsanto Company CR240

- 13U1 122 of 374 No statistically significant differences were observed for ferulic acid or p

- coumaric acid (Table VII

- 6). T he data demonstrated that MON 87411 was not a major contributor to variation in ferulic acid and p

- coumaric acid levels in maize grain and confirmed the compositional equivalence of MON 87411 to the conventional control in levels of these

components.

VI I .A .4 . Nutrient Levels in Maize Forage

Forage samples were assessed for levels of ash, protein, total fat, carbohydrates by calculation, fiber (ADF and NDF), and minerals (calcium and phosphorus). The levels of these components can vary widely depending on local growing conditions ( Harrigan et al.

2009 ; Ridley et al. 2011 ) . This naturally occurring variability is imp ortant in assessing the biological relevance of statistically significant differences in composition.

With the exception of ash, there were no statistically significant differences in levels of proximates, fiber, or minerals (Table VII

- 7). The mean ash value was 5.57% dw for

MON 87411 and 5.95% dw for the conventional control, a difference of

- 0.39% dw . This difference was evaluated in the context of the range of the conventional control values,

5.04% dw , calculated from the minimum (4.51% dw ) and maxim um (9.55% dw ) values.

The mean difference in ash values between MON 87411 and the conventional control was less than the range of the conventional control values, indicating that MON 87411 does not impact levels more than natural variation within the conv entional control grown at multiple locations. The mean difference was also less than the variation seen in the reference values (ranged 3.53 to 8.33% dw , a magnitude of 4.80% dw ), and the

MON 87411 mean value was within the 99% tolerance interval, the val ues observed in the literature, and/or the ILSI

- CCDB values (Table VII

- 8).

The data demonstrated that MON 87411 was not a major contributor to variation in ash levels in maize forage and confirmed the compositional equivalence of MON 87411 to

the conventional control in levels of these components. Also, the mean value of this component was within the 99% tolerance interval, the values observed in the literature,

and/or the ILSI

- CCDB values. These data confirmed that the observed significant

diffe rence in mean value of ash was not compositionally meaningful from a food and feed safety perspective.Monsanto Company CR240

- 13U1 123 of 374 Table VII

- 1. Summary of Maize Grain Protein and Amino Acids for MON 8 7411, Conventional Control , and Reference Hybrids

Difference (Test minus Control)

Component (% dw)1 MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value % Relative 6

Protein 10.71 (0.49) 10.28 (0.49) (7.21

- 14.59) 4.54 0.43 (0.17) 0.023 4.19

8.03

- 13.10 8.06

- 12.60 5.66, 15.13

Alanine 0.84 (0.044) 0.81 (0.044) (0.51

- 1.20) 0.42 0.027 (0.019) 0.166 3.34

0.62

- 1.03 0.61

- 1.03 0.37, 1.24

Arginine 0.48 (0.013) 0.47 (0.013) (0.40

- 0.66) 0.15 0.0088 (0.0055) 0.133 1.86

0.41

- 0.54 0.40

- 0.54 0.35, 0.66

Aspartic Acid 0.68 (0.028) 0.66 (0.028) (0.49

- 0.91) 0.28 0.022 (0.011) 0.068 3.41

0.54

- 0.80 0.52

- 0.80 0.42, 0.91

Cystine/Cysteine 0.21 (0.0061) 0.21 (0.0061) (0.16

- 0.30) 0.07 0.0010 (0.0034) 0.768 0.50

0.17

- 0.25 0.17

- 0.24 0.12, 0.32

Glutamic Acid 2.03 (0.11) 1.95 (0.11) (1.23

- 2.87) 1.06 0.078 (0.045) 0.108 3.98

1.44

- 2.51 1.43

- 2.49 0.87, 3.02Monsanto Company CR240

- 13U1 124 of 374 Table VII

- 1 (continued)

. Summary of Maize Grain Protein and Amino Acids for MON 87411, Conventional Control, and

Reference Hybrids

Difference (Test minus Control)

Component (% dw)1 MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value % Relative 6

Glycine 0.38 (0.0099) 0.38 (0.0099) (0.31

- 0.51) 0.12 0.0024 (0.0044) 0.591 0.64

0.34

- 0.43 0.32

- 0.44 0.27, 0.52

Histidine 0.28 (0.010) 0.27 (0.010) (0.20

- 0.46) 0.12 0.0096 (0.0041) 0.033 3.54

0.23

- 0.32 0.21

- 0.34 0.13, 0.45

Isoleucine 0.40 (0.020) 0.38 (0.020) (0.25

- 0.54) 0.21 0.018 (0.0084) 0.050 4.77

0.29

- 0.49 0.27

- 0.48 0.19, 0.56

Leucine 1.41 (0.086) 1.35 (0.086) (0.79

- 2.01) 0.81 0.061 (0.034) 0.100 4.50

0.96

- 1.79 0.95

- 1.76 0.51, 2.14

Lysine 0.28 (0.0057) 0.27 (0.0057) (0.24

- 0.34) 0.09 0.0057 (0.0042) 0.197 2.12

0.24

- 0.32 0.22

- 0.31 0.22, 0.35

Methionine 0.21 (0.0074) 0.21 (0.0074) (0.15

- 0.27) 0.08

- 0.00024 (0.0040) 0.952

- 0.11

0.18

- 0.25 0.17

- 0.25 0.13, 0.28Monsanto Company CR240

- 13U1 125 of 374 Table VII

- 1 (continued)

. Summary of Maize Grain Protein and Amino Acids for MON 87411, Conventional Control, and

Reference Hybrids

Difference (Test minus Control)

Component (% dw)1 MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value % Relative 6

Phenylalanine 0.56 (0.032) 0.54 (0.032) (0.34

- 0.78) 0.31 0.023 (0.014) 0.130 4.20

0.41

- 0.70 0.40

- 0.70 0.24, 0.83

Proline 0.97 (0.045) 0.95 (0.045) (0.64

- 1.39) 0.41 0.019 (0.018) 0.321 1.98

0.74

- 1.18 0.74

- 1.15 0.44, 1.47

Serine 0.48 (0.023) 0.47 (0.023) (0.33

- 0.67) 0.26 0.017 (0.0093) 0.095 3.53

0.35

- 0.60 0.33

- 0.59 0.25, 0.69

Threonine 0.37 (0.015) 0.36 (0.015) (0.27

- 0.50) 0.14 0.010 (0.0062) 0.131 2.74

0.30

- 0.44 0.29

- 0.44 0.22, 0.51

Tryptophan 0.071 (0.0016) 0.071 (0.0016) (0.053

- 0.086) 0.02 0.00018 (0.00070) 0.805 0.25

0.064

- 0.080 0.059

- 0.082 0.053, 0.091

Tyrosine 0.42 (0.020) 0.40 (0.020) (0.27

- 0.57) 0.22 0.018 (0.0082) 0.046 4.42

0.31

- 0.52 0.30

- 0.52 0.22, 0.58Monsanto Company CR240

- 13U1 126 of 374 Table VII

- 1 (continued)

. Summary of Maize Grain Protein and Amino Acids for MON 87411, Conventional Control, and

Reference Hybrids

Difference (Test minus Control)

Component (% dw ) 1 MON 87411 Mean (S.E.)2 Range Conventional

Control

Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value % Relative 6

Valine 0.49 (0.021) 0.48 (0.021) (0.34

- 0.70) 0.22 0.015 (0.010) 0.167 3.04

0.39

- 0.58 0.37

- 0.59 0.27, 0.70

1dw = dry weight.

2Mean (S.E.) = least

- square mean (standard error). 3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4 With 95% confidence, the tolerance interval contains 99% of the values expressed in the population of reference hybrids. Negative limits were

set to zero.

5 Maximum value minus minimum value for the conventional control maize hybrid.

6 The relative magnitude of the difference in mean values between MON 87411 and the conventional control, expressed as a percent of the

conventional control

.Monsanto Company CR240

- 13U1 127 of 374 Table VII

- 2. Summary of Maize Grain Fat and Fatty Acids for MON 87411, Conventional Control, and Reference Hybrids

Difference (Test minus Control)

Component MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value
Total Fat (% dw) 1 3.79 (0.047) 3.83 (0.047) (2.65

- 5.60) 0.68

- 0.036 (0.063) 0.573

3.42

- 4.15 3.43

- 4.10 1.55, 6.69

16:0 Palmitic 6 13.61 (0.045) 13.62 (0.045) (9.98

- 12.71) 0.73

- 0.0063 (0.032) 0.842

13.31

- 13.97 13.32

- 14.05 8.50, 14.14

18:0 Stearic 1.68 (0.032) 1.70 (0.032) (1.56

- 2.58) 0.43

- 0.021 (0.018) 0.249

1.57

- 1.88 1.53

- 1.96 0.93, 2.98

18:1 Oleic 21.89 (0.15) 21.70 (0.15) (20.52

- 42.88) 2.67 0.19 (0.091) 0.040

20.86

- 22.96 20.81

- 23.49 7.74, 50.71

18:2 Linoleic 60.90 (0.22) 61.06 (0.22) (42.82

- 64.10) 3.30

- 0.17 (0.099) 0.095

59.38

- 61.96 59.08

- 62.38 33.63, 77.43

18:3 Linolenic 1.09 (0.0093) 1.09 (0.0093) (0.85

- 1.41) 0.14 0.0035 (0.0059) 0.552

1.04

- 1.17 1.02

- 1.16 0.57, 1.65Monsanto Company CR240

- 13U1 128 of 374 Table VII

- 2 (continued)

. Summary of Maize Grain Fat and Fatty Acids for MON 87411, Conventional Control, and Reference Hybrids

Difference (Test minus Control)

Component MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value

20:0 Arachidic 0.41 (0.0068) 0.42 (0.0068) (0.33

- 0.63) 0.09

- 0.0024 (0.0042) 0.571

0.39

- 0.44 0.37

- 0.47 0.21, 0.70

20:1 Eicosenoic 0.26 (0.0018) 0.26 (0.0018) (0.19

- 0.34) 0.03 0.0038 (0.0022) 0.101

0.24

- 0.28 0.24

- 0.27 0.12, 0.38

22:0 Behenic 0.16 (0.0016) 0.16 (0.0016) (0.055

- 0.25) 0.03

- 0.0016 (0.0020) 0.434

0.14

- 0.17 0.15

- 0.18 0.0065, 0.31

1dw = dry weight.

2Mean (S.E.) = least

- square mean (standard error). 3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4 With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference ma ize hybrids. Negative limits

set to zero.

5 Maximum value minus minimum value for the conventional control maize hybrid.

6 Fatty acid means and ranges are e xpressed as a % of total fatty acid. Prefix numbers refer to number of carbon atoms and number of carbon

carbon double bonds in the fatty acid molecule; 16:0 means sixteen carbon atoms and zero double bonds.

Numbers are not included in text discussion for reasons of clarity. The followi ng fatty acids with

more than 50% of observations below the assay LOQ were excluded from statistical analysis: caprylic acid, capric acid, lauric acid, myristic acid, myristoleic acid, pentadecanoic acid, pentadecenoic acid, palmitoleic acid, heptadecanoic acid, heptadecenoic acid, gamma linolenic acid,

eicosadienoic acid, eicosatrienoic acid, and arachidonic acid.Monsanto Company CR240

- 13U1 129 of 374 Table VII

- 3. Summary of Maize Grain Carbohydrates by Calculation and Fiber for MON 87411, Conventional Control, and Reference Hybrids

Difference (Test minus Control)

Component (% dw)1 MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value Carbohydrates by 84.13 (0.51) 84.53 (0.51) (78.76

- 87.56) 4.77

- 0.40 (0.20) 0.068

Calculation 81.84

- 86.85 82.18

- 86.95 77.72, 90.40

Acid Detergent Fiber 3.06 (0.083) 3.26 (0.083) (1.89

- 5.16) 1.26

- 0.20 (0.10) 0.074

2.50

- 3.62 2.73

- 4.00 2.18, 4.98

Neutral Detergent Fiber 8.26 (0.17) 8.74 (0.17) (7.41

- 14.80) 2.94

- 0.48 (0.20) 0.018

7.12

- 12.14 7.36

- 10.30 6.04, 13.44

Total Dietary Fiber 11.50 (0.20) 11.82 (0.20) (10.33

- 17.11) 4.60

- 0.33 (0.27) 0.247

10.19

- 12.90 10.43

- 15.03 9.83, 16.84

1dw = dry weight.

2Mean (S.E.) = least

- square mean (standard error). 3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4 With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference maize hybrids. Negative limits

set to zero.

5 Maximum value minus minimum value for the conventional control maize hybrid.Monsanto Company CR240

- 13U1 130 of 374 Table VII

- 4. Summary of Maize Grain Ash and Minerals for MON 87411, Conventional Control , and Reference Hybrids

Difference (Test minus Control)

Component MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value
Ash (% dw) 1 1.36 (0.026) 1.35 (0.026) (1.07

- 1.75) 0.55 0.0098 (0.030) 0.746

1.13

- 1.51 1.05

- 1.60 1.07, 1.80

Calcium (% dw) 0.0031 (0.00007) 0.0030 (0.00007) (0.0026

- 0.0056) 0.00 0.00009 (0.00005) 0.063

0.0024

- 0.0037 0.0026

- 0.0033 0.0019, 0.0062

Copper (mg/kg dw) 1.33 (0.056) 1.41 (0.056) (1.10

- 3.23) 0.65

- 0.077 (0.030) 0.021

1.09

- 1.88 1.13

- 1.78 0.28, 3.75

Iron (mg/kg dw) 16.84 (0.41) 16.33 (0.41) (13.80

- 24.48) 4.67 0.51 (0.18) 0.013

14.59

- 19.70 13.72

- 18.39 10.71, 28.62

Magnesium (% dw) 0.11 (0.0031) 0.12 (0.0031) (0.097

- 0.15) 0.03

- 0.00050 (0.0011) 0.657

0.094

- 0.13 0.094

- 0.13 0.086, 0.16

Manganese (mg/kg dw) 6.16 (0.26) 5.99 (0.26) (4.93

- 10.42) 2.68 0.17 (0.070) 0.033

4.64

- 7.13 4.63

- 7.32 2.28, 12.14Monsanto Company CR240

- 13U1 131 of 374 Table VII

- 4 (continued)

. Summary of Maize Grain Ash and Minerals for MON 87411, Conventional Control, and Reference Hybrids

Difference (Test minus Control)

Component MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value Phosphorus (% dw) 0.31 (0.0071) 0.31 (0.0071) (0.24

- 0.39) 0.11

- 0.0018 (0.0033) 0.591

0.25

- 0.33 0.23

- 0.34 0.22, 0.43

Potassium (% dw) 0.34 (0.0061) 0.35 (0.0061) (0.30

- 0.43) 0.09

- 0.0055 (0.0034) 0.127

0.32

- 0.39 0.31

- 0.40 0.28, 0.46

Zinc (mg/kg dw) 21.44 (0.72) 20.93 (0.72) (16.40

- 33.92) 6.45 0.50 (0.22) 0.038

18.54

- 26.54 17.76

- 24.21 11.63, 36.32

1dw = dry weight.

2Mean (S.E.) = least

- square mean (standard error). 3Range is the minimum and maximum raw values for the conventional reference hybrids.

4 With 95% confidence, the tolerance interval contains 99% of the values expressed in the population of conventional reference hybrids. Negative

limits were set to zero.

5 Maximum value minus minimum value for the conventional control maize hybrid.Monsanto Company CR240

- 13U1 132 of 374 Tabl e VII

- 5. Summary of Maize Grain Vitamins for MON 87411, Conventional Control, and Reference Hybrids

Difference (Test minus Control)

Component (mg/kg dw)1 MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value

Folic Acid 0.28 (0.0071) 0.28 (0.0071) (0.19

- 0.52) 0.17 0.0041 (0.0074) 0.580

0.23

- 0.39 0.21

- 0.37 0.084, 0.56

Niacin 17.33 (0.75) 18.78 (0.75) (14.90

- 38.07) 20.69

- 1.45 (0.61) 0.021

14.86

- 20.70 13.73

- 34.41 4.69, 42.03

Vitamin A 1.29 (0.058) 1.38 (0.058) (0.32

- 4.76) 2.94

- 0.095 (0.064) 0.145

0.75

- 1.57 0.65

- 3.59 0, 4.91

Vitamin B1 3.44 (0.065) 3.56 (0.065) (2.79

- 4.96) 0.85

- 0.12 (0.047) 0.021

3.04

- 3.85 3.10

- 3.94 1.86, 5.07

Vitamin B2 1.53 (0.048) 1.64 (0.048) (1.15

- 2.54) 1.01

- 0.11 (0.052) 0.058

1.22

- 2.03 1.28

- 2.30 0.94, 2.37

Vitamin B6 6.16 (0.11) 6.10 (0.11) (5.09

- 12.13) 3.49 0.060 (0.13) 0.648

5.15

- 7.44 5.07

- 8.56 3.84, 10.03Monsanto Company CR240

- 13U1 133 of 374 Table VII

- 5 (continued)

. Summary of Maize Grain Vitamins for MON 87411, Conventional Control, and Reference Hybrids

Difference (Test minus Control)

Component (mg/kg dw)1 MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value

Vitamin E 10.53 (0.24) 10.28 (0.24) (6.37

- 31.91) 3.78 0.25 (0.29) 0.400

6.99

- 15.33 8.94

- 12.72 0, 30.69

1dw = dry weight.

2Mean (S.E.) = least

- square mean (standard error). 3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4 With 95% confidence, the tolerance interval contains 99% of the values expressed in the population of conventional reference hybrids. Negative

limits were set to zero.

5 Maximum value minus minimum value for the conventional control maize hybrid.Monsanto Company CR240

- 13U1 134 of 374 Table VII

- 6. Summary of Maize Grain Anti

- nutrients and Secondary Metabolites for MON 87411, Conventional Control , and Reference Hybrids

Difference (Test minus Control)

Component MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value

Anti

- nutrient (% dw1) Phytic Acid 0.99 (0.029) 0.98 (0.029) (0.60

- 1.25) 0.52 0.012 (0.022) 0.584

0.75

- 1.27 0.67

- 1.19 0.56, 1.41

Raffinose 0.25 (0.0081) 0.24 (0.0081) (0.093

- 0.40) 0.19 0.0066 (0.0058) 0.256

0.14

- 0.31 0.10

- 0.29 0, 0.45

Secondary Metabolite (μg/g dw)

Ferulic Acid 1846.74 (28.24) 1896.61 (28.24) (1337.21

- 3286.55) 392.34

- 49.87 (29.30) 0.110

1528.09

- 2031.96 1700.68

- 2093.02 749.39, 3421.84

p

- Coumaric Acid 148.56 (2.91) 148.27 (2.91) (62.24

- 387.51) 33.33 0.28 (3.31) 0.932

132.88

- 197.75 131.03

- 164.37 0, 461.05

1dw = dry weight.

2Mean (S.E.) = least

- square mean (standard error). 3Range is the minimum and maximum raw values for the conventional reference hybrids.

4 With 95% confidence, the tolerance interval contains 99% of the values expressed in the population of conventional reference hybrids. Negative

limits were set to zero.

5 M aximum value minus minimum value for the conventional control maize hybrid.Monsanto Company CR240

- 13U1 135 of 374 Table VII

- 7. Summary of Maize Forage Proximates, Fiber and Minerals for MON 87411, Conventional Control , and Reference Hybrids

Difference (Test minus Control)

Component (% dw)1 MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value

Ash 5.57 (0.22) 5.95 (0.22) (3.53

- 8.33) 5.04

- 0.39 (0.16) 0.018

3.95

- 7.01 4.51

- 9.55 3.38, 7.59

Carbohydrates by 84.96 (0.56) 84.57 (0.56) (79.74

- 90.37) 7.09 0.38 (0.25) 0.128

Calculation 81.65

- 88.55 80.91

- 88.00 80.32, 89.79

Protein 7.58 (0.46) 7.42 (0.46) (4.19

- 11.38) 5.03 0.17 (0.16) 0.321

5.27

- 10.25 4.63

- 9.66 3.12, 11.55

Total Fat 1.91 (0.17) 2.07 (0.17) (0.66

- 3.60) 2.40

- 0.16 (0.15) 0.296

0.49

- 2.93 0.99

- 3.39 0.67, 3.58

Acid Detergent Fiber 25.45 (0.94) 25.07 (0.94) (18.64

- 37.68) 12.65 0.39 (0.66) 0.558

16.58

- 34.25 17.89

- 30.55 14.75, 38.41

Neutral Detergent Fiber 44.68 (1.49) 42.84 (1.49) (34.97

- 67.39) 19.17 1.84 (1.19) 0.145

28.32

- 55.35 32.16

- 51.33 28.74, 62.39Monsanto Company CR240

- 13U1 136 of 374 Table VII

- 7 (continued) . Summary of Maize Forage Proximates, Fiber and Minerals for MON 87411, Conventional Control,

and Reference Hybrids

Difference (Test minus Control)

Component (% dw)1 MON 87411 Mean (S.E.)2 Range Conventional

Control Mean (S.E.) Range Reference Hybrids (Range)3

Tolerance Interval 4 Conventional Control Range

Value 5 Mean (S.E.) p

- Value

Calcium 0.17 (0.016) 0.18 (0.016) (0.074

- 0.37) 0.34

- 0.013 (0.0078) 0.112

0.093

- 0.26 0.084

- 0.42 0.015, 0.32

Phosphorus 0.19 (0.011) 0.19 (0.011) (0.11

- 0.26) 0.13

- 0.00002 (0.0058) 0.997

0.095

- 0.24 0.13

- 0.25 0.082, 0.26

1dw = dry weight.

2Mean (S.E.) = least

- square mean (standard error). 3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4 With 95% confidence, the tolerance interval contains 99% of the values expressed in the population of conventional reference hybrids. Negative

limits were set to zero.

5 Maximum value minus minimum value for the conventional control maize hybrid.Monsanto Company CR240

- 13U1 137 of 374 Table VI I

- 8

. Literature and ILSI Database Ranges for Components in Maize Forage and Grain

Grain Tissue Components 1 Literature Range 2 ILSI Range 3

Grain Nutrients Proximates (% dw)

Ash 1.17

– 2.01 a ; 1.27

– 1.63 b 0.616

– 6.282 Carbohydrates by calculation 81.31

– 87.06 a ; 82.10

– 85.98 b 77.4

– 89.5 Fat, total 2.95

– 4.40 a ; 3.1 8

– 4. 23 b 1.742

– 5. 900

Prot ein 8.27

– 13.33 a ; 9.17

– 12.19 b 6.15

– 17.26 Fiber (% dw) Acid detergent fiber 1.82

– 4.48 a ; 1.83

– 3.39 b 1.82

– 11.34 Neutral detergent fiber 6.51

– 12.28 a ; 6.08

– 10.36 b 5.59

– 22.64 Total dietary fiber 10.65

– 16.26 a ; 10.57

– 14.56 b 9.01

– 35.31

Amino Acids (% dw)

Alanine 0.60

– 1.04 a ; 0.68

– 0.96 b 0.44

- 1.39 Arginine 0.34

– 0.52 a ; 0.34

– 0.50 b 0.12

- 0.64 Aspartic acid 0.52

– 0.78 a ; 0.59

– 0.76 b 0.33

– 1.21 Cystine 0.19

– 0.26 a ; 0.20

– 0.26 b 0.13

– 0.51

Glutamic acid 1.54

– 2.67 a ; 1.71

– 2.44 b 0.97

– 3.54 Glycine 0.33

– 0.43 a ; 0.33

– 0.42 b 0.18

– 0.54

Histidine 0.25

– 0.37 a ; 0.27

– 0.34 b 0.14

– 0.43

Isoleucine 0.30

– 0.48 a ; 0.32

– 0.44 b 0.18

– 0.69

Leucine 1.02

– 1.87 a ; 1.13

– 1.65 b 0.64

– 2.49 Lysine 0.2 6

– 0.33 a ; 0.2 8

– 0.3 1 b 0.17

– 0.67

Methionine 0.17

– 0.26 a ; 0.16

– 0.30 b 0.12

– 0.47 Phenylalanine 0.43

– 0.72 a ; 0.45

– 0.63 b 0.24

– 0.93

Proline 0.74

– 1.21 a ; 0.78

– 1.11 b 0.46

– 1.63

Serine 0.39

– 0.67 a ; 0.43

– 0.60 b 0.24

– 0.77

Threonine 0.29

– 0.45 a ; 0.31

– 0.39 b 0.22

– 0.67 Tryptophan 0.047

– 0.085 a ; 0.04 2

– 0.0 70 b 0.027

– 0.215 Tyrosine 0.13

– 0.43 a ; 0.12

– 0.41 b 0.10

– 0.64

Valine 0.42

– 0.62 a ; 0.45

– 0.58 b 0.27

– 0.86 Fatty Acids (% Total FA)

16:0 Palmitic 8.80

– 13.33 a ; 9.84

– 12.33 b 7.94

– 20.71

18:0 Stearic 1.36

– 2.14 a ; 1.30

– 2.10 b 1.02

– 3.40

18:1 Oleic 19.50

– 33.71 a ; 19.59

– 29.13 b 17.4

– 40.2

18:2 Linoleic 49.31

– 64.70 a ; 56.51

– 65.65 b 36.2

– 66.5

18:3 Linolenic 0.89

– 1.56 a ; 1.03

– 1.38 b 0.57

– 2.25

20:0 Arachidic 0.30

– 0.49 a ; 0.30

– 0.41 b 0.279

– 0.965

20:1 Eicosenoic 0.17

– 0.29 a ; 0. 17

– 0. 27 b 0.170

– 1.917

22:0 Behenic 0.069

– 0.28 a ; 0. 059

– 0. 18 b 0.110

– 0.349

Minerals Calcium (% dw ) 0.0036

– 0.0068 a ; 0.00 35

– 0.00 70 b 0.00127

– 0.02084 Copper (mg/kg dw ) 1.14

– 3.43 a ; 1. 39

– 2.7 6 b 0.73

– 18.50 Iron (mg/kg dw ) 14.17

– 23.40 a ; 15.90

– 24.66 b 10.42

– 49.07 Magnesium (% dw ) 0.091

– 0.14 a ; 0.10

– 0.14 b 0.0594

– 0.194 Manganese (mg/kg dw ) 4.83

– 8.34 a ; 4.78

– 9.35 b 1.69

– 14.30 Phosphorous (% dw ) 0.24

– 0.37 a ; 0.2 7

– 0. 38 b 0.147

– 0.533 Potassium (% dw ) 0.29

– 0.39 a ; 0.3 6

– 0.4 3 b 0.181

– 0.603 Zinc (mg/kg dw ) 16.78

– 28.17 a ; 18.25

– 30.44 b 6.5

– 37.2Monsanto Company CR240

- 13U1 138 of 374 Table VII

- 8 (continued)

. Literature and ILSI Database Ranges for Components in Maize Forage and Grain

Grain Tissue Components 1 Literature Range 2 ILSI Range 3

Vitamins (mg/kg dw )

Folic acid 0.19

– 0.35 a ; 0.2 3

– 0. 42 b 0.147

– 1.464
Vitamin A [ **β**

– Carotene] 122

– 4 74 0 c 0.19

– 46

. 81

Vitamin B 1 [Thiamine] 2.33

– 4.17 a ; 2.71

– 4.33 b 1.26

– 40.00

Vitamin B 2 [ Riboflavin] 0.94

– 2.42 a ; 1.64

– 2.81 b 0.50

– 2.36

Vitamin B 3 [Niacin] 15.07

– 32.38 a ; 13.64

– 42.06 b 10.37

– 46.94

Vitamin B 6 [ Pyridoxine] 4.93

– 7.53 a ; 4.97

– 8.27 b 3.68

– 11.32
Vitamin E [ **α**

– Tocopherol] 5.96

– 18.44 a ; 2.84

– 15.53 b 1.5 37

– 68.672

Grain Anti

– Nutrients (% dw) Phytic acid 0

. 69

– 1.09 a ; 0.60

– 0.94 b 0.111

– 1.570

Raffinose 0.079

– 0.22 a ; 0.061

– 0.15 b 0.020

– 0.320

Ferulic acid Grain Secondary Metabolites ( **μ** g/g dw )

1205.75

– 2873.05 a ; 1011.40

– 2539.86 b 291.9

– 3885.8

p

– Coumaric acid 94.77

– 327.39 a ; 66.48

– 259.68 b 53.4

– 576.2

Forage Tissue Components 1 Literature Range 2 ILSI Range 3

Forage Nutrients

Proximates (% dw )

Ash 2.67

– 8.01 a ; 4.59

– 6.90 b 1.527

– 9.638 Carbohydrates by calculation 81.88

– 89.26 a ; 84.11

– 87.54 b 76.4

– 92.1 Fat, total 1.28

– 3.62 a ; 0.20

– 1.76 b 0.296

– 4.570

Protein 5.80

– 10.24 a ; 5.56

– 9.14 b 3.14

– 11.57

Fiber (% dw ) Acid detergent fiber 19.11

– 30.49 a ; 20.73

– 33.39 b 16.13

– 47.39 Neutral detergent fiber 27.73

– 49.62 a ; 31.81

– 50.61 b 20.29

– 63.71

Minerals (% dw)

Calcium 0.12

– 0.33 a ; 0.21

– 0.41 b 0.071 39

– 0.576 79 Phosphorous 0.090

– 0.26 a ; 0.13

– 0.21 b 0.0936 2

– 0.3704 1

1 dw = dry weight; FA = fatty acids.

2 Literature range references: a US and b Chile ( Harrigan et al. 2009 )

, c ( Safawo et al. 2010 )

. 3 ILSI range is from ILSI Crop Composition Database, 20 11 [Accessed 9 January 2013] ( ILSI 2011 )

.Monsanto Company CR240

- 13U1 139 of 374 V I I.B. Compositional Assessment of MON 87411 Conclusion

Compositional analysis was conducted on grain and forage of MON 87411 treated with glyphosate grown at eight sites in a 2011/2012 field production in Argentina that a re representative of commercial agricultural regions for maize production. The compositional analysis, based on the OECD consensus document for maize, included measurement of nutrients, anti

- nutrients and secondary metabolites in conventional commercial reference hybrids to provide dat a on the natural variability of each compositional component analyzed.

Of the 60 components statistically assessed for MON 87411, only 12 components (protein, histidine, tyrosine, oleic acid, neutral detergent fiber, copper, iron, manganese, zinc, niacin , and vitamin B1 in grain and ash in forage) showed a significant difference

between MON 87411 and the conventional control in the combined

- site analysis . For these 12 components, the mean difference in component values between MON 87411 and

the conventio nal control was less than the range of the conventional control values and the reference hybrid values. For all components analyzed, the MON 87411 mean component values were within the tolerance intervals, the values observed in the literature, and/or the ILSI

- CCDB values.

These results support the overall conclusion that MON 87411 is not a major contributor to variation in component levels in maize grain and forage and confirmed the compositional equivalence of MON 87411 to the conventional control in l evels of these components. These data confirmed that the components with a statistically significant differences (p<0.05) between MON 87411 and the conventional control were not compositionally meaningful from a food and feed safety perspective.Monsanto Company CR240

- 13U1 140 of 374 VI I I

. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides a comparative assessment of the phenotypic, agronomic, and

environmental interaction characteristics of MON 87411 compared to the conventional control. The data supp ort a conclusion that MON 87411 is not meaningfully different from the conventional control with the exception of the insect

- protected and glyphosate

tolerant traits, and therefore, is not expected to pose a plant pest risk compared to

conventional maize. These conclusions are based on the results of multiple evaluations from laboratory and field assessments.

Phenotypic, agronomic, and environmental interaction characteristics of MON 87411 were evaluated in a comparative manner to assess plant pest potenti al. These assessments included evaluation of seed germination characteristics, plant growth and development characteristics, observations of plant responses to abiotic stressors, plant

- disease and plant

- arthropod interactions, and pollen characteristics. Results from these assessments

demonstrate that MON 87411 does not possess a) increased weediness characteristics; b) increased susceptibility or tolerance to specific abiotic stress ors , diseases, or arthropods; or c) characteristics that would confer a p lant pest risk compared to the conventional

control.

VI I I.A. Characte ristics Measured for Assessment

In the phenotypic, agronomic, and environmental interactions assessment of

MON 87411, data were collected to evaluate altered plant pest potential. A de tailed description of the regulated article phenotype is requested as part of the petition for determination of nonregulated status in 7 CFR § 340.6 including differences from the unmodified recipient organism that would “substantiate that the regulated ar ticle is unlikely to pose a greater plant pest risk than the unmodified organism from which it was derived”. As part of the characterization of MON 87411, data were collected to provide a detailed description of the phenotypic, agronomic, and environmenta l interaction

characteristics of MON 87411. A subset of these data were included in an evaluation of specific characteristics related to altered plant pest potential ( e.g. seed dormancy, lodging, ear drop, and environmental interactions data).

The plant characterization of MON 87411 encompassed five general data categories: 1) seed germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive development (including pollen characteristics); 4) lodging and seed retention on the plant ; 5) plant response to abiotic stress and interactions with diseases and arthropods. An overview of the characteristics assessed is presented in Table VIII

- 1

.

The phenotypic, agronomic, and environmental interactions data were evaluated from a

basis of fa miliarity ( OECD 1993 ) and were comprised of a combination of field and laboratory studies conducted by scientists who are familiar with the production and evaluation of maize. In each of these assessments, MON 87411 was compared to an appropriate conventional control ( i.e

., M PA640B ) that had a genetic background similar

to MON 87411 but did not possess insect

- protected or glyphosate

- toleran ce traits. InMonsanto Company CR240

- 13U1 141 of 374 addition, multiple commercial conventional maize reference hybrids developed through conventional breeding and selection (se e Appendix H , I, and J , and Tables H

- 1, I

- 1, and

J

- 1) were included to provide a range of comparative values for each characteristic that are representative of the variability in existing commercial maize hybrids. Data collected

for the various characteri stics from the commercial reference hybrids provide context for interpreting experimental results.Monsanto Company CR240

- 13U1 142 of 374 Table VI I I

- 1. Phenotypic, Agronomic

, and Environmental Interaction

Characteristics Evaluated in U.S. Field Trials and Laborator y Studies

Data category Characteristics

measured (associated section where discussed) Evaluation timing (Setting of evaluation) 1 Evaluation description (measurement endpoints)
Germination, dormancy, and

emergence Normal germinated (VIII.C.1) Day 4 and 7 : 20/30°C (Laboratory) Percentage of seed producing seedlings exhibiting normal developmental characteristics

Abnormal germinated (VIII.C.1) Day 7 : 20/30°C (Laboratory) Percentage of seed producing seedlings that could not be classified

as normal germinated

Germinated (VIII.C.1) Day 4, Day 7, and Day 12 : 5, 10, 20, 30, 10/20 and

10/30°C (Laboratory) Percentage of seed that had germinated (both normally or abnormally)

Dead (VIII.C.1) Day 4 and 7: 5, 10, 20, 30, 10/20, 10/30, and 20/30°C. Day 12: 5, 10, 20, 30, 10/20 and

10/30°C (Laboratory) Percentage of seed that had visibly

deteriorated and become soft to the touch (also included non

- viable hard

and non

- viable firm

- swollen seed)

Viable hard (VIII.C.1) Day 7:

20/30°C Day 12: 5, 10, 20, 30, 10/20 and 10/30°C (Laboratory) Percentage of seed that did not

imbibe water and remained hard to the touch (viability determined by a

tetrazolium test 2 )

Viable firm

- swollen (VIII.C.1) Day 7:

20/30°C Day 12: 5, 10, 20, 30, 10/20 and

10/30°C (Laboratory) Percentage of seed that imbibed

water and were firm to the touch but did not germinate (viability determined by a tetrazolium test 2 )

Early stand count (VIII.C.2.1) V2

– V 5 growth stage (Field) Number of emerged plants in two

rows Final stand count (VIII.C.2.1) Pre

- harvest (Field) Number of plants in two rows

Vegetative growth Plant vigor (VIII.C.2.1) V2

– V 4 growth stage (Field) Rated on a 1

- 9 scale, where 1 = excellent vigor and 9 = poor vigor Stay green (VIII.C.2.1) Maturity (Field) Rated on a 1

- 9 scale, 1 = 90 to 100%; 2 = 80

- 89%; 3 = 70

- 79%; 4 =

60

- 69%; 5 = 50

- 59%; 6 = 40

- 49%;

7 = 30

- 39%; 8 = 20

- 29%; and 9 = 0

– 19% green tissue Ear height (VIII.C.2.1) R1

– Maturity (Field) Distance from the soil surface at the base of the plant to the ear

attachment node on five plants per plot Plant height (VIII.C.2.1) R1

– Maturity (Field) Distance from the soil surface at the base of the plant to the flag leaf

collar on five plants per plotMonsanto Company CR240

- 13U1 143 of 374 Table VII I

- 1 (continued). Phenotypic, Agronomic and Environmental Interaction Characteristics Evaluated in U.S. Field Trials and Laboratory Studies

Data category Characteristics

measured Evaluation timing (Setting of evaluation) 1 Evaluation description Reproductive growth Days to 50% pollen

shed (VIII.C.2.1) Pollen shed (Field) Days from planting until 50% of the plants have begun to shed pollen Days to 50% silking (VIII.C.2.1) Silking (Field) Days from planting until 50% of the plants have multiple silks exposed Pollen viability (VIII.C.3) Tasseling (Laboratory) Percentage of viable pollen based on pollen grain staining

characteristics Pollen morphology (VIII.C.3) Tasseling (Laboratory) Diameter of viable pollen grains

and observations

Grain moisture (VIII.C.2.1) Harvest (Field) Percentage moisture of harvested shelled grain
Test weight (VIII.C.2.1) Harvest (Field) Test weight (kilogram/hecto liter) of harvested shelled grain

Yield (VIII.C.2.1) Harvest (Field) Calculated in Mg/ha

, adjusted to 15.5% grain moisture content Lodging and

Seed retention Stalk lodged plants (VIII.C.2.1) Pre

- harvest (Field) Number of plants per plot

broken below the ear Root lodged plants (VIII.C.2.1) Pre

- harvest (Field) Number of plants per plot leaning at the soil surface at

>30° from the vertical Dropped ears (VIII.C.2.1) Pre

- harvest (Field) Number of mature ears dropped from plants

Environmental

interactions Abiotic stress

response (VIII.C.2.2.1) Four times during growing season (Field) Qualitative assessment of each plot, with categorical scale of increasing severity (none, slight, moderate, severe) Disease damage (VIII.C.2.2.1) Four times during growing season (Field) Qualitative assessment of each plot, with categorical scale of increasing severity (none, slight, moderate, severe) Arthropod damage (VIII.C.2.2.1) Four times during growing season (Field) Qualitative assessment of each plot, with categorical scale of increasing severity (none, slight, moderate, severe)

Stalk rot disease (VIII.C.2.2.1) Harvest (Field) Qualitative assessment of each plot, with categorical scale of increasing severity (none, slight, moderate, severe)

Ear/kernel rot

disease (VIII.C.2.2.1) Harvest (Field) Qualitative assessment of each plot, with categorical scale of increasing severity (none, slight, moderate, severe)Monsanto Company CR240

- 13U1 144 of 374 Table VII I

- 1 (continued). Phenotypic, Agronomic and Environmental Interaction Characteristics Evaluated in U.S. Field Trials and Laboratory Studies

Data category Characteristics

measured Evaluation timing (Setting of evaluation) 1 Evaluation description

Environmental

interactions Corn earworm damage (VIII.C.2.2.2) R5 growth stage (Field) Quantitative assessment on 10 plants per plot by examining ears for damaged area using a plastic film grid (each grid cell = 0.5

cm 2 ) European corn borer damage (VIII.C.2.2.2) R6 growth stage (Field) Quantitative assessment on 10 plants per plot by counting number of feeding galleries and total length of feeding galleries

in each stalk Arthropod

abundance (VIII.C.2.2.2) Five collection times during growing season (Field) Quantitative assessment of arthropod abundance via sticky

traps collections and visual

counts

1 Plant growth stages were determined using descriptions and guidelines outlined in Corn Growth and Development ( Ritchie, et al. 1997 )

. 2 Viability of hard and firm

- swollen seed were determined by a tetrazolium test ( AOSA/SCST 2010 )

.

V I II.B. Interpretation of Phenotypic and Environmental Interaction Data

Pla nt pest risk assessments for biotechnology

- derived crops are comparative assessments, and are considered from a basis of familiarity. The concept of familiarity is based on the fact that the biotechnology

- derived plant is developed from a well

- characteriz ed

conventional crop whose biological properties and plant pest potential are well

- known

. Familiarity considers the biology of the crop, the introduced trait s , the receiving environment and the interaction of these factors, and provides a basis for compar ative environmental risk assessment between a biotechnology

- derived plant and its conventional counterpart.

Expert knowledge and experience with conventionally bred maize was the basis for selecting appropriate endpoints and estimating the range of respo nses that would be considered typical for maize . As such, MON 87411 was compared to the conventional control in the assessment of phenotypic, agronomic, and environmental interaction characteristics. An overview of the characteristics assessed is present ed in Table

- VII I

- 1

. A subset of the data relating to well

- understood weedy characteristics ( e.g ., seed dormancy, ear drop, and lodging) was used to assess whether there was an increase in weediness potential of MON 87411 compared to a conventional maize

. Evaluation of environmental interaction characteristics ( e.g ., plant abiotic stress, plant

- disease, and plant

- arthropod interactions) was also considered in the plant pest assessment. Prior to analysis, the overall dataset was evaluated for possible evi dence of biologically

- relevant changes and unexpected plant responses. No unexpected observations or issues were identified. Based on all of the data collected, an assessment was made to determine if

MON 87411 could be expected to pose an increased plant pest risk compared to

conventional maize.Monsanto Company CR240

- 13U1 145 of 374 VI I I. B.1. Interpretation of Detected Difference s Criteria

Comparative plant characterization data between a biotechnology

- derived crop and the conventional control are interpreted in the context of contributions to increased plant pest/weed potential. Under the framework of familiarity, characteristics for which no differences are detected support a conclusion of no increased plant pest/weed potential.

Characteristics for which differences are dete cted are considered in a step

- wise method (Figure VIII

- 1) or in a similar fashion. All detected differences for a characteristic are considered in the context of whether or not the difference would increase the crop’s plant pest/weed potential. Ultimatel y, a weight of evidence approach considering all

characteristics and data is used for the overall risk assessment of differences and their significance. In detail, Figure VIII

- 1 illustrates the stepwise assessment process employed:

Figure VIII

- 1. Sche matic Diagram of Agronomic and Phenotypic Data Interpretation Methods
Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological change for the crop in terms of plant pest/weed potential and subsequent steps are not considered. If the answer is “yes” or “uncertain”, the subsequent step is considered.Monsanto Company CR240

- 13U1 146 of 374 Steps 1 and 2

– Evaluate Detected Statistically Significant Differences

Data on each measured characteristic are statistically analyzed, where appropriate, within

each individual site and in a combined

- site analysis, in which the data are pooled among sites. All statistically significant differences are evaluated and considered in the context of a change in plant pest/weed potential. Differences detected in individual

- site analyses that are not detected when data across multiple environments are pooled in the combined

site analysis are considered not biologically meaningful in terms of plant pest/weed potential and, therefore, are not further considered in s ubsequent steps. Any difference

detected in the combined

- site analysis is further assessed.

Step 3

– Evaluate differences in the context of commercial reference materials included in the Study

If a difference for a characteristic is detected in the combin ed

- site analysis across multiple environments, then the mean value of the biotechnology

- derived crop for the characteristic is assessed relative to the range of variation of the commercial reference materials included in the study ( e.g. , reference range).

Step 4

– Evaluate Differences in the Context of the Crop

If the mean value of the characteristics for a biotechnology

- derived crop is outside the variation of the commercial reference materials included in the study, the mean value of the biotechnology

- der ived crop is assessed relative to known values common for the crop ( e.g. , published values).

Step 5

– Relevance of Difference to Plant Pest/Weed Potential

If the mean value of the characteristics for a biotechnology

- derived crop is outside the range of v alues common for the crop, the difference is then assessed for whether or not it is meaningful in terms of plant pest/weed potential.

Step 6

– Conduct Risk Assessment on Identified Hazard

If an adverse effect (hazard) is identified, risk assessment on the difference is conducted. The risk assessment considers contributions to enhanced plant pest/weed potential of the crop itself, the impact of differences detected in other measured characteristics, and potential for and effects of trait introgression i nto any populations growing outside of cultivated environments or into a sexually

- compatible species.

V I II.B.2. Interpretation of Environmental Interactions Data

For the qualitative assessments of abiotic stress response, disease damage, and arthropod

da mage, the biotechnology

- derived crop and conventional control are considered different in susceptibility or tolerance if the range of injury symptoms of each did not overlap across all four replications. Any observed differences are assessed for biologica l significance in the context of the range of the commercial reference materials, and for consistency in other observation times and sites. Differences that are not consistently observed in multiple environments are considered not biologically meaningful in terms of plant pest potential.Monsanto Company CR240

- 13U1 147 of 374 Quantitative assessments of corn earworm and European corn borer damage are analyzed within individual sites and pooled across sites in a combined site analysis. Statistically significant differences detected between the biotechnology

- derived crop and conventional

control s are evaluated using the method outlined in Figure VIII

- 1.

Quantitative assessments of arthropod abundance are only analyzed within each

individual site. Statistically significant differences between the biotechnology

- derived crop and conventional control are assessed for biological significance in the context of the range of the commercial reference hybrids , and for consistency in other collection times and collection sites and in the context of pest pot ential. Differences that are not consistently detected in multiple environments are considered not biologically meaningful in terms of plant pest potential.

VI I I. C

. Comparative Assessments of the Phenotypic, Agronomic

, and

Environmental Interaction Characteristics of MON 87411

This section provides the results of comparative assessments conducted in replicated laboratory and/or multi

- site field experiments to provide a detailed phenotypic, agronomic, and environmental interaction s description of MON 87411 . The

characteristics for MON 87411 evaluated in these assessments included: seed germination and dormancy characteristics (Section VII I .C.1), plant phenotypic, agronomic, and environmental interaction observations under field conditions (Section

VII I.C.2), and pollen characteristics (Section VIII.C.3). Additional details for each assessment are provided in Appendices G, H, and I.

VI I I. C .1. Seed Dormancy and Germination Characteristics

USDA

- APHIS considers the potential for weediness to constitute a plant pest factor (7 CFR § 340.6). Seed germination and dormancy mechanisms vary with species and their genetic basis tends to be complex. Seed dormancy ( e.g ., hard seed) is an important characteristic that is often associated with plants that are con sidered weeds ( Anderson

1996 ; Lingenfelter and Hartwig 2007 )

. Information on germination and dormancy characteristics is therefore useful when assessing a plant for increased weediness potential. To assess germination characteristics, s tandardized germination assays are available and routinely used. The Association of Official Seed Analysts (AOSA), an internationally recognized seed testing organization, recommends a temperature range of

20/ 30 °C as optimal for testing the germination a nd dormancy characteristics of maize

seed ( AOSA 2012b ; a )

. Additional temperature regimes were also eval uated.

A comparative assessment of seed germination and dormancy characteristics was

conducted on MON 87411 and the conventional control. The seed lots to be used for the germination testing (selfed F 2 grain) of MON 87411, the conventional control, and th e reference hybrids (4 per site, 11 unique across all locations) were produced in replicated field trials during 2012 in Iowa (IA), Illinois (IL), and Kansas (KS). These geographic areas represent a broad range of environmental conditions for maize produc tion for this product. The plots were not treated with glyphosate. The germination and dormancy

characteristics (as noted in Table VIII

- 1) were assessed in seven separate split

- plot experiments with four replications, one for each temperature regime.Monsanto Company CR240

- 13U1 148 of 374 D escriptions of the evaluated germination and dormancy characteristics and the timing of the evaluations for all temperature regimes are listed in Table VII I

- 1. Additional details on the materials and experimental methods used in this evaluation are presented in Appendix H

.

In the combined

- site analysis, in which data were pooled across the three seed production sites, no statistically significant differenc es (α=0.05) were detected between MON 87411 and the conventional control for any characteristic at the AOSA temperature regime (20/30 °C), or at the temperature regimes of 5, 10, 20, 30, 10/20, 10/30 °C (Table VII I

- 2). In addition, no hard seed were observ ed at any temperature.

The germination and dormancy characteristics evaluated were used to assess MON 87411 in the context of plant pest risk. The results of this assessment, particularly the lack of increased hard seed, and no changes in other germinat ion and dormancy characteristics, support the conclusion that the introduction of insect

- protected and glyphosate

- tolerant

traits is not expected to result in increased plant pest/weed potential compared to

conventional maize.Monsanto Company CR240

- 13U1 149 of 374 Table VI I I

- 2. Germinatio n Characteristics of MON 87411 and the Conventional

Control

Temperature Assessment Mean % (S.E.) 1 Reference Range 2
(°C) Category MON 87411 Control

5 Germinated 0.3 (0.14) 0.2 (0.11) 0.0

- 0.3

Viable Hard † 0.0 (0.00) 0.0 (0.00) 0.0

- 0.0

Dead 3.0 (0.62) 2.2 (0.47) 1.3

- 5.3

Viable Firm

- Swollen 96.7 (0.68) 97.7 (0.47) 94.5

- 98.6

10 Germinated 82.0 (2.40) 83.1 (2.30) 86.3

- 97.5

Viable Hard † 0.0 (0.00) 0.0 (0.00) 0.0

- 0.0

Dead 0.9 (0.36) 1.0 (0.28) 0.5

- 3.8

Viable Firm

- Swollen 17.1 (2.14) 15.9 (2.14) 0.0

- 11.3

20 Germinated 98.6 (0.38) 99.0 (0.25) 95.5

- 99.5

Viable Hard † 0.0 (0.00) 0.0 (0.00) 0.0

- 0.0

Dead 1.3 (0.36) 1.0 (0.25) 0.5

- 4.5

Viable Firm

- Swollen 0.1 (0.08) 0.0 (0.00) 0.0

- 0.0

30 Germinated 99.1 (0.23) 98.6 (0.42) 92.5

- 99.3

Viable Hard † 0.0 (0.00) 0.0 (0.00) 0.0

- 0.0

Dead 0.9 (0.23) 1.4 (0.42) 0.8

- 7.5

Viable Firm

- Swollen † 0.0 (0.00) 0.0 (0.00) 0.0

- 0.0

10/20 Germinated 98.7 (0.26) 98.3 (0.47) 94.8

- 99.3

Viable Hard † 0.0 (0.00) 0.0 (0.00) 0.0

- 0.0

Dead 0.7 (0.22) 0.9 (0.29) 0.8

- 4.5

Viable Firm

- Swollen 0.7 (0.28) 0.8 (0.28) 0.0

- 0.8

10/30 Germinated 98.8 (0.37) 99.3 (0.25) 96.3

- 99.5

Viable Hard † 0.0 (0.00) 0.0 (0.00) 0.0

- 0.0

Dead 1.1 (0.31) 0.8 (0.25) 0.5

- 3.8

Viable Firm

- Swollen 0.1 (0.08) 0.0 (0.00) 0.0

- 0.3

20/30 Normal Germinated 98.2 (0.37) 98.3 (0.33) 93.8

- 99.8
(AOSA) Abnormal Germinated 0.8 (0.24) 0.3 (0.14) 0.0

- 2.8

Viable Hard † 0.0 (0.00) 0.0 (0.00) 0.0

- 0.0

Dead 1.0 (0.28) 1.3 (0.36) 0.3

- 4.3

Viable Firm

- Swollen 0.0 (0.00) 0.0 (0.00) 0.0

- 0.1

Note: The experimental design was a split

- plot with four replications.

No statistically significant difference between MON 87411 and the conventional control were detected (α=0.05) using ANOVA

. † No statistical comparison could be made due to lack of variability in the data.

1 MON 87411 and the conventional control values represent means with standard error (S.E.) in parentheses. n = 12. In some instances, the total percentage of both MON 87411 and the conventional control did not equal 100% due to numerical rounding of the means.

2 Reference range is the minimum and maximum mean values observed among the 11 unique reference hybrids

.Monsanto Company CR240

- 13U1 150 of 374 V I II. C .2

. Field Phenotypic, Agronomic, and Environmental Interactions

Characteristics

Phenotypic and agronomic characteristics , and environmental interactions were evaluated under field conditions as part of the plant characterization assessment of MON 87411

. These data were developed to provide USDA

- APHIS with a detailed description of

MON 87411 relative to the conventional control and reference hybrids . According to 7 CFR § 340.6, as part of the petition to seek deregulation, a petitioner must submit “a

d etailed description of the phenotype of the regulated article.” This information is being provided to assess whether there are phenotypic differences between MON 87411 and the conventional control that may impact its plant pest/weed potential. Specific

c haracteristics that are related to weediness ( e.g. , lodging and ear drop) were used to assess whether there is a potential increase in weediness of MON 87411 compared to

conventional maize

. Environmental interactions including plant response to abiotic

st ress, disease damage, specific arthropod

- related damage, and arthropod abundance were also assessed as an indicator of changes to MON 87411 and are also considered in the plant pest assessment.

The results of the assessments of agronomic and phenotypic c haracteristics demonstrated

that the introduction of the glyphosate tolerant and insect protected trait s did not meaningfully alter the plant pest/weed potential of MON 87411 compared to

conventional maize . Furthermore, the lack of meaningful differences in plant response to abiotic stress, disease damage, arthropod

- related damage, and arthropod abundance also support the conclusion that the introduction of the glyphosate tolerant and insect protected trait s is not expected to result in increased plant pes t/weed potential for

MON 87411 compared to conventional maize

.

VI I I. C .2.1. Field Phenotypic and Agronomic Characteristics

Data were collected at nine sites in the U.S. during 2012 to evaluate phenotypic and agronomic characteristics of MON 87411 compared to the conventional control (Table

VII I

- 3)

. The se nine sites provided a diverse range of environmental and agronomic conditions representative of commercial maize production areas in North America

. The experimental design at each s ite was a randomized complete block with four replications

. At all sites, MON 87411 , the conventional control, and four reference hybrids were evaluated. A total of 22 unique reference hybrids were evaluated among the nine sites (Appendix I

, Table I

- 1). T he planted plot dimensions varied between sites, due to variability in available planting equipment (Appendix I

, Table I

- 2). All plots of

MON 87411 , the conventional control, and the reference hybrids at each site were uniformly managed in order to assess whether the introduction of insect

- protected and glyphosate

- tolerant traits altered the phenotypic and agronomic characteristics of

MON 87411 compared to the conventional control.

Descriptions of the evaluated phenotypic characteristics and the timing of the evaluations

are listed in Table VII I

- 1. The materials, methods, details concerning the timing ofMonsanto Company CR240

- 13U1 151 of 374 phenotypic assessments, and detailed results of the individual

- site data comparisons are presented and discussed in Appendix I (Table I

- 4 ). The results of the combined

- site analyses are summarized below.

In a combined

- site analysis in which the data were pooled among the sites, no statistically significant differences were detected (α=0.05) between MON 87411 and the conventional

control for any of the a ssessed characteristics, including early stand count, 50% p ollen shed, days to 50% silking, stay green rating, ear height, plant height, dropped ear count, stalk lodged plants, root lodged plants, final stand count, grain m oisture, test weight, and yield ( Table VII I

- 4). Thus, the phenotypic and agronomic characteristics of MON 87411 were not altered in terms of pest/weed potential compared to conventional maize ( Figure

VII I

- 1, step 2 , answer “no ” ).

Plant vigor data were summarized as ranges within individ ual sites. MON 87411 and the conventional control were considered different if the range of vigor values did not overlap across all four replications. There were no differences observed between

MON 87411 and the conventional control in plant vigor a cross sites (Appendix I , Table I

4).

