## Petition 10-336-01p



# E NVIRONMENTAL 

# R EPORT 

# Petition for Determination of Nonregulated Status of Event
5307 Corn

OECD Unique Identifier: SYN-Ø53Ø7-1

Prepared for Syngenta Biotechnology, Inc

. Research Triangle Park, North Carolina

Prepared by /Vanasse Hangen Brustlin, Inc.
Watertown, Massachusetts



## Syngenta Reference No. 5307-USDA-3a

October 31, 2011Event 5307 Corn Environmental Report

i Contents 

# Table of Contents

Acronyms and Abbreviations
1 Purpose and Need ........................................................................................................ 1-1

1.1 Regulatory Authority ............................................................................................... 1-1
1.2 Regulated Organisms ............................................................................................. 1-2
1.3 Petition for Determination of Nonregulated Status: Syngenta Event 5307 Corn .... 1-3
1.4 Purpose of and Need for Product ........................................................................... 1-4
1.5 APHIS Response to Petition for Nonregulated Status ............................................ 1-4
1.6 Coordinated Framework Review ............................................................................. 1-5
1.7 Public Involvement .................................................................................................. 1-6 1.8 Issues Considered .................................................................................................. 1-6

2 Affected Environment ................................................................................................. 2- 1

2.1 Corn Biology ........................................................................................................... 2-1
2.2 Corn Production ...................................................................................................... 2-4

2.2.1 Production and Yield .................................................................................. 2-4

2.2.2 Agronomic Practices .................................................................................. 2-6

2.2.3 Specialty S ystems .................................................................................... 2-18

2.2.4 Raw and Processed Corn Commodities .................................................. 2-22

2.2.5 Persistence in the Environment/Weediness Potential .............................. 2-22
2.3 Physical Environment ........................................................................................... 2-23

2.3.1 Water Quality and Use ............................................................................. 2-23

2.3.2 Soil ........................................................................................................... 2-25

2.3.3 Air Quality ................................................................................................. 2-25

2.3.4 Climate ..................................................................................................... 2-26
2.4 Biological Environment ......................................................................................... 2-26

2.4.1 Animals .................................................................................................... 2-27

2.4.2 Plants ....................................................................................................... 2-28

2.4.3 Biodiversity ............................................................................................... 2-28

2.4.4 Gene Movement in the Natural Environment ........................................... 2-28
2.5 Threatened and Endangered Species .................................................................. 2-29
2.6 Public Health ......................................................................................................... 2-30

2.6.1 Human Health .......................................................................................... 2-30

2.6.2 Animal (Livestock) Health ........................................................................ 2-31

2.6.3 Worker Safety .......................................................................................... 2-32Event 5307 Corn Environmental Report

ii Contents 2.7 Socioeconomics .................................................................................................... 2-32

2.7.1 Domestic Economy .................................................................................. 2-33

2.7.2 Trade Economy ........................................................................................ 2-34

2.7.3 Social Environment .................................................................................. 2-35

3 Alternatives ............................................................................................................ ....... 3-1

3.1 No Action: Continuation as a Regulated Article ...................................................... 3-1
3.2 Proposed Action: Determination that Event 5307 Corn is No Longer a Regulated Article ....................................................................................................................... .. 2 3.3 Alternatives Considered But Rejected from Further Evaluation .............................. 3-3

3.3.1 Prohibit Any Event 5307 Corn from Being Released ................................. 3-3

3.3.2 Establish an Isolation Distance Between Event 5307 Corn and NonTransgenic Corn ........................................................................................ 3-4

3.3.3 Establish Geographic Restrictions for Event 5307 Corn ............................ 3-5

3.3.4 Require Testing for Event 5307 Corn ......................................................... 3-5
3.4 Comparison of Alternatives ..................................................................................... 3-5
4 Environmental Consequences ................................................................................... 4-1

4.1 Introduction ............................................................................................................. 4-1

4.1.1 Scope of the Environmental Analysis ......................................................... 4-1

4.1.2 Methodology ............................................................................................... 4-2

4.1.3 Assumptions ............................................................................................... 4-3
4.2 Corn Production ...................................................................................................... 4-4

4.2.1 Agronomic Practices .................................................................................. 4-4

4.2.2 Specialty Corn Systems ........................................................................... 4-11
4.3 Physical Environment ........................................................................................... 4-16

4.3.1 Water Quality and Use ............................................................................. 4-16

4.3.2 Soil ........................................................................................................... 4-18

4.3.3 Air Quality ................................................................................................. 4-19

4.3.4 Climate ..................................................................................................... 4-19
4.4 Biological Environment ......................................................................................... 4-20

4.4.1 Animals .................................................................................................... 4-20

4.4.2 Plants ....................................................................................................... 4-27

4.4.3 Soil Microorganisms ................................................................................. 4-28

4.4.4 Biodiversity ............................................................................................... 4-30

4.4.5 Gene Movement in the Natural Environment ........................................... 4-32
4.5 Threatened and Endangered Species .................................................................. 4-33

4.5.1 APHIS Section 7 Process ........................................................................ 4-34

4.5.2 Potential Effects ....................................................................................... 4-34
4.6 Public Health ......................................................................................................... 4-37

4.6.1 Human Health .......................................................................................... 4-37

4.6.2 Animal (Livestock) Health ........................................................................ 4-39

4.6.3 Worker Safety .......................................................................................... 4-41
4.7 Socioeconomics .................................................................................................... 4-42

4.7.1 Domestic Economic Environment ............................................................ 4-42

4.7.2 Trade Economic Environment .................................................................. 4-43Event 5307 Corn Environmental Report

iii Contents 4.7.3 Social Environment .................................................................................. 4-44
4.8 Cumulative Effects ................................................................................................ 4-45

4.8.1 Methodology and Assumptions ................................................................ 4-45

4.8.2 Reasonably Foreseeable Future Actions ................................................. 4-46

4.8.3 Cumulative Effects Evaluation ................................................................. 4-49
5 Consideration of Executive Orders and Other Federal Laws Relating to .............
Environmental Impacts ....................................................................................... 5-1

5.1 Executive Orders with Domestic Implications ......................................................... 5-1

5.1.1 Executive Order 12898: Environmental Justice ......................................... 5-1

5.1.2 Executive Order 13045: Protection of Children .......................................... 5-2

5.1.3 Executive Order 13112: Invasive Species ................................................. 5-3

5.1.4 Executive Order 13186: Migratory Birds .................................................... 5-3
5.2 Executive Order with International Implications ...................................................... 5-3
5.3 Other Federal Laws ................................................................................................ 5-5

5.3.1 Clean Water Act ......................................................................................... 5-6

5.3.2 Clean Air Act .............................................................................................. 5-6

5.3.3 National Historic Preservation Act .............................................................. 5-6

5.3.4 Federal Laws Regarding Unique Characte ristics of Geographic Areas ..... 5-7
6 List of Preparers ....................................................................................................... .... 6-1

7 References .............................................................................................................. ....... 7-1
Appendix A Public Inte rest Asse ssment ........................................................................... 1
Appendix B Safety and Nutritional Assessment ................................................................ 1
Appendix C Data Tables ...................................................................................................... 1

APPENDICES

A Public Interest Assessment

B Safety and Nutritional Assessment

C Data TablesEvent 5307 Corn Environmental Report

iv Contents 

# List of Tables

Table No. Description Page

2-1 Deregulated Transgenic Corn Cultivars Containing

Bt-derived Proteins ................................................................................ 2-3

2-2 Corn Insect Pests .................................................................................. 2-9 2-3 National List of Approved Insecticides for Organic Production Systems .............................................................................................. 2-11 2-4 Threatened or Endangered Coleopteran Species Occurring in Corn-Growing Regions ........................................................................ 2-30 2-5 List of Corn Mycotoxins Toxic to Animals Consuming

Contaminated Feed ............................................................................. 2-32 2-6 Corn and Crop Cash Receipts ............................................................ 2-33 3-1 Summary of Environmental Consequences .......................................... 3-6 4-1 Planned Stacking Combinations ......................................................... 4-46 4-2 Commercially Available Corn Breeding Stack Combination with

Insect Control Traits ............................................................................ 4-47Event 5307 Corn Environmental Report

i Acronyms and Abbreviations ACRONYMS AND ABBREVIATIONS

μg/g Micrograms per gram
ACHP Advisory Council on Historic Properties
AIA Advanced Informed Agreement
AMS Agricultural Marketing Service (also USDA-AMS)
AOSCA Association of Official Se ed Certifying Agencies
APHIS Animal and Plant Health Inspection Service (also USDA-APHIS)
ARMS Agricultural Resource Management Survey
ARS Agricultural Research Services (also USDA-ARS)
ATTRA Appropriate Technology Transfer for Rural Areas
BRS Biotechnology Regulatory Service
B t Bacillus thuringiensis
C Centigrade
C3 Three-carbon molecule photosynthetic pathway
C4 Four-carbon molecule photosynthetic pathway
CBD Convention on Biological Diversity CEQ Council on Environmental Quality
CFR Code of Federal Regulations
CO 2 Carbon dioxide
CRP Conservation Reserve Program Cry Crystal proteins
EA Environmental Assessment
EIS Environmental Impact Statement
EO Executive Order
EPA Environmental Protection Agency (also USEPA)
ER Environmental Report
ERS Economic Research Service (also USDA-ERS)
ESA Endangered Species Act
FATUS Foreign Agricultural Trad e of the United States
FAS Foreign Agricultural Service (also USDA-FAS)
FB 1 Fumonisin B 1 FDA Food and Drug Administra tion (also USDHHS-FDA)
FFP Food, feed, or processing
FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
FESTF FIFRA - Endangered Species Task Force
FONSI Finding of No Significant Impact
FR Federal Register
FWS Fish and Wildlife Service (also USFWS)
GHG Greenhouse gases
IMS Information Management System
IP Identity Preservation
IPM Integrated Pest Management
IPCC Intergovernmental Panel on Climate Change
IPPC International Plant Protection Convention
IRM Insect Resistance Management
LMO Living modified organisms LOQ Limit of quantification MJD Multi-Jurisdictional Database
N 2 O Nitrous oxide
NAPPO North American Plant Protection OrganizationEvent 5307 Corn Environmental Report

ii Acronyms and Abbreviations NASS National Agricultural Statistics Service (also USDA-NASS)
NCGA National Corn Growers Association
NEPA National Environmental Policy Act
NHPA National Historic Preservation Act
NOP National Organic Program
NPS Nonpoint source
NPTN National Pesticide Telecommunications Network
NRC National Research Council
NRCA Natural Resources Conservation Service (also USDA-NRCS)
OECD Organization for Economic Cooperation and Development
OTA Organic Trade Association
PIPs Plant-incorporated protectants

p mi Phosphomannose isomerase gene (also known as manA )
PMI Phosphomannose isomerase protein
PPA Plant Protection Act
PPRA Plant Pest Risk Assessment
PRA Pest Risk Analysis
RR2 Roundup-Ready© Corn 2
RSPM Regional Standards for Phytosanitary Measures
SCGO Seed Corn Growers of Ontario
TSCA Toxic Substances Control Act
US United States
USACOE United States Army Corps of Engineers
USDA United States Department of Agriculture
USDHHS United States Department of Health and Human Services
USEPA United States Environmental Protection Agency (also EPA)
USFWS United States Fish and Wi ldlife Service (also FWS)
USGC U.S. Grains Council Vip Vegetative insecticidal proteinEvent 5307 Corn Environmental Report

1-1 Purpose and Need 

# 1

Purpose and Need

This Chapter describes the purpose of and need for determining a nonregulated status for Event 5307 corn, and includes an explanation of the regulatory context of
the decision.



## 1.1 Regulatory Authority

"Protecting American agriculture" is the basi c charge of the United States Department of Agriculture (USDA) Animal and Plant He alth Inspection Service (APHIS). APHIS provides leadership in ensuring the health and care of plants and animals. The agency improves agricultural productivity and competitiveness, and contributes to the national economy and public health. USDA policy states that all methods of agricultural production (resulting in conven tional, genetically engineered [transgenic], and organic varieties) can provide benefits to the environment, consumers, and producers.

Since 1986, the United States government ha s regulated genetically engineered organisms pursuant to a regulatory framework known as the Coordinated Framework for the Regulation of Biotechnology 1 (Coordinated Framework). The Coor dinated Framework describes the comprehensive federal regulatory policy for en suring the safety of biotechnology research and products and explains how federal agencies will use existing federal statutes in a manner that ensures public health and enviro nmental safety while maintaining regulatory flexibility to avoid impeding the growth of the biotechnology industry.

The Coordinated Framework is based on several important guiding principles:

(1) Agencies should define those transgenic organisms subject to review to the extent permitted by their respective statutory authorities;


1 Executive Office of the President, O ffice of Science and Technology Policy. 1986.Event 5307 Corn Environmental Report

1-2 Purpose and Need (2) Agencies are required to focus on the characteristics and risks of the biotechnology product, not the process by which it is created; and

(3) Agencies are mandated to exercise oversight of genetically engineered organisms only when there is evidence of “unreasonable” risk.

The Coordinated Framework explains the re gulatory roles and authorities for the three major federal agencies involved in regulating genetically engineered organisms: APHIS, the US Food and Dr ug Administration (FDA), and the US Environmental Protection Agency (EPA):

 APHIS is responsible for regulating gene tically engineered organisms pursuant to the plant pest authorities in the Plant Protection Act 2 (PPA) to ensure that genetically engineered organisms do not pose a plant pest risk to the

environment.

 The FDA regulates genetically engineered organisms under the authority of the Federal Food, Drug, and Cosmetic Act

. 3 The FDA is responsible for ensuring the safety and proper labeling of all plant- derived foods and feeds, including those that are genetically engineered. To help developers of food and feed derived from genetically engineered crops comply with their obligations under federal food safety laws, FDA encourages them to participate in a voluntary consultation process. In the FDA policy statement 4 concerning regulation of products derived from new plant varieties, including thos e genetically engineered, FDA uses this consultation process to ensure that human food and animal feed safety issues or other regulatory issues (e.g., labeling) are resolved prior to commercial distribution of bioengineered food. All food and feed derived from genetically engineered crops currently on the market in the United States have successfully completed this consultation process.

 The EPA regulates plant-incorporated protectants (PIPs) under the Federal Insecticide, Fungicide, and Rodenticide Act 5 (FIFRA) and certain biological control organisms under the Toxic Substances Control Act 6 (TSCA). The EPA is responsible for regulating the sale, distri bution and use of pesticides, including pesticides that are produced by an organism through biotechnology.



## 1.2 Regulated Organisms

The mission of APHIS Biotechnology Regulatory Service (BRS) is to protect America’s agriculture and environment using a dy namic and science-based regulatory



2 Plant Protection Act . 2000. 3 Federal Food, Drug, and Cosmetic Act . 1938. 4 USDHHS-FDA. 1992. 5 Federal Insecticide, Fungicide, and Rodenticide Act. 1947. 6 Toxic Substances Control Act . 1976.Event 5307 Corn Environmental Report

1-3 Purpose and Need framework that allows for the safe develo pment and use of genetically engineered organisms. APHIS regulations 7 promulgated pursuant to authority granted by the PPA govern the introduction (importation, interstate movement, or release into the environment) of certain genetically engin eered organisms. A genetically engineered organism is no longer subject to the plant pe st provisions of the PPA or the applicable regulatory requirements after APHIS determines that it is unlikely to pose a plant pest risk. A genetically engineered organism is considered a regulated article if the donor organism, recipient organism, vector, or vect or agent used in engineering the organism belongs to one of the taxa listed in the regulation 8 and is also considered a plant pest. A genetically engineered organism is also re gulated when APHIS has reason to believe that the genetically engineered organism ma y be a plant pest or APHIS does not have information to determine if the genetically engineered organism is unlikely to pose a plant pest risk.

A person may petition the agency for a de termination that a particular regulated article is unlikely to pose a plant pest risk , and, therefore, is no longer regulated under the plant pest provisions of the PPA or the applicable regulations. The petitioner is required 9 to provide information related to plant pest risk that the agency may use to determine whether the regulated article is unlikely to present a greater plant pest risk than the unmodi fied organism. A genetically engineered organism is no longer subject to the re gulatory requirements or the plant pest provisions of the PPA when APHIS determines that it is unlikely to pose a plant pest
risk.



## 1.3 Petition for Determination of Nonregulated Status: Syngenta Event 5307 Corn

Syngenta Biotechnology, Inc. submitted an in itial petition (10-336-01p) to APHIS in 2010, which was revised in 2011, 10 for determination of nonregulated status for Event 5307 rootworm-resistant corn (hereafter referred to as Event 5307 or 5307 corn). Nonregulated status would include 5307 co rn, progeny from crosses between 5307 corn and conventional corn, and progeny from crosses of 5307 corn with other transgenic corn that has be en deregulated. Event 5307 co rn is currently regulated
under the PPA. Interstate movements and field trials of 5307 corn have been conducted under permits issued or notifica tions acknowledged by APHIS since 2005. Data resulting from these field trials are described in the Syngenta petition.



7 USDA-APHIS. 1987. 8 USDA-APHIS. 1995. 7 CFR Section 340.2. 9 USDA-APHIS. 1995. 7 CFR Section 340.6(c)(4). 10 Vlachos and Huber. 2011.Event 5307 Corn Environmental Report

1-4 Purpose and Need 

## 1.4 Purpose of and Need for Product

Corn rootworm ( Diabrotica ) larvae feed on the roots of growing corn plants and are widespread and major pests of US corn. Corn ( Zea mays L., maize) derived from Syngenta’s transformation Event 5307 cont ains a unique engineered insecticidal protein, eCry3.1Ab, that is active against three economically important corn rootworm species that cause significant damage to the US corn crop annually. This engineered pesticide provides corn plants with high resistance to larval feeding damage by western corn rootworm ( Diabrotica virgifera virgifera ), northern corn rootworm ( D. longicornis barberi ) and Mexican corn rootworm ( D. virgifera zeae ). Event 5307 corn demonstrates excellent efficacy in controlling these damaging pests.

Corn varieties containing the transgene ecry3.1Ab have the potential to displace applications of conventional rootworm inse cticides that are of concern due to human and environmental risk factors. The new prot ein, eCry3.1Ab, acts on target pests via a unique mode of action, reducing the selection pressure on pest populations to evolve resistance to other Cry proteins an d control methods. Growers are expected to
derive benefits from the convenience and ease -of-use of 5307 corn as an alternative to the application of conventional insecticides , as well as realize economic benefits through increased crop yield under conditions of pest pressure, and reduced costs of insecticide applications. The availability of 5307 corn is expected to contribute to the significant trend in reduced use of hazardous insecticides and extends the useful life of other commercially availa ble corn rootworm-protected Bt cultivars (Cry3Bb1, Cry34Ab1/Cry35Ab1, and mCry3A corn).

The benefits of 5307 corn include reduced insecticide use, improved worker safety, reductions in the use of fossil fuels to apply chemical insecticides, economic benefits for growers, improved insect resistance management, and increased competition in the marketplace for insect-protected seed products.



## 1.5 APHIS Response to Petition for Nonregulated Status

Under the authority of the plant pest pr ovisions of the PPA, APHIS has issued regulations for the safe development and use of genetically engineered organisms. APHIS must respond to petitioners that request a determination of the regulated status of genetically engineered organisms, 11 including transgenic plants such as Syngenta’s 5307 corn. When a petition for nonregulated status is submitted, APHIS must make a determination if the genetically engineered organism is unlikely to pose a plant pest risk.



11 USDA- APHIS 1987. 7 CFR Section 340.6.Event 5307 Corn Environmental Report

1-5 Purpose and Need APHIS will prepare an environm ental assessment (EA) that will consider the potential environmental effects as part of an agen cy determination of nonregulated status
consistent with the National Environmental Policy Act 12 (NEPA) and implementing regulations from the Council on Environmental Quality (CEQ) 13 and APHIS, 14 as well as APHIS BRS procedures. This Environmental Report (ER) has been prepared in order to specifically evaluate the effects that ma y result from deregulation of Syngenta’s 5307 corn on the physical, biological, and human environment.



## 1.6 Coordinated Framework Review

Syngenta has obtained an Experimental Use Permit (67979-EUP-8) from EPA that allows for broad-scale field testing of 5307 corn and various breeding stack combinations that include 5307 corn. The Experimental Use Permit 15 was initially granted on June 1, 2010 with effect through February 28, 2012 and was extended 16 on March 3, 2011 with effect through December 31, 2013. In connection with this Experimental Use Permit, EPA established 17 and extended 18 a temporary exemption from the requirement of a tolerance for eCry3.1Ab residues in corn commodities, pursuant to §408(d) of the Federal Food, Drug, and Cosmetic Act . Phosphomannose isomerase (PMI), the selectable marker protein produced by 5307 corn plants, is exempt from food and feed tolerances. 19

In April 2011 Syngenta submitted applications to the EPA for registration of the eCry3.1Ab PIP in 5307 corn (Appendix A) and in two breeding stacks including 5307 corn, specifically Bt11 × MIR604 × TC1507 × 5307 × GA21 and Bt11 × MIR162 ×
MIR604 × TC1507 × 5307 × GA21 corn. In addition to the corn rootworm control provided by 5307 corn, the controls pr ovided by other constituents of these
combinations are:

 A Cry1Ab protein for lepidopteran control (Bt11);

 A modified Cry3A protein for co rn rootworm control (MIR604);

 A Cry1F protein for lepido pteran control (TC1507);

 A double mutated 5-enolpyruvylshikimate-3-phosphate synthase enzyme for glyphosate tolerance (GA21);

 A Vip3Aa20 protein for lepido pteran control (MIR162); and

 A phosphinothricin acetyl transferase enzyme for glufosinate tolerance (Bt11 and TC1507).



12 National Environmental Policy Act . 1970. 13 CEQ. 1978. 14 USDA-APHIS. 1995. 15 USEPA. 2010c. 16 USEPA. 2011c. 17 USEPA. 2010a. 18 USEPA. 2011a. 19 USEPA. 2007b.Event 5307 Corn Environmental Report

1-6 Purpose and Need The registration sought for the PIP in 5307 corn as a stand-alone cultivar (i.e., not part of a breeding stack) will be for a manufacturing-use product; Syngenta will not seek an end-use product registration from EPA for 5307 corn. Rather, commercial registrations will be sought from EPA fo r the two breeding stack products that include 5307 corn. Concurrently, Syngenta also submitted a petition (Petition No. 1F7857) to the EPA to establish a none xpiring exemption from the requirement of a tolerance for eCry3.1Ab residues in food and feed commodities.

Event 5307 corn falls within the scope of the FDA policy statement concerning regulation of food products derived fr om new plant varieties, including those developed by genetic engineering. 20 Syngenta initiated a voluntary pre-market consultation process with FDA and submitted a Safety and Nutritional Assessment for 5307 corn in January 2011 (Appendix B). 21



## 1.7 Public Involvement

APHIS routinely seeks public comment on draft environmental assessments prepared in response to petitions to dere gulate genetically engineered organisms. APHIS does this through a notice published in the Federal Register. This ER, the EA, the petition submitted by Syngenta, and APHIS’ Plant Pest Risk Assessment of 5307 corn will be available for public comme nt for a period to be specified by APHIS. Comments received by the end of the period will be analyzed and used to inform APHIS’ determination decision of the regu lated status of 5307 corn and to assist APHIS in determining whether an Environmental Impact Statement (EIS) is required prior to the determination decision of th e regulated status of this corn line.



## 1.8 Issues Considered

The list of issues considered in this ER were developed in accordance with the requirements of NEPA, and through coordination with APHIS. The list was developed through experience in considering potential environmental impacts relevant to transgenic field crops like 5307 co rn, as well as concerns and issues raised in public comments submitted for other EAs of transgenic organisms. The issues
considered also address concerns raised in previous and unrelated court decisions, as well as questions that have been raised by various stakeholders in the past. The
issues considered in this ER are listed below.

 Corn Production 

###  Acreage and areas of production 

###  Agronomic practices



20 USDHHS-FDA. 1992. 21 Vlachos and Ward. 2011. (Appendix B)Event 5307 Corn Environmental Report

1-7 Purpose and Need 

###  Specialty systems 

###  Raw and processed agricultural commodities 

###  Persistence in the environment and weediness potential

 Physical and Natural Environment 

###  Water quality and use 

###  Soil 

###  Air quality 

###  Climate 

###  Animals 

###  Plants 

###  Soil microorganisms 

###  Biodiversity 

###  Gene movement in the natural environment 

###  Threatened and endangered species

 Public Health 

###  Human health 

###  Animal (livestock) health 

###  Worker safety

 Socioeconomic Factors 

###  Domestic economic environment 

###  Trade economic environment 

###  Social environment

 Cumulative EffectsEvent 5307 Corn Environmental Report

1-8 Purpose and Need This page intentionally left blank.Event 5307 Corn Environmental Report

2-1 Affected Environment 

# 2

Affected Environment

This Chapter describes corn biology, co rn production, and the environmental and human resources that could potentially be affected by deregulation of Event 5307

corn.



## 2.1 Corn Biology

Maize, or corn, is a member of the Maydeae tribe of the grass family Poaceae. 22 The western hemisphere genera Zea and Tripsacum are included in the tribe Maydeae . The Asian genera of Maydeae are Coix

, Polytoca

, Chionachne

, Schlerachne and Trilobachne

. 23

Corn is a robust monoecious (i.e., having separate male and female flowers for each plant) annual plant, which was developed through artificial selection and does not occur in natural ecosystems outside of cultivation. The corn plant is adapted for high productivity because it has a large leaf ar ea and a modified photosynthetic pathway that allows it to survive extended periods of drought. 24 The corn plant, along with a limited number of other plants like sugarcane, millet, and sorghum, uses the C4 photosynthetic pathway. At a biochemi cal level, these plants convert carbon dioxide (CO 2 ) into a four-carbon molecule in contrast to the more common photosynthetic pathway in which CO 2 is converted into a three-carbon molecule. 25 C4 photosynthesis allows the corn plant to produce more dry matter (i.e., in various combinations of carbohydrates, proteins, oils, and mineral nutrients) per unit of water transported than the C3 photosynthetic pathway. Because of their high photosynthetic efficiency, the C4 crops like corn and sugarcane are favored for ethanol production as well.

Zea mays is an allogamous plant (i.e., cross-fertilizes by pollen from one flower to stigmas on another) that propagates through seed produced predominantly by cross pollination. Pollen is transferred by wind and, in controlled breeding situations, by

 22 Organization for Economic Co-operat ion and Development. 2003. Pg. 11. 23 Ibid. Pg. 14. 24 Frost and Gilman

. 2011. Pg. 1. 25 Ibid. Pg. 1.Event 5307 Corn Environmental Report

2-2 Affected Environment humans. Corn can only be crosse d experimentally with the genus Tripsacum ; however, wild member species of its own genus (teosinte: Z. diploperennis, Z. perennis, Z. luxurians, Z. nicaraguanensis ) may hybridize with corn under natural conditions (see Section 2.4.4, Gene Movement in the Natural Environment ). Corn is not weedy, and does not persist outside of cultivation. There are no reports in which corn propagated vegetatively under field conditions, and the only known propagation method for corn is through seed germination. Corn is primarily grown in warm temperate
climates. Corn seed is sensitive to cold an d typically does not survive freezing winter
conditions. 26 Consequently, corn has no innate dormancy. 27

Humans have been selectively breeding co rn for thousands of years to emphasize desired characteristics such as increased yield. Beginning in 1996, transgenic corn products were introduced in the US market by seed companies, thereby adding important new genetic traits to modern corn varieties. Corn cultivars representing
30 different transformation events have been deregulated by APHIS 28 in connection with 26 petitions for deregulation; these cultivars were genetically engineered to offer insect resistance, herbicide tolerance, and other traits. Sixteen of these cultivars, as listed in Table 2-1, contain proteins derived from Bacillus thuringiensis ( Bt ) and are
known as Bt corn cultivars. Bt corn cultivars assist grow ers in preventing insect damage that would otherwise cause yield loss. The Bt “crystal” proteins (referred to as Cry proteins) in these cultivars are insecticidal against certain Coleoptera or Lepidoptera and exert their insecticidal activity when they:

 Are ingested by the insect and solubilized in the insect gut;
 Are activated by specific proteolytic cleavage by midgut enzymes;
 Bind to specific receptors on the surface of the insect midgut; and
 Form ion channels in the gut membrane.

 26 Organization for Economic Co-operat ion and Development. 2003. Pg. 23. 27 Ibid. Pg. 25. 28 USDA-APHIS. 2011b.Event 5307 Corn Environmental Report

2-3 Affected Environment 

### Table 2-1 Deregulated Transgen ic Corn Cultivars Containing Bt- derived Proteins

Cultivar Filed By/Petition Number Transgenic feature NEPA Status (by APHIS)

Event 176 Ciba Seeds (petition 94-319-01p) Lepidopteran (European corn borer) resistant, expressing Cry1Ab FONSI and EA issued June 13, 1995

MON 80100 Monsanto (petition 95-093-01p) Lepidopteran resistant, expressing Cry1Ab FONSI and EA issued August 28, 1995

Bt11 Northrup King Company (petition 95-195-01p) Lepidopteran resistant, expressing Cry1Ab, and glufosinate tolerant FONSI and EA issued January 22, 1996

DBT418 DeKalb (petition 96-291-01p) Lepidopteran resistant, expressing Cry1Ac, and glufosinate tolerant FONSI and EA issued April 9, 1997

MON 809 and
MON 810 Monsanto (petition 96-017-01p) Lepidopteran resistant, expressing Cry1Ab FR notice published March 15, 1996

MON 802 Monsanto (petition 95-317-01p) Lepidopteran resistant, expressing Cry1Ab, and glyphosate tolerant FONSI and EA issued May 27, 1997

CBH-351 AgrEvo (petition 97-265-01p) Lepidopteran resistant, expressing Cry9C, and glufosinate tolerant FONSI and EA issued May 11, 1998

Line 1507 Mycogen (petition 00-136-01p) Lepidopteran resistant, expressing Cry1F, and glufosinate tolerant FONSI and EA issued August 2, 2001

MON 863 Monsanto (petition 01-137-01p ) Corn rootworm resistant, expressing Cry3Bb1 FONSI and EA issued October 17, 2002

DAS 6275 Dow (petition 03-181-01p) Lepidopteran resistant, expressing Cry1F, and glufosinate tolerant FONSI and EA issued October 20, 2004

DAS 59122 Dow (petition 03-353-01p) Corn rootworm resistant, expressing Cry34/35Ab1, and glufosinate tolerant FONSI and EA issued September 23, 2005

MON 88017 Monsanto (petition 04-125-01p) Corn rootworm resistant, expressing Cry3Bb1, and glyphosate tolerant FONSI and EA issued December 14, 2005

MIR604 Syngenta (petition 04-362-01p) Corn rootworm resistant, expressing modified Cry3A FONSI and EA issued March 16, 2007

MON 89034 Monsanto (petition 06-298-01p) Lepidopteran resistant, expressing Cry1A.105 and Cry2Ab2 FONSI and EA issued July 15, 2008

MIR162 Syngenta (petition 07-253-01p) Lepidopteran resistant, expressing Vip3Aa20 FONSI and EA issued April 9, 2010Event 5307 Corn Environmental Report

2-4 Affected Environment 

## 2.2 Corn Production

This section describes corn production and yield in the US, current agronomic practices, specialty corn production syst ems, raw and processed corn commodities, and corn’s persistence in the environment and weediness potential. Corn is grown for animal feed, human food, vegetable oil, high fructose corn syrup, starch, fermentation into ethanol, and many other uses.



### 2.2.1 Production and Yield

Corn is usually cultivated in temperate regions that provide sufficient moisture and an adequate number of frost-free days to reach maturity. US corn production is primarily focused in the Corn Belt, an area that includes Iowa, Illinois, Nebraska, and Minnesota, and parts of Indiana, South Dakota, Kansas, Ohio, Wisconsin, and
Missouri. The Corn Belt has a combination of seasonal warm weather, rainfall, and favorable soil conditions for corn growth. Approximately 68.7 million acres of corn, representing approximately 78 percent of the US total of 88.2 million acres, was planted in these ten states in 2010. 29 The planted acres of corn for each county in selected states in 2010 are depicted below.

Source: USDA-NASS. 2011c.

 29 USDA-NASS. 2011d. Pg. 8.Event 5307 Corn Environmental Report

2-5 Affected Environment Nationally, planted corn acreage has increased from approximately 75 million acres in 1990 to a peak of nearly 95 million acres in 2008, as shown in the graph below. Corn growers were projected to plant more than 92 million acres of corn in 2011. Harvested acreage lags planted acreage by 5 to 7 million acres each year due to losses from adverse weather and insect damage.

Source: USDA-NASS. 2011a

.

The majority of domestic corn is produced for livestock feed (grain and silage) and fuel (ethanol), comprising 39 and 36 percent, respectively of the market. Corn for grain production in 2010 was estimated at 12.4 billion bushels, 5 percent below the record high production of 13.1 billion bushels set in 2009. 30 Grain yield was approximately 152.8 bushels per acre, 11.9 bushels below the record high yield of 164.7 bushels per acre set in 2009. Since 1990, corn yields have increased by about 2.0 bushels per acre per year. 31

The USDA-Economic Research Service (ERS) provides 10-year projections of supply and utilization for major field crops, including corn, grown in the US. ERS projects that an average of 89.5 million acres of corn will be planted each year through 2019. 32 Output will increase during this period, according to ERS:

Expanding output is attributable to yi eld growth. The projected 2010/11 corn yield is based on the simple linear tren d since 1990. The longer term trend for 2011/12 and later years reflects an annual yield increase of 2.0 bushels per acre per year, resulting in record corn production in 2011/12 and beyond.



30 USDA-NASS. 2011d. 31 Savage. 2011. Pg. 1. 32 USDA-ERS. 2011b. Pg. 6.Event 5307 Corn Environmental Report

2-6 Affected Environment Increases in corn yields have been driven by improvements in plant genetics, machinery, and cultivation practices that have allowed for faster, more precise planting and earlier harvesting. The latest round of advances in genetics and planting technology is expe cted to be fully adopted by the early years of the projections. Thus, longer term yield gains are expected to be somewhat slower than during the late 1990s and early 2000s. Gains continue to be supported by improved genetics, including advances in plant
utilization of water and fertilizer.

In 2011, approximately 88 percent of the US corn fields were planted with transgenic crops for pest management. 33 As described in Section 2.2.2.3, Insect Management , and Section 2.2.2.5, Weed Management , the transgenic crops provide insect resistance and herbicide tolerance, as well as production -related traits. Insect -resistant varieties containing Bt -derived transgenes comprise about 16 percent of the US corn acreage, and herbicide-tolerant varieties some 23 percent; products combining both traits were planted on 49 percent of the corn acreage. 34



### 2.2.2 Agronomic Practices

Corn growers choose agronomic practices that maximize grain yield. Grain yield can be affected by cropping practices such as crop rotation and tillage techniques, supplemental irrigation based on soil and climate conditions, and methods to manage insects, disease, and weeds. This section describes common agronomic practices for corn.



### 2.2.2.1 Cropping Practices

Crops grown in rotation typically have higher yields, lower weed biomass, and lower insect populations than those grown continuously. 35 Corn is typically rotated with legumes such as soybeans or alfalfa. Corn/soybean rotations have been found to have 9 percent higher yields than corn/corn rotations. 36 Weed population density and biomass may be reduced using crop rotation (temporal diversification) and intercropping (spatial diversification) strategies. 37 The risk of yield losses due to damage from some insects, particularly corn rootworm, decreases when corn is rotated with other crops

. 38

Legumes provide the greatest rotation benefits for corn; the benefit is principally attributed to the nitrogen-fixing property of legumes but other factors (such as increased organic matter and reduced weed seed banks 39 ) may also contribute to

 33 USDA-NASS. 2011a. Pg. 22. 34 Ibid. Pg. 22. 35 Hicks and Thomison. 2004. Pgs. 504-505. 36 Hicks and Hoverstad. 2007. Pg. 1. 37 Liebman and Dyck. 1993. Pg. 92. 38 Bessin. 2004. Pg. 1. 39 Eisenthal. 2011. Pg. 1.Event 5307 Corn Environmental Report

2-7 Affected Environment higher yield. Corn is rotated with soybeans annually in much of the Corn Belt, but oats and alfalfa may also be rotated with corn and soybeans or during fallow years in semi-annual to five-year cycles. 40 In 2010, 71 percent of the acres planted in corn were in a rotation program in the last three years. 41

Tilling the soil reduces weeds that would otherwise crowd or compete with the corn

crop. 42 Until recently, conventional tillage was commonplace, using moldboard plows prior to and after planting. Reduced or conservation tillage methods have largely replaced conventional methods. Redu ced tillage uses chis el plows and results
in less soil disturbance. Conservation tilla ge also uses chisel plows and includes notill and focused methods such as strip, ridge, or mulch tillage. The intensive plowing of conventional tillage results in less than 15 percent crop residue (unharvested plant material); reduced tillage is associated with 15 to 30 percent crop residue; and conservation tillage is associated with at least 30 percent crop residue and substantially less soil erosion than other tillage practices. 43 Because of its low cost and positive impact on soil quality, cons ervation tillage is currently and widely practiced in the Midwestern US. In 2010, 62 percent of the acres planted in corn used
a no-till or minimu m tillage method. 44

Herbicides are used to kill weeds, as described in Section 2.2.2.5, Weed Management

, in all of these tillage methods. In no-till systems, the herbicide is applied directly to the last season's crop residue. In the other methods, soil is tilled before the herbicide is applied. The amount of herbicide used is somewhat independent of tillage method, 45 although transgenic crops may facilitate no-till methods by reducing the need for soil application of herbicides.



### 2.2.2.2 Irrigation

Soil and water (rainfall or irrigation) are the key resources in all crop production. Soil supports the basic physical, chemical, and bi ological processes for corn to grow, and specific soil characteristics regulate water flow between infiltration, root-zone storage, deep percolation, and storm water runoff. 46 Corn Belt soils are deep, fertile, and rich in organic material and nitrogen, and the land is relatively level. The warm nights, hot days, and well-distributed rain fall of the region during the growing season are ideal conditions for raising corn. Irrigation early in the season helps establish a uniform stand and water availability, whether by rainfall or irrigation, during flowering is critical to achieve good seed set. 47



40 Hicks and Thomison. 2004. Table 3.2.4.4. 41 USDA-NASS. 2011b. Pg. 2. 42 USEPA. 2009. Pg. 1. 43 Ibid. Pg. 1. 44 USDA-NASS. 2011b. Pg. 2. 45 USEPA. 2009. Pg. 1. 46 Christensen. 2002. Pg. 9. 47 Beck. 2004. Pg. 577.Event 5307 Corn Environmental Report

2-8 Affected Environment Corn grown for grain has substantially more irrigated area than any other single crop in the US, about 10.6 million acres in 1997, but only about 15 percent of the total corn acreage is irrigated. 48 Irrigated corn yields are about 29 percent higher than unirrigated corn yields. Irrigated corn is geographically distributed in the Corn Belt: eastern states irrigate only about 3 percent of the total corn area, while western states irrigate about 50 percent.



### 2.2.2.3 Insect Management

Corn is susceptible to attack by a variety of in sects throughout its life cycle. Insects can be categorized as major and consistent pests, major and sporadic pests, and moderate to minor pests, based on annual destructiveness and their geographic distribution. The most economically signif icant corn pests include Diabrotica species (the corn rootworm complex) and Ostrinia nubilalis (European corn borer). The damage inflicted by rootworm larvae can significantly reduce grain yield by interfering with photosynthetic rates, limiting the uptake of water and nutrients, an d by increasing the plant's susceptibility to lodging. 49 Lodging further reduces the effective grain yield by making the plants more susceptible to breaking, reduci ng their access to sunlight, and increasing the difficulty with which the grain can be harvested efficiently . Table 2-2 lists corn insect pests found in the US in several corn growth stages.

Insect pest management consists of several practices:

 Crop Rotation: Crop rotation is the most utilized method for insect management and is often the lowest cost tactic. Rotation with a non-grass crop reduces the levels of many pests through starvation or eliminating insect reproduction. Section 2.2.2.1, Cropping Practices , describes crop rotation practices.

 Selection of Planting and Harvest: Plant phenology can be manipulated to disrupt synchronization with the phenology of the pest insects. This can be achieved by either delaying or advancin g planting dates. Early-planted corn has shown lower susceptibility to corn earworm ( Helicoverpa zea ) and southwestern corn borer ( Diatraea grandiosella ) damage than late-planted crops. 50 The female D. grandiosella tends to lay fewer eggs on more mature plants and the plants have already passed their critical developmental stage. Early harvest often produces phenological asynchronies with a pest’s life cycle, allowing harvest before the damaging phase occurs. Early-planted corn can be harvested before many fully grown pre-diapause larvae have girdle d the mature plants and caused yield losses through lodging of the plants. 51

 48 Christensen. 2002. Pg. 31. 49 Olesen et al . 2005. Pg. 1. 50 Koul et al. 2004. Pg. 29. 51 Ibid. Pg. 29.Event 5307 Corn Environmental Report

2-9 Affected Environment 

### Table 2-2 Corn Insect Pests

Growth Stage Description of Damage Pests
From planting to full emergence Seedlings are pulled up and eaten
Seeds are bored or completely hollowed out Seedcorn maggots (Delia platura)

Seedcorn beetle (Stenolophus lecontei)

Wireworms (Melanotus spp. )

Emergence to knee-high corn Stunting and wilting
Unnatural growth (stem twisting or excessive tillering)
Sandblasted leaves (leaves with speckled appearance)
Removal of plant tissue (chunks of leaves eaten, plants cut off near base, etc.) White grubs ( Cyclocarpa spp., Papillia japonica

, Phyllophaga spp.)
Grape colaspis (Colaspis brunnea)
Chinch bug (Blissus leucopterus)

Black cutworm (Agrotis ipsilon)
Stink bugs, several species

Stalk Borer (Papaipema nebris)
Thrips, several species

Corn flea beetle (Chaetocnema ectypa)
Sod webworm, several species

Southern corn leaf beetle (Myochorus denticollis)
Billbugs, several species
Armyworm (Pseudaletia unipunctata)
Knee-high to tasseling corn Leaf tissues are removed (margin feeding or ragged holes in leaves, elongated lesions)
Stalks malformed (gooseneck growth)

Holes bored in the stalk Corn rootworms (Diabrotica spp. )
Fall armyworm (Spodoptera frugiperda)
Grasshopper, several species
European corn borer (Ostrinia nubilalis)

Southwestern corn borer (Diatraea grandiosella)
Stalk borer ( Papaipema nebris )
Tasseling to corn maturity Leaf tissue removed (chunks of leaf removed)

Stalks malformed or broken
Tassel damaged (tassel broken, eaten in whorl or discolored)
Silks clipped
Ear damaged (chunks of kernel removed, chewing damage, ear drop) Fall armyworm
Grasshoppers
European corn borer

Southwestern corn borer
Corn rootworm (especially western corn rootworm)
Corn leaf aphid (Rhopalosiphum maidis)

Corn earworm (Helicoverpa zea)
Japanese beetle (Popillia japonica)

Source: Summarized from O’Day et al . 1998.Event 5307 Corn Environmental Report

2-10 Affected Environment  Hybrid selection: Hybrids vary in their ability to withstand and resist insect pests such as European corn borer. Rapi d germination, early vigor, strong ear shanks, tight husks, resistance to stalk rots and other pests, strong stalks, and uniform performance over a wide populati on range are all factors influenced by hybrid genetics that may influence losses to insects. 52 Seedling insects, stalk borers, and ear feeding insects are most influenced by hybrid traits. Some hybrids have European corn borer resistance traits that reduce susceptibility to this pest. 53

 Chemical Management: Insecticides can be used selectively through spot treatments by placing the chemical in the area occupied only by the pest to be controlled. This also helps protect non- target organisms and natural enemies. Recently developed insecticides are more selective in their toxicity spectrum. Naturally derived products like Bacillus thuringiensis and spinosad affect only certain orders of insect pests and work only if they are ingested. Other products are selective because of the way they are presented to the pest. Imidacloprid, for example, works because it is absorbed and incorporated into the corn vascular system and selectively kills insects such as aphids and stink bugs that suck plant juices. Chemical management of corn rootworm includes preventive seed treatments and in-furrow soil applications. Foliar applications are made if they
are needed for adult corn rootworm beetles later in the season. In areas where corn rootworm is common, grower s basically treat routinely and prophylactically, not selectively.

 Insecticides Used in Organic Production: There are a number of insecticides approved for use in certified organic production systems, mainly non-synthetic compounds or biocontrols. Conditions for use of an insecticide must be documented under the National Organic Standard. The Organic Crops Workbook 54 lists the approved classes of insecticides used for organic production (Table 2-3) based on the USDA’s regulatory lists of approved and prohibited synthetic and non-synthetic substances. 55

 Transgenic Products: Corn seed companies have developed a number of transgenic products that incorporate insect resistance traits, as described in Section 2.1, Corn Biology . These products, alone or in combinations with other traits (such as herbicide tolerance), provide corn growers with an additional tool to combat insect pests.


52 VanDuyn et al. 2005. Pg. 2. 53 Ibid. Pg. 2. 54 ATTRA. 2003. Pgs. 28-29. 55 USDA-AMS. 2007.Event 5307 Corn Environmental Report

2-11 Affected Environment 

### Table 2-3 National List of Approved Insecticides for Organic Production Systems

Class General Description Examples Notes Botanicals Derived from plants pyrethrum, rotenone, sabadilla, neem Strychnine and nicotine are also botanicals, but are expressly prohibited under the National List; Rotenone, an insecticide approved for use in organic production, is highly toxic to fish. Biologicals Insecticides containing disease organisms or toxins derived from disease organisms Bacillus thuringiensis, Beauveria bassiana, Trichoderma harzianum, Spinosad Much like synthetic insecticides, insect pests have been observed to develop resistance to biological insecticides.

Spray Oils Vegetable or animal derived oilsPetroleum derived oils (narrowrange oils) - Commonly used to control scale and mite pests

Insecticidal

soaps Fatty acid insecticidal soaps - Although synthetic insecticides, they are allowed in organic production; Used on predatory mites

Minerals Mineral-based insectic ides Sulfur, copper products,
diatomaceous earth and kaolin clay Natural minerals like arsenic, lead, and sodium fluoaluminate are prohibited; Sulfur can reduce populations of beneficial insects; Diatomaceous earth can cause respiratory problems in animals and humans Pheromones Hormones used in products
called mating disrupters - -

Source: ATTRA. 2003.

Farmers have increasingly turned to inte grated pest management practices (IPM), which allow them to reduce energy use, environmental risk, and production costs while maintaining the quality of agricultural products and helping improve water, air, and soil quality. IPM was introduced in the late 1960s and shifted the emphasis in pest control from a single-tactic, chemically based approach to a multi-tactic, economically based system. 56 IPM is site-specific in nature, and includes prevention, avoidance, monitoring and suppression of weeds, insects, diseases, and other pests. IPM includes insect scouting or monitoring to determine pest populations; applying compatible alternative biological, cultural , mechanical and chemical controls; and establishing action thresholds for agricultur al inputs. Timely and targeted delivery of pest management interventions is key to successful IPM.



56 Koul et al . 2004. Pg. 29.Event 5307 Corn Environmental Report

2-12 Affected Environment 

### Corn Rootworm - Biology, Feeding Behavior and Economic Loss

Corn rootworms are important insect pests in the Corn Belt. There are at least four
different corn rootworm taxa in the US: western corn rootworm (D. virgifera virgifera ),
the northern corn rootworm (D. longicornis barberi ), the Mexican corn rootworm (D. virgifera zeae ), and the southern corn rootworm (D. undecimpunctata howardi ; also known as the spotted cucumber beetle).

Western and northern corn rootworms have similar life cycles. Both have a single generation each year, and corn is the only economic host. In general, corn rootworms cannot complete their life cycle without the food supplied by corn plants. Beginning in July, females lay eggs in the soil at a de pth of two to four inches near the base of the corn plant. 57 The eggs overwinter, and the onset of hatch ranges from late May to mid June. Rootworm larvae feed on corn roots for three to four weeks, passing through three growth stages (instars). The second instar larvae are often the first detected because first instars are very small (only 1/16th of an inch long). 58

Western and northern corn rootworm larvae f eed first on roots near the soil surface; when these are consumed, the next lower no de is attacked. First and second instars leave brown feeding scars (lesions) as they tunnel from root tips to the plant base, destroying root hairs and small roots. Th ird instars cause the majority of root damage and they generally feed on the la rge primary roots near the stalk. Larval corn rootworm injury results in yield losses in three ways:

 Root pruning and tunneling disrupt nutrie nts and water transport from the root system;

 Lack of root support causes goosenecking and lodging, which may limit sunlight capture by the plants and complicate harvesting; and

 Root feeding promotes invasions by seco ndary pathogens such as bacteria and fungi which increase the incidence of corn rots. 59

Additionally, adult corn rootworm beetles can cause silk clipping injury to corn plants, resulting in poor pollination and incomplete kernel set.

The Mexican corn rootworm ha s similar biology to the western corn rootworm (both

are D. virgifera subspecies), but is primarily an economic pest in Texas. 60

The corn rootworm genus is one of the most damaging corn pests. In the US, approximately 20 to 25 million acres of corn are treated annually with soil insecticides to protect crops from feeding damage caused by corn rootworm larvae. 61

 57 O’Day et al .1998. Pg. 39. 58 Ibid. Pg. 39. 59 Ibid. Pg. 40. 60 Rice. 2004. Pg. 1. 61 Roehrdanz et al . 2003. Pg. 901.Event 5307 Corn Environmental Report

2-13 Affected Environment Corn rootworm larval feeding can disrup t the movement of water and nutrients, which can slow development and stunt plants, ultimately leading to yield loss. During dry periods, when conditions suppr ess root generation, rootworm damage is amplified. In 2006 (prior to widespread adoption of transgenic crops for rootworm control), the USDA estimated that corn rootworms caused $1 billion in lost revenue each year, 62 which includes $800 million in yield loss and $200 million in treatment costs for corn growers. 63



### Corn Rootworm Management

Corn rootworm can be managed by crop rotation, insecticide application, and transgenic corn products. Aspects of each method unique to corn rootworm are described in the following paragraphs.

About 29 percent of US corn acreage is not rotated but rather is planted to continuous

corn. 64 Insecticide application rates often increase when crop rotation is decreased. 65 Until recently corn rootworm caused damage almost exclusively in fields where corn was grown at least two years in a row. In addition, new strains of corn rootworm have evolved that survive intervening years. Corn producers have used crop rotation (usually with soybeans) to control corn rootworm, but this method has some economic and biological limitations. Rotations have become less effective for the following reasons:

 A new biotype of western corn rootworm (the western variant) has appeared in central Illinois, northern Indiana, and pa rts of Michigan that can lay eggs in soybean fields. The eggs hatch in the following season, coinciding with the corn

rotation. 66 This strain has spread rapidly si nce it was first observed in 1992.

 A new northern corn rootworm biotype (the northern variant) has exhibited extended diapause in which some eggs can survive through a non-corn rotation to attack corn in a subsequent season. In South Dakota, Minnesota, Iowa, and Nebraska, the new northern corn rootworm biotype can diapause for two winters, which allows the eggs to bypass the rotated crop and hatch in time to feed on the next corn crop. 67

 Southern corn rootworm overwinters as an adult and has a varied diet, reducing the effectiveness of crop rotation in controlling this pest. 68 Larvae of southern
corn rootworm can attack more than 200 plant species, including soybeans.



62 USDA-ARS. 2006. 63 Fyksen. 2003. Pg. 1. 64 Gianessi et al . 2002. Pg. 4. 65 USDA-ERS. nd. Pg. 146. 66 Gray et al . 2009. Pg. 303. 67 Gianessi et al . 2002. Pgs. 9-10. 68 Kuepper. 2002. Pg. 8.Event 5307 Corn Environmental Report

2-14 Affected Environment The geographic distribution of the northern and western corn rootworms, and their variant populations, over the US is shown in the following map.

Source: Syngenta. 2010.

Much like resistance to crop rotation techniques, corn rootworm has developed
resistance to some chemical insecticides. 69 The EPA believes that resistance to corn rootworm insecticides may result in increased chemical use and a greater dependence on insecticides. 70

Before corn rootworm-protected Bt corn varieties were introduced in 2003, an estimated 14 million acres were treated annually with conventional insecticides to
control corn rootworms. 71 This translated to the annual application of more than 7.7 million pounds of insecticide active ingredient to corn fields for corn rootworm
control. 72 Controlling Diabrotica rootworms accounted for the largest single use of
conventional insecticides in the US at that time



### .

The EPA has registered numerous insectic ide products for the control of corn rootworm (a list of representative products is provided in Table C-1, Appendix C).
The insecticides used to control corn root worm in conventionally grown corn consist mainly of organophosphates, carbamates, synthetic pyrethroids and phenyl benzyl classes of chemistry. The majority of these products are classified as “Restricted Use.” The EPA requires application control measures for such insecticides to limit human and environmental exposure.



69 Meinke et al . 1998. Pg. 598. 70 Vlachos and Ward. 2011. Pg. 14. 71 Ibid. Pg. 14. 72 Ibid. Pg. 14.Event 5307 Corn Environmental Report

2-15 Affected Environment In organic production systems, Bt foliar sprays may be used as a natural insecticide.
However, t h e use of foliar sprays of Bt has been limited be cause of lack of persistence, limited coverage, relatively high cost, and the difficulty in determining the proper dose. 73

Several varieties of transgenic rootworm-resistant corn have been developed by seed producers; these products provide several benefits:

 Increased Root Protection : Field studies indicate that transgenic rootwormprotected corn provides as good as or better efficacy than soil insecticides in protecting corn roots from signific ant corn rootworm larval injury. 74 In an Iowa study, transgenic corn hybrids were 100 percent effective in protecting roots from economic damage (i.e., damage that would result in yield loss), whereas the insecticide was only 63 percent effective and the untreated nontransgenic hybrid offered no protection from insect damage. 75 An added benefit of transgenic rootworm-protected corn is that root pr otection does not depend upon planting time, weather influences, calibration of application equipment, or soil conditions for optimum performance.

 Reduced insecticide use. Replacing insecticides with transgenic plants reduces insecticide use against the target pest. In the period from 1996 to 2008, the volume of active ingredients in insecticides applied to corn fields was reduced by 35 percent as a result of insect resistant transgenic corn use 76 for control of European corn borer and other lepidopter an pests (beginning in 1996) and corn rootworm (beginning in 2003). In 2008 alone, the annual savings in the volume of insectide active ingredient use was 4 million kilograms. 77

 Increased farm worker safety. The majority of corn ro otworm insecticides (both granular and liquid formulations) are labeled as Restricted Use Pesticides by the EPA. These insecticides are products that, without additional regulatory restrictions, could cause unreasonable adve rse effects on wildlife and/or injury to the applicator. Transgenic insect-control technologies pose no such safety risks to applicators or the environment. Use of transgenic rootworm-protected corn decreases exposure of farm workers to ch emical insecticides. Planting transgenic rootworm-protected corn on 10 millio n acres would avoid the application of 5.3 million pounds of insecticide. 78

Transgenic corn varieties may be limited in controlling corn rootworm if the insects develop resistance to these products. To de lay evolution of resistance to transgenic crops producing Bt toxins, nearby refuges of corn not producing Bt toxins for control of the same pests are required by the EPA. The refuges promote survival of


73 Rhoush. 1994. Pg. 504. 74 Rice et al . 2004. Pg. 2. 75 Ibid. Pg. 2. 76 Brookes and Barfoot. 2010. Pg. 98. 77 Ibid. Pg. 98. 78 Rice. 2004. Pg. 5.Event 5307 Corn Environmental Report

2-16 Affected Environment susceptible pests, which mate with resistant insects and slow the evolution of

resistance. 79 Such refuges are expected to be mo st effective in slowing resistance when the toxin concentration in the adjacent Bt crops is high enough to kill all or nearly all target insects. 80

The widespread use of transgenic Bt corn could generate selection pressures for

insect resistance. 81 Although one recent study indica tes that western corn rootworm in a localized area may have developed resistance to one Cry protein under intense cultivation, 82 combining Bt proteins in the same plant that bind to different binding sites in the target pest limits the potential for resistance to develop to any one product. 83 Other insect pest resistance that has been found may be related to insufficiently high Cry protein concentrations in the transgenic crop. 84 Nonetheless,

insect resistance to Bt crops has not caused widespread failure of control measures, in part due to insect resistance management (IRM) strategies, including supplemental pesticide use and refuges. 85 In the case of Bt corn grown in the Corn Belt, refuge acres are typically 5 to 20 percent of th e grower’s corn area, depending on the product’s requirements. Greenhouse and labo ratory tests suggest that insects under intense selection pressure by Cry proteins over multiple generations may develop resistance rapidly in the absence of a refuge to sustain susceptible populations. These data in combination with the report of field resistance to a Bt product further emphasize the importance of effective refuges for resistance management. 86 Resistance management strategies, wh ich are mandated by EPA’s terms of Bt corn product registrations, have been developed for all Bt corn products to mitigate the risk of pest resistance and to implement additional measures if resistance occurs.



### 2.2.2.4 Disease and Other Pest Management

In addition to direct damage caused by feeding on plant tissue, insects play an important role in the transmission and dissemination of pathogenic organisms during corn development. Soil contains microorganisms, particularly fungi, that may infect plant parts injured by soil-dwelling insects. Primary roots of the seedling and the radical and seminal roots are commonly infected with Fusarium species after the roots have served their function and become senescent. Feeding by corn rootworms has been associated with increased frequencies of Fusarium infection; 87 rootworm feeding may also lead to increased incidences of stalk rots. These pathogen infections can reduce crop quality, harvestability, and yield.



79 Tabashnik et al . 2008. Pg. 199. 80 Meihls et al 2008. Pg. 19177. 81 Tabashnik et al . 2008. Pg. 199. 82 Gassmann et. al . 2011. Pg. 22629. 83 Tabashnik et al . 2008. Pg.201. 84 Ibid. Pg. 201. 85 Tabashnik et al . 2008. Pg. 201. 86 Meihls et al . 2008. Pg. 19177. 87 Dicke and Guthrie. 1988. Pg. 813.Event 5307 Corn Environmental Report

2-17 Affected Environment Crop disease reduces both the quantity an d quality of grain harvested. Disease loss estimates for corn production in the US range between 2 and 15 percent each year and reached 20 percent in 1970 with the southern corn leaf blight epidemic. 88 Losses estimated for diseases are difficult to determine due to the following factors:

 Variation in yield potential between different years;
 Differences in genetic background of germplasm being grown; and
 Potential for unfavorable environmental conditions.

Table C-2 (Appendix C) lists corn diseases, their pathogens, the conditions that favor the spread of disease, and current management practices. In addition, mycotoxins
such as aflatoxins and fumoni sins may accumulate in corn as it matures in the field, and inappropriate storage conditions may facilitate additional fungal deterioration
and contribute to the accumulation of aflatoxins. 89 A transgenic corn product has not yet been developed specifically to combat mycotoxins, although Bt corn used for control of ear-feeding lepidopteran pests ha s been shown to reduce the incidence of some mycotoxins in grain. 90 Minimizing insect dama ge through pest control
measures such as Bt corn can reduce the incidence of fungal infection and accumulation of the associated mycotoxins.



### 2.2.2.5 Weed Management

Weeds compete with corn for light, nutrients, and water, especially during the first 3 to 5 weeks following emergence of the crop. 91 Late-season weed infestations do not reduce corn yield nearly as much as earl y weed competition, but late-season weeds can harbor destructive insect pests. Competitive weeds include nightshade, smartweed, nutsedge, foxtail, velvetleaf , lambs-quarters, pigweed, and waterhemp. 92

Weeds can survive in crop production system s because of natural herbicide tolerance and because of growth types or life cycles th at help them avoid being treated, such as some winter annual weed species. Industry practice includes weed resistance management training to reduce the pote ntial for weeds to develop tolerance to
herbicides. 93

Weed control methods differ depending on a number of factors. No single weed control regime is effective for all growing conditions. The management practices utilized by a grower will depend on the types of pests in their field, level of infestation, cropping system, type of so il, cost, weather, time, and labor. An integrated weed management program utilizes a combination of cultural (planting),


88 Jeffers. 2004. Pg. 670. 89 Rooney et al . 2004. Pgs. 290-291. 90 Council for Biotechnology Information. 2001. Pgs. 1 and 2. 91 Wright et al . 2009. Pg. 1. 92 Hager et al . 1998. Pg. 3. 93 NCGA. 2011. Slide 2.Event 5307 Corn Environmental Report

2-18 Affected Environment mechanical (tillage), and chemical (herbici de) methods for consistent, effective weed
control. 94 Integrated weed management is a USDA policy. 95 An integrated weed management program can help prevent th e development of weed resistance to herbicides and the emergence of dominant weeds.

In 2010, herbicide active ingredients were applied to 98 percent of acres planted to

corn. 96 The most widely used herbicide was glyphosate, applied to 66 percent of the planted acreage. Atrazine was applied to 61 percent of the planted acres and acetochlor was applied to 25 percent of the planted acres. Between 1996 and 2009, the rate of glyphosate application per crop year rose 39 percent for corn 97 while broadbased herbicide usage decreased. 98

Herbicide-tolerant corn products have been genetically engineered to allow use of herbicides without harming the crop. Herb icide-tolerant corn has been widely adopted by growers in North America and of fers enhanced weed control. In 2010, approximately 70 percent of the US corn crop was herbicide-tolerant. 99 Currentlyavailable transgenic herbicide-tolerant corn cultivars include three glyphosatetolerant cultivars and four glufosinate- (phosphinothricin) tolerant cultivars. 100 However, over-reliance on herbicide-tolerant crops may under certain conditionscontribute to the development of herbicide-tolerant weeds. Before glyphosatetolerant crops were introduced, only three weed species in the world were known to have developed resistance to glyphosate. Glyphosate- or glufosinate-tolerant weeds now found in the US include common waterhemp ( Amaranthus rudis ), horseweed ( Conzya spp.), giant ragweed ( Ambrosia trifida ), common ragweed ( Ambrosia artemisiifolia ), palmer amaranth ( Amaranthus palmeri ), hairy fleabane ( Conzya

conariensis ), Italian ryegrass ( Lolium multiflorum ), rigid ryegrass ( Lolium perenne ), and Johnsongrass ( Sorghum halpense ). 101



### 2.2.3 Specialty Systems

Specialty systems include organic corn an d specialty products such as white and waxy corn, sweet corn and popcorn. These corn products comprise about 8 percent
of the US corn market. 102


94 Wright et al . 2009. Pg. 1. 95 USDA. 1990. Pg. 1. 96 USDA-NASS. 2011b. Pg. 2. 97 Benbrook. 2009. Pg. 5. 98 USEPA. 2011d. Pg. 28. 99 USDA-ERS. 2011a. 100 USDA-APHIS. 2011b. 101 Knezevic. 2010. Pg. 3. 102 USGC. 2006. Pg. 12.Event 5307 Corn Environmental Report

2-19 Affected Environment 

### 2.2.3.1 Organic Crop Production

Organic farming as defined in this report includes any production system that falls within the USDA Agricultural Marketing Service (AMS) National Organic Program (NOP) as a certified organi c production system. The NOP was established by the Organic Foods Production Act of 1990 103 and implementing USDA regulations. 104

Organic production operator s must develop and maintain an organic system plan approved by an accredited certifying agent. 105 The plan describes how the operation will achieve and document compliance with the NOP’s National Organic Standards. The NOP requires organic farming operations to have distinct, defined boundaries and buffer zones to prevent unintended contact with excluded methods from adjoining land that is not under organic management. 106 Use of synthetic insecticides, fertilizers, and transgenic crop s is excluded. Natural products, including

some Btfoliar spray insecticides, are allowed. Since organic certification is processbased, the presence of genetically modified organism residues (e.g., transgenic corn) does not by itself constitute a violation of the NOP regulations. 107 Growers use a variety of methods, including temporal and physical isolation, to prevent the commingling of organic crops with non-orga nic crops. For example organic corn may be planted at a different time than neig hboring non-organic corn to avoid crosspollination due to overlapping flowering periods, or a buffer zone may place the organic corn farther away from non-organi c corn than wind-borne non-organic corn pollen will typically travel.

An integrated strategy 108 for controlling selected insect pests in corn using organic practices might be comprised of:

 Monitoring to determine pest pressure an d need for treatment and, if necessary;

 Direct treatment of each ear with a microbial or botanical insecticide carried in vegetable oil to control corn earworm;

 Trichogramma releases and/or foliar applications of Bt or spinosad to control European corn borer; and/or

 Foliar applications of Bt or spinosad for fall armyworm control.

In 2008 (the most recent data available 109 ), approximately 168,000 acres of certified organic corn were planted (for seed, grain, or silage), 110 representing 0.20 percent of



103 Organic Foods Production Act of 1990, as amended. 104 USDA. 2011a. 7 CFR Part 205. 105 USDA. 2011a. 7 CFR Part 205, Section 201. 106 USDA. 2011a. 7 CFR Part 205, Paragraph 205.202(c). 107 USDA-AMS. 2011. Pg. 1. 108 Hazzard and Westgate. 2004. Pg. 1.Event 5307 Corn Environmental Report

2-20 Affected Environment the 93.6 million acres of corn planted in the US that year. 111 The top three states planted with organic corn for grain or seed were Wisconsin (33,619 acres), Minnesota (27,565 acres) and Iowa (25,419 acres). Organic corn grown for grain or seed produced 15,749,401 bushels from 143,432 harvested acres, 112 or an average of 109.8 bushels per acre. Comparatively, non-organic corn yielded an average of 153.9 bushels per acre in 2008. 113 On a bushels-per-acre basi s, organic corn yield is 72 percent of non-organic corn. 114 To produce the volume of organic corn that was generated in 2008 via non-organic methods would have required an additional 32,000,000 acres of planted organic corn, a 40 percent increase. Although both organic and non-organic yields have been increasing an average of 2.0 bushels per acre per year in recent decades, organic corn yields currently lag non-organic corn yields by about 21.5 years. Organic corn yields in 2011 are similar to the non-organic corn yields of 1980.



### 2.2.3.2 Seed Production

Seed corn production differs from commercial grain production because seed companies impose strict requirements to maintain seed identity and high levels of genetic purity of the final product. As described in Section 2.2.3.3, Other Specialty Production Systems , seed purity is accomplished using contracts, tracking and traceability systems, quality assurance pr ocesses, record maintenance, auditing, proper labeling, appropriate sampling and te sting and identity preservation systems.

Seed increase for a new corn variety encompasses larger and larger acreage for each successive generation. The initial generation of the new inbred line consists of one or only a few plants. These plants will produce a few ears. The seeds from these few ears are used to plant a small plot, approximately 0.1 acres. Early generation plants are self pollinated and pollen control is generally achieved by using bags over the tassels and ears. Using this system of pollen control allows many lines to be grown in a small area with an extremely low possibility of cross pollination with other lines grown in the immediate area. The seeds harvested are used to plant several acres for the next increase. Plants at this stage are also self pollinated to increase the seed; however, the area is likely too large for bagging as a means of pollen control. Pollen control is achieved by means of temporal or physical isolation from other lines. This increase produces inbred line seed. In the next stage, which produces single cross hybrid seed to be used for planting co mmercial corn grain fields, two different inbred lines (both are from the previous stage increase) are planted in the same field. One inbred line is considered the male line providing the pollen and the second line

109 The USDA-NASS conducted a detailed survey of organ ic farming in 2008. USDA-ERS and USDA-NASS both prepare annual reports of overall crop production that do not provide as detailed information about organic farms. This summary therefore compares organic and non-organic farming in 2008, using USDA-NASS and USDA-ERS data as cited. 110 USDA-NASS. 2008a. Pgs. 51-52. 111 USDA-ERS. 2010. Pg. 1. 112 USDA-NASS. 2008a. Pg. 2. 113 USDA-NASS. 2009b. Pg. 1. 114 Savage. 2011. Pg. 1.Event 5307 Corn Environmental Report

2-21 Affected Environment is the female line, from which the hybrid seed will be harvested. Procedures must be used to prevent these plants of the female line from producing viable fertile pollen.

The field sizes used for any one corn vari ety at each step of increase can vary considerably depending on the present or expected market share of each variety, the level of testing to be conducted during the increase phase, and the expected yield of the inbred lines. In the case of corn, based on current production data, the amount of seed of all varieties combined must be sufficient to plant over 90 million acres each

year.



### 2.2.3.3 Other Specialty Production Systems

Approximately 8 percent of corn grown in the US is specialty corn, which includes sweet corn, popcorn, white, waxy, hard endosperm, high oil, non-transgenic, and organic corn. 115 These corn varieties are specified by buyers and end-users of corn for production, and premiums are paid for delivering a product that meets purity and quality standards for the corn variet y. Product differentiation and market segmentation in the specialty corn industry includes mechanisms to keep track of the grain (traceability) for Identity Preservation (IP) and quality assurance processes (e.g., ISO9001-2000 certification), as well as contracts between growers and buyers that specify delivery agreements. 116 Systems used by specialty corn growers and end-users to maintain identity of the production include:

 Contracts – written agreements detailing responsibilities and duties of both parties including premiums for reaching goals and penalties for failing to attain specifications.

 Tracking and Traceability Systems – correct labeling of all products (planting seeds and harvested material) and test ing procedures for identifying and detecting acceptability of materials.

 Quality Assurance Processes – oversigh t on handling procedures, testing planting seeds, and testing harvested materials to determine acceptability of use and product requirements, and assuring testing procedures are appropriate.

 Closed-Loop Systems – the end-user supp lies the planting seeds and guarantees to purchase final products. This may also require that the end-user conduct intermediate procedures such as plan ting, providing oversight during the growing season, harvesting, and transportation to processing plant.

 Identity Preservation Systems – using syst ems of identity preservation that have been shown to be successful in the past such as the seed certification systems conducted by members of the Association of Official Seed Certifying Agencies

 115 USGC. 2006. Pg.12. 116 Sundstrom et al. 2002. Pg. 14.Event 5307 Corn Environmental Report

2-22 Affected Environment (AOSCA). 117 To maintain the purity of the corn product, this production system is based on controlling, tracking and documenting each step from seed production to end use (processing plants).



### 2.2.4 Raw and Processed Corn Commodities

Corn is processed to make it more palatable or isolate functional ingredients suitable for specific purposes. 118 Dry or wet milling is used to separate the bran and germ from the endosperm; wet milling further se parates the endosperm into its chemical components, principally starch and protein. Breeding and genetic engineering for certain traits have improved the concentrat ions of desired ingredients, but there are no differences in handling requirements for processing transgenic and non-transgenic corn except for certain products like Syngenta’s alpha-amylase (“Enogen”) corn, which requires special handling procedures to ensure that this corn is not used for unintended purposes.



### 2.2.5 Persistence in the Environment/Weediness Potential

Corn, a highly domesticated plant, 119 is dependent upon humans for survival. 120 It does not persist in the environment outside of cultivated areas and does not have a potential to develop as a weed. Corn is not listed on the Federal noxious weed list. 121 It is grown throughout the world without any report that it is a weed or that it forms persistent feral populations, although corn seed from a previous year’s crop can overwinter in fields and germinate the follo wing year in warmer areas. Manual or chemical measures are often applied to remo ve these volunteers, but the plants that are not removed do not result in feral populations in following years.

Transgenic insect-resistant corn plants are no better at establishing feral populations than non-transgenic corn. 122 A field study of transgenic insect-resistant hybrids, nontransgenic hybrids, and native Mexican la ndraces planted and allowed to naturally propagate for two years resulted in no viable plants, indicating that the populations
had died out. 123 There were no differences in the replacement capacity of transgenic corn hybrids and the nontransgenic control hybrids.

 117 AOSCA. 2011. Pg. 2. 118 Orthoefer and Eastman. 2004. Pg. 868. 119 Troyer. 2004. Pg. 134. 120 Hallauer. 2004. Pgs 899-900. 121 USDA-APHIS. 2010a. 122 Raybould et al , 2011. Pg 8. 123 Raybould et al , 2011. Pg 7.Event 5307 Corn Environmental Report

2-23 Affected Environment 

## 2.3 Physical Environment

Water, soil, and air affect, and are affe cted by, corn agriculture; the methods described in Section 2.2.2, Agronomic Practices , may reduce some adverse impacts. This section describes water quality and use, soil characteristics, and air quality impacted by corn agriculture. Climatic conditions, in the context of potential climate change, are also discussed.



### 2.3.1 Water Quality and Use

Agriculture can affect water quality and use in irrigation. The following subsections describe corn agriculture’s impact to water quality and use.



### 2.3.1.1 Water Quality

Agricultural nonpoint source (NPS) pollution is the leading source of water quality impacts to rivers and lakes, the second la rgest source of impairments to wetlands, and a major contributor to contamination of estuaries and groundwater. 124 The primary cause of NPS pollution is increased sedimentation from soil erosion. Soil erosion can introduce sediments, fertilizers, and insecticides to nearby lakes and streams when they are carried from corn fields by rain or irrigation water. 125 Insecticides or their degradates have been detected in many of the nation’s

streams, 126 and agricultural stream sites in the Corn Belt have been documented with
chemical herbicide concentrations that exceed the human-health benchmark established by the EPA. 127

Certain agronomic practices, including conservation tillage methods and reduced fertilizer or insecticide application rates, may reduce adverse impacts. The EPA
recommends 128 several Best Management Practice s for protecting water quality:

 Conservation Tillage - leaving crop residue (plant materials from past harvests) on the soil surface reduces runoff and soil erosion, conserves soil moisture, helps keep nutrients and insecticides on the field, and improves soil, water, and air quality;

 Crop Nutrient Management - fully managing and accounting for all nutrient inputs helps ensure nutrients are available to meet crop needs while reducing
nutrient movements off fields. It also helps prevent excessive buildup in soils and helps protect air quality;

 124 USEPA. 2005. Pg.1. 125 Ibid. Pg. 1. 126 Gilliom et al . 2007. Pg. 4. 127 Ibid. Pg. 6. 128 USEPA. 2008. Pg. 1.Event 5307 Corn Environmental Report

2-24 Affected Environment  Pest Management - varied methods for keeping insects, weeds, disease, and other pests below economically harmful levels while protecting soil, water, and air quality; and

 Conservation Buffers - from simple grassed waterways to riparian areas, buffers provide an additional barrier of protecti on by capturing potential pollutants that might otherwise move into surface waters.



### 2.3.1.2 Water Use

Corn requires a steady supply of moisture, totaling approximately 4,000 gallons through the growing season, to produce one bushel of grain. 129 Rainfall, stored soil moisture from precipitation before the growing season, and supplemental irrigation during the growing season provide water for corn, as described in Section 2.2.2.2, Irrigation . Supplemental irrigation was used on 12 million acres of corn fields in the US in 2008, reflecting 15 percent of all corn acres harvested for grain. 130 Groundwater is the major source for irrigation, used on almost 90 percent of irrigated corn acreage
in the US.



### Water Use by Corn Plant in th e First 100 Days After Planting

Source: Rhoades and Yonts. 2011

.
The total amount of water used by corn vari es from season to season and location to location, and is dependent on temperature, humidity, soil fertility, wind, solar radiation, total leaf area of the crop and the interaction of these factors. 131 The typical water use by corn in the first 100 days after planting, as calculated from rainfall and irrigation, is illustrated in the graph above.



129 South Dakota Corn Grower s Association. 2010. Pg. 1. 130 Christensen. 2002. Pg. 31. 131 Krantz et al . 2008. Pg. 3.Event 5307 Corn Environmental Report

2-25 Affected Environment Corn does not extract water uniformly throug hout its rooting depth. Generally, more water is extracted from shallow depths and less from deeper depths. If water is applied to the soil surface, the typical extraction pattern follows the 4-3-2-1 rule: 40 percent of the water comes from the top quarter of the root zone, 30 percent comes from the second quarter, and so on. 132



### 2.3.2 Soil

The interaction between the below-grou nd community of microorganisms and arthropods, plant-root structure, and organi c residues in the soil is central to soil ecological processes including decomposin g organic material, su bsequent nutrient cycling and release, and maintaining soil structure and composition. Cultivating corn directly impacts these biological attributes . Agronomic practices such as crop type, tillage, and pest management regime have greater effects on the biology of the soil than the type of corn that is cultivated. 133 For example, conven tional tillage and mechanized harvesting machinery may distur b and expose the top soil surface layer, leaving the land prone to degradation. Soil degradation can lead to a decline in water quality and contribute to the greenhouse effect. 134 A decline in soil quality and soil resilience enhances the greenhouse effect through emissions of radiatively active gases (CO 2 , nitrous oxide [NO X ]). 135 Land that is prone to degradation is also more likely to adversely impact water resource quality and communities of organisms dependent on those water resources.

Conservation tillage methods, including no-till, ridge-till, low-till and minimum-till, leave a crop mulch on the ground to provid e a protective cover to the soil between seasons and improve soil fertility by maintaining nutrient-rich organic matter on the
field. 136 Organic matter builds up in the soil, absorbing CO 2 and helping to reduce a significant amount of greenhouse gas.



### 2.3.3 Air Quality

Air quality may potentially be directly affe cted by agricultural activities such as burning, tilling, harvesting, spraying , and fertilizing. Smoke from burning agricultural waste releases particulates. Tilling and harvesting with motorized equipment release emissions that include ca rbon monoxide, nitrogen oxides, reactive organic gases, particulate matter, sulfur oxides, and greenhouse gases (GHG). 137 Tillage also releases GHG because of the loss of CO 2 to the atmosphere, and the exposure and oxidation of soil organic matter. 138 Aerial application of insecticides may cause impacts from drift and diffusion. Insecticides may volatilize after



132 Krantz et al. 2008. Pg. 4. 133 Griffiths et al . 2007. Pg. 196. 134 Lal and Bruce. 1999. Pg. 177. 135 Ibid. Pg. 178. 136 NCGA. 2007. Pg. 1. 137 USEPA. 2011b. Pgs. 6-17. 138 Baker et al . 2005. Pg. 11260.Event 5307 Corn Environmental Report

2-26 Affected Environment application to soil or plant surfaces and move following wind erosion. 139 NO X may also be released following nitrogen fertilizer application. 140 Agriculture, including land-use changes for farming, is responsibl e for an estimated 6 percent of all humaninduced GHG emissions in the US. 141 NO X emissions from agricultural soil management comprise 68 percent of all US N 2 O emissions. 142



### 2.3.4 Climate

Corn agriculture may potentially affect or be affected by climate change. 143, 144 Agriculture-related activities are recogniz ed as both direct (e.g., exhaust from motorized equipment) and indirect (e.g., agricultural-related soil disturbance)
sources of GHGs. 145 Greenhouse gases collectively function as retainers of solar radiation and contribute to climate change. The agricultural sector is identified as the second largest contributor to GHG emissions in the US, ranking only behind the energy sector. 146

Climate change may also potentially affect agricultural crop production. These potential impacts on the agro-environment and individual crops may be direct, including changing patterns in precipitation, temperature, and duration of growing season, or may cause indirect impacts ex tending the ranges of weeds and other
pests. 147 One recent study 148 of aggregate North American impacts on agriculture from climate change projects yield increases of 5 to 20 percent for this century, while other data suggest a reduction in yields. 149 Certain regions of the US are likely to be more heavily impacted than others beca use water resources may be substantially reduced. North American production is expected to adapt with improved cultivars and responsive farm management 150



## 2.4 Biological Environment

The biological environment described in this section includes animals, plants, biodiversity, and corn gene movement. Threatened and endangered species are
discussed in Section 2.5.


139 Vogel et al . 2008. Pg. 1101. 140 Aneja et al . 2009. Pg. 4236. 141 USEPA. 2011b. Pg. 6-1. 142 Ibid. Pg. 6-1. 143 Iserman. 1994. Pg. 106. 144 Aneja et al . 2009. Pg. 4238. 145 USEPA. 2011b. Pg. 6-17. 146 USEPA. 2011b. Pg. 2-23. 147 IPCC. 2007. Pg. 48. 148 Ibid. Pg. 48. 149 Gillis. 2011. Pg. 7. 150 IPCC. 2007. Pg. 52.Event 5307 Corn Environmental Report

2-27 Affected Environment 

### 2.4.1 Animals

Animals that may be found in corn fields include birds, large and small mammals, and invertebrates. Reptiles and amphibians, although potentially present, have little reported effect on corn.

Birds can be beneficial or detrimental to the agro-environment. Although many birds visit row crop fields such as corn, resident numbers are low and few nest there, likely due to overlap between nesting phenology and mechanized harvest. 151 Bird species that have been observed in row crop fiel ds during the growing season include redwinged blackbirds ( Agelaius phoeniceus ), brown-headed cowbirds ( Molothrus ater ), and vesper sparrows ( Pooecetes gramineus ). 152 Red-winged blackbirds are often initially attracted to corn fields to feed on insect pests, but then also feed on corn seeds. Red-winged blackbirds can destroy mo re than 360,000 tons of field corn and
substantial amounts of sweet corn annually. 153 Other species, such as horned larks ( Eremophila alpestris ), may use fallow fields as foraging habitat. 154

Throughout the latter half of the growing season, field corn functions as food and cover for white-tailed deer ( Odocoileus virginianus ). 155 Deer can significantly damage or completely destroy small corn fields that are surrounded by woody or brushy areas. Deer damage to large corn fields is often limited to a few rows closest to the
wooded areas.

Corn fields are utilized for feeding and cover by small mammals such as raccoons ( Procyon lotor ), woodchuck ( Marmota monax ), deer mice ( Peromyscus maniculatus ), meadow voles ( Microtus pennsylvanicus ), and thirteen-lined ground squirrels ( Spermophilus tridecemlineatus ). Raccoons can damage fiel d corn. The deer mouse is
the most common small mammal in some corn production regions. Deer mice feed on a wide variety of plant and animal matter, but feed primarily on seeds and insects. They are considered beneficial in agroecosystems because they consume both weed and pest insect species. The meadow vole feeds primarily on fresh grass, sedges, and herbs, but also on seeds and grains. Meadow voles also can be considered beneficial for their role in the consumption of weeds, but can be an agricultural pest where abundant. The thirt een-lined ground squirrel feeds primarily on seeds of weeds and crops such as corn and wheat. Thirteen-lined ground squirrels have the potential to damage agricultural cr ops, although they can also be beneficial when eating pest insects such as grasshoppers and cutworms.

Many of the invertebrate organisms found in corn-producing areas are considered pests, such as corn earworm, European corn borer, fall armyworm, and corn rootworm. Many other invertebrates are considered beneficial. Numerous


151 Patterson and Best. 1996. Pg. 153. 152 Best et al . 1990. Pg. 89. 153 Dolbeer. 1990. Pg. 309. 154 Best et al . 1990. Pg. 89. 155 Vercauteren and Hygnstrom. 1993. Pg. 218.Event 5307 Corn Environmental Report

2-28 Affected Environment invertebrates perform valuable functions such as pollinating plants (e.g., bees), contributing to the decay of organic matter and cycling soil nutrients (e.g., earthworms), and attacking other pest insects and mites (e.g., ladybird beetles). Other beneficial insects include lacewing, mi nute pirate bug, syrp hid fly, damselbug, ground beetle, praying mantis, spined soldier bug, tachinid fly, and parasitic

wasps. 156



### 2.4.2 Plants

Corn fields can be bordered by other ag ricultural fields (including other corn varieties), woodlands, or pasture and grasslands. From an agronomic perspective,
the most relevant members of a surrounding plant community are those that can behave as weeds. Corn agronomic performa nce can be reduced by weed competition for water, nutrients, and light. US corn yi elds are threatened by more than 200 weed species annually. 157

Common corn field weeds include giant foxtail ( Setaria faberi ), giant ragweed, velvetleaf ( Abutilon theophrasti ), common cocklebur ( Xanthium strumarium ), Canada thistle ( Cirsium arvense ), common lambsquarters ( Chenopodium album ), Johnsongrass, fall panicum ( Panicum dichotomiflorum ), and hairy fleabane. 158 Weeds such as giant foxtail and barnyard grass ( Echinochloa crusgalli ) have been shown to reduce corn yields by up to 14 159 and 35 160 percent, respectively. Weed management strategies that corn growers use, including strategies to address weed resistance to herbicides, are discussed in Section 2.2.2.5, Weed Management

.



### 2.4.3 Biodiversity

Biodiversity within agricultural ecosystems is strongly impacted by agricultural practices, including the type of cultivated plant and crop-specific management practices. Species diversity and abundance in corn agroecosystems may differ between transgenic, non-transgenic, and organic corn. Relative to any natural ecosystem, species abundance and richness will generally be less in intensively managed agroecosystems.



### 2.4.4 Gene Movement in the Natural Environment

Corn is self-compatible and wind-pollinated. In the US, there are no native plant species that can be pollinated by corn pollen without human intervention. However, teosinte (the wild progenitor of corn) can sometime s be found as introduced populations in botanical gardens in the US and as feral populations of Zea mexicana in Florida, Alabama,

 156 SCGO. 2007. Pgs. 1-4. 157 Heap. 2008. Pg.1. 158 Childs. 1996. Pg. 1. 159 Fausey et al . 1997. Pg.256. 160 Bosnic and Swanton. 1997. Pg. 281.Event 5307 Corn Environmental Report

2-29 Affected Environment and Maryland, 161 and Zea perennis in South Carolina. 162 Evidence of introgression of genes from corn into US teosinte populations has no t been sought but complex mechanisms of incompatibility have been described that are barriers to this potential. 163



## 2.5 Threatened and Endangered Species

This section identifies the coleoptera n species protected under the federal Endangered Species Act (ESA) 164 that occur within the region wher e corn is grown. The consideration of protected species is limited to Coleoptera, as this is the insect group to which corn rootworms belong, and the insecticidal prot ein produced in 5307 corn (eCry3.1Ab) has been shown to be active only on certain Co leoptera and not on other invertebrate or vertebrate species. The proposed action’s la ck of potential impacts to these species and their critical habitat is discussed in Section 4.5, Threatened and Endangered Species

.

Syngenta obtained data on listed coleopteran species and critical habitat from the US Fish
and Wildlife Service 165 (FWS). County-level species location information was obtained from a work product of the FIFRA Endangered Species Task Force (FESTF) including the Information Management System (IMS) an d the NatureServe Multi-Jurisdictional Database (MJD) licensed by FESTF. 166 This database contains county-level species data provided from the FWS and crop data from the USDA Census of Agriculture. This provides the best available aggregated data on federally listed species habitat and occurrence. Syngenta Crop Protection LLC is a full member of FESTF and has access to this licensed dataset. This database provides information only for extant species
locations.

