Monsanto Company CR262-14U1 1 of 260

Petition for the Determination of Nonregulated Status for Increased Ear Biomass MON 87403 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 August 1, 2014 Revised September 12, 2014 USDA Petition Number #14-213-01p OECD Unique Identifier: MON-87403-1

Monsanto Petition Number: CR262-14U1

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

Prepared by:
D. Anstrom, M.S., T. Bhakta, Ph.D., P. Chinnadurai, M.S., M.Phil., J.M. Cordts, M.S., M.B.A., T. Edrington, Ph.D., G.E. Frierdich, B.S., DABT, M. Leibman, Ph.D., K. Nemali, Ph.D., E. Park, Ph.D., K. Skottke, Ph.D., G.B. Tilton, Ph.D., M. Wang, Ph.D., E.G. Webb, M.S.
Contributors and/or Principal Investigators:
P. Asiimwe, Ph.D., G. Brown, B.S., C. Garnaat, M.S., J. Hart, M.S., K. Howard, Ph.D., K. Klug, M.S., D. Kovalic, Ph.D., K.W. Lawry, M.S., M. McPherson, Ph.D., S.G. Riordan, B.S., B. Sammons, Ph.D., A. Silvanovich, Ph.D., S.B. Storrs, Ph.D., Q. Tian, M.D., Ph.D., R. Wang, M.S., J.M. Ward., Ph.D.,

T. Werk, M.S., J. Whitsel, M.S., Y. Yan, Ph.D.Monsanto Company CR262-14U1 2 of 260 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, pursuant to Executive Order 12600, the Privacy Act and other applicable authorities, 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 a CBI-deleted copy of 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.

© 2014 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.CERTIFICATIO N

Th e undersigne d certifie s that , t o th e bes t knowledg e an d belie f

o f th e undersigned , thi s petitio n include s al l informatio n an d view s o n whic h t o bas e a determination , an d tha t i t include s al l relevan t

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

.



# Ml 

# l^M^

Joh n M

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

. Regulator y Affair s Manage r

Address : Monsant o Compan y 80 0 Nort h Lindberg h Blvd. , Mai l Sto p C3S D St

. Louis , M O 6316 7

Tel : (314 ) 694-483 1 Fax : (314 ) 694-308 0

Monsant o Compan y CR262-14U 1 3 of26 0Monsanto Company CR262-14U1 4 of 260 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 87403, any progeny derived from crosses between MON 87403 and conventional maize, and any progeny derived from crosses of MON 87403 with biotechnology-derived maize that have previously been granted nonregulated status under 7 CFR Part 340. Product Description Monsanto Company has developed biotechnology-derived maize MON 87403 that has increased ear biomass at an early reproductive phase (R1) compared to conventional control maize. MON 87403 was produced through insertion of the coding region of the full-length Arabidopsis thaliana ATHB17 gene through Agrobacterium -mediated transformation . ATHB17 is a member of the HD-Zip family of plant transcription factors, which are proteins that bind to specific DNA sequences and regulate gene expression. The HD-Zip family of proteins is found broadly across plant species and specific HD-Zip proteins have been shown to play an important role in the modulation of plant growth and development. The HD-Zip family consists of four subfamilies and ATHB17 is a member of the class II subfamily. HD-Zip II proteins form either homodimers or heterodimers with other HD-Zip II proteins within the same subfamily and function as repressors of gene expression. In MON 87403, maize-specific splicing of the ATHB17 t r a n s c r i p t r e s u l t s i n a t r u n c a t e d p r o t e i n , A T H B 1 7 Δ 1 1 3 , w h i c h i s m i s s i n g t h e first 113 N-terminal amino acids that are expressed in Arabidopsis thaliana . The A T H B 1 7 ∆ 1 1 3 p r o t e i n r e t a i n s t h e a b i l i t y t o f

o r m h o m o
- a n d h e t e r o
- d i m e r s a n d b i n d t o t a r g e t D N A s e qu e n c e s l i k e t h e f u l l

- l e n g t h p r o t e i n

. T h e A T H B 1 7 ∆ 1 1 3 p r o t e i n i s , however, unable to function as a transcriptional repressor because the protein lacks a f u n c t i o n a l r e p r e s s i o n d o m a i n

. B y a d o m i n a n t

- n e g a t i v e m e c h a n i s m , A T H B 1 7 ∆ 1 1 3 c a n alter the activity of endogenous maize HD-Zip II proteins, which are predominantly
e x p r e s s e d i n e a r t i s s u e

. T h u s , t h e A T H B 1 7 ∆ 1 1 3 p r o t e i n l i k e l y m o d u l a t e s H D

- Z i p I I

regulated pathways in the ear, which leads to increased ear growth at the early reproductive phase. This increased ear growth is associated with increased partitioning of dry matter (photosynthate) from the source (vegetative) tissue to the sink (ear) tissue in MON 87403 compared to control plants. Early reproductive stages in maize are a critical period of maize growth at which the maximum ear biomass (sink size) is determined by a combination of genetics andMonsanto Company CR262-14U1 5 of 260

environmental conditions. Dry matter (photosynthate) produced by the plant during reproductive stages is allocated to the ear for its growth after the sink size is determined. Thus, ear biomass, which is set during early reproductive stages, is considered an important determinant of reproductive success and a larger ear biomass at early reproductive stages is associated with increased grain yield at harvest. Consistent with this, multiple years of field testing showed that MON 87403 out-yielded its comparators at a majority of locations tested. MON 87403 will be combined with other deregulated biotechnology-derived traits through traditional breeding methods to create commercial products with increased yield opportunity as well as protection against maize pests and tolerance to multiple herbicides. These next generation combined-trait maize products will continue to offer broader grower choice and continued pest control durability. Adoption of improved maize hybrids with increased yield opportunity that results in incremental increases in national average grain yield can positively impact production, exports and economic welfare.

Data and Information Presented Confirms the Lack of Plant Pest Potential and the Food and Feed Safety of MON 87403 Compared to Conventional Maize The data and information presented in this petition demonstrate MON 87403 is agronomically, phenotypically, and compositionally comparable to commercially cultivated maize, with the exception of the introduced trait. Moreover, the data and information presented demonstrate MON 87403 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 87403 was confirmed based on multiple, well-established lines of evidence:

 Maize does not possess any of the attributes commonly associated with weeds, has a history of safe consumption, and serves as an appropriate basis of comparison for MON 87403.

 A detailed molecular characterization of the inserted DNA demonstrates a single, intact copy of the T-DNA insert in a single locus within the maize genome.

 E x t e n s i v e e v a l u a t i o n o f t h e t r u n c a t e d A T H B 1 7 Δ 1 1 3 p r o t e i n e x p r e s s e d i n MON 87403 confirms that it is unlikely to be a toxin or allergen.

 A compositional assessment supports the conclusion that MON 87403 grain and forage is compositionally equivalent to grain and forage of conventional maize.

 An extensive evaluation of MON 87403 phenotypic and agronomic characteristics and environmental interactions demonstrates MON 87403 has no increased plant pest risk potential compared to conventional maize.

 An assessment of potential impact to non-target organisms (NTOs) including organisms beneficial to agriculture and endangered species indicates thatMonsanto Company CR262-14U1 6 of 260 MON 87403 is not expected to have an effect on other organisms compared to conventional maize under normal agricultural practices.

 Evaluation of the agronomic and phenotypic characteristics of MON 87403, using current maize cultivation and management practices, leads to the conclusion that deregulation of MON 87403 would not have an effect on maize agronomic practices. Maize is a Familiar Crop Lacking Weedy Characteristics 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. Because of the importance of maize in agriculture, plant breeders continuously strive to improve commercial maize yields. Initial improvements in maize yield were due to the domestication of varieties with desirable traits like larger ear biomass. Maize ears became larger over time during this domestication. During the hybrid era (1939 to present), commercial maize yield in the U.S. increased nearly six-fold with an average 99 kg ha -1 increase every year. The major factor that contributed to yield increase during this era is a favorable response of hybrids to increased plant population density which resulted in an increase in the number of ears per hectare. Whereas increased planting densities resulted in a net increase in grain yields, they resulted in a decrease in ear size at the individual plant level. Commercial varieties with MON 87403 can provide increased yield opportunity as this trait increases ear biomass during early reproductive stages at production planting densities. 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 in a rotation with soybeans or 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 maize, such as hybrids containing MON 87403, 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.Monsanto Company CR262-14U1 7 of 260

Conventional Maize MPA640B and EXP257 are Appropriate Comparators to MON 87403 Conventional control materials developed for use as comparators in safety assessment studies were based on the appropriate fit for various studies and seed availability. The conventional control materials included the original transformation line (LH244) and LH244 crossed to two conventional lines (LH287 and LH295) to create F 1 starting control materials. Both MPA640B (LH244 × LH287) and EXP257 (LH244 × LH295) were used as controls in molecular characterization studies. MPA640B was used as the conventional control in compositional analysis studies and in phenotypic, agronomic and environmental interactions assessments. Where appropriate, commercial hybrid maize materials (reference hybrids) were also used to establish a range of variability or responses representative of commercial maize in the U.S. Molecular Characterization Verified the Integrity and Stability of the Inserted DNA in MON 87403 MON 87403 was developed through Agrobacterium- mediated transformation of maize immature embryos from inbred line LH244 utilizing plasmid PV-ZMAP5714. PV-ZMAP5714 is approximately 11.7 kb in size and contains three cassettes: one T-DNA, delineated by Left and Right Border regions, contains the ATHB17 expression cassette, the plasmid backbone contains the cp4 epsps selectable marker cassette, and the aadA expression cassette. PV-ZMAP5714 employs a tandem T-DNA approach to generate marker-free plants. In this tandem T-DNA approach, a single right border and a single left border were used to achieve separate, unlinked insertion of the T-DNA as well as the cp4 epsps selectable marker gene located in the plasmid backbone. After initial selection of transformants for glyphosate tolerance, the cp4 epsps cassette was segregated away by conventional breeding and molecular analysis was used to identify plants containing only the intended T-DNA (and not the cp4 epsps ). Characterization of the DNA insert in MON 87403 was conducted using a combination of sequencing, PCR, and bioinformatics. The results of this characterization demonstrate that MON 87403 contains one copy of the intended transfer DNA (T-DNA) containing the ATHB17 expression cassette 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 87403 by Next Generation Sequencing and Junction Sequence Analysis (NGS/JSA) demonstrated that MON 87403 contained a single intended DNA insert. These whole-genome sequence analyses provided a comprehensive assessment of MON 87403 to determine the presence and identity of sequences derived from PV-ZMAP5714 and demonstrated that MON 87403 contained a single T-DNA insert with no detectable backbone sequences.

 Directed sequencing (locus-specific PCR, DNA sequencing and analyses) performed on MON 87403 was used to determine the complete sequence of theMonsanto Company CR262-14U1 8 of 260 single DNA insert from PV-ZMAP5714, the adjacent flanking DNA, and the 5' and 3' insert-to-flank junctions. This analysis confirmed that the sequence and organization of the inserted DNA is identical to the corresponding region in the PV-ZMAP5714 T-DNA. Furthermore, the genomic organization at the insertion site was assessed by comparing the sequences flanking the T-DNA insert in MON 87403 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 87403 upon DNA integration.

 Generational stability analysis by NGS/JSA demonstrated that the single PV-ZMAP5714 T-DNA insert in MON 87403 has been maintained through five breeding generations, thereby confirming the stability of the T-DNA in MON 87403.

 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 87403 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 87403.

D a t a C o n f

i r m s A T H B 1 7 Δ 1 1 3 P r o t e i n S a f e t y Characterization of the introduced protein(s) in a biotechnology-derived crop is important to establishing food, feed, and environmental safety and a multistep approach was used to
c h a r a c t e r i z e a n d a s s e s s t h e s a f e t y o f t h e A T H B 1 7 Δ 1 1 3 p r o t e i n e x p r e s s e d i n M O N 8 7 4 0 3

r e s u l t i n g f

r o m t h e g e n e t i c m o d i f

i c a t i o n

. T h e e x p r e s s i o n l e v e l o f t h e A T H B 1 7 Δ 1 1 3 protein in selected tissues of MON 87403 was determined and exposure to humans and animals through diet was evaluated. In addition, the donor organism for the A T H B 1 7 Δ 1 1 3 p r o t e i n c o d i n g s e qu e n c e , Arabidopsis thaliana , is ubiquitous in the environment and is not commonly known for human or animal pathogenicity or allergenicity.

A s s e s s i n g t h e s a f

e t y o f t h e A T H B 1 7 ∆ 1 1 3 p r o t e i n r e qu i r e s a c o n s i d e r a t i o n o f b o t h t h e hazard associated with the protein and the potential exposure to it. Data presented demonstrate that the expression of this protein in maize grain is below the limit of detection (LOD) and is extremely low in other tissues tested, hence exposure to A T H B 1 7 ∆ 1 1 3 p r o t e i n i s n e g l i g i b l e

. B i o i n f o r m a t i c s e a r c h e s u s i n g t h e A T H B 1 7 ∆ 1 1 3 amino acid sequence as the query identified homologous sequences from several different food plants, including soybean, rice, maize, tomato, potato, orange, papaya, grape, and
c r u c i f e r o u s v e g e t a

b l e s

. O v e r a l l t h e p r o t e i n s e qu e n c e i d e n t i t y o f A T H B 1 7 ∆ 1 1 3 t o homologs in these species ranges from ~58-83%, with the highest identity to the homologs in the Brassica species Brassica rapa (a species including crops such as turnip and napa cabbage) and Brassica oleracea (a species including crops such as cabbage and B r u s s e l s s p r o u t s )

. T h e a m i n o a c i d s e qu e n c e a l i g n m e n t b e t w e e n A T H B 1 7 ∆ 1 1 3 a n d i t sMonsanto Company CR262-14U1 9 of 260

f o o d c r o p h o m o l o g s s p a n s t h e l e n g t h o f t h e A T H B 1 7 ∆ 1 1 3 p r o t e i n

. T h u s A T H B 1 7 ∆ 1 1 3 shares sequence identity and structural similarity with proteins present in plants currently consumed, establishing that humans and animals are exposed to this class of proteins and that no adverse effects have been attributed to this class of proteins. Bioinformatics analysis also determined that the A T H B 1 7 Δ 1 1 3 protein lacks structural similarity to known allergens, gliadins, glutenins, or protein toxins. Testing also showed that the A T H B 1 7 Δ 1 1 3 protein is rapidly digested in pepsin and pancreatin suggesting that the negligible amount of protein expressed is further reduced by proteolysis during ingestion thereby reducing the already negligible exposure. Additionally, a mouse gavage study demonstrated no acute oral toxicity with a No Observable Adverse Effect Level (NOAEL) for A T H B 1 7 Δ 1 1 3 of 1335 mg/kg, the highest dose tested. Undetectably low expression levels in grain coupled with rapid digestibility and no evidence of any toxic or allergenic concerns supports the conclusion that consumption of the A T H B 1 7 Δ 1 1 3 protein from MON 87403 or its progeny poses no meaningful risk to human and animal health or an increased plant pest risk.
MON 87403 is Compositionally Equivalent to Conventional Maize Compositional analysis was conducted on grain and forage of MON 87403 grown at eight sites representative of typical agricultural regions for maize production in the U.S. in 2012. The evaluation of MON 87403 followed considerations relevant to the compositional quality of maize as defined by a 2002 OECD consensus document. 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, sodium, and furfural had more than 50% of observations below the assay limit of quantitation (LOQ) and were excluded from statistical analysis. Moisture in 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 comparisons were based on compositional data combined across all field sites. Statistically significant differences were identified at the 5 % l e v e l ( α = 0

. 0 5 )

. T h e c o m p o s i t i o n a l d a t a f r o m t h e r e f

e r e n c e h y b r i d s w e r e c o m b i n e d across all field sites to calculate a 99% tolerance interval for each component to estimate the natural variability of each component in maize. Of the 60 components statistically assessed for MON 87403, none of the components showed a significant difference between MON 87403 and the conventional control. These results support the overall conclusion that MON 87403 was not a major contributor to variation in component levels in maize grain and forage and confirmed the compositional equivalence of MON 87403 to the conventional control in levels of these components. These results support the overall food and feed safety and lack of plant pest risk of MON 87403.Monsanto Company CR262-14U1 10 of 260

MON 87403 Does Not Change Maize Plant Pest Potential or Environmental Interactions 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 riskassessment. 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 biotechnology-derived plant and the conventional control. Thus, the phenotypic, agronomic, and environmental interaction assessment of MON 87403 included genetically similiar conventional controls as comparators. This evaluation used a weight of evidence approach and considered statistical differences between MON 87403 and the conventional control with respect to reproducibility, magnitude, and directionality. The observations were taken on plants not treated with herbicides in order to evaluate only the impact of the introduced trait in MON 87403. Comparison to a range of commercial references grown concurrently established the range of natural variability for maize, and provided a context from which to further evaluate any statistical differences. Characteristics assessed included: seed dormancy and germination, pollen morphology, plant phenotypic observations and environmental interaction evaluations. The phenotypic, agronomic, and environmental interaction assessment demonstrated that MON 87403 is comparable to the conventional control. Thus, MON 87403 is not expected to have increased weediness or plant pest potential compared to conventional maize. Seed dormancy and germination characterization indicated that MON 87403 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 87403 compared to the conventional control. For pollen characteristic assessments, there were no
s t a t i s t i c a l l y s i g n i f

i c a n t d i f f e r e n c e s ( α = 0

. 0 5 ) d e t e c t e d b e t w e e n M O N 8 7 4 0 3 a n d t h e 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 87403 is not likely to have increased weediness or plant pest potential compared to conventional maize. Data were collected at 13 sites in the U.S. during 2012 to evaluate phenotypic and agronomic characteristics of MON 87403 compared to the conventional control and four reference hybrids. These 13 sites provided a diverse range of environmental and agronomic conditions representative of commercial maize production areas in North America. In the combined-site analysis, no statistically significant differences were detected between MON 87403 and the conventional control for 12 of 13 characteristics including early stand count, days to 50% pollen shed, days to 50% silking, stay green rating, plant height, dropped ear count, stalk lodged plants, root lodged plants, final stand count, grain moisture, test weight and yield. One significant difference was detected for ear height in the combined-site analysis.Monsanto Company CR262-14U1 11 of 260

MON 87403 had increased ear height (111.1 vs. 107.5 cm) compared to the conventional control. However, the mean value of MON 87403 for ear height was within the reference range for this characteristic Ear height is not a reported weediness characteristic and whereas large increases in ear height might be expected to increase stalk lodging, there was no increase in stalk lodging in these trials. In addition, a small change in ear height would not be expected to change agronomic practices. Thus, the measured phenotypic characteristics of MON 87403 were not altered in terms of pest/weed potential compared to conventional maize. In an assessment of abiotic stress, no differences in the range of responses were observed between MON 87403 and the conventional control for any of the 143 comparisons of plant response to abiotic stressors. In an assessment of disease responses, no differences in the range of responses were observed between MON 87403 and the conventional control for any of the 176 comparisons. Finally, in an assessment of arthropod damage, no differences in the range of responses were observed between MON 87403 and the conventional control for any of the 150 comparisons for plant damage caused by arthropods. In summary, the phenotypic, agronomic, and environmental interaction data were evaluated to characterize MON 87403, and to assess whether the trait introduced in MON 87403 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 87403 and the conventional control, and comparison to the range of the commercial reference hybrids. Results from the phenotypic, agronomic, and environmental interactions assessment indicated that MON 87403 does not possess enhanced weediness characteristics, increased susceptibility or tolerance to specific abiotic stress, diseases, or arthropods, or characteristics that would confer a plant pest risk compared to conventional maize. MON 87403 Will Not Negatively Affect NTOs Including Those Beneficial to Agriculture An evaluation of the impacts of MON 87403 on non-target organisms (NTOs) is a component of the plant pest risk assessment. Because MON 87403 does not possess pesticidal activity, all organisms that interact with MON 87403 are considered to be NTOs. Data from 2012 U.S. phenotypic and agronomic studies and observational data on environmental interactions such as plant-disease interactions, arthropod damage and arthropod abundance, were collected at select sites for MON 87403 and conventional controls. Results from these extensive studies support conclusions of no adverse impacts to non-target arthropod populations and no changes to plant-disease interactions. The biochemical information and experimental data for evaluation of MON 87403 included molecular characterization, ATHB17  113 protein safety assessment, data from the environmental interactions assessment, demonstration of compositional equivalence to conventional maize, and demonstration of agronomic and phenotypic equivalence to
c o n v e n t i o n a l m a i z e ( w i t h t h e e x c e p t i o n o f p r e s e n c e o f A T H B 1 7 ∆ 1 1 3 p r o t e i n i n MON 87403). Taken together, these data support the conclusion that MON 87403 has noMonsanto Company CR262-14U1 12 of 260

reasonable mechanism to harm NTOs, nor does it pose an additional risk to threatened and endangered species compared to the cultivation of conventional maize. Deregulation of MON 87403 is Not Expected to Have Effects on Maize Agronomic Practices An assessment of current maize agronomic practices was conducted to determine whether the cultivation of MON 87403 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. MON 87403 was developed to provide increased ear biomass at an early reproductive stage in maize hybrids through expression of the A T H B 1 7 Δ 1 1 3 protein. As such, there are no additional phenotypes associated with expression of the intended T-DNA in this product. As phenotypic evaluations, evaluations of stress responses, and pest/disease susceptibility showed no differences between MON 87403 and reference hybrids 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 87403. MON 87403 is similar to conventional maize in its agronomic, phenotypic, environmental, and compositional characteristics and has naturally occurring levels of protection against pests and diseases comparable to and typical of conventional commercial maize hybrids. Based on this assessment, the introduction of MON 87403 is not expected to result in changes or impacts to current maize agronomic practices.

Conclusion Based on the data and information presented in this petition, it is concluded that MON 87403 is not expected to be a plant pest. Therefore, Monsanto Company requests a determination from APHIS that MON 87403 and any progeny derived from crosses between MON 87403 and conventional maize or deregulated biotechnology-derived maize be granted nonregulated status under 7 CFR Part 340.Monsanto Company CR262-14U1 13 of 260 TABLE OF CONTENTS

RELEASE OF INFORMATION.........................................................................................2 CERTIFICATION ...............................................................................................................3 EXECUTIVE SUMMARY .................................................................................................4 TABLE OF CONTENTS...................................................................................................13 LIST OF TABLES.............................................................................................................17 LIST OF FIGURES ...........................................................................................................20 ABBREVIATIONS AND DEFINITIONS........................................................................22 I. RATIONALE FOR THE DEVELOPMENT OF MON 87403 .............................24 I.A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR § 340.6 ...............................................................................................................24 I.B. Rationale for the Development of MON 87403..................................................24 I.C. Submissions to Other Regulatory Agencies........................................................26 I.C.1. Submission to FDA ..................................................................................26 I.C.2. Submissions to Foreign Government Agencies .......................................27 II. THE BIOLOGY OF MAIZE .................................................................................28 II.A. Maize as a Crop ...............................................................................................28 II.B. Characteristics of the Recipient Plant..............................................................29 II.C. Maize as a Test System in Product Safety Assessment...................................29 III. DESCRIPTION OF THE GENETIC MODIFICATION ......................................31 III.A. The Plasmid Vector PV-ZMAP5714...............................................................31 III.B. Description of the Transformation System......................................................32 III.C. The ATHB17 Coding Sequence and the ATHB17 Protein (T-DNA)..............32 III.D. Regulatory Sequences......................................................................................35 III.E. T-DNA Borders ...............................................................................................35 III.F. Genetic Elements Outside of the T-DNA Borders ..........................................35 IV. CHARACTERIZATION OF THE GENETIC MODIFICATION........................39 IV.A. Determining the Number and Identity of DNA Inserts in MON 87403..........43 IV.A.1. Next Generation Sequencing of MON 87403 and Conventional Control Genomic DNA..........................................................................................43 IV.A.2. Characterization of insert number in MON 87403 using Bioinformatic Analysis ..........................................................................................44 IV.B. Organization and Sequence of the Insert and Adjacent DNA in MON 87403...................................................................................................................52 IV.C. Sequencing of the MON 87403 Insertion Site.................................................54Monsanto Company CR262-14U1 14 of 260 IV.D. Determination of Insert Stability over Multiple Generations of MON 87403...................................................................................................................56 IV.E. Inheritance of the Genetic Insert in MON 87403 ............................................57 IV.F. Characterization of the Genetic Modification Summary and Conclusion.....................................................................................................................61 V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE A T H B 1 7 Δ 1 1 3 P R O T E I N P R O D U C E D I N M O N 8 7 4 0 3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 V.A. I d e n t i t y a n d F u n c t i o n o f t h e A T H B 1 7 Δ 1 1 3 P r o t e i n f r o m M O N 8 7 4 0 3

. . . . . . . . . 6 2 V.A.1. S e qu e n c e I d e n t i t y o f t h e A T H B 1 7 Δ 1 1 3 P r o t e i n f r o m MON 87403 ...........................................................................................................62 V.A.2. S t r

u c t u r e a n d F u n c t i o n o f t h e A T H B 1 7 Δ 1 1 3 P r o t e i n f r o m MON 87403 ...........................................................................................................64 V.B. C h a r a c t e r i z a t i o n a n d E qu i v a l e n c e o f A T H B 1 7 Δ 1 1 3 P r o t e i n f r o m MON 87403...................................................................................................................64 V.C. E x p r e s s i o n L e v e l s o f A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3

. . . . . . . . . . . . . . . . . . . . . . . . . 6 5 V.D. A s s e s s m e n t o f P o t e n t i a l A l l e r g e n i c i t y o f t h e A T H B 1 7 Δ 1 1 3 P r o t e i n

. . . . . . . . . . . . . 6 8 V.E. S a f

e t y A s s e s s m e n t S u m m a r y o f A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3

. . . . . . . . 6 8 V.E.1. The Donor Organism has a History of Safe Use ..................................68 V.E.2. A T H B 1 7 Δ 1 1 3 P r o t e i n B e l o n g s t o a C o m m o n C l a s s o f P l a n t Proteins ..............................................................................................................69 V.E.3. A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3 i s N o t H o m o l o g o u s t o Known Allergens or Toxins...................................................................................69 V.E.4. A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3 i s S u s c e p t i b l e t o P e p s i n and Pancreatin Digestion .......................................................................................70 V.E.5. A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3 i s N o t A c u t e l y T o x i c

. . . . . . . . . . . . . . 7 0 V.E.6. H u m a n a n d A n i m a l E x p o s u r e t o t h e A T H B 1 7 Δ 1 1 3 P r o t e i n s

. . . . . . . . . . . . . . 7 0 V.F. A T H B 1 7 Δ 1 1 3 P r o t e i n C h a r a c t e r i z a t i o n a n d S a f e t y C o n c l u s i o n

. . . . . . . . . . . . . . . . . . . . . 7 1 VI. COMPOSITIONAL ASSESSMENT OF MON 87403.........................................73 VI.A. Compositional Equivalence of MON 87403 Grain and Forage to Conventional Maize ......................................................................................................74 VI.B. Compositional Assessment of MON 87403 Conclusion.................................92 VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL INTERACTIONS ASSESSMENT....................................................................................93 VII.A. Characteristics Measured for Assessment ...................................................93 VII.B. Interpretation of Phenotypic and Environmental Interaction Data ..............98 VII.B.1. Interpretation of Criteria for Detected Differences ..............................98 VII.B.2. Interpretation of Vigor and Environmental Interactions Data............101Monsanto Company CR262-14U1 15 of 260 VII.C. Comparative Assessments of the Phenotypic, Agronomic, and Environmental Interaction Characteristics of MON 87403 ........................................101 VII.C.1. Seed Dormancy and Germination Characteristics..............................101 VII.C.2. Field Phenotypic, Agronomic, and Environmental Interactions Characteristics......................................................................................................104 VII.C.3. Pollen Characteristics .........................................................................115 VII.D. Conclusions for Phenotypic, Agronomic, and Environmental Interactions Evaluation................................................................................................117 VIII. U.S. AGRONOMIC PRACTICES ...................................................................118 VIII.A. Introduction................................................................................................118 VIII.B. Overview of U.S. Maize Production..........................................................118 VIII.B.1. Maize Production...............................................................................118 VIII.C. Management of Insect Pests.......................................................................120 VIII.D. Management of Diseases and Other Pests .................................................120 VIII.E. Weed Management ....................................................................................120 VIII.F. Crop Rotation Practices in Maize ..............................................................120 VIII.G. Maize Volunteer Management...................................................................121 VIII.H. Stewardship of MON 87403 ......................................................................121 VIII.I. Impact of the Introduction of MON 87403 on Agricultural Practices.......122 IX. PLANT PEST ASSESSMENT............................................................................123 IX.A. Characteristics of the Genetic Insert and Expressed Product ........................123 IX.A.1. Genetic Insert......................................................................................123 IX.B. Expression and Characterization of Gene Product ........................................124 IX.B.1. Protein Safety and Expression Levels ................................................124 IX.C. Compositional Characteristics.......................................................................124 IX.D. Phenotypic, Agronomic, and Environmental Interaction Characteristics .............................................................................................................125 IX.D.1. Seed Dormancy and Germination.......................................................125 IX.D.2. Plant Growth and Development .........................................................125 IX.D.3. Response to Abiotic Stressors ............................................................126 IX.D.4. Pollen Morphology and Viability.......................................................126 IX.E. Impact to Non-target Organisms Including Those Beneficial to Agriculture or Threatened and Endangered Species ...................................................126 IX.F. Weediness Potential of MON 87403.............................................................127 IX.G. Potential for Pollen Mediated Gene Flow and Introgression ........................127 IX.G.1. Hybridization with Cultivated Maize .................................................128Monsanto Company CR262-14U1 16 of 260 IX.G.2. Hybridization with Wild Annual Species of Zea mays subsp. mexicana ............................................................................................................132 IX.G.3. Hybridization with the Wild Perennial Species of Subgenus Tripsacum ............................................................................................................132 IX.G.4. Transfer of Genetic Information to Species with which Maize Cannot Interbreed (Horizontal Gene Flow) .........................................................132 IX.H. Potential Impact on Maize Agronomic Practices ..........................................133 IX.I. Conventional Breeding with Other Biotechnology-derived or Conventional Maize ....................................................................................................133 IX.J. Summary of Plant Pest Assessments.............................................................134 X. ADVERSE CONSEQUENCES OF INTRODUCTION .....................................135 REFERENCES ................................................................................................................136 APPENDICES .................................................................................................................146 Appendix A: USDA Notifications and Permits...............................................................147 A p p e n d i x B : M o d e o f A c t i o n o f A T H B 1 7 ∆ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3

. . . . . . . . . . . . . . . . . . . . . . . . 1 5 3 Appendix C: Overview, Materials, Methods, and Supplementary Results for Molecular Analyses of MON 87403................................................................................167 Appendix D: Materials, Methods and Results for Characterization of A T H B 1 7 Δ 1 1 3 P r o t e i n P r o d u c e d i n M O N 8 7 4 0 3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 4 Appendix E: Materials and Methods Used for the Analysis of the Levels of A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 3 Appendix F: Materials and Methods for Compositional Analysis of MON 87403 Maize Grain and Forage...................................................................................................196 Appendix G: Materials, Methods, and Individual Site Results for Seed Dormancy and Germination Assessment of MON 87403.................................................................212 Appendix H: Materials, Methods, and Individual Site Results from Phenotypic, Agronomic, and Environmental Interaction Assessment of MON 87403 under Field Conditions...............................................................................................................218 Appendix I: Materials and Methods for Pollen Morphology and Viability Assessment.......................................................................................................................257Monsanto Company CR262-14U1 17 of 260 LIST OF TABLES
Table I-1. Differences in R1 Ear Weight between MON 87403 and the Conventional Control in 2012 U.S. Field Trials 1 ...............................................................26 Table III-1. Summary of Genetic Elements in Plasmid PV-ZMAP5714 .........................36 Table IV-1. Summary of Genetic Elements in MON 87403 ............................................45 Table IV-2. Unique Junction Sequence Class Results......................................................50 Table IV-3. Junction Sequence Classes Detected.............................................................56 Table IV-4. Segregation of the Expression Cassette During the Development of MON 87403 .......................................................................................................................60 T a b l e V

- 1

. S u m m a r y o f A T H B 1 7 Δ 1 1 3 P r o t e i n L e v e l s i n T i s s u e s f r o m MON 87403 Grown in 2012 United States Field Trials....................................................67 Table VI-1. Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and Conventional References........................................................76 Table VI-2. Summary of Maize Grain Total Fat and Fatty Acids for MON 87403, Conventional Control, and Conventional References........................................................80 Table VI-3. Summary of Maize Grain Carbohydrates by Calculation and Fiber for MON 87403, Conventional Control, and Conventional References .................................82 Table VI-4. Summary of Maize Grain Ash and Minerals for MON 87403, Conventional Control, and Conventional References........................................................83 Table VI-5. Summary of Maize Grain Vitamins for MON 87403, Conventional Control, and Conventional References ..............................................................................85 Table VI-6. Summary of Maize Grain Anti-nutrients and Secondary Metabolites for MON 87403, Conventional Control, and Conventional References............................87 Table VI-7. Summary of Maize Forage Proximates, Fiber, and Minerals for MON 87403, Conventional Control, and Conventional References .................................88 Table VI-8. Literature and ILSI Database Ranges for Components in Maize Forage and Grain................................................................................................................90 Table VII-1. Phenotypic, Agronomic, and Environmental Interaction Characteristics Evaluated in U.S. Field Trials, and Laboratory Studies............................95 Table VII-2. Germination Characteristics of MON 87403 and the Conventional Control .............................................................................................................................103 Table VII-3. Field Phenotypic Evaluation Sites for MON 87403 during 2012..............106 Table VII-4. Combined-Site Comparison of MON 87403 to Conventional Control for Phenotypic and Agronomic Characteristics During 2012.............................107 Table VII-5. Summary of Qualitative Environmental Interactions Assessments during 2012 ......................................................................................................................110 Table VII-6. Combined-Site Comparison of CEW and ECB Damage to MON 87403 Compared to the Conventional Control during 2012 .................................112Monsanto Company CR262-14U1 18 of 260

Table VII-7. Summary of Arthropod Abundance Assessments and Detected Differences during 2012 ..................................................................................................113 Table VII-8. Pollen Characteristics of MON 87403 Compared to the Conventional Control during 2012 ..................................................................................116 Table VIII-1. Maize Production in the U.S., 2008-2013 ................................................119 Table IX-1. Summary of Published Literature on Maize Cross Pollination...................129 Table A-1. USDA Notifications and Permits Approved for MON 87403 and Status of Trials Planted ....................................................................................................148 Table B-1. Combined Site Analysis of R1 Ear Weight, R1 Stover Weight, Total Plant Weight, and Partitioning in MON 87403 and the Conventional Control from 2012 U.S. Field Trials......................................................................................................161 Table C-1. Sequencing (NGS) Conducted for MON 87403 and Control Genomic DNA.................................................................................................................................179 Table C-2. Summary of NGS Data for the Conventional Control DNA Sample Spiked with PV-ZMAP5714............................................................................................180 Table D-1. Summary of the Tryptic Masses Identified for the MON 87403 - p r o d u c e d A T H B 1 7 Δ 1 1 3 u s i n g M A L D I

- T O F M S

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 7 Table D-2. Molecular Weight Comparison Between the MON 87403- and E. coli- p r o d

u c e d A T H B 1 7 Δ 1 1 3 P r o t e i n s

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 1 T a b l e E

- 1

. A T H B 1 7 Δ 1 1 3 E x t r a c t i o n M e t h o d s 1 for Tissue Samples..............................194 Table F-1. Conventional Commercial Reference Maize Hybrids ...................................196 Table F-2. Re-expression Formulas for Statistical Analysis of Composition Data........206 Table G-1. Starting Seed of MON 87403, Conventional Control and Commercial Maize Reference Hybrids Used in Dormancy Assessment .............................................214 Table G-2. Dormancy and Germination Characteristics of MON 87403 and the Conventional Control Seed Produced at each of the Three Field Sites ...........................215 Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental Interaction Assessment ....................................................................................................220 Table H-2. Field and Planting Information.....................................................................222 Table H-3. Data Missing or Excluded from Analysis.....................................................231 Table H-4. Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials ..................................233 Table H-5. Abiotic Stressor Evaluations Using a Categorical Scale for MON 87403 and the Conventional Control in 2012 U.S. Field Trials............................238 Table H-6. Disease Damage Evaluations Using a Categorical Scale for MON 87403 and the Conventional Control in 2012 U.S. Field Trials............................239 Table H-7. Arthropod Damage Evaluations Using a Categorical Scale for MON 87403 and the Conventional Control in 2012 U.S. Field Trials............................240Monsanto Company CR262-14U1 19 of 260

Table H-8. Individual Site Analysis: Corn Earworm Damage and European Corn Borer Damage to MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials...............................................................................................................241 Table H-9. Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials......................................................................................................242 Table H-10. Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials ................................................................................................................................250 Table I-1. Starting Seed for Pollen Morphology and Viability Assessment....................258Monsanto Company CR262-14U1 20 of 260 LIST OF FIGURES
Figure III-1. Circular Map of PV-ZMAP5714 .................................................................33 Figure III-2. Schematic of the Development of MON 87403...........................................34 Figure IV-2. Junctions and Junction Sequences ...............................................................42 Figure IV-3. Two Unique Junction Sequence Classes are Produced by the Insertion of a Single Plasmid Region.................................................................................42 Figure IV-4. Schematic Representation of the Insert and Flanking Sequences in MON 87403 .......................................................................................................................47 Figure IV-5. Breeding History of MON 87403 .................................................................48 Figure IV-6. Junction Sequences Detected by NGS/JSA .................................................51 Figure IV-7. Overlapping PCR Analysis Across the Insert in MON 87403 ....................53 Figure IV-8. PCR Amplification of the MON 87403 Insertion Site.................................55 Figure IV-9. Breeding Path for Generating Segregation Data for MON 87403...............59 Figure V-1. Insert map of MON 87403 and schematic diagram of ATHB17 transcription, mRNA processing, and predicted translation ..............................................63 Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods.......................................................................................................99 Figure VIII-1. Planted Maize Acres by County in the U.S. in 2012...............................119 Figure B-1. Maize developmental stages.........................................................................154 Figure B-2. US maize yields from 1866 to 2013 in bushels per acre ..............................154 Figure B-3. Relationship between ear biomass during early reproductive stage and kernel number at harvest..................................................................................................156 Figure B-4: Domain structure of ATHB17......................................................................158 F i g u r e B

- 5 : M o d e l f

o r d o m i n a n t n e g a t i v e m e c h a n i s m o f A T H B 1 7 ∆ 1 1 3

. . . . . . . . . . . . . . . . . . . . . . . 1 5 9 Figure B-6. Ear growth and ear partitioning in MON 87403 and the control at different developmental stages in a greenhouse ..............................................................162 F i g u r e B

- 7

. S u m m a r y o f m o d e
- o f

- a c t i o n o f A T H B 1 7 Δ 1 1 3 p r o t e i n i n M O N 8 7 4 0 3 maize................................................................................................................................163 Figure C-1. Sequencing and Sequence Selection ...........................................................169 Figure C-2. Junctions and Junction Sequences...............................................................170 Figure C-3. Two Unique Junction Sequence Classes are Produced by the Insertion of a Single Plasmid Region...............................................................................172 Figure D-1. MALDI-TOF MS Coverage Map of the MON 87403-produced A T H B 1 7 Δ 1 1 3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 7 Figure D-2. Western Blot Analysis of MON 87403- and E. coli-produced A T H B 1 7 Δ 1 1 3 P r o t e i n s

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 0Monsanto Company CR262-14U1 21 of 260

Figure D-3. Molecular Weight and Purity Analysis of the MON 87403-produced A T H B 1 7 Δ 1 1 3 P r o t e i n

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 2 Figure I-1. General Morphology of Pollen from MON 87403, the Conventional Control, and Commercially Available Conventional Reference Materials under 200X Magnification.........................................................................................................259Monsanto Company CR262-14U1 22 of 260 ABBREVIATIONS AND DEFINITIONS 1

~ approximately AA amino acid ADF acid detergent fiber ANOVA analysis of variance APHIS Animal and Plant Health Inspection Service ATHB17 a member of the class II family of homeodomain-leucine zipper (HDZip) transcription factors A T H B 1 7 Δ 1 1 3 the protein expressed from the ATHB17 gene in MON 87403 BLASTX Basic Local Alignment Search Tool B-Left Border Region DNA region from Agrobacterium tumefaciens containing the left

border sequence B-Right Border Region DNA region from Agrobacterium tumefaciens containing the right

border sequence BSA bovine serum albumin CaMV cauliflower mosaic virus CEW corn earworm CFR Code of Federal Regulations CTAB cetyltrimethylammonium bromide DAS days after sowing
DEEM-FCID Dietary Exposure Evaluation Model - Food Commodity Intake

Database DTT dithiothreitol DWCF dry weight conversion factor dwt dry weight of tissue E. coli Escherichia coli ECB European corn borer EDTA ethylenediaminetetraacetic ELISA enzyme-linked immunosorbent assay EPA Environmental Protection Agency FA fatty acid FDA Food and Drug Administration FIFRA Federal Insecticide, Fungicide and Rodenticide Act FMOC fluorenylmethyl chloroformate Gb gigabase GC gas chromatography GE genetically engineered GLP Good Laboratory Practice GRIN Germplasm Resources Information Network HD homeodomain HD-Zip homeodomain leucine zipper class family of transcription factors HPLC high-performance liquid chromatography

1 Alred, G.J., C.T. Brusaw, and W.E. Oliu. 2003. Handbook of Technical Writing, 7th edn., pp. 2-7. Bedford/St. Martin's, Boston, MA.Monsanto Company CR262-14U1 23 of 260

HRP horseradish peroxidase ICP inductively coupled plasma IgG immunoglobulin G ILSI-CCDB International Life Sciences Institute Crop Composition Database JSA junction sequence analysis JSC junction sequence class LB loading buffer LOD limit of detection LOQ limit of quantitation LZ leucine zipper
MALDI TOF MS matrix assisted laser desorption /ionization - time of flight mass

spectrometry

MIXED a statistical tool developed by the SAS Institute that uses a mixedmodel ANOVA to analyze data MMT million metric tons MOE margin of exposure MW molecular weight

n number of samples NDF neutral detergent fiber NFDM non-fat dry milk NGS next generation sequencing NHANES National Health and Nutrition Examination Survey NOAEL no observable adverse effect level OECD Organisation for Economic Co-operation and Development OGTR Office of the Gene Technology Regulator (Australia) OPA o-phthalaldehyde OSL over season leaf OSR over season root PBST phosphate-buffered saline with Tween- 20 PCR polymerase chain reaction PMSF phenylmethylsulfonyl fluoride ppm parts per million PRESS predicted residual sum of squares PVP polyvinylpyrrolidone QC quality control RT-PCR reverse transcription polymersae chain reaction SD standard deviation SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SE standard error SGF simulated gastric fluid SIF simulated intestinal fluid SOP standard operating procedure TDF total dietary fiber TFA trifluoroacetic acid UTR untranslated region v/v volume to volume ratio UV ultraviolet w/v weight to volume ratioMonsanto Company CR262-14U1 24 of 260 I. RATIONALE FOR THE DEVELOPMENT OF MON 87403

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 87403, any progeny derived from crosses between MON 87403 and conventional maize, and any progeny derived from crosses of MON 87403 with biotechnology-derived maize that have previously been granted nonregulated status under 7 CFR Part 340.

I.B. Rationale for the Development of MON 87403 Monsanto Company has developed biotechnology-derived maize MON 87403 that has increased ear biomass at an early reproductive stage (R1) compared to conventional control maize. MON 87403 was produced through insertion of the coding region of the full-length Arabidopsis thaliana ATHB17 gene through Agrobacterium -mediated transformation . The ATHB17 protein is a member of the HD-Zip family of plant transcription factors, which are proteins that bind to specific DNA sequences and regulate gene expression. The HD-Zip family of proteins is found broadly across plant species and specific HD-Zip proteins have been shown to play an important role in the modulation of plant growth and development. The HD-Zip family consists of four subfamilies and ATHB17 is a member of the class II subfamily. HD-Zip II proteins form either homodimers or heterodimers with other HD-Zip II proteins and function as repressors of gene expression. In MON 87403, maize-specific splicing of the ATHB17 t r a n s c r i p t r e s u l t s i n a t r

u n c a t e d p r o t e i n , A T H B 1 7 Δ 1 1 3 , w h i c h i s m i s s i n g t h e f i r s t 1 1 3 N

t e r m i n a l a m i n o a c i d s t h a t a r e e x p r e s s e d i n A r a b i d o p s i s

. T h e A T H B 1 7 ∆ 1 1 3 p r o t e i n retains the ability to form homo- and hetero-dimers and bind to target DNA sequences l i k e t h e f

u l l

- l e n g t h p r o t e i n

. A T H B 1 7 ∆ 1 1 3 i s , h o w e v e r , u n a b l e t o f u n c t i o n a s a transcriptional repressor because the protein lacks a functional repression domain. By a d o m i n a n t

- n e g a t i v e m e c h a n i s m , A T H B 1 7 ∆ 1 1 3 c a n a l t e r t h e a c t i v i t y o f e n d o g e n o u s m a i z e HD-Zip II proteins, which are predominantly expressed in ear tissue. Thus, the A T H B 1 7 ∆ 1 1 3 p r o t e i n l i k e l y m o d u l a t e s H D

- Z i p I I

- r e g u l a t e d p a t h w a y s i n t h e e a r , w h i c h leads to increased ear growth at an early reproductive stage (R1). This increased ear growth is associated with increased partitioning of dry matter (photosynthate) from the source (vegetative) tissue to the sink (ear) tissue in MON 87403 compared to conventional control plants. Supplemental information on the function of theMonsanto Company CR262-14U1 25 of 260

A T H B 1 7 ∆ 1 1 3 p r o t e i n i n M O N 8 7 4 0 3 i s d e s c r i b e d i n A p p e n d i x B : a n d i s a l s o p ub l i s h e d in Rice et al. (2014). Maize is one of the largest U.S. crops based on acreage and quantity harvested each year. In 2013 maize was planted on 35.48 million ha in the United States (USDA-FAS 2014). Because of its importance, plant breeders continuously strive to improve commercial maize yield. Initial improvements in maize yield were due to the domestication of varieties with desirable traits like larger ear biomass. Maize ears became larger over time during the domestication era (University of Utah 2014) . During the hybrid era (1939 to present), commercial maize yield in the U.S. increased nearly six-fold with an average 99 kg ha -1 increase every year (Lee and Tollenaar 2007). The major factor that contributed to yield increase during the hybrid era is a favorable response of hybrids to increased plant population density which resulted in an increase in the number of ears per hectare (Bruns and Abbas 2003). However, at the individual plant level, yield increase was associated with a decrease in ear size. Commercial varieties with MON 87403 can provide increased yield opportunity as this trait increases ear biomass during early reproductive stages. Because of the expected increase in human population growth over time, increased agricultural output will be required to come from increased productivity ( i.e ., yield per unit area) as opposed to an increase in area under production (OECD-FAO 2008). Agricultural biotechnology provides tools to help address the increasing demand for food and feed due to this population growth. Augmenting gains in yield opportunity from biotechnology as well as with continual gains from breeding would have a positive impact on the U.S. economy. For example, increasing the average rate of yield gain by just 1 bu/ac/year, over and above historical average yield gains for the next ten years, would have a net economic impact of $16B USD (Leibman et al. 2014).
Early reproductive stages in maize have been identified as a crucial phase during maize growth when a determination of maximum ear size is set based on plant genetics and environmental conditions (Borrás and Westgate 2006; Jones et al. 1996). Dry matter produced by the plant during reproductive stages is allocated to the ear for its growth (Ritchie et al. 1997) after the sink size is determined. Thus increase in ear size that is set during early reproductive stages is considered an important determinant of reproductive success (Borrás et al. 2004; Zaidi et al. 2003). Published literature suggests that a larger ear size at early reproductive stages can result in increased kernels per hectare (Fisher and Palmer 1983; Severini et al. 2011) thus potentially increasing agronomic benefit to farmers.
Efficacy of MON 87403 was demonstrated by directly comparing its R1 (silking stage) ear weight to a conventional control with the same genetic background. Ear weight is the total weight of the primary ear including the husk, shank, cob, silk and ovules at the R1 stage. MON 87403 and the control were grown at 13 field locations within U.S. maize production regions in 2012. MON 87403 and the control were planted at each site in randomized complete block designs with four replications. Measurements were collected from all of the plants at the R1 stage in a 1 m length of the designated row. Statistical comparisons were made between MON 87403 and the control across all 13 sitesMonsanto Company CR262-14U1 26 of 260

(combined-site analyses). The level of statistical significance for all statistical
c o m p a r i s o n s w a s p r e d e t e r m i n e d t o b e 5 % ( α = 0

. 0 5 )

. There was a statistically significant increase in R1 ear weight between MON 87403 and the control in the combined site analysis (Table I-1). The R1 ear weight in MON 87403 was 11.7% higher than the control (Table I-1). Table I-1. Differences in R1 Ear Weight between MON 87403 and the Conventional Control in 2012 U.S. Field Trials 1
Characteristic (units) MON 87403

(Mean ± SE) Control

(Mean ± SE) Change (%) p-value

R1 ear weight 2 (g) 144.50 (±8.47) 129.30 (±8.13) 11.7 0.004*

1 Locations included in the combined-site analysis: Jackson, Arkansas; Vermilion, Illinois; Warren, Illinois; Boone, Indiana; Greene, Iowa; Jefferson, Iowa; Pawnee, Kansas; (2 sites) Polk, Nebraska; York, Nebraska; Perquimans, North Carolina; Berks, Pennsylvania; Lehigh, Pennsylvania. 2 R1 ear weight is the dry weight of the entire primary ear at silking stage from all plants in the 1 m sampling.
* D e n o t e s s t a t i s t i c a l d i f

f e r e n c e b e t w e e n M O N 8 7 4 0 3 a n d t h e c o n t r o l ( α = 0

. 0 5 )

. N = 5 1

. In summary, MON 87403 provides a potential benefit to growers and the food and feed industry by conferring an increase in yield opportunity for maize.

I.C. Submissions to Other Regulatory AgenciesUnder 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 87403 by USDA constitutes only one component of the overall regulatory oversight and review of this product. As a practical matter, MON 87403 cannot be released and marketed until FDA and USDA have completed their reviews and assessments under their respective jurisdictions. As MON 87403 does not contain a PIP, no submission will be made to the EPA.

I.C.1. Submission to FDA MON 87403 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 accordance with this policy, Monsanto will be submitting a food/feed safety and nutritional assessment summary document to FDA in the near future.Monsanto Company CR262-14U1 27 of 260

I.C.2. Submissions to Foreign Government Agencies Consistent with our commitments to the Excellence Through Stewardship ® (ETS) Program 2 , Monsanto intends to obtain import approvals from all key maize import markets with functioning regulatory systems prior to commercial release of hybrids containing MON 87403.

® Excellence Through Stewardship is a registered trademark of Excellence Through Stewardship, Washington, DC. 2 http://www.excellencethroughstewardship.org/

.Monsanto Company CR262-14U1 28 of 260 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 87403 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 VIII; volunteer management of maize in Section VIII.H; and inter-species/genus introgression potential in Section IX.G.

