1 

# Pioneer Hi

- Bred International, Inc. High Oleic 305423 Soybean

OECD Unique Identifier: DP

- 3 Ø 5423

- 1



# Final Environmental Assessment

Ma y 2010

Agency Contact Cynthia Eck Document Control Officer Biotechnology Regulatory S ervices USDA, APHIS Riverdale, MD 20737

The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, sex, religion, age, disability, political beliefs, sexual orientation, or marital or family sta tus. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’S TARGET Center at (202) 720

– 2600 (voice a nd TDD).

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– W, Whitten Building, 1400 Independence Avenue, SW, Washington, DC 20250

– 9410 or call (202) 720

– 5964 (voice and TDD). USDA is an equal opportunity prov ider and employer.

________________________________ ______________________ Mention of companies or commercial products in this report does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned. USDA neither guarantees nor warrants the standard of any product mentioned. Product names are mentioned solely to report factually on available data and to provide specific information.

________________________________ ______________________ This publication reports research involving pesticides. All uses of pesticides must be registered by appropriate State and/or Feder al agencies before they can be recommended.

________________________________ ______________________2 

### Table of Contents

I. PURPOSE & NEED

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

II. INTRODUCTION

................................ ................................ ................................ .. 5

III. AFFECTED ENVIRO NMENT

................................ ................................ ............ 7

A. Soybean

................................ ................................ ................................ ................................ ............................. 7

B. Agricultural Produc tion of Soybean

................................ ................................ ................................ ............ 7

C. Soybean Composition

................................ ................................ ................................ ................................ ..... 9

IV. ALTERNATIVES

................................ ................................ ............................... 11

A . No Action: Continuation as a regulated article

................................ ................................ ....................... 11

B. Grant nonregulated status to Pioneer 305423 soybean, “in whole”

- Preferred Alternative: Determination that Pioneer 305423 soybean is no longer a regulated article.

................................ ......... 12

Alternatives Considered but Rejected from Further Consideration

................................ ......................... 12

V. ENVIRONMENTAL CON SEQUENCES

................................ ............................ 13

A. No Action

................................ ................................ ................................ ................................ ........................ 13 A. Soybean

................................ ................................ ................................ ................................ ....................... 13 B. Agricultural Production of S oybean

................................ ................................ ................................ .......... 14 C. Soybean Composition

................................ ................................ ................................ ................................ 15

B. Preferred Alternative

................................ ................................ ................................ ................................ .... 16 A. Soybean

................................ ................................ ................................ ................................ ....................... 16 B. Agricultural Production of Soybean

................................ ................................ ................................ .......... 17 C. Soybean Composition

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

Cumulative effects

................................ ................................ ................................ ................................ .............. 24

Potential impact on non

- target organisms, including beneficial organisms and threatened or endangered species

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

Consideration of Executive Orders, Standards and Treaties relating to environmental impacts

........ 28

VI. REFERENCES

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

VII. PREPARERS AND R EVIEWERS

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

VIII. APPEN DIX 1

- BIOTECHNOLOGY CONSUL TATION NOTE TO THE FILE BNF NO. 000110

................................ ................................ ............................. 403 I. PURPOSE & NEED

"Protecting American agriculture" is the basic charge of the U.S. Department of Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS). APHIS provides leadership in ensuring the health and care of plants and animals. The a gency improves agricultural productivity and competitiveness, and contributes to the national economy and the public health. USDA asserts that all methods of agricultural production (conventional, organic, or the use of genetically engineered varieties) ca n provide benefits to the environment, consumers, and farm income.

In 1986, the Federal Government’s Office of Science and Technology Policy (OSTP) published a policy document known as the Coordinated Framework for the Regulation of Biotechnology. This do cument specifies three Federal agencies that are responsible for regulating biotechnology in the U.S.: USDA’s APHIS, the U.S. Department of Health and Human Services’ Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). APHIS regulates genetically engineered (GE) organisms under the Plant Protection Act of 2000. FDA regulates GE organisms under the authority of the Federal Food, Drug, and Cosmetic Act. The FDA policy statement concerning regulation of products derived from n ew plant varieties, including those genetically engineered, was published in the Federal Register on May 29, 1992 (57 FR 22984

- 23005). Under this policy, FDA uses what is termed a consultation process to ensure that human food and animal feed safety issue s or other regulatory issues (e.g., labeling) are resolved prior to commercial distribution of bioengineered food. The EPA regulates plant

- incorporated protectants under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and certain biologica l control organisms under the Toxic Substances Control Act (TSCA). Together, these agencies ensure that the products of modern biotechnology are safe to grow, safe to eat, and safe for the environment. USDA, EPA, and FDA enforce agency

- specific regulation s to products of biotechnology that are based on the specific nature of each GE organism. Products are regulated according to their intended use and some products are regulated by more than one agency.

The APHIS Biotechnology Research Service’s (BRS) mission is to protect America’s agriculture and environment using a dynamic and science

- based regulatory framework that allows for the safe development and use of genetically engineered organisms. APHIS regulations at 7 Code of Federal Regulations (CFR) p art 340, which were promulgated pursuant to authority granted by the Plant Protection Act, as amended (7 United States Code (U.S.C.) 7701

– 7772), regulate the introduction (importation, interstate movement, or release into the environment) of certain GE org anisms and products. A GE organism is no longer subject to the regulatory requirements of 7 CFR part 340 when APHIS determines that it is unlikely to pose a plant pest risk. A GE 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 regulation (7 CFR 340.2) and is also considered a plant pest. A GE organism is also regulated under Part 340 when APHIS has reason to believe that the GE organism may be a plant pest or APHIS does not have sufficient information to determine if the GE organism is unlikely to pose a plant pest risk.4 A person may petition the agency to evaluate submitted data and determine that a particular regulated article is unlikely to pose a plant pest risk, and, therefore, should no longer be regulated under 7 CFR 340.6 entitled “Petition for d etermination of

n onregulated s tatus.” The petitioner is required to provide information under § 340.6(c)(4) related to p lant pest risk that the agency use s to determine whether the regulated article is unlikely to present a greater plant pest risk than the unmodified organism. A GE organism is no longer subject to the regulatory requirements of 7 CFR part 340 when APHIS de termines that it is unlikely to pose a plant pest risk.

Pioneer Hi

- Bred International, Inc. (Pioneer) of Johnston, IA submitted a petition to APHIS seeking a determination of non

- regulated status for their transgenic high oleic acid soybean event DP

- 3Ø5 423

- 1 (hereafter referred to as Pioneer 305423 s oybean) (Pavely

, 2007)

. According to Pioneer, their 305423 soybean is engineered to produce increased amounts of monounsaturated fatty acid ( o leic) and decreased amounts of polyunsaturated fatty acids (linol eic and linolenic) and to lesser extent, saturated fatty acid (palmitic acid) . This soybean is also engineered to express a new protein, a modified soybean acetolactate synthase. The modified soybean acetolactate synthase was used as a selectable marker for transformation. The Pioneer 305423 soybean is currently regulated under 7 CFR p art 340. Interstate movements and field trials of Pioneer 305423 soybean have been conducted under permits issued or notifications acknowledged by APHIS.

Under the autho rity of 7 CFR part 340, APHIS has the responsibility for the safe development and use of genetically engineered organisms under the provisions of the Plant Protection Act. APHIS must respond to petitioners that request a determination of the regulated sta tus of genetically engineered organisms, including genetically engineered crop plants such as Pioneer 305423 soybean

. If 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.

As a Federal agency subject to compliance with the National Environmental Policy Act (NEPA) (42 U.S.C. 4321 et seq. ), APHIS has prepared this environmental assessment (EA) to consider the potential environmental effects of this pr oposed action (granting nonregulated status) and the reasonable alternatives to that action consistent with NEPA regulations (40 CFR parts 1500

- 1508, 7 CFR 1b, and 7 CFR part 372 ) and the USDA and APHIS NEPA implementing regulations and procedures

. This E A has been prepared in order to specifically evaluate the effects on the quality of the human environment 1

The Pioneer 305423 soybean is designed for human and animal consumption and as such

, may also be subject to regulation by Food and Drug Administration (FDA)

. FDA policy uses what is termed a consultation process to ensure that human food and animal feed safety issues or other regulatory issues (e.g., labeling) are resolved prior to comm ercial distribution of biotechnology

- derived food. Pioneer submitted a summary of its safety that may result from the deregulation of Pioneer 305423 soybean

.

1 Under NEPA regulations, the “human environment” includes “the natural and physical environment and the relationship of people with that environment” (40 CFR §1508.14)5 and nutritional assessment to FDA for Pioneer 305423 soybean in 2007. Pioneer concluded that, with the exception of the intended change in fatty acid composition , the 305423 soybean and the foods and feeds derived from it are not materially different in composition, safety, or any other relevant parameter from soybeans now grown, marketed, and consumed. In January 2009, FDA considered Pioneer's consultation on th e 305423 soybean to be completed regarding the safety and nutritional assessment for Pioneer 305423 soybean and had no further questions regarding the safety of Pioneer 305423 soybean (FDA, 2009). T he text of the FDA’s scientific and regulatory

assessmen t response for Pioneer 305423 is available at the FDA website (FDA, 2009). Because Pioneer 305423 soybean does not contain any GE pesticides or the genetic machinery necessary to produce them, or tolerance to herbicides, EPA consultation is not required.

Public Involvement

APHIS

- BRS r outinely seeks public comment on draft environmental assessments prepared in response to petitions to deregulate GE organisms. APHIS

- BRS does this through a notice published in the Federal Register. This EA, the petition submitted by Pioneer, and APHIS’s plant pest risk assessment, were made available for public
comment for 120 days, from September 2, 2009 until December 28, 2009 . Comments that were received within the comment period were fully analyzed and used by APHI S to determine if the petition to deregulate the Pioneer 305423 soybean should be granted.

Decision to Be Made

APHIS will also use the information from this EA, and the comments received, to inform and to assist APHIS’ decisionmaker to determine whether to grant nonregulated status, or
to continue to regulate Pioneer 305423 soybean under the regulations at 7 CFR part 340

, or that an Environmental Impact Statement for Pioneer 305423 soybean is necessary prior to the decision of whether to grant nonregulat ed status to this soybean variety.



# I I . Introduction

Pioneer has developed a transgenic soybean line “Pioneer 305423 Soybean” that produces soybean seeds with increased levels of monounsaturated (oleic) fatty acid

, decreased levels of polyunsaturated fa tty acids (linoleic and linolenic) and decreased levels of palmitic acid ( Pavely

, 2007 )

. In addition, Pioneer 305423 soybean also contains

a slightly modified version of a soybean acetolactate synthase gene

. The expression of the modified version of a so ybean acetolactate synthase protein can increase the inherent tolerance level to the ALS

- inhibiting class of herbicides

. This trait is intended for selecting and identifying thi s high oleic soybean bioengineered event, rather than as a separate commercial trait as this version of the gene does not confer commercial levels of herbicide tolerance in Pioneer 305423 soybean

. Pioneer indicated there is no plan to commercially promote Pioneer 305423 soybean as tolerant to sulfonylurea herbicides ( Pavely

, 2007 )

.6 Genetic modification of the fatty acid composition of soybean oil has b een one of the major goals of many soybean breeders over the last 50 years. The level s of polyunsaturated fatty acid s are one of the major factors influencing the quality of vegetab le oils. Soybean oils rich in monounsaturated fatty acid s could provide improved commercial value. Pioneer identifies users of the modified oil as both food manufacturers and industrial product manufacturers, especially those who are presently using soyb ean oil products.

Unmodified s oybean oil has poor oxidative stability due to its unstable c hemical structure and naturally occurring levels of polyunsaturated fatty acids. Polyunsaturated fatty acids increase rancidity compared with saturated and monouns aturated fatty acids, especially after prolonged contact with oxygen, light or heat. This characteristic reduces product stability and shelf life. Hydrogenation is a chemical process that improves stability and shelf life necessary for food application ; h owever, hydrogenation has the undesirable
consequences of creating trans

- fatty acids

.

In recent years, trans

- fatty acid have come under considerable scrutiny because of the ir negative affect s on human health

. On July 9, 2003, the FDA issued a regulatio n requiring manufacturers to list trans

- fatty acids, or trans fat, on the Nutrition Facts panel of foods and some dietary supplements ( FDA

, 2003 )

. With this rule, consumers have more information to make food choices that could lower their consumption of t rans

- fat as part of a heart

- healthy diet.

USDA APHIS has previously granted nonregulated status to a high oleic soybean variety ( USDA

- APHIS

, 1997 ) developed by DuPont in which the high oleic phenotype was conferred by introduction of the soybean omega

- 6 desaturase gene 1 ( fad2

- 1 gene). Th o se DuPont high oleic soybean varieties received regulatory approval in Australia, Canada, Japan, New Zealand, and the United States.