Syngenta’s analysis identified a total of 17 coleopteran species listed by the FWS asEndangered or Threatened under the ESA, two of which do not occur in corngrowing regions. One coleopteran species was subsequently listed as endangered (Casey’s June beetle, Dinacoma caseyi ) but does not occur in corn-growing regions. 167 Table 2-4 lists the 15 threatened or endang ered species of Coleoptera found within corn-growing regions. Critical habitat, where established by the FWS, is also
indicated.

 161 USDA-NRCS. 2011a. Pg. 1. 162 USDA-NRCS. 2011b. Pg. 1. 163 Kermicle and Evans. 2010. Pg. 737. 164 Endangered Species Act of 1973 , as amended. 165 USFWS. 2011a. Pgs. 1-3. 166 See the FESTF website at http://www.festf.org/visitors/default.asp

. 167 USFWS. 2011b. Pg. 58954.Event 5307 Corn Environmental Report

2-30 Affected Environment 

### Table 2-4 Threatened or Endangered Coleopteran Species Occurring in Co rn-Growing Regions

Scientific Name Family Common Name ESA Status Critical Habitat

Batrisodes texansus Staphylinidae Coffin Cave mold beetle Endangered None
Batrisodes venyivi Staphylinidae Helotes mold beetle Endangered Bexar Co., TX
Brychius hungerfordii Halipidae Hungerford’s crawling water beetle Endangered None

Cicindela dorsalis dorsalis Carabidae Northeastern beach tiger beetle Threatened None

Cicindela nevadica lincolniana Carabidae Salt Creek tiger beetle Endangered Lancaster and Saunders Co., NE
Cicindela puritan Carabidae Puritan tiger beetle Threatened None
Desmocerus californicus dimorphus Cerambycidae Valley elderberry longhorn beetle Threatened Sacramento Co., CA
Elaphus viridus Carabidae Delta green ground beetle Threatened Solano Co., CA

Heterelmis comalensis Byrrhoidae Comal Springs riffle beetle Endangered Hays and Comal Co., TX
Nicrophorus americanus Silphidae American burying beetle Endangered None

Rhadine exilis Carabidae None Endangered Bexar Co., TX

Rhadine infernalis Carabidae None Endangered Bexar Co., TX
Rhadine Persephone Carabidae Tooth Cave ground beetle Endangered None
Stygoparnus comalensis Dryopidae Comal Springs dryopid beetle Endangered Hays and Comal Co., TX
Texamaurops reddelli Staphylinidae Kretschmarr Cave mold beetle Endangered none

Source: FESTF



## 2.6 Public Health

Public health concerns related to corn stem from human consumption of corn and corn products, animal (livestock) consumpt ion of feed corn and corn products, and
the indirect effect on human health and wo rker safety from laborers’ exposure to agricultural chemicals.



### 2.6.1 Human Health

Corn has no known human health risks except allergenicity. The Food Allergen Labelling and Consumer Protection Act 168 requires disclosure of the presence of eight specific food groups which are designated as “major food allerg ens” on the package
labels. 169 These eight food groups (milk, eggs, fish, crustacean shel lfish, tree nuts, peanuts, wheat and soybean) account for 90 percent of food allergic reactions in the

US. 170 Corn is not designated as a “major food allergen” and the FDA guidance on compliance with the Act does not include reference to corn or refined corn products. 171 There are no known human health e ffects from the novel proteins in

Bt corn.



168 Food Allergen Labelling and Consumer Protection Act of 2004. 169 USDHHS-FDA. 1992. Pg. 1. 170 Ibid. Pg. 1. 171 Corn Refiners Association. 2006. Pg. 1.Event 5307 Corn Environmental Report

2-31 Affected Environment At present, the prevalence of corn food allergy in the US is exceedingly low, estimated to affect no more than 0.016 percent of the general population. 172 Exposure to corn allergens can occur externally (direc t skin contact) or internally (ingestion). Some attributed symptoms of corn allerg y include dermatitis, asthma, urticaria, migraine headache, ulcerative colitis, irritable bowel disease, celiac sprue, and anaphylaxis under severe exposure. 173 Currently there is no cure for corn allergy. The only mechanism for managing the hypersensitivity is to avoid consuming foods that contain the allergen.



### 2.6.2 Animal (Livestock) Health

Corn has no known animal (livestock) health risks except from the presence of mycotoxins, which are secondary metabolites produced by certain fungi. Mycotoxins are considered unavoidable contaminants in food as there is no known technology that can completely eliminate their presence in crops. Insect damage is one factor that predisposes corn to mycotoxin contaminatio n, as insect herbivory creates stalk or kernel wounds that encourage fungal colonization. 174 Mycotoxins are of concern
worldwide because of their toxic and carc inogenic effects in humans and animals feeding on infected corn. Table 2-5 provides a list of corn mycotoxins; the two categories of most concern ar e fumonisins and aflatoxins.

Fumonisins are found exclusively in corn, wh ile aflatoxins are found in a variety of crops including corn, cotton, peanuts and some nuts. 175 Fumonisins are produced by the fungi Fusarium verticillioides and F. proliferatum. Since their first discovery in 1988, over 28 types of fumonisins have been isolated, of which fumonisin B 1 (FB 1 ) is the

most common in corn. 176 Fumonisins can be highly toxi c to animals, causing diseases such as leukoencephalomalacia in horses and pulmonary edema in swine. 177 Lepidopteran-protected Bt corn hybrids have re duced fumonisin levels. 178

Aflatoxins are produced by the fungi Aspergillus flavus and A. parasiticus , and are the most potent chemical liver carcinogens. 179 In poultry, aflatoxin consumption results in liver damage, impaired productivity and reproductive efficiency, decreased egg production in hens, inferior eggshell quality, inferior carcass quality, and increased susceptibility to disease. 180 In cattle, the primary symptoms are reduced weight gain, liver and kidney damage, and reduced milk production. 181 Extensive testing and use have identified no animal (livestock) he alth risks for transgenic corn products.


172 Ibid. Pg. 1. 173 USDHHS-FDA. 1992. Pg. 2. 174 Wu. 2006a. Pg. 121. 175 Wu. 2008. Pg. 1. 176 Wu. 2006b. Pg. 278. 177 Ibid. Pg. 278. 178 Munkvold et al . 1999. Pg. 133. 179 Wu. 2006b. Pg. 278. 180 Ibid. Pg. 278. 181 Ibid. Pg. 278.Event 5307 Corn Environmental Report

2-32 Affected Environment 

### Table 2-5 List of Corn Mycotoxins Toxic to Animals Consuming Contaminated Feed

Mycotoxin Fungi Associated Symptoms/Toxicology

Aflatoxin Aspergillus flavus, A. parasiticus liver necrosis, liver tumors, reduced growth, depressed immune response, carcinogen

Fumonisin Fusarium moniliforme, F. proliferatum equine leukoencephalomalacia, porcine pulmonary edema
Deoxynivalenol F. graminearum feed refusal, reduced weight gain, diarrhea, vomiting

Trichothecenes F. graminearum, F. culmorum, F. poae alimentary toxic aleukia, necrosis, hemorrhage, oral lesions in broiler chickens

Ochratoxins Penicillium verrucosum, Aspergillus ochraceus porcine nephropathy; various symptoms in poultry

Citrinin Penicillium spp ., Aspergillus spp

. kidney damage
Cyclopiazonic acid Penicillium spp ., Aspergillus spp

. neurotoxin
Sterigmatocystin Aspergillus spp

. , and others carcinogen, mutagen
Source: Koennig and Payne. 1999. Pg. 4.



### 2.6.3 Worker Safety

Workers engaged in corn production may encounter insecticides, herbicides, fungicides or fertilizers that may pose a work er health or safety risk unless used in accordance with the EPA-established agricult ure-specific requirements in the Worker
Protection Standard 182 that protect field workers from the hazards of chemical exposure. The Occupational Safety and Heal th Administration requires all employers to protect their employees from hazards as sociated with agricultural chemicals.

Pesticides are used on most corn acreage in the US, and changes in acreage, crops, or farming practices can affect the amounts an d types of pesticides used and thus the risks to workers. Registered pesticides, in cluding the representative products listed in Table C-1 (Appendix C), must have use restrictions that, if followed, have been determined to be protective of worker health.



## 2.7 Socioeconomics

Corn agriculture can affect socioeconomic resources such as the domestic economy, international trade economy, and the social environment. This section describes key current issues within each of these topics.



182 USEPA. 1992.Event 5307 Corn Environmental Report

2-33 Affected Environment 

### 2.7.1 Domestic Economy

Domestic demand for corn in the US comes from its use for feed, ethanol production, food, and seed, and totaled 11.1 billion bushels in the 2009/2010 marketing year. 183 Exports (as described in Section 2.6.2, Trade Economy ) added another 2 billion bushels
to total US corn demand. Demand is sati sfied almost entirely by domestic supply with few imports, the US being largely a net exporter of corn. In the 2009/2010 marketing year, feed was approximately 40 percent of US corn production; food, seed, and industrial uses were approx imately 45 percent (including ethanol production, at about 36 percent); and exports the remaining 15 percent. 184

US corn farms have increased cash receipts from $34.1 billion in 2007 to a projected $62.0 billion in 2011, representing an incr ease from 22.7 percent of the US crop market to 30.0 percent. 185 Annual cash receipts for corn as compared to all crops are
shown in Table 2-6.



### Table 2-6 Corn and Crop Cash Receipts

Year Cash Receipts ($ billions)
Corn All Crops

2007 

### 34.1 150.1

2008 

### 48.4 175.0

2009 

### 42.5 168.3

2010 

### 44.8 172.9

2011 

### 62.0 206.5
Source: USDA-ERS. 2011c. Pg. 1.

Net returns for corn farmers fluctuated between $125 and $200 per acre from the mid-1990s until the mid-2000s, but more than doubled in price to nearly $400 per
acre in the late 2000s. 186 Net returns are expected to remain near $350 per acre for the next decade. USDA-ERS attributes the recent increase and projected future high prices to ethanol demand, in part due to the renewable fuel standard component of
the Energy Policy Act

. 187 The recent average annual market price of corn 188 in the US has been relatively high, due in part to the ethanol demand:

 2008-- $4.06/bushel
 2009-- $3.55/bushel
 2010-- $5.40/bushel

 183 USDA. 2011b. Pg. 70. 184 Ibid. Pgs. 61 and 70. 185 USDA-ERS. 2011c. Pg. 1. 186 USDA-ERS. 2011b. Pg. 3. 187 Energy Policy Act of 2005

. 188 USDA-NASS. 2011d. Pg. 17.Event 5307 Corn Environmental Report

2-34 Affected Environment There is a niche market for non-transgenic food and feed in the US, as is evident from private labeling initiatives such as the Non- GMO Project. This initiative offers thirdparty product verification and labeling for non-transgenic products. 189 There also is a niche market for organic products in the US. Sales of organic products have been growing quickly, from $1 billion in 1990 to $26.7 billion in 2010, with a 7.7 percent
increase between 2009 and 2010 alone. 190 To satisfy the demand for organic corn, producers have had to adopt specific production practices to maintain and prevent the use of excluded methods as dictated by the NOP. To offset the increase in investment related to these more extensive practices, premiums are often paid for non-transgenic or organic corn. For example, August 201 1 non-organic corn in selected markets averaged $7.65 per bushel, whereas or ganic corn averaged $13.00 per bushel. 191



### 2.7.2 Trade Economy

Agribusiness is one of the world’s largest industries, employing 1.3 billion people and producing $1.3 trillion worth of goods each year. 192 The US is the largest world exporter of corn, averaging nearly 50 mil lion metric tons per year between 1990 and

2010. 193 US corn exports peaked at 61 million metric tons in 2007/08. 194 In 2010, the total value of US corn exports was nearly $8 billion. 195 During the last half decade, the US share of world corn exports averaged 60 percent; the second largest exporter is Argentina. Japan is the world’s largest corn importer, typically followed by South Korea, Mexico, Egypt, and Taiwan. 196

The primary US corn export destinations ar e also the largest world importers of corn and do not have major barriers for im porting transgenic products. Data on international trade in organic corn are not readily available but trade in organic corn is likely to be a very small share of the total corn trade.

The USDA Interagency Agricultural Proj ections Committee forecasts near-term production increases for grain products in general, in response to recent global supply-and-demand conditions. 197 Prices are projected to decline globally in the short term as production expands. Steady gr owth in production and historically high prices are projected long term, through 20 20. Assuming that current subsidies and tariffs remain in effect, corn is expected to remain the primary feedstock for US ethanol production, comprising about 36 percent of the total corn use.


189 See Non-GMO-Project website: http://www.nongm oproject.org/

. 190 OTA. 2011. Pg. 1. 191 Rodale Institute. 2011. Pg. 1. 192 Shelton et al . 2002. Pg. 870. 193 USDA. 2011b. Pg. 26. 194 USDA-ERS. 2009. Pg. 2. 195 USDA-ERS. 2011d. Pg. 1. 196 USDA-FAS. 2011. Pg. 1. 197 USDA. 2011b. Pg. 26.Event 5307 Corn Environmental Report

2-35 Affected Environment 

### 2.7.3 Social Environment

Social issues related to corn include farm er and consumer choice, as well as the structure of US corn farms. Farmers have a range of options in agronomic practices, seed products (non-transgenic, transgenic, and organic), and markets for their products. Consumers have a range of corn products to choose from in a free market system such as the US.

Genetic engineering has exerted a downward pressure on food prices by increasing agricultural productivity. Relatively lower food prices presumably allow consumers to choose between a greater variety of products that are now more affordable. Transgenic crops can positively contribute to sustainability for farmers. Traits such as herbicide tolerance and insect resistance, as described in Section 2.2, Corn Production

, offer opportunities for making agriculture more sustainable. 198 The advent of the NOP in the US has also increased consumer choice, as is evident by the rapid growth of the organic market segment.

US corn farms and their operators are similar in many respects to those of other feed grain crops. According to data from the 2003 Agricultural Resource Management Survey (ARMS), the majority of feed grain farms (84 percent) raised corn; 199 overall feed grain data are therefore generally app licable to corn farms when grain-specific data are not available. Feed grain farms operate more acres per farm, have higher gross and net incomes per farm, and have high er values of farm equity per farm than nonfeed grain farms. Feed grain operators were much more likely to list farming as their occupation and were more likely th an operators of nonfeed grain farms to operate a farm organized as a partnership or family corporation. 200 Only 29 percent of all feed grain farms specialized in feed grains, but those farms accounted for 51 percent of all feed grain production, an d most of the specialized farms were corn
farms. 201

In 2003, feed grain farms’ average annual net cash income was $45,916, compared to $8,875 for nonfeed grain crops. 202 The ratio of cash expense to gross cash income was 75 percent for feed grain farms, compared to 85 percent for nonfeed grainfarms. 203 The total household income (from all sources) for corn farm operators averaged about $70,000 in 2003, nearly $10,000 abov e the US average household income that year but nearly equal to the average income from nonfeed grain farm families. 204 Offfarm income sources include off-farm em ployment, investment income, pensions, Social Security payments, and gifts. 205 Additionally, there are four types of government payments to feed grain operators: direct; counter-cyclical and loan



198 Franke et al . 2011. Pg. 88. 199 Hoffman et al . 2007. Pg. 22. 200 Hoffman et al . 2007. Pg. 23. 201 Hoffman et al . 2007. Pg. 24 202 Hoffman et al . 2007. Pg. 22. 203 Hoffman et al . 2007. Pg. 22. 204 Hoffman et al . 2007. Pg. 24. 205 Hoffman et al. 2007. Pg. 24.Event 5307 Corn Environmental Report

2-36 Affected Environment deficiency; conservation reserve, wetl and reserve, and environmental quality incentives program payments; and other. 206 For corn operators, these programs contribute some 15 percent of their ca sh income. Direct government payments accounted for about 8 percent of average gross cash income for corn farms in 2003 207 while the other three categories comprised another 7 percent of cash income for corn
farmers. 208



206 Hoffman et al . 2007. Pg. 22. 207 Hoffman et al . 2007. Pg. 22. 208 Hoffman et al . 2007. Pg. 23.Event 5307 Corn Environmental Report

3-1 Alternatives 

# 3

Alternatives

This chapter describes the alternatives considered regarding deregulation of Event 5307 corn. To approve Syngenta’s petition for nonregulated status, APHIS
must first determine that 5307 corn is unlik ely to pose a plant pest risk. If the Plant Pest Risk Assessment (PPRA) determines that 5307 corn is unlikely to pose a plant pest risk, APHIS must conclude that 5307 corn is no longer subject to Chapter 7, Part 340 of the Code of Federal Regulations (7 CF R Part 340), or the plant pest provisions
of the PPA.



## 3.1 No Action: Continuation as a Regulated Article

Under the No Action alternative, APHIS would deny the petition and the status quo would be maintained. Event 5307 corn and progeny derived from 5307 corn would continue to be regulated articles. Event 5307 corn would be grown under USDA notification or permit and confined release conditions. It would have restricted availability to growers. Measures to ensure physical and reproductive confin ement would continue to be implemented. Growers would continue to use deregulated transgenic corn, currently comprising some 88 percent of the US corn market, or non-tran sgenic corn (including organic corn) that is
available without restrictions. APHIS could choose this alternative if there were insufficient evidence to demonstrate the lack of plant pe st risk from the unconfined cultivation of

5307 corn.

Event 5307 corn is unlikely to pose a plant pe st risk. Choosing this alternative would not satisfy the purpose and need of making a determination of plant pest risk status and responding to the petition for nonregulated status. Additionally, th is alternative would not satisfy the product’s purpose of providin g growers with an additional product to control corn rootworm. This product is expected to contribute to the trend in reduced use
of hazardous insecticides and extend the useful life of other commercially available corn rootworm-protected Bt products (Cry3Bb1, Cry34Ab1/Cry35Ab1, and modified Cry3A corn), improve worker safety, reduce the use of fossil fuels to apply chemical insecticides, provide economic benefits for growers, and increase competition in the marketplace for insect-protected seed products.Event 5307 Corn Environmental Report

3-2 Alternatives 

## 3.2 Proposed Action: Determination that Event 5307 Corn is No Longer a Regulated Article

This alternative would deregulate 5307 corn, offering growers a new cultivar that is highly resistant to larval feeding damage by three coleopteran pests:

 Western corn rootworm ( Diabrotica virgifera virgifera );
 Northern corn rootworm ( D. longicornis barberi ); and
 Mexican corn rootworm ( D. virgifera zeae ).

Corn plants derived from transformation Event 5307 will contain the gene ecry3.1Ab encoding an eCry3.1Ab protein and the gene pmi (also known as manA ) encoding the enzyme phosphomannose isomerase (PMI). The eCry3.1Ab protein is an engineered chimera of modified Cry3A (mCry3A) and Cry1Ab proteins, each of which is derived from a native gene of Bacillus thuringiensis ( Bt ), a soil bacterium. The mCry3A protein is active among certain Coleoptera, whereas Cry1Ab is active on certain Lepidoptera. The gene pmi
was obtained from Escherichia coli strain K-12 and the PMI protei n it encodes was utilized as a plant selectable marker during development of 5307 corn.

As an engineered protein, eCry3.1Ab has si milarities to other well-characterized Cry (crystal) proteins, namely mCry3A and Cry1 Ab. Cry proteins exert their insecticidal activity when they:

 Are ingested by the insect and solubilized in the insect gut;
 Are activated by specific proteolytic cleavage by midgut enzymes;
 Bind to specific receptors on the surface of the insect midgut; and
 Form ion channels in the gut membrane.

Collectively, these four processes result in disruption of the normal function of the midgut leading to the death of the insect . The eCry3.1Ab protein exhibits the same behavior as other coleopteran-active Bt Cry proteins, including alkaline solubility, cleavage by chymotrypsin, specificity of brush border membrane binding, and ion channel formation. Although mCry3A and eCry3.1Ab act on corn rootworms by the same general mechanism, eCry3.1Ab has a unique binding site in the target pest (different from mCry3A). 209 Combining eCry3.1Ab and mCry3A in a single product will not result in these proteins competing for the same binding site in the pest gut
membrane.

The mode of action of eCry3.1Ab, like th at of most other Cry proteins, is highly specific to insects and is ineffectual in mammalian or other vertebrate species. Studies conducted by Syngenta demonstrate that eCry3.1Ab is not active on species



209 Walters et al . 2010. Pg. 3086.Event 5307 Corn Environmental Report

3-3 Alternatives outside the Chrysomelidae family of Coleop tera. It demonstrates no lepidopteran activity, despite containing sequences from a lepidopteran-active protein (Cry1Ab).

Event 5307 corn will not be sold as a stand-alone product. Rather, Syngenta will “stack” the 5307 corn transgenes with other transgenes by conventional plant breeding to deliver two modes of action against corn rootworm and (at least) two modes of action against lepidopteran pests as well as herbicide tolerance. The two breeding stacks planned that include 5307 corn are Bt11 × MIR604 × TC1507 × 5307 ×
GA21 and Bt11 × MIR162 × MIR604 × TC1507 × 5307 × GA21 corn. In addition to the corn rootworm control provided by 5307 corn, the controls provided by other
constituents of these combinations are:

 A Cry1Ab protein for lepidopteran control (Bt11);
 A modified Cry3A protein for co rn rootworm control (MIR604);
 A Cry1F protein for lepido pteran control (TC1507);
 A double mutated 5-enolpyruvylshikimate-3-phosphate synthase enzyme for glyphosate tolerance (GA21);
 A Vip3Aa20 protein for lepido pteran control (MIR162); and
 A phosphinothricin acetyltransferase enzyme for glufosinate tolerance (Bt11 and TC1507).

Under the Proposed Action alternative, 5307 corn and its progeny would no longer be regulated articles. Permits issued or notifications acknowledged by APHIS would no longer be required for introducti ons of 5307 corn and its progeny.



## 3.3 Alternatives Considered But Rejected from Further Evaluation

Other alternatives were considered but rejected from further evaluation. These alternatives were considered in light of APHIS's authority under the plant pest provisions of the PPA and the regulations at 7 CFR Part 340, with respect to the purpose and need for 5307 corn. APHIS has determined that it has no regulatory authority to impose restrictions on a produc t once it concludes that the product does not pose a plant pest risk. These alternatives are discussed briefly below along with the specific reasons for rejecting each.



### 3.3.1 Prohibit Any Event 5307 Corn from Being Released

Under this alternative, APHIS would declin e to deregulate 5307 corn and prohibit the release of 5307 corn by denying any permits associated with its field testing. No such restriction has been imposed on other Bt corn products.Event 5307 Corn Environmental Report

3-4 Alternatives In enacting the Plant Protection Act , Congress found that

[D]ecisions affecting imports, exports, and interstate movement of products regulated under [the Plant Protection Act ] shall be based on sound

science.... 210

The White House Emerging Technologies Interagency Policy Coordination Committee has developed broad principles, 211 consistent with Executive Order 13563, 212 to guide the development and implementation of policies for oversight of emerging technolo gies (such as genetic engineering) at the agency level. Agencies should adhere to Executive Order 13563 and the following principle to the extent permitted by law, when re gulating emerging technologies:

[D]ecisions should be based on the best reasonably obtainable scientific, technical, economic, and other information, within the boundaries of the authorities and mandates of each agency.

Based on the PPRA and the scientific data ev aluated therein, 5307 corn is unlikely to pose a plant pest risk. Accordingly, there is no basis in science for prohibiting the release of Event 5307 corn, and this alternative is not appropriate for further
consideration.



### 3.3.2 Establish an Isolation Distance Between Event 5307 Corn and Non-Transgenic Corn

Under this alternative, APHIS would establish an isolation distance separating 5307 corn from non-transgenic corn. Because 5307 corn is unlikely to pose a plant pest risk, any alternative requiring isolation distances would be inconsistent with the APHIS’s statutory authority under the pl ant pest provisions of the PPA and regulations in 7 CFR Part 340.

The imposition of isolation distances also would not meet APHIS’s purpose and need to respond appropriately to a petition for nonreg ulated status based on the requirements in 7 CFR Part 340 and the agency’s authority un der the plant pest provisions of the PPA. Additionally, this alternative would not satisf y the product’s purpose of providing growers with an additional product to control corn rootworm. Individuals may choose on their own to use isolation distances and other management practices to minimize possible gene
movement between corn fields.



210 Plant Protection Act § 402(4). 211 White House Emerging Technologies Interagency Policy Coordination Committee. 2011. 212 Executive Order 13563. 2011.Event 5307 Corn Environmental Report

3-5 Alternatives 

### 3.3.3 Establish Geographic Rest rictions for Event 5307 Corn

Under this alternative, APHIS would restrict the production of 5307 corn to certain geographic areas based on the location of organic production systems or production systems for markets sensitive to transgenic products. This approach might or might not address concerns over possible gene movement between transgenic and non-transgenic plants. Because 5307 corn does not pose a plant pest risk, however, imposing geographic restrictions where ther e is no greater plant pest risk would not be consistent with APHIS’s statutory author ity under the plant pest provisions of the PPA and regulations in 7 CFR Part 340.

The imposition of geographic restrictions also would not meet APHIS’s purpose and need to respond appropriately to a petiti on for nonregulated status based on the requirements in 7 CFR Part 340 and the agency’s authority under the plant pest provisions of the PPA. Additionally, this alternative would not satisfy the product’s purpose of providing growers with an addi tional product to control corn rootworm. Individuals may choose on their own to ge ographically isolate their non-transgenic corn production systems from 5307 corn or to use other management practices to minimize possible gene movement between corn fields.



### 3.3.4 Require Testing for Event 5307 Corn

Under this alternative, APHIS would requ ire and provide testing to identify the presence of transgenic products in systems that produce non-transgenic products. There are no nationally established regulation s involving testing, cr iteria, or limits of transgenic material in systems that pr oduce non-transgenic products. Such a requirement would be extremely difficult to implement and maintain. Additionally, because 5307 corn does not pose a plant pest ri sk, the imposition of any type of testing requirement is inconsistent with the pl ant pest provisions of the PPA and the regulations at 7 CFR Part 340. Therefore, imposing such a requirement for 5307 corn would not meet APHIS’s purpose and need to respond appropriately to the petition in accordance with its regulatory authorities. Additionally, this alternative would not satisfy the product’s purpose of providing growers with an additional product to
control corn rootworm.



## 3.4 Comparison of Alternatives

Table 3-1 summarizes the potential environmental consequences, including cumulative effects, associated with selectio n of either the No Action or the Proposed Action alternatives. Based on the resources described in Chapter 2, Affected

Environment , an objective evaluation of the No Action and Proposed Action alternatives’ potential impacts is provided in Chapter 4, Environmental Consequences

.Event 5307 Corn Environmental Report

3-6 Alternatives 

### Table 3-1 Summary of Envi ronmental Consequences

Resource Alternative No Action Proposed Action

Corn Production Acreage and areas of corn production Unchanged Unchanged Cropping practices Unchanged Unchanged Pesticide use Unchanged Reduced Organic farming Unchanged Unchanged Specialty corn production Unchanged Unchanged
Physical Environment
Water quality and use Unchanged Unchanged Soil Unchanged Unchanged Air quality Unchanged Unchanged Climate Unchanged Unchanged
Biological Environment Animals Unchanged Unchanged Plants Unchanged Unchanged Biodiversity Unchanged Improved Gene movement Unchanged Unchanged Threatened and Endangered Species Unchanged Unchanged

Public Health Human health Unchanged Unchanged Animal (livestock) health Unchanged Unchanged Worker safety Unchanged Improved

Socioeconomics Domestic economy Unchanged Improved Trade economy Unchanged Unchanged Social environment Unchanged Unchanged

Cumulative Effects Agronomic practices Unchanged Reduced pesticide use Physical environment Unchanged Unchanged Biological environment Unchanged Improved biodiversity Threatened and endangered species Unchanged Unchanged Public health Unchanged Improved worker safety Socioeconomics Unchanged Improved domestic economyEvent 5307 Corn Environmental Report

4-1 Environmental Consequences 

# 4

Environmental

Consequences

This Chapter describes the potential en vironmental consequences of the two
alternatives under consideration: No Acti on and the Proposed Action. Under the No Action alternative, Event 5307 corn woul d continue to be a regulated article as defined by the PPA

. 213 Environmental releases of 5307 corn would be under APHIS regulation, as they have been since 2005. 214 Limited field tests would be allowed, but the product would not be marketed. Under the Proposed Action alternative, APHIS would determine that 5307 corn does not pose a plant pest risk. Event 5307 corn would no longer be regulated under the PPA and Syngenta would market this product in stacks with other transgenic co rn traits; there are no plans to market 5307 corn as a stand-alone product.



## 4.1 Introduction

This Section 4.1 describes the scope and methodology of the analysis, and assumptions adopted in Chapter 4. Sections 4.2 through 4.7 describe the potential direct and indirect impacts to environm ental and human resources from the No Action and the Proposed Action alternat ives for 5307 corn. A cumulative effects analysis for Event 5307 corn, stacked with other transgenic traits, is provided in

Section 4.8.



### 4.1.1 Scope of the Environmental Analysis

The environmental consequences of selecting either alternative may directly, indirectly, or cumulatively affect certain resources in some areas. As required by regulations implementing the National Environmental Policy Act (NEPA) promulgated by the Council on Environmental Quality 215 (CEQ), an analysis of the environmental



213 Plant Protection Act . 2000. Section 403, Definitions

. 214 Appendix A of the Petition lists permits/ authorizations in chronological order. 215 CEQ. 1978. 40 CFR Part 1500; Section 1502.16, Environmental Consequences

.Event 5307 Corn Environmental Report

4-2 Environmental Consequences consequences discusses the direct and indire ct effects of a proposed action, and their significance. APHIS’s agency-specific NEPA regulations refer to CEQ guidance in defining environmental consequences. 216 Direct effects are defined by the CEQ as those “which are caused by the action and occur at the same time and place.” 217 Indirect effects are defined as those “which are caused by the action and are later in time or farther removed in distance, but ar e still reasonably foreseeable. Indirect effects may include growth inducing effe cts and other effects related to induced changes in the pattern of land use, population density or growth rate, and related effects on air and water and other natural systems, including ecosystems.” 218 The CEQ’s definition of cumulative effects is provided in Section 4.8, Cumulative Effects

.

The potential impacts to the resources described in Chapter 2 were analyzed, including corn production practices as well as physical, biological, public health, and socioeconomic resources. The resources analyzed are of public interest or are potentially affected by the Proposed Action and are commonly addressed in APHIS’s NEPA evaluations of their plant pest risk de cisions. Resources that have no potential to be affected by the Proposed Action, such as coastal zones or noise levels, are not addressed in this report. The Proposed Acti on’s potential direct or indirect effects from 5307 corn as a stand-alone product on the analyzed resources were compared to how those resources may be affected if the No Action alternative is selected. The scope of the cumulative effects analysis is provided in Section 4.8, Cumulative Effects

.

The geographic scope of this analysis included any US land currently producing corn or producing crops that could incorporat e a corn rotation, land that could be converted from inactive cropland to acti ve cropland, and land currently in the Conservation Reserve Program (CRP) that could be removed from the program and farmed for corn. Geographic areas outside of the US were not considered in this report, except to address international trade impacts.



### 4.1.2 Methodology

The potential direct and indirect effects of the No Action alternative were evaluated by a review and analysis of readily avai lable published literature (peer-reviewed technical articles, industry information, and agency data), as well as Syngenta’s

Petition 219 and Public Interest Assessment 220 for Event 5307 corn. The potential direct and indirect effects of the Propos ed Action alternative to physical and biological resources were determined by laboratory analyses and field studies conducted by Syngenta, as described in the Petition and Public Interest Assessment, supported by literature review and analysis. Potential direct and indirect effects of the Proposed Action to public health and socioeconomic issues were determined by Syngenta’s studies described in the Public Interest Assessment, supported by


216 USDA-APHIS. 1995. 7 CFR Part 372, Section 372.4, Definitions

. 217 CEQ. 1978. 40 CFR Part 1508, Section 1508.8, Effects , Paragraph (a). 218 CEQ. 1978. 40 CFR Part 1508, Section 1508.8, Effects , Paragraph (b). 219 Vlachos and Huber. 2011. 220 Vlachos and Ward. 2011. (Appendix A.)Event 5307 Corn Environmental Report

4-3 Environmental Consequences literature review and analysis. The method ology used for the cumulative effects analysis is provided in Section 4.8, Cumulative Effects . All information sources are cited and full references are provided in Chapter 7.



### 4.1.3 Assumptions

The analysis of the potential environm ental consequences of the No Action alternative was based on the assumption that regulated field trials of 5307 corn on small plots of land, as described in the Peti tion, would be continued. As noted above, the analysis of direct or indirect effects of the Proposed Action ar e based on 5307 corn in the absence of stacking. The cumulati ve effects analysis was based on an assumption of unlimited marketing of 5307 corn as a product stacked with other transgenic traits. The consequences of bo th alternatives were evaluated using the 5307 corn phenotype described in the Petition. Laboratory, greenhouse, growth chamber, and field investigations with 5307 corn confirmed that there were no changes in seed, pollen, plant phenotype, or composition parame ters suggestive of increased plant pest risk. Assessments of the grain and forage from multiple US field sites demonstrate that 5307 corn is nutritionally and compositionally equivalent to, and as safe and nutritious as, its non-transgenic counterpart.

The environmental consequences of the two alternatives were analyzed under the assumption that the majority of farmers who produce transgenic, non-transgenic, or organic corn are using reasonable, common ly accepted best management practices for their chosen system and varieties du ring corn production. These management practices include standard planting dates, seeding rates, harvest times, and pest management methods as well as preser vation and coexistence measures. The analyses also allow for effects that could re sult if some farmers do not follow best management practices.

The environmental consequences of both the No Action and the Proposed Action
alternatives were evaluated in the contex t of existing transgenic seed products. Sixteen other transgenic corn cultivars containing Bt -derived proteins (Cry or Vip) that have a similar mode of action as the eCry3.1Ab protein of 5307 corn have been deregulated by APHIS 221 (see Table 2-1). Some of thes e are marketed as single trait products while others are marketed as st acked products; some of the cultivars are currently on the market and some have been discontinued or were never commercialized. Four include corn rootworm resistance traits (MON 863, DAS 59122-7, MON 88017 and MIR604 corn); th ese four cultivars are currently on the
market.

As of October 12, 2011, one additional petition for deregulating a corn rootworm-resistant corn cultivar (Petitio n 11-244-01p for Event DP-004114-3 corn) is currently pending an APHIS decision; this cultivar is also resistant to Lepidoptera



221 USDA-APHIS. 2011b.Event 5307 Corn Environmental Report

4-4 Environmental Consequences and tolerant of glufosinate herbicide. Additi onally, petitions for deregulation of three herbicide-tolerant and one drought-tolerant corn cultivar (MON 87427-7, DAS 40278-9, HCEM 485, and MON 87460) are pending APHIS’s determination of plant pest risk. 222



## 4.2 Corn Production

This section describes the potential impacts to agronomic practices, specialty corn systems, and raw and processed corn commo dities that may result from the No Action and Proposed Action alternatives.



### 4.2.1 Agronomic Practices

Potential changes in agronomic practices, including acreage and areas of corn production, cropping practice s, irrigation, and insect management, are described for the No Action and Proposed Action alternatives in the following paragraphs.



### 4.2.1.1 Acreage and Areas of Corn Production

The amount of transgenic corn planted in the US is increasing. Of the total corn acres planted in 2011, 88 percent were transgenic varieties, 223 up from 61 percent in

2006. 224 As described in Chapter 2, Section 2.2.1, Production and Yield , most increases in corn production are expected from yiel d growth rather than increases in planted areas. Substantive changes in corn production acreage and areas are more likely to result from market conditions and changes in federal policies than any other source.

Arable land for increased corn planting acreage could result from the return of CRP lands to agricultural production. The 2008 US Farm Bill 225 reduced CRP lands from
39.2 million acres to 32 million acres. 226 Available acreage for corn production could increase by this federal policy change. The conversion of CRP acres, if it were to occur, would be independent of the ad option of transgenic corn products.



### No Action Alternative

The acreage and areas of corn production are not likely to change as a direct or
indirect result of the No Action alternativ e. Conventional production practices that use transgenic varieties would continue to increase without granting nonregulated
status to 5307 corn under the No Action al ternative, based on current acreage trends.



222 USDA-APHIS. 2011b. 223 USDA-NASS, 2011d. Pg.25. 224 USDA-NASS. 2006. Pg. 24. 225 Food, Conservation, and Energy Act of 2008 (FCEA). 226 Ibid. Section 2103, Paragraph (3).Event 5307 Corn Environmental Report

4-5 Environmental Consequences It is anticipated that seed with transgen ic traits and non-transgenic hybrids would
continue to be available under the No Action alternative. Corn is currently produced in 49 states (all states but Alaska, accord ing to the 2002 Census of Agriculture) and under the No Action alternative the rang e of production would be unchanged. CRP acreage converted to corn production would not be affected by this alternative. Current trends in the acreage and areas of production are likely to continue to be driven by market conditions and federal policy even if 5307 corn continues to remain a regulated article. Regulated field trials of 5307 corn would only impact small plots
of land with no effect on the ac reage and areas of corn production.



### Proposed Action Alternative

The acreage and areas of corn production are not likely to change as a direct or indirect result of the Proposed Action alte rnative. In 2011, transgenic corn accounted for 88 percent of all corn acres in production in the US and, as noted above, the use of transgenic corn seed has been increasing. Mo st corn is planted in fields that have been in crop production for years, rath er than converted CRP lands. Granting nonregulated status to 5307 corn is not expe cted to alter the range of corn cultivation as the new transgenic trait (rootworm resistance) does not otherwise change the plant’s agronomic performance compared to non-transgenic varieties. 227

Event 5307 corn has normal agronomic char acteristics: there were no statisticallysignificant differences in most agronomic traits between 5307 corn and a nontransgenic, near-isogenic control hybrid. 228 Syngenta studies identified small but statistically significant differences in grain moisture, plant height, the number of heat units to 50 percent pollen shed, and grain yield. 229 However, there were no observed deficits in agronomic performance of 5307 corn.

Growers are not expected to change the acreage or areas of corn production as a result of deregulating 5307 corn. CRP acreage converted to corn production would not be affected by this alternative. Current trends in the acreage and areas of corn production are likely to continue to be driven by market conditions and federal policy if 5307 corn is deregulated.



### 4.2.1.2 Cropping Practices

Corn growers choose from many corn hybrids (including transgenic varieties) marketed by seed companies. Hybrids genera lly differ in agronomic characteristics, including disease and pest resistance and length of growing period. 230 The optimum planting dates for corn in the Corn Belt usually are in April or May but are influenced by factors such as growing locality, environmental conditions, seed


227 Vlachos and Huber. 2011. Pg. 99. 228 Vlachos and Huber. 2011. Pg. 98. 229 Vlachos and Huber. 2011. Pg. 99. 230 Olson and Sander. 1988. Pg. 648.Event 5307 Corn Environmental Report

4-6 Environmental Consequences growing period, and seed variety. Harvesting corn in the Corn Belt generally occurs from mid-to-late September through November.

Crop rotations are used to optimize soil nu trition and fertility, and reduce pathogen loads. As described in Chapter 2, Section 2.2.2.1, Cropping Practices , crop rotation is an effective measure for controlling corn rootworm, although in some areas variant rootworm populations display behavioral changes that circumvent rotation strategies. Some northern corn rootworm populations have an extended diapause that allows eggs to hatch when the crop rotation returns to corn rather than in the non-corn rotation crop in the growing season that follows corn. A variant western corn rootworm population now lays its eggs in soybean fields rather than corn fields, allowing eggs to hatch in fields rotating to corn.

Corn rootworm-resistant products may redu ce the need to rotate crops for this purpose, but other benefits of crop rotati on (e.g., increased yield) would remain. Corn-on-corn (“continuous corn”) is used by some growers in response to market demands and expectations of higher economic returns. 231, 232

Changes in rotation practices are more likely to result from market conditions than
other factors. A recent increase in corn-to- corn rotations has been attributed to the increase in corn prices due to higher demand, driven primarily by ethanol production. 233, 234 In fact, corn-on-corn rotation was used prior to the increase in
corn for ethanol. 235 Corn-on-corn rotations may provide a continual host
environment for some insects and diseases. However, in a farm on a corn-soybean rotation, continuously growing corn for multiple seasons can decrease populations of soybean pests, such as soybean cyst nematode. In some areas, corn-on-corn rotation has increased levels of fertilizer inputs. 236

Tillage practices are unlikely to change, although conservation methods have been increasingly adopted for other reasons. The use of tilling for weed control and impacts to soil moisture and carbon content, air quality, and erosion are unaffected by transgenic varieties for insect management.



### No Action Alternative

Cropping practices are not likely to change as a direct or indirect result of the No Action alternative. Cropping practices are li kely to continue to be driven by market
conditions even if 5307 corn continues to remain a regulated article. The current economics of corn production are driving the change or perceived change in crop rotation practices. Growers make choices to plant certain corn varieties and use


231 Erickson and Lowenberg-DeBoer. 2005. Pg. 1. 232 Malcolm et al . 2009. Pg. 24. 233 Hart. 2006. Pg. 5. 234 Vyn. 2006. Pg. 1. 235 Erickson and Lowenberg-DeBoer. 2005. Pgs. 1-2. 236 Sawyer. 2007. Pg. 27.Event 5307 Corn Environmental Report

4-7 Environmental Consequences certain crop rotation practices based on factors such as yield, weed and disease pressures, cost of seed and other inputs, technology fees, worker safety, potential for crop injury, and ease and flexib ility of the production system. 237, 238 Under the No Action alternative, the demands and price of corn would continue to depend on the market for field corn, and corn-to-corn ro tations would continue to be used by farmers if this cropping pr actice meets the economic and marketing strategy for the particular farmer. Regulated field trials of 5307 corn would only impact small plots of
land with no effect on cropping practices.



### Proposed Action Alternative

Cropping practices such as rotation are not likely to change as a direct or indirect result of the Proposed Action alternative. Introduction of Event 5307 corn for corn
rootworm control would not result in addi tional corn-on-corn rotation, but could allow farmers to implement corn-on-corn rotation more easily. The current economics of corn production are driving changes in crop rotation practices as farmers seek the most profitab le production method. Granting nonregulated status to 5307 corn is unlikely to change the price of corn commodities in the US. Prices would continue to be set by market demand, withou t regard to the number or type of corn varieties available on the market. Deregula ting 5307 corn is unlikely to affect a farmer’s decision to either stop using a corn-on-corn rotation, or to increase the overall use of corn-on-corn rotation as a cropping strategy with the US farming community. Deregulating 5307 co rn would not directly or indirectly impact tillage or weed control practices, and therefore soil moisture, carbon content, air quality, and
soil erosion would not be affected either



### 4.2.1.3 Irrigation

As described in Chapter 2, Section 2.2.2.2, Irrigation , supplemental irri gation is used in some corn production areas, such as the western portion of the Corn Belt during the growing season and during flowering. Irrigation rates are affected by local climate conditions (seasonal rainfall), and climate change could increase or decrease irrigation rates in some areas.



### No Action Alternative

Irrigation practices are not likely to change as a direct or indirect result of the No Action alternative. Even if 5307 corn continues to remain a regulated article, irrigation rates are likely to continue to be driven by climate conditions. Regulated field trials of 5307 corn would only impact small plots of land with no effect on irrigation rates.

 237 Olson and Sander. 1988. Pgs. 646-647. 238 Gianessi. 2005. Pg. 1.Event 5307 Corn Environmental Report

4-8 Environmental Consequences 

### Proposed Action Alternative

Irrigation practices are not likely to change as a direct or indirect result of the Proposed Action alternative. The damage inflicted by corn rootworm larvae can significantly reduce grain yield by interf ering with photosynthetic rates and by limiting the uptake of water and nutrients. 239 Reducing rootworm-caused damage could decrease, and would likely not increa se, irrigation rates. If 5307 corn is deregulated, irrigation rates are likely to continue to be affected by climate conditions. Because the eCry3.1Ab protei n prevents the negative physiological impact of root pruning by rootworms, 53 07 corn may allow more efficient use of
water and fertilizer. 240



### 4.2.1.4 Pesticide Use

As described in Chapter 2, Section 2.2.2.3, Insect Management , corn growers use a variety of methods, including applying chemical pesticides (specifically, insecticides), to control insect pests. Broad-spectrum insecticides may impact agriculturally important non-target organisms, including beneficial insects such as honeybees or insects that prey on other insects. Bt -derived products have a limited activity spectrum and affect only certain orders of insect pests. Bt -derived products are active only if they are ingested and activated by certain organisms, and they are selective because only organisms with gut receptors that bind specific Bt proteins are affected. Use of broad-spectrum or Bt -derived insecticides, however, can result in insects developing resistance to the insecticide.

Yield losses in all crops due to weeds, diseases, and insects were substantial and widespread until the introduction and adopti on of crop protection chemicals in the 1960s. Weeds compete with crops for light, nutrients, water, and other growth factors. If weeds are left uncontrolled, corn cannot be grown economically. Estimates of corn yield loss caused by pathogens have ranged from 2 to 17 percent. 241 A corn crop is susceptible to attack by a variety of insects from the time it is planted until it
is consumed as food or feed.

Before the introduction of Bt -derived corn varieties, the tools available to growers for insect control consisted of insecticide app lications, agronomic practices, and, to a limited extent, the use of corn varieties with a degree of native pest resistance. The
introduction of the first Bt corn varieties in 1996 provided growers with an effective means of limiting damage caused by the European corn borer. Prior to the introduction of corn rootworm-protected Bt corn varieties in 2003, an estimated 14 million acres were treated annually with conventional insecticides to control corn rootworms. This equated to applications of more than 7.7 million pounds of


239 Vlachos and Huber. 2011. Pg. 20. 240 Vlachos and Ward. 2011. Pg. 37. 241 Smith and White. 1988. Pg. 687.Event 5307 Corn Environmental Report

4-9 Environmental Consequences insecticide active ingredients annually in corn fields for the control of Diabrotica species. 242 Control of Diabrotica accounted for the largest si ngle use of conventional
insecticides in the US at that time.

Increased adoption of corn rootworm-prote cted corn products is of environmental importance because many of the chemical in secticides registered for corn rootworm control present potential risks to applicators, other agricultural workers, and wildlife. The soil-applied and foliar chemical insectic ides for larval and adult corn rootworm control subject to the greatest use reductio ns following the adoption of transgenic rootworm-protected corn varieties are or ganophosphates, carbamates, pyrethroids, and pyrazoles. Adoption of additional r ootworm-protected corn varieties is not expected to markedly reduce the use of in secticidal seed treatments also used to control other soil pests such as wireworms and grubs; however, these seed treatment products are applied at a lower rate per acre than the soil-applied or foliar
insecticides used for co rn rootworm control.

Corn rootworm populations have developed resistance to some insecticides and to
non-chemical control methods. 243 Resistance to some corn rootworm insecticides may result in increased chemical use. 244 As an alternative, crops engineered to produce Bt toxins that target specific pest taxa have had favorable environmental effects, particularly when replacing broad-spectrum insecticides. 245, 246 Insectresistant transgenic corn, including stacked-trait varieties with herbicide tolerance, accounted for 65 percent of corn acres planted in 2011

. 247

Rootworm-protected Bt corn hybrids have been available to US growers since 2003. The current PIP proteins registered for co rn rootworm control in field corn are Cry3Bb1 (in MON 863 and MON 88017), Cry34Ab1/Cry35Ab1 (in DAS 59122-7), and mCry3A (in MIR604). As the industry trend is towards combined-trait hybrid offerings, these Cry proteins are available in multiple combinations. From the perspective of preventing or mitigating resistance in target pest populations, the deployment of multiple corn rootworm traits in a single corn hybrid is essential to the durability of the registered PIPs in corn.

Transgenic varieties for corn rootworm cont rol are as effective, or more effective,
than chemical insecticides used for rootworm control. 248 Available data indicate that broad-spectrum insecticide use in corn agriculture has declined since the
introduction of insect-resistant corn varietie s. Some estimates indicate that reductions
in chemical insecticide use on the order of 70 249 to 75 250 percent could result from widespread adoption of corn rootworm-prote cted varieties. For example, insecticide



242 Ward et al . 2005. Pgs. 242-243. 243 Arthropod populations having pesticide resistance ar e listed on Arthropod Pesticide Resistance Database. 244 USEPA. 2010b. Pg. 122. 245 NRC. 2010. 246 Carpenter. 2011. Pg. 12. 247 USDA-ERS. 2011a. Pg. 2. 248 Gianessi. 2005. Pg. 245. 249 Oehme and Pickrell. 2003. Pg. 20. 250 Rice. 2004. Pg. 4.Event 5307 Corn Environmental Report

4-10 Environmental Consequences use for corn rootworm control could be reduced by over 5 million pounds per year if 10 million acres of transgenic corn were planted 251 (compared to approximately 92 million acres of corn planted in the US in 2011).

The widespread use of transgenic Bt corn could generate selection pressures for

insect resistance. 252 One recent study indicates that western corn rootworm in a localized area may have developed resistance to one Cry protein under intense
cultivation. 253 Nonetheless, insect resistance to Bt crops has not caused widespread failure of control measures, in part due to insect resistance management (IRM) strategies, including supplemental pesticid e use and requirements for growers to plant refuges of crop varieties lacking similar Bt traits. 254

The EPA requires refuges to minimize the potential for corn rootworms to develop resistance. The refuge requirements are product-specific and range from 5 to 20 percent of each corn field, and can be sp atial (percentage of the field area) or as a portion of the seed mix (“refuge in a bag”), depending upon the specific product. 255 Varieties with multiple transgenic traits acting against a particular pest typically have smaller percentage requirements fo r refuges than single-trait products. Additionally, the National Corn Growers Association promotes EPA-mandated IRM strategies for all Bt corn varieties, including corn rootworm-protected cultivars, to minimize the potential for the insects to develop such resistance. 256



### No Action Alternative

Trends in insecticide use are not likely to change as a direct or indirect result of the No Action alternative. Corn production, and pesticide use in corn, would remain as it is practiced today by the farming community. Insecticide use rates are likely to continue to decline even if 5307 corn continues to remain a regulated article, as growers will continue to have access to ex isting deregulated insect-resistant corn varieties. Growers make choices to use certain pesticides based on weed, insect and disease pressures, cost of seed and other inputs, technology fees, fuel costs, worker safety, potential for crop injury, and ease and flexibility of the system. 257, 258

Any environmental effects due to pesticide use in the agricultural production of corn would remain the same under the No Action alternative. The availability of other
corn rootworm-resistant varieties would continue if 5307 corn continues to remain a regulated article. Corn growers are likely to continue to use the wide spectrum of available conventional pesticides or any of the four other corn rootworm-resistant cultivars that are currently available, as listed in Table 2-1, Deregulated Transgenic


251 Rice. 2004. Pg. 4. 252 Tabashnik et al . 2008. Pg. 199. 253 Gassmann et. al . 2011. Pg. 5. 254 Tabashnik et al . 2008. Pg. 201. 255 USEPA. 2007a. Pg. 140. 256 NCGA. nd. 257 Olson and Sander. 1988. Pgs. 646-647. 258 Gianessi. 2005. Pg. 241.Event 5307 Corn Environmental Report

4-11 Environmental Consequences Corn Cultivars Containing Bt-derived Proteins , or any that may be developed and marketed in the future. Regulated field trials of 5307 corn would only impact small plots of land with little e ffect on insecticide use.



### Proposed Action Alternative

If 5307 corn is deregulated, trends in redu cing insecticide use are likely to continue. Corn growers would have a new corn rootworm-control option to use in addition to the four other corn rootworm-resistant products that are currently available, as listed in Table 2-1, Deregulated Transgenic Corn Co ntaining Bt-derived Proteins , or any that may be developed and marketed in the future. Th ere is concern about the ability of corn
rootworms to evolve resistance to contro l mechanisms that include crop rotation, chemical insecticides, and rootworm-protected Bt corn products. As 5307 corn has demonstrated efficacy against corn rootworm and the eCry3.1Ab protein operates via a novel mode of action, 5307 corn is expect ed to extend the useful life of other commercially available co rn rootworm-protected Bt corn cultivars (i.e., containing Cry3Bb1, Cry34Ab1/Cry35Ab1 or mCry3A). The availability of eCry3.1Ab as an
additional tool for rootworm control woul d also reduce the selection pressure on rootworm populations to develop resistance to other methods of control. The potential environmental consequences of reduced pesticide use from 5307 corn stacked with other transgenic corn cultivars are described in Section 4.8, Cumulative Effects

.

Granting nonregulated status to 5307 corn would provide growers with an alternative to other transgenic corn rootwo rm-protected varieties as well as use of conventional insecticides. The availability of 5307 corn is expected to contribute to
the trend in reduced use of chemical insecticides.



### 4.2.2 Specialty Corn Systems

Potential changes in specialty corn systems, such as organic farming, seed production, and other specialty production, are described for the No Action and Proposed Action alternatives in the following sections.



### 4.2.2.1 Organic Farming

Organic farming operations coexist with farming operations using non-transgenic and transgenic varieties for insect re sistance. As described in Chapter 2, Section 2.2.3.1, Organic Crop Production , the National Organic Program (NOP) requires that organic farms have distin ct, defined boundaries and buffer zones separating adjoining land not under organic management to prevent unintended contact with prohibited substances. Orga nic production operations must also develop and maintain an organic producti on system plan. Excluded production methods include methods used to genetically modify organisms or otherwiseEvent 5307 Corn Environmental Report

4-12 Environmental Consequences influence their growth and development by means not possible under natural conditions or processes.

In organic systems, the use of synthetic pesticides and fertilizers is strictly limited, and transgenic crops and inputs are prohibited. Event 5307 corn would not be approved for use in organic systems becaus e it is transgenic. Practices growers may use to exclude transgenic products include planting only organic seed, planting earlier or later than neighboring farmers who may be using transgenic crops so that the crops will flower at different times (t emporal isolation), and employing adequate isolation distances between the organic field and the fields of neighbors to minimize the chance that pollen will be carried betw een the fields (physical isolation). Organic growers must also maintain records to show that production and handling procedures comply with USDA organic standards.

Certified organic corn acreage is a small but increasing percentage of overall corn production. The most recent data indicates that the certified organic corn acreage in 2008 was approximately 195,000 acres, representing 0.21 percent of the total field

corn acreage 259 in the US. This was an increase of nearly 50 percent from the 131,000 acres in organic corn production in 2005, which represented 0.16 percent of the total field corn acreage that year. Comp aratively, transgenic corn accounted for 80 percent of the 87.3 million acres of field corn planted in 2008, 260 about 69.8 million acres. That number represents an increase in plantings for transgenic corn since 2005, when transgenic corn accounted for 52 percen t of the 81.6 million acres of field corn planted that year, 261 about 42.4 million acres.

Organic corn growers may benefit from tr ansgenic crops grown nearby. Reducing populations of insect pests in transgenic crop fields can help lower population pressure in adjacent fields, organic or not, as is evident from economic analyses of avoided costs. For example, one study found that cumulative yield losses from

Bt corn-suppressed European corn borer that would have occurred if the populations had remained at historic levels have been valued at over $4.3 billion in a 5-state region over 14 years. 262 Such area-wide pest suppression could conceivably occur following sustained use of rootworm-resistant corn varieties, thus resulting in benefits to organic corn growers.

Growers may choose to grow transgenic or non-transgenic corn, and obtain price premiums for growing varieties of corn fo r particular markets (e.g., using organic methods for corn production or producing a specialty corn variety for particular processing needs). For example, in August, 2011, non-organic corn averaged $7.65 per bushel, 263 whereas organic corn averaged $13.00 per bushel. 264 Transgenic,



259 USDA-ERS. 2010. 260 USDA-NASS. 2008b. Pg. 32. 261 USDA-NASS. 2005. Pg. 24. 262 Hutchison et al . 2011. Pg. 224. 263 USDA-NASS. 2009b. Pg.15. 264 Rodale Institute. 2011. Pg. 1.Event 5307 Corn Environmental Report

4-13 Environmental Consequences non-transgenic, and organic production sy stems can all provide benefits to the environment, consumers, and/or farm income.

Gene movement into and out of organic pr oduction systems has been managed using various types of isolation practices, such as differences in planting time (which result in differences in flowering time) or making sure that fields are distant from other compatible crops (by using isolation buffers) to minimize gene movement by pollen

transmission.

The commonly used corn production practi ces and the practical methods typically used by organic corn farmers reduce the likelihood of incidental gene movement between fields of transgenic and non-transgenic corn. These practices protect organic crops and thus maximize profits and price premiums accorded to corn under organic production. It is assumed that farmers are al ready using, or have the ability to use, these common practices.



### No Action Alternative

Organic farming practices, including fiel d management to avoid excluded methods and gene movement through pollen drift or other means, would not change as a
direct or indirect result of the No Action alternative. Organic corn seed availability would be unaffected by the No Action al ternative. Organic production systems and non-organic production systems would contin ue to coexist in accordance with NOP requirements. Organic corn production would continue to be driven by market conditions if 5307 corn continues to remain a regulated article.

The amount of transgenic corn and organic corn in the US is increasing, and current trends suggest that both production practice s will likely continue to increase. The use of coexistence measures (e.g., isolation of the farm, physical barriers or buffer zones between organic production and non-orga nic production, as well as formal communications between neighboring farms) are expected to continue. Organic
farms would continue to benefit from the insect suppression provided by proximate farms with transgenic crops. The availability of seed for corn varieties that are transgenic, non-transgenic, or used for or ganic production would remain the same. Regulated field trials of 5307 corn would only impact small plots of land with no effect on organic farming.



### Proposed Action Alternative

Organic farming practices, including fiel d management to avoid excluded methods and gene movement through pollen drift or ot her means, are not likely to change as a direct or indirect result of the Proposed Action alternative. Organic corn seed availability would be unaffect ed by deregulation of 5307 corn. Consistent with NOP standards and practices, organic and non-organic corn production systems would continue to coexist. Organic farms would continue to benefit from the insect suppression provided by proximate farms with transgenic crops. Organic cornEvent 5307 Corn Environmental Report

4-14 Environmental Consequences production would continue to be regulated by the NOP and driven by market conditions if 5307 corn is deregulated. Nonregulated status for 5307 corn would not change current practices of organic corn producers or the growth of organic corn production.

Currently, the use of transgenic corn variet ies and the use of organic corn production systems are both increasing due to market demands, and these markets would likely continue to increase under the Proposed Action alternative. Event 5307 corn incorporating the eCry3.1Ab protein would not present new and different issues than existing insect-resistant Bt corn cultivars (see Table 2-1) with respect to impacts on organic farmers. APHIS has granted nonreg ulated status to the products listed in Table 2-1 based on a finding that these other products do not pose a plant pest risk to organic or non-transgenic corn.



### 4.2.2.2 Seed Production

As described in Chapter 2, Section 2.2.3.3, Other Specialty Production Systems , seed purity is accomplished using contracts, tracking and traceability systems, quality assurance processes, closed loop systems, and identity preservation systems. Seed corn production differs from commercial grain production because seed companies impose strict requirements to maintain seed identity and high levels of genetic purity of the final product. The practices used to maintain seed purity do not vary substantively between transgenic and non-transgenic varieties.



### No Action Alternative

The availability of methods used to produce seed corn under the No Action Alternative would be the same as in current seed corn production systems.



### Proposed Action Alternative

Seed corn production methods are not likely to change as a direct or indirect result of the Proposed Action alternative. If 5307 corn is deregulated, specialty products would continue to be protected by the iden tity systems established in the industry. Event 5307 corn would be produced in a manner similar to other seed corn inbred lines and resulting hybrids. The inbreds and resulting hybrids are typically produced under identity preservation systems that in clude contracts with growers, traceability, product tracking, and process verification. Syngenta and other seed corn companies take precautions to ensure that inbred pa rent lines are not misa ppropriated by third parties. These procedures greatly minimize any chances of commingling of 5307 corn seed with other seed and, ultimately, commercial grain.Event 5307 Corn Environmental Report

4-15 Environmental Consequences 

### 4.2.2.3 Other Specialty Corn Production Systems

As with the purity processes described above for seed production, specialty corn growers and end users maintain the identity of their products by contracts, tracking and traceability systems, quality assuranc e programs, closed loop systems, and identity preservation systems.



### No Action Alternative

Other specialty corn production systems are not likely to change as a direct or indirect result of the No Action alternative. Specialty products are likely to continue to be protected by the identity-preservatio n systems established in the industry even if 5307 corn continues to remain a regulated article. Regulated field trials of 5307 corn would only impact small plots of land wi th no effect on other specialty corn production systems.



### Proposed Action Alternative

Other specialty corn production systems are not likely to change as a direct or indirect result of the Proposed Action al ternative. If 5307 corn is deregulated, specialty products would continue to be protected by the identity-preservation systems established in the industry.



### 4.2.2.4 Raw and Processed Commodities

As described in Chapter 2, Section 2.2.4, Raw and Processed Corn Commodities , thereare no differences in handling requirements for processing transgenic and nontransgenic corn.



### No Action Alternative

Raw and processed corn commodities are not likely to change as a direct or indirect
result of the No Action alternative. Raw and processed corn commodities would
continue to be handled in accordance with regulatory standards and industry practices even if 5307 corn continues to re main a regulated article. Regulated field trials of 5307 corn would only impact small plots of land with no effect on raw or processed commodities.



### Proposed Action Alternative

Raw and processed corn commodities are not likely to change as a direct or indirect result of the Proposed Action alternative. Neither a direct nor indirect plant pestEvent 5307 Corn Environmental Report

4-16 Environmental Consequences effect is foreseen on any raw or processed 5307 corn co mmodity. Based on Syngenta’s field trials, compositional profiles of grain and forage from 5307 corn hybrids are equivalent to those of the corresponding non-transgenic hybrids and commercial hybrids. 265, 266 Apart from the expected insect control benefits and associated benefits in crop yield and crop health, 5307 corn hybrids are agronomically equivalent to their non-transgenic counterparts. 267 Channeling/stewardship plans documenting product purity would remain unchanged under the Proposed Action. If 5307 corn is deregulated, raw and proc essed corn commodities would continue to be handled in accordance with regulato ry standards and industry practices.



## 4.3 Physical Environment

This section describes the potential impacts to water quality and use, soil, air quality, and climate that may result from the No Action and Proposed Action alternatives.



### 4.3.1 Water Quality and Use

Potential changes to water quality and use are described for the No Action and Proposed Action alternatives in the following sections.



### 4.3.1.1 Water Quality

As described in Chapter 2, Section 2.3.1.1, Water Quality , the primary cause of agricultural NPS pollution is increased sedimentation from soil erosion, which can introduce sediments, fertilizers, and pesticides to nearby lakes and streams (Transgenic corn is neither sediment nor fertilizer.) Agronomi c practices such as conservation tillage, crop nutrient mana gement, pest management, and conservation buffers help protect water quality from agricultural runoff.

Corn pollen may be deposited into water bodi es adjacent to corn fields, contributing to turbidity or suspended solids. However, corn pollen is heavy and wind-borne pollen densities decrease rapidly from the source. 268 Corn anthesis lasts for up to 14 days, and any pollen deposited into water bodies from adjacent fields during this period is unlikely to remain in suspension. 269 Corn plant matter could be transferred to water bodies after field harvest, pote ntially accumulating in stream or lake
sediments. 270 There are no federal water quality standards for the insecticidal proteins present in transgenic insect-resistant plant varieties.


265 Vlachos and Huber. 2011. Pg. 99. 266 Vlachos and Huber. 2011. Pg. 102. 267 Vlachos and Huber. 2011. Pg. 127. 268 Luna et al . 2001. Pg. 1556. 269 Webster et al . 1999. Pg. 698. 270 Rosi-Marshall et al . 2007. Figure 1, Potential Fate of Corn Byproducts in Streams Adjacent to Corn Fields.Event 5307 Corn Environmental Report

4-17 Environmental Consequences 

### No Action Alternative

Water quality is not likely to change as a direct or indirect result of the No Action alternative. Water quality would continue to be regulated by federal programs (some of which have been delegated to certain states) and agronomic practices to protect water quality would continue to be implemented even if 5307 corn continues to remain a regulated article. Chemical insecticide use would continue to be reduced as transgenic corn products with insect resistance traits are developed, marketed, and adopted. Regulated field trials of 5307 corn would only impact small plots of land with no effect on water quality.



### Proposed Action Alternative

Water quality is not likely to change as a direct or indirect result of the Proposed Action alternative. Water quality would continue to be regulated by federal programs (some of which have been dele gated to certain states) and agronomic practices to protect water quality would cont inue to be implemented if 5307 corn is deregulated. Event 5307 corn does not contain any substances which have the potential to affect water quality. The eCry 3.1Ab protein concentrations in 5307 corn are lowest at senescence, when grain is harvested, and any residual eCry3.1Ab protein is likely to degrade significantl y during the period between harvest and transfer of any plant matter to the waterways. 271 Chemical insecticide use would continue to be reduced as transgenic corn products with insect-resistance traits are developed, marketed, and adopted, potentially improving water quality. Chemical herbicide use is not likely to change as a result of deregulating 5307 corn; water quality would not be affected by the Proposed Action.



### 4.3.1.2 Water Use

As described in Chapter 2, Section 2.3.1.2, Water Use , the production of a bushel of corn requires some 4,000 gallons of water, by either rainfall or supplemental irrigation. Section 4.2.1.3, Irrigation , explains that transgenic corn products do not affect irrigation rates.



### No Action Alternative

Water use is not likely to change as a direct or indirect result of the No Action
alternative. Even if 5307 corn continues to remain a regulated article, irrigation rates would continue to driven by local conditions (e.g., climate, water availability, water pumping capacity, and fuel and electrical co sts). Regulated field trials of 5307 corn would only impact small plots of la nd with no effect on water use.


271 Vlachos and Huber. 2011. Pg. 139.Event 5307 Corn Environmental Report

4-18 Environmental Consequences 

### Proposed Action Alternative

Water use is not likely to change as a direct or indirect result of the Proposed Action alternative. As described in Section 4.2.1.3, Irrigation , 5307 corn does not change corn’s water requirements. If 5307 corn is deregulated, irrigation rates would continue to driven by local conditions.



### 4.3.2 Soil

As described in Chapter 2, Section 2.3.2, Soil , agronomic practices such as crop type, tillage, and pest management regimes have greater effects on the biology of the soil than the type of corn cultivated. Degraded soil structure and composition may lead to decreased water retention, a decrease in soil carbon aggregation and net positive carbon sequestration, and increased greenho use gas emissions. Conservation tillage methods can reduce these adverse effects and have been increasingly adopted by

corn growers.

Most proteins do not persist or accumulate in soil because they are inherently degradable in soils that have normal microbial populations. 272, 273 Cry proteins from B. thuringiensis are rapidly degraded in a variety of soil types and these proteins do
not accumulate. 274, 275, 276



### No Action Alternative

Soil characteristics are not likely to change as a direct or indirect result of the No Action alternative. The adoption of insect-r esistant corn varieties would be expected to continue. Corn growers would continue current agronomic practices and further adoption of conservation tillage methods is expected even if 5307 corn continues to remain a regulated article. Regulated field trials of 5307 corn would only impact small plots of land with no ef fect on soil characteristics.



### Proposed Action Alternative

Soil characteristics are not likely to change as a direct or indirect result of the Proposed Action alternative. Event 5307 corn production would not change land acreage or cultivation practices for transgenic or non-transgenic corn. Corn growers would continue current agronomic practice s and further adoption of conservation tillage methods is expected regardless of whether 5307 corn is deregulated or not. Deregulating 5307 corn would not adversely impact soil.


272 Burns. 1982. Pg. 425. 273 Marx et al . 2005. Pg. 35. 274 Mendelsohn et al . 2003. Pg.1007. 275 Dubelman et al . 2005. Pg. 915. 276 Head et al . 2002. Pg. 34.Event 5307 Corn Environmental Report

4-19 Environmental Consequences 

### 4.3.3 Air Quality

As described in Chapter 2, Section 2.3.3, Air Quality , agricultural activities such as burning, tilling, harvesting, spraying pesticides, and fertilizing, including the emissions from farm equipment, can direct ly affect air quality. With regard to transgenic corn with insect resistance tr aits, tilling, harvesting, and fertilizing practices do not vary between transgenic and non-transgenic agricultural varieties. Air quality impacts from aerial application of insecticides may vary between transgenic corn varieties with insect resi stance traits and non-transgenic varieties because less insecticide application would be needed for transgenic varieties. Aerial application of insecticides may impact air quality from drift, diffusion, and volatilization of the chemicals, as well as motor vehicle emissions from airplanes or helicopters. Section 4.2.1.4, Pesticide Use , explains that insecticide use can be reduced by transgenic insect-resistant products , with resultant air quality benefits.



### No Action Alternative

Air quality is not likely to change as a direct or indirect result of the No Action alternative. Corn growers would continue cu rrent trends in agricultural activities even if 5307 corn continues to remain a regulated article; application of insecticides
would continue to be reduced as additional insect-resistant products are adopted by growers. Regulated field trials of 5307 corn would only impact small plots of land with no effect on air quality.



### Proposed Action Alternative

Air quality is not likely to change as a direct result of the Proposed Action alternative. However, air quality would be indirectly improved if aerial application of corn rootworm pesticides is reduced. Corn growers would continue current trends in agricultural activities if 5307 corn is deregulated. Spray application of insecticides
could continue to be reduced as additional insect-resistant varieties are adopted by growers because 5307 corn would give growers another option to combat corn rootworm. Event 5307 corn production woul d not change land acreage or cultivation practices for transgenic or non-transgenic corn.



### 4.3.4 Climate

As described in Chapter 2, Section 2.3.4, Climate , agriculture-related activities are recognized as both direct (e.g., exhaus t from motorized equipment) and indirect (e.g., agriculture-related soil disturbance, fertilizer production) sources of greenhouse gases (GHGs). Transgenic crops in general have reduced the release of GHGs from agriculture, equivalent to removing 5 million cars from the roads eachEvent 5307 Corn Environmental Report

4-20 Environmental Consequences year, 277 although the portion attributable directly to insect-resistant corn products is
not known.

The climate can also affect agricultural crop production, and climate change could affect corn yields either positively or negatively. Transgenic corn products with insect resistance traits are not expected to differ from non-transgenic corn in response to climate change.



### No Action Alternative

The climate is not likely to change as a direct or indirect result of the No Action alternative. Corn growers would continue cu rrent trends in agricultural activities
even if 5307 corn continues to remain a regulated article. GHG emission reductions would continue as other transgenic varieties are adopted by growers and they seek more energy-efficient methods of agricultur e. Regulated field trials of 5307 corn would only impact small plots of land with no effect on the climate. Increases or decreases in corn yields resulting from climate change would not be affected by the
No Action alternative.



### Proposed Action Alternative

The climate is not likely to change as a direct or indirect result of the Proposed Action alternative. Event 5307 corn prod uction would not change land acreage or cultivation practices for transgenic or non-transgenic corn. Corn growers would continue current trends in agricultural ac tivities if 5307 corn is deregulated. GHG emission reductions would continue as other transgenic varieties are adopted by growers and they seek more energy-efficient methods of agriculture. Increases or decreases in corn yields resulting from climate change would not be affected by the Proposed Action alternative.



## 4.4 Biological Environment

This section describes the potential impacts to animals, plants, biodiversity, gene movement in the natural environment, and threatened and endangered species that may result from the No Action an d Proposed Action alternatives.



### 4.4.1 Animals

As described in Chapter 2, Section 2.4.1, Animals , corn fields may be temporarily or permanently occupied by invertebrates, birds, reptiles and amphibians, and mammals. Aquatic organisms may reside in water bodies adjacent to corn fields.


277 Brookes and Barfoot. 2005. Pg.195.Event 5307 Corn Environmental Report

4-21 Environmental Consequences Animals can have positive, negative, or no effect on corn production; insect-resistant corn varieties are targeted to certain invert ebrate species that negatively impact corn. Table 2-1, Deregulated Transgenic Corn Cultivars Containing Bt-derived Proteins , lists the transgenic corn cultivars containing Cry or Vip proteins for insect resistance that have been deregulated by APHIS. These cu ltivars are lepidopteran (European corn borer and other species) or coleopteran (corn rootworm) resistant.

As described in Chapter 2, Section 2.2.2.3, Insect Management , some species of corn rootworm have developed resistance to some chemical pesticides. Similarly, insects may develop resistance to the insecticidal proteins in transgenic Bt crops. However, refuges of non-transgenic corn, and stacking traits to provide multiple modes of action against the target insect, minimize the potential for insects to develop

resistance.

The following paragraphs summarize potential exposure pathways and available information on the effects of Cry proteins on non-target invertebrates, aquatic organisms, birds, and mammals. 278 Conventional broad-spectrum insecticides are potentially toxic to inverteb rates and vertebrates (including humans) as discussed in Section 2.2.2.3, Insect Management . The EPA requires application control measures for certain Restricted Use insecticides, includin g several used for corn rootworm control, to limit human and environmental exposure. Bt foliar sprays contain Cry proteins and many are approved insecticides for use in organic production systems to control moths, beetles, mosquitoes, and flies (Lepidoptera, Coleoptera, and Diptera), and exhibit low toxicity to non-target organisms. 279

Non-Target Invertebrates

Transgenic Bt crops, including corn, containing Cr y proteins could potentially affect non-target invertebrates that directly consume Bt plant material or are exposed via Cry protein residues in soil, water, or pr ey species. However, for a Cry protein to exert toxicity, the appropriate activating enzyme(s) and receptor binding sites would need to exist in the midgut of the non- target species, and sufficiently high concentrations of active Cry protein wo uld have to reach these binding sites.

Non-target arthropods typically do not feed on corn plants: most species that feed on corn plants are, by definition, pests. The more likely route of exposure to transgenic proteins is consumption of prey that have fed on transgenic corn plants or consumption of transgenic pollen if prey is scarce. 280 The concentration of Cry proteins in the prey of non-target arthropods will vary depending on the prey species, its developmental stage, and the concentration of Cry protein in plant parts on which they are feeding. Several studies have examined the concentration of a representative Cry protein (Cry1Ab) in herbivores relative to th e concentration in plants on which they are


278 Potential impacts to threatened and endangered spec ies are separately discussed in Section 4.4.5. 279 NPTN. 2000. Pgs 2-3. 280 Harwood et al . 2005. Pg. 2820.Event 5307 Corn Environmental Report

4-22 Environmental Consequences feeding. In general, the results show th at herbivorous arthropods contain lower
concentrations of Bt toxin than the plants on which they are feeding:

 Sucking insects, such as aphids, contain only trace amounts of Cry1Ab when feeding on Bt corn; 281, 282, 283, 284

 Lepidopteran larvae contain between 0.1 and 0.25 times the concentration of Cry1Ab in Bt corn on which they are feeding; 285, 286

 Thrips ( Frankliniella tenuicornis ) contain up to 0.35 times the concentration of Cry1Ab in Bt corn, although adults contain about half this amount and pupae less than 1/40th the concentration in larvae; 287 and

 Spider mites ( Tetranychus urticae ) have been found to contain between 0.7 and approximately 4.6 times the concentration of Cry1Ab in Bt corn. 288, 289, 290, 291

Multiple indicator species have been exposed to purified Bt proteins in direct feeding studies; these studies have typically not shown any hazard to the tested species, despite exposure to very high test conc entrations under “no choice” conditions where the species was continually exposed through its diet. Some laboratory studies have found an effect, but concluded thro ugh refinement of exposure models and estimates, or via field studies, that the effe ct was not adverse or not representative of
field conditions. 292

Corn rootworm predators could ingest Cry proteins when consuming corn
rootworms that have consumed Bt corn plant material. A study of a transgenic corn product expressing the Cry3Bb1 protein foun d no significant effe cts on predators for any measured parameter (including larval development, physical characteristics, and mortality). 293

Honey bees could potentially forage for corn pollen and therefore could be exposed to Cry proteins. 294 Honey bees can successfully rear young on a diet of 100 percent corn pollen; however, it is unlikely that corn pollen regularly comprises more than 50 percent of their diet. 295 Laboratory studies indicate that Cry proteins have no adverse effects on honey bees. 296



281 Head et al . 2001. Pg. 38. 282 Raps et al . 2001. Pg. 531. 283 Dutton et al . 2002. Pg. 444. 284 Obrist et al . 2006a. Pg. 42. 285 Dutton et al . 2002. Pg. 445. 286 Obrist et al 2006b. Pgs. 148-149. 287 Obrist et al . 2005. Pg. 413. 288 Dutton et al . 2002. Pg. 444. 289 Obrist et al . 2006a. Pg. 44. 290 Obrist et al, 2006b. Pg. 146. 291 Alvarez-Alfageme et al . 2008. Pg. 951. 292 Duan et al. 2010. Pg. 76. 293 Ahmad et al . 2006. Pg. 10693. 294 Severson and Parry. 1981. Pg. 99. 295 Babendreier et al . 2004. Pg. 294. 296 Duan et al . 2008. Pg. 1415.Event 5307 Corn Environmental Report

4-23 Environmental Consequences A meta-analysis of 74 laboratory studies of Cry protein toxicity and 52 field studies
of Bt crops containing the same Cry protei ns revealed that laboratory studies correctly predicted the reduced field abundance of certain non-target Lepidoptera exposed to lepidopteran-active proteins. 297 In the case of predators consuming prey that had fed on lepidopteran-active Bt plants, some laboratory tri-trophic studies
identified reduced abundances that were , nevertheless, not observed under field conditions; thus, the laboratory studies overestimated risk. However, laboratory studies incorporating tri-trophic interactions of lepidopteran-active Bt plants, herbivores, and parasitoids were better correlated with the decreased field abundance of parasitoids than were direct-exposure assays. This result is not surprising because many parasitoids are asso ciated specifically with target pests of

Bt crops. Control of pest species by Bt crops can be expected to indirectly affect the abundance of their specialist parasitoids under field conditions. In similar studies with coleopteran-acti ve Cry proteins and Bt plants, the meta-analysis revealed no
adverse effects on survival in laborato ry studies or field abundance for any functional group of non-target arthropods examined, including coleopteran and noncoleopteran species.

Transgenic insect-resistant products may reduce broad-spectrum insecticide use, as described in Section 4.2.1.4, Pesticide Use . Since the commercialization of Bt crops, there have been a substantial number of fi eld studies that have demonstrated that non-target invertebrates are generally more abundant in Bt cotton and Bt corn fields than in non-transgenic fields managed with chemical insecticides. 298 These studies demonstrate that, not only are the Bt crops not causing any unreasonable adverse effects in the environment, but arthropod prevalence and diversity is greater in

Bt crop fields.

Aquatic Organisms

A potential route of exposure of aquatic organisms to insecticidal proteins from

Bt corn is through pollen deposited into water bodies adjacent to corn fields. As explained in Section 4.3.1.1, Water Quality , corn pollen is he avy and wind-borne pollen densities decrease rapidly from the source. Any pollen deposited into water bodies from adjacent fields during the corn anthesis period (up to 14 days) is unlikely to remain in suspension. In addition, the bioactivity of transgenic proteins is likely to degrade in pollen grains deposited in water.

Unharvested corn material deposited in wate r bodies as litter could also potentially introduce Cry proteins to aquatic organism s. However, significant degradation of protein is likely to occur during the pe riod between harvest and transfer to the waterways 299 and between when the material is deposited in waterways and when it becomes palatable to aquatic organisms.



297 Duan et al. 2010. Pg. 76. 298 USEPA. 2007a. Pg. 6. 299 Vlachos and Huber. 2011. Pg. 139.Event 5307 Corn Environmental Report

4-24 Environmental Consequences Commercially raised fish may also be expose d to corn grain containing Cry proteins. About 30 percent corn grain by weight is typical in commercial fish feeds used in aquaculture. 300 Fish feed is heat-treated during preparation and it is likely that Cry proteins in feed prepared from insect-res istant corn would be at least partially denatured by heat and lose activity. Corn in fish feed is unlikely to comprise 100 percent insect-resistant corn grain. 301 The mode of action of most Cry proteins is highly specific to insects and is not biologic ally active in vertebrate species, including
fish. 302

Birds

The principal potential route of exposure of birds to Cry proteins is through ingestion of kernels. Some birds such as crows ( Corvus brachyrhynchos ), grackles ( Quiscalus quiscula ), and sandhill cranes ( Grus canadensis ) uproot sprouting corn to feed on the germinating kernels. 303, 304, 305 Red-winged blackbirds typically slit open husks with their bills and puncture kernels in the milk stage. 306 Blackbirds also forage for spilled kernels and weed seeds in corn stubble. 307 Wild birds are unlikely to consume a diet of 100 percent corn kern els. The diets of red-winged blackbirds and common grackles, for example, are comprised of up to 50 percent corn
kernels. 308, 309 The mode of action of most Cry proteins is highly specific to insects and these proteins are not biologically acti ve in vertebrate species, including birds. 310 Cry proteins are therefore are not expected to adversely affect non-target
vertebrates. 311

Mammals

The principal potential route of exposure of wild mammals to Cry proteins from

Bt corn is through ingestion of corn kernels. Rodents such as thirteen-lined ground squirrels, deer mice, house mi ce, prairie and meadow voles ( Microtus spp.), and woodchucks feed on germinating corn seed s. Larger mammals such as white-tailed deer typically nip off ear tips and raccoons chew through husks. Wild mammals are unlikely to consume a diet of 100 percent corn kernels. For example, the proportion
of corn kernels in wild rodent diets varies greatly according to species, but can be up to 73 percent. 312 The mode of action of most Cry proteins is highly specific to insects and these proteins are not biologically ac tive in vertebrate species, including

 300 NRC. 1983. Pg. 48. 301 USDA-NASS. 2011a. Pg. 25. 302 Vlachos and Huber. 2011. Pg. 79. 303 Steffey et al . 1999. Pg. 21. 304 Blackwell et al . 2001. Pg. 65. 305 Sterner et al . 2003. 306 Steffey et al . 1999. Pg. 21. 307 Linz et al . 2003. Pg. 263. 308 McNichol et al . 1979. Pg. 276. 309 Homan et al . 1994. Pg. 381. 310 Vlachos and Huber. 2011. Pg. 79. 311 Shimada et al . 2006. Pg. 1115. 312 Vlachos and Huber. 2011. Pg. 137.Event 5307 Corn Environmental Report

4-25 Environmental Consequences mammals. 313 Cry proteins are therefore are not expected to adversely affect nontarget vertebrates. 314



### No Action Alternative

Non-target species of animals would not be affected as a direct or indirect result of the No Action alternative. Animal exposure to genetically modifi ed crops containing Cry proteins would be nearly identical to current conditions even if 5307 corn continues to remain a regulated article. Approved genetically modified crops containing Cry proteins would continue to be used by growers and there would be no change in animal exposure rates outs ide of controlled environments. Pesticide usage would continue to have the same effects on animal species. Chemical insecticides would continue to be applied, following current trends in application rates, and organic corn growers would continue to apply microbial Bt foliar sprays. Regulated field trials of 5307 corn would only impact small plots of land with
controlled animal incursions.



### Proposed Action Alternative

Non-target species of animals are not likely to be affected as a direct or indirect result of the Proposed Action alternative. If 5307 corn is deregulated, animal exposure to genetically modified crops containing Cr y proteins would be similar to current
conditions. Event 5307 corn has been de monstrated to not adversely impact nontarget species.

Widespread cultivation of 5307 corn would not substantively change the overall usage of transgenic corn products with insect resistance traits. Other approved genetically modified crops containing Cry proteins have been demonstrated to not have adverse effects on animals and would continue to be used by growers. Pesticide usage would continue to have the same effects on animal species. Chemical insecticides would continue to be applied, following current trends in application rates, and organic corn growers would continue to apply microbial Bt foliar sprays. Adoption of 5307 corn may reduce the use of broad-spectrum insecticides, with a consequent reduction of potential impacts on the diversity of non-target insects. 315,

316 This potential impact is therefore addressed as a cumulative effect in Section 4.8.3.2, Biodiversity . Additionally, because it represents a new tool for corn rootworm control, the use of 5307 corn could help reduce the likelihood that rootworm resistance to chemical pesticides would increase. 317 This could help avoid future increases in the use rates of chemical insecticides that might otherwise result if current use rates became less effective in controlling rootworms.


313 Vlachos and Huber. 2011. Pg. 79. 314 Shimada et al . 2006. Pg. 1115. 315 Dively. 2005. Pg. 1267. 316 Marvier et al . 2007. Pg. 1476. 317 Vlachos and Huber. 2011. Pg. 154.Event 5307 Corn Environmental Report

4-26 Environmental Consequences Event 5307 corn is not substantively differen t in its effects on non-target species from other approved insect-resistant corn pr oducts with Cry proteins. The eCry3.1Ab protein present in 5307 corn has a unique bind ing site in the target pest but acts by the same general mechanism (i.e., pore formation in the target pest gut) as the approved Cry protein products. Syngenta’s pl ant tissue studies determined that, on a fresh-weight basis, the concentrations of eCry3.1Ab in individual samples across all locations and plant stages ranged from less than the lower limit of quantification (< LOQ 318 ) to 71.21 micrograms per gram (μg/g) in leaves, 0.40 μg/g to 9.29 μg/g in roots, 1.60 μg/g to 7.29 μg/g in kernels, < LOQ 319 to 0.09 μg/g in pollen and 1.70 μg/g to 28.64 μg/g in whole plants. 320 Activity spectrum data indicate that the insecticidal effects of eCry3.1Ab are limited to certain species of the Chrysomelidae family of Coleoptera. The eCry3.1Ab protei n demonstrates no lepidopteran activity, despite containing sequences from a lepidopteran-active protein, 321 because of the specificity of the eCry3.1Ab protein. There would be no additional risk to animals from widespread cultivation of 5307 corn.