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 2013a). 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).Monsanto Company CR262-14U1 29 of 260

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 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 maize varieties grown in the U.S. are hybrids, 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 (Duvick 1999). 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 87403 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 F 2 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 hybrid development.

II.C. Maize as a Test System in Product Safety Assessment Hybrid maize MPA640B (LH244 × LH287) and EXP257 (LH244 × LH295) were used as near isogenic, conventional controls for this submission (hereafter referred to as conventional controls), based on seed availability. As noted, one parent of each of these control maize lines is LH244, the inbred from which MON 87403 is derived, while the other parents (LH287 and LH295) are other maize inbreds. As such, both of these maizeMonsanto Company CR262-14U1 30 of 260

hybrids constitute relevant comparators for MON 87403. 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 regions. Both MPA640B and EXP257 were used in molecular characterization studies, while MPA640B was used as the conventional control in compositional analysis and 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 87403 test materials were generated for the molecular characterization (Sections III and IV), protein characterization and expression analysis (Section V), compositional analysis (Section VI), and phenotypic, agronomic and environmental interactions assessment (Section VII). The full molecular characterization studies (NGS/JSA) were conducted with the R 3 generation (Figure IV-4). Initiation of commercial breeding efforts was conducted with the R 4 generation (Figure IV-4). Protein characterization and expression analysis, composition analysis, and phenotypic, agronomic and environmental interactions assessment were conducted with various MON 87403 breeding generations as noted in the Breeding Tree (Figure IV-4).Monsanto Company CR262-14U1 31 of 260 III. DESCRIPTION OF THE GENETIC MODIFICATION
MON 87403 was developed through Agrobacterium tumefaciens- mediated transformation of maize immature embryos from line LH244 utilizing plasmid PV-ZMAP5714. This section describes the plasmid vector, the donor genes, and the regulatory elements used in the development of MON 87403. In this section, transfer DNA (T-DNA) refers to DNA that is transferred to the plant during transformation and retained through the development of MON 87403. An expression cassette is comprised of sequences to be transcribed and the genetic regulatory elements necessary for the expression of those sequences.

III.A. The Plasmid Vector PV-ZMAP5714 Plasmid vector PV-ZMAP5714 was used for the transformation of maize to produce MON 87403 and its plasmid map is shown in Figure III-1. The elements included in this plasmid vector are described in Table III-1. PV-ZMAP5714 is approximately 11.7 kb in length and contains three cassettes: one T-DNA element, delineated by Left and Right Border regions, contains the ATHB17 expression cassette, and the plasmid backbone contains the cp4 epsps selectable marker cassette and the aadA expression cassette. PV-ZMAP5714 employs a tandem T-DNA approach to generate marker-free plants (Huang et al. 2004). In this tandem T-DNA approach, a single right border and a single left border were used to achieve separate, unlinked insertion of the T-DNA as well as the cp4 epsps selectable marker gene located in the plasmid backbone. After initial selection of transformants for glyphosate tolerance, the cp4 epsps cassette was segregated away by conventional breeding and molecular analysis was used to identify plants containing only the intended T-DNA (and not the cp4 epsps ).

The T-DNA contains the ATHB17 expression cassette. The ATHB17 expression cassette is regulated by the e35s/Ract1 chimeric promoter from the 35S RNA of cauliflower mosaic virus (CaMV) and the act1 gene from Oryza sativa , the 5 ́ untranslated leader sequence of the Cab gene from Triticum aestivum, the act1 intron from Oryza sativa, and the 3 ́ untranslated region of heat shock protein 17 ( Hsp17 ) of Triticum aestivum . The plasmid backbone contains the cp4 epsps and aadA expression cassettes. The cp4 epsps expression cassette is regulated by the act1 promoter from Oryza sativa , the act1 intron from Oryza sativa , the CTP2 targeting sequence from Arabidopsis thaliana , and the nos 3 ′ u n t r a n s l a t e d r e g i o n f

r o m Agrobacterium tumefaciens . The aadA expression cassette isregulated by the bacterial promoter, and 3' untranslated region of an aminoglycosidemodifying enzyme, 3''(9)- O -nucleotidyltransferase from the transposon Tn7. During transformation, both the T-DNA and the cp4 epsps expression cassette were inserted into the maize genome (Section III.B). Subsequently, traditional breeding, segregation, selection and screening were used to isolate those plants that contain the ATHB17 expression cassette (T-DNA) and do not contain the cp4 epsps and aadA expression cassettes (backbone). The backbone region of PV-ZMAP5714, located outside of the T-DNA, contains two origins of replication for maintenance of the plasmid vector in bacteria ( ori-V , ori-pUC ), a bacterial selectable marker gene ( aadA ), and a coding sequence for repressor of primerMonsanto Company CR262-14U1 32 of 260

(ROP) protein for the maintenance of the plasmid vector copy number in Escherichia coli ( E. coli ). The backbone also contains the cp4 epsps expression cassette. A description of the genetic elements and their prefixes (e.g., B-, P-, L-, I-, CS-, T-, and OR-) in PV-ZMAP5714 is provided in Table III-1.

III.B. Description of the Transformation System MON 87403 was developed through Agrobacterium tumefaciens -mediated transformation of immature maize embryos based on the method described by Sidorov and Duncan (2009), utilizing PV-ZMAP5714. 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 , respectively. Once transformed callus developed, the callus was placed on media conducive to shoot and root development. The rooted plants (R 0 ) with normal phenotypic characteristics were selected and transferred to soil for growth and further assessment. As demonstrated in this petition (Section VII), the use of disarmed Agrobacterium tumefaciens strain ABI, a designated plant pest, as the transformation vector has not imparted plant pest characteristics to MON 87403.

The R 0 plants generated through the transformation process described above had already been exposed to glyphosate in the selection medium and demonstrated glyphosate tolerance. The R 0 plants containing the cp4 epsps expression cassette, as well as the TDNA cassette from the plasmid vector backbone, were self-pollinated to produce R 1 seed and R 1 plants (Huang et al. 2004). Subsequently, R 1 plants that were positive for the T-DNA and negative for the cp4 epsps expression cassette were identified by a polymerase chain reaction (PCR) based analysis (Huang et al. 2004). The R 1 plants homozygous for the T-DNA were selected for further development and their progenies were subjected to further molecular and phenotypic assessments. As is typical of a commercial event production and selection process, hundreds of different transformation events (regenerants) were generated in the laboratory using PV-ZMAP5714. After many months of careful selection and evaluation of these hundreds of events in the laboratory, greenhouse and field, MON 87403 was selected as the lead event based on superior agronomic, phenotypic, and molecular characteristics (Prado et al. 2014). Studies on MON 87403 were initiated to further characterize the genetic insertion and the expressed product, and to establish the food, feed, and environmental safety relative to commercial maize. The major steps involved in the development of MON 87403 are depicted in Figure III-2.

III.C. The ATHB17 Coding Sequence and the ATHB17 Protein (T-DNA)

The ATHB17 coding sequence is from Arabidopsis thaliana and encodes the ATHB17 protein (Figure III-3). For additional information about the truncated ATHB17 protein produced in MON 87403, see Section V.Monsanto Company CR262-14U1 33 of 260

Figure III-1. Circular Map of PV-ZMAP5714 A circular map of PV-ZMAP5714 used to develop MON 87403 is shown. PV-ZMAP5714 contains a single T-DNA. Genetic elements are shown on the exterior of the map.Monsanto Company CR262-14U1 34 of 260

Figure III-2. Schematic of the Development of MON 87403

1 MIKLLFTYIC TYTYKLYALY HMDYACVCMY KYKGIVTLQV CLFYIKLRVF 51 LSNFTFSSSI LALKNPNNSL IKIMAILPEN SSNLDLTISV PGFSSSPLSD 101 EGSGGGRDQL RLDMNRLPSS EDGDDEEFSH DDGSAPPRKK LRLTREQSRL 151 LEDSFRQNHT LNPKQKEVLA KHLMLRPRQI EVWFQNRRAR SKLKQTEMEC 201 EYLKRWFGSL TEENHRLHRE VEELRAMKVG PTTVNSASSL TMCPRCERVT 251 PAASPSRAVV PVPAKKTFPP QERDR

Figure III-3. Deduced Amino Acid Sequence of the ATHB17 Protein The amino acid sequence of the ATHB17 protein was deduced from the full-length coding nucleotide sequence present in PV-ZMAP5714. The amino-terminal 113 amino acids (highlighted in gray) are not predicted to be translated in MON 87403 as a result of mRNA splicing. Transformed LH244 (a maize line for more efficient transformation) immature embryos with PV-ZMAP5714 in Agrobacterium tumefaciens

Selected transformants containing the selectable marker ( cp4 epsps expression cassette) and generated rooted shoots from the transformed callus tissues

Identified MON 87403 as lead event and further evaluated its progeny in laboratory and field assessments for T-DNA insert integrity, absence of all other vector DNA including cp4 epsps, and superior phenotypic characteristics Assembled Agrobacterium binary plasmid vector PV-ZMAP57147 and transferred to Agrobacterium tumefaciens strain ABI

Evaluated by PCR and selected the transformed plants for the homozygous presence of the T-DNA and absence of the vector backbone that includes

the cp4 epsps expression cassette

Evaluated plants for insert integrity using molecular analysesMonsanto Company CR262-14U1 35 of 260

III.D. Regulatory Sequences

The ATHB17 coding sequence in MON 87403 is under the regulation of the e35S/Ract1 promoter, the chlorophyll a/b binding protein (CAB) leader, the Ract1 intron, and the heat shock protein 17 ( Hsp17) 3' untranslated region. The e35S/Ract1 promoter, which directs transcription in plant cells, is a chimeric promoter consisting of the duplicated enhancer region from the cauliflower mosaic virus 35S RNA promoter (CaMV) (Kay et al. 1987) combined with the promoter of the act1 gene from Oryza sativa that encodes Actin 1 (McElroy et al. 1990). The Cab leader sequence 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.

III.E. T-DNA Borders PV-ZMAP5714 contains Left and Right Border regions (Figure III-1 and Table III-1) that were derived from A. tumefaciens plasmids. The border regions each contain a 24-25 bp 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 (Section VII), the use of genetic elements from A. tumefaciens , a designated plant pest, has not imparted plant pest characteristics to MON 87403.

III.F. Genetic Elements Outside of the T-DNA Borders Genetic elements that exist outside of the T-DNA border regions are those that are essential for the maintenance or selection of PV-ZMAP5714 in bacteria and are referred to as plasmid backbone. 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-pUC , is required for the maintenance of the plasmid in E. coli and is derived from the plasmid vector pUC (Vieira and Messing 1987). Coding sequence rop encodes the repressor of primer (ROP) protein which is necessary for the maintenance of plasmid vector copy number in E. coli (Giza and Huang 1989). The backbone also contains the cp4 epsps expression cassette that codes for the CP4 EPSPS protein (conferring tolerance to glyphosate) that was used as the selectable marker during transformation (Huang et al. 2004). The cp4 epsps expression cassette is regulated by the Ract1 promoter from Oryza sativa , the Ract1 intron from Oryza sativa , the CTP2 targeting sequence from Arabidopsis thaliana , and the nos 3 ′ u n t r a n s l a t e d region from Agrobacterium tumefaciens . The absence of the backbone and other unintended plasmid sequence in MON 87403 was confirmed by sequencing and bioinformatic analyses (see Section IV.A).Monsanto Company CR262-14U1 36 of 260

Table III-1. Summary of Genetic Elements in Plasmid PV-ZMAP5714

Genetic Element Location in Plasmid Function (Reference)
T-DNA B 1 -Right Border Region 1-357 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) Intervening Sequence 358-375 Sequence used in DNA cloning P 2

- e35S/Ract1 376-1556 Chimeric promoter consisting of the duplicated enhancer region from the cauliflower mosaic virus 35S RNA promoter (CaMV) (Kay et al. 1987) combined with the promoter of the act1 gene from Oryza sativa that encodes Actin 1 (McElroy et al. 1990) that directs transcription in plant cells Intervening Sequence 1557-1561 Sequence used in DNA cloning L 3

- Cab 1562-1622 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 1623-1638 Sequence used in DNA cloning I 4

- Ract1 1639-2118 Intron and flanking UTR sequence of the act1 gene from Oryza sativa (rice) encoding rice Actin 1 protein. This sequence is involved in regulating gene expression (McElroy et al. 1990) Intervening Sequence 2119-2130 Sequence used in DNA cloning CS 5

- ATHB17 2131-2958 Coding sequence of the ATHB17 gene from Arabidopsis thaliana encoding a member of the class II homeodomain-leucine zipper gene family (HD-Zip II) that is thought to act as a transcription factor (Ariel et al. 2007) Intervening Sequence 2959-2971 Sequence used in DNA cloning T 6

- Hsp17 2972-3181 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 3182-3234 Sequence used in DNA cloning B-Left Border Region 3235-3676 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA (Barker et al. 1983)Monsanto Company CR262-14U1 37 of 260

Table III-1 (continued). Summary of Genetic Elements in Plasmid PV-ZMAP5714

Genetic Element Location in Plasmid Function (Reference)
Backbone Intervening Sequence 3677-3682 Sequence used in DNA cloning P- Ract1 3683-4603 Promoter and leader of the act1 gene from Oryza sativa (rice) encoding the rice Actin 1 protein (McElroy et al. 1990) that directs transcription in plant cells I- Ract1 4604-5081 Intron and flanking UTR sequence of the act1 gene from Oryza sativa (rice) encoding rice Actin 1 protein (McElroy et al. 1990). This sequence is involved in regulating gene expression Intervening Sequence 5082-5090 Sequence used in DNA cloning TS 7

- CTP2 5091-5318 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 5319-6686 Coding sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein that provides glyphosate tolerance (Barry et al. 2001); (Padgette et al. 1996) Intervening Sequence 6687-6701 Sequence used in DNA cloning T- nos 6702-6954 3' UTR sequence of the nopaline synthase ( nos ) gene from Agrobacterium tumefaciens pTi encoding NOS (Bevan et al. 1983; Fraley et al. 1983), that directs polyadenylation of the mRNA Intervening Sequence 6955-7005 Sequence used in DNA cloning OR 8

- ori V 7006-7402 Origin of replication from the broad host range plasmid RK2 , used for maintenance of plasmid in Agrobacterium (Stalker et al. 1981) Intervening Sequence 7403-8910 Sequence used in DNA cloning CS- rop 8911-9102 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 9103-9529 Sequence used in DNA cloning OR- ori-pUC 9530-10118 Origin of replication from plasmid pUC for maintenance of plasmid in E. coli (Vieira and Messing 1987)Monsanto Company CR262-14U1 38 of 260

Table III-1 (continued). Summary of Genetic Elements in Plasmid PV-ZMAP5714

Genetic Element Location in Plasmid Function (Reference) Intervening Sequence 10119-10648 Sequence used in DNA cloning aadA 10649-11537 Bacterial promoter, coding sequence, and 3' UTR for an aminoglycoside-modifying enzyme, 3''(9)- O -nucleotidyltransferase from the transposon Tn7 (Fling et al. 1985). This sequence confers spectinomycin and streptomycin resistance Intervening Sequence 11538-11673 Sequence used in DNA cloning 1 B, Border 2 P, Promoter 3 L, Leader 4 I, Intron 5 CS, Coding Sequence 6 T, Transcription Termination Sequence 7 TS, Targeting Sequence 8 OR, Origin of ReplicationMonsanto Company CR262-14U1 39 of 260 IV. CHARACTERIZATION OF THE GENETIC MODIFICATION
Characterization of the DNA insert in MON 87403 was conducted using a combination of sequencing, PCR, and bioinformatics. The results of this characterization demonstrate that MON 87403 contains one copy of the intended transfer DNA (T-DNA) containing the ATHB17 expression cassette 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:

1. Molecular characterization of MON 87403 by Next Generation Sequencing and Junction Sequence Analysis (NGS/JSA) demonstrated that MON 87403 contained a single intended DNA insert. These whole-genome sequence analyses provided a comprehensive assessment of MON 87403 to determine the presence and identity of sequences derived from PV-ZMAP5714 (DuBose et al. 2013; Kovalic et al. 2012), demonstrating that MON 87403 contained a single T-DNA insert with no detectable backbone sequences.

2. Directed sequencing (locus-specific PCR, DNA sequencing and analyses) performed on MON 87403 was used to determine the complete sequence of the single DNAinsert from PV-ZMAP5714, the adjacent flanking DNA, and the 5' and 3' insert-toflank junctions. This analysis confirmed that the sequence and organization of the inserted DNA is identical to the corresponding region in the PV-ZMAP5714 T-DNA. Furthermore, the genomic organization at the insertion site was assessed by comparing the sequences flanking the T-DNA insert in MON 87403 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 87403 upon DNA integration.

3. Generational stability analysis by NGS/JSA demonstrated that the single PV-ZMAP5714 T-DNA insert in MON 87403 has been maintained through five breeding generations, thereby confirming the stability of the T-DNA in MON 87403.

4. 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 87403 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 87403. 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. Appendix C provides an additional overview of these techniques, their use in DNA characterization in crop plants and the materials and methods.Monsanto Company CR262-14U1 40 of 260

Figure IV-1. Molecular Characterization using Sequencing and Bioinformatics Genomic DNA from the test and the conventional control was sequenced using technology that produces a set of short, randomly distributed sequence reads (each approximately 100 bp long) that comprehensively cover both genomes (Step 1). Utilizing these genomic sequence reads, bioinformatics search tools were used to select all sequence reads that were significantly similar to the transformation plasmid (Step 2) for use in read mapping to determine the presence/absence of backbone sequences and Junction Sequence Analysis (JSA) bioinformatics to determine the insert and copy number (Step 3). Overlapping PCR products are also produced which span any inserts and their wild type locus (Step 4 and Step 5 respectively); these overlapping PCR products are sequenced to allow for detailed characterization of the inserted DNA and insertion site(s). The NGS/JSA method characterized the genomic DNA from MON 87403 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. 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). To confirm sufficient sequence coverage of the genome, the 100-mer sequence reads are analyzed to determine the coverage of a known single-copy endogenous gene, this demonstrates the depth of coverage (the median number of times each base of the genome is independently sequenced). The level of sensitivity of this method was demonstrated by detection of a positive control spiked at 1 and 1/10 th copy-per-genome equivalent, thus confirming the Step 1: Next GenerationSequencing (NGS) of genomic DNA samples. A collection of 100-mer sequences are generatedwhich comprehensively cover the test andcontrol sample genomes

Step 2: Selection of all 100-mers containing sequence significantly similar to that of the transformation plasmid

Step 3: Bioinformatic analysis to detect and characterize all selected100-mersequences originating from the transgenic insertions

Step 4: Directed sequencing across the insertion from the 5' flank to the 3' flank

Step 5: Directedsequencing across the wildtype insertion site 1) Presence or absence of backbone insertions: No unintendedbackbone sequences detected (Read mapping) 2) Insert and copy number determined: Junctionsequence pairs detected (JSA)

3) Exact sequence of insert(s) is determined 4) Organization and intactness of genetic elements in the insert sequence is confirmed

5) Integrity andorganization of insertionsite(s) Experimental Stage Resultant MolecularCharacterization

NGS/JSA

Directed SequencingMonsanto Company CR262-14U1 41 of 260

method’s ability to detect any sequences derived from the transformation plasmid. Bioinformatics analysis was then used to select sequencing reads that contained sequences similar to the transformation plasmid, and these were analysed in depth to determine the number and the identity of sequence in the DNA inserts. NGS/JSA was run on all 5 generations of MON 87403 samples and the conventional controls. Results of NGS/JSA are shown in Sections IV.A and IV.D. The number of DNA inserts was determined by analyzing sequence reads for novel junctions, while the identity of the inserted DNA and absence of backbone was assessed by sequence read mapping. The junctions of the DNA insert and the flanking DNA are unique for each insertion (Kovalic et al. 2012). An example is shown in Figure IV-2. 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, one at the 5' end of the insert (Figure IV-3, named junction sequence class A, or JSC-A, in this case) and one at the 3' end of the insert (junction sequence class B, JSC-B) (Kovalic et al. 2012).Monsanto Company CR262-14U1 42 of 260

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. A group of junction sequences which share the same junction point and common flanking sequence is called a Junction Sequence Class (or JSC).

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. ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACG ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACGTGT ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACGTGTCG ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACGTGTCGGG ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACGTGTCGGGAT

FlankingDNA Plasmid DNA Junction



## DNA insert 5’ Flank 3’ Flank Insert Junction Regions Junction Sequences: Class A

Junction Sequences: Class BMonsanto Company CR262-14U1 43 of 260

By evaluating the number of 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, each originating from one end of the insert, both containing portions of plasmid DNA insert and flanking sequence. Directed sequencing (locus-specific PCR and DNA sequencing analyses, Figure IV-1, step 5) complements the NGS/JSA analyses. Sequencing of the insert and flanking genomic DNA determined the complete sequence of the insert and flanks. This analysis evaluates if the sequence of the insert is identical to the corresponding sequence from the T-DNA in PV-ZMAP5714, if each genetic element in the insert is intact, if the T-DNA sequence is inserted as a single copy, and further confirms that no vector backbone or other unintended plasmid sequences were present within the T-DNA insert in MON 87403. Results are described in Sections IV.B and IV.C; methods are presented in Appendix C: . The stability of the T-DNA present in MON 87403 across multiple generations was evaluated by NGS/JSA analyses 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. All integrated sequences are expected to align to the T-DNA region of the plasmid. Results are described in Section IV.D.; methods are presented in Appendix C: . Segregation analysis of the T-DNA was conducted to determine the inheritance and stability of the insert in MON 87403. Segregation analysis corroborates the insert stability demonstrated by NGS/JSA and independently establishes the genetic behavior of the T-DNA. Results and methods are described in Section IV.E.

IV.A. Determining the Number and Identity of DNA Inserts in MON 87403 The number of insertion sites of PV-ZMAP5714 DNA in MON 87403 was assessed by performing NGS/JSA on MON 87403 genomic DNA. A plasmid map of PV-ZMAP5714 is shown in Figure III-1. Table IV-1 provides descriptions of the genetic elements present in MON 87403. A schematic representation of the insert and flanking sequences in MON 87403 is shown in Figure IV-4. For full details on materials and methods see Appendix C.

IV.A.1. Next Generation Sequencing of MON 87403 and Conventional Control Genomic DNA Genomic DNA from five generations of MON 87403 and the appropriate conventional control (Figure IV-5) was isolated from seed and prepared for sequencing according to the manufacturer’s protocol (Illumina, TruSeq library protocol. For material and method details see Appendix C: ). These genomic DNA libraries were used to generate short (~100 bp) randomly distributed sequence fragments (sequencing reads) of the maize genome (see Figure IV-1, Step 1).Monsanto Company CR262-14U1 44 of 260

To demonstrate sufficient sequence coverage the 100-mer sequence reads were analyzed by mapping all reads to a known single-copy endogenous gene ( Zea mays pyruvate decarboxylase (

pdc3 ), GenBank accession version: AF370006.2). The analysis of sequence coverage plots showed that the depth of coverage ( i.e. , the median number of times any base of the genome is expected to be independently sequenced) was 75× or greater for the five generations of MON 87403 (R 3 , R 4 , R 5 , R 4 F 1 and R 5 F 1 ) and the conventional control. 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). To demonstrate the method’s ability to detect any sequences derived from the PV-ZMAP5714 transformation plasmid, a sample of conventional control DNA spiked with PV-ZMAP5714 DNA at 1 and 1/10 th genome equivalent was analyzed by NGS and bioinformatics. The level of sensitivity of this method was demonstrated to a level of 1 genome equivalent, 100% nucleotide identity was observed over 100% of PV-ZMAP5714 (Table C-2). 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 1/10 th genomic equivalent (99.97% coverage at 98.83% identity for the 1/10 th genome equivalent spiked control sample, Table C-2) and, hence, a detection level of at most 1/10 th genome equivalent was achieved for the plasmid DNA sequence assessment.

IV.A.2. Characterization of insert number in MON 87403 using Bioinformatic Analysis

The number of insertion sites of DNA from PV-ZMAP5714 in MON 87403 was assessed by performing NGS/JSA on MON 87403 genomic DNA using the R 3 generation (Figure IV-5).Monsanto Company CR262-14U1 45 of 260

Table IV-1. Summary of Genetic Elements in MON 87403 Genetic Element 1 Location in Sequence 2 Function (Reference) 5' Flanking DNA 1-1345 DNA sequence flanking the 5' end of the insert B 3 -Right Border Region r1 1346-1369 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) Intervening Sequence 1370-1387 Sequence used in DNA cloning P 4 -e35S/Ract1 1388-2568 Chimeric promoter consisting of the duplicated enhancer region from the cauliflower mosaic virus 35S RNA promoter (CaMV) (Kay et al. 1987) combined with the promoter of the act1 gene from Oryza sativa that encodes Actin 1 (McElroy et al. 1990) that directs transcription in plant cells Intervening Sequence 2569-2573 Sequence used in DNA cloning L 5 -Cab 2574-2634 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 2635-2650 Sequence used in DNA cloning I 6 -Ract1 2651-3130 Intron and flanking UTR sequence of the act1 gene from Oryza sativa (rice) encoding Actin 1 protein. This sequence is involved in regulating gene expression (McElroy et al. 1990)Monsanto Company CR262-14U1 46 of 260

Table IV-1 (continued). Summary of Genetic Elements in MON 87403

Genetic Element 1 Location in Sequence 2 Function (Reference) Intervening Sequence 3131-3142 Sequence used in DNA cloning CS 7 -ATHB17 3143-3970 Coding sequence of the ATHB17 gene from Arabidopsis thaliana encoding a member of the class II homeodomain-leucine zipper gene family (HD-Zip II) that is thought to act as a transcription factor (Ariel et al. 2007) Intervening Sequence 3971-3983 Sequence used in DNA cloning T 8 -Hsp17 3984-4193 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 4194-4246 Sequence used in DNA cloning B-Left Border Region r1 4247-4477 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA (Barker et al. 1983) 3' Flanking DNA 4478-5744 DNA sequence flanking the 3' end of the insert
1 Although flanking sequences and intervening sequences are not functional genetic elements; they comprise a portion of the sequence. 2 Numbering refers to the sequence of the insert in MON 87403 and adjacent DNA. 3 B, Border 4 P, Promoter 5 L, Leader Sequence 6 I, Intron Sequence 7 CS, Coding Sequence 8 T, Transcriptional Terminator r1 Superscript in Left and Right Border Regions indicate that the sequence in MON 87403 was truncated compared to the sequences in PV-ZMAP5714.Monsanto Company CR262-14U1 47 of 260

Figure IV-4. Schematic Representation of the Insert and Flanking Sequences in MON 87403 DNA derived from T-DNA of PV-ZMAP5714 integrated in MON 87403. Right-angled arrows indicate the ends of the integrated T-DNA and the beginning of the flanking sequence. Genetic elements within the insert are identified on the map. 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 87403 was truncated compared to the sequences in PV-ZMAP5714.Monsanto Company CR262-14U1 48 of 260

Figure IV-5. Breeding History of MON 87403 R 0 corresponds to the transformed plant, F# is the filial generation,  designates self-pollination. 1 Generation used for molecular characterization 2 Generations used to confirm insert stability 3 Generation used for commercial development of MON 87403 4 Generation used for agronomic/phenotypic and compositional analysis studies



### LH244 R 0



### LH244 R 1



### LH244 R 2



### LH244 R 3 1,2



### LH244 R 4 2,3



### LH244 R 5 2 

### R 5 

### F 1 

### (LH244 x LH287 ) 2,4 

### R 4 

### F 1 

### (LH244 x LH295

) 2Monsanto Company CR262-14U1 49 of 260

IV.A.2.1. Selection of Sequence Reads Containing Sequence of the PV-ZMAP5714 PV-ZMAP5714 was transformed into the parental variety LH244 to produce MON 87403. Consequently, any DNA inserted into MON 87403 will consist of sequences that are similar to the PV-ZMAP5714 DNA sequence. Therefore, to fully characterize the DNA from PV-ZMAP5714 inserted in MON 87403, it is sufficient to completely analyze only the sequence reads that have similarity to the transformation plasmid (Figure IV-1, Step 2). Using established criteria (described in the materials and methods, Appendix C: ), sequence reads similar to the transformation plasmid were selected from MON 87403 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 plasmid sequence as a query to determine the DNA insertion site number. Any inserted transformation plasmid sequence, regardless of origin, either T-DNA or backbone, can be identified by mapping reads to the transformation plasmid sequence while the number of inserted DNA molecules can be determined using JSA. Therefore unlike the traditional Southern blot analysis that separately hybridizes T-DNA or backbone probes, in NGS/JSA the determination of the T-DNA insert number and of the absence of backbone or unintended sequences are simply represented by the identification of sequence reads that match the transformation plasmid, 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). If MON 87403 contains a single T-DNA insert, two junction sequence classes (JSCs), each containing portions of T-DNA sequence and flanking sequence, will be detected. To determine the insert number in MON 87403, 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 Table IV-2.Monsanto Company CR262-14U1 50 of 260

Table IV-2. Unique Junction Sequence Class Results

Sample Junction Sequence

Classes Detected MON 87403 2 LH244 0
The location and orientation of the junction sequences relative to the T-DNA insert determined for MON 87403 (as described in Section IV.B) are illustrated in Figure IV-6. As shown in the figure, there are two junction sequence classes identified in MON 87403. Junction Sequence Class A and Class B (JSC-A and JSC-B) both contain the T-DNA border sequence joined to genomic flanking sequence, indicating that they represent the sequences at the junctions of the intended T-DNA insert and genomic 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-ZMAP5714 T-DNA at a single locus in the genome of MON 87403. JSC-A represents the junction of the T-DNA Left Border sequence to the 5' flank and JSC-B represents the junction of the T-DNA Right Border sequence to the 3' flank. Complete alignment of the JSCs to the full flank/insert sequence confirms that both of these JSCs originate from the same locus of the MON 87403 genome and are linked by contiguous, known and expected DNA that makes up the single insert.

IV.A.2.3. Determination of the Inserted DNA Identity To determine the identity of inserted DNA, all selected sequences decribed in section IV.A.2.1 were mapped to the transformation plasmid sequence. While thousands of sequence reads from the R 3 generation mapped to the plasmid T-DNA sequence, only four reads mapped to the plasmid backbone. From this result it was determined that MON 87403 does not contain any sequence from the transformation plasmid backbone. Based on the comprehensive NGS/JSA study, it was concluded that MON 87403 contains one T-DNA inserted into a single locus, as shown in Figure IV-6 and is devoid of backbone sequence. The identity of the DNA insert was confirmed by the sequencing and analysis of overlapping PCR products from this locus as described below in Section IV.B., which showed that the DNA insert contained only the T-DNA elements from the plasmid.Monsanto Company CR262-14U1 51 of 260

Figure IV-6. Junction Sequences Detected by NGS/JSA Linear map of MON 87403 illustrating the relationship of the detected junction sequences to the insert locus. The individual junction sequences detected by JSA are illustrated as stacked bars.
r1 Superscript in Left and Right Border Regions indicate that the sequence in MON 87403 was truncated compared to the sequences in PV-ZMAP5714.Monsanto Company CR262-14U1 52 of 260

IV.B. Organization and Sequence of the Insert and Adjacent DNA in MON 87403 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 5). PCR primers were designed to amplify three overlapping regions of the MON 87403 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 87403 insert is 3,132 bp and that each genetic element within the T-DNA is intact compared to the transformation plasmid PV-ZMAP5714, with the exception of the 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-ZMAP5714. The sequence and organization of the insert was also shown to be identical to the corresponding T-DNA of PV-ZMAP5714, confirming that a single copy of the T-DNA was inserted as intended. This analysis also shows that only T-DNA elements (described in Table IV-1) were present. Moreover, the result, together with the conclusion of single DNA insert detected by NGS/JSA, demonstrated that no PV-ZMAP5714 backbone elements are present in MON 87403.Monsanto Company CR262-14U1 53 of 260

Figure IV-7. Overlapping PCR Analysis Across the Insert in MON 87403 PCR was performed on both conventional control genomic DNA and MON 87403 genomic DNA using three pairs of primers to generate overlapping PCR fragments from MON 87403 for sequencing analysis. To verify the PCR products, a portion of each PCR was loaded on a 1% (w/v) agarose gel and visualized by ethidium bromide staining. The expected product size for each amplicon is provided in the illustration. Lane designations are as follows: Lane Lane 1 1 Kb Plus DNA Ladder 7 PV-ZMAP5714 2 MON 87403 8 Conventional Control 3 No Template Control 9 MON 87403 4 Conventional Control 10 No Template Control 5 MON 87403 11 Conventional Control 6 No Template Control 12 1 Kb Plus DNA Ladder Arrows next to the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained from the 1 Kb DNA Plus 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 87403 was truncated compared to the sequences in PV-ZMAP5714. 1 2 3 4 5 6 7 8 9 10 11 12 12

3 2 1.6 1

0.2 12

3 2 1.6 1

0.2 Product A Product B Product CMonsanto Company CR262-14U1 54 of 260

IV.C. Sequencing of the MON 87403 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 6). The PCR was performed with one primer specific to the genomic DNA sequence flanking the 5' end of the MON 87403 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 87403 indicates that 149 bases of maize genomic DNA were deleted during integration of the T-DNA. The remainder of the flanks in MON 87403 are 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 Agrobacterium -mediated transformation process (Salomon and Puchta 1998).Monsanto Company CR262-14U1 55 of 260

Figure IV-8. PCR Amplification of the MON 87403 Insertion Site PCR analysis was performed to evaluate the 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 ′ f l a n k i n g s e qu e n c e o f t h e i n s e r t i n M O N 8 7 4 0 3

. T h e D N A g e n e r a t e d f r o m t h e c o n v e n t i o n a l control PCR was used for sequencing analysis. This illustration depicts the MON 87403 insertion site in the conventional control (upper panel) and the MON 87403 insert (lower panel). To verify the PCR products, a portion of each PCR was loaded on the gel. Lane designations are as follows: Lane 1 1 Kb Plus DNA Ladder 2 Conventional Control 3 No template DNA control Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Plus 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 87403 was truncated compared to the sequences in PV-ZMAP5714. Primer A Primer A Primer B

Primer B 1 2 3

12

3 2 1.6 1

0.2Monsanto Company CR262-14U1 56 of 260

IV.D. Determination of Insert Stability over Multiple Generations of MON 87403 In order to demonstrate the stability of the T-DNA present in MON 87403 through multiple generations, NGS/JSA analysis was performed using DNA obtained from five breeding generations of MON 87403. The breeding history of MON 87403 is presented in Figure IV-5, and the specific generations tested are indicated in the figure legend. The MON 87403 R 3generation was used for the molecular characterization analyses discussed in Sections IV.AIV.C. and shown in Figure IV-5. 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 R 3 generation. The conventional controls used for the generational stability analysis included LH244, which included similar background genetics to the R 3 , R 4 and the R 5 generations and represents the original transformation line; LH244 × LH295, a hybrid with similar background genetics to the R 4 F 1 hybrid; and LH244 × LH287, a hybrid with similar background genetics to the R 5 F 1 hybrid. Genomic DNA isolated from each of the selected generations of MON 87403 and conventional control was used for NGS/JSA analysis. To determine the insert number in the MON 87403 generations, the sequences selected as described in Section IV.A.2.1 were analyzed using JSA (Kovalic et al. 2012). Table IV-3 shows the number of resultant JSCs containing PV-ZMAP5714 DNA sequence determined by this analysis. Table IV-3. Junction Sequence Classes Detected
Sample Junction Sequence

Classes Detected MON 87403 (R 3 ) 2 MON 87403 (R 4 ) 2 MON 87403 (R 4 F 1 ) 2 MON 87403 (R 5 ) 2 MON 87403 (R 5 F 1 ) 2 LH244 0 LH244 × LH295 0 LH244 × LH287 0
Alignment of the JSCs from each of the assessed MON 87403 generations (R 4 , R 5 , R 4 F 1, and R 5 F 1 ) to the full flank/insert sequence and JSCs determined for the MON 87403 R 3 generation, confirms that the pair of JSCs originates from the same region of the MON 87403 genome and is linked by contiguous, known and expected DNA sequence. This single identical pair of JSCs is observed as a result of the insertion of PV-ZMAP5714 T-DNA at a single locus in the genome of MON 87403. The consistency of these JSC data across all generations tested demonstrates that this single locus was stably maintained throughout the MON 87403 breeding process. These results demonstrate that the MON 87403 single integration locus was maintained through several generations of breeding MON 87403; thereby confirming the stability of the insert.Monsanto Company CR262-14U1 57 of 260

Based on this comprehensive sequence data and bioinformatic analysis (NGS/JSA), it is concluded that MON 87403 contains a single and stable T-DNA insertion.

IV.E. Inheritance of the Genetic Insert in MON 87403 The MON 87403 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 lines containing MON 87403, genotypic segregation data were recorded to assess the inheritance and
s t a b i l i t y o f t h e M O N 8 7 4 0 3 T

- D N A u s i n g C h i

- s qu a r e ( χ 2 ) analysis over several generations. The χ 2 analysis is based on comparing the observed segregation ratio to the expected segregation ratio according to Mendelian principles. The MON 87403 breeding path for generating segregation data is described in Figure IV-9. The transformed R 0 plant was self-pollinated to generate R 1 seed. An individual plant homozygous for the MON 87403 T-DNA was identified in the R 1 segregating population via an End-Point TaqMan  PCR assay. The homozygous positive R 1 plant was self-pollinated to give rise to R 2 seed. The R 2 plants were self-pollinated to produce R 3 seed. The R 3 plants were self-pollinated to produce R 4 seed. R 4 plants homozygous for the MON 87403 T-DNA were crossed via traditional breeding techniques to a Monsanto proprietary recurrent parent that does not contain the ATHB17 coding sequence to produce hemizygous R 4 F 1 seed. The R 4 F 1 plants were crossed with the recurrent parent to produce BC 1 F 1 seed. The BC 1 F 1 generation was tested for the presence of the T-DNA by End-Point TaqMan PCR to select for hemizygous MON 87403 plants. BC 1 F 1 plants hemizygous for MON 87403 T-DNA were crossed with the recurrent parent to produce the BC 2 F 1 plants. BC 2 F 1 plants hemizygous for MON 87403 T-DNA were crossed with the recurrent parent to produce the BC 3 F 1 plants.

The inheritance of the MON 87403 T-DNA was assessed in the BC 1 F 1 , BC 2 F 1 , and BC 3 F 1 generations. At the BC 1 F 1 , BC 2 F 1 , and BC 3 F 1 generations, the MON 87403 T-DNA was predicted to segregate at a 1:1 ratio (hemizygous positive: homozygous negative) according to Mendelian inheritance principles.

A P e a r s o n ’ s c h i

- s qu a r e ( χ 2 ) analysis was used to compare the observed segregation ratios of the ATHB17 coding sequence to the expected ratios. The Chi-square was calculated as:
χ 2 = ∑ [ ( | o

– e | ) 2 / e]

 TaqMan is a registered trademark of Roche Molecular Systems, Inc.Monsanto Company CR262-14U1 58 of 260

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

. 0 5 )

. T h e r e s u l t s o f t h e χ 2 analysis of the segregating progeny of MON 87403 are presented in Table I V

- 4

. T h e χ

2 value in the BC 1 F 1 , BC 2 F 1 , and BC 3 F 1 generations indicated no statistically significant difference between the observed and expected 1:1 segregation ratio (hemizygous positive: homozygous negative) of MON 87403 T-DNA. These results support the conclusion that the MON 87403 T-DNA resides at a single locus within the maize genome and is inherited according to Mendelian principles of inheritance. These results are also consistent with the molecular characterization data indicating that MON 87403 contains a single intact copy of the ATHB17 expression cassette inserted at a single locus in the maize genome.Monsanto Company CR262-14U1 59 of 260

Figure IV-9. Breeding Path for Generating Segregation Data for MON 87403 Chi-square analysis was conducted on segregation data from BC 1 F 1 , BC 2 F 1 , and BC 3 F 1 generations (bolded text). TI: T rait I ntegration: Replacement of genetic background of MON 87403 by recurrent background except inserted gene. RP: Recurring parent BC: Back-Cross.  : Self- Pollinated.

LH244 R 1

Breeding path continued LH244 R 4 LH244 R 3 LH244 R 2 

  Transformed LH244 R 0 Plant



R 4 F 1 (LH244 R 4 x RP)

TI:BC 1 F 1

TI:BC 2 F 1 x RP

x RP x RP

TI:BC 3 F 1

x RP (Expected segregation 1:1) (Hemizygous positive: Homozygous negative)

(Expected segregation 1:1) (Hemizygous positive: Homozygous negative)

(Expected segregation 1:1) (Hemizygous positive: Homozygous negative)Monsanto Company CR262-14U1 60 of 260

Table IV-4. Segregation of the Expression Cassette During the Development of MON 87403

Generation Number of

plants Observed

Positives Observed

Negatives Expected

Positives Expected

Negatives χ 2 Probability

BC 1 F 1 180 88 92 90.00 90.00 0.09 0.766

BC 2 F 1 178 82 96 89.00 89.00 1.10 0.294

BC 3 F 1 181 101 80 90.50 90.50 2.44 0.119Monsanto Company CR262-14U1 61 of 260

IV.F. Characterization of the Genetic Modification Summary and Conclusion Molecular characterization of MON 87403 by NGS/JSA and directed sequencing demonstrated that a single copy of the intended transfer DNA (T-DNA) containing the ATHB17 expression cassette from PV-ZMAP5714 was integrated into the maize genome at a single locus. These analyses also showed no PV-ZMAP5714 backbone elements were present in the event. Directed sequence analyses performed on MON 87403 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 ATHB17 expression cassette. Analysis of the T-DNA insertion site in maize showed that the 5' and 3' genomic DNA flanking the T-DNA insert in MON 87403 are identical to the conventional control, except for a 149 base pair deletion of genomic DNA at the insertion site in MON 87403. This deletion is not expected to affect food or feed safety. Generational stability analysis by NGS/JSA demonstrated that the T-DNA in MON 87403 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 genetic behavior of the T-DNA in MON 87403 at a single chromosomal locus.Monsanto Company CR262-14U1 62 of 260 V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE A T H B 1 7 Δ 1 1 3 PROTEIN PRODUCED IN MON 87403
Characterization of the introduced protein(s) in a biotechnology-derived crop is important to establishing food, feed, and environmental safety. As described in Sections IV and V, MON 87403 contains an ATHB17 expression cassette that, when transcribed and translated, results in the expression of the A T H B 1 7 Δ 1 1 3 protein. This section summarizes: 1) the identity and function of the A T H B 1 7 Δ 1 1 3 protein produced in MON 87403; 2) assessment of equivalence between the plant-produced and E. coli -produced proteins; 3) the level of the A T H B 1 7 Δ 1 1 3 protein in plant tissues from MON 87403; 4) assessment of the potential allergenicity of the A T H B 1 7 Δ 1 1 3 protein produced in MON 87403; and 5) the food and feed safety assessment of the A T H B 1 7 Δ 1 1 3 protein produced in MON 87403. The data support a conclusion that the A T H B 1 7 Δ 1 1 3 protein produced in MON 87403 is safe for human or animal consumption based on several lines of evidence summarized below.

V.A. Identity and Function of the A T H B 1 7 Δ 1 1 3 Protein from MON 87403

V.A.1. S e q u e n c e I d e n t i t y o f t h e A T H B 1 7 Δ 1 1 3 P r o t e i n f r o m M O N 8 7 4 0 3
As described in Section IV, the ATHB17 gene is derived from Arabidopsis thaliana

. Compared to the wild type sequence of the ATHB17 protein, the amino acid sequence of the ATHB17-encoded protein expressed in MON 87403 lacks 113 amino acids due to
a l t e r n a t i v e m R

N A s p l i c i n g , a n d h a s t h u s b e e n d e s i g n a t e d A T H B 1 7 ∆ 1 1 3

. T h e ATHB17 expression cassette in MON 87403 includes the ATHB17 coding sequence and the I- Ract1 intron (Table IV-1). Typically, pre-mRNA processing includes mRNA splicing, which consists of the removal of the introns from pre-mRNA transcripts and ligation of exons to produce mature mRNA (Simpson and Filipowicz 1996). To determine the sequence of the mature ATHB17 mRNA transcript in MON 87403, total RNA was extracted from leaf tissue and then PolyA + RNA was enriched. mRNA transcripts of ATHB17 were amplified by RT-PCR and then analyzed by sequence analysis. In MON 87403, the ATHB17 pre-mRNA is spliced to remove the majority of the I- Ract1 intron as well as a portion of the ATHB17 gene (Figure V-1). In order to predict the amino acid sequence of the protein produced from the ATHB17 mRNA, a Basic Local Alignment Search Tool (BLASTX) search was performed to compare the ATHB17 mRNA consensus sequence against the GenBank_Protein_Preferred protein sequence database. The top result of this homology search was the Arabidopsis thaliana ATHB17 protein sequence (GenBank ID 179876107). The first methionine (M) amino acid in the BLASTX alignment provides a predicted translation start site for the ATHB17-derived p r o t e i n i n M O N 8 7 4 0 3 , a n d r e s u l t s i n t h e p r o t e i n A T H B 1 7 ∆ 1 1 3 , w h i c h i s l a c k i n g t h e N-terminal 113 amino acids found in wild type ATHB17 (Figure V-2). To confirm the p r e s e n c e o f t h e A T H B 1 7 Δ 1 1 3 p r o t e i n i n M O N 8 7 4 0 3 , w e s t e r n b l o t a n a l y s i s w a s conducted on MON 87403 with an anti-ATHB17 antibody. Western blots revealed the presence of an immunogenic band corresponding to the expected molecular weight of the p r e d i c t e d A T H B 1 7 ∆ 1 1 3 p r o t e i n ( F i g u r e D

- 2 )

.Monsanto Company CR262-14U1 63 of 260

Figure V-1. Insert map of MON 87403 and schematic diagram of ATHB17 transcription, mRNA processing, and predicted translation The nucleotide sequence of the mature ATHB17 mRNA was determined using RT-PCR followed by Sanger sequencing. The mRNA sequence that codes for the ATHB17 protein in MON 87403 was determined. This analysis allowed for the identification of the splice junction in MON 87403 ATHB17 mRNA. The maize splicing of MON 87403 ATHB17 mRNA results in the excision of the I- Ract1 element (splice junction site 2662) and a portion of the CS-

ATHB17 coding region (splice junction site 3417). The observed CS- ATHB17 coding region is predicted to produce a version of theATHB17 protein in MON 87403 without the first 113 amino acids at the N-terminus compared to the sequence of ATHB17 found in Arabidopsis (the translation initiation site is denoted by an *). ATHB17 mRNA 2662

Spliced Region

A

T H B 1 7 Δ 1 1 3 Translation Transcription & mRNA Processing

3417
*Monsanto Company CR262-14U1 64 of 260 1 MNRLPSSEDG DDEEFSHDDG SAPPRKKLRL TREQSRLLED SFRQNHTLNP KQKEVLAKHL 61 MLRPRQIEVW FQNRRARSKL KQTEMECEYL KRWFGSLTEE NHRLHREVEE LRAIKVGPTT 121 VNSASSLTMC PRCERVTPAA SPSRAVVPVP AKKTFPPQER DR
Figure V-2. Predicted Amino Acid Sequence of the Protein Produced by MON 87403 ATHB17 mRNA The consensus sequence of MON 87403 mature ATHB17 mRNA was used in a BLASTX 2.2.23 search of the GenBank_Protein_Preferred database. The predicted amino acid
s e q u e n c e o f M O N 8 7 4 0 3 A T H B 1 7 ∆ 1 1 3 , b e g i n n i n g w i t h t h e f i r s t m e t h i o n i n e a m i n o a c i d in the top BLAST result is shown above.

V.A.2. Structure and Function of the A T H B 1 7 Δ 1 1 3 Protein from MON 87403

The Arabidopsis thaliana ATHB17 protein is a member of the HD-Zip family of transcription factors. In plants, the family of HD-Zip proteins is further segregated into four distinct subfamilies designated as I, II, III, and IV, based upon the sequences of conservative structural domains and motifs that convey DNA specificity and physiological function (Ariel et al. 2007). As described in Section V.A.1, expression of the Arabidopsis thaliana ATHB17 gene in MON 87403 results in production of the A T H B 1 7 Δ 1 1 3 p r o t e i n , w h i c h c o n s i s t s o f a s i n g l e p o l y p e p t i d e c h a i n o f 1 6 2 a m i n o a c i d s and has a predicted molecular weight of ~22 kDa. HD-Zip subfamily II members are characterized by highly conserved domains that include a homeodomain (HD) that recognizes a 9 bp DNA sequence CAAT(C/G)ATTG (Sessa et al. 1993), a leucine-zipper (LZ) domain responsible for protein dimerization, and a repression domain (Rice et al. 2014)

. A T H B 1 7 Δ 1 1 3 r e t a i n s t h e H D a n d L Z d o m a i n s f o u n d i n w i l d

- t y

p e A T H B 1 7 b u t lacks the repression domain.

V.B. Characterization and Equivalence of A T H B 1 7 Δ 1 1 3 Protein from MON 87403 The safety assessment of crops derived through biotechnology includes characterization of the physicochemical and functional properties of the protein produced from the inserted DNA, and confirmation of the safety of the protein. For safety data generated using E. coli -produced protein to be applied to plant-produced protein, the equivalence of the plant- and E. coli -produced proteins must be assessed. As reported in Section V.C, the expression level of the ATHB17 Δ 1 1 3 protein in MON 87403 is very low in all tissues evaluated. Due to this low level expression, the MON 87403-produced ATHB17 Δ 1 1 3 protein was not isolated to a high level of purity, but instead limited amounts of the protein were enriched in leaf extract to a purity of approximately 3% (Appendix D: ). Characterization of this preparation enabled the demonstration of equivalence between the MON 87403-produced ATHB17 Δ 1 1 3 protein and the E. coli- produced A T H B 1 7 Δ 1 1 3 p r o t e i n

. A s u m m a r y o f t h e s e a n a l y t i c a l r e s u l t s a r e s h o w n b e l o w a n d t h e details of the materials, methods, and results are described in Appendix D: .

