Petition 10-336-01p 

ENVIRONMENTAL REPORT

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, 2011 
Event 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 Systems .................................................................................... 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 .......................................................................................... 2-32
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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 .................................................................. 4-43
Event 5307 Corn 

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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 Characteristics of Geographic Areas ..... 5-7

6 List of Preparers ........................................................................................................... 6-1

7 References ..................................................................................................................... 7-1

Appendix A Public Interest Assessment ........................................................................... 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 Tables 
Event 5307 Corn 

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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 ............................................................................ 4-47 
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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 Seed 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 

Bt 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 

CO2 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 Trade of the United States 

FAS Foreign Agricultural Service (also USDA-FAS) 

FB1 Fumonisin B1

FDA Food and Drug Administration (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

N2O Nitrous oxide 

NAPPO North American Plant Protection Organization
Event 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 

pmi 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 Wildlife Service (also FWS) 

USGC U.S. Grains Council 

Vip Vegetative insecticidal protein 
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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 basic charge of the United States Department 

of Agriculture (USDA) Animal and Plant Health 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 conventional, genetically engineered [transgenic], 

and organic varieties) can provide benefits to the environment, consumers, and 

producers. 

Since 1986, the United States government has regulated genetically engineered organisms 

pursuant to a regulatory framework known as the Coordinated Framework for the Regulation 

of Biotechnology1 (Coordinated Framework). The Coordinated Framework describes the 

comprehensive federal regulatory policy for ensuring 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 environmental 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, Office 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 regulatory roles and authorities for the 

three major federal agencies involved in regulating genetically engineered 

organisms: APHIS, the US Food and Drug Administration (FDA), and the US 

Environmental Protection Agency (EPA): 

 APHIS is responsible for regulating genetically engineered organisms pursuant 

to the plant pest authorities in the Plant Protection Act2 (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 statement4 concerning regulation of products derived 

from new plant varieties, including those 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 Act5 (FIFRA) and certain biological control 

organisms under the Toxic Substances Control Act6 (TSCA). The EPA is 

responsible for regulating the sale, distribution 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 dynamic 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. 
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1-3 Purpose and Need 

framework that allows for the safe development and use of genetically engineered 

organisms. APHIS regulations7 promulgated pursuant to authority granted by the PPA 

govern the introduction (importation, interstate movement, or release into the 

environment) of certain genetically engineered organisms. A genetically engineered 

organism is no longer subject to the plant pest 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 vector agent used in engineering the organism 

belongs to one of the taxa listed in the regulation8 and is also considered a plant pest. A 

genetically engineered organism is also regulated when APHIS has reason to believe 

that the genetically engineered organism may 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 determination 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 required9 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 unmodified organism. A genetically engineered 

organism is no longer subject to the regulatory 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 initial 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 corn, progeny from crosses between 5307 

corn and conventional corn, and progeny from crosses of 5307 corn with other 

transgenic corn that has been deregulated. Event 5307 corn is currently regulated 

under the PPA. Interstate movements and field trials of 5307 corn have been 

conducted under permits issued or notifications 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. 
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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 contains 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 insecticides that are of concern due to human 

and environmental risk factors. The new protein, 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 and 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 available 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 provisions 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 environmental assessment (EA) that will consider the potential 

environmental effects as part of an agency determination of nonregulated status 

consistent with the National Environmental Policy Act12 (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 may 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 Permit15 was initially 

granted on June 1, 2010 with effect through February 28, 2012 and was extended16 on 

March 3, 2011 with effect through December 31, 2013. In connection with this 

Experimental Use Permit, EPA established17 and extended18 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 provided by other constituents of these 

combinations are: 

 A Cry1Ab protein for lepidopteran control (Bt11); 

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

 A Cry1F protein for lepidopteran control (TC1507); 

 A double mutated 5-enolpyruvylshikimate-3-phosphate synthase enzyme for 

glyphosate tolerance (GA21); 

 A Vip3Aa20 protein for lepidopteran 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. 
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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 for the two breeding stack products that 

include 5307 corn. Concurrently, Syngenta also submitted a petition (Petition 

No. 1F7857) to the EPA to establish a nonexpiring 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 from 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 deregulate 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 comment 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 regulated status of 5307 corn and to assist 

APHIS in determining whether an Environmental Impact Statement (EIS) is required 

prior to the determination decision of the 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 corn, 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) 
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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 Effects 
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1-8 Purpose and Need 

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2-1 Affected Environment 

2

Affected Environment 

This Chapter describes corn biology, corn 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 area 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 biochemical level, these plants convert carbon 

dioxide (CO2) into a four-carbon molecule in contrast to the more common 

photosynthetic pathway in which CO2 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-operation 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 crossed 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 and typically does not survive freezing winter 

conditions.26 Consequently, corn has no innate dormancy.27

Humans have been selectively breeding corn 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 APHIS28 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 growers 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-operation 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 Transgenic 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, 2010 
Event 5307 Corn 

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2.2 Corn Production 

This section describes corn production and yield in the US, current agronomic 

practices, specialty corn production systems, 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. 
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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 yield growth. The projected 2010/11 corn 

yield is based on the simple linear trend 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. 
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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 expected 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 banks39) 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. 
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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. Reduced tillage uses chisel plows and results 

in less soil disturbance. Conservation tillage 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, conservation 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 minimum 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 biological 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 rainfall 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. 
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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 insects 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 significant 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, and by increasing the plant's susceptibility to 

lodging.49 Lodging 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. 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 advancing 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 girdled 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. 
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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. 
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 Hybrid selection: Hybrids vary in their ability to withstand and resist insect 

pests such as European corn borer. Rapid germination, early vigor, strong ear 

shanks, tight husks, resistance to stalk rots and other pests, strong stalks, and 

uniform performance over a wide population 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, growers 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 Workbook54

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. 
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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 oils 

Petroleum 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 insecticides 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 integrated 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 agricultural inputs. Timely and targeted delivery of 

pest management interventions is key to successful IPM. 



56 Koul et al. 2004. Pg. 29. 
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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 depth 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 feed first on roots near the soil surface; 

when these are consumed, the next lower node 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. Third instars cause the majority of root 

damage and they generally feed on the large primary roots near the stalk. Larval 

corn rootworm injury results in yield losses in three ways: 

 Root pruning and tunneling disrupt nutrients 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 secondary 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 has 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. 
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Corn rootworm larval feeding can disrupt the movement of water and nutrients, 

which can slow development and stunt plants, ultimately leading to yield loss. 

During dry periods, when conditions suppress 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 parts 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 since 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. 
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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 insecticide 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 rootworm 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. 
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2-15 Affected Environment 

In organic production systems, Bt foliar sprays may be used as a natural insecticide. 

However, the use of foliar sprays of Bt has been limited because 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 significant 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 protection 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 use76 for control of 

European corn borer and other lepidopteran 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 rootworm 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 adverse 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 chemical insecticides. Planting transgenic 

rootworm-protected corn on 10 million 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 delay 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. 
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2-16 Affected Environment 

susceptible pests, which mate with resistant insects and slow the evolution of 

resistance.79 Such refuges are expected to be most 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 indicates 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 the grower’s corn area, depending on the 

product’s requirements. Greenhouse and laboratory 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, which 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. 
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Crop disease reduces both the quantity and 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 fumonisins 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 has been shown to reduce the incidence of 

some mycotoxins in grain.90 Minimizing insect damage 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 early 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 systems because of natural herbicide tolerance 

and because of growth types or life cycles that help them avoid being treated, such as 

some winter annual weed species. Industry practice includes weed resistance 

management training to reduce the potential 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 soil, 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. 
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mechanical (tillage), and chemical (herbicide) methods for consistent, effective weed 

control.94 Integrated weed management is a USDA policy.95 An integrated weed 

management program can help prevent the 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 corn97 while broadbased herbicide usage decreased.98

Herbicide-tolerant corn products have been genetically engineered to allow use of 

herbicides without harming the crop. Herbicide-tolerant corn has been widely 

adopted by growers in North America and offers enhanced weed control. In 2010, 

approximately 70 percent of the US corn crop was herbicide-tolerant.99 Currently 

available 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 conditions 

contribute 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 and 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. 
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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 organic production system. The NOP was established by the

Organic Foods Production Act of 1990103 and implementing USDA regulations.104

Organic production operators 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 crops is excluded. Natural products, including 

some Bt foliar 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-organic crops. For example organic corn may 

be planted at a different time than neighboring non-organic corn to avoid crosspollination due to overlapping flowering periods, or a buffer zone may place the 

organic corn farther away from non-organic corn than wind-borne non-organic corn 

pollen will typically travel. 

An integrated strategy108 for controlling selected insect pests in corn using organic 

practices might be comprised of: 

 Monitoring to determine pest pressure and 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 available109), 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. 
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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 basis, 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 processes, record maintenance, auditing, 

proper labeling, appropriate sampling and testing 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 commercial 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 organic 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. 
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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 variety 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 variety. 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 testing procedures for identifying and 

detecting acceptability of materials. 

 Quality Assurance Processes – oversight 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 supplies the planting seeds and guarantees 

to purchase final products. This may also require that the end-user conduct 

intermediate procedures such as planting, providing oversight during the 

growing season, harvesting, and transportation to processing plant. 

 Identity Preservation Systems – using systems 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. 
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(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 separates the endosperm into its chemical 

components, principally starch and protein. Breeding and genetic engineering for 

certain traits have improved the concentrations 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 following year in warmer areas. Manual or 

chemical measures are often applied to remove 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 landraces 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. 
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2.3 Physical Environment 

Water, soil, and air affect, and are affected 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 largest 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 

recommends128 several Best Management Practices 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. 
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 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 protection 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 the First 100 Days After Planting 

Source: Rhoades and Yonts. 2011. 

The total amount of water used by corn varies 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 Growers Association. 2010. Pg. 1. 

130 Christensen. 2002. Pg. 31. 

131 Krantz et al. 2008. Pg. 3. 
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Corn does not extract water uniformly throughout 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-ground community of microorganisms and 

arthropods, plant-root structure, and organic residues in the soil is central to soil 

ecological processes including decomposing organic material, subsequent 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, conventional tillage and 

mechanized harvesting machinery may disturb 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 (CO2, nitrous oxide [NOX]).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 provide 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 CO2 and helping to reduce a 

significant amount of greenhouse gas. 

2.3.3 Air Quality 

Air quality may potentially be directly affected 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 carbon monoxide, nitrogen oxides, reactive 

organic gases, particulate matter, sulfur oxides, and greenhouse gases (GHG).137

Tillage also releases GHG because of the loss of CO2 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. 
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application to soil or plant surfaces and move following wind erosion.139 NOX may 

also be released following nitrogen fertilizer application.140 Agriculture, including 

land-use changes for farming, is responsible for an estimated 6 percent of all humaninduced GHG emissions in the US.141 NOX emissions from agricultural soil 

management comprise 68 percent of all US N2O emissions.142

2.3.4 Climate 

Corn agriculture may potentially affect or be affected by climate change.143, 144

Agriculture-related activities are recognized 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 extending the ranges of weeds and other 

pests.147 One recent study148 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 because water resources may be substantially 

reduced. North American production is expected to adapt with improved cultivars 

and responsive farm management150

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. 
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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 fields 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 more 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 field 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 thirteen-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 crops, 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. 
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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, minute pirate bug, syrphid 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 agricultural 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 performance can be reduced by weed competition 

for water, nutrients, and light. US corn yields 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 14159 and 35160 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 sometimes 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 

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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 not 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 coleopteran species protected under the federal Endangered 

Species Act (ESA)164 that occur within the region where 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 protein produced in 5307 corn (eCry3.1Ab) has 

been shown to be active only on certain Coleoptera and not on other invertebrate or 

vertebrate species. The proposed action’s lack 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 Service165 (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) and 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 as 

Endangered 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 endangered 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. 
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2-30 Affected Environment 

Table 2-4 Threatened or Endangered Coleopteran Species Occurring in Corn-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) consumption of feed corn and corn products, and 

the indirect effect on human health and worker 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 Act168 requires disclosure of the presence of eight 

specific food groups which are designated as “major food allergens” on the package 

labels.169 These eight food groups (milk, eggs, fish, crustacean shellfish, 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 effects 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. 
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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 (direct skin contact) or internally (ingestion). 

Some attributed symptoms of corn allergy 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 contamination, as insect herbivory creates stalk or 

kernel wounds that encourage fungal colonization.174 Mycotoxins are of concern 

worldwide because of their toxic and carcinogenic effects in humans and animals 

feeding on infected corn. Table 2-5 provides a list of corn mycotoxins; the two 

categories of most concern are fumonisins and aflatoxins. 

Fumonisins are found exclusively in corn, while 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 B1 (FB1) is the 

most common in corn.176 Fumonisins can be highly toxic to animals, causing diseases 

such as leukoencephalomalacia in horses and pulmonary edema in swine.177

Lepidopteran-protected Bt corn hybrids have reduced 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) health 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. 
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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 worker health or safety risk unless used in 

accordance with the EPA-established agriculture-specific requirements in the Worker 

Protection Standard182 that protect field workers from the hazards of chemical 

exposure. The Occupational Safety and Health Administration requires all employers 

to protect their employees from hazards associated 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 and types of pesticides used and thus the 

risks to workers. Registered pesticides, including 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. 
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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 satisfied 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 approximately 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 increase 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 corn188 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. 
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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 2011 non-organic corn in selected markets 

averaged $7.65 per bushel, whereas organic 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 million 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 are also the largest world importers of corn 

and do not have major barriers for importing 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 Projections 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 growth in production and historically high 

prices are projected long term, through 2020. 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.nongmoproject.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. 
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2-35 Affected Environment 

2.7.3 Social Environment 

Social issues related to corn include farmer 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 applicable 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 higher 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 than 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, and 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 above 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 employment, 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. 
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2-36 Affected Environment 

deficiency; conservation reserve, wetland reserve, and environmental quality 

incentives program payments; and other.206 For corn operators, these programs 

contribute some 15 percent of their cash income. Direct government payments 

accounted for about 8 percent of average gross cash income for corn farms in 2003207

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 unlikely 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 CFR 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 confinement 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-transgenic 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 pest risk from the unconfined cultivation of 

5307 corn. 

Event 5307 corn is unlikely to pose a plant pest 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, this alternative would 

not satisfy the product’s purpose of providing 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 protein it encodes was utilized as 

a plant selectable marker during development of 5307 corn. 

As an engineered protein, eCry3.1Ab has similarities to other well-characterized Cry 

(crystal) proteins, namely mCry3A and Cry1Ab. 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 that 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 Coleoptera. 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 corn rootworm control (MIR604); 

 A Cry1F protein for lepidopteran control (TC1507); 

 A double mutated 5-enolpyruvylshikimate-3-phosphate synthase enzyme for 

glyphosate tolerance (GA21); 

 A Vip3Aa20 protein for lepidopteran 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 introductions 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 product 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 decline 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 technologies (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 regulating 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 evaluated 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 plant 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 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 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 

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3-5 Alternatives 

3.3.3 Establish Geographic Restrictions 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 there is no greater plant pest risk would not 

be consistent with APHIS’s statutory authority 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 petition 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 additional product to control corn rootworm. 

Individuals may choose on their own to geographically 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 require and provide testing to identify the 

presence of transgenic products in systems that produce non-transgenic products. 

There are no nationally established regulations involving testing, criteria, or limits of 

transgenic material in systems that produce non-transgenic products. Such a 

requirement would be extremely difficult to implement and maintain. Additionally, 

because 5307 corn does not pose a plant pest risk, the imposition of any type of testing 

requirement is inconsistent with the plant 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 selection 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 Environmental 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 economy 
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4-1 Environmental Consequences 

4

Environmental 

Consequences 

This Chapter describes the potential environmental consequences of the two 

alternatives under consideration: No Action and the Proposed Action. Under the No 

Action alternative, Event 5307 corn would 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 corn 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 environmental and human resources from the No 

Action and the Proposed Action alternatives 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 Quality215 (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. 
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4-2 Environmental Consequences 

consequences discusses the direct and indirect 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 are still reasonably foreseeable. Indirect 

effects may include growth inducing effects 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 decisions. 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 Action’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 incorporate a corn rotation, land that could be 

converted from inactive cropland to active 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 available published literature (peer-reviewed 

technical articles, industry information, and agency data), as well as Syngenta’s 

Petition219 and Public Interest Assessment220 for Event 5307 corn. The potential 

direct and indirect effects of the Proposed 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.) 
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4-3 Environmental Consequences 

literature review and analysis. The methodology 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 environmental 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 Petition, would be continued. As noted above, 

the analysis of direct or indirect effects of the Proposed Action are based on 5307 corn 

in the absence of stacking. The cumulative effects analysis was based on an 

assumption of unlimited marketing of 5307 corn as a product stacked with other 

transgenic traits. The consequences of both 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 parameters 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, commonly accepted best management practices 

for their chosen system and varieties during corn production. These management 

practices include standard planting dates, seeding rates, harvest times, and pest 

management methods as well as preservation and coexistence measures. The 

analyses also allow for effects that could result 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 context 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 APHIS221 (see Table 2-1). Some of these are marketed as single trait 

products while others are marketed as stacked 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); these four cultivars are currently on the 

market. 

As of October 12, 2011, one additional petition for deregulating a corn 

rootworm-resistant corn cultivar (Petition 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. 
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4-4 Environmental Consequences 

and tolerant of glufosinate herbicide. Additionally, 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 commodities 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 practices, 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 yield 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 Bill225 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 adoption 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 alternative. Conventional production practices that 

use transgenic varieties would continue to increase without granting nonregulated 

status to 5307 corn under the No Action alternative, 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). 
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4-5 Environmental Consequences 

It is anticipated that seed with transgenic 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, according to the 2002 Census of Agriculture) and 

under the No Action alternative the range 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 acreage 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 alternative. 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. Most corn is planted in fields that have 

been in crop production for years, rather than converted CRP lands. Granting 

nonregulated status to 5307 corn is not expected 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 characteristics: there were no statistically 

significant 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 generally 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. 
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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 nutrition 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 reduce the need to rotate crops for this 

purpose, but other benefits of crop rotation (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 likely 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. 
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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 flexibility 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 rotations would continue to be used by 

farmers if this cropping practice 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 additional 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 profitable 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, without regard to the number or type of corn 

varieties available on the market. Deregulating 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 corn 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 irrigation 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. 
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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 interfering with photosynthetic rates and by 

limiting the uptake of water and nutrients.239 Reducing rootworm-caused damage 

could decrease, and would likely not increase, irrigation rates. If 5307 corn is 

deregulated, irrigation rates are likely to continue to be affected by climate 

conditions. Because the eCry3.1Ab protein prevents the negative physiological 

impact of root pruning by rootworms, 5307 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 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 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 applications, 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. 
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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 single use of conventional 

insecticides in the US at that time. 