Well-characterized modes of action, physicoc hemical properties, and results of safety studies (as summarized below) demonstrate that the eCry3.1Ab protein present in 5307 corn presents no evidence of risk of harm for avian, fish, or mammalian species. Laboratory testing has similarly shown no ad verse effects on survival associated with exposures of eCry3.1Ab in a range of non-target invertebrate species. 322

Non-Target Invertebrates

Syngenta has conducted laboratory tests on six species representing non-target invertebrates potentially exposed to eCry3.1Ab via cultivation of 5307 corn. In summary, there were no statistically significant differences in survival between treatment and control groups of:

 Honey bees ( Apis mellifera ), 323

 Spotted ladybird beetle larvae ( Coleomegilla maculata ), 324

 Second-instar flower bugs ( Orius laevigatus ), 325

 Larvae of Poecilus cupreus , a carabid beetle, 326

 Adult rove beetles ( Aleochara bilineata ), 327 or  Adult earthworms ( Eisenia fetida ). 328

Aquatic Organisms

 318 LOQ for leaves was 0.02 μg/g; Vlachos and Huber. 2011. Pg. 88. 319 LOQ for pollen was 0.08 μg/g; Vlachos and Huber. 2011. Pg. 88. 320 Vlachos and Huber, 2011. Pg. 87. 321 Vlachos and Huber. 2011. Pgs. 79 and 129. 322 Vlachos and Huber. 2011. Pg. 151. 323 Vlachos and Huber. 2011. Pg. 145. 324 Vlachos and Huber. 2011. Pg. 145. 325 Vlachos and Huber. 2011. Pg. 146. 326 Vlachos and Huber. 2011. Pg. 146. 327 Vlachos and Huber. 2011. Pg. 147. 328 Vlachos and Huber. 2011. Pg. 147.Event 5307 Corn Environmental Report

4-27 Environmental Consequences Syngenta has conducted laboratory test s on two species representing aquatic organisms potentially exposed to eCry3.1Ab via cultivation of 5307 corn. Aquatic concentrations of eCry3.1Ab are expected to be far below those at which biological activity is observed among known eCry3. 1Ab-sensitive species. In summary, there were no statistically significant differences between treatment and control groups of adult gammarid freshwater shrimp ( Gammarus fasciatus ) 329 or juvenile channel catfish ( Ictalurus punctatus ). 330

Birds

Syngenta has conducted laboratory tests on two species representing birds potentially exposed to eCry3.1Ab via cultivation of 5307 corn. In summary, there were no statistically significant differences in survival or overall performance between treatment and control groups of juvenile bobwhite quail ( Colinus virginianus ) 331 or broiler chickens ( Gallus gallus domesticus ). 332

Mammals

Syngenta has conducted laboratory tests on one species representing mammals potentially exposed to eCry3.1Ab via cultivation of 5307 corn. There were no statistically significant differences between treatment and control groups of mice ( Mus musculus ). 333

Syngenta initiated a voluntary pre-mark et consultation process with FDA and submitted a Safety and NutritionalAassessment for 5307 corn in January 2011. 334 The
assessment demonstrated that the introduced proteins, eCry3.1Ab 335 and phosphomannose isomerase (PMI), 336 in 5307 corn are not toxic to human and animal consumers and have minimal allergenic potential. Additionally, Syngenta’s assessment showed that 5307 corn is nutritionally equivalent to non-transgenic corn
and other corn in commerce.



### 4.4.2 Plants

As described in Chapter 2, Section 2.4.2, Plants , corn fields can be bordered by other agricultural fields (including other corn varieties), woodlands, or pasture and grasslands. The most agronomically impo rtant members of a surrounding plant community are those that behave as weeds. Chapter 2, Section 2.2.2.5, Weed Management , explains the range of methods that growers use for weed control, including integrated weed management. These methods may be cultural (planting),


329 Vlachos and Huber. 2011. Pg. 148. 330 Vlachos and Huber. 2011. Pg. 149. 331 Vlachos and Huber. 2011. Pg. 142. 332 Vlachos and Huber. 2011. Pg. 143. 333 Vlachos and Huber. 2011. Pg. 143. 334 Zeph and Vlachos. 2011. (Appendix B.) 335 EPA has issued a temporary tolerance exemption for eC ry3.1Ab in corn (Federal Register 76:180; Pgs. 57653- 57657; USEPA. 2011a) and Syngenta has petitioned for a non-expiring tolerance exemption. 336 EPA has granted a permanent tolerance exemption for PM I in all crops (40 CFR Part 180.1252; USEPA. 2007b).Event 5307 Corn Environmental Report

4-28 Environmental Consequences mechanical (tillage), and chemical (herbici de), or some combin ation thereof. As described in Section 2.2.5, Persistence in the Environment/ Weediness Potential

, cultivated corn is not a weed and transgenic cultivars currently on the market have not changed corn’s weediness potential.



### No Action Alternative

Plants would not be affected as a direct or in direct result of the No Action alternative. Weed management methods would continue following current trends even if 5307 corn continues to remain a regulated ar ticle. Regulated field trials of 5307 corn would only impact small plot s of land with controlled plant exposures. No impacts to plant species compared to any effects from current agronomic practices are anticipated.



### Proposed Action Alternative

Plants would not be affected as a direct or indirect result of the Proposed Action alternative. Event 5307 corn production would not change land acreage or cultivation practices for transgenic or non-transgenic corn. Event 5307 corn does not have increased persistence in the environment or weediness potential compared with non-transgenic corn. 337 No changes in impacts to non-weed plant species compared to any effects from current agronomic practi ces are anticipated. A description of the potential impacts to plants from a biodiver sity perspective from 5307 corn in stacked combinations is provided in Section 4.8, Cumulative Effects

.



### 4.4.3 Soil Microorganisms

Plant type and root exudates influence the microorganisms that colonize the rhizosphere. 338 While B. thuringiensis occurs naturally in soil, growing transgenic

Bt corn increases the amount of Cry endotoxins present in agroecosystems. 339 Cry proteins are typically expressed in all or most tissues throughout the plant’s life cycle, potentially creating routes of ex posure for soil organisms through root exudation, trophic level interactions, plant decomposition after harvest, and toxin persistence in the soil. 340 However, different effects of Bt plants on microbial communities in soil, ranging from no effects to minor and statistically significant effects, have been reported. 341

Soil bacterial communities are influenced by plant species and cultivars as well as other environmental factors, such as soil type and agricultural practices. 342 One study concluded that plant variety (but not the presence of Cry proteins) had a


337 Vlachos and Huber. 2011. Pg. 152. 338 Icoz et al . 2008. Pg. 658. 339 Blackwood and Buyer. 2004. Pg. 832. 340 Devare et al . 2004. Pg. 837. 341 Icoz et al . 2008. Pg. 657. 342 Icoz et al . 2008. Pg. 659.Event 5307 Corn Environmental Report

4-29 Environmental Consequences significant but transient effect on the numb ers of microorganisms and the activities of enzymes involved in the degradation of plant biomass, 343 while another study found no deleterious effect of growing corn-rootworm-resistant Bt corn on microbial biomass, activity, or bacterial community structure. 344

As noted in Section 4.3.2, Soil , most proteins do not persist or accumulate in soil because they are inherently degradable in soils that have normal microbial populations. 345, 346 Cry proteins derived from Bt are rapidly degraded in a variety of soil types and the proteins typically do not accumulate in soil. 347, 348, 349 Soil type has a large effect on the microbial community and availability of Cry proteins. 350 Certain Cry proteins may adsorb rapidly to clay minerals, on the clay-sized fraction of soil, on humic soils, and on complexes of montmorillonite-humic acids-aluminum hydroxypolymers. 351 Some field studies on the persistence of Cry proteins released by transgenic plants showed that Cry prot eins do not persist and degrade rapidly in soil, although a small fraction may be pr otected from biodegradation in the plant matrix or bound on surface-active particles. 352

The numbers of microorganisms and the activity of some enzymes involved in the degradation of plant biomass differ significantly by season, probably as a result of
differences in the water content of soils , ambient temperatures, and plant stage growth at the time of sampling. 353 Cry protein concentrations in the rhizosphere vary during the growth of the plant and can be affected by microbial activity, which depends in part on soil temperature and humidity. 354 A comparison of a cornrootworm-resistant Bt corn variety and a non-transgenic control corn treated with a
conventional insecticide concluded that the addition of the conventional insecticide had greater effects on the microbial func tion in soil and decaying roots than

Bt corn. 355

The EPA has concluded that Bt crops have a positive effect on soil flora compared to non-selective synthetic chemical pesticides. 356 Even though Bt is naturally ubiquitous in soil, the presence and release of Bt toxins from the aboveground and belowground parts of Bt plants may influence microbial diversity. Bt toxins have been found to be present in most tissues of Bt plants. 357 However, studies have found no differences in



343 Icoz et al . 2008. Pg. 658. 344 Devare et al . 2004. Pg. 842. 345 Burns. 1982. Pg. 425. 346 Marx et al . 2005. Pg. 35. 347 Mendelsohn et al . 2003. Pg. 1007. 348 Head et al . 2002. Pg. 34. 349 Dubelman et al . 2005. Pg. 915. 350 Blackwood and Buyer. 2004. Pg. 835. 351 Saxena and Stotzky. 2001. Pg. 1225. 352 Icoz et al . 2008. Pg. 660. 353 Icoz et al . 2008. Pg. 650. 354 Baumgarte and Tebbe. 2005. Pg. 2547. 355 Lawhorn et al . 2009. Pg. 367. 356 USEPA. 2001. Pg. IIC53. 357 Sivasupramaniam et al . 2008. Pg.547.Event 5307 Corn Environmental Report

4-30 Environmental Consequences microbial biodiversity or activity between fields cultivated with Bt corn or the corresponding non- Bt corn. 358

The Cry proteins released in root exudates of Bt corn or from the degradation of
biomass of Bt corn are not toxic to earthworms, nematodes, protozoa, bacteria, or fungi. 359 The EPA has determined that Cry proteins do not have any measurable adverse effect on microbial populations in the soil and that horizontal transfer of genes from transgenic plants to soil bacteria has not been demonstrated. 360



### No Action Alternative

Soil microorganisms would not be affected as a direct or indirect result of the No Action alternative. The use of transgenic corn producing Cry proteins and the use of
other Bt -derived insecticides would continue following current trends even if 5307 corn continues to remain a regulated ar ticle. Regulated field trials of 5307 corn would only impact small plot s of land with controlled plant exposures. No impacts to soil microorganisms compared to any effects from current agronomic practices are anticipated.



### Proposed Action Alternative

Soil microorganisms would not be affected as a direct or indirect result of the Proposed Action alternative. Syngenta conducted a laboratory study to examine the fate of eCry3.1Ab in live soil. A rapid dec line in eCry3.1Ab bioactivity was observed: no biological activity above background cont rol levels was detected after 14 days of eCry3.1Ab incubation in live soil. 361 The Cry protein present in 5307 corn does not bioaccumulate and a laboratory study demonstrated that eCry3.1Ab biological activity is rapidly degraded in healthy soils. No changes in impacts to soil microorganisms compared to any effects from current agronomic practices are anticipated.



### 4.4.4 Biodiversity

Biodiversity in an agroecosystem depends on the diversity of vegetation within and around the agroecosystem; permanence of crops within the agroecosystem; intensity of management; and the extent of isolation from natural vegetation. 362 Determining the level of biological diversity associated within any crop agroecosystem is complex because biological diversity can be defined and measured in many ways. Another difficulty with biodiversity studies is separating expected impacts from indirect impacts. For example, reductions of biological control organisms are seen in some



358 Icoz et al . 2008. Pg. 660. 359 Saxena and Stotzky. 2001. Pg. 1228. 360 USEPA. 2001. Pg. IIC15. 361 Vlachos and Huber. 2011. Pg. 135. 362 Altieri. 1999. Pg. 21.Event 5307 Corn Environmental Report

4-31 Environmental Consequences Bt- expressing transgenic crops but are caused by reduction of the pest host population rather than as a direct effect. 363

Many studies over the last 10 years have investigated the differences in biological diversity and abundance between transgenic and non-transgenic crop fields, particularly those transgenic crops that are resistant to insects (e.g., Bt crops) or herbicides (e.g., glyphosate- or glufosinate-tolerant). Conflicting results are often reported. Some studies have found negligible to modest decreases in biological diversity or abundance due to crops geneti cally engineered to produce insecticidal proteins or tolerate herbicide application for weed management. 364, 365, 366 Other studies compared Bt crops to non-transgenic crops that were unsprayed or sprayed
with insecticides and found that Bt crops do not cause any overall changes in arthropod abundance or diversity. 367, 368, 369, 370 A review of over 360 research papers concluded that there is no evidence of landscape-level effects from Bt

crops. 371 Compared to the use of broad-spectrum insecticides in agriculture, Bt crops may increase biological diversity in agroecosystems by reducing broad-spectrum insecticide use, thus allowing more non-target species to survive. 372, 373 Most transgenic crops increase the productivity of cultivated lands, so biodiversity is protected because additional land is no t needed for the same volume of crop production. 374



### No Action Alternative

Biodiversity would not be affected as a dire ct or indirect result of the No Action alternative. Biodiversity within agroecosys tems would continue to be affected by agricultural practices following current trends. Growers would continue to have access to existing deregulated insect-resis tant corn varieties, and adoption of new transgenic corn varieties would be expected to continue even if 5307 corn continues to remain a regulated article. Regulated fiel d trials of 5307 corn would only impact small plots of land with controlled animal and plant incursions. No changes in biodiversity compared to effects from current agronomic practices are anticipated.



### Proposed Action Alternative

Biodiversity would be improved as a direct result of the Proposed Action alternative, as chemical pesticide use rates would decrease because of the introduction of this insect-resitant cultivar. More non-target species would survive. Event 5307 corn

 363 USDA-APHIS. 2011a. Pg. 16. 364 Marshall et al. 2003. Pg.85. 365 Pilcher et al. 2005. Pg. 1312. 366 Ponsard et al . 2002. Pg. 1204. 367 Chen et al. 2008. Pg. 4. 368 Romeis et al . 2006. Pgs. 67-68. 369 Torres and Ruberson. 2005. Pg. 1254. 370 Wolfenbarger et al . 2008. Pg. 8. 371 Carpenter. 2011. Pg. 6. 372 Marvier et al. 2007. Pg. 1476. 373 Romeis et al. 2006. Pg. 67. 374 Raven. 2010. Pg. 528.Event 5307 Corn Environmental Report

4-32 Environmental Consequences production would not change land acreage or cultivation practices for transgenic or non-transgenic corn. Event 5307 corn incorporates a Cry protein with a similar receptor-specific mode of action as ot her Cry proteins present in approved insect-resistant corn cultivars and does not have increased persistence in the environment or weediness potential over non-transgenic corn. 375 As described in Section 4.2, Corn Production , 5307 corn does not exhibit any different agronomic traits, apart from resistance to corn rootworms, or require different agronomic practices. As described in Section 4.4.1, Animals , and Section 4.4.2, Plants , animal and plant species that typically inhabit corn production areas would be managed the same as other for other insect-resistant corn products on th e market. The potential biodiversity effects of 5307 corn when stacked with other transgenic traits are described in Section 4.8, Cumulative Effects

.



### 4.4.5 Gene Movement in the Natural Environment

Corn is a highly domesticated plant with limited gene movement in the natural environment, and gene movement from transg enic corn is not any different than that
of other cultivated corn varieties. 376 As described in Chapter 2, Section 2.2.5, Persistence in the Environment/Weediness Potential , corn is dependent upon humans for survival. Cultivated corn has limited sexual compatibility with its closest relative, teosinte, but no wild populations of teosin te exist in the US outside of some feral populations in Alabama, Florida, and Maryland. 377 Corn can only be crossed experimentally with the genus Tripsacum , and does not persist outside of cultivation. The only known propagation method for corn is through seed germination. Like many domesticated crops, corn seed from a previous year’s crop can overwinter and germinate the following year. Corn seedlings may appear in soybean fields following a corn crop in a corn-on-soybean rotation. These plants do not result in sustained populations in subsequent year s. Corn does not possess th e suite of traits that are
characteristic of successful weeds.

An extensive review of information relevant to the potential risks of horizontal gene
transfer for Bt crops to soil microbes was conducted by the EPA. No evidence of horizontal gene transfer under field co nditions was found, and there was only equivocal evidence for horizontal gene tran sfer under laboratory conditions designed to maximize the recovery of transformants. 378



### No Action Alternative

Corn’s characteristic of limited gene movement in the natural environment would
not be affected as a direct or indirect re sult of the No Action alternative. Corn breeding by traditional or transgenic means would continue even if 5307 corn


375 Vlachos and Huber. 2011. Pg. 152. 376 USDA-APHIS. 2011a. Pg. 53. 377 USDA-NRCS. 2011a. 378 USEPA. 2001. Pg. IIC16.Event 5307 Corn Environmental Report

4-33 Environmental Consequences continues to remain a regulated article. Regu lated field trials of 5307 corn would only impact small plots of land with minima l potential for exposure to sexually compatible plants (i.e., other corn pl ants). No changes in gene movement characteristics of current corn varieties are anticipated.



### Proposed Action Alternative

Corn’s characteristic of limited gene movement in the natural environment would
not be affected as a direct or indirect re sult of the Proposed Action alternative. Event 5307 corn has no more potential for gene introgression to sexually compatible wild relatives than other transgenic or non-transgenic corn varieties. 379 The codons
in the ecry3.1Ab gene are optimized for expression in plants, and hence the gene is likely to have low sequence homology with genes of soil microbes. 380 Horizontal gene transfer of ecry3.1Ab from 5307 corn to soil microbes is highly unlikely. The probability of spread of ecry3.1Ab outside corn cultivation by horizontal gene transfer is negligible. No changes in gene movement characteristics compared to current corn varieties are anticipated.



## 4.5 Threatened and Endangered Species

The Endangered Species Act 381 (ESA) establishes the federal program to protect threatened and endangered species. Once an animal or a plant is added to the list of threatened or endangered species, protecti ve measures apply to the species and its habitat. These measures include protection from adverse effects of federal activities, such as APHIS’s determination of nonregulated status for transgenic crops. Section 7(a)(2) of the ESA requires that federal agencies, in consultation with the US Fish and Wildlife Service (USFWS), ensure that any action they authorize, fund, or carry out is not likely to jeopardize the co ntinued existence of a listed species or result in the destruction or adverse modification of designated critical habitat. It is the responsibility of the federal agency taking the action to assess the effects of their action and to consult with the USFWS if it is determined that the action “may affect” listed species or critical habitat.

This section evaluates the potential for effects from cultivation of 5307 corn and its progeny on federally listed threatened and endangered species and species proposed for listing, as well as designated critical habitat and habitat proposed for designation, as required under Section 7 of the ESA. Direct effects are analyzed by considering the response that these species could have if exposed to 5307 corn. Indirect effects are
those that could result from the use of 5307 corn which would occur later in time but are still reasonably foreseeable.


379 Vlachos and Huber. 2011. Pg. 152. 380 Vlachos and Huber. 2011. Pg. 153. 381 Endangered Species Act of 1973, as amended.Event 5307 Corn Environmental Report

4-34 Environmental Consequences 

### 4.5.1 APHIS Section 7 Process

Working with the USFWS, APHIS has developed a process for conducting an effects determination for petitions for no nregulated status. This process 382 is used by APHIS to fulfill their obligations and responsibilities under Section 7 of the ESA for biotechnology regulatory actions. APHIS considers the following information in its analysis of transgenic plants:

 The biology, taxonomy, and weediness potential of the crop plant and its sexually compatible relatives;

 Characteristics of each transgene with re spect to its structure and function and the nature of the organism from which it was obtained;

 Where the new transgene and its products are produced in the plant and their quality;

 The agronomic performance of the plant, including disease and pest susceptibilities, weediness potential, and agronomic and environmental impact;

 Determination of the concentration of known plant toxicants; and

 Determination whether the transgenic pl ant is sexually compatible with any threatened or endangered plant species.



### 4.5.2 Potential Effects

Based on the process described above, the following paragraphs describe the potential effects to threatened or endanger ed species that may result from the No Action or Proposed Action alternatives.



### No Action Alternative

The No Action alternative would not affect an y listed threatened or endangered species. Under this alternative, 5307 corn could continue to be grow n as a regulated article in
limited field trials. Corn that would be widely cultivated consists of non-transgenic hybrids and a range of deregulated transgenic corn varieties that are herbicide-tolerant or which are insecticidal to certain Lepidopt era (e.g., European corn borer and corn earworm) or Coleoptera (corn rootworm) (see Table 2-1, Deregulated Transgenic Corn Cultivars Containing Bt-derived Proteins ). APHIS has determined, as documented in prior Environmental Assessments, that none of the deregulated transgenic corn cultivars have an adverse effect on species protected under the ESA. 383, 384, 385



382 USDA-APHIS. nd. 383 USDA-APHIS. 2007. Pg. 14. 384 USDA-APHIS. 2009. Pg. 15. 385 USDA-APHIS. 2010b. Pg. 18.Event 5307 Corn Environmental Report

4-35 Environmental Consequences 

### Proposed Action Alternative

Wide cultivation of 5307 corn is not expect ed to differ from practices normally used for commercial corn production. Event 5307 corn production would not change land acreage or cultivation practices for transgenic or non-transgenic corn. The potential environmental impacts on listed threatened or endangered species from 5307 corn are those associated with typical commercial corn production, in areas where commercial corn is typically produced, an d therefore would not affect the natural habitat of any listed species. The analysis of potential effects considered the potential for 5307 corn to extend the range of corn production and the potential to expand agricultural production into new areas.

Event 5307 corn does not express any phenotypic traits that would allow it to be commercially viable outside of the existing range of corn cultivation, and is targeted specifically for use in the regions where co rn rootworm is a recognized agricultural pest (see Geographic Distribution of Northern and Western Corn Rootworm and their

Variants , in Corn Rootworm Biology, Feeding Behavior and Economic Loss ; Chapter 2, Section 2.2.2.3, Insect Management ). As described in Section 4.2.1, Agronomic Practices

, considering that 88 percent of the corn currently grown in the US is genetically modified, it is expected that 5307 corn, if deregulated, would be planted in areas where corn is currently grown. The agrono mic analyses show that 5307 corn would use the same agronomic practices – including the same fertilizers, herbicides, fungicides, irrigation, crop rotations, and tillage – as non-transgenic corn hybrids.
There is no hazard associated with the cultivation of 5307 corn that would be different from production of non-transgenic corn. There is no evidence that deregulating 5307 corn would affect any listed species or critical habitat protected
under the ESA.

Due to the selectivity of eCry3.1Ab for certain coleopteran species, potential exposure to threatened or endangered species is restricted to the order Coleoptera. Chapter 2, Section 2.5, Threatened and Endangered Species , describes the coleopteran species listed by the USFWS as endangered or threatened under the ESA. At present, there are no additional coleopteran species proposed for listing. A geospatial analysis was conducted for the listed coleopteran species; 15 were found within corn production counties. Refined geospatial analysis show ed that only three of these co-occurred with potential corn production locations:

 Kretschmarr Cave mold beetle ( Texamaurops reddelli ),
 American burying beetle ( Nicrophorus americanus ), and
 Salt Creek tiger beetle ( Cicindela nevadica lincolniana ).

The analysis showed that there would be no exposure of two of these species to
5307 corn. The Kretschmarr Cave mold beetle inhabits small isolated caves within the Edwards Limestone formation in Texas, 386 making contact with corn unlikely. The

 386 USFWS. 1988. Pg. 36030.Event 5307 Corn Environmental Report

4-36 Environmental Consequences American burying beetle relies on carrion (mammals, birds, reptiles) as a food

source 387 and would not be expected to ingest 5307 corn or its insecticidal protein
because it does not eat corn or insects. The Salt Creek tiger beetle is a predatory beetle that feeds on other insects, and due to its observed proximity to corn production areas it may conceivably be exposed to very low eCry3.1Ab concentrations (compared to levels measured in 5307 corn) via predation. While predation upon arthropods containing trace amounts of eCry3.1Ab is possible, the beetle’s predation habits would greatly limi t the opportunity for such exposure. Salt Creek tiger beetle larvae are known to prey on insects from the entrance of permanent burrows built in saline stream banks and barren salt flats of saline wetlands, habitats which do not occur in cornfields. 388 The Salt Creek tiger beetle is a
member of the Carabidae fa mily. Biosafety analyses documented in the Petition 389 showed no effects of eCry3.1Ab on the surv ival of carabid beetles when tested at levels 62 times greater than the worst-ca se estimated environmental concentration. 390 Therefore, any exposure of Salt Creek tige r beetles to trace amounts of eCry3.1Ab via their prey would be unlikely. Further, if such exposure were to occur, no measurable hazard is predicted.

A safety assessment 391 conducted on 5307 corn showed that the insecticidal activity of eCry3.1Ab is limited to certain Chrysomelidae species within the order Coleoptera. The chrysomelids shown to be sensitive to the insecticidal effects of eCry3.1Ab are the larvae of the western corn rootworm ( Diabrotica virgifera virgifera ),
northern corn rootworm (D. longicornis barberi) , Mexican corn rootworm ( D. virgifera zeae), and the Colorado potato beetle (Leptinotarsa decemlineata)

. 392

Apart from the transgene that encodes eCry3.1Ab in 5307 corn, 5307 corn also contains the gene pmi (also known as manA ) that encodes the selectable marker PMI enzyme, which is not a toxin. Event 5307 corn does not express additional proteins, natural toxicants, allelochemicals, pheromon es, or hormones, etc. that could directly or indirectly affect a listed species or spec ies proposed for listing. The high specificity of the eCry3.1Ab protein makes it unlikel y that non-target or threatened or endangered insects would be affected.

As indicated by the studies conducted by Syngenta, the eCry3.1Ab protein is not insecticidal to species outside the Chrysomelidae family of Coleoptera. The eCry3.1Ab protein demonstrates no lepi dopteran activity, despite containing sequences from a lepidopteran-active protei n (Cry1Ab). The absence of lepidopteran activity is consistent with the fact that, although eCry3.1Ab contains a portion of Cry1Ab, that portion does not include th e region responsible for lepidopteran activity.

 387 USFWS. 2011a. Pg. 2. 388 USFWS. 2005. Pg. 2. 389 Vlachos and Huber. 2011. Pg. 150. 390 The worst-case estimate assumes that the organisms’ diet is comprised of 100 percent 5307 corn material. 391 Vlachos and Huber. 2011. Pg. 150. 392 The Colorado potato beetle is not a pest of corn, but it is sensitive to eCry3.1Ab in direct laboratory feeding studies.Event 5307 Corn Environmental Report

4-37 Environmental Consequences Corn, including 5307 corn, has no sexually compatible relatives found in natural areas, and is only able to reproduce with other corn plants in the US. Corn cannot naturalize (as discussed in Chapter 2, Section 2.1, Corn Biology ) and has no potential to become weedy. The biosafet y studies conducted by Syngenta 393 show no toxic effects to birds, earthworms, fish, freshwater shrimp, non-target insects, or mammals,
and show that the insecticidal effects of 5307 corn are limited to corn rootworm larvae, which are not pollinators. As described in the Petition, Syngenta evaluated the composition and nutritional quality of 5307 corn and compared its composition to the composition of a corresponding non- transgenic corn variety and other corn varieties for which composition data were avai lable. The data suggest that there is no difference in composition and nutritional quality between 5307 corn and commercial corn varieties. It is therefore not anticipated that 5307 corn would have an adverse effect on non-target animals that consume it, or on listed animal species, including animals such as insects, bats or birds that may be pollinators of plants, or on listed plant species.



## 4.6 Public Health

This section describes the potential impacts to human health, animal (livestock) health, and worker safety that may result from the No Action and Proposed Action
alternatives.



### 4.6.1 Human Health

The general public is often concerned abou t the potential impacts that transgenic products could have on human health becaus e of possible toxic or nutritional effects of the products, or how the product might change the allergenicity of food products. Insecticidal Cry proteins from B. thuringiensis have a long history of safe use in food crops. Their modes of action are highly specific within narrow ranges of related insect species, and are not relevant to mammals or other vertebrates. PMI, the selectable marker protein produced by 5307 corn plants, is exempt from food and
feed tolerances. 394

The EPA requires seed registrants to submit tests of potential toxicity and allergenicity of the transgenic proteins in Bt corn cultivars before they can be approved for human consumption. All test s that have been performed for adverse mammalian impact from ingesting Cry prot eins have been negative, even at extremely high doses. 395 The toxicity of insecticidal Bt proteins depends on binding to specific receptors present in the insect midgut. With regard to the specific Cry


393 Vlachos and Huber. 2011. Pg. 151. 394 USEPA. 2007b. 395 Wu. 2006. Pg. 121.Event 5307 Corn Environmental Report

4-38 Environmental Consequences proteins produced in Bt crops, research demonstrates that this specificity limits each protein’s toxic effect to certain insect species.

As described in Chapter 1, Section 1.1, Regulatory Authority , it is the responsibility of
food and feed manufacturers to ensure that the products they market are safe and properly labeled. Food and feed derived from transgenic products must be in compliance with all applicable legal an d regulatory requirements. Transgenic products for food and feed may undergo a voluntary consultation process with the FDA prior to release onto the market. To date, all transgenic crops marketed in the US have been the subject of pre-marketing consultations with the FDA.

Certain Bt corn cultivars can reduce mold infestation on corn grain. 396 One study found that transgenic hybrids with cry genes for lepidopteran resistance had fumonisin concentrations as low as 10 pe rcent of those found in non-transgenic
counterparts. 397 Any reduction in mold toxins resulting from use of Bt corn can provide direct benefits to people and co rn-fed livestock by reducing exposure to mycotoxins. In a variety of field studies, lepidopteran-protected corn varieties expressing Bt proteins have been shown to have significantly lower levels of the common mycotoxins that are produced by fungal pathogens. 398



### No Action Alternative

Human health is not likely to be affected as a direct or indirect result of the No Action alternative. Previously deregulated transgenic corn would continue to be used as food for human consumptio n. However, the Cry proteins of Bt corn products
are not toxic to humans and do not have any known allergenic properties for humans. Human exposure to 5307 corn wo uld be limited to those individuals involved in cultivation under regulated co nditions even if 5307 corn continues to remain a regulated article. Regulated field trials of 5307 corn would only impact small plots of land with no potential for ex posure to the general public. Exposure to existing transgenic and non-transgenic corn would not change under this alternative.



### Proposed Action Alternative

Human health is not likely to be affected as a direct or indirect result of the Proposed Action alternative. If 5307 corn is deregulated, the general public would primarily come in contact with the introduced transgenic proteins (i.e., eCry3.1Ab and PMI) through dietary exposure to food and prod ucts derived from 5307 corn, although most processed products would contain no detectable eCry3.1Ab or PMI residues. However, the Cry proteins of Bt corn products, including 5307 corn, are not toxic to humans and do not have any known allerg enic properties for humans. The donor organism for the source genes used to create eCry3.1Ab is B. thuringiensis , a


396 Carpenter et al . 2002. Section 1, Pg. 3. 397 Munkvold et al . 1999. Pg. 133. 398 Wu. 2006. Pgs. 404-410.Event 5307 Corn Environmental Report

4-39 Environmental Consequences ubiquitous soil bacterium. The eCry3.1Ab protein is derived from a family of

Bt proteins that has a long history of safe use in food crops. Similarly, the PMI marker protein present in 5307 corn raises no human health concerns with respect to toxicity or allergenicity. Exposure to ex isting transgenic and non-transgenic corn would not change under this alternative.

A discussion on the mechanism of action for eCry3.1Ab, its spectrum of activity, and its lack of toxicity to non-coleopteran species is presented in the Petition 399 and the Safety and Nutritional Assessment. 400 A comprehensive assessment of the safety of eCry3.1Ab demonstrated that the protein is nontoxic to mammals and unlikely to be a food allergen. The eCry3.1Ab protein is considered nontoxic because it does not share significant amino acid similarity with known protein toxins, is nontoxic to mice at a very high dose, is rapidly degraded in simulated mammalian gastric fluid, and the insecticidal mode of action is not relevant to mammals. The eCry3.1Ab protein is not likely to become a food allergen because it is not derived from a known source of allergenic proteins, it does not have sign ificant amino acid sequence identity to known allergenic proteins, it is rapidly degraded in simulated mammalian gastric fluid, it is not glycosylated, and it is labile upon heating at temperatures of 37oC and above. The PMI protein is also consider ed nontoxic because it does not share significant amino acid homology with known protein toxins, it is nontoxic to mice at a very high dose, and it is rapidly degraded in simulated mammalian gastric fluid. PMI is not likely to become a food allergen because it is not derived from a known source of allergenic proteins, it does not have any significant amino acid sequence identity to known allergenic proteins with implications for its allergenic potential, it is rapidly degraded in simulated mammalian gastric fluid, it is not glycosylated, and it is labile upon heating at temperatures of 37oC and above.

Syngenta initiated a voluntary pre-mark et consultation process with FDA and submitted a Safety and Nutritional Assessment for 5307 corn in January 2011. 401 The assessment demonstrated a lack of toxicity and allergenicity of 5307 corn for human and animal consumption. With the exception of the worker safety benefits described in Section 4.6.3, Worker Safety, no impacts to humans, either directly or indirectly, are expected from deregulating 5307 corn; effe cts would be similar to the No Action
alternative.



### 4.6.2 Animal (Livestock) Health

As described in Chapter 2, Section 2.5.2, Animal (Livestock) Health, livestock ingestion of feed from transgenic crops, with subsequent human ingestion of livestock food products, is a potential concern about transgenic crops.


399 Vlachos and Huber. 2011. Pgs. 134-139. 400 Zepha and Vlachos, 2011. (Appendix B) 401 Zeph and Vlachos. 2011. (Appendix B)Event 5307 Corn Environmental Report

4-40 Environmental Consequences Chapter 2, Section 2.2.1, Production and Yield , explains that approximately 39 percent of the corn produced in the US is used fo r livestock feed and most of the corn used currently for livestock feed is transgenic. As discussed in Section 4.6.1, Human Health

, Cry proteins are not expected to be allerg enic, toxic, or pathogenic in mammals or poultry. Additionally, no gene transfer to gastrointestinal flora is expected. Insecticidal Cry proteins from B. thuringiensis have a long history of safe use in feed

crops. 402 Their modes of action are highly specific within narrow ranges of related insect species, and are not relevant to mammals, including domestic livestock, or other vertebrates. Cry proteins also have a history of safe consum ption in the context
of other food and feeds. 403 Additionally, the selectable marker PMI protein produced by 5307 corn plants is exempt from food and feed tolerances. 404



### No Action Alternative

Animal (livestock) health would not be affected as a direct or indirect result of the No
Action alternative. Livestock would not be exposed to 5307 corn if it continues to remain a regulated article. Previously deregulated transgenic corn will continue to be used as feed for animal consumption, an d adoption of transg enic corn varieties would be expected to continue. Exposure to existing transgenic and non-transgenic corn would not change under this alternative. Regulated field trials of 5307 corn would only impact small plots of land with no potential for exposure to livestock.



### Proposed Action Alternative

Animal (livestock) health is not likely to be affected as a direct or indirect result of the Proposed Action alternative. If 5307 corn is deregulated, livestock would primarily come in contact with the introduced eCry3.1Ab and PMI proteins in 5307 corn through feed products derived from 5307 corn. The eCry3.1Ab protein is derived from a family of Bt proteins that has a long history of safe use in food crops. There are no animal health or environmental concerns with respect to toxicity orallergenicity of eCry3.1Ab or PMI. Exposure to existing transgenic and nontransgenic corn would not change under this alternative. A compositional analysis concluded that forage and grain from 5307 corn hybrids are considered similar in composition to forage and grain from bo th the non-transgenic comparator and conventional corn hybrids. 405 Event 5307 corn will be as safe and nutritious as
conventional corn for livestock.

Syngenta initiated a voluntary pre-mark et consultation process with FDA and submitted a Safety and Nutritional Assessment for 5307 corn in January 2011. 406 The assessment demonstrated a lack of toxicity and allergenicity of the eCry3.1Ab and

 402 USEPA. 2001. Pg. 23. 403 USDHHS-FDA. 2010. 404 USEPA. 2007b. 405 Vlachos and Huber. 2011. Pg. 108. 406 Zeph and Vlachos. 2011. (Appendix B)Event 5307 Corn Environmental Report

4-41 Environmental Consequences PMI proteins in 5307 corn for human and animal consumption. No adverse impacts to livestock, either directly or indirectly, are expected from deregulating 5307 corn.



### 4.6.3 Worker Safety

As described in Chapter 2, Section 2.6.3, Worker Safety, pesticides, including insecticides and herbicides, are used on most corn acreage in the US. The EPA’s
Worker Protection Standard 407 requires employers to take actions to reduce the risk of pesticide poisonings and injuries among agricultural workers and pesticide handlers. The restrictions and precautions fo r worker safety associated with several
conventional insecticides for rootworm control include:

 Protective clothing (chemical-resistant gloves and other sk in protection, eye protection, respirators, etc. ) or other measures (closed-system applications) to minimize applicator exposure;

 Minimum worker reentry intervals post application; and

 Minimum preharvest intervals post application.

Large-scale cultivation of transgenic Bt corn has increased since its introduction in 1996. A number of studies indicate that the application of pesticides has decreased since the introduction of transgenic crops, 408 reducing exposure of agricultural workers to the hazards of pesticide mixing, loading and application.

EPA requires several types of data for Bt PIPs to provide a reasonable certainty that
no harm to workers will result from the a ggregate exposure of these proteins. None
of the Bt proteins registered as plant pesticides in the US are toxic or have been shown to have any significant effect on humans. 409



### No Action Alternative

Agricultural workers and pesticide applicators would continue to be exposed to a variety of EPA-registered pesticides such as those approved for control of corn rootworm (Table C-1, Appendix C) under the No Action alternative. Chemical insecticide use rates would likely continue to decrease as insect-resistant transgenic corn continues to be adop ted. Approved transgenic Bt corn cultivars would continue to be used even if 5307 corn continues to remain a regulated article. Regulated field trials of 5307 corn would only impact small plots of land with limited potential for exposure to agricultural workers. Under the No Action alternative, agricultural workers and pesticide applicators would continue to be exposed to a variety of
chemicals.


407 USEPA. 1992. 40 CFR Part 170.1, Scope and Purpose

. 408 Kleter et al . 2007. Pg. 111. 409 USEPA. 2010b. Pg. 23.Event 5307 Corn Environmental Report

4-42 Environmental Consequences 

### Proposed Action Alternative

Similar to the No Action alternative, EPA-registered pesticides that are currently used for corn production would continue to be used by growers under the Proposed Action alternative. As described in Section 4.2.1.4, Pesticide Use , trends in reduced insecticide use are likely to continue as a direct result of the Proposed Action alternative. The safety and convenience of planting insect-protected corn compared to the application of conventional insecticides are consistently cited by growers as benefits of transgenic crops. The further ad option of transgenic varieties, including 5307 corn, would continue to extend these benefits to workers. Worker exposure to insecticides would continue to decline. If 5307 corn is deregulated, agricultural workers and pesticide applicators would likely benefit from the use of 5307 corn due to a reduction in the use of corn rootworm insecticide applications and the number of acre-treatments per year.



## 4.7 Socioeconomics

This section describes the potential impacts to the domestic economic environment, trade economic environment, and social en vironment that may result from the No Action and Proposed Action alternatives.



### 4.7.1 Domestic Economic Environment

As described in Chapter 2, Section 2.7.1, Domestic Economy, domestic demand for corn in the US comes primarily from its use for feed, ethanol production, food, and seed. In 2011, transgenic products comprised 88 percent of the planted corn to satisfy
this demand. 410 The transgenic traits include herbicide tolerance, insect resistance, and other traits. Approximately 65 percent of the US corn crop in 2011 is comprised
of Bt corn. 411 Overall, harvest security and quality is better with Bt corn. Farm income is positively impacted by Bt corn by reducing production costs or increasing revenues. Pest-resistant corn generally has a positive impact on farm income due to cost savings from reduced pesticide use.

Section 2.2.2.3, Insect Management , explains that the corn r ootworm family is one of the most damaging corn pests, and was ofte n referred to as the “billion dollar bug” due to annual yield losses and control cost s prior to the introduction of transgenic cultivars for rootworm control, beginning in 2003. 412 Seed companies have developed transgenic crops with corn rootworm resistance specifically to address
this economic loss. Four corn rootworm-r esistant cultivars (MON 863, DAS 59122-7, MON 88017 and MIR604 corn) are currently available to growers to control this economically important pest.

 410 USDA-ERS. 2011b. Pg. 2. 411 Ibid. Pg. 2. 412 Metcalf. 1986. Pg. vii.Event 5307 Corn Environmental Report

4-43 Environmental Consequences 

### No Action Alternative

The domestic economic environment would no t be affected as a direct or indirect
result of the No Action alte rnative. Growers would continue to make choices to plant certain corn varieties and use certain crop ro tation practices based on factors such as yield, weed and disease pressures, cost of seed and other inputs, technology fees, worker safety, potential for crop injury, and ease and flexibility of the production system. 413, 414 The No Action alternative would not affect available options for growers and therefore not affect the dome stic economy. Event 5307 corn would remain a regulated article and would require an APHIS permit or notification for release into the environment. Regulated field trials of 5307 corn would only impact small plots of land with no im pact on the domestic economy.



### Proposed Action Alternative

The domestic economic environment would be positively affected as a direct and indirect result of the Proposed Action alternative. Growers would have an additional tool to use against corn rootworms if 5307 corn is deregulated, directly reducing economic loss from this pest. The Propos ed Action could indirectly result in economic benefit from increased competition in the seed market. The availability of multiple corn rootworm-resistant products would increase grower choice and price competition, potentially resulting in lower seed prices for growers, and assist in managing insect resistance to existing products for corn rootworm control. Specific economic projections are not available.

To the extent that the planting of 5307 corn results in a decrease of insecticide applications for corn rootworm, or an incr ease in yields, farms adopting 5307 corn might experience an increase in net income. Event 5307 corn hybrids demonstrated an average grain yield advantage of 63 bushels per acre over control hybrids in the presence of intense larval rootworm pressure during field tests. 415 Growers are expected to realize a real-world increase in yield and would therefore likely realize direct economic gain from this product. 416 A description of the potential impacts to
the domestic economic environment from 5307 corn in stacked combinations is provided in Section 4.8, Cumulative Effects

.



### 4.7.2 Trade Economic Environment

As described in Chapter 2, Section 2.7.2, Trade Economy, the primary US corn export destinations are also the largest world importers of corn and do not have major barriers for importing food or feed commod ities produced from transgenic crops, including those with insect resistance trai ts. Developing countries’ demand for corn

 413 Olson and Sander. 1988. Pgs. 646-647. 414 Gianessi. 2005. Pg. 241. 415 Vlachos and Ward. 2011. Pg. 37. 416 Ibid. Pg. 37.Event 5307 Corn Environmental Report

4-44 Environmental Consequences has increased steadily for the past three decades, propelling the global trade market above 70 million metric tons every year since 1999/2000. 417 Corn imports to European Union countries have declined steadily since the Common Agricultural Policy limited grain imports and EU membership has expanded. 418



### No Action Alternative

The trade economic environment would not be affected as a direct or indirect result
of the No Action alternative. Additional genetically modified corn varieties would continue to be developed and available in the trade market. Event 5307 corn would remain a regulated article and would require an APHIS permit or notification for release into the environment. Regulated field trials of 5307 corn would only impact small plots of land with no impact on the worldwide corn trade.



### Proposed Action Alternative

The trade economic environment would not be affected as a direct or indirect result of the Proposed Action alternative. Worl dwide market conditions and destination country approval of transgenic crop commo dities would continue to be factors for international corn prices, without regard to the presence or absence of 5307 corn on the market. Deregulating 5307 corn woul d not adversely impact the trade economy and may potentially enhance it through more efficient production of corn supplies
worldwide.

Syngenta has applied to Canadian agencies for approval of the unconfined
environmental release and food and feed use of corn commodities and processed goods containing 5307 corn. To avoid adversely affecting international trade in corn commodities exported from the US (and Ca nada), regulatory filings for 5307 corn import approvals have been made in Ja pan, South Korea, Taiwan, Australia/New Zealand, South Africa, Colombia, and the European Union. Applications are planned for additional countries including Mexico, China, the Philippines, Indonesia, and Russia. The trade economic impa cts associated with the dere gulation of 5307 corn are anticipated to be very similar to the No Action alternative.



### 4.7.3 Social Environment

As described in Chapter 2, Section 2.7.3, Social Environment, farmers have a range of options in agronomic practices, seed products to choose from, and opportunities for sale to customers. Consumers have a range of corn products to choose from. Section 4.2.2.1, Organic Farming , explains that growers may choose to grow transgenic, non-transgenic, or organic corn, and obtain price premiums for growing varieties of corn for particular markets. Regulatory agency requirements (under the

 417 USDA-ERS. 2009. Pg. 3. 418 Ibid. Pg. 3.Event 5307 Corn Environmental Report

4-45 Environmental Consequences Federal Food, Drug, and Cosmetic Act and the NOP, for example) and consumer advocacy groups promote food product safety and consumer choice.



### No Action Alternative

The social environment would not be affected as a direct or indire ct result of the No Action alternative. Event 5307 corn would remain a regulated article and would require an APHIS permit or notification for release into the environment. Regulated field trials of 5307 corn would only impact small plots of land with no impact on the
social environment.



### Proposed Action Alternative

The social environment would not be affected as a direct or indirect result of the Proposed Action alternative. Regulatory programs and consumer choicewould be unchanged by granting nonregulated status to 5307 corn.



## 4.8 Cumulative Effects

This section describes the potential cumulative effects that may result from the Proposed Action alternative. In accordance with CEQ regulations, this evaluation considers the potential effects that, although individually minor, could have a collectively significant impact on the enviro nment when added to other past, present, and reasonably foreseeable future actions.



### 4.8.1 Methodology and Assumptions

The CEQ regulations define a cumulative ef fect as “the impact on the environment which results from the incremental impact of the action when added to other past, present, and reasonably foreseeable future actions regardless of what agency (Federal or non-Federal) or person unde rtakes such other actions. Cumulative impacts can result from individually minor but collectively significant actions taking place over a period of time.” 419

Based on the information provided in Sections 4.2 through 4.7, which is summarized in Table 3-1, the Proposed Action would not adversely impact the physical, biological, or socioeconomic environment. Fu rthermore, as discussed in this section, there are no past, present or reasonably foreseeable actions that, in aggregate with the Proposed Action alternative, would adve rsely affect these resources. Based on the evaluation provided in previous sections of this Chapter, if 5307 corn is deregulated corn production practices would contin ue unchanged and most environmental
resources would be unaffected. There would be no cumulative adverse effects to corn


419 CEQ. 1978. 40 CFR Part 1508, Section 1508.7, Cumulative impact

.Event 5307 Corn Environmental Report

4-46 Environmental Consequences production, the physical environment, the bi ological environment, public health, or
the socioeconomic environment. Event 5307 corn, in stacked combinations, is likely
to have beneficial cumulative effects on pesticide usage, biodiversity, and the domestic economy.



### 4.8.2 Reasonably Foreseeable Future Actions

Transgenic corn varieties marketed in the US today typically contain multiple transgenic traits, some of which have been combined by traditional breeding between different deregulated cultivars. In the event that APHIS reaches a determination of nonregulated status, 5307 corn would potentially be combined with non-transgenic and transgenic corn cultivars by traditional breeding techniques, resulting in a plant variety that, for example, may be resistant to one or more
herbicides and contain other insect-resista nce traits. APHIS’s current regulations at 7 CFR Part 340 do not provide for agency oversight of transgenic corn varieties previously deregulated pursuant to Part 340 and the PPA, nor over stacked varieties combining deregulated transgenic cultivars, unless it can be positively shown that such stacked varieties were likely to pose a plant pest risk.

There is no certainty that 5307 corn will be stacked with any particular deregulated transgenic variety, as company plans and market demands play a significant role in those business decisions. Predicting all potential combinations of stacked varieties that could be created using both deregu lated transgenic corn cultivars and nontransgenic corn cultivars is hypothetical and purely speculative. However, Syngenta intends to offer 5307 corn for sale to US growers in two combinations comprised of the insect protection and herbicide tolerance traits; these combinations are listed as A and B in Table 4-1. Syngenta has requeste d authorization of these stacked products from the EPA, with regard to the specific combinations of insecticidal traits present in each product. 420



### Table 4-1 Planned St acking Combinations

Cultivar Traits Combination

5307 Corn rootworm resistant A, B
Bt11 Lepidopteran resistant and glufosinate tolerant A, B

MIR604 Corn rootworm resistant A, B
TC1507 Lepidopteran resistant and glufosinate tolerant A, B
GA21 Glyphosate tolerant A, B
MIR162 Lepidopteran resistant B



420 Vlachos and Ward. 2011.Event 5307 Corn Environmental Report

4-47 Environmental Consequences The combination hybrids A and B listed in Table 4-1 will combine two corn rootworm-active proteins (eCry3.1Ab fr om 5307 corn and mCry3A from MIR604 corn) that each provide control of wester n, northern, and Mexican corn rootworms. The hybrids will also combine other Bt -derived proteins that will deliver broad-spectrum control of economically important lepidopteran pests and traits that confer tolerance to glyphosate and glufosinate herbicide applications as additional weed-control options. Apart from 5307 corn , all of the cultivars derived from other transgenic events in these breeding stacks have already been deregulated by APHIS. Breeding stacks for insect control currently approved for sale or use in the US
combine various insect resistance and herbicid e tolerance traits, as listed in Table 4-2.Event 5307 Corn Environmental Report

4-48 Environmental Consequences 

### Table 4-2 Commercially Available Corn Breeding Stack Combinations with Insect Control Traits

Product Event Names Resist ance/Tolerance Traits

Agrisure® GT/CB/LL Bt11/GA21 Lepidoptera, glyphosate, glufosinate

Agrisure Viptera TM

3110 Bt11/MIR162/GA21 Lepidoptera, glyphosate, glufosinate
Agrisure CB/LL/RW Bt11/MIR604 Lepidoptera, rootworm, glufosinate

Agrisure 3000GT Bt11/MIR604/GA21 Lepidoptera, rootworm, glyphosate, glufosinate
Agrisure Viptera 3111 Bt11/MIR604/MIR162/GA21 Lepidoptera, rootworm, glyphosate, glufosinate
Syngenta GT/RW MIR604/GA21 Rootworm, glyphosate

YieldGard® Corn Borer with Roundup Ready® Corn 2 (RR2) MON 810/NK603 Lepidoptera, glyphosate

YieldGard Rootworm/RR2 MON 863/NK603 Rootworm, glyphosate

Genuity TM

VT Double PRO TM

MON 89034/NK603 Lepidoptera, glyphosate

YieldGard Plus MON 810/MON 863 Lepidoptera, rootworm
YieldGard Plus/RR2 MON 810/MON 863/NK603 Lepidoptera, rootworm, glyphosate

YieldGard VT Triple® and GenuityTM VT Triple MON 810/MON 88017 Lepidoptera, rootworm, glyphosate

(Genuity) Smart StaxTM MON 89034 /MON 88017/ TC1507/DAS-59122-7 Lepidoptera, rootworm, glyphosate, glufosinate
Genuity VT Triple PRO MON 89034/MON 88017 Lepidoptera, rootworm, glyphosate
Herculex® I/RR2 TC1507/NK603 Lepidoptera, glyphosate, glufosinate

Herculex RW/RR2 DAS 59122-7/NK603 Rootworm, glyphosate, glufosinate
Herculex XTRA TC1507/DAS 59122-7 Lepidoptera, rootworm, glufosinate

Herculex XTRA/RR2 TC1507/DAS 59122-7/NK603 Lepidoptera, rootworm, glyphosate, glufosinate

Optimum® AcreMax® 1 Seed blend of 90% TC1507/DAS-59122- 7/NK603 and 10% TC1507/NK603 refuge seed 90% Lepidoptera, rootworm, glyphosate; 10% Lepidoptera, glyphosate

Optimum AcreMax RW Seed blend of 90% DAS-59122-7/NK603 and 10% NK603 refuge seed 90% rootworm, glyphosate; 10% glyphosate

Sources: NCGA website http://ncga.com/know -before-you-grow/ and Pioneer website http://www.pioneer.com/home/site /about/products/product-traits -technology/optim um-acre-max/

For purposes of cumulative impact analysis, reasonably foreseeable future actions include the potential for stacking certain already approved transgenic corn cultivars with 5307 corn, or for creating new stac ks with similar combinations of traits.Event 5307 Corn Environmental Report

4-49 Environmental Consequences 

### 4.8.3 Cumulative Effects Evaluation

This section considers the potential cumulative effects resulting from including 5307 corn in multi-trait (stacked) combinations. Syngenta intends to commercialize
the 5307 corn rootworm-resistance trait in combination with other insect-resistance traits (effective in controlling corn rootworm and Lepidoptera) and herbicide-tolerance traits, as listed in Tabl e 4-1. Although APHIS has found that none of the 30 currently approved transgenic corn cultivars (as of October 12, 2011) presents a significant environmental impa ct alone or cumulatively, APHIS has not specifically addressed the cumulative im pact of stacking these products with 5307 corn. The first step in evaluating the cumulative impacts of deregulating 5307 corn is to identify the resources that might be affected by that action. Areas of concern with regard to these stacked co mbinations include agronomic practices (insecticide use and potential development of resistant insect populations; herbicide use and potential development of herbic ide-tolerant weeds), biodiversity, and domestic economic issues (cost to farmers).

Potential cumulative effects of the Prop osed Action on these resources were evaluated by comparing the results of the direct and indirect impacts analyses with data from a literature review. The analysis provided below discusses the potential cumulative effects of the Proposed Action al ternative in reference to these potentially affected resources. The potential cumulati ve effects of the No Action alternative would be a combination of the impacts of the existing approved transgenic corn cultivars and the impacts from limited field trials of 5307 corn as a regulated article. The potential cumulative effects of the Proposed Action alternative would be a combination of the impacts from existing a pproved transgenic co rn products and the impacts of the incorporation of 5307 corn in new stacking combinations.



### 4.8.3.1 Pesticide Use

As noted in Table 3-1 and di scussed in Section 4.2.1, Agronomic Practices , the Proposed Action is expected to have the effect of reducing insecticide use. The Proposed Action is not expected to chan ge overall herbicide use. The past and current actions potentially contributing to cu mulative effects on this resource are pest management strategies including conventional insecticide and herbicide use, crop rotation practices, and the introduction of transgenic corn varieties and their increasing use. The array of transgenic corn cultivars currently available for insect pest management is listed in Table 2-1. The future actions potentially contributing to
this cumulative effect are the combination of 5307 corn stacked with transgenic corn cultivars exhibiting insect resistance and/or herbicide tolerance, as listed in
Table 4-1.Event 5307 Corn Environmental Report

4-50 Environmental Consequences 

### Insecticide Use

Corn rootworm has evolved resistance to chemical insecticides and crop rotation practices that have historically been used to control these economically destructive pests. As described in Chapter 2, Section 2.2.2.3, Insect Management , use of conventional (chemical) pesticides to cont rol corn rootworm has been reduced since the introduction of transgenic Bt corn for insect control.

A key factor in the continued use of Bt corn for insect control is product durability (i.e., delaying or avoiding the development of resistance to Bt proteins in insect pests). The widespread use of transgenic Bt corn could generate selection pressures
for insect resistance. 421 Nonetheless, insect resistance to Bt crops has not caused widespread failure of control measures, in part due to IRM strategies, including rescue applications of insecticides and nearby refuges of crop varieties lacking the insecticidal trait. Although one recent study indicates that, in one localized area, western corn rootworm may have developed resistance to a Cry protein under intense cultivation, 422 stacking two transgenic corn traits with different modes of action against the target pest limits the pote ntial for resistance to develop to any one product. 423

Refuge strategies include planting a designated percentage of corn refuge acres (in the Corn Belt, typically 5 to 20 percent of a grower’s corn field area 424 ), either in a designated area that is a percent of the total area (a block) or a percent of the total seed planted (“refuge-in-a-bag”). Refuge sizes are determined for each specific insect-resistant corn or stacked product, and are mandated by the EPA as part of product registration. IRM practices are required by seed companies as a condition of seed purchase, and implemented through agreements signed by growers.

Corn rootworm-resistant cultivars with a different mode of action that may be
stacked with 5307 corn in the future include Syngenta’s previously approved

rootworm-resistant Bt corn variety, MIR604 corn. APHIS granted nonregulated status for this product in March 2007. 425 APHIS determined that cultivating MIR604 corn would not necessarily result in the development of resistant rootworm populations. EPA has mandated in the term s of registration that growers use IRM strategies for MIR604 and other Bt corn varieties expressing PIPs for the control of insect pests.



### No Action Alternative

As described in Section 4.2.1.4, Pesticide Use , insecticide use would not be affected as
a direct or indirect result of the No Acti on alternative. For the cumulative effects



421 Tabashnik et al . 2008. Pg. 199. 422 Gassmann et al . 2011. Pg. 4. 423 Tabashnik et al . 2008. Pg. 201. 425 USDA-APHIS. 2007. 425 USDA-APHIS. 2007.Event 5307 Corn Environmental Report

4-51 Environmental Consequences analysis of the No Action alternative, th e reasonably foreseeable future would not include stacking 5307 corn with other transgenic corn cultivars. Event 5307 corn would remain a regulated article and would require an APHIS permit or notification
for release into the environment. Corn root worms are not likely to develop resistance

to Bt corn as a result of the continued regu lated status of 5307 corn. Regulated field trials of 5307 corn would only impact small plots of land with no impact on
insecticide use or corn rootworm resistance to Bt corn.

Insect-resistant transgenic corn varieties wo uld continue to be available, and insects could potentially develop resistance to thes e varieties. When combined with the past, present, and reasonably foreseeable future actions, there would be no cumulative
effect from the No Action alternative on in secticide use or corn rootworm resistance

to Bt corn. However, without the additional rootworm control option offered by 5307 corn, fewer PIP traits would be availa ble to growers to combat the evolution of

insect resistance.



### Proposed Action Alternative

As described in Section 4.2.1.4, Pesticide Use , if 5307 corn is deregulated, trends of reduced insecticide use are likely to continue. Corn growers would have a new corn
rootworm-control cultivar in addition to the four other corn rootworm-control cultivars that are currently available. This would allow for more effective IRM strategies. Because 5307 corn has excelle nt efficacy against corn rootworm and because the eCry3.1Ab protein operates via a unique mode of action, introduction of the 5307 corn breeding stacks is expected to further extend the useful life of other commercially available corn rootworm-protect ed products (i.e., the PIPs Cry3Bb1, Cry34Ab1/Cry35Ab1, and mCry3A). 426

The combination of eCry3.1Ab from 5307 corn and mCry3A via Syngenta’s deregulated MIR604 corn in the same corn hybrids may also justify a reduction in the size of the required on-farm refuge from a minimum of 20 percent of a grower’s corn acres to 5 percent. 427 Appropriate refuge requirements would be implemented in the context of a comprehensive IRM program ma ndated by the EPA. IRM would include insect scouting or monitoring to dete rmine pest populations, considering and applying compatible alternative biological, cultural, mechanical and chemical controls, and establishing action thresholds for agricultural inputs. The timely and targeted delivery of pest management in terventions is key to successful IRM. Planting 5307 corn stacks would eliminate the need to target and time applications of rootworm insecticides, due to the sustained production of the insect control proteins (eCry3.1Ab and mCry3A) in vulnerable root tissues. 428 The 5307 corn breeding stacks are consistent with the concept of IRM for corn.


426 Vlachos and Ward. 2011. Pg. 30. 427 Vlachos and Huber. 2011. Pg. 154. 428 Vlachos and Ward. 2011. Pgs. 31-32.Event 5307 Corn Environmental Report

4-52 Environmental Consequences With a reduced minimum refuge requirement (i.e., from 20 to 5 percent of a grower’s corn acres), the use of transgenic Bt corn may be expected to increase. Increased use of transgenic Bt corn is correlated with reduced reliance on, and use of, insecticides
to control corn rootworm. It is also corr elated with higher corn yields than can typically be achieved with insecticides alone. Thus, when combined with past, present, and reasonably foreseeable future actions, the cumulative effects of the Proposed Action alternative would likely result in increased corn yields (on what previously would have been refuge acres), further reduction of insecticide use, and a reduced likelihood that corn rootworm po pulations will develop resistance to Cry proteins, extending the effe ctiveness of these tools.



### Herbicide Use

Herbicide-tolerant transgenic corn cultivars have been developed to allow use of herbicides without harming the crop. As described in Chapter 2, Section 2.2.2.5, Weed Management , herbicide-tolerant corn has been widely adopted by growers in North
America and offers enhanced weed control. Currently available transgenic herbicidetolerant corn cultivars include multiple glyphosate- or glufosinate- (phosphinothricin) tolerant cultivars. 429

In 2011, approximately 72 percent of the US corn crop was planted to transgenic
varieties that were herbicide tolerant. 430 However, over-reliance on herbicidetolerant crops may under certain conditio ns promote the development of herbicidetolerant weeds. Weeds can potentially survive in crop production systems because of natural tolerance to the chemical(s) and beca use of growth types or life cycles that help them avoid being treated, such as some winter annual weed species. As described in Chapter 2, Section 2.2.2.5, Weed Management , some weeds have developed herbicide tolerance and the increased use of herbicide-tolerant corn may increase the prevalence of herbicide-tolerant weeds. Industry practice includes weed resistance management training to redu ce the potential for weeds to develop
tolerance to herbicides. 431 Because there are several herbicides available, and transgenic corn varieties tolerant to differe nt herbicides availabl e, growers have the ability to rotate herbicide use, rather than relying on a single herbicide.

The adoption of herbicide-tolerant crops al so has been associated with the increased adoption of conservation tillage, which decreases runoff, increases water infiltration,
and reduces soil erosion. 432



429 USDA-APHIS. 2011b. 430 USDA-ERS. 2011a. Pg. 2. 431 NCGA. 2011. Slide 2. 432 Carpenter. 2011. Pg. 8.Event 5307 Corn Environmental Report

4-53 Environmental Consequences 

### No Action Alternative

Herbicide use would not be affected as a dire ct or indirect result of the No Action alternative. The reasonably foreseeable fu ture for the No Action Alternative would not include stacking 5307 corn with other transgenic corn cultivars. Event 5307 corn would remain a regulated article and would require an APHIS permit or notification for release into the environment. Transgenic corn varieties with herbicide tolerance would continue to be available, and weeds could continue to develop resistance to herbicides. Thus, when combined with pa st, present, and reasonably foreseeable future actions, there would be no cumulative effect from the No Action alternative on
herbicide use or on the evolution of weed tolerance to herbicides.



### Proposed Action Alternative

Although 5307 corn does not have an herb icide tolerance trait, under the Proposed Action alternative it will be stacked with other transgenic corn cultivars that do have herbicide tolerance traits. Syngenta woul d market 5307 corn stacked with its ownherbicide-tolerant cultivars. If 5307 corn is deregulated and stacked with herbicidetolerant cultivars, weed ma nagement methods may be altered to a minor degree but would generally continue following current trends.

In APHIS’s recent analysis of the DP-0981 40-6 herbicide-tolerant corn, it was noted that the acres of transgenic corn planted with herbicide-tolerant varieties declined in 2008 but that corn varieties with insect resistance, whether alone or stacked with an herbicide tolerance trait, are planted more readily than those varieties conferring
tolerance to herbicides alone. 433 Stacked products combining insect resistance and herbicide tolerance (and pote ntially other traits) are likely to become more common regardless of the regulatory status of 5307 corn. The Proposed Action would likely
continue this trend.

As described in Chapter 2, Section 2.2.2.5, Weed Management , glyphosate application rates have increased recently and it is reasonable to conclude that they will continue to do so. Weeds may continue to deve lop tolerance to herbicides. Given the widespread adoption of herb icide-tolerant corn that has already occurred, stacking 5307 corn with herbicide tolerance traits is unlikely to significantly change current trends in herbicide use and increasing weed tolerance to herbicides. When combined with the past, present, and reasonably fo reseeable future acti ons, the Proposed Action would have a negligible cumulative effect on herbicide use or weed tolerance
to herbicides.

 433 USDA-APHIS. 2009. Pg. 5.Event 5307 Corn Environmental Report

4-54 Environmental Consequences 

### 4.8.3.2 Biodiversity

The past and current actions potentially contributing to a cumulative effect on biodiversity are the introduction of transgen ic corn varieties and their increasing use. The array of transgenic corn varieties cu rrently available for pest management is outlined above. The future actions potentia lly contributing to cumulative effects on biodiversity are any combination of 5307 corn stacked with transgenic corn cultivars exhibiting insecticidal properties or herbicide tolerance.

This cumulative effects analysis focuses on potential changes in biodiversity that may result from impacts to non-target plants and animals within the agroecosystem. Other aspects of biodiversity would not be affected by the Proposed Action, as described in previous sections of this Chapter. Insect biodiversity within agricultural production areas could increase with the us e of transgenic crops that reduce the use of broad-spectrum insecticides.

Several different Cry proteins are incorporated in various Bt corn varieties to provide insect resistance. EPA conducted a compre hensive environmental assessment of the registered Bt PIPs in 2001. 434 Although other Bt corn cultivars incorporating Cry proteins have been introduced in the interv ening 10 years, and many of the PIPs in earlier cultivars were re-registered in 2010, the 2001 EPA review provides a general
assessment of the risks to bi odiversity associated with Bt corn varieties. Summarizing then-existing published studies and EPA’s reviews of submitted studies on potential Cry protein impacts to non-target specie s (vertebrates and invertebrates), EPA concluded that “the weight of evidence from the reviewed data indicate that there is no hazard to non-target wildlife from the continued registration of Bt crops.” 435 Minimal to undetectable adverse impacts, and in some cases beneficial impacts, to non-target insect populations were shown. 436 EPA also noted that Bt crops have a positive effect on soil flora compared to non-selective synthetic chemical pesticides. 437 More recent studies have also concluded that the use of Bt crops, rather than broad-spectrum insecticides, could allow larger populations of beneficial insects and non-pest herbivores to persist in crop fields. 438

Cultivars expressing other Bt -derived proteins may be stacked with 5307 corn in the future, including Syngenta’s most recently approved lepidopteran-resistant cultivar, MIR162 corn. APHIS granted nonregulated status for this cultivar in April 2010. 439 APHIS determined that there were no past, present, or reasonably foreseeable actions that would aggregate with effects of granting nonregulated status to MIR162 corn to create cumulative impacts or reduce the lo ng-term productivity or sustainability of any of the resources associated with the ecosystem in which the MIR162 corn is

 434 USEPA. 2001. Pg. IIC1. 435 USEPA. 2001. Pg. IIC81. 436 USEPA. 2001. Pg. IIC84. 437 USEPA. 2001. Pg. IIC53. 438 Pilson and Prendeville. 2004. Pg. 164. 439 USDA-APHIS. 2010b.Event 5307 Corn Environmental Report

4-55 Environmental Consequences planted. 440 APHIS also noted that transgenic corn varieties with Bt traits have been available on the market and the body of ev idence in peer-reviewed literature does not suggest any negative effect on biodiversity.

In general, applying less toxic herbicides (e.g., glyphosate) may be more environmentally beneficial than traditional herbicides. 441 However, animals that consume the weeds targeted by the herbicides could potentially be impacted if the weeds are eradicated. These relationships an d the actual impact on biodiversity are difficult to determine. For example, one st udy suggested that reductions in monarch butterfly ( Danaus plexippus ) populations overwintering in Mexico might be attributed in part to loss of host milkweed ( Asclepias syriaca ) plants in the Corn Belt from the extensive use of glyphosate. 442 However, other studies 443 suggest that monarch populations are very dynamic because of the high reproductive potential of this species, and no similar reduction was obse rved in populations studied in the US.



### No Action Alternative

As described in Section 4.4.4, Biodiversity , the No Action alternative would not directly or indirectly affect biodiversity. Fo r the cumulative effect s analysis of the No Action alternative, stacking 5307 corn with other transgenic corn cultivars would not occur under the No Action alternative. Event 5307 corn would remain a regulated article and would require an APHIS permit or notification for release into the environment. Non-target plants and animals would not be affected as a result of the continued regulated status of 5307 corn. Re gulated field trials of 5307 corn would only impact small plots of land with no impact on biodiversity. When combined with the past, present, and reasonably foreseea ble future actions, there would be no
cumulative effect from the No Ac tion alternative on biodiversity.



### Proposed Action Alternative

As described in Section 4.4.4, Biodiversity , the Proposed Action alternative would not adversely affect biodiversity. Event 5307 co rn would only be stacked with approved transgenic cultivars that have been demonstrated by APHIS review to have insignificant impacts to animals. The hybrids under consideration by Syngenta would combine two rootworm-active proteins that each provide control of western, northern, and Mexican corn rootworms but do not compete for the same binding site in western corn rootworm gut membranes. 444 If 5307 corn is deregulated and stacked with herbicide-tolerant products, weed management methods may be altered to a minor degree, but would generally continue following current trends. Event 5307 corn would only be stacked with approved transgenic cultivars that have been demonstrated to have insignificant impacts to non-weed plants.



440 USDA-APHIS. 2010b. Pg. 28. 441 Pilson and Prendeville. 2004. Pg. 155. 442 Brower et al. 2011. Pg. 2. 443 Davis. 2011. Pg. 3. 444 Vlachos and Huber. 2011. Pg. 154.Event 5307 Corn Environmental Report

4-56 Environmental Consequences Corn growers would have a new corn rootworm-control product, stacked with other control products, to add to the tools available for pest management. Representative non-target animal species were not harmed when exposed to high concentrations of eCry3.1Ab, the protein expressed in 5307 corn. 445 APHIS has determined that the other cultivars would not have a significant adverse impact on biodiversity and, in some cases could have a beneficial impact . Stacking 5307 corn with the existing transgenic corn cultivars would only add in crementally to these beneficial impacts. Section 4.8.3.1, Pesticide Use , explains that stacking 5307 corn with herbicide-tolerance traits may incrementally increase the use ra tes or displacement of certain herbicides, potentially contributing to a negligible degree to increased weed tolerance to herbicides. Stacking with other insect-resistance traits would potentially decrease broad-spectrum insecticide use, cumulatively reducing the selection pressure for insect resistance and adverse impacts to non- target insects. On balance, these effects are expected to improve biodiversity. When combined with the past, present, and reasonably foreseeable future actions, the cumulative effects of the Proposed Action alternative would likely improve bi odiversity within agroecosystems.



### 4.8.3.3 Domestic Economy

The past and current actions potentially contributing to cumulative effects on the domestic economy are the introduction of transgenic corn varieties and their increasing use. The array of transgenic co rn varieties currently available for pest management is outlined above. The futu re actions potentially contributing to
cumulative effects to the domestic econ omy are any combination of 5307 corn stacked with transgenic corn with PIPs exhibiting herbicide tolerance, or other
desirable traits.

Farmers are expected to economically benefit from corn rootworm-resistant transgenic corn products. Ch apter 2, Section 2.2.2.3, Insect Management , explains that the corn rootworm family is one of the most damaging corn pests, affecting as much as 32 million acres each year. Prior to the introduction of rootworm-protected transgenic corn varieties to the market, corn rootworms cost US farmers nearly $1 billion annually in crop losses and control costs. 446 Using corn rootwormprotected varieties offsets these costs by in creasing yield and reducing the risk, cost,
and time associated with insecticide use. 447 Seed companies and growers could also see an economic benefit, from increased profits.

Similar benefits are expected from inse ct-resistant crops targeted at other economically significant pests. For example, lepidopteran-active Bt corn has remained effective against the European corn borer for more than a decade, yielding


445 Vlachos and Huber. 2011. Pg. 149. 446 Rice. 2004. Pg. 1. 447 Rice. 2004. Pg. 3.Event 5307 Corn Environmental Report

4-57 Environmental Consequences billions of dollars of estimated benefits to farmers in the midwestern US. The cumulative economic benefit to corn grower s in a five-state region over a 14-year period ending in 2009 has b een estimated at $6.9 billion. 448 These growers include many who did not plant Bt corn varieties, but nevertheless benefited from area-wide suppression of European corn borer populations as a result of sustained Bt corn use in the region.

Herbicide-tolerant transgenic corn also has benefits for the domestic economy. For farmers, the general increase in yield, redu ction in some input costs, improvement in pest control, increase in worker safety, and time-management benefits have generally outweighed the additional costs of transgenic seed. 449 Herbicide-tolerant crops have not greatly increased yields but have generally improved weed control and improved farmers’ incomes by saving time.

Combining insect resistance traits and herb icide tolerance traits in a single stacked product would likely convey the individual economic benefits of each trait to farmers and seed companies. Available information do es not allow specific quantification of the economic benefit that any given product would provide because of the complexity and variability of the market. Corn farmers’ profitability is subject to resource costs (seeds, fuel), environmenta l factors (weather, pests), and labor costs (planting, harvesting, and processing). Corn commodity prices fluctuate in accordance with supply and demand, the latter of which can be driven by food, feed, or fuel needs as well as federal government policy.



### No Action Alternative

As described in Section 4.7.1, Domestic Economic Environment , the No Action alternative would not directly or indirectly affect the domestic environment, and existing trends would continue. For the cumulative effects analysis of the No Action alternative, stacking 5307 corn with other transgenic corn cultivars, would not occur. Event 5307 corn would remain a regulated article and would require an APHIS permit or notification for release into the environment. The commercial market would not be affected as a result of the continued regulated status of 5307 corn. Regulated field trials of 5307 corn would only impact small plots of land with no impact on the domestic economy. However, this would not increase the number of different stacked corn varieties available, and growers would not have an increased choice about what to plant. When combined with the past, present, and reasonably foreseeable future actions, there would be no cumulative effect from the No Action alternative on the domestic economy.



448 Hutchison et al . 2011. Pg. 224. 449 NRC. 2010. Pg. 174.Event 5307 Corn Environmental Report

4-58 Environmental Consequences 

### Proposed Action Alternative

As described in Section 4.7.1, Domestic Economic Environment , the Proposed Action alternative would positively affect, both directly and indirectly, the domestic economy. Growers would have a broader rang e of tools to use to combat insects and weeds if 5307 corn is deregulated, directly reducing economic loss from economically important pests. For stacked varieties in particular, the combination of eCry3.1Ab and modified Cry3A (via Syngenta’s dere gulated MIR604 corn) in the same corn hybrids could also justify a reduction in the size of the required on-farm refuge from a minimum of 20 percent of a grower’s corn acres to 5 percent. 450 This would have economic benefits for the grower from increased yield, and would further reduce insecticide use for rootworm control. Although the specific domestic economy
benefits cannot be calculated because of th e variability and complexity of the market, it is assumed that growers will make rati onal decisions to maximize economic gain from their operations, and 5307 corn stacked with other transgenic corn traits will increase the varieties available to growers.


450 Vlachos and Huber. 2011. Pg. 154.Event 5307 Corn Environmental Report

5-1 Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts 

# 5

Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts

As required by the CEQ, this Chapter considers the Executive Orders (EOs) and other federal laws related to the potential environmental impacts of the Proposed

Action.



## 5.1 Executive Orders with Domestic Implications

Four EOs have domestic implications that are relevant to the environmental assessment of the petition to deregulate 5307 corn.



### 5.1.1 Executive Order 12898: Environmental Justice

EO 12898, Federal Actions to Address Environmental Justice in Minority Populations and Low-Income Populations

, 451 requires federal agencies to conduct their programs, policies, and activities that substantially a ffect human health or the environment in a manner so as not to exclude persons and populations from participation in or benefiting from such programs. It also enfo rces existing statutes to prevent minority or low-income communities from being subjected to disproportionately high and
adverse human health or environmental effects.

Event 5307 corn is not significantly different from other transgenic or non-transgenic corn. As described in Chapter 4, Section 4.4.1, Animals , the eCry3.1Ab and PMI proteins do not pose a hazard to humans . A voluntary FDA consultation for food

 451 Executive Order 12898 of February 11, 1994.Event 5307 Corn Environmental Report

5-2 Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts and feed use of 5307 corn was initiated in January 2011 (Appendix B). Data and information provided by Syngenta support the safe use of 5307 corn and indicate that
it will be as nutritious and wholesome as other corn currently used as food and feed. Based on these analyses, 5307 corn is not ex pected to have a disproportionate adverse effect on minorities or low-income populations.

As described in Chapter 4, Section 4.2.1, Agronomic Practices , the cultivation of previously deregulated corn varieties with similar insect resistance traits has been
associated with a decrease in insecticide a pplications. If insecticide applications are reduced, there may be a beneficial effect on minority populations. These populations might include farm workers and their familie s, and other rural dwelling individuals who are potentially exposed to insecticides through aerial application, groundwater, or other routes of exposure.



### 5.1.2 Executive Order 13045: Pr otection of Children

EO 13045, Protection of Children from Environmental Health Risks and Safety Risks

, 452 acknowledges that children may suffer disproportionately from environmental health and safety risks because of thei r developmental stage, greater metabolic activity levels, and behavior patterns, as co mpared to adults. This EO requires each federal agency (to the extent permitted by law and consistent with the agency’s mission) to identify, assess, and address en vironmental health risks and safety risks that may disproportionately affect children.

Event 5307 corn is not significantly different from other transgenic or non-transgenic corn. As described in Chapter 4, Section 4.4.1, Animals , the eCry3.1Ab and PMI proteins do not pose a hazard to humans . A voluntary FDA consultation for food and feed use of 5307 corn was initiated in January, 2011 (Appendix B). Data and information provided by Syngenta support the safe use of 5307 corn and indicate that
it will be as nutritious and wholesome as other corn current used as food and feed. Based on these analyses, 5307 corn is not ex pected to have a disproportionate adverse
effect on children.

As described in Chapter 4, Section 4.2.1, Agronomic Practices , the cultivation of previously deregulated corn varieties with similar insect resistance traits has been
associated with a decrease in insecticide a pplications. If insecticide applications are reduced, there may be a beneficial effect on children that might be exposed to the chemicals. Similar to minority populations, these children might include families of farm workers and other rural dwelling indi viduals who are exposed to insecticides through aerial application, groundwa ter contamination, or other routes.


452 Executive Order 13045 of April 21, 1997.Event 5307 Corn Environmental Report

5-3 Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts 

### 5.1.3 Executive Order 13112: Invasive Species

EO 13112, Invasive Species, 453 requires federal agencies to take action to prevent the introduction of invasive species, to provide for their control, and to minimize the economic, ecological, and human health impacts that invasive species cause.

Corn is a highly domesticated plant and is not an invasive species. Both nontransgenic and transgenic corn varieties that have been granted nonregulated status are widely grown in the US and have not developed weedy or invasive characteristics. As described in Section 4.4.5, Gene Movement in the Natural

Environment , 5307 corn plants are very similar in agronomic characteristics to other corn varieties that are currently grown and are not expected to become weedy or invasive. Accordingly, the Proposed Action would not raise concerns addressed by EO 13112, Invasive Species

.



### 5.1.4 Executive Order 13186: Migratory Birds

EO 13186, Responsibilities of Federal Agen cies to Protect Migratory Birds, 454 requires each federal agency to avoid and minimize, to the extent practicable, adverse impacts on migratory bird populations when conducting agency actions.

Although migratory birds forage in corn fields, as described in Section 4.4.1, Animals

, 5307 corn is not expected to have any adve rse impacts on migratory birds because the eCry3.1Ab protein is not biologically active in avian species. To confirm the absence of any impact on avian species, Syngenta has conducted an eCry3.1Ab toxicity study on juvenile bobwhite quail and a 5307 corn feed ing study on broiler chickens, in which no harmful effects to quail or chickens were observed. Granting nonregulated status to this corn cultivar therefore is not expected to have a negative effect on migratory bird populations. The Proposed Action accordin gly would be in compliance with EO 13186, Responsibilities of Federal Agen cies to Protect Migratory Birds

.



## 5.2 Executive Order with International Implications

EO 12114, Environmental Effects Abroad of Major Federal Actions, 455 requires federal officials to take into consideration any potential environmental effects outside the US, its territories, and possessions that may result from actions being taken.

All of the existing national and international regulatory authorities and phytosanitary regimes that currently apply to introduction of new corn cultivars internationally apply equally to those covered by an APHIS determination of


453 Exectuive Order 13112 of February 3, 1999. 454 Executive Order 13186 of January 10, 2001. 455 Executive Order 12114 of January 4, 1979.Event 5307 Corn Environmental Report

5-4 Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts nonregulated status under 7 CFR Part 340. International trade of 5307 corn subsequent to a determination of nonregulat ed status for the product would be fully subject to national phytosanitary requ irements and be in accordance with phytosanitary standards developed under the International Plant Protection Convention (IPPC). 456

The purpose of the IPPC “is to secure a common and effective action to prevent the spread and introduction of pests of plants and plant products and to promote appropriate measures for their control.” 457 The protection it affords extends to natural flora and plant products and includ es both direct and indirect damage by pests, including weeds. The IPPC set a st andard for the reciprocal acceptance of phytosanitary certification among the 177 nations that have signed or acceded to the

convention. 458 In April 2004, a standard for pest risk analysis (PRA) of living modified organisms (LMOs) was adopted at a meeting of the governing body of the IPPC as a supplement to the existing International Standard for Phytosanitary Measure

No. 11

. 459 The standard acknowledges that LMOs will not present a pest risk and that a determination needs to be made early in the PRA for importation as to whether the LMO poses a potential pest risk resulting from the genetic modification. APHIS pest risk assessment procedures for genetically engineered organisms are consistent with the guidance developed under the IPPC. In addition, issues that may relate to commercialization and transboundary movement of particular agricultural commodities produced through biotechnology are being addressed in other international forums and through national regulations.

The Cartagena Protocol on Biosafety (the Protocol) is a treaty under the United Nations Convention on Biological Diversity (CBD) that established a framework for the safe transboundary movement, with respect to the environment and biodiversity, of LMOs, which includes those modified th rough biotechnology. The Protocol came into force on September 11, 2003, and 161 countries are currently parties to it. 460 Although the US is not a party to the CBD and thus not a party to the Protocol, US exporters will still need to comply with domestic regulations that importing countries that are parties to the Protocol have put in place to comply with their obligations.

The first intentional transboundary moveme nt of LMOs intended for environmental release (field trials or commercial planting) will require consent from the importing country under an advanced informed agr eement (AIA) provision, which includes a requirement for a risk assessment consiste nt with Annex III of the Protocol, and the required documentation. LMOs imported for food, feed, or processing (FFP) are exempt from the AIA procedure, and are covered under Article 11 and Annex II of the Protocol. Under Article 11, parties must post decisions to the Biosafety



456 IPPC. 1997. 457 IPPC. 1997. Pg.1. 458 IPPC. 2011. 459 IPPC. 2004. 460 CBD. 2011.Event 5307 Corn Environmental Report

5-5 Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts Clearinghouse database on domestic use of LMOs for FFP that may be subject to transboundary movement. To facilitate comp liance with obligations to this protocol, the US government has developed a website that provides the status of all regulatory reviews completed for different uses of bioengineered products. 461 These data will be available to the Biosafety Cl earinghouse. International trade of 5307 corn would be conducted in compliance with the Protocol.

Biosafety and biotechnology consensus docu ments, guidelines, and regulations, are managed by APHIS in accordance with the requirements of the North American Plant Protection Organization (NAPPO) and the Organization for Economic Cooperation and Development (OECD). NAPPO has completed three modules of the Regional Standards for Phytosanitary Measures (RSPM) No. 14, Importation and Release (into the environment) of Transgenic Plants in NAPPO Member Countries

. 462 The Proposed Action is not expected to affect APHIS’s participation in NAPPO or the

OECD.

North American Biotechnology Initiative is a forum for information exchange and cooperation on agricultural biotechnology issues for the US, Mexico, and Canada. Bilateral discussions on biotechnology regulatory issues are also held regularly with other countries including Argentina, Brazil, Japan, China, and South Korea. Several countries including Argentina, Brazil, Australia, Canada, China, Colombia, Japan, Mexico, New Zealand, South Korea, the Phil ippines, South Africa, and the European Union have already approved Bt corn varieties to be grown or imported for food or
feed.

As described in Section 4.7.2, Trade Economic Environment , the Proposed Action is not expected to affect the US corn export ma rket since Syngenta is actively pursuing regulatory approvals for 5307 corn in coun tries with functioning regulatory systems for genetically modified organisms and that import corn from the US or Canada. Regulatory filings for 5307 co rn import approvals have been made in Australia, Japan, New Zealand, South Africa, South Korea, Taiwan, and the European Union. Applications are planned for additional countries including Mexico, China, the Philippines, Indonesia, and Russia.



## 5.3 Other Federal Laws

In addition to the National Environmental Policy Act and the Plant Protection Act , three other federal environmental laws are pote ntially relevant to the environmental assessment of the petition to deregulate 5307 corn. Other federal land management laws and regulations address the unique characteristics of certain geographic areas, and are also potentially relevant to deregulation of 5307 corn.



461 See http://usbiotechreg.nbii.gov 462 NAPPO. 2003.Event 5307 Corn Environmental Report

5-6 Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts 

### 5.3.1 Clean Water Act

The Federal Water Pollution Control Act 463 (commonly referred to as the Clean Water Act) and implementing regulations 464, 465 require entities that discharge regulated
materials to certain surface wa ter bodies (including wetlands) obtain authorization to do so from federal or state agenci es under various permit programs.

As described in Section 4.3.1, Water Quality and Use , water quality is not likely to change as a direct or indirect result of the Proposed Action. De regulating 5307 corn would not result in a change of agricultural practices or any new discharge of pollutants to surface water bodies. Water qu ality would continue to be regulated and agronomic practices to protect water quality would continue to be implemented if 5307 corn is deregulated. Chemical insecticide use would continue to be reduced as transgenic corn products with insect-resis tant traits are developed, marketed, and adopted. Cultivating 5307 corn would not require Clean Water Act permits different than those already required for agricultural activities.



### 5.3.2 Clean Air Act

The Clean Air Act Amendments of 1990 466 (commonly abbreviated as the Clean Air Act) and implementing regulations 467 require entities that discharge regulated materials into the atmosphere obtain authorization to do so from federal or state agencies under various permit programs.

As described in Section 4.3.3, Air Quality , the Proposed Action is not likely to directly change air quality. Event 5307 corn production would not change land acreage or cultivation practices for transgenic or non-transgenic corn. Air quality would be indirectly improved if aerial application of corn rootworm pesticides is reduced. Corn growers would continue current trends in agricultural activities if 5307 corn is deregulated. Spray application of insecticides could continue to be reduced as additional insect-resistant products are ad opted by growers because 5307 corn would give growers another option to combat co rn rootworm. Cultivating 5307 corn would not require Clean Air Act permits different than those already required for agricultural activities.



### 5.3.3 National Historic Preservation Act

The National Historic Preservation Act 468 (NHPA) and its implementing regulations 469 require federal agencies to:



463 Federal Water Pollution Control Act, as amended. 33 USC 1251-1376

. 464 USACOE. Various dates. 33 CFR Parts 320 through 332. 465 USEPA. Various dates. 40 CFR Parts 230 through 233. 466 Clean Air Act Amendments of 1990 , as amended. 40 USC 7401-7671. 467 USEPA. Various dates. 40 CFR Parts 50 through 99. 468 National Historic Preservation Act of 1966 , as amended. 16 USC 470 et seq

. 469 ACHP. Various dates. 36 CFR Parts 800 through 801.Event 5307 Corn Environmental Report

5-7 Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts 1) Determine whether activities they propos e constitute "undertakings" that have the potential to cause effects on historic properties, and

2) If so, to evaluate the effects of such un dertakings on such historic resources and consult with the Advisory Council on Hist oric Preservation (i.e., State Historic Preservation Offices, Tribal Historic Pr eservation Officers), as appropriate.

