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 - RevisedMaize Line HCEM485 USDA Extension Petition

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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 sub

stitutions. 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 gly

phosate-tolerant form of the enzyme and are also present in the modified EPSPS enzyme pro

duced 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 maizefollowing 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 heter

ologous 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 suscepti

bility 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 USDA Extension Petition

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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, orother 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 ser

ine mutation at position 106, relative to the amino acid sequence of the native maize EPSP synthase enzyme. USDA United States Department of AgricultureMaize Line HCEM485 USDA Extension Petition

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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

.......................................... 50Maize Line HCEM485 USDA Extension Petition

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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 hybrid

ization.

...................................................................................................................... 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 anti

body.

......................................................................................................................... 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 hy

brids.

......................................................................................................................... 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.

................. 42Maize Line HCEM485 USDA Extension Petition

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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. R ati o nale f o r S ubmissi o n o f R e q uest f o r Ex tensi o n o f No nre g ulated S tatus

I.1 B as

i s for the R equest 

### The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agri

culture (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 author

ity, 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 non

regulated status to additional articles, upon finding that the additional articles do not pose a po

tential 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 un

changed 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 appropri

ate. 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 previ

ous 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 riskMaize Line HCEM485 USDA Extension Petition

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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 G A21

Characteristic HCEM485 Event G A21 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 Cla I + Eco RV restriction frag

ment ( ca . 6.0 kb) Not I 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 se

quences from Z. mays EPSPSencoding gene Optimized chloroplast transit sequences derived from Z. mays and Helianthus annus (sunflower)

ribulose-1,5-bisphosphate car

boxylase genes Promoter and source 5’ region of the maize ( Z. mays ) EPSPS-encoding gene containing native promoter sequences 5’ region of the rice ( Oryza sa

tiva ) 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 R at i onale for the D evelopment of M a

i ze L i ne H C EM 485 

### 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.Maize Line HCEM485 USDA Extension Petition

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### II. T he M ai z e F amil y
There are no changes from Section II of the previously approved petition number 97-099- 01p.



### III. D escripti o n o f the T ransf o rmati o n Sy stem
The antecedent organism, maize line GA21, was produced using a particle acceleration meth

od. 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 D onor G enes an d

R e g ulatory S

equen c es 

### The antecedent organism, maize line GA21, was generated using a particle acceleration transformation system with a gel-isolated Not I 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, origi

nally 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 Cla I and Eco RV restriction endonuclease sites was chosen for further characterization (Figure 1). Nucleotide sequenc

ing 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 (Hu

man Genome Center, Institute for Medical Science, University of Tokyo).

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Stine Seed Farm Page 12 of 127 Figure 1: Map of 6.0 kb maize gen omic fragment and developmental steps in creating maize line HCEM485. III.2 T he V

e c tor p H C EM 

### The 6.0 kb maize genomic fragment was cloned into the Cla I and Eco RV sites of pBlueScript vector and subjected to site-directed mutagenesis using the QuikChange Site-Directed Muta

genesis 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 mu

tations 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.Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 13 of 127 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 a l., 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 mutat

ed 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.Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 14 of 127 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 P lant T

ransformat i on an d

R e g enerat i on 

### For DNA introduction, pHCEM was digested with Cla I and Eco RV, subjected to agarose gel electrophoresis (1 percent agarose), and the 6.0 kb band was excised and purified using Qia

gen’s Qiaquick gel extraction kit. The purified maize DNA fragment was introduced into im

mature 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 maizefollowing 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 A nal y sis o f M ai z e L ine HCEM485

I V .1 M ole c ular C hara c ter i zat i on 

### 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 Cla I and Acc 65I (cor

responding to positions 1–2346); and b) A/E – obtained from a double digest of the pHCEM plasmid with Acc 65I and Eco RV (corresponding to positions 2347–6010). Probes ( ca . 50 ng each) were labeled with 50 μCi of (α- 32 

### P)-dCTP (3000 Ci/mmol) using a random labeling system (RediprimeTM II, Amersham Piscataway, NJ). Genomic DNA (7 μg) isolated fromMaize Line HCEM485 USDA Extension Petition

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## CBI Deleted

HCEM485 and control Stine 963 maize was digested (37oC, overnight) with different restric

tion endonuclease enzymes and restriction fragments were separated by agarose gel electro

phoresis 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
32 

### P-labeled probes followed by autoradiography. [

M 1 2 3 4 5 6 7 8 9

A

/ C p r o b e B a m H I H i n d I I I A c c 6 5 I

2 3

. 1

9

. 4 6

. 6

4

. 4

2

. 3 2

. 0 M 1 2 3 4 5 6 7 8 9 B a m H I H i n d I I I A c c 6 5 I

A / E p r o b e 2 3

. 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 Hin dIII digestion (Figure 4 A and B, lane 7) indicated the presence of a single >=23 kb hybrid

izing 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 frag

ment. Digestion with Bam HI 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 Bam HI 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 correspondingMaize Line HCEM485 USDA Extension Petition