Increased adoption of corn rootworm-protected corn products is of environmental 

importance because many of the chemical insecticides registered for corn rootworm 

control present potential risks to applicators, other agricultural workers, and wildlife. 

The soil-applied and foliar chemical insecticides for larval and adult corn rootworm 

control subject to the greatest use reductions following the adoption of transgenic 

rootworm-protected corn varieties are organophosphates, carbamates, pyrethroids, 

and pyrazoles. Adoption of additional rootworm-protected corn varieties is not 

expected to markedly reduce the use of insecticidal 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 corn 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 corn 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 control 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 varieties. Some estimates indicate that reductions 

in chemical insecticide use on the order of 70249 to 75250 percent could result from 

widespread adoption of corn rootworm-protected varieties. For example, insecticide 



242 Ward et al. 2005. Pgs. 242-243. 

243 Arthropod populations having pesticide resistance are 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. 
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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 planted251 (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 pesticide 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 spatial (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 for 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 existing 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. 
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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 effect on insecticide use. 

Proposed Action Alternative 

If 5307 corn is deregulated, trends in reducing 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 Containing Bt-derived Proteins, or any that may be 

developed and marketed in the future. There is concern about the ability of corn 

rootworms to evolve resistance to control 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 expected to extend the useful life of other 

commercially available corn 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 would 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 rootworm-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 resistance. As described in Chapter 2, 

Section 2.2.3.1, Organic Crop Production, the National Organic Program (NOP) 

requires that organic farms have distinct, defined boundaries and buffer zones 

separating adjoining land not under organic management to prevent unintended 

contact with prohibited substances. Organic production operations must also 

develop and maintain an organic production system plan. Excluded production 

methods include methods used to genetically modify organisms or otherwise 
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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 because 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 (temporal isolation), and employing adequate 

isolation distances between the organic field and the fields of neighbors to minimize 

the chance that pollen will be carried between 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 acreage259 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. Comparatively, 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 percent of the 81.6 million acres of field corn 

planted that year,261 about 42.4 million acres. 

Organic corn growers may benefit from transgenic 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 for 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. 
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non-transgenic, and organic production systems can all provide benefits to the 

environment, consumers, and/or farm income. 

Gene movement into and out of organic production 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 practices 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 already using, or have the ability to use, 

these common practices. 

No Action Alternative 

Organic farming practices, including field 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 alternative. Organic production systems and 

non-organic production systems would continue 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 practices 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-organic 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 organic 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 field management to avoid excluded methods 

and gene movement through pollen drift or other means, are not likely to change as a 

direct or indirect result of the Proposed Action alternative. Organic corn seed 

availability would be unaffected 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 corn 
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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 varieties 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 nonregulated 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 identity 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 include contracts with growers, traceability, 

product tracking, and process verification. Syngenta and other seed corn companies 

take precautions to ensure that inbred parent lines are not misappropriated by third 

parties. These procedures greatly minimize any chances of commingling of 5307 corn 

seed with other seed and, ultimately, commercial grain. 
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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 assurance 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-preservation 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 with 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 alternative. 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, there 

are 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 remain 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 pest 
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4-16 Environmental Consequences 

effect is foreseen on any raw or processed 5307 corn commodity. 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 processed corn commodities would continue to 

be handled in accordance with regulatory 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.) Agronomic practices such as 

conservation tillage, crop nutrient management, pest management, and conservation 

buffers help protect water quality from agricultural runoff. 

Corn pollen may be deposited into water bodies 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, potentially 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.
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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 delegated to certain states) and agronomic 

practices to protect water quality would continue 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 eCry3.1Ab protein concentrations in 5307 corn 

are lowest at senescence, when grain is harvested, and any residual eCry3.1Ab 

protein is likely to degrade significantly 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 costs). Regulated field trials of 5307 corn 

would only impact small plots of land with no effect on water use. 



271 Vlachos and Huber. 2011. Pg. 139. 
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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 greenhouse 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-resistant 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 effect 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 practices 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. 
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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 directly affect air quality. With regard to 

transgenic corn with insect resistance traits, 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 resistance 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 current 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 would 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., exhaust 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 each 
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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 current 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 agriculture. 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 production would not change land acreage or 

cultivation practices for transgenic or non-transgenic corn. Corn growers would 

continue current trends in agricultural activities 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 and 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. 
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Animals can have positive, negative, or no effect on corn production; insect-resistant 

corn varieties are targeted to certain invertebrate 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 cultivars 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 invertebrates 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, including 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 Cry proteins could potentially affect 

non-target invertebrates that directly consume Bt plant material or are exposed via 

Cry protein residues in soil, water, or prey 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 would 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 the concentration in plants on which they are 



278 Potential impacts to threatened and endangered species are separately discussed in Section 4.4.5. 

279 NPTN. 2000. Pgs 2-3. 

280 Harwood et al. 2005. Pg. 2820. 
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4-22 Environmental Consequences 

feeding. In general, the results show that 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 concentrations under “no choice” conditions 

where the species was continually exposed through its diet. Some laboratory studies 

have found an effect, but concluded through refinement of exposure models and 

estimates, or via field studies, that the effect 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 found no significant effects 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. 
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A meta-analysis of 74 laboratory studies of Cry protein toxicity and 52 field studies 

of Bt crops containing the same Cry proteins 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 associated 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-active Cry proteins and Bt plants, the meta-analysis revealed no 

adverse effects on survival in laboratory 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 field 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 heavy 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 water bodies as litter could also potentially 

introduce Cry proteins to aquatic organisms. However, significant degradation of 

protein is likely to occur during the period between harvest and transfer to the 

waterways299 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. 
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Commercially raised fish may also be exposed 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-resistant 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 biologically 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 kernels. 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 active 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 mice, prairie and meadow voles (Microtus spp.), and 

woodchucks feed on germinating corn seeds. 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 active 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. 
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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 modified 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 outside 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 Cry proteins would be similar to current 

conditions. Event 5307 corn has been demonstrated 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. 
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Event 5307 corn is not substantively different in its effects on non-target species from 

other approved insect-resistant corn products with Cry proteins. The eCry3.1Ab 

protein present in 5307 corn has a unique binding 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 plant 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 

(< LOQ318) 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, < LOQ319 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 protein 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, physicochemical 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 adverse 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. 
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4-27 Environmental Consequences 

Syngenta has conducted laboratory tests 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-market consultation process with FDA and 

submitted a Safety and NutritionalAassessment for 5307 corn in January 2011.334 The 

assessment demonstrated that the introduced proteins, eCry3.1Ab335 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 important 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 eCry3.1Ab 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 PMI in all crops (40 CFR Part 180.1252; USEPA. 2007b). 
Event 5307 Corn 

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4-28 Environmental Consequences 

mechanical (tillage), and chemical (herbicide), or some combination 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 indirect result of the No Action alternative. 

Weed management methods would continue following current trends even if 

5307 corn continues to remain a regulated article. Regulated field trials of 5307 corn 

would only impact small plots 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 practices are anticipated. A description of the 

potential impacts to plants from a biodiversity 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 exposure 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 

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4-29 Environmental Consequences 

significant but transient effect on the numbers 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 proteins do not persist and degrade rapidly in 

soil, although a small fraction may be protected 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 function 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 

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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 article. Regulated field trials of 5307 corn 

would only impact small plots 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 decline in eCry3.1Ab bioactivity was observed: 

no biological activity above background control 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. 
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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 genetically 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 not needed for the same volume of crop 

production.374 

No Action Alternative 

Biodiversity would not be affected as a direct or indirect result of the No Action 

alternative. Biodiversity within agroecosystems would continue to be affected by 

agricultural practices following current trends. Growers would continue to have 

access to existing deregulated insect-resistant 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 field 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 

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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 other 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 the 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 transgenic 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 teosinte 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 years. Corn does not possess the 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 conditions was found, and there was only 

equivocal evidence for horizontal gene transfer 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 result 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. 
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4-33 Environmental Consequences 

continues to remain a regulated article. Regulated field trials of 5307 corn would only 

impact small plots of land with minimal potential for exposure to sexually 

compatible plants (i.e., other corn plants). 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 result 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 Act381 (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, protective 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 continued 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.
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4.5.1 APHIS Section 7 Process 

Working with the USFWS, APHIS has developed a process for conducting an effects 

determination for petitions for nonregulated status. This process382 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 respect 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 plant 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 endangered species that may result from the No 

Action or Proposed Action alternatives. 

No Action Alternative 

The No Action alternative would not affect any listed threatened or endangered species. 

Under this alternative, 5307 corn could continue to be grown 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 Lepidoptera (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. 
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Proposed Action Alternative 

Wide cultivation of 5307 corn is not expected 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, and 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 corn 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 agronomic 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 showed 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. 
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American burying beetle relies on carrion (mammals, birds, reptiles) as a food 

source387 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 limit 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 family. Biosafety analyses documented in the Petition389

showed no effects of eCry3.1Ab on the survival of carabid beetles when tested at 

levels 62 times greater than the worst-case estimated environmental concentration.390

Therefore, any exposure of Salt Creek tiger 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 assessment391 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, pheromones, or hormones, etc. that could directly 

or indirectly affect a listed species or species proposed for listing. The high specificity 

of the eCry3.1Ab protein makes it unlikely 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 lepidopteran activity, despite containing 

sequences from a lepidopteran-active protein (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 the 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 

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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 biosafety studies conducted by Syngenta393 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 available. 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 about the potential impacts that transgenic 

products could have on human health because 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 tests that have been performed for adverse 

mammalian impact from ingesting Cry proteins 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. 
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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 and 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 percent 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 corn-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 consumption. 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 would be limited to those individuals 

involved in cultivation under regulated conditions 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 exposure 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 products 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 allergenic 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. 
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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 existing 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 Petition399 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 significant 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 considered 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-market 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; effects 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) 
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Chapter 2, Section 2.2.1, Production and Yield, explains that approximately 39 percent 

of the corn produced in the US is used for 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 allergenic, 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 consumption 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, and adoption of transgenic 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 or 

allergenicity 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 both 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-market 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) 
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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 Standard407 requires employers to take actions to reduce the risk 

of pesticide poisonings and injuries among agricultural workers and pesticide 

handlers. The restrictions and precautions for worker safety associated with several 

conventional insecticides 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; 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 aggregate 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 adopted. 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. 
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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 adoption 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 environment 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 rootworm family is one of 

the most damaging corn pests, and was often referred to as the “billion dollar bug” 

due to annual yield losses and control costs 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-resistant 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. 
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No Action Alternative 

The domestic economic environment would not be affected as a direct or indirect 

result of the No Action alternative. Growers would continue to make choices to plant 

certain corn varieties and use 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 flexibility of the production 

system.413, 414 The No Action alternative would not affect available options for 

growers and therefore not affect the domestic 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 impact 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 Proposed 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 increase 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 commodities produced from transgenic crops, 

including those with insect resistance traits. 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. 
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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. Worldwide market conditions and destination 

country approval of transgenic crop commodities 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 would 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 Canada), regulatory filings for 5307 corn 

import approvals have been made in Japan, 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 impacts associated with the deregulation 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. 
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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 indirect 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 environment when added to other past, present, 

and reasonably foreseeable future actions. 

4.8.1 Methodology and Assumptions 

The CEQ regulations define a cumulative effect 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 undertakes 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. Furthermore, as discussed in this section, 

there are no past, present or reasonably foreseeable actions that, in aggregate with 

the Proposed Action alternative, would adversely affect these resources. Based on the 

evaluation provided in previous sections of this Chapter, if 5307 corn is deregulated 

corn production practices would continue 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. 
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4-46 Environmental Consequences 

production, the physical environment, the biological 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-resistance 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 deregulated 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 requested 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 Stacking 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. 
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The combination hybrids A and B listed in Table 4-1 will combine two corn 

rootworm-active proteins (eCry3.1Ab from 5307 corn and mCry3A from MIR604 

corn) that each provide control of western, 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 herbicide tolerance traits, as listed in Table 4-2. 
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Table 4-2 Commercially Available Corn Breeding Stack Combinations with 

Insect Control Traits 

Product Event Names Resistance/Tolerance Traits 

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

Agrisure VipteraTM 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 

GenuityTM VT Double PROTM 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/optimum-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 stacks with similar combinations of traits. 
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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 Table 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 impact alone or cumulatively, APHIS has not 

specifically addressed the cumulative impact 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 combinations include agronomic practices 

(insecticide use and potential development of resistant insect populations; herbicide 

use and potential development of herbicide-tolerant weeds), biodiversity, and 

domestic economic issues (cost to farmers). 

Potential cumulative effects of the Proposed 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 alternative in reference to these potentially 

affected resources. The potential cumulative 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 approved transgenic corn 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 discussed 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 change overall herbicide use. The past and 

current actions potentially contributing to cumulative 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. 
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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 control 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 potential 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 area424), 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 terms 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 Action 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. 
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analysis of the No Action alternative, the 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 rootworms are not likely to develop resistance 

to Bt corn 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 

insecticide use or corn rootworm resistance to Bt corn. 

Insect-resistant transgenic corn varieties would continue to be available, and insects 

could potentially develop resistance to these varieties. When combined with the past, 

present, and reasonably foreseeable future actions, there would be no cumulative 

effect from the No Action alternative on insecticide use or corn rootworm resistance 

to Bt corn. However, without the additional rootworm control option offered by 

5307 corn, fewer PIP traits would be available 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 excellent 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-protected 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 mandated by the EPA. IRM would include 

insect scouting or monitoring to determine 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 interventions 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. 
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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 correlated 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 populations will develop resistance to Cry 

proteins, extending the effectiveness 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 conditions promote the development of herbicidetolerant weeds. Weeds can potentially survive in crop production systems because of 

natural tolerance to the chemical(s) and because 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 reduce the potential for weeds to develop 

tolerance to herbicides.431 Because there are several herbicides available, and 

transgenic corn varieties tolerant to different herbicides available, growers have the 

ability to rotate herbicide use, rather than relying on a single herbicide. 

The adoption of herbicide-tolerant crops also 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. 
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No Action Alternative 

Herbicide use would not be affected as a direct or indirect result of the No Action 

alternative. The reasonably foreseeable future 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 past, 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 herbicide tolerance trait, under the Proposed 

Action alternative it will be stacked with other transgenic corn cultivars that do have 

herbicide tolerance traits. Syngenta would market 5307 corn stacked with its own 

herbicide-tolerant cultivars. If 5307 corn is deregulated and stacked with herbicidetolerant cultivars, weed management methods may be altered to a minor degree but 

would generally continue following current trends. 

In APHIS’s recent analysis of the DP-098140-6 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 potentially 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 develop tolerance to herbicides. Given the 

widespread adoption of herbicide-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 foreseeable future actions, the Proposed 

Action would have a negligible cumulative effect on herbicide use or weed tolerance 

to herbicides. 



433 USDA-APHIS. 2009. Pg. 5. 
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4.8.3.2 Biodiversity 

The past and current actions potentially contributing to a cumulative effect on 

biodiversity are the introduction of transgenic corn varieties and their increasing use. 

The array of transgenic corn varieties currently available for pest management is 

outlined above. The future actions potentially 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 use 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 comprehensive environmental assessment of the 

registered Bt PIPs in 2001.434 Although other Bt corn cultivars incorporating Cry 

proteins have been introduced in the intervening 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 biodiversity 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 species (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 long-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. 
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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 evidence 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 and the actual impact on biodiversity are 

difficult to determine. For example, one study 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 studies443 suggest that monarch 

populations are very dynamic because of the high reproductive potential of this 

species, and no similar reduction was observed 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. For the cumulative effects 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. Regulated 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 foreseeable future actions, there would be no 

cumulative effect from the No Action 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 corn 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. 
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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 incrementally 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 rates 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 biodiversity 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 corn varieties currently available for pest 

management is outlined above. The future actions potentially contributing to 

cumulative effects to the domestic economy 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. Chapter 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 increasing 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 insect-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. 
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billions of dollars of estimated benefits to farmers in the midwestern US. The 

cumulative economic benefit to corn growers in a five-state region over a 14-year 

period ending in 2009 has been 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, reduction 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 herbicide tolerance traits in a single stacked 

product would likely convey the individual economic benefits of each trait to farmers 

and seed companies. Available information does 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), environmental 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. 
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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 range 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 deregulated 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 the variability and complexity of the market, 

it is assumed that growers will make rational 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. 
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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 affect 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 enforces 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. 
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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 expected 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 applications. If insecticide applications are 

reduced, there may be a beneficial effect on minority populations. These populations 

might include farm workers and their families, 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: Protection 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 their developmental stage, greater metabolic 

activity levels, and behavior patterns, as compared 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 environmental 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 expected 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 applications. 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 individuals who are exposed to insecticides 

through aerial application, groundwater contamination, or other routes. 



452 Executive Order 13045 of April 21, 1997. 
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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 Agencies 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 adverse 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 feeding 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 accordingly would be in compliance with EO 13186, 

Responsibilities of Federal Agencies to Protect Migratory Birds. 

5.2 Executive Order with International 

Implications 

EO 12114, Environmental Effects Abroad of Major Federal Actions,455requires 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. 
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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 nonregulated status for the product would be fully 

subject to national phytosanitary requirements 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 includes both direct and indirect damage by 

pests, including weeds. The IPPC set a standard 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 through 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 movement of LMOs intended for environmental 

release (field trials or commercial planting) will require consent from the importing 

country under an advanced informed agreement (AIA) provision, which includes a 

requirement for a risk assessment consistent 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 compliance 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 Clearinghouse. International trade of 5307 corn would be 

conducted in compliance with the Protocol. 

Biosafety and biotechnology consensus documents, 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 Philippines, 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 market since Syngenta is actively pursuing 

regulatory approvals for 5307 corn in countries with functioning regulatory systems 

for genetically modified organisms and that import corn from the US or Canada. 

Regulatory filings for 5307 corn 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 potentially 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 Act463 (commonly referred to as the Clean Water 

Act) and implementing regulations464, 465 require entities that discharge regulated 

materials to certain surface water bodies (including wetlands) obtain authorization to 

do so from federal or state agencies 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. Deregulating 5307 corn 

would not result in a change of agricultural practices or any new discharge of 

pollutants to surface water bodies. Water quality 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-resistant 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 1990466 (commonly abbreviated as the Clean Air 

Act) and implementing regulations467 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 adopted by growers because 5307 corn would 

give growers another option to combat corn 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 Act468(NHPA) and its implementing regulations469

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 propose constitute "undertakings" that have 

the potential to cause effects on historic properties, and 

2) If so, to evaluate the effects of such undertakings on such historic resources and 

consult with the Advisory Council on Historic Preservation (i.e., State Historic 

Preservation Offices, Tribal Historic Preservation Officers), as appropriate. 

