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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 part 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. Bachman, Ph.D., B.A. Comstock, 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 of 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 

Monsanto'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 

authority 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 

document 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 regulation. 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 materials, 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.
CERTIFICATION 

The undersigned certifies that, to the best knowledge and behef of the undersigned, this 

petition includes all information and views on which to base a determination, and that it 

includes all relevant data and information known to the petitioner that are unfavorable to 

the petition. 

John M . Cordts, M.S., M.B.A. 

Regulatory Affairs Manager 

Monsanto Company 

800 North Lindbergh Blvd., Mail Stop C3SD 

St. Louis, MO 63167 

Tel: (314)-694-4831 

Fax: (314)-694-3080 

Monsanto Company CR240-13U1 3 of374 
Monsanto 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 

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 

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 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 

have previously been granted nonregulated status under 7 CFR part 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 designed to match the sequence of western corn 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 

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

machinery resulting in down-regulation of the targeted DvSnf7 gene leading to CRW 

mortality. 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 MOA that offers enhanced control of 

target insect pests and prolonged durability of existing Bt technologies designed to 

control 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 water conservation. MON 87411 is also glyphosate tolerant and will continue to 

agricultural herbicides. 

 

® Roundup and Roundup Ready are registered trademarks of Monsanto Technology LLC
Monsanto 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 biotechnologyderived traits to provide protection against both above-ground and below-ground maize 

pests as well as tolerance to multiple herbicides. These next generation combined-trait 

maize products will offer broader grower choice, improved production efficiency, 

increased pest control durability, and enhanced grower profit potentials.

The data and information presented in this petition demonstrate that MON 87411 is 

agronomically, phenotypically, and compositionally 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 Information 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 present

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

millions of acres in the 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 and 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 grain 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 conclusion 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 almost 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 report 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 characteristics 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 

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

differences in several factors such as flowering time, geographical separation, and 

development timings make natural crosses in the U.S. highly unlikely. 

Based on seed availability and the appropriate fit for various studies, conventional control 

materials were developed for use as comparators in safety assessment studies. The 

conventional control materials included the original transformation line (LH244) and two 

hybrid conventional control lines (hybrids MPA640B and/or NL6169), both of which 

have similar genetic backgrounds 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) were 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 assessments. 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 Comparators for

MON 87411

MON 87411 was developed through Agrobacterium-mediated transformation of maize 

immature embryos from line LH244 utilizing plasmid vector PV-ZMIR10871. 

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 87411
Monsanto 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 TubA 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 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 DNA from

PV-ZMIR10871 DNA was integrated at a single locus 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' insertto-flank junctions. 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 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 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 intended 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 

demonstrates that a single copy of the intended T-DNA was stably integrated at a single 
Monsanto 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 proteins expressed in MON 87411. The expression levels of the 

CP4 EPSPS and Cry3Bb1 proteins in selected tissues of MON 87411 were determined

and exposure to humans and animals through diet was evaluated. In addition, the donor 

organisms for the CP4 EPSPS and Cry3Bb1 protein coding sequences, 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 lack structural similarity to known allergens or protein toxins. As has been 

previously shown in safety assessments of other Roundup Ready and Cry3Bb1-

containing crops, the 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 western 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. The 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 expression studies demonstrated that MON 87411 DvSnf7 RNA was expressed 

at mean levels ranging from 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 suppression 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 

RNAi 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 Safety
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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. 

corn belt and Argentina maize-growing regions occurs at relatively similar latitudes with 

an approximate 6 month offset. The average 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 Argentina 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 consensus 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 the 78 components assayed, 18 had more than 50% of 

observations that were below the assay limit of quantitation and were therefore excluded 

from statistical analysis. 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 within the 

conventional control and reference hybrid values. Additionally, MON 87411 mean 

component values were within the tolerance intervals of the reference hybrids, the values 

for maize observed in the literature, and/or the International Life Sciences Institute Crop 

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 confirmed the 

compositional equivalence of grain and forage from MON 87411 to conventional 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 biotechnologyderived 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 Potential or Environmental 

Interactions
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biotechnology-derived plant and the conventional control. Thus, the phenotypic, 

agronomic, and environmental interaction assessment of MON 87411 included a

genetically similar 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 directionality. 

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. Characteristics assessed included: 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 with 

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 

conventional 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 likely 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 well 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 statistically 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 damage, 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 arthropodrelated 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 that the introduced traits in 
Monsanto 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 traps, no statistically 

significant differences were 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 of 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 abundance 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 associated 

with the traits 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 damage 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 introduced 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 comparison 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 weediness characteristics, increased 

susceptibility or tolerance to specific abiotic stressors, 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 to 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 Organisms Including Those 

Beneficial to Agriculture 
Monsanto 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 proteins have been 

granted non-regulated status by USDA-APHIS. After both extensive testing and 

widescale commercial cultivation, in no instance have adverse impacts to NTOs been 

associated with exposure to Cry3Bb1 or CP4 EPSPS proteins from these biotechnologyderived crops. 

As noted previously, the suppression cassette in MON 87411 contains the CaMV e35S

promoter, maize hsp70 intron, the two DvSnf7p 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 dsRNA. 

When consumed by CRW, this 240 bp DvSnf7 dsRNA activates the RNAi process 

leading to suppression of the targeted CRW Snf7 gene. 

To address potential impacts to NTOs, specific 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 

four Lepidopteran species. No impacts to survival, growth, or development of these 

species were noted when fed extremely high doses (relative to levels present in 

MON 87411) of DvSnf7_240 dsRNA over multi-day bioassay periods. 

Additional NTO assessments were conducted on a battery of organisms based on 

recommendations published 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 greater 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 could 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 restricted activity of the DvSnf7 RNA, that 

cultivation of MON 87411 will have no effect 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 highly 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 MOAs against the target corn 

rootworm pests of maize in the U.S. 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 that have been widely grown in the 

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

current agronomic or management practices for maize. As phenotypic evaluations, 

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

between MON 87411 and reference hybrids (other than protection from CRW larval 

feeding), no changes are anticipated in crop rotations, tillage practices, planting practices, 

fertility management, 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 

protection against pests (other than CRW) and diseases comparable to and typical of 

conventional commercial maize hybrids. Based 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 biotechnologyderived maize be granted nonregulated status under 7 CFR part 340.

Conclusion
Monsanto Company CR240-13U1 14 of 374

TABLE OF CONTENTS

RELEASE 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 and 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 Safety Assessment .......................................39

III. DESCRIPTION OF THE GENETIC MODIFICATION ....................................41

III.A. The Plasmid Vector PV-ZMIR10871.................................................................41

III.B. Description of the Transformation System.........................................................41
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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. DvSnf7p 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 87411.................................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. Expression 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...................................................................................................................91
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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 Labile 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...........100

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 Insects......................................................................................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 in Maize Grain ...................................................121

VII.A.3. Secondary Metabolites Levels in Maize Grain ....................................121

VII.A.4. Nutrient Levels in Maize Forage..........................................................122
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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. Characteristics of the Genetic Insert and Expressed Products ..........................176

X.A.1. Genetic Insert ..........................................................................................176

X.B. Mode-of-Action .................................................................................................177

X.C. Expression and Characterization of Gene Products...........................................177

X.C.1. Protein Safety and Expression Levels.....................................................177
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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 with 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.........................................................................294
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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-Generation Sequencing and Junction Sequence Analysis 

for the Characterization of DNA Inserted into Crop Plants......................................368
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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 ...............102

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 margins of exposure (MOEs) for DvSnf7 RNA..................................111

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

MON 87411, Conventional Control, and Reference Hybrids....................................123

Table VII-2. Summary of Maize Grain Fat and Fatty Acids for MON 87411, 

Conventional Control, and Reference 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....................................135
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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

Table 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 87411 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 Notifications....................................................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 Cry3Bb1Proteins.......................................................................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 MALDI-TOF MS....................................269

Table C-8. Comparison of Immunoreactive Signal Between MON 87411-

and E coli-produced CP4 EPSPS Protein...................................................................273
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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 Methods1 for Tissue Samples...................281

Table D-2. CP4 EPSPS Protein Extraction Methods1 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 Compared 

to the Conventional Control..........................................................................................336

Table I-5. Qualitative Assessment: Abiotic Stressor Evaluations Using a 

Categorical Scale for MON 87411 and the Conventional Control............................341

Table I-6. Qualitative Assessment: Disease Damage Evaluations Using a 

Categorical Scale 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 Corn 

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 Seed for Pollen Morphology and Viability Assessment.............359
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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 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). ...............34

Figure III-1. Circular Map of PV- ZMIR10871...........................................................43

Figure III-2. Schematic of the 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 Amplification of the MON 87411 Insertion Site ...........................72

Figure IV-9. Junction sequences detected by JSA. Junction Sequence Class 

A Alignment (All Generations Tested)...........................................................................75

Figure IV-10. Junction sequences detected by JSA. Junction Sequence Class 

B Alignment (All Generations Tested)...........................................................................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 MON 87411- and E. coli -produced 

Cry3Bb1 Proteins...........................................................................................................257
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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 MON 87411 DvSnf7 cDNA and 

DvSnf7_968 cDNA .........................................................................................................290

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

dsRNA in DvSnf7_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 ...........................................................................372
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ABBREVIATIONS AND DEFINITIONS1

~ approximately

ADF acid detergent fiber

ANOVA analysis 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-enolpyruvylshikimate-3-phosphate synthase protein from 

Agrobacterium tumefaciens strain CP4

CRW corn rootworm

CTAB hexadecyltrimethylammonium bromide

CV coefficient of variation

DAP days after planting

dATP deoxyadenosine triphosphate

DDI daily dietary intake

DEEM-FCID 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 designed to match the western corn rootworm (WCR; 

Diabrotica virgifera virgifera) DvSnf7 gene

DvSnf7_240 the active insecticidal RNA in MON 87411

DvSnf7_968 an in vitro transcribed DvSnf7 single stranded RNA

DvSnf7p

partial coding sequence of the Snf7 gene from Diabrotica virgifera

virgifera encoding the Snf7 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.
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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 acid

FDA U.S. Food and Drug Administration

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FMOC fluorenylmethyl chloroformate

FSE 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 kilograms per hectoliter

LOD limit of detection

LOQ limit of quantitation

MEEC maximum expected environmental concentration

MESA

4-Morpholinepranesulfonic acid - ethylenediaminetetraacetic 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

NHANES National Health and Nutrition Examination Survey

NOAEL no observable adverse effect level

NOEC no observable effect concentration

nt nucleotide

NTO non-target organism
Monsanto Company CR240-13U1 27 of 374

OECD Organisation 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 Plant Protection Act

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 ribonuclease

RT room temperature

SAP Scientific Advisory Panel

SBV soybean variant

SCR southern corn rootworm

SD standard deviation

SDS sodium dodecyl sulfate

S.E. standard error

SGF simulated gastric fluid

SIF simulated intestinal fluid

siRNA small interfering RNA

sp. species

TDF total dietary fiber

T-DNA transfer DNA

TFA trifluoroacetic acid

TSSP tissue-specific site pool 

TTC threshold of toxicological concern

Tz tetrazolium

USDA United States Department of Agriculture

UTR untranslated region

UV ultraviolet

v/v volume to volume

WCR western corn rootworm
Monsanto 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 

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 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 

have previously been granted nonregulated status under 7 CFR Part 340.

I.B. Rationale 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 acres and grain harvested from 

87.4 million acres (USDA-NASS 2013a). Average yields in the previous five 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, 

with a total production of about 10.8 billion bushels (USDA-NASS 2013a), valued at 

approximately $77 billion (USDA-NASS 2013c). 

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

containing insecticidal 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. Estimates 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

The introduction of insect-protected and herbicide tolerant (HT) biotechnology-derived 

maize hybrids 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% of 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 losses in corn. 

Insect-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 of 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 complex2

Chandler, et al. 2008

(Diabrotica spp., 

Coleoptera: Chrysomelidae) ( ). The corn rootworm complex 

includes Diabrotica species that are significant pests of maize including western CRW 

(D. virgifera virgifera), northern CRW (D. barberi), and southern CRW (D. 

undecimpunctata howardi). These insect larvae damage maize by feeding on the roots, 

reducing the ability of the plant to absorb water and nutrients from the soil, and causing 

harvesting difficulties because of plant lodging (Riedell 1990; Spike and Tollefson 1991). 

CRW 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-protection traits has increased from its 

first plantings in 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 insecticides 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 virgifera), northern CRW (D. barberi), and southern CRW (D. 

undecimpunctata howardi).
Monsanto Company CR240-13U1 30 of 374

I.B.2. Introduction of Insect-Protection Traits 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 AgroSciences (Herculex®/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 Cry3Bb1 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 while Monsanto’s 

Genuity VT Triple PRO (MON 88017 × MON 89034) provides two Bt proteins with two 

MOAs (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 effective 

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 

products ( ; Roush 1998). All of these noted CRW-protected products

have provided highly effective control of CRW across wide growing 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 offers 

increased control of target insect pests and will prolong the durability of existing CRWcontrolling 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 Triple 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 

designed to match the sequence in western corn 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 dsRNA 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 mortality (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 will provide benefits to growers similar to those obtained by use of existing 

CRW-protected maize hybrids, which includes 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-alone product, but will be 

combined, through traditional breeding methods, with other deregulated biotechnologyderived traits to provide protection against both above-ground and below-ground maize 

pests as well as tolerance to multiple herbicides. These next 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 RNAi in Plants

Naturally occurring RNA-mediated gene suppression (RNAi) in plants has been 

previously documented and includes 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 result from production 

of a dsRNA and concomitant suppression of glutelin genes (Kusaba, et al. 2003). RNAmediated 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 

including 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). 

Safety assessments have been conducted (Parrott et al. 2010; Petrick, et al. 2013) and 

global regulatory 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 greatly 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. Modes-of-Action of the Inserted Genetic Components

I.D.1. Mode-of-Action of the RNAi Component of MON 87411

MON 87411 contains a DvSnf7 suppression cassette that expresses an inverted repeat 

sequence designed to match the sequence in WCR and thereby utilizes the RNAi pathway 

to control CRW (Diabrotica spp.). 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 dsRNA is recognized 

by the pest’s RNAi machinery, resulting in the down-regulation of the targeted DvSnf7

gene leading to WCR mortality (Bolognesi et al. 2012) (Figure I-1). 
Monsanto Company CR240-13U1 33 of 374

Figure I-1. Diagram of MON 87411 dsRNA Oral Delivery 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). The dsRNA 

molecule that activates the mechanism is first processed by a class of RNase III enzymes 

called Dicers into small interfering RNAs (siRNAs, ~21-25 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 and mRNA cleavage that leads to specific 

suppression of the target mRNA (Hammond 2005; Tomari and Zamore 2005). In the 

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

DvSnf7 was selected as the target mRNA in CRW due to its vital cellular 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 

involved 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 have shown that suppression of DvSnf7 in WCR leads
Monsanto Company CR240-13U1 34 of 374

to accumulation of ubiquitinated proteins3

Ramaseshadri, et al. 2013

destined for lysosomal degradation 

( ). Sorting of transmembrane proteins is critical to regulate 

signal transduction in cells and as such, suppression of this sorting through the ESCRTIII complex impairs cell homeostasis and functioning, 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 phenotypes 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 ubiquitination 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 concentration 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 activity are 

highlighted (Ramaseshadri et al. 2013).

Induction of RNAi-mediated gene suppression in insects via an oral route of exposure 

requires efficient uptake of dsRNAs by midgut 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 long dsRNA (e.g., DvSnf7 240-mer) is taken up by the insect

and significant biological activity was only observed with dsRNA sequences ≥ 60 bp 

(Bolognesi et al. 2012). Finally, several key points have been identified in demonstrating

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 of DvSnf7 

expression beyond WCR midgut tissues, and 4) growth inhibition and WCR mortality 

(Bolognesi et al. 2012).

I.D.2. Modes-of-Action of the CP4 EPSPS and Cry3Bb1 Proteins

MON 87411 contains 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 relative to endogenous plant EPSPS 

(Padgette, et al. 1996). In MON 87411, as in other Roundup Ready plants, aromatic 

amino acids and other metabolites necessary for plant growth and development 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 Cry protein, processing to the active form, binding 

to midgut receptors and insertion of the toxin into 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 biotechnology-derived MON 89034 expressing two Cry proteins
Monsanto 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 therefore do not

impact root 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 plants 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 evaluations were 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 locations for 

control 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 Regulatory Agencies

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

protectants (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 compliance 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 Insecticide, 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 

commodities 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 requirement of a tolerance for residues of nucleic acids that are part of a plantincorporated 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 production 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 Registration for MON 87411 and the genetic material (PV-ZMIR10871) 

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) Program4

 

® 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 biotechnology-derived crops.