The A T H B 1 7 Δ 1 1 3 protein purified from leaf tissue of MON 87403 was characterized and equivalence of the physicochemical properties between the MON 87403-producedMonsanto Company CR262-14U1 65 of 260

and E. coli -produced proteins was established using a panel of analytical techniques (Appendix D: ), including: 1) SDS-PAGE analysis, to establish equivalence of apparent molecular weight between the MON 87403-produced and E. coli -produced A T H B 1 7 Δ 1 1 3 proteins; 2) western blot analysis with antibodies specific for the A T H B 1 7 Δ 1 1 3 protein to establish immunoreactive equivalence between the MON 87403-produced and E. coli -produced A T H B 1 7 Δ 1 1 3 proteins; and 3) MALDI TOF MS analysis of MON 87403-produced A T H B 1 7 Δ 1 1 3 to establish protein identity. Due to the low quantity and purity of the enriched MON 87403-produced ATHB17 Δ 1 1 3 , direct assessment of the ATHB17 Δ 1 1 3 N

- t e r m i n a l s e qu e n c e , s p e c i f i c D N A b i n d i n g activity, and glycosylation status were not feasible to be conducted for the MON 87403-produced ATHB17 Δ 1 1 3 p r o t e i n

. Instead, these characteristics of ATHB17 Δ 1 1 3 i n M O N 8 7 4 0 3 w e r e a s s e s s e d i n d i r e c t l y

. The N-terminal sequence of the E. coli -produced ATHB17 Δ 1 1 3 p r o t e i n was directly analyzed and shown to match the N-terminus predicted by the RT-PCR analysis of MON 87403 ATHB17 RNA as detailed in Section V.A.1. An SDS-PAGE assessment demonstrated that the apparent molecular weights of the MON 87403-produced and E. coli

- p r o d u c e d A T H B 1 7 ∆ 1 1 3 w e r e equivalent, which supports a conclusion that the N-termini are equivalent (Figure D-2). Analysis of the DNA binding activity of the E. coli -produced ATHB17 Δ 1 1 3 demonstrated that the protein specifically bound to the target DNA sequence CAAT(C/G)ATTG, as expected. While functional activity could not be directly measured for the isolated MON 87403-produced ATHB17 Δ 1 1 3 p r o t e i n , t h e a c t i v i t y measured for E. coli -producted ATHB17 ∆ 1 1 3 i s c o n s i s t e n t w i t h t h e m e c h a n i s m o f a c t i o n for ATHB17 ∆ 1 1 3 i n M O N 8 7 4 0 3

. There are no predicted N-terminal targeting or signal sequences in the A T H B 1 7 Δ 1 1 3 amino acid sequence, which is expected due to the truncation of the N-terminus of the protein. Thus, although the A T H B 1 7 Δ 1 1 3 protein sequence contains a consensus potential N-glycosylation sequence (NHT, starting at amino acid position 45), it lacks the N-terminal signal sequence required for transport to the endoplasmic reticulum, the site of N-glycosylation (Pattison and Amtmann 2009; Vitale and Denecke 1999). In total, this supports a conclusion that the MON 87403-produced ATHB17 Δ 1 1 3 p r o t e i n is not glycosylated. This is similar to the CP4-EPSPS protein, which contains potential N-glycosylation sites but has been shown to not be glycosylated (Harrison et al. 1996). Taken together, these data provide a characterization of the MON 87403-produced A T H B 1 7 Δ 1 1 3 protein and establish its equivalence to the E. coli -produced A T H B 1 7 Δ 1 1 3 protein.

V.C. E x p r e s s i o n L e v e l s o f A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3

A T H B 1 7 Δ 1 1 3 p r o t e i n l e v e l s i n v a r i o u s t i s s u e s o f M O N 8 7 4 0 3 r e l e v a n t t o t h e r i s k assessment were determined by a validated enzyme-linked immunosorbent assay (ELISA). Tissues of MON 87403 were collected from four replicate plots planted in a randomized complete block field design during the 2012 growing season from the following five field sites in The United States: Jackson County, ArkansasMonsanto Company CR262-14U1 66 of 260

(site code ARNE); Story County, Iowa (site code IALL); Jefferson County, Iowa (site code IARL); Pawnee County, Kansas (site code KSLA) and Lehigh County, Pennsylvania (site code PAGR). The field sites were representative of maize-producing regions suitable for commercial production. OSL1 3 , OSR1 4 , forage, and grain tissue samples were collected from each replicated plot at all field sites. Determining the protein expression levels serves to characterize MON 87403 and the levels are then used to assess food, feed and environmental exposure. Protein expression levels themselves do not provide information relevant to the assessment of the plant pest potential of MON 87403. Therefore, it is deemed appropriate to determine the A T H B 1 7 Δ 1 1 3 p r o t e i n l e v e l s f o r O S L 1 , O S R 1 , f

o r a g e , a n d g r a i n t i s s u e s a m p l e s , w h i c h were collected from each replicated plot at all field sites. Leaf and root tissues are distinct above and below ground plant tissues that may be important to environmental exposure. Forage and grain levels were determined to evaluate food and feed exposure in humans and animals and are required for margin of exposure calculations for both. 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 A p p e n d i x E

. T h e i n d i v i d u a l A T H B 1 7 Δ 1 1 3 p r o t e i n l e v e l s i n M O N 8 7 4 0 3 a c r o s s a l l samples analyzed from all sites ranged from <LOD (<0.00028 μg/g dwt) t o 0

. 0 1 7 μ g / g d w t

. T h e m e a n A T H B 1 7 Δ 1 1 3 p r o t e i n l e v e l a m o n g a l l t i s s u e t y p e s w a s highest in OSL1 at 0.014 μg/g dwt and lowest in grain at <LOD μg/g dwt.

3 Over season leaf 1 4 Over season root 1Monsanto Company CR262-14U1 67 of 260

T a b l e V

- 1

. S u m m a r y o f A T H B 1 7 Δ 1 1 3 P r o t e i n L e v e l s i n T i s s u e s f r o m M O N 8 7 4 0 3 Grown in 2012 United States Field Trials

Tissue Type 1 Development Stage 2 Mean(SD)

Range ( μ g / g d w t ) 3 LOQ/LOD 4

( μ g / g d w t )

OSL1 V3-V4 0.014 (0.0020) 0.00109/0.00049 0.0096 – 0.017

OSR1 V3-V4 0.0023 (0.0016) 0.00078/0.00065 0.00083 – 0.0058
Forage R5 0.0018 (0.00064) 0.00078/0.00063 0.0011 – 0.0035

Grain R6 <LOD (N/A) 0.00156/0.00028 N/A – N/A

1 OSL= over season leaf OSR= over season root 2 The crop development stage each tissue was collected. 3 Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram ( μ g ) o f p r o t e i n p e r g r a m ( g ) o f t i s s u e o n a d r y w e i g h t b a s i s ( d w t )

. T h e m e a n s , S D , a n d r a n g e s (minimum and maximum values) were calculated for each tissue across all sites. (n=20, except OSR1 where n=16 due to four samples resulting in inconclusive levels) 4 LOQ=limit of quantitation; LOD=limit of detection.Monsanto Company CR262-14U1 68 of 260

V.D. Assessment of Potential Allergenicity of the A T H B 1 7 Δ 1 1 3 Protein The allergenic potential of an introduced protein is assessed by comparing the biochemical characteristics of the introduced protein to biochemical characteristics of known allergens (Codex Alimentarius 2009). Using a weight of evidence approach, a protein is not likely to be associated with allergenicity if: 1) the protein is from a non-allergenic source; 2) the protein represents a small portion of the total plant protein; 3) the protein does not share structural similarities to known allergens based on the amino acid sequence; and 4) the protein does not show resistance to pepsin digestion. The A T H B 1 7 Δ 1 1 3 protein has been assessed for its potential allergenicity according to these safety assessment guidelines.
1) The A T H B 1 7 Δ 1 1 3 protein originates from Arabidopsis thaliana an organism that has not been reported to be a source of known allergens.
2) The A T H B 1 7 Δ 1 1 3 protein level in grain is < the limit of detection of the assay and, therefore, conservatively is less than 0.0000003% of the total protein in the grain that could be consumed from MON 87403 maize.
3) Bioinformatics analyses demonstrated that the A T H B 1 7 Δ 1 1 3 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 A T H B 1 7 Δ 1 1 3 protein demonstrate that the protein is rapidly digested by pepsin in simulated gastric fluid (SGF) and in simulated intestinal fluid (SIF). Taken together, these data support the conclusion that the A T H B 1 7 Δ 1 1 3 protein does not pose a significant allergenic risk to humans.

V.E. Safety Assessment Summary of A T H B 1 7 Δ 1 1 3 Protein in MON 87403

V.E.1. The Donor Organism has a History of Safe Use

The Arabidopsis genus is a member of the mustard (Brassicaceae) family, which includes many well known food crops including cabbage, cauliflower, radish, turnip and canola/rapeseed. The most well known member of the Arabidopsis genus is a small annual flowering plant Arabidopsis thaliana (L.) which is also known under the common name of mouse-ear cress (Meyerowitz and Somerville 1994). Arabidopsis thaliana offers important advantages for basic research in genetics and molecular biology due to the small size of its sequenced genome, short life cycle, well developed classical genetics, and many naturally occurring mutants. Arabidopsis thaliana thus serves as an excellent model system for studying complex processes in plants. Arabidopsis thaliana is not purposely consumed as a food source by humans. Camelina sativa , an emerging oilseed crop, is reported to be the cultivated species most closely related to Arabidopsis (Flannery et al. 2006). Camelina sativa leaves are consumed as fresh greens by humans in the country of Georgia (Facciola 1998).Monsanto Company CR262-14U1 69 of 260

Arabidopsis thaliana is generally not considered an allergenic or toxic source organism. Although Arabidopsis thaliana contains homologs of proteins previously described as allergens in other plant species (e.g., germins, lipid transfer protein, profilins, and small molecular weight calcium binding proteins), no Arabidopsis proteins have been reported in a peer-reviewed database of known allergens (FARRP 2013). One case of occupational asthma has been reported in a laboratory worker due to exposure to Arabidopsis pollen (Yates et al. 2008). In a recent internal bioinformatic assessment, none of the HD-Zip family of proteins in Arabidopsis, including the ATHB17 protein, showed any homology to any known allergens, toxins, or any other protein with known adverse effects.

V.E.2. A T H B 1 7 Δ 1 1 3 P r o t e i n B e l o n g s t o a C o m m o n C l a s s o f P l a n t P r o t e i n s
The Arabidopsis thaliana HB17 ( ATHB17 ) gene encodes a member of the HD-Zip protein family of transcription factors (Ariel et al. 2007). The HD-Zip protein family is characterized by the presence of a leucine zipper (LZ) domain adjacent to the C-terminus of a homeodomain (HD) (Mukherjee et al. 2009; Ruberti et al. 1991). These two domains, HD and LZ, are individually present in transcription factors found across eukaryotic organisms, however such combination of HD and LZ domains in a single transcription factor is unique to plants; HD-Zip genes are found in all plant groups, but not in animals or fungi (Ariel et al. 2007). A bioinformatic search for homeobox genes (i.e. genes encoding proteins containing a HD) performed across several plant genomes (Mukherjee et al. 2009) yielded a comprehensive list of HD-Zip family members present in several plant species. It identified 48 HD-Zip genes in the Arabidopsis genome, 47 genes in the rice genome, and 70 genes in the maize genome. The HD-Zip family comprises 63% of all homeobox genes in maize and 40% - 60% of the homeobox genes in other plant species, making it the most abundant class of homeobox genes in many plant species.

B i o i n f o r m a t i c s e a r c h e s u s i n g t h e A T H B 1 7 ∆ 1 1 3 a m i n o a c i d s e qu e n c e a s t h e q u e r y identify homologous sequences from several different plant species, including the food crops soybean, rice, corn tomato, potato, orange, papaya, grape, and cruciferous

v e g e t a b l e s

. O v e r a l l t h e p r o t e i n s e qu e n c e i d e n t i t y o f A T H B 1 7 ∆ 1 1 3 t o h o m o l o g s i n t h e s e species range from ~58-83%, with the highest identity to the homologs in the Brassica species Brassica rapa (a species including common food crops such as turnip and napa cabbage) and Brassica oleracea (a species including common food crops such as cabbage
a n d B r u s s e l s s p r o u t s )

. T h e a m i n o a c i d s e qu e n c e a l i g n m e n t b e t w e e n A T H B 1 7 ∆ 1 1 3 a n d i t s f o o d c r o p h o m o l o g s s p a n s t h e l e n g t h o f t h e A T H B 1 7 ∆ 1 1 3 p r o t e i n

. T h u s A T H B 1 7 ∆ 1 1 3 s h a r e s s e qu e n c e i d e n t i t y a n d s t r u c t u r a l s i m i l a r i t y w i t h p r o t e i n s p r e s e n t i n plants currently consumed, establishing that humans and animals are exposed to this class of proteins and that no adverse effects have been attributed to this class of proteins.

V.E.3. A T H B 1 7 Δ 1 1 3 Protein in MON 87403 is Not Homologous to Known Allergens or Toxins Bioinformatics analyses were performed to assess the potential for allergenicity, toxicity, or biological activity of A T H B 1 7 Δ 1 1 3 . The analysis demonstrated that A T H B 1 7 Δ 1 1 3Monsanto Company CR262-14U1 70 of 260

protein does not share amino acid sequence similarity with known allergens, gliadins, glutenins, or protein toxins which could have adverse effects to human or animal health.

V.E.4. A T H B 1 7 Δ 1 1 3 Protein in MON 87403 is Susceptible to Pepsin and Pancreatin Digestion

A T H B 1 7 Δ 1 1 3 was readily digestible by pepsin in simulated gastric fluid and pancreatin in simulated intestinal fluid. Rapid degradation of A T H B 1 7 Δ 1 1 3 b y p e p s i n a n d pancreatin makes it highly unlikely that A T H B 1 7 Δ 1 1 3 i n M O N 8 7 4 0 3 would be absorbed in the digestive tract and have any adverse effects on human or animal health.

V.E.5. A T H B 1 7 Δ 1 1 3 Protein in MON 87403 is Not Acutely Toxic

A n a c u t e o r a l t o x i c o l o g y s t u d y w a s c o n d u c t e d w i t h t h e A T H B 1 7 Δ 1 1 3 p r o t e i n

. R e s u l t s i n d i c a t e d t h a t A T H B 1 7 Δ 1 1 3 d i d n o t c a u s e a n y a d v e r s e e f

f e c t s i n m i c e , w i t h a N o Observable Adverse Effect Level (NOAEL) of 1335 mg/kg body weight, the highest dose tested. As described below, this NOAEL ( i.e ., known safe dose) is approximately one billion times (1 x 10 9 ) higher than a conservative estimate of high end exposure through dietary consumption of MON 87403.

V.E.6. Human and Animal Exposure to the A T H B 1 7 Δ 1 1 3 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 appropriate animal toxicity study and an estimate of human exposure. Since no evidence of mammalian toxicity has been reported for A T H B 1 7 Δ 1 1 3 , and the expression levels in grain are extremely low (2.5 x 10 -4  g/g fwt 5 ), dietary risk assessments would normally not be considered necessary. Nevertheless, a dietary risk assessment was still conducted for this protein in order to provide further assurances of safety by calculating a MOE between the NOAEL (1335 mg/kg body wt/day) for the A T H B 1 7 Δ 1 1 3 protein in a mouse acute oral toxicity study (Section V.E.5 and 95 th percentile consumption estimate 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, 95 th percentile exposure to A T H B 1 7 Δ 1 1 3 for the general U.S. population was estimated to be 0.51 ng/kg body weight (bw). For non-nursing infants in the U.S., the most highly exposed sub-population, 95 th percentile exposure to A T H B 1 7 Δ 1 1 3 was estimated to be 1.21 ng/kg bw. For the A T H B 1 7 Δ 1 1 3 protein, MOEs for acute dietary intake were estimated to be 2.6 x 10 9 and 1.1  10 9 for the general

5 Fresh weight basis was calculated by multiplying the dry weight value of 2.8 x 10 -4 μg/gram (Section V.C) by a dry weight correction factor of 0.882 to account for 11.8% moisture content in the grain.Monsanto Company CR262-14U1 71 of 260

population and non-nursing infants, respectively. The MOEs reflect that a human would have to eat millions of kilograms of maize in one day to achieve exposures to the expressed proteins in MON 87403 that were not toxic to mice, which would be a physical impossibility. These very large MOEs further indicate that there is no meaningful risk to human health from dietary exposure to the A T H B 1 7 Δ 1 1 3 protein produced by MON 87403. Actual MOEs will likely be much higher because the exposure estimates utilized are conservative (95 th percentile, assume 100% market penetration of MON 87403) and as described in Section V.E.4., A T H B 1 7 Δ 1 1 3 is rapidly digested, further minimizing exposures. In addition, 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 (Hammond and Jez 2011). Like other proteins, the A T H B 1 7 Δ 1 1 3 protein in MON 87403 is 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 87403. Thus, there are likely to be significantly lower exposures to the functionally active form of this protein through consumption of MON 87403 than the already negligible levels estimated above. The potential A T H B 1 7 Δ 1 1 3 protein exposure to animals from consumption of MON 87403 in feeds was evaluated by calculating an estimate of daily dietary intake (DDI) and relating that value to total daily dietary protein intake. The highest percentage of A T H B 1 7 Δ 1 1 3 protein was in the lactating dairy cow, which was 0.000001% (g/g) of the total daily dietary protein intake (6.8 x 10 -8 g of A T H B 1 7 Δ 1 1 3 / k g b w divided by 5.9 g/kg bw total daily dietary protein intake for lactating dairy cow). The percentage of the A T H B 1 7 Δ 1 1 3 protein consumed as part of the daily protein intake for chickens and pigs is much less than for the lactating dairy cow. Therefore, in the maximal intake scenario, poultry, swine and lactating dairy cattle would be consuming 0.000001% (g/g) or less of their total protein as A T H B 1 7 Δ 1 1 3 protein from MON 87403 maize. In summary, there is no significant risk to human and animal health associated with dietary exposure to the A T H B 1 7 Δ 1 1 3 protein in food and feed products derived from MON 87403.

V.F. A T H B 1 7 Δ 1 1 3 Protein Characterization and Safety Conclusion The data and information provided in this section address the questions important to the food and feed safety of the A T H B 1 7 Δ 1 1 3 protein in MON 87403, including the potential
a l l e r g e n i c i t y a n d t o x i c i t y

. T o s u m m a r i z e , A T H B 1 7 ∆ 1 1 3 b e l o n g s t o t h e H D

- Z i p c l a s s o f plant transcription factors, and has high amino acid sequence homology to HD-Zip proteins encoded in several common food crops. The donor organism for the A T H B 1 7 Δ 1 1 3 c o d i n g s e qu e n c e , Arabidopsis thaliana , is ubiquitous in the environment and not commonly known for human or animal pathogenicity, or allergenicity. The physicochemical characteristics of the A T H B 1 7 Δ 1 1 3 protein from MON 87403 haveMonsanto Company CR262-14U1 72 of 260

been characterized and shown to be equivalent to those of E. coli- produced A T H B 1 7 Δ 1 1 3 , w h i c h w a s u s e d f o r t h e d e s c r i b e d s a f

e t y s t u d i e s . The expression level of A T H B 1 7 Δ 1 1 3 protein in selected tissues of MON 87403 were determined and shown to be below the limit of detection of a sensitive assay in grain. An assessment of the allergenic potential of the protein supports the conclusion that the A T H B 1 7 Δ 1 1 3 protein does not pose a meaningful safety risk to humans and animals or an allergenic risk to humans. The A T H B 1 7 Δ 1 1 3 protein lacks structural similarity to allergens, toxins or other proteins known to have adverse effects on mammals. In addition, the A T H B 1 7 Δ 1 1 3 protein is rapidly digested in simulated digestive fluids and demonstrated no oral toxicity in mice at a test level that corresponds to a very large margin of exposure when considering the protein is undetectable in grain. Based on the above information, the consumption of the A T H B 1 7 Δ 1 1 3 protein from MON 87403 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 87403 or derived from MON 87403 are as safe as maize currently on the market for human and animal consumption.Monsanto Company CR262-14U1 73 of 260 VI. COMPOSITIONAL ASSESSMENT OF MON 87403
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 furtherdocumented 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 87403 compared to that of a conventional control grown and harvested under similar conditions, as appropriate. In addition, conventional commercial maize reference varieties (hereafter refered to as reference hybrids) were included in the composition analyses to establish a range of natural variability for each component, defined by the 99% tolerance interval. 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 87403 is expected to be grown.Monsanto Company CR262-14U1 74 of 260

VI.A. Compositional Equivalence of MON 87403 Grain and Forage to Conventional Maize Grain and forage samples were collected from MON 87403, a conventional control maize, and a total of 17 different reference hybrids (Table F-1) grown in the United States during a 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. The field sites were planted in a randomized complete block design with four blocks per site. MON 87403, the conventional control, and reference hybrids were grown in areas of the United States that were typical for maize production and under normal agronomic field conditions for their respective geographic regions. The evaluation of MON 87403 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 quantitation (LOQ) and were excluded from statistical analysis. Moisture in 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 87403 and the conventional control was based on compositional data combined across all field sites. Statistically significant differences

w e r e i d e n t i f

i e d a t t h e 5 % l e v e l ( α = 0

. 0 5 )

. T h e c o m p o s i t i o n a l d a t a f r o m t h e r e f

e r e n c e 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. A statistically significant difference between MON 87403 and the conventional control does not necessarily imply biological relevance from a food and feed safety perspective. Therefore, statistically significant differences observed are typically evaluated in the context of natural variability to determine whether a detected difference indicates a biologically relevant compositional change. However, in this study, no significant differences between MON 87403 and the conventional control were observed in any of the measured components. Measurements of grain nutrients, including protein, amino acids (18 components), total fat, fatty acids (22 components), carbohydrates by calculation, fiber (3 components), ash, minerals (9 components), vitamins (7 components), antinutrients (phytic acid and raffinose), and secondary metabolites (furfural, ferulic acid, and p-coumaric acid) demonstrated that values in MON 87403Monsanto Company CR262-14U1 75 of 260

were not statistically significantly different from those in the conventional control (Table VI-1 to Table VI-6). Forage component levels, including ash, protein, total fat, carbohydrates by calculation, fiber (ADF and NDF), and minerals (calcium and phosphorus) were also not statistically significantly different between MON 87403 and the conventional control (Table VI-7). International Life Sciences Institute Crop Composition Database (ILSI-CCDB) and published literature values for all analytes are provided in Table VI-8. 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 87403 and the conventional control. The lack of any statistically significant differences between MON 87403 and the conventional control demonstrated that MON 87403 was not a major contributor to variation in nutrient, antinutrient, or secondary metabolite component levels in maize grain or forage and confirmed the compositional equivalence of MON 87403 to the conventional control in levels of these components.Monsanto Company CR262-14U1 76 of 260

Table VI-1. Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component (% dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value % Relative 6 Protein 10.13 (0.34) 10.15 (0.34) (8.58 - 12.63) 4.01 -0.016 (0.14) 0.911 -0.16 8.41 - 11.90 8.49 - 12.50 7.72, 12.67

Alanine 0.77 (0.030) 0.77 (0.030) (0.63 - 0.99) 0.36 0.00036 (0.012) 0.975 0.05 0.63 - 0.91 0.62 - 0.98 0.55, 1.01

Arginine 0.48 (0.0093) 0.48 (0.0093) (0.43 - 0.61) 0.13 -0.00007 (0.0051) 0.988 -0.01 0.41 - 0.53 0.41 - 0.54 0.41, 0.59

Aspartic Acid 0.63 (0.018) 0.63 (0.018) (0.56 - 0.79) 0.21 0.00058 (0.0080) 0.943 0.09 0.54 - 0.72 0.52 - 0.74 0.52, 0.78

Cystine/Cysteine 0.21 (0.0041) 0.21 (0.0041) (0.17 - 0.26) 0.06 0.0017 (0.0032) 0.589 0.83 0.18 - 0.23 0.18 - 0.24 0.16, 0.25

Glutamic Acid 1.85 (0.077) 1.86 (0.077) (1.50 - 2.47) 0.91 -0.0056 (0.033) 0.870 -0.30 1.48 - 2.26 1.43 - 2.34 1.26, 2.52Monsanto Company CR262-14U1 77 of 260

Table VI-1 (continued). Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component (% dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value % Relative 6 Glycine 0.37 (0.0065) 0.37 (0.0065) (0.32 - 0.44) 0.08 0.00016 (0.0036) 0.966 0.04 0.32 - 0.41 0.33 - 0.42 0.32, 0.44

Histidine 0.28 (0.0068) 0.28 (0.0068) (0.22 - 0.35) 0.09 0.0016 (0.0034) 0.648 0.55 0.24 - 0.33 0.24 - 0.33 0.20, 0.35

Isoleucine 0.36 (0.014) 0.36 (0.014) (0.28 - 0.44) 0.17 -0.00044 (0.0061) 0.944 -0.12 0.29 - 0.43 0.28 - 0.46 0.26, 0.46

Leucine 1.27 (0.059) 1.27 (0.059) (0.98 - 1.65) 0.71 0.0015 (0.026) 0.956 0.12 0.98 - 1.58 0.93 - 1.64 0.81, 1.73

Lysine 0.27 (0.0048) 0.27 (0.0048) (0.23 - 0.33) 0.07 0.0013 (0.0027) 0.625 0.50 0.24 - 0.30 0.23 - 0.31 0.24, 0.31

Methionine 0.21 (0.0063) 0.20 (0.0063) (0.16 - 0.26) 0.08 0.00071 (0.0028) 0.803 0.35 0.17 - 0.23 0.17 - 0.25 0.15, 0.26Monsanto Company CR262-14U1 78 of 260

Table VI-1 (continued). Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component (% dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value % Relative 6 Phenylalanine 0.52 (0.022) 0.52 (0.022) (0.42 - 0.65) 0.24 0.0024 (0.0093) 0.801 0.47 0.41 - 0.63 0.40 - 0.64 0.36, 0.68

Proline 0.95 (0.030) 0.95 (0.030) (0.73 - 1.12) 0.43 0.0032 (0.015) 0.830 0.34 0.80 - 1.10 0.76 - 1.18 0.64, 1.17

Serine 0.44 (0.015) 0.45 (0.015) (0.37 - 0.58) 0.18 -0.0068 (0.0072) 0.356 -1.51 0.35 - 0.51 0.37 - 0.55 0.34, 0.57

Threonine 0.36 (0.0098) 0.35 (0.0098) (0.31 - 0.44) 0.11 0.0025 (0.0043) 0.570 0.69 0.30 - 0.40 0.30 - 0.41 0.28, 0.43

Tryptophan 0.077 (0.0015) 0.077 (0.0015) (0.067 - 0.096) 0.01 -0.00019 (0.00085) 0.827 -0.25 0.063 - 0.086 0.069 - 0.084 0.064, 0.093

Tyrosine 0.41 (0.016) 0.41 (0.016) (0.34 - 0.55) 0.20 0.00043 (0.0081) 0.959 0.10 0.32 - 0.48 0.32 - 0.52 0.30, 0.53Monsanto Company CR262-14U1 79 of 260

Table VI-1 (continued). Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component (% dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value % Relative 6 Valine 0.46 (0.013) 0.46 (0.013) (0.38 - 0.57) 0.17 -0.00092 (0.0061) 0.880 -0.20 0.38 - 0.52 0.39 - 0.56 0.35, 0.57

1dwt = 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 control maize hybrid.
6 The relative magnitude of the difference in mean values between MON 87403 and the control, expressed as a percent of the control.Monsanto Company CR262-14U1 80 of 260

Table VI-2. Summary of Maize Grain Total Fat and Fatty Acids for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Total Fat (% dwt) 1 3.56 (0.072) 3.54 (0.072) (2.49 - 4.70) 0.98 0.026 (0.060) 0.682 3.10 - 3.90 3.11 - 4.09 1.93, 5.49

16:0 Palmitic 6 12.77 (0.14) 12.68 (0.14) (9.90 - 13.12) 2.99 0.097 (0.072) 0.219 12.27 - 13.50 10.59 - 13.59 7.90, 14.94

18:0 Stearic 2.04 (0.022) 2.06 (0.022) (1.46 - 2.42) 0.46 -0.025 (0.019) 0.206 1.87 - 2.14 1.82 - 2.28 1.05, 2.72

18:1 Oleic 21.84 (0.30) 21.77 (0.30) (21.13 - 34.04) 7.59 0.070 (0.19) 0.716 20.77 - 23.46 20.50 - 28.08 14.77, 38.25

18:2 Linoleic 61.23 (0.30) 61.39 (0.30) (51.19 - 62.88) 6.25 -0.15 (0.16) 0.372 59.60 - 62.78 57.12 - 63.36 45.88, 70.60

18:3 Linolenic 1.31 (0.014) 1.30 (0.014) (0.78 - 1.48) 0.24 0.012 (0.010) 0.267 1.22 - 1.40 1.16 - 1.40 0.69, 1.67Monsanto Company CR262-14U1 81 of 260

Table VI-2 (continued). Summary of Maize Grain Total Fat and Fatty Acids for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value 20:0 Arachidic 0.45 (0.012) 0.45 (0.012) (0.30 - 0.52) 0.14 -0.0017 (0.0034) 0.634 0.41 - 0.52 0.40 - 0.54 0.26, 0.55

20:1 Eicosenoic 0.20 (0.0030) 0.20 (0.0030) (0.19 - 0.30) 0.05 -0.0020 (0.0017) 0.243 0.18 - 0.22 0.18 - 0.23 0.14, 0.33

22:0 Behenic 0.16 (0.0081) 0.16 (0.0081) (0.055 - 0.23) 0.13 0.0029 (0.0045) 0.544 0.062 - 0.21 0.069 - 0.20 0, 0.26

1dwt = 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 control maize hybrid.
6 Expressed as % total fatty acid. Prefix numbers refer to number of carbon atoms and number of carbon-carbon double bonds in the fatty acid molecule; 16:0 means sixteen carbon atoms and zero double bonds. Numbers are not included in text discussion for reasons of clarity. The 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.Monsanto Company CR262-14U1 82 of 260

Table VI-3. Summary of Maize Grain Carbohydrates by Calculation and Fiber for MON 87403, Conventional Control, and Conventional References

Difference (Test minus Control)

Component (% dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Carbohydrates by 84.98 (0.34) 84.98 (0.34) (82.55 - 86.97) 3.84 0.0021 (0.12) 0.986 Calculation 83.09 - 86.73 82.77 - 86.61 81.80, 87.71

Acid Detergent Fiber 3.63 (0.068) 3.62 (0.068) (2.52 - 4.42) 1.49 0.0087 (0.062) 0.893 3.16 - 4.07 2.91 - 4.40 2.36, 4.43

Neutral Detergent Fiber 9.47 (0.19) 9.41 (0.19) (6.86 - 12.18) 3.48 0.063 (0.16) 0.696 8.11 - 10.67 7.53 - 11.01 5.32, 12.85

Total Dietary Fiber 13.04 (0.15) 12.95 (0.15) (9.83 - 17.30) 4.19 0.098 (0.22) 0.657 11.53 - 14.70 11.50 - 15.69 10.05, 15.51

1dwt = 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 control maize hybrid.Monsanto Company CR262-14U1 83 of 260

Table VI-4. Summary of Maize Grain Ash and Minerals for MON 87403, Conventional Control, and Conventional References

Difference (Test minus Control)

Component MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Ash (% dwt) 1 1.31 (0.026) 1.33 (0.026) (1.02 - 1.53) 0.42 -0.017 (0.019) 0.371 1.09 - 1.52 1.10 - 1.51 1.08, 1.60

Calcium (% dwt) 0.0037 (0.00021) 0.0037 (0.00021) (0.0011 - 0.0058) 0.00 0.00006 (0.00006) 0.321 0.0026 - 0.0049 0.0025 - 0.0049 0.0011, 0.0059

Copper (mg/kg dwt) 1.57 (0.11) 1.55 (0.11) (0.92 - 6.11) 1.87 0.023 (0.12) 0.852 1.13 - 3.35 1.04 - 2.91 0.29, 3.17

Iron (mg/kg dwt) 19.33 (0.82) 19.60 (0.82) (14.66 - 25.54) 8.09 -0.27 (0.21) 0.240 15.60 - 24.15 15.81 - 23.90 10.87, 27.03

Magnesium (% dwt) 0.12 (0.0033) 0.12 (0.0033) (0.093 - 0.14) 0.04 -0.00010 (0.0013) 0.939 0.10 - 0.14 0.096 - 0.14 0.092, 0.15

Manganese (mg/kg dwt) 6.17 (0.31) 6.14 (0.31) (4.02 - 9.46) 3.99 0.029 (0.11) 0.787 4.36 - 7.85 4.55 - 8.55 2.59, 10.23Monsanto Company CR262-14U1 84 of 260

Table VI-4 (continued). Summary of Maize Grain Ash and Minerals for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Phosphorus (% dwt) 0.30 (0.0068) 0.30 (0.0068) (0.24 - 0.36) 0.10 -0.0017 (0.0041) 0.683 0.25 - 0.33 0.24 - 0.34 0.24, 0.38

Potassium (% dwt) 0.33 (0.0076) 0.33 (0.0076) (0.27 - 0.42) 0.10 -0.0023 (0.0044) 0.611 0.30 - 0.40 0.30 - 0.40 0.23, 0.42

Zinc (mg/kg dwt) 20.52 (0.83) 20.98 (0.83) (15.56 - 30.10) 10.65 -0.46 (0.30) 0.165 16.30 - 25.20 16.49 - 27.14 9.09, 32.95

1dwt = 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 control maize hybrid.Monsanto Company CR262-14U1 85 of 260

Table VI-5. Summary of Maize Grain Vitamins for MON 87403, Conventional Control, and Conventional References

Difference (Test minus Control)

Component (mg/kg dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Vitamin A (  -Carotene) 1.16 (0.039) 1.14 (0.039) (0.48 - 2.85) 0.49 0.025 (0.023) 0.319 0.89 - 1.37 0.88 - 1.37 0, 3.10

Vitamin B1 (Thiamin) 3.48 (0.13) 3.52 (0.13) (2.54 - 4.99) 1.41 -0.046 (0.042) 0.277 2.72 - 4.19 2.82 - 4.23 1.73, 5.12

Vitamin B2 (Riboflavin) 1.83 (0.059) 1.71 (0.059) (1.35 - 2.35) 1.00 0.12 (0.053) 0.057 1.48 - 2.43 1.22 - 2.22 1.25, 2.22

Vitamin B3 (Niacin) 16.47 (0.78) 16.59 (0.78) (12.77 - 30.15) 14.27 -0.12 (0.46) 0.788 12.74 - 21.74 12.61 - 26.88 7.36, 30.18

Vitamin B6 (Pyridoxine) 7.11 (0.17) 6.89 (0.17) (4.66 - 8.80) 3.51 0.22 (0.19) 0.255 5.50 - 10.63 5.85 - 9.36 4.51, 8.98

Vitamin B9 (Folic Acid) 0.39 (0.016) 0.39 (0.016) (0.22 - 0.77) 0.20 0.00065 (0.0086) 0.940 0.27 - 0.50 0.28 - 0.47 0.038, 0.69Monsanto Company CR262-14U1 86 of 260

Table VI-5 (continued). Summary of Maize Grain Vitamins for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component (mg/kg dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Vitamin E (  -Tocopherol) 17.87 (0.65) 18.33 (0.65) (8.68 - 25.90) 8.20 -0.47 (0.24) 0.095 14.70 - 20.76 15.03 - 23.23 2.50, 27.12

1dwt = 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 control maize hybrid.Monsanto Company CR262-14U1 87 of 260

Table VI-6. Summary of Maize Grain Anti-nutrients and Secondary Metabolites for MON 87403, Conventional Control, and Conventional References

Difference (Test minus Control)

Component MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Anti-nutrient (% dwt1) Phytic Acid 0.89 (0.017) 0.87 (0.017) (0.68 - 1.15) 0.40 0.023 (0.016) 0.164 0.70 - 1.04 0.67 - 1.07 0.68, 1.18

Raffinose 0.22 (0.012) 0.23 (0.012) (0.062 - 0.35) 0.14 -0.0030 (0.0041) 0.487 0.14 - 0.30 0.17 - 0.31 0.00088, 0.40

Secondary Metabolite (μg/g dwt) Ferulic Acid 2262.60 (61.27) 2213.54 (61.27) (1381.65 - 2990.97) 1350.33 49.06 (48.06) 0.341 1833.71 - 2587.97 1344.15 - 2694.48 827.07, 3473.40

p-Coumaric Acid 216.73 (5.25) 212.58 (5.25) (103.35 - 383.41) 86.91 4.15 (5.09) 0.441 160.59 - 242.25 173.81 - 260.72 6.62, 433.65

1dwt = 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 control maize hybrid.Monsanto Company CR262-14U1 88 of 260

Table VI-7. Summary of Maize Forage Proximates, Fiber, and Minerals for MON 87403, Conventional Control, and Conventional References

Difference (Test minus Control)

Component (% dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Ash 4.00 (0.35) 4.14 (0.35) (2.05 - 7.98) 4.10 -0.14 (0.11) 0.199 2.17 - 5.87 2.14 - 6.24 0.67, 7.56

Carbohydrates by 86.98 (0.43) 86.76 (0.43) (82.09 - 90.15) 6.01 0.22 (0.28) 0.449 Calculation 84.01 - 90.21 83.73 - 89.74 81.04, 92.37

Protein 7.08 (0.27) 7.19 (0.27) (4.14 - 10.27) 4.92 -0.12 (0.19) 0.548 5.60 - 8.92 4.42 - 9.34 3.56, 10.69

Total Fat 1.96 (0.12) 1.98 (0.12) (0.62 - 3.18) 2.41 -0.023 (0.12) 0.851 0.92 - 2.89 0.53 - 2.94 0.81, 3.33

Acid Detergent Fiber 23.01 (0.89) 22.44 (0.89) (16.01 - 37.25) 12.85 0.57 (0.83) 0.513 16.18 - 33.12 17.33 - 30.17 17.89, 28.94

Neutral Detergent Fiber 36.83 (1.06) 37.23 (1.06) (27.09 - 54.66) 26.57 -0.40 (1.33) 0.768 28.67 - 44.62 29.77 - 56.33 30.85, 44.85Monsanto Company CR262-14U1 89 of 260

Table VI-7 (continued). Summary of Maize Forage Proximates, Fiber, and Minerals for MON 87403, Conventional Control, and Conventional References Difference (Test minus Control)

Component (% dwt)1 MON 87403 Mean (S.E.)2 Range Control

Mean (S.E.) Range Conventional

Reference (Range)3 Tolerance Interval 4 Control Range Value 5 Mean

(S.E.) p-Value Calcium 0.22 (0.015) 0.23 (0.015) (0.12 - 0.40) 0.17 -0.013 (0.0083) 0.150 0.12 - 0.34 0.16 - 0.33 0.10, 0.36

Phosphorus 0.16 (0.0058) 0.16 (0.0058) (0.10 - 0.30) 0.09 0.0011 (0.0049) 0.828 0.12 - 0.22 0.13 - 0.22 0.045, 0.30

1dwt = 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 control maize hybrid.Monsanto Company CR262-14U1 90 of 260

Table VI-8. Literature and ILSI Database Ranges for Components in Maize Forage and Grain Grain Tissue Components 1 Literature Range 2 ILSI Range 3 Grain Nutrients Proximates (% dwt) Ash 1.17 – 2.01 a ; 1.27 – 1.63 b 0.616 – 6.282 Carbohydrates by calculation 81.31 – 87.06 a ; 82.10 – 85.98 b 77.4 – 89.5 Fat, total 2.95 – 4.40 a ; 3.18 – 4.23 b 1.742 – 5.900 Protein 8.27 – 13.33 a ; 9.17 – 12.19 b 6.15 – 17.26 Fiber (% dwt) Acid detergent fiber 1.82 – 4.48 a ; 1.83 – 3.39 b 1.82 – 11.34 Neutral detergent fiber 6.51 –12.28 a ; 6.08 – 10.36 b 5.59 – 22.64 Total dietary fiber 10.65 – 16.26 a ; 10.57 – 14.56 b 9.01 – 35.31 Amino Acids (% dwt) Alanine 0.60 – 1.04 a ; 0.68 – 0.96 b 0.44 - 1.39 Arginine 0.34 – 0.52 a ; 0.34 – 0.50 b 0.12 - 0.64 Aspartic acid 0.52 – 0.78 a ; 0.59 – 0.76 b 0.33 – 1.21 Cystine 0.19 – 0.26 a ; 0.20 – 0.26 b 0.13 – 0.51 Glutamic acid 1.54 – 2.67 a ; 1.71 – 2.44 b 0.97 – 3.54 Glycine 0.33 – 0.43 a ; 0.33 – 0.42 b 0.18 – 0.54 Histidine 0.25 – 0.37 a ; 0.27 – 0.34 b 0.14 – 0.43 Isoleucine 0.30 – 0.48 a ; 0.32 – 0.44 b 0.18 – 0.69 Leucine 1.02 – 1.87 a ; 1.13 – 1.65 b 0.64 – 2.49 Lysine 0.26 – 0.33 a ; 0.28 – 0.31 b 0.17 – 0.67 Methionine 0.17 – 0.26 a ; 0.16 – 0.30 b 0.12 – 0.47 Phenylalanine 0.43 – 0.72 a ; 0.45 – 0.63 b 0.24 – 0.93 Proline 0.74 – 1.21 a ; 0.78 – 1.11 b 0.46 – 1.63 Serine 0.39 – 0.67 a ; 0.43 – 0.60 b 0.24 – 0.77 Threonine 0.29 – 0.45 a ; 0.31 – 0.39 b 0.22 – 0.67 Tryptophan 0.047 – 0.085 a ; 0.042 – 0.070 b 0.027 – 0.215 Tyrosine 0.13 – 0.43 a ; 0.12 – 0.41 b 0.10 – 0.64 Valine 0.42 – 0.62 a ; 0.45 – 0.58 b 0.27 – 0.86 Fatty Acids (% Total FA) 16:0 Palmitic 8.80 – 13.33 a ; 9.84 – 12.33 b 7.94 – 20.71 18:0 Stearic 1.36 – 2.14 a ; 1.30 – 2.10 b 1.02 – 3.40 18:1 Oleic 19.50 – 33.71 a ; 19.59 – 29.13 b 17.4 – 40.2 18:2 Linoleic 49.31 – 64.70 a ; 56.51 – 65.65 b 36.2 – 66.5 18:3 Linolenic 0.89 – 1.56 a ; 1.03 – 1.38 b 0.57 – 2.25 20:0 Arachidic 0.30 – 0.49 a ; 0.30 – 0.41 b 0.279 – 0.965 20:1 Eicosenoic 0.17 – 0.29 a ; 0.17 – 0.27 b 0.170 – 1.917 22:0 Behenic 0.069 – 0.28 a ; 0.059 – 0.18 b 0.110 – 0.349 Minerals Calcium (% dwt) 0.0030 – 0.0083 c 0.00127 – 0.02084 Copper (mg/kg dwt) 0.85 – 3.54 c 0.73 – 18.50 Iron (mg/kg dwt) 10.58 – 30.97 c 10.42 – 49.07 Magnesium (% dwt) 0.085 – 0.15 c 0.0594 – 0.194 Manganese (mg/kg dwt) 3.67 – 9.39 c 1.69 – 14.30 Phosphorous (% dwt) 0.25 – 0.38 c 0.147 – 0.533 Potassium (% dwt) 0.29 – 0.47 c 0.181 – 0.603 Zinc (mg/kg dwt) 16.67 – 31.38 c 6.5 – 37.2Monsanto Company CR262-14U1 91 of 260

Table VI-8 (continued). Literature and ILSI Database Ranges for Components in Maize Forage and Grain Grain Tissue Components 1 Literature Range 2 ILSI Range 3 Vitamins (mg/kg dwt) Folic acid 0.19 – 0.35 a ; 0.23 – 0.42 b 0.147 – 1.464 Vitamin A [  –Carotene] 122 – 4740 d 0.19 – 46.81 Vitamin B 1 [Thiamine] 2.33 – 4.17 a ; 2.71 – 4.33 b 1.26 – 40.00 Vitamin B 2 [Riboflavin] 0.94 – 2.42 a ; 1.64 – 2.81 b 0.50 – 2.36 Vitamin B 3 [Niacin] 15.07 – 32.38 a ; 13.64 – 42.06 b 10.37 – 46.94 Vitamin B 6 [Pyridoxine] 4.93 – 7.53 a ; 4.97 – 8.27 b 3.68 – 11.32 Vitamin E [  –Tocopherol] 5.96 – 18.44 a ; 2.84 – 15.53 b 1.537 – 68.672
Grain Anti–Nutrients (% dwt) Phytic acid 0.69 – 1.09 a ; 0.60 – 0.94 b 0.111 – 1.570 Raffinose 0.079 – 0.22 a ; 0.061 – 0.15 b 0.020 – 0.320

Grain Secondary Metabolites (  g/g dwt) Ferulic acid 1205.75 – 2873.05 a ; 1011.40 – 2539.86 b 291.9 – 3885.8 p–Coumaric acid 94.77 – 327.39 a ; 66.48 – 259.68 b 53.4 – 576.2

Forage Tissue Components 1 Literature Range 2 ILSI Range 3 Forage Nutrients Proximates (% dwt) Ash 2.67 – 8.01 a ; 4.59 – 6.90 b 1.527 – 9.638 Carbohydrates by calculation 81.88 – 89.26 a ; 84.11 – 87.54 b 76.4 – 92.1 Fat, total 1.28 – 3.62 a ; 0.20 – 1.76 b 0.296 – 4.570 Protein 5.80 – 10.24 a ; 5.56 – 9.14 b 3.14 – 11.57

Fiber (% dwt) Acid detergent fiber 19.11 – 30.49 a ; 20.73 – 33.39 b 16.13 – 47.39 Neutral detergent fiber 27.73 – 49.62 a ; 31.81 – 50.61 b 20.29 – 63.71

Minerals (% dwt) Calcium 0.12 – 0.33 a ; 0.21 – 0.41 b 0.07139 – 0.57679 Phosphorous 0.090 – 0.26 a ; 0.13 – 0.21 b 0.09362 – 0.37041

1 dwt=dry weight; FA = fatty acids. 2 Literature range references: a US and b Chile (Harrigan et al. 2009), c France (Ridley et al. 2011), d (Safawo et al. 2010). 3 ILSI range is from ILSI Crop Composition Database, 2011 [Accessed 9 January 2013] (ILSI 2011).Monsanto Company CR262-14U1 92 of 260

VI.B. Compositional Assessment of MON 87403 Conclusion Compositional analysis was conducted on grain and forage of MON 87403 grown at eight sites in a 2012 field production in the United Sates that are representative of typical 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 reference hybrids to provide data on the natural variability of each compositional component analyzed. Of the 60 components statistically assessed for MON 87403, none of the components showed a significant difference between MON 87403 and the conventional control. These results support the overall conclusion that MON 87403 was not a major contributor to variation in component levels in maize grain and forage and confirmed the compositional equivalence of MON 87403 to the conventional control in levels of these components.Monsanto Company CR262-14U1 93 of 260 VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT
This section provides a comparative assessment of the phenotypic, agronomic, and environmental interaction characteristics of MON 87403 compared to the conventional control. The data support a conclusion that MON 87403 is not meaningfully different from the conventional control with the exception of the increased ear biomass trait, 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 87403 were evaluated in a comparative manner to assess plant pest potential. These assessments included evaluation of seed germination characteristics, plant growth and developmentcharacteristics, observations of plant responses to abiotic stress, plant-disease and plantarthropod interactions, and pollen characteristics. Results from these assessments demonstrate that MON 87403 does not possess a) increased weediness characteristics; b) increased susceptibility or tolerance to specific abiotic stresses, diseases, or arthropods; or c) characteristics that would confer a plant pest risk compared to the conventional control.

VII.A. Characteristics Measured for Assessment In the phenotypic, agronomic, and environmental interactions assessment of MON 87403, 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. That description includes 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 87403, data were collected to provide a detailed description of the phenotypic, agronomic, and environmental interaction characteristics of MON 87403. 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 87403 encompassed six 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; and 6) intended phenotype (increased ear biomass). An overview of the characteristics assessed is presented in Table VII-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 87403 was compared to an appropriate conventional control that had a genetic background similar to MON 87403 but did not possess increased ear biomass trait. In addition, multiple commercial maize hybrids developed through conventional breeding and selection (see Appendices G, H,Monsanto Company CR262-14U1 94 of 260

and I and Tables G-1, H-1, and I-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 provided context for interpreting experimental results.Monsanto Company CR262-14U1 95 of 260

Table VII-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 1

(Setting of evaluation) Evaluation description (measurement endpoints) Germination, dormancy, and emergence Normal germinated

(VII.C.1) Day 4 and 7:

20/30°C (Laboratory) Percentage of seed producing seedlings exhibiting normal developmental characteristics Abnormal germinated (VII.C.1) Day 7:

20/30°C (Laboratory) Percentage of seed producing seedlings that could not be classified as normal germinated Germinated (VII.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 (VII.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 (VII.C.1) Day 7:

20/30°C Day 12: 5, 10, 20, 30, 10/20 and 10/30°C (Laboratory) Percentage of seed that did not imbibe water and remained hard to the touch (viability determined by a tetrazolium
test 2 )

Viable firm-swollen (VII.C.1) Day 7:

20/30°C Day 12: 5, 10, 20, 30, 10/20 and 10/30°C (Laboratory) Percentage of seed that imbibed water and were firm to the touch but did not germinate (viability determined by a tetrazolium test 2 )

Early stand count (VII.C.2.1) V2 – V5 1 growth stage

(Field) Number of emerged plants in two rows

Vegetative growth Final stand count

(VII.C.2.1) Pre-harvest (Field) Number of plants in two rows

Plant vigor (VII.C.2.1) V2 – V5 1 growth stage

(Field) Rated on a 1-9 scale, where 1 = excellent vigor and 9 = poor vigor Stay green (VII.C.2.1) Maturity 1 (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 (VII.C.2.1) R1 – Maturity 1 (Field) Distance from the soil surface at the base of the plant to the ear attachment node on five plants per plot Plant height (VII.C.2.1) R1 – Maturity 1 (Field) Distance from the soil surface at the base of the plant to the flag leaf collar on five plants per plotMonsanto Company CR262-14U1 96 of 260

Table VII-1 (continued). Phenotypic, Agronomic and Environmental Interaction Characteristics Evaluated in U.S. Field Trials and Laboratory Studies

Data category Characteristics

measured Evaluation timing 1

(Setting of evaluation) 1 Evaluation description Reproductive growth Days to 50% pollen

shed (VII.C.2.1) Pollen shed 1 (Field) Days from planting until 50% of the plants have begun to shed pollen Days to 50% silking (VII.C.2.1) Silking 1 (Field) Days from planting until 50% of the plants have multiple silks exposed Pollen viability (VII.C.3) Tasseling (Laboratory) Percentage of viable pollen based on pollen grain staining characteristics Pollen morphology (VII.C.3) Tasseling (Laboratory) Diameter of viable pollen grains and visual observations Grain moisture (VII.C.2.1) Harvest (Field) Percentage moisture of harvested shelled grain Test weight (VII.C.2.1) Harvest (Field) Test weight (kilogram/hecto liter) of harvested shelled grain Yield (VII.C.2.1) Harvest (Field) Calculated in Mg/ha, adjusted to 15.5% grain moisture content Lodging and Seed retention Stalk lodged plants

(VII.C.2.1) Pre-harvest (Field) Number of plants per plot broken below the ear Root lodged plants (VII.C.2.1) Pre-harvest (Field) Number of plants per plot leaning at the soil surface at >30° from the vertical Dropped ears (VII.C.2.1) Pre-harvest (Field) Number of mature ears dropped from plants Environmental interactions Abiotic stress

response (VII.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 (VII.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 (VII.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 (VII.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 (VII.C.2.2.1) Harvest (Field) Qualitative assessment of each plot, with categorical scale of increasing severity (none, slight, moderate, severe)Monsanto Company CR262-14U1 97 of 260

Table VII-1 (continued). Phenotypic, Agronomic and Environmental Interaction Characteristics Evaluated in U.S. Field Trials and Laboratory Studies

Characteristics measured Evaluation timing 1

(Setting of evaluation) 1 Evaluation description Environmental interactions Corn earworm

damage (VII.C.2.2.2) R5 growth stage 1 (Field) Quantitative assessment on 10 plants per plot by examining ears for damaged area using a plastic film grid (each grid cell = 0.5 cm 2 ) European corn borer damage (VII.C.2.2.2) R6 growth stage 1 (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 (VII.C.2.2.2) Five collection times during

growing season (Field) Quantitative assessment of arthropod abundance via sticky traps collections and visual counts Intended Phenotype R1 ear biomass

(VII.C.2.1) R1 Growth Stage 1 (Field) Dry weight in grams of whole ears from 1 m in length of row
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).Monsanto Company CR262-14U1 98 of 260

VII.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 trait, 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 87403 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 VII-1. A subset of the data relating to well-understood weedy characteristics (e.g., seed dormancy, pre-harvest seed loss characteristics, and lodging) was used to assess whether there was an increase in weediness potential of MON 87403 compared to 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 87403 could be expected to pose an increased plant pest risk compared to conventional maize.