The Proposed Action is not expected to adversely 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 tribe’s request. The tribes would have control 

over any potential conflict with cultural resources on tribal properties. The Proposed 

Action would also have no impact on districts, 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 undertaking that may directly or indirectly cause 

alteration in the character or use of historic properties protected under the NHPA. In 

general, common agricultural activities conducted under this action do not have the 

potential to introduce new visual, atmospheric, 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 regulations 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 existing 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 determination of nonregulated status of Event 5307 

corn. This action would not convert agricultural land use to nonagricultural use and 

therefore would have no adverse impact on prime farm land. Standard agricultural 

practices for land preparation, planting, pest 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 

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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 preparing 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 Systematics, 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 

documents 
Event 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 

technology industry

Dr. Lawrence Zeph 

Senior Regulatory Manager 

Ph.D., Molecular and Cell Biology, Pennsylvania 

State University 

B.A., Biology, Indiana University 

 22 years of experience in regulatory policy for 

transgenic crops 

 
Event 5307 Corn 

Environmental Report 

7-1 References 

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Pesticide Programs, Biopesticides and Pollution Prevention Division.
Event 5307 Corn 

Environmental Report 

7-19 References 

_______. 2007b. Phosphomannose isomerase in all plants; exemption from the requirement of

a tolerance. 40 CFR § 174.527.

_______. 2008. Agriculture Management Practices for Water Quality Protection. USEPA website:

http://www.epa.gov/owow/watershed/wacademy/acad2000/agmodule/.

Accessed June 23, 2011.

_______. 2009. Soil Preparation. USEPA Ag 101 website:

http://www.epa.gov/agriculture/ag101/cropsoil.html. Accessed June 22, 2011.

_______. 2010a. Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption

from the requirement of a tolerance. 40 CFR § 174.532.

_______. 2010b. Biopesticides Registration Action Document: Cry1Ab and Cry1F Bt Plant‐

Incorporated Protectants. Washington, DC: EPA Office of Pesticide Programs,

Biopesticides and Pollution Prevention Division.

_______. 2010c. Issuance of an Experimental Use Permit. Published in 75 FR 57778 on

September 22, 2010.

_______. 2011a. Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption

from the requirement of a tolerance. 40 CFR § 174.532.

_______. 2011b. Inventory of U.S. Greenhouse Gas Emissions and Sinks: 1990‐2009.

USEPA Report 430‐R‐11‐05. Washington, DC: USEPA.

_______. 2011c. Pesticide Experimental Use Permit, receipt of extension application;

comment request. Published in 76 FR 3135 on January 19, 2011.

_______. 2011d. Pesticides Industry Sales and Usage: 2006 and 2007 Market Estimates.

Washington, DC: USEPA Office of Chemical Safety and Pollution Prevention,

Office of Pesticide Programs, Biological and Economic Analysis Division.

United States Fish and Wildlife Service (USFWS). 1988. Endangered and Threatened

Wildlife Plants; Final Rule to Determine Five Texas Cave Invertebrates to be

Endangered Species. Department of the Interior, 50 CFR Part 17, Vol. 53,

No. 180. Published at 53 FR 36029 on September 16. 1988.

_______. 2005. Service Lists Salt Creek Tiger Beetle as Endangered. Department of the

Interior News Release published on October 6, 2005.

_______. 2011a. American Burying Beetle (Nicrophorous americanus). Species summary.

_______. 2011b. Endangered and Threatened Wildlife and Plants; Determination of

Endangered Status for Casey’s June Beetle and Designation of Critical Habitat.

Department of the Interior, 50 CFR Part 17. Published at 76 FR 58954 on

September 22, 2011.
Event 5307 Corn 

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7-20 References 

U.S. Grains Council (USGC). Value Enhanced Grain Report 2006. USGC website:

http://www.grains.org/images/stories/technical_publications/USGC_Report_

_Final__04_28_06.pdf. Accessed June 30, 2011.

Van Duyn, J., et al. 2005. Organic Insect Pest Management: Field Corn. North Carolina

State University.

Vercauteren, K.C., and S.E. Hygnstrom. 1993. White‐Tailed Deer Home Range

Characteristics and Impacts Relative to Field Corn Damage. Wildlife Damage

Management. Great Plains Wildlife Damage Control Workshop Proceedings.

University of Nebraska, Lincoln.

Vlachos, D., and D. Ward. 2011. 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. Research Triangle

Park, NC: Syngenta Seeds, Inc. – Field Crops—NAFTA.

Vlachos, D., and S.A. Huber. 2011. Petition for Determination of Nonregulated Status for

Rootworm‐Resistant Event 5307 Corn. Research Triangle Park, NC: Syngenta

Biotechnology, Inc.

Vogel, J.R., et al. 2008. Pesticides in rain in four agricultural watersheds in the United

States. Journal of Environmental Quality 37:1101‐1115.

Vyn, T.J. 2006. Meeting the Ethanol Demand: Consequences and Compromises Associated

with More Corn on Corn in Indiana. Purdue University. ID‐336: 1‐4.

Walters, F.S., C.M. deFontes, H. Hart, G.W. Warren, and J.S. Chen. 2010.

Lepidopteran‐active variable‐region sequence imparts coleopteran activity in

eCry3.1Ab, an engineered Bacillus thuringiensis hybrid insecticidal protein.

Applied and Environmental Microbiology 76:3082‐3088.

Ward, D., T. DeGooyer, T. Vaughn, G. Head, M. McKee, J. Astwood, and J. Pershing.

2005. Genetically enhanced maize as a potential management option for corn

rootworm: YieldGard® rootworm maize case study. In Western Corn

Rootworm: Ecology and Management. S. Vidal, U. Kuhlmann, and C. Edwards

(eds.). CAB International, Wallingford, Oxfordshire.

Webster, J.R., E.F. Benfield, T.P. Ehrman, M.A. Schaeffer, J.L. Tank, J.J. Hutchens, and

D.J. D’Angelo. 1999. What happens to allochthonous material that falls into

streams? A synthesis of new and published information from Coweeta.

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Office of Information and Regulatory Affairs.

Wolfenbarger, L.L., S.E. Naranjo, J.G. Lundgren, R.J. Bitzer, and L.S. Watrud. 2008.

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regulatory impacts. Transgenic Research 15:277‐289.

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5307 Corn. OECD Unique Identifier: SYN‐Ø53Ø7‐1.
Event 5307 Corn 

Environmental Report 

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)

Authors

Demetra Vlachos, M.S.

Dennis Ward, Ph.D.

Date

March 31, 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 41
SSB-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 contained in PR Notice 

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 31, 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 by the Good Laboratory Practice (GLP) 

Standards 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 Manager

Syngenta Seeds, Inc. – Field Crops - NAFTA

P.O. Box 12257

3054 East Cornwallis Road

Research Triangle Park, NC 27709

Date: March 31, 2011
SSB-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. Transgenes 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 assessed by adult emergence counts... 22

c) Northern corn rootworm efficacy as assessed by adult emergence counts.. 23

d) Mexican corn rootworm efficacy as assessed by root damage ratings......... 25

2. Summary of efficacy trials of 5307 corn against target pests ........................... 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 .............................................................................................................. 38
SSB-201-10 Page 5 of 41

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 products labeled for use in field 

corn to control corn rootworm larvae and/or adults.......................................33
SSB-201-10 Page 6 of 41

List of Figures

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 2006 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 efficacy 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. ........................................................................................... 28
SSB-201-10 Page 7 of 41

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 registrations for two new products that 

include the 5307 corn PIP in breeding combinations with other registered PIPs: Bt11 

× MIR604 × TC1507 × 5307 corn and Bt11 × MIR162 × MIR604 × TC1507 × 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 control 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. The 

deployment of 5307 corn, in Bt11 × MIR604 × TC1507 × 5307 corn and Bt11 × 

MIR162 × MIR604 × TC1507 × 5307 corn, will also offer these qualitative benefits. 

Reduction of broad-spectrum insecticide use by growers is of special importance 

because several conventional pesticide products registered for corn rootworm control 

have use restrictions or label warnings related 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 reduce chemical insecticide use by corn 

growers. Based on an extensive battery of safety studies provided for review by the 

U.S. Environmental Protection Agency, the 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 demonstrate 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 rootworm), 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 control 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 and 
SSB-201-10 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 targets

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 reducing 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 cultivars, 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 MIR604 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 refuge 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-201-10 Page 9 of 41

I. Introduction

At 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 

introduction, plant-incorporated protectant (PIP) registrations have only been granted 

under special circumstances. FIFRA authorizes the Administrator of the 

Environmental Protection Agency (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 adverse 

effect on human health or 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, adoption of crops with insect and herbicide tolerance traits has increased 

dramatically since their commercial introduction. Between 1996 and 2008, the use of 

transgenic crops boosted global farm incomes by $52 billion, of which $7.1 billion 

was directly attributable the use of insect-resistant corn hybrids in the U.S. alone 

(Brookes and Barfoot, 2010). Improved insect protection and weed control provided 

by transgenic crops have led to increased crop yields and reductions 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.

Event 5307 corn was developed by Syngenta Biotechnology, Inc. to provide U.S.

growers with corn hybrids that are resistant to feeding damage caused by coleopteran

insect pests, specifically corn rootworms. There is no herbicide tolerance trait in 5307 

corn; the selectable marker used during transformation was phosphomannose 

isomerase (PMI), which 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 approved PIPs to provide control of multiple lepidopteran and coleopteran 

pests as well as herbicide tolerance. The specific products will represent PIP 

combinations1 contained in breeding crosses of transgenic Bt11 × MIR604 × TC1507 

× 5307 corn and Bt11 × MIR162 × MIR604 × TC1507 × 5307 corn. 

This assessment characterizes several anticipated benefits of 5307 corn and the two 

5307 stacked-trait products to support a determination that the associated PIP 

registrations will be in the public interest.

 1Bt11, TC1507 and MIR162 corn produce the lepidopteran-active PIPs Cry1Ab, 

Cry1F and Vip3Aa20, respectively. MIR604 and 5307 corn produce the coleopteranactive PIPs mCry3A and eCry3.1Ab. 
SSB-201-10 Page 10 of 41

II. Overview of Corn

Zea mays Linnaeus, known as maize throughout the world and as corn in the United 

States, is one of the few major crop species indigenous to the western hemisphere. It 

has been cultivated in the Americas since early historic times. Corn is the leading 

production crop globally; the 2008/2009 growing season yielded approximately 

792 million metric tons of grain (USDA, 2010a).

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. accounted for 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 billion bushels (335 metric tons) (USDA, 2010b). 

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 segment 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 significantly 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, 2009 (Baker et al., 2010). 

50.8%

19.1%

18.3%

11.8%
SSB-201-10 Page 11 of 41

Total U.S. agricultural exports in 2010 were estimated 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. cropland moved into 

export channels in 2008 (USDA, 2010c). On 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 agricultural 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 grown. The bulk of corn is 

produced between latitudes 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 production 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 precipitation ranges from 25 to 

over 500 cm. Summer rainfall of 15 cm is approximately the lower limit for corn

production without irrigation.

The upper midwest region 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, Missouri, Kansas, and South Dakota are major corngrowing 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 hundreds 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 yields 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 (NCGAa, 2010).
SSB-201-10 Page 12 of 41

Figure 2. Geographic distribution by county of acres 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 or feed. 

Insect pests can be categorized as major and consistent pests, major and sporadic, and 

moderate to minor based on annual 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 Ostrinia nubilalis (European corn borer).
SSB-201-10 Page 13 of 41

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 (Tetranychus 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 (Striacosta 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 

crambidoides)

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 uptake 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 during 
SSB-201-10 Page 14 of 41

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 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 infections 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 Bt corn

varieties in 1996 provided growers with an 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 conventional 

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 

accounted 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 

tolerance, accounted for 63% of corn acres planted in 2009 (USDA, 2009a). 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 beneficial 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 that corn rootworm populations have 

developed resistance to some insecticides and to non-chemical control methods. 

Resistance to some corn rootworm insecticides may result in increased chemical use 

(EPA, 2010). Although crop rotation has historically been an effective control tool, 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. 
SSB-201-10 Page 15 of 41

Rootworm-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 863 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 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 key to the 

durability of the registered PIPs in corn. 

III. Characteristics 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 contains the transgene ecry3.1Ab encoding an eCry3.1Ab protein and the 

transgene 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, 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 development 

of 5307 corn. PMI allows 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 range 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 protein. 

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 for 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 toxicity 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 Diabrotica spp.: 

western, northern and Mexican corn rootworms. 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. 

Although the eCry3.1Ab protein contains a portion of mCry3A in its amino acid 
SSB-201-10 Page 16 of 41

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−endotoxins 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 processes 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 insect 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 5307 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 

results 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 insecticidal spectrum of a given Cry protein. Individual Cry proteins are

usually active against only a few related species within any phylogenetic Order. Most 

of the insect-specific toxins identified to date have shown activity among the Orders 

of Lepidoptera and/or Diptera, or Coleoptera. A few Bt crystal toxins have shown 

activity against nematodes (Marroquin et al., 2000; Wei et al., 2003). 

Accompanying the registration applications for the 5307 corn breeding stacks (Bt11 × 

MIR604 × TC1507 × 5307 corn and Bt11 × 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 that 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 

potentially exposed to plant-incorporated eCry3.1Ab has been conducted. These 
SSB-201-10 Page 17 of 41

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 observed 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 properties 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 stacked-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 ingredient” 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 corn 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 from 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 larvae. 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 entries 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 in 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 each 
SSB-201-10 Page 18 of 41

location in the 2008 trials, three replicate plots of each trial entry were planted 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 (silking 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 customary 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 pruned 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 Equivalent 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 roots pruned

3.00 Equivalent of 3.00 nodes of roots pruned

Western corn rootworm larvae were very destructive to the nontransgenic, nearisogenic 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 the 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 ranged 
SSB-201-10 Page 19 of 41

from 1.42 to 2.81. The 5307 hybrids 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., 2009)
SSB-201-10 Page 20 of 41

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 

western 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 

Minnesota 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 whether 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 lower than the control hybrid. Although both 
SSB-201-10 Page 21 of 41

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 the MIR604 hybrid (0.17). By 

comparison, the control hybrid had mean damage ratings of 1.75. 

Figure 6. Rootworm efficacy of 5307, MIR604 × 5307, and MIR604 corn hybrids; 

Illinois and Minnesota trials, 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 locations
SSB-201-10 Page 22 of 41

b) Western corn rootworm efficacy as assessed by adult emergence counts

Root 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 emerge 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 assessments

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 location 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, etc.) 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 erected 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 outside 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 survived 

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 the 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 MIR604 corn. By comparison, the percent survival in the single-event 
SSB-201-10 Page 23 of 41

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

Nontransgenic 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 corn rootworm efficacy as assessed by adult emergence 

counts

When considered across the US Corn Belt, northern corn rootworms do not typically 

cause as much economic damage as do western corn rootworms, the primary target 
SSB-201-10 Page 24 of 41

pest of 5307 corn. Nevertheless, northern corn rootworms remain a significant pest in 

many corn-growing areas, particularly in cooler regions. 

It is 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 trials 

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 differences 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, MIR604 × 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 northern 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 NCRW 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 rootworm
SSB-201-10 Page 25 of 41

d) Mexican corn rootworm 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 efficacy 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 experimental 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 appropriate for a randomized complete block design, and 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), all 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 

significant 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 both trials, mean root damage ratings in the 5307 and MIR604 ×

5307 plots were very low, and ranged from 0.01 to 0.045. By contrast, the control 

plants had mean root damage ratings of 0.71 and 0.69.

Although the statistical power of this small dataset of Mexican 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-201-10 Page 26 of 41

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, 2007
SSB-201-10 Page 27 of 41

2. Summary of efficacy trials of 5307 corn against target pests

Multi-year and multi-location field assessments of the efficacy of 5307 corn against 

western 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 was 

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 significant 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 eCry3.1Ab 

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 resistance 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 5307 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 planted 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 nearisogenic (i.e., in the same germplasm background). In most 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 trials (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, the 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 yield 
SSB-201-10 Page 28 of 41

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 recently compiled, more extensive set of trial data for MIR604 hybrids 

(Agrisure® RW corn). These data demonstrate a 25 to 57 bushels/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 performance of 5307 corn and MIR604 × 5307 corn under corn 

rootworm pressure. 

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 < 0.0001
SSB-201-10 Page 29 of 41

IV. Public Interest Factors Applicable to 5307 Corn and the 

Breeding Stacks Thereof

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 consideration 

of these factors, as they relate to 5307 corn and the breeding stacks thereof (Bt11 ×

MIR604 × TC1507 × 5307 and Bt11 × MIR162 × MIR604 × TC1507 × 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 can 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 the 

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 EPA, 

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 × TC1507 × 5307 and Bt11 × MIR162 × MIR604 × TC1507 × 5307 corn)

compared to the application of conventional insecticides, along with the opportunity to 

extract an economic benefit through increased crop yield, are expected to be attractive 

to growers.

A. Need Factors

Prior to the introduction of rootworm-protected Bt corn varieties in 2003, the primary 

tactics for corn rootworm control were crop rotation (e.g., corn/soybean rotation), soilapplied insecticides, foliar insecticides and, to a lesser extent, insecticidal seed 

treatments. The current PIP proteins registered for corn rootworm 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 infestation (acreage and degree of infestation), the comparative 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, costing 
SSB-201-10 Page 30 of 41

$172 million ($12.29/acre), were applied to 14 million acres, or 17% of U.S. corn 

acreage. Between 2005 and 2013, corn-rootworm-infested acreage was projected to 

increase from approximately 31.8 million acres 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, reaching 18 to 19 million acres by the year 2013. Product 

efficacy data provided herein 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 applicators, 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 reductions 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 rootwormprotected corn products is not expected to markedly reduce the use 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 control, 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 corn 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 the 5307 corn breeding stacks is expected to extend the useful life of other 

commercially available 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 resistance to other methods of control. 

B. Composition Factors

The active insecticidal component of 5307 corn plants, eCry3.1Ab, is plantincorporated, thus it fundamentally differs from the composition of conventional pest 

control products. The characteristics 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 public interest. 
SSB-201-10 Page 31 of 41

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 expected 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, from 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 the 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 

(see Part III). The eCry3.1Ab protein is present in all tissues 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 it 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 

monitoring 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 of 
SSB-201-10 Page 32 of 41

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) in 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 control measures such as crop rotation. The 5307 corn 

breeding stacks will fit seamlessly into the concept of IPM for corn. 
SSB-201-10 Page 33 of 41

Table 5. Representative conventional insecticide products labeled for use in field corn to control corn rootworm larvae and/or adults. 