4 http://www.excellencethroughstewardship.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) provides 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 Ageing (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 relative 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-species/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 854 million metric tons (MMT) (USDA-FAS 

2013). The top five production regions in 2012 were: USA (274 MMT), China (208 

MMT), Brazil (73 MMT), EU-27 (55 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 industrialized 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 industrial 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 maize 
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likely occurred in southern 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 contributing 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 inflorescence (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 likely 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 positive 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 LH244, 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. corn belt. LH244 was 

initiated from a single cross of LH197 × LH199 followed by a backcross to LH197. The 

F2 combination ((LH197 × 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 

developed 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. 

II.C. Maize as a Test System in Product Safety Assessment

Based on seed availability and appropriate fit for various studies, hybrid maize lines

MPA640B (LH244 × LH287) and NL6169 (LH244 × HCL645) were used as near 

isogenic, conventional controls for this submission (hereafter referred to as conventional 

controls). As noted, one parent of each of these control maize lines is LH244, the inbred 

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

other maize inbreds. As such, both of these maize lines constitute relevant comparators 

for MON 87411. In addition, other commercial maize hybrids (hereafter referred to as 

reference hybrids) were used to establish ranges of natural variability representative of 

commercial maize hybrids. Reference hybrids used at each field trial location were 

selected based on their availability and agronomic fit for the respective geographic 
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regions. 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 phenotypic, 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, appropriate 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), compositional analysis (Section VII), and phenotypic, 

agronomic and environmental interactions assessment (Section VIII). The full molecular 

characterization studies (NGS/JSA) were conducted with the R4 generation (Figure IV-4). 

Initiation of commercial breeding efforts was conducted with the R5 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 the Breeding Tree 

(Figure IV-4). 
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III. 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) refers 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.

III.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 contains 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 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 

TubA 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, located 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 plasmid 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 

containing 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 plants (R0) with normal phenotypic characteristics were selected and 

transferred to soil for growth and further assessment. As demonstrated in this petition, 
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the use of disarmed Agrobacterium tumefaciens strain ABI, a designated plant pest, as the 

transformation vector has not imparted plant pest characteristics to MON 87411.

The R0 plants generated through the transformation process described above had already 

been exposed to glyphosate in the selection medium and demonstrated glyphosate 

tolerance. The R0 plants self-pollinated to produce R1 seed and R1 plants were evaluated 

for the presence of the T-DNA via quantitative polymerase chain reaction (PCR) analysis. 

The R1 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 laboratory using 

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

hundreds of events in the laboratory, greenhouse and field, MON 87411 was selected as 

the lead event based on superior agronomic, phenotypic, and molecular characteristics.

Studies on MON 87411 were 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 III-2.
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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 DvSnf7 indicates partial sequence.
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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 tissues

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 polymerase chain reaction 

(PCR) analyses

Evaluated plants for insert integrity by Southern blot analysis 
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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 the 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 similar 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. 

III.E. DvSnf7p sequence

The DvSnf7p sequence is the partial coding sequence of the Snf7 gene 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). The DvSnf7 suppression cassette contains 

two 240 bp DvSnf7p sequences in an inverted orientation. There is an intervening 

sequence of 150 nucleotides between the two DvSnf7p sequences (noted on Tables III-1 

and IV-1). When the suppression cassette is transcribed, the RNA expressed forms a 

hairpin loop thereby allowing the formation of double stranded DvSnf7 RNA. The 

DvSnf7p sequences 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 

heat 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, is 
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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 DvSnf7p 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 Borders

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 origin 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 promoter and coding sequence for an enzyme from transposon Tn7 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 the 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).
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Table III-1. Summary of Genetic Elements in PV-ZMIR10871

Genetic Element Location in 

Plasmid 

Vector

Function (Reference)

T-DNA

B1-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

T2-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

DvSnf7p 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

DvSnf7p 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 expression 

(Brown and Santino 1997)

P4-e35S 2618-3238 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 3239-3264 Sequence used in DNA cloning
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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

L5-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

CS6-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 

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 9206-9209 Sequence used in DNA cloning
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Table III-1 (continued). Summary of Genetic Elements in PV-ZMIR10871

Genetic Element Location in 

Plasmid 

Vector

Function (Reference)

TS7-CTP2 9210-9437 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 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)

Vector 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 Tn7 (Fling et al. 1985)

that confers spectinomycin and 

streptomycin resistance

Intervening Sequence 12769-13298 Sequence used in DNA cloning

OR8-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 cloning
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Table III-1 (continued). Summary of Genetic Elements in PV-ZMIR10871

Genetic Element Location in 

Plasmid 

Vector

Function (Reference)

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, Transcription 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.
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 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 GVTKVDFSQY 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 SVSTAC

121 TRITGLLEGE DVINTGKAMQ AMGARIRKEG DTWIIDGVGN GGLLAPEAPL DFGNAATGCR

MLHG ASSRPATARK SSGLSGTVRI PGDKSISHRS FMFGGLASGE

181 LTMGLVGVYD FDSTFIGDAS LTKRPMGRVL NPLREMGVQV KSEDGDRLPV TLRGPKTPTP

241 ITYRVPMASA QVKSAVLLAG 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-ZMIR10871. 
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IV. 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 multiple 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 whole-genome sequence analyses provided 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.

• Directed sequencing (locus-specific PCR, DNA sequencing and analyses) of

MON 87411 was 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 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 

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 occurred 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 chromosomal 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 present 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 Appendices B and L. 
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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 insert(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 number 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 

Sequencing
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Figure IV-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. 

Each insertion 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 

insert, 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 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.

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCGGG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCGGGAT

Plasmid DNA Flanking DNA

Junction

5’ Flank DNA insert 3’ Flank

Insert Junction Regions

Junction Sequences: Class A

Junction Sequences: Class B
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By evaluating the number of unique junction classes detected, the number of insertion 

sites of the plasmid sequence can be determined. A longer description of the molecular 

methods used to characterize MON 87411 can be found in Appendices B and L. For a 

single insert two junction sequence classes are expected, one each originating from either 

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 

contains 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 sample 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 1/10th 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 site. 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 element (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 insert 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 native 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 stably 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 stability of the insert is as expected across 
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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-DNA 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 maintained 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 

behavior of the T-DNA as a single chromosomal locus.
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Figure IV-4. Breeding History of MON 87411

R0 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 compositional analysis and RNA expression studies. 5 Generation used for agronomic/phenotypic studies

LH244 R1

LH244 R5

2,3

LH244 R4

1,2

LH244 R3

LH244 R2









R4F1 (LH244 × HCL645)2,4

LH244 R0



LH244 R6

2

R5F1 (LH244 × LH287)2,5



× HCL645

× LH287
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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 into 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.
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Table IV-1. Summary of Genetic Elements in MON 87411

Genetic Element1 Location in 

Sequence2

Function (Reference)

5′ Flank 1-1460 Sequence flanking the 5′ end of the insert

B3-Left Border Regionr1 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

T4-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

DvSnf7P 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 ESCRT-III complex (Babst et al. 2002)

that forms part of the suppression cassette

Intervening Sequence 2669-2818 Sequence used in DNA cloning

DvSnf7P 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 ESCRT-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)

P6-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 cells
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Table IV-1 (continued). Summary of Genetic Elements in MON 87411

Genetic Element1 Location in 

Sequence2

Function (Reference)

Intervening Sequence 5495-5500 Sequence used in DNA cloning

L7-Cab 5501-5561 5' UTR leader sequence from chlorophyll a/bbinding (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 6058-6066 Sequence used in DNA cloning

CS8-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, 

HSP17, 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

TS9-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 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 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 

mRNA
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Table IV-1 (continued). Summary of Genetic Elements in MON 87411

Genetic Element1 Location in 

Sequence2

Function (Reference)

Intervening Sequence 12676-12693 Sequence used in DNA cloning

B-Right Border Regionr1 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 sequence 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 summarized in 

Section IV.A.1, Section IV.A.2 and Table IV-2. The sensitivity of the method is 

demonstrated in Section IV.A.1 with data shown in Table IV-3. The JSA analysis of the 

R4 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 were 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 materials and methods see 

Appendix B.
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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 isolated 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 sequence 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 sequence 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×

coverage 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 conventional control maize DNA 

spiked with PV-ZMIR10871 DNA at 1 and 1/10th 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 that 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 1/10th genomic equivalent (99.64% coverage at 

100% identity for the 1/10th genome equivalent spiked control sample, Table IV-3) and, 

hence, a detection level of at least 1/10th genome equivalent was achieved for the plasmid 

DNA sequence assessment.
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Table IV-2. Sequencing (NGS) Conducted for MON 87411 and Control Genomic 

DNA

Sample Total Nucleotides (Gb) Effective Median Depth of 

Coverage (x-fold)

LH244 346.9 126x

LH244 × HCL645 309.5 107x

LH244 × LH287 342.3 118x

R4 334.9 125x

R4F1 338.9 113x

R5F1 346.6 126x

R5 352.3 140x

R6 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 genome. The median 

effective depths 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 spikein 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

1/10th copy Spike 1 copy Spike

Extent of coverage1 of PV-ZMIR10871 99.64% 100%

Percent identity of coverage2 of PV-ZMIR10871 100% 100%
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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 R4

generation. A single genomic DNA insertion is expected to produce two junction 

sequence classes and any additional integration sites would produce additional junction 

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 107 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 transformation 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 of 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 determined (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.
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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 

number 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 sequences 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 

intended PV-ZMIR10871 T-DNA at a single locus in the genome of MON 87411. JSCA 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 DNA 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 analysis 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 present in MON 87411.
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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 braces.

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.
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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 (continued). 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, respectively. 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.
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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 sequencing 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 Right and Left border regions. The border regions both contain 

small terminal deletions 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 of 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.
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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 R4 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 illustration. 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

18 19 20 21 22 23 24 25 26

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 H
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Lane designations are as follows:

Lane Sample Lane Sample

1 1 kb DNA Extension Ladder 18 1 kb 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 kb DNA Extension Ladder 34 1 kb 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 kb DNA Extension Ladder 

(Invitrogen, Grand Island, NY) on the ethidium bromide stained gel.
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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 primer 

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 

indicates 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 presumably resulted from double-stranded break repair mechanisms in the plant 

during the Agrobacterium-mediated transformation process (Salomon and Puchta 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 generated 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 each of the PCR reactions was loaded 

on the gel. Lane designations are as follows:

Lane Sample

1 1 kb DNA Extension Ladder

2 Conventional Control LH244

3 No template DNA control

4 1 kb DNA Extension Ladder

Arrows on the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained 

from the 1 kb DNA Extension Ladder (Invitrogen, Grand Island, NY) on the ethidium bromide 

stained gel.

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.

5.0

4.0

40

3.0

2.0

1.6

1.0

0.5

1 2 3 4 

1.6

3.0

2.0

5.0

4.0

40

1.0

0.5

Primer A Primer B

Primer A Primer B
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IV.D. Determination of Insert Stability over Multiple Generations of MON 87411

In order to demonstrate the stability 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 (R4) 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 generations were evaluated by NGS/JSA 

analysis as previously described in Section IV.A, and compared to the fully characterized 

MON 87411 (R4) generation. The conventional controls used for the generational 

stability analysis included LH244, which has a genetic background similar to the 

MON 87411 (R4), MON 87411 (R5) and MON 87411 (R6) generations and represents the 

original transformation line. The conventional control hybrid LH244 × HCL645 has a 

genetic background similar to the MON 87411 R4F1 hybrid. In addition, the conventional 

control hybrid LH244 × LH287, has a genetic background similar to the 

MON 87411 R5F1 hybrid. Genomic DNA isolated from each of the selected generations 

of MON 87411 and conventional controls were used for NGS/JSA analysis. The results 

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 JSCs Detected

MON 87411 (R4) 2

MON 87411 (R4F1) 2

MON 87411 (R5) 2

MON 87411 (R5F1) 2

MON 87411 (R6) 2

LH244 0

LH244 × HCL645 0

LH244 × LH287 0
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Figure IV-9 and Figure IV-10, below, identify the presence of two, and only two, 

identical junction sequence classes in each of the five assessed MON 87411 generations 

(R4, R5, R6, R4F1 and R5F1) 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 results, 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 

bioinformatic analysis (NGS/JSA), it is concluded that MON 87411 contains a single and 

stable T-DNA insertion.
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R4 -AATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

Directed Seq. GAATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R6 GAATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R4F1 -AATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R5 GAATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

R5F1 GAATTGAAAAAAAATTGGTAATTACTCTTTCTTTTTCTCCATATTGACCATCATACTCAT

 

R4 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

Directed Seq. TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

R6 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

R4F1 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

R5 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

R5F1 TGCTGATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

 

R4 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC

Directed Seq. TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC

R6 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC

R4F1 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACR5 TTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG----

R5F1 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 their generation 

(R4, R5, R6, R4F1 and R5F1). 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.
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R5F1 AGCGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

Directed Seq. AGCGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R5 --CGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R4F1 -GCGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R4 ---GGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

R6 AGCGGCTAGAGCCACACCCAAGTTCCTAACTATGATAAAGTTGCTCTGTAACAGAAAACA

 

R5F1 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

Directed Seq. CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

R5 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

R4F1 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

R4 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

R6 CCATCTAGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

 

R5F1 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT----

Directed Seq. ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R5 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R4F1 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R4 ATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R6 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 sequences for each of the five generations are labeled according to their generation 

(R4, R5, R6, R4F1 and R5F1). 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.
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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 (χ2

) 

analysis over several generations. The χ2 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 transformed R0 plant was self-pollinated to generate R1 seed. An 

individual homozygous positive plant was identified in the R1 segregating population via 

a Real-Time TaqMan

The homozygous positive R1 plant was self-pollinated to give rise to R2 seed. The R2

plants were self-pollinated to produce R3 seed. The R3 plants were self-pollinated to 

produce R4 seed. Homozygous positive R4 plants were crossed via traditional breeding 

techniques to a recurrent parent (HCL645) that does not contain the DvSnf7p, cp4 epsps, 

or cry3Bb1 coding sequences to produce hemizygous R4F1 seed. The R4F1 plants were 

crossed with the recurrent parent to produce BC1F1 seed. The BC1F1 generation was 

tested for the presence of the T-DNA by End-Point TaqMan PCR to select for 

hemizygous MON 87411 plants. BC1F1 plants hemizygous for MON 87411 T-DNA 

were crossed with the recurrent parent to produce the BC2F1 plants. BC2F1 plants 

hemizygous for MON 87411 T-DNA were self-pollinated to produce the BC2F2 plants. 

BC2F1 plants hemizygous for MON 87411 T-DNA were crossed with the recurrent parent 

to produce the BC3F1 plants. 

PCR assay. 

The inheritance of the MON 87411 T-DNA was assessed in the BC2F1, BC2F2, and 

BC3F1 generations. At the BC2F1 and BC3F1 generations, the MON 87411 T-DNA was 

predicted to segregate at a 1:1 ratio (hemizygous positive: homozygous negative) 

according to Mendelian inheritance principles. At the BC2F2 generation, the MON 87411 

T-DNA was predicted to segregate at a 1:2:1 ratio (homozygous positive: hemizygous

positive: homozygous negative) according to Mendelian inheritance principles. 

A Pearson’s Chi-square (χ2) analysis was used to compare the observed segregation ratios 

of the MON 87411 T-DNA to the expected ratios. The Chi-square (χ2) analysis used the 

statistical 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 χ2 analysis of the MON 87411 segregating progeny are presented in 

Table IV-6 and Table IV-7. The χ2 values in the BC2F1 and BC3F1 generations indicated 

no statistically significant difference between the observed and expected 1:1 segregation 

ratio (hemizygous positive: homozygous negative) of the MON 87411 T-DNA. The χ2

value for the BC2F2 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 contains a 

single intact copy of the T-DNA inserted at a single locus in the maize genome.
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Figure IV-11. Breeding Path for Generating Segregation Data for MON 87411

*Chi-square analysis was conducted on segregation data from the BC2F1, BC2F2, and BC3F1 generations (bolded text).

TI: Trait Integration: Replacement of genetic background of MON 87411 by recurrent background except inserted gene.

RP: Recurring parent. 

