Request for Extension of Determination of Nonregulated Status to the Additional Regulated 

Article:

Maize Line HCEM485

The undersigned submits this request under 7 CFR Part 340.6(e) to request an extension of 

determination of nonregulated status that the article should not be regulated under 

7 CFR Part 340.

Submitted by:

Harry H. Stine

President

Stine Seed Farm, Inc.

22555 Laredo Trail

Adel, Iowa 50003

USA

Revised Document Submitted:

14 February 2011

Contributors:

B. Held, Ph.D., V. Sekar, Ph.D., J.D. Behn, J.T. Mason and K. Muir

Stine Seed Farm Petition Number 09-063-01p_a3 - Revised
Maize Line HCEM485

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Summary

Glyphosate herbicide-tolerant maize line HCEM485 was produced by introducing a 6.0 kb 

maize genomic fragment, originally isolated from a bacterial artificial chromosome (BAC) 

library derived from the maize inbred line B73, containing a modified form of the endogenous 

maize EPSPS encoding gene. DNA introduction was via aerosol beam injector, which is a 

naked DNA delivery method.

The 6.0 kb fragment contained the endogenous maize epsps expression cassette including native 

promoter, coding sequence, intron, and termination regions. The maize EPSPS coding sequence 

was specifically modified by site-directed mutagenesis to introduce two single-nucleotide substitutions. These two point mutations resulted in a codon change from threonine→isoleucine 

at position 102 (relative to the amino acid sequence of the native maize EPSPS enzyme) and 

a proline→serine change at position 106. These two amino acid substitutions result in a glyphosate-tolerant form of the enzyme and are also present in the modified EPSPS enzyme produced in the antecedent organism, transgenic maize event GA21. Except for the amino acid 

substitutions at positions 102 and 106, the amino acid sequence of the double-mutated EPSPS 

(2mEPSPS) enzyme is identical to the native maize EPSPS sequence.

The only DNA sequences introduced into maize line HCEM485 were those derived from maize 

following the introduction of two point-mutations resulting in the expression of a glyphosatetolerant form of the native EPSPS enzyme. Maize line HCEM485 does not contain any heterologous DNA sequences, either coding or non-coding, from any other species, including those 

that could be considered a plant pest. In addition, the genetic modification process resulting 

in maize line HCEM485 did not employ any organism (e.g., Agrobacterium tumefaciens) that 

could be considered a plant pest.

The introduced sequences in maize line HCEM485 are contained within a single genetic locus 

within the maize genome as demonstrated by Southern blot analysis and Mendelian inheritance 

studies. The modified maize EPSPS protein expressed in maize line HCEM485 is intact, of 

the expected molecular weight and there was no evidence of truncated forms of the enzyme. 

The modified maize EPSPS expressed in HCEM485 maize is also immunochemically crossreactive with the modified maize EPSPS expressed in the antecedent organism, GA21, and the 

enzymes from both sources express the same mutations responsible for conferring glyphosate 

herbicide tolerance.

Agronomic and phenotypic characteristics of an HCEM485 maize hybrid and three control 

hybrids were evaluated in a series of field trials across 15 United States Corn Belt locations in 

2007. The agronomic characteristics chosen for comparison were those typically observed by 

professional maize breeders and agronomists and represented a broad range of characteristics 

throughout the development of the maize plant. Results of these trials suggest that there were 

no biologically significant unintended effects on plant growth habit and general morphology, 

vegetative vigor, flowering and pollination, grain yield, grain test weight, or disease susceptibility as a result of the genetic modification introduced into maize line HCEM485. These data 

support the conclusion that HCEM485-derived hybrids are unlikely to form feral persistent 

populations, or to be more invasive or weedy than conventional maize hybrids, and would not 

display higher rates of outcrossing than unmodified maize.
Maize Line HCEM485

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Levels of key nutrients, minerals, antinutrients, and secondary metabolites were determined 

in samples of maize grain and forage derived from HCEM485 and control hybrids collected 

from up to four field trial locations in 2007. For most analyses, there were no statistically 

significant differences and in cases where statistically significant differences were observed, 

the magnitudes of the differences were small and in every case, mean values determined for 

both HCEM485 and control samples were within the ranges of natural variation as reported in 

the literature. Overall, no consistent patterns emerged to suggest that biologically significant 

changes in composition of the grain or forage had occurred as an unintended consequence of 

the genetic modification resulting in maize line HCEM485. The conclusion based on these data 

was that grain and forage from HCEM485 maize were substantially equivalent in composition 

to both the control hybrids included in this study, and to other commercial maize hybrids.

In conclusion, there is no expectation that cultivation of maize line HCEM485 would have 

any environmental effects different from the cultivation of the antecedent organism, GA21, or 

other maize lines exhibiting glyphosate tolerance that have also been deregulated by USDAAPHIS (e.g., NK603; MON 88017; and MON 802). Therefore, on the basis of the substantial 

phenotypic equivalence between maize line HCEM485 and the antecedent organism, GA21, 

Stine Seed Farm requests that an extension of nonregulated status be granted to maize line 

HCEM485, any progeny derived from crosses between HCEM485 and conventional maize, 

and any progeny derived from crosses of HCEM485 with other deregulated maize lines.

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Certification

The undersigned certifies, that to the best knowledge and belief of the undersigned, this petition 

includes all information and views on which to base a determination, and that it includes all 

relevant data and information known to the petitioner which are unfavorable to the petition.

_______________________________

Harry H. Stine

President

Stine Seed Farm, Inc.

22555 Laredo Trail

Adel, Iowa 50003

USA

Phone: 515-677-2605

Fax: 515-677-2716

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Abbreviations Used in This Petition

2mEPSPS double-mutated EPSP synthase; native maize EPSPS containing 

Thr-102→Ile and Pro-106→Ser substitutions.

AI active ingredient

APHIS Animal and Plant Health Inspection Service

BAC bacterial artificial chromosome

bp base pairs

CBI confidential business information

CTP chloroplast transit peptide

DW dry weight

ELISA enzyme-linked immunosorbent assay

EPSP 5-enolpyruvylshikimate-3-phosphate

EPSPS 5-enolpyruvylshikimate-3-phosphate synthase

FDA Food and Drug Administration

FW fresh weight

g gram

GRAS generally recognized as safe

HRP horseradish peroxidase

ILSI International Life Sciences Institute

kb kilobases

kg kilogram

LOQ limit of quantification

μg microgram

mg milligram

μm micrometer

MW molecular weight

ND not determined

NOS nopaline synthase

OECD Organization for Economic Cooperation and Development

OTP optimized transit peptide

PEP phosphenolpyruvate

S3P shikimate-3-phosphate

SD standard deviation

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

TIPS threonine to isoleucine mutation at position 102 and proline to serine mutation at position 106, relative to the amino acid sequence of 

the native maize EPSP synthase enzyme.

USDA United States Department of Agriculture
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Contents

I. Rationale for Submission of Request for Extension of Nonregulated Status ............9

I.1 Basis for the Request...............................................................................................9

I.2 Rationale for the Development of Maize Line HCEM485 ...................................10

II. The Maize Family ....................................................................................................11

III. Description of the Transformation System...............................................................11

III.1 Donor Genes and Regulatory Sequences..............................................................11

III.2 The Vector pHCEM...............................................................................................12

III.3 Plant Transformation and Regeneration................................................................14

IV. Genetic Analysis of Maize Line HCEM485 ............................................................14

IV.1 Molecular Characterization...................................................................................14

IV.2 Inheritance and Stability .......................................................................................18

IV.3 Gene Expression....................................................................................................20

IV.3.1 2mEPSPS Integrity and Equivalence to mEPSPS in Event GA21 ....................20

IV.4 2mEPSPS Protein Safety.......................................................................................22

IV.5 Conclusions...........................................................................................................23

V. Phenotypic and Agronomic Characteristics .............................................................23

V.1 Trial Design and Evaluation..................................................................................24

V.1.1 Plant Material.....................................................................................................24

V.1.2 Trial Locations...................................................................................................25

V.1.3 Agronomic Traits Assessed................................................................................25

V.1.4 Experimental Design and Statistical Analysis ...................................................27

V.2 Growth Habit.........................................................................................................27

V.3 Vegetative Vigor....................................................................................................28

V.4 Reproductive Characteristics ................................................................................28

V.5 Yield and Grain Characteristics ............................................................................29

V.6 Disease Observations............................................................................................30

V.7 Pollen Morphology and Viability..........................................................................30

V.8 Compositional Analysis ........................................................................................31

V.8.1 Proximates..........................................................................................................32

V.8.2 Minerals .............................................................................................................34

V.8.3 Vitamins.............................................................................................................36

V.8.4 Amino Acids ......................................................................................................37

V.8.5 Fatty Acids.........................................................................................................38

V.8.6 Secondary Metabolites and Antinutrients..........................................................40

V.8.7 Phytosterols........................................................................................................42

V.8.8 Nutritional Impact..............................................................................................42

V.9 Conclusions...........................................................................................................43

VI. Environmental Impact of Introduction of Maize Line HCEM485...........................43

VII. Adverse Consequences of Introduction....................................................................44

VIII. References................................................................................................................44

IX. Appendix 1: HCEM Nucleotide Sequence..............................................................47

X. Appendix 2: USDA APHIS Release Notifications 

Relevant to the Field Testing of Maize Line HCEM485..........................................50
Maize Line HCEM485

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List of Figures

Figure 1: Map of 6.0 kb maize genomic fragment and developmental steps in creating 

maize line HCEM485...............................................................................................12

Figure 2: Amino acid sequence alignments of maize EPSP synthase enzymes. .....................13

Figure 3: Linear diagram of pHCEM plasmid showing all probes used for Southern hybridization.......................................................................................................................14

Figure 4: Southern blot hybridisation of HCEM485 with A/E and A/C probes. ....................15

Figure 5: Putative organization of inserted DNA in HCEM485..............................................17

Figure 6: Southern analysis for the presence of vector sequences in HCEM485...................18

Figure 7: Breeding tree for maize line HCEM485..................................................................19

Figure 8: Western immunoblot analysis of 2mEPSPS protein with specific monoclonal antibody. .........................................................................................................................20

Figure 9: Western immunoblot analysis with polyclonal antisera. ..........................................21

Figure 10:The reaction catalyzed by EPSP synthase...............................................................22

Figure 11:Pedigree chart of HCEM485 seed used in 2007 agronomic trials...........................24

Figure 12:Morphology of pollen from HCEM485 and control hybrids. .................................31

List of Tables

Table 1: Comparison of maize line HCEM485 with event GA21 .........................................10

Table 2: Observed vs. expected hybridising fragments unique to HCEM485 DNA .............16

Table 3: Observed vs. expected segregants for F1 hybrid and F2 selfed generations derived 

from HCEM485 maize.............................................................................................19

Table 4: Trial locations and dates...........................................................................................25

Table 5: List and definitions of agronomic traits assessed in the field trials..........................26

Table 6: Comparison of growth habit characteristics of HCEM485 and control hybrids......28

Table 7: Comparison of vegetative growth characteristics of HCEM485 and control hybrids..........................................................................................................................28

Table 8: Comparison of reproductive characteristics of HCEM485 and control hybrids......29

Table 9: Comparison of yield and grain characteristics of HCEM485 and control hybrids..30

Table 10: Comparison of disease ratings for HCEM485 and control hybrids. ........................30

Table 11: Pollen viability and diameter measurements............................................................31

Table 12: Proximate composition of grain from HCEM485 and control hybrids....................33

Table 13: Proximate composition of forage from HCEM485 and control hybrids..................34

Table 14: Mineral composition of grain from HCEM485 and control hybrids. ......................35

Table 15: Mineral composition of forage from HCEM485 and control hybrids. ....................36

Table 16: Vitamin analysis of grain from HCEM485 and control hybrids. .............................37

Table 17: Amino acid composition of grain from HCEM485 and control hybrids. ................38

Table 18: Fatty acid composition of grain from HCEM485 and control hybrids. ...................40

Table 19: Secondary metabolites and antinutrients in grain from HCEM485 and control 

hybrids......................................................................................................................41

Table 20: Phytosterol composition of grain from HCEM485 and control hybrids..................42
Maize Line HCEM485

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Annexes

Note: The following annexes are included as attachments. 

