Plant Pest Risk Assessment for Pioneer 4114 Maize

Background

Pioneer Hi-Bred International, Inc. (referred to as “Pioneer” hereafter) has petitioned APHIS 

(USDA-APHIS Petition Number #11-244-01p, referred to as “Petition”) for a determination that 

genetically engineered (GE) corn (Zea mays) event DP-ØØ4114-3 (referred to as “4114 maize”

or “4114”) is unlikely to pose a plant pest risk and, therefore, is no longer a regulated article 

under regulations at 7 CFR part 340. APHIS administers 7 CFR part 340 under the authority of 

the plant pest provisions of the Plant Protection Act of 20011. This plant pest risk assessment was 

conducted to determine whether event DP-ØØ4114-3 maize is unlikely to pose a plant pest risk.

History of Development of Insect-Resistant and Herbicide-Tolerant 4114 

maize

The United States is the largest producer of corn in the world (USDA-ERS, 2010). Corn is the 

primary feed grain in the United States, accounting for more than 90 percent of the total value of 

feed grains (USDA-ERS 2010). Corn is also considered as a major biofuel crop in the U.S. 

Recently strong demand for ethanol production has resulted in increased corn demand and higher 

corn prices and has provided incentives to increase corn acreage. Corn production in the U.S. has 

risen over time and is currently about 80 million acres annually. USDA has projected that U.S. 

corn cultivation may continue to increase in the coming years (USDA-NASS 2011). 

Maize producers face substantial economic losses from insect damage every year. For centuries, 

scientists have searched for crop plants that can survive and produce in spite of insect pests. 

Mycogen Seeds introduced the first genetically engineered corn to express the insecticidal 

protein that occurs naturally in the soil bacterium Bacillus thuringiensis (Bt) in 1996. Now, Bt 

corn is one of the most widely adopted genetically engineered crops. In 2009, 63% of field corn 

planted in the United States was transgenic for Bt traits. 

Bt is a naturally-occurring soil borne bacterium that is found worldwide. This bacterium 

produces crystal-like proteins (“Cry” proteins) that selectively kill specific groups of insects. 

Once eaten by the insects, Cry proteins bind to specific “receptors” on the intestinal lining and 

rupture the cells. If enough toxins are eaten, the specific insects will die. Bt corn was created by 

inserting selected cry genes into the corn plant. 

The 4114 maize is an insect-resistant and herbicide tolerant product. Pioneer used recombinant 

DNA techniques to develop 4114 maize event which contains cry1F, cry34Ab1, cry 35Ab1 and 

pat genes. The cry1F gene derived from the bacterium Bacillus thuringiensis subsp. aizawai.

This gene encodes a Cry1F insecticidal protein that controls specific lepidopteran pests of corn. 

The Cry1F and associated genetic elements from 4114 maize are identical to those in 1507 

maize, which was given non-regulated status by USDA-APHIS in 2001 (Petition 00-136-01p). 

 

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Section 403 (14) of the Plant Protection Act (7USC Sec 7702(14) defines plant pest as: “Plant Pest - The term “plant pest” means any living 

stage of any of the following that can directly or indirectly injure, cause damage to, or cause disease in any plant or plant product: (A) A 

protozoan. (B) A nonhuman animal. (C) A parasitic plant. (D) A bacterium. (E) A fungus. (F) A virus or viroid. (G) An infectious agent or other 

pathogen. (H) Any article similar to or allied with any of the articles specified in the preceding subparagraphs.”
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

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Both cry34Ab1 and cry35Ab1 genes are derived from the bacterium Bacillus thuringiensis strain 

PS149B1. The Cry34Ab1 and Cry35Ab1 proteins together comprise an active binary insecticidal 

crystal protein that confers resistance to certain corn rootworm pests. The cry34Ab1 and 

cry35Ab1 and associated genetic elements from 4114 are identical to those in 59122 maize, 

which was deregulated by USDA-APHIS in 2005 (Petition 03-353-01p). The 4114 also 

expresses the pat gene, which is derived from the bacterium Streptomyces viridochromogenes. 

This gene encodes a phosphinothricin-N-acetyltransferase (PAT) enzyme. PAT detoxifies 

glufosinate and thereby confers tolerance to herbicides based on this active ingredient. The 

herbicide tolerance provides a weed management tool for farmers and a method of selecting for 

transgenic corn in the laboratory. 

The 4114 maize is a new maize line that combines genes and traits from two maize lines that had 

previously been given non-regulated status: 1507 maize deregulated by USDA in 2001, which 

expresses the Cry 1F and PAT proteins, and 59122 maize deregulated by USDA in 2005, which 

expresses the Cry 34Ab1, Cry 35Ab1 and PAT proteins. From the breeding stack of these two 

lines (1507 maize and 59122 maize), 1507 x 59122 maize was developed and is grown widely in 

the U.S. Globally, many countries and the European Union have approved and used 1507 maize 

as food/feed since 2002 and have approved and used 59122 since 2005 (ISAAA Briefs 39 2008). 