Pioneer 305423 soybean has been field tested in the United States since 2005 as
aut horized by APHIS

. Associated notifications acknowledged and permits issued by APHIS are listed in Figure 23 ( Pavely

, 2007

, p

. 67 ). The list compiles a total of 13 test sites in diverse regions of the U.S. and Canada including the major soybean growing are a of the Midwest and winter nurseries in Hawaii. Field tests conducted under APHIS oversight allow for evaluation in agricultural settings under confinement measures designed to minimize the likelihood of persistence in the environment after completion of the field trial. Under confined field trial conditions, data are gathered on multiple parameters and used by applicants to evaluate agronomic characteristics and product performance. Th ese data are also valuable to APHIS as the agency assesses the poten tial for a new variety to pose a plant pest risk. The evaluated data may be found in the APHIS plant pest risk assessment (USDA

- APHIS

, 2009)

.7 

# III. Affected Environment

A

. S oybean The soybean ( Glycine max (L.) Merr.) is an economically important legumin ous crop, providing oil and protein. Soybean plants are grown for their seed, which is further processed to yield oil and meal. Soybean is ranked number one in oil production ( 56 %) among the major oil seed crops production in the world ( Soy Stats

, 2008 ). Other expanding uses for soybeans in the U.S. include s oy biodiesel, animal agriculture, exports, and edible soybean oil (USB

, 2007). Increased public focus on data supporting the human health benefits of soybeans could create more consumer demand, and will be examined further in the Environmental Consequences section of this EA.

The OECD Consensus Document (OECD

, 2000) provides detailed information about the crop biology of soybean. The genus Glycine is divided into two subgenera, g lycine and

s oja

. T he subgenus s oja consists of three annual species: G. soja Sieb. and Zucc., the wild form of soybean, G. gracilis Skvortz., the weedy form of soybean and G. max, which is the cultivated soybean. These species do not exist naturally in the United States ( USDA

- NRCS

, 2008 ). Soybean lacks sexually compatible wild relatives in the United States and its territories. Therefore, there is no potential for gene flow from cultivated soybean plants to wild soybean relatives in the U.S.

Transgenes in crops have th e potential to move within a population

. The potential for outcrossing can be defined as the ability of gene escape to other soybean fields. Soybean is a highly self

- pollinating species with a cross

- pollination rate of less than one percent in plants gro wn in close proximity ( OECD

, 2000; Caviness

, 1966). Cross

- pollination greater than 4.6 m from a pollen source has been rarely observed although it has been reported that insects can sometimes transfer the pollen that distance or more (Caviness

, 1996). Ev en if gene flow occurred, the nature of this trait (oil composition changes) would not confer a selective advantage. T he only known propagation method for soybean is through seed germination (i.e., there are no reports of vegetative propagation under fiel d conditions in the United States) . Mature soybean seeds have no innate dormancy, are sensitive to cold, and are not expected to survive in freezing winter conditions (Raper et al.

, 1987). Volunteer plants that might grow under certain environmental cond itions can be easily controlled mechanically or with herbicides (Zollinger

, 2005 ). Soybean is not weedy ( Holm et al.

, 1977) , is not found outside of cultivated areas, and does not compete well with other cultivated plants (Hymowitz et al.

, 1987).

B. Agri cultural Production of Soybean
To determine the areas of soybean production, APHIS used data from the National Agricultural Statistics Service (NASS) 2002 /2007 Census of Agriculture to determine where soybean is produced in the United States ( USDA

- NASS 20 09 )

. Only 17 states in 2008 produced more than 1 million acres among the 31 that produced soybean according to the 2002 Census of Agriculture. These states include IA, IL, MN, IN, MO, NE, OH, SD, AR, ND, KS, MI, MS, WI, NC, KY, TN (USDA

- NASS, 2008). In t h e U.S.

, soybean s were harvested on 72.1 million acres in 2008 (USDA

- NASS, 2008)

. T his8 harvest ed soybean acreage was only one percent below the record high acreage in 2006 (USDA

- NASS, 2008a)

. Pioneer states that this variety will “offer growers another va lue

- added specialty soybean option” and do not expect that soybean market dynamics will change with the availability of this product (Pavely

, 2007). Properties of soybean oil from this variety will be preferred over those commodity soybeans with less stab ility, and over the inferior flavor properties that currently characterize hydrogenated commodity oils (Pavely, 2007). Thus, products derived from Pioneer 305423 soybean will likely only replace commodity soybean products, and not create new demand. APHIS concludes that i t is
not likely that “new

, ” previously uncultivated land will be brought into soybean production beyond that currently or historically used for such production

. Processed soybeans are the largest source of protein feed and the second la rgest source of vegetable oil in the world. Soybeans represent about 90 percent of U.S. total oilseed production, while other oilseeds

— such as cottonseed, sunflower seed, canola, and peanuts

— account for the remainder (USDA

- ERS

, 2008).

Agricultural p roduc tion of g enetically engineered herbicide tolerant soybean Based on USDA survey data, adoption of genetically engineered herbicide

- tolerant (HT) soybeans increased from 17 percent of U.S. soybean acreage in 1997 to 68 percent in 2001 and 92 percent in 2008 (Fernandez

- Cornejo

, 2008)

. Use of herbicide

- resistant crops has been a major development in agriculture over the past 20 years

. Weed control had been one of the biggest challenges for soybean growers. I nfestation with weeds during an entire growing sea son have result ed in soybean yield losses ranging from 12 to 80% (Barrentine

, 1989). By the early 1990’s, Gianessi et al. (2002) reported over 70 individual herbicides or combination products registered for weed control in soybean

. Along with the increa sed use of herbicides, biotypes of various plant species developed resistance to certain herbicide modes of action (Heap

, 200 7 ). With the 1996 commercial introduction of glyphosate tolerant soybean, a major shift occurred with an increased use of glyphosa te accompanying the increased planting of glyphosate tolerant soybean (92% of all soybean planted in the United States in 2008 ) and a decrease in use of other soybean herbicides(Gianessi et al.

, 2002). According to USDA’s Agricultural and Resource Managem ent Surveys (ARMS) in 2001

- 03, growers who used glyphosate

tolerant soybean technology cited the simplicity in weed control as the most common reason for growing HT soybean varieties. The popularity of glyphosate

- tolerant soybean is due to advantages of t he technology over conventional weed control practice.

Agricultural p roduction of conventional soybean and genetically

- engineered

, non herbicide tolerant soybean According to the report
, Adoption of Genetically Engineered C rops in the U.S.

,

“ approximatel y 8 % of total soybean acres in 200 8 were planted with non

- herbicide tolerant varieties (Fernandez

- Cornejo

, 2008)

. ” A portion of this area (about 0.1 3 % of U.S. soybean production in 2008 ) was devoted to the production of organic soybean (USDA

- NASS

, 2010 ).9 Weed control is one of the biggest challenges for all soybean farmers. United States soybean farmers began switching from the use of tillage for weedcontrol to herbicides in the late1950s. Herbicides were estimated to be used on more than 97 percent of the total soybean acreage in 1997 in the U.S. (Fernandez

- Cornejo et al.

, 2002). As a result,

s oybean is a major market for pesticides in general and for herbicides in particular.

For Pioneer 305423 soybean (a non

- herbicide tolerant soybean ) , there are no anticipated changes in pesticide use for weed management compared to conventional soybean varieties

. The potential herbicide use in growing conventional soybean or Pioneer 305423 soybean should be very similar. Currently, there are at least 70 registr ations for herbicides for weed management in soybean, plus numerous mixtures (Crop Protection Reference

, 2009).

Organic soybean production The production of organic soybeans represents about 0.1 3 % of U.S. soybean production (USDA

- NASS

, 20 10 ). In 200 8

, 98 ,199 acres of certified or exempt organic soybean were harvested in United States. Under the USDA National Organic Program guideline

, the use of synthetic pesticides, fertilizers, and genetically engineered crops is strictly limited. Pioneer 305423 soybe an is not approved for use in organic systems because it is genetically engineered.

Maintaining the integrity of the organic production process is important to producers of organic soybeans. There are many practices organic producers use to prevent movem ent of GE soybean or the transfer of pollen from GE soybean into their organic production fields (Organic Farming Research Foundation (2002)

. G rowers may choose to plant only organic seed ; plant earlier or later than neighboring farmers who may be using G E crops

, ensuring that the flowering times between GE and organically produced crops will differ

, thus min im izing the change of pollen movement between fields ; and also employ adequate isolation distances between the organic field and the fields of neighbo rs to minimize the chance that pollen will be carried between the fields. Additionally, o rganic growers must maintain records to show that production and handling procedures comply with USDA organic standards.

C . Soybean C omposition Generally, soybean see d consists of oil (about 20%), protein (about 40%), carbohydrate (about 35%) and ash (about 5%) (Liu

, 1997). Soybean oil is currently the predominant plant oil produced in the world

, and is used in a wide variety of food applications (FAO, 2009 ; Lusas, 20 04 )

. After the oil is extracted the remaining solid materials in the form of flakes are toasted and ground to produce soybean meal.

Oil For total world vegetable oil production

, soybean is ranked number one among the major oilseed crops, and in the US s upplies 71% of all fats and oils consumed ( Soy Stats

, 2008 )

. Conventional s oybean oil is composed of a mixture of several fatty acids

. The major unsaturated fatty acids in conventional soybean oil are 7% linolenic acid; 51% linoleic acid ; and 23% oleic a cid. It also contains saturated fatty acids 4% ste a ric a ci d and 10%10 palmitic acid ( Codex standard for edible fats and oil

, 1996 )

. Soybean oil has a relatively high proportion of oxidation prone linolenic acid, which reduces product stability and shelf lif e and which is an undesirable property for the food industry. The hydrogenation process (chemical addition of hydrogen) is used to enhance the oil’s stability by reducing its polyunsaturated fatty acid content. But this process has undesirable consequenc es including the formation of trans fatty acid isomers and a characteristic “hydrogenated flavor” (Fernandez

, 1995). Partially hydrogenated oils are used by the food industry because they extend product shelf life and have a desirable taste and texture ( T he Pennsylvania State University

, 2006 ). Although use of soybean oil in food is a large part of the market for soybean products, soy oil is also used for industrial products as Pioneer notes, and modified oils may be used for biodegradable products such a s certain types of lubricants.

Meal Soybean meal

, which contains about 50% protein by dry weight

, remain s the primary product from soybean, and of this more than 95% of domestic soybean meal is consumed by livestock (ABG, 2007)

. Only a small proportion of the soybean crop is consumed directly by humans. Soybeans are considered to be a source of complete protein. A complete protein is one that contains significant amounts of all the essential amino acids that must be provided to the human body because of the body’s inability to synthesize them. The ten essential amino acids are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (Kuiken et al

. , 194 9 )

. Cystine is also an important amino acid as i t can partially substitute for methionine. Protein Digestibility Corrected Amino Acids Score (PDCAAS) is considered the gold standard for measuring protein quality for human s since 1990. The PDCAAS rating has been adopted by the FDA and FAO/WHO as the pr eferred method to determine protein quality (FAO/WHO

, 1990)

. By this criterion

, soybean protein was reported to be equivalent to animal protein with the highest rating of 1.0 ( Hasler

, 2002). Humans can produce 10 of the 20 amino acids. Soybean contains t he other ten essential amino acids (Kuiken, et al

. , 1949) that are necessary for human nutrition and are not produced naturally in the body. The essential amino acid composition of soybean is included in the USDA N utrition database ( USDA

- ARS

, 2006)

. Isof lavones Soybeans naturally contain isoflavone compounds which are reported to possess biochemical activity

, including estrogenic and hypocholesterolemic effects. The major is o flavones in soybeans include genistein and daidzein

. Isoflavones are polyphenol compounds, produced primarily by beans and other legumes, including peanuts and chickpeas

. Is oflavones are closely related to the antioxidant flavonoids found in other plants, vegetables and flowers (Manach et al., 2004)

.

Antinutrients Soybean grain contains several key anti nutrients, such as oligosaccharides, lectins, phytic acid and protease inhibitors (OECD

, 2001)

. The trypsin and chymotrypsin inhibitors can inhibit digestion of proteins (OECD, 2001)

. Others such as lectins are sugar

- binding proteins , preventing digestio n of proteins resulting in decreased animal growth (OECD,11 2001)

. The activity of these inhibitors is destroyed during the heat treatment processing of the soybean product s

. The low molecular weight carbohydrates (e.g. stachyose, raffinose) can cause flat ulence when consumed (Rackis

, 1974). Phytic acid binds most of the phosphorus in soybean and can chelate various minerals as well (OECD, 2001)

. Livestock producers common ly add phy tic acid degrading enzymes to animal feed formula s ( V ats and Ban erjee, 200 4 )

.



# IV. Alternatives

This EA analyzes the potential environmental consequences of a proposal to grant nonregulated status to Pioneer 305423 soybean. In order for Pioneer 305423 soybean to be granted nonregulated status, APHIS must determine that Pion eer 305423 soybean is unlikely to pose a plant pest risk. The analysis by APHIS in its plant pest risk assessment (USDA

- APHIS, 2009) demonstrates that there was sufficient data to determine that Pioneer 305423 soybean is unlikely to pose a plant pest risk and therefore is eligible for nonregulated status.