Annex 1 Agronomic analysis of maize line HCEM485. 

Laboratory Study ID: SSF-07-323.

Annex 2 Morphology and viability of pollen collected from HCEM485 maize. 

Laboratory Study ID: SSF-07-288.

Annex 3 Compositional analysis of grain and forage derived from HCEM485 

hybrid maize grown during 2007 in the USA. 

Report Number: SSF-08-098.

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I. Rationale for Submission of Request for Extension

of Nonregulated Status

I.1	 Basis for the Request

The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA) has been given the responsibility, under the Federal Plant Pest Act (7 U.S.C. 

150aa–150jj) and the Plant Quarantine Act (7 U.S.C. 151–167), to prevent the introduction and 

dissemination into the United States or interstate movement of plant pests. Under this authority, APHIS has published regulations found at 7 CFR Part 340 pertaining to the introduction 

(importation, interstate movement, and release into the environment) of genetically engineered 

organisms and products derived from known plant pests (regulated articles). An organism is 

not subject to the regulations when the organism is demonstrated not to present a plant pest 

risk.

Section 340.6(e) of the regulations provides that APHIS may extend a determination of nonregulated status to additional articles, upon finding that the additional articles do not pose a potential for plant pest risk, and should therefore not be regulated. Such a finding would be made 

based on an evaluation of the similarity of the additional articles to an antecedent organism, 

i.e., an organism that has already been the subject of a determination of nonregulated status by 

APHIS under Section 340.6, and that is used as a reference for comparison to the subject article 

under consideration. 

In its guidance, APHIS has provided the following example of a molecular manipulation that 

is unlikely to pose new risk issues beyond those that would have been considered in the initial 

determination of nonregulated status:

• Modifications in which the amino acid sequence of any encoded proteins is unchanged with respect to the corresponding sequence in the antecedent organism 

(i.e., synonymous codon changes).

When applying this guidance it is clear that a request for an extension of determination of 

nonregulated status for maize line HCEM485 as based upon the previous determination of 

nonregulated status for Roundup Ready® maize line GA21 (petition 97-099-01p) is appropriate. The glyphosate tolerance of maize line GA21 was imparted by the insertion of a doublemutated form of the maize (Zea mays L.) EPSPS encoding gene into the maize genome. In the 

same manner, the glyphosate tolerance in maize line HCEM485 is also based on expression 

of the same modified EPSPS enzyme derived from Z. mays, with the notable difference that 

expression of the modified EPSPS enzyme is regulated by endogenous DNA sequences also 

derived from Z. mays rather than regulatory sequences derived from other species. 

The specific differences between HCEM485 and its progeny, and the event GA21 in the previous petition are discussed in the appropriate sections and also summarized in Table 1.

As a further basis for this request for a determination of nonregulated status, the petitioner 

notes that glyphosate tolerant maize line HCEM485 contains only DNA sequences derived 

from the recipient organism, Z. mays, which is not considered a plant pest; it does not contain 

DNA sequences derived from any organism that could be considered to pose a plant pest risk 
Maize Line HCEM485

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nor was it produced using any organism that could be considered to pose a plant pest risk. On 

this basis, it can be concluded that maize line HCEM485 is unlikely to pose a plant pest risk 

and may, therefore, be afforded nonregulated status.

Table 1: Comparison of maize line HCEM485 with event GA21

Characteristic HCEM485 Event GA21

Crop maize maize

Genus and species name Zea mays Zea mays

Parent line Stine 963 Unspecified

Transformation method Aerosol beam direct DNA transfer Microparticle acceleration direct 

DNA transfer

Trait Tolerance to glyphosate herbicide Tolerance to glyphosate herbicide

Gene product double-mutated EPSPS 

(2mEPSPS)

double-mutated EPSPS

Vector pHCEM pDPG434

Transforming DNA ClaI + EcoRV restriction fragment (ca. 6.0 kb)

NotI restriction fragment (ca. 3.4 

kb)

Gene and source Modified EPSPS-encoding gene 

from Z. mays including native 

introns and exons

Modified EPSPS-encoding gene 

from Z. mays

Targeting sequences Native chloroplast transit sequences from Z. mays EPSPSencoding geneOptimized chloroplast transit 

sequences derived from Z. mays

and Helianthus annus (sunflower) 

ribulose-1,5-bisphosphate carboxylase genes

Promoter and source 5’ region of the maize (Z. mays) 

EPSPS-encoding gene containing 

native promoter sequences

5’ region of the rice (Oryza sativa) actin 1 gene containing the 

promoter and first intron

Terminator and source 3’ nontranslated region of the 

native maize (Z. mays) EPSPSencoding gene

3’ nontranslated region from the 

nopaline synthase (nos) gene 

derived from the Ti plasmid of 

Agrobacterium tumefaciens

I.2	 Rationale for the Development of Maize Line HCEM485

There are no changes in rationale from Section I.A of the previously approved petition number 

97-099-01-p, which briefly discusses the benefits of glyphosate tolerant maize.

Prior to commercialization of maize line HCEM485, Stine Seed Farm will seek the following 

regulatory approvals in the United States:

1. Extension of the existing determination of nonregulated status granted for maize line 

GA21 (97-099-01p) to maize line HCEM485 and all progenies from crosses between 

this line and other maize varieties.

2. Maize line HCEM485 is within the scope of the FDA policy statement concerning 

products derived from new plant varieties, including those genetically engineered, 

published in the Federal Register on May 29, 1992. 
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II. The Maize Family

There are no changes from Section II of the previously approved petition number 97-099-

01p.

III. Description of the Transformation System

The antecedent organism, maize line GA21, was produced using a particle acceleration method. For the production of maize line HCEM485, DNA introduction was via aerosol beam 

injector (Held et al., 2004), which is a naked DNA delivery method.

III.1	 Donor Genes and Regulatory Sequences

The antecedent organism, maize line GA21, was generated using a particle acceleration 

transformation system with a gel-isolated NotI DNA restriction fragment of plasmid vector 

pDPG434 containing the modified EPSPS encoding gene. In comparison, glyphosate-tolerant 

maize line HCEM485 was produced by introducing a 6.0 kb maize genomic fragment, originally isolated from a bacterial artificial chromosome (BAC) library derived from the maize 

inbred line B73, containing a modified form of the endogenous maize EPSPS encoding gene 

(Held et al., 2006). 

The maize BAC library was screened with a DNA probe complementary to a portion of the 

maize EPSP synthase gene (GenBank Accession No. X63374) and one of the resultant BAC 

clones containing a 6.0 kb genomic fragment flanked by unique ClaI and EcoRV restriction 

endonuclease sites was chosen for further characterization (Figure 1). Nucleotide sequencing of the 6.0 kb fragment revealed that it contained an epsps 5’ regulatory sequence (before 

position 1868), an EPSP synthase coding region (positions 1868–5146) comprised of 8 exons 

(labelled a–h in Figure 1) and 7 introns, and a 3’ untranslated region (after position 5146). The 

EPSP synthase coding region also contained sequences encoding an endogenous N-terminal 

chloroplast transit peptide (position 1868–2041) as predicted using the PSORT algorithm (Human Genome Center, Institute for Medical Science, University of Tokyo).

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Figure 1: Map of 6.0 kb maize genomic fragment and developmental steps in creating maize line HCEM485.

III.2	 The Vector pHCEM

The 6.0 kb maize genomic fragment was cloned into the ClaI and EcoRV sites of pBlueScript 

vector and subjected to site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Two mutations were introduced into the EPSPS coding sequence: a 

cytosine to thymine substitution at position 2886 and a second cytosine to thymine substitution 

at position 2897. These two point-mutations resulted in two amino acid changes within the 

sequence of the mature EPSPS protein, a Thr-102→Ile and Pro-106→Ser substitution. The 

introduction of the T102I/P106S (TIPS) mutations was based on previous work demonstrating 

that Class I EPSP synthase variants containing TIPS mutations resulted in functional tolerance 

to glyphosate-containing herbicides (Spencer et al., 2000; Lebrun et al., 2003). These two mutations are the same mutations as introduced into the modified maize EPSP synthase encoding 

gene introduced into the antecedent organism, maize line GA21. An amino acid alignment of 

maize EPSP synthase enzymes illustrating these changes is included in Figure 2.
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Figure 2: Amino acid sequence alignments of maize EPSP synthase enzymes.

HCEM485 is the amino acid sequence of the mutated EPSP synthase expressed in maize line HCEM485 (Held et al., 

2006). GA21 is the amino acid sequence of the mutated EPSP synthase expressed in maize event GA21 (Spencer et al., 

2000). GenBank Accession No. X63374 corresponds to an EPSP synthase encoding sequence from a maize cell culture 

(Lebrun et al., 1991). GenBank Accession No. AY106729 was identified from a maize bacterial artificial chromosome (BAC) 

library as part of a project of expressed sequence tag (EST) assemblies (Gardiner et al., 2004). Positions of the threonine 

to isoleucine and proline to serine substitutions at positions 102 and 106 (relative to the native enzyme), respectively, are 

shown. The serine residue at position 89 of the HCEM485 EPSP synthase sequence is identical to the sequence of the 

native enzyme from GenBank Accession No. AY106729.

The pBlueScript vector containing the 6.0 kb maize genomic fragment with double-mutated 

EPSPS-encoding gene is designated pHCEM (Figure 3). Nucleotide sequencing of the mutated maize 6.0 kb fragment contained in pHCEM confirmed that no other alterations in sequence 

had been inadvertently introduced. This sequence is presented in Appendix 1.
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Figure 3: Linear diagram of pHCEM plasmid showing all probes used for Southern hybridization.

All restriction enzyme sites corresponding to those enzymes used for Southern hybridizations are indicated.

III.3	 Plant Transformation and Regeneration

For DNA introduction, pHCEM was digested with ClaI and EcoRV, subjected to agarose gel 

electrophoresis (1 percent agarose), and the 6.0 kb band was excised and purified using Qiagen’s Qiaquick gel extraction kit. The purified maize DNA fragment was introduced into immature maize embryos derived from the elite inbred line Stine 963 by aerosol beam injection 

(Held et al., 2004). After 5 days of culture on non-selective medium, embryos were transferred 

onto medium containing glyphosate (100 mg/l). After two 14-day passages, embryos were 

transferred onto medium containing successively greater glyphosate concentrations, up to 540 

mg/l, and regeneration was carried out as previously described (Held et al., 2004).

The only DNA sequences introduced into maize line HCEM485 were those derived from maize 

following the introduction of two point-mutations resulting in the expression of a glyphosateresistant form of the native maize EPSP synthase. Except for the introduced TIPS mutations, 

the amino acid sequence of the double-mutated maize EPSPS (2mEPSPS) enzyme expressed 

in maize line HCEM485 is identical to the native wild-type maize EPSPS sequence reported 

by Gardiner et al., 2004 (Figure 2). Maize line HCEM485 does not contain any heterologous 

DNA sequences, either coding or non-coding, from any other species. 