4114 and 1507 x 59122 have identical genes and their genetic elements, but the cry and pat

genes for1507 x 59122 maize are located in two unlinked loci. The purpose in developing 4114 

is to have all these inserted genes on a single transformation construct that has been integrated at 

a single genetic locus in the maize genome. 4114 will have an advantage over1507 x 59122 

maize because having three linked traits at a single locus will simplify breeding efforts. 

APHIS BRS completed plant pest risk assessments and Environmental Assessments (EAs) for 

1507 maize and 59122 maize (http://www.aphis.usda.gov/biotechnology/not_reg.html). The EAs 

fully addressed all resource areas of potential concern. In both petitions, APHIS concluded on 

the basis of the EA that the impacts would not be significant. The agency issued Findings of No 

Significant Impacts (FONSI) and made determinations of nonregulated status for each. The 

Cry1F, Cry34Ab1, Cry35Ab1and PAT proteins have a history of safe use in agricultural crop 

commodities. Cry1F protein and PAT protein have been present in commercial maize varieties 

such as 1507 since 2003. Cry34Ab1, Cry35Ab1 and PAT protein have been present in 

commercial maize varieties such as 59122 and 1507 x 59122 maize since 2006. PAT protein has 

been present in a numerous commercial GE crops planted in the U.S since 1996.

Description of the Modification—Genetic material inserted and protein 

produced

4114 maize was produced using Agrobacterium tumefaciens mediated transformation of Pioneer 

proprietary inbred line PHWWE (Pioneer 2011, pp. 30-34). The A. tumefaciens strain

LBA4404 that was used to develop 4114 maize was made nonpathogenic by removing tumor 

inducing (Ti) DNA sequences normally present in A. tumefaciens (Koncz and Schell 1986). The 

disarmed A. tumefaciens carried a binary plasmid vector PHP27118 (Pioneer 2011, Figure 3 and 

4, pp. 30-31). In vitro selection of transformation events was based on tolerance to the herbicide 
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

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bialaphos which is rendered non-toxic to plant tissues by the presence of the PAT protein. The 

size of the T-DNA was 11,978 base pairs (bp) and contained a single T-DNA delineated by left 

and right border regions in which there were four expression cassettes: cry1F gene cassette, 

cry34Ab1 gene cassette, cry35Ab1 gene cassette and the pat gene cassette. 

The cry1F expression cassette consisted of the following genetic elements (Pioneer 2011, Table

2, p.32): 

 Promoter, 5’untranslated region (UTR) and intron from the maize polyubiquitin gene 

(Christensen et al., 1992).

 Truncated version of the cry1F gene from Bacillus thuringiensis var. aizawai

 Terminator sequence from the Agrobacterium tumefaciens pTi15955 ORF 25 (Barker et 

al., 1983). 

The cry34Ab1 expression cassette consisted of the following genetic elements (Pioneer 2011, 

Table 2, page 33):

 Promoter, 5’untranslated region (UTR) and intron from the maize polyubiquitin gene 

(Christensen et al., 1992).

 Codon-optimized version of the cry34Ab1gene encoding 14KDa Delta-endotoxin 

parasporal crystal protein form Bacillus thuringiensis strain PS149B1 (Ellis et al., 2002).

 Terminator region from Solanum tuberosum proteinase inhibitor II gene (pinII) (Keil et 

al., 1986).

The cry 35Ab1 expression cassette consisted of the following genetic elements (Pioneer 2011, 

Table 2, page 34):

 Promoter from Triticum aestivum peroxidase including leader sequences (Hertig et al., 

1991).

 Codon-optimized version of the cry35Ab1 gene encoding a 44kDa delta-endotoxin 

parasporal crystal protein from Bacillus thuringiensis strain PS149B1 (Ellis et al., 2002).

 Terminator region from Solanum tuberosum proteinase inhibitor II gene (Keil et al., 

1986).

The pat gene expression cassette consisted of the following genetic elements (Pioneer 2011, 

Table 2, page 34):

 Promoter (CaMV 35S) from Cauliflower Mosaic Virus (Franck et al., 1980).

 Codon-optimized phosphinothricin acetyltransferase (pat) gene from Streptomyces 

viridochromogenes. 

 35S terminator from Cauliflower Mosaic Virus (Franck et al., 1980)

In addition to the above-mentioned genetic elements, the inserted T-DNA also contains short 

noncoding DNA sequences called polylinkers. The polylinkers contain restriction enzyme 
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

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recognition sites and are used for cloning purposes. Also, the T-DNA left and right border 

sequences (T-DNA borders) contain 25 base pairs of DNA that are leftover from the Ti plasmid 

of A. tumefaciens.