The phenotypic and agronomic characteristics evaluated in this study were used to provide a detailed description of MON 87411 compared to the conventional control. A

subset of these characteristics was used to assess the we ediness of MON 87411

. The results of the agronomic and phenotypic assessment demonstrate that there were no unexpected changes in the phenotype of MON 87411 compared to the conventional control when managed under the agronomic practices for maize producti on. Thus, the

introduction of insect

- protected and glyphosate

- tolerant traits is not expected to result in increased plant pest/weed potential from MON 87411 compared to conventional maize.Monsanto Company CR240

- 13U1 152 of 374 Table V I II

- 3. Field Phenotypic Evaluation Sites for MON 87411 during 2012

Site Code County, State

IABG Greene, Iowa

IARL Jefferson, Iowa

ILMN Warren, Illinois

INSH Boone, Indiana

KSLA Pawnee, Kansas

NCBD Perquimans, North Carolina

NEDC Butler, Nebraska

NEYO York, Nebraska

PAHM Berks, PennsylvaniaMonsanto Company CR240

- 13U1 153 of 3 74 Table V I II

- 4. Combined

- Site Comparison of MON 87411 to Conventional Control for Phenotypic and Agronomic Characteristics During 2012

Mean (S.E.) 1 Reference Range 2 Phenotypic Characteristic (units) MON 87411 Control
Early stand count (#/plot) 82.1 (1.59) 83.4 (1.20) 72.0

– 92.5
Days to 50% pollen shed 63.3 ( 0.64 ) 63.1 (0.59) 60.0

– 67.0
Days to 50% silking 63.4 (0.62) 63.3 (0.54) 59.3

– 66.5
Stay green rating (1

- 9 scale) 5.6 (0.42) 5.6 (0.40) 2.8

– 9.0
Ear height (cm) 104.1 (1.99) 103.6 (2.07) 87.4

– 132.2
Plant height (cm) 238.3 (4.13) 239.1 (4.32) 204.4

– 262.6
Dropped ears (#/plot) 0.7 (0.29) 1.0 (0.42) 0.0

– 10.9
Stalk lodged plants (#/plot) 1.2 (0.29) 1.1 (0.39) 0.0

– 5.7
Root lodged plants (#/plot) 0.1 (0.06) 0.2 (0.13) 0.0

– 1.3
Final stand count (#/plot) 3 65.0 (0.96) 64.3 (0.81) 60.5

– 70.8
Grain moisture (%) 4 17.6 (0.38) 17.9 (0.42) 12.6

– 22.4
Test Weight (kg/hl) 5 72.8 (0.57) 73.2 (0.64) 68.2

– 77.7

Yield (Mg/ha) 6 11.1 (0.44) 10.9 (0.44) 7.4

– 15.8

Note: The experimental design was a randomized complete block with four replicates per site. No statistically significant difference s were detected between MON 87411 and the conventional

control (α=0.05) using ANOVA.

1 MON 87411 and the conventional control values represent means with standard error (S.E.) in parentheses. n = 36, except where noted.

2 Reference range is calculated from the minimum and maximum mean values from among the 22 unique reference hybrids

. 3 Final stand count was excluded from a single replication of MON 87411 at the NEDC site

because it was identified as an outlier. n = 35 for MON 87411; n = 36 for the conventional

control.

4 Grain moisture was excluded from a single replication of the conventional control at the IARL site because it was collected incorrectly. n = 36 for MON 87411; n = 35 for the conventional

control.

5 Test weight (kg/hl = kilogram/hectoliter) was excluded from a single rep lication of the conventional control at two sites (IARL and PAHM). n = 36 for MON 87411; n = 34 for the

conventional control.

6 Yield (Mg/ha = megagram/hectare) data was dropped from a single replication of the conventional control at the IARL site because the shelled plot weight was collected incorrectly.

n = 36 for MON 87411; n = 35 for the conventional control.Monsanto Company CR240

- 13U1 154 of 374 VI I I. C .2. 2 . Environmental Interaction Characteristics

USDA

- APHIS considers the environmental interaction of the biotechnology

- derived crop compared to its conventional control to determine the potential for increased plant pest

characteristics. Evaluations of environmental interactions were c onducted as part of the plant characterization for MON 87411 . In the 2012 U.S. field trials conducted to evaluate the phenotypic and agronomic characteristics of MON 87411 , data were also collected on plant response to abiotic stress ( e .g

. , drought, wind, nutrient deficiency, etc.), disease damage, arthropod

- related damage, and arthropod abundance (Table s VII I

- 5 and

VIII

- 6 ; Appendix I ; Tables I

- 5

, I

- 6

, I

- 7

, I

- 8

, I

- 9 , and I

- 10 , respectively). These data were used as part of the plant pest/ environmental an alysis (Section X ) to assess plant pest potential compared to the conventional control and provide confimatory data for the conclusion of laboratory NTO testing data ( Section VI .E.). The results of the field

evaluations showed that insect

- protected and glyphosate

- tolerant traits did not unexpectedly alter the assessed environmental interactions of MON 87411 compared to

the conventional control. The lack of significant biological differences in plant response s to abiotic stress, disease damage, arthropod

- related damage, and pest

- and beneficial

arthropod abundance support the conclusion that the introduction of the insect

- protected and glyphosate

- tolerant traits is not expected to result in increased plant pest potential

from MON 87411 compared to commerc ial maize

.

VI I I.C.2. 2 .1. Qualitative Environmental Interactions Assessment

Plant responses to abiotic stressors, disease damage, and arthropod damage were assessed at natural levels, i.e ., no artificial infestation or imposed abiotic stress; therefore the se levels typically varied between observations at a site and among sites. Plant responses to abiotic stress, disease damage, and arthropod damage data were collected from each plot using a categorical scale (none, slight, moderate, and severe) of increas ing severity of observed damage for each stressor. This scale was utilized to allow for the evaluation of the wide variety of potential abiotic stressor, disease damage, and arthropod damage symptoms potentially occurring across the season and across site s. These data were categorical and therefore were summarized and not subjected to ANOVA. For a particular stressor, all comparisons of the range of responses for MON 87411 to the range of responses for the conventional control across all observation time s and sites are reported.

Descriptions of the evaluated environmental interactions characteristics and the timing of

the evaluations are listed in Table VII I

- 1. The materials, methods, additional details concerning the qualitative environmental intera ctions assessments, and detailed results of the qualitative data comparisons are presented and discussed in Appendix I (Tables I

- 5 through I

- 7 ).

In the qualitative assessment, no differences in the range of responses were observed

between MON 87411 and the conventional control for any of the 100 comparisons of plant response to abiotic stressors, including cold, drought, flood, frost, hail, heat, nutrient deficiency, soil compaction, sunscald, and wind (Table VII I

- 5 and Appendix I ; Table I

- 5 ). Addi tionally, no differences in the range of responses were observed betweenMonsanto Company CR240

- 13U1 155 of 374 MON 87411 and the conventional control for any of the 119 comparisons for plant damage caused by diseases, including anthracnose, bacterial leaf spot, ear rot, eyespot,

Fusarium sp., Goss’s bacterial wilt, gray leaf spot, leaf blight, maize rough dwarf virus, Pythium sp., Rhizoctonia sp., rust, seedling blight, smut, stalk rot, and Stewart’s bacterial

wilt (Table VII I

- 5 and Appendix I ; Table I

- 6 ). Finally, no differences in the range of responses were observed between MON 87411 and the conventional control for any of

the 102 comparisons for plant damage caused by arthropods, including aphid

, armyworm
, billbug

, cutworm , corn earworm, corn flea beetle, rootworm beetle, European corn borer, grasshopper, Japanese beetle, sap beetle, spider mite, stink bug, and wireworm adult (Table VII I

- 5 and Appendix I ; Table I

- 7 ).

The lack of differences observed between MON 87411 and the conventional control for plant responses to abiotic stressors, disease damage, and arthropod

- related damage in multiple environments across the U.S. supports the conclusion that the introduction of the

insect

- protected and glyphosate

- tolerant traits is not expected to cause a biologically meaningful change in terms of plant pest potential compared to the conventional control (See Section VII I .B.2.)

.Monsanto Company CR240

- 13U1 156 of 374 Table VI I I

- 5 . Summary of Qualitative Environmental Interactions Assessments during 2012

Note: The experimental design was a randomized complete block with four replicates per site.

No differences were observed between MON 87411 and the conventional control during any observation for damage caused by any of the assessed stressors.

1 MON 874 11 and the conventional control were considered different in susceptibility or tolerance if the range of injury symptoms across four replications did not overlap between MON 87411 and the

conventional control.

VI I I.C.2. 2 .2. Quantitative Environmental Interactions Assessment

Quantitative arthropod assessments on corn earworm (CEW: Helicoverpa zea ) damage, European corn borer (ECB: Ostrinia nubilalis ) damage , and arthropod abundance were conducted at four sites (IABG, NEYO, NCBD, and PAHM)

. CEW and ECB damage was assessed once during the growing season at each site . Arthropod abundance was assessed from collections performed five times during the growing season at each site using sticky traps and visual counts.

Damage data were collected for CEW an d ECB at the four sites in 2012. Both individual

and combined

- site (the four site pooled data) analys e s were conducted. Descriptions of the evaluated environmental interactions characteristics and the timing of the evaluations

are listed in Table VII I

- 1. The materials, methods, additional details concerning the specific arthropod damage assessments, and detailed results of the individual

- site data comparisons are presented and discussed in Appendix I (Table I

- 8 ). The results of the

combined

- site analysi s are summarized below.

In the combined

- site analysis, no statistical ly significant differences ( α=0.05 ) were detected between MON 87411 and the conventional control for plant damage caused by

corn earworm or European corn borer (Tables VII I

- 6 ). Conside ring th is lack of significant differences, the results indicate no changes in susceptibility or resistance of

MON 87411 to these common maize pests. (See Figure VII I

- 1, Step 2, answer “no”).

Arthropod abundance was assessed from collections performed using sticky traps and

visual counts at the four sites in 2012. Variations in temporal activity and geographical distribution of arthropod taxa occurr ed between sites, therefore, only individual

- site Stressor Number of

observations

across all sites Number of observations with

no differences between

MON 87411 and the

conventional control across

all sites 1

Abiotic stressors 100 100

Disease damage 1 1 9 1 1 9

Arthropod

- related damage 102 102

Total 321 321Monsanto Company CR240

- 13U1 157 of 374 analyses were cond ucted for arthropod abundance data. Statistical analyses and significance testing of differences were only performed for the arthropods present in sufficient numbers to allow more robust statistical analysis. An inclusion criterion was

established where a given arthropod must have an average count per plot per collection time (across all materials) of ≥ 1. Additional details of the arthropod abundance

assessments and detailed results of the individual

- site data comparisons are provided in Appendix I (Tab les I

- 9 and I

- 10 ). The results of these analyses are summarized below

and in Table VII I

- 7.

In an assessment of arthropod abundance from sticky traps, a total of 108 statistical comparisons were made between MON 87411 and the conventional control for arthr opod abundance involving the following arthropods: aphid, corn flea beetle, d elphacid plant hopper, green lacewing, ladybird beetle, leaf hopper, micro

- parasitic hymenoptera,

macro

- parasitic hymenoptera, sap beetle, minute pirate bug

, and spider. Lack of

su fficient arthropod abundance precluded statistical comparisons between MON 87411

and the conventional control for 79 additional comparisons; however, descriptive statistics were provided for these comparisons ( Appendix I ; Table I

- 9 )

.

No statistically significant differences ( α=0.05 ) were detected between MON 87411 and

the conventional control for 104 out of 108 comparisons. The four differences detected

between MON 87411 and the conventional control included aphid, delphacid planthopper, minute pirate bug, and spider (Table VII I

- 7 and Appendix I ; Table I

- 9)

. The abundance of aphid s was higher in MON 87411 than the conventional control in Collection 3 (1.8 vs. 0.0 per plot) at the NEYO site. T he mean value for aphid abundance

on MON 87411 w as within the range of the reference hybrids. The abundance of delphacid planthopper s was lower in MON 87411 than the conventional control in Collection 1 at the IABG site (0.0 vs. 1.8 per plot). MON 87411 had higher abundance of spiders than the conventio nal control (3.3 vs. 0.3 per plot) in Collection 5 and lower

abundance than the conventional control for minute pirate bugs (1.0 vs. 3.0 per plot) in

Collection 1 at the PAHM site. T he mean value s for delphacid planthopper, minute pirate bug, and spider a bundance on MON 87411 were outside the respective ranges of reference hybrids. However, t he se differences were not consistently detected across

collections or sites (Table VII I

- 7 and Appendix I ; Table I

- 9)

. Thus, the se differences in aphid, delphacid pla nthopper, minute pirate bug, and spider abundance were not indicative of a consistent response associated with the trait and are not considered biologically meaningful in terms of increased pest potential of MON 87411 compared to

the conventional control (See Section VII I .B.2.)

.

In an assessment of arthropod abundance from visual counts

, a total of 61 statistical comparisons were made between MON 87411 and the conventional control for arthropod abundance involving the following pest and beneficial arthropo ds: ant

- like flower beetle, click beetle, corn flea beetle, ladybird beetle adult, ladybird beetle larvae, minute pirate bug , sap beetle, shining flower beetle, and spider

. Lack of sufficient arthropod abundance precluded statistical comparisons between M ON 87411 and the conventional control for 152 additional comparisons; however, the descriptive statistics were provided for these comparisons ( Appendix I

, Table I

- 10 )

.Monsanto Company CR240

- 13U1 158 of 374 No statistically significant differences ( α=0.05 ) were detected between MON 87411 and

th e conventional control for 60 out of 61 comparisons. One difference was detected

between MON 87411 and the conventional control for ant

- like flower beetle (Table

VII I

- 7 and Appendix I ; Table I

- 10). The abundance of ant

- like flower beetle s was lower

on MON 87411 than the conventional control in Collection 2 at the IABG site (0.8 vs. 4.3 per plot). The mean value for ant

- like flower beetle abundance on MON 87411 was lower than the range of reference hybrids (1.8

– 3.3 per plot). However, the difference

for ant

- like flower beetle was not consistently detected across collections at the IABG

site (Table VII I

- 7 and Appendix I ; Table I

- 10). Thus, this difference in ant

- like flower beetle abundance was not indicative of a consistent response associated with t he trait and

is not considered biologically meaningful in terms of increased pest potential

of MON 87411 compared to the conventional control ( See Section VII I .B.2.)

.Monsanto Company CR240

- 13U1 159 of 374 Table VI I I

- 6 Combined

- Site Comparison of Pest Damage to MON 87411 Compared to the Conventional Control during 2012

Pest Arthropod Damage Assessment Mean (S.E.) 1 Reference

range 2 MON 87411 Control

Corn earworm ( H. zea ) Damage area (cm 2 ) of 10 plants per plot 1.9 (0.47) 1.3 (0.30) 0.2

– 3.2

European corn borer
( O. nubilalis ) Number of stalk galleries of 10 plants per plot 0.4 (0.16) 0.5 (0.21) 0.0

– 1.8

European corn borer
( O. nubilalis ) Stalk gallery length (cm) of 10 plants per plot 1.6 (0.72) 2.0 (0.94) 0.0

– 8.3

Note: The experimental design was a randomized complete block with four replicates per site. No statistically significant differences were detected between MON 87411 and the conventional control (α=0.05) using ANOVA.

1 MON 87411 and the conventional contr ol values represent means with standard error (S.E.) in parentheses. n = 16.

2 Reference range is calculated from the minimum and maximum mean values from among 15 unique reference hybrids across four sit es (IABG, NCBD, NEYO, and PAHM).Monsanto Company CR240

- 13U1 160 of 374 Table VIII

- 7. Summary of Arthropod Abundance Assessments and Detected Differences during 2012

Summary of Statistical Comparisons 1 Summary of Detected Differences 2

Arthropod

Abundance

Assessment Number

of sites Number of comparisons

across sites Number of comparisons where

no differences were

detected Arthropod Site Collection

Number Within

reference range? Consistently

detected across

collections or

sites?

Sticky Traps 4 108 104 Aphids NEYO 3 Yes No Delphacid planthopper IABG 1 No No

Minute pirate bug PAHM 1 No No
Spiders PAHM 5 No No

Visual Counts 4 61 60 Ant

- like flower

beetle IABG 2 No No

1 Quantitative arthropod abundance assessments were statistically analyzed at α=0.05 using ANOVA. Lack of sufficient arthropod abundance precluded statistical comparisons between MON 87411 and the conventional control for additional 79 comparisons (sticky traps) and 1 52 comparisons (visual counts) ; however, descriptive statistics were provided for thes e comparisons in Appendix I (Tables I

- 9 and I

- 10 ).

2 Five statistically significant differences were detected. These differences are further discussed in Section VIII.C.2.2.2 using the approach outlined in Section

VII I .B.2

.Monsanto Company CR240

- 13U1 161 of 374 VI I I. C .3. Pollen Characteristics

USDA

- APHIS considers the potential for gene flow and introgression of the biotechnology

- derived trait(s) into sexually compatible plants and wild relatives to determine the potential for increased weedy or invasive characteristics of the receiving species. Pollen morphology and viability information are pertinent to this assessment and, therefore, were assessed for MON 87411. In addition, morphological

char acterization of pollen produced by MON 87411 and the conventional control is relevant to the plant pest risk assessment because it adds to the detailed description of the phenotype of MON 87411 compared to the conventional control.

The viability and morpho logy of pollen collected from MON 87411 compared to that of

the conventional control was also assessed. Pollen was collected from MON 87411, the conventional control, and four commercial references grown under similar agronomic

conditions in a field trial in Iowa, a geographic area that represents environmentally relevant conditions for maize production for this product. The trial was arranged in a randomized complete block design with four replications. Once all plants across the replications reached th e flowering stage, pollen was collected from three non

systematically selected plants per plot and stained for assessment. Descriptions of the

evaluated pollen viability and morphology characteristics and the timing of the

evaluations are listed in Table VIII

- 1. The details of the materials and experimental methods used in this evaluation are presented in Appendix J

.

No statistically significant differences (α=0.05) were detected between MON 87411 and the conventional control for percent viable pollen or pollen grain diameter (Table VIII

- 8). Furthermore, no visual differences in general pollen morphology were observed between

MON 87411 and the conventional control (Appendix J , Figure J

- 1).

The pollen characterization data contribute to the detailed phen otypic description of

MON 87411 compared to the conventional control. Based on the assessed characteristics, the results support a conclusion that neither pollen viability nor morphology of MON 87411 were altered compared to conventional maize.Monsanto Company CR240

- 13U1 162 of 374 Table V I II

- 8

. Pollen Characteristics of MON 87411 Compared to the Conventional Control during 2012

Pollen

Characteristic (unit) Mean (S.E.) 1
Reference Range 2

MON 87411 Control

Viability 3 (%) 98.7 (0.43) 98.9 (0.21) 99.0

– 99.2

Diameter 4 (μm) 87.0 (1.02) 88.9 (1.29) 80.4

– 88.9

Note: The experimental design was a randomized complete block design with four replications. No significant differences were detected between the MON 87411 and the conventional control (α=0.05) using ANOVA

. 1 MON 87411 and the conventional control values represent means with standard error (S.E.) in parentheses. n =4

2 Reference range is the minimum and maximum mean value observed among the four reference hybrid s.

3 Evaluated from three sub

- samples per replication at 100X magnification

. 4 Evaluated from 1 0 representative viable pollen grains per replication at 200X magnification

.

VI I I. D

. Conclusions for Phenotypic, Agronomic, and Environmental Interactions

Evaluation

Comparative plant characterization data between a biotechnology

- derived crop and the conventional control are interpreted in the context of contributions to increased plant pest potential as assessed by USDA

- APHIS. Under the framework of familiarity,

char acteristics for which no differences are detected support a conclusion of no increased plant pest potential of the biotechnology

- derived crop compared to the conventional crop. Ultimately, a weight

- of

- evidence approach that considers all characteristics a nd data is used for the overall risk assessment of differences and their significance.

An extensive and robust set of agronomic, phenotypic, and environmental interactions data, including specific weedy and plant pest potential characteristics, were used to assess
whether the introduction of insect

- protected and glyphosate

- tolerant traits altered the plant pest potential of MON 87411 compared to the conventional control, considered within the context of the variation among commercial reference hybrids . T hese

assessments included five general data categories: 1) seed germination, dormancy, and emergence; 2) vegetative growth; 3 ) reproductive development ( including pollen characteristics); 4) lodging and seed retention on the plant; 5) plant response to abi otic

stress and interactio ns with diseases and arthropods

. Within these data categories, data

relevant to underst an d ing specific characteristics associated with weediness w ere also assessed to determine whether there was a potential increase in weediness of MON 87411 compared to conventional maize

.Monsanto Company CR240

- 13U1 163 of 374 Results from the se assessments comparing MON 87411 and the conventional control

demonstrate that MON 87411 does not possess: 1) i ncreased weediness characteristics; 2) increased susceptibility or tolerance to specific abiotic stress, diseases, or arthropods; or 3) characteristics that would confer a plant pest risk compared to conventional maize

. Therefore, based on the results of multiple assessments discussed above and presented in the appendices , the weight

- of

- evidence indicates that MON 87411 is not meaningfully

different from conventional maize with the exception of insect

- protected and glyphosate

tolerant traits and is not exp ected to pose a plant pest risk compared to conventional

maize

.Monsanto Company CR240

- 13U1 164 of 374 I X

. U.S. AGRONOMIC PRACTICES

I X .A

. Introduction

As part of the plant pest assessment required by 7 CFR § 340.6(c)( 4), impacts of deregulation on agricultural and cultivation practices must be considered. This section provides a summary of current agronomic practices in the U.S. and North America for producing maize, and is included in this petition as a baseline to a ssess possible impacts to agricultural practices due to the cultivation of MON 87411. Discussions include maize production, plant growth and development, general management practices during the season, management of weeds, insects and diseases, maize rota tional crops, and volunteer management. Information presented in the previous section demonstrated that

MON 87411 is no more susceptible to diseases or pests than commercially cultivated maize. Additionally, data presented in Section VIII show that MON 8 7411 is not expected to pose a plant pest risk compared to conventional maize. Current CRW

- control products available in the U.S. were grown on over half of the maize acres in 2011 ( Marra

et al. 2012 ) . MON 87411 is protected from CRW feeding using two different modes of action derived from Cry3Bb1 protein and DvSnf7 dsRNA. Thus, there are no expected changes to the inputs needed for MON 87411, and no expected impacts to agronomic practices employed for production of maize compared to the current situation.

I X .B. Over view of U.S. Maize Production

I X .B.1. Maize Production

The U.S., China, Brazil, Argentina, and Mexico are the top five countries pr oducing maize globally ( USDA

- FAS 2013 ) . As noted in Section I.B., maize ( Zea mays L.) is the largest crop grown in the U.S. in terms of acreage planted (97.2 million acres in 2012) ( USDA

- NASS 2013d )

, exceeding soybean and wheat with acreages of 77.2 and 55.7 million acres, respectively ( USDA

- NASS 2013a ) . The value of maize reached $77.4

billion in the United States in 2012, exceeding soybeans and wheat with values of $43.2 and $17.9 billion, respectively ( USDA

- NASS 2013c ) . The principal uses of maize are feed and residual, ethanol fuel, export, and high

- fructose corn syrup ( Capehart, et al.

2012 )

.

The planting of 97.2 million acres of maize in 2012 was the largest acreage since 1936 (Table IX

- 1) ( USDA

- NASS 2013d ) and was up over 5.2 million acres from 2011. Much of that production occurs in upper Midwest states (Figure IX

- 1). Of the 2012 acreage, approximately 87.4 million acres were h arvested for grain and 7.4 milli ( Gassmann, et al.

2012 ) on acres were harvested for silage ( USDA

- NASS 2013d ) . Total production was approximately 10.8 billion bushels with an average yield of 123 bu shels per acre (Table

IX

- 1). The value of maize grain production in the U.S. has ranged from $46.7 to $77.4 billion in the past 6 years (Table IX

- 1) ( USDA

- NASS 2013d )

.Monsanto Company CR240

- 13U1 165 of 374 Table IX

- 1. Maize Production in the U.S., 2007

- 2012

Year Acres

Planted
(×1000) Acres

Harvested
(×1000) Average

Yield
(bushels/acre) Total

Production
(×1000 bushels) Value (billions $)

2012 97,155 87,375 123.4 10,780,296 77.35

2011 91,936 83,989 147.2 12,359,612 76.94

2010 88,192 81,446 152.8 12,446,865 64.64

2009 86,382 79,490 164.7 13,091,862 46.73

2008 85,982 78,570 153.9 12,091,648 49.31

2007 93,527 86,520 150.7 13,037,875 54.67

Source: ( U SDA

- NASS 2013d )

Figure IX

- 1. Planted Maize Acres by County in the U.S. in 2012

Source: ( USDA

- NASS 2012 )Monsanto Company CR240

- 13U1 166 of 374 I X .C. Produ ction Management Considerations

Other than the specific insertion of a partial coding sequence of the DvSnf7 gene that provides another MOA for protection against CRW, MON 87411 is similar to several

other events present in CRW

- protected maize hybrids being grown in the U.S. ( e.g. , Genuity SmartStax, Genuity VT Triple PRO, etc). Herbic id e tolerant maize has been in use commercially in the U.S. since 1997 and constituted 73% of the total maize crop in

2012 ( USDA

- ERS 2013 ) . CRW

- protected maize has been planted commercially since

2003 and in 2010 constituted 53% of the total U.S. cr op ( Brookes and Barfoot 2012 )

. Given the widespread use of these HT and insect

- protected maize hybrids , Monsanto anticip ates no specific production management changes from introduction of

MON 87411 above and beyond those in current use on a majority of U.S. maize acres

.

I X

. D . Management of Insect Pests

Maize is subject to attack by numerous insects and global production l osses from arthropod pests are minimally estimated at 12%, with potential losses being much higher ( Culy 2000 ; Vaadia 1985 ) . Approximately 100 species of insects cause most of the pest damage to maize production in the U.S. ( O'Day, et al. 1998 )

. Certain insect pests are abundant almost every year and tend to be chronic problems while other pest infestations are sporadic and occur only every 5 to 10 years with a severity great enough to reach

economic thresholds ( O'Day et al. 1998 )

. For migratory pests, weather events must favor

the movement into maize

- producing regions. In addition, maize or other suitable host plants must be available and in the preferred growth stage when the insects a rrive and local weather conditions must also be favorable for the insects to survive and reproduce. Some of the most economically important migratory maize pests in the U.S. include black cutworm, fall armyworm and corn earworm ( O'Day et al. 1998 )

. Other economically important pests such as European corn borer and corn rootworm overwinter

in the field ( O'Day et al. 1998 )

. Table IX

- 2 lis ts the most important insect pests in maize

in the North Central states of the U.S.Monsanto Company CR240

- 13U1 167 of 374 Table IX

- 2 . Insect Pests in Maize in the North Central States and Typical Time of Damage

Typical Time of Damage Growth Stage Common Name of Insect Pest

Planting to Full Emergence Planting to V2 Seed corn maggot

Seed corn beetles

Wireworms Emergence to Knee

- High VE to V8 Wireworms White grubs Grape colaspis larva Cinch bugs

Black cutworm

Stalk borer Thrips

Corn leaf beetle

Sod webworms

Southern corn leaf beetle Billbugs Armyworms

Knee

- High to Tasseling V8 to VT Fall armyworm Grasshoppers European corn borer

Southwestern corn borer

Corn rootworm Tasseling to Maturity VT to R6 Corn leaf aphid

Corn earworm

Source: ( Dicke and Guthrie 1988 ; O'Day et al. 1998 )

Insect pests are best managed through the use of integrated pest management (IPM) programs where all viable control strategies are considered and appropriate strategies are

selected for us e against specific insect pests ( Bradley, et al. 2013 ) . Control strategies employed in IPM programs may include chemical, cultural, mechanical, biological and genetic options ( i.e. , use of biotechnology

- derived or naturally occurring resistant or tolerant crop varieties). Almost all maize seed includes a treatment of an insecticide such as thiamethoxam or clothianidin for control or suppression of several seedling pests including w hite grubs, wireworms, seed corn maggots, cinch bugs, black cutworm, and billbugs ( Bradley et al. 2013 ; University of Tennessee 2013 ) . Applications of insecticide

for co ntrol of various maize insect pests has ranged from 12% to 31% of the planted acreage over the past 20 years ( USDA

- NASS 2013b ) In 2010, however, only 12% of planted acres were treated with an insecticide ( USDA

- NASS 2013b ) . Numerous insecticidal active ingredients are registered for use in maize for the control of insect pests. However, only seven insecticide active ingredients (bifenthrin (2%), c hlorpyrifos (1%), cyfluthrin (2%), lambda

- cyhalothrin (2%), permethrin (1%), tebupirimphos (2), and tefluthrin (3%)) represent almost all of the total treated maize acres in the U.S. ( USDA

- NASS 2013b )

. These insecticides are effective in managing insect pests that may cause damage to maize from emergence through maturity (Tabl e IX

- 3 ) ( Bradley et

al. 2013 ) . Other IPM practices that are used include rotation to non

- host crops to break pest life cycles, soil tillage to manage weed, arthropod and disease pes ts, and removal of crop residues that can harbor both arthropod and disease organisms ( Vasileiadis, et al.Monsanto Company CR240

- 13U1 168 of 374 2011 ) . Additionally, some researchers have evaluated and found effective the use of specific beneficial nematodes for biological control of specific maize pests ( Kurtz, et al.

2007 )

.

Table IX

- 3 . Insecticide Applications in Maize in 2010 in the U.S.

Herbicide Chemical Family Mode of Action
(MOA) Percent of

Maize Acres

Treated Quantity
Applied
(1000 lbs ai)

Bifenthrin Pyrethroids

Sodium channel

modulators 2.0 68

**β**

- Cyfluthrin Pyrethroids 2.0 15

Lambda

Cyhalothrin Pyrethroids 2.0 24

Permethrin Pyrethroids 1.0 72

Tefluthrin Pyrethroids 3.0 242

Z

- Cypermethrin Pyrethroids 2

Chlorpyrifos Organophosphates

Acetylcholin

esterase inhibitors 1.0 478

Tebupirimphos Organophosphates 2.0 195

Tebufos Organophosphates 137

Dimethoate Organophosphates 52

Propargite Propargite Inhibitors of

mitochondrial ATP
synthase 109

Spiromesifen Tetronic & tetramic

acid derivatives Inhibitors of acetyl
CoA carboxylase 59

Total 12 1631

Source: ( USDA

- NASS 2013b )

.

The most damaging root

- feeding pests of maize in the major U.S. maize growing regions are larvae of the corn rootworm complex ( CRW: Diabrotica ssp., Coleoptera; Chrysomelidae) ( Chandler et al. 2008 ) . There are three primary species of CRW in the U.S., namely western corn rootworm (WCR: D iabrotica virgifera virgifera ), northern corn rootworm (NCR: Diabrotica barberi ) and southern corn rootworm (SCR: Diabrotica undecimpunctata howardi ) and all are considered major insect pests of maize ( Culy 2000 ) . In the Midwest region, the WCR is the dominant species while the SCR is not considered an economic pest there ( O'Day et al. 1998 )

. Corn rootworms can cause serious injury and yield losses in the following ways: 1) root pruning and tunnelingMonsanto Company CR240

- 13U1 169 of 374 disrupting the transport of nutrients and water from the root system, 2) lack of root support causing gooseneck lodging, which can complicate harv esting, and 3) root feeding promotes invasion by secondary pathogens such as bacteria and fungi, increasing the

incidence of root rots ( O'Day et al. 1998 ) . Yield losses depend on the number of lar vae per plant and on plant maturity, soil fertility, and amount of moisture following peak injury, as well as the ability of the plant to regenerate root tissue ( O'Day et al. 1998 )

. Historically, populations of the NCR and WCR had been estimated to result in annual yield losses and control costs that exceeded $1 billion ( Metcalf 1986 ) . Researchers have reported yield losses of 0.8 to 2.5% per larva per plant ( Petty, et al. 1969 ; Smith 1979 )

. In addition, adult rootworm beetles can cause stripping of the upper layer of tissu e from the leaves and silk clipping, although this rarely has an impact on maize yields ( O'Day et

al. 1998 )

.

In past years, various management practices have been employed to manage CRW pests
incl uding crop rotation, insecticide application at planting to manage larvae, and foliar insecticide application to manage adult beetles. In 2003, maize hybrids containing Bt proteins to protect against CRW feeding became available and have been widely adopt ed on broad acreages in the U.S. since then (Figure IX

- 2). Prior to introduction of these

corn rootworm

- protected Bt hybrids, two variants of WCR and NCR were identified that made crop rotation as a means to manage rootworm populations less effective. The soybean variant (SBV) of the WCR adapted its egg

- laying behavior to lay eggs in crops other than maize, namely soybe an ( Levine and Oloumi

- Sadeghi 1996 ) . The SBV has been reported to occ ur in Illinois, Indiana, Michigan, Ohio, Minnesota, Iowa, and

Missouri ( Gray, et al. 2009 ; Onstad, et al. 1999 ) . The extended diapause variant (EDV) of the NCR has adapted to crop rotations as well. Although most NCR eggs hatch the following spring, some of the eggs of EDV hatch after two winter s and the larval stages

are able to feed on maize roots even in rotated maize ( Krysan, et al. 1984 )

. The EDV variant is most prevalent in portions of South Dakota, Nebraska, Iowa, and Minnesota ( Alston, et al. 2002 ) . While these variant CRW populations do exist, corn rootworm

protected Bt maize products (both single and dual MOA) available to growers have provided and continue to provide excellent control of corn rootworm

- com plex pests on a vast majority of acres.

The introduction of biotechnology

- derived insect

- protected maize has offered growers alternative and highly effective solutions for control of major insect pests in maize. As noted in Section I (Product Rationale), in 2012, approximately 85 million acres in the U.S. (or 88% of the total U.S. maize acreage) were planted with biotechnology

- derived maize hybrids, and approximately 64 million acres (or 67% of the total maize acreage) were planted with maize hybrids cont aining insecticidal crystal (Cry) proteins derived

from Bt ( USDA

- ERS 2013 ) . Of the 97.2 million acres of maize planted in the U.S. in 2012, approximately 50 million contained Bt proteins for protection against corn rootworm (Figure IX

- 2)

.Monsanto Company CR240

- 13U1 170 of 374 Figure IX

- 2. Maize acres in the U.S. planted to hybrids containing Bt proteins for corn rootworm protection

Source: ( Marra et al. 2012 ) (updated with 2012 internal Monsanto data )

MON 87411 was developed to build upon current Bt protein

- based CRW control technology by introducing a new MOA based on RNAi that will continue to effectively

control corn rootworm

- complex pests in maize fields and is expected to prolong the durability of existing Bt technologies. By incorporating two distinct MOAs, MON 87411 is expected to offer increased control of target insect pests and will continue to provide benefits to growers similar to those obtained with the use of existing CRW

- protected maize hybrids. Thus, no changes to agronomic practices are expected related to grower

use of MON 87411.

IX.D.1. Insect Resistance Management

MON 87411 will not be offered for commercial use as a stand

- alone single

- event product

. Thus, no specific insect resistance management (IRM) program is being develope d for MON 87411 alone

. Instead MON 87411 is intended to be commercialized

as combined

- trait breeding stacks with previously deregulated biotechnology

- derived

traits. As these selected insect

- protected combined

- trait products are identified and developed, Monsanto (or other developers) will develop appropriate IRM program(s) for these products and submit them to U.S. EPA as part of its registration package(s). EPA has required IRM proposals for each of the insect

- protected corn products it has previously registered. IRM plans , which are included as conditions of U.S. EPA registration , are designed to delay the development of insect resistance to specific PIPs

and prolong the useful life of these products. 0 10 20 30 40 50 60

2003 2004 2005 2006 2007 2008 2009 2010 2011 2012Millions of Acres

YearMonsanto Company CR240

- 13U1 171 of 374 I X .E. Management of Diseases and Other Pests

Man agement of diseases during maize growth and development is important for protecting the yield of harvested grain. Diseases can cause stand loss, reduce photosynthesis, increase lodging, and lower yields and grain quality ( Mueller and Pope

2009 )

. The incidence of diseases varies from year to year because they ar e highly influenced by climate and other environmental factors. Estimates for annual yield losses because of disease have ranged from 7 to 17% ( Shurtleff 1980 )

. More than 60 diseases occur in maize in the U.S., but m any occur infrequently or are not prevalent enough to

cause measurable loss ( Stuckey, et al. 1993 )

. Maize diseases can be grouped into six categories: 1) seed and seedling diseases, 2) leaf diseases, 3) stalk rots, 4) ear rots, 5) viral diseases, and 6) nematode diseases. Most maize seed is treated with fungicide for the prevention of seed and seedling diseases ( Stuckey et al. 1993 ) . Other diseases can be managed to varying degrees with use of resistant hybrids, crop rotation, tillage, foliar fungicides, and various cultural practices ( Mueller and Pope 2009 ; Stuckey et al. 1993 )

. In addition, several nematode species have been known to cause disease in maize ( Smith

and White 1988 ) . The best methods for management of pest nematodes are primarily crop rotation and stress protection ( Stuckey et al. 1993 )

. O ther than use as part of a seed treatments, fungicides were applied on just 8% of U.S. maize acreage in 2010 ( USDA

NASS 2013b )

. Fungicides currently used on maize plants in the U.S. include azoxystrobin, propiconazole, pyraclostrobin, and trifloxystrobin ( USDA

- NASS 2013b )

.

Environmental observations in field studies have demonstrated no apparent impact of

MON 87411 on diseases of maize (Section VIII). Therefore, no changes in current disease management practices are anticipated from the introduction of MON 87411.

I X

. F

. Weed Management

Weed control in maize is essential to optimizing yield because weeds compete wit h maize for light, nutrients, and moisture and can lead to reductions in yield ( Knake et al.

1990 ) . Weed control the first several weeks after maize emergence is the most critical

per iod to avoid yield losses in maize ( Bosnic and Swanton 1997 ; Carey and Kells 1995 ; Hall, et al. 1992 ) . Some weeds can tolerate cold, wet conditions better than maize, and can gain an advantage prior to planting. Annual weed species such as giant foxtail ( Setaria spp.), barnyardgrass ( Echinochloa crus

- galli (L.) Beauv .) and Palmer pigweed ( Amaranthus spp.) can reduce maize yields by up to 13, 35 and 74%, respectively ( Bosnic and Swanton 1997 ; Fausey, et al. 1997 ; Gianessi, et al. 2002 ; Knake and Slife

1965 ) . In a study of mixed weed populations competing with maize, yields were reduced by up to 20% when the weeds reached a height of eight inches ( Carey and Kells 1995 ; Gianessi et al. 2002 )

.

A survey of Extension Service weed scientists solicited estimates of the percent of maize acreage infested with individual weed species by state or region, as well as the potential impact on maize yields if the species were left uncontrolled. In this survey, twelve annual broadleaf, nine annual grass, and seven perennial species were identified as troublesome weeds (Table IX

- 4) ( Gianessi et al. 2002 ) . Estimates of yield loss ranged from a low of 15% due to wirestem muhly and sandburs to a high of 48% from

burcucumber.Monsanto Company CR240

- 13U1 172 of 374 Table IX

- 4

. Troublesome Weeds in Maize

Weed Species

Annuals

Burcucumber Broadleaves

Cocklebur

Jimsonweed

Kochia Lambsquarters Morningglory Nightshade Pigweeds/Waterhemp Ragweed

, Common Ragweed, Giant

Smartweeds

Velvetleaf

Barnyardgrass Grasses

Bermudagrass Crabgrass spp Cupgrass

, Woolly

Foxtail spp Millet

, Wild

- Pro so Panicum, Fall

Sandburs

Shattercane

Perennials

Bindweed

, Field Dogbane

, Hemp Johnsongrass Muhly, Wire stem Nutsedge

, Yellow Quackgrass

Thistle

, Canada Infestation Area 11

PA/ OH/TN/SE

MW/NP / SE

MW / CO

NP/NW

MW / SE/NE / CA

MW / SE / SP

MW/NP / CA

US

MW / SE/NE

MW/NP

MW/ SD/NE / SE

MW/NE/NP

SP/NW / CA

MD / SE/UT/CA

MW / SE/NE

IA/WI

MW/NE/NP

UT/WY/ CO/ID

MW / SE/NE/NP

NP / UT / WY

MW / SP

ND / SW / CA

IL/MO

MW / SE / SW / CA

PA

MW / SE/NE/NP / CA

MW/NE / UT

NE/MW/NP / CO (%) Acreage Infested

5

- 10

20

- 60

5

- 20

10

- 70

15

- 80

20

- 75

25

- 50

30

- 90

20

- 70

10

- 45

30

- 70

25

- 70

80

- 90

10

- 20

20

- 80

15

- 20

50

- 90

15

- 40

15

- 80

5

- 30

5

- 40

40

- 80

2

- 20

20

- 60

2

10

- 70

10

- 70

5

- 25 Potential Yield

Loss (%)

48

33

17

33

33

33

26

36

30

28

22

28

23

47

29

29

31

31

30

15

33

18

21

45

15

21

27

26 Adap ted from ( Gianessi et al. 2002 )

11 Area Abbreviations:

PA : P e nnsyl v an i a OH : Ohio US: United St a te s MD: Maryland CA: Ca liforni a UT: Utah

TN: Tennessee SE: Southeast WY: Wyoming

MW: Midwest NW: Northwest SW: Southwest

SP : Southern Plains SD: South Dakota ND: North Dakota

NP: Northern Plains NE: Northeast IA : Iowa

WI: Wisconsin MO : Missouri CO: Colorado

ID: IdahoMonsanto Company CR240

- 13U1 173 of 374 Until the early 1950s, tillag e and cultivation practices were primarily used for weed control in maize, but they have been largely replaced by the use of herbicides. Herbicide use in maize became widespread by the end of the 1970s. In 2010, herbicides were applied to 98% of the plan ted maize acreage ( USDA

- NASS 2013b )

. Currently,

g lyphosate is the most w idely applied herbicide in maize followed by atrazine at 78% and 61%, respectively, of planted acreage. The chloracetamide herbicides (long chain fatty acid inhibitors such as acetochlor, s

- metolachlor, dimethenamid, and flufenacet) were applied to 56% of the planted maize acreage in 2010 ( USDA

- NASS 2013b )

.

The introduction of biotechnology

- derived HT maize (like MON 87411) continues to offer growers an effective in

- crop solution for control of weeds. In 2012, approximately 73% of the total maize acreage in the U.S. was planted with hybrids possessing HT traits. Although glyph osate provides effective broad

- spectrum control of numerous annual and perennial weed species, pre

- emergence residual herbicides are also recommended components of weed control programs for Roundup Ready maize ( Monsanto Company

2013 ) . Pre

- emergence residual herbicides provide early season control to reduce e arly weed competition, improve control of certain hard to control broadleaf weed species ( e.g ., morning glory spp.), and can provide control of glyphosate

- resistant weeds.

IX.F.1. Weed Resistance Management

As MON 87411 uses the same gene ( cp4 epsps ) as other Roundup Ready maize products,

no separate or additional recommendations for managing glyphosate

- resistant weeds have been developed for MON 87411. As described in its Technology Use Guide ( Monsanto Company 2013 ) , Monsanto continues to recommend that growers implement diversified weed management pr ograms. Recommendations include starting with clean weed

- free fields, use of a diverse set of weed control tools, including residual herbicides and multiple MOA, use of other cultural practices, as needed, and control/removal of weed

escapes. Guidelines have been developed by a wide variety of stakeholders to help facilitate the effective management of herbicide resistance ( HRAC 2011 )

.

I X

. G . Crop Rotat ion Practices in Maize

Crop rotation is a well

- established farming practice and a useful management tool for maize production. Crop rotations are used to diversify farm income, spread labor requirements throughout the year, and spread the crop loss risk associated with weather and pest damage across two or more crops. In terms of soil and pest management, rotations are used to 1) manage weed, insect, and disease pests, 2) reduce soil erosion by wind and water, 3) maintain or increase soil organic matter, 4) provide biologically fixed nitrogen when legumes are used in th e rotation, and 5) manage excess nutrients ( Singer

and Bauer 2009 ) . Studies in U.S. c orn b elt states indicate maize yield is about 10

- 15% higher in m aize grown following soybean than maize grown following maize ( Singer and

Bauer 2009 )

. Despite the many benefits of crop rotations, crop price fluctu ations, input costs, rental agreements, government price supports, weather, choice of farming system

and on

- farm resources, and other factors all contribute to decisions regarding crop

rotations. Market conditions such as U.S. government

- mandated ethanol use and record high commodity maize prices have increased the demand for maize grain and resulted in increases in maize acreage in recent years ( Singer and Bauer 2009 ; USDA

- NASS 2013d )

.Monsanto Company CR240

- 13U1 174 of 374 Introduction of MON 87411 is not , however, expected to impact crop rotation practices any more so than current CRW

- protected and HT pro ducts available to growers.

I X

. H

. Maize Volunteer Management

Volunteer maize commonly occurs in rotational crops in the season following cultivation of conventional or biotechnology

- derived maize. Viable grain is not produced on the approximately 8% of U .S. maize acres that are cultivated for the production of silage, and volunteer maize plants typically do not occur in the rotational crops that follow maize harvested as silage. In the warmer climates of the Southeast and Southwest, volunteer

maize is ra re because maize grain remaining after harvest is likely to germinate in the fall and the resulting plants can usually be controlled by tillage or by freezing temperatures in

the winter. In northern maize

- growing regions, volunteer maize does not always o ccur in rotational crops because of seed decomposition over the winter, efficient harvest procedures, and tillage prior to planting rotational crops. None of the agronomic or phenotypic data collected for MON 87411 (Section VIII.C.), including data regard ing germination and seed dormancy, indicates that MON 87411 is any more likely to volunteer than existing commercial maize hybrids.

Management of volunteer maize in rotational crops involves minimizing or reducing the potential for volunteers through prac tices that include: 1) adjusting harvest equipment to minimize the amount of grain lost in the field; 2) planting hybrids that reduce the extent of ear drop; 3) choosing hybrids with superior stalk strength and reduced lodging; and 4) practicing no

- till p roduction to significantly reduce the potential for volunteer growth in rotational crops. If volunteer maize does occur in subsequent crops, pre

- plant tillage, in

crop cultivation and the use of selective herbicides are very effective management tools.

I ntroduction of MON 87411 is not expected to impact maize volunteer management practices any more so than current CRW

- protected and HT products available to growers.

I X

. I

. Stewardship of MON 87411

Monsanto develops effective products and technologies that deliver value to growers and conserve resources that agriculture depends on, and is committed to assuring that its products and technologies are safe and environmentally responsible. Monsanto demonstrates this commitment by implementing product stew ardship processes throughout the lifecycle of a product and by participation in the Excellence Through Stewardship ® (ETS) Program ( BIO 2010 )

. ETS policies and practices include rigorous field compliance and q uality management systems and verification through auditing. Monsanto’s Stewardship Principles are also articulated in Technology Use Guides ( Monsanto Company 2013 ) and Monsanto Technology Stewardship Agreements that are signed by growers who utilize Monsanto branded traits, to ensure stewardship compliance

.

As an integral action of fulfilling this stewardship commitment, Monsanto will seek biotechnology regulatory approvals for MON 87411 in all key maize importing countries with functioning regulatory systems to assure global compliance and support the flow of international trade. These actions will be consistent with the Biotechnology Industry Organization Policy on Product Launch ( BIO 2010 ) . Monsanto continues to monitorMonsanto Company CR240

- 13U1 175 of 374 other countries that are key importer s of maize from the U.S., for the development of formal biotechnology approval processes. If new functioning regulatory processes are developed, Monsanto will make appropriate and timely regulatory submissions.

Monsanto also commits to industry best pra ctices on seed quality assurance and control to ensure the purity and integrity of MON 87411 seed. As with all of Monsanto’s products, before commercializing MON 87411 products in any country, the appropriate

detection methods will be made available to ma ize producers, processors, and buyers.

IX. J . Impact of the Introduction of MON 87411 on Agricultural Practices

MON 87411 has been developed to provide two MOA against the primary CRW

complex pests of maize in the corn belt as well as glyphosate tolerance in a single product. As tolerance to glyphosate and protection from corn rootworm complex pests are present in many currently available maize hybrids and are widely grown in the U.S.,

the introduction of MON 87411 is not e xpected to have major impact s on current agronomic, cultivation and management practices for maize. No changes are anticipated in crop rotations, tillage practices, planting practices, fertility management, weed and disease management, and volunteer manag ement from the introduction of MON 87411.

MON 87411 has been shown to be comparable to conventional maize in its compositional, phenotypic, and agronomic characteristics (Sections VII and VIII).

CRW

- protected and glyphosate tolerant maize products have b een widely grown in the

U.S. since 2003 and the introduction of MON 87411, with its new MOA, will improve the durability of CRW

- protection traits and extend the useful lifetime of these products.

Biotechnology

- derived CRW

- protected maize has been cultivat ed and consumed in the U.S. since 2003 and has reduced insecticide applications directed at this pest ( U.S. EPA

2011 ) . MON 87411 introduces a new MOA based on RNAi that offers increased control of CRW pests and is expected to prolong the durability of existing CRW

- control technologies. As such, it will continue to provide benefits to growers similar to those obtained by use of existing CRW

- protected maize hybrids, including reduced use of insecticides, increased yield protection, water conservation, an d increased worker safety.Monsanto Company CR240

- 13U1 176 of 374 X. PLANT PEST ASSESSMENT

This section provides a brief review and assessment of the plant pest potential of

MON 87411 and its impact on agronomic practices and the environment. USDA

- APHIS has responsibility, under the Plant Protection Act (PPA) (7 U.S.C. § 7701

- 7772), to prevent the introduction and dissemination of plant pests into the U.S. Regulation

7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, ther eby allowing unrestricted introduction

of the article.

According to the PPA, the definition of “plant pest” includes the living stage of any of the following that can directly or indirectly injure, cause damage to, or cause disease in any plant or plant pr oduct: (A) a protozoan; (B) a nonhuman animal; (C) a parasitic plant; (D) a bacterium; (E) a fungus; (F) a virus or viroid; or (G) an infectious agent or other pathogen; (H) Any article similar to or allied with any of the articles specified in the precedi ng subparagraphs (7 U.S.C. § 7702[14]).

The regulatory endpoint under the PPA for biotechnology

- derived crop products is not zero risk, but rather a determination that deregulation of the article in question is not expected to pose a greater plant pest ri sk than its unmodified comparator . Information in this petition related to plant pest risk characteristics includes: 1) mode

- of

- action and changes to plant metabolism; 2) composition; 3) expression and characteristics of the gene product; 4) potential fo r weediness of the regulated article; 5) impacts to NTOs; 6) disease and pest susceptibilities; 7) impacts on agronomic practices; and 8) impacts on the weediness of any other plant with which it can interbreed, as well as the potential for gene flow. Usi ng the assessment above, the data and analysis presented in this petition

lead to a conclusion that MON 87411 is not expected to be a plant pest, and therefore should no longer be subject to regulation under 7 CFR § 340.

X

. A . C haracteristics of the Geneti c Insert and Expressed Products

X. A .1. Genetic Insert

As described in Section III, MON 87411 was developed by Agrobacterium tumefaciens

mediated transformation of maize embryos using plasmid vector PV

- ZMIR 10871.

Characterization of the DNA insert in MON 87411 was conducted using a combination of sequencing, PCR, and bioinformatics methods. The results of this characterization

demonstrate that MON 87411 contains one copy of the intended T

- DNA containing the

DvSnf7 suppression cassette and the cry3Bb1 and cp4 epsps expression cassettes that is stably integrated at a single locus and is inherited according to Mendelian principles over multiple generations. These methods also confirmed that no vector backbone or othe r unintended plasmid sequences are present in MON 87411. Additionally, the genomic organization at the insertion site was assessed by comparing the sequences flanking the

T

- DNA insert in MON 87411 to the sequence of the insertion site in conventional maiz e. This analysis determined that no major DNA rearrangement occurred at the insertion site in MON 87411 upon DNA integration.Monsanto Company CR240

- 13U1 177 of 374 X. B . Mode

- of

- Action

As noted in Section I. D .2., MON 87411 contains the same CP4 EPSPS and Cry3Bb1 proteins as those found in MON 88017 maize (APHIS petition No. 04

- 125

- 01p) that was granted non

- regulated status by USDA

- APHIS in 2006 ( USDA

- APHIS 2013 ) . The MOA of the CP4 EPSPS protein, which is found in numerous other approved Roundup Ready crops, is well understood ( Padgette et al. 1996 )

. Similarly, the MOA of Bt proteins such as Cry3Bb1 has been extensively studied and is well understood ( Gill et al.

1992 ; Whalon and Wingerd 2003 )

.

As previously described in Section I.C.1., the RNAi mechanism is a natural process in eukaryotic organisms for the regulation of gene expressio n. Double

- stranded RNA molecules that activate the mechanism are first processed into siRNAs which are then incorporated into multi

- protein RNA

- induced silencing complexes (RISC). These RISCs facilitate target sequence recognition and mRNA cleavage, lead ing to specific suppression of the targeted mRNA. The targeted DvSnf7 is a vacuolar sorting protein in

the ESCRT

- III complex and is involved in sorting transmembrane proteins enroute to lysomsomal degradation. Snf7 is an essential gene in CRW with signif icant roles in intracellular trafficking of proteins and because of its key cellular function, its suppression resulting from the expression of specific DvSnf7 dsRNAs

, results in CRW mortality.

C haracterization of the insecticidal mode of action of DvSnf7 dsRNA has demonstrated

that Snf7 is an essential gene in WCR that plays a significant role in intracellular protein trafficking and its function is consistent with its known function in other eukaryotes ( Bolognesi et al. 2012 ; Ramaseshadri et al. 2013 )

. T he spectrum of activity for DvSnf7

dsRNA has been shown to be narrow and activity is only evident in a subset of beetles within the Galerucinae subfamily of Chrysomelidae within the Order Coleoptera (Section VI.E.) ( Bachman et al. 2013 )

. For the NTO assessment of MON 87411

, in addition to the activity spectrum testing (Sections VI.E. 2 . and VI.E. 3 .), an additional battery of beneficial organisms was tested that included re presentative pollinators ( honey bee ), a detritivore (ear thworm ) and insect predators/parasitoids (ladybird beetle, c arabid beetle,

Orius , parasitic wasp). The testing and assessment for DvSnf7 RNA lead to the

conclusion that no negative impacts to NTOs, including those beneficial to agriculture,

will result fr om the cultivation of MON 87411.

X. C

. Expression and Characterization of Gene Products

X. C .1. Protein Safety and Expression Levels

The safety and expression of the Cry3Bb1 and CP4 EPSPS proteins are detailed in

Section V. Expression levels were determ ined from 19 tissue types from trials conducted in Argentina and are presented in Section V.C. The expression in the various tissues ranged from 3.0 to 460 μg/g dw for Cry3Bb1 and <LOQ to 76 μg/g dw for CP4 EPSPS. These proteins are present at a very sma ll percentage of the total protein in maize seed (no more than 0.004% and 0.002% for Cry3Bb1 and CP4 EPSPS, respectively). Both proteins also have establishe d histories of safe use, having been assessed by USDA, FDA and U.S. EPA on multiple occasions (Sec tion V.E.). Neither protein originates from anMonsanto Company CR240

- 13U1 178 of 374 organism known to be a source of allergens

, a bioinformatic assessment of each shows no shared amino acid sequence similarities to known allergens, and each protein is rapidly digested in a simulated gastric fluid assay (Sections V.D.1 and V.D.2). Taken together, the results of these analyses suppo rt a determination that MON 87411 is no more likely to pose a plant pest risk than conventional maize.

X. C .2 . RNA Safety and Expression Levels

The safety and expression of the DvSnf7 RNA expressed in MON 87411 is detailed in Section VI. Expression leve ls were determined from 19 tissue types from trials conducted in Argentina and are presented in Section VI. C

. The expression in the various tissue types ranged from <LOQ to 213 × 10 - 3 μg/g dw. As RNAi is a naturally occurring, ubituitous process in eukar yotes, there is a long history of safe consumption of the RNA molecules mediating this process

. Further, there is no evidence suggesting that dietary consumption of nucleic acids

, like RNA

, is associated with toxicity or allergenicity ( Petrick et al. 2013 ; U.S. FDA 1992 )

. Total nucleic acid consumption in the human diet

for DNA and RNA is estimated to be 1

- 2 grams per day ( Suchner et al. 2000 ) which equates to an approximate maximal intake of 0.2 g/kg/day fo r a non

- nursing infant (the highest consumer per body weight). Given these parameters, consumption of DvSnf7

RNA from MON 87411 would be extremely low ( ≤ 0.4 ng/kg/day) relative to the total estimated dietary RNA intake. Taken together, the results of th ese assessments, along with the demonstrated specificity of DvSnf7 RNA to CRW and the history of safe consumption of RNA, support a determination that MON 87411 is no more likely to pose a plant pest risk than conventional maize.

X. D . Compositional Chara cteristics

Compositional comparisons based on OECD guidance were presented in Section VII to assess whether levels of nutrients, anti

- nutrients, and secondary metabolites in grain and forage derived from MON 87411 are comparable to levels in the conventio nal control and several reference hybrids for which there is an established history of safe consumption. Nutrients assessed in this analysis included proximates , carbohydrates by

calcula tion, acid detergent fiber , neutral detergent fibe r
, total dietary fi ber, minerals, amino acids, and vitamins. Anti

- nutrients assessed in grain included phytic acid and raffinose. Secondary metabolites assessed in grain included furfural, ferulic acid, and p

coumaric acid. Forage samples were assessed for levels of proxi mates, fiber, minerals (calcium and phosphorus) , and carbohydrates by calculation . In all, 78 different components were assayed (9 in forage and 69 in grain). While 78 components were assayed, 16 of these had more than 50% of observations below the assay LOQ and were excluded from statistical analysis.