The regulations at 7 CFR 340.6(d)(3)(i) state that APHIS may "approve the petition in whole or in part." Because APHIS has found that Pioneer 305423 soybean is unlikely to pose a plant pest risk, the o nly action alternative considered in this EA is to grant nonregulated status “in whole” to Pioneer 305423 soybean. An “ in part ” deregulation can be given if there is a plant pest risk associated with some, but not all lines requested in a petition. The p etition for Pioneer 305423 soybean only requested APHIS to grant nonregulated status to one soybean event, therefore, an “in part” determination is not an appropriate consideration. Thus, only two alternatives will be considered in this EA: (1) no action and (2) to grant nonregulated status to Pioneer 305423 soybean, “in whole.”

APHIS has assessed the potential for environmental impacts for each alternative in the

“

Environmental Consequences ” sections below.

A

. No Ac tion: Continuation as a regulated ar ticle

Under the "no action" alternative, APHIS would deny the petition. Pioneer 305423 soybeans and progeny derived from them would continue to be regulated articles under the regulations at 7 CFR part 340. Permits issued or notifications acknowledged b y APHIS would still be required for introductions of Pioneer 305423 soybeans and measures to ensure physical and reproductive confinement would continue to be implemented. APHIS might choose this alternative if there were insufficient evidence to demonstr ate the lack of plant pest risk from the unconfined cultivation of Pioneer 305423 soybeans.

Soybean breeders have achieved soybean oil compositional changes by both conventional breeding and genetic engineering (Fehr 2007). Under this no action alternat ive, growers and other parties who are involved in production, handling, processing or consumption of soybean would continue to have access to existing deregulated GE high oleic acid12 soybean products as well as conventional high or mid level oleic soybean varieties. However, growers would not have widespread access to soybean varieties based on Pioneer 305423 soybean since it would continue to be regulated under Part 340. There is no potential for human consumption of Pioneer 305423 soybean high oleic aci d soybean under this alternative.

This alternative is not the preferred alternative because APHIS has already determined through a plant pest risk assessment (USDA

- APHIS, 2009) that Pioneer 305423 soybean is unlikely to pose a plant pest risk. Choosing this alternative would hinder the purpose and need of APHIS to allow for the safe development and use of GE organisms given that Pioneer 305423 soybean is unlikely to pose a plant pest risk.

B. Grant nonregulated status to Pioneer 305423 soybean, “in whol e”

- Preferred Alternative: Determination that Pioneer 305423 soybean is no longer a regulated article

.
Under this alternative, Pioneer 305423 soybeans and progeny derived from them would no longer be regulated articles under the regulations at 7 CFR part 340. Pioneer 305423 soybean is eligible for nonregulated status because APHIS has determined that this GE organism is unlikely to pose a plant pest risk (USDA

- APHIS, 2009). Permits issued or notifications acknowledged by APHIS would no longer be required for introductions of high oleic acid soybeans derived from this event. APHIS might choose this alternative if there was sufficient evidence to demonstrate the lack of plant pest risk from the unconfined cultivation of high oleic acid soybeans derived fro m this event

. Under this alternative, growers may have future access to Pioneer 305423 soybean and progeny derived from this variety if the developer decides to commercialize Pioneer 305423. In addition, growers and other parties that are involved in pr oduction, handling, processing or consumption of soybean would continue to be able to use the current high or mid level oleic soybean products by conventional breeding as well as the genetically engineered soybean variety. Consumers may benefit by having access to a greater range of potentially healthful food products. By granting nonregulated status to Pioneer 305423 soybean , the purpose and need to allow the safe development and use of GE organisms is met.

APHIS has chosen Alternative B as the prefer red alternative for the proposed action because APHIS has determined that Pioneer 305423 soybean is unlikely to pose a plant pest risk ( USDA

- APHIS, 2009 ).



### Alternatives Considered but Rejected from Further Consideration

Geographic restrictions APHIS con sidered geographic restrictions based upon geographic variation in plant pest risk. As presented in APHIS plant pest risk assessment for Pioneer 305423 soybean

, there is no geographic differences in the plant pest risks for Pioneer 305423 soybean13 (USDA

- AP HIS 2009). This alternative was rejected and not analyzed in detail because Pioneer 305423 soybean is unlikely to pose a plant pest risk and therefore, APHIS will have no regulatory authority over Pioneer 305423 soybean and will be unable to impose regula tory restrictions on this GE soybean variety.


# V. Environmental Consequences

According to APHIS regulations at 7 CFR p art 340, an organism is no longer subject to regulatory requirements when it is demonstrated not to present a plant pest risk ( USDA

APHIS

, 2009 ). Under the regulations, APHIS is required to render a determination on a petition for nonregulated status. The a nalysis of p otential e nvironmental c onsequences in the following sections address the potential impact s to the human environment from the alternatives analyzed in this EA, namely taking no action and g rant ing nonregulated status to Pioneer 305423 soybean, “in whole”

.

S COPE OF THE E NVIRONMENTAL A NALYSIS Although the preferred alternative would allow for plantings of Pioneer 305423 soybea n to occur anywhere in the U.S., APHIS limited the environmental analysis to those areas that currently support soybean production. To determine areas of soybean production, APHIS used data from the National Agricultural Statistics Service (NASS) 2002 Cen sus of Agriculture to determine where soybean is produced in the United States ( USDA

NASS, 2009 ). Only 17 states in 2008 produced more than 1 million acres among the 31 that produced soybean according to the 2002 Census of Agriculture. These states inclu de IA, IL, MN, IN, MO, NE, OH, SD, AR, ND, KS, MI, MS, WI, NC, KY, TN (USDA

NASS, 2008). Because the oil derived from this Pioneer 305423 soybean will likely replace that from existing soybean varieties, it is not likely that new land will be planted
to s oybean by growers. The most recently harvested area of soybean production (72.1 million acres) is already only one percent below the record high acreage of 2006 (USDA

NASS, 2008a).



### A. No Action
Under the “no action” alternative, Pioneer 305423 soybean wou ld continue to be a regulated article

. APHIS’ assessment of environmental consequences under the no action alternative is described below.


### A. Soybean Under the ‘no action’ alternative, flowering and reproduction in soybean production fields will remain unchanged. DuPont’s GE high oleic soybean variet y remains eligible for commercial production and Pioneer 305423 will remain a regulated article.

The food/feed nutritional and safety assessment for Pioneer 305423 soybean has been reviewed by FDA. Under F ederal Food, Drug, and Cosmetic Act (FFDCA), 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 Pioneer 305423 soybean must be14 in compliance with all ap plicable legal and regulatory requirements. In January 2009, FDA completed the safety and nutritional assessment for this product and had no further questions regarding the safety of Pioneer 305423 soybean (FDA, 2009).

APHIS assessment of the safety of this product focuses on its potential to pose a plant pest risk, and that analysis, is based on the comparison of the GE

- soybean to its non

- GE counterpart (USDA

- APHIS, 2009). B ased on the assessment of the laboratory evidence provided by Pioneer (Pavely, 2007) and accompanying scientific literature (Reference section), APHIS has concluded that Pioneer 305423 soybean would have no significant impacts on human or animal health



### B. Agricultural Production of Soybean Most of the soybean acreage in the U.S. is planted to GE varieties. Of the total soybean acres planted in 2008, 92% were GE glyphosate tolerant soybean varieties (USDA

- NASS

, 2008). In 200 8 , t he production of organic soybeans represented about 0.1 3 % of U.S. soybean production ( USDA

- NASS , 2010 ). C onventional production practices that use GE varieties (such as herbicide tolerant varieties and new GE traits planned by four companies ) will likely still dominate in terms of acreage, or perhaps increase in acreage ( Bonny, 2009)

, even without granting no nregulated status to Pioneer 305423 soybean under the “no action” alternative. A vailable seed for both GE and conventional varieties will remain the same under the “no action” alternative

. Pioneer 305423 soybean variety will remain unavailable for commer cial production

. Soybean is currently produced in more than 30 states according to the 200 8 State Soy C rop Statistics ( USDA

- NASS

, 2008 ) , and under the “no action” alternative, this area for production will likely be unchanged.

Weed control is one of th e biggest challenges for conventional soybean farmers because poorly controlled weeds drastically decrease crop yield and quality. Full season infestation of weeds can result in soybean yield losses ranging from 12 to 80% (Barrentine

, 1989). Weeds growin g with soybeans compete with the crop for light, moisture, and nutrients. Uncontrolled weeds reduce soybean yields and interfere with harvest. Soybeans are very competitive with weeds once they develop a canopy, but early emerging weeds can cause serious problems. Thus, early

- season weed control is the key to providing the soybeans with a competitive advantage and minimizing the effect of weeds. Crop rotation is one of the most effective ways to manage certain weed problems specific to a crop. Herbicid es, when used properly, are a safe and effective method to control certain weeds in soybeans.

Under the “no action” alternative, herbicides will still be used alone or in combination and selected on the basis of their effectiveness on the different we ed species in the soybean field. Different herbicides have different modes of action; the correct herbicide rate must be used for each in order to obtain good weed control results and to minimize soybean plant injury

. APHIS has no authority under the Pla nt Protection Act to regulate herbicide use. The use of herbicides is regulated by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) restrictions administered by the EPA, which mandate registration and use of all pesticides. EPA includes ins tructions and restrictions15 on how herbicides can be applied, and has determined that there is no unreasonable environmental risk if the user adheres to the directions. Directions include application restrictions that minimize impacts on nearby environment s. Violators of the regulations are liable for all negative consequences of their actions; therefore, farmers who use herbicides are very likely to follow its label restrictions, and thereby limiting any potential adverse impacts

. The large scale commerc ial cultivation of GE glyphosate tolerant soybean crop acreage has steadily increased from 1996, accounting for 92 % of soybean acreage in 2008. The primary reason that farmers have switched to glyphosate

- tolerant soybean is the simplicity of the weed man agement programs. Glyphosate is a highly effective, nonselective, broad

- spectrum herbicide and in general, considered “environmentally friendly” when compared to other herbicides ( Cerdeira and Duke, 2006)

. U nder the ‘no action’ alternative , APHIS expects no change to the availability or use of GE glyphosate tolerant soybean vari e ties

. These GE varieties will remain available for commercial use. Thus, it is likely that under the ‘no action’ alternative, greater than 90% of the soybean acres produced in t he U.S. will continue to be GE varieties and production of organic varieties will remain unchanged (Bonny, 2009)

.

If APHIS choose s the no action alternative there would be no direct impact on organic or other non

- transgenic soybean farmers. The current cultivation practices are unlikely to change and 92% of the soybeans produced would likely be planted with the current herbicide tolerant bi otech soybean varieties.



### C. Soybean Composition

Soybean production for modified fatty acid composition The g enet ic changes in soybeans that resulted in soybean oil composition changes have been achieved by both conventional and genetic engineering techniques . Mutagenesis was used to develop the major genes for reduced palmitic and linolenic acids that are in the cu ltivars currently grown for commercial production (Fehr

, 2007). Conventional soybean breeders have obtained varieties with greater than 70% oleic acid by intercrossing (Alt et al.

, 2005). As described in this EA, DuPont (USDA

- APHIS

, 1997) has used geneti c engineering techniques to increase oleic acid composition in its commercially available soybean to near 80%. Therefore, under the ‘no action’ alternative, soybean varieties that have modified oil content, either through conventional breeding techniques or through genetic engineering, will still be available and on the market. Additionally, other seed high in oleic oil (e.g. GE sunflower oil) will also remain in commerce.

A variety of soybean oil choices are presented to food manufacturers, particular ly in response to the requirement by FDA that those oils with trans

- fats be labeled. These choices may be altered linolenic acid varieties or interesterified soybean oils (Eckel et al., 2007). In response to any soybean oils with altered fatty acid ratio s that are used in food products, substantial efforts in reformulating the entire product may be needed (Eckel et al., 2007). Although these specialty oils are marketed exclusively as identity preserved16 (see Summary and Section I.B, Taveley, 2007) and are not likely to be found in commodity soybean oils, food manufacturers need to be especially attentive to altered fatty acid ratios that may be present in commodity oils. Pioneer proposes to use current industry standard mechanisms to maintain product ident ity, from seed production to delivery to customers after oil refining ( Pioneer Public Comment APHIS Docket 2007

- 0156

- 0045 ; Regulations.gov (2009). These methods include sales of seed for planting to growers under contracts to deliver produced specialty oi lseed at defined locations, and also to meet standards for oil quality. Other agreements by oilseed crushers and refiners could specify precisely the acceptable range of fatty acids that would meet established standards for the commodity oil. The Biotechn ology Industry Organization in cooperation with its member companies is also developing stewardship standards for any specialty oilseed products that may differ substantially from commodity oils (Jeff Barach, Grocery Manufacturers Association, personal

com munication, 2009).