=Self-Pollinated

LH244 R1

Breeding path 

continued

LH244 R4

LH244 R3

LH244 R2







Transformed LH244 R0 Plant



R4F1 (LH244 x HCL645)

TI:BC1F1

TI:BC2F1

× RP

× RP

× RP

TI:BC3F1

x RP

(Expected segregation 1:1) 

(Hemizygous positive: Homozygous negative)

(Expected segregation 1:1)

(Hemizygous positive: Homozygous negative)



TI:BC2F2

(Expected segregation 1:2:1)

(Homozygous positive: Hemizygous positive: Homozygous negative)
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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 Probability2 Negative Negative

BC2F1

1 351 172 179 175.50 175.50 0.14 0.709

BC3F1

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 Probability2

BC2F2

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).
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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 shows the flanks in MON 87411 are 

identical to the conventional control, excepting a 118 bp deletion of genomic DNA at the 

insertion site 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 insert. 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 single chromosomal locus.
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V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE Cry3Bb1 

and CP4 EPSPS PROTEINS PRODUCED IN MON 87411

Characterization of the introduced protein(s) in a biotechnology-derived crop is important 

to establishing food, feed, and environmental safety. As described in Section IV, 

MON 87411 contains cry3Bb1 and cp4 epsps expression cassettes that, when transcribed 

and translated, result in the expression of the Cry3Bb1 and CP4 EPSPS proteins. The 

characterization and safety assessment for the DvSnf7 suppression 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 equivalence 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 human 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 Proteins from 

MON 87411

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

Cry3Bb1 protein originates from Bacillus thuringiensis (Bt), a ubiquitous gram-positive 

soil bacterium that accumulates crystal proteins during sporulation. Cry3Bb1 protein is a 

member of the 3D-Cry family of insecticidal proteins (Crickmore 2012). Proteins within 

the 3D-Cry proteins are subdivided into different groups based on the high specificity 

they have for their target category of insects. Because of their narrow spectrum of 

activity, they lack an impact on broader insect populations or other organisms. For 

example, Cry3 proteins have insecticidal activity specifically against coleopteran insects, 

while Cry1A proteins have insecticidal activity specifically against 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 activated 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. While 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).
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Cry3Bb1 protein in MON 87411 is a protein consisting of a single polypeptide of 652

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. 2007). 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 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). Each 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 CP4 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 plant 

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 phosphoenolpyruvate (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 substrate 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 epsps coding sequence encodes an 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 endogenous 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. 1996). 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, requirements 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 expressed 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. 
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V.B. Characterization and Equivalence of Cry3Bb1 and CP4 EPSPS Proteins from 

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 safety 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 functional 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 shown below and the 

details of the materials, methods, and results are described in Appendix C.

The Cry3Bb1 and CP4 EPSPS proteins purified from grain of MON 87411 were

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 analysis of the MON 87411-produced proteins

established identity; 2) MALDI-TOF MS analysis yielded peptide masses consistent with 

the expected peptide masses from the theoretical trypsin digest of the 

MON 87411-produced sequences; 3) MON 87411-produced Cry3Bb1 and CP4 EPSPS 

proteins were detected on western blots probed with their respective protein-specific 

antibodies and the immunoreactive properties of the MON 87411-produced and 

E. coli-produced proteins were shown to be equivalent; 4) the apparent molecular weights

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 in MON 87411, summarized in section V.E.

V.C. Expression Levels of Cry3Bb1 and CP4 EPSPS Proteins 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 assay 

(ELISA). Tissues of MON 87411 were collected from four replicate plots planted in a 

randomized complete block field design during the 2011 - 2012 growing season from the 

following 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. corn belt and Argentina growing regions 
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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 over season leaf (OSL1 through OSL4), over season root (OSR1 through 

OSR4), 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 conditions 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.

The details of the materials and methods are described in Appendix D. The individual 

Cry3Bb1 protein levels in MON 87411 across all samples analyzed from all sites ranged 

from 3.0 to 460 μg/g dw. The mean Cry3Bb1 protein level among all tissue types was

highest in OSWP1 at 340 μg/g dw and lowest in grain at 4.0 μg/g dw.
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Table V-1. Summary of Cry3Bb1 Protein Levels in Tissues from MON 87411 

Grown in 2011 – 2012 Argentina Field Trials

Tissue1

Development 

Stage2

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 - 390

OSL2 V6-V8 35-44 40 (7.8) 210 (40) 0.035/0.006

26 – 56 120 – 270

OSL3 V10-V13 50-55 40 (7.9) 170 (35) 0.035/0.006

21 – 52 92 – 220

OSL4 V14-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 V6-V8 35-44 16 (4.0) 120 (24) 0.035/0.028

9.4 – 25 67 – 170

OSR3 V10-V13 50-55 15 (4.0) 84 (21) 0.035/0.028

9.6 – 24 54 – 130

OSR4 V14-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 V6-V8 35-44 30 (5.3) 190 (30) 0.035/0.008

21 – 40 130 – 270

OSWP3 V10-V13 50-55 20 (6.8) 140 (39) 0.035/0.008

9.2 – 33 59 – 210

OSWP4 V14-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 – 50
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Table V-1. (continued) Summary of Cry3Bb1 Protein Levels in Tissues from MON 87411 

Grown in 2011 – 2012 Argentina Field Trials

Tissue1

Development 

Stage2

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 R6 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 The crop development stage each tissue was collected. 3 The number of days 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). 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 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 samples 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 types was highest in OSWP1 at 63 μg/g dw 

and lowest in grain at 1.9 μg/g dw.
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Table V-2. Summary of CP4 EPSPS Protein Levels in Tissues from MON 87411

Grown in 2011 – 2012 Argentina Field Trials

Tissue1

Development 

Stage2

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 V6-V8 35-44 7.0 (0.64) 36 (3.1) 0.137/0.071

6.0 – 7.9 29 – 39

OSL3 V10-V13 50-55 7.4 (0.78) 32 (3.8) 0.137/0.071

6.4 – 8.9 27 – 42

OSL4 V14-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 V6-V8 35-44 5.2 (1.0) 37 (7.0) 0.068/0.033

3.8 – 7.1 23 – 48

OSR3 V10-V13 50-55 5.6 (0.84) 31 (4.7) 0.068/0.033

4.0 – 7.1 24 – 37

OSR4 V14-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 V6-V8 35-44 5.6 (0.94) 36 (5.8) 0.137/0.070

3.4 – 7.4 21 – 46

OSWP3 V10-V13 50-55 4.6 (1.1) 33 (6.2) 0.137/0.070

2.3 – 6.6 21 – 45

OSWP4 V14-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 – 11
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Table V-2. (continued) Summary of CP4 EPSPS Protein Levels in Tissues from 

MON 87411 Grown in 2011 – 2012 Argentina Field Trials 

Tissue1

Development 

Stage2

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 R5 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 R6 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 days 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 quantitation; LOD=limit of detection.

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

Proteins

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 nonallergenic source; 2) the protein represents a small portion of the total plant protein; 

3) the protein does not share structural similarities to known allergens based 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 originates from Bt, 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 share 

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 Cry3Bb1protein does not 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 protein originates 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 demonstrated 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. 
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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 not 

pose a significant allergenic risk to humans or animals. 

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

MON 87411

A comprehensive set of factors have been considered and assessed in the safety 

assessment of the Cry3Bb1 and CP4 EPSPS proteins in food and feed or 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 

50 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 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). 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 

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 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 United States EPA has approved commercial use of the 
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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 scale cultivation of these 

Cry3Bb1-expressing crops without any indication of harmful impact on the environment, 

non-target organisms, 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 environment.

V.E.1.3. Cry3Bb1 Protein Demonstrates Specificity

Cry3Bb1 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 this donor organism for USDA-APHIS deregulations, as 

well as completed Monsanto consultations with the FDA regarding Roundup Ready 

varieties of soybean (1995 and 2007), canola (1995, 2002, and 2012), maize (1998 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 EPSPS 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 in 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 

EPSPS proteins, and their enzymatic activity, pose no hazards to humans, animals, 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 plants (U.S. EPA 1996). 
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V.E.2.3. CP4 EPSPS Catalyzes a Specific Enzyme Reaction

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. 1996) 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 in MON 87411 are Not Homologous to 

Known Allergens or Toxins

Bioinformatics analyses were performed to assess the potential for allergenicity, toxicity, 

or biological activity of Cry3Bb1 and CP4 EPSPS. The analyses 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 animal health.

V.E.4. Cry3Bb1 and CP4 EPSPS Proteins in MON 87411 are Labile in in vitro

Digestion Assays

As has been described in previous regulatory submissions for a number of Roundup 

Ready crops and MON 88017 (APHIS Petition No. 04-125-01), both the Cry3Bb1 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 was 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 protein 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. 1996) and for Cry3Bb1 at 1930 mg/kg bw (FSANZ 2006), the 

highest 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 Proteins

A common approach used to assess potential health risks for potentially toxic materials is 

to calculate a Margin of Exposure (MOE) between the lowest NOAEL from an 
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appropriate animal toxicity study and an estimate of human exposure. Since no evidence 

of mammalian toxicity 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 calculating a MOE between the NOAELs for the Cry3Bb1 and CP4 EPSPS 

proteins in mouse acute oral toxicity studies (Section V.E.5) and 95th percentile 

consumption estimates of acute dietary exposure determined using the Dietary Exposure 

Evaluation Model - Food Commodity 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 levels of expressed 

protein on a fresh weight basis (μg/g) discussed above (Sections V.C.1 and V.C.2), 95th

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-population, 95th 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 × 105

) 

and 110,000 (1.1 × 105) 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 × 105) and 69,000 (6.9 × 104) for the general population and non-nursing infants, 

respectively. Actual MOEs will likely be much higher because: 1) the exposure estimates 

utilized are conservative (95th percentile, assume 100% MON 87411) and 2) as described 

in section V.E.4, Cry3Bb1 and CP4 EPSPS are rapidly digested, further minimizing 

exposures. These very large MOEs5

There are a number of steps in the processing of maize to make food ingredients, 

including high 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 to the expressed proteins in MON 87411 that were not toxic to mice, which 

would be a physical impossibility.
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protein consumed was in the lactating dairy cow, 0.014% (g/g) and 0.0034% (g/g) of the 

total dietary protein intake (0.000876 g Cry3Bb1/kg bw divided by 6.2 g dietary protein, 

and 0.000210 g CP4 EPSPS/kg bw divided by 6.2 g dietary protein which is the total 

dietary protein intake 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 scenario, per kg body weight, 

poultry, swine and lactating dairy cattle would be consuming 0.02% (g/g) and 0.004%

(g/g) or 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 glyphosate 

tolerance, 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 present in

MON 88017 and the tissue expression of the Cry3Bb1 protein is comparable to the 

previously registered maize products MON 88017 and MON 863. For MON 863, a 
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series of NTOs were evaluated to assess for potential negative impacts of 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 previous 

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 (USDAAPHIS 2013)(USDA-APHIS Petition No. 01-137-01p), USDA-APHIS considered the 

potential impact of the Cry3Bb1 protein on NTOs, including beneficial organisms and 

threatened or endangered species concluding: 

“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 88017 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. Field observations, compositional 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 environmental hazard assessment of Cry3Bb1-

producing maize based on studies conducted with similar Cry3Bb1 proteins on 

representative species of bird, fish, and terrestrial non-target insects, including 

endangered species, to support the registration of MON 863 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 critical habitats.” (U.S. EPA 

2010)
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V.F. Cry3Bb1 and CP4 EPSPS Proteins Characterization and Safety Conclusion

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 proteins in 

MON 87411, including their potential allergenicity and toxicity. To summarize, the 

physicochemical characteristics of the Cry3Bb1 and the CP4 EPSPS proteins from 

MON 87411 were determined and shown to be equivalent to those of their

E. coli-produced counterparts. Given the very large MOEs for Cry3Bb1 and CP4 EPSPS 

proteins 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 the proteins 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. strain CP4, respectively, are ubiquitous in the 

environment and not commonly known for human or animal pathogenicity, or 

allergenicity. The Cry3Bb1 and CP4 EPSPS proteins 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 digestive 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 proteins 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 market 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 MON 87411 are also considered as safe for the environment as other maize 

products. 
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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. Endogenous RNAmediated 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. This lack of toxicity or allergenicity for ingested 

RNA also extends 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 extensive sequence-independent physiological and 

biochemical barriers 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 ingestion of large doses of a particular small (micro)

RNA (miRNA) from rice led to some absorption of the miRNA, 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 (miR168a) regulating animal gene expression – may be a common phenomenon; 

and that miRNAs in food may regulate specific genes in animals based upon sequence 

identity between plant miRNAs and mammalian genes. A second publication 

(Heinemann, et al. 2013), a review article relying almost exclusively on the Zhang 

(2012a) study, suggests that the current safety/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 appropriate 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 animal 
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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) involving Monsanto scientists (Zhang, 

et al. 2012b) revealed confounding factors which likely explain, in part, the 

unexpected findings in the 2012 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 

in part, from cross-contamination during the sequencing procedure, thus calling

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 claims of dietary miRNA uptake and 

physiological impact (Dickinson, 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 observations (Zhang 

et al. 2012a) were due to variability in gene expression and protein expression 

data and nutritional 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 

primate 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; Jain 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 presented below in this petition regarding specificity and lack of adverse 

impacts on NTOs (Section VI.E.) supports the safety of the consumption of 

MON 87411 and the expressed construct-derived RNA molecules contained 

within it. 
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Finally, these numerous points regarding 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 appropriate for 

assessing this product.

VI.B. Characterization 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 DvSnf7 transcript required large amounts of 

DvSnf7 RNA as the test substance. Because of the extremely low expression of DvSnf7 

RNA in MON 87411 (Table VI-1), it was necessary to produce RNA through in vitro

transcription methods in order to obtain sufficient quantities of DvSnf7 RNA 

(DvSnf7_968 RNA6

Regarding the overall potential for toxicity and allergenicity, RNA is different from 

proteins. While certain rare proteins are capable of demonstrating toxicity (usually 

occurring acutely) (

) for subsequent safety studies. In order to demonstrate that the 

in vitro-produced RNA was 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 those of in vitroproduced RNA, DvSnf7_968 RNA. A summary 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 toxicity 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 If

7

 

6 DvSnf7_968 is the predominant RNA transcript produced from the DvSnf7 suppression cassette and, in 

addition to the DvSnf7p sequences, contains portions of the CaMV 35S promoter, maize hsp70 intron, and 

pea E9 3’ untranslated region from PV-ZMIR10871. 

digestion followed by northern blot analysis 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 If specifically digests single stranded RNA, leaving dsRNA intact
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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

DvSnf7 RNA levels in various tissues of MON 87411 were determined by a validated 

QuantiGene® Plex 2.0 Assay. Plant material from MON 87411 was 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 for 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 

analyzed from all sites ranged from lower than the limit of detection (<LOD) to 

213 × 10-3 μg/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 expression level <LOD or lower than the limit of quantitation (<LOQ) were not 

included in the mean determinations.
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Table VI-1. Summary of DvSnf7 RNA Levels in Maize Tissues Collected from 

MON 87411 Produced in Argentina Field Trials during 2011-2012

Tissue 

Type1

Development 

Stage2

Mean

(SD)

Range

(μg/g fw)3

Mean

(SD)

Range

(μg/g dw)4

LOD/

LOQ5

(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 × 10-3
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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 

Type1

Development

Stage2

Mean

(SD)

Range

(μg/g fw)3,6

Mean

(SD)

Range

(μg/g dw)4

LOD/

LOQ5

(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 pollen 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) 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 pollen samples <LOD and from the rest of the samples for 

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

The potential dietary intake of DvSnf7 RNA from the consumption of food derived from 

MON 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 DvSnf7 RNA in the included maize fractions

(Determined using DEEM-FCID8

Total nucleic acid consumption in the diet for DNA and 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 levels of expressed 

DvSnf7 RNA in grain from MON 87411 on a fresh weight basis (μg/g) discussed above 

(Table VI-1), 95th 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 ng/kg body weight per day, respectively. These exposure 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 million 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 information, the exposure assessment described above, rather than a dietary risk 

assessment is considered most 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 Dietary Exposure Evaluation Model– Food Commodity Intake Database. 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.
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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 indicates that 

there would be no significant risk to human and animal health associated with 

consumption 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 RNAdependent 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 bioamplification 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 lifestage (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 greater 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 dsRNA 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 expressed in 

MON 87411 maize and are described below (Table VI-3).
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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 taxa that includes the target organism as well as nontarget 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 bioassays to 

characterize the concentration-effect relationship and to estimate the LC50

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 effects with mean 12-

day LC50 values of 4.4 ng DvSnf7_240 dsRNA/ml diet and 1.2 ng DvSnf7_240 

dsRNA/ml 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 

bioassays 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 complements 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) evaluated 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, and 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 insensitive to dietary dsRNA without the use of a 

 

9 The LC50 value is the measure of toxicity of a substance (Lethal Concentration), that when fed to a test 

organism over a given time period, results in death of 50% of those organisms. 
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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 in total, 7 species across four 

families in this Order were tested with the goal to more fully characterize the 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/ml diet and exceed 

DvSnf7 dsRNA expression values 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 DvSnf7_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 tested, two species L. decemlineata and Tribolium castaneum have, 

however, been shown 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).
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Table VI-2. Susceptibility to DvSnf7_240 dsRNA in laboratory bioassays

Order Family Subfamily Species Common Name

Bioassay 

Duration 

(days)

Endpoints1

LC50 2 or No 

Observed Effect 

Concentration3

 

(ng/ml or g diet)

Coleoptera Chrysomelidae Galerucinae Diabrotica virgifera virgifera Western corn rootworm 12 S 4.42

Chrysomelidae Galerucinae Diabrotica undecimpunctata howardi Southern corn rootworm 12 S 1.22

Chrysomelidae Chrysomelinae Leptinotarsa decemlineata Colorado potato beetle 12 S, G 5,0003

Tenebrionidae Tenebrioninae Tribolium castaneum Red flour beetle 30 S, G 5,0003

Coccinellidae Coccinellinae Coleomegilla maculata Pink-spotted lady beetle 24 S, G, D 3,0003

Coccinellidae Epilachninae Epilachna varivestis Mexican bean beetle 28 S, G, D 3,0003

Carabidae Harpalinae Poecilus chalcites Carabid beetle 35 S, G, D 5,0003

Hemiptera Anthocoridae Anthocorinae Orius insidiosus Insidious Flower bug 9 S, D 5,0003

Hymenoptera Eulophidae Entedoninae Pediobius foveolatus Eulophid wasp 21 S 3,0003

Pteromalidae Pteromalinae Nasonia vitripennis Jewel wasp 20 S 5,0003

Lepidoptera Noctuidae Noctuinae Spodoptera frugiperda Fall armyworm 8 S, G 5003

Noctuidae Heliothinae Helicoverpa zea Corn earworm 12 S, G 5,0003

Crambidae Pyraustinae Ostrinia nubilalis European corn borer 12 S, G 5,0003

Bombycidae Bombycinae Bombyx mori Silkworm 14 S, G 5,0003

1 S: Survival; G: Growth; D: Development

2 LC50 survival data reported in (Bachman et al. 2013) 3 No observed effect concentration (NOEC) is equal to the maximum concentration tested in these bioassays. 
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VI.E.4. Non-Target Organism Assessment for DvSnf7

Evaluation of the potential risks to NTOs is an important component of APHIS’s plant 

pest risk assessment of a biotechnology-derived crop. Assessment of the potential risks 

to NTOs 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 evaluation 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 insectprotected 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). 