IV. Genetic Analysis of Maize Line HCEM485

IV.1	 Molecular Characterization

Southern analysis of HCEM485 maize DNA was performed in order to estimate the number 

of sites of insertion of the introduced DNA. Two probes were used that together spanned the 

entire 6.0 kb maize DNA fragment introduced into HCEM485. These probes were designated: 

a) A/C – obtained from a double digest of the pHCEM plasmid with ClaI and Acc65I (corresponding to positions 1–2346); and b) A/E – obtained from a double digest of the pHCEM 

plasmid with Acc65I and EcoRV (corresponding to positions 2347–6010). Probes (ca. 50 

ng each) were labeled with 50 μCi of (α-32P)-dCTP (3000 Ci/mmol) using a random labeling 

system (RediprimeTM II, Amersham Piscataway, NJ). Genomic DNA (7 μg) isolated from 
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CBI Deleted

HCEM485 and control Stine 963 maize was digested (37oC, overnight) with different restriction endonuclease enzymes and restriction fragments were separated by agarose gel electrophoresis followed by transfer onto Hybond N+ nylon membrane. A third sample of DNA 

comprising the control Stine 963 with an amount of the plasmid pHCEM equivalent to a single 

copy per genome (12.58 pg) was treated in a similar manner to the other samples and included 

on the gels. Southern hybridizations were performed according to standard procedures using 

32P-labeled probes followed by autoradiography. [

M 1 2 3 4 5 6 7 8 9

A/C probe

Acc65I BamHI HindIII

23.1

9.4

6.6

4.4

2.3

2.0

M 1 2 3 4 5 6 7 8 9

Acc65I BamHI HindIII

A/E probe

23.1

9.4

6.6

4.4

2.3

2.0

A B

Figure 4: Southern blot hybridisation of HCEM485 with A/E and A/C probes.

Samples of genomic DNA (7μg) from HCEM (lanes 1, 4 and 7), negative control Stine 963 maize (lanes 2, 5 and 8) and 

negative control Stine 963 with 1 copy equivalent of plasmid pHCEM (lanes 3, 6 and 9) were subjected to digestion with the 

restriction enzymes indicated followed by gel eletrophoresis and transfer onto nylon membranes. In panel A, the membrane 

was hybridized with the A/C probe (Figure 3) and in panel B the membrane was hybridised with the A/E probe. Molecular 

size markers were included in lanes marked M and the size of these fragments in kb is indicated on the left hand side of 

each panel.

Southern analysis of HCEM485 genomic DNA using both the A/C and A/E probes following 

HindIII digestion (Figure 4 A and B, lane 7) indicated the presence of a single >=23 kb hybridizing fragment that was unique to HCEM485 (i.e., not present in digests of control Stine 963 

maize DNA). This was expected as this enzyme does not cut within the inserted DNA fragment. Digestion with BamHI followed by hybridization with the A/C probe (Figure 4A, lane 4) 

produced a single band of the same size as that obtained in the control DNA. Again, this is as 

expected given the BamHI sites in the inserted DNA (Figure 3), however the greater intensity 

of this band suggested that multiple copies of the inserted DNA was present. Hybridization 

with the A/E probe (Figure 4B, lane 4) produced three unique bands and one corresponding 
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CBI Deleted

to the control DNA. Digestion with Acc65I (an isoschizomer of KpnI which digests DNA at 

the same recognition site) revealed three hybridising bands unique to HCEM485 with both 

probes (Figure 4 A and B, lane 1). The observed bands are summarised in Table 2 below, 

together with expected fragment sizes based on the putative organization discussed below. 

Bands equivalent to those observed in the control DNA lanes are not included in this table. 

Hybridising fragments from the plasmid DNA includes as a positive control can be seen in the 

Acc65I and BamHI digests, however these are not clearly visible in the HindIII digested lanes. 

As HindIII does not cut within the plasmid it is likely that there could be multiple bands in this 

lane corresponding to different forms of the plasmid (supercoiled, relaxed circles and linear) 

and, together with the low amount (12.58 pg - equivalent to a single copy per haploid genome), 

can mean that the bands in this lane are not intense enough to be visualised in the background 

hybridisation seen on these blots.

Sizes of fragments observed on the Southern blots shown in Figure 4, given in kb by comparison to the 

marker lane fragments. Expected sizes are derived from the putative insert organization detailed in Figure 5. Expected fragments listed greater than a certain size are those where only one restriction enzyme 

site occurs in the inserted DNA, with the other site somewhere in the flanking genomic DNA.

Based on this information, a putative organization of the inserted DNA in HCEM485 has been 

developed, illustrated in Figure 5. This putative organization suggests 4 complete copies of the 

fragment are inserted at a single location. The expected fragment sizes included in Table 2 are 

derived from the putative organization and compared to the observed bands.]

By comparison, the inserted DNA within event GA21 was comprised of two complete copies 

of the herbicide tolerant gene cassette (Act promoter + intron/OTP/mEPSPS/NOS) and one 

incomplete fragment within a single DNA ca. 18.5 kb segment, which was shown to be stably 

inherited across multiple generations as a single genetic locus (Monsanto, 1997).

Due to the fact that the inserted DNA in HCEM485 was comprised exclusively of sequences 

derived from the host organism, maize, and because of the repetitive nature of the DNA insert 

(i.e., multiple copies of a 6 kb fragment), more elaborate molecular characterization (e.g., 

nucleotide sequencing) of the insert was not practically feasible. Further evidence supports the 

single insertion site (segregation analysis) and lack of any truncated coding regions (western 

blot analysis of expressed proteins).

Furthermore, again considering that the introduced DNA was exclusively derived from Z. 

mays, the consequences of any potential genetic rearrangements (e.g., deletions, truncations, 

A/C probe A/E probe

Observed Expected Observed Expected

Acc65I ~4.7

~6

~8

4.7

6.01

 >2.350

~7.3

~6

~5

7.326

6.010

>3.661

BamHI

~2 2.042

~7.8

~4

~15

7.738

3.969

>3.871

HindIII ~24 >24 ~24 >24
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rearrangement, or the potential production of chimeric open reading frames) arising from the 

genetic modification resulting in maize line HCEM485 are not materially different from the 

consequences of potential genetic rearrangements arising from natural genetic recombination 

events or mutations during sexual reproduction in maize. 

[

A

B

Figure 5: Putative organization of inserted DNA in HCEM485.

Panel A shows the putative organization of the inserted DNA in HCEM485 by comparison with the hybridising bands observed with the enzymes KpnI and BamH and the A/C probeI. These fragments can be observed in panel A of Figure 4.

Panel B shows a similar comparison based on hybridization with the A/E probe. ]

In order to confirm the absence of any plasmid backbone sequences within the HCEM485 

genome, samples of genomic DNA (7 μg) isolated from HCEM485, the control Stine 963 

maize and control Stine 963 maize DNA with an amount of the plasmid pHCEM equivalent to 

a single copy per genome (12.58 pg) were digested (37oC, overnight) with different restriction 
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endonuclease enzymes and restriction fragments were separated by agarose gel electrophoresis 

followed by transfer onto Hybond N+ nylon membrane. Southern hybridization was perfomed 

using two probes complementary to the plasmid backbone sequences in vector pHCEM (VP1 

and VP2 Figure 3). There were no detectable hybridization signals from samples derived 

from maize line HCEM485 (Figure 6 A and B, lanes 1,4,7 and 10), consistent with the lack of 

incorporation of any vector backbone derived sequences in the maize genome. Hybridization 

signals from the plasmid DNA added to the control Stine 963 DNA (Lanes 3, 6, 9 and 12) are 

clearly seen, demonstrating the sensitivity of the hybridization. As HindIII does not cut the 

plasmid, this band represents a circular form which does not migrate according to its molecular 

weight.

M 1 2 3 4 5 6 7 8 9 M 10 11 12

BamHI HindIII KpnI Acc65I

VP1 probe

23.1

9.4

6.6

4.4

2.3

2.0

M 1 2 3 4 5 6 7 8 9 M 10 11 12

BamHI HindIII KpnI Acc65I

VP2 probe

23.1

9.4

6.6

4.4

2.3

2.0

Figure 6: Southern analysis for the presence of vector sequences in HCEM485.

Samples of genomic DNA (7μg) from HCEM (lanes 1, 4, 7 and 10), negative control Stine 963 maize (lanes 2, 5, 8 and 11) 

and negative control Stine 963 with 1 copy equivalent of plasmid pHCEM (lanes 3, 6, 9 and 12) were subjected to digestion 

with the restriction enzymes indicated followed by gel eletrophoresis and transfer onto nylon membranes. In panel A, the 

membrane was hybridized with the vector probe VP1 (Figure 3) and in panel B the membrane was hybridised with the Vector probe VP2. Molecular size markers were included in lanes marked M and the size of these fragments in kb is indicated 

on the left hand side of each panel.

IV.2	 Inheritance and Stability

The inheritance pattern of the glyphosate-tolerance trait has been investigated in F1 hybrid 

and F2 segregating plant populations derived from maize line HCEM485. The breeding 

tree for HCEM485 maize is shown below (Figure 7), indicating the derivation of (9289xHCEM485)9032 F1 and (9289xHCEM485)9032 S1F2 plants that were tested for segregation of 

the herbicide-tolerance trait.

Segregation analysis was conducted on F1 hybrid and F2 segregating plant populations derived 

from maize line HCEM485 (Figure 7) by screening for glyphosate tolerance. Progeny plants 

of each generation were grown in the greenhouse and treated with 2.5X the recommended field 
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application rate of glyphosate at approximately the V4 stage of plant development and visually 

scored for herbicide susceptibility. Numbers of trait positive and trait negative plants from 

each generation are shown in Table 3. The data in Table 3 were used to assess the goodness-offit of the observed ratios to the expected ratios using Chi Square analysis with Yates correction 

factor (Little and Jackson Hills, 1978).

χ2

= ∑[Observed-expected- 0.5]2/expected

This analysis tested the hypothesis that the introduced trait segregated as a single locus in a 

Mendelian fashion. The critical value to reject the hypothesis at the 5% level is 3.84. Since the 

Chi squared value was less than 3.84 (Table 2), the hypothesis that the genetic trait behaved in 

a Mendelian fashion was accepted.

Figure 7: Breeding tree for maize line HCEM485.

Table 3: Observed vs. expected segregants for F1 hybrid and F2 selfed generations derived from HCEM485 

maize.

(9289xHCEM485)9032 F1 (9289xHCEM485)9032 S1F2

Observed Expected Observed Expected

Trait Positive1 129 124.5 107 108

Trait Negative 120 124.5 37 36

Total 249 249 144 144

Expected Segregation Ratio 1:1 3:1

Observed Segregation Ratio 1.036:0.964 2.972:1.028

χ2 0.930 0.624

1. Differentiation of trait positive and trait negative plants was based on tolerance to glyphosate. Plants 

were sprayed at the V4 stage of development with 2.5X the normal rate of glyphosate application (1X = 

32 oz/acre).

2. For significance at the 95% confidence level (p<0.05), the Chi square value should be >= 3.841. Chi 

square values <3.841 indicate that the null hypothesis (i.e., observed and expected segregation ratios are 

not significantly different) should not be rejected at the 95% confidence level.
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IV.3	 Gene Expression

IV.3.1 2mEPSPS Integrity and Equivalence to mEPSPS in Event GA21

A western blot analysis was conducted with a monoclonal antibody specific to 2mEPSPS to assess the integrity of this protein as expressed in maize line HCEM485 and to assess its equivalence with the modified EPSPS protein expressed in the antecedent organism, GA21, in both 

leaf and seed tissues. 