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/Insecticide

(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 
SSB-201-10 Page 34 of 41

Product Active Ingredient(s);

% of Formulation

Signal Word/

Classification

Environmental Hazards 

and/or Reasons for Restricted Use Classification

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

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 lambda-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

chlorpyrifos; 44.9% WARNING

Restricted Use

Toxic to fish, aquatic invertebrates, birds and small mammals, highly toxic to 

bees 
SSB-201-10 Page 35 of 41

Product Active Ingredient(s);

% of Formulation

Signal Word/

Classification

Environmental Hazards 

and/or Reasons for Restricted Use Classification

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 

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 

Sevin® 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-cypermethrin; 

9.2%

WARNING 

Restricted Use

Extremely toxic to fish, aquatic invertebrates, oysters and shrimp; highly 

toxic to bees
SSB-201-10 Page 36 of 41

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 classified 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 keeping 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 battery of mammalian toxicity studies were conducted for the eCry3.1Ab 

protein produced in5307 corn. These studies found no evidence of eCry3.1Ab proteininduced 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 

insecticidal protein is plant-incorporated, the opportunity for exposure when handling and 

planting seed is insignificant. Planting of 5307 corn breeding stacks (Bt11 × MIR604 × 

TC1507 × 5307 and Bt11 × MIR162 × MIR604 × TC1507 × 5307 corn) by growers will 

essentially eliminate the environmental and occupational health risks currently associated 

with chemical controls for corm rootworm pests (via the rootworm-control PIPs in 5307 

and MIR604 corn) as well as for the lepidopteran pests that will otherwise be controlled 

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 has 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 

exposure levels exceeding expected environmental 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 Fusarium spp. after they have served their function and become senescent. Feeding 
SSB-201-10 Page 37 of 41

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 infections can lead to reductions 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 resulting 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 more 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/acre 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 rootworm 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. The cost of chemical insecticide control 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, thereby 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 rates. Additionally, the availability of a new and reliable pest-control 

option that promotes higher yield, more efficient use of water and fertilizer, healthier 

plants, and better grain quality, while reducing the environmental impacts of agriculture, 

will operate 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-201-10 Page 38 of 41

V. References

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submission for EPA registration.)

Seastrum, L. (2009). In vitro Digestibility of eCry3.1Ab Protein as Contained in Test 

Substance ECRY3.1AB-0208 Under Simulated Mammalian Intestinal Conditions. 

Unpublished Syngenta Report No. SSB-015-09. (Included as a data volume in 

submission for EPA registration.)

Seastrum, L., K. Ward, and A. Raybould (2010). Investigation of the Potential 

Interaction between Lepidopteran-active (Cry1Ab + Vip3Aa20 + Cry1F) and 

Coleopteran-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 registration.)

Song, S. (2010). In vitro digestibility of eCry3.1Ab Protein under simulated mammalian 

gastric conditions. Unpublished Syngenta Report 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-201-10 Page 41 of 41

National Agricultural Statistics Service. Washington, DC. Accessed May 12, 2010:

http://usda.mannlib.cornell.edu/usda/current/Acre/Acre-06-30-2009.pdf.

USDA (2009b). Data and statistics: Quick stats. Washington, DC: U.S. Department 

ofAgriculture - 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

u-01-12-2010.pdf

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. Warren and J.S. Chen (2010). Lepidopteranactive variable region sequence imparts coleopteran activity in eCry3.1Ab, an 

engineered Bacillus thuringiensis hybrid insecticidal protein. Appl. Env. Microbio.

76: 3082–3088. (Included as a data volume in submission for EPA registration.)

Ward, D., T. DeGooyer, T. Vaughn, G. Head, M. McKee, J. Astwood, and J. Pershing. 

(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 Western 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 Food and Drug Administration

Safety and Nutritional Assessment of Event 5307 Corn

OECD Unique Identifier: SYN-Ø53Ø7-1

Submitted By:

Demetra Vlachos

Syngenta Seeds, Inc.

P.O. Box 12257

3054 East Cornwallis Road

Research Triangle Park, NC 27709-2257

Date:

January 27, 2011

Authors:

Lawrence Zeph, Ph.D. and Demetra Vlachos, M.S.

Syngenta Seeds, Inc.

© Syngenta 2011

Page 1 of 168
5307-FDA-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-FDA-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 Toxicity 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 Pepsin 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 Protein 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 Potential....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-FDA-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
5307-FDA-1 Page 5 of 168

I.B. List of Figures

Figure IV-1. Plasmid map for vector pSYN12274. ..................................................................17

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.......................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 KpnI, NcoI, SmaI, and PmeI 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, using restriction enzymes KpnI, NcoI, and 

SmaI + PmeI ........................................................................................................31

Figure IV-7. Location of the BstEII, SpeI, SmaI, and PmeI restriction sites and position of 

the 1176 bp pmi-specific probe in the T-DNA region of the transformation 

plasmid pSYN12274............................................................................................33

Figure IV-8. Southern blot analysis of 5307 corn for copy number of functional elements: 

1176-bp pmi-specific probe, using restriction enzymes BstEII, SpeI, and SmaI 

+ PmeI..................................................................................................................35

Figure IV-9. Location of the KpnI, SpeI, SmaI, and PmeI 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 KpnI, SpeI, 

and SmaI + PmeI..................................................................................................39

Figure IV-11. Location of the BstEII, SpeI, SmaI, and PmeI 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 BstEII, 

SpeI, and SmaI + PmeI.........................................................................................44

Figure IV-13. Location of the KpnI, NcoI, SmaI, and PmeI 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 KpnI, NcoI, 

and SmaI + PmeI..................................................................................................48

Figure IV-15. Location of the BstEII, SpeI, SmaI, and PmeI 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-FDA-1 Page 6 of 168

Figure IV-16. Genetic stability Southern blot analysis of 5307 corn with the 6423-bp fulllength T-DNA-specific probe, using restriction enzymes BstEII, SpeI, and 

SmaI + PmeI.........................................................................................................53

Figure IV-17. Location of the BstEII, SpeI, SmaI, and PmeI restriction sites and position of 

the 5312-bp plasmid backbone-specific probe in the transformation plasmid 

pSYN12274..........................................................................................................59

Figure IV-18. Genetic stability Southern blot analysis of 5307 corn with the 5312-bp 

plasmid backbone-specific probe, using restriction enzymes BstEII, SpeI, and 

SmaI + PmeI.........................................................................................................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
5307-FDA-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 studies. ............................................25

Table IV-3. Expected and observed hybridization band sizes in Southern blot analysis of 

5307 corn, using an ecry3.1Ab-specific probe and restriction enzymes KpnI, 

NcoI, and SmaI + PmeI. ............................................................................................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 BstEII, SpeI, and 

SmaI + PmeI..............................................................................................................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 KpnI, 

SpeI, and SmaI + PmeI..............................................................................................38

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 

BstEII, SpeI, and SmaI + PmeI..................................................................................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 enzymes 

KpnI, NcoI, and SmaI + PmeI. ..................................................................................47

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 

BstEII, SpeI, and SmaI + PmeI..................................................................................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 

restriction enzymes BstEII, SpeI, and SmaI + PmeI..................................................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 LC50 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 densitometry analysis................................................79

Table VI-3. Effect of temperature on insecticidal activity of eCry3.1Ab in diet 

incorporation bioassay with Colorado potato beetle larvae: LC50 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 dryweight (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 measured 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
5307-FDA-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 nontransgenic corn. ..........................................................................................113
5307-FDA-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, FDA 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 concerns). 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.
5307-FDA-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-FDA-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-FDA-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 Bacillus thuringiensis-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 insecticidal 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 exempt 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 indicate allergenic potential. The large body of data 

and information described 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.

Analyses 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 similar in composition to forage and grain from 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 control 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 nutritionally comparable to and as safe as conventional corn.
5307-FDA-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: 

Demetra Vlachos

Senior Regulatory Affairs Manager 

Tel. 919-226-7383; Mobile 919-433-7642; Fax 919-226-7462

demetra.vlachos@syngenta.com

II.B. Subject of This Consultation and the Plant Species from Which it is 

Derived

The subject of this consultation is coleopteran-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, an 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 Unique Identifier SYN-Ø53Ø7-1.

II.D. Description of the Genetic Materials 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 isomerase (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-FDA-1 Page 14 of 168

bacterium. The eCry3.1Ab1 protein is insecticidally active against certain Coleoptera, 

including western corn rootworm (Diabrotica virgifera virgifera), northern 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. In 

breeding combinations with other insect-resistant corn varieties, 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 available corn in 

the US. In 2010, there were an estimated nearly 88 million acres planted to corn in the US,

producing over 13 billion bushels of grain (USDA-NASS 2010). 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 exported 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 array 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 official Cry protein designation under the formal nomenclature 

scheme for B. thuringiensis Cry proteins (Crickmore et al. 2010). 
5307-FDA-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 coleopteran 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 requirement of food and 

feed tolerances 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, 

concurrently 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 §174.527

for PMI in all plants, in connection with its use as a selectable marker in pesticidal 

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.
5307-FDA-1 Page 16 of 168

IV. Transformation and Development 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 grass 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 (Hitchcock 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 introduced 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 

(International 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 an 

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 the annual 

teosintes into two subspecies of Z. mays, ssp. mexicana (including races Chalco, Central 

Plateau and Nobogame) and ssp. parviglumis 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 the 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, Randolph 

proposed that corn was domesticated, independently, in the southwestern US, Mexico, and 
5307-FDA-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 is 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 5307 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 gene 

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 curling 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 schematic of the 

plasmid vector is shown in 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-FDA-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 Virus promoter region (Hohn et al. 2007; 

Stavolone 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 gene (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 translated 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 corn (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 cornoptimized gene was then modified to incorporate a consensus cathepsin-G 

protease recognition 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 recognition 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). 
5307-FDA-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 polyadenylation 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
5307-FDA-1 Page 20 of 168

Table IV-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 is 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, plantassociated bacteria (Heeb et al. 2000)

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 Tiplasmid (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)
5307-FDA-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 TAT GAC GGC CGA

eCry3.1Ab M T S N G R Q C A G I R P Y D G R 

mcry3A .....................................................ATG ACG GCC GAC

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 sequence. 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 unique to eCry3.1Ab (see Figure IV-2B).

(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 

amino 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-FDA-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 via 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 positive 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 (T0 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-FDA-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 containing plasmid vector pSYN12274

Selection of transformation events 

on medium containing mannose

Regeneration of transformed 

cells into corn plants

Evaluation of transformants for 

insecticidal efficacy

Introgression of transformation events into elite inbred 

lines for evaluation of agronomic performance

Selection of Event 5307 corn for development

 

Regulatory studies to assess safety 

and nutritional composition

Introgression of Event 5307 

transgenes into commercial breeding 

lines and field evaluations
5307-FDA-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 diagram in Figure IV-4 indicates how each of the 5307 genotypes was 

derived via conventional breeding crosses from the original Event 5307 T0 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 Syngenta studies described in this submission 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 × BC5F3.” As indicated in Figure IV-4, it was produced by crossing a 

nontransgenic inbred parent, NP2171, with the F3 (third generation) transgenic progeny of 

self-pollinated plants following five successive generations of backcross (BC) breeding of 

the transgenes into to a recurrent NP2460 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 identical 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 studies) required seed from multiple generations in the 5307 

breeding pedigree, as indicated in Table IV-2.
5307-FDA-1 Page 25 of 168

Table IV-2. Event 5307 and control seed materials used in studies. 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 Description

(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)(BC5F3)*

None

Southern blots for 

copy no. of functional 

elements (Part IV.E.2)

D NP2171 × 

NP2460(5307)(BC5F3)*

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

F1

BC6

BC7

NP2171 × 

NP2460(5307)(BC5F3)*

E

Parental inbred lines NP2222, 

NP2460, and NP2171 (not 

shown in Fig. IV-4)

NP2171 × NP2460

Mendelian inheritance 

analysis (Part IV.E.4)

A

B

C

F1

BC6

BC7

None

Determination of 

insert flanking 

sequences 

(Part IV.E.5)

D NP2171 × 

NP2460(5307)(BC5F3)*

None

Equivalence of 

eCry3.1Ab produced 

in 5307 corn and 

recombinant E. coli 

(Part VI.C)

D NP2171 × 

NP2460(5307)(BC5F3)*

E NP2171 × NP2460

eCry3.1Ab and PMI 

concentrations in 

5307 corn tissues 

(Part VI.N)

D NP2171 × 

NP2460(5307)(BC5F3)*

E NP2171 × NP2460

Compositional 

assessment of forage 

and grain 

(Part VII.C)

D NP2171 × 

NP2460(5307)(BC5F3)*

E NP2171 × NP2460

Broiler chicken 

feeding study with 

grain (Part VII.D)

D NP2171 × 

NP2460(5307)(BC5F3)*

E NP2171 × NP2460

* This 5307 hybrid is alternatively referred to as “NP2171 x BC5F3” or “NP2171 x NP2640(5307).”
5307-FDA-1 Page 26 of 168

Figure IV-4. Pedigree diagram of Event 5307 and control seed materials.

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 initial Event 5307 F1 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)(BC5F3)

 ×NP2460*

 ×NP2460*

× NP2460*

× NP2460*

NP2222* × 5307 

F1

BC1

BC5

× NP2460*

⊗

× NP2222*

5307 T0 (NP2222*)

× NP2460*

BC2

BC3

× NP2460*

BC4

BC5F2

BC5F3

NP2171* × homozygotes

⊗ homozygotes

BC6 BC7

A

B

 C

Nontransgenic

Control Hybrid 

NP2171 × NP2460

NP2171* × NP2460*

× NP2460*

NP2171* × homozygotes 

D

E

T0 = original transformant

x = cross

BC = backcross

⊗ = self-pollination

* = field corn inbred
5307-FDA-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 

ecry3.1Ab 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. Characterization 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 that 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 5307 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. Analysis 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 conducted by identifying the T-DNA insertion site 
5307-FDA-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 sequence of the PCR amplification product in the 

resulting plasmid preparations. 

An alignment of the nucleotide 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

Southern blot analyses demonstrated that the T-DNA insert in 5307 corn contains single 

copies of ecry3.1Ab, pmi, the CMP promoter sequence, and the ZmUbiInt promoter 

sequence 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 displayed in table format (Table IV-3, Table IV-4, Table IV-5,

Table IV-6, and Table IV-7, respectively) for each Southern blot.

IV.E.3.a. Copy Number of Functional Elements: ecry3.1Ab-Specific Probe

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 KpnI, NcoI, SmaI, and PmeI 

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 KpnI (Figure IV-6A, Lane A3) produced a single 

hybridization band at approximately 8.5 kb, corresponding to a single copy of ecry3.1Ab. 

Genomic 5307 corn DNA digested with NcoI (Figure IV-6B, Lane B3) 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 SmaI + PmeI (Figure IV-6C, 

Lane C3) produced a single hybridization band at approximately 6.3 kb, corresponding to a 

single copy of ecry3.1Ab. 
5307-FDA-1 Page 29 of 168

The negative control corresponding to each digest showed no hybridization (KpnI, Figure 

IV-6A, Lane A4; NcoI, Figure IV-6B, Lane B4; and SmaI + PmeI, Figure IV-6C, Lane C4). 

One hybridization band of the expected size was present in all lanes (Lanes A5, B5 and C5 

of Figure IV-6A, B and C, respectively) containing positive control DNA from the plasmid 

pSYN12274. 

For Southern blot analyses 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 bands were detected, indicating that there were no extraneous DNA fragments of 

ecry3.1Ab in the 5307 corn genome.

Figure IV-5. Location of the KpnI, NcoI, SmaI, and PmeI 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 digestion. 

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)

KpnI NcoI

PmeI SmaI

CMP promoter (346 bp)

NOS terminator (253 bp)

KpnI

> 2.8 kb

> 3.9 kb

SmaI + PmeI

6339 bp

NcoI

RB (25 bp)
5307-FDA-1 Page 30 of 168

Table IV-3. Expected and observed hybridization band sizes in Southern blot analysis of 

5307 corn, using an ecry3.1Ab-specific probe and restriction enzymes KpnI, NcoI, and SmaI 

+ PmeI.

Figure &

Lane No.

Source of DNA Restriction 

enzyme(s)

Expected 

number of 

bands

Expected 

band size 

(kb)

Observed 

band size 

(kb)

Figure IV-6A, 

Lane A3

5307 NP2171 × BC5F3 KpnI 1 >2.8 ~8.5

Figure IV-6A, 

Lane A4

NP2171 × NP2460 KpnI none none none

Figure IV-6A,

Lane A5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with KpnI)

SmaI + PmeI 1 ~6.3 ~6.3

Figure IV-6B, 

Lane B3

5307 NP2171 × BC5F3 NcoI 1 >3.9 ~19

FigureIV-6B, 

Lane B4

NP2171 × NP2460 NcoI none none none

Figure IV-6B, 

Lane B5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with NcoI)

SmaI + PmeI + 

NcoI

1 1 ~3.9 ~3.9

Figure IV-6C, 

Lane C3

5307 NP2171 × BC5F3 SmaI + PmeI 1 ~6.3 ~6.3

Figure IV-6C,

Lane C4

NP2171 × NP2460 SmaI + PmeI none none none

Figure IV-6C, 

Lane C5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with SmaI + PmeI)

SmaI + PmeI 1 ~6.3 ~6.3

1 Digestion of pSYN12274 with NcoI was the result of addition to NP2171 × NP2460 digested with NcoI 
5307-FDA-1 Page 31 of 168

Figure IV-6. Southern blot analysis of 5307 corn for copy number of functional elements: 1962-bp ecry3.1Ab-specific probe, using 

restriction enzymes KpnI, NcoI, and SmaI + PmeI

(A) KpnI

Lane A1 = molecular weight markers

Lane A2 = blank

Lane A3 = 5307 NP2171 × BC5F3 digested with KpnI

Lane A4 = NP2171 × NP2460 digested with KpnI

Lane A5 = Positive control (NP2171 × NP2460

digested with KpnI and 16.53 pg of 

pSYN12274 digested with SmaI + PmeI)

(B) NcoI

Lane B1 = molecular weight markers

Lane B2 = blank

Lane B3 = 5307 NP2171 × BC5F3 digested with NcoI

Lane B4 = NP2171 × NP2460 digested with NcoI

Lane B5 = Positive control (NP2171 × NP2460 

digested with NcoI and 16.53 pg of 

pSYN12274 digested with SmaI + PmeI + 

NcoI)

(C) SmaI + PmeI

Lane C1 = molecular weight markers

Lane C2 = blank

Lane C3 = 5307 NP2171 × BC5F3 digested with 

SmaI + PmeI

Lane C4 = NP2171 × NP2460 digested with SmaI + 

PmeI

Lane C5 = Positive control (NP2171 × NP2460

digested with SmaI + PmeI and 16.53 pg 

of pSYN12274 digested with SmaI + 

PmeI)
5307-FDA-1 Page 32 of 168

IV.E.3.b. Copy Number of Functional Elements: pmi-Specific Probe

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 analysis, are shown in

Figure IV-7. The results of this analysis are shown in Table IV-4 and Figure IV-8. 