High oleic oils are found to be useful for industrial products, such as biodegradable lubricants (National Ag

- Based Lubricants Center, 2008; Machinery Lubrication (2010). Such products are used in food processing applications or farm tractor transmissions. High oleic oils are used in the industrial sector for base oils that are produced for hydraulic or gear oils (National Ag

- Based Lubricants Center, 2008). Because properties of oils are dependent upon properties of constituent fatty acids, the specific content deriving from differing soybean varieties is of interest to these manufacturers. Again, specialty soy oils (See Section I.B., Taveley, 2007) are marketed as identity preserved products, and are not likely to be found in commod ity soybean oils.



### B. Prefe r red Alternative

Under this alternative, Pioneer 305423 soybean would no longer be a regulated article under 7 CFR part 340. Permits issued and/or notifications acknowledged by APHIS would no longer be required for introduction s of Pioneer 305423 soybean. APHIS has chosen the preferred alternative for the proposed action because the Pioneer 305423 soybean lacks plant pest characteristics , as determined in APHIS’ Plant Pest Risk Assessment (USDA

- APHIS

, 2009)

. APHIS’ assessment of environmental consequences under the preferred alternative is described below.


### A. Soybean Under this alternative, Pioneer 305423 soybean would be available to growers. A potential environmental impact to consider as a result of planting this soybean v ariety is the potential for gene flow to sexually compatible plant species

. Based on the plant pest risk assessment (USDA

- APHIS, 2009) , APHIS has determined that Pioneer 305423 soybean is not a plant pest and that gene flow between this product and weedy and wild relatives will not occur in the United States. W eedy and wild relative species do not exist naturally in the United States (USDA

- NRCS, 2008). Therefore, s oybean lacks sexually compatible wild relatives in the United States and its territories. APHIS does note that gene flow can take place between a field planted with Pioneer 305423 soybean and a neighboring soybean crop. The potential for outcrossing can be defined as the ability of17 gene escape to other soybean fields. Soybean is a highly sel f

- pollinating species with a

cross

- pollination rate of less than one percent in plants grown in close proximity (OECD, 2000; Caviness, 1966). Cross

- pollination greater than 4.6 m from a pollen source has been rarely observed although it has been reported that insects can sometimes transfer the pollen that distance or more (Caviness, 1996). Although the biology of the soybean crop limits the amount of gene flow that may occur between two soybean fields

, soybean farmers may implement certain management prac tices to minimize gene flow between production fields (e.g. , plant at different dates than neighboring soybean, to encourage differing flowering times, establish isolation distance s to avoid mechanical mixing and short

- distance pollen flow )

.

The food/fee d nutritional and safety assessment for Pioneer 3 05423 soybean has been reviewed by FDA. Under Federal Food, Drug, and Cosmetic Act ( FFDCA ) , it is the responsibility of food and feed manufacturers to ensure that the products they market are safe and prope rly labeled. Food and feed derived from Pioneer 305423 soybean must be in compliance with all applicable legal and regulatory requirements

. In January 2009, FDA completed the safety and nutritional assessment for this product and had no further questions regarding the safety of Pioneer 305423 soybean (FDA

, 2009).

APHIS assessment of the safety of th is product focuses on its potential to pose a plant pest risk, and that analysis, is based on the comparison of the GE

- soybean to its non

- GE counterpart (US DA

- APHIS

, 2009). B ased on the as sessment of the laboratory evidence provided by Pioneer (Pavely

, 2007) and accompanying scientific literature (Reference section), APHIS has concluded that Pioneer 305423 soybean would have no significant impacts on human o r animal health with overall impacts similar to the no action alternative.



### B. Agricultural Production of Soybean In 2008, GE soybean wa s planted on 92 % of all soybean acres in the US, and the use of GE soybean has been steadily increasing over the last 3 years (USDA

- NASS 2006, 2007, 2008). Conventional and GE soybean production occurs on land that is dedicated to crop production. Most soybean is planted in agricultural fields that have been in crop production for multiple years.

Pioneer 305423 soybea n has been field tested under permits issued or notifications acknowledged by APHIS in the United States since 2002. F ield test sites included the major soybean growing areas of the Midwest and the winter nursery areas in Hawaii. Agronomic and phenotypic data were collected by the applicant to assess agronomic comparability to conventional soybean

. To evaluate the agronomic and phenotypic characteristics of Pioneer 305423 soybean, the data were collected to address specific characteristics that influence reproductive and survival biology. Base d on these data, APHIS agronomists evaluated the potential for weediness as compared to conventional soybean. The evaluation concluded that t here were no biological ly significant differences in weediness potential between Pioneer 305423 soybean and other soybean varieties (see Section VIII

- A

- D and X

- C in Pavely, 2007)

. No differences were found in dormancy or germination potential, in responses to diseases or insects, seedling vigor, plant maturity,18 plant height, a nd the lowered linolenic acid content would be correlated with reduced cold tolerance. These results show that the agronomic and phenotypic characteristics of Pioneer 305423 soybean were either not altered when compared to nontransgenic (conve n tional) soy bean or were characteristic of less fit ness (cold tolerance potential)

. Therefore, g ranting nonregulated status to Pioneer 305423 soybean under the “preferred” alternative is not expected to substa ntial ly alter the range of soybean cultivation as the

new GE trait ( h igh o leic a cid ) does not change the production needs or growth habits of GE soybean varieties compared to conventional varieties (USDA

- APHIS

, 200 9 ).

Pioneer 305423 soybean oil will be marketed as “ TREUS TM 2 ” high oleic soybean oil

. This soybean variety will likely be introduced to areas where soybean is currently grown for oil production as a n alternative to other choices ( such as conventional and GE varieties producing altered fatty acids ) already available in the market (see Summary, Pavely (2 007))

. Therefore, the current distribution of soybean production acreage will likely remain unchanged as a result of APHIS granting nonregulated status to Pioneer 305423 soybean.

Pioneer 305423 soybean was genetically engineered to express a modified so yb ean acetolactate synthase. However, t his modified soybean acetolactate synthase was used as a selectable marker for transformation and does not confer commercial levels of herbicide tolerance in this event. Also, Pioneer 305423 soybean is not genetical ly engineered for tolerance to glyphosate. Because of the herbicide usage similarity of Pioneer 305423 soybean to conventional soybean, there is no expected change in herbicide use with Pioneer 305423 soybean under the preferred alternative

. Therefore, po tential impacts from the use of EPA registered herbicides are the same as those from the no action alternative.

Production of mid and high oleic soybean In 2003, Dupo n t Protein T echnologies provided grower contracts for producti o n of G

- 94

- 1, G

- 94

- 19 or G

- 168 GE high oleic soybean. Production over several thousand acres through 2005 revealed no environmental or agronomic issues

. Beginning in 2007, growers in Iowa planted mid

- oleic, low linolenic producing varieties (Stockhausen, 2008). These varieti es, a vailable through the Iowa State University Research Foundation, such as IA3036 and IA3039 have oleic acid content of between 49 and 53% (Iowa State University, 2010) . These lines were grown und er contracts with commercial oilseed crushers (J. J.G. Minot, personal communication, IA State Univ. Res. Fndtn). No agronomic issues were identified, nor impacts on agricultur al production noted.

O rganic and Conventional Soybean Production Organic farming operations as described by the National Organic Program , wh ich is administered by USDA’s Agricultural Marketing Service

, requires organic production operations to have distinct, defined boundaries and buffer zones to prevent unintended contact with excluded methods from adjoining land that is not under organic

ma nagement. Organic production operations must also develop and maintain an organic production system plan approved by their accredited certifying agent. This plan enables

2 TREUS TM is a trademark of Pioneer Hi

- Br ed International, Inc.19 the production operation to achieve and document compliance with the National Organi c Standards, including the prohibition on the use of excluded methods. Excluded methods include a variety of methods used to genetically modify organisms or influence their growth and development by means that are not possible under natural conditions or
processes.
Organic certification involves oversight by an accredited certifying agent of the materials and practices used to produce or handle an organic agricultural product. This oversight includes an annual review of the certified operation’s organic system plan and on

- site inspections of the certified operation and its records. Although the National Organic Standards prohibit the use of excluded methods, they do not require testing of inputs or products for the presence of excluded methods. The pre sence of a detectable residue of a product of excluded methods alone does not necessarily constitute a violation of the National Organic Standards (USDA

- AMS

, 2007). The unintentional presence of the products of excluded methods will not affect the status of an organic product or operation when the operation has not used excluded methods and has taken reasonable steps (such as isolation zones, use of buffer rows surrounding the organic crops or adjusting planting dates and appropriate cleaning of planting a nd harvesting equipment) to avoid contact with the products of excluded methods as detailed in their approved organic system plan. Organic certification of a production or handling operation is a process claim, not a product claim.

It is not likely that f armers , including organic and conventional farmers, who choose not to plant transgenic soybean varieties or sell transgenic soybeans varieties, will be significantly impacted by the commercial use of Pioneer 305423 soybean

. Non

transgenic soybean will lik ely still be sold and will be r eadily available to those who w ish to plant it. An internet search of “ S oybean Seed Company ” identifies vendors that offer all types of soybean seeds for purchase including conventional and transgenic (Vendors, 2009)

.

If P ioneer receives regulatory approval from all appropriate agencies, Pioneer 305423 soybean will likely be widely available to growers and b reeders. O ther farmer s who choose not to plant or sell Pioneer 305423 soybean or other transgenic soybeans will not l ikely be significantly impacted by the expected commercial use of this product because:

(a) non

- transgenic soybeans will likely still be sold and will be readily available to those who wish to plant these varieties ;

( b ) soybean is a highly self

- pollinated plant and therefore buffer requirements to isolate GE and organic varieties would be minimal;

( c ) 92% of the 2008 soybean acreage in the United States is already planted to transgenic herbicide tolerant varieties with organic soybean currently coexisting in the largely biotech agricultural environment ; and20 (d) APHIS expects that Pioneer 305423 soybean may replace some of the presently available GE soybean varieties without significantly affecting the overall total soybean acreage so organic farmers will be able to coexist with biotech soybean producers as they do now.

If Pioneer 305423 soybean is granted nonregulated status, similar to the no action alternative there would be no direct impact on organic or other non

- transgenic soybean farmers as the ma rket share of transgenic soybean are unlikely to change by the introduction of Pioneer 305423 soybean.



### C. Soybean Composition Soybean is one of the leading agricultural products in the United States. Soybean is class ified as an oilseed and is characteriz ed by its high (38

– 45%) protein content as well as its high (20%) oil content. The m olecular a nalysis d ata supplied in the Petition show that Pioneer 305423 soybean contains multiple copies of soybean fatty acid desaturase gene ( Gm

-- fad2

- 1 ) and one modified version of the soybean acetolactate synthase gene ( Gm

- hra )

. Pioneer 305423 soybean is a transgenic soybean product that provides soybean seeds with increased levels of monounsaturated (o leic) fatty acid s and decreased levels of polyunsaturated fatty acid s (linoleic and linolenic). The compositional assessment was conducted in accordance with the Organization of Economic Co

operati o n and Development ( OECD ) consensus document on compositio nal considerations for new varieties of soybean (OECD

, 2001) ( Pavely

, 200 7 , p 76).

On July 9, 2003, the Food and Drug Administration (FDA) announced that it would require the labeling of all food products for their trans

- fat content beginning January 1, 2006. As a result of the federal regulation, the food industry in the United States has been actively pursuing alternatives to hydrogenated oils so that their products can be labeled as containing 0 g of trans

- fat. The oil from Pioneer 305423 soybean w ith high oleic acid could be adopted as one of the alternatives to hydrogenated oil.

Soybean seeds both genetically engineered and conventionally breed for altered fatty acid composition of the soybean oil have been sold commercially and have a history of safe

use. The commercialization of Pioneer 305423 soybean could be beneficial for the consumer’s health. Increased intake of oils high in monounsaturated fatty acids, such as oleic acid have been shown to have positive effects on total cholesterol level s when compared to equal intakes of hydrogenated oils (Lichtenstein et al.

, 2006). Likewise, increased intake of oils high in oleic acid can decrease LDL

- cholesterol levels compared to equal intakes of saturated oils (Mensink et al.

, 1989) and increased H DL

- cholesterol levels compared to an equal intake of polyunsaturated oil (Mata et al.

, 1992). Moderate consumption of oil high in oleic acid has also demonstrated decreases in systolic blood pressure (Bondia

- Pons et al.

, 200 7 ).

Similar to the no action a lternative, a variety of soybean oil choices are presented to food manufacturers and for use in industral products

. T hese specialty oils are marketed exclusively as identity preserved (see Summary and Section I.B, Taveley, 2007) and will21 not likely be fou nd in commodity soybean oils. Pioneer 305423 soybaen would be an additional soybean variety available to this specility food oil market.