A combined

- site analysis of the data was conducted to determine statistically significant

diffe rences (at 5% ) between MON 87411 and the conventional control. The biological significance of difference s from the data w as reviewed using considerations relevant to food and feed safety and nutritional quality. These considerations included: 1) a

determination of the mean differences of nutrient and anti

- nutrient components of

MON 87411 and the conventional control; 2) consideration of the mean differences

between MON 87411 and the conventional control in the context of the range of valuesMonsanto Company CR240

- 13U1 179 of 374 for the conventional control, 3) comparison of the mean differences between MON 87411

and the conventional control to va riation in conventional maize as estimated by in

- study reference hybrid values and assessing whether the mean values for MON 87411 were

within 99% tolerance intervals

, literature values, and /or ILSI Crop Composition Database

values. These evaluations of v ariation within the conventional control and conventional

maize , including reference hybrids, are important as crop composition is known to be influenced by environment and germplasm.

The compositional analysis provided a comprehensive comparative assess ment of the levels of key nutrients, anti

- nutrients, and secondary metabolites in maize grain and forage of MON 87411 and the conventional control. Of the 60 components statisticall y assessed there were no statis tically significant differences in 48 compo nents. Only 12 components (protein, histidine, tyrosine, oleic acid, neutral detergent fiber, copper, iron, manganese, zinc, niacin, vitamin B1 in gr ain and ash in forage) showed significant

difference s between MON 87411 and conventional control. For the se 12 components, the mean difference in component values between MON 87411 and the conventional control was less than the range of the conventional control values and the reference hybrid values. Overall, MON 87411 mean component values were within 99% t olerance

interval s, values observ ed in the literature, and/or ILSI

- CCDB values. These data

indicate that the components with significant differences were not compositionally meaningful from a food and feed safety or nutri ti onal perspective. These results further support the overall conclusion that MON 87411 was not a major contributor to variation in component levels in maize grain and forage and confirmed the equivalence of

MON 87411 to the conventional control in levels of these components.

X. E

. Phenotypic

, Agronomic

, and Environmental Interaction Characteristics

An extensive set of comparative plant characterization data were used to assess whether

the introductio n of the insect

- protection and glyphosate tolerance traits altered the plant

pest p otential of MON 87411 compared to the conventional control (Section VIII). Phenotypic, agronomic, and environmental interaction characteristics of MON 87411 were evaluated and compared to those of the conventional control

. As described below,

these asses sments included: seed dormancy and germination characteristics; agronomic

and plant phenotypic characteristics; observations for abiotic stress response, disease damage, arthropod

- related damage, arthropod abundance, and pollen characteristics.

Results fr om all phenotypic, agronomic, and environmental interaction assessments

demonstrated that MON 87411 does not possess weedy characteristics, or increased susceptibility or tolerance to specific diseases, insects, or abiotic stressors compared to

the convent ional control. Taken together, the results of the analysis suppor t a
determination that MON 87411 is no more likely to pose a plant pest risk than

conventional maize.

X. F

. W eediness Potential of MON 87411

Maize is not listed as a weed in the major weed r eferences ( Crockett 1977 ; University of

Montana 2011 ) , nor is it present on the lists of noxious weed species distributed by the federal government (7 CFR § 360). In addition, maize has been grown throughout the world without any report that it is a serious weed. During domestication of maize, traitsMonsanto Company CR240

- 13U1 180 of 374 often associated with weediness, such as, seed dormancy, a seed dispersal mechanism, or the ability to form reproducing populations outside of cultivation, have not been selected. Even if individual kernels of maize were distributed within a field or along tra nsportation routes from the fields to storage or processing facilities, sustainable volunteer maize populations are not found growing in fence rows, ditches, or road sides. Maize is poorly suited to survive without human assistance and is not capable of s urviving as a weed ( Galinat 1988 ; Keeler 1989 )

.

In c omparative studies between MON 87411 and a conventional control, phenotypic, agronomic and environmental interaction data were evaluated (Section VIII) for changes that would impact the plant pest potential, in particular, plant weediness potential.

Resul ts of these evaluations show that there is no fundamental difference between

MON 87411 and the conventional control for traits potentially associated with weediness. Furthermore, comparative field observations between MON 87411 and its conventional

contro l and their response to abioti c stressors indicated no differences and, therefore, no increased weediness potential. Collectively, these findings support the conclusion that

MON 87411 has no increased weed potential compared to conventional maize and it i s no more likely to become a weed than conventional maize.

X. F

. 1. Seed Dormancy and Germination

A comparative assessment of seed germination and dormancy characteristics was

conducted on MON 87411 and the conventional control. The results of this assess ment, particularly the lack of increased hard seed, and no changes in other germination and dormancy characteristics, support the conclusion that the introduction of MON 87411 is not expected to result in increased plant pest/weed potential compared to con ventional

maize.

X. F

. 2. Plant Growth and Development

Evaluations of plant growth and development characteristics in the field are useful for assessing potential weediness characteristics such as stalk and root lodging. Phenotypic

characteristics such as early stand count, days to 50% pollen shed and silking, stay green, ear height, plant height, dropped ears, stalk lodged plants, root lodged plants, final stand count, grain moisture, test weight, and yield were assessed. In the combined

- site analysis

in which the data were pooled among the sites, no statistically signif icant differences

were detected between MON 87411 and the conventional control for any of the assessed

characteristics

. Thus, the phenotypic and agronomic characteristics of MON 87411 wer e not altered in terms of pest/weed potential compared to conventional maize

.Monsanto Company CR240

- 13U1 181 of 374 X. F .3 . Pollen Morphology and Viability

Evaluations of pollen morphology and viability from field

- grown plants provide useful information in a plant pest assessment as it relates to the potential for gene flow to, and possible introgression of a biotechnology

- derived trait into sexually

- compatible plants

and wild relatives. No statist ically significant differences were detected between

MON 87411 and the conventional contro l for percent viable pollen or pollen grain

diameter . Furthermore, no visual differences in general pollen morphology were

observed between MON 87411 and the conventional control . Based on the assessed characteristics, the results support a conclusion th at neither pollen viability nor morphology of MON 87411 were altered compared to conventional maize

.

X. G . Impact to Non

- Target Organisms

, Including Those Beneficial to Agriculture

Information presented previously in S ection s V.E. and VI.E. is relevant to the plant pest

risk assessment for MON 87411 because it describes the spectrum of activity for the CP4 EPSPS protein, the Cry3Bb1 protein, and the DvSnf7 RNA. According to 7 CFR Part 340.6, this information is part of the required information needed for evaluation of plant pest potential. Given that the safety of CP4 EPSPS and Cry3Bb1 proteins as well as the

CRW

- resistance and glyphosate

- tolerance traits have previously been evaluated by both

USDA

- APHIS in regulatory submissions for MON 863 and MON 88017 ( USDA

- APHIS

2013 ) and EPA ( U.S. EPA 2010 ) , the primary focus of t he NTO section (Section VI.E.)

w as on DvSnf7 RNA.

The NTO assessmen t for MON 874 11 has taken into consideration the MOA s , the spectrum of insecticidal activity, and exposure levels to the CP4 EPSPS and Cry3Bb1 proteins and the DvSnf7 RNA produced by MON 87411 . The data, analyse s and assessments presented support a conclu sion that MON 87411 will not negatively impact NTOs, including those beneficial to agriculture, and does not present a n increased plant pest risk compared to conventional maize.

X. G .1. Impact on Threatened and Endangered Species

Threatened and endangere d species risk assessments were previously conducted by

USDA ( USDA

- APHIS 2013 ) and EPA ( U.S. EPA 2010 ) for Cry3Bb1

- containing maize products MON 8 63 and MON 88017, and indicat ed no direct or indirect effects to threatened or endangered species

.

Because of the specificity of the DvSnf7 RNA for coleopteran species within the family Chrysomelidae ( Bachman et al. 2013 )

, endan gered species concerns for this trait are properly focused on the order Coleoptera. Currently , there are 18 listed Coleoptera on the threatened and endangered species list , however none of these species are members of the family Chrysomelidae ( USFWS 2013a )

. Additionally

, the listed threatened or endangered coleopteran species are not expected to be in or near maize fields a s many of the endangered and threatened coleopteran species occur in caves or aquatic habitats ( U.S. EPA 2010 )

.Monsanto Company CR240

- 13U1 182 of 374 Threatened and endangered coleopteran species in the U.S. have restrictive habitat

ranges, which generally do not include maize fields ( U.S. EPA 2010 )

. However, some endangered and threatened coleopteran habitats may occur near agricultural production

areas. An analysis of the county level distributions of threatened or endangered terrestrial coleopteran spe cies indicates that the potential concern regarding range overlap with maize production was restricted to the American burying beetle ( Nicrophorus

americanus ). The American burying beetle is the largest carrion beetle in North America ( USFWS 1991 ) and is only found in limited areas encompassing parts of 9 12

USFWS 2013b states, including Arkansas, Kansas, Massachusetts, Nebraska, Oh io, Oklahoma, Rhode Island, South Dakota, and Texas ( ) . Adults feed on carrion and occasionally

other insects ( USFWS 1991 ) , while larvae feed exclusively on buried carr ion or carrion regurgitations provided by their parents ( U.S. EPA 2010 ; USFWS 2008 ) ). The American burying beetle’s habitat is variable and includ es mature forests, shrub

- covered areas, and some grassland habitats and the beetle’s preferred habitat has been correlated with an

abundance of small vertebrate biomass ( USFWS 2008 ) . Considering that both larvae and adult beetles are carrion feeders, exposure to the DvSnf7 RNA in MON 87411 is highly unlikely due to their fee ding ecology.

There are several threatened and endangered coleopteran species that occur in aquatic

habitat s . As discussed above, however, exposure of aquatic organisms to biotech nology

derived crops has been shown to be limited temporally and spatially and the U.S. EPA

has previously concluded that potential exposure of aquatic organisms is low to negligible ( U. S. EPA 2010 )

.

Due to the lack of proximity to maize cultivation, lack of relevant exposure because of feeding ecology and the restricted activity of the DvSnf7 RNA , it is concluded that

cultivation of MON 87 4 11 will have no e ffect on threatened and endangered species, including coleopteran species.

X. H . Environmental Fate of CRW Products Expressing DvSnf7_968

The two components of the insecticidal activity of MON 87411 are the Cry3Bb1 protein and the DvSnf7_968 RNA. The environmental safety of the Cry3Bb1 protein has been demonstrated in multiple environmental studies and assessed extensively as part of many previous EPA product registrations, e.g. , MON 863, EPA Reg. # 524

- 528; MON 863 x MON 810, EPA Reg. # 524

- 545; MON 88017, EPA Reg. # 524

- 551, MO N 88017 x MON 810, EPA Reg. # 524

- 552 and MON 89034 x MON 88017, EPA Reg. # 524

- 576 among others ( U.S. EPA 2010 ) . Thus, the current environmental assessment pertains exclusively to the RNA component of the insecticidal activity, DvSnf7_968.

Soil organisms may be exposed to DvSnf7_968 by contact with roots and with above

ground plant residues or pollen reaching t he soil

. Soil microorganisms may be exposed to DvSnf7_968 by degrading living or decaying maize biomass or by ingestion or absorption of the dsRNA after its release into the soil. Assessment of t he environmental

12 An experimental population that is listed as non

- essential exists in Missouri ( USFWS 2013a )

.Monsanto Company CR240

- 13U1 183 of 374 fate of DvSnf7_968 and any other nucleic a cid (DNA or RNA) from biotechnology

derived crops should consider the amount of nucleic acids already in the environment

fro m conventional sources. Thousands of tons of nucleic acids are released into the environment every year from conventional plant biomass ( roots, leaves, pollen, etc.), in

addition to the nucleic acid contribution of other decaying animal and microbial matter ( Dale, et al. 2002 )

. It is unlikely that nucleic acids originating from b iotechnology

derived plants will persist in soil or interact differently than nucleic acids from non

engineered organisms , or persist by being incorporated into microbes via soil uptake ( Dale, et al. 2001 )

.

There are currently no published laboratory or field monitoring studies that directly

address the persistence or accumulation of RNA in maize fields. However, a laboratory soil study of DNA that encodes recombinant neomycin phosphotransferase II (rNPT

- II), a marker used in early plant genetic engin eering, showed rapid dissipation of the target

DNA ( Widmer, et al. 1996 ) . The study showed that after the rNPT

- II DNA w as added to soil by incorporating ground tissue of transgenic tobacco containing the rNPT

- II sequence and by addition to soil of the purified plasmid DNA, both the tissue

- derived

rNPT

- II sequence and the plasmid DNA degraded rapidly. Only a small proporti on of the DNA was detectable for longer periods. Up to 0.08% of the plasmid DNA was detectable at 40 days, and up to 0.14% of tissue

- derived DNA sequence was detectable for 120 days.

X

. I

. Potentia l for Pollen M ediated Gene Flow and Introgression

Pollen mediated gene flow is a process whereby one or more genes successfully integrate into the genome of a recipient plant. Introgression is affected by both biotic and abiotic factors such as plant biology, pollen biology/volume, plant phenology, overl ap of flowering times, proximity of the pollen source and sink, ambient conditions such as temperature and relative humidity, and field architecture. Because gene introgression is a natural biological process, it does not constitute an environmental risk in and of itself. Gene introgression must be considered in the context of the transgenes inserted into the biotechnology derived plant, and the likelihood that the presence of the transgenes and their subsequent transfer to recipient plants will result in increased plant pest potential. The potential for g ene introgression from MON 87411 is discussed below

.

X. I .1. Hybridization with Cultivated Maize

Maize morphology fosters cross pollination

, therefore, high levels of pollen mediated gene flow can occur in this species. In addition, researchers recognize that (1) the amount of gene flow that occurs can be high because of open pollination; (2) the percent gene flow can vary by population, hybrid or inbred; (3) the level of gene flow decreases with greate r distance between the source and recipient plants; (4) environmental factors affect the level of gene flow; (5) maize pollen is viable for a short period of time under field conditions; (6) maize produces ample pollen over an extended period of time; and, (7) maize is almost exclusively wind pollinated.

Based on several studies conducted on the extent of pollen mediated gene flow between maize fields, results were found to vary depending on the experimental design,Monsanto Company CR240

- 13U1 184 of 374 environmental conditions, and detection m ethod, as expected. In general, the percent of gene flow diminished with increasing distance from the source field, generally falling below 1% at distances >200 m (~660 feet) (Table X

- 1). This information is useful for managing gene flow during maize bree ding, seed production, identity preservation or other applications; in addition, it forms the basis for the USDA

- APHIS performance standards for maize. All testing and production of regulated MON 87411 seed or grain

have been conducted under USDA notificat ion according to these standards. Gene flow from fields planted with MON 87411 to other maize would not be of concern because of the lack of potential to cause harm to humans and to the environment.Monsanto Company CR240

- 13U1 185 of 374 Table X

- 1. Summary of Published Literatur e on Maize Cross Pollination

Pollinator

Distance (m) Reported Outcrossing (%) Comments Country Reference

0 28.6 Frequencies by distance investigated. Three year study. Single male and female. Pollen source was a yellow

dent and the female was a white sweet corn.. USA ( Jones and Brooks 1950 )

25 14.2

75 5.8

125 2.3

200 1.2

300 0.5

400 0. 2

500 0.2

1

- Frequencies by distance investigated. Single yellow sweet corn hybrid was used as a pollen source and as pollen recipient. To measure levels of outcrossing, pollen recipient plants were detasseled. UK ( Haskell and Dow 1951 )

3.6 95

4.8

-

6

-
7.3

-
8.5

-
9.8

-
11

-
12

-
13.4

-

18 10Monsanto Company CR240

- 13U1 186 of 374 Table X

- 1 (continued). Summary of published literature on maize outcrossing

Pollinator

Distance
(m) Reported
Outcrossing
(%) Comments Country Reference

1 2.25 Dispersal of maize pollen investigated. Single hybrid.
Gene flow decreased with greater distance from the source.

Closer correlation of number of plants with gene flow than

physical distance. Data reported in this table represent

means from two of four fields Brazil ( Paterniani and

Stort 1974 ) 10 0.02

20 0.008

30 0.005

34 0.003

2

- 4 0.01 Gene flow in isolated and crossing blocks was evaluated.
Two year study. Single male and female. Bt female hybrid

was detasseled. Mexico ( Garcia, et al.

1998 )

30 1.04 Frequencies by distance investigated. Two year study.
Single RR male and non

- RR female. Data reported in this
table represent one of two years. USA ( Jemison and
Vayda 2001 ) 40 0.03

350 0Monsanto Company CR240

- 13U1 187 of 374 Table X

- 1 (continued). Summary of published literature on maize outcrossing

Pollinator

Distance
(m) Reported
Outcrossing
(%) Comments Country Reference

100 0.01 Frequencies by distance and pollen viability investigated.
Two year study. Single male and female. A purple gene
marker was utilized to measure pollen mobility. Pollen
viability lasted one hour in the driest

- hottest year and two

hours in the most humid, less hot year. Mexico ( Luna, et al. 2001 )

150

-
200 0.01

300

-
400

-

1 30

- 40 Frequencies by distance investigated. Two sites/one year.
Six hybrid pairs. Six Bt and six near isogenic non

- Bt
hybrids. Hybridization was assessed by measuring the
expression of Bt gene in kernels collected from
neighboring plants. Alternatively, sampled kernels were
grown and seedlings tested for expression of Bt gene. Data
reported in this table represent estimates from a graph. USA ( Chilcutt and

Tabashnik 2004 ) 3 18

- 22

8 9

- 12

16 3

- 5

24 0

- 2

32 2

- 4

1 9.7

- 19.0 Frequency by distance investigated. Three year, three sites.
Single male and female/location. Canada ( Ma, et al. 2004 )

5 1.3

- 2.6

10 0.7

- 2.0

14 0.3

- 0.6

19 0.4

24 0

- 0.3

28 0.1

- 0.5

33 0

- 0.3

36 0

- 0.1Monsanto Company CR240

- 13U1 188 of 374 Table X

- 1 (continued). Summary of published literature on maize outcrossing

Pollinator

Distance
(m) Reported
Outcrossing
(%) Comments Country Reference

200 0.03 Detasseling efficiency on pollen containment investigated.
Four inbreds were used as a source of pollen; yellow
inbred, two GM inbreds ( Bt and RR) and an IT
(imidazolinone tolerant) inbred. The recipient pollen traps

were two white inbreds and a male

- ster ile hybrid. Two
year/three locations study. USA ( Stevens, et al.

2004 ) 300 0.02

24

- 32 0.01

- 0.7 Frequencies by distance investigated. Single male and 7
females with different RM used. The male parent source of
pollen contains the genetic markers P1

- rr and R1

- nj. When
male pollen pollinated female yellow plants a purple
coloration occurred in the fertilized yellow kernels. Two
year/two site study. Data reported in this table represent

results from one site. USA ( Halsey, et al.

2005 ) 60

- 62 0.01

- 0.2

123

- 125 0.001

- 0.08

244

- 254 0

- 0.02

486

- 500 0

- 0.005

743

- 745 0

- 0.002

1.8 1.0

- 2.5 Isolation distance investigated. The objectives were (i) to
evaluate current industry isolation practices to produce
hybrid seed that meets higher levels of genetic purity and

(ii) to identify practices that will improve reproductive
isolation in hybrid seed fields. Three year/315 fields.
Multiple hybrids from 24 seed companies tested. Data
reported in this table represent estimates from a graph. USA ( Ireland, et al.

2006 ) 9.4 1.2

- 2.5

20.6 1.0

- 2.2

35.8 0.5

- 2.3

200 0.6

- 1.4Monsanto Company CR240

- 13U1 189 of 374 Table X

- 1 (continued). Summary of published literature on maize outcrossing

Pollinator

Distance
(m) Reported
Outcrossing
(%) Comments Country Reference

1 17.0

- 29.9 Frequencies of cross

- pollination by distance investigated.
Pollination was quantified by measuring out

- crossing from
a transgenic hybrid plot into a conventional grain
production field. A combination of three marker genes was

utilized to detect outcrosses: y1 (seed color gene), Bt and
RR. Two years/two sites. Single male and female. USA ( Goggi, et al.

2006 )

10 1.5

- 2.5

35 0.4

100 0.03

- 0.05

150 0.01

- 0.03

200 0.007

- 0.03

250 0.002

- 0.03

0 < 0.9% at

distances < 20

m Efficiency of border rows and isolation distance on cross

pollination investigated. Available datasets were utilized

to make predictions for reducing out

- crossing to levels

below 0.9%. USA ( Gustafson, et al.

2006 )

4.6

18.3

0 3

- 13 Frequencies of cross

- pollination with a PCR based method
investigated. The main objective of the study was to
compare a PCR based method to real cross

- fertilization

rates as determined by phenotypic analysis. Four Bt
hybrids and a single non

- Bt hybrid we re used as a male and
female respectively. One year/one site. Spain ( Pla, et al. 2006 )

2 0.2

- 10

5 0.1

- 2.3

10 0.2

- 3.7

20 0.1

- 0.8

40 0

- 0.7

80 0.1

- 0.2Monsanto Company CR240

- 13U1 190 of 374 Table X

- 1 (continued). Summary of published literature on maize outcrossing

Pollinator

Distance
(m) Reported
Outcrossing
(%) Comments Country Reference

0 10.5 Frequency of cross pollination (expressed as %GM DNA)
by distance investigated. The study was conducted in large
farm scale evaluation (FSE) across the UK. Data reported

here are maximum raw values UK ( Weekes, et al.

2007 ) 2 34.9

5 9.9

10 12.2

15 0.5

20 8.2

25 4

40 3.7

50 5.9

70 0.13

75 0.28

80 0.12

100 2.3

120 0.16

142 0.06

147 0

150 5.4

160 0

200 0.24Monsanto Company CR240

- 13U1 191 of 374 Table X

- 1 (continued). Summary of published literature on maize outcrossing

Pollinator

Distance
(m) Reported
Outcrossing
(%) Comments Country Reference

52 0.009 Cross

- pollination investigated using occurrence of yellow

kernels in 13 white maize fields. In no case, the cross

pollination of the whole field was > than 0.02%. In every

field some cross

- pollination with a low rate, on an average
of 1.8% of the sampled ears, could be found. These

p ollinations were mostly single cross

- pollinations on the

ear. Switzerland ( Bannert and
Stamp 2007 ) 85 0.015

105 0.003

125 0.01

149 0.016

150 0.007

200 0.009

287 0.005

371 0.008

402 0.005

458 0.0002

4125 0.006

4440 0.0005

1 42.2 Frequencies of cross

- pollination by distance investigated.
The pollen source was a stacked RR/ Bt yellow hybrid. The
recipient was a nontransgenic white hybrid. Higher
outcrossing detected when white hybrid used detasseled. USA ( Goggi, et al.

2007 ) 10 6.3

35 1.3

100 0.1

12 4.2 Frequency of cross pollination and coexistence by distance
investigated. Two crops were used as barriers to determine

their usefulness as buffer crops in maize. Three genetic
markers to measure outcrossing were used: GM Bt maize, a

kernel color maize and a molecular marker test EU ( Langhof, et al.

2008 ) 12 11.7

12 3.8Monsanto Company CR240

- 13U1 192 of 374 X. I .2. Hybridization with Wild Annual Species of Zea mays subsp. mexicana

For gene flow to occur by normal sexual transmission, the following conditions must exist: (1) the two parents must be sexually compatible; (2) there must be overlapping flowering times ; and (3) a suitable factor (such as wind or insects) must be present a nd capable of transferring pollen between the two parents.

Maize and annual teosinte ( Zea mays subsp. mexicana ), are genetically compatible, wind

pollinated and teosinte pollen can pollinate maize silks when in close proximity to each

other

, e.g. in areas of Mexico and Guatemala ( Wilkes 1972 )

. Maize crosse s with teosinte; however, teosinte is not present in the U.S. other than as an occasional botanical garden specimen or small feral populations in Florida, Alabama and Maryland. In experimental studies where maize and teosinte species were planted together , very low hybridization rates were observed for maize and Zea mays subsp. mexicana ( Baltazar, et

al. 2005 ; Ellstrand, et al. 2007 )

. Differ ences in factors such as flowering time, geographical distribution , and development factors make natural crosses in the U.S. highly unlikely.

X. I

. 3. Hybridization with the Wild Perennial Species of Subgenus Tripsacum

In contrast with maize and teosinte, w hich hybridize under certain conditions, it is only with extreme difficulty and special techniques that maize and the related perennial species, Tripsacum dactyloides (gamma grass) hybridize. Furthermore, hybrids of the cross are male sterile, even after several backcrosses to maize ( Russell and Hallauer

1980 )

.

A single species, Tri psacum floridanum (Florida gamma grass), found in the extreme

southern Florida counties of Miami

- Dade, Collier and Monroe has been categorized as a threatened species by the state of Florida and listed on the USDA Natural Resources

Conservation Service (USDA

- NRCS) database ( USDA

- NRCS 2012 )

. Another species, Tripsacum dactyloides (Eastern gamma grass), found primarily throughout the eastern U.S., has been categorized as endangered in Massachusetts and Pennsylvania , and as

threatened in New York ( USDA

- NRCS 2013 )

. However, given the level of difficulty for natural hybridization between species of Tripsacum and Zea as mentioned above, the

occurrence of T. floridanum primarily in both highly urbanized and non

- agricultural, swampy areas of the state where commercial maize is not typically grown, as well as the preference of T. dactyloides for wet habitats where hybrid maize production would not occur, it is very unlikel y there would be any impact on this species due to the

introduction of MON 87411

.

X. J

. Transfer of Genetic Information to Species with which Maize Cannot Interbreed (Horizontal Gene Flow)

Monsanto is aware of no reports confirming the transfer of genetic material from maize to other species with which maize cannot interbreed. The probability for horizontal gene flow to occur is judged to be exceedingly small. Even if it were to occur, the consequences would be negligible since the CP4 EPSPS and Cry3Bb1 proteins producedMonsanto Company CR240

- 13U1 193 of 374 in MON 87411 are the same proteins as in commercial MON 863 and MON 88017 maize products and have been shown to have no meaningful toxicity to humans and NTOs.

Similar to the situation with transfer of the protein genes, the likelihood of horizontal transfer of the partial DvSnf7 gene from MON 87411 is also exceed ingly small . T he consequence of such transfer , given the known lack of toxicity to all but a very small

subset of organisms (CRW species), would also be expected to be inconsequential. In either case, the presence of these genes would not be expected to increase the pest potential of the recipient species.

X. K . Potential Impact on Maize Agronomic Practices

An assessment of current maize agronomic practices was conducted to determine whether

the cultivation of MON 87411 has the potential to impact current maize management practices (Section IX). Maize fields are typically highly managed agricultural areas th at are dedicated to crop production. Other than the specific insertion of the partial DvSnf7 gene that provides another MOA for protection against CRW, MON 87411 is similar to many other maize hybrids being grown in the U.S. ( e.g. , Genuity SmartStax, Genu ity VT Triple PRO, etc). Herbic id e tolerant maize has been in use commercially in the U.S. since 1997 and constituted 73% of the total maize crop in 2012 ( USDA

- ERS 2013 )

. CRW

- protected maize has been planted commercially since 2003 and in 2010 constituted 53% of the total U.S. crop ( Brookes and Barfoot 2012 ) . CRW

- protected maize hybrids have been grown o n tens of millions of acres in the U.S. since their introduction. Given the widespread use of these HT and insect

- protected maize hybrids , Monsanto anticipates no specific changes to agronomic practices from introduction of MON 87411 above and beyond thos e in current use.

MON 87411 is similar to commercially cultivated maize in its agronomic, phenotypic, ecological, and compositional characteristics, and has levels of resistance to insect pests and diseases comparable t o other commercially cultivated maiz e

. Based on this assessment, the introduction of MON 87411 is not like ly to impact current U.S. maize agronomic or cultivation pract ices or lead to an increased plant pest potential compared to other maize hybrids widely available to growers

.

X. L

. Conventional Breeding with Other Biotechnology

- derived or Conventional

Maize

Numerous biotechnology

- derived maize products have been deregulated or are under consideration for deregulation by APHIS

. Once deregulated, MON 87411 may be bred

with these d ereg ulated maize products, as well as with conventional maize , creating new improved hybrids

. APHIS has determined that none of the individual biotechnology

derived maize products it has previously deregulated displays increased plant pest

characteristics. A PHIS has also concluded that progeny derived from crosses of these deregula ted maize products with conventional or previously deregula ted maize are unlikely to exhibit new plant pest properties. This presumption, that combined

- trait biotechnology products are unlikely to exhibit new characteristics that would pose n ew plant pest risks not observed in the single event biotech product, is based upon several

facts. Namely: 1) stability of the genetic inserts is confirmed in each approved biotech

-Monsanto Company CR240

- 13U1 194 of 374 derived maiz e product across multiple genera tions ; 2) stability of each of the introduced traits is continually and repeatedly assessed as new combined

- trait hybrids are created by plant breeders and tested over multiple seasons prior to commercialization; 3) combined

trait products are developed using conventional breeding that has been safely used for thousands of years to generate new varieties ( Steiner, et al. 2013 ; WHO 1995 ) ; 4) worldwide organizations, such as World Health Organization, Food and Agriculture Organization/ World Health Organization, International Seed Federation, CropLife International and U.S. FDA, conclude that the safety of the combined

- trait product can be based on the safety of the parental GE events ( CLI 2005 ; FAO

- WHO 1996 ; ISF 2005 ; WHO 1995 ) ; and 5) practical applications in the field have shown that two unr elated biotechnology traits combined together by conventional breeding do not display new characteristics or properties distinct from those present in the single event biotech products ( Pilacinski, et al. 2011 )

.

Therefore, based on the considerations above and the conclusion that MON 87411 is no more likely to pose a plant pest risk than commercially cultivated maize

, it can be concluded that any progeny derived from crosses between MON 87411 and conventional maize or deregulated biotechnology

- derived maize are no more likely to pose a plant risk than commercially cultivated maize.

X

. M

. Su mmary of Plant Pest Assessments

A p lant pest, as defined in the PPA, is the living stage of any of the following, or a similar article, that can directly or indirectly injure, damage, or cause disease in any plant or plant product: (A) a protozoan; (B) a nonhuman animal; (C) a parasitic plant; (D) a bacterium; (E) a fung u s; (F) a virus or viroid; (G) an infectious agent or other pathogen

,

or (H) a ny article similar to or allied with any of the articles specified in the preceding subparagraphs (7 U.S. C. § 7702[14]). Characterization data presented in Sections I V through VII I and Section X of this petition confirm that MON 87411 , with the exception

of protection from CRW larval feeding and glyphosate tolerance , is not fundamentally

different from conve ntional maize , in terms of plant pest potential. Monsanto is not aware of any study results or observations associated with MON 87411 that would suggest an increased plant pest risk would result from its introduction.

The plant pest assessment was based on multiple lines of evidence developed from a

detailed characterization of MON 87411 compared to conventional maize , followed by a risk assessment on detected differences. The plant pest risk assessment in this petition was based on the following lines of evidence: 1) insertion of a single functional copy of

the Cry3Bb1 and CP4 EPSPS genes and DvSnf7 suppression casse tte; 2) characterization

and safety of the expressed product s ; 3) compositional equivalence of MON 87411 forage and grain compared to a conventional control; 4) phenotypic

, agronomic , and

environmental characteristics demonstrating no increased plant pest potential compared

to conventional maize ; 5) negligible risk to NTOs

, including organisms beneficial to agriculture; 6) familiarity with maize as a cultivated crop and 7) no greater likelihood to impact agronomic practices

, cultivation practices, or the ma nagement of weeds, diseases and insects, than conventional maize

.Monsanto Company CR240

- 13U1 195 of 374 Based on the data and information presented in this petition, it is concluded that, like

conventional maize and previously deregulated biotechnology

- derived maize

, MON 87411 is not expecte d to be a plant pest. Results also support a conclusion of no increased weediness potential of MON 87411 compared to conventional maize

. Therefore, Monsanto Company requests a determination from USDA

- APHIS that

MON 87411 and any progeny derived from cros ses between MON 87411 and other

commercial maize be granted nonregulated status under 7 CFR part 340.Monsanto Company CR240

- 13U1 19 6 of 374 X I . AD VER SE CONSEQUENCES OF INTRODUCTION

Monsanto knows of no study results or observations associated with MON 87411 indicating that there would be adverse consequences from its introduction. MON 87411 produces the Cry3Bb1 and CP4 EPSPS proteins and the DvSnf7 RNA

. The Cry3Bb1

and CP4 EPSPS pr otein s produced in MON 87411 are identical to the same protein s

presen t in MON 88017 that was previously granted a determination of nonregulated

statu s by USDA

- APHIS

. The unique DvSnf7 RNA in MON 87411 has been thoroughly characterized and its safety has been thoroughly assessed in this submission. As demonstrated by field results and laboratory tests, the only phenotypic difference s between MON 87411 and conventional maize are protection from CRW larval feeding

and glyph osate tolerance

.

The data and info rmation presented in this pet ition demonstrate that MON 87411 is unlikely to pose an increased plant pest risk compared to conventional maize. This conclusion is reached based on multiple lines of evidence developed from a detailed

characterization of the product compared to conventional maize, followed by risk

assessment on detected differences . The characterization evaluations included molecular analyses, which confirmed the insertion of one copy of the intended DNA containing the DvSnf7 suppression cas sette and the cry3Bb1 and cp4 epsps expression cassettes that is stably integrated at a single locus and is inherited according to Mendelian principles over multiple generations.

A nalysis of key nutrients, anti

- nutrients, and secondary meta bolites of MON 87411

demonstrate that MON 87411 is compositionally equivalent to conventional maize. The phe notypic evaluations , including an assessment of seed germination and dormancy characteristics, plant growth and development characteristics, pollen characteristi cs, ecological interaction characteristics, and environmental inter actions also indicated

MON 87411 is unchanged compared to conventional maize. There is no indication that

MON 87411 would have an adverse impact on beneficial or non

- target organisms, including th reatened or endangered species . Therefore, based on the lack o f increased pest potential compared to conventional maize, the risks for humans, animals, and other

NTO s from MO N 87411 are neglig ible

.

The introduction of MON 874 11 will not adversely impact cultivation practices or the management of weeds, diseases, and insects in maize production systems. Farmers

familiar with CRW

- protected maize products ( e.g ., Genuity VT Tripl e Pro or Genuity SmartStax) currently available w ill be advised to continue to employ the same crop rotational practices, weed control practices and/or volunteer control measures c urrently in place for these products.Monsanto Company CR240

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- 8828.Monsanto Company CR240

- 13U1 213 of 374 APPENDICESMonsanto Company CR240

- 13U1 214 of 374 Appendix A: USDA Notifications and Permits

Field trials of MON 87411 have been conducted in the U.S. since 2010 . The protocols for these trials include field performance, breeding and observation, agronomics, and generation of field materials and data necessary fo r this petition. In addition to the

MON 87411 phenotypic assessment data, observational data on pest and disease stressors were collected from these product development trials. The majority of the final reports have been submitted to the USDA. However, some final reports, mainly from the 2012

- 2013 seasons, are still in preparation. A list of trials conducted under USDA notifications or permits and the status of the final reports for these trials are provided in Table A

- 1.Monsanto Company CR240

- 13U1 215 of 374 Table A

- 1. USDA Notificatio ns and Permits Approved for MON 87411 and Status

of Trials Planted under These Notifications

USDA No. Effective date Release State (# of sites) Trial Status

2010 Field Trials

10

- 050

- 101n 21

- Mar

- 2010 IL (4) Submitted

10

- 050

- 105n 23

- Mar

- 2010 IA (4) Submitted

10

- 054

- 141n 25

- Mar

- 2010 OH (1) Submitted

10

- 081

- 101n 16

- Apr

- 2010 HI (1) Submitted

2011 Field Trials

11

- 014

- 104n 13

- Feb

- 2011 HI (2) Submitted

10

- 351

- 113rm 15

- Feb

- 2011 HI (1) Submitted

11

- 034

- 104n 02

- Mar

- 2011 IA (10) Submitted

11

- 034

- 102n 02

- Mar

- 2011 IL (12) Submitted

11

- 034

- 107n 02

- Mar

- 2011 OH (6) Submitted

11

- 034

- 105n 02

- Mar

- 2011 KS (6) Submitted

11

- 039

- 101n 09

- Mar

- 2011 IA (2), IL (4), NE (1) Submitted

11

- 041

- 107n 11

- Mar

- 2011 KS (5) Submitted

11

- 045

- 106n 13

- Mar

- 2011 IA (7) Submitted

10

- 351

- 117rm 15

- Mar

- 2011 IL (1), IN (1) Submitted

11

- 045

- 103n 16

- Mar

- 2011 IL (8) Submitted

11

- 045

- 109n 16

- Mar

- 2011 NE (1) Submitted

11

- 047

- 102n 17

- Mar

- 2011 OH (4) Submitted

11

- 056

- 102n 27

- Mar

- 2011 IA (2), IL (2), NE (1) Submitted

11

- 062

- 101n 01

- Apr

- 2011 IN (1) Submitted

11

- 152

- 104n 01

- Jul

- 2011 PR (1) Submitted

11

- 123

- 104rm 01

- Sep

- 2011 HI (2) Submitted

11

- 265

- 104n 24

- Oct

- 2011 HI (2) Submitted

11

- 292

- 101n 18

- Nov

- 2011 HI (1) Submitted

11

- 322

- 102n 18

- Dec

- 2011 HI (1) Submitted

11

- 322

- 101n 18

- Dec

- 2011 HI (1) Submitted

2012 Field Trials

12

- 006

- 101n 05

- Feb

- 2012 GA (1) Submitted

11

- 291

- 111rm 15

- Feb

- 2012 IA (4), IL (1), IN (1 ), KS (3) Submitted

11

- 305

- 105rm 01

- Mar

- 2012 HI (3) Submitted

12

- 038

- 103n 08

- Mar

- 2012 IA (1) Submitted

11

- 320

- 105rm 15

- Mar

- 2012 IA (5), IL (3), NE (4), SD (1) Submitted

12

- 051

- 101n 20

- Mar

- 2012 IA (2), NE (3) Submitted

12

- 051

- 102n 20

- Mar

- 2012 IA (1), IL (9) Submitted

12

- 051

- 103n 20

- Mar

- 2012 IA (4), IL (4) SubmittedMonsanto Company CR240

- 13U1 216 of 374 Table A

- 1 (continued). USDA Notifications and Permits Approved for MON 87411

and Status of Trials Planted under These Notifications

USDA No. Effective date Release State (# of sites) Trial Status

12

- 051

- 104n 20

- Mar

- 2012 IA (4), KS (2) Submitted

11

- 326

- 108rm 22

- Mar

- 2012 IA (11), IL (20) Submitted

12

- 059

- 120n 28

- Mar

- 2012 IL (1), NE (1) Submitted

12

- 059

- 109n 28

- Mar

- 2012 HI (1) Submitted

12

- 060

- 110n 30

- Mar

- 2012 IA (1), IL (1), IN (1 ) Submitted

12

- 061

- 105n 31

- Mar

- 2012 IA (1), IL (1), NC (1), NE (1), PA (1) Submitted

12

- 062

- 111n 01

- Apr

- 2012 IA (1), IL (1), IN (1 ), KS (1) Submitted

12

- 065

- 109n 04

- Apr

- 2012 IL (2), NE (1) Submitted

12

- 074

- 110n 13

- Apr

- 2012 IA (1) Submitted

12

- 075

- 114n 14

- Apr

- 2012 IA (1), IL (1), NE (1), SD (1) Submitted

12

- 116

- 105n 07

- May

- 2012 CO (2), IA (2), IL (1), KS (5), NE (1), SD (1) Submitted

12

- 143

- 104n 20

- Jun

- 2012 HI (4), PR (1) Submitted

12

- 125

- 106rm 01

- Sep

- 2012 HI (3) Submitted

12

- 251

- 101n 07

- Oct

- 2012 AR (1), IL (1), KS (1), NE (1) Submitted

12

- 312

- 103n 07

- Dec

- 2012 HI (2) In Progress

2013 Field Trials

12

- 320

- 125rm 07

- Mar

- 2013 KS (32), NE (20) In Progress

13

- 044

- 101rm 07

- Mar

- 2013 HI (2), PR (2) In Progress

12

- 320

- 109rm 08

- Mar

- 2013 IA (32), NE (7), SD (10), TN (3) In Progress

12

- 312

- 109rm 15

- Mar

- 2013 HI (8), PR(2) In Progress

12

- 320

- 114rm 15

- Mar

- 2013 IA (1), IL (37), IN (3), KS (10), MS (1) In Progress

13

- 039

- 102n 15

- Mar

- 2013 HI (11), PR (3) In Progress

13

- 051

- 101n 22

- Mar

- 2013 IA (8), IL (23), KS (12), NE (7) In Progress

13

- 052

- 105n 23

- Mar

- 2013 IA (2), IL (1), MN (12), SD (1) In Progress

13

- 053

- 109n 24

- Mar

- 2013 IA (8), IL (8), MS (1), NE (4), SD (4) In Progress

13

- 053

- 110n 24

- Mar

- 2013 IA (8), IL (8), MS (1), NE (4), SD (4) In Progress

13

- 059

- 103n 30

- Mar

- 2013 IA (5), IL (5), NE (4) In Progress

13

- 064

- 123n 04

- Apr

- 2013 IA (3) In Progress

13

- 066

- 105n 06

- Apr

- 2013 AR (1), CA (1), IA (5), IL (10), IN (3), KS (3), LA (1), MI (1), MO (3), MS (3), NC (2), NE (5), OH (1), PA (2), TX (2), WI (1) In ProgressMonsanto Company CR240

- 13U1 217 of 374 Table A

- 1 (continued). USDA Notifications and Permits Approved for MON 87411

and Status of Trials Planted under These Notifications

USDA No. Effective date Release State (# of sites) Trial Status

13

- 066

- 109n 06

- Apr

- 2013 IA (11), IL (6), NE (4), SD (5) In Progress

13

- 119

- 103n 29

- May

- 2013 HI (11), PR (3) In Progress

13

- 191

- 102n

13

- 301

- 101n 09

- Aug

- 2013

04

- Dec

- 2013 GA (1) HI (8), PR (3) In Progress In ProgressMonsanto Company CR240

- 13U1 218 of 374 Appendix B : Materials

, Methods , and Supplementary Results for Molecular Analyses of MON 87411

B.1. Materials and Methods

B.1.1 Test Substance

The test substance in this study was MON 87411 . Genomic DNA for use in this study

was extracted from seed tissue listed in the table below.

Generation Seed Lot Number

R 4 11308817

R 4 F 1 11320173

R 5 11326313

R 5 F 1 11333176

R 6 11338858

B.1.2 Control Substance

The control substances were the conventional maize lines which have similar genetic background as the generations as shown in the table below. Genomic DNA for use in

this study was extracted from seed tissue listed in the table below.

Control Substance Generations Seed Lot Number

LH244 R 4 , R 5 , R 6 11264747

LH244 × HCL645 R 4 F 1 (hybrid) 11320031

LH244 × LH287 R 5 F 1 (hybrid) 11333170

B.1.3 Reference Substance

The reference substance was plasmid vector PV

- ZMIR10871, which was used to develop

MON 87411. Whole plasmid served as a positive control for sequencing and bioinformatic analyses. The identity of the reference plasmid was confirmed by restriction enzyme digestion pri or to the study. Documentation of the confirmation of the plasmid vector identity was archived with the raw data. Appropriate molecular size markers from commercial sources were used for size estimations on agarose gels. The unique identity of the molec ular weight markers was documented in the raw data.

B.1.4 Characterization of Test, Control, and Reference Substances

The seed for the test and control substances used in this study were obtained from Monsanto Trait Development. The synthesis records for these materials are located in the ORION system. The identities of the test substance and the conventional control substance were confirmed by the sequencing in the study. No certificates of analysis (COA) or verification of identity (VOI) certificates were generated for these materials.Monsanto Company CR240

- 13U1 219 of 374 The Study Director reviewed the chain of custody documentation to confirm the identity of the test and control substances prior to the use of these materials in the study.

Test, control and reference DNA substance were considered stable during storage if they yielded interpretable signals in sequencing experiments and/or did not appear visibly degraded on the stained gels.

B.1.5 Genomic DNA Isolation

For sequencing library construction and PCR reactions, genomic DNA wa s isolated from seed tissues of the test and control substances. First the seeds were decontaminated by vigorously agitating them by hand for 30 seconds with 0.05% (v/v) Tween

- 20, followed by a tap water rinse. The seeds were then vigorously agitated wit h 0.5% (w/v) NaOCl,

allowed to stand for one minute at room temperature, and rinsed with tap water. The seeds were then vigorously agitated with 1% (v/v) HCl, allowed to stand for one minute at room temperature, and rinsed with tap water. The 1% (v/v) HC l rinse was repeated one time, and then the seeds were rinsed with distilled water and placed in a drying oven

at 75°C

- 80°C to dry. The dried seeds were ground to a fine powder in a Harbil paint shaker. Genomic DNA was extracted using a hexadecyltrimethy lammonium bromide (CTAB) extraction protocol. Briefly, 16 ml CTAB buffer (1.5% (w/v) CTAB, 75 mM Tris HCl (pH 8.0), 100 mM EDTA (pH 8.0), 1.05 M NaCl, and 0.75% (w/v) PVP) and

RNase A was added to ground seed tissue. The samples were incubated at ~65 **°** fo r 60 minutes with intermittent mixing. The samples were cooled to room temperature and subjected to three rounds of chloroform:isoamyl alcohol (24:1) extraction. Approximately 1.6 ml of 10% CTAB solution (10% (w/v) CTAB and 0.7 M NaCl) was

added to the s amples, mixed by inversion and extracted once with chloroform:isoamyl alcohol (24:1). Approximately 15 ml of CTAB precipitation buffer (1% (w/v) CTAB,

50 mM Tris HCl (pH 8.0), 10 mM EDTA (pH 8.0)) was added to the samples, mixed by inversion, and allowed to stand at room temperature for 50

- 70 minutes. Following centrifugation to precipitate the DNA, the samples were dissolved in high salt TE buffer (10 mM Tris HCl (pH 8.0), 1 mM EDTA (pH 8.0), 1 M NaCl). The DNA was precipitated with 3 M sodium acetate ( pH 5.2) and 100% (v/v) ethanol. The DNA pellets were washed with 70% ethanol, air dried and resuspended in TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0).All extracted DNA was stored in a 4°C refrigerator.Monsanto Company CR240

- 13U1 220 of 374 B.1.6 DNA Quantification

PV

- ZMIR10871 DNA and extracted genomic DNA were quantified using a QubitTM Fluorometer (Invitrogen, Carlsbad, CA). For directed sequencing, genomic DNA was quantified using a NanodropTM Spectrophotometer (Thermo Scientific, Wilmington, DE) according to manufacturer’s instructio ns.

B.1.7 Agarose Gel Electrophoresis

After quantification, approximately 0.5

- 1 microgram (μg) of the extracted DNA for NGS/JSA sequencing library construction was run on a 1% (w/v) agarose gel to check the quality.

B.1.8 Shearing of DNA

Approximately 1 μg of DNA from the test, control and reference substances were sheared using a Covaris S

- 220 ultrasonicator. The DNA was diluted to ~ 18

- 20 ng/μl in Buffer EB (Qiagen Inc., Valencia, CA) and fragmented using the following settings to create approximately 325 bp fragments with 3' or 5' overhangs: duty cycle of 10; peak incident power of 175; intensity of 5.0, 200 bursts per cycle, in the frequency sweeping mode at ~

6°C for 80 seconds for test and control DNA or 60 seconds for reference DNA.

B.1.9 Bioanaly zer Analysis

One microliter of sheared genomic DNA was diluted 1:10 in Buffer EB and run on a DNA High Sensitivity chip on an Agilent 2100 Bioanalyzer to check the quality of the shearing. After preparing the chip according to the manufacturer’s instructi ons, 1 μl of each diluted DNA sample or water was added to individual wells and the chip was run on the Bioanalyzer using the dsDNA, High Sensitivity Assay reagents.

B.1.10 Paired End Library Preparation

Paired end genomic DNA libraries were prepared for the test, control, and reference substances using the Illumina TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions for the low

- throughput procedure with the following exception: a Sage Science Pippin Pre p DNA Size Selection system (Sage Science Inc., Beverly, MA) was used to size select the DNA fragments instead of agarose gel electrophoresis.

First, the 3' and 5' overhangs of the DNA fragments generated by the shearing process

were converted into blunt e nds by adding 10 μl of Illumina Resuspension Buffer and

40 μl of Illumina End Repair mix to each sample and mixing thoroughly by pipette.

Then the libraries were incubated for 30 minutes at 30°C. The end

- repaired samples were purified using AMPure XP bead s (Beckman Coulter, Inc., Brea, CA) and resuspended in 17.5 μl of Illumina Resuspension Buffer.Monsanto Company CR240

- 13U1 221 of 374 Fifteen microliters of each library was transferred to a new tube for adenylation, which adds a single adenosine nucleotide to the 3' ends of the blunt fragmen ts. Then 2.5 μl of Illumina Resuspension Buffer and 12.5 μl of Illumina A

- Tailing Mix were added to each library and mixed thoroughly by pipetting. The libraries were incubated for 30 minutes at 37°C. After incubation, 2.5 μl each of individual adapter index, Illumina Resuspension Buffer, and Illumina DNA Ligase Mix was immediately added to each tube, and mixed thoroughly by pipetting to begin ligation of each library. The libraries were incubated for

10 minutes at 30°C. Then 5 μl of Illumina Stop Liga se Buffer was added to each tube and mixed thoroughly by pipetting to stop the ligation reaction. Next, another

AMPure XP bead cleanup was performed on the libraries which were then resuspended

in 32.5 μl of Illumina Resuspension Buffer prior to size sele ction.

The libraries were run on the Sage Science Pippin Prep Size Selection system using

2% gel cassettes according to the manufacturer’s instructions. Ten microliters of loading

solution were added to 30 μl of each of the purified libraries and mixed th oroughly by pipetting. Forty microliters of Marker B was loaded in the cassette well designated for the reference sample, and 40 μl of each DNA library was loaded in the remaining wells for analysis. After elution of the desired size range (~445 bp) of D NA fragments, the DNA sample in the elution chamber of the cassette was removed from the cassette by pipette and transferred into PCR strip tubes.

After removal from the Pippin Prep, the libraries were again put through the AMPure XP bead cleanup procedu re and resuspended in 22.5 μl of Illumina Resuspension Buffer. Twenty μl of the resuspended library was added to five microliters of Illumina PCR

Primer Cocktail and 25 μl of Illumina PCR Master Mix and mixed thoroughly by pipetting. The DNA fragments we re enriched through PCR using the following cycling conditions: 1 cycle at 98°C for 30 seconds; 10 cycles at 98°C for 10 seconds, 60°C for

30 seconds, 72°C for 30 seconds; 1 cycle at 72°C for 5 minutes. Following PCR amplification, a final AMPure XP bead cleanup was performed on the libraries which were resuspended in 32.5 μl of Illumina Resuspension Buffer. Finally, 1 μl of each DNA library was diluted 1:10 in Buffer EB for running in a DNA High Sensitivity chip on an Agilent 2100 Bioanalyzer as describe d above. All purified library DNA was stored in a

- 20°C freezer.

B.1.11 Next

- Generation Sequencing

The library samples described above were sequenced by The Genome Analysis Center (TGAC, Monsanto) using Illumina HiSeq technology that produces short seque nce reads (~100 bp long). Sufficient numbers of these sequence fragments were obtained (>75x genome coverage) to comprehensively cover the genomes of the test event and the

conventional control (Kovalic et al., 2012) . Sequencing runs performed by the TGA C were assessed versus standard QC criteria.

B.1.12 Junction Sequence Analysis Bioinformatics

High

- throughput sequence reads were enriched by mapping to the PV

- ZMIR10871 transformation plasmid sequence using the local alignment software BlastAll (V2.2.21 ) inMonsanto Company CR240

- 13U1 222 of 374 order to collect all reads that were sourced from the plasmid as well as reads with sequences representing integration point. All collected reads were further refined by removing sequencing artefacts of sequencing adapters, redundant reads and low qu ality read ends. All quality refined reads were then used to identify junction points with custom developed bioinformatics tools as detailed below. All significant junctions are reported for both the test and the control samples. All software versions w ere documented in the archived data package and the software versions which were used in this study have been archived.

B.1.12.1 Sequencing Read Enrichment

The transformation plasmid PV

- ZMIR10871 sequence was used as reference to find all

reads that were either fully matched to the insert plasmid fragments or partially matched with junction sequences. A junction sequence is characterized by a combination of transformation plasmid sequence and flanking sequence that is likely to be host genome flanking seq uence or any other co

- inserted sequence. Local alignment with BlastAll (V2.2.21) was performed to collect all sequencing reads with an e

- Score of less than 1e

- 5 and at least 30 bases match of greater than 96.7% identity to the transformation plasmid (Kova lic et al., 2012) . Both reads of the paired

- end sequences were collected in all cases.

B.1.12.2 Read Quality Refinement

In order to identify all duplicate read pairs, a high quality segment (bases 3

- 42) of all collected pairs was compared to all others with short sequence alignment software (Bowtie v.0.12.3) allowing up to 1 mismatch. If multiple read pairs were matched at both paired reads, such read pairs were deemed redundant and only the best quality pair of reads was kept for further analysis.

Computer software Novoalign (v.2.06.09) was used to remove any adapter sequences at either end of the sequencing reads. Low q uality read ends (with phred scores of 12 or lower) were trimmed. Only reads of 30 bases or longer after adapter and quality trimming were collected. A custom developed Perl script "farm_gen_sm_bucket.pl" was used to perform read enrichment and read qual ity refinement as described above.

B.1.12.3 Junction Detection

Enriched and quality refined reads of both test and control samples were aligned against

the whole PV

- ZMIR10871 transformation plasmid sequence in order to detect junction sequences using cust om developed Perl script "farm_blast_map.pl". Reads with partial match to the transformation plasmid of at least 30 bases match and 96.7% identity were collected as potential junction sequences (Kovalic et al., 2012) . The collected reads were also aligne d against the genomic sequence collection of the host genome in order to remove junction reads sourced from the plant endogenous homologues. Custom developed Perl script “junctions_by_bn.pl” was used to identify the junction position on

the transformation plasmid and their supporting junction reads. For each junction position, all supporting junction reads were aligned at the 30 plasmid bases proximal toMonsanto Company CR240

- 13U1 223 of 374 the junction position. The remaining bases of these reads were sorted to show the alignment and the c onsensus of the flanking junction sequences past the junction point.

B.1.12.4 Effective Sequencing Depth Determination

A single copy locus from the native plant genome (Pyruvate decarboxylase ( pdc3 ), GenBank accession.version: AF370006.2) was selected fro m the Zea mays genome and used to determine the effective sequence depth coverage. All reads with at least 30 bases match and 96.7% identity were considered as reads sourced from this locus. A custom developed Perl script “farm_match_reads.pl” was used t o perform such alignment and calculate the actual depth distribution at this locus.

B.1.12.5 Positive Spike

- in Controls

To produce “spike

- in” positive control samples for sequencing, plasmid DNA libraries

were created as described above (B.1.8

– B.1.10) a nd then diluted to 1 and 1/10 maize genome equivalents (representation of the plasmid DNA at concentrations equivalent to single copy or 1/10 copy per genome) before pooling with samples produced from the control materials (as described above).

B.1.13 PC R and DNA Sequence Analyses to Examine the Insert and Flanking Sequences in MON 87411

Overlapping PCR products, denoted as Product A, Product B, Product C, Product D, Product E, Product F, Product G and Product H were generated that span the insert and

ad jacent 5 ′and 3′ flanking DNA sequences in MON 87411. For each fragment generation experimental conditions were chosen to successfully produce on

- target amplifications. These products were analyzed to determine the nucleotide sequence of the insert in

MON 87411, as well as that of the DNA flanking the 5 ′ and 3′ ends of the insert.

The PCR analyses for Product A, Product D, Product E and Product H were each conducted using 50 ng of genomic DNA template in a 50 **μ** l reaction volume. The

reaction contained a f inal concentration of 0.5 **μ** M of each primer and 1x concentration of Phusion High Fidelity PCR Master Mix with HF Buffer (NEB, Ipswich, MA).