Genomic 5307 corn DNA digested with BstEII (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 SpeI (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 SmaI + PmeI (Lane 7) produced a single hybridization band at the expected 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 (BstEII Lane 4, SpeI 

Lane 6, and SmaI + PmeI Lane 8). One hybridization band 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-FDA-1 Page 33 of 168

Figure IV-7. Location of the BstEII, SpeI, SmaI, and PmeI restriction sites and position of the 

1176 bp pmi-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)

RB (25 bp) LB (25 bp)

pmi-specific probe (1176 bp)

PmeI SmaI BstEII

SpeI

CMP promoter (346 bp) NOS terminator (253 bp)

NOS terminator (253 bp) ZmUbiInt promoter (1993 bp)

BstEII

> 4.0 kb

> 5.9 kb

SmaI + PmeI

6339 bp

SpeI
5307-FDA-1 Page 34 of 168

Table IV-4. Expected and observed hybridization band sizes in Southern blot analysis of 5307 

corn, using a pmi-specific probe and restriction enzymes BstEII, SpeI, and SmaI + PmeI.

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 × BC5F3 BstEII 1 > 4.0 kb ~ 7.2 kb

Figure IV-8, 

Lane 4

NP2171 × NP2460 BstEII none none none

Figure IV-8, 

Lane 5

5307 NP2171 × BC5F3 SpeI 1 > 5.9 kb ~ 7.0 kb

Figure IV-8,

Lane 6

NP2171 × NP2460 SpeI none none none

Figure IV-8, 

Lane 7

5307 NP2171 × BC5F3 SmaI + PmeI 1 ~6.3 kb ~6.3 kb

Figure IV-8, 

Lane 8

NP2171 × NP2460 SmaI + PmeI none none none

Figure IV-8, 

Lane 9

Positive control (16.53 pg of 

pSYN12274 mixed with NP2171

× NP2460 digested with SmaI + 

PmeI)

SmaI + PmeI 1 ~6.3 kb ~6.3 kb
5307-FDA-1 Page 35 of 168

Figure IV-8. Southern blot analysis of 5307 corn for copy number of functional elements: 

1176-bp pmi-specific probe, using restriction enzymes BstEII, SpeI, and SmaI + PmeI.

Lane 1 = molecular weight markers

Lane 2 = blank

Lane 3 = 5307 NP2171 × BC5F3 digested with BstEII

Lane 4 = NP2171 × NP2460 digested with BstEII

Lane 5 = 5307 NP2171 × BC5F3 digested with SpeI

Lane 6 = NP2171 × NP2460 digested with SpeI

Lane 7 = 5307 NP2171 × BC5F3 digested with SmaI + PmeI

Lane 8 = NP2171 × NP2460 digested with SmaI + PmeI

Lane 9 = Positive control (NP2171 × NP2460 digested with SmaI + PmeI and 16.53 pg of pSYN12274 digested with 

SmaI + PmeI)
5307-FDA-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 probe and restriction sites for KpnI, SpeI, 

SmaI, and PmeI. Figure IV-10 depicts the results of the corresponding Southern 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 KpnI and probed with the CMP 

promoter-specific probe, one hybridization band of approximately 8.5 kb was observed as 

expected (Figure IV-10A, Lane A3) (Table IV-5). For Southern blot analysis with genomic 

DNA digested with SpeI and probed with the CMP promoter-specific probe, one hybridization 

band of approximately 2.6 kb was observed as expected (Figure IV-10B, Lane B3) (Table IV-5). 

For Southern blot analysis with genomic DNA digested with SmaI + PmeI and probed with the 

CMP promoter-specific probe, one hybridization band of approximately 6.3 kb was observed as 

expected (Figure IV-10C, Lane C3) (Table IV-5). 

No CMP promoter-specific hybridization band was present in DNA extracted from the control 

substance plants (Figure IV-10A, B, and C, Lanes A4, B4 and C4, respectively) and therefore

hybridization with this probe was specific to the 5307 corn insert. As expected, one 

hybridization band of approximately 6.3 kb was observed in the lane containing the positive 

control, plasmid pSYN12274 DNA, digested with SmaI + PmeI (Figure IV-10A, B, and C, Lanes

A5, 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 promoter 

sequence. No unexpected bands were detected, indicating that there were no extraneous DNA 

fragments of the CMP promoter sequence in the 5307 corn genome. 
5307-FDA-1 Page 37 of 168

Figure IV-9. Location of the KpnI, SpeI, SmaI, and PmeI restriction sites 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)

PmeI SpeI SmaI

CMP promoter (346 bp)

NOS terminator (253 bp)

KpnI

KpnI

> 2.8 kb

> 0.6 kb

SmaI + PmeI

6339 bp

SpeI
5307-FDA-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 KpnI, SpeI, and SmaI + 

PmeI.

Figure &

Lane No. Source of DNA Restriction enzyme(s)

Expected 

number of 

bands

Expected 

band size 

(kb)

Observed 

band size 

(kb)

Figure IV-10A, 

Lane A3

5307 NP2171 × BC5F3 KpnI 1 >2.8 ~8.5

Figure IV-10A, 

Lane A4

NP2171 × NP2460 KpnI none none None

Figure IV-10A, 

Lane A5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with KpnI)

SmaI + PmeI 1 ~6.3 ~6.3

Figure IV-10B, 

Lane B3

5307 NP2171 × BC5F3 SpeI 1 >0.6 ~2.6

Figure IV-10B, 

Lane B4

NP2171 × NP2460 SpeI none none none

Figure IV-10B, 

Lane B5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with SpeI)

SmaI + PmeI 1 ~6.3 ~6.3

Figure IV-10C, 

Lane C3

5307 NP2171 × BC5F3 SmaI + PmeI 1 ~6.3 ~6.3

Figure IV-10C, 

Lane C4

NP2171 × NP2460 SmaI + PmeI none none none

Figure IV-10C, 

Lane C5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with SmaI + PmeI)

SmaI + PmeI 1 ~6.3 ~6.3
5307-FDA-1 Page 39 of 168

Figure IV-10. Southern blot analysis of 5307 corn for copy number of functional elements: 346-bp CMP promoter-specific probe, 

using restriction enzymes KpnI, SpeI, and SmaI + PmeI

(A) KpnI

Lane A1 = molecular weight markers

Lane A2 = blank

Lane A3 = 5307 NP2171 × BC5F3 digested with KpnI

Lane A4 = NP2171 × NP2460 digested with KpnI

Lane A5 = Positive control (NP2171 × NP2460 digested 

with KpnI and 16.53 pg of pSYN12274 digested 

with SmaI + PmeI)

(B) SpeI

Lane B1 = molecular weight markers

Lane B2 = blank

Lane B3 = 5307 NP2171 × BC5F3 digested with SpeI

Lane B4 = NP2171 × NP2460 digested with SpeI

Lane B5 = Positive control (NP2171 × NP2460 digested 

with SpeI and 16.53 pg of pSYN12274 digested 

with SmaI + PmeI)

(C) SmaI + PmeI

Lane C1 = molecular weight markers

Lane C2 = blank

Lane C3 = 5307 NP2171 × BC5F3 digested with SmaI + 

PmeI

Lane C4 = NP2171 × NP2460 digested with SmaI + PmeI

Lane C5 = Positive control (NP2171 × NP2460 digested 

with SmaI + PmeI and 16.53 pg of pSYN12274 

digested with SmaI + PmeI)
5307-FDA-1 Page 40 of 168

IV.E.3.d. Copy Number of Functional Elements: ZmUbiInt PromoterSpecific Probe

Figure IV-11 shows a map of the T-DNA of 5307 corn transformation plasmid pSYN12274, 

indicating the location of the ZmUbiInt promoter-specific probe and restriction sites for BstEII, 

SpeI, SmaI, and PmeI. Figure IV-12 depicts 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 BstEII and probed with the 

ZmUbiInt promoter-specific probe, one hybridization band of approximately 7.2 kb was 

observed as expected (Figure IV-12A, Lane A3) (Table IV-6).

No hybridization band was present in DNA extracted from the control substance plants (Figure 

IV-12A, Lanes A4, A5, A6, and A7) and therefore hybridization was specific to the 5307 corn

insert. As expected, three 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 SmaI + PmeI + BstEII

(Figure IV-12A, Lane A8).

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 approximately 12 kb and 18 kb corresponding to the 

hybridization bands observed in lanes containing DNA extracted from control plants (Figure 

IV-12A, Lanes A5 and A6), respectively, were also observed in the DNA extracted from 5307 

corn plants (Figure IV-12A, Lane A3). Two hybridization bands of approximately 8.4 kb and 18 

kb corresponding to the hybridization bands observed in lanes containing DNA extr acted from 

NP2460 plants (Figure IV-12A, Lane A7) and NP2171 plants (Figure IV-12A, Lane A5), 

respectively, were observed in the lane containing DNA extracted from NP2171 × NP2460 

plants (Figure IV-12A, Lane A4) and the lane containing the positive control (Figure IV-12, 

Lane A8). 

For Southern blot analysis with genomic DNA digested with SpeI 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-12B, Lane B3) 

(Table IV-6). No hybridization band was present in the DNA extracted from the control 

substance plants (Figure IV-12B, Lanes B4, B5, B6, and B7) and therefore hybridization was

specific to the 5307 corn insert. As expected, three hybridization bands of approximately 6.3 kb, 

14 kb, and 20 kb were observed in the lane containing the positive control DNA digested with 

SmaI + PmeI (Figure IV-12B, Lane B8). 

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 approximately 25 kb and 14 kb corresponding to the 

hybridization bands observed in lanes containing DNA extracted from control substance plants 

(Figure IV-12B, Lanes B5 and B6) were also observed in the DNA extracted from 5307 corn

plants (Figure IV-12B, Lane B3). Finally, two hybridization 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-12B, Lane B7) and NP2171 plants (Figure IV-12B, Lane B5), 

respectively, were observed in the lane containing DNA extracted from NP2171 × NP2460 

plants (Figure IV-12B, Lane B4) and the lane containing the positive control (Figure IV-12B, 

Lane B8). 

For Southern blot analysis with genomic DNA digested with SmaI + PmeI and probed with the 

ZmUbiInt promoter-specific probe, one hybridization band of approximately 6.3 kb was 

observed in the DNA extracted from 5307 corn plants as expected (Figure IV-12C, Lane C3) 

(Table IV-6). This hybridization band was absent in the lane containing DNA extracted from the 

control substance plants NP2171 × NP2460 (Figure IV-12C, Lanes C4, C5, C6, and C7) and

therefore hybridization was specific to the 5307 corn insert. A 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 DNA (Figure IV-12C, Lane C8). 

At least one additional band resulting from cross-hybridization of the ZmUbiInt promoterspecific 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-12C, Lane C6) was also observed in the lane containing DNA extracted from 5307 

corn plants (Figure IV-12C, Lane C3). An additional high molecular weight band (greater than 

30 kb) was observed in lanes containing DNA extracted from control substance plants (Figure 

IV-12C, Lanes C5 and C7). 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-12C, Lane C3), 

control substance plants (Figure IV-12C, Lane C4), and the lane containing the positive control 

(Figure IV-12C, Lane C8). 

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 demonstrated that 

5307 corn contains a single copy of the ZmUbiInt promoter sequence. No unexpected bands 

were detected, indicating that there were no extraneous DNA fragments of the ZmUbiInt 

promoter sequence in the 5307 corn genome. 
5307-FDA-1 Page 42 of 168

Figure IV-11. Location of the BstEII, SpeI, SmaI, and PmeI restriction sites and position of the 

1993-bp ZmUbiInt promoter-specific probe in the T-DNA region of the transformation plasmid 

pSYN12274.
5307-FDA-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 BstEII, SpeI, and 

SmaI + PmeI.

Figure & 

Lane No. Source of DNA Restriction enzyme(s)

Expected no.

of bands

Expected 

band size (kb)

Observed 

band size (kb)

Figure IV-12A,

Lane A3

5307 NP2171 × BC5F3

BstEII

1 5307 insert

x endogenous

> 4.0

unknown

~ 7.2

~ 12 (endogenous)

~ 18 (endogenous)

Figure IV-12A,

Lane A4

NP2171 × NP2460 BstEII x endogenous unknown ~ 8.4 (endogenous)

~ 18 (endogenous)

Figure IV-12A,

Lane A5

NP2171 BstEII x endogenous unknown ~ 18 (endogenous)

Figure IV-12A,

Lane A6

NP2222 BstEII x endogenous unknown ~ 12 (endogenous)

Figure IV-12A,

Lane A7

NP2460 BstEII x endogenous unknown ~ 8.4 (endogenous)

Figure IV-12A,

Lane A8

Positive control (16.53 pg 

of pSYN12274 mixed 

with NP2171 × NP2460 

digested with BstEII)

SmaI + PmeI

+ BstEII1

1 pSYN12274

x endogenous

~ 3.9

unknown

~ 3.9

~ 8.4 (endogenous)

~ 18 (endogenous)

Figure IV-12B,

Lane B3

5307 NP2171 × BC5F3

SpeI

1 5307 insert

x endogenous

> 5.9

unknown

~ 7.0

~ 14 (endogenous)

~ 25 (endogenous)

Figure IV-12B,

Lane B4

NP2171 × NP2460 SpeI x endogenous unknown ~ 14 (endogenous)

~ 20 (endogenous)

Figure IV-12B,

Lane B5

NP2171 SpeI x endogenous unknown ~ 14 (endogenous)

Figure IV-12B,

Lane B6

NP2222 SpeI x endogenous unknown ~ 25 (endogenous)

Figure IV-12B, 

Lane B7

NP2460 SpeI x endogenous unknown ~ 20 (endogenous)

Figure IV-12B,

Lane B8

Positive control (16.53 pg 

of pSYN12274 mixed 

with NP2171 × NP2460 

digested with SpeI)

SmaI + PmeI

1 pSYN12274

x endogenous

~ 6.3

unknown

~ 6.3

~ 14 (endogenous)

~ 20 (endogenous)

Figure IV-12C, 

Lane B3

5307 NP2171 × BC5F3

SmaI + PmeI

1 5307 insert

x endogenous

~ 6.3

unknown

~ 6.3

~ 18 (endogenous)

> 30 (endogenous)

Figure IV-12C, 

Lane C4

NP2171 × NP2460 SmaI + PmeI x endogenous unknown > 30 (endogenous)

Figure IV-12C,

Lane C5

NP2171 SmaI + PmeI x endogenous unknown > 30 (endogenous)

Figure IV-12C,

Lane C6

NP2222 SmaI + PmeI x endogenous unknown ~ 18 (endogenous)

Figure IV-12C, 

Lane C7

NP2460 SmaI + PmeI x endogenous Unknown > 30 (endogenous)

Figure IV-12C,

Lane C8

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with SmaI + PmeI)

SmaI + PmeI 1 pSYN12274

x endogenous

~ 6.3

unknown

~ 6.3

> 30 (endogenous)

x = unknown number 1 Digestion of pSYN12274 with BstEII was the result of addition to NP2171 × NP2460 digested with BstEII
5307-FDA-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 BstEII, SpeI, and SmaI + PmeI. 

(A)BstEII

Lane A1 = molecular weight markers

Lane A2 = blank

Lane A3 = 5307 NP2171 × BC5F3 digested with BstEIlI

Lane A4 = NP2171 × NP2460 digested with BstEII

Lane A5 = NP2171 digested with BstEII

Lane A6 = NP2222 digested with BstEII

Lane A7 = NP2460 digested with BstEII

Lane A8 = Positive control (NP2171 × NP2460 digested 

with BstEII and 16.53 pg of pSYN12274 

digested with SmaI + PmeI + BstEII)

(B) SpeI

Lane B1 = molecular weight markers

Lane B2 = blank

Lane B3 = 5307 NP2171 × BC5F3 digested with SpeI

Lane B4 = NP2171 × NP2460 digested with SpeI

Lane B5 = NP2171 digested with SpeI

Lane B6 = NP2222 digested with SpeI

Lane B7 = NP2460 digested with SpeI

Lane B8 = Positive control (NP2171 × NP2460 digested 

with SpeI and 16.53 pg of pSYN12274 digested 

with SmaI + PmeI)

(C) SmaI + PmeI

Lane C1 = molecular weight markers

Lane C2 = blank

Lane C3 = 5307 NP2171 × BC5F3 digested with SmaI + 

PmeI

Lane C4 = NP2171 × NP2460 digested with SmaI + PmeI

Lane C5 = NP2171 digested with SmaI + PmeI

Lane C6 = NP2222 digested with SmaI + PmeI

Lane C7 = NP2460 digested with SmaI + PmeI

Lane C8 = Positive control (NP2171 × NP2460 digested 

with SmaI + PmeI and 16.53 pg of pSYN12274 

digested with SmaI + PmeI)
5307-FDA-1 Page 45 of 168

IV.E.3.e. Copy Number of Functional Elements: NOS Terminator-Specific Probe

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 KpnI, NcoI, 

and SmaI + PmeI. Figure IV-14 depicts the results of the corresponding Southern 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 KpnI and probed with the NOS 

terminator-specific probe, two hybridization 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-14A, Lane A3) (Table IV-7). No hybridization bands were present in DNA extracted from 

the control substance plants (Figure IV-14A, Lane A4). Therefore, 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 band of 

approximately 6.3 kb was observed in the lane containing the positive control, as expected

(Figure IV-14A, Lane A5). 

For Southern blot analysis with genomic DNA digested with NcoI and probed with the NOS 

terminator-specific probe, two hybridization bands of approximately 16 kb and 19 kb were 

observed in the lane containing DNA extracted from 5307 corn plants as expected (Figure 

IV-14B, Lane B3) (Table IV-7). These hybridization bands were absent in the lane containing 

DNA extracted from the control substance plants (Figure IV-14B, Lane B4) and were, therefore, 

specific to the two copies of the NOS terminator sequence in the 5307 corn insert. As expected, 

two hybridization bands of approximately 2.5 kb and 3.9 kb were observed in the lane containing 

the positive control digested with SmaI + PmeI + NcoI (Figure IV-14B, Lane B5). 

For Southern blot analysis with genomic DNA digested with SmaI + PmeI and probed with the 

NOS terminator-specific probe, one hybridization band of approximately 6.3 kb was observed in 

the lane containing DNA extracted from 5307 corn plants as expected (Figure IV-14C, Lane C3) 

(Table IV-7). This hybridization band was absent in the lane containing DNA extracted from the 

control substance plants (Figure IV-14C, Lane C4) 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-14C, Lane C9). 

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 

demonstrated 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 were no extraneous DNA fragments of 

the NOS terminator sequence in the 5307 corn genome.
5307-FDA-1 Page 46 of 168

Figure IV-13. Location of the KpnI, NcoI, SmaI, and PmeI 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 KpnI, NcoI, and SmaI + 

PmeI.

