II.A. CORN AS A CROP Corn is the major feed grain grown by farmers in the U.S., leading all other crops in value and volume of production. It is produced on every continent of the world with the exception of Antarctica. (Ohio Corn Marketing Program, 2000) (Table 1). Although corn is produced throughout the U.S., the major production area (accounting for over 50 percent of the corn grown) is located in the states of Iowa, Illinois, Nebraska and Minnesota. Other major corn growing states are Indiana, Ohio, Wisconsin, South Dakota, Michigan, Missouri, Kansas and Kentucky. All of these states together are collectively referred to as the Corn Belt (Ohio Corn Marketing Program, 2000).
17 Revised September 7, 2004
The primary use of the corn produced each year is as an animal feed (50.1%). The remaining corn is exported (22.6%), held as ending stock (10.3%), processed into corn syrup for use as a sweetener (8.0%), converted into ethanol (5.0%), extracted for starch (2.6%), used in processed foods (1.2%) and grown as seed (0.2%) (Ohio Corn Marketing Program, 2000). The yellow dent type of corn is used for feed and this is grown as a commodity crop in North America. Wet milling to produce starch and sweetener products for foods accounts for the greatest volume of corn that is processed (Table 2). Non-food products such as industrial starches, corn gluten feed, and corn gluten meal are also manufactured (White and Pollak, 1995). The primary products derived from the dry milling process are corn grits, cornmeals, and corn flours. The largest food/feed product volume of the dry-milling industry is animal feed followed by brewing and food uses. Table 1. Corn Production in Specified Countriesa
1999/00 Country Argentina Brazil Canada China, Peoples Republic of European Union Egypt Hungary Indonesia India Mexico Nigeria Romania Philippines South Africa, Republic of Thailand Ukraine Others Subtotal United States World Total
2000/01 15,400 41,536 6,827 106,000 37,823 5,636 5,000 5,900 12,068 17,917 4,000 4,800 4,508 8,040 4,700 3,848 52,719 336,722 251,854 588,576
2001/02 14,700 35,501 8,389 114,088 40,006 6,160 7,600 6,000 13,510 20,400 5,000 7,000 4,505 10,050 4,500 3,641 56,814 357,864 241,485 599,349
2002/03 15,500 45,000 8,975 121,300 40,089 5,880 6,000 6,100 11,100 18,800 5,200 7,300 4,300 9,200 4,200 4,200 60,495 373,639 228,805 602,444
Production 17,200b 31,641 9,161 128,086 36,404 5,678 7,000 6,200 11,470 19,240 5,100 10,500 4,449 11,455 3,900 1,737 58,601 367,822 239,549 607,371
USDA-FAS 2003. http://www.fas.usda.gov/psd/complete_tables/GF-table9-81.htm Numbers are 1,000 metric tons.
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Table 2. Food and Industrial Corn Use: 1993-2002a Crop Yearb HFCSc Glucose Starch & Dextrose Fuel Beverage Cereals Alcohol Alcohol and Other Products Total

1994 1993

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Table 3. Genetics elements of the plasmid PHP17662. Genetic element RB Size (bp) 25 Location on PHP17662 (bp) 18072-18096 18327-20312 Function Right Border ubiquitin promoter (plus ubiquitin 5UTR and intron) (Christensen et al., 1992) from Zea mays synthetic version of the cry34Ab1 gene encoding the 14 kDa delta-endotoxin parasporal crystal protein from the nonmotile strain PS149B1 (NRRL B21553)] of Bacillus thuringiensis terminator sequence from Solanum tuberosum proteinase inhibitor II (An et al., 1989) promoter from Triticum aestivum peroxidase; bases 45-1342 from GenBank X53675 (Hertig et al., 1991) synthetic version of the cry35Ab1 gene encoding a 44 kDa delta-endotoxin parasporal crystal protein from the nonmotile strain PS149B1 (NRRL B21553) of Bacillus thuringiensis terminator sequence from Solanum tuberosum proteinase inhibitor II (An et al., 1989) 35S promoter from Cauliflower Mosaic Virus, Strasbourg strain (Hohn et al., 1982) synthetic, plant-optimized phosphinothricin acetyltransferase coding sequence from Streptomyces viridochromogenes 35S terminator from Cauliflower Mosaic Virus Left Border