Data from Southern blot analyses provided to and reviewed by APHIS, demonstrated that a 

single, intact PHP27118 T-DNA (Pioneer 2011, Figure 13, page 47) was inserted into the 

genome of 4114 maize and that no region from the backbone of plasmid PHP27118 was inserted

(Pioneer 2011, page 43-74). The stability of the introduced genes was determined by eventspecific and gene-specific endpoint PCR analyses for several generations ((Pioneer 2011, pp. 75-

76). The stability integration was further confirmed by the Mendelian inheritance of the T-DNA 

in 4114 maize over four generations (Pioneer 2011, pp. 75-76).

Potential for 4114 Maize to have Altered Disease and Pest Susceptibilities

USDA-APHIS assessed whether 4114 maize is likely to have significantly increased disease and 

pest susceptibility. This assessment encompasses a thorough consideration of the transformation 

process, introduced genes and their genetic elements, and their expression products to cause 

interactions with pests and diseases. 

Transformation Process

APHIS considered the potential for the transformation process to cause or aggravate disease 

symptoms in 4114 maize or other plants or to cause the production of plant pathogens. Wild type 

Agrobacterium tumefaciens carries a tumor-inducing (Ti) plasmid that can be transferred to 

broadleaf plants and cause crown gall disease. Agrobacterium tumefaciens strain LBA4404 

contains a disarmed Ti plasmid that is incapable of inducing tumor formation due to the deletion 

of the phytohormone genes originally present in the Agrobacterium plasmid (Koncz and Schell 

1986). The 4114 maize was produced by an Agrobacterium tumefaciens strain LBA4404 

mediated transformation with plasmid PHP27118 (Pioneer 2011, Page 38). This transformation 

process should not lead to crown gall disease in 4114 maize. Instead, in the T-DNA region 

maize-optimized cry1F, cry34Ab1 and cry35Ab1 genes, as well as the synthetic pat gene and 

their regulatory components necessary for their expression in the maize genome were introduced 

into maize. Agrobacterium-mediated plant transformation has been used widely for decades, has 

not been implicated in causing plant disease, and is highly unlikely to pose a plant pest risk.

Introduced genes and their genetic elements 

APHIS reviewed Southern blot analysis data that demonstrates that 4114 maize plants 

regenerated from the transformation event contain single copy, intact plasmid PHP27118 TDNA. No region from the backbone of plasmid PHP27118 was inserted (Pioneer 2011, pp. 43-

73). The stability of the inserted DNA was also evaluated over several generations (Pioneer 

2011, pp. 75-76). Plant phenotypes were evaluated to ensure stability of the traits during the 

plant breeding process. Mendelian segregation of the inserted genes was analyzed using Chisquare analysis over 5 generations (Pioneer 2011, p. 76). Each generation of plants was treated 

with glufosinate-ammonium to eliminate those plants that were not herbicide-tolerant, reflecting 

a lack of inheritance of the pat gene. Table 7 of the Petition shows the expected and observed 
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

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segregation for the five generations tested. There was no significant deviation from the expected 

1:1 ratio. 

The donor organisms for the cry1F, cry34Ab1, cry35Ab1 and pat genes are soil-inhabiting 

bacteria. Neither of these bacteria are plant or human pathogens, and the Cry1F, Cry34Ab1, 

Cry35Ab1 and PAT proteins encoded by these genes do not cause disease or the production of 

infectious agents in plants. The synthetic, maize-optimized cry34Ab1, cry35Ab1 and pat coding 

sequences were modified for optimal expression in maize, in part, by changing their codon for 

improved expression in maize. The promoter and terminator for the pat gene are derived from 

cauliflower mosaic virus (CaMV) which is a plant viral pathogen (Cauliflower mosaic virus 

causes disease primarily in cruciferous plants). The CaMV 35S promoter and terminator 

sequences are non-coding, regulatory sequences of known function and do not cause disease 

symptoms in plants nor encode for an infectious agent. 

Compositional Analysis

Compositional analyses were conducted to assess whether the composition and nutrient levels in 

grain and forage derived from 4114 maize were comparable to those in the conventional maize 

variety. Compositional comparisons between 4114 maize and conventional controls were 

performed using the principles and analytes outlined in the OECD consensus documents for 

maize composition (OECD, 2002 ) for new varieties of maize. The comparators were nontransgenic near-isoline maize lines with about 99% genetic similarity to 4114 maize in 

compositional comparisons. Grain and forage samples were also collected from non-modified 

maize hybrids in two different experiments. Four hybrid lines were analyzed in 2003 and an 

additional four hybrid lines were analyzed in 2007. The conventional commercial maize hybrids 

selected by Pioneer for both studies were normally planted commercially in their adapted 

geographic region (Pioneer 2011, p. 101). A total of eight conventional maize reference varieties 