Figure &

Lane No. Source of DNA

Restriction 

enzyme(s)

Expected 

number of 

bands

Expected 

band size 

(kb)

Observed 

band size 

(kb)

Fig. IV-14A, 

Lane A3 5307 NP2171 × BC5F3 KpnI 2 > 2.8

> 3.6

~ 6.4

~ 8.5

Fig. IV-14A, 

Lane A4 NP2171 × NP2460 KpnI none none none

Fig. IV-14A, 

Lane A5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with KpnI)

SmaI + PmeI 1 ~6.3 ~ 6.3

Fig. IV-14B, 

Lane B3 5307 NP2171 × BC5F3 NcoI 2 > 2.5

> 3.9

~ 16

~ 19

Fig. IV-14B, 

Lane B4 NP2171 × NP2460 NcoI none none None

Fig. IV-14B, 

Lane B5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with NcoI)

SmaI + PmeI + 

NcoI

1 2 ~ 2.5

~ 3.9

~ 2.5

~ 3.9

Fig. IV-14C, 

Lane C3 5307 NP2171 × BC5F3 SmaI + PmeI 1 ~ 6.3 ~ 6.3

Fig. IV-14C, 

Lane C4 NP2171 ×NP2460 SmaI + PmeI none none none

Fig. IV-14C, 

Lane C5

Positive control (16.53 pg of 

pSYN12274 mixed with 

NP2171 × NP2460 digested 

with SmaI + PmeI)

SmaI + PmeI 1 ~ 6.3 ~ 6.3

1 Digestion of pSYN12274 with NcoI was the result of addition to NP2171 × NP2460 digested with NcoI
5307-FDA-1 Page 48 of 168

Figure IV-14. Southern blot analysis of 5307 corn for copy number of functional elements: 253-bp NOS terminator-specific probe, 

using restriction enzymes KpnI, NcoI, and SmaI + PmeI.

A. KpnI

Lane A1 = molecular weight markers

Lane A2 = blank

Lane A3 = 5307 NP2171 × BC5F3 digested with KpnI

Lane A4 = NP2171 × NP2460 digested with KpnI

Lane A5 = Positive control (NP2171 × NP2460

digested with KpnI and 16.53 pg of 

pSYN12274 digested with SmaI + PmeI)

B. NcoI

Lane B1 = molecular weight markers

Lane B2 = blank

Lane B3 = 5307 NP2171 × BC5F3 digested with NcoI

Lane B4 = NP2171 × NP2460 digested with NcoI

Lane B5 = Positive control (NP2171 × NP2460 

digested with NcoI and 16.53 pg of 

pSYN12274 digested with SmaI + PmeI + 

NcoI)

C. SmaI + PmeI

Lane C1 = molecular weight markers

Lane C2 = blank

Lane C3 = 5307 NP2171 × BC5F3 digested with SmaI 

+ PmeI

Lane C4 = NP2171 × NP2460 digested with SmaI + 

Pme

Lane C5 = Positive control (NP2171 × NP2460

digested with SmaI + PmeI and 16.53 pg of 

pSYN12274 digested with SmaI + PmeI)
5307-FDA-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-DNAspecific 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 TDNA inserted elsewhere in the 5307 corn genome. Identical hybridization patterns 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 plasmid 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 insert 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 F1, 5307 BC6, 

5307 BC7, and 5307 NP2171 × BC5F3) 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 polymorphism 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 these 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 were located in the corn

genome flanking the 5307 corn insert. This first strategy was used twice with two different 

enzymes (BstEII and SpeI) to determine the copy number of the 5307 corn insert and the 

presence or absence of extraneous DNA fragments of the plasmid pSYN12274 T-DNA in other 
5307-FDA-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 either 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 SmaI + PmeI, which cut within 

the 5307 corn insert such that a DNA fragment of predictable size 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 BstEII, 

SpeI, SmaI, and PmeI. 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 BstEII, SpeI, SmaI, and PmeI 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-FDA-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 BstEII, SpeI, and SmaI 

+ PmeI.

Figure & 

Lane No. Source of DNA Restriction enzyme(s)

Expected 

number of bands

Expected 

band size (kb)

Observed 

band size (kb)

Figure IV-16A, 

Lane A3

5307 F1 BstEII 2 5307 insert

x endogenous

>2.4

>4

unknown

~2.9 

~7.2 

~8.4 (endogenous)

~12 (endogenous)

Figure IV-16A, 

Lane A4

5307 BC6 BstEII 2 5307 insert

x endogenous

>2.4

>4

unknown

~2.9

~7.2

~8.4 (endogenous)

~12 (endogenous)

Figure IV-16A, 

Lane A5

5307 BC7 BstEII 2 5307 insert

x endogenous

>2.4

>4

unknown

~2.9

~7.2

~8.4 (endogenous)

~12 (endogenous)

Figure IV-16A, 

Lane A6

5307 NP2171 × BC5F3 BstEII 2 5307 insert

x endogenous

>2.4

>4

unknown

~2.9

~7.2

~12 (endogenous)

~18 (endogenous)

Figure IV-16A, 

Lane A7

NP2171 × NP2460 BstEII x endogenous unknown ~8.4 (endogenous)

~18 (endogenous)

Figure IV-16A, 

Lane A8

NP2222 BstEII x endogenous unknown ~12 (endogenous)

Figure IV-16A, 

Lane A9

NP2460 BstEII x endogenous unknown ~8.4 (endogenous)

Figure IV-16A, 

Lane A10

NP2171 BstEII x endogenous unknown ~18 (endogenous)

Figure IV-16A, 

Lane A11

5307 F1 SpeI 2 5307 insert

x endogenous

>0.6

>5.8

unknown

~2.6

~7 

~20 (endogenous)

~25 (endogenous)

Figure IV-16A, 

Lane A12

5307 BC6 SpeI 2 5307 insert

x endogenous

>0.6

>5.8

unknown

~2.6

~7

~20 (endogenous)

~25 (endogenous)

Figure IV-16A, 

Lane A13

5307 BC7 SpeI 2 5307 insert

x endogenous

>0.6

>5.8

unknown

~2.6

~7

~20 (endogenous)

~25 (endogenous)

Figure IV-16A, 

Lane A14

5307 NP2171 × BC5F3 SpeI 2 5307 insert

x endogenous

>0.6

>5.8

unknown

~2.6

~7

~14 (endogenous)

~25 (endogenous)

Figure IV-16A, 

Lane A15

NP2171/NP2460 SpeI x endogenous unknown ~14 (endogenous)

~20 (endogenous)

Figure IV-16A, 

Lane A16

NP2222 SpeI x endogenous unknown ~25 (endogenous)

Figure IV-16A, 

Lane A17

NP2460 SpeI x endogenous unknown ~20 (endogenous)

Figure IV-16A, 

Lane A18

NP2171 SpeI x endogenous unknown ~14 (endogenous)

Figure IV-16A, 

Lane A19

Positive control 

(NP2171/NP2460 and 

16.53 pg of 

SpeI 1 pSYN12274

x endogenous

11.8

unknown

~11.8

~14 (endogenous)

~20 (endogenous)
5307-FDA-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 BstEII, SpeI, and 

SmaI + PmeI 

Figure & 

Lane No.

Source of 

DNA

Restriction 

enzyme(s)

Expected 

number of bands

Expected band 

size (kb)

Observed band 

size (kb)

Figure IV-16B,

Lane B3

5307 F1 SmaI + PmeI 1 5307 insert

x endogenous

6.3

unknown

~6.3

~18 (endogenous)

Figure IV-16B, 

Lane B4

5307 BC6 SmaI + PmeI 1 5307 insert

x endogenous

6.3

unknown

~6.3

~18 (endogenous)

Figure IV-16B, 

Lane B5

5307 BC7 SmaI + PmeI 1 5307 insert

x endogenous

6.3

unknown

~6.3

~18 (endogenous)

Figure IV-16B, 

Lane B6

5307 NP2171 × 

BC5F3

SmaI + PmeI 1 5307 insert

x endogenous

6.3

unknown

~6.3

~18 (endogenous)

Figure IV-16B, 

Lane B7

NP2171 ×

NP2460

SmaI + PmeI x endogenous unknown >30 (endogenous)

Figure IV-16B, 

Lane B8

NP2222 SmaI + PmeI x endogenous unknown ~18 (endogenous)

Figure IV-16B, 

Lane B9

NP2460 SmaI + PmeI x endogenous unknown >30 (endogenous)

Figure IV-16B,

Lane B10

NP2171 SmaI + PmeI x endogenous unknown >30 (endogenous)

Figure IV-16B, 

Lane B11

Positive control 

(NP2171 ×

NP2460 and 

16.53 pg of 

pSYN12274)

SmaI + PmeI 1 pSYN12274

x endogenous

6.3

unknown

~6.3 

>30 (endogenous)
5307-FDA-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 BstEII, SpeI, and SmaI + PmeI.

(A) BstEII or SpeI

Lane A1 = molecular weight markers

Lane A2 = blank

Lane A3 = 5307 F1 digested with BstEII

Lane A4 = 5307 BC6 digested with BstEII

Lane A5 = 5307 BC7 digested with BstEII

Lane A6 = 5307 NP2171 × BC5F3 digested with BstEII

Lane A7 = NP2171 × NP2460 digested with BstEII

Lane A8 = NP2222 digested with BstEII 

Lane A9 = NP2460 digested with BstEII

Lane A10 = NP2171 digested with BstEII

Lane A11 = 5307 F1 digested with SpeI

Lane A12 = 5307 BC6 digested with SpeI

Lane A13 = 5307 BC7 digested with SpeI

Lane A14 = 5307 NP2171 × BC5F3 digested with SpeI

Lane A15 = NP2171 × NP2460 digested with SpeI

Lane A16 = NP2222 digested with SpeI

Lane A17 = NP2460 digested with SpeI

Lane A18 = NP2171 digested with SpeI

Lane A19 = Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274 digested with SpeI)
5307-FDA-1 Page 54 of 168

Figure IV-16 (continued). Genetic stability Southern blot analysis of 5307 corn with the 6423-bp 

full-length T-DNA-specific probe, using restriction enzymes BstEII, SpeI, and SmaI + PmeI.

B. SmaI + PmeI

Lane B1 = molecular weight markers

Lane B2 = blank

Lane B3 = 5307 F1 digested with SmaI + PmeI

Lane B4 = 5307 BC6 digested with SmaI + PmeI

Lane B5 = 5307 BC7 digested with SmaI + PmeI

Lane B6 = 5307 NP2171 × BC5F3 digested with SmaI + PmeI

Lane B7 = NP2171 × NP2460 digested with SmaI + PmeI

Lane B8 = NP2222 digested with SmaI + PmeI

Lane B9 = NP2460 digested with SmaI + PmeI

Lane B10 = NP2171 digested with SmaI + PmeI

Lane B11 = Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274 digested with SmaI + PmeI)
5307-FDA-1 Page 55 of 168

For Southern blot analysis with genomic DNA digested with BstEII and probed with the fulllength 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 F1, 5307 BC6, 5307 BC7, and 5307 

NP2171 × BC5F3 plants, as expected (Figure IV-16A, Lanes A3, A4, A5, and A6) (Table IV-8). 

These hybridization bands were absent in lanes containing DNA extracted from the control 

plants (Figure IV-16A, Lanes A7, A8, A9, and A10) 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 SpeI and loaded with DNA extracted from NP2171 × NP2460 plants) (Figure 

IV-16A, Lane A19).

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-16A, Lane A9) and NP2222 plants (Figure IV-16A, 

Lane A8), respectively, were observed in lanes containing DNA extracted from 5307 F1, 5307 

BC6, and 5307 BC7 plants (Figure IV-16A, Lanes A3, A4, and A5, respectively). Two 

hybridization bands of approximately 12 kb and 18 kb, corresponding to the hybridization bands 

observed in lanes containing DNA extracted from NP2222 plants (Figure IV-16A, Lane A8) and 

NP2171 plants (Figure IV-16A, Lane A10), respectively, were observed in the lane containing 

DNA extracted from 5307 NP2171 × BC5F3 plants (Figure IV-16A, Lane A6). 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-16A, Lane A9) and 

NP2171 plants (Figure IV-16A, Lane A10), respectively, were observed in lanes containing 

DNA extracted from NP2171 × NP2460 plants (Figure IV-16A, Lane A7).

For Southern blot analysis with genomic DNA digested with SpeI and 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 F1, 5307 BC6, 5307 BC7, and 5307 

NP2171 × BC5F3 plants, as expected (Figure IV-16A, Lanes A11, A12, A13, and A14) (Table 

IV-8). These bands were absent in lanes containing DNA extracted from the control plants 

(Figure IV-16A, Lanes A15, A16, A17, and A18) 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 

SpeI and loaded with DNA extracted from NP2171 × NP2460 plants) (Figure IV-16A, Lane 

A19). 

Additional bands resulting from cross-hybridization of the ZmUbiInt promoter sequence present 

in 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-FDA-1 Page 56 of 168

extracted from NP2460 plants (Figure IV-16A, Lane A17) and NP2222 plants (Figure IV-16A, 

Lane A16), respectively, were observed in lanes containing DNA extracted from 5307 F1, 5307 

BC6, and 5307 BC7 plants (Figure IV-16A, Lanes A11, A12, and A13). 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-16A, Lane A18) and NP2222 

plants (Figure IV-16A, Lane A16), respectively, were observed in the lane containing DNA 

extracted from 5307 NP2171 × BC5F3 plants (Figure IV-16A, Lane A14). 

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-16A, Lane A18) and NP2460 plants (Figure IV-16A, Lane A17), respectively, were observed 

in lanes containing DNA extracted from NP2171 × NP2460 plants (Figure IV-16A, Lane A15). 

For Southern blot analysis with genomic DNA digested with SmaI + PmeI 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 F1, 5307 BC6, 5307 BC7, and 5307 NP2171 × 

BC5F3 plants as expected (Figure IV-16B, Lanes B3, B4, B5, and B6) (Table IV-8). This 

hybridization band was absent in lanes containing DNA extracted from the control plants (Figure 

IV-16B, Lanes B7, B8, B9, and B10) and was, therefore, 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 SmaI + PmeI and loaded with DNA extracted from NP2171 × NP2460 

plants) (Figure IV-16B, Lane B11). At least one additional band resulting from crosshybridization of the ZmUbiInt 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 

hybridization band observed in the lane containing DNA extracted from NP2222 plants (Figure 

IV-16B, Lane B8), was observed in lanes containing DNA extracted from 5307 F1, 5307 BC6, 

5307 BC7 and 5307 NP2171 × BC5F3 plants (Figure IV-16B, Lanes B3, B4, B5, and B6). An 

additional high molecular weight band (greater than 30 kb) was observed in lanes containing 

DNA extracted from NP2460 plants (Figure IV-16B, Lane B9) and NP2171 plants (Figure 

IV-16B, Lane B10). 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 F1, 5307 BC6, 5307 BC7, and NP2171 × NP2460) (Figure IV-16B, Lanes 

B3, B4, B5, and B7) and/or the NP2171 polyubiquitin promoter allele (5307 NP2171 × BC5F3

and NP2171 × NP2460) (Figure IV-16B, Lanes B6 and B7). 
5307-FDA-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 BstEII and probed with a full length TDNA-specific probe and only two hybridization bands specific to the 5307 corn insert were 

observed when the genomic DNA was digested with SpeI and probed with a full length T-DNAspecific 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 consistent with the genetic 

make-up of the various generations analyzed. As expected, the 5307 F1, 5307 BC6, and 5307 

BC7 generations carry the maize polyubiquitin promoter allelic forms present in NP2222 and 

NP2460, the 5307 NP2171 × BC5F3 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 SmaI + PmeI was the predicted size. The 

approximately 18 kb band observed 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 F1, 5307 BC6, 5307 BC7, and 5307 NP2171 × BC5F3). The faint and high 

molecular weight 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 F1, 5307 BC6, 5307 BC7, and 

NP2171 × NP2460) and/or the NP2171 polyubiquitin promoter allelic form (5307 NP2171 × 

BC5F3 and NP2171 × NP2460). Because no additional bands were observed (other than those 

associated with the 5307 corn insert and the corn endogenous sequence), Southern 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 identical in lanes containing DNA extracted 

from plants grown from all generations (5307 F1, 5307 BC6, 5307 BC7, and 5307 NP2171 × 

BC5F3); 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 

generations of 5307 corn analyzed in this study indicates that the insert is stably inherited from 

one generation to the next. 
5307-FDA-1 Page 58 of 168

IV.E.4.b. Stability of the T-DNA Using a Plasmid Backbone-Specific 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 of 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 F1, 5307 BC6, 5307 BC7, 5307 NP2171 × BC5F3) 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 BstEII, SpeI, SmaI, and 

PmeI. 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 BstEII and probed with the plasmid 

pSYN12274 backbone-specific probe, no hybridization bands were observed in the lanes 

containing DNA extracted from plants grown from the test and control substances (5307 F1, 

5307 BC6, 5307 BC7, 5307 NP2171 × BC5F3, NP2171 × NP2460, NP2222, NP2460, and 

NP2171) (Figure IV-18A, Lanes 3, 4, 5, 6, 7, 8, 9, and 10) as expected. The positive control 

(16.53 pg of plasmid pSYN12274 DNA digested with SpeI and loaded with DNA extracted from 

NP2171 × NP2460 plants) produced the expected 11.8 kb band (Figure IV-18A, Lane 19). For 

Southern blot analyses with genomic DNA digested with SpeI and probed with the plasmid 

pSYN12274 backbone-specific probe, no hybridization bands were observed in the test and 

control substances (5307 F1, 5307 BC6, 5307 BC7, 5307 NP2171 × BC5F3, NP2171 × NP2460, 

NP2222, NP2460, and NP2171) (Figure IV-18A, Lanes A11, A12, A13, A14, A15, A16, A17, 

and A18) as expected. The positive control (16.53 pg of plasmid pSYN12274 DNA digested 

with SpeI and loaded with DNA extracted from NP2171 × NP2460 plants) produced the 

expected 11.8 kb band (Figure IV-18A, Lane A19). For Southern blot analyses with genomic 

DNA digested with SmaI + PmeI and probed with the backbone-specific probe, no hybridization 

bands were observed in the test and control substances (5307 F1, 5307 BC6, 5307 BC7, 5307 

NP2171 × BC5F3, NP2171 × NP2460, NP2222, NP2460, and NP2171) (Figure IV-18B, Lanes 

B3, B4, B5, B6, B7, B8, B9, and B10). The positive control (16.53 pg of plasmid pSYN12274 

DNA digested with SmaI + PmeI and loaded with DNA extracted from NP2171 × NP2460 

plants) produced the expected 4.2 kb and 1.2 kb bands (Figure IV-18B, Lane B11).

The data from the three Southern blot analyses demonstrated that all the 5307 corn generations 

analyzed are free of plasmid pSYN12274 backbone sequence.
5307-FDA-1 Page 59 of 168

Figure IV-17. Location of the BstEII, SpeI, SmaI, and PmeI restriction sites and position of the 

5312-bp plasmid backbone-specific probe in the transformation plasmid pSYN12274.
5307-FDA-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 BstEII, 

SpeI, and SmaI + PmeI.