UBI1ZM PRO 1,986

cry34Ab1

20342-20710

PINII TERM

20735-21052

TA 1,299 PEROXIDASE

21079-22377

cry35Ab1

22393-23544

PINII TERM CaMV35S PRO PAT CaMV35S TERM LB
23566-23883 23885-24433 24434-24985 24998-25196 25439-25463
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V. GENETIC CHARACTERIZATION OF EVENT DAS-59122-7 Molecular characterization by Southern blot analysis concluded that the insert in B.t. Cry34/35Ab1 event DAS-59122-7 occurred as a simple integration of a single intact T-DNA from plasmid PHP17662. Two Southern blot studies were completed to determine copy number and intactness of the insert, determine the stability of the insert across multiple generations, and to provide a detailed restriction enzyme map of the insertion region. In the first study that compared two distinct generations of B.t. Cry34/35Ab1 event DAS-59122-7, digestion with Xho I, an enzyme that cuts once in the T-DNA, produced one hybridizing band for all three gene probes, cry34Ab1, cry35Ab1, and pat, indicating one T-DNA had inserted. Hybridization of Sac I digested DNA with the cry35Ab1 and pat gene probes resulted in bands of the expected size for an intact TDNA insertion and hybridization with the cry34Ab1 gene probe produced one border fragment substantiating the Xho I results of a single T-DNA insertion. An additional digestion with Hind III, an enzyme that releases all three gene transcription units as one DNA fragment, revealed that the T-DNA unit was inserted intact. Hybridization with five probes located outside of the T-DNA on plasmid PHP17662 did not detect any gene fragments, indicating the absence of the tetracycline and spectinomycin resistance genes, the virG gene, and the absence of regions immediately adjacent to the Left and Right T-DNA Borders in transgenic corn event DAS-59122-7. Identical fragment sizes were observed in all cases for two distinct generations of transgenic corn event DAS-59122-7, indicating stability of inheritance across generations (Locke and Igo, 2003; Appendix 2, Section 6). In the second study, a detailed restriction enzyme map of the insertion was hypothesized based on Southern blot analysis of the DNA inserted into event DAS-59122-7 (Figure 3). The Southern hybridization results indicated that a single, intact T-DNA inserted into the corn genomic DNA to produce event DAS-59122-7 and sites for Bsa I, Nco I, Sac I and Xho I were placed relative to the Right and Left Border regions of the T-DNA insertion. In addition, the results did not indicate that rearrangements of the T-DNA had occurred, as all internal restriction enzyme sites appeared to be intact and produced hybridizing fragments of the expected size (Locke et al., 2003; Appendix 2, Section 7). Identical fragment sizes were observed in all cases for four distinct generations of B.t. Cry34/35Ab1 event DAS-59122-7 analyzed in the two studies, indicating stability of inheritance across generations.

Lane Sample Hi-II + 7 copies PHP17662 Hi-II + 3 copies PHP17662 DIG VII Hi-II DAS-59122-7T27 DAS-59122-7T30 DAS-59122-7T31 DAS-59122-7T32 DAS-59122-7T34 DAS-59122-7T35 Lane Sample DAS-59122-7T36 DAS-59122-7T37 DAS-59122-7T38 DAS-59122-7T39 DAS-59122-7T42 DAS-59122-7T43 DAS-59122-7T45 DAS-59122-7T47 DAS-59122-7T49 DAS-59122-7T50 Lane Sample DAS-59122-7T51 DAS-59122-7T52 DAS-59122-7T11 (s) DAS-59122-7T22 (s) DAS-59122-7T23 (s) Blank Hi-II DIG VII Hi-II + 3 copies PHP17662 Hi-II + 7 copies PHP17662
1.95 1.88 1.51 1.48 1.2 0.99 0.72
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Dow AgroSciences/Pioneer Hi-Bred International USDA Petition for Nonregulated Status Cry34/35Ab1 Line 59122 CBI-Deleted Version Figure 40. Southern blot analysis of Gel 3; cry34Ab1 probe. DNA isolated from transgenic corn event DAS-59122-7 (BC2S1) and 581 unmodified corn was digested with Sac I and probed with the cry34Ab1 gene probe. Approximately 3 g of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP17662 at the indicated approximate gene copy number equivalents and 3 g of unmodified 581 DNA. Fragment sizes in kb of the DIG-labeled DNA Molecular Weight Marker VII (DIG VII marker) are indicated adjacent to the blot image. Lane Assignments:
Lane Sample Lane Sample Lane Sample 581 + 7 copies PHPDAS-59122-7TDAS-59122-7T+ 3 copies PHPDAS-59122-7TDAS-59122-7T79 DIG VII 13 DAS-59122-7TDAS-59122-7T14 DAS-59122-7TDAS-59122-7T28 (s) Blank 15 DAS-59122-7TDAS-59122-7T33 (s) DAS-59122-7TDAS-59122-7TDAS-59122-7T40 (s) DAS-59122-7TDAS-59122-7TBlank DAS-59122-7TDAS-59122-7TDIG VII DAS-59122-7TDAS-59122-7T581 + 3 copies PHP17662 DAS-59122-7TDAS-59122-7T581 + 7 copies PHP17662 (s) indicates a sensitive or null segregant that was negative for expression of both Cry34Ab1 and PAT proteins.

3.6 2.8

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Dow AgroSciences/Pioneer Hi-Bred International USDA Petition for Nonregulated Status Cry34/35Ab1 Line 59122 CBI-Deleted Version Figure 41. Southern blot analysis of Gel 4; cry34Ab1 probe. DNA isolated from transgenic corn event DAS-59122-7 (BC2S1) and Hi-II unmodified corn was digested with Sac I and probed with the cry34Ab1 gene probe. Approximately 3 g of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP17662 at the indicated approximate gene copy number equivalents and 3 g of either unmodified Hi-II or 581 DNA. Fragment sizes in kb of the DIG-labeled DNA Molecular Weight Marker VII (DIG VII marker) are indicated adjacent to the blot image. Note: Approximately 0.5 g of DAS-59122-7 T66 DNA was loaded in lane 16. Lane Assignments:
Lane Sample Hi-II + 7 copies PHP17662 Hi-II + 3 copies PHP17662 DIG VII Hi-II Blank DAS-59122-7T56 Blank DAS-59122-7T29 (s) DAS-59122-7T41 (s) DAS-59122-7T44 (s) Lane Sample DAS-59122-7T46 DAS-59122-7T53 DAS-59122-7T57 DAS-59122-7T64 DAS-59122-7T65 DAS-59122-7T66 DAS-59122-7T67 DAS-59122-7T70 DAS-59122-7T71 DAS-59122-7T74 (s) (s) (s) (s) (s) (s) (s) (s) (s) (s) Lane Sample DAS-59122-7T75 (s) DAS-59122-7T11 (s) DAS-59122-7T22 (s) Blank DAS-59122-7T78 Blank DIG VII 581 + 3 copies PHP+ 7 copies PHP17662 Blank