The PCR analyses for Product B, Product C and Product F were each conducted using

50 ng of genomic DNA template in a 50 **μ** l reaction volume. The reaction contained a

final concentration of 0.2 **μ** M of each primer and 1.25 units/reaction of Ex Taq Polymerase (Takara Bio Inc., Shiga, Japan).

The PCR analysis for Product G was each conducted using 50 ng of genomic DNA

temp late in a 20 μl reaction volume. The reaction contained a final concentration of 0.5 μM of each primer and 1x concentration of Phusion High Fidelity PCR Master Mix with HF Buffer (NEB, Ipswich, MA).Monsanto Company CR240

- 13U1 224 of 374 The amplification of Product A, Product D, Product F and Product H were performed under the following cycling conditions: 1 cycle at 98 **°** C for 30 seconds; 25 cycles at 98 **°** C

for 10 seconds, 72 **°** C for 2 minutes 20 seconds; 1 cycle at 72 **°** C for 5 minutes.

The amplification of Product B and Product C were performed un der the following cycling conditions: 1 cycle at 98 **°** C for 10 seconds; 10 cycles at 70 **°** C, decreasing 1 ° C per cycle for 30 seconds, 72 **°** C for 2 minutes; 25 cycles at 98 **°** C for 10 seconds, 60 **°** C for 30
seconds, and 72 **°** C for 2 minutes.

The amplification of Product E was performed under the following cycling conditions: 1 cycle at 98 **°** C for 30 seconds; 25 cycles at 98 **°** C for 10 seconds, 65 ° C for 20 seconds,

72 **°** C for 2 minutes; 1 cycle at 72 **°** C for 5 minutes.

The amplification of Product G was performed under th e following cycling conditions: 1 cycle at 98 **°** C for 30 seconds; 25 cycles at 98 **°** C for 10 seconds, 71 ° C for 20 seconds,

72 **°** C for 2 minutes; 1 cycle at 72 **°** C for 5 minutes.

Aliquots of each PCR product were separated on a 1.0% (w/v) agarose gel and visualize d by ethidium bromide staining to verify that the products were the expected size. Prior to sequencing, each verified PCR product was either purified using the QIAquick PCR Purification Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s inst ructions and quantified using the Nanodrop spectrophotometer according to the manufacturer’s instructions or purified using Exo

- SAP IT (Affymetrix, Santa Clara, CA). The purified PCR products were sequenced using multiple primers, including primers used f or PCR amplification. All sequencing was performed by Monsanto TGAC (The Genome Analysis Center) using BigDye terminator chemistry (Applied Biosystems, Foster City, CA).

A consensus sequence was generated by compiling sequences from multiple sequencing

re actions performed on the overlapping PCR products. This consensus sequence was aligned to the PV

- ZMIR10871 sequence to determine the integrity and organization of the integrated DNA and the 5 ′ and 3′ insert

- to

- flank DNA junctions in MON 87411.

B.1.14 PCR and DNA Sequence Analyses to Examine the Integrity of the DNA

Insertion Site in MON 87411.

To examine the MON 87411 T

- DNA insertion site in control maize, PCR and sequence analyses were performed on genomic DNA from the conventional control maize. The

pr imers used in this analysis were designed from the DNA sequences flanking the insert

in MON 87411. A forward primer specific to the DNA sequence flanking the 5 ′ end of the insert was paired with a reverse primer specific to the DNA sequence flanking the 3 ′

end of the insert.

The PCR reactions were conducted using 50 ng of genomic DNA template in a 50 **μ** l

reaction volume. The reaction contained a final concentration of 0.5 **μ** M of each primer and 1x concentration of Phusion High Fidelity PCR Master Mix with HF Buffer (NEB). The amplification was performed under the following cycling conditions: 1 cycle at 98 **°** CMonsanto Company CR240

- 13U1 225 of 374 for 30 seconds; 25 cycles at 98 **°** C for 10 seconds, 72 **°** C for 2 minutes 20 seconds; 1 cycle

at 72 **°** C for 5 minutes.

A small aliquot of each PCR product wa s separated on a 1.0% (w/v) agarose gel and visualized by ethidium bromide staining to verify that the PCR products were the expected size prior to sequencing. Only the verified PCR product from the conventional control LH244 was either purified using the QIAquick PCR Purification Kit (Qiagen, Inc.) according to the manufacturer’s instructions and quantified using the Nanodrop spectrophotometer according to the manufacturer’s instructions or purified using Exo

SAP IT (Affymetrix, Santa Clara, CA). The pur ified PCR product was sequenced using multiple primers, including primers used for PCR amplification. All sequencing was performed by TGAC using BigDye terminator chemistry (Applied Biosystems).

A consensus sequence was generated by compiling sequences fr om multiple sequencing reactions performed on the verified PCR product. This consensus sequence was aligned

to the 5 ′ and 3′ sequences flanking the MON 87411 insert to determine the integrity and organization of the insertion site.Monsanto Company CR240

- 13U1 226 of 374 B.2 Supplementary Results

Junction Sequence Class A:

**R 4**  Plasmid DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAG CCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA ACTATTCATTAGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA ^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGC CA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTMonsanto Company CR240

- 13U1 227 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGT TTTGCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATC CATGTAGATTTCCCGGACATGAAGCCA^CTGAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATG TAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATC CATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT TTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGGGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCA TTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATG TAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CT TAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC CTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT TAATAAATAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCMonsanto Company CR240

- 13U1 228 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGACTTTTTATTTTCCTTTTAATAAATAATCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAA TAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA ATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT TCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAA TAAATAATCCATCACTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT TGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAA

ATCCATGTAGATTTCCCGGACATG AAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT CCATCACTTTAAATGA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACT ATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT AAATGAACC

**R 4**  **F 1**  Plasmid DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTMonsanto Company CR240

- 13U1 229 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATG AAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

TTCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGTCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGC CA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGA CATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGACTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

AT CCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT GTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTT CCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT

ATCCATGTAGATTTCCCGGACATG AAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT CCATCAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT TCCTTTTAATAAATAATCCATCACTTT

ATCCTTGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTMonsanto Company CR240

- 13U1 230 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC ATTAGTCTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATT TCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAA TAAATAATCCATCACTTTAAATGAAC

**R 5**  Plasmid DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA ACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^C TTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAMonsanto Company CR240

- 13U1 231 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCAT GTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTT TTGC

AGCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAG CCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTGTTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTT

ATCCATGTAGATTTCCCGG ACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA ^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CT TAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACAT GAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCC A^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATMonsanto Company CR240

- 13U1 232 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT TTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCC GGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CT TAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTA GTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGT TTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC ATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAG CCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTATATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT TATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGTCATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

**R 5**  **F 1**  Plasmid DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAMonsanto Company CR240

- 13U1 233 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTAT T

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCC A^CTTAACTATTCATTAGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAG ATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCG GACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCC ATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCGTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC TATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT TTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT TTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT TCCTTTTAATAAATAATCCATCACMonsanto Company CR240

- 13U1 234 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT GTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

**R 6**  Plasm id DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCA TGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCA TT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACAT GAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT CATTAGTMonsanto Company CR240

- 13U1 235 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT CATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAA CTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT TT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTCGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATG TAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATT TCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATN

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATG TAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC CTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTMonsanto Company CR240

- 13U1 236 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC TATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT TTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACAT GAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGATTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCTATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTT TTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATA A

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTT CCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTATATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAG CCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTACTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCC ATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGGAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA

ATCCA TGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA TC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATA AATAATCCATCAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTMonsanto Company CR240

- 13U1 237 of 374 ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTT TTATTTTCCTTTTAATAAATAATCCATCACTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAA CTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCC ATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT TTCCTTTTAATAAATAATCCATCACTTTAAATGA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGG ACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC

Junction Sequence Class B:

**R 4**  Plasmid DNA sequence ^Flank

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTA TCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAG TGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACMonsanto Company CR240

- 13U1 238 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

A GAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTG TTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG AATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AG AGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAG TGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGT TTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC CCT

AGAGCGGCCGCGTTTAAACTATCAGCGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC ACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTMonsanto Company CR240

- 13U1 239 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTT T^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGA TGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAG AACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCG GCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACT

AGAGCGGCCGCGTTTA AACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTT AAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCG TTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGGATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT

AGAGCG GCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT CACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAA TCTACCCTAGAACTAGTTCACTTTTGTGTGCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AG AGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTMonsanto Company CR240

- 13U1 240 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCA TACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACA AACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

**R 4**  **F 1**  Plasmid DNA sequence ^Flank

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGC GTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTACACTATCAGTGTTT^AGAGAATCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGA AGCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATTACAAACCTCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTA

AGAGCGGCTGCGTTTAAACTATCAGTGTTT^AGAGAATCAC AAACCTCTAGATGTAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

A GAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGC GTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTMonsanto Company CR240

- 13U1 241 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTA

AGAGCGGCCG CGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAA TCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AG AGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAAC TATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGCACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTA TCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTA AACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG CA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGA TGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

**R 5**  Plasmid DNA sequence ^Flank

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAG CGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCAMonsanto Company CR240

- 13U1 242 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAAC TATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTG TTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGAT

AGAGCGGCCGCGTTTAAACTATCAGTG TTT^AGAGAATCACAAACCTCTAGAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

A GAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAACTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCG GCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTA TTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTMonsanto Company CR240

- 13U1 243 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACA AACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTA

AGAGCGGCCGCGTTTAAACTA TCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTA CCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT ^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTA CCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTA TCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTA TCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGA GAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACT

AGAGCGGCCGCGTTTAAACTATCAGGGTTT^AGAGAATCACAAACCTCTAGATGAATTAATCTACCCTAGAACTAGTTCACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCA CAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCAC AAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA TCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGMonsanto Company CR240

- 13U1 244 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGT TT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGT TTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG TGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAA TCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC CCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AG AGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT T

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTA TTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTC

AGAGCGGCCG CGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT ACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

**R 5**  **F 1**  Plasmid DNA sequence ^Flank

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAMonsanto Company CR240

- 13U1 245 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGTGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA TCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTAT

AGACCGGCCGCGTTTAAACTACCAGTGTTT^AGAGAATCACAACCCTCTAGATGTATAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AG AGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCNN

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTAT TAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^ AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA ACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCG TTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG TGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC CTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT

**R 6**  Plasmid DNA sequence ^FlankMonsanto Company CR240

- 13U1 246 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCC GCGTTTAAACTATCAGTGTTT^AGAGAATCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA ACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCGCAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAG

AGAGC GGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGAT

AGCGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA ACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTA TCAGTGTTT^AGAGAATCCCAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGT GTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCCAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACMonsanto Company CR240

- 13U1 247 of 374 AGAGCGGCCGCGTTTAAACTATCA GTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGC CGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTAT TAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGGGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTT T^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCG TTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACT

AGAGCGGCCGCGTTTAAACTA TCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATC AGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAA CTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCC GCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT G

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT AGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT AATCTACCCTAGAACTAGTTCACTTTTGTGTGMonsanto Company CR240

- 13U1 248 of 374 AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCTTTTAAACTATCAGTGTTT^AGAGAATCA CAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT AGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA TCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGA TGTACTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCTMonsanto Company CR240

- 13U1 249 of 374 Figure B

- 1. Junction sequences of MON 87411 detected by NGS/JSA

Trimmed nucleotide alignment of the detected junction s equences. The detected junction sequences are ordered by junction sequence class (Kovalic et a., 2012) and then by length. The junction point between the T

- DNA border and flanking sequence is indicated by the “^” character. Both alignments are trimmed t o include only the 30 plasmid bases proximal to the junction point, as well as all sequence of the flank. Both alignments are

shown 5 ′

- >3 ′ beginning with the detected plasmid sequenc e.

References for Appendix B

Kovalic, D., C. Garnaat, L. Guo, Y. Yan, J. Groat, A. Silvanovich, L. Ralston, M. Huang, Q. Tian, A. Christian, N. Cheikh, J. Hjelle, S. Padgette and G. Bannon. 2012. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops i mproved through modern biotechnology. The Plant

Genome Journal 5:149

- 163.Monsanto Company CR240

- 13U1 250 of 374 Appendix C : Materials, Methods and Results for Characterization of Cry3Bb1

and CP4 EPSPS Protein s Produced in MON 87411

C. 1 Characterization of Cry3Bb1 Protein in MON 87411

C.1. 1 Materials for Cry3Bb1 Characterization

The MON 87411

- produced Cry3Bb1protein (lot 11355394) was purified from the grain

of MON 87411 (lot 11330153). The MON 87411

- produced Cry3Bb1 protein was stored

in a

- 80 oC freezer in a buffer solution containing 10 mM sodium carbonate/bicarbonate, 0.1 mM EDTA, pH 10.0.

The E. Coli

- produced Cry3Bb1 protein (lot 11309151) was used as the reference substance. The Cry3Bb1 protein reference substance was generated from cell paste produced by large

- scale fermentation of E. coli containing the pMON70855 expression plasmid. The coding sequence for cry3Bb1 contained on the expression plasmid (pMON70855) was confirmed prior to and after fermentation. The E. coli

- produced Cry3Bb1 protein was characterized previously.

C. 1.2 Cry3Bb1 Protein Purification

The plant

- produced Cr y3Bb1 protein was purified from grain of MON 87411. The purification procedures were not performed under a GLP plan; however, all procedures were documented on worksheets and, where applicable, SOPs were followed. Cry3Bbl protein was extracted from seven batches (~ 3 kg each) of MON 87411 grain with 50 mM sodium carbonate/bicarbonate, pH 10.8, 1 mM EDTA, 1mM Benzamidine HCl, 0.5 mM PMSF, 0.1% Triton X

- 100 (1: 5 (w/v) grain to buffer ratio). The extracts were clarified by centrifugation. The clarified ext ract was brought to 40% saturation by the addition of ammonium sulfate and clarified by centrifugation. The resulting supernatant was brought to 50% saturation by the addition of ammonium sulfate, clarified by centrifugation, and the pellet resuspended in one

- tenth the original volume in Capto Q Buffer A (50 mM sodium carbonate/bicarbonate, pH 10.8, 1 mM EDTA, 1mM Benzamidine HCl, 0.5 mM PMSF). The dissolved pellets were then diluted 1:1 with CaptoQ buffer A and loaded onto a Capto Q (GE Healthcare, Piscat away, NJ) anion exchange column that was pre

equilibrated with CaptoQ buffer A. The column was washed with 20% buffer B (50 mM sodium carbonate/bicarbonate, pH 10.8, 1 mM EDTA, 1 mM Benzamidine HCl, 0.5 mM PMSF, 500 mM NaCl). The Cry3Bbl protein was eluted with 40% buffer B. The eluted protein was diluted 1:1 with 100mM Tris

- HCl pH 8, 1M ammonium sulfate, 1 mM EDTA, 1 mM Benzamidine HCl, 0.5 mM PMSF. The diluted sample was loaded onto a Phenyl Sepharose HP (GE Healthcare) column pre

- equilibrated with Pheny l Sepharose buffer A (50 mM Tris

- HCl pH 8, 0.5 M ammonium sulfate, 1 mM EDTA, 1 mM Benzamidine HCl, 0.5 mM PMSF). The column was washed with Phenyl Sepharose buffer A and then eluted with 38% Phenyl Sepharose buffer B (50 mM Tris

- HCl pH8, 1 mM EDTA, 1 mM B enzamidine HCl, 0.5 mM PMSF). As a final purification step, the Phenyl Sepharose 38% buffer B pool containing the Cry3Bb1 passed at a slow flow rateMonsanto Company CR240

- 13U1 251 of 374 through a PBS equilibrated immunoaffinity column. The column consisted of Protein L Agarose cross

- linked t o an anti

- Cry3Bb1 monoclonal antibody. The bound Cry3Bb1 protein was eluted from the column using 100 mM glycine/150 mM NaCl, pH 2.5. Fractions were collected, immediately neutralized to pH 8.0 using 1 M Tris

- HCl, pH 8.0. Elution fractions were pooled an d concentrated using Spin X spin concentrators (Corning, Corning NY) with a 10K MWCO pretreated with 5% PEG (average molecular weight 3350). This MON 87411 Cry3Bb1 purified from the grain of MON 87411 was aliquoted and stored in a

- 80 °C freezer.

The fina l buffer composition of the sample was 10 mM sodium carbonate/bicarbonate, 0.1 mM EDTA, pH 10.0. The purified MON 87411

- produced Cry3Bb1 protein was aliquoted, assigned lot 11355394 , and stored at in a

- 80°C freezer.

C. 1.3 N

- Terminal Sequencing of Cry3Bb 1

C. 1.3 .1 Methods

N

- terminal sequencing by automated Edman degradation chemistry was carried out in an attempt to confirm the identity of MON 87411

- produced Cry3Bb1

MON 87411

- produced Cry3Bb1 was separated by SDS

- PAGE and transferred to PVDF

membrane. Th e blot was stained using Coomassie Blue R

- 250. The major band s at 77

and ~65 kDa containing MON 87411

- produced Cry3Bb1 w ere excised from the blot and

used for N

- terminal sequence analysis. The analysis was performed for 15 cycles using automated Edman degradation chemistry (Hunkapiller et al., 1983) using an Applied Biosystems 494 Procise Sequencing System equipped with 140C Microgradient system a
Perkin Elmer Series 200 UV/VIS Absorbance Detector with Procise **** Control Software (version 2.1) . Chromatographic data were collected using SequencePro (Applied Biosystems, Foster City CA; version 2.1). A phenylthiohydantoin

- amino a cid (PTH

- AA) standard solution (Applied Biosystems) was used to chromatographically calibrate the instrument for the analysis

. A control protein, **β**

- lactoglobulin, (Applied Biosystems) was analyzed before and after the sequence analysis of the MON 87411

- pr oduced Cry3Bb1 protein to verify that the sequencer met performance criteria for repetitive yield and sequence identity.

C .1

. 3 .2 Results of the N

- terminal Sequence Analysis

N

- terminal sequencing analysis was performed on the MON 87411

- produced Cry3Bb1

p rotein. The reaction did not yield any observable sequence for the

~ 77. 0 kDa band

, presumably because the N

- terminus was blocked. It is well documented that the

N

- terminal residue of many proteins is blocked due to post

- translational modification in

viv o ( Polevoda and Sherman, 2000)

. Blocked N

- terminal amino acids cannot be directly sequenced by Edman degradation.

N

- terminal sequencing of the first 15 amino acids was perfor med on the ~65 kDa band of

MON 87411

- produced Cry3Bb1

. A sequence for the Cry3Bb1 protein deduced from the cry3Bb1 gene present in grain of MON 87411 was observed. The data obtainedMonsanto Company CR240

- 13U1 252 of 374 correspond to the deduced Cry3Bb1 protein beginning at amino acid position

- 50 ( Table

C

- 1). Hence, the sequence information c onfirms the identity of the

~ 6 5 kDa Cry3Bb1

band isolated from the grain of MON 87411

.

Table C

- 1. N

- Terminal Sequence of the MON 87411

- produced Cry3Bb1

MW Expected Sequence Experimental Results Position

77 kDa MANPNNRSEHDTIKV No Sequence Observed

65 kDa TEDSSTEVLDNSTVK TEDSSTEVLDNSTVK 50

- 64 The expected amino acid sequence of the N

- terminus of the ~65 kDa Cry3Bb1 protein was

deduced from t he cry3Bb1 gene present in MON 87411

. The experimental sequences obtained

from the MON 87411

- produced Cry3Bb1 were compared to the expected sequence. The single

letter IUPAC

- IUB amino acid code is A, alanine; D, aspatic acid; E, glutamic acid; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; R, argini ne; S, serine; T, threonine; V, valine

.

C. 1.4 MALDI

- TOF Tryptic Mass Map Analysis of Cry3Bb1

C. 1.4 .1 Methods

MALDI

- TOF MS mass fingerprint analysis was used to confirm the identity of the

MON 87411

- produced Cry3Bb1 protein. A MON 87411

- produced Cry3Bb 1 protein sample was separated by SDS

- PAGE and the gel was stained using Brilliant Blue G

Colloidal stain (Sigma

- Aldrich, St. Louis, MO)

. Each ~77 kDa band was excised and

destained with 40% (v/v) methanol / 10% (v/v) acetic acid. The excised bands were washed in 100 mM ammonium bicarbonate and treated with 10 mM dithiothreitol (DTT)

at 37°C for 2 h followed by incubation for 20 minutes with 10 mM iodoacetic acid in the

dark. The excised bands were then washed with 25 mM ammonium bicarbonate, dried using vacuum centrifugation and rehydrated with 20 μl 20 μg/ml trypsin (Promega, Madison, WI). After 1 h, excess liquid was removed and the excised bands were incubated at 38°C overnight in 40 μl of 10% (v/v) acetonitrile in 25 mM ammonium

bicarbonate. The ex cised bands were sonicated for 5 min and the resulting extract was transferred to new microcentrifuge tube labeled Extract 1 and dried using vacuum centrifugation. The excised bands were then extracted two more times, each with 30 μl

of a 60% (v/v) aceton itrile, 0.1% (v/v) trifluoroacetic acid, 0.1% (v/w) β

- octyl

- glucopyranoside solution and sonicated for 5 min. These two extracts were pooled into a new tube labeled Extract 2 and dried using vacuum centrifugation. Extract

1 and 2 were then treated with 0 .1% trifluoroacetic acid (TFA) and dried. The extracts were solubilized in 50% (v/v) acetonitrile, 0.1% TFA and sonicated for 5 min. Extracts were spotted to wells on an analysis plate and mixed with either 2,5

- dihydroxybenzoic acid (DHB, Thermo Fisher S cientific Inc. Waltham, MA), α

- cyano

- 4

- hydroxycinnamic acid (α

- Cyano, Thermo Fisher Scientific Inc.), or 3,5

- dimethoxy

- 4

- hydroxycinnamic acid (Sinapinic acid, Thermo Fisher Scientific Inc.). The samples in DHB, α

- Cyano, and Sinapinic acid matrix were anal yzed in the 300 to 5000 Da, the 500 to 5000 Da, and 500

to 7000 Da range, respectively. CalMix 2 TM was used as the external calibrant (Sequazyme TM Peptide Mass Standards kit, AB SciEx, Foster City, CA). The analysis was performed on a Voyager TM DE Pro Biospectrometry TM workstation (AppliedMonsanto Company CR240

- 13U1 253 of 374 Biosystems) using Voyager Instrument Control Panel software (version 5.10.2) and Data Explorer data analysis software (version 4.0.0.0). Protonated peptide masses were isotopically resolved in reflector mode (Aebersold 1993; Billeci and Stults 1993)

. GPMAW32 software (Lighthouse Data, Odense M, Denmark) was used to generate an in silico digest of the Cry3Bb1 protein sequence. Masses within 1 Da of the monoisotopic mass were matched against the in silico dige st of the MON 87411

- produced Cry3Bb1 sequence. All matching masses were tallied and a coverage map was generated for the mass fingerprint.

C. 1.4 .2 Results of MALDI

- TOF Tryptic Mass Map Analysis

The identity of the MON 87411

- produced Cry3Bb1 protein was c onfirmed by MALDI

TOF MS analysis of peptide fragments produced by the trypsin digestion of the MON

87411

- produced Cry3Bb1 protein. There were 41 unique peptides identified that corresponded to the expected masses (Table C

- 2 ). The identified masses were u sed to assemble a peptide map of the Cry3Bb1 protein ( Figure C

- 1 ). The experimentally determined coverage of the Cry3Bb1 protein was 73% ( 478 out of 652 amino acids). This analysis further confirms the identity of MON 87411

- produced Cry3Bb1 protein.

As p art of the mass fingerprint analysis, a mass was identified that corresponded to the predicted mass of an acetylated N

- terminal tryptic fra gment. The N

- terminus of the

~ 77. 0

kDa MON 87411

- produced Cry3Bb1 protein begins with alanine at position two.Monsanto Company CR240

- 13U1 254 of 374 Table C

- 2. Summary of the Tryptic Masses Identified for the

MON 87411

- Produced Cry3Bb1 Using MALDI

- TOF MS

Observed Mass 1 Expected Mass Diff. 2 Fragment Sequence 3

564.33 564.31 0.02 45

- 48 EFLR

619.35 619.34 0.01 299

- 303 TELTR

686.45 686.42 0.03 155

- 160 TPLSLR

727.37 727.35 0.02 2

- 7 4 Ac

- ANPNNR

794.44 794.40

794.48 0.04

0.04 394

- 400

292

- 298 LSFDGQK*

LYSKGVK*

877.13 876.47 0.66 161

- 167 SKRSQDR

897.61 897.58 0.03 513

- 520 ITQLPVVK

925.51 925.47 0.04 573

- 580 YASTTNLR

937.56 937.53 0.03 348

- 355 LRPGYFGK

958.58 958.56 0.02 646

- 653 IEFIPVQL

1026.52 1026.49 0.03 258

- 266 GSTYD AWVK

1067.58 1067.52 0.06 181

- 190 NSMP...AVSK

1128.55 1128.56 0.01 377

- 386 TITS...YGDK

1185.67 1185.70 0.03 556

- 566 VTLN...LLQR

1350.67 1350.64 0.03 170

- 180 ELFS...SHFR

1362.72 1362.70 0.02 490

- 500 GTIP...WTHR

1385.68 1385.66 0.02 501

- 512 SDVF.... DAEK

1457.84 1457.74 0.10 404

- 417 TIAN.... PNGK

1460.93 1460.86 0.07 554

- 566 FKVT.... LLQR

1496.82 1496.77 0.05 319

- 331 YGPT.... NSIR

1590.97 1590.91 0.06 641

- 653 IYID..... PVQL

1619.90 1619.83 0.07 168

- 180 IREL.... SHFR

1649.96 1649.84 0.12 626

- 640 NELI.... SNEK

1765.05 1764.98 0.07 304

- 318 DIFT.... TLQK

2001.13 2001.03 0.10 332

- 347 KPHL.... FHTR

2041.22 2041.08 0.14 581

- 597 LFVQ.... YINK

2147.03 2146.93 0.10 472

- 488 AYSH.... MQDR

2298.38 2298.25 0.13 273

- 291 EMTL.... YDIR

2394.23 2394.10 0.13 356

- 376 DSFN.... GSSK

2402.48 2402.35 0.13 191

- 211 FEVL.... LLLK

2483.41 2483.32 0.09 521

- 545 AYAL... LFLK

2484.26 2484.06 0.20 212

- 232 DAQV...

. EFYR

2499.18 2499.08 0.10 425

- 445 VDFS.... YDSK

2552.36 2552.20 0.16 237

- 257 LTQQ.... NGLR

2637.54 2637.29 0.25 131

- 153 ALAE.... NSWK

2655.39 2655.18 0.21 425

- 446 VDFS.....DSKR

2734.57 2734.28 0.29 447

- 471 NNGH.... PLEK

3444.04 3443.64

3443.71 0.40

0.33 15

- 44

404

- 434 VTPN.....LNYK*

TIAN...... DDQK*

3988.68 3988.93 0.25 15

- 48 VTPN..... EFLR

1 The observed mass was collected from at least one of three matrices including **α**

- cyano, DHB and sinapinic acid. The observed mass shown is the mass closest to the expected mass.

2 The data represent the calculated difference between the expected mass and the observed mass

3 For peptide matches greater than nine amino acids in length the first 4 residues and last 4 residues are shown separated by three dots (...)

4 AC is the abbreviati on for acetylation

* The expected peptide masses are nearly identical (< 1 dalton). Because this analysis could not determine with certainty which expected peptide was actually observed, the peptides with an asterisk (*) were not included in determining seq uence coverage (Figure C

- 1).Monsanto Company CR240

- 13U1 255 of 374 001 M ANPNNR SEH DTIK VTPNSE LQTNHNQYPL ADNPNSTLEE LNYKEFLR MT

051 EDSSTEVLDN STVKDAVGTG ISVVGQILGV VGVPFAGALT SFYQSFLNTI

101 WPSDADPWKA FMAQVEVLID KKIEEYAKSK ALAELQGLQN NFEDYVNALN

151 SWK K TPLSLR SKRSQDRIRE LFSQAESHFR NSMPSFAVSK FEVLFLPTYA

201 QAANTHLLLL KDAQVFGEEW GYSSEDVAEF YR RQLK LTQQ YTDHCVNWYN

251 VGLNGLRGST YDAWVK FNRF R R EMTLTVLD LIVLFPFYDI R LYSKGVK TE

301 LTRDIFTDPI FLLTTLQKYG PTFLSIENSI RKPHLFDYLQ GIEFHTRLRP

351 GYFGKDSFNY WSGNYVETRP SIGSSKTITS PFYGDK STEP VQK LSFDGQK

401 VYR TIANTDV AAWPNGK VYL GVTK VDFSQY DDQKNETSTQ TYDSKRNNGH

451 VSAQDSIDQL PPETTDEPLE KAYSHQLNYA ECFLMQDR R G TIPFFTWTHR

501 SVDFFNTIDA EKITQLPVVK AYALSSGASI IEGPGFTGGN LLFLK ESSNS

551 IAK FKVTLNS AALLQR YRVR IR YASTTNLR LFVQNSNNDF LVIYINK TMN

601 KDDDLTYQTF DLATTNSNMG FSGDK NELII GAESFVSNEK IYIDKIEFIP

651 VQL Figure C

- 1. MALDI

- TOF MS Coverage Map of the MON 87411

- produced Cry3Bb1 The amino acid sequence of the MON 87411

- pro duced Cry3Bb1 protein was deduced from t he cry3Bb1 gene present in MON 87411

. Boxed regions correspond to tryptic peptides that were

identified from the MON 87411

- produced Cry3Bb1 protein sample using MALDI

- TOF MS. In total, 73% ( 478 out of 652 amino acids) of the expected protein sequence was identified.

C. 1.5 Western Blot Analysis

- Immunoreactivity of Cry3Bb1

C .1

. 5 .1 Methods

Western blot analysis was performed as follows to confirm the identity of the

MON 87411

- produced Cry3Bb1 protein and to compare the immunoreactivity of the

MON 87411

- produced and E. coli

- produced Cry3Bb1 protein. MON 87411

- produced

and E. coli

- produced Cry3Bb1 protein were diluted in 1× LB ( 62 mM Tris

- HCl, 5% (v/v)

2

- mercaptoethanol, 2% (w/v) sodiu m dodecyl sulfate (SDS), 0.005% (w/v) bromophen ol blue, 10% (v/v) glycerol, pH 6.8) and heated to 100 ° C for 3 min utes . Each protein sample was loaded in duplicate at

~ 1, 2, and 3 ng purity corrected Cry3Bb1 protein per lane onto a pre

- cast Tris

- glycine ( 4

- 20 %) polyacrylamide gradient mini

- gel ( Invitrogen ).

Pre

- stained molecular weight standards (Precis ion Plus Protein Standards

, Bio

- Rad) were loaded on the gel for molecular weight reference and to verify electrotransfer of the proteins to the membrane. Following electrophoresis at a constant voltage, proteins were

electrotransferred to a PVDF membrane (Invitrogen).

The membrane was blocked with 5 % nonfat dry milk (NFDM) in 1 **×** phosphate buffered saline containing 0.05% (v/v) Tween

- 20 (PBST) and incubated with a goat anti

- Cry3Bb1Monsanto Company CR240

- 13U1 256 of 374 antibody (lot G839074) at a dilution of 1:2500 in 1 % NFDM in PBST . After washing with PBST, the membrane was incubated with horseradish peroxidase (HRP)

- conjugated

horse anti

- goat IgG (H+L) IgG (Vector Labs, Burlingame CA) at a dilution of 1:5000 in

1 % NFDM in PBST and washed again, with PBST. Immunoreactive bands w ere detected using the ECL TM detection system (GE Healthcare) and Amersham Hyperfilm (GE Healthcare). The film was developed using a Konica SRX

- 101A automated film processor (Konica Minolta Medical & Graphic, Inc., Tokyo, Japan).

Quantification of the band s on the blot was performed on a GS

- 800 densitometer with the supplied Quantity One **** software (version 4.6.7, Bio

- Rad) using the using the lane selection and contour tool. The signal intensities of the immunoreactive bands migrating at the expected positi on for the Cry3Bb1 protein were quantified as “ contour quantity” values. The immunoreactivity was reported in OD × mm 2

.

C .1

. 5 .2 Results of Cry3Bb1 Protein Immunoreactivity Equivalence

Western blot analysis was conducted using goat anti

- Cry3Bb1 polyclonal antibody as additional means to confirm the identity of the Cry3Bb1 protein isolated from the grain of

MON 87411 and to assess the equivalence of the immunoreactivity of the

MON 87411

- produced and E. coli

- produced Cry3Bb1 proteins.

The results showed that immunoreactive bands migrating at the expected apparent MW were present in all lanes loaded with the MON 87411

- produced (Figure C

- 2 , lanes 10

- 15 )

or E. coli

- produced (Figure C

- 2 , lanes 3

- 8 ) Cry3Bb1 proteins. For each amount loaded, comparable signal inte nsi ty was observed between the MON 87411

- and E. coli

- produced Cry3Bb1 protein bands. As expected, the signal intensity increased with increasing load

amounts of the MON 87411

- produced and E. coli

- produced Cry3Bb1 proteins, thus supporting identification of MON 87411

- produced Cry3Bb1 protein.

To compare the immunoreactivity of the MON 87411

- and the E. coli

- produced Cry3Bb1 proteins, densitometric analysis was conducted on bands that migrated to the expected apparent MW for Cry3Bb1 proteins (~7 7

- 55 kDa). The signal intensity (reported in

OD × mm 2 ) of the i mmunore active bands identified by anti

- Cry3Bb1 antibodies and migrating b etween the full

- length protein at 7 7.0 kDa and the insecticida l tryptic core (at

~55 kDa) were included in the mean signal intensit ies (Table C

- 3 )

. Because the mean signal intensity of the MON 87411

- produced Cry3Bb1 protein band was within 35% of

the mean signal intensity of the E. coli

- produced Cry3Bb1 protein, the

MON 87411

- produced and E. coli

- produced Cry3Bb1 proteins were determ ined to have equivalent immunoreactivity.Monsanto Company CR240

- 13U1 257 of 374 Figure C

- 2. Western Blot Analysis of MON 87411

- and E. coli

- p roduced Cry3Bb1

Proteins Aliquots of the MON 87411

- produced Cry3Bb1 protein and the E. coli

- produced Cry3Bb1 protein were subjected to SDS

- PAGE and electrotransferred to a PVDF membrane. Proteins were detected using anti

- Cry3Bb1 antibodies as the primary antibodies. Immunoreactive bands were visualized using HRP

- conjugated secondary antibodies and an ECL system. The

MWs (kDa) of the standards are s hown on the left. Lane 1 and 2 were cropped from the image. The 15 second exposure is shown. Lane designations are as follows:

Lane Sample Amount (ng)

1 Precision Plus Protein Standards Dual color

2 Blank

3 E. coli

- Produced Cry3Bb1 protein 1

4 E. coli

- Produced Cry3Bb1 protein 1

5 E. coli

- Produced Cry3Bb1 protein 2

6 E. coli

- Produced Cry3Bb1 protein 2

7 E. coli

- Produced Cry3Bb1 protein 3

8 E. coli

- Produced Cry3Bb1 protein 3

9 Blank

10 MON 87411

- produced Cry3Bb1 protein 1

11 MON 87411

- produced Cry3Bb1 protein 1

12 MON 87411

- produced Cry3Bb1 protein 2

13 MON 87411

- produced Cry3Bb1 protein 2

14 MON 87411

- produced Cry3Bb1 protein 3

15 MON 87411

- produced Cry3Bb1 protein 3 kDa

10 15 20 25 37 50 75 100 150 250 Lane 3 4 5 6 7 8 9 10 11 12 13 14 15Monsanto Company CR240

- 13U1 258 of 374 T able C

- 3. Comparison of Immunoreactive Signal Between MON 87411

- and E

coli

- produced Cry3Bb1 Proteins

Mean Signal intensity from

MON 87411

- produced Cry3Bb1 1

(OD x mm 2 ) Mean Signal intensity from

E. coli

- produced Cry3Bb1 1

(OD x mm 2 ) Acceptance limits 2 for

MON 87411

- produced Cry3Bb1 (OD x mm 2 )

7.35 7.02 4.56

- 9.48

1 Each value represents the mean of six values (n=6)

2 The acceptance limits are for the MON 87411

- produced Cry3Bb1 protein and are based on the interval
between +35% (7.02 **×** 1.35) and

**−** 35% (7.02 **×** 0.65) of the mean of the E. Coli

- produced Cry3Bb1 signal intensity across all loads.

C .1

. 6 Molecular Weight Estimation of Cry3Bb1 using SDS

- PAGE

C .1

. 6 .1 Methods

MON 87411

- produced and E. coli

- produced Cry3Bb1 protein were diluted in 1× LB and

heated to 95

- 105 °C for 3

- 5 min. The MON 8 7411

- produced Cry3Bb1 protein was loaded in duplicate at 1, 2, and 3 μg onto a pre

- cast Tris

- glycine ( 4

- 20 %) polyacrylamide gradient mini

- gel ( Invitrogen ) . The E. coli

- produced Cry3Bb1 protein was loaded at 1 μg total protein in a single lane. Broad Ran ge Molecular Weight Standards (Bio

- Rad) were prepared and loaded on the gel in parallel. Following electrophoresis at a constant voltage, proteins were briefly fixed in 40% (v/v) methanol, 7% (v/v) acetic acid and

stained for 17 h ours with Brilliant Blue G

- Colloidal stain (Sigma

- Aldrich, St. Louis, MO). Gels were briefly destained in 10% (v/v) acetic acid , 25% (v/v) methanol followed by 6.5 h ours in 25% (v/v) methanol. Analysis of the gel was performed using a Bio

- Rad

GS

- 800 densitometer supplied with Qu antity One software (4.6.7). Apparent MW was reported as an average of a ll six lanes containing the MON 87411

- produced Cry3Bb1 protein.

C .1

. 6 .2 Results of Cry3Bb1 Protein Molecular Weight Equivalence

The intact MON 87411

- p roduced Cry3Bb1 protein (Figure C

- 3 , lanes 3

- 8) migrated to the same position on the gel as the E. coli

- p roduced Cry3Bb1 protein (Figure C

- 3 , lane 2) and the apparent MW was calculated to be 77. 0 kDa (Table C

- 4 ). Because the experimentally determined apparent MW of the MON 87411

- produced Cry3Bb1 protein was within the acceptance li mits for equivalence, the MON 87411

- and E. coli

- produced Cry3Bb1 proteins were determined to have equivale nt apparent molecular weights.

Table C

- 4. Molecular Weight Comparison Between the MON 87411

- a nd

E. coli

- produced Cry3Bb1 Proteins

Apparent MW of

MON 87411

- Produced Cry3Bb1Protein (kDa) Apparent MW of

E. coli

- Produced Cry3Bb1

Protein 1
(kDa) Preset Acceptance Limits

for the MON 87411

- Produced Cry3Bb1Protein(kDa)

77.0 74.5 72.9

- 81.7

1 As reported on the Certificate of Analysis for lot 11309151Monsanto Company CR240

- 13U1 259 of 374 Figure C

- 3. Molecular Weight and Purity Analysis of the MON 87411

- produced Cry3Bb1 Protein Aliquots of the MON 87411

- produced Cry3Bb1 and the E. coli

- produced Cry3Bb1 proteins were subjected to SDS

- PAGE and gel was stained with Brilliant Blue G

- Colloidal stain. The MWs (kDa) are shown on the left and correspond to the standards loaded in Lanes 1 and 9. Lane designations are as follows:

Lane Sample Amount (μg)

1 Broad Range MW Standards 4.5

2 E. coli

- produced Cry3Bb1 protein 1

3 MON 87411

- produced Cry3Bb1 protein 1

4 MON 87411

- produced Cry3Bb1 protein 1

5 MON 87411

- produced Cry3Bb1 protein 2

6 MON 87411

- produced Cry3Bb1 protein 2

7 MON 87411

- produced Cry3Bb1 protein 3

8 MON 87411

- produced Cry3Bb1 protein 3

9 Broad Range MW Standards 4.5

10 Blank kDa

14.4

6.5 21.5

31 45 66.2 97.4 116.3

200 Lane 1 2 3 4 5 6 7 8 9 10Monsanto Company CR240

- 13U1 260 of 374 C .1

. 7 Glycosylation Analysis of Cry3Bb1

C .1

. 7 .1 Methods

ECL Glycoprotein Detection Kit (GE Healthcare) was used for glycoprotein detection.

The MON 87411

- produced Cry3Bb1 protein, the E. coli

- produced Cry3Bb1 protein, and a positive control, transferrin ( Sigma

- Aldrich ), were diluted in 1× LB and heated to

100 °C for 4 min. Two amounts (~100 and ~200 ng) of the intact the

MON 87411

- produced Cry3Bb1 protein (purity corrected) and the E. coli

- produced Cry3Bb1 protein (purity corrected) were loaded onto a pre

- cast Tris

- glycine 4

- 20% polyacrylamide gradient mini

- gel (Invitrogen). Four amounts (~50, ~100, ~150, and

~200 ng) of the positive control were loaded on the gel. Protein MW Standa rds ( Precision Plus Protein TM Standards Dual color, Bio

- Rad ) were also loaded for molecular weight reference and to verify electrotransfer of the proteins to the membrane. Following electrophoresis at a constant voltage, proteins were electrotransferred t o a PVDF membrane (Invitrogen).

Glycosylation analysis was performed on the PVDF membrane at room temperature using the Amersham ECL TM glycoprotein Detection Module (GE Healthcare) as directed by the manufacturer. Glycosylated proteins were detected usin g ECL TM reagents (GE Healthcare) and Amersham Hyperfilm (GE Healthcare). The film was developed using a

Konica SRX

- 101A automated film processor (Konica Minolta). An identical gel was run

and electrotransferred to a PVDF membrane in parallel. Proteins we re stained with

Coomassie Brilliant Blue R

- 250 staining solution (Bio

- Rad) and then destained with

1 **×** Coomassie Brilliant Blue R

- 250 Destaining Solution (Bio

- Rad). After washing with water, the blot was scanned using Bio

- Rad GS

- 800 densitometer.

C .1

. 7 .2 Results of Glycosylation Analysis

Eukaryotic proteins can be post

- translationally modified with carbohydrate moieties (Rademacher et al., 1988) . To test whether Cry3Bb1 protein was glycosylated when expressed in the grain of MON 87411, the MON 87411

- produ ced Cry3Bb1 protein was analyzed using an ECL TM Glycoprotein Detection Module (GE Healthcare). To assess equivalence of the MON 87411

- and E. coli

- produced Cry3Bb1 proteins, the

E. coli

- produced Cry3Bb1 protein was also analyzed.

A clear glycosylation signal was observed at the expected in the lanes containing t he positive control (transferrin) and the band intensity increased with increasing concentration (Figure C

- 4 panel A, lanes 1

- 4 ). In contrast, no glycosylation signals were observed at the expec ted molecular weight for Cry3Bb1 in the lanes containing the

MON 87411

- produced Cry3Bb1 protein (Figure C

- 4 panel A, lanes 7 and 8) or

E. coli

- produced Cry3Bb1 protein (Figure C

- 4 panel A, lanes 9 and 10). The band

observed in the E. coli

- produced Cry3Bb1 sample was at a lower molecular weight than the Cry3Bb1 protein. This band likely represents a naturally biotinylated protein that co

purified with the E. coli

- produced Cry3Bb1 protein (Choi

- Rhee and Cronan 2003)

, because binds to the str eptavidin

- HRP conjugate and therefore is detectable by the ECL

system.Monsanto Company CR240

- 13U1 261 of 374 To confirm that MON 87411

- and E. coli

- produced Cry3Bb1 proteins were appropriately loaded for glycosylation analysis, a second membrane with identical loadings and transfer

time was st ained with Coomassie Blue R250 for protein detection (Figure C

- 4 Panel B).

Both the MON 87411

- produced Cry3Bb1 (Figure C

- 4 panel B, lanes 7 and 8) and

E. coli

- produced Cry3Bb1 (Figure C

- 4 panel B, lanes 9, and 10) proteins were detected.

These data indic ate that the glycosylation status of MON 87411

- produced Cry3Bb1 protein is equivalent to that of the E. coli

- produced Cry3Bb1 protein and that neither is glycosylated.Monsanto Company CR240

- 13U1 262 of 374 Figure C

- 4. Glycosylation Analysis of the MON 87411

- produced Cry3Bb1 Protein Aliquots of the transferrin (positive control), E. coli

- produced Cry3Bb1 and MON

87411

- produced Cry3Bb1 were subjected to SDS

- PAGE and electrotransferred to PVDF membranes. The MWs (kDa) correspond to the Precision Plus Protein TM Dual Color Standards.

Th e arrows show the expected migration of the MON 87411

- and E. coli

- produced Cry3Bb1 protein. (A) Where present, labeled carbohydrate moieties were detected by addition of streptavidin conjugated to HRP followed by a luminol

- based the detection using ECL r eagents and exposure to Hyperfilm ® . (B) An equivalent blot was stained with Coomassie Blue R250 to confirm the presence of proteins. Lane designations and the approximate mass loaded are as

follows:

Lane Sample Amount (ng)

1 Transferrin (positive control) 50

2 Transferrin (positive control) 100

3 Transferrin (positive control) 150

4 Transferrin (positive control) 200

5 Precision Plus Protein TM Standards

6 Blank

7 MON 87411

- produced Cry3Bb1 protein 100

8 MON 87411

- produced Cry3Bb1 protein 200

9 E. coli

- produced Cry3Bb1 protein 100

10 E. coli

- produced Cry3Bb1 protein 200 kDa

10 15 20 25 37 50 75 100 150 250 1 2 3 4 5 6 7 8 9 10 Lane

kDa

10 15 20 25 37 50 75 100 150 250 1 2 3 4 5 6 7 8 9 10 Lane A. B.Monsanto Company CR240

- 13U1 263 of 374 C .1

. 8 Cry3Bb1 Functional Activity Analysis

C .1

. 8 .1 Methods

I nsects. CPB eggs were obtained from French Agricultural Research, Inc. (Lamberton, MN). The eggs were incubated at temperatures ranging from 15 °C to 27 °C, to achieve

the desired hatch time.

Bioassays. CPB larvae ( ≤ 30 hours old) were used to measure biological activity of the

MON 87411

- produced and E. coli

- produced Cry3Bb1 protein samples in accordance

with the current version of Monsanto SOP BR

- ME

- 10 4 8

. The bioassay was replicated three times on separate days , each with a separate batch of insects. The

MON 87411

- produced and E. coli

- produced substances were run in parallel during each bioassay. Each bioassay replicate consisted of a series o f six dilutions yielding a dose

series with a two

- fold separation factor ranging from 0.0 78

– 2.5 **μ** g Cry3Bb1 protein/ml

diet for the E. coli

- produced and MON 87411

- produced Cry3Bb1 and a single buffer

control

. Th e Cry3Bb1 protein dosing solutions were prepared by diluting the respective protein with purified water and in corporating the dilution into a Bio

- Serv CPB agar

- based

insect diet ( pre

- mix #F9380B ; Frenchtown , NJ ). This dose series in diet was chosen to

ade quately characterize the dose

- effect relationship on CPB mortality for the

E. coli

- produced and MON 87411

- produced Cry3Bb1 proteins. The diet mixture was then dispensed in 0.5 ml aliquots into a 128 well tray (# BAW 12 8, Bio

- Serv, Frenchtown, NJ). Individual i nsect larvae were placed on these diets using a fine paintb rush, with a target number of 24 insects per treatment

. The i nfested wells were covered by a ventilated adhesive cover (# BACV16, Bio

- Serv, Frenchtown, NJ) and the insects were

allowe d to feed for a period of approximately 7 days in an environmental chamber programmed at 27 **°** C, 60% relative humidity and a lighting regime of 14 light : 10 dark.

The number of insects infested and the number of surviving insects at each dose level

w as re corded at the end of the 7 day incubation period. For a bioassay to be accepted, control mortality cannot be > 20%.

C .1

. 8 .2 Results of Functional Activity

The functional activity of the MON 87411

- produced and E. c oli

- produced Cry3Bb1 protein w as determin ed Colorado potato beetle diet

- incorporation bioassays . In this assay, activity is expressed as LC 50, μg/ml diet

. The MON 87411

- and E. coli

- produced Cry3Bb1 proteins were considered functionally equivalent if the LC 50 , of both were within acceptance limits of 0.37 μg/ml of diet to 0.97 μg/ml ; the prediction interval

calculated from data obtained for the E. coli

- produced Cry3Bb1 protein activity. The

LC 50 of the MON 87411

- and E. coli

- produced Cry3Bb1 proteins were deter mined to be

0.77 μg/ml diet and 0.67 μg/ml diet respectively (T able C

- 5). Because the LC 50 of

MON 87411

- produced and E. coli

- produced Cry3Bb1 proteins were within the acceptance limits (Table C

- 5 ), the protein s were determined to have equivalent functional activity

.Monsanto Company CR240

- 13U1 264 of 374 Table C

- 5. Cry3Bb1 Functional Activity Assay

MON 87411

- produced Cry3Bb1 Protein 1

(LC 50, μg/ml diet) E. coli

- produced Cry3Bb1

Protein 1

(LC 50, μg/ml diet) P reset Acceptance Limits

for MON 87411

- produced Cry3Bb1 Protein 2

(LC 50, μg/ml diet )

0.77 0.67 0.37

– 0.97

1 Value refers to mean based on n = 3

2 Data obtained for the E. coli

- produced protein was used to generate the acceptance limits

C.2 Characterization of CP4 EPSPS Protein in MON 87411

C .2 .1 Materials for CP4 EPSPS Characterization

The MON 87411

- p roduced CP4 EPSPS protein (lot 11 351676 ) was purified from the grain of MON 87411 (lot 11330153 ). The MON 87411

- produced CP4 EPSPS protein

was stored in a

- 80 oC freezer in a buffer solution containi ng 50 mM Tris

- HCl, pH 7.5,

50 mM KCl, 2 mM dithiothreitol (DTT), 1 mM benzamidine

- HCl, and 25% glycerol.

The E. coli

- produced CP4 EPSPS protein (lot 10000739) was used as the reference

substance. The CP4 EPSPS protein reference substance was generated f rom cell paste produced by large

- scale fermentation of E. coli containing the pMON21104 expression plasmid. The coding sequence for cp4 epsps contained on the expression plasmid (pMON21104) was confirmed prior to and after fermentation. The E. coli

- produ ced

CP4 EPSPS protein was characterized previously

.

C .2

. 2 . CP4 EPSPS Protein Purification

The plant

- produced CP4 EPSPS protein was purified from grain of MON 87411. The purification procedures were not performed under a GLP plan; however, all procedure s were documented on worksheets and, where applicable, SOPs were followed. Three

CP4 EPSPS protein purification batches were prepared to gene rate a sufficient amount of

MON 87411 produc ed CP4 EPSPS protein. All purification steps were performed at

~4 °C. CP4 EPSPS was purified from an extract of ground grain using a combination of purification methods. The purification process for the first two batches used ammonium sulfate fractionation, hydrophobic interaction chromatography, anion

- exchange chromatogr aphy, and a second hydrophobic interaction chromatography. The purification process for the third batch used ammonium sulfate fractionation, hydrophobic interaction chromatography, anion

- exchange chromatography, cellulose phosphate affinity chromatography , and hydroxyapatite chromatography.

Chromato graphy fractions containing CP4 EPSPS protein were identified by western blot analysis or SDS

- PAGE staine d gels. The final purified CP4 EPSPS protein product from all three batches was combined, concentrated a nd buff er

- exchanged to prepare the

CP4 EPSPS protein lot used in the equivalence study. Each of the three purification procedures are briefly described below and a detailed description of the purification procedures are filed under Lot 11351676 in the Mon santo Regulatory Archives.Monsanto Company CR240

- 13U1 265 of 374 CP4 EPSPS protein Batch 1 . Grain of MON 87411 was ground in the presen ce of dry ice

and stored at

- 80 °C until use. A total of 920 g of ground powder was mixed with EX buffer (100 mM Tris

- HCl pH 7.5, 2 mM EDTA, 2 mM benzamidine

- HCl, 4 mM DTT,

1 mM phenylmethylsulfonyl fluoride, 1 % polyvinyl

- polypyrrolidone and 10 % glycerol) at a sample weight (g) to buffer volume (ml) of approximately 1:10 fo r ~1.5 hr. The slurry was centrifuged to clarify, and the supernatant was collected, filtered, and brought

to 40 % ammonium sulfate saturation by addition of solid ammonium sulfate. Following equilibration, the 40 % saturated solution was centrifuged to pellet precipitated proteins. The supernatant was brought to 70 % ammonium sulfate sa turation by addition of the solid salt. Following equilibration, the precipitated protein containing the majority of

CP4 EPSPS protein was collected by centrifugation. The 40

- 70 % ammonium sulfate pellet was re

- suspended in PS

- A buffer (50 mM Tris

- HCl, p H 7.5, 1 mM DTT, 10 % glycerol (v/v), and 1.5 M ammonium sulfate). The dissolved suspension was centrifuged, filtered, and loaded onto a Phenyl Sepharose Fast Flow (GE Healthcare, Piscataway, NJ) column equilibrated with PS

- A buffer. Proteins were eluted with a linear salt gradient

that decreased from 1.5 M to 0 M ammonium sulfate over 6 column volumes . Fractions containing the CP4 EPSPS protein were pooled and desalted by dia lysis against S30Q

- A buffer (50 mM Tris

- HCl, pH 7.5, 1 mM EDTA, 1 mM benzamidin e

- HCl, and 4 mM DTT). The desalted sample was loaded onto a Source 30Q (GE Healthcare) column equilibrated wit h S30Q

- A buffer. The bound CP4 EPSPS was eluted with a linear salt gradient that increased from 0 M to 0.4 M KCl over 15 column vol umes. Fracti ons containing CP4 EPSPS were pooled and dialyzed against PS

- A buffer. The dialyzed sample was then loaded onto a Source 15PHE (GE Healthcare) column equilibrated with

PS

- A buffer. Proteins were eluted with a linear salt g radient that decreased from 1.5 M to

0 M ammonium sulfate over 20 column volumes . Fractions containing the CP4 EPSPS protein were pooled and buffer e xchanged by dialysis against 50 mM Tris

- HCl pH 7.5,

50 mM KCl, 1 mM benzamidine

- HCl, 2 mM DTT, and 10 % glycerol. The buffer exchanged sa mple was initially concentrated within the dialysis tubing by packing in Aquacide II (EMD Chemicals, San Diego, CA), then further concentrated by centrifugation in an Amicon Ultra spin concentrator (ULTRACEL

- 10K, 10,000 MWCO, regenerated cellulose membrane ). The sample was then buffer exchanged into 1.2X formulation buffer (60 mM Tris

- HCl pH 7.5, 60 mM KCl, 1.2 mM benzamidine

- HCl,

2.4 mM DTT, and 10 % glycerol), yielding a final volume of ~500 μl. This Batch 1 purified CP4 EPSPS sample was frozen and stored in a

- 80 **°** C freezer.

CP4 EPSPS protein Batch 2 . The purification procedure used for the Batch 2 isolation of

CP4 EPSPS from the grain of MON 87411 followed the same sequence of fracti onation steps as used for Batch 1, except that the initial amou n t of grain extracted for Batch 2 was

~3 kg. Because a larger amount of grain was extracted for Batch 2, the bed size of chromatography columns, the column gradient volumes and the dialysis volumes used were increased, as appropriate, to accommodate the la rger amounts of sample handled at each step. The final volume of Batch 2 protein sample was ~350 μl. This Batch 2 purified CP4 EPSPS sample was frozen and stored in a

- 80 **°** C freezer.