Based on the results 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 testing against a soil decomposer [earthworm (Eisenia andrei)], and five

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 

Table VI.3. 

Exposure information was developed to determine the maximum expected environmental 

concentration (MEEC) for the DvSnf7 RNA produced in MON 87411 to NTOs. Test 

concentrations were based on the measured DvSnf7 RNA expression in the tissue(s) 

types that the NTO would most likely be exposed to in the environment. A targeted 

MOE of greater than 10-times the MEEC 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 to 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, the maximum expression value from the leaf development stage with the highest 

expression (V14-R1) was used to represent worst-case scenario to calculate the MOE for 
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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, MOEs were calculated based on the ratio of the 

NOECs to the MEECs. 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 protein, 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 restricted 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 crop 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). Since there is no meaningful ecologicallyrelevant exposure to aquatic organisms from maize, other than through purposeful 

feeding of processed maize products, effects tests on aquatic species were not conducted 

for MON 87411. Additionally, 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, successful RNAi 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. 
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Table VI-3. Maximum expected environmental concentrations (MEECs), no observed effect concentrations (NOECs) from nontarget 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 ng4 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 Maximum expression levels determined from MON 87411. 2 MOE values were calculated based on the ratio of the NOEC to MEEC. The MOE was determined based on the maximum expression level of the DvSnf7 RNA 

in the tissue from MON 87411deemed 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 μg)). 5 The NOECrepresents the concentration of the test solution used for dosing individual larval cells. A single dose of 10 μl 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.
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VI.F. Characterization and Safety Conclusions

DvSnf7 RNA from MON 87411 is a dsRNA that upon consumption by 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 justifies 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 low 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 bw per day). Anticipated 

dietary exposures to DvSnf7 in animals are also very low, the highest anticipated levels 

being in lactating 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 Chrysomelidae 

within the order Coleoptera. Specific testing on a battery of NTOs (honeybee, 

earthworm, ladybird beetle, and others) further supports 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 animal consumption and the environment.
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VII. 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. Compositional 

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 OECD 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 composition. 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, antinutrients, 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 comparison 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 of 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.
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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). 

VII.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 provide 

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 reference 

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 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 

maize hybrids developed in the U.S. are often used directly by farmers in Argentina. 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 lbs 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 (caprylic, 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 statistically 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. 
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A statistically significant difference 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 and the conventional 

control that was used in steps two and three, below. For protein and amino acids 

only10

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 is 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 

compares the mean difference between MON 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 Composition 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 used 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 if 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 protein. 

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. 
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whether the change in composition would have an impact from a food and 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 equivalence.

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 statistically 

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) showed a significant 

difference between MON 87411 and the conventional control in the combined site 

analysis. For these 12 components, the mean differences 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% 

tolerance 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 

confirmed 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 

the following section. These data confirmed that the components with observed 

significant differences were not compositionally meaningful from a food and feed safety 

perspective. 

VII.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 

components). 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 associated 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 significant 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 conventional 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 maximum 
Monsanto Company CR240-13U1 117 of 374

(12.60% dw) protein values. The mean difference in protein 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 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 acid 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). Correspondingly, 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 magnitudes 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 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 intervals, the 

values observed in the literature, and/or the ILSI-CCDB values. 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 occurring 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 conventional 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 within 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 values 
Monsanto 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 variation 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 calculation 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 

statistically 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 maximum (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 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 variation in 

carbohydrates by calculation and fiber 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 NDF was not 

compositionally meaningful from a food and feed safety perspective. 
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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 composition. 

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 calcium, 

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 MON 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 within 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 was 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 variation 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 87411 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), 

indicating 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 was 
Monsanto Company CR240-13U1 120 of 374

within the 99% tolerance interval, the values observed in the literature, and/or the ILSICCDB values (Table VII-8). 

For zinc, the mean value was 21.44 mg/kg dw for MON 87411 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 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 zinc values was also less 

than the variation seen in the reference hybrid values (ranged 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 not 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 perspective. 

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 conditions (Harrigan et al. 2009; Ridley et al. 

2011; Safawo, et al. 2010). This naturally occurring variability is important in assessing 

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 differences 

(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 difference 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 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 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 values (Table VII8). 
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 was 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 literature, and/or the ILSICCDB 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 vitamins were not compositionally meaningful from a 

food and feed safety perspective. 

VII.A.2. Anti-Nutrient Levels in Maize Grain

Anti-nutrients assessed included phytic acid and raffinose. Phytic acid, the major storage 

form of phosphorus in maize grain, is considered an anti-nutrient due to its mineralchelating properties and the sequestration of phosphorus in phytic acid, reducing 

phosphorus bioavailabity. Raffinose 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 were 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 conventional control in levels of these 

components. 

VII.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 secondary 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). The 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. 

VII.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 important 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 maximum (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 conventional 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 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 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 

difference 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 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 Interval4

Conventional 

Control Range

Value5

Mean

(S.E.) p-Value % Relative6

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, 3.02
Monsanto 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 Interval4

Conventional 

Control Range

Value5

Mean

(S.E.) p-Value % Relative6

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, 0.28
Monsanto 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 Interval4

Conventional 

Control Range

Value5

Mean

(S.E.) p-Value % Relative6

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, 0.58
Monsanto 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 Interval4

Conventional 

Control Range

Value5

Mean

(S.E.) p-Value % Relative6

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.
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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 Interval4

Conventional 

Control Range

Value5

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 Palmitic6 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, 1.65
Monsanto 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 Interval4

Conventional 

Control Range

Value5

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 maize 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 expressed as a % of total fatty acid. Prefix numbers refer to number of carbon atoms and number of carboncarbon 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 following 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.
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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 Interval4

Conventional 

Control Range

Value5

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 Interval4

Conventional 

Control Range

Value5

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, 12.14
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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 Interval4

Conventional 

Control Range

Value5

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.
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Table 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 Interval4

Conventional 

Control Range

Value5

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, 10.03
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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 Interval4

Conventional 

Control Range

Value5

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.
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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 Interval4

Conventional 

Control Range

Value5

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

Maximum value minus minimum value for the conventional control maize hybrid.
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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 Interval4

Conventional 

Control Range

Value5

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, 62.39
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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 Interval4

Conventional 

Control Range

Value5

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.
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Table VII-8. Literature and ILSI Database Ranges for Components in Maize

Forage and Grain

Grain Tissue Components1 Literature Range2 ILSI Range3

Grain Nutrients

Proximates (% dw)

Ash 1.17 – 2.01a; 1.27 – 1.63b 0.616 – 6.282

Carbohydrates by calculation 81.31 – 87.06a; 82.10 – 85.98b 77.4 – 89.5

Fat, total 2.95 – 4.40a; 3.18 – 4.23b 1.742 – 5.900

Protein 8.27 – 13.33a; 9.17 – 12.19b 6.15 – 17.26

Fiber (% dw)

Acid detergent fiber 1.82 – 4.48a; 1.83 – 3.39b 1.82 – 11.34

Neutral detergent fiber 6.51 –12.28a; 6.08 – 10.36b 5.59 – 22.64

Total dietary fiber 10.65 – 16.26a; 10.57 – 14.56b 9.01 – 35.31

Amino Acids (% dw)

Alanine 0.60 – 1.04a; 0.68 – 0.96b 0.44 - 1.39

Arginine 0.34 – 0.52a; 0.34 – 0.50b 0.12 - 0.64

Aspartic acid 0.52 – 0.78a; 0.59 – 0.76b 0.33 – 1.21

Cystine 0.19 – 0.26a; 0.20 – 0.26b 0.13 – 0.51

Glutamic acid 1.54 – 2.67a; 1.71 – 2.44b 0.97 – 3.54

Glycine 0.33 – 0.43a; 0.33 – 0.42b 0.18 – 0.54

Histidine 0.25 – 0.37a; 0.27 – 0.34b 0.14 – 0.43

Isoleucine 0.30 – 0.48a; 0.32 – 0.44b 0.18 – 0.69

Leucine 1.02 – 1.87a; 1.13 – 1.65b 0.64 – 2.49

Lysine 0.26 – 0.33a; 0.28 – 0.31b 0.17 – 0.67

Methionine 0.17 – 0.26a; 0.16 – 0.30b 0.12 – 0.47

Phenylalanine 0.43 – 0.72a; 0.45 – 0.63b 0.24 – 0.93

Proline 0.74 – 1.21a; 0.78 – 1.11b 0.46 – 1.63

Serine 0.39 – 0.67a; 0.43 – 0.60b 0.24 – 0.77

Threonine 0.29 – 0.45a; 0.31 – 0.39b 0.22 – 0.67

Tryptophan 0.047 – 0.085a; 0.042 – 0.070b 0.027 – 0.215

Tyrosine 0.13 – 0.43a; 0.12 – 0.41b 0.10 – 0.64

Valine 0.42 – 0.62a; 0.45 – 0.58b 0.27 – 0.86

Fatty Acids (% Total FA)

16:0 Palmitic 8.80 – 13.33a; 9.84 – 12.33b 7.94 – 20.71

18:0 Stearic 1.36 – 2.14 a; 1.30 – 2.10b 1.02 – 3.40

18:1 Oleic 19.50 – 33.71a; 19.59 – 29.13b 17.4 – 40.2

18:2 Linoleic 49.31 – 64.70a; 56.51 – 65.65b 36.2 – 66.5

18:3 Linolenic 0.89 – 1.56a; 1.03 – 1.38b 0.57 – 2.25

20:0 Arachidic 0.30 – 0.49a; 0.30 – 0.41b 0.279 – 0.965

20:1 Eicosenoic 0.17 – 0.29a; 0.17 – 0.27b 0.170 – 1.917

22:0 Behenic 0.069 – 0.28a; 0.059 – 0.18b 0.110 – 0.349

Minerals

Calcium (% dw) 0.0036 – 0.0068a; 0.0035 – 0.0070b 0.00127 – 0.02084

Copper (mg/kg dw) 1.14 – 3.43a; 1.39 – 2.76b 0.73 – 18.50

Iron (mg/kg dw) 14.17 – 23.40a; 15.90 – 24.66b 10.42 – 49.07

Magnesium (% dw) 0.091 – 0.14a; 0.10 – 0.14b 0.0594 – 0.194

Manganese (mg/kg dw) 4.83 – 8.34a; 4.78 – 9.35b 1.69 – 14.30

Phosphorous (% dw) 0.24 – 0.37a; 0.27 – 0.38b 0.147 – 0.533

Potassium (% dw) 0.29 – 0.39a; 0.36 – 0.43b 0.181 – 0.603

Zinc (mg/kg dw) 16.78 – 28.17a; 18.25 – 30.44b 6.5 – 37.2
Monsanto Company CR240-13U1 138 of 374

Table VII-8 (continued). Literature and ILSI Database Ranges for Components in 

Maize Forage and Grain

Grain Tissue Components1 Literature Range2 ILSI Range3

Vitamins (mg/kg dw)

Folic acid 0.19 – 0.35a; 0.23 – 0.42b 0.147 – 1.464

Vitamin A [β–Carotene] 122 – 4740c 0.19 – 46.81

Vitamin B1 [Thiamine] 2.33 – 4.17a; 2.71 – 4.33b 1.26 – 40.00

Vitamin B2 [Riboflavin] 0.94 – 2.42a; 1.64 – 2.81b 0.50 – 2.36

Vitamin B3 [Niacin] 15.07 – 32.38a; 13.64 – 42.06b 10.37 – 46.94

Vitamin B6 [Pyridoxine] 4.93 – 7.53a; 4.97 – 8.27b 3.68 – 11.32

Vitamin E [α–Tocopherol] 5.96 – 18.44a

; 2.84 – 15.53b 1.537 – 68.672

Grain Anti–Nutrients (% dw)

Phytic acid 0.69 – 1.09a; 0.60 – 0.94b 0.111 – 1.570

Raffinose 0.079 – 0.22a; 0.061 – 0.15b 0.020 – 0.320

Ferulic acid

Grain Secondary Metabolites (μg/g dw)

1205.75 – 2873.05a; 1011.40 – 2539.86b 291.9 – 3885.8

p–Coumaric acid 94.77 – 327.39a; 66.48 – 259.68b 53.4 – 576.2

Forage Tissue Components1 Literature Range2 ILSI Range3

Forage Nutrients

Proximates (% dw)

Ash 2.67 – 8.01a; 4.59 – 6.90b 1.527 – 9.638

Carbohydrates by calculation 81.88 – 89.26a; 84.11 – 87.54b 76.4 – 92.1

Fat, total 1.28 – 3.62a; 0.20 – 1.76b 0.296 – 4.570

Protein 5.80 – 10.24a; 5.56 – 9.14b 3.14 – 11.57

Fiber (% dw)

Acid detergent fiber 19.11 – 30.49a; 20.73 – 33.39b 16.13 – 47.39

Neutral detergent fiber 27.73 – 49.62a; 31.81 – 50.61b 20.29 – 63.71

Minerals (% dw)

Calcium 0.12 – 0.33a; 0.21 – 0.41b 0.07139 – 0.57679

Phosphorous 0.090 – 0.26a; 0.13 – 0.21b 0.09362 – 0.37041

1

dw=dry weight; FA = fatty acids. 2

Literature range references: aUS and bChile (Harrigan et al. 2009), c(Safawo et al. 2010). 3

ILSI range is from ILSI Crop Composition Database, 2011 [Accessed 9 January 2013] (ILSI 2011).
Monsanto Company CR240-13U1 139 of 374

VII.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 are 

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 data 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 conventional 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 levels 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

VIII. 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 support a conclusion that MON 87411 is not meaningfully different 

from the conventional control with the exception of the insect-protected and glyphosatetolerant 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 potential. 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 stressors, diseases, or arthropods; 

or c) characteristics that would confer a plant pest risk compared to the conventional 

control. 

VIII.A. Characteristics Measured for Assessment

In the phenotypic, agronomic, and environmental interactions assessment of 

MON 87411, data were collected to evaluate altered plant pest potential. A detailed 

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 article 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 environmental 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 familiarity (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., MPA640B) that had a genetic background similar 

to MON 87411 but did not possess insect-protected or glyphosate-tolerance traits. In 
Monsanto Company CR240-13U1 141 of 374

addition, multiple commercial conventional maize reference hybrids developed through 

conventional breeding and selection (see 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 characteristics from the commercial reference hybrids provide context for 

interpreting experimental results.
Monsanto Company CR240-13U1 142 of 374

Table VIII-1. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics Evaluated in U.S. Field Trials and Laboratory 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 test2)

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 test2)

Early stand count 

(VIII.C.2.1)

V2 – V5 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 – V4 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 plot
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Table VIII-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)
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Table VIII-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 

cm2)

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). 

VIII.B. Interpretation of Phenotypic and Environmental Interaction Data

Plant 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-characterized 

conventional crop whose biological properties and plant pest potential are well-known. 