VII.B.1. Interpretation of Criteria for Detected Differences 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 of the biotechnology-derived crop compared to the conventional crop. Characteristics for which differences are detected are considered in a step-wise method (Figure VII-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 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 VII-1 illustrates the stepwise assessment process employed:Monsanto Company CR262-14U1 99 of 260

Figure VII-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 potential and subsequent steps are not considered. If the answer is “yes” or “uncertain,” the subsequent step is considered. Steps 1 and 2 - Evaluate Detected Statistically Significant Differences Data on each measured characteristic are statistically analyzed, where appropriate, within each individual site and in a combined-site analysis, in which the data are pooled among sites. All statistically significant differences are evaluated and considered in the context of a change in plant pest/weed potential. Differences detected in individual-site analyses that are not detected when data across multiple environments are pooled in the combined-site analysis are considered not biologically meaningful in terms of plant pest/weed potential and, therefore, are not further considered in subsequent steps. Any difference detected in the combined-site analysis is further assessed. Step 3 - Evaluate Differences in the Context of Reference Hybrids 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 reference hybrids included in the study (e.g., reference range).

Hazard identification and risk assessment on difference Outside variation for crop? No

Yes No

Yes Adverse in terms of plant pest/weed potential?

Yes Outside variation of study references? No Not adverse; the direction or magnitude of the detected difference in the measured characteristic does not contribute to a biological change for the crop in terms of plant pest/weed potential Yes Statistical differences detected in combined-site analysis? No Differences detected in the combined

- site and individual site analyses are evaluated
Step 2

Step 3

Step 4 The measured characteristic does not contribute to a biological change for the crop in terms of plant pest/weed potential

Step 5

Step 6 Step 1Monsanto Company CR262-14U1 100 of 260

Step 4 - Evaluate Differences in the Context of the Crop If the mean value of the characteristic for a biotechnology-derived crop is outside thevariation of the reference hybrids in the study, the mean value of the biotechnologyderived crop is assessed relative to known values common for the crop (e.g., published values). Step 5 - Relevance of Differences to Plant Pest/Weed Potential If the mean value of the characteristic 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, a 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.Monsanto Company CR262-14U1 101 of 260

VII.B.2. Interpretation of Vigor and Environmental Interactions Data For the qualitative assessments of vigor, abiotic stress response, disease damage, and arthropod damage, the biotechnology-derived crop and the 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 reference hybrids, and for consistency in other observation times and sites. Differences that are not consistently observed in multiple environments are not considered to be biologically meaningful in terms of plant pest potential. 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 the conventional control are evaluated using the method outlined in Figure VII-1. Quantitative assessments of arthropod abundance are only analyzed within each individual site. Statistically significant differences between the biotechnology-derived crop and the conventional control were assessed for biological significance in the context of the range of the reference hybrids, and for consistency with other collection times and collection sites and in the context of pest potential. Differences that are not consistently detected in multiple environments are not considered biologically meaningful in terms of plant pest potential.

VII.C. Comparative Assessments of the Phenotypic, Agronomic, and Environmental Interaction Characteristics of MON 87403 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 87403. The characteristics for MON 87403 evaluated in these assessments included: seed dormancy and germination characteristics (Section VII.C.1), plant phenotypic, agronomic, and environmental interaction observations under field conditions (Section VII.C.2), and pollen characteristics (Section VII.C.3). Additional details for each assessment are provided in Appendices G, H, and I.

VII.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.Monsanto Company CR262-14U1 102 of 260

A comparative assessment of seed germination and dormancy characteristics was conducted on MON 87403 and the conventional control. The seed lots used for the germination testing of MON 87403 (selfed F2 grain), the conventional control, and the reference hybrids (4 per site, 9 unique across all locations) were produced in replicated field trials during 2012 in Story County, Iowa (IA), Warren County, Illinois (IL), and Lehigh County, Pennsylvania (PA). These geographic areas represent a broad range of environmental conditions for maize production for this product. The experiments were arranged as separate split-plot experiments with four replications for each temperature regime. Descriptions of the evaluated germination and dormancy characteristics and the timing of the evaluations for all temperature regimes are listed in Table VII-1. Additional details on the materials and experimental methods used in this evaluation are presented in Appendix G: . In the combined-site analysis, in which data were pooled across the three seed production
s i t e s , n o s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s ( α = 0

. 0 5 ) w e r e d e t e c t e d b e t w e e n M O N 8 7 4 0 3 and the conventional control for any characteristic at the AOSA temperature regime (20/30 °C), or at the temperature regimes of 5, 10, 20, 30, 10/20, 10/30 °C (Table VII-2). In addition, no hard seed were observed at any temperature. The germination and dormancy characteristics evaluated were used to assess MON 87403 in the context of plant pest/weed potential. 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 increased ear biomass trait is not expected to result in increased plant pest/weed potential compared to conventional maize.Monsanto Company CR262-14U1 103 of 260

Table VII-2. Germination Characteristics of MON 87403 and the Conventional Control Temperature
(°C) Assessment

Category Mean % (S.E.) 1 Reference Range 2 MON 87403 Control 5 Germinated 5.7 (0.90) 6.2 (1.24) 2.3 – 9.5

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 Dead 4.1 (0.60) 3.5 (0.69) 1.5 – 6.1 Viable Firm-Swollen 90.3 (1.21) 90.3 (1.15) 86.8 – 94.8 10 Germinated 95.0 (0.73) 94.0 (0.79) 95.0 – 99.0

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 Dead 1.8 (0.58) 2.0 (0.51) 0.5 - 3.6 Viable Firm-Swollen 3.2 (0.66) 4.0 (0.98) 0.0 – 3.8 20 3 Germinated 99.3 (0.26) 99.1 (0.23) 96.3 - 99.8

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 Dead 0.7 (0.26) 0.9 (0.23) 0.3 – 3.7 Viable Firm- Swollen† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 30 Germinated 99.4 (0.23) 99.6 (0.19) 97.5 - 99.8

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 Dead 0.6 (0.23) 0.4 (0.19) 0.3 - 2.5 Viable Firm- Swollen† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 10/20 Germinated 99.4 (0.19) 99.5 (0.19) 96.0 - 100.0

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 Dead 0.4 (0.15) 0.5 (0.19) 0.0 – 3.8 Viable Firm- Swollen 0.2 (0.11) 0.0 (0.00) 0.0 - 0.5 10/30 Germinated 99.6 (0.19) 99.5 (0.19) 97.0 – 100.0

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 Dead 0.4 (0.19) 0.5 (0.19) 0.0 - 3.0 Viable Firm- Swollen† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 20/30 Normal Germinated 98.9 (0.31) 98.8 (0.32) 95.6 – 99.8 (AOSA) Abnormal Germinated 0.8 (0.25) 0.7 (0.22) 0.0 - 2.0

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 Dead 0.3 (0.13) 0.5 (0.23) 0.3 – 2.5 Viable Firm- Swollen 0.1 (0.08) 0.0 (0.00) 0.0 - 0.1 Note: The experimental design was a split-plot with four replications. No statistically significant differences were detected between MON 87403 and the
c o n v e n t i o n a l c o n t r o l ( α = 0

. 0 5 ) u s i n g A N O V A

. 1 MON 87403 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 87403 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 9 commercially available reference hybrids. 3 Three replicates of the reference Midland Phillips 799 were used from sites ILMN and PAGR. †No statistical comparison could be made due to lack of variability in the data.Monsanto Company CR262-14U1 104 of 260

VII.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 87403. These data were developed to provide USDA-APHIS with a detailed description of MON 87403 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 87403 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 87403 compared to conventional maize. Environmental interactions including plant response to abiotic stress, disease damage, arthropod-related damage, and arthropod abundance were also assessed as an indirect indicator of changes to MON 87403 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 increased ear biomass trait did not meaningfully alter the plant pest/weed potential of MON 87403 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 increased ear biomass trait is not expected to result in increased plant pest/weed potential for MON 87403 compared to conventional maize.

VII.C.2.1. Field Phenotypic and Agronomic Characteristics Data were collected at 13 sites (Table VII-3) in the U.S. during 2012 to evaluate phenotypic and agronomic characteristics of MON 87403 compared to the conventional control. These 13 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 87403, the conventional control, and four reference hybrids were evaluated. A total of 21 unique reference hybrids were evaluated among the 13 sites (Table H-1). The planted plot dimensions varied between sites due to variability in available planting equipment (Table H-2). All plots of MON 87403, the conventional control, and the reference hybrids at each site were uniformly managed in order to assess whether the introduction of the increased ear biomass maize trait altered the phenotypic and agronomic characteristics of MON 87403 compared to the conventional control. Descriptions of the evaluated phenotypic characteristics and the timing of the evaluations are listed in Table VII-1. The materials, methods, details concerning the timing of phenotypic assessments, and detailed results of the individual-site data comparisons are presented and discussed in Appendix H (Table H-4). The results of the combined site analyses are summarized below. In the combined-site analysis, no statistically significant differences were detected between MON 87403 and the conventional control for 12 of 13 characteristics includingMonsanto Company CR262-14U1 105 of 260

early stand count, days to 50% pollen shed, days to 50% silking, stay green rating, plant height, dropped ear count, stalk lodged plants, root lodged plants, final stand count, grain moisture, test weight and yield (Table VII-4). One significant difference was detected for ear height in the combined-site analysis. MON 87403 had increased ear height (111.1 vs. 107.5 cm) compared to the conventional control. However, the mean value of MON 87403 for ear height was within the reference range for this characteristic and a small ear height difference would not be expected to influence plant weediness. Thus, the measured phenotypic characteristics of MON 87403 were not altered in terms of pest/weed potential compared to conventional maize (Figure 1, step 2, “no” answer) and (Figure 1, step 3, “no” answer). Plant vigor data were summarized as ranges within individual sites. MON 87403 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 betweenMON 87403 and the conventional control in the plant vigor ratings across sites (Table H4). The phenotypic and agronomic characteristics evaluated in this study were used to provide a detailed description of MON 87403 compared to the conventional control. A subset of these characteristics was used to assess the weediness of MON 87403. The results of the agronomic and phenotypic assessment demonstrate that there were no changes indicating altered weediness/pest potential of MON 87403 compared to the conventional control. Thus, the introduction of the increased ear biomass trait is not expected to result in increased plant pest/weed potential from MON 87403 compared to conventional maize. Evaluation of Intended Phenotypic Characteristic: In other specifically designed experiments, ear biomass data were collected at 13 sites in the U.S. during 2012 to evaluate the intended phenotype of MON 87403 compared to the conventional control (Table VII-1). These 13 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 87403 and the conventional control were evaluated. The harvested area within each plot was 1 m in length. All plots of MON 87403 and the conventional control were uniformly managed in order to assess whether the introduction of the increased ear biomass maize trait demonstrated the intended phenotypic characteristic. Descriptions of the evaluated phenotypic characteristic and the timing of the evaluation are listed in Table VII-1). As noted previously (Section I.B), in the combined-site analysis, a statistically significant difference was detected between MON 87403 and the conventional control for increased ear biomass (Table I-1). MON 87403 had increased ear biomass (mean 144.5 g/m of row vs. 129.3 g/m of row) compared to the conventional control thereby demonstrating the intended phenotype.Monsanto Company CR262-14U1 106 of 260

Table VII-3. Field Phenotypic Evaluation Sites for MON 87403 during 2012 Site Code County, State ARNE Jackson, Arkansas IABG Greene, Iowa IARL Jefferson, Iowa ILCX Vermilion, Illinois ILMN Warren, Illinois INSH Boone, Indiana KSLA Pawnee, Kansas NCBD Perquimans, North Carolina NEDC Butler, Nebraska NESH Polk, Nebraska NEYO York, Nebraska PAGR Lehigh, Pennsylvania PAHM Berks, PennsylvaniaMonsanto Company CR262-14U1 107 of 260

Table VII-4. Combined-Site Comparison of MON 87403 to Conventional Control for Phenotypic and Agronomic Characteristics During 2012 Mean (S.E.) 1 Reference Range 2 Phenotypic Characteristic (units) MON 87403 Control Min Max Early stand count (#/plot) 83.0(0.92) 83.6 (0.90) 75.4 96.8 Days to 50% pollen shed 63.5 (0.67) 63.0 (0.65) 56.4 71.3 Days to 50% silking 63.9 (0.65) 64.0 (0.70) 56.0 68.8 Stay green rating (1-9 scale) 5.3 (0.36) 5.2 (0.35) 1.0 9.0 Ear height (cm) 111.1 (2.24)* 107.5 (2.25) 86.9 133.2 Plant height (cm) 240.5 (3.69) 239.5 (3.48) 184.2 260.3 Dropped ears (#/plot) 1.2 (0.45) 0.9 (0.36) 0.0 6.1 Stalk lodged plants (#/plot) 2.4 (0.58) 2.2 (0.53) 0.0 10.8 Root lodged plants (#/plot) 0.7 (0.26) 0.5 (0.17) 0.0 1.0 Final stand count (#/plot) 72.5 (0.72) 72.0 (0.70) 63.8 76.0 Grain moisture (%) 19.1 (0.61) 19.0 (0.58) 14.0 28.7 Test weight (kg/hl) 72.6 (0.69) 72.7 (0.67) 67.9 79.2 Yield (Mg/ha) 10.1 (0.45) 10.3 (0.52) 6.3 14.3

* I n d i c a t e s s t a t i s t i c a l d i f

f e r e n c e b e t w e e n M O N 8 7 4 0 3 a n d t h e c o n v e n t i o n a l c o n t r o l ( α = 0

. 0 5 )

. Note: The experimental design was a randomized complete block with four replicates per site. 1 Test and control values represent means with standard error in parentheses. N = 51. 2 Reference range is calculated from the minimum and maximum mean values from among 21 unique reference materials.Monsanto Company CR262-14U1 108 of 260

VII.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 87403. In the 2012 U.S. field trials conducted to evaluate the phenotypic and agronomic characteristics of MON 87403, 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 VII-5 through VII-7 and H-5 through H-10). These data were used to assess plant pest potential (Section IX) compared to the conventional control. The results of the field evaluations showed that the increase ear biomass trait did not unexpectedly alter the assessed environmental interactions of MON 87403 compared to the conventional control. The lack of significant biological differences in plant responses to abiotic stress, disease damage, arthropod-related damage, and arthropod abundance support the conclusion that the introduction of the increased ear biomass trait is not expected to result in increased plant pest potential from MON 87403 compared to commercial maize.

VII.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 stressors, 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 87403 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 VII-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 H (Tables H-5 through H-7). In the qualitative assessment, no differences in the range of responses were observed between MON 87403 and the conventional control for any of the 143 comparisons of plant response to abiotic stressors, including cold, drought, flood, frost, hail, heat, nutrient deficiency, soil compaction, sunscald, and wind (Tables VII-5 and H-5). Additionally, no differences in the range of responses were observed between MON 87403 and the conventional control for any of the 176 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,Monsanto Company CR262-14U1 109 of 260

Pythium sp., Rhizoctonia sp., rust, seedling blight, smut, stalk rot, and Stewart’s bacterial wilt (Tables VII-5 and H-6). Finally, no differences in the range of responses were observed between MON 87403 and the conventional control for any of the 150 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 (Tables VII-5 and H-7).

The lack of differences observed between MON 87403 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 increased ear biomass trait is not expected to cause a biologically meaningful change in terms of plant pest potential compared to the conventional control (See Section VII.B.2).Monsanto Company CR262-14U1 110 of 260

Table VII-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 87403 and the conventional control during any observation for damage caused by any of the assessed stressors. 1 MON 87403 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 87403 and the conventional control. Stressor Number of observations across all sites Number of observations with no differences between MON 87403 and the conventional control across all sites 1

Abiotic stressors 143 143 Disease damage 176 176 Arthropod-related damage 150 150 Total 469 469Monsanto Company CR262-14U1 111 of 260

VII.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 (Table VII-3: 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. Descriptions of the evaluated environmental interactions characteristics and the timing of the evaluations are listed in Table VII-1. The materials, methods, additional details concerning the specific arthropod damage assessments, sticky traps, and visual counts with detailed results of the individual-site data comparisons are presented and discussed in Appendix H (Tables H-8 through H-10). The results of the combined-site analysis are summarized below.

I n t h e c o m b i n e d

- s i t e a n a l y s i s , n o s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s ( α = 0

. 0 5 ) w e r e detected between MON 87403 and the conventional control for plant damage caused by CEW or ECB (Table VII-6). Considering this lack of significant differences, the results indicate no changes in susceptibility or resistance of MON 87403 to these common maize pests (See Figure VII-1, Step 2, answer “no”). In an assessment of arthropod abundance from sticky traps, no statistically significant differences were detected between MON 87403 and the conventional control for 130 out of 144 comparisons among the collections at the four sites (Tables VII-7 and H-9). The mean abundance values for MON 87403 were within the respective ranges of commercial reference hybrids for seven of the 14 detected differences. For the remaining seven differences, the mean abundance values from MON 87403 were outside the reference range; however, these differences were not consistently detected across collections or sites. Thus, these differences 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 87403 compared to the conventional control (See Section VII.B.2) In an assessment of arthropod abundance from visual counts, no statistically significant differences were detected between MON 87403 and the conventional control for 61 out of 66 comparisons among the collections at the four sites (Tables VII-7 and H-10). The mean abundance values for MON 87403 were within the respective ranges of commercial reference hybrids for three of the five detected differences. For the remaining two differences, the mean abundance values from MON 87403 were outside the reference range; however, these differences were not consistently detected across collections or sites. Thus, these differences 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 87403 compared to the conventional control (See Section VII.B.2).Monsanto Company CR262-14U1 112 of 260

Table VII-6. Combined-Site Comparison of CEW and ECB Damage to MON 87403 Compared to the Conventional Control during 2012 Pest Arthropod Assessment Timing Damage assessment Mean (S.E.) 1 Reference range 2 MON 87403 Control Corn earworm ( H. zea ) R5 Damage area of 10 plants per plot (cm 2 ) 1.8 (0.41) 2.7 (0.78) 0.0 – 5.0

European corn borer ( O. nubilalis ) R6 Number of stalk galleries of 10 plants per plot 0.3 (0.12) 0.3 (0.11) 0.0 – 1.2

European corn borer ( O. nubilalis ) R6 Stalk gallery length (cm) of 10 plants per plot 1.3 (0.57) 1.4 (0.50) 0.0 – 4.7

Note: The experimental design was a randomized complete block with four replicates per site. N o s t a t i s t i c a l l y s i g n i f

i c a n t d i f f e r e n c e s w e r e d e t e c t e d b e t w e e n M O N 8 7 4 0 3 a n d t h e c o n v e n t i o n a l c o n t r o l ( α = 0

. 0 5 ) u s i n g A N O V A

. 1 MON 87403 and the conventional control values represent means with standard error in parentheses. N = 16. 2 Reference range is calculated from the minimum and maximum mean values from among 11 unique reference materials across all sites (IABG, NCBD, NEYO, and PAHM).Monsanto Company CR262-14U1 113 of 260

Table VII-7. Summary of Arthropod Abundance Assessments and Detected Differences during 2012
Summary of Statistical Comparisons 1 Summary of Detected Differences 2

Arthropod Abundance Assessment Number

of sites Number of

comparisons across sites Number of

comparisons where no differences were detected Arthropod Site Collection Number Within

reference range? Consistently

detected

across collections or sites? Sticky Traps 4 144 130 Lacewings IABG 1 Yes No Lacewings NEYO 3 Yes No Lacewings NEYO 5 No No Macro-parasitic hymenoptera PAHM 4 No No Micro-parasitic hymenoptera NCBD 2 Yes No Aphids NEYO 5 Yes No Corn flea beetles IABG 3 & 5 No No Corn rootworm beetles NEYO 3 Yes No Thrips NCBD 2 Yes No Thrips NEYO 2 & 4 No No Thrips NEYO 5 Yes No Thrips PAHM 3 No No

Visual Counts 4 66 61 Minute pirate bugs PAHM 4 No No Corn rootworm beetles PAHM 1 No No Sap beetles NCBD 5 Yes No Sap beetles NEYO 3 Yes No Shining flower beetles NCBD 2 Yes NoMonsanto Company CR262-14U1 114 of 260

1 Q u a n t i t a t i v e a rt h r o p o d a b u n d a n c e a s s e s s m e n t s w e r e s t a t i s t i c a l l y a n a l y z e d a t α = 0

. 0 5 u s i n g A N O V A

. L a c k o f s u f f i c i e n t a rt h r o p o d abundance precluded statistical comparisons between MON 87403 and the conventional control for additional 92 comparisons (sticky traps) and 171 comparisons (visual counts); however, descriptive statistics were provided for these comparisons in Appendix H (Tables H-9 and H-10).

2 Nineteen statistically significant differences were detected. These differences are further discussed in Section VII.C.2.2 using the approach outlined in Section VII.B.2.Monsanto Company CR262-14U1 115 of 260

VII.C.3. Pollen Characteristics USDA-APHIS considers the potential for gene flow and introgression of the biotechnology-derived trait 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 87403. In addition, morphological characterization of pollen produced by MON 87403 and the conventional control is relevant to the plant pest risk assessment because it adds to the detailed description of the phenotype of MON 87403 compared to the conventional control. The viability and morphology of pollen collected from MON 87403 compared to that of the conventional control were assessed. Pollen was collected from MON 87403, the conventional control, and four commercial references (Table I-1) grown under similar agronomic conditions at the field site in Story County, 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 non-systematically selected plants per plot and stained for assessment. Descriptions of the evaluated pollen viability and morphology characteristics and the timing of the evaluations are listed in Table VII-1. The details of the materials and experimental methods used in this evaluation are presented in Appendix I: .

N o s t a t i s t i c a l l y s i g n i f

i c a n t d i f f e r e n c e s ( α = 0

. 0 5 ) w e r e d e t e c t e d b e t w e e n M O N 8 7 4 0 3 a n d the conventional control for percent viable pollen or pollen grain diameter (Table VII-8). Furthermore, no visual differences in general pollen morphology were observed between MON 87403 and the conventional control (Figure I-1). The pollen characterization data contribute to the detailed phenotypic description of MON 87403 compared to the conventional control. Based on the assessed characteristics, the results support a conclusion that neither pollen viability nor morphology of MON 87403 were altered compared to conventional maize.Monsanto Company CR262-14U1 116 of 260

Table VII-8. Pollen Characteristics of MON 87403 Compared to the Conventional Control during 2012

Pollen Characteristic (unit) Mean (S.E.) 1 Reference Range 2

MON 87403 Control

Viability 3 (%) 97.8 (0.70) 98.7 (0.36) 97.3 – 99.3

Diameter 4 (μm) 81.8 (1.20) 84.6 (2.06) 80.5 – 83.7

Note: No significant differences were detected between the MON 87403 and the
c o n v e n t i o n a l c o n t r o l ( α = 0

. 0 5 )

. 1 MON 87403 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 40X magnification. 4 Evaluated from 10 representative viable pollen grains per replication at 200X magnification.Monsanto Company CR262-14U1 117 of 260

VII.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 characteristics, were used to assess whether the introduction of the increased ear biomass trait altered the plant pest potential of MON 87043 compared to the conventional control, considered within the context of the variation among the reference hybrids. These assessments included six 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; and 6) intended phenotype (increased ear biomass). 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 87403 compared to conventional maize. Results from these assessments comparing MON 87403 and the conventional control demonstrate that MON 87403 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 87403 is not meaningfully different from conventional maize with the exception of the increased ear biomass trait and is not expected to pose a plant pest risk compared to conventional maize.Monsanto Company CR262-14U1 118 of 260 VIII. U.S. AGRONOMIC PRACTICES

VIII.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 87403. 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 87403 is no more susceptible to diseases or pests than commercially cultivated maize. Additionally, data presented in this section show that MON 87403 is not expected to pose a plant pest risk compared to conventional maize. MON 87403 provides an i n c r e a s e d e a r b i o m a s s a t t h e R 1 r e p r o d u c t i v e s t a g e d e r i v e d f

r o m t h e A T H B 1 7 ∆ 1 1 3 protein and hence an increased yield opportunity. Thus, there are no expected changes to the inputs needed for MON 87403, and no expected impacts to agronomic practices employed for production of maize compared to the current situation.

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

VIII.B.1. Maize Production The U.S., China, Brazil, Ukraine, and Argentina are the top five countries producing maize globally (USDA-FAS 2014). As noted in Section I.B, maize ( Zea mays L.) is the largest crop grown in the U.S. in terms of acreage planted (95.4 million acres in 2013) (Table VIII-1) (USDA-NASS 2014c), exceeding soybean and wheat with acreages of 76.5 (USDA-NASS 2014b) and 56.2 million acres (USDA-NASS 2014a), respectively. The value of maize reached $62.7 billion in the United States in 2013, exceeding soybeans and wheat with values of $41.8 and $14.4 billion, respectively (USDA-NASS2013a). The principal uses of maize are feed and residual, ethanol fuel, export, and highfructose corn syrup (Capehart et al. 2012).
The planting of 95.4 million acres of maize in 2013 was down 1.8 million acres from 2012 (Table VIII-1). Much of that production occurs in upper Midwest states (Figure VIII-1). Of the 2013 acreage planted, approximately 87.7 million acres were harvested for grain and 6.3 million acres were harvested for silage (USDA-NASS 2013b). Total production was approximately 13.9 billion bushels with an average yield of 158.8 bushels per acre (Figure VIII-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 VIII-1).Monsanto Company CR262-14U1 119 of 260

Table VIII-1. Maize Production in the U.S., 2008-2013

Year Acres Planted (x 1,000) Acres

Harvested (x 1,000) Production

(x 1,000 $) Production

(x 1,000 bushels) Yield

(bushels/acre)

2013 95,365 87,668 62,716,048 13,925,147 158.8 2012 97,155 87,375 74,330,610 10,780,296 123.4 2011 91,936 83,989 76,939,603 12,359,612 147.2 2010 88,192 81,446 64,643,295 12,446,865 152.8 2009 86,382 79,490 46,734,066 13,091,862 164.7 2008 85,982 78,570 49,312,615 12,091,648 153.9
Source: (USDA-NASS 2013b)

Figure VIII-1. Planted Maize Acres by County in the U.S. in 2012 Source: (USDA-NASS 2012)Monsanto Company CR262-14U1 120 of 260

VIII.C. Management of Insect Pests Monsanto summarized major issues associated with the management of insect pests in its petition for nonregulated status for Corn Rootworm-Protected Maize MON 87411 (Petition #13-290-01p) (Monsanto 2013). None of the information on this subject has changed in any substantive way and is incorporated here by reference (Monsanto 2013) (Section IX.D). In brief, insect pests continue to cause damage to maize and are commonly addressed by insecticide treatment of seeds, soil, or over-the-top application of insecticides, or use of a number of crop rotation or integrated pest management practices. MON 87403 was developed to increase the yield opportunity of commercial maize hybrids and has no unique pest control attributes. Thus, no changes to insect pest control practices are expected from use of MON 87403.

VIII.D. Management of Diseases and Other Pests Monsanto summarized major issues associated with the management of diseases and other pests in its petition for nonregulated status for Corn Rootworm-Protected Maize MON 87411 (Petition #13-290-01p) (Monsanto 2013). None of the information on this subject has changed in any substantive way and is incorporated here by reference (Monsanto 2013) (Section IX.E). Briefly, management of diseases and pests of maize are important to protecting the yield of harvested grain. Disease and pest incidence varies from year to year and growers may choose to use pesticides or a variety of management practices to control problem diseases or pests. Environmental observations in field studies have demonstrated no apparent impact of MON 87403 on diseases of maize (Section VII.C.2). Therefore, no changes in current disease management practices are anticipated from the introduction of MON 87403.

VIII.E. Weed Management Monsanto summarized major issues associated with the management of weeds in its Petition for nonregulated status for corn rootworm-protected MON 87411 (Petition #13-290-01p) (Monsanto 2013). None of the information on this subject has changed in any substantive way and is incorporated here by reference (Monsanto 2013) (Section IX.F). Briefly, weed control in maize is essential to optimizing yield because weeds compete with maize for light, nutrients, and moisture. MON 87403 was developed to increase the yield opportunity of commercial maize hybrids and has no unique herbicide tolerance traits. Thus, no changes to herbicide use or weed resistance management practices are expected from use of MON 87403.

VIII.F. 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 weatherMonsanto Company CR262-14U1 121 of 260

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 2013b). Introduction of MON 87403 is not, however, expected to impact crop rotation practices any more so than current biotechnology-derived products available to growers.

VIII.G. 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 87403 (Section VIII.C), including data regarding germination and seed dormancy, indicates that MON 87403 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 inrotational 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 87403 is not expected to impact maize volunteer management practices any more so than existing commercial maize hybrids.

VIII.H. Stewardship of MON 87403 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 processesMonsanto Company CR262-14U1 122 of 260

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 87403 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 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 87403 seed. As with all of Monsanto’s products, before commercializing MON 87403 products in any country, the appropriate detection methods will be made available to maize producers, processors, and buyers.

VIII.I. Impact of the Introduction of MON 87403 on Agricultural Practices MON 87403 has been developed to provide an increased yield opportunity in maize hybrids that will be grown in the U.S. The introduction of MON 87403 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 87403. MON 87403 has been shown to be comparable to conventional maize in its compositional, phenotypic, and agronomic characteristics (Sections VI and VII). When introgressed into existing biotechnology-derived maize hybrids that contain insect protection and herbicide tolerance traits, MON 87403 is expected to continue to provide benefits to growers, that include reduced use of insecticides , increased yield protection and opportunity, water conservation , and increased worker safety.Monsanto Company CR262-14U1 123 of 260 IX. PLANT PEST ASSESSMENT
This section provides a brief review and assessment of the plant pest potential of MON 87403 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 PPA, the definition of “plant pest” includes 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; or (G) an infectious agent or other pathogens (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 plant pest risk. 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 87403 is not expected to be a plant pest, and therefore should no longer be subject to regulation under 7 CFR § 340.

IX.A. Characteristics of the Genetic Insert and Expressed Product

IX.A.1. Genetic Insert As described in Section III, MON 87403 was developed by Agrobacterium -mediated transformation of maize embryos using plasmid vector PV-ZMAP5714. Characterization of the DNA insert in MON 87403 was conducted using a combination of sequencing, PCR, and bioinformatics methods. The results of this characterization demonstrate that MON 87403 contains one copy of the intended T-DNA containing the ATHB17 expression cassette 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 87403. Additionally, the genomic organization at the insertion site was assessed by comparing the sequences flanking the T-DNA insert in MON 87403 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 87403 upon DNA integration.Monsanto Company CR262-14U1 124 of 260

IX.B. Expression and Characterization of Gene Product

IX.B.1. Protein Safety and Expression Levels

T h e s a f e t y a n d e x p r e s s i o n o f t h e A T H B 1 7 Δ 1 1 3 p r o t e i n i s d e t a i l e d i n S e c t i o n V

. Expression levels were determined from four tissue types from trials conducted in 2012 in the United States and are presented in Section V.C. The expression in the various t i s s u e s r a n g e d f

r o m < L O D ( < 0

. 0 0 0 2 8 μ g / g d w ) t o 0

. 0 1 7 μ g / g d w

. A T H B 1 7 Δ 1 1 3 represents a very small percentage of the total protein in maize seed (no more than 0

. 0 0 1 % )

. T h e A T H B 1 7 Δ 1 1 3 p r o t e i n o r i g i n a t e s f r o m Arabidopsis thaliana, and no Arabidopsis proteins have been reported in the peer-reviewed database of known allergens (FARRP 2013). Futhermore, bioinformatic assessments show that the A T H B 1 7 Δ 1 1 3 p r o t e i n d o e s n o t s h o w h o m o l o g y t o a n y kn o w n a l l e r g e n s , t o x i n s , o r a n y other protein with known adverse effects. These assessments also showed that the amino
a c i d s e qu e n c e o f t h e A T H B 1 7 Δ 1 1 3 p r o t e i n s h a r e s 5 8 %

- 8 3 % s e qu e n c e i d e n t i t y t o proteins present in a variety of food crops including corn, rice, sorghum, cruciferous

v e g e t a b l e s , t o m a t o , p o t a t o , p a

p a y a , o r a n g e a n d g r a

p e , i n d i c a t i n g t h a t A T H B 1 7 ∆ 1 1 3 i s very similar to proteins that have a robust history of safe use. Finally, the protein is rapidly digested in simulated gastric fluid and simulated intestinal fluid assays (Section V.D) and a mouse gavage study demonstrated no acute oral toxicity with a NOAEL for A T H B 1 7 Δ 1 1 3 of 1335 mg/kg, further supporting a conclusion that this protein is safe for consumption.

IX.C. Compositional Characteristics Compositional comparisons based on OECD guidance were presented in Section VI to assess whether levels of nutrients, anti-nutrients, and secondary metabolites in grain and forage derived from MON 87403 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 andraffinose. 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 (nine in forage and 69 in grain). Of those 78 components, 14 fatty acids, sodium, and furfural had more than 50% of observations below the assay limit of quantitation (LOQ) and were excluded from statistical analysis. Moisture in grain and forage was measured for conversion of components to dry weight, but was not statistically analyzed. Therefore, 60 components were statistically analyzed. A combined-site analysis of the data was conducted to determine statistically significant differences (at 5%) between MON 87403 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 87403 and the conventional control; 2) consideration of the mean differencesMonsanto Company CR262-14U1 125 of 260

between MON 87403 and the conventional control in the context of the range of values for the conventional control, 3) comparison of the mean differences between MON 87403 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 87403 were within 99% tolerance intervals, literature values, and/or ILSI-CCDB 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 87403 and the conventional control. Of the 60 components statistically assessed there were no statistically significant differences in any component. These results support the overall conclusion that MON 87403 was compositionally equivalent to the conventional control.

IX.D. Phenotypic, Agronomic, and Environmental Interaction Characteristics An extensive set of comparative plant characterization data were used to assess whether the introduction of an increased ear biomass trait altered the plant pest potential of MON 87403 compared to the conventional control (Section VII). Phenotypic, agronomic, and environmental interaction characteristics of MON 87403 were evaluated and compared to those of the conventional control. As described previously, 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 87403 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 87403 is no more likely to pose a plant pest risk than conventional maize.

IX.D.1. Seed Dormancy and Germination A comparative assessment of seed germination and dormancy characteristics was conducted on MON 87403 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 87403 is not expected to result in increased plant pest/weed potential compared to conventional maize.

IX.D.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,Monsanto Company CR262-14U1 126 of 260

ear height, plant height, dropped ears, stalk lodged plants, root lodged plants, final stand count, grain moisture, test weight, R1 ear biomass, and yield were assessed. In the combined-site analysis in which the data were pooled among the sites, no statistically significant differences, except for ear height, were detected between MON 87403 and the conventional control for any of the assessed characteristics. Thus, the phenotypic and agronomic characteristics of MON 87403 were not altered in terms of pest/weed potential compared to conventional maize.

IX.D.3. Response to Abiotic Stressors No differences were observed during comparative field observations between MON 87403 and the conventional control with respect to responses to abiotic stressors such as drought, mineral and nutrient toxicity, or temperature stress. The lack of significant differences in the MON 87403 response to abiotic stress supports the conclusion that the introduction of the ATHB17 Δ 1 1 3 p r o t e i n is unlikely to result in increased pest/weed potential compared to conventional maize.

IX.D.4. 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 87403 and the conventional control for percent viable pollen or pollen grain diameter. Additionally, no visual differences in general pollen morphology were observed between MON 87403 and the conventional control. Based on the assessed characteristics, the results support a conclusion that neither pollen viability nor morphology of MON 87403 were altered and therefore do not contribute to an increased pest/weed potential compared to conventional maize.

IX.E. Impact to Non-target Organisms Including Those Beneficial to Agriculture or Threatened and Endangered Species Data from 2012 U.S. phenotypic and agronomic studies and observational data on environmental interactions such as plant-disease interactions, arthropod damage and arthropod abundance, were collected at select sites for MON 87403 and conventional controls. Results from these extensive studies support conclusions of no adverse impacts to non-target arthropod populations and no changes to plant-disease interactions.
As also noted, the ATHB17 Δ 1 1 3 p r o t e i n b e l o n g s t o a c o m m o n c l a s s o f p l a n t p r o t e i n transcription factors (HD-Zip) and shares protein sequence homology with a number of common food crops that are consumed widely by humans and animals without noted allergenic or toxic effects. Bioinformatics analyses also assessed the potential for
a l l e r g e n i c i t y , t o x i c i t y , o r b i o l o g i c a l a c t i v i t y o f A T H B 1 7 Δ 1 1 3

. T h o s e a n a l y s e s d e m o n s t r a t e d t h a t A T H B 1 7 Δ 1 1 3 p r o t e i n d o e s n o t s h a r e a m i n o a c i d s e q u e n c e s i m i l a r i t y with known allergens, gliadins, glutenins, or protein toxins which could have adverse effects to human or animal health. Additionally, a mouse gavage study usingMonsanto Company CR262-14U1 127 of 260

ATHB17 Δ 1 1 3 p r o t e i n a l s o s u p p o r t s a c o n c l u s i o n o f s a f e t y f r o m c o n s u m p t i o n o f MON 87403 by vertebrate organisms. Taken together, these data, along with the compositional equivalence data noted above (Section VI.A), support the conclusion that MON 87403 has no reasonable mechanism for harm to NTOs, or impact on threatened and endangered species compared to cultivation of conventional maize.

IX.F. Weediness Potential of MON 87403 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 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 87403 and a conventional control, phenotypic, agronomic and environmental interaction data were evaluated (Section VII) 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 87403 and the conventional control for traits potentially associated with weediness. The only difference noted related to ear height and that characteristic is not known to contribute to weediness. Furthermore, comparative field observations between MON 87403 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 87403 has no increased weed potential compared to conventional maize and it is no more likely to become a weed than conventional maize.

IX.G. 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 87403 is discussed below.Monsanto Company CR262-14U1 128 of 260

IX.G.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, 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 IX-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 87403 seed or grain have been conducted under USDA notification according to these standards. Gene flow from fields planted with MON 87403 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 CR262-14U1 129 of 260

Table IX-1. Summary of Published Literature on Maize Cross Pollination

Pollinator Distance (m) Reported

Outcrossing (%) Comments Country Reference

~1 28.6 Frequencies of outcrossing by distance. Three year study. Pollen source was a yellow dent and the pollen recipient

was a white sweet maize. USA (Jones and Brooks 1950) 25 14.2 75 5.8 125 2.3 200 1.2 300 0.5 400 0.2 500 0.2 100 0.01 Frequencies of outcrossing by distance and pollen viability. Two year study. A purple gene marker was utilized to measure pollen mobility. Mexico (Luna et al. 2001) 150 0.00 200 0.01 300 0.00 400 0.00 1 9.7-19.0 Frequency of outcrossing by distance. Conducted over three years and three sites. Single male and female per

location. Canada (Ma et al. 2004) 5 1.3-2.6 10 0.7-2.0 14 0.3-0.6 19 0.4 24 0-0.3 28 0.1-0.5 33 0-0.3 36 0-0.1Monsanto Company CR262-14U1 130 of 260

Table IX-1 (continued). Summary of Published Literature on Maize Cross Pollination Pollinator Distance (m) Reported

Outcrossing (%) Comments Country Reference

24-32 0.01-0.7 Frequencies of outcrossing by distance. One pollen donor and 7 pollen recipients had different relative maturities. The pollen donor had the genetic markers P1-rr and R1-nj. Pollination of the pollen recipient caused the female with typically a yellow kernel to produce a kernel with a purple coloration. Conducted over two years/two sites. Values reported here are from one site. USA (Halsey et al. 2005) 60-62 0.01-0.2 123-125 0.001-0.08 244-254 0-0.02 486-500 0-0.005
743-745 0-0.002

1 17.0-29.9 Frequency of outcrossing by distance. Pollination was quantified by measuring outcrossing from a transgenic hybrid plot to a conventional grain production field. A combination of three marker genes were utilized to detect outcrosses: y1 (seed color gene), Bt and glyphosate tolerance. Two years/two sites. 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 ~1 3-13 The main objective of the study was to compare a PCR based method to measure outcrossing rates determined by phenotypic analysis. Four Bt hybrids and a single non- Bt hybrid were used as a pollen donor and reciepient, respectively. Conducted in

one year/one site. Spain (Pla et al. 2006) 2 0.2-10 5 0.1-2.3 10 0.2-3.7 20 0.1-0.8 40 0-0.7 80 0.1-0.2Monsanto Company CR262-14U1 131 of 260

Table IX-1 (continued). Summary of Published Literature on Maize Cross Pollination Pollinator Distance (m) Reported

Outcrossing (%) Comments Country Reference

2 34.9 Frequency of outcrossing (expressed as % GM DNA) by distance. The study was conducted in a large farm scale evaluation across the UK. Values reported here are

maximum raw values UK (Weekes et al. 2007) 5 9.9 10 12.2 20 8.2 25 4 50 5.9 150 5.4 200 0.24 52 0.009 Frequency of outcrossing by distance. Outcrossing was measured using the occurrence of yellow kernels in 13

white kernel maize fields. Switzerland (Bannert and Stamp 2007) 85 0.015 105 0.003 125 0.01 149 0.016 150 0.007 200 0.009 287 0.005 371 0.008 402 0.005 458 0.0002 4125 0.006 4440 0.0005Monsanto Company CR262-14U1 132 of 260

IX.G.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.

IX.G.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 87403.

IX.G.4. 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, theMonsanto Company CR262-14U1 133 of 260

consequences would be negligible since the A T H B 1 7 Δ 1 1 3 p r o t e i n is similar to other HDZip proteins found in maize that are not known to have meaningful toxicity to humans and NTOs. Similar to the situation with transfer of the protein genes, the likelihood of horizontal transfer of the A T H B 1 7 Δ 1 1 3 c o d i n g s e qu e n c e from MON 87403 is also exceedingly small. The consequence of such transfer, given the known lack of toxicity of these classes of proteins would also be expected to be inconsequential. In either case, the presence of this coding sequence would not be expected to increase the pest/weed potential of the recipient species.

IX.H. Potential Impact on Maize Agronomic Practices An assessment of current maize agronomic practices was conducted to determine whether the cultivation of MON 87403 has the potential to impact current maize management practices (Section VIII). Maize fields are typically highly managed agricultural areas that are dedicated to crop production. Other than the specific insertion of the ATHB17 coding sequence that provides the potential for increased ear biomass at an early reproductive phase and hence increased yield opportunity, MON 87403 is similar to other high-yielding maize hybrids available commercially in the U.S. The data presented demonstrate that MON 87403 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 87403 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.

IX.I. Conventional Breeding with Other Biotechnology-derived or Conventional Maize Numerous biotechnology-derived maize products have been deregulated or are under consideration for deregulation by USDA-APHIS. Once deregulated, MON 87403 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 biotechnology-derived 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 severalfacts. Namely: 1) stability of the genetic inserts is confirmed in each approved biotechderived 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 byplant 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, CropLifeMonsanto Company CR262-14U1 134 of 260

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 87403 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 87403 and conventional maize or deregulated biotechnology-derived maize are no more likely to pose a plant risk than commercially cultivated maize.