were included to provide data for the development of a 99% tolerance interval for each 

experiment. For each compositional experiment, a 99% tolerance interval was calculated. This 

interval is expected to contain, with 95% confidence, 99% of the values obtained from the 

population of commercial reference varieties. By comparison to the 99% tolerance interval, any

statistically significant differences between 4114 maize and the comparators may be put into 

perspective, and can be assessed for biological relevance in the context of the natural variability

in maize. In addition, based on published literature (e.g. Codex 1996; Codex 2005; ILSI 2006) 

for maize, a combined literature range has been established. The 4114 maize analyte ranges that 

fell within the tolerance interval and/or combined literature range for that analyte were 

considered to be within the range of normal variability of commercial maize hybrids. 

Pioneer’s compositional analyses of 4114 maize and a near-isoline control were based on forage 

and grain harvested from 2010 at six field locations in the U.S. and Canada (Pioneer 2011, Table 

28, p. 131). Compositional analyses of grain samples included protein, fat, acid detergent fiber, 

neutral detergent fiber, ash, carbohydrates, fatty acid, vitamins and minerals, key anti-nutrients, 

and key secondary metabolites. Compositional analyses of forage samples included protein, fat, 

acid detergent fiber, neutral detergent fiber, ash, carbohydrates, calcium, and phosphorus. 
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

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Pioneer also measured the concentrations of all four expressed proteins in various growth stages

(Pioneer 2011, Table 8, p. 78). The Cry1F, Cry34Ab1, and Cry35Ab1 protein concentrations in 

4114 maize in each tissue were compared to respective protein concentrations in 1507, 59122, 

and/or 1507 x 59122 maize (Pioneer 2011, Table 10-13). The results for each study on 

environmental effects for Cry1F and Cry34/35Ab1 are summarized in US EPA Biopesticides 

Registration Action Documents (BRAD) (US-EPA 2010a; US-EPA 2010b). Complete reviews 

of each study can be found in the individual EPA Data Evaluation Reports. Cry1F and binary

Cry34Ab1/Cry35ab1 proteins have been well established (US-EPA 2010a; US-EPA 2010b;

USDA-APHIS 2001; USDA-APHIS 2005). The Cry1F, Cry34Ab1, Cry35Ab1 and PAT proteins 

expressed in 4114 maize are generally lower than or comparable to protein expressed in 

previously approved 1507, 59122 and 1507 x 59122 maize lines. The only notable exceptions 

were concentrations of Cry1F in 4114 maize pollen. The concentration of Cry1F protein in 4114 

maize pollen (35μg/g tissue dry weight) is approximately 1.5 times that of 1507 or 1507 x 59122 

maize (Pioneer 2011, Table10). Based on the Expected Environmental Concentration (EEC) of 

Cry1F in 4114 maize pollen (35μg/g tissue dry weight), the calculated Margin of Exposure 

(MOE) from 4114 maize still indicates a sufficient margin of safety for the expected 

environmental exposure to the Cry1F protein from 4114 maize pollen (Pioneer 2011,Table 12).

Overall, a comprehensive evaluation of 4114 maize and the controls showed no biologically

meaningful differences for grain and forage compositions either for major nutrients (Pioneer 

2011, Table 15-19, pp. 104-117) or key anti-nutrients in maize grain (Pioneer 2011, Table 20, p. 

119). The few detected differences were either exceedingly small in magnitude or the mean 

component values of 4114 maize and the control were within the 99% tolerance interval. 

Therefore, based on the data presented by Pioneer on forage and grain, it is reasonable to assume 

that the foods and feeds derived from 4114 maize can be considered compositionally and 

nutritionally equivalent to those derived from conventional maize. 

Agronomic Properties

Pioneer conducted agronomic evaluations on 4114 maize and conventional and /or control 

maize. These evaluations are used to determine whether 4114 maize is agronomically

comparable to conventional maize and they provide reasonable scientific measures as to whether 

4114 maize has plant pest potential. These assessments included 14 agronomic parameters and 

three seed germination parameters, two pollen characteristics (shape and color), and several 

observations on pest response (disease incidence and insect damage) (Pioneer 2011, Table 30

and 31, pp. 135-140). The agronomic data showed no significant differences between 4114 

maize and control maize. These data support Pioneer’s claim that 4114 maize is agronomically

comparable to conventional maize except for the intended traits and that 4114 maize does not 

possess characteristics that constitute a plant pest risk compared to conventional maize. 

The 4114 maize and its control lines have been field tested in the U.S. and Puerto Rico since 

2006. For each field trial, a plant pathologist, entomologist and plant breeder surveyed the field 

at least every four weeks. The observations of naturally occurring insects, diseases and any

unexpected differences between 4114 maize and control lines were summarized in Table 8.2 of 

the Petition. APHIS has reviewed the data and found that 4114 maize did not exhibit any
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

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meaningful differences compared with its non-transgenic counterparts for naturally-occurring 

insect or disease infestations. 