Familiarity considers the biology of the crop, the introduced traits, the receiving 

environment and the interaction of these factors, and provides a basis for comparative 

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 responses 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 presented in Table- VIII-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 evidence 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. 
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VIII.B.1. Interpretation of Detected Differences 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 detected 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. Ultimately, 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. Schematic 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.
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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 combinedsite analysis are considered not biologically meaningful in terms of plant pest/weed 

potential and, therefore, are not further considered in subsequent 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 combined-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-derived 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 values 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 into any populations growing outside of 

cultivated environments or into a sexually-compatible species.

VIII.B.2. Interpretation of Environmental Interactions Data

For the qualitative assessments of abiotic stress response, disease damage, and arthropod 

damage, 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 biological 

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.
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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 

controls 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 potential. Differences that are not 

consistently detected in multiple environments are considered not biologically 

meaningful in terms of plant pest potential.

VIII.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 interactions description of MON 87411. The 

characteristics for MON 87411 evaluated in these assessments included: seed 

germination and dormancy characteristics (Section VIII.C.1), plant phenotypic, 

agronomic, and environmental interaction observations under field conditions (Section 

VIII.C.2), and pollen characteristics (Section VIII.C.3). Additional details for each 

assessment are provided in Appendices G, H, and I. 

VIII.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 considered 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, standardized 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 and dormancy characteristics of maize 

seed (AOSA 2012b; a). Additional temperature regimes were also evaluated.

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 F2 grain) of MON 87411, the conventional control, and the 

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 production 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. 
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Descriptions of the evaluated germination and dormancy characteristics and the timing of 

the evaluations for all temperature regimes are listed in Table VIII-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 differences (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 VIII-2). 

In addition, no hard seed were observed 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 germination 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. 
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Table VIII-2. Germination Characteristics of MON 87411 and the Conventional 

Control

Temperature Assessment Mean % (S.E.)1

Reference Range2

(°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.
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VIII.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 

detailed 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 

characteristics 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 

stress, 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 characteristics demonstrated 

that the introduction of the glyphosate tolerant and insect protected traits 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 traits is not expected to result in increased plant pest/weed potential for 

MON 87411 compared to conventional maize.

VIII.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 

VIII-3). These 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 site 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). The 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 VIII-1. The materials, methods, details concerning the timing of 
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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 assessed characteristics, including early stand count, 50% pollen 

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 moisture, test weight, and 

yield (Table VIII-4). Thus, the phenotypic and agronomic characteristics of MON 87411

were not altered in terms of pest/weed potential compared to conventional maize (Figure 

VIII-1, step 2, answer “no”).

Plant vigor data were summarized as ranges within individual 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 across sites (Appendix I, Table I4).

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 weediness 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 production. 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.
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Table VIII-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, Pennsylvania
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Table VIII-4. Combined-Site Comparison of MON 87411 to Conventional Control 

for Phenotypic and Agronomic Characteristics During 2012

Mean (S.E.)1 Reference 

Range2 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 differences 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 replication 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.
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VIII.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 conducted 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 (Tables VIII-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 analysis (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 responses

to abiotic stress, disease damage, arthropod-related damage, and pest- and beneficialarthropod 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 commercial maize.

VIII.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 these 

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 increasing 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 sites. 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 times and sites are 

reported. 

Descriptions of the evaluated environmental interactions characteristics and the timing of 

the evaluations are listed in Table VIII-1. The materials, methods, additional details 

concerning the qualitative environmental interactions 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 VIII-5 and Appendix I; 

Table I-5). Additionally, no differences in the range of responses were observed between 
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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 VIII-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 VIII-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 VIII.B.2.).
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Table VIII-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 87411 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. 

VIII.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 and ECB at the four sites in 2012. Both individual 

and combined-site (the four site pooled data) analyses were conducted. Descriptions of 

the evaluated environmental interactions characteristics and the timing of the evaluations 

are listed in Table VIII-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 analysis are summarized below. 

In the combined-site analysis, no statistically 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 VIII-6). Considering this lack of 

significant differences, the results indicate no changes in susceptibility or resistance of 

MON 87411 to these common maize pests. (See Figure VIII-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 occurred 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 sites1

Abiotic stressors 100 100

Disease damage 119 119

Arthropod-related damage 102 102

Total 321 321
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analyses were conducted 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 (Tables I-9 and I-10). The results of these analyses are summarized below 

and in Table VIII-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 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. Lack of 

sufficient 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 VIII-7 and Appendix I; Table I-9). The 

abundance of aphids 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 planthoppers 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 conventional 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. 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 VIII-7 and Appendix I; Table I-9). Thus, these differences in 

aphid, delphacid planthopper, 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 VIII.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 arthropods: 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 MON 87411 and the conventional 

control for 152 additional comparisons; however, the descriptive statistics were provided 

for these comparisons (Appendix I, Table I-10).
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No statistically significant differences (α=0.05) were detected between MON 87411 and 

the 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 

VIII-7 and Appendix I; Table I-10). The abundance of ant-like flower beetles 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 VIII-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 the trait and 

is not considered biologically meaningful in terms of increased pest potential 

of MON 87411 compared to the conventional control (See Section VIII.B.2.).
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Table VIII-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 (cm2) 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 control 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 sites (IABG, 

NCBD, NEYO, and PAHM).
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Table VIII-7. Summary of Arthropod Abundance Assessments and Detected Differences during 2012

Summary of Statistical Comparisons1 Summary of Detected Differences2

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 152 comparisons (visual counts); 

however, descriptive statistics were provided for these 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 

VIII.B.2 . 
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VIII.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 

characterization 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 morphology 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 the flowering stage, pollen was collected from three nonsystematically 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 phenotypic 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. 
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Table VIII-8. Pollen Characteristics of MON 87411 Compared to the Conventional 

Control during 2012 

Pollen 

Characteristic

(unit)

Mean (S.E.)1 

Reference Range2

MON 87411 Control

Viability3 (%) 98.7 (0.43) 98.9 (0.21) 99.0 – 99.2

Diameter4 (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 

hybrids.

3

Evaluated from three sub-samples per replication at 100X magnification. 4

Evaluated from 10 representative viable pollen grains per replication at 200X magnification.

VIII.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, 

characteristics 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 and 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. These 

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 abiotic 

stress and interactions with diseases and arthropods. Within these data categories, data 

relevant to understanding specific characteristics associated with weediness were also 

assessed to determine whether there was a potential increase in weediness of MON 87411

compared to conventional maize. 
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Results from these assessments comparing MON 87411 and the conventional control 

demonstrate that MON 87411 does not possess: 1) increased 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 glyphosatetolerant traits and is not expected to pose a plant pest risk compared to conventional 

maize. 
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IX. U.S. AGRONOMIC PRACTICES

IX.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 assess 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 rotational 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 87411 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. 

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

IX.B.1. Maize Production

The U.S., China, Brazil, Argentina, and Mexico are the top five countries producing 

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 harvested 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 bushels 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).
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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: (USDA-NASS 2013d)

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

Source: (USDA-NASS 2012)
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IX.C. Production 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). Herbicide 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). 

Given the widespread use of these HT and insect-protected maize hybrids, Monsanto 

anticipates 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. 

IX.D. Management of Insect Pests

Maize is subject to attack by numerous insects and global production losses 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 arrive 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 lists the most important insect pests in maize 

in the North Central states of the U.S. 
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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 use 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 white grubs, wireworms, seed corn maggots, cinch bugs, black cutworm, and 

billbugs (Bradley et al. 2013; University of Tennessee 2013). Applications of insecticide 

for control 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%), chlorpyrifos 

(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 (Table 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 pests, and removal of 

crop residues that can harbor both arthropod and disease organisms (Vasileiadis, et al. 
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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

LambdaCyhalothrin

Pyrethroids 2.0 24

Permethrin Pyrethroids 1.0 72

Tefluthrin Pyrethroids 3.0 242

Z-Cypermethrin Pyrethroids 2

Chlorpyrifos Organophosphates

Acetylcholinesterase 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: Diabrotica 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 tunneling 
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disrupting the transport of nutrients and water from the root system, 2) lack of root 

support causing gooseneck lodging, which can complicate harvesting, 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 larvae 

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 tissue 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 

including 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 adopted 

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 soybean (Levine and Oloumi-Sadeghi 1996). The SBV has 

been reported to occur 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 winters 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 rootwormprotected Bt maize products (both single and dual MOA) available to growers have 

provided and continue to provide excellent control of corn rootworm-complex 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 containing 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). 
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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 

developed 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 2012

Millions of Acres

Year
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IX.E. Management of Diseases and Other Pests

Management 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 are 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 many 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). Other than use as part of a seed 

treatments, fungicides were applied on just 8% of U.S. maize acreage in 2010 (USDANASS 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.

IX.F. Weed Management

Weed control in maize is essential to optimizing yield because weeds compete with 

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 

period 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.
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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-Proso

Panicum, Fall

Sandburs

Shattercane

Perennials

Bindweed, Field

Dogbane, Hemp

Johnsongrass

Muhly, Wire stem

Nutsedge, Yellow

Quackgrass

Thistle, Canada

Infestation Area11

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

Adapted from (Gianessi et al. 2002)

 

11 Area Abbreviations: 

PA: Pennsylvania OH: Ohio US: United States 

MD: Maryland CA: California 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: Idaho
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Until the early 1950s, tillage 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 planted maize acreage (USDA-NASS 2013b). Currently, 

glyphosate is the most widely 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 glyphosate 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 early 

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 programs. 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). 

IX.G. Crop Rotation 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 the rotation, and 5) manage excess nutrients (Singer 

and Bauer 2009). Studies in U.S. corn belt states indicate maize yield is about 10-15% 

higher in maize grown following soybean than maize grown following maize (Singer and 

Bauer 2009). Despite the many benefits of crop rotations, crop price fluctuations, 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). 
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Introduction of MON 87411 is not, however, expected to impact crop rotation practices 

any more so than current CRW-protected and HT products available to growers.

IX.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 rare 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 occur 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 regarding 

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 practices 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 production to significantly reduce the potential for volunteer growth in 

rotational crops. If volunteer maize does occur in subsequent crops, pre-plant tillage, incrop cultivation and the use of selective herbicides are very effective management tools. 

Introduction 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. 

IX.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 stewardship 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 quality 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 monitor 
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other countries that are key importers 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 practices 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 maize 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 CRWcomplex 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 expected to have major impacts 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 management 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 been 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 cultivated 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, and increased worker safety. 
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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, thereby 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 product: (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 

preceding 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 risk 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 for 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. Using 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. Characteristics of the Genetic Insert and Expressed Products

X.A.1. Genetic Insert

As described in Section III, MON 87411 was developed by Agrobacterium tumefaciensmediated 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 other 

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 maize. 

This analysis determined that no major DNA rearrangement occurred at the insertion site 

in MON 87411 upon DNA integration.
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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 expression. 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, leading 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 significant 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. 

Characterization 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). 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 (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 representative pollinators (honeybee), a 

detritivore (earthworm) and insect predators/parasitoids (ladybird beetle, carabid 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 from 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 determined 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 small 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 established histories of safe use, having been assessed by USDA, FDA 

and U.S. EPA on multiple occasions (Section V.E.). Neither protein originates from an 
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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 support 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 levels 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 eukaryotes, 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 for 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 these 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 Characteristics 

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 conventional control 

and several reference hybrids for which there is an established history of safe 

consumption. Nutrients assessed in this analysis included proximates, carbohydrates by 

calculation, acid detergent fiber, neutral detergent fiber, total dietary fiber, 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 pcoumaric acid. Forage samples were assessed for levels of proximates, 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 

differences (at 5%) between MON 87411 and the conventional control. The biological 

significance of differences from the data was 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 values 
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for the conventional control, 3) comparison of the mean differences between MON 87411 

and the conventional control to variation 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 variation 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 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 statistically 

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) showed significant 

differences between MON 87411 and conventional control. For these 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% tolerance 

intervals, values observed 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 nutritional 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 introduction of the insect-protection and glyphosate tolerance traits altered the plant 

pest potential 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 assessments 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 from 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 conventional control. Taken together, the results of the analysis support a 

determination that MON 87411 is no more likely to pose a plant pest risk than 

conventional maize.

X.F. Weediness Potential of MON 87411

Maize is not listed as a weed in the major weed references (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, traits 
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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 transportation 

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 surviving as a weed 

(Galinat 1988; Keeler 1989). 

In comparative 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. 

Results 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 

control and their response to abiotic 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 is 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 assessment, 

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 conventional 

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 significant 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 were 

not altered in terms of pest/weed potential compared to conventional maize.
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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 statistically significant differences were detected between 

MON 87411 and the conventional control 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 that 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 Sections 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 the NTO section (Section VI.E.) 

was on DvSnf7 RNA. 

The NTO assessment for MON 87411 has taken into consideration the MOAs, 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, analyses and 

assessments presented support a conclusion that MON 87411 will not negatively impact 

NTOs, including those beneficial to agriculture, and does not present an increased plant 

pest risk compared to conventional maize. 

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

Threatened and endangered species risk assessments were previously conducted by 

USDA (USDA-APHIS 2013) and EPA (U.S. EPA 2010) for Cry3Bb1-containing maize 

products MON 863 and MON 88017, and indicated 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), endangered 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 as many of 

the endangered and threatened coleopteran species occur in caves or aquatic habitats

(U.S. EPA 2010). 
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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 species 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 912

USFWS 2013b

states, 

including Arkansas, Kansas, Massachusetts, Nebraska, Ohio, Oklahoma, Rhode Island, 

South Dakota, and Texas ( ). Adults feed on carrion and occasionally 

other insects (USFWS 1991), while larvae feed exclusively on buried carrion or carrion 

regurgitations provided by their parents (U.S. EPA 2010; USFWS 2008)). The American 

burying beetle’s habitat is variable and includes 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 feeding ecology. 

There are several threatened and endangered coleopteran species that occur in aquatic 

habitats. As discussed above, however, exposure of aquatic organisms to biotechnologyderived 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 87411 will have no effect 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, MON 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 aboveground plant residues or pollen reaching the 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 the 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 acid (DNA or RNA) from biotechnologyderived crops should consider the amount of nucleic acids already in the environment 

from 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 biotechnologyderived plants will persist in soil or interact differently than nucleic acids from nonengineered 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 engineering, showed rapid dissipation of the target 

DNA (Widmer, et al. 1996). The study showed that after the rNPT-II DNA was 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 proportion 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. Potential for Pollen Mediated 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, overlap 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 gene 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 greater 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 method, 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 breeding, 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 notification 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 Literature 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 10
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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 

10 0.02 Stort 1974)

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 

40 0.03 Vayda 2001)

350 0
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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 

3 18-22 Tabashnik 2004)

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 0-0.1
Monsanto 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-sterile hybrid. Two 

year/three locations study. 

USA (Stevens, et al. 

300 0.02 2004)

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. 

60-62 0.01-0.2 2005)

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. 

9.4 1.2-2.5 2006)

20.6 1.0-2.2

35.8 0.5-2.3

200 0.6-1.4
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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 crosspollination 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 were 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 0.1-0.2
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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. 

2 34.9 2007)

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 0.24
Monsanto 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 crosspollination 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 

pollinations were mostly single cross-pollinations on the 

ear.

Switzerland (Bannert and 

85 0.015 Stamp 2007)

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. 

10 6.3 2007)

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. 

12 11.7 2008)

12 3.8
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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 and 

capable of transferring pollen between the two parents.

Maize and annual teosinte (Zea mays subsp. mexicana), are genetically compatible, windpollinated 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 crosses 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). Differences 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, which 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, Tripsacum 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 unlikely 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 produced 
Monsanto 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 exceedingly small. The 

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 that 

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, Genuity VT 

Triple PRO, etc). Herbicide 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 on 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 those 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 to other commercially cultivated maize. Based on this 

assessment, the introduction of MON 87411 is not likely to impact current U.S. maize

agronomic or cultivation practices 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 deregulated maize products, as well as with conventional maize, creating new 

improved hybrids. APHIS has determined that none of the individual biotechnologyderived maize products it has previously deregulated displays increased plant pest 

characteristics. APHIS has also concluded that progeny derived from crosses of these 

deregulated maize products with conventional or previously deregulated 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 new 

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 maize product across multiple generations; 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) combinedtrait 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 unrelated 

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. Summary of Plant Pest Assessments

A plant 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 fungus; (F) a virus or viroid; (G) an infectious agent or other pathogen, 

or (H) any 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 IV

through VIII 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 conventional 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 cassette; 2) characterization 

and safety of the expressed products; 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 management 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 expected 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 crosses between MON 87411 and other 

commercial maize be granted nonregulated status under 7 CFR part 340. 
Monsanto Company CR240-13U1 196 of 374

XI. ADVERSE 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 proteins produced in MON 87411 are identical to the same proteins

present in MON 88017 that was previously granted a determination of nonregulated 

status 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 differences 

between MON 87411 and conventional maize are protection from CRW larval feeding 

and glyphosate tolerance.

The data and information presented in this petition 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 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. 