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## CBI Deleted

to the control DNA. Digestion with Acc 65I (an isoschizomer of Kpn I 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 Acc 65I and Bam HI digests, however these are not clearly visible in the Hin dIII digested lanes. As Hin dIII 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 or ganization detailed in Fig

ure 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 Acc 65I ~4.7 ~6 ~8 4.7 6.01 >2.350 ~7.3 ~6 ~5 7.326 6.010 >3.661 Bam HI ~2 2.042 ~7.8 ~4 ~15 7.738 3.969 >3.871 Hin dIII ~24 >24 ~24 >24Maize Line HCEM485 USDA Extension Petition

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## CBI Deleted

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 ob

served with the enzymes Kpn I and Bam H 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 restrictionMaize Line HCEM485 USDA Extension Petition

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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 Hin dIII 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 1 0 1 1 1 2 B a m H I H i n d I I I K p n I A c c 6 5 I

V P 1 p r o b e 2 3

. 1

9

. 4 6

. 6

4

. 4

2

. 3 2

. 0 M 1 2 3 4 5 6 7 8 9 M 1 0 1 1 1 2 B a m H I H i n d I I I K p n I A c c 6 5 I

V P 2 p r o b e 2 3

. 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 Vec

tor 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.

I V .2 I nher i tan c e an d

S ta bi

l

i ty 

### 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 (9289xH

CEM485)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 fieldMaize Line HCEM485 USDA Extension Petition

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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 fromeach 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).
χ

2 

### = ∑[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: O bserved vs . expected segregants for F1 hybrid and F2 selfed generations derived from HCEM485 maize. (9289xHCEM485)9032 F1 (9289xHCEM485)9032 S1F2 O bserved Expected O bserved Expected Trait Positive 1 129 124.5 107 108 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 χ

2 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.Maize Line HCEM485 USDA Extension Petition

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## CBI Deleted

I V .3 G ene E xpress i on 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 as

sess the integrity of this protein as expressed in maize line HCEM485 and to assess its equiva

lence 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, indicat

ing 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 bacte

rial sample of the protein (Figure 8, lanes 1 and 5) retained the His-tag used to isolate the pro

tein 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. 

### ]Maize Line HCEM485 USDA Extension Petition

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## CBI Deleted

In summary, the 2mEPSPS protein expressed in maize line HCEM485 is equivalent with re

spect 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 M r

2 1 6
1 3 2
7 8
4 5

. 7
3 2

. 5
1 8

. 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 un

necessary 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 na

tive regulatory sequences; (2) qualitatively, from western blot analysis, the level of expressionMaize Line HCEM485 USDA Extension Petition

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### of 2mEPSPS in HCEM485 is less than that observed in the antecedent organism, GA21 (Figure9); 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).

I V .4 2 m EPSPS P rote i n

S

afety 

### 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 ac

ids (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 biologi

cal 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 modi

fied EPSPS expressed in event GA21, the 2mEPSPS protein expressed in maize line HCEM485Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 23 of 127 

### has the same safety characteristics as the modified EPSPS protein expressed in the antecedent organism, GA21.

I V .5 C on c lus i ons 

### 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. P hen o t y pic and Ag r o n o mic C haracteristics
It was not possible to evaluate hybrids of HCEM485 in direct comparison to hybrids of the an

tecedent organism. However as GA21 was determined to be not significantly different to com

mercially 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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Stine Seed Farm Page 24 of 127 Figure 11: Pedigree chart of HCEM485 seed used in 2007 agronomic trials.
V

.1 T r

i al D es

ig

n an d

E valuat i on 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.Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 25 of 127 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 se

lected 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 ir

rigation 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 de

velop 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 plant

ing. EMRGR Seedling Vigor V3 Early emergence vigor rating. Data col

lected prior to V3 stage of maize develop

ment. 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, respec

tively, for resistance to root lodging). GMSTP Grain Moisture Per

cent Harvest Percent grain moisture measured at harvest.