Lane Sample 581 + 7 copies PHP+ 3 copies PHP17662 DIG VII 581 Blank DAS-59122-7T54 DAS-59122-7T55 DAS-59122-7T56 DAS-59122-7T58 DAS-59122-7T59 Lane Sample DAS-59122-7T60 DAS-59122-7T61 DAS-59122-7T62 DAS-59122-7T63 DAS-59122-7T68 DAS-59122-7T69 DAS-59122-7T72 DAS-59122-7T73 DAS-59122-7T76 DAS-59122-7T77 Lane Sample DAS-59122-7T78 DAS-59122-7T79 DAS-59122-7T80 DAS-59122-7T28 (s) DAS-59122-7T33 (s) DAS-59122-7T40 (s) Blank DIG VII 581 + 3 copies PHP+ 7 copies PHP17662
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Dow AgroSciences/Pioneer Hi-Bred International USDA Petition for Nonregulated Status Cry34/35Ab1 Line 59122 CBI-Deleted Version Figure 45. Southern blot analysis of Gel 4; cry35Ab1 probe. DNA isolated from transgenic corn event DAS-59122-7 (BC2S1) and Hi-II unmodified corn was digested with Sac I and probed with the cry35Ab1 gene probe. Approximately 3 g of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP17662 at the indicated approximate gene copy number equivalents and 3 g of either unmodified Hi-II or 581 DNA. Fragment sizes in kb of the DIG-labeled DNA Molecular Weight Marker VII (DIG VII marker) are indicated adjacent to the blot image. Note: Approximately 0.5 g of DAS-59122-7 T66 DNA was loaded in lane 16. Lane Assignments:

Lane 9

Sample Hi-II + 7 copies PHP17662 Hi-II + 3 copies PHP17662 DIG VII Hi-II Blank DAS-59122-7T56 Blank DAS-59122-7T29 (s) DAS-59122-7T41 (s)
Sample DAS-59122-7T46 DAS-59122-7T53 DAS-59122-7T57 DAS-59122-7T64 DAS-59122-7T65 DAS-59122-7T66 DAS-59122-7T67 DAS-59122-7T70 DAS-59122-7T71
(s) (s) (s) (s) (s) (s) (s) (s) (s)

Lane 29

Sample DAS-59122-7T75 (s) DAS-59122-7T11 (s) DAS-59122-7T22 (s) Blank DAS-59122-7T78 Blank DIG VII 581 + 3 copies PHP+ 7 copies PHP17662
DAS-59122-7T44 (s) 20 DAS-59122-7T74 (s) 30 (s) indicates a sensitive or null segregant that was negative for expression of both Cry34Ab1 and PAT proteins.
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Dow AgroSciences/Pioneer Hi-Bred International USDA Petition for Nonregulated Status Cry34/35Ab1 Line 59122 CBI-Deleted Version Figure 46. Southern blot analysis of Gel 1; pat probe. DNA isolated from transgenic corn event DAS-59122-7 (BC2S1) and PH09B unmodified corn was digested with Sac I and probed with the pat gene probe. Approximately 3 g of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP17662 at the indicated approximate gene copy number equivalents and 3 g of unmodified PH09B DNA. Fragment sizes in kb of the DIG-labeled DNA Molecular Weight Marker VII (DIG VII marker) are indicated adjacent to the blot image. Lane Assignments:

Figure 57. SDS-PAGE gel of immuno-purified Cry34/35Ab1 ICP from event DAS-59122-7 corn stained with GelCode Glycoprotein Stain (Panel A) and GelCode Blue Total Protein Stain (Panel B).