CP4 EPSPS protein Batch 3 . The initial purification steps used for the Batch 3 isolation

of CP4 EPSPS from the grain of MON 87411 followed the same sequence of fractionation steps as used for Batch 1 through the So urce 30Q step, starting with ~2 kgMonsanto Company CR240

- 13U1 266 of 374 of gro und grain. Following Source 30 Q fraction ation, fractions containing CP 4 EPSPS were pooled, brought to 25 % glycerol (v/v) and stored frozen and

- 20 °C until the next step. The pooled sample was thawed, concentrated and buffer exchanged into CPE

bu ffer (10 mM MES pH 5.9, 1 mM benzamidine

- HCl, 1 mM DTT, 10 μM Na

- tungstate,
and 10 % glycerol) using 15 ml Amicon Ultracell

- 10K centrifugal concentrators. The buffer exchanged and concentrated sample was diluted with CPE buffer and loaded on a

column of P11 Cellulose Phosphate (Whatman/GE Healthcare, Maidstone, UK) equilibrated in C PE buffer. The column was washed with CPE buffer followed by CPE

buffer with 0.5 mM phosphoenolpyruvate (PEP). CP4 EPSPS was then eluted with CPE

buffer with 0.5 mM PEP and 0.5 mM shikimate

- 3

- phosphate (S

- 3

- P). Fractions containing CP4 EPSPS were pooled and concentrated using a Vivaspin 15R centrifugal

conc entrator equipped with a 10,000 MWCO membrane (Sartorius Stedim Biotech, Goettingen, Germany). The glycerol concentration was increased to 25 % and the sample

frozen and held at

- 20 °C until the next step. The sample was thawed, diluted with

CHT

- A buffer (50 mM HEPES pH 7.0, 1 mM benzamidine

- HCl, 1 mM DTT, and 10 % glycerol), and loaded on a ceramic hydroxyapatite (CHT Type 1, Bio

- Rad Laboratories, Hercules, CA) column. Protein was eluted with linear phosphate gradient from 0 to

50 mM sodium phosphate in CHT

- A buffer over 20 column vol umes. Fractions containing CP4 EPSPS were pooled and concentrated using an Amicon Ultracel

- 10K centrifugal concentration device. The pooled sample was buffer exchange d into 1.2X formulation buffer (60 mM Tris

- HCl, pH 7.5, 60 mM KCl, 2.4 mM DTT, 1.2 mM

benzamidine

- HCl, 10 % glycerol), yielding a final volume of ~0.5 ml of buffer exchanged protein concentrate.

Preparation of the final pooled protein product. The Batch 1 and 2 samples were thawed and pooled w ith purified protein from Batch 3. The pooled protein sample was

concentrated to ~0.5 ml by centrifugation in a 4 ml Amicon Ultracel

- 10K centrifugal

concentration device, equipped with a 10,000 MWCO membrane. Buffer exchange was conducted in the same unit using 1.2X formulation buffer (60 mM Tris

- HCl, pH 7.5,

60 mM KCl, 2.4 mM DTT, 1.2 mM benzamidine

- HCl, 10 % glycerol), to a final volume

of 300 μl, then brought to 500 μl volume with a 200 μl buffer rinse of the spin

concentrator. Glycerol was added bringing the final protein volume to 600 μl in 1X

formulation buffer and 25 % glycerol. Final buffer composition of the sample was:

50 mM Tris

- HCl pH 7.5, 50 mM KCl, 2 mM DTT, 1 mM benzamidine

- HCl and 25 % glycerol. Thi s CP4 EPSPS protein purified from the grain of MON 87411 was aliquoted, assigned lot 11351676, and stored in a

- 80 °C freezer.

C .2

. 3 N

- Terminal Sequencing of CP4 EPSPS

C .2.3 .1 Methods

N

- terminal sequencing, carried out by automated Edman degradation chem istry, was used to confirm the identity of the MON 87411

- produced CP4 EPSPS.

A MON 87411

- produced CP4 EPSPS protein sample was separated by SDS

- PAGE and

transferred to a PVDF membrane. The blot was stained using Coomassie Blue R

- 250 and

destained with 1× Coomassie Brilliant blue R

- 250 Destaining Solution (Bio

- Rad) . TheMonsanto Company CR240

- 13U1 267 of 374 major band at ~44 kDa containing the test protein was excised f rom the blot and was used

for N

- terminal sequence analysis. The analysis was performed for 15 cycles u sing automated Edman degradation chemistry (Hunkapiller et al., 1983)

. An Applied Biosystems 494 Procise Sequencing System with a 140C Microgradient system and a Perkin Elmer Series 200 UV/VIS Absorbance Detector and Procise TM Control Software (version 2. 1 ) were used . Chromatographic data were collected using SequencePro software (Applied Biosystems, Foster City CA; version 2.1). A PTH

- AA standard solution (Applied Biosystems) was used to chromatographically calibrate the instrument for the analysis. A control protein, **β**

- lactoglobulin , (Applied Biosystems ) was analyzed

before and after analysis of the MON 87411

- produced CP4 EPSPS protein

.

C .2 .3.2 Results of the N

- terminal Sequence Analysis

Fifteen cycles of N

- terminal sequencing was performed on the MO N 87411

- produced

CP4 EPSPS protein. The expected sequence for the CP4 EPSPS protein deduced from

the cp4 epsps gene present in MON 87411 was observed. The data obtained correspond

to the deduced CP4 EPSPS protein beginning at amino acid position 2 ( Table C

- 6)

. Hence, the sequence information confirms the identity of the CP4 EPSPS protein isolated

from the seed of MON 87411

.

Table C

- 6 . N

- Terminal Sequence of the MON 87411

- produced CP4EPSPS

Amino acid

residue # from

the N

- terminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Expected Sequence → M L H G A S S R P A T A R K S S

│ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Experimental

Sequence →

– L H G A S S R P A T A R K X X

The experimental sequences obtained from the MON 87411

- produced CP4 EPSPS protein were compared to the expected seq uence. The single letter IUPAC

- IUB amino acid code is M, methionine; L, leucine; H, histidine; G, glycine; A, alanine, S, serine; R, arginine; P, proline; T, threonine; K, lysine; (X) indicates that the residue was not ide ntifiable.

C 2.4 MALDI

- TOF Tryptic Mass Map Analysis of CP4 EPSPS

C 2.4 .1 Methods

MALDI

- TOF MS tryptic mass fingerprint analysis was used to confirm the identity of the

MON 87411

- produced CP4 EPSPS protein. A MON 87411

- produced CP4 EPSPS
prot ein sample wa s separated by SDS

- PAGE and the gel was stained using Brilliant Blue

G

- Colloidal stain (Sigma

- Aldrich, St. Louis, MO) . The major band at ~44 kDa band was excised, transferred to a microcentrifuge tube , and destained . The gel band was washed

in 200 μl of 100 mM ammonium bicarbonate

, and then incub ated in 100 μl of 10 mM

DTT at 37 °C for 2 h to reduce protein disulfide bonds . The protein was then alkylated inMonsanto Company CR240

- 13U1 268 of 374 the dark for 2 0 min with 100 μl of 20 mM iodo acetic acid and washed with 200 μl of

25 mM ammonium bicarbonate for 3 × 15 min washes. The gel band was dried with a Speed

- Vac **** concentrator (Thermo Fisher Scientific, Waltham, MA) and then rehydrated

with 20 μl of trypsin solution (20 μg/ml). After 1 h, excess liquid was removed and the gel was incubated overnight at 37 °C in 40 μl of 10 % acetonitrile in 25 mM ammonium bicarbonate. The gel band was sonicated for 5 min to further elute proteolytic fragments. The resulting extract was transferred to a new tube labeled Extract 1 and dried using a Speed

- Va c concentrator. The gel band was re

- extracted twice with 30 μl of a

60 % acetonitrile, 0.1 % trifluoroacetic acid, 0.1 % β

- octyl

- glucopyranoside solution and

sonicated for 5 min. These samples were pooled into a new tube labeled Extract 2 and

dried with a Speed

- Vac concentrator. A solution of 0.1 % trifluoroacetic acid (TFA) was

added to all Extract 1 and 2 tubes and they were dried as before. A solution of

50 % acetonitrile, 0.1 % TFA was added to each tube and all were sonicated for 5 min.

Each extra ct (0.3 μl) was spotted to three wells on an analysis plate. For each extract

0.75 μl of 2, 5

- dihydroxybenzoic acid (DHB), α

- cyano

- 4

- hydroxycinnamic acid (α Cyano), or 3,5

- dimethoxy

- 4

- hydroxycinnamic acid (sinapinic acid) ( Thermo Fisher

Scientific ) was ad ded to one of the spots. The samples in DHB matrix were analyzed in

the 300 to 5 000 Da range. Samples in α

- Cyano and Sinapinic acid were analyzed in the

500 to 5000 and 500 to 7000 Da range, respectively. CalMix 2 TM was used as the external calibrant (S equazyme TM Peptide Mass Standard s kit, AB SciEx ). The analysis was performed on a Voyager TM DE Pro Biospectrometry TM workstation (Applied Biosystems) using Voyager Instrument Control Panel software ( version 5.10.2 ) and Data Explorer data analysis software ( version 4.0.0.0 ). Protonated peptide masses were isotopically resolved in reflector mode (Aebersold 1993; Billeci and Stults 1993)

. GPMAW32 software ( Lighthouse Data ) was used to generate an in silico digest of the

CP4 EPSPS protein sequence. Masses w ithin 1 Da of the monoisotopic mass were matched against the in silico digest of the MON 87411

- produced Cry3Bb1 sequence. All matching masses were tallied and a coverage map was generated for the mass fingerprint.

C 2.4 .2 Results of MALDI

- TOF Tryptic Mass Map Analysis

T he identity of the MON 87411

- produced CP4 EPSPS protein was confirmed by

MALDI

- TOF MS analysis of peptide fragments produced by the trypsin digestion of the

MON 87411

- produced CP4 EPSPS protein. There were 23 unique peptides identified that corresponded to the masses expected to be produ ced by trypsin digestion of the

CP4 EPSPS protein (Table C

- 7 ). The identified masses were used to assemble a coverage map of the entire CP4 EPSPS protein (Figure C

- 5 ). The experimentally determined mass coverage of the CP4 EPSPS protein was 67.3 % (3 06 out of 455 amino acids).Monsanto Company CR240

- 13U1 269 of 374 Table C

- 7 . Summary of the Tryptic Masses Identified for the

MON 87411

- produced CP4 EPSPS Using MALDI

- TOF MS

Observed Mass 1 Expected Mass Difference 2 Fragment Sequence 3

389.20 389.25 0.05 225

- 227 TIR

474.23 474.27 0.04 228

- 231 LEGR

599.33 599.33 0.00 29

- 33 SISHR

616.33 616.34 0.01 128

- 132 RPMGR

629.34 629.29 0.05 201

- 205 DHTEK

711.45 711.45 0.00 133

- 138 VLNPLR

733.44 733.38 0.06 352

- 357 VKESDR

835.39 835.39 0.00 62

- 69 AMQAMGAR

863.47 863.46 0.01 15

- 23 SSGLS GTVR

872.45

872.46 872.45

872.52 0.00

0.06 313

- 320

358

- 366 GVTVPEDR*

LSAVA NGLK*

930.49 930.51 0.02 169

- 177 VPMAS AQVK

948.51 948.52 0.01 161

- 168 TPTPITYR

991.56 991.55 0.01 14

- 23 KSSG

... GTVR

1115.57 1115.57 0.00 295

- 305 LAGG...ADLR

1357.72 1357.71 0.01 146

- 157 SEDG...VTLR

1359.67

1359.6 3 1359.72

1359. 64 0.05

0.0 1 354

- 366

34

- 46 ESDR...NGLK*

SFMF...GETR*

1558.82 1558.83 0.01 47

- 61 ITGL...NTGK

1646.85 1646.84 0.01 389

- 405 GLGN...LDHR

1763.83 1763.81 0.02 367

- 382 LNGV...LVVR

1993.9 7 1993.97 0.00 206

- 224 MLQG...DGVR

2183.1 9 2183.17 0.0 2 275

- 294 TGLI...INPR

2367.33 2367.33 0.00 178

- 200 SAVL...IMTR

2450.24 2450.23

2450.2 2 0.01

0.0 2 24

- 46

105

- 127 IPGD...GETR*

LTMG...SLTK*

3244.52 3244.52 0.00 73

- 104 EGDT...TGCR

3249.59 3249.62 0.03 321

- 351 APSM...EELR

4188.47 4188.26 0.21 234

- 274 LTGQ...NPTR

1 The observed mass was obtained from at least one of three matrices including **α**

- cyano, DHB, and sinapinic acid. The observed mass shown is the mass closest to the expected mass.

2 The data represents the calculated difference between the expected mass and the observed mass.

3 For peptide matches greater than nine amino acids in length the first 4 residues and last 4 res idues are show separated by three dots (...) *The expected peptide masses are nearly identical (<1 dalton). Because this analysis did not determine with certainty which expected peptide was actually observed, the peptides with an asterisk (*) were not

inclu ded in determining sequence coverage (Figure C

- 5).Monsanto Company CR240

- 13U1 270 of 374 Figure C

- 5 . MALDI

- TOF MS Coverage Map of the MON 87411

- P roduced CP4

EPSPS Protein The amino acid sequence of the CP4 EPSPS protein was deduced from the cp4 epsps gene present in MON 87411. Boxed regions correspond to regions covered by tryptic peptides that were unambiguously identified from the MON 87411

- produced CP4 E PSPS protein sample using

MALDI

- TOF MS. In total, 67.3 % (306 of 455 total amino acids) of the expected protein sequence was covered by the identified peptides.

C .2

. 5 Western Blot Analysis

- Immunoreactivity of CP4 EPSPS

C .2

. 5 .1 Methods

Western blot analysis was performed to confirm the identity of the MON 87411

- produced

CP4 EPSPS protein and to compare the immunoreactivity of the MON 87411

- and

E. coli

- produced proteins.

The MON 87411

- and E. coli

- produced CP4 EPSPS proteins were analyzed concurrently on the same gel using three protein loadings of 1, 2 and 3 ng. Loadings of the three

concentr ations were made in duplicate on the gel. In preparation for analysis the

E. coli

- produced CP4 EPSPS was diluted with storage buffer to the same purity corrected

concentration as the MON 87411

- produced protein (0.2 63 mg CP4 EPSPS /ml). Aliquots of each pr otein were subsequently diluted in water and 1 **×** (LB), and heated at 95 °C for

4 min. S amples were further diluted in 5 **×** LB to a final concentration of 0.25 ng/μl, and applied to a pre

- cast Tris

- glycine 4

- 20 % polyacrylamide gradient gel (Invitrogen). Pre

stained molecular weight markers (Precision Plus Protein Standards Dual C olor;

Bio

- Rad) were loaded to verify electrotransfer of the proteins to the membrane and to

estimate the size of the immunoreactive ba nds observed. Following electrophoresis,

electrotransfer to a PVDF membrane (Invitrogen) was performed.

After electrotransfer, the membrane was blocked for 1 h with 5 % (w/v) NFDM in PBST)

at room temperature . The membrane was then probed with a 1:10 ,0 00 dilution of goat

anti

- CP4 EPSPS antibody ( lot G

- 848018 ) in 1 % NFDM in PBST for 60 min at room

001 MLHGASSRPA TARKSSGLSG TVRIPGDKSI SHRSFMFGGL ASGETRITGL

051 LEGEDVINTG KAMQAMGARI RKEGDTWIID GVGNGGLLAP EAPLDFGNAA

101 TGCRLTMGLV GVYDFDSTFI GDASLTKRPM GRVLNPLREM GVQVKSEDGD

151 RLPVTLRGPK TPTPITYRVP MASAQVKSAV LLAGLNTPGI TTVIEPIMTR

201 DHTEKMLQGF GANLTVETDA DGVRTIRLEG RGKLTGQVID VPGDPSSTAF

251 PLVAALLVPG SDVTILNVLM NPTRTGLILT LQEMGADIEV INPRLAGGED

301 VADLRVRSST LKGVTVPEDR APSMIDEYPI LAVAAAFAEG ATVMNGLEEL

351 RVKESDRLSA VANGLKLNGV DCDEGETSLV VRGRPDGKGL GNASGAAVAT

401 HLDHRIAMSF LVMGLVSENP VTVDDATMIA TSFPEFMDLM AGLGAKIELS

451 DTKAAMonsanto Company CR240

- 13U1 271 of 374 temperature

. Excess antibody was removed using washes with PBST. Finally, the membrane was probed with HRP

- conjugated rabbit anti

- goat IgG (Thermo, Rockford , IL)

at a dilution of 1: 5 ,000 in 1 % NFDM in PBST for 1 h at room temperature. Excess

HRP

- conjugate was removed using washes with PBST. All washes were performed at
room temperature. Immunoreactive bands were visualized using the ECL detection system ( GE, Healthcare) with exposure to Amersham Hyperfilm ECL (GE, Healthcare). The film was developed using a Konica SRX

- 101A automated film processor.

Quantification of the bands on the blot was performed using a Bio

- Rad GS

- 800 densitometer with the supplied Quantity One software (version 4.4.0) using the band volume tool. The signal intensities of the immunoreactive b ands observed for the

MON 87411

- and E. coli

- produced proteins migrating at the expected position on the blot film were quantified as “adjusted volume” values (OD **×** mm 2 ).

C .2

. 5 .2 Results of CP4EPSPS Protein Immunoreactivity Equivalence

Western blot analysi s was conducted using goat anti

- CP4 EPSPS polyclonal antibody as an additional means to confirm the identity of the CP4 EPSPS protein isolated from the grain of MON 87411 and to assess the equivalence of the immunoreactivity of the

MON 87411

- and the E. coli

- produced CP4 EPSPS proteins.

The results demonstrated that the anti

- CP4 EPSPS antibody recognized the

MON 87411

- produced CP4 EPSPS protein that migrated to an identical position as the

E. coli

- produced CP4 EPSPS protein (Figure C

- 6 ). Furthermore, the immunoreactive signal increased with increasing amounts of CP4 EPSPS protein loaded.

To compare the immunoreactivity of the MON 87411

- produced CP4 EPSPS protein and

the E. coli

- produced CP4 EPSPS proteins, densitometric analysis was conducted on bands that migrated to the expected apparent MW for CP4 EPSPS proteins (~44 kDa). The signal intensity (reported in OD × mm 2 ) of the band of i nterest in lanes loaded with

the MON 87411

- produced and the E. coli

- produced CP4 EPSPS protein was measure d (Table C

- 8 ). Because signal intensity of the MON 87411

- produced CP4 EPSPS protein

band was within 35% of the E. coli

- produced CP4 EPSPS protein, the

MON 87411

- produced and E. coli

- produced CP4 EPSPS proteins were determined to have equivalent immunoreactivity.Monsanto Company CR240

- 13U1 272 of 374 Figure C

- 6 . Western Blot Analysis of MON 87411

- and E. coli

- P roduced

CP4 EPSPS Proteins Aliquots of the MON 87411

- produc ed CP4 EPSPS protein and the E. coli

- produced

CP4 EPSPS protein were separated by SDS

- PAGE and electrotransferred to a PVDF

membrane. The membrane was incubated with anti

- CP4 EPSPS antibodies as the primary antibodies and immunoreactive bands were visuali zed using HRP

- conjugated secondary antibodies and an ECL system (GE Healthcare). Approximate molecular weights (kDa) are shown on the left and correspond to the markers loaded in lane 1. Lane 1 was cropped from the image. The 3 min exposure is shown. L ane designations

are as follows:

Lane Sample Amount (ng)

1 Precision Plus Protein Standards Dual color (cropped from the image) -

2 E. coli

- produced CP4 EPSPS protein 1

3 E. coli

- produced CP4 EPSPS protein 1

4 E. coli

- produced CP4 EPSPS protein 2

5 E. coli

- produced CP4 EPSPS protein 2

6 E. coli

- produced CP4 EPSPS protein 3

7 E. coli

- produced CP4 EPSPS protein 3

8 Blank

- 9 MON 87411

- produced CP4 EPSPS protein 1

10 MON 87411

- produced CP4 EPSPS protein 1

11 MON 87411

- produced CP4 EPSPS protein 2

12 MON 87411

- produced CP4 EPSPS protein 2

13 MON 87411

- produced CP4 EPSPS protein 3

14 MON 87411

- produced CP4 EPSPS protein 3

15 Blank

- 2 3 4 5 6 7 8 9 10 11 12 13 14 15

250
150
100

75

50

37

25
20

15

10 Lane #

MW
( kDa )Monsanto Company CR240

- 13U1 273 of 374 Table C

- 8 . Comparison of Immunoreactive Signal Between MON 87411

- and E

coli

- produced CP4 EPSPS Protein

Mean Signal Intensity from

MON 87411

- Produced

CP4 EPSPS 1

(OD **×** mm 2 ) Mean Signal Intensity from

E. coli

- Produced CP4 EPSPS 1

(OD **×** mm 2 ) Acceptance Limits 2 for

MON 87411

- Produced

CP4 EPSPS
(OD **×** mm 2 )

6.0 6.0 3.9

– 8.1

1 Ea ch value represents the mean of six values (n=6).

2 The acceptance limits are for the MON 87411

- produced CP4 EPSPS protein and are based on the interval
between +35% (6.0 **×** 1.35) and

**−** 35% (6.0 **×** 0.65) of the mean of the E. coli

- produced CP4 EPSPS signal intensity across all loads.

C .2

. 6 Molecular Weight Estimation of CP4 EPSPS using SDS

- PAGE

C .2

. 6 .1 Methods

MON 87411

- produced CP4 EPSPS protein and E. coli

- produced CP4 EPSPS protein , as well as Bio

- Rad B road R ange m olecular w eight s tandards

, were diluted in water and

5 **×** LB

. The diluted s amples were heated to 95 °C for 3

- 5 min. The

MON 87411

- produced CP4 EPSPS protein was analyzed in duplicate at 1, 2, and 3 μg protein per lane. The E. coli

- produced CP4 EPSPS reference standard was analyzed at

1 μg total protein in a single lane. The sa mples were loaded onto a pre

- cast Tris glycine

4

- 20 % polyacrylamide gradient mini

- gel (Invitrogen) and electrophoresis was performed at a constant voltage . Proteins were fixed by placing the gel in a solution of 40 % (v/v)

methanol and 7 % (v/v) acetic a cid for 30 min, and stained for 18 h with Brilliant Blue G

C olloidal stain

. Gels were destained for 30 to 45 sec with a solution containing

10 % (v/v) acetic acid and 25 % (v/v) methanol, and for 8 h with 25 % (v/v) methanol. Analysis of th e gel was performed using a Bio

- Rad GS

- 800 densitometer with the supplied Quantity One software (version 4.4.0). Apparent MW was reported as an average of a ll six lanes containing the MON 87411

- produced Cry3Bb1 protein.

C .2

. 6 .2 Results of CP4EPSPS Protein Molecul ar Weight Equivalence

For molecular weight and purity analysis, the MON 87411

- produced CP4 EPSPS protein was separated using SDS

- PAGE. Following electrophoresis, the gel was stained with

Brilliant Blue G

- Colloidal stain and analyzed by densitometry (Figur e C

- 7). The

MON 87411

- produced CP4 EPSPS protein (Figure C

- 7 , lanes 3

- 8) migrated to the same position on the gel as the E. coli

- produced CP4 EPSPS protein (Figure C

- 7 , lane 2) and the apparent molecular w eight was calculated to be 42.9 kDa (Table C

- 9 ). Because the experimentall y determined apparent MW of MON 87411

- produced CP4 EPSPS protein was within the pre

- set acceptance limits for equivalence (Table C

- 9; 42.6

– 45.1 kDa),

the MON 87411

- produced and E. coli

- produced CP4 EPSPS proteins were determined to have equivalent apparent molecular weights.Monsanto Company CR240

- 13U1 274 of 374 Table C

- 9 . Molecular Weight Comparison Between the MON 87411

- and

E. coli

- produced CP4 EPSPS Proteins

Apparent MW

of MON 87411

- Produced

CP4 EPSPS Protein (kDa) Apparent MW of

E. coli

- Produced

CP4 EPSPS Protein 1

(kDa) Preset Acceptance Limits

for MON 87411

- Produced

CP4 EPSPS (kDa)

42.9 43.8 42.6

- 45.1

1 As reported on the Certificate of Analysis for lot 10000739.

Figure C

- 7 . Molecular Weight and Purity Analysis of the MON 87411

- produced

CP4EPSPS Protein Aliquots of the MON 87411

- produced and the E. coli

- produced CP4 EPSPS proteins were subjected to SDS

- PAGE and the gel was stained with Brilliant Blue G

- colloidal stain. The MWs (kDa) are shown on the left and correspond to the standards loaded in Lanes 1 and 9. Lane 10 (blank lane) was cropped from the image. Lane designations are

as follows:

Lane Sample Amount (μg)

1 Broad Range MW Standards 4.5

2 E. coli

- produced CP4 EPSPS protein 1

3 MON 87411

- produced CP4 EPSPS protein 1

4 MON 87411

- produced CP4 EPSPS protein 1

5 MON 87411

- produced CP4 EPSPS protein 2

6 MON 87411

- produced CP4 EPSPS protein 2

7 MON 87411

- produced CP4 EPSPS protein 3

8 MON 87411

- produced CP4 EPSPS protein 3

9 Broad Range Molecular Weight Markers 4.5

10 Blank (cropped from the image)

-- 200.0

116.25

97.4
66.2

45.0

31.0

21.5
14.4

6.5 1 2 3 4 5 6 7 8 9 Lane #

MW
( kDa )Monsanto Company CR240

- 13U1 275 of 374 C .2

. 7 Glycosylation Analysis of CP4 EPEPS

C .2

. 7 .1 Methods ECL Glycoprotein Detection Kit (GE Healthcare) was used for glycoprotein detection.

The MON 87411

- produced CP4 EPSPS protein, the E. coli

- produced CP4 EPSPS protein, and a positive control, transferrin ( Sigma

- Aldrich ), were diluted in 1× LB and

heated to 100 °C for 4 min. Two amounts (~100 and ~200 ng) of the intact the

MON 87411

- produced CP4 EPSPS protein (purity corrected) and the E. coli

- produced

CP4 EPSPS protein (purity corrected) were loaded onto a pre

- cast Tris

- glycine 4

– 20 % polyacrylamide gradient mini

- gel (Invitrogen). Four amounts (~50, ~100, ~150, and

~200 ng) of the positive control were loaded on the gel. Protein MW Standards ( Precision Plus Protein TM Standards Dual color, Bio

- Rad

, ) were also loaded for MW reference and to verify electrotransfer of the proteins to the membrane. Following electrophoresis at a constant voltage, proteins were electrotransferred to a PVDF membrane (Invitrogen).

Glycosyl ation analysis was performed on the PVDF membrane at room temperature using the Amersham ECLTM glycoprotein Detection Module (GE Healthcare) as directed by the manufacturer. Glycosylated proteins were detected using ECLTM reagents (GE Healthcare) and Amersh am Hyperfilm (GE Healthcare). The film was developed using a Konica SRX 101A automated film processor (Konica Minolta). An identical gel was run and electrotransferred to a PVDF membrane in parallel. Proteins were stained with

Coomassie Brilliant Bl ue R 250 staining solution (Bio

- Rad) and then destained with 1 × Coomassie Brilliant Blue R 250 Destaining Solution (Bio

- Rad). After washing with water, the bl ot was scanned using Bio Rad GS

- 800 densitometer

.

C

. 2.7 .2 Results of Glycosylation Analysis

To test whether the CP4 EPSPS protein was glycosylated when expressed in the seed of

MON 87411 , the MON 87411

- produced CP4 EPSPS protein was analyzed using an ECL TM Gl ycoprotein Detection Module (GE Healthcare, Piscataway, NJ). To assess equivalence of the MON 87 411

- and E. coli

- produced CP4 EPSPS proteins, the

E. coli

- produced CP4 EPSPS protein, previously been shown to be free of glycosyl ation (Harrison et al. 1996) was also analyzed.

A clear glycosylation signal was observed at the expected molecular weight i n the lanes containing t he positive control (transferrin) and the band intensity increased with increasing concentration (Figure C

- 8 , Panel A, lanes 2

- 5). In contrast, signals were not observed in the lanes containing the MON 87411

- or E. coli

- produced protein at the expected molecular weight for the CP4 EPSPS protein (Figure C

- 8 panel A, lanes 6

- 9).

To confirm that sufficient MON 87411

- and E. coli

- produced CP4 EPSPS proteins were present for glycosylation analysis, a second membrane (with identical lo adings and transfer times) was stained with Coomassie Blue R250 for protein detection (Figure C

- 8

Panel B). Both the MON 87411

- and E. coli

- produced CP4 EPSPS proteins were clearly detected (Figure C

- 8 , Panel B, Lanes 6

- 9).Monsanto Company CR240

- 13U1 276 of 374 These data indicate that the glycosylation status of MON 87411

- produced CP4 EPSPS protein is equivalent to that of the E. coli

- produced CP4 EPSPS protein and that neither is glycosylated

.

Figure C

- 8 . Glycosylation Analysis of the MON 87411

- produced CP4 EPSPS

Protein Aliquots of tran sferrin (positive control), E. coli

- produced CP4 EPSPS protein and

MON 87411

- produced CP4 EPSPS protein were subjected to SDS

- PAGE and electrotransferred to PVDF membranes. The MWs (kDa) correspond to the Precision

Plus Protein TM Standards. The arrows sh ow the expected migration of the

MON 87411

- produced and E. coli

- produced CP4 EPSPS proteins. (A) Where present, labeled carbohydrate moieties were detected by addition of streptavidin conjugated to HRP followed by a luminol

- based detection using ECL reage nts and exposure to Hyperfilm **** . (B) An equivalent blot was stained with Coomassie Blue R250 to confirm the presence of proteins. Lane designations and the approximate mass loaded are as

follows:

Lane Sample Amount (ng)

1 Precision Plus Protein TM Standards

- 2 Transferrin (positive control) 50

3 Transferrin (positive control) 100

4 Transferrin (positive control) 150

5 Transferrin (positive control) 200

6 E. coli

- produced CP4 EPSPS protein (negative control) 100

7 E. coli

- produced CP4 EPSPS protein (negative control) 200

8 MON 87411

- produced CP4 EPSPS protein 100

9 MON 87411

- produced CP4 EPSPS protein 200

10 Blank

- Lane # 2 3 4 5 6 7 8 9 10 Lane # 1 2 3 4 5 6 7 8 9 10

250

150 100 75

50

37

25 20
15 10 250

150 100 75

50

37

25 20
15 10 A. B.Monsanto Company CR240

- 13U1 277 of 374 C .2

. 8 CP4 EPSPS Functional Activity Analysis

C .2

. 8 .1 Methods

In preparation for analysis the E. coli

- produced CP4 EPSPS was diluted with storage

buffer to the same purity corrected concentration as the MON 87411

- produced protein (0.2 63 mg CP4 EPSPS /ml). Prior to functional activity analysis, both MON 87411

- and

E. coli

- produced proteins were diluted with 50 mM HEPES, pH 7.0 buffer to a purity

corrected concentration of ~50 μg/ml. Assays for both proteins were conducted in triplicate. The reactions were performed in 50 mM HEPES, pH 7.0, 0.1 mM ammonium molybdate, 1 mM P EP and 5 mM potassium fluoride with or without 2 mM S

- 3

- P for

2 min at ~25 **°** C. The reactions were initiated by the addition of PEP. After 2 min, the reactions were quenched with phosphate assay reagent (0.033% malachite green,

1.1% ammonium molybdate) an d then fixed with 33% (w/v) sodium citrate. A standard curve was prepared using 0 to 10 nmoles of inorganic phosphate in water treated with the phosphate assay reagent and 33% (w/v) sodium citrate. The absorbance of each reaction

and each standard was me asured in duplicate at 660 nm using a PowerWave Xi **** microplate reader. The amount of inorganic phosphate released from PEP in each reaction was determined using the standard curve. For CP4 EPSPS, the specific activity was defined in unit per mg of protei n (U/mg), where a unit (U) is defined as 1 μmole of inorganic phosphate released from PEP per min at 25 **°** C. Calculations of the specific activities were performed using Microsoft Excel (2007).

C .2

. 8 .2 Results of Functional Activity

The functio nal activ ities of the MON 87411

- and E. coli

- produced CP4 EPSPS proteins were determined using a colorimetric assay that measures formation of inorganic phosphate (Pi) from the EPSPS

- catalyzed rea ction between shikimate

- 3

- phosphate (S

- 3

- P) and PEP. In this assay, protein

- specific activity is expressed as units per milligram of protein (U/mg), where a unit is defined as one μmole of inorganic phosphate released from PEP per mi nute at 25 °C. The MON 87411

- and E. coli

- produced

CP4 EPSPS proteins were considered to h ave equivalent functional activity if the specific activities of both were within the acceptance limits of 1.96 to 7.90 U/mg.

The experimentally determined specific activities for the MON 87411

- and

E. coli

- produced CP4 EPSPS pro teins are presented in Table C

- 10 . The specific

activities of MON 87411

- and E. coli

- produced CP4 EPSPS proteins were 5.78 U/mg and

5.00 U/mg of CP4 EPSPS protein, respectively. Because the specific activity of the

MON 87411

- produced CP4 EPSPS protein falls within the prese t a cceptance criterion (Table C

- 10 ), the MON 87411

- produced CP4 EPSPS protein was considered to have equivalent functional activity to that of the E. coli

- produced CP4 EPSPS protein.Monsanto Company CR240

- 13U1 278 of 374 Table C

- 10

. CP4 EPSPS Functional Activity Assay

MON 87411

- Produced

CP4 E PSPS Protein 1
(U/mg) E. coli

- Produced

CP4 EPSPS Protein 1
(U/mg) Preset Acceptance Limits for

MON 87411

- Produced CP4 EPSPS (U/mg)

5.78 ± 0.19 5.00 ± 0.38 1.96

– 7.90

1 Value refers to mean and standard deviation calculated based on n=6 which includes three replicate assays spectrophometrically read in duplicate plate wells.Monsanto Company CR240

- 13U1 279 of 374 References for Appendix C

Aebersold, R. 1993. Mass spectrometry of proteins and peptides in biotechnology. Current Opinion in Biotechnology 4: 412

- 419.

Billeci, T.M. and J.T. Stults. 1993. Tryptic mapping of recombinant proteins by matrix

assisted laser desorption/ionization mass spectrometry. Analytical Chemistry 65: 1709

- 1716.

Choi

- Rhee, E. and J.E. Cronan. 2003. The biotin carboxylase

- biotin carboxyl carrier protein complex of Escherichia coli acetyl

- CoA carboxylase. J Biol Chem 278: 30806

- 30812.

Harrison, L.A., M.R. Bailey, M.W. Naylor, J.E. Ream, B.C. Hammond, D.L. Nida, B.L

. Burnette, T.E. Nickson, T.A. Mitsky, M.L. Taylor, R.L. Fuchs, and S.R. Padgette. 1996. The expressed protein in glyphosate

- tolerant soybean, 5

- enolpyruvylshikimate

- 3

- phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and i s not toxic to acutely gavaged mice. The Journal Of Nutrition 126: 728

- 740.

Hunkapiller, M.W., R.M. Hewick, L.E. Hood and W.J. Dreyer. 1983. High

- sensitivity sequencing with gas

- phase sequenator. Methods in Enzymology 91: 399

- 413.

Polevoda, B. and F. She rman. 2000. N α

- terminal acetylation of eukaryotic proteins. J

Biol Chem 275: 36479

- 36482.

Rademacher, T.W., R.B. Parekh an d R.A. Dwek. 1988. Glycobiology. Annual Review of Biochemistry 57: 785

- 838.Monsanto Company CR240

- 13U1 280 of 374 Appendix D : Materials and Methods Used for the Analysis of the Levels of Cry3Bb1 and CP4 EPSPS Protein s in MON 87411

D.1. Materials

Over season leaf (OSL1

- 4), over season root (OSR1

- 4), over season whole plant (OSWP1

- 4), stover, senescent root, forage root, forage , grain, pollen and silk tissue samples from glyphosate

- treated MON 87411 were harvested from five field sites in Argentina during the 2011

– 2012 growing season from starting seed lot 11320173.

E coli

- produced Cry3Bb1 (lot 10000775) and CP4 EPSPS protein (lot 10000739) were used as the analytical reference standards.

D.2. Characterization of the Materials

The identity of MON 87411 was confirmed by conducting MON 87411 event

- specific polymerase chain reaction (PCR) analyses on the harvested grain from eac h site.

D.3. Field Design and Tissue Collection

Field trials were initiated during the 2011

– 2012 planting season to generate

MON 87411 at various maize growing locations in Argentina. OSL1

- 4, OSR1

- 4,

OSWP1

- 4, stover, senescent root, forage root, fora ge, grain, pollen and silk tissue samples from the following field sites were analyzed: Pergamino, Buenos Aires (Site Code BAFO); Hunter, Buenos Aires (Site Code BAHT); Pergamino, Buenos Aires (Site Code BAPE); Sarasa, Buenos Aires (Site Code BASS) and Sal to, Buenos Aires (Site Code BATC). At each site, four replicated plots of plants containing MON 87411 were planted using a randomized complete

- block field design. Tissue samples were collected from each replicated plot at all field sites. See Tables V

- 1 and V

- 2 for detailed descriptions of when the samples were collected.

D.4. Tissue Processing and Protein Extraction

All tissue samples harvested were shipped to Monsanto’s processing facility, and were ground, except pollen, by the Monsanto Sample Manage ment Team to facilitate protein extraction. The ground tissue samples, and pollen, were stored in a

- 80 °C freezer until transferred on dry ice to the analytical facility.

D.4.1. Cry3Bb1 Protein

The Cry3Bb1 protein was extracted from maize tissues as described in Table D

- 1. The protein extracts were aliquoted and stored frozen in a

- 80 °C freezer until analysis.Monsanto Company CR240

- 13U1 281 of 3 74 Table D

- 1. Cry3Bb1 Protein Extraction Methods 1 for Tissue Samples

Sample Type Tissue

- to

- Buffer

Ratio Extraction Buffer

Leaf 2 /Grain/Pollen/Silk/Root 3 /Forage 4 1:100 50 mM sodium

carbonate

- bicarbonate

+ 0.1% (w/v) BSA 5

1 Cry3Bb1 protein was extracted from each tissue by adding the appropriate volume of extraction buffer and

chrome steel beads, and shaking in a Harbil mixer (Harbil Industries Inc., Compton, CA). The extracted samples were clarified using a serum filter.

2 Over season leaf (OSL1, OSL2, OSL3, and OSL4).

3 Over season root (OSR1, OSR2, OSR3, and OSR4), forage root a nd senescent root.

4 Forage, stover and over season whole plant (OSWP1, OSWP2, OSWP3, and OSWP4).

5 50 mM sodium carbonate

- bicarbonate with 0.1 % bovine serum albumin (BSA) [0.015 M Na 2 CO 3 ,

0.035 M NaHCO 3 , and 0.1% (w/v) BSA].

D .4.2. CP4 EPSPS Protein

CP4 EPSPS protein was extracted from maize tissues samples as described in Table D

- 2. The protein extracts were aliquoted and stored frozen in a

- 80 °C freezer until analysis.

Table D

- 2. CP4 EPSPS Protein Extraction Methods 1 for Tissue Samples

Sample Type Tissue

- to

- Buffer

Ratio Extraction Buffer

Leaf 2 /Pollen/Silk/Forage 3 1:100 1× PBST + 0.1% (w/v) BSA 5

Grain 1:100 1× TBA + 10 mM DCA 6

Root 4 1:50 1× PBST + 0.1% (w/v) BSA

1 CP4 EPSPS protein was extracted from each tissue by adding the appropriate volume of extraction buffer

and chrome steel beads, and shaking in a Harbil mixer (Harbil Industries Inc., Compton, CA). The extracted samples were clarified using a serum fi lter.

2 Over season leaf (OSL1, OSL2, OSL3, and OSL4).

3 Forage, stover and over season whole plant (OSWP1, OSWP2, OSWP3, and OSWP4).

4 Over season root (OSR1, OSR2, OSR3, and OSR4), forage root and senescent root.

5 Phosphate buffered saline buffer with Tween

- 20 and 0.1% bovine serum albumin [0.001 M KH 2 PO 4 ,

0.01 M Na 2 HPO 4 , 0.137 M NaCl, and 0.0027 M KCl with 0.05% (v/v) Tween

- 20 and 0.1% (w/v) BSA].

6 Tris

- borate buffer (pH 7.8) with L

- ascorbic acid and 10 mM deoxycholic acid [0.1 M Tris,

0.1 M Na 2 B 4 O 7 , 0.005 M MgCl 2 , 0.05% (v/v) Tween

- 20, 0.2% (w/v) L

- ascorbic acid and 0.01 M DCA].Monsanto Company CR240

- 13U1 282 of 374 D.5. Cry3Bb1 and CP4 EPSPS Antibodies

D.5.1. Cry3Bb1 Antibodies

Goat polyclonal antibodies specific for the Cry3Bb1 protein were purified using

Protein G affinity chromatography. The concentration of the purified IgG was

determined to be 4.5 mg/ml by spectrophotometric methods. The purified antibody was stored in (1×PBS) (0.001 M KH 2 PO 4 , 0.01 M Na 2 HPO 4 , 0.137 M NaCl, and

0.0027 M KCl).

Purified Cry3Bb1 antibodies were coupled with biotin (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The detection reagent was NeutrAvidin (Thermo Fisher Scientific) conjugated to horseradish peroxidase.

D.5.2. CP4 EPSPS Antibodies

Mouse monocl onal antibody clone 39B6.1 (IgG2a isotype, kappa light chain) specific for

the CP4 EPSPS protein was purified from mouse ascites fluid using Protein A affinity chromatography. The concentration of the purified IgG was determined to be 2.3 mg/ml by spectro photometric methods. Production of the 39B6.1 monoclonal antibody was performed by Strategic Biosolutions (Newark, DE). The purified antibody was stored in a buffer (0.02 M NaH 2 PO 4 , 0.15 M NaCl, and 15 ppm Proclin 300). The detection

reagent was goat an ti

- CP4 EPSPS antibody, otherwise known as anti

- protein 4 (Sigma

Aldrich, Saint Louis, MO) conjugated to horseradish peroxidase.

D.6. Cry3Bb1 and CP4 EPSPS ELISA Methods

D.6.1. Cry3Bb1 Protein

Goat anti

- Cry3Bb1 capture antibodies were diluted in a coating buffer (0.015 M Na 2 CO 3 , 0.035 M NaHCO 3 ,) and immobilized onto 96

- well microtiter plates at 5 **μ** g/ml followed by incubation in a 4 **°** C refrigerator for **≥** 12 hours. Prior to each step in the assay, plates were washed with 1× PBS containing 0.05 % (v/v) Tween 20 (1× PBST) . Plates were

blocked with the addition of 150 μl per well of 1× PBS with 0.25% (w/v) casein for

60 to 70 minutes at 37 **°** C. Cry3Bb1 protein standard or sample extract was added at

100 **μ** l per well and incubated for 60 to 70 minutes at 37 **°** C. Biotinylated goat

anti

- Cry3Bb1 antibodies were added at 100 **μ** l per well and incubated for

60 to 70 minutes at 37 **°** C. NeutraAvidin

- horseradish peroxidase conjugate was added at

100 **μ** l per well and incubated for 30 to 35 minutes at 37 **°** C. Plates were deve loped by adding 100 μl per well of horseradish peroxidase substrate, 3,3',5,5'

- tetramethylbenzidine (TMB, Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction was terminated by the addition of 100 μl per well of 6 M H 3 PO 4 . Quantification of the Cry3Bb1 protein was accomplished by interpolation from a Cry3Bb1 protein standard curve that ranged

from 0.35

- 11.2 ng/ml.Monsanto Company CR240

- 13U1 283 of 374 D.6.2. CP4 EPSPS Protein

The CP4 EPSPS ELISA was performed using plates commercially coated at 2 μg/ml (Agri Diagnostic Manufac turing, Rickreall, OR). CP4 EPSPS protein standard or sample

extract was added at 100 **μ** l per well and incubated for 60 to 70 minutes at 37 **°** C. Prior to each step in the assay, plates were washed with 1× PBST. Goat anti

- CP4 EPSPS conjugated to horseradis h peroxidase was added at 100 **μ** l per well and incubated for

60 to 70 minutes at 37 **°** C. Plates were developed by adding 100 μl per well of horseradish peroxidase substrate, TMB. The enzymatic reaction was terminated by the

addition of 100 μl per well of 6 M H 3 PO 4 . Quantification of the CP4 EPSPS protein was accomplished by interpolation from a CP4 EPSPS protein standard curve that ranged

from 0.456

- 14.6 ng/ml.

D.7. Moisture Analysis

Tissue moisture content was determined using a Mettler Toledo HR83 Moi sture Analyzer System (Mettler

- Toledo, Inc. Columbus, OH). A homogeneous tissue

- specific site pool (TSSP) was prepared consisting of samples of a given tissue type grown at a specific site. The mean percent moisture for each TSSP was calculated from trip licate analyses. A TSSP dry weight conversion factor (DWCF) was calculated using MoistureDirect version

4.0 software as follows:

DWCF = 1

− � Mean % TSSP Moisture

100 �

The DWCF was used to convert protein levels assessed on a μg/g fresh weight (fw) basis

into lev els reported on a μg/g dry weight (dw) basis using the following calculation:

Protein Level in Dry Weight = Protein Level Fresh Weight

DWCF

The protein levels (ng/ml) that were reported to be less than or equal to the limit of detection (LOD) or less than t he limit of quantitation (LOQ) on a fresh weight basis were not reported on a dry weight basis.

D.8. Data Analyses

ELISA plates were analyzed on a SPECTRAmax Plus 384 (Molecular Devices, Sunnyvale, CA) microplate spectrophotometer, using a dual wavelength detection method. Protein concentrations were determined by optical absorbance at a wavelength

of 450 nm with a simultaneous reference reading of 620 nm. Data reduction analyses were performed using Molecular Devices SOFTmax PRO GxP version 5.4 software

. Absorbance readings and protein standard concentrations were fitted with a four

parameter logistic curve fit. Following the interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was reported on a “μg/g fw” basis for data th at were greater than or equal to the LOQ. This conversion utilized a sample dilution factor,

and tissue

- to

- buffer ratio. The protein values in “μg/g fw” were also converted toMonsanto Company CR240

- 13U1 284 of 374 “μg/g dw” by applying the DWCF. Microsoft Excel 2007 (Microsoft, Redmond, WA) was used to calculate the protein levels in all maize tissues. The sample means, standard deviations (SDs), and ranges were also calculated using Microsoft Excel 2007. All protein expression levels were rounded to two significant figures.

A test substan ce extract that resulted in unexpectedly negative result by ELISA analysis

was re

- extracted twice for the protein of interest and re

- analyzed by ELISA to confirm the result. The sample with the confirmed unexpected result was omitted from all

calculations

.Monsanto Company CR240

- 13U1 285 of 374 Appendix E : Materials, Methods and Results for Characterization of the

DvSnf7 RNA Produced in MON 87411

E .1 Materials

The MON 87411 RNA was extracted from ~V6 maize MON 87411 leaf tissue. The

MON 87411 RNA was resuspended in water and stored in a

- 80 ° C freezer. The in vitro

produced DvSnf7 RNA (Dvsnf7_968 RNA; lot 11331164 ) was used as the reference

substance. The DvSnf7_968 RNA was in vitro transcribed from the plasmid pMON149601 and purified using acid phenol:chloroform, precipitated with isop ropanol and resuspended in UltraPure Water (Invitrogen). The in vitro transcribed DvS n f7_968 RNA was previously characterized.

E

. 2. RNA extraction and Poly(A) enrichment

The MON 87411 RNA was extracted and enriched from maize MON 87411 leaf tissue. The extraction and enrichment procedure was not performed under a GLP plan; however, all procedures were documented on worksheets and, where applicable, SOPs were

followed.

MON 87411 RNA was extracted from MON 87411 leaf tissue. Prior to extraction, l eaf tissue was processed into a fine powder using a Harbil paint shaker. Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the

m anufacturer’s instructions

. All extracted RNA was eluted by water and stored in a

- 80°C freezer.

MON 87411 poly(A) RNA was enriched from MON 87411 RNA using the Ambion MicroPoly (A) Purist MAG kit (Life Technologies, Carlsbad, CA) as per manufacturer’s specifications. All MON 87411 poly(A) RNA was resuspended in nuclease

- free water
and stored in a

- 80 °C freezer.

E .3 . Sequence analyses

E .3 .1. Methods

DNA sequence analysis was performed to confirm the identity of MON 87411 DvSnf7 RNA and to compare its sequence to that of DvSnf7_968 RNA

.

MON 87411 DvSnf7 RNA was reverse transcribed us ing a sequence specific primer to produce cDNA (complementary DNA) utilizing an Omniscript Reverse Transcription kit from Qiagen (Valencia, CA). The manufacturer’s specifications were followed with the exception of the incubation time, which was run for 3 0 min. The resulting cDNA was

stored in a

- 20 °C freezer.

Two overlapping PCR products (Product A and Product B) were generated that span the expected primary transcript sequence of MON 87411 DvSnf7 RNA (Figure E

- 1). These products were analyzed to deter mine the nucleotide sequences of MON 87411 DvSnf7

RNA.Monsanto Company CR240

- 13U1 286 of 374 The PCR amplification of Product A was performed on 1 μl of diluted MON 87411

DvSnf7 cDNA in a 20 **μ** l reaction volume. Each reaction contained a final concentration

of 0.2 **μ** M of each primer, 1 mM MgCl 2 , 0.2 mM dNTP mix, and 0.5 units of PrimeSTAR® GXL Polymerase (Takara Bio Inc., Shiga, Japan). The following cycling

conditions were followed: 38 cycles at 98 **°** C for 10 seconds; 60 **°** C for 15 seconds;

68 **°** C for 5 seconds with an initial denaturation step at 98 **°** C for 2 min and a final

extension cycle for 5 min at 68 **°** C.

The PCR amplification of Product B was performed on 1 μl of diluted MON 87411

DvSnf7 cDNA in a 20 **μ** l reaction volume. Each reaction contained a final concentration

of 0.2 **μ** M of each prime r, 1 mM MgCl 2 , 0.2 mM dNTP mix, and 0.5 units of PrimeSTAR® GXL Polymerase (Takara Bio Inc., Shiga, Japan). The following cycling

conditions were followed: 35 cycles at 98 **°** C for 10 seconds; 60 **°** C for 15 seconds;

68 **°** C for 30 seconds with an initial dena turation step at 98 **°** C for 2 min and a final

extension cycle for 5 min at 68 **°** C.

Aliquots of each PCR product w ere separated on a 1.2% (w/v) agarose gel and visualized by ethidium bromide staining to verify that the products were the expected size. Prior to sequencing, each verified PCR product was purified using the QIAquick Gel Extraction

Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions and quantified using a Nanodrop spectrophotometer according to the manufacturer’s

instructions. The purified PCR products were sequenced using multiple primers, including primers used for PCR amplification.

Al l sequencing was performed by Monsanto TGAC (The Genome Analysis Center ) using BigDye TM terminator chemistry (Applied Biosystems, Foster City, CA). A consensus sequence was generated by compiling sequences from multiple sequencing reactions performed on th e overlapping PCR products. This consensus sequence was obtained to determine the identity of MON 87411 DvSnf7 RNA. The consensus sequence was also aligned to the sequence of DvSnf7_968 cD NA which was established during its characterization (lot 11331164 ). This alignment established the equivalence between

MON 87411 DvSnf7 RNA and DvSnf7_968 RNA.

E .3 .2. Results of Sequence Analyses

In order to characterize the MON 87411 DvSnf7 RNA, and to demonstrate molecular equivalence between it and DvSnf7_968 RNA, the sequence of MON 87411 DvSnf7

cDNA (complementary DNA) was determined. MON 87411 DvSnf7 RNA was reverse transcribed to cDNA and amplified by PCR. The amplified MON 87411 DvSnf7 cDNA was sequenced to obtain the primary transcript sequence which contain s the predicted transcription start and polyadenylation sites (Coruzzi et al. 1984) , and most importantly,

the 240 bp dsRNA functional region (inverted repeat) (Bolognesi et al. 2012)

. The consensus sequence of MON 87411 DvSnf7 cDNA was aligned to the seq uence of cDNA

from DvSnf7_968 RNA ( DvSnf7_968 cDNA) which was previously characterized under APS characterization plan 11331164 . In this APS characterization, DvSnf7_968 RNA was shown to consist of 968 nucleotides encompassing the predicted transcription start,Monsanto Company CR240

- 13U1 287 of 374 the polyadenylation site, and the 240 bp dsRNA

. This analysis demonstrated that the sequenced regions between MON 87411 DvSnf7 cDNA and DvSnf7_968 cDNA are identical. More importantly, it was shown that t here is a 100% sequence identity in the inverted repeat regions between the MON 87411 DvSnf7 cDNA consensus sequence and the DvSnf7_968 cDNA reference sequence (Figure E

- 2 ). This analysis serves as identity

confirmation of the MON 87411 DvSnf7 RNA as well as demonstrating the molecular equivale nce between MON 87411 DvSnf7 RNA and DvSnf7_968 RNA

.

E .4 . Northern Blot Analysis

E .4 .1. Methods

Northern blot analysis was carried out to characterize MON 87411 DvSnf7 RNA and to demonstrate the molecular equivalence between MON 87411 DvSnf7 RNA and

DvSn f7_968 RNA. The dsRNA regions of MON 87411 DvSnf7 RNA and DvSnf7_968 RNA were specifically targeted through RNase I f treatment of the RNAs. RNase I f preferentially digests single stranded RNA leaving behind double stranded region s such as the active regio n of DvSnf7. It is this 240 bp dsRNA , that is efficiently taken up by WCR and is processed by the RNAi machinery, ultimately leading to mRNA degradation. Therfore, RNase I f digestion facilitates a direct comparison of the size equivalence between the activ e dsRNA regions of MON 87411 DvSnf7 RNA and DvSnf7_968 RNA.

DvSnf7_968 RNA and poly (A) RNA from MON 87411 plant tissues were digested with

RNase I f (New England Biolabs, Ipswich, MA) using the manufacturer’s suggested

reaction conditions for 5 minutes. Following digestion, the samples were heat inactivated

at 70 °C for 20 minutes, precipitated, and resuspended in nuclease

- free water.

RNase I f digested MON 87411 poly (A) RNA and DvSnf7_968 RNA were resolved on a

~6.6% (w/v) formaldehyde/2.5% ag arose gel buffered by 1x MESA. The gel was run at

100V for ~2.5 hours. An appropriate molecular size marker was included on the gel. After running, the gel was stained with SYBR Gold (Life Technologies, Carlsbad, CA ) and the RNA visualized and photograp hed.

The RNA resolved on the formaldehyde/agarose gel was transferred to a neutral nylon

membrane. The ladder and wells were marked before UV light cross

- linking the RNA to

the membrane.

The n orthern blot was hybridized with a [α

32 P]

- dATP

- labeled probe specific to the 240 bp dsRNA region of DvSnf7 . Prior to hybridization, the DvSnf7 probe template was generated by PCR using plasmid PV

- ZMIR10871 as the template and primers specific to

the DvSnf7 240 bp dsRNA . The PCR products were separated by agarose g el electrophoresis and purified from the gel using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer's specifications. After purification, the probe template was quantified using a Qubit Fluorometer (Life Technologies, Carls bad,

CA ). Approximately 25 ng of probe template was labeled with [α

32 P]

- dATPMonsanto Company CR240

- 13U1 288 of 374 (~6000 Ci/mmol) using the RadPrime DNA Labeling System (Life Technologies, Carlsbad, CA ).

The hybridization was carried out at 55 °C and the membrane was washed at the same

tem perature. Two exposures of the blot were generated using Kodak Biomax MS film (Eastman Kodak, Rochester, NY) in conjunction with two Kodak Biomax MS intensifying screen in a

- 80 °C freezer. The banding in the MON 87411 DvSnf7 RNA and DvSnf7_968 RNA lanes was visually compared

, after a one hour exposure, to determine their size similarity and the identity of MON 87411 DvSnf7 RNA.

E .4 .2. Results of the Northern Blot Analysis

P oly (A) RNA from MON 87411 plant tissues (MON 87411 poly (A) RNA) and DvSnf7_968 RNA were digested by RNase I f which preferentially digests single stranded RNA leaving dsRNA regions intact. Each RNase I f digested sample was loaded in triplicate and subjected to n orthern blot analyses. The n orthern blot was hybridized with a probe specific to the 240 bp dsRNA region of DvSnf7 in order to characterize

MON 87411 DvSnf7 RNA by identifying the presence of the functionally active double

stranded region of DvSnf7 RNA

. Additionally, a comparison of the dsRNA regions of

MON 87 411 DvSnf7 RNA and DvSnf7_968 RNA was conducted using this n orthern blot analysis to determine equivalence

. The results are shown in Figure E

- 3. MON 87411

DvSnf7 RNA contains the expected ~240 bp dsRNA region of DvSnf7 as indicated by

the

~ 240 base band on the blot (Lanes 4

- 6). This is the expected size of the double stranded functionally active region of DvSnf7 (Bolognesi et al. 2012)

. Furthermore, DvSnf7_968 RNA (lanes 1

- 3) exhibits a single band at approximately 240 bases, similar

to MON 87411 DvSnf7 RNA (lanes 4

- 6), indicating that MON 87411 DvSnf7 RNA and DvSnf7_968 RNA are equivalent with respects to their dsRNA functionally active regions.Monsanto Company CR240

- 13U1 289 of 374 Figure E

- 1

. PCR Amplification of MON 87411 DvSnf7 cDNA PCR was performed on MON 87411 DvSnf7 cDNA using two pairs of primers to generate overlapping PCR fragments for sequencing analysis. To verify the PCR products, the PCR reactions were run on a gel. The expected product size for each amplicon is provided in the

illustration of the MON 87411 DvSnf7 transcript (A region of I

- Hsp70 is spliced out.) The

MON 87411 DvSnf7 suppression cassette is depicted above the DvSnf7 transcript. This figure is a representative of the data generated in the study and the ill ustrations are not to scale. Lane designations are as follows:

Lane Sample

1 1 Kb DNA Plus Ladder

2 MON 87411 DvSnf7 cDNA

3 Reverse transcriptase reaction minus reverse transcriptase

4 No template control

5 MON 87411 DvSnf7 cDNA

6 Reverse transcriptase reaction minus reverse transcriptase

7 No template control Arrows next to the agarose gel image denote the size of the DNA, in kilobase pairs, obtained from the 1kb DNA Plus Ladder (Invitrogen) on the ethidium bromide stained gel

. Product B: ~ 609 bp

Product A: ~ 425 bp 0.1 0.2 0.3 0.4 0.5 0.65 0.85 1.0 1.65 2.0 3.0 12.0 1 2 3 4 5 6 7 Product A Product BTranscription

DvSnf7 p DvSnf7 pDvSnf7pDvSnf7pMonsanto Company CR240

- 13U1 290 of 374 DvSnf7 87411 1 ACACGCTGAACCGTCTTCGATACCAAGCGGGAGCTCGACGTCCCTCAGCAGTCGCTGTGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 1 ACACGCTGAACCGTCTTCGATACCAAGCGGGAGCTCGACGTCCCTCAGCAGTCGCTGTGC

DvSnf7 87411 61 GATACCATCCATGATATCGTGAACATCATCTACATTCAAATTCTTATGAGCTTTCTTAAG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 61 GATACCATCCATGATATCGTGAACATCATCTACATTCAAATTCTTATGAGCTTTCTTAAG

DvSnf7 87411 121 GGCATCTGCAGCATTTTTCATAGAATCTAATACAGCAGTATTTGTGCTAGCTCCTTCGAG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 121 GGCATCTGCAGCATTTTTCATAGAATCTAATACAGCAGTATTTGTGCTAGCTCCTTCGAG

DvSnf7 87411 181 GGCTTCCCTCTGCATTTCAATAGTTGTAAGGGTTCCATCTATTTGTAGTTGGG TCTTTTC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 181 GGCTTCCCTCTGCATTTCAATAGTTGTAAGGGTTCCATCTATTTGTAGTTGGGTCTTTTC

DvSnf7 87411 241 CAATCGTTTCTTCTTTTTGAGGGCTTGGAGTGCAACTCTTTT ATTTTTCGACGCATTTTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 241 CAATCGTTTCTTCTTTTTGAGGGCTTGGAGTGCAACTCTTTTATTTTTCGACGCATTTTT

DvSnf7 87411 301 CTTTGCAAGTACTGCGATCGCGTTAACGCTTTATCACGATACCTTCTACCACATATCACT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 301 CTTTGCAAGTACTGCGATCGCGTTAACGCTTTATCACGATACCTTCTACCACATATCACT

DvSnf7 87411 361 AACAACATCAACACTCATCACTCTCGACGACATCCACTCGATCACTACTCTCACACGACC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 361 AACAACATCAACACTCATCACTCTCGACGACATCCACTCGATCACTACTCTCACACGACC

DvSnf7 87411 421 GATTAACTCCTCATCCACGCGGCCGCCTGCAGGAGCGCAAAGAAAAATGCGTCGAAAAAT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 421 GATTAACTCCTCATCCACGCGGCCGCCTGCAGGAGCGCAAAGAAAAATGCGTCGAAA AAT

DvSnf7 87411 481 AAAAGAGTTGCACTCCAAGCCCTCAAAAAGAAGAAACGATTGGAAAAGACCCAACTACAA ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DvSnf7_968 481 AAAAGAGTTGCACTCCAAGCCCTCAAAAAGAAGAAACGATTGGAAAAGACCCAACTACAA

DvSnf7 87411 541 ATAGATGGAACCCTTACAACTATTGAAATGCAGAGGGAAGCCCTCGAAGGAGCTAGCACA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 541 ATAGATGGAACCCTTACAACTATTGAAATGCAGAGGGAAGCCCTCGAAGGAGCTAGCACA

DvSnf7 87411 601 AATACTGCTGTATTAGATTCTATGAAAAATGCTGCAGATGCCCTTAAGAAAGCTCATAAG ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Dv Snf7_968 601 AATACTGCTGTATTAGATTCTATGAAAAATGCTGCAGATGCCCTTAAGAAAGCTCATAAG

DvSnf7 87411 661 AATTTGAATGTAGATGATGTTCACGATATCATGGATTAGATCGCCAGCGGTACTCGCTGA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 661 AATTTGAATGTAGATGATGTTCACGATATCATGGATTAGATCGCCAGCGGTACTCGCTGA

DvSnf7 87411 721 GGCCTAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATG CATCAGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 721 GGCCTAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGT

Figure E

- 2 . Sequence Alignment between MON 87411 DvSnf7 cDNA and DvSnf7_968 cDNAMonsanto Company CR240

- 13U1 291 of 374 DvSnf7 87411 781 TTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 781 TTCATTGCGCACACACCAGAATCCTACTGAGTT TGAGTATTATGGCATTGGGAAAACTGT

DvSnf7 87411 841 TTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTAT ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DvSnf7_968 841 TTTTCTTGTACCATTTGTTGTGCT TGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTAT

DvSnf7 87411 901 CGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| DvSnf7_968 901 CGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCA

DvSnf7 87411 961 TTCTCAAA 968 |||||||| DvSnf7_968 961 TTCTCAAA 968

Figure E

- 2 ( c ontinued )

. Sequence Alignment between MON 87411 DvSnf7 cDNA and DvSnf7_968 cDNA The consensus sequence of MON 87411 DvSnf7 cDNA(top sequence labeled DvSnf7 87411) aligned to that of the previously characterized DvSnf7_968 cDNA (bottom sequence labeled DvSnf7_968) s equence. The inverted repeat regions are underlined. The numbering indicates base position.Monsanto Company CR240

- 13U1 292 of 374 Figure E

- 3 . Northern Blot Analysis to Confirm the Equivalence between the dsRNA in DvSnf7_968 RNA and MON 87411 DvSnf7 RNA The blot was hybridized with a 32P

- labled, DvSnf7 probe specific to the 240 bp dsRNA

. Lanes

1

- 3 contain 75 pg of DvSnf7_968 RNA that was RNase I f digested and lanes 4

- 6 contain 200 ng

of MON 87411 poly (A) RNA also subjected to RNase I f digestion. Lane d esignations are as

follows:

Lane Sample

1 DvSnf7_968 RNA (RNase I f digested )

2 DvSnf7_968 RNA (RNase I f digested)

3 DvSnf7_968 RNA (RNase I f digested )

4 MON 87411 poly (A) RNA (RNase I f digested)

5 MON 87411 poly (A) RNA (RNase I f digested )

6 MON 87411 poly (A) RNA (RNase I f digested ) Arrows denote the size of the RNA, in bases, obtained from the Low Range Ribo Ruler ladder on the SYBR Gold stained gel.Monsanto Company CR240

- 13U1 293 of 374 References for Appendix E

Bolognesi, R., P. Ramaseshadri, J. Anderson, P. Bachman, W. Clinton, R. Flannagan, O. Ilagan, C. Lawrence, S. Levine, W. Moar, G. Mueller, J. Tan, J. Uffman, E. Wiggins, G. Heck and G. Segers. 2012. Characterizing the mechanism of action of double

- stranded RNA activity against Western corn rootworm ( Diabrotica vir gifera virgifera LeConte).

PLoS ONE 7:e47534.

Coruzzi, G., R. Broglie, C. Edwards and N.

- H. Chua. 1984. Tissue

- specific and light

regulated expression of a pea nuclear gene encoding the small subunit of ribulose

- 1, 5

- bisphosphate carboxylase. EMBO Journal 3:1671

- 1679.Monsanto Company CR240

- 13U1 294 of 374 Appendix F : Materials and Methods Used for the Analysis of Expression Levels

of DvSnf7 RNA in MON 87411

F .1 Materials

Over season leaf (OSL1, OSL2, OSL3, and OSL4), over season root (OSR1, OSR2, OSR3, and OSR4), over season whole plant ( OSWP1, OSWP2, OSWP3, and OSWP4), forage root, forage, senescent root, stover, pollen, silk, and grain tissue samples from glyphosate treated plants grown from starting seed lot 11320173 were collected at five field sites in Argentina during 2011

- 2012 growi ng season. MON 87411 plots were

treated at the 2

- 4 leaf stage with glyphosate herbicide at the label rate (0.95 kg active ingredient [a.i.]/ha). An in vitro transcribed DvSnf7 RNA, DvSnf7_968 (lot: 11331164), was used as an analytical reference standard for the assessment of DvSnf7 RNA levels

from MON 87411.

F .2 Characterization of the Materials

The identity of MON 87411 was confirmed by analysis of the harvested grain DNA by an

event

- specific polymerase chain reaction (PCR) method.

F .3. Field Design and Tissue Collection

Field trials were initiated during the 2011 planting season to generate MON 87411 over season leaf (OSL1 through OSL4), over season root (OSR1 through OSR4), over season whole plant (OSWP1 through OSWP4), forage root, forage, senescen t root, stover, pollen, silk, and grain at various maize growing locations in Argentina. The tissue samples from the following field sites were analyzed: Pergamino, Buenos Aires (site code BAFO); Hunter, Buenos Aires (site code BAHT); Pergamino, Buenos Ai res (site code BAPE); Sarasa, Buenos Aires (BASS); and Salto, Buenos Aires (site code BATC). These field sites were representative of maize producing regions suitable for commercial production. At each site, four replicated plots of plants containing MON 87411 were planted using a randomized complete

- block field design. Over season leaf (OSL1 through OSL4), over season root (OSR1 through OSR4), over season whole plant (OSWP1 through OSWP4), forage root, forage, senescent root, stover, pollen , silk, and

g rain samples were collected from each replicated plot at all field sites. See Table VI

1 for detailed descriptions of when the samples were collected.

F .4. Tissue Processing and Total RNA Extraction

Tissue samples were shipped on dry ice to Monsanto Compa ny (Saint Louis, Missouri)

and stored in

- 80° C freezers. All tissue samples except for pollen, were roughly ground with dry ice by the Monsanto Sample Management Team. The roughly ground samples and pollen samples were further processed to fine powder u sing liquid nitrogen . The prepared tissue samples were stored in

- 80° C freezers before and after transferred on dry ice to the analytical facility.Monsanto Company CR240

- 13U1 295 of 374 Total RNA from leaf (OSL1 through OSL4), root (OSR1 through OSR4), whole plant (OSWP1 through OSWP4), for age root, senescent root, and silk tissues was extracted using a Trizol method (Chomczynski and Sacchi 1987) and is briefly described below. On dry ice, approximately 0.1 grams of frozen processed tissue were transferred into a tube and weighed. Trizol r eagent (Ambion Biotechnology, Austin, TX) was added to the tube and mixed by vortexing. Following an incubation at RT, chloroform (Fisher Scientific, Pittsburgh, PA) was added and mixed. Following another incubation at RT, the tube was centrifuged at 12, 000 x g and ~4°C for 10 minutes. The aqueous phase was transferred to a new tube, and the RNA was precipitated with an equal volume of isopropyl alcohol (Sigma, Saint Louis, MO). To pellet the RNA, the tube was centrifuged at 12,000 x g and ~4°C for 20 m inutes. The RNA pellet was washed using 70% (v/v) ethanol and resuspended in nuclease

- free water (Gibco by Life Technologies, Grand Island, NY). All extracted RNA samples were stored in a

- 80°C freezer.

Total RNA from forage, stover, pollen, and grain ti ssues was extracted as follows. On dry ice, approximately 0.1 grams of frozen processed tissue were transferred to a tube and weighed. RNA extraction buffer (25 mM Tris

- HCl pH 8.0, 25 mM EDTA, 75 mM NaCl,

1% SDS, and 1% β

- mercaptolethanol) was added to the tube and mixed by vortexing. An equal volume of acid phenol (pH 4.3): chloroform [2:1 (v/v)] (Sigma, Saint Louis, MO; Fisher Scientific, Pittsburgh, PA, respectively) was added to the tube and mixed. The tube was centrifuged at 12,000 x g and 2

- 6 °C for 15 minutes to separate the phases. The aqueous phase was transferred to a new tube and an equal volume of acid phenol (pH 4.3): chloroform [1:1 (v/v)] was added to the tube and mixed. The tube was centrifuged at 12,000 x g and 2

- 6 °C for 10 minutes t o separate the phases. The aqueous phase was transferred to a new tube and 1⁄2 volume of 15 M LiCl was added to the tube while gently mixing. The tube was incubated on ice for ~ 2 hours to precipitate RNA. To pellet the RNA, the tube was centrifuged at 20 ,000 x g and 2

- 6 °C for 30 minutes. The RNA pellet was washed with 75% (v/v) ethanol and resuspended in nuclease

- free water (Gibco by Life Technologies). All extracted RNA samples were stored in a

- 80°C

freezer.

All extracted RNA samples were quantified using a NanoDrop

- 8000 (Thermo Scientific, Wilmington, DE) according to the manufacturer’s instructions. Prior to the analysis, each RNA sample was normalized in water to an appropriate concentration manually and/or using a Biomek FX P Laboratory Automatic Workstation (Beckman Coulter Inc., Brea, CA) according to the manufacturer’s instructions . All normalized RNA samples were stored in

a

- 80 **°** C freezer before analysis.

F .5. Analytical Reference Standard DvSnf7_968

The Analytical Reference Standard DvSnf7_ 968 was produced by in vitro transcription.

This in vitro transcribed DvSnf7 RNA corresponds to the expected in planta DvSnf7 RNA transcribed from the DvSnf7 suppression cassette in MON 87411. The sequence of the DvSnf7_968 was confirmed by sequencing an alysis. The DvSnf7_968 concentration

was 2.21 mg/ml as determined by spectrophotometric analysis.Monsanto Company CR240

- 13U1 296 of 374 F .6. QuantiGene® Plex 2.0 Assay

The normalized total RNA (Section D.2.4) was analyzed using a validated QuantiGene ®

Plex 2.0 Assay (Affymetrix Inc., Santa Clara, CA). This bead

- based assay relies on a series of oligonucleotide probes that recognize and hybridize specifically to a portion of the DvSnf7 RNA. Capture probes conjugated to a magnetic polystyrene microbead are designed to capture the DvSnf7 RNA , while blocking probes act to prevent non

- specific hybridization to unrelated RNAs, thereby increasing assay specificity. The assay also includes oligonucleotide probes which hybridize to the DvSnf7 RNA and the branched DNA molecules that mediate the sig nal amplification. Detection of the DvSnf7 RNA is by fluorescence detection of the hybridization complex while it passes through a signal detector. The fluorescence intensity is proportional to the number of the DvSnf7 RNA molecules present in the sample allowing the DvSnf7 RNA to be quantified by including a reference standard in the assay. The DvSnf7_968 ( Section F .5 ) was used as an analytical reference standard. To ensure quality, each assay also includes a background control, a negative control (QC

- ), and a positive control (QC+).

F .6.1. Target Specific Probe Set Design and Generation

The probe set used in the QuantiGene ® Plex 2.0 Assay (Affymetrix Inc.) was designed to detect transcripts containing DvSnf7 RNA (Affymetrix Inc. 2010) . The probe set was generated and supplied by Affymetrix and stored in a

- 20 **°** C freezer upon receipt.

Three RNA targets, DvSnf7, actin, and EF

- 1α, were included in this assay. The DvSnf7 RNA was to be measured and reported. Actin and EF

- 1α were included in the assay to meet Affymetrix’s requirements for the QuantiGene ® Plex 2.0 Assay design, which has to be at least three targets. However, actin and EF

- 1α will be neither analyzed nor reported since they are not relevant to the DvSnf7 RNA quantification.

F .6.2. Backg round Control, Negative Quality Control (QC

- ), and Positive Quality Control (QC+)

Nuclease

- free water was used as a background control for each assay since all RNA samples, including the DvSnf7_968 and the total RNA extracted from the plant tissues,

were dissolved and normalized in nuclease

- free water.

Total RNA extracted from conventional maize NL6169 leaf tissue was used as a QC

- for each assay. The l eaf tissue sample was collected from NL6169 plants grown from seed

lot 11320471. The absence of the transgene containing the DvSnf7 expression cassette was confirmed by analysis of the leaf DNA extracted from individual plant leaf punches using an endpoint Taqman ® PCR analysis method.

The DvSnf7_968 was spiked to an appropriate concentration in a separa te aliquot of the same conventional maize RNA to serve as a QC+ for each assay.Monsanto Company CR240

- 13U1 297 of 374 F .6.3. Reference Standard Curve Generation

The DvSnf7_968 was spiked in separate aliquots of the same conventional maize NL6169 RNA at eight different concentrations to se rve as reference standards for each assay. A standard curve was generated for each assay plate.

F .6.4. Execution of the QuantiGene ® Plex 2.0 Assay

The QuantiGene ® Plex 2.0 Assay (Affymetrix Inc.) was performed according to the

manufacturer’s instructions (Affymetrix Inc. 2010) with a modification to introduce a denaturation step. Briefly, each sample, including the nuclease

- free water background control, QC

- , QC+, DvSnf7_968 reference standards, and the test samples, was assayed in triplicate wells in a volume of 20 microliters (μl). For the test samples, the total RNA was normalized to a concentration such that an equal amount of RNA for each tissue type was added to the assay. To ensure the capture of the DvSnf7 RNA during hybridization,

a denaturatio n step was introduced: the sample was mixed with the target

- specific oligonucleotide probe set in a well of a 96

- well PCR microplate (Axygen Scientific, Union City, CA) and heated for 5 minutes at 95 °C using a thermocycler (Applied Biosystems, Foster, CA ). After heating, the samples were kept in the thermocycler at 46°C until use. Before removing the plate from the thermocycler, the hybridization buffer containing the rest of the components for the capture of the DvSnf7 RNA were added to each sample wel l. The PCR microplate was then removed from the thermocycler and the content of each well (~100 μl) was transferred to the corresponding well of a Hybridization Plate (Affymetrix Inc.) followed by overnight hybridization (Affymetrix Inc. 2010)

.

After si gnal amplification following the overnight hybridization, all QuantiGene ®

Plex 2.0 Assay plates were read by a Luminex LX200 analyzer (Luminex Corp., Austin, TX) according to the manufacturer’s instructions. The net median fluorescence intensity (MFI) fro m each assay well, which is the MFI value subtracted by the MFI from the

nuclease

- free water background control, was reported digitally.

F .7. Moisture Analysis

Tissue moisture content was determined using an IR

- 200 Moisture Analyzer (Denver

Instrument Com pany, Arvada, CO). A homogeneous tissue

- specific site pool (TSSP) was prepared consisting of samples of a given tissue type grown at a given site. The average percent moisture for each TSSP was calculated from triplicate analyses. A TSSP Dry Weight Conv ersion Factor (DWCF) was calculated as follows:

DWCF = 1

− � Mean % TSSP Moisture

100 �Monsanto Company CR240

- 13U1 298 of 374 The DWCF was used to convert DvSnf7 levels assessed on a μg/g fresh weight (fw) basis into levels reported on a μg/g dry weight (dw) basis using the following calculation:

DvSnf 7 Level in Dry Weight = DvSnf 7 Level in Fresh Weight

DWCF

The DWCFs were only applied to test samples with DvSnf7 RNA levels greater than or equal to the assay’s limit of quantitation (LOQ).

F .8. Data Analyses

Each net MFI value was converted to a log2 value. The sample mean (mean), standard deviation (SD), and coefficient of variation (CV) from the three replicate wells of each sample were calculated and retained in the raw data. For each analysis, a standard curve was generated based on the log2 values of the theoretical concentrations (ng/ml) of the eight DvSnf7_968 reference standards and the log2 values of their net MFI, which established the correlation between the DvSnf7_968 concentration in the assay and the log2 value of the net MFI obtain ed from the assay. Using this standard curve, the net MFI of the test samples was converted to a DvSnf7 concentration (ng/ml). Each test sample with a DvSnf7 RNA level greater than or equal to the LOQ was converted to a

**μ** g/g fw value using the total RNA/ tissue ratio and dilution factor. All the **μ** g/g fw values

were converted to **μ** g/g dw values by applying the DWCFs. The test samples with the DvSnf7 RNA level lower than or equal to the assay’s LOD or less than the assay’s LOQ were reported as ≤ LOD or < LO Q. The mean, SD, and range of the DvSnf7 RNA levels

in MON 87411 were determined on both fw and dw basis for each tissue type across all

five sites.Monsanto Company CR240

- 13U1 299 of 374 References for Appendix F

Affymetrix Inc. 2010. QuantiGene® Plex 2.0 Assay: Using a magnetic plate wash er. Affymetrix, Inc., Santa Clara, California.

Chomczynski, P. and N. Sacchi. 1987. Single

- step method of RNA isolation by acid guanidinium thiocyanate

- phenol

- chloroform extraction. Analytical Biochemistry

162:156

- 159.Monsanto Company CR240

- 13U1 300 of 374 Appendix G : Materials

, Methods , and Individual Site Results for

Compositional Analysis of MON 87411 Maize Grain and Forage

Compositional comparisons between MON 87411 and the conventional control maize hybrid were performed using the principles and analytes outlined in the OECD consensus documents for maize composition (OECD, 2002) . These principles are accepted globally

and have been employed previously in assessments of maize products derived through biotechnology. The compositional assessment was conducted on grain and forage samples harvested from a single growing season conducted in Argentina during

2011/2012 under norma l agronomic practices.

G .1. Materials

Harvested grain and forage from MON 87411, a conventional control that has similar genetic background to that of MON 87411, and conventional, commercial reference maize hybrids were compositionally assessed. The refe rence hybrids are listed in

Table G

- 1.

Table G

- 1. Conventional Commercial Reference Maize Hybrids

Material Name Seed Lot # Field Site Codes

Dow AgroSciences Mill 527 11319757 BABE, BATC
Phillips 717 11300073 BABE, BAFO, BATC

Stewart S518 11226920 BABE
Syngenta NK 880 11319758 BABE, BAGH, BASS

ACA 2002 11319751 BAFO

NC+ 4443 11226700 BAFO, BALN

Nidera AX 878 11319754 BAFO, BAHT

ACA 430 11319753 BAGH
Gateway 6116 11227211 BAGH
Midland Phillips 7B15P 11226702 BAGH

Stewart S602 11226919 BAHT

Stine 9724 11298951 BAHT
La Tijereta LT 625 11319756 BALN, BATC
Legacy L7671 11226598 BALN, BATC
Syngenta NK 940 11319760 BALN

H

- 9180 11226704 BAPE

LG2597 11226862 BAPE
Syngenta NK 910 11319759 BAPE

Dekalb DK747 11319755 BASS
Mycogen 2M746 11226705 BASSMonsanto Company CR240

- 13U1 301 of 374 G .2. Characterization of the Materials

The identities of MON 87411, the conventional control, and reference hybrids were confirmed prior to use in the compositional assessment.

G. 3. Field Production of the Samples

Grain and forage samples from MON 87411, the conventional control, and the reference hybrids were collected from eight replicated sites in Argentina during the 2011/2012 growing season. The field sites were located in: Berdier, Buenos Aires (BABE); three sites in Pergamino, Buenos Aires (BAFO, BAPE, and BATC); Gahan, Buenos Aires (BAGH); Hunter, Buenos Aires (BAHT); Los Indios, Buenos Aires (BALN); and Sarasa, Buenos Aires (BASS). Starting seeds were planted in a randomized complete block design with fou r plots for each of MON 87411, the conventional control, and the reference hybrids. The production was conducted under normal agronomic field conditions for their respective geographic regions that are typical areas for maize production in Argentina. MON 87411 plots were treated with glyphosate at approximately 0.95 kg a.i./hectare at approximately the V2 growth stage to generate samples under conditions of the intended use of the product.

Forage was collected at early dent (R5) and grain was collected at physiological maturity. Forage samples were shipped on dry ice and grain was shipped at ambient temperature from the field sites to Monsanto Company (Saint Louis, Missouri). Subsamples were ground to a powder, stored in a freezer set to maintain

- 20°C l ocated at Monsanto Company (Saint Louis, Missouri). Subsamples were shipped on dry ice to Covance Laboratories Inc. (Madison, Wisconsin) for compositional analysis.

G .4. Summary of Analytical Methods

Nutrients analyzed in this study included moisture, a sh, protein, total fat, carbohydrates by calculation, acid detergent fiber (ADF), neutral detergent fiber (NDF), total dietary fiber (TDF), amino acids (18 components), fatty acids (22 components), minerals (calcium, copper, iron, magnesium, manganese, pho sphorus, potassium, sodium, and zinc) and vitamins [β

- carotene (referred to as vitamin A), B1, B2, B6, E (α

- tocopherol), niacin, and folic acid], in the grain, and moisture, ash, protein, total fat, carbohydrates by calculation, ADF, NDF, calcium and phosp horus in the forage. The anti

- nutrients assessed in grain included phytic acid and raffinose. Secondary metabolites assessed in grain included furfural, ferulic acid, and p

- coumaric acid.

All compositional analyses were performed at Covance Laboratorie s, Inc. (Madison, Wisconsin). Methods for analysis were based on internationally

- recognized procedures and literature publications. Brief descriptions of the methods utilized for the analyses are

described below.

G .4.1. 2

- Furaldehyde (Furfural)

The ground samples were extracted with 4% trichloroacetic acid and injected directly on a high

- performance liquid chromatography (HPLC) system for quantitation of freeMonsanto Company CR240

- 13U1 302 of 374 furfurals by ultraviolet detection (Albala

- Hurtado, et al., 1997) . The limit of quantit ation was 0.500 ppm.

Reference Standard:

**•** ACROS Organics, 2

- Furaldehyde, 99.5%, Lot Number A0296679

G .4.2. Acid Detergent Fiber

The ANKOM2000 Fiber Analyzer automated the process of removal of proteins, carbohydrates, and ash. Fats and pigments were removed with an acetone wash prior to analysis. The fibrous residue that was primarily cellulose and lignin and insoluble protein co mplexes remained in the Ankom filter bag, and was determined gravimetrically (Goering and Van Soest, 1970; Komarek, et al., 1993) . The limit of quantitation was

0.100%.

G .4.3. Amino Acid Composition

The following 18 amino acids were analyzed:

**•** Total alani ne **•** Total lysine

**•** Total arginine **•** Total methionine

**•** Total aspartic acid (including asparagine) **•** Total phenylalanine

**•** Total cystine (including cysteine) **•** Total proline

**•** Total glutamic acid (including glutamine) **•** Total serine

**•** Total glycine **•** Total threonine

**•** Total histidine **•** Total tryptophan

**•** Total isoleucine **•** Total tyrosine

**•** Total leucine **•** Total valine

The samples were hydrolyzed in 6N hydrochloric acid for approximately 24 hours at approximately 106

- 118oC. Phenol was added to the 6N hydrochloric acid to prevent

h alogenation of tyrosine. Cystine and cysteine were converted to S

- 2

- carboxyethylthiocysteine by the addition of dithiodipropionic acid. Tryptophan was hydrolyzed from proteins by heating at approximately 110oC in 4.2N sodium hydroxide for approximately 2 0 hours.

The samples were analyzed by HPLC after pre

- injection derivatization. The primary

amino acids were derivatized with o

- phthalaldehyde (OPA) and the secondary amino acids were derivatized with fluorenylmethyl chloroformate (FMOC) before injection ( AOAC, 2012f; Barkholt and Jensen, 1989; Henderson and Brooks, 2010; Henderson, et al., 2000; Schuster, 1988) . The limit of quantitation for this study was 0.100 mg/g.Monsanto Company CR240

- 13U1 303 of 374 Reference Standards:

Component Manufacturer Lot No. Purity (%)

L

- Alanine Sigma

- Aldrich BCBC5470 99.8

L

- Arginine Monohydrochloride Sigma

- Aldrich 1361811 100

L

- Aspartic Acid Sigma

- Aldrich BCBB9274 100.6

L

- Cystine Sigma

- Aldrich 1451329 100

L

- Glutamic Acid Sigma

- Aldrich 1423805 100.2
Glycine Sigma

- Aldrich 1119375 100

L

- Histidine Monohydrochloride
Monohydrate Sigma

- Aldrich BCBB1348 99.9

L

- Isoleucine Sigma

- Aldrich 1423806 100

L

- Leucine Sigma

- Aldrich BCBC6907 99.9

L

- Lysine Monohydrochloride Sigma

- Aldrich 1362380 100.2

L

- Methionine Sigma

- Aldrich 1423807 99.9

L

- Phenylalanine Sigma

- Aldrich BCBC5774 100

L

- Proline Sigma

- Aldrich 1414414 99.7

L

- Serine Sigma

- Aldrich 1336081 99.9

L

- Threonine Sigma

- Aldrich 1402329 100

L

- Tyrosine Sigma

- Aldrich BCBC2417 100

L

- Valine Sigma

- Aldrich 1352709 100

L

- Tryptophan Sigma

- Aldrich BCBB1284 99.8

G .4.4. Ash

All organic matter was driven off when the samples were ignited at approximately 550oC in a muffle furnace for at least 5 hours. The remaining inorganic material was determined gravimetrically and referred to as ash (AOAC, 2012p) . The limit of quantitati on was 0.100%.

G .4.5. Beta Carotene (Vitamin A)

Samples were saponified and extracted with hexane. The samples were then injected on

a reverse

- phase HPLC system with ultraviolet light detection. Quantitation was achieved with a linear regression analy sis (AOAC, 2012q; Quackenbush, 1987) . The limit of quantitation was 0.0200 mg/100g.

Reference Standard:

**•** Sigma

- Aldrich, Beta

- Carotene Type I, 98.7% (based on E1% = 2280 for Lambda Maximum of 478 nm to 479 nm in Hexane), Lot Number 021M1304V

G .4.6. Carbohy drate

The total carbohydrate level was calculated by difference using the fresh weight

- derived data and the following equation (USDA, 1973) :Monsanto Company CR240

- 13U1 304 of 374 % carbohydrates = 100 %

- (% protein + % fat + % moisture + % ash)

The limit of quantitation was calculated as 0.100%.

G .4.7. Fat by Acid Hydrolysis

The samples were hydrolyzed with hydrochloric acid. The fat was extracted using ether and hexane. The extracts were dried down and filtered through a sodium sulfate column. The remaining extracts were then evaporat ed, dried, and weighed (AOAC, 2012t; u)

. The limit of quantitation was 0.100%.

G .4.8. Fat by Soxhlet Extraction

The samples were weighed into a cellulose thimble containing sodium sulfate and dried to remove excess moisture. Pentane was dripped through the samples to remove the fat. The extract was then evaporated, dried, and weighed (AOAC, 2012v; a) . The limit of quantitation was 0.100%.

G.4.9. Fatty Acids

The following 22 fatty acids were analyzed:

**•** 8:0 Caprylic **•** 18:0 Stearic

**•** 10:0 Capric **•** 18:1 Oleic

**•** 12:0 Lauric **•** 18:2 Linoleic

**•** 14:0 Myristic **•** 18:3 gamma

- Linolenic

**•** 14:1 Myristoleic **•** 18:3 Linolenic

**•** 15:0 Pentadecanoic **•** 20:0 Arachidic

**•** 15:1 Pentadecenoic **•** 20:1 Eicosenoic

**•** 16:0 Palmitic **•** 20:2 Eicosadienoic

**•** 16:1 Palmitoleic **•** 20:3 Eicosatrienoic

**•** 17:0 Heptadecanoic **•** 20:4 Arachidonic

**•** 17:1 Heptadecenoic **•** 22:0 Behenic

The lipid was extracted and saponified with 0.5N sodium hydroxide in methanol. The saponification mixture was methylated with 14% boron trifluoride in methanol. The resulting methyl esters were extracted with heptane containing an internal standard. The methyl esters of the fatty acids were analyzed by gas chromatography (GC) using external standards for quantitation (AOCS 2009) . The limit of quantitation was

0.00400%.Monsanto Company CR240

- 13U1 305 of 374 References :

Manu facturer Lot No. Component Weight
(%) Purity
(%)

JY10

- W MA7

- W

Nu

- Chek Prep

GLC Reference

Standard

Covance 1

Covance 2 JY10

- W

MA7

- W Methyl Octanoate 3.0 1.25 99.7
Methyl Decanoate 3.25 1.25 99.6

Methyl Laurate 3.25 1.25 99.8
Methyl Myristate 3.25 1.25 99.8
Methyl Myristoleate 1.0 1.25 99.5
Methyl Pentadecanoate 1.0 1.25 99.6
Methyl Pentadecenoate 1.0 1.25 99.4
Methyl Palmitate 10.0 15.75 99.8
Methyl Palmitoleate 3.0 1.25 99.7
Methyl Heptadecanoate 1.0 1.25 99.6
Methyl 10

- Heptadecenoate 1.0 1.25 99.5

Methyl Stearate 7.0 14.00 99.8
Methyl Oleate 10.0 15.75 99.8
Methyl Linoleate 10.0 15.75 99.8
Methyl Gamma

Linolenate 1.0 1.25 99.4

Methyl Linolenate 3.0 1.25 99.5
Methyl Arachidate 2.0 1.25 99.8
Methyl 11

- Eicosenoate 2.0 1.25 99.6
Methyl 11

- 14

Eicosadienoate 1.0 1.25 99.5

Methyl 11

- 14

- 17

Eicosatrienoate 1.0 1.25 99.5

Methyl Arachidonate 1.0 1.25 99.4

Methyl Behenate 1.0 1.25 99.8

G.4.10. Folic Acid

The samples were hydrolyzed in a potassium phosphate buffer with the addition of ascorbic acid to protect the folic acid during autoclaving. Following hydrolysis by autoclaving, the samples were treated with a chicken

- pancreas enzyme and incubated approxi mately 18 hours to liberate the bound folic acid. The amount of folic acid was determined by comparing the growth response of the samples, using the bacteria

Lactobacillus casei , with the growth response of a folic acid standard. This response was measured turbidimetrically (AOAC, 2012c; d; Infant Formula Council, 1985) . The limit of quantitation was 0.0600 μg/g.

Reference Standard:

**•** USP, Folic acid, 98.9%, Lot Number Q0G151Monsanto Company CR240

- 13U1 306 of 374 G.4 .11. Minerals/ ICP Emission Spectrometry

The following nine minerals were analyzed:

**•** Calcium **•** Phosphorus

**•** Copper **•** Potassium

**•** Iron **•** Sodium

**•** Magnesium **•** Zinc

**•** Manganese

The samples were dried, precharred, and ashed overnight in a muffle furnace set to maintain 500oC. The ashed samples were re

- ashed with nitric acid, treated with hydrochloric acid, taken to dryness, and put into a solution of 5% hydrochloric acid. The

am ount of each element was determined at appropriate wavelengths by comparing the emission of the unknown samples, measured on the inductively coupled plasma spectrometer, with the emission of the standard solutions (AOAC, 2012e; g)

.

Reference Standard:

In organic Ventures Reference Standards and Limits of Quantitation:

Mineral Lot Numbers Concentration
(μg/mL) LOQ (ppm)

Calcium F2

- MEB417082MCA, F2

- MEB417084 200, 1000 20.0

Copper F2

- MEB417082MCA, F2

- MEB417083MCA 2.00, 10.0 0.500

Iron F2

- MEB417082MCA, F2

- MEB417085 10.0, 50.0 2.00

Magnesium F2

- MEB417082MCA, F2

- MEB417083MCA 50.0, 250 20.0

Manganese F2

- MEB417082MCA, F2

- MEB417083MCA 2.00, 10.0 0.300

Phosphorus F2

- MEB417082MCA, F2

- MEB417084 200, 1000 20.0

Potassium F2

- MEB417082MCA, F2

- MEB417084 200, 1000 100

Sodium F2

- MEB417082MCA, F2

- MEB417084 200, 1000 100

Zinc F2

- MEB417082MCA, F2

- MEB417083MCA 10.0, 50.0 0.400

G.4.12. Moisture

The samples were dried in a vacuum oven at approximately 100oC. The moisture weight loss was determined and converted to percent moisture (AOAC, 2012h; i) . The limit of quantitation was 0.100%.

G.4.13. Neutral Detergent Fiber

The ANKOM2000 Fiber Analyzer automated the process of the removal of protein, carbohydrate, and ash. Fats and pigments were removed with an acetone wash prior to analysis. Hemicellulose, cellulose, lignin and insoluble protein fraction were left in theMonsanto Company CR240

- 13U1 307 of 374 f ilter bag and determined gravimetrically (AACC, 1998; Goering and Van Soest, 1970; Komarek, et al., 1994) . The limit of quantitation was 0.100%.

G.4.14. Niacin

The samples were hydrolyzed with sulfuric acid and the pH was adjusted to remove

interferences . The amount of niacin was determined by comparing the growth response of the samples, using the bacteria Lactobacillus plantarum , with the growth response of a niacin standard. This response was measured turbidimetrically (AOAC, 2012j; c) . The

limit of quantitation was 0.300 μg/g.

Reference Standard:

**•** USP, Niacin, 99.8%, Lot Number J0J235

G.4.15. p

- Coumaric Acid and Ferulic Acid

The ground samples were extracted with methanol followed by alkaline hydrolysis and buffering prior to injection on an analyti cal HPLC system for quantification of p

coumaric acid and ferulic acid by ultra violet (UV) detection (Hagerman and Nicholson, 1982) . The limit of quantitation for the p coumaric acid and ferulic acid was 33.3 ppm.

Reference Standards:

Component Manufacturer Lot No. Purity (%)

p

- Hydroxycinnamic Acid
(p

- Coumaric Acid) Sigma

- Aldrich 091M1197V 99.6

4

- Hydroxy

- 3

- methoxycinnamic Acid
(Ferulic Acid)

4

- Hydroxy

- 3

- methoxycinnamic Acid
(Ferulic Acid) ACROS Organics

ACROS Organics A0261354

A0294716 99.4

99.4

G.4.16. Phytic Acid

The samples were extracted using hydrochloric acid and sonication, purified using a silica based anion exchange column, concentrated and injected onto a HPLC system with

a refractive index detector (Lehrfeld, 1989; Lehrfeld, 1994) . The limit of quantitation

was 0.100%.

Reference Standard:

**•** Sigma

- Aldrich, Phytic Acid Sodium Salt Hydrate, 97.9%, Lot Number

BCBH8701VMonsanto Company CR240

- 13U1 308 of 374 G .4.17. Protein

The protein and other organic nitrogen in the samples were converted to ammonia by digesting the samples with sulfuric acid containing a catalyst mixture. The acid digest was made alkaline. The ammonia was distilled and then titrated with a previously

st andardized acid. Instrumentation was used to automate the digestion, distillation and titration processes. The percent nitrogen was calculated and converted to equivalent protein using the factor 6.25 (AOAC, 2012k; l) . The limit of quantitation was 0.10 0%.

G.4.18. Raffinose

Sugars in the samples were extracted with a 50:50 water:methanol solution. Aliquots were taken, dried under inert gas, and then reconstituted with a hydroxylamine hydrochloride solution in pyridine containing phenyl

- β

- D

- glucopyranos ide as the internal standard. The resulting oximes were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid treatment, and then analyzed by GC using a

flame ionization detector (Brobst, 1972; Mason and Slover, 197 1) . The limit of quantitation was 0.0500%.

Reference Standard:

**•** Sigma

- Aldrich, D

- (+)

- Raffinose pentahydrate, 99.6%, Lot Number 019K1156

G.4.19. Total Dietary Fiber

Duplicate samples were gelatinized with α

- amylase and digested with enzymes to break

down s tarch and protein. Ethanol was added to each sample to precipitate the soluble fiber. The sample was filtered, and the residue was rinsed with ethanol and acetone to remove starch and protein degradation products and moisture. Protein content was

determ ined for one of the duplicates; ash content was determined for the other. The total dietary fiber in the sample was calculated using protein and ash values (AOAC, 2012m)

. The limit of quantitation was 1.00%.

G.4.20. Vitamin B1 (Thiamine Hydrochloride)

T he samples were autoclaved under weak acid conditions to extract the thiamine. The resulting solutions were incubated with a buffered enzyme solution to release any bound thiamine. The solutions were purified on a cation

- exchange column. Aliquots were

r eacted with potassium ferricyanide to convert thiamine to thiochrome. The thiochrome was extracted into isobutyl alcohol, measured on a fluorometer, and quantitated by comparison to a known standard (AOAC, 2012n; o; r) . The limit of quantitation was

0.01 0 mg/100g.

Reference Standard:

**•** USP, Thiamine Hydrochloride, 99.7%, Lot Number P0K366Monsanto Company CR240

- 13U1 309 of 374 G.4.21. Vitamin B2 (Riboflavin)

The samples were hydrolyzed with dilute hydrochloric acid and the pH was adjusted to remove interferences. The amount of riboflavin was determined by comparing the growth response of the samples, using the bacteria Lactobacillus rhamnosus , with the growth response of multipoint riboflavin standards. The growth response was measured turbidimetrically (AOAC, 2012s; c) . The limit of quantitation was 0.200 μg/g.

Reference Standard:

**•** USP, Riboflavin, 99.7%, Lot Number N1J079

G.4.22. Vitamin B6 (Pyridoxine Hyd rochloride)

The samples were hydrolyzed with dilute sulfuric acid in the autoclave and the pH was adjusted to remove interferences. The amount of pyridoxine was determined by comparing the growth response of the samples, using the yeast Saccharomyces

cere visiae , with the growth response of a pyridoxine standard. The response was measured turbidimetrically. Results were reported as pyridoxine hydrochloride (AOAC, 2012b; Atkins, et al., 1943) . The limit of quantitation was 0.0700 μg/g.

Reference Standard:

**•** USP, Pyridoxine hydrochloride, 99.8%, Lot Number Q0G409

G.4.23. Vitamin E ( α

- Tocopherol)

The samples were saponified to break down any fat and release vitamin E. The saponified mixtures were extracted with ethyl ether and then quantitated by high

performance liquid chromatography using a silica column (Cort, et al., 1983; McMurray, et al., 19 80; Speek, et al., 1985) . The limit of quantitation was 0.00500 mg/g.

Reference Standard:

**•** USP, Alpha Tocopherol, 98.9%, Lot Number N0F068

G.5. Data Processing and Statistical Analysis

After compositional analyses were performed, data spreadsheets contain ing individual values for each analysis were sent to Monsanto Company for review. Data were then transferred to Certus International, Inc., where they were converted into the appropriate units and statistically analyzed. The following formulas were used for re

- expression of composition data for statistical analysis (Table G

- 2):Monsanto Company CR240

- 13U1 310 of 374 Table G

- 2. Re

- expression Formulas for Statistical Analysis of Composition Data

Component From (X) To Formula 1

Proximates (excluding Moisture),

Fiber, Anti

- nutrients % fwt % dw X/d

Amino Acids (AA) mg/g fwt % dw X/(10d)
Secondary Metabolites ppm fwt μg/g dw X/d
Copper, Iron, Manganese, Zinc ppm fwt mg/kg dw X/d
Calcium, Magnesium, Phosphorus,

Potassium ppm fwt % dw X/(10 4 d)

Folic Acid, Niacin, Vitamin B2,

Vitamin B6 μg/g fwt mg/kg dw X/d

Vitamin A, Vitamin B1 mg/100g fwt mg/kg dw 10X/d

Vitamin E mg/g fwt mg/kg dw 10 3 X/d

Fatty Acids (FA) % fwt % Total FA (100)X j / **Σ** X, for

each FA j where **Σ** X

is over all the FA

1 ‘X’ is the individual sample value; d is the fraction of the sample that is dry matter.

In order to complete a statistical analysis for a compositional constituent in this compositional assessment, at least 50% of all the values for an analyte in grain or forage had to be greater than the assay limit of quantitati on (LOQ). Analytes with more than 50% of observations below the assay LOQ were excluded from summaries and analysis. The following 16 analytes in grain with more than 50% of observations below the assay LOQ were excluded from statistical analysis: 8:0 c aprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gamma linolenic acid, 20:2 eicosadi enoic acid, 20:3 eicosatrienoic acid, 20:4 arachidonic acid, sodium, and furfural.

Otherwise, individual results below the LOQ were assigned a value equal to one

- half the quantitation limit. Fourteen observations for 22:0 Behenic were assigned a value e qual to

one

- half of the LOQ (0.002% fwt).

The data were assessed for potential outliers using a studentized PRESS residuals calculation. A PRESS residual is the difference between any value and its value predicted from a statistical model that excludes th e data point. The studentized version

scales these residuals so that the values tend to have a standard normal distribution when outliers are absent. Thus, most values are expected to be between **±** 3. Extreme data points that are also outside of the **±** 6 studentized PRESS residual ranges are considered for exclusion, as outliers, from the final analyses. Seven components had PRESS

residual values outside of the **±** 6 range.

Of the seven flagged values, only the vitamin E value from a conventional reference was removed from further analysis as an outlier. The remaining values were not removedMonsanto Company CR240

- 13U1 311 of 374 because they were not extreme values or they were deemed sufficiently close to neighboring values to lack sufficient evidence for removal.

The outlier test procedure was reapplied to the remaining vitamin E data to detect potential outliers that were masked in the first analysis. One vitamin E value from a commercial reference was identified as an outlier, but the value was not an extreme value

and was not removed as an outlier.

Maize compositional components were statistically analyzed using a mixed

- model analysis of variance with the SAS MIXED procedure.

Analyses of the combined replicated sites were performed using model (1).

(1) Yijk = U + Ti + Lj + B(L)jk + LTij + eijk,

where Yijk = unique individual observation, U = overall mean, Ti = substance effect, Lj = random site effect, B(L)jk = random block within site effect, LTij = random site by substance interaction effect, and eijk = residual error.

For each compo nent analysis, individual mean comparison tests of MON 87411 vs.

conventional control were conducted.

A range of observed values from the reference hybrids was determined for each analytical component. Additionally, data from the reference hybrids were used to develop 99% tolerance intervals. A tolerance interval is an interval that one can claim, with a specified degree of confidence, contains at least a specified proportion, p, of an entire sampled population for the parameter measured.

For each compo sitional component analyzed, two

- sided 99% tolerance intervals were calculated that are expected to contain, with 95% confidence, 99% of the quantities expressed in the population of reference hybrids. Each estimate was based upon the average of all obser vations per unique reference hybrid. Because negative quantities are not possible, negative calculated lower tolerance bounds were set to zero.Monsanto Company CR240

- 13U1 312 of 374 References for Appendix G

A ACC . 1998. Approved methods of the American Association of Cereal Chemists.

Ameri can Association of Cereal Chemists, Inc., St. Paul, Minnesota.

Albala

- Hurtado, S., M.T. Veciana

- Nogues, M. Izquierdo

- Pulido and M.C. Vidal

- Carou. 1997. Determination of free and total furfural compounds in infant milk formulas by high

- performance liquid chromatography. Journal of Agricultural and Food Chemistry

45: 2128

- 2133.

AOAC . 2012a. AOAC official method 960.39: Fat (crude) or ether extract in meat. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012b. AOAC offic ial method 961.15: Vitamin B 6 (pyridoxine, pyridoxal, pyridoxamine) in food extracts. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012c. AOAC official method 960.46: Vitamin assays. Association of Analytical

Communit ies International, Gaithersburg, Maryland.

AOAC . 2012d. AOAC official method 992.05: Total folate (Pteroylglutamic acid) in infant formula. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012e. AOAC official method 984 .27: Calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc in infant formula. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012f. AOAC official method 988.15: Tryptophan in foods and foo d and feed ingredients. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012g. AOAC official method 985.01: Metals and other elements in plants and pet foods. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012h. AOAC official method 925.09: Solids (total) and loss on drying (moisture) in flour. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012i. AOAC official method 926.08: Loss on drying (moisture) in cheese. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012j. AOAC official method 944.13: Niacin and niacinamide (nicotinic acid and nicotinamide) in vitamin preparations. Association of Analytical Commu nities International, Gaithersburg, Maryland.Monsanto Company CR240

- 13U1 313 of 374 AOAC . 2012k. AOAC official method 955.04: Nitrogen (total) in fertilizers Kjeldahl method. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012l. AOAC official method 979.09 : Protein in grains. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012m. AOAC official method 985.29: Total dietary fiber in foods. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2 012n. AOAC official method 942.23: Thiamine (Vitamin B 1 ) in human and pet foods. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012o. AOAC official method 953.17: Thiamine (Vitamin B 1 ) in grain products.

Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012p. AOAC official method 923.03: Ash of flour. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012q. AOAC official method 2005.07 β

· carotene in su pplements and raw

materials reversed

- phase high pressure liquid chromatographic method. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012r. AOAC official method 957.17: Thiamine (Vitamin B 1 ). Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012s. AOAC official method 940.33: Riboflavin (Vitamin B 2 ) in vitamin preparations. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012t. AOAC official method 922.06: Fat in flour. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012u. AOAC official method 954.02: Fat (crude) or ether extract in pet food. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC . 2012v. AOAC official method 948.22: Fat (crude) in nuts and nut products. Association of Analytical Communities International, Gaithersburg, Maryland.

AOCS. 2009. Preparation of methyl esters of fatty acids. Method Ce 2

- 66, American Oil

C hemists' Society, Champaign, Illinois.

Atkins, L., A.S. Schultz, W.L. Williams and C.N. Frey. 1943. Yeast microbiological methods for determination of vitamins. Industrial and Engineering Chemistry, Analytical

Edition 15: 141

- 144.Monsanto Company CR240

- 13U1 314 of 374 Barkholt, V. and A.L. Je nsen. 1989. Amino acid analysis determination of cysteine plus

half

- cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Analytical Biochemistry 177: 318

- 322.

Brobst, K.H. 1972. Gas

- liquid chromatography of trimeth ylsilyl derivatives. Pages 3

- 8 in Methods in Carbohydrate Chemistry. Volume 6. Academic Press, New York, New York.

Cort, W., T.S. Vicente, E.H. Waysek and B.D. Williams. 1983. Vitamin E content of feedstuffs determined by high

- performance liquid chromatog raphic fluorescence. Journal of Agriculture and Food Chemistry 31: 1330

- 1333.

Goering, H.K. and P.J. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agriculture Handbook No. 379. U.S. Department of Agricult ure, Agricultural Research Service, Washington, D.C.

Hagerman, A.E. and R.L. Nicholson. 1982. High

- performance liquid chromatographic determination of hydroxycinnamic acids in the maize mesocotyl. Journal of Agricultural and Food Chemistry 30: 1098

- 1102.

Henderson, J.W. and A. Brooks. 2010. Improved amino acid methods using Agilent ZORBAX Eclipse Plus C18 columns for a variety of Agilent LC instrumentation and separation goals. Agilent Technologies, Inc., Wilmington, Delaware.

Henderson, J.W., R.D. Ricke r, B.A. Bidlingmeyer and C. Woodward. 2000. Rapid, accurate, sensitive, and reproducible HPLC analysis of amino acids. Agilent Technologies, Inc., Wilmington, Delaware.

Infant Formula Council. 1985. Methods of analysis for infant formulas. Section C

- 2,

In fant Formula Council, Atlanta, Georgia.

Komarek, A.R., J.B. Robertson and P.J. Van Soest. 1993. A comparison of methods for determining ADF using the Filter Bag Technique versus conventional filtration. Journal

of Animal Science 72: 114.

Komarek, A.R., J .B. Robertson and P.J. Van Soest. 1994. Comparison of the filter bag technique to conventional filtration in the Van Soest NDF analysis of 21 feeds. National Conference on Forage Quality, Evaluation, and Utilization, American Society of Agronomy, Inc., Cro p Science Society of America, Inc., Soil Society of America, Inc., Madison, Wisonsin.

Lehrfeld, J. 1989. High

- performance liquid chromatography analysis of phytic acid on a pH

- stable macroporous polymer column. Cereal Chemistry 66: 510

- 515.

Lehrfeld, J. 1994. HPLC separation and quantitation of phytic acid and some inositol phosphates in foods: Problems and solutions. Journal of Agricultural and Food Chemistry

42: 2726

- 2731.Monsanto Company CR240

- 13U1 315 of 374 Mason, B.S. and H.T. Slover. 1971. A gas chromatographic method for the determin ation of sugars in foods. Journal of Agricultural and Food Chemistry 19: 551

- 554.

Mcmurray, C.H., W.J. Blanchflower and D.A. Rice. 1980. Influence of extraction techniques on determination of α

- Tocopherol in animal feedstuffs. Journal of the

Association o f Official Analytical Chemists 63: 1258

- 1261.

O ECD . 2002. Consensus document on compositional considerations for new varieties of maize ( Zea mays ): Key food and feed nutrients, antinutrients and secondary plant metabolites. ENV/JM/MONO(2002)25. Organisati on for Economic Co

- operation and Development, Paris, France.

Quackenbush, F.W. 1987. Reverse phase HPLC separation of cis and trans

- carotenoids and its application to β

- carotenes in food materials. Journal of Liquid Chromatography

10: 643

- 654.

Schuster, R. 1988. Determination of amino acids in biological pharmaceutical plant and food samples by automated precolumn derivatization and high

- performance liquid chromatography. Journal of Chromatography 431: 271

- 284.

Speek, A.J., J. De Schrijver and W.H.P. Sch reurs. 1985. Vitamin E composition of some seed oils as determined by high

- performance liquid chromatography with fluorometric

detection. Journal of Food Science 50: 121

- 124.

USDA . 1973. Energy value of foods: Basis and derivation. Agricultural Handbook N o. 74. US Department of Agriculture, Agricultural Research Service, Washington, D.C.Monsanto Company CR240

- 13U1 316 of 374 Appendix H : Materials

, Methods
, and Individual Site Results for Seed Dormancy and Germination Assessment of MON 87411

H .1. Materials

Seed germination and dormancy characteristics were assessed on seed from MON 87411

, the conventional control, and reference hybrids produced in replicated field trials during 2012 at the following site s : Jefferson County, IA (IARL), Warren County, IL (ILM N), and Pawnee County, KS (KSLA)

.

H .2. Characterization of the Materials

The identities of the MON 87411 and the conventional control starting seed were verified by event

- specific polymerase chain reaction (PCR) analyses. During the growing season,

the field planting order of MON 87411 and the conventional control plots was confirmed by event

- specific PCR analyses. Chain

- of

- custody documentation for all starting seed for dormancy and germination study was maintained from harvest through shipment to the performing laboratory with the use of packaging labels and plant sample transfer forms.

H .3. Germination Testing Facility and Experimental Methods

G ermination and d ormancy evaluations were conducted at BioDiagnostics, Inc. in River Falls, WI. The principal investigator was qualified to conduct seed germination and dormancy testing consistent with the standards established by the Association of Official Seed Analysts (AOSA), a seed trade association (AOSA/SCST 2010 ; AOSA, 2012a;

2012 b ).

The seed lots (Selfed F 2 grain) of MON 87411, the conventional control, and four reference hybrids from each location were tested under seven different temperature regimes. Seven germ ination chambers were used in the study and each chamber was maintained dark under one of the following seven temperature regimes: constant temperature of approximately 5, 10, 20, or 30 °C or alternating temperatures of approximately 10/20, 10/30, or 20/3 0° C. The alternating temperature regimes were maintained at the lower temperature for 16 hours and the higher temperature for 8 hours. The temperature inside each germination chamber was monitored and recorded throughout the duration of the study.

Appr oximately 100 seeds each of MON 87411, the conventional control, and the reference hybrids were placed on pre

- moistened germination towels using a vacuum planting system. Additional pre

- moistened germination towels were placed on top of the

seed. The ger mination towels were then rolled up in a wax cover. All rolled germinat ion

towels were labeled and placed into an appropriately labeled bucket. Each bucket within a temperature regime represented a replicate per site. There were 4 reps per site for a

to tal of 12 buckets for each temperature regime. Each bucket contained 6 towels or 1 towel per entry. Buckets were then placed in the appropriate germination chambers.Monsanto Company CR240

- 13U1 317 of 374 Each temperature regime constituted a separate split

- plot experiment with four replicati ons where the whole

- plot was the location where starting seed (Selfed F 2 grain) was produced and the sub

- plot was the material type ( i.e. , MON 87411, the conventional

control or reference material). Whole

- plots and sub

- plots were randomized within each

tem perature regime.

A description of each germination characteristic evaluated and the timing of evaluations are presented in Table VII I

- 1. The types of data collected depended on the temperature regime. Each rolled germination towel in the AOSA

- recommend ed temperature regime ( i.e.