HAVPN Final Stand Count Harvest Harvest population (plants per acre). HUP5N Heat units to 50% pol

len shed Flowering (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, respec

tively, for resistance to root lodging). STKLR Stalk Lodging Rating Harvest This is a 1-9 rating where a higher score in

dicates 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.Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 27 of 127 V.1.4 Experimental Design and Statistical Analysis 

### Each of the agronomic trials utilized a randomized complete block design with three replica

tions 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, identi

fied 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:
Y

ij 

### = U + T i 

### + L j 

### + LT ij 

### + e ij 

### where Y ij 

### is the observed response for genotype i at location j , U is the overall mean, T i 

### is the treatment (HCEM485 vs. control genotypes) effect, L j 

### is the location effect, LT ij 

### is the location x treatment (genotype) interaction effect and e ij 

### is the residual error (Annicchiarico, 2002). For each variate, the statistical significance of the genotype effect ( i.e. , HCEM485 vs. control hy

brids) 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 G rowth H a

bi t 

### 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 statisti

cal 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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## CBI Deleted

[ ERTLR a 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 N b 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 V e g etat i ve V ig

or 

### 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 be

tween 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.

[ EMRGR a (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

N b 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 R epro d u c t i ve C hara c ter i st ic s 

### 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 variatesMaize Line HCEM485 USDA Extension Petition

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## CBI Deleted

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.

[ EMRGP a (%) 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

N b 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 Y i el d an d G ra i n C hara c ter i st ic s 

### 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 anal

ysis, 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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## CBI Deleted

[ YGSMN a (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 N b 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 D i sease O b servat i ons 

### 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 Septem

ber. 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).

[ SRDI a 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 N b 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 inci

dence; 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 P ollen M orpholo g y an d

V i a

bi

l

i ty 

### 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 HCEM485Maize Line HCEM485 USDA Extension Petition

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## CBI Deleted

maize, pollen morphology and viability were investigated by microscopically examining pol

len 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 refer

ence 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 read

ily 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 indi

cated in each photomicrograph. 

### ]

V

.8 C ompos i t

i onal A nalys i s 

### 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 sam

ples 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). IndividualMaize Line HCEM485 USDA Extension Petition

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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 ex

ception 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 vari

ance 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 cul

tivated maize forage and grain.

V.8.1 Proximates 

### Analysis of the major constituents of maize, or proximates, was used to determine the nutri

tional 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 re

ported 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 (mois

ture, 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 hybridMaize Line HCEM485 USDA Extension Petition

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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 differ

ences 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 statisti

cally significant differences, average values were within the ranges reported in the literature.Maize Line HCEM485 USDA Extension Petition

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Comparison of the calcium and phosphorus composition of the HCEM485 forage and the con

trol 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.Maize Line HCEM485 USDA Extension Petition

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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 veg

etable, 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 dif

ferences between HCEM485 and control sample means were observed for levels of tocopher

ols, 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 ribo

flavin (B2) were below the limit of quantification in all samples. For all of the quantifiable ana

lytes, 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.Maize Line HCEM485 USDA Extension Petition

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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 con

sidered 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.Maize Line HCEM485 USDA Extension Petition

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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%) ac

ids. 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.Maize Line HCEM485 USDA Extension Petition

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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 de

termined 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 dif

ferences 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, antinu

trients, is responsible for deleterious effects related to the absorption of nutrients and micronu

trients 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, fur

fural, inositol, phytic acid, raffinose and trypsin inhibitor. These secondary metabolites and antinutrients were analyzed in grain samples from HCEM485 and control hybrids (T able 19).

Phenolic acids — may have beneficial health effects because of their anti-oxidant proper

ties. 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 cere

als. 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 (Clif

ford, 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).Maize Line HCEM485 USDA Extension Petition

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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 (Rick

ard 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 phospho

rous 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 con

sidered 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 soy

beans. Trypsin inhibitors in soybean give rise to inactivation and loss of trypsin in the smallMaize Line HCEM485 USDA Extension Petition

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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 chymotryp

sin 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 concentra

tions occurring in vegetable oils. They are absorbed only in trace amounts but have the ben

eficial 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 con

trol hybrids (Table 20). Since phytosterols are not commonly included in compositional analy

ses 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 de

termine 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 conven

tional 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 perfor

mance (Flachowsky et al. , 2005). Therefore, once compositional and phenotypic equivalenceMaize Line HCEM485 USDA Extension Petition

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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 C on c lus i ons 

### 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, dis

ease 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 com

position to both the control hybrids included in this study, to other commercial maize hybrids and - by extension - to the antecedent organism.