kDa 18 kDa 18 -

Panel A
kDa -- 220 -- 120 -- 70 -- 50 -- 30 -- 20 -- 10

Panel B

110 Revised September 7, 2004
and 25 L, respectively, and 20 L of each sample was removed for MALDI-TOF peptide mass fingerprint analysis. The concentrated proteins were mixed with 10 L of Laemmli sample buffer and heated for 5 minutes at 100 C. After a brief centrifugation, the supernatants were loaded directly on the gel. SDS-PAGE was performed with a Bio-Rad Ready gel fitted in a Ready Gel module. After separation the gel was stained with Pierce GelCode Blue protein stain according to the manufacturers protocol. Finally, the gel was scanned with a Molecular Dynamics densitometer to obtain a permanent visual record of the gel. The respective Cry34Ab1 and Cry35Ab1 bands were excised from the gel, placed into siliconized Eppendorf microcentrifuge tubes, and destained with 50% acetonitrile in 25 mM NH4HCO3. The gel pieces were dried using vacuum centrifugation, and digested with sequencing grade trypsin overnight (approximately 17 hours) at 37 C. The peptides were extracted with 50% acetonitrile in 0.5% TFA. After brief centrifugation to pellet the gel pieces, the supernatant containing the peptides was decanted and dried in a Savant Speed-Vac and the samples were stored at 20 C until MALDI-TOF MS analysis. For clarity not all of the molecular weight markers were labeled. The lanes contained: Lane 1 Lane 2 Lane 3 BenchMark Molecular Weight Standard 5 L Immuno-purified Cry34Ab1 (from event DAS-59122-7) 30 L Immuno-purified Cry35Ab1 (from event DAS-59122-7) 30 L
Figure 58. SDS-PAGE gel of corn-derived Cry34Ab1 and Cry35Ab1 (event DAS-59122-7) proteins for MALDI-TOF peptide mass fingerprinting. Cry34Ab1 and Cry35Ab1 Lot #2 was concentrated to ~1000 L
1 kDa 220 --120 --70 ---50 ---30 ---20 ----

Cry35Ab1

Cry34Ab----
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Dow AgroSciences/Pioneer Hi-Bred International USDA Petition for Nonregulated Status Cry34/35Ab1 Line 59122 CBI-Deleted Version Figure 59. Expected amino acid sequence and peptide fragments of corn-derived Cry35Ab1. Molecular weight: 43.8 kDa. Trypsin Cleavage (at K and R) of corn-derived Cry35Ab1. Alternating blocks of upper and lower case letters within the amino acid sequence are used to differentiate the potential peptides after trypsin digestion. The numbers on the left and right sides indicate the amino acid residue numbers.
Figure 60. Expected amino acid sequence and peptide fragments of corn-derived Cry34Ab1. Molecular weight: 13.6 kDa. Trypsin Cleavage (at K and R) of corn-derived Cry34Ab1. Alternating blocks of upper and lower case letters within the amino acid sequence are used to differentiate the potential peptides after trypsin digestion.

Protein

Crude Fiber

Cry34/35

control

Carbohydrates

In the proximate analysis of grain, no statistically significant differences were observed for crude fat, crude fiber, ADF, or NDF in the across location summary analysis (Table 26). Crude protein, ash and carbohydrates means across locations in the modified hybrid were within less than 9.2% of the control entry, and significantly different (P<0.05). Significant differences for these analytes were observed in 1 of 6 locations for each of the analytes. The across location mean values for all grain proximate, fiber, and carbohydrate analytes for the modified and control entries were within reported literature ranges (Figure 67).
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Table 26. Summary of proximates and fiber analysis for DAS-59122-7 and control grain: Across sites BU01, BU02, LI01, LI02, VI01, and VI02 in Chile.
Means3 Analyte1 Literature Range16.1 1.2 18.8 1.6 5.5 1.82 11.3 3.0 22.6 0.62 6.28 63.3 89.8 DAS-59122-7 10.0* 4.69 2.3 3.5 10.8 1.55* 83.8* CONTROL 9.61 4.49 2.3 3.5 10.3 1.42 84.5 STANDARD 0.140, 0.136 0.099, 0.095 0.113, 0.109 0.047, 0.045 0.301, 0.290 0.039, 0.038 0.195, 0.188
Crude Protein Crude Fat Crude Fiber ADF

NDF6 Ash Carbohydrates7

Percent dry weight Watson, 1982 and 1987; Jugenheimer, 1976; OECD, 2002; ILSI, 2003; Essner, 2003. Least square means 4 Standard error of DAS-59122-7 is followed by the control sample missing at location VIO2. 5 Acid Detergent Fiber 6 Neutral Detergent Fiber 7 Carbohydrates are calculated using the following formula = 100% - % protein - % fat - % ash *Means in row differ (P<0.05).
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Dow AgroSciences/Pioneer Hi-Bred International USDA Petition for Nonregulated Status Cry34/35Ab1 Line 59122 CBI-Deleted Version Figure 67. Proximate analysis of grain (% dry weight) from corn line 59122 and the control line. The data was generated from analysis of samples collected from six field trials conducted in Chile in 2002/2003. Means at each location shown: diamond = BU01, square = BU02, triangle = LI01, X = LI02, open circle = VI01, solid circle = VI02. Literature ranges are shaded. ADF=acid detergent fiber. NDF= neutral detergent fiber.