Oil Composition

Pioneer 305423 soybean was generated by the insertion of a second copy of a soybean fatty acid desat urase gene ( Gm

- fad2

- 1 ) in to a publicly available soybean cultivar “ Jack ”

. The fatty acid desaturase gene is responsible for the synthesis of linoleic acid, which is the major polyunsaturated fatty acid present in soybean oil

. By silencing 3

APHIS has previously granted nonregulated status to a GE high oleic soybean (APHIS Petition # 97

- 008

- 01p) (USDA

- APHIS

, 1997) developed by DuPont in which high oleic phenotype was conferred by introduction of the soybea n omega

- 6 desaturase gene 1 ( fad2

- 1 gene). DuPont high oleic soybean varieties have received regulatory approval for food use in the Australia, Canada, Japan, New Zealand, and the United States (AgBios, 2010)

. Australia New Zealand Food Authority (ANZFA) developed a draft “risk analysis report” ( ANZFA

, 2000) for the DuPont high oleic product. ANZFA concluded that high oleic acid soybeans do not raise any public health and safety concerns. Health Canada gave DuPont a “no objection to the food use of high oleic soybeans” in 2000 (AgBios, 2010)

. the fatty acid desaturase gene , it prevent s linoleic acid fr om being synthesized and leads to the accumulation of oleic acid in the seeds. An overview of fatty acid biosynthesis in soybean is illustrated in Figure 11, page 41 of the Petition ( Pavely

, 200 7 )

. The intend ed change to Pioneer 305423 soybeans is to greatly increase oleic acid content in the seed (Heppard

, 1996 ). Multiple copies of the Gm

- fad2

- 1 gene appear to be necessary for effective co

- suppression of the endogenous fatty acid desaturase gene 1 ( El

- Shemy et al.

, 2004; Mishra et al.

, 2005)

.

Data that support the safety of GmFad2

- 1 gene was reviewed by FDA resulting in their issuing a FDA Biotech Consultation BNF #00003 9 ( FDA

, 199 6 b )

. FDA has evaluated the food safety of the new proteins in bioengineered pla nts since 1992 and provided recommendations concerning its food safety ( FDA

, 2004 )

. In January, 2009, FDA completed the voluntary consultation of the Pioneer 305423 soybean for “Food and Feed Safety and Nutritional Assessment ” and had no further questions concerning Pioneer 305423 soybean based on the information provided by Pioneer ( FDA

, 2009 )

. Fatty acid compositional data was collected on Pioneer 305423 soybean and comparisons were made to conventional control lines and a set of reference soybean varieties (OECD

, 2001). A total of 25 fatty acids were analyzed in Pioneer 305423 soybean and control lines. Eleven fatty acid concentrations were near or below the detection limits of the
assay. These fatty acids are listed i n Petition ( Pavely

, 200 7

, page 79). The analys es of other 14 fatty acids are presented in Table 5, Petition ( Pavely

, 200 7

, page 81

- 82). Six of the 14 fatty acids were statistically significantly different from control soybean lines : myristic acid, palmit oleic acid, stearic acid, arachidic acid, eicosenoic acid and lignoceric acid ( Pavely

, 200 7

, page 79

- 82 ). However, the levels of these fatty acids are not

3 Gene silencing refers to a technique for selectively turn off specific genes within a cell.22 biologically significant; they are relatively minor fatty acids (together comprising less than 6% of fatty acid content ) and are common fatty acids in vegetable oils at similar levels ( USDA

- ARS

, 2006 )

. Two of the 14 fatty acids w ere not statistically significantly different : behenic acid and linoleic acid isomer (9,15)

. The remaining six fatty acids ( o leic acid, linoleic acid, linolenic acid, palmitic acid, heptadecanoic acid, and heptadecenoic acid) are discussed below.

F atty acids analys es confirmed that Pioneer 305423 soybean has significant ly more oleic acid (from an average of 21% to 76% of total fatty acid content )

, less in linoleic acid (from an average of 52% down to 3.6%) , less in linolenic (9.4% to 5.4%) and less in palmitic acid (from an average of 10.3% down to 6.28%) than conventional soybeans

. A mid

- oleic acid soybean variety is offered b y Iowa State Research Foundation producing

m

ore than 50% oleic acid (Stockhausen, 2008). High oleic acid levels are found in other commonly consumed premium edible oils (e.g., olive oil 55

- 83% oleic, Vossen (2007) and high oleic sunflower ( at least 75% o leic

, CODEX, 2009) and high oleic canola oil (70% oleic, Canolainfo.org, 2010 ). The consumption of high levels of oleic acid is not considered to pose any safety concerns (Lunn, 2007 ; FDA, 2009 )

. Linolenic acid is produced directly from conversion of lin oleic acid and therefore the decrease (from an average of 9.3% down to 5.4%) in the linole n ic acid was directly related to decreased linoleic acid levels

. Linoleic acid and alpha

- linolenic acid are fatty acids the human body requires , and can not be construct ed from other components by known chemical pathways

. They therefore must be obtain ed from the diet

. T hese two fatty acids are widely distributed in plant oils at (e.g. safflower oil, poppy seed oil, walnut oil, olive oil) and other food sources ( USDA

- ARS

, 2006)

. The decrease in palmitic acid content in Pioneer 305423 soybean is one of the intended effects. Palmitic acid is one of the most common saturated fatty acids found in animals and plants. Palmitic acid constitutes between 2 2 and 30 percent of most animal fats and is also an important constituent of most vegetable fats ( 2

- 17 pe rcent, but 35

- 45 percent of palm oil) ( Table 1, Nishina et al., 1993 ; Beare

- Rogers et al., 2001 )

.

Pioneer also noted increase d levels of two minor fatty acids, heptadecanoic acid and heptadecenoic acid ( Pavely

, 200 7

, pp

. 80

- 82). Th ese increase s likely r esult from change s in the GM

- HRA protein that shifts a metabolic pathway leading to production of the C17 4 fatty acids. Pioneer de s cribes th is complex oil biosynthesis in the Petition ( Pavely

, 200 7

, Appendix 7, pp

. 171

- 172). The combined value of heptade canoic and heptadecenoic fatty acids in control soybean line is less than 0.2% of total fatty acids. In Pioneer 305423 soybean, the combined value of these two fatty acids is about 2% of total fatty acids. Heptadecanoic acid is commonly found in meat, to fu and butter (USDA

- ARS

, 2006) , and h eptadecenoic acid is commonly found in meat (Senaratne

, 2009) and tofu ( USDA

- ARS

, 2006 ) . The levels of these two fatty acids in Pioneer 305423 soybean are comparable to those already found in the diet and there is no evidence to indicate that exposure to eith er fatty acid through di etar y sources w ould have adverse effects in humans.

4 C17 fatty acids are fatty acids containing 17 carbon atoms (e.g. heptadecanoic acid and heptadecenoic acid).23 Soybean Meal C omposition

Amino Acids Soybean meal is fed to animals primarily as a source of protein. Amino acid content in Pioneer 30 5423 soybean was determined for 18 am i no acids

. APHIS reviewed and analyzed the data presented in the petition, and concludes that there are no significant differences in amino acid composition between Pioneer 305423 soybean and control (non

- GE) soybean l ines ( Pavely

, 2007)

.
Novel protein The inserted G m

- hra gene is a slightly modified version of the soybean acetolactate synthase gene ( als gene) that is responsible for toleran ce to ALS inhibiting herbicide s

. The Gm

- hra gene encodes the GM

- HRA protein wit h two amino acid residues modified from the endogenous enzyme. In Pioneer 305423 soybean, GM

- HRA is the only novel protein expressed and was solely used as a selectable marker during the transformation procedure. The HRA fragment in Pioneer 305423 soybea n does not confer commercial levels of sulfonylurea or other ALS ( acetolactate synthase ) inhibitor herbicide tolerance. Pioneer has no plan to commercially market Pioneer 305423 as a sulfonylurea herbicide tolerant variety

.
ALS ( acetolactate synthase ) pr oteins are present in nature, as als genes have been isolated from bacteria, fungi, algae and plants (Friden et al.

, 1985; Falco et al.

, 1985; Reith et al.

, 1995; Mazur et al.

, 1987). Several commercialized non

- GE crops (Clearfield trade mark) with the he rbicide tolerant als gene are available in the current market. Data that support the safety of modified soybean acetolactate synthase proteins have been reviewed by FDA in the FDA Biotech Consultation BNF #000110 ( FDA

, 2009) for GE Pioneer soybeans Petiti on #06

- 354

- 01p (Pavely

, 2007 ). FDA Biotech Consultation BNF #00 0050 (FDA

, 1998) for GE flax, FDA Biotech Consultation BNF #000030 ( FDA

, 1996 a ) for GE
cotton
, FDA Biotech Consultation BNF #000108 ( FDA

, 2007) for GE Pioneer soybeans Petition #06

- 271

- 01p (US DA

- APHIS

, 2006)

. Pioneer also assessed the acute toxicity in mice of a dose of 2000 mg purified GM

- HRA protein per kilogram of body weight ( Pavely 2007, pp 62

- 63 )

. Even at this high dosage, no clear threshold of acute toxicity in mice was reached. Sinc e GM

- HRA protein is expressed at such a low level in Pioneer 305423 soybean, the amount of these soybeans that would need to be consumed to cause harm in mammals would be outside the limits of any realistic scenario for consumption. It is clear there is a wide margin of safety for GM

- HRA.

Based on Pioneer's d ata and information in Petition

, the recently published Environmental Assessment for APHIS Petition # 06

- 271

- 01p for GE Pioneer soybeans ( USDA

- APHIS 2006) and results of multiple previous FDA consulta tions

, t here is no indication of risk from GM

- HRA protein due to exposure or other environmental safety

concerns under the preferred alternative

. GM

- HRA protein was also assessed by the applicant for possible allergenicity and toxicity using international ly accepted guidance from the Codex Alimentarius Commission. APHIS reviewed this information (Pavely

,24 2007, p62) and concludes that GM

- HRA proteins are unlikely to be either allergenic or toxic to humans or animals.

Proximate, isoflavones and antinutrient s

Data on p roximate and fiber in soybean grain was provided to APHIS in the petition (Pavely

, 2007)

. Proximate analysis is a chemical method of assessing and expressing the nutritional value of a feed. Soybeans are occasionally used as an alternative fo rage source when alfalfa or clovers are in short supply. In 2001 , an OECD consensus document on compositional considerations for new soybean varieties, suggest ed parameters that soybean developers should measure. The proximate nutrient content

, includin g crude protein, c rude fat, fiber, and ash content of soybean meal is one of the parameter s

. APHIS reviewed th is information and concludes that t he ranges for crude protein, fat, ash, neutral detergent fiber (NDF) and a cid detergent f iber (ADF) of Pioneer 305423 soybean are comparable to nontransgenic soybean lines or reference soybean lines. APHIS also concludes that p roximate and fiber analysis of soybean forage and grain samples demonstrate that there is no unexpected difference between Pioneer 305423 s oybean and control (non

- GE) soybean lines.

Antinutrients are compounds which decrease the nutritional value, usually by making an essential nutrient unavailable or indigestible when consumed by humans/animals. Soybean seeds are known to contain different compounds displaying antinutrient effects. Soybean grain contains several key antinutrients, such as oligosaccharides, trypsin inhibitors, lectins and phytic acid. APHIS reviewed the information on the key antinutrients stachyose , raffinose, lectins, phy tic acid and trypsin inhibitor presented in the petition for Pioneer 305423 soybean and the control (non

- GE) soybean lines and concludes there were no statistically significant differences were seen between Pioneer 305423 soybean and control (non

- GE) soybe an lines.

APHIS also reviewed the information presented on i soflavones and concludes there were no statistically significant differences observed between Pioneer 305423 soybean and control (non

- GE) soybean lines.

APHIS has concluded that Pioneer 305423 soybean, and the foods and feeds derived from it are not materially different in safety, composition, or any other relevant parameter from soybeans now grown, marketed, and consumed. Results of these comparisons indicate that Pioneer 305423 soybean is co mpositionally and nutritionally equivalent to conventional soybean va r ieties currently in commerce except for the intended changes in the fatty acid profiles

. 

### Cumulative effects

APHIS considered wheth er the proposed action could lead to significant cumula tive impacts, when considered in light of other past, present, and reasonably foreseeable future actions, regardless of what agency or person initiated such actions. As mentioned above, Pioneer 305423 soybean is not the first high oleic soybean product to be granted25 nonregulated status. APHIS has previously made determinations of nonregulated status for DuPont high oleic soybean in the market currently.

APHIS has evaluated the potential cumulative impacts of granting nonregulated status to Pioneer 30542 3 soybean. In 2008, GE soybean was planted on 92% of all soybean acres in the US, and the use of GE soybean has been steadily increasing over the last 3 years (USDA

- NASS

, 2006, 2007, 2008). Conventional and GE soybean production occurs on land that is de dicated to crop production. Most soybean is planted on agricultural fields that have been in crop production for years. Pioneer 305423 soybean will not a ffect the amount of acreage devoted to GE varieties, nor will Pioneer 305423 soybean alter the growin g regions available for soybean production. T here would be no direct impact on organic or other non

- transgenic soybean farmers as the market share of transgenic soybean are unlikely to change by the introduction of Pioneer 305423 soybean. Pioneer 305423 is not herbicide tolerant at commercial herbicide application rates. Therefore, APHIS has no reason to believe that there will be any substantive change in current herbicide use rates as a result of granting nonregulated status to Pioneer 305423

.