[The western blot analysis demonstrated that the 2mEPSPS protein expressed in maize leaf 

tissue from line HCEM485 was intact, with no significant difference in apparent molecular 

weight between the bacterial and plant-produced forms of the protein (Figure 8, lanes 1 and 3). 

In addition, the immunoreactive protein detected in samples from HCEM485 corresponded in 

size to the modified EPSPS protein expressed in event GA21 (Figure 8, lanes 3 and 4). There 

were no cross-reacting species detected in control samples of parental Stine 963 maize, indicating that the monoclonal antibody used for detection was specific for the modified form of the 

maize EPSP synthase (Figure 8, lane 2). Similarly, seed tissue of line HCEM485 expressed 

immunoreactive protein of the same size as that from GA21 (Figure 8, lanes 6 and 8), with no 

cross-reacting species detected in the control hybrid 9289/5056 (Figure 8, lane 7). The bacterial sample of the protein (Figure 8, lanes 1 and 5) retained the His-tag used to isolate the protein and is thus larger than the plant produced forms. This size difference is less noticeable on 

the left hand panel (lanes 1 - 4) as the gel used was of lower resolution than the gel used for the 

right hand panel (4-20% acrylamide compared to 10-20%).

Figure 8: Western immunoblot analysis of 2mEPSPS protein with specific monoclonal antibody.

Samples containing purified E. coli-expressed 2mEPSPS protein (50 ng; lane 1), leaf tissue extracts prepared from negative 

control Stine 963 maize (20 μg protein; lane 2), HCEM485 maize (20 μg protein; lane 3), transgenic GA21 maize (20 μg 

protein; lane 4), E. coli-expressed 2mEPSPS protein (25 ng; lane 5), and seed extracts from transgenic GA21 maize (20 μg 

protein; lane 6), negative control Stine 9289/5056 maize (20 μg protein; lane 7), HCEM485 maize (20 μg protein; lane 8). 

Samples were subjected to SDS-PAGE followed by electroblotting onto PVDF membrane. The membrane was incubated 

sequentially with mouse anti-2mEPSPS monoclonal antibody and HRP-conjugated anti-mouse IgG followed by enhanced 

chemiluminescent detection of bound labeled antibody. The positions of pre-stained MW markers are indicated.]
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In summary, the 2mEPSPS protein expressed in maize line HCEM485 is equivalent with respect to molecular weight and immunochemical cross-reactivity to the modified EPSPS protein 

expressed in maize line GA21.

[A further western blot was performed with a polyclonal antisera to 2mEPSPS raised in rabbits 

to determine if there were any novel polypeptides produced from the inserted DNA (Figure 9). 

From this blot it can be seen the only cross-reacting protein in HCEM485 (lane 3) corresponds 

to the protein present in the control lines 9032 and 963 (lanes 2 and 4, respectively). GA21 

(lane 1) produced a large amount of cross-reacting protein, as expected due to the use of a 

highly active heterologous promoter in this trangenic event. HCEM485 appears to contain a 

greater amount of the EPSPS protein, as compared to the control samples, due to the presence 

of an estimated four extra copies of the gene with a native promoter.

1 2 3 4 1 2 3 4 Mr

216

132

78

45.7

32.5

18.4

7.6

A B

Figure 9: Western immunoblot analysis with polyclonal antisera.

Leaf tissue extracts (20μg protein per lane) were prepared from GA21 (lane 1), control Stine 9032 maize (lane 2), HCEM485 (lane 3), 

and control Stine 963 maize (lane 4). These samples were subjected to SDS-PAGE and stained with Coomassie Blue stain (panel 

A). A similar gel was electro-blotted onto PVDF membrane and incubated with rabbit polyclonal antisera to 2mEPSPS (panel B). 

Kaleiodoscope pre-stained molecular weight markers were used (lane Mr) with molecular weights in kDa as shown ]

More detailed analysis of 2mEPSPS protein expression in various plant tissues was judged unnecessary as it would contribute little to the risk assessment. This determination was based on 

the rationale that: (1) levels of expression in target tissues are likely to be of the same order of 

magnitude as the endogenous native EPSPS enzyme since expression is driven by the same native regulatory sequences; (2) qualitatively, from western blot analysis, the level of expression 
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of 2mEPSPS in HCEM485 is less than that observed in the antecedent organism, GA21 (Figure 

9); and (3) the lack of any realistically attainable level of exposure of humans, animals, or nontarget organisms to 2mEPSPS protein in plant material or products derived from HCEM485 

likely to result in an adverse effect given the demonstrated lack of acute toxicity of modified 

EPSPS proteins from both plant and bacterial sources (Monsanto, 1997; Monsanto, 2000). 

IV.4	 2mEPSPS Protein Safety

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; 3-phophoshikimate 

1-carboxyvinyl-transferase; EC2.5.1.19) (Steinrücken and Amrhein, 1980) is the sixth enzyme 

of the shikimic acid pathway, which is essential for the biosynthesis of aromatic amino acids (L-Phe, L-Tyr and L-Trp) and chorismate-derived secondary metabolites in algae, higher 

plants, bacteria, and fungi (Kishore and Shah, 1988). EPSPS catalyzes the reaction between 

phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) to yield EPSP and inorganic 

phosphate (Pi) (Figure 10, Haslam, 1974; Geiger and Fuchs, 2002). EPSPS enzymes identified 

from plants and bacteria (Class I EPSPS) have been the most studied with respect to enzyme 

kinetics and active site analysis. 

Figure 10: The reaction catalyzed by EPSP synthase.

EPSPS has been identified as the primary target of glyphosate [N-(phosphonomethyl) Gly], 

which is a nonselective, broad-spectrum, foliar-applied herbicide first commercialized in 1974 

and widely used for the management of annual, perennial, and biennial herbaceous species of 

grasses, sedges, and broadleaf weeds, as well as woody brush and tree species (Bradshaw et 

al., 1997; Baylis, 2000). In addition to being highly effective on a broad spectrum of annual 

and perennial weed species common to many cropping systems, glyphosate has very favorable 

environmental characteristics, such as lack of residual soil activity and very low toxicity to 

mammals, birds, and fish (Smith and Oehme, 1992; Padgette et al., 1996). 

The maize EPSPS enzyme and those from various other plant and microbial food sources have 

been part of the protein component of human and animal diets over thousands of years, and are 

not associated with any known health concerns. As the mutations introduced into the maize 

enzyme involve substitutions with standard amino acids common to all proteins of biological origin, and do not alter the functional properties of the enzyme except for its affinity for 

glyphosate, the 2mEPSPS protein in HCEM485 maize is not considered to be inherently toxic.

Based on its source (Zea mays), deduced amino acid sequence, and equivalence with the modified EPSPS expressed in event GA21, the 2mEPSPS protein expressed in maize line HCEM485 
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has the same safety characteristics as the modified EPSPS protein expressed in the antecedent 

organism, GA21.

IV.5	 Conclusions

Maize line HCEM485 contains additional copies of the maize EPSP synthase encoding gene, 

modified to express tolerance to glyphosate herbicide, and associated regulatory sequences 

(e.g., 5’ promoter sequences and 3’ non-coding termination sequences) also derived from 

maize, inserted at a single genetic locus within the maize genome as demonstrated by Southern 

blot analysis and Mendelian inheritance studies. The modified maize EPSP synthase expressed 

in maize line HCEM485 is intact, of the expected molecular weight and there was no evidence 

of truncated forms of the enzyme. The modified maize EPSPS expressed in HCEM485 maize 

is also immunochemically cross-reactive with the modified maize EPSPS expressed in the 

antecedent organism, GA21, and the enzymes from both sources contain the same mutations 

responsible for conferring glyphosate herbicide tolerance.

V. Phenotypic and Agronomic Characteristics

It was not possible to evaluate hybrids of HCEM485 in direct comparison to hybrids of the antecedent organism. However as GA21 was determined to be not significantly different to commercially available corn varieties in agronomic and phenotypic characteristics, a comparison 

of HCEM485 and existing commercial hybrids will be used to establish that HCEM485 is not 

significantly differnt to these hybrids and thus demonstrate the similarity to GA21.

Agronomic and phenotypic characteristics of an HCEM485 maize hybrid and three control 

hybrids were evaluated in a series of field trials across 15 United States Corn Belt locations in 

2007. The material used for the field trials was developed from the initial HCEM485 event 

as detailed in Figure 11. The agronomic characteristics chosen for comparison were those 

typically observed by professional maize breeders and agronomists and represented a broad 

range of characteristics throughout the life cycle of the maize plant. Comparisons were made 

between HCEM485 and control hybrids without glyphosate treatment. Separate plots of 

HCEM485 were grown with glyphosate treatment at recommended field rates (32 oz/acre), but 

these were not used for the statistical comparison as it is not valid to compare plants with such 

different treatments.

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Figure 11: Pedigree chart of HCEM485 seed used in 2007 agronomic trials.

V.1	 Trial Design and Evaluation

V.1.1 Plant Material

Agronomic equivalence trials were conducted using the following hybrid lines:

HCEM485 hybrid (((HCEM485)2/9289/9032)3/5056) [trait positive]

Control hybrid 9289x5056 [trait negative]

Control hybrid 9032x5056 [trait negative]

Control hybrid 963x5056 [trait negative]

A pedigree map showing the derivation of the HCEM485 hybrid is shown in Figure 11. The 

control hybrids were produced by crossing the inbred lines Stine 963, 9289 or 9032, each of 

which were used as parental lines in the breeding of HCEM485, with inbred line 5056, which 

was also used in creating the HCEM485 hybrid.
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V.1.2 Trial Locations

Field trials were conducted at 15 locations in 2007 under USDA notification # 07-046-110n. 

These 15 locations covered nine states in the United States Corn Belt (Table 4) and were selected to represent a range of diverse growing environments where HCEM485 maize hybrids 

are expected to be commercially grown. Field husbandry at all of the trial sites (including irrigation use, fertilization rate, and pest control methods) was consistent with best agronomic 

practices in the area. Agronomic practices for all genotypes within a trial at a single location 

were identical.

Table 4: Trial locations and dates.

Location Code City State Planting Date Harvest Date

ADL1 Adel IA 5-Jun-2007 NA

ADL2 Adel IA 5-Jun-2007 17-Oct-2007

ATL Atlantic IA 6-Jun-2007 23-Oct-2007

LAU Laurel NE 7-Jun-2007 24-Oct-2007

LEN Lennox SD 7-Jun-2007 25-Oct-2007

SMI Smithshire IL 7-Jun-2007 NA

MAR Marion AR 5-Jun-2007 20-Oct-2007

EDM Edmondson AR 6-Jun-2007 20-Oct-2007

BLO Blomkest MN 5-Jun-2007 26-Oct-2007

BIR Bird Island MN 5-Jun-2007 26-Oct-2007

FIT Fithian IL 7-Jun-2007 15-Oct-2007

LIN Lincoln IL 4-Jun-2007 15-Oct-2007

DUR Durand MI 11-Jun-2007 16-Oct-2007

SHE Sheridan IN 4-Jun-2007 9-Oct-2007

OH Spencerville OH 12-Jun-2007 12-Oct-2007

NA = not applicable. Trial was destroyed prior to harvest.

V.1.3 Agronomic Traits Assessed

Up to 17 separate agronomic characteristics were assessed at each location, but not all traits 

were assessed at all locations. These agronomic traits covered a broad range of characteristics 

encompassing the entire life cycle of the maize plant and included data assessing germination 

and seedling emergence, growth habit, vegetative vigor, days to pollen shed, days to maturity, 

and yield parameters (Table 5).