136 Revised September 7, 2004
Dow AgroSciences/Pioneer Hi-Bred International USDA Petition for Nonregulated Status Cry34/35Ab1 Line 59122 CBI-Deleted Version Table 28. Potency of microbially-expressed Cry34/35Ab1 ICP against several insect pests of corn. Microbially-expressed Cry34Ab1 (TSN102172) and Cry35Ab1 (TSN102171) proteins were used in a 1:3 mass ratio in diet overlay (for chewing insects) bioassays and a membrane feeding system for corn leaf aphid. Potency of Microbial ICP Against Several Insect Pests of Maize Insect GI50 (95% Confidence Limits) LC50 (95% Fiducial Limits) 2 (g ai/cm ) (g ai/cm2) northern corn rootworm (nCRW) larvae western corn rootworm (wCRW) larvae southern corn rootworm (sCRW) larvae European corn borer (ECB) larvae corn earworm (CEW) larvae black cutworm (BCW) larvae western corn rootworm (wCRW) adult 0.100 (0.0077-1.30) 2.35 (1.12-4.91) 0.224 (0.089-0.563) 18.7 (5.58-62.5) a 42.7 (26.4-69.2) >400 -(g ai/mL) -5.56 (1.76-19.6) 44.5 (18.5-165) a 343 (190-796) >400 >400 >400 >400 (g ai/mL) >4,000
corn leaf aphid (CLA) mixed
Results from all concentrations were not used in calculations.
Field Efficacy Efficacy of the Cry34/35Ab1 corn line 59122, along with lines 45216 and 45214, were evaluated for resistance to western corn rootworm (wCRW) at 4 locations in 4 states (IA, NE, WI, IN) in 2003 under USDA APHIS notification #03-035-15n. At each location the efficacy trial was planted into ground that was previously planted with corn to ensure that high numbers of western corn rootworm eggs were present. Additionally, western corn rootworm eggs were infested into the plot at a rate of approximately 1000 eggs per linear foot of row. This artificial infestation of western corn rootworm eggs occurred between the V2 and V4 (2-4 leaf) corn stage. Also at this time, the plots containing Cry34/35Ab1 corn were sprayed with a glufosinate-ammonium herbicide. Cry34/35Ab1 corn is also tolerant to glufosinate-ammonium herbicide, and this tolerance allows for easy identification and subsequent removal of null plants from the plots. Following infestation and removal of null plants, the plots were maintained using corn production methods typical for each location. Just prior to anthesis, the aerial portion of the plants were removed and discarded. The roots were dug from the soil, washed to remove the soil, and then evaluated for western corn rootworm damage using the Iowa State University 0-3 corn rootworm rating scale (J. D. Oleson 1998). The root rating data from each location were pooled across the 4 locations. These trials were designed as randomized complete blocks with three replicates. Data were analyzed using analysis of variance (ANOVA). Data were pooled and analyzed across locations using a mixed factor ANOVA. All means were separated using a protected LSD test (P=0.05). As expected, corn line 59122 (event DAS-59122-7) as well as corn events A and B expressing the Cry34Ab1/Cry35Ab1 bICP, controlled Diabrotica larvae (Table 29). Root ratings for the non-transgenic control plants exposed to western corn rootworm averaged 1.58 (0-3 scale), while the root ratings for the transgenic events ranged from 0.02-0.10 for

155.3 167.7 142.4 139.0 176.1 189.2 148.8
127.6 128.3 138.0 127.9 170.1 176.2 139.8
174.2 157.1 149.8 132.4 190.9 203.9 165.8
121.3 135.3 149.4 131.8 185.5 178.4 166.7
156.5 167.9 147.2 153.3 183.2 218.5 190.7
1 Hybrid contains event DAS-59122-7. 2 Hybrid does not contain event DAS-59122-7.
V.G. Secondary Metabolites, Anti-Nutrients and the Allergenic Potential of Proteins in Line 59122 Secondary metabolites are neither nutrients nor anti-nutrients (OECD, 2002). Characteristic secondary metabolites in corn are furfural and phenolic acids (ferulic and p-coumaric). Furfural is a heterocyclic aldehyde and is generally recognized as safe (GRAS). The phenolic acids are structural components of plant cells and act as a natural pesticide against insects and fungi. Ferulic acid and p-coumaric acid have weak antioxidative properties. Anti-nutrients in corn include phytic acid, raffinose, trypsin and chymotrypsin inhibitors. Raffinose is a non-digestible oligosaccharide and is considered an anti-nutrient due to the gas production and resulting flatulence caused by its consumption. The levels of trypsin and chymotrypsin inhibitors in corn are considered insignificant. Phytic acid binds about 60-75% of phosphorus in the form of phytate
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which results in less than 15% bioavailability of phosphorus for nonruminant animals It is common practice for swine and poultry producers to add phytase to the diet formulation to improve the utilization of phosphorus for these nonruminants. Grain of line 59122 was analyzed for secondary metabolites and anti-nutrients common to corn. Grain samples were collected from the field trial conducted in Chile in 2002/2003 as stated in Section V.E. of this petition. The results of these analyses are presented in Table 40 and Figure 68 and show that line 59122 is comparable to the nontransgenic control.
Table 40. Summary of secondary metabolites and anti-nutrients for DAS-59122-7 and control grain: Across sites BU01, BU02, LI01, LI02, VI01, and VI02 in Chile.
Means3 Analyte1 Literature Range2 DAS-59122-7 Control Standard Error4
Secondary Metabolites and Anti-Nutrients Inositol Raffinose Furfural P-Coumaric Acid Ferulic Acid Phytic acid Trypsin Inhibitor (TIU/g)

NR5 0.08 0.31

0.022 0.13 ND 0.014 0.177 0.877 2.82
0.021 0.12 ND 0.015 0.182 0.798 2.84
0.001 0.007 ND 0.001 0.012 0.040, 0.039 0.065, 0.063
0.003 0.058 0.02 0.37 0.29 1.29 1.1 7.185
Percent dry weight Watson, 1982; OECD, 2002; ILSI, 2003. Least square means 4 Standard error of DAS-59122-7 is followed by the control sample missing at location VIO2. *Means in row differ (P<0.05).
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Dow AgroSciences/Pioneer Hi-Bred International USDA Petition for Nonregulated Status Cry34/35Ab1 Line 59122 CBI-Deleted Version Figure 68. Resultsiof lsecondary metabolite and anti-nutrient analysis of grain (%maric Acid except Ino s to Raffinose P-Cou dry weight as indicated) from Cry34/35Ab1 line 59122 and the control line. The data was generated from analysis No literature range available of samples collected from six field trials conducted in Chile in 2002/2003. Means at each location shown: diamond = BU01, square = BU02, triangle = LI01, X = LI02, open circle = VI01, solid circle = VI02. Literature ranges are shaded. Furfural was not detected in the samples.