The Proposed Action is not expected to adve rsely impact cultural resources on tribal properties. Any farming activity that may be taken by farmers on tribal lands would be undertaken by the tribe or at the trib e’s request. The tribes would have control over any potential conflict with cultural re sources on tribal properties. The Proposed Action would also have no impact on distri cts, sites, highways, structures, or objects listed in or eligible for listing in the National Register of Historic Places, nor would it cause any loss or destruction of significant scientific, cultural, or historical resources.

The Proposed Action is not an undertakin g that may directly or indirectly cause
alteration in the character or use of histor ic properties protected under the NHPA. In general, common agricultural activities conducted under this action do not have the potential to introduce new visual, atmospheri c, or noise elements to areas in which they are used that are different from current agricultural practices and could result in
effects on the character or use of historic properties. The cultivation of 5307 corn alone or in approved stacks is not expected to change any of these agronomic practices that would result in an adverse impact under the NHPA.



### 5.3.4 Federal Laws Regarding Unique Characteristics of Geographic Areas

Other federal land management laws and re gulations protect park lands, prime farm lands, wetlands, wild and scenic areas, or ecologically critical areas. A determination of nonregulated status for 5307 corn is not expected to affect unique characteristics of these geographic areas, and the common agricultural practices that would be carried out in the cultivation of 5307 corn are not expected to deviate from current practices. As described in Section 4.2.1, Agronomic Practices , 5307 corn, when stacked with other deregulated corn traits, is expected to be deployed on agricultural land currently suitable for corn production and replace ex isting varieties, and is not expected to increase the acreage of corn production.

The Proposed Action would not include any major new ground disturbances; new physical destruction or damage to property; alterations of property, wildlife habitat, or landscapes; or prescribed sale, lease, or transfer of ownership of any property. The Proposed Action is limited to a determinat ion of nonregulated status of Event 5307 corn. This action would not convert agricult ural land use to nonagricultural use and therefore would have no adverse impact on prime farm land. Standard agricultural practices for land preparation, planting, pe st control, irrigation, and harvesting of plants would be used on agricultural lands planted to 5307 corn, alone or when stacked with other deregulated corn traits.Event 5307 Corn Environmental Report

5-8 Consideration of Executive Orders and Other Federal Laws Relating to Environmental Impacts This page intentionally left blank.Event 5307 Corn Environmental Report

6-1 List of Preparers 

# 6

List of Preparers

Vanasse Hangen Brustlin, Inc. Jonathan Feinstein
Senior Vice President
Senior Technical Advisor MCP Community Planning and Area Development, University of Rhode Island

B.S., Natural Resources Development, University of Rhode Island

Over 30 years of experience in environmental consulting and prepar ing NEPA documents

Lisa A. Standley, Ph.D.
Chief Environmental Scientist
Technical Document Lead Ph.D., Botany, University of Washington M.S., Botany, Cornell University B.S., Ecology and Systemat ics, Cornell University

25 years of experience in environmental consulting and 20 years of experience in preparing NEPA
documents

Christopher Rife Senior Project Manager
Lead Document Author Masters of Environmental Policy and Management, University of Denver B.A., Humboldt State University

20 years of experience in environmental consulting and 15 years of experience in preparing NEPA
documents

Devleena Ghosh
Environmental Scientist
Researcher B.S., Biology and Environment, Pine Manor College

5 years of experience in environmental consulting and 2 years of experience in preparing NEPA
documentsEvent 5307 Corn Environmental Report

6-2 List of Preparers Rhodes Life Sciences, LLC

Dr. Richard C. Rhodes III Associate Director, Rhode Island Agricultural Experiment Station and Rhode Island Cooperative

Extension Associate Dean, College of
the Environment and Live

Sciences Ph.D., Texas A&M University M.S., Texas A&M University B.S., University of Delaware

32 years of research experience in endocrinology, physiology and agriculture

Syngenta Biotechnology

Demetra Vlachos Global Crop Lead, Seeds Regulatory Affairs M.S., Genetics, Rutgers University B.A., Biological Sciences, Douglass College

19 years of experience in agricultural biotechnology regulatory affairs and environmental safety assessment of transgenic crops; 30 years experience in mammalian toxicology and risk assessment of pesticides

Dr. Dennis Ward US Crop Lead, Seeds Regulatory Affairs Ph.D., Pharmacology and Technology, University of
California B.S., Biochemistry, University of California

25 years of experience in toxicology, risk assessment and regulatory affairs within the agricultural
technolo gy industr y

Dr. Lawrence Zeph Senior Regulatory Manager Ph.D., Molecular and Cell Biology, Pennsylvania State University B.A., Biology, In diana University

22 years of experience in regulatory policy for transgenic cropsEvent 5307 Corn Environmental Report

7-1 References 

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A-1 Appendix A: Public Interest Assessment 

##  

# Appendix A Public Interest Assessment

### Title



## Public Interest Assessment Supporting US EPA Registration of 5307 Corn and the Breeding Stacks Bt11 × MIR604 × TC1507 × 5307 and Bt11 × MIR162 × MIR604 × TC1507 × 5307 Corn

Data Requirement

Federal Register 51 :7626 (1986)

Author s

Demetr a Vlachos , M.S.

Dennis Ward , Ph.D.

Date

March 3 1 , 2011

Submitter

Syngenta Seeds, Inc.

– Field Crops

- NAFTA P.O. Box 12257 3054 East Cornwallis Road Research Triangle Park, NC 27709

Report ID

SSB

- 201

- 10

Page 1 of 41SSB

- 201

- 10 Page 2 of 41 

## Statement of No Data Confidentiality Claim

No claim of confidentiality is being made for information in this report on the basis of its falling within the scope of FIFRA § 10(d)(1)(A), (B), or (C).

Syngenta submits this material to the United States Environmental Protection Agency specifically under provisions contained in FIFRA

, as amended, and consents to use and disclosure of this material by EPA according to FIFRA. In submitting this material to EPA according to method and format requirements co ntained in PR Notic e 86

- 5 and 40 CFR §158.33, Syngenta does not waive any protection of rights involving this material that would have been claimed by the company if this material had not been submitted to the EPA.

Company: Syngenta Seeds , Inc.

– Field Crops

- NAFTA

Submitter :

Demetra Vlachos , M.S. Regulatory Affairs Manager

Date: March 3 1

, 2011

© 2011. Syngenta. All Rights Reserved.SSB

- 201

- 10 Page 3 of 41 

## Statement Concerning Good Laboratory Practices

This assessment is not a study as defined b y the Good Laboratory Practice (GLP) Standard s as set forth in 40 CFR §160. Thus, the GLP Standards do not apply.

Study Director: There was no GLP Study Director for this assessment.

Submitter :

Demetra Vlachos, M.S

. Regulatory Affairs Manag er Syngenta Seeds, Inc.

– Field Crops

- NAFTA P.O. Box 12257 3054 East Cornwallis Road Research Triangle Park, NC 27709

Date: March 3 1 , 2011SSB

- 201

- 10 Page 4 of 41 

## Table of Contents

Page

Statement of No Data Confidentiality Claim

................................ ................................ ...... 2
Statement Concerning Good Laboratory Practices

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

Table of Contents

................................ ................................ ................................ ................ 4
Summary

................................ ................................ ................................ ............................. 7

I. Introduction

................................ ................................ ................................ .................. 9

II. Overview of Corn

................................ ................................ ................................ ... 10

A. Importance of Corn to the U.S. Economy

................................ ......................... 10

B. Corn Agronomics

................................ ................................ .............................. 11

C. Pests of Corn

................................ ................................ ................................ ..... 12

D. Current Insect Control Practices

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

III. Characteristics of 5307 Corn

................................ ................................ .................. 15

A. Transgene s and Novel Proteins in 5307 Corn

................................ ................... 15

B. Field Performance of 5307 Corn

................................ ................................ ....... 17

1. Efficacy studies of 5307 corn and stacked MIR604 × 5307 corn

..................... 17

a) Western corn rootworm efficacy as assessed by root damage ratings

......... 17 b) Western corn rootworm efficacy as assess ed by adult emergence counts

... 22 c) Northern corn rootworm efficacy as assessed by adult emergence counts

.. 23 d) Mexican corn root worm efficacy as assessed by root damage ratings

......... 25

2. Summary of e fficacy t rials of 5307 c orn a gainst t arget p ests

........................... 27

3. Grain yield studies of 5307 corn and stacked MIR604 × 5307 corn

................. 27

IV. Public Interest Factors Applicable to 5307 Corn and the Breeding Stacks Thereof

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

A. Need Factors

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

B. Composition Factors

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

C. Usage Factors

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

D. Performance Factors

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

E. Risk Factors

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

1. Reduced

- risk alternative to conventional insecticides

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

2. Safety profile of 5307 corn and 5307 corn breeding stacks

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

F. Economic Factors

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

V. References

................................ ................................ ................................ .............. 38SSB

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### List of Tables

Page

Table 1. Categorization of corn insect pests based on their potential for causing economic losses (modified from Gray and Luckmann, 1994).

...................... 13

Table 2. Damage rating scale used to assess root damage by corn rootworm larvae.

. 18

Table 3. Surviving adult western corn rootworm beetles recovered in five artificially infested field trials in Missouri, 2007

- 2009

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

Table 4. Surviving adult northern corn rootworm beetles recovered in two naturally infested trials in Missouri

................................ ................................ 24

Table 5. Representative conventional insecticide prod ucts labeled for use in field corn to control corn rootworm larvae and/or adults.

................................ ...... 33SSB

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### List of Figur es

Page

Figure 1. U.S. corn usage by segment, 2009 (Baker et al., 2010).

................................ ... 10

Figure 2. Geographic distribution by county of acres harvested for corn grain in 2008 (source USDA/NASS Charts & Maps).

................................ ............................ 12

Figure 3. Root damage ratings from western corn rootworm field efficacy trials conducted in 200 6 with 5307 corn ( Haney et al., 2008)

................................ ... 19

Figure 4. Root damage ratings from western corn rootworm field efficacy trials conducted in 2008 with 5307 corn ( Haney et al., 2009)

................................ ... 19

Figure 5. Root mass of a 5307 hybrid compared to a control hybrid under high western corn rootworm pressure.

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

Figure 6

. Rootworm e fficacy of 5307, MIR604 × 5307, and MIR604 corn hybrids; Illinois and Minnesota trials, 2005

- 2009.

................................ ......................... 21

Figure 7. Efficacy of 5307 corn against Mexican corn rootworm in Texas, 2006

........... 26

Figure 8. Efficacy of 5307 corn against Mexican corn rootworm in Texas, 2007

........... 26

Figure 9. Grain yield performance of 5307 corn and MIR604 × 5307 corn under corn rootworm pressure.

................................ ................................ ........................... 28SSB

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## Summary

Syngenta is seeking a manufacturing

- use registration under FIFRA Section 3 for a new plant

- incorporated protectant ( PIP) , the eCry3.1Ab protein, as produced in corn derived from transformation event 5307 (“5307 corn” or “Event 5307 corn” )

. Additionally, Syngenta seeks commercial registration s for two new products that include the 5307 corn PIP in breeding combinations with other registered PIPs: Bt11

× MIR604 × TC150 7 × 5307 corn and Bt11 × MIR162 × MIR604 × TC150 7 × 5307

corn. Data and information are provided herein to support a finding that issuance of these registrations will be in the public interest.

Corn is the most widely cultivated U.S. crop, in terms of acreage planted and net value. Transgenic corn hybrids that produce Bacillus thuringiensis

- derived Cry proteins for targeted rootworm control have been available in the U.S. since 2003. Prior to their introduction, control of Diabrotica rootworms accounted for the largest single use of conventional insecticides in the U.S. Transgenic corn hybrids currently available to U.S. growers for rootworm control have significantly improved growers’ ability to co ntrol these pests effectively and easily, while dramatically reducing growers’ use of broad

- spectrum soil

- applied insecticides. The attendant benefits of the current PIP rootworm

- control traits on the market include higher and more consistent grain yield, economic benefits to growers, healthier plants, improved worker safety, and reduced use of fossil fuels to apply insecticide treatments. T he deployment of 5307 corn, in Bt11 × MIR604 × TC1507 × 5307 corn and Bt11 × MIR162 × MIR604 × TC1507 × 5307 corn, w ill also offer these qualitative benefits.

Reduction of broad

- spectrum insecticide use by growers is of special importance because several conventional pesticid e products registered for corn rootworm control have use restrictions or label warnings relat ed to their potential to be highly toxic to humans and/or wildlife. Registration of the PIP in 5307 corn and the breeding combinations thereof is expected to further reduc e chemical insecticide use by corn
growers. Based on an extensive battery of safety studies provided for review by the U.S. Environmental Protection Agency, t he eCry3.1Ab protein poses no significant human health or environmental risks , either in 5307 corn alone or in the PIP breeding combinations described above

.

Field studies demonstr ate that the eCry3.1Ab protein in 5307 corn is highly effective in controlling the larvae of three important U.S. rootworm pests: Diabrotica virgifera virgifera LeConte (western corn rootworm) , D. longicornis barberi Smith and Lawrence (northern corn root worm)

, and D. virgifera zeae Krysan and Smith (Mexican corn rootworm). In the presence of high rootworm infestations, 5307 corn hybrids have also demonstrated a significant yield advantage in side

- by

- side comparisons with nontransgenic, near

- isogenic cont rol hybrids.

Event 5307 corn

, offered in the breeding combinations Bt11 × MIR604 × TC1507 × 5307 corn and Bt11 × MIR162 × MIR604 × TC1507 × 5307 corn, will provide important new choices in pest control, thus promoting marketplace competition andSSB

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- 10 Page 8 of 41 

### higher adoption rates of biotechnology

- derived corn varieties

. Moreover, studies demonstrate that the eCry3.1Ab protein in 5307 corn has unique properties and target s a different gut binding site in corn rootworm larvae than does mCry3A (in Syngenta’s registered MIR604 corn, a component of both breeding stacks above). The concurrent deployment of both eCry3.1Ab and mCry3A in the same hybrid offerings to growers is expected to help preserve target pest susceptibility to both Cry proteins. Additionally, by reduci ng the selection pressure on rootworm populations to evolve resistance to any single method of control, this strategy is predicted to help prolong pest susceptibility to other B. thuringiensis (Bt)

- derived rootworm

- control proteins in transgenic corn culti vars, as well as to other established control methods , including conventional insecticides and crop rotation strategies

.
Current insect resistance management strategies for Bt corn products are centered around the planting of a structured refuge that can provide a source of susceptible adult insects with which rare resistant insects can mate , thereby preventing or delaying the establishment of resistance genes in a population. The size and configuration of the structured refuge are determined by toxin dose and insect biology. A second important aspect of combining eCry3.1Ab and mCry3A in a single product is that the minimum size of the on

- farm refuge required to delay resistance can be reduced from 20% of a grower’s total corn acreage (e.g., for MIR60 4 corn or other corn products with single rootworm

- control traits) to 5% of a grower’s acreage. This will offer additional pest control benefits on what would previously have been refuge acres, will reduce the environmental impact of insecticide use on re fuge acres, and will facilitate grower compliance with refuge requirements. Corn hybrids producing both eCry3.1Ab and mCry3A will represent an important and effective new tool in the corn grower’s arsenal of rootworm control mechanisms.SSB

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- 10 Page 9 of 41 

## I. Introduction

A t the time of application for a pesticide registration under Section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) it is often not possible to determine if the registration will be granted under special circumstances. Since their in troduction

, plant

- incorporated protectant (PIP) registrations have only been granted under special circumstances

. FIFRA authorizes the Administrator of the
E nvironmental P rotection A gency (EPA) to conditionally register a pesticide containing a new active ingredient for a period sufficient to generate and submit additional data. Conditional registrations are granted if the Administrator determines that use of the pesticide during such period will not cause any unreasonable ad verse effect on human health o r the environment and that use of the pesticide is in the public interest.

The introduction of crops improved through modern biotechnology has been the single most important technological innovation in United States (U

. S

. ) agriculture in

recent years. As a result of the benefits growers derive from genetically modified

crops, a doption of crops with insect and herbicide tolerance traits has increased dramatically since their commercial introduction

. Between 1996 and 2008, the use of transgenic crops boost ed global farm incomes by $52 billion, of which $ 7.1 billion

w as directly attributable the use of insect

- resistant c orn hybrids in the U.S. alone (Brookes and Barfoot, 2010)

. Improved insect protection and weed control provided by transgenic crops have le d to increased crop yields and reduct ions in conventional pesticide applications (Marra et al. , 2002) . The continued development and introduction of such products are expected to benefit growers

, agricultural workers,

consumers , and the environment

.

Even t 5307 corn was developed by S yngenta Biotechnology , Inc. to provide U

. S

. growers with corn hybrids that are resistant to feeding damage caused by coleopteran insect pests , specifically corn rootworm s

. There is no herbicide tolerance trait in 5307
corn; t he selectable marker used during transformation was phosphomannose isomerase (PMI)

, w hich has no agronomic utility. Event 5307 corn will not be offered as a stand

- alone product, but will be offered to U.S. growers in breeding combinations with other appro ved PIPs to provide control of multiple lepidopteran and coleopteran pests as well as herbicide tolerance. The s pecific products will represent PIP combin ations 1 

### contained in breeding crosses of transgenic Bt11 × MIR604 × TC150 7 × 5307 corn and Bt11 × MIR 162 × MIR604 × TC150 7 × 5307 corn.

This assessment characterize s several anticipated benefits of 5307 corn and the two 5307 stacked

- trait products to support a determination that the associated PIP registrat ion s will be in the public interest.

1 

### Bt11, TC1507 and MIR162 corn produce the lepidopteran

- active PIPs Cry1Ab, Cry1F and Vip3Aa20, respectively. MIR604 and 5307 corn produce the coleopteran

active PIPs mCry3 A and eCry3.1Ab.SSB

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## II. Overvie w of Corn

Zea mays Linnaeus , know n as maize throughout the world and as corn in the U nited States , is one of the few major crop spe cies indigenous to the western h emisphere. It has been cultivated in the Americas since early historic times. Corn is the leading production crop glob ally ; the 200 8 /200 9 growing season yielded approximately 792 million metric tons of grain (USDA, 20 10 a).

A. Importance of Corn to the U.S. Economy

Corn is the largest crop grown in the U.S. in terms of both volume and value. The U.S. accoun t ed fo r nearly 39 % of global corn production in 2008/2009 ( USDA, 2010a ) , while utilizing only 20% of the global corn area harvested (NCGA, 2010b)

. In 2009, nearly 80 million acres of corn were harvested for grain in the U.S. , yielding 13.2 bill ion bushels (335 metric tons) (USDA, 20 10b ).

Corn is grown for animal feed, human food, vegetable oil, high fructose corn syrups (HFCS), starch, fermentation into ethanol, and a multitude of industrial and consumer uses. U.S. corn usage by market segme nt is shown in Figure 1. Corn grain used as a feedstock for fuel ethanol production has increased dramatically in recent years ; this trend is expected to continue as the U.S. seeks renewable sources of energy. Domestic corn production contributes signifi cantly to the positive U.S. trade balance in agricultural products.

Feed/Residual
Export

Ethanol

HFCS

Starch

Sweetners

Cereal/Other



### Figure 1

. U.S. corn usage by segment, 200 9 ( Baker et al. , 2010 ). 50.8 %

19.1 % 18.3 % 11.8 %SSB

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### Total U.S. agricultural exports in 2010 were estima ted to be valued at about $100 billion, and over 10 % were attributable to corn . Agricultural exports generate employment, income, and purchasing power in both farm and nonfarm sectors of the

economy. The production equivalent of over one

- fourth of U.S. c ropland moved into export channels in 2008 (USDA, 2010c). O n a raw crop basis , the U

. S

. exported 40 % of food

- grain production, 15 % of feed grains, and more than 43 % of oilseeds. The value of all agricultural exports increased more than imports ; net agric ultural exports in 2008 contributed $35.0 billion to the overall U.S. economy, an increase of $16.4
billion over 2007. Technology advances increase agricultural productivity and promote the competitiveness of U.S. growers in the global market. Moreover, such advances are becoming increasingly important to global food security (Flavell, 2010)

.

B. Corn Agronomics

Z. mays is a large, annual monoecious grass ; the duration of its life cycle depends on the cultivar and the environment in which the cultivar is grow n . The bulk o f corn is produced between l atitudes 30 ° and 47 ° . Practically no corn is grown where the mean midsummer temperature is less than 19 ° C or where the average nighttime temperature during the summer months falls much below 13 ° C. The greatest pr oduction occurs where the warmest month isotherms range between 21 ° and 27 ° C and the freeze

- free season lasts 120 to 180 days. Corn is grown in areas where annual p recipitation ranges from 25 to

over 500 cm. Summer rainfall of 15 cm is approx imately the lower limit for corn production without irrigation.

The upper m idwest regi on of the U.S. provides an ideal combination of temperature, rainfall, and soil type for the cultivation of corn . Iowa, Illinois, Nebraska, Minnesota, Indiana, Ohio, Wisconsin, Mi ssouri, Kansas, and South Dakota are major corn

growing states. Production in these ten states accounted for over 80 % of total production in 2009 (USDA, 2010b). Figure 2 displays the geographic distribution of acres planted in 2008.

Growers have hundred s of corn hybrids from which to choose. Available varieties differ widely in agronomic characteristics, including length of growing period and yield . Technology providers continue to develop varieties with desirable traits and increasing yield. Corn yie lds have increased an average of 3.5 bushels per year over the past decade. The average yield reported for the 2008 U.S. growing season was 153.9 bushels/acre (NCGA a , 2010).SSB

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### Figure 2

. Geographic distribution by county of acr es harvested for corn grain in 2008 (source USDA/NASS Charts & Maps).



### C. Pests of Corn

Yield losses in all crops due to weeds, diseases

, and insects were substantial and widespread until the introduction and adoption of crop protection chemicals in the 1960s

. Weeds compete with crops for light, nutrients, water

, and other growth factors. If weeds are left uncontrolled, corn simply cannot be grown successfully . Estimates of corn yield loss caused by pathogens have ranged from 2 to 17% (Smith and White, 1988) . In addition, a corn crop is susceptible to attack by a variety of insects from the time it is planted until it is consumed as food o r feed

.
Insect pests can be categorized as major and consistent pests, major and sporadic, and moderate to minor based on an nual destructiveness and their geographic distribution. Table 1 categorizes most of the insect pests of corn found in the U.S. The most economically significant of these corn are Diabrotica spp

. ( the corn rootworm complex ) and O strinia nubilalis ( E uropean corn borer )

.SSB

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### Table 1

. Categorization of corn insect pests based on their potential for causing economic losses ( modified from Gray and Luckmann, 1994).

Major & Consistent Insect Pests Moderate to Minor Insect Pests (cont. )

Western corn rootworm ( Diabrotica virgifera virgifera ) Cinch bug ( Blissus leucopterus )

Northern corn rootworm ( Diabrotica longicornis

barberi ) Southern corn rootworm ( Diabrotica undecimpunctata )

European corn borer ( Ostrinia nubilalis ) Other cutworms , many species

Corn earworm ( Helicoverpa zea ) Seedcorn beetle ( Stenolophus lecontei )

Fall armyworm ( Spodoptera frugiperda ) Seedcorn maggot ( Delia platura )

Banks grass mite ( Oligonychus pratensis )

Major Sporadic Insect Pests Two

- spotted spider mite ( Te tranychus urticae )

Black cutworm ( Agrotis ipsilon ) Billbugs, many species

Corn leaf aphid ( Rhopalosiphum maidis ) White grubs, many species

Southwest corn borer ( Diatraea grandiosella ) Stalk borer ( Papaipema nebris )

Sugarcane borer ( Diatraea saccharalis ) Garden symphylan ( Scutigerella immaculata )

Lesser cornstalk borer ( Elasmopalpus lignosellus ) Japanese beetle ( Popillia japonica )

Western bean cutworm ( St r iacosta albicosta ) Sod webworms, several species

Grape colaspis ( Colaspis brunnea )

Moderate to Minor Insect Pests Thrips, several species

Wireworms, many species Dusky sap beetle ( Carpophilus lugubris )

Armyworm ( Pseudaletia unipunctata ) Stink bugs, several species

White

- fringed beetles ( Graphognathus spp. ) Southern cornstalk borer ( Diatraea

cramb idoides )

Grasshoppers, many species Corn root aphid ( Anuraphis maidiradicis )

Corn flea beetle ( Chaetocnema pulicaria )



### The damage inflicted by rootworm larvae can significantly reduce grain yield by interfering with photosynthetic rates, limiting the u ptake of water and nutrients, and by increasing the plant's susceptibility to lodging (Oleson et al.

, 2005). Lodging (leaning) further reduces the effective grain yield by making the plants more susceptible to breaking, reducing their access to sunlight, and increasing the difficulty with which the grain can be harvested efficiently.

In addition to direct damage caused by feeding on plant tissue, insects play an important role in the transmission and dissemination of pathogenic organisms duringSSB

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### corn dev elopment. Soil contains microorganisms, particularly fungi, that may infect plant parts injured by soil

- dwelling insects. Primary roots of the seedling and the radical and seminal roots are commonly infected with Fusarium spp. after the roots have served their function and become senescent. Feeding by corn rootworms has been associated with increased frequencies of Fusarium infection (Dicke and Guthrie, 1988); rootworm feeding may also lead to increased incidences of stalk rots. These pathogen infection s can reduce crop quality, harvestability, and yield.

D. Current Insect Control Practices

The most widespread and damaging insects of corn in the U.S. Corn Belt have been Diabrotica species and O. nubilalis

. Before the introduction of Bacillus thuringiensis (Bt)

- derived corn varieties

, the tools available to growers for pest control consisted of insecticide applications, agronomic practices , and to a limited extent, the use of varieties with a degree of native pest resistance. The introduction of the first B t corn varieties in 1996 provided growers with a n effective means of limiting damage caused by O. nubilalis

. Prior to the introduction of corn rootworm

- protected Bt corn varieties in 2003, an estimated 14 million acres were treated annually with conventio nal insecticides to control corn rootworms. This equated to applications of more than 7.7 million pounds of insecticide active ingredient annually in corn fields for the control of Diabrotica species (Ward et al. , 2005). Control of Diabrotica rootworms

a ccounted for the largest single use of conventional insecticides in the U.S. at that time.

Insect

- resistant transgenic corn , including stacked

- trait varieties with herbicide toler ance, accounted for 63% of corn acres planted in 2009 (USDA, 2009 a )

. 

### Crops engineered to produce Bt toxins that target specific pest taxa have had favorable environmental effects, particularly when replacing the use of broad

- spectrum insecticides that may also impact agriculturally important nontarget organisms, including benefic ial insects such as honeybees or natural enemies that prey on other insects (NRC, 2010; Carpenter, 2011).

Prior to the advent of insect protected field corn, corn rootworm was controlled through the use of crop rotation (e.g., corn/soybean rotation) and insecticides applied to the soil, plant or seed

. It is significant th at corn rootworm population s have developed resistance to some insecticides and to non

- chemical control methods

. R esistance to some corn rootworm insecticides may result in increased c hemical use (EPA, 20 10 ). Although crop rotation has historically been an effective control tool , in

some areas , variant rootworm populations dis play behavioral changes that circumvent rotation strategies. Some northern corn rootworm populations have an e xtended diapause that allows eggs to hatch when the crop rotation returns to corn rather than in the non

- corn rotation crop in the growing season that follows corn. A variant western corn rootworm population now lays its eggs in soybean fields rather than corn fields, allowing eggs to hatch in fields rotating to corn.SSB

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### R ootworm

- protected Bt corn hybrids have been available to U.S. growers since 2003

. The current PIP proteins registered for corn rootworm control in field corn are Cry3Bb1 (in events MON 8 63 and MON 88017 corn), Cry34Ab1/Cry35Ab1 (in event DAS

- 59122

- 7 corn) and mCry3A (in Syngenta’s event MIR604 corn). As the industry trend is towards combined

- trait hybrid offerings, it is important to note that the aforementioned Cry proteins are availabl e in multiple combinations

. From the perspective of preventing or mitigating resistance in target pest populations, the deployment of multiple corn rootworm traits in a single corn hybrid is key to the durability of the registered PIP s in corn

.



## III. Characte ristics of 5307 Corn

A. Transgenes and Novel Proteins in 5307 Corn
Using the techniques of modern molecular biology, Syngenta has transformed corn ( Zea mays L. ) to produce 5307 corn , a new cultivar that produces an insecticidal protein with activity against certain corn rootworm ( Diabrotica ) species. Event 5307

corn contain s the trans gene ecry3.1Ab encoding an eCry3.1Ab protein and the

trans gene pmi (also known as manA ) encoding the enzyme phosphomannose isomerase (PMI). The eCry3.1Ab protein is an engineer ed chimera of modified Cry3A (mCry3A) and Cry1Ab proteins , the native forms of which occur in B. thuringiensis . The gene pmi was obtained from Escherichia coli strain K

- 12 and the
PMI protein it encodes was utilized as a plant selectable marker during dev elopment of 5307 corn

. PMI al lows cultured plant cells to utilize mannose as the primary carbon source. It does not confer herbicide tolerance or other agronomic characteristics

.
Syngenta conducted mortality bioassays with the eCry3.1Ab protein in a r ange of insect species to identify those that are susceptible. The results of these bioassays demonstrate that the activity of eCry3.1Ab protein is limited to species within the Order Coleoptera, yet not all coleopteran species are sensitive to the protei n. Although part of the amino acid sequence of the eCry3.1Ab protein is derived from Cry1Ab, a lepidopteran

- active protein, the portion of Cry1Ab represented in eCry3.1Ab does not include the N

- terminal lepidopteran

- active region. This likely accounts fo r why eCry3.1Ab demonstrates no activity against several lepidopteran species tested (Nelson, 2010).

The native Cry3A protein from B. thuringiensis is highly toxic to Colorado potato beetle larvae ( Leptinotarsa decemlineata ), but has very little or no tox icity to related beetles in the genus Diabrotica

. By modifying the native gene cry3A , Syngenta introduced a protease recognition site into the native Cry3A protein, thereby creating a modified Cry3A (mCry3A) that was active on the larvae of three Diabroti ca sp p. : western, northern and Mexican corn rootworm s . Modified Cry3A is the registered PIP in Syngenta’s MIR604 corn (Agrisure® RW corn). L. decemlineata and the same Diabrotica spp. that are sensitive to mCry3A are also sensitive to eCry3.1Ab. Althou gh t he eCry3.1Ab protein contains a portion of mCry3A in its amino acidSSB

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### sequence, it is significant that eCry3.1Ab and mCry3A have unique biochemical properties and do not compete for the same binding site in the guts of corn rootworm larvae.

The species specificity of each Bt **δ**

- endotoxin is the result of the efficiency of the various steps involved in producing an active protein toxin and its subsequent interaction with the epithelial cells in the insect midgut. To be insecticidal, most
known Bt **δ−** endoto xins must: (1) be ingested by the insect and solubilized in the gut, (2) be activated by specific proteolytic cleavages, (3) bind to specific receptors on the surface of the insect midgut, and (4) form ion channels. The completion of all these four proce sses results in disruption of the normal function of the midgut leading to the death of the insect.

The eCry3.1Ab protein induces toxicity via the four steps noted above (Jones, 2010). Data support the conclusion that eCry3.1Ab recognizes different inse ct midgut receptor binding sites than the activated form of mCry3A in the brush border membrane of western corn rootworm larvae, the primary target pest of 5307 corn. This differential binding, in conjunction with ion channel profiles, is indicative of a unique mode of action for eCry3.1Ab relative to the related mCry3A protein (Jones, 2010; Walters et al.

, 2010).

Syngenta conducted or sponsored an extensive battery of studies to support an environmental safety assessment for the eCry3.1Ab protein and 530 7 corn. No harmful effects were observed in studies with a range of nontarget organisms, which were exposed to levels of eCry3.1Ab protein at or above expected environmental concentrations. A comprehensive environmental risk assessment describing the

res ults of these studies has been submitted for EPA review (Nelson, 2010).

The absence of adverse effects in nontarget species is consistent with the known mechanism by which Cry proteins exert their selective toxicity and the relatively narrow insecticida l spectrum of a given Cry protein. Individual Cry protein s are usually active against only a few related species within any phylogen et ic Order. Most of the insect

- specific toxins identified to date have shown activity among the O rders of L epidoptera and/ or Diptera, or Coleoptera. A few Bt crystal toxins have shown activity against nematodes (Marroquin et al. , 2000; Wei et al. , 2003).

A ccompanying the registration applications for the 5307 corn breeding stacks ( Bt11 × MIR604 × TC1507 × 5307 corn and Bt 11 × MIR162 × MIR604 × TC1507 × 5307

corn )

, Syngenta is providing studies that demonstrate the absence of synergistic effects among the combined PIP proteins in these products (Seastrum et al., 2010; Seastrum

, 2010 ) . These studies support a conclusion tha t there will not be adverse effects on nontarget species as a result of exposure to the combined PIPs in the stacked

- trait corn hybrids ( Raybould, 2011a, 2011b)

.
Additionally, a standard battery of studies to identify hazards for mammalian species poten tially exposed to plant

- incorporated eCry3.1Ab has been conducted. TheseSSB

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### eCry3.1Ab studies include an acute oral toxicity study with mice ( Korgaonkar , 2009),
in vitro digestive fate assays ( Song, 2010 ; Seastrum, 2009), a comparison of amino acid sequence to that of known toxins and allergens ( Harper, 2011 ; McClain, 2011 ), and a characterization of the biochemical properties of the eCry3.1Ab protein (Nelson

, 2008 ; Nelson

, 2009). The results of the safety studies demonstrated that no adverse effects were ob served in mice exposed to a maximum attainable oral dose, that the protein is rapidly degraded in a simulated gastric environment, that the protein does not share sequence homology with known mammalian toxins or allergens, and that it does not possess prop erties suggestive of food allergen potential. A temporary exemption from the requirement of food and feed tolerances has been established for eCry3.1Ab ( 40 CFR §174. 532) in connection with the current Experimental Use Permit for 5307 corn and associated s tacked

- trait corn (67979

- EUP

- 8). Concurrently with its application for registration of the eCry3.1Ab PIP in 5307, Syngenta has submitted a petition for a permanent tolerance exemption.

The PMI marker protein in 5307 corn is considered an “inert ingredi ent” with respect to pesticidal activity . PMI is exempt from tolerances in all plants (40 CFR §174 .527); a battery of safety studies previously submitted by Syngenta support this exemption.

B. Field Performance of 5307 Corn

Other than its resistance to co rn rootworm larval damage, 5307 corn is phenotypically equivalent to conventional corn

. In the presence of populations of western, northern or Mexican corn rootworm larvae, production of eCry3.1Ab in the roots of 5307 corn plants will protect the plants f rom significant damage by rootworm larvae and will preserve the inherent yield potential of the corn varieties that contain the trait

.

1. Efficacy studies of 5307 corn and stacked MIR604 × 5307 corn

a) Western corn rootworm efficacy as assessed by root damage ratings
Syngenta conducted field trials at two Illinois locations in 2006 ( Haney et al. , 2008 ) and four locations in Illinois and Minnesota in 2008 ( Haney et al. , 2009 ) to assess the efficacy of 5307 corn hybrids in controlling western corn rootworm larva e . Cultural practices used in the trials (tillage, fertilization, herbicide application, etc.) were typical of the recommended agricultural procedures for each area, except that all insecticides (including seed treatments) were avoided.

The trial entrie s were planted in a randomized complete block design in fields that had been planted to a trap crop of cucurbits the previous season to attract corn rootworm beetles for increased egg accumulation (Branson and Sutter, 1989). In some locations, additional western corn rootworm eggs were also applied to artificially infest corn seedlings. These methods were used to assess 5307 corn efficacy at high levels of pest pressure.

At both locations i n the 2006 trials, four replicate plots of each trial entry were planted in four

- row plots, with 25 plants per row. For each plot, root masses were evaluated from four random plants in the center two rows, avoiding the row ends. At eachSSB

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### location in the 2008 trials, three replicate plots of each trial entry were plant ed in three

- row plots, with 15 plants per row. For each plot, root masses were evaluated from six random plants in the center row, avoiding the row ends.
Damage ratings were assessed on roots collected and washed at the R1 stage of plant development (si lking stage, beginning when silks are visible outside husks). Ratings were made on a scale of 0.01 (no damage) to 3.0 (heavy damage) using a modification of the Iowa State University node

- injury linear (0

– 3) scale for rating root damage by

corn rootworm larvae (Oleson et al., 2005). The root damage rating scale used is described in Table 2. For each set of trials (2006 and 2008), mean damage ratings were compared across entries by analysis of variance, and statistical significance was assigned at the c ustomary 5% level.

Table 2 . Damage rating scale used to assess root damage by corn rootworm larvae.
Rating scale was modified from the 0

– 3 scale of Oleson et al. (2005).

Rating Description of Rootworm Damage

0.01 No damage to 1

– 2 light surface scars on roots

0.02 3+ light surface scars ≤ 4 moderate scars (combined across all roots on a plant)

0.05 5+ heavy scars (long, deep scars), but no root pruning (pruning ≤ 1.5 inches from crown)

0.10 1 root pruned to ≤ 1.5 inches accompanied by heavy scars

0.25 2+ roots pr uned to ≤ 1.5 inches (up to 1⁄4 nodes, equivalent, pruned)

0.50 Equivalent of 0.50 node of roots pruned

0.75 Equivalent of 0.75 node of roots pruned

1.00 Equivalent of 1.00 node of roots pruned

1.25 Equivalent of 1.25 nodes of roots pruned

1.50 Equivale nt of 1.50 nodes of roots pruned

1.75 Equivalent of 1.75 nodes of roots pruned

2.00 Equivalent of 2.00 nodes of roots pruned

2.25 Equivalent of 2.25 nodes of roots pruned

2.50 Equivalent of 2.50 nodes of roots pruned

2.75 Equivalent of 2.75 nodes of r oots pruned

3.00 Equivalent of 3.00 nodes of roots pruned



### Western corn rootworm larvae were very destructive to the nontransgenic, near

isogenic control hybrids at all trial locations in both years, but caused significantly less injury to the 5307 corn hybrids. Mean root damage ratings in the 2006 trials (Figure 3) ranged from 0.12 to 0.20 for the 5307 hybrids across both locations and were 1.53 and 1.94 for the controls. These ratings demonstrated that t he 5307 hybrids sustained only 6.3% to 12.3% of the damage observed in the control hybrids in both locations. In 2008 (Figure 4) , mean root damage ratings ranged from 0.04 to 0.06 for the 5307 hybrid entries across the four test locations, while the control ratings rangedSSB

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### from 1.42 to 2.81. The 5307 h ybrids sustained only 1.5% to 4.2% of the damage observed in the control hybrids across the four test locations. In all cases, the differences between the 5307 and control hybrids were statistically significant.
The photograph in Figure 5 illustrates how destructive western corn rootworm larvae were to the control hybrid under high pest pressure and how highly effective the 5307 hybrid was in preventing damage under the same pest pressure.

Figure 3 . Root damage ratings from western corn rootworm field efficacy trials conducted in 2006 with 5307 corn ( Haney et al. , 2008)

Figure 4 . Root damage ratings from western corn rootworm field efficacy trials conducted in 2008 with 5307 corn ( Haney et al. , 20 09)SSB

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### Figure 5 . Root mass of a 5307 hybrid compared to a control hybrid under high
western corn rootworm pressure

. A typical root mass of a 5307 corn hybrid is shown on the left (Shirley, IL, 2008). Note the loss of entire nodes of roots from the control hybrid on the right due to extensive feeding by corn rootworm larvae.



### The efficacy of 5307 corn in preventing root feeding damage was also examined in a broader set of field trials conducted in 2005 through 2009, at a total of 21 trial locations in Illinois and Minnesota. In addition to comparing the performance of a 5307 corn hybrid to a nontransgenic control hybrid, comparisons were also made to a MIR604 × 5307 hybrid and a MIR604 hybrid. All hybrids tested were in the same background genotype. The trials were conducted under natural rootworm infestation, which was facilitated by planting a cucurbit trap crop the previous season. The design and methods used in these trials were otherwise similar to those employed in the

we stern corn rootworm trials described above, and root damage ratings were assigned using the same 0.01

– 3.0 rating scale (Table 2 ). At the Illinois trial locations, the predominant rootworm species was western corn rootworm, whereas the trials in Minnesot a had mixed infestations of western and northern corn rootworms.

The results of these five years of trials are summarized in Figure 6 and again demonstrate the high rootworm control efficacy of the eCry3.1Ab protein in 5307 hybrids. This was evident wh ether the trait was present alone via 5307 corn or in combination with the mCry3A trait in MIR604 corn . The combined statistical analysis across this set of trials indicated that the root damage ratings for all the transgenic hybrids were significantly lo wer than the control hybrid. Although bothSSB

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### the 5307 hybrid and the MIR604 × 5307 hybrid had the lowest mean root damage ratings across all locations (0.05 on a scale of 0.01

– 3.0), these ratings were not statistically lower than the damage ratings for th e MIR604 hybrid (0.17). By comparison, the control hybrid had mean damage ratings of 1.75.

Figure 6 . Rootworm e fficacy of 5307, MIR604 × 5307, and MIR604 corn hybrids; Illinois and Minnesota tr ials, 2005

- 2009.

Western corn rootworm was the predominant rootworm species in the Illinois trials, while mixed infestations of western and northern corn rootworms were present in the Minnesota trials. 

## 0.05 0.05 0.17 1.75

0.00 0.50 1. 00 1.50 2.00 2.50

5307 MIR604x5307 MIR604 Negative Control Root damage ratings, 0.01

– 3.0 rating scale 

### Natural rootworm infestation, preceded by trap cucurbit crop in prior season 

## 5 years, 21 locationsSSB

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### b) Western corn rootworm efficacy as assessed by adult emergence counts

R oot damage ratings are commonly used to measure the efficacy of insect control tactics against corn rootworms, however, several other methods are also employed (Oleson et al.

, 2005). Collecting and counting rootworm adults as they emerg e from the soil represent an alternative method (Hein et al.

, 1985). Studies of adult rootworm emergence were conducted in controlled field experiments using 5307 corn , MIR604

corn , MIR604 × 5307 corn , and nontransgenic control corn

. These field assessme nts were conducted in multiple locations in Missouri in 2007 to 2009 by Bruce Hibbard, Ph.D. (Plant Genetics Research Unit; USDA Agricultural Research Service; Columbia, Missouri), as part of an insect resistance management research effort to characterize 5307 corn performance (Hibbard, 2009).

The trials were planted in a randomized complete block design in one location with three replications in 2007, in one location with five replications in 2008, and in two locations with five replications and one loc ation with four replications in 2009. Due to prior cropping practices, the plots were not expected to already contain western corn rootworms at the time of planting. Cultural practices used in the trials (tillage, fertilization, herbicide application, et c.) were typical of the recommended agricultural procedures for each area, except that all insecticides (including seed treatments) were avoided . Following artificial infestation of the test plots with western corn rootworm eggs, individual tents were ere cted over each replicate plot to collect and count the adult rootworm beetles as they emerged. The Missouri locations where the trials were conducted do not have the rotation

- resistant (extended diapause) variant of western corn rootworm that lays eggs ou tside of corn (in soybeans and other crops). Thus, it was assumed that all western corn rootworm adults recovered in these trials survived from eggs infested in these trials.

As shown in Table 3 , the mean number of western corn rootworm adults that sur vived to emerge from the transgenic corn plots was markedly and statistically significantly lower than in the control plots in all individual trials. Reflecting the increased statistical power of the combined data across all trials in 2007

- 2009, both th e 5307

corn hybrid and the MIR604 × 5307 stacked hybrid were statistically significantly more efficacious than the MIR604 hybrid alone. The mean number of beetles recovered per tent for these hybrids were 1.9, 0.8, and 19, respectively, compared with 879 beetles per tent in the control plots. Although only half the number of beetles emerged from the MIR604 × 5307 stacked hybrid as emerged from the 5307 hybrid, the difference between them was not statistically significant; both hybrids provided high levels of control. Because so few western corn rootworm adults emerged from the 5307 corn plots as well as the MIR604 × 5307 corn plots, this likely indicates that the eCry3.1Ab trait alone in 5307 corn is highly effective in controlling the larvae. Using 100% control survival as a comparator, the mean percent survival ranged from only 0.03%

– 1.92% across all five trials for hybrids containing the eCry3.1Ab trait, whether as a single 5307 hybrid or in a breeding stack combination with the mCry3A trait in MIR60 4 corn . By comparison, the percent survival in the single

- eventSSB

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### MIR604 hybrid plots ranged from 1.15%

- 3.12%; this demonstrated slightly lower but, nevertheless, very good efficacy of the mCry3A trait against the target pest.

Table 3 . Surviving adult western corn rootworm beetles recovered in five artificially infested field trials in Missouri, 2007

- 2009

Location

- Year Corn Hybrid Adult WCRW Beetles Recovered
Mean No. per
Tent ± SEM % Relative
Survival

Site 1

- 2007 5307 2.7 ± 0.7 b 1.92

MIR604 x 5307 0.3 ± 0.3 b 0.24

MIR604 4.3 ± 3.4 b 3.12
Nontransgenic control 139 ± 44 a 100

Site 2

- 2008 5307 0.6 ± 0.4 b 0.06

MIR604 x 5307 0.6 ± 0.2 b 0.06

MIR604 14.0 ± 3.4 b 1.45
Nontransg enic control 964 ± 147 a 100

Site 2

- 2009 5307 5.2 ± 1.6 b 0.48

MIR604 x 5307 2.2 ± 0.9 b 0.20

MIR604 22.4 ± 6.2 b 2.07
Nontransgenic control 1081 ± 116 a 100

Site 3

- 2009 5307 0.4 ± 0.2 b 0.19

MIR604 x 5307 0.2 ± 0.2 b 0.10

MIR604 2.4 ± 1.4 b 1.15
Nontransgenic control 208 ± 45 a 100

Site 4

- 2009 5307 0.5 ± 0.5 b 0.03

MIR604 x 5307 0.5 ± 0.5 b 0.03

MIR604 52.8 ± 29.4 b 2.75
Nontransgenic control 1916 ± 295 a 100

All 5307 1.9 ± 0.6 c 0.21

MIR604 x 5307 0.8 ± 0.3 c 0.09

MIR604 19.0 ± 6.3 b 2.16
Nontransgenic control 879 ± 149 a 100
Data from Hibbard (2009)
WCRW = western corn rootworm Means followed by the same letter are not statistically different (i.e., p > 0. 05). All = combined data from all trial sites and years.



### c) Northern c orn r ootworm efficacy as assessed by adult emergence
counts

When considered across the US Corn B elt, n orthern corn rootworms do not typically cause as much economic damage as do western cor n rootworms, the primary targetSSB

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### pest of 5307 corn . Nevertheless, northern corn rootworms remain a significant pest in

many corn

- growing areas, particularly in cooler regions.

I t i s difficult to successfully mass rear northern corn rootworms in culture, hence it is not feasible to artificially infest test plots with eggs of this species to conduct controlled trials under high population pressure. However, natural infestations of northern corn rootworm larvae were evident in two of the five Missouri tria ls conducted by Bruce Hibbard (Hibbard, 2009). At one trial location in 2007 and another location in 2008, sufficient northern corn rootworm beetles were captured in the tents placed over the corn plots to observe 5307 corn efficacy against this pest.

As shown in Table 4, a mean of over 21 northern corn rootworm beetles per plot survived to emerge from the control corn plots, whereas a mean of less than one beetle per plot survived to emerge from the plots containing the 5307 and/or MIR604 traits; the dif ferences between the control and the transgenic plots were significant for all comparisons ( p < 0.05). Although not a single surviving adult northern corn rootworm beetle emerged in any of the MIR604 × 5307 corn plots, the differences between the 5307, MI R604 × 5307, and MIR604 hybrids were not statistically significant. This may reflect the limited statistical power afforded by the data for the two trial sites. Nevertheless, these trials demonstrated high efficacy of eCry3.1Ab and 5307 corn against nort hern corn rootworms, both alone and in combination with mCry3A in MIR604 corn

.

Table 4 . Surviving adult northern corn rootworm beetles recovered in two naturally infested trials in Missouri

Location

- Year Corn Hybrid Adult N CRW Beetles Recovered

Mean No. per Tent
± SEM % Relative
Survival

Site 1

- 2007 5307 0.3 ± 0.3 b 1.49

MIR604 x 5307 0.0 ± 0.0 b 0.00

MIR604 0.3 ± 0.3 b 1.49

Nontransgenic control 22.3 ± 5.5 a 100

Site 2

- 2008 5307 0.2 ± 0.2 b 0.93

MIR604 x 5307 0.0 ± 0.0 b 0.00

MIR604 0.8 ± 0.4 b 3.74

Nontransgenic control 21.4 ± 2.5 a 100

Data from Hibbard (2009) Means followed by the same letter are not statistically different ( i.e., p > 0.05 )

. NCRW = northern corn rootwormSSB

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### d) Mexican c orn r ootworm efficacy as assessed by root damage ratings
While western and northern corn rootworms are pests of corn throughout the Midwestern states, the Mexican corn rootworm occurs in Texas, Oklahoma and New Mexico (Krysan, 1986). The efficac y of 5307 corn hybrids in controlling Mexican corn rootworm was investigated in 2006 and 2007 under naturally infested conditions at one Texas location where corn had been grown for several consecutive seasons. The performance of 5307 corn was compared to that of MIR604 corn , MIR604 × 5307

corn , one or two corn rootworm insecticide treatments applied at the label rate to nontransgenic corn , and an untreated nontransgenic control corn hybrid. All hybrids were of near

- isogenic genotypes and were planted in four replicates of two

- row plots in a randomized complete block design. The trials were managed under typical local agricultural practices, however, insecticide applications (including seed treatments) were not applied, except where noted as the experimen tal variable. Six root masses per replicate plot were evaluated for feeding damage according to the 0.01 to 3.00 rating scale described in Table 2 . The ratings were compared by an analysis of variance a ppropriate for a r andomized complete block design

, a nd statistical significance was assigned at the customary 5% level.

In the 2006 trial (Figure 7 ), all entries had significantly less root damage than the control hybrid, but none was statistically different from another. In the 2007 trial (Figure 8 ), a ll entries again had significantly less root damage than the control. The 5307 hybrid was significantly more efficacious than the nontransgenic hybrid treated with Force® 3G (tefluthrin granular) insecticide. However, there were no statistically signific ant differences in root ratings among the 5307 hybrid, the MIR604 × 5307 hybrid, the MIR604 hybrid, the MIR604 hybrid treated with Force 3G insecticide, or the MIR604 hybrid treated with Cruiser® 0.25 insecticide (a thiamethoxam seed treatment). Across bo th trials, mean root damage ratings in the 5307 and MIR604 × 5307 plots were very low, and ranged from 0.01 to 0.045 . B y contrast, the control plants had mean root damage ratings of 0.71 and 0.69.

Although the statistical power of this small dataset of M exican corn rootworm trials

to

- date is limited, the trials clearly demonstrated that 5307 corn , either alone or in a breeding stack with MIR604 corn , was highly effective in controlling this damaging pest.SSB

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### Figure 7

. Efficacy of 5307 corn against Mexican corn rootworm in Texas, 2006

Figure 8

. Efficacy of 5307 corn against Mexican corn rootworm in Texas, 2007SSB

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### 2. Summary of efficacy t rials of 5307 c orn against target p ests

M ulti

- year and multi

- location field assessments of the efficacy of 5307 corn against

w estern corn rootworm larvae were conducted by measuring feeding damage to corn roots and by counting the numbers of adult beetles that survived to emerge from infested corn plots. Both methods independently demonstrated that 5307 corn provides excellent control of this primary target pest. Where 5307 corn was also tested in a breeding stack with MIR604 corn , as MIR604 × 5307 hybrids, efficacy w as also excellent.

Although the efficacy of 5307 corn against northern and Mexican corn rootworm larvae has not been as extensively studied, the trials to

- date also indicate that 5307

corn is highly efficacious in reducing root feeding damage by these s ignificant pests. Similarly

, the MIR604 × 5307 breeding stack also appears to be highly effective.

Data in some trials suggested a possible efficacy advantage of combining the eCry3.1Ab trait in 5307 corn with the mCry3A trait in MIR604 corn , however, the numerical differences in efficacy ratings were not statistically significant. This may reflect the fact that the eCry3.1Ab protein in 5307 corn alone is highly effective in controlling rootworm larvae; incremental efficacy afforded by stacking the eCr y3.1Ab and mCry3A traits may, therefore, be difficult to discern . Expanded pre

- commercial product testing will more precisely define the relative efficacy profiles of 5307 and MIR604 × 5307 hybrids under a variety of growing conditions and levels of pest pressure. Nevertheless, as detailed in the discussion of insect resista nce management benefits of 5307 corn there are compelling reasons to combine the traits in 5307 and MIR604 corn in breeding stacks for commercial deployment.

3. Grain yield studies of 5 307 corn and stacked MIR604 × 5307 corn

Syngenta conducted field studies of 5307 corn grain yield over three years in 13 U.S. locations across the Corn Belt. The trial entries were planted in a randomized complete block design in fields that had been pla nted to a trap crop of cucurbits the previous season to attract corn rootworm beetles for increased egg accumulation (Branson and Sutter, 1989). The test and control corn hybrids evaluated were near

isogenic (i.e., in the same germplasm background). In m ost locations , 5307 corn was also compared to MIR604 corn, a MIR604 x 5307 corn breeding stack, as well as nontransgenic corn treated with Force® 3G insecticide, a granular soil applied tefluthrin (pyrethroid) formulation. Cultural practices used in the t rials (tillage, fertilization, herbicide application, etc.) were typical of the recommended agricultural procedures for each area, except that all other insecticides (including seed treatments) were avoided.
Under these conditions of rootworm pressure, t he average yield advantage of 5307 corn was 63 bushels/acre compared to untreated control corn and 25 bushels/acre compared to control corn treated with Force 3G insecticide (Figure 9). Grain yieldSSB

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### was not statistically different between the 5307, MIR604 and MIR604 x 5307 corn test plots.

The 63 bushels/acre mean yield advantage for 5307 corn compares very favorably with a r ecently compiled

, more extensive set of trial data for MIR604 hybrids (Agrisure® RW corn) . These data demo nstrate a 25 to 57 bushe ls/acre yield advantage of MIR604 corn over untreated control hybrids under conditions where rootworm populations reached economically damaging levels (Syngenta Agronomy Research, 2010).

Figure 9 . Grain yield p erformance of 5307 corn and MIR604 × 5307 corn under corn rootworm p ressure
.
Means and standard errors are shown; means not labeled with the same letter differ statistically ( p <
0.05 )

.

191 185 184

166

128

0 50 100 150 200 250

5307 MIR604x5307 MIR604 Negative Control +

Force 3G Negative Control Grain Yield (bushels/acre)Grain Yield (bushels/acre) 

### 2007 to 2009: Grain Yield Under Corn Rootworm Pressure of 5307, MIR604x5307 stack, and MIR604 vs. Negative Control with Force 3G and Negative Control with no insecticide in 2 genetic backgrounds 3 years, 13 locations

A

B AB AB

C

p

- value < 0.0001SSB

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## IV. Public Interest Factors Applicable to 5307 Corn and the Breeding Stacks Thereo f

The U.S. EPA’s 1986 policy notice (EPA, 1996) regarding conditional registrations indicates that EPA will consider a variety of factors pertaining to the need for a new pesticide active ingredient, specifically its comparative benefits, risks, and costs . EPA policy (EPA, 1986) states: “The Agency must determine that (1) there is a need for the new chemical that is not being met with other currently registered pesticides or

non

- pesticide alternatives; (2) the new pesticide is comparatively less risky to health or the environment than currently registered pesticides; or (3) the benefits (including economic benefits) from the use of the new chemical exceed those of alternative registered pesticides and other available non

- chemical techniques.” A considera tion of these factors, as they relate to 5307 corn and the breeding stacks thereof ( Bt11 × MIR604 × TC150 7 × 5307 and Bt11 × MIR162 × MIR604 × TC150 7 × 5307 corn)

, clearly demonstrates that the associated PIP registrations will be in the public interest.

Registration of the Bt

- derived eCry3.1Ab protein encoded by the pSYN12274 vector in 5307 corn c an be presumed to be in the public interest because it meets the criteria for a conditional registration

. Registration of a new pesticide is presumed to be in t he public interest if it is a replacement for another pesticide that is of continuing concern
to EPA. Corn varieties containing the 5307 transgenes have the potential to displace applications of conventional rootworm insecticides that are of concern to EP A

, growers , and the public due to human and environmental risk factors

. Additionally, the safety, convenience, and simplicity of the 5307 corn breeding stacks ( Bt11 × MIR604 × TC150 7 × 5307 and Bt11 × MIR162 × MIR604 × TC150 7 × 5307 corn) compared to the application of conventional insecticides, along with the opportunity to extract an economic benefit through increased c rop yield, are expected to be attractive to growers.

A. Need Factors

Prior to the introduction of rootworm

- protected Bt corn varieties in 2 003 , the primary tactics for corn rootworm control were crop rotation (e.g., corn/soybean rotation), soil

applied insecticides, foliar insecticides and, to a lesser extent, insecticidal seed treatments. The current PIP proteins registered for corn rootwor m control are Cry3Bb1 (in events MON 863 and MON 88017 corn), Cry34Ab1/Cry35Ab1 (in event DAS

- 59122

- 7 corn) and mCry3A (in Syngenta’s event MIR604 corn).

Grower demand for corn rootworm control technologies is influenced by the level of corn rootworm in festation (acreage and degree of infestation), the comparat ive cost

vs. benefit of competing corn rootworm control technologies, U.S. and global market acceptance and approval of a technology, and other regulatory constraints ( e.g. , refuge requirements). In the year 2000, prior to the introduction of corn rootworm

- protected transgenic hybrids, almost 8 million pounds of corn rootworm insecticide, costingSSB

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### $172 million ($12.29/acre), were applied to 14 million acres, or 17% of U.S. corn

acreage. B etween 200 5 and 2013, corn

- rootworm

- infested acreage was projected to increase from approximately 31.8 million acre s to 39 million acres (EPA, 2010 ). EPA estimated that the market for transgenic in

- plant corn rootworm protection would increase by 2.6% per year, rea ching 18 to 19 million acres by the year 2013 . P roduct efficacy data provided here in indicate that hybrids containing the eCry3A.1Ab trait from 5307 corn will provide excellent protection against corn rootworm feeding damage , and that this protection will likely meet or exceed that of current control alternatives.

Increased adoption of corn rootworm

- protected corn products is of special importance because many of the chemical insecticides registered for corn rootworm control present potential risks to a pplicators

, other agricultural workers , and wildlife, and bear Restricted Use labels. The chemical insecticides for larval and adult corn rootworm control subject to the greatest use reduction s following the adoption of transgenic

rootworm

- protected corn varieties are organophosphates, carbamates, pyrethroids, and pyrazoles

. The availability of 5307 corn breeding stacks is expected to contribute to this significant trend in reduced use of hazardous insecticides. Adoption of rootworm

protected corn produc ts is not expected to markedly re duce the us e of insecticidal seed treatments; however, seed treatment products are applied at a lower rate per acre than soil

- applied or foliar insecticides . (In addition to use of seed treatments for larval

rootworm contr ol, they are also used to control other soil pests such as wireworms and grubs.)

There is concern about the ability of corn rootworms to evolve resistance to control mechanisms, including crop rotation, chemical insecticides, and rootworm

- protected
Bt cor n products. Because 5307 corn has excellent efficacy against corn rootworm and because the eCry3.1Ab protein operates via a unique mode of action, introduction of th e 5307 corn breeding stacks is expected to extend the useful life of other commercially av ailable corn rootworm

- protected Bt corn products ( i.e. , the PIPs Cry3Bb1, Cry34Ab1/Cry35Ab1 and mCry3A). The availability of eCry3.1Ab as an additional tool for rootworm control will also reduce the selection pressure on rootworm populations to evolve res istance to other methods of control.

B. Composition Factors

The active insecticidal component of 5307 corn plants, eCry3.1Ab, is plant

incorporated, thus it fundamentally differs from the composition of conventional pest control products. The characteristi cs of PIPs virtually eliminate the occupational and environmental risks currently associated with the application of chemical controls for corn insect pests. These product characteristics support a conclusion that registration of 5307 corn is in the publi c interest.SSB

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### C. Usage Factors

The safety, convenience, and simplicity of planting insect

- protected corn compared to the application of conventional insecticides, along with the opportunity to extract an economic benefit through increased crop yield, are expec ted to make the 5307 breeding stack products highly attractive to growers. The restrictions and precautions associated with several conventional insecticides (Table 5) for rootworm control include:

**•** protective clothing (chemical

- resistant gloves and other skin protection, eye protection, respirators, etc.) or other measures (closed

- system applications) to minimize applicator exposure

**•** minimum worker reentry intervals post application

**•** minimum preharvest intervals post application

**•** restrictions against applying the product near bodies of water, when bees are present, or when spray drift is likely

**•** ensuring that granular insecticides are covered with soil post

- application to protect wildlife

**•** recommendations regarding insect resistance management

By contrast, fro m the grower’s perspective, the use of 5307 breeding stacks for insect control warrants only restrictions and obligations related to insect resistance management, including planting and managing refuge acres.

Registration of the PIPs in 5307 corn and th e 5307 corn breeding stacks will help reduce the manufacture, transportation, storage, and disposal of millions of pounds of hazardous chemicals annually and reduce the fossil fuel consumption and greenhouse gas emissions associated with these activities.

D. Performance Factors

Multi

- year and multi

- location field efficacy trials demonstrate the substantial rootworm protection and yield advantages of 5307 corn, both alone and in combination with Syngenta’s existing PIP rootworm control product, MIR604 corn (s ee Part III). The eCry3.1Ab protein is present in all ti ssues of 5307 corn plants, although the concentrations in pollen are extremely low (Nelson, 2010). Production of eCry3.1Ab in roots throughout the corn plant’s development ensures protection where i t is needed and eliminates the risk of insecticide failures associated with timing of soil

- applied or foliar insecticide applications or unfavorable environmental conditions.

Integrated pest management (IPM) in agriculture includes insect scouting or

mo nitoring to determine pest populations, consideration and application of compatible alternative biological, cultural, mechanical and chemical controls, and the establishment of action thresholds for agricultural inputs. The timely and targeted delivery of pest management interventions is key to successful IPM. The planting ofSSB

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### 5307 corn stacks eliminates the need to target and time applications of rootworm insecticides, due to the sustained production of the insect control proteins (eCry3.1Ab and mCry3A) i n vulnerable root tissues. The planting of 5307 corn stacks is compatible with current insect scouting and monitoring programs that provide data upon which to base crop management decisions. These seed products are also fully compatible with cultural con trol measures such as crop rotation. The 5307 corn breeding stacks will fit seamlessly into the concept of IPM for corn.SSB

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### Table 5

. Representative c onventional insecticide products labeled for use in field corn to control corn ro otworm larvae and/or adults

.

Product Active Ingredient(s); % of Formulation Signal Word/ Classification Environmental Hazards and/or Reasons for Restricted Use Classification

Ambush ® Insecticide p ermethrin ; 25.6% WARNING Restricted Use E xtremely to xic to fish and aquatic invertebrates, highly toxic to bees

Avicta® Duo Nematicide/Insecticide (seed treatment) abamectin; 12.4% thiamethoxam; 28.1% WARNING Restricted Use Toxic to fish and wildlife, highly toxic to aquatic invertebrates, exposed
treat ed seeds may be hazardous to wildlife

Aztec ® 2.1% Granular Insecticide t ebupirimfos ; 2.0% cyfluthrin ; 0.1% WARNING Restricted Use Toxic to fish and wildlife

Baythroid ® 2 Emulsifiable Pyrethroid Insecticide c yfluthrin ; 25% DANGER Restricted Use E xtre mely toxic to fish and aquatic invertebrates, highly toxic to bees

Capture ® 2EC Insecticide/Miticide b ifenthrin ; 25.1% WARNING Restricted Use E xtremely toxic to fish and aquatic invertebrates, highly toxic to bees

Capture ® LFR Insecticide b ifenthrin ; 17.15% WARNING Restricted Use E xtremely toxic to fish and aquatic invertebrates, highly toxic to bees

Counter ® 15G Systemic Insecticide Nematicide t erbufos ; 15% DANGER Restricted Use Highly toxic to fish and wildlife, known to cause fish kills, birds and mammals may be killed if granules not covered with soil. Fatal if swallowed, inhaled or absorbed through skin , c orrosive , c auses irreversible eye damage. Counter 20G Systemic Insecticide

- Nematicide t erbufos ; 20% DANGER Restricted Use Highly toxic to fish and wildlife , known to cause fish kills, b irds and mammals may be killed if granules not covered with soil

. Acute dermal, oral and inhalation toxicity. Cruiser ® 5FS (seed treatment) thiamethoxam; 47.6% CAUTION Toxic to wildlife, highly toxic to aqu atic invertebrates, t reated seeds exposed on soil surface may be hazardous to wildlife DuPont Asana ® XL I nsecticide

- 0.66

e mulsible concentrate e sfenvalerate ; 8.4% WARNING Restricted Use E xtremely toxic to fish and aquatic invertebrates, highly toxic to bees

Declare ® Emulsifiable Insecticide Concentrate methyl parathion ; 45.11% DANGER. Restricted Use F atal if swallowed, inhaled or absorbed through skin, highly toxic to aquatic invertebrates and wildlife, highly toxic to bees Dimethoate 4 EC Systemi c Insecticide d imethoate ; 44.8% WARNING Toxic to wildlife and aquatic invertebrates, highly toxic to bees

Dimethoate 400 Systemic Insecticide

- Miticide d imethoate ; 43.5% WARNING Toxic to wildlife and aquatic invertebrates, highly toxic to beesSSB

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Forc e ® 3G Insecticide t efluthrin ; 3% CAUTION Restricted Use very highly toxic to freshwater and estuarine fish and invertebrates

F

orce® ST Insectici de for corn seed treatment t efluthrin ; 26.8% CAUTION very highly toxic to freshwater and estuarine fish and in verteb rates ; exposed treated seeds may be hazardous to birds and other wildlife Force ® CS Insecticide t efluthrin ; 23.4% WARNING Restricted Use very highly toxic to freshwater and estuarine fish and invertebrates

Fortress ® 2.5G granular insecticide c hlor ethoxyfos ; 2.5% WARNING Restricted Use toxic to wild mammals, birds, fish and aquatic invertebrates ; harmful if absorbed through the skin Fortress ® 5G granular insecticide c hlorethoxyfos ; 5% DANGER Restricted Use toxic to wild mammals, birds, fish an d aquatic invertebrates ; fatal if swallowed; may be fatal if inhaled; harmful if absorbed through skin Furadan ® 4F insecticide/ nematicide c arbofuran ; 44% DANGER Restricted Use toxic to fish, birds and other wildlife ; highly toxic to bees ; can seep or leach through soil and can contaminate groundwater; poisonous if swallowed or inhaled; may be fatal or harmful as a result of skin or eye contact or breathing spray mist. Furadan ® LFR insecticide/ nematicide c arbofuran ; 40.64% DANGER Restricted Use toxi c to fish, birds and other wildlife; highly toxic to bees; can seep or leach through soil and can contaminate groundwater; poisonous if swallowed or inhaled; may be fatal or harmful as a result of skin or eye contact or breathing spray mist. Gaucho® 600 F lowable (seed treatment) imidacloprid; 48.7% CAUTION Highly toxic to birds and aquatic invertebrates

Gaucho® 75 ST Insecticide (seed treatment) imidacloprid; 7 5 % CAUTION Highly toxic to birds and aquatic invertebrates

Lam b da 25 CS lam b da

- cyhalothrin; 23.6% WARNING Restricted Use Extremely toxic to fish and aquatic invertebrates; toxic to wildlife; highly toxic to bees Lannate ® LV insecticide m ethomyl ; 29% DANGER Restricted Use F atal if swallowed, toxic to fish, aquatic invertebrates and mammals, hig hly toxic to bees, known to leach through soil into groundwater Lannate ® SP insecticide m ethomyl ; 90% DANGER Restricted Use F atal if swallowed, may cause blindness, toxic to fish, aquatic invertebrates and mammals, highly toxic to bees, known to leach through soil into groundwater Lorsban ® 15G Granular Insecticide c hlorpyrifos ; 15% CAUTION Toxic to fish, aquatic invertebrates, birds and small mammals, highly toxic to bees Lorsban ®

- 4E Insecticide c hlorpyrifos ; 44.9% WARNING Restricted Use Toxic t o fish, aquatic invertebrates, birds and small mammals, highly toxic to beesSSB

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Lorsban ® 75WG I nsecticide c hlorpyrifos ; 75% WARNING Toxic to fish, aquatic invertebrates, birds and small mammals, highly toxic to bees

Mocap ® 1 5 % G ranular N ematicide / I nsectic ide e thoprop ; 1 5 % DANGER Restricted Use Toxic to aquatic organisms and wildlife ; extremely toxic to birds; toxic to bees; fatal if absorbed through skin; may be fatal if swallowed; causes irreversible eye damage; harmful if inhaled Penncap

- M ® Microenc apsulated Insecticide methyl parathion ; 22 .0 % WARNING Restricted Use H ighly toxic to aquatic invertebrates and wildlife

Phorate 20 G Organophosphate Insecticide phorate; 20.0% DANGER Restricted Use Very highly toxic to fish and wildlife. Birds and m ammals may be killed if granules are not properly covered with soil. Fatal if swallowed, inhaled or absorbed through the skin; corrosive; causes irreversible eye damage Poncho® 600 (seed treatment) clothianidin; 48 % CAUTION Toxic to aquatic invertebrate s

Pounce ® 3.2 EC Insecticide p ermethrin ; 38.4% CAUTION. Restricted Use E xtremely toxic to fish and aquatic invertebrates, highly toxic to bees

Pounce ® 25 WP Insecticide p ermethrin ; 25% WARNING. Restricted Use E xtremely toxic to fish and aquatic inv ertebrates, highly toxic to bees

Sevin ® 80 S olupak c arbaryl ; 80% WARNING Extremely toxic to aquatic invertebrates, highly toxic to bees Sevin ® brand XLR PLUS Carbaryl Insecticide c arbaryl ; 44.1% CAUTION Extremely toxic to aquatic invertebrates, hig hly toxic to bees

Tundra® Max Insecticide chlorpyrifos; 28.6% bifenthrin; 9.0% WARNING Restricted Use Extremely toxic to fish, aquatic invertebrates, small mammals, and birds. Highly toxic to bees. Warrior II with Zeon Technology ® Insecticide lambda

- cy halothrin ; 22.8 % WARNING. Restricted Use E xtremely toxic to fish and aquatic organisms and toxic to wildlife, highly toxic to bees

Zeta

- Cype 0.8EW Insecticide zeta

- cypermethrin; 9.2% WARNING Restricted Use Extremely toxic to fish, aquatic invertebrates, oysters and shrimp; highly toxic to beesSSB

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### E. Risk Factors

1. Reduced

- risk alternative to conventional insecticides

Representative conventional insecticide products registered for control of corn rootworm in field corn are listed in Table 5. Several are classi fied as Restricted Use Pesticides due to human and/or environmental risk concerns. The Restricted Use classification, imposed due to adverse environmental effects under normal use practices or applicator safety concerns [40 CFR 152.171(a)], limits the use of these chemicals to certified pesticide applicators who have received special training needed for safe handling and application of these products. Personal protective equipment to reduce occupational exposure, special labeling, and special record keepi ng are required for the sale and use of Restricted Use Pesticides. Without these restrictions, EPA has determined that use of these products may cause unreasonable adverse effects on humans and/or the environment. This classification is an indication of EPA concern about the safety of these products.

Event 5307 corn has the potential to displace the use of many of the Restricted Use Pesticides that are currently being used for control of corn rootworms. Based on this consideration alone, the plant

- incorporated eCry3.1Ab pesticidal protein encoded in 5307 corn is entitled to a presumption of public interest.

2. Safety profile of 5307 corn and 5307 corn breeding stacks

A standard batter y of mammalian toxicity studies were conducted for the eCry3.1Ab protein produced in5307 corn . These studies found no evidence of eCry3.1Ab protein

induced adverse effects. The protein is rapidly degraded in mammalian digestive systems and bears no amino acid sequence similarities to known toxins or allergens. Because the in secticidal protein is plant

- incorporated, the opportunity for exposure when handling and planting seed is insignificant . Planting of 5307 corn breeding stacks ( Bt11 × MIR604 × TC150 7 × 5307 and Bt11 × MIR162 × MIR604 × TC150 7 × 5307 corn) by growers will essentially eliminate the environmental and occupational health risks currently associated with che mical controls for corm rootworm pests (via the rootworm

- control PIP s in 5307 and MIR604 corn) as well as for the lepidopteran pests that will otherwise be c ontrolled by the PIP traits in one or both of the 5307 breeding stacks (i.e., the PIPs in Bt11, TC1507, and MIR162 corn)

.
The selectivity of eCry3.1Ab for certain coleopteran pests minimizes risk for nontarget organisms. A series of hazard identification studies ha s been conducted with nontarget indicator species, including many species that are part of the corn ecosystem. No adverse effects attributable to eCry3.1Ab proteins were observed in these studies, even at

e xposure levels exceeding expected envi ronmenta l concentrations ( Nelson, 2010 ). Additionally, no adverse effects are predicted from use of the 5307 corn breeding stacks (Raybould, 2011a, 2011b).

Primary roots of the seedling and the radical and seminal roots are commonly infected with Fusariu m spp. after they have served their function and become senescent. FeedingSSB

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### by corn r ootworms has been associated with increased frequencies of Fusarium infection (Dicke and Guthrie, 1988) . R ootworm feeding may also lead to increased incidences of stalk r ots. These pathogen infections can lead to reduc tions in crop quality, harvestability, and yield. Due to the superior protection from damage that will be afforded by planting 5307 corn there is a potential health benefit for the livestock industry result ing from reduced Fusarium levels in livestock feed.

F. Economic Factors

The adoption of new varieties improved through biotechnology has added greatly to farm productivity and profits since their introduction (Brookes and Barfoot, 2010). Varieties containing herbicide tolerance and insect protection traits have been widely adopted by corn, soybean, and cotton growers because they protect the inherent yield potential of these crops and typically reduce grower input costs. Insect

- protected varieties, including stacked

- trait varieties that offer insect protection and herbicide tolerance, accounted for 63% of corn planted in 2009 (USDA, 2009a). The adoption of transgenic corn varieties is estimated to have reduced the application of conventional pesticides by mo re than 20 million pounds annually (NCGA, 2010b; Doane, 2010).

Event 5307 corn demonstrates excellent efficacy against western, northern and Mexican

corn rootworms . Moreover, 5307 corn hybrids demonstrated an average grain yield advantage of 63 bushels/a cre over the corresponding control hybrids in the presence of significant larval rootworm pressure

. Growers will realize significant economic gain due to the insect

- control efficacy and associated yield advantage of the 5307 corn breeding stacks under roo tworm pressure.

In breeding stacks with MIR604 corn, a justified reduction in the size of the previously required 20% structured corn rootworm refuge to 5% of a grower’s corn acres will have further economic benefits . T he cost of chemical insecticide con trol on the previous refuge acres will be reduced, while grain yields for these acres will potentially increase.

Because the eCry3.1Ab protein in 5307 corn does not compete for the same larval gut binding site as mCry3A in MIR604 corn (Walters et al.

, 2010), it offers key advantages for insect resistance management when stacked with MIR604 corn. Moreover, it can be expected to extend the durability of other commercially available corn rootworm PIPs as well as other methods of corn rootworm control, the reby helping to maintain lower overall costs for pest control.

The availability of multiple rootworm

- protected corn products will increase grower choice and price competition, potentially resulting in lower seed prices for growers and higher adoption rate s. Additionally, the availability of a new and reliable pest

- control option that promote s higher yield, more efficient use of water and fertilizer, healthier plants, and better grain quality , while reducing the environmental impacts of agriculture, will o perate to reduce the costs of producing corn and contribute to global food security. These substantial economic benefits indicate that registration of 5307 corn is in the public interest.SSB

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## V. References

Baker, A., Allen, E

. and H. Lutman (2010). Feed out look. A Report from the Economic Research Service, United States Department of Agriculture. FDS

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Branson, T.F. and G.R. Sutter ( 1989 ) . Evaluating and Breeding for Maize Resistance to the Rootworm Complex. In : Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects. Mexico, D., F.: CIMMYT.

Brookes, G. and P. Barfoot (20 10 ). GM crops: global socio

- economic and environmental impacts 1996

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. PG Economics Ltd : Dorchester, UK . A cc es sed March 29, 2011 : http://www.pgeconomics.co.uk/pdf/2010

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- impact

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April

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Carpenter, J. (2011). Impacts of GM crops on biodiversity. GM Crops 2:1, 1

- 17; January/February/March 2011. published online in advance of print edition. https://www.landesbioscience.com/journals/gmcrops/article/15086/
Dicke, F. F. and W. D. Guthrie (1988). The most important corn insects. Pp 767

- 867. In Corn and Corn Improvement. Third Edition. Sprague, G. F. and J. W. Dudley (eds.). American Society of Agronomy, Crop Science Society of America and Soil Science Society of America, Madison, Wisconsin.

Doane (2010). 2009 AgroTrak. The US agrochemical usage study. A Doane Marketing Research syndicated tracking study. St. Louis, Missouri.

E PA (1986). U.S. Environmental Protection Agency. Conditional registrati on of new pesticides. Federal Register 51 :7628.

EPA (2010 ). U.S. Environmental Protection Agency. Biopesticides Registration Action
Document

- Modified Cry3A Protein and the Genetic Material Necessary for its Production (via Elements of pZM26) in Event MIR604 Corn SYN

- IR604

- 8. Office of Pesticide Programs ; Biopesticides and Pollution Prevention Division. Accessed March 30, 2011: http://www.regulations.gov/#!documen tDetail;D=EPA

- HQ

- OPP

2010

- 0607

- 0027

. Flavell, R. (2010). Knowledge and technologies for sustainable intensification of food production. New Biotechnology 27 : 505

- 516. T ransgenic P lants for F ood S ecurity in the Context of D evelopment

– P roceedings of a Study Week of the Pontifical
A cademy of Sciences , 30 November 2010

. http://www.sciencedirect.com/science/article/B8JG4

- 507CS31

- 1/2/2f9dde686fc1d cc2390c7e2106d21004
Gray, M. and W. Luckmann (1994). Integrating the cropping system for corn insect pest

manage ment. p p 507

- 541 In Introduction t o Insect Pest Management. 3rd Ed

. Metcalf, R. L. and W. H. Luckmann (eds.). John Wiley & Sons, Inc., New York,SSB

- 201

- 10 Page 39 of 41 

### New York.
Haney, L., J. White and M. Meghji (200 8 )

. Efficacy of 5307 Maize Hybrids Against Western Corn Rootworm in the Field

. Unpublished Syngenta R eport No. SSB

- 505

- 08. US EPA MRID 47734718.
Haney L.

, J. White and M. Meghji (2009). Efficacy of a 5307 Maize Hybrid Against Western Corn Rootworm in the Field

. Unpublished Syngenta R eport No. SSB

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- 09. US EPA MRID 47734719.
Harper, B. ( 2011 ). eCry3.1Ab (Entrez® Database Accession Number ADC30135): Assessment of Amino Acid Sequence Similarity to Known or Putative Allergens

. Unpublished Syngenta report, No. SSB

- 11 2

- 1 1

. (Included as a data volume in submission for EPA registration.)
Hein, G.L., M.K. Bergman, R.G. Bruss, and J.J. Tollefson (1985). Absolute sampling technique for corn rootworm (Coleoptera: Chrysomelidae) adult emergence that adjusts to common

- row spacing. J

. Econ

. Entomol

. 78: 1503

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Hibbard, B. (2009) Selection intensity of SYN101 and its stack with MIR604

. Columbia, MO. Unpublished report to Syngenta Biotechnology, Inc.
Jones, W. (2009) Characterization and Safety of eCry3.1Ab Protein and Corn Plants Derived from Event 5307. Unpublished Syngenta Document No. 5307

- EPA

- EUP. US EPA MRID 47734701.
Korgaonkar, C. K. (2009) ECRY3.1AB

- 0208: Single

- Dose Oral (Gavage) Toxic ity Study in Mice with a 14

- Day Observation Period. Unpublished Syngenta Study No.
WIL

- 639031. US EPA MRID 47753901.
Krysan, J.L. (1986). Introduction: biology, distribution, and identification of pest Diabrotica , pp. 1

- 24, In J.L. Krysan and T.A. Mil ler (eds.), Methods for the Study of
Pest Diabrotica . Springer

- Verlag, New York.
Marra, M., P. Pardey, and J. Alston (2002). The payoffs to transgenic field crops: An assessment of the evidence. AgBioForum 5:43

- 50. http://www.agbioforum.org/v5n2/v5n2a02

- marra.htm
Marroquin, L.D., D. Elyassnia, J.S. Griffitts, J.S. Feitelson, R.V. Aroian (2000) Bacillus th u ringiensis susceptibility and isolation of resistance mutants in the nematode
Caenor habditis elegans

. Genetics 155: 1693

- 1699.
McClain, S. (2011). eCry3.1Ab (Entrez® Database Accession Number ADC30135): Assessment of Amino Acid Sequence Similarity to Known or Putative Toxins. Unpublished Syngenta Report No. SSB

- 165

- 11. (Included as a data volume in submission for EPA registration.)

N ational C orn G rowers A ssociation (2010a). Corn Production Trends. Acc essed February 28, 2010: http://www.ncga.com/corn

- production

- trends

Natio nal Corn Growers Association (2010b). 2010 World of corn. Innovation i n Action. National Corn Grower’s Association. www.worldofcorn.com. Accessed May 10, 2010: http://ncga.com/files/pdf/WOC2010.pdfSSB

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### N e lson, A. (2008) Characterization of Test Substance ECRY3.1AB

- 0208 and Certificate of Analysis. Unpublished Syngenta Report No. SSB

- 010

- 08. MRID 47734704

. Nelson, A. (2009) Comparison of eCry3.1Ab Protein Produced in Event 5307

- Derived Maize Plants and eCry3.1Ab Protein Produced in Recombinant Escherichia coli

. Unpublished Syngenta Report No. SSB

- 002

- 09. MRID 47734703.
Nelson, A. (2010). The Environmental Fate and Effects of eCry3.1Ab in Event 5307 Maize: Estimated Environmental Concentrations, Marg ins of Exposure in Nontarget Organism Studies, and US Endangered Species Assessment. Unpublished Syngenta Report No. SSB

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Oleson, J.D., Y. Park, T.M. Nowatzki and J.J. Tollefson. (20 05). Node

- injury scale to evaluate root injury by corn rootworms. J. Econ

. Entomol. 98(1): 1

- 8.
Raybould, A. (2011a). Non

- Target Organism Risk Assessment for Bt11 x MIR604 x TC1507 x 5307 x GA21 Stacked Maize (Field Corn) Hybrids. Unpublished Syngenta Report No. NRA

- 115

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Raybould, A. (2011b). Non

- Target Organism Risk Assessment for Bt11 x MIR162 x MIR604 x TC1507 x 5307 x GA21 Stacked Maiz e (Field Corn) Hybrids. Unpublished Syngenta R eport No. NRA

- 114

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Seastrum, L. (2009)

. In v itro Digestibility of eCry3.1Ab Protein as Contained in Test Substance ECRY3.1AB

- 0208 Under Simulated Mammalian Intestinal Conditions. Unpubli shed Syngenta Report No. SSB

- 015

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Seastrum, L., K. Ward, and A. Raybould (2010). Investigation of the Potential Interaction between Lepidopteran

- active (Cry1Ab + Vip3Aa20 + Cry1F) and Cole opteran

- active (mCry3A + eCry3.1Ab) Insecticidal Bt Proteins Using Target
Insects Ostrinia nubilalis and Leptinotarsa decemlineata

. Unpublished Syngenta Report No. TK0026520. (Included as a data volume in submission for EPA registration.)

Seastrum, L. ( 2010). Investigation of the Potential Interaction between Insecticidal Proteins mCry3A + eCry3.1Ab Using Colorado Potato Beetle ( Leptinotarsa decemlineata ). Unpublished Syngenta Report No. TK0026862. (Included as a data volume in submission for EPA regis tration.)
Song, S. (2010). In vitro digestibility of eCry3.1Ab Protein under simulated mammalian gastric conditions. Unpublished Syngenta R eport No. TK0028111

. (Included as a data volume in submission for EPA registration.)
Syngenta Agronomy Research ( 2010). Corn rootworm management guide. Accessed March 20, 2011: http://www.syngenta.com/country/us/en/ Seeds/PDF%20and%20Other%20doc ument%20Storage%20Library/Ag_Reports/CornRootworm_Syngenta_FINAL.pdf

. USDA (2009a). Acreage. June 30. Cr Pr 2

- 5 (6

- 09). U.S. Department of Agriculture

–SSB

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### National Agricultural Statistics Service. Washington, DC. Accessed May 12, 2010 : http://usda.mannlib.cornell.edu/usda/current/Acre/Acre

- 06

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- 2009.pdf

. USDA (2009b). Data and statistics: Quick stats. Washington, DC: U.S. Department ofAgricu lture

- National Agricultural Statistics Service. Accessed May 12, 2010 : http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp
USDA (2010a). World agricultural production. Foreign Agricultural Service, United States Department of Agriculture. February 2010. Accessed March 13, 2010: http://www.fas.usda.gov/wap/circular/2010/ 10

- 02/productionfull02

- 10.pdf
USDA (2010b). Crop production 2009 summary. National Agricultural Statistics Service, United States Department of Agriculture. Report Cr Pr 2

- 1(10), January 2010. Accessed March 13, 2010: http://usda.mannlib.cornell.edu/usda/nass/CropProdSu//2010s/2010/CropProdS

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USDA (2010c). Agricultural Trade Multipliers: Effects of Trade on the U.S. Economy. Updated February 8, 2010. Accessed April 29, 2010 : http://www.ers.usda.gov/Data/TradeMultiplier/econeffects/2008overview.aspx

Walters, F.S., C.M. deFontes, H. Hart, G.W. W arren and J.S. Chen (2010)

. Lepidopteran

a ctive variable region s equence i mparts c oleopteran a ctivity in eCry3.1Ab, a n

e ngineered Bacillus t huringiensis h ybrid i nsecticidal p rotein

. Appl

. Env

. Microbio

. 76 : 3082

– 3088

. (Included as a data volume in submissi on for EPA registration.)
Ward, D., T. DeGooyer, T. Vaughn, G. Head, M. McKee, J. Astwood, and J. P er s h ing. (2005). Genetically enhanced maize as a potential management option for corn rootworm: YieldGard ®



### Wei, J.

- Z., K. Hale, L. Carta, E. Platzer, C. Wong, S

- C Fang, and R.V. Aroian (2003)

. Bacillus thuringiensis crystal proteins that target nematodes. Proc. Natl. Acad. Sci.
USA 100: 2760

- 2765. rootworm maize case study. Pp 239

- 262. In Wes tern Corn Rootworm: Ecology and Management. Vidal, S., U. Kuhlmann, and C. Edwards (eds.). CAB International, Wallingford, Oxfordshire.Event 5307 Corn Environmental Report

B-1 Appendix B: Safety and Nutritional Assessment 

##  

# Appendix B Safety and Nutritional Assessment

### Title Page 

## Biotechnology Consultation Document for the
United States F ood and D rug A dministration

Safety and Nutritional Assessment of Event 5307 Corn

OECD Unique Identifier: SYN

- Ø 53 Ø 7

- 1

Submitted By :

Demetra Vlachos Syngenta Seeds, Inc.
P.O. Box 12257
3054 East Cornwallis Road Research Triangle Park, NC 27709

- 2257

Date :

January 27 , 201 1

Author s :

Lawrence Zeph, Ph.D. and Demetra Vlachos, M.S.