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Table 5: List and definitions of agronomic traits assessed in the field trials.

Abbreviation Trait Timing Description

BRRNP Percent Barren Plants Harvest Percent of plants per plot that do not develop an ear.

DROPP Percent Dropped Ears Harvest Percent of plants per plot that have dropped 

a developed ear prior to harvest. 

EAGRR Early Growth Rating V6 Early growth rating recorded at V6 on a 

scale of 1–9, with 9=most vigorous growth.

EMRGP Early Stand Count V3 Percent of sowed kernels that resulted in 

emerged plants within 14 days after planting.

EMRGR Seedling Vigor V3 Early emergence vigor rating. Data collected prior to V3 stage of maize development. Rated on a 0-9 scale, where 0=dead 

and 9=most vigorous growth.

ERHTN Ear Height After anthesis Ear height from base of plant to node where 

ear connects to plant (cm). Taken at R2-R6 

stage of maize development.

ERTLP Early Root Lodging A 1-9 rating where a higher score indicates 

less root lodging potential (1 is very poor, 5 

is intermediate, and 9 is very good, respectively, for resistance to root lodging).

GMSTP Grain Moisture PercentHarvest Percent grain moisture measured at harvest.

HAVPN Final Stand Count Harvest Harvest population (plants per acre).

HUP5N Heat units to 50% pollen shedFlowering 

(anthesis)

Heat units to 50% of plants shedding pollen.

HUS5N Heat units to 50% 

silking

Flowering 

(anthesis)

Heat units to 50% of plants extruding silks.

LFCLR Leaf Color Rating After anthesis Leaf color rating taken between R4 and R6 

stage of maize development. 5=same as 

commercial check. 1=darker, 9=severely 

chlorotic.

PLHTN Plant Height After anthesis Plant height from base of plant to collar of 

flag leaf (cm). Taken between R2 and R6 

stage of maize development.

RTLDR Root Lodging Rating Harvest A 1-9 rating where a higher score indicates 

less root lodging potential (1 is very poor, 5 

is intermediate, and 9 is very good, respectively, for resistance to root lodging).

STKLR Stalk Lodging Rating Harvest This is a 1-9 rating where a higher score indicates less stalk lodging potential (1 is very 

poor, 5 is intermediate, and 9 is very good, 

respectively, for resistance to stalk lodging).

TWSMN Test Weight Harvest Grain test weight (pounds/bushel) converted 

to standard 15% moisture.

YGSMN Grain Yield Harvest Grain yield (bushels/acre) converted to 

standard 15% grain moisture.
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V.1.4 Experimental Design and Statistical Analysis

Each of the agronomic trials utilized a randomized complete block design with three replications per location. Plot size was ca. 0.002 acres, using 2-row plots, 17.5 feet long with 30 

inches between the rows. Each plot was planted to contain approximately 62 plants of the same 

genotype and there were 12 plots at each location - three replicates of the HCEM485 hybrid 

and the three control hybrids. Full data for the individual plots is included in Annex 1

Data from each of the three control hybrid lines were treated as a single treatment group, identified as control hybrids, in comparisons with the HCEM485 hybrid. Data for the variates (traits) 

were subjected to an analysis of variance across locations using the generalized linear model:

Yij = U + Ti

 + Lj + LTij + eij

where Yij is the observed response for genotype i at location j, U is the overall mean, Ti is the 

treatment (HCEM485 vs. control genotypes) effect, Lj is the location effect, LTij is the location 

x treatment (genotype) interaction effect and eij is the residual error (Annicchiarico, 2002). For 

each variate, the statistical significance of the genotype effect (i.e., HCEM485 vs. control hybrids) was determined using a standard F-test. An F-test probability of < 0.05 indicates that the 

difference between the genotypes was statistically significant with 95% confidence. An F-test 

was also used to assess the significance of the location x genotype interaction – a significant 

outcome (F-test probability < 0.05) indicates that the effect of genotype was not consistent 

across all locations, in which case the comparison of genotypes averaged across locations may 

not be meaningful.

The data for several variates did not lend themselves to formal statistical analysis because they 

did not conform to the assumptions upon which the validity of the analysis depends. In some 

cases, the problem was that the data were too discrete, with values taking one of a very limited 

range of options. In other cases the dataset contained too few non-zero data points on which 

to base a reasonable estimate of residual error. Consequently, results for such variates are 

presented as means. Full results and statistical calculations are included in Annex 1 and only 

summary tables are included with discussion of the results below.

V.2	 Growth Habit

In addition to the agronomic characteristics discussed in other sections below, the following 

parameters are discussed here as indicators of basic morphology and growth habit: ERTLR 

(early root lodging rating); STKLR (stalk lodging rating); RTLDR (late season root lodging 

rating); and LFCLR (leaf color rating). None of these variates were suitable for formal statistical analysis and are presented as a summary of genotype means (Table 6). Overall, there were 

no remarkable differences indicative of an alteration in plant growth habit.

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[

ERTLRa STKLR RTLDR LFCLR

(1-9 rating) (1-9 rating) (1-9 rating) (1-9 rating)

HCEM485 hybrid 8.71 8.17 8.55 4.93

Control hybrids 8.69 8.15 8.56 4.91

Mean Difference -0.02 -0.02 0.01 -0.02

Nb 15 14 14 15

a. ERTLR = early root lodging rating; STKLR = stalk lodging rating; RTLDR = late root lodging rating; LFCLR = 

leaf color rating.

b. N = number of locations with data.]

V.3	 Vegetative Vigor

Comparisons of vegetative vigor between HCEM485-derived and control hybrids were based 

on assessments of: EMRGR (seedling vigor); EAGRR (early growth rating); ERHTN (ear 

height); and PLHTN (plant height). The only variates suitable for statistical analysis were 

ERHTN and PLHTN, both of which showed small but statistically significant increases between HCEM485 and control hybrids (Table 7). The magnitudes of these increases were 2.3% 

and 2.7%, respectively which is small in relation to the range of values for these traits seen in 

commercial corn hybrids released by Stine Seed Farms. Ratings of seedling vigor and early 

growth were similar between HCEM485 and control hybrids.

[

EMRGRa

(0-9 rating)

EAGRR

(1-9 rating)

ERHTN

(cm)

HCEM485 hybrid 7.20 7.60 96.4 ± 16.3

7.46 7.54 94.2 ± 15.4

Mean Difference -0.26 0.06 2.2

Nb 15 15 15

Range observed in Stine 

Seed Farms hybrids 71 - 152

* = indicates that the difference between the genotypes was statistically significant at the 95 percent confidence 

level (p < 0.05).

a. EMRGR = seedling vigor; EAGRR = early growth rating; ERHTN = ear height; PLHTN = plant height. Mean 

values are shown. For ERHTN and PLHTN, the mean standard deviation is indicated.

b. N = number of locations with data.]

V.4	 Reproductive Characteristics

The relevant field indicators of potential changes to seed dormancy, pollination or fertility 

were: EMRGP (percent of emerged plants); HUS5N (heat units to 50 percent silking; HUP5N 

(heat units to 50 percent pollen shed); and BRRNP (percent barren plants). For those variates 
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suitable for statistical analysis, there were small but statistically significant decreases in both 

heat units to 50% silking (-2.5%) and heat units to 50% pollen shed (-2.0%) (Table 8). Once 

again, these are small differences representing only 1-2 days in the timing of these particular 

traits and are within the range of commercial hybrids. Furthermore, these are traits which are 

addressed during breeding process and which will be defined for commercial hybrids produced 

with HCEM485. Although there was no significant difference in the percent germinated plants 

(EMRGP) between HCEM485 and control hybrids, the values for both these groups were 

lower than expected, which was likely due to problems with greenhouse seed production for 

the trials. Data on percent barren plants (BRRNP) were not suitable to statistical analysis but 

mean values were not markedly different between HCEM485 and control hybrids.

[

EMRGPa

(%)

HUS5N

(heat units)

HUP5N

(heat units)

BRRNP

(%)

HCEM485 hybrid 49.0 ± 12.1 1358.0 ± 170.3 1467.7 ± 237.3 0.75

Control hybrids 48.4 ± 12.5 1393.5 ± 169.6 1498.4 ± 233.3 0.35

Mean Difference 0.6 -35.5 -30.7 0.4

Nb 15 15 10 14

Range observed in Stine 

Seed Farms hybrids 1012 - 1868 1100 - 1930

* = indicates that the difference between the genotypes was statistically significant at the 95 percent confidence 

level (p < 0.05).

a. EMRGP = percent emergence; HUS5N = heat units to 50% silking; HUP5N = heat units to 50% pollen shed; 

BRRNP = percent barren plants. Mean values are shown. For EMRGP, HUS5N and HUP5N, the mean standard 

deviation is indicated.

b. N = number of locations with data.]

V.5	 Yield and Grain Characteristics

Parameters used to evaluate yield and grain characteristics included: YGSMN (grain yield); 

HAVPN (plant population at harvest); DROPP (percent dropped ears); TWSMN (grain test 

weight); and GMSTP (grain moisture percent). Among the variates suitable for statistical analysis, there were no significant differences in average yield, plant population at harvest, grain 

moisture, or grain test weight between HCEM485 and control hybrids (Table 9). For both yield 

and plant population at harvest, there were significant genotype x location interactions. Both 

of these traits are subject to large variance based on initial planting density and germination so 

a meaningful range of values for commercial hybrids is not available. The mean value seen in 

HCEM485 is slightly higher in both cases, indicating no deleterious effects from the insertion 

of the modified EPSPS. Although not subject to statistical analysis, there were no remarkable 

differences in percent dropped ears between HCEM485 and control genotypes (Table 9).

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[

YGSMNa

(bu/acre)

HAVPN

(plants/acre)

DROPP

(%)

GMSTP

(%)

TWSMN

(lb/bu)

HCEM485 hybrid 115.4 ± 51.2 14976 ± 3410 0.06 18.8 ± 7.7 55.2 ± 1.9

Control hybrids 113.9 ± 50.7 14888 ± 3622 0.04 18.3 ± 7.1 55.2 ± 2.3

Mean Difference 1.5 88 0.02 0.5 0.1

0.463

Nb 13 14 14 13 13

** = indicates that the effect of genotype was not consistent across all locations, in which case the comparison of 

genotype averaged across locations is questionable.

a. YGSMN = grain yield; HAVPN = final stand count at harvest; DROPP = percent dropped ears; GMSTP = grain 

moisture percent; TWSMN = grain test weight. Mean values are shown. For YGSMN, HAVPN and GMSTP, the 

mean standard deviation is indicated.

b. N = number of locations with data.]

V.6	 Disease Observations

Natural disease infections were rated in trials where the disease incidence was sufficiently high 

to warrant assessment. Observations were made at four sites (Adel, Iowa; Laurel, Nebraska; 

Blomkest, Minnesota; and Lincoln, Illinois) on two separate dates during August and September. These sites were chosen in order to provide a broad range of ecological sites within the 

maturity range for the hybrids used. Disease incidence was generally low, with no littledisease 

noted during the first insepctions. At the time of the second inspection only two sites showed 

sufficient levels to be scored for Southern rust disease (SRDI) and gray leaf spot (GLSDR), 

with one site scored for Northern maize leaf blight (NCLBR), common rust (CMRR) and smut 

(SMTR). Although not suitable for statistical analysis, there were no remarkable differences 

in any of these ratings between HCEM485 and control hybrids (Table 10).