### VI. E nvir o nmental I mpact o f I ntr o ducti o n o f M ai z e L ine 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 poten

tial 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).Maize Line HCEM485 USDA Extension Petition

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### VII. A dverse Co nse q uences o f I ntr o ducti o n
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 introduc

tion. 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. R eferences

Adams, T.B., Doull, J. and Goodman, J.I. (1997). The FEMA GRAS assessment of furfural used as a flavour ingredient. Food and Chemical Toxicology 35(8) : 739–751. Alexander, M.P. (1969). Differential staining of aborted and non-aborted pollen. Stain Technology 41 : 117–122.ANZFA (2000). Final Risk Assessment Report – Application A362: Food derived from glyphosatetolerant corn line GA21. Annicchiarico, 2002. Genotype x Environment Interactions. FAO Plant Protection and Production Papers, 174. FAO. Baylis, A. (2000). Why glyphosate is a global herbicide: Strengths, weaknesses and prospects. Pest Management Science 56(4) : 299–308. Bradshaw, L.D., Padgette, S.R., Kimball, S.L. and Wells, B.H. (1997). Perspectives on glyphosate re

sistance. Weed Technology 11 : 189–198. Clifford, M.N. (1999). Chlorogenic acids and other cinnamates – nature, occurrence and dietary bur

den. Journal of the Science of Food and Agriculture 79(3) : 362–372. EFSA (2006). Guidance document of the scientific panel on genetically modified organisms for the risk assessment of genetically modified plants and derived food and feed. European Food Safety Agency. EFSA Journal 99 : 1–100. EuropaBio (2003). Safety Assessment of GM Crops, Document 1.1 Substantial Equivalence–Maize, Issue 2. The European Association for Bioindustries. Flachowsky, G., Chesson, A. and Aulrich, K. (2005). Animal nutrition with feeds from genetically modified plants. Archives of Animal Nutrition 59(1) : 1–40. Gardiner, J., Schroeder, S., Polacco, M.L., Sanchez-Villeda, H., Fang, Z., Morgante, M., Landewe, T., Fengler, K., Useche, F., Hanafey, M., Tingey, S., Chou, H., Wing, R., Soderlund, C. and Coe, E.H. (2004). Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization. Plant Physiology 134(4) : 1317–1326. Geiger, D.R. and Fuchs, M.A. (2002). Inhibitors of aromatic amino acid biosynthesis (glyphosate). In : Böger P, Wakabayashi K, Hirai K (Eds.). Herbicide Classes in Development. Springer, Heidel

berg, Germany, pp 59–85.Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 45 of 127 Haslam, E. (1974). The Shikimate Pathway. John Wiley and Sons, New York, USA. Held, B.M., Wilson, H.M., Dykema, P.E., Lewnau, C.J. and Eby, J.C. (2006). Glyphosate resistant plants. US Patent No. 7,045,684. Held, B.M., Wilson, H.M., Hou, L., Lewnau, C.J. and Eby, J.C. (2004). Methods and compositions for the introduction of molecules into cells. US Patent No. 6,809,232. ILSI (2006). Crop Composition Database Ver. 3.0. International Life Sciences Institute. (http://www. cropcomposition.org). Kishore, G.M. and Shah, D.M. (1988). Amino acid biosynthesis inhibitors as herbicides. Annual Re

view of Biochemistry 57 : 627–663. Lebrun, M., Freyssinet, G., Sailland, A., Rolland, A. and Freyssinet, G. (1991). Molecular basis of resistance to shikimic herbicides in adapted maize cell cultures. Genbank Accession X63374. Lebrun, M., Sailland, A., Freyssinet, G. and Degryse, E. (2003). Mutated 5-enolpyruvylshikimate-3- phosphate synthase, gene coding for said protein and transformed plants containing said gene. U.S. Patent 6566587. Little, T.M. and Jackson Hills, F. (1978). Agricultural experimentation: design and analysis. Wiley, New York. Linus Pauling Institute (2004). Micronutrient Information Center, Oregon State University. (http://lpi. oregonstate.edu/infocenter/vitamins/vitaminE/). Monsanto (1997). USDA Petition 97_09901p. Monsanto (2000). USDA Petition 00_01101p. Naczk, M., Amarowicz, R. and Shahidi, F. (1997). α-Galactosides of sucrose in foods: composition, flatulence-causing effects and removal. In : Shahidi,F. (Ed.). Anti-nutrients and Phytochemicals in Food. American Chemical Society Symposium Series 662. Oxford University Press, Cary, NC, USA, pp 127-151. NRC (1998). Nutrient Requirements of Swine (10th Revised Edition). National Research Council, National Academy Press, Washington DC, USA. OECD (2002). Consensus Document on Compositional Considerations for New Varieties of Maize ( Zea mays ): Key Food and Feed Nutrients, Anti-Nutrients and Secondary Plant Metabolites. (ENV/ JM/MONO(2002)25). Organization for Economic Cooperation and Development (OECD), Paris, France. OECD (2003). Considerations for the Safety Assessment of Animal Feedstuffs derived from Geneti