Syngenta Seeds, Inc.

© Syngenta 201 1

Page 1 of 168

### 5307

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- 1 Page 2 of 168 

## I. Administrative

I.A. Table of Contents

I. Administrative

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

I.A. Table of Contents

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

I.B. List of Figures

................................ ................................ ................................ ...................... 5
I.C. List of Tables

................................ ................................ ................................ ....................... 7

I.D. Release of Information

................................ ................................ ................................ ........ 9

I.E. Abbreviations and Acronyms

................................ ................................ ............................ 10

I.F. Summary of the Safety and Nutritional Assessment of Event 5307 Corn

......................... 12

II. Synopsis of the Safety and Nutritional Assessment of Event 5307 Corn

............................. 13

II.A. Name and Address of the Submitter

................................ ................................ .................. 13

II.B. Subject of This Consultation and the Plant Species from Which it is Derived

................. 13
II.C. Designation of Transformation Event

................................ ................................ ................ 13

II.D. Description of the Genetic Materials Introduced Into Event 5307 Corn

........................... 13

II.E. The Intended Technical Effect of Event 5307 Corn

................................ .......................... 13

II.F. Application and Uses of Event 5307 Corn

................................ ................................ ........ 14
II.G. Applications for Which Event 5307 Corn is Not Suitable

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

III. Status with Other Regulatory Agencies

................................ ................................ ................. 15

III.A. Submissions to US Agencies

................................ ................................ ............................. 15
III.A.1. Environmental Protection Agency

................................ ................................ ............ 15
III.A.2. Department of Agriculture

................................ ................................ ........................ 15

III.B. Submissions to Foreign Agencies

................................ ................................ ...................... 15

IV. Transformation and Development of Event 5307 Corn

................................ ........................ 16

IV.A. Characterization of the Parent Plant

– Corn

................................ ................................ ...... 16
IV.A.1. Description of Zea mays L. (Corn; Maize)

................................ ............................... 16
IV.A.2. Origin of the Species Zea mays L.

................................ ................................ ............ 16

IV.B. Description of the Genetic Materials Introduced into 5307 Corn

................................ ...... 17
IV.C. Description of the Transformation System

................................ ................................ ........ 22
IV.C.1. Development of Event 5307 Corn

................................ ................................ ............. 22
IV.C.2. Development of Test and Control Seed Materials

................................ .................... 24

IV.D. Quality Control Testing of Seed Materials

................................ ................................ ........ 27

IV.E. Characterization of the Genetic Material in Event 5307 Corn

................................ .......... 27
IV.E.1. DNA Insert Sequencing

................................ ................................ ............................ 27
IV.E.2. Analysis of DNA Insertion Site in Corn Genome

................................ ..................... 27
IV.E.3. Copy Number of Functional Elements

................................ ................................ ...... 28
IV.E.3.a. Copy Number of Functional Elements: ecry3.1Ab

- Specific Probe

..................... 28
IV.E.3.b. Copy Number of Functional Elements: pmi

- Specific Probe

................................ 32
IV.E.3.c. Copy Number of Functional Elements: CMP promoter

- specific probe

.............. 36
IV.E.3.d. Copy Number of Functional Elements: ZmUbiInt PromoterSpecific Probe

....... 40
IV.E.3.e. Copy Number of Functional Elements: NOS Terminator

- Specific Probe

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

### 5307

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- 1 Page 3 of 168 IV.E.4. Generational Stability

................................ ................................ ............................... 49
IV.E.4.a. Stability of the T

- DNA Using a Full

- Length T

- DNA

- specific Probe

.................. 49
IV.E.4.b. Stability of the T

- DNA Using a Plasmid Backbone

- Specific Probe

.................... 58
IV.E.5. Mendelian Inheritance of the DNA Insert

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

IV.E.6. Flanking Sequence and Analysis to Identify Putative Open Reading Frames

.......... 65

IV.E.7. Summary of the Genetic Characterization of Event 5307 Corn

................................ 67

V. Absence of Genes That Encode Resistance to Antibiotics

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

VI. Safety Assessment of the Introduced eCry3.1Ab and PMI Proteins

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

VI.A. Identity and Function of the eCry3.1Ab Protein in Event 5307 Corn

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

VI.B. Production of eCry3.1Ab Test Substance for Safety Studies

................................ ............ 70
VI.C. Characterization of the Plant

- and Escherichia coli

- produced eCry3.1Ab Proteins

.......... 70

VI.D. Assessment of eCry3.1Ab Protein Toxicity

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

VI.D.1. Assessment of eCry3.1Ab Amino Acid Sequence Similarity to Known Toxins

...... 72
VI.D.2. Acute Oral T oxicity Study of eCry3.1Ab

................................ ................................ . 73
VI.D.3. Conclusion of the eCry3.1Ab Toxicity Assessment

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

VI.E. Assessment of eCry3.1Ab Allergenic Potential

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

VI.E.1. Source of the eCry3.1Ab Protein

................................ ................................ .............. 75
VI.E.2. Assessment of eCry3.1Ab Amino Acid Sequence Similarity to Known Allergens

. 75
VI.E.3. In vitro P epsin Digestibility of eCry3.1Ab

................................ ............................... 76
VI.E.4. Susceptibility of eCry3.1Ab to Inactivation by Heat

................................ ................ 82
VI.E.5. Conclusion of the Assessment of eCry3.1Ab Allergenic Potential

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

VI.F. eCry3.1Ab History of Safe Use

................................ ................................ ......................... 83
VI.G. Conclusion of the eCry3.1Ab Protein Safety Assessment

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

VI.H. Identity and Function of the PMI P rotein in Event 5307 Corn

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

VI.I. Regulatory Status and Established Safety of PMI

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

VI.J. Characterization of the Plant

- and Escherichia coli

- produced PMI Proteins

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

VI.K. Updated Bioinformatic Assessment of PMI Amino Acid Sequence Similarity

to Known Toxins

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

VI.L. Updated Bioinformatic Assessment of PMI Amino Acid Sequence Similarity

to Known Allergens

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

VI.M. PMI History of Safe Use

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

VI.N. Conclusion of the PMI Safety Assessment

................................ ................................ ........ 89
VI.O. eCry3.1Ab and PMI Protein Expression Levels in 5307 Corn and Exposure Potent ial

.... 89
VI.O.1. Concentrations of eCry3.1Ab and PMI

................................ ................................ ..... 89
VI.O.2. Mammalian Exposure Potential

................................ ................................ ................ 89

VII. Food and Feed Nutritional Assessment of Event 5307 Corn

................................ ............ 92

VII.A. Justification for Comparators

................................ ................................ ............................. 92

VII.B. Historic Use of Corn in the U.S.

................................ ................................ ........................ 92
VII.C. Compositional Assessment of 5307 Forage and Grain

................................ ...................... 93
VII.C.1. Design and methods used in compositional analysis study

................................ ...... 93
VII.C.1.a. Statistical analysis for across

- location comparisons

................................ ............. 93
VII.C.1.b. Statistical analysis for individual

- location comparisons

................................ ....... 94

### 5307

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- 1 Page 4 of 168 VII.C.1.c. Comparison with ILSI Crop Composition Database

................................ ....... 94
VII.C.2. Results of statistical analysis for forage

................................ ................................ .... 96
VII.C.3. Results of statistical analysis for grain

................................ ................................ ...... 96
VII.C.4. Conclusion of compositional analyses

................................ ................................ ...... 96

VII.D. Other Information Relevant to the Nutritional Assessment of Event 5307 Corn: Broiler Chicken Feeding Study

................................ ................................ ....................... 115

VII.E. Summary of the Food and Feed Nutritional Assessment

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

VIII. References

................................ ................................ ................................ ........................... 117

Appendix A: Materials and Methods Used in the Genetic Characterization of Event 5307
Corn

................................ ................................ ................................ ........................ 124

Appendix B: Materials, Methods and Results of Characterization Studies on the eCry3.1Ab and PMI Proteins

................................ ................................ ............... 133

Appendix C: Quantification of eCry3.1Ab and PMI in 5307 Corn

................................ ......... 151

Appendix D: Compositional Analysis of 5307 Forage and Grain

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

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- 1 Page 5 of 168 I.B. List of Figures

Figure IV

- 1. Plasmid map for vector pSYN12274.

................................ ................................ .. 17
Figure I V

- 2. A. Schematic illustrating the origin of the amino acid residues present in eCry3.1Ab.
B. Nucleotide alignment between ecry3.1Ab and mcry3A and corresponding amino acids present at the N

- termini of eCry3.1Ab and mCry3A.

...................... 21
Figure IV

- 3. Steps in the development of 5307 corn.

................................ ............................... 23
Figure IV

- 4

. Pedigree diagram of Event 5307 and control seed materials.

.............................. 26
Figure IV

- 5. Location of the Kpn I, Nco I, Sma I, and Pme I restriction sites and position of the 1962 bp ecry3.1Ab

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274.

................................ ................................ .. 29
Figure IV

- 6. Southern blot analysis of 5307 corn for copy number of functional elements: 1962

- bp ecry3.1Ab

- specific probe, us ing restriction enzymes Kpn I , Nco I , and Sma I + Pme I

................................ ................................ ................................ ........ 31
Figure IV

- 7. Location of the Bst EII, Spe I, Sma I, and Pme I restriction sites and position of the 1176 bp pmi

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274

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

Fi gure IV

- 8. Southern blot analysis of 5307 corn for copy number of functional elements: 1176

- bp pmi

- specific probe, using restriction enzymes Bst EII, Spe I, and Sma I

+ Pme I.

................................ ................................ ................................ ................. 35
Figure IV

- 9. Location of the Kpn I, Spe I, Sma I, and Pme I restriction sites and position of the 346 bp CMP promoter

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274.

................................ ................................ .. 37
Figure IV

- 10. Southern blot analysis of 5307 corn for copy number of functional elements: 346

- bp CMP promoter

- specific probe, using restriction enzymes Kpn I, Spe I, and Sma I + Pme I

................................ ................................ ................................ .. 39
Figure IV

- 11. Location of the Bst EII, Spe I, Sma I, and Pme I restriction sites and position of the 1993

- bp ZmUbiInt promoter

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274.

................................ ................................ .. 42
Figure IV

- 12. Southern blot analysis of 5307 corn for copy number of functional elements: 1993

- bp ZmUbiInt promoter

- specific probe, using restriction enzymes Bst EII, Spe I, and Sma I + Pme I.

................................ ................................ ........................ 44
Figure IV

- 13. Location of the Kpn I, Nco I, Sma I, and Pme I restriction sites and position of the 253

- bp NOS terminator

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274.

................................ ................................ .. 46
Figure IV

- 14. Southern blot analysis of 5307 corn for copy number of functional elements: 253

- bp NOS terminator

- specific probe, using restriction enzymes Kpn I, Nco I, and Sma I + Pme I.

................................ ................................ ................................ . 48
Figure IV

- 15. Location of the Bst EII, Spe I, Sma I, and Pme I restriction sites and position of the 6423

- bp full

- length T

- DNA

- specific probe in the
T

- DNA region of the transformation plasmid pSYN12274.

................................ 50

### 5307

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- 1 Page 6 of 168 Figure IV

- 16. Genetic stability Southern blot analysis of 5307 corn with the 6423

- bp full

length T

- DNA

- specific probe, using res triction enzymes Bst EII, Spe I, and
Sma I + Pme I.

................................ ................................ ................................ ........ 53
Figure IV

- 17. Location of the Bst EII, Spe I, Sma I, and Pme I restriction sites and position of the 5312

- bp plasmid backbone

- specific probe in the transformation plasmid pSYN12274.

................................ ................................ ................................ ......... 59
Figure IV

- 18. Genetic stabilit y Southern blot analysis of 5307 corn with the 5312

- bp plasmid backbone

- specific probe, using restriction enzymes Bst EII, Spe I, and Sma I + Pme I.

................................ ................................ ................................ ........ 61
Figure VI

- 1. SDS

- PAGE analysis of eCry3.1Ab following digestion in simulated gastric fluid.

................................ ................................ ................................ ..................... 78
Figure VI

- 2. Western blot analysis of eCry3.1Ab following digestion in simulated gastric fluid.

................................ ................................ ................................ ..................... 81

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- 1 Page 7 of 168 I.C. List of Tables

Table IV

- 1. Description of genetic elements in vector pSYN12274.

................................ ........... 18

Table IV

- 2. Event 5307 and control seed materials used in st udies.

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

Table IV

- 3. Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn, using an ecry3.1Ab

- sp ecific probe and restriction enzymes Kpn I,
Nco I, and Sma I + Pme I.

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

Table IV

- 4. Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn, using a pmi

- specific probe and restriction enzymes Bst EII, Spe I, and Sma I + Pme I.

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

Table IV

- 5. Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn, using a CMP promoter

- specific probe and restriction enzymes Kpn I, Spe I, and Sma I + Pme I.

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

Table IV

- 6. Expected and observed hybridiz ation band sizes in Southern blot analysis of 5307 corn, using a ZmUbiInt promoter

- specific probe and restriction enzymes
Bst EII, Spe I, and Sma I + Pme I.

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

Table IV

- 7. Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn, using a NOS terminator

- specific probe and restriction enzym es Kpn I, Nco I, and Sma I + Pme I.

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

Table IV

- 8. Expected and observed hybridization band sizes in Southern blot analysis of 5307 c orn, using a full length T

- DNA

- specific probe and restriction enzymes
Bst EII, Spe I, and Sma I + Pme I.

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

Table IV

- 9. Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn, using a plasmid pSYN12274 backbone

- specific probe and

restr iction enzymes Bst EII, Spe I, and Sma I + Pme I.

................................ ................. 60

Table IV

- 10. Observed versus expected frequencies for ecry3.1Ab across generations.

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

Table IV

- 11. Observed versus expected frequencies for pmi across generations.

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

Table VI

- 1. Comparison of Colorado potato beetle larvae LC 50 

### values for eCry3.1Ab from 5307 plants and microbial eCry3.1Ab test substance.

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

Table VI

- 2. The eCry3.1Ab remaining at each time point during exposure to simulated gastric fluid as determined by densitometr y analysis.

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

Table VI

- 3. Effect of temperature on insecticidal activity of eCry3.1Ab in diet incorporation bioassay with Colorado potato beetle larvae: LC 50 

### values and 95% confidence intervals at 144 hours.

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

Table VI

- 4. Concentrations of eCry3.1Ab in 5307 corn tissues at four stages on a dry

weight (DW) and a fresh

- weight (FW) basis.

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

Table VI

- 5. Concentrations of PMI in 5307 corn tissues at four stages on a dry

- weight (DW) and a fresh

- weight (FW) basis.

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

Table VII

- 1. Forage and grain components m easured in 5307 and conventional corn.

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

Table VII

- 2. Proximate composition of forage from 5307 corn and nontransgenic corn.

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

Table VII

- 3. Calcium and phosphorus composition of forage from 5307 corn and nontransgenic corn.

................................ ................................ ................................ ... 99

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- 1 Page 8 of 168 Table VII

- 4. Proximate and starch composition of grain from 5307 corn and nontransgenic

corn.

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

Table VII

- 5. Mineral composition of grain from 5307 corn and nontransgenic corn.

................. 103

Table VII

- 6. Vitamin composition of grain from 5307 corn and nontransgenic corn.

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

Table VII

- 7. Amino acid composition of grain from 5307 corn and nontransgenic corn.

........... 107

Table VII

- 8. Fatty acid composition of grain from 5307 corn and nontransgenic corn.

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

Table VII

- 9. Secondary metabolite and antinutrient composition of grain from 5307 corn and nontransgen ic corn.

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

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- 1 Page 9 of 168 I.D. Release of Information

Syngenta is submitting the information in this consultation document for review by the United States Food and Drug Administration (FDA) as part of a federal regulatory process. By submitting this information to FDA, Syngenta does not authorize its release to any third
party. In responding to a request made under the Freedom of Information Act (FOIA), 5 U.S.C. §552, covering all or some of the information in this consultation document, Syngenta expects that, in advance of the release of the information, F DA will provide Syngenta with a copy of the FOIA request and the material proposed to be released, and the opportunity to object to the release of information based on appropriate legal grounds (e.g., responsiveness, trade secret, and/or commercial concern s). Except in accordance with the foregoing, Syngenta does not authorize the release, publication, or other distribution of this information (including internet posting) without prior notice and Syngenta consent.

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- 1 Page 10 of 168 I.E. Abbreviations and Acronyms

5307 Event 5307; derived from corn transformation Event 5307
ADF acid detergent fiber AEBSF 4

- (2

- aminoethyl)

- benzenesulfonylfluoride HCl ANOVA analysis of variance AOAC Association of Analytical Communities APHIS Animal and Plant Health Inspection Service BC backcross BCA bicinchoninic acid
BME beta

- mercaptoethanol bp base pairs BSA bovine serum albumin
B.t. Bacillus thuringiensis

cm centimeter(s) CMP cestrum yellow leaf curling virus promoter CFR U.S. Code of Federal Regulations Cry1 crystal protein from Bacillus thuringiensis CTAB hexadecyltrimethylammonium bromide
Da daltons DIG digoxigenin
DNA deoxyribonucleic acid dsDNA double

- stranded deoxyribonucleic acid
DTT dithiothreitol
DW dry weight
EDTA ethylenediamine tetraacetic acid ELISA enzyme

- linked immunosorbent assay
EPA U.S. Environmental Protection Agency
F1 first generation of progeny from a breeding cross
FARRP Food Allergy Research and Resource Program
FDA Food and Drug Administration FFDCA Federal Food, Drug and Cosmetic Act
FIFRA Federal Insecticide, Fungicide and Rodenticide Act FOIA Freedom of Information Act
FW fresh weight G6PDH glucose 6

- phosphate dehydrogenase gdw grams dry weight IgG immunoglobulin G ILSI International Life Sciences Institute kb kilobase(s) kDa kilodaltons
LB left border LC50 median lethal concentration; a statistically derived estimate of the concentration resulting in mortality of 50% of the test organisms LOD limit of detection LOQ limit of quantification
LTR long terminal repeat

### 5307

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- 1 Page 11 of 168 MES 2

- (N

- morpholino)ethanesulfonic acid
MW molecular weight
N number of samples
NADP β

- nicotinamide adenine dinucleotide phosphate
NADPH β

- nicotinamide adenine dinucleotide phosphate reduced NCBI National Center for Biotechnology Information
NDF neutral detergent fiber
No. number NOS nopaline synthase OD optical density OECD Organisation for Economic Co

- operation and Development ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction
PMI phosphomannose isomerase
PVDF polyvinylidene difloride
PVP polyvinylpyrrolidone
RB right border SD standard deviation SDS sodium dodecylsulfate SDS

- PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis SEM standard error of the mean

spec streptomycin adenylyltransferase gene from Escherichia coli SSC saline

- sodium citrate buffer
T

- DNA transferred deoxyribonucleic acid
TMB tetramethylbenzidine US United States U.S.C. United States Code USDA U.S. Department of Agriculture vir virulence regulon in Agrobacterium tumefaciens v/v volume per volume w/v weight per volume w/w weight per weight ZmUbiInt Zea mays ubiquitin promoter with intron

### 5307

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- 1 Page 12 of 168 I.F. Summary of the Safety and Nutritional Assessment of Event 5307 Corn

The proteins encoded by the transgenes ecry3.1Ab and pmi in Syngenta’s Event 5307 corn have been evaluated for their food and feed safety, and additional studies have demonstrated that grain and forage from Event 5307 corn are nutritionally equivalent to conventional corn. The gene ecry3.1Ab encodes a chimeric protein , eCry3.1Ab, that represents a fusion
between two B acillus t huringiensis

- derived proteins : modified Cry3A (mCry3A) and Cry1Ab

. The gene pmi encodes the enzyme phosphomannose isomerase (PMI) , which was used as a selectable marker in the production of Event 5307 corn.

The species

- specific insect icidal mode of action of eCry3.1Ab and its similarity to other Cry proteins for which human safety has previously been established (e.g., mCry3A

, Cry3A, and Cry1Ab ) support the prediction that no adverse health effects will result from exposure to the eCry3.1Ab protein present in 5307 corn

. The safety of PMI has been established previously, and it is e xempt from food and feed tolerances in all crops.

No toxicity to mammals was observed following oral exposure to high doses of eCry3.1Ab

, and the properties of this protein do not indicat e allergenic potential. The large body of data
and information de scribed herein support the conclusion that there is reasonable certainty that no harm will result from mammalian (including human) exposure to eCry3.1Ab and PMI produced in 5307 corn

.
Analyse s of key nutritional components of forage and grain from 5307 cor n showed that no biologically significant changes in composition occurred as an unintended result of the transformation process or expression of the transgenes in 5307 corn . Forage and grain from
5307 corn are similar in composition to forage and grain fr om conventional corn . In addition, a 49

- day feeding study demonstrated that there were no adverse dietary effects on broiler chickens consuming diets prepared with 5307 corn grain when compared with those consuming diets prepared with nontransgenic contro l corn grain, either as a direct effect of the transgenic proteins in the diet or as a result of any unintended compositional changes in the grain that may have altered its nutritional value. These results support the conclusion
that 5307 corn is nutritio nally comparable to and as safe as conventional corn

.

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- 1 Page 13 of 168 

## II. Synopsis of the Safety and Nutritional Assessment of Event 5307 Corn

II.A. Name and Address of the Submitter

Submitting company:

Syngenta Seeds, Inc.
P.O. Box 12257
3054 East Cornwallis Road
Research Triangle Park

, North Carolina
27709

- 2257

Company representative:

Dem etra Vlachos
Senior Regulatory Affairs Manager
Tel. 919

- 226

- 7383 ; Mobile 9 19

- 433

- 7642; Fax 919

- 226

- 7462
d emetra.vlachos @syngenta.com

II.B. Subject of This Consultation and the Plant Species f rom Which it is
Derived

The s ubject of this consultation is coleo pteran

- resistant corn ( maize; Zea mays L.), derived
from transformation Event 5307 (hereafter “Event 5307 corn” or “5307 corn ”). The variety that was transformed to produce Event 5307 corn was NP2222, a n elite Syngenta inbred field corn variety.

II.C. Designation of Transformation Event

The designation of the transformation event is Event 5307 corn, which has been assigned the

OECD U nique I dentifier SYN

- Ø 53 Ø 7

- 1

.

II.D. Description of the Genetic Materi als Introduced Into Event 5307 Corn

See description in Part IV below.

II.E. The Intended Technical Effect of Event 5307 Corn

Event 5307 corn contains the transgenes ecry3.1Ab and pmi (also known as manA ) , which
encode the proteins eCry3.1Ab and phosphomannose i somerase ( PMI ) , respectively

. The eCry3.1Ab protein is a chimeric protein comprised largely of portions of a modified Cry3A (mCry3A) protein and a Cry1Ab protein derived from Bacillus thuringiensis , a ubiquitous

### 5307

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- 1 Page 14 of 168 bacterium

. The eCry3.1Ab 1 

### protein is insecticidally active against certain C oleoptera

, including w estern corn rootworm (Diabrotica virgifera virgifera)

, n orthern corn rootworm ( D

. longicornis barberi) and Mexican corn rootworm ( D

. virgifera zeae ) , which are significant pests of US corn

. PMI was utilized as a selectable marker during the development of Event 5307 corn , and serves no agronomic or other purpose in the plant

. I n breeding combination s with other insect

- resistant corn varietie s , 5307 corn will allow growers to have optimal broad

- spectrum control of several significant corn pests

. No intended compositional changes are expected as a result of the genetic modification in Event
5307 corn

. Event 5307 corn is as safe and nutritious as food and feed derived from
conventional corn varieties.

II.F. Application and Uses of Event 5307 Corn

Event 5307 corn will be grown for the same uses as current commercially av ailable corn in
the US. In 20 10 , there were an estimated nearly 8 8 million acres planted to c orn in the US

, producing over 1 3 billion b ushels of grain (USDA

- NASS 20 10 ). Corn grown in the US is predominantly of the yellow dent type, a commodity crop largely used to feed domestic animals, either as grain or silage. The remainder of the crop is exp orted or processed by wet or dry milling to yield products such as high fructose corn syrup and starch or oil, grits and
flour. These processed products are used extensively in the food industry. For example,

corn starch serves as a raw material for an a rray of processed foods, and in industrial manufacturing processes. Since the early 1980s, a significant amount of corn grain has also been used for fuel ethanol production. The by

- products from these distilling processes are
often used in animal feeds.

II.G. Applications for Which Event 5307 Corn is Not Suitable

Event 5307 corn is suitable for all uses applicable for conventional corn

.

1 The descriptor “eCry3.1Ab” was assigned by Syngenta; the “e” denotes that it was engineered, and the

“ Cry3” and “1Ab” descriptors relate to the respective source Cry (crystal) proteins, mCry3A and Cry1Ab. The eCry3.1Ab protein has not been assigned an off icial Cry protein designation under the formal nomenclature scheme for B. thuringiensis Cry pro teins (Crickmore et al. 2010 ).

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- 1 Page 15 of 168 

## III. Status with Other Regulatory Agencies

III.A. Submissions to US Agencies

III.A.1. Environmental Protection Agency

Substances that are pesticides, as defined under the Federal Insecticide, Fungicide, and
Rodenticide Act ( FIFRA ) , are subject to regulation by the US Environmental Protection Agency (EPA). The eCry3.1Ab protein encoded by the genetic insert in Event 5307 corn has insecticidal activity against several significant cole opteran pests and is, therefore, regulated by the EPA as a plant

- incorporated protectant

. Pursuant to § 408(d) of the Federal Food, Drug and Cosmetic Act, a temporary exemption from the requir ement of food and
feed tolerance s was established, effective June 10, 2010, under 40 CFR §174. 532 for eCry3.1Ab in corn. This exemption is currently set to expire June 1, 2012, however, a petition to renew the exemption is currently pending at the EPA. A petition for a permanent tolerance exemption for eCry3.1Ab will be submitted by Syngenta to the EPA in early 2011,

concur rently with a FIFRA §3 registration application for use of eCry3.1Ab in Event 5307

corn.

A permanent exemption from the requirement of a tolerance exists under 40 CFR §1 74

. 527 for PMI in all plants , in connection with its use as a selectable marker in pest icidal transgenic plants

.

III.A.2. Department of Agriculture

A petition (number 10

- 336

- 01p ) 

### for the determination of nonregulated status under 7 CFR
Part 340.6 for Event 5307 corn was submitted to the US Department of Agriculture (USDA)

on December 1, 2010

.

III.B. Submissions to Foreign Agencies

Syngenta also intends to commercialize Event 5307 corn for cultivation in Canada and will seek the necessary food, feed and environmental approvals to do so. Syngenta will also pursue regulatory approvals for importation of Event 5307 corn in key export markets for

US and Canadian corn ( including Japan, Taiwan, South Korea, the Philippines, and Mexico) where functioning systems exist for regulating imports of genetically modified crops

.
Syngenta will additionally examine commercial opportunities in other countries where corn

rootworm species are significant economic pests of corn

. In countries where commercial opportunities exist, Syngenta may apply for cultivation approvals in the future

.

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## IV. Transformation and Dev elopment of Event 5307 Corn

IV.A. Characterization of the Parent Plant

– Corn

IV.A.1. Description of Zea mays L. ( Corn; Maize)

Zea is a genus of the family Graminae (Poaceae), commonly known as the grass family.

Corn ( Zea mays L. ; maize ) is a tall, monecious annual gra ss with overlapping sheaths and broad conspicuously distichous blades. Plants have staminate spikelets in long spike

- like racemes that form large spreading terminal panicles (tassels) and pistillate inflorescences in the leaf axils, in which the spikelets occur in 8 to 16 rows, approximately 30 cm long, on a thickened, almost woody axis (cob). The whole structure (ear) is enclosed in numerous large foliaceous bracts and long styles (silks) protrude from the tip of the ear as a mass of silky threads (Hitch cock and Chase 1971). Pollen is produced entirely in the staminate
inflorescence

, and eggs entirely in the pistillate inflorescence. Corn is wind

- pollinated and
both self

- and cross

- pollination are usually possible. Shed pollen usually remains viable for 10 to 30 minutes, but can remain viable for longer durations under favorable conditions (Coe
et al . 1988). Cultivated corn is presumed to have been derived from teosinte ( Z. mexicana ) and is thought to have been intr oduced into the old world in the sixteenth century. Corn is cultivated worldwide and represents a staple food for a significant proportion of the world’s population. No significant native toxins are reported to be associated with the genus Zea (Internati onal Food Biotechnology Council 1990).

IV.A.2. Origin of the Species Zea mays L.

It is generally agreed that teosinte ( Z. mexicana ) is an ancestor of corn , although opinions vary as to whether corn is a domesticated version of teosinte (OECD 2003). Teosinte is a n ancient wild grass found in Mexico and Guatemala. Because it has differentiated into various races, species and plant habits, taxonomic classification is still a matter of
controversy. Doebley and Iltis (1980) and Iltis and Doebley (1980) classified th e annual teosintes into two subspecies of Z. mays

, ssp. mexicana (including races Chalco, Central Plateau and Nobogame) and ssp. pa rviglumis var. parviglumis (race Balsas) and var. huehuetenangensis (race Huehuetenango)

, and the species Z. luxurians (race Guatemala). The perennial teosintes from Jalisco, Mexico are separated into two more species according to ploidy, Z. perennis and Z. diploperennis

. The Meso

- American region located within middle South Mexico and Central America is recognized as one of th e main centers of origin and development of agriculture as well as center of origin and diversification of more than one hundred crops ( OECD 2003 ). At the present time, there is no agreement about where exactly corn was domesticated and there are several proposals in this regard. Based on the findings of archaeological materials from the corn plant (pollen, cobs, husks, and other remnants) in the US and Mexico that are older than those found in South America, Randol ph proposed that corn was domesticated, independently, in the southwestern US, Mexico, and

### 5307

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- 1 Page 17 of 168 Central America (OECD 2003)

. Mangelsdorf proposed that “corn had not one origin but
several in both Mexico and South America

, ” because the archaeological evidence i s found in Mexico and several morphological characteristics of extant populations are found in the corn races of South America (Andes region) in comparison to those races of Meso

- America (OECD 2003)

.

IV.B. Description of the Genetic Materials Introduced into 530 7 Corn

Event 5307 corn was produced by Agrobacterium tumefaciens

- mediated transformation of
immature corn embryos using the transformation plasmid vector pSYN12274. The DNA region between the left and right borders of the transformation plasmid included g ene expression cassettes for ecry3.1Ab and pmi (also known as manA ) ; this T

- DNA was
transferred into the corn genome during transformation. The ecry3.1Ab expression cassette
consisted of the ecry3.1Ab coding region regulated by a cestrum yellow leaf curli ng virus (CMP) promoter and a nopaline synthase (NOS) polyadenylation (terminator) sequence.
The pmi expression cassette consisted of the pmi coding region regulated by a Zea mays polyubiquitin (ZmUbiInt) promoter and the NOS terminator sequence. A schem atic of the plasmid vector is shown i n Figure IV

- 1 . The size and description of each genetic element in
the vector are shown in Table IV

- 1 below.

Figure IV

- 1

. Plasmid map for vector pSYN12274.



## pSYN12274

11769 bp

pmi (1176 bp) spec (789 bp) virG (726 bp) repA (1074 bp) ecry3.1Ab (1962 bp) CMP promoter (346 bp)

VS1 ori (405 bp) ColE1 ori (807 bp)

NOS terminator (253 bp) NOS terminator (253 bp)

LB (25 bp) RB (25 bp)

ZmUbiInt promoter (1993 bp)

### 5307

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- 1 Page 18 of 168 Table IV

- 1

. Description of genetic elements in vector pSYN12274.

Genetic
element Size (bp) Position Description

Active ingredient cassette

Intervening

sequence 203 26 to 228 Intervening sequence with restriction sites used for cloning

CMP promoter 346 229 to 574 Cestrum Yellow Leaf Curling Vir us promoter region (Hohn et al. 2007;
S tavolone et al. 2003). Provides constitutive expression in corn

.

Intervening

sequence 9 575 to 583 Intervening sequence with restriction sites used for cloning

ecry3.1Ab 1962 584 to 2545 An engineered Cry gene active against certain corn rootworm ( Diabrotica ) species (Entrez® Accession No. GU327680 [NCBI 2010a ]). The gene ecry3 .1Ab (Walters et al. 2010) consists of a fusion between the 5′ end (Domain I, Domain II and 15 amino acids of Domain III) of a modified Cry3A gene ( mcry3A ) and the 3′ end (Domain III and Variable Region 6 [Höfte and Whiteley 1989]) of a synthetic Cry1Ab ge ne ( see descriptions of mcry3A and cry1Ab and Figure IV

- 2 below). Upstream of the mcry3A domain, the gene ecry3.1Ab carries a 67

- bp

- long oligomer extension at its 5′ end, which was introduced during the engineering of the variable regions and is tr anslated into the following 22 amino acid residues: MTSNGRQCAGIRPYDGRQQHRG. The next 459 amino acid residues are identical to a portion of mCry3A, followed by 172 residues that are identical to a portion of Cry1Ab. Figure IV

- 2 illustrates the origins of the corresponding amino acid sequences in eCry3.1Ab.

Description of mcry3A : a corn

- optimized cry3A was synthesized to accommodate the preferred codon usage for cor n (Murray et al. 1989). The synthetic sequence was based on the native Cry3A protein sequence from Bacillus thuringiensis subsp . tenebrionis (Sekar et al. 1987). The corn

optimized gene was then modified to incorporate a consensus cathepsin

- G protease re cognition site within the expressed protein. The amino acid sequence of the encoded mCry3A corresponds to that of the native Cry3A, except that (1) its N

- terminus corresponds to methionine 48 of the native protein and (2) a cathepsin G protease recognitio n site has been introduced, beginning at amino acid residue 155 of the native protein. This cathepsin

- G recognition site has the sequence alanine

- alanine

- proline

- phenylalanine, and has replaced the amino acids valine

- 155, serine

- 156, and serine

- 157 in the native protein (Chen and Stacy 2003).

Description of cry1Ab : The gene cry1Ab was originally cloned from Bacillus thuringiensis subsp. kurstaki strain HD

- 1 (Geiser et al. 1986). Its sequence
was codon

- optimized (Koziel et al. 1997) to accommodate the preferred codon usage for corn (Murray et al. 1989).

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- 1 Page 19 of 168 Table IV

- 1 ( Continued ). Description of genetic elements in vector pSYN12274.

Genetic
element Size (bp) Position Description

Intervening

sequence 30 2546 to 2575 Intervening sequence with restriction sites used for cloning

NOS

253 2576 to 2828 Terminator sequence from the nopaline synthase gene of Agrobacterium tumefaciens (Entrez Accession Number V00087 [NCBI 2010a]). This sequence provides a polyadenylation site (Depicker et al. 1982).

Selectable marker cassette

Intervening

sequence 25 2829 to 2853 Intervening sequence with restriction sites used for cloning

ZmUbiInt promoter 1993 2854 to 4846 Promoter region from the maize polyubiquitin gene which contains the first intron (Entrez® Accession Number S94464 [NCBI, 2010a ]). Provides constitutive expression in monocots (Christensen et al. 1992)

Intervening

sequence 12 4847 to 4858 Intervening sequence with restriction sites used for cloning

pmi 1176 4859 to 6034 Escherichia coli strain K

- 12 gene pmi encoding the enzyme phosphomannose isomerase (PMI) (Entrez Accession Number M15380 [NCBI, 2010a ]); this gene is also known as manA

. Catalyzes the
isomerization of mannose

- 6

- phosphate to fructose

- 6

- phosphate (Negrotto et al., 2000).

Intervening

sequence 60 6035 to 6094 Intervening sequence with restriction sites used for cloning

NOS 253 6095 to 6347 Terminator sequence from the nopaline synthase gene of Agrobacterium tumefaciens (Entrez Accession Number V00087 [NCBI,
2010a ]). This sequence provides a polyad enylation site (Depicker et
al. 1982).

Intervening

sequence 88 6348 to 6435 Intervening sequence with restriction sites used for cloning

Plasmid backbone

Left border (LB) 25 6436 to 6460 Left border region of T

- DNA from Agrobacterium tumefaciens nopaline
Ti

- plasmid (Entrez® Accession Number J01825 [NCBI, 2010a]). Short direct repeat sequence that flanks the T

- DNA and is required for the
transfer of the T

- DNA into the plant cell (Zambryski et al. 1982)

Intervening

sequence 349 6461 to 6809 Intervening sequence with restriction sites used for cloning sequence

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- 1 ( Continued ). Description of genetic elements in vector pSYN12274.

Genetic
element Size (bp) Position Description

spec 789 6810 to 7598 Streptomycin adenylyltransferase gene, aadA , from Escherichia coli transposon Tn7 (similar to Entrez® Accession Number X03043 [NCBI 2010a]). Confers resistance to streptomycin and spectinomycin and is used as a bacterial selectable marker (Fling et al. 1985) Intervening

sequence 299 7599 to 7897 Intervening sequence with restriction sites used for cloning

virG 726 7898 to 8623 The VirGN54D gene ( virG ) from pAD1289 (similar to Entrez® Accession Number AF242881 [NCBI, 2010a). The N54D substitution results in a
constitutive virG phenotype. VirG i s part of the two

- component regulatory system for the virulence ( vir ) regulon in Agrobacterium tumefaciens (Hansen et al. 1994). Intervening

sequence 29 8624 to 8652 Intervening sequence with restriction sites used for cloning

repA 1074 8653 to 9726 Gene encoding the pVS1 replication protein from Pseudomonas aeruginosa (similar to Entrez® Accession Number AF133831 [NCBI 2010a]), which is a part of the minimal pVS1 replicon that is functional in Gram

- negative, plant

associated bacteria (Heeb et al. 200 0) Intervening

sequence 42 9727 to 9768 Intervening sequence with restriction sites used for cloning

VS1 ori 405 9769 to 10173 Consensus sequence for the origin of replication and partitioning region from plasmid pVS1 of Pseudomonas aeruginosa (Entrez® Accession Number U10487 [NCBI 2010a]). Serves as origin of replication in Agrobacterium
tumefaciens host (Itoh et al. 1984) Intervening

sequence 677 10174 to 10850 Intervening sequence with restriction sites used for cloning

ColE1 ori 807 10851 to 11657 Origin of replication (similar to Entrez® Accession Number V00268 [NCBI 2010a]) that permits replication of plasmids in Escherichia coli (Itoh and Tomizawa 1979) Intervening

sequence 112 11658 to 11769 Intervening sequence with restriction sites used for cloning

Right border (RB) 25 1 to 25 Right border region of T

- DNA from Agrobacterium tumefaciens nopaline Ti

plasmid (Entrez® Accession Number J01826 [NCBI 2010a]). Short direct repeat that flanks the T

- DNA and is required for the transfer of the T

- DNA into the plant cell (Wang et al . 1984)

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- 1 Page 21 of 168 Figure IV

- 2

. A . Schematic illustrating the origin of the amino acid residues present in eCry3.1Ab. B. Nucleotide alignment between ecry3.1Ab and mcry3A and corresponding amino acids present at the N

- termini of eCry3.1Ab and mCry3A.

A

.

B

.

**ecry3.1Ab ATG**  **ACT AGT**  **AAC GGC**  **CGC CAG**  **TGT GCT**  **GGT ATT CGC**  **CCT T**  AT GAC GGC CG A **eCry3.1Ab M**  **T S**  **N G R**  **Q C A**  **G I R**  **P** Y D G R

**mcry3A**

............................................ ......... ATG ACG GCC G AC **mCry3A** M T A D

**ecry3.1Ab** CAA CAA CAC CGA GGC C

**-** TG GAC AGC AGC ACC ACC AAG GAC GTG **eCry3.1Ab** Q Q H R G **L D S S T T K D**  **V**

**mcry3A** AAC AAC ACC GAG GCC CTG GAC AGC AGC ACC ACC AAG GAC GTG **mCry3A** N N T E A **L D S S T T K D V** 

(A) The black rectangles represent the variable regions 1 through 6 (V1 through V6) of Cry1Ab; the rectangles with diagonal lines represent the conserved blocks 1 through 5 (CB1 through CB5) of Cry1Ab; the gray rectangles represent the Cry1Ab tail sequenc e. The white rectangles represent the variable regions 1 through 5 of mCry3A; the rectangles with crosshatch lines represent the conserved blocks 1 through 5 of mCry3A. The vertically striped portion of eCry3.1Ab represents the N

- terminal amino acids uniq ue to eCry3. 1Ab (see Figure IV

- 2 B ).

(B) The dotted line symbolizes sequence absent from mCry3A; the dash indicates a one

- base

- pair deletion in ecry3.1Ab

. The first 40 nucleotides of ecry3.1Ab

, and the 13 amino acids they encode

, are in bold text to highlight their addition during the engineering of the gene. The 9 amino acids LDSSTTKDV shown for both proteins in bold text indicate where the

ami no acid sequence of eCry3.1Ab is restored to that of mCry3A. 

## V1 V2 V3 V4 V5 V6

CB1 CB2 CB3 CB4 CB5 Domain I Domain II Domain III

V1 V2 V3 V4 V5

CB1 CB2 CB3 CB4 CB5

V1 V2 V3 V4 V5

CB1 CB2 CB3 CB4 CB5 

# Cry1Ab

mCry3A

eCry3.1Ab V6

### 5307

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- 1 Page 22 of 168 IV.C. Description of the Transformation System

IV.C.1. Development of Event 5307 Corn

Transformation of Z. mays to produce Event 5307 corn was accomplished using immature embryos of a proprietary corn line v ia Agrobacterium tumefaciens

- mediated transformation (Negrotto et al . 2000; Grimsley and Bisaro 1987; Ishida et al. 1996). Using this method,
DNA within the left border (LB) and right border (RB) elements of a transformation plasmid, referred to as the transferred DNA (T

- DNA), is integrated into the genome of infected cells, while genetic elements outside of the plasmid borders are generally not. Plants posi tive for both the pmi and the ecry3.1Ab genes and negative for the spec gene were transferred to the greenhouse for further propagation.

Event 5307 transformation employed a binary vector system (de Framond et al

. 1983). Plasmid pSYN12274, which contained the pmi gene from E. coli and the coding sequence
for ecry3.1Ab between the right and left borders of the T

- DNA, was placed into A. tumefaciens strain LBA4404 (licensed from Japan Tobacco, Inc.); see Figure IV

- 1

.
Progeny of the original transformant (T 0 

### plant) were field tested for resistance to insect feeding damage and for agronomic performance after introgression of the transgenes into multiple elite lines of corn . A schematic showing the steps in development of Event 5307

corn is shown in Figure IV

- 3

.

### 5307

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- 1 Page 23 of 168 Figure IV

- 3

. Steps in the development of 5307 corn
.



### Synthetic gene ecry3.1Ab engineeered from parts of mcry3A and cry1Ab

Assembly of ecry3.1Ab and pmi genes and regulatory elements in

vector pSYN12274 in E. coli

Transformation of elite inbred NP2222 embryos with A

. tumefaciens co ntaining plasmid vector pSYN12274

Selection of transformation events

on medium containing mannose

Regeneratio n of transformed
cells into corn plants

Evaluation of transformants for insecticidal efficacy

Intro gression of transformation events into elite inbred lines for evaluation of agronomic performance

Selection of Event 5307 corn for development

Re gulatory studies to assess safety and nutritional composition Introgression of Event 5307
transgenes into commercial breeding
lines and field evaluations

### 5307

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- 1 Page 24 of 168 IV.C.2. Development of Test and Control Seed Materials

Table IV

- 2 lists the genotypes and descriptions of the various 5307 corn and nontransgenic control corn seed genotypes used in the studies described within this submission . The breeding pedigree diagra m in Figure IV

- 4 indicates how each of the 5307 genotypes was derived via conventional breeding crosses from the original Event 5307 T 0 

### transformant. Seed genotypes used in studies described herein are identified in the pedigree diagram by a single letter code (A through F ); these correspond to seed lot identifiers in Table IV

- 2

. In Figure IV

- 4 , genotypes enclosed in ovals represent transgenic 5307 corn genotypes that
were used in studies described herein.

Several Synge nta studies described in this s ubmission were conducted using 5307 hybrid corn of genotype NP2171 × NP2460(5307), which was hemizygous for the transgenes; a hemizygous genotype is representative of future hybrids containing the 5307 transgenes that would be grown commercially. This 5307 hybrid is indicated by the letter code “ D ” in the breeding diagram ( Figure IV

- 4 ), and is alternatively referred to within this submission as “NP2171 × BC5F 3. 

### ” As indicated in Figure IV

- 4 , it was produced by crossing a nontransgenic inbred parent, NP2171, with the F 3 

### (third generation) transgenic progeny of
self

- pollinated plants following five successive generations of backcross (BC) breeding of the transgenes into to a recurrent NP 2460 inbred. This backcross process fully introgressed the transgenes from the initial transformed germplasm (inbred line NP2222) into NP2460 germplasm. The corresponding nontransgenic control seed material (indicated by the letter
code “ E ” in Figure IV

- 4 ) was produced by crossing nontransgenic inbreds NP2171 and NP2640. Except for the presence of the transgenes, control hybrid NP2171 × NP2460 is nearly genetically i dentical to the corresponding transgenic hybrid; thus, this control is
considered near

- isogenic to the NP2171 × NP2460(5307) hybrid.

Two genetic characterization studies described in this submission (genetic stability studies
and Mendelian inheritance s tudies) required seed from multiple generations in the 5307 breeding pedi gree, as indicated in Table IV

- 2

.

### 5307

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- 1 Page 25 of 168 Table IV

- 2

. Event 5307 and control seed materials used in st udies

. The female parent germplasm is shown first in each hybrid genotype description; the male parent germplasm is listed second. The pedigree diagram in Figure IV

- 4 indicates the origin of Event 5307 seed and control seed materials of these genotypes by seed lot identifier (Lot ID).

Study Descri ption (Relevant Part of
Submission ) Event 5307 Seed Material Nontransgenic Control Seed Material

Lot
ID Genotype Lot
ID Genotype

Nucleotide sequence inserted ( Part IV.E.1 ) D NP2171 × NP2460(5307)(BC5F 3 )* None

Southern blots for copy no. of functional elements (Part IV.E.2 ) D NP2171 × NP2460(5307)(BC5F 3 )* E NP2171 × NP2460 + parental inbred lines
NP2222

, NP2460

, NP2171 (not
shown in Fig. IV

- 4)

Southern blot analysis of genetic stability (Part IV.E.3 ) A
B
C
D F 1 BC6
BC7
NP2171 × NP2460(5307)(BC5F 3 )* E P arental inbred lines NP2222

, NP2460 , and NP2171 (not shown in Fig. IV

- 4)

NP2171 × NP2460

Mendelian inheritance analysis (Part IV.E.4 ) A
B
C F 1 BC6
BC7 None

Determination of insert flanking

sequences (Part IV.E.5 ) D NP2171 × NP2460(5307)(BC5F 3 )* None

Equivalence of eCry3.1Ab produced
in 5307 corn and
recombinant E. coli (Part VI.C ) D NP2171 × NP2460(5307)(BC5F 3 )* E NP2171 × NP2460

eCry3.1Ab and PMI
concentrations in
5307 corn tissues (Part VI.N ) D NP2171 × NP2460(5307)(BC5F 3 )* E NP2171 × NP2460

Compositional assessment of forage and grain (Part VII.C ) D NP2171 × NP2460(5307)(BC5F 3 )* E NP2171 × NP2460

Broiler chicken feeding stud y with grain (Part VII.D ) D NP2171 × NP2460(5307)(BC5F 3 )* E NP2171 × NP2460

* This 5307 hybrid is a lternatively referred to as “NP2171 x BC5F 3 ” or “NP2171 x NP2640 (5307). ”

### 5307

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- 1 Page 26 of 168 Figure IV

- 4

. Pedigree diagram of Event 5307 and control seed material s.
Generations in ovals were Event 5307 materials used in studies described in this submission. Generations in rectangles were

used as controls. Boxed letter codes correspond to specific seed lots used in studies listed in Table IV

- 2 . A backcross (BC)
is a cross of an individual with one of its parents. The init ial Event 5307 F 1 generation was crossed with one of its inbred
parents, NP2460, to create generation BC1. Likewise, BC2 originated from a cross of the BC1 with the parental line NP2460.
Successive backcrosses with the recurrent parent followed similarly to introgress the transgenes into NP2460 germplasm.



### NP2171 x NP2460(5307) (BC5F 3 

### ) × NP2460 * × NP2460 * 

### × NP2460* × NP2460* NP2222* × 5307

F 1



### BC1

BC5 × NP2460*

**⊗** × NP2222* 5307 T 0 

### ( NP2222 * )

× NP2460* BC2

BC3

× NP2460*

BC4

BC5F 2



### BC5F 3



### NP2171* × h omozygo tes **⊗** homozygo tes 

### BC6 BC7 

## A

B

C

Non transgenic
Control Hybrid

NP2171 × 

### NP2460 NP2171* × NP2460* × NP2460*

NP2171* × homozygotes



## D

E T 0 = original transformant

x = cross
BC = backcross

**⊗** = self

- pollination

* = field corn inbred

### 5307

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- 1 Page 27 of 168 IV.D. Quality Control Testing of Seed Materials

All test and control seed lots were analyzed for the presence of Event 5307 DNA and
adventitious DNA from other transformation events using the real

- time PCR method
described in Appendix A

. All Event 5307 seed lots were confirmed to contain the genes

ecry 3.1Ab and pmi based on nucleotide sequence . Additionally, all Event 5307 seed lots
were confirmed to contain Event

- 5307

- specific DNA based on nucleotide sequences at the junction of the T

- DNA insert and the corn genome. The analyses did not detect these components in control seed lots. All test and control seed lots had no detectable sequences
that would be indicative of DNA from other regulated events under development at Syngenta, or deregulated events for which testing methodology is available.

IV.E. Chara cter ization of the Genetic Material in Event 5307 Corn

IV.E.1. DNA Insert Sequencing

Two overlapping DNA fragments that span the 5307 corn insert were amplified from genomic DNA extracted from 5307 corn using a polymerase chain reaction method. These fragments were cloned, and sequences of the clones were aligned to create a consensus of
the T

- DNA sequence. The consensus nucleotide sequence data for the 5307 corn insert were compared to the sequence of the transformation plasmid pSYN12274 ( Figure IV

- 1 ). The
data demonstrated th at the insert was intact and that the organization of the functional elements within the insert, as present in plasmid pSYN12274, was maintained. The
functional elements ecry3.1Ab

, pmi, the CMP promoter, the ZmUbiInt promoter, and the
NOS terminators in 5 307 corn were identical to those in the transformation plasmid pSYN12274.

One nucleotide change was identified in the 5307 corn insert 48 bp upstream of the CMP promoter in a non

- coding region of the T

- DNA ( Figure IV

- 1 ). This nucleotide change has no effect on the transgenes encoded by 5307 corn

. Sequence analysis revealed that some truncation occurred at the right border (RB) and left border (LB) ends of the T

- DNA during the transformation process. The entire RB, three bp
of non

- coding sequence at the 5′ end of the insert, and eight bp of the LB were truncated. These deletions had no effect on the functionality of the insert; similar deletions have previously been observed in transformations with Agrobacterium tumefaciens (Tinland and Hohn 1995; Brunaud et al. 2002; Chilton and Que 2003).

IV.E.2. Analysi s of DNA Insertion Site in Corn Genome

Sequence analysis of the 5307 maize genomic insertion site was conducted to determine whether any corn genomic DNA was deleted when the 5307 corn T

- DNA insert integrated
into the corn genome. This analysis was conduc ted by identifying the T

- DNA insertion site

### 5307

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- 1 Page 28 of 168 using PCR analysis of extracted genomic DNA, followed by cloning the PCR fragment into
a vector and determining the nucleotide sequenc e of the PCR amplification product in the resulting plasmid preparations.

A n alignment of the nu cleotide sequence of the corn genomic sequence flanking the 5307

corn T

- DNA insert with the sequence of the corn genomic insertion site, as determined from the nontransgenic control maize line used for transformation (Syngenta inbred NP2222),
revealed that 33 base pairs of genomic corn DNA were deleted during integration of the

5307 T

- DNA into the corn genome. This deletion has had no apparent deleterious effect on the phenotype or properties of Event 5307 corn.

IV.E.3. Copy Number of Functional Elements

South ern blot analyses demonstrated that the T

- DNA insert in 5307 corn contains single

cop ies of ecry3.1Ab, pmi , the CMP promoter sequenc e, and the ZmUbiInt promoter

se q u ence and two copies of the NOS terminator sequence, as expected for a single insertion
site . Results also indicated that there are no extraneous DNA fragments of the functional
elements elsewhere in the 5307 corn genome, and that 5307 corn is free of backbone sequence from the transformation plasmid pSYN12274.

For each Southern blot, there is a map showing the location of the specific probe and the locations of the restriction enzyme sites used in that analysis. These can be found in Figure

IV

- 5

, Figure IV

- 7

, Figure IV

- 9

, Figure IV

- 11 , and Figure IV

- 13

. The results of these
Southern blot analyses are shown in Figure IV

- 6

, Figure IV

- 8

, Figure IV

- 10

, Figure IV

- 12

, and Figure IV

- 14

, respectively. In addition, comparisons of the expected and observed hybridization bands are di splayed in table format ( Table IV

- 3

, Table IV

- 4

, Table IV

- 5

, Table IV

- 6 , and Table IV

- 7 , respectively ) for each Southern bl ot.

IV.E.3.a. Copy Number of Functional Elements: ecry3.1Ab

- S pecific P robe

A map of the T

- DNA of 5307 corn transformation plasmid pSYN12274, indicating the
location of the ecry3.1Ab

- specific probe and restriction sites for Kpn I , Nco I , Sma I, and Pme I
is shown in Figure IV

- 5

. The results of this analysis are shown in Table IV

- 3 and Figure

IV

- 6

.

Genomic 5307 corn DNA digested with Kpn I ( Figure IV

- 6 A, Lane A 3) produced a single hybridization band at approximately 8.5 kb, corresponding to a single copy of ecry3.1Ab

. Genomic 5307 corn DNA digested with Nco I ( Figure IV

- 6 B, Lane B 3) produced a single hybridization band at the expected size of approximately 19 kb, corresponding to a single copy of ecry3.1Ab

. Genomic 5307 corn DNA digested with Sma I + Pme I ( Figure IV

- 6 C,

Lane C 3) produced a single hybridization band at approximately 6.3 kb, corresponding to a single copy of ecry3.1Ab

.

### 5307

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- 1 Page 29 of 168 The negative control corresponding to each digest showed no hybridization ( Kpn I

, Figure

IV

- 6 A, Lane A 4 ; Nco I

, Figure IV

- 6 B, Lane B 4 ; and Sma I + Pme I

, Figure IV

- 6 C, Lane C 4 ). One hybridization band of the expected size was present in all lanes (Lanes A5, B5 and C5
of Figure IV

- 6 A, B and C, respectively) containing positive control DNA from the plasmid pSYN12274.

For Southern blot analy ses with the ecry3.1Ab

- specfic probe, detection of only one hybridization band of the expected size for each restriction enzyme digestion strategy
demonstrated that the T

- DNA insert in 5307 corn contains a single copy of ecry3.1Ab . No unexpected b ands were detected, indicating that there were no extraneous DNA fragments of ecry3.1Ab in the 5307 corn genome.

Figure IV

- 5

. Location of the Kpn I, Nco I, Sma I, and Pme I restriction sites and position of the 1962 bp ecry3.1Ab

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274

.

The vertical arrows indicate the site of restriction dige stion. Sizes of the expected restriction fragments are indicated. pmi (1176 bp) ecry3.1Ab (1962 bp)

LB (25 bp)

ecry3.1Ab

- specific probe (1962 bp) ZmUbiInt promoter (1993 bp)

NOS terminator (253 bp)

**Kpn** I **Nco** I **Pme** I **Sma** I CMP promoter (346 bp) NOS terminator (253 bp)

Kpn I > 2.8 kb

> 3.9 kb

Sma I + Pme I 6339 bp Nco I RB (25 bp)

### 5307

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- 1 Page 30 of 168 Table IV

- 3

. Expected and observed hybridization band sizes in S outhern blot analysis of 5307 corn , using a n ecry3.1Ab

- specific probe and restricti on enzymes Kpn I, Nco I, and Sma I

+ Pme I

.

Figure & Lane N o. Source of DNA Restriction
enzyme (s) Expected number of

bands Expected band size (kb) Observed band size (kb)

Figure IV

- 6 A,
Lane A 3 5307 NP2171 × BC5F 3 Kpn I 1 >2.8 ~8.5

Figure IV

- 6 A,
Lane A 4 NP2171 × NP2460 Kpn I none none none

Figure IV

- 6 A,
Lane A 5 Positive control (16.53 pg of pSYN12274 mixed with
NP2171 × NP2460 digested
with Kpn I) Sma I + Pme I 1 ~6.3 ~6.3

Figure IV

- 6 B,
Lane B 3 5307 NP2171 × BC5F 3 Nco I 1 >3.9 ~19

Figure IV

- 6 B,
Lane B 4 NP2171 × NP2460 Nco I none none none

Figure IV

- 6 B,
Lane B 5 Positive control (16.53 pg of pSYN12274 mixed with
NP2171 × NP2460 digested
with Nco I ) Sma I + Pme I +
Nco I 1 1 ~3.9 ~3.9

Figure IV

- 6 C,
Lane C 3 5307 NP2171 × BC5F 3 Sma I + Pme I 1 ~6.3 ~6.3

Figure IV

- 6 C,
Lane C 4 NP2171 × NP2460 Sma I + Pme I none none none

Figure IV

- 6 C,
Lane C 5 Positive control (16.53 pg of pSYN12274 mixed with
NP2171 × NP2460 digested
with Sma I + Pme I ) Sma I + Pme I 1 ~6.3 ~6.3

1 Digestion of pSYN12274 with Nco I was the result of addition to NP2171 × NP2460 digested with Nco I

### 5307

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- 1 Page 31 of 168 Figure IV

- 6

. Southern blot analysi s of 5307 corn for copy number of functional elements : 1962

- bp ecry3.1Ab

- specific probe, using restriction enzymes Kpn I , Nco I , and Sma I + Pme I

(A ) Kpn I

Lane A1 = molecular weight markers Lane A2 = blank Lane A3 = 5307 NP2171 × BC5F 3 digested with Kpn I Lane A4 = NP2171 × NP2460 digested with Kpn I Lane A5 = Positive control ( NP2171 × NP2460 digested with Kpn I and 16.53 pg of pSYN12274 digested with Sma I + Pme I) (B) Nco I

Lane B1 = molecular weight markers Lane B2 = blank Lane B3 = 5307 NP2171 × BC5F 3 digested with Nco I Lane B4 = NP2171 × NP2460 digested with Nco I Lane B5 = Positive control ( NP2171 × NP2460 digested with Nco I and 16.53 pg of pSYN12274 digested with Sma I + Pme I + Nco I) (C) Sma I + Pme I

Lane C1 = molecular weight markers Lane C2 = blank Lane C3 = 5307 NP2171 × BC5F 3 digested with Sma I + Pme I Lane C4 = NP2171 × NP2460 digested with Sma I + Pme I Lane C5 = Positive control ( NP2171 × NP2460 digested with Sma I + Pme I and 16.53 pg of pSYN12274 digested with Sma I + Pme I)

### 5307

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- 1 Page 32 of 168 IV.E.3.b. Copy Number of Functional Elements: pmi

- S pecific P robe

A map of the T

- DNA region in the 5307 corn transformation vector pSYN12274 indicating the
location of the pmi

- specific probe, and the restriction enzymes used in this an alysis, are shown in Figure IV

- 7 . The r esults of this analysis are shown in Table IV

- 4 and Figure IV

- 8

.

Genomic 5307 corn DNA digested with Bst EII ( Figure IV

- 8

, Lane 3) produced a single hybridization band of approximately 7.2 kb, corresponding to a single copy of pmi

. Genomic
5307 corn DNA digested with Spe I (Lane 5) produced a single hybridization band at approximately 7.0 kb, corresponding to a single copy of pmi . Genomic 5307 corn DNA digested
with Sma I + Pme I (Lane 7) produced a single hybridization band at the exp ected size of approximately 6.3 kb, corresponding to a single copy of pmi and confirming the intactness of the
insert.

The negative control corresponding to each digest showed no hybridization ( Bst EII Lane 4, Sp e I

Lane 6

, and Sma I + Pme I Lane 8). One hy bridization ba nd of the expected size was present in
the lane (Lane 9) containing positive control DNA from the plasmid pSYN12274.

For the pmi

- specific probe , the restriction enzyme digests resulted in a single hybridization signal in each case, demonstrating that 5307 corn contains a single copy of pmi . No unexpected bands were detected, indicating that 5307 corn does not contain any additional pmi coding regions
other than that associated with the DNA insert.

### 5307

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- 1 Page 33 of 168 Figure IV

- 7

. Location of the Bst EII, Spe I, Sma I, and Pme I restriction site s and position of the 1176 bp pmi

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274

T he vertical arrows indicate the site of restriction digestion. Sizes of the expected restriction fragments are indicated. pmi (1176 bp) ecry3.1Ab (1962 bp)

LB (25 bp) RB (25 bp)

pmi

- specific probe (1176 bp) **Pme** I **Sma** I **Bst** EII **Spe** I CMP promoter (346 bp) NOS terminator (253 bp) NOS terminator (253 bp) ZmUbiInt promoter (1993 bp)

Bst EII > 4.0 kb

> 5.9 kb

Sma I + Pme I 6339 bp Spe I

### 5307

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- 1 Page 34 of 168 Table IV

- 4

. Expected and observed hybridization band sizes in Southern blot anal ysis of 5307

corn , using a pmi

- specific probe and restriction enzymes Bst EII, Spe I, and Sma I + Pme I

.

Figure & Lane No. Source of DNA Restriction
enzyme (s) Expected number of

bands Expected band size (kb) Observed band size (kb)

Figure IV

- 8

, Lane 3 5307 NP2171 × BC5F 3 Bst EII 1 > 4.0 kb ~ 7.2 kb

Figure IV

- 8

, Lane 4 NP2171 × NP2460 Bst EII none none none

Figure IV

- 8

, Lane 5 5307 NP2171 × BC5F 3 Spe I 1 > 5.9 kb ~ 7.0 kb

Figure IV

- 8

, Lane 6 NP2171 × NP2460 Spe I none none none

Figure IV

- 8

, Lane 7 5307 NP2171 × BC5F 3 Sma I + Pme I 1 ~6.3 kb ~6.3 kb

Figure IV

- 8

, Lane 8 NP2171 × NP2460 Sma I + Pme I none none none

Figure IV

- 8

, Lane 9 Positive control (16.53 pg of pSYN12274 mixed with NP2171

× NP2460 digested with Sma I +
Pme I ) Sma I + Pme I 1 ~6.3 kb ~6.3 kb

### 5307

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- 1 Page 35 of 168 Figure IV

- 8

. Southern blot analysis of 5307 corn for c opy number of functional elements: 1176

- bp pmi

- specific probe, using restriction enzymes Bst EII, Spe I, and Sma I + Pme I

.

Lan e 1 = molecular weight markers
Lane 2 = blank
Lane 3 = 5307 NP2171 × BC5F 3 dig e sted with Bst EII
Lane 4 = NP2171 × NP2460 dig e sted with Bst EII
Lane 5 = 5307 NP2171 × BC5F 3 dig e sted with Spe I
Lane 6 = NP2171 × NP2460 dig e sted with Spe I
Lane 7 = 5307 NP2171 × BC5F 3 dig e sted with Sma I + Pme I
Lane 8 = NP2171 × NP2460 dig e sted with Sma I + Pme I
Lane 9 = Positive control ( N P2171 × NP2460 digested with Sma I + Pme I and 16. 5 3 pg of pSYN12274 digested with
S ma I + Pme I )

### 5307

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- 1 Page 36 of 168 IV.E.3.c. Copy Number of Functional Elements: CMP promoter

- specific probe

Figure IV

- 9 shows a map of the T

- DNA of 5307 corn transformation plasmid pSYN12274, indicating the location of the CMP promoter

- specific prob e and restriction sites for Kpn I, Spe I

, SmaI , and Pme I. Figure IV

- 10 depicts the results of the corresponding Sou thern blot analyses,
and Table IV

- 5 outlines the expected and observed sizes of the hybridization bands.

For Southern blot analysis with genomic DNA digested with Kpn I and probed with the CMP
promoter

- specific probe, one hybridization band of approximately 8.5 kb was observed as expected ( Figure IV

- 10 A , Lane A 3) ( Table IV

- 5 ). For Southern blot analysis with genomic DNA digested with Spe I and probed with the CMP promoter

- specific probe, one hybridization band of approximately 2.6 kb was observed as expected ( Figure IV

- 10 B, Lane B 3) ( Table IV

- 5 ). For Southern blot analysis with genomic DNA digested with Sma I + Pme I and pr obed with the CMP promoter

- specific probe, one hybridization band of approximately 6.3 kb was observed as

expec ted ( Figure IV

- 10 C, Lane C 3) ( Table IV

- 5 ).

No CMP promoter

- specific hybridization band was present in DNA extracted fr om the control substance plants ( Figure IV

- 10 A, B, and C, Lane s A 4 , B4 and C4 , respectively ) and therefore hybridization with this probe was specific to the 5307 corn insert. As expected, o ne hybridization band of approximately 6.3 kb was observed in the lane containing the positive
control

, plasmid pSYN12274 DNA

, digested with Sm a I + Pme I ( Figure IV

- 10 A, B, and C, Lane s A 5 , B5 and C5, respectively ).

For Southern blot analyses with the CMP promoter

- specfic probe, detection of only one hybridization band of the expected size for each restriction enzyme digestion strategy
demonstrated that the 5307 corn T

- DNA insert contains a single copy of the CMP prom oter sequence. No unexpected bands were detected, indicating that there w ere no extraneous DNA fragments of the CMP promoter sequence in the 5307 corn genome.

### 5307

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- 1 Page 37 of 1 68 Figure IV

- 9

. Location of the Kpn I, Spe I, Sm a I, and Pme I restriction site s and position of the 346 bp CMP promoter

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274

.

The vertical arrows indicate the site of restriction digestion. Sizes of the expected restriction fragments are indicated. pmi (1176 bp) ecry3.1Ab (1962 bp)

LB (25 bp) RB (25 bp)

CMP promoter

- specific probe (346 bp) ZmUbiInt promoter (1993 bp)

NOS terminator (253 bp)

**Pme** I **Sma** I **Spe** I CMP promoter (346 bp) NOS terminator (253 bp) **Kpn** I

Kpn I > 2.8 kb

> 0.6 kb

Sma I + Pme I 6339 bp Spe I

### 5307

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- 1 Page 38 of 168 Table IV

- 5

. Expected and observed hybridization band sizes in Southern blot analysis of 5307

corn , using a CMP promoter

- specific probe and restriction enzymes Kpn I, Spe I, and Sma I +
Pme I

.

Figure & Lane No. Source of DNA Restriction
enzyme (s) Expected number of

bands Expected band size (kb) Observed band size (kb)

Figure IV

- 10 A,
Lane A 3 5307 NP2171 × BC5F 3 Kpn I 1 >2.8 ~8.5

Figure IV

- 10 A,
Lane A 4 NP2171 × NP2460 Kpn I none none None

Figure IV

- 10 A,
Lane A 5 Positive control (16.53 pg of pSYN12274 mixed with
NP2171 × NP2460 digested
with Kpn I ) Sma I + Pme I 1 ~6.3 ~6.3

Figure IV

- 10 B,
Lane B 3 5307 NP2171 × BC5F 3 Spe I 1 >0.6 ~2.6

Figure IV

- 10 B,
Lane B 4 NP2171 × NP2460 Spe I none none none

Figure IV

- 10 B,
Lane B 5 Positive control (16.53 pg of pSYN12274 mixed with
NP2171 × NP2460 digested
with Spe I ) Sma I + Pme I 1 ~6.3 ~6.3

Figure IV

- 10 C,
Lane C 3 5307 NP2171 × BC5F 3 Sma I + Pme I 1 ~6.3 ~6.3

Figure IV

- 10 C,
Lane C 4 NP2171 × NP2460 Sma I + Pme I none none none

Figure IV

- 10 C,
Lane C 5 Positive control (16.53 pg of pSYN12274 mixed with
NP2171 × NP2460 digested
with Sma I + Pme I ) Sma I + Pme I 1 ~6.3 ~6.3

### 5307

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- 1 Page 39 of 168 Figure IV

- 10 . Southern blot analysis of 5307 corn for c opy number of functional elements: 346

- bp CMP promoter

- specific probe, using restriction enzymes Kpn I, Spe I, and Sma I + Pme I

(A) Kpn I

Lane A1 = molecular weight markers Lane A2 = blank Lane A3 = 5307 NP2171 × BC5F 3 digested with Kpn I Lane A4 = NP2171 × NP2460 digested with Kpn I Lane A5 = Positive control ( NP2171 × NP2460 digested with Kpn I and 16.53 pg of pSYN12274 digested with Sma I + Pme I) (B) Spe I

Lane B1 = molecular weight markers Lane B2 = blank Lane B3 = 5307 NP2171 × BC5F 3 digested with Spe I Lane B4 = NP2171 × NP2460 digested with Spe I Lane B5 = Positive control ( NP2171 × NP2460 digested with Spe I and 16.53 pg of pSYN12274 digested with Sma I + Pme I) (C) Sma I + Pme I

Lane C1 = molecular weight markers Lane C2 = blank Lane C3 = 5307 NP2171 × BC5F 3 digested with Sma I + Pme I Lane C4 = NP2171 × NP2460 digested with Sma I + Pme I Lane C5 = Positive control ( NP2171 × NP2460 digested with Sma I + Pme I and 16.53 pg of pSYN12274 digested with Sma I + Pme I)

### 5307

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- 1 Page 40 of 168 IV.E.3.d. Copy Number of Functional Elements: ZmUbiInt P romoter S pecific Probe

Figure IV

- 11 shows a map of the T

- DNA of 5307 corn tra nsformation plasmid pSYN12274, indicating the location of the ZmUbiInt promoter

- specific probe and restriction sites for Bst EII, Spe I, Sma I, and Pme I. Figure IV

- 12 d epicts the results of the corresponding Southern blot analyses, and Table IV

- 6 outlines the expected and observed sizes of the hybridization bands.

For Southern blot analysis with genomic DNA digested with Bst EII and probed with the ZmUbiInt promoter

- specific probe, one hybridization band of approximately 7.2 kb was observed as expected ( Figure IV

- 12 A, Lane A 3) ( Table IV

- 6 )

. No hybridization band was present in DNA extracted from the con trol substance plants ( Figure

IV

- 12 A, Lane s A 4

, A 5, A 6, and A 7) and therefore hybridization was specific to the 5307 corn insert. As expected, t hree hybridization bands of approximately 3.9 kb, 8.4 kb, and 18 kb were observed in the lane containing the positive control DNA digested with Sma I + Pme I + Bst EII ( Figure IV

- 12 A, Lane A 8).

Additional bands resulting from cross

- hybridization of the ZmUbiInt promoter

- specific probe with the endogenous maize polyubiquitin promoter sequence were also detected in all material analyzed. Two hybridization bands of approxim ately 12 kb and 18 kb corresponding to the hybridization bands observed in lanes containing DNA extracted from control plants ( Figure

IV

- 12 A, Lane s A 5 and A 6 ) , respectively, were also observed in the DNA extracted from 5307

corn p lants ( Figure IV

- 12 A, Lane A 3). Two hybridization bands of approximately 8.4 kb and 18 kb corresponding to the hybridization bands observed in lanes containing DNA extr act ed from NP2460 plants ( Figure IV

- 12 A, Lane A 7) and NP2171 plants ( Figure IV

- 12 A, Lane A 5), respectively, were observed in the lane contain ing DNA extracted from NP2171 × NP2460 plants ( Figure IV

- 12 A, Lane A 4) and the lane containing the positive control ( Figure IV

- 12

, Lane A 8).

For Southern blot analysis with genomic DNA digested with Spe I and probed with the ZmUbiInt
promoter

- specific probe, one hybridization band of approximately 7.0 kb was observed in the
lane containing DNA extracted from 5307 corn plants as expected ( Figure IV

- 12 B, Lane B 3) ( Table IV

- 6 ). No hybridization band was present in the DNA extracted from the con trol
subs tance plants ( Figure IV

- 12 B, Lane s B 4

, B 5, B 6, and B 7) and therefore hybridization was specific to the 5307 corn insert

. As expected, t hree hybridization bands of ap proximately 6.3 kb, 14 kb, and 20 kb were observed in the lane containing the positive control DNA digested with

Sma I + Pme I ( Figure IV

- 12 B, Lane B 8).

Additional bands resulting from cross

- hybridization of the ZmUbiInt promoter

- specific probe with the endogenous maize polyubiquitin promoter sequence were also detected in all m aterial analyzed. Two hybridization bands of approximately 25 kb and 14 kb corresponding to the hybridization bands observed in lanes containing DNA extracted from control substance plants ( Figure IV

- 12 B, Lane s B 5 and B 6) were also observed in the DNA extracted from 5307 corn plants ( Figure IV

- 12 B, Lane B 3). Finally, two hybridiza tion bands of approximately 20 kb and

### 5307

- FDA

- 1 Page 41 of 168 14 kb corresponding to the hybridization bands observed in lanes containing DNA extracted
from NP2460 plants ( Figure IV

- 12 B, Lane B 7) and NP2171 plants ( Figure IV

- 12 B, Lane B 5), respectively, were observed in the lane contain ing DNA extracted from NP2171 × NP2460 plants ( Figure IV

- 12 B, Lane B 4) and the lane containing the positive control ( Figure IV

- 12 B,

Lane B 8).

For Southern blot analysis with genomic DNA digested with Sma I + Pme I and probed with the ZmUbiInt promoter

- specific probe, one hybridization band of approximately 6.3 kb was
observed in t he DNA extracted from 5307 corn plants as expected ( Figure IV

- 12 C, Lane C 3) ( Table IV

- 6 ). This hybridization band was absent in the lane containing DNA extracted from the control substance plants NP2171 × NP2460 ( Figure IV

- 12 C, Lane s C 4

, C 5, C 6, and C 7) and
therefore hybridization was specific to the 5307 corn insert. A hybridizat ion band of approximately 6.3 kb and a high molecular weight band (greater than 30 kb) was observed in the lane containing the positive control DNA ( Figure IV

- 12 C, Lan e C 8).

At least one additional band resulting from cross

- hybridization of the ZmUbiInt promoter

specific probe with the endogenous maize polyubiquitin promoter sequence was also detected in all material analyzed. One hybridization band of approximately 18 kb corresponding to the hybridization band observed in the lane containing DNA extracted from control substance plants ( Figure IV

- 12 C, Lane C 6) was also observed in the lane containing DNA extracted from 5307

corn plants ( Figure IV

- 12 C, Lane C 3). An additional high molecular weight band (greater than 30 kb) was observed in lanes containing DNA extracted from control substance plants ( Figure

IV

- 12 C, Lane s C 5 and C 7). This faint high molecular weight band (greater than 30 kb) was also observed in the lane containing DNA extracted from 5307 corn plants ( Figure IV

- 12 C, Lane C 3),
control substance plants ( Figure IV

- 12 C, Lane C 4), and the lane containing the positive control ( Figure IV

- 12 C, Lane C 8).

For Southern blot analyses with the ZmUbiInt promoter

- specfic probe, detection of only one hybridization band specific to 5307 corn for each restriction enzyme digestion demons trated that
5307 corn contains a single copy of the ZmUbiInt promoter sequence. No unexpected bands
were det ected, indicating that there were no extraneous DNA fragments of the ZmUbiInt promoter sequence in the 5307 corn genome.

### 5307

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- 1 Page 42 of 168 Figure IV

- 11

. Location of the Bst EII, Spe I, Sma I, and Pme I restriction sites and position of the 1993

- bp ZmUbiInt promoter

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274.

### 5307

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- 1 Page 43 of 168 Table IV

- 6 . Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn, using a ZmUbiInt promoter

- specific probe and restriction enzymes Bst EII, Spe I, and Sma I + Pme I.
Figure & Lane No. Source of DNA Restr iction
enzyme (s) Expected n o. of bands Expected band size (kb) Observed band size (kb)
Figure IV

- 12 A,
Lane A 3 5307 NP2171 × BC5F 3 Bst EII 1 5307 insert x endogenous > 4.0 unknown ~ 7.2 ~ 12 (endogenous) ~ 18 (endogenous) Figure IV

- 12 A,
Lane A 4 NP2171 × NP2460 Bst EII x endogenous unknown ~ 8.4 (endogenous)

~ 18 (endogenous) Figure IV

- 12 A,
Lane A 5 NP2171 Bst EII x endogenous unknown

~ 18 (endogenous)

Figure IV

- 12 A,
Lane A 6 NP2222 Bst EII x endogenous unknown

~ 12 (endogenous)

Figure IV

- 12 A,
Lane A 7 NP2460 Bst EII x endogenous unknown

~ 8.4 (endogenous)

Figure IV

- 12 A,
Lane A 8 Positive control (16.53 pg of pSYN12274 mixed
with NP2171 × NP2460 digested with Bst EII ) Sma I + Pme I

+ Bst EII 1 1 pSYN12274 x endogenous ~ 3.9 unknown ~ 3.9

~ 8.4 (endogenous)

~ 18 (endogenous)

Figure IV

- 12 B,
Lane B 3 5307 NP2171 × BC5F 3 Spe I 1 5307 insert x endogenous > 5.9 unknown ~ 7.0

~ 14 (endogenous)

~ 25 (endogenous) Figure IV

- 12 B,
Lane B 4 NP2171 × NP2460 Spe I x endogenous unknown ~ 14 (endogenous)

~ 20 (endogenous) Figure IV

- 12 B,
Lane B 5 NP2171 Spe I x endogenous unknown

~ 14 (endogenous)

Figure IV

- 12 B,
Lane B 6 NP2222 Spe I x endogenous unknown

~ 25 (endogenous)

Figure IV

- 12 B,
Lane B 7 NP2460 Spe I x endogenous unknown

~ 20 (endogenous)

Figure IV

- 12 B,
Lane B 8 Positive control (16.53 pg of pSYN12274 mixed
with NP2171 × NP2460 digested with Spe I ) Sma I + Pme I 1 pSYN12274 x endogenous ~ 6.3 unknown ~ 6.3

~ 14 (endogenous)

~ 20 (endogenous)

Figure IV

- 12 C,
Lane B 3 5307 NP2171 × BC5F 3 Sma I + Pme I 1 5307 insert x endogenous ~ 6.3 unknown ~ 6.3

~ 18 (endogenous)

> 30 (endogenous) Figure IV

- 12 C,
Lane C 4 NP2171 × NP2460 Sma I + Pme I x endogenous unknown > 30 (endogenous)

Figure IV

- 12 C,
Lane C 5 NP2171 Sma I + Pme I x endogenous unknown > 30 (endogenous)

Figure IV

- 12 C,
Lane C 6 NP2222 Sma I + Pme I x endogenous unknown

~ 18 (endogenous)

Figure IV

- 12 C,
Lane C 7 NP2460 Sma I + Pme I x endogenous Unknown > 30 (endogenous)

Figure IV

- 12 C,
Lane C 8 Positive control (16.53 pg of pSYN12274 mixed with
NP2171 × NP2460 digested
with Sma I + Pme I ) Sma I + Pme I 1 pSYN12274 x endogenous ~ 6.3 unknown ~ 6.3

> 30 (endogenous)

x = unknown number

1 Digestion of pSYN12274 with Bst EII was the result of addition to NP2171 × NP2460 digested with Bst EII

### 5307

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- 1 Page 44 of 168 Figure IV

- 12 . Southern blot analysis of 5307 corn for copy number of functional elements: 1993

- bp ZmUbiInt promoter

- specific probe, using restriction enzymes Bst EII, Spe I, and Sma I + Pme I.

(A) Bst EII

Lane A1 = molecular weight markers Lane A2 = blank Lane A3 = 5307 NP2171 × BC5F 3 digested with Bst EIlI Lane A4 = NP2171 × NP2460 digested with Bst EII Lane A5 = NP2171 digested with Bst EII Lane A6 = NP2222 digested with Bst EII Lane A7 = NP2460 digested with Bst EII Lane A8 = Positive control ( NP2171 × NP2460 digested with Bst EII and 16.53 pg of pSYN12274 digested with Sma I + Pme I + Bst EII) (B) Spe I

Lane B1 = molecular weight markers Lane B2 = blank Lane B3 = 5307 NP2171 × BC5F 3 digested with Spe I Lane B4 = NP2171 × NP2460 digested with Spe I Lane B5 = NP2171 digested with Spe I Lane B6 = NP2222 digested with Spe I Lane B7 = NP2460 digested with Spe I Lane B8 = Positive control ( NP2171 × NP2460 digested with Spe I and 16.53 pg of pSYN12274 digested with Sma I + Pme I) (C) Sma I + Pme I

Lane C1 = molecular weight markers Lane C2 = blank Lane C3 = 5307 NP2171 × BC5F 3 digested with Sma I + Pme I Lane C4 = NP2171 × NP2460 digested with Sma I + Pme I Lane C5 = NP2171 digested with Sma I + Pme I Lane C6 = NP2222 digested with Sma I + Pme I Lane C7 = NP2460 digested with Sma I + Pme I Lane C8 = Positive control ( NP2171 × NP2460 digested with Sma I + Pme I and 16.53 pg of pSYN12274 digested with Sma I + Pme I)

### 5307

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- 1 Page 45 of 168 IV.E.3.e. Copy Number of Functional Elements: NOS T erminator

- S pecific P robe

Figure IV

- 13 shows a map of the T

- DNA of 5307 corn transformation plasmid pSYN12274, indicating the location of the NOS terminator

- specific probe and restriction sites for Kpn I, Nco I,
and Sma I + Pme I. Figure IV

- 14 depicts the results of the corresponding Sou thern blot analyses,
and Table IV

- 7 outlines the expected and observed sizes of the hybridization bands.

For Southern blot analysis with genomic DNA digested with Kpn I and probed with the NOS
terminator

- specific probe, two hybr idization bands of approximately 6.4 kb and 8.5 kb were observed in the lane containing DNA extracted from 5307 corn plants as expected ( Figure

IV

- 14 A, Lane A 3) ( Table IV

- 7 ). No hybridization bands were present i n DNA extracted from the control substance plants ( Figure IV

- 14 A, Lane A 4)

. T herefore, two copies of the NOS terminator sequence are present in the 5307 corn insert (one NOS terminator sequence regulating ecry3.1Ab and one NOS terminator sequence regulating pmi ). One hybridization ban d of approximately 6.3 kb was observed in the lane containing the positive control

, as expected ( Figure IV

- 14 A, Lane A 5).

For Southern blot analysis with genomic DNA digested with Nco I and probed with the NOS
terminator

- specific probe, two hybridization bands of approximately 16 kb and 19 kb were

o bserved in the lane containing DNA extracted from 5307 corn plants as expected ( Figure

IV

- 14 B, Lane B 3) ( Table IV

- 7 ). These hybridization bands were absent in the lane containing DNA extracted from the control substance plants ( Figure IV

- 14 B, Lane B 4) and were

, therefore, specific to the two copies of the NOS terminator sequence in the 5307 corn insert. As expected,

t wo hybridization bands of approximately 2.5 kb and 3.9 kb were observed in the lane containing the positive control digested w ith Sma I + Pme I + Nco I ( Figure IV

- 14 B, Lane B 5).

For Southern blot analysis with genomic DNA digested with Sma I + Pme I and probed with the
NOS terminator

- specific probe, one hybridization band of approximately 6.3 kb was obser ved in the lane containing DNA extracted from 5307 corn plants as expected ( Figure IV

- 14 C, Lane C 3) ( Table IV

- 7 ). This hybridization band was absent in the lane containing DNA extracted from the control substance plants ( Figure IV

- 14 C, Lane C 4 ) and was

, therefore, specific to the 5307 corn insert. One hybridization band of approximately 6.3 kb was observed in the lane containing the positive control

, as expected ( Figure IV

- 14 C, Lane C 9).

For Southern blot analyses with the NOS terminator

- specfic probe, detection of two hybridization bands of the expected size for each restriction enzyme digestion strategy
demons trated that 5307 corn contains two copies of the NOS terminator sequence (one NOS terminator sequence regulating ecry3.1Ab and one NOS terminator sequence regulating pmi ). No unexpected bands were detected, indicating that there w ere no extraneous DNA fra gments of the NOS terminator seq uence in the 5307 corn genome.

### 5307

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- 1 Page 46 of 168 Figure IV

- 13 . Location of the Kpn I, Nco I, Sma I, and Pme I restriction sites and position of the 253

- bp NOS terminator

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274.

### 5307

- FDA

- 1 Page 47 of 168 Table IV

- 7 . Expected and observed hybridization band sizes in Southern blot analysis of 5307

corn , using a NOS terminator

- specific probe and restriction enzymes Kpn I , Nco I, and Sma I + Pme I

.