IX.J. Summary of Plant Pest Assessments A plant pest, as defined in the PPA, is the living stage of any of the following 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 III through VII and Section IX of this petition confirm that MON 87403, with the exception of the increased ear biomass at R1 phenotype, 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 87403 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 87403 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 A T H B 1 7 Δ 1 1 3 cassette; 2) characterization and safety of the expressed products; 3) compositional equivalence of MON 87403 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. Based on the data and information presented in this petition, it is concluded that, like conventional maize and previously deregulated biotechnology-derived maize, MON 87403 is not expected to be a plant pest. Results also support a conclusion of no increased weediness potential of MON 87403 compared to conventional maize. Therefore, Monsanto Company requests a determination from USDA-APHIS that MON 87403 and any progeny derived from crosses between MON 87403 and other commercial maize be granted nonregulated status under 7 CFR Part 340.Monsanto Company CR262-14U1 135 of 260 X. ADVERSE CONSEQUENCES OF INTRODUCTION
Monsanto knows of no study results or observations associated with MON 87403 indicating that there would be adverse consequences from its introduction. MON 87403 produces a truncated A T H B 1 7 Δ 1 1 3 p r o t e i n , w h i c h h a s b e e n f u l l y c h a r a c t e r i z e d and its safety has been thoroughly assessed in this submission. As demonstrated by field test results and laboratory tests, the only phenotypic differences between MON 87403 and conventional maize are related to an increased ear biomass phenotype at the R1 growth stage. The data and information presented in this petition demonstrate that MON 87403 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 A T H B 1 7 Δ 1 1 3 cassette 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 87403 demonstrate that MON 87403 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 87403 is unchanged compared to conventional maize. There is no indication that MON 87403 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 87403 are negligible. The introduction of MON 87403 will not adversely impact cultivation practices or the management of weeds, diseases, and insects in maize production systems. Farmers familiar with commercial maize hybrids 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 CR262-14U1 136 of 260 REFERENCES
Anderson, W.P. 1996. Weed ecology. Pages 27-38 in Weed Science: Principles and Applications. Third Edition. West Publishing Company, St. Paul, Minnesota.
AOSA. 2012a. AOSA Rules for testing seeds. Volume 4: Seedling evaluation, Association of Official Seed Analysts, Ithaca, New York.
AOSA. 2012b. AOSA Rules for testing seeds. Volume 1: Principles and procedures, Association of Official Seed Analysts, Ithaca, New York.
AOSA/SCST. 2010. Tetrazolium testing handbook. Association of Official Seed Analysts and the Society of Commercial Seed Technologists, Ithaca, New York.
Ariel, F.D., P.A. Manavella, C.A. Dezar and R.L. Chan. 2007. The true story of the HDZip family. Trends in Plant Science 12:419-426.
Baltazar, B.M., J. de Jesús Sánchez-Gonzalez, L. de la Cruz-Larios and J.B. Schoper. 2005. Pollination between maize and teosinte: An important determinant of gene flow in Mexico. Theoretical and Applied Genetics 110:519-526.
Bannert, M. and P. Stamp. 2007. Cross-pollination of maize at long distance. European Journal of Agronomy 27:44-51.
Barker, R.F., K.B. Idler, D.V. Thompson and J.D. Kemp. 1983. Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Molecular Biology 2:335-350.
Barry, G.F., G.M. Kishore, S.R. Padgette and W.C. Stallings. 2001. Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases. Patent 6,248,876, U.S. Patent Office, Washington, D.C.
Bevan, M., W.M. Barnes and M.-D. Chilton. 1983. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Research 11:369-385.
BIO. 2010. Product launch stewardship policy. Biotechnology Industry Organization, Washington, D.C. http://www.bio.org/articles/product-launch-stewardship-policy [Accessed May 31, 2012].
Borrás, L., G.A. Slafer and M.E. Otegui. 2004. Seed dry weight response to source-sink manipulations in wheat, maize and soybean: A quantitative reappraisal. Field Crops Research 86:131-146.
Borrás, L. and M.E. Westgate. 2006. Predicting maize kernel sink capacity early in development. Field Crops Research 95:223-233.Monsanto Company CR262-14U1 137 of 260

Bruns, H.A. and H.K. Abbas. 2003. Effects of plant populations on maize hybrids in the sub-tropical Mid South USA. Maydica 48:21-27.
Capehart, T., E. Allen and J. Bond. 2012. Feed outlook. FDS-12I. U.S. Department of Agriculture, Economic Research Service, Washington, D.C.
Clarke, L. and J. Carbon. 1976. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell 9:91-99.
CLI. 2005. Position paper: Regulation of plant biotechnology products containing two or more traits combined by conventional plant breeding. CropLife International, Brussels, Belgium.
Codex Alimentarius. 2009. Foods derived from modern biotechnology. Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations, Rome, Italy.
Crockett, L. 1977. Contents. Pages vii-ix in Wildly Successful Plants: A Handbook of North American Weeds. Macmillan Publishing Co., Inc., New York, New York.
Depicker, A., S. Stachel, P. Dhaese, P. Zambryski and H.M. Goodman. 1982. Nopaline synthase: Transcript mapping and DNA sequence. Journal of Molecular and Applied Genetics 1:561-573.
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.
Duvick, D.N. 1999. Heterosis: Feeding people and protecting natural resources. Pages 19-29 in The Genetics and Exploitation of Heterosis in Crops. J.G. Coors and S. Pandey (eds.). American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.
Ellstrand, N.C., L.C. Garner, S. Hegde, R. Guadagnuolo and L. Blancas. 2007. Spontaneous hybridization between maize and teosinte. Journal of Heredity 98:183-187.
Facciola, S. 1998. Brassicaceae. Pages 52-61 in Cornucopia II: A Source Book of Edible Plants. Kampong Publications, Vista, California.
FAO-WHO. 1996. Joint FAO/WHO expert consultation on biotechnology and food safety. Paper 61. Food and Agriculture Organization of the United Nations, World Health Organization, Rome, Italy.
FARRP. 2013. Allergen database, version 13. University of Nebraska, Food Allergy Research and Resource Program, Lincoln, Nebraska. http://www.allergenonline.org/

.Monsanto Company CR262-14U1 138 of 260

Fisher, K.S. and A.F.E. Palmer. 1983. Maize. Pages 155-248 in Potential Productivity of Field Crops Under Different Environments. International Rice Research Institute, Manila, Philippines.
Flannery, M.L., F.J. Mitchell, S. Coyne, T.A. Kavanagh, J.I. Burke, N. Salamin, P. Dowding and T.R. Hodkinson. 2006. Plastid genome characterisation in Brassica and Brassicaceae using a new set of nine SSRs. Theoretical and Applied Genetics 113:1221- 1231.
Fling, M.E., J. Kopf and C. Richards. 1985. Nucleotide sequence of the transposon Tn 7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)- O -nucleotidyltransferase. Nucleic Acids Research 13:7095-7106.
Fraley, R.T., S.G. Rogers, R.B. Horsch, P.R. Sanders, J.S. Flick, S.P. Adams, M.L. Bittner, L.A. Brand, C.L. Fink, J.S. Fry, G.R. Galluppi, S.B. Goldberg, N.L. Hoffmann and S.C. Woo. 1983. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences of the United States of America 80:4803-4807.
Galinat, W.C. 1988. The origin of corn. Pages 1-31 in Corn and Corn Improvement. Third Edition. G.F. Sprague and J.W. Dudley (eds.). American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.
Giza, P.E. and R.C.C. Huang. 1989. A self-inducing runaway-replication plasmid expression system utilizing the Rop protein. Gene 78:73-84.
Goggi, A.S., P. Caragea, H. Lopez-Sanchez, M. Westgate, R. Arritt and C. Clark. 2006. Statistical analysis of outcrossing between adjacent maize grain production fields. Field Crops Research 99:147-157.
Goodman, M.M. 1988. The history and evolution of maize. Critical Reviews in Plant Sciences 7:197-220.
Halsey, M.E., K.M. Remund, C.A. Davis, M. Qualls, P.J. Eppard and S.A. Berberich. 2005. Isolation of maize from pollen-mediated gene flow by time and distance. Crop Science 45:2172-2185.
Hammond, B.G. and J.M. Jez. 2011. Impact of food processing on the safety assessment for proteins introduced into biotechnology-derived soybean and corn crops. Food and Chemical Toxicology 49:711-721.
Harrigan, G.G., D. Lundry, S. Drury, K. Berman, S.G. Riordan, M.A. Nemeth, W.P. Ridley and K.C. Glenn. 2010. Natural variation in crop composition and the impact of transgenesis. Nature Biotechnology 28:402-404.Monsanto Company CR262-14U1 139 of 260

Harrigan, G.G., W.P. Ridley, K.D. Miller, R. Sorbet, S.G. Riordan, M.A. Nemeth, W. Reeves and T.A. Pester. 2009. The forage and grain of MON 87460, a drought-tolerant corn hybrid, are compositionally equivalent to that of conventional corn. Journal of Agricultural and Food Chemistry 57:9754-9763.
Harrison, L.A., M.R. Bailey, M.W. Naylor, J.E. Ream, B.G. 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. Journal of Nutrition 126:728-740.
Herrmann, K.M. 1995. The shikimate pathway: Early steps in the biosynthesis of aromatic compounds. The Plant Cell 7:907-919.
Huang, S., L.A. Gilbertson, T.H. Adams, K.P. Malloy, E.K. Reisenbigler, D.H. Birr, M.W. Snyder, Q. Zhang and M.H. Luethy. 2004. Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors. Transgenic Research 13:451-461.
ILSI. 2011. Crop Composition Database, Version 4.2. International Life Sciences Institute, Washington, D.C. http://www.cropcomposition.org/

. ISF. 2005. Genetically modified crops and plant breeding. International Seed Foundation, Nyon, Switzerland.
Jones, M.D. and J.S. Brooks. 1950. Effectiveness of distance and border rows in preventing outcrossing in corn. Oklahoma Agricultural Experiment Station Technical Bulletin No. T-38. Oklahoma State Univeristy, Stillwater, Oklahoma.
Jones, R.J., B.M.N. Schreiber and J.A. Roessler. 1996. Kernel sink capacity in maize: Genotypic and maternal regulation. Crop Science 36:301-306.
Kay, R., A. Chan, M. Daly and J. McPherson. 1987. Duplication of CaMV 35 S promoter sequences creates a strong enhancer for plant genes. Science 236:1299-1302.
Keeler, K.H. 1989. Can genetically engineered crops become weeds? Bio/Technology 7:1134-1139.
Klee, H.J., Y.M. Muskopf and C.S. Gasser. 1987. Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: Sequence analysis and manipulation to obtain glyphosate-tolerant plants. Molecular and General Genetics 210:437-442.
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 achieveMonsanto Company CR262-14U1 140 of 260

molecular characterization of crops improved through modern biotechnology. The Plant Genome 5:149-163.
Lamppa, G.K., G. Morelli and N.-H. Chua. 1985. Structure and developmental regulation of a wheat gene encoding the major chlorophyll a/b-binding polypeptide. Molecular and Cellular Biology 5:1370-1378.
Lee, E.A. and M. Tollenaar. 2007. Physiological basis of successful breeding strategies for maize grain yield. Crop Science 47:S202-S215.
Leibman, M., J.J. Shryock, M.J. Clements, M.A. Hall, P.J. Loida, A.L. McClerren, Z.P. McKiness, J.R. Phillips, E.A. Rice and S.B. Stark. 2014. Comparative analysis of maize (Zea mays) crop performance: natural variation, incremental improvements and economic impacts. Plant Biotechnology Journal 1:1-10.
Lingenfelter, D.D. and N.L. Hartwig. 2007. Introduction to weeds and herbicides. Penn State University Agricultural Research and Cooperative Extension, University Park, Pennsylvania.
Luna, S.V., J.M. Figueroa, B.M. Baltazar, R.L. Gomez, R. Townsend and J.B. Schoper. 2001. Maize pollen longevity and distance isolation requirements for effective pollen control. Crop Science 41:1551-1557.
Ma, B.L., K.D. Subedi and L.M. Reid. 2004. Extent of cross-fertilization in maize by pollen from neighboring transgenic hybrids. Crop Science 44:1273-1282.
McElroy, D., W. Zhang, J. Cao and R. Wu. 1990. Isolation of an efficient actin promoter for use in rice transformation. The Plant Cell 2:163-171.
McElwain, E.F. and S. Spiker. 1989. A wheat cDNA clone which is homologous to the 17 kd heat-shock protein gene family of soybean. Nucleic Acids Research 17:1764.
Meyerowitz, E.M. and C.R. Somerville. 1994. Arabidopsis . Cold Spring Harbor Laboratory Press, New York, New York.
Monsanto. 2013. Petition for determination of nonregulated status for corn rootworm protected and glyphosate tolerant MON 87411 maize. 13-290-01p. Monsanto Company, St. Louis, Missouri.
Monsanto Company. 2013. U.S. technology use guide. Monsanto Company, St. Louis, Missouri.
Morris, M.L. 1998. Overview of the world maize economy. Pages 13-34 in Maize Seed Industries in Devoloping Countries. M.L. Morris (ed.). Lynne Rienner Publishers, Inc., Boulder, Colorado.Monsanto Company CR262-14U1 141 of 260

Mukherjee, K., L. Brocchieri and T.R. Bürglin. 2009. A comprehensive classification and evolutionary analysis of plant homeobox genes. Molecular Biology and Evolution 26:2775-2794.
OECD-FAO. 2008. Agricultural outlook 2008-2017. Organisation for Economic Cooperation and Development, Food Agriculture Organization of the United Nations, Paris, France.
OECD. 1993. Safety considerations for biotechnology: Scale-up of crop plants. Organisation for Economic Co-operation and Development, Paris, France. www.biosafety.be/CU/BSL_Ressources/PDF/M00034525.pdf [Accessed January 9, 2009].
OECD. 2002a. Report of the OECD workshop on the toxicological and nutritional testing of novel foods. SG/ICGB(1998)1/FINAL. Organisation for Economic Co-operation and Development, Paris, France.
OECD. 2002b. 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.
OECD. 2003. Consensus document on the biology of Zea mays subsp. mays (maize). ENV/JM/MONO(2003)11. Organisation for Economic Co-operation and Development, Paris, France.
OGTR. 2008. The biology of Zea mays L. ssp mays (maize or corn). Version 1, September 2008, Australian Government, Department of Health and Ageing, Office of the Gene Technology Regulator, Canberra, Australia.
Padgette, S.R., D.B. Re, G.F. Barry, D.E. Eichholtz, X. Delannay, R.L. Fuchs, G.M. Kishore and R.T. Fraley. 1996. New weed control opportunities: Development of soybeans with a Roundup Ready TM gene. Pages 53-84 in Herbicide-Resistant Crops: Agricultural, Environmental, Economic, Regulatory and Technical Aspects. S.O. Duke (ed.). CRC Press, Inc., Boca Raton, Florida.
Pattison, R.J. and A. Amtmann. 2009. N-glycan production in the endoplasmic reticulum of plants. Trends in Plant Science 14:92-99.
Pilacinski, W., A. Crawford, R. Downey, B. Harvey, S. Huber, P. Hunst, L.K. Lahman, S. MacIntosh, M. Pohl, C. Rickard, L. Tagliani and N. Weber. 2011. Plants with genetically modified events combined by conventional breeding: An assessment of the need for additional regulatory data. Food and Chemical Toxicology 49:1-7.
Pla, M., J.-L. La Paz, G. Peñas, N. García, M. Palaudelmàs, T. Esteve, J. Messeguer and E. Melé. 2006. Assessment of real-time PCR based methods for quantification of pollen-Monsanto Company CR262-14U1 142 of 260

mediated gene flow from GM to conventional maize in a field study. Transgenic Research 15:219-228.
Prado, J.R., G. Segers, T. Voelker, D. Carson, R. Dobert, J. Phillips, K. Cook, C. Cornejo, J. Monken, L. Grapes, T. Reynolds and S. Martino-Catt. 2014. Genetically engineered crops: From idea to product. Annual Review of Plant Biology 65:769-790.
Rice, E.A., A. Khandelwal, R.A. Creelman, C. Griffith, J.E. Ahrens, J.P. Taylor, L.R. Murphy, S. Manjunath, R.L. Thompson, M.J. Lingard, S.L. Back, H. Larue, B.R. Brayton, A.J. Burek, S. Tiwari, L. Adam, J.A. Morrell, R.A. Caldo, Q. Huai, J.-L.K. Kouadio, R. Kuehn, A.M. Sant, W.J. Wingbermuehle, R. Sala, M. Foster, J.D. Kinser, R. Mohanty, D. Jiang, T.E. Ziegler, M.G. Huang, S.V. Kuriakose, K. Skottke, P.P. Repetti, T.L. Reuber, T.G. Ruff, M.E. Petracek and P.J. Loida. 2014. Expression of a truncated ATHB17 protein in maize increases ear weight at silking. PLoS ONE 9:e94238.
Ridley, W.P., G.G. Harrigan, M.L. Breeze, M.A. Nemeth, R.S. Sidhu and K.C. Glenn. 2011. Evaluation of compositional equivalence for multitrait biotechnology crops. Journal of Agricultural and Food Chemistry 59:5865-5876.
Ritchie, S.W., J.J. Hanway and G.O. Benson. 1997. How a corn plant develops. Special Report No. 48. Iowa State University of Science and Technology, Cooperative Extension Service, Ames, Iowa.
Ruberti, I., G. Sessa, S. Lucchetti and G. Morelli. 1991. A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif. EMBO Journal 10:1787-1791.
Russell, W.A. and A.R. Hallauer. 1980. Corn. Pages 299-312 in Hybridization of Crop Plants. W.R. Fehr and H.H. Hadley (eds.). American Society of Agronomy, Inc., Crop Science Society of America, Inc., Madison, Wisconsin.
Safawo, T., N. Senthil, M. Raveendran, S. Vellaikumar, K.N. Ganesan, G. Nallathambi, S. Saranya, V.G. Shobhana, B. Abirami and E.V. Gowri. 2010. Exploitation of natural

v a r i a b i l i t y i n m a i z e f

o r β

- c a r o t e n e c o n t e n t u s i n g H P L C a n d g e n e s p e c i f i c m a r k e r s

. Electronic Journal of Plant Breeding 1:548-555.
Salomon, S. and H. Puchta. 1998. Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO Journal 17:6086-6095.
Sessa, G., G. Morelli and I. Ruberti. 1993. The Athb-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. EMBO Journal 12:3507-3517.
Severini, A.D., L. Borrás, M.E. Westgate and A.G. Cirilo. 2011. Kernel number and kernel weight determination in dent and popcorn maize. Field Crops Research 120:360- 369.Monsanto Company CR262-14U1 143 of 260

Sidorov, V. and D. Duncan. 2009. Agrobacterium -mediated maize transformation: Immature embryos versus callus. Pages 47-58 in Methods in Molecular Biology: Transgenic Maize - Methods and Protocols. M.P. Scott (ed.). Humana Press, Inc, Totowa, New Jersey.
Simpson, G.G. and W. Filipowicz. 1996. Splicing of precursors to mRNA in higher plants: Mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery. Plant Molecular Biology 32:1-41.
Singer, J. and P. Bauer. 2009. Soil management practices: Crop rotations for row crops. Iowa State University Extension, Ames, Iowa. http://soilquality.org/practices/row_crop_rotations.html [Accessed March 11, 2013].
Stalker, D.M., C.M. Thomas and D.R. Helinski. 1981. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Molecular and General Genetics 181:8-12.
Steiner, H.-Y., C. Halpin, J.M. Jez, J. Kough, W. Parrott, L. Underhill, N. Weber and L.C. Hannah. 2013. Editor's choice: Evaluating the potential for adverse interactions within genetically engineered breeding stacks. Plant Physiology 161:1587-1594.
U.S. EPA. 2013. Dietary exposure evaluation model - Food commodity intake database. Version 3.18/9.14. U.S. Environmental Protection Agency, Washington, D.C. http://www.epa.gov/pesticides/science/deem/ [Accessed July 17, 2013].
U.S. FDA. 1992. Statement of policy: Foods derived from new plant varieties. Federal Register 57: 22984-23005.
University of Montana. 2011. Noxious weeds - Summary. University of MontanaMissoula, Missoula, Montana. http://invader.dbs.umt.edu/Noxious_Weeds/noxlist.asp [Accessed May 3, 2011].
University of Utah. 2014. The evolution of corn. University of Utah, Salt Lake City, Utah. http://learn.genetics.utah.edu/content/selection/corn/ [Accessed February 18, 2014].
USDA-APHIS. 1986. Coordinated framework for regulation of biotechnology. Federal Register 51:23302.
USDA-ARS. 2013. GRIN taxonomy for plants: Zea mays L., U.S. Department of Agriculture, Agricultural Research Service, Washington, D.C. http://www.arsgrin.gov/cgi-bin/npgs/html/taxon.pl?42207 [Accessed March 18, 2013].
USDA-FAS. 2013. World agricultural production. WAP 3-13. U.S. Department of Agriculture, Foreign Agricultural Service, Washington, D.C.Monsanto Company CR262-14U1 144 of 260

USDA-FAS. 2014. World agricultural production. WAP 4-14. U.S. Department of Agriculture, Foreign Agricultural Service, Washington, D.C.
USDA-NASS. 2012. Corn for all purposes 2012: Planted acres by county for selected states. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.
USDA-NASS. 2013a. Crop values: 2012 summary, February 2013. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.
USDA-NASS. 2013b. National statistics for corn. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C. http://www.nass.usda.gov/Statistics_by_Subject/result.php?8D643273-366F-3C5C-A47D [Accessed March 18, 2013].
USDA-NASS. 2014a. Wheat, US total. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.
USDA-NASS. 2014b. Soybeans, US total. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.
USDA-NASS. 2014c. Corn, US total. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.
USDA-NRCS. 2012. The PLANTS database. U.S. Department of Agriculture, Natural Resources Conservation Service, Washington, D.C. http://www.plants.usda.gov/index.html

. USDA-NRCS. 2013. Threatened & endangered: Protected plants for scientific name = Tripsacum . U.S. Department of Agriculture, Natural Resources Conservation Service, Washington, D.C. http://www.plants.usda.gov/core/profile?symbol=TRDA3 [Accessed September 7, 2013].
Vieira, J. and J. Messing. 1987. Production of single-stranded plasmid DNA. Methods in Enzymology 153:3-11.
Vitale, A. and J. Denecke. 1999. The endoplasmic reticulum - Gateway of the secretory pathway. The Plant Cell 11:615-628.
Weekes, R., T. Allnutt, C. Boffey, S. Morgan, M. Bilton, R. Daniels and C. Henry. 2007. A study of crop-to-crop gene flow using farm scale sites of fodder maize ( Zea mays L.) in the UK. Transgenic Research 16:203-211.
WHO. 1995. Application of the principles of substantial equivalence to the safety evaluation of foods or food components from plants derived by modern biotechnology. World Health Organization, Geneva, Switzerland.Monsanto Company CR262-14U1 145 of 260

Wilkes, H.G. 1972. Maize and its wild relatives. Science 177:1071-1077.
Wych, R.D. 1988. Production of hybrid seed corn. Pages 565-607 in Corn and Corn Improvement. Third Edition. G.F. Sprague and J.W. Dudley (eds.). American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.
Yates, B., A. De Soyza, R. Harkawat and C. Stenton. 2008. Occupational asthma caused by Arabidopsis thaliana : A case of laboratory plant allergy. European Respiratory Journal 32:1111-1112.
Zaidi, P.H., G. Srinivasan and C. Sanchez. 2003. Morpho-physiological traits associated with variable field performance of different types maize germplasm across multiple environments. Maydica 48:207-220.
Zambryski, P., A. Depicker, K. Kruger and H.M. Goodman. 1982. Tumor induction by Agrobacterium tumefaciens : Analysis of the boundaries of T-DNA. Journal of Molecular and Applied Genetics 1:361-370.
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.Monsanto Company CR262-14U1 146 of 260 APPENDICESMonsanto Company CR262-14U1 147 of 260 Appendix A: USDA Notifications and Permits

Field trials of MON 87403 have been conducted in the U.S. since 2007. 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 87403 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 - 2014 seasons, are still in preparation. A list of trials conducted under USDA notifications or permits and the status of the final reports for these trials are provided in Table A-1.

.Monsanto Company CR262-14U1 148 of 260

Table A-1. USDA Notifications and Permits Approved for MON 87403 and Status of Trials Planted

Field Trial Year USDA No. Effective Date Trial Status Release State Sites 2007 07-126-102n 6/8/2007 Submitted HI 2

07-126-102n 6/8/2007 Submitted HI 1 08-036-109n 3/6/2008 Submitted IA 9 2008 08-036-112n 3/21/2008 Submitted IL 4 IN 2 08-036-115n 3/20/2008 Submitted IL 6 08-036-117n 3/6/2008 Submitted KS 3 08-106-111rm 6/30/2008 Submitted HI 1 08-330-103rm 2/27/2009 Submitted IL 8 09-034-101n 3/5/2009 Submitted IA 8 09-035-102n 3/25/2009 Submitted KS 2 2009 OH 4

09-035-103n 3/25/2009 Submitted IA 1 IL 1 09-142-101rm 8/31/2009 Submitted HI 1 09-266-101n 10/23/2009 Submitted HI 1 09-317-101rm 2/12/2010 Submitted CA 5 IA 12 IL 11 IN 3 KS 10 OH 6 10-054-140n 3/24/2010 Submitted KS 13 10-056-112n 3/26/2010 Submitted NE 15 2010 10-063-105rm 4/17/2010 Submitted HI 2

10-067-112n 4/7/2010 Submitted AR 1 IL 3 MO 1 OH 1 WI 1 10-067-113n 4/7/2010 Submitted IA 9 IL 6 IN 1 10-068-104n 4/8/2010 Submitted IA 6 IL 7 OH 5 10-068-116n 4/8/2010 Submitted IA 4 IL 2 IN 3 KS 1 2011 NE 2

10-068-117n 4/8/2010 Submitted IL 2 NE 1 10-069-121n 4/9/2010 Submitted IL 1 10-078-109n 4/18/2010 Submitted IA 1 10-123-102n 6/2/2010 Submitted HI 3 10-131-103rm 6/29/2010 Submitted HI 2 10-264-101n 10/20/2010 Submitted HI 1Monsanto Company CR262-14U1 149 of 260 10-131-103rm 6/29/2010 Submitted HI 1 10-264-101n 10/20/2010 Submitted HI 1 10-322-103n 12/20/2010 Submitted HI 2 10-350-104rm 1/18/2011 Submitted HI 1 10-350-109rm 2/15/2011 Submitted CA 3 IA 6 IL 8 IN 1 KS 6 OH 6 TX 1 10-351-114rm 3/15/2011 Submitted CA 2 IA 9 IL 7 IN 1 KS 5 OH 2 11-012-106n 2/6/2011 Submitted KS 5 MS 1 2011 11-012-107n 2/6/2011 Submitted IA 5

11-014-103n 2/6/2011 Submitted IL 3 11-034-115n 3/5/2011 Submitted CA 5 KS 5 11-040-108n 3/11/2011 Submitted IA 2 IL 3 IN 1 11-040-109n 3/11/2011 Submitted NE 13 11-041-101n 3/18/2011 Submitted KS 14 11-041-126n 3/11/2011 Submitted MS 1 11-045-102n 3/16/2011 Submitted IL 8 11-045-104n 3/16/2011 Submitted OH 6 TX 1 11-046-101rm 5/18/2011 Submitted HI 2 11-048-102n 3/19/2011 Submitted IL 11 IN 1 11-048-110n 3/19/2011 Submitted IA 11 11-066-102n 4/6/2011 Submitted HI 1 11-067-109n 4/7/2011 Submitted IL 2 OH 5 11-068-101n 4/7/2011 Submitted KS 4 11-068-104n 4/8/2011 Submitted IL 6 11-068-106n 4/7/2011 Submitted IA 6 11-097-105n 4/20/2011 Submitted IL 1 11-123-104rm 9/1/2011 Submitted HI 2 11-154-110n 7/3/2011 Submitted HI 1 11-154-111n 7/3/2011 Submitted HI 1 11-291-108rm 2/15/2012 Submitted IA 3 IL 2 IN 1 2012 KS 21

11-305-104rm 3/1/2012 Submitted HI 2 11-320-102rm 3/15/2012 Submitted IA 9 IL 4Monsanto Company CR262-14U1 150 of 260 IN 1 KS 3 NE 11 SD 3 11-322-101n 12/18/2011 Submitted HI 2 11-322-102n 12/18/2011 Submitted HI 2 11-326-105rm 3/22/2012 Submitted IA 12 IL 23 11-342-104rm 12/23/2011 Submitted HI 1 IL 1 12-032-117rm 6/1/2012 Submitted HI 1 PR 1 12-058-101n 3/28/2012 Submitted MN 6 WI 7 12-059-120n 3/28/2012 Submitted IL 1 NE 1 2012 12-061-105n 3/31/2012 Submitted IA 1 IL 1 NC 1 NE 1 PA 1 12-062-111n 4/1/2012 Submitted AR 1 IA 1 IL 3 IN 1 KS 1 MS 1 OH 1 PA 1 12-065-109n 4/4/2012 Submitted IL 3 MO 1 NE 3 12-072-104n 4/11/2012 Submitted TX 2 12-074-110n 4/13/2012 Submitted IA 1 12-125-106rm 9/1/2012 Submitted HI 1 PR 2 12-143-104n 6/20/2012 Submitted HI 4 12-214-105rm 12/1/2012 Submitted HI 1 12-251-101n 10/7/2012 Submitted AR 1 IL 1 KS 1 NE 1 12-143-104n 6/20/2012 Submitted HI 2 12-214-104rm 12/1/2012 Submitted HI 1 12-312-103n 12/7/2012 Submitted HI 2 12-312-106rm 3/1/2013 Submitted HI 1 12-312-109rm 3/7/2013 Submitted PR 1 2013 12-320-109rm 3/15/2013 Submitted IA 19 SD 4 TN 1 12-320-114rm 3/15/2013 In Progress IL 28 IN 1 KS 8Monsanto Company CR262-14U1 151 of 260 MS 1

12-320-125rm 3/15/2013 In Progress KS 7 NE 7 13-037-101rm 6/1/2013 In Progress HI 2 13-037-102rm 6/1/2013 In Progress HI 2 13-037-104rm 6/1/2013 In Progress PR 1 13-037-105rm 6/1/2013 In Progress HI 1 13-037-106rm 6/1/2013 In Progress HI 2 PR 1 13-039-102n 3/8/2013 Submitted HI 3 13-044-101rm 3/7/2013 Submitted HI 1 13-052-105n 3/23/2013 Submitted IA 2 MN 9 SD 1 2013 13-059-103n 3/30/2013 Submitted IL 1

13-066-105n 4/6/2013 Submitted AR 1 IA 1 IL 1 LA 1 13-119-103n 5/29/2013 In Progress HI 2 PR 1 13-120-102rm 9/1/2013 In Progress HI 1 PR 1 13-120-103rm 9/1/2013 In Progress HI 1 PR 1 13-120-104rm 9/1/2013 In Progress HI 1 13-120-105rm 9/1/2013 In Progress HI 1 13-120-106rm 9/1/2013 In Progress HI 1 13-120-107rm 9/1/2013 In Progress HI 1 13-220-103n 9/7/2013 Submitted PR 1 13-301-101n 12/4/2013 In Progress HI 1 13-213-101rm 12/1/2013 In Progress HI 1 13-213-102rm 12/1/2013 In Progress HI 1 13-213-104rm 12/1/2013 In Progress HI 1 13-213-105rm 12/1/2013 In Progress HI 1 13-213-106rm 12/1/2013 In Progress HI 1 13-297-103rm 3/1/2014 In Progress HI 1 13-297-106rm 3/1/2014 In Progress HI 1 13-297-108rm 3/1/2014 In Progress HI 2 13-297-109rm 3/1/2014 In Progress HI 1 13-301-101n 12/4/2013 In Progress HI 3 2014 13-305-101rm 3/1/2014 In Progress IL 1

13-305-102rm 3/1/2014 In Progress IL 23 IN 1 13-305-103rm 3/1/2014 In Progress IL 2 13-305-105rm 3/1/2014 In Progress IL 17 IN 1 13-305-106rm 3/1/2014 In Progress KS 10 NE 8 13-305-108rm 3/1/2014 In Progress KS 8 NE 6 13-305-109rm 3/1/2014 In Progress KS 8 NE 6Monsanto Company CR262-14U1 152 of 260 13-305-113rm 3/1/2014 In Progress IA 24 MS 1 TN 2 13-305-114rm 3/1/2014 In Progress IA 15 13-305-115rm 3/1/2014 In Progress IL 1 NE 1 2014 13-305-116rm 3/1/2014 In Progress IA 17

14-031-117rm 6/1/2014 In Progress HI 1 14-038-103n 3/9/2014 In Progress IL 1 MI 1 MN 11 NE 1 14-065-101n 4/5/2014 In Progress IL 1 NE 1 14-065-102n 4/5/2014 In Progress IL 1 NE 1 Grand Total 849Monsanto Company CR262-14U1 153 of 260 Appendix B: M o d e o f A c t i o n o f A T H B 1 7 ∆ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3
Grain yield results from a sequential growth and development process - first the plant grows in the vegetative phase and produces photosynthetic tissue, followed by flowering, the production of seeds, and finally seed filling and maturation ( Figure B-2 ). MON 87403 was produced through insertion of the coding region of the full-length ATHB17 gene through Agrobacterium -mediated transformation into maize . ATHB17 is a member of the HD-Zip family of proteins that are plant transcription factors; i.e. proteins that bind to specific DNA sequences and are essential for regulation of gene expression. This family of proteins is found broadly across plant species (Ariel et al, 2007) and is thought to play an important role in the regulation of plant growth and development. Expression of ATHB17 in MON 87403 results in increased biomass of the ear at the early reproductive phase which increases yield opportunity in maize. The purpose of this appendix is to describe further details on the mechanism of action of ATHB17 in MON 87403.
Maize ( Zea mays subsp. mays ) is one of the world’s most important cereal crops with a global production forecast of ~960 million MT in 2013-14. The United States is the leading maize producing country in the world (USDA-FAS, 2014). In 2013 maize was planted on 35.48 million ha in the United States (USDA-FAS, 2014). Improvements in crop yield have been a primary focus of conventional breeding. Maize breeders have been extremely successful at improving grain yields in the last 70 years ( Figure B-2 ). Commercial maize yield in the United States increased nearly six-fold in the last 70 years of genetic improvement, with an approximately 99 kg ha -1 increase every year from 1939 (Lee and Tollenaar, 2007). This continuous yield increase was due to many factors including genetic improvement. Yield has increased despite the fact that the ear size per plant has declined (Bruns and Abbas, 2003). Since MON 87403 results in increased ear biomass, it has the potential to address some of the challenges faced by maize breeders in their efforts to continue to improve yield and the efficiency of maize production.Monsanto Company CR262-14U1 154 of 260

Figure B-1. Maize developmental stages. Vegetative stages are identified based on the number of visible leaf collars. Note the ear with silks at the R1 stage. Reproductive stages are mainly characterized by kernel development as shown in the insert. By the R6 stage, kernels attain physiological maturity. (Nafziger 2012)

Figure B-2. US maize yields from 1866 to 2013 in bushels per acre (compiled from USDA-NASS, 2014)

.
B.1. Maize growth stages, source and sink tissues:
In order to understand the mode of action of ATHB17 in maize, a review of maize developmental stages and the impact of partitioning on reproductive tissue developmentMonsanto Company CR262-14U1 155 of 260

is needed. Maize vegetative growth begins with germination and seedling emergence (VE, Figure B-1 ) and ends when ‘tassel’ or male inflorescence has completely emerged from the plant (VT, Figure B-1 ). Pollen from anthers on the tassel is shed normally after the VT stage. Ear tissue is initiated at a node by approximately the V5 growth stage and the ear grows rapidly as the plant approaches the flowering stage. The reproductive growth stage in maize starts with R1 when silks (receptors of pollen) are visible on the ear (female inflorescence), and ends at R6 or physiological maturity (Ritchie et al., 1997). At the R1 stage- ovules on the ear are completely developed, and after pollination they will mature during reproductive growth stages as grain for harvest ( Figure B-1 ). Vegetative tissue (mostly leaves) is the primary source tissue for production of reproductive tissue biomass. The ear is considered the major sink tissue during reproductive stages in maize (Ritchie et al., 1997). Sink tissues lack the ability to produce biomass but accumulate the biomass that is produced by the source tissue (Ho, 1988).

B.2. Partitioning of assimilates to sink tissue in maize: The transport and distribution of photosynthetic assimilates produced by the source tissue to sink tissue is referred to as ‘partitioning’ (Gifford and Evans, 1981). As the maize ear grows, it generates the driving force needed to transport assimilates produced by the source tissue to the sink tissue (Tang and Boyer, 2013). Increased ear partitioning involves preferential accumulation of biomass in the ear tissue relative to the other growing vegetative organs. Grain yield in maize is a function of total assimilates (biomass) produced by the source tissue and the fraction of produced assimilates partitioned to the sink or ear tissue (Lee and Tollenaar, 2007). Thus, increased partitioning to the ear can result in higher yield regardless of an associated increase in total assimilates produced by the plant. The increased assimilate partitioning to the ear during the R1 stage has been one of the factors that has contributed to yield increase in North American maize hybrids (Echarte et al., 2004).

B.3. Sink size during early reproductive stage can affect grain yield in maize: The size of the sink (ear) during the early reproductive stages can have a profound effect on grain yield in maize (Lizaso et al., 2007). In maize, the maximum sink size is determined during early reproductive stages, and is influenced both by the plant’s genetics and by prevailing environmental conditions (Jones et al., 1996; Borras and Westgate, 2006). An increase in ear biomass at early reproductive stages can result in larger kernel number at harvest (Fisher and Palmer, 1983; Severini et al, 2011) (Figure B-3) and is considered an important determinant of reproductive success (Zaidi et al, 2003). Thus, the early reproductive stages are crucial phases in maize development, and a greater ear biomass during the early reproductive stages provides increased yield opportunity in maize.Monsanto Company

Figure B-3 . Relationship between ear biomass during early reproductive stage and kernel number at harvest et al., 2011).

B.4. Grain yield i mprovemen achieved by s election and The genetic changes that breeding were typically achieved through the se encoding transcriptional regulator proteins (Doebley et al., 2006). For example, selection for maize plants with single stalk and short branches tipped by ears during the domestication of maize from teosinte indirectly of the tb1 gene, which encodes a transcription factor (Doebley examples of indirect selection for specific variants during conventional breeding can be found in crops like rice, wheat, tomato expression of a transcription factor using agricultural biotechnology is an extension conventional breeding and the introduction of new genetic elements that use or modify existing pathways in the plant.

B.5. ATHB17 is a member of the HD

HD- Zip proteins are a family of plant sequences and regulate gene expression. This family of proteins plant species and is thought to play an important role in and development (Ariel et al,

su bfamilies, or classes designated as I, II, III, and IV (Ariel et al, 2007

CR262-14U1 . Relationship between ear biomass during early reproductive stage and kernel number at harvest (graph made from the data on dent corn in Table 1,

mprovemen t through domestication and b reeding has been election and safe use of transcriptional regulator p roteins: The genetic changes that have resulted in yield improvement through conventional breeding were typically achieved through the se lection and safe use of plant genes encoding transcriptional regulator proteins (Doebley et al., 2006). For example, selection for maize plants with single stalk and short branches tipped by ears during the domestication of maize from teosinte indirectly resulted in selection of specific variants

gene, which encodes a transcription factor (Doebley et al., examples of indirect selection for specific variants during conventional breeding can be found in crops like rice, wheat, tomato and cauliflower (Doebley et al., expression of a transcription factor using agricultural biotechnology is an extension

and provides an opportunity to further enhance crop yields through the introduction of new genetic elements that use or modify existing pathways in the

ATHB17 is a member of the HD -Zip family of transcription f actors Zip proteins are a family of plant transcription factors which bind to specific DNA sequences and regulate gene expression. This family of proteins is found broadly across

and is thought to play an important role in the regulation of plant growth and development (Ariel et al, 2007). The HD- Zip family comprises four distinct bfamilies, or classes designated as I, II, III, and IV (Ariel et al, 2007

156 of 260 . Relationship between ear biomass during early reproductive stage and

(graph made from the data on dent corn in Table 1, Severini

reeding has been largely roteins: resulted in yield improvement through conventional

lection and safe use of plant genes encoding transcriptional regulator proteins (Doebley et al., 2006). For example, selection for maize plants with single stalk and short branches tipped by ears during the resulted in selection of specific variants

et al., 2006). Other examples of indirect selection for specific variants during conventional breeding can be et al., 2006). Thus expression of a transcription factor using agricultural biotechnology is an extension of an opportunity to further enhance crop yields through the introduction of new genetic elements that use or modify existing pathways in the

actors : which bind to specific DNA is found broadly across the regulation of plant growth Zip family comprises four distinct bfamilies, or classes designated as I, II, III, and IV (Ariel et al, 2007 ). All HD-ZipMonsanto Company CR262-14U1 157 of 260

proteins are characterized by the presence of a homeodomain (HD) with an immediately adjacent leucine zipper (LZ) domain. HD-Zip proteins have been shown to dimerize with members of their subfamily (as homo- or heterodimers), but not with other subfamilies of HD-Zip proteins to bind to target DNA sequences (Sessa et al., 1993). Many HD-Zip proteins have been shown to function as repressors of gene expression (Sessa et al., 1993; Steindler et al., 1999; Ohgishi et al., 2001; Henriksson et al. 2005), including down regulating the transcription of genes within the HD-Zip family (Ohgishi et al., 2001; Sorin et al., 2009). Transcriptional repressors are proteins that bind to DNA sites to prevent RNA polymerase from initiating transcription (Lewin, 2000). ATHB17 is a member of the class II subfamily. The protein consists of 275 amino acids and contains five domains; a homeodomain (HD), a leucine zipper (LZ), an N-terminal domain, a repression domain and a C-terminal domain. It has been shown that HD-Zip II proteins in Arabidopsis recognize a 9 bp DNA sequence CAAT(G/C)ATTG (Sessa et al 1993) which is distinct from the consensus sequence for other HD-Zip proteins. In maize 18 HD-Zip II genes have been identified through a systematic bioinformatic analysis (Zhao et al., 2011). Characterization of ATHB17 and of the maize HD-Zip II proteins tested showed that they are all able to function as transcriptional repressors (Rice et al., 2014). Based on the reported literature, HD-Zip II proteins have diverse functions throughout plant growth and development. For example, overexpression of several HD-Zip II genes has been shown to increase shade avoidance (or avoiding competition for light from neighboring plants) responses (ex: elongation to intercept light) in Arabidopsis (Ciarbelli et al., 2008; Sawa et al., 2002; Sorin et al., 2009; Steindler et al., 1999). In addition, multiple Arabidopsis lines over-expressing ATHB17 suggested a role in regulating photosynthetic capacity (Hymus et al., 2013). Interestingly, some HD-Zip II proteins are involved in regulation of reproductive growth and development (Meijer et al 1997). In the following sections, the mode-of-action of ATHB17 expression in MON 87403 is described through an assessment of the molecular and phenotypic characteristics of MON 87403.

B.6. MON 87403 expresses a truncated ATHB17 protein: MON 87403 was produced through insertion of the coding region of the full-length ATHB17 gene into maize through Agrobacterium -mediated transformation . Western blot analysis of leaf extracts revealed that MON 87403 and other ATHB17-transformed maize line express a truncated ATHB17 protein of ~20 kDa, in comparison to the predicted size ~32 kDa (Rice et al., 2014). Sequence analysis of the ATHB17 transcript produced in MON 87403 confirms that a truncated transcript is produced, and as a result of splicing is predicted to encode a version of the ATHB17 protein that lacks the first 113 amino acids ( A T H B 1 7 ∆ 1 1 3 ) c o r

r e s p o n d i n g t o a n e x p e c t e d m o l e c u l a r w e i g h t o f
~ 2 2 k D a

. T h e domains that comprise ATHB17 are shown in Figure B-4, which also depicts the t r u n c a t i o n o f t h e r e p r e s s i o n d o m a i n i n t h e A T H B 1 7 ∆ 1 1 3 p r o t e i n p r o d u c e d i n MON 87403.Monsanto Company CR262-14U1 158 of 260

Figure B-4: Domain structure of ATHB17. ATHB17 contains the characteristic Homeodomain (HD) and Leucine Zipper (LZ) domains of HD-Zip family members. HD is required for DNA binding. LZ domain is responsible for homodimerization and hetero-dimerization with other HD-Zip II proteins. A repression domain is present upstream of the HD. ATHB17 protein expressed in MON 87403 lacks the first 113 amino acids resulting in truncation of the repression domain.

B.7. A T H B 1 7 ∆ 1 1 3 r e t a i n s t h e f u n c t i o n a l b i n d i n g p r o p e r t i e s o f A T H B 1 7 :
T h e s ub c e l l u l a r l o c a l i z a t i o n o f A T H B 1 7 ∆ 1 1 3 w a s e v a l u a t e d i n m a i z e p r o t o p l a s t s

. T h e
r e s u l t s i n d i c a t e d t h a t A T H B 1 7 ∆ 1 1 3 i s t r a n s p o r t e d i n t o t h e n u c l e u s , c o n s i s t e n t w i t h activity as a transcription factor (Rice et al., 2014). In vitro DNA binding studies showed t h a t l i k e o t h e r H D

- Z i p I I p r o t e i n s , A T H B 1 7 ∆ 1 1 3 b i n d s e f f i c i e n t l y t o c o n s e n s u s D N A targets for class I and class II HD-Zips, with slightly higher affinity for the class II binding sites (Rice et al., 2014). Studies conducted to identify maize proteins that interact with ATHB17 identified several HD-Zip II proteins but did not identify HD-Zip p r o t e i n s f r o m o t h e r c l a s s e s

. ( R i c e e t a l

. , 2 0 1 4 )

. T h e r e s u l t s s h o w t h a t A T H B 1 7 ∆ 1 1 3retains DNA binding and protein-protein interaction (ability to form homo- and heterodimers) properties that are characteristic of the full-length protein.

B.8. A T H B 1 7 ∆ 1 1 3 r e l i e v e s r e p r e s s i o n o f H D

- Z i p I I p r o t e i n s :
S i n c e A T H B 1 7 Δ 1 1 3 l a c k s a l a r g e p o rt i o n o f t h e r e p r e s s i o n d o m a i n o f A T H B 1 7 , i t w o u l d not be expected to act as a transcriptional repressor. To determine whether A T H B 1 7 Δ 1 1 3 p r o t e i n c a n a c t a s a t r a n s c r i p t i o n a l r e p r e s s o r i n m a i z e , a n in-vitro assay system was established that allows for detection of repression of reporter gene expressionin maize protoplasts. The results showed that the repression activity observed when fulll e n g t h A T H B 1 7 w a s a d d e d w a s n o t o b s e r v e d w h e n A T H B 1 7 Δ 1 1 3 w a s a d d e d

. B a s e d o n t h e s e r e s u l t s , i t w a s c o n c l u d e d t h a t A T H B 1 7 Δ 1 1 3 l a c k s t h e a b i l i t y t o a c t a s a transcriptional repressor (Rice et al., 2014).

A l t h o u g h A T H B 1 7 Δ 1 1 3 d o e s n o t f u n c t i o n a s a r e p r e s s o r , t h e p r o t e i n r e t a i n s d i m e r i z a t i o n and DNA binding properties (Rice et al., 2014). Therefore, the likely action of A T H B 1 7 Δ 1 1 3 e x p r e s s e d i n m a i z e i s t o a t t e n u a t e t h e a c t i v i t y o f e n d o g e n o u s H D

- Z i p I I proteins through a dominant-negative mechanism. The dominant-negative mechanism can occur either through formation of non-functional homodimers that compete for DNA binding sites, or formation of less active heterodimers through competition for DNA binding (see Figure B-5).

T o e v a l u a t e t h e a

b i l i t y o f A T H B 1 7 ∆ 1 1 3 t o a c t a s a d o m i n a n t n e g a t i v e r e g u l a t o r , t h e a b i l i t y o f A T H B 1 7 ∆ 1 1 3 t o r e l i e v e r e p r e s s i o n a c t i v i t y o f t h e f u l l

- l e n g t h A T H B 1 7 p r o t e i nMonsanto Company

was examined using the maize protoplast system described above repor ter gene expression caused by the full relieved as increasing amounts of suggests that A T H B 1 7 Δ 1 1 3 protein on ge ne repression.

Because ATHB17 ∆ 1 1 3 c a n f o r m h e t e r o d i m e r s w i t h m a i z e H D factors and bind to the same DNA sequence, ATHB17 dominant negative regulator of endogenous maize HD Experiments were conducted in al., 2014). Dose- dependent relief of the HD amounts of ATHB17  113 was observed for all tested HD suggest that ATHB17 ∆ 1 1 3 p r o t e i n proteins, that repress transcription sites. Thus, it is likely that ATHB17 pathways associated with maize HD

Figure B-5 : Model for dominant negative mechanism of ATHB17 ATHB17 is expressed as domain. Maize endogenous HD ATHB17  113 can interact with endogenous HD ATHB17  113 and endogenous HD for DNA binding resulting in altered target expression due repression. In this view, only one repression domain only one of the two repression domains

CR262-14U1 was examined using the maize protoplast system described above

. The repression of the ter gene expression caused by the full

- length ATHB17 protein was gradually relieved as increasing amounts of A T H B 1 7 Δ 1 1 3 w e r e added (Rice et al., 2014).

A T H B 1 7 Δ 1 1 3 has the ability to counteract the effect of the full ne repression.

∆ 1 1 3 c a n f

o r m h e t e r o d i m e r s w i t h m a i z e H D

- Zip II transcriptional factors and bind to the same DNA sequence, ATHB17 ∆ 1 1 3 c o u l d b e e x p e c t e d t o a c t a s a dominant negative regulator of endogenous maize HD

- Zip II transcription facto Experiments were conducted in maize leaf protoplasts to evaluate this possibility (Rice et

dependent relief of the HD -Zip II repression activity by increasing 113 was observed for all tested HD -Zip II proteins ∆ 1 1 3 p r o t e i n can act as a regulator of endogenous maize HD that repress transcription of genes with promoters containing class Thus, it is likely that ATHB17 ∆ 1 1 3 h a s t h e p o t e n t i a l t o a f fect activities and pathways associated with maize HD -Zip II proteins.

: Model for dominant negative mechanism of ATHB17 ∆ 1 1 3 ATHB17 is expressed as a truncated protein in maize lacking part of the repression

Maize endogenous HD -Zip IIs function as transcriptional repressors. 113 can interact with endogenous HD -Zip IIs to form 113 and endogenous HD -Zip IIs, or ATHB17  113 homodimers can compete for DNA binding resulting in altered target expression due to inability to cause active

In this view, only one repression domain is on each face of the DNA so that only one of the two repression domains is visible (Rice et al., 2014).

159 of 260 The repression of the length ATHB17 protein was gradually added (Rice et al., 2014). This has the ability to counteract the effect of the full -length

Zip II transcriptional ∆ 1 1 3 c o u l d b e e x p e c t e d t o a c t a s a Zip II transcription facto rs. leaf protoplasts to evaluate this possibility (Rice et

II repression activity by increasing

II proteins . These results can act as a regulator of endogenous maize HD -Zip II

promoters containing class II binding

fect activities and

∆ 1 1 3 truncated protein in maize lacking part of the repression

IIs function as transcriptional repressors.

to form heterodimers of 113 homodimers can compete to inability to cause active is on each face of the DNA so thatMonsanto Company CR262-14U1 160 of 260

B.9. A T H B 1 7 ∆ 1 1 3 l i k e l y m o d u l a t e s H D

- Z i p I I s t h r o u g h a d o m i n a n t

- n e g a t i v e mechanism to impact reproductive growth: Based on the molecular mechanism proposed above, it is likely that expression of A T H B 1 7 ∆ 1 1 3 i m p a c t s m a i z e e a r g r o w t h t h r o u g h i n t e r a c t i o n w i t h f u n c t i o n s o f endogenous maize HD-Zip II proteins. HD-Zip II proteins are involved in many processes related to reproductive development (Zuniga-Mayo et al., 2012). For example, a knock-out of a HD-Zip II gene in Arabidopsis showed no altered phenotypes during the vegetative growth phase but showed altered reproductive and fruit development compared to the wild type (Zuniga-Mayo et al., 2012). To investigate whether the HD-Zip II proteins might be involved in the regulation of ear growth in maize, the expression patterns of HD-Zip II genes were evaluated in two different maize hybrids (Rice et al., 2014). The results showed that HD-Zip II genes were expressed in all sampled maize tissue types, as well as across developmental stages. There were eight HD-Zip II genes that were predominantly expressed in the ear tissue, suggesting that they might be actively involved in the regulation of plant reproductive or ear growth (Rice et al., 2014).

O u r a n a l y s i s i n d i c a t e s t h a t t h e l i k e l y a c t i o n o f A T H B 1 7 Δ 1 1 3 e x p r e s s e d i n m a i z e i s t o attenuate the activity of endogenous HD-Zip II proteins through a dominant-negative mechanism. As several HD-Zip II genes were predominantly expressed in the ear tissue,

w e b e l i e v e t h a t A T H B 1 7 ∆ 1 1 3 p r o t e i n i n m a i z e l i k e l y m o d u l a t e s H D

- Z i p I I r e g u l a t e d pathways in the ear, leading to changes in ear growth.

B.10. MON 87403 maize has increased ear biomass during the early reproductive stages: Based on the role of specific HD-Zip II proteins in growth and development (Ciarbelli et al., 2008; Zuniga-Mayo et al., 2012; Bou-Torrent et al., 2012), expression patterns of H D

- Z i p I I g e n e s i n m a i z e d u r i n g t h e V 1 6

- R 1 s t a g e s , a n d l i k e l y a c t i o n o f A T H B 1 7 Δ 1 1 3 through a dominant-negative mechanism, a hypothesis was developed that the MON 87403 event in maize results in increased ear biomass at the R1 stage compared to a conventional maize hybrid. The ear biomass at the R1 growth stage was measured in MON 87403 and conventional control plants which were grown at 13 field locations within the maize production regions of the United States in 2012. Characteristics measured included stover or vegetative biomass and ear biomass at the R1 growth stage (~60-70 days after planting). Stover and ear biomass per 1 meter length of row were determined by cutting plants at the ground level and measuring the dry weight of the vegetative material and top-most ear, respectively. Total biomass per 1 meter length of row was calculated as the sum of stover and ear biomass. From these measurements, biomass partitioning to the ear was calculated as the ratio of ear biomass to total biomass (Marcelis, 1996). Statistical comparisons were made between MON 87403 and the control across all 13 sites (combined-site analyses) for the above characteristics. Statistical analysis indicated no significant differences in the R1 stover biomass and the R1 total biomass between MON 87403 and the control. There was a statisticallyMonsanto Company CR262-14U1 161 of 260

significant increase in R1 ear biomass (p < 0.05) in MON 87403 compared to the control in the combined site analysis (Table B-1). The R1 ear biomass in MON 87403 was approximately 12% higher than the control. Moreover, partitioning to the ear was significantly increased in MON 87403 compared to the control. These findings support t h a t A T H B 1 7 ∆ 1 1 3 p r o t e i n i n m a i z e r e g u l a t e s e a r g r o w t h

. Table B-1. Combined Site Analysis of R1 Ear Weight, R1 Stover Weight, Total Plant Weight, and Partitioning in MON 87403 and the Conventional Control from 2012 U.S. Field Trials Characteristic (units) MON 87403 (Mean ± SE) Control

(Mean ± SE) Change (%) p-value
R1 ear biomass (g) 144.50 (±8.47) 129.30 (±8.13) 11.7 0.004* R1 stover biomass (g) 768.13 (±20.57) 772.32 (±20.45) -0.5 0.791 R1 total plant biomass (g) 912.63 (±25.47) 901.62 (±26.02) 1.2 0.563 Partitioning to ear 0.16 (±0.0064) 0.14 (±0.0058) 10.0 0.001*

Locations included in the combined-site analysis: Jackson, Arkansas; Vermilion, Illinois; Warren, Illinois; Boone, Indiana; Greene, Iowa; Jefferson, Iowa; Pawnee, Kansas; (2 sites) Polk, Nebraska; York, Nebraska; Perquimans, North Carolina; Berks, Pennsylvania; Lehigh, Pennsylvania. (Partitioning to ear = R1 ear biomass / R1 total plant biomass). All measurements are dry weight per 1 meter length of row.
* D e n o t e s a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e b e t w e e n M O N 8 7 4 0 3 a n d t h e c o n t r o l ( α = 0

. 0 5 )
In MON 87403, the expression of the ATHB17  113 transcription factor results in an increase of ear biomass and partitioning to ear at the R1 growth stage. Increased ear biomass and improvement in partitioning to the ear during the R1 stage in maize are traits that have been improved through breeding (Echarte et al., 2004). Thus, increasing ear biomass through introduction of the ATHB17  113 transcription factor in maize is similar to genetic improvements that have been achieved through conventional breeding. The increased ear biomass at the R1 growth stage can provide the opportunity for a yield advantage (Fisher and Palmer, 1983; Zaidi et al., 2003; Severini et al, 2011). Ear biomass advantage in MON 87403 occurs during early ear development and accumulates through the silking stage
A greenhouse study was conducted to confirm field results and to assess the growth stage when an ear biomass advantage is seen in MON 87403 compared to the control. Plants were grown in pots under optimal growing conditions in a greenhouse. Plants were sampled at 4 stages; V16 (late vegetative), VT (tasseling), early R1, and late R1 (silking). The ear biomass of MON 87403 was approximately 18% greater (29.4 g vs. 25.0 g) than that of the control at the late R1 stage (Figure B-6). Statistical analysis indicated that ear biomass was significantly greater in MON 87403 than the control at early R1 and late R1 stages. Throughout the time course during which biomass was measured (V16 – late R1), ears of MON 87403 plants accumulated approximately 24 % more biomass (26.8 g vs. 21.6 g), on average, than the ears of control plants. In addition to supporting the ear biomass advantage in MON 87403 compared to the control that was observed in the fieldMonsanto Company CR262-14U1 162 of 260

trial, these results also suggest that the increased early ear biomass of MON 87403 accumulates gradually over the period from V16 to late R1.

Figure B-6. Ear growth and ear partitioning in MON 87403 and the control at different developmental stages in a greenhouse. Symbol ‘*’ indicates statistical
s i g n i f i c a n c e a t a l e v e l o f 5 % ( α = 0

. 0 5 )

. ( A ) E a r b i o m a s s ( d r y w e i g h t ) ( B ) E a r partitioning (ear dry weight/total dry weight). “DAS” is days after sowing. Similar to ear biomass advantage, partitioning to ear was significantly higher in MON 87403 compared to the control at the early and the late R1 growth stages (6). These data support the ear biomass advantage in MON 87403 compared to the control was due to increased partitioning to the ear, similar to the findings observed in the field trial.