The data (transformation method, introduced genes and their genetic elements, compositional 

analysis and agronomic properties) cited by Pioneer and reviewed by APHIS indicate that 4114 

maize is not biologically different from non-transgenic conventional maize lines (with the 

exceptions of intended introduced genetic constructs and traits) and 4114 maize is no more 

susceptible to pests and diseases compared to conventional maize. The introduced genetic 

constructs and traits (certain lepidopteran insect and corn rootworm insect resistance and 

tolerance to glufosinate herbicides) are not expected to alter disease and pest susceptibilities. 

Potential of 4114 maize to Impact the Weediness of Other Plants with Which 

It can Interbreed.

In the U.S., corn is not listed as a weed (Crockett 1977; Muenscher 1980), nor is it present in the 

Federal Noxious Weed List (7 CFR part 3602). Furthermore, corn is grown throughout the world 

without any report that it is a serious weed or that it forms persistent feral populations (Gould 

1968). Like many domesticated crops, corn seed from a previous year’s crop can overwinter and 

germinate the following year. Manual or chemical measures are often applied to remove these 

volunteers, but the plants that are not removed do not typically result in feral populations in 

following years because maize is incapable of sustained reproduction outside of domestic 

cultivation and maize are non-invasive in natural habitats (Gould 1968). Corn possesses few of 

the characteristics of those plants that are notably successful as weeds (Baker 1965; Keeler 

1989).

APHIS assessed whether 4114 maize is any more likely to become a weed than the isogenic

non-transgenic corn line or other corn varieties currently under cultivation. The assessment

encompasses a thorough consideration of the basic biology of corn and an evaluation of the 

unique characteristics of 4114 maize. Pioneer has been conducting agronomic evaluations in 

both laboratory experiments and field trials. 

The germination and dormancy evaluations were tested under laboratory conditions. Seed 

dormancy is an important characteristic that is often associated with plants that are considered 

weeds (Anderson 1996). Although dormancy is not associated with modern maize cultivars,

maize seed dormancy tests can be used to determine whether 4114 maize is agronomically 

comparable to conventional maize and determine whether 4114 maize is more likely to pose a 

plant pest risk when compared to conventional maize. Standardized germination assays of the 

Association of Official Seed Analysts (AOSA 2007) are used as a baseline to measure the 

germination potential. These assays evaluate various germination parameters at the optimum 

temperature for growth and at a few other temperature regimes to assess other seed germination 

properties (Pioneer 2011, Table 23, page 126). A near-isoline control (approximately 99% 

similar) was used for comparison and two commercial maize lines were evaluated in the study to 

establish a reference range for germination and dormancy. No statistically significant differences 

 

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were detected. The results (Pioneer 2011, Table 24-27, pp. 127-128) support the conclusion that 

4114 maize is comparable to conventional maize with respect to germination and potential 

dormancy and 4114 maize does not exhibit characteristics that would cause it to be weedier than 

the parental lines or conventional varieties.

A total of 14 different agronomic phenotypic characteristics, as well as observations for plant 

responses to plant-disease and plant-insect interactions were evaluated by Pioneer (Pioneer 2011,

Table 29, pp. 133-134) at 15 U.S. locations and 2 Canadian locations across maize production 

regions in 2010 (Pioneer 2011, Table 28, page 131). At these 17 locations, the range of values 

for agronomic parameters was within the range of values expected for non-modified commercial

maize hybrids. Agronomic characteristic evaluated (Pioneer 2011, Table 29, pp. 133-134) 

included: early population, seedling vigor, plant height, ear height, stalk lodging, root lodging, 

final population, stay green, time to silking, time to pollen shed, yield, pollen viability, disease 

incidence and insect damage. No differences in phenotypic characteristics that might contribute 

to enhance the fitness 4114 maize, including weediness traits, were identified. The mean values

of these agronomic traits observed for 4114 maize fell within the range of values observed for 

the commercial maize varieties. Field trial data (Pioneer 2011, Table 30 and Table 31, pp. 135-

140) indicated that 4114 maize does not exhibit characteristics that would cause it to be weedier 

than the near-isoline control and non-modified conventional maize. 

In addition, agronomic field observation data showed no significant differences between 4114 

maize and the non-transgenic counterparts for disease incidence and insect damage (other than 

resistance to the targeted lepidopteran and corn rootworm pests). The introduced traits, European 

corn borer, western corn rootworm resistance, and glufosinate ammonium herbicide tolerance, 

are not expected to cause 4114 maize to become a weed. The 1507 maize, which expresses 

Cry1F protein confers resistance to certain lepidopteran pests (including European corn borer) 

and was given non-regulated status by USDA-APHIS in 2001 (USDA-APHIS 2001). The 59122 

maize, which expresses Cry34Ab1/ Cry35Ab1 protein together, confers resistance to corn 

rootworm pests and was also given non-regulated status by USDA-APHIS in 2005 (USDAAPHIS 2005). The PAT protein provides tolerance to glufosinate-ammonium herbicides and has 

been in various commercially grown crops given non-regulated status by USDA-APHIS since 

1996 (e.g., Petition 96-068-01p, Petition 97-205-01p, Petition 98-238-01p, etc. found here: 

http://www.aphis.usda.gov/biotechnology/not_reg.html ). There is no linkage between these

proteins’ expressions and any increased survival or overwintering capacity that would alter the 

prevalence of volunteer plants in subsequent growing seasons. The 1507 x 59122 maize hybrids

have been on the market since 2005. In 2010, 1507 x 59122 maize was grown commercially on 

approximately 16% of U.S. maize acres (Pioneer 2011, page 32). After many years of 

commercialization , 1507 maize, 59122 maize and 1507 x 59122 maize hybrids have not 

exhibited characteristics that would indicate weediness. There is no reason to expect that 4114 

maize would result in increased weediness, since 4114 maize contains the same Cry1F, 

Cry34Ab1, Cry35Ab1 and PAT proteins and will provide similar insect resistance and herbicide 

tolerance to that of1507x59122 maize. The above considerations, together with the fact that the 

novel traits have no intended effect on weediness, leads USDA-APHIS to conclude that 4114 

maize has no altered weediness potential compared to current commercialized varieties. Field 

evaluation of phenotypic and agronomic characteristics showed no differences relative to its 
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

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comparator(s), and supports the conclusion that 4114 maize is not likely to have increased 

weediness compared to conventional maize varieties. 

Potential for Gene Flow and Gene Introgression from 4114 Maize into 

Sexually-Compatible Relatives

APHIS evaluated the potential for gene introgression to occur from 4114 maize to sexually

compatible wild relatives and considered whether such introgression would result in increased 

weediness. Introgression is a process whereby gene(s) successfully incorporate into the genome 

of a recipient plant. 

Maize belongs to the grass family, Poaceae. The genus Zea has five species: diploperennis HH, 

luxurians, mays, nicaraguensis, and perennis (OGTR 2008). Zea mays (2n=20) has two 

subspecies of which Z. mays ssp mays is the only cultivated species. Zea mays is common in the 

US and is known only from cultivation. Occasionally it is found in abandoned fields or on 

roadsides. The closest wild relatives of maize are the teosintes which are sexually compatible 

with Zea mays. All teosinte members can be crossed with cultivated corn to produce fertile first 

generation hybrids (Doebley1990a; Wilkes 1967). However maize teosinte hybrids exhibit low 

fitness and have little impact on gene introgression in subsequent generation (Galinat, 1988). 

Additionally, teosintes are normally confined to the tropical and subtropical regions of Mexico, 

Guatemala, and Nicaragua. A fairly rare, sparsely dispersed feral population of teosinte has been 

reported in Florida (USDA PLANTS Database3, accessed 11/18/2011).

Maize is a predominantly an outcrossing plant species. The rate of self-pollination is 5% (Sleper 

and Poehlman, 2006). The short viability period of pollen grains limits the possibility of 

outcrossing. Since 4114 maize does not exhibit characteristics that can cause it to be any weedier

than other cultivated corn, its potential impact due to the limited potential for gene introgression 

into teosinte is not expected to be any different from that of other cultivated maize varieties.

Tripsacum is a genus of seven species, three of which occur in the US (Gould and Shaw 1983). 

Although, it is difficult, Tripsacum can be successfully hand crossed with maize to form hybrids. 

However these hybrids have a high degree of sterility (Doebley 1990a; Wilkes 1967) and are 

generally unstable because of differences in chromosome number and lack of pairing between 

chromosomes (Eubanks 1997). First generation hybrids are much less fit for survival and 

dissemination in the wild and typically show reduced reproductive capacity. Furthermore, gene 

flow from maize to Tripsacum is virtually impossible because of several factors including 

distribution, genetic incompatibility, temporal separation of flowering time, etc. (Galinat 1988).

None of the sexually compatible relatives of maize in the U.S. are considered to be weeds in the 

U.S. (Holm et al., 1979), therefore, the unlikely acquisition of an herbicide tolerance gene and 

three insect tolerance cry genes would not be expected to transform them into weeds. Therefore, 

USDA has concluded that adverse consequences of gene flow from 4114 maize to wild or weedy

species in the U.S. are highly unlikely.

 

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Potential Effects on Non-target Organisms, Including those Beneficial to 

Agriculture.

APHIS evaluated the potential for 4114 maize to have damaging or toxic effects directly or 

indirectly on non-target organisms. Non-target organisms considered were representatives of the 

exposed species in the agriculture environment. 

For previous EPA registrations, Pioneer/DuPont submitted the Safety Assessments evaluating

the effect of Cry1F, Cry34/35Ab1, and Cry1F x Cry34/35Ab1maize events on required and 

voluntary host range species. EPA reviewed and concluded that the levels of Cry1F, Cry34Ab1, 

Cry35Ab1 and Cry1F x Cry34/35Ab1 proteins in 1507, 59122 and 1507 x 59122 maize will not 

pose unreasonable adverse effects to corn field flora and fauna (US-EPA, 2010a; US-EPA 

2010b; US-EPA, 2010c). Available data also indicate that there should be minimal short-term 

accumulation of Cry proteins in agricultural soil. No evidence of synergy between the 

lepidopteran-active and coleopteran-active insecticidal proteins expressed by 1507 x 59122 

maize was found in laboratory studies of target and non-target insects evaluated for the

registration. The proteins expressed in 4114 maize are identical to those in previously approved

events 1507, 59122 and 1507 x 59122 maize (Pioneer 2011, Section 2 and Appendix 2).

Therefore, previous safety studies conducted in 1507 and 59122 and 1507x59122 maize should 

be relevant for 4114 maize. 

In addition, these Cry proteins do not share any amino acid sequence similarities with known 

protein toxins which have adverse effects on mammals. Regarding allergenicity potential, all 

expressed Cry proteins originate from a non-allergenic source. Cry proteins have no sequence 

similarities or homology compared with known allergens; the Cry proteins will only be present at 

low levels in food; and the proteins expressed in 4114 are not glycosylated. The potential for any 

of the expressed proteins to be food allergens is minimal.

The pat gene comes from Streptomyces viridochromogenes and encodes the enzyme 

phosphinothricin acetyltransferase. The PAT protein homologs are likely ubiquitous in the 

environment. PAT protein shares no significant homology with any protein known to be toxic or 

allergenic (OECD 1999; ILSI 2011). A dose equivalent to 5000 mg/kg body weight was used for 

the PAT toxicity study. No signs of toxicity were seen in any of the groups of mice tested over a 

period of 14 days. Acute toxicity studies in mice show the PAT protein has no toxicity even at 

doses much higher than mice could encounter due to exposure to GE plants expressing the PAT 

protein (Herouet et al. 2005). In addition, the PAT protein is rapidly digested in experiments 

simulating the gastric environment (Herouet et al. 2005). Eight plant species expressing PAT 

proteins have been approved for environmental release in 11 different countries and have been 

well documented (ILSI 2011). Data from peer-reviewed literature show that the PAT protein 

expressed in GE plants has negligible impact on the phenotype of those plants (ILSI 2011). Since 

PAT protein is widespread in the environment, Canadian, Australian, Japanese and U.S. 

regulatory authorities have concluded that the expression of PAT protein in GE plants does not 

have any significant potential to adversely impact other organisms (CFIA 1995; OGTR 2003; 

Japanese BCH 2010; USDA APHIS 1998).
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

11

Pioneer also assessed the non-target impact of 4114 on beneficial organisms in the corn 

agroecosystem. At present, APHIS is not aware of any identified significant adverse effects of 

Cry1F, Cry34/35Ab1 and PAT proteins on the abundance of non-target beneficial organisms in 

the field. Field testing reports submitted to APHIS showed minimal to undetectable changes in 

the beneficial insect abundance or diversity. To date, available field test data show that compared 

to crops treated with conventional chemical pesticides, the transgenic crops have no detrimental 

effect on the abundance of non-target insect populations.

The 4114 maize is comparable in agronomic characteristics and compositional characteristics to 

non-transgenic conventional maize except for the presence of the introduced proteins. As 

described earlier, the Cry1F and PAT proteins are identical to those in previously approved 1507 

maize; Cry34Ab1, Cry35Ab1 and PAT are identical to those in previously approve 59122 maize 

and PAT protein has been approved across several crops to use commercially in U.S. since 1996. 

In 2010, 1507 x 59122 maize was grown on approximately 16% of U.S. maize acres. Many years 

of data on these proteins demonstrate the lack of toxicity to humans and animals, and the absence 

of adverse effects on non-target organisms and the environment.

Potential for Transfer of Genetic Information to Organisms with which 4114 

maize Cannot Interbreed.

APHIS examined the potential for the new genetic material inserted into 4114 maize to be 

horizontally transferred to other organisms without sexual reproduction and whether such an 

event could lead directly or indirectly to disease, damage, injury or harm to plants. Horizontal 

gene transfer and expression of DNA from a plant species to other species is highly unlikely to 

occur based on the following reasons.

The horizontal gene transfer (HGT) between unrelated organisms is one of the most intensively

studied fields of science. Horizontal gene transfer and expression of DNA from a plant species to 

bacteria or animal species is unlikely to occur. A number of points support this conclusion:

1. Many genomes (or parts thereof) from bacteria that are closely associated with plants

have been sequenced including Agrobacterium and Rhizobium (Kaneko et al., 2000; Wood et al., 

2001; Kaneko et al., 2002). There is no evidence that these organisms contain genes derived 

from plants. Also, Bacillus thuringiensis species are generally common in soil and therefore 

various cry genes have been available for long periods of time for horizontal transfer from 

Bacillus thuringiensis species to plants or soil microorganisms and decaying plant material. 

Therefore the likelihood of any impact or new horizontal gene transfer that is not already capable 

of taking place in the soil is extremely unlikely.

2. No evidence has been identified for any mechanism by which maize genes could be 

transferred to humans or animals, or any evidence that such gene transfer has occurred for any

plant species during evolutionary history, despite animals and humans eating large quantities of 

plant DNA. In cases where review of sequence data implied that horizontal gene transfer 

occurred, these events are inferred to occur on an evolutionary time scale on the order of millions 

of years (Koonin et al., 2001; Brown 2003). 
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

12

3. Transgene DNA promoters and coding sequences are optimized for plant expression, not 

prokaryotic bacterial expression. Thus even if horizontal gene transfer occurred, proteins 

corresponding to the transgenes are not likely to be produced. 

4. FDA has evaluated horizontal gene transfer from the use of antibiotic resistance marker 

genes, and concluded that the likelihood of transfer of antibiotic resistance genes from plant 

genomes to microorganisms in the gastrointestinal tract of humans or animals, or in the 

environment, is extremely unlikely (FDA 1998). 

5. APHIS also considered whether horizontal transfer of DNA from 4114 maize to plant

viruses was likely to occur and would lead to the creation or selection of a more virulent plant 

pathogen through recombination with other plant viruses. This issue has been considered before 

by other science review panels and government regulatory bodies (Keese 2008). Although 

sequences of the Cauliflower Mosaic Virus are contained within 4114 maize, those sequences are 

limited to the regulatory elements. Regulatory elements such as promoters and terminators have 

not been implicated in viral recombination. 

Finally, under natural conditions; no transfer of an intact functional gene has been demonstrated 

to date (Miki and McHugh, 2004).Therefore APHIS concludes that horizontal gene transfer is 

unlikely to occur and thus poses no plant pest risk.

Potential Changes to Agricultural or Cultivation Practices.

APHIS considered potential impacts associated with the cultivation of European corn borerresistant, corn rootworm-resistant and glufosinate-ammonium tolerant 4114 maize on current 

agricultural practices in corn and other crops.

Pioneer has provided data which indicate that 4114 maize expresses the Cry1F, Cry34Ab1 and 

Cry35Ab1 and PAT proteins to allow control of major corn pests and weeds. Using seeds 

containing 4114 maize is expected to be less expensive and likely to replace commercial 

products containing 1507 x 59122 maize. Compared to currently-available Bt and herbicide 

tolerant products containing 1507 x 59122 maize, no increased use of insecticides or herbicides 

from the use of 4114 maize is expected. 

Bt corn has been available since 1996. Planting of Bt corn grew from about 8 percent of U.S. 

corn acreage in 1997 to 65 percent in 2011 (USDA-ERS 2011).The NASS June Agriculture 

Survey (USDA-ERS 2011) indicated the increases in acreage share in recent years may be 

largely due to the commercial introduction of a new variety of Bt corn that controls corn 

rootworm, a pest that may be more destructive to corn yield than the European corn borer. The 

4114 maize contains both rootworm and European corn borer resistance. Some reduction of 

chemical insecticide and herbicide use by growers is expected (vs. conventional corn). The 

reduced chemical pesticide use will benefit the environment directly and can mean less exposure 

to people who apply chemical pesticides to corn. The availability of multiple Bt corn products 

will also increase grower choice and price competition.
Plant Pest Risk Assessment for Insect-Resistant and Herbicide-Tolerant Maize 4114

13

Finally, given that 4114 maize is highly likely to constitute a replacement product for similar 

corn lines that are already grown on significant acres in the U.S. and which do not present plant 

pest risks, APHIS concludes that 4114 maize also does not present a plant pest risk. 

Conclusion

APHIS has prepared the plant pest risk assessment in order to determine if 4114 maize is 

unlikely to pose a plant pest risk. Based on the information provided by the applicant and the

lack of plant pest risk from the inserted genetic material, weedy characteristics, atypical 

responses to disease, insects or plant pests in the field, effects on non-targets or beneficial 

organisms in the agro-ecosystem, changes to agricultural practices, and horizontal gene transfer, 

APHIS has concluded that 4114 maize is highly unlikely to pose a plant pest risk. 

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