In 200 3, several thousand acres of DuPont high oleic soybeans were grown under
contract in Iowa for DuPont Protein Technologies. There were no known or reported environmental impacts due to the production of this high oleic soybean variety. This DuPont high oleic soybean variety has not been planted since 2005 due to changes in market strategy for DuPont.

Based on this information, APHIS has determined that there are no past, present, or reasonably foreseeable actions that would aggregate with effects of the proposed action to create cumulative impacts or reduce the long

- term productivity or sustainability of any of the resources associated with the ecosystem in which Pioneer 305423 soybean is planted.


### Potential impact on non

- target organisms, including b eneficial organisms and threatened or endangered species

Un i ntended effects on non

- target organisms are one of the safety concerns considered for genetically engineered foods

, including the potential for altered expression of plant produced toxicants and anti

- nutrients or the formation of novel toxins. Generally, unintended effects can be predicted or explained through our current knowledge of plant biology and metabolic pathways. Molecular and biochemical analysis data can also help the risk assessors t o determine the levels of transcriptional and translational changes. APHIS reviewed and analyzed the data presented for Pioneer 305423 soybean and evaluated the potential of this GE soybean variety to cause damage or have toxic effects directly or indirec tly on non

- target organisms.

Toxcity and Allergencity The only novel protein expressed in Pioneer 305423 soybean is GM

- HRA. The potential toxicity of the GM

- HRA protein was assessed by comparing the amino acid sequence of the GM

- HRA protein with known pr otein toxins in the bioinformative database ( NIH

NCBI, 2010) as well as in an acute toxicity feeding study in mice ( Pavely

, 200 7 , p 63)

.26 The study found no evide nce of acute toxicity in mice fed with high dose s of purified GM

- HRA protein ( Pavely

, 200 7 , p

. 63)

. Since Pioneer 305423 soybean was proposed for

use as a food/feed product

, there is a wide safety margin taken into account (by the dosage range) in the acute toxicity feeding study

. APHIS concludes, after reviewing the data presented, including FDA’ s food safety and nutritional assessment, that there will not be any toxic effect at any feasible level of consumption

.

The information on the possible allergenicity of Pioneer 305423 soybean was reviewed and analyzed by APHIS

. GM

- HRA protein is not kno wn to be a protein toxin/allergen based on the information in the database of the Food Allergy Research and Resource Program (FARRP), University of Nebraska, Allergen Database (Version 6.0, January 2006)

. Likewise

, the rapid proteolytic degradation (less than 30 seconds) under simulated mammalian digestion condition s provide s further evidence to confirm the safety of the protein (FAO

, 1996). In addition, Pioneer provided a detailed assessment of human and animal safety assessment of the GM

- HRA protein to FDA as part of the consultation on food and feed safety for Pioneer 305423 soybean (FDA

, 2009)

. FDA had no further questions on the safety of Pioneer 305423 soybean.

Nutrition Nutritional studies on targeted animal s are performed in case s where the compo sition of the GE plant differs significantly from the non

- GE counterpart. Pioneer conducted a 42

- day feeding study on broiler chickens comparing Pioneer 305423 soybean and non

transgenic soybean as the main diets. Broilers are the choice animal for feedi ng studies because they are fast growing and are particularly sensitive to the presence of toxic elements in their feed. APHIS evaluated the results of feeding tests on broilers (Pavely

, 2007) and concluded that there is no difference between Pioneer 3054 23 soybean and traditional soybean variet y counterparts, and Pioneer 305423 soybean can be considered

as nutritionally equal to the non

- GE counterparts (Kuiper et al

. , 1999). Therefore, when

c ompare d to conventional soybean varieties, Pioneer 305423 soybe an is not expect ed to have any adv er s e effects on birds and animals feeding in the field

.

Pest and Disease Plant protease inhibitors have been determined to play a potent defensive role against certain pests and pathogens of soybeans (Boulter

, 1993 ; Hild er et al.

, 1987 ; Williamson et al.

, 1996 ; Joshi et al. 1998) as described in the Plant Pest Risk Assessment ( USDA

APHIS

, 2009 , p 3

- 5)

. The reduced protease activity could potentially result in Pioneer 305423 soybean being more susceptible to insect damage or fungal disease than the non

GE control lines. Pioneer provided data (Pavely

, 2007, pp

. 158

- 160) to support the conclusion that Pioneer 305423 soybean is comparable to non

- GE control lines in environmental fitness and defense against pests and diseases

. The mean value for trypsin inhibitor was significantly different (lower ; adjusted P

- value < 0.05 ) in Pioneer 305423 soybean as compared to the control line although remaining within the statistical tolerance interval and the “combined literature range

. ” APHIS reviewed the information and concludes that r educing the protease activities of the Pioneer 305423 soybean is not expected to have any effect on insects or threatened and endangered insect species feeding i n a soybean field (Pavely, 2007, pp

. 158

- 1 60)

.27 Soil Communities APHIS also evaluated the effects of production of Pioneer 305423 soybean on soil communities in agri cultur al settings

. In this case the genetic modification does not confer herbicide tolerance or insect resistance

. Therefore, t he potential pesticide ( herbicide and insecticide) usage should be similar as that for conventional soybean plantings. Currently, there are over 70 registrations for herbicides for weed management in soybean (Gianessi et al.

, 2002), plus numerous mixture s (C rop Protection Reference

, 200 9 ). In planting Pioneer 305423 soybean, t he pesticide or herbicide runoff into ground and surface water should have no more deleterious effects on non

- target organisms, including aquatic animals or aquatic invertebrates in pon ds and streams than conventional planting s

. Reports issued by the OECD (OECD

, 1986) and a working group coordinated by the Royal Society of London (Royal Society

, 2000) have indicated that environmental risks of biotechnology

- derived crops are not fundame ntally different from risks of conventionally derived products. Therefore, cultivation of Pioneer 305423 soybean should have no effects on either the microbial or invertebrate communities of soil ecosystems when compared to current agricultural practices for soybean

. Threatened and Endangered Species Using the information pertaining to the potential impacts to non

- target organisms described above, APHIS also considered the potential impact of deregulating Pioneer 305423 soybean on federally listed threa tened or endangered species (TES) and species proposed for listing [http://ecos.fws.gov/tess_public/SpeciesReport.do (accessed April 10, 2009)] , as well as designated critical habitat and habitat proposed for designation, as required under Section 7 of the Endangered Species Act. In this analysis, APHIS considered the biology of Pioneer 305423, and how its production would differ from typical agricultural practices associated with cultivation of soybean.

To identify negative effects or significant impact s on TES animal species, APHIS evaluated the risks to TES animals from consuming Pioneer 305423 soybean. Risk is a combination of hazard and exposure. APHIS first conducted hazard identification for Pioneer 305423 soybean. APHIS reviewed and analyzed th e composition and nutritional quality of Pioneer 305423 soybean submitted in the petition, and compared the composition of Pioneer 305423 to the composition of a non

- genetically engineered control soybean line and the natural variation found in commercial soybean varieties (Pavely

, 2007) . If the composition of Pioneer 305423 soybean is similar to other commercial soybean plants, it is unlikely that Pioneer 305423 poses a hazard to TES animal species. If no hazards are identified, then the risk of Pioneer 305423 soybean harming TES animal species is also unlikely, regardless of exposure.

As discussed in this EA, the proteins produced by the inserted genes and the changes in fatty acid composition do not raise safety issues. As noted above in this sectio n, consump tion of GM

- HRA protein has shown no toxicity in lab testing with mice, and no measurable change in nutritional response on broiler chickens. The Pioneer 305423 soybeans do not express additional proteins, natural toxicants, allelochemicals, pher omones, hormones, etc. that could directly or indirectly affect a listed TES or species28 proposed for listing. Data submitted on the composition of the Pioneer 305423 soybeans indicate that these soybeans are not significantly different from non

- transgenic soybeans and would not be expected to have any effect on TES that would be different from non

transgenic soybeans. The Pioneer 305423 soybean is not sexually compatible with a federally listed TES or a species proposed for listing. APHIS has not identif ied any stressor caused by the production of Pioneer 305423 that could affect the reproduction, numbers, or distribution of a listed TES or species proposed for listing. Consequently, an exposure analysis for individual species is not necessary.

Soybeans do not grow and persist in unmanaged habitats and would not be expected to invade and/or persist in the natural environment. Soybean fields are typically highly managed agricultural areas that can be expected to be dedicated to crop production for
many y ears and cultivation of Pioneer 305423 soybean is not expected to differ from typical soybean cultivation. After reviewing possible effects of deregulating Pioneer 305423 soybean, APHIS expects Pioneer 305423 soybean to replace some of the presently avail able soybean varieties, but APHIS does not expect that Pioneer 305423 soybean will cause new soybean acres to be planted in areas that are not already devoted to agriculture. APHIS has considered the effect of Pioneer 305423 soybean production on habitat that could be used by TES, including critical habitat, and could identify no difference from affects that would occur from the production of other soybean varieties.

Based on the above information, APHIS has determined that the preferred alternative, der egulating Pioneer 305423 soybean would have no effect on federally listed threatened or endangered species and species proposed for listing, or on designated critical habitat or habitat proposed for designation. Consequently, a written concurrence or form al consultation with the USFWS is not required for this action.



### Consideration of Executive Orders, Standards and Treaties relating to environmental i mpact s

Executive Order (EO) 12898 (US

- NARA , 2008), “Federal Actions to Address Environmental Justice in M inority Populations and Low

- Income Populations”

, 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 participa tion 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.

EO 13045 (US

- NARA , 2008), “Prote ction of Children from Environmental Health Risks and Safety Risks” , acknowledges that children may suffer disproportionately from environmental health and safety risks because of their developmental stage, greater metabolic activity levels, and behavior p atterns, as compared to adults. The EO (to the extent permitted by law and consistent with the agency’s mission) required each Federal agency to identify, assess, and address environmental health risks and safety risks that may disproportionately affect c hildren.29 Each alternative was analyzed with respect to EO 12898 and 13045. Based on the information submitted by the applicant and assessed by APHIS, Pioneer 305423 soybean is not significantly different than conventional soybean and has successfully com pleted the FDA voluntary consultation for food and feed use. Therefore, Pioneer 305423 soybean is not expected to have a disproportionate adverse effect on minorities, low

income populations, or children.

EO 13112 (US

- NARA , 2008), “Invasive Species”, st ates that Federal agencies 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. Both non

- GE and GE soybean varieties that have been granted nonregulated status are widely grown in the U.S. Based on historical experience with soybean and the data submitted by the applicant and assessed by APHIS, Pioneer 305423 soybean plants are very similar in fitness characteristics to oth er soybean varieties currently grown and are not expected to become weedy or invasive [see (USDA

- APHIS

, 200 9 ) for the plant pest risk assessment of Pioneer 305423 soybean ].

EO 13186 (US

- NARA , 2008), “Responsibilities of Federal Agencies to Protect Migrat ory Birds” , states that Federal agencies taking actions that have, or are likely to have, a measurable negative effect on migratory bird populations are directed to develop and implement, within 2 years, a Memorandum of Understanding (MOU) with the Fish

an d Wildlife Service that shall promote the conservation of migratory bird populations. Data submitted by the applicant has shown no difference in compositional and nutritional quality of Pioneer 305423 soybean compared to conventional soybean , apart from th e presence of Pioneer 305423 soybean

. Pioneer also conducted feeding experiments on broiler chickens to evaluate the effects of Pio ne er 305423 soybean on birds (page 15 of EA ). The applicant reported no harmful effects to chickens from Pioneer 305423
soy bean

. The migratory birds that occasionally forage in soybean fields are unlikely to contain high amounts of Pioneer 305423 soybean as soybean availability is limited by seed germination and harvest. Based on APHIS’ assessment of Pioneer 305423 soybean i t is unlikely that granting nonregulated status to this soybean variety will have a negative effect on migratory bird populations.

INTERNATIONAL IMPLICATIONS

EO 12114 (US

- NARA , 2008), “Environmental Effects Abroad of Major Federal Actions”, requires Federal officials to take into consideration any potential environmental effects outside the U.S., its territories , and possessions that result from actions being taken. APHIS has given this due consideration and does not expect a significant environmental i mpact outside the U.S. should nonregulated status be granted to Pioneer 305423 soybean

. It should be noted that all the considerable, existing national and international regulatory authorities and phytosanitary regimes that currently apply to introduction s of new soybean cultivars internationally, apply equally to those covered by an APHIS determination of nonregulated status under 7 CFR part 340. Any international trade of Pioneer 305423 soybean subsequent to a determination of nonregulated status for th e product would be fully subject to national phytosanitary requirements and be in accordance with phytosanitary standards developed under the30 International Plant Protection Convention (IPPC

, 2008 ).

The purpose of the IPPC “is to secure a common and effect ive action to prevent the spread and introduction of pests of plants and plant products and to promote appropriate measures for their control” (IPP C, 2008); 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 nations that have signed or acceded to the Convention (169 countries as of September 2008). In April 2004, a standar d 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 an existing standard, International Standard for Phytosanitary Measure No. 11 (ISPM

- 11, Pest Risk Analysis for Quarantine Pests). The standard acknowledges that all 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 commoditie s produced through biotechnology are being addressed in other international forums and through national regulations.