Conclusion:
Based on the studies described above, t he A T H B 1 7 Δ 1 1 3 p r o t e i n c a n b i n d t o t a r g e t D N A sequences but lacks transcriptional repression activity. By a dominant-negative

m e c h a n i s m , A T H B 1 7 Δ 1 1 3 p r o t e i n i n M O N 8 7 4 0 3 m a i z e c a n a l t e r t h e a c t i v i t y o f endogenous HD-Zip IIs. HD-Zip IIs have been shown to play a role in reproductive growth in Arabidopsis (Ciarbelli et al., 2008; Zuniga-Mayo et al., 2012; Bou-Torrent et al., 2012). In maize, several HD-Zip IIs are predominantly expressed in ear tissue. The A T H B 1 7 ∆ 1 1 3 p r o t e i n l i k e l y m o d u l a t e s H D

- Z i p I I

- r e g u l a t e d p a t h w a y s i n t h e e a r , l e a d i n g to changes in ear growth. The result is an increase in ear partitioning and ear biomass at the R1 growth stage in MON 87403 maize. A larger ear at the R1 growth stage increases

V 1 6 ( 4 9 D A S )

V T ( 5 1 D A S )

E a r l y R 1 ( 5 6 D A S )

L a t e R 1 ( 6 1 D A S )EarBiomass(g/plant)

0 5 10 15 20 25 30 35

MON 87403

Control

V 1 6 ( 4 9 D A S )

V T ( 5 1 D A S )

E a r l y R 1 ( 5 6 D A S )

L a t e R 1 ( 6 1 D A S )EarPartitioning

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22

MON 87403 Control

* *

* *

A BMonsanto Company

yield opportunity in MON MON 87403 is summari zed

Figure B-7 . Summary of maize

CR262-14U1 yield opportunity in MON 87403 maize (Leibman et al., 2014). The mode of action of

zed in Figure B-7.

. Summary of mode-of- a c t i o n o f A T H B 1 7 Δ 1 1 3 p r o t e i n i n M O N

163 of 260 (Leibman et al., 2014). The mode of action of

a c t i o n o f A T H B 1 7 Δ 1 1 3 p r o t e i n i n M O N 87403Monsanto Company CR262-14U1 164 of 260 References for Appendix BAriel, F.D., P.A. Manavella, C.A. Dezar and R.L. Chan. 2007. The true story of the HDZip family. Trends in Plant Science 12: 419-426. Borrás, L. and M.E. Westgate. 2006. Predicting maize kernel sink capacity early in development. Field Crops Research 95: 223-233. Bruns, H.A. and H.K. Abbas. 2003. Effects of plant populations on maize hybrids in the sub-tropical mid south USA. Maydica 48: 21-27.Bou-Torrent, J., M. Salla-Martret, R. Brandt, T. Musielak, J.C. Palauqui, J.F. MartinezGarcia and S. Wenkel. 2012. ATHB4 and HAT3, two class II HD-ZIP transcription factors, control leaf development in Arabidopsis. Plant Signaling and Behavior 7: 1382- 1387.
Ciarbelli, A.R., A. Ciolfi, S. Salvucci, V. Ruzza, M. Possenti, M. Carabelli, A. Fruscalzo, G. Sessa, G. Morelli and I. Ruberti. 2008. The Arabidopsis homeodomain-leucine zipper II gene family: Diversity and redundancy. Plant Molecular Biology 68: 465-478. Doebley, J.F., B.S. Gaut and B.D. Smith. 2006. The molecular genetics of crop domestication. Cell 127: 1309-1321. Echarte, L., F.H. Andrade, C.R.C. Vega and M. Tollenaar. 2004. Kernel number determination in Argentinean maize hybrids released between 1965 and 1993. Crop Science 44: 1654-1661. Fisher, K.S. and A.F.E. Palmer. 1983. Maize. Pages 155-248 in Potential Productivity of Field Crops Under Different Environments. International Rice Research Institute, Manila, Philippines. Gifford, R.M. and L.T. Evans. 1981. Photosynthesis, carbon partitioning, and yield. Annual Review of Plant Physiology 32: 485-509. Henriksson, E., A.S.B. Olsson, H. Johannesson, H. Johansson, J. Hanson, P. Engström and E. Söderman. 2005. Homeodomain leucine zipper class I genes in Arabidopsis. Expression patterns and phylogenetic relationships. Plant Physiology 139: 509-518. Ho, L.C. 1988. Metabolism and compartmentation of imported sugars in sink organs in relation to sink strength. Annual Review of Plant Physiology and Plant Molecular Biology 39: 355-378. Hymus, G.J., S. Cai, E.A. Kohl, H.E. Holtan, C.M. Marion, S. Tiwari, D.R. Maszle, M.R. Lundgren, M.C. Hong, N. Channa, P. Loida, R. Thompson, J.P. Taylor, E. Rice, P.P. Repetti, O.J. Ratcliffe, T.L. Reuber and R.A. Creelman. 2013. Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity. Journal of Experimental Botany 64: 4479-4490.Monsanto Company CR262-14U1 165 of 260

Jones, R.J., B.M.N. Schreiber and J.A. Roessler. 1996. Kernel sink capacity in maize: Genotypic and maternal regulation. Crop Science 36: 301-306. Lee, E.A. and M. Tollenaar. 2007. Physiological basis of successful breeding strategies for maize grain yield. Crop Science 47: S202-S215. Leibman, M., J.J. Shryock, M.J. Clements, M.A. Hall, P.J. Loida, A.L. McClerren, Z.P. McKiness, J.R. Phillips, E.A. Rice and S.B. Stark. 2014. Comparative analysis of maize ( Zea mays ) crop performance: natural variation, incremental improvements and economic impacts. Plant Biotechnology Journal 1:1-10. Lewin, B. 2000. The operon. Pages 273-318 in Genes VII. Oxford University Press, Oxford, United Kingdom.Lizaso, J.I., A.E. Fonseca and M.E. Westgate. 2007. Simulating source-limited and sinklimited kernel set with CERES-maize. Crop Science 47: 2078-2088. Marcelis, L.F.M. 1996. Sink strength as a determinant of dry matter partitioning in the whole plant. Journal of Experimental Botany 47: 1281-1291. Meijer, A.H., E. Scarpella, E.L. van Dijk, L. Qin, A.J.C. Taal, S. Rueb, S.E. Harrington, S.R. McCouch, R.A. Schilperoort and J.H.C. Hoge. 1997. Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice. The Plant Journal 11: 263-276. Nafziger, E. 2012. Corn. Pages 13-26 in Illinois Agronomy Handbook. University of Illinois Extension, Champaign, Illinois. Ohgishi, M., A. Oka, G. Morelli, I. Ruberti and T. Aoyama. 2001. Negative autoregulation of the Arabidopsis homeobox gene ATHB-2. The Plant Journal 25: 389- 398. Rice, E.A., A. Khandelwal, R.A. Creelman, C. Griffith, J.E. Ahrens, J.P. Taylor, L.R. Murphy, S. Manjunath, R.L. Thompson, M.J. Lingard, S.L. Back, H. Larue, B.R. Brayton, A.J. Burek, S. Tiwari, L. Adam, J.A. Morrell, R.A. Caldo, Q. Huai, J.L. Kouadio, R. Kuehn, A.M. Sant, W.J. Wingbermuehle, R. Sala, M. Foster, J.D. Kinser, R. Mohanty, D. Jiang, T.E. Ziegler, M.G. Huang, S.V. Kuriakose, K. Skottke, P.P. Repetti, T.L. Reuber, T.G. Ruff, M.E. Petracek and P.J. Loida. 2014. Expression of a truncated ATHB17 protein in maize increases ear weight at silking. PLoS ONE 9:e94238. Ritchie, S.W., J.J. Hanway and G.O. Benson. 1997. How a corn plant develops. Special Report No. 48. Iowa State University of Science and Technology, Cooperative Extension Service, Ames, Iowa. Sawa, S., M. Ohgishi, H. Goda, K. Higushi, Y. Shimada, S. Yoshida and T. Koshiba. 2002. The HAT2 gene, a member of the HD-Zip gene family, isolated as an auxin inducible gene by DNA microarray screening, affects auxin response in Arabidopsis. The Plant Journal 32: 1011-1022.Monsanto Company CR262-14U1 166 of 260

Sessa, G., G. Morelli and I. Ruberti. 1993. The Athb-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. EMBO Journal 12: 3507-3517. Severini, A.D., L. Borrás, M.E. Westgate and A.G. Cirilo. 2011. Kernel number and kernel weight determination in dent and popcorn maize. Field Crops Research 120: 360- 369. Sorin, C., M. Salla-Martret, J. Bou-Torrent, I. Roig-Villanova and J.F. Martínez-García. 2009. ATHB4, a regulator of shade avoidance, modulates hormone response in Arabidopsis seedlings. The Plant Journal 59: 266-277. Steindler, C., A. Matteucci, G. Sessa, T. Weimar, M. Ohgishi, T. Aoyama, G. Morelli and I. Ruberti. 1999. Shade avoidance responses are mediated by the ATHB-2 HD-Zip protein, a negative regulator of gene expression. Development 126: 4235-4245. Tang, A.C. and J.S. Boyer. 2013. Differences in membrane selectivity drive phloem transport to the apoplast from which maize florets develop. Annals of Botany 111:551- 562. USDA-FAS. 2014. World agricultural production, January 2014. WAP 1-14. U.S. Department of Agriculture, Foreign Agricultural Service, Washington, D.C. USDA-NASS (2014). Corn, US total. Washington, D.C., U.S. Department of Agriculture, National Agricultural Statistics Service. Zaidi, P.H., G. Srinivasan and C. Sanchez. 2003. Morpho-physiological traits associated with variable field performance of different types maize germplasm across multiple environments. Maydica 48: 207-220. Zhao Y., Y. Zhou, H. Jiang, X. Li, D. Gan, X. Peng, S. Zhu, and B. Cheng. (2011) Systematic Analysis of Sequences and Expression Patterns of Drought-Responsive Members of the HD-Zip Gene Family in Maize. PLoS ONE 6(12): e28488.
Zúñiga-Mayo, V.M., N. Marsch-Martínez and S. de Folter. 2012. JAIBA, a class-II HDZIP transcription factor involved in the regulation of meristematic activity, and important for correct gynoecium and fruit development in Arabidopsis. The Plant Journal 71: 314- 326.Monsanto Company CR262-14U1 167 of 260 Appendix C: Overview, Materials, Methods, and Supplementary Results for

Molecular Analyses of MON 87403

C.1. NGS/JSA Overview 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:

 the number of insertion sites;

 the presence/absence plasmid backbone;

 insert copy number at each insertion site;

 DNA sequence of each inserted DNA;

 sequence of the native locus at each insertion site.Monsanto Company CR262-14U1 168 of 260

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. 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 C-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 C-1, box 1). Sufficient numbers of sequence fragments are obtained ( ≥ 7 5 × g e n o m e c o v e r a g e ) t o c o m p r e h e n s i v e l y c o v e r t h e g e n o m e s o f t h e s e qu e n c e d samples (Ajay et al. 2011; Clarke and Carbon 1976; Wang et al. 2008). A previous study with a variety of transformation events demonstrate that 75× 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). Notwithstanding known 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 singlec o p y e n d o g e n o u s g e n e , t h i s a n a l y s i s d e m o n s t r a t e s c o v e r a g e a t ≥ 7 5 × m e d i a n d e p t h i n 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 75× or greater 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).Monsanto Company CR262-14U1 169 of 260

Figure C-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
c o m p r e h e n s i v e l y c o v e r t h e g e n o m e s o f e a c h s a m p l e a t ≥ 7 5 × m e d i a n c o v e r a g e

. U s i n g t h e s e 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 lack of detectable sequences, including the use of suitable controls for experimental comprehensiveness and sensitivity. 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 a query sequence 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. 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 eachMonsanto Company CR262-14U1 170 of 260

insertion and an example is shown in Figure C-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 T-DNA copy number and 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. The identity of inserted DNA was confirmed by mapping of sequence reads. The selected sequences, a subset of which are junction sequences, represent plasmid sequence integrated into the genome of the transformation event. These sequences were compared to the transformation plasmid to determine which region(s), T-DNA or backbone, of the transformation plasmid was (were) integrated during transformation.

Figure C-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). The next step in the molecular characterization is confirmation of the integrity of the insert 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 confirms that the sequence of the insert is identical to the corresponding sequence in the plasmid vector and if each genetic element in the insert is intact. Furthermore, the genomic organization at the insertion site is ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACG ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACGTGT ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACGTGTCG ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACGTGTCGGG ATCCATGTAGATTTCCCGGACATGAAGCCA AGAACTAGGAAACGACGACGTGTCGGGAT

FlankingDNA Plasmid DNA JunctionMonsanto Company CR262-14U1 171 of 260

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 well as the identity of the inserted sequence, as described above. This information is used to determine the number and identity of insertion sites. For a single T-DNA insert, two junction sequence classes are expected, both containing portions of T-DNA and flankingsequence (Figure C-2), with one each originating from either end of the insert (Figure C3). All the integrated sequences align to the T-DNA portion of the plasmid. 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 and all the integrated sequences align to the T-DNA portion of the plasmid.Monsanto Company CR262-14U1 172 of 260

Figure C-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. 

## DNA insert 5’ Flank 3’ Flank Insert Junction Regions Junction Sequences: Class A

Junction Sequences: Class BMonsanto Company CR262-14U1 173 of 260

C.2. Materials and Methods

C.2.1. Test Substance The test substance in this study was MON 87403. Genomic DNA for use in this study was extracted from tissue listed in the table below.

Generation Seed ORION 6 ID

R 3 11346584

R 4 11346578

R 5 11346585

R 4 F 1 11346581

R 5 F 1 11346575

6 ORION is a proprietary database used at Monsanto Company to track Regulatory plant samples.Monsanto Company CR262-14U1 174 of 260

C.2.2. Control Substance The control substance is the conventional maize variety LH244, which has similar genetic background as the test substances. Genomic DNA for use in this study was extracted from tissue listed in the table below.

Control Substance Generations Seed ORION ID

LH244 R 3 , R 4 , R 5 11264747

LH244 x LH295 R 4 F 1 (hybrid) 11266870

LH244 x LH287 R 5 F 1 (hybrid) 11333170

C.2.3. Reference Substance The reference substance was plasmid vector PV-ZMAP5714, which was used to develop MON 87403. 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.

C.2.4. 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 MIDAS7 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. 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.

7 MIDAS is a proprietary database used at Monsanto Company to track plant synthesis records.Monsanto Company CR262-14U1 175 of 260

C.2.5. 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 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 pellet 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.

C.2.6. DNA Quantification PV-ZMAP5714 DNA and extracted genomic DNA were quantified using a Qubit TM Fluorometer (Invitrogen, Carlsbad, CA). For directed sequencing, genomic DNA was quantified using a Nanodrop TM Spectrophotometer (Thermo Scientific, Wilmington, DE) according to manufacturer’s instructions.

C.2.7. Agarose Gel Electrophoresis After quantification, approximately 0.5-1 μg of the extracted DNA for NGS/JSA sequencing library construction was run on a 1% (w/v) agarose gel to check the quality.

C.2.8. 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 ~ 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 incidentMonsanto Company CR262-14U1 176 of 260

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.

C.2.9. 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.

C.2.10. 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. 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.Monsanto Company CR262-14U1 177 of 260

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 microliters of the resuspended library was added to five microliters of Illumina PCR Master Mix and 25 μl of Illumina PCR Primer Cocktail 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.

C.2.11. 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 ( ≥ 7 5 x g e n o m e c o v e r a g e ) t o c o m p r e h e n s i v e l y c o v e r t h e e n t i r e g e n o m e s o f t h e t e s t e v e n t , the conventional control and the spiked-in control (Kovalic et al. 2012). Sequencing runs performed by the TGAC passed standard QC criteria. No sequence data in this study failed to meet these QC acceptance criteria.

C.2.12. Junction Sequence Analysis Bioinformatics High-throughput sequence reads were enriched by mapping to the PV-ZMAP5714 transformation plasmid sequence using the local alignment software BlastAll (V2.2.21) in 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. Sequencing Read Enrichment The transformation plasmid PV-ZMAP5714 sequence was used as reference to find all reads that were either fully matched to the insert plasmid fragments or partially matched as junction sequences. The sequence used was obtained from the MEGA 8 system. A junction sequence is characterized by a combination of transformation plasmid sequence

8 MEGA is a proprietary database used at Monsanto Company to track sequences and annotations.Monsanto Company CR262-14U1 178 of 260

and flanking sequence that is likely to be host genome flanking sequence or any other coinserted sequence, or a discontinuity in plasmid sequence caused by duplication or deletion. 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. 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.

Junction Detection Enriched and quality refined reads of both test and control samples were aligned against the whole PV-ZMAP5714 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 and their match cutoff position on the plasmid were noted (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 map reads and 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 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. Effective Sequencing Depth Determination A single copy locus ( Zea mays 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. The analysis showed that pdc3 displayed 75× or greater median coverage for each sample, as listed in Table C-1.Monsanto Company CR262-14U1 179 of 260

Table C-1. Sequencing (NGS) Conducted for MON 87403 and Control Genomic DNA

Sample Total Nucleotides (Gb) Effective Median Depth of

Coverage (x-fold) LH244 264.5 106x LH244 x LH295 296.1 108x LH244 x LH287 294.7 115x R 3 246.4 113x R 4 280.7 110x R 4 F 1 310.1 120x R 5 281.5 113x R 5 F 1 311.0 121x 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 within the maize genome ( pdc3 ). The median effective depths of coverage are shown for all samples.

Positive Spike-in Controls and Experimental Limit of Detection To produce “spike-in” positive control samples for sequencing, plasmid DNA libraries were created as described above 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). At 1 genome equivalent, 100% nucleotide identity was observed over 100% of PV-ZMAP5714 (Table C-2). 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 least 1/10th genomic equivalent (98.83% coverage at 99.97% identity for the 1/10th genome equivalent spiked control sample) and, hence, a detection level of at most 1/10th genome equivalent was achieved for the plasmid DNA sequence assessment.Monsanto Company CR262-14U1 180 of 260

Table C-2. Summary of NGS Data for the Conventional Control DNA Sample Spiked with PV-ZMAP5714

1 Extent of coverage is calculated as the percent of all PV-ZMAP5714 bases observed in the sequencing of the spike-in samples:

Extent of coverage = number of spike in bases detected

total length ( bp ) of spike in plasmid × 100

2 Percent identity of coverage is calculated as the percent of all PV-ZMAP5714 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

C.2.13. PCR and DNA Sequence Analyses to Examine the Insert and Flanking Sequences in MON 87403Overlapping PCR products, denoted as Product A, Product B, and Product C (Figure IV7 ) w e r e g e n e r a t e d t h a t s p a n t h e i n s e rt a n d a d j a c e n t 5 ′ a n d 3 ′ f

l a n k i n g D N A s e qu e n c e s i n MON 87403. 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 87403, as well as that of the D N A f l a n k i n g t h e 5 ′ a n d 3 ′ e n d s o f t h e i n s e r t

. The PCR analyses for Product A, Product B, and Product C were each conducted using 84 ng of genomic DNA template in a 50  l reaction volume. The reaction contained a final concentration of 0.5  M of each primer and 1x concentration of Phusion High Fidelity PCR Master Mix with HF Buffer (Thermo Scientific). The amplification of Product A and Product B were performed under the following cycling conditions: 1 cycle at 98  C for 30 seconds; 35 cycles at 98  C for 30 seconds, 65  C for 30 seconds, 72  C for 2 minutes 30 seconds; 1 cycle at 72  C for 5 minutes. The amplification of Product C was performed under the following cycling conditions: 1 cycle at 98  C for 30 seconds; 35 cycles at 98  C for 30 seconds, 72  C for 30 seconds, 72  C for 2 minutes; 1 cycle at 72  C for 5 minutes. 1/10 th copy Spike 1 copy Spike Extent of coverage 1 of PV-ZMAP5714 98.83% 100%
Percent identity of coverage 2 of PV-ZMAP5714 99.97% 100%Monsanto Company CR262-14U1 181 of 260

Aliquots of each PCR product were separated on an 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 Exo-SAP IT (Affymetrix, Santa Clara, CA) and a MinElute 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. 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-ZMAP5714 sequence to determine the integrity and organization of the i n t e g r a t e d D N A a n d t h e 5 ′ a n d 3 ′ i n s e rt

- t o
- f l a n k D N A j u n c t i o n s i n M O N 8 7 4 0 3

. C.2.14. PCR and DNA Sequence Analyses to Examine the Integrity of the DNA Insertion Site in MON 87403. To examine the MON 87403 T-DNA insertion site in control maize, PCR and sequence analyses were performed on genomic DNA from the conventional control maize LH244. The primers used in this analysis were designed from the DNA sequences flanking the insert in MON 87403. A forward primer specific to the DNA sequence flanking the 5 ′ e n d o f t h e i n s e r t w a s p a i r e d w i t h a r e v e r s e p r i m e r s p e c i f i c t o t h e D N A s e qu e n c e f l a n k i n g t h e 3 ′ e n d o f t h e i n s e r t

. The PCR reactions were conducted using 84 ng of genomic DNA template in a 50  l reaction volume. The reaction contained a final concentration of 0.5  M of each primer and 1x concentration of Phusion High Fidelity PCR Master Mix with HF Buffer (Thermo Scientific). The amplification was performed under the following cycling conditions: 1 cycle at 98  C for 30 seconds; 35 cycles at 98  C for 20 seconds, 71  C for 20 seconds, 72  C for 2 minutes; 1 cycle at 72  C for 5 minutes. A small aliquot of each PCR product was separated on an 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 purified using Exo-SAP IT (Affymetrix, Santa Clara, CA) and a MinElute 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. 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 t o t h e 5 ′ a n d 3 ′ s e qu e n c e s f l a n k i n g t h e M O N 8 7 4 0 3 i n s e rt t o d e t e r m i n e t h e i n t e g r i t y a n d organization of the insertion site.Monsanto Company CR262-14U1 182 of 260 References for Appendix C Ajay, S.S., S.C.J. Parker, H. Ozel Abaan, K.V. Fuentes Fajardo and E.H. Margulies. 2011. Accurate and comprehensive sequencing of personal genomes. Genome Research 21:1498-1505. Clarke, L. and J. Carbon. 1976. A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome. Cell 9:91-99. Codex Alimentarius. 2009. Foods derived from modern biotechnology. Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations, Rome, Italy. DuBose, A.J., S.T. Lichtenstein, N. Narisu, L.L. Bonnycastle, A.J. Swift, P.S. Chines and F.S. Collins. 2013. Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene. Nucleic Acids Research 41:e70-e70. Kovalic, D., C. Garnaat, L. Guo, Y. Yan, J. Groat, A. Silvanovich, L. Ralston, M. Huang, Q. Tian, A. Christian, N. Cheikh, J. Hjelle, S. Padgette and G. Bannon. 2012. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology. The Plant Genome Journal 5:149-163. Miller, J.R., S. Koren and G. Sutton. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95:315-327.Minoche, A.E., J.C. Dohm and H. Himmelbauer. 2011. Evaluation of genomic highthroughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems. Genome Biology 12:R112. Shendure, J. and H. Ji. 2008. Next-generation DNA sequencing. Nature Biotechnology 26:1135-1145. Wang, J., W. Wang, R. Li, Y. Li, G. Tian, L. Goodman, W. Fan, J. Zhang, J. Li, J. Zhang, Y. Guo, B. Feng, H. Li, Y. Lu, X. Fang, H. Liang, Z. Du, D. Li, Y. Zhao, Y. Hu, Z. Yang, H. Zheng, I. Hellmann, M. Inouye, J. Pool, X. Yi, J. Zhao, J. Duan, Y. Zhou, J. 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 diploid genome sequence of an Asian individual. Nature 456:60-65. Wei, H., C. Therrien, A. Blanchard, S. Guan and Z. Zhu. 2008. The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases. Nucleic Acids Research 36:e50.Monsanto Company CR262-14U1 183 of 260

Zhang, J., R. Chiodini, A. Badr and G. Zhang. 2011. The impact of next-generation sequencing on genomics. Journal of Genetics and Genomics 38:95-109. Zhang, R., Y. Yin, Y. Zhang, K. Li, H. Zhu, Q. Gong, J. Wang, X. Hu and N. Li. 2012. Molecular characterization of transgene integration by next-generation sequencing in transgenic cattle. PLoS ONE 7:e50348.Monsanto Company CR262-14U1 184 of 260 Appendix D: Materials, Methods and Results for Characterization of

A T H B 1 7 Δ 1 1 3 P r o t e i n P r o d u c e d i n M O N 8 7 4 0 3

D.1. C h a r a c t e r i z a t i o n o f A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3

D.1.1. Materials

T h e M O N 8 7 4 0 3

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s p u r i f i e d f r o m l y o p h i l i z e d l e a f ( l o t 1 1 3 4 7 2 5 5 ) o f M O N 8 7 4 0 3

. T h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s stored in a -80 oC freezer in a buffer solution containing 20 mM NaPO4, pH 7.4, 0.5 M NaCl, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic (EDTA), 2 μM leupeptin, 2 μM E-64 and 1% IGEPAL CA-630.

The E. coli

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n ( l o t 1 1 3 8 0 0 0 3 ) w a s u s e d a s t h e r e f e r e n c e s ub s t a n c e

. T h e A T H B 1 7 Δ 1 1 3 p r o t e i n r e f e r e n c e s ub s t a n c e w a s g e n e r a t e d f r o m c e l l p a s t e produced by large-scale fermentation of E. coli containing the pMON109241 expression plasmid. The coding sequence for ATHB17 contained on the expression plasmid (pMON109241) was confirmed prior to and after fermentation. Records pertaining to the production of the E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n a r e a r c h i v e d u n d e r O r i o n lot 11359538 in the Monsanto Regulatory Archives.

D.1.2. A T H B 1 7 Δ 1 1 3 P r o t e i n P u r i f i c a t i o n
T h e A T H B 1 7 Δ 1 1 3 p r o t e i n w a s p u r i f i e d f r o m l y o p h i l i z e d l e a f ( l o t # 1 1 3 4 7 2 5 5 ) o f MON 87403. The purification procedure was not performed under a Good Laboratory Practice (GLP) protocol, however, all procedures were documented and, where
a p p l i c a b l e , S t a n d a r d O p e r a t i o n P r o c e d u r e s w e r e f

o l l o w e d

. T h e A T H B 1 7 Δ 1 1 3 w a s purified from an extract of lyophilized leaf using a combination of techniques, including anionic exchange and immunoaffinity chromatography. The purification procedure is briefly described below.

A T H B 1 7 Δ 1 1 3 w a s e n r i c h e d f r o m l y o p h i l i z e d M O N 8 7 4 0 3 l e a f t i s s u e

. A p p r o x i m a t e l y 1 kg of lyophilized tissue was resuspended in a total of 44 L of chilled extraction buffer (100 mM NaPO 4 , pH 7.4, 100 mM NaCl, 5 mM DTT, 2 mM MgCl 2 , 1 mM EDTA, 1 mM benzamidine-HCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 μM leupeptin, 2 μM pepstatin A, 2 μM E-64, 2 μM bestatin, 10U/ml benzonase and 1% IGEPAL CA-630) and homogenized with an Ultra-Turrax T-50 dispersing instrument (IKA Work Inc., Wilmington, NC) equipped with a medium grind probe at 6400 rpm for 30 seconds. The suspension was then transferred to 4°C and ~560 g of poly(vinylpolypyrrolidone) equilibrated in 4 L of extraction buffer, and 1.25 kg of Amberlite IRA-402 resin equilibrated in 4 L of extraction buffer were added yielding concentrations of ~1% (w/v) and ~2.5 (w/v), respectively, in a final volume of 52 L (approximately 1:50 tissue to buffer ratio). The extraction slurry was incubated with stirring at 4°C for 2 hours before the addition of 1 kg of Celpure P100 filter agent (Imerys Filtration Minerals, San Jose, CA). The extraction slurry was then filtered using a 14-inch plate and frame filter press (Ertel Alsop, Kingston, NY) with MicroMedia cellulose and diatomaceous earth filter sheets (Ertel Alsop). The filtrate was chased through the filter press with 75 L of chaseMonsanto Company CR262-14U1 185 of 260

buffer (1 mM DTT, 1 mM EDTA, 1 mM PMSF, 2 μM leupeptin, 2 μM pepstatin A, 2 μM E-64, 2 μM bestatin and 1% IGEPAL CA-630). The eluting chase filtrate was combined with the extraction filtrate until a total volume of 100 L was reached and the remaining chase filtrate was discarded. The resulting clarified extract solution was loaded onto a 2.2 L (7 cm x 20 cm) Sulfopropyl

- Sepharose Fast Flow cation exchange resin (GE Healthcare, Piscataway, NJ) column pre-equilibrated with Equilibration Buffer (20 mM NaPO 4 , pH 7.4, 1 mM DTT, 1 mM EDTA, 1 mM MgCl 2 and 1% IGEPAL CA-630). The loaded column was washed with Buffer A (20 mM NaPO 4 , pH 7.4, 1 mM DTT, 1 mM EDTA, 2 μM leupeptin and 2 μ M E

- 6 4 ) a n d t h e b o u n d A T H B 1 7 Δ 1 1 3 w a s e l u t e d u s i n g a 4

- s t e p s a l t g r a d i e n t o f 50 mM, 500 mM, 600 mM and 1.0 M NaCl in Buffer A. Elution fractions containing the M O N 8 7 4 0 3

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w e r e i d e n t i f i e d b y w e s t e r

n b l o t a n a l y s i s a n d p o o l e d

. T h e A T H B 1 7 Δ 1 1 3 i n t h e s e f r a c t i o n s w a s p r e c i p i t a t e d b y t h e a d d i t i o n o f (NH 4 ) 2 SO 4 at a final saturation of 60% followed by overnight incubation on an orbital
s h a k e r a t 4 ° C

. F o l l o w i n g o v e r n i g h t i n c ub a t i o n , t h e p r e c i p i t a t e d A T H B 1 7 Δ 1 1 3 w a s pelleted by centrifugation and the resulting supernatants were discarded.

T h e r e s u l t i n g p r e c i p i t a t e d M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s i m m e d i a t e l y resuspended in 240 ml of Immuno Equilibration Buffer (20 mM NaPO 4 , pH 7.4, 0.5 M NaCl, 1 mM DTT, 1 mM EDTA, 2 μM leupeptin, 2 μM E-64 and 1% IGEPAL CA-630). The resuspended solution was clarified by centrifugation and the resulting supernatant was loaded onto a 1.0 ml (1.3 cm x 1.0 cm) Protein G Fast Flow (GE Healthcare) column to which a monoclonal anti-ATHB17 antibody had been conjugated. The loaded column was washed with successive 10 column volume washes of Immuno Wash Buffer (20 mM NaPO 4 , pH 7.4, 0.1 M NaCl, 1 mM DTT, 1 mM EDTA, 2 μM leupeptin and 2 μM E-64), Immuno Wash Buffer with 1.0 M NaCl and Immuno Wash Buffer with 10% propylene g l y c o l

. T h e b o u n d A T H B 1 7 Δ 1 1 3 w a s t h e n e l u t e d w i t h I g G E l u t i o n B u f f e r ( T h e r m o Scientific, Rockford, IL) containing 2 μM leupeptin and 2 μM E-64. Elution fractions
c o n t a i n i n g t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w e r e i d e n t i f i e d b y w e s t e r

n b l o t a n a l y s i s

. T h e A T H B 1 7 Δ 1 1 3 i n t h e s e f r a c t i o n s w a s f

u rt h e r c o n c e n t r a t e d b y t h e addition of 25 μl of UNOsphere S cation exchange support resin (Bio-Rad, Hercules, CA), pre-equilibrated in IgG Elution Buffer, to each fraction. Following overnight i n c ub a t i o n a t 4 ° C w i t h s h a k i n g , t h e A T H B 1 7 Δ 1 1 3 b o u n d r e s i n w a s p e l l e t e d b y centrifugation and the resulting supernatant was discarded. The resulting resin pellets were washed with 15 ml of Immuno Wash Buffer followed by centrifugation and combined in a filter paper spin cup microfuge tube (Thermo Scientific). The bound A T H B 1 7 Δ 1 1 3 w a s t h e n e l u t e d w i t h I m m u n o E qu i l i b r a t i o n B u f f e r

. E l u t i o n f

r a c t i o n s c o n t a i n i n g t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w e r e i d e n t i f i e d b y w e s t e r

n blot analysis and pooled. The final buffer composition of the sample was 20 mM NaPO 4 , pH 7.4, 0.5 M NaCl, 1 mM DTT, 1 mM EDTA, 2 μM leupeptin, 2 μM E-64 and 1% IGEPAL CA-630. The p u r i f i e d M O N 8 7 4 0 3

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s a l i qu o t e d , a s s i g n e d lot 11361368 and stored at in a -80°C freezer.Monsanto Company CR262-14U1 186 of 260

D.1.3. MALDI-TOF Tryptic Mass Map Analysis

D.1.3.1. Methods MALDI-TOF tryptic mass fingerprint analysis was used to confirm the identity of the M O N 8 7 4 0 3

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n

. T h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s d i l u t e d i n 1 × l o a d i n g b u f f e r ( L B ; 6 2 m M T r i s
- H C l , 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). The sample was heated to 99°C for 5 minutes and loaded at ~150 ng in four lanes of a pre-cast Tris-glycine (4-20 %) polyacrylamide gradient mini-gel (Invitrogen). Pre-stained MW standards (Precision Plus Protein Standards, Bio-Rad) were loaded on the gel for molecular weight reference. Following electrophoresis, proteins were briefly fixed in 40% (v/v) methanol, 7% (v/v) acetic acid and stained for 16 hours with Brilliant Blue G-Colloidal stain (Sigma-Aldrich). Gels were briefly destained in 10% (v/v) acetic acid, 25% (v/v) methanol followed by 3 hours in 25% (v/v) methanol. Each ~22 kDa band was excised and destained with 40% (v/v) methanol/10% (v/v) acetic acid. The excised bands were incubated in 100 mM ammonium bicarbonate for 1 hour and treated with 10 mM DTT at 37°C for 2 hours followed by incubation for 20 minutes with 10 mM iodoacetic acid in the dark. The excised bands were then washed 3 times with 25 mM ammonium bicarbonate, dried using vacuum centrifugation and rehydrated with 20 μl of 20 μg/ml trypsin (Promega, Madison, WI). After 1 hour, excess liquid was removed and the excised bands were incubated overnight at 37°C in 40 μl of 10% (v/v) acetonitrile in 25 mM ammonium bicarbonate. The excised bands were then sonicated for 5 minutes and the resulting extract was transferred to new microcentrifuge tube labeled Extract 1. The excised bands were then extracted two more times, each with 30 μl of a 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid (TFA) solution and sonicated for 5 min. These two extracts were combined with the first extract and dried using vacuum centrifugation. The extracts were solubilized in 5 μl of 50% (v/v) acetonitrile, 0.1% TFA and sonicated for 5 min. Extract was spotted to wells on an analysis plate and mixed with
α
- c y a n o
- 4

- h y d r o x y c i n n a m i c a c i d ( α
- C y a n o , T h e r m o S c i e n t i f

i c )

. T e s t s a m p l e s w e r eanalyzed using AB Sciex TOF/TOF 5800 MS in reflector positive ion mode. Signal-toN o i s e w a s s e t t o ≥ 1 0

. O n l y m o n o i s o t o p i c i o n s w e r e a s s i g n e d i n a m a s s l i s t

. A p l a t e model/default calibration was performed using TOF/TOF calibration mixture standards ( A B S c i e x , F o s t e r C i t y , C A )

. T h e s a m p l e s i n α
- C y a n o m a t r i x w e r e a n a l y z e d i n t h e 5 0 0 t o 5 0 0 0 D a r a n g e

. T h e m a s s s p e c t r a w e r e s e a r c h e d a g a i n s t t h e A T H B 1 7 Δ 1 1 3 p r o t e i n sequence using Mascot and ProteinPilot protein identification tools. Search parameter criteria included: Peptide Mass Tolerance ± 0.5 Da, Fixed Modifications- Carboxymethyl (C). Peptide mass fingerprint was generated by Mascot and ProteinPilot. All matching masses were tallied and a coverage map was generated for the mass fingerprint.

D.1.3.2. Results of MALDI-TOF Tryptic Mass Map Analysis Peptide mass fingerprint analysis is a standard technique used for confirming the identity
o f p r o t e i n s

. T h e i d e n t i t y o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s confirmed by MALDI-TOF MS analysis of peptide fragments produced by the trypsin d i g e s t i o n o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n

.Monsanto Company CR262-14U1 187 of 260

There were 8 unique peptides identified that corresponded to the expected masses (Table D-1). The identified masses were used to assemble a peptide map of the A T H B 1 7 Δ 1 1 3 p r o t e i n ( F i g u r e D

- 1 )

. T h e e x p e r i m e n t a l l y d e t e r m i n e d c o v e r a g e o f t h e A T H B 1 7 Δ 1 1 3 p r o t e i n w a s 5 0 % ( 8 1 o u t o f 1 6 2 a m i n o a c i d s )

. T h i s a n a l y s i s c o n f i r m s t h e i d e n t i t y o f M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n

.

Table D-1. Summary of the Tryptic Masses Identified for the MON 87403 - p r o d u c e d A T H B 1 7 Δ 1 1 3 u s i n g M A L D I

- T O F M S
Experimental Mass 1 Calculated Mass 2 Diff. 3 Fragment 4 Sequence 5

873.46 873.43 0.03 154-160 TFPPQER 878.47 878.45 0.02 37-43 LLEDSFR 1001.56 1001.53 0.03 153-160 KTFPPQER 1218.65 1218.61 0.04 66-74 QIEVWFQNR 1374.67 1374.63 0.04 93-103 WFGS...ENHR 1378.68 1378.68 0.00 33-43 EQSR...DSFR 2224.17 2223.99 0.18 116-135 VGPT...RCER 2358.02 2357.95 0.07 4-25 LPSS...APPR
1 Only experimental masses that matched calculated masses are listed in the table. 2 The calculated mass is the relative molecular mass calculated from the matched peptide sequence. 3 The calculated difference between the experimental mass and the calculated mass. 4 Fragment numbering is based on the predicted N-terminus of the protein. 5 For peptide matches greater than nine amino acids in length the first 4 residues and last 4 residues are shown separated by dots (...).

001 MNRLPSSEDG DDEEFSHDDG SAPPRKKLRL TREQSRLLED SFRQNHTLNP 051 KQKEVLAKHL MLRPRQIEVW FQNRRARSKL KQTEMECEYL KRWFGSLTEE
101 NHRLHREVEE LRAIKVGPTT VNSASSLTMC PRCERVTPAA SPSRAVVPVP 151 AKKTFPPQER DR Figure D-1. MALDI-TOF MS Coverage Map of the MON 87403-produced A T H B 1 7 Δ 1 1 3 T h e a m i n o a c i d s e q u e n c e o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s d e d u c e d f r o m the ATHB17 gene present in MON 87403 and RT-PCR analysis of MON 87403 ATHB17 RNA

. Boxed regions correspond to peptides that were identified from the MON 87403-produced A T H B 1 7 Δ 1 1 3 p r o t e i n s a m p l e u s i n g M A L D I

- T O F M S

. I n t o t a l , 5 0 % ( 8 1 o u t o f 1 6 2 a m i n o a c i d s ) of the expected protein sequence was identified.Monsanto Company CR262-14U1 188 of 260

D.1.4. Western Blot Analysis-Immunoreactivity

D.1.4.1. Methods Quantitative western blot analysis was used to confirm the identity of the MON 87403 p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n , d e t e r m i n e t h e c o n c e n t r a t i o n o f M O N 8 7 4 0 3 p r o d u c e d A T H B 1 7 Δ 1 1 3 i n t h e e n r i c h e d s a m p l e , a n d t o a s s e s s t h e e qu i v a l e n c e o f t h e immunoreactivity of the MON 87403 produced and E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 proteins. MON 87403-produced and E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n s w e r e d i l u t e d i n t o 1× LB and heated to 99°C for 7 minutes. The E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 w a s u s e d to prepare a standard curve ranging between ~0.5 and ~3.0 ng (purity corrected). The E. coli

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n a n d M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 protein (two dilutions of 1:40 and 1:80 in duplicate) were loaded onto a pre-cast Tris-glycine (4-20%) polyacrylamide mini-gel (Invitrogen). Precision Plus Protein TM Molecular Dual color Standards (Bio-Rad) were loaded on the gel in parallel 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 nitrocellulose membrane (Invitrogen, Grand Island, NY).
The membrane was blocked with 5% non-fat dry milk (NFDM) in 1  phosphate bufferedsaline containing 0.05% (v/v) Tween-20 (PBST) and incubated with a polyclonal antiA T H B 1 7 Δ 1 1 3 a n t i b o d y ( l o t G 8 5 8 5 0 2 B ) a t a d i l u t i o n o f 1 : 4 0 0 0 i n 1 % N F D M i n P B S T for 1 hour at RT. After washing with PBST, the membrane was next incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (Thermo Scientific) at a dilution of 1:8,000 in 1% NFDM in PBST for 30 minutes at RT and washed again, with PBST. Immunoreactive bands were detected using the ECL TM detection system (GE Healthcare) and Amersham Hyperfilm (GE Healthcare). The film was developed using a Konica SRX-101A automated film processor (Konica Minolta Medical & Graphic, Inc., Tokyo, Japan).

T h e p r o m i n e n t i m m u n o r e a c t i v e b a n d i n e a c h l a n e , r e p r e s e n t i n g A T H B 1 7 Δ 1 1 3 , w a s quantified. Quantification of the bands on the blot was performed on a GS 800 densitometer with the supplied Quantity One® software (Bio-Rad). The concentration of M O N 8 7 4 0 3

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 i n t h e s a m p l e w a s c a l c u l a t e d a s t h e m e a n o f t h e c o n c e n t r a t i o n s o f M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 i n e a c h l a n e r e l a t i v e t o t h e standard curve.

D.1.4.2. R e s u l t s o f A T H B 1 7 Δ 1 1 3 P r o t e i n I m m u n o r e a c t i v i t y E q u i v a l e n c e
W e s t e r n b l o t a n a l y s i s w a s c o n d u c t e d u s i n g r a

b b i t a n t i

- A T H B 1 7 Δ 1 1 3 p o l y c l o n a l
a n t i b o d i e s t o 1 ) a s s e s s t h e i d e n t i t y o f M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 , 2 ) d e t e r m i n e t h e c o n c e n t r a t i o n o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 i n t h e e nr i c h e d s a m p l e from the leaf tissue of MON 87403; and 3) assess the relative immunoreactivity of the MON 87403 produced and the E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n s

.Monsanto Company CR262-14U1 189 of 260

The results demonstrated that immunoreactive bands with the same electrophoretic mobility were present in lanes loaded with the MON 87403-produced (Figure D-2, lanes 8-11) or the E. coli

- p r o d u c e d ( F i g u r e D

- 2 , l a n e s 2

- 7 ) A T H B 1 7 Δ 1 1 3 p r o t e i n

. A s expected, the signal intensity increased with increasing load amounts of the MON 87403-produced and E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n s , s u p p o r t i n g i d e n t i f i c a t i o n o f M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n

. Q u a n t i t a t i v e w e s t e r n b l o t analysis was conducted to determine the concentration of MON 87403-produced A T H B 1 7 Δ 1 1 3 i n t h e e n r i c h e d s a m p l e

. T h e c o n c e n t r a t i o n o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s d e t e r m i n e d t o b e 0

. 0 0 8 m g / m l

. B e c a u s e t h e MON 87403-produced and the E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n s w e r e b o t h i m m u n o r e a c t i v e t o a n a n t i

- A T H B 1 7 Δ 1 1 3 a n t i b o d y , t h e M O N 8 7 4 0 3

- p r o d u c e d a n d E. coli

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n s w e r e d e t e r m i n e d t o h a v e e qu i v a l e n t immunoreactivity.Monsanto Company CR262-14U1 190 of 260

Figure D-2. Western Blot Analysis of MON 87403- and E. coli-produced A T H B 1 7 Δ 1 1 3 P r o t e i n s A l i qu o t s o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 a n d t h e E. coli -produced A T H B 1 7 Δ 1 1 3 p r o t e i n s w e r e s ub j e c t e d t o S D S

- P A G E a n d e l e c t r o t r a n s f e r r e d t o a
n i t r o c e l l u l o s e m e m b r a n e

. P r o t e i n s w e r e d e t e c t e d u s i n g a n t i

- A T H B 1 7 Δ 1 1 3 a n t i b o d i e s a s the primary antibodies. Immunoreactive bands were visualized using HRP-conjugated secondary antibodies and an ECL system. The molecular weights (kDa) of the standards are shown on the left. The 10 minute exposure is shown. Lanes 1 and 12 were cropped from the image. Lane designations are as follows.

Lane Sample Amount (ng) Dilution 1 Precision Plus Protein TM Standards

- - 2 E. coli

- P r o d

u c e d A T H B 1 7 Δ 1 1 3 0.4

- 3 E. coli

- P r o d

u c e d A T H B 1 7 Δ 1 1 3 0.7

- 4 E. coli

- P r o d

u c e d A T H B 1 7 Δ 1 1 3 1.1

- 5 E. coli

- P r o d

u c e d A T H B 1 7 Δ 1 1 3 1.4

- 6 E. coli

- P r o d

u c e d A T H B 1 7 Δ 1 1 3 1.8

- 7 E. coli

- P r o d

u c e d A T H B 1 7 Δ 1 1 3 2.1

- 8 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3

- 1:80 9 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3

- 1:80 10 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3

- 1:40 11 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3

- 1:40 12 Precision Plus Protein TM Standards

- -

kDa 250.0

100.0 75.0

50.0

37.0

25.0 20.0

15.0

10.0 150.0 2 3 4 5 6 7 8 9 10 11Monsanto Company CR262-14U1 191 of 260

D.1.5. Apparent Molecular Weight and Purity Estimation using SDS-PAGE

D.1.5.1. Methods MON 87403-produced and E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n s w e r e d i l u t e d i n t o 1 × L B a n d h e a t e d t o 9 5 ° C f o r 5 m i n

. T h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n
w a s l o a d e d i n d

u p l i c a t e a t ~ 8

. 0 , ~ 1 2

. 0 , a n d
~ 1 6

. 0 n g , b a s e d o n t h e A T H B 1 7 Δ 1 1 3 p r o t e i n concentration, onto a pre-cast (4-20%) polyacrylamide mini-gel (Invitrogen). The E. coli

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s l o a d e d a t ~ 4 n g , b a s e d o n t h e p u r i t y c o r r e c t e d A T H B 1 7 Δ 1 1 3 p r o t e i n c o n c e n t r a t i o n , i n a s i n g l e l a n e

. B r o a d R a n g e M o l e c u l a r Weight Standards (Bio-Rad) were prepared and loaded on the gel at ~90 ng. Following electrophoresis at a constant voltage, proteins were stained using a ProteoSilver TM Silver Stain Kit (Sigma-Aldrich, St. Louis, MO) according to manufacturer’s recommended protocol. Analysis of the gel was performed using a Bio-Rad GS-800 densitometer supplied with Quantity One software (version 4.6.7). Apparent MW and purity were reported as an average of all six lanes containing the MON 87403-produced A T H B 1 7 Δ 1 1 3 p r o t e i n

. D.1.5.2. R e s u l t s o f A T H B 1 7 Δ 1 1 3 P r o t e i n M o l e c u l a r W e i g h t E q u i v a l e n c e
T h e M O N 8 7 4 0 3

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n ( F i g u r e D

- 3 , l a n e s 3

- 8 ) m i g r a t e d t o t h e same position on the gel as the E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n ( F i g u r e D

- 3 , l a n e 2) and the apparent MW was calculated to be 22.4 kDa (Table D-2). Because the
e x p e r i m e n t a l l y d e t e r

m i n e d a p p a r e n t M W o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 protein was within the pre-set acceptance limits for equivalence (Table D-2), the MON 87403-produced and E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n s w e r e d e t e r m i n e d t o have equivalent apparent molecular weights.

T h e p u r i t y o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 p r o t e i n w a s c a l c u l a t e d b a s e d o n the six loads on the gel (Figure D-3, lanes 3-8). The average purity was determined to be 3%.

Table D-2. Molecular Weight Comparison Between the MON 87403- and E. coli- p r o d u c e d A T H B 1 7 Δ 1 1 3 P r o t e i n s

Apparent MW of MON 87403-produced A T H B 1 7 Δ 1 1 3 P r o t e i n ( k D a ) Apparent MW of E. coli -produced A T H B 1 7 Δ 1 1 3 P r o t e i n 1 (kDa) Preset Acceptance

Limits for the MON 87403-produced A T H B 1 7 Δ 1 1 3 P r o t e i n 2 (kDa) 22.4 22.2 22.2 – 23.5
1 As reported on the Certificate of Analysis for lot 11380003 2 Calculated lower and upper bounds for one future assay based on two-tailed 95% prediction interval derived from apparent MW determinations for E. coli

- p r o d u c e d A T H B 1 7 Δ 1 1 3

.Monsanto Company CR262-14U1 192 of 260

Figure D-3. Molecular Weight and Purity Analysis of the MON 87403-produced A T H B 1 7 Δ 1 1 3 P r o t e i n A l i qu o t s o f t h e M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 a n d t h e E. coli -produced A T H B 1 7 Δ 1 1 3 p r o t e i n s w e r e s ub j e c t e d t o S D S

- P A G E a n d t h e g e l w a s s t a i n e d w i t h s i l v e r stain. The molecular weights (kDa) of the standards are shown on the left. Lane 10 was cropped from the image. Lane designations are as follows:

Lane Sample Amount (ng) 1 Broad Range MW Standards 90 2 E. coli

- p r o d

u c e d A T H B 1 7 Δ 1 1 3 4 3 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 8 4 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 8 5 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 12 6 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 12 7 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 16 8 M O N 8 7 4 0 3

- p r o d u c e d A T H B 1 7 Δ 1 1 3 16 9 Broad Range MW Standards 90 10 Blank

-

1 2 3 4 5 6 7 8 9 kDa 200.0
116.3 97.4

66.2

45.0

31.0

21.5

14.4 6.5 ATHB17 Δ 113Monsanto Company CR262-14U1 193 of 260 Appendix E: Materials and Methods Used for the Analysis of the Levels of

A T H B 1 7 Δ 1 1 3 P r o t e i n i n M O N 8 7 4 0 3

E.1. Materials Over season leaf (OSL1), over season root (OSR1), forage, and grain tissue samples from MON 87403 were harvested from five field sites in The United States during the 2012 growing season from starting seed lot 11332602. E coli- p r o d u c e d A T H B 1 7 Δ 1 1 3 (lot 11280374) was used as the analytical reference standard.

E.2. Characterization of the Materials The identity of MON 87403 was confirmed by conducting MON 87403 event-specific polymerase chain reaction (PCR) analyses on the starting seed.

E.3. Field Design and Tissue Collection Field trials were initiated during the 2012 planting season to generate tissues of MON 87403 at various maize growing locations in The United States. OSL1, OSR1, forage, and grain, tissue samples from the following field sites were analyzed: Jackson County, Arkansas (site code ARNE); Story County, Iowa (site code IALL); Jefferson County, Iowa (site code IARL); Pawnee County, Kansas (site code KSLA) and Lehigh County, Pennsylvania (site code PAGR). At each site, four replicated plots of plants containing MON 87403 were planted using a randomized complete block field design. Tissue samples were collected from each replicated plot at all field sites. See Table V-1 for detailed descriptions of when the samples were collected.