Figure & Lane Number Source of DNA Restriction 

enzyme(s)

Expected 

number of 

bands

Expected 

band size 

(kb)

Observed 

band size 

(kb)

Figure IV-18A, Lane A3 5307 F1 BstEII none none none

Figure IV-18A, Lane A4 5307 BC6 BstEII none none none

Figure IV-18A, Lane A5 5307 BC7 BstEII none none none

Figure IV-18A, Lane A6 5307 NP2171 × BC5F3 BstEII none none none

Figure IV-18A, Lane A7 NP2171 × NP2460 BstEII none none none

Figure IV-18A, Lane A8 NP2222 BstEII none none none

Figure IV-18A, Lane A9 NP2460 BstEII none none none

Figure IV-18A, Lane A10 NP2171 BstEII none none none

Figure IV-18A, Lane A11 5307 F1 SpeI none none none

Figure IV-18A, Lane A12 5307 BC6 SpeI none none none

Figure IV-18A, Lane A13 5307 BC7 SpeI none none none

Figure IV-18A, Lane A14 5307 NP2171 × BC5F3 SpeI none none none

Figure IV-18A, Lane A15 NP2171 × NP2460 SpeI none none none

Figure IV-18A, Lane A16 NP2222 SpeI none none none

Figure IV-18A, Lane A17 NP2460 SpeI none none none

Figure IV-18A, Lane A18 NP2171 SpeI none none none

Figure IV-18A, Lane A19 Positive control 

(NP2171×NP2460 and 

16.53 pg of pSYN12274)

SpeI 1 11.8 ~11.8

Figure IV-18B, Lane B3 5307 F1 SmaI + PmeI none none none

Figure IV-18B, Lane B4 5307 BC6 SmaI + PmeI none none none

Figure IV-18B, Lane B5 5307 BC7 SmaI + PmeI none none none

Figure IV-18B, Lane B6 5307 NP2171 × BC5F3 SmaI + PmeI none none none

Figure IV-18B, Lane B7 NP2171 × NP2460 SmaI + PmeI none none none

Figure IV-18B, Lane B8 NP2222 SmaI + PmeI none none none

Figure IV-18B, Lane B9 NP2460 SmaI + PmeI none none none

Figure IV-18B,Lane B10 NP2171 SmaI + PmeI none none none

Figure IV-18B,Lane B11 Positive control 

(NP2171×NP2460 and 

16.53 pg of pSYN12274)

SmaI + PmeI 2 4.2

1.2

~4.2

~1.2
5307-FDA-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 BstEII, SpeI, and SmaI + PmeI.

(A) BstEII or SpeI

Lane A1 = molecular weight markers

Lane A2 = blank

Lane A3 = 5307 F1 digested with BstEII

Lane A4 = 5307 BC6 digested with BstEII

Lane A5 = 5307 BC7 digested with BstEII

Lane A6 = 5307 NP2171 × BC5F3 digested with BstEII

Lane A7 = NP2171 × NP2460 digested with BstEII

Lane A8 = NP2222 digested with BstEII

Lane A9 = NP2460 digested with BstEII

Lane A10 = NP2171 digested with BstEII

Lane A11 = 5307 F1 digested with SpeI

Lane A12 = 5307 BC6 digested with SpeI

Lane A13 = 5307 BC7 digested with SpeI

Lane A14 = 5307 NP2171 × BC5F3 digested with SpeI

Lane A15 = NP2171 × NP2460 digested with SpeI

Lane A16 = NP2222 digested with SpeI

Lane A17 = NP2460 digested with SpeI

Lane A18 = NP2171 digested with SpeI

Lane A19 = Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274 digested with SpeI)
5307-FDA-1 Page 62 of 168

Figure V-18. (Continued)

(B) SmaI + PmeI

Lane B1 = molecular weight markers

Lane B2 = blank

Lane B3 = 5307 F1 digested with SmaI + PmeI

Lane B4 = 5307 BC6 digested with SmaI + PmeI

Lane B5 = 5307 BC7 digested with SmaI + PmeI

Lane B6 = 5307 NP2171 × BC5F3 digested with SmaI + PmeI

Lane B7 = NP2171 × NP2460 digested with SmaI + PmeI

Lane B8 = NP2222 digested with SmaI + PmeI

Lane B9 = NP2460 digested with SmaI + PmeI

Lane B10 = NP2171 digested with SmaI + PmeI 

Lane B11 = Positive control (NP2171 × NP2460 and 16.53 pg of pSYN12274 digested with SmaI + PmeI)
5307-FDA-1 Page 63 of 168

IV.E.5. Mendelian Inheritance of the DNA Insert

A Chi-square (Χ2) 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 this analysis were grown from seeds of the 

5307 F1, 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 

expected 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 goodnessof-fit of the observed genotypic ratios to the expected genotypic ratios using Chi-square analysis 

with Yates’ correction factor as in Armitage and Berry (1987). 

Χ2 = ∑ [|(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. This 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.
5307-FDA-1 Page 64 of 168

Table IV-10. Observed versus expected frequencies for ecry3.1Ab across generations. 

Trait

F1 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

Χ2 value 0.098 1.099 0.100

Table IV-11. Observed versus expected frequencies for pmi across generations. 

Trait

F1 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

Χ2 value 0.098 1.099 0.100
5307-FDA-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 similarity 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 Information 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 parameters 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 measure 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 are 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 insert 

were not annotated as a gene. The corn genomic sequences flanking the 5ʹ region and the 3ʹ 
5307-FDA-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 AY664415.1. 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 sequences 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 5ʹ region and the 3ʹ region of the 5307 insert are likely to be repeated 

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 Zea 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 similar 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 region 

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, was 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 between 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 3ʹ 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 allergen similarity searches are described in Appendix A. 
5307-FDA-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 terminator 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 that 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 of 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 endogenous 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 screened 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 similarity 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.
5307-FDA-1 Page 68 of 168

V. Absence of Genes That Encode Resistance to Antibiotics

As indicated in Figure IV-1 and described in 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 

corn genome during transformation. Southern blot analyses (Figure IV-18) described in Part 

IV.E.3.b. demonstrated no hybridization of Event 5307 corn DNA with the pSYN12274 

backbone-specific probe (Figure IV-17). This confirms that, as expected, no spec DNA 

sequences were incorporated into Event 5307 corn DNA. 
5307-FDA-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) information 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 protein, (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 Event 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 coleopteranactive eCry3.1Ab protein 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 Nterminus 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 172 residues of Cry1Ab at the C-terminus. The 

eCry3.1Ab protein is 653 amino acid residues in size and has a molecular weight of 

approximately 73.7 kDa. 

The eCry3.1Ab protein is insecticidally active against the larvae of certain coleopteran pests in the 

family Chrysomelidae, including western 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 multiple other organisms, including lepidopteran 

insects, other nontarget insects, and avian, mammalian, and aquatic species.

The spectrum of activity of any specific insecticidal 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 the 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 disruption of the normal function of the midgut leading to the death of the insect. The 
5307-FDA-1 Page 70 of 168

eCry3.1Ab protein exhibits 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 feasible 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 produce the identical eCry3.1Ab protein 

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 residues 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 spectrometry, 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 analytical

methods were used to characterize the eCry3.1Ab protein produced in Event 5307 corn and to 

assess whether the microbially produced eCry3.1Ab 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 characterization 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 
5307-FDA-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 LC50 values (Table VI-1). 

Table VI-1. Comparison of Colorado potato beetle larvae LC50 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

LP5307a LP-NEGb + ECRY3.1AB-0208

μg eCry3.1Ab/ml insect diet

LC50 95% CI LC50 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 identities 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 microbiallyproduced 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 

Chrysomelidae 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. 
5307-FDA-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 amino 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 National 

Center for Biotechnology Information (NCBI 2010b) 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 are conserved across different proteins from different species. The 

BLASTP algorithm is optimized to identify these domains or shorter sequence similarities 

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 significance 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 sequence. 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 

proteins 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 0.32. 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. 
5307-FDA-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 (Berks 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. The 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 have 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 no 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 Toxicity Study of eCry3.1Ab

The potential toxicity of the eCry3.1Ab protein was evaluated in an acute oral toxicity study 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 

weight. 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 period 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 administration (study day 0) and once daily on nondosing 

days (study days 1-13). Detailed physical examinations were performed weekly. Individual 
5307-FDA-1 Page 74 of 168

body weights and food consumption were recorded daily during the study. Complete necropsies 

were conducted on all animals, 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-related 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 was conducted on all animals. The 

necropsies included, but were not limited to, examination 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. 

These 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 remaining 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; these 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 observable

adverse effect level (NOAEL) for eCry3.1Ab protein in male and female mice was > 2000 mg/kg 

body weight.
5307-FDA-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 test-substance-related effects were observed in mice administered a 

single high dose (2000 mg/kg body weight) of eCry3.1Ab protein. Therefore, eCry3.1Ab is 

considered nontoxic. 

VI.E. Assessment of eCry3.1Ab 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, there 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 produce 

allergenic proteins.

VI.E.2. Assessment of eCry3.1Ab Amino Acid Sequence Similarity to Known Allergens

Amino acid sequence comparisons between novel proteins and known allergens are part of the 

weight-of-evidence approach to assessing potential allergenicity. 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 eCry3.1Ab 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, allergenic 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. Proteins 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 AllergenOnline database using the
5307-FDA-1 Page 76 of 168

FASTA search algorithm1 (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-aminoacid 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. 

The eCry3.1Ab protein 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 acid identity that 

might indicate the presence of common T-cell binding epitopes. There were no matches of eight 

contiguous amino 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 (SGF). Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), 

Western blot and densitometry analyses were used to evaluate the in vitro digestibility of 

eCry3.1Ab in SGF over a 15-minute time course at 37°C (Figure 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 

assessed 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 (Lane 9). Two very faint, diffuse bands with molecular weights of approximately 4 kDa 

and 5 kDa, respectively, were visible after incubation in SGF for 15 seconds (Lane 8). These 

two bands diminished in intensity over the time course and were no longer detectable after 

incubation in SGF for 10 minutes (Lane 18).

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).
5307-FDA-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 μg (Lane 22). Therefore, the limit of detection (LOD) of eCry3.1Ab for 

the SDS-PAGE used in this study was determined to be 0.025 μg. 

Further 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). 
5307-FDA-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 kDa1. 

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 zero2

Lane 8: in vitro digestibility assay - 15 seconds2, 3, 4

Lane 9: in vitro digestibility assay - 30 seconds2

Lane 10: in vitro digestibility assay - 45 seconds2

Lane 11: in vitro digestibility assay - 1 minute2

Lane 12: in vitro digestibility assay - 1 minute 15 seconds2

Lane 13: in vitro digestibility assay - 1 minute 30 seconds2

Lane 14: in vitro digestibility assay - 1 minute 45 seconds2

Lane 15: in vitro digestibility assay – 2 minutes2

Lane 16: in vitro digestibility assay – 3 minutes2

Lane 17: in vitro digestibility assay – 5 minutes2

Lane 18: in vitro digestibility assay – 10 minutes2

Lane 19: in vitro digestibility assay – 15 minutes2

Lane 20: molecular weight standard

Lane 21: 0.1 μg eCry3.1Ab for LOD determination

Lane 22: 0.025 μg eCry3.1Ab for LOD determination4

Lane 23: 0.0063 μg eCry3.1Ab for LOD determination

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 MW 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 the 

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 scanner 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
5307-FDA-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 

remaining1

Amount (μg) of 

eCry3.1Ab 

remaining2

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 eCry3.1Ab remaining = amount of eCry3.1Ab at time zero (0.5 μg) × percent (%) of eCry3.1Ab remaining. 

This calculation was based on the fact that equivalent volumes of each SGF digestibility assay sample were 

subjected to SDS-PAGE.
5307-FDA-1 Page 80 of 168

The Western blot analysis results (Figure VI-2) confirm that eCry3.1Ab 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 were 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). This protein cross-reacted with the antibody capable of detecting eCry3.1Ab and displayed a 

mobility consistent with the molecular weight of two eCry3.1Ab molecules (150 kDa). 

Therefore, it most likely represents a dimer of eCry3.1Ab. An additional faint band with an 

approximate molecular weight of 47 kDa was also visible in the time zero digestibility assay 

sample (Lane 7). This 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 corresponds to a minor eCry3.1Ab hydrolysis product derived from the sample and is

not related to pepsin digestion. Both bands (150 kDa and 47 kDa) were no longer detectable

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 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 

applied to the SDS-PAGE and electroblotted. The lowest amount of eCry3.1Ab visible on the 

blot (Lanes 21 through 25) was 0.4 ng (Lane 22). Therefore, the LOD of eCry3.1Ab for the 

Western blot used in this study was determined to be 0.4 ng.
5307-FDA-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 zero1

Lane 5: eCry3.1Ab control (eCry3.1Ab in SGF without pepsin) – 15 minutes1

Lane 6: molecular weight standard

Lane 7: in vitro digestibility assay - time zero1

Lane 8: in vitro digestibility assay - 15 seconds2

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 digestibility 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 determination2

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, the 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-FDA-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, 37oC, and 65oC, eCry3.1Ab retained bioactivity against Colorado potato beetle larvae

when compared with that observed at 4oC (Table VI-2). However, after treatment at 95oC for 

30 minutes, LC50 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 

inactivated, and therefore, denatured upon heating at temperatures of 95oC and above.

Table VI-3. Effect of temperature on insecticidal activity of eCry3.1Ab in diet incorporation 

bioassay with Colorado potato beetle larvae: LC50 values and 95% confidence intervals at 144 

hours.

Temperature 

[°C]

LC50

[μg eCry3.1Ab/ml]

95% Confidence Interval

[μg 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 not 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-FDA-1 Page 83 of 168

VI.F. eCry3.1Ab History of Safe Use

Prior dietary exposure to the eCry3.1Ab protein has not occurred because it is a novel protein 

engineered by Syngenta. Effective June 16, 2010, a temporary tolerance exemption was 

established for eCry3.1Ab (40 CFR 174.532) in connection with an Experimental Use Permit 

granted by the EPA. 

Although there 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 MIR604 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 crops (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 lepidopteran-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 tolerance 

exemptions exist for Cry1Ab and other Cry1 proteins used in transgenic crops (including fulllength 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 have been identified during the use history of the associated products. 
5307-FDA-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 considered 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-related 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 upon heating at temperatures of 95oC and above.
5307-FDA-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 manA1) 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 produce 

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 Status and Established Safety of PMI

PMI proteins are present as selectable markers 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 is 

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 allergen. 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:

• PMI 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 their ubiquitous occurrence in nature

and previous use in transgenic corn. 

 

1 Entrez Nucleotide Database Accession No. M15380 (NCBI 2010a). 
5307-FDA-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 does 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 described in 

Part VI.L, below.) 

• PMI is rapidly degraded in simulated mammalian gastric fluid containing pepsin. 

• PMI is labile upon heating at temperatures of 65oC and 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 bioinformatic 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

Sequencing of the inserted T-DNA in 5307 plants confirmed 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, is 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 test 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 proteins from 

both sources were demonstrated to have the predicted molecular weight of approximately 42.8 

kDa, and both immunologically cross-reacted with the same anti-PMI antibodies, as determined 

by Western blot analysis, thus confirming their identity and integrity. It was also confirmed that 
5307-FDA-1 Page 87 of 168

the PMI proteins from both sources catalyzed the same chemical reaction1 and had similar 

specific activity (455.67 U/mg PMI for the plant-produced PMI and 526.26 U/mg PMI for the 

microbially produced PMI; see also Table 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 equivalent. Therefore, the microbially produced 

PMI is a suitable surrogate for PMI produced in Event 5307 corn.

VI.K. Updated Bioinformatic Assessment of PMI Amino Acid Sequence 

Similarity to Known Toxins

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) program2 (Altschul et al. 1997). This procedure determined: 

1. whether any proteins in the database shared significant similarity to the PMI amino acid 

sequence, indicating they may be closely related to PMI, 

2. whether any proteins demonstrating significant sequence similarity to the PMI amino 

acid sequence were known or putative 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 Evalues approaching zero, whereas the probability that amino acid sequence similarities occurred 

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 required 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 similarity matrix was Blocks Substitution Matrix62 

(BLOSUM62). 
5307-FDA-1 Page 88 of 168

VI.L. Updated Bioinformatic Assessment of PMI Amino Acid Sequence 

Similarity to Known Allergens

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 protein sequences in the AllergenOnline database using the 

FASTA search algorithm1 (Pearson and Lipman 1988). Additionally, the PMI sequence was 

also examined for matches of eight contiguous amino acids (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 contiguous 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 (Codex 2009)

demonstrated no cross-reactivity between PMI and the allergen α-parvalbumin, using serum 

from the single individual known to have demonstrated IgE-mediated allergy to this specific αparvalbumin. The allergic patient’s serum IgE did not recognize any portion of PMI as an 

allergenic 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 between 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 history 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 that 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

(Malvolti et al. 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. 2002) and have been demonstrated to be 

essential for many organisms, 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-FDA-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 tolerances in all plants by the EPA (40 CFR 

174.527) based on data demonstrating that PMI is nontoxic and that PMI is not likely to be a 

food allergen. 

VI.O. eCry3.1Ab and PMI Protein 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 senescence) 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 locations 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 grain or 

grain by-products. The average eCry3.1Ab concentration measured in kernels from 5307 maize 

(4.45 μg eCry3.1Ab/g dry weight [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 concentration 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 PMI concentration measured in kernels at maturity was 2.08 μg PMI/gdw (Table VI-4), 

representing approximately 0.002% of the total protein. (These calculations are based 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 

proteins, 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-FDA-1 Page 90 of 168

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.

μg/g DW μg/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 effects 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-FDA-1 Page 91 of 168

Table VI-5. Concentrations of PMI in 5307 corn tissues at four stages on a dry-weight (DW) 

and a fresh-weight (FW) basis.

μg/g DW μg/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 effects 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 Limit of 

Detection (LOD); 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-FDA-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 and the corresponding 

conventional control corn were harvested from six locations in the USA during 2008. 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 49-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. Historic 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 in 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. Roughly 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 products 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 processes. 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-FDA-1 Page 93 of 168

VII.C. Compositional Assessment of 5307 Forage and Grain

Compositional analyses of 5307 corn were performed to identify any changes in nutrient or antinutrient 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 undertaken 

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 metabolites. 

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 are 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 hybrids were harvested from six locations in the U.S.

during 2008: 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 according 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 components measured in this study were selected based on recommendations of the 

Organisation 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, analyte 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 subjected to analysis of variance using the following mixed 

model: 

Yijk = U + Ti + Lj + B(L)jk + LTij + eijk

In this model, Yijk is the observed response for genotype i at location j block k, U is the overall 

mean, Ti is the genotype effect, Lj is the location effect, B(L)jk is the effect of block within a 
5307-FDA-1 Page 94 of 168

location, LTij is the location-by-genotype interaction effect, and eijk is the residual error. 