[

SRDIa GLSDR NCLBR CMRR SMTR

(0-5 rating) (0-5 rating) (0-5 rating) (0-5 rating) (0-5 rating)

HCEM485 hybrid 0.93 1.21 0.22 1.00 1.00

Control hybrids 0.90 1.22 0.15 1.08 1.00

Mean Difference 0.03 -0.01 0.07 -0.08 0.0

Nb 2 2 1 1 1

a. SRDI = Southern rust disease rating; GLSDR = gray leaf spot disease rating; NCLBR = northern maize leaf 

blight rating; CMRR = common rust rating; and SMTR = smut rating. All ratings were on a 0–5 scale: 0= no incidence; 1= 2-9%; 2= 10-24%; 3= 25-49%; 4= 50-74%; and 5= 75-100%.

b. N = number of locations with data.]

V.7	 Pollen Morphology and Viability

In order to assess whether the presence of the mutated EPSP synthase encoding gene, the gene 

product, or the genetic modification process altered the pollen characteristics of HCEM485 
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maize, pollen morphology and viability were investigated by microscopically examining pollen grains that had been fixed and stained according to the method described by Alexander 

(1969).

Although the viability of HCEM485 pollen (94.6%) was statistically significantly greater than 

the control (86.0%), both values were within the range that has been reported for other reference samples of maize pollen (Monsanto 2004) and the observed difference was, therefore, 

considered small and unlikely to be of biological significance (Table 11). There were no readily discernible differences in HCEM485 and control pollen morphology (Figure 11) and no 

significant difference in average cell diameter was detected between HCEM485 and control 

pollen samples.

[

Genotype Mean Pollen Viability ± SD (%)a Mean Pollen Diameter ± SD (μm)

HCEM485 94.6 ± 2.9 105.5 ± 6.5

Control (9289x5056) 86.0 ± 4.2 104.8 ± 5.2

N 5 60

Figure 12: Morphology of pollen from HCEM485 and control hybrids.

Representative photomicrographs of control (left) and HCEM485 (right) pollen samples. Pollen samples were stained with 

Alexander’s stain and examined under light microscopy (80X magnification). The scale bar representing 100 μm is indicated in each photomicrograph.]

V.8	 Compositional Analysis

For compositional analysis, grain samples from HCEM485 and control hybrids were obtained 

from four trial locations (ADL2, FIT, BLO and OH, as identified in (Table 4) and forage samples were obtained from two locations (ADL2 and FIT). HCEM485 samples were from plants 

treated with glyphosate at the normal commercial application rate (32 oz/acre). Individual 
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samples of grain and forage from each of the three negative control hybrids were respectively 

combined into a single composite grain and forage sample from each replicated plot. Triplicate 

samples obtained from each plot of HCEM485 hybrid and the composited negative controls 

were analyzed for up to 87 components in grain and 8 components in forage. With the exception of grain total dietary fiber, starch, chromium and selenium, which were analyzed by 

Eurofins (Des Moines, IA), all compositional analyses were performed by EPL Bio-Analytical 

Services (Niantic, IL), according to standard methods. 

Data for analytes above the limit of quantification (LOQ) were subjected to analysis of variance across all locations with genotype and location as factors. Average values for each analyte 

were compared to data for forage and grain composition published in both the International 

Life Sciences Institute (ILSI) crop composition database (ILSI, 2006) and the Organization 

for Economic Co-operation and Development consensus document on new maize varieties 

(OECD, 2002) to assess whether any observed variation was within the natural range for cultivated maize forage and grain.

V.8.1 Proximates

Analysis of the major constituents of maize, or proximates, was used to determine the nutritional properties of maize grain and forage from different hybrids. The major constituents of 

maize grain and forage are carbohydrates, protein, fat and ash. Fiber is the predominant form 

of carbohydrate present in forage and starch is the major carbohydrate in maize grain. Fiber 

is measured by the neutral detergent fiber method (NDF), which measures the insoluble fiber: 

lignin, cellulose and hemicellulose. Total dietary fiber (TDF) consists of the insoluble and 

soluble fiber (pectin). The soluble fiber fraction in maize is negligible, so the NDF value in 

maize grain is comparable to that of TDF. The acid detergent fiber (ADF) method solubilizes 

hemicellulose, measuring only cellulose and lignin (Watson, 1987).

Comparison of the proximate composition of the HCEM485 grain and the negative control 

grain samples is shown in Table 12. No statistically significant differences were observed for 

protein, fat, carbohydrates, ADF, NDF, ash, starch or carbohydrate. A statistically significant 

difference was observed for TDF, however, the magnitude of the difference was small (ca. 

6.4%). The average values for all proximates measured in grain were within the ranges reported in the literature.

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Comparison of the proximate composition of the HCEM485 forage and the negative control 

forage is shown in Table 13. No statistically significant differences were found in five (moisture, ash, carbohydrates, ADF, and NDF) of the seven analytes tested. The only statistically 

significant differences observed were a higher (ca. 6.8%) mean protein content, which was not 

consistent across locations, and a lower (ca. 13.2%) level of total fat in the HCEM485 hybrid 
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samples than in the control samples. The average values for all proximates in forage, including 

protein and fat, were within the ranges reported in the literature.

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V.8.2 Minerals

Several mineral ions are recognized as essential plant nutrients and are required by the plant 

in significant quantities. These macronutrients include calcium, phosphorous, potassium and 

sodium. The micronutrient minerals, iron, copper and zinc are incorporated in plant tissues in 

only trace amounts. Maize is an important source of selenium in animal feed (Watson, 1987), 

and this analyte was also included in the analyses of grain.

Comparison of the mineral composition of the HCEM485 grain and the negative control grain 

is shown in Table 14. No statistically significant differences were observed for levels of iron, 

magnesium, manganese, phosphorus, sodium or zinc. Small but statistically significant differences were noted for calcium, copper, and potassium. For selenium, values that were below 

the limit of quantification (<LOQ) were distributed equally between the HCEM485 hybrid and 

control hybrids, where 5 out of 12 total values for each set of samples were <LOQ. Analytes 

with values <LOQ were not suitable for statistical analysis but quantifiable levels of selenium 

in the HCEM485 samples (ranging from 0.11–0.21 mg/kg dry weight) were all within ranges 

reported in the literature. Levels of chromium in HCEM485 and control samples were all 

<LOQ. For all minerals that were statistically analyzed, including those that showed statistically significant differences, average values were within the ranges reported in the literature.
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Comparison of the calcium and phosphorus composition of the HCEM485 forage and the control forage samples is shown in Table 15. Only calcium was statistically significantly higher 

(ca. 13%) in HCEM485 samples than control samples, and mean levels of both calcium and 

phosphorus were within the ranges reported in the literature.
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V.8.3 Vitamins

Although animal feed formulations are usually supplemented with additional vitamins to 

achieve nutritional balance, maize contains two fat-soluble vitamins, vitamin-A (β-carotene) 

and vitamin E, and most of the water-soluble vitamins. Vitamin A occurs in two forms in 

nature. Its true form, retinol, is present in foods of animal origin such as fish oils and liver. 

Provitamin A, in the form of the carotenoids β-carotene and cryptoxanthin are found in plants 

and converted in the body to vitamin A. Vitamin E (tocopherol) occurs in a variety of vegetable, nut, and oilseed crops, and of the various structural isomers (alpha-, beta-, delta- and 

gamma-tocopherol), α-tocopherol is the most biologically important as a natural antioxidant. 

Alpha-tocopherol is the only form of vitamin E that is actively maintained in the human body, 

and has the greatest nutritional significance (Linus Pauling Institute, 2004). The water-soluble 

vitamins B1 (thiamine) and B6 (pyridoxine) are present in maize grain at quantities sufficient 

to be important in animal rations (Watson, 1987).

Comparison of the vitamin analysis of grain is shown in Table 16. Statistically significant differences between HCEM485 and control sample means were observed for levels of tocopherols, thiamine (B1), pyridoxine (B6) and folic acid (B9). The magnitudes of these differences 

were small, ranging from ca. 7–18%, and in some cases (e.g., B6, B9, and α-tocopherol), the 

differences were not consistent across growing locations. Levels of β-cryptoxanthine and riboflavin (B2) were below the limit of quantification in all samples. For all of the quantifiable analytes, the mean values were within the ranges reported in the literature, including those where 

significant differences were observed between samples from HCEM485 and control hybrids.
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V.8.4 Amino Acids

The quality of protein produced by different maize hybrids can be determined by measuring 

the content of different amino acids. Eighteen amino acids commonly found in maize are considered to be important for compositional analysis (EuropaBio, 2003). Levels of methionine 

and cysteine are important for formulation of animal feed, as are lysine and tryptophan, which 

cannot be produced by non-ruminant animals such as swine and poultry and are present at low 

concentrations in maize.

Comparison of the amino acid composition of HCEM485 grain and the control grain is shown in 

Table 17. The only significant difference was in mean methionine content between HCEM485 

and control samples, however, this difference was not consistent across all growing locations. 
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Average levels of all amino acids, including methionine, were within the ranges reported in the 

literature.

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V.8.5 Fatty Acids

Five fatty acids account for nearly 98 percent of the total fatty acids in maize grain (ILSI, 2006), 

with the most abundant being linoleic (C18:2 Δ9,12; 57.6%) and oleic (C18:1 Δ9; 26.0%) acids. Less abundant, but occurring at measurable levels are palmitic (C16:0; 11.03%), stearic 

(C18:0; 1.8%) and α-linolenic (C18:3 Δ9,12,15; 1.13%) acids. 
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The desaturation of oleic acid to form linoleic acid, and its subsequent desaturation to form 

α-linolenic acid, occurs only in plants, hence both linoleic and α-linolenic acids are essential 

fatty acids for mammals. For this reason, it was desirable to measure for any unintended 

changes in the levels of linoleic and α-linolenic acids, and their key precursors, palmitic, stearic 

and oleic acids, in grain from HCEM485. 

Other polyunsaturated and longer chain polyunsaturated fatty acids, such as γ-linolenic (C18:3 

Δ6,9,12), eicosatrienoic (C20:3 Δ8,11,14) and arachidonic (C20:4 Δ5,8,11,14) acids can all be 

synthesized by mammals from dietary sources of α-linolenic and linoleic acid. Hence, small 

changes in the levels of these trace fatty acids in HCEM485-derived grain would have little or 

no biological significance to either humans or animals consuming HCEM485 grain products. 

The synthesis of palmitoleic (C16:1 Δ9) and saturated fatty acids with chain lengths greater 

than 18 (e.g., C20:0, C22:0, C24:0), can be accomplished in mammals through de novo fatty 

acid synthesis without dietary requirements for palmitic and stearic acids, respectively. 

The complete fatty acid profile of maize grain from HCEM485 and control hybrids was determined and the results are summarized in Table 18. The concentrations of the following 

fatty acids were below the limit of quantification in one or more samples and not included in 

the analysis: caprylic (C8:0); capric (C10:0); lauric (C12:0); myristic (C14:0); myristoleic 

(C14:1); pentadecanoic (C15:0); pentadecenoic (C15:1); palmitoleic (C16:1); heptadecanoic 

(C17:0); heptadecenoic (C17:1); gamma-linolenic (C18:3); eicosadienoic (C20:2); arachidonic 

(C20:4); eicosatrienoic (C20:3); behenic (C22:0); and erucic (C22:1). Statistically significant 

differences observed for quantifiable fatty acids were for palmitic (C16:0), stearic (C18:0), 

oleic (C18:1), linolenic (C18:3) and eicosenoic (C20:1), however, the magnitude of these differences was small, ranging between ca. 1% and 4.4%. Average levels of all quantifiable fatty 

acids, including those where significant differences were observed, were within the ranges 

reported in the literature.