Figure & Lane No. Source of DNA Restriction
enzyme (s) Expected number of

bands Expected band size (kb) Observed band size (kb)

Fig. I V

- 1 4 A

, Lane A 3 5307 NP2171 × BC5F 3 Kpn I 2 > 2.8

> 3.6 ~ 6.4

~ 8.5
Fig. IV

- 14 A, Lane A 4 NP2171 × NP2460 Kpn I none none none

Fig. IV

- 14 A, Lane A 5 Positive control (16.53 pg of pSYN12274 mixed with NP2171 × NP2460 digested with Kpn I ) Sma I + Pme I 1 ~6.3

~ 6.3

Fig. I V

- 1 4 B, Lane B 3 5307 NP2171 × BC5F 3 Nco I 2 > 2.5

> 3.9 ~ 16

~ 19
Fig. IV

- 14 B, Lane B 4 NP2171 × NP2460 Nco I none none None

Fig. IV

- 14 B, Lane B 5 Positive control (16.53 pg of pSYN12274 mixed with NP2171 × NP2460 digested with Nco I ) Sma I + Pme I + Nco I 1 2 ~ 2.5

~ 3.9 ~ 2.5

~ 3.9

Fig. IV

- 14 C, Lane C 3 5307 NP2171 × BC5F 3 Sma I + Pme I 1

~ 6.3

~ 6.3

Fig. IV

- 14 C, Lane C 4 NP2171 × NP2460 Sma I + Pme I none none none

Fig. IV

- 14 C, Lane C 5 Positive control (16.53 pg of pSYN12274 mixed with NP2171 × NP2460 digested with Sma I + Pme I ) Sma I + Pme I 1

~ 6.3

~ 6.3

1 Digestion of pSYN12274 with Nco I was the result of addition to NP2171 × NP2460 digested with Nco I

### 5307

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- 1 Page 48 of 168 Figure IV

- 14

. Southern blot analysis of 5307 corn for c opy number of functional elements: 253

- bp NOS terminator

- specific probe, using restriction enzymes Kpn I , Nco I, and Sma I + Pme I

.

A. KpnI

Lane A1 = molecular weight markers
Lane A2 = blank
Lane A3 = 5307 NP2171 × BC5F 3 digested with Kpn I
Lane A4 = NP2171 × NP2460 digested with Kpn I
Lane A5 = Positive control ( NP217 1 × NP2460 digested with Kpn I and 16.53 pg of pSYN12274 digested with Sma I + Pme I) B. Nco I

Lane B1 = molecular weight markers
Lane B2 = blank
Lane B3 = 5307 NP2171 × BC5F 3 digested with Nco I
Lane B4 = NP2171 × NP2460 digested with Nco I Lane B5 = Positive control (NP2171 × NP2460 digested with Nco I and 16.53 pg of pSYN12274 digested with Sma I + Pme I + Nco I) C. Sma I + Pme I

Lane C1 = molecular weight markers Lane C2 = blank Lane C3 = 5307 NP2171 × BC5F 3 digested with Sma I

+ Pme I Lane C4 = NP2171 × NP2460 digested with Sma I + Pme Lane C5 = Positive control ( NP2171 × NP2460 digested with Sma I + Pme I and 16.53 pg of pSYN12274 digested with Sma I + Pme I)

### 5307

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- 1 Page 49 of 168 IV.E.4. Generational Stability

Molecular analyses were performed to demonstrate the genetic stability of the 5307 corn insert over four generations. Southern blot analyses were performed using standard molecular biology techniques. Each Southern blot contained a positive control and a negative control. The positive control, representing one copy of a fragment of known size in the corn genome, was included to demonstrate the sensitivity of each experiment; the negative control, DNA extracted from plants grown from nontransgenic corn seed, was included in order to identify possible endogenous DNA sequences that hybridize with the probe. Two probes were used: a full

- length T

- DNA

specific probe containing every base of the plasmid pSYN12274 T

- DNA and a plasmid pSYN12274 backbone

- specific probe containing every base of plasmid pSYN12274 present
outside of the T

- DNA region.

These Southern blot analyses demonstrated that 5307 corn contains a single, complete copy of
the T

- DNA insert and that there are no extraneous DNA fragments of plasmid pSYN12274 T

DNA inserted elsewhere in the 5307 corn genome. Identical hybridization p atterns across all generations of 5307 corn analyzed in this study indicate that the insert is stably inherited from one generation to the next. Additionally, every generation of 5307 corn examined was free of backbone sequence from the transformation pla smid pSYN12274. Descriptions and results of these Southern blot analyses of 5307 corn are provided below . Details of the methods used are provided in Appendix A

.

IV.E.4.a. Stability of the T

- DNA Using a Full

- Length T

- DNA

- specific Probe

Genetic stability of the ins ert during conventional breeding of 5307 corn was determined by Southern blot analyses using a full

- length T

- DNA

- specific probe. The Southern blot analyses included genomic DNA extracted from 5307 plants of four generations (5307 F 1 

### , 5307 BC6, 5307 BC7, and 5307 NP2171 × BC5F 3 

### ) and the corresponding nontransgenic, near

- isogenic control genotypes (NP2171 × NP2460, NP2222, NP2460, and NP2171) (see Figure IV

- 4 breeding pedigree). The full

- length T

- DNA

- specific probe, which contains sequence of the
maize polyubiquitin promoter (ZmUbiInt), cross

- hybridizes to genomic DNA fragments of
different sizes in the different corn lines due to restriction fragment length polymorphi sm of the genomic DNA that carries the endogenous maize polyubiquitin promoter. Control genomic DNA from inbred lines NP2222, NP2460, and NP2171 was needed because the 5307 corn generations analyzed were created by crossing with these corn lines. For the se experiments, genomic DNA was analyzed using two restriction enzyme digestion strategies.

In the first strategy, the corn genomic DNA was digested with an enzyme that cut once within
the 5307 corn insert. The other recognition sites for this enzyme we re located in the corn genome flanking the 5307 corn insert. This first strategy was used twice with two different enzymes ( Bst EII and Spe I) to determine the copy number of the 5307 corn insert and the presence or absence of extraneous DNA fragments of th e plasmid pSYN12274 T

- DNA in other

### 5307

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- 1 Page 50 of 168 regions of the 5307 corn genome. These digests were expected to result in only two hybridization bands corresponding to the 5307 corn insert when a full length T

- DNA

- specific probe was used. More than two bands with eit her digest would have indicated that there were multiple copies of the insert in the plant genome.

In the second strategy, the corn genomic DNA was digested with Sma I + Pme I, which cut within
the 5307 corn insert such that a DNA fragment of predictable s ize was released. This strategy was used to determine the presence of any closely linked extraneous DNA fragments of the plasmid pSYN12274 T

- DNA.

Figure IV

- 15 shows a map of the T

- DNA of the 5307 corn transformation plasmid pSYN12274, indicating the location of the full

- length T

- DNA

- specific probe and restriction sites for Bst EII

, Spe I , Sma I, and Pme I. Figure IV

- 16 depicts the results of the corresponding Southern blot analyses, and Table IV

- 8 provides the expected and observed sizes of the hybridization bands.

Figure IV

- 15

. Location of the Bst EII, Spe I, Sma I, and Pme I restriction sites and position of the 6423

- bp full

- length T

- DNA

- specific probe in the T

- DNA region of the transformation plasmid pSYN12274

.

### 5307

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- 1 Page 51 of 168 Table IV

- 8 . Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn, using a full length T

- DNA

- specific probe and restriction enzymes Bst EII, Spe I, and Sma I

+ Pme I.
Figure & Lane No. Source of DNA Restriction
enzyme (s) Expected number of bands Expected band size (kb) Observed band size (kb)

Figure IV

- 16 A,
L ane A 3 5307 F 1 Bst EII 2 5307 insert

x endogenous >2.4 >4 unknown ~2.9 ~7.2 ~8.4 (endogenous) ~12 (endogenous) Figure IV

- 16 A

, Lane A 4 5307 BC6 Bst EII 2 5307 insert

x endogenous >2.4 >4 unknown ~2.9 ~7.2 ~8.4 (endogenous) ~12 (endogenous) Figure IV

- 16 A,
Lane A 5 5307 BC7 Bst EII 2 5307 insert

x endogenous >2.4 >4 unknown ~2.9 ~7.2 ~8.4 (endogenous) ~12 (endogenous) Figure IV

- 16 A,
Lane A 6 5307 NP2171 × BC5F 3 Bst EII 2 5307 insert

x endogenous >2.4 >4 unknown ~2.9 ~7.2 ~12 (endogenous) ~18 (endogenous) Figure IV

- 16 A,
Lane A 7 NP2171 × NP2460 Bst EII x endogenous unknown ~8.4 (endogenous) ~18 (endogenous)

Figure IV

- 16 A,
Lane A 8 NP2222 Bst EII x endogenous unknown ~12 (endogenous)

Figure IV

- 16 A,
Lane A 9 NP2460 Bst EII x endogenous unknown ~8.4 (endogenous)

Figure IV

- 16 A,
Lane A 10 NP2171 Bst EII x endogenous unknown ~18 (endogenous)

Figure IV

- 16 A,
Lane A 11 5307 F 1 Spe I 2 5307 insert

x endogenous >0.6 >5.8 unknown ~2.6 ~7 ~20 (endogenous) ~25 (endogenous) Figure IV

- 16 A,
Lane A 12 5307 BC6 Spe I 2 5307 insert

x endogenous >0.6 >5.8 unknown ~2.6 ~7 ~20 (endogenous) ~25 (endogenous) Figure IV

- 16 A,
Lane A 13 5307 BC7 Spe I 2 5307 insert

x endogenous >0.6 >5.8 unknown ~2.6 ~7 ~20 (endogenous) ~25 (endogenous) Figure IV

- 16 A,
Lane A 14 5307 NP2171 × BC5F 3 Spe I 2 5307 insert

x endogenous >0.6 >5.8 unknown ~2.6 ~7 ~14 (endogenous) ~25 (endogenous) Figure IV

- 16 A,
Lane A 15 NP2171/NP2460 Spe I x endogenous unknown ~14 (endogenous) ~20 (endogenous)

Figure IV

- 16 A,
Lane A 16 NP2222 Spe I x endogenous unknown ~25 (endogenous)

Figure IV

- 16 A,
Lane A 17 NP2460 Spe I x endogenous unknown ~20 (endogenous)

Figure IV

- 16 A,
Lane A 18 NP2171 Spe I x endogenous unknown ~14 (endogenous)

Figure IV

- 16 A,
Lane A 19 Positive control (NP2171/NP2460 and 16.53 pg of Spe I 1 pSYN12274 x endogenous 11.8 unknown ~11.8 ~14 (endogenous) ~20 (endogenous)

### 5307

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- 1 Page 52 of 168 Table IV

- 8 ( continued ). Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn , using a full length T

- DNA

- specific probe and restriction enzymes Bst EII, Spe I, and Sma I + Pme I

Figure & Lane No

. Source of DNA Restriction
enzyme ( s ) Expected number of bands Expected band size (kb) Observed band size (kb)

Figure IV

- 16 B,
Lane B 3 5307 F 1 Sma I + Pme I 1 5307 insert

x endogenous 6.3

unknown ~6.3

~18 (endogenous)

Figure IV

- 16 B,
Lane B 4 5307 BC6 Sma I + Pme I 1 5307 insert

x endogenous 6.3

unknown ~6.3

~18 (endogenous)

Figure IV

- 16 B,
Lane B 5 5307 BC7 Sma I + Pme I 1 5307 insert

x endogenous 6.3

unknown ~6.3

~18 (endogenous)

Figure IV

- 16 B,
Lane B 6 5307 NP2171 ×
BC5F 3 Sma I + Pme I 1 5307 insert

x endogenous 6.3

unknown ~6.3

~18 (endogenous)

Figure IV

- 16 B,
Lane B 7 NP2171 × NP2460 Sma I + Pme I x endogenous unknown >30 (endogenous)

Figure IV

- 16 B,
Lane B 8 NP2222 Sma I + Pme I x endogenous unknown ~18 (endogenous)

Figure IV

- 16 B,
Lane B 9 NP2460 Sma I + Pme I x endogenous unknown >30 (endogenous)

Figure IV

- 16 B,
Lane B 10 NP2171 Sma I + Pme I x endogenous unknown >30 (endogenous)

Figure IV

- 16 B,
Lane B 11 Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274) Sma I + Pme I 1 pSYN12274

x endogenous 6.3

unknown ~6.3

>30 (endogenous)

### 5307

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- 1 Page 53 of 168 Figure IV

- 16

. Genetic stability Southern blot analysis of 5307 corn with the 6423

- bp full

- length T

- DNA

- specific probe, using restriction enzymes Bst EII, Spe I, and Sma I + Pme I

.

(A) Bst EII or Spe I

Lane A1 = molecular weight markers Lane A2 = blank Lane A3 = 5307 F 1 digested with Bst EII Lane A4 = 5307 BC6 digested with Bst EII Lane A5 = 5307 BC7 digested with Bst EII Lane A6 = 5307 NP2171 × BC5F 3 digested with Bst EII Lane A7 = NP2171 × NP2460 digested with Bst EII Lane A8 = NP2222 digest ed with Bst EII Lane A9 = NP2460 digested with Bst EII Lane A10 = NP2171 digested with Bst EII Lane A11 = 5307 F 1 digested with Spe I Lane A12 = 5307 BC6 digested with Spe I Lane A13 = 5307 BC7 digested with Spe I Lane A14 = 5307 NP2171 × BC5F 3 digested with Spe I Lane A15 = NP2171 × NP2460 digested with Spe I Lane A16 = NP2222 digested with Spe I Lane A17 = NP2460 digested with Spe I Lane A18 = NP2171 digested with Spe I Lane A19 = Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274 digested with Spe I)

### 5307

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- 1 Page 54 of 168 Figure IV

- 1 6 (continued ). Genetic stability Southern blot analysis of 5307 corn with the 6423

- bp full

- length T

- DNA

- specific probe, using restriction enzymes Bst EII, Spe I, and Sma I + Pme I

.

B

. Sma I + Pme I

Lane B1 = molecular weight markers
Lane B2 = blank
Lane B3 = 5307 F 1 digested with Sma I + Pme I Lane B4 = 5307 BC6 digested with Sma I + Pme I Lane B5 = 5307 BC7 digested with Sma I + Pme I
Lane B6 = 5307 NP2171 × BC5F 3 digested with Sma I + Pme I
Lane B7 = NP2171 × NP2460 digested with Sma I + Pme I Lane B8 = NP2222 digested with Sma I + Pme I Lane B9 = NP2460 digested with Sma I + Pme I
Lane B10 = NP2171 digested with Sma I + Pme I Lane B11 = Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274 digested with Sma I + Pme I)

### 5307

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- 1 Page 55 of 168 For Southern blot analysis with genomic DNA digested with Bst EII and probed with the full

length T

- DNA

- specific probe, two hybridization bands of approximately 2.9 kb and 7.2 kb were observed in lanes containing DNA extracted from 5307 F 1 

### , 5307 BC6, 5307 BC7, and 5307

NP2171 × BC5F 3 

### plants

, as expected ( Figure IV

- 16 A , Lanes A 3, A 4, A 5, and A 6) ( Table IV

- 8 ). These hybridization bands were absent in lanes containing DNA extracted from the con trol plants ( Figure IV

- 16 A , Lanes A 7, A 8, A 9, and A 10) and were, therefore, specific to the 5307

corn insert. Three hybridization bands of approximately 11.8 kb, 14 k b, and 20 kb were observed in the lane containing the positive control (16.53 pg of plasmid pSYN12274 DNA digested with Spe I and loaded with DNA extracted from NP2171 × NP2460 plants) ( Figure

IV

- 16 A , Lane A 19).

Additional bands resulting from cross

- hybridization of the ZmUbiInt promoter sequence present on the full length T

- DNA

- specific probe with the endogenous maize polyubiquitin promoter sequence were also detected in all material analyzed. Two hybridization bands of approximately 8.4 kb and 12 kb, corresponding to the hybridization bands observed in lanes containing DNA extracted from NP2460 plants ( Figure IV

- 16 A , Lane A 9) and NP2222 plants ( Figure IV

- 16 A

, Lane A 8), respectively, were observed in lanes containing DNA extracted from 5307 F 1 

### , 5307 BC6, and 5307 BC7 plants ( Figure IV

- 16 A , Lanes A 3, A 4, and A 5 , respectively ). Two hybridization bands of approximately 12 kb and 18 kb, corresponding to the hy bridization bands observed in lanes containing DNA extracted from NP2222 plants ( Figure IV

- 16 A , Lane A 8) and NP2171 plants ( Figure IV

- 16 A , Lane A 10), respectively, were observed in the lane containing
DNA extracted from 5307 NP2171 × BC5F 3 

### plants ( Figure IV

- 16 A , Lane A 6). Finally, two hybridization bands of approximately 8.4 kb and 18 kb, corresponding to the hybridization bands observed in lanes containing DNA extracted from NP2460 plants ( Figure IV

- 16 A , Lane A 9) and NP2171 plants ( Figure IV

- 16 A , Lane A 10), respectively, were observed in lanes containing
DNA extracted from NP2171 × NP2460 plants ( Figure IV

- 16 A , Lane A 7).

For Southern blot analysis with genomic DNA digested with Spe I an d probed with the full length

T

- DNA

- specific probe, two hybridization bands of approximately 2.6 kb and 7.0 kb were observed in lanes containing DNA extracted from 5307 F 1 

### , 5307 BC6, 5307 BC7, and 5307
NP2171 × BC5F 3 

### plants

, as expected ( Figure IV

- 16 A , Lanes A 11, A 12, A 13, and A 14) ( Table

IV

- 8 ). These bands were absent in lanes containing DNA extracted from the control plants ( Figure IV

- 16 A , L anes A 15, A 16, A 17, and A 18) and were, therefore, specific to the 5307 corn insert. Three hybridization bands of approximately 11.8 kb, 14 kb, and 20 kb were observed in the lane containing the positive control (16.53 pg of plasmid pSYN12274 DNA digested with Spe I and loaded with DNA extracted from NP2171 × NP2460 plants) ( Figure IV

- 16 A, Lane

A 19).

Additional bands resulting from cross

- hybridization of the ZmUbiInt promoter sequence present

i n the full length T

- DNA

- specific probe with the endogenous maize polyubiquitin promoter sequence were also detected in all material analyzed . Two hybridization bands of approximately 20 kb and 25 kb, corresponding to the hybridization bands observed in lanes containing DNA

### 5307

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- 1 Page 56 of 168 extracted from NP2460 plants ( Figure IV

- 16 A , Lane A 17) and NP2222 plants ( Figure IV

- 16 A

, Lane A 16), respectively, were observed in lanes containing DNA extracted from 5307 F 1 

### , 5307 BC6, and 5307 BC7 plants ( Figure IV

- 16 A , Lanes A 11, A 12, and A 13). Two hybridization bands of approximately 14 kb and 25 kb, corresponding to the hybridization bands observed in lanes containing DNA extracted from NP2171 plants ( Figure IV

- 16 A , Lane A 18) and NP2222 plants ( Figure IV

- 16 A , Lane A 16), respectively, were observed in the lane containing DNA
extracted from 5307 NP2171 × BC5F 3 

### plants ( Figure IV

- 16 A , Lane A 14).

Finally, two hybridization bands of approximately 14 kb and 20 kb, corresponding to the hybridization bands observed in lanes containing DNA extracted from NP2171 plants ( Figure

IV

- 16 A , Lane A 18) and NP246 0 plants ( Figure IV

- 16 A , Lane A 17), respectively, were observed
in lanes contain ing DNA extracted from NP2171 × NP2460 plants ( Figure IV

- 16 A , Lane A 15).

For Southern blot analysis with genomic DNA digested with Sma I + Pme I and probed with the full length T

- DNA

- specific probe, one hybridization band of approximately 6.3 kb was observed in lanes containing DNA extracted from 5307 F 1 

### , 5307 BC6, 5307 BC7, and 5307 NP2171 ×
BC5F 3 

### plants as expected ( Figure IV

- 16 B , Lanes B 3, B 4, B 5, and B 6) ( Table IV

- 8 ). This hybridization band was absent in lanes containing DNA extracted from the control plants ( Figure

IV

- 16 B , Lanes B 7, B 8, B 9, and B 10) and was, the refore, specific to the 5307 corn insert. One hybridization band of approximately 6.3 kb and a high molecular weight band (greater than 30 kb) was observed in the lane containing the positive control (16.53 pg of plasmid pSYN12274 DNA digested with Sma I + Pme I and loaded with DNA extracted from NP2171 × NP2460 plants) ( Figure IV

- 16 B , Lane B 11). At least one additional band resulting from cross

hybridization of the Zm UbiInt promoter sequence present on the full length T

- DNA

- specific probe with the endogenous maize polyubiquitin promoter sequence was also detected in all material analyzed. One hybridization band of approximately 18 kb, corresponding to the hybridizatio n band observed in the lane containing DNA extracted from NP2222 plants ( Figure

IV

- 16 B , Lane B 8), was observed in lanes containing DNA extracted from 5307 F 1 

### , 5307 BC6

, 5307 BC7 and 5307 NP2171 × BC5F 3 

### plants ( Figure IV

- 16 B , Lanes B 3, B 4, B 5, and B 6). An additional high molecular weight band (greater than 30 kb) was observed in lanes containing DNA extracted from NP2460 plants ( Figure IV

- 16 B , Lane B 9) and NP2171 plants ( Figure

IV

- 16 B , Lane B 10). This faint high molecular weight band (greater than 30 kb) was observed in all lanes containing DNA extracted from plants carrying either the NP2460 polyubiquitin promoter allele (5307 F 1 

### , 5307 BC6, 5307 BC7, and NP2171 × NP2460) ( Figure IV

- 16 B , Lanes

B 3, B 4, B 5, and B 7) and/or the NP2171 polyubiquitin promoter allele (5307 NP2171 × BC5F 3 

### and NP2171 × NP2460) ( Figure IV

- 16 B , Lanes B 6 and B 7).

### 5307

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- 1 Page 57 of 168 Data from these Southern blot analyses demonstrated that the 5307 corn insert integrated into a single locus of the corn genome as only two hybridization bands specific to the 5307 corn insert were observed when genomic DNA was digested with Bst EII and probed with a full length T

DNA

- specific probe and only two hybridization bands specific to the 5307 corn insert were
obser ved when the genomic DNA was digested with Spe I and probed with a full length T

- DNA

specific probe. These hybridization bands were specific to the 5307 corn insert and corresponded to each side of the restriction site of the enzyme used for Southern blot analysis. Additional hybridization bands observed resulted from cross

- hybridization between the ZmUbiInt promoter sequence present on the full length T

- DNA

- specific probe with the maize endogenous polyubiquitin promoter sequence; these bands were consiste nt with the genetic
make

- up of the various generations analyzed. As expected, the 5307 F 1 

### , 5307 BC6, and 5307 BC7 generations carry the maize polyubiquitin promoter allelic forms present in NP2222 and NP2460, the 5307 NP2171 × BC5F 3 

### generation carries the maize polyubiquitin promoter allelic forms present in NP2171 and NP2222, and the control NP2171 × NP2460 material carries the maize polyubiquitin promoter allelic forms present in NP2171 and NP2460.

Data from these Southern blot analyses also demonstrated that a complete copy of the 5307 corn insert integrated into the corn genome as the hybridization band specific to the 5307 corn insert observed when the genomic DNA was digested with Sma I + Pme I was the predicted size. The approximately 18 kb band obser ved on this Southern blot was present in lanes containing DNA extracted from NP2222 plants and all generations carrying the NP2222 polyubiquitin promoter allelic form (5307 F 1 

### , 5307 BC6, 5307 BC7, and 5307 NP2171 × BC5F 3 

### ). The faint and high
molecular wei ght band (greater than 30 kb) observed on this Southern blot was present in lanes containing DNA extracted from NP2460 plants and NP2171 plants and all generations carrying either the NP2460 polyubiquitin promoter allelic form (5307 F 1 

### , 5307 BC6, 5307 BC7, and

NP2171 × NP2460) and/or the NP2171 polyubiquitin promoter allelic form (5307 NP2171 ×
BC5F 3 

### and NP2171 × NP2460). Because no additional bands were observed (other than those
associated with the 5307 corn insert and the corn endogenous sequence), Sout hern blot analyses indicated that there were no extraneous DNA fragments of plasmid pSYN12274 T

- DNA in other regions of the 5307 corn genome. The data depicted in the Southern blot analyses showed that the hybridization bands specific to the insert were i dentical in lanes containing DNA extracted from plants grown from all generations (5307 F 1 

### , 5307 BC6, 5307 BC7, and 5307 NP2171 ×

BC5F 3 

### ); these results indicated that the 5307 corn insert is stably inherited from one generation
to the next. Southern blot analyses demonstrated that 5307 corn contains a single, complete copy of the insert and that there are no extraneous DNA fragments of plasmid pSYN12274 T

- DNA
inserted elsewhere in the 5307 corn genome. Identical hybridization patterns across all generatio ns of 5307 corn analyzed in this study indicates that the insert is stably inherited from one generation to the next.

### 5307

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- 1 Page 58 of 168 IV.E.4.b. Stability of the T

- DNA Using a Plasmid Backbone

- S pecific Probe

The absence of plasmid backbone sequence in 5307 corn was assessed by Southern blot analyses using plasmid pSYN12274 backbone sequence as a probe on Southern blots of DNA subjected to the two restriction enzyme digestion strategies described above. This plasmid backbone

- specific probe contained every base o f the plasmid pSYN12274 backbone present outside of the T

- DNA region. With both restriction enzyme digestion strategies, no hybridization bands were expected.

The Southern blot analyses included genomic DNA from four generations of 5307 corn plants (5307 F 1 

### , 5307 BC6, 5307 BC7, 5307 NP2171 × BC5F 3 

### ) and the corresponding nontransgenic control plants (NP2171 × NP2460, NP2222, NP2460, and NP2171) (see pedigree diagram, Figure

IV

- 4 ). Figure IV

- 17 shows a map of the plasmid pSYN12274 indicating the location of the plasmid pSYN12274 backbone

- specific probe and restriction sites for Bst EII, Spe I, Sma I, and

Pme I. Figure IV

- 18 depicts the results of the corresponding Southern blot analyses, and Table

IV

- 9 provides the expected and observed sizes of the hybridization bands.

For Southern blot analyses with genomic DNA digested with Bst EII and probed with the plasmid pSYN12274 backbone

- specific probe, no hybridiza tion bands were observed in the lanes containing DNA extracted from plants grown from the test and control substances (5307 F 1 

### , 5307 BC6, 5307 BC7, 5307 NP2171 × BC5F 3 

### , NP2171 × NP2460, NP2222, NP2460, and NP2171) ( Figure IV

- 18 A , Lanes 3, 4, 5, 6, 7, 8, 9, and 10) as expected. The positive control (16.53 pg of plasmid pSYN12274 DNA digested with Spe I and loaded with DNA extracted from

NP2171 × NP2460 plants) produced the expected 11.8 kb band ( Figure IV

- 18 A, Lane 19). For Southern blot analyses with genomic DNA digested with Spe I and probed with the plasmid pSYN12274 backbone

- specific probe, no hybridization bands were observed in the test and control substances (5307 F 1 

### , 5307 BC6, 5307 BC7, 5307 NP2171 × BC5F 3 

### , NP2171 × NP 2460, NP2222, NP2460, and NP2171) ( Figure IV

- 18 A, Lanes A 11, A 12, A 13, A 14, A 15, A 16, A 17,
and A 18) as expected. The positive control (16.53 pg of plasmid pSYN12274 DNA digested
with Spe I and loaded with DNA extracted from NP2171 × NP2460 plants) produced the expected 11.8 kb band ( Figure IV

- 18 A, Lane A 19). For Southern blot analyses with genomic DNA digested with Sma I + Pme I and probed with the backbone

- specific probe, no hybridization bands were observed in the test and control substances (5307 F 1 

### , 5307 BC6, 5307 BC7, 5307
NP2171 × BC5F 3 

### , NP2171 × NP2460, NP2222, NP2460, and NP2171) ( Figure IV

- 18 B, Lanes

B 3, B 4, B 5, B 6, B 7, B 8, B 9, and B 10). The positive control (16.53 pg of plasmid pSYN12274 DNA digested with Sma I + Pme I and loaded with DNA extracted from NP2171 × NP2460 plants) produced the expected 4.2 kb and 1.2 kb bands ( Figure IV

- 18 B, Lane B 11).

The data from the three Southern blot analyses demonstrated that all the 5307 corn generations analyzed are free of plasmid pSYN12274 backbone sequence.

### 5307

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- 1 Page 59 of 168 Figure IV

- 17 . Location of the Bst EII, Spe I, Sma I, and Pme I restriction sites and position of the 5312

- bp plasmid backbone

- specific probe in the transformation plasmid pSYN12274.

### 5307

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- 1 Page 60 of 168 Table IV

- 9 . Expected and observed hybridization band sizes in Southern blot analysis of 5307 corn, using a plasmid pSYN12274 backbone

- specific probe and restriction enzymes Bst EII, Spe I, and Sma I + Pme I.

Figure & Lane Number Source of DNA Restriction
enzyme (s) Expected number of

bands Expected band size (kb) Observed band size (kb)
Figure IV

- 18 A, Lane A 3 5307 F 1 Bst EII none none none

Figure IV

- 18 A, Lane A 4 5307 BC6 Bst EII none none none
Figure IV

- 18 A, Lane A 5 5307 BC7 Bst EII none none none
Figure IV

- 18 A, Lane A 6 5307 NP2171 × BC5F 3 Bst EII none none none

Figure IV

- 18 A, Lane A 7 NP2171 × NP2460 Bst EII none none none
Figure IV

- 18 A, Lane A 8 NP2222 Bst EII none none none
Figure IV

- 18 A, Lane A 9 NP2460 Bst EII none none none

Figure IV

- 18 A, Lane A 10 NP2171 Bst EII none none none
Figure IV

- 18 A, Lane A 11 5307 F 1 Spe I none none none
Figure IV

- 18 A, Lane A 12 5307 BC6 Spe I none none none

Figure IV

- 18 A, Lane A 13 5307 BC7 Spe I none none none
Figure IV

- 18 A, Lane A 14 5307 NP2171 × BC5F 3 Spe I none none none

Figure IV

- 18 A, Lane A 15 NP2171 × NP2460 Spe I none none none

Figure IV

- 18 A, Lane A 16 NP2222 Spe I none none none

Figure IV

- 18 A, Lane A 17 NP2460 Spe I none none none

Figure IV

- 18 A, Lane A 18 NP2171 Spe I none none none

Figure IV

- 18 A, Lane A 19 Positive control ( NP2171 × NP2460 and 16.53 pg of pSYN12274) Spe I 1 11.8 ~11.8

Figure IV

- 18 B, Lane B 3 5307 F 1 Sma I + Pme I none none none
Figure IV

- 18 B, Lane B 4 5307 BC6 Sma I + Pme I none none none

Figure IV

- 18 B, Lane B 5 5307 BC7 Sma I + Pme I none none none
Figure IV

- 18 B, Lane B 6 5307 NP2171 × BC5F 3 Sma I + Pme I none none none
Figure IV

- 18 B, Lane B 7 NP2171 × NP2460 SmaI + PmeI none none none

Figure IV

- 18 B, Lane B 8 NP2222 SmaI + PmeI none none none
Figure IV

- 18 B, Lane B 9 NP2460 SmaI + PmeI none none none
Figure IV

- 18 B,Lane B 10 NP2171 SmaI + PmeI none none none

Figure IV

- 18 B,Lane B 11 Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274) SmaI + PmeI 2 4.2
1.2 ~4.2
~1.2

### 5307

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- 1 Page 61 of 168 Figure IV

- 18 . Genetic stability Southern blot analysis of 5307 corn with the 5312

- bp plasmid backbone

- specific probe, using restriction enzymes Bst EII, Spe I, and Sma I + Pme I.

(A) Bst EII or Spe I

Lane A1 = molecular weight markers

Lane A2 = blank

Lane A3 = 5307 F 1 digested with Bst EII Lane A4 = 5307 BC6 digested with Bst EII Lane A5 = 5307 BC7 digested with Bst EII
Lane A6 = 5307 NP2171 × BC5F 3 digested with Bst EII

Lane A7 = NP2171 × NP2460 digested with Bst EII Lane A8 = NP2222 digested with Bst EII Lane A9 = NP2460 digested with Bst EII Lane A10 = NP2171 digested with Bst EII

Lane A11 = 5307 F 1 digested with Spe I Lane A12 = 5307 BC6 digested with Spe I Lane A13 = 5307 BC7 digested with Spe I
Lane A14 = 5307 NP2171 × BC5F 3 digested with Spe I

Lane A15 = NP 2171 × NP2460 digested with Spe I Lane A16 = NP2222 digested with Spe I Lane A17 = NP2460 digested with Spe I Lane A18 = NP2171 digested with Spe I Lane A19 = Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274 digested with Spe I)

### 5307

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- 1 Page 62 of 168 Figure V

- 1 8

. ( Con tinued )

(B) Sma I + Pme I

Lane B1 = molecular weight markers

Lane B2 = blank

Lane B3 = 5307 F 1 digested with Sma I + Pme I Lane B4 = 5307 BC6 digested with Sma I + Pme I Lane B5 = 5307 BC7 digested with Sma I + Pme I

Lane B6 = 5307 NP2171 × BC5F 3 digested with Sma I + Pme I

Lane B7 = NP2171 × NP2460 digested with Sma I + Pme I Lane B8 = NP2222 digested with Sma I + Pme I Lane B9 = NP2460 digested with Sma I + Pme I Lane B10 = NP2171 digested with Sma I + Pme I

Lane B11 = Positive control ( NP2171 × NP2460 and 16.53 pg of pSYN12274 digested with Sma I + Pme I )

### 5307

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- 1 Page 63 of 168 IV.E.5. Mendelia n Inheritance of the DNA Insert

A Chi

- square ( Χ

2 

### ) analysis of ecry3.1Ab and pmi inheritance data over three generations of Event
5307 corn was performed to test the hypothesis that the transgenes are inherited in accordance with the laws of Mendelian genetics. The Chi

- square analysis was based on a comparison of observed and expected gene segregation ratios from each generation. Real

- time PCR analyses (Ingham et al. 2001) were conducted on DNA extracted from leaf tissue of three generations of
5307 corn plants to determine the number of plants that were positive or negative for both the
transgenes ecry3.1Ab and pmi . The plants used for thi s analysis were grown from seeds of the
5307 F 1 

### , 5307 BC6 and 5307 BC7 generations; these genotypes are also indicated in the list of seed materials used ( Table IV

- 2 ) and the pedigree diagram ( Figure IV

- 4 ). During breeding, only progeny that tested positive for the two transgenes were selected for further crossing; thus, the
expect ed inheritance ratio for positive to negative plants was 1:1 in each generation.

Genotypic data generated for the three 5307 corn generations were used to assess the goodness

of

- fit of the observed genotypic ratios to the expected genotypic ratios using Ch i

- square analysis with Yates’ correction factor as in Armitage and Berry (1987).

Χ

2 

### = ∑ [|(observed

– expected)|

- 0.5] 2 

### / expected

All plants tested positive for the control assay targeting the endogenous corn gene adh1, confirming that DNA was present in all real

- time PCR reactions. The expected and observed frequencies of ecry3.1Ab and pmi for each generation are presented in Table IV

- 10 and Table

IV

- 11 . The critical value for rejection of the null hypothesis at the 5% level was 3.84 (Strickberger 1976). The Chi

- square values for each generation tested were found to be less than
3.84. Thi s analysis demonstrates that both the ecry3.1Ab and pmi are inherited in a predictable manner according to Mendelian principles. These results are consistent with the genetic
characterization data for 5307 corn , which indicate stable integration of the T

- DNA at a single locus in the genome.

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- 1 Page 64 of 168 Table IV

- 10

. Observed versus expected frequencies for ecry3.1Ab across generations.

T rait F 1 BC6 BC7

Observed Expected Observed Expected Observed Expected

Positive 44 46 40 45.5 43 45

Negative 48 46 51 45.5 47 45

Total 92 92 91 91 90 90

Χ

2 value 0.098 1.099 0.100



### Table IV

- 11

. Observed versus expected frequencies for pmi across generations.

Trait F 1 BC6 BC7

Observed Expected Observed Expected Observed Expected

Positive 44 46 40 45.5 43 45

Negative 48 46 51 45.5 47 45

Total 92 92 91 91 90 90

Χ

2 value 0.098 1.099 0.100

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- 1 Page 65 of 168 IV.E.6. Flanking Sequence and Analysis to Identify Putative Open Reading Frames

Nucleotide sequences flanking the 5 **′** and 3 **′** ends of the T

- DNA in 5307 corn were screened for similarity with sequences found in public databases. This comparison provided an indication of
whether the 5307 corn T

- DNA inserted into a known plant gene. A sequence simi larity analysis was performed using Basic Local Alignment Search Tool for Nucleotides (BLASTN) software, version 2.2.19 (Altschul et al. 1997) which compared the flanking sequences with nucleotide sequences in the National Center for Biotechnology Informat ion Nucleotide Collection (nr/nt) database (NCBI 2010a). The nr/nt database contains all sequences from the National Institutes of Health genetic sequence database (GenBank®), RefSeq Nucleotides, the European Molecular Biology Laboratory, the DNA Database of Japan, and nucleotide sequences derived from the
three

- dimensional structures from the Brookhaven Protein Data Bank. At the time the BLASTN analysis was run (May 7, 2010), the nr/nt database contained over 11 million sequences. Details of the paramet ers used in the BLASTN analysis are provided in Appendix A

.
BLASTN analysis was conducted using the 1000 bp of corn genomic sequence flanking the 5ʹ

end of the 5307 T

- DNA insert as the query sequence. The search identified all alignments to sequences in the nr/nt database with search results yielding an E

- value of 10 or lower. This analysis was repeated using the 1000 bp of corn genomic sequence flanking the 3ʹ end of the

5307 T

- DNA as the query sequence. The E

- value, or “expectation value,” is a measu re of the probability that matches between sequences occurred by chance. Search results involving comparisons between nucleotide sequences with highly similar sequences yield E

- values approaching zero. The probability that sequence similarities occurred by chance increases with higher E

- values (Ponting 2001).

Using the results of each BLASTN query, the 20 alignments with the lowest E

- values were examined for this analysis. The corn genomic sequences flanking the 5ʹ region and the 3ʹ region

of the 5307 corn insert aligned to multiple bacterial artificial chromosome (BAC) corn sequences that were annotated as various corn chromosome numbers ( i.e., corn chromosome numbers 4, 5, 6, 8, 9, and 10). This suggests that the 5307 corn insert is located in a repetitive region of the

corn genome. Both flanking sequences aligned to NCBI nucleotide database accession numbers AY530950.1, AY530951.1 and AY530952.1, which were annotated as containing gene sequences. However, the regions of AY530950.1, AY530951.1, and AY530952.1 that were
similar to the corn genomic sequences flanking the 5ʹ region and the 3ʹ region of the 5307 insert were not annotated as a gene. Both flanking sequences also aligned to accession numbers AY664416.1, AY664413 .1, AY664419.1, and AY664415.1. (However, alignments of corn genomic sequence flanking the 3′ region of the 5307 insert to accession numbers AY664419.1 and AY664415.1 were not among those with the 20 highest similarity scores.) These accession
numbers ar e all annotated as containing sequences of various genes and repeat regions. However, the regions of AY664416.1, AY664413.1, AY664419.1, and AY664415.1 that were
similar to the corn genomic sequences flanking the 5ʹ region and the 3ʹ region of the 5307 in sert were not annotated as a gene. The corn genomic sequences flanking the 5ʹ region and the 3ʹ

### 5307

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- 1 Page 66 of 168 region of the 5307 insert aligned to a region of AY664413.1 that was annotated as repeat region Giepumx19002_LTR retrotransposon. Additionally, short regions of both flanking sequences ( i.e., ~20 to 40 base pairs in length) aligned to multiple regions of multiple sequences in the database. Some of these regions were also annotated as repeat regions in accession numbers AY664416.1, AY664413.1, AY664419.1, and A Y664415.1. These repeat regions were also associated with retrotransposons. Approximately 49 to 78% of the corn genome is suggested to be comprised of retrotransposons (San Miguel et al. 1998). However, only approximately 5% of the retrotransposon seque nces in the corn genome are predicted to produce proteins (Meyers et
al. 2001). Because retrotranposons are repetitive, the repeat regions that align to corn genomic sequences flanking the 5 ʹ region and the 3ʹ region of the 5307 insert are likely to be re peated
elsewhere in the corn genome.

BLASTN analysis indicated that the corn genomic sequence flanking the 3′ region of the 5307

corn insert is also similar to accession number EU954153.1, which was annotated as a hypothetical protein mRNA sequence from Ze a mays . The region of the flanking sequence that is similar to EU954153.1 was located 109 bp downstream of the genome

- to

- insert junction. BLASTN analysis also indicated that the corn genomic sequence flanking the 3′ region of the

5307 corn insert is simi lar to accession number EZ054274.1, annotated as mRNA sequence from Zea mays . The region of the flanking sequence that is similar to EZ054274.1 was located 376 bp downstream of the genome

- to

- insert junction. The corn genomic sequence flanking the 5ʹ regi on of the 5307 corn insert did not align to sequence EU954153.1 or EZ054274.1. None of the alignments retrieved indicates that a known endogenous corn gene was interrupted by the Event
5307 T

- DNA insert.

The Vector NTI AdvanceTM program, version 10.3.0, wa s used to identify any putative open reading frames (ORFs) that span the junctions between the corn genomic sequence and the 5307

corn T

- DNA insert. For this analysis, putative ORFs were defined as DNA sequences in any reading frame that are contained bet ween a putative start codon (ATG) and a putative stop codon (TAG, TAA, or TGA), and have a minimum translation size of 30 amino acids. This analysis identified one putative ORF of 243 bp; it spanned the junction between the corn genomic sequence and the 3 ʹ region of the 5307 corn insert.

Bioinformatic analyses demonstrated that the translated 81

- amino

- acid sequence of this putative ORF had no significant sequence similarity to known or putative toxins or allergens. The
methods used for these toxin and al lergen similarity searches are described in Appendix A

.

### 5307

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- 1 Page 67 of 168 IV.E.7. Summary of the Genetic Characterization of Event 5307 Corn

Southern blot analyses demonstrate that Event 5307 corn (1) contains, at a single locus within the

corn genome, a single copy each of the gene ecry3.1Ab , its CMP promoter sequence, the marker

gene pmi , its ZmUbiInt promoter sequence, and the two expected copies of the NOS terminator sequence, one NOS terminator sequence regulating ecry3.1Ab and one NOS term inator sequence regulating pmi ; (2) does not contain any extraneous DNA fragments of these functional elements
inserted elsewhere in the corn genome; (3) and does not contain plasmid backbone sequence from the transformation plasmid, pSYN12274.

Nucleotide sequence analysis of the entire Event 5307 insert confirms that the insert is intact and that the organization of the functional elements within the insert is identical to their organization within pSYN12274. One nucleotide change compared to the sequence of pSYN12274 was identified 48 base pairs (bp) upstream of the CMP promoter in a noncoding region of the insert
in Event 5307 corn . However, this nucleotide change had no effect on the functionality of the insert. Additionally, the analysis indicates th at some truncation of the nucleotide sequence
occurred at the 5′ and 3′ ends of the T

- DNA during the transformation process that resulted in
Event 5307 corn ; such truncation occurs commonly in transformation via Agrobacterium . The entire RB and three bp o f noncoding sequence at the 5′ end of the insert, and eight bp of the LB were truncated; however, these deletions had no effect on the functionality of the insert.

Sequence analysis of the Event 5307 insertion site demonstrates that 33 bp of corn genomic sequence were deleted when the Event 5307 insert integrated into the corn genome. BLASTN analyses comparing the corn genomic sequence flanking the Event 5307 insert to sequences in public databases indicate that the insert does not disrupt any known endog enous corn gene.

A putative 243

- bp novel open reading frame (ORF) spanning the junction between corn genomic sequence and the 3′ region of the 5307 corn insert was identified. The translated 81

- amino

- acid sequence encoded by the putative ORF was screene d for amino acid sequence similarity to known or putative allergens or toxins. Comparisons to the FARRP AllergenOnline database indicate that the amino acid translation of the identified putative ORF shows no biologically relevant amino acid sequence simi larity to any known or putative protein allergens. Additionally, the results of a comprehensive amino acid similarity search of the NCBI Entrez® Protein Database indicate that the amino acid translation of the identified putative ORF shows no biologically relevant amino acid sequence similarity to any known or putative toxins. These data collectively demonstrate that there are no deleterious changes in the 5307 corn genome as a result of the T

- DNA insertion.

The ecry3.1Ab and pmi segregation ratios over several generations of Event 5307 corn plants are consistent with linkage of these transgenes at a single locus in the corn nuclear genome. These data and the results from Southern blot analyses of multiple generations of Event 5307 corn indicate that the transgenic locus is stably inherited during conventional breeding.

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- 1 Page 68 of 168 

## V. Absence of Genes That Encode Resistance to Antibiotics

As indicated in Figure IV

- 1 and described i n Table IV

- 1 , the intended T

- DNA between the left and right borders of plasmid pSYN12257 does not contain any sequences that encode antibiotic
resistance markers. Therefore, no genes encoding resistance to antibiotics are predicted to occur
in Event 5307 corn. Although the backbone DNA of the pSYN12257 transformation plasmid (i.e., the region not included between the left and right borders) contains the antibiotic resistance

gene spec ( see Table IV

- 1 ), it is predicted that this gene would not have been transferred to the

co rn genome during transformation. Southern blot an alyses ( Figure IV

- 18 ) described in Part
IV.E.3.b

. demonstrated no hybridization of Event 5307 corn DNA with the pSYN1227 4
backbone

- specific probe ( Figure IV

- 17 ) . This confirms that, as expected, no spec DNA sequences were incorporated into Event 5307 corn DNA.

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- 1 Page 69 of 168 

## VI. Safety Assessment of the Introduced eCry3.1Ab and PMI Proteins

This section provides a summary of the safety assessment for the eCry3.1Ab and PMI proteins produced in Event 5307 corn . This section includes (1) information about the identity and function of each protein, (2) i nformation characterizing the plant

- produced proteins and demonstrating equivalence to microbially produced test substances used in safety studies, (3) information about the toxicity of each protein, (4) information about the allergenic potential of each p rotein, (5) information about history of safe use, and (6) information about eCry3.1Ab and

PMI protein levels in plant tissues and exposure potential.

VI.A. Identity and Function of the eCry3.1Ab Protein in Event 5307 Corn

The eCry3.1Ab protein produced in E vent 5307 corn is a chimeric Cry protein comprised largely of portions of modified Cry3A (mCry3A) protein and Cry1Ab protein , which are derived
from Bacillus thuringiensis ( B.t .) (see Table IV

- 1 ). As illustrated in Figure IV

- 2 , the coleopteran

active e Cry3.1Ab pro tein was produced by exchange of the variable regions (V1 to V6) between mCry3A (a coleopteran

- active protein) and Cry1Ab (a lepidopteran

- active protein )

. At the N

terminus of eCry3.1Ab are 22 amino acid residues that are not derived from either mCry3A or Cry1Ab, per se, but were the result of a PCR

- induced mutation that resulted in a reading

- frame shift in the nucleotide sequence of a portion of the gene mcry3A . The next 459 residues are identical to those of mCry3A, followed by 17 2 residues of Cry1Ab at the C

- terminus . The eCry3.1Ab protein is 65 3 amino acid residues in size and has a molecular weight of approximately 73.7 kDa.

The eCry3.1Ab protein i s insecticidally active against the larvae of 

### certain coleopteran pests in 

### the family Chrysomelidae, including w estern corn rootworm ( 

### Diabrotica virgifera virgifera ), northern corn rootworm ( D

. longicornis barberi ), Mexican corn rootworm ( D. virgifera zeae ), and Colorado potato beetle ( Leptinotarsa decemlineata )

. 

### The rootworm species are all pests of corn, whereas Colorado potato beetle is a pest of other crops. The eCry3.1Ab protein has not demonstrated insecticidal activity among Coleoptera outside the family Chrysomelidae, and no biological activity has been observed in tests of mul tiple other organisms, including lepidopteran insects, other nontarget insects

, and avian, mammalian, and aquatic species.

The spectrum of activity of a ny specific insecticid al Cry protein is quite narrow , and is usually restricted to a few related species . One given Cry protein is usually active against only a few species within any phylogenic Order. The specificity of each B.t

. Cry protein ( **δ**

- endotoxin ) is the result of the efficiency of the various steps involved in producing an active protein toxin and its subsequent interaction with the epithelial cells in the insect midgut. To be insecticidal, most

known B.t

. **δ**

- endotoxins must: (1) be ingested by t he insect and solubilized in the insect gut, (2) be activated by specific proteolytic cleavages, (3) bind to specific receptors on the surface of the insect midgut and (4) form ion channels. The completion of all these four processes results in disruptio n of the normal function of the midgut leading to the death of the insect. The

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- 1 Page 70 of 168 eCry3.1Ab protein ex hibits the same behavior as other coleopteran active B.t

. **δ**

- endotoxins including alkaline solubility, cleavage by chymotrypsin, specificity of brush border membrane binding and ion channel formation. The mode of action of eCry3.1Ab

, like that of other Cry proteins

, is highly specific to insects and is not relevant to mammalian species.

VI.B. Production of eCry3.1Ab Test Substance for Safety Studies

It was not feas ible to extract sufficient eCry3.1Ab protein from Event 5307 corn plants to conduct safety studies, because such studies require relatively large amounts of purified test protein. Syngenta undertook extensive efforts to produc e the identical eCry3.1Ab pro tein
present in 5307 plants via a microbial production system, for use as a test substance. However, these efforts did not yield sufficient quantities of bioactive protein, and it proved necessary to produce eCry3.1Ab with an N

- terminal tag of histidine r esidues as a purification aid. This is a common procedure for protein synthesis and purification. Except for the N

- terminal tag consisting of one methionine and six histidine residues, the eCry3.1Ab produced in recombinant
E. coli has the identical amino acid sequence as eCry3.1Ab produced in 5307 plants. The resulting lyophilized test substance was designated ECRY3.1AB

- 0208; it was determined to be 89.6% pure eCry3.1Ab by weight and the intact mass of the eCry3.1Ab protein, as measured by
mass spectrome try, was 74.8 kDa. The methods used to produce and characterize this test
substance are described in Appendix B. Studies comparing this microbially produced eCry3.1Ab test substance to eCry3A.1Ab from 5307 plants are described below , and demonstrate that microbially produced eCry3.1Ab is a suitable surrogate for eCry3.1Ab in 5307 plants

.

VI.C. Characterization of the Plant

- and Escherichia coli

- produced eCry3.1Ab
Proteins

The amino acid sequence of the plant

- produced eCry3.1Ab can be deduced from the observation that the nucleotide sequence of ecry3.1Ab recovered from 5307 plants is identical to the intended sequence (see Part IV.E.1

. DNA Insert Sequencing)

. Nevertheless, a series of analytic al
methods were used to characterize the eCry3.1Ab protein produced in Event 5307 corn and to
assess whether the microbially produced e Cry3.1A b test substance , ECRY3.1AB

- 0208, is a suitable surrogate for use in food and feed safety studies. A description of the results of the protein characterization is summarized below. A detailed description of the methods and results for the protein charact erization can be found in Appendix B.

The identities of the plant

- produced and microbially produced eCry3.1Ab proteins were confirmed by immunoreactivity, apparent molecular weight, glycosylation status, peptide mass mapping analysis, and N

- terminal amino acid sequence. Both proteins were shown to have the predicted molecular weight and cross

- reacted with the same antibodies , as shown by Western blot analysis. There was no evidence of post

- translational glycosylation of eCry3.1Ab from the plant

- produced protein or the microbially produced protein. Peptide mass mapping identified 76% and 87% of the predicted eCry3.1Ab amino acid sequence for the plant

- produced protein

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- 1 Page 71 of 168 and microbially produced protein, respectively, confirming the identity of the insecticidal protein
from both sources.

In addition to the biochemical analyses, the eCry3.1Ab proteins from both sources were evaluated for biological activity

. In bioassays against first

- instar Colorado potato beetle

, 1 

### both the plant

- produced protein and microbially produced protein were insecticidally active

, with comparable LC 50 

### values ( Table VI

- 1 )

.

Table VI

- 1

. Comparison of Colorado potato beetle larvae LC 50 

### values for eCry3.1Ab from 5307 plants and microbial eCry3.1Ab test substance.

First instars were exposed to diets containing eCry3.1Ab from 5307 plants or test substance ECRY3.1AB

- 0208. Leaf extract from control plants was included in the bioassays with ECRY3.1AB

- 0208 to control for leaf matrix effects.

Replicate LP5307 a LP

- NEG b + ECRY3.1AB

- 0208
μg eCry3.1Ab/ml insect diet

LC 50 95% CI LC 50 95% CI

#1 1.316 0.631

- 3.187 1.780 0.924

- 3.190

#2 1.669 0.875

- 3.279 1.113 0.113

- 4.001

#3 2.888 1.765

- 4.655 3.226 1.955

- 5.134

a Leaf protein extract of 5307 plants containing eCry3.1Ab

. b Leaf protein extract of control plants, tested in combination with eCry3.1Ab from test substance ECRY3.1AB

- 0208 to control for effects of the leaf protein matrix

.



### Based on these results, the identitie s of the plant

- and microbially produced eCry3.1Ab proteins
have been verified and it can be concluded that eCry3.1Ab produced in recombinant E. coli and

Event 5307 corn are biochemically and functionally equivalent. Therefore, the microbially

produced test substance is a suitable surrogate for eCry3.1Ab expressed in Event 5307 corn

.

1 Although Colorado potato beetle larvae are not corn pests, they are sensitive to eCry3.1Ab, and are members of the Chrysomelid ae family of Coleoptera, the same family that includes corn rootworms. Colorado potato beetle larvae were used in these bioassays because they are more amenable than rootworm larvae to laboratory testing.

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- 1 Page 72 of 168 VI.D. Assessment of eCry3.1Ab Protein Toxicity

VI.D.1. Assessment of eCry3.1Ab Amino Acid Sequence Similarity to Known Toxins

An a mino acid sequence comparison between a novel protein and known protein toxins can be a useful predictor of toxicity. To determine whether the eCry3.1Ab protein had any significant amino acid sequence similarity to proteins identified as toxins, the protein sequence (Entrez Accession No. ADC30135) was systematically compared to the latest posting of the Nationa l Center for Biotechnology Information (NCBI 2010 b ) Entrez Protein Database. The Basic Local Alignment Search Tool for Proteins program (BLASTP) was used to search the NCBI database. This process identified (1) whether any proteins in the database showed significant similarity to the eCry3.1Ab amino acid sequence ( i.e ., alignments with BLASTP Expectation values [ E

- values] below an established threshold), indicating that the amino acid sequence might be closely related to the eCry3.1Ab amino acid sequence , and (2) whether any proteins showing sequence similarity to the eCry3.1Ab amino acid sequence were known or putative toxins.

Most proteins are modular in nature and contain repeating functional domains within the protein. Similarly, functional domains a re conserved across different proteins from different species. The BLASTP algorithm is optimized to identify these domains or shorter sequence simil arities present within the full

- length query sequence; as a result, this approach detects more similarities than would a search started by aligning two sequences over their entire length. The search conservatively identified all sequences in the database with search results yielding an E

- value of
10 or lower.

A threshold below which similarity to the query sequence is considered significant, and not the result of random similarity in amino acid composition, is required for meaningful analysis of database alignments. To assess the signif icance of sequence similarity to the eCry3.1Ab amino acid sequence, additional searches were conducted with shuffled versions of the eCry3.1Ab amino acid sequence. Five shuffled sequences were created through random shuffling of the eCry3.1Ab amino acid s equence. The resulting shuffled sequences all had the same amino acid composition as eCry3.1Ab ( i.e., the same number of residues of each specific amino acid), but were unlikely to have amino acid sequences similar to those of either eCry3.1Ab or other

pr oteins found in the NCBI Entrez® Protein Database. Searches using these shuffled sequences provided an estimate of the background incidence of alignments that would be expected for any sequence with the same amino acid composition as eCry3.1Ab. Searches with the five shuffled versions of the eCry3.1Ab amino acid sequence identified alignments yielding E

- values that ranged from 0.32 to 2.1; therefore, the threshold E

- value for significant amino acid sequence similarity to eCry3.1Ab was considered to be 0.3 2. Proteins with significant amino acid

sequence similarity to eCry3.1Ab ( i.e., with E

- values less than 0.32) were evaluated for identity, source and biological function , if known . The NCBI Entrez® Protein Database records and supporting literature were accessed, if needed, using the Entrez® Accession Numbers.

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- 1 Page 73 of 168 The NCBI Entrez® Protein Database search identified 495 sequences with significant similarity to the eCry3.1Ab amino acid sequence ( i.e., E

- values less than 0.32). Of these, 449 were
identified as known or putative delta

- endotoxin proteins (also known as Cry proteins or insecticidal crystal proteins) from 14 species or synthetic gene constructs. The E

- values for alignments between these sequences and the eCry3.1Ab amino acid sequence ranged from 0 .0 to 0.24. An additional 31 sequences were identified as hypothetical proteins of unspecified function from six species. Two additional entries were similar to a twin arginine translocation pathway signal from Vibrio angustum and Ralstonia eutropha (Ber ks et al. 2000). These sequences were not known or putative toxins.

Additionally, 13 proteins or hypothetical proteins from Bacillus thuringiensis were described in the database supporting information as being part of the parasporin family of proteins. T he term parasporin is defined as “ Bacillus thuringiensis and related bacterial parasporal proteins that are

non

- hemolytic but capable of preferentially killing cancer cells” (Yamashita et al. 2005). A
classification and nomenclature scheme similar to that of Cry proteins has now been developed for parasporins (Ohba et al. 2006); however, several proteins described in this search still retain their “Cry” nomenclature names. The parasporins have not been shown to have insecticidal activity to date. They ha ve been shown to have strong in vitro cytocidal activity against sensitive cells derived from cancer cell lines, but low or no toxicity to other normal human or other mammalian cell lines (Mizuki et al. 2000; Katayama et al. 2005; Yamashita et al. 2005). There are no published reports of in vivo parasporin toxicity to humans or other mammals.

The results of a comprehensive amino acid similarity search of the NCBI Entrez® Protein Database support the conclusion that the eCry3.1Ab amino acid sequence shows n o significant similarity with any known or putative toxins other than known or putative delta

- endotoxin proteins (also described as Cry proteins or insecticidal crystal proteins).

VI.D.2. Acute Oral Tox icity Study of eCry3.1Ab

The potential toxicity of the eCry3.1Ab protein was eval u ated in an acute oral toxicity s tudy in the mouse conducted by WIL Research Laboratories, LLC (Ashland, OH, USA). The microbially produced, lyophilized test substance ECRY3.1AB

- 0208 containing eCry3.1Ab protein (89.6% purity

, w/ w ; see Part VI.B ) was administered as a single oral dose via gavage to groups of five male and five female Crl:CD

- 1 (ICR) mice at 0 or 2000 mg eCry3.1Ab/ kg body

we ight. The dosing vehicle, 0.5% (w/v) aqueous carboxymethylcellulose

, was administered to the control group. The dosing formulations were administered at a dose volume of 10 mL/kg for all groups. All animals were euthanized after a 14

- day observation per iod following dosing.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed at the time of dosing, approximately 1

- 2 hours post

- dosing and approximately 4

- 5 hours post

- dosing on the day of dose administr ation (study day 0) and once daily on nondosing days (study days 1

- 13). Detailed physical examinations were performed weekly. Individual

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- 1 Page 74 of 168 body weights and food consumption were recorded daily during the study. Complete necropsies
were conducted on all an imals, and selected tissues were examined microscopically from all
animals.

All animals survived the 14

- day observation period to the scheduled necropsy. There were no observed clinical signs of distress or impairment. There were no test

- substance

- relate d clinical observations. All clinical findings in the test

- substance

- treated groups were limited to single animals and/or were common findings for laboratory mice of this age and strain. At the end of
the 14

- day observation period, a complete necropsy wa s conducted on all animals. The necropsies included, but were not limited to, e xamination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities, including viscera. Histopathology
evaluations were conducted on all the gastrointestinal tissues of all the animals , as well as the spleen, thymus, mandibular and mesenteric lymph nodes, and any gross lesions

.
Body weights and food consumption were unaffected by test substance administration. Statistically significantly higher mean body weight gain was noted for the 2000 mg/kg group males on study days 3

- 4 and 13

- 14 compared to the control group. Statistically significantly lower mean body weight gain was noted for the 2000 mg/kg females on study days 2

- 3 and 9

- 10.
The se differences in body weight gain were considered incidental and not related to test substance administration because the magnitude of the change was very small.

Statistically significantly higher mean food consumption was noted for the 2000 mg/kg group males on study days 8

- 9 and 12

- 13. This difference in food consumption was considered incidental and not related to test substance administration because the magnitude of the change was small and no other changes in food consumption were noted for the re maining study
intervals.

Review of the gross necropsy observations revealed no observations that were considered to be
associated with administration of the test substance. Occasional histologic changes were noted in both treated and control animals; thes e were all considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance.
There was no test

- substance

- related alteration in the prevalence, severity

, or histologic character
of those incidental tissue alterations.

In summary, test substance ECRY3.1AB

- 0208, containing the active ingredient eCry3.1Ab protein (89.6% purity w/w), administered as a single oral dose at 2000 mg eCry3.1Ab/ kg body weight followed by a 14

- day nondosing observation period was well tolerated in male and
female CD

- 1 mice. No toxicity was observed in mice given an acute oral gavage dose of 2000 mg eCry3.1Ab/kg body weight. Based on the results of this study, the estimated no observ able
adverse effect level (NOAEL) for eCry3.1Ab protein in male and female mice was > 2000 mg/kg body weight.

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- 1 Page 75 of 168 VI.D.3. Conclusion of the eCry3.1Ab Toxicity Assessment

The eCry3.1Ab protein does not share significant amino acid sequence similarity to known protein toxins and no adverse te st

- substance

- related effects were observed in mice administered a single high dose (2000 mg/kg body weight) of eCry3.1Ab protein

. T herefore, eCry3.1Ab is
considered nontoxic.

VI.E. Assessment of eCry3.1 Ab Allergenic Potential

VI.E.1. Source of the eCry3.1Ab Protein

The eCry3.1Ab protein is a synthetic protein (chimera) containing regions from both mCry3A and Cry1Ab. The source of the native Cry3A and Cry1Ab proteins is Bacillus thuringiensis (see
Table IV

- 1 )

. Bacteria have no history of allergenicity (Taylor and Hefle 2001; FAO/WHO 2001). Additionally, during decades of widespread use of Bacillus thuringiensis formulations as insecticides , the re have been no confirmed reports of immediate or delayed allergic reactions to the Cry protein components of these products, despite significant oral, dermal and inhalation

exposure (EPA 2010 ). Therefore, eCry3.1Ab is not derived from a source known to p roduce allergenic proteins.

VI.E.2. Assessment of eCry3.1Ab Amino Acid Sequence Similarity to Known Allerge ns

Amino acid sequence comparisons between novel proteins and known allergens are part of the weight

- of

- evidence approach to assessing potential allergenic ity. For example, in combination with other supporting data, the presence of significant sequence amino acid similarity to known allergens could indicate that the novel protein might elicit an allergic cross

- reaction in sensitized
individuals.

The eCry3.1 Ab amino acid sequence was systematically compared to the protein sequences in the Food Allergy Research and Resource Program (FARRP) AllergenOnline database, version 10.0 (FARRP 2010), located online at www.allergenonline.org . The FARRP AllergenOnline database contains the amino acid sequences of known and putative protein allergens. It is a curated, peer

- reviewed database containing proteins identified as food allergens, respiratory allergens, allergeni c venom proteins, contact allergens, gliadins, and glutenins. Entries were compiled primarily from searches of publicly available protein databases using the National Center for Biotechnology Information (NCBI) Entrez® search and retrieval system. Protei ns are classified as known or putative allergens according to predetermined criteria set by the FARRP expert review panel.

The FAARP AllergenOnline database (2010) contained 1,471 non

- redundant entries at the time the searches were performed. Sequential 80

- amino

- acid peptides of the eCry3.1Ab sequence were compared to the protein sequences in the FAARP Allergen Online database using the

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- 1 Page 76 of 168 FASTA search algorit hm 1 

### (Pearson and Lipman 1988). Any 80

- amino

- acid peptide in the query sequence having greater than 35% amino acid identity to an allergen sequence would be considered to have significant identity to the allergen sequence, in accordance with the recommendations of the Codex Alimentarius Commission ( Codex 2009). The results of this analysis revealed that there was no significant identity between any of the sequential 80

- amino

acid peptides of eCry3.1Ab and any entry in the FAARP AllergenOnline database. Therefore, eCry3.1Ab does not share overall sequence similarity with any known allergenic protein.

T he eCry3.1Ab prot ein sequence was also examined for matches of eight contiguous amino acids (Hileman et al. 2002) between the eCry3.1Ab sequence and the allergen sequences in the FAARP AllergenOnline database to screen for short, local regions of amino aci d identity that might indicate the presence of common T

- cell binding epitopes. There were no matches of eight contiguous a mino acids between eCry3.1Ab and any proteins in the FAARP AllergenOnline
database. These results support the conclusion that eCry3. 1Ab shows no biologically relevant amino acid sequence similarity to any known or putative protein allergens.

VI.E.3. In vitro Pepsin Digestibility of eCry3.1Ab

A study was conducted to assess the in vitro digestibility of eCry3.1Ab in simulated mammalian gastric fluid (SG F). Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS

- PAGE), Western blot and densitometry analyse s were used to evaluate the in vitro digestibility of eCry3.1Ab in S G F over a 15

- minute time course at 37°C ( Figu re VI

- 1 )

.
The eCry3.1Ab protein degraded rapidly upon exposure to the pepsin enzyme in SGF. Intact eCry3.1Ab (molecular weight 74.8 kDa) was readily digested in less than 30 seconds

, as

as sessed by SDS

- PAGE analysis. A very faint band corresponding to intact eCry3.1Ab (74.8 kDa) was visible following incubation in SGF for 15 seconds (Lane 8). However, this band was no longer visible in the digestibility assay sample taken following incubation in SGF for 30
seconds (La ne 9). Two very faint, diffuse bands with molecular weight s of approximately 4 kDa and 5 kDa, respectively, were visible after incubation in SGF for 15 seconds (Lane 8). These
two bands diminished in in tensity over the time course and were no longer detectable after
incubation in SGF for 10 minutes (Lane 1 8 ).

The eCry3.1Ab protein incubated in SGF without pepsin (eCry3.1Ab control) showed no significant degradation over 15 minutes (Lanes 4 and 5), which indicates that the hydrolysis of eCry3.1Ab, seen in the SGF samples (Lanes 7 through 19), can be attributed to pepsin. Similar
band intensities were visualized for the time zero digestibility assay sample (Lane 7) and the eCry3.1Ab time zero control (Lane 4), confirming that equal amounts of eCry3.1Ab were

1 FASTA version 3.45 was used with the following parameters: extension penalty of 2 and gap creation penalty of 12. The scoring matrix was the Blocks Substitution Matrix 50 (BLOSUM50).

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- 1 Page 77 of 168 applied to the SDS

- PAGE. The lowest amount of eCry3.1Ab visible on the gel (Lanes 21 through 25) was 0.025 μ g (Lane 22). Therefore, t he limit of detection ( LOD ) of eCry3.1Ab for
the SDS

- PAGE used in th is study was determined to be 0.025 μg.

Fur th er analysis of the eCry3.1Ab protein bands in the SDS

- PAGE gel by densitometry revealed
that 3% and 0% of the eCry3.1Ab remained after incubation in SGF for 15 and 30 seconds, respectively ( Table VI

- 2 )

.

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- 1 Page 78 of 168 Figure VI

- 1

. SDS

- PAGE analysis of eCry3.1Ab following digestion in simulated gastric fluid

. The molecular weight of eCry3.1Ab is 74.8 kDa. The molecular weight of pepsin is 34.6 kDa 1

.

Lane 1: molecular weight standard
Lane 2: SGF control

– time zero
Lane 3: SGF control

– 15 minutes
Lane 4: eCry3.1Ab control (eCry3.1Ab in SGF without pepsin)

– time zero
Lane 5: eCry3.1Ab control (eCry3.1Ab in SGF without pepsin)

– 15 minutes
Lane 6: molecular weight standard
Lane 7: in vitro digestibility assay

- time zero 2

Lane 8: in vitro digestibility assay

- 15 seconds 2, 3 , 4

Lane 9: in vitro digestibility assay

- 30 seconds 2

Lane 10: in vitro digestibility assay

- 45 seconds 2

Lane 11: in vitro digestibility assay

- 1 minute 2

Lane 12: in vitro digestibility assay

- 1 minute 15 seconds 2

Lane 13: in vitro digestibility assay

- 1 minute 30 sec onds 2

Lane 14: in vitro digestibility assay

- 1 minute 45 seconds 2

Lane 15: in vitro digestibility assay

– 2 minutes 2

Lane 16: in vitro digestibility assay

– 3 minutes 2

Lane 17: in vitro digestibility assay

– 5 minutes 2

Lane 18: in vitro digestibility assay

– 10 minutes 2

Lane 19: in vitro digestibility assay

– 15 minutes 2 Lane 20: molecular weight standard Lane 21: 0.1 μg eCry3.1Ab for LOD determination Lane 22: 0.025 μg eCry3.1Ab for LOD determination 4 Lane 23: 0.0063 μg eCry3.1Ab for LOD determi nation Lane 24: 0.0016 μg eCry3.1Ab for LOD determination Lane 25: 0.0004 μg eCry3.1Ab for LOD determination

1 The 34.6 kDa pepsin band showed slightly lower mobility and therefore appeared to have a higher apparent molecular weight (MW) when compared to the MW standards on the gel. The difference between the expected and observed MWs can be explained by the limitations of SDS

- PAGE for accurate MW determination. Dube and Flynn (1988) reviewed the reliability of SDS

- PAGE for M W determinations and concluded that the apparent MW of a protein by this method is typically within 10% of its true MW. This depends greatly on the similarity between the properties of the protein of interest and the proteins in th e standard set (Sadeghi et al

. 2003).

2 The lanes selected for the eCry3.1Ab band detection and quantification by densitometry analysis ( Table VI

- 2 ).
3 Due to the resolution limits of the sca nner and the printer, the very faint bands (approximately 4 kDa and 5 kDa) visible on the actual gel might not be visible on the printed image that appears here.
4 Due to the resolution limits of the scanner and the printer, the very faint band (74.8 kDa) visible on the actual gel might not be visible on the printed image that appears here. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Mol. Wt [kDa]

188

----

62

---- 49

---- 38

---- 28

----

17

---- 14

----

6

----

3

---- 98

----

eCry3.1Ab

Pepsin eCry3.1Ab

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- 1 Page 79 of 168 Table VI

- 2

. The eCry3.1Ab remaining at each time point during exposure to simulated gastric fluid as determined by densitometry analysis

.

Lane in 

### Figure VI

- 1 Time point Densitometric
volume Percent (%) of eCry3.1Ab remaining 1 

### Amount (μg) of eCry3.1Ab remaining 2



### 7 Time zero 373.9 100 0.50

8 15 seconds 12.3 3 0.02

9 30 seconds 0.7 0 0

10 45 seconds 1.0 0 0

11 1 minute 0.6 0 0

12 1 minute

, 15 seconds 1.1 0 0

13 1 minute

, 30 seconds 0.7 0 0

14 1 minute

, 45 seconds 1.2 0 0

15 2 minutes 0.7 0 0

16 3 minutes 0.8 0 0

17 5 minutes 0.6 0 0

18 10 minutes 1.4 0 0

19 15 minutes 0.9 0 0

1 Percent (%) of eCry3.1Ab remaining relative to the amount of eCry3.1Ab at time zero as determined by densitometry.

2 The amount of eCry3.1Ab at time zero was equivalent to 0.5 μg. The amount of remaining eCry3.1Ab at each time point was calculated as follows: amount of eCry 3

. 1 Ab remaining = amount of eCry 3

. 1 Ab at time zero ( 0

. 5 μg ) × percent (%) of eCry 3

. 1 Ab remaining

. This calculation was based on the fact that equivalent volumes of each SGF digestibility assay sample were subjected to SDS

- PAGE.

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- 1 Page 80 of 168 The Western blot analy sis results ( Figure VI

- 2 ) confirm that eC ry 3

. 1Ab is readily digested in
SGF in less than 30 seconds. After incubation of eCry3.1Ab in SGF for 30 seconds (Lane 9), no protein bands representing either intact eCry3.1Ab or eCry3.1Ab

- derived fragments w ere visible.

A very faint band with an approximate molecular weight of 150 kDa was visible in the eCry3.1Ab control samples (Lanes 4 and 5) and the time

- zero digestibility assay sample ( Lane

7 ). Th is protein cross

- reacted with the antibody capable of detecting eCry3.1Ab and display ed a mobility consistent with the molecular weight of two eCry3.1Ab molecules ( 150 kDa)

. T herefore

, it most likely represents a dimer of eCry3.1Ab

. An additional faint band with an

appro ximate molecular weight of 47 kDa was also visible in the time zero digestibility assay sample (Lane 7 )

. Th is band cross

- reacted with the antibody capable of detecting eCry3.1Ab and

was also present in the eCry3.1Ab control (Lanes 4 and 5)

. This suggests that the 47 kDa band most likely correspond s to a mino r eCry3.1Ab hydrolysis product derived from the sample and is not related to pepsin digestion. Both bands (150 kDa and 47 kDa) w ere no longer detect able following exposure to SGF for 15 seconds (Lane 8)

.
The eCry3.1Ab protein incubated in SGF without pepsin (eCry3.1Ab control) showed no significant degradation over 15 minutes (Lanes 4 and 5), which indicates that the hydrolysis of eCry3.1Ab, seen in the SGF samples (Lanes 7 through 19), can be attribut ed to pepsin. Similar
band intensities were visualized for the time

- zero digestibility assay sample (Lane 7) and the eCry3.1Ab time

- zero control (Lane 4), confirming that equal amounts of eCry3.1 Ab were applied to the SDS

- PAGE and electro blotted

. The lowest amount of eCry3.1Ab visible on the blot (Lanes 21 through 25) was 0.4 ng (Lane 22). Therefore, t he LOD of eCry3.1Ab for the
Western blot used in this study was determined to be 0.4 ng.

### 5307

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- 1 Page 81 of 168 Figure VI

- 2

. Western blot analysis of eCry3.1Ab following digestion in simulated gastric fluid

.

The molecular weight of eCry3.1Ab is 74.8 kDa.

Lane 1: molecular weight standard
Lane 2: SGF control

– time zero
Lane 3: SGF control

– 15 minutes Lane 4: eCry3.1Ab control (eCry3.1Ab in SGF without pepsin)

– time zero 1 Lane 5: eCry3.1Ab control (eCry3.1Ab in SGF without pepsin)

– 15 minutes 1
Lane 6: molecular weight standard
Lane 7: in vitro digestibility assay

- time zero 1

Lane 8: in vitro digestibility assay

- 15 seconds 2

Lane 9: in vitro digestibility assay

- 30 seconds
Lane 10: in vitro digestibility assay

- 45 seconds
Lane 11: in vitro digestibility assay

- 1 minute
Lane 12: in vitro digestibility assay

- 1 minute 15 seconds
Lane 13: in vitro digestibil ity assay

- 1 minute 30 seconds
Lane 14: in vitro digestibility assay

- 1 minute 45 seconds
Lane 15: in vitro digestibility assay

– 2 minutes
Lane 16: in vitro digestibility assay

– 3 minutes
Lane 17: in vitro digestibility assay

– 5 minutes
Lane 18: in vitro digestibility assay

– 10 minutes
Lane 19: in vitro digestibility assay

– 15 minutes Lane 20: molecular weight standard Lane 21: 1.6 ng eCry3.1Ab for LOD determination Lane 22: 0.4 ng eCry3.1Ab for LOD determination 2 Lane 23: 0.1 ng eCry3.1Ab for LOD determination Lane 24: 0.024 ng eCry3.1Ab for LOD determination Lane 25: 0.006 ng eCry3.1Ab for LOD determination

1 Due to the resolution limits of the scanner and the printer, the very faint bands (47 kDa and 150 kDa) visible on the actual blot might not be visible on the printed image that appears here.

2 Due to the resolution limits of the scanner and the printer, t he very faint band (74.8 kDa) visible on the actual blot might not be visible on the printed image that appears here. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Mol. Wt [kDa]

188

----

62

---- 49

---- 38

---- 28

----
17

---- 14

----

6

----

3

---- 98

---- eCry3.1Ab eCry3.1Ab

### 5307

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- 1 Page 82 of 168 VI.E.4. Susceptibility of eCry3.1Ab to Inactivation by Heat

The temperature stability of eCry3.1Ab was evaluated by incubating the aliquots of an aqueous
solution of the test substance at various temperatures [4oC (control), 25oC, 37oC, 65oC and 95oC] for 30 minutes and determining the loss of insecticidal activity in an insect bioassay with Colorado potato beetle ( Leptinotarsa decemlineata ) larvae , a sensitive coleopteran species

. At 25oC, 37 oC, and 65oC

, eCry3.1Ab retained bioactivity against Colorado potato beetle larvae when compared with that observed at 4oC ( Table VI

- 2 )

. However, a fter treatment at 95oC for 30 minutes, LC 50 

### values and 95% confidence intervals could not be estimated due to the low mortality in these samples at all concentrations tested. These data demonstrate that eCry3.1Ab is
inac tivated, and therefore, denatured upon heating at temperatures of 95oC and above.

Table VI

- 3 . Effect of t emperature on i nsecticidal a ctivity of eCry3.1Ab in d iet i ncorporation b ioassay with Colorado potato beetle l arvae: LC 50 

### v alues and 95% confidence i ntervals at 144 hours.



### Temperature [° C ] LC 50 

### [ μ g eCry3.1Ab/ml ] 95% Confidence Interval [μ g eCry3.1Ab/ml]

4 0.918 0.419

- 1.617

25 1.802 1.066

- 2.714

37 1.814 0.411

- 4.714

65 4.682 1.321

- 10.092

95 >100 Not estimable

VI.E.5. Conclusion of the Assessment of eCry3.1Ab Allergenic Potential

The weight

- of

- evidence indicates that eCry3.1Ab is not likely to be a food allergen because:

**•** eCry3.1Ab is not derived from a known source of allergenic proteins,

**•** eCry3.1Ab does n ot have any significant amino acid sequence similarity to known or putative allergenic proteins,

**•** eCry3.1Ab is rapidly degraded in simulated mammalian gastric fluid, and

**•** eCry3.1Ab is inactivated, and thus denatured, at temperatures of 95oC and above.

### 5307

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- 1 Page 83 of 168 VI.F. eCry3.1Ab History of Safe Use

Prior dietary exposure to t he eCry3.1Ab protein has not occurred because it is a nove l protein engineered by Syngenta. Effective June 16, 2010, a temporary tolerance exemption was established for eCry3.1Ab (40 CFR 174.532) i n connection with an Experimental Use Permit granted by the EPA.

Although ther e is no history of prior exposure to Cry3.1Ab , per se, the protein is similar to other
well

- characterized Cry proteins with a history of safe use

. It is comprised largely (96.6%) of portions of mCry3A and Cry1Ab proteins (see Figure IV

- 2 ) , which have been safely used in
other genetically modified crops. The mCry3A protein is produced by Syngenta’s MIR6 04 corn (the subject of BNF No. 99) , a commercial product, and is a modified version of the native Cry3A protein from B.t. subsp. tenebrionis

. Cry3A is produced in transgenic B

. t

. (NewLeaf)
potatoes and is present in microbial B.t. formulations used for control of coleopteran pests in food crops

. Pursuant to § 408(d) of the Federal Food, Drug and Cosmetic Act, food and feed tolerance exemptions have been established for mCry3A

, Cry3A, and other Cry3 proteins used in transgenic crop s ( mCry3A, 40 CFR 174.505; Cry3A, 40 CFR 174. 509; and Cry3Bb1, 40 CFR 174.518) , reflecting their demonstrated mammalian safety

.

The C

- terminal portion of the native, full

- length Cry1Ab B.t. protein contained in the eCry3.1Ab amino acid sequence produced 5307 corn is also present in Syngenta’s transgenic COT67B

cotton (the subject of BNF No. 112) , which produces a full

- length Cry1Ab. (Syngenta’s Bt11 corn and Monsanto’s MON810 corn produce truncated Cry1Ab proteins representing primarily
the N

- terminal le pidopteran

- active region of the native Cry1Ab protein; this region is not present in eCry3.1Ab.) Native Cry1Ab is present in multiple microbial B.t . subsp. kurstaki

- based insecticides used for lepidopteran control in food crops

. Accordingly , multiple tol erance exemptions exist for Cry1Ab and other Cry1 proteins used in transgenic crops ( including full

length Cry1Ab, 40 CFR 174.529; Cry1Ac, 40 CR 174.510; and Cry1F, 40 CFR 174.520). Spore preparations of multiple strains of B.t., producing a variety of Cry proteins, are exempt from food and feed tolerances on food crops (40 CFR 180

- 1011). These multiple tolerance exemptions for Cry proteins are supported by extensive safety studies, and no documented food or feed safety
issues ha ve been identified during the use history of the associated products.

### 5307

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- 1 Page 84 of 168 VI.G. Conclusion of the eCry3.1Ab Protein Safety Assessment

A substantial body of data exists to support the safety of eCry3.1Ab protein produced in 5307

corn

.
The eCry3.1Ab protein is c onsidered nontoxic:

**•** The mode of action for the insecticidal activity of eCry3.1Ab is not relevant to mammals,

**•** eCry3.1Ab does not have significant amino acid sequence similarity to known protein toxins,

**•** eCry3.1Ab was nontoxic to mice with no treatment

- relat ed effects at a single dose of 2000 mg eCry3.1Ab/kg body weight, and

**•** Cry1 and Cry3 proteins have a history of safe use in agriculture.

The weight

- of

- evidence supports the conclusion that eCry3.1Ab is not likely to be a food allergen:

**•** eCry3.1Ab is not derived from a known source of allergenic proteins,

**•** eCry3.1Ab does not have significant amino acid sequence similarity to known or putative allergenic proteins,

**•** eCry3.1Ab is rapidly degraded in simulated mammalian gastric fluid, and

**•** eCry3.1Ab is labile up on heating at temperatures of 95oC and above.

### 5307

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- 1 Page 85 of 168 VI.H. Identity and Function of the PMI Protein in Event 5307 Corn

Event 5307 corn contains the gene pmi (also known as manA 1 

### ) from Escherichia coli strain K

- 12 ( a nonpathogenic strain ) (Miles and Guest 1984)

, as a selectable marker ; it serves no agronomic
or other purpose in 5307 plants. This gene encodes a phosphomannose isomerase (PMI) protein
of 391 amino acids having a molecular weight of approximately 42.8 kDa. PMI enzymes serve
an essential function in many organisms, including humans. PMI catalyzes the reversible
interconversion of mannose 6

- phosphate and fructose 6

- phosphate and has utility as a selectable marker for transformation of many plant species ( Negrotto et al. 2000). Plant cells that produ ce sufficient PMI are able to survive and grow on media containing mannose as the only or primary energy source (Miles and Guest 1984) , whereas plant cells that do not produce PMI cannot

.

VI.I. Regulatory S tatus and Established Safety of PMI

PMI proteins are present as selectable marker s in three other Syngenta transgenic corn cultivars
for which FDA consultations have been completed : MIR162 corn ( BNF No. 113) , MIR604 corn ( BNF No. 99 ) and Event 3272 corn ( BNF No. 95 ). The PMI protein encoded in 5307 corn i s identical to that in MIR162 corn and 3272 corn . (The PMI variant in MIR604 corn differs by
two amino acid substitutions from the PMI produced in MIR162 corn, 3272 corn, and 5307 corn.)

PMI has been granted an exemption from food and feed tolerances in all plants by the EPA (40 CFR 174.527). This tolerance exemption is supported by extensive data and information

, submitted by Syngenta and previously reviewed by the EPA and FDA

, demonstrating that PMI is nontoxic and is unlikely to become a food alle rgen. Therefore, detailed descriptions of these
studies are not reiterated herein. However, the substantial body of data that exists to support the safety of PMI protein produced in 5307 maize may be summarized as follows:

PMI is considered nontoxic:

**•** PM I does not share significant amino acid similarity to known protein toxins . (See also the description of the updated search for amino acid sequence similarity to known toxins
described in Part VI.K , below.)

**•** PMI was nontoxic to mice with no treatment

- related effects at high acute doses

.

**•** PMI proteins have a history of safe exposure due to the ir ubiquitous occurrence in nature and previous use in transgenic corn

.

1 Entrez Nucleotide Database Accession No. M15380 ( NCBI 2010a ).

### 5307

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- 1 Page 86 of 168 A standard weight

- of

- evidence analysis for allergenic potential (Codex 2009) indicates that PMI is unlikely to be a food allergen and is unlikely to be cross

- reactive to known allergens :

**•** PMI is not derived from a known source of allergenic proteins

.

**•** PMI do es not have any significant amino acid sequence similarity to known or putative allergenic proteins with implications for its allergenic potential . (See also the description of the updated search for amino acid sequence similarity to known allergens descr ibed in

Part VI.L , below.)

**•** PMI is rapidly degraded in simulated mammalian gastric fluid containing pepsin

.

**•** PMI is labile upon heating at temperatures of 65 oC a nd above.

**•** PMI is not glycosylated in corn.

To supplement the safety data summarized above, however, the following additional information
is described herein:

**•** A characterization study (see Part VI.J , below) demonstrating that microbially produced

PMI used as a test substance for safety studies is equivalent to, and a suitable surrogate for, PMI as produced in 5307 plants;

**•** Updated bioin formatic analyses , conducted in 2010, confirming that PMI does not share biologically significant amino acid sequence similarity to known toxins or allergens (see

Part VI.K and Part VI.L , below).

VI.J. Characterization of the Plant

- and Escherichia coli

- produced PMI Proteins

S equencing of the inserted T

- DNA in 5307 plants confirme d the presence of the exact nucleotide sequence of pmi , as intended (see Part IV.E.1 ). This indicates that the intact PMI protein ( 391 amino acids), as intended, i s encoded in 5307 plants.

Additionally, a characterization and comparison of PMI extracted from Event 5307 plants and

PMI in a test substance produced in recombinant E. coli was conducted to assess whether the microbially produced PMI t est substance (PMI

- 0105 , containing 89.5 % PMI by weight ) used for safety studies is a suitable surrogate for PMI produced in 5307 corn plants. PMI protein s from
both sources were demonstrated to have the predicted molecular weight of approximately 42.8 kDa, and both im munologically cross

- reacted with the same anti

- PMI antibodies, as determined by Western blot analysis , thus confirming the ir identity and integrity. It was also confirmed that

### 5307

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- 1 Page 87 of 168 the PMI proteins from both sources catalyzed the same chemical reaction 1 

### and ha d similar specific activity ( 455.67 U/mg PMI for the plant

- produced PMI and 526.26 U/mg PMI for the microbially produced PMI ; s ee also Tabl e B

- 1 in Appendix B ).