Genotype was regarded as a fixed effect, while the effects of location, block within location, and 

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 Kenward-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 ILSI 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 (2008). The ILSI database is the most comprehensive and 

current source of crop composition data for most nutritional components. 
5307-FDA-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 B1 (thiamine) 20:1 eicosenoic

Moisture Selenium Isoleucine (Ile) Vitamin B2 (riboflavin) 22:0 behenic

Neutral detergent 

fiber (NDF) Sodium Leucine (Leu) Vitamin B3 (niacin) Anti-nutrients

Protein Zinc Lysine (Lys) Vitamin B6 (pyridoxine) Phytic acid

Proximates Methionine (Met) Vitamin B9 (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 (2008).

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.
5307-FDA-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 VII-2 and Table VII-3). 

When analyzing the results at each individual location, statistically significant 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 fell within the range of natural variation 

of corn reported in the ILSI database (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 B1, B2, B3 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, B6, and B9 (Table 

VII-6), as well as 16:0 palmitic, 18:0 stearic, 18:3 linolenic, and 20:1 eicosenoic acids (Table 

VII-8). However, 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 variation 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 the range of natural variation for corn reported in the ILSI database (ILSI 2008), with the 

exception of starch and Vitamin B2 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, forage and 

grain from 5307 corn hybrids are considered 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.
5307-FDA-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 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 Moisture Protein Fat Ash Carbohydrates 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

Na 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-FDA-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 Carbohydrates 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
5307-FDA-1 Page 99 of 168

Table VII-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
5307-FDA-1 Page 100 of 168

Table VII-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
5307-FDA-1 Page 101 of 168

Table VII-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 Carbohydrates 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 applicable a Grain was mechanically dried after harvest; moisture levels were not subject to ANOVA
5307-FDA-1 Page 102 of 168

Table VII-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 Carbohydrates 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
5307-FDA-1 Page 103 of 168

Table VII-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 Sea,c Nab,c Zn

Across Event 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 (2008) 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

Nd 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-FDA-1 Page 104 of 168

Table VII-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 Sea Naa 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 Event 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 Event 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
5307-FDA-1 Page 105 of 168

Table VII-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; the range of values among these replicates is provided. 

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

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-FDA-1 Page 106 of 168

Table VII-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-FDA-1 Page 107 of 168

Table VII-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
5307-FDA-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
5307-FDA-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
5307-FDA-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
5307-FDA-1 Page 111 of 168

Table VII-8. Fatty acid compositiona 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 

Palmitoleicb 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 performed. 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
5307-FDA-1 Page 112 of 168

Table 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 

Palmitoleica 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
5307-FDA-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 replicates is provided. 

Ferulic acid ρ-Coumaric 

acid Inositol Phytic acid Trypsin inhibitor Furfurala,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

Nc 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-FDA-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)

Furfurala

(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

5307-FDA-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 feeding 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 49-day feeding study was performed to evaluate 

whether standard poultry 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 each of the grain sources 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 49 consecutive days. 

Parameters evaluated in the study included survival, 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, nearisogenic 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 expressed on an absolute weight basis. A few 

statistically significant differences 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 consuming 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 parts including fat pads, drums, wings, and pectoralis major muscles.

Overall, diets 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 yield. 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 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, control 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 nutritionally comparable to and as safe as conventional corn.
APPENDIX A

5307-FDA-1 Page 117 of 168

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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 conclusions 

of these experiments are described in Part IV. 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 Biotechnology, 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 the descriptions of the studies in Part IV. 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 list 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 each 

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 and 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.
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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 into 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 buffer (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 

was 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 clean bottle, 
APPENDIX A

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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. DNA 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 TBS-380 MiniFluorometer.

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 × BC5F3 generation, as identified in the pedigree chart of plant materials 

(Figure IV-4). PCR amplification was carried out using the ExpandTM Long Template PCR 

System. Table A-3 lists the primers used to amplify the insert fragments; Tables A-4 and A5 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
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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. The plasmid DNA was then

independently extracted, and the resulting plasmid preparations, which contained the PCR 

amplification products, were subsequently sequenced.

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 generated 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.
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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, representing 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 μg 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 

calculated by the following formula with a corn genome size of 2.67 × 109 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 agarose gels, and the 

DNA fragments were then separated by electrophoresis in 1X Tris-acetate-EDTA 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 transferred 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-generated probes and the molecular weight marker-specific probe were labeled 

with phosphorus-32-deoxycytidine triphosphate ([α-32P]-dCTP) by random priming using 

the MegaprimeTM DNA labeling system. Unincorporated label ([α-32P]-dCTP) was removed 

using the Micro Bio-Spin® Chromatography Columns. 
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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 and 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 

The 5ʹ and 3ʹ 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

Table 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 extracted, 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).
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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 sequences 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 similar 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 10 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 and 

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 

accumulation 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 

sequence 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 Database 

(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 BLASTP 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:
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5307-FDA-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 matches 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 (Ponting 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 identified.

A.10. Amino Acid Sequence Comparison of Query Peptide to Known or Putative 

Allergens

The 81-amino-acid query peptide sequence representing the translated sequence of the 

putative 243-bp ORF was screened for biologically relevant amino acid sequence similarity 

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 allergens, 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 international 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 
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5307-FDA-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. The 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 translated 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 exceeding 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 sequence in the allergen database 

indicates the potential for immunologically relevant sequence similarity (Codex 2009).
APPENDIX B

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Appendix B: Materials, Methods and Results of Characterization 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 studies. 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 leaf 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 analysis 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 analyses. 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 collected 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 containing 100 mM sodium borate (pH 

10.0), 0.2% polyvinylpyrrolidone (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 Homogenizer (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 Buffer (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

5307-FDA-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 blender 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 through 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 the 5307 and control corn leaves were designated LP5307 and LPNEG, 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 immunoreactivity, 

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 prepared 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.

Extracts 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 was 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 buffer (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 cheesecloth and centrifuged at approximately 3100 rpm for 
APPENDIX B

5307-FDA-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 column 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 mM 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 stored 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 Nterminus. 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 

centrifugation. 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 purified 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. The 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 Beacon 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 analysis, 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

5307-FDA-1 Page 136 of 168

analyzed with BioMetallics DeltaSoft PC Microplate Analysis Software, v. 1.71.2, using a 

four-parameter fit of the standard curve. 

Immunoreactivity and Molecular Weight Determination

Western blot analysis was used to investigate the integrity of eCry3.1Ab in ECRY3.1AB0208, 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 loaded on the gel for LP5307 (29.6 μg total 

protein), was included in the analysis as a negative control. The molecular-weight standard 

was SeeBlue 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 

phosphatase 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 diet 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 treatments 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 of ECRY3.1AB-0208 was prepared in 10 mM 

ammonium bicarbonate buffer (pH 10.0) to a concentration of 5 mg eCry3.1Ab/ml. From 

this, a 50 μg eCry3.1Ab/ml solution was prepared and serially diluted 1:1 (v/v) in 10 mM 

ammonium bicarbonate buffer (pH 10.0) to produce eight solutions with concentrations 

ranging from 50 to 0.390 μg eCry3.1Ab/ml. The dilution series was then 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-FDA-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 

(treatment 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 potato 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 freshly 

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, aliquots 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) and from ECRY3.1AB0208 were analyzed by peptide mass mapping. Aliquots containing 2.5 to 5 μg 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 

polyacrylamide 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 

iodoacetamide, 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-FDA-1 Page 138 of 168

flight mass spectrometer (Waters/Micromass Q-TOF Premier) connected to a Waters CapLC 

capillary 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 

modifications, which have previously been reported to occur in plants. Specifically, the 

modifications investigated included α-N-acetylation, protein N-formylation and protein Nmethylation.

N-Terminal Amino Acid Sequence Analysis

To determine the N-terminal amino acid 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 corresponding 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 LC50 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 + 

ECRY3.1AB-0208, LP5307, and IAP5307 revealed immunoreactive bands consistent with 

the predicted molecular weight1of 74.8 kDa for samples containing eCry3.1Ab from 

ECRY3.1AB-0208 (Figure B-1, Lanes 2, 3 and 6) and 73.7 kDa for samples containing 

plant-produced eCry3.1Ab (Lanes 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 expected and 

observed mw on the gels can be explained by the limitations of SDS-PAGE for accurate determination of mw. Dube and 

Flynn (1988) have reviewed the reliability of SDS-PAGE for mw 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 the standard set (Sadeghi et al., 2003). Additionally, the intact mass of eCry3.1Ab in 

ECRY3.1AB-0208 waspreviously measured as 74.8 kDa.
APPENDIX B

5307-FDA-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 (10 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-terminal 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 Table 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 B2, 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-FDA-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-0208) 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 from the 

microbially produced ECRY3.1AB-0208 test substance (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 microbially produced nor the plant-produced eCry3.1Ab 

protein was glycosylated. 
APPENDIX B

5307-FDA-1 Page 141 of 168

Figure B-2. Glycosylation analysis of eCry3.1Ab in IAP5307 (immunopurified plant-produced 

protein) and ECRY3.1AB-0208 (microbially produced test substance).

Lane 1: Transferrin (positive control), 100 ng

Lane 2: Transferrin (positive control), 50 ng

Lane 3: Transferrin (positive control), 25 ng

Lane 4: Transferrin (positive control), 10 ng

Lane 5: Creatinase (negative control), 2 μg.

Lane 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-FDA-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 acid 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 analysis 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 IAP5307.

Identified eCry3.1Ab protein fragments are bold and underlined.

MTSNGRQCAGIRPYDGRQQHRGLDSSTTKDVIQKGISVVGDLLGVVGFPFGGALVSFYTNF

LNTIWPSEDPWKAFMEQVEALMDQKIADYAKNKALAELQGLQNNVEDYVSALSSWQKNPAA

PFRNPHSQGRIRELFSQAESHFRNSMPSFAISGYEVLFLTTYAQAANTHLFLLKDAQIYGE

EWGYEKEDIAEFYKRQLKLTQEYTDHCVKWYNVGLDKLRGSSYESWVNFNRYRREMTLTVL

DLIALFPLYDVRLYPKEVKTELTRDVLTDPIVGVNNLRGYGTTFSNIENYIRKPHLFDYLH

RIQFHTRFQPGYYGNDSFNYWSGNYVSTRPSIGSNDIITSPFYGNKSSEPVQNLEFNGEKV

YRAVANTNLAVWPSAVYSGVTKVEFSQYNDQTDEASTQTYDSKRNVGAVSWDSIDQLPPET

TDEPLEKGYSHQLNYVMCFLMQGSRGTIPVLTWTHKSVDFFNMIDSKKITQLPLTKSTNLG

SGTSVVKGPGFTGGDILRRTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGR

PINQGNFSATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVYIDRIEFV

PAEVTFEAEYDLERAQKAVNELFTSSNQIGLKTDVTDYHIDQV 

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 ECRY3.1AB0208.

Identified eCry3.1Ab protein fragments are bold and underlined.

MHHHHHHMTSNGRQCAGIRPYDGRQQHRGLDSSTTKDVIQKGISVVGDLLGVVGFPFGGAL

VSFYTNFLNTIWPSEDPWKAFMEQVEALMDQKIADYAKNKALAELQGLQNNVEDYVSALSS

WQKNPAAPFRNPHSQGRIRELFSQAESHFRNSMPSFAISGYEVLFLTTYAQAANTHLFLLK

DAQIYGEEWGYEKEDIAEFYKRQLKLTQEYTDHCVKWYNVGLDKLRGSSYESWVNFNRYRR

EMTLTVLDLIALFPLYDVRLYPKEVKTELTRDVLTDPIVGVNNLRGYGTTFSNIENYIRKP

HLFDYLHRIQFHTRFQPGYYGNDSFNYWSGNYVSTRPSIGSNDIITSPFYGNKSSEPVQNL

EFNGEKVYRAVANTNLAVWPSAVYSGVTKVEFSQYNDQTDEASTQTYDSKRNVGAVSWDSI

DQLPPETTDEPLEKGYSHQLNYVMCFLMQGSRGTIPVLTWTHKSVDFFNMIDSKKITQLPL

TKSTNLGSGTSVVKGPGFTGGDILRRTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQF

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 during translation (Walling 2006). Secondly, the nature 

of the N-terminal block found for the plant-produced protein, as described under NTerminal Amino Acid Sequence Analysis, below, was identified. The analysis of the Nterminal 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 (Martinez et al. 2008).

N-Terminal Amino Acid Sequence Analysis

The N-terminal sequence results confirmed that eCry3.1Ab in the microbially produced test 

substance ECRY3.1AB-0208 had the predicted N-terminal amino acid sequence:

Predicted sequence: MHHHHHHMTS

eCry3.1Ab 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 Nterminus. However, the N-terminal peptide was 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

5307-FDA-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 

functionally 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 transformation vectors 

encoded the same PMI enzyme. 

The conclusions of these analyses are described in Part VI.Iof this submission, 

Characterization of the 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 suspended 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 Leaf 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 preparation, 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 Extracts of 5307 and Control Corn Leaf Tissue). This sample was designated 

LP-NEG + PMI-0105.
APPENDIX B

5307-FDA-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 fortified with test substance PMI-0105 (LPNEG + 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 plates 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 plant 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 plates 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 plates were washed and subsequently incubated with 100 μl/well rabbit anti-mouse IgG 

conjugated 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 monohydrate, 

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 Determination

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 with 

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 (LPNEG + PMI-0105) and the microbially produced test substance PMI-0105. Aliquots 

equivalent to 5 ng PMI from sample LP5307, LPNEG + PMI-0105 and test substance PMI0105 diluted in 10X Sample Buffer (as described by Laemmli (1970) containing 8% 
APPENDIX B

5307-FDA-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 

(Invitrogen) 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 electroblotting, 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 by 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-phosphate 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. Microbially-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 salt 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

5307-FDA-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 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. Results are reported as the mean and standard deviation of 

triplicate enzymatic assays.
APPENDIX B

5307-FDA-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 PMI0105 revealed a dominant immunoreactive band consistent with the predicted molecular 

weight of PMI (Figure B-5, Lanes 2, 3, and 4). The 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 relatively

diminished intensity of the PMI bands in the presence of the plant matrix was most likely 

due to matrix effects. The matrix effect 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 comigration of the matrix with PMI or PMI fragments may have limited access of the antibody 

to epitopes of the target protein and therefore diminished the signal. 

The Western blot analysis also revealed some faint bands combined with a diffuse 

background signal in the molecular weight range above 62 kDa in both LP5307 and LPNEG + 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 protein and most likely represents a nonspecific 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 revealed a very faint protein band with a molecular weight of approximately 30 

kDa in the microbially produced test substance PMI-0105. Because this protein crossreacted with the anti-PMI antibody it is most likely a PMI degradation product. The 

degradation 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-FDA-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-produced test substance PMI-0105

Lane 5: Plant extract sample LP-NEG

Lane 6: Molecular weight standard SeeBlue Plus2
APPENDIX B

5307-FDA-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 PMI0105 (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, LPNEG (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 Activity1(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

5307-FDA-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 growth 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, the 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 weight 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 extracts were analyzed by ELISA (Tijssen 1985) to 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 for each ELISA plate. Nontransgenic 

plant tissue extracts were analyzed in parallel to evaluate any impact of the plant matrix on 

the ELISA. 
APPENDIX C

5307-FDA-1 Page 152 of 168

C.4. eCry3.1Ab Quantification – Extraction and ELISA Procedures

Buffers

The buffers used for extraction and ELISA 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 ELISA 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 the 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 

were 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

5307-FDA-1 Page 153 of 168

provided in the kit 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 

measured 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 detected, respectively. Extraction efficiencies ranged from 77% to 88% across the tissue 

types analyzed; the reported tissue concentrations have been adjusted for extraction 

efficiency. 
APPENDIX C

5307-FDA-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 OmniPrep 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. 

PMI Quantification

Rabbit polyclonal anti-PMI antibody was diluted in carbonate-bicarbonate buffer and added 

to each well of a 96-well microtiter 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 added 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-FDA-1 Page 155 of 168

tablet per 10 ml of citrate-phosphate buffer) and incubated 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 analyzed 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 efficiencies ranged from 72% to 80% across the tissue types 

analyzed; the reported tissue concentrations have been adjusted for extraction efficiency. 
APPENDIX D

5307-FDA-1 Page 156 of 168

Appendix D: Compositional Analysis of 5307 Forage and Grain

This appendix describes the methods used to conduct the compositional analysis study described 

in Part VII.Cof this submission, wherein the results are also provided. References cited for 

individual methods in this appendix are listed at the end of the appendix.

Study Design

Forage and grain for compositional 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 chipper-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 maturity (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 collected 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 

shipped on dry ice to the contract testing facililty. The samples were stored at –20°C ± 10°C 

until they were analyzed. 
APPENDIX D

5307-FDA-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 analytical 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 Standards for Compositional Analyses

2-Furaldehyde (Albala-Hurtado et al. 1997)

The ground sample was extracted with 4% trichloroacetic acid and injected directly on a highperformance 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 freshweight 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 positioned 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%, calculated on a freshweight 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

5307-FDA-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 hydroxide. 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 through 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.

Reference 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

Sigma, 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%, calculated 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 detection. 

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 spectrophotometrically), 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-FDA-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 evaporated 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. 

Fatty 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 heptane 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 Reference 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 JA16-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 approximately 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 turbidimetrically. 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-weight 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 of 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 inositol 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, carbohydrate, 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 niacin 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 with 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 accomplished 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%, calculated on a 

fresh-weight basis. 

Reference Standard: Aldrich, Phytic Acid, Dodecasodium Salt Hydrate, 95%, 

Lot Number 077K0693
APPENDIX D

5307-FDA-1 Page 162 of 168

Protein (AOAC 2005h; Bradstreet 1965; Kalthoff and Sandell 1948)

Nitrogenous compounds in the sample were reduced in the presence of 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 using 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 interferences. 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 freshweight basis. 

Reference Standard: USP, Pyridoxine hydrochloride, 99.8%, Lot Number Q0G409

Raffinose (Brobst 1972; Mason and Stover 1971)

The sample 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 hexamethyldisilazane 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 amyloglucosidase. 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 spectrophotometer 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 column. 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 calculated 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 the 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 

with 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 was 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~nitroanilide 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 measured 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-FDA-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 highperformance 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 Methods 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: Gaithersburg, 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 INTERNATIONAL, 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

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AOAC. 2005k. Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method 

982.30. AOAC International: Gaithersburg, Maryland. 

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 Practices 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. 

AOCS. 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 vitamins. Industrial and Engineering Chemistry, Analytical Edition 

15:141-144.

Bradstreet RB. 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

JN 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 determination 

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 range 

(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 chromatography analysis of phytic acid on a pHstable, 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 Agricultural 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

5307-FDA-1 Page 168 of 168

Quackenbush FW. 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 fluorometric 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. 2-11. 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 Tables 
Event 5307 Corn 

Environmental Report 

1 Appendix C 

Table C-1: Representative Conventional Insecticide 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 

hybrids 
Event 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 

hybrids 
Event 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 Johnson 
Event 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.