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V.8.6 Secondary Metabolites and Antinutrients

Secondary metabolites are defined as those natural products which do not function directly in 

the primary biochemical activities that support growth, development and reproduction of the 

organism in which they occur (EuropaBio, 2003). One class of secondary metabolites, antinutrients, is responsible for deleterious effects related to the absorption of nutrients and micronutrients from foods (Shahidi, 1997). There are generally no recognized antinutrients in maize at 

levels considered to be harmful, but for the purposes of safety assessment OECD recommends 

testing for the following secondary metabolites in maize: ferulic acid, ρ-coumaric acid, furfural, inositol, phytic acid, raffinose and trypsin inhibitor. These secondary metabolites and 

antinutrients were analyzed in grain samples from HCEM485 and control hybrids (Table 19).

Phenolic acids — may have beneficial health effects because of their anti-oxidant properties. Ferulic acid and p-coumaric acid are weak anti-oxidants. In vitro tests are equivocal as 

to whether ferulic acid enhances or inhibits the effects of mutagenic substances (Sasaki et al., 

1989; Stich, 1992). Ferulic acid and ρ-coumaric acid are found in vegetables, fruit and cereals. They are also used as flavoring in foods, as supplements and in traditional Chinese herbal 

medicine. Daily intake of phenolic acids by humans is estimated to be 0.2–5.2 mg/day (Clifford, 1999; Radtke et al., 1998).

There were no significant differences in mean ferulic acid or ρ-coumaric acid between grain 

samples from HCEM485 and control hybrids (Table 19).
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Furfural — is a heterocyclic aldehyde which occurs in several vegetables, fruits and cereals. 

It is used as a pesticide, but also in foodstuff as flavoring. Furfural is generally recognized as 

safe (GRAS) under conditions of intended use as a flavor ingredient. Field maize generally 

contains < 0.01 ppm (< 0.001 mg/100g) furfural (Adams et al., 1997). Furfural was below the 

lower limit of quantification in all grain samples (Table 19).

Phytic acid — (myo-inositol 1,2,3,4,5,6-hexakis[dihydrogenphosphate]) is considered to be 

an antinutrient due to its ability to bind minerals, proteins and starch at physiological pH (Rickard and Thompson, 1997). Phytic acid is present in maize germ and binds 60–75 percent of 

phosphorous in the form of phytate (NRC, 1998), decreasing the bioavailability of phosphorous in maize for non-ruminant animals. Phytic acid levels in maize grain vary from 0.45–1.0 

percent of dry matter (Watson, 1982).

There was no significant difference in mean phytic acid level between grain samples from 

HCEM485 or control hybrids, although average inositol levels were significantly lower (ca. 

-13%) in HCEM485 grain samples (Table 19). In both cases, the average values were well 

within the ranges reported in the literature for these two analytes.

Alpha-galactosides — of sucrose, including raffinose, are widely distributed in higher plants 

(Naczk et al., 1997). Due to the absence of alpha-galactosidase activity in human and animal 

mucosa, raffinose cannot be broken down by enzymes in the gastrointestinal tract and is considered an antinutrient, although it is not toxic. No statistically significant differences were 

detected in raffinose levels between the HCEM485 and control grain samples and all values 

were within ranges reported in the literature (Table 19).

Protease inhibitors — are found in abundance in raw cereals and legumes, especially soybeans. Trypsin inhibitors in soybean give rise to inactivation and loss of trypsin in the small 
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intestine, triggering the induction of excess trypsin in the pancreas at the expense of sulfurcontaining amino acids (Shahidi, 1997). Maize contains low levels of trypsin and chymotrypsin inhibitors, neither of which is considered nutritionally significant (White and Pollak, 1995). 

A small, but statistically significant increase (ca. 3.4%) in mean trypsin inhibitor activity was 

observed from HCEM485 grain samples compared with control samples (Table 19), but this 

difference was not consistent across all growing locations and levels of trypsin inhibitor for all 

samples were within the range reported in the literature. 

V.8.7 Phytosterols

Phytosterols are cholesterol-like molecules found in all plant foods, with the highest concentrations occurring in vegetable oils. They are absorbed only in trace amounts but have the beneficial effect of inhibiting the absorption of dietary cholesterol (Ostlund, 2002). Phytosterols 

are not endogenously synthesized in the body but are derived solely from the diet (Rao and 

Koratkar, 1997).

There were no significant differences in mean levels of cholesterol, campesterol, stigmsterol, 

β-sitosterol, stigmastanol, or total phytosterols between grain samples from HCEM485 or control hybrids (Table 20). Since phytosterols are not commonly included in compositional analyses of Z. mays, there no ranges for these given in the OECD concensus document for maize 

(OECD, 2002). One reference (Ryan et al, 2007) gives a single value (mean +/- standard error 

of three independant extractsions on the same sample) and so does not provide a range to determine biological significance of the different values seen in the control maize hybrids.

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V.8.8 Nutritional Impact

Compositional analysis is the cornerstone of the nutritional assessment of a food derived from 

a new plant variety. When compositional equivalence between the new food and its conventional counterpart has been established, the results of numerous published livestock feeding 

trials with genetically modified varieties of maize, soybean, canola, cotton, or sugar beet, have 

confirmed no significant differences in digestibility of nutrients, animal health or animal performance (Flachowsky et al., 2005). Therefore, once compositional and phenotypic equivalence 
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has been established, nutritional equivalence may be assumed, and livestock feeding trials add 

little to the safety assessment (OECD, 2003; EFSA, 2006). As the HCEM485-derived hybrid 

was determined to be compositionally equivalent to control parental lines, additional livestock 

feeding studies are not necessary.

V.9	 Conclusions

The agronomic performance and phenotypic data generated for the HCEM485-derived hybrid 

and control hybrids suggest that the genetic modification resulting in maize line HCEM485 

did not have any biologically significant unintended effect on plant growth habit and general 

morphology, vegetative vigor, flowering and pollination, grain yield, grain test weight, disease susceptibility, or pollen morphology. These data support the conclusion that HCEM485-

derived hybrids are unlikely to form feral persistent populations, or to be more invasive or 

weedy than conventional maize hybrids, and would not display higher rates of out crossing 

than unmodified maize. With this conclusion, similarity to GA21, the antecedent organism, is 

demonstrated.

Levels of key nutrients, minerals, antinutrients, and secondary metabolites were determined 

in samples of maize grain and forage derived from HCEM485 and control hybrids collected 

from up to four field trial locations in 2007. For most analyses, there were no statistically 

significant differences and in cases where statistically significant differences were observed, 

the magnitudes of the differences were small and in every case, mean values determined for 

both HCEM485 and control samples were within the ranges of natural variation as reported in 

the literature. Overall, no consistent patterns emerged to suggest that biologically significant 

changes in composition of the grain or forage had occurred as an unintended consequence of 

the genetic modification resulting in maize line HCEM485. The conclusion based on these 

data was that grain and forage from HCEM485 maize were substantially equivalent in composition to both the control hybrids included in this study, to other commercial maize hybrids 

and - by extension - to the antecedent organism.

VI. Environmental Impact of Introduction of Maize

Line HCEM485

There are no changes from Section VI of the previously approved petition 97-099-01p in terms 

of the description of glyphosate herbicide, current uses of maize herbicides, weediness potential of glyphosate tolerant maize, cross pollination to wild and cultivated related species and 

transfer of genetic material to species to which maize cannot interbreed (e.g., horizontal gene 

transfer). There is no expectation that cultivation of maize line HCEM485 would have any 

environmental effects different from the cultivation of the antecedent organism, GA21, or other 

maize lines exhibiting glyphosate tolerance that have also been deregulated by USDA-APHIS 

(e.g., NK603; MON 88017; and MON 802).
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VII. Adverse Consequences of Introduction

Stine Seed Farm knows of no study results or observations associated with maize line HCEM485 

that would be anticipated to result in adverse environmental consequences from its introduction. Therefore, on the basis of the substantial phenotypic equivalence between maize line 

HCEM485 and the antecedent organism, GA21, Stine Seed Farm requests that an extension of 

nonregulated status be granted to maize line HCEM485.

VIII. References

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ANZFA (2000). Final Risk Assessment Report – Application A362: Food derived from glyphosatetolerant corn line GA21.

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Baylis, A. (2000). Why glyphosate is a global herbicide: Strengths, weaknesses and prospects. Pest 

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the national food consumption survey. Zeitschrift fur Ernahrungswissenschaft 37(2): 190–197.

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tocopherol content and fatty acid profile of selected seeds grains and legumes. Plant Foods Hum 

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products, composition and nutritive values. Cereal Foods World 40(10): 756–762.
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CBI Deleted

IX. Appendix 1: HCEM Nucleotide Sequence

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No CBI 

Maize Line HCEM485 

USDA Extension Petition 

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IX. APPENDIX 1: HCEM NUCLEOTIDE SEQUENCE 

LOCUS HCEM 6010 bp DNA linear 

DEFINITION Zea mays fragment containing EPSPS encoding sequences, following sitedirected mutagenesis 

ACCESSION Not-Specified. 

VERSION Not-Specified. 

KEYWORDS

SOURCE Zea mays 

ORGANISM

FEATURES Location/Qualifiers 

 source 1..6010 

 5’ UTR 1..1867 

 /note=”epsps regulatory sequences” 

 transit_peptide 1868..2041 

 /gene="CTP" 

 CDS 1868..2167 

 /gene="epsps exon 1" 

 CDS 2696..2944 

 /gene="epsps exon 2" 

 CDS 3044..3193 

 /gene="epsps exon 3" 

 CDS 3467..3679 

 /gene="epsps exon 4" 

 CDS 4189..4302 

 /gene="epsps exon 5" 

 CDS 4463..4675 

 /gene="epsps exon 6" 

 CDS 4756..4818 

 /gene="epsps exon 7" 

 CDS 4928..5146 

 /gene="epsps exon 8" 

 gene 2042..5146 

 /gene="2mEPSPS" 