Based on these results, the identities of the plant

- and microbially produced PMI proteins have been verified and it can be concluded that the PMI produced in recombinant E. coli and Event
5307 corn are biochemically and functionally equivale nt. Therefore, the microbially produced PMI is a suitable surrogate for PMI produc ed in Event 5307 corn

.

VI.K. Updated Bioinformatic Assessment of PMI Amino Acid Sequence Similarity to Known Toxi ns

Bioinformatic analyses demonstrated that the PMI amino acid sequence (391 amino acids) has no significant sequence similarity to any toxins. This comparison was conducted using a 2010 posting of the National Center for Biotechnology Information (NCBI) Entrez® Protein Database (NCBI 2010b), and searching the database using the Basic Local Alignment Search Tool for Proteins (BLASTP) program 2 

### (Altschu l et al. 1997). This procedure determine d :

1. w hether any proteins in the database shared significant similarity to the PMI amino acid sequence, indicating they may be closely related to PMI,

2. w hether any proteins demonstrating significant sequence simila rity to the PMI amino acid sequence were known or putat ive toxins, indicating possible implications for the toxic potential of PMI.

The NCBI Entrez® database search identified all amino acid sequences with Expectation values ( E

- values) of 10 or lower . The E

- value is a measure of the probability that amino acid matches between sequences occurred by chance. Comparisons between highly similar sequences yield E

values approaching zero, whereas the probability that amino acid sequence similarities occurr ed only by chance increases with higher E

- values (Ponting 2001). The PMI query sequence showed no significant sequence similarity to any known or putative toxins.

1 One unit (U) of PMI activity is defined as the amount of enzyme require d to catalyze the conversion of 1 μmol of
mannose 6

- phosphate to fructose 6

- phosphate per minute (equivalent to 1 μmol NADP reduced per min). Reaction:
Mannose 6

- P (catalyzed by PMI)  Fructose 6

- P (catalyzed by phosphoglucose isomerase)  Glucose 6

- P

+ NADP (catalyzed by glucose 6

- P dehydrogenase)  6

- Gluconolactone + NADPH

2 BLASTP version 2.2.8 was used with the following parameters: no complexity filter; expectation score = 10; word size = 3; gap costs: existence = 11 and extension = 1. The simila rity matrix was Blocks Substitution Matrix62 (BLOSUM62).

### 5307

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- 1 Page 88 of 16 8 VI.L. Updated Bioinformatic Assessment of PMI Amino Acid Sequence Similarity to Known Allergen s

Bioinformatic analyses showed that the PMI amino acid sequence has no biologically relevant similarity to the sequences of known or putative allergens in the current posting of FARRP AllergenOnline database, version 10.0 (FARRP 2010). Sequential 80

- amino

- acid peptides of the PMI sequence were compared to the pr otein sequences in the Allergen Online database using the FASTA search algorithm 1 

### (Pearson and Lipman 1988). Additionally, the PMI sequence was also examined for matches of eight contigu ous amino aci ds (Hileman et al. 2002) with any allergen sequences, to screen for short, local regions of amino acid identity that might indicate the presence of common T

- cell binding epitopes. As previously identified, there was one sequence identity match of eight co ntiguous identical amino acids between PMI and a known allergen, α

- parvalbumin from Rana species CH2001 (unidentified edible frog) ( Hilger et al. 2002). Further investigation using IgE

- specific serum screening methodology (C odex 2009)
demonstrated no cros s

- reactivity between PMI and the allergen α

- parvalbumin, using serum from the single individual known to have demonstrated IgE

- mediated allergy to this specific α

parvalbumin. The allergic patient’s serum IgE did not recognize any portion of PMI as an
all ergenic epitope. These results support the conclusion that the eight

- amino

- acid sequence identity between the PMI protein and α

- parvalbumin from Rana species CH2001 is not biologically relevant and has no implications for the potential cross

- reactivity be tween PMI and

α

- parvalbumin. Therefore, the short sequence identity match with α

- parvalbumin from Rana species CH2001 has no implications for the potential allergenicity of PMI.

VI.M. PMI History of Safe Use

Data and information are available to support a hist ory of safe use of PMI proteins. PMI proteins are present as the selectable marker in commercially available transgenic corn products ( Syngenta’s MIR162 corn and MIR604 corn )

. It is conceivable th at small amounts of PMI proteins from various sources have always been present in the food and feed supply due to the ubiquitous occurrence of PMI proteins in nature, including food plants and animals. PMI proteins have been found in such diverse plant species as tobacco (Barb et al. 2002), walnut (Malvo l ti et a l. 1993), and Brassica species (Chen et al. 1989 )

, as well as in seeds of soybeans and other legumes (Lee and Matheson 1984)

. Genes encoding putative PMI proteins have been purified and characterized from many other organisms, including bacteria, yeast, rats, pigs, and
humans (Proudfoot et al. 1994a, 1994b; Davis et al. 2 002) and have been demonstrated to be essential for many or ganisms, including humans.

1 FASTA version 3.45 was used with the following parameters: extension penalty of 2 and gap creation penalty of 12. The scoring matrix was the Blocks Substitution Matrix 50 (BLOSUM50).

### 5307

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- 1 Page 89 of 168 VI.N. Conclusion of the PMI Safety Assessment

A substantial body of data exists to support the safety of PMI produced in Event 5307 corn

. PMI has been granted an exemption from food and feed tolerance s in all plants by the E PA ( 40 CFR 174.527) based on data demonstrating that PMI is nontoxic and that PMI is no t likely to be a food allergen

.

VI.O. eCry3.1Ab and PMI P rotein Expression Levels in 5307 Corn and Exposure
Potential

VI.O.1. Concentrations of eCry3.1Ab and PMI

The concentrations of eCry3.1Ab and PMI in various 5307 plant tissues were quantified using
ELISA. The concentrations of these proteins were measured in leaves and kernels as well as whole plants at four growth stages (whorl, anthesis, maturity and senesc ence) from 5307 hybrid maize hybrid grown at four US locations in 2008. The means across all four locations for eCry3.1Ab ( Table VI

- 3 ) and PMI ( Table VI

- 4 ) concentrations were determined on a dry

- and
fresh

- weight basis , and represent the levels of these proteins in 5307 corn in relevant tissue types
across four different location s throughout the life of the plant. Details of the methods used to quantify eCry3.1Ab and PMI in 5307 plants are provided in Appendix C.

VI.O.2. Mammalian Exposure Potential

Kernels from 5307 maize are the most likely tissue to enter the food supply , either as gr ain or grain by

- products. The average eCry3.1Ab concentration measured in kernels from 5307 maize (4.45 μg eCry3.1Ab/g d ry w eight [gdw] at senescence, Table VI

- 3 ) represents approximately 0.004% of the total protein in kernels

. Humans would potentially consume corn grain at the senescence stage of plant development, whereas livestock would be more likely to consume the kernels at plant maturity. The average eCry3. 1Ab concentration measured in kernels at maturity
was 6.19 μg eCry3.1Ab/ gdw ( Table VI

- 3 ), representing approximately 0.006% of the total protein.

The average PMI con centration measured in kernels from 5307 corn (1.11 μg PMI/gdw at

senescence, Table VI

- 4 ) represents approximately 0.001% of the total kernel protein. The average PM I concentration measured in kernels at maturity was 2.08 μg PMI/gdw ( Table VI

- 4 ), representing approximately 0.002% of the total protein

. (T hese calculations are base d on corn grain /kernels containing 10% total protein by weight .)

Given the low levels of eCry3.1Ab and PMI in 5307 kernels, dietary exposure potential can be
considered minimal. Because no health hazards have been identified for the eCry3.1Ab or PMI

pro teins, specific dietary exposure estimates for 5307 grain or grain by

- products used in human food products or 5307 grain, grain by

- products, forage or silage used as animal feed are not necessary to support a conclusion regarding the safety of 5307 corn

.

### 5307

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- 1 Page 90 of 168 Table VI

- 4

. C oncentrations of eCry3.1Ab in 5307 corn tissues at four stages on a dry

- weight (DW) and a fresh

- weight (FW) basis

.

μ g/g DW μ g/g FW
Stage Location Mean ± SD Range Mean ± SD Range

Whorl Leaves 142.96 ± 53.44 88.65

– 279.79 23.75 ± 3.16 16.81

– 33.80
Whole plants 111.08 ± 38.36 75.16

– 178.22 15.78 ± 2.47 11.41

– 28.64

Anthesis Leaves 84.34 ± 9.85 61.37

– 112.62 20.23 ± 2.59 13.83

– 27.59
Whole plants 38.14 ± 8.72 14.18

– 55.67 8.11 ± 2.11 3.10

– 13.12

Maturity Leaves 49.04 ± 31.79 1.46

– 105.60 25.33 ± 17.99 0.89

– 71.21
Whole plants 16.03 ± 5.45 6.37

– 38.94 8.86 ± 3.93 3.36

– 21.96

Kernels 6.19 ±1.87 2.37

– 9.64 4.56 ± 1.40 1.60

– 7.29

Senescence Leaves

– < LOQ

– 26.50

– < LOQ

– 20.29
Whole plants 8.27 ± 2.90 3.41

– 25.46 3.60 ± 0.94 1.70

– 10.65

Kernels 4.45 ± 0.82 2.92

– 6.76 3.24 ± 0.41 2.38

– 4.66

Means represent data from 4 locations. At each location, 5 plants were analyzed at each sampling stage. All data are corrected for extraction efficiency. Tissues of near

- isogenic, nontransgenic control plants were assayed in parallel to assess any effec ts of the plant matrix on the ELISA.

– = Not Applicable. It was not possible to calculate the mean as some values were below the limit of quantification ( LOQ )

. LOQ = 0.10 μg/g DW and 0.02 μg/g FW

.

### 5307

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- 1 Page 91 of 168 Table VI

- 5

. Co ncentrations of PMI in 5307 corn tissues at four stages on a dry

- weight (DW) and a fresh

- weight (FW) basis

.

μ g/g DW μ g/g FW
Stage Location Mean ± SD Range Mean ± SD Range

Whorl Leaves 4.83 ± 1.47 2.97

– 8.33 0.81 ± 0.10 0.59

– 1.13
Whole plants 4.23 ± 1.38 2.59

– 7.69 0.62 ± 0.22 0.34

– 1.13

Anthesis Leaves 2.91 ± 0.33 1.74

– 5.20 0.70 ± 0.08 0.43

– 1.28
Whole plants 4.38 ± 2.43 2.00

– 8.83 0.93 ± 0.47 0.44

– 2.13

Maturity Leaves

– < LOQ

– 3.50

– < LOQ

– 1.66
Whole plants 1.83 ± 0.51 0.68

– 2.56 0.96 ± 0.31 0.41

– 1.57

Kernels 2.08 ± 0.49 1.04

– 3.82 1.36 ± 0.29 0.74

– 2.38

Senescence Leaves

– < LOD

– 0.54

– < LOD

– 0.42
Whole plants 0.97 ± 0.37 0.39

– 2.02 0.43 ± 0.14 0.15

– 0.71

Kernels 1.11 ± 0.05 0.70

– 1.62 0.82 ± 0.06 0.50

– 1.28

Means represent data from 4 locations. At each location, 5 plants were analyzed at each sampling stage. All data are corrected for extraction efficiency. Tissues of near

- isogenic, nontransgenic control plants were assayed in parallel to assess any effec ts of the plant matrix on the ELISA.

– = Not Applicable. It was not possible to calculate the mean as some values were below the LOQ or L imit of Detection (L OD ); LOQ = 0.06 μg/g DW and 0.03 μg/g FW for leaves at maturity ; LOQ = 0.05 μg/g DW and 0.03 μg/g

FW for leaves at senescence ; LOD = 0.01 μg/g DW and FW for leaves at senescence

.

### 5307

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- 1 Page 92 of 168 

## VII. Food and Feed Nutritional Assessment of Event 5307 Corn

VII.A. Justification for Comparators

To confirm that Event 5307 corn and food and feed derived from Event 5307 corn are nutritionally comparable to and as safe as that derived from conventional corn , the composition of forage and grain from Event 5307 corn was compared to the composition of forage and grain from nontransgenic, near

- isogenic corn

. The Event 5307 corn a nd the corresponding
conventional control corn were harvested from six locations in the USA during 200 8

. The locations were selected to be representative of the range of environmental conditions under which the hybrid varieties were expected to be grown. All plants were grown using local agronomic practices for the respective regions.

In addition, a 4 9

- day broiler chicken feeding study was performed to evaluate whether standard poultry diets prepared with grain from Event 5307 corn supported broiler chicken survival, growth, and feed conversion to body weight that was not significantly different from survival, growth, and feed conversion of chickens consuming diets prepared with grain from nontransgenic, near

- isogenic corn grown in the same location during the same growing season.

VII.B. Histor ic Use of Corn in the U.S.

Field corn or maize ( Zea mays L.) is the most widely distributed cereal grain grown worldwide.
Cultivated corn is presumed to have been derived from teosinte ( Z. mexicana ) and is thought to have been introduced into the old world in the sixteenth century. Corn represents a staple food for a significant proportion of the world's population. The modern era of corn hybrid production began in the US where research conducted i n the early part of the 1900s proved that hybrid corn could produce a yield superior to open

- pollinated varieties (Sprague and Eberhart 1977). Gradually, hybrid varieties replaced the open

- pollinated types in the 1930s and 1940s. Almost
all corn grown in the US now comes from hybrid seed that is obtained every planting season from private enterprises; the older open

- pollinated varieties are virtually unknown in commerce (Hallauer et al. 1988). Corn grown in the US is predominantly of the yellow dent type , a commodity crop

. R oughly 60% of the crop is fed to livestock either as grain or silage.
Livestock that feed on corn include cattle, pigs, poultry, sheep and goats. The remainder of the crop is exported or processed by wet or dry milling to yield prod ucts such as high fructose corn

syrup , starch

, oil, grits and flour. These processed products are used extensively in the food industry. For example, corn starch serves as a raw material for an array of processed foods and in industrial manufacturing pro cesses. Since the early 1980s a significant amount of grain has also been used for fuel ethanol production. The by

- products from these processes are often used
in animal feeds.

### 5307

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- 1 Page 93 of 168 VII.C. Compositional Assessment of 5307 Forage and Grain

Compositional analyses o f 5307 corn were performed to identify any changes in nutrient or anti

nutrient content of the new crop in the context of its use as food or feed and to assess its biochemical equivalence and familiarity to conventional corn. This assessment was undertake n by performing quantitative analyses of 59 biochemical components of 5307 hybrid corn forage and grain including key food and feed nutrients, antinutrients, and secondary plant met abolites. An identical set of analyses was performed on nontransgenic, near

- isogenic control hybrid corn.

VII.C.1. Design and methods used in compositional analysis study

The 5307 hybrid plants were genotype NP2171 × NP2460(5307) and the control plants were genotype NP2171 × NP2460. The seed materials used to plant these composition trials ar e also
identified in Table IV

- 2 and in the pedigree diagram in Figure IV

- 4 . The trials were conducted
under USDA APHIS notification 08

- 051

- 104n.

Forage and grain from the 5307 and control hybrid s were harvested from six locations in the U

. S

. during 200 8: Stanton, MN

, Janesville, WI

, New Haven, IN

, Shirley, IL

, Marshall , MO , and Bloomington, IL

. These locations are representative of major corn growing regions of the U.S. At each location, the hybrids were planted in a randomized complete block design, with three replicates for each genotype. All plots were managed acc ording to local agronomic practices for the respective regions. Plants were self

- pollinated by hand and the developing ears were bagged
to avoid cross

- pollination.

The component s measured in this study were selected based on r ecommendations of the Organis ation for Economic Co

- operation and Development (OECD 2002 ) for comparative assessment of composition of new varieties of corn . The components analyzed are listed in
Table VII

- 1 below.

All analyses were conducted using methods published and approved by the Association of Analytical Communities (AOAC) International or other industry

- standard analytical methods. Based on the moisture content of each sample, anal yte levels were converted to equivalent units of dry weight. A detailed description of the methodology for the compositional analyses is provided in Appendix D

.

VII.C.1.a. Statistical analysis for across

- location comparisons

The data for each component were subject ed to analysis of variance using the following mixed
model:

Y ijk 

### = U + T i 

### + L j 

### + B(L)jk + LT ij 

### + e ijk



### In this model, Y ijk 

### is the observed response for genotype i at location j block k

, U is the overall

mean, T i 

### is the genotype effect, L j 

### is the location effect, B(L) jk 

### is the effect of block within a

### 5307

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- 1 Page 94 of 168 location, LT ij 

### is the location

- by

- genotype interaction effect, and e ijk 

### is the residual error. Genotype was regarded as a fixed effect, while the effects of location, block within location, an d
location

- by

- genotype were regarded as random.

For each quantifiable component, an F test was used to assess the statistical significance of the genotype effect with an alpha level of 0.05 and with the denominator degrees of freedom
determined using the K enward

- Roger method (Kenward and Roger 1997). Moisture content of grain was not statistically analyzed because the samples had been mechanically dried.



### VII.C.1.b. Statistical analysis for individual

- location comparisons

The data for each component at each location were subjected to an analysis of variance with genotype and block included in the statistical model. Significance was based on an alpha level
of 0.05.

Statistical analyses were performed using SAS v. 9.2 (SAS Institute, Inc.; Cary, NC).

VII.C.1.c. Comparison with I LSI Crop Composition Database

The mean levels of each component for each location and across locations were calculated and compared nonstatistically with means and ranges for forage and grain composition published in the ILSI Crop Composition Database (200 8). The ILSI database is the most comprehensive and current source of crop composition data for most nutritional components.

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- 1 Page 95 of 168 Table VII

- 1

. Forage and grain components measured in 5307 and conventional corn.

Forage Grain

Minerals Minerals Amino acids Secondary Metabolites Fatty acids

Calcium Calcium Alanine (Ala) ρ

- Coumaric acid 16:0 palmitic

Phosphorus Copper Arginine (Arg) Ferulic acid 18:0 stearic

Proximates Iron Aspartic acid (Asp) Furfural 18:1 oleic
Acid detergent fiber (ADF) Magnesium Cystine (Cys) Inositol 18:2 linoleic

Ash Manganese Glutamic acid (Glu) Vitamins 18:3 linolenic

Carbohydrates Phosphorus Glycine (Gly) Vitamin A (β

- carotene) 20:0 arachidic

Fat Potassium Histidine (His) Vitamin B 1 (thiamine) 20:1 eicosenoic

Moisture Selenium Isoleucine (Ile) Vitamin B 2 (riboflavin) 22:0 behenic
Neutral detergent fiber (NDF) Sodium Leucine (Leu) Vitamin B 3 (niacin) Anti

- nutrients

Protein Zinc Lysine (Lys) Vitamin B 6 (pyridoxine) Phytic acid

Proximates Methionine (Met) Vitamin B 9 (folic acid) Raffinose

ADF Phenylalanine (Phe) Vitamin E (α

- tocopherol) Trypsin inhibitor

Ash Proline (Pro)

Carbohydrates Serine (Ser)

Fat Threonine (Thr)

Moisture Tryptophan (Trp)

NDF Tyrosine (Tyr)

Protein Valine (Val)

Starch
Total Dietary Fiber (TDF)



### Table VII

- 2 through Table VII

- 9 report the statistical comparisons of nutritional component levels in forage and grain between 5307 corn and nontransgenic corn, the mean levels for each genotype across locations and at each location, a range of individual replicate values, and the levels for conventional hybrid corn reported in the ILSI Crop Composition Database (200 8).

All data were compared with the ranges reported in the ILSI database to establish whether the results were within the range of natural variation and to provide an indication of whether the results were likely to be of biological significance.

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- 1 Page 96 of 168 VII.C.2. Results of statistical analysis for forage

When analyzed across all six locations, there were no statistically significant differences in any of the measured forage components (proximates, calcium, and phosphorus) between genotypes ( Table VI I

- 2 and Table VII

- 3 ).

When analyzing the results at each individual location, s tatistically signi ficant differences were observed in moisture, protein, calcium, and phosphorus values , but all of these components were only different at one location (out of six) and all means fe l l within the range of natural variation
of corn reported in the ILSI databa se (ILSI 2008)

.

VII.C.3. Results of statistical analysis for grain

When analyzed across all six locations, there were no statistically significant differences in 52 of the 59 components including: proximates, starch, and fiber components ( Table VII

- 4 ), minerals ( Table VII

- 5 ), vitamins B 1 

### , B 2 

### , B 3 

### and E ( Table VII

- 6 ), amino acids ( Table VII

- 7 ), oleic, linoleic, arachidic, and behenic fatty acids ( Table VII

- 8 ), antinutrients, or secondary metabolites ( Table

VII

- 9 ).

There were a few statistically significant differences observed in vitamins A, B 6 

### , and B 9 

### ( Table

VII

- 6 ), as well as 16:0 palmitic, 18:0 stearic, 18:3 linolenic, and 20:1 eicosenoic acids ( Table

VII

- 8 ). Ho wever, the differences observed were small and the mean values observed for these
vitamins and all but one fatty acid (18:3 linolenic) were all within the ranges of values observed for the nontransgenic grain

, and all means fell within the natural variatio n of corn reported in the ILSI database (ILSI 2008).

Some statistically significant differences were observed in values for individual locations but all of these components were only different at one or two locations and all per

- location means fell
within th e range of natural variation for corn reported in the ILSI database (ILSI 2008) , with the exception of starch and Vitamin B 2 

### in the nontransgenic grain, for which the values exceeded the reported range

.

VII.C.4. Conclusion of compositional analyses

For all 59 chemical components that were measured in 5307 forage and grain , including those for which statistically significant differences were observed, the average values (when quantifiable) were within the ranges of natural variation reported in the ILSI database (2008). No biologically significant changes in composition were found to have occurred as an unintended result of the transformation process or expression of the transgenes in 5307 corn

. In conclusion, f orage and grain from 5307 corn hybrids are conside red similar in composition to forage and grain from both the nontransgenic comparator and conventional corn hybrids.

These data support the conclusion that 5307 corn will be as safe and nutritious as conventional

corn.

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- 1 Page 97 of 168 Table VII

- 2 . Proximate composition of forage from 5307 corn and nontransgenic corn.

Proximate levels are shown in % DW, except for moisture (% FW). Results significant ly different at P < 0.05 are shown in bold italic type.
For across

- location analyses, N = 18; the range of values among these replicates is provided.

Location Data source Statistic Moisture Protein Fat Ash Carbo

hydrates ADF NDF

Across Event 5307 mean 73.0 7.72 1.90 4.12 86.3 29.1 44.9

all range 66.5

– 79.5 5.91

– 10.3 0.893

– 2.81 2.89

– 5.35 82.9

– 88.9 22.3

– 40.1 35.5

– 56.1

Nontransgenic mean 72.3 7.57 1.89 4.34 86.2 28.6 45.4

range 66.7

– 78.0 6.27

– 10.0 0.843

– 2.63 3.43

– 6.18 82.3

– 89.0 19.0

– 41.5 32.3

– 57.4

ANOVA ( F test)
Genotype effect P 0.126 0.525 0.893 0.076 0.895 0.696 0.785

SEM 1.49 0.449 0.118 0.295 0.64 1.33 1.91

ILSI (2008) mean 70.2 7.78 2.039 4.628 85.6 27.00 41.51

range 49.1

– 81.3 3.14

– 11.57 < LOQ

– 4.570 1.527

– 9.638 76.4

– 92.1 16.13

– 47.39 20.29

– 63.71

N

a 945 945 921 945 945 945 945

a N is the number of ILSI values used to calculate the mean and excludes values < LOQ

### 5307

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- 1 Page 98 of 168 Table VII

- 2 ( Continued ). Proximate composition of forage from 5307 corn and nontransgenic corn. Proximate levels are shown in % DW, except for moisture (% FW). Results significantly different at P < 0.05 are shown in bold italic type. For individual location means, N = 3.

Location Data source Statistic Moisture Protein Fat Ash Carbo

hydrates ADF NDF

L1 Event 5307 mean 70.2 7.98 2.23 3.50 86.3 26.9 36.4
Nontransgenic mean 68.8 7.28 1.73 3.67 87.3 26.4 40.1

P 0.208 0.286 0.415 0.162 0.162 0.887 0.480

SEM 0.53 0.340 0.346 0.055 0.35 2.48 3.04

L2 Event 5307 mean 72.1 6.96 2.01 4.59 86.4 29.7 44.1
Nontransgenic mean 71.4 6.77 2.11 4.82 86.3 33.8 48.9

P 0.630 0.731 0.772 0.363 0.961 0.433 0.563

SEM 0.96 0.340 0.207 0.139 0.43 2.98 4.97

L4 Event 5307 mean 72.5 7.46 2.15 3.64 86.8 28.1 46.7
Nontransgenic mean 71.3 6.96 2.30 4.01 86.7 26.4 41.1

P 0.105 **0.033** 0.795 0.417 0.771 0.738 0.295

SEM 0.31 0.066 0.350 0.258 0.21 3.13 2.82

L6 Event 5307 mean 76.0 8.40 1.41 4.64 85.5 29.1 47.7
Nontransgenic mean 76.7 9.07 1.77 4.41 84.8 27.0 45.5

P 0.654 0.521 0.192 0.646 0.349 0.092 0.077

SEM 0.99 0.610 0.131 0.300 0.41 0.49 0.46

L7 Event 5307 mean 78.5 9.35 1.73 5.05 84.0 33.8 48.6
Nontransgenic mean 76.7 8.80 1.83 5.47 83.9 32.4 52.9

P **0.007** 0.141 0.695 0.377 0.804 0.672 0.541

SEM 0.10 0.162 0.162 0.265 0.42 2.06 4.19

L8 Event 5307 mean 68.6 6.19 1.89 3.33 88.6 26.7 45.7
Nontransgenic mean 68.7 6.54 1.57 3.65 88.3 25.5 43.7

P 0.225 0.057 0.323 0.162 0.625 0.688 0.594

SEM 0.04 0.061 0.174 0.104 0.33 1.88 2.33

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- 3 . Calcium and phosphorus composition of forage from 5307 corn and nontransgenic corn.

Calcium and phosphorus levels shown in mg/kg DW. Results significantly different at P < 0.05 are shown in bold italic type.
For across

- location analyses, N = 18; the range of values among these replicates is provided.

Location Data source Statistic Ca P

Across Event 5307 mean 2346 1906

all range 1450

– 3470 1420

– 2870

Nontransgenic mean 2354 1953

range 1660

– 3350 1390

– 2890

ANOVA ( F test)
Genotype effect P 0.886 0.491

SEM 209.3 163.9

ILSI (2008) mean 2028.6 2066.1

range 713.9

– 5767.9 936.2

– 3704.1

N 481 481

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- 3 (Continued) . Calcium and phosphorus composition of forage from 5307 corn and nontransgenic corn.
Calcium and phosphorus levels shown in mg/kg DW. Results significantly different at P < 0.05 are shown in bold italic type. For individual location means, N =3

.

Location Data source Statistic Ca P

L1 Event 5307 mean 2370 1723
Nontransgenic mean 2210 1523

P 0.208 **0.003**

SEM 61.6 8.2

L2 Event 5307 mean 2130 1500
Nontransgenic mean 2223 1457

P 0.711 0.694

SEM 154.5 67.4

L4 Event 5307 mean 2217 1783
Nontransgenic mean 2303 1813

P 0.087 0.869

SEM 19.3 113.1

L6 Event 5307 mean 2497 2493
Nontransgenic mean 2407 2677

P 0.771 0.662

SEM 191.4 255.6

L7 Event 5307 mean 3287 1947
Nontransgenic mean 3190 2043

P **0.032** 0.585

SEM 12.5 105.9

L8 Event 5307 mean 1573 1990
Nontransgenic mean 1793 2203

P 0.138 0.076

SEM 64.8 44.0

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- 4 . Proximate and starch composition of grain from 5307 corn and nontransgenic corn.

Proximate and starch levels shown in % DW, except moisture (% FW). Results significantly different at P < 0.05 are shown in bold italic type

. For across

- location analyses, N = 18; the range of values among these replicates is provided.

Location Data source Statistic Moisturea Protein Fat Ash Carbo

hydrates ADF NDF TDF Starch

Across Event 5307 mean 10.13 10.86 4.54 1.46 83.1 2.74 8.85 11.8 69.4

all range 9.54

– 11.4 9.12

– 12.6 3.85

– 4.93 1.22

– 1.60 81.0

– 85.3 2.23

– 3.34 7.68

– 9.52 10.8

– 13.4 62.0

– 73.7

Nontransgenic mean 10.18 10.92 4.72 1.40 83.0 2.85 8.83 11.7 70.3

range 9.21

– 12.2 9.20

– 13.0 4.43

– 5.09 1.09

– 1.67 80.7

– 84.7 2.47

– 3.48 7.79

– 10.2 10.6

– 13.5 63.1

– 77.3

ANOVA ( F test)
Genotype effect P

– 0.737 0.053 0.138 0.515 0.281 0.930 0.700 0.589

SEM

– 0.375 0.067 0.044 0.44 0.069 0.128 0.19 1.21

ILSI (2008) mean 11.3 10.30 3.555 1.439 84.6 4.05 11.23 16.43 57.7

range 6.1

– 40.5 6.15

– 17.26 1.742

– 5.823 0.616

– 6.282 77.4

– 89.5 1.82

– 11.34 5.59

– 22.64 8.85

– 35.31 26.5

– 73.8

N 1434 1434 1174 1410 1410 1350 1349 397 168

– = not ap plicable

a Grain was mechanically dried after harvest; moisture levels were not subject to ANOVA

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- 4 (Continued). Proximate and starch composition of grain from 5307 corn and nontransgenic corn.
Proximate and starch levels shown in % DW, except moisture (% FW). Results significantly different at P < 0.05 are shown in bold italic type. For individual location means, N = 3.

Location Data source Statistic Moisturea Protein Fat Ash Carbo

hydrates ADF NDF TDF Starch

L1 Event 5307 mean 10.54 10.93 4.38 1.43 83.2 2.63 8.66 11.2 69.4 Nontransgenic mean 11.13 10.60 4.79 1.29 83.3 2.89 9.27 11.9 71.7

P

– 0.405 0.068 **0.038** 0.578 0.390 0.623 0.456 0.401

SEM

– 0.225 0.080 0.019 0.14 0.169 0.754 0.54 1.54

L2 Event 5307 mean 10.73 9.38 4.53 1.34 84.8 2.89 9.01 11.7 71.9 Nontransgenic mean 11.00 9.97 4.68 1.25 84.1 2.67 8.73 11.2 74.7

P

– **0.011** 0.358 0.594 0.070 0.152 0.407 0.431 0.090

SEM

– 0.044 0.090 0.094 0.13 0.069 0.188 0.41 0.64

L4 Event 5307 mean 9.77 10.97 4.84 1.42 82.8 2.84 9.20 11.6 72.5 Nontransgenic mean 9.48 10.50 4.84 1.39 83.3 2.73 8.65 11.9 66.0

P

– 0.630 0.974 0.628 0.621 0.391 0.406 0.710 **0.019**

SEM

– 0.586 0.126 0.037 0.65 0.076 0.372 0.50 0.65

L6 Event 5307 mean 9.79 12.37 4.59 1.54 81.5 2.85 9.10 12.5 67.3 Nontransgenic mean 9.56 12.50 4.77 1.62 81.1 2.87 8.89 12.4 67.8

P

– 0.801 0.257 0.159 0.593 0.962 0.442 0.978 0.940

SEM

– 0.327 0.080 0.025 0.41 0.217 0.154 0.75 3.87

L7 Event 5307 mean 10.20 10.60 4.25 1.52 83.6 2.54 8.55 12.0 65.9 Nontransgenic mean 10.27 10.67 4.56 1.38 83.4 2.97 8.45 11.4 68.9

P

– 0.868 0.384 0.112 0.805 0.188 0.842 0.135 0.222

SEM

– 0.249 0.198 0.037 0.42 0.156 0.322 0.19 1.23

L8 Event 5307 mean 9.74 10.90 4.68 1.50 82.9 2.67 8.57 11.7 69.7 Nontransgenic mean 9.64 11.27 4.66 1.47 82.6 2.95 8.99 11.4 72.9

P

– 0.053 0.792 0.159 0.057 0.479 0.519 0.189 0.283

SEM

– 0.062 0.047 0.012 0.05 0.229 0.389 0.11 1.59

– = not applicable. a Grain was mechanically dried after harvest; moisture levels were not subject to ANOVA

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- 5 . Mineral composition of grain from 5307 corn and nontransgenic corn.

Mineral levels shown in mg/kg DW. Results significant at P < 0.05 are shown in bold italic type.
For across

- location analyses N = 18; the range of values among these replicates is provided.

Location Data source Statistic Ca Cu Fe Mg Mn P K Se a,c Na b,c Zn

Across E vent 5307 mean 43.9 1.52 23.7 1323 5.65 3228 3758

– – 23.0

all range 38.6

– 49.3 0.89

– 4.20 21.2

– 28.0 1150

– 1430 4.69

– 6.61 2620

– 3520 3400

– 4010 <LOQ

– 0.363 < LOQ 19.5

– 26.9

Nontransgenic mean 44.0 1.89 23.3 1336 5.43 3307 3776

– – 23.4

range 40.3

– 50.1 1.02

– 4.36 20.3

– 28.1 1220

– 1450 4.43

– 6.38 2650

– 3600 3240

– 4150 <LOQ

– 0.400 < LOQ 20.5

– 27.9

ANOVA ( F test)
Genotype effect P 0.891 0.058 0.308 0.401 0.131 0.110 0.707

– – 0.355

SEM 1.28 0.253 0.85 21.2 0.249 94.7 81.0

– – 0.78

ILSI (2 008) mean 46.4 1.75 21.81 1193.8 6.18 3273.5 3842 0.20 31.75 21.6

range 12.7

– 208.4 <LOQ

– 18.50 10.42

– 49.07 594.0

– 1940.0 1.69

– 14.30 1470.0

– 5330.0 1810.0

– 6030.0 <LOQ

– 0.75 <LOQ

– 731.54 6.5

– 37.2

N d 1344 1249 1255 1257 1256 1349 1257 89 223 1257

– = not applicable

a The LOQ for selenium was 0.055

– 0.056 mg/kg DW

b The LOQ for sodium was 110

– 114 mg/kg DW

c Where some or all values were < LOQ, calculation of the mean and statistical comparison were not possible, thus only the range is shown

d N is the number of ILSI values used to calculate the mean and excludes values < LOQ

### 5307

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- 5 (Continued) . Mineral composition of grain from 5307 corn and nontransgenic corn. Mineral levels shown in mg/kg DW. Results significant at P < 0.05 are shown in bold italic type. For individual location means, N = 3.

Location Data source Statistic Ca Cu Fe Mg Mn P K Se a Na a Zn

L1 Event 5307 mean 47.6 2.66 23.6 1347 5.66 3163 3803 <LOQ

– 0.086 <LOQ 22.3
Nontransgenic mean 45.4 2.89 22.0 1330 5.03 3103 3680 0.079 <LOQ 22.1

P 0.198 0.556 0.187 0.755 0.096 0.712 0.381

– – 0.915

SEM 0.81 0.232 0.55 33.0 0.149 99.7 78.1

– – 0.78

L2 Event 5307 mean 41.6 1.35 22.4 1307 5.16 2857 3473 0.113 <LOQ 20.4
Nontransgenic mean 41.0 1.88 23.1 1337 5.31 2903 3403 0.125 <LOQ 22.1

P 0.657 0.200 0.482 0.644 0.669 0.866 0.724 0.211

– 0.138

SEM 0.87 0.198 0.63 39.4 0.219 172.4 121.7 0.0046

– 0.49

L4 Eve nt 5307 mean 39.7 1.45 27.2 1297 5.09 3293 3657 0.348 <LOQ 25.7
Nontransgenic mean 41.2 1.28 27.5 1283 5.11 3413 3760 0.364 <LOQ 26.8

P 0.200 0.453 0.701 0.732 0.910 0.230 0.446 0.569

– **0.045**

SEM 0.59 0.133 0.42 23.9 0.128 49.7 77.7 0.0160

– 0.16

L6 Event 5307 mean 46.7 0.95 22.5 1347 6.44 3383 3920 <LOQ

– 0.058 <LOQ 22.1
Nontransgenic mean 47.7 1.09 22.5 1353 6.25 3487 3930 <LOQ

– 0.063 <LOQ 21.4

P 0.118 0.271 1.000 0.900 0.369 0.335 0.946

– – 0.559

SEM 0.27 0.066 0.27 33.2 0.117 58.1 92.7

– – 0.78

L7 Eve nt 5307 mean 46.8 1.36 24.3 1243 6.32 3227 3750 <LOQ

– 0.063 <LOQ 24.1
Nontransgenic mean 45.5 2.09 23.4 1297 6.14 3417 3883 <LOQ

– 0.107 <LOQ 23.9

P 0.156 0.302 0.459 **0.047** 0.366 **0.033** 0.231

– – 0.841

SEM 0.40 0.372 0.73 8.5 0.108 24.8 55.4

– – 0.83

L8 Event 5307 mean 40.8 1.34 22.1 1400 5.25 3443 3947 <LOQ

– 0.061 <LOQ 23.5
Nontransgenic mean 42.9 2.14 21.0 1417 4.73 3520 3997 <LOQ

– 0.062 <LOQ 24.2

P 0.152 0.156 0.200 0.588 **0.018** 0.323 0.286

– – 0.417

SEM 0.66 0.252 0.39 18.4 0.049 41.7 24.5

– – 0.51

– = not applicable

a Where some or all values were < LOQ, calculation of the mean and statistical comparison were not possible, thus only the range is shown

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- 6 . Vitamin composition of grain from 5307 corn and nontransgenic corn.

Vitamin levels shown in mg/100 g DW except as indicated for vitamin E (mg/g). Results significant at P < 0.05 are shown in bold italic type.
For across

- location analyses, N = 18; th e range of values among these replicates is provided.

Location Data source Statistic Vitamin A **β**

- carotene Vitamin B 1 Thiamine Vitamin B 2 Riboflavin Vitamin B 3 Niacin Vitamin B 6 Pyridoxine Vitamin B 9 Folic Acid Vitamin E a

α - tocopherol

Across Event 5307 mean 0.155 0.449 0.198 3.13 0.692 0.0397 0.0093

all range 0.133

– 0.185 0.399

– 0.511 0.156

– 0.264 2.53

– 4.11 0.587

– 0.769 0.0305

– 0.0460 0.00719

– 0.0111

Nontransgenic mean 0.176 0.458 0.198 3.18 0.737 0.0382 0.0090

range 0.155

– 0.216 0.408

– 0.518 0.152

– 0.318 2.51

– 3.70 0.621

– 0.815 0.0289

– 0.0463 0.00607

– 0.0110

ANOVA ( F test)

Genotype effect P **<0.001** 0.146 0.941 0.674 **0.005 0.031**  0.074

SEM 0.0049 0.0126 0.0096 0.104 0.0167 0.00199 0.00055

ILSI (2008) mean 0.684 0.530 0.125 2.376 0.644 0.0651 0.0103

range 0.019

– 4.681 0.126

– 4.000 0.050

– 0.236 1.037

– 4.694 0.368

– 1.132 0.0147

– 0.1464 0.0015

– 0.0687

N 276 894 704 415 415 895 863

a Original units of mg/100 g reported by the testing laboratory were converted to mg/g

### 5307

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- 6 (Continued) . Vitamin composition of grain from 5307 corn and nontransgenic corn.

Vitamin levels shown in mg/100 g DW except as indicated for vitamin E (mg/g). Results significant at P < 0.05 are shown in bold italic type. For individual location means, N = 3.

Location Data source Statistic Vitamin A **β**

- carotene Vitamin B1
Thiamine Vitamin B2 Riboflavin Vitamin B3
Niacin Vitamin B6 Pyridoxine Vitamin B9
Folic Acid Vitamin Ea

α - tocopherol

L1 Event 5307 mean 0.152 0.473 0.219 3.27 0.611 0.0448 0.0075 Nontransgenic mean 0.171 0.476 0.205 3.29 0.716 0.0434 0.0069

P 0.297 0.939 0.612 0.963 0.105 0.399 0.176
SEM 0.0096 0.0220 0.0162 0.225 0.0261 0.00091 0.00022

L2 Event 5307 mean 0.151 0.456 0.175 3.53 0.660 0.0422 0.0082 Nontransgenic mean 0.167 0.476 0.202 3.33 0.684 0.0424 0.0078

P 0.082 0.232 0.587 0.657 0.645 0.807 0.313
SEM 0.0035 0.0083 0.0294 0.279 0.0321 0.00059 0.00018

L4 Event 5307 mean 0.141 0.488 0.197 3.16 0.739 0.0393 0.0093 Nontransgenic mean 0.159 0.504 0.169 3.20 0.748 0.0360 0.0090

P **0.016** 0.548 0.222 0.903 0.609 0.093 0.289
SEM 0.0017 0.0164 0.0112 0.239 0.0098 0.00078 0.00011

L6 Event 5307 mean 0.172 0.432 0.213 2.89 0.699 0.0427 0.0095 Nontransgenic mean 0.188 0.424 0.246 2.85 0.749 0.0407 0.0094

P 0.061 0.218 0.403 0.826 0.484 0.506 0.743
SEM 0.0029 0.0033 0.0222 0.104 0.0421 0.00176 0.00028

L7 Event 5307 mean 0.157 0.434 0.183 3.16 0.721 0.0317 0.0107 Nontransgenic mean 0.176 0.435 0.172 3.25 0.759 0.0299 0.0109

P 0.218 0.808 0.519 0.709 0.076 0.346 0.733
SEM 0.0076 0.0009 0.0100 0.153 0.0079 0.00104 0.00030

L8 Event 5307 mean 0.160 0.410 0.203 2.77 0.723 0.0377 0.0105 Nontransgenic mean 0.195 0.432 0.191 3.17 0.767 0.0368 0.0098

P **0.025** 0.077 0.560 0.498 **0.003** 0.744 0.454

SEM 0.0040 0.0045 0.0123 0.345 0.0018 0.00176 0.00056

a Original units of mg/100 g reported by testing laboratory were converted to mg/g

### 5307

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- 7 . Amino acid composition of grain from 5307 corn and nontransgenic corn.

Amino acid levels shown in mg/g DW. Results significant at P < 0.05 are shown in bold italic type.
For across

- location analyses, N = 18; the range of values among these replicates is provided.

Location Data source Statistic Asp Thr Ser Glu Pro Gly Ala Cys Val

Across Event 5307 mean 6.93 3.80 5.14 20.4 9.23 3.95 8.21 2.33 5.12

all range 5.82

– 8.15 3.19

– 4.39 4.10

– 6.10 16.4

– 24.9 7.55

– 11.0 3.52

– 4.39 6.64

– 9.97 2.07

– 2.50 4.33

– 6.08

Nontransgenic mean 6.88 3.79 5.17 20.6 9.24 3.97 8.24 2.36 5.13

range 6.00

– 8.20 3.36

– 4.47 4.44

– 6.28 17.4

– 25.3 7.84

– 10.9 3.61

– 4.35 7.06

– 10.0 2.14

– 2.59 4.28

– 6.01

ANOVA ( F test)

Genotype effect P 0.625 0.908 0.736 0.715 0.973 0.761 0.846 0.284 0.877

SEM 0.236 0.123 0.203 0.90 0.375 0.087 0.345 0.043 0.179

ILSI (2008) mean 6.88 3.75 5.12 20.09 9.51 3.85 7.90 2.21 4.90

range 3.35

– 12.08 2.24

– 6.66 2.35

– 7.69 9.65

– 35.36 4.62

– 16.32 1.84

– 5.39 4.39

– 13.93 1.25

– 5.14 2.66

– 8.55

N 1350 1350 1350 1350 1350 1350 1350 1350 1350

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- 1 Page 108 of 168 Table VII

- 7 (Continued) . Amino acid composition of grain from 5307 corn and nontransgenic corn.
Amino acid levels shown in mg/g DW. Results significant at P < 0.05 are shown in bold italic type. For individual location means, N = 3.

Location Data source Statistic Asp Thr Ser Glu Pro Gly Ala Cys Val

L1 Event 5307 mean 7.07 3.82 5.09 21.1 9.25 3.86 8.42 2.33 5.25 Nontransgenic mean 6.74 3.71 4.98 19.9 8.51 3.80 7.97 2.34 5.00

P 0.133 0.197 0.431 0.239 0.408 0.059 0.212 0.707 0.179

SEM 0.096 0.043 0.082 0.51 0.499 0.011 0.176 0.016 0.088

L2 Event 5307 mean 5.94 3.26 4.28 16.8 7.78 3.56 6.78 2.12 4.40 Nontransgenic mean 6.27 3.48 4.63 18.3 8.18 3.75 7.30 2.26 4.66

P **0.030 0.018**  0.118 **0.010** 0.380 **0.028 0.010**  0.148 **0.012**

SEM 0.041 0.021 0.092 0.10 0.255 0.024 0.037 0.045 0.020

L4 Event 5307 mean 6.96 3.81 5.25 20.5 9.08 4.04 8.23 2.36 5.17 Nontransgenic mean 6.97 3.84 5.32 20.9 9.78 4.13 8.40 2.36 5.30

P 0.961 0.833 0.761 0.591 0.247 0.485 0.591 0.940 0.367

SEM 0.128 0.088 0.149 0.48 0.307 0.075 0.186 0.028 0.079

L6 Event 5307 mean 7.89 4.31 5.94 24.0 10.90 4.28 9.60 2.47 5.82 Nontransgenic mean 7.73 4.22 5.95 23.9 10.14 4.18 9.48 2.54 5.72

P 0.747 0.668 0.976 0.967 0.301 0.606 0.849 0.277 0.820

SEM 0.306 0.123 0.206 1.01 0.390 0.124 0.393 0.033 0.272

L7 Event 5307 mean 6.69 3.68 4.98 19.3 8.82 3.95 7.85 2.32 4.91 Nontransgenic mean 6.48 3.61 4.93 19.0 8.92 3.86 7.68 2.32 4.74

P 0.514 0.680 0.885 0.772 0.763 0.554 0.725 0.840 0.533

SEM 0.192 0.109 0.186 0.78 0.219 0.084 0.292 0.021 0.167

L8 Event 5307 mean 7.03 3.89 5.30 20.9 9.55 4.02 8.36 2.39 5.16 Nontransgenic mean 7.09 3.88 5.19 21.5 9.89 4.08 8.59 2.36 5.38

P 0.835 0.933 0.519 0.661 0.584 0.594 0.648 0.311 0.508

SEM 0.189 0.075 0.097 0.83 0.368 0.064 0.306 0.019 0.198

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- 1 Page 109 of 168 Table VII

- 7 ( Continued ). Amino acid composition of grain from 5307 corn and nontransgenic corn.

Amino acid levels shown in mg/g DW. Results significant at P < 0.05 are shown in bold italic type.
For across

- location analyses, N = 18; the range of values among these replicates is provided.

Location Data source Statistic Met Ile Leu Tyr Phe Lys His Arg Trp

Across Event 5307 mean 2.29 3.92 13.8 3.18 5.50 3.10 2.99 4.81 0.570

all range 1.97

– 2.51 3.19

– 4.77 10.8

– 17.1 1.57

– 4.18 4.34

– 6.68 2.76

– 3.36 2.57

– 3.44 3.72

– 5.56 0.381

– 0.704

Nontransgenic mean 2.36 3.91 13.8 3.26 5.52 3.09 3.01 4.82 0.557

range 2.08

– 2.56 3.23

– 4.71 11.5

– 17.3 1.67

– 3.98 4.73

– 6.70 2.74

– 3.38 2.57

– 3.43 4.20

– 5.32 0.380

– 0.700

ANOVA ( F test)

Genotype effect P 0.102 0.947 0.789 0.711 0.883 0.902 0.684 0.892 0.722

SEM 0.049 0.163 0.66 0.153 0.239 0.059 0.088 0.144 0.0298

ILSI (2008) mean 2.09 3.68 13.41 3.36 5.25 3.15 2.96 4.33 0.627

range 1.24

– 4.68 1.79

– 6.92 6.42

– 24.92 1.03

– 6.42 2.44

– 9.30 1.72

– 6.68 1.37

– 4.34 1.19

– 6.39 0.271

– 2.150

N 1350 1350 1350 1350 1350 1350 1350 1350 1350

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- 1 Page 110 of 168 Table VII

- 7 ( Continued ). Amino acid composition of grain from 5307 corn and nontransgenic corn.
Amino acid levels shown in mg/g DW. Results significant at P < 0.05 are shown in bold italic type. For individual location means, N = 3.

Location Data source Statistic Met Ile Leu Tyr Phe Lys His Arg Trp

L1 Event 5307 mean 2.32 4.03 14.3 2.88 5.63 3.08 3.01 4.43 0.497 Nontransgenic mean 2.32 3.74 13.3 3.47 5.26 3.06 2.92 4.61 0.601

P 0.915 0.150 0.231 0.334 0.283 0.319 0.175 0.429 0.552

SEM 0.039 0.092 0.39 0.330 0.177 0.014 0.031 0.132 0.1039

L2 Event 5307 mean 2.02 3.26 11.1 2.57 4.49 2.81 2.62 4.19 0.494 Nontransgenic mean 2.23 3.49 12.2 3.02 4.86 2.95 2.80 4.44 0.539

P 0.144 0.066 **0.007** 0.228 0.079 0.072 **0.009** 0.161 0.656

SEM 0.061 0.043 0.06 0.187 0.077 0.029 0.012 0.083 0.0605

L4 Event 5307 mean 2.28 3.95 13.8 3.16 5.50 3.19 3.03 5.02 0.680 Nontransgenic mean 2.36 4.01 14.1 3.52 5.63 3.22 3.11 5.29 0.588

P 0.279 0.535 0.589 0.622 0.515 0.762 0.402 0.343 0.169

SEM 0.037 0.057 0.30 0.441 0.114 0.068 0.058 0.157 0.0307

L6 Event 5307 mean 2.42 4.56 16.4 3.54 6.46 3.31 3.34 5.23 0.624 Nontransgenic mean 2.52 4.46 16.3 3.06 6.31 3.21 3.28 4.92 0.658

P 0.115 0.793 0.931 0.524 0.724 0.578 0.721 0.523 0.427

SEM 0.027 0.229 0.72 0.446 0.255 0.100 0.109 0.279 0.0238

L7 Event 5307 mean 2.37 3.74 13.0 3.43 5.27 3.08 2.91 5.02 0.554 Nontransgenic mean 2.39 3.58 12.7 3.39 5.18 2.97 2.82 4.84 0.502

P 0.823 0.538 0.761 0.895 0.781 0.333 0.589 0.555 0.403

SEM 0.037 0.154 0.61 0.189 0.193 0.063 0.100 0.178 0.0350

L8 Event 5307 mean 2.32 3.98 14.0 3.48 5.68 3.12 3.04 4.95 0.573 Nontransgenic mean 2.32 4.21 14.5 3.08 5.88 3.15 3.13 4.80 0.454

P 0.919 0.477 0.644 0.665 0.592 0.607 0.509 0.703 0.134

SEM 0.021 0.187 0.61 0.562 0.231 0.031 0.086 0.236 0.0342

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- 8 . Fatty acid composition a 

### of grain from 5307 corn and nontransgenic corn.

Fatty acids shown as % of total fatty acids. Results significant at P < 0.05 are shown in bold italic type.
For across

- location analyses, N = 18; the range of values among these replicates is provided.

Location Data source Statistic 16:0 Palmitic 16:1 Palmitoleic b 18:0 Stearic 18:1 Oleic 18:2 Linoleic 18:3 Linolenic 20:0 Arachidic 20:1 Eicosenoic 22:0 Behenic

Across Event 5307 mean 15.7

– 1.74 24.5 55.6 1.60 0.392 0.250 0.220

all range 15.1

– 16.1 <LOQ

– 0.137 1.50

– 2.04 22.0

– 27.0 53.2

– 58.1 1.48

– 1.71 0.353

– 0.453 0.238

– 0.265 0.186

– 0.252

Nontransgenic mean 15.2

– 1.81 24.9 55.7 1.50 0.387 0.242 0.213

range 14.6

– 15.9 <LOQ

– 0.450 1.54

– 2.17 22.6

– 26.4 53.8

– 58.4 1.40

– 1.57 0.361

– 0.437 0.232

– 0.261 0.194

– 0.247

ANOVA ( F test)

Genotype effect P **<0.001**

– **0.038** 0.108 0.599 **<0.001** 0.186 **<0.001** 0.243

SEM 0.07

– 0.059 0.54 0.60 0.017 0.0098 0.0029 0.0056

ILSI (2008) mean 11.50 0.154 1.82 25.8 57.6 1.20 0.412 0.297 0.176

range 7.94

– 20.71 <LOQ

– 0.447 1.02

– 3.40 17.4

– 40.2 36.2

– 66.5 0.57

– 2.25 0.279

– 0.965 0.170

– 1.917 <LOQ

– 0.349

N 1344 596 1344 1344 1344 1344 988 987 924

– = not applicable

a Where some or all values were <LOQ, % of total fatty acids could not be calculated and statistical analysis could not be perf ormed. Levels <LOQ were observed for all replicates at all locations for 8:0 caprylic, 10:0 capric, 12:0 lauric, 14:0 myristic, 14:1 myristoleic, 15:0 pentadecanoic, 15:1 pentadecenoic, 17:0 heptadecanoic, 17:1 heptadecenoic, 20:2 eicosadienoic, 20:3 eicosatrienoic, and 20:4 arachidonic fatty acids
b Some values were <LOQ, therefore, only the range is shown

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- 1 Page 112 of 168 T able VII

- 8 ( Continued ). Fatty acid composition of grain from 5307 corn and nontransgenic corn. Fatty acids shown as % of total fatty acids. Results significant at P < 0.05 are shown in bold italic type. For individual location means, N = 3.

Location Data source Statistic 16:0 Palmitic 16:1 Palmitoleic a 18:0 Stearic 18:1 Oleic 18:2 Linoleic 18:3 Linolenic 20:0 Arachidic 20:1 Eicosenoic 22:0 Behenic

L1 Event 5307 mean 15.5 <LOQ 1.53 22.4 58.1 1.63 0.374 0.247 0.210 Nontransgenic mean 15.1 <LOQ 1.58 22.7 58.2 1.51 0.371 0.237 0.204

P 0.225

– **0.013** 0.286 0.199 0.122 0.641 0.061 0.730

SEM 0.16

– 0.004 0.15 0.06 0.033 0.0043 0.0019 0.0107

L2 Event 5307 mean 15.8 <LOQ 1.77 23.5 56.4 1.67 0.397 0.242 0.216 Nontransgenic mean 15.5 <LOQ

– 0.450 1.93 24.3 55.8 1.54 0.388 0.238 0.202

P 0.149

– 0.202 0.120 0.406 **0.017** 0.467 0.476 0.100

SEM 0.10

– 0.060 0.22 0.43 0.012 0.0077 0.0038 0.0035

L4 Event 5307 mean 15.7 0.134 1.76 25.3 54.8 1.57 0.391 0.245 0.200 Nontransgenic mean 15.2 0.132 1.86 25.5 55.0 1.47 0.391 0.240 0.212

P **0.013** 0.372 **0.023** 0.319 0.478 **0.019** 0.960 **0.047** 0.373

SEM 0.04 0.0017 0.011 0.14 0.16 0.010 0.0042 0.0009 0.0075

L6 Event 5307 mean 15.8 <LOQ

– 0.132 1.68 24.5 55.5 1.60 0.368 0.246 0.228 Nontransgenic mean 15.1 <LOQ

– 0.134 1.72 24.7 56.1 1.54 0.370 0.239 0.206

P 0.069

– 0.270 0.560 0.210 0.203 0.606 0.242 0.072

SEM 0.14

– 0.019 0.17 0.25 0.023 0.0027 0.0029 0.0045

L7 Event 5307 mean 15.5 <LOQ 1.98 26.4 53.6 1.55 0.441 0.263 0.242 Nontransgenic mean 15.4 <LOQ

– 0.143 1.96 26.1 54.1 1.47 0.427 0.253 0.235

P **0.038**

– 0.701 0.493 0.291 0.086 0.336 0.132 0.678

SEM 0.02

– 0.032 0.26 0.23 0.017 0.0077 0.0030 0.0098

L8 Event 5307 mean 15.7 <LOQ

– 0.137 1.71 24.9 55.1 1.59 0.380 0.254 0.221 Nontransgenic mean 15.0 <LOQ

– 0.124 1.80 25.9 54.9 1.45 0.378 0.247 0.220

P 0.053

– 0.082 0.211 0.678 0.093 0.878 0.294 0.946

SEM 0.13

– 0.020 0.39 0.29 0.032 0.0054 0.0037 0.0061

– = not applicable

a Where some or all values were < LOQ, % of total fatty acids could not be calculated, statistical analysis could not be performed, and only the range is shown

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- 1 Page 113 of 168 Table VII

- 9 . Secondary metabolite and antinutrient composition of grain from 5307 corn and nontransgenic corn.

Analyte units as in column headings. Results significant at P < 0.05 are shown in bold italic type.
For across

- location analyses, N = 18; the range of values among these repli cates is provided.

Ferulic acid ρ - Coumaric acid Inositol Phytic acid Trypsin inhibitor Furfural a,b Raffinose

Location Data source Statistic (mg/kg DW) (mg/kg DW) (ppm DW) (% DW) (TIU/mg DW) (mg/kg DW) (% DW)

Across Event 5307 mean 1906 186 2510 0.910 3.34

– 0.156

all range 1670

– 2190 153

– 229 2120

– 3160 0.671

– 1.03 2.39

– 4.42 < LOQ 0.115

– 0.199

Nontransgenic mean 1889 186 2504 0.942 3.46

– 0.163

range 1620

– 2090 148

– 226 1980

– 3060 0.729

– 1.06 2.22

– 3.94 < LOQ 0.119

– 0.188

ANOVA ( F test) Genotype effect P 0.691 0.926 0.951 0.216 0.393

– 0.066

SEM 52.4 9.1 86.1 0.0261 0.118

– 0.0087

ILSI (2008) mean 2201.1 218.4 1331.5 0.745 2.73 3.697 0.312

range 291.9

– 3885.8 53.4

– 576.2 89.0

– 3765.4 0.111

– 1.570 <LOQ

– 7.18 <LOQ

– 6.340 <LOQ

– 0.320

N c 817 817 504 1196 696 14 701

– = not applicable

a The LOQ for furfural was 0.55

– 0.57 mg/kg DW

b All values were <LOQ and therefore statistical comparison was not possible

c N is the number of ILSI values used to calculate the mean and excludes values < LOQ

### 5307

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- 1 Page 114 of 168 Table VII

- 9 ( Continued ). Secondary metabolite and antinutrient composition of grain from 5307 corn and nontransgenic corn.

Analyte units as in column headings. Results significant at P < 0.05 are shown in bold italic type. For individual location means, N = 3.

Location Data source Statistic Ferulic acid (mg/kg DW) ρ - Coumaric acid (mg/kg DW) Inositol (ppm DW) Phytic acid (% DW) Trypsin inhibitor (TIU/mg DW) Furfural a (mg/kg DW) Raffinose (% DW)

L1 Event 5307 mean 1770 169 2323 0.903 3.31 <LOQ 0.179 Nontransgenic mean 1883 187 2160 0.878 3.58 <LOQ 0.174

P 0.579 0.494 0.062 0.577 0.124

– 0.707

SEM 122.2 15.9 30.1 0.0268 0.073

– 0.0082

L2 Event 5307 mean 1747 197 2517 0.840 2.90 <LOQ 0.166 Nontransgenic mean 1827 207 2230 0.848 3.48 <LOQ 0.178

P 0.463 0.375 0.323 0.938 0.111

– 0.607

SEM 62.8 6.5 155.8 0.0592 0.149

– 0.0133

L4 Event 5307 mean 2137 226 2790 1.000 3.58 <LOQ 0.164 Nontransgenic mean 1977 209 2760 1.006 3.73 <LOQ 0.175

P 0.186 0.193 0.896 0.818 0.702

– 0.093

SEM 57.2 6.3 143.5 0.0171 0.241

– 0.0026

L6 Event 5307 mean 2017 177 2527 0.842 3.44 <LOQ 0.167 Nontransgenic mean 1990 185 2523 0.965 3.64 <LOQ 0.173

P 0.829 0.702 0.959 0.156 0.412

– 0.234

SEM 76.6 12.8 40.9 0.0390 0.140

– 0.0027

L7 Event 5307 mean 1800 159 2470 0.912 3.40 <LOQ 0.139 Nontransgenic mean 1753 154 2627 0.984 3.28 < LOQ 0.156

P 0.340 0.225 0.691 0.053 0.861

– 0.091

SEM 26.6 2.0 241.1 0.0123 0.403

– 0.0039

L8 Event 5307 mean 1967 186 2433 0.964 3.41 <LOQ 0.122 Nontransgenic mean 1903 175 2727 0.971 3.02 <LOQ 0.123

P 0.604 0.601 0.081 0.864 0.311

– 0.893

SEM 73.5 13.0 62.8 0.0230 0.205

– 0.0031

– = not applicable

a All values were < LOQ and therefore statistical comparison was not possible

### APPENDIX A

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- 1 Page 115 of 168 VII.D. Other Information Relevant to the Nutritional Assessment of Event
5307 Corn : Broiler Chicken Feeding Study

Chickens consume large quantities of corn grain in commercial feeds. Broiler chickens, in particular, have relatively high corn consumption because conventional feed ing regimens have been designed to provide maximal body weight gain in the shortest amount of time. In addition, broiler chickens are highly sensitive to small nutrient changes within their diets because of their rapid growth rates. A 4 9

- day feeding stud y was performed to evaluate whether standard po ultry diets prepared with Event 5307 corn grain had any effect on male or female broiler chicken survival, growth, feed conversion (an indicator of how efficiently a bird converts feed to live body weight) , or carcass yield when compared with control corn grain . Three lots of corn grain were used to prepare poultry diets as follows:

**•** diets prepared with grain from 5307 corn

**•** diets prepared with grain from nontransgenic, near

- isogenic control corn

**•** diets prepared with a commercially available lot of North Carolina corn

The specific seed varieties planted to generate the 5307 and nontransgenic, near

- isogenic control corn grain are specified in Table IV

- 2 and Figure IV

- 4

. The poultry diets were formulated based on the individual nutrient analyses for eac h of the grain s ources to meet
standard nutritional recommendations for growing chickens (diets prepared with 55% to 63% maize) and were fed to groups of 90 male and 90 female birds for 4 9 consecutive days.

Parameters evaluated in the study included surv ival, body weight, feed conversion and carcass yield. Broiler chickens fed diets prepared with Event 5307 grain did not show any adverse effects compared to chickens fed diets prepared with either the nontransgenic, near

isogenic grain or the commercially available grain. There were no statistically significant differences between groups for body weight, feed consumption, survival, overall feed conversion, and carcass yield when expres sed on an absolute weight basis

. A few statistically significant diffe renc es were noted in carcass yield when expressed as a percentage of total body weight . Male broilers fed 5307 maize grain had decreased thigh weights compared with male broilers fed nontransgenic diets, but they were not different
from the males consumin g the commercially available control diets. Female broilers fed 5307 diets had decreased thigh and pectoralis minor weights compared with female broilers fed the nontransgenic and NCSU 2007 diets. There were no differences noted in the other
carcass part s including fat pads, drums, wings, and pectoralis major muscles.

Overall, d iets containing 5307 maize grain supported rapid broiler chicken growth at low mortality rates and excellent feed conversion ratios and there were no adverse effects on carcass yie ld. There were no adverse dietary effects on broiler chickens consuming diets prepared with Event 5307 corn grain when compared with those consuming diets prepared with nontransgenic corn grain, either as a direct effect of the transgenic proteins in the diet

### APPENDIX A

5307

- FDA

- 1 Page 116 of 168 or as a result of any unintended compositional changes in the grain that may have altered its
nutritional value. The results of this study support the conclusion that Event 5307 corn is nutritionally comparable to and as safe as conventional corn for consumers.

VII.E. Summary of the Food and Feed Nutritional Assessment

Analysis of key nutritional components of forage and grain from 5307 corn showed that no biologically significant changes in composition occurred as an unintended result of the transformation process or expression of the transgenes in 5307 corn . Forage and grain from
5307 corn are considered similar in composition to forage and grain from conventional corn

. In addition, a 4 9

- day feeding study demonstrated that there were no adverse dietary effects on broiler chickens consuming diets prepared with 5307 corn grain when compared with those consuming diets prepared with nontransgenic, control corn grain, either as a direct
effec t of the transgenic proteins in the diet or as a result of any unintended compositional changes in the grain that may have altered its nutritional value. These results support the
conclusion that 5307 corn is nutritionally comparable to and as safe as con ventional corn

.

### APPENDIX A

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- 1 Page 117 of 168 

## VIII. References

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, Lipman DJ. 1997. Gapped BLAST and PSI

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Armitage P

, Berry G. 1987

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Arumuganathan K, Earle ED. 1991. Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 9:208

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Barb AW, Williamson JD

, Pharr DM. 2002. Selection for mannose

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Christensen AH, Sharrock RA , Quail PH. 1992. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol

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Coe EH Jr

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### APPENDIX A

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## Appendix A: Materials and Methods Used in the Genetic
Characterization of Event 5307 Corn

This appendix provides details of the materials and methods used in the various studies performed to genetically characterize Event 5307 corn. The design, results and conclusion s of these experiments are described in Part I V . E. of this submission ( Characterization of
the Genetic Material in Event 5307 Corn ).

A.1. Plant Material

Event 5307 corn and nontransgenic control corn seeds were planted and grown in a Syngenta Biotechno logy, Inc. greenhouse in Research Triangle Park, North Carolina, USA under standard greenhouse conditions and then processed to extract genomic DNA. The specific generations of 5307 plants used and the corresponding control plants used are specified in th e descriptions of the studies in Part I V . C.2. of this submission

, Development
of Test and Control Seed Materials

. Appropriate quality control methods were used to verify the purity and identity of the plant material used in each study. The pedigree showing the genotypes of Event 5307 seed for the various studies is provided in Figure IV

- 4 . Table

IV

- 2 provides a li st of the sources of plant material used for each study.

A.2. Real

- Time PCR Analysis

All plants grown for genetic characterization studies were individually analyzed for the presence of ecry3.1Ab and pmi by real

- time PCR analysis (Ingham et al. 2001). A control assay targeting the endogenous corn alcohol dehydrogenase gene 1 ( adh1 ) was used to confirm the presence of DNA in each reaction. Leaf discs were sampled from each individual plant. Deoxyribonucleic acid (DNA) was isolated from leaf discs of ea ch individual plant using a method adapted from the Wizard® Magnetic 96 DNA Plant System
for real

- time PCR analysis.

Table A

- 1 lists the primers and probes used to detect ecry3.1Ab, pmi and adh1. Figure A

- 1
shows the locations of the ecry3.1Ab

- specific an d pmi

- specific primers and probes in the transferred DNA (T

- DNA) of plasmid pSYN12274, the transformation plasmid used to generate 5307 corn.

The following cycling parameters were used for this reaction: 95°C for five minutes, followed by 40 cycles of 95 °C for five seconds and 60°C for 30 seconds.

### APPENDIX A

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- 1 Page 125 of 168 Table A

- 1. Real

- time PCR primers and probes used for the detection of ecry3.1Ab , pmi , and adh1

Amplicon of interest Forward primer sequence (5ʹ to 3ʹ) Reverse primer sequence (5ʹ to 3ʹ) Probe sequence (5ʹ to 3ʹ)

ecry3.1Ab TACGAGAGCTGGGTG
AACTTCA CGATCAGGTCCAGCA
CGG CCGCTACCGCCGCG
AGATGA

pmi CCGGGTGAATCAGCG
TTT GCCGTGGCCTTTGAC
AGT TGCCGCCAACGAATC
ACCGG

adh1 GAACGGTGTTGGGTTT
GCAT TGCAGCCTAACCATG
CGCAGGGTA TCCAGCAATCCTTGC
ACCTT



### Figure A

- 1. Locations of real

- time PCR primers and probes in the plasmid pSYN12274
T

- DNA



### A.3. Genomic DNA Extraction

Following verification of the plants’ identity by real

- time PCR analysis (see above), leaf tissue for 10 plants of each genotype was pooled i nto a sampling bag and stored at

- 80°C ± 10°C. Genomic DNA used for nucleotide sequencing and Southern blot analyses was isolated from the pooled leaf tissue from 10 plants per genotype using a modification of the method described in Saghai

- Maroof et al . (1984). Pooled leaf tissue was ground into a fine powder using a pre

- chilled mortar and pestle, with liquid nitrogen, and then placed into a bottle for storage. For each DNA extraction, approximately 40 g of this tissue and 200 ml of prewarmed CTAB buf fer (100 mM Tris pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 2% CTAB [w/v], 0.2% [v/v] β

- mercaptoethanol) were combined in a bottle; the sample was then mixed gently and incubated for 90 minutes at 65°C ± 5°C. An equal volume of chloroform: isoamyl alcohol (24 :1) was then added, followed by gentle mixing and centrifugation for 10
minutes at 7277 × g at room temperature. The resulting aqueous phase was trans

- ferred to a clean container, and 10 μg of ribonuclease per ml of aqueous phase was added. The sample

wa s mixed and incubated for 30 minutes at 37°C ± 2°C. An equal volume of chloroform: isoamyl alcohol (24:1) was then added, followed by gentle mixing and centrifu

- gation for 10
minutes at 7277 × g at room temperature. The aqueous phase was collected in a c lean bottle,

### APPENDIX A

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- 1 Page 126 of 168 and the DNA was precipitated with a 0.8 volume of isopropanol. The DNA was then pelleted by centrifugation at 291 × g and washed once with 70% ethanol. The DNA pellet was air dried and dissolved in 2.5 ml of prewarmed 0.1X Tris

- EDTA.

A.4. D NA Quantitation

The concentration of DNA was measured using a Quant

- iTTM PicoGreen® dsDNA kit. A

two

- point standard curve was generated using a Lambda DNA standard. Genomic DNA was quantified by interpolation from the two point standard curve using the TB S

- 380 Mini

Fluorometer.

A.5. Nucleotide Sequence of the T

- DNA Insert

Two overlapping fragments that span the 5307 corn insert were amplified from genomic DNA using PCR analysis (Figure A

- 2 ). The 5307 plants used for this analysis were from
the NP2171 × BC5F 3 

### generation, as identified in the pedigree chart of plant materials ( Figure IV

- 4 ). PCR amplification was carried out using the ExpandTM Lo ng Template PCR System. Table A

- 3 lists the primers used to amplif y the insert fragments; Tables A

- 4 and A

5 contain the thermal cycling parameters.

Figure A

- 2. Map of the 5307 corn insert and location of PCR

- amplified fragments from 5307 corn to determine insert sequence

Table A

- 2. Primers used to amplify the insert of 5307 corn

### APPENDIX A

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- 1 Page 127 of 168 Table A

- 3. Cycling parameters for PCR amplification of insert Fragment 1

Table A

- 4. Cycling parameters for PCR amplification of insert Fragment 2



### The PCR fragments were cloned into pCR®4

- TOPO® vector , and three colonies for each PCR product were randomly selected and grown . T he plasmid DNA was then independently extracted , and t he resulting plasmid preparations , which contained the PCR amplification products

, were subsequently seque nced.

Dye

- terminator sequencing, a modification of the dideoxynucleotide chain

- terminator sequencing method, was carried out using the ABI3730XL analyzer with ABI BigDye® 3.1 terminator chemistry. The sequence analysis was done using the Phred, Phrap, and Consed package (from the University of Washington), and was carried out to an error rate of less than 1 in 10,000 bases (Ewing and Green 1998).

Three individual clones for each PCR product were sequenced individually, and a consensus
sequence was generat ed for each clone. These sequences were aligned using AlignX

, TM a component of Vector NTI Advance

, TM version 10.3.0, to obtain the final consensus sequence for each segment of the insert sequence.

### APPENDIX A

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- 1 Page 128 of 168 A. 6 

### . Southern Blot Analyses

Southern blot analyses were performed using standard molecular biology techniques (Chomczynski 1992). Each lane contained 7.5 μg of genomic DNA that was digested with the appropriate restriction enzyme(s) for 8 to 16 hours.

A positive control, represent ing one copy of a fragment of known size in the corn genome, was included on each Southern blot. The positive control for these Southern blot analyses was digested DNA from plasmid pSYN12274. This positive control was loaded in a well together with 7.5 μ g of digested DNA from NP2171 × NP2460 plants, so that the migration of this positive control DNA reflected, more accurately, the migration of the restriction fragment in the corn genome. The amount of positive control (picograms for one copy) was
calcula ted by the following formula with a corn genome size of 2.67 × 10 9 

### bp (Arumuganathan and Earle 1991).

The molecular weight marker (serving as the reference substance), the digested genomic DNA, and the positive control were loaded onto 1% SeaKem® Gold a garose gels, and the DNA fragments were then separated by electrophoresis in 1X T ris

- acetate

- E DTA buffer.

Following a 10 minute depurination in 0.25 N HCl, the DNA in the gel was denatured in 0.5
M NaOH and 1.5 M NaCl for 30 minutes. The DNA was then tr ansferred to a Zeta

- Probe GT membrane, by downward alkaline transfer, for 90 minutes using a Bio

- Rad Appligene Vacuum Blotter. After rinsing the membrane briefly in 2X SSC, the DNA was cross

- linked to the membrane using ultraviolet light.

All PCR

- genera ted probes and the molecular weight marker

- specific probe were labeled with phosphorus

- 32

- deoxycytidine tr iphosphate ([α

- 32 

### P]

- dCTP) by random priming using the MegaprimeTM DNA labeling system. Unincorporated label ([α

- 32 

### P]

- dCTP) was removed using the Micro Bio

- Spin® Chromatography Columns.

### APPENDIX A

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- 1 Page 129 of 168 Membranes were incubated in 30 ml of PerfectHybTM Plus Hybridization Buffer (which contained 100 μg/ml denatured Calf Thymus DNA) for at least 30 minutes at 65°C ± 5°C. Both the molecular weight marker

- specific probe an d either the full length T

- DNAspecific probe or backbone

- specific probe were added to the hybridization solution, and the membranes were incubated for 16 hours at 65°C ± 5°C. Incubation was followed by a
combination of washes at 65°C ± 5°C in 2X SSC with 0.1% SDS and washes at 65°C ± 5°C in 0.1X SSC with 0.1% SDS. Finally, the membranes were subjected to imaging using a Molecular Dynamics Storm 860® phosphorimager.

A .7. Nucleotide Sequencing of T

- DNA Flanking Regions in Corn Genome

T he 5 ʹ and 3 ʹ corn genomic sequences flanking the 5307 corn insert were previously recovered. This preliminary sequence was used to design primers for amplification of the flanking regions from 5307 corn. The flanking regions were amplified from genomic DNA
extracted from 5307 corn using the ExpandTM High

- Fidelity PCR System. Table A

- 5 lists the primers used to amplify the flanking regions; Table A

- 6 contains the thermal cycling
parameters.

Table A

- 5. Primers used to amplify the flanking regions of 5307 DNA insert

Tab le A

- 6. PCR cycling parameters for the flanking regions of 5307 DNA insert



### The PCR fragments were cloned into pCR ® 

### 4

- TOPO ® 

### vector, and three colonies for each PCR product were randomly selected and grown. The plasmid DNA was then independently extracte d, and the resulting plasmid preparations, which contained the PCR amplification products, were subsequently sequenced as described above ( see Nucleotide Sequence of the T

- DNA Insert).

### APPENDIX A

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- 1 Page 130 of 168 A.8. Flanking Sequence Analysis to Determine if T

- DNA Inserted into a Known Corn
Gene

The following parameters were used for the BLASTN analysis against the National Center for Biotechnology Information (NCBI) Nucleotide Database (NCBI 2010a) to identify corn genomic sequences having identity or high similarity to sequence s flanking the 5′ and 3′

ends of the 5307 T

- DNA insert:

**•** 

### Expect = 10. The expectation value ( E

- value) is a measure of the probability that matches between sequences occurred by chance. Search results involving comparisons between nucleotides with highly s imilar sequences yield E

- values approaching zero; the probability that sequence similarities occurred by chance increases with higher E

- values (Ponting 2001). The search identified all sequences in the database with search results yielding an E

- value of 1 0 or lower; this is the conservative default search setting for this parameter.

**•** 

### The scoring scheme used was the default for nucleotides: +1 for a match and

- 3 for a mismatch.

**•** 

### The following gap penalties were used for this scoring matrix: Existence = 5 a nd Extension = 2. A gap is a space introduced into an alignment to compensate for insertions and/or deletions in one sequence relative to another. The introduction of a gap causes the deduction of a fixed value from the alignment score to prevent the

acc umulation of excessive gaps in an alignment. Extension of the gap to encompass additional nucleotides is also penalized in determining the score of an alignment. The resultant score is derived from the number of identical matches between the query

sequen ce and the database entry, with higher scores indicating more identity between the two sequences.

**•** 

### A low complexity filter was used for this search.

A.9. Amino Acid Sequence Comparison of Query Peptide to Known or Putative
Toxins

The Basic Local Alignment Search Tool for Proteins (BLASTP) program, version 2.2.19, (Altschul et al

. 1997) was used to compare the query peptide representing the translated sequence of the putative 243

- bp ORF to all entries in the NCBI Entrez® Protein Dat abase (containing over 10 million amino acid sequences) (NCBI 2010b). Information associated with the sequences having the highest similarity to the query sequence was examined to determine if any of the sequences was a toxin or putative toxin.

The BLAS TP algorithm is optimized to identify regions of local similarity between protein sequences. This approach detects more similarities than would a search that aligns two sequences over their entire length. The following default parameters were used in the BLASTP comparisons:

### APPENDIX A

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- 1 Page 131 of 168 **•** Expectation value ( E

- value) = 10.

**•** Word size = 3

**•** Gap costs: existence = 11 and extension = 1

**•** Similarity matrix: Blocks Substitution Matrix62 (BLOSUM62)

**•** No complexity filter

The E

- value is a measure of the probability that match es between sequences occurred by chance. Search results involving comparisons between proteins with highly similar sequences yield E

- values approaching zero; the probability that sequence similarities occurred by chance increases with higher E

- values (Pon ting 2001). The search identified all sequences in the database with search results yielding an E

- value of 10 or lower. These sequences were evaluated for source and biological function. Any sequences described as toxins or putative toxins were identifi ed.

A.10. Amino Acid Sequence Comparison of Query Peptide to Known or Putative Allergens

T he 81

- amino

- acid query peptide sequence representing the translated sequence of the putative 243

- bp ORF was screened for biologically relevant amino acid sequence s imilarity to any of the known or putative protein allergens within the FARRP AllergenOnline database (FARRP 2010). The FARRP AllergenOnline database is a curated, peer

- reviewed database containing proteins identified as food allergens, respiratory allerge ns, allergenic venom proteins, contact allergens, gliadins, and glutenins. Entries were compiled primarily from searches of publicly available protein databases using the NCBI Entrez® search and retrieval system, most recently searched in 2009 (NCBI 2009) . The NCBI dataset was screened by searches for entries associated with allergy or celiac disease; duplicate entries were removed, and additional entries were identified from publications. The list of candidate entries was then reviewed by an internation al panel of allergy experts who reviewed published clinical and laboratory evidence to support the candidate sequences as allergens. Proteins are classified as known or putative allergens according to predetermined criteria set by the FARRP expert review panel. The latest version of the FARRP AllergenOnline database (2010) contains 1,471 nonredundant entries. Similarity searches were performed using an exact copy of the entire list of sequences in the current version of the FARRP AllergenOnline database (2010) (maintained at Syngenta Biotechnology, Inc., Research Triangle Park, NC, USA). Two different sequence searches were performed to compare the translated junction sequence with sequences in the FARRP AllergenOnline database. In the first search, the FASTA search algorithm, version 3.45 (Pearson and Lipman 1988), was used to assess overall sequence similarity by comparing sequential 80

- amino

- acid peptides of the query sequence with the sequences in the FARRP

### APPENDIX A

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- 1 Page 132 of 168 AllergenOnline database. Each successive “ window” of 80 amino acids was offset from the previous window by one residue, such that each peptide overlapped the previous peptide by 79 amino acids. The default FASTA settings used include an extension penalty of two and gap creation penalty of 12. Th e scoring matrix for FASTA was the Blocks Substitution Matrix50 (BLOSUM50), the same scoring matrix used by the authors of the FARRP AllergenOnline database. The BLOSUM50 matrix is weighted to favor identical amino acids likely to impact protein structure . In the second search, the query sequence was screened for matches of eight or more contiguous amino acids (Hileman et al

. 2002) using a program developed by Syngenta; this program compared every possible peptide of eight contiguous amino acids of the tr anslated putative ORF with the sequences in the FARRP AllergenOnline database.

The FASTA search produces alignments between the 80

- amino

- acid peptides of the query sequence and the sequences in the allergen database. The evaluation of each query peptide sequence alignment utilizes the minimum criterion of 80 amino acids of alignment length with greater than 35% shared amino acid identity. Any alignments excee ding this criterion for shared sequence similarity indicate the potential for immunologically relevant sequence similarity (Codex 2009). Additionally, any match of eight (or more) identical contiguous amino acids between any query sequence and any sequenc e in the allergen database indicates the potential for immunologically relevant sequence similarity (Codex 2009).

### APPENDIX B

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- 1 Page 13 3 of 168 

## Appendix B: Materials, Methods and Results of C haracterization Studies

on the eCry3.1Ab and PMI Proteins

This appendix presents materials, methods and detailed results of analyses of the biochemical properties and biological activity of the eCry3.1Ab and PMI proteins in 5307 corn and in the corresponding eCry3.1Ab and PMI test substances used in safety studie s. A summary of the results and conclusions of these analyses is provided in Part VI of this
submission

, titled Safety Assessment of the Introduced eCry3.1Ab and PMI Proteins

.



### B .1. Materials and Methods for Comparison of eCry3.1Ab Produced in 5307 Corn
Plants and Recombinant E. coli

Three preparations containing eCry3.1Ab were evaluated in this study: (1) LP5307,
extracted from leaf material of 5307 corn plants ; ( 2 ) IAP5307, immunopurified eCry3.1Ab
derived from lea f material of 5307 corn plants ; and (3 ) microbial test substance

ECRY3.1AB

- 0208, prepared from a recombinant E

. coli overexpression system

. The sample designated LP5307 was used for Western blot analy sis and insecticidal activity assays. The sample designated IAP5307 was used as the source of purified plant

- produced eCry3.1Ab for Western blot, glycosylation, N

- terminal sequencing and peptide mass mapping analyse s. Nontransgenic, near

- isogenic plants were used as negative control plant material, the source of the material designated LP

- NEG.

Event 5307 Corn Leaf Tissue and Negative Control Corn Leaf Tissue

Young leaves from greenhouse

- grown 5307 plants and nontransgenic, near

- isogenic plants
were collec ted 4

- 6 weeks after emergence, frozen at

– 80°C ± 10°C, and ground into a fine powder using a Grindomix Knife Mill (Retsch).

Extracts of 5307 Corn and Control Corn Leaf Tissue for Western Blot Analysis

Leaf powder was resuspended in extraction buffer cont aining 100 mM sodium borate (pH 10.0), 0.2% p olyvinylpyrrolidone ( PVP), 7.69 mM sodium azide, 0.5% Tween 20, and supplemented with one Complete Protease Inhibitor Cocktail tablet/50 ml of buffer (Roche). The mixture was homogenized with an Omni

- Prep Homog enizer (Omni International), incubated for 30 minutes on ice and centrifuged at 3,000 rpm for 15 minutes at 4°C (Sorvall Legend RT). The resulting supernatants were stored overnight at 2°C to 8°C and then stored

at

- 20°C ± 8°C in 4X NuPage LDS Sample Buff er (Invitrogen) containing NuPage Sample Reducing Agent (Invitrogen) for subsequent Western blot analysis. The sample extracts from the 5307 and control corn leaves were designated LP5307 and LP

- NEG, respectively.

### APPENDIX B

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- 1 Page 134 of 168 Extracts of 5307 Corn and Control Corn Leaf Tissue for Insect Bioassays

Extracts for bioassays were prepared by resuspending leaf powder from 5307 and control plants as described above, in a 10 mM ammonium bicarbonate, pH 10.0 buffer. The extraction mixtures were homogenized in a Waring blend er for 45 seconds, incubated on ice for 1.5 hours and centrifuged (Sorvall RC5B) at 8,000 rpm for 30 minutes at 4°C. The resulting supernatant was filtered through cheesecloth, centrifuged for an additional 30 minutes at 8,000 rpm at 4°C, and filtered thr ough cheesecloth again. The resulting clear supernatant was concentrated using centrifugal filter devices (Millipore). The sample was then stored overnight at 2

- 8°C for subsequent insect diet incorporation and ELISA analysis. The sample extracts from th e 5307 and control corn leaves were designated LP5307 and LP

NEG, respectively.

Extracts of Control Leaf Tissue Fortified with Test Substance ECRY3.1AB

- 0208 for Western Blot Analysis

To determine whether the plant matrix affects eCry3.1Ab mobility or immun oreactivity,

ECRY3.1AB

- 0208 was added to control leaf extract. This sample allowed for comparison of the microbially produced eCry3.1Ab and plant

- produced eCry3.1Ab in the same matrix. For Western blot analysis, ECRY3.1AB

- 0208 was added to LP

- NEG, as pre pared for above Western blot analysis, such that the total protein and amount of eCry3.1Ab loaded on the gel was equivalent to that estimated for sample LP5307, as prepared for Western blot analysis. This sample was designated LP

- NEG + ECRY3.1AB

- 0208.

Ext racts of Control Leaf Tissue Fortified with Microbially Produced Test Substance
ECRY3.1AB

- 0208 for Insect Bioassays

To determine if the plant matrix affects bioactivity, ECRY3.1AB

- 0208 was added to control leaf extract. This sample allowed for comparison of the microbially produced eCry3.1Ab and plant

- produced eCry3.1Ab in the same matrix. For the bioassays, ECRY3.1AB

- 0208
was added to LP

- NEG, as prepared for bioassays, such that when incorporated into the diet the concentration of eCry3.1Ab in the diet w as equivalent to the eCry3.1Ab concentration in the diet containing only the ECRY3.1AB

- 0208 test substance. This sample was designated

LP

- NEG + ECRY3.1AB

- 0208.