The Cartagena Protocol on Biosafety is a treaty under the United Nations Convention on Biological Diversity (CBD) that established a fram ework 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 150 countries are Parties to it as of Januar y, 2009 (CBD

- UN

, 2008). Although the U.S. is not a party to the CBD, and thus not a party to the Cartagena Protocol on Biosafety, U.S. 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 ag reement (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 un der Article 11 and Annex II of the Protocol. Under Article 11 Parties must post decisions to the Biosafety 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 U.S. Government has developed a website that provides the status of all regulatory reviews completed for different uses of bioengineered products (NBII

, 2008). These data will be available to the Biosafety Clearinghouse. APHIS continue s to work toward harmonization of biosafety and biotechnology consensus documents, guidelines, and regulations, including within the North American Plant Protection Organization (NAPPO), which includes Mexico, Canada, and the U.S., and within the Organizat ion for Economic Cooperation and Development. NAPPO has completed three modules of a standard for the Importation and Release into the31 Environment of Transgenic Plants in NAPPO Member Countries (NAPPO

, 2008).

APHIS also participates in the North American Biotechnology Initiative (NABI) , a forum for information exchange and cooperation on agricultural biotechnology issues for the U.S., Mexico and Canada. In addition, bilateral discussions on biotechnology regulatory issues are held regularly with other cou ntries including: Argentina, Brazil, Japan, China, and Korea.

COMPLIANCE WITH CLEAN WATER ACT AND CLEAN AIR ACT This Environmental Assessment evaluated the changes in soybean production due to the unrestricted use of Pioneer 305423 soybean

. Pioneer 305 423 soybean will not lead to the increased production of soybean in U.S. agriculture. There is no expected change in water use due to the production of Pioneer 305423 soybean compared to current soybean production regimes , nor is it expected that air quali ty will change due to do the production of Pioneer 305423 soybean

.



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# VII . PREPARERS AND REVIEWERS

Michael Gregoire, Deputy Administrator Biotechnology Regulatory Services

Beverly Simmons, Associate Deputy Administrator for Emerging and International Programs Sidney Abel, Assistant Deputy Adminis trator

BRS, Environmental Risk Analysis Division Michael Watson , Ph.D., Division Director Margaret Jones , Ph.D., Senior Biotechnologist ( Reviewer ) Karen Green, Biotechnologist ( Preparer of Environmental Assessment ) Luc Hebou , Biotechnologist (Review er)

BRS, Policy and Coordination Division40 John Turner, Ph.D., Director Andrea Huberty, Ph. D., Branch Chief (Reviewer) Richard Coker, Regulatory Analys t Michael Blanchette, Environmental Protection Specialist (Reviewer)

AGENCY CONTACT

Ms. Cynthia Ec k, Document Control Officer USDA, APHIS, BRS 4700 River Road, Unit 147 Riverdale, MD 20737

- 1237 Phone: (301) 734

- 0667 Fax: (301) 734

- 8669 Cynthia.A.Eck@aphis.usda.gov 

# VIII. Appendix 1

- Bio techn ology Consultation Note to the File BNF No. 000110

Date: January 14, 2009

Subject: High Oleic 305423 Soybean

Keywords: soybean, Glycine max , high oleic 305423 soybean, TREUS TM , OECD unique identifier DP

- 3Ø5423

- 1, omega

- 6 desaturase, FAD2

- 1

, gm

- fad2

- 1 , see d

- specific silencing, gm

- hra , soybean acetolactate synthase

1. Introduction

In a submission dated December 27, 2006, Pioneer Hi

- Bred International, Inc. (Pioneer), a DuPont Company, submitted to the Food and Drug Administration (FDA) a safety and nutrition al assessment of the bioengineered high oleic 305423 soybean line containing the transformation event DP

- 3Ø5423

- 1. Pioneer provided additional information on August 31, 2007, January 30, February 19, March 18, November 21, December 12, and December 19, 200 8. Pioneer concluded that food and feed derived from the 305423 soybean are as safe and nutritious as food and feed derived from conventional soybean varieties currently being marketed.

2. Intended Effect

The intended effect of the modification in 305423 s oybean is to produce soybean seeds with increased levels of monounsaturated fatty acid (oleic) and decreased levels of polyunsaturated fatty acids (linoleic and linolenic). To accomplish this objective, Pioneer inserted a fragment of the soybean microsomal omega

- 6 desaturase gene ( FAD2

- 1 ) into41 soybean. The fragment of the FAD2

- 1 gene does not code for a protein. Transcription of the gene fragment under the control of a seed

- preferred KTi3 promoter acts to silence the expression of the endogenous soybean ome ga

- 6 desaturase, which results in an increased level of oleic acid and decreased levels of linoleic and linolenic acids in the soybean seed. A gene ( gm

- hra ) encoding a modified version of the soybean acetolactate synthase ( als gene) that confers tolerance to a sulfonylurea herbicide was used as a selectable marker for the transformation.

3. Genetic Modifications and Characterization

3.1. Parental Variety

The publicly available soybean variety "Jack" was used as the recipient in the DNA transformation to cre ate 305423 soybean.

3.2. Transformation DNA and Method

Pioneer used microprojectile bombardment to co

- transform secondary plant cell embryos with two purified linear DNA fragments: a 2924 base pair fragment containing the gm

fad2

- 1 cassette, and the 4512 b ase pair fragment containing the gm

- hra cassette. The gm

fad2

- 1 cassette includes the promoter region from the soybean Kunitz trypsin inhibitor gene (KTi3), a fragment of the FAD2

- 1 gene that corresponds to approximately 40% of the middle portion of the FA D2

- 1 gene, and the 3' untranslated region of the KTi3 gene (KTi3 terminator).

The gm

- hra cassette includes a promoter and an intron from the 5'regulatory region of the S

- adenosyl

- L

- methionine synthetase (SAMS) gene from soybean, the gm

- hra gene that encode s the GM

- HRA protein, and the terminator from the endogenous als gene. 1

3.3. Characterization, Stability, and Inheritance of the Introduced DNA

In order to character ize the introduced DNA, Pioneer conducted Southern blot and sequencing analyses of the DNA inserted into the 305423 soybean. Pioneer reports that the 305423 soybean contains four genetically linked insertions. The 305423 soybean has multiple intact and par tial copies of the gm

- fad2

- 1 cassette that contain, in total, eight copies of the KTi3 promoter, seven copies of the gm

- fad2

- 1 gene fragment, and five copies of the KTi3 terminator. Pioneer states that it appears that multiple copies of the
gm

- fad2

- 1 gene fragment are necessary for effective co

- suppression of the endogenous gene. One copy of the KTi3 promoter is associated with a small non

- functional fragment of plasmid backbone DNA. Pioneer determined that a single intact gm

- hra cassette is inserted into t he genome of 305423 soybean. Pioneer noted that further analysis of 305423 soybean for plasmid backbone sequence using Southern hybridization showed that 305423 soybean contains neither the hygromycin gene nor the bacterial plasmid origin of replication pr esent on the plasmids from which the gm

- fad2

- 1 cassette and the
gm

- hra cassette are derived. Pioneer sequenced the inserted DNA and reported that the sequence confirmed the results of the Southern analysis.42 Each of the four insertions in 305423 soybean was screened for the presence of open reading frames (ORFs) containing both a start and stop codon that spanned any novel junctions. Pioneer identified two such ORFs. Pioneer reports that neither ORF contains the necessary regulatory elements for transcriptio n. Northern blot analysis detected no transcripts of the ORFs in developing seeds from either 305423 soybean or the control soybean. Pioneer concluded that it is very unlikely that a protein is expressed from either ORF. Pioneer also reports that screening of the ORFs against a database containing known protein toxins 2 and to a database of known allergens 3 showed no biologically significant identities to known protein toxins or allergens. Based upon their analyses, Pioneer concluded that there are no safety concerns resulting from these ORFs.

Pioneer reports that Southern blot an alyses across three generations showed that the inserted DNA in 305423 soybean is stably integrated into the genome. The same event

specific hybridization pattern was observed for all but one plant that apparently lost the
gm

- hra cassette due to a recombin ation event. Pioneer investigated the frequency of recombination in 305423 soybean by examination of 1000 additional segregating 305423 soybean plants by PCR

- based assays and found no other recombinants.

Pioneer conducted chi square analysis of trait inher itance data. They report that the expected segregation ratios were observed in crosses showing the Mendelian inheritance and stability of the introduced trait. Pioneer reports that when both traits were analyzed in the same plants, data confirmed co

- segreg ation of the gm

- fad2

- 1 gene fragment and the
gm

- hra gene.

4. Introduced Protein

– GM

- HRA

4.1. Identity, Function, and Characterization

Pioneer describes the GM

- HRA protein as a modified version of the endogenous soybean acetolactate synthase (ALS) enzyme a nd that this modified ALS enzyme confers tolerance to ALS

- inhibiting herbicides. ALS enzymes are widely distributed in nature and have been isolated from bacteria, fungi, algae, and plants. ALS

- inhibiting herbicides inhibit plant growth by blocking the act ion of the ALS enzyme thereby inhibiting branched

- chain amino acid biosynthesis. The mature GM

- HRA protein differs from the endogenous ALS enzyme at two specific amino acids and is responsible for GM

- HRA insensitivity to ALS

- inhibiting herbicides. The gm

- h ra gene was used only as a selectable marker for the transformation.

Pioneer characterized 305423 soybean

- produced GM

- HRA protein using various methodologies 4 and de monstrated its equivalence with Escherichia coli

- produced GM

HRA protein. The E. coli

- derived GM

- HRA protein was used for in vitro and in vivo biochemical and toxicological studies.

The GM

- HRA protein levels in 305423 soybean were measured in replicated sa mples of leaf, root, forage and grain tissues using a quantitative enzyme

- linked immunosorbent assay (ELISA). Pioneer reports that the mean GM

- HRA protein concentrations in 305423 soybean leaf, root, forage, and grain were 4.0, 0.18, 5.7, and 2.5 nanograms per milligram43 (ng/mg) tissue dry weight respectively. Pioneer concluded that the above results confirm that the expression of the GM

- HRA protein in 305423 soybean is constitutive.

4.2. Assessment of Potential Toxicity and Allergenicity

Pioneer reports the results of a global sequence similarity search of the GM

- HRA amino acid sequence against the National Center for Biotechnology Information (NCBI) Protein dataset. The search was conducted using the BLASTP algorithm. 5 GM

- HRA showed sequence similarity to other ALS proteins. None of the proteins returned by the search was identified as a toxin. Pioneer concluded that the GM

- HRA protein did not share relevant sequence s imilarities with known protein toxins.

Pioneer conducted an acute oral toxicity study in mice. A single dose of 582 mg per kilogram of body weight (mg/kg bw) of GM

- HRA protein was administered by oral gavage to five male and five female mice. No clinical s ymptoms of toxicity, body weight loss, gross organ lesions or mortality were observed. Pioneer concluded that the result of this study shows that the GM

- HRA protein does not cause acute toxicity.

Pioneer reports that while soybean is one of the major food allergens, none of the identified allergens is a member of the ALS family and the ALS protein from soybean has not been characterized as a soy allergen. Pioneer compared the amino acid sequence of the GM

- HRA protein to the amino acid sequences of known all ergens in the Food Allergy Research and Resource Program (FARRP, version 6.0) database using the FASTA34 sequence alignment program. 6 None of the identified alignmen ts met or exceeded the threshold of greater than or equal to 35% identity over 80 amino acids and no contiguous stretches of 8 or greater amino acids were shared between the GM

- HRA protein and the proteins in the allergen database. Additionally, the GM

- HRA protein is not glycosylated.

Pioneer also reports that the GM

- HRA protein is rapidly (< 30 seconds) hydrolyzed in simulated gastric fluid (SGF) to fragments of < 3 kDa; and when subjected to simulated intestinal fluid (SIF), the GM

- HRA protein, including the low weight molecular fragments seen in SGF, is completely and rapidly (< 2 minutes) hydrolyzed.

Pioneer concluded that the GM

- HRA protein is unlikely to be a toxin and is not a potential allergen.

5. Evidence for Silencing of FAD2

- 1 Gene Expression

Pio neer determined that a 597 base pair gm

- fad2

- 1 gene fragment, which is identical to a portion of the coding sequence of the endogenous soybean microsomal omega

- 6 desaturase gene ( FAD2

- 1 ), resulted in the down regulation of expression of the endogenous FAD2

- 1 gene. Pioneer examined the expression of the endogenous FAD2

- 1 gene in the leaf and developing seed of ten 305423 soybean plants and five control Jack soybean plants. Northern blots indicated that transcripts of the FAD2

- 1 gene in developing seeds of 30 5423 soybeans were greatly diminished when compared to the44 control soybean. This greatly decreased level of transcription confirms that the endogenous FAD2

- 1 gene is effectively silenced.

6. Food and Feed Uses of Soybean

Pioneer describes historical and cu rrent uses of soybean varieties in food and animal feed. The primary use of commodity soybeans is for soybean meal that is consumed by animals. Raw soybeans contain several antinutritional factors (trypsin inhibitors, urease, and hemagglutinins). Heat trea tment is the most common processing method used to minimize the activity of such factors. Soybean oil is the major soybean fraction consumed by humans. Soybean oil accounts for 80% of total United States consumption of oils and fats.

Pioneer states that th e 305423 soybean variety is intended to be used for the production of high oleic soybean oil. The oil is intended to be a highly stable vegetable oil suitable for frying applications without the need for hydrogenation which produces trans fatty acids and " hydrogenated flavor." Pioneer states that it is aware of no food or feed uses of current soybean varieties for which the 305423 soybean variety would not be equally suitable.

7. Overview of Compositional Analysis

Pioneer assessed the composition of grain a nd forage from the 305423 soybean and a null segregant (non

- transgenic isoline) control. Pioneer states that the null segregant plants are an appropriate control because they are almost genetically identical to the corresponding 305423 soybean plants with the exception that they do not carry the transgenic DNA. Both the transgenic and control soybeans were grown at six field locations in soybean

growing areas of North America using a randomized complete block design with three replicates at each location. P ioneer also analyzed grain and forage from four different commercial soybean varieties.

Pioneer measured 52 components in grain and 5 in forage. Pioneer used the composition data obtained from the commercial varieties to calculate 99% tolerance intervals w ith 95% confidence for all measured components. To interpret the composition results for 305423 soybeans, Pioneer used the confidence intervals and established a combined literature range using data from published literature and databases on soybean compos ition.

Pioneer performed statistical analyses of composition data obtained for 305423 soybean and control soybean using mean values calculated from data aggregated from all tests sites. Pioneer used a linear mixed model analysis of variance (ANOVA). In ord er to hold the rate of false positive results to 5% or less, Pioneer employed the false discovery rate (FDR) approach (Benjamini and Hochberg (1995) and Westfall et al. (1999)). Pioneer reported the composition data by providing mean values, ranges, FDR

- ad justed P

- values, unadjusted P

- values, tolerance intervals, and literature ranges, as available. Pioneer discussed analytical results in the context of FDR

- adjusted P

- values. Unless so indicated,45 statistical analyses using the unadjusted and adjusted P

- valu es are in agreement. Pioneer used a P

- value of 0.05 to denote a statistically significant difference between the control and the 305423 soybean.

Pioneer analyzed grain samples for proximates (protein, fat, and ash), acid detergent fiber (ADF), neutral dete rgent fiber (NDF), fatty acids, amino acids, isoflavones, and antinutrients. Compositional analysis of forage samples included proximates, ADF, and NDF. Table 1 contains the complete list of all measured components.

Table 1. Components measured in grain an d forage

Proximates * & Fiber * Fatty Acids ** Amino Acids Isoflavones + Antinutrients

ash fat protein acid detergent fiber (ADF) neutral dete rgent fiber (NDF) myristic (14:0) palmitic (16:0) palmitoleic (16:1) heptadecanoic (17:0) heptadecenoic (17:1) stearic (18:0) oleic (18:1) linoleic (18:2) linoleic (18:2)

isomer (9,15) linolenic (18:3) arachidic (20:0) eicosenoic (20:1) behenic (22:0) li gnoceric (24:0) methionine cystine lysine tryptophan threonine isoleucine histidine valine leucine arginine phenylalanine glycine alanine aspartic acid glutamic acid proline serine tyrosine genistin genistein malonylgenistin daidzin daidzein malonyldaidzin glycitin glycitein malonylglycitin stachyose raffinose lectins phytic acid trypsin inhibitor

* Proximates and fiber were measured in both soybean grain and forage. All other components were measured in grain only.

** The levels of eleven additional fatty acids were near or below the lower limit of quantitation.

+ The levels of acetylgenistin, acetyldaidzin, and acetylglycitin were below the limit of quantitation.

7.1 Compositional Analysis of Soybean Grain

Intended Compositional Change

– Fatty Acids

Pion eer analyzed the fatty acid composition of the oil extracted from the grain of the 305423 and control soybeans. Pioneer provides the levels of 14 fatty acids (see Table 1) calculated as percentages of the total fatty acids. Pioneer states that the fatty ac id analysis confirmed the expected high oleic acid phenotype as shown by a substantial increase in the mean level of oleic acid to 76.5% in 305423 soybean as compared to a mean level of 21% in the control soybean. As expected, the increase in the level of oleic acid was accompanied by a decrease in the level of linoleic acid. The mean level of linoleic acid in the control soybean was 52.5% and that in the 305423 soybean was 3.6%. The level of46 linolenic acid also decreased since linolenic acid is formed in s oybeans directly from linoleic acid. Pioneer also notes that the levels of two minor fatty acids, heptadecanoic and heptadecenoic, increased in the 305423 soybean to 0.8% and 1.2% of the total fatty acids, respectively. Pioneer explains that the increase i n the levels of heptadecanoic and heptadecenoic fatty acids is not unexpected because the GM

- HRA enzyme most likely results in the increased concentration of 2

- ketobutyrate, which is the substrate in the biosynthesis of hepta fatty acids in soybeans. Pione er also reported changes in the levels of all remaining fatty acids, which are not biologically significant.

Proximates and Amino Acids

Pioneer reports that the mean levels of protein and fiber in grain from the 305423 and control soybean are not statistic ally significantly different. While the mean level of fat was statistically significantly lower in the 305423 soybean than in the control using the unadjusted P

- value, no statistical difference was detected using the FDR

- adjusted P

value. The mean level of ash was statistically significantly lower in the 305423 soybean as compared to the control soybean. Mean levels of the proximates and fiber in grain from 305423 and control soybean lines were within the 99% tolerance intervals for the reference varieties and within the combined literature range.

Pioneer reports that there are no statistically significant differences observed in the mean levels of amino acids between 305423 and control soybean grain with the exception of threonine and glutamic acid. The mea n levels of these two amino acids were statistically significantly increased in 305423 soybean when the unadjusted P

- values were used, but not when the FDR

- adjusted P

- values were used. All these levels were within the 99% tolerance intervals.

Isoflavones

P ioneer analyzed grain from the 305423 and control soybeans for twelve isoflavones, of which nine were quantified (see Table 1). The mean levels for daidzin, malonylgenistin, and malonyldaidzin were statistically significantly increased in the 305423 versus the control soybean. Mean genestin levels were only shown to be statistically significantly increased when the unadjusted P

- value was used. Mean values for all the measured isoflavones were within the 99% tolerance intervals.

In both the control and 30542 3 soybean the mean levels of genistin, glycitin, malonylgenistin, malonylglycitin, and malonyldaidzin were above the combined literature range. Daidzin levels were only above the combined literature range in the 305423 soybean. No literature data were avai lable for the level of glycitein. The values for the other two isoflavones (genistein and daidzein) were within the combined literature
range.47 Antinutrients

Pioneer measured several antinutrients in soybean grain including non

- digestible oligosaccharides s tachyose and raffinose, lectins, phytic acid, and trypsin inhibitor (see Table 1). No statistically significant differences were observed between the 305423 and control soybean in mean values for raffinose, lectins, and phytic acid. The mean value for tryp sin inhibitor was statistically significantly lower for 305423 soybean as compared to the control soybean. The reduction in the mean value of trypsin inhibitor in 305423 soybean was expected as Pioneer reported that the promoter used to drive expression of the FAD2

- 1 gene, the Kunitz trypsin inhibitor promoter, has been shown to silence the KTi3 gene which encodes the Kunitz trypsin inhibitor. The mean value for stachyose was statistically significantly increased when the unadjusted P

- value was used, but no t when the FDR

- adjusted P

- value was used. Mean values for all the measured antinutrients were within the 99% tolerance intervals and within the combined literature ranges.

7.2 Compositional Analysis of Soybean Forage

Pioneer analyzed forage for protein, fa t, ash, ADF, and NDF. Pioneer reports that no statistically significant differences were observed between the mean levels of these components in forage from the 305423 and control soybeans with the exception of the level of fat. The mean level of fat was s tatistically significantly decreased in 305423 using the unadjusted P

- value, but not when using the FDR

- adjusted P

- value. All mean levels were within the 99% tolerance intervals and combined literature ranges, with the exception of NDF. The mean level of N DF in both the control and 305423 soybean was above the combined literature range.

7.3 Endogenous Allergens

Pioneer conducted a study to assess whether the transformation process may have increased the overall allergenicity of 305423 soybean compared to co nventional soybean. Using sera from clinically reactive soy allergic patients, Pioneer conducted IgE immunoblot and ELISA studies using protein extracts from 305423 soybean and conventional soybean. Pioneer reports that the SDS

- PAGE Coomassie blue

- stained protein profiles for 305423 and control soybean extracts appeared to be the same; they are similar in their IgE binding profile, and showed the same IgE binding capacity for 305423 and control soy extracts. Pioneer concluded that the levels of endogenous

a llergens in and the allergic potential of 305423 soybean are comparable to those in nontransgenic control soybean.

8. Fatty Acids Intake

8.1 Human Diet

Pioneer generated estimates of dietary exposure to various fatty acids from the consumption of soy oil d erived from conventional soybeans, as well as 305423 soybeans. Pioneer concluded that the intake of oleic acid would increase, while the intakes of48 linoleic acid and trans fatty acid would decrease. Pioneer states that based on very conservative intake estimates calculated on the assumption that high oleic soybean oil would replace all soybean oil in commercial applications, the dietary intake of linoleic acid would still fall within the current intake levels. Pioneer also notes that a variety of other oils used by the food industry would provide significant amounts of linoleic acid in the diet. Other noted changes in dietary intakes of fatty acids would result in small increases in the consumption of minor fatty acids, heptadacanoic acid (C17:0), heptadecen oic acid (C17:1), and the (9, 15) isomer of linoleic acid (cis

- 9, cis

- 15

- octadecadienoic acid).

Pioneer states that the 17

- carbon fatty acids, heptadecanoic and heptadecenoic acids, occur at low levels in commonly consumed foods. For example, heptadecanoic acid is commonly found in meat (lamb, beef, pork), butter, and tofu, while heptadecenoic acid is found in foods such as tofu, beef, cheese, and olive oil. Pioneer stated that odd

- chain fatty acids such as 17

- carbon fatty acids are readily metabolized.

8.2 Animal Diets

When oil is removed from the soybean a defatted meal is generated that is used as a primary protein supplement for animal feed. Pioneer provided examples of swine and poultry diets to demonstrate that the reduced intake of linoleic acid, an e ssential fatty acid, would not lead to a nutritional deficit for animals consuming feeds containing meal derived from 305423 soybeans. Corn, the major ingredient of animal diets, is the primary source of linoleic acid in such diets. Even though soybean mea l derived from 305423 soybeans would have reduced amounts of linoleic acid, the quantity of linoleic acid provided by corn is several

- fold above the animals' requirement.

9. Common or Usual Name of the Oil Product

Pioneer concluded that based on the intend ed increase in the level of oleic acid and decrease in the levels of linoleic and linolenic acids, a new common or usual name is appropriate for the oil from 305423 soybeans to distinguish this oil from the conventional soybean oil as defined in the Food C hemicals Codex (FCC). Pioneer proposed the name "high oleic soybean oil" for the oil that will be produced from 305423 soybeans.

10. Conclusion

Pioneer has concluded that, with the exception of the intended change in fatty acid composition, the 305423 soyb ean and the foods and feeds derived from it are not materially different in composition, safety, or any other relevant parameter from soybeans now grown, marketed, and consumed. At this time, based on Pioneer's data and information, the agency considers Pi oneer's consultation on the 305423 soybean to be complete.

Mary D. Ditto, Ph.D.49 1 The gm

- hra cassette also contains three Flp recombinase target sequences; however, these sequences were not used in the development of the 305423 soybean. The presence of thes e sites does not cause recombination. Recombination requires the presence of the specific Flp recombinase enzyme that is not present in plants.

2 National Center for Biotechnology Information (NCBI) Protein Dataset release 156.0. The NCBI dataset incorporat es all non

- redundant entries from all GenBank nucleotide translations along with protein sequences from SWISS

- PROT, PIR, PRF, and PDB databases.

3 Allergen database derived from the Food Allergy Research and Resource Program (FARRP, version 6.0).

4 Sodium do decyl sulfate polyacrylamide gel electrophoresis (SDS

- PAGE), Western blot analysis, glycoprotein staining, mass determination of the tryptic peptides by matrix assisted laser desorption ionization mass spectrometry (MALDI

- MS), and N

- terminal amino acid seq uence analysis.

5 The E

- score was set at 1.0 to ensure that proteins even with limited similarity would not be overlooked.

6 University of Nebraska Allergen Database, version 6, January 2006; www.allergenonline.com.

From FDA website page: http://www.fda.gov/ Food/Biotechnology/Submissions/ucm155595.htm