 3’UTR 5147..6010 

BASE COUNT 1581 a 1375 c 1288 g 1766 t 

ORIGIN

 1 atcgattgca accttcaaat tctcttcgat cttccttcca aattcatcta atatttcatc 

 61 ctcggcaagg aaatcttcta acgtgtaaac ttcactggga tccaactcga aaggatcaaa 

 121 ctctccctct ggttcgtttg acattgtgga tggagtgact aacctgctaa caccctgcaa 

 181 caatttatac aggagcatat cctcatgcac acgcaaaact gatgttgtcc acaagacacg 

 241 cacaggacac gcacaggaca cgcaaacagt ttcagactca tgcacacgca catcagtttc 

 301 agactcaggc acacgcacat caaatcacct tcgcttgtcg atgagtcgca gccgcatcgt 

 361 acaatggcga ttttaccgac gataaggcat gggagcacga gccgtcgccg tcgccttgcg 

 421 agacgacggg agcgatctct cccttcattt aatctcttcc acgtcaggtt attttgctga 

 481 gatggcagta tacagacggc aaagttaatg ccgttgtaca tgcccttaga ctcttccgtc 

 541 accaactcac ttagattttt acaacggaac ataaggttcg cttgcagact tacatataag 

 601 gtatagttgc ataataatcg ccttatgctg tacattgcga cacccgtaaa tattcgatga 

 661 aatattagta cacaatatta aataagaacg aacaatacat atattatcat tgatcttagt 

 721 atctcctttt gctcctcgta gaacaattct gtgtaaatta tgcgtaaaat tcgaggacca 

 781 aaacattggc tagaaaaata cctaaaatca gttttgcaat tgtttctgat tttcctcata 

 841 ttttcttgct tataaagttt tccaaaagta ccattttgga tgaaaaaacg gaaaacaacg 

 901 ctggtctact tgtaaatttg gtagtgacat ttgggaccgt ctagacacga cctaaaaata 

 961 gtagtctaaa acatagtctg acacgatgcc ttaaaaatag acgacaaagc acaacacgat 

 1021 tagatgtgtc gtgttttgac cgacacgaca caaagtaagg cacgatttaa aacccaataa 

 1081 ataatatttt aatggttatt ttatgttcca ataattttca tctcttcaaa aaaatgttat 

 1141 agaaatcatt gatacttagt tgaatatcct aacacaatat atatatatat attaatatat 

 1201 atatatatca attttaagtc actttgctag acatagtaat atattttaaa tattttctct 

 1261 ttcttgtata tttttaaaat acacatcagt ttttatatgt gtcgtgcttg aaccgacacg 

 1321 atataatcat cggttcgccg tacttctaga tcatgatgtt cctaggtttt aatattaaga 

 1381 gacggtctat attaactcaa aactatttcg tgaaaggcta actcgaaaaa aaaatgaatg 

 1441 taatcacggc ccgtcctgga ttcgagattc taacgtttca ttcgtgtcca gtgtgcacac 

 1501 ttgtggaaaa ggaagacgaa gaaaaaaacc aacaactaac tccggcccgc cggatgcgcc 

 1561 cacctacttc cccctcgccc ctctcatggt ctctctcgcg cccagatctg ctactagacg 

 1621 gcaccgctgc agcgcgtcgt gtcgcggggg ttggtggcag gcagcgagag cttgccgttc 

 1681 ctctctctca gttgtcaggt cctaggctca cctcaccggc tcccagcccg cttctatttc 

 1741 ttcctccccg accccgtgca ggtggcagtc cagtccacgc caccaaccgc gaggcgaacc 
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 1801 aaaccaaccc actctcccca accccgcgcg cccaggccgc ccgccctacc aaccatcggc 

 1861 gtcggcaatg gcggccatgg cgaccaaggc cgccgcgggc accgtgtcgc tggacctcgc 

 1921 cgcgccgtcg cgccgccacc accgcccgag ctcggcgcgc ccgcccgccc gccccgccgt 

 1981 ccgcgggctg cgggcgcctg ggcgccgcgt gatcgccgcg ccgccggcgg cggcagcggc 

 2041 ggcggcggtg caggcgggtg ccgaggagat cgtgctgcag cccatcaagg agatctccgg 

 2101 caccgtcaag ctgccggggt ccaagtcgct ttccaaccgg atcctcctgc tcgccgccct 

 2161 gtccgaggtg agcgattttg gtgcttgctg cgctgccctg tctcactgct acctaaatgt 

 2221 tttgcctgtc gaataccatg gattctcggt gtaatccatc tcacgatcag atgcaccgca 

 2281 tgtcgcatgc ctagctctct ctaatttgtc tagtagtttg tatacggatt aatattgata 

 2341 aatcggtacc gcaaaagcta ggtgtaaata aacactagaa aattggatgt tcccctatcg 

 2401 gcctgtactc ggctactcgt tcttgtgatg gcatgctgtc tcttcttggt gtttggtgaa 

 2461 caaccttatg aaatttgggc gcaaagaact cgccctcaag ggttgatctt atgccatcgt 

 2521 catgataaac agtggagcac ggacgatcct ttacgttgtt tttaacaaac tttgtcagaa 

 2581 aactagcatc attaacttct taatgacgat ttcacaacaa aaaaaggtaa cctcgctact 

 2641 aacataacaa aatacttgtt gcttattaat tatatgtttt ttaatctttg atcaggggac 

 2701 aacagtggtt gataacctgt tgaacagtga ggatgtccac tacatgctcg gggccttgag 

 2761 gactcttggt ctctctgtcg aagcggacaa agctgccaaa agagctgtag ttgttggctg 

 2821 tggtggaaag ttcccagttg aggattctaa agaggaagtg cagctcttct tggggaatgc 

 2881 tggaattgca atgcggtcat tgacagcagc tgttactgct gctggtggaa atgcaacgta 

 2941 tgtttcctct ctttctctct acaatacttg ctggagttag tatgaaaccc atgggtatgt 

 3001 ctagtggctt atggtgtatt ggtttttgaa cttcagttac gtgcttgatg gagtaccaag 

 3061 aatgagggag agacccattg gcgacttggt tgtcggattg aagcagcttg gtgcagatgt 

 3121 tgattgtttc cttggcactg actgcccacc tgttcgtgtc aatggaatcg gagggctacc 

 3181 tggtggcaag gttagctact aagggccaca tgttacattc ttctgtaaat ggtacaacta 

 3241 ttgtcgagct tttgcatttg taaggaaagc attgattgat ctgaatttga tgctacacca 

 3301 caaaatatcc tacaaatggt catccctaac tagcaaacaa tgaagtaata cttggcatgt 

 3361 gtttatcaaa ttaatttcca tcttctgggg cattgcctgt tttctagtct aatagcattt 

 3421 gtttttagca ttaattagct cttacaattg ttatgttcta caggtcaagc tgtctggctc 

 3481 catcagcagt cagtacttga gtgccttgct gatggctgct cctttggctc ttggggatgt 

 3541 ggagattgaa atcattgata aattaatctc cattccctac gtcgaaatga cattgagatt 

 3601 gatggagcgt tttggtgtga aagcagagca ttctgatagc tgggacagat tctacattaa 

 3661 gggaggtcaa aaatacaagt aagctctgta atgtatttca ctactttgat gccaatgttt 

 3721 cagttttcag ttttccaaac agtcgcatca atatttgaat agatgcactg tagaaaaaaa 

 3781 atcattgcag ggaaaaacta gtactgagta ttttgactgt aaattatttt accagtcgga 

 3841 atatagtcag tctattggag tcaagagcgt gaaccgaaat agccagttaa ttatcccatt 

 3901 atacagagga caaccatgta tactattgaa acttggttta taagagaatc taggtagctg 

 3961 gactcgtagc tgcttggcat ggataccttc ttatctttag gaaaagacac ttgatttttt 

 4021 ttttctgtgg ccctctatga tgtgtgaacc tgcttctcta ttgctttaga aggatatatc 

 4081 tatgtcgtta tgcaacatgc ttcccttagc catttgtact gaaatcagtt tcataagttc 

 4141 gttagtggtt ccctaaacga aaccttgttt ttctttgcaa tcaacaggtc ccctaaaaat 

 4201 gcctatgttg aaggtgatgc ctcaagcgca agctatttct tggctggtgc tgcaattact 

 4261 ggagggactg tgactgtgga aggttgtggc accaccagtt tgcaggtaaa gatttcttgg 

 4321 ctggtgctac aataactgct tttgtctttt tggtttcagc attgttctca gagtcactaa 

 4381 ataacattat catctgcaaa tgtcaaatag acatacttag gtgaattcat gtaaccgttt 

 4441 ccttacaaat ttgctgaaac ctcagggtga tgtgaagttt gctgaggtac tggagatgat 

 4501 gggagcgaag gttacatgga ccgagactag cgtaactgtt actggcccac cgcgggagcc 

 4561 atttgggagg aaacacctca aggcgattga tgtcaacatg aacaagatgc ctgatgtcgc 

 4621 catgactctt gctgtggttg ccctctttgc cgatggcccg acagccatca gagacggtaa 

 4681 aacattctca gccctacaac catgcctctt ctacatcact acttgacaag actaaaaact 

 4741 attggctcgt tggcagtggc ttcctggaga gtaaaggaga ccgagaggat ggttgcgatc 

 4801 cggacggagc taaccaaggt aaggctacat acttcacatg tctcacgtcg tctttccata 

 4861 gctcgctgcc tcttagcggc ttgcctgcgg tcgctccatc ctcggttgct gtctgtgttt 

 4921 tccacagctg ggagcatctg ttgaggaagg gccggactac tgcatcatca cgccgccgga 

 4981 gaagctgaac gtgacggcga tcgacacgta cgacgaccac aggatggcca tggccttctc 

 5041 ccttgccgcc tgtgccgagg tccccgtgac catccgggac cctgggtgca cccggaagac 

 5101 cttccccgac tacttcgatg tgctgagcac tttcgtcaag aattaataaa gcgtgcgata 

 5161 ctaccacgca gcttgattga agtgataggc ttgtgctgag gaaatacatt tcttttgttc 

 5221 tttctattct ctttcacggg attaagtttt gagtctgtaa cgttagttgt ttgtagcaag 

 5281 tttctatttc ggatcttaag tttgtgcact gtaagccaaa tttcatttca agagtggttc 

 5341 gttggaataa taagaataat aaattacgtt tcagtggctg tcaagcctgc tgctacgttt 

 5401 taggagatgg cattagacat tcatcatcaa caacaataaa accttttagc ctcaaacaat 

 5461 aatagtgaag ttatttttta gtcctaaaca agttgcatta ggatatagtt aaaacacaaa 

 5521 agaagctaaa gttagggttt agacatgtgg atattgtttt ccatgtatag tatgttcttt 

 5581 ctttgagtct catttaacta cctctacaca taccaacttt agtttttttt ctacctcttc 

 5641 atgttactat ggtgccttct tatcccactg agcattggta tatttagagg tttttgttga 

 5701 acatgcctaa atcatctcaa tcaacgatgg acaatctttt cttcgattga gctgaggtac 

 5761 gtcatctaca ggataggacc ttgagaatat gtgtccgtca atagctaacc ctctactaat 

 5821 tttttcaatc aagcaaccta ttggcttgac tttaattcgt accggcttct actacttcta 

 5881 cagtattttg tctctataaa ttgcagctac aacagtcaga acggctggct ttaaaatcaa 
Maize Line HCEM485

USDA Extension Petition

Stine Seed Farm Page 49 of 127

CBI Deleted

]

No CBI 

Maize Line HCEM485 

USDA Extension Petition 

Stine Seed Farm Page 46 of 47 

 5941 atggcctaag gatcattgaa aggcatctta gcaatgtcta aaattattac cttctctaga 

 6001 cgttgatatc 

//
Maize Line HCEM485

USDA Extension Petition

Stine Seed Farm Page 50 of 127

X. Appendix 2: USDA APHIS Release Notifications

Relevant to the Field Testing of Maize Line HCEM485

USDA # Internal ID County and State of Release

05-060-09n SSF2005-006 Dallas, IA; Marshall, IA; Madison, IA; Lincoln, IL; Warren, 

IL; Vermillion, IL; Tipton, IN; Hamilton, IN; Boone, IN; Blue 

Earth, MN; Clinton, MO; Saline, MO; Valley, NE; Dodge, NE; 

Paulding, OH.

06-047-09n SSF2006-001 Dallas, IA; Cass, IA; Logan, IL; Vermillion, IL; Tipton, IN; 

Hamilton, IN; Boone, IN; Lincoln, SD; Renville, MN; Valley, 

NE; Arkansas, AR; Paulding, OH.

07-046-110n SSF2007-003 Dallas, IA; Tipton, IN; Hamilton, IN; Vermillion, IL; Logan, IL; 

Defiance, OH; Shiawassee, MI; Renville, MN; Kandiyohi, MN; 

Crittenden, AR; Lincoln, SD; Cedar, NE; Cass, IA; Warren, IL.

08-046-109n SSF2008-003 Dallas, IA.

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Maize Line HCEM485

USDA Extension Petition

Stine Seed Farm Page 51 of 127

CBI Deleted

Pages 51 through 127 are Annexes 1 through 3 and contain confidential 

business information on each page. They have therefore been removed 

from this document

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