Analysis of key nutrients, anti-nutrients, and secondary metabolites of MON 87411 

demonstrate that MON 87411 is compositionally equivalent to conventional maize. The 

phenotypic evaluations, including an assessment of seed germination and dormancy 

characteristics, plant growth and development characteristics, pollen characteristics, 

ecological interaction characteristics, and environmental interactions 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 threatened or endangered species. Therefore, based on the lack of increased 

pest potential compared to conventional maize, the risks for humans, animals, and other 

NTOs from MON 87411 are negligible.

The introduction of MON 87411 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 Triple Pro or Genuity 

SmartStax) currently available will be advised to continue to employ the same crop 

rotational practices, weed control practices and/or volunteer control measures currently in 

place for these products.
Monsanto Company CR240-13U1 197 of 374

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Zhang, Y., B.E. Wiggins, C. Lawrence, J. Petrick, S. Ivashuta and G. Heck. 2012b. Analysis of plantderived miRNAs in animal small RNA datasets. BMC Genomics 13:381.

Zhou, J., G.G. Harrigan, K.H. Berman, E.G. Webb, T.H. Klusmeyer and M.A. Nemeth. 2011. Stability in 

the composition equivalence of grain from insect-protected maize and seed from glyphosate-tolerant 

soybean to conventional counterparts over multiple seasons, locations, and breeding germplasms. Journal 

of Agricultural and Food Chemistry 59:8822-8828.
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APPENDICES
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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 for 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. 
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Table A-1. 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

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) Submitted 
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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 Progress
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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 Progress
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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

R4 11308817

R4F1 11320173

R5 11326313

R5F1 11333176

R6 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 R4, R5, R6 11264747

LH244 × HCL645 R4F1 (hybrid) 11320031

LH244 × LH287 R5F1 (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 prior 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 molecular 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. 
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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 was 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 with 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) HCl 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 hexadecyltrimethylammonium 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° for 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 samples, 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.
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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 instructions.

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 Bioanalyzer 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 instructions, 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 Prep 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 ends 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 beads (Beckman Coulter, Inc., Brea, CA) and 

resuspended in 17.5 μl of Illumina Resuspension Buffer. 
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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 fragments. 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 Ligase 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 selection.

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 thoroughly 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 DNA 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 procedure 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 were 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 described 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 sequence 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 TGAC 

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) in 
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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 quality 

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 were 

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 sequence 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 

(Kovalic 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 quality 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 quality 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 custom 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 aligned 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 to 
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the junction position. The remaining bases of these reads were sorted to show the 

alignment and the consensus 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 from 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 to 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) and 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 PCR 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 

adjacent 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 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). 

The PCR analyses for Product B, Product C and Product F were each conducted using 

50 ng of genomic DNA template in a 50 μl reaction volume. The reaction contained a 

final concentration of 0.2 μM 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 

template 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).
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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 under 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 the 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 visualized 

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 instructions 

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 for 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 

reactions 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 

primers 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 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). 

The amplification was performed under the following cycling conditions: 1 cycle at 98°C 
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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 was 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 ExoSAP IT (Affymetrix, Santa Clara, CA). The purified 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 from 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. 
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B.2 Supplementary Results 

Junction Sequence Class A:

R4

Plasmid DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGT

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

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT
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ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTGAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGGGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC
Monsanto Company CR240-13U1 228 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGACTTTTTATTTTCCTTTTAATAAATAATCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC

R4F1

Plasmid DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT
Monsanto Company CR240-13U1 229 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

TTCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGTCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGACTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCTTGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT
Monsanto Company CR240-13U1 230 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTCTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

R5

Plasmid DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACT

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^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTA
Monsanto Company CR240-13U1 231 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

AGCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTGTTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT
Monsanto Company CR240-13U1 232 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTATATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGTCATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

R5F1

Plasmid DNA sequence ^Flank

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA
Monsanto Company CR240-13U1 233 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCGTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCAC
Monsanto Company CR240-13U1 234 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

R6

Plasmid 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

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGT
Monsanto Company CR240-13U1 235 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTCGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATN

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT
Monsanto Company CR240-13U1 236 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGATTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCTATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTATATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTACTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCC

ATCCATGGAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACT
Monsanto Company CR240-13U1 237 of 374

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAAT

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATG

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGA

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAAC

ATCCATGTAGATTTCCCGGACATGAAGCCA^CTTAACTATTCATTAGTGTTTTGCCTTTTTATTTTCCTTTTAATAAATAATCCATCACTTTAAATGAACC

Junction Sequence Class B:

R4

Plasmid DNA sequence ^Flank

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC
Monsanto Company CR240-13U1 238 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGCGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT
Monsanto Company CR240-13U1 239 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGGATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTT
Monsanto Company CR240-13U1 240 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R4F1

Plasmid DNA sequence ^Flank

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTACACTATCAGTGTTT^AGAGAATCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAGCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATTACAAACCTCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTA

AGAGCGGCTGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT
Monsanto Company CR240-13U1 241 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGCACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R5

Plasmid DNA sequence ^Flank

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCA
Monsanto Company CR240-13U1 242 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAACTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT
Monsanto Company CR240-13U1 243 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACT

AGAGCGGCCGCGTTTAAACTATCAGGGTTT^AGAGAATCACAAACCTCTAGATGAATTAATCTACCCTAGAACTAGTTCACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG
Monsanto Company CR240-13U1 244 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

R5F1

Plasmid DNA sequence ^Flank

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACA
Monsanto Company CR240-13U1 245 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTC

AGAGCGGCCGTGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTAT

AGACCGGCCGCGTTTAAACTACCAGTGTTT^AGAGAATCACAACCCTCTAGATGTATAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCNN

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT

R6

Plasmid DNA sequence ^Flank
Monsanto Company CR240-13U1 246 of 374

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCGCAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGAT

AGCGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCCCAAACCTCTAGATGTATTA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCCAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTAC
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AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGGGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCAC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG
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AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTG

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCTTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCAT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATA

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTC

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTATTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT

AGAGCGGCCGCGTTTAAACTATCAGTGTTT^AGAGAATCACAAACCTCTAGATGTACTAATCTACCCTAGAACTAGTTCACTTTTGTGTGCATACTTTTCT
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Figure B-1. Junction sequences of MON 87411 detected by NGS/JSA

Trimmed nucleotide alignment of the detected junction sequences. 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 to 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 sequence.

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 improved through modern biotechnology. The Plant 

Genome Journal 5:149-163.
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Appendix C: Materials, Methods and Results for Characterization of Cry3Bb1 

and CP4 EPSPS Proteins 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 Cry3Bb1 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 extract 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, Piscataway, NJ) anion exchange column that was preequilibrated 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 Phenyl 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 Benzamidine 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 rate 
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through a PBS equilibrated immunoaffinity column. The column consisted of Protein L 

Agarose cross-linked to 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 and 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 final 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 Cry3Bb1

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. The blot was stained using Coomassie Blue R-250. The major bands at 77 

and ~65 kDa containing MON 87411-produced Cry3Bb1 were 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 acid (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-produced 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 

protein. 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 

vivo (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 performed 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 obtained 
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correspond to the deduced Cry3Bb1 protein beginning at amino acid position- 50 (Table

C-1). Hence, the sequence information confirms the identity of the ~65 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 the 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, arginine; 

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 Cry3Bb1 protein 

sample was separated by SDS-PAGE and the gel was stained using Brilliant Blue GColloidal 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 excised 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) acetonitrile, 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 Scientific 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 analyzed in the 300 to 5000 Da, the 500 to 5000 Da, and 500 

to 7000 Da range, respectively. CalMix 2TM was used as the external calibrant 

(SequazymeTM Peptide Mass Standards kit, AB SciEx, Foster City, CA). The analysis 

was performed on a VoyagerTM DE Pro BiospectrometryTM workstation (Applied 
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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 digest 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 confirmed by MALDITOF 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 used 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 part of the mass fingerprint analysis, a mass was identified that corresponded to the

predicted mass of an acetylated N-terminal tryptic fragment. The N-terminus of the ~77.0

kDa MON 87411-produced Cry3Bb1 protein begins with alanine at position two. 
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Table C-2. Summary of the Tryptic Masses Identified for the 

MON 87411-Produced Cry3Bb1 Using MALDI-TOF MS 

Observed Mass1 Expected Mass Diff.2 Fragment Sequence3

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 4Ac-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 GSTYDAWVK

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 abbreviation 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 sequence coverage (Figure C-1).
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001 MANPNNRSEH DTIKVTPNSE LQTNHNQYPL ADNPNSTLEE LNYKEFLRMT 

051 EDSSTEVLDN STVKDAVGTG ISVVGQILGV VGVPFAGALT SFYQSFLNTI 

101 WPSDADPWKA FMAQVEVLID KKIEEYAKSK ALAELQGLQN NFEDYVNALN

151 SWKKTPLSLR SKRSQDRIRE LFSQAESHFR NSMPSFAVSK FEVLFLPTYA

201 QAANTHLLLL KDAQVFGEEW GYSSEDVAEF YRRQLKLTQQ YTDHCVNWYN

251 VGLNGLRGST YDAWVKFNRF RREMTLTVLD LIVLFPFYDI RLYSKGVKTE

301 LTRDIFTDPI FLLTTLQKYG PTFLSIENSI RKPHLFDYLQ GIEFHTRLRP

351 GYFGKDSFNY WSGNYVETRP SIGSSKTITS PFYGDKSTEP VQKLSFDGQK

401 VYRTIANTDV AAWPNGKVYL GVTKVDFSQY DDQKNETSTQ TYDSKRNNGH

451 VSAQDSIDQL PPETTDEPLE KAYSHQLNYA ECFLMQDRRG TIPFFTWTHR

501 SVDFFNTIDA EKITQLPVVK AYALSSGASI IEGPGFTGGN LLFLKESSNS 

551 IAKFKVTLNS AALLQRYRVR IRYASTTNLR LFVQNSNNDF LVIYINKTMN 

601 KDDDLTYQTF DLATTNSNMG FSGDKNELII 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-produced Cry3Bb1 protein was deduced from the 

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) sodium dodecyl sulfate (SDS), 0.005% (w/v) bromophenol 

blue, 10% (v/v) glycerol, pH 6.8) and heated to 100°C for 3 minutes. 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 (Precision 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-Cry3Bb1 
Monsanto 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 were 

detected using the ECLTM 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 bands 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 position 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 intensity 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 (~77-55 kDa). The signal intensity (reported in 

OD × mm2

) of the immunoreactive bands identified by anti-Cry3Bb1 antibodies and 

migrating between the full-length protein at 77.0 kDa and the insecticidal tryptic core (at 

~55 kDa) were included in the mean signal intensities (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 determined to have 

equivalent immunoreactivity.
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Figure C-2. Western Blot Analysis of MON 87411- and E. coli -produced 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 shown 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 15
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Table C-3. Comparison of Immunoreactive Signal Between MON 87411- and E

coli-produced Cry3Bb1Proteins

Mean Signal intensity from 

MON 87411 -produced Cry3Bb11

(OD x mm2)

Mean Signal intensity from 

E. coli-produced Cry3Bb11

(OD x mm2)

Acceptance limits2for 

MON 87411-produced 

Cry3Bb1(OD x mm2)

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 87411-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 Range 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 hours 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 hours in 25% (v/v) methanol. Analysis of the gel was performed using a Bio-Rad 

GS-800 densitometer supplied with Quantity One software (4.6.7). Apparent MW was

reported as an average of all six lanes containing the MON 87411-produced Cry3Bb1 

protein.

C.1.6.2 Results of Cry3Bb1 Protein Molecular Weight Equivalence

The intact MON 87411-produced Cry3Bb1 protein (Figure C-3, lanes 3-8) migrated to 

the same position on the gel as the E. coli-produced 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 limits for equivalence, the MON 87411- and E. coli-produced 

Cry3Bb1 proteins were determined to have equivalent apparent molecular weights.

Table C-4. Molecular Weight Comparison Between the MON 87411-and 

E. coli-produced Cry3Bb1 Proteins

Apparent MW of 

MON 87411-Produced 

Cry3Bb1Protein

(kDa)

Apparent MW of 

E. coli-Produced Cry3Bb1 

Protein1

(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 11309151
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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 10
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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 Standards 

(Precision Plus ProteinTM 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 to a PVDF 

membrane (Invitrogen). 

Glycosylation 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 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 were 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-produced 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 the 

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 expected 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 copurified with the E. coli-produced Cry3Bb1 protein (Choi-Rhee and Cronan 2003), 

because binds to the streptavidin-HRP conjugate and therefore is detectable by the ECL 

system.
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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 stained 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 indicate 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.
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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 ProteinTM Dual Color Standards. 

The 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 reagents 

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 ProteinTM 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.
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C.1.8 Cry3Bb1 Functional Activity Analysis

C.1.8.1 Methods

Insects. 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-1048. 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 of six dilutions yielding a dose 

series with a two-fold separation factor ranging from 0.078 – 2.5 μg Cry3Bb1 protein/ml

diet for the E. coli-produced and MON 87411-produced Cry3Bb1 and a single buffer 

control. The Cry3Bb1 protein dosing solutions were prepared by diluting the respective 

protein with purified water and incorporating the dilution into a Bio-Serv CPB agar-based 

insect diet (pre-mix #F9380B; Frenchtown, NJ). This dose series in diet was chosen to 

adequately 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 (# BAW128, Bio-Serv, Frenchtown, 

NJ). Individual insect larvae were placed on these diets using a fine paintbrush, with a 

target number of 24 insects per treatment. The infested wells were covered by a 

ventilated adhesive cover (# BACV16, Bio-Serv, Frenchtown, NJ) and the insects were 

allowed 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 

was recorded 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. coli-produced Cry3Bb1 

protein was determined Colorado potato beetle diet-incorporation bioassays. In this 

assay, activity is expressed as LC50, μg/ml diet. The MON 87411- and E. coli-produced 

Cry3Bb1 proteins were considered functionally equivalent if the LC50, 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 

LC50 of the MON 87411- and E. coli-produced Cry3Bb1 proteins were determined to be 

0.77 μg/ml diet and 0.67 μg/ml diet respectively (Table C-5). Because the LC50 of 

MON 87411-produced and E. coli-produced Cry3Bb1 proteins were within the 

acceptance limits (Table C-5), the proteins 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 Protein1

(LC50, μg/ml diet)

E. coli-produced Cry3Bb1 

Protein1

(LC50, μg/ml diet)

Preset Acceptance Limits 

for MON 87411-produced 

Cry3Bb1 Protein2

(LC50, 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-produced CP4 EPSPS protein (lot 11351676) 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 containing 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 from 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-produced 

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 procedures 

were documented on worksheets and, where applicable, SOPs were followed. Three 

CP4 EPSPS protein purification batches were prepared to generate a sufficient amount of 

MON 87411 produced 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 

chromatography, 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. 

Chromatography fractions containing CP4 EPSPS protein were identified by western blot 

analysis or SDS-PAGE stained gels. The final purified CP4 EPSPS protein product from 

all three batches was combined, concentrated and buffer-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 Monsanto Regulatory Archives.
Monsanto Company CR240-13U1 265 of 374

CP4 EPSPS protein Batch 1. Grain of MON 87411 was ground in the presence 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 for ~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 saturation 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, pH 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 dialysis against S30Q-A 

buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM benzamidine-HCl, and 4 mM 

DTT). The desalted sample was loaded onto a Source 30Q (GE Healthcare) column 

equilibrated with 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 volumes. Fractions 

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 gradient 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 exchanged 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 sample 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 fractionation 

steps as used for Batch 1, except that the initial amount 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 larger 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 Source 30Q step, starting with ~2 kg 
Monsanto Company CR240-13U1 266 of 374

of ground grain. Following Source 30Q fractionation, fractions containing CP4 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 

buffer (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 CPE 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 

concentrator 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 volumes. Fractions 

containing CP4 EPSPS were pooled and concentrated using an Amicon Ultracel -10K 

centrifugal concentration device. The pooled sample was buffer exchanged 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 with 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. This 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 chemistry, 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). The 
Monsanto Company CR240-13U1 267 of 374

major band at ~44 kDa containing the test protein was excised from the blot and was used 

for N-terminal sequence analysis. The analysis was performed for 15 cycles using 

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 ProciseTM 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 MON 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 sequence. 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 identifiable.

C2.4 MALDI-TOF Tryptic Mass Map Analysis of CP4 EPSPS

C2.4.1 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 

protein sample was 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 incubated in 100 μl of 10 mM 

DTT at 37 °C for 2 h to reduce protein disulfide bonds. The protein was then alkylated in 
Monsanto Company CR240-13U1 268 of 374

the dark for 20 min with 100 μl of 20 mM iodoacetic 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-Vac 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 extract (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 added to one of the spots. The samples in DHB matrix were analyzed in 

the 300 to 5000 Da range. Samples in α-Cyano and Sinapinic acid were analyzed in the 

500 to 5000 and 500 to 7000 Da range, respectively. CalMix 2TM was used as the 

external calibrant (Sequazyme

TM Peptide Mass Standards kit, AB SciEx). The analysis 

was performed on a VoyagerTM DE Pro BiospectrometryTM 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 within 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.