, alternating 20/30 °C) was assessed periodically during the study for normally germinated, abnormally germinated, hard (viable and nonviable), dead, and firm swollen (viable and nonviable) seed as defined by AOSA guidelines ( AOS A, 2012a; 2012b ). AOSA only provides guidelines for testing seed under optimal temperatures, whereas additional temperature regimes were included to test diverse environmental conditions. Therefore, e ach rolled germination towel in the additional temperature regimes ( i.e. , 5, 10, 20, 30, alternating 10/20

, and 10/30 °C) was assessed periodically during the study for germinated, hard (viable and nonviable), dead, and firm swollen (viable and nonviable) seed. Because temperature extremes could affec t the development of seedlings, AOSA standards were not applied and no distinction was made between normal or abnormal germinated seed. Therefore, any seedling with a radical of 1 mm or more was classified as germinated.

The calculation of percent seed in each assessment category was based on the actual number of seeds evaluated ( e.g. , 99 or 101). Across temperature regimes, the total number of seeds evaluated from each germination towel was approximately 100.

Within both AOSA and the additional temperat ure regimes, hard and firm

- swollen seeds remaining at the final evaluation date were subjected to a tetrazolium (Tz) test for evaluation of viability according to AOSA standards (AOSA/SCST 2010) . The number

of nonviable hard and nonviable firm

- swollen see d was added to the number of dead seed counted on all collection dates to determine the total percent dead seed. Total counts for percent viable hard and viable firm

- swollen seed were determined from the Tz test.

H .4. Statistical Analysis

An analysis of variance was conducted using SAS ****

**** SAS is a registered trademark of SAS Ins titute, Inc. (SAS 20 08 ) according to a split

- plot design ( production site as the whole plot and starting seed material as the sub

- plot ) with four replications

. MON 87411 was compared to the conventional control for germination

and dor mancy characteristics of seed produced within each site ( i.e ., i ndividual site analysis) and in a combined

- site analysis in which the data were pooled across all three

sites. The seed germination and dormancy characteristics analyzed included percent germinated seed , percent viable hard seed, percent dead seed , and percent viable firm

swollen seed. The percent germinated seed were categorized as either normal germinatedMonsanto Company CR240

- 13U1 318 of 374 or abnormal germinated for the AOSA tem perature regime. The level of statistical significance was predetermined to be 5% ( **α** =0.05). MON 87411 was not statistically compared to the reference hybrids , nor were comparisons made across temperature regimes. The minimum and maximum mean values were determined from the reference

materials across all sites to provide a range of values ( i.e ., reference range) representative of commercial maize hybrids

. Results from the combined

- site analysis are presented in

Table VII I

- 2

.

H .5. Individual

- Site Seed Do rmancy and Germination Analysis

In the individual site analyses, five statistically significant differences in total were

detected between MON 87411 and the conventional control for the measured characteristics ( i.e. , percent germinated, viable hard, dead , or viable firm

- swollen seed) of the seed (Selfed F 2 grain) produced at the IARL, ILMN, and KSLA sites (Table H

- 2).

MON 87411 had fewer dead seed than the conventional control at 10/20°C (0.7% vs. 2.0%) and more viable firm swollen seed than the conven tional control at 20°C (0.3% vs. 0.0%) for the seed produced at the IARL site. MON 8 7411 had more germinated seed than the conventional control at 20/30°C (99.3% vs. 97.8%) and fewer dead seed than the conventional control at 20/30°C (0.3% vs. 1.8%) for th e seed produced at the ILMN site.

MON 87411 had more germinated seed than the conventional control at 5°C (0.5% vs. 0.0%) for the seed produced at the KSLA site. Statistically significant differences

between MON 87411 and the conventional control for germi nation amd dormancy characteristics in the individual site analyses were not consistently detected across temperature regimes or the individual sites. These differences were not detected in the combined site analysis (Section VIII

- 1, Table VIII

- 2) and are unlikely to be biologically meaningful in terms of increased pest/weed potential (See Figure VIII

- 1, Step 2, answer “no”).Monsanto Company CR240

- 13U1 319 of 374 Table H

- 1. Starting Seed of MON 87411

, Conventional Control and Commercial

Maize Reference Hybrids Used in Dormancy Assessment

Site Code Material Name Phenotype Monsanto Lot

Number

IARL MON 87411 Glyphosate

- Tolerant

and Insect

- Protect ed 11354899

IARL MPA640B 1 Conventional Control 11354894

IARL Legacy L7671 Conventional Reference 11354895

IARL Lewis 7007 Conventional Reference 11354896

IARL Gateway 6116 Conventional Reference 11354897

IARL Phillips 717 Conventional Reference 11354898

KSLA MON 87411 Glyphosate

- Tolerant

and Insect

- Protect ed 11354905

KSLA MPA640B Conventional Control 11354900

KSLA Lewis 7007 Conventional Reference 11354901

KSLA Midland Phillips 799 Conventional Reference 11354902

KSLA NC+ 5220 Conventional Reference 11354903

KSLA LG2540 Conventional Reference 11354904

ILMN MON 87411 Glyphosate

- Tolerant

and Insect

- Protect ed 11354911

ILMN MPA640B Conventional Control 11354906

ILMN Gateway 4148 Conventional Reference 11354907

ILMN H

- 9180 Conventional Reference 11354908

ILMN Stewart S588 Conventional Reference 11354909

ILMN LG2548 Conventional Reference 11354910

1 MPA640B = LH244 × LH287Monsanto Company CR240

- 13U1 320 of 374 Table H

- 2. Germination and Dormancy Characteristics of MON 87411 and the Conventional Control Seed (Selfed F 2 Grain)

Produced at each of the Three Field Sites

IARL 1 ILMN 1 KSLA 1

Mean % (S.E.) 2 Mean % (S.E.) 2 Mean % (S.E.) 2

Temperature Regime Assessment Category MON 87411 Control MON 87411 Control 3 MON 87411 Control

5 °C Germinated 0.0 (0.00) 0.3 (0.25) 0.5 (0.29) 0.3 (0.25) 0.5 (0.29) * 0.0 (0.00)

Viable Hard 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)

Dead 2.0 (0.91) 1.0 (0.41) 4.3 (1.38) 2.3 (0.48) 2.8 (0.75) 3.3 (1.11)

Viable Firm Swollen 98.0 (0.91) 98.8 (0.25) 95.3 (1.49) 97.5 (0.65) 96.8 (0.85) 96.8 (1.11)

10 °C Germinated 73.5 (2.60) 75.9 (1.34) 90.5 (1.55) 91.0 (1.22) 82.0 (2.48) 82.5 (4.09)

Viable Hard 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)

Dead 2.0 (0.82) 2.0 (0.41) 0.0 (0.00) 0.8 (0.25) 0.8 (0.25) 0.3 (0.25)

Viable Firm Swollen 24.5 (2.10) 22.1 (1.28) 9.5 (1.55) 8.3 (1.03) 17.3 (2.43) 17.3 (3.88)

20 °C Germinated 98.0 (0.71) 99.0 (0.41) 99.3 (0.25) 99.3 (0.48) 98.5 (0.87) 98.8 (0.48)

Viable Hard 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)

Dead 1.8 (0.63) 1.0 (0.41) 0.8 (0.25) 0.8 (0.48) 1.5 (0.87) 1.3 (0.48)

Viable Firm Swollen 0.3 (0.25) * 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.0(0.00) 0.0 (0.00)

30 °C Germinated 98.8 (0.48) 97.8 (0.95) 99.3 (0.48) 99.8 (0.25) 99.3 (0.25) 98.3 (0.48)

Viable Hard 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)

Dead 1.3 (0.48) 2.3 (0.95) 0.8 (0.48) 0.3 (0.25) 0.8 (0.25) 1.8 (0.48)

Viable Firm Swollen 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)Monsanto Company CR240

- 13U1 321 of 374 Table H

- 2 (continued) . Germination and Dormancy Characteristics of MON 87411 and the Conventional Control Seed (Selfed F 2 Grain) Produced at each of the Three Field Sites

IARL 1 ILMN 1 KSLA 1

Mean % (S.E.) 2 Mean % (S.E.) 2 Mean % (S.E.) 2

Temperature Regime Assessment Category MON 87411 Control MON 87411 Control 3 MON 87411 Control

10/20 °C Germinated 98.0 (0.41) 97.0 (0.58) 99.3 (0.25) 100.0 (0.00) 98.8 (0.48) 98.0 (0.71)

Viable Hard 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)

Dead 0.7 (0.47) * 2.0 (0.00) 0.5 (0.29) 0.0 (0.00) 0.8 (0.48) 0.8 (0.48)

Viable Firm Swollen 1.3 (0.63) 1.0 (0.58) 0.3 (0.25) 0.0 (0.00) 0.5 (0.50) 1.3 (0.48)

10/30 °C Germinated 98.5 (0.29) 98.3 (0.25) 99.0 (1.00) 99.5 (0.29) 99.0 (0.58) 100.0 (0.00)

Viable Hard 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)

Dead 1.5 (0.29) 1.8 (0.25) 0.8 (0.75) 0.5 (0.29) 1.0 (0.58) 0.0 (0.00)

Viable Firm Swollen 0.0 (0.00) 0.0 (0.00) 0.3 (0.25) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

20/30 °C Normal Germinated 97.5 (0.29) 98.5 (0.65) 99.3 (0.48) * 97.8 (0.63) 97.8 (0.75) 98.8 (0.48)

(AOSA) Abnormal Germinated 0.8 (0.48) 0.0 (0.00) 0.5 (0.29) 0.5 (0.29) 1.3 (0.48) 0.5 (0.29)

Viable Hard 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)

Dead 1.8 (0.48) 1.5 (0.65) 0.3 (0.25) * 1.8 (0.85) 1.0 (0.41) 0.8 (0.25)

Viable Firm Swollen 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)

Note: The experimental design was a split

- plot with four replications.

* Indicates a statistically significant difference between MON 87411 and the conventional control (α=0.05) using ANOVA

. † No statistical comparison could be made due to lack of variability in the data.

1 Site codes are as follows: IARL = Jefferson County, Iowa, KSLA = Pawnee County, Kansas, and ILMN = Warren County Illinois.

2 MON 87411 and the conventional control values represent means with standard error (S.E.) in parentheses. N = 4. In some instances, the total percentage of

both MON 87411 and the conventional control did not equal 100% due to numerical rounding of the means.Monsanto Company CR240

- 13U1 322 of 374 References for Appendix H

AOSA/SCST. 2010. Tetrazolium testing handbook. Association of Official Seed Analysts and the Society of Commercial Seed Technologists, Ithaca, New York.

SAS. 20 08 . SAS/STAT software version 9. 2 . SAS Institute, Inc., Cary, North Carolina.Monsanto Company CR240

- 13U1 323 of 374 Appendix I : Material s , Methods
, and Individual Site Results from Phenotypic,

Agronomic

, and Environmental Interaction Assessment o f MON 87411 under

Field Conditions

I .1. Materials

Agronomic, phenotypic, and environmental interaction characteristics were assessed for

MON 87411 , the conventional control, and 22 reference hybrids grown under similar agronomic conditions

. Four reference hybrids were planted per site (Table I

- 1).

I .2. Characterization of the Materials

The presence or absence of the MON 87411 event in the starting seed of MON 87411 and the conventional control was verified by event

- specific polymerase chain reaction ( PCR ) analyses. No molecular analyses were performed on the reference starting seed.

I

. 3 . Field Sites and Plot Design

Field trials were established in 20 12 at nine sites that provided a range of environmental and agronomic conditions representative of U.S. maize growing regions ( Section VIII,

Table VII I

- 3 ). The Principal Investigator at each site was familiar with the growth, production, and evaluation of maize characteristics.

At all sites, seed of MON 87411 , the conventional control, and four conventional

reference hybrids were planted in a randomized complete block design with four replications. The planted plot dimensions varied between sites, due to variability in available planting equipment and data collection ( Table I

- 2 ). At IABG (Iowa), NEYO (Nebraska), NCBD (North Carolina), and PAHM (Pennsylvania ) sites

, e ach replicated plot consisted of 16 rows of maize spaced approximately 0.76

- 0.96 m apart and approximately 6 m long . Phenotypic and qualitative environmental interactions data

were collected from rows 2 and 3 (rows 14 and 15 at NCBD site). Rows six and eight were used to collect arthropod samples using sticky traps. Rows 9, 10, 11, and 12 were designated for visual counts of arthropod abundance. Rows 14 and 15 (rows 2 and 3 at NCBD site) were used to assess plant damage caused by corn earworm ( Helicoverpa

zea ) and European corn borer ( Ostrinia nubilalis ).

At IARL (Iowa), ILMN (Illinois), INSH (Indiana), KSLA (Kansas), and NEDC (Nebraska) sites, each replicated plot consisted of six rows of maize spaced

appro ximately 0.76 m apart and approximately 6 m long. The plots were separated by two rows of conventional maize along their length. Phenotypic and qualitative environmental interactions data were collected from rows 4 and 5 except one plot at ILMN and two p lots at NEDC.

I .4. Planting and Field Operations

Planting information, soil description, and cropping history of the trial area are listed in

Table I

- 2. Prior to planting, the Principal Investigator at each site prepared the plot area with a proper seed bed according to local agronomic practices, including tillage,Monsanto Company CR240

- 13U1 32 4 of 374 fertilization, and pest management. During the growing season, all plots were assessed for agronomic conditions and pest populations, including pest arthropods , diseases and weeds. Fertilizer, irrigation, agricultural chemicals, and other management practi ces were applied as necessary. M aintenance operations were performed uniformly across all plots.Monsanto Company CR240

- 13U1 325 of 374 Table I

- 1. Starting Seed for Phenotypic , Agronomic, and Environmental

Interaction Assessment

Site Code 1 Material Name Monsanto Lot Number Phenotype

All MON 87411 11333176 Glyphosate

- Tolerant and

Insect

- Protected

All MPA 640 B 2 11333170 Conventional Control

IABG Jacobsen Seed JS4431 11267096 Coventional Reference Phillips 713 11300072 Coventional Reference

LG2615CL 11226863 Coventional Reference

Pioneer 32B81 11226578 Coventional Reference

IARL Legacy L7671 11226598 Coventional Reference

Lewis 7007 11226559 Coventional Reference Gateway 6116 11227211 Coventional Reference Phillips 717 11300073 Coventional Reference

ILMN Gateway 4148 11273005 Coventional Reference

H

- 9180 11226704 Coventional Reference

Stewart S588 11226918 Coventional Reference

LG2548 11266731 Coventional Reference

INSH Gateway 6158 11273006 Coventional Reference

LG2620 11226861 Coventional Reference

Stewart S518 11226920 Coventional Reference Legacy L7671 11226598 Coventional Reference

KSLA Lewis 7007 11226559 Coventional Reference
Midland Phillips 799 11226703 Coventional Reference

NC+ 5220 11226701 Coventional Reference

LG2540 11266730 Coventional Reference

NCBD LG2548 11266731 Coventional Reference

Stewart S602 11226919 Coventional Reference Midland Phillips 799 11226703 Coventional Reference Gateway 6158 11273006 Coventional Reference

NEDC Gateway 4148 11273005 Coventional Reference Midland Phillips 799 11226703 Coventional Reference

NC+ 4443 11226700 Coventional Reference

LG2540 11266730 Coventional Reference

NEYO LG2540 11266730 Coventional Reference

NC+ 5220 11266701 Coventional Reference Mycogen 2M746 11226705 Coventional Reference Gateway 6158 11273006 Coventional Reference

PAHM Phillips 717 11300073 Coventional Reference

Stewart S588 11226918 Coventional Reference

Pioneer 33T56 11226580 Coventional Reference Gateway 4148 11273005 Coventional Reference

1 Site code: IABG = Greene County, IA; IARL = Jefferson County, IA; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NEYO = York County, NE; PAHM = Berks County, PA.

2 MPA640 B = LH244 × LH287.Monsanto Company CR240

- 13U1 326 of 374 Table I

- 2. Field and Planting Information

Site 1 Planting

Date 2 Harvest

Date 2 Approximate Planting Rate

(seeds/m) Approximate

Plot Size (m × m) Rows

per Plot Soil Type % OM 3 Previous Crop

2011

IABG 05/09/2012 10/05/2012 7.2 6.1 × 12.2 16 Loam 4.0 Soybean

IARL 05/11/2012 10/12/2012 7.2 6.1 × 4.6 6 Silty Clay Loam 3.4 Soybean

ILMN 05/10/2012 10/01/2012 6.9 6.2 × 4.6 6 Silty Clay Loam 4.5 Soybean

INSH 05/11/2012 11/02/2012 7.2 6.1 × 4.6 6 Silt Loam 2.5 Maize

KSLA 05/11/2012 09/21/2012 7.2 6.1 × 4.6 6 Silt Loam 2.6 Sorghum

NCBD 05/11/2012 09/20/2012 6.6 6.1 × 15.5 16 Sandy Loam 2.6 Cotton

NEDC 05/05/2012 09/11/2012 7.2 6.2 × 4.6 6 Silt Loam 2.6 Soybean

NEYO 05/08/2012 10/09/2012 7.2 6.1 × 12.2 16 Silt Loam 3.0 Soybean

PAHM 05/19/2012 10/19/2012 8.2 6.1 × 12.2 16 Loam 1.6 Vegetables 4

Note: All plots were thinned to a uniform density of approximately 31 plants (IABG, INSH, KSLA, NCBD, NEYO, and PAHM) and 38 plants (IARL, ILMN, and NEDC) per 6.0 m row.

1 Site code: IABG = Greene County, IA; IARL = Jefferson County, IA; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = P awnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NEYO = York County, NE; PAHM = Berks County, PA.

2 Planting and Harvest Date = mm/dd/yyyy.

3 % OM = Percent Organic Matter.

4 Vegetables = peppers, tomatoes, potatoes, cabbage, maize.Monsanto Company CR240

- 13U1 327 of 374 I

. 5 . Phenotypic Observations

The description of the characteristics measured and the designated developmental stages where observations occurred are listed in Section VIII, Table VIII

- 1.

I

. 6

. Environmental Observations

Environmental interaction s ( i.e. , interactions between the crop plants and their receiving environment) were used to characterize MON 87411 by evaluating plant response to abiotic stressors, disease damage, and arthropod

- related damage using qualitative

methods described in section I

. 7

. In addition, specific arthropod damage (corn earworm damage and European corn borer) and arthropod abundance were evaluated using the quantitative methods described in Section I

. 8

.

I

. 7

. Plant Response to Abiotic Stress, Disease Damage, and Arthropod

- Rela ted Damage

MON 87411 and the conventional control were evaluated at all sites for plant response to abiotic stressors, disease damage, and arthropod damage. A target of t hree abiotic stressors, three diseases, and three arthropod pests were evaluated four times during the following four crop developmental stages: V 6

– V 8 ; V1 2

- V T ; R1

- R3; and R5

- R6.

Abiotic stressor, disease damage and arthropod damage observations were collected from each plot using the categorical scale of increasing severity listed below :

Category Severity of plant damage

None No symptoms observed

Slight Symptoms not damaging to plant development ( e.g

. , minor feeding or minor lesions); mitigation likely not required

Moderate Intermediate between slight and severe; likely requires mitigation

Severe Symptoms damaging to plant development ( e.g

. , stunting or death); mitigation unlikely to be effective

Method used for selecting stressors at each field site:

1. Prior to each data collection, maize was surveyed in proximity to the study area or the border rows of the study for abiotic stressors ( e.g. , drought), diseases ( e.g. , gray leaf spot), and arthropod damage ( e.g. , corn flea beetle).

2. The Principal Investigator chose three abiotic stressors, three diseases, and three arthropod specie s that are actively causing damage for subsequent evaluation in the study plots. The Principal Investigator s were requested to select additional stressors if present.

3. If fewer than three abiotic stressors, diseases, or arthropod species were present, th e cooperator chose additional abiotic stressors, diseases, and arthropod species that are known to commonly occur in that geographical region and cause damage at the study

site at that time.Monsanto Company CR240

- 13U1 328 of 374 4. All plots at a site were rated for the same abiotic stressors, d iseases, and arthropod pests at a given observation, even if that selected stressor was not present in some or all of the plots.

5. If a selected stressor was not present, the cooperator recorded the rating as “none”.

As indicated above, t he Principal Invest igator at each field site chose abiotic stressors, diseases, and arthropod pests that were either actively causing plant injury in the study area or were likely to occur in maize during the given observation period. Therefore, the type of abiotic stressors, diseases, and arthropod pests assessed varied between

observations at a site and between sites.

In addition, ear and kernel rot disease and stalk rot disease were evaluated at harvest (R6 growth stage) using the above categorical scale . Ear and kernel rot disease data were collected by evaluating five non

- systematically selected ears (one per plant) from each plot. The husks were pulled back and each ear was examined for disease. To evaluate stalk rot, five non

- systematically selecte d stalks in each plot were cut longitudinally. The

stalks were then examined for disease

.

I

. 8 . Arthropod Abundance

Specific arthropod (corn earworm and European corn borer) damage and arthropod

abundance were assessed quantitatively from observations/col lections performed at IABG, NEYO, NCBD, and PAHM sites.

Corn earworm damage was evaluated at R5 growth stage by examining ears from ten plants (5 consecutive plants per row) in each plot

. The husks were pulled back and each

ear was examined for corn earworm damage using a plastic film grid (size of each grid

0.5 cm 2 )

. Damage (cm 2 ) per plant was calculated as the total number of grid cells matching the damage area multiplied by 0.5 (each grid cell = 0.5 cm 2 ).

European corn borer damage was eval uated at R6 growth stage by examining ten plants (5 consecutive plants per row) in each plot

. Damage was assessed by splitting each of ten plants and counting the number of feeding galleries per plant and length of feeding gallery (cm.) in each stalk.

A r thropods were collected using yellow sticky traps five times during the growing season at the following intervals: late vegetative

– VT, R1, R2, R3, and R4 growth stage. Sticky traps (two per plot) were deployed in rows 6 and 8 of each plot at the approximate midpoint between the ground level and the top of the plant canopy for all arthropod collections. At each specified collection, traps were deployed for approximately 7 days. Sticky traps were then sent to the University of Arkansas, Fayetteville, AR for arthropod

identification and enumeration. A maximum of twelve arthropods were enumerated for

each collection. Seven preselected arthropods (or arthropod groups), namely aphid

, corn flea beetle, leafhopper, spider, micro pa rasitic hymenoptera, ladybird beetle, and minute pirate bug were enumerated at all sites for each collection time. Additionally, for each individual collection ( e.g. , Collection 1, IABG site), four non

- systematically selected samples were examined to deter mine the most abundant arthropods to obtain a total of upMonsanto Company CR240

- 13U1 329 of 374 to twelve arthropods to be enumerated for that particular collection and site. Thus, the suite of arthropods assessed often varied between collections from a site and between

sites due to difference s in temporal activity and geographical distribution of arthropod

taxa.

Five visual counts were conducted during the growing season at approximately VT

- R1, R1, R2, R3, and R4

- R5 from five non

- systematically selected plants per plot to collect

abundance da ta per plot

. Visual counts were made by examining the stalk, the leaf blade,

the leaf collar

, the ear tip, the silk, and the tassel of each plant.

I .9. Data Assessment

Experienced scientists familiar with the experimental design and evaluation criteria were involved in all components of data collection, summarization, and analysis. Study personnel assessed that measurements were taken properly, data were consistent with expectations based on experience with the crop, and the experiment was carefully monitored. Prior to analysis, the overall dataset was evaluated for evidence of biologically relevant changes and for possible evidence of an unexpected plant response. Any unexpected observations or issues during the trials that would impact the trial objectives were noted. Data were then subjected to data summarization or statistical analysis as indicated I .10.

I

. 10

. Environmental Interactions Evaluation Criteria for Qua litative Data

I .1 0 .1 Agronomic and Phenotypic Data

Plant vigor data were summarized but not subjected to an analysis of variance ( ANOVA )

. MON 87411 was considered different from the conventional control in vigor if the ranges of vigor of MON 87411 did no t overlap with the range of vigor of the conventional control across all replications. Any observed differences between the MON 87411 and

conventional control were further assessed in the context of the range of the reference materials, and for consistenc y at other sites

.

An ANOVA was conducted according to a randomized complete block design using

SAS ****

**** SAS is a registered trademark of SAS Institute, Inc. to compare MON 87411 and the conventional control for the 13 phenotypic

c haracteristics listed in Table VII I

- 1 , with the exception of plant vigor

. The level of statistical significance was predetermined to be 5 % (α = 0.05). C omparisons of

MON 87411 and the conventional control were conducted within site (individual site analysis) and in a combined

- site analysis, in which the data were pooled across sites.

MON 87411 and the conventional control materials were not statistically compared to the

reference materials. The reference range for each measured phenotypic characteristic

was determined from the minimum and maximum mean values from the 22 conventional

reference hybrids that were included across all sites

.Monsanto Company CR240

- 13U1 330 of 374 Data excluded from analysis and the reasons for exclusion are listed in Table I

- 3

.

I .1 0

. 2 Environmental Interaction Data

The environmental interaction data ( i.e.

, plant respons e to abiotic stressors, disease damage, and arthropod damage ) are categorical and were not subjected to ANOVA.

MON 87411 and conventional control were considered different in susceptibility or tolerance if the range of injury symptoms of each did not over lap across all four replications. Any observed differences were further assessed in the context of the range of the reference materials, and for consistency at other sites

.

An ANOVA was conducted according to a randomized complete block design SAS ®

(SAS 2008; 2012) for corn earworm (CEW) damage, European corn borer (ECB) damage, and arthropod abundance. The level of statistical significance was predetermined to be 5 % (α = 0.05). MON 87411 was compared to the conventional control at each site (individua l

- site analysis) for CEW damage, ECB damage, and the arthropod abundance. Additionally, corn earworm damage and European corn borer damage data were pooled across sites (combined

- site analysis) for statistical comparison

between MON 87411 and the conventi onal control. Minimum and maximum mean

values were calculated for CEW damage and ECB damage from 15 reference hybrids that

were included at IABG, NEYO, NCBD, and PAHM sites. The reference range for arthropod abundance evaluated from a given collection an d site was determined from the

minimum and maximum mean values collected from the reference maize at the site.

For the arthropod abundance data, s tatistical analyses and significance testing of

differences between MON 87411 and the conventional control m aterials were only performed for the arthropods present in sufficient numbers to estimate the material mean arthropod counts and the variation of the means. An inclusion criterion was established where a given arthropod must have an average count per plot per collection time ( across all materials ) of ≥ 1.

Data excluded from analysis and the reasons for exclusion are listed in Table I

- 3

.

I

. 11 . Statistical Analysis

In individual site assessments of plant vigor, MON 87411 and the conventional control were considered different if the range of vigor values did not overlap across all four replications. There were no differences observed between MON 87411 and the conventional control in plant vigor at all sites (Tabl e I

- 4)

.

In the individual

- site analysis, a total of 11 statistically significant differences were detected out of 107 comparisons between MON 87411 and the conventional control (Table I

- 4 ). These differences were distributed among eight of the 13 pheno typic

characteristics. MON 87411 had a lower early stand count than the conventional control at the IABG (82.3 vs. 86.3 plants per plot) and PAHM (72.5 vs. 78.8 plants per plot) sites but a higher early stand count at the NEDC site (95.3 vs. 89.8 plants p er plot).

MON 87411 had fewer days to 50% pollen shed than the conventional control at the NCBD site (59.3 vs. 60.5 days) and more days to 50% silking than the conventionalMonsanto Company CR240

- 13U1 331 of 374 control at the NE YO site (66.5 vs. 65.5 days). MON 87411 exhibited a higher stay green rating (less green tissue) than the conventional control at the KSLA site ( 1.8 vs. 1 .0 rating). MON 87411 had a higher final stand count than the conventional control at the KSLA site (77.3 vs.74.0 plants/plot). Grain moisture percentage was lower for

MON 87411 than the conventional control at the IARL (16.9 vs. 17.7 %) and PAHM (21.1 vs. 22.3 %) sites. The test weight was higher for MON 87411 than the conventional control at the PAHM site (68.5 vs. 65.8 kg/hl). MON 87411 had a higher yield than t he conventional control at the INSH site (11.5 vs. 9.2 Mg/ha). The statistical

differences between MON 87411 and the conventional control detected in the individual

site analysis for early stand count, days to 50% pollen shed, days to 50% silking, stay

gr een, final stand count, grain moisture percentage, test weight, and yield were not

detected in the combined

- site analysis. Thus, the differences detected for these phenotypic characteristics do not indicate a consistent response associated with the trait and are unlikely to be biologically meaningful, in terms of increased pest/weed potential

of MON 87411 compared to the conventional maize (See Figure VII I

- 1, S tep 2 , answer “no”).

I

. 12 . Individual Field Site Plant Growth

, Development , and Environmental

Interactions Results and Discussion

Plant Response to Abiotic Stressor, Disease Damage, and Arthropod

- related Damage:

In the individual

- site assessment, n o differences were observed between MON 87411 and the conventional control for any of th e 100 comparisons for the assessed abiotic stressors, including cold, drought, flood, frost, hail, heat, nutrient deficiency, soil compaction, sunscald, and wind (Table I

- 5).

In the individual

- site assessment, no differences were observed between MON 87411 and the conventional control for any of the 119 comparisons for the assessed diseases, including anthracnose, bacterial leaf spot, ear rot, eyespot, Fusarium sp., Goss’s bacterial wilt, gray leaf spot, leaf blight, maize rough dwarf virus, Pythium sp., Rh izoctonia sp., rust, seedling blight, smut, stalk rot, and Stewart’s bacterial wilt (Table I

- 6).

In the individual

- site assessment, no differences were observed between MON 87411 and the conventional control for any of the 102 comparisons for the assessed arthropods, including aphid, armyworm , billbug, cutworm, corn earworm, corn flea beetle, rootworm beetle, European corn borer, grasshopper, Japanese beetle, sap beetle, spider mi te, stink bug, and wireworm adult (Table I

- 7).

Corn Earworm and European Corn Borer Damage:

In the individual

- site analysis, one statistically significant difference was detected out of 12 comparisons between MON 87411 and the conventional control for CEW and E CB among all observations at all four sites (Table I

- 8). C EW damage was higher for

MON 87411 than the conventional control at the NCBD site (3.3 vs. 1.5 cm 2 damaged area per plot). This statistical difference in the individual

- site analysis was not detected in

the combined

- site analysis . Thus, this difference was not indicative of a consistent plantMonsanto Company CR240

- 13U1 332 of 374 response associated with the trait and is unlikely to be biologically meaningful in terms of increased pest potential of MON 87411 compared to the conve ntional control (See Figure

VIII

- 1, Step 2, answer “no”).

Sticky Trap:

A total of 108 statistical comparisons were made between MON 87411 and the conventional control for arthropod abundance involving the following arthropods: aphid, corn flea beetle, delphacid planthopper, green lacewing, ladybird beetle, leafhopper,

micro

- parasitic hymenoptera, macro

- parasitic hymenoptera, sap beetle, minute pirate bug, and spider (Table I

- 9). Lack of sufficient arthropod abundance precluded statistical comparisons b etween MON 87411 and the conventional control for 79 additional comparisons; however, descriptive statistics were provided for these comparisons (Table

I

- 9).

No statistically significant differences were detected between MON 87411 and the

conventional co ntrol for 104 out of 108 comparisons (Table I

- 9). The abundance of aphid s was higher in MON 87411 than the conventional control in Collection 3 (1.8 vs. 0.0 per plot) at the NEYO site. The mean value for aphid abundance on MON 87411 was within the range of the reference hybrids. The abundance of delphacid planthopper s was lower in MON 87411 than the conventional control in Collection 1 at the IABG site (0.0 vs. 1.8 per plot). MON 87411 had higher abundance than the conventional control for spider s (3.3 vs. 0.3 per plot) in Collection 5 and lower abundance than the conventional control for minute pirate bug s (1.0 vs. 3.0 per plot) in Collection 1 at the PAHM site. The mean values for delphacid planthopper, minute pirate bug, and spider abundance on MON 87411 were outside the respective ranges of reference hybrids. However, these differences were not consistently detected across collections or sites (Table I

- 9). Thus, these differences in aphid, delphacid planthopper, minute pirate bug, and spide r abundance were not indicative of a consistent response associated with the trait and are not considered biologically meaningful in terms of increased pest potential

of MON 87411 compared to conventional maize (See Section VII I .B.2.).

Visual counts:

A t otal of 61 statistical comparisons were made between MON 87411 and the conventional control for arthropod abundance involving the following pest and beneficial arthropods: ant

- like flower beetle, click beetle, corn flea beetle, ladybird beetle adult, ladyb ird beetle larvae, minute pirate bug, sap beetle, shining flower beetle, and spider (Table I

- 10). Lack of sufficient arthropod abundance precluded statistical comparisons

between MON 87411 and the conventional control for 152 additional comparisons;

howev er, the descriptive statistics were provided for these comparisons (Table I

- 10).

No statistically significant differences were detected between MON 87411 and the conventional control for 60 out of 61 comparisons (Table I

- 10). The abundance of ant

like flo wer beetle s was lower in MON 87411 than the conventional control in Collection 2 at the IABG site (0.8 vs. 4.3 per plot). The mean value for ant

- like flower beetle

abundance on MON 87411 was lower than the range of reference hybrids (1.8

– 3.3 perMonsanto Company CR240

- 13U1 333 of 374 plot). However, the difference for ant

- like flower beetle was not consistently detected

across collections at the IABG site (Table I

- 10). Thus, this difference in ant

- like flower beetle abundance was not indicative of a consistent response associated with the t rait and is not considered biologically meaningful in terms of increased pest potential

of MON 87411 compared to conventional maize (See Section VIII.B.2.)

.Monsanto Company CR240

- 13U1 334 of 374 Table I

- 3

. Data Missing or Excluded from Analysis

Site Code 1 Material Name Material Type Plots Characteristics Reason for Exclusion

IALL , ILCY, ILPH, and

ILWY All All All Phenotypic and environmental interactions

data E xtreme weather conditions (strong

wind or drought) that caused extensive damage across the plots

IARL MON 87411 Test 406 Root lodged plants Wind damage

IARL MPA640B Legacy L7671 Control

Reference 106

107 Moisture (%), shelled plot weight, and test weight Incorrect data collection;

Mechanical errors

INSH Legacy L7671 Reference 205 Days to 50% silking and days to 50% pollen

shed Identified as an outlier

NEDC MON 87411

Midland Phillips 799 Test

Reference 104

106 Final stand count Identified as an outlier

PAHM MON 87411 Phillips 717

Pioneer 33T56

MPA640B Test

Reference

Reference

Control 106

206

306

406 Sticky trap data and visual count Missing data

PAHM MPA640B Control 406 Test weight Missing data

NEDC All All All Environmental interactions evaluation # 4 ( Animal damage ) Improper selection of stressor

NEYO All All All Environmental interaction evaluation # 4 ( Black stem disease ) Improper selection of stressor

NEYO All All All Environmental interaction evaluation # 3 ( abiotic stressor ) Missing data for two abiotic

stressors

IARL All All All Environmental interaction evaluation # 2 Incorrect data collection

IARL All All All Environmental interaction evaluation # 3 and 4 ( Bean leaf beetle ) Improper selection of stressor

KSLA All All All Environmental interaction evaluation # 2 ( Velvetbean caterpillar ) Improper selection of stressorMonsanto Company CR240

- 13U1 335 of 374 Table I

- 3 (continued). Data Missing or Excluded from Analysis

Site Code 1 Material Name Material Type Plots Characteristics Reason for Exclusion

NCBD All All All Environmental interaction evaluation # 1 ( Other arthropod ) Improper selection of stressor

IABG All All All Environmental interaction evaluation # 4 ( Other arthropod ) Improper selection of stressor

IABG All All All Visual count

– Collection # 1 Incorrect data collection

INSH LG2620 Reference 101 Environmental interaction evaluation # 1 ( Bacterial blight

- Pseudomonas ) Incorrect data collection; not rated for all plots

1 Site code: IABG = Greene County, IA; IALL = Story, IA; IARL = Jefferson County, IA; ILCY = Clinton, IL; ILPH = Champaign, IL; ILWY = Stark, IL ; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NEYO = York County, NE; PAHM = Berks County, PA.Monsanto Company CR240

- 13U1 336 of 374 Table I

- 4

. Individual Site Phenotypic Comparison of MON 87411 Compared to the Conventional Control

Phenotypic Characteristics
Plant Vigor (1

- 9 rating) 2 Early stand count (#/plot) Days to 50% pollen shed

Range Mean (S.E.) 3 Mean (S.E.) 3

Site

Code 1 MON 87411 Control MON 87411 Control MON 87411 Control

IABG 2

– 3 1

– 3 82.3 (1.11)* 86.3 (1.44) 65.3 (0.25) 65.3 (0.48)

IARL 7

– 8 5

– 7 84.8 (2.25) 82.3 (3.12) 62.8 (0.63) 62.0 (0.41)

ILMN 2

– 3 1

– 3 83.0 (0.41) 82.8 (1.18) 62.0 (0.00)† 62.0 (0.00)

INSH 4

– 6 3

– 4 76.5 (3.52) 83.5 (1.44) 66.5 (1.66) 64.8 (0.75)

KSLA 2 2 97.3 (0.75) 96.0 (0.82) 56.0 (0.00) 56.0 (0.00)

NCBD 3

– 4 3

– 4 71.3 (3.04) 72.3 (1.65) 59.3 (0.25)* 60.5 (0.50)

NEDC 2 2 95.3 (1.49)* 89.8 (1.70) 68.0 (0.41) 68.8 (0.25)

NEYO 1 1 76.0 (0.82) 78.8 (1.44) 66.3 (0.48) 65.5 (0.29)

PAHM 3

– 4 3

– 5 72.5 (1.89)* 78.8 (1.55) 63.5 (0.50) 63.0 (0.41)Monsanto Company CR240

- 13U1 337 of 374 Table I

- 4 (c ontinued)

. Individual Site Phenotypic Comparison of MON 87411 Compared to the Conventional Control

Phenotypic Characteristics
Days to 50% silking Stay

- green rating (1

- 9 scale) Ear height (cm)

Mean (S.E.) 3 Mean (S.E.) 3 Mean (S.E.) 3

Site

Code 1 MON 87411 Control MON 87411 Control MON 87411 Control

IABG 64.3 (0.25) 65.3 (0.63) 7.3 (1.03) 7.0 (0.00) 96.8 (2.19) 101.4 (1.22)

IARL 61.0 (0.58) 61.0 (0.00) 3.0 (0.00) 3.5 (0.29) 97.7 (3.95) 90.8 (2.40)

ILMN 62.0 (0.00)† 62.0 (0.00) 4.0 (0.00) 4.0 (0.00) 107.9 (1.03) 109.5 (0.81)

INSH 67.3 (1.49) 66.0 (0.82) 4.5 (0.29) 5.5 (0.87) 97.1 (2.21) 93.6 (2.51)

KSLA 58.0 (0.00) 58.0 (0.00) 1.8 (0.25)* 1.0 (0.00) 103.2 (1.45) 102.2 (1.13)

NCBD 58.8 (0.63) 59.8 (0.25) 5.5 (0.50) 6.0 (0.00) 126.9 (2.69) 130.1 (1.73)

NEDC 68.3 (0.25) 68.3 (0.48) 9.0 (0.00) 9.0 (0.00) 113.5 (0.83) 107.8 (4.33)

NEYO 66.5 (0.50)* 65.5 (0.29) 6.3 (0.25) 6.0 (0.00) 107.7 (1.05) 104.3 (1.20)

PAHM 64.8 (0.63) 64.3 (0.25) 8.8 (0.25) 8.0 (0.41) 85.9 (3.05) 93.3 (5.68)Monsanto Company CR240

- 13U1 338 of 374 Table I

- 4 (c ontinued)

. Individual Site Phenotypic Comparison of MON 87411 Compared to the Conventional Control

Phenotypic Characteristics
Plant height (cm) Dropped ears (#/plot) Stalk lodged plants (#/plot)

Mean (S.E.) 3 Mean (S.E.) 3 Mean (S.E.) 3

Site

Code 1 MON 87411 Control MON 87411 Control MON 87411 Control

IABG 259.8 (2.51) 266.0 (6.25) 0.0 (0.00)† 0.0 (0.00) 0.3 (0.25) 0.0 (0.00)

IARL 200.6 (9.72) 202.7 (3.79) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)† 0.0 (0.00)

ILMN 257.5 (1.56) 263.7 (3.37) 0.0 (0.00) 0.0 (0.00) 0.3 (0.25) 0.0 (0.00)

INSH 214.0 (9.56) 204.2 (7.99) 0.5 (0.29) 1.3 (0.48) 3.3 (0.63) 3.3 (0.85)

KSLA 223.7 (3.50) 221.9 (1.02) 0.0 (0.00)† 0.0 (0.00) 0.0 (0.00)† 0.0 (0.00)

NCBD 259.2 (0.67) 262.2 (1.09) 5.3 (0.95) 7.8 (0.95) 0.3 (0.25) 0.0 (0.00)

NEDC 252.4 (4.75) 247.3 (5.50) 0.0 (0.00) 0.0 (0.00) 1.8 (0.85) 1.0 (0.41)

NEYO 260.4 (1.63) 257.1 (2.47) 0.3 (0.25) 0.0 (0.00) 1.0 (0.71) 0.3 (0.25)

PAHM 217.2 (3.54) 227.2 (7.10) 0.0 (0.00) 0.3 (0.25) 4.3 (0.85) 5.8 (2.06)Monsanto Company CR240

- 13U1 339 of 374 Table I

- 4 (c ontinued)

. Individual Site Phenotypic Comparison of MON 87411 Compared to the Conventional Control

Phenotypic Characteristics
Root lodged plants (#/plot) Final stand count (#/plot) Grain moisture (%)

Mean (S.E.) 3 Mean (S.E.) 3 Mean (S.E.) 3

Site

Code 1 MON 87411 Control MON 87411 Control MON 87411 Control

IABG 0.3 (0.25) 1.0 (1.00) 60.3 (0.25) 60.8 (0.48) 17.0 (0.79) 16.9 (0.59)

IARL 0.0 (0.00) 0.8 (0.48) 61.5 (0.50) 61.8 (0.25) 16.9 (0.15)* 17.7 (0.33)

ILMN 0.0 (0.00)† 0.0 (0.00) 62.0 (0.71) 61.5 (0.50) 19.7 (0.28) 19.5 (0.17)

INSH 0.5 (0.50) 0.0 (0.00) 70.0 (1.47) 69.3 (2.95) 18.2 (0.26) 18.3 (0.14)

KSLA 0.0 (0.00)† 0.0 (0.00) 77.3 (0.48)* 74.0 (0.00) 15.1 (0.19) 15.4 (0.43)

NCBD 0.0 (0.00) 0.3 (0.25) 67.8 (1.03) 67.0 (0.58) 20.0 (0.19) 20.0 (0.19)

NEDC 0.0 (0.00) 0.0 (0.00) 62.0 (0.00) 61.3 (0.25) 14.8 (0.33) 15.6 (0.35)

NEYO 0.0 (0.00)† 0.0 (0.00) 61.0 (1.35) 60.8 (0.75) 15.4 (0.22) 15.2 (0.20)

PAHM 0.0 (0.00)† 0.0 (0.00) 62.3 (0.63) 62.5 (0.29) 21.1 (0.27)* 22.3 (1.01)Monsanto Company CR240

- 13U1 340 of 374 Table I

- 4 (c ontinued)

. Individual Site Phenotypic Comparison of MON 87411 Compared to the Conventional Control

Phenotypic Characteristics
Test weight (kg/hl) Yield (Mg/ha)

Mean (S.E.) 3 Mean (S.E.) 3

Site

Code 1 MON 87411 Control MON 87411 Control

IABG 76.9 (0.80) 77.8 (0.33) 11.7 (1.18) 9.5 (0.42)

IARL 76.6 (0.64) 75.8 (0.64) 8.2 (1.00) 8.3 (0.43)

ILMN 69.6 (0.18) 69.9 (0.18) 14.4 (0.29) 14.9 (0.04)

INSH 71.6 (1.01) 73.2 (1.18) 11.5 (0.66)* 9.2 (0.77)

KSLA 74.8 (0.36) 75.5 (0.40) 14.1 (0.48) 14.0 (0.51)

NCBD 69.0 (0.41) 70.1 (0.78) 9.5 (0.28) 9.3 (0.59)

NEDC 72.1 (1.00) 73.5 (1.07) 7.5 (1.16) 8.0 (0.52)

NEYO 76.2 (0.28) 76.3 (0.18) 12.5 (0.07) 13.0 (0.17)

PAHM 68.5 (0.46)* 65.8 (0.88) 10.4 (0.27) 11.1 (0.48)

Note: The experimental design was a randomized complete block with four replicates per site.

* Indicates statistical ly significant difference between MON 87411 and the conventional control (α =0.05) using ANONA

. † Indicates p

- values could not be generated due to lack of variability in the data.

1 Site code: IABG = Greene County, IA; IARL = Jefferson County, IA; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = P awnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler Count y, NE; NEYO = York County, NE; PAHM = Berks County, PA.

2 Data were not subjected to statistical analysis. Plant vigor rating range (minimum

- maximum); the range of plant vigor ratings for the references

is as follows: IABG 1

– 3; IARL 5

– 8 ; ILMN 1

– 3; INSH 3

– 5 ; KSLA 2 ; NCBD 3

– 4 ; NEDC 2 ; NEYO 1 ; PAHM 2

– 4

3 MON 87411 and the conventional control values represent means with standard error in parentheses. N = 4 except where noted in Ta ble I

- 3.Monsanto Company CR240

- 13U1 341 of 374 Table I

- 5

. Qualitative Assessment : Abiotic Stressor Evaluations Using a Categorical

Scale for MON 87411 and the Conventional Control

Abiotic Stressor Number of

Observations across

Sites 1 Number of Observations where No

Differences were Observed between

MON 87411 and the Conventional

Control

Total 100 100

Cold 1 1 Drought 2 22 22

Flood 3 5 5

Frost 2 2

Hail 8 8

Heat 20 20

Nutrient deficiency 12 12 Soil compaction 3 3

Sunscald 5 5

Wind 2 2 2 2

Note: The experimental design was a randomized complete block with four replicates per site.

No differences were observed between MON 87411 and the conventional control during any observation for damage caused by any of the assessed abiotic stressors. Data were not subjected to statistical analysis. Observational data collected at four crop development stages: V6

– V8; V12

- VT; R1

- R3; and R5

- R6.

1 Site code: IABG = Greene County, IA; IARL = Jefferson County, IA; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NEYO = York County, NE; PAHM = Berks County, PA.

2 In cludes dryness, dry/heat.

3 Includes wet soil.Monsanto Company CR240

- 13U1 342 of 374 Table I

- 6

. Qualitative Assessment : Disease Damage Evaluations Using a Categorical

Scale for MON 87411 and the Conventional Control

Disease Number of

Observations

across Sites 1 Number of Observations where No

Differences were Observed between

MON 87411 and the Conventional

Control

Total 119 119

Anthracnose 6 6 Bacterial leaf spot 1 1

Ear rot 2 12 12 Eyespot 5 5

Fusarium sp. 8 8

Goss’s bacterial wilt 10 10 Gray leaf spot 20 20 Leaf blight 3 16 16 Maize rough dwarf virus 2 2 Pythium sp. 2 2
Rhizoctonia sp. 2 2

Rust 4 7 7 Seedling blight 2 2

Smut 8 8

Stalk rot 5 10 10

Stewart’s bacterial wilt 8 8

Note: The experimental design was a randomized complete block with four replicates per site.

No differences were observed between MON 87411 and the conventional control during any observation for damage caused by any of the assessed diseases. Data were not subjected to statistical analysis. Observational data collected at four crop development stages: V6

– V8; V12

- VT; R1

- R3; and R5

- R6.

Additional assessments of ear rot disease and stalk rot disease were made on 5 plant/plots at
harvest.

1 Site code: IABG = Greene County, IA; IARL = Jefferson County, IA; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NEYO = York County, NE; PAHM = Berks County, PA.

2 Assessed on 5 non

- syste matically selected plants. At ILMN and INSH, stalk rot data were collected both on a per plot basis and on 5 plant/plots.

3 Includes northern and southern.

4 Includes common and southern

. 5 Assessed on 5 non

- systematically selected plants. At NEDC, stalk rot data were collected both on a per plot basis and on 5 plant/plots.Monsanto Company CR240

- 13U1 343 of 374 Table I

- 7

. Qualitative Assessment : Arthropod Damage Evaluations Using a Categorical Scale for MON 87411 and the Conventional Control

Arthropod Number of

Observations

across Sites 1 Number of Observations

where No Differences were

Observed between

MON 87411 and the

Conventional Control

Total 102 102

Aphid (Aphididae) 3 3 Armyworm (Noctuidae) 2 18 18 Billbugs ( Sphenophorus parvulus ) 2 2 Cutworm (Noctuidae) 3 6 6

Corn earworm ( Helicoverpa zea ) 5 5 Corn flea beetle ( Chaetocnema pulicaria ) 5 5 Rootworm beetle ( Diabrotica spp. ) 16 16 European corn borer ( Ostrinia nubilalis ) 9 9 Grasshopper ( Melanoplus spp. ) 14 14 Japanese beetle ( Popillia japonica ) 9 9 Sap beetle (Nitidulidae) 3 3 Spider mites ( Tetranychus spp.) 3 3 Stink bugs (Pentatomidae) 8 8 Wireworm beetle ( Elateridae ) 1 1

Note: The experimental design was a randomized complete block with four replicates per site.

No differences were observed between MON 87411 and the conventional control during any observation for damage caused by any of the assessed arthropods. Data were not subjected to statistical analysis. Observational data collected at four crop development stages: V6

– V8; V12

- VT; R1

- R3; and R5

- R6.

1 Site code: IABG = Greene County, IA; IARL = Jefferson County, IA; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NEYO = York County, NE; PAHM = Berks County, PA.

2 Includ es beet armyworm and fall armyworm.

3 Includes black cutworm.Monsanto Company CR240

- 13U1 344 of 374 Table I

- 8

. Individual

- Site Analysis: Quantitative Assessment of Corn Earworm and European Corn Borer Damage to MON 87411 Compared to the Conventional

Control

Pest Damage assessment 1 Site 2 MON 87411 Control

Corn earworm ( H. zea ) 3 Mean (S.E.) damage area of 10 plants per plot (cm 2 ) IABG 0.7 (0.38) 0.5 (0.28)

NCBD 3.3 (1.25)* 1.5 (0.39)

NEYO 3.2 (0.13) 3.0 (0.22)

PAHM 0.3 (0.23) 0.2 (0.11)

European corn borer
( O. nubilalis ) 4 Mean (S.E.) number of stalk galleries of 10 plants per plot IABG 0.0 (0.03) 0.0 (0.00)

NCBD 0.1 (0.03) 0.1 (0.04)

NEYO 0.0 (0.00) 0.0 (0.00)

PAHM 1.4 (0.24) 1.8 (0.33)

European corn borer
( O. nubilalis ) 4 Mean (S.E.) stalk gallery length (cm) of 10 plants per plot IABG 0.1 (0.08) 0.0 (0.00)

NCBD 0.5 (0.19) 0.2 (0.14)

NEYO 0.0 (0.00) 0.0 (0.00)

PAHM 5.9 (1.46) 7.9 (1.46)

Note: The experimental design was a randomized complete block with four replicates per site.

*Indicates a statistically significant difference between MON 87411 and the conventional control (α=0.05) using ANOVA.

1 MON 87411and the conventional control values represent means with standard error in parentheses.

N = 4.