159 Revised September 7, 2004
Appendix 2, Section 11). Although not directly comparable to a dietary endpoint, this environmental concentration is quite low, indicating low risk to fish. There may not be an actual exposure route for fish to ingest the ICP in nature. Nontarget Arthropods Honeybees. There was no effect on mean survival to emergence for honeybee exposed to either 2 mg pollen from a Cry34/35Ab1-expressing event (from an earlier higher expressing event than the subject of this petition) or to 5.6 g/larvae Cry34/35Ab1 ICP (100X the amount consumed in pollen of the higher expressing event), 3.2 g/larva Cry34Ab1 protein, or 2.4 g/larva Cry35Ab1 protein (Maggi, 2001; Appendix 2, Section 25). The LC50 is >3.2 ug Cry34Ab1 protein/larva. For the Event DAS-59122-7 pollen HEEE of 75.29 g/g (Table 41), the equivalent exposure level is 0.07529 g/mg x 2 mg fed = 0.15 g/larva. Green Lacewing. The dietary LC50 for green lacewing (Chrysoperla carnea) larvae exposed to Cry34/35Ab1 has been investigated in a series of studies with microbiallyproduced protein administered in a diet of moth eggs (Sindermann et al., 2001; Appendix 2, Section 35). The single limit concentration of 280 g of Cry34/35Ab1 is 10X the empirical concentration in pollen of a high expressing corn event (16 g/g Cry34Ab1 + 12 g/g Cry35Ab1 = 28 g/g Cry34/35Ab1). The LC50 was determined to be greater than 16 g Cry34Ab1/mL. The target multiple of 10X field exposure for dosing was based on the assumption of a Cry34Ab1 expression level of 16 g/g in pollen. For Event DAS59122-7, the HEEE pollen value is 75.29 g/g (Table 41), resulting in an actual multiple of 16/75.29 x 10 = 2.1X. Field exposure of green lacewing to Cry34/35Ab1 proteins is restricted to larvae feeding on aphids. When the Event DAS-59122-7 Cry34Ab1 HEEE for leaf tissue is multiplied by a conservative estimate of protein transmission efficiency from leaf tissue to aphid, the exposure estimate is 235.03 g/g x 0.0087 = 2 g/g (Table 41; Poletika, 2003; Appendix 2, Section 11). Parasitic Wasp. Parasitic Hymenoptera adults (Nasonia vitripennis) were exposed to a single limit concentration of Cry34/35Ab1 in sugar water for up to 10 days. There were no significant differences in mortality between the treatment groups and a sugar water control. The LC50 was greater than 160 g Cry34Ab1/mL of the microbially-derived proteins (Sindermann et al., 2001; Appendix 2, Section 35). The exposure level represents 10X the empirical concentration in pollen of a high expressing corn event. The target multiple for field exposure was based on the assumption of a Cry34Ab1 expression level of 16 g/g in pollen. For Event DAS-59122-7, the HEEE pollen value is 75.29 g/g (Table 41), resulting in an actual multiple of 16/75.29 x 10 = 2.1X. Ladybird Beetle. Adult ladybird beetles (Hippodamia convergens) were exposed to a single limit dose concentration (280 g/mL Cry34/35Ab1 ICP) in sugar water (Bryan, R.L. et al., 2000; Appendix 2, Section 20). The dose concentration was 10X the empirical expression of the Cry34/35Ab1 ICP in pollen (based on the expression of a high-expressing corn event). Ladybird beetle adults fed ad libitum over 15 days and were

161 Revised September 7, 2004
Table 41. Summary of Guideline Hazard Tests for Effect of Cry34/35Ab1 ICP.
Guideline OECD 401 Study Title Acute ToxicityMouse Test Substance Cry35Ab1 microbiallyproduced protein (TSN102171; 37% pure) Cry34Ab1 microbiallyproduced protein (TSN102172; 54% pure) Cry34/35Ab1 pollen from event TC5639; Pollen from nontransgenic isoline (5XH}); Cry34Ab1 microbiallyproduced protein (TSN102172; 54% pure); Cry35Ab1 microbiallyproduced protein (TSN102171; 37% pure) Cry34Ab1 microbiallyproduced protein (TSN102172; 54% pure); Cry35Ab1 microbiallyproduced protein (TSN102171; 37% pure) Results LD50 > 2700 mg Cry34Ab1/kg LD50 > 1850 mg Cry35Ab1/kg LD50 > 2000 mg Cry34/35Ab1/kg

OPPTS 885.4380

Acute Dietary Toxicity LD50 Honeybees
NOECpollen = 2 mg/larvae (0.056 g Cry34/35Ab1 ICP/larvae) NOECICP = 20 g/larvae NOECCry34Ab1 = 3.2 g/larvae NOECCry35Ab1 = 2.4 g/larvae