cally Modified Plants. ENV/JM/MONO(2003)10. Organization for Economic Cooperation and Development (OECD), Paris, France. Ostlund, R.E. Jr. (2002). Phytosterols in human nutrition. Annual Review of Nutrition 22 : 533–549. Padgette, S.R., Re, D.B., Barry, G.F., Eichholtz, D.E., Delannay, X., Fuchs, R.L., Kishore, G.M. and Fraley, R.T. (1996). New weed control opportunities: Development of soybeans with a Roundup ReadyTM gene. In : Duke, S.O. (Ed). Herbicide-Resistant Crops: Agricultural, Environmental, Economic, Regulatory, and Technical Aspects. CRC Press, Inc., Boca Raton, FL, USA, pp 53–84.Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 46 of 127 Radtke, J., Lineisen, J. and Wolfram, G. (1998). Phenolic intake of adults in a Bavarian subgroup of the national food consumption survey. Zeitschrift fur Ernahrungswissenschaft 37(2) : 190–197. Rao, A.V. and Koratkar, R. (1997). Anticarcinogenic effects of saponins and phytosterols. In : Shahidi,F. (Ed.). Anti-nutrients and Phytochemicals in Food. American Chemical Society Symposium Se

ries 662. Oxford University Press, Cary, NC, USA. Rickard, S.E. and Thompson, L.U. (1997). Interactions and biological effects of phytic acid. In : Shahidi,F. (Ed.). Anti-nutrients and Phytochemicals in Food. American Chemical Society Sym

posium Series 662. Oxford University Press, Cary, NC, USA. Ryan, E., Galvin, K., O’Connor, T.P., Maguire, A.R. and O’Brien, N.M. (2007). Phytosterol, squalene, tocopherol content and fatty acid profile of selected seeds grains and legumes. Plant Foods Hum Nutr. 63: 85-91 Sasaki, Y.F., Imanihi, H., Ohta, T. and Shirasu, Y. (1989). Modifying effects of components of plant essence on the induction of sister-chromatid exchanges in cultured Chinese hamster ovary cells. Mutation Research 226 : 103–110. Shahidi, F. (1997). Beneficial health effects and drawbacks of antinutrients and phytochemicals in foods. In : Shahidi,F. (Ed.). Anti-nutrients and Phytochemicals in Food. American Chemical Society Symposium Series 662. Oxford University Press, Cary, NC, USA. Smith, E.A. and Oehme, F.W. (1992). The biological activity of glyphosate to plants and animals: A literature review. Veterinary and Human Toxicology 34 : 531–543. Spencer, M., Mumm, R. and Gwyn, J. (2000). Glyphosate resistant maize lines. U.S. Patent 6040497. Steinrücken, H., and Amrhein, N. (1980). The herbicide glyphosate is a potent inhibitor of 5-enolpyr

uvylshikimic acid-3-phosphate synthase. Biochemical and Biophysical Research Communica

tions 94 : 1207–1212. Stich, H.F. (1992). Teas and tea components as inhibitors of carcinogen formation in model systems and
man. Preventive Medicine 21(3) : 377–384. Watson, S.A. (1982). Corn, amazing maize, general properties. In : Wolff, I.A. (Ed.). CRC Handbook of Processing and Utilisation in Agriculture, Vol. II, Part I, Plant Products. CRC Press, Inc, Boca Raton, FL, USA, pp 3-29. Watson, S.A. (1987). Structure and composition. In : Watson, S.A. and Ranstead, P.E. (Eds.). Corn: Chemistry and Technology. American Association of Cereal Chemists, Minnesota. White, P.J. and Pollak, L.M. (1995). Corn as a food source in the United States: Part II. Processes, products, composition and nutritive values. Cereal Foods World 40(10) : 756–762.Maize Line HCEM485 USDA Extension Petition

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I X
. A ppendi x 1: HCEM N ucle o tide S e q uence

[ No CBI Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 44 of 47 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 gaggcgaaccMaize Line HCEM485 USDA Extension Petition

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## CBI Deleted No CBI Maize Line HCEM485 USDA Extension Petition

Stine Seed Farm Page 45 of 47 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 ttaaaatcaaMaize Line HCEM485 USDA Extension Petition

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## 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

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. A ppendi x 2: USDA APHIS R elease No tificati o ns R elevant t o the F ield T estin g o f M ai z e L ine 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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## 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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