E.4. Tissue Processing and Protein Extraction All tissue samples harvested were shipped to Monsanto’s processing facility and were ground by the Monsanto Sample Management Team to facilitate protein extraction. All ground tissue samples were then lyophilized by the Monsanto Sample Management Team. The lyophilized tissue samples were stored in a -80  C freezer until transferred on dry ice to the analytical facility.

T h e A T H B 1 7 Δ 1 1 3 p r o t e i n w a s e x t r a c t e d f r o m m a i z e t i s s u e s a s d e s c r i b e d i n T a b l e E

- 1

. The protein extracts were aliquoted and stored frozen in a -80°C freezer until analysis.Monsanto Company CR262-14U1 194 of 260

T a b l e E

- 1

. A T H B 1 7 Δ 1 1 3 E x t r a c t i o n M e t h o d s 1 for Tissue Samples

Sample Type Tissue-to-Buffer Ratio Extraction Buffer

Leaf 2 /Grain/Root 3 /Forage 1:100 1 × TB + 0.1% BSA 4

1 A T H B 1 7 Δ 1 1 3 p r o t e i n w a s e x t r a c t e d f r o m e a c h t i s s u e b y a d d i n g t h e a p p r o p r i a t e v o l u m e o f extraction buffer, beads, and shaking in a Harbil mixer (Harbil Industries Inc., Compton, CA). The extracted samples were clarified using centrifugation and a serum filter. 2 Over season leaf (OSL1). 3 Over season root (OSR1). 4 0.1 M Tris, 0.1 M Na 2 B 4 O 7 , 0.005 M MgCl 2 , 0.05% (v/v) Tween-20 and 0.1 % (w/v) BSA.

E.5. A T H B 1 7 Δ 1 1 3 A n t i b o d i e s
M o u s e m o n o c l o n a l a n t i b o d y ( L o t G

- 8 6 9 0 5 5 ) s p e c i f i c f o r t h e A T H B 1 7 Δ 1 1 3 p r o t e i n w a s purified using Protein G column. The concentration of the purified antibody was determined to be 3.79 mg/ml by spectrophotometric methods. The purified antibody was stored in a phosphate buffered saline (0.001 M KH 2 PO 4 , 0.01 M Na 2 HPO 4 , 0.137 M NaCl, 0.0027 M KCl and 0.05 % NaN 3 ).

G o a t p o l y c l o n a l a n t i b o d i e s s p e c i f i c f o r t h e A T H B 1 7 Δ 1 1 3 p r o t e i n w e r e p u r i f i e d u s i n g affinity chromatography with a Protein G column. The concentration of the purified antibodies was determined to be 10.72 mg/ml by spectrophotometric methods. The purified antibodies (lot G-869057) were stored in a phosphate buffered saline (0.001 M KH 2 PO 4 , 0.01 M Na 2 HPO 4 , 0.137 M NaCl, 0.0027 M KCl and 0.05% NaN 3 ). The goat polyclonal antibodies were coupled with biotin (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions and assigned lot G-869059. The detection reagent was Poly-HRP conjugate (Thermo Fisher Scientific).

E.6. A T H B 1 7 Δ 1 1 3 E L I S A M e t h o d

M o u s e a n t i

- A T H B 1 7 Δ 1 1 3 c a p t u r e a n t i b o d y w a s d i l u t e d i n t o a c o a t i n g b u f f e r (0.015 M Na 2 CO 3 and 0.035 M NaHCO 3 ) and immobilized onto 96-well microtiter plates at 1  g/ml followed by incubation in a 4  C r e f r i g e r a t o r f

o r ≥ 1 2 h o u r s

. P r i o r t o e a c h s t e p in the assay, plates were washed with 1 × phosphate buffered saline containing 0

. 0 5 % ( v / v ) T w e e n 2 0

. P l a t e s w e r e b l o c k e d w i t h t h e a d d i t i o n o f 3 0 0 μ l p e r w e l l o f Blocker Casein in PBS (Thermo Fisher Scientific, catalog number 37528) for 60 to 90 minutes at 37  C

. A T H B 1 7 Δ 1 1 3 p r o t e i n s t a n d a r d o r s a m p l e e x t r a c t w a s a d d e d a t 200  l per well and incubated for 60 to 65 minutes at 37  C. Biotinylated goat

a n t i

- A T H B 1 7 Δ 1 1 3 a n t i b o d i e s w e r e a d d e d a t 2 0 0  l per well and incubated for 60 to 65 minutes at 37  C. Poly-HRP conjugate was added at 200  l per well andMonsanto Company CR262-14U1 195 of 260

incubated for 30 to 35 minutes at 37  C. Plates were developed by adding 200  l per well of horseradish peroxidase substrate, 3,3',5,5'-tetramethylbenzidine (Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction was terminated by the addition of 100  l per well of 6 M H 3 PO 4 . Q u a n t i f i c a t i o n o f t h e A T H B 1 7 Δ 1 1 3 p r o t e i n w a s a c c o m p l i s h e d b y i n t e r p o l a t i o n f

r o m a n A T H B 1 7 Δ 1 1 3 p r o t e i n s t a n d a r d c u r v e t h a t r a n g e d f

r o m 0 - 0.5 ng/ml.

E.7. 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 fiveparameter logistic curve fit. Following the interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was reported on a “  g/g dwt” basis for data that were greater than or equal to the LOQ. This conversion utilized a sample dilution factor and a tissue-to-buffer ratio. Microsoft Excel 2007 (Microsoft, Redmond, WA) was used to calculate the protein levels in 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. Any test substance extract that resulted in unexpectedly negative or positive results by ELISA analysis were re-extracted twice for the protein of interest and re-analyzed by ELISA to confirm the results. Samples with confirmed unexpected results were omitted from all calculations. Samples that were not confirmed to be either positive or negative were reported as inconclusive and omitted from all calculations.Monsanto Company CR262-14U1 196 of 260

Appendix F: Materials and Methods for Compositional Analysis of MON 87403 Maize Grain and Forage
Compositional comparisons between MON 87403 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 the United States during 2012 under typical agronomic practices.

F.1. Materials Harvested grain and forage from MON 87403, a conventional control (MPA640B) that has similar genetic background to that of MON 87403, and conventional, commercial reference maize hybrids were compositionally assessed. The reference hybrids are listed in Table F-1. Table F-1. Conventional Commercial Reference Maize Hybrids

Material Name Seed Lot # Field Site Codes Burrus 645 11227210 ILMN, NESH Gateway 4148 11273005 ILMN, PAGR Gateway 6158 11273006 ARNE, INSH H-9180 11226704 IARL Lewis 6442 11226558 NESH Lewis 7007 11226559 IALL, IARL, KSLA LG2540 11226898 PAGR 11266730 ARNE, IARL, ILMN, INSH LG2620 11226861 KSLA Midland Phillips 799 11226703 ARNE, ILMN, INSH, NESH, PAGR Mycogen 2M746 11226705 IALL NC+ 4443 11226700 ARNE NC+ 5220 11226701 IALL, KSLA Phillips 713 11300072 KSLA Phillips 717 11300073 IALL, NESH Stewart S588 11226918 PAGR Stewart S602 11226919 IARL Stine 9724 11298951 INSHMonsanto Company CR262-14U1 197 of 260

F.2. Characterization of the Materials The identities of MON 87403, the conventional control, and reference hybrids were confirmed prior to use in the compositional assessment.

F.3. Field Production of the Samples Grain and forage samples from MON 87403, the conventional control, and the reference hybrids were collected from eight replicated sites in United States during the 2012 growing season. The field sites were located in: Jackson, Arkansas (ARNE); Story, Iowa (IALL); Jefferson, Iowa (IARL); Warren, Illinois (ILMN); Boone, Indiana (INSH); Pawnee, Kansas (KSLA); Polk, Nebraska (NESH); and Lehigh, Pennsylvania (PAGR). Starting seeds were planted in a randomized complete block design with four plots for each of MON 87403, 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 the United States. 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.

F.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

z i n c ) a n d v i t a m i n s [ β

- c a r o t e n e ( r e f e r r e d t o a s v i t a m i n A ) , B 1 , B 2 , B 6 , E ( α
- t o c o p h e r o l ) , 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.

F.4.1. 2-Furaldehyde The ground samples were extracted with 4% trichloroacetic acid and injected directly on a high-performance liquid chromatography system for quantitation of free furfurals by ultraviolet detection (Albala-Hurtado, et al. 1997). The limit of quantitation was 0.500 ppm.Monsanto Company CR262-14U1 198 of 260

Reference Standard : ACROS Organics, 2 Furaldehyde, 99.5%, Lot Number A0296679

F.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%.

F.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 are derivatized with fluorenylmethyl chloroformate (FMOC) before injection (AOAC 2012d; Barkholt and Jensen 1989; Henderson and Brooks 2010; Henderson, et al. 2000; Schuster 1988). The limit of quantitation for this study was 0.100 mg/g.Monsanto Company CR262-14U1 199 of 260

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

F.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 2012h). The limit of quantitation was 0.100%.

F.4.5. Vitamin A (Beta Carotene) The samples were saponified and extracted with hexane. The samples were then injected on a reverse phase high-performance liquid chromatography (HPLC) system with ultraviolet light detection. Quantitation was achieved with a linear regression analysis (AOAC 2012a; Quackenbush 1987). The limit of quantitation was 0.0200 mg/100g.

Reference Standard: Manufacturer Analyte Lot No. Purity (%) Sigma-Aldrich Beta Carotene 091M1417V 98.2* * Based on E1%=2280 for Lambda Maximum of 478 to 479 nm in Hexane.

F.4.6. Carbohydrate The total carbohydrate level was calculated by difference using the fresh weight-derived data and the following equation (USDA 1973): % carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)Monsanto Company CR262-14U1 200 of 260

The limit of quantitation was calculated as 0.100%.

F.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 2012k; l). The limit of quantitation was 0.100%.

F.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 2012m; n). The limit of quantitation was 0.100%.

F.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 using external standards for quantitation (AOCS 2009b; a). The limit of quantitation was 0.00400%.Monsanto Company CR262-14U1 201 of 260 Reference Standards:

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

F.4.10. Vitamin B9 (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 2012p; q; Infant Formula Council 1985). The limit
o f q u a n t i t a t i o n w a s 0

. 0 6 0 0 μ g / g

. Reference Standard: USP, Folic acid, 98.9%, Lot Number Q0G151Monsanto Company CR262-14U1 202 of 260

F.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 500  C. 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 2012s; o). The limits of quantitation were as follows: Inorganic Ventures Reference Standards and Limits of Quantitation:

Mineral Lot Numbers Concentration (μg/mL) LOQ (ppm) Calcium F2-MEB453071MCA, F2-MEB453073 200, 1000 20.0 Copper F2-MEB453071MCA, F2-MEB453072MCA 2.00, 10.0 0.500
Iron F2-MEB453071MCA, F2-MEB453074 10.0, 50.0 2.00 Magnesium F2-MEB453071MCA, F2-MEB453072MCA 50.0, 250 20.0 Manganese F2-MEB453071MCA, F2-MEB453072MCA 2.00, 10.0 0.300 Phosphorus F2-MEB453071MCA, F2-MEB453073 200, 1000 20.0
Potassium F2-MEB453071MCA, F2-MEB453073 200, 1000 100
Sodium F2-MEB453071MCA, F2-MEB453073 200, 1000 100
Zinc F2-MEB453071MCA, F2-MEB453072MCA 10.0, 50.0 0.400

F.4.12. Moisture
The samples were dried in a vacuum oven at approximately 100  C. The moisture weight loss was determined and converted to percent moisture (AOAC 2012r; j). The limit of quantitation was 0.100%.

F.4.13. Neutral Detergent Fiber The ANKOM2000 Fiber Analyzer automated the process of the removal of protein, carbohydrate, and ash. Fats and pigments were removed with an acetone wash prior to analysis. Hemicellulose, cellulose, lignin and insoluble protein fraction were left in theMonsanto Company CR262-14U1 203 of 260

filter bag and determined gravimetrically (AACC 1998; Komarek, et al. 1994; USDA 1970). The limit of quantitation was 0.100%.

F.4.14. Vitamin B3 (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 2012i; p). The l i m i t o f qu a n t i t a t i o n w a s 0

. 3 0 0 μ g / g

. Reference Standard:
USP, Niacin, 99.8%, Lot Number J0J235

F.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 p-coumaric acid and ferulic acid by ultra violet (UV) detection (Hagerman and Nicholson 1982). The limit of quantitation for the p-coumaric acid and ferulic acid was 33.3 ppm. Reference Standards:

Component Manufacturer Lot No. Purity (%) p-Hydroxycinnamic Acid (p-Coumaric Acid) Sigma-Aldrich 091M1197V 99.6
4-Hydroxy-3- methoxycinnamic Acid (Ferulic Acid) 4-Hydroxy-3- methoxycinnamic Acid (Ferulic Acid) ACROS Organics

ACROS Organics A0261354

A0294716 99.4

99.4

F.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 high-performance liquid chromatography (HPLC) system with a refractive index detector (Lehrfeld 1989; Lehrfeld 1994). The limit of quantitation was 0.100%. Reference Standard:
Sigma-Aldrich, Phytic Acid Sodium Salt Hydrate, 97.9%, Lot Number BCBH8701VMonsanto Company CR262-14U1 204 of 260

F.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 2012g; f). The limit of quantitation was 0.100%.

F.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 h y d r o c h l o r i d e s o l u t i o n i n p y r i d i n e c o n t a i n i n g p h e n y l

- β

- D

- g l u c o p y r a n o s i d e a s t h e i n t e r

n a l standard. The resulting oximes were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid treatment, and then analyzed by gas chromatography 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

F.4.19. Total Dietary Fiber
Duplicate samples were gelatinized with  -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 2012c). The limit of quantitation was 1.00%.

F.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 2012b; t; u). The limit of quantitation was 0.010 mg/100g. Reference Standard:
USP, Thiamine Hydrochloride, 99.7%, Lot Number P0K366Monsanto Company CR262-14U1 205 of 260

F.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 t u r b i d i m e t r i c a l l y ( A O A C 2 0 1 2 e ; p ; c )

. T h e l i m i t o f qu a n t i t a t i o n w a s 0

. 2 0 0 μ g / g

. Reference Standard:
USP, Riboflavin, 99.7%, Lot Number N1J079
The United States Pharmacopeia, Twenty-Ninth Revision, p. 1913, United States Pharmacopeial Convention, Inc., Rockville, MD (2005).

F.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 2 0 1 2 v ; A t k i n s , e t a l

. 1 9 4 3 )

. T h e l i m i t o f qu a n t i t a t i o n w a s 0

. 0 7 0 0 μ g / g

. Reference Standard:
USP, Pyridoxine hydrochloride, 99.8%, Lot Number Q0G409

F.4.23. V i t a m i n E ( α - T o c o p h e r o l ) The samples were saponified to break down any fat and release vitamin E. Thesaponified 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.

Note: Alpha tocopherol is part of a mixed standard which also includes beta, delta, and gamma isomers. The reference standard material for those isomers may contain small amounts of alpha tocopherol. All reference standards that contributed to the alpha tocopherol concentration are listed below. Reference Standard:

Manufacturer Component Lot No. Purity (%) USP Alpha Tocopherol O0K291 99 Acros Organics D-gamma-Tocopherol A0083534 99.3 Sigma-Aldrich ( + )

- δ

- T o c o p h e r o l 090M1916V 92Monsanto Company CR262-14U1 206 of 260

F.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 F-2): Table F-2. Re-expression Formulas for Statistical Analysis of Composition Data

Component From (X) To Formula 1 Proximates (excluding Moisture), Fiber, Anti-nutrients % fwt % dw X/d Amino Acids (AA) mg/g fwt % dw X/(10d) Secondary Metabolites ppm fwt μ g / g d w X/d Copper, Iron, Manganese, Zinc ppm fwt mg/kg dw X/d Calcium, Magnesium, Phosphorus, Potassium ppm fwt % dw X/(10 4 d)
Vitamins B2, B3, B6, B9 μ g / g f w t mg/kg dw X/d Vitamin A, Vitamin B1 mg/100g fwt mg/kg dw 10X/d Vitamin E mg/g fwt mg/kg dw 10 3 X/d

Fatty Acids (FA) % fwt % Total FA (100)X j /  X, for each FA

j where  X is over all the FA
1 ‘X’ is the individual sample value; d is the fraction of the sample that is dry matter.

In order to complete a statistical analysis for a compositional constituent in this compositional assessment, at least 50% of all the values for an analyte in grain or forage had to be greater than the assay limit of 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. Forty-five observations for 22:0 behenic acid and one value for calcium were assigned a value equal to one-half of the LOQ (0.002% fwt and 10.00 ppm fwt, respectively). 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 versionMonsanto Company CR262-14U1 207 of 260

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. Eleven results had PRESS residual values outside of the  6 range. Of the eleven flagged values, only the copper values from two conventional references were removed from further analysis as outliers. The remaining values were not removed 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 copper data to detect potential outliers that were masked in the first analysis. One copper value from a commercial reference was identified as a potential 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 87403 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 CR262-14U1 208 of 260 References for Appendix F 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.

A O A C

. 2 0 1 2 a

. A O A C o f f i c i a l m e t h o d 2 0 0 5

. 0 7 : β
· c a r o t e n e i n s u p p l e m e n t s a n d r a w materials reversed-phase high pressure liquid chromatographic method. Association of Analytical Communities International, Gaithersburg, Maryland.
AOAC. 2012b. AOAC official method 942.23: Thiamine (Vitamin B 1 ) in human and pet foods. Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012c. AOAC official method 985.29: Total dietary fiber in foods. Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012d. AOAC official method 988.15: Tryptophan in foods and food and feed ingredients. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland.
AOAC. 2012e. AOAC official method 940.33: Riboflavin (Vitamin B 2 ) in vitamin preparations. Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012f. AOAC official method 979.09: Protein in grains. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012g. AOAC official method 955.04: Nitrogen (total) in fertilizers Kjeldahl method. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012h. AOAC official method 923.03: Ash of flour. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland.
AOAC. 2012i. AOAC official method 944.13: Niacin and niacinamide (nicotinic acid and nicotinamide) in vitamin preparations. Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012j. AOAC official method 926.08: Loss on drying (moisture) in cheese. Association of Analytical Communities International, Gaithersburg, Maryland.Monsanto Company CR262-14U1 209 of 260

AOAC. 2012k. AOAC official method 922.06: Fat in flour. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012l. AOAC official method 954.02: Fat (crude) or ether extract in pet food. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012m. AOAC official method 948.22: Fat (crude) in nuts and nut products. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland.
AOAC. 2012n. AOAC official method 960.39: Fat (crude) or ether extract in meat. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012o. AOAC official method 985.01: Metals and other elements in plants and pet foods. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012p. AOAC official method 960.46: Vitamin assays. Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012q. AOAC official method 992.05: Total folate (Pteroylglutamic acid) in infant formula. Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012r. AOAC official method 925.09: Solids (total) and loss on drying (moisture) in flour. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland. AOAC. 2012s. AOAC official method 984.27: Calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc in infant formula. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland.
AOAC. 2012t. AOAC official method 953.17: Thiamine (Vitamin B 1 ) in grain products. Association of Analytical Communities International, Gaithersburg, Maryland.
AOAC. 2012u. AOAC official method 957.17: Thiamine (Vitamin B 1 ). Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012v. AOAC official method 961.15: Vitamin B 6 (pyridoxine, pyridoxal, pyridoxamine) in food extracts. Association of Analytical Communities International, Gaithersburg, Maryland. AOCS. 2009a. Preparation of methyl esters of fatty acids. Method Ce 2-66, American Oil Chemists' Society, Champaign, Illinois.Monsanto Company CR262-14U1 210 of 260

AOCS. 2009b. Determination of saturated, cis- monounsaturated, and cis -polyunsaturated fatty acids in marine and other oils containing long chain polyunsaturated fatty acids (PUFAs) by capillary GLC3. Official Methods and Recommended Practices of the American Oil Chemists' Society, 5th Edition. Method Ce 1i-07, 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. 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. 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 ofMonsanto Company CR262-14U1 211 of 260

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. 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 

 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, anti-nutrients and secondary plant metabolites. OECD ENV/JM/MONO (2002)25. Organisation of Economic Co-Operation and Development, Paris, France. Quackenbush, F.W. 1987. Reverse phase HPLC separation of cis and trans-carotenoids and its application to  -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. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agricultural Handbook No. 379. U.S. Department of Agriculture, Washington, DC. 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 CR262-14U1 212 of 260 Appendix G: Materials, Methods, and Individual Site Results for Seed Dormancy and Germination Assessment of MON 87403

G.1. Materials Seed germination and dormancy characteristics were assessed on seed from MON 87403, the conventional control, and reference hybrids produced in replicated field trials during 2012 at the following sites: Story County, Iowa (IALL); Warren County, Illinois (ILMN); and Lehigh County, Pennsylvania (PAGR).

G.2. Characterization of the Materials The identities of the MON 87403 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 87403 and the conventional control plots was confirmed by event-specific PCR analyses. Chain-of-custody documentation for all starting seed for this 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.

G.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 and Society of Commercial Seed Technologists (SCST) (AOSA/SCST 2010; AOSA, 2012a; 2012b).
The seed lots (Selfed F 2 grain) of MON 87403, 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 87403, the conventional control, and thereference hybrids were placed on pre-moistened germination towels. Additional premoistened germination towels were placed on top of the seed. 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 1 towel per entry. Buckets were then placed in the appropriate germination chambers. Each temperature regime constituted a separate split-plot experiment with four replications. A description of each germination characteristic evaluated and the timing of evaluations areMonsanto Company CR262-14U1 213 of 260

presented in Table VII-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 100). 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.

G.4. Statistical Analysis An analysis of variance was conducted using SAS ® version 9.3 (SAS 2010) according toa split-plot design (production site as the whole plot and starting seed material as the subplot) with four replications. MON 87403 was compared to the conventional control for dormancy and germination 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 dormancy and germination characteristics analyzed included percent germinated seed, percent viable hard seed, percent dead seed, and percent viable firm swollen seed. The percent germinated seed were categorized as either normal germinated or abnormal germinated for the AOSA temperature regime. The level
o f s t a t i s t i c a l s i g n i f

i c a n c e w a s p r e d e t e r

m i n e d t o b e 5 % ( α = 0

. 0 5 )

. M O N 8 7 4 0 3 w a s n o t 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 the study sites ( i.e., reference range) representative of maize hybrids. Results from the combined-site analysis are presented in Table VII-2.

G.5. Individual-Site Seed Dormancy and Germination Analysis In the individual site analyses, no statistically significant differences were detected at sites IALL and ILMN. Two statistically significant differences were detected betweenMonsanto Company CR262-14U1 214 of 260

MON 87403 and the conventional control at the PAGR site for the measured characteristics abnormal germinated seed and viable firm-swollen seed. MON 87403 hadsignificantly fewer abnormal germinated seed (0.3% vs. 1.3%) and more viable firmswollen seed (0.3% vs. 0.0%) than the conventional control at the 20/30 °C. Statistically significant differences between MON 87403 and the conventional control for both germination characteristics in the individual site analyses were not detected in the combined site analysis (Table VII-2) and are unlikely to be biologically meaningful in terms of increased pest/weed potential (See Figure VII-1, Step 2, answer “no”).

Table G-1. Starting Seed of MON 87403, Conventional Control and Commercial Maize Reference Hybrids Used in Dormancy Assessment

Site 1 Material Type Material Name Phenotype Material ID IALL Control MPA640B Conventional 11354876 IALL Reference Lewis 7007 Conventional 11354877 IALL Reference Mycogen 2M746 Conventional 11354878 IALL Reference NC+ 5220 Conventional 11354879 IALL Reference Phillips 717 Conventional 11354880
IALL Test MON 87403 Increased Ear Biomass Maize 11354881
ILMN Control MPA640B Conventional 11354882 ILMN Reference Gateway 4148 Conventional 11354883 ILMN Reference Midland Phillips 799 Conventional 11354884 ILMN Reference Burrus 645 Conventional 11354885 ILMN Reference LG2540 Conventional 11354886
ILMN Test MON 87403 Increased Ear Biomass Maize 11354887
PAGR Control MPA640B Conventional 11354888 PAGR Reference Gateway 4148 Conventional 11354889 PAGR Reference Midland Phillips 799 Conventional 11354890 PAGR Reference Stewart S588 Conventional 11354891 PAGR Reference LG2540 Conventional 11354892
PAGR Test MON 87403 Increased Ear Biomass Maize 11354893

1 IALL = Story County, Iowa, ILMN = Warren County, Illinois, and PAGR = Lehigh County, Pennsylvania

.Monsanto Company CR262-14U1 215 of 260

Table G-2. Dormancy and Germination Characteristics of MON 87403 and the Conventional Control Seed Produced at each of the Three Field Sites

IALL 1 ILMN 1 PAGR 1 Temperature Assessment Mean % (S.E.) 2 Mean % (S.E.) 2 Mean % (S.E.) 2 (°C) Category MON 87403 Control MON 87403 Control MON 87403 Control
5 Germinated 8.8 (0.95) 10.8 (1.80) 4.5 (1.04) 5.0 (1.29) 3.8 (1.44) 2.8 (0.85)

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 3.8 (0.48) 2.3 (1.11) 4.0 (0.71) 3.3 (0.75) 4.5 (1.76) 5.0 (1.47)
Viable Firm Swollen 87.5 (1.32) 87.0 (2.12) 91.5 (1.55) 91.8 (1.31) 91.8 (2.87) 92.3 (1.65)
10 Germinated 93.8 (1.18) 92.5 (1.94) 97.0 (0.58) 95.5 (0.87) 94.3 (1.44) 94.0 (0.91)

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 (1.50) 1.8 (1.11) 1.0 (0.41) 1.8 (0.85) 3.0 (0.71) 2.5 (0.87)
Viable Firm Swollen 4.8 (1.38) 5.8 (2.63) 2.0 (0.71) 2.8 (1.18) 2.8 (1.03) 3.5 (0.87)
20 3 Germinated 100.0 (0.00) 99.8 (0.25) 99.3 (0.25) 99.0 (0.41) 98.8 (0.63) 98.5 (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 0.0 (0.00) 0.3 (0.25) 0.8 (0.25) 1.0 (0.41) 1.3 (0.63) 1.5 (0.29)
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)
30 Germinated 99.8 (0.25) 99.8 (0.25) 99.8 (0.25) 99.5 (0.50) 98.8 (0.48) 99.5 (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 0.3 (0.25) 0.3 (0.25) 0.3 (0.25) 0.5 (0.50) 1.3 (0.48) 0.5 (0.29)
Viable Firm Swollen 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00)Monsanto Company CR262-14U1 216 of 260

Table G-2 (continued). Dormancy and Germination Characteristics of MON 87403 and the Conventional Control Seed Produced at each of the Three Field Sites

IALL 1 ILMN 1 PAGR 1 Temperature Assessment Mean % (S.E.) 2 Mean % (S.E.) 2 Mean % (S.E.) 2 (°C) Category MON 87403 Control MON 87403 Control MON 87403 Control
10/20 Germinated 99.5 (0.29) 100.0 (0.00) 99.5 (0.50) 99.8 (0.25) 99.3 (0.25) 98.8 (0.25)

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 0.3 (0.25) 0.0 (0.00) 0.3 (0.25) 0.3 (0.25) 0.8 (0.25) 1.3 (0.25)
Viable Firm Swollen 0.3 (0.25) 0.0 (0.00) 0.3 (0.25) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)
10/30 Germinated 99.5 (0.29) 99.5 (0.29) 99.8 (0.25) 99.8 (0.25) 99.5 (0.50) 99.3 (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 0.5 (0.29) 0.5 (0.29) 0.3 (0.25) 0.3 (0.25) 0.5 (0.50) 0.8 (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)
20/30 Normal Germinated 99.5 (0.50) 100.0 (0.00) 98.0 (0.41) 98.3 (0.25) 99.3 (0.48) 98.3 (0.63) (AOSA) Abnormal Germinated 0.5 (0.50) 0.0 (0.00) 1.5 (0.29) 0.8 (0.25) 0.3 (0.25)* 1.3 (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 0.0 (0.00) 0.0 (0.00) 0.5 (0.29) 1.0 (0.41) 0.3 (0.25) 0.5 (0.50)
Viable Firm- Swollen 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.3 (0.25)* 0.0 (0.00)

Note: The experimental design was a split-plot with four replications.
* I n d i c a t e s a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n M O N 8 7 4 0 3 a n d t h e c o n v e n t i o n a l c o n t r o l ( α = 0

. 0 5 ) u s i n g A N O V A

. 1 Site codes are as follows: IALL = Story County, Iowa, ILMN = Warren County, Illinois and PAGR = Lehigh County, Pennsylvania. 2 MON 87403 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 87403 and the conventional control did not equal 100% due to numerical rounding of the means. 3 Three replicates of the reference Midland Phillips 799 were used from sites ILMN and PAGR. †No statistical comparison could be made due to lack of variability in the data.Monsanto Company CR262-14U1 217 of 260 References for Appendix G AOSA/SCST (2010). Tetrazolium testing handbook. Ithaca, New York, Association of Official Seed Analysts and the Society of Commercial Seed Technologists.
Association of Official Seed Analysts. 2012a. AOSA Rules for testing seeds. Volume 1. Principles and Procedures. Association of Official Seed Analysts, Ithaca, NY.
Association of Official Seed Analysts. 2012b. AOSA Rules for testing seeds. Volume 4. Seedling Evaluation. Association of Official Seed Analysts, Ithaca, NY.
SAS. (2010). SAS/STAT software version 9.3. SAS Institute, Inc., Cary, North Carolina.Monsanto Company CR262-14U1 218 of 260

Appendix H: Materials, Methods, and Individual Site Results from Phenotypic, Agronomic, and Environmental Interaction Assessment of MON 87403 under Field Conditions

H.1. Materials Agronomic, phenotypic, and environmental interaction characteristics were assessed for MON 87403, the conventional control, and 21 reference hybrids grown under similar agronomic conditions. Four reference hybrids were planted per site (Table H-1).

H.2. Characterization of the Materials The presence or absence of the MON 87403 event in the starting seed of MON 87403 and the conventional control was verified by event-specific polymerase chain reaction (PCR) analyses. No molecular analyses were performed on the reference starting seed.

H.3. Field Sites and Plot Design Field trials were established in 2012 at 13 sites that provided a range of environmental and agronomic conditions representative of U.S. maize growing regions (Table VII-3). The Principal Investigator at each site was familiar with the growth, production, and evaluation of maize characteristics. At all sites, seed of MON 87403, the conventional control, and four reference hybrids were planted in a randomized complete block design with four replications. At all sites except IABG, ILCX, NEDC, NEYO, NCBD, and PAHM, each replicated plot consisted of 8 rows of maize spaced approximately 0.76 m apart and approximately 6 m long. Phenotypic and qualitative environmental interaction data were targeted to be collected from rows 4 and 5. The remaining rows were used for other purposes. At sites: IABG, NEYO, NCBD, and PAHM 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 interaction data were targeted to be collected from rows 2 and 3. Rows 6 and 8 were targeted to collect arthropod samples using sticky traps (rows 8 and 10 at NCBD site). Rows 9, 10, 11, and 12 were targeted for visual counts of arthropod abundance. Rows 13 and 14 were targeted to assess plant damage caused by corn earworm ( Helicoverpa zea ) and European corn borer ( Ostrinia nubilalis ). The remaining rows were used for other purposes. At sites: ILCX, and NEDC, each replicated plot consisted of 6 rows of maize spaced approximately 0.76 m apart and approximately 6 m long. Phenotypic and qualitative environmental interaction data were collected from rows 2 and 3. The remaining rows were used for other purposes.

H.4. Planting and Field Operations Planting information, soil description, and cropping history of the trial area are listed in Table H-2. Prior to planting, the Principal Investigator at each site prepared the plot areaMonsanto Company CR262-14U1 219 of 260

with a proper seed bed according to local agronomic practices, including tillage, 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 CR262-14U1 220 of 260

Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental Interaction Assessment

Site Code 1 Material Name Regulatory Lot Number Phenotype T/C/R 2

All MON 87403 11332602 Increased Ear Biomass Maize T
MPA640B 3 11332601 Conventional C

ARNE Gateway 6158 11273006 Conventional R Midland Phillips 799 11226703 Conventional R NC+ 4443 11226700 Conventional R LG2540 11266730 Conventional R

IABG Gateway 6158 11273006 Conventional R LG2620 11226861 Conventional R Burrus 645 11227210 Conventional R Legacy L7671 11226598 Conventional R

IARL Lewis 7007 11226559 Conventional R H-9180 11226704 Conventional R Stewart S602 11226919 Conventional R LG2540 11266730 Conventional R

ILCX Gateway 4148 11273005 Conventional R Mycogen 2M746 11226705 Conventional R NC+ 4443 11226700 Conventional R LG2548 11266731 Conventional R

ILMN Gateway 4148 11273005 Conventional R Midland Phillips 799 11226703 Conventional R Burrus 645 11227210 Conventional R LG2540 11266730 Conventional R

INSH Gateway 6158 11273006 Conventional R Midland Phillips 799 11226703 Conventional R Stine 9724 11298951 Conventional R LG2540 11266730 Conventional R

KSLA Lewis 7007 11226559 Conventional R LG2620 11226861 Conventional R NC+ 5220 11226701 Conventional R Phillips 713 11300072 Conventional R

NCBD Pioneer 32T16 11226579 Conventional R Mycogen 2M746 11226705 Conventional R Stewart S588 11226918 Conventional R Legacy L7671 11226598 Conventional RMonsanto Company CR262-14U1 221 of 260

Table H-1 (continued). Test, Control, and Reference Starting Seed Site Code 1 Material Name Regulatory Lot Number Phenotype T/C/R 2

NEDC Gateway 4148 11273005 Conventional R LG2620 11226861 Conventional R Stewart S588 11226918 Conventional R Phillips 717 11300073 Conventional R

NESH Lewis 6442 11226558 Conventional R Midland Phillips 799 11226703 Conventional R Burrus 645 11227210 Conventional R Phillips 717 11300073 Conventional R

NEYO Pioneer 32B81 11226578 Conventional R Mycogen 2M746 11226705 Conventional R Burrus 645 11227210 Conventional R LG2540 11266730 Conventional R

PAGR Gateway 4148 11273005 Conventional R Midland Phillips 799 11226703 Conventional R Stewart S588 11226918 Conventional R LG2540 11226898 Conventional R

PAHM Gateway 4148 11273005 Conventional R Midland Phillips 799 11226703 Conventional R Stewart S588 11226918 Conventional R LG2540 11226898 Conventional R

1 Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA. 2 T/C/R = Test/Control/Reference. 3 MPA640B = LH244+LH287.Monsanto Company CR262-14U1 222 of 260

Table H-2. Field and Planting Information

Site 1 Planting

Date 2 Harvest

Date 2 Approximate

Planting Rate (seeds/m) Approximate

Plot Size (m × m) Rows

per Plot Soil Type % OM 3 Previous Crop 2011 ARNE 05/15/2012 9/20/2012 7.2 6.1 × 6.1 8 Sandy Loam 1.3 Soybean IABG 05/09/2012 10/05/2012 7.2 6.1 × 12.2 16 Loam 4.0 Soybean IARL 05/11/2012 10/11/2012 7.2 6.1 × 6.1 8 Silt Clay Loam 3.4 Soybean ILCX 05/11/2012 09/24/2012 7.2 6.2 × 4.6 6 Silt Clay Loam 3.1 Soybean ILMN 05/10/2012 10/01/2012 6.9 6.2 × 6.3 8 Silt Clay Loam 4.5 Soybean INSH 05/16/2012 11/01/2012 7.2 6.1 × 6.1 8 Silt Loam 1.8 Soybean KSLA 05/11/2012 09/20/2012 8.2 6.1 × 6.1 8 Silt Loam 2.6 Sorghum NCBD 05/11/2012 09/20/2012 6.6 6.1 × 15.4 16 Sandy Loam 2.6 Cotton NEDC 05/05/2012 09/10/2012 7.2 6.2 × 4.6 6 Silt Loam 2.6 Soybean NESH 05/09/2012 09/25/2012 7.2 6.2 × 6.1 8 Silt Loam 1.5 Wheat NEYO 05/09/2012 10/09/2012 7.2 6.1 × 12.2 16 Silt Loam 3.0 Soybean PAGR 05/18/2012 10/10/2012 7.2 6.1 × 6.1 8 Loam 2.4 Soybeans PAHM 05/19/2012 10/22/2012 8.2 6.1 × 12.2 16 Sandy Loam 1.6 Vegetables 4

1 Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA. 2 Planting and Harvest Date = mm/dd/yyyy. 3 % OM = Percent Organic Matter. 4 Vegetables = peppers, tomatoes, potatoes, cabbage, maize.Monsanto Company CR262-14U1 223 of 260

H.5. Phenotypic Observations The description of the characteristics measured and the designated developmental stages where observations occurred are listed in Table VII-1.

H.6. Environmental Observations
Environmental interactions ( i.e ., interactions between the crop plants and their receiving environment) were used to characterize MON 87403 by evaluating plant response to abiotic stressors, disease damage, and arthropod-related damage using qualitative methods described in Section H.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 H.8.

H.7. Plant Response to Abiotic Stress, Disease Damage, and Arthropod-Related Damage MON 87403 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 CR262-14U1 224 of 260

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.

H.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 cm 2 ). Damage (cm 2 ) per plant was calculated as the total number of grid cells matching the damage area multiplied by 0.5 (each grid cell = 0.5 cm 2 ). European corn borer damage was 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 Kansas State University, Manhattan, Kansas, for arthropod identification and enumeration. For pre-processing, four sticky traps from each sampling date at each site were non-systematically selected to prepare a list of pest and beneficial arthropods for enumeration. A maximum of twelve arthropods were enumerated for each collection. From the selected samples, up to six pest and six beneficial arthropods were counted based on their abundance and likelihood of their presence in maize. Thus, the suite of arthropods assessed often varied betweenMonsanto Company CR262-14U1 225 of 260

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.

H.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 Section H-10.

H.10. Statistical Analysis Plant Growth and Development
Plant vigor data were summarized but not subjected to an analysis of variance (ANOVA); due to the qualitative and subjective nature of the rating. MON 87403 was considered different from the conventional control in vigor if the ranges of vigor of MON 87403 did not overlap with the range of vigor of the conventional control across all replications. Any observed differences between the MON 87403 and conventional control were further assessed in the context of the range of the commercial reference materials, and for consistency at other sites.
An ANOVA was conducted according to a randomized complete block design using SAS® (SAS Software Release 9.3 (TS1M0), 2010) to compare MON 87403 and the conventional control for the phenotypic characteristics listed in Table VII-1, with the exception of plant vigor. The level of statistical significance was predetermined to be 5% ( α = 0

. 0 5 )

. C o m p a r i s o n s o f M O N 8 7 4 0 3 a n d t h e c o n v e n t i o n a l c o n t r o l w e r e c o n d u c t e d within site (individual site analysis) and in a combined-site analysis, in which the data were pooled across sites. MON 87403 and the conventional control materials were not statistically compared to the commercial reference materials. The reference range for each measured phenotypic characteristic was determined from the minimum and maximum mean values from the 21 conventional commercial reference maize hybrids planted among the sites. Data excluded from the study and the reasons for their exclusion are listed in Table H-3.Monsanto Company CR262-14U1 226 of 260

H.11. 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 87403 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 using SAS® (SAS Software Release 9.3 (TS1M0), 2010) for corn earworm damage, European corn borer damage, and arthropod abundance. The level of statistical significance was p r e d e t e r m i n e d t o b e 5 % ( α = 0

. 0 5 )

. M O N 8 7 4 0 3 w a s c o m p a r e d t o t h e c o n v e n t i o n a l control at each site (individual-site analysis) for corn earworm damage, European corn borer 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 87403 and the conventional control. Minimum and maximum mean values were calculated for corn earworm damage and European corn borer damage from 11 conventional commercial reference maize 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 conventional commercial reference maize hybrids at the site. For the arthropod abundance data, statistical analyses and significance testing of differences between MON 87403 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
a l l m a t e r i a l s ) o f ≥ 1

. Data excluded from the study and the reasons for their exclusion are listed in Table H-3.

H.12. Individual Field Site Plant Growth, Development, and Environmental Interactions Results and Discussion

H.12.1. Plant Growth Development In the individual-site analysis, a total of 14 statistically significant differences were detected out of 155 comparisons between MON 87403 and the conventional control (Table H-4). These differences were distributed among eight of the 13 phenotypic characteristics. MON 87403 had higher early stand count than the conventional control at the ILMN site (82.8 vs. 79.8 plants). MON 87403 reached days to 50% pollen shed later than the conventional control at the PAGR site (68.8 vs. 65.8 days). MON 87403 reached days to 50% silking earlier than the conventional control at the NESH site (68.8 vs. 71.5 days) but reached days to 50% silking later than the conventional control at the PAGR site (69.3 vs. 66.8 days). MON 87403 had higher stay green (more green tissue)Monsanto Company CR262-14U1 227 of 260

than the conventional control at the NESH site (9.0 vs. 8.0 rating). MON 87403 had higher final stand count than the conventional control at the NESH site (77.5 vs. 74.8 plants). MON 87403 had lower test weight than the conventional control at the NCBD site (70.2 vs. 71.9 kg/hL). MON 87403 had a lower yield than the conventional control at the ILMN site (15.5 vs. 17.0 Mg/ha), at the NCBD site (8.7 vs. 10.0 Mg/ha), and at the PAGR site (12.1 vs. 13.9 Mg/ha). The statistical differences between MON 87403 and the conventional control detected in the individual-site analyses for early stand count, days to 50% pollen shed, days to 50% silking, stay green, final stand count, test weight, and yield were not detected in the combined-site analysis. Thus, the differences detected for these phenotypic characteristics at individual sites 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 87403 compared to conventional control (Figure VII-1, step 2, “no” answer). MON 87403 had increased ear height compared to the conventional control at the ILMN site (125.3 vs. 119.9 cm), NEDC site (121.8 vs. 111.4 cm), NESH site (124.3 vs. 118.2 cm) and PAHM site (89.3 vs. 78.0 cm). While statistical differences were detected for ear height in both the individual site and thecombined-site analyses, the mean values of MON 87403 for ear height in the combinedsite analysis were within the range of values for commercial maize reference hybrids. Therefore, these differences in ear height were not indicative of a consistent plant response associated with the trait and are unlikely to be biologically meaningful in terms of increased plant pest/weed potential of MON 87403 compared to conventional control (Figure VII-1, step 3, “no” answer). In individual site assessments of plant vigor, MON 87403 and the conventional control were considered different if the range of values did not overlap between MON 87403 and the conventional control across all four replications. There were no differences observed between MON 87403 and the conventional control in plant vigor (Table H-4). Plant Response to Abiotic Stressor, Disease Damage, and Arthropod-related Damage:
In the individual-site assessment, no differences were observed between MON 87403 and the conventional control for any of the 143 comparisons for the assessed abiotic stressors, including cold, drought, flood, frost, hail, heat, nutrient deficiency, soil compaction, sunscald, and wind (Table H-5).
In the individual-site assessment, no differences were observed between MON 87403 and the conventional control for any of the 176 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, northern leaf spot, Pythium sp., Rhizoctonia sp., rust, seedling blight, smut, stalk rot, and Stewart’s bacterial wilt (Table H-6).
In the individual-site assessment, no differences were observed between MON 87403 and the conventional control for any of the 150 comparisons for the assessed arthropods, including aphids, armyworms, bean leaf beetles, billbugs, cutworms, corn earworms, corn flea beetles, corn rootworm beetles, European corn borers, grasshoppers, JapaneseMonsanto Company CR262-14U1 228 of 260

beetles, June beetles, sap beetles, slugs, spider mites, stink bugs, and wireworm beetles (Table H-7). Corn Earworm and European Corn Borer Damage: In the individual-site analysis, no statistically significant difference was detected out of 12 comparisons between MON 87403 and the conventional control for corn earworm and European corn borer among all observations at all four sites (Table H-8). Sticky Trap: A total of 144 statistical comparisons were made between MON 87403 and the conventional control for arthropod abundance involving the following arthropods: aphids, billbugs, corn flea beetles, corn rootworm beetles, delphacid planthoppers, grasshoppers, lacewings, ladybird beetles, leafhoppers, macro-parasitic hymenoptera, micro-parasitic hymenoptera, minute pirate bugs, damsel bugs, sap beetles, seedcorn beetles, spiders, syrphid flies, tachinid flies, tarnished plant bugs, thrips and click beetles (Table H-9). Lack of sufficient arthropod abundance precluded statistical comparisons between MON 87403 and the conventional control for 92 additional comparisons; however, descriptive statistics were provided for these comparisons. No statistically significant differences were detected between MON 87403 and the conventional control for 130 out of 144 comparisons. The mean abundance of lacewings was higher in MON 87403 than the conventional control in Collection 1 (2.0 vs. 0.3 per plot) at the IABG site. The mean abundance of lacewings was lower in MON 87403 than the conventional control in Collection 3 (2.5 vs. 8.0 per plot) and higher than the conventional control in Collection 5 (3.0 vs. 0.8 per plot), at the NEYO site. The mean abundance of macro-parasitic hymenoptera was lower in MON 87403 than the conventional control in Collection 4 (0.3 vs. 3.3 per plot) at the PAHM site. The mean abundance of micro-parasitic hymenoptera was higher in MON 87403 than the control in Collection 2 (21.0 vs. 8.3 per plot) at the NCBD site. The mean abundance of aphids was higher in MON 87403 than the conventional control in Collection 5 (22.5 vs. 13.8 per plot) at the NEYO site. The mean abundance of corn flea beetles was lower in MON 87403 than the conventional control in Collection 3 (0.0 vs. 1.5 per plot) at the IABG site and higher in MON 87403 than the conventional control in Collection 5 (1.3 vs. 0.3 per plot) at the IABG site. The mean abundance of corn rootworm beetles was lower in MON 87403 than the conventional control in Collection 3 (23.0 vs. 34.3 per plot) at the NEYO site. The mean abundance of thrips was higher in MON 87403 than the conventional control in Collection 2 (32.5 vs. 18.5 per plot) at the NCBD site, in Collection 2 (183.0 vs. 71.5 per plot), Collection 4 (134.3 vs. 60.3 per plot), and Collection 5 (138.0 vs. 62.3 per plot) at the NEYO site and Collection 3 (9.7 vs. 2.8 per plot) at the PAHM site. The mean abundance values for MON 87403 were within the reference ranges for all differences detected in arthropod abundance with the exception of the difference detected for lacewing abundance in Collection 5 at the NEYO site (MON 87403 mean = 3.0 per plot; reference range = 0.5 – 2.8 per plot), macro-parasitic hymenoptera abundance inMonsanto Company CR262-14U1 229 of 260

Collection 4 at the PAHM site (MON 87403 mean = 0.3 per plot; reference range = 1.5 – 2.3 per plot), corn flea beetles abundance in Collection 3 (MON 87403 mean = 0.0 per plot; reference range = 1.8 – 2.0 per plot) and Collection 5 (MON 87403 mean = 1.3 per plot; reference range = 2.0 – 3.8 per plot) at the IABG site, thrips abundance in Collection 2 (MON 87403 mean = 183.0 per plot; reference range = 66.8 – 159.0 per plot) and Collection 4 (MON 87403 mean = 134.3 per plot; reference range = 56.3 – 125.0 per plot) at the NEYO site, and in Collection 3 at the PAHM site (MON 87403 mean = 9.7 per plot; reference range = 2.3 – 4.8 per plot). However, these differences were not consistently detected across collections or sites. Thus, these differences in lacewings, macro-parasitic hymenoptera, micro-parasitic hymenoptera, aphids, corn flea beetles, corn rootworms, and thrips 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 87403 compared to conventional maize (Section VII.B.2).

Visual Counts: A total of 66 statistical comparisons were made between MON 87403 and theconventional control for arthropod abundance involving the following arthropods: antlike flower beetles, corn flea beetles, Japanese beetles, lacewing adults, lacewing larvae, ladybird beetle adults, ladybird beetle larvae, minute pirate bugs, corn rootworm beetles, sap beetles, shining flower beetles, spiders, stink bugs and click beetles (Table H-10). Lack of sufficient arthropod abundance precluded statistical comparisons between MON 87403 and the conventional control for 171 additional comparisons; however, the descriptive statistics were provided for these comparisons. No statistically significant differences were detected between MON 87403 and the conventional control for 61 out of 66 comparisons. The mean abundance of minute pirate bugs was lower in MON 87403 than the conventional control in Collection 4 (0.0 vs. 1.0 per plot) at the PAHM site. The mean abundance of corn rootworm beetles was lower in MON 87403 than the conventional control in Collection 1 (0.3 vs. 2.0 per plot) at the PAHM site. The mean abundance of sap beetles was lower in MON 87403 than the conventional control in Collection 5 (1.3 vs. 4.5 per plot) at the NCBD site and Collection 3 (1.0 vs. 4.5 per plot) at the NEYO site. The mean abundance of shining flower beetles was lower in MON 87403 than the conventional control in Collection 2 (7.0 vs. 12.3 per plot) at the NCBD site. The mean abundance values for MON 87403 were within the reference ranges for all differences detected in arthropod abundance with the exception of the difference detected for minute pirate bug abundance in Collection 4 at the PAHM site (MON 87403 mean = 0.0 per plot; reference range = 0.5 – 3.7 per plot) and corn rootworm beetle abundance in Collection 1 at the PAHM site (MON 87403 mean = 0.3 per plot; reference range = 1.0 – 2.0 per plot). However, these differences were not consistently detected across collections or sites.Monsanto Company CR262-14U1 230 of 260

Thus these differences in minute pirate bugs, corn rootworm beetles, sap beetles and shining flower beetles 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 87403 compared to conventional maize (Section VII.B.2).Monsanto Company CR262-14U1 231 of 260

Table H-3. Data Missing or Excluded from Analysis

Site Code 1 Material Name Material Type Plots Characteristics Reason for Exclusion

All All All All Data was both collected & analyzed for Anthesis Silking Interval, but not reported for combined site or individual site analysis. Not relevant to the mode of action for this trait.

IALL, ILCY, ILPH, ILRD, OHTR, MSST, ILWY All All All All phenotypic and environmental interactions data. Extreme weather conditions (strong wind or drought) that caused extensive damage.

ILCX All All 401thru 407 All phenotypic and environmental

interactions data. Drought stress

PAHM Midland Phillips 799

MON 87403 Gateway 4148 Midland Phillips 799 Reference

Test Reference Reference 106, 206,

306, 406 Arthropod data collection Planting error

PAHM NEDC IABG MON 87403

Burrus 645 Test

Reference 206, 407,

405, 401 First sub-sample of ear height and/or

plant height are missing. Electronic data collection errorMonsanto Company CR262-14U1 232 of 260

Table H

- 3 (continued) . Data Missing or Excluded from Study

Site Code 1 Material Name Material Type Plots Characteristics Reason for Exclusion

NESH Midland Phillips 799 Reference 203 Test Weight Data collection error

PAGR MON 87403 Test 204 Early Stand Count Statistical outlier

NCBD Legacy L7671 Reference 207 Moisture & Yield Data collection error

ARNE NEYO LG2540

Mycogen 2M746 Reference 301, 103, 203, 204, 304, 403 Days to 50% Pollen Fewer than 10 plants shed pollen.