C2.4.2 Results of MALDI-TOF Tryptic Mass Map Analysis

The 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 produced 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% (306 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 Mass1 Expected Mass Difference2 Fragment Sequence3

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 SSGLSGTVR

872.45

872.46

872.45

872.52

0.00

0.06

313-320

358-366

GVTVPEDR*

LSAVANGLK*

930.49 930.51 0.02 169-177 VPMASAQVK

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

1359.63

1359.72

1359.64

0.05

0.01

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

1993.97 1993.97 0.00 206-224 MLQG...DGVR

2183.19 2183.17 0.02 275-294 TGLI...INPR

2367.33 2367.33 0.00 178-200 SAVL...IMTR

2450.24 2450.23

2450.22

0.01

0.02

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 residues 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 

included 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-Produced 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 EPSPS 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 

concentrations 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 (0.263 mg CP4 EPSPS/ml). Aliquots 

of each protein were subsequently diluted in water and 1× (LB), and heated at 95 °C for 

4 min. Samples 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). Prestained molecular weight markers (Precision Plus Protein Standards Dual Color; 

Bio-Rad) were loaded to verify electrotransfer of the proteins to the membrane and to 

estimate the size of the immunoreactive bands 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 1:10,000 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 DTKAA
Monsanto 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 bands 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 analysis 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 × mm2

) of the band of interest in lanes loaded with 

the MON 87411-produced and the E. coli-produced CP4 EPSPS protein was measured 

(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-Produced 

CP4 EPSPS Proteins

Aliquots of the MON 87411-produced 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 visualized 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. Lane 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 EPSPS1

(OD × mm2)

Mean Signal Intensity from 

E. coli-Produced CP4 EPSPS1

(OD × mm2

)

Acceptance Limits2 for 

MON 87411-Produced 

CP4 EPSPS 

(OD × mm2)

6.0 6.0 3.9–8.1

1

Each 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 Broad Range molecular weight standards, were diluted in water and

5× LB. The diluted samples 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 samples 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 acid for 30 min, and stained for 18 h with Brilliant Blue GColloidal 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 the 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 all six lanes containing the MON 87411-produced Cry3Bb1 protein.

C.2.6.2 Results of CP4EPSPS Protein Molecular 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 (Figure 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 weight was calculated to be 42.9 kDa (Table C-9). Because the 

experimentally 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 Protein1

(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

Lane # 1 2 3 4 5 6 7 8 9

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 ProteinTM 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). 

Glycosylation 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 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 were 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.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 

ECLTM Glycoprotein Detection Module (GE Healthcare, Piscataway, NJ). To assess 

equivalence of the MON 87411- and E. coli-produced CP4 EPSPS proteins, the 

E. coli-produced CP4 EPSPS protein, previously been shown to be free of glycosylation 

(Harrison et al. 1996) was also analyzed. 

A clear glycosylation signal was observed at the expected molecular weight in the lanes 

containing the 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 loadings 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 transferrin (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 ProteinTM Standards. The arrows show 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 reagents 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 ProteinTM 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 

(0.263 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 PEP 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) and 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 measured 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 protein (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 functional activities 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 reaction 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 minute at 25 °C. The MON 87411- and E. coli-produced 

CP4 EPSPS proteins were considered to have 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 proteins 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 preset acceptance 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 EPSPS Protein1

(U/mg)

E. coli-Produced 

CP4 EPSPS Protein1

(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 matrixassisted 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 is 

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. Sherman. 2000. Nα

-terminal acetylation of eukaryotic proteins. J 

Biol Chem 275: 36479-36482.

Rademacher, T.W., R.B. Parekh and R.A. Dwek. 1988. Glycobiology. Annual Review of 

Biochemistry 57: 785-838.
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Appendix D: Materials and Methods Used for the Analysis of the Levels of 

Cry3Bb1 and CP4 EPSPS Proteins 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 each 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, forage, 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 Salto, 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 Management 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.
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Table D-1. Cry3Bb1 Protein Extraction Methods1 for Tissue Samples 

Sample Type Tissue-to-Buffer 

Ratio

Extraction Buffer

Leaf2/Grain/Pollen/Silk/Root3/Forage4 1:100

50 mM sodium 

carbonate-bicarbonate 

+ 0.1% (w/v) BSA5

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 and 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 Na2CO3, 

 0.035 M NaHCO3, 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 Methods1 for Tissue Samples 

Sample Type Tissue-to-Buffer 

Ratio

Extraction Buffer

Leaf2/Pollen/Silk/Forage3 1:100 1× PBST + 0.1% (w/v) BSA5

Grain 1:100 1× TBA + 10 mM DCA6

Root4 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 filter. 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 KH2PO4, 

 0.01 M Na2HPO4, 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 Na2B4O7, 0.005 M MgCl2, 0.05% (v/v) Tween-20, 0.2% (w/v) L-ascorbic acid and 0.01 M DCA].
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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 KH2PO4, 0.01 M Na2HPO4, 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 monoclonal 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 spectrophotometric 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 NaH2PO4, 0.15 M NaCl, and 15 ppm Proclin 300). The detection 

reagent was goat anti-CP4 EPSPS antibody, otherwise known as anti-protein 4 (SigmaAldrich, 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 Na2CO3, 

0.035 M NaHCO3,) and immobilized onto 96-well microtiter plates at 5 μg/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 μl per well and incubated for 60 to 70 minutes at 37 °C. Biotinylated goat 

anti-Cry3Bb1 antibodies were added at 100 μl per well and incubated for 

60 to 70 minutes at 37 °C. NeutraAvidin-horseradish peroxidase conjugate was added at 

100 μl per well and incubated for 30 to 35 minutes at 37 °C. Plates were developed 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 H3PO4. Quantification of the Cry3Bb1 protein 

was accomplished by interpolation from a Cry3Bb1 protein standard curve that ranged 

from 0.35 - 11.2 ng/ml. 
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D.6.2. CP4 EPSPS Protein

The CP4 EPSPS ELISA was performed using plates commercially coated at 2 μg/ml 

(Agri Diagnostic Manufacturing, Rickreall, OR). CP4 EPSPS protein standard or sample 

extract was added at 100 μl 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 horseradish peroxidase was added at 100 μl 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 H3PO4. 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 Moisture 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 triplicate 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 levels 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 the 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 fourparameter 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 that 

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 to 
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“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 substance 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. 
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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 vitroproduced 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 isopropanol 

and resuspended in UltraPure Water (Invitrogen). The in vitro transcribed DvSnf7_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, leaf 

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 

manufacturer’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 using 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 30 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 determine the nucleotide sequences of MON 87411 DvSnf7 

RNA.
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The PCR amplification of Product A was performed on 1 μl of diluted MON 87411 

DvSnf7 cDNA in a 20 μl reaction volume. Each reaction contained a final concentration 

of 0.2 μM of each primer, 1 mM MgCl2, 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 μl reaction volume. Each reaction contained a final concentration 

of 0.2 μM of each primer, 1 mM MgCl2, 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 denaturation step at 98 °C for 2 min and a final 

extension cycle for 5 min at 68 °C. 

Aliquots of each PCR product were 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. 

All sequencing was performed by Monsanto TGAC (The Genome Analysis Center) using 

BigDyeTM terminator chemistry (Applied Biosystems, Foster City, CA). A consensus 

sequence was generated by compiling sequences from multiple sequencing reactions 

performed on the 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 cDNA 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 contains 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 sequence 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, 
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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 there 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 

equivalence 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 

DvSnf7_968 RNA. The dsRNA regions of MON 87411 DvSnf7 RNA and DvSnf7_968 

RNA were specifically targeted through RNase If treatment of the RNAs. RNase If 

preferentially digests single stranded RNA leaving behind double stranded regions such 

as the active region 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 If digestion facilitates a direct comparison of the size 

equivalence between the active 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 If (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 If digested MON 87411 poly (A) RNA and DvSnf7_968 RNA were resolved on a 

~6.6% (w/v) formaldehyde/2.5% agarose 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 photographed.

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 northern blot was hybridized with a [α32P]-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 gel 

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, Carlsbad, 

CA). Approximately 25 ng of probe template was labeled with [α32P]-dATP 
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(~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 

temperature. 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

Poly (A) RNA from MON 87411 plant tissues (MON 87411 poly (A) RNA) and 

DvSnf7_968 RNA were digested by RNase If which preferentially digests single stranded 

RNA leaving dsRNA regions intact. Each RNase If digested sample was loaded in 

triplicate and subjected to northern blot analyses. The northern 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 87411 DvSnf7 RNA and DvSnf7_968 RNA was conducted using this northern 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.
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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 illustrations 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 B Transcription

DvSnf7p DvSnf7p DvSnf7p DvSnf7p
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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 GGCTTCCCTCTGCATTTCAATAGTTGTAAGGGTTCCATCTATTTGTAGTTGGGTCTTTTC

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 181 GGCTTCCCTCTGCATTTCAATAGTTGTAAGGGTTCCATCTATTTGTAGTTGGGTCTTTTC

DvSnf7 87411 241 CAATCGTTTCTTCTTTTTGAGGGCTTGGAGTGCAACTCTTTTATTTTTCGACGCATTTTT

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

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 GATTAACTCCTCATCCACGCGGCCGCCTGCAGGAGCGCAAAGAAAAATGCGTCGAAAAAT

DvSnf7 87411 481 AAAAGAGTTGCACTCCAAGCCCTCAAAAAGAAGAAACGATTGGAAAAGACCCAACTACAA

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 481 AAAAGAGTTGCACTCCAAGCCCTCAAAAAGAAGAAACGATTGGAAAAGACCCAACTACAA

DvSnf7 87411 541 ATAGATGGAACCCTTACAACTATTGAAATGCAGAGGGAAGCCCTCGAAGGAGCTAGCACA

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 541 ATAGATGGAACCCTTACAACTATTGAAATGCAGAGGGAAGCCCTCGAAGGAGCTAGCACA

DvSnf7 87411 601 AATACTGCTGTATTAGATTCTATGAAAAATGCTGCAGATGCCCTTAAGAAAGCTCATAAG

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 601 AATACTGCTGTATTAGATTCTATGAAAAATGCTGCAGATGCCCTTAAGAAAGCTCATAAG

DvSnf7 87411 661 AATTTGAATGTAGATGATGTTCACGATATCATGGATTAGATCGCCAGCGGTACTCGCTGA

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 661 AATTTGAATGTAGATGATGTTCACGATATCATGGATTAGATCGCCAGCGGTACTCGCTGA

DvSnf7 87411 721 GGCCTAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGT

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 721 GGCCTAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGT

Figure E-2. Sequence Alignment between MON 87411 DvSnf7 cDNA and 

DvSnf7_968 cDNA
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DvSnf7 87411 781 TTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGT

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 781 TTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGT

DvSnf7 87411 841 TTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTAT

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 841 TTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTAT

DvSnf7 87411 901 CGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCA

 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

DvSnf7_968 901 CGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCA

DvSnf7 87411 961 TTCTCAAA 968

 ||||||||

DvSnf7_968 961 TTCTCAAA 968

Figure E-2 (continued). 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) sequence. The inverted repeat regions are underlined. The numbering indicates 

base position.
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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 If digested and lanes 4-6 contain 200 ng 

of MON 87411 poly (A) RNA also subjected to RNase If digestion. Lane designations are as 

follows:

Lane Sample

1 DvSnf7_968 RNA (RNase If digested)

2 DvSnf7_968 RNA (RNase If digested)

3 DvSnf7_968 RNA (RNase If digested)

4 MON 87411 poly (A) RNA (RNase If digested)

5 MON 87411 poly (A) RNA (RNase If digested)

6 MON 87411 poly (A) RNA (RNase If digested)

Arrows denote the size of the RNA, in bases, obtained from the Low Range Ribo Ruler ladder on 

the SYBR Gold stained gel.
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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 virgifera virgifera LeConte). 

PLoS ONE 7:e47534.

Coruzzi, G., R. Broglie, C. Edwards and N.-H. Chua. 1984. Tissue-specific and lightregulated 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 growing 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, senescent 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 Aires (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 

grain samples were collected from each replicated plot at all field sites. See Table VI1for 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 Company (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 using liquid nitrogen. The 

prepared tissue samples were stored in -80° C freezers before and after transferred on dry 

ice to the analytical facility. 
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Total RNA from leaf (OSL1 through OSL4), root (OSR1 through OSR4), whole plant 

(OSWP1 through OSWP4), forage 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 reagent (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 minutes. 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 tissues 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 to 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 FXP 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 analysis. The DvSnf7_968 concentration 

was 2.21 mg/ml as determined by spectrophotometric analysis.
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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 signal 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. Background 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 leaf 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 separate aliquot of the 

same conventional maize RNA to serve as a QC+ for each assay. 
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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 serve 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 denaturation 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 well. 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 signal 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) from 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 Company, 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 Conversion Factor (DWCF) was calculated as follows:

DWCF = 1 − �

Mean% TSSP Moisture

100 �
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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:

DvSnf7 Level in Dry Weight = DvSnf7 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 obtained 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 

μg/g fw value using the total RNA/tissue ratio and dilution factor. All the μg/g fw values 

were converted to μg/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 < LOQ. 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 washer. 

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.
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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 normal 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 reference 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 BASS
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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 four 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 located 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, ash, 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, phosphorus, 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 phosphorus 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 Laboratories, 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 free 
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furfurals by ultraviolet detection (Albala-Hurtado, et al., 1997). The limit of quantitation 

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 complexes 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 alanine • 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 

halogenation 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 20 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.
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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 

quantitation 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 analysis (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. Carbohydrate

The total carbohydrate level was calculated by difference using the fresh weight-derived 

data and the following equation (USDA, 1973):
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% 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 evaporated, 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%.
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References:

Manufacturer 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 

approximately 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 Q0G151
Monsanto 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 

amount 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:

Inorganic 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 the 
Monsanto Company CR240-13U1 307 of 374

filter 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 analytical HPLC system for quantification of pcoumaric 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 

BCBH8701V
Monsanto 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 

standardized 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 0.100%.

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-glucopyranoside 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, 1971). 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 starch 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 

determined 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)

The 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 

reacted 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 

0.010 mg/100g.

Reference Standard:

• USP, Thiamine Hydrochloride, 99.7%, Lot Number P0K366
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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 Hydrochloride)

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 

cerevisiae, 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 highperformance liquid chromatography using a silica column (Cort, et al., 1983; McMurray, 

et al., 1980; 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 containing 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):
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Table G-2. Re-expression Formulas for Statistical Analysis of Composition Data

Component From (X) To Formula1

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/(104

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 103X/d

Fatty Acids (FA) % fwt % Total FA

(100)Xj/ΣX, for 

each FAj where ΣX 

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 quantitation (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 caprylic 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 eicosadienoic 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 equal 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 the 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 removed 
Monsanto 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 component 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 compositional 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 observations 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

AACC. 1998. Approved methods of the American Association of Cereal Chemists. 

American 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 official method 961.15: Vitamin B6 (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 

Communities 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 food 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 Communities 

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. 2012n. AOAC official method 942.23: Thiamine (Vitamin B1) in human and pet 

foods. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012o. AOAC official method 953.17: Thiamine (Vitamin B1) 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 supplements 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 B1). Association of 

Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012s. AOAC official method 940.33: Riboflavin (Vitamin B2) 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 

Chemists' 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. Jensen. 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 trimethylsilyl 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 chromatographic 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 

Agriculture, 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. Ricker, 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, 

Infant 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., Crop 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.
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Mason, B.S. and H.T. Slover. 1971. A gas chromatographic method for the determination 

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 of Official Analytical Chemists 63: 1258-1261.

OECD. 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. Organisation 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. Schreurs. 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 No. 

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 sites: Jefferson County, IA (IARL), Warren County, IL (ILMN), 

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

Germination and dormancy 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;

2012b).

The seed lots (Selfed F2 grain) of MON 87411, the conventional control, and four 

reference hybrids from each location were tested under seven different temperature 

regimes. Seven germination 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 20/30° 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. 

Approximately 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 germination towels were then rolled up in a wax cover. All rolled germination 

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 

total 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 

replications where the whole-plot was the location where starting seed (Selfed F2 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 

temperature regime. 

A description of each germination characteristic evaluated and the timing of evaluations 

are presented in Table VIII-1. The types of data collected depended on the temperature 

regime. Each rolled germination towel in the AOSA-recommended 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 (AOSA, 2012a; 2012b). 

AOSA only provides guidelines for testing seed under optimal temperatures, whereas 

additional temperature regimes were included to test diverse environmental conditions. 

Therefore, each 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 affect 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 temperature 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 seed 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 Institute, Inc.

(SAS 2008) 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 dormancy characteristics of seed produced within each site (i.e., individual 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 firmswollen seed. The percent germinated seed were categorized as either normal germinated 
Monsanto Company CR240-13U1 318 of 374

or abnormal germinated for the AOSA temperature 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 VIII-2.

H.5. Individual-Site Seed Dormancy 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 F2 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 conventional control at 20°C (0.3% vs. 

0.0%) for the seed produced at the IARL site. MON 87411 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 the 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 germination 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-Protected 11354899 

IARL MPA640B1 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-Protected 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-Protected 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 × LH287
Monsanto Company CR240-13U1 320 of 374

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

 IARL1 ILMN1 KSLA1

 Mean % (S.E.)2 Mean % (S.E.)2 Mean % (S.E.)2

Temperature 

Regime Assessment Category MON 87411 Control MON 87411 Control3 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)
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Table H-2 (continued). Germination and Dormancy Characteristics of MON 87411 and the Conventional Control Seed 

(Selfed F2 Grain) Produced at each of the Three Field Sites

 IARL1 ILMN1 KSLA1

 Mean % (S.E.)2 Mean % (S.E.)2 Mean % (S.E.)2

Temperature 

Regime Assessment Category MON 87411 Control MON 87411 Control3 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. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.
Monsanto Company CR240-13U1 323 of 374

Appendix I: Materials, Methods, and Individual Site Results from Phenotypic,

Agronomic, and Environmental Interaction Assessment of 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 2012 at nine sites that provided a range of environmental 

and agronomic conditions representative of U.S. maize growing regions (Section VIII, 

Table VIII-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, each 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 

approximately 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 plots 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, 
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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 practices 

were applied as necessary. Maintenance 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 Code1 Material Name Monsanto Lot Number Phenotype

All MON 87411 11333176 Glyphosate-Tolerant and 

 Insect-Protected

All MPA640B2 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

MPA640B = LH244 × LH287.
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Table I-2. Field and Planting Information

Site1

Planting 

Date2

Harvest 

Date2

Approximate

Planting Rate 

(seeds/m)

Approximate 

Plot Size

(m × m)

Rows 

per Plot Soil Type % OM3

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 Vegetables4

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 = Pawnee 

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.
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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 interactions (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-Related

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 three abiotic 

stressors, three diseases, and three arthropod pests were evaluated four times during the 

following four crop developmental stages: V6–V8; V12-VT; 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 species that are actively causing damage for subsequent evaluation in the 

study plots. The Principal Investigators were requested to select additional stressors 

if present. 

3. If fewer than three abiotic stressors, diseases, or arthropod species were present, the 

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, diseases, 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, the Principal Investigator 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 selected 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/collections 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 cm2

). Damage (cm2) per plant was calculated as the total number of grid cells 

matching the damage area multiplied by 0.5 (each grid cell = 0.5 cm2

). 

European corn borer damage was evaluated 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.

Arthropods 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 parasitic 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 determine the most abundant arthropods to obtain a total of up 
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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 differences 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 data 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 Qualitative Data

I.10.1 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 not 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 consistency 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 

characteristics listed in Table VIII-1, with the exception of plant vigor. The level of 

statistical significance was predetermined to be 5 % (α = 0.05). Comparisons 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.10.2 Environmental Interaction Data

The environmental interaction data (i.e., plant response 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 overlap 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 (individual-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 conventional 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 and site was determined from the 

minimum and maximum mean values collected from the reference maize at the site. 

For the arthropod abundance data, statistical analyses and significance testing of 

differences between MON 87411 and the conventional control materials 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 (Table 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 phenotypic 

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 per 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 conventional 
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control at the NEYO 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 the 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 individualsite analysis for early stand count, days to 50% pollen shed, days to 50% silking, stay 

green, 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 VIII-1, Step 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, no differences were observed between MON 87411 and 

the conventional control for any of the 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., Rhizoctonia 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 mite, 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 ECB

among all observations at all four sites (Table I-8). CEW damage was higher for 

MON 87411 than the conventional control at the NCBD site (3.3 vs. 1.5 cm2 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 plant 
Monsanto 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 conventional 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 between 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 control for 104 out of 108 comparisons (Table I-9). The abundance of 

aphids 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 planthoppers 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 

spiders (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. 

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 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 conventional maize (See Section VIII.B.2.). 

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 

arthropods: 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 

(Table I-10). Lack of sufficient arthropod abundance precluded statistical comparisons 

between MON 87411 and the conventional control for 152 additional comparisons; 

however, 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 antlike flower beetles 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 per 
Monsanto 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 trait 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 Code1 Material Name Material Type Plots Characteristics Reason for Exclusion

IALL, ILCY, 

ILPH, and 

ILWY

All All All Phenotypic and environmental interactions

 data

Extreme 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 stressor 
Monsanto Company CR240-13U1 335 of 374

Table I-3 (continued). Data Missing or Excluded from Analysis

Site Code1 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 

Code1 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 (continued). 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 

Code1 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 (continued). 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 

Code1 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 (continued). 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 

Code1 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 (continued). 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 

Code1 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 statistically 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 = Pawnee 

County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, 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 Table 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 

Sites1

Number of Observations where No 

Differences were Observed between 

MON 87411 and the Conventional 

Control

Total 100 100

Cold 1 1

Drought2 22 22

Flood3 5 5

Frost 2 2

Hail 8 8

Heat 20 20

Nutrient deficiency 12 12

Soil compaction 3 3

Sunscald 5 5

Wind 22 22

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 Includes 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 Sites1

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 rot2 12 12

Eyespot 5 5

Fusarium sp. 8 8

Goss’s bacterial wilt 10 10

Gray leaf spot 20 20

Leaf blight3 16 16

Maize rough dwarf virus 2 2

Pythium sp. 2 2

Rhizoctonia sp. 2 2

Rust4 7 7

Seedling blight 2 2

Smut 8 8

Stalk rot5 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-systematically 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 Sites1

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

Includes 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 assessment1 Site2 MON 87411 Control

Corn earworm 

(H. zea)

3

Mean (S.E.) damage area 

of 10 plants per plot (cm2)

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 = Perquimans, 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 

range4

Mean (S.E.)3 Reference 

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 – 12.0
Monsanto Company CR240-13U1 346 of 374

Table I-9. (continued). 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 

range4

Mean (S.E.)3 Reference 

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 — — —
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Table I-9. (continued). 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 

range4

Mean (S.E.)3 Reference 

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 – 229.8
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Table I-9. (continued). 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 

range4

Mean (S.E.)3 Reference 

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 – 6.8
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Table I-9. (continued). 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 

Range4 Coll.1 Site2 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.10.2).

1

Arthropods were enumerated at five crop development stages: Collection 1 = late vegetative – VT growth stage; Collection 2 = R1 growth stage; 

Collection 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 sites); N= 

3 (PAHM site). 

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. 
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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 

range4

Mean (S.E.)3 Reference 

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 – 7.3
Monsanto Company CR240-13U1 352 of 374

Table I-10. (continued). 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 

range4

Mean (S.E.)3 Reference 

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 – 0.0
Monsanto Company CR240-13U1 353 of 374

Table I-10. (continued). 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 

range4

Mean (S.E.)3 Reference 

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 – 0.5
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Table I-10. (continued). 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 

range4

Mean (S.E.)3 Reference 

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 – 0.3
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Table I-10. (continued). 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

range4

Mean (S.E.)3 Reference 

range4 Coll.1 Site2 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 

= R2 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 in 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. 
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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.
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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 was 

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 pollen 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 collection, 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 shaken 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 suspended pollen/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 installed 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 microscope. 

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 VIII-8.

 

 Olympus Corporation.

 Windows XP is a registered trademark of Microsoft Corporation.
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J.3.2. Pollen Diameter

For a single predetermined subsample per plot, pollen 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. General pollen 

morphology was qualitative; therefore, no statistical analysis was conducted on these 

observations. 

Material Phenotype Monsanto Lot Number

MPA640B1 Conventional Control 11333170

MON 87411 Glyphosate -Tolerant and Insect-Protected 11333176

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.
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Figure J-1. General Morphology 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 degree of hydration.
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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.
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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 vitroproduced DvSnf7 RNA, referred to as DvSnf7_968 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 study, 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 at a single test concentration and compared with a water-treated 

assay control. Treatments were mixed into an 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 replicated 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% survival in both treatments), was observed with E. andrei treated with 

DvSnf7_968 RNA at a concentration equivalent to 5000 ng RNA/g soil dry weight.

Additionally, the biomass of worms exposed to DvSnf7_968 RNA (-8.4 ± 1.4% change) 

was not significantly reduced compared to the assay control (-9.4 ± 2.4% change) (p > 

0.05) and there were no adverse observed effects on worm behavior. Based on the results 

of assessments for mortality, behavior and change in biomass, the NOEC for 

DvSnf7_968 RNA was concluded to be 5000 ng RNA/g soil dry weight.

K.2. Evaluation of the Potential Dietary Effects of DvSnf7_968 RNA on Honeybee 

Larvae (Apis mellifera L.). 

The purpose of this study was to evaluate the potential dietary effects 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 dose 
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administered to the brood cell. A single dose 1000 ng/g solution, in a 10 μl 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/cell13. In addition, an assay control treated with 10 μL of

30% (w/v) sucrose/purified water was included as well as a positive control (2000 μg

potassium 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 cells 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 27 to 30oC and 

47.3 to 72.6% relative humidity (RH). The endpoints measured 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 initiated 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 achieved 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. 

Survival 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, the NOEC of the DvSnf7_968 RNA for honeybee larvae was ≥11.3

ng/larvae. 

K.3. Evaluation of the Potential Dietary Effects of DvSnf7_968 RNA on Honeybee 

Adults (Apis mellifera L.). 

The purpose of this study was to evaluate the potential dietary effects of DvSnf7_968 

RNA on the survival of honey bee (Apis mellifera L.) adults in 14-day continuous feeding 

study. Honey bee adults, <1 day post-emergence, 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 μg 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 20 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 hours. Test bees for each treatment group were observed 

 

13 A single dose of 10 μl 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.
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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 assessments were conducted. Environmental conditions 

were maintained at approximately 26 to 32oC and 41 to 80% relative humidity (RH). The 

positive control produced 100% mortality, confirming the validity of the test system. 

Though the study was designed to 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 exposure is considered to be appropriate exposure 

duration for a chronic feeding study for adult honey bees (EFSA, 2013). There 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%. The NOEC of the DvSnf7_968 RNA for adult honeybees 

was ≥1000 ng/g.

351BK.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 lady 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 (120 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 27° C, a relative humidity (RH) of 70 % and a 14L: 10D photoperiod. There were no 

significant differences (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 assay 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 emergence 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 the biomass of 

females averaging 10.7 mg and 10.9 mg (p > 0.05) and males averaging 9.7 mg and 9.5 

mg (p > 0.05) for test and control treatments respectively. The positive control group had 

17% survival and none of the insects in the positive control group developed to the pupa

stage, confirming the validity of the test system. The NOEC of the DvSnf7_968 RNA

for C. maculata was concluded to be ≥1000 ng/g of diet. 
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K.5. Evaluation of Potential Dietary Effects of DvSnf7_968 RNA on the Carabid 

ground beetle, Poecilus chalcites (Say) (Coleoptera: Carabidae)

The purpose of 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. Larvae 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 27° C, a RH of 70 % and a 14L: 10D photoperiod. The endpoints 

measured were survival, development to the adult stage, and development time (days) to 

adult emergence, and adult biomass. There was no significant difference for survival 

between the DvSnf7_968 RNA (93%) and the assay control (92%) treatments and all of 

the sublethal endpoints (p > 0.05). For the test or control diets, the percentage that 

developed to the adult stage averaged 70% and 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%,

survival and none of the insects in the positive control group developed to the pupal or 

adult stage, confirming the validity of the test system. The NOEC of the DvSnf7_968 

RNA for P. chalcites was ≥1000 ng/g of diet. 

35BK.6. Evaluation of Potential Dietary Effects of DvSnf7_968 RNA on the Insidious 

Flower Bug, Orius insidiosus (Say) (Heteroptera: Anthocoridae)

The purpose of this study was to evaluate potential dietary effects of DvSnf7_968 RNA 

on the survival and development of O. insidiosus nymphs over 10 days of continuous 

exposure following the methodology described in Tan et al., (2011).Three treatment diets

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 μg potassium arsenate/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 diets 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 16L: 8D

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 the 
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test system was effective in detecting toxic effects through the dietary exposure. The 

NOEC of the DvSnf7 RNA for the insidious flower bug, O. insidiosus, was ≥1000 ng/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 μg 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 

exposures 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 

maintained in an incubator at a target temperature of 25° C, a target RH of 70 % and a 

16L: 8D 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.
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References for Appendix K

EFSA. 2013. EFSA Guidance Document on the risk assessment 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.
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Appendix L: Next-Generation Sequencing and Junction Sequence Analysis for 

the Characterization of DNA Inserted into Crop Plants

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 relied 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 bioinformatics 

(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. 

There are multiple advantages to using next-generation 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 method 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 32P-labeled probes. This new method provides higher reproducibility, 

because it is less dependent on complex lab based procedures. The method described 

here is essentially identical 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 characterization 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 analysis, which demonstrates the stable heritability of inserted 

DNA sequences over a number of breeding generations, is also routinely conducted.
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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 obtained (≥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 methods 

(Clarke and Carbon 1976; Lander and Waterman 1988), to provide genome coverage that 

would be expected to not miss a single basepair in complex genomes (Kovalic et al. 

2012). Furthermore, even with know biases in next-generation sequencing techniques, 

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 known singlecopy endogenous gene, this analysis demonstrates coverage at ≥75x median depth in each

sample. Furthermore, in 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, which 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 

limitation 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). 
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Using bioinformatics tools, the sequence reads that are derived from the plasmid vector 

are selected for further analysis out of the comprehensive genomic sequence dataset 

produced from the transformation event. To determine the insert number, the known 

sequence of the transformation vector plasmid is used as an “e-probe” in the 

bioinformatics analysis to search for and select the sequences that contain any portion of 

sequence of the plasmid. The DNA sequencing reads with a match 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 

at least 96.7% sequence identity are collected. The results of a parameter optimization 

study that systematically evaluated many different potential parameter sets established

these selection criteria as providing the best possible combination of sensitivity and 

specificity. 

Figure L-1. Sequencing and Sequence Selection

Genomic DNA from the test and control material were sequenced using Illumina 

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 

coverage. Using these genome sequence reads, bioinformatics search tools were used to 

select all sequence reads that are significantly similar (as defined in the text) to the 

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 by 
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lack 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 flanking 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 junction 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 

sequences of all unique junction classes detected, the number of insertion sites of the 

plasmid sequence can be determined. For a single insert, two junction sequence classes 

are expected, one each originating from either 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 JSC).

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCGGG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCGGGAT

Plasmid DNA Flanking DNA

Junction
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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 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 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, locus-specific PCR and DNA sequencing analyses, which 

complements the NGS/JSA analyses, and is common to both the Southern-based and the 

NGS/JSA characterization methods. Directed sequencing (locus-specific PCR and DNA 

sequencing analyses) of the transformation event determines the complete 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 single 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, the 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. 

This 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 generations, two identical junction sequence classes are expected in all the 

generations tested.

5’ Flank DNA insert 3’ Flank

Insert Junction Regions

Junction Sequences: Class A

Junction Sequences: Class B
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References for Appendix L

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Clarke, L. and J. Carbon. 1976. A colony bank containing synthetic CoI EI hybrid 

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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 

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Lander, E.S. and M.S. Waterman. 1988. Genomic mapping by fingerprinting random 

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Miller, J.R., S. Koren and G. Sutton. 2010. Assembly algorithms for next-generation 

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Minoche, A.E., J.C. Dohm and H. Himmelbauer. 2011. Evaluation of genomic highthroughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems. 

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Shendure, J. and H. Ji. 2008. Next-generation DNA sequencing. Nature Biotechnology

26:1135-1145.

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Qin, L. Ma, G. Li, Z. Yang, G. Zhang, B. Yang, C. Yu, F. Liang, W. Li, S. Li, D. Li, P. 

Ni, J. Ruan, Q. Li, H. Zhu, D. Liu, Z. Lu, N. Li, G. Guo, J. Zhang, J. Ye, L. Fang, Q. 

Hao, Q. Chen, Y. Liang, Y. Su, A. san, C. Ping, S. Yang, F. Chen, L. Li, K. Zhou, H. 

Zheng, Y. Ren, L. Yang, Y. Gao, G. Yang, Z. Li, X. Feng, K. Kristiansen, G.K.-S. Wong, 

R. Nielsen, R. Durbin, L. Bolund, X. Zhang, S. Li, H. Yang and J. Wang. 2008. The 

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Wei, H., C. Therrien, A. Blanchard, S. Guan and Z. Zhu. 2008. The Fidelity Index 

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