2 Site code: IABG = Greene, IA; NCBD = Perq uimans, NC; NEYO = York, NE; PAHM = Berks, PA.

3 Damage assessment for H. zea was conducted at R5 growth stage.

4 Damage assessments for O. nubilalis were conducted at R6 growth stage.Monsanto Company CR240

- 13U1 345 of 374 Table I

- 9

. Individual

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411 Compared to

the Conventional Control

Aphid ( Aphididae) Brown Lacewing ( Hemerobiidae) Corn Flea Beetle (Chrysomelidae)

Pest Arthropod Beneficial Arthropod Pest Arthropod

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control MON 87411 Control

1 IABG 1.5 (0.87) 1.0 (0.41) 0.5

– 3.3

— — — 0.3 (0.25)† 0.5 (0.50) 0.0

– 0.8

NCBD 0.8 (0.75)† 0.3 (0.25) 0.3

– 1.8 0.5 (0.29)† 0.3 (0.25) 0.0

– 0.5 0.5 (0.29)† 0.0 (0.00) 0.0

– 4.3

NEYO 0.5 (0.29)† 0.0 (0.00) 0.0

– 2.0

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM 0.7 (0.33)† 0.7 (0.33) 0.3

– 1.0 0.7 (0.33)† 0.3 (0.33) 0.0

– 0.3 3.7 (1.45) 2.3 (1.33) 3.7

– 11.3

2 IABG 1.3 (0.75) 1.0 (0.41) 1.3

– 3.5 0.3 (0.25)† 0.3 (0.25) 0.3

– 1.0 0.5 (0.50)† 0.5 (0.50) 0.0

– 0.3

NCBD 0.8 (0.25) 0.3 (0.25) 0.3

– 4.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00) 2.0 (1.08) 0.5

– 5.3

NEYO 0.5 (0.29)† 0.3 (0.25) 0.0

– 2.3

— — — 0.5 (0.29)† 0.0 (0.00) 0.0

– 0.8

PAHM 1.0 (1.00)† 0.7 (0.67) 0.0

– 0.8

— — — 3.0 (1.53) 6.7 (0.88) 5.7

– 12.5

3 IABG 1.5 (0.29) 0.5 (0.29) 1.0

– 3.8

— — — 0.3 (0.25)† 0.0 (0.00) 0.5

– 1.0

NCBD 1.5 (0.65) 1.0 (0.71) 0.0

– 2.3

— — — 0.8 (0.48) 1.3 (0.95) 0.3

– 16.0

NEYO 1.8 (0.48)* 0.0 (0.00) 1.0

– 2.5

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM 0.3 (0.33)† 0.0 (0.00) 0.0

– 0.3

— — — 9.3 (4.48) 5.0 (1.73) 7.7

– 20.8

4 IABG 4.0 (1.73) 0.8 (0.25) 1.5

– 5.5 0.0 (0.00)† 0.0 (0.00) 0.3

– 0.8 0.5 (0.29) 0.3 (0.25) 1.3

– 4.8

NCBD 1.3 (0.25)† 0.0 (0.00) 0.0

– 1.8

— — — 1.0 (0.71) 3.3 (1.93) 0.3

– 19.8

NEYO 1.8 (0.48) 0.8 (0.25) 2.5

– 7.0

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

PAHM 0.3 (0.33)† 1.0 (0.58) 0.0

– 1.7

— — — 7.7 (3.53) 6.7 (3.28) 12.3

– 23.3

5 IABG 7.5 (4.25) 2.8 (1.11) 6.3

– 12.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.8 0.3 (0.25)† 0.5 (0.29) 0.5

– 1.5

NCBD 1.0 (0.71) 1.0 (0.41) 0.5

– 1.8

— — — 3.3 (1.11) 6.8 (1.03) 4.3

– 27.8

NEYO 21.0 (5.58) 19.3 (7.06) 15.8

– 30.0

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

PAHM 1.7 (0.88)† 0.7 (0.33) 0.0

– 1.0

— — — 5.0 (1.73) 3.0 (1.53) 3.0

– 12.0Monsanto Company CR240

- 13U1 346 of 374 Table I

- 9

. ( c ontinued). Individual

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411 Compared to the Conventional Control

Delphacid Planthopper ( Delphacidae ) Grasshopper (Acrididae) Green Lacewings (Chrysopidae)
Pest Arthropod Pest Arthropod Beneficial Arthropod

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control MON 87411 Control

1 IABG 0.0 (0.00)* 1.8 (0.85) 1.5

– 3.5

— — — 3.8 (1.25) 2.8 (1.31) 1.5

– 4.8

NCBD 51.0 (5.58) 64.0 (6.92) 63.8

– 106.0

— — — 0.0 (0.00)† 0.8 (0.25) 0.0

– 0.3

NEYO

— — — — — — 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.3

PAHM 6.7 (1.45) 3.0 (1.53) 3.8

– 10.0

— — — — — —
2 IABG 0.8 (0.48) 0.5 (0.29) 1.3

– 3.0

— — — 1.8 (0.85) 2.5 (1.19) 0.0

– 3.0

NCBD 10.0 (4.02) 11.8 (2.29) 15.3

– 23.0

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

NEYO 0.3 (0.25)† 0.5 (0.29) 0.3

– 0.5

— — — 0.8 (0.75)† 0.3 (0.25) 0.5

– 1.5

PAHM 4.7 (2.73) 5.0 (1.73) 2.3

– 10.5

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.5

3 IABG

— — — — — — — — —
NCBD 8.0 (1.08) 5.0 (2.52) 9.3

– 12.8

— — — — — —
NEYO 0.8 (0.25)† 0.3 (0.25) 0.3

– 0.5

— — — 4.0 (1.68) 2.5 (0.29) 1.5

– 4.8

PAHM 6.0 (2.00) 4.7 (1.20) 2.7

– 8.8

— — — — — —
4 IABG 1.3 (0.75)† 0.3 (0.25) 0.5

– 0.8

— — — 4.3 (1.11) 7.0 (1.22) 5.8

– 6.5

NCBD 4.8 (1.11) 6.0 (0.41) 7.5

– 8.3

— — — — — —
NEYO

— — — — — — 0.5 (0.29) 0.8 (0.25) 0.8

– 7.3

PAHM 4.7 (1.20) 2.3 (0.33) 0.7

– 10.3

— — — — — —
5 IABG

— — — — — — 6.3 (2.75) 5.3 (1.11) 4.5

– 9.8

NCBD 3.3 (1.25) 4.3 (1.44) 3.8

– 8.3

— — — — — —
NEYO

— — — — — — 3.0 (0.71) 1.5 (1.19) 0.3

– 2.0

PAHM 1.0 (0.58)† 0.7 (0.33) 0.0

– 2.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

— — —Monsanto Company CR240

- 13U1 347 of 374 Table I

- 9

. ( c ontinued). Individual

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411 Compared to the Conventional Control

Ladybird Beetle (Coccinellidae) Leafhopper (Cicadellidae) Micro

- Parasitic Hymenoptera
Beneficial Arthropod Pest Arthropod Beneficial Arthropod

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control MON 87411 Control

1 IABG 1.3 (0.75) 0.5 (0.50) 1.0

– 3.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 33.8 (2.66) 45.5 (3.40) 34.3

– 67.3

NCBD 3.5 (0.87) 5.3 (3.20) 2.5

– 3.5 37.8 (4.53) 40.0 (5.43) 37.3

– 53.0 129.8 (8.43) 141.5 (20.97) 214.5

– 278.0

NEYO 0.8 (0.25) 0.3 (0.25) 0.0

– 7.5 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 7.5 (1.94) 9.0 (1.47) 8.8

– 13.0

PAHM 15.3 (0.88) 14.3 (1.33) 21.5

– 32.3 4.3 (1.20) 4.0 (2.08) 3.5

– 7.3 128.0 (18.0) 104.0 (18.36) 145.7

– 189.3

2 IABG 0.8 (0.25) 1.3 (0.25) 1.0

– 5.8 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.0 36.3 (7.72) 44.8 (10.46) 48.0

– 88.3

NCBD 7.5 (1.85) 10.0 (2.58) 9.3

– 16.5 40.5 (8.91) 42.3 (15.10) 33.5

– 79.8 92.3 (15.37) 97.8 (12.25) 153.3

– 187.5

NEYO 0.0 (0.00)† 0.8 (0.48) 0.5

– 1.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 14.3 (3.28) 17.3 (4.35) 14.3

– 17.5

PAHM 39.0 (11.14) 33.7 (6.36) 26.3

– 36.0 3.0 (1.53) 5.0 (1.15) 3.0

– 10.3 102.7 (12.81) 119.0 (8.96) 114.0

– 141.8

3 IABG 0.0 (0.00)† 0.8 (0.25) 0.5

– 1.5 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.0 30.0 (4.78) 28.8 (3.88) 38.3

– 102.3

NCBD 5.8 (1.44) 6.5 (0.50) 4.0

– 7.0 40.8 (9.00) 43.3 (10.66) 48.0

– 69.5 66.8 (7.96) 62.5 (13.12) 61.3

– 87.3

NEYO 0.3 (0.25)† 0.8 (0.48) 0.3

– 1.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 17.8 (1.03) 12.8 (1.65) 14.3

– 33.0

PAHM 20.7 (5.90) 13.0 (2.08) 10.3

– 14.5 2.3 (0.33) 3.0 (1.00) 1.5

– 4.5 78.7 (22.51) 61.3 (2.96) 59.3

– 104.0

4 IABG 1.3 (0.48) 0.5 (0.50) 1.3

– 4.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 47.0 (5.15) 47.5 (3.30) 44.5

– 89.8

NCBD 8.5 (1.85) 7.0 (1.68) 5.5

– 8.0 27.5 (0.87) 33.5 (7.60) 28.3

– 45.0 65.8 (11.53) 56.5 (15.54) 61.5

– 104.3

NEYO 0.5 (0.29)† 0.8 (0.75) 0.3

– 1.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 12.5 (1.50) 9.3 (2.29) 13.8

– 69.0

PAHM 7.3 (0.67) 5.7 (1.20) 2.7

– 5.0 3.7 (0.88) 4.3 (1.33) 4.0

– 11.0 118.3 (24.44) 85.0 (20.43) 100.0

– 177.0

5 IABG 2.0 (0.82) 2.0 (2.00) 1.3

– 4.5 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 17.5 (4.33) 13.8 (2.39) 20.0

– 58.5

NCBD 1.5 (0.29) 3.8 (0.48) 1.8

– 5.0 17.3 (2.25) 20.3 (4.71) 19.5

– 27.3 92.0 (25.01) 65.8 (18.86) 86.8

– 107.5

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 11.3 (0.75) 13.5 (2.10) 9.8

– 20.0

PAHM 5.7 (0.67) 4.3 (2.33) 2.5

– 3.7 5.0 (1.00) 3.3 (2.03) 4.0

– 9.8 182.7 (21.84) 152.0 (33.56) 136.7

– 229.8Monsanto Company CR240

- 13U1 348 of 374 Table I

- 9

. ( c ontinued). Individual

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411 Compared to the Conventional Control

Macro

- parasitic hymenoptera Sap Beetle ( Nitidulidae ) Minute Pirate Bug ( Anthocoridae)
Beneficial Arthropod Pest Arthropod Beneficial Arthropod

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control MON 87411 Control

1 IABG

— — — — — — 1.0 (0.71) 0.8 (0.48) 0.8

– 3.8

NCBD

— — — — — — 1.3 (0.63)† 0.3 (0.25) 0.5

– 0.8

NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM

— — — — — — 1.0 (0.58)* 3.0 (0.58) 2.0

– 7.5

2 IABG

— — — — — — 1.5 (0.96) 3.3 (0.25) 3.3

– 9.5

NCBD

— — — 1.3 (0.48)† 1.0 (0.71) 0.0

– 1.0 1.3 (0.95) 1.3 (0.48) 0.0

– 1.8

NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

PAHM 0.7 (0.33) 1.0 (0.58) 0.7

– 2.5 0.7 (0.33)† 1.3 (0.33) 0.3

– 1.8 2.7 (1.20) 4.7 (2.19) 5.0

– 10.0

3 IABG

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 1.3 (0.48) 1.0 (0.41) 0.8

– 3.5

NCBD 0.5 (0.50)† 0.3 (0.25) 0.0

– 0.3 0.8 (0.25)† 0.3 (0.25) 0.3

– 1.0 0.3 (0.25) 0.5 (0.29) 1.0

– 1.8

NEYO 0.0 (0.00)† 0.0 (0.00) 0.3

– 0.8

— — — 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.3

PAHM 0.3 (0.33) 1.7 (0.33) 0.5

– 1.7 0.3 (0.33)† 0.0 (0.00) 0.7

– 0.8 4.0 (1.53) 2.0 (0.00) 2.3

– 10.0

4 IABG

— — — — — — 0.3 (0.25) 1.0 (0.41) 2.3

– 6.3

NCBD

— — — — — — 1.0 (0.58) 0.8 (0.25) 1.5

– 4.0

NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

PAHM

— — — — — — 0.3 (0.33) 0.3 (0.33) 2.3

– 4.0

5 IABG

— — — 0.0 (0.00)† 0.3 (0.25) 0.0

– 1.0 0.5 (0.29) 1.8 (1.44) 1.3

– 3.5

NCBD

— — — 1.5 (0.65) 1.0 (0.71) 0.3

– 1.5 1.3 (0.75)† 0.5 (0.29) 0.5

– 1.5

NEYO 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.3

— — — 1.0 (0.41)† 0.0 (0.00) 0.5

– 0.8

PAHM 0.3 (0.33)† 0.0 (0.00) 0.0

– 0.5

— — — 2.0 (0.58) 1.0 (1.00) 4.0

– 6.8Monsanto Company CR240

- 13U1 349 of 374 Table I

- 9

. ( c ontinued). Individual

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411 Compared to the Conventional Control

Spider (Araneae)
Beneficial Arthropod

Mean (S.E.) 3 Reference Range 4 Coll. 1 Site 2 MON 87411 Control

1 IABG 0.5 (0.29)† 0.3 (0.25) 0.5

– 0.8

NCBD 3.3 (0.48) 1.0 (0.41) 1.5

– 2.3

NEYO 0.5 (0.29)† 0.3 (0.25) 0.0

– 1.3

PAHM 0.7 (0.33)† 1.7 (0.33) 0.5

– 1.3

2 IABG 0.0 (0.00)† 0.3 (0.25) 0.0

– 1.0

NCBD 1.5 (0.29) 2.5 (1.50) 1.5

– 3.3

NEYO 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.5

PAHM 1.3 (0.33)† 0.0 (0.00) 1.0

– 1.5

3 IABG 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.3

NCBD 1.8 (0.75) 1.3 (0.25) 1.3

– 3.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

PAHM 2.3 (1.45) 0.7 (0.33) 1.3

– 2.5

4 IABG 0.8 (0.75)† 0.3 (0.25) 0.0

– 1.0

NCBD 1.0 (0.58) 2.0 (1.00) 1.5

– 4.0

NEYO 0.0 (0.00)† 0.3 (0.25) 0.3

– 0.3

PAHM 2.0 (2.00) 2.0 (0.00) 1.0

– 1.8

5 IABG 0.3 (0.25)† 0.3 (0.25) 0.3

– 0.5

NCBD 1.5 (0.29) 0.8 (0.48) 1.5

– 2.8

NEYO 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.5

PAHM 3.3 (0.88)* 0.3 (0.33) 0.3

– 1.3

Note: The experimental design was a randomized complete block with four replicates per site.

* Indicates a statistically significant difference between MON 87411 and the conventional control (α = 0.05) using ANOVA

.Monsanto Company CR240

- 13U1 350 of 374 † Indicates p

- values could not be generated where the taxa did not meet inclusion criteria (see appendix I .1 0 .2).

1 Arthropods were enumerated at five crop development stages: Collection 1 = late vegetative

– VT growth stage; Collection 2 = R1 growth stage;

C ollection 3 = R2 growth stage; Collection 4 = R3 growth stage; Collection 5 = R4 growth stage.

2 Site code: IABG = Greene, IA; NCBD = Perquimans, NC; NEYO = York, NE; PAHM = Berks, PA.

3 MON 87411 and the conventional control values represent means with standard error in parentheses. N = 4 (IABG, NCBD, and NEYO si tes); N= 3 (PAHM site).

4 Reference range is calculated from the minimum and maximum mean values from among reference hybrids a t each site. A dash (

— ) indicates data not available.Monsanto Company CR240

- 13U1 351 of 374 Table I

- 10. Individual

- Site Analysis: Arthropod Abundance in Visual Counts from MON 87411 Compared to the

Conventional Control

Ant

- like Flower Beetle (Anthicidae) Click Beetle Corn Flea Beetle

Pollen Feeder Pest Arthropod Pest Arthropod

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control MON 87411 Control

1 IABG

— — — — — — — — —

NCBD

— — — 1.0 (0.71) 1.8 (0.85) 0.8

– 1.5 2.5 (1.26) 3.3 (1.97) 1.0

– 5.0

NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM

— — — — — — 6.3 (4.37) 4.7 (1.45) 7.8

– 10.3

2 IABG 0.8 (0.48)* 4.3 (2.29) 1.8

– 3.3

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

NCBD

— — — 1.3 (0.75) 1.3 (0.75) 1.0

– 1.8 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.8

NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM

— — — — — — 8.0 (5.13) 7.3 (0.88) 7.5

– 11.0

3 IABG 0.5 (0.50) 1.0 (0.71) 0.8

– 2.0

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

NCBD

— — — 0.0 (0.00)† 0.8 (0.48) 0.5

– 0.8 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM

— — — — — — 12.0 (3.51) 7.7 (3.84) 7.8

– 9.8

4 IABG 1.3 (0.95) 2.8 (1.49) 1.5

– 3.0

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

NCBD

— — — 0.5 (0.50)† 0.0 (0.00) 0.0

– 0.5 0.3 (0.25)† 0.3 (0.25) 0.0

– 0.8

NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM

— — — — — — 3.7 (1.76) 2.0 (0.58) 4.0

– 6.0

5 IABG 1.8 (1.11) 1.8 (1.44) 0.5

– 1.8

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

NCBD

— — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 1.3 (1.25) 3.3 (1.89) 0.8

– 3.0

NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM

— — — — — — 2.3 (0.67) 7.3 (3.93) 5.5

– 7.3Monsanto Company CR240

- 13U1 352 of 374 Table I

- 10

. ( c ontinued). Individual

- Site Analysis: Arthropod Abundance in Visual Counts from MON 87411 Compared to the

Conventional Control

Japanese Beetle ( Scarabaeidae) Lacewing Adult (Chrysopidae) Lacewing Larvae (Chrysopidae)
Pest Arthropod Beneficial Arthropod Beneficial Arthropod

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control MON 87411 Control

1 IABG

— — — — — — — — —
NCBD 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.5 (0.50)† 0.5 (0.50) 0.3

– 0.5 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.5

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.3 (0.33) 0.0

– 0.5 0.0 (0.00)† 0.0 (0.00) 0.0

– 1.0

2 IABG 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.8 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

NCBD 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.0 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.0 0.8 (0.48)† 0.5 (0.50) 0.0

– 0.5

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.7 0.3 (0.33)† 0.0 (0.00) 0.0

– 0.8

3 IABG 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.3 (0.25)† 1.3 (0.48) 0.0

– 0.3

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.3 0.5 (0.29)† 0.3 (0.25) 0.3

– 0.5

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.3 (0.33) 0.0

– 0.7 0.3 (0.33)† 0.0 (0.00) 0.0

– 0.7

4 IABG 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.5

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.7 (0.67)† 0.0 (0.00) 0.0

– 0.0

5 IABG 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.3

– 0.5 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.3

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.5 0.0 (0.00)† 0.5 (0.50) 0.0

– 0.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.5 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0Monsanto Company CR240

- 13U1 353 of 374 Table I

- 10

. ( c ontinued). Individual

- Site Analysis: Arthropod Abundance in Visual Counts from MON 87411 Compared to the

Conventional Control

Ladybird Beetle Adult (Coccinellidae) Ladybird Beetle Larvae (coccinellidae) Lygus Bug (Miridae)
Beneficial Arthropod Beneficial Arthropod Pest Arthropod

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control MON 87411 Control

1 IABG

— — — — — — — — —

NCBD 0.5 (0.29)† 0.5 (0.29) 0.3

– 1.0 0.5 (0.50) 0.5 (0.50) 0.3

– 1.8

— — —
NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

— — —
PAHM 3.7 (1.45) 7.0 (2.08) 2.5

– 3.3 0.7 (0.67)† 0.0 (0.00) 0.0

– 0.5

— — —
2 IABG 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

— — —
NCBD 0.5 (0.50)† 0.5 (0.50) 0.3

– 1.0 2.8 (0.95) 3.5 (0.50) 1.5

– 2.8

— — —
NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

— — —
PAHM 3.0 (2.52) 5.0 (4.04) 1.5

– 2.5 0.3 (0.33)† 0.7 (0.67) 0.0

– 1.3 0.0 (0.00)† 1.0 (1.00) 0.0

– 0.3

3 IABG 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

— — —
NCBD 1.0 (0.41)† 0.3 (0.25) 0.0

– 1.5 1.3 (0.63)† 0.3 (0.25) 0.8

– 1.3

— — —
NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

— — —
PAHM 1.3 (0.33)† 0.3 (0.33) 0.0

– 0.8 1.3 (0.67)† 0.0 (0.00) 0.0

– 1.7 0.0 (0.00)† 0.7 (0.67) 0.0

– 0.3

4 IABG 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

— — —
NCBD 0.8 (0.48)† 0.0 (0.00) 0.0

– 0.8 0.5 (0.50)† 0.0 (0.00) 0.0

– 0.3

— — —
NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

— — —
PAHM 0.0 (0.00)† 0.3 (0.33) 0.0

– 0.7 0.7 (0.67)† 0.0 (0.00) 0.0

– 0.3 0.7 (0.67)† 1.0 (0.00) 0.0

– 1.3

5 IABG 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

— — —
NCBD 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.5 (0.29)† 0.3 (0.25) 0.3

– 0.5

— — —
NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

— — —
PAHM 0.0 (0.00)† 0.7 (0.33) 0.0

– 0.8 0.7 (0.33)† 0.7 (0.67) 0.0

– 1.0 0.7 (0.33)† 0.3 (0.33) 0.0

– 0.5Monsanto Company CR240

- 13U1 354 of 374 Table I

- 10

. ( c ontinued). Individual

- Site Analysis: Arthropod Abundance in Visual Counts from MON 87411 Compared to the

Conventional Control

Minute Pirate Bug (Anthocoridae) Sap Beetle ( Nitidulidae ) Shining Flower Beetle ( Phalacridae)
Beneficial Arthropod Pest Arthropod Pollen Feeder

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control MON 87411 Control

1 IABG

— — — — — — — — —
NCBD 1.5 (0.65) 0.3 (0.25) 1.0

– 3.5 4.5 (2.63) 5.5 (2.96) 3.8

– 7.5 6.3 (1.49) 4.3 (1.84) 5.8

– 10.5

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

— — —
PAHM 6.3 (2.33) 10.3 (5.49) 8.3

- 13.3 6.0 (2.08) 4.3 (1.45) 5.3

– 12.8 2.0 (0.58) 5.3 (3.18) 1.5

– 5.3

2 IABG 5.0 (0.41) 4.3 (1.11) 1.5

- 4.5 6.3 (1.44) 10.8 (4.77) 4.0

– 7.5

— — —
NCBD 2.0 (0.71) 2.3 (0.63) 0.5

- 3.3 4.8 (4.09) 3.5 (1.55) 2.8

– 7.8 9.0 (2.74) 15.3 (3.09) 7.8

– 17.8

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.3

— — —
PAHM 5.3 (1.45) 6.0 (0.00) 4.0

– 10.0 8.0 (1.53) 5.0 (2.52) 1.3

– 7.3 1.7 (0.88) 4.0 (1.53) 2.0

– 3.5

3 IABG 1.5 (0.65) 0.8 (0.48) 0.5

– 1.8 2.0 (0.91) 2.0 (0.58) 0.3

– 4.3

— — —
NCBD 1.3 (0.48) 1.5 (1.19) 0.5

– 1.0 2.8 (1.80) 1.8 (1.18) 1.3

– 3.8 5.3 (0.85) 3.8 (1.11) 2.0

– 2.5

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 4.8 (0.75) 4.8 (0.85) 1.8

– 3.0

— — —
PAHM 3.3 (1.20) 9.3 (2.73) 4.0

– 9.0 3.3 (1.45) 2.3 (0.88) 1.3

– 5.3 1.3 (0.88) 1.3 (0.67) 0.0

– 2.8

4 IABG 0.8 (0.48) 1.3 (0.75) 1.3

– 1.8 4.0 (1.47) 6.8 (3.61) 2.3

– 6.0

— — —
NCBD 0.3 (0.25)† 0.3 (0.25) 0.0

– 0.8 0.8 (0.75)† 0.3 (0.25) 0.0

– 1.3 1.0 (0.58)† 1.3 (0.63) 0.0

– 1.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 1.5 (0.87) 2.5 (0.96) 0.8

– 2.8

— — —
PAHM 5.0 (1.53) 7.3 (0.67) 1.7

– 6.5 2.7 (2.19) 5.0 (2.31) 0.3

– 2.3 1.3 (0.67)† 0.7 (0.67) 0.0

– 0.7

5 IABG 1.3 (0.48) 1.0 (0.58) 0.8

– 1.8 14.0 (7.39) 8.8 (4.85) 2.5

– 4.0

— — —
NCBD 0.8 (0.75)† 0.8 (0.48) 0.3

– 1.0 3.5 (0.96) 4.8 (1.11) 1.8

– 5.0 0.5 (0.29)† 0.5 (0.29) 0.3

– 0.8

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 1.8 (0.85)† 1.8 (1.11) 0.0

– 0.5

— — —
PAHM 2.0 (1.00) 4.7 (1.45) 1.3

– 3.5 0.3 (0.33) 1.7 (1.20) 1.7

– 2.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3Monsanto Company CR240

- 13U1 355 of 374 Table I

- 10

. ( c ontinued). Individual

- Site Analysis: Arthropod Abundance in Visual Counts from MON 87411 Compared to the

Conventional Control

Spider (Araneae) Stink bug (Pentatomidae)
Beneficial Arthropod Pest Arthropod

Mean (S.E.) 3 Reference

range 4 Mean (S.E.) 3 Reference

range 4 Coll. 1 Site 2 MON 87411 Control MON 87411 Control

1 IABG

— — — — — —

NCBD 7.3 (1.60) 4.0 (1.08) 3.0

- 5.8 1.0 (0.00)† 0.3 (0.25) 0.3

– 1.8

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM 1.0 (0.58) 1.7 (0.88) 0.8

– 3.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 5.7

2 IABG 1.8 (0.85) 1.5 (0.96) 0.5

– 2.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

NCBD 6.8 (2.69) 4.8 (1.25) 3.5

– 5.0 1.0 (0.71)† 1.5 (0.87) 0.3

– 1.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM 1.7 (1.20) 0.3 (0.33) 1.0

– 2.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3

3 IABG 0.3 (0.25)† 0.3 (0.25) 0.0

– 0.5 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.5

NCBD 4.5 (1.32) 5.0 (1.47) 3.3

– 5.8 0.5 (0.29)† 0.3 (0.25) 0.0

– 1.8

NEYO 0.5 (0.29)† 0.8 (0.48) 0.3

– 1.8 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.5

PAHM 1.7 (0.33) 1.0 (0.58) 1.5

– 2.7 0.0 (0.00)† 0.0 (0.00) 0.0

– 2.3

4 IABG 0.3 (0.25)† 0.3 (0.25) 0.0

– 0.8 0.5 (0.29)† 0.5 (0.50) 0.3

– 0.8

NCBD 2.3 (1.44) 3.3 (2.29) 3.0

– 4.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.5

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.3 0.0 (0.00)† 0.3 (0.25) 0.0

– 0.3

PAHM 2.0 (1.15) 0.3 (0.33) 1.3

– 2.3 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

5 IABG 0.3 (0.25)† 1.0 (0.71) 0.3

– 0.8 0.5 (0.29)† 0.5 (0.29) 0.3

– 0.5

NCBD 3.8 (1.18) 2.3 (0.48) 1.5

– 5.3 0.3 (0.25)† 0.0 (0.00) 0.0

– 0.3

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0 0.0 (0.00)† 0.0 (0.00) 0.0

– 0.0

PAHM 1.0 (0.00) 2.7 (1.45) 1.5

- 3.3 0.0 (0.00)† 0.3 (0.33) 0.0

– 0.0

Note: The experimental design was a randomized complete block with four replicates per site.Monsanto Company CR240

- 13U1 356 of 374 * Indicates a statistically significant difference between MON 87411 and the conventional control (α = 0.05) using ANOVA.

† Indicates p

- values could not be generated where the taxa did not meet inclusion criteria (see appendix I.10 .2).

1 Arthropods were enumerated at five crop development stages: Collection 1 = VT

- R1 growth stage; Collection 2 = R1 growth stage; Collection 3

= R 2 growth stage; Collection 4 = R3 growth stage; Collection 5 = R4

- R5 growth stage.

2 Site code: IABG = Greene, IA; NCBD = Perquimans, NC; NEYO = York, NE; PAHM = Berks, PA.

3 MON 87411 and the conventional control values represent means with standard error i n parentheses. N = 4.

4 Reference range is calculated from the minimum and maximum mean values from among reference hybrids at each site. A dash (

— ) indicates data not available.Monsanto Company CR240

- 13U1 357 of 374 References for Appendix I

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.

SAS. 2012. SAS/STAT software version 9.3. SAS Institute, Inc., Cary, North Carolina.Monsanto Company CR240

- 13U1 358 of 374 Appendix J : Materials and Methods for Pollen Morphology and Viability

Assessment

J .1. Plant Production

MON 87411 , the conventional control, and four reference hybrids were grown under similar agronomic conditions in a field trial in Story County, Iowa

. The trial wa s arranged in a randomized complete block design with four replications. Each plot

consisted of six rows approximately 6 m in length.

J .2. Flower Collection and Pollen Sample Preparation

Tassel bags were placed on three non

- systematically selected plants during polle n shed. The following morning, pollen was collected from three plants per plot and transferred to a uniquely labeled tube. Pollen collected from each plant in a plot represented a subsample

. Within approximately 30 minutes of collecti on, Alexander’s stain solution (Alexander 1980)

, in a 1:5 dilution with distilled water

, was added to each tube (at least 2:1 (v/v) stain to pollen) to fix and stain the pollen, rendering the pollen non

- viable. The

tubes were closed and the contents shake n until thoroughly mixed. Subsamples were placed on wet ice within 30 minutes of pollen collection and maintained under those conditions until receipt at the performing laboratory.

J

. 3 . Data Collection

Slides were prepared by aliquoting s uspended polle n /stain solution onto a slide. Pollen

characteristics were assessed under an Olympus **** BX53 light microscope equipped with an Olympus © DP72 digital color camera. The microscope and camera were connected to a computer running Microsoft Windows XP ****

J

. 3 .1. Pollen Viability and inst alled with an Olympus © cellSens (version 1.4.1) software.

When pollen grains were exposed to the staining solution, viable pollen grains stained red to purple due to the presence of living cytoplasmic content. Non

- viable pollen grains stained light blue to green or colorless , and the shape appeared round to collapsed depending on the degree of hydration

. For each pollen sample, the number of viable and

non

- viable pollen grains was counted from a random field of view under the mi croscope. A minimum of 100 pollen grains were counted for each of the three subsamples per plot

. Mean pollen viability for each replicate was calculated from the subsamples as shown in

Table VII I

- 8

.

**** Olympus Corporation.

**** Windows XP is a registered trademark of Microsoft Corp oration.Monsanto Company CR240

- 13U1 359 of 374 J

. 3 .2. Pollen Diameter

For a single predetermined su bsample per plot, p ollen grain diameter was measured along two perpendicular axes for 10 representative pollen grains per replication. Mean pollen diameter for each replicate was calculated from the total of 20 diameter measurements as
shown in Table VIII

- 8.

J

. 3 .3. General Pollen Morphology

General pollen morphology of MON 87411 , the conventional control, and the reference hybrids was observed as shown in Figure J

- 1.

J

. 4 . Statistical Analysis

An analysis of variance was conducted according to a randomized complete block design

SAS ****

Table J

- 1. Starting Seed for Pollen Morphology and Viability Assessment (SAS 2012) . The level of statistical significance was predetermined to be 5% (α=0.05). MON 87411 was compared to the conventional control for percent viable pollen and pollen grain diameter. MON 87411 and the conventional control were not statistically compared to the reference hybrids . Minimum and maximum mean values

were calculated for each characteristic from the four reference hybrids . Ge neral pollen morphology was qualitative; therefore, no statistical analysis was conducted on these

observations.

Material Phenotype Monsanto Lot Number

MPA640B 1 Conventional Control 11333170

MON 87411 Glyphosate

- T olerant and I nsect

- P rotect ed 1133317 6

Gateway 6158 Conventional Reference 11273006

Mycogen 2M746 Conventional Reference 11226705

LG2597 Conventional Reference 11226862

Phillips 713 Conventional Reference 11300072

1 MPA640B = LH244 × LH287

.

**** SAS is a registered trademark of SAS Institute, Inc.Monsanto Company CR240

- 13U1 360 of 374 Figure J

- 1

. General Mo rphology of Pollen from MON 87411

, the Conventional Control, and the Reference Hybrids under 200X Magnification The maize pollen samples were stained with Alexander’s stain diluted 1:5 with distilled water. Viable pollen grains stained red to purple, while non

- viable pollen grains stained blue to green and the shape appeared round to collapsed depending on the degr ee of hydration.Monsanto Company CR240

- 13U1 361 of 374 References for Appendix J

Alexander, M.P. 1980. A versatile stain for pollen fungi, yeast and bacteria. Stain Technology 55:13

- 18.

SAS. 2012. SAS/STAT software version 9.3. SAS Institute, Inc., Cary, North Carolina.Monsanto Company CR240

- 13U1 362 of 374 Appendix K : Summary of Non

- target Organism Studies

This appendix provides a summary of the studies conducted to assess the potential effects

of DvSnf7 RNA on non

- target organisms. All studies were conducted using in vitro

produced DvSnf7 RNA, referred to as DvSnf7_96 8 RNA. With the exception of the earthworm study, all studies utilizing the in vitro

- produced test substance included a diet analysis using a sensitive insect ( Diabrotica undecimpunctata howardi ; Southern corn rootworm) to confirm that the DvSnf7_968 RNA was biologically active and had the expected level of biological activity in diet. Additionally, where appropriate based upon the diet matrix, the homogeneity of the test material and stability over the period of storage was also confirmed.

K. 1. Evaluation of Potential Effects of DvSnf7_968 RNA to the Earthworm Eisenia

andrei in an Acute Exposure Study in an Artificial Soil Substrate.

The purpose of this study was to evaluate the potential effects of dietary and contact exposure of DvSnf7_968 RNA on adults of the earthworm Eisenia andrei (Oligochaeta: Lumbricidae) over a 14 day exposure period. For this stud y, a dilution of DvSnf7_968 RNA was prepared in purified water and incorporated into a standard artificial soil medium following the standard methodology described in OECD guideline 207. The test

substance was evaluated a t a single test concentration and c ompared with a water

- treated

assay control. Treatments were mixed into a n artificial soil in replicated 1 L glass jars (test arenas/chambers). At initiation of the test, ten adult E. andrei were placed on the soil surface of each jar. Each treatment was re plicated four times for a total of 40 earthworms exposed per treatment. All jars were maintained in an incubator under a 24L: 0D photoperiod at 20° C. The survival , biomass and behavior of the worms were assessed at 7 and 14 days after commencement of the bioassay. In lieu of a positive control, the sensitivity of the E. andrei worm culture used for this study was previously confirmed in a separate testing at the Test Facility, in accordance with the guideline, and conducted within 12 months from the start of the study in

- life phase. After 14 days, no effect on survival (100% sur vival in both treatments), was observed with E

. andrei treated with DvSnf7_968 RNA at a concentration equivalent to 5 000 ng RNA/g soil dry weight

. Additionally, the biomass of worms exposed to DvSnf7_968 RNA (

- 8.4 ± 1.4% change) was not significantly redu ced compared to the assay control (

- 9.4 ± 2.4% change) (p > 0.05) and there were no adverse observed effects on worm behavior. B ased on the results of assessments for mortality, behavior and change in biomass, the NOEC for DvSnf7_968 RNA was concluded to be 5000 n g RNA / g soil dry weight.

K. 2. Evaluation of the Potential Dietary Effect s of DvSnf7_968 RNA on Honeybee Larvae ( Apis mellifera L.).

The purpose of this study was to evaluate the potential dietary effect s of DvSnf7_968

RNA on the survival and development of honey bee ( Apis mellifera L.) larvae

. Larvae

of A. mellifera

, 2 to 3 days old, were exposed to DvSnf7_968 RNA in a single doseMonsanto Company CR240

- 13U1 363 of 374 administered to the brood cell. A single dose 1000 ng/g solution , in a 10 **μ** l aliquot of 30% (w /v) sucrose /purified water

, was added to each larval cell at test initiation for a total mass of 11.3 ng DvSnf7/cell 13

. In addition, a n assay control treated with 10 μL of 30% (w/v) sucrose /purified water was included as well as a positive control ( 2000 μg
pot assium arsenate /ml) in a 30% (w/v) sucrose /purified water solution to confirm that the

test honey bee larvae were feeding

. For each treatment there were four replicate frames

with a target number of 20 larvae assessed per frame

. Treatments were administered to
the cell s and frames were returned to the hives for incubation. Treated brood cells were mapped in each treated frame and identified on acetate overlay maps to indicate the study, hive, replicate, and treatment group numbers. Post

- capping and prior to emergence, all treated frames were removed from the hives, placed into screened hive boxes in a growth

chamber and maintained under a 0L: 24D photoperiod at approximately 2 7 to 3 0 oC and

47.3 to 72.6 % relative humidity (RH). The endpoints mea sured were survival at two different life stages: dosing to cell

- capping (larval stage) and cell

- capping to test termination (pupal stage). There was 100% survival in both the DvSnf7_968 RNA and assay control treatments. Additionally, emergence was initia ted in the test and control on the same day for the DvSnf7_968 RNA treatment and the assay control (day 14), approximately 50% emergence occurred on the same day (day 15) for the DvSnf7_968 RNA treatment and the assay control and 100% emergence was achieve d on the same day (day 17) for the DvSnf7_968 RNA treatment and the assay control. Behavioral observations at emergence indicated no adverse behavior or morphological effects.

S urvival for the positive control treatment was 0%, confirming the validity of the test system. Based on no differences in survival and development between the test and control treatments, t he NOEC of the DvSnf7_968 RNA for honeybee larvae was ≥ 11.3 ng/larvae

.

K. 3. Evaluation of the Potential Dietary Effect s of DvSnf7_968 RNA on Honeybee Adults ( Apis mellifera L.).

The purpose of this study was to evaluate the potential dietary effect s of DvSnf7_968

RNA on the survival of honey bee ( Apis mellifera L.) adults in 14

- day continuous feeding study

. H oney bee adults

, < 1 day post

- eme rgence, were exposed to three treatments that included: 1000 ng/g DvSnf7_968 RNA in a 50% (w/v) sucrose/purified water solution, an assay control with 50% (w/v) sucrose/purified water solution and a positive control
(200 **μ** g potassium arsenate /ml) in a 50% (w/v) sucrose/purified water solution to confirm

that the test bees are feeding . For each of the three treatments there were four cages (replicates) containing a target number 2 0 adult bees per replicate. Each cage was provided with approximately 10 ml of appropriate treatment diet solution in a vial inserted on the top of each cage. The vial was replaced every two days with fresh

treatment diets over the study duration of 14 days

. Prior to test initiation, adult bees were

starved for a maximum of two hour s

. Test bees for each treatment group were observed

13 A single dose of 10 **μ** l of 1000 ng/g solutio n was added to each larval cell for a total mass of 11.3 ng

DvSnf7/cell. The concentration of 1000 ng/g DvSnf7_968 RNA in the diet solution is calculated based on the density of the 30% sucrose/water (w/v) solution of 1.1270 g/ml

.Monsanto Company CR240

- 13U1 364 of 374 daily for mortality, abnormal behavior, and appearance. During the test period, adult bees

were maintained under a 0L: 24D photoperiod, with the exception of the time during which mortality and behavior a ssessments were conducted. Environmental conditions

were maintained at approximately 2 6 to 32oC and 41 to 8 0% relative humidity (RH)

. The positive control produced 100% mortality, confirming the validity of the test system. Though the study was designed t o be a 14

- day exposure, the mortality in the assay control exceeded 20% on day 10 and continued to increase until test termination at day 14. Therefore, day 10 survival data from the DvSnf7_968 RNA treatment and assay control were analyzed. A 10

- day expos ure is considered to be appropriate exposure duration for a chronic feeding study for adult honey bees (EFSA, 2013). The re were no significant differences (p > 0.05) in mean survival between the DvSnf7_968 RNA ( 79%)

and the assay control ( 76% ) treatments after 10 days of continuous dietary exposure

. Mean survival on day 14 was not analyzed and survival in the DvSnf7_968 RNA and the

assay control was both 30%. T he NOEC of the DvSnf7_968 RNA for adult honeybees

was ≥ 1000 ng/g

.

351B K. 4. Evaluation of Potential Dietary Effects of DvSnf7_968 RNA on the Lady Beetle, Coleomegilla maculata (DeGeer) (Coleoptera: Coccinellidae).

The purpose of this study was to evaluate potential dietary effects of the DvSnf7_968

RNA on the survival, development and growth of the la dy beetle, Coleomegilla

maculata

. The test substance dissolved in water was incorporated into an agar based

artificial C. maculata diet at a single concentration of 1000 ng DvSnf7_968 RNA/g of

diet. In addition, appropriate assay control (purified water) and a positive control (1 20 μg potassium arsenate/g diet) were included in the study. Each treatment was replicated three times and with a target of 20 insects per diet treatment . Larvae were exposed for a

maximum of 18 days. C. maculata larvae originating from the same egg batch were used for each replicate. All diet treatments were maintained in an incubator with a temperature

of 2 7 **°** C , a relative humidity (RH ) of 70 % and a 14 L : 10 D photoperiod

. There were no significant differ ences (p > 0.05) for survival between the DvSnf7_968 RNA (92%) and the assay control (90%) treatments. Additionally, there were no significant differences (p > 0.05) for the mean percent that developed to adults between the DvSnf7_968 RNA (92%) and the as say control (90%) treatments. There was no significant difference for development days to adult emergence between the larvae fed the test or control diet (p > 0.05). For the test and the assay control diet treatment groups the development time to

adult em ergence was 15 days. There was no significant difference with adult biomass

between the test and control diet (p > 0.05). Adults that developed from larvae fed test or control diet averaged 10.2 mg for both treatments. Because of significant differences in body mass between males and females in C. maculata populations, separate analyses were performed by gender. There was no significant difference between t he biomass of females averag ing 10.7 mg and 10.9 mg (p > 0.05) a nd males averag ing 9.7 mg and 9.5 mg (p > 0.05) for test and control treatments respectively. The positive control group had

17% surviva l and n one of the insects in the positive control group developed to the pupa stage

, confirming the validity of the test system. T he NOEC of the DvSnf7_968 RNA

for C. m aculata was concluded to be ≥ 1000 n g/g of diet.Monsanto Company CR240

- 13U1 365 of 374 K. 5. Evaluation of Potential Dietary Effects of DvSnf7_968 RNA on the Carabid ground beetle, Poecilus chalcites (Say) (Coleoptera: Carabidae)

The purpose o f this study was to evaluate potential dietary toxicity of the DvSnf7_968 RNA against larvae of the carabid beetle, Poecilus chalcites

. The test substance dissolved

in water was incorporated into an agar

- based artificial diet at a single concentration of 1000 ng DvSnf7_968 RNA/g of diet. In addition, appropriate an assay controls (purified water) and a positive control (220 μg potassium arsenate/g diet) were included in the study. Each treatment was replicated three times and each replicate was initiated with a target of 20 insects per diet treatment. L arvae were exposed for a maximum of 35 days. All dietary exposures were initiated with first instar larvae and larvae were individually housed throughout the study. All diet treatments were maintained in an incubator with a temperature of 2 7 **°** C , a RH of 70 % and a 14 L : 10 D photoperiod

. The endpoints measured were survival, development to the adult stage, and development time (days) to adult emergence, and adult biomass. The re was no significant difference for survival

between the DvSnf7_968 RNA ( 93%) and the assay control (92% ) treatments and a ll of the sublethal endpoints (p > 0.05). For the test or control diets, the percentage that developed to the adult stage averaged 70% a nd 75%, respectively, adult biomass averaged 31.9 mg and 32.3 mg, respectively, and the development time to adult emergence averaged 33 days for both treatments. The positive control group had 65%,

surviva l and n one of the insects in the positive control g roup developed to the pupal or adult stage

, confirming the validity of the test system. T he NOEC of the DvSnf7_968

RNA for P. chalcites was ≥ 1000 n g/g of diet.

35B K. 6. Evaluation of Potential Dietary Effects of DvSnf7_968 RNA on the Insidious Flower Bug, O rius insidiosus (Say) (Heteroptera: Anthocoridae)

The purpose of this study was to evaluate potential dietary effect s of DvS nf7_968 RNA on the survival and development of O. insidiosus nymphs over 10 days of continuous exposure following the methodology de scribed in Tan et al., (2011)

. Three treatment diet s were prepared for separate feeding exposures to O. insidiosus nymphs, including an assay control diet, a DvSnf7_968 RNA treatment diet at 1000 ng /g diet, and a positive control
diet (100 **μ** g potassium ars enate/g diet). All dietary exposures were initiated with 5

- day old nymphs and each treatment consisted of a total of 40 nymphs. All test nymphs were individually housed in test arenas and supplied with appropriate treatment diet in two

domes. The dome diet s were replaced every two days. All test nymphs were allowed to

feed ad libitum on the treated diet for 10 days. All diet treatments were maintained in an incubator with a temperature of 25 ± 5 **°** C , a RH of 70 ± 10 % and a 16 L : 8 D photoperiod

. The test nymphs were observed every day to record mortality and development. Survival of the nymphs was 93% in both the assay control and DvSnf7_968 RNA treatment diets after 10 days of continuous feeding. Nymph development to the adult stage was not significantly different (p > 0.05) at 95% and 98% in the assay control and DvSnf7_968 RNA treatment, respectively. The average development time of the test nymphs was not significantly different (p > 0.05) between the control and test groups, with mean development times of 11.11 ± 0.15 days in the assay control treatment and

10.87 ± 0.13 days in DvSnf7_968 RNA treatment. In contrast, the O. insidiosus nymphs

fed the positive control treatment diet had 100% mortality by day 10, indicating that theMonsanto Company CR240

- 13U1 366 of 374 test system was effecti ve in detecting toxic effects through the dietary exposure. T he

NOEC of the DvSnf7 RNA for the insidious flower bug

, O. insidiosus

, wa s **≥** 1000 n g/ g diet.

K. 7. Evaluation of Potential Dietary Effects of DvSnf7_968 RNA on the Parasitic Wasp, Pediobius foveolatus (Hymenoptera: Eulophidae)

The purpose of this study was to evaluate potential dietary effects of DvSnf7_968 RNA on the survival of adult parasitic wasp, Pediobius foveolatus Crawford over 20 days of continuous exposure. Three treatment diets were prepared for separate feeding exposures

to P. foveolatus adults. An assay control diet was 30% honey/water (v/v) solution, the DvSnf7_968 RNA treatment diet prepared at 1000 ng/g in a 30% honey/water (v/v) solution and a positive control ( 200 μ g potassium arsenate /g ) in a 30% honey/water (v/v) solution. Exposure of P. foveolatus adults to the three treatment diets was replicated four times with 10 adult wasps per replicate for a total of 40 wasps per treatment. All dietary

exposur es were initiated with newly emerged adults after approximately 24 hours of acclimation. The adult wasps in each replicate were housed together in a single arena and

allowed to feed ad libitum on the treated diet for 20 days. All diet treatments were

maint ained in an incubator at a target temperature of 25 ° C, a target RH of 70 % and a

16 L : 8 D photoperiod

. Mortality was observed every two days at diet replacement and the dead wasps, if any, were removed from the test arenas at the time of observation. There was no mortality of P. foveolatus adults fed the DvSnf7_968 RNA treatment diet or the assay control diet after 20 days. In contrast, the P. foveolatus adults fed the positive

control treatment diet showed 100% mortality at day 12, indicating that the test system was effective in detecting toxic effects through the dietary exposure. The NOEC of the

DvSnf7 RNA for the parasitic wasp, P. foveolatus

, was ≥ 1000 ng/g diet.Monsanto Company CR240

- 13U1 367 of 374 References for Appendix K

EFSA. 2013. EFSA Guidance Document on the risk assessme nt of plant protection products on bees ( Apis mellifera

, Bombus spp. and solitary bees). EFSA Journal 11:266

.

Tan, J., M.S. Paradise, S.L. Levin, P.M. Bachman, J.P. Bachman, J.P. Uffman, C. Jiang, D.B. Carson. 2011. Development and survival of Orius insidiosus (Say) nymphs on encapsulated bee pollen

- based diet in a Tier

- I toxicity assay. Environmental Entomology

40:1613

- 1621.Monsanto Company CR240

- 13U1 368 of 374 Appendix L: Next

- Generation Sequencing and Junction Sequence Analysis for

the C haracterization of DNA I nserted into C ro p P lants

Background

Safety assessments of biotechnology

- derived crops include a detailed molecular characterization of the inserted DNA sequence and its location within the genome (Codex Alimentarius 2009) . Typically, molecular characterization has relie d on Southern blot analysis to establish locus and copy number along with targeted sequencing of polymerase chain reaction products spanning any inserted DNA to complete the characterization process. With the advent of next

- generation sequencing (Shendure and Ji 2008; Zhang et al. 2011)

, improvements in sequencing technologies have enabled alternative methods for molecular characterizations which do not require Southern blot analysis. Next

- Generation Sequencing and Junction Sequence Analysis bioinformatic s (NGS/JSA) utilizes sequencing (both next

- generation technologies and traditional methods) and bioinformatics to produce characterizations equivalent to those achieved by

current Southern blot based methods.

Th ere are multiple advantages to using next

- g eneration sequencing and bioinformatics, most notably the robustness, simplicity and consistency of the method compared with

Southern blot studies, which require customized experimental design for every

transformation event. The new sequencing

- based metho d overcomes many technical challenges inherent in Southern blot analyses ( e.g. , false positive hybridization bands resulting from incomplete digestion or star activity (Wei et al. 2008) ) and the need for

radioactive 32 P

- labeled probes. Th is new method provides higher reproducibility, because it is less dependent on complex lab based procedures. T he method described here is essentially ide ntical for all transformation events and it robustly establishes

molecular characteristics of genetically engineered crops (Kovalic et al. 2012)

. Additionally, similar techniques are being used to characterize transgene integration sites and insert molecular anatomy in mammalian systems (DuBose et al. 2013; Zhang et al. 2012)

.

Method Synopsis

Molecular characterizat ion of the inserted DNA and associated native flanking sequences consists of a multistep approach to determine:

1. the number of insertion sites;

2. the presence/absence plasmid backbone;

3. insert copy number at each insertion site;

4. DNA sequence of each inserted DNA;

5. sequence of the native locus at each insertion site.

Additionally, current methods also establish a description of any genetic rearrangements that may have occurred at the insertion site as a consequence of transformation. Generational stability ana lysis, which demonstrates the stable heritability of inserted DNA sequences over a number of breeding generations, is also routinely conducted.Monsanto Company CR240

- 13U1 369 of 374 The first step of the molecular characterization, determination of number of insert sites

, is conducted using a combination of next

- generation sequencing technologies (NGS) and Junction Sequence Analysis (JSA) bioinformatics (DuBose et al. 2013; Kovalic et al. 2012)

. A schematic representation of the basis of the characterization

, including the NGS/JSA methodology and the directed sequencing

, is presented in Figure 1 (Kovalic et al. 2012)

.

Genomic DNA from the transformation event and the conventional control are used to generate short (~100 bp) randomly distributed sequence fragments (sequencing reads) in sufficient numbers to ensure comprehensive coverage of the genomes (Shendure and Ji 2008) (Figure 1 box 1)

. Sufficient numbers of sequence fragments are obtain ed ( ≥ 75x effective genome coverage) to comprehensively cover the genomes of the sequenced samples (Ajay et al. 2011; Clarke and Carbon 1976; Wang et al. 2008) . Previous studies with a variety of transformation events demonstrate that 75x coverage of the genome is adequate to provide comprehensive coverage and ensure detection of inserted DNA, producing results equivalent to Southern blot analysis (Kovalic et al. 2012) . The 75x coverage used in this method is predicted

, based on established and accepted m ethods (Clarke and Carbon 1976; Lander and Waterman 1988)

, to provide genome coverage that would be expected to not miss a single basepair in complex genome s (Kovalic et al. 2012) . Furthermore, even with know biases in next

- generation sequencing technique s, including the Illumina sequencing by synthesis method employed here (Minoche et al. 2011) , it has previously been established experimentally that given deep next

- generation sequencing

, it is possible to achieve comprehensive coverage of complex genomes that form the foundation for accurate whole genome studies (Ajay et al. 2011; Wang et al. 2008)

.

To confirm sufficient sequence coverage in both the transformation event and the control,

the 100 bp sequence reads are analyzed to determine the coverage of a kn own single

copy endogenous gene, this analysis demonstrates coverage at ≥ 75x median depth in each sample. Furthermore, i n order to confirm the method’s ability to detect any sequences derived from the transformation plasmid, plasmid DNA is spiked into conventional control DNA at a single copy genome equivalent ratio and 1/ 10 copy genome equivalent

ratio. This analysis demonstrates that any portion of the plasmid may be detected at a single copy per genome level and 1/10 copy genome equivalent level, wh ich is adequate sensitivity to observe any inserted fragment

.

Also of note is that although the method presented here provides ≥ 75x coverage of the genomes under study, accurate assembly of complete genome sequences for the

transformation event and conventional control is not technically possible using currently available sequence assembly tools. This is due to the nature of the sequences generated in this study, short reads of a single short insert length (Miller et al. 2010)

, in addition to

li mitation on available sequence assembly algorithms (Zhang et al. 2011) . The sequences generated with this method represent datasets sufficient for achieving precise molecular

characterization of transformed DNA in transformation events where reference to a template sequence (plasmid DNA) is utilized for comparison (Kovalic et al. 2012)

.Monsanto Company CR240

- 13U1 370 of 374 U sing bioinformatics tools, the sequence reads that are derived from the plasmid vector

are selected for further analysis o ut of th e comprehensive genomic sequence datase t produced from the transformation event. To determine the insert number, the known sequence of the transformation vector plasmid is used as a n “e

- probe” in the bioinformatics analysis to search for and select the sequences that contain any portion of

seq uence of the plasmid. The DNA sequencing reads with a mat ch to the query sequence having an e

- value of 1 × 10 −5 or less and having a match length of at least 30 bases with

a t least 96.7% sequence identity are collected. The results of a par ameter optimiz ation study that systematically evaluated many diff erent potential parameter sets established

these selecti on criteria as providing th e best possible combination of sensitivity and specifi city

.

Figure L

- 1. Sequencing and Sequence Selection

Genomic D NA from the test and control material were sequenced using I llumina HiSeq/TruSeq technology (Illumina, Inc.) that produces large numbers of short sequence reads approximately 100 bp in length. Sufficient numbers of these sequence fragments were obtained to comprehensively cover the genomes of each sample at ≥ 75x average

covera ge. Using these genome sequence reads, bioinformatics search tools were used to select all sequence reads tha t are signifi cantly similar ( as defined in the text) to th e transformation plasmid. Only the selected sequence reads were used in further bioinformatics analysis to determine the insert number by detecting and characterizing all junction sequences and the presence or absence of the plasmid backbone sequences byMonsanto Company CR240

- 13U1 371 of 374 l ack of detectable sequences, including the use of suitable controls for experimental comprehensiveness and sensitivity.

The number of DNA inserts is determined by analyzing the selected sequences for novel junctions. The junctions of the DNA insert and fl anking DNA are unique for each

insertion and an example is shown in Figure 2 below (Kovalic et al. 2012) . Therefore, insertion sites can be recognized by analyzing for sequence reads containing such junctions. Each insertion will produce two unique junct ion sequence classes characteristic of the genomic locus, with one at the 5' end of the insert, in this case named Junction Sequence Class A (JSC

- A), and similarly one at the 3' end of the insert, JSC

- B ( as illustrated in Figure 3 from (Kovalic et al. 2012 ) )

. By evaluating the number and the

sequence s of all unique junction classes detected, the number of insertion sites of the plasmid sequence can be det ermined. For a single insert

, two junction sequence classes are expected, one each originating from ei ther end of the insert, both containing portions

of T

- DNA and flanking sequence.

Figure L

- 2

. Junctions and Junction Sequences Depicted above are five example junction sequences formatted and labeled to indicate the plasmid/flanking DNA portions of the sequences and with the junction point indicated (plasmid DNA is shown in bold, underlined text and flank DNA is shown in plain text). Junctions are detected by examining the NGS data for sequences having portions of plasmid sequences that span less than the full read. Detected junctions are typically characteristic of plasmid insertions in the genome. A group of junction sequences which share the same junction point and common flanking sequence (as shown above) is called a Junction Sequence Class (or JS C).

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACG

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACGTGT

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACGTGTCG

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACGTGTCGGG

**ATCCATGTAGATTTCCCGGACATGAAGCCA** AGAACTAGGAAACGACGACGTGTCGGGAT

Flanking DNA Plasmid DNA JunctionMonsanto Company CR240

- 13U1 372 of 374 Figure L

- 3 . Two Unique Junction Sequence Classes are Produced by the Insertion of a Single Plasmid Region A schematic representation of a single DNA insertion within the genome showing the inserted DNA, the 5' and 3' flanks (depicted as areas bound ed by dotted lines), and the two distinct regions spanning the junctions between inserted DNA and flanking DNA (shaded boxes). The group of

~100

- mer sequences in which each read contains sequences from both the DNA insert and the adjacent flanking DNA at a given junction is called a Junction Sequence Class. In this example, two distinct junction sequence classes (in this case: Class A at the 5’ end and Class B at the 3’ end) are represented.

The next step in the molecular characterization is determination of the insert copy number, integrity of the insert

, lack of backbone or other unintended plasmid sequences, and flanking sequence of the native locus at the insertion site. This analysis is conducted using directed sequencing, l ocus

- specific PCR and DNA sequencing analyses

, which

comp lements the NGS/JSA analyses

, and is common to both the Southern

- based and the

NGS/JSA characterization method s . Directed sequencing (l ocus

- specific PCR and DNA sequencing analyses ) of the transformation event determines the complet e sequence of

the insert and flanks. This determines if the sequence of the insert is identical to the corresponding sequence in plasmid vector, if each genetic element in the insert is intact

, if the plasmid vector sequence is inserted as a singl e copy, and establishes no vector backbone or other unintended plasmid sequences were inserted in the event

. This comparison allows a determination of whether the T

- DNA elements are present in the

intended order. Furthermore, the genomic organization at the insertion site is assessed by comparing the insert and flanking sequence to the sequence of the insertion site in

conventional control genome.

Finally, t he stability of the T

- DNA across multiple generations is evaluated by NGS/JSA analyses. Genomic DNA from multiple generations of the transformation event is assayed for the number and sequences of all unique junction classes, as described above.

T his information is used to determine the number and identity of insertion sites. For a single insert

, two junction sequence classes are expected, both containing portions of

T

- DNA and flanking sequence (Figure 2), with one each originating from either end of

the insert (Figure 3)

. In the case of an event where a single locus is stably inherited over multiple generatio ns, two identical junction sequence classes are expected in all the generations tested. 

## DNA insert 5’ Flank 3’ Flank Insert Junction Regions

Junction Sequences: Class A

Junction Sequences: Class BMonsanto Company CR240

- 13U1 373 of 374 References for Appendix L

Ajay, S.S., S.C.J. Parker, H. Ozel Abaan, K.V. Fuentes Fajardo and E.H. Margulies.

2011. Accurate and comprehensive sequencing of personal genomes. Genome Research

21:1498

- 1505.

Clarke, L. and J. Carbon. 1976. A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome. Cell 9:91

- 99.

Codex Alimentarius. 2009. F oods derived from modern biotechnology. Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations, Rome, Italy.

DuBose, A.J., S.T. Lichtenstein, N. Narisu, L.L. Bonnycastle, A.J. Swift, P .S. Chines and F.S. Collins. 2013. Use of microarray hybrid capture and next

- generation sequencing to identify the anatomy of a transgene. Nucleic Acids Research 41:e70

- e70.

Kovalic, D., C. Garnaat, L. Guo, Y. Yan, J. Groat, A. Silvanovich, L. Ralston, M. Huang, Q. Tian, A. Christian, N. Cheikh, J. Hjelle, S. Padgette and G. Bannon. 2012. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology. T he Plant

Genome Journal 5:149

- 163.

Lander, E.S. and M.S. Waterman. 1988. Genomic mapping by fingerprinting random clones: A mathematical analysis. Genomics 2:231

- 239.

Miller, J.R., S. Koren and G. Sutton. 2010. Assembly algorithms for next

- generation

seque ncing data. Genomics 95:315

- 327.

Minoche, A.E., J.C. Dohm and H. Himmelbauer. 2011. Evaluation of genomic high

throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems. Genome Biology 12:R112.

Shendure, J. and H. Ji. 2008. Next

- ge neration DNA sequencing. Nature Biotechnology

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