PAGR INSH Stewart S588

LG2540 Reference 102, 202, 206 Phenotypic data, stalk rot and ear rot Plants were 10% below final stand count for site

IABG All All All Environmental interaction evaluation # 4 (Other) Improper selection of stressor

NEYO All All All Environmental interaction evaluation # 3 (2 arthropod, 1 abiotic and1 disease stressor) Missing data for five stressors

INSH All All All Environmental interaction evaluation # 3 (Drought) Missing data for 8 stressors

KSLA All All All Insect stressor observation 2 (velvet bean caterpillar) Improper selection of stressor Note: The study also included additional test entry not relevant to the objectives of this report, which was included in the statistical analysis input file. However, no statistical comparisons were made using this additional test entry. 1 Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA.Monsanto Company CR262-14U1 233 of 260

Table H-4. Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials Phenotypic Characteristics (units)

Plant Vigor (1-9 scale) 2 Early stand count (#/plot) Days to 50% pollen shed Range Mean (S.E.) 3 Mean (S.E.) 3 Site Code 1 MON 87403 Control MON 87403 Control MON 87403 Control

ARNE 3 – 4 3 – 4 83.5 (0.96) 83.5 (0.87) 54.5 (0.87) 53.5 (0.65) IABG 1 – 2 2 – 3 82.5 (2.87) 84.5 (0.87) 65.0 (0.00) 65.0 (0.00) IARL 3 3 – 5 83.3 (2.43) 79.0 (2.68) 61.0 (0.00) 61.3 (0.48) ILCX 1 1 82.7 (1.33) 83.0 (0.58) 60.3 (0.33) 60.7 (0.33) ILMN 1 – 2 1 – 3 82.8 (0.63)* 79.8 (0.48) 63.0 (0.00) † 63.0 (0.00) INSH 2 – 4 3 – 5 81.3 (0.48) 82.0 (1.08) 65.0 (1.08) 63.5 (0.87) KSLA 2 2 94.0 (1.22) 95.0 (1.63) 57.5 (0.96) 57.0 (0.58) NCBD 3 – 4 3 72.3 (2.50) 72.8 (1.70) 59.3 (0.25) 59.8 (0.25) NEDC 2 2 89.0 (1.35) 86.5 (0.65) 71.0 (0.00) 70.5 (0.50) NESH 2 2 88.5 (1.19) 92.8 (2.59) 68.0 (0.00) 68.0 (0.00) NEYO 1 1 78.3 (1.55) 79.3 (1.11) 68.0 (0.00) 68.0 (0.00) PAGR 3 – 4 1 – 8 73.0 (1.00) 80.0 (1.83) 68.8 (0.75)* 65.8 (0.75) PAHM 2 – 4 2 – 4 86.0 (2.20) 88.5 (0.87) 63.0 (0.71) 63.0 (0.41)Monsanto Company CR262-14U1 234 of 260

Table H-4 (continued). Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials Phenotypic Characteristics (units) Days to 50% silking Stay-green rating (1-9 scale) Ear height (cm) Mean (S.E.) 3 Mean (S.E.) 3 Mean (S.E.) 3 Site Code 1 MON 87403 Control MON 87403 Control MON 87403 Control

ARNE 55.3 (1.18) 54.0 (0.58) 4.3 (0.25) 4.5 (0.50) 136.1 (3.99) 133.1 (3.66) IABG 63.8 (0.63) 63.8 (0.25) 8.0 (0.41) 7.8 (0.75) 98.7 (1.66) 104.6 (2.52) IARL 60.5 (0.50) 60.5 (0.50) 3.5 (0.29) 4.0 (0.41) 96.4 (4.00) 91.7 (4.85) ILCX 64.0 (1.00) 66.0 (0.00) 3.3 (1.45) 3.3 (0.67) 108.6 (0.92) 105.1 (3.64) ILMN 63.0 (0.00) † 63.0 (0.00) 4.3 (0.25) 4.3 (0.25) 125.3 (1.28)* 119.9 (1.26) INSH 66.0 (1.35) 64.5 (1.19) 4.0 (0.00) 4.0 (0.00) 107.2 (6.34) 108.9 (3.69) KSLA 58.5 (0.50) 59.0 (0.58) 1.0 (0.00) 1.0 (0.00) 100.5 (3.36) 95.8 (3.40) NCBD 59.3 (0.25) 59.5 (0.29) 6.3 (0.25) 6.3 (0.25) 127.1 (5.15) 128.7 (3.14) NEDC 70.0 (0.58) 71.0 (0.00) 7.8 (0.48) 8.0 (0.41) 121.8 (3.99)* 111.4 (2.40) NESH 68.8 (0.75)* 71.5 (1.66) 9.0 (0.00)* 8.0 (0.00) 124.3 (1.17)* 118.2 (1.74) NEYO 68.0 (0.00) 68.3 (0.25) 6.0 (0.00) 5.5 (0.29) 113.2 (3.21) 103.6 (1.90) PAGR 69.3 (0.75)* 66.8 (0.75) 2.0 (0.41) 1.5 (0.29) 95.9 (6.04) 98.8 (7.60) PAHM 64.0 (0.71) 64.3 (0.63) 8.5 (0.29) 8.5 (0.29) 89.3 (5.39)* 78.0 (1.95)Monsanto Company CR262-14U1 235 of 260

Table H-4 (continued). Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials Phenotypic Characteristics (units) Plant height (cm) Dropped ears (#/plot) Stalk lodged plants (#/plot) Mean (S.E.) 3 Mean (S.E.) 3 Mean (S.E.) 3 Site Code 1 MON 87403 Control MON 87403 Control MON 87403 Control

ARNE 265.0 (2.39) 268.6 (0.83) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) IABG 257.1 (3.96) 257.3 (3.51) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) IARL 213.5 (4.32) 205.7 (3.93) 0.3 (0.25) 0.0 (0.00) 1.8 (1.75) 0.0 (0.00) ILCX 209.5 (9.34) 205.3 (5.74) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) ILMN 265.4 (2.74) 269.5 (3.41) 0.0 (0.00) 0.0 (0.00) 0.5 (0.29) 1.0 (0.58) INSH 223.4 (8.86) 230.2 (5.59) 3.8 (0.63) 2.3 (0.48) 10.3 (3.64) 11.3 (2.02) KSLA 213.1 (8.54) 212.8 (4.04) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) NCBD 264.7 (6.83) 261.1 (3.03) 8.5 (4.33) 7.3 (3.20) 0.5 (0.29) 0.3 (0.25) NEDC 257.8 (4.28) 251.8 (2.14) 0.0 (0.00) 0.5 (0.29) 3.5 (1.55) 3.8 (1.31) NESH 258.8 (2.49) 249.2 (4.03) 0.0 (0.00) † 0.0 (0.00) 0.3 (0.25) 0.8 (0.25) NEYO 265.3 (2.60) 260.1 (0.87) 0.0 (0.00) † 0.0 (0.00) 0.3 (0.25) 0.3 (0.25) PAGR 214.4 (6.66) 224.4 (4.12) 2.0 (0.00) 1.0 (0.41) 7.5 (1.85) 6.3 (2.39) PAHM 210.8 (7.96) 209.3 (5.87) 1.0 (0.71) 1.0 (0.41) 6.0 (1.78) 4.8 (1.03)Monsanto Company CR262-14U1 236 of 260

Table H-4 (continued). Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials Phenotypic Characteristics (units)

Root lodged plants (#/plot) Final stand count (#/plot) Grain moisture (%) Mean (S.E.) 3 Mean (S.E.) 3 Mean (S.E.) 3
Site Code 1 MON 87403 Control MON 87403 Control MON 87403 Control

ARNE 0.3 (0.25) 0.0 (0.00) 75.3 (0.25) 75.0 (0.00) 16.1 (0.32) 16.1 (0.14) IABG 0.8 (0.75) 1.3 (0.75) 72.3 (1.55) 72.3 (1.11) 16.6 (0.10) 17.5 (0.66) IARL 4.3 (1.89) 1.8 (1.03) 74.8 (0.75) 73.5 (0.96) 17.4 (0.13) 17.7 (0.17) ILCX 0.3 (0.33) 0.3 (0.33) 76.0 (0.00) † 76.0 (0.00) 25.9 (0.58) 26.4 (1.22) ILMN 2.3 (1.93) 1.8 (1.44) 75.3 (0.25) 75.5 (0.29) 20.9 (1.55) 19.1 (1.18) INSH 0.8 (0.48) 1.0 (0.71) 71.5 (1.04) 70.8 (1.89) 19.3 (0.27) 19.5 (0.48) KSLA 0.0 (0.00) † 0.0 (0.00) 76.8 (1.60) 76.3 (0.25) 16.4 (0.34) 16.0 (0.32) NCBD 0.0 (0.00) † 0.0 (0.00) 68.8 (1.60) 68.8 (1.89) 19.5 (0.25) 19.6 (0.21) NEDC 0.0 (0.00) † 0.0 (0.00) 75.8 (0.63) 73.8 (1.31) 15.5 (0.27) 15.4 (0.55) NESH 0.0 (0.00) 0.8 (0.48) 77.5 (1.26)* 74.8 (1.44) 16.7 (0.60) 16.4 (0.27) NEYO 0.0 (0.00) † 0.0 (0.00) 61.5 (0.65) 60.0 (1.41) 15.7 (0.13) 15.7 (0.14) PAGR 0.0 (0.00) † 0.0 (0.00) 63.8 (0.63) 65.8 (0.75) 30.6 (0.46) 29.4 (1.09) PAHM 0.0 (0.00) † 0.0 (0.00) 73.8 (0.25) 74.5 (0.65) 19.9 (0.52) 20.2 (0.46)Monsanto Company CR262-14U1 237 of 260

Table H-4 (continued). Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials Phenotypic Characteristics (units) Test weight (kg/hl) Yield (Mg/ha) Mean (S.E.) 3 Mean (S.E.) 3
Site Code 1 MON 87403 Control MON 87403 Control

ARNE 74.6 (0.69) 74.5 (0.27) 10.2 (0.17) 10.7 (0.17) IABG 75.8 (0.36) 77.4 (0.46) 7.5 (1.35) 8.8 (1.19) IARL 75.4 (0.95) 75.7 (0.84) 7.6 (0.51) 6.4 (0.75) ILCX 72.7 (0.66) 71.8 (2.82) 7.9 (1.38) 4.9 (1.41) ILMN 70.1 (0.35) 71.4 (0.61) 15.5 (0.44)* 17.0 (0.44) INSH 71.1 (0.34) 70.6 (0.86) 10.5 (0.88) 11.1 (0.53) KSLA 78.4 (0.33) 77.8 (0.57) 14.0 (0.41) 13.5 (0.46) NCBD 70.2 (0.64)* 71.9 (0.73) 8.7 (0.64)* 10.0 (0.54) NEDC 74.8 (0.53) 74.7 (0.37) 7.0 (0.12) 7.7 (0.11) NESH 76.4 (0.38) 75.3 (0.13) 5.0 (0.16) 4.6 (0.56) NEYO 76.0 (0.40) 75.8 (0.27) 13.3 (0.47) 12.9 (0.40) PAGR 59.3 (0.38) 60.3 (0.98) 12.1 (0.73)* 13.9 (0.86) PAHM 68.6 (0.50) 68.2 (0.26) 11.7 (0.44) 11.0 (0.34)
Note: The experimental design was a randomized complete block with four replicates per site.
* I n d i c a t e s s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e b e t w e e n M O N 8 7 4 0 3 a n d t h e c o n v e n t i o n a l c o n t r o l ( α = 0

. 0 5 ) u s i n g A N O V A

. † Indicates p-values could not be generated due to lack of variability in the data. 1 Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE; PAGR = Lehigh County, PA; 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: ARNE 1– 4; IABG 1 – 3; IARL 2 – 4; ILCX 1; ILMN 1 – 3; INSH 3 – 4; KSLA 2; NCBD 3 – 4; NEDC 2; NESH 2; NEYO 1; PAGR 1 – 8; PAHM 2 – 5 3 MON 87403 and the conventional control values represent means with standard error in parentheses. N = 4, except where noted in Table H-5.Monsanto Company CR262-14U1 238 of 260

Table H-5. Abiotic Stressor Evaluations Using a Categorical Scale for MON 87403 and the Conventional Control in 2012 U.S. Field Trials

Abiotic Stressor Number of Observations across Sites 1 Number of Observations where No

Differences were Observed between MON 87403 and the Conventional

Control Total 143 143

Cold 2 2 Drought 2 36 36 Flood 3 7 7 Frost 2 2 Hail 9 9 Heat 27 27 Nutrient deficiency 24 24 Soil compaction 4 4 Sunscald 4 4 Wind 28 28

Note: The experimental design was a randomized complete block with four replicates per site. No differences were observed between MON 87403 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: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA. 2 Includes dryness, dry/heat. 3 Includes wet soil.Monsanto Company CR262-14U1 239 of 260

Table H-6. Disease Damage Evaluations Using a Categorical Scale for MON 87403 and the Conventional Control in 2012 U.S. Field Trials

Disease Number of Observations across Sites 1 Number of Observations where No Differences were Observed between MON 87403 and the Conventional

Control Total 176 176

Anthracnose 8 8 Bacterial leaf spot 2 2 Ear rot 2 19 19 Eyespot 6 6 Fusarium sp. 8 8 Goss’s bacterial wilt 11 11 Gray leaf spot 23 23 Leaf blight 3 24 24 Maize rough dwarf virus 3 3 Northern leaf spot 7 7 Pythium sp. 2 2 Rhizoctonia sp. 2 2 Rust 4 12 12 Seedling blight 2 2 Smut 15 15 Stalk rot 5 17 17 Stewart’s bacterial wilt 15 15

Note: The experimental design was a randomized complete block with four replicates per site. No differences were observed between MON 87403 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: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA. 2 Assessed on 5 non-systematically selected plants. At ARNE, ILCX, ILMN, INSH, NEDC, and NESH ear 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 ILCX, NEDC, NESH, and PAGR stalk rot data were collected both on a per plot basis and on 5 plant/plots.Monsanto Company CR262-14U1 240 of 260

Table H-7. Arthropod Damage Evaluations Using a Categorical Scale for MON 87403 and the Conventional Control in 2012 U.S. Field Trials

Arthropod Number of Observations across Sites 1 Number of Observations

where No Differences were Observed between MON 87403 and the Conventional Control Total 150 150

Aphids (Aphididae) 5 5 Armyworms (Noctuidae) 2 24 24 Bean leaf beetles ( Cerotoma trifurcata ) 2 2 Billbugs ( Sphenophorus parvulus ) 2 2 Cutworms (Noctuidae) 3 10 10 Corn earworms ( Helicoverpa zea ) 13 13 Corn flea beetles ( Chaetocnema pulicaria ) 4 7 7 Corn rootworm beetles ( Diabrotica sp. ) 14 14 European corn borers ( Ostrinia nubilalis ) 17 17 Grasshoppers ( Melanoplus spp.) 20 20 Japanese beetles ( Popillia japonica ) 7 7 June beetles (Scarabaeidae) 2 2 Sap beetles (Nitidulidae) 6 6 Slugs 5 (Gastropoda) 1 1 Southwestern corn borer ( Diatraea grandiosella ) 2 2 Spider mites ( Tetranychus sp.) 5 5 Stink bugs (Pentatomidae) 11 11 Western bean cutworm ( Richia albicosta ) 1 1 Click beetles (Elateridae) 6 1 1

Note: The experimental design was a randomized complete block with four replicates per site. No differences were observed between MON 87403 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: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA. 2 Includes beet armyworm and fall armyworm 3 Includes black cutworm
4 Includes flea beetle 5 Slugs are not arthropods but are one of the common stressor in Maize.
6 Wireworm adultsMonsanto Company CR262-14U1 241 of 260

Table H-8. Individual Site Analysis: Corn Earworm Damage and European Corn Borer Damage to MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Pest Damage assessment Site 1 Mean (S.E.) 2

MON 87403 Control
Corn earworm ( H

. zea ) 3 Damage area of 10 plants per plot (cm 2 ) IABG 0.6 (0.12) 1.2 (0.25)
NCBD 3.4 (0.85) 6.4 (2.01)
NEYO 2.9 (0.38) 3.3 (0.44)

PAHM 0.4 (0.19) 0.1 (0.06)
European corn borer ( O

. nubilalis ) 4 Number of stalk galleries of 10 plants per plot IABG 0.0 (0.00) 0.0 (0.03)
NCBD 0.1 (0.08) 0.2 (0.09)

NEYO 0.0 (0.03) 0.1 (0.03)

PAHM 1.0 (0.27) 1.0 (0.25)

European corn borer ( O

. nubilalis ) 4 Stalk gallery length (cm) of 10 plants per plot IABG 0.0 (0.00) 0.1 (0.08)
NCBD 0.6 (0.39) 1.3 (0.64)
NEYO 0.1 (0.10) 0.2 (0.10)

PAHM 4.6 (1.27) 4.0 (1.04)

Note: The experimental design was a randomized complete block with four replicates per site. 1 Site code: IABG = Greene, IA; NCBD = Perquimans, NC; NEYO = York, NE; PAHM = Berks, PA. 2 MON 87403 and the conventional control values represent means with standard error in parentheses. 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 CR262-14U1 242 of 260

Table H-9. Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Spiders (Araneae) Ladybird beetles (Coccinellidae) Lacewings (Chrysopidae) Beneficial Arthropod Beneficial Arthropod Beneficial Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG

— — — 0.0 (0.00)† 0.3 (0.25) 0.0 – 3.0 2.0 (0.41)* 0.3 (0.25) 1.0 – 3.3 NCBD 0.3 (0.25)† 1.0 (1.00) 0.5 – 1.0 1.0 (0.41) 1.5 (0.87) 1.0 – 2.0

— — — NEYO 0.0 (0.00)† 0.3 (0.25) 0.3 – 0.5 0.3 (0.25)† 0.0 (0.00) 0.3 – 2.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.0 PAHM 0.0 (0.00)† 1.3 (0.48) 0.0 – 0.7 12.3 (2.40) 14.5 (0.87) 11.3 – 25.5

— — — 2 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5 0.0 (0.00)† 0.3 (0.25) 0.3 – 1.5 1.8 (0.48) 2.3 (1.11) 2.5 – 4.0 NCBD 0.3 (0.25)† 0.5 (0.29) 0.3 – 1.3 5.0 (1.08) 5.3 (1.65) 5.5 – 6.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 NEYO 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.5 0.3 (0.25)† 0.0 (0.00) 0.3 – 1.0 0.8 (0.75)† 0.5 (0.29) 0.0 – 1.0 PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 7.3 (4.06) 4.3 (1.84) 2.5 – 11.5

— — — 3 IABG 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3

— — — 0.0 (0.00)† 0.3 (0.25) 0.0 – 1.0 NCBD 0.5 (0.29)† 0.5 (0.29) 0.0 – 0.8 4.5 (0.96) 3.0 (0.71) 2.8 – 4.5 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 NEYO 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 2.5 (1.04)* 8.0 (1.73) 1.0 – 3.3 PAHM 2.0 (0.58)† 0.3 (0.25) 0.0 – 0.5 1.7 (0.67) 2.8 (1.38) 2.5 – 4.3

— — — 4 IABG

— — — 0.0 (0.00)† 0.3 (0.25) 0.3 – 1.5 2.3 (0.85) 0.5 (0.29) 3.5 – 6.3 NCBD 0.5 (0.29)† 0.5 (0.29) 0.0 – 0.8 6.0 (1.47) 6.0 (0.82) 3.0 – 7.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 NEYO

— — — — — — 1.5 (0.65) 3.3 (1.97) 0.5 – 2.8 PAHM 1.3 (0.88)† 0.5 (0.29) 0.0 – 0.8 2.3 (1.20) 0.8 (0.25) 0.3 – 2.7

— — — 5 IABG

— — — 0.3 (0.25) 0.5 (0.50) 0.3 – 2.8 5.3 (2.29) 6.5 (2.53) 5.5 – 9.3 NCBD 1.0 (0.41)† 1.0 (0.41) 0.3 – 1.8 2.0 (1.41) 1.5 (0.65) 0.5 – 3.5

— — — NEYO 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.5

— — — 3.0 (0.41)* 0.8 (0.48) 0.5 – 2.8 PAHM

— — — 0.0 (0.00)† 0.5(0.50) 0.5 – 2.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3Monsanto Company CR262-14U1 243 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Macro-parasitic hymenoptera Micro-parasitic hymenoptera Damsel bugs 5 (Nabidae) Beneficial Arthropod Beneficial Arthropod Beneficial Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG 6.5 (1.19) 4.5 (1.04) 6.0 – 9.0 15.0 (2.12) 12.0 (3.58) 13.3 – 18.0

— — — NCBD 0.5 (0.50)† 0.0 (0.00) 0.0 – 0.3 33.3 (1.93) 44.5 (1.32) 50.8 – 61.8 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.8 NEYO 2.0 (0.91) 0.8 (0.25) 1.5 – 1.8 15.3 (2.14) 13.3 (2.06) 14.0 – 19.8

— — — PAHM 0.7 (0.33)† 1.0 (0.41) 0.0 – 1.8 24.7 (2.33) 27.0 (4.80) 22.0 – 37.5

— — — 2 IABG 7.3 (3.20) 4.8 (0.85) 5.3 – 11.0 20.8 (1.44) 17.8 (4.11) 15.5 – 20.8

— — — NCBD 0.8 (0.48)† 0.3 (0.25) 0.0 – 1.5 21.0 (6.47)* 8.3 (1.80) 14.3 – 25.8

— — — NEYO 0.3 (0.25) 1.0 (0.58) 0.5 – 1.8 15.5 (2.60) 12.3 (2.50) 15.5 – 24.0

— — — PAHM 1.0 (1.00) 1.3 (0.95) 1.0 – 2.3 19.3 (1.67) 25.5 (6.64) 16.0 – 29.8

— — — 3 IABG 3.8 (0.75) 3.3 (1.11) 3.3 – 5.3 11.3 (2.10) 10.5 (5.12) 12.8 – 15.5

— — — NCBD

— — — 18.8 (2.95) 19.8 (2.78) 13.5 – 26.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 NEYO 2.3 (1.11) 2.3 (0.75) 1.8 – 3.0 15.5 (1.85) 13.5 (1.19) 12.5 – 25.3

— — — PAHM 1.3 (0.33) 2.0 (0.41) 0.3 – 2.8 25.3 (5.49) 23.5 (6.22) 21.0 – 29.3

— — — 4 IABG 6.0 (0.91) 5.3 (0.95) 3.0 – 6.5 19.5 (1.50) 15.0 (7.75) 10.5 – 21.5

— — — NCBD

— — — 17.8 (1.49) 17.0 (0.91) 9.0 – 22.8

— — — NEYO 1.8 (0.75) 3.8 (1.65) 2.0 – 2.8 12.3 (3.12) 12.5 (4.21) 10.5 – 12.0

— — — PAHM 0.3 (0.33)* 3.3 (1.03) 1.5 – 2.3 51.0 (12.74) 32.0 (6.47) 27.0 – 47.5

— — — 5 IABG 2.0 (0.71) 2.5 (0.50) 1.5 – 2.3 5.8 (1.25) 9.0 (3.51) 7.5 – 10.5

— — — NCBD 0.5 (0.29)† 0.3 (0.25) 0.0 – 0.5 14.5 (2.33) 13.5 (3.23) 10.0 – 15.5

— — — NEYO 0.8 (0.48) 1.0 (0.41) 1.5 – 2.0 12.8 (4.70) 7.5 (1.26) 7.5 – 14.0

— — — PAHM 2.0 (0.58) 3.5 (0.50) 3.5 – 5.0 37.0 (10.97) 29.3 (5.28) 32.5 – 41.0

— — —Monsanto Company CR262-14U1 244 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Minute pirate bugs (Anthocoridae) Syrphid flies (Syrphidae) Aphids (Aphididae) Beneficial Arthropod Beneficial Arthropod Pest Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG 0.8 (0.48)† 1.0 (0.41) 0.3 – 1.0

— — — 0.0 (0.00)† 0.0 (0.00) 0.3 – 1.5 NCBD

— — — 1.3 (0.75) 1.5 (0.50) 1.3 – 4.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 NEYO

— — — 0.8 (0.48)† 0.8 (0.48) 0.3 – 1.0

— — — PAHM 0.7 (0.67)† 0.5 (0.29) 0.0 – 2.3 4.0 (1.15) 6.8 (1.84) 3.5 – 6.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.0 2 IABG 1.0 (0.58) 2.0 (1.08) 1.3 – 3.5

— — — — — — NCBD

— — — 6.0 (1.00) 4.3 (1.31) 3.5 – 6.5

— — — NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

— — — — — — PAHM 1.0 (0.58) 1.3 (0.48) 1.0 – 1.5 5.3 (2.33) 6.0 (2.74) 4.0 – 10.3

— — — 3 IABG 0.8 (0.25) 1.0 (0.41) 0.8 – 1.3 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.5 (0.29)† 0.3 (0.25) 0.3 – 1.0 NCBD

— — — 11.5 (2.40) 7.5 (2.40) 9.3 – 16.3

— — — NEYO

— — — 0.5 (0.29) 0.5 (0.50) 0.8 – 2.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.5 PAHM 2.0 (0.58) 2.0 (0.71) 1.3 – 4.0 2.3 (1.86) 1.8 (0.48) 1.8 – 4.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 4 IABG 0.5 (0.29) 0.8 (0.25) 0.8 – 3.3

— — — 1.0 (0.71) 1.3 (0.95) 1.0 – 2.8 NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 22.3 (5.95) 14.8 (2.78) 13.3 – 22.8

— — — NEYO

— — — 1.8 (0.75) 0.8 (0.75) 1.5 – 2.8 1.3 (0.25) 1.0 (0.71) 1.0 – 2.3 PAHM 0.3 (0.33)† 0.3 (0.25) 0.0 – 1.0 9.3 (2.96) 8.3 (1.38) 8.0 – 14.5

— — — 5 IABG 1.5 (0.65) 0.8 (0.48) 0.8 – 4.0 1.5 (0.96)† 0.5 (0.50) 0.5 – 1.5 1.3 (0.48) 3.5 (1.26) 2.0 – 2.5 NCBD 0.8 (0.25)† 0.3 (0.25) 0.3 – 0.8 72.5 (17.50) 49.0 (6.77) 62.5 – 102.8 0.5 (0.50)† 0.5 (0.29) 0.5 – 1.5 NEYO

— — — 0.8 (0.48) 0.8 (0.25) 1.3 – 2.0 22.5 (3.28)* 13.8 (3.07) 14.5 – 32.0 PAHM 0.3 (0.33)† 0.8 (0.48) 0.0 – 1.3 6.7 (1.33) 4.0 (1.41) 5.0 – 12.0

— — —Monsanto Company CR262-14U1 245 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Billbugs (Curculionidae) Corn flea beetles ( Chaetocnema sp.) Corn rootworm beetles ( Diabrotica sp.) Pest Arthropod Pest Arthropod Pest Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG

— — — — — — 2.3 (0.63) 1.5 (0.65) 0.8 – 1.8 NCBD

— — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

— — — NEYO

— — — 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.8 1.0 (0.41) 0.8 (0.48) 1.3 – 2.8 PAHM

— — — 6.0 (1.73) 6.0 (2.16) 5.3 – 13.8 0.7 (0.33) 1.3 (1.25) 0.7 – 3.5 2 IABG

— — — 1.5 (0.29) 2.8 (1.03) 2.5 – 5.0 2.8 (0.75) 1.8 (0.75) 0.5 – 2.8 NCBD 0.5 (0.50)† 0.5 (0.29) 0.5 – 1.3

— — — 0.3 (0.25)† 0.0 (0.00) 0.0 - 0.8 NEYO

— — — 0.5 (0.29)† 0.8 (0.48) 0.5 – 1.0 9.3 (0.63) 9.8 (3.07) 7.8 – 13.8 PAHM

— — — 7.7 (1.45) 4.3 (1.25) 5.0 – 19.3 1.0 (0.58)† 0.5 (0.29) 0.3 – 1.0 3 IABG

— — — 0.0 (0.00)* 1.5 (0.65) 1.8 – 2.0 0.8 (0.25) 0.5 (0.50) 1.0 – 2.0 NCBD

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 NEYO

— — — — — — 23.0 (3.00)* 34.3 (3.75) 15.0 – 27.8 PAHM

— — — 17.7 (1.76) 16.5 (2.72) 10.5 – 24.5 1.0 (0.00)† 1.0 (0.41) 0.0 – 2.0 4 IABG

— — — 1.0 (0.71) 1.0 (0.71) 0.8 – 3.8 1.3 (0.63) 0.3 (0.25) 0.3 – 2.0 NCBD

— — — 7.0 (2.27) 1.8 (0.48) 6.8 – 15.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 NEYO

— — — — — — 5.3 (1.03) 9.0 (2.48) 2.3 – 7.5 PAHM

— — — 16.3 (2.19) 7.3 (1.93) 6.0 – 34.3 0.3 (0.33)† 1.0 (0.58) 0.0 – 1.0 5 IABG

— — — 1.3 (0.25)* 0.3 (0.25) 2.0 – 3.8 0.5 (0.29)† 0.3 (0.25) 0.3 – 1.3 NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 18.3 (2.84) 11.3 (3.12) 21.3 – 29.3

— — — NEYO

— — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.3 – 0.5 PAHM

— — — 6.3 (2.73) 4.3 (0.63) 4.0 – 11.0

— — —Monsanto Company CR262-14U1 246 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Delphacid planthoppers (Delphacidae) Grasshoppers (Orthoptera) Leafhoppers (Cicadellidae)

Pest Arthropod Pest Arthropod Pest Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG

— — — — — — 8.3 (0.63) 7.5 (1.89) 8.5 – 14.0 NCBD 24.0 (3.49) 28.8 (6.70) 27.3 – 41.0

— — — 43.5 (5.81) 50.0 (10.06) 43.0 – 70.5
NEYO

— — — — — — 1.8 (0.48) 0.8 (0.75) 0.8 – 1.5 PAHM

— — — — — — 16.3 (1.20) 12.8 (3.33) 14.0 – 33.5 2 IABG

— — — — — — 8.8 (2.10) 10.3 (1.11) 7.5 – 11.3 NCBD

— — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 30.3 (4.82) 24.3 (2.46) 21.8 – 53.3 NEYO

— — — — — — 1.0 (0.41)† 1.0 (0.41) 0.3 – 1.0 PAHM

— — — 0.3 (0.33)† 0.3 (0.25) 0.0 – 0.5 10.3 (3.71) 11.8 (2.02) 9.0 – 26.8 3 IABG

— — — — — — 2.3 (0.63) 4.5 (0.50) 2.3 – 3.8 NCBD 2.8 (1.38) 1.3 (0.63) 0.5 – 2.3

— — — 46.5 (6.28) 37.3 (2.72) 35.8 – 59.3 NEYO

— — — — — — 1.0 (0.71)† 1.3 (0.63) 0.0 – 1.5 PAHM

— — — 0.3 (0.33)† 1.0 (0.41) 0.0 – 0.8 17.0 (4.58) 15.8 (4.39) 7.0 – 26.0 4 IABG

— — — — — — 3.8 (0.75) 6.5 (1.04) 2.8 – 6.8 NCBD 1.3 (0.25)† 0.8 (0.48) 0.5 – 1.3

— — — 24.0 (2.12) 24.0 (2.58) 21.3 – 37.0 NEYO

— — — — — — 1.3 (0.95)† 1.0 (0.41) 0.5 – 1.5 PAHM

— — — 0.7 (0.67)† 0.0 (0.00) 0.0 – 1.0 12.3 (1.45) 13.5 (4.33) 6.5 – 26.5 5 IABG

— — — — — — 4.5 (1.19) 2.5 (0.65) 2.0 – 6.0 NCBD

— — — — — — 16.3 (2.66) 21.0 (3.11) 15.0 – 22.3 NEYO

— — — — — — 0.8 (0.48) 0.5 (0.29) 0.0 – 2.3 PAHM

— — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5 10.7 (2.85) 9.3 (2.56) 6.5 – 24.0Monsanto Company CR262-14U1 247 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Sap beetles ( Nitidulidae) Seedcorn beetles ( Stenolophus sp.) Tachinid flies (Tachinidae) Pest Arthropod Pest Arthropod Pest Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG 0.0 (0.00)† 0.8 (0.48) 0.8 – 1.5

— — — — — — NCBD

— — — — — — — — — NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

— — — — — — PAHM

— — — — — — — — — 2 IABG 2.0 (0.71)† 0.5 (0.50) 0.0 – 1.0

— — — — — — NCBD

— — — — — — — — — NEYO 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.8

— — — 0.5 (0.50)† 0.0 (0.00) 0.0 – 0.3 PAHM

— — — — — — — — — 3 IABG 1.0 (0.58)† 0.5 (0.29) 0.0 – 1.3

— — — — — — NCBD

— — — 0.3 (0.25)† 0.5 (0.50) 0.0 – 0.3

— — — NEYO 1.0 (0.71) 0.5 (0.29) 0.5 – 1.5

— — — — — — PAHM

— — — — — — — — — 4 IABG 2.0 (1.68) 1.5 (0.87) 0.8 – 2.3

— — — — — — NCBD

— — — — — — — — — NEYO 0.8 (0.48) 1.0 (0.00) 1.0 – 3.0

— — — — — — PAHM

— — — — — — — — — 5 IABG 0.5 (0.50) 0.3 (0.25) 1.0 – 1.5

— — — — — — NCBD

— — — — — — — — — NEYO 1.3 (0.75) 2.5 (1.04) 0.3 – 2.0

— — — — — — PAHM

— — — — — — — — —Monsanto Company CR262-14U1 248 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Thrips (Thripidae) Tarnished plant bugs ( Lygus sp.) Click beetle 5 (Elateridae) Pest Arthropod Pest Arthropod Pest Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG 31.5 (11.59) 41.5 (7.79) 26.8 – 50.8 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.5

— — — NCBD 50.5 (10.90) 40.5 (5.01) 37.5 – 76.3

— — — 2.0 (0.71) 3.8 (1.11) 2.0 – 5.0 NEYO 4.8 (2.25) 1.0 (0.58) 4.0 – 8.0 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.0

— — — PAHM 12.0 (5.77) 8.8 (5.01) 9.5 – 13.5 1.7 (0.88) 0.0 (0.00) 0.8 – 2.5

— — — 2 IABG 86.3 (23.97) 79.3 (27.29) 40.3 – 95.5 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3

— — — NCBD 32.5 (5.85)* 18.5 (4.03) 20.8 – 34.5

— — — 3.0 (0.41) 2.8 (1.31) 3.0 – 7.5 NEYO 183.0 (54.68)* 71.5 (13.76) 66.8 – 159.0

— — — — — — PAHM 1.3 (0.88) 2.5 (1.04) 1.8 – 6.7 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.5

— — — 3 IABG 38.5 (4.50) 39.0 (18.18) 24.0 – 130.0

— — — — — — NCBD 39.3 (6.30) 29.8 (6.17) 46.0 – 59.3

— — — 2.0 (0.71) 0.8 (0.25) 2.0 – 3.0 NEYO 319.3 (102.23) 207.3 (58.63) 171.8 –

511.0 — — — — — — PAHM 9.7 (4.26)* 2.8 (2.14) 2.3 – 4.8

— — — — — — 4 IABG 95.5 (6.96) 139.3 (82.00) 44.8 – 167.8

— — — — — — NCBD 33.8 (5.27) 26.5 (2.90) 27.8 – 85.8

— — — 0.8 (0.25) 0.8 (0.48) 0.8 – 3.3 NEYO 134.3 (36.50)* 60.3 (14.02) 56.3 – 125.0

— — — — — — PAHM 11.0 (5.29) 7.5 (3.30) 6.0 – 12.3

— — — 0.0 (0.00)† 0.8 (0.48) 0.0 – 0.5 5 IABG 30.0 (14.57) 15.3 (2.87) 17.8 – 41.5

— — — — — — NCBD 33.3 (5.50) 44.3 (15.58) 18.5 – 42.8

— — — 1.3 (0.25) 0.8 (0.25) 1.5 – 2.3 NEYO 138.0 (10.12)* 62.3 (15.52) 102.3 – 173.8 — — — — — — PAHM 1.7 (0.88) 1.3 (0.48) 0.7 – 3.5 0.7 (0.67)† 0.0 (0.00) 0.0 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5Monsanto Company CR262-14U1 249 of 260

Note: The experimental design was a randomized complete block with four replicates per site.

* I n d i c a t e s a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e b e t w e e n M O N 8 7 4 0 3 a n d t h e c o n v e n t i o n a l c o n t r o l ( α = 0

. 0 5 ) u s i n g A N O V A

. † Indicates p-values could not be generated where the taxa did not meet inclusion criteria. Among taxa that did not meet inclusion criteria, centipedes, millipedes, and corn earworms data are not presented due to low probability of capture on sticky traps. (—)A dash indicates data not available. 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 87403 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 materials at each site. 5 Click beetle = wireworm adults; Damsel bug = Nabis bugMonsanto Company CR262-14U1 250 of 260

Table H-10. Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Ant-like flower beetles (Anthicidae) Click beetles (Elateridae) Corn flea beetles ( Chaetocnema sp.)

Pollen Feeder Pest Arthropod Pest Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 NCBD

— — — 1.0 (0.71) 2.3 (1.31) 2.0 – 4.3 0.5 (0.29)† 0.0 (0.00) 0.3 – 2.0 NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 PAHM

— — — — — — 8.7 (3.53) 6.5 (2.40) 3.0 – 8.5 2 IABG 1.0 (0.41) 1.8 (0.75) 1.3 – 5.0

— — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 NCBD

— — — 1.5 (0.87) 0.5 (0.29) 0.5 – 2.0 0.3 (0.25)† 0.3 (0.25) 0.0 – 1.0 NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 PAHM

— — — — — — 9.3 (2.73) 9.3 (3.64) 6.5 – 9.5 3 IABG 2.0 (1.00) 2.3 (1.03) 1.8 – 4.3

— — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 NCBD

— — — 0.8 (0.25)† 0.3 (0.25) 0.3 – 0.5 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

— — — — — — 6.7 (4.06) 6.3 (1.65) 8.8 – 11.0 4 IABG 3.8 (0.85) 3.5 (0.87) 3.5 – 4.5

— — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 NCBD

— — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 0.5 (0.29)† 0.0 (0.00) 0.0 – 1.0 NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 PAHM

— — — — — — 3.3 (0.88) 3.3 (1.11) 4.0 – 8.3 5 IABG 1.0 (0.71)† 0.8 (0.48) 0.5 – 1.3

— — — 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 1.0 (0.71) 0.5 (0.29) 0.5 – 3.0 NEYO

— — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 PAHM

— — — — — — 3.3 (1.33) 3.8 (2.25) 3.7 – 4.8Monsanto Company CR262-14U1 251 of 260

Table H-10 (continued). Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Japanese beetles (Scarabaiedae) Lacewing adults (Chrysopidae) Lacewing larvae (Chrysopidae) Pest Arthropod Beneficial Arthropod Beneficial Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 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 0.5 (0.29)† 0.5 (0.29) 0.3 – 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 – 0.3 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 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 (0.25) 0.0 – 0.5 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 2 IABG 0.8 (0.75)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.3 (0.25) 0.0 – 1.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 1.0 (0.41) 0.5 – 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 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 0.3 (0.33)† 0.3 (0.25) 0.0 – 0.5 0.3 (0.33)† 0.0 (0.00) 0.0 – 0.7 3 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 NCBD 0.5 (0.50)† 0.0 (0.00) 0.0 – 0.5 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3 0.5 (0.50)† 0.5 (0.29) 0.0 – 0.3 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 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 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.5 (0.50) 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 – 0.0 0.5 (0.29)† 0.3 (0.25) 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 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.0 0.3 (0.33)† 0.3 (0.25) 0.0 – 0.3 5 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.3 (0.25) 0.3 – 1.3 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 – 0.3 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 – 0.3 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 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0Monsanto Company CR262-14U1 252 of 260

Table H-10 (continued). Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Ladybird beetles adult (Coccinellidae) Ladybird beetles Larvae (Coccinellidae) Minute pirate bugs (Anthocoridae) Beneficial Arthropod Beneficial Arthropod Beneficial Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG 0.3 (0.25)† 0.0 (0.00) 0.3 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.0 (0.00) 0.3 – 0.5 NCBD 0.0 (0.00)† 0.5 (0.50) 0.3 – 1.3 0.0 (0.00)† 0.5 (0.29) 0.3 – 1.3 2.3 (1.03) 2.5 (1.32) 1.8 – 4.8 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 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 PAHM 1.0 (0.58) 2.5 (1.32) 2.3 – 6.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5 6.7 (1.67) 9.5 (2.40) 8.5 – 11.7 2 IABG 0.3 (0.25)† 0.5 (0.29) 0.0 – 0.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 6.3 (1.65) 4.5 (0.96) 3.0 – 11.0 NCBD 0.3 (0.25)† 0.0 (0.00) 0.3 – 0.8 1.3 (0.75) 2.0 (0.58) 0.5 – 2.3 1.0 (0.41) 1.8 (0.63) 0.8 – 3.8 NEYO 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 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 2.7 (0.33) 3.5 (1.76) 0.8 – 3.7 0.3 (0.33)† 0.8 (0.48) 0.0 – 0.5 5.7 (2.03) 6.0 (2.20) 5.3 – 8.0 3 IABG 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 1.8 (0.75) 2.5 (0.96) 2.0 – 4.0 NCBD 0.5 (0.29)† 0.3 (0.25) 0.0 – 1.0 1.3 (0.95)† 0.8 (0.25) 0.5 – 1.0 1.0 (1.00)† 0.3 (0.25) 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 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 PAHM 0.0 (0.00)† 0.5 (0.50) 0.0 – 1.3 1.7 (0.33) 1.8 (0.25) 1.3 – 2.0 8.7 (4.67) 5.8 (1.55) 6.3 – 11.5 4 IABG 0.5 (0.29)† 0.3 (0.25) 0.0 – 0.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 2.3 (1.44) 2.5 (1.55) 1.5 – 3.3 NCBD 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.5 (0.50)† 0.5 (0.50) 0.5 – 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 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 PAHM 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.8 0.7 (0.67)† 2.0 (0.71) 0.3 – 1.5 0.0 (0.00)* 1.0 (0.41) 0.5 – 3.7 5 IABG 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 1.5 (0.87) 1.0 (0.58) 0.8 – 2.5 NCBD 0.0 (0.00)† 0.5 (0.50) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5 0.0 (0.00)† 0.5 (0.29) 0.0 – 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 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 PAHM 0.3 (0.33)† 0.3 (0.25) 0.0 – 0.5 0.7 (0.33)† 0.8 (0.48) 0.5 – 1.0 1.0 (0.58) 1.8 (0.85) 1.3 – 5.5Monsanto Company CR262-14U1 253 of 260

Table H-10 (continued). Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Corn rootworm beetles (Chrysomelidae) Sap beetles (Nitidulidae) Shining flower beetles (Phlacridae)

Pest Arthropod Pest Arthropod Pollen Feeder Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control MON 87403 Control 1 IABG

— — — 0.5 (0.29)† 0.3 (0.25) 0.3 – 0.8

— — — NCBD 1.0 (0.58) 1.3 (0.75) 1.0 – 2.0 4.3 (3.92) 4.5 (2.87) 2.5 – 5.3 5.0 (0.91) 6.0 (0.71) 3.8 – 6.8 NEYO 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

— — — PAHM 0.3 (0.33)* 2.0 (0.71) 1.0 – 2.0 4.3 (0.67) 8.0 (1.08) 1.8 – 8.5 1.3 (0.88) 3.5 (1.55) 1.7 – 3.0 2 IABG 3.0 (1.29) 2.5 (0.65) 1.5 – 6.0 4.5 (0.96) 9.5 (3.97) 8.8 – 19.0

— — — NCBD 0.3 (0.25)† 0.5 (0.50) 0.0 – 1.0 6.3 (5.27) 7.3 (3.59) 4.3 – 9.8 7.0 (3.19)* 12.3 (1.70) 6.8 – 10.5 NEYO 1.0 (0.41) 1.5 (0.65) 1.3 – 2.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

— — — PAHM 0.3 (0.33) 1.3 (0.63) 0.7 – 1.8 2.7 (0.33) 4.3 (0.75) 2.5 – 6.0 2.3 (0.33) 1.8 (0.75) 1.0 – 2.0 3 IABG 0.5 (0.29)† 1.0 (0.41) 0.5 – 1.3 4.8 (1.03) 2.8 (0.85) 3.5 – 7.5

— — — NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 1.5 (0.87) 2.5 (1.19) 1.5 – 3.8 1.5 (0.65) 1.0 (0.00) 1.0 – 3.0 NEYO 3.8 (1.75) 6.8 (1.03) 1.8 – 6.0 1.0 (0.00)* 4.5 (0.96) 0.3 – 1.3

— — — PAHM 1.0 (0.58)† 0.0 (0.00) 0.0 – 1.0 2.3 (2.33) 1.0 (1.00) 1.0 – 3.3 2.0 (1.53) 1.0 (0.41) 0.3 – 2.7 4 IABG 1.3 (0.95) 0.8 (0.48) 0.8 – 2.3 3.3 (1.38) 7.5 (1.89) 4.3 – 9.0

— — — NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.3 (0.25) 0.3 – 1.3 0.8 (0.25)† 0.0 (0.00) 0.0 – 2.0 NEYO 0.8 (0.25)† 1.3 (0.48) 0.3 – 1.0 2.0 (1.08) 0.8 (0.48) 0.3 – 2.3

— — — PAHM 0.0 (0.00)† 0.8 (0.48) 0.0 – 0.5 1.3 (0.88) 1.8 (0.75) 1.5 – 4.0 0.0 (0.00)† 0.5 (0.29) 0.0 – 1.3 5 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 5.3 (2.72) 5.0 (1.78) 1.8 – 5.8

— — — NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 1.3 (0.48)* 4.5 (1.19) 1.0 – 5.5 0.8 (0.48)† 0.3 (0.25) 0.0 – 0.8 NEYO

— — — 0.0 (0.00)† 1.0 (0.71) 0.0 – 1.0

— — — PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 1.3 (0.67) 1.5 (0.65) 1.5 – 4.8 1.0 (1.00)† 0.5 (0.29) 0.0 – 0.3Monsanto Company CR262-14U1 254 of 260

Table H-10 (continued). Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the Conventional Control in 2012 U.S. Field Trials

Spiders (Araneae) Stink bugs (Pentatomidae) Beneficial Arthropod Pest Arthropod Mean (S.E.) 3 Reference range 4 Mean (S.E.) 3 Reference range 4 Coll. 1 Site 2 MON 87403 Control MON 87403 Control 1 IABG 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 NCBD 4.8 (1.25) 3.0 (0.91) 3.5 – 4.5 0.8 (0.48)† 1.3 (0.48) 0.0 – 1.0 NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.0 PAHM 0.7 (0.33) 1.5 (0.50) 0.0 – 2.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 2 IABG 1.3 (0.75)† 0.0 (0.00) 0.0 – 1.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 NCBD 3.8 (0.75) 2.8 (1.18) 3.5 – 5.0 1.5 (0.50)† 0.5 (0.29) 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 (0.88) 2.3 (1.44) 0.7 – 2.0 0.3 (0.33)† 0.0 (0.00) 0.0 – 0.3 3 IABG 1.5 (0.50) 0.8 (0.25) 0.8 – 1.0 0.3 (0.25)† 0.3 (0.25) 0.3 – 0.5 NCBD 3.0 (1.22) 3.8 (1.11) 4.3 – 6.0 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.5 NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 PAHM 4.0 (1.53) 3.5 (2.22) 2.5 – 3.7 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 4 IABG 0.5 (0.29) 0.8 (0.25) 0.8 – 2.3 1.0 (0.71)† 0.5 (0.29) 0.0 – 1.0 NCBD 2.5 (0.65) 3.0 (1.29) 2.8 – 5.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 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 2.0 (0.58) 1.5 (0.65) 1.0 – 3.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 5 IABG 0.3 (0.25)† 0.0 (0.00) 0.3 – 0.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3 NCBD 3.0 (0.71) 2.3 (0.48) 2.0 – 3.3 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 – 0.0 PAHM 2.7 (0.33) 2.8 (0.75) 1.5 – 5.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5Monsanto Company CR262-14U1 255 of 260

Note: The experimental design was a randomized complete block with four replicates per site.

* I n d i c a t e s a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e b e t w e e n M O N 8 7 4 0 3 a n d t h e c o n v e n t i o n a l c o n t r o l ( α = 0

. 0 5 ) u s i n g A N O V A

. † Indicates p-values could not be generated where the taxa did not meet inclusion criteria (—) A dash indicates data not available. 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 87403 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 materials at each site.Monsanto Company CR262-14U1 256 of 260 References for Appendix H SAS Software Release 9.3 (TS1M0). 2010. Copyright 2002-2010 by SAS Institute, Inc., Cary, North Carolina.Monsanto Company CR262-14U1 257 of 260 Appendix I: Materials and Methods for Pollen Morphology and Viability Assessment

I.1. Plant Production MON 87403, the conventional control, and reference hybrids were grown under similar agronomic conditions in a field trial in Story County, Iowa (Table I-1). The trial was arranged in a randomized complete block design with four replications. Each plot consisted of eight rows approximately 6 m in length.

I.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 immediately after pollen collection and maintained under those conditions until receipt at the performing laboratory.

I.3. Data Collection Slides were prepared by aliquoting suspended pollen/stain solution onto a slide. Pollen characteristics were assessed under an Olympus ©9 BX53 light microscope equipped with an Olympus © DP72 digital color camera. The microscope and camera were connected to a computer running Microsoft Windows XP ®10 and installed with an Olympus © cellSens (version 1.4.1) software.

I.3.1. Pollen Viability 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 VII-8.

 Olympus Corporation.

 Windows XP is a registered trademark of Microsoft Corporation.Monsanto Company CR262-14U1 258 of 260

I.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 VII-8.

I.3.3. General Pollen Morphology General pollen morphology of MON 87403, the conventional control, and the reference hybrids was observed Figure I-1.

I.4. Statistical Analysis An analysis of variance was conducted according to a randomized complete block design using SAS © (SAS, 2012). The level of statistical significance was predetermined to be 5 % ( α = 0

. 0 5 )

. M O N 8 7 4 0 3 w a s c o m p a r e d t o t h e c o n v e n t i o n a l c o n t r o l m a t e r i a l f o r percent viable pollen and pollen grain diameter. MON 87403 and 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. Table I-1. Starting Seed for Pollen Morphology and Viability Assessment
Material Name Material Type Phenotype Monsanto Lot Number

MPA640B Control Conventional 11332601 Lewis 7007 Reference Conventional 11226559 Mycogen 2M746 Reference Conventional 11226705 NC+ 5220 Reference Conventional 11226701 Phillips 717 Reference Conventional 11300073

MON 87403 Test Increased Ear Biomass Maize 11332602Monsanto Company CR262-14U1 259 of 260

Figure I-1. General Morphology of Pollen from MON 87403, the Conventional Control, and Commercially Available Conventional Reference Materials 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.Monsanto Company CR262-14U1 260 of 260 References for Appendix I 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.