OPPTS 885.4340

Non-target Insect Green Lacewing
Non-target Insect Parasitic Hymenoptera
Cry34Ab1 microbiallyproduced protein (TSN102172; 54% pure); Cry35Ab1 microbiallyproduced protein (TSN102171; 37% pure)
1)Non-target Insect Ladybird Beetle (adults; Hippodamia convergens)
1) Cry34Ab1 microbiallyproduced protein (TSN102172; 54% pure); Cry35Ab1 microbiallyproduced protein (TSN102171; 37% pure)
LC50 ICP > 280 g a.i./mL NOECICP > 280 g a.i./mL LC50 Cry34Ab1 > 160 g a.i./mL NOEC Cry34Ab1 > 160 g a.i./mL LC50 Cry35Ab1 > 120 g a.i./mL NOEC Cry35Ab1 > 120 g a.i./mL LC50 ICP > 280 g a.i./mL NOECICP > 280 g a.i./mL LC50 Cry34Ab1 > 160 g a.i./mL NOEC Cry34Ab1 > 160 g a.i./mL LC50 Cry35Ab1 > 120 g a.i./mL NOEC Cry35Ab1 > 120 g a.i./mL 1) LC50 ICP > 280 g a.i./mL NOECICP > 280 g a.i./mL LC50 Cry34Ab1 > 160 g a.i./mL NOEC Cry34Ab1 > 160 g a.i./mL LC50 Cry35Ab1 > 120 g a.i./mL NOEC Cry35Ab1 > 120 g a.i./mL 2) LC50 ICP > 902 g a.i./g NOECICP > 902 g a.i./g LC50 Cry34Ab1 > 900 g a.i./g NOEC Cry34Ab1 > 900 g a.i./g LC50 Cry35Ab1 > 2 g a.i./g NOEC Cry35Ab1 > 2 g a.i./g 3)Weight gain, delay in development NOECICP >58.52 g a.i./g NOEC Cry34Ab1 > 58.5 g a.i./g NOEC Cry35Ab1 > 0.02 g a.i./g LC50 ICP > 12.7 mg a.i./kg diet NOECICP > 12.7 mg a.i./kg diet LC50 Cry34Ab1 > 3.2 mg a.i./kg diet NOEC Cry34Ab1 > 3.2 mg a.i./kg diet LC50 Cry35Ab1 > 9.5 mg a.i./kg diet NOEC Cry35Ab1 > 9.5 mg a.i./kg diet

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insecticides or insecticide-treated seed. If the Cry34/35Ab1 rootworm-protected corn is planted on acreage that was in corn the previous year, then the refuge corn should also be planted on acreage that was in corn the previous year to ensure adequate production of susceptible beetles in the refuge. Management of pests other than rootworms can follow local integrated pest management guidelines; refuge and Cry34/35Ab1 rootwormprotected corn can be managed independently. Computer models predict that this product durability plan will protect the durability of Cry34/35Ab1 rootworm-protected corn. Several tools are currently available and effective for managing rootworms, including soil-applied insecticides, foliar-applied insecticides, insecticide-treated seed, crop rotation and other transgenic options. However, there are resistance concerns with many of these tools. Corn rootworms have evolved resistance to broadcast soil insecticides (cyclodienes), aerial insecticides (methyl parathion) and crop rotation (through extended diapause and altered oviposition behavior). Some believe that the existing commercial transgenic option, expressing the Cry3Bb Bt protein, is at high risk for resistance evolution. The availability of novel corn rootworm protection tools lessens the selection pressure for pest adaptation to existing rootworm control methods. There is no significant sequence homology of either Cry34Ab1 or Cry35Ab1 with other registered PIPs (Cry1Ab, Cry1Ac, Cry1F), including the Cry3 family which is exploited in the only commercialized PIP (Cry3Bb) targeting corn rootworms. Cry34/35 is not expected to exhibit cross-resistance to any other currently available rootworm-control technology. By providing an additional option to corn growers for managing rootworms, Cry34/35Ab1 rootworm-protected corn will add to the sustainability of all tools. VI.G. POTENTIAL CHANGES IN AGRICULTURAL PRACTICES ASSOCIATED WITH THE USE OF HERBICIDE TOLERANT CORN LINES Approximately 80 million acres of corn are planted annually in the United States, and nearly all of these acres are treated with herbicide. Products are applied pre-plant, preemergence and post-emergence. In general, corn receives a soil applied herbicide application followed by a post-emergence application. Corn line 59122 is glufosinateammonium tolerant, and as such, provides an alternative weed management tool to growers. Glufosinate-ammonium is a broad spectrum, post-emergence herbicide. Such an herbicide could provide growers the opportunity to move away from pre-emergence, residually active compounds and could increase the amount of conservation and no-till acres of corn planted in the United States. Corn line 59122 is still susceptible to other herbicides normally used to control corn should it appear as a volunteer weed in other crops. For example, in soybean, the crops most commonly rotated with corn, herbicides based on sulfonylurea, lipid biosynthesis inhibitors or Fluazifop/fomesafen could be used to control corn volunteers. It is estimated that approximately 10 million acres of herbicide tolerant corn will be planted in 2000-2001. This includes other glufosinate- and glyphosate-tolerant corn

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developed through recombinant technology as well as imidizolinone tolerant corn developed through selected mutagenesis and traditional plant breeding. If line 59122 crosses with corn lines expressing resistance to herbicides with different modes of action, corn volunteers with multiple herbicide resistance may emerge. However, the competition from the pollen load within a given field would keep the incidence of this very low. Additionally, various agronomic practices including appropriate variety selections and crop rotation, and rotation of herbicides with different modes of action can be used to avoid or manage volunteer corn resistant to one or a few herbicides. Corn lines that contain both B.t. and herbicide tolerance have been on the market since 1997 and have had a positive impact on agricultural practices. These products have provided simple, inexpensive yet highly effective means of controlling lepidopteran pests; have been environmentally benign so they preserve beneficial insects, decrease cultivation needs, and require less total pounds of active pesticidal compounds (both insecticidal and herbicidal). Corn line 59122 is coleopteran specific and herbicide tolerant, therefore, it is anticipated that it will also provide the same benefits as the lepidopteran B.t. plants.
VI.H. VERTICAL TRANSFER OF THE INTRODUCED GENETIC MATERIAL Non-cultivated Zea mays species are not found in the United States. The genus most closely related to Zea is Tripsicum, a genus of eleven species. Three Tripsicum species occur in the U.S. Crosses can be made between Z. mays and T. dactyloides, but these require human intervention and progeny are frequently sterile or genetically unstable. Therefore, cross-pollination between Z. mays and T. dactyloides in the natural environment is not expected to occur. If outcrossing to cultivated corn should occur, the frequency with which this would occur is expected to be very low due to the short distances corn pollen will travel and the limited window of viability (Raynor et al., 1972). Additionally, the outcrossing potential to cultivated corn is also diminished in seed production fields due to traditional containment practices to ensure seed genetic purity. Seed production fields are located in isolation to prevent introgression of genetic material from unwanted sources of corn pollen.
VI.I. HORIZONTAL TRANSFER OF THE INTRODUCED GENETIC MATERIAL There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to microbes (Nap et al., 1992; Redenbaugh et al., 1994). Even if such a transfer were to take place, transfer of cry34/35Ab1 or pat from line 15344 would not present a human health or plant pest risk. Genes encoding the PAT enzyme and similar acetyl transferases are found in nature. Similarly, B.t. Cry34/35Ab1 ICP was isolated from Bacillus

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Vaeck, M., Reynaerts, A., Hofte, H., Jansens, S., De Beuckeleer, M., Dean, C., Zabeau, M., Van Montagu, M., and Leemans, J. 1987. Transgenic plants protected from insect attack. Nature 328:33-37 Watson, S. A. 1982. Corn: Amazing maize. General Properties. pp 3-29 in CRC Handbook of Processing and Utilization in Agriculture, vol II, Part 1 Plant Products I. A. Wolf (ed.) CRC Press Inc., Florida Watson, S. A. 1987. Structure and Composition. pp 53-82 in Corn: Chemistry and Technology. S. A. Watson and P. E. Ransted (eds.) American Association of Cereal Chemists, Inc., Minnesota Weber, N. and Igo, E. 2003. Characterization of transgenic corn event DAS-59122-7 to investigate the genetic equivalence of the inserted DNA within a single generation. Pioneer Hi-Bred International, Inc. Unpublished internal report. 43p. 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 40:756762. Zupan, J.R., and Zambryski, P. 1995. Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol. 107: 1041-1047. Zupan, J. , and Zambryski, P. 1997. The Agrobacterium DNA transfer complex. Crit. Rev. Plant Science 16: 279-295.
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APPENDIX 1. CRY34/35AB1: DIETARY RISK ASSESSMENT
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APPENDIX 2. STUDY REPORTS SUPPORTING THE REGULATORY APPROVAL OF B.T. CRY34/35AB1 MAIZE LINE 59122.
CBI-Deleted COPY All study reports contained in this appendix are Confidential Business Information.
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Appendix 2 Section 1 [CBI-Deleted] Narva, K., Schnepf, H.E. and Wolt, J.D. 2003. Cry34/35 Protein Distribution and Familiarity. Study ID: GH-C 5702. Dow AgroSciences LLC unpublished internal report. 26p.
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Appendix 2 Section 2 [CBI-Deleted] Essner, R. 2003. Agronomic Characteristics, Quantitative ELISA and Nutrient Composition Analysis of Hybrid Maize Lines Containing cry34Ab1, cry35Ab1 and pat genes. Study ID: PHI-2002-050. Pioneer Hi-Bred unpublished internal report. 1971p.
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Appendix 2 Section 3 [CBI-Deleted] Herman, R.A. and Babcock, J.M. 2003. Biological Equivalency of Cry34/35Ab1 Insecticidal Crystal Protein in Transgenic Plants Derived from Transgenic Pseudomonas fluorescens. Study ID: GH-C 5696. Dow AgroSciences unpublished internal report. 17p.
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Appendix 2 Section 4 [CBI-Deleted] Schafer, B.W., Collins, R.A., Schwedler, D.A. and Xu, X.U. 2003. Characterization of Cry34Ab1 and Cry35Ab1 proteins from transgenic maize event E4497.59.1.22 (DAS59122-7). Study ID: 030033. Dow AgroSciences unpublished internal report. 50p.