Immunoaffinity

- Purified Plant

- Produced Protein

Leaf powder from 5307 corn, prepared as described above, was resuspended in extraction buffer (pH 7.5) containing 100 mM sodium borate, 0.2% PVP, 7.69 mM sodium azide, 1.2% concentrated hydrochloric acid, 0.5% Tween 20, supplemented with one Complete Protease
Inhibitor Cocktail tablet/50 ml of b uffer (Roche). The mixture was homogenized with an
Omni

- Prep Homogenizer and incubated for up to 2 hours on ice. The mixture was then centrifuged at approximately 2700 rpm for 10 minutes at 4°C (Sorvall Legend RT). The supernatant was filtered through c heesecloth and centrifuged at approximately 3100 rpm for

### APPENDIX B

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- 1 Page 135 of 168 15 minutes at 4°C (Sorvall Legend RT). After a second centrifugation step (10,000 rpm for
12 minutes ; Sorvall RC5B) the clarified supernatant was then loaded onto an equilibrated immunoaffinity colu mn with mouse anti

- mCry3A antibodies bound to the matrix. To remove any proteins not bound to the antibodies, the column was washed with a 50 mM sodium bicarbonate buffer pH 8.0 containing 150 mM sodium chloride. After an additional wash step with a 10 m M sodium phosphate buffer pH 6.8, eCry3.1Ab was eluted in 100 mM glycine buffer (pH 2.5), neutralized, and fractions were analyzed for eCry3.1Ab protein by ELISA. Fractions containing eCry3.1Ab protein were pooled, concentrated by ultrafiltration, and sto red at 2°C to 8°C until further use. The resulting sample, designated IAP5307, was used as the source of purified plant

- produced eCry3.1Ab for Western blot, glycosylation, N

- terminal sequencing and peptide mass mapping analysis.

Microbially Produced Test Substance ECRY3.1AB

- 0208

Test substance ECRY3.1AB

- 0208 was prepared from an E. coli overexpression system. The eCry3.1Ab protein in test substance ECRY3.1AB

- 0208 is identical to that expressed in 5307 corn except that it contains one additional methionine and six histidine residues at the N

terminus. The intended additional seven amino acids aid in purification from the E. coli overexpression system. The ecry3.1Ab gene used for microbial expression was linked to the
bacterial tac promoter in a vector derived from pET24a (Novagen) and transformed into E.
coli strain DH5α (New England Biolabs ). ECRY3.1AB

- 0208 was prepared from pooled
batches of E. coli cell paste. E. coli cells were ruptured and the cell debris removed by centrifug ation. The soluble material was filtered, applied to an immobilized metal affinity column (GE Healthcare Nickel Sepharose Fast Flow column), and eluted using an imidazole step gradient. Fractions containing the eCry3.1Ab protein were then further purifie d via anion exchange chromatography and eCry3.1Ab was eluted with a sodium chloride gradient. The eluted eCry3.1Ab

- containing fractions were pooled, concentrated and the buffer was exchanged. The solution was lyophilized and designated ECRY3.1AB

- 0208. T he test
substance was stored at

- 20°C ± 8°C until further use. ECRY3.1AB

- 0208 was determined to contain 89.6% eCry3.1Ab by weight and the intact mass of the eCry3.1Ab protein, as measured by mass spectrometry, was 74.8 kDa.

eCry3.1Ab Quantification

The Be acon Analytical Systems (BAS) eCry3.1Ab ELISA kit was used as described in Appendix C ., section C .4., to quantify eCry3.1Ab.

Total Protein Determination

Total protein in samples, LP5307, LP

- NEG + ECRY3.1AB

- 0208, and LP

- NEG as prepared
for Western blot an alysis, was quantified via the bicinchoninic acid method (Hill and Straka 1988), using bovine serum albumin as the reference protein standard. The results were

### APPENDIX B

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- 1 Page 136 of 168 analyzed with BioMetallics DeltaSoft PC Microplate Analysis Software, v. 1.71.2

, using a
four

- p arameter fit of the standard curve.

Immunoreactivity and Molecular Weight Determination

Western blot analysis was used to investigate the integrity of eCry3.1Ab in ECRY3.1AB

0208, LP5307, LP

- NEG + ECRY3.1AB

- 0208 and IAP5307. Aliquots containing 10 ng of eCry3.1Ab prepared in NuPage LDS Sample Buffer (Invitrogen) were subjected to SDS polyacrylamide gel electrophoresis (SDS

- PAGE) with a NuPAGE 4

- 12% Bis

- Tris polyacrylamide gradient gel (Invitrogen) using 3

- (N

- morpholino)propane

- sulfonic acid (MOPS) running buffer (Bio

- Rad). An aliquot of the control plant sample LP

- NEG, equivalent in total protein to the amount lo aded on the gel for LP5307 (29.6 μg total protein), was included in the analysis as a negative control. The molecular

- weight standard
was SeeBlu e Plus2 pre

- stained standard (Invitrogen). After electroblotting, the membrane was probed with polyclonal goat antibodies capable of detecting eCry3.1Ab protein. Alkaline phosphatase

- conjugated donkey anti

- goat IgG (Jackson ImmunoResearch) diluted

to 1:3 ,000 in Tris

- buffered saline with Tween 20 (Sigma

- Aldrich) and 5% normal donkey serum was used to bind to the primary antibody and was visualized by development with 5

- bromo

- 4

- chloro

- 3

- indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) alkaline phosphatas e substrate solution (Sigma

- Aldrich). The Western blot was examined for the presence of intact immunoreactive eCry3.1Ab or immunoreactive eCry3.1Ab fragments.

Insecticidal Activity

The insecticidal activity of eCry3.1Ab was assessed in feeding assays with freshly hatched
first

- instar Colorado potato beetles (L. decemlineata) in three independent bioassays

. The insect diet was prepared by blending a boiling mixture of 2.6 grams of agar and 169 ml of

Milli

- Q water with 28.1 grams of Colorado potato beetle d iet powder mix and 1 gram of potassium hydroxide as per the manufacturer’s instructions (Bio

- Serv). The diet mixture was cooled to approximately 55°C in a water bath. Antibacterial and antifungal agents were
each added to the cooled diet.

Bioassay treat ments consisted of (1) test substance ECRY3.1AB

- 0208, (2) eCry3.1Ab extracted from 5307 corn leaves; LP5307, (3) control leaf extract fortified with test
substance ECRY3.1AB

- 0208; LP

- NEG + ECRY3.1AB

- 0208, and (4) control corn leaf tissue extract; LP

- NEG. A a stock solution o f ECRY3.1AB

- 0208 was prepared in 10 mM ammonium bicarbonate buffer (pH 10.0) to a concentration of 5 mg eCry3.1Ab/ml. Fr om th is , a 50 μg eCry3.1Ab/ml solution was prepared and serially diluted 1:1 (v/v) in 10 mM
ammonium bicarbonate bu ffer (pH 10.0) to produce eight solutions with concentrations ranging from 50 to 0.390 μg eCry3.1Ab/ml. The dilution series was t hen mixed 1:1 (v/v)
with the freshly prepared Colorado potato beetle diet to produce eight diets with eCry3.1Ab
concentrations ranging from 25 to 0.195 μg/ml diet. Additional treatments containing

### APPENDIX B

5307

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- 1 Page 137 of 168 eCry3.1Ab extracted from 5307 corn leaves, LP5307 (treatment 2), control leaf extract
fortified with the microbially

- produced test substance

, LP

- NEG + ECRY3.1AB

- 0208 (tr eatment 3) and control leaf tissue extract LP

- NEG (treatment 4) as prepared for bioassays were also serially diluted 1:1 (v/v) in 10 mM ammonium bicarbonate buffer (pH 10.0) and subsequently mixed 1:1 (v/v) with freshly prepared Colorado potato beetle diet . Each of these treatments was analyzed as a series of eight dilutions. Water and buffer controls were prepared in the same manner for each bioassay. The water control was prepared by mixing 1:1 (v/v) of purified water with freshly prepared Colorado pot ato beetle diet. The buffer control was prepared by mixing 1:1 (v/v) of 10 mM ammonium bicarbonate buffer (pH 10.0) with freshly prepared Colorado potato beetle diet.

The bioassays were conducted in 24

- well culture plates. Each well contained one freshl y hatched L. decemlineata insect larva and 100 μl of insect diet. Larvae were transferred to each well manually using a small paint brush. The wells were covered with silicone stoppers and stored at ambient laboratory conditions. Mortality readings were taken periodically starting at 72 hours and continued until at least 144 hours.

Glycosylation Analysis

To determine whether eCry3.1Ab in ECRY3.1AB

- 0208 and eCry3.1Ab immuno

- affinity purified from 5307 corn leaf extract (IAP5307) were glycosylated, aliquo ts equivalent to 1 and 2 μg of eCry3.1Ab were analyzed with the DIG Glycan Detection Kit (Roche), in accordance with the manufacturer’s instructions. The positive control was transferrin (a glycosylated protein) at 100, 50, 25 and 10 ng, and the negative control was creatinase (a nonglycosylated protein) at 2 μg. Samples were separated by SDS

- PAGE with a

NuPAGE4

- 12% Bis

- Tris polyacrylamide gradient gel and NuPAGE MES SDS running buffer and electroblotted to nitrocellulose membrane (Invitrogen). While on the membrane, glycan moieties were oxidized with periodate, labeled with digoxigenin (DIG), and detected
with an alkaline

- phosphatase

- linked anti

- DIG antibody.

Peptide Mass Mapping Analysis

eCry3.1Ab purified from an extract of 5307 corn leaves (IAP5307) a nd from ECRY3.1AB

0208 were analyzed by peptide mass mapping. Aliquots containing 2.5 to 5 μ g of eCry3.1Ab purified from 5307 corn leaf extract (IAP5307) and from test substance

ECRY3.1AB

- 0208 were subjected to SDSPAGE using a NuPAGE 4

- 12% Bis

- Tris polyac rylamide gradient gel and NuPAGE MES SDS running buffer. The gel was stained with Coomassie G250 (Invitrogen), the protein band corresponding to the molecular weight of eCry3.1Ab was excised from the gel, and the protein was reduced, alkylated with
iodoac etamide, and independently digested with trypsin and chymotrypsin. The mass analysis of the eCry3.1Ab

- produced peptides was performed using a quadrupole time

- of

-

### APPENDIX B

5307

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- 1 Page 138 of 168 flight mass spectrometer (Waters/Micromass Q

- TOF Premier) connected to a Waters CapLC capillar y liquid chromatography instrument. The detected peptide masses were searched using Mascot Software (Matrix Science) against a protein database containing the eCry3.1Ab protein sequence. The Mascot search parameters included likely N

- terminal
modificatio ns, which have previously been reported to occur in plants. Specifically, the
modifications investigated included α

- N

- acetylation, protein N

- formylation and protein N

methylation.

N

- Terminal Amino Acid Sequence Analysis

To determine the N

- terminal amino a cid sequence of eCry3.1Ab from test substance

ECRY3.1AB

- 0208 and eCry3.1Ab purified from 5307 corn leaf extract (IAP5307) were both subjected to SDS

- PAGE followed by electroblotting to a PVDF membrane. The blot was stained with amido black, and the band c orresponding to eCry3.1Ab was excised and subjected to N

- terminal amino acid sequence analysis using automated Edman

- based chemistry (Brauer et al. 1984).

Statistical Methods

The LC 50 

### values determined in the insecticidal activity assay were calculated using the U.S. EPA Probit Analysis Program, version 1.5.

B .2. Results of Comparison of eCry3.1Ab Produced in 5307 Corn Plants and
Recombinant E. coli

Immunoreactivity and Molecular Weight

Western blot analysis of eCry3.1Ab in test substance ECRY3.1AB

- 0208, LP

- NEG +

E CRY3.1AB

- 0208, LP5307, and IAP5307 revealed immunoreactive bands consistent with the predicted molecular weight 1 

### o f 74.8 kDa for samples containing eCry3.1Ab from

ECRY3.1 AB

- 0208 ( Figure B

- 1, Lanes 2, 3 and 6) an d 73. 7 kDa for samples containing plant

- produced eCry3.1Ab (L anes 4 and 5).

1 Although the eCry3.1Ab protein band showed slightly higher mobility (and therefore an apparent lower molecular weight [mw] ) in comparison to the mw standards on the Western blot ( Figure B

- 1), the difference between the exp ected and
observed mw on the gels can be ex plained by the limitations of SDS

- PAGE for accurate determination of m w

. Dube and Flynn (1988) have reviewed the reliability of SDS

- PAGE for m w determinations and concluded that the apparent m w of a protein by this method is typically within 10% of its true m w

. This depends greatly on the similarity between the properties of the protein of interest and the proteins in the standard set (Sadeghi et al

. , 2003). Additionally, the intact mass of eC ry3.1Ab in
ECRY3.1AB

- 0208 waspreviously me asured as 74.8 kDa

.

### APPENDIX B

5307

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- 1 Page 139 of 168 Figure B

- 1. Western blot analysis of eCry3.1Ab in test substance ECRY3.1AB

- 0208, LP

- NEG + ECRY3.1AB

- 0208, LP5307, IAP5307 and the negative control LP

- NEG.

Lane 1: Molecular

- weight markers
Lane 2: Microbially produced test substance ECRY3.1AB

- 0208 (10 ng eCry3.1Ab)
Lane 3: Control corn leaf extract, LP

- NEG (29.6 μg total protein) with the addition of test substance
ECRY3.1AB

- 0208 (1 0 ng eCry3.1Ab)
Lane 4: 5307 corn leaf extract, LP5307 (10 ng eCry3.1Ab/29.6 μg total protein)
Lane 5: Immunopurified eCry3.1Ab protein from 5307 corn leaf extract, IAP5307 (10 ng eCry3.1Ab)
Lane 6: Test substance ECRY3.1AB

- 0208 (10 ng eCry3.1Ab)
Lane 7 : Control corn leaf extract, LP

- NEG (29.6 μg total protein)
Lane 8: Molecular

- weight markers



### The slight difference in molecular weight between the microbial and plant sources of eCry3.1Ab was consistent with the presence of the seven additional N

- termi nal amino acids in the microbially produced protein. As expected, no immunoreactive bands were observed
in the plant

- produced control substance

, LP

- NEG (Figure B

- 1, Lane 7).

Insecticidal Activity

The results of the insect bioassays were presented in Tabl e VI

- 1 and discussed in Part VI.C
of this submission

.

Glycosylation Analysis

The positive control protein, transferrin, at 10 ng generated a clearly visible band (Figure B

2, Lane 4). Transferrin has a molecular weight of approximately 80,000 Da and contains approximately 5% glycan moieties by weight. This corresponds to approximately 25 glucose equivalents per molecule (based on a calculated molecular weight of 162 Da for the

### APPENDIX B

5307

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- 1 Page 140 of 168 glycan moiety). Of the 10 ng of transferrin loaded on the gel, 0.5 ng could be attributed to glycan moieties and was clearly detectable. The highest concentration of eCry3.1Ab from both plant and microbial sources (IAP5307 and ECRY3.1AB

- 02 08) loaded on the blot was 2 μg (2,000 ng). If 0.5 ng of glycan were detected in eCry3.1Ab, this would correspond to 0.025% by weight (0.5/2,000 ng), or 0.115 glucose equivalents per molecule. In other words, if eCry3 .1Ab bands were stained as strongly as 10 ng of transferrin in Lane 4, this would indicate glycosylation of about 1 in 8.7 of the eCry3.1Ab molecules. No bands corresponding to glycosylated eCry3.1Ab were visible for the sample prepared fro m the microbially produced ECRY3.1AB

- 0208 test subs tance ( Figure B

- 2, Lanes 9 and 10) or immunopurified plant

- produced eCry3.1Ab, IAP5307 (Lanes 7 and 8). Therefore, the
results indicate that neither the mic robially produced nor the plant

- produced eCry3.1Ab protein was glycosylated.

### APPENDIX B

5307

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- 1 Page 141 of 168 Figure B

- 2. Glycosylation analysis of eCry3.1Ab i n IAP5307 (immunopurified p lant

- produced protein ) and ECRY3.1AB

- 0208 ( microbially produced test substance ).

Lane 1: Transferrin (positive control), 100 ng Lane 2: Tr ansferrin (positive control), 5 0 ng Lane 3: Tr ansfer rin (positive control), 25 ng Lane 4: Transferrin (positive control), 10 ng Lane 5: Creatinase (negative control), 2 μg

. Lan e 6: Molecular

- weight markers.
Lane 7: Immunopurified eCry3.1Ab protein from Event 5307 corn leaf extract, IAP5307; 1 μg Lane 8: Immunopurified eCry3.1Ab protein from Event 5307 corn leaf extract, IAP5307; 2 μg Lane 9: eCry3.1Ab from microbially produced test substance ECRY3.1AB

- 0208; 1 μg
Lane 10: eCry3.1Ab from microbially produced test substance ECRY3.1AB

- 0208; 2 μg

.

### APPENDIX B

5307

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- 1 Page 142 of 168 Peptide Mass Mapping

Analysis of the plant

- produced eCry3.1Ab yielded coverage equivalent to 76% of the total predicted eCry3.1Ab amino acid sequence, as shown in Figure B

- 3. Analysis of the microbially produced eCry3.1Ab yielded coverage equivalent to 87 % of the total predicted eCry3.1Ab amino aci d sequence, as shown in Figure B

- 4. The identified peptides corresponded to regions throughout the sequence of eCry3.1Ab including the N

- termini of both proteins. The results of the peptide mass mapping analysi s confirmed the identity of the purified proteins from both sources as eCry3.1Ab.

Figure B

- 3. Predicted amino acid sequence of eCry3.1Ab and sequence identified by peptide mass mapping analysis of eCry3.1Ab from immunopurified plant

- produced sample IAP530 7.

Identified eCry3.1Ab protein fragments are bold and underlined.



### M **TSNGRQCAGIRPY** DGRQQHR **GLDSSTTKDVIQK** GISVVGDLLGVVGFPFGGALVSFYTNF

LNTIWPSEDPW **KAFMEQVEALMDQK** IADYAKNKALAELQGLQNNVEDYVSALSSWQK **NPAA**
**PFRNPHSQGRIRELFSQAESHFRNSMPSFAISGYEVLFLTTYAQAANTHLFLLKDAQIYGE**

**EWGYEKEDIAEFYKR** QLK **LTQEYTDHCVKWYNVGLDKLRGSSYESWVNFNR** YRR **EMTLTVL**

**DLIALFPLYDVRLYPKEVKTELTRDVLTDPIVGVNNLRGYGTTFSNIENYIRKPHLFDYLH**

**R** IQF **HTRFQPGYY** GNDSFNYWSGNY **VSTRPSIGSNDIITSPFYGNKSSEPVQNLEFNGEK** V

Y **RAVANTN LAVWPSAVYSGVTKVEFSQYNDQTDEASTQTYDSKR**  NVGAVSWDSIDQLPPET

TDEPLEK **GYSHQLNYVMCFLMQGSRGTIPVLTWTHKSVDFFNMIDSKKITQLPLTKSTNLG**

**SGTSVVKGPGFTGGDILR** R **TSPGQISTLRVNITAPLSQR** YRVRIR **YASTTNLQFHTSIDGR**
**PINQGNFSATMSSGSNLQSGSFR** TVGFTTPFNFSNGSSVF **TLSAHVFNSGNEVYIDR** IEF **V**

**PAEVTF** EAEY **DLERAQKAVN ELFTSSNQIGLKTDVTDYHID**  QV

Figure B

- 4. Predicted amino acid sequence of eCry3.1Ab and sequence identified by peptide mass mapping analysis of eCry3.1Ab from microbially produced test substance ECRY3.1AB

0208.

Identified eCry3.1Ab protein fragments are bold and underlined.



### MHHHHHHMTSNGRQCAGIRPY DGRQQHR **GLDSSTTKDVIQK** GISVVGDLLGVVGFPFGGAL

VSFYTNFLNTIWPSEDPW **KAFMEQVEALMDQK** IADY **AKNKALAELQGLQNNVEDYVSALSS**
**WQKNPAAPFRNPHSQGRIRELFSQAESHFRNSMPSFAISGYEVLFLTTYAQAANTHLFLLK**
**DAQIYGEEWGYEKEDIAEFYKR** QLK **LTQEYTDHCVKWYNVGLDKLRGSSYESWVNFN R**  YRR

**EMTLTVLDLIALFPLYDVRLYPKEVKTELTRDVLTDPIVGVNNLRGYGTTFSNIENYIRKP**
**HLFDYLHRIQF** HTRFQPGYYGNDSFNYWSGNY **VSTRPSIGSNDIITSPFYGNKSSEPVQNL**

**EFNGEK** VYR **AVANTNLAVWPSAVYSGVTKVEFSQYNDQTDEASTQTYDSKRNVGAVSWDSI**
**DQLPPETTDEPLEKGYSHQLNYVMCFLMQGSRGTIPVLTWTHKSVDFFNMIDSKKITQLPL**

**TKSTNLGS GTSVVKGPGFTGGDILRRTSPGQISTLRVNITAPLSQR**  YRVRIR **YASTTNLQF**
**HTSIDGRPINQGNFSATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY**
**IDRIEFVPAEVTFEAEYDLERAQKAVNELFTSSNQIGLKTDVTDYHIDQV**

### APPENDIX B

5307

- FDA

- 1 Page 143 of 168 The peptide mass analysis provided two additional results regarding the structure of the proteins. Firstly, the intact N

- terminus of the plant

- produced protein could be identified. The analysis showed that the N

- terminal methionine was removed leaving the penultimate amino acid, threonine, at the N

- terminus of the eCry3 .1Ab protein. This is a common process for many proteins occurring d uring translation (Walling 2006). Secondly, the nature of the N

- ter minal block found for the plant

- produced protein, as described under N

Terminal Amino Acid Sequence Analysis , below, wa s identified. The analysis of the N

terminal peptide of the plant

- produced protein suggested the addition of an acetyl

- residue at the primary amino group of the N

- terminal threonine

. This is a common modification known for plant

- expressed proteins (Marti nez et al. 2008).

N

- Terminal Amino Acid Sequence Analysis

The N

- terminal sequence results confirmed that eCr y3.1Ab in the microbially produced test substance ECRY3.1AB

- 0208 had the predicted N

- terminal amino acid sequence:

Predicted sequence 

### : MHHHHHHMTS

e Cry3.1Ab in ECRY3.1AB

- 0208 

### : MHHHHHHMTS

N

- terminal sequencing analysis of eCry3.1Ab immunoaffinity

- purified from 5307 corn leaves (IAP5307) revealed that the majority of the protein was naturally blocked at the N

terminus. However, the N

- terminal peptide w as identified by peptide mass mapping (see Peptide Mass Mapping , above) and confirmed the expected sequence for the eCry3.1Ab protein, starting at threonine as described above. The analysis of the N

- terminal peptide of the plant

- produced protein suggested the addition of an acetyl residue at the primary amino group of the N

- terminal threonine

.

### APPENDIX B

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- 1 Page 144 of 168 B .3. Materials and Methods for Comparison of PMI Produced in 5307 Corn Plants
and Recombinant E. coli

The purpose of these analyses was to compare PMI from 5307 corn plants with PMI from
test substance PMI

- 0105, which was produced by overexpressing the gene pmi in
recombinant E. coli . The PMI proteins from both sources were compared biochemically and
func tionally to justify use of PMI in PMI

- 0105 as a surrogate for PMI in 5307 corn plants, for safety testing purposes. Both sources of PMI were predicted to have the identical amino acid sequence, because the gene pmi in both the plant and E. coli transforma tion vectors encoded the same PMI enzyme.

The conclusions of these analyses are described in Part VI.I of this submission

, Characterization of th e Plant

- and Escherichia coli

- produced PMI Proteins

.

Event 5307 Corn Leaf Tissue and Negative Control Corn Leaf Tissue

Young leaves from greenhouse

- grown 5307 plants and nontransgenic, near

- isogenic plants
were collected 4

- 6 weeks after emergence, frozen at

– 80°C ± 10°C, and ground into a fine powder using a Grindomix Knife Mill (Retsch). The leaf powder was then lyophilized and
stored at

- 80 ± 10°C.

Extracts of 5307 and Control Corn Leaf Tissue

Approximately 100 mg of lyophilized leaf powder was suspende d in 3 ml extraction buffer (pH 7.0) containing 50 mM Tris, 2 mM DTT), 1 mM AEBSF, and 1 μM leupeptin. The mixture was then homogenized using an Omni

- Prep Homogenizer (Omni International) and centrifuged for 15 minutes at 10,000 rpm at approximately 4°C. The supernatant was desalted using PD

- 10 columns (GE Healthcare Life Sciences) and eluted in extraction buffer. The resulting supernatant was then concentrated using the Millipore Amicon Ultra centrifugal filter devices (Millipore) and Tween 20 was added to achieve a final concentration of 0.1%. The samples derived from the 5307 and control corn tissues were designated LP5307 and LP

- NEG, respectively. They were stored at 2

- 8°C overnight prior to ELISA and enzymatic activity assays.

Extract of Control L eaf Tissue Fortified with Test Substance PMI

- 0105

PMI from test substance PMI

- 0105 was added to the extraction buffer with an aliquot of control leaf tissue to determine if the plant matrix or extraction procedure has an effect on PMI. For this sample pre paration, 600 ng PMI from test substance PMI

- 0105 was added to 3 ml of extraction buffer containing 100 mg of control leaf tissue prior to homogenization. The sample was then homogenized, centrifuged, desalted and concentrated as described above (see Extr acts of 5307 and Control Corn Leaf Tissue) . This sample was designated

LP

- NEG + PMI

- 0105.

### APPENDIX B

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- 1 Page 145 of 168 PMI Protein Quantification

The concentration of PMI in the plant extract samples (LP5307), negative control samples (LP

- NEG), and the negative control sample fortifi ed with test substance PMI

- 0105 (LP

NEG + PMI

- 0105) was determined by ELISA (Tijssen 1985). Polyclonal rabbit antibody generated against PMI protein was diluted to 2 μg/ml in a buffer containing 35 mM sodium bicarbonate and 15 mM sodium carbonate (pH 9.5) and used to coat a Nunc MaxiSorp 96

- well plate (ThermoFisher Scientific) at a volume of 100 μl/well. The plates were incubated overnight at 5°C ± 3°C. The plate contents were then emptied and tapped on paper towels to remove residual solution. The plat es were blocked with phosphate buffered saline (PBS) pH 7.4 containing 1% nonfat milk for at least 30 minutes at room temperature. The plates were washed five times with PBS containing 0.05% Tween 20 and incubated for 2 hours at 20°C ± 2°C with diluted pl ant extract samples and PMI

- 0105 standards at a volume of 100 μl/well

. Dilutions were made in ELISA dilution buffer (PBS plus 1% nonfat milk and 0.05% Tween 20). The plant extract samples and standards were assayed in triplicate. After washing, the plat es were incubated with 100 μl/well monoclonal mouse antibody (1 μg/ml) generated against PMI protein diluted in ELISA dilution buffer for 1 hour at 20°C ± 2°C. The p lates were washed and subsequently incubated with 100 μl/well rabbit anti

- mouse IgG conjug ated with horseradish peroxidase (Sigma

- Aldrich ) diluted 1:20,000 in ELISA dilution
buffer for 1 hour at 20°C ±2°C. The plates were washed again and incubated in 0.1 mg/ml
TMB substrate solution (Sigma

- Aldrich ) in citrate buffer (24 mM citric acid monohyd rate, 60 mM dibasic sodium phosphate

, 

### pH 5.0 containing 0.006% hydrogen peroxide) at a volume of 100 μl/well. Color was allowed to develop for 30 minutes in the dark at room temperature. The reaction was stopped by the addition of 50 μl 3 M sulfuric acid per well, and absorbance at 450 nm was measured with a Tecan Sunrise microplate reader (Tecan US). The results were analyzed with BioMetallics DeltaSoft PC Microplate Analysis Software, v. 1.71.2 using a four

- parameter algorithm.

Total Protein Determinat ion

Total protein of the plant extract samples (LP5307, LP

- NEG + PMI

- 0105 and LP

- NEG) were quantified via the bicinchoninic acid (BCA) method (Hill and Straka 1988), using bovine serum albumin as the reference protein standard. The results were analyzed w ith BioMetallics DeltaSoft PC Microplate Analysis Software, v. 1.71.2 using a 4

- parameter fit
of the standard curve.

Immunoreactivity and Molecular Weight Determination

Western blot analysis was used to investigate the integrity of PMI in the plant extract sample LP5307, the control leaf extract fortified with the microbially produced test substance (LP

NEG + PMI

- 0105) and the microbially produced test substance PMI

- 0105. Aliquots equivalent to 5 ng PMI from sample LP5307, LPNEG + PMI

- 0105 and test substan ce PMI

0105 diluted in 10X Sample Buffer (as described by Laemmli (1970) containing 8%

### APPENDIX B

5307

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- 1 Page 146 of 168 glycerol, 1% BME, 2% SDS, 65 mM Tris, and 0.01% bromophenol blue) were subjected to SDS polyacrylamide gel electrophoresis (SDS

- PAGE) with a NuPAGE 4

- 12% Bis

- Tris gel (I nvitrogen) and NuPAGE MES SDS running buffer (Invitrogen). An aliquot of the negative control sample (LP

- NEG); equivalent to 92.8 μg total protein ( the total amount of protein loaded on the gel for LP5307 ) , was included in the analysis as a negative control. Additionally, based on total protein, LP

- NEG + PMI

- 0105 was supplemented with
additional LP

- NEG extract to equal 86.8 μg total protein. The molecular

- weight standard was SeeBlue Plus2 pre

- stained standard (Invitrogen). After electroblo tting, the polyvinylidene difluoride (PVDF) membrane was probed with immunoaffinity

- purified polyclonal goat antibody generated against PMI diluted to 1 μg/ml in Tris

- buffered saline, pH 8.0 with 3% nonfat milk. Alkaline phosphatase

- conjugated donkey anti

- goat IgG (Jackson ImmunoResearch) diluted 1:3000 in Tris

- buffered Saline with Tween 20, pH 8.0) was used to bind to the primary antibody and was visualized by development with BCIP/NBT alkaline phosphatase substrate solution (Sigma

- Aldrich). The Western blot was examined for the presence of intact immunoreactive PMI or immunoreactive PMI fragments.

Enzymatic Activity

The enzymatic activity of PMI was measured in triplicate using a continuous coupled spectrophotometric assay based on the method described b y Gracy and Noltmann (1968)
and Gill et al. (1986). PMI activity was measured by linking the formation of fructose 6

- phosphate (resulting from the isomerization of mannose 6

- phosphate) to the reduction of

NADP via phosphoglucose isomerase and glucose 6

- ph osphate dehydrogenase (as shown in
the diagram below). The molar reduction of NADP in this system can be directly converted
into the molar isomerization of mannose 6

- phosphate via PMI. The enzymatic reactions
were conducted in 96

- well plates. Microbiall y

- produced and plant produced PMI were diluted in 50 mM Tris buffer, pH 7.0 containing 0.1% Tween 20. The reaction was initiated by adding 5 ng PMI from triplicate plant extract samples LP5307, LP

- NEG + PMI

- 0105, or
PMI from test substance PMI

- 0105 to an assay mixture containing 10 mM mannose 6

- phosphate (Sigma

- Aldrich ), 1 mM β

- nicotinamide adenine dinucleotide phosphate (NADP)
sodium sal t hydrate (Sigma

- Aldrich ), 2 U/ml phosphoglucose isomerase (Sigma

- Aldrich ) and 2 U/ml glucose 6

- phosphate dehydrogenase (G6PDH) (Sigma

- Aldrich ) in 50 mM Tris
buffer

, pH 7.0. The total volume of the reaction mixture was 200 μl.

### APPENDIX B

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- 1 Page 147 of 168 The assay mixture was preincubated at 25°C ± 2°C for 10 minutes prior to the addition of PMI. Following the 10 minute preincubation, PMI was added to the assay mixture and the plate was read by a SpectraMax Plus384 spectrophotometer (Molecular Devices) to determine the pathlength of the sample in each well. The reduction of NADP was monitored spectrophotometrically at 340 nm at 25oC ± 2oC over 10 minutes with readings taken every 15 seconds. The change in absorbance over time was monitored using SoftMax Pro version 5.2. The extinction coefficient of NADPH, 6.22 cm

- 1 

### mM

- 1 

### , was used for calculating the amount of NADPH formed. One unit (U) of P MI activity is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of mannose 6

- phosphate to
fructose 6

- phosphate per min (equivalent to 1 μmol NADP reduced per min) under the described reaction conditions. Results are reported a s the mean and standard deviation of triplicate enzymatic assays.

### APPENDIX B

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- 1 Page 148 of 168 B .4. Results of Comparison of PMI Produced in 5307 Corn and Recombinant E. coli

Immunoreactivity and MolecularWeight Determination

Western blot analysis of PMI in the plant extract sample LP5307, the control leaf extract
fortified with PMI test substance PMI

- 0105 (LP

- NEG + PMI

- 0105), and test substance PMI

0105 reveal ed a dominant immunoreactive band consistent with the predicted molecular weight of PMI ( Figure B

- 5, Lanes 2, 3, and 4). Th e intensity of the PMI bands in the presence of the plant matrix (Lanes 2 and 3) was diminished compared to the intensity of the PMI band in the absence of the matrix (Lane 4). However, there was similar intensity
between the PMI bands from test substance PMI

- 0105 in the presence of plant matrix (Figure B

- 5, Lane 3) and PMI from 5307 corn extract (Lane 2). Thus, the relative ly diminished intensity of the PMI bands in the presence of the plant matrix was most likely
due to matrix effects. The matrix effec t can be attributed to the nature of the sample. Crude plant extract preparations contain all soluble cell proteins of varying molecular weights that may interfere with the mobility of the analyzed protein and with antibody binding. The co

migration of t he matrix with PMI or PMI fragments may have limit ed access of the antibody to epitopes of the target protein and therefore diminish ed the signal.

The Western blot analysis also revealed some faint bands combined with a diffuse background signal in the m olecular weight range above 62 kDa in both LP5307 and LP

NEG + PMI

- 0105 (Figure B

- 5, Lanes 2 and 3). As this effect (with exactly the same band pattern) was also detected in the negative plant extract sample LP

- NEG (Lane 5) it is not related to PMI protei n and most likely represents a non specific response on the Western blot. As expected, no immunoreactive band corresponding to the molecular weight of PMI was observed in the negative plant extract sample LP

- NEG (Lane 5). The Western blot analysis also re vealed a very faint protein band with a molecular weight of approximately 30 kDa in the microbially produced test substance PMI

- 0105. Because this protein cross

reacted with the anti

- PMI antibody it is most likely a PMI degradation product. The degradati on product was not visible in the sample with the control leaf extract fortified with the microbially produced test substance (LP

- NEG + PMI

- 0105) due to the matrix effect
described above.

### APPENDIX B

5307

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- 1 Page 149 of 168 Figure B

- 5. Western blot analysis for PMI in LP5307, LP

- NEG + PMI

- 0105, test substance
PMI

- 0105, and LP

- NEG.

Lane 1: Molecular weight standard SeeBlue Plus2 (Invitrogen) Lane 2: Plant extract sample LP5307 Lane 3: Plant extract sample LP

- NEG + PMI

- 0105 Lane 4: Microbially

- p roduced test substance PMI

- 0105 Lane 5: Plant extract sample LP

- NEG Lane 6: Molecular weight standard SeeBlue Plus2

### APPENDIX B

5307

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- 1 Page 150 of 168 The average PMI enzymatic activity was 455.67 U/mg PMI in the triplicate plant extract samples LP5307 and 526.26 U/mg PMI in the microbially produced test substance PMI

0105 ( Table B

- 1). The control leaf extract fortified with the microbially produced test substance (LP

- NEG + PMI

- 0105) had a specific activity of 518.81 U/mg PMI (Table B

- 1 ), demonstrating that the plant matrix had little effect on the enzymatic activity of PMI. As expected, no PMI activity was detected in the negative control plant extract sample, LP

NEG ( Table B

- 1)

.

Table B

- 1. Enzymatic activity of PMI in 5307 corn leaf extract, control corn leaf extract fortified with test substance PMI

- 0105, test substance PMI

- 0105, and control corn leaf extract.

LP5307 is leaf extract of 5307 corn plants. LP

- NEG + PMI

- 0105 is control corn leaf extract fortified with microbially produced PMI test substance PMI

- 0105. LP

- NEG is control corn leaf extract.

1 One unit of PMI activity is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of mannose 6

- phosphate to fructose 6

- phosphate per min (equivalent to 1 μmol NADP reduced per min) under the described reaction
conditions

.

2 LP5307

- 1,

- 2, and

- 3 represent three independent extractions. Assay Replicate PMI Activity 1 (U/mg PMI) Average PMI Activity of

Assay Replicates (U/mg PMI) SD of Assay Replicate s (U/mg PMI) CV of Assay Replicates (%) Overall Average PMI Activity (U/mg PMI) Overall SD (U/mg PMI) Overall CV (%)

1 469.58
2 490.03 3 479.21
1 478.77
2 474.93
3 439.47
1 418.44
2 448.30
3 402.34
1 514.24
2 534.33 3 507.86
1 519.00
2 531.87 3 527.90
1 below LOD
2 below LOD
3 below LOD LP5307

- 1 2

LP5307

- 2

LP5307

- 3

LPNEG + PMI

- 0105 423.03

518.81

PMI

- 0105

LPNEG 526.26

below LOD 10.23

21.67

23.32

13.82

6.59

not applicable 479.60

464.39 6.7%

1.3%

not applicable 455.67 30.37

2.1%

4.7%

5.5%

2.7%

### APPENDIX C

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- 1 Page 151 of 168 

## Appendix C: Quantification of eCry3.1Ab and PMI in 5307 Corn

This appendix provides details of the materials and methods used to determine the concentrations of eCry3.1Ab and PMI in the tissues of 5307 plants at various gro wth stages.
Refer to Part VI.N.1 of this submission for a description of the study and results.

C .1. Plant Tissue Production

Plants were grown for the collection of tissues in Bloomington, IL; Sadorus, IL; Shirley, IL; and Stanton, MN in two plots per location; one for the 5307 hybrid and one for the control hybrid.

C .2. Plant Tissue Processing

In the presence of dry ice, th e leaf, kernel and whole

- plant samples were individually processed to a fine powder. A subsample from each homogeneous, powdered sample was lyophilized and stored at

– 80 oC ± 10oC. The percent dry weight of each sample was determined from the sample weig ht before and after lyophilization.

C .3. Tissue Extraction and Analysis

Protein extractions were performed on representative aliquots of the lyophilized leaf, kernel

, and whole

- plant samples. The e xtracts were analyzed by ELISA (Tijssen 1985) t o quantify the amount of eCry3.1Ab and PMI in each sample. Sample extracts were analyzed in triplicate, and standard curves were generated with known amounts of the corresponding reference protein. Standard curves were generated fo r each ELISA plate. Nontransgenic plant tissue extracts were analyzed in parallel to evaluate any impact of the plant matrix on
the ELISA.

### APPENDIX C

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- 1 Page 152 of 168 C .4. eCry3.1Ab Quantification

– Extraction and ELISA Procedures

Buffers

The buffers used for extraction and ELI SA analysis of eCry3.1Ab are listed in the following table:

Name of buffer Constituents

Phosphate

- buffered saline (PBS) 140 mM sodium chloride, 8.24 mM sodium phosphate dibasic, 1.81mM sodium phosphate monobasic, pH 6.75 Borate buffer (leaves, kernels,

whole plants ) 0.1 M Sodium tetraborate decahydrate, 0.2% PVP

- 360, 7.69 mM sodium azide, 0.5% Tween 20; titrated to pH 10.0. Complete Protease Inhibitor Cocktail (Roche Applied Science) added on day of extraction

Dilution buffer PBS, 0.05% Tween 20, 1% BSA, 0.02% sodium azide

Wash buffer 10 mM Tris, 0.05% Tween 20, 0.02% sodium azide



### eCry3.1Ab Extraction from Leaves, Kernels , and Whole Plants

A ratio of 3 ml of borate buffer pH 10.0 was added to 100 mg of lyophilized tissue. The samples were vortexed, placed on wet ice for at least 30 minutes, homogenized using an
Omni

- Prep Homogenizer, and centrifuged at 2°C to 8°C to form a pellet. The supernatants were removed and stored at 2°C to 8°C until analysis.

eCry3.1Ab Quantitation

The eCry3.1Ab EL ISA kit was manufactured at Beacon Analytical Systems (BAS), Portland, ME. The assay is a double

- antibody sandwich assay in which the eCry3.1Ab protein is affixed to the wells of a microtiter plate using a monoclonal, anti

- mCry3A antibody that binds to th e mCry3A domains of the eCry3.1Ab protein. The primary antibody was diluted and added to each well of a 96

- well microtiter plate. The plate was then blocked using a proprietary method. Dilutions of each tissue extract and appropriate
serial dilutions of eCry3.1Ab reference protein (ECRY3.1AB

- 0208), prepared in dilution buffer, were applied to the pre

- coated plates at a total volume of 100 μl/well. The plates were incubated at room temperature on a titre plate shaker at 400 rpm for 1 hour. The plates

we re washed five times with wash buffer in a BioTek ELx405 Microplate Washer. After washing the plates, a secondary, rabbit polyclonal anti

- Cry1Ab antibody (provided in the kit) was then used to bind the Cry1Ab domain of the eCry3.1Ab protein at 100 μl/well . The plates were incubated at room temperature on a titre plate shaker at 400 rpm for one hour
and washed five times as described above.

After the plates were washed, a tertiary donkey anti

- rabbit conjugated with alkaline phosphatase diluted in dilution buffer was added to each of the wells (100 μl/well) and incubated at room temperature on a titre plate shaker at 400 rpm for one hour. The plates were then washed five times as described above, and alkaline phosphatase substrate solution

### APPENDIX C

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- 1 Page 153 of 168 provided in the k it was added at a volume of 100 μl/well. The plates were incubated for 30 minutes at room temperature on a titre plate shaker at 400 rpm. The reaction was stopped by the addition of 3N sodium hydroxide (100 μl/well), and absorbance of the reaction was

me asured at a dual wavelengths (405 and 492 nm) with a Tecan Sunrise Microplate Reader. The results were analyzed with BioMetallics DeltaSoft PC Microplate Analysis Software, v. 1.71.2, using a four

- parameter algorithm. The results for kernel samples were analyzed with SoftMax Pro, v. 5.2, using a four

- parameter algorithm.

The lower limit of quantification (LOQ) or limit of detection (LOD) of eCry3.1Ab is shown

on Table VI

- 2 (Part VI.C.) for any tissue type in which eCry3.1Ab could not be quantified
or dete cted, respectively. E xtraction efficiencies ranged from 7 7 % to 88% across the tissue types analyzed ; the reported tissue concentrations have been adjusted for extraction efficiency.

### APPENDIX C

5307

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- 1 Page 154 of 168 

### C .5. PMI Quantification

– Extraction and ELISA Procedures

Buffers

The buffers used for extraction and ELISA analysis of PMI are listed in the following table :

Name of buffer Constituents

PBS 138 mM sodium chloride, 2.7 mM potassium chloride, 10.1 mM disodium phosphate, 1.8 mM potassium dihydrogen phosphate, pH 7.4 Blocking buffer PBS, 1% powdered milk

Borate buffer 0.1 M sodium tetraborate decahydrate, 0.2% PVP

- 360, 7.69 mM sodium azide, 1.2% concentrated hydrochloric acid, 0.5% Tween 20; pH will be approximately 7.5. Complete Protease Inhibitor Cocktail (Roche Applied Science) added on day of extraction

Carbonate

- bicarbonate buffer 34.9 mM sodium bicarbonate, 15.0 mM sodium carbonate, pH 9.5

Citrate

- phosphate buffer 23.8 mM citric acid, 59.9 mM disodium phosphate, pH 5.0

Dilution buffer PBS, 0.05% Tween 20, 1% powdered milk

Wash buffer PBS, 0.05% Tween 20



### PMI Extraction from Leaves, Kernels , andWhole Plants

A ratio of 3 ml of borate buffer, pH 7.5 was added to 100 mg of lyophilized tissue. The samples were mixed, placed on wet ice for at least 30 minutes, homogenized using an Omni

Prep Homogenizer, and cent rifuged at 2°C to 8°C to form a pellet. The supernatants were removed and stored at 2°C to 8°C until analysis.

PMI Quantification

Rabbit polyclonal anti

- PMI antibody was diluted in carbonate

- bicarbonate buffer and added
to each well of a 96

- well microti ter plate at a volume of 100 μl/well. The plates were stored overnight in a refrigerator set at 2°C to 8°C. The antibody solution was removed and blocking buffer was added to the plate at a volume of 250 μl/well and then incubated at room temperature for at least 30 minutes. After blocking incubation, the plates were washed five times with wash buffer in a BioTek ELx405 microplate washer and dilutions of each tissue extract and appropriate serial dilutions of PMI reference protein (PMI

- 0105) prepared

in dilution buffer were applied to the plates at a total volume of 100 μl/well. The plates were incubated at 18°C to 22°C for 2 hours. After incubation, plates were washed five times
as described above and a monoclonal anti

- PMI antibody diluted in dilution buffer was added to the plate at a volume of 100 μl/well and incubated at 18°C to 22°C for one hour.

The plates were washed five times after incubation and a horseradish

- peroxidase

- conjugated
rabbit anti

- mouse immunoglobin diluted in dilution buffer was ad ded at a volume of 100 μl/well and incubated at 18°C to 22°C for one hour. After incubation, the plates were washed five times, and TMB substrate solution was added at a volume of 100 μl/well (one

### APPENDIX C

5307

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- 1 Page 155 of 168 tablet per 10 ml of citrate

- phosphate buffer) and incubate d at room temperature in the dark for 30 minutes. The reaction was stopped by addition of 3 M sulfuric acid at a volume of 50 μl/well, and the absorbance of the reaction was measured at 450 nm with a Tecan Sunrise microplate reader. The results were ana lyzed with BioMetallics DeltaSoft PC Microplate Analysis Software, v. 1.71.2, using a four

- parameter algorithm.

The lower limit of quantification (LOQ) or limit of detection (LOD) of PMI is shown on
Table VI

- 3 (Part VI.C.) for any tissue type in which PMI could not be quantified or detected, respectively. PMI extraction ef ficiencies ranged from 72% to 80 % across the tissue types analyzed; the reported tissue concentrations have been adjusted for extraction efficiency.

### APPENDIX D

5307

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- 1 Page 156 of 168 

## Appe ndix D: Compositional Analysis of 5307 Forage and Grain

This a ppendix describes the methods used to conduct the compositional analysis study described

in Part VII.C of this submission , wherein the results are also provided. References cited for
individual methods in this a ppendix are listed at the end of the a ppendix.

Study Design

Forage and grain for co mpositional analyses were harvested from multiple locations planted in the U.S. in 2008. The locations chosen were representative of major corn producing regions in the country. For all locations, trials were planted with a 5307 hybrid and near

- isogenic, nontransgenic hybrid in a randomized complete block design with three replicated plots, and were managed following local agronomic practices. The plants were self

- pollinated by hand and the developing ears were bagged to avoid cross

- pollination. Trials were planted in eight locations in an effort to ensure that grain and forage from at least six locations could be harvested in the event of loss due to adverse environmental conditions (early freeze, drought,

etc

. ). Six locations that produced sufficient grain and forage were selected for this study (see

Part VII.C.1 ).

Forage Sampling and Processing

For each genotype, the entire above

- ground portion of five plants from each of the three replicate plots at each location was harvested at dough stage (R4), the stage at which silage typically is prepared. Plants were pooled to create a composite sample for each replicate plot, then ground using a chippe r

- shredder. A subsample from each well

- mixed composite sample was shipped overnight on ice packs to Syngenta Crop Protection, Inc. (Greensboro, NC). The samples were
stored at

– 20°C ± 10°C, then finely ground and shipped on dry ice to a contract research laboratory, where they were stored at

– 20°C ± 10°C until they were analyzed.

Grain Sampling and Processing

For each genotype, ears were collected from 15 plants from each replicate plot at each location. Ears were harvested after reaching physiological m aturity (R6) and then mechanically dried to approximately 9% to 12% moisture content. (Mechanical drying after harvest is standard agronomic practice for improving storage characteristics of corn grain.) Each sample consisted of grain shelled from ears c ollected from 15 plants from one replicate plot. A well

- mixed subsample of approximately 500 g of grain from each plot was shipped at ambient temperature to Syngenta Crop Protection, Inc., where it was stored at

– 20°C ± 10°C, then finely ground and shippe d on dry ice to the contract testing facililty. The samples were stored at

– 20°C ± 10°C until they were analyzed.

### APPENDIX D

5307

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- 1 Page 157 of 168 Compositional Analyses

As detailed in Table VII

- 1 (Part VII of this submission ), forage was analyzed for proximates and the minerals calcium and phosphorus. Grain was analyzed for major constituents (proximates, including starch), minerals, amino acids, fatty acids, vitamins, and selected anti

- nutrients and secondary metabolites.

All compositional analyses were conducted using methods published and approved by AOAC International, or other industry

- standard analyti cal methods

, described below

. Based on the moisture content of each sample, analyte levels were converted to equivalent units of dry weight.

Analytical Methods and Reference Sta ndards for Compositional Analyse s

2

- Furaldehyde (Albala

- Hurtado et al. 1997)

The ground sample was extracted with 4% trichloroacetic acid and injected directly on a high

performance liquid chromatography system for quantitation of free furfurals by ultraviolet detection. The limit of quantitation (LOQ) for this study was 0.500 ppm , calculated on a fresh

weight basis.

Reference Standard : Acros 2

- Furaldehyde, 99.7%, Lot Number A0219180

Acid Detergent Fiber (ADF) (USDA 1970)

The sample was washed with acetone to remove fats and pigments. It was then placed in a filter bag and posit ioned in an Ankom analyzer where it was washed with an acidic boiling detergent solution that dissolved the protein, carbohydrate, and ash. The lignocellulose fraction remaining was determined gravimetrically. The LOQ for this study was 0.100%, calculate d on a fresh

weight basis.

Amino Acid Composition (AOAC 2005k)

Total aspartic acid (including asparagine)
Total threonine
Total serine Total glutamic acid (including glutamine) Total proline Total glycine
Total alanine
Total valine
Total isoleucine
Total leucine Total tyrosine Total phenylalanine
Total histidine

### APPENDIX D

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- 1 Page 158 of 168 Total lysine Total arginine Total tryptophan
Sulfur

- containing amino acids: Total methionine Total cystine (including cysteine)

The sample was assayed by three methods to obtain the full profile. Tryptophan required a base hydrolysis with sodium hy droxide. The sulfur

- containing amino acids required an oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the samples for the remaining amino acids was accomplished t hrough direct acid hydrolysis with hydrochloric acid. Once hydrolyzed, the individual amino acids were then quantitated using an automated amino acid analyzer. The LOQ for each amino acid assay was 0.100 mg/g, calculated on a fresh

- weight
basis.

Referen ce Standards : ThermoScientific K18, 2.5 μmol/mL per constituent except cystine (1.25 μmol/mL), Lot Number JK126327 Sigma, L

- Tryptophan, 100%, Lot Number 076K0075 Sigma/BioChemika, L

- Cysteic Acid Monohydrate, >99% (used as 100%),
Lot Number 1305674 Sig ma, L

- Methionine Sulfone, 100%, Lot Number 047K1321

Ash (AOAC 2005b)

The sample was placed in an electric furnace at 550°C and ignited to drive off all volatile organic matter. The nonvolatile matter remaining was quantitated gravimetrically and calculated to determine percent ash. The LOQ for this study was 0.100%, calcu lated on a fresh

- weight basis.

Beta

- Carotene (AOAC 2005e; Quackenbush 1987)

The sample was saponified and extracted with hexane. The sample was then injected on a reverse phase high

- performance liquid chromatography system with ultraviolet light detectio n. Quantitation was achieved with a linear regression analysis. The LOQ for beta

- carotene was 0.0200 mg/100 g, calculated on a fresh

- weight basis.

Reference Standard : Sigma

- Aldrich, Beta

- carotene, Type I, 100% (stock standard concentration determined spe ctrophotometrically), Lot Number 068K2561

Carbohydrates (USDA 1973)

The total carbohydrate level was calculated by difference using the fresh weight

- derived data and the following equation:

% carbohydrates = 100 %

- (% protein + % fat + % moisture + % ash )

The LOQ for this study was 0.100%, calculated on a fresh

- weight basis.

### APPENDIX D

5307

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- 1 Page 159 of 168 Fat by Acid Hydrolysis (AOAC 2005a)

The sample was hydrolyzed with hydrochloric acid at an elevated temperature. The fat was extracted with ether and hexane. The extract was evaporat ed on a steambath, redissolved in hexane and filtered through a sodium sulfate column. The hexane extract was then vaporated again on a steambath under nitrogen, dried, and weighed. The LOQfor this study was 0.1%, calculated on a fresh weight basis.

Fa tty Acids (AOAC 2005l; AOCS 1997b and 2001)

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 h eptane containing an internal standard. The methyl esters of the fatty acids were analyzed by gas chromatography using external standards for quantitation. The LOQ was 0.00500%, calculated on a fresh

- weight basis.

Reference Standards : Nu Chek Prep GLC R eference Standard Hazelton No. 1, Lot Number AU18

- S Nu Chek Prep GLC Reference Standard Hazelton No. 2, Lot Number M13

- O Nu Chek Prep GLC Reference Standard Hazelton No. 3, Lot Number MA18

- S Nu Chek Prep GLC Reference Standard Hazelton No. 4, Lot Number JA 16

- T Nu Chek Prep Methyl Gamma Linolenate, used as 100%, Lot Number U

- 63M

- JY12

- R Nu Chek Prep Methyl Tridecanoate, used as 100%, Lot Number N

- 13M

- JA16

- T

Folic acid (AOAC 2005i; Infant Formula Council 1985)

The sample was hydrolyzed in a potassium phosphate buffer with the addition of ascorbic acid to protect the folic acid during autoclaving. Following hydrolysis by autoclaving, the sample was
treated with a chicken

- pancreas enzyme and incubated approximate ly 18 hours to liberate the bound folic acid. The amount of folic acid was determined by comparing the growth response of the sample, using the bacteria Lactobacillus casei , with the growth response of a folic acid standard. This response was measured tu rbidimetrically. The LOQ was 0.00600 mg/100 g,
calculated on a fresh

- weight basis.

Reference Standard : USP, Folic acid, 98.9%, Lot Number Q0G151

ICP Emission Spectrometry (AOAC 2005m)

The sample was dried, precharred, and ashed overnight in a muffle set to maintain 500°C. The ashed sample was 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 sample, measured on the inductively coupled plasma spectrometer, with the emission of the standard solutions. The LOQs (Table D

- 1) were calculated on a fresh

- weigh t basis.

### APPENDIX D

5307

- FDA

- 1 Page 160 of 168 Table D

- 1. Inorganic Ventures Reference Standards and Limits of Quantitation

Mineral Lot Numbers Calibration Standard Concentration (μg/ml) LOQ (ppm)

Calcium B2

- MEB280039
B2

- MEB266040 200
1000 20.0

Copper B2

- MEB280039
B2

- MEB280036 2
10 0.50

Iron B2

- MEB280039
B2

- MEB280035 10
50 2.00

Magnesium B2

- MEB280039
B2

- MEB280036 50
250 20.0

Manganese B2

- MEB280039
B2

- MEB280036 2
10 0.30

Phosphorus B2

- MEB280039
B2

- MEB266040 200
1000 20.0

Potassium B2

- MEB280039
B2

- MEB266040 200
1000 100

Sodium B2

- MEB280039
B2

- MEB266040 200
1000 100

Zinc B2

- MEB280039
B2

- MEB280036 10
50 0.40



### ICP

- Mass Spectrometry (AOAC 2005o)

The sample was wet

- ashed with nitric acid using microwave digestion. Using inductively coupled plasma mass spectrometry, the amount of each element was determined by comparing the counts generated by the unknowns to those generated by standard solutions o f known concentrations. The LOQ for this study was 50.0 ppb, calculated on a fresh

- weight basis.

Reference Standard : SPEX, Selenium, 100 mg/L, Lot Number 6

- 74GS

Inositol (Infant Formula Council 1985b; Atkins et al. 1943)

The inositol sample was extracted with dilute hydrochloric acid at a high temperature. The amount of inositol was determined by comparing the growth response of the sample, using the
yeast Saccharomyces carlsbergenesis , with the growth response of an inos itol standard. The response was measured turbidimetrically. The LOQ for this study was 40.0 μg/g, calculated on a

fresh

- weight basis.

Reference Standard : Sigma

- Aldrich, Myo

- Inositol, 100%, Lot Number 065K0018

### APPENDIX D

5307

- FDA

- 1 Page 161 of 168 Moisture (AOAC 2005c)

The sample was dried in a vacuum oven at approximately 100°C to a constant weight. The moisture weight loss was determined and converted to percent moisture. The LOQ for this study was 0.100%, calculated on a fresh

- weight basis.

Neutral Detergent Fiber (NDF), Enzyme Method (AACC 1998; USDA 1970)

The sample was washed with acetone to remove fats and pigments. It was then placed in a filter bag and positioned in an Ankom analyzer where it was washed with a neutral boiling detergent solution that dissolved the protein, carboh ydrate, enzyme, and ash. The remaining hemicellulose, cellulose, and lignin fractions were determined gravimetrically. The LOQ for this study was 0.100%.

Niacin (AOAC 2005g)

The sample was 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 sample, using the
bacteria Lactobacillus plantarum, with the growth response of a nia cin standard. This response was measured turbidimetrically. The LOQ for this study was 0.0300 mg/100 g.

Reference Standard: USP, Niacin, 99.8%, Lot Number I0E295

p - Coumaric Acid and Ferulic Acid (Hagerman and Nicholson 1982)

The sample was extracted w ith methanol using ultrasonication, hydrolyzed using 4N sodium hydroxide, buffered using acetic acid/sodium hydroxide, acidified with 3N hydrochloric acid, and filtered. The levels of p

- coumaric and ferulic acids in the extract were determined by

reverse phase high

- performance liquid chromatography with ultraviolet detection. The LOQ for

p

- coumaric acid and ferulic acid was 50.0 ppm, calculated on a fresh

- weight basis.

Reference Standards : Acros Organics, 4

- Hydroxy

- 3

- methoxycinnamic acid (ferulic acid), 99.4%, Lot Number A0248008
Acros Organics, p

- Hydroxycinnamic acid (p

- coumaric Acid), 99.4%, Lot Number A0236839

Phytic Acid (Lehrfeld 1989 and 1994)

The sample was extracted using 0.5 M HCl with ultrasonication. Purification and concentration

were a ccomplished on a silica

- based anion

- exchange column. The sample was analyzed on a polymer high

- performance liquid chromatography column PRP

- 1, 5 μm (150 x 4.1 mm) with a refractive index detector. The LOQ for this study was approximately 0.100%, calculat ed on a
fresh

- weight basis.

Reference Standard : Aldrich, Phytic Acid, Dodecasodium Salt Hydrate, 95%,
Lot Number 077K0693

### APPENDIX D

5307

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- 1 Page 162 of 168 Protein (AOAC 2005h; Bradstreet 1965; Kalthoff and Sandell 1948)

Nitrogenous compounds in the sample were reduced in the presence o f boiling sulfuric acid and a mercury catalyst mixture to form ammonia. The acid digest was made alkaline. The ammonia was distilled and then titrated with a previously standardized acid. The percent nitrogen was calculated and converted to protein usin g the factor 6.25. The LOQ for this study was 0.100%,
calculated on a fresh

- weight basis.

Pyridoxine Hydrochloride (AOAC 2005j; Atkins et al. 1943)

The sample was hydrolyzed with dilute sulfuric acid in the autoclave and the pH was adjusted to
remove int erferences. The amount of pyridoxine was determined by comparing the growth response of the sample, using the yeast Saccharomyces carlsbergensis, with the growth response of a pyridoxine standard. The response was measured turbidimetrically. Results were reported as pyridoxine hydrochloride. The LOQ for this study was 0.00700 mg/100 g, calculated on a fresh

weight basis.

Reference Standard : USP, Pyridoxine hydrochloride, 99.8%, Lot Number Q0G409

Raffinose (Brobst 1972; Mason and Stover 1971)

The sampl e was extracted with deionized water and the extract treated with a hydroxylamine hydrochloride solution in pyridine, containing phenyl

- β

- D

- glucoside as an internal standard. The resulting oximes were converted to silyl derivatives by treatment with hexam ethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization detector. The acceptable LOQ for this study was 0.100%, calculated on a fresh weight basis.

Reference Standards : Sigma, D(+)

- Raffinose Pentahydrate, 99% ( 84.0% after correction for degree of hydration), Lot Number 037K1059

Starch (AOAC 2005p)

The sample was extracted with alcohol to remove carbohydrates other than starch, i.e. sugars. Then it was hydrolyzed into glucose with α

- amylase and amyloglucosidas e. Glucose was oxidized with glucose oxidase to form peroxide, which reacted with a dye in the presence of peroxidase to give a stable colored product proportional to glucose concentration. The glucose concentration was quantitated by measurement on a sp ectrophotometer at 540 nm. Percent starch was then calculated from the glucose concentration. The LOQ for this study was 0.05%,
calculated on a fresh

- weight basis.

Reference Standard : Sigma D(+)

- Glucose, 99.9%, Lot Number 123K0095

### APPENDIX D

5307

- FDA

- 1 Page 163 of 168 Thiamine Hydrochloride (AOAC 2005f)
The sample was autoclaved under weak acid conditions to extract the thiamine. The resulting solution was incubated with a buffered enzyme solution to release any bound thiamine. The solution was purified on a cation

- exchange co lumn. An aliquot was 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. The limit of quantitation was c alculated and reported on a fresh weight basis. The LOQ for this study was 0.01 mg/100 g. Results were reported as thiamine hydrochloride.

Reference Standard : USP, Thiamine Hydrochloride, Purity 99.8% (used as 95.9% after
correction for moisture content ), Lot Number 01F236

Total Dietary Fiber (TDF) (AOAC 2005n)

Duplicate samples were gelatinized with α

- amylase and digested with enzymes to break down starch and protein. Ethanol was added to each sample to precipitate the soluble fiber. The samples were 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 t he sample was calculated using the protein and ash values. The LOQ for this study was 1.00%, calculated on a
fresh

- weight basis.

Trypsin Inhibitor (AOCS 1997a)

The sample was ground and defatted with petroleum ether. A sample of matrix was extracted
wit h 0.01N sodium hydroxide. Varying aliquots of the sample suspension were exposed to a known amount of trypsin and benzoy1

- DL

- arginine~p~nitroanilide hydrochloride. The sample was allowed to react for 10 minutes at 37°C. After 10 minutes, the reaction wa s halted by the addition of acetic acid. The solution was centrifuged, then the absorbance was determined at 410 nm. Trypsin inhibitor activity was determined by photometrically measuring the inhibition of trypsin’s reaction with benzoyl

- DL

- arginine~p~ni troanilide hydrochloride. The LOQ for this study was 1.00 Trypsin Inhibitor Units (TIU)/mg, calculated on a fresh

- weight basis.

Vitamin B2 (Riboflavin) (AOAC 2005d; US Pharmacopeia 2005)

The sample was 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 sample, using the bacteria Lactobacillus casei , with the growth response of multipoint riboflavin standards. The growth response was mea sured turbidimetrically. The LOQ for this study was 0.0200 mg/100 g, calculated on a fresh

- weight basis.

Reference Standard : USP, Riboflavin, 100%, Lot Number N0C021

### APPENDIX D

5307

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- 1 Page 164 of 168 Vitamin E (Cort et al. 1983; McMurray et al. 1980; Speek et al. 1983)

The sample was saponified to break down any fat and release vitamin E. The saponified mixture was extracted with ethyl ether and then quantitated by high

- performance liquid chromatography using a silica column. The LOQ for this study was 0.500 mg/100 g, calculated on a fresh weight
basis.

Reference Standard : USP, Alpha

- Tocopherol, 100%, Lot Number M

### APPENDIX D

5307

- FDA

- 1 Page 165 of 168 

### References for Appendix D

AACC

. 1998. Approved Methods of the American Association of Cereal Chemists , 9th Ed., Method 32.20. AACC International: St. Paul, Minnesota.

Albala

- Hurtado S , Veciana

- Nogues MT , Izquierdo

- Pulido M , Vidal

- Carou MC. 1997. Determination of free and total furfural compounds in infant milk formulas by high

performance liquid chromatography. Journal of Agricultural and Food Chemistry 45:
2128

- 2133.

AOAC

. 2005a

. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods
Methods 922.06 and 954.02

. AOAC International: Gaithersburg, Maryland.

AOAC. 2005b

. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
923.03

. AOAC International: Gaithersburg, Maryland.

AOAC

. 2005c. Official Method s of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods
926.08 and 925.09. AOAC International: Gaithersburg, Maryland.

AOAC. 2005d. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods
940.33 and 960.46

. AOAC International: Gaithersbur g, Maryland.

AOAC

. 2005e

. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
941.15

. AOAC International: Gaithersburg, Maryland.

AOAC. 2005f

. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods 942.23, 953.17, and 957.17

. AOAC International: Gaithersburg, Maryland.

AOAC

. 2005g

. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
944.13

. AOAC International: Gaithersburg, Maryland.

AOAC. 2005h

. Official Methods of Analysis of AOAC INTERNATION AL, 18th Ed., Methods
955.04 and 979.09

. AOAC International: Gaithersburg, Maryland.

AOAC

. 2005i. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods
960.46 and 992.05

. AOAC International: Gaithersburg, Maryland.

AOAC. 2005j. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
961.15. AOAC International: Gaithersburg, Maryland.

### APPENDIX D

5307

- FDA

- 1 Page 166 of 168 AOAC

. 2005k

. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
982.30. AOAC International: Gaithersburg, Maryla nd.

AOAC. 2005l. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
983.23

. AOAC International: Gaithersburg, Maryland.

AOAC

. 2005m

. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods
984.27 and 985.01

. AOAC International: Gaithersburg, Maryland.

AOAC. 2005n. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
985.29. AOAC International: Gaithersburg, Maryland.

AOAC

. 2005o. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
993.14

. AOAC International: Gaithersburg, Maryland.

AOAC. 2005p

. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method
996.11

. AOAC International: Gaithersburg, Maryland.

AOCS. 1997a. Official Methods and Recommended Prac tices of the AOCS , 5th Ed. Method Ba

12

- 75. American Oil Chemists’ Society: Champaign, Illinois.

AOCS. 1997b. Official Methods and Recommended Practices of the AOCS , 5th Ed. Method Ce

2

- 66. American Oil Chemists’ Society: Champaign, Illinois.

AOC S. 2001

. Official Methods and Recommended Practices of the AOCS , 5th Ed. Method Ce

1e

- 92. American Oil Chemists’ Society: Champaign, Illinois.

Atkins L , Schultz AS , Williams WL , Frey CN. 1943. Yeast microbiological methods for
determination of vitam ins. Industrial and Engineering Chemistry, Analytical Edition
15: 141

- 144.

Bradstreet R B. 1965. The Kjeldahl Method for Organic Nitrogen. Academic Press: New York,
New York.

Brobst KM. 1972 . Gas

- liquid chromatography of trimethylsilyl derivatives. In: Methods in Carbohydrate Chemistry, Volume 6: General Carbohydrate Methods . RN Whistler and

J N BeMiller, Eds. Academic Press: New York, New York.

### APPENDIX D

5307

- FDA

- 1 Page 167 of 168 Cort WM , Vincente TS , Waysek EH , Williams BD. 1983 . Vitamin E content of feedstuffs determined by high

- performance liquid chromatographic fluorescence. Journal of Agricultural and Food Chemistry

. 31: 1330

- 1333.

Hagerman AE, Nicholson RL. 1982 . High

- performance liquid chromatographic determina tion of hydroxycinnamic acids in maize mesocotyl. Journal of Agricultural and Food Chemistry 30: 1098

- 1102

ILSI. 2008

. International Life Sciences Institute Crop Composition Database, v. 3.0. http://www.cropcomposition.org/cgi

- perl/search_ora.cgi; search parameters: crop type = “CORN_FIELD,” tissue type = “FORAGE” or “GRAIN,” crop year = “All”, country = “All,” and state = “All”; searching not restricted by analysis method or value rang e (accessed February 9, 2010).

Infant Formula Council . 1985a

. Methods of Analysis for Infant Formulas. Section C

- 2. Atlanta, Georgia.

Infant Formula Council. 1985b

. Methods of Analysis for Infant Formulas. Section C

- 7. Atlanta, Georgia.

Kalthoff IM, Sandell EB. 1948

. Quantitative Inorganic Analysis. MacMillan: New York.

Kenward MG

, Roger JH. 1997. Small sample inference for fixed effects from restricted
maximum likelihood. Biometrics 53: 983

– 997.

Lehrfeld J. 1989 . High

- performance liquid ch romatography 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: Problem and solutions. Journal of Ag ricultural and Food Chemistry 42: 2726

- 2731.

Mason BS

, Slover HT. 1971. A gas chromatographic method for the determination of sugars in
foods. Journal of Agricultural and Food Chemistry. 19: 551

- 554.

McMurray CH , Blanchflower WJ , Rice DA. 1980 . Influence of extraction techniques on determination of alpha

- tocopherol in animal feedstuffs. Journal of the Association of Official Analytical Chemists 63: 1258

- 1261.

### APPENDIX D

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- 1 Page 168 of 168 Quackenbush F W. 1987. Reverse phase HPLC separation of cis

- and trans

- carotenoids and its application to β

- carotenes in food materials. Journal of Liquid Chromatography 10: 643

- 653.

Speek AJ

, Schijver J , Schreurs WHP. 1983

. Vitamin E composition of some seed oils as determined by high

- performance liquid chromatography with fluorometri c quantitation. Journal of Food Science 50 : 121

- 124.

USDA. 1970

. Forage fiber analyses. Agriculture Handbook No. 379 . United States Department of Agriculture: Washington, DC.

USDA. 1973 . Energy value of foods. Agriculture Handbook No. 74, pp. 2

- 1 1

. United States Department of Agriculture: Washington, DC.

US Pharmacopeia

. 2005. The United States Pharmacopeia, Twenty

- Ninth Revision, p. 1913, United States Pharmacopeial Convention, Inc.: Rockville, Maryland.Event 5307 Corn Environmental Report

C-1 Appendix C: Data Tables 

##  

# Appendix C Data TablesEvent 5307 Corn Environmental Report

1 Appendix C Table C-1: Representative Conventional Insectic ide Products to Control Corn Rootworm

Product Active Ingredient(s);

% of Formulation Signal

Word/Classification Environmental Hazards and/or Reasons for Restricted

Use Classification

Ambush® Insecticide permethrin; 25.6% WARNING/Restricted

Use Extremely toxic to fish and aquatic invertebrates, highly toxic

to bees.

Avicta® Duo

Nematicide/lnsecticide (seed treatment) abamectin; 12.4%
thiamethoxam;

28.1% WARNING/Restricted

Use Toxic to fish and wildlife, highly toxic to aquatic invertebrates, exposed treated seeds may be hazardous to wildlife.

Aztec® 2.1% Granular

Insecticide tebupirimfos; 2.0% cyfluthrin;0.1% WARNING/Restricted

Use Toxic to fish and wildlife.

Baythroid® 2 Emulsifiable Pyrethroid Insecticide cyfluthrin; 25% DANGER/Restricted

Use Extremely toxic to fish and aquatic invertebrates, highly toxic

to bees.
Capture® 2EC

Insecticide/Miticide bifenthrin;25.1% WARNING/Restricted

Use Extremely toxic to fish and aquatic invertebrates, highly toxic

to bees
Capture® LFR Insecticide bifenthrin; 17.15% WARNING/Restricted

Use Extremely toxic to fish and aquatic invertebrates, highly toxic

to bees.
Counter® 15G Systemic

Insecticide Nematicide terbufos; 15% DANGER/Restricted

Use Highly toxic to fish and wildlife, known to cause fish kills, birds and mammals may be killed if granules not covered with soil. Fatal if swallowed, inhaled or absorbed through skin, corrosive, causes irreversible eye damage.
Counter 20G Systemic

Insecticide - Nematicide terbufos; 20% DANGER/Restricted

Use Highly toxic to fish and wildlife, known to cause fish kills, birds and mammals may be killed if granules not covered with soil. Acute dermal, oral and inhalation toxicity. Cruiser® 5FS (seed treatment) thiamethoxam;

47.6% CAUTION Toxic to wildlife, highly toxic to aquatic invertebrates, treated seeds exposed on soil surface may be hazardous to wildlife.

DuPont Asana®XL

Insecticide - 0.66 emulsible

concentrate esfenvalerate; 8.4% WARNING/Restricted

Use Extremely toxic to fish and aquatic invertebrates, highly toxic

to bees.

Declare® Emulsifiable

Insecticide Concentrate methyl parathion;

45.11% DANGER/Restricted

Use Fatal if swallowed, inhaled or absorbed through skin, highly toxic to aquatic invertebrates and wildlife, highly toxic to bees.

Dimethoate 4 EC Systemic Insecticide dimethoate; 44.8% WARNING Toxic to wildlife and aquatic invertebrates, highly toxic to

bees.
Dimethoate 400 Systemic

Insecticide -Miticide dimethoate; 43.5% WARNING Toxic to wildlife and aquatic invertebrates, highly toxic to

bees.

Force® 3G Insecticide tefluthrin; 3% CAUTION/Restricted

Use Very highly toxic to freshwater and estuarine fish and

invertebrates.

Force® ST Insecticide for

corn seed treatment tefluthrin; 26.8% CAUTION Very highly toxic to freshwater and estuarine fish and invertebrates; exposed treated seeds may be hazardous to

birds and other wildlife.

Force® CS Insecticide tefluthrin; 23.4% WARNING/Restricted

Use Very highly toxic to freshwater and estuarine fish and

invertebrates.
Fortress® 2.5G granular

insecticide chlorethoxyfos;

2.5% WARNING/Restricted

Use Toxic to wild mammals, birds, fish and aquatic invertebrates; harmful if absorbed through the skin.Event 5307 Corn Environmental Report

2 Appendix C Product Active Ingredient(s);

% of Formulation Signal

Word/Classification Environmental Hazards and/or Reasons for Restricted

Use Classification

Fortress® 5G granular

insecticide chlorethoxyfos; 5% DANGER/Restricted

Use Toxic to wild mammals, birds, fish and aquatic invertebrates; fatal if swallowed; may be fatal if inhaled; harmful if absorbed through skin.

Furadan® 4F insecticide/

nematicide carbofuran; 44% DANGER/Restricted

Use Toxic to fish, birds and other wildlife; highly toxic to bees; can seep or leach through soil and can contaminate groundwater; poisonous if swallowed or inhaled; may be fatal or harmful as a result of skin or eye contact or breathing spray mist.

Furadan® LFR

insecticide/ nematicide carbofuran; 40.64% DANGER/Restricted

Use Toxic to fish, birds and other wildlife; highly toxic to bees; can seep or leach through soil and can contaminate groundwater; poisonous if swallowed or inhaled; may be fatal or harmful as a result of skin or eye contact or breathing spray mist.

Gaucho® 600 Flowable (seed treatment) imidacloprid; 48.7% CAUTION Highly toxic to birds and aquatic invertebrates.

Gaucho® 75 ST Insecticide (seed treatment) imidacloprid; 75% CAUTION Highly toxic to birds and aquatic invertebrates.

Lambda 25 CS lam bda-cyhalothrin;

23.6% WARNING/Restricted

Use Extremely toxic to fish and aquatic invertebrates; toxic to wildlife; highly toxic to bees

Lannate® LV insecticide methomyl; 29% DANGER/Restricted

Use Fatal if swallowed, toxic to fish, aquatic invertebrates and mammals, highly toxic to bees, known to leach through soil into groundwater.

Lannate® SP insecticide methomyl; 90% DANGER/Restricted

Use Fatal if swallowed, may cause blindness, toxic to fish, aquatic invertebrates and mammals, highly toxic to bees, known to leach through soil into groundwater.

Lorsban® 15G Granular

Insecticide chlorpyrifos; 15% CAUTION Toxic to fish, aquatic invertebrates, birds and small mammals, highly toxic to bees.

Lorsban® -4E Insecticide chiorpyrifos; 44.9% WARNING/Restricted

Use Toxic to fish, aquatic invertebrates, birds and small mammals, highly toxic to bees.

Lorsban® 75WG

Insecticide chlorpyrifos; 75% WARNING Toxic to fish, aquatic invertebrates, birds and small mammals, highly toxic to bees.
Mocap® 15% Granular

Nematicide/ Insecticide ethoprop; 15% DANGER/Restricted

Use Toxic to aquatic organisms and wildlife; extremely toxic to birds; toxic to bees; fatal if absorbed through skin; may be fatal if swallowed; causes irreversible eye damage; harmful if inhaled.
Penncap-M® Microencapsulated

Insecticide methyl parathion;

22.0% WARNING/Restricted

Use Highly toxic to aquatic invertebrates and wildlife.

Phorate 20 G Organophosphate

Insecticide phorate; 20.0% DANGER/Restricted

Use Very highly toxic to fish and wildlife. Birds and mammals may be killed if granules are not properly covered with soil. Fatal if swallowed, inhaled or absorbed through the skin; corrosive; causes irreversible eye damage.Event 5307 Corn Environmental Report

3 Appendix C Product Active Ingredient(s);

% of Formulation Signal

Word/Classification Environmental Hazards and/or Reasons for Restricted

Use Classification

Poncho® 600 (seed treatment) clothianidin; 48% CAUTION Toxic to aquatic invertebrates.

Pounce® 3.2 EC

Insecticide permethrin; 38.4% CAUTION/Restricted

Use Extremely toxic to fish and aquatic invertebrates, highly toxic

to bees.

Pounce® 25 WP

Insecticide permethrin; 25% WARNING/Restricted

Use Extremely toxic to fish and aquatic invertebrates, highly toxic

to bees.
Sewn® 80 Solupak carbaryl; 80% WARNING Extremely toxic to aquatic invertebrates, highly toxic to bees.

Sevin® brand XLR PLUS Carbaryl Insecticide carbaryl; 44.1% CAUTION Extremely toxic to aquatic invertebrates, highly toxic to bees.

Tundra® Max Insecticide chlorpyrifos; 28.6%
bifenthrin; 9.0% WARNING/Restricted

Use Extremely toxic to fish, aquatic invertebrates, small mammals, and birds. Highly toxic to bees.

Warrior II with Zeon Technology® Insecticide lambda-cyhalothrin;

22.8% WARNING/Restricted

Use Extremely toxic to fish and aquatic organisms and toxic to wildlife, highly toxic to bees.
Zeta-Cype 0.8EW

Insecticide zeta-cy perm ethrin;

9.2% WARNING/Restricted

Use Extremely toxic to fish, aquatic invertebrates, oysters and shrimp; highly toxic to bees.

Source: Vlachos and Ward. 2011.Event 5307 Corn Environmental Report

1 Appendix C Table C-2: Corn Diseases

Disease

Name Pathogen Conditions Management

Seed Rot Fungi and Bacteria. Pythium, Fusarium, Diplodia, Rhizoctonia,
Penicillium spp., various soil borne
bacteria Favored by prolonged wet and cold soil conditions in the spring. Soil temperatures 50 F or lower
favor seed rots Fungicide seed treatment. Plant
when soil conditions are warmer and drier, use the proper planting depth Gray Leaf Spot Fungus. Cercospora zeae-maydis Infection is favored by extended warm, wet, humid weather Select hybrids with resistance (tolerance based on risk), two year crop rotation, cleanly plow
under infected residue

Anthracnose Leaf Blight Fungus. Colletotrichum graminicola Favored by cool to warm, wet, humid weather, continuous corn with reduced tillage. Resistant hybrids, rotate corn with nongrass crops. Cleanly plow under infected residue.

Common
Corn Rust Fungus. Puccinia sorghi Disease favored by cool (66 F optimum) humid weather Resistant hybrids. Foliar fungicides may be useful in seed production fields Eyespot Fungus. Kabatiella zeae Disease favored by cool moist
conditions. Crop rotation, tillage practices, resistant genotypes Brown Spot Fungus. Physoderma maydis Favored by hot, humid tropical
conditions Crop rotation and conventional tillage

Southern
Corn Rust Fungus. Puccinia polysora Favored by high humidity and temperatures around 80 F. Resistant hybrids. Foliar fungicides may be useful in seed production fields.

Northern Corn Leaf Blight Fungus. Exserohilum turcicum Favored by extended wet, cool, humid weather, minimum tillage, continuous corn. Usually occurs during or after pollination Resistant hybrids. Foliar fungicides may be useful in seed production fields. Cleanly plow
under infected residue

Northern Leaf Spot Fungus. Helminthosporium
carbonum (Race 3) Favored by moderate temperatures and high relative humidity, minimum tillage,

continuous corn Resistant hybrids. Disease is primarily a problem in seed production fields with certain highly susceptible inbreds. Foliar fungicides may be useful in seed production fields. Cleanly plow
under infected residue

Southern
Corn Leaf Blight Fungus. Bipolaris maydis Favored by extended warm, wet, humid weather, minimum tillage,
and continuous corn. Resistant hybrids. Foliar fungicides may be useful in seed production fields. Cleanly plow
under infected residue

Stewart's Wilt Bacterium. Erwinia stewartii Favored by high infestation levels of flea beetles in April through late

June Apply insecticides early to control corn flea beetles, resistant hybridsEvent 5307 Corn Environmental Report

2 Appendix C Table C-2: Corn Diseases (continued)

Disease

Name Pathogen Conditions Management

Anthracnose Leaf Blight Fungus. Colletotrichum graminicola Favored by cool to warm, wet, humid weather, minimum tillage, continuous corn, stresses that result in early senescence Resistant hybrids (full season hybrids tend to have more resistance than short season), two year crop rotation with non-grass crops, cleanly plow under infected residue, balanced soil fertility.

Goss’s Wilt Bacteria. Clavibacter michiganenis Temperatures between 12°C to 40°C (ideal temperature 27°C) Deep plowing of residue following harvest and crop rotation.

Common

Smut Fungus. Ustilago maydis Rainy and Humid Conditions. Use of high levels of nitrogen or barnyard manure Crop rotation, fungicide treatment, resistant hybrids.

Head Smut Fungus. Sphacelotheca reliana Favored by soil temperatures of 21
to 28°C and low soil moisture under dryland conditions, sandy
soils Fungicide seed treatment, in furrow treatment, application of ammonium sulphate or urea
fertilizer Sorghum Downy
Mildew Fungus. Peronosclerospora sorghi Period of free moisture or saturated atmosphere of 4 hours needed for germination and

infection Fungicide seed treatment

Diplodia Stalk

Rot Fungus. Diplodia maydis Warm, moist weather in late summer (2-3 wks after silking), stresses that result in early

senescence. Resistant hybrids (full season hybrids tend to have more
resistance than short season hybrids), balanced soil fertility, recommended plant population.

Gibberella
Stalk Rot Fungus. Gibberella zeae Warm, moist weather in late summer (2-3 wks after silking). More prevalent when plants are subjected to stresses that result in early senescence and a reduction of sugar to roots and stalks. Resistant hybrids. Full season hybrids tend to have more
resistance than short season. Balanced soil fertility. Do not exceed recommended plant population

Fusarium
Stalk Rot Fungus. Fusarium moniliforme Warm, moist weather shortly after pollination. More prevalent when plants are subjected to stresses (such as dry weather) that result in early senescence and a reduction of sugar to roots and stalks. Resistant hybrids. Full season hybrids tend to have more
resistance than short season. Balanced soil fertility. Do not exceed recommended plant populations Stenocarpella
Stalk Rot Stenocarpella maydis and S.macrospora Warm, moist conditions Crop rotation, tillage and resistant hybridsEvent 5307 Corn Environmental Report

3 Appendix C Table C-2: Corn Diseases (continued)

Disease

Name Pathogen Conditions Management

Charcoal Rot Macrophomina phaseolina Post flowering drought stress with soil temperatures between 30-
42°C Management practices that reduce crop stress and reduce soil temperatures Diplodia Ear

Rot Fungus. Diplodia maydis Dry weather prior to silking, followed by wet conditions within first 30 days after silking. Resistant hybrids, crop rotation, clean plowing, harvest early to prevent weathering. Dry corn to
15% moisture content and below to prevent further mold growth in storage.

Gibberella Ear

Rot Fungus. Gibberella zeae Cool wet weather within first 21 days after silking favors the development of this disease. Resistant hybrids, crop rotation, harvest early to prevent continued mold growth in the field, clean plowing. Dry corn to
15% moisture content and below to prevent further mold growth in storage.

Fusarium Ear

Rot Fungus. Fusarium moniliforme Dry, warm weather. Infection occurs through injury by insects or
environmental stress. Resistant hybrids (avoid sowing hybrids with weak seed coats or poor husk cover), crop rotation, clean plowing, harvest grain early, dry corn to 15% moisture content and below to prevent further mold growth in storage

Aspergillus

Ear Rot Fungi. Aspergillus flavus, A. glaucus, A. niger A. flavus is more common in Indiana following hot, dry weather, injury by drought stress and insect damage. Mold growth in storage when moisture is higher than 18%.
A. flavus can produce a carcinogenic secondary metabolite

known as aflatox In storage, controlled by drying
corn to a moisture content below
15% as soon after harvest as possible. In the field, avoid insect or mechanical damage to ears.

Maize Dwarf
Mosaic Virus. Maize dwarf mosaic virus (MDMV) strain A or B Those favorable to aphids and growth of Johnson grass in fields Resistant hybrids, control rhizome Johnsongrass or other overwintering weed hosts.

Maize
Chlorotic

Dwarf Virus. Maize chlorotic dwarf virus (MCDV) Those that favor leafhopper reproduction and growth of Johnson grass in fields. Resistant varieties. Sow early in the growing season to avoid large leaf hopper populations. Control perennial JohnsonEvent 5307 Corn Environmental Report

4 Appendix C Table C-2: Corn Diseases (continued)

Disease

Name Pathogen Conditions Management

Crazy Top Fungus. Scleropthora macrospora Saturated soil conditions for 24-48

hours Provide adequate soil drainage, control grassy weeds, avoid sowing in low, wet spots.

Maize
Chlorotic

Mottle Virus. MCMV - Crop rotation, resistant hybrids

High Plains
Disease Virus. High plains virus and wheat

streak mosaic virus Disease occurs when corn is planted adjacent to small grains field that are beginning to dry
down Modification in planting dates, Resistant hybrids

Sources: Ruhl 2007 and Smith et al . 2004.