Monsanto Company 07-CR-191U Page 2 of 544 Release of Information

Monsanto is submitting the information in this petition for review by the USDA as part of the regulatory process. By submitting this information, Monsanto does not authorize its release to any third party. In the event the USDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552 and 7 CFR Part 1, covering all or some of this information, Monsanto expects that, in adva nce of the release of the document(s), USDA will provide Monsanto with a copy of the material proposed to be released and the opportunity to object to the release of a ny information based on appropriate legal grounds, e.g., responsiveness, c onfidentiality, and/or compet itive concerns. Monsanto expects that no information that has been identified as CBI (confidential businessinformation), will be provided to any third party. Monsanto understands that a CBIdeleted copy of this information may be made available to the public in a reading room and by individual request, as part of a public comment period. Except in accordance with the foregoing, Monsanto does not authorize the release, public ation or other distribution of this information (including website pos ting) without Monsanto's prior notice and

consent.

© 2009 Monsanto Company. All Rights Reserved.

This document is protected under copyright law. This document is for use only by the regulatory authority to which this has been submitted by Monsanto Comp any, and only in support of actions requested by Monsanto Company. Any other use of this material, without prior written consent of Monsanto, is strictly prohibited. By submitting this document, Monsanto does not grant any party or entity any right or license to the information or intellectual property described in this document.Monsanto Company 07-CR-191U Page 4 of 544 Petition for the Determination of Non-Regulated Status for MON 87460

Summary

The Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) has resp onsibility under the Plant Protection Act (PPA) (Title IV Pub. L. 106-224, 114 Stat. 438 , 7 U.S.C. § 7701-7772) to prevent the introduction and dissemination of plant pests into the U.S. The APHIS regulation at 7 CFR § 340.6 provides that an applicant may pe tition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, ther eby allowing unrestrict ed introduction of the

article.

Monsanto Company is submitting this request to APHIS for a determination of nonregulated status for the ne w biotechnology-derived corn ( Zea mays L.) product, MON 87460, any progeny derived from cr osses between MON 87460 and conventional corn, and any progeny derived from crosse s of MON 87460 with other biotechnologyderived corn which has been granted non-regulated stat us under 7 CFR Part 340.

Drought Tolerance Trait Corn is a versatile crop that provides food, feed, and fuel to a global economy. Recently, a surge in demand for corn has been created by growing economies in the developing

world and its use as an alternative fuel so urce in the developed world. These demands are exceeding production, leading to diminished grain reserves worldwide. In addition, climate change may have variable impact s on crop yields, potentia lly creating further supply disruptions. The combination of th ese factors places a premium on corn yield stability in sub-optimal environments.

Drought stress is the major cause of yield reduction in corn and its effects have far reaching global socio-economic implications. In North America alone, it is estimated that 40% of annual crop losses are due to sub-optimal water availability. Consequently, increasing drought tolerance in corn is a major goal of breeding selection and biotechnology. Advances in conventional plant breeding a nd agronomic practices have made contributions to increasing corn drought tolerance and yield potential. Nearly all conventional corn hybrids curren tly on the market have been bred to exhibit some degree of drought tolerance. Biotechnology provides a significant new tool th at can be used in combination with conventional breeding and ag ronomic practices to increase corn yield stability under wate r-limited conditions.

Monsanto has developed MON 87460 that re duces yield loss un der water-limited conditions compared to conventional corn. Under well-watered c onditions, grain yield

for MON 87460 is not different from conve ntional corn. Like conventional corn, MON 87460 is still subject to yield loss unde r water-limited conditions, particularly during flowering and grainfill periods when corn yield potential is most sensitive to stressMonsanto Company 07-CR-191U Page 5 of 544 by disrupting kernel development. Under se vere water deficit, corn grain yield for MON 87460, as well as conventional corn, can be reduced to zero.

MON 87460 expresses a cold shock protein B (CSPB) produced from the inserted

Bacillus subtilis -derived gene. In bacteria, the CSPB protein helps preserve normal cellular functions during certain stre sses by binding cellular RNA and unfolding nontranslatable secondary structures affecting R NA stability and translat ion. As in bacteria, the CSPB protein in MON 87460 binds RNA and appears to help maintain plant cellular functions. Data suggest that MON 87460 reduc es yield loss, primarily through increased kernel number per ear, under water-limited conditions by minimizing the effect of water limitation on photosynthesis, stomatal conducta nce, and carbon fixation. On a plant level, corn yield losses associated with drought stress occur as a result of reduced synchrony between anthesis and silking, embr yo loss, and/or reduced grain filling in

viable kernels. Studies on conventional germplasm with enhanc ed drought tolerance show that yield improvements are attain ed through improvements in all of these endpoints. Therefore, the enhanced yi eld stability of MON 87460 under water-limited conditions, conferred by the expressed CSPB protein, appears to be the result of improvements in the natural stress res ponse mechanisms in conventional corn.

Water Management Regimes Employed for Field Studies Because MON 87460 reduces yield loss under water-limited conditions, field studies were designed to assess the plant pest pot ential of MON 87460 across a broad range of soil moisture and environmental conditions relevant to where commercial production would be expected. In each study, MON 87460 was compared to a conventional control, which had a genetic background similar to MON 87460 but did not possess the drought

tolerance trait. In additi on, multiple conventional corn hybrids (references) that possess a range of drought tolerance imparted through conventional breeding were included in the analysis to establish a range of natural vari ability for each measured characteristic. The commercial reference hybrids selected for each study were adapted to the geographic region relevant to each study site; as such, each reference hybrid included some degree of drought tolerance conferred th rough conventional breeding.

Field studies were established using three di fferent water management regimes: (1) wellwatered, (2) both well-watered a nd water-limited treatments established in the same field, or (3) water managed according to typical or standard agronomic practices, which included typical amounts of s upplemental irrigation at rele vant sites. Field studies

established under well-watered conditions allowed an evaluation of MON 87460 in the absence of trait bias, where no statistical differences were expected between MON 87460 and the control. Field studies managed to establish both well-watered and water-limited

conditions allowed an evaluation of MON 87460 under limited soil moisture conditions where it is expected to provide a yield bene fit. Finally, field studies managed according to typical agronomic practices allowed an evaluation of M ON 87460 under a natural range of environmental conditions relevant to commercial corn production regions. Data and plant tissue samples generated from these field studies with various water management regimes were used to assess the safety of MON 87460 under different soil moisture conditions including: (1) expression le vels of the inserted proteins in severalMonsanto Company 07-CR-191U Page 6 of 544 tissue types, (2) compositional analysis of forage and grain, (3) phenotypic and agronomic characteristics at several plant development stages, and (4) environmental

interactions. The variati on in yield response observed among MON 87460, the control, and commercial reference hybrids in these field studies is within normal levels expected for conventional corn. Therefore, the data fr om these field studies are relevant to the plant pest potential assessmen t of MON 87460. In the water-limited sites, the data are also relevant to demonstrate the efficacy of MON 87460. MON 87460 is expected to provide reduced yield loss under water-limite d conditions compared to conventional corn. Reduced yield loss is a desirable agronomic characteristic and is not, per se , considered to be associated with plant pest potential. In the case of MON 87460, the data and analysis presented in this petition suppor t the conclusion that MON 87460 is unlikely to be a plant pest.

Data and Information Presented to A ssess Plant Pest Potential of MON 87460 The data and information presented in this application demonstrate the familiarity of MON 87460 as compared to conventional corn and, moreover, show that MON 87460 is unlikely to be a plant pest. This conclusion is based on eight major lines of evidence. The first is that modern corn has inherently low plant pest potentia l because it is poorly suited to survive without human assistance and is not capable of surviving as a weed due to intense selection for domestication purposes during its evolution as a crop. The second

is the molecular characterization of the in serted DNA in MON 87460 which confirms the insertion of a single functional copy of the cspB and (neomycin phosphotransferase II ( nptII ) expression cassettes at a single locus w ithin the corn genome. The third is a

detailed biochemical charac terization of the CSPB and NPTII proteins produced in MON 87460, which shows very low levels of th ese proteins and demonstrates that the proteins have a history of safe use. The fourth line of ev idence is an assessment of the allergenicity and toxicity potential of the CSPB and NPTII proteins based on extensive information collected and studies performed on the two proteins. The results demonstrate with reasonable certainty that the CSPB and NPTII proteins are unlikely to be allergens or toxins. The fifth is the compositional a nd nutritional assessment which confirms that MON 87460 forage and grain are not compositio nally different from conventional corn.

The sixth line of evidence is the extensive evaluation of the MON 87460 phenotypic and agronomic characteristics and environmenta l interactions, which demonstrates that MON 87460 poses no increased pl ant pest potential, includi ng weediness potential, and no adverse environmental impact compared to conventional corn. The seventh is an assessment on the potential impact on non-targ et organisms (NTO) and threatened or endangered species, which concludes that MO N 87460 is unlikely to have any effect on these organisms under the conditions of use. Finally, the eighth line of evidence is an assessment of agronomic practices confirmi ng that the introduction of MON 87460 is no more likely to have an impact on land use, cu ltivation practices, or the management of weeds, diseases, and insects th an the use of conventionally br ed drought tolerant corn.

Plant Pest Potential of Modern Corn In the U.S., corn is not listed as a weed in the major weed referen ces, nor is it present on the lists of noxious weed species distributed by the federal government (7 CFR Part 360).

Modern corn cannot survive as a weed due to intense selection for domesticationMonsanto Company 07-CR-191U Page 7 of 544 purposes during its evolution as a crop. Du ring domestication of corn, traits often

associated with weediness such as seed dor mancy, a dispersal mechanism, or the ability to form reproducing populations outside of cultivation, have not been selected. For example, the corn ear is enclosed with husks . Consequently, seed dispersal of individual

kernels is limited. Even if individual kernels of corn were distributed within a field or along transportation routes from the fields to storage or proc essing facilities, sustainable volunteer corn populations are not found growing in fence rows, ditches, and road sides. Although corn seed can overwinte r and emerge as volunteer plan ts in rotational crops, the populations do not persist, and agronomic ma nagement practices, including mechanical and chemical measures, can be used to control the volunteer plants. Therefore, the plant pest potential of modern corn is inherently low.

Molecular Characterization of the Inserted DNA MON 87460 was produced by Agrobacterium -mediated transformation of corn with PVZMAP595, a binary vector containing a si ngle transfer DNA (T-DNA). The T-DNA contains two expression cassettes. The firs t expression cassette produces CSPB and the second expression cassette produces NPTII, a se lectable marker that was used during product development. During transformation, the T-DNA was inserted into the genome.

Molecular characterization of MON 87460 by Southern blot analyses demonstrate that the DNA inserted into the corn genome is pr esent at a single lo cus and contains one functional copy of the cspB and the nptII expression cassettes. All genetic elements are present in the inserted DNA as expected. No backbone plasmid DNA sequences were detected. PCR and DNA sequence analyses pr ovided the complete DNA sequence of the insert and confirmed the organization of the elements within the insert.

The stability of the integrated DNA is demons trated by the fact that the Southern blot fingerprint of MON 87460 was maintained for se ven generations tested in the breeding history. Furthermore, these generations ha ve been shown not to contain any backbone sequence from plasmid PV-ZMAP595. The stab ility was further confirmed by the fact

that the inheritance of the T-DNA in MON 87460 follows Mendelian patterns of segregation.

Characterization of th e CSPB and NPTII Proteins The expression levels of CSPB and NPT II proteins were determined in MON 87460 tissues produced during two growing seasons fr om multiple field sites in the major corn production regions of the U.S. and Chile. In the U.S. during 2006, six sites were established using typical water management practices as a standard assessment under a natural range of environmental conditions. In Chile during 2006/2007, the experimental design at three sites included well-watered and water-limited treatments to evaluate the

effect of soil moisture level on CSPB and NP TII protein levels. The results demonstrate that both CSPB and NPTII proteins were expr essed in all tissues co llected, including leaf, root, forage, silk, pollen, grai n, and stover, the e xpression levels declined over the growing season, and were similar under both well-watered and water-limited conditions. Results from the U.S. 2006 study show the mean CSPB protein levels (μg/g dwt) across all test sites were 0.072 in grain, 0.10 in fora ge, 13 in pollen, 3.1 in leaves of plants at V2-V4 stage, 0.47 in leaves of plants at pre-VT stage, 0.029 in forage root, and 0.042 inMonsanto Company 07-CR-191U Page 8 of 544 stover. In tissues harvested throughout the growing season, mean CSPB protein levels (μg/g dwt) across all test s ites varied from 0.47-3.1 in leaf , 0.24-1.4 in root, and 0.67-2.8 in whole plant. In general, levels of the CSPB protein declined ove r the growing season. The mean NPTII protein levels (μg/g dwt) across all test sites were less than the limit of quantitation in grain, 0.12 in forage, 2.6 in l eaves of plants at V2-V4 stage, and 0.47 in roots of plants at V2-V4 stage. Results from the Chile 2006/2007 study show the mean CSPB protein levels (μg/g dwt) across all te st sites under well-wat ered and water-limited treatments, respectively, were 0.048 and 0.038 in grain, 0.11 and 0.15 in forage, 25 and 27 in pollen, 2.8 and 2.8 in l eaves of plants at V2-V4 st age, 0.39 and 0.44 in leaves of plants at pre-VT stage, 0.039 and 0.076 in forage root, and 0.033 and 0.072 in stover. In tissues harvested throughout the growing seas on, mean CSPB protei n levels (μg/g dwt)

across all test sites under well-watered and wa ter-limited treatments, respectively, varied from 0.39-2.8 and 0.44-2.8 in leaf, 0.31-1.3 and 0.40-1.5 in root, and 0.67-3.2 and 0.70- 2.9 in whole plant. In general, levels of the CSPB protein declined over the growing

season and were similar between the well-wat ered and water-limited treatments. The mean NPTII protein levels (μg/ g dwt) across all test sites under well-watered and waterlimited treatments, respectively, were less than the limit of quantitation in grain, 0.16 and 0.17 in forage, 2.4 and 2.6 in leaves of plants at V2-V4 stage, and 0.51 and 0.48 in roots of plants at V2-V4 stage. The results show that the level of CSPB protein was very low in all tissue types, the leve l declined over the growing season, and there were no obvious differences in expression level in tissues co llected from plants grown under well-watered

or water-limited conditions. The level of NPT II protein in grain was below the Limit of Quantitation (LOQ) of the method, and there were no obvious differences observed in NPTII protein levels in the other three tissue types collec ted from plants grown under

well-watered or wate r-limited conditions.

Detailed safety assessments of CSPB a nd NPTII and their resp ective donor organisms establish that these proteins are safe for hu man consumption. CSPB has a history of safe consumption and NPTII is present in seve ral biotechnology-derived crops that have undergone previous safety assessments. The hi story of safe use and data from multiple studies demonstrate the safety of MON 87460 and the CSPB and NPTII proteins. The donor organism of the CSPB protein, B. subtilis , is not pathogenic, is present in many fermented foods, and has a hist ory of safe consumption. Pr oteins containing cold shock domains are ubiquitous in nature, being pr esent in many plants and common bacteria including species that are normally present in ga strointestinal flora. Cold shock proteins (CSPs) have no known toxicity and are not associated with pathogenicity. The CSPB protein is homologous to the CSPs found in Lactobacillus

, Lactococcus

, Bifidobacterium

, and E. coli , which are normally present in gastrointe stinal flora and, therefore, considered to be safe. The amino acid identity ranges from 35% to 98.5% across different plant and bacterial species. The strains of lactic acid bacteria, Bifidobacterium and Lactobacillus

, are the most common type of bacteria used in the dairy industry for preparation of probiotic products containing live ba cterial cultures. In addition, Bacillu s, Lactobacillus,

and Lactococcus species containing CSPs are i nvolved in many food fermentation processes of milk, meats, cereals, and vegetables.Monsanto Company 07-CR-191U Page 9 of 544 Allergenicity and Toxicity Poten tial of the CSPB and NPTII Proteins CSPB protein does not share any amino acid se quence similarities with known allergens, gliadins, glutenins, or protei n toxins which have adverse effects on mammals. This has been shown by extensive assessments with bioi nformatic tools, such as FASTA sequence alignment search and an eight-amino acid sliding window searc h. Digestive fate experiments conducted with the CSPB protein demonstrate that the full-length protein is rapidly digested in simulate d gastric fluid (SGF), a char acteristic shared among many proteins with a history of safe consumpti on. A small transiently stable CSPB protein fragment was very quickly degraded during sh ort exposure to simulated intestinal fluid (SIF). Rapid digestion of th e full-length CSPB protein in SGF and SIF, together with rapid degradation of the small transiently stable fragment in SIF, indicates that it is highly unlikely that the CSPB protein and its frag ment will reach absorptive cells of the intestinal mucosa. Proteins that are rapi dly digestible in mammalian gastrointestinal systems are unlikely to be allergens when consumed. Finally , the CSPB protein represents no more than 0.00007% of the tota l protein in the grai n of MON 87460. Acute oral toxicity studies with mice demonstrate that CSPB protein is not acutely toxic and does not cause any adverse effects even at th e highest dose levels tested, which was 4.7 mg/kg body weight. The dietary safety assessm ent of CSPB based on the acute toxicity data and corn product dietary pattern establishes that the margin of exposure (MOE) for the overall U.S. population is 26,700. For chil dren aged 1-6 years old, an age group with the highest corn consumption on a body weight basis, the MOE was gr eater than or equal to 11,400 for CSPB. Dietary expos ure in animals will also be low with chickens, swine, and dairy cows consuming only nanogram quan tities of each protein per kilogram of body weight. Taken together, these data in dicate that food and feed derived from MON 87460, which contain the CSPB pr otein, are safe for consumption.

The safety of NPTII has been extensively ev aluated through several lines of experimental evidence, and several products containing NP TII have been approved by regulatory agencies on a global basis. NPTII is the most commonly used antibiotic resistance marker in several commercially grow n biotechnology-derived crops including

YieldGard ® Rootworm corn (MON 863), Bollgard ® cotton (MON 531), Bollgard ® II cotton (MON 15985), and Roundup Ready cott on (MON 1445). FDA evaluated NPTII as part of a petition for FLAVR SAVR ® tomatoes and approved its use as a food additive. Additionally, EPA established an exemption from the requirement of a tolerance for

NPTII for use as a selectable marker in raw agricultural commodities. In 2007, the European Food Safety Authority (EFSA) affi rmed its conclusion th at the presence of nptII does not pose a threat to human health or the environment. Extensive bioinformatic assessments with tools such as FASTA seque nce alignment search and an eight-amino acid sliding window search demonstrate th at NPTII does not share any amino acid sequence similarities with known allergens, glia dins, glutenins, or protein toxins which

have adverse effects on mammals. Numerous st udies have suggested th at the presence of

this antibiotic-resist ance gene in any crop or crop pr oducts will have no impact on food safety. Studies using purifie d NPTII protein revealed that NPTII degrades rapidly in simulated gastric and intestinal fluids suggest ing that the protein is unlikely to cause an allergic response. Acute oral toxicity studies with mice de monstrate that NPTII is not acutely toxic and does not cause any adverse effects even at the highest dose levelsMonsanto Company 07-CR-191U Page 10 of 544 tested, 5,000 mg/kg body weight . A dietary safety asse ssment for MON 87460 based on the acute toxicity data and a corn product diet ary consumption pattern established that the MOEs are very high for the overall U.S. popul ation and for children aged 1-6 years old, an age group with the highest corn consum ption on a body weight basis. NPTII is ubiquitous in E. coli, and, therefore, is normally present within the human gastrointestinal tract. Moreover, USDA previ ously evaluated the safety ofNPTII in other biotechnologyderived products, including corn. Similar to these products, there is negligible risk for the production of NPTII in MON 87460 to resu lt in a plant pest risk. A published assessment of the ecological im pact of NPTII in crops repo rted that the amount of free kanamycin accumulating in soils, through the ac tion of microorganisms or animal feces, is restricted by absorption to soil components so that no direct selection pressure for kanamycin resistant plants can occur. Al so, enhanced physiologi cal fitness resulting from potential pleiotropic effects of nptII gene expression has not been observed. Thus, based on all the available evidence, it can be concluded that the NPT II protein is safe for

use as a selectable marker in biotechnology-derived plants.

Composition and Nutrition of Forage and Grain A detailed compositional assessment of MON 87460 confirmed that it is as safe and

nutritious as conventional co rn. The composition of MON 87460 was determined from forage and grain tissues produced during two gr owing seasons from mu ltiple field sites in the major corn production regions of th e U.S. and Chile. In each assessment, MON 87460 was compared to an appropriate conventional control, which had a genetic background similar to MON 87460 but did not possess the drought tolerance trait. In addition, multiple conventional corn hybrids (references) that possess a range of drought tolerance imparted through conventional bree ding were included in the analysis to establish a range of natural va riability for each analyte, wher e the range of variability is defined by a 99% tolerance interval for that particular analyte. In the U.S. during 2006, six sites were established using typical wa ter management practices as a standard assessment under a natural range of enviro nmental conditions. In Chile during 2006/2007, the experimental design included well -watered and water-limited treatments

to evaluate the effect of soil moisture leve l on component levels. Across both years of

data there were no consiste nt differences between MON 87460 and the control. Within the U.S. 2006 combined-site analysis, there were no significant di fferences (p>0.05) for 59 (95.2%) of 62 comparisons. Furthermore, results from the U.S. 2006 study show that there were no significant differences (p >0.05) for 407 (93.7% ) of the 434 total comparisons made between the MON 87460 te st and the control. The 27 detected

differences were not consistent across site s, were small in magnitude and the mean component values of MON 87460 and the control were within the 99% tolerance interval established from the commercial reference hybr ids that were produced at the same time

and field sites as MON 87460 and the control. Within the combined site analysis of the well-watered plots from the Chile study, ther e were no significant differences (p>0.05) for 59 (96.7%) of 61 comparisons. Furthermore, results from well-wa tered plots in the Chile study show that there were no signi ficant differences (p>0.05) for 230 (94.3%) of the 244 total comparisons made between M ON 87460 and the control. The 14 detected

differences were not consistent across site s, were small in magnitude, and the mean component values of the test and control substances were within the 99% toleranceMonsanto Company 07-CR-191U Page 11 of 544 interval established from the commercial reference hybrids that were produced at the

same time and field sites as MON 87460 and th e control. Within the combined site analysis of the water-limited plots from the Chile study, there were no significant differences (p>0.05) for 59 ( 96.7%) of 61 comparisons. Furt hermore, results from the water-limited plots in the Chile study show that there were no significant differences (p>0.05) for 233 (95.5%) of the 244 total comparisons made between MON 87460 and

the control. The nine detected differences we re not consistent across sites, were small in magnitude and the mean component values of th e test and control subs tances were within the 99% tolerance interval established from the commercial reference hybrids. Samples from the Chile study were also analyzed for 11 secondary metabolites that are potentially associated with drought stress. Results from this additional analysis further confirm that MON 87460 is not compositionally different from conventional corn. All compositional analyses, therefore, suppor t the conclusion that MON 87460 is not different from conventional corn when grown under a broad range of environmental conditions.

Phenotypic and Agronomic Characteri stics and Environmental Interactions The plant characterization and environmenta l interactions assessment of MON 87460 demonstrates that it poses no increased plant pest potential, including weediness potential. Data were collected to evaluate the likelihood of altered plant pest potential based on requirements of USDA-APHIS . The phenotypic, agronomic, and environmental interactions data were evaluated from a basis of familiarity and were comprised of a combination of field, greenhous e, and laboratory studies. Phenotypic and agronomic characteristics of MON 87460 were evaluated relative to an appropriate control under a broad range of environmental conditions to assess plant pest potential and potential environmental impact. These asse ssments included evaluations among 14 plant growth and development characteristics, fi ve seed germination parameters, two pollen characteristics, observations for plant-inse ct and plant-disease interactions, abiotic stressors, volunteer potential, and survival outside cultivat ion. Each of the measured

characteristics is useful to assess fam iliarity and plant pest potential of MON 87460 compared to conventional corn. In addition, cer tain characteristics can be used to assess weediness potential, including seed germina tion and dormancy (hard seed), pre-harvest seed loss characteristics (lodging and ear drop), and the potential to volunteer in

cultivated areas or surv ive outside cultivation.

The phenotypic field studies were conducte d during 2006 and 2007 in the major corn production regions of the U.S. and Chile. Six field studies totaling 31 sites were established using the three water management regimes described above: (1) well-watered (17 sites); (2) well-watered and water-limited treatments (9 sites); and (3) water managed according to typical local agronomic pract ices and water conditions (5 sites).

Results from the combined-site analyses within each of the six studies detected only four instances of a phenotypic difference between MON 87460 and the control. For the wellwatered regime, two separate studies totaling 17 sites were established in the U.S. during 2006 (8 sites) and 2007 (9 sites). In the co mbined-site analyses of these data no

differences were detected between MON 87460 and the control in the 2007 study. In the 2006 study, an increase in root lodged plants was detected for MON 87460 compared toMonsanto Company 07-CR-191U Page 12 of 544 the control (5.6 vs. 1.5, respectively). Fo r the well-watered and water-limited regime, three different studies totaling nine field sites (six sites included in combined-site analyses) were established in Chile (3 sites in 2006/2007) and the U.S. (3 sites in 2007 from two separate studies). In the Chile 2006/2007 study, no phenotypic differences were detected with the exception of the expected increase in yield for MON 87460 compared to the control under water-lim ited conditions (114.5 vs . 86.7 bushels/acre, respectively). In the U.S. 2007 Study-1, no phenotypic differences were detected in

either the well-watered or wa ter-limited treatments. In the U.S. 2007 Study-2, stay green rating was lower (more green tissue) for MON 87460 compared to the control in both the well-watered (5.8 vs. 6.7) and water-limited tr eatments (6.3 vs. 8.3), respectively. For the typical agronomic practices regime, one st udy with five sites was established in the U.S. during 2006 and no differences between MON 87460 and the control were detected in this study. In summary, the phenotypi c data support the conclusion that MON 87460 possesses no characteristics that would confer increased plant pest potential, including weediness potential, or result in advers e environmental impacts compared toconventional corn. With the exception of the expected differences in yield under waterlimited conditions, the magnitude of the detected differences was small, the differences did not represent a trend in the data across studies and years, and the mean values of MON 87460 were within the range of values observed for the commercial references.

No biologically meaningful differences were detected in the germination and dormancy of seed from MON 87460. In particular, the absence of hard seed supports a conclusion of no increased weediness pot ential of MON 87460 compared to conventional corn for germination and dormancy characteristics. No differences were detected in pollen morphology or viability between MON 87460 and the control. Results from the

environmental interactions assessments also support the conclusi on that MON 87460 has no increased susceptibility or tolerance to specific diseases, arthropods, or abiotic stressors, with the exception of drought . Finally, MON 87460 was not altered in its ability to volunteer in cultivated fields or survive in areas not managed for agricultural production compared to conventional corn.

Non-Target Organisms and Thre atened or Endangered Species

The environmental assessment of MON 87460 dem onstrates that it poses negligible risk to non-target organisms and will not affect threatened or endangered species under the

conditions of use. The assessment took into consideration the familia rity with CSPB and NPTII protein modes of action and their expression levels in MON 87460. Although CSPB is not known to exert any effects on pe st and non-pest organisms, studies were conducted to examine the potential effects of MON 87460 on biotic stressors that may

affect corn. Studies demonstrate a lack of any effects observed in various species exposed to MON 87460. Additional data and information presented demonstrate that

MON 87460 is not different from conventiona l corn with respect to persistence, invasiveness, or gene flow.

MON 87460 is unlikely to outcross with sexually compatible species in the U.S. Corn and annual teosinte ( Zea mays subsp. mexicana ) are genetically compatible, windpollinated, and may hybridize when in close pr oximity to each other, e.g., in areas ofMonsanto Company 07-CR-191U Page 13 of 544 Mexico and Guatemala. However, teosinte is not present in the U.S. other than as an occasional botanical garden specimen and a few small feral populations of Zea mexicana in Florida, Alabama, and Maryland and Zea perennis in South Carolina. Differences in

factors such as flow ering time, geographical separati on, and development factors make

natural crosses in the U.S. hi ghly unlikely. In contrast w ith corn and teosinte which easily hybridize under certain conditions, speci al techniques are required to hybridize

corn and Tripsacum and the open literature indicates that the offspring of the cross show varying levels of ster ility. The species Tripsacum floridanum , found in extreme southern Florida, has been categorized as a threatened species by the state of Florida and is listed

on the USDA’s Natural Resources Conservation Service Database. However, given the level of difficulty for natural hybridization between species of Tripsacum and Zea , and

the occurrence of T. floridanum primarily in both highly urbanized and non- agricultural, swampy areas of the state, it is very unlikel y there would be any im pact on this species

due to the introduction of MON 87460.

Corn Agronomic Practices Finally, an assessment of current corn agronomic practices confirmed that the

introduction of MON 87460 is no mo re likely to impact land us e, cultivation practices, or the management of weeds, diseases, and in sects than the use of conventionally bred drought tolerant corn. The introduction and rapid adoption of biotechnology-derived corn products in the past decade have had no significant impact on land use or cropland acreage in the U.S. The total crop area in the U.S. has remained relatively steady as is the case for field corn acreage. Cumulative impacts to seed production, crop rotation and tillage practices, or weed, di sease, and insect management practices are not expected

from the introduction of MON 87460. Conve ntionally bred, drought tolerant hybrids have been planted in the U.S. for decades without any documentation of such effects. Because MON 87460 reduces yield loss under wa ter-limited conditions, it is foreseeable that continued improvements in drought tolera nce for corn, includi ng the introduction of MON 87460, may result in the cumulative be nefit of decreasing water demands for irrigation in the Great Plains. This tre nd is already underway due to the continued improvements in conventionally bred drought tolerant corn, and the introduction of MON 87460 is not anticipated to have a significant impact. Thus, MON 87460 is environmentally benign and beneficial to corn growers in areas suitable for commercial corn production, but prone to frequent drought stress.

Conclusion Based on the data and information presented in this petition, it is concluded that MON 87460 is unlikely to be a plant pest. The adoption of MON 87460 may increase economic and environmental benefits, primar ily in the Western Dryland region of the Great Plains, due to the protection of corn yield under water-limited conditions, but is not expected to have a significant environmen tal impact. Therefore, Monsanto Company requests a determination from APHIS th at MON 87460 and any progeny derived from

crosses between MON 87460 and other commercial corn be granted non-regulated status

under 7 CFR Part 340.Monsanto Company 07-CR-191U Page 14 of 544 Table of Contents

Release of Information ......................................................................................................2

Certification ................................................................................................................. .......3

Summary ....................................................................................................................... ......4

Table of Contents ............................................................................................................. 14

List of Tables ................................................................................................................ ....17

List of Figures ............................................................................................................... ....28

Abbreviations, Acronyms, and Definitions ....................................................................31

I. Rationale for the Development of MON 87460 ..........................................................37
I.A. Basis for the Request for a Determ ination of Nonregulated Status under

7 CFR Part 340.6....................................................................................... 37 I.B. Product Concept and Benefits of MON 87460 .................................................... 37

I.C. Intended Function of the Genetic Modification ................................................... 42

I.D. Mode of Action of th e Drought Tolerance Trait ................................................. 43 I.E. Adoption, Estimated Use, and Global Trade Flows of MON 87460 ................... 76 I.F. Submissions to Other Regulatory Agencies ......................................................... 76

I.G. References ............................................................................................................ 77

II. The History, Biology, and Global Use of Corn ........................................................80

II.A. Introduction ........................................................................................................ 80 II.B. History of Corn ................................................................................................... 80 II.C. Taxonomy and Phylogenetics ............................................................................. 81 II.D. Biology of Corn and Environm ental Effects on its Development ...................... 83

II.E. Global Use of Corn ............................................................................................. 89

II.F. Conclusions ......................................................................................................... 91

II.G. References .......................................................................................................... 91

III. Description of the Transformation System .............................................................96
III.A. Transformation System ..................................................................................... 96

III.B. References ......................................................................................................... 96

IV. Donor Genes and Regulatory Sequences .................................................................99

IV.A. Vector PV-ZMAP595 ....................................................................................... 99

IV.B. T-DNA .............................................................................................................. 99

IV.C. Genetic Elements Outside the T-DNA Borders .............................................. 103

IV.D. References ....................................................................................................... 104

V. Genetic Analysis ........................................................................................................106
V.A. Insert and Copy Number .................................................................................. 113

V.B. Presence or Absence of Plasmid PV-ZMAP595 Backbone ............................. 114

V.C. Intactness of cspB and nptII Expression Cassettes ........................................... 118 V.D. Southern Blot Analyses of MON 87460 across Multiple Generations ............ 130 V.E. Organization of the Inserted Genetic Elements of MON 87460 ...................... 135Monsanto Company 07-CR-191U Page 15 of 544 V.F. Inheritance of the Gene tic Insert in MON 87460 ............................................. 135

V.G. Conclusions of Molecular Characterization ..................................................... 136

V.H. References ........................................................................................................ 137

VI. Characterization of the Introd uced CSPB and NPTII Proteins .........................138

VI.A. CSPB Protein .................................................................................................. 138

VI.B. NPTII Protein .................................................................................................. 140 VI.C. Expression Levels of CSPB and NPTII Proteins in MON 87460 ................... 141 VI.D. Food and Feed Safety Assessme nt Summary of the CSPB and NPTII

Proteins ................................................................................................... 147

VI.E. References ....................................................................................................... 148

VII. Compositional Assessment ....................................................................................151
VII.A. U.S. 2006 Composition Study under Typical Agronomic Conditions .......... 151VII.B. Chile 2006/2007 Composition Study under Well-Watered and WaterLimited Conditions ................................................................................. 159 VII.C. Additional Secondary Meta bolites from the Chile 2006/2007 Composition Study under Well-Watered and Water-Limited

Conditions ............................................................................................... 169

VII.D. Overall Conclusions from Compositional Analysis of MON 87460

from the U.S. 2006 and Chile 2006/2007 Studies .................................. 174

VII.E. References ...................................................................................................... 175

VIII. Phenotypic, Agronomic, and Envi ronmental Interactions Assessment ..........176

VIII.A. Characteristics Measured for Assessment .................................................... 176 VIII.B. Interpretation of Assessment Data ............................................................... 179 VIII.C. Phenotypic and Agronomic Assessment ...................................................... 185 VIII.D. Seed Germination and Dormancy Assessment ............................................ 204 VIII.E. Pollen Morphology and Viability Assessment ............................................. 209

VIII.F. Environmental Interactions Assessment ....................................................... 210

VIII.G. Overall Conclusions of Phenot ypic, Agronomic, and Environmental

Interactions Assessment of MON 87460 ................................................ 230

VIII.H. References .................................................................................................... 230

IX. Environmental Consequences and Impact on Agronomic Practices ..................232

IX.A. Characterization of MON 87460 and the expressed CSPB and NPTII

Proteins ................................................................................................... 233

IX.B. Weediness Potential ........................................................................................ 238

IX.C. Gene Flow ....................................................................................................... 238 IX.D. Corn Production, Current Agr onomic Practices, and Land Use ..................... 240 IX.E. Corn Acreage, Land Use, and the Conservation Reserve Program ................ 262 IX.F. Mitigation and Remediation Measures Before and After Deregulation of

MON 87460 in the U.S. .......................................................................... 262

IX.G. Conclusions ..................................................................................................... 263

IX.H. References ....................................................................................................... 265

X. Adverse Consequences of Introduction...................................................................271Monsanto Company 07-CR-191U Page 16 of 544 Appendix A. Materials and Methods Us ed for Molecular Analyses of

MON 87460 ..............................................................................................................272

Appendix B. Materials, Methods, and Resu lts for Characterization of CSPB

Protein ......................................................................................................................2 76

Appendix C. Materials, Methods, and Re sults for Characterization of NPTII

Protein ......................................................................................................................2 94

Appendix D. Materials and Methods for Ex pression Levels of CSPB and NPTII

Proteins ....................................................................................................................30 0

Appendix E. Materials and Methods fo r Forage and Grain Compositional Analysis ....................................................................................................................31 0

Appendix F. Phenotypic and Agronomic Assessment: Individual Site Results

from 2006 and 2007 Field Studies .........................................................................388

Appendix G. Phenotypic Data from U.S. and Chile Field Sites Excluded from Combined-Site Analyses .........................................................................................418

Appendix H. Environmental Interactions Da ta for Individual Field Sites in U.S. and Chile Field Studies during 2006 and 2007 .....................................................421

Appendix I. Water Management and Environmental Data for U.S. and Chile Field Studies during 2006 and 2007 ......................................................................470

Appendix J. Materials, Metho ds and Individual Site Results for Seed Dormancy

and Germination .....................................................................................................504

Appendix K. Materials and Methods fo r Pollen Morphology and Viability Analysis ....................................................................................................................50 8

Appendix L. Materials and Methods fo r Volunteer Potential Assessment ..............510

Appendix M. Materials and Methods for Survival Outside Cultivation

Assessment ...............................................................................................................513

Appendix N. USDA Notificati ons Approved for MON 87460 ...................................519

Appendix O. Petitioner’s Environmental Assessment ................................................524Monsanto Company 07-CR-191U Page 17 of 544 List of Tables

Table I-1. In vitro Melting Activity of CSPB and CSPB Variants .................................. 46 Table I-2. Yield Component and Physiol ogy Data from a Water-Limited Kansas

Field Trial in 2003 ....................................................................................... 47 Table I-3. Yield Component Data fr om a Well-Watered and Water-Limited

California Field Trial in 2007 ...................................................................... 48 Table I-4. Average Relative Water Conten t of Transgenic CSPB Plants and the

Control Grown under Water-Limited Conditions in the Greenhouse ......... 49

Table II-1. Taxonomic Classification of Corn and its Close Relatives ........................... 82 Table IV-1. Summary of Genetic Elemen ts in Plasmid Vector PV-ZMAP595 ............ 100 Table V-1. Summary of Genetic Elements in MON 87460 ........................................... 108 Table V-2. Summary Chart of the Exp ected DNA Fragments using Combinations of Restriction Enzymes and Probes ........................................................... 113 Table V-3. Segregation Patterns of cspB between Generations of MON 87460 ........... 136

Table VI-1. CSPB Protein Levels in Tissues Collected from MON 87460 Produced in the U.S. during 2006 under Typical Agronomic

Conditions ................................................................................................. 144

Table VI-2. CSPB Protein Levels in Tissues Collected from MON 87460 Produced in Chile during 2006/2007 under Well-Watered and

Water-Limited Conditions ......................................................................... 145

Table VI-3. NPTII Protein Levels in Tissues Collected from MON 87460 Produced in the U.S. during 2006 under Typical Agronomic

Conditions ................................................................................................. 146

Table VI-4. NPTII Protein Levels in Tissues Collected from MON 87460 Produced in Chile during 2006/2007 under Well-Watered and

Water-Limited Conditions ......................................................................... 146 Table VII-1. Monthly Temperature and Accumulated Water Data for the U.S. 2006 Study ................................................................................................. 156 Table VII-2. Summary of Significant Differences (p<0.05) Comparing MON 87460 to the Conventional Control in the U.S. during 2006 under Typical Agronomic Conditions ................................................................. 157 Table VII-3. Monthly Temperature and Accumulated Water Data for the Chile 2006/2007 Study ....................................................................................... 163 Table VII-4. Summary of Significant Differences (p<0.05) Comparing MON

87460 to the Conventional Contro l in Chile during 2006/2007 under

Well-Watered Conditions .......................................................................... 164 Table VII-5. Summary of Significant Differences (p<0.05) Comparing MON

87460 to the Conventional Contro l in Chile during 2006/2007 under

Water-Limited Conditions ......................................................................... 168 Table VII-6. Summary of Significant Differences in Additional Secondary Metabolite Composition (p<0.05) Comparing MON 87460 to the

Conventional Control in Chile during 2006/2007 under WellWatered Conditions ................................................................................... 171Monsanto Company 07-CR-191U Page 18 of 544 Table VII-7. Summary of Significant Differences in Additional Secondary Metabolite Composition (p<0.05) Comparing MON 87460 to the

Conventional Control in Ch ile during 2006/2007 under WaterLimited Conditions .................................................................................... 173 Table VIII-1. Characteristics Measured for Phenotypic, Agronomic and

Environmental Interactions Assessment of MON 87460 .......................... 178

Table VIII-2. U.S. and Chile Field St udies Established under Three Different Water Management Regimes for Phenotypic Evaluation of MON 87460 during 2006 and 2007 .......................................................... 189

Table VIII-3. Site Inclus ion Assessment for U.S. and Chile Field Studies during

2006 and 2007 ........................................................................................... 190 Table VIII-4. U.S. 2006: Phenotypic and Agronomic Comparison of MON 87460 to the Control in the Combined-Site Analysis from a 2006 U.S. Field Study Conducted under Well-Watered Conditions ................................... 192 Table VIII-5. U.S. 2007: Phenotypic and Agronomic Comparison of MON 87460 to the Control in the Combined-Site Analysis from a 2007 U.S. Field Study Conducted under Well-Watered Conditions ................................... 193

Table VIII-6. Chile 2006/2007: Phe notypic and Agronomic Comparison of

MON 87460 to the Control under Well-Watered Conditions in the Combined-Site Analysis in 2006/2007 Chilean Field Trials

Established with Well-Watered an d Water-Limited Treatments .............. 195

Table VIII-7. Chile 2006/2007: Phe notypic and Agronomic Comparison of

MON 87460 to the Control under Water-Limited Conditions in the Combined-Site Analysis in 2006/2007 Chilean Field Trials

Established with Well-Watered an d Water-Limited Treatments .............. 196 Table VIII-8. U.S. 2007 Study-1: Phe notypic and Agronomic Comparison of

MON 87460 to the Control under Well-Watered Conditions in the Combined-Site Analysis in 2007 U.S. Field Trials Established with

Well-Watered and Water-Limited Treatments .......................................... 199 Table VIII-9. U.S. 2007 Study-1: Phe notypic and Agronomic Comparison of

MON 87460 to the Control under Water-Limited Conditions in the Combined-Site Analysis in 2007 U.S. Field Trials Established with

Well-Watered and Water-Limited Treatments .......................................... 200 Table VIII-10. U.S. 2007 Study-2: Ph enotypic and Agronomic Comparison of

MON 87460 to the Control under Well-Watered Conditions in the Combined-Site Analysis in 2007 U.S. Field Trials Established with

Well-Watered and Water-Limited Treatments .......................................... 201 Table VIII-11. U.S. 2007 Study-2: Ph enotypic and Agronomic Comparison of

MON 87460 to the Control under Water-Limited Conditions in a

2007 Texas Field Trial Established with Well-Watered and WaterLimited Treatments ................................................................................... 202 Table VIII-12. Phenotypic and Agrono mic Comparison of MON 87460 to the

Control in the Combined-Site An alysis from a 2006 U.S. Field Study Conducted under Typical Agronomic Conditions .......................... 204

Table VIII-13. Seed Germination and Dormancy Parameters Evaluated ...................... 207Monsanto Company 07-CR-191U Page 19 of 544 Table VIII-14. Germination of MON 87460 and a Conventiona l Control Corn

across Sites ................................................................................................ 208 Table VIII-15. Viability and Diameter of Pollen Collected from MON 87460 and

a Conventional Control Corn under Well-Watered Conditions in

2007 ........................................................................................................... 209 Table VIII-16. Viability and Diameter of Pollen Collected from MON 87460 and

a Conventional Control Corn under Watered-Limited Conditions in

2007 ........................................................................................................... 210 Table VIII-17. Comparison of MON 87460 to the Control Prior to Drought Treatment and at 7 and 15 Days after Treatment ...................................... 216 Table VIII-18. Comparison of MON 87460 to the Control Prior to Cold

Treatment and at 4 and 8 Days after Treatment ........................................ 219 Table VIII-19. Comparison of MON 87460 to the Control Prior to Heat

Treatment and at 3 and 5 Days after Treatment ........................................ 222 Table VIII-20. Comparison of MON 87460 to the Control Prior to Salt Treatment and at 9 and 12 Days after Treatment ....................................................... 225

Table VIII-21. Observed Volunteer Corn Plants of MON 87460 Compared to the

Control and References in a 2006/2007 U.S. Field Trial .......................... 227 Table VIII-22. Survival Outside of Cultivation Phenotypic Comparison of

MON 87460 to the Control in 2007 U.S. Field Trials ............................... 229

Table IX-1. Field Corn Producti on in the U.S. (1996 – 2008) ...................................... 242

Table IX-2. Field Corn Pr oduction by Regions and States in the U.S. (2008) .............. 243 Table IX-3. Comparison of Field Corn Pr oduction in the Midwest vs. the Great

Plains ......................................................................................................... 245

Table IX-4. Greatest Increases in No-Till Corn in 2004 ............................................... 249 Table IX-5. Changes to Irrigated Co rn Acreage in the Great Plains ............................. 254

Table IX-6. Diseases of Corn ......................................................................................... 259

Table IX-7. Insect Pests of Corn .................................................................................... 260

Table IX-8. Troublesome Weeds in U.S. Corn Fields ................................................... 261

Table B-1. N-terminal Amino Acid Se quence Analysis of the CSPB Protein

Purified from Grain Tissue of MON 87460 .............................................. 284 Table B-2. Summary of the Tryptic Masses Identified for the MON 87460- Produced CSPB Protein Using MALDI-TOF Mass Spectrometry ........... 285 Table B-3. CSPB Functional Assay Results .................................................................. 292

Table D-1. Protein Extraction Meth ods for U.S. 2006 Tissue Samples ........................ 302

Table D-2. CSPB Protein Levels in Tissues Collected from MON 87460 Produced at the QUI site in Chile during 2006/2007 under WellWatered and Water-Limited Conditions ................................................... 308

Table D-3. NPTII Protein Levels in Tissues Collected from MON 87460 Produced at the QUI site in Chile during 2006/2007 under WellWatered and Water-Limited Conditions ................................................... 309

Table E-1. Reference Substances for U.S. 2006 Composition Study ............................ 311 Table E-2. Reference Substances for Chile 2006/2007 Composition Study ................. 311 Table E-3. Reference Calibration Range s and Limits of Quantitation .......................... 315

Table E-4. Unit Conversions .......................................................................................... 320Monsanto Company 07-CR-191U Page 20 of 544 Table E-5. Comparison of Proximates, Fi ber, and Mineral Content in Forage

from MON 87460 and Conventional C ontrol for Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions .................... 328 Table E-6. Comparison of the Proximate s and Fiber Content in Grain from

MON 87460 and Conventional Control for Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions .......................... 329 Table E-7. Comparison of the Mineral Content in Grain from MON 87460 and

Conventional Control for Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions ....................................................... 330 Table E-8. Comparison of the Amino Acid Content in Grain from MON 87460

and Conventional Control for Comb ined Sites in the U.S. during 2006 under Typical Agronomic Conditions .............................................. 331 Table E-9. Comparison of the Fatty Acid Content in Grain from MON 87460 and

Conventional Control for Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions ....................................................... 334 Table E-10. Comparison of the Vitamin Content in Grain from MON 87460 and

Conventional Control for Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions ....................................................... 335 Table E-11. Comparison of the Anti-nut rient and Secondary Metabolite Content

in Grain from MON 87460 and Conventional Control for Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions ....... 336 Table E-12. Comparison of Proximates, Fi ber, and Mineral Content in Forage

from MON 87460 and Conventional C ontrol for Combined Sites in Chile during 2006/2007 under Well-Watered Conditions ........................ 337 Table E-13. Comparison of the Proximate s and Fiber Content in Grain from

MON 87460 and Conventional Control for Combined Sites in Chile during 2006/2007 under Well-Watered Conditions .................................. 338 Table E-14. Comparison of the Mineral Content in Grain from MON 87460 and

Conventional Control for Combined Sites in Chile during 2006/2007

under Well-Watered Conditions ................................................................ 339 Table E-15. Comparison of the Amino Acid Content in Grain from MON 87460

and Conventional Control for Co mbined Sites in Chile during

2006/2007 under Well-Watered Conditions ............................................. 340 Table E-16. Comparison of the Fatty Acid Content in Grain from MON 87460

and Conventional Control for Co mbined Sites in Chile during

2006/2007 under Well-Watered Conditions ............................................. 343 Table E-17. Comparison of the Vitamin Content in Grain from MON 87460 and

Conventional Control for Combined Sites in Chile during 2006/2007

under Well-Watered Conditions ................................................................ 344 Table E-18. Comparison of the Anti-nut rient and Secondary Metabolite Content

in Grain from MON 87460 and Conventional Control for Combined Sites in Chile during 2006/2007 unde r Well-Watered Conditions ........... 345 Table E-19. Comparison of Proximates, Fi ber, and Mineral Content in Forage

from MON 87460 and Conventional C ontrol for Combined Sites in Chile during 2006/2007 under Water-Limited Conditions ....................... 346Monsanto Company 07-CR-191U Page 21 of 544 Table E-20. Comparison of the Proximate s and Fiber Content in Grain from

MON 87460 and Conventional Control for Combined Sites in Chile during 2006/2007 under Water-Limited Conditions ................................. 347 Table E-21. Comparison of the Mineral Content in Grain from MON 87460 and

Conventional Control for Combined Sites in Chile during 2006/2007

under Water-Limited Conditions .............................................................. 348 Table E-22. Comparison of the Amino Acid Content in Grain from MON 87460

and Conventional Control for Co mbined Sites in Chile during

2006/2007 under Water-Limited Conditions ............................................ 349 Table E-23. Comparison of the Fatty Acid Content in Grain from MON 87460

and Conventional Control for Co mbined Sites in Chile during

2006/2007 under Water-Limited Conditions ............................................ 352 Table E-24. Comparison of the Vitamin Content in Grain from MON 87460 and

Conventional Control for Combined Sites in Chile during 2006/2007

under Water-Limited Conditions .............................................................. 353 Table E-25. Comparison of the Anti-nut rient and Secondary Metabolite Content

in Grain from MON 87460 and Conventional Control for Combined Sites in Chile during 2006/2007 unde r Water-Limited Conditions .......... 354 Table E-26. Comparison of the Additiona l Secondary Metabolite Composition ofForage from MON 87460 and Conventional Control for CombinedSites from the 2006/2007 Chile Production Conducted under WellWatered Conditions ................................................................................... 355 Table E-27. Comparison of the Additiona l Secondary Metabolite Composition of

Grain from MON 87460 and Conven tional Control for CombinedSites from the 2006/2007 Chile Production Conducted under WellWatered Conditions ................................................................................... 356 Table E-28. Comparison of the Additiona l Secondary Metabolite Composition ofForage from MON 87460 and Conventional Control for CombinedSites from the 2006/2007 Chile Pr oduction Conducted under WaterLimited Conditions .................................................................................... 357 Table E-29. Comparison of the Additiona l Secondary Metabolite Composition of

Grain from MON 87460 and Conven tional Control for CombinedSites from the 2006/2007 Chile Pr oduction Conducted under WaterLimited Conditions .................................................................................... 358

Table E-30. Literature and ILSI Database Ranges of Components of Corn Forage

and Grain ................................................................................................... 359 Table E-31. Monthly Temper ature and Monthly Accumula ted Water Data for the QUI Site in the Chile 2006/2007 Study .................................................... 363 Table E-32. Summary of Significant Differences (p<0.05) Comparing MON 87460 to the Conventional Control from the QUI Site in Chile during 2006/2007 under Well-Watered and Water-Limited

Conditions ................................................................................................. 364 Table E-33. Comparison of Proximates, Fi ber, and Mineral Content in Forage

from MON 87460 and Conventional C ontrol from the QUI Site in Chile during 2006/2007 under Well-Watered Conditions ........................ 365Monsanto Company 07-CR-191U Page 22 of 544 Table E-34. Comparison of the Proximate s and Fiber Content in Grain from

MON 87460 and Conventional Control from the QUI Site in Chile during 2006/2007 under Well-Watered Conditions .................................. 366 Table E-35. Comparison of the Mineral Content in Grain from MON 87460 and Conventional Control from the QUI Site in Chile during 2006/2007

under Well-Watered Conditions ................................................................ 367 Table E-36. Comparison of the Amino Acid Content in Grain from MON 87460

and Conventional Control from the QUI Site in Chile during

2006/2007 under Well-Watered Conditions ............................................. 368 Table E-37. Comparison of the Fatty Acid Content in Grain from MON 87460

and Conventional Control from the QUI Site in Chile during

2006/2007 under Well-Watered Conditions ............................................. 371 Table E-38. Comparison of the Vitamin Content in Grain from MON 87460 and Conventional Control from the QUI Site in Chile during 2006/2007

under Well-Watered Conditions ................................................................ 372 Table E-39. Comparison of the Anti-nut rient and Secondary Metabolite Content

in Grain from MON 87460 and Conve ntional Control from the QUI Site in Chile during 2006/2007 under Well-Watered Conditions ............. 373 Table E-40. Comparison of Proximates, Fi ber, and Mineral Content in Forage

from MON 87460 and Conventional C ontrol from the QUI Site in Chile during 2006/2007 under Water-Limited Conditions ....................... 374 Table E-41. Comparison of the Proximate s and Fiber Content in Grain from

MON 87460 and Conventional Control from the QUI Site in Chile during 2006/2007 under Water-Limited Conditions ................................. 375 Table E-42. Comparison of the Mineral Content in Grain from MON 87460 and Conventional Control from the QUI Site in Chile during 2006/2007

under Water-Limited Conditions .............................................................. 376 Table E-43. Comparison of the Amino Acid Content in Grain from MON 87460

and Conventional Control from the QUI Site in Chile during

2006/2007 under Water-Limited Conditions ............................................ 377 Table E-44. Comparison of the Fatty Acid Content in Grain from MON 87460

and Conventional Control from the QUI Site in Chile during

2006/2007 under Water-Limited Conditions ............................................ 380 Table E-45. Comparison of the Vitamin Content in Grain from MON 87460 and Conventional Control from the QUI Site in Chile during 2006/2007

under Water-Limited Conditions .............................................................. 381 Table E-46. Comparison of the Anti-nut rient and Secondary Metabolite Content

in Grain from MON 87460 and Conve ntional Control from the QUI Site in Chile during 2006/2007 under Water-Limited Conditions ............ 382 Table E-47. Summary of Significant Differences in Additional Secondary Metabolite Composition (p<0.05) Comparing MON 87460 to the Conventional Control from the QUI Site in Chile during 2006/2007

under Well-Watered and Water-Limited Conditions ................................ 383 Table E-48. Comparison of the Additio nal Secondary Metabolite Content in Forage from MON 87460 and Conventional Control from the QUI Site in Chile during 2006/2007 under Well-Watered Conditions ............. 384Monsanto Company 07-CR-191U Page 23 of 544 Table E-49. Comparison of the Additio nal Secondary Metabolite Content in

Grain from MON 87460 and Conven tional Control from the QUI Site in Chile during 2006/2007 under Well-Watered Conditions ............. 385 Table E-50. Comparison of the Additio nal Secondary Metabolite Content in Forage from MON 87460 and Conventional Control from the QUI Site in Chile during 2006/2007 under Water-Limited Conditions ............ 386 Table E-51. Comparison of the Additio nal Secondary Metabolite Content in

Grain from MON 87460 and Conven tional Control from the QUI Site in Chile during 2006/2007 under Water-Limited Conditions ............ 387 Table F-1. Field Phenotypic Evaluati on Sites for MON 87460 during 2006 and

2007 ........................................................................................................... 394 Table F-2. Test, Control, and Reference Substances in the 2006 U.S. Field Study

under Well-Watered Conditions ................................................................ 395 Table F-3. Test, Control, and Reference Substances in the 2007 U.S. Field Study

under Well-Watered Conditions ................................................................ 396 Table F-4. Test, Control, and Refere nce Corns used in the 2006/2007 Chilean Field Study Established with Well-Watered and Water-Limited

Treatments ................................................................................................. 397 Table F-5. Study-1, Test, C ontrol, and Reference Corns used in the 2007 U.S.

Field Studies Established with Well-Watered and Water-Limited

Treatments ................................................................................................. 397 Table F-6. Study-2, Test, C ontrol, and Reference Corns used in the 2007 U.S.

Field Studies Established with Well-Watered and Water-Limited

Treatments ................................................................................................. 398 Table F-7. Test, Control, and Reference Substances in the 2006 U.S. Field Study under Typical Agronomic Conditions ....................................................... 399 Table F-8. Test Site Locations, Plan ting Information, Soil Description, and Cropping History in a 2006 Fiel d Study under Well-Watered

Conditions ................................................................................................. 400 Table F-9. Test Site Locations, Plan ting Information, Soil Description, and Cropping History in 2007 U.S. Fi eld Studies under Well-Watered

Conditions ................................................................................................. 401 Table F-10. Test Site Locations, Plan ting Information, Soil Description, and Cropping History in a 2006/2007 Ch ilean Field Study Established

with Well-Watered and Water-Limited Treatments .................................. 402 Table F-11. Study-1, Test Site Locations, Planting Information, Soil Description, and Cropping History in 2007 U.S. Field Studies Established with

Well-Watered and Water-Limited Treatments .......................................... 403 Table F-12. Study-2, Test Site Locations, Planting Information, Soil Description, and Cropping History in 2007 U.S. Field Studies Established with

Well-Watered and Water-Limited Treatments .......................................... 403 Table F-13. Test Site Locations, Plan ting Information, Soil Description, and Cropping History in a 2006 U.S. Fi eld Study Established with Water Managed According to Local Agronomic Practices ................................. 404 Table F-14. Phenotypic Comp arison of MON 87460 to the Control at Each Site in the 2006 U.S. Field Study Under Well-Watered Conditions ................ 405Monsanto Company 07-CR-191U Page 24 of 544 Table F-15. Phenotypic Comp arison of MON 87460 to the Control at Each Site in the 2007 U.S. Field Study under Well-Watered Conditions ................. 408 Table F-16. Phenotypic Comp arison of MON 87460 to the Control at Each Site in a 2006/2007 Chilean Field Study Established with Well-Watered

and Water-Limited Treatments in 2006/2007 ........................................... 411 Table F-17. Phenotypic Comp arison of MON 87460 to the Control at Each Site in a 2006/2007 Chilean Field Study Established with Well-Watered

and Water-Limited Treatments ................................................................. 412 Table F-18. Study-1, Phenotypic Comparis on of MON 87460 to the Control at

Each Site in 2007 Field Studies Es tablished with Well-Watered and

Water-Limited Treatments ........................................................................ 413 Table F-19. Study-1, Phenotypic Comparis on of MON 87460 to the Control at

Each Site in 2007 Field Studies Es tablished with Well-Watered and

Water-Limited Treatments ........................................................................ 414 Table F-20. Study-2, Phenotypic Comparis on of MON 87460 to the Control at

Each Site in 2007 U.S. Field Studi es Established with Well-Watered

and Water-Limited Treatments ................................................................. 415 Table F-21. Phenotypic Comp arison of MON 87460 to the Control at Each Site in a 2006 U.S. Field Study Established with Water Managed According to Local Agronomic Practices ................................................. 416 Table G-1. Individual Site Analysis of Phenotypic Characteristics of MON 87460 to the Control for the QUI Site in Chile during 2006/2007 under

Well-Watered Conditions .......................................................................... 418

Table G-2. Individual Site Analysis of Phenotypic Characteristics of MON 87460 to the Control for the QUI site in Chile During 2006/2007 under

Water-Limited Conditions ......................................................................... 419 Table G-3. U.S. 2007 Study-2: Wa ter-Limited Treatment – Phenotypic Comparison of MON 87460 to the Control at Each Site .......................... 420

Table H-1. Abiotic Stressor Evaluations using an Observational Severity Scale for MON 87460 Compared to the Co ntrol and References in a 2006 U.S. Field Study under Well-Watered Conditions .................................... 431 Table H-2. Disease Damage Evaluations using an Observational Severity Scale for MON 87460 Compared to the Co ntrol and References in a 2006 U.S. Field Study under Well-Watered Conditions .................................... 432 Table H-3. Arthropod Damage Evaluations using an Observational Severity Scale for MON 87460 Compared to the Co ntrol and References in a 2006 U.S. Field Study under Well-Watered Conditions .................................... 433 Table H-4. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87460 Compared to the Control and References in a 2006 U.S. Field Study under Well-Watered Conditions ........................ 434 Table H-5. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87460 Compared to the Control and References atthe IL1, KS, and NE Sites in a 2006 U.S. Field Study under WellWatered Conditions ................................................................................... 435Monsanto Company 07-CR-191U Page 25 of 544 Table H-6. Arthropod Pest Abundance on MON 87460 Compared to the Control and References at the IL1, KS, a nd NE Sites in a 2006 U.S. Field Study under Well-Watered Conditions ..................................................... 436 Table H-7. Beneficial Arthropod Ab undance on MON 87460 Compared to the Control and References at the IL1, KS, and NE Sites in a 2006 U.S. Field Study under Well-Watered Conditions ............................................ 439

Table H-8. Abiotic Stressor Evaluations using an Observational Severity Scale for MON 87460 Compared to the Co ntrol and References in a 2007 U.S. Field Study under Well-Watered Conditions .................................... 442

Table H-9. Disease Stressor Evaluations using an Observational Severity Scale for MON 87460 Compared to the Co ntrol and References in a 2007 U.S. Field Study under Well-Watered Conditions .................................... 443 Table H-10. Arthropod Stresso r Evaluations using an Observational Severity Scale for MON 87460 Compared to the Control and References in a 2007 U.S. Field Study under Well-Watered Conditions ........................... 444 Table H-11. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87460 Comp ared to the Control and References Combined across IA1, IL 1, and IN Sites in a 2007 U.S. Field Study under Well-Watered Conditions ............................................ 445 Table H-12. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87460 Comp ared to the Control, and References at the IA1, IL1, and IN sites in a 2007 U.S. Field Study

under Well-Watered Conditions ................................................................ 446 Table H-13 Arthropod Pest Abundance on MON 87460 Compared to the Control and References at the IA1, IL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered Conditions ..................................................... 447

Table H-14. Beneficial Arthropod Abundance on MON 87460 Compared to the Control and References at the IA1, IL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered Conditions ............................................ 450

Table H-15. Abiotic Stressor Incidence of MON 87460 Compared to the Control

and References in a 2006 U.S. Fi eld Study Established with Water Managed According to Local Agronomic Practices ................................. 453

Table H-16. Disease Stressor Incidence of MON 87460 Compared to the Control

and References in a 2006 U.S. Fi eld Study Established with Water Managed According to Local Agronomic Practices ................................. 454 Table H-17. Arthropod Stressor Incide nce of MON 87460 Compared to the

Control and References in a 2006 U. S. Field Study Established with Water Managed According to Lo cal Agronomic Practices ...................... 455

Table H-18. Chile 2006/2007: Abiotic Stressor Incidence of MON 87460 Compared to the Control and th e References in a Field Study

Established with Well-Watered an d Water-Limited Treatments .............. 456

Table H-19. Chile 2006/2007: Disease Stressor Incidence of MON 87460 Compared to the Control and References in a Field Study

Established with Well-Watered an d Water-Limited Treatments .............. 457Monsanto Company 07-CR-191U Page 26 of 544 Table H-20. Chile 2006/2007: Arthropod Stressor Incidence of MON 87460 Compared to the Control and References in a Field Study

Established with Well-Watered an d Water-Limited Treatments .............. 458 Table H-21. U.S. 2007 Study-1: Abiotic Stressor Evaluations using an Observational Severity Scale for MON 87460 Compared to the

Control and References in Fiel d Studies Established with WellWatered and Water-Limited Treatments ................................................... 459 Table H-22. U.S. 2007 Study-1: Diseas e Damage Evaluations Using an Observational Severity Scale for MON 87460 Compared to the

Control and References in Fiel d Studies Established with WellWatered and Water-Limited Treatments ................................................... 460 Table H-23. U.S. 2007 Study-1: Arth ropod Damage Evaluated using an Observational Severity Scale for MON 87460 Compared to the

Control and References in Fiel d Studies Established with WellWatered and Water-Limited Treatments ................................................... 461 Table H-24. U.S. 2007 Study-1: Quantita tive Assessment for Corn Earworm and European Corn Borer Damage to MON 87460 Compared to the

Control and References at TX in Field Studies Established with

Well-Watered and Water-Limited Treatments .......................................... 462 Table H-25. U.S. 2007 Study-1: Arth ropod Pest abundance on MON 87460 Compared to the Control and Refe rences at TX in Field Studies

Established with Well-Watered an d Water-Limited Treatments .............. 463 Table H-26. U.S. 2007 Study-1: Ar thropod Beneficial Abundance on MON 87460 Compared to the Control and References at TX in Field

Studies Established with Well-Watered and Water-Limited

Treatments ................................................................................................. 465 Table H-27. U.S. 2007 Study-2: Abiotic Stressor Evaluations using an Observational Severity Scale for MON 87460 Compared to the

Control and References in Fiel d Studies Established with WellWatered and Water-Limited Treatments ................................................... 467 Table H-28. U.S. 2007 Study-2: Diseas e Damage Evaluations using an Observational Severity Scale for MON 87460 Compared to the

Control and References in Fiel d Studies Established with WellWatered and Water-Limited Treatments ................................................... 468 Table H-29. U.S. 2007 Study-2: Arthr opod Damage Evaluations using an Observational Severity Scale for MON 87460 Compared to the

Control and References in Fiel d Studies Established with WellWatered and Water-Limited Treatments ................................................... 469

Table I-1. U.S. 2006 Well-Watered S ites - Monthly Average Minimum and Maximum Temperatures and Temperature Range .................................... 499

Table I-2. U.S. 2007 Well-Watered S ites - Monthly Average Minimum and Maximum Temperatures and Temperature Range .................................... 500 Table I-3. U.S. 2006 Typical Local Practice Sites - Monthly Average Minimum and Maximum Temperatures and Temperature Range ............................. 501Monsanto Company 07-CR-191U Page 27 of 544 Table I-4. Chile 2006/2007 Well-Watere d and Water-Limited Sites - Monthly Average Minimum and Maximum Temperatures and Temperature Range ......................................................................................................... 502 Table I-5. U.S. 2007 Study-1 Well-Watere d and Water-Limited Sites - Monthly Average Minimum and Maximum Temperatures and Temperature Range ......................................................................................................... 502 Table I-6. U.S. 2007 Study-2 Well-Watere d and Water-Limited Sites - Monthly Average Minimum and Maximum Temperatures and Temperature Range ......................................................................................................... 503 Table J-1. Individual Site Analysis for Germination of MON 87460 and the

Control in 2006 .......................................................................................... 507 Table L-1. Planting, Soil type, and Croppi ng History Information for Volunteer Potential Study .......................................................................................... 512 Table M-1. Test, Control, and Referen ce Starting Seed for Survival Outside Cultivation Study ....................................................................................... 514 Table M-2. Study Area Planting Informati on and Soil Description for Survival Outside Cultivation study .......................................................................... 516 Table M-3. Monthly Temp erature and Rainfall Amount s for Survival Outside Cultivation Study ....................................................................................... 517

Table M-4. Characteristics Evaluated at Each Site for Survival Outside Cultivation Study ....................................................................................... 518 Table N-1. USDA Notifications Approve d for MON 87460 and Status of Trials

Conducted Under these Notifications. ...................................................... 520 Table O-1. Comparison of Likely Envi ronmental Impacts Between “No Action” and “Approval in Whole” Alternatives ..................................................... 525 Table O-2. Certified Organic and Total U.S. Acreage of Selected Crops ..................... 530 Table O-3. Organic and Conventional Seed Sources ..................................................... 537Monsanto Company 07-CR-191U Page 28 of 544 List of Figures

Figure I-1. Percent Yield Advantage fo r MON 87460 Compared to the Control against Percent Yield Loss of the References in U.S. and Chilean Field Studies during 2006 and 2007 ............................................................ 41 Figure I-2. Percent Yield Advantage for MON 87460 Compared to the Control in U.S. and Chilean Field Studies during 2006 and 2007 ............................... 42 Figure I-3. Co-Immunopreci pitation of RNA from MON 87460 using CSPB and the CSPB_F30A Variant ............................................................................. 50 Figure I-4. Gel Shift Analysis of To tal RNA Isolated from MON 87460 ....................... 51 Figure I-5. Gel Shift Analysis of rRNA and Luciferase mRNA ...................................... 52 Figure I-6. Co-Immunopreci pitation of CSPB-FLAG:RN A Complexes from Leaf

Tissue .......................................................................................................... 53 Figure I-7. Differential A ccumulation of CSPB acro ss Leaves of MON 87460 ............. 54 Figure I-8. Expression and Accumulati on of CSPB Protein in Germinating MON 87460 Seedlings ................................................................................ 55 Figure I-9. Expression of CSPB in Tissues of MON 87460 Grown in a

Greenhouse .................................................................................................. 56 Figure I-10. Immunohistochemical Localizat ion of CSPB in Plant Tissue ..................... 57 Figure I-11. Quantum Efficiency of P hotosystem II-Mediated Electron Transfer,

or Φ II, for MON 87460 and the Control over Time ................................... 58 Figure I-12. CO 2 Assimilation over Time for MON 87460 and the Control ................... 59 Figure I-13. Stomatal Conductance over Time for MON 87460 and the Control ........... 60 Figure I-14. Leaf 5 Daily LER Pre-Dr ought (T0-3 to T0) and Post-Drought Initiation (T1-T4) ........................................................................................ 61 Figure I-15. Leaf 6 Daily LER Pre-Dr ought (T0-3 to T0) and Post-Drought Initiation (T1-T4) ........................................................................................ 62 Figure I-16. Leaf 7 Daily LER Pre-Dr ought (T0-3 to T0) and Post-Drought Initiation (T1-T4) ........................................................................................ 63 Figure I-17 Leaf 8 Daily LER Pre-Dr ought (T0-3 to T0) and Post-Drought Initiation (T1-T4) ........................................................................................ 64 Figure I-19. Automated LER Data and Pl ant Temperature for an Approximately 48 Hour Growth Period during Well-Watered Conditions ......................... 66 Figure I-20. Automated LER Data and Pl ant Temperature for an Approximately 48 Hour Growth Period during Water-Limited Conditions ........................ 67 Figure I-21. Automated LER Data under Well-Watered and Water-Limited

Conditions for 5- and 15-min Windows ...................................................... 68 Figure I-22. Water Potential of Transg enic CSPB Plants and the Control throughout the Duration of Stress Imposition ............................................. 69 Figure I-23. Osmotic Potential of Tran sgenic CSPB Plants and the Control throughout the Duration of Stress Imposition ............................................. 70 Figure I-24. Average Abscisic Acid Cont ent in Leaves from Transgenic CSPB

Plants and the Control Taken over the Course of Two Days under

Water-Limited Conditions ........................................................................... 71 Figure I-25. Leaf Fructose and Glucose C ontent in Transgenic CSPB Plants and

the Control ................................................................................................... 72Monsanto Company 07-CR-191U Page 29 of 544 Figure I-26. Leaf Raffinose and Inositol C ontent in Transgenic CSPB Plants and

the Control ................................................................................................... 73 Figure I-27. Choline and Proline of Tran sgenic CSPB Plants and the Control throughout the Duration of Stress Imposition ............................................. 74 Figure I-28. Relative Glycine Betaine Levels of Transgen ic CSPB Plants and the Control throughout the Duration of Stress Imposition ................................ 75 Figure II-1. Growth Stages of Corn fr om Early Vegetative (V4) through Late Grainfill (R5) ............................................................................................... 84 Figure III-1. Circular Map of Plasmid PV-ZMAP595..................................................... 97 Figure III-2. Schematic Representation of the Development of MON 87460 .................. 98 Figure IV-1. Deduced Amino Acid Seque nce of the Full Length CSPB Protein

Present in MON 87460 .............................................................................. 102 Figure IV-2. Nucleotide Sequence Encodi ng the NPTII Protein in PV-ZMAP595 ...... 102 Figure IV-3. Deduced Amino Acid Seque nce of the Full Length NPTII Protein

Present in MON 87460 .............................................................................. 102 Figure V-1. Schematic Representation of the Insert and Genomic Flanking Sequences in MON 87460 ......................................................................... 110 Figure V-2. Circular Map of Plasmi d PV-ZMAP595 Showing Probes 1-6 .................. 111 Figure V-3. Circular Map of Plasmi d PV-ZMAP595 Showing Probes 7-13 ................ 112 Figure V-4. Southern Blot Analysis of MON 87460: In sert and Copy Number .......... 116 Figure V-5. Southern Blot Analysis of MON 87460: PV-ZMAP595 Backbone ......... 117 Figure V-6. Southern Blot Analysis of MON 87460: P- Ract1 ...................................... 123 Figure V-7. Southern Blot Analysis of MON 87460: I- Ract1 ....................................... 124 Figure V-8. Southern Blot Analysis of MON 87460: CS- cspB ..................................... 125 Figure V-9. Southern Blot Analysis of MON 87460: T- tr7 .......................................... 126 Figure V-10. Southern Blot Analysis of MON 87460: loxP + P- 35S ............................ 127 Figure V-11. Southern Blot Analysis of MON 87460: CS- nptII ................................... 128 Figure V-12. Southern Blot Analysis of MON 87460: T- nos + loxP + B-Left

Border ........................................................................................................ 129 Figure V-13. MON 87460 Breeding Diagram ............................................................... 132 Figure V-14. Generational Stability of MON 87460: In sert and Copy Number .......... 133 Figure V-15. Generational Stability of MON 87460: PV-ZMAP595 Backbone ......... 134 Figure VI-1. Protein Sequence of Bacillus subtilis CSPB as Expressed in

MON 87460 ............................................................................................... 139 Figure VIII-1. Decision Diagram for Inte rpretation of Detected Differences ............... 181 Figure VIII-2. Decision Diagram for Site Inclusion in Plan t Characterization Assessments Requiring Defined Well-Watered and Water-Limited

Treatments ................................................................................................. 184 Figure IX-1. U.S. Conservation Tillage Acreage by Watershed ................................... 250 Figure IX-2. U.S. Acres of Irrigated Corn Harvested for Grain .................................... 252 Figure B-1. MALDI-TOF MS Coverage Map of the CSPB Protein Isolated from

MON 87460 ............................................................................................... 286 Figure B-2. Wester n Blot Analysis of MON 87460- and E. coli -produced CSPB

Proteins ...................................................................................................... 287 Figure B-3. SDS-PAGE of E. coli - and MON 87460-produced CSPB Proteins ........... 289 Figure B.4. Glycosylation Analysis of the MON 87460-produced CSPB Protein ........ 290Monsanto Company 07-CR-191U Page 30 of 544 Figure C-1. Western Blot Analysis of the MON 87460- and E. coli -produced

NPTII Protein ............................................................................................ 298 Figure I-1. Calculated and Accumulated Soil Water Values at IA1 in 2006 ................. 471 Figure I-2. Calculated and Accumulated Soil Water Values at IA2 in 2006 ................. 472 Figure I-3. Calculated and Accumulated Soil Water Values at IL1 in 2006 ................. 473 Figure I-4. Calculated and Accumulated Soil Water Values at IL2 in 2006 ................. 474 Figure I-5. Calculated and Accumulated Soil Water Values at IN1 in 2006 ................. 475 Figure I-6. Calculated and Accumulated Soil Water Values at IN2 in 2006 ................. 476 Figure I-7. Calculated a nd Accumulated Soil Water Values at KS in 2006 .................. 477 Figure I-8. Calculated and Accumulated Soil Water Values at NE in 2006 .................. 478 Figure I-9. Calculated and Accumulated Soil Water Values at IA1 in 2007 ................. 479 Figure I-10. Calculated and Accumulated Soil Water Values at IA2 in 2007 ............... 480 Figure I-11. Calculated and Accumulated Soil Water Values at IL1 in 2007 ............... 481 Figure I-12. Calculated and Accumulated Soil Water Values at IL2 in 2007 ............... 482 Figure I-13. Calculated and Accumulated Soil Water Values at IL3 in 2007 ............... 483 Figure I-14. Calculated and Accumulated Soil Water Values at IN in 2007 ................. 484 Figure I-15. Calculated and Accumulated Soil Water Values at NE in 2007 ................ 485 Figure I-16. Calculated and Accumulate d Soil Water Values at OH in 2007 ............... 486 Figure I-17. Calculated and Accumulate d Soil Water Values at PA in 2007 ................ 487 Figure I-18. Accumulated Water at IAE in 2006 ........................................................... 488 Figure I-19. Accumulated Wa ter at IAW in 2006 ......................................................... 488 Figure I-20. Accumulated Water at IL in 2006 .............................................................. 489 Figure I-21. Accumulated Water at IN in 2006 ............................................................. 489 Figure I-22. Accumulated Water at NE in 2006 ............................................................ 490 Figure I-23. Accumulated Wa ter at CL in 2006/2007 ................................................... 491 Figure I-24. Accumulated Wa ter at CT in 2006/2007 ................................................... 492 Figure I-25. Accumulated Wa ter at LUM in 2006/2007 ............................................... 493 Figure I-26. Accumulated wa ter at QUI in 2006/2007 .................................................. 493 Figure I-27. Calculated and Accumulate d Soil Water Values at CA Study-1 in

2007 ........................................................................................................... 494 Figure I-28. Calculated and Accumulate d Soil Water Values at TX Study-1 in

2007 ........................................................................................................... 495 Figure I-29. Calculated and Accumulate d Soil Water Values at KS Study-2 in

2007 ........................................................................................................... 496 Figure I-30. Calculated and Accumulate d Soil Water Values at NE Study-2 in

2007 ........................................................................................................... 497 Figure I-31. Calculated and Accumulate d Soil Water Values at TX Study-2 in

2007 ........................................................................................................... 498Monsanto Company 07-CR-191U Page 31 of 544 Abbreviations, Acronyms, and Definitions

Note: Standard abbreviations, e.g., units of measure, are used according to the format

described in ‘Instructions to Authors’ in the Journal of Biological Chemistry

.

1 × LB Laemmli Buffer [62.5mM Tris-H Cl, 5% (v/v) 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulf ate, 0.005% (w/v) bromophenol blue, 10% (v/v) glycerol, pH 6.8]

5 × LB Five times concentrated 1 × LB

6-FAM 6-carboxyfluorescein

35S Promoter and leader from the Ca uliflower mosaic virus (CaMV)

35S RNA

AA Amino acid

AACC American Association of Cereal Chemists

aadA Bacterial promoter and coding sequence for an aminoglycosidemodifying enzyme, 3'(9)-O-nuc leotidyltransferase from the transposon Tn7

AD8 Allergen, gliadin, and gluten in protein sequence database

ADF Acid detergent fiber

AEC buffer Buffer solution of 20 mM Tris-HCl, pH 7.0

ALLERGENSEARCH Computer program for the s earch against known allergens

ALLPEPTIDES Protein sequence database comprised of NRAA and SwissProt

databases

AOAC Association of Offici al Analytical Chemists

AOCS American Oil Chemists Society

AOSA Association of Offi cial Seed Analysts

AOSCA Association of Official Seed Certifying Agencies

APHIS Animal and Plant Health Inspection Service

APS Analytical protein standard

ATP Adenosine triphosphate

B Border

Blp I Restriction enzyme isolated from Xanthomonas badrii

bp Base pair

BSA Bovine serum albumin

B. subtilis Bacillus subtilis

bu/ac Bushels per acreMonsanto Company 07-CR-191U Page 32 of 544 bw Body weight

CBI Confidential business information

CFIA Canadian Food Inspection Agency

CFR Code of Federal Regulations

CI Confidence interval

CIMMYT International Maize a nd Wheat Improvement Center

CRP Conservation Reserve Program

CS Coding sequence

CSD Cold shock domain

CSP Cold shock protein

cspB Coding sequence for CSPB from B. subtilis

CSPB Cold shock protein B from B. subtilis

CTAB Cetyltrimethylammonium bromide

DAP Days after planting

DAT Days after treatment

dATP Deoxyadenosine triphosphate

dCTP Deoxycytidine triphosphate

DLP Dual labeled probe

DNA Deoxyribonucleic acid

dNTP Deoxyribonucleotide triphosphate , a generic term referring to the four deoxyribonucleotides: dATP , dCTP, dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidine triphosphate)

dT Deoxythymidine

DW or dw Dry weight

DWCF Dry weight conversion factor

dwt Dry weight of tissue

EC Electrica l conductivity

E. coli Escherichia coli

Eco O109 I Restriction enzyme isolated from E. coli

EcoR V Restriction enzyme isolated from E. coli

ECL Enhanced chemil uminescence

EDTA Ethylenediaminetetraacetic acid

EFSA European Food Safety Authority

ELISA Enzyme-linked immunosorbent assay

EPA Environmental Protection AgencyMonsanto Company 07-CR-191U Page 33 of 544 F15 Phenylalanine 15

F27 Phenylalanine 27

F30 Phenylalanine 30

FA Fatty acid

FASTA Algorithm used to find local high scoring alignments between a pair of protein or nucleotide sequences

FDA Food and Drug Administration

FONSI Finding of No Significant Impact

FW or fw Fresh weight

fwt Fresh weight of tissue

GRAS Generally re cognized as safe

H29 Histidine 29

HC Health Canada

HI Harvest index

HMM Health Ministry of Mexico

Hin d III Restriction enzyme isolated from Haemophilus influenzae

HPLC High-performance liquid chromatography

HRP Horseradish peroxidase

I Intron

ILSI International Life Sciences Institute

ILSI CCD International Life Sciences Institute Crop Co mposition Database

I- Ract1 Intron from the rice actin gene

kb kilobase

L Leader

L2V Amino acid change in MON 87460–produced CSPB that substitutes lysine in position two to valine

Left Border DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA

LOD Limit of detection

LOQ Limit of quantitation

loxP Sequence from Bacteriophage P1 for the recombination site recognized by Cre recombinase

MAFF Ministry of Agriculture, Forestry and Fisheries (Japan)

MALDI-TOF Matrix Assisted Laser Desorption Ionizat ion - Time of Flight

MH+ Protonated mass ionMonsanto Company 07-CR-191U Page 34 of 544 MHLW Ministry of Health, Labor, and Welfare (Japan)

MOE Margin of exposure

MON 863 A Monsanto corn produc t, producing the insecticidal Bacillus thuringiensis Cry3Bb1 protein and NPTII protein

MON 87460 A Monsanto corn product, and the subject of this application, which produces CSPB and NPTII proteins

MON 89034 A Monsanto corn produc t, producing the insecticidal Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins

mRNA Messenger RNA

MS Mass spectrometry

MT/ha Metric tons per hectare

MWCO Molecular weight cut-off

MW Molecular weight

n Number of observations

N/A Not applicable

NA Not available

NCGA National Corn Grower’s Association

NDF Neutral detergent fiber

NEPA National Environmental Policy Act

NFDM Non-fat dry milk

NK603 A Monsanto corn product pr oducing the glyphosate-tolerant CP4 EPSPS protein

NOEL No observable effect level

NOP National Organic Program

Not Restriction enzyme isolated from Nocardia otitidis

nptII Coding sequence of neomycin phos photransferase II gene that confers resistance to neomycin and kanamycin

NPTII Neomycin phosphotransferase II

NTO Non-target organism

OECD Organization for Economic Co-operation and Development

OFPA Organic Foods Production Act

OR Origin of replication

ori-pBR322 Origin of replication from pBR 322 for maintenance of plasmid in

E. coli

ori V Origin of replication for Agrobacterium derived from the broad host range plasmid RK2Monsanto Company 07-CR-191U Page 35 of 544 OSL Overseason leaf

OSR Overseason root

OSWP Overseason whole plant

P Promoter

PAR Photosynthetically active radiation

PBS Phosphate buffered saline

PBST Phosphate buffered sa line containing Tween-20

PCR Polymerase chain reaction

PDB Protein data bank

ppm Parts per million

PRESS Predicted residual sums of squares

PTH Phenylthiohydantoin

PVDF Polyvinylidene difluoride

PVP Polyvinyl pyrrolidone

PV-ZMAP595 Plasmid vector used to develop MON 87460

Ract1 The rice actin gene

Right Border DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA

RNA Ribonucleic acid

RNP Ribonucleoprotein

RK2 Broad host range bacterial plasmid

rop Coding sequence for repressor of primer protein for maintenance of plasmid copy number in E. coli

ROP Repressor of primer protein

SAS ® Originally an acronym for Statis tical Analysis System, now an integrated system of software products provided by the SAS Institute, Inc. headquartered in Cary, North Carolina, USA

SD Standard deviation

SDS-PAGE Sodium dodecyl sulfate- polyacrylamide gel electrophoresis

S.E. Standard error

SGF Simulated gastric fluid

SIF Simulated intestinal fluid

SOP Standard operating procedure

sp

. Species

SPAD Single photon avalanche diodeMonsanto Company 07-CR-191U Page 36 of 544 ss Single stranded

subsp

. Subspecies

Taq Thermus aquaticus , a thermophilic bacterium

T Terminator (where used as a prefix to a gene sequence)

TCA Trichloroacetic acid

T/C/R Test/Control/Reference materials

T-DNA Transfer DNA

TDF Total dietary fiber

TE buffer Tris-EDTA buffer (10 mM Tr is HCl, pH 8.0, 1 mM EDTA, pH 8.0, 1M NaCl)

TFA Trifluoroacetic acid

TMB 3,3’,5,5’-Tetramethylbenzidene

Tn5 Prokaryotic E. coli transposon from which the nptII coding sequence is derived.

T-nos 3’ nontranslated transcript te rmination sequence of the nopaline synthase gene from Agrobacterium tumefaciens

Tris Tris(hydroxymethyl)aminomethane

T- tr7 3' nontranslated sequence of the transcript 7 gene from Agrobacterium tumefaciens that directs polyadenylation

TSSP Tissue-specific site pool

Tween-20 Polyoxyethylenesorbitan monolaurate

USDA United States Department of Agriculture

USDA-APHIS United States Department of Agriculture – Animal and Plant Health Inspection Service

USDA-ERS United States Department of Agriculture – Economic Research

Service

USDA-NASS United States Department of Agriculture – National Agricultural

Statistics Service

U.S. EPA United States Environmental Protection Agency

UV Ultraviolet

v/v Volume to volume ratio

w/v Weight to volume ratio

Xba Restriction enzyme isolated from Xanthomonas badriiMonsanto Company 07-CR-191U Page 37 of 544 I. Rationale for the Development of MON 87460

I.A. Basis for the Request for a Determin ation of Nonregulated Status under 7 CFR

Part 340.6

The Animal and Plant Health Inspection Serv ice (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility, under the Plant Protection Act (7 U.S.C. § 7701- 7772) and the Plant Quarantine Act (7 U.S.C. § 151-167), to preven t the introduction and dissemination of plant pests into the United States. The APHIS regulation 7 CFR Part 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, ther eby allowing unrestrict ed introduction of the

article.

I.B. Product Concept and Benefits of MON 87460

I.B.1. Product Concept

Monsanto has developed MON 87460 that re duces yield loss un der water-limited conditions compared to conventional corn. Under well-watered c onditions, grain yield

for MON 87460 is not different from conve ntional corn. Like conventional corn, MON 87460 is still subject to yield loss unde r water-limited conditions, particularly during flowering and grainfill periods when corn yield pote ntial is most sensitive to stress, by disrupting kernel development (C laassen and Shaw, 1970; Boyer and Westgate, 2004). Under severe water deficit, corn grain yield for MON 87460, as well as conventional corn, can be reduced to zero.

Knowing that stress response proteins allow organisms to adapt to and survive adverse environments, Monsanto scien tists hypothesized th at inserting a stress response protein into plants could impart a de sirable phenotype. As part of a broad screening effort, the gene encoding CSPB from the soil bacterium B. subtilis was inserted into corn using Agrobacterium -mediated transformation. Transformed pl ants were tested for their ability to yield more grain than control plants unde r water-limited conditions. The CSPB protein expressed in MON 87460 exhibits key behaviors and properties th at are similar to what is reported for bacterial cold s hock proteins and cold shock domain-containing proteins in plants.

I.B.2. Product Benefits

MON 87460 is expected to provi de significant value to corn producers and consumers. Corn is the largest crop grown in the U.S. in terms of acreage planted and net value (USDA-NASS, 2007). Limited water availability is the single most im portant factor that reduces global crop yields. In North Ameri ca alone, it is estimated that 40% of annual crop losses are due to sub-optimal water av ailability (Boyer, 1982) . In both temperate and tropical regions suitable for commercial corn production globally, the average annual corn yield loss attributable to moderate wate r deficits is approximately 15% (Barker et al., 2005). During periods of severe drought, these losses can be much higher and can potentially result in complete crop failure.Monsanto Company 07-CR-191U Page 38 of 544 Improved yields under water-limited conditions w ill help to ensure a stable grain supply, even in years with low rainfall. Growers are expected to adopt MON 87460 in regions suitable for corn production that are most prone to frequent drought stress. In the U.S., the major area of adoption is li kely to be the Western Dryla nd region of the Corn Belt. This region has also been defined as the Grea t Plains and includes po rtions of ten states (Colorado, Kansas, Montana, Nebraska, Ne w Mexico, North Dakot a, Oklahoma, South Dakota, Texas and Wyoming) contributi ng 26% of the national corn production (Riebsame, 1990). Annual precip itation in this region averag es between 12 to 20 inches (30 to 50 cm) (http://prism.oregonstate.edu). Other corn producing geographies with similar conditions, such as parts of Africa, Europe, and Latin America, may also benefit

from this trait.

The adoption of MON 87460 is expected to provi de benefits due to reduced yield loss under water-limited conditions. While much pr ogress has been made to improve corn yield in water-limited environments thr ough breeding, selec tion, and agronomic management practices, there remains potentia l for additional improvement. Higher corn yield per acre may help conserve the total number of acres needed to meet the needs for food, feed, and biofuel uses or produce more corn grain on the same number of acres already used for corn production. Positive im pacts on yield and im proved yield stability will provide value to producers, consumers, and the environment.

Efficacy of MON 87460 can be demonstrated by directly comparing its grain yield to a

conventional control under water-limited co nditions. A detailed assessment of the agronomic and phenotypic characterization of MON 87460, including yield, is presented

in Section VIII. A sub-set of these data , including field trials conducted under waterlimited conditions, are useful to demonstrate efficacy of MON 87460.

Because MON 87460 reduces yield loss under water-limited conditions, field studies were designed to evaluate the environmen tal safety of MON 87460 across a broad range

of soil moisture and environmental conditions relevant to where commercial production would be expected. Field studies that are relevant to demonstrate the efficacy of MON 87460 were established using two diffe rent experimental designs and water management regimes: (1) well-watered only, or (2) both well-watered and water-limited treatments established in the same field arrange d in either a strip- or split-plot design.

Field studies managed to impose only well-w atered conditions were established at 17 sites in the U.S. during 2006 and 2007. The well-watered treatment was intended to provide optimal grain yield and allowed an evaluation of MON 87460 in the absence of trait bias, with no differences in yield e xpected between MON 87460 and the control. The water treatment in these studies required av ailable soil moisture to be maintained at

> 50% of field capacity for th e duration of the study to a void drought stress conditions. Water was provided by natural rainfall and supplemental irrigation as needed.

Field studies managed to impose both well- watered and water-limited conditions were

established at nine sites in the U.S. and Chile during 2006 and 2007. The water-limitedMonsanto Company 07-CR-191U Page 39 of 544 treatment allowed an evaluation of MON 87460 under conditions where it is expected to provide a yield benefit compared to the cont rol. The well-watered treatment in these studies required available soil mois ture to be maintained at > 50% of field capacity for the duration of the study to avoid drought stress conditions and provide optimal grain yield. The water-limited treatment was managed the same as the well-watered treatment, with the exception that available soil moisture be reduced to <50% of field capacity to impose a moderate drought stress during the late vegeta tive through early grain fill growth stages (~V10 – R3) when corn yield potential is most sensitive to stress (Claassen and Shaw, 1970; Boyer and Westgate, 2004).

For the nine field sites established with both well-watered and water-limited treatments, criteria were established to identify sites that were managed appropriately to impose the defined treatment levels (Section VIII.C). Sp ecifically, for a site to be considered waterlimited, commercial reference hybrids in the water-limited plots had to exhibit a minimum 15% reduction in yield compared to the same reference hybrids planted in the well-watered plots. The 15% yield loss criteria represents a meaningful stress level, as moderate water deficits result in approxima tely a 15% yield loss annually for corn grown in both temperate and tropical regions (Barker et al., 2005). Reductions in plant height, ear height, and days to 50% silking are also phenotypic i ndicators of moisture deficit stress in corn (Campos et al ., 2006); therefore, these charact eristics were assessed to

confirm the inclusion of sites that met the 15% yield reduction requirement. Due to untimely rainfall during the imposed water-limitation treatments and complications in water management techniques, only six of th e nine established sites met the inclusion criteria of having both well-watered and water-limited treatments for plant characterization assessments requiring both treatments (Section VIII.C, Table VIII-3).

The efficacy of MON 87460 under water-limited conditions is demonstrated by plotting the yield advantage of MON 87460 versus th e relative stress leve l imposed among the nine field sites (Figure I-1). Yield a dvantage of MON 87460 was calculated as the average yield of MON 87460 compared to the control within each site. Relative stress level is represented by the yield loss ofthe commercial references grown under waterlimited conditions compared to well-watered tr eatments. Yield loss of references was calculated as the average yield of the references in the well-watered treatment compared to the average yield under the water-limited treatment within each site. The presenteddata represent the nine sites that were established with both well-watered and waterlimited treatments (Section VIII.C, Table VIII-3 ). As expected, the yield advantage for

MON 87460 tended to increase with higher levels of water de ficit stress, but there was large variation. The three data points in the lower left portion of Figure I-1 represent the

three sites that did not meet the inclusion criteria (i.e., QUI site in Chile 2006/2007 and

the KS and NE sites in U.S. 2007 Study-2) (Table VIII-3). Yi eld loss of the references at these three sites did not meet the require d minimum 15% reduction due to the imposed water-limitation treatment, indicating the appropriate water stress level was not

established. Consistent with the data for MON 87460 gr own under well-watered field conditions, MON 87460 did not pr ovide a yield advantage comp ared to the control at

these three sites when water stress was minimal. As described previously (Section I.B.1), MON 87460 is still subject to yield loss unde r water-limited conditi ons, and under severeMonsanto Company 07-CR-191U Page 40 of 544 water deficit, yield can be reduced to zero. In tuitively, as stress levels (i.e., yield loss of the references) increase beyond the range presen ted in Figure I-1, the yield advantage of MON 87460 compared to the control should be e xpected to decrease a nd be eliminated as the grain yield for both MON 87460 and the control falls to zero.

A comparison of the average percent yiel d advantage of MON 87460 compared to the control under both field trial designs also demonstrates the efficacy of MON 87460 under water-limited conditions (Figure I-2). Percent yield advantage values were calculated as the average yield of MON 87460 versus the co ntrol, averaged across all sites for each treatment. Reference yield values presented at the top of the graph represent the average yield of the commercial references averaged across all sites for each treatment. The wellwatered and water-limited treatments include only the six sites that met the required inclusion criteria (Section VIII.C, Table VIII-3 ). In the well-watered only experimental design, yield potential was 179 bushels per acre (bu/ac), calculated as the average yield of

the references across all 17 sites. In the stri p- and split-plot expe rimental designs, yield potentials were 207 and 144 bu/ac for the well -watered and water-limited treatments, respectively. As expected, MON 87460 provide d a yield benefit compared to the control under water-limited conditions and similar yield to the control under well-watered conditions. Yield advantage values for M ON 87460 in the water-lim ited treatment were highly variable, but are represen tative of the natural varia tion expected for corn grown under sub-optimal soil moisture conditions.

It is known that corn hybrids are variable in their respons e to drought stress (Campos et al., 2006). Breeders have been selecting for improved drought tolerance in conventional corn through traditional breeding and select ion methods for decades. The variation in yield response observed among MON 87460, th e control, and commercial reference hybrids in the field studies discussed above is within normal levels expected for conventional corn grown under sub-optimal soil moisture environments. Therefore, the

data from these field studies are relevant to the safety assessment of MON 87460. In the water-limited sites, the data are also releva nt to demonstrate the efficacy of MON 87460

Taken together, these data support that MON 87460 produces yields similar to the control under well-watered conditions and provides a yi eld advantage (i.e., reduced yield loss)under water-limited conditions. This yield advantage occurs over a range of waterlimited conditions but will decrease as water deficit stress becomes too severe. Among the broad range of geographical and environm ental conditions suited for commercial corn production, it is expected that MON 87460 w ill produce a range of responses based on the timing, severity, and dura tion of water-limited conditi ons experienced during the growing season. On average, under wa ter-limited conditions, MON 87460 hybrids are expected to provide a 6% or greater yield advantage compared to commercial hybrids.

Monsanto and other seed companies commonl y evaluate their commercial hybrids under a range of environmental conditions each year and assign drought tolerance ratings as a performance characteristic for each hybrid (http://www.asgrowanddekalb.com/ seedresourceguide/search/seeds). Commerci al hybrids of MON 87460 will be evaluated for drought tolerance in a manner consistent with the breeding and selection of otherMonsanto Company 07-CR-191U Page 41 of 544 commercial corn hybrids. As per standard process for all commercial products, both conventional and biotechnology-derived, Mons anto will conduct yield trials under relevant conditions to ensure that hybrids released for sale meet appropriate yield performance standards across several evaluation and selection criteria.

0 10 20 30 40 50 60 -20

0 20 40 60

y = 0.66x + 1.98 R-square = 0.54 p = 0.0235

95% Confidence Band

Figure I-1. Percent Yield Advantage fo r MON 87460 Compared to the Control against Percent Yield Loss of the References in U.S. and Chilean Field Studies during 2006 and 2007

Yield advantage of MON 87460 was calculated as the average yield of MON 87460 compared to

the control within each site. Yield loss of refe rences was calculated as the average yield of the

reference materials in the well-watered treatmentcompared to the average yield under the waterlimited treatment within each site. Data presente d represent the nine sites that were established with both well-watered and water-limited treatments (Section VIII.C, Table VIII-3).Yield advantage of MON 87460 vs.control(% difference)

Yield loss of references grown under water-limited vs. well-watered treatments (% difference)Monsanto Company 07-CR-191U Page 42 of 544 Treatment Type WWonly WWsp WLspPercent Difference (Yield):MON 87460 vs. Control

0 10 20 30

Figure I-2. Percent Yield Advantage for MON 87460 Compared to the Control in U.S. and Chilean Field Studies during 2006 and 2007

Experimental design and treatment type: WWonl y = well-watered only design (n = 17 sites);WWsp = well-watered treatment in either a strip- or split-plot design (n = 6 sites); WLsp = waterlimited treatment in either a strip- or split-plot design (n = 6 sites); Error bars represent standard

error.

I.C. Intended Function of the Genetic Modification

MON 87460 was chosen for development ba sed on its yield advantage under waterlimited conditions compared to the control and absence of ne gative pleiotropic effects on plant performance. The insertion of the cspB gene in MON 87460 confers tolerance to

water-limited conditions that would othe rwise negatively impact yield. The npt II gene was inserted to facilitate selection of plants containing cspB during early product development. The presence of NPTII does not pose any safety concerns (EFSA, 2007; FDA 1994 and 1998; Flavell et al., 1992; Fuchs et al., 1993a & 1993b; and Nap et al., 1992). Reference yield (bu/acre)

179 207 144Yield advantage of MON 87460 vs. control(% difference)

Experimental design and treatment typeMonsanto Company 07-CR-191U Page 43 of 544 I.D. Mode of Action of the Drought Tolerance Trait

Cold shock proteins confer environm ental stress tolerance in bacteria. Efficacy in MON 87460 is derived by expression of the inserted Bacillus subtilis CSPB protein. The CSPB protein in MON 87460 bel ongs to the CSP family, which has been extensively studied in bacteria . Early investigations of bacterial responses to coldinduced stress led to the disc overy of CSPs, a group of sma ll proteins that contain a highly conserved RNA-binding sequence iden tified as a cold shock domain (CSD).

In bacteria, a variety of environmenta l stresses are known to disrupt normal cell physiology, in part due to the production of RNA secondary structur es which leads to a reduction in protein synthesis. Under envi ronmental stress, CSD-containing proteins have been shown to bind many types of R NA (Cristofari and Dar lix, 2002), leading to sustainable translation, maintenance of mR NA levels, and improved cellular function.

While some members of the bacterial CSP fa mily accumulate strictly in response to temperature shift (Etchegaray et al., 1996), others, including the B. subtilis CSPB protein, are also involved in maintaining normal ce llular functions at both optimal temperatures (Graumann et al., 1997) and under nutrient limitation (Anderson et al., 2006). In actively transcribing B. subtilis cells, CSPB is localized around the nucleoid, co-l ocalizing with the ribosomes (Mascarenhas et al., 2001; Weber et al., 2001). In st ationary-phase cells CSPB is distributed throughout the cell, indi cating that specific localization of CSPB depends on cell development stage (Weber et al., 2001). It has been suggested that accumulation of the CSPB protein in B. subtilis cells can be triggered under several stress conditions that share a common signal such as inactivation of ribosomes (Schindler et al., 1999; Graumann et al., 1997).

CSD-containing proteins also confer en vironmental stress tolerance in plants. Similar to bacteria, CSD-containing proteins in plants also bind RNA, unfold RNA secondary structures caused by environmental st ress, and help maintain cellular functions under stress. These plant CSD proteins share a high level of similarity to the bacterial

CSPs and have been shown to share in vitro and in vivo functions with bacterial CSPs (Karlson and Imai, 2003; Kim et al., 2007; Nakaminami et al., 2005 and 2006; Chaikam and Karlson, 2008; Fusaro et al., 2007). Pl ant CSD proteins have been reported to respond to abiotic stresses in Arabidopsis (Fusaro et al., 2007), wheat (Karlson et al., 2002), and rice (Chaikam and Karlson, 2008), and to play an important role in various aspects of plant development (Fusaro et al., 2007; Chaikam and Karlson, 2008). Direct relationships between the ability of CSD-c ontaining proteins to bind RNA and/or ssDNA

and stress tolerance have been established (Nakaminami et al., 2006; Castiglioni et al., 2008) and results of in vitro experiments show that plant CSD proteins can bind RNA, synthetic mRNA, and ssDNA (Sasaki et al., 2007). The apparent absence of binding sequence specificity indicates that plant CS D proteins could be involved in a more general response to stress by binding RNAs and, therefore, helping cells to maintain cellular functions following the stre ss. CSD proteins from rice and Arabidopsis have been shown to be highly expressed in ap ical meristems, ovules, embryos, and seeds (Fusaro et al., 2007; Chaikam and Karlson, 2008) and, therefore, could potentially affect growth rate, flowering time, and seed deve lopment. The CSD proteins have been

localized both in th e cytoplasm and the nuclei (Sasaki et al., 2007; Fusaro et al., 2007)Monsanto Company 07-CR-191U Page 44 of 544 indicating that these proteins can potential ly be involved in multiple steps of RNA metabolism including localization, transla tion and stability.

CSPB protein expressed in MON 87460 has a si milar structure and function to other CSD-containing proteins. As with bacterial and other plant CSD-c ontaining proteins, the CSPB protein from B.

subtilis , which is expressed in MON 87460, bi nds RNA, unfolds RNA secondary structures, and accumulates in actively growing tissues. Data from in vitro and in vivo experiments indicate that CSPB preferentially binds plant RNA, but not dsDNA (Figures I.3 - 5, 6). CSPB was also eff ective in unfolding secondary RNA structures in
vitro, while variants of the CSPB protein (C SPB_F30R and CSPB_F30 A) with impaired RNA binding functions were unable to bind and unfold RNA (Table I-1). In vitro coimmunoprecipitation experiment s with CSPB, CSPB_F30A and RNA from MON 87460

further confirm that CSPB interacts with RNA while a variant la cking a functional RNA binding site will not interact with RNA in vitro (Figure I-3). Finally, gel shift experiments demonstrate that CSPB is ca pable of binding both ribosomal RNA (rRNA) and messenger RNA (mRNA) (Figures I-4 and I-5). To demonstrate the in vivo interaction between the CSPB protein and RNA in corn plants expressing CSPB, a CSPB/RNA complex was co-immun oprecipitated from leaf tissue, confirming that CSPB

interacts with corn RNA in vivo (Figure I-6).

In MON 87460, the expression of the CSPB pr otein is under contro l of the rice actin promoter which enables constitutive expres sion of the protein and decouples expression of CSPB protein from the cold shock res ponse in bacteria. Using a CSPB specificEnzyme-Linked Immunosorbent Assay (ELISA), the pattern of CSPB accumulation in MON 87460 leaf tissue, seedlings and deve loping reproductive tissues was evaluated (Figures I-7 – I-9). CSPB accumulation is hi ghest in rapidly growing areas of the leaf and seedlings and tends to decl ine as the tissue matures. CSPB concentrations tend to increase over time in developing ears and de cline over time in silks. Likewise, CSPB

concentrations increase over time in immature tassels and either remain the same or decline in pollen. Sub-cellu lar localization of the CSPB protein was evaluated by immunohistochemistry. In MON 87460 coleop tiles, CSPB was distributed between cytoplasm and nucleus (Figur e I-10). Similar sub-cellular localization of the CSPB protein was previously observed in corn protoplasts (Castig lioni et al., 2008).

Taken together, the data on RNA binding, CS PB accumulation and CSPB localization in MON 87460 are consistent with the pattern of RNA binding, accumulation, localization, and functions described for plan t CSD-containing proteins (Fus aro et al., 2007; Sasaki et al., 2007; Chaikam and Karlson, 2008).

CSPB protein expression improves yield and vegetative productivity under water-limited

conditions. Water limitation during the growing season can diminish corn productivity and yield, particularly during flowering and grainfill periods when co rn yield potential is most sensitive to stress by disrupting fertilizati on and kernel development (Claassen and Shaw, 1970; Boyer and Westgate, 2004; Campos et al., 2006).Monsanto Company 07-CR-191U Page 45 of 544 Using a high through-put biotechnology approac h, Castiglioni et al. (2008) demonstrated that bacterial CSPs can confer improved stress adaptation to multiple plant species. CSPB-containing events were evaluated in wate r-limited field trials in environments that received no rainfall during th e 10 to 14 days immediately pr ior to flowering. The waterlimited treatment resulted in an average reduction in growth rates to 50% of the wellwatered rate. Using an across-event analysis , the CSPB-containing events demonstrated increases in leaf extension rates relative to the controls, improvements in chlorophyll content and improvements in photosynthetic rates. These measures of vegetative performance indicated that the CSPB prot ein has a positive impact on overall plant productivity and, therefore, yield potential. When plants were grown under well-watered conditions in both the greenhouse and field, no appreciable difference between CSPBexpressing lines and the control were detected.

In field trials under water-limited conditions, MON 87460 demonstrated improvements in yield and yield components through trends toward increased yiel d (16.5%), kernels per ear (13.1%), and kernel weight (3.9%) (Table I-2). Simila r results were observed in a subsequent study, where differen ces were detected in yield (9.3%), kernels per ear (8.5%) and a trend toward increased kernel weight (2.5%) (Table I-3). Results from these studies demonstrate that the major compone nt contributing to the improved yield of

MON 87460 under water-limited conditions is th e increased number of kernels per ear,

which is consistent with the current understand ing of the effect of drought stress on corn yield potential (Westgate et al., 2004; Ca mpos et al., 2006; Welcker et al., 2007).

In a greenhouse study conducted under water-limited conditions, young MON 87460 plants showed a trend toward increased leaf gr owth rate relative to the control plants and overall better vegetative performance as measured by photosynthetic rate, carbon fixation, and stomatal conducta nce (Figures I-11 – I-13, re spectively). The improved vegetative productivity is believed to be co rrelated to the high accumulation of CSPB in the rapidly growing tissues (F igures I-14 – I-21). Leaf gr owth and improved vegetative performance provide the physiological capacity necessary for the development of reproductive organs such as silks and pollen especially under drought stress. In turn, improvements in plant physiological capacity leads to increased numbers of kernels per ear and overall improved yield potential (A ndrade et al., 2002; Bruce et al., 2002; Campos et al., 2006; Welcker et al., 2007; Fuad -Hassan et al., 2008; Chenu et al., 2008).

CSPB protein expression does not affect co mmon drought tolerant responses in corn plants Common mechanisms of plant response to dro ught stress are not al tered in transgenic CSPB-containing corn plants. Greenhouse experiments were conducted with CSPB events that are genetically and phenotypically similar to MON 87460 to evaluate if these characteristics had been altered when compar ed with controls. Th e plants containing CSPB protein show broadly the same relativ e water content (Table I-4), leaf water potential (Figure I-22), and l eaf osmotic potential (Figure I-23) as the control plant. CSPB-containing plants accumulate similar levels of abscisic acid (ABA) (Figure I-24) and osmotically active solutes (sucrose, fruc tose, glucose, choline, proline, glycineMonsanto Company 07-CR-191U Page 46 of 544 betaine) as observed in controls (Figures I-25 - 28). Results from these experiments suggest that common mechanisms of plant response to drought stress are not altered in transgenic CSPB-containing corn plants.

CSPB protein function: Conclusion In summary, expression of th e CSPB protein in MON 87460 re sults in reduced yield loss under drought conditions when compared to conventional corn grown under identical conditions. The major component contri buting to the improved yield of MON 87460

under water-limited conditions is the increased number of kernels per plant, which is consistent with the current understanding of the effect of drought stress on corn yield potential. CSD-containing prot eins moderate stress respon ses in bacteria and plants, primarily through stabilization of RNA. Li ke endogenous CSD proteins found in plants, the CSPB protein in MON 87460 interacts wi th RNA and accumulates and localizes to rapidly growing tissues and in developing reproductive organs, thereby helping to maintain normal cellular function in those tissues critical to yield. Under water-limited conditions, there is a trend toward improved growth rate , photosynthetic rate, carbon fixation, and stomatal conductance for M ON 87460 compared to the control, but accumulation of ABA and osmotically active solu tes are not altered in transgenic CSPBcontaining corn plants.

Table I-1. In vitro Melting Activity of CS PB and CSPB Variants

Protein Specific Activity (pmoles opened DLP 1 /μg CSPB)

CSPB 0.905 ± 0.033

CSP_F30R <LOD 2

CSPB_F30A <LOD

1 DLP – Dual-Labeled Probe

2 LOD – Limit of Detection

The melting activities of CSPB and the diffe rent variants were measured for all proteins using 3 μg of protein for each replicate. The activity represents the average of three replicates. In this ass ay, a hairpin-shaped (stem-loop) molecular beacon is labeled with a fluorophore at th e 5’end and quencher at the 3’-terminus. Due to the close proximity of the fluorescent tag and quencher in the hairpin conformation, the fluorescence is efficiently quenched. When a CSPB protein “melts” the hairpin conformation, the fluorescent tag and quencher are spatially separated which permits fluorescence. CSPB is cold shock protein B. CSPB_F30A contains alanine instead of phenylalanine in position 30 and CSPB_F30R contains an arginine instead of phenylalanine in the same position. CSPB, CSPB_F30A and CSPB_F30R were produced in E. coli

.Monsanto Company 07-CR-191U Page 47 of 544 Table I-2. Yield Component and Physiology Data from a Water-Limited Kansas

Field Trial in 2003

Endpoint MON 87460 Mean (S.E.) 1 Control Mean (S.E.) Difference,

MON 87460

minus control Diff (%) pvalue

Yield 2 (bu/ac) 3 80.0 (10.9) 68.7 (10.9) 11.3 16.4 0.153

Kernels per ear 4 289 (36.9) 256 (36.9) 33 12.9 0.233

200 kernel weight (g) 5 72.6 (2.7) 69.9 (2.7) 2.7 3.9 0.283

Ears per plot 6 33.8 (1.3) 33.5 (1.3) 0.3 0.9 0.834

Stomatal

conductance (mmol/m 2 /s) 262.7 (20.78) 235.8 (20.78) 26.9 11.4 0.064

Photosynthetic rate (μmol CO 2 /m 2 /s) 7 37.2 (2.24) 34.1 (2.24) 3.1 9.1 0.066

Transpiration rate (mmol/m 2 /s) 6.1 (0.35) 5.8 (0.35) 0.3 5.2 0.126

Leaf extension rate (cm/5 d) 8 21.2 (1.79) 17.4 (1.79) 3.8 21.8 0.008

1 S.E. – standard error

2 Yield data were normalized to 15.5% moisture.

3 bu/ac – bushels per acre

4 Kernel per ear measurements were collected from a subsample out of each plot.

5 Kernel weights were taken from 200 kern el samples from a subsample of ears.

6 The number of harvestable ears was counted from each of the plots at the end of the season.

MON 87460 and the control were planted in two row plots, 34 plants per row, at a density of 32,000 plants per acre. Twelve paired-plot repli cates were planted in a randomized block design.

7 Photosynthesis measurements were made using a PP Systems Ciras-1 Portable Photosynthesis System.

8 Photosynthesis and leaf extension rate measur ements were collected on six plants each of

MON 87460 and the control.Monsanto Company 07-CR-191U Page 48 of 544 Table I-3. Yield Component Data fr om a Well-Watered and Water-Limited

California Field Trial in 2007

Endpoint MON 87460 Mean (S.E.) 1 Control Mean (S.E.) Difference,

MON 87460

minus control Diff (%) pvalue

Well-watered yield (bu/ac) 2, 3 297 (4.08) 295 (3.98) 2 0.7 0.568

Water-limited yield (bu/ac) 206 (12.7) 188 (12.7) 18 9.6 0.038

Water-limited kernels

per ear 4 483.1 (20.50) 445.2 (20.50) 37.9 8.5 0.004

Water-limited 50 kernel weight (g) 5 17.4 (0.4) 16.9 (0.4) 0.5 3.0 0.068

Water-limited leaf extension rate (cm/8 d) 6 27.8 (1.20) 24.7 (1.20) 3.1 12.6 0.034

Water-limited plant height (cm) 6 220 (1.77) 215 (1.73) 5 2.3 0.046

Water-limited plant

biomass increase (g/8 d) 6 24.0 (1.62) 22.0 (1.70) 2.0 9.1 0.376

1 S.E. – standard error

2 Yield data were normalized to 15.5% moisture.

3 bu/ac – bushels per acre

4 Kernel per ear measurements were collected from a subsample out of each plot.

5 Kernel weights were taken from 50 kernel samples from a subsample of ears.

6 Leaf extension, plant height and plant biomass m easurements were collected from three plants in each of the ten water-limited replicates.

The well-watered treatment contained five repli cates. The water-limited treatment contained 10 replicates. Each replicate consisted of four six -row plots with 20.5 foot rows planted with 50 kernels per row.Monsanto Company 07-CR-191U Page 49 of 544 Table I-4. Average Relative Water Content of Transgenic CSPB Plants and the

Control Grown under Water-Limited Conditions in the Greenhouse
Entry Ave RWC (%) Std Dev RWC

Control 78 4.9 ZM_M38245 82 5.7 ZM_M38705 77 5.7 ZM_M38727 75 5.0 ZM_M38835 1 75 5.0 ZM_M38840 76 4.8 ZM_M38862 80 5.3

1 Entry ZM_M38835 is MON 87460

Other entries contain the same construct as MON 87460.

All plants were subjected to a 12 day peri od of moderate stress based on pot weight.Monsanto Company 07-CR-191U Page 50 of 544 Figure I-3. Co-Immunoprecipitation of RNA from MON 87460 using CSPB and the CSPB_F30A Variant

(A) Total RNA (2.5 μg) isolated from MON 87460 leaf tissue was incubated with CSPB (5 μg) or the CSPB_F30A variant (F30A) (5 μg). CSPB was immunoprecipitated with an affinity purified anti-CSPB antibody (30 μg). Any RNA that co -precipitated was quantified using fluorescent probes. Immunoprecipitation conditions are describe d in the table below the graph. CSPB and CSPB_F30A were produced in E. coli

. (B) Aliquots of total RNA from MON 87460 were mixed with CSPB (Lane 4) or the CSPB_F30A variant (F30A) (Lane 5). The CSPB-RNA complex, if present, was immunoprecipitated with an affinity purified anti-CSPB antibody (CSPB-Ab). Samples were mixed with ethidium bromide and run on a 1% ag arose gel. Lane 1 contains a 1 kb molecular weight ladder. Lane 2 contains MON 87460 RNA alone in the absence of immunoprecipitation as a positive control. Lane 3 is the immunopr ecipitate of MON 87460 RNA and CSPB-Ab and

demonstrates that CSPB-Ab with RNA alone produces no signal. CSPB and CSPB_F30A were produced in E. coli . Results confirm that CSPB binds detectable amounts of RNA but CSPB_F30A does not. CSPB +

F30A +

RNA + + +
CSPB-Ab + + +RNA (μg/ml)

CSPB +

F30A +

RNA + + + +

CSPB-Ab + + +

Lanes 1 2 3 4 5

Lanes 1 2 3 4 5 

## A BMonsanto Company 07-CR-191U Page 51 of 544 Figure I-4. Gel Shift Analysis of Total RNA Isolated from MON 87460

(A) Aliquots of total RNA (2.5 μg) purified from MON 87460 leaf tissue were incubated with different amounts of CSPB, mixed with ethidium bromide and run on an agarose gel. A shift in the RNA banding pattern is seen with increasing concentrations of CSPB. Lanes 8 and 9 do not contain RNA, demonstrating no signal was observed for CSPB alone. (B) BSA was used as a control and mixed with total RNA (2.5 μg) purif ied from MON 87460. No shifts in the banding pattern are observed for increasing amounts of BSA. Lane 1 in each gel contains a 1Kb molecular weight ladder. The amount of protein added to each reaction is described in the table below each image. Lanes 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7

Lanes 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 CSPB(μg) 0 5 10 20 50 100 50 100 0 50 0 0 0 0 BSA (μg) 0 0 0 0 0 0 0 0 0 0 25 50 100 200 

## A BMonsanto Company 07-CR-191U Page 52 of 544 Figure I-5. Gel Shift Analysis of rRNA and Luciferase mRNA

(A) Aliquots of total RNA (0.5 μg) purified from MON 87460 leaf tissue and in vitro transcribed Luciferase mRNA (Luc mRNA, 0.5 μg) were in cubated with increasing amounts of CSPB and mixed with ethidium bromide. The shifting in the banding of the RNA was observed on 10% agarose gels under UV light. (B) BSA was used as a control and mixed with total RNA (0.5 μg) purified from MON 87460 and Luciferase mRNA (Luc mRNA, 0.5 μg) and compared to shifts produced by CSPB. No shifts are observed for increasing amounts of BSA. Lane 1 in each gel contains a 1Kb molecular weight ladder. Lanes 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 

## A B

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

CSPB(μg) 0 1 3 5 10 50 0 1 3 5 10 0 10 50 0 0 10 50 0 BSA (μg) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 0 0 0 50

Total RNA + + + + + - - - - - - + + + + - - - -

Luc mRNA - - - - - + + + + + + - - - - + + + +Monsanto Company 07-CR-191U Page 53 of 544 Figure I-6. Co-Immunoprecipitation of CSPB-FLAG:RNA Complexes from Leaf

Tissue

Leaf tissue from corn plants expressing a FLAG-tagged version of CSPB was ground and extracted. The FLAG-CSPB:RNA complexes were immunoprecipitated using anti-FLAG antibodies and analyzed on an Agilent Bioana lyzer. The results show the nucleic acid precipitated compared to a conventional leaf (A ) and the digestion of the Co-IP sample with RNAse (B) and DNAse(C). The 25nt Marker show s where a 25 nucleotide (nt) sequence would

elute. Results confirm that CSPB binds RNA in vitro but not DNA.

CSPB-FLAG corn plants are transgenic events containing the CSPB coding region with an additional sequence which adds 24 nucleotides to the 3’ end resulting in an eight peptide Cterminal addition, called FLAG, to the translated CSPB protein. This eight peptide group allows the FLAG-tagged protein to be co-immunoprecipitate d with a commercial kit (Sigma, St. Louis, MO). The CSPB-FLAG corn expression construct contained the same promoter and terminator as those used to drive CSPB expression in M ON 87460 (i.e., rice actin promoter and Tr7 terminator). The event used for the Co-IP expe riments had a single copy transgene insertion and it expressed the expected protein as confirme d by western blot and MALDI-TOF Mass Spectrometry. Plants were kept well watered a nd samples were taken 20 days after planting by cutting the plant at the leaf collar of the V4 or V5 leaf, removing the leaf and harvesting the basal 1/8th of the next three youngest leaves from test and control plants. 

## A B C

25nt Marke r 25nt Marke r 25nt Marke r

FLAG-CSPB Co-IP

N egative Control Co - IP FLAG-CSPB Co-IP

FLAG-CSPB Co-IP + RNase FLAG-CSPB Co-IP

FLAG-CSPB Co-IP + DNaseMonsanto Company 07-CR-191U Page 54 of 544 Figure I-7. Differential Accumulation of CSPB across Leaves of MON 87460

CSPB levels in different V10 leaf sections of corn MON 87460 sampled according to the leaf diagram (A) were determined using a validated CSPB-specific ELISA (B). The values represent the means of three leaf sections from two different V10 leaves. The sample locations from leaf base to leaf tip shown in panel A correspond with the numbers in the x-axis of panel B. The basal, rapidly growing, portion of the leaf contai ned a significantly higher level of CSPB than the rest of the leaf segments, with the distal portion of the leaf having the lowest concentrations of

CSPB. 0.0 10.0 20.0 30.0 40.0 50.0

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46pg CSPB/ μg Total Protein

Basal to Distal Sampling 

## BMonsanto Company 07-CR-191U Page 55 of 544 Figure I-8. Expression and Accumulati on of CSPB Protein in Germinating MON 87460 Seedlings

Plants harvested were dissected into basal and distal leaf (A). The expression of the CSPB mRNA was determined by northern blot using a CSPB-specific probe. The total rRNA was used as a control for the amount of RNA loaded on the gel (B). The level of CSPB (C) and total protein (D) in both tissue types were determin ed from tissue homogenates using a validated ELISA and a colorimetric assay, respectively. 

## A

B 1 kB

0.5 kB Control

d3 d5



## D C

0 200 400 600 800 1000

3 5 6 7 10 11ng/g fwt

Days after germination Basal-Leaf Distal-Leaf Coleoptile

0 10 20 30 40 50

3 5 6 7 10 11mg/g fwt

Days After Germination Basal Leaf Distal Leaf Coleoptile CSPB mRNA

28S rRNALevel of CSPB (ng /g fwt)Total Protein (ng /g fwt) MON 87460

d3 d5 d6 d7 d10 d11

Days After Planting Days After PlantingMonsanto Company 07-CR-191U Page 56 of 544 Figure I-9. Expression of CSPB in Ti ssues of MON 87460 Grown in a Greenhouse

MON 87460 plants were grown under well-watered conditions in a greenhouse and tissues were sampled on different days after planting. CSPB levels were determined by a validated ELISA and are based on fresh weight of the tissue. (A) Immature ears and silks (B) Immature tassels and pollen. CSPB levels increased in developing ears and tassels. Highest CSPB levels were detected in pollen whereas silks had the lowest levels. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

25 30 35 40 45 50 55 60CSPB (ppm)

Days After Planting (DAP) Immature Tassel Pollen

4.0 5.5



## ≈ 4.5 5.0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

25 30 35 40 45 50 55 60CSPB (ppm)

Days After Planting (DAP) Immature Ear Silk 

# A

BMonsanto Company 07-CR-191U Page 57 of 544 Figure I-10. Immunohistochemical Loca lization of CSPB in Plant Tissue

Shoot (coleoptiles) sections from 3-day post-germin ation plants were incubated with affinity purified goat anti-CSPB. The CSPB antibody w as detected using a fluorescently labeled small fragment secondary antibody. The control tissue (A) had no specific fluorescent signal, while in MON 87460 (B) the fluorescent signal can be clearly seen; both images are overlays with bright light to view the cells at 20X magnification. Observation of just the fluorescent signal under 20X (C) and 60X (D) magnification shows that the CSPB is localized in the nucleus and cytoplasm. 

## A B

C DMonsanto Company 07-CR-191U Page 58 of 544 Figure I-11. Quantum Efficiency of Phot osystem II-Mediated Electron Transfer, or

Φ II, for MON 87460 and the Control over Time

Measurements of the maximum fluorescence quant um yield of photosystem II (PS2) mediated electron transfer ( Φ II ) were obtained with an OS30P chlorophyll fluorometer as a measure of photosystem II activity. Readings were made on le aves that were at the top of the canopy and whose surface was visibly exposed to illuminati on. Results were obtained from six paired replicate plants of MON 87460 and the control that were subjected to six days of water-limited conditions based on pot we ight beginning at the V5 growth stage. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0.5

PM AM PM PM PM PM

5/16/2008 5/19/2008 5/20/2008 5/21/2008 5/22/2008

T0 T1 T2 T3 T4PS2QuantumEfficiency(ΦII;Fm'‐Fs/Fm')

Control

MON 87460Monsanto Company 07-CR-191U Page 59 of 544 Figure I-12. CO 2 Assimilation over Time for MON 87460 and the Control

Measurements of CO 2 assimilation (A N ) were obtained using a LICOR 6400 instrument. Measurements were made on small sections of mature V5 leaves midway between the base and the tip of the leaf that had reached steady-stat e in the measurement chamber. Gas exchange parameters were obtained under steady-state illumina tion. Results were obtained from six paired replicate plants of MON 87460 and the control that were subjected to six days of water-limited conditions based on pot weight begi nning at the V5 growth stage. 0 5 10 15 20 25 30 35 40 45

PM AM PM PM PM PM

5/16/2008 5/19/2008 5/20/2008 5/21/2008 5/22/2008

T0 T1 T2 T3 T4CO2assimilation(AN;μmolCO2m‐2s‐1) Control

MON 87460Monsanto Company 07-CR-191U Page 60 of 544 Figure I-13. Stomatal Conductance ov er Time for MON 87460 and the Control

Measurements of stomatal conductance (g

s ) were obtained using a LICOR 6400 instrument. Measurements were made on small sections of mature V5 leaves midway between the base and the tip of the leaf that had reached steady-stat e in the measurement chamber. Gas exchange parameters were obtained under steady-state illumina tion. Results were obtained from six paired replicate plants of MON 87460 and the control that were subjected to six days of water-limited conditions based on pot weight begi nning at the V5 growth stage. 0 0.05

0.1 0.15

0.2 0.25

0.3

PM AM PM PM PM PM

5/16/2008 5/19/2008 5/20/2008 5/21/2008 5/22/2008

T0 T1 T2 T3 T4StomatalConductance(gs;mmolH2Om‐2s‐1)

Control

MON 87460Monsanto Company 07-CR-191U Page 61 of 544 Figure I-14. Leaf 5 Daily LER Pre-Drought (T0-3 to T0) and Post-Drought Initiation (T1-T4)

T1 leaf extension rate (LER) is an average b ased on three-day height difference between T1 and T0. T0-1, -2 & -3 are all well-watered measuremen ts prior to treatment initiation at T0. Leaves were measured from the soil to the tip of the leaf. Fifteen plants each of MON 87460 and the

control were measured. Water-l imited conditions were imposed after the last watering on the morning of May 16 based on pot weight. For leaf 5, measurements were terminated after emergence of the ligule (May 19) because little growth accumulates after that stage. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

T0

‐ 3 T0

‐ 2 T0

‐ 1 T0 T1 T2 T3 T4LER(cm) Control

MON 87460Monsanto Company 07-CR-191U Page 62 of 544 Figure I-15. Leaf 6 Daily LER Pre-Drought (T0-3 to T0) and Post-Drought Initiation (T1-T4)

T1 LER is an average based on three-day height difference between T1 and T0. T0-1, -2 & -3 are all well-watered measurements prior to treatme nt initiation at T0. Leaves were measured from the soil to the tip of the leaf. Fifteen plants each of MON 87460 and the control were measured. Water-limited conditions were imposed after the last watering on the morning of May 16 based on pot weight. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

T0

‐ 3 T0

‐ 2 T0

‐ 1 T0 T1 T2 T3 T4LER(cm) Control

MON 87460Monsanto Company 07-CR-191U Page 63 of 544 Figure I-16. Leaf 7 Daily LER Pre-Drought (T0-3 to T0) and Post-Drought Initiation (T1-T4)

T1 LER is an average based on three-day height difference between T1 and T0. T0-1, -2 & -3 are all well-watered measurements prior to treatme nt initiation at T0. Leaves were measured from the soil to the tip of the leaf. Fifteen plants each of MON 87460 and the control were measured. Water-limited conditions were imposed after the last watering on the morning of May 16 based on pot weight. 0.0 2.0 4.0 6.0 8.0 10.0 12.0

T0

‐ 3 T0

‐ 2 T0

‐ 1 T0 T1 T2 T3 T4LER(cm) Control

MON 87460Monsanto Company 07-CR-191U Page 64 of 544 Figure I-17 Leaf 8 Daily LER Pre-Drou ght (T0-3 to T0) and Post-Drought Initiation (T1-T4)

T1 LER is an average based on three-day height difference between T1 and T0. T0-1 is a wellwatered measurement prior to treatment initiation at T0. Leaves were measured from the soil to the tip of the leaf. Fifteen plants each of MON 87460 and the control were measured. Waterlimited conditions were imposed after the last watering on the morning of May 16 based on pot weight. 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

T0

‐ 3 T0

‐ 2 T0

‐ 1 T0 T1 T2 T3 T4LER(cm) Control

MON 87460Monsanto Company 07-CR-191U Page 65 of 544 Figure I-18. Leaf 9 Daily LER durin g the Drought Progression (T1-T4)

T1 LER is an average based on three-day heig ht difference between T1 and T0. The waterlimited treatment initiated at T0. Leaves were measured from the soil to the tip of the leaf. Fifteen plants each of MON 87460 and the cont rol were measured. Water-limited conditions were imposed after the last watering on the morning of May 16 based on pot weight. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

T0

‐ 3 T0

‐ 2 T0

‐ 1 T0 T1 T2 T3 T4LER(cm) Control

MON 87460Monsanto Company 07-CR-191U Page 66 of 544 TIMESTAMP 0 10 20 30 40 50 60LER mV/hr

Figure I-19. Automated LER Data and Plant Temperature for an Approximately 48 Hour Growth Period during Well-Watered Conditions

Blue line is the control, the red line is MON 87460 and the black line is temperature, as indicated by the arrows.

LER measurements were ma de using a draw wire sensor potenti ometer mounted above the plants. The device continuously outputs a range of voltage linearly dependent on the retraction length of each wire. 1 mV/hr = 0.1556 mm/hr. Voltage was sampled every 5 seconds, averaged and logged every minute. Four plants each of MON 87460 and the control were measured. Well-watered conditions were based on pot weight. Control

MON 87460 TemperatureMonsanto Company 07-CR-191U Page 67 of 544LER mV/hr

TIMESTAMP 0 10 20 30 40 50 60

Figure I-20. Automated LER Data and Plant Temperature for an Approximately 48 Hour Growth Period during Water-Limited Conditions

Blue line is the control, the red line is MON 87460 and the black line is temperature, as indicated by the arrows.

LER measurements were ma de using a draw wire sensor potenti ometer mounted above the plants. The device continuously outputs a range of voltage linearly dependent on the retraction length of each wire. 1 mV/hr = 0.1556 mm/hr. Voltage was sampled every 5 seconds, averaged and logged every minute. Four plants each of MON 87460 and the control were measured. Water-limited conditions were based on pot weight. Temperature

MON 87460

ControlMonsanto Company 07-CR-191U Page 68 of 544 Figure I-21. Automated LER Data under Well-Watered and Water-Limited

Conditions for 5- and 15-min Windows

LER measurements were ma de using a draw wire sensor potenti ometer mounted above the plants. The device continuously outputs a range of voltage linearly dependent on the retraction length of each wire. 1 mV/hr = 0.1556 mm/hr. Voltage was sampled every 5 seconds, averaged and logged every minute. LER was calculated as the difference of mV output over a 5-minute period or a 15- minute period. Four plants each of MON 87460 and the control were measured. Water treatments were based on pot weight. Control

MON 87460

Control

MON 87460 Control

MON 87460 Control

MON 87460Monsanto Company 07-CR-191U Page 69 of 544 Figure I-22. Water Potential of Transgenic CSPB Plants and the Control throughout the Duration of Stress Imposition

The study was conducted using three CSPB-contai ning events and a control. All events

contained the same construct as MON 87460. Entr ies were planted in two row plots with three replications per experiment. Plots were irriga ted weekly to the V8 stage and then water was withheld until the R2 developmental stage. Physiological parameters were assessed on three separate days during the water-limitation treatment cycle initiating at the onset of drought, before visible wilting. Water potential measurements were taken on three plants per plot every two hours between 6 a.m. and 6 p.m. An asterisk i ndicates a statistically significant difference at a p

value of less than 0.100 usi ng an analysis of variance. -16.0

-14.0

-12.0

-10.0

-8.0

-6.0

-4.0

-2.0

0.06:00 AM8:00 AM10:00 AM12:00 PM2:00 PM4:00 PM6:00 PM6:00 AM8:00 AM10:00 AM12:00 PM2:00 PM4:00 PM6:00 PM6:00 AM8:00 AM10:00 AM12:00 PM2:00 PM4:00 PM6:00 PM

June 25 June 30 July 6Total Water Potential (bars) Total Water Potential: CSPB Events

Total Water Potential: Control

* *Monsanto Company 07-CR-191U Page 70 of 544 Figure I-23. Osmotic Potential of Tran sgenic CSPB Plants and the Control throughout the Duration of Stress Imposition

The study was conducted using three CSPB-contai ning events and a control. All events

contained the same construct as MON 87460. Entr ies were planted in two row plots with three replications per experiment. Plots were irriga ted weekly to the V8 stage and then water was withheld until the R2 developmental stage. Physiological parameters were assessed on three separate days during the water-limitation treatment cycle initiating at the onset of drought, before visible wilting. Osmotic potential measurements were taken on three plants per plot every two hours between 6 a.m. and 6 p.m. An asterisk i ndicates a statistically significant difference at a p

value of less than 0.100 usi ng an analysis of variance. -20.0

-18.0

-16.0

-14.0

-12.0

-10.0

-8.0

-6.0

-4.0

-2.0

0.06:00 AM8:00 AM10:00 AM12:00 PM2:00 PM4:00 PM6:00 PM6:00 AM8:00 AM10:00 AM12:00 PM2:00 PM4:00 PM6:00 PM6:00 AM8:00 AM10:00 AM12:00 PM2:00 PM4:00 PM6:00 PM

June 25 June 30 July 6Osmotic Potential (bars) Osmotic Potential: CSPB Events Osmotic Potential: Control

* * *Monsanto Company 07-CR-191U Page 71 of 544 Figure I-24. Average Abscisic Acid Co ntent in Leaves from Transgenic CSPB Plants and the Control Taken over the Course of Two Days under Water-Limited

Conditions

The study was conducted using three CSPB-containi ng events (identified with codes beginning ‘ZM’) and a control. All events contained th e same construct as MON 87460. All plants were grown for approximately five weeks prior to th e experiment. The water-limited treatment was established based on pot weight. Samples were taken at four time-points over the course of two days, tracking with the onset of stress. One gram leaf samples were taken from 12 control plants and six replicates for each of the two transgenic entries at each time-point. For each plant, two samples were taken from two different leaves (young leaves and old leaves). 0 1000 2000 3000 4000 5000 6000 7000negZM M38840ZM M39872negZM M38840ZM M39872negZM M38840ZM M39872negZM M38840ZM M39872negZM M38840ZM M39872negZM M38840ZM M39872negZM M38840ZM M39872negZM M38840ZM M39872

old leaf young leaf old leaf young leaf old leaf young leaf old leaf young leaf

T0 (9:30 AM) T1 (3:00 PM) T2 (9:30 AM) T3 (3:00 PM)

February 15 February 16Average ABA Content (ng/g DW)Monsanto Company 07-CR-191U Page 72 of 544 Figure I-25. Leaf Fructose and Glucose Co ntent in Transgenic CSPB Plants and the

Control

The study was conducted using three CSPB-containi ng events (identified with codes beginning ‘ZM’) and a control. All events contained the same construct as MON 87460. Plants were produced under well-watered and water- limited conditions in a greenhouse. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

ZM_M38714 ZM_M38721 ZM_M40946 ZM_M38714 ZM_M38721 ZM_M40946

Well-watered Water-limitedSugar Content (mmoles/gram FW)

Fructose Content: Control Fructose Content: Test Glucose Content: Control Glucose Content: TestMonsanto Company 07-CR-191U Page 73 of 544 Figure I-26. Leaf Raffinose and Inositol Content in Transgenic CSPB Plants and

the Control

The study was conducted using three CSPB-containi ng events (identified with codes beginning ‘ZM’) and a control. All events contained the same construct as MON 87460. Plants were produced under well-watered and water- limited conditions in a greenhouse. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

ZM_M38714 ZM_M38721 ZM_M40946 ZM_M38714 ZM_M38721 ZM_M40946

Well-watered Water-limitedSugar Content (mmoles/gram FW)

Raffinose Content: Control Raffinose Content: Test Inositol Content: Control Inositol Content: TestMonsanto Company 07-CR-191U Page 74 of 544 Figure I-27. Choline and Proline of Tr ansgenic CSPB Plants and the Control throughout the Duration of Stress Imposition

The study was conducted using MON 87460, five ot her events (identified with codes beginning ‘ZM’) containing the same construct as MON 87460 and a control. All plants were subjected to a 12 day period of moderate stress based on pot weight in the greenhouse. Six leaf punches from 12 plants per entry were collected 12 days after the initiation of stress. Samples were analyzed for choline and proline. Prior to sample extr action, deuterated homologues (d9 choline and d7 proline) were added as internal standards to contro l for extraction efficiency. Data are reported as the ratio of the analyte recovered relative to the recovery of the corresponding internal standard. 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

MON 87460

Control ZM_M 38245 ZM_M 38705 ZM_M 38727 ZM_M 38835 ZM_M 38840 ZM_M 38862Relative Metabolite Ratios(endogenous metabolite/internal standard) Relative choline levels Relative proline levelsMonsanto Company 07-CR-191U Page 75 of 544 Figure I-28. Relative Glycine Betaine Leve ls of Transgenic CSPB Plants and the Control throughout the Dura tion of Stress Imposition

The study was conducted using MON 87460, five ot her events (identified with codes beginning ‘ZM’) containing the same construct as MON 87460 and a control. All plants were subjected to a 12 day period of moderate stress based on pot weight in the greenhouse. Six leaf punches from 12 plants per entry were collected 12 days after the initiation of stress. Samples were analyzed for glycine betaine. Prior to sample extraction, the deuterated homologue (d9 glycine betaine)

was added as an internal standard to control fo r extraction efficiency. Data are reported as the ratio of the analyte recovered relative to the recovery of the internal standard. 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50

MON 87460

Control ZM_M 38245 ZM_M 38705 ZM_M 38727 ZM_M 38835 ZM_M 38840 ZM_M 38862Relative Metabolite Ratios(endogenous metabolite/internal standard) Relative glycine betaine levelsMonsanto Company 07-CR-191U Page 76 of 544 I.E. Adoption, Estimated Use, and Global Trade Flows of MON 87460

As described previously (Sect ion I.B.2), growers are expe cted to adopt MON 87460 in regions suitable for corn production that are most prone to frequent drought stress. In the U.S., the major area of adoption is likely to be the Western Dryland region of the Corn Belt known as the Great Plains, which cont ributes 25% of the U.S. corn production (Riebsame, 1990). Other corn producing geog raphies with similar conditions, such as parts of Africa, Europe, and Latin America, may also benefit from this technology. The successful adoption of MON 87460 is expected to increase economic and environmental benefits to producers and consumers due to the protection of corn yields under waterlimited conditions. Increased corn yield per ac re will help conserve the total number of acres needed to meet the needs for food, fee d, and biofuel uses. It is expected that MON 87460 will enter commerce as part of the normal trade flows of yellow corn and it

will be utilized in the same manner as any other yellow corn.

I.F. Submissions to Other Regulatory Agencies

I.F.1. Submission to the FDA

MON 87460 falls within the scope of the Food and Drug Administration’s (FDA) policy statement concerning regulation of products de rived from new plant varieties, including those produced through genetic engineering. Monsanto has voluntarily initiated and will complete a consultation process with FDA prior to commercial distribution of this product. A safety and nutr itional assessment of food and feed derived from MON 87460 was submitted to the FDA dated December 19, 2008.

I.F.2. Submissions to Foreign Government Agencies

Regulatory submissions for im port and production approvals wi ll be made to countries that import U.S. corn grain and have regulatory approval processes in place. These will

include submissions to a number of forei gn government regulatory agencies, including Japan’s Ministry of Agricultu re, Forestry and Fisheries (M AFF) and the Ministry of Health, Labor and Welfare (MHLW); the Cana dian Food Inspection Agency (CFIA) and Health Canada; the Health Ministry of Mexico (HMM); the European Food Safety Authority (EFSA), and the regulatory au thorities in many ot her countries. As appropriate, notifications of im port will be made to importing countries that do not have a formal approval process.Monsanto Company 07-CR-191U Page 77 of 544 I.G. References

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Andrade, F. H., L. Echarte, R. Rizzalli, A. Della Maggiora, and M. Casanovas. 2002. Kernel number prediction in maize under nitrog en or water stress. Crop Science.

42:1173-1179.

Barker, T., H. Campos, M. Cooper, D. Dolan, G. Edmeades, J. Habben, J. Schlusser, D. Wright, and C. Zinselmeier. 2005. Improvi ng Drought Tolerance in Maize. Pages 173-253 in Plant Breeding Reviews. Vol 25, J. Janick, (ed.) John Wiley and Sons, Inc., Hoboken, New Jersey.

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Campos, H., M. Cooper, G.O. Edmeades, C. Loffler, J.R. Schussl er, and M. Ibanez. 2006. Changes in drought tolerance in ma ize associated with fifty years of breeding for yield in the U.S. Corn Belt. Maydica. 51:369-381.

Castiglioni, P., D. Warner, R.J. Bensen, D. C. Anstrom, J. Harrison, M. Stoecker, M. Abad, G. Kumar, S. Salvador, R. D’Ordine , S. Navarro, S. Back, M. Fernandes, J. Targolli, S. Dasgupta, C. Bonin, M.H. Luethy, and J.E. Heard. 2008. Bacterial RNA chaperones confer abiotic stress to lerance in plants and improved grain yield in maize under water-limited conditions. Plant Physiology. 147:446-455.

Chaikam, V., and Karlson, D. 2008. Functio nal characterization of two cold shock domain proteins from Oryza sativa . Plant Cell and Environment. 31: 995-1006.

Chenu K, Chapman SC, Hammer GL, McLean G, Salah HB, Tardieu F. 2008. Shortterm responses of leaf growth rate to water deficit scale up to whole-plant and crop levels: an integrated modeling approach in maize. Plant Cell and

Environment. 31: 378-391.

Claassen, M. M. and R. H. Shaw. 1970. Wa ter deficit effects on corn. II. Grain components. Agronomy Journal. 62: 652-655.

Cristofari, G., and J.L. Darlix. 2002. The ubi quitous nature of RNA chaperone proteins. Progress in Nucleic Acid Resear ch and Molecular Biology. Vol 72 72:223-268.

EFSA. 2007. Statement of the Scientific Pane l on Genetically Modified Organisms on the

safe use of the npt II antibiotic resistance marker gene in genetically modified plants. European Food Safety Authority. Brussels, Belgium http://www.efsa.europa.eu.Monsanto Company 07-CR-191U Page 78 of 544 Etchegaray, J.P., Jones, P.G., and Inouye, M. 1996. Differential thermoregulation of two highly homologous cold shock genes, cspA and cspB , of Escherichia coli . Genes

to Cells. 1:171-178.

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Antibiotic Resistance Marker Genes in Transgenic Plants (Draft Guidance released September 4, 1998).

Flavell, R.B., Dart, E., Fuchs, R.L., and Fral ey, R.T. 1992. Selectable marker genes: safe for plants? BioTechnology. New York. 10:141-144.

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31:1349-1360.

Fuchs, R.L., R.A. Heeren, M.E. Gustaf son, G.J. Rogan, D.E. Bartnicki, R.M. Leimgruber, R.F. Finn, A. Hershman, a nd S.A. Berberich. 1993a. Purification and

characterization of microbi ally expressed neomycin phosphotransferase II (NPTII) protein and its equivalence to the plant expressed protein. BioTechnology. 11:1537-1542.

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Karlson, D., and Imai, R. 2003. Conservation of the cold shock domain protein family in plants. Plant Physiology. 131:12-15

Karlson, D., K. Nakaminami, T. Toyomasu, and R. Imai. 2002. A cold-regulated nucleic acid-binding protein of winter wheat shar es a domain with bacterial cold shock proteins. Journal of Biological Chemistry. 277:35248-35256.

Kim, J.Y., S.J. Park, B.S. Jang, C.H. Jung, S. J. Ahn, C.H. Goh, K. Cho, O. Han, and H.S. Kang. 2007. Functional characterization of a glycine-rich R NA-binding protein 2 in Arabidopsis thaliana under abiotic stress conditions. Plant Journal. 50:439-451.Monsanto Company 07-CR-191U Page 79 of 544 Mascarenhas, J., Weber, M.H., and Graumann, P.L. 2001. Specific polar localization of ribosomes in Bacillus subtilis depends on active transcription. EMBO Reports.

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water deficit. Journal of E xperimental Botany. 58:339 - 349.

Westgate, M.E., M.E. Otegui, and F.H. Andrade. 2004. Physiology of the Corn Plant. in Corn: Origin, History, Technology, and Production, C.W. Smith, J. Betran and E.C.A. Runge, (eds.) John Wiley and Sons, Hoboken, New Jersey.Monsanto Company 07-CR-191U Page 80 of 544 II. The History, Biology, and Global Use of Corn

This section summarizes the history, biology, and global use of corn based on: (1) the consensus document on the biology of corn ( Zea mays L.) developed as part of the Organization for Economic Co-operation and Development (OECD, 2003a); (2) a summary prepared by the Biotechnology Regul atory Services of the United States Department of Agriculture-Animal and Plan t Health Inspection Service (USDA-APHIS, 2009); (3) information provided in the USDA petition for MON 89034 (Monsanto Company petition No. 06-CR-166U); a nd (4) other published literature.

II.A. Introduction

Corn is a versatile crop that provides food, feed, and fuel to a global economy. Recently, a surge in demand for corn has been created by growing economies in the developing

world and its use as an alternative fuel so urce in the developed world. These demands are exceeding production, leading to diminished grain reserves worldwide. In addition, climate change may have variable impact s on crop yields, potentia lly creating further supply disruptions. The combination of th ese factors places a premium on corn yield stability in sub-optimal environments.

Drought stress is the major cause of yield reduction in corn and its effects have far reaching global socio-economic implications. In North America alone, it is estimated that 40% of annual crop losses are due to su b-optimal water availabi lity (Boyer, 1982). Consequently, increasing drought tolerance in corn is a major goal of agricultural breeding and biotechnology. Advances in breed ing and agronomic practices have made significant contributions to increasing corn yield potential and drought tolerance. Biotechnology provides a dditional tools that can be used in combination with breeding and agronomic practices to improve productivity.

Monsanto has developed drought tolerant co rn MON 87460 that pr ovides reduced yield loss under water-limited conditions compared to conventional corn. Efficacy in MON 87460 is derived by e xpression of the inserted Bacillus subtilis cold shock protein B (CSPB). Understanding th e applications, benefits, a nd function of MON 87460 in meeting the global demands on corn production requires an understanding of corn’s origins and history, its biol ogy and cultivation, and its im portance to global trade. A summary of the history, taxonomy, and biology of corn, including gene flow between cultivated corn and its wild relatives, a nd global use of corn is presented below.

II.B. History of Corn

From its likely origin as a wild grass, corn has undergone continuous breeding, modification, and selection for properties th at suit the needs of consumers and growers who wish to produce corn grain. Corn origin ated in the highlands of Mexico 7,000 to 10,000 years ago. European contact introduced corn to the rest of the world and has

allowed it to become an essential cr op for food, feed, and fuel (Goodman, 1988).

One of the most striking differences between cultivated corn and its wild relatives is the obvious emphasis that millennia of breeding and selection efforts have placed on grainMonsanto Company 07-CR-191U Page 81 of 544 yield. All modern corn hybrids produce multi -rowed ears containing hundreds of kernels. Corn’s closest wild relative, teosinte, produces a cluster of spikes, each with one or two rows of seeds. The transformation of spikes into a single enclosed ear is one of the key achievements in the development of corn as a crop (Wilkes, 2004). Since the earliest cultivation efforts in Mexico until the early 20 th century, corn existed primarily in the form of open pollinated varieties. The discovery of corn hybridization led to the development of modern-day dent corn, better adaptation to previously adverse environments, and significant yiel d increases (Duvick et al., 2004).

In the 1940s, when hybrid corn began to predom inate, average U.S. yi elds were 30 – 40 bushels/acre (bu/ac, 2.2 metr ic tons/hectare (MT/ha)) (Duvi ck et al., 2004). By 2000, average U.S. yields were over 130 bu/ac (8.6 MT/ha). Adva nces in breeding and the widespread availability of fertilizers and pe sticides made significant contributions to these advancements (Troyer, 2004). Biotec hnology provides additional tools to improve productivity by reducing the inputs needed to control weeds and insects (Hicks and Thomison, 2004; Kaeppler, 2004).

In 2007, herbicide tolerance and insect resistan ce traits were grown on 73% of U.S. corn acres. Combined trait products, with combin ations of herbicide tolerance and insect resistance, were the largest category at 28% of U.S. co rn acres (NCGA, 2008). As a complement to these existing traits, MO N 87460 provides yield stability under waterlimited conditions that otherwise limit plant pe rformance. Increasing demand for corn in the food, feed, and fuel sectors places a pr emium on technologies that stabilize yield and allow more corn to be produced on existing acres.

II.C. Taxonomy and Phylogenetics

Several hypotheses exist on the origin of corn but the preponderance of evidence supports the hypothesis that corn descended from teosinte (Galinat, 1988). The teosinte genome is similar to corn; teosin te easily crosses with corn, and teosinte has several morphological traits similar to corn. Teosinte has a more weedy appearance and more tillers than modern corn hybrids. The one major distingu ishing difference between corn and teosinte is the female inflorescence, or ear. Modern corn hybrids have one to three lateral

branches that terminate in an ear with 8 to 24 rows of kernels that are enclosed in

modified leaves or husks. Teosinte also ha s lateral branches, but they terminate in tworowed spikes of perhaps 12 fruit cases, with each fruit case having one seed enclosed by an indurated glume (Goodman and Brown, 1988).

Corn ( Zea mays L.) is a member of the tribe Maydae, which is included in the subfamily Panicoideae of the grass family Gramineae. Table II-1 summarizes the taxonomic

classification of corn a nd its close relatives.Monsanto Company 07-CR-191U Page 82 of 544 Table II-1. Taxonomic Classification of Corn and its Close Relatives

Family - Gramineae Subfamily - Panicoideae

Tribe - Maydae Western Hemisphere:

I. Genus - Zea A. Subgenus - Luxuriantes

1. Zea luxurians (2n = 20)

2. Zea perennis (2n = 40)

3. Zea diploperennis (2n = 20) B. Subgenus - Zea

1. Zea mays (2n = 20) ( corn) Subspecies

1. Z. mays parviglumis (2n = 20)

2. Z. mays huehuetenangensis (2n = 20)

3. Z. mays mexicana (Schrad.) (2n = 20) ( teosinte )

II. Genus – Tripsacum

A. Section – Tripsacum B. Section – Fasciculata Species Species

1. T. andersomii (2n = 64) 1. T. jalapense (2n = 72)

2. T. australe (2n = 36) 2. T. lanceolatum (2n = 72)

Varieties 3. T. fasciculatum (2n = 36) a) T. australe var. australe 4. T. maizar (2n = 36, 72) b) T. australe var. hirstum 5. T. pilosum (2n = 72)

3. T. bravum (2n = 36, 72) Varieties

4. T. cundinamarce (2n = 36) a) T. pilosum var. guatemalense

5. T. dactyloides (2n = 72) b) T. pilosum var. pilosum

Varieties a) T. dactyloides var. hispidum b) T. dactyloides var. dactyloides c) T. dactyloides var. meridonale d) T. dactyloides var. mexicanum

6. T. floridanum (2n = 36)

7. T. intermedium (2n = 72

8. T. manisuroides (2n = 72)

9. T. latifolium (2n = 36)

10. T. percuvianum (2n = 72, 90, 108)

11. T. zopilotense (2n = 36, 72)

Asia:

I. Genera—

Chionachne (2n = 20) Schlerachne (2n = 20)

Coix (2n = 10, 20) Trilobachne (2n = 20) Polytoca (2n = 20)

Tribe— Andropogoneae

I. Genus - ManisurisMonsanto Company 07-CR-191U Page 83 of 544 The genera included in the tribe Maydae include Zea and Tripsacum in the Western Hemisphere, and Coix

, Polytoca

, Chionachne

, Schlerachne , and Trilobachne in Asia. Although some researchers have implicated the Asian genera in the origin of corn, the evidence for them is not as extensive and convincing as for the genera located in the Western Hemisphere.

The genus Zea includes two sub-genera: Luxuriantes and Zea . Corn ( Zea mays L.) is a separate species within the subgenus Zea, along with three subspeci es. All species within the genus Zea , except corn, are different species of te osinte. Until recently, the teosinte species were included in the genus Euchlaena rather than the genus Zea

.

II.D. Biology of Corn and Environmental Effects on its Development

II.D.1. Genetics

Corn is genetically one of the best developed and best charac terized of the higher plants. Because of the separation of male and female inflorescence, number of seeds produced on the female inflorescence, ease in handlin g (growing and hand polli nating), nature of the chromosomes, and low basic chromosome number (n = 10), corn has been accessible for study at all levels of genetics.

Corn was one of the first crop species studied in genetics laborator ies to obtain a basic understanding of mitosis, meiosis, chromo some segregation, linkage and effects of crossing-over, and transposable elements. B ecause of the importance of corn in the U.S. and world economies, and the genetic information obtained since 1900, corn has continued to receive extensive study in modern genetics laboratories.

A key concept in breeding is quantitative tr ait loci (QTLs). QTLs are regions of chromosomes that are known to play a role in imparting physiological traits of interest and represent a significant ar ea of advancement in modern crop breeding. Identifying QTLs of interest and combining them into a single line of germplasm can produce progeny with increased yield potential and tolerance to multiple stresses (Takeda and Matsuoka, 2008). Corn has QTLs related to flowering synchrony under drought stress on chromosomes 1, 2, 5, 6, 8, and 10. Physiological traits associated with tolerance of other stresses are also controlled by multiple Q TLs, indicating that stress responses are multigenic traits rather than the effect of a single gene or pathway (Chinnusamy et al., 2005). A key challenge for relying on QTLs to impart abiotic stre ss tolerance is the difficulty in imposing targeted stresses under field conditions to select for desirable phenotypes (Takeda and Matsuoka, 2008). In a ddition, selecting for a particular trait through QTLs or other approaches, even when the gene has been identified, does not necessarily mean the exact function of the gene is known (Takeda and Matsuoka, 2008).

II.D.2. Growth and Development

Corn development is measured in vegetative (V) and reproductive (R) stages (Figure II1). The V number corresponds to the number of leaves with a visibl e collar or ligule. Leaves initiate at the growing point, or meristem, which in corn and other grasses is at the base of the plant. At V5, ear shoot formati on is complete and all le aves have initiated.Monsanto Company 07-CR-191U Page 84 of 544 Until V6, the growing point of the plant is below ground. Beginning at V10, corn plants accumulate a reserve of nutrients and dry weight to support reproductive development and grain production. V stages proceed thro ugh VT (tassel emergence or anthesis) and the R stages begin at silking (R1). R2 and R3 represent early to mid grainfill while R4 and R5 represent mid to late grainfill. R6 is physiological maturity (Hanway, 1966).

Figure II-1. Growth Stages of Corn from Early Vegetative (V4) through Late Grainfill (R5)

Corn yield is driven by both kernel numbe r and kernel weight. Kernel number is a function of how many ovules are fertilized and supported through maturity and is the primary determinant of overall yield (Westgat e et al., 2004). Kernel weight is a function of the amount of dry matter available to support kernel development and the amount of water available during grainfill. Harvest inde x (HI) is the ratio of grain biomass to shoot biomass and is used to quantify the relationshi p between sink tissue and source tissue. In modern hybrids HI is approximately 0.5 (W estgate et al., 2004). The amount of shoot

biomass available at the end of the season to support kernel developm ent is a function of the amount of photosynthetically active radiation (PAR) the plan t is able to intercept.

This in turn is a function of the amount of leaf area exposed to sunlight, which is determined by the rate of leaf expansion. Sufficiently severe, a dverse conditions during any growth stage can limit plant growth and therefore limit yield (Westgate et al., 2004).

II.D.3. Environmental Effects on Growth and Development

Corn has been adapted to a wide geographi cal range, but its growth, development, and yield potential are still cl osely related to environm ental conditions, including temperature, nutrients, and water. Soil temper atures must be above 10 °C for corn seed to germinate. Corn cannot survive temperatures below 0 ° C for more than six to eight hours after the growing point is above ground, which occurs at V6. Likewise, soil moisture must be sufficient for germination to occur. Once plants emerge, nutrient supplies, particularly nitrogen must be suffici ent to allow rapid growth. Water limitation during these growth stages can impair growth, but the effects are often reversible (Barker et al., 2005). Until V6, cold soil temperatur es will adversely effect corn growth and development. As canopy closure begins in the V6 growth stage, high plant density (moreMonsanto Company 07-CR-191U Page 85 of 544 than 30,000 plants per acre) can limit PAR inte rception, reducing growth rate (Westgate et al., 2004) and limiting the supply of photosynthate. Shading caused by high planting

densities also reduces the amount of availa ble photosynthate, reducin g the ability of a plant to support developing ke rnels (Setter et al., 2001). As corn reaches reproductive stages (VT-R1), periods of extended high temperatures can reduce pollen viability (Schoper et al., 1987) and yield.

Among all of the environmental stressors that affect corn development and yield, water availability is by far the most important. In North America alone, it is estimated that 40% of annual crop losses are due to sub-optimal water availab ility (Boyer, 1 982). As a C4 plant, corn is inherently more water use effi cient and transpires only half as much water as C3 plants (Taiz, 1991), e.g., rice, soybea n, and cotton. Despite this key advantage, water limitation still adversely impacts corn growth, development, and yield, particularly during reproductive growth stages. In genera l, water limitation cause s plants to lose turgor pressure and stomata close to limit water loss. Water limitation also causes corn leaves to roll, or curl upon themselves, reducing surface area, decreasing photosynthesis and, ultimately, growth. Water limitation mani fests itself visually in corn as reduced plant height and yield. Lim iting water during later reproductiv e growth stages results in scorching and leaf firing (Hsia o, 1973; Westgate et al., 2004).

The flowering growth stages (V T – R1) are a particularly vuln erable period for corn plant development and the resulting yields. Four da ys of visible wilting in the growth stages just prior to VT can reduce corn yields by up to 25 percent. Four days of visible wilting between the final V stages and R2 can re duce yield by 50 percent (McWilliams, 2002). An important mechanism by which drought reduces corn yield is by preventing synchronous flowering. Optimal yields depend upon silking (R1) occu rring within a day of anthesis (VT). Synchrony is measured as the anthesis-silking interval (ASI). As ASI increases, silks emerge too late to intercept pollen and yields decrease dramatically. As silks are dependent upon a sufficient supply of water to extrude at the correct time, water limitation is a primary cause of large ASI valu es (Westgate et al., 2004 ). Silks are more sensitive to low water potential than leaves or roots, low water potential being associated with poor solute accumulation and an inabil ity to retain turgor (Maiti and WescheEbeling, 1998). Failure to intercept pollen is not the sole cause of poor kernel number, however. Abortion of fertilized kernels may al so be a significant factor in yield loss under water limitation (Westgate et al., 2004). Water limitati on during late vegetative stages through flowering will reduce kernel number and increase the number of plants without ears (barrenness). Water limitation during grain filling wi ll result in smaller kernels (Campos et al., 2006). Kernels will not accumulate dry matter once their

moisture level falls below 30%.

Breeders have sought to improve drought tolerance in corn for decades. Breeding under water-limited conditions has led to improved germplasm that is ab le to produce better yields than non-drought tolerant hybrids. The primary mechanism for improvement was by selection for synchronous flowering. Br eeders conducted this selection simply by self-pollinating plants under drought stress. Those plants that were silking while pollen was being shed could be selfed, in effect sel ecting for plants that were able to maintainMonsanto Company 07-CR-191U Page 86 of 544 normal function under drought stress. A retros pective study of advances in drought tolerance over 50 year s found that the greatest breeding yield gains were obtained under flowering stress at slightly more than 0.1 MT/ha/year. These breeding advances also improved yields under optimal conditions, alth ough sensitivity to mid- to late-grainfill stress was also increased (Campos et al., 2006).

II.D.4. Cultivation

Corn is currently grown on over 150 million hectares globally. In 2007-2008 the U.S., China, Brazil, the European Union, and Me xico were the top five producers (USDAFAS, 2008). Crop inputs play a significant role in determining yield and will vary based on climate and soil type. Water is a key i nput with 50-60 cm of water needed under temperate conditions. Corn rapidly depl etes nitrogen and phos phorous and these essential nutrients must be replenished with each crop. Insect pressure and competition

from weeds are also central concerns in corn management and must be managed to minimize yield losses (Olson and Sander, 1988) . As with crop input s, the appropriate choice of hybrid is essential for optimal yield and will vary with planting location.

Hybridization is a f undamental concept used in globa l corn breeding and production. Corn was originally developed as an open-pol linated (cross-fertilizi ng) crop species, and until the beginning of the 20 th century, only open-pollinated corn varieties were grown. The fundamental concepts for development of hybrid corn were defined by 1920 (Sprague, 1946). Continuous selfing of indi viduals for multiple generations results in pure (inbred) lines within which every plant has similar traits. In these inbred lines, heterozygous loci are eliminat ed by inbreeding to homozygous loci of either one of the

two alleles at each locus. The fixation of alleles in pure lines causes a general reduction in vigor and productivity. Crossing two inbred lines restores vigor (h eterosis). Selecting for inbred lines and crosses that possess desira ble traits forms the basis of modern corn breeding. Recent techniques such as marker-a ssisted breeding have reduced the time and number of generations required to pr oduce pure inbreds (Yousef and Juvik, 2001).

Since 1996, biotechnology has provided growers with an array of traits to further improve yields by reducing competition from weeds and insects. Herbicide tolerance and insect resistance traits increase productivity wh ile decreasing inputs (Brookes and Barfoot, 2008; Hicks and Thomison, 2004; Kaeppler, 2004). Globally, between 1996 and 2006,

herbicide tolerant corn allowe d a 3.9% reduction in the use of herbicide active ingredients

and insect resistant corn al lowed a 5.0% reduction in th e use of insecticide active ingredients. Notably, over this same peri od, an additional $1.1 bil lion and an additional $3.6 billion of global farm income can be attrib uted to the adoption of herbicide tolerant and insect resistant corn, resp ectively. Improved yields are responsible for 43% of these economic gains (Brookes and Barfoot, 2008).

Rotation with other crops is t ypically advantageous for corn yield. Rotation benefits corn production by allowing alternate weed, insect and disease management strategies and improving soil structure (Hicks and Thomison, 2004) . In the U.S. Corn Belt, soybean is a common rotational crop although so rghum is also used. In the Western Dryland region, wheat, sorghum and soybean may be plante d in rotation with corn. Wheat-fallowMonsanto Company 07-CR-191U Page 87 of 544 rotation is also used to store rainfall during the fallow se ason. Recent research has shown that wheat-fallow rotations may actually reduc e the amount of soil mo isture available to wheat crops because fallow season management re quires herbicides or tillage, the cost of which can exceed profits from the subsequent wheat crop. In order to maximize the return on any stored water, a more intensive crop such as corn, sorghum or millet should be grown in rotation with wh eat (Croissant et al., 1998).

II.D.5. Potential Gene Transfer

II.D.5.1. Outcrossing with Wild Zea and Tripsacum Species

Gene flow between corn and its closest relative, teosinte ( Z. mays mexicana Schrad.) is specific to Mexico and Central America. In the Central Plateau and Valley of Mexico, corn can grow sympatrically with teosinte providing the opportunity for hybridization (Wilkes, 1967; Sanchez et al., 1998). The geneti c exchange between corn and teosinte is dependant on (1) the spatial isolation of the two species, (2) the seasonal isolation of the two species and (3) the fitness of the hybrid s combined with the types of selection operating in the teosinte populat ions. Wilkes (1967) suggest ed the presence of hybrids

under natural conditions in Central Plateau and Valley of Mexico as an unequivocal indication of cross compatibili ty between corn and teosinte . Baltazar et al. (2005) and Ellstrand et al. (2007) pres ented further evidence of hybr idization between corn and teosinte. Hybrid and open-pollinated corn ears produced a mean of 8 and 11 seeds per ear, respectively, when hand-pollinated with teosinte pollen, whic h is approximately 1– 2% of the ovules normally produced on a hybr id corn ear (Balta zar et al., 2005). Hybridization in the other dir ection is difficult but teosinte ears can produce up to 0.2–0.3 seeds per ear when pollinated with corn pol len (Baltazar et al., 2005; Ellstrand et al., 2007).

With the exception of Tripsacum floridanum , it is difficult to cross Tripsacum with corn, and the offspring of the cross show varying levels of sterility. Tripsacum -corn hybrids have not been observed in the field, and Tripsacum -teosinte hybrids have not been produced (Wilkes, 1972).

II.D.5.2. Outcrossing with Cu ltivated Zea Varieties

Gene flow in corn is closely associated w ith the biology of the st aminate and pistillate

inflorescences. Corn is a cr oss- and wind-pollinated crop th at produces pollen in copious quantities. A hybrid tassel of normal si ze can produce up to 25 million pollen grains (Kiesselbach, 1999). Corn pollen has a m ean diameter of approximately 100-106 microns (Rodriguez et al., 2006). Dispersal of corn pollen is determined by a diversity of environmental and physical factors. Wind dir ection, turbulence and ve locity are directly linked to pollen movement (Jones and Brook s, 1950; Di-Giovanni and Kevan, 1991). Other factors such as pollen density, air density and viscosity, pollen sedimentation velocity, and pollen radius seem to influen ce pollen transport and deposition (Paterniani and Stort, 1974; Di-Giovanni et al., 1995; Aylor, 2002). On ce in the atmosphere, pollen grains must remain viable long enough to be ab le to reach a viable silk to complete the pollination process. On average, corn pol len loses 100% viability after two hours of atmospheric exposure (Luna et al., 2001; Aylor, 2003).Monsanto Company 07-CR-191U Page 88 of 544 II.D.5.3. Survival Capacity and Weediness of Corn

Modern-day corn cannot survive outside of cultivation (Gould, 1968). Volunteer corn is not typically observed growing in fence ro ws, ditches, and roadsides as a weed. Although corn from the previous crop year can overwinter and germinate the following year, it cannot persist as a weed. The appearan ce of corn in soybean fields following the corn crop from the previous year is a common occurrence. Measures often are taken to eliminate either the plants with a hoe or to use herbicides to kill the corn plants in soybean fields, but the plants that remain and produce seed usually do not persist in the following years.

It is difficult for corn to survive as a weed because of past selecti on in the evolution of corn. In contrast with weedy plants, corn ha s a polystichous female inflorescence (or ear) on a stiff central spike (or cob) enclosed w ith husks (modified leaves). Consequently, seed dispersal of individual kernels does not occur naturally because of the structure of

the ears of corn. Individual kernels of cor n, however, can be distributed during grain harvest and transportation to storage facilities. In neither instance (natural or mechanical harvesting) does corn become a weed. Corn cannot surviv e without human assistance and is not capable of surviving as a weed.

II.D.6. Natural Habitat of Corn and Significant Ecological Interactions

As corn is the product of domestication it does not have a natural habi tat. Corn’s closest wild relative, teosinte, is native to Mexico a nd Guatemala. Teosinte prefers areas that are seasonally dry and receive summer rain (Gonzalez and Corral, 1997; Wilkes, 1972).

Water requirements for corn growth vary over the season, reaching a peak at VT. Prior to VT, corn requires approximately 0.1 inches of water per day (0.25 cm/day). From VT to R3, corn requires approximately 0.35 inches per day (0.9 cm/day) or about 7 inches (18 cm) total. (McWilliams, 2002). While modern-day corn hybrids have been developed through breeding and biotechnolo gy to increase drought tole rance, this basic water requirement has not been surpassed and will not allow corn to be grow n or persist outside of current corn growing regions

Corn is the target of a variety of microbial pa thogens and insect pests. Rusts, smuts, leaf blights, and stalk rots are among the more co mmon diseases of microbial origin. Their prevalence and economic importance vary fr om country to country (Smith and White, 1988). Fungi such as Aspergillus sp. and Fusarium sp. produce mycotoxins that can adversely impact humans and livestock that consume contaminated grain. Insect damage and abiotic stresses such as drought ca n exacerbate fungal in fections (Dowd, 2001).

The primary insect pests of corn belong to the orders Lepidoptera and Coleoptera. Lepidopterans feed on leaves and stalks as larvae and ears as adults. Examples include the European corn borer ( Ostrinia nubialis ), the Asian corn borer ( O. furnacalis ), the spotted stem borer ( Chillo partellus ), fall army worm ( Spodoptera frugiperda ), and members of the genus Diatrea . Coleopterans feed on roots and stalks as larvae and silks, pollen, and leaves as adults. Examples include the northern corn rootworm ( DiabroticaMonsanto Company 07-CR-191U Page 89 of 544 barberi ), the western corn rootworm ( D. vergifera vergifera ), and the southern corn rootworm ( D. undecimpunctata ) (Dicke and Guthrie, 1988).

II.D.7. Corn as a Test System in this Petition

The transformation for MON 87460 was conducte d with LH59, an inbred corn line. A single transformed plant was then self-crossed to increase se ed supplies. A homozygous inbred line was developed though self-crossi ng and selection, and th en used to produce other lines used for product testing, regul atory studies, and co mmercial production.

II.E. Global Use of Corn

Corn is used globally for food, feed, and fuel , and in recent years th e demand for corn has increased, leading to higher prices for raw grain and its derivatives (NCGA, 2008). OECD-FAO’s joint 2008-2017 Agricultural Outl ook forecasted that corn prices will remain 40-60% higher in the next decade than they have been for the last decade. The report also concluded that increased yields on existing agricultural land will be more important to improving commodity supplies than bringing new land into cultivation. In developing countries, economic growth, cha nging diets, and growing populations are driving added demand. In developed countries, fuel uses are the largest source of new demand. These factors along with diminished stocks and climate change will lead to variability in agricultural pr oduct supply and possibly result in price spikes (OECD-FAO, 2008).

Food uses include sweet corn, popcorn, and processed field co rn, which are all hybrids of Zea mays . Of the corn used for food and industria l uses, the majority is processed by wet milling to produce starch and sweetener products (e.g., high fructose corn syrup) for use in foodstuffs. Non-food products such as indu strial starches, corn gluten feed and corn gluten meal are also manufactured thr ough the wet mill process (May, 1987; Watson, 1988). The primary products derived from the dry milling process are corn meal, corn flour, and ethanol.

Because of its high starch content, corn is used as a valuable energy source in animal feed for domestic livestock, such as cattle, pigs and poultry. This star ch content is also amenable to fermentation, providing ethanol for use as fuel. Whole corn is usually ground and mixed with a high-protein feed compound and with vitamin and mineral supplements to balance the ration according to the nutritional requirements of the animals being fed (Leath and Hill, 1987). Corn is al so used for processing and the production of derivatives, which have a wide range of food, feed and industrial applications. Some of the processed fractions are used for animal f eed, such as corn gluten, a resource that is rich in corn protein. Corn is also used for the production of feed additives. Ethanol production from the dry mill process provides dried distiller’s gr ain solubles (DDGS) which are another source of animal feed (RFA, 2008).

II.E.1. Historical Uses of Corn a nd its Major Processed Products

Corn and its processed products have been in use for at least the last 7,000 years. The earliest uses were likely as flour or dough. This continues to be the primary use inMonsanto Company 07-CR-191U Page 90 of 544 developing countries throughout Latin America and Africa. In industrialized countries and developing Asian countries, the primary us e for corn grain is animal feed although ethanol production has been rising stead ily (NCGA, 2008; OECD, 2003b; OECD-FAO, 2008).

II.E.2. Global Cultivation and Trade Flow s for Corn and its Major Processed

Products

From 2003 to 2007, worldwide corn grain prod uction averaged 702 million metric tons (MMT) per year. During this same period, the top corn grain producers were the U.S., China, the European Union (EU), Brazil, and Mexico, accounting for 74% of average annual global corn production. Also during this period, corn produc tion trended upwards

from 627 MMT in 2003 to 769 MMT in 2007 (USDA-FAS, 2008). During 2000 to 2005, the latest period for which data are availabl e, silage and forage production averaged 372 MMT and corn oil production averaged 2 MMT per year. (FAOSTAT, 2007).

From 2003 to 2007, the top corn grain export ers were the United States, Argentina, Brazil, China, and Ukraine. Together, th ese countries accounted for 93% of average annual exports (USDA-FAS, 2008). The majo r exported processed corn products are gluten feed, flour, oil, bran, and starch. In recent years, annual exports of DDGS from the U.S. have increased significantly fr om approximately 0.7 MMT to just under 1.8 MMT, second only to gluten feed in terms of processed co rn product exports from the U.S. (ProExporter, 2008). Annual expor ts increased between 2003 and 2007 from 79 MMT to 92 MMT. From 2003 to 2007, the top fi ve grain importers were Japan, Korea, Mexico, EU and Egypt, accounting for 51% of average annual imports (FAOSTAT, 2007).

Between 1997 and 2007, global de mand for cereals (coarse grains, wheat and rice) was equal to or greater th an production (FAO, 2008). This ha s led to reduced overall cereal supply with stocks at a 25 year low. Prices of cereals have increased globally, with U.S. corn prices rising 38% betw een 2007 and 2008. Similarly, wheat and rice prices have doubled in some markets. For corn, the pri ce change is largely the result of increased demand while for the other cereals, particular ly wheat, price increases are the result of both increased demand and reduced supply. The reduced wheat supply is the result of persistent drought in wheat production areas such as Australia (FAO, 2008).

In the U.S., demand for corn is driven by th e demand for feed and fuel. In 2007 animal feed accounted for over 45% of corn consumption followed by nearly 25% for ethanol, and approximately 10% for food and industrial uses with the remainder being exported (NCGA, 2008). Globally, feed uses account for much of th e demand. Global demand for

animal feed rose 20% between 2007 and 2008. Expanding economies in China, India and Brazil between 1990 and 2006 have led to gr eater consumption of meat and dairy products (von Braun, 2007).

Within this context, the impacts of climate change take on a new ur gency with respect to corn production. In a 2008 report, the U.S. C limate Change Science Program stated that climate change is already impacting agricultur al productivity. The report concluded thatMonsanto Company 07-CR-191U Page 91 of 544 a combination of increased temperature vari ability, altered rainfall patterns and the resulting increases in drought frequency will magnify yiel d variability (CCSP, 2008; Hatfield et al., 2008).

II.F. Conclusions

Corn is a versatile crop that provides food, f eed and fuel. This versatility, coupled with growing economies in the deve loping world and the need to rely on alternative fuel sources in the developed world have led to a surge in demand. These demands are exceeding production, leading to diminished corn grain reserves worldwide. Diminished reserves magnify the impacts of supply disr uptions. Climate change will have variable impacts on crop yields, potentially creating s upply disruptions. The combination of these factors places a premium on corn yield st ability in sub-optimal environments.

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Baltazar, B.M., J.J. Sánchez-Gonzalez, L. De la Cruz-Larios, and J.B. Schoper. 2005.

Pollination between maize and teosinte: an important determinant of gene flow in

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Barker, T., H. Campos, M. Cooper, D. Dolan, G. Edmeades, J. Habben, J. Schlusser, D. Wright, and C. Zinselmeier. 2005. Improvi ng Drought Tolerance in Maize. Pages 173-253 in Plant Breeding Reviews. Vol 25, J. Janick, (ed.) John Wiley and Sons, Inc., Hoboken, New Jersey.

Boyer, J.S. 1982. Plant productivity and environment. Science. 218:443-448.

Brookes, G. and P. Barfoot. 2008. GM Crops: Global Socio-economic and Environmental Impacts 1996-2006. P.G. Economics http://www.pgeconomics.co.uk.

Campos, H., M. Cooper, G.O. Edmeades, C. Loffler, J.R. Schussl er, and M. Ibanez. 2006. Changes in drought tolerance in ma ize associated with fifty years of breeding for yield in the U.S. Corn Belt. Maydica. 51:369-381.

CCSP. 2008. The effects of climate change on agriculture, land resources, water resources, and biodiversity. A Report by the U.S. Climate Change Science Program and the Subcommittee on Globa l Change Research. U.S. Climate Change Science Program and the Subcommittee on Global Change Research., U.S. Environmental Protection Agency, Washington, D.C.

Chinnusamy, V., L. Xiong, and J.K. Zhu. 2005. Use of genetic engineering and molecular biology approaches for crop improvement for stress environments. Pages 47-89 in

Abiotic Stresses: Plant Resistance through Breedi ng and Molecular Approaches, M. Ashraf and P.J.C. Harris, (eds.) Food Products Press, New York, New York.Monsanto Company 07-CR-191U Page 92 of 544 Croissant, R.L., G.A. Pete rson, and D.G. Westfall. 1998. Dryland Cropping Systems. Colorado State University http://www.ext.colostate.edu/.

Dicke, F.F., and W.D. Guthrie. 1988. The most important corn inse cts. Pages 769-880 in Corn and Corn Improvement. 3 rd ed., Sprague, G.F. and J.W. Dudley, (eds.). American Society of Agronomy, Crop Sc ience Society of America and Soil Science Society of America, Madison, WI.

Di-Giovanni, F. and P.G. Kevan. 1991. Fact ors affecting pollen dynamics and its importance to pollen contamination: a review. Canadian Journal of Forest

Research. 21: 1155-1170.

Di-Giovanni, F., P.G. Kevan, and M.E. Nasr. 1995. The variability in settling velocities of some pollen and spores. Grana. 34: 39-44.

Dowd, P.F. 2001. Biotic and abio tic factors limiting efficacy of Bt corn in indirectly reducing mycotoxin levels in commer cial fields. Journal of Economic Entomology. 94:1067-1074.

Duvick, D.N., J.S.C. Smith, and M. Cooper. 2004. Changes in performance, parentage, and genetic diversity of successful co rn hybrids, 1930-2000. in Corn: Origin, History, Technology, and Production, C.W. Smith, J. Betran and E.C.A. Runge, (eds.) John Wiley and Sons, Hoboken, New Jersey.

Ellstrand, C.N., L.C. Garner, S. Hegde , R. Guadagnuolo, and L. Blancas. 2007. Spontaneous hybridization be tween maize and teosinte. Journal of Heredity.

98:183-187.

FAO. 2008. Crop Prospects and Food Situation. Global Information and Early Warning System on Food and Agriculture. No. 2, April 2008.

FAOSTAT. 2007. FAOSTAT Database. http:// faostat.fao.org [Accessed December 15, 2007].

Galinat, W.C. 1988. The origin of corn. Pages 1-31 in Corn and Corn Improvement. 3 rd
ed., Sprague, G.F. and J.W. Dudley, (eds.) . American Society of Agronomy, Crop Science Society of America, and Soil Sc ience Society of America, Madison, WI.

Gonzalez, J., and J. Corral. 1997. Teosinte distribution in Mexico. Proceedings of a forum: gene flow among maize landrac es, improved maize, varieties and Teosinte: implications fo r transgenic maize:18-39.

Goodman, M. M. 1988. The history and evolu tion of maize. CRC Critical Reviews in

Plant Sciences. 7:197-220.

Goodman, M.M., and W.L. Brown. 1988. Races of corn. Pages 33-79 in Corn and Corn Improvement. 3 rd ed., Sprague, G.F. and J.W. D udley, (eds.). American Society of Agronomy, Crop Science Society of Am erica, and Soil Science Society of America, Madison, WI.

Gould, F.W. 1968. Grasses and man. Page 382 in Grass Systematics McGraw Hill, New York, New York.Monsanto Company 07-CR-191U Page 93 of 544 Hanway, J.J. 1966. How a corn plant develops . Special Report. Iowa State university cooperative extension service, Ames, IA 48.

Hatfield, J., K. Boote, P. Fay, L. Hahn, C. Izaurralde, B.A. Kimball, T. Mader, J. Morgan, D. Ort, W. Polley, A. Thomson, and D. Wolfe. 2008. Agriculture. in The Effects of Climate Change on Agriculture , Land Resources, Water Resources, and Biodiversity. A Report by the U.S. Clim ate Change Science Program and the Subcommittee on Global Change Research. U.S. Climate Change Science Program and the Subcommittee on Global Change Research, Washington, D.C.

Hicks, D.R., and P.R. Thomison. 2004. Corn management. in Corn: Origin, History, Technology, and Production, C.W. Smith, J. Betran and E.C.A. Runge, (eds.) John Wiley and Sons, Hoboken, New Jersey.

Hsiao, T.C. 1973. Plant responses to water st ress. Annual Review of Plant Physiology.

24:519-570.

Jones, J.M., and J.S. Brooks. 1950. Effectiv eness and distance of border rows in preventing outcrossing in corn. Oklahom a Agricultural Experiment Station

Technical Bulletin. No. T-38.

Kaeppler, S. 2004. Biotechnology: new horizons. in Corn: Origin, History, Technology, and Production, C.W. Smith, J. Betran a nd E.C.A. Runge, (eds.) John Wiley and Sons, Hoboken, New Jersey.

Kiesselbach, T.A. 1999. The stru cture and reproduction of corn. 50 th Anniversary Edition. Cold Spring Harbor Laboratory Pr ess, Cold Spring Harbor, New York.

Leath, M.N., and L.D. Hill. 1987. Economics of production, marketing and utilization, Pages 210-219 in Corn: Chemistry and Technology., S.A. Watson and R.E. Ramstad, (eds.) American Association of Cereal Chemists, St. Paul, MN.

Luna, V. S., J.M. Figueroa, B.M. Baltazar, R.L. Gomez, R. Townsend and J.B. Schoper 2001. Maize pollen longevity and distance isolation requ irements for effective pollen control. Crop Science. 41:1551-1557.

Maiti, R.K. and P. Wesche-Ebeling. 1998. Maize Science. Pages 132-133. Science Publishers, Inc. Enfield, New Hampshire.

May, J.B. 1987. Wet milling: processes and pr oducts. Pages 377-397 in Corn: Chemistry and Technology, S.A. Watson and P.E. Rams tad, (eds.) American Association of Cereal Chemists, St. Paul, MN.

McWilliams, D. 2002. Drought Strategies for Corn and Grain Sorghum. New Mexico State University http://www.cahe.nmsu.edu.

NCGA. 2008. The World of Corn 2008. Na tional Corn Growers Association http://www.ncga.com/WorldOfCorn/main/index.asp Accessed May 15, 2008.Monsanto Company 07-CR-191U Page 94 of 544 OECD. 2003a. Consensus document on the biology of Zea mays subsp. mays (Maize). Series on Harmonisation of Regul atory Oversight in Biotechnology. ENV/JM/MONO(2003)11, Organization fo r Economic Co-operation and Development, Paris, France http://www.olis.oecd.org/olis/2003d oc.nsf/LinkTo/NT0000426E/$FILE/JT00147 699.PDF Accessed January 9, 2009.

OECD. 2003b. Considerations for the safety assessment of animal feedstuffs derived from genetically modified plants. Series on the Safety of Novel Foods and Feeds. ENV/JM/MONO(2003)10, Organization for Economic Co-operation and Development, Organization for Economic Co-operation and Development, Paris, France http://www.olis.oecd.org/olis/ 2003doc.nsf/LinkTo/env-jm-mono(2003)10 Accessed February 5, 2007.

OECD-FAO. 2008. OECD-FAO Agricultura l Outlook 2008-2017. OECD-FAO Paris, France http://www.agri-outl ook.org/dataoecd/44/18/40713249.pdf.

Olson, R.A., and D.H. Sander. 1988. Corn Production. Pages 639-686 in Corn and Corn Improvement. 3 rd ed., Sprague, G.F. and J.W. Dudl ey, (eds.). American Society of Agronomy, Crop Science Society of Am erica and Soil Science Society of America, Madison, WI.

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Rodriguez, J.G.F., J.J. Sanchez G. Baltazar, M.B. De la Cruz L., Santacruz-Ruvalcaba, E., Ron, J.P. and Schoper, J.B. 2006. Ch aracterization of floral morphology and synchrony among Zea species in Mexico. Maydica. 51: 383-398.

Sanchez, J.J., G., T.A. Kato Y., M. Aguilar san Miguel, J.M. Hernandez C., A. Lopez R.,

and J.A Ruiz C. 1998. Ditrib ucion y caracterizacion del teocintle . Libro Tecnico No. 2 del Instituto Nacional de Investigac iones Forestales, Agricolas y Pecuarias (INIFAP).

Schoper, J. B., R.J. Lambert, B.L. Vasila s, and M.E. Westgate. 1987. Plant factors controlling seed set in maize. Plant Physiology. 83:121-125.

Setter, T.L., B.A. Flannigan, and J. Mel konian. 2001. Loss of kernel set due to water deficit and shade in maize: carbohydrate supplies, abscisic ac id, and cytokinins. Crop Science. 41:1530-1540.

Smith, D.R., and D.G. White. 1988. Diseases of corn. Pages 687-766 in Corn and Corn Improvement. 3 rd ed., Sprague, G.F. and J.W. Dudl ey, (eds.). American Society of Agronomy, Crop Science Society of Am erica and Soil Science Society of America, Madison, WI.Monsanto Company 07-CR-191U Page 95 of 544 Sprague, G.F. 1946. The experimental ba sis for hybrid maize. Biology Review. 21:101-

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41-645-655.Monsanto Company 07-CR-191U Page 96 of 544 III. Description of the Transformation System

III.A. Transformation System

MON 87460 was developed through Agrobacterium -mediated transformation of corn line LH59 embryos and expresses cold shock protein B (CSPB) from Bacillus subtilis (Kingdom: Bacteria, Phylum: Firmicutes, Cl ass: Bacilli). The transformation was performed with the binary plasmid vector PV-ZMAP595 (Figure III-1). Agrobacterium tumefaciens strain ABI contains a modified Ti pl asmid that is incapable of inducing

tumor formation due to the de letion of the phytohormone gene s originally present in the Agrobacterium plasmid (Koncz and Schell, 1986). The vector, PV-ZMAP595, contains both the left and right border sequences fla nking the transfer DNA (T-DNA) to facilitate

transformation.

The Agrobacterium -mediated corn transformation to produce MON 87460 was based on the method described by Armstrong and Phillip s (Armstrong and Phil lips, 1988). Briefly, freshly isolated immature corn embryos we re used for callus initiation. After coculturing with Agrobacterium carrying the transformati on vector, the calli were transferred from filter paper to callus initi ation medium containing carbenicillin to

eliminate Agrobacterium and paromomycin to eliminate cells that were not transformed, so that only cells containing the T-DNA surv ived. The resulting tr ansformed cells were

then subcultured several times on a selec tion medium and regenerated into plants.

The R 0 plants generated through the above tran sformation were self-pollinated, and the subsequent R 1 plants were screened for the pres ence of CSPB protein, tolerance to kanamycin, and homozygosity of the inserted gene. Only the plants that were homozygous for the cspB insert and tolerant to ka namycin were advanced for development, and their progenies were subjec ted to further molecular (Southern blot) and phenotypic assessments. MON 87460 was selected as the lead event based on its reduced yield loss under water-limited conditions, phe notypic characteristics, and molecular profile. Additional studies were conducted with MON 87460 to further characterize the genetic insertion and the expressed protei n, and to establis h the food, feed, and environmental safety relative to conventional corn. Th e major steps involving the development of MON 87460 are depicted in Figure III-2.

III.B. References

Armstrong, C.L. and R.L. Phillips. 1988. Geneti c and cytogenetic va riation in plants regenerated from organogenic and friable, embryogenic tissue cu ltures of maize. Crop Science 28:363-369.

Koncz, C. and J. Schell. 1986. The promoter of Tl-DNA gene 5 controls the tissue specific expression of chimeric genes car ried by a novel type of Agrobacterium binary vector. Molecular and General Genetics. 204:383-396.Monsanto Company 07-CR-191U Page 97 of 544 Figure III-1. Circular Map of Plasmid PV-ZMAP595

Plasmid PV-ZMAP595 containing the T-DNA used in Agrobacterium -mediated transformation to produce MON 87460. Approximate locations of the genetic elements are indicated on the map. CS- rop

OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos

loxP B-Left Border OR- ori V

PV- ZMAP595

9379 bp CS- rop

OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos

loxP B-Left Border OR- ori V

PV- ZMAP595

9379 bp CS- rop

OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos

loxP B-Left Border OR- ori V

PV- ZMAP595

9379 bp CS- rop

OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos

loxP B-Left Border OR- ori V

PV- ZMAP595

9379 bpMonsanto Company 07-CR-191U Page 98 of 544 Figure III-2. Schematic Representati on of the Development of MON 87460 Assembled Agrobacterium binary plasmid vector

PV-ZMAP595 and transferred to Agrobacterium

tume f

aciens, strain ABI

Transformed LH59 (a conventional corn variety) callus

tissue with the vector PV-ZMAP595 in Agrobacterium tumefaciens

Selected transformants and ge nerated rooted shoots from

the transformed callus tissues

Evaluated plants for insert integrity and tolerance to kanamycin

Evaluated progeny generations in laboratory and field

studies. Identified MON 87460 as lead candidate through

field studies

Introgressed MON 87460 into other germplasm and evaluated these for commercial performance Selected transformed plants tolerant to paromomycin

Screened homozygous plants with quantitative polymerase

chain reaction method

Conducted characterization and safety assessment studiesMonsanto Company 07-CR-191U Page 99 of 544 IV. Donor Genes and Regulatory Sequences

This section describes the donor genes and re gulatory sequences used in the development of MON 87460 and the deduced amino acid seque nces of the CSPB and NPTII proteins produced in MON 87460. In this section, T-DNA refers to DNA that is transferred to the plant during transformation. An expression cassette is composed of a coding sequence and the regulatory elements necessary for the expression of the coding sequence.

IV.A. Vector PV-ZMAP595

The PV-ZMAP595 vector used for the tran sformation of corn embryos to produce MON 87460 is shown in Figure III-1 and its el ements described in Table IV-1. This vector is approximately 9.4 kb and contains a single T-DNA delineated by left and right border regions that contains two expression cassettes: a cspB gene expression cassette, which contains coding sequence for CSPB from Bacillus subtilis and a neomycin phosphotransferase II ( nptII ) expression cassette, which confer s resistance to kanamycin. The T-DNA that is expected to incorporate in to the corn genome is approximately 4.6 kb and the DNA backbone region that is not incorporated into the corn genome is approximately 4.8 kb.

The cspB expression cassette contains the cspB coding sequence under the regulation of

the Ract1 promoter and leader, Ract1 intron, and the tr7 3' nontranslated sequence. The nptII expression cassette contains the nptII coding sequence under the regulation of the

35S promoter and the nos 3' nontranslated sequence.

The backbone region outside of the T-DNA c ontains two origins of replication for maintenance of plasmid in bacteria (OR- oriV, OR- ori-pBR322 ), a bacterial selectable marker gene ( aadA ), and a coding sequence for repr essor of primer protein for maintenance of plasmid copy number in E. coli (CS- rop ). A description of the genetic elements and their prefixes (e.g. P-, L-, I-, TS -, OR-, B-, CS-, and T-) in PV-ZMAP595 is provided in Table IV-1.

IV.B. T-DNA

IV.B.1. The cspB Coding Sequence and CSPB Protein

MON 87460 expresses the CSPB protei n, an RNA chaperone protein from Bacillus

subtilis , which is associated with reduced yi eld loss under water limitation through the unfolding of RNA secondary structure thereby, facilitating RNA translation (Phadtare et al., 2002). The amino acid sequence of th e CSPB protein produced in MON 87460 is identical to the native CSPB protei n (Willimsky et al., 1992) produced in Bacillus subtilis with the exception of one amino acid change in the second position from leucine to valine, designated as CSPB-L2V. This ami no acid change was implemented to facilitate the assembly of the plasmid vector PV-Z MAP595 for plant transformation. The fulllength amino acid sequence is shown in Figure IV-1.Monsanto Company 07-CR-191U Page 100 of 544 Table IV-1. Summary of Genetic Elem ents in Plasmid Vector PV-ZMAP595

Genetic Element Location in

Plasmid Function (Reference)

Vector Backbone Intervening Sequence 1 – 52 Sequence used in DNA cloning

CS 1

- rop 53 – 244 Coding sequence for repressor of primer protein for maintenance of plasmid copy number in E.

coli (Giza and Huang, 1989) Intervening Sequence 245 – 671 Sequence used in DNA cloning

OR 2

- ori.pBR322 672 – 1260 Origin of replication from pBR322 for the maintenance of the plasmid in E. coli (Sutcliffe, 1978) Intervening Sequence 1261 – 1790 Sequence used in DNA cloning

aadA 1791 – 2679 Bacterial promoter and coding sequence for anaminoglycoside-modifying enzyme, 3’(9)-Onucleotidyltransferase from the transposon Tn7 that confers spectinomycin and streptomycin resistance(Fling et al., 1985) (GenBank accession X03043) Intervening Sequence 2680 – 2815 Sequence used in DNA cloning

T-DNA

B 3 -Right Border 2816 – 3172 DNA from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA (Depicker et al., 1982) Intervening Sequence 3173 – 3204 Sequence used in DNA cloning

P 4

- Ract1 3205 – 4128 Promoter and leader from the rice actin gene,
act1, of Oryza sativa (McElroy et al., 1990)

I 5

- Ract1 4129 – 4605 Intron from the rice actin gene, act1, of Oryza

sativa (McElroy et al., 1991) Intervening Sequence 4606 – 4607 Sequence used in DNA cloning

CS- cspB

4608 – 4811 Codon modified coding sequence of the cspB gene from Bacillus subtilis encoding CSPB (Willimsky et al., 1992) Intervening Sequence 4812 – 4841 Sequence used in DNA cloning

T 6

- tr7

4842 – 5349 3' nontranslated sequence of transcript 7 gene

from Agrobacterium tumefaciens that directs polyadenylation (Dhaese et al., 1983) Intervening Sequence 5350 – 5423 Sequence used in DNA cloning

1 CS

– Coding Sequence; 2 OR

– Origin of Replication; 3 B

– Border; 4 P

– Promoter; 5 I

– Intron.

Table IV-1 continues on next page.Monsanto Company 07-CR-191U Page 101 of 544 Table IV-1 (cont.). Summary of Genetic Elements in Plasmid Vector PV-ZMAP595 T-DNA (cont.)

T 6

- tr7

4842 – 5349 3' nontranslated sequence of transcript 7 gene from Agrobacterium tumefaciens that directs polyadenylation (Dhaese et al., 1983) Intervening Sequence 5350 – 5423 Sequence used in DNA cloning

loxP 5424 – 5457 Sequence from Bacteriophage P1 for the recombination site recognized by Cre recombinase (Russell et al., 1992) Intervening Sequence 5458 – 5483 Sequence used in DNA cloning

P- 35S 5484 – 5776 Promoter for the 35S RNA of the Cauliflower Mosaic Virus (Odell et al., 1985) Intervening Sequence 5777 – 5840 Sequence used in DNA cloning

CS- nptII 5841 – 6635 Coding sequence from Tn5 (Beck et al., 1982) in E. coli encoding neomycin and kanamycin resistance (Fraley et al., 1983) Intervening Sequence 6636 – 6666 Sequence used in DNA cloning

T- nos 6667 – 6919 3' nontranslated sequence of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens which terminates and directs polyadenylation (Bevan et al., 1983) Intervening Sequence 6920 – 6944 Sequence used in DNA cloning

loxP 6945 – 6978 Sequence from Bacteriophage P1 for the recombination site recognized by Cre recombinase (Russell et al., 1992) Intervening Sequence 6979 – 6998 Sequence used in DNA cloning

B-Left Border 6999 – 7440 DNA from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA (Barker et al., 1983)

Vector Backbone Intervening Sequence 7441 – 7526 Sequence used in DNA cloning

OR- ori V 7527 – 7923 Origin of replication from the broad host range plasmid RK2 for maintenance of the plasmid in Agrobacterium (Stalker et al., 1981) Intervening Sequence 7924 – 9379 Sequence used in DNA cloning

6 T

– 3' nontranslated transcriptiona l termination sequence and polya denylation signal sequences.Monsanto Company 07-CR-191U Page 102 of 544 1 MVEGKVKWFN SEKGFGFIEV EGQDDVFVHF SAIQGEGFKT LEEGQAVSFE
51 IVEGNRGPQA ANVTKEA

Figure IV-1. Deduced Amino Acid Seque nce of the Full Length CSPB Protein

Present in MON 87460

The amino acid sequence of the CSPB was deduced from the full-length cspB coding sequence present in PV-ZMAP595.

1 ATGATTGAAC AAGATGGATT GCACGCAGGT TCTCCGGCCG CTTGGGTGGA

51 GAGGCTATTC GGCTATGACT GGGCACAACA GACAATCGGC TGCTCTGATG

101 CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG

151 ACCGACCTGT CCGGTGCCCT GAATGAACTG CAGGACGAGG CAGCGCGGCT

201 ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG

251 TCACTGAAGC GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG

301 GATCTCCTGT CATCTCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC

351 TGATGCAATG CGGCGGCTGC ATACGCTTGA TCCGGCTACC TGCCCATTCG

401 ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG GATGGAAGCC

451 GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC

501 AGCCGAACTG TTCGCCAGGC TCAAGGCGCG CATGCCCGAC GGCGAGGATC

551 TCGTCGTGAC GCATGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT

601 GGCCGCTTTT CTGGATTCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG

651 CTATCAGGAC ATAGCGTTGG CTACCCGTGA TATTGCTGAA GAGCTTGGCG

701 GCGAATGGGC TGACCGCTTC CTCGTGCTTT ACGGTATCGC CGCTCCCGAT

751 TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT TCTGA

Figure IV-2. Nucleotide Sequence Encodi ng the NPTII Protein in PV-ZMAP595

1 MIEQDGLHAG SPAAWVERLF GYDWAQQTIG CSDAAVFRLS AQGRPVLFVK
51 TDLSGALNEL QDEAARLSWL ATTGVPCAAV LDVVTEAGRD WLLLGEVPGQ
101 DLLSSHLAPA EKVSIMADAM RRLHTLDPAT CPFDHQAKHR IERARTRMEA
151 GLVDQDDLDE EHQGLAPAEL FARLKARMPD GEDLVVTHGD ACLPNIMVEN
201 GRFSGFIDCG RLGVADRYQD IALATRDIAE ELGGEWADRF LVLYGIAAPD
251 SQRIAFYRLL DEFF

Figure IV-3. Deduced Amino Acid Seque nce of the Full Length NPTII Protein

Present in MON 87460

The amino acid sequence of the NPTI I was deduced from the full-length nptII coding sequence present in PV-ZMAP595.Monsanto Company 07-CR-191U Page 103 of 544 IV.B.2. The cspB Regulatory Sequences

Adjacent to the right border region of plasmid PV-ZMAP595 is the cspB expression

cassette. The cspB coding sequence is under the regulatory control of the Ract1 promoter and leader from the actin gene, act1, of Oryza sativa (McElroy et al., 1990). Located

between the Ract1 promoter and the cspB coding sequence is the I- Ract1 nontranslated intron from the actin gene, act1, of Oryza sativa (McElroy et al., 1991). Following the cspB coding sequence is the 3' nontranslated sequence of transcript 7 gene from Agrobacterium tumefaciens (T- tr7 ) that directs polyadenylat ion (Dhaese et al., 1983).

IV.B.3. The nptII-loxP Coding Sequence and NPTII Protein

The nptII cassette contains the nptII coding sequence flanked by loxP sites. The NPTII protein in MON 87460 confers resistance to kanamycin, which was used to facilitate the selection process. The loxP sites were inserted to facilita te the potential excision of the nptII cassette using CRE recombinase. The DNA sequence of the nptII coding region

from the vector PV-ZMAP595 is shown in Figure IV-2. The deduced full-length amino acid sequence is show n in Figure IV-3.

IV.B.4. The nptII Regulatory Sequences

Adjacent to the left border region of plasmid PV-ZMAP595 is the nptII expression

cassette. The nptII coding sequence is under the regulatory control of the 35S promoter
from the Cauliflower Mosaic Viru s (Odell et al., 1985). Following the nptII coding sequence is the 3' nontra nslated sequence of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens (T- nos ) which terminates transcription and directs polyadenylation (Bevan et al., 1983).

IV.B.5. T-DNA Borders

Plasmid PV-ZMAP595 contains right border and left border regions that delineate the

T-DNA to be transferred into corn and are invo lved in the efficient transfer of the T-DNA into the corn genome. These border regions (Figure III-1 and Table IV-1) were derived

from Agrobacterium tumefaciens plasmids (Depicker et al ., 1982; Barker et al., 1983).

IV.C. Genetic Elements Outside the T-DNA Borders

Four genetic elements exist outside of th e T-DNA borders that are essential for the

maintenance and selection of the vector PV-ZMAP595 in bacteria. They include: ORori V, origin of replication for the maintenance of the plasmid in Agrobacterium (Stalker et al., 1981); CS- rop, coding sequence of repressor of primer (ROP) protein for the maintenance of plasmid copy number in E. coli (Giza and Huang, 1989); OR-oripBR322, origin of replication from pBR322 fo r the maintenance of the plasmid in E. coli (Sutcliffe, 1979); and aadA , a bacterial promoter and coding sequence of an enzyme from transposon Tn7 that confers spectinomycin and streptomycin resistance for molecular cloning and selection purposes (F ling et al., 1985) prior to pl ant transformation. As these elements are outside of the border regions, they are not expected to be transferred into the corn genome. The absence of the backbone sequence in MON 87460 has been confirmed by Southern blot analyses (Sections V.B and V.D.2).Monsanto Company 07-CR-191U Page 104 of 544 IV.D. References

Barker, R.F., K.B. Idler, D.V. Thompson and J.D. Kemp. 1983. Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTil5955. Plant Molecular Biololgy.2:335-350.

Beck, E., G. Ludwig, E.A. Auerswald, B. Reiss and H. Schaller. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from

transposon Tn5 . Gene. 19:327-336.

Bevan, M., W.M. Barnes and M. Chilton. 1983. Structure and tr anscription of the nopaline synthase gene region of T-DNA. Nucleic Acids Research. 11:369-385.

Depicker, A., S. Stachel, P. Dhaese, P. Zambryski and H.M. Goodman. 1982. Nopaline synthase: transcript mapping and DNA sequence. Journal of Molecular and Applied Genetics. 1:561-573.

Dhaese, P., H. d.Greve, J. Gielen, J. Se urinck, M. v.Montague and J. Schell. 1983. Identification of sequences involved in polyadenylati on of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models. EMBO Journal . 2:419-

426.

Fling, M.E., J. Kopf and C. Richards. 1985. Nucleotide sequence of the transposon Tn 7 gene encoding an aminoglycoside-modifying enzyme, 3 ′ (9)- O

nucleotidyltransferase. Nucl eic Acids Research. 13:7095-7106.

Fraley, R.T., S.G. Rogers, R.B. Horsch, P. R. Sanders, J.S. Flick, S.P. Adams, M.L. Bittner, L.A. Brand, C.L. Fink, J.S. Fr y, G.R. Galluppi, S.B. Goldberg, N.L. Hoffmann and S.C. Woo. 1983. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences USA. 80:4803-4807.

Giza, P.E. and R.C.C. Huang. 1989. A self -inducing runaway-replication plasmid expression system utilizing the Rop protein. Gene. 78:73-84.

McElroy, D., W. Zhang, J. Cao and R. Wu. 1990. Isolation of an efficient actin promoter

for use in rice transformation. Plant Cell. 2:163-171.

McElroy, D., A.D. Blowers, B. Jenes a nd R. Wu. 1991. Construction of expression vectors based on the rice actin 1( Act1 ) 5 ′ region for use in monocot

transformation. Molecular a nd General Genetics. 231:150-160.

Odell, J.T., F. Nagy and N-H. Chua. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature. 313:810-812.

Phadtare, S., M. Inouye and K. Severinov. 2002. The nucleic acid melting activity of

Escherichia coli CspE is critical for transcription antitermination and cold acclimation of cells. Journal of Biological Chemistry 277:7239-7245.

Russell, S.H., J.L. Hoopes and J.T. Odell. 1992. Directed excision of a transgene from the plant genome. Molecular and General Genetics. 234:49-59.Monsanto Company 07-CR-191U Page 105 of 544 Stalker, D.M., C.M. Thomas and D.R. Helin ski. 1981. Nucleotide sequence of the region of the origin of replication of the br oad host range plasmid RK2. Molecular and

General Genetics. 181:8-12.

Sutcliffe, J.G. 1979. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symposium. 43:77-103.

Willimsky, G., H. Bang, G. Fischer and M. A. Marahiel. 1992. Characterization of cspB , a Bacillus subtilis inducible cold shock gene a ffecting cell viability at low temperatures. Journal of Bacteriology. 174:6326-6335.Monsanto Company 07-CR-191U Page 106 of 544 V. Genetic Analysis

Molecular analyses were performed to ch aracterize the integrated DNA in MON 87460. Southern blot analyses were used to determ ine the number of integration sites within the corn genome (insert number), the number of copies within one insert (copy number), and the absence of plasmid backbone sequences in th e plant. The Southern strategy was used

to insure that all detectable fragments would have been detect ed in the analysis. The corn genome was assayed with probes that spanned the entire transforma tion plasmid and all probes were less than 2 kb in length in order to retain a high level of sensitivity. The high level of sensitivity was demonstrated for each blot by including and detecting a 1/10 th
genome equivalent of the positive control. Two restriction enzyme sets were specifically chosen to independently confir m the presence of the insert and minimize the possibility that two DNA fragments could co migrate on the gel. In addi tion, at least one enzyme cut in each of the known regions of flanking DNA to insure that at least one of the 5 ́ border fragments and one of the 3 ́ border fragment s were sequenced. The results of these analyses show that a single copy of the T- DNA inserted at a single locus of the genome.

The stability of the DNA insert across multip le generations was also demonstrated by Southern blot fingerprint an alysis. Seven generations of MON 87460 were digested with one of the enzyme sets utilized for the c opy number analysis and were hybridized with probes that would detect the en tire insert (two hybridizatio n bands). This fingerprint strategy consists of two border fragments that assay not only th e stability of the insert, but also the stability of genomic DNA directly adjacent to the insert.

The DNA sequencing analyses compliment the S outhern analyses. While Southern blot data demonstrated the presence of a single insert in MON 87460, the sequencing of the insert and the genomic DNA directly adjacent to the insert determined exactly what DNA was inserted during transformation. In additi on, genomic rearrangements at the insertion site were assessed by comparing the insert and flanking sequence to the insertion site in conventional corn. Taken together, the data confirms that a single copy of the T-DNA inserted in a single locus in the corn genom e and was associated with a 22 base pair

deletion.

Genomic DNA from MON 87460 was digested with appropriate restriction enzymes and subjected to Southern blot analyses to ch aracterize the T-DNA that was integrated into the corn genome. For each digest, there were duplicated samples wh ich consisted of an equal amount of digested DNA with one set of samples run for a longer period of time (Long Run) than the second set (Short Run). The long run allows for greater resolution of high molecular weight DNA, whereas the short run allows the detection of low molecular weight DNA. Genomic DNA samples from conventional corn were used as the negative controls on the blots to determine potentia l non-specific hybridization signals. The positive controls for Southern blots were generated by digestion of plasmid DNA with enzyme combinations to produce the DNA banding patterns that were most

relevant to the molecular assessment of MON 87460. Probe templates generated from the plasmid DNA were also used as positive controls. In addition, DNA markers were included to provide size estimation of the hybr idized bands on Southern blots. MinorMonsanto Company 07-CR-191U Page 107 of 544 differences in the sizes of the hybridizat ion bands according to the DNA marker were observed compared to the expected sizes. The altered migrations may be due to the

difference in salt concentrations between the DNA sample and the molecular weight marker (Sambrook and Russell, 2001).

The genetic elements present in MON 87460 are listed in Table V-1. The insert is identical to the T-DNA sequence of PV-ZMAP595, from the proximal end of the promoter to the Left Border. The informa tion and results derived from the molecular analyses were used to construct a linear ma p of the insert in MON 87460. This linear map depicts restriction sites identified in the insert and the flanking corn genomic DNA flanking the insert, and provide s information on the expected banding patterns and sizes of the DNA fragments after restriction enzy me digestion. The linear map is shown in Figure V-1. Based on the insert linear map a nd the plasmid map, a ta ble summarizing the expected DNA fragments for Southern analyses is presented in Table V-2. The probes used in the Southern analyses and the map of PV-ZMAP595 are presented in Figure V-2 and Figure V-3. The materials and methods used in the analyses are presented in Appendix A.Monsanto Company 07-CR-191U Page 108 of 544 Table V-1. Summary of Genetic Elements in MON 87460

Genetic Element 1 Location in Sequence 2 Function (Reference) Sequence flanking 5'

end of the insert 1-1121 Corn genomic DNA

P 3

- Ract1 87460 1122-1312 Truncated promoter and leader from the rice actin gene, act1, of Oryza sativa (McElroy et al., 1990)

I 4

- Ract1 1313-1789 Intron from the rice actin gene, act1, of Oryza sativa (McElroy et al., 1991) Intervening Sequence 1790-1791 Sequence used in DNA cloning

CS 5

- cspB 1792-1995 Codon optimized coding sequence of the cspB gene from Bacillus subtilis encoding CSPB (Willimsky et al., 1992) Intervening Sequence 1996-2025 Sequence used in DNA cloning

T 6

- tr7 2026-2533 3' nontranslated sequence of transcript 7 gene from Agrobacterium tumefaciens that directs polyadenylation (Dhaese et al., 1983) Intervening Sequence 2534-2607 Sequence used in DNA cloning

loxP 2608-2641 Sequence from Bacteriophage P1 for the recombination site recognized by Cre recombinase (Russell et al., 1992) Intervening Sequence 2642-2667 Sequence used in DNA cloning

P- 35S 2668-2960 Promoter for the 35S RNA of the Cauliflower mosaic virus (Odell et al., 1985) Intervening Sequence 2961-3024 Sequence used in DNA cloning

CS- nptII 3025-3819 Coding sequence from Tn5 (Beck et al., 1982) in E. coli encoding neomycin and kanamycin resistance (Fraley et al., 1983) Intervening Sequence 3820-3850 Sequence used in DNA cloning

1 Flanking sequences and inte rvening sequences are not functional genetic elements

2 Numbering includes the insert in MON 87460 and adjacent genomic DNA

3 P

– Promoter

4 I

– Intron

5 CS

– Coding Sequence

6 T

– 3' nontranslated transcriptiona l termination sequence and pol yadenylation signal sequences

Table V-1 continues on next page.Monsanto Company 07-CR-191U Page 109 of 544 Table V-1 (cont.). Summary of Genetic Elements in MON 87460

Genetic Element Location in Sequence Function (Reference)

T- nos 3851-4103 3' nontranslated sequence of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens which terminates and directs polyadenylation (Bevan et al., 1983) Intervening Sequence 4104-4128 Sequence used in DNA cloning

loxP 4129-4162 Sequence from Bacteriophage P1 for the recombination site recognized by Cre recombinase (Russell et al., 1992) Intervening Sequence 4163-4182 Sequence used in DNA cloning

B 7 -Left Border 87460 4183-4430 DNA from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA (Barker et al., 1983) Sequence flanking 3'

end of the insert 4431-5214 Corn genomic DNA

7 B

– BorderMonsanto Company 07-CR-191U Page 110 of 544Figure V-1. Schematic Representation of the Insert and Genomic Flanking Sequences in MON 87460A linear map of the insert and known genomic DNA flanking the insert in MON 87460 is shown. Identified on the map are geneticelementswithin the insert, as well as restriction sites with positions relative to the size of the linearmap for enzymes used in the Southern analyses. Shownon the lower portion of the map are the expected sizes of the DNA fragments after digestions with respective restriction enzymeor combination ofenzymes. The dotted line indicates the additional DNA fragment that would be present if partial digestion of the internalEcoO109 I restrictionsite occurs. Arrows with dotted lines indicate the end of the insert and the beginning of corn genomic flanking sequence.EcoO109 I64EcoRVHind IIIEcoO109I/NotIEcoO109 I 875EcoRV2225NotI2553EcoRV4915Hind III4506>2.2 kb 2.7 kb>4.5 kb>2.7 kb1.7 kb0.8 kb P -Ract1

87460 I -Ract1 CS- cspB T

- tr7 loxP loxP

P -35S

CS

- nptII T

- nos B-Left Border 874605' Genomic Flank DNA3' Genomic Flank DNA15214EcoO109 I64EcoRVHind IIIEcoO109I/NotIEcoO109 I 875EcoRV2225NotI2553EcoRV4915Hind III4506>2.2 kb 2.7 kb>4.5 kb>2.7 kb1.7 kb0.8 kb P -Ract1

87460 I -Ract1 CS- cspB T

- tr7 loxP loxP

P -35S

CS

- nptII T

- nos B-Left Border 874605' Genomic Flank DNA3' Genomic Flank DNA15214EcoO109 I64EcoRVHind IIIEcoO109I/NotIEcoO109 I 875EcoRV2225NotI2553EcoRV4915Hind III4506>2.2 kb 2.7 kb>4.5 kb>2.7 kb1.7 kb0.8 kb P -Ract1

87460 I -Ract1 CSEcoO109 I64EcoRVHind IIIEcoO109I/NotIEcoO109 I 875EcoRV2225NotI2553EcoRV4915Hind III4506>2.2 kb 2.7 kb>4.5 kb>2.7 kb1.7 kb0.8 kb P -Ract1

87460 I -Ract1 CS- cspB T

- tr7 loxP loxP

P -35S

CS

- nptII T

- nos B-Left Border 874605' Genomic Flank DNA3' Genomic Flank DNA15214EcoO109 I64EcoRVHind IIIEcoO109I/NotIEcoO109 I 875EcoRV2225NotI2553EcoRV4915Hind III4506>2.2 kb 2.7 kb>4.5 kb>2.7 kb1.7 kb0.8 kb P -Ract1

87460 I -Ract1 CS- cspB T

- tr7 loxP loxP

P -35S

CS

- nptII T

- nos B-Left Border 874605' Genomic Flank DNA3' Genomic Flank DNA15214Monsanto Company 07-CR-191U Page 111 of 544 Probe DNA Probe Start Position Stop Position Total Length (~kb)

1 T-DNA Probe 1 2816 4782 2.0

2 T-DNA Probe 2 4670 6085 1.4

3 T-DNA Probe 3 5839 7440 1.6

4 Backbone Probe 1 7441 66 2.0

5 Backbone Probe 2 9241 1245 1.4

6 Backbone Probe 3 1094 2815 1.7

Figure V-2. Circular Map of Plasmi d PV-ZMAP595 Showing Probes 1-6

Plasmid PV-ZMAP595 containing the T-DNA used in Agrobacterium-mediated transformation to produce MON 87460. Locations of the genetic elements are depicted by arrows on the interior of the map with their annotations shown on the exteri or of the map. Restriction sites for enzymes used in Southern analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map. The overlapping T- DNA and backbone probes used in the Southern analyses (labeled 1-6 within the interior of th e map) are detailed in the accompanying table. PV- ZMAP595

9379 bp Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V



### T-DNA 1 3

2 4 5

6 PV- ZMAP595

9379 bp Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V

Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 CS- rop

OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V



### T-DNA 1 3

2 4 5

6 PV- ZMAP595

9379 bp Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V



### T-DNA 1 3

2 4 5

6 PV- ZMAP595

9379 bp Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V

Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 CS- rop

OR- ori.pBR322

aadA

B-Right Border

P-

Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V



### T-DNA 1 3

2 4 5

6Monsanto Company 07-CR-191U Page 112 of 544 Probe DNA Probe Start Position Stop Position Total Length (~kb)

7 P- Ract1 Probe 2816 4128 1.3

8 I- Ract1 Probe 4129 4607 0.5

9 CS- cspB Probe 4608 4811 0.2

10 T- tr7 Probe 4842 5354 0.5

11 loxP + P- 35S Probe 5424 5785 0.36

12 CS- nptII Probe 5839 6635 0.8

13 T- nos + loxP + Left Border

Probe 6667 7440 0.8

Figure V-3. Circular Map of Plasmi d PV-ZMAP595 Showing Probes 7-13

Plasmid PV-ZMAP595 containing the T-DNA used in Agrobacterium-mediated transformation to produce MON 87460. Locations of the genetic elements are depicted by arrows on the interior of the map with their annotations shown on the exteri or of the map. Restriction sites for enzymes used in Southern analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map. The genetic element pr obes used in the Southern analyses (labeled 7-13 within the interior of the map) are detailed in the accompanying table. PV- ZMAP595

9379 bp Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P- Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V



### T-DNA 7

9 8 10 13

12

11 PV- ZMAP595

9379 bp Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P- Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V

Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P- Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V



### T-DNA 7

9 8 10 13

12

11 CS- rop

PV- ZMAP595

9379 bp Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P- Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V



### T-DNA 7

9 8 10 13

12

11 PV- ZMAP595

9379 bp Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P- Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V

Blp I 2140

Eco O109 I 3198 Eco O109 I 8956 Eco O109 I 8998

EcoR V 5041 Not I 5369 Xba I 5376 OR- ori.pBR322

aadA

B-Right Border

P- Ract1

I- Ract1

CS- cspB T- tr7 loxP P- 35S CS- nptII T- nos loxP B-Left Border OR- ori V



### T-DNA 7

9 8 10 13

12

11 CS- ropMonsanto Company 07-CR-191U Page 113 of 544 Table V-2. Summary Chart of the Exp ected DNA Fragments using Combinations of Restriction Enzymes and Probes

Probes used 1, 2, 3 4, 5, 6 7 8 9 10 11 12 13

Southern

blot in Figure V-4 V-5 V-6 V-7 V-8 V-9 V-10 V-11 V-12

Plasmid

Blp I + Xba I 3.2 kb +

6.1 kb 3.2 kb +

6.1 kb 3.2 kb 3.2 kb 3.2 kb 3.2 kb 6.1 kb 6.1 kb 6.1 kb

Probe
templates 1 1.4 kb +

1.6 kb +

2.0 kb 1.4 kb +

1.7 kb +

2.0 kb -- 2

-- -- -- -- -- --

MON 87460

Hind III > 4.5 kb -- -- -- -- -- -- -- --

Eco R V 2.7 kb

+ > 2.2 kb no band > 2.2

kb > 2.2

kb > 2.2

kb 2.7 kb

+
> 2.2 kb 2.7 kb 2.7 kb 2.7 kb

Eco O109 I

and Not I -- no band 1.7 kb 1.7 kb 1.7 kb 1.7 kb 1.7 kb > 2.7

kb >2.7

kb

1 Probe templates were spiked when multiple pr obes were used in Southern blot analysis.

2 ‘--’ Indicates that the particular restriction en zyme or the combination of the enzymes was not used in the analysis.

V.A. Insert and Copy Number

The number of T-DNA inserts (insert num ber) in the MON 87460 genome was evaluated by digesting the test and control DNA with Hind III, a restriction enzyme that does not cleave within the T-DNA. Therefore, Hind III releases a restriction fragment containing the entire T-DNA and adjacent plant genomic DNA (Figure V-1). Therefore, the number of restriction fragments detect ed indicates the nu mber of inserts present in MON 87460. The number of copies of the T-DNA (copy nu mber) integrated at a single locus was determined by digesting test and control genomic DNA samples with the restriction

enzyme EcoR V, which cleaves once within the insert (Figure V-1). If MON 87460 contains one copy of the T-DNA, probing wi th the entire T-DNA will result in two bands, each representing a po rtion of the T-DNA along with adjacent plant genomic

DNA.

The Southern blot used to determine insert and copy number of the T-DNA (Figure V-4)

contained several controls. To determin e if any endogenous background hybridization bands were detected when probing with the T-DNA, the blot contained conventional corn genomic DNA digested with Hind III (Figure V-4, lanes 1 and 8) or EcoR V (Figure V-4, lanes 3 and 10). The conventional control DNA digested with Hin d III or Eco R V produced several hybridization signals. Th ese hybridization signals result from theMonsanto Company 07-CR-191U Page 114 of 544 probes hybridizing to endogenous sequences re siding in the corn genome and are not specific to the inserted DNA. These signals were produced in both test and control lanes,

and therefore the bands are consid ered to be endogenous background.

To ensure that each of the T-DNA probes was ab le to hybridize to thei r respective targets, probe template spikes (Figure V-2, probes 1- 3) that were generated from plasmid PVZMAP595 and mixed at different concentrations (1 copy: one genomic copy equivalence; 0.1 copy: one tenth genomic c opy equivalence) with the control DNA pre-digested with

Eco R V were included on the blot (Figure V-4, lanes 5-6). The expected hybridization bands at approximately 1.4, 1.6, and 2.0 kb were detected. The approximately 0.1 and 1 copies of the 1.4 kb band were faint in comp arison to the 1.6 and 2.0 kb bands, but were clearly detectable. The det ection of the probe template positive hybridization controls demonstrates that all three probes are hybridizin g to the target DNA. To ensure that the T-DNA probes hybridize to the plasmid used for transformation, plasmid PV-ZMAP595 digested with a combination of Blp I and Xba I was spiked in the control DNA predigested with Eco R V. The expected hybridization bands of approximately 3.2 and 6.1 kb (Figure V-4, lane 7) were detected (re fer to Figure V-2 and Figure V-3 plasmid map).

MON 87460 DNA digested with Hin d III (Figure V-4, lanes 2 a nd 9) and hybridized with the T-DNA probes produced a single unique band of approximately 6.8 kb. This is consistent with the expected band being gr eater than 4.5 kb (Figure V-1) and confirms

that MON 87460 contains one insert located within a 6.8 kb Hin d III restriction fragment. MON 87460 DNA digested with Eco R V and hybridized with the T-DNA probes produced two bands (Figure V-4, lanes 4 and 11) of approximately 2.7 and 7.2 kb. The approximately 2.7 kb band is the expected size for the border fragment containing the 3' end of the inserted DNA (T-DNA) along with the adjacent genomic DNA flanking the 3' end of the insert (Figure V-1). The approximately 7.2 kb band is consistent with the expected band being greater than 2.2 kb (Figure V-1). This band represents the 5' border fragment containing the 5' end of the in serted DNA along with the adjacent genomic DNA flanking the 5' end of the insert.

The results of MON 87460 presented in Fi gure V-4 show that MON 87460 contains a single copy of the T-DNA that resides at a single locus of in tegration on an approximately 6.8 kb Hin d III restriction fragment. Sout hern blot analyses described below using a combination of Eco O109 I and Not I which produced an expected 5 ́ border fragment further confirmed this conclusion.

V.B. Presence or Absence of Plasmid PV-ZMAP595 Backbone

Test and control DNAs were digested with either a combination of the restriction

enzymes Eco O109 I and Not I or the restriction enzyme Eco R V to determine if PV-ZMAP595 sequences, other than the T-DN A region, were inserted in MON 87460. The samples were electrophoresed, blotted, a nd the blot was hybridized simultaneously with three overlapping probe s (Figure V-2, probes 4-6) that spanned the backbone sequence of PV-ZMAP595. The re sults are shown in Figure V- 5. Probe template spikes (Figure V-2, probes 4-6) generated from th e plasmid PV-ZMAP595 were mixed with theMonsanto Company 07-CR-191U Page 115 of 544 pre-digested control genomic DNA to se rve as a positive hybridization control. Additionally, plasmid PV-ZMAP595 DNA previous ly digested with the combination of Blp I and Xba I was mixed with control genomic DNA digested with Eco R V and loaded on the gel to serve as a positive hybridization control.

The Southern blot used to determine the presence or absence of plasmid PV-ZMAP595 backbone sequences (Figure V- 5) contained several controls. To determine if any endogenous background hybridization bands we re detected when probing with the backbone sequences, the blot contained conve ntional corn genomic DNA digested with a combination of Eco O109 I and Not I (Figure V-5, lanes 1 and 8) or Eco R V (Figure V-5, lanes 3 and 10). The conventional corn genomic DNA digested with Eco O109 I and

Not I (Figure V-5, lanes 1 and 8) or Eco R V (Figure V-5, lanes 3 and 10) produced several hybridization signals . These hybridization signals result from the probes hybridizing to endogenous sequences residing in the corn genome and are not specific to the inserted DNA. These signals were pr oduced in both test and control lanes, and therefore are considered to be endogenous background.

To ensure that each backbone probe was capab le of hybridizing to its respective target, probe template spikes (Figure V-2, probes 4-6) that were generated from plasmid

PV-ZMAP595 and mixed at different concentr ations with the cont rol DNA pre-digested

with Eco R V were included on the bl ot (Figure V-5, lanes 5 and 6). The expected hybridization bands at 1.4, 1.7, and 2.0 kb were de tected. The results show that the three probes hybridized, as exp ected, to the target DNA. To ensure that the backbone probes hybridize to the plasmid used for transfor mation, plasmid PV-ZMAP595 digested with Blp I /Xba I was mixed with conventiona l control DNA digested with Eco R V (Figure V5, lane 7). The expected hybridization bands at 3.2 and 6.1 kb were detected, in addition to the endogenous background produced by th e conventional control DNA (Figure V-5, lanes 3 and 10).

The results presented in Figure V-5 show that MON 87460 DNA digested with the

combination of Eco O109 I and Not I (Figure V-5, lanes 2 and 9) or Eco R V (Figure V-5, lanes 4 and 11) showed no detectable hybr idization signal besi des the endogenous background, indicating that MON 87460 does not contain any detectable backbone sequence from the transformation vector PV-ZMAP595.Monsanto Company 07-CR-191U Page 116 of 544 Figure V-4. Southern Blot Analysis of MON 87460: Insert and Copy Number

The blot was hybridized simultaneously with three overlapping 32 P-labeled T-DNA probes that span the insert (Figure V-2, prob es 1-3). Each lane contains ~10 μ g of digested genomic DNA isolated from seed. Lane designations are as follows: 1: Conventional ( Hin d III) 2: MON 87460 ( Hin d III) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with probe templates [~0.1 copy] 6: Conventional ( Eco R V) spiked with probe templates [~1 copy] 7: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 8: Conventional ( Hin d III) 9: MON 87460 ( Hin d III) 10: Conventional ( Eco R V) 11: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20 40

8.1

6.1 15 1 2 3 4 5 6 7 8 9 10 11

LONG RUN SHORT RUN

8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20 40

8.1

6.1 15

4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20

4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20 40

8.1

6.1 15 1 2 3 4 5 6 7 8 9 10 11

LONG RUN SHORT RUNMonsanto Company 07-CR-191U Page 117 of 544 Figure V-5. Southern Blot Analysis of MON 87460: PV-ZMAP595 Backbone

The blot was hybridized simultaneously with three overlapping 32 P-labeled probes that span the entire backbone sequence (Figure V-2, probes 4-6) of plasmid PV-ZMAP595. Each lane contains ~10 μg of digested genomic DNA isolated fro m seed. Lane designations are as follows: 1: Conventional ( Eco O109 I/ Not I) 2: MON 87460 ( Eco O109 I/ Not I) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with probe templates [~0.1 copy] 6: Conventional ( Eco R V) spiked with probe templates [~1 copy] 7: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 8: Conventional ( Eco O109 I/ Not I) 9: MON 87460 ( Eco O109 I/ Not I) 10: Conventional ( Eco R V) 11: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 23.1

2.0 2.3 4.4 6.6 9.4 23.1

2.0

0.5 6.6

4.4

2.3 9.4 1 2 3 4 5 6 7 8 9 10 11 LONG RUN SHORT RUN

23.1

2.0 2.3 4.4 6.6 9.4 23.1

2.0 2.3 4.4 6.6 9.4 23.1

2.0 2.3 4.4 6.6 9.4 23.1

2.0

0.5 6.6

4.4

2.3 9.4 23.1

2.0

0.5 6.6

4.4

2.3 9.4 23.1

2.0

0.5 6.6

4.4

2.3 9.4 1 2 3 4 5 6 7 8 9 10 11 LONG RUN SHORT RUNMonsanto Company 07-CR-191U Page 118 of 544 V.C. Intactness of cspB and nptII Expression Cassettes

The copy number of the inserted cspB and nptII coding sequences and each of the associated genetic elements were asse ssed by digesting MON 87460 genomic DNA with restriction enzyme Eco R V or a combination of Eco O109 I and Not I and hybridizing Southern blots with probes covering th e inserted cspB and nptII cassettes. The size of the genomic fragments and the T-DNA elements exp ected to be contained in each of those fragments is indicated below a nd summarized in Table V-2.

Digestion of MON 87460 genomic DNA with the combination of Eco O109 I and Not I was expected to generate two border fragment s with expected sizes of 1.7 kb and greater than 2.7 kb (Figure V-1). The 1.7 kb re striction fragment contains genomic DNA flanking the 5' end of the insert, Ract1 87460 promoter and Ract1 leader, Ract1 intron, cspB coding sequence, and the tr7 3' nontranslated sequence. Th e restriction fragment greater

than 2.7 kb contains the 5' loxP sequence, 35S promoter, nptII coding sequence, nos 3' nontranslated sequence, 3' loxP sequence, left border and genomic DNA flanking the 3'

end of the insert.

Digestion of MON 87460 genomic DNA with Eco R V was expected to release two border fragments with expected sizes of 2.7 kb and greater than 2.2 kb (Figure V-1). The restriction fragment greater than 2.2 kb contai ns genomic DNA flanking the 5' end of the insert, Ract1 87460 promoter and Ract1 leader, Ract1 intron, cspB coding sequence, and a portion of the tr7 3' nontranslated sequence. The approximately 2.7 kb restriction fragment contains the remaining portion of the tr7 3' nontranslated sequence, 5' loxP

sequence, 35S promoter, nptII coding sequence, nos 3' nontranslated sequence, 3' loxP sequence, left border, and genomic DNA flanking the 3' end of the insert.

Individual Southern blots were hybridized with the following probes: Right Border +

Ract1 promoter and leader probe, Ract1 intron probe, cspB coding sequence probe, tr7 3' nontranslated sequence probe, loxP + 35S promoter probe, nptII coding sequence probe,

or nos 3 ′ nontranslated + loxP + Left Border sequence probe (Figure V-3, probes 7 – 13, respectively).

V.C.1. Right Border + Ract1 Promoter and Leader (Probe 7)

The Southern blot used to confirm the copy number of the Right Border + Ract1 promoter and leader sequences (Figure V-6) co ntained several controls . To determine if any endogenous background hybridization bands were detected when probing with probe 7, the blot contained conventional corn genom ic DNA digested with a combination of

Eco O109 I and Not I (Figure V-6, lanes 1 and 7) or Eco R V (Figure V-6, lanes 3 and 9). The conventional corn genomic DNA produced several hybridization signals resulting from the probes hybridizing to endogenous sequences residing in the corn genome and are not specific to the inserted DNA. Th ese signals were produc ed in both test and control lanes, and therefor e the bands are considered to be endogenous background.

As a positive hybridization control, the blot contained plasmid PV-ZMAP595 that was digested with a combination of Blp I and Xba I and mixed with pre-digested controlMonsanto Company 07-CR-191U Page 119 of 544 DNA. Results of this analysis show the expected hybridization band of approximately 3.2 kb (Figure V-6, lanes 5 and 6).

MON 87460 DNA digested with a combination of Eco O109 I and Not I (Figure V-6, lanes 2 and 8) produced the expected single unique band of approximately 1.7 kb (Figure V-1). The hybridization band in the long run (F igure V-6, lane 8) appears slightly larger than the corresponding band in the short run (Figure V-6, la ne 2), most likely due to

better resolution of the band in the longer run. MON 87460 DNA digested with Eco R V (Figure V-6, lanes 4 and 10) produced a si ngle unique band of approximately 7.5 kb.

This is consistent with the expected band being greater th an 2.2 kb (Figure V-1). This band in the long run (Figure V-6, lane 10) app ears slightly larger than the corresponding band in the short run (Figure V-6, lane 4), mo st likely due to better resolution of the band in the longer run. There we re no additional bands detect ed using the Right Border, promoter and leader sequence probe. Base d on the results presen ted in Figure V-6,

MON 87460 contains no additiona l, detectable Right border, Ract1 promoter and leader

elements other than those associated with the cspB cassette.

V.C.2. Ract1 Intron (Probe 8)

The Southern blot used to confirm the copy number of the Ract1 Intron sequence (Figure V-7) contained several controls. To determine if any endogenous background hybridization bands were detected when probing with probe 8, the blot contained conventional corn genomic DNA di gested with a combination of Eco O109 I and Not I (Figure V-7, lanes 1 and 7) or Eco R V (Figure V-7, lanes 3 and 9). The results of this analysis show no detectab le hybridization bands.

As a positive control, the blot contained plasmid PV-ZMAP595 that was digested with a

combination of Blp I and Xba I and mixed with Eco R V pre-digested control DNA. Results of this experiment produced an expe cted band which migrated at approximately 3.1 kb (Figure V-7, lanes 5 and 6).

MON 87460 DNA digested with a combination of Eco O109 I and Not I (Figure V-7, lanes 2 and 8) that was electrophoresed, blot ted, and hybridized with probe 8 produced the expected single unique band of approximate ly 1.7 kb (Figure V-1). This band in the long run appears slightly larger than the corresponding band in the short run, most likely

due to better resolution of the band in the long run. MON 87460 DNA digested with

Eco R V (Figure V-7, lanes 4 and 10) produced the single unique band of approximately 7.2 kb. This is consistent with the expected band being greater than 2.2 kb (Figure V-1). There were no additional hybridiza tion bands detected using the Ract1 intron probe. Based on the results presented in Figur e V-7, MON 87460 contains no additional,

detectable Ract1 intron elements other than those associated with the cspB cassette

.

V.C.3 cspB Coding Sequence (Probe 9)

The Southern blot used to confirm the copy number of the cspB Coding Sequence (Figure V-8) contained several controls. To determine if any endogenous background hybridization bands were detected when probing with probe 9, the blot contained conventional corn genomic DNA di gested with a combination of Eco O109 I and Not IMonsanto Company 07-CR-191U Page 120 of 544 (Figure V-8, lanes 1 and 7) or Eco R V (Figure V-8, lanes 3 and 9). The results of this analysis show no detectable hybridization ba nds, as expected for the negative control (Figure V-8, lanes 1, 3, 7, and 9).

As a positive hybridization control, the blot contained plasmid PV-ZMAP595 that was digested with a combination of Blp I and Xba I and mixed with pre-digested control DNA. Results of this experiment produced an expected band which migrated at approximately 3.1 kb (Figure V-8, lanes 5 and 6).

MON 87460 DNA digested with a combination of Eco O109 I and Not I (Figure V-8, lanes 2 and 8) and hybridized with probe 9 produced the expected single unique band of approximately 1.7 kb (Figure V-1). This band in the long run appears slightly larger than the corresponding band in the short run, most lik ely due to better resolution of the band in the long run. MON 87460 DNA digested with Eco R V (Figure V-8, lanes 4 and 10) and hybridized with probe 9 produced the single unique band of approximately 7.2 kb. This is consistent with the expect ed band being greater than 2.2 kb (Figure V-1). There were no additional hybridization bands detected using the cspB coding sequence probe. Based on the results presented in Figure V-8, M ON 87460 contains no additional, detectable cspB coding sequence elements other than those associated with the cspB cassette

.

V.C.4. tr7 3' Nontranslated Sequence (Probe 10)

The Southern blot used to confirm the copy number of the tr7 3' Nontranslated Sequence (Figure V-9) contained several controls. To determine if any endogenous background hybridization bands were det ected when probing with pr obe 10, the blot contained conventional corn genomic DNA di gested with a combination of Eco O109 I and Not I (Figure V-9, lanes 1 and 7) or Eco R V (Figure V-9, lanes 3 and 9). The results of this analysis show no detectable hybridization ba nds, as expected for the negative control (Figure V-9, lanes 1, 3, 7, and 9).

As a positive hybridization control, the blot contained plasmid PV-ZMAP595 that was digested with a combination of Blp I and Xba I and mixed with pre-digested control DNA. Results of this experiment produced an expected band which migrated at approximately 3.1 kb (Figure V-9, lanes 5 and 6).

MON 87460 DNA digested with a combination of Eco O109 I and Not I (Figure V-9, lanes 2 and 8) and hybridized with probe 10 produced the expe cted band of 1.7 kb (Figure V-1). Although difficult to observe in Figure V-9, overexposure of the Southern blots show a faint unexpected band of appr oximately 2.5 kb that is likely a result of partial digestion. The 2.5 kb band is consiste nt with partial dige stion of genomic DNA

because there is an Eco O109 I site at position 875 in the 5' flanking genomic DNA (Figure V-1). MON 87460 DNA digested with Eco R V (Figure V-9, lanes 4 and 10) and hybridized with probe 10 produced the expect ed bands of approximately 2.7 and 7.5 kb. The approximately 7.5 kb band is consistent with the expected band being greater than 2.2 kb and the approximately 2.7 kb band is the e xpected size for the 3 ́ border fragment (Figure V-1). The 2.7 kb band is less in tense than the approximately 7.5 kb band probably due to a smaller portion of the tr7 probe hybridizing to the 2.7 kb fragment.Monsanto Company 07-CR-191U Page 121 of 544 There were no additional ba nds detected using the tr7 3' nontranslated probe. Based on the results presented in Figure V-9, MON 8746 0 contains no additional, detectable tr7 3' nontranslated sequence elements other than those associated with the intact cspB cassette.

V.C.5. loxP + 35S Promoter (Probe 11)

The Southern blot used to confirm the copy number of the loxP + 35S Promoter sequences (Figure V-10) cont ained several controls. To determine if any endogenous background hybridization bands were detected when probing with probe 11, the blot contained conventional corn genomic DNA digested with a combination of Eco O109 I

and Not I (Figure V-10, lanes 1 and 7) or Eco R V (Figure V-10, lanes 3 and 9). The results of this analysis show no detectable hybridization bands, as expected for the negative control (Figure V-10, lanes 1, 3, 7, and 9).

As a positive hybridization control, the blot contained plasmid PV-ZMAP595 that was digested with a combination of Blp I and Xba I and mixed with pre-digested control DNA. Results of this experiment produced the expected size band at 6.1 kb (Figure V10, lanes 5 and 6).

MON 87460 DNA digested with a combination of Eco O109 I and Not I (Figure V-10, lanes 2 and 8) and hybridized with probe 11 produced the expected single unique band of approximately 3.2 kb. This is consistent with the expected band being greater than 2.7 kb (Figure V-1). MON 87460 DNA digested with Eco R V (Figure V-10, lanes 4 and 10) and hybridized with probe 11 produced the expe cted single unique band of 2.7 kb (Figure V-1). As there were no unexpected bands de tected, the results presented in Figure V-10 show that MON 87460 contains no additional, detectable loxP sequence or 35S promoter
elements other than those a ssociated with the intact nptII cassette

.

V.C.6. nptII Coding Sequence (Probe 12)

The Southern blot used to confirm the copy number of the nptII Coding Sequence (Figure V-11) contained several controls. To determine if any endogenous background hybridization bands were det ected when probing with pr obe 12, the blot contained conventional corn genomic DNA di gested with a combination of Eco O109 I and Not I (Figure V-11, lanes 1 and 7) or Eco R V (Figure V-11, lanes 3 and 9). The results of this analysis show no detectab le hybridization bands.

As a positive hybridization control, the blot contained plasmid PV-ZMAP595 that was digested with a combination of Blp I and Xba I and mixed with pre- digested control DNA and hybridized with probe 12. Results of th is experiment produced an expected band which migrated at approximately 5.5 kb (Figure V-11, lanes 5 and 6).

MON 87460 DNA digested with a combination of Eco O109 I and Not I (Figure V-11, lanes 2 and 8) and hybridized with probe 12 produced the expected single unique band of approximately 3.2 kb (Figure V-1). This band in the long run a ppears slightly larger than the corresponding band in the short run, most lik ely due to better resolution of the band in the long run. This band size is consistent with the expected band being greater than 2.7 kb. MON 87460 DNA digested with Eco R V (Figure V-11, lanes 4 and 10) andMonsanto Company 07-CR-191U Page 122 of 544 hybridized with probe 12 produ ced the expected single uni que band of 2.7 kb (Figure V1). There were no additional bands detected using the nptII coding sequence probe. Based on the results presented in Figur e V-11, MON 87460 contains no additional,

detectable nptII coding sequence elements other than those associated with the intact nptII cassette

.

V.C.7. nos 3' Nontranslated sequence + loxP + Left Border Sequence (Probe 13)

The Southern blot used to confirm the copy number of the nos 3' Nontranslated sequence

+ loxP + Left Border Sequence (Figure V-12) cont ained several controls. To determine if any endogenous background hybridization bands were detected when probing with probe 13, the blot contained conventional corn ge nomic DNA digested with a combination of

Eco O109 I and Not I (Figure V-12, lanes 1 and 7) or Eco R V (Figure V-12, lanes 3 and 9). The results of this analysis show no detectable hybridization bands.

As a positive hybridization control, the blot contained plasmid PV-ZMAP595 that was digested with a combination of Blp I and Xba I and mixed with pre-digested control DNA. Results of this experiment produced the expected size band at 6.1 kb (Figure V12, lanes 5 and 6).

MON 87460 DNA digested with a combination of Eco O109 I and Not I (Figure V-12, lanes 2 and 8) and hybridized with probe 13 produced the expected single unique band of approximately 3.2 kb (Figure V-1). This is consistent with the expected band being greater than 2.7 kb (Figure V- 1). MON 87460 DNA digested with Eco R V (Figure V-12, lanes 4 and 10) and hybridized with probe 13 produced the expected single unique band of 2.7 kb (Figure V-1). As there were no unexpected bands detected, the results presented in Figure V-12 show that MON 87460 contains no additional, detectable nos 3' nontranslated sequence, loxP sequence or left border sequence elements other than those

associated with the intact nptII cassette

.Monsanto Company 07-CR-191U Page 123 of 544 Figure V-6. Southern Blot Analysis of MON 87460: P- Ract1

The blot was hybridized with a 32 P-labeled probe that spanned the Right Border, Ract1 promoter and leader (Figure V-3, probe 7). Each lane c ontains approximately 10 μg of digested genomic DNA isolated from seed. Lane designations are as follows: 1: Conventional ( Eco O109 I/ Not I) 2: MON 87460 ( Eco O109 I/ Not I) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~0.1 copy] 6: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 7: Conventional ( Eco O109 I/ Not I) 8: MON 87460 ( Eco O109 I/ Not I) 9: Conventional ( Eco R V) 10: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

7.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 6.1 4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20 40

8.1

6.1 15 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

7.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 6.1 7.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 6.1 7.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 6.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 6.1 4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20 40

8.1

6.1 15

4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20 40

8.1

6.1 15

4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20

4.1

2.0 10

1.6 7.1

5.1

3.1

1.0

0.5 20 40

8.1

6.1 15Monsanto Company 07-CR-191U Page 124 of 544 Figure V-7. Southern Blot Analysis of MON 87460: I- Ract1

The blot was hybridized with a 32 P-labeled probe that spanned the Ract1 intron (Figure V-3, probe 8). Each lane contains approximately 10 μg of digested genomic DNA isolated from seed. Lane designations are as follows: 1: Conventional ( Eco O109 I/ Not I) 2: MON 87460 ( Eco O109 I/ Not I) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~0.1 copy] 6: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 7: Conventional ( Eco O109 I/ Not I) 8: MON 87460 ( Eco O109 I/ Not I) 9: Conventional ( Eco R V) 10: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 8.1

6.1 40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 20 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 15

6.1

4.1 40

8.1 1 2 3 4 5 6 7 8 9 10

LONG RUN SHORT RUN

8.1

6.1 40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 20

8.1

6.1 40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 20

8.1

6.1 40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 20 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 15

6.1

4.1 40

8.1 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 15

6.1

4.1 40

8.1 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 15

6.1

4.1 40

8.1 1 2 3 4 5 6 7 8 9 10

LONG RUN SHORT RUNMonsanto Company 07-CR-191U Page 125 of 544 Figure V-8. Southern Blot Analysis of MON 87460: CS- cspB

The blot was hybridized with a 32 P-labeled probe that spanned the cspB coding sequence (Figure V-3, probe 9). Each lane contains approximately 10 μg of digested genomic DNA isolated from seed. Lane designations are as follows: 1: Conventional ( Eco O109 I/ Not I) 2: MON 87460 ( Eco O109 I/ Not I) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~0.1 copy] 6: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 7: Conventional ( Eco O109 I/ Not I) 8: MON 87460 ( Eco O109 I/ Not I) 9: Conventional ( Eco R V) 10: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 15

8.1

6.1 4.1 10

20 6.1 7.1 40 20

0.5 2.0

1.6

1.0 3.1 5.1 8.1

40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

15

8.1

6.1 4.1 10

20 6.1 7.1 40 20

0.5 2.0

1.6

1.0 3.1 5.1 8.1

40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

8.1

6.1 4.1 10

20 6.1 7.1 40 20

0.5 2.0

1.6

1.0 3.1 5.1 8.1

40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

20 6.1 7.1 40 20

0.5 2.0

1.6

1.0 3.1 5.1 8.1

40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

7.1 40 20

0.5 2.0

1.6

1.0 3.1 5.1 8.1

40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 40 20

0.5 2.0

1.6

1.0 3.1 5.1 8.1

40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUNMonsanto Company 07-CR-191U Page 126 of 544 Figure V-9. Southern Blot Analysis of MON 87460: T- tr7

The blot was hybridized with a 32 P-labeled probe that spanned the tr7 3' nontranslated sequence (Figure V-3, probe 10). Each lane contains approximately 10 μg of digested genomic DNA isolated from seed. Lane designations are as follows: 1: Conventional ( Eco O109 I/ Not I) 2: MON 87460 ( Eco O109 I/ Not I) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~0.1 copy] 6: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 7: Conventional ( Eco O109 I/ Not I) 8: MON 87460 ( Eco O109 I/ Not I) 9: Conventional ( Eco R V) 10: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 20

7.1 8.1 40

10 15

5.1

4.1

3.1

2.0

1.6 6.1 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 15

6.1

4.1 40

8.1 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

20

7.1 8.1 40

10 15

5.1

4.1

3.1

2.0

1.6 6.1 20

7.1 8.1 40

10 15

5.1

4.1

3.1

2.0

1.6 6.1 20

7.1 8.1 40

10 15

5.1

4.1

3.1

2.0

1.6 6.1 8.1 40

10 15

5.1

4.1

3.1

2.0

1.6 6.1 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 15

6.1

4.1 40

8.1 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 15

6.1

4.1 40

8.1 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 7.1 10 20

0.5 2.0

1.6

1.0 3.1 5.1 15

6.1

4.1 40

8.1 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUNMonsanto Company 07-CR-191U Page 127 of 544 Figure V-10. Southern Blot Analysis of MON 87460: loxP + P- 35S

The blot was hybridized with a 32 P-labeled probe that spanned the loxP sequence and 35S promoter (Figure V-3, probe 11). Each lane c ontains approximately 10 μg of digested genomic DNA isolated from seed. Lane designations are as follows: 1: Conventional ( Eco O109 I/ Not I) 2: MON 87460 ( Eco O109 I/ Not I) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~0.1 copy] 6: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 7: Conventional ( Eco O109 I/ Not I) 8: MON 87460 ( Eco O109 I/ Not I) 9: Conventional ( Eco R V) 10: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 15

6.1 40

8.1

8.1

6.1 40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 20

1.0 5.1 7.1 10 20

0.5 2.0
1.6

1.0 3.1 4.1 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

15

6.1 40

8.1

8.1

6.1 40

15

10

7.1

5.1

4.1

3.1

2.0

1.6 20

1.0 5.1 7.1 10 20

0.5 2.0
1.6

1.0 3.1 4.1 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUNMonsanto Company 07-CR-191U Page 128 of 544 Figure V-11. Southern Blot Analysis of MON 87460: CS- nptII

The blot was hybridized with a 32 P-labeled probe that spanned the nptII coding sequence (Figure V-3, probe 12). Each lane contains approximately 10 μg of digested genomic DNA isolated from seed. Lane designations are as follows: 1: Conventional ( Eco O109 I/ Not I) 2: MON 87460 ( Eco O109 I/ Not I) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~0.1 copy] 6: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 7: Conventional ( Eco O109 I/ Not I) 8: MON 87460 ( Eco O109 I/ Not I) 9: Conventional ( Eco R V) 10: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 8.1 20 40

10 15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 10

5.1

2.0

1.6 7.1

3.1

1.0

0.5 20

8.1 40

4.1 6.1 15 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

8.1 20 40

10 15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 8.1 20 40

10 15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 8.1 20 40

10 15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 40

10 15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 10

5.1

2.0

1.6 7.1

3.1

1.0

0.5 20

8.1 40

4.1 6.1 15 10

5.1

2.0

1.6 7.1

3.1

1.0

0.5 20

8.1 40

4.1 6.1 15 10

5.1

2.0

1.6 7.1

3.1

1.0

0.5 20

10

5.1

2.0

1.6 7.1

3.1

1.0

0.5 20

5.1

2.0

1.6 7.1

3.1

1.0

0.5 20

8.1 40

4.1 6.1 15 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUNMonsanto Company 07-CR-191U Page 129 of 544 Figure V-12. Southern Blot Analysis of MON 87460: T- nos + loxP + B-Left Border

The blot was hybridized with a 32 P-labeled probe that spanned the nos 3' nontranslated sequence,

loxP sequence and Left Border sequence (Figur e V-3, probe 13). Each lane contains approximately 10 μg of digested genomic DNA isolated from seed. Lane designations are as

follows: 1: Conventional ( Eco O109 I/ Not I) 2: MON 87460 ( Eco O109 I/ Not I) 3: Conventional ( Eco R V) 4: MON 87460 ( Eco R V) 5: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~0.1 copy] 6: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 7: Conventional ( Eco O109 I/ Not I) 8: MON 87460 ( Eco O109 I/ Not I) 9: Conventional ( Eco R V) 10: MON 87460 ( Eco R V) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 4.1

2.0 7.1 10

1.6 5.1

3.1

1.0

0.5 20 40

8.1

6.1 15 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUN

8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 8.1 40

10 20

15

5.1

4.1

3.1

2.0

1.6

1.0 7.1

6.1 4.1

2.0 7.1 10

1.6 5.1

3.1

1.0

0.5 20 40

8.1

6.1 15

4.1

2.0 7.1 10

1.6 5.1

3.1

1.0

0.5 20 40

8.1

6.1 15

4.1

2.0 7.1 10

1.6 5.1

3.1

1.0

0.5 20

4.1

2.0 7.1 10

1.6 5.1

3.1

1.0

0.5 20

10

1.6 5.1

3.1

1.0

0.5 20 40

8.1

6.1 15 1 2 3 4 5 6 7 8 9 10 LONG RUN SHORT RUNMonsanto Company 07-CR-191U Page 130 of 544 V.D. Southern Blot Analyses of MON 87460 across Multiple Generations

In order to assess the stab ility of the inserted DNA in MON 87460 across generations, Southern blot analyses were performed usi ng DNA obtained from multiple generations of MON 87460. For reference, the breeding diagram of MON 87460 is presented in Figure V-13. The specific generations tested are indicated in the legends of Figures V13, V-14 and V-15.

V.D.1. Generational Stability of the Insert

DNA samples from seven generations of MON 87460 were isolated and subjected to digestion with Eco R V (refer to generations indicated in bold in Figure V-13). Digestion

of the test materials with Eco R V was expected to releas e two border fragments with expected sizes of 2.7 kb and >2.2 kb (F igure V-1). The blot was hybridized simultaneously with three radiolabeled probe s that span the entire T-DNA sequence of plasmid PV-ZMAP595 (Figure V-2, probes 1-3) . The hybridization bands detected in each generation are compared to the fully characterized R3F1 [(LH59 R3×LH244)F 1 ] generation to determine insert stability.

The Southern blot used to confirm genera tional stability of the T-DNA (Figure V-14)

contained several controls. To determin e if any endogenous background hybridization bands were detected when hybridizing with th e three radiolabeled probes that span the entire T-DNA, the blot contained c onventional control DNA digested with Eco R V (Figure V-14, lane 1). The re sults of this analys is show several detectable hybridization bands. These hybridization signals result from the probes hybridizing to endogenous targets residing in the corn genome and are not specific to the inserted DNA.

To ensure that each probe was capable of hybridizing to its respective target, the blot contained probe template spik es (Figure V-2, probes 1-3) that were generated from plasmid PV-ZMAP595 and mixed at differe nt concentrations with control DNA pre-digested with Eco R V (Figure V-14, lanes 2 and 3). When hybridized with three overlapping 32 P-labeled probes that span the entire T-DNA (Figure V-2, pr obes 1-3), the expected hybridization bands at approxima tely 1.4, 1.6, and 2.0 kb we re detected. The 0.1 and 1 copies of the 1.4 kb band are faint in comparis on to the 1.6 and 2.0 kb bands, but were clearly detectable. The detection of the probe template positive hybridization controls demonstrates that all three probes ar e hybridizing to the target DNA. To ensure that the probes were capable of hybridizing to the plasmid used for transformation, the blot contained plasmid PV-ZMAP595 that was digested with a combination of Blp I and

Xba I and mixed with control DNA pre-digested with Eco R V (Figure V-14, lane 4). Hybridization with probes 1-3 produced the e xpected size bands at approximately 3.2 and 6.1 kb in addition to the endogenous background.

MON 87460 DNA isolated from multiple ge nerations of MON 87460 (Figure V-13), digested with restriction enzyme Eco R V, and hybridized w ith three overlapping 32 Plabeled probes that span the entire T- DNA (Figure V-2, probes 1-3) produced two hybridization bands at 2.7 kb and approxima tely 7.2 kb (Figure V-14, lanes 5-11). The approximately 7.2 kb band is consistent with the 5' border fragment which was expected to be greater than 2.2 kb (Figure V-1). The 2.7 kb band is the expected size for theMonsanto Company 07-CR-191U Page 131 of 544 border fragment containing the 3' end of the insert and adjacent flanking genomic DNA (Figure V-1). This is the same restriction pattern observed for the F 1 generation (LH59 R3 x LH244) shown in Figure V-4 (lanes 4 and 11). There were no additional unexpected bands detected, in dicating that the single c opy of T-DNA in MON 87460 is stable across the selected generations.

V.D.2. Confirmation of the Absence of PV-ZMAP595 Backbone Sequence

The generations of MON 87460 utilized to assess st ability of the insert were also tested for the presence of backbone sequence by Southern blot analysis. Test and control DNA samples were digested with Eco R V and the blot was hybri dized simultaneously with three radiolabeled probes that span th e entire backbone sequence of plasmid PVZMAP595 (Figure V-2, probes 4-6).

The Southern blot used to confirm the ab sence of the PV-ZMAP 595 backbone sequences (Figure V-15) contained several controls. To determine if any endogenous background hybridization bands were detected when hybridiz ing with the three radiolabeled backbone probes, the blot contained conven tional control DNA digested with Eco R V (Figure V15, lane 1). Several hybridization bands were detected. These signals were produced in all lanes, including those lanes containing the conventional cont rol DNA material, and therefore they are consider ed endogenous background. These hybridization signals result from the probes hybridizing to endogenous targ ets residing in the corn genome and are not specific to the inserted DNA.

To ensure that each of the backbone probes was capable of hybridizing to its respective target, the blot contained probe template spikes (Figure V-2, pr obes 4-6) that were generated from plasmid PV-ZMAP595 and mi xed with the control DNA pre-digested

with Eco R V (Figure V-15, lanes 2 and 3). The exp ected sizes of the three bands are 1.4, 1.7, and 2.0 kb; however, the migrations of the approximately 1.7, 2.1 and 2.5 kb fragments as indicated by the molecular wei ght markers are slightly higher than expected. The altered migra tions may be due to the diffe rence in salt concentrations between the DNA sample and the molecula r weight marker (Sambrook and Russell, 2001). The results show that the three probes hybridized to the target DNA. To ensure the probes were capable of hybridizing to the plasmid used for transformation, the blot contained plasmid PV-ZMAP595 that wa s digested with a combination of Blp I and Xba I and mixed with control DNA pre-digested with Eco R V (Figure V-15, lane 4). Hybridization with probes 4-6 produced the e xpected size bands at approximately 3.2 and 6.1 kb, in addition to the endogenous backgr ound produced by the conventional control

DNA.

MON 87460 DNA isolated from multiple gene rations of MON 87460 (refer to Breeding History of MON 87460, Figure V-13), di gested with restriction enzyme Eco R V, and hybridized with th ree overlapping 32 P-labeled probes that span the backbone sequences of PV-ZMAP595 (Figure V-2, probes 4-6) show no detectable hybridization signals, besides the endogenous background bands (Figure V-15, lanes 5-11). Consistent with results depicted in Figure V-5 (lanes 4 and 11), these results indicate that the generations testedMonsanto Company 07-CR-191U Page 132 of 544 do not contain any detectable backbone se quence from the transformation vector

PV-ZMAP595.

Figure V-13. MON 87460 Breeding Diagram

The (LH59 R 3 × LH244)F 1 generation was used for the molecular characterization of MON 87460. The (LH59 R 2 × LH244) F 1 , (LH59 R 3 × LH244) F 1 , (LH59 R 3 × LH244) F 2 , LH59 R 4 , (LH59 R 4 × HCL301) F 1 , (LH59 R 4 × HCL301) F 2 , and (RP×BC 2 F 1 ) BC 3 F 1 generations were used for generational stability (indicated in bold). The (LH59 R 3 × LH244) F 2 and (LH59

R 4 × HCL301) F 2 generations were used for expr ession and composition analyses. R0 = transformed plant; F(#) = filial generation; ⊗ = self-pollination; BC(#) = backcross generation; RP = recurrent parent for commercial seed development; TI = trait integrati on for commercial seed development. R 0

TI: (RP × LH59 R 4 )BC 0 F 1

TI: (RP × BC 2 F 1 )BC 3 F 1 LH59 R 0

LH59 R 1

LH59 R 2

LH59 LH59 R 3

LH59 R 5 ⊗

⊗

⊗ ⊗

⊗ × LH59

(LH59 R 2 × LH244)F 1

(LH59 R 3 × LH244)F 1

(LH59 R 3 × LH244)F 2

(LH59 R 4 × HCL301)F 1

(LH59 R 4 × HCL301)F 2 × RP

× RP (3 i ) ⊗

⊗Monsanto Company 07-CR-191U Page 133 of 544 Figure V-14. Generational Stability of MON 87460: Insert and Copy Number

The blot was hybridized with three overlapping 32 P-labeled probes that spanned the entire backbone sequence (Figure V-2, probes 1-3). Ea ch lane contains approximately 10 μg of digested genomic DNA isolated from seed. The breeding history of MON 87460 is illustrated in Figure V-13. Lane designations are as follows: 1: Conventional ( Eco R V) 2: Conventional ( Eco R V) spiked with probe templates [~0.1 copy] 3: Conventional ( Eco R V) spiked with probe templates [~1 copy] 4: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 5: MON 87460 [(LH59 R 2 x LH244)F 1 , Eco R V] 6: MON 87460 [(LH59 R 3 x LH244)F 1 , Eco R V] 7: MON 87460 [(LH59 R 3 x LH244)F 2 , Eco R V] 8: MON 87460 (LH59 R 4 , Eco RV) 9: MON 87460 [(LH59 R 4 x HCL301)F 1 , Eco R V] 10: MON 87460 [(LH59 R 4 x HCL301)F 2 , Eco R V] 11: MON 87460 [(RP x BC 2 F 1 )BC 3 F 1 , Eco R V] Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 8.1 40

10 20
15

5.1

4.1

3.1

2.0

0.5 1.0 7.1
6.1

1.6 1 2 3 4 5 6 7 8 9 10 11

8.1 40

10 20
15

5.1

4.1

3.1

2.0

0.5 1.0 7.1
6.1

1.6 8.1 40

10 20
15

5.1

4.1

3.1

2.0

0.5 1.0 7.1
6.1

1.6 8.1 40

10 20
15

5.1

4.1

3.1

2.0

0.5 1.0 7.1
6.1

1.6 1 2 3 4 5 6 7 8 9 10 11Monsanto Company 07-CR-191U Page 134 of 544 Figure V-15. Generational Stability of MON 87460: PV-ZMAP595 Backbone

The blot was hybridized with three overlapping 32 P-labeled probes that spanned the T-DNA (Figure V-2, probes 4-6). Each lane contains approximately 10 μg of digested genomic DNA isolated from seed. The breeding history of M ON 87460 is illustrated in Figure V-13. Lane designations are as follows: 1: Conventional ( Eco R V) 2: Conventional ( Eco R V) spiked with probe templates [~0.1 copy] 3: Conventional ( Eco R V) spiked with probe templates [~1 copy] 4: Conventional ( Eco R V) spiked with PV-ZMAP595 ( Blp I /Xba I) [~1 copy] 5: MON 87460 [(LH59 R 2 x LH244)F 1 , Eco R V] 6: MON 87460 [(LH59 R 3 x LH244)F 1 , Eco R V] 7: MON 87460 [(LH59 R 3 x LH244)F 2 , Eco R V] 8: MON 87460 (LH59 R 4 , Eco RV) 9: MON 87460 [(LH59 R 4 x HCL301)F 1 , Eco R V] 10: MON 87460 [(LH59 R 4 x HCL301)F 2 , Eco R V] 11: MON 87460 [(RP x BC 2 F 1 )BC 3 F 1 , Eco R V] Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 4.4 23.1

6.6 9.4

2.0

0.5 2.3 1 2 3 4 5 6 7 8 9 10 11

4.4 23.1

6.6 9.4

2.0

0.5 2.3 4.4 23.1

6.6 9.4

2.0

0.5 4.4 23.1

6.6 9.4

2.0

0.5 2.3 1 2 3 4 5 6 7 8 9 10 11Monsanto Company 07-CR-191U Page 135 of 544 V.E. Organization of the Insert ed Genetic Elem ents of MON 87460

The organization of the genetic elements wi thin the insert of MON 87460 was confirmed by DNA sequence analyses. Several polymera se chain reaction (PCR) primers were designed to amplify overlapping DNA fragments sp anning the entire length of the insert. The amplified DNA fragments were subjected to DNA sequence analyses. The DNA sequence of the insert contains 3309 base pairs beginning at base 3938 of PV-ZMAP595

located in the P- Ract1 element region, and ending at ba se 7246 in the Left Border region of PV-ZMAP595. There are 733 base pairs of the P- Ract1 element region of PVZMAP595 (base 3205-3937) absent in the M ON 87460 insert and this molecular rearrangement presumably resulted from doubl e-strand break repair mechanisms in the plant during the Agrobacterium- mediated transformation process (Salomon and Puchta, 1998). In addition to the insert DNA sequence, 1121 base pairs of corn genomic DNA flanking the 5' end of the insert and 784 base pairs of corn genomic DNA flanking the 3'

end of the insert were also determined. Results confirm the presence and that the organization of the insert genetic elem ents is as depicted in Table V-1.

V.F. Inheritance of the Genetic Insert in MON 87460

During the development of the MON 87460, tra it segregation data were generated and analyzed. Chi-square analysis was perf ormed over two generati ons to confirm the segregation and stability of the cspB gene in MON 87460. The Ch i-square analysis is based on testing the observed segregation ra tio to the expected segregation ratio according to Mendelian principles. The R 0 plant was self-pollinated to produce R 1 seed, which is expected to segregate 1:2:1 (1 homozygote: 2 hemizygous: 1 null segregant) for the gene. A homozygous selection (R 1 plant) was identified from the segregating population by using an NPTII based Invader assay. The selected R 1 plant was selfpollinated again to produce R 2 seed, which was expected to be fixed for the trait, meaning, all seed are homozygous for the gene.

In additional tests, plants were backcrossed to produce BC 3 F 1 , BC 3 F 2 , BC 3 F 3 , BC 4 F 1 , and

BC 5 F 1 seed. These generations are derived from the R4 generation identified in Figure V-13. The BC 3 F 1 , BC 4 F 1 , and BC 5 F 1 generations were expected to segregate 1:1 (1 positive:1 negative). The BC3F2 genera tion was expected to segregate 1:2:1 (1 homozygote:2 hemizygous:1 null segregant) for the gene. The BC 3 F 3 generation was expected to be fixed for the trait.

The Chi-square test was computed as:



## ∑ −

=


## e e o

x ) ( 2

2

where o = observed frequency of the genotype and e = e xpected frequency of the genotype. The critical Chi-square value at α = 0.05 and 1 degree of freedom is 3.841.Monsanto Company 07-CR-191U Page 136 of 544 The segregation patterns reported in Table V-3 are based on PCR-based assays. The Chisquare values for the R 1 , BC 3 F 1 , BC 3 F 2 , BC 4 F 1 , and BC 5 F 1 generations indicated no significant differences between the observed an d expected segregatio n ratios. The data

for the R 2 and BC 3 F 3 generations confirmed that the popul ations were fixed and that all plants tested positive for the cspB gene. These results are consistent with molecular characterization data indicating single insertion site of the gene and confirm that the cspB/nptII cassette within MON 87460 follows th e expected Mende lian pattern of segregation.

Table V-3. Segregation Patterns of cspB between Generations of MON 87460

Generation Number

of Plants Observed

Positives Observed Negatives Expected

Positives Expected Negatives Chi-Square* Probability
( α = 0.05)

R 1 36 26 10 27 9 0.1481 NS

R 2 89 89 0 89 0 Fixed

⎯

BC 3 F 1 178 84 94 89 89 0.562 NS

BC 3 F 2 154 124 30 115.5 38.5 2.502 NS

BC 3 F 3 474 474 0 474 0 Fixed

⎯

BC 4 F 1 80 44 36 40 40 0.800 NS

BC 5 F 1 82 44 38 41 41 0.439 NS

* The critical Chi-square value at α = 0.05 and 1 degree of freedom is 3.841. cspB – Gene encoding cold shock protein B from Bacillus subtilis

. NS – not significant.

V.G. Conclusions of Molecular Characterization

Molecular analyses show that one intact copy of the cspB and nptII expression cassette was integrated at a single chromosomal locus contained within a ~6.8 kb Hind III restriction fragment. No additional elem ents from the transformation vector PVZMAP595, linked or unlinked to the intact DNA insert, were detected in the genome of MON 87460. Additionally, backbone sequenc e from PV-ZMAP595 was not detected. Generational stability analysis demonstrate that the expected Southern blot fingerprint of

MON 87460 has been maintained across se ven generations of breeding, thereby confirming the stability of the DNA insert over multiple generations. These generations were also shown not to contain any det ectable backbone sequence from plasmid PVZMAP595. In addition, DNA sequence analys es confirmed the sequence identity between the MON 87460 insert and the por tion of the T-DNA from PV-ZMAP595 that was integrated into the corn genome. Thes e results also confirmed the organization of the genetic elements within the cspB and nptII expression cassett es of MON 87460, which was identical to that in plasmid PV -ZMAP595. Analysis of the T-DNA insertionMonsanto Company 07-CR-191U Page 137 of 544 site indicates that there is a 22-bp deleti on of genomic DNA at th e insert-to-plant DNA junction. Segregation analyses show heritability and stability of the cspB and nptII genes occurred as expected across multiple generati ons, which corroborates the molecular insert stability analysis and establishes the gene tic behavior of the DNA insert at a single

chromosomal locus.

V.H. References

Barker, R.F., K.B. Idler, D.V. Thompson and J.D. Kemp. 1983. Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTil5955. Plant Molecular Biology. 2:335-350.

Beck, E., G. Ludwig, E.A. Auerswald, B. Reiss and H. Schaller. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from

transposon Tn5 . Gene. 19:327-336.

Bevan, M., W.M. Barnes and M. Chilton. 1983. Structure and tr anscription of the nopaline synthase gene region of T-DNA. Nucleic Acids Research. 11:369-385.

Dhaese, P., H. d.Greve, J. Gielen, J. Se urinck, M. v.Montague and J. Schell. 1983. Identification of sequences involved in polyadenylati on of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models. The EMBO Journal.

2:419-426.

Fraley, R.T., S.G. Rogers, R.B. Horsch, P. R. Sanders, J.S. Flick, S.P. Adams, M.L. Bittner, L.A. Brand, C.L. Fink, J.S. Fr y, G.R. Galluppi, S.B. Goldberg, N.L. Hoffmann and S.C. Woo. 1983. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences. USA. 80:4803-4807.

McElroy, D., W. Zhang, J. Cao and R. Wu. 1990. Isolation of an efficient actin promoter

for use in rice transformation. Plant Cell. 2:163-171.

McElroy, D., A.D. Blowers, B. Jenes a nd R. Wu. 1991. Construction of expression vectors based on the rice actin 1( Act1 ) 5 ′ region for use in monocot

transformation. Molecular a nd General Genetics. 231:150-160.

Odell, J.T., F. Nagy and N-H. Chua. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature. 313:810-812.

Russell, S.H., J.L. Hoopes and J.T. Odell. 1992. Directed excision of a transgene from the plant genome. Molecular and General Genetics. 234:49-59.

Salomon, S. and H. Puchta. 1998. Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO Journal. 17:6086-6095.

Sambrook, J. and D. Russell. 2001. Chapter 5 Protocol 1: Agarose gel electrophoresis. Pages 5.4 to 5.13 in Molecular cloning: a laboratory manual. 3 rd edition. Cold Spring Harbor Laboratory Pre ss, Cold Spring Harbor, NY.

Willimsky, G., H. Bang, G. Fischer and M. A. Marahiel. 1992. Characterization of cspB , a Bacillus subtilis inducible cold shock gene a ffecting cell viability at low temperatures. Journal of Bacteriology. 174:6326-6335.Monsanto Company 07-CR-191U Page 138 of 544 VI. Characterization of the Intr oduced CSPB and NPTII Proteins

Characterization of the intr oduced proteins is important to establishing the basic components of the food, feed and environm ental safety assessments. This section

summarizes the evaluation of the CSPB and NPTII proteins produced in MON 87460 including: (a) equivalence of the in planta -produced proteins to the recombinant E. coli

produced proteins used in protein safety studi es, (b) the expression levels of the proteins determined in corn tissues, and (c) a summar y of the food and feed safety assessment of the CSPB and NPTII proteins.

VI.A. CSPB Protein

VI.A.1. Identity and Function of the CSPB Protein

The CSPB protein in MON 87460 belongs to th e cold-shock protein (CSP) family and is identical in amino acid sequence to th e native CSPB protein produced in B. subtilis with the exception of one amino acid change in the second position from leucine to valine that was necessary for cloning purpos es. Bacterial CSPs are composed of approximately 67- 73 amino acid residues (Graumann et al., 1997) and although typically acidic in nature, contain several positively charged amino acid residues that may facilitate binding to the negatively charged backbones of polynucleotides . Accumulation of the CSPB protein in

B. subtilis cells occurs after tran sition from exponential grow th to stationary phase (Graumann et al., 1997; Graumann and Ma rahiel, 1999), indicating that CSPB

accumulation in cells can be triggered under several stress conditions that share a common signal such as inactivat ion of ribosomes (Schindler et al., 1999; Graumann et al., 1997). Stability of the protein both in vivo and in vitro depends on the protein’s ability to form a complex with nucleic acids, most likely mRNAs (Schindler et al., 1999). In the absence of polynucleic acids, the C SPB protein has a very low thermodynamic stability and is susceptible to rapid proteo lytic degradation (Schi ndler et al., 1999).

The structure of CSPB protein has been previously described (PDB accession number 1NMF) (Schindelin et al., 1993; Schindelin et al., 1994). The CSPB protein in

MON 87460 consists of 66 amino acids and has an isoelectric point of 4.31. The protein is composed of five antiparallel β -strands forming a five-strand β -barrel similar to the structure of CSPA protein from E. coli (PDB accession number 1MJC ) (Schindelin at al., 1993; Newkirk et al., 1994). All CSPs possess binding site s for single stranded nucleic acids called RNA-binding ri bonucleoprotein (RNP) motifs (Newkirk et al., 1994; Schröder et al., 1995). CSPB protein, like other CSPs, contains two conserved RNP motifs: RNP1 and RNP2 (Figure VI-1, shown in boxes). Within the CSPB RNP domains four aromatic amino acids F15, F27, H29, and F30 (Figure VI-1, highlighted in bold and underlined) are required for the double-stra nded polynucleotide unw inding or “melting” activity exhibited by CSPB. These amino acids are conserved in all CSPs and thought to be essential for protein function in bacteria (Phadtare et al., 2002). In vitro studies suggest that by binding to RNA secondary structures, CSPs reduce the free energy required for misfolded RNA to unfold and a dopt the correct configuration (Herschlag, 1995). Experimental evidence suggests that CSPs bind at the single-stranded mRNA loop and then progressively cover this region, forcing the stem to ope n (Phadtare et al., 2002). It was suggested that CSPs bind to single stranded nucleic acids, RNA andMonsanto Company 07-CR-191U Page 139 of 544 ssDNA, but do not appear to bind to dsDNA (Max et al., 2006). The stable association of

CSPs with nucleic acids has been co nfirmed by co-crystallization of the B. subtilis CSPB protein in a complex with single stranded pol ynucleotides (Bienert et al., 2004; Max et al., 2007). The crystal structure data re vealed the stoichio metry and sequence determinants of the binding of single-stranded nucleic acids to a preformed site on CSPB. These findings together with the described m echanism of RNA destabilization led to the classification of CSPs as RNA chaperones.

The nucleic acid unfolding (“melting”) function of CSPB can be demonstrated in an in

vitro assay using a molecular beacon system (Phadtare and Severinov, 2005). The hairpin-shaped molecule beacon is labeled with a fluorophore at the 5’- and quencher at

the 3’-terminus. Due to the close proximity of the fluores cent tag and quencher in the hairpin conformation, the fluorescence is effi ciently quenched. When a CSPB protein “melts” the hairpin conformation, the fluorescen t tag and quencher are spatially separated which permits fluorescence. This assay has been broadly utilized to characterize the specificity of a variety of CSPs including cold-shock domain (CSD)-c ontaining proteins identified in bacteria and plants (Karlson et al., 2002; Kim et al., 2007; Phadtare et al., 2002).

Figure VI-1. Protein Sequence of Bacillus subtilis CSPB as Expressed in

MON 87460

The relative position of the β− sheets regions and the conserved RNA binding motifs

RNP1 and RNP2 are indicated.

VI.A.2. Characterization of CSPB Protein Produced in MON 87460

The expression levels of CSPB protein in di fferent tissues of MON 87460 are relatively low. Therefore, it was necessary to produce the protein in a high-expressing, recombinant microorganism in order to obtai n sufficient quantities of the protein for safety studies. A recombinant CSPB protein was produced in E. coli , the sequence of which was engineered to match that of CSPB protein produced in MON 87460. The equivalence of the physicochemical charact eristics and functional activity between the MON 87460-produced and E. coli -produced CSPB protein was confirmed by a panel of analytical techniques, in cluding sodium dodecyl su lfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis, matrix assisted laser RNP2 RNP1 VEGKVKWFNSEKG F GFIEVEGQDDV F V HF SAIQGEGFKTLEEGQAVSFE IVEGNRGPQAANVTKEA 

### β 1 β 2 β 3 β 4 β 5Monsanto Company 07-CR-191U Page 140 of 544 desorption/ionization time-of-flight mass spect rometry (MALDI-TOF MS), glycosylation analysis, and assay of biologi cal activity. The details of the materials, methods, and results are described in Appendix B

, while the conclusions are summarized as follows.

A comparison of the MON 87460 -produced CSPB to the E. coli -produced CSPB reference protein standard confirmed th e identity of the MON 87460-produced CSPB protein and established the equivalence of the plant produced protein to the E. coli

produced CSPB reference protein standard. The molecular weight of the MON 87460-

and E. coli -produced CSPB proteins was esti mated by SDS-PAGE. The SDS-PAGE demonstrates that the proteins migrated at the same molecular weight, indicating that the CSPB proteins from both sources are equi valent in their molecular weight. The electrophoretic mobility and immunoreactiv e properties of the MON 87460-produced CSPB protein were shown to be equivalent to those of the E. coli -produced CSPB reference standard. The N-terminus of the MON 87460-produced CSPB was consistent with the predicted amino acid sequence translated from the cspB coding sequence, and the MALDI-TOF mass spectrometry analysis yi elded peptide masses consistent with the expected peptide masses from the translated cspB coding sequence. The MON 87460-

and the E. coli -produced CSPB reference standard were also found to be equivalent based

on the functional activi ties and the lack of glycosylati on. Taken together, these data provide a detailed characterization of the CSPB protein isolated from MON 87460 and established its equivalence to the E.coli -produced CSPB refere nce protein standard.

VI.B. NPTII Protein

VI.B.1. Identity and Function of the NPTII Protein

The NPTII protein functions as a selectable marker in the initial laboratory stages of plant cell selection following transf ormation (Horsch et al., 1984; DeBlock et al., 1984). The NPTII enzyme uses ATP to phosphorylate neomycin and related aminoglycoside antibiotics, thereby inactivating them. Cells that produce the NPTII enzyme selectively survive exposure to these aminoglycosides. The nptII coding sequence is derived from the prokaryotic E. coli transposon Tn5 (Beck et al., 1982). The purpose of inserting the gene encoding the NPTII protein into corn cells along with CSPB was to have an effective method for selecting ce lls after transformation. In general, the efficiency of plant cells transformation is often low, ranging from 1x10 -5 to lx10 -4 of cells treated (Fraley et al., 1983). Therefore, the selectable marker, NPTII, was used to facilitate the screening process.

VI.B.2. Characterization of the NP TII Protein Produced in MON 87460

The NPTII protein produced in MON 87460 was characterized, and its equivalence to a previously characterized E. coli -produced NPTII reference substance was demonstrated. Demonstration of the equivalence between E. coli - and MON 87460-produced NPTII proteins allows utilization of previ ous safety assessmen t data performed on E. coliproduced NPTII to confirm the safety of th e NPTII protein in MON 87460. The analyses employed for the characterization of MON 87460-produced NPTII protein and establishment of the equivalence between MON 87460- and E. coli -produced proteinsMonsanto Company 07-CR-191U Page 141 of 544 included western blot and SDS-PAGE analyses . The details of the materials, methods, and results are described in Appendix C.

The NPTII protein from MON 87460 was characterized and compared to the E. coli

produced NPTII reference protein standard. Th e results of this analysis confirmed the identity of the MON 87460-produced NPTII prot ein and established the equivalence of the plant produced protein to the E. coli -produced NPTII referen ce protein standard. A western blot analysis was utilized to compare the immunoreactivity and apparent molecular weight of the MON 87460-produced NP TII protein to that of the previously

characterized E. coli -produced NPTII reference protei n standard. Th e MON 87460- and

E. coli -produced NPTII proteins displayed sim ilar immunoreactivity with NPTII-specific antibody and had identical electromobility on SDS-PAGE. Taken together, these data establish equivalence betw een the MON 87460-produced and E.coli -produced NPTII reference protein standard.

VI.C. Expression Levels of CSPB and NPTII Proteins in MON 87460

The levels of the CSPB and NPTII protei ns in various tissues of MON 87460 were determined using enzyme-linked immunosorbe nt assays (ELISA). The materials and methods for the ELISA analysis, as well as a de scription of the tissue types, are provided in Appendix D. To produce the tissues for analysis, MON 87460 and conventional corn were each planted in field studies conducted during two different growing seasons. The

first season was conducted at six sites in the U.S. durin g 2006 under typical agronomic practices and water conditions. The second season was conducted at four sites in Chile during 2006/2007 using a st rip-plot design to establish tw o water treatment levels (wellwatered and water-limited) to assess for any changes in CSPB and NPTII protein levels

under different soil moisture conditions. Th e sites were located in the major corngrowing regions of the U.S. and Chile. Addi tional information on the water management for each study is provided in Section VIII. C. Forage, stover, silk, pollen, and grain samples were collected at appropriate times of plant development. Over-season leaf (OSL), over-season root (OSR), and over-s eason whole plant (OSW P) samples were collected four times (1-4) over the season corresponding to plant growth stages V2-V4, V6-V8, V10-V12, and pre-VT (p re-tasseling), respectively. The expression levels of CSPB and NPTII proteins in these tissue s are shown in Tables VI-1 to VI-4.

CSPB expression in MON 87460 is driven by the rice actin constitutive promoter and thus is expected to occur in all plant tissues at various levels. The protein was detected in all tissue types with the highest level of e xpression in pollen, followed by leaf, root, silk, forage, grain, stover, senescent root, and forage root. In general, the levels of CSPB protein declined over the growing season in samp les from both field studies. Consistent

with the constitutive nature of the rice actin promoter, no obvious difference was observed in CSPB protein levels in tissu es collected from plants grown under wellwatered or water-limited conditions.

NPTII expression in MON 87460 is driven by the constitutive CaMV 35S promoter, which directs expression across all plant tissues at various le vels. The NPTII protein was

detected in three out of four analyzed tissue types, with the highest level determined inMonsanto Company 07-CR-191U Page 142 of 544 leaves, followed by roots and forage. The level of NPTII protein in grain was below the Limit of Quantitation (LOQ) of the method. As was observed with the CSPB levels and

consistent with the constitutive nature of the CaMV 35S promoter, no obvious difference was observed in NPTII protein levels in tissues collected from plants grown under wellwatered or water-limited conditions.

VI.C.1. CSPB Expression Levels in MON 87460

Results from the U.S. 2006 study show the mean CSPB protein levels across all six sites were highest in pollen (13 μg/g dwt), followed by young leaf (OSL-1, 3.1 μg/g dwt), young root (OSR-1, 1.4 μg/g dwt), silk (1.2 μg /g dwt), forage (0.10 μg/g dwt), grain (0.072 μg/g dwt), stover (0.042 μg/g dwt), se nescent root (0.041 μg/g dwt), and forage root (0.029μg/g dwt) (Table VI-1). In ti ssues harvested throughout the growing season, mean CSPB protein levels in MON 87460 acr oss all sites ranged from 0.47 – 3.1 μg/g dwt in leaf, 0.24 – 1.4 μg/g dwt in root, and 0.67 – 2.8 μg/g dwt in whole plant.

Results from the Chile 2006/2007 study represen t combined-site data from three (CL, CT, LUM) of the four sites. For a site to be included in the combined-site analysis, commercial reference hybrids in the water- limited plots had to exhibit a minimum 15% reduction in yield compared to the same re ference hybrids planted in the well-watered plots. A detailed description of the site inclusion criteria applied to these data is provided

in Section VIII.B.2

. As described in Section VIII.C and Table VIII-3, the QUI site in the Chile 2006/2007 study was not estab lished with the appropriate water stress treatments; therefore, data and analysis for the QUI site are presented in Appendix D. Results show the mean CSPB protein levels across the thr ee sites that were subjected to either wellwatered or water-limited conditions were hi ghest in pollen (25 μg /g dwt well-watered and 27 μg/g dwt water-limited conditions), followed by young leaf (OSL1, 2.8 μg/g dwt

for both well-watered and water-limited c onditions), young root (OSR1, 1.3 μg/g dwt well-watered and 1.5 μg/g dwt water-limite d conditions), silk (0.82 μg/g dwt wellwatered and 1.1 μg/g dwt μg/g dwt water-limite d conditions), forage (0.11 μg/g dwt μg/g dwt well-watered and 0.15 μg/g dwt water-li mited conditions), grain (0.048 μg/g dwt μg/g dwt well-watered and 0.038 μg/g dwt wa ter-limited conditions), stover (0.033 μg/g dwt well-watered and 0.072 μg/g dwt water-limited conditions), senescent root (0.031 μg/g dwt well-watered and 0.052 μg/g dwt wa ter-limited conditions), and forage root (0.039 μg/g dwt μg/g dwt well-watered and 0.076 μg/g dwt water-limited conditions) (Table VI-2). In tissues harvested thr oughout the growing season, mean CSPB protein levels in MON 87460 across all sites ranged from 0.39 – 2.8 μg/g dwt in leaves harvested from plants grown under well-watered and fr om 0.44 – 2.8 μg/g dwt in leaves harvested from plants grown under water-limited condi tions. The mean CSPB protein levels in roots harvested from MON 87460 plants acro ss all sites ranged from 0.031 – 1.3 μg/g dwt in well-watered and 0.052 – 1.5 μg/g dwt in water-limited conditions. In whole plants of MON 87460, the mean CSPB protei n levels were 0.67 – 3.2 μg/g dwt in wellwatered and 0.70 – 2.9 μg/g dwt in water-limited conditions.

VI.C.2. NPTII Expression Levels in MON 87460

Results from the U.S. 2006 study show the m ean NPTII protein levels across all sites under typical agronomic practices were hi ghest in young leaf (OSL1, 2.6 μg/g dwt),Monsanto Company 07-CR-191U Page 143 of 544 followed by roots (OSR1, 0.47 μg/g dwt), and fora ge (0.12μg/g dwt) (T able VI-3). The levels of NPTII protein in grain were be low the NPTII assay Limit of Quantitation (LOQ), which was 0.0047 μ g/g fwt for grain. The range in NPTII protein levels for MON 87460 in leaf, forage, and grain were 0.21 – 0.63, 0.017 – 0.053, and <LOQ μ g/g fresh weight, respectively.

Results from the Chile 2006/2007 study show the mean NPTII protein levels across the three sites were highest in young leaf (OSL 1, 2.4 μg/g dwt well-wate red and 2.6 μg/g dwt water-limited), followed by root (OSR1, 0.51 μg/g dwt in well-watered and 0.48 μg/g dwt water-limited conditions), and forage (0.16 μg/g dwt in well- watered and 0.17 μg/g dwt water-limited conditions) (Table VI-4). Th e levels of NPTII pr otein in grain were below the NPTII assay LOQ (0.0047 μ g/g fwt for grain) for tissue collected from plants

under both well-watered and water-limited cond itions. The NPTII protein levels in

MON 87460 were lower than NPTII levels dete rmined for the equivalent tissue types in

MON 863 that also relied on NPTI I as a selectable marker. The range of NPTII protein levels in MON 863 leaf, forage, and grain were 0.74 – 1.4, 0.17 – 0.23 and <LOQ μ g/g fwt, respectively. The range in NPTII prot ein levels for MON 87460 in leaf, forage, and grain were 0.84-5.0 μg/g dwt well-watered and 0.98-4.0 μg/g dwt water-limited, 0.13- 0.19 μg/g dwt well-watered and 0.14-0.22 μg /g dwt water-limited, and LOD/LOQ μ g/g fresh weight for well-watered and water-limited, respectively.Monsanto Company 07-CR-191U Page 144 of 544 Table VI-1. CSPB Protein Levels in Tissues Collected from MON 87460 Produced in the U.S. during 2006 under Typical Agronomic Conditions

Tissue Type 1 Mean (SD) 2

( μ g/g fwt) 3 Range 4

( μ g/g fwt) Mean (SD) ( μ g/g dwt) 5 Range ( μ g/g dwt) LOQ / LOD ( μ g/g fwt)

OSL-1 0.45 (0.14) 0.24 – 0.77 3.1 (0.93) 2.0 – 5.1 0.015 / 0.0069

OSL-2 0.40 (0.21) 0.18 – 0.80 2.2 (1.2) 1.0 – 4.4 0.015/ 0.0069

OSL-3 0.21 (0.067) 0.10 – 0.29 1.0 (0.30) 0.54 – 1.5 0.015 / 0.0069

OSL-4 0.10 (0.047) 0.034 – 0.19 0.47 (0.25) 0.16 – 0.96 0.015 / 0.0069

OSR-1 0.13 (0.052) 0.060 – 0.24 1.4 (0.55) 0.55 – 2.4 0.0020 / 0.0018

OSR-2 0.11 (0.052) 0.030 – 0.21 1.0 (0.43) 0.25 – 1.6 0.0020 / 0.0018

OSR-3 0.059 (0.026) 0.015 – 0.11 0.43 (0.20) 0.077 – 0.84 0.0020 / 0.0018

OSR-4 0.035 (0.015) 0.012 – 0.063 0.24 (0.10) 0.098 – 0.42 0.0020 / 0.0018

OSWP-1 0.30 (0.13) 0.11 – 0.46 2.8 (1.4) 1.1 – 5.1 0.0045 / 0.0043

OSWP-2 0.18 (0.075) 0.096 – 0.31 1.9 (0.87) 0.86 – 3.4 0.0045 / 0.0043

OSWP-3 0.11 (0.041) 0.067 – 0.22 0.88 (0.29) 0.45 – 1.4 0.0045 / 0.0043

OSWP-4 0.091 (0.049) 0.015 – 0.18 0.67 (0.36) 0.11 – 1.3 0.0045 / 0.0043

Forage Root 0.0055 (0.0034) 0.0020 – 0.015 0.029 (0.013) 0.014 – 0.055 0.0020 / 0.0018

Senescent

Root 0.0040 (0.0013) 0.0022 – 0.0068 0.041 (0.084) 0.014 – 0.36 0.0020 / 0.0018

Forage 0.027 (0.0095) 0.011 – 0.046 0.10 (0.032) 0.041– 0.17 0.0045 / 0.0043

Stover 0.018 (0.013) 0.0056 – 0.047 0.042 (0.024) 0.013 – 0.090 0.0045 / 0.0043

Silk 0.099 (0.024) 0.031 – 0.13 1.2 (0.32) 0.31 – 1.8 0.0075 / 0.0047

Pollen 7.3 (1.6) 5.3 – 10 13 (2.3) 10 – 17 0.050 / 0.045

Grain 0.063 (0.014) 0.040 – 0.089 0.072 (0.015) 0.045 – 0.10 0.0038 / 0.0017

1 Over-season leaf (OSL), over-season root (OSR), and over-season whole plant (OSWP) samples were

collected four times (1-4) over th e season corresponding to plant growth stages V2-V4, V6-V8, V10- V12, and pre-VT (pre-tasseling), respectively.

2 The arithmetic mean and standard deviation (SD) we re calculated for each tissue type across all sites

(n=18 for all tissues except OSL-2, OSWP-2 and forage where n=17, and senescent root where n=16).

3 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a fresh weight (fwt)

basis.

4 Minimum and maximum values were determin ed for each tissue type across all sites.

5 Protein levels are expressed as “ μ g/g” of tissue on a dry weight (dwt) basis. The dry weight values were

calculated by dividing the fresh weight values by the dry weight conversion factors obtained from moisture analysis data.Monsanto Company 07-CR-191U Page 145 of 544 Table VI-2. CSPB Protein Levels in Tissues Collected from MON 87460 Produced in Chile during 2006/2007 under Well-Wat ered and Water-Limited Conditions

Well-Watered Water-Limited

Tissue Typ e 1 Mean (SD) 2

Range 3

( μ g/g fwt) 4 Mean (SD) Range ( μ g/g dwt) 5 Mean (SD) Range ( μ g/g fwt) Mean (SD) Range ( μ g/g dwt) LOQ / LOD ( μ g/g fwt)

OSL-1 0.50 (0.19) 2.8 (1.0) 0.50 (0.20) 2.8 (0.95) 0.015/0.0069 0.28 - 0.80 1.7 - 4.5 0.26 - 0.80 1.7 - 4.2

OSL-2 0.48 (0.18) 2.6 (1.2) 0.47 (0.15) 2.6 (1.0) 0.015/0.0069 0.21 - 0.69 0.96 - 3.8 0.23 - 0.62 1.1 - 3.6

OSL-3 0.13 (0.10) 0.56 (0.48) 0. 11 (0.073) 0.45 (0.32) 0.015/0.0069 0.023 - 0.33 0.10 - 1.5 0.023 - 0.25 0.086 - 1.1

OSL-4 0.10 (0.041) 0.39 (0.13) 0. 11 (0.054) 0.44 (0.17) 0.015/0.0069 0.040 - 0.14 0.18 - 0.58 0.050 - 0.20 0.22 - 0.69

OSR-1 0.13 (0.029) 1.3 (0.29) 0. 14 (0.034) 1.5 (0.43) 0.0020/0.0018 0.079 - 0.18 0.79 - 1.8 0.10 - 0.20 0.95 - 2.2

OSR-2 0.086 (0.025) 0.86 (0.25) 0. 10 (0.015) 0.82 (0.092) 0.0020/0.0018 0.070 - 0.13 0.70 - 1.4 0.082 - 0.12 0.74 - 0.95

OSR-3 0.061 (0.012) 0.49 (0.12) 0. 054 (0.012) 0.41 (0.13) 0.0020/0.0018 0.035 - 0.075 0.27 - 0.62 0.036 - 0.076 0.24 - 0.63

OSR-4 0.045 (0.012) 0.31 (0.076) 0. 058 (0.016) 0.40 (0.087) 0.0020/0.0018 0.032 - 0.067 0.22 - 0.45 0.036 - 0.084 0.28 - 0.52

OSWP-1 0.32 (0.11) 3.2 (0.98) 0. 30 (0.092) 2.9 (0.84) 0.0045/0.0043 0.18 - 0.52 1.8 - 4.8 0.20 - 0.42 1.8 - 3.8

OSWP-2 0.19 (0.036) 2.3 (0.54) 0. 18 (0.046) 2.2 (0.61) 0.0045/0.0043 0.12 - 0.24 1.4 - 3.0 0.12 - 0.25 1.4 - 3.1

OSWP-3 0.10 (0.042) 0.89 (0.34) 0. 091 (0.032) 0.71 (0.25) 0.0045/0.0043 0.065 - 0.17 0.59 - 1.4 0.067 - 0.15 0.44 - 1.1

OSWP-4 0.11 (0.026) 0.67 (0.16) 0. 13 (0.037) 0.70 (0.16) 0.0045/0.0043 0.076 - 0.17 0.48 - 0.98 0.10 - 0.20 0.55 - 1.0

Forage Root 0.0052 (0.0018) 0.039 (0.015) 0. 011 (0.0039) 0.076 (0.029) 0.0020/0.0018 0.0026 - 0.0088 0.017 - 0.068 0.0056 - 0.016 0.035 - 0.12

Senescent

Root 0.0040 (0.0017) 0.031 (0.015) 0. 0067 (0.0051) 0.052 (0.040) 0.0020/0.0018 0.0026 - 0.0073 0.020 - 0.061 0.0026 - 0.017 0.019 - 0.14

Forage 0.026 (0.0041) 0.11 (0.018) 0. 035 (0.0078) 0.15 (0.040) 0.0045/0.0043 0.018 - 0.034 0.077 - 0.14 0.022 - 0.047 0.087 - 0.22

Stover 0.011 (0.0023) 0.033 (0.0070) 0. 021 (0.010) 0.072 (0.033) 0.0045/0.0043 0.0071 - 0.014 0.018 - 0.040 0.011 - 0.036 0.035 - 0.12

Silk 0.073 (0.019) 0.82 (0.28) 0. 13 (0.048) 1.1 (0.38) 0.0075/0.0047 0.050 - 0.12 0.50 - 1.5 0.054 - 0.22 0.49 - 1.8

Pollen 18 (5.6) 25 (7.4) 18 (6.5) 27 (10) 0.050/0.045 7.0 - 24 8.9 - 33 12 - 31 18 - 48

Grain 0.041 (0.012) 0.048 (0.014) 0. 033 (0.0067) 0.038 (0.0079) 0.0038/0.0017 0.028 - 0.065 0.033 - 0.075 0.021 - 0.045 0.024 - 0.053

1 Over-season leaf (OSL), root (OSR), and whole plant (OSWP) samples were collected four times (1-4)

corresponding to plant growth stages V2-V4, V6-V8, V10-V12, and pre-VT (pre-tasseling), respectively.

2 The arithmetic mean and standard devi ation (SD) were calculated for each tissue type across all sites (n=9 for

well-watered and n=9 for water-limited, except OSR-2 where n=6 for under both well-watered and waterlimited conditions and senescent root where n=6 for well-watered).

3 Minimum and maximum values were determin ed for each tissue type across all sites.

4 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a fresh weight (fwt) basis.

5 Protein levels are expressed as “ μ g/g” of tissue on a dry weight (dwt) basis. The dry weight values were

calculated by dividing the fresh weight values by the dry weight conversion factors obtained from moisture analysis data.Monsanto Company 07-CR-191U Page 146 of 544 Table VI-3. NPTII Protein Levels in Tissues Collected from MON 87460 Produced in the U.S. during 2006 under Typical Agronomic Conditions

Tissue Type 1 NPTII ( μ g/g fwt) Mean (SD) 2 Range ( μ g/g fwt) 3 NPTII ( μ g/g dwt) Mean (SD) 4 Range ( μ g/g dwt) LOQ / LOD ( μ g/g fwt)

OSL-1 0.37 (0.12) 0.21–0.63 2.6 (0.92) 1.3–4.2 0.0047 / 0.0090

OSR-1 0.041 (0.011) 0.024–0.068 0.47 (0.12) 0.30–0.85 0.0075 / 0.0043

Forage 0.034 (0.011) 0.017–0.053 0.12 (0.049) 0.053–0.20 0.0056/ 0.0024

Grain <LOQ N/A 5 0.0047 / 0.0024

1 Over-season leaf (OSL-1) and over-season root (O SR-1) samples were collected at the V2-V4 plant

growth stage.

2 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a fresh weight (fwt)

basis. The arithmetic mean and standard deviati on (SD) were calculated for each tissue type across all sites (n=18).

3 Minimum and maximum values were determ ined for each tissue type across sites.

4 Protein levels are expressed as μ g/g of tissue on a dry weight (dwt) basis. The dry weight values were

calculated by dividing the fresh weight values by the dry weight conversion factors obtained from moisture analysis data.

5 N/A – Not applicable; dry weight values were not calcula ted if fresh weight values were less than the LOQ.

Table VI-4. NPTII Protein Levels in Tissues Collected from MON 87460 Produced in Chile during 2006/2007 under Well-Wat ered and Water-Limited Conditions

Well-Watered Water-Limited

Tissue Typ e 1 Mean (SD) 2

Range 3

( μ g/g fwt) 4 Mean (SD) Range ( μ g/g dwt) 5 Mean (SD) Range ( μ g/g fwt) Mean (SD) Range ( μ g/g dwt) LOQ / LOD ( μ g/g fwt)

OSL-1 0.42 (0.23) 2.4 (1.3) 0.46 (0.18) 2.6 (0.98) 0.047/0.0090 0.15 - 0.85 0.84 - 5.0 0.16 - 0.68 0.98 - 4.0

OSR-1 0.051 (0.0083) 0.51 (0.083) 0.046 (0.0075) 0.48 (0.097) 0.0075/0.0043 0.041 - 0.064 0.41 - 0.64 0.035 - 0.057 0.39 - 0.64

Forage 0.037 (0.0041) 0.16 (0.020) 0.039 (0.0048) 0.17 (0.028) 0.0056/0.0024 0.031 - 0.044 0.13 - 0.19 0.034 - 0.048 0.14 - 0.22

Grain <LOQ(N/A 6 ) N/A (N/A) <LOQ (N/A) N/A (N/A) 0.0047/0.0024 <LOD-0.0057 N/A <LOD-0.0051 N/A

1 Over-season leaf (OSL-1) and root (OSR-1) samples were collected at the V2 -V4 plant growth stage.

2 The mean and standard deviation (SD) were calculated across sites (n=9 for well-watered and n=9 for

water-limited, except OSL-1 where n=8 for water-limited).

3 Minimum and maximum values were determ ined for each tissue type across sites.

4 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a fresh weight (fwt)

basis.

5 Protein levels are expressed as μ g/g on a dry weight (dwt.) basis. The dry weight values were calculated by

dividing the fwt by the dry weight conversion factors obtained from moisture analysis data.

6 N/A – Not applicable; dry weight values were not calcula ted if fresh weight values were less than the LOQ

or LOD.Monsanto Company 07-CR-191U Page 147 of 544 VI.D. Food and Feed Safety Assessment Sum mary of the CSPB and NPTII Proteins

Numerous factors have been c onsidered in the safety assess ment of the CSPB and NPTII proteins that are expressed in MON 87460. A comprehensive assessment of safety of these proteins was submitted to the FDA. The assessment leads to the following

conclusions.

a) The donor organism of the CSPB protein, B. subtilis , is not pathogenic, is often used as a food additive, is present in many fermented foods, and has a history of safe consumption. In 1999, FDA designate d enzyme preparations from this organism as GRAS (generally recogni zed as safe, FDA, 1999). Proteins containing cold shock domains are ubiquito us in nature, being present in many plants and common bacteria including species that are normally present in gastrointestinal flora. Cold shock pr oteins have no known toxicity and are not associated with pathogenicity.

b) The B. subtilis CSPB shares a high percent of iden tity with CSPs present in other bacterial species broadly used by the food industry and with CSD-containing proteins in plant species us ed as a food. Many foods pr epared with the help of B.

subtilis have been consumed for a long time with no documented history of any

adverse effects to human health. CSPB protein present in MON 87460 shares amino acid identity to othe r naturally occurring CSD-c ontaining proteins found in food and food products. The amino acid identity ranges from 35% to 98.5% across different plant and b acterial species. The CSPB protein is homologous to the CSP proteins found in the genera Lactobacillus

, Lactococcus and Bifidobacterium , and E. coli , which are normally present in gastrointestinal flora and, therefore, considered to be safe. The strains of lac tic acid bacteria, Bifidobacterium and Lactobacillus , are the most common type of bacteria used in the dairy industry for preparation of probi otic products contai ning live bacterial cultures. These bacteria resist gastric aci d, bile salts and pancre atic enzymes, and, thus, readily colonize the intestin al tract (Rolfe, 2000). In addition, Bacillu s, Lactobacillus, and Lactococcus species containing CSPs are involved in many fermentation processes of milk, meats, cereals and vegetables.

c) A dietary safety assessment was conducted to evaluate the risks to humans and animals from the CSPB and NPTII proteins present in the foods and feeds derived from MON 87460. Risks are quantified as a margin of exposure (MOE), which is

defined as the ratio of the No Observab le Effect Level (NOEL) from an acute mouse gavage study to estimates of the diet ary intake of the respective proteins. Acute oral toxicity studies with mice demonstrated that the two proteins are not acutely toxic and do not cause any observed adverse eff ects even at the highest tested dose levels, which are 4.7 a nd 5,000 mg/kg body weight for CSPB and NPTII proteins, respectively. The dietar y safety assessment showed that the MOEs for the overall U.S. population were greater than or equal to 26,700 and 454,000,000 for the CSPB and NPTII proteins, respectively. For children aged 1- 6 years old, an age group with the highe st corn consumption on a body weight basis, the MOEs were greater th an or equal to 11,400 and 208,000,000 for the CSPB and NPTII proteins, respectively. Di etary exposures in animals will also beMonsanto Company 07-CR-191U Page 148 of 544 low, with chickens, swine, and dairy cows consuming only nanogram quantities of each protein per kilogram of body weight.

d) Digestive fate experiments conducted with the CSPB protein demonstrated that the full-length protein is rapidly digested in SGF, a characteristic shared among many proteins with a history of safe c onsumption. A small transiently stable CSPB protein fragment was very quickly degraded during short exposure to SIF. Rapid digestion of the full-length CSPB pr otein in SGF and SIF, together with rapid degradation of the small transiently st able fragment in SIF, indicates that it is highly unlikely that the CSPB protei n and its fragment will reach absorptive cells of the intestinal mucosa. Proteins that are rapidly digestible in mammalian gastrointestinal systems are unlikely to be allergens when consumed. Finally, the CSPB protein represents no more than 0.00007% of the total protein in the grain

of MON 87460.

e) CSPB and NPTII proteins do not share any amino acid sequence similarities with known allergens, gliadins, glutenins, or pr otein toxins which ha ve adverse effects to mammals. This has been shown by extensive assessments with bioinformatic tools, such as FASTA sequence alignmen t tool and an eight-amino acid sliding

window search.

f) The safety of the NPTII pr otein and its donor organism, E. coli , have been recognized by regulatory agencies and well documented. All existing data suggest that the NPTII protein represents a negligible hazard to human health and is safe for consumption.

Using the guidance provided by the FDA, a conclusion of “no concern” is reached for the donor organisms and the CSPB and NPTII proteins. The food and feed products containing MON 87460 or ma de of MON 87460 are safe for human and animal consumption.

VI.E. References

Beck, E., G. Ludwig, E.A. Auerswald, B. Reiss and H. Schaller. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from

transposon Tn5 . Gene. 19:327-336.

Bienert, R., M. Zeeb, L. Dostál, A. Feske, C. Magg, K. Max, H. Welfle, J. Balbach and U. Heinemann. 2004. Single-stranded DNA bound to bacterial cold-shock proteins: preliminary crystallogr aphic and Raman analysis. Acta Crystallographica Section D. 60:755-757.

DeBlock, M., L. Herrera-Estrella, M.V. M ontagu, J. Schell and P. Zambryski. 1984. Expression of foreign genes in regenera ted plants and in their progeny. EMBO

Journal. 3:1681-1689.Monsanto Company 07-CR-191U Page 149 of 544 FDA. 1999. Carbohydrase and protease en zyme preparations derived from Bacillus

subtilis or Bacillus amyloliquefaciens ; affirmation of GRAS status as direct food ingredients, Final Rule . Federal Register 64:198 87-19895, U.S. Food and Drug Administration, Washington, D.C.

Fraley, R.T., S.G. Rogers, R.B. Horsch, P. R. Sanders, J.S. Flick, S.P. Adams, M.L. Bittner, L.A. Brand, C.L. Fink, J.S. Fr y, G.R. Galluppi, S.B. Goldberg, N.L. Hoffmann and S.C. Woo. 1983. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences USA. 80:4803-4807.

Graumann, P.L., T.M. Wendrich, M.H.W. Webe r, K. Schroder and M.A. Marahiel. 1997. A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures. Molecular Microbiology. 25:741-756.

Graumann, P.L. and M.A. Marahiel. 1999. Cold shock proteins CspB and CspC are major stationary-phase-indu ced proteins in Bacillus subtilis . Archives of Microbiology.

171:135-138.

Herschlag, D. 1995. RNA chaperones and th e RNA folding problem. Journal of Biological Chemistry 270(36): 20871-20874.

Horsch, R.B., R.T. Fraley, S.G. Rogers, P.R. Sanders, A. Lloyd and N. Hoffmann. 1984.

Inheritance of functional fore ign genes in plants. Science. 223:496-498.

Karlson, D., K. Nakaminami, T. Toyomasu and R. Imai. 2002. A cold-regulated nucleic acid-binding protein of winter wheat shar es a domain with bacterial cold shock proteins. Journal of Biological Chemistry. 277:35248-35256.

Kim, J.S., S.J. Park, K.J. Kwak, Y.O. Kim, J.Y. Kim, J. Song, B. Jang, C.-H. Jung and H. Kang. 2007. Cold shock domain proteins a nd glycine-rich RNA-binding proteins

from Arabidopsis thaliana can promote the cold adaptation process in Escherichia

coli . Nucleic Acids Research. 35:506-516.

Max, K.E.A., M. Zeeb, R. Bienert, J. Ba lbach and U. Heinemann. 2006. T-rich DNA single strands bind to a preformed site on the bacterial cold shock protein Bs

CspB. Journal of Molecular Biology. 360:702-714.

Max, K.E.A., M. Zeeb, R. Bienert, J. Ba lbach and U. Heinema nn. 2007. Common mode of DNA binding to cold shock domains. Crystal structure of hexathymidine bound to the domain-swapped form of a major cold shock protein from Bacillus aldolyticus . FEBS Journal 274:1265-1279.

Newkirk, K., W. Feng, W. Jiang, R. Teje ro, S.D. Emerson, M. Inouye and G.T.

Montelione. 1994. Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli : identification of a binding epitope for DNA. Proceedings of the National Academy of Sciences USA. 91:5114-5118.

Phadtare, S., and K. Severinov. 2005. Nucleic acid melting by Escherichia coli CspE.

Nucleic Acids Research. 33:5583-5590.Monsanto Company 07-CR-191U Page 150 of 544 Phadtare, S., S. Tyagi, M. Inouye and K. Severinov. 2002. Thr ee amino acids in

Escherichia coli CspE surface-exposed aromatic pa tch are critical for nucleic acid melting activity leading to transcription antitermination and cold acclimation of cells. Journal of Biological Chemistry. 277:46706-46711.

Rolfe, R.D. 2000. The role of probiotic cultures in the control of gastrointestinal health.

Journal of Nutrition. 130:396S-402S.

Schindelin, H., M.A. Marahiel and U. Heinemann. 1993. Univ ersal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein.

Nature. 364:164-168.

Schindelin, H., W. Jiang, M. Inouye and U. Heinemann. 1994. Crystal structure of CspA, the major cold shock protein of Escherichia coli . Proceedings of the National Academy of Sciences USA. 91:5119-5123.

Schindler, T., P.L. Graumann, D. Perl, S.F. Ma, F.X. Schmid and M.A. Marahiel. 1999. The family of cold shock proteins of Bacillus subtilis : stability and dynamics in

vitro and in vivo . Journal of Biological Chemistry. 274:3407-3413.

Schröder, K., P.L. Graumann, A. Schnuchel, T.A. Hlak and M.A. Marahiel. 1995. Mutational analysis of the putative nuclei c acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing th e Y-box motif. Molecu lar Microbiology.

16:699-708.Monsanto Company 07-CR-191U Page 151 of 544 VII. Compositional Assessment

Compositional analyses were conducted to assess the nutrient, anti-nutrient and key secondary metabolite levels in the forage and grain tissues deri ved from MON 87460. Because MON 87460 reduces yield loss under water-limited conditions, field studies were designed to evaluate the composition of MON 87460 across a broad range of soil

moisture and environmental conditions rele vant to where commer cial production would be expected. In each assessment, MON 87460 was compared to an appropriate conventional control, which had a geneti c background similar to MON 87460 but did not possess the drought tolerance trait. In addition, multiple conventional corn hybrids (references) were included in the analysis to establish a range of natural variability for each analyte, where the range of variability is defined by a 99 % tolerance interval for that particular analyte. Results of the comparisons indicate that MON 87460 is compositionally and nutritionally equivalent to conventional corn hybrids that have a history of safe consumption th at are currently in commerce.

The compositional assessment was conducted on forage and grain samples harvested from two different growing seasons using two different water management regimes. The

first season was conducted in the U.S. duri ng 2006 under typical agronomic practices and water conditions (Section VII.A). The second season was conducted in Chile during 2006/2007 using a strip-plot design to establish two water treatment levels (well-watered and water-limited) to assess for any changes in compositional equivalence under different soil moisture conditions (Section VII.B). Re sults from these assessments were published by Harrigan et al. (2009). Samples from the Chile 2006/2007 study were also analyzed for 11 additional secondary metabol ites that are potentially a ssociated with drought stress (Section VII.C). Square brackets in the tables presented in this section denote ranges for

the test and control or 99% tolerance interval s for the reference materials. Materials and methods for the compositional analyses are provided in Appendix E.

The commercial reference hybrids selected for each study were adapted to the geographic region in which they were grown with selec tions based on agronomic characteristics such as relative maturity and drought tolerance ratings. Relative maturity was an important

consideration in order to ensure that the test , control and reference materials would be at comparable stages of development at each data or sample collection time point. Drought tolerance ratings typically spanned a range of ratings exhibited by conventional hybrids.

VII.A. U.S. 2006 Composition Study under Typical Agronomic Conditions

Forage and grain tissues of MON 87460 and c ontrol corn were harvested from plants grown under typical agronomic prac tices with three replicates at each of six field sites in the U.S. within corn production regions. Tw o sites were in Iowa (IAE, IAW), one each was in Illinois (IL), Indiana (IN), Kansas (KS) and Nebraska (NE). Each site received water as is typical of the growing area. F our sites were rainfed (IAE, IAW, IL, IN) and two (KS, NE) received supplemental irrigati on. These conditions provide a comparison

of MON 87460 and the control under conditions common to corn production. Table VII1 presents temperature data and applied water from the production period. Three

different conventional commercial corn hybrids were also grown at each of the six sites.Monsanto Company 07-CR-191U Page 152 of 544 This allowed harvest of forage and grain fr om a total of 18 commercial references to provide information on natural variation in the levels of analyzed nutrients and antinutrients. Compositional analysis included the significant nutrients, anti-nutrients, and key secondary metabolites, consistent with OECD guidelines (OECD, 2002).

The experiment was arranged in a randomized complete block design with three replicates per block. Tissue was collected from MON 87460 and the control from all three blocks; tissue from the three different co mmercial references grown at each site was collected from a single block. Forage was coll ected at the early dent (R5) plant growth stage, and grain was collected at physiological maturity.

The compositional data set was examined for evidence of statistically significant

differences between MON 87460 and the control. Seven sets of statis tical analyses were made, six based on the data from each of the replicated field sites and the seventh based

on data from a combination of a ll six field sites. Statisticall y significant differences were determined at the 5% level of significance (p <0.05) using established statistical methods.

Commercial references were included to provide data for the development of a 99%

tolerance interval for each co mponent analyzed. This interv al is expected to contain, with 95% confidence, 99% of the values obtained from the population of commercial

corn. The tolerance interval illustrates the compositional variability that currently occurs in corn grown commercially. It allows statistically significant differences between MON 87460 and the control to be placed in bi ological perspective. This comparative

evaluation also considers natu ral ranges in corn component levels published in the

literature or in the International Life Sciences Institute (ILSI) Crop Composition Database (http:// www.cropcomposition.org).

VII.A.1. Assessment of Nutrients, Anti-N utrients, and Key Secondary Metabolites

Forage and grain samples were harvested from all plots an d analyzed for nutritional and anti-nutrient components. Compositional an alyses of the forage samples included measurement of proximates (moisture, fat, protein, ash), carbohydr ates by calculation, acid detergent fiber (ADF), neutral detergent fiber (NDF), calcium, and phosphorus. Compositional analyses of the grain samples included measurement of proximates (moisture, fat, protein, ash), carbohydrates by calculation, ADF, NDF, total dietary fiber (TDF), total amino acid composition, fa tty acid composition (C8-C22), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc), vitamins (vitamin B1 [thiamine], vitamin B2 [riboflavin], vita min B6 [pyridoxine], vitamin E, niacin, folic acid) , anti-nutrients (phytic acid , raffinose), and secondary metabolites (ferulic acid, furfural, p -coumaric acid). Methods for analysis were based on internationally-recognized procedures a nd literature publications (Appendix E).

In total, 77 different an alytical components were measured (9 in forage, 68 in grain). Of these evaluated components, 15 had more than 50% of the observations below the assay limit of quantitation (LOQ). Components with more than 50% of observations below the assay LOQ were excluded from statistical analysis. These included 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14 :1 myristoleic acid, 15:0Monsanto Company 07-CR-191U Page 153 of 544 pentadecanoic acid, 15:1 pentadecenoic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gamma linolen ic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, 20:4 arachidonic acid, sodium, and furfural. These components are known to be present at low levels in corn grain (OECD, 2002). Therefore, 62 components (9 in forage and 53 in grain) we re statistically assesse d using a mixed model analysis of variance method.

Statistical evaluation of the composition data involved a comparison of the forage and grain from MON 87460 to those of the control. There were a tota l of 434 comparisons made (seven sets of comparisons × 53 co mponents from grain and seven sets of comparisons × nine components from forage).

A summary of significant differences (p<0.05 ) between test and control in both the combined-site and individual site (total of 6 s ites) analyses is pres ented in Table VII-2. Mean values, ranges, standard error values and statistical an alyses for the combined-site data are presented in Appendix E (Tables E- 5 through E-11). The standard error values presented in Appendix E are calculated from the ANOVA. Thus, each test and control mean for a given analyte mean has the same standard error. Literature and ILSI Crop Composition Database ranges for corn components are presented in Table E-30.

The combined-site analysis show that there were no statistically si gnificant differences (p>0.05) for 59 (95.2%) of the 62 comparisons between the mean component values of

MON 87460 and the control. Of the three significant differences, mean component

values of the test and cont rol substances were within the 99% tolerance interval

established from the commercial references. Th e individual site anal ysis show that there were no statistically significant differe nces (p>0.05) for 348 (93.5%) of the 372 comparisons. Individual site differences we re not reproducible across multiple sites and, where differences were observed, mean comp onent values of the test and control

substances were within the 99% tolerance in terval (Table VII-2). Therefore, these

differences were within the natural va riability of corn for these components.

VII.A.2. Levels of Nutrients

Corn forage and grain contain a variety of ke y nutrients that provide much of this crop’s

value as a food and feed. The OE CD consensus document on compositional

considerations for corn describes the nutrients present in corn grai n or processed corn products and includes proximates, fiber, mine rals, total amino acids, fatty acids (FA) and vitamins (OECD, 2002). A comprehensive comparison of MON 87460 and the control confirms that the two materials are compositi onally equivalent with respect to nutrients.

In the combined-site analysis of forage, no significant differences were found between

MON 87460 and the control. In the combin ed-site analysis of grain, 50 of the 53 comparisons were not significantly different (p>0.05). The three differences were detected in the values for ash, 18:0 stearic acid and 20:1 eicosenoic ac id (Table VII-2). However, values for 18:0 stearic acid were not significantly different (p>0.05) at any of the individual sites. The magnitude of th e combined-site difference in the 18:0 stearic acid value was small (0.069% of total FA) and the mean component values for the testMonsanto Company 07-CR-191U Page 154 of 544 and control substances were within the 99% tolerance interval established from the commercial reference hybrids grown at the same sites. Only one out of the six individual site comparisons for ash and 20:1 eicosenoic acid values showed a significant difference (p<0.05). The magnitude of the differences in ash (0.082 % DW) and 20:1 eicosenoic acid (0.0078% of total FA) was extremely sm all, and the mean values for these two components were within the 99% tolerance in terval established from the commercial references grown at the same sites. Furthe rmore, this lack of reproducibility across multiple sites established that the differences observed in the combined-site analysis in values for these two components were not biologically meaningful. These findings

confirmed that these minor differences reflec ted the natural variab ility of conventional

corn.

In the individual-site analysis of forage, 51 of the 54 comparisons were not significantly different (p>0.05). The three differences were in the values for carbohydrates by calculation, moisture, and prot ein, with each component di fference being observed at only a single site. This lack of reproducibili ty across multiple sites indicated that there were no meaningful trends in differences in the values for these three components and

that this limited number of differences consti tuted no biological significance. For grain nutrients, individual site differences in com ponents not recorded in the combined-site analysis included values for moisture (two sites), and cystine, histidine, lysine, methionine, valine, 18:1 oleic acid, 18:2 lin oleic acid, 18:3 linolenic acid, 22:0 behenic acid, ADF, TDF, thiamine, folic ac id, and riboflavin (each at a si ngle site only). The lack of reproducibility in differences across multiple sites and the fact that the mean values for these components at these sites were within the 99% tolerance inte rval established from

the commercial references confirmed that the limited number of site differences in values for these components were not biologically meaningful.

In summary, statistical analysis highlighted no consistent differences across sites in the levels of nutrient components from MON 8746 0 and the control. The limited number of differences observed in this study reflected th e natural variation of conventional corn and supported the compositional equivalence of MON 87460 to conventional corn.

VII.A.3. Levels of Key Anti-Nutri ents and Secondary Metabolites

The OECD consensus document on compositiona l considerations for corn describes the anti-nutrients and secondary metabolites presen t in corn grain or pr ocessed corn products (OECD, 2002). The anti-nutrients assessed included phytic acid and raffinose. Phytic acid is widely distributed in plants and can lim it the uptake of minerals such as calcium in higher animals (Lott et al., 2000; Nova k and Haslberger, 2000). Raffinose is a nondigestible oligosaccharide that is consid ered to be an anti-nutrient due to gas production and the resulting flatulence cause d by its consumption (Voragen, 1998). The secondary metabolites included ferulic acid, p -coumaric acid, and furfural. Ferulic acid

and p -coumaric acid are derived from the aromatic amino acids, phenylalanine and tyrosine (Douglas, 1999), and serve as precu rsors for a large group of phenylpropanoid compounds. The non-starch polysaccharide pent osans are a major source of furfural (Adams et al., 1997).Monsanto Company 07-CR-191U Page 155 of 544 No combined-site differences (p>0.05) between values for grain anti-nutrient components and secondary metabolites in MON 87460 and the control were recorded. Individual site differences (p<0.05) were observed in values for raffinose, phytic acid, and ferulic acid. For each component, these differences were observed at a single site only. As only one out of six individual site comparisons record ed a significant difference for each of these components, these differences represented no me aningful trend and were considered to be not biologically meaningful. The limited numb er of differences recorded in this study

reflected the natural varia tion of conventional corn.

In summary, statistical analysis highlighted no consistent differences across sites in the levels of anti-nutrient components and secondary metabolites in MON 87460 and the control. Thus, a comprehensive evalua tion of anti-nutrient components and key secondary metabolites supported the compositional equivalence of MON 87460 to

conventional corn.

VII.A.4. Conclusions for U.S. 2006 under Typical Agronomic Practices

The overall dataset was evaluated for evidence of biologically rele vant changes. The combined-site analysis show that there we re no statistically si gnificant differences (p>0.05) for 59 (95.2%) of the 62 comparisons between the mean component values of

MON 87460 and the control. Of the three significant differences observed, mean component values of the test and control substances were within the 99% tolerance

interval established from the commercial refe rences. The individual site analysis show

that there were no statistical ly significant differences (p >0.05) for 348 (93.5%) of the 372 comparisons. Individual site differences we re not reproducible across multiple sites and, where differences were observed, mean compon ent values test and control substances

were within the 99% tolerance interval. Fu rthermore, the limited number of component differences was characterized by small differences in magnitude.

These factors established that the limited nu mber of differences observed in this study

were within the natural variab ility of corn for these compone nts, reflected no meaningful trends, and were of no biological significance.

Therefore, the corn grain and forage de rived from MON 87460, and consequently the foods and feeds derived from MON 87460, can be considered compositionally equivalent to those derived from conventional corn with a history of safe consumption.Monsanto Company 07-CR-191U Page 156 of 544Table VII-1. Monthly Temperature and Accumulated Water Data for the U.S. 2006 StudySite1Measurement May June JulyAugust September OctoberIAEAccumulated water (in.) 2.5 2.3 2.3 6.2 2.4 2.1Avg Max temp (°F) 72 81 86 86 76 59Avg Min temp (°F) 51 60 65 62 49 37Range2(°F) 30 - 98 45 - 97 54 - 95 55 - 96 32 - 94 19 - 94IAWAccumulated water (in.) 1.8 0.4 2.8 4.9 8.7 1.5Avg Max temp (°F) 72 84 88 83 71 60Avg Min temp (°F) 50 59 63 66 48 38Range2(°F) 34 - 91 49 - 93 52 - 99 58 - 93 34 - 86 23 - 90ILAccumulated water (in.) 2.1 1.5 3.7 3.1 2.8 2.7Avg Max temp (°F) 72 81 88 82 75 60Avg Min temp (°F) 51 57 64 62 48 36Range2(°F) 36 - 94 46 - 92 46 - 97 51 - 96 34 - 88 19 - 92INAccumulated water (in.) 4.8 5.5 3.4 5.6 2.4 5.9Avg Max temp (°F) 71 80 86 83 75 63Avg Min temp (°F) 51 58 65 63 53 41Range2(°F) 40 - 92 52 - 89 50 - 96 55 - 93 37 - 87 25 - 90KSAccumulated water (in.) 5.3 8.7 8 10.9 2 1.9Avg Max temp (°F) 81 92 96 92 80 68Avg Min temp (°F) 53 62 66 67 51 42Range2(°F) 37 - 100 52 - 102 54 - 109 52 - 107 36 - 92 23 - 96NEAccumulated water (in.) 1.9 3.8 5.6 9.5 4.8 0.8Avg Max temp (°F) 79 88 91 86 76 63Avg Min temp (°F) 51 61 65 63 50 37Range2(°F) 37 - 96 52 - 101 55 - 103 52 - 100 36 - 93 18 - 961Site codes are as follows: IAE = Benton County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; KS= Pawnee County, KS;NE = York County, NE.2The range is the absolute maximum and minimum temperature in each month.Monsanto Company 07-CR-191U Page 157 of 544Table VII-2. Summary of Significant Differences (p<0.05) Comparing MON 87460 tothe Conventional Control in the U.S.during 2006 under Typical Agronomic ConditionsTissue/Site/Components (Units)1MeanMON 87460MeanControlMean Diff(% of Control)Signif.(p-value) MON 87460 (Range)99% ToleranceInterval2ForageILCarbohydrates (% DW)87.31 85.75 1.81 0.007 (86.23 - 88.01) [82.09, 90.80]INMoisture (% FW)66.23 67.80 -2.31 0.008 (64.70 - 67.40) [59.32, 81.14]Protein (% DW) 6.75 7.23 -6.600.031 (6.68 - 6.90) [4.92, 10.30]GrainCombination of all sitesAsh (% DW)1.54 1.46 5.60 0.041 (1.33 - 1.83) [0.55, 2.30]18:0 Stearic (% Total FA)2.05 1.98 3.50 0.024 (1.88 - 2.34) [1.00, 2.51]20:1 Eicosenoic (% Total FA)0.18 0.19 -4.05 0.007 (0.17 - 0.19) [0.15, 0.33]IAEMoisture (% FW)9.78 9.22 6.08 0.049 (9.57 - 10.00) [7.58, 12.13]Histidine (% DW)0.31 0.32 -2.68 0.032 (0.30 - 0.32) [0.20, 0.36]Methionine (% DW)0.21 0.20 2.45 0.035 (0.20 - 0.21) [0.14, 0.28]Valine (% DW)0.51 0.53 -3.71 0.028 (0.48 - 0.52) [0.33, 0.62]18:3 Linolenic (% Total FA)1.22 1.25 -2.45 0.040 (1.17 - 1.25) [0.39, 1.85]Total Dietary Fiber (% DW)12.23 11.60 5.40 0.029 (11.94 - 12.52) [8.11, 17.95]Raffinose (% DW)0.20 0.15 27.28 0.009 (0.19 - 0.20) [0.039, 0.26]IAW22:0 Behenic (% Total FA)0.24 0.21 12.99 0.015 (0.20 - 0.25) [0, 0.37]Thiamine HCl (mg/kg DW)2.85 2.48 14.81 0.011 (2.77 – 2.9) [1.84, 4.94]Ferulic Acid (μg/g DW) 1753.10 1847.90 -5.13 0.003 (1661.13 - 1914.78) [395.96, 3485.38]Table VII-2 continues on the next page.Monsanto Company 07-CR-191U Page 158 of 544Table VII-2 (cont.). Summary of Significant Differences(p<0.05) Comparing MON 87460 to the Conventional Controlin the U.S. during 2006 under Typical Agronomic ConditionsTissue/Site/Components (Units)1MeanMON 87460MeanControlMean Diff(% of Control)Signif.(p-value) MON 87460 (Range)99% ToleranceInterval2ILMoisture (% FW)9.94 10.43 -4.73 0.013 (9.71 - 10.30)[7.58, 12.13]Folic Acid (mg/kg DW)0.28 0.27 4.19 0.033 (0.27 - 0.29) [0.13, 0.38]INCystine (% DW)0.19 0.20 -5.55 0.039 (0.19 - 0.20) [0.15, 0.27]18:1 Oleic (% Total FA)20.05 20.63 -2.83 0.025 (19.96 - 20.13) [11.92, 39.78]18:2 Linoleic (% Total FA)63.81 63.03 1.24 0.026 (63.67 - 63.95) [45.91, 72.47]Acid Detergent Fiber (% DW)2.55 3.63 -29.68 0.036 (2.52 - 2.60) [0.62, 5.72]Riboflavin/Vitamin B2 (mg/kgDW)1.52 1.09 39.64 0.039 (1.45 - 1.57) [0.047, 2.91]Phytic Acid (% DW)0.63 0.77 -17.45 0.007 (0.60 - 0.66) [0.50, 1.11]KSLysine (% DW)0.33 0.31 4.51 0.045 (0.31 - 0.34) [0.22, 0.36]20:1 Eicosenoic (% Total FA)0.18 0.19 -7.81 0.043 (0.17 - 0.18) [0.15, 0.33]NEAsh (% DW)1.57 1.38 14.01 0.007 (1.49 - 1.62) [0.55, 2.30]1DW= dry weight; FW=fresh weight, FA = fatty acid.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Tables E-5 through E-11 in Appendix E present the full results of the combined site analysis for the compositional analyses from the U.S. 2006 productionincluding means, standard error values and p-values.Monsanto Company 07-CR-191U Page 159 of 544 VII.B. Chile 2006/2007 Composition Study under Well-Watered and Water-Limited

Conditions

Forage and grain samples of MON 87460 and its conventional control we re harvested from plants grown in three replicates at each of f our field sites in Chile in 2006 - 2007. These field

sites were located in commercia l corn production regions of Chile. The sites were Calera de Tango (CT), Colina (CL), Lumbreras (LUM) and Qu illota (QUI). These sites are well-suited for corn production, but as they typically do not receive any rainfall during the growing season, all water rece ived at each site occurred through cont rolled irrigation. At each site, a strip-plot design was used allo wing comparisons of the test a nd control substa nces under two separate irrigation regimens, well-watered and water-limited. The well-watered treatment was managed to provide optimal grain yield. The water-limited treatment was managed to impose a drought stress by withholdi ng irrigation during approxima tely the V10 – R2 growth stages, which represents the growth stages when corn grain yield potentia l is most susceptible to drought stress (Campos et al., 2006). Table VII-3 presents temperature data and applied water from the production period. In additi on to MON 87460 and its conventional control,

four different conventional commercial corn h ybrids were also grown at each site. This allowed harvest of forage and grain from a total of 16 commercial references from each water treatment to provide information on natural variation in the levels of analyzed nutrient

and anti-nutrients under well-watered and wate r-limited conditions. Compositional analysis included components consiste nt with OECD guidelines.

The experiment was arranged in a strip-plot design with three replicates per site, with irrigation treatment (well-watered or water-limite d) as the whole plot and substance type as the sub-plot. The whole plot factor was arranged as a randomized complete block design. The strip-plot factor consisted of the test, control, and reference substances.

Tissue was collected from MON 87460 and the control from all three blocks for each treatment; tissue from the four different co mmercial references grown at each site was collected from a single block for each treatment. Forage was collected at the early dent (R5) plant growth stage; grain was co llected at physiological maturity.

Within each treatment, the composition of fora ge and grain of MON 87460 was compared to

that of the conventional control across sites (combined-site analysis) and within site (individual site analysis). Results from the Chile 2006/2007 study represent combined-sitedata from three (CL, CT, LUM) of the four sites. For a site to be included in the combinedsite analysis, commercial reference hybrids in the water-limited plots had to exhibit a minimum 15% reduction in yield compared to th e same reference hybrids planted in the wellwatered plots. A detailed description of the si te inclusion criteria applied to these data is provided in Section VIII.B.2. As described in Section VIII.C and Table VIII-3, the QUI site in the Chile 2006/2007 study was not establis hed with the appropriate water stress treatments; therefore, data for the QUI site are presented in Appendix E, Section E-8.

Statistical comparisons between MON 87460 and the control were performed within each irrigation treatment. A range of component values and a statistical population were

determined for the commercial references within each irrigation treatment. Thus, four sets of statistical analyses were made for each treatment, three based on the data from each of theMonsanto Company 07-CR-191U Page 160 of 544 replicated field sites and the fourth based on da ta from the combined sites. Statistically significant differences were determined at the 5% level of significance ( α = 0.05) using

established statistical methods.

Commercial references were included to provide data for the development of a 99%

tolerance interval for each com ponent analyzed. This interval is expected to contain, with 95% confidence, 99% of the values obtained from the population of commercial corn. The

tolerance interval illustrates th e compositional variability that o ccurs in corn currently grown commercially. It allows statistically sign ificant differences be tween MON 87460 and the control to be placed in biological perspective. This comp arative evaluation can also consider natural ranges in co rn component levels publishe d in the literature or in the International Life Sciences Institute (ILSI) Crop Composition Database (http://www.cropcomposition.org).

VII.B.1. Assessment of Nutrients, Anti-N utrients, and Key Secondary Metabolites

under Well-Watered Conditions

The well-watered plots provide a compos itional comparison between MON 87460 and a conventional control grown under conditions that are optimal for corn growth and development. Results confirm that MON 87460 and the control are compositionally equivalent when produced unde r well-watered conditions.

Forage and grain samples were harvested fr om all well-watered plots and analyzed for

nutritional and anti-nutrient co mponents as described in Sec tion VII.A.1. In total, 77 different analytical components were measured (nine in forage, 68 in grain). Of these evaluated components, 16 had more than 50% of the observations be low the assay limit of quantitation (LOQ). Components with more than 50% of observations below the assay LOQ were excluded from statistical analysis. These included 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gamma linolenic acid, 20:2 eicosadie noic acid, 20:3 eicosa trienoic acid, 20:4 arachidonic acid, sodium, and furf ural. These components are known to be present at low levels in corn grain (OECD, 2002). Therefore, 61 components (9 in fora ge, 52 in grain) were statistically assessed using a mixed model analysis of variance method.

Statistical evaluation of the composition data involved a comparison of the forage and grain

from MON 87460 to those of the control. Ther e were a total of 244 comparisons made (four sets of comparisons × 52 components from gr ain and four sets of comparisons × nine components from forage).

A summary of significant differences (p<0.05 ) between test and co ntrol in both the

combined-site and individual site (total of 3 sites) analyses for the well-watered treatment is presented in Table VII-4. Mean values, ranges, standard error values and statistical analyses for the combined-site data are presented in Appendix E (Tables E-12 through E-18). The standard error values presented in Appendix E are calculated from the ANOVA. Thus, each test and control mean for a given analyte mean has the same standard error. Literature and ILSI Crop Composition Database ranges for corn components are presented in Table E-30.Monsanto Company 07-CR-191U Page 161 of 544 The combined-site analysis show that there were no statistically significant differences (p>0.05) for 59 (96.7%) of the 61 comparisons between the mean component values of MON 87460 and the control. Of the two sign ificant differences observed, mean component

values of the test and control substances were within the 99% tolerance interval established

from the commercial references. The individu al site analysis show that there were no statistically significant differences (p>0.05) for 171 (93.4%) of the 183 comparisons. Individual site differences were not reproducib le across multiple sites and, where differences were observed, mean component values of the te st and control substances were within the 99% tolerance interval (Table VII-4). Therefore, these differences were within the natural variability of corn for these components.

VII.B.2. Levels of Nutrients under Well-Watered Conditions

A description of nutrients presen t in corn grain is provided in the OECD consensus document on compositional considerations for corn (O ECD, 2002). A comparative assessment of levels of proximates, fiber, minerals, total amino acids, fatty acids, and vitamins follows.

In the combined-site analysis of forage, no significant differences were found between

MON 87460 and the control. In the combin ed-site analysis of grain, 50 of the 52 comparisons were not significantly different (p >0.05). Differences included values for total fat and magnesium. Individual site comparisons between values for total fat and magnesium in MON 87460 and the control grain show a si gnificant difference (p< 0.05) only at a single site. This lack of reproducibility across mu ltiple sites established that the differences

observed in the combined-site analysis in values for thes e two components were of no biological significance. The ma gnitude of the differences in total fat (0.17% DW) and magnesium (0.01% of DW) were extremely sm all, and the mean values for these two components were within the 99% tolerance in terval established from the commercial references grown at the same s ites. These findings confirme d that these minor differences

reflected the natural variab ility of conventional corn.

In the individual-site analysis of forage, 23 of the 27 comparisons were not significantly different (p>0.05). Differences included valu es for carbohydrates by calculation, moisture, ADF, and calcium, with each component difference being observed at only a single site. This lack of reproducibility acro ss all sites established that there were no meaningful trends in values for these components and that this limited number of differences constituted no biological significance. For grai n nutrients, individua l site differences in components not recorded in the combined-site analysis incl uded values for serine, threonine, 18:0 stearic acid, 18:2 linoleic acid, 18:3 lin olenic acid, and vitamin E. For each component, these

differences were observed at a si ngle site only. This lack of reproducibility in observing these differences across multiple sites, and the fact that the mean values for these components at these sites were within the 99% tolerance in terval established from the commercial references, support a conclusion that th e limited number of site differences in values for these components were of no biological significance.

In summary, statistical analysis highlighted no consistent differences across sites in the levels of nutrient components from MON 87460 and the control. The limited number ofMonsanto Company 07-CR-191U Page 162 of 544 differences recorded in this study reflected th e natural variation of corn and supported the compositional equivalence of MON 87460 and conventional corn.

VII.B.3. Levels of Anti-Nutrients and Ke y Secondary Metabolites under Well-Watered

Conditions

A description of the anti-nutrients and seco ndary metabolites presen t in corn grain is provided in the OECD consensus document on compositional considerations for corn (OECD, 2002). The anti-nutrients and key secondary metabolites analyzed in this study are

the same as those listed in Section VII.A.3

.

The statistical analysis highlighted no differences within or across sites in the levels of antinutrient components and secondary metabolite s in MON 87460 and the control. Thus, a comprehensive evaluation of anti-nutrient components and key secondary metabolites supported the compositional equivalence of MON 87460 and conventional corn.

VII.B.4. Conclusions for Chile 2006/2007 Well-Watered Conditions

The overall dataset was evaluated for evidence of biologically relevant changes. The combined-site analysis show that there were no statistically significan t differences (p>0.05) for 59 (96.7%) of the 61 comparisons between the mean component values of MON 87460 and the control. Of the two significant diffe rences observed, mean component values of the

test and control substances we re within the 99% tolerance in terval established from the commercial references. The individual site analysis show that there were no statistically significant differences (p>0.05) for 171 (93.4%) of the 183 co mparisons. Individual site differences were not reproducible across multiple sites and, where differences were observed, mean component values test and control substan ces were within the 99% tolerance interval. Furthermore, the limited number of compone nt differences was characterized by small differences in magnitude

These factors established that the limited number of differences observed in this study were within the natural variability of corn for these components, re flected no meaningful trends, and were of no biological significance.

Therefore, the corn grain and forage de rived from MON 87460 grow n under well-watered conditions, and consequently the foods a nd feeds derived from MON 87460, can be considered compositionally equivalent to thos e derived from conventional corn grown under the same conditions. MON 87460 grown under we ll-watered conditions is as safe as

conventional corn with a hist ory of safe consumption.Monsanto Company 07-CR-191U Page 163 of 544Table VII-3. Monthly Temperature and Accumulated Water Data for the Chile 2006/2007 StudySite1Measurement December January February March April MayCLAccumulated water (in.),well-watered 0.9 10.3 8.5 9.4 2.8 0.0Accumulated water (in.),water-limited2, 30.9 10.3 4.7 5.6 2.8 0.0Avg Max temp (°F) NA588 85 82 74 67Avg Min temp (°F) NA553 49 47 41 33Range4(°F) NA546 - 97 42 - 94 41 - 94 33 - 89 26 - 78CTAccumulated water (in.),well-watered 2.8 9.4 8.5 10.3 2.8 0.0Accumulated water (in.),water-limited2, 33.8 9.4 2.8 6.6 2.8 0.0Avg Max temp (°F) NA584 79 79 71 66Avg Min temp (°F) NA552 50 50 41 37Range4(°F) NA546 - 91 42 - 90 42 - 88 31 - 87 29 – 77LUMAccumulated water (in.),well-watered 2.8 9.4 8.5 10.3 1.9 0.0Accumulated water (in.),water-limited2, 32.8 9.4 2.8 6.6 2.8 0.0Avg Max temp (°F) NA581 78 79 73 67Avg Min temp (°F) NA552 50 49 42 37Range4(°F) NA547 - 89 42 - 89 42 - 94 32 - 87 28 - 771Site codes are as follows: CL = Colina;CT = Calera de Tango; LUM = Lumbreras.2Water limitation began at the V10 growth stage which occurred at approximately February 7.3Water limitation ended at the R2 growth stage which occurred at approximately March 13.4The range is the absolute maximum and minimum temperature in each month.5Temperature data are available from January 6 through May 25;planting occurred in late December and early January.Rainfall did not occur during the production period.Monsanto Company 07-CR-191U Page 164 of 544Table VII-4. Summary of SignificantDifferences (p<0.05) Comparing MON 87460 to the Conventional Control in Chileduring 2006/2007 under Well-Watered ConditionsTissue/Site/Components (Units)1MeanMON 87460MeanControlMean Diff(% of Control)Signif.(p-value) MON 87460 (Range)99% ToleranceInterval2ForageCTMoisture (% FW)72.53 76.10 -4.69 <0.001 70.90 - 75.00 [69.22, 81.25]Carbohydrates (% DW)87.22 86.14 1.26 0.048 86.98 - 87.46 [82.51, 92.09]Acid Detergent Fiber (% DW)22.84 29.47 -22.51 0.002 17.95 - 31.28 [16.01, 45.98]Calcium (% DW)0.26 0.32 -18.99 0.047 0.25 - 0.28 [0.043, 0.46]GrainCombination of all sitesTotal Fat (% DW)3.89 3.72 4.52 0.029 3.45 - 4.23 [2.47, 4.68]Magnesium (% DW)0.12 0.11 8.64 0.012 0.10 - 0.14 [0.064, 0.16]CLVitamin E (mg/kg DW)12.46 10.50 18.66 0.002 12.37 - 12.51 [0, 19.32]CT18:2 Linoleic (% Total FA)64.64 63.82 1.28 0.048 64.11 - 65.10 [49.61, 73.18]LUMTotal Fat (% DW)3.96 3.61 9.54 0.010 3.80 - 4.23 [2.47, 4.68]Magnesium (% DW)0.13 0.11 15.54 0.022 0.11 - 0.14 [0.064, 0.16]Serine (% DW)0.50 0.44 13.33 0.024 0.47 - 0.54[0.32, 0.65]Threonine (% DW)0.35 0.32 10.08 0.047 0.33 - 0.37 [0.23, 0.42]18:0 Stearic (% Total FA)1.83 1.71 6.83 0.024 1.74 - 1.97 [0.60, 2.58]18:3 Linolenic (% Total FA)1.20 1.23 -2.41 0.042 1.17 - 1.23 [0.72, 1.66]1DW = dry weight; FW = fresh weight; FA = fatty acid.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Tables E-12 through E-18 in Appendix E present the full resultsof the combined site analysis for the compositional analyses from the Chile 2006/2007production including means, standard error values and p-values.Monsanto Company 07-CR-191U Page 165 of 544 VII.B.5. Assessment of Nutrients, Anti-N utrients, and Key Secondary Metabolites

under Water-Limited Conditions

The water-limited treatment allowed a compositional comparison between MON 87460 and the conventional control with similar backgroun d genetics but lacking the introduced trait. Both were grown under conditions intended to impose drought stress by withholding irrigation during approximately the V10 – R2 gr owth stages, which represents the growth stages when corn grain yield potential is mo st susceptible to drough t stress. Irrigation management in this treatment was intended to provide well-watered conditions before and after the V10 - R2 growth stages.

Forage and grain samples were harvested from all water-limited plots and analyzed for the

same nutritional and anti-nutrien t components assessed in the evaluation of samples from the well-watered plots. A summary of significant differences (p< 0.05) between test and control

in both the combined-site and indi vidual site (total of 3 sites) analyses for the water-limited treatment is presented in Table VII-5. Mean values, ranges, standard error values, and statistical analyses for the combined-site da ta are presented in Appendix E (Tables E-19 through E-25). Literature and ILSI Crop Co mposition Database ranges for corn components are presented in Table E-30.

The combined-site analysis show that there were no statistically significant differences (p>0.05) for 59 (96.7%) of the 61 comparisons between the mean component values of MON 87460 and the control. Of the two sign ificant differences observed, mean component

values of the test and control substances were within the 99% tolerance interval established

from the commercial references. The individu al site analysis show that there were no statistically significant differences (p>0.05) for 174 (95.1%) of the 183 comparisons. Individual site differences were not reproducib le across multiple sites and, where differences were observed, mean component values of the te st and control substances were within the 99% tolerance interval (Table VII-5). Therefore, these differences were within the natural variability of corn for these components.

VII.B.6. Levels of Nutrient s under Water-Limited Conditions

In the combined-site analysis of forage, ei ght of the nine comparisons between MON 87460 and the control were not significantly different (p>0.05). The single difference was in total fat values. However, the mean values for total fat in the test and control substances were within the 99% tolerance interval established from the commercial reference hybrids grown at the same sites, indicating that the difference was within the natural variability of corn for this component. Values for to tal fat were not observed to be significantly different (p>0.05) at any of the individual sites. In the combined-site analysis of grain, 51 of the 52 comparisons were not significantly different (p>0.05). The single di fference was in 20:1

eicosenoic acid values. However, the mean values for 20:1 eicosenoic acid in the test and

control substances were within the 99% tolerance interval established from the commercial references grown at the same sites, indicati ng that the difference was within the natural variability of corn for this component. Values for 20:1 eicosenoic acid were observed to be significantly different (p<0.05) at on ly one of the individual sites.Monsanto Company 07-CR-191U Page 166 of 544 For forage, 26 of the 27 individual site comparisons were not si gnificantly different (p>0.05).

Individual site differences included only a single value fo r moisture. This lack of reproducibility across multiple sites establishe d that there are no meaningful trends in differences in values for this component and that the limited number of differences constituted no biological significance. For grai n nutrients, individual site differences in components not recorded in th e combined-site analysis incl uded values for iron, phosphorus, 18:1 oleic acid, 22:0 behe nic acid, folic acid, vitamin E, and phytic acid. For each component, these differences were observed at a single site only. The fact that the mean values for these components at multiple site s were within the 99% tolerance interval

established from the commercial references an d the lack of reproducibility in differences

across these sites confirmed that the limited number of site differences in values for these components were within the natural vari ability of corn fo r these components.

In summary, statistical analysis highlighted no consistent differences across sites in the levels of nutrient components from MON 87460 and the control. Those limited number of differences observed in this study reflected th e natural variation of conventional corn and supported the compositional equivalence of MON 87460 and conventional corn.

VII.B.7. Levels of Anti-Nutri ents and Key Secondary Meta bolites under Water-Limited

Conditions

No combined-site differences between values for grain anti-nutrient components (phytic acid and raffinose) and secondary metabolites ( p -coumaric acid and ferulic acid) in MON 87460 and the control were recorded. Individual site differences were observed for a single value for phytic acid. As only one out of six individual site comparisons was recorded for this component, this difference represented no m eaningful trend and reflected the natural

variation observed with conventional corn.

In summary, statistical analysis highlighted no consistent differences across sites in the levels of anti-nutrient components and secondary metabolites in MON 87460 and the conventional control. Thus, a comprehens ive evaluation of anti-nutrien t components and key secondary metabolites supported the compositional equivalence of MON 87460 and conventional corn.

VII.B.8. Conclusions for Chile 2006/2007 Water-Limited Conditions

The overall dataset was evaluated for evidence of biologically relevant changes. The combined-site analysis show that there were no statistically significan t differences (p>0.05) for 59 (96.7%) of the 61 comparisons between the mean component values of MON 87460 and the control. Of the two significant diffe rences observed, mean component values of the

test and control substances we re within the 99% tolerance in terval established from the commercial references. The individual site analysis show that there were no statistically significant differences (p>0.05) for 174 (95.1%) of the 183 co mparisons. Individual site differences were not reproducible across multiple sites and, where differences were observed, mean component values of the test and contro l substances were within the 99% tolerance interval. Furthermore, the limited number of component differences was characterized by small differences in magnitude.Monsanto Company 07-CR-191U Page 167 of 544 These factors established that the limited number of differences observed in this study were within the natural variability of corn for these components, re flected no meaningful trends, and were of no biological significance.

Therefore, the corn grain and forage de rived from MON 87460 grown under water-limited conditions, and the intended foods and feeds derived from MON 87460, can be considered compositionally equivalent to those derived from conventional corn grown under the same conditions. MON 87460 grown under limited water av ailability is as safe as conventional corn with a history of safe consumption.Monsanto Company 07-CR-191U Page 168 of 544Table VII-5. Summary of SignificantDifferences (p<0.05) Comparing MON 87460 to the Conventional Control in Chileduring 2006/2007 under Water-Limited ConditionsTissue/Site/Components (Units)aMeanMON 87460MeanControlMean Diff(% of Control)Signif.(p-value)MON 87460 (Range) 99% ToleranceIntervalbForageCombination of all sitesTotal Fat (% DW)1.32 0.84 56.03 0.045 0.50 - 1.92 [0, 3.25]CTMoisture (% FW)72.13 74.23 -2.83 0.005 72.00 - 72.30 [70.85, 80.94]GrainCombination of all sites20:1 Eicosenoic (% Total FA)0.1763 0.1845 -4.43 0.042 0.1617 - 0.1916 [0.11, 0.34]CL22:0 Behenic (% Total FA) 0.17 0.11 57.68 0.029 0.13 - 0.20 [0, 0.32]Vitamin E (mg/kg DW) 13.34 11.16 19.54 0.001 12.57 - 14.24 [0, 22.61]Phytic Acid (% DW) 0.87 0.69 25.47 0.012 0.84 - 0.89 [0.40, 1.12]CTIron (mg/kg DW)17.61 18.81 -6.34 0.046 17.06 -18.24 [7.05, 30.38]Phosphorus (% DW)0.32 0.35 -8.35 0.027 0.32 - 0.32 [0.25, 0.42]20:1 Eicosenoic (% Total FA) 0.17 0.18 -9.16 0.016 0.16 - 0.17 [0.11, 0.34]Folic Acid (mg/kg DW)0.30 0.25 19.36 0.046 0.25 - 0.37 [0.098, 0.58]LUM18:1 Oleic (% Total FA)20.38 20.89 -2.46 0.030 20.20 - 20.48 [12.15, 35.55]aDW= dry weight; FA=fatty acid.cWith 95% confidence, the interval contains99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Tables E-19 through E-25 in Appendix E present the full resultsof the combined site analysis for the compositional analyses from the Chile 2006/2007production including means, standard error values and p-values.Monsanto Company 07-CR-191U Page 169 of 544 VII.C. Additional Secondary Metabolit es from the Chile 2006/2007 Composition Study under Well-Watered and Water-Limited Conditions

As part of the comparative assessment approach described previously, the OECD consensus documents suggest that additional components relevant to the trait can be

considered for characterization of food a nd feed derived from new products (OECD, 2002). Because MON 87460 is expected to be grown in regions subjected to frequent drought stress, additional secondary metabolites selected for further comparative

evaluation included those known to be generally associated w ith stress responses in a range of plants and tissues, and thus possibly relevant in corn. There is no evidence in the literature for such compone nts that are unique to corn.

Eleven additional secondary metabolites were selected for comparison between

MON 87460 and the control. These selected me tabolites included: os moprotectants, such as sugars and polyols (sucrose, glucose, fructo se, sorbitol, mannitol, and glycerol), free proline, glycine betaine and choline (Yan cey, 2004 and 2005), as well as metabolites that are generally associated with stress responses such as salicylic acid (Yuan and Lin, 2008), and abscisic acid (Wasilewska, et al., 2008). These 11 metabolites were measured in forage and grain of MON 87460 and the control. No safety issues are evident for these metabolites and most represent an extrem ely minor fraction of corn biomass.

Field design and sampling are de scribed in Section VII.B. Samples from test, control,

and reference substances from all four s ites in Chile were subjected to additional compositional analysis. This section describe s results of combined-site data from three (CL, CT, LUM) of the four s ites established. For a site to be included in the combinedsite analysis, commercial refe rence hybrids in the water-lim ited plots had to exhibit a minimum 15% reduction in yield compared to the same reference hybrids planted in the well-watered plots. A detailed description of the site inclus ion criteria applied to these data is provided in Section VIII.B.2. As described in Section VIII.C and Table VIII-3, the QUI site in the Chile 2006/2007 study was not established with the appropriate water stress treatments; therefore, data for th e QUI site are presented in Appendix E.

Statistical comparisons between the test and control substances we re performed within each irrigation treatment. A range of compone nt values and a sta tistical population were

determined for the reference substances within each irrigation treatment. Thus, four sets of statistical analyses were made for each trea tment, three based on the data from each of the replicated field sites and th e fourth based on data from a combination of all three field sites. Statistically significant differences we re determined at the 5% level of significance ( α = 0.05) using established statistical methods.

VII.C.1. Levels of Additional Seco ndary Metabolites under Well-Watered

Conditions

Two of the 11 metabolites (sor bitol, mannitol) had more th an half of the observations below the assay limit of quantitation (LOQ). Metabolites with more than half of the observations below the assay LOQ were excluded from statistical analysis. Therefore, nine metabolites in both forage and grain we re statistically assessed using a mixed model analysis of variance method.Monsanto Company 07-CR-191U Page 170 of 544 There were a total of 72 comparisons made (f our sets of comparisons × nine components from grain and four sets of comparisons × nine components fr om forage). A summary of significant differences (p<0.05) between test and control in both the combined-site and individual site (total of 3 sites) analyses for the well-watered treatment is presented in Table VII-6. Mean values, ranges, standard error values and statistical analyses for the combined-site data are presented in Appendi x E (Tables E-26 and E-27). The standard error values presented in Appendix E are cal culated from the ANOVA. Thus, each test and control mean for a given analyte mean has the same standard error. Literature and ILSI Crop Composition Database ranges for co rn components are presented in Table E30.

The combined-site analysis show that there were no statistically si gnificant differences (p>0.05) for 17 (94.4%) of the 18 comparisons between the mean component values of

MON 87460 and the control. The single difference was for absc isic acid. However, the magnitude of the combined-site difference in the abscisic acid values was exceedingly small (21.37 ppb FW) and therefore considered to be within the natural variability of corn for this component. The indivi dual site analysis show that there were no statistically significant differences (p>0.05) for 51 (94.4 %) of 54 comparisons. Individual site differences were not reproducible across mu ltiple sites and, where differences were observed, with the exception of the highly variable forage ab scisic acid, mean component

values test and control substan ces were within the 99% tolera nce interval (Table VII-6). Therefore, these differences were within the natural variability of corn for these

components.

VII.C.2. Conclusions for Chile 2006/2007 Addi tional Secondary Metabolites WellWatered Conditions

In summary, statistical analysis highlighted no consistent differences across sites in the levels of the additional secondary metabo lites from MON 87460 and the control. The

limited number of differences obs erved in this study reflecte d the natural variation of corn and support the conclusion that levels of key osmoprotectants and metabolites generally associated with stress do not differ between MON 87460 and conventional corn. This supports the assessment of MON 87460 as compositiona lly equivalent to

conventional corn.Monsanto Company 07-CR-191U Page 171 of 544Table VII-6. Summary of SignificantDifferences in AdditionalSecondary Metabolite Composition (p<0.05) ComparingMON 87460 to the Conventional Control in Chileduring 2006/2007 under Well-Watered ConditionsTissue/Site/Components (Units)1MeanMON 87460MeanControlMean Diff(% of Control)Signif.(p-value)MON 87460 (Range) 99% ToleranceInterval2ForageCombination of all sitesAbscisic Acid (ppb FW)37.03 15.66 136.54 0.040 (11.90 - 122.00) [1.22, 33.02]CLAbscisic Acid (ppb FW)75.23 15.63 381.24 0.003 (18.50 - 122.00) [1.22, 33.02]CTFree Proline (% DW)0.027 0.019 40.22 0.025 (0.023 - 0.031) [0, 0.042]Choline (ppm FW)135.67 108.97 24.50 <0.001 [114.00 - 148.00] [76.96, 179.64]1DW= dry weight; FW=fresh weight.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limitswere set to zero.Tables E-26 and E-27 in Appendix E present the full results of the combined site analysis for the compositional analyses from the Chile 2006/2007 productionincluding means, standard error values and p-values.Monsanto Company 07-CR-191U Page 172 of 544 VII.C.3. Levels of Additional Seco ndary Metabolites under Water-Limited

Conditions

A summary of significant differences (p<0.05 ) between test and control in both the combined-site and individual site (total of 3 sites) analyses for the water-limited treatment is presented in Table VII-7. Mean values, ranges, standa rd error values and statistical analyses for the combined-site da ta are presented in Appendix E (Tables E-28 and E-29). The standard error values pres ented in Appendix E are calculated from the ANOVA. Thus, each test and control mean for a given analyte mean has the same

standard error. Literature and ILSI Crop Composition Database ranges for corn components are presented in Table E-30.

The combined-site analysis show that there were no statistically si gnificant differences (p>0.05) for 17 (94.4%) of the 18 comparisons between the mean component values of MON 87460 and the control. For the singl e significant differe nce observed, mean component values of the test and control substances were within the 99% tolerance

interval established from the commercial refe rences. The individual site analysis show that there were no statistically significant differences (p>0.05) fo r 44 (81.5%) of the 54 comparisons. Individual site differences we re not reproducible across multiple sites and, where differences were observed, mean compon ent values test and control substances were within the 99% tolerance interval (Table VII-7). Therefore, these differences were

within the natural va riability of corn fo r these components.

VII.C.4. Conclusions for Chile 2006/2007 Addi tional Secondary Metabolites WaterLimited Conditions

In summary, statistical analysis highlighted no consistent differences across sites in the levels of metabolite components from MON 87460 and the control. The limited number of differences observed in this study reflects the natural variation of corn and supports the conclusion that levels of key osmoprotectants and metabolites potentially associated with stress do not differ between MON 87460 and conventional corn. This supports the conclusion that MON 87460 is compositionally equivalent to conventional corn.Monsanto Company 07-CR-191U Page 173 of 544Table VII-7. Summary of SignificantDifferences in AdditionalSecondary Metabolite Composition (p<0.05) ComparingMON 87460 to the Conventional Control in Chile during 2006/2007 under Water-Limited ConditionsTissue/Site/Components (Units)1MeanMON 87460MeanControlMean Diff(% of Control)Signif.(p-value)MON 87460 (Range) 99% ToleranceInterval2ForageCLGlycine Betaine ppm FW)102.40 147.00 -30.34 0.016 [73.20 - 122.00] [0, 357.15]Salicylic Acid (ppm FW)0.11 0.33 -68.43 0.002 [0.10 - 0.11] [0, 0.82]CTAbscisic Acid (ppb FW)42.27 23.57 79.35 0.043 [32.90 - 54.30] [0, 94.59]Choline (ppm FW)157.00 144.00 9.03 0.024 [153.00 - 160.00] [66.54, 217.46]LUMCholine (ppm FW)167.67 151.67 10.55 0.035 [153.00 - 181.00] [66.54, 217.46]Salicylic Acid (ppm FW)0.41 0.26 57.57 0.016 [0.27 - 0.58] [0, 0.82]GrainCombination of all sitesSucrose (% DW)1.63 1.86 -12.40 0.008 [1.33 - 1.86] [0.61, 2.84]CLAbscisic Acid (ppb FW)10.03 16.59 -39.52 0.041 [8.78 - 11.90] [0, 176.41]Glycerol (% DW) 0.030 0.023 31.03 0.045 [0.025 - 0.034] [0, 0.060]CTSucrose (% DW) 1.71 2.03 -15.85 0.010 [1.44 - 1.86] [0.61, 2.84]LUMSucrose (% DW) 1.41 1.58 -10.55 0.040 [1.33 - 1.54] [0.61, 2.84]1DW= dry weight; FW=fresh weight.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limitswere set to zero.Tables E-28 and E-29 in Appendix E present the full results of the combined site analysis for the compositional analyses from the Chile 2006/2007 productionincluding means, standard error values and p-values.Monsanto Company 07-CR-191U Page 174 of 544 VII.D. Overall Conclusions from Compos itional Analysis of MON 87460 from the

U.S. 2006 and Chile 2006/2007 Studies

The compositional analyses of MON 87460 were based on forage and grain harvested from two different growing seasons, the first during 2006 in the U.S. conducted under typical agronomic practices and water conditions, and the second during 2006/2007 in

Chile under well-watered and water-limited conditions. Thus, the multi-year study

allowed a determination of whether food and feed derived from MON 87460 exhibits compositional equivalence to conventional co rn under a broad range of environmental

conditions.

Components evaluated in samples harvested from both studies incl uded (1) moisture, protein, carbohydrates by calculation fat, fiber, and ash in a proximate analysis, (2)

essential macro- and micro-nutrients in a nutritional analysis, and (3) known endogenous

toxicants and anti-nutrients.

Overall, a comprehensive evaluation of ke y nutrient, anti-nutrients and secondary metabolites from MON 87460 and the control showed no biologically meaningful

differences. The statistical differences were small in magnitude and not reproducible across multiple sites. All mean component valu es of the test and control substances were within the 99% tolerance interval established from commercial references. Therefore, the forage and grain from MON 87460 and the foods and feeds derived from such, can be considered compositionally equivalent to those derived from conventional corn.

A supplementary analysis of secondary metabol ites associated with stress tolerance was conducted for samples from the Chile 2006/2007 study. Statistical comparisons between the test and control substances were perfor med within each water treatment and showed very similar results. The few detected differences were either exceedingly small in magnitude or the mean component values of MON 87460 and the contro l were within the

99% tolerance interval. Therefor e, these differences were with in the natural variability of corn for these components. The evaluati on of these additional metabolites further supports the compositional equivalence of MON 87460 to conventional corn, which has a history of safe consumption.Monsanto Company 07-CR-191U Page 175 of 544 VII.E. References

Adams, T. B., J. Doull, J.I. Goodman, I.C. Munro, P. Newberne, P.S. Portoghese, R.L. Smith, B.M. Wagner, C.S. Weil, L.A. Woods and R.A. Ford. 1997. The FEMA

GRAS assessment of furfural used as a flavour ingredient. Food and Chemical Toxicology. 35: 739-751.

Campos, H., M. Cooper, G.O. Edmeades, C. Loffler, J.R. Schussler, and M. Ibanez. 2006. Changes in drought tolerance in ma ize associated with fifty years of breeding for yield in the U.S. Corn Belt. Maydica. 51:369-381.

Douglas, C.J. 1999. Phenylpropanoid metabolism and lignin biosynthesis: from weeds to

trees. Trends in Plant Science. 1: 171-178.

Harrigan, G.G., W.P. Ridley, K.D. Miller, R. Sorbet, S.G. Riordan, M.A. Nemeth, W. Reeves, T.A. Pester. 2009. The fora ge and grain of MON 87460, a droughttolerant corn hybrid, are compositionally e quivalent to that of conventional corn. J. Agric. Food Chem. 57 : 9754–9763.

Lott, J.N.A., I. Ockenden, V. Raboy and G.D. Batten. 2000. Phytic acid and phosphorus in crop seeds and fruits: a global estimate. Seed Science Research. 10: 11-33.

Novak, W.K. and A.G. Haslberger. 2000. Substa ntial equivalence of antinutrients and inherent plant toxins in geneticall y modified novel foods. Food Chemical. Toxicology. 38: 473-483.

OECD. 2002. Consensus document on compositiona l considerations for new varieties of maize ( Zea mays ): key food and feed nutrients, an ti-nutrients and secondary plant metabolites. ENV/JM/MONO(2002)25. Organi zation for Economic Co-operation and Development, Series on the Safety of Novel Foods and Feeds, No. 6, Paris,

France.

Voragen, A.G.J. 1998. Technological aspects of functional food-related carbohydrates. Trends in Food Science and Technology. 9: 328-335.

Wasilewska, A., F. Vlad, C. Sirichandra, Y. Redko, F. Jammes, C. Valon, N.F. dit Frey, and J. Leung. 2008. An update on abscisic acid signaling in plants and more...

Molecular Plant. 1:198-217.

Yancey, P.H. 2004. Compatible and counteracting solutes: protecting cells from the Dead Sea to the deep sea. Sc ience Progress. 87:1-24.

Yancey, P.H. 2005. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and ot her stresses. Journal of Experimental Biology. 208:2819-2830.

Yuan, S. and Lin, H.H. 2008. Role of salicylic acid in plant abiotic stress. Zeitschrift Fur Naturforschung Section C-a Jour nal of Bioscience. 63:313-320.Monsanto Company 07-CR-191U Page 176 of 544 VIII. Phenotypic, Agronomic, and Envi ronmental Interactions Assessment

This section provides an assessment of the phenotypic and agronomic characteristics, and

the environmental interactions of MON 87460. As an introduction to Monsanto’s approach to the plant pest ri sk assessment of MON 87460, the first two sections provide a

list of the characteristics measured among the assessment studies conducted (Section VIII.A) and the criteria used to inte rpret the data generated (Section VIII.B). The remaining Sections (VIII.C through VIII.F) present the results of several field, greenhouse, and laboratory studies that co mprise the comprehensive assessment of

MON 87460.

Phenotypic and agronomic characteristic s of MON 87460 were evaluated in a comparative manner to assess plant pest pot ential (OECD, 1993). These assessments included 14 plant growth and development characteristics, five seed germination parameters, two pollen characteristics, and obs ervations for plant-insect and plant-disease interactions and plant responses to abiotic stressors. Results from the phenotypic and agronomic assessments indicate that MON 87460 does not demonstr ate increased plant pest characteristics compared to conventional corn. Data on enviro nmental interactions

indicate that MON 87460 does not confer any increased suscep tibility or tolerance to specific diseases, insects, or abiotic stresso rs, with the exception of the drought tolerance

trait.

VIII.A. Characteristics Measured for Assessment

In the phenotypic, agronomic, and envi ronmental interactions assessment of MON 87460, data were collected to evaluate specific aspects of altered plant pest potential based on requirements of USDA -APHIS set forth at 7 CFR § 340.6. The assessment encompasses five general data categories: (1) phenotypic growth and development, including vegetative and reproductive growth; (2) germination and dormancy; (3) pollen viability and morphology; (4) plant interactio ns with insects, diseases, and abiotic stressors; and (5) persistence in cultivated fields or areas outside of

cultivation. An overview of the characteristics assessed is presented in Table VIII-1.

The phenotypic, agronomic, and environmental interactions data were evaluated on the basis of familiarity (OECD, 1993) and were comprised of a combination of field, greenhouse, and laboratory studies conducted by scientists who are familiar with the production and evaluation of corn. In e ach of these assessments, MON 87460 was compared to an appropriate conventional c ontrol, which had a ge netic background similar to MON 87460 but did not possess the drought tolerance trait. In addition, multiple commercial corn hybrids (references) were includ ed to provide a range of baseline values that are common to the existing commercial corn hybrids for each measured phenotypic, agronomic, and environmental interaction characteristic. The commercial reference hybrids selected for each study we re adapted to the geographic region in which they were grown with selections based on agronomic char acteristics such as relative maturity and drought tolerance ratings. Relative maturity wa s an important consideration in order to ensure that the test, control and reference ma terials would be at comparable stages of development at each data or sample collection time point. Data collected from theMonsanto Company 07-CR-191U Page 177 of 544 commercial references reflect a range of selection and breeding for desirable characteristics including dr ought tolerance and can ther efore provide context for interpreting experimental results.

The commercial reference hybrids selected for each study were adapted to the geographic region relevant to each study site. It is know n that corn hybrids ar e variable in their response to drought stress (Campos et al., 2006). Breeders have been selecting for improved drought tolerance in conventional corn through traditional breeding and

selection methods for decades. Monsanto a nd other seed companies commonly evaluate their commercial hybrids under a range of envi ronmental conditions each year and assign drought tolerance ratings as a performance characteristic for each hybrid (http://www.asgrowanddekalb.com/seedresour ceguide/search/seeds) . Approximately 75% of the conventionally bred hybrids offere d for planting in the dr yer western areas of the Great Plains are rated as having very good to excellent drought tolerance and have been recommended for commercial production fiel ds that regularly experience drought stress. The variation in yield respons e observed among MON 87460, the control, and commercial reference hybrids in the field stud ies discussed below is within normal levels expected for conventional corn grown under s ub-optimal soil moisture environments. The characteristics measured among the fi eld, greenhouse, and laboratory studies are listed in Table VIII-1. The standard phe notypic, agronomic, and environmental

interactions characteristics are well known to experts fam iliar with corn breeding and agronomic performance. MON 87460 is expe cted to provide redu ced yield loss under water-limited conditions compared to convent ional corn. Reduced yield loss is a desirable agronomic char acteristic and is not per se considered to be associated with plant pest potential. Additional plant characteristics assessed included germination and dormancy, pollen characteristics collected under well-watered and water-limited conditions, abiotic stress tolerance to multip le levels of drought, cold, heat, and salt conditions, volunteer potential in a subsequent season under cultivated so il conditions, and survival ability under environments not cultivated for agricultural production. These

characteristics are useful to assess plant pest potential of MON 87460. Specifically,

certain characteristics can be used to a ssess weediness potential, including seed germination and dormancy (hard seed), pre-harv est seed loss characteristics (lodging and ear drop), and the potential to volunteer in cultivated areas or survive outside cultivation.

Because MON 87460 reduces yield loss under water-limited conditions, field studies were designed to evaluate the relevant characteristics of M ON 87460 across a broad range of soil moisture and environmental conditions relevant to where commercial production would be expected. Agronomic a nd phenotypic characteristics data were collected from plants grown under a combina tion of three different water management regimes: (1) well-watered, (2) well-watered and water-limited treatments in the same field arranged in either a strip- or split -plot design, or (3) water managed according to typical local agronomic practices and water conditions. A detailed description of the three water management regimes is provided in Section VIII.C. In total, these data included more than 400 phenotypic and agro nomic evaluations and more than 800 observations for plant-arthropod, plant-disease, and plant responses to abiotic stressor

interactions.Monsanto Company 07-CR-191U Page 178 of 544 Table VIII-1. Characteristics Measu red for Phenotypic, Agronomic and

Environmental Interactions Assessment of MON 87460

Characteristic Characteristics measured Evaluation timing Evaluation description (measurement endpoints)

Plant phenotypic and agronomic

characteristics Dormancy, Germination After 4, 7, and 12 days Percent normally germinated, abnormally germinated, viable hard (dormant), dead, and

viable firm swollen seed

Seedling vigor* Stage V2 – V4 Rated as: 1-3 = above average vigor, 7-9 = below average vigor (2007 scale) or, where 0 = dead, and 9 = above average vigor (2006 scale)

Early stand count Stage V2 – V4 Nu mber of emerged plants per plot

Final stand count Pre-harvest Number of plants per plot

Stay green* Maturity Rated as: 1 = 90-100% green tissue, 5 = 50- 59% green tissue, 9 = 0-19% green tissue (2007 rating scale) or, 0 = entire plant dried, and 9 = entire plant green (2006 rating scale)

Ear height Maturity Distance from the soil surface at the base of the plant to the ear attachment node

Plant height Maturity Distance from the soil surface at the base of the plant to the flag leaf collar

Stalk lodged plants Pre-harvest Number of plants per plot broken below the ear

Root lodged plants Pre-harvest Number of plants per plot leaning at the soil

surface at >30 ° from the vertical

Days to 50% pollen shed Pollen shed Days from planting until 50% of the plants have begun to shed pollen

Days to 50% silking Silking Days from planting until 50% of the plants have silks exposed

Pollen viability Tasseling Viable and nonviable pollen based on pollen grain staining characteristics

Pollen morphology Tasseling Diameter of viable pollen grains

Grain moisture Harvest Moisture per centage of harvested shelled grain

Test weight (lb/bu) Harvest Test weight of harvested shelled grain

Yield (bu/ac) Harvest Harvested weight of shelled grain, adjusted to

15.5% moisture

Dropped ears Pre-harvest Number of mature ears dropped from plants

Plant

environmental

interactions Insect, disease and

abiotic stressors Variable, from planting to harvest Qualitative assessment of each plot, with rating on a 0-9 scale for plant-insect, plant-disease, and plant response to abiotic stressor

interactions

Abiotic stress tolerance to drought, cold, heat,

and salt Stage V2 – V6 Conducted in greenhouse and growth chamber experiments. Measur ements included plant height, growth stage, vigor, chlorophyll content, and biomass.

Volunteer potential After fall planting and following spring Number of plants present as volunteer corn in plots

Survival outside of

cultivation Variable, from planting to harvest Variable, phenotyp ic assessments from planting to harvest that includes early and final stand counts, vigor ratings, pl ant height, and number of ears and seed per plot

*Rating scale changed in 2007 to be consistent with assessments used by corn breedersMonsanto Company 07-CR-191U Page 179 of 544 VIII.B. Interpretation of Assessment Data

Plant pest risk assessments for biotechnology- derived crops are, by standard, comparative assessments. The concept of familiarity is useful when designing a risk assessment and evaluating the plant pest pot ential of a biotechnology-derive d plant compared to the conventional crop. The concept of familiarity is based on the fact that the biotechnologyderived plant is developed from a conventi onal plant variety whos e biological properties and plant pest potential are known to experts. Familiarity considers the biology of the crop, the introduced trait, th e receiving environment, a nd the interaction among thesefactors, and provides a basis for comparative risk assessment between a biotechnologyderived plant and its conventional counterpart.

Expert knowledge and experience with c onventionally bred corn was the basis for selecting appropriate endpoints and estimatin g the range of responses that would be considered typical for corn. Thus, assessment of phenotypic and agronomic characteristics and environmental interactions was essential to compare the biotechnology-derived plant to the conven tional counterpart. An overview of the characteristics assessed is presented in Table VIII-1. A subset of the data relating to well-understood weediness criteria (e.g., dorma ncy, lodging or pre-harvest seed loss characteristics, volunteer poten tial and survival out side cultivation) was used to assess whether there is an increased weediness pote ntial, an element of APHIS’s plant pest

determination. Data on abiotic stress tole rance from the greenhouse and growth chamber assays were used to characterize the extent of stress tolerance imparted by the insertion of

the cspB gene and determine whether any pote ntial changes in tolerance required additional evaluation as a compone nt of the plant pest risk a ssessment. Based on all of the data collected, an assessment was made whether the biotechnology -derived plant is likely to pose an increased plant pest risk compared to the co nventional counterpart

.

Agronomists familiar with the experimental design, evaluation criteria, and expected outcomes were involved in all steps of data collection, summari zation, and analyses. This supervision ensured that measuremen ts were taken appropr iately, data were consistent with expectations based on experi ence with the crop, and that the experimental and field sites were carefully monitored. Th ese scientists were expected to report any unexpected observations or issu es during the course of the studies. The overall dataset was evaluated for evidence of biologically rele vant changes and for possible evidence of an unexpected plant response and the data we re subsequently submitted for statistical analysis.

VIII.B.1. Interpretation of Detected Differences Criteria

Comparative plant characteriz ation data between a biotec hnology-derived crop and the control are interpreted in the context of contri butions to increased pl ant pest potential as assessed by APHIS. Under the framework of familiarity, characteristics for which no differences are detected suppor t a conclusion of no increase d plant pest potential of the biotechnology-derived crop compared to th e conventional crop. Characteristics for

which differences are detected are considered in the step-w ise method (Figure VIII-1).

All detected differences for a characteristic are considered in the context of whether or not the difference would increase the plan t pest potential of the biotechnology-derivedMonsanto Company 07-CR-191U Page 180 of 544 crop. Ultimately, a weight of evidence appr oach considering all characteristics and

studies was used for the overall risk assessmen t of differences and th eir significance. In detail, Figure VIII-1 illustrates the st epwise assessment process employed:

• Steps 1 & 2. Evaluate Detected Statistical Differences . Combined-site and individual-site statistical analyses are conducted and evaluated on each

measured characteristic. Differences de tected in the individual-site analysis must be observed in the combined-site analysis to be considered further for plant pest potential. A difference in th e combined-site analysis is further assessed regardless of whether or not the difference is detected in the individual-site analysis.

• Step 3. Evaluate Differences Relative to Reference Range. If a difference is detected in the combined-site analysis across multiple environments, then the test substance mean value is assessed relative to the range of values of the

reference substances.

• Step 4. Evaluate Difference in the Context of the Crop. If the test substance

mean is outside the variation of the re ference substances (e .g., reference range or tolerance interval), the test substance mean is considered in the context of known values common for the crop.

• Step 5. Evaluate Difference in the Context of Plant Pest Potential . If the

test substance mean is outside the range of values common for the crop, the

detected difference is then a ssessed for plant pest potential.

• Step 6. Conduct Risk Assessment on Identified Hazard. If a hazard is identified, risk assessment on the difference is conducted. The risk assessment

considers contributions to enhanced plant pest potent ial of the crop itself, the impact of differences detected in other measured characteristics, and potential for, and effects of trait transfer to feral populations of th e crop or a sexually compatible species.Monsanto Company 07-CR-191U Page 181 of 544 Note: A “no” answer at any step indicates th at the characteristic does not contribute to a biological or ecological change for the crop in te rms of plant pest potential and subsequent steps are not considered. If the answer is “yes” or uncertain the subsequent step is considered.

Figure VIII-1. Decision Diagram for In terpretation of Detected Differences

VIII.B.2. Interpretation of Site Inclusion Criteria

Water management was a critical aspect in the field-based plant characterization

evaluation of MON 87460. Because MON 87460 reduces yield loss under water-limited conditions, field studies were designed to evaluate the en vironmental consequences of MON 87460 across a broad range of soil moisture and environmental conditions relevant to where commercial producti on would be expected. Tw o distinct hypotheses were

tested in this risk assessment: (1) no phenotypic differences between MON 87460 and the

control under well-watered conditions, and (2) no pheno typic differences between

MON 87460 and the control under water-limited conditions except for grain yield. As such, field studies were designed to enable assessment of the effects of defined water treatments on the phenotypic and agronomic characteristics of MON 87460 compared to the conventional control, and sites established with a defined water treatment had to meet set criteria to qualify as either well-watered (non-stressed) or water-limited (stressed) to

be included in the assessment. Hazard identification & risk

assessment on difference Outside variation for crop? ( “ nonfamiliar ” ) No

Y es

No

Yes Plant pest potential? Yes Outside variation of study references? No Not a plant pest; the direction

of the detected difference in the

measured characteristic does not contribute to a biological or ecological change for the crop in terms of plant pest potential Yes Statistical differences detected in combined-site analysis? No Differences detected in the combined-site and individual-site analyses are evaluated* Step 1

Step 2

Step 3

Step 4 The measured characteristic

does not contribute to a biological or ecological change

for the crop in terms of plant pest potential

Step 5

Step 6

*See text for interpretation of differences detected in the individual-site analysisMonsanto Company 07-CR-191U Page 182 of 544 The well-watered treatment allowed a comp arison of MON 87460 and the control in the absence of trait bias, where no statistical differences were expected between MON 87460 and the conventional control. The water-limited treatment imposed during susceptible growth stages (i.e., approximately V 10 through R3) allowed a comparison of

MON 87460 and the control under conditi ons where MON 87460 was expected to provide a yield benefit. Water treatment le vels were managed by using a combination of either a water budgeting program (KanSche d software, supported by Kansas State University Research and Extension), physical assessments for soil moisture (Appearance and Feel Test; Black and Rogers, 1989), or by applying water on a calendar basis following local agronomic practices.

Criteria were established to identify sites that were managed appropriately to impose the defined well-watered and water-limited treatment levels. Specifically, commercial reference hybrids in the water-limited plots had to exhibit a mini mum 15% reduction in yield compared to the same reference hybrids planted in the well-wate red plots. The 15% yield loss criteria represents a m eaningful stress level, as mode rate water deficits result in approximately a 15% yield loss annually for corn grown in both te mperate and tropical regions (Barker et al., 2005). Reductions in plant height, ear height, and days to 50% silking are also phenotypic indi cators of moisture deficit in corn (Campos et al., 2006); therefore, these characte ristics were assessed to confirm the inclusion of sites that met the 15% yield reduction requirement.

Field sites designed to assess the effects of water limitation were established with defined well-watered and water-limited treatments. Each site was evaluated in a step-wise method (Figure VIII-2) using se t criteria to determ ine if a differential water treatment

was achieved and if a water treatment effect was observed in the plant responses of the reference substances. Only sites that met the inclusion criteria were included in the

assessment. Data from sites that did not meet the inclusion criteria are presented in the appendices. In detail, Figure VIII-2 illustrates the stepwise assessment process employed:

• Step 1. Confirm Differenti al Water Treatments Imposed . A review of the water management practices and rainfall data is conducted for each site. All rainfall events and irrigation amount s during the water stress period are evaluated for compliance with soil mois ture requirements. Precipitation and irrigation records should support the expectation that differential water treatments were imposed at the field site.

• Step 2. Confirm Target Soil Moisture Levels Imposed.

Well-watered treatment : available moisture should be > 50% of field capacity for the duration of the study to avoid drought stress conditions and provide optimal grain yield.

Water-limited treatment : available moisture should be <50% of field capacity during targeted stress treatment (e.g., V10 – R3).Monsanto Company 07-CR-191U Page 183 of 544 At sites where soil moisture data were not collected, evidence of water treatment effect is necessary.

• Step 3. Confirm Phenotypic Responses to Water Treatments . A weight of evidence approach is taken when compar ing the references in the well-watered

treatment to the references in the water-limited treatment. Indicators of water limitation included yield reduction (minim um of 15%), reduced plant height, reduced ear height, and delayed silking.

No differences in these phenotypic criteria may indicate that the site is not useful for assessments requiring well-w atered and water-limited treatments.

• Step 4. Confirm Site Meets Inclusion Criteria . A “yes” answer indicates that a site is applicable for assessments requiring defined well-watered and waterlimited treatments. At a minimum, the re quirement for a 15% yield reduction in the reference substances due to the impos ed water stress treatment must be metfor a site to be considered applicable for assessments that require defined wellwatered and water-limited treatments. This requirement ensures assessments are conducted under conditions of signifi cant stress as evidenced by yield reduction and irrigation records.Monsanto Company 07-CR-191U Page 184 of 544 Note: A “no” answer at any step indicates that the site is not useful for assessments requiring

well-watered and water-limited treatments. If the answer is “yes” or uncertain, the subsequent step is considered. Other asse ssments may be appropriate under th e treatment levels achieved in the study. All comparisons of plant responses to assess for a water treatment effect at each site are made using the reference substances.

Figure VIII-2. Decision Diagram for Site Inclusion in Plant Characterization Assessments Requiring Defined Well- Watered and Water-Limited Treatments Site useful for assessments requiring

defined well-watered and water-limited

treatments. Phenotypic responses of reference substances indicate the expected

differences between well-watered and

water-limited treatments? Water monitoring data confirm that the

desired well-watered and water-limited

treatment levels were established? No Site not useful for plant assessments requiring defined

well-watered and water-limited treatments. Data may be

relevant to assessments conducted using the stress

levels achieved. Water managed appropriately to impose

differential water treatments? Step 1

Step 2 Site not useful for assessing

water treatment effect.

Step 3

Step 4 Yes

In the absence of one or more phenotypic indicators of moisture stress, the site is not useful for assessments requiring

well-watered and water-limited

treatments. No

No Yes

YesMonsanto Company 07-CR-191U Page 185 of 544 Note The preceding two sections provid e an introduction to Monsanto’s approach to the plant pest risk a ssessment of MON 87460 by presenting a

list of the characteristics measured (VIII.A) and the methods used to interpret the assessment data (VIII.B). The following sections (VIII.C through VIII.F) present the results of several field, greenhouse, and laboratory studies used as the basis for the assessment of MON 87460

VIII.C. Phenotypic and Agronomic Assessment

The purpose of these studies was to assess the phenotypic and agronom ic characteristics of MON 87460 compared to a conventional control, which had a genetic background similar to MON 87460 but did not possess th e drought tolerance trait. In addition, multiple conventional corn hybrids (references) we re included in the analysis to establish a range of natural va riability for each characteristic. Because MON 87460 reduces yield

loss under water-limited condi tions, field studies were designed to evaluate the environmental consequences of MON 87460 across a broad range of soil moisture and

environmental conditions relevant to wher e commercial production would be expected. Field studies were established using three di fferent water management regimes: (1) wellwatered, (2) well-watered and water-limited tr eatments in the same field arranged in either a strip- or split-plot design, or (3) water managed according to typical local agronomic practices and water conditions.

Well-Watered Treatment

Field studies established under well-watered conditions were intended to provide optimal grain yield and a llowed an evaluation of MON 87460 in the absence of trait bias, where no st atistical differences were expected between MON 87460 and the control. Field studies managed to impose only well-watered conditions were established at 17 sites in the U.S. during 2006 and 2007 (Table VIII-2). The water treatment in these studies required available soil moisture to be maintained at > 50% of field capacity for the duration of the study to avoid drought stress conditions. Water was provided by natural rainfall and s upplemental irrigation as needed.

Well-Watered and Water-Limited Treatments

Field studies established under both well-watered and water-limited

conditions allowed an evaluation of MON 87460 under conditions where it is expected to reduce yield loss. Field studies managed to impose both

well-watered and water-limited conditions were established at nine sites inthe U.S. and Chile during 2006 and 2007 (Table VIII-2). The wellwatered treatment in these studies requi red available soil moisture to be

maintained at > 50% of field capacity for the duration of the study to avoid drought stress conditions and provide optimal grain yield. The waterlimited treatment was managed the same as the well-watered treatment, with the exception that av ailable soil moisture be reduced to <50% of field capacity to impose a moderate drought stress during the late vegetativeMonsanto Company 07-CR-191U Page 186 of 544 through early grain fill growth stag es (~V10 – R3) when corn yield potential is most sensitive to stre ss (Claassen and Shaw, 1970; Boyer and Westgate, 2004). See Section II.D .3 for additional information on the effects of drought stress on corn grow th and development. For the nine

field sites established with both well-watered and water-limited treatments (Table VIII-2), the inclusion criteria were used to identify sites that were managed appropriately to impose the defined treatment levels (see previous Section VIII.B.2). Due to untimely rainfall during the imposed water-limitation treatments and complications in water management techniques, only six of the nine sites established met the required inclusion criteria of having both well-watered and water-limited treatments for plant characterization assessments requiring both treatments (Table VIII-3). The Chile 2006/2007 study was also used to determine CSPB and NPTII expression levels (Section VI.C) a nd composition of forage and grain (Section VII).

Typical Agronomic Practices Treatment A field study established under wate r conditions typical of local agronomic practices allowed an ev aluation of MON 87460 under a broad range of environmental conditions rele vant to commercial corn production regions. The study was established at six sites in the U.S. during 2006 (Table VIII-2). Each site received water as is typical of the growing area. Four sites were rainfed (IAE, IAW, IL, IN) and two (KS, NE) received normal amounts of supplemental irri gation as needed to produce normal yields. Only five of the sites were useful to collect phenotypic data due to a plot staking error at the KS site in the plots used exclusively for phenotypic data collection. All six sites were used to determin e CSPB and NPTII expression levels (Section VI .C) and composition of forage and grain (Section VII) as the staking e rror did not affect the rows used exclusively for tissue sampling.

Total of 31 Field Sites for Phenotypic Assessment among the Three Water Regimes Phenotypic and agronomic data were collect ed from 31 field site locations over two years: 13 U.S. sites in 2006, 4 Chilean site s in 2006/2007, and 14 U. S. sites in 2007 (Table VIII-2). As noted previously, phenotypic data were not collected at the KS site in the U.S. 2006 typical agronomic practices stud y. These field site locations provided a broad range of environmental and agronomic conditions representati ve of U.S. corngrowing regions. The California and Chile loca tions selected for a sub-set of these sites provide environments that are well-suited fo r corn production but typically do not receive any rainfall during the growing season. These e nvironments were used to establish sites with well-watered and water-limited treatments to ensure all water applied to each site occurred through controlled irrigation. Plots were established at each field site as a randomized complete block design with three rep lications or as strip- or split-plot designs with either three or four replic ations. Each plot consisted of four to eight rows of corn spaced approximately 30 inches apart and approximately 20 ft in length. Plant growth stage was assessed several times during the growing season.Monsanto Company 07-CR-191U Page 187 of 544 All combined-site data and sites that met the inclusion criteria for plant characterization assessments requiring both well-watered and water-limited treatments are summarized below in Sections V.C.1 through V.C.3. The following 14 phenotypic and agronomic

characteristics were evaluated in each fiel d study: seedling vigor, ea rly stand count, days to 50% pollen shed, days to 50% silking, stay green, ear height, plant height, dropped ears, stalk lodging, root lodging, final stand coun t, grain moisture, test weight, and yield. The evaluations and timing of plant assessmen ts are described in Table VIII-1. The location of each test site along with year of establishment and water management details are described in Table VIII-2. The phenotypic and agronomic data were analyzed using SAS. Means were calculated across-sites (ref erred to as combined-site analysis), in which data were pooled from all sites and anal yzed statistically, or data are presented for an individual location (referred to as by-s ite analysis) where appropriate. For each characteristic, MON 87460 was compared to th e control. Differences were considered significant at the 5% level (p ≤ 0.05). Additional details on ma terials, methods, and results from the individual site analyses, data from sites excluded from analyses, environmental interactions data from individual sites, and water and temperature water data from individual sites are provided in Appe ndices F, G, H, and I, respectively.

Data collection methods for the agronomic and phenotypic field trials varied by the type of data that were being collected. Seedling vigor, date of 50% pollen shed, date of 50% silking and stay green were re ported based on a whole plot vi sual assessment. Early and final stand counts, dropped ears, stalk lodgi ng and root lodging were reported on a whole plot basis. Plant height and ear height were measured on five representative plants from two rows per plot. Plot wei ght is a direct measurement of all grain harvested from two rows per plot while grain moisture and test weight are based on a subsample of grain from each plot. Yield is calculated using plot weight, grain moisture and test weight. Abiotic stressors, arthropod damage and dis ease damage were assessed from two rows on a whole plot basis. Stalk and ear/kern el rot assessments were made on five nonsystematically selected plants in two rows from each plot. Arthropods were collected using a sticky trap in the middle of a row in e ach plot. The sticky trap s were left in place for seven days per deployment. European corn borer and co rn earworm damage assessments were made on five non-systematica lly selected plants in three rows from each plot in 2006 and on ten non-systematically selected plants in three rows from each plot in 2007. Tables F-8 through F-13 in Ap pendix F show the number of replicates planted at each site. For the combined-site analyses, the sample size (n) is the number of replicates multiplied by the number of sites. For the individual site analyses, n is the number of replicates at the site. All sites were planted with three replicates per site except U.S. 2007 Study 1 which had four replicates per site. The standard error values presented in this section and in Appendix F are calculated fr om the raw data. Thus, each

test and control mean has its own individua l standard deviation and hence a different

standard error.

Regardless of the water regime establishe d, the following studies demonstrate that no phenotypic or agronomic differences were obs erved for MON 87460 that would lead to a conclusion of increased plant pest potential. MON 87460 is expected to provide reducedMonsanto Company 07-CR-191U Page 188 of 544 yield loss under water-limited conditions compared to conventional corn. Reduced yield loss is a desirable agronomic characteristic and is not per se considered to be associated with plant pest potential.Monsanto Company 07-CR-191U Page 189 of 544 Table VIII-2. U.S. and Chile Field Studi es Established under Three Different Water Management Regimes for Phenotyp ic Evaluation of MON 87460 during 2006

and 2007
Water Management Treatment / (Experimental Design) Country/

Year Location Site

Code

Well-watered U.S. 2006 Jefferson Co., Iowa IA1

(Randomized complete block) Benton Co., Iowa IA2
Stark Co., Illinois IL1
Warren Co., Illinois IL2
Boone Co., Indiana IN1
Parke Co., Indiana IN2

Pawnee Co., Kansas KS
York Co., Nebraska NE

(Randomized complete block) U.S. 2007 Jefferson Co., Iowa IA1

Van Horne Co., Iowa IA2
Stark Co., Illinois IL1
Warren Co., Illinois IL2
Clinton Co., Illinois IL3
Boone Co., Indiana IN
York Co., Nebraska NE
Fayette Co., Ohio OH
Berks Co., Pennsylvania PA

Well-watered and water-limited Chile 2006/2007 Calera de Tango, Chile CT

(Strip-plot design) Colina, Chile CL
Lumbreras, Chile LUM
Quillota, Chile QUI

(Split-plot design) U.S. 2007 Sutter Co., California CA
Study-1 Carson Co., Texas TX

(Strip-plot design) U.S. 2007 Pawnee Co., Kansas KS
Study-2 York Co., Nebraska NE

Carson Co., Texas TX

Typical agronomic practices U.S. 2006 Benton Co., Iowa IAE

(Randomized complete block) Greene Co., Iowa IAW
Stark Co., Illinois IL
Parke Co., Indiana IN

Pawnee Co., Kansas KS*
York Co., Nebraska NE

*Phenotypic data were not collected from the KS site in the U.S. 2006 typical agronomic practices study.Monsanto Company 07-CR-191U Page 190 of 544Table VIII-3. Site Inclusion Assessment for U.S. and Chile Field Studies during 2006 and 2007Yield ± S.E. (bu/ac)Plant height ± S.E.(in)Ear height ± S.E.(in)Days to 50% silking± S.E.Study, Location,and YearSitecode1WellWateredWater-LimitedReduction(%)2Well-WateredWater-LimitedWell-WateredWater-LimitedWell-WateredWater-LimitedChile 2006/2007CL185.5± 10.7282.3*± 11.1856110.7± 2.7479.7*± 7.2463.4± 2.3850.9*± 3.9463.1± 0.1663.8± 0.21CT236.5± 4.95152.3*± 9.8236105.9± 2.9192.1± 2.1755.0± 2.0846.0± 1.7966.2± 0.4067.3± 0.16LUM213.9± 4.2894.4*± 4.815697.9± 1.9675.0*± 1.2250.4± 1.6341.8*± 0.9070.3± 0.3673.7*± 0.24QUI203.1± 5.27196.3± 2.513112.0± 1.72112.8± 2.5463.5± 2.1263.4± 2.0667.7± 0.3667.1± 0.96U.S. 2007 CA215.9±2.01178.8±7.9117100.4±1.01101.1±1.1651.7±0.4252.1±0.6860.3±0.2760.5±0.18Study-1TX212.2±2.86165.2*±3.782272.9±0.5862.6*±0.3332.5±0.2329.6*±0.4857.0±0.0057.3*±0.16U.S. 2007 KS141.0± 3.79138.3± 7.55288.1± 2.1588.8± 2.1542.1± 2.2942.5± 1.9163.2± 0.2962.7± 0.24Study-2NE223.0± 1.59214.1± 1.88491.0± 3.1687.7*± 3.5641.5± 1.7142.1± 1.4168.4± 0.2168.7± 0.14TX233.7± 10.50191.8*± 8.621876.1± 1.7277.1± 2.0927.9± 0.6728.2± 0.9459.0± 0.0059.0± 0.00Note: Phenotypic data are mean values for the commercialreferences planted at each site; S.E. = standard error.* Indicates statistical difference within site between reference means in the well-watered and water-limited treatments (p≤.0.05) using analysis of variance.1CL = Colina; CT = Calera de Tango; LUM = Lumbreras; QUI = Quillota; CA = California, Study-1; TX = Texas, Study-1 and Study-2;KS = Kansas;NE = Nebraska.2Percent yield reduction calculated as difference between reference mean under well-watered and water-limited conditions, divided by reference meanunder well-watered conditions. Sites QUI, KS, and NE did not meet the inclusion criteria of a minimum 15% yield reduction in references as an effect ofthe water-limited treatment. The lack of differences in plant height, ear height, and days to 50% silking between the water treatments at these sites alsoconfirmed that the plants did not experience meaningful water stress. Consequently, these three sites were not included in assessments that required waterlimited conditions.Monsanto Company 07-CR-191U Page 191 of 544 VIII.C.1. Field Studies Establishe d under Well-Watered Conditions

Field studies established under well-watered co nditions were intended to provide optimal grain yield and allowed an ev aluation of MON 87460 in the ab sence of trait bias, where

no statistical differences were expected between MON 87460 and the control.

U.S. 2006 Well-Watered Assessment In 2006, a well-watered field study was estab lished at eight sites (Table VIII-2). Comparative assessments of phenotypic and ag ronomic characteristics were conducted

on MON 87460 and the conventional control. In addition, 19 commercial corn hybrids

were included as references. In the co mbined-site analyses, no differences between

MON 87460 and the conventional control were detected for seedling vigor, early stand count, days to 50% pollen shed, days to 50% silking, stay green, ear height, plant height, dropped ears, stalk lodged plants , final stand count, grain mois ture, test weight, or yield (Table VIII-4). The only significant differen ce detected was an increase in root lodged plants for MON 87460 compared to the contro l (5.6 vs. 1.5, respectively). Root lodging is a characteristic that may be associated with weediness, because increased lodging can contribute to viable seed being returned to the soil. However, this was not observed consistently for MON 87460. Although the numbe r of root lodged plan ts was greater for MON 87460 compared to the control, th e mean value observed for MON 87460 was within the range of values observed in the co mmercial references. The difference in root lodged plants does not represent a trend in the data across studies and years and is not considered to be biologically meaningful in terms of increased plant pest potential (Figure VIII-1, Step 3).

U.S. 2007 Well-Watered Assessment In 2007, a well-watered field study was established at nine sites (Table VIII-2). Comparative assessments of phenotypic and ag ronomic characteristics were conducted

on MON 87460 and the conventional control. In addition, 11 commercial corn hybrids

were included as references. In the combin ed-site analysis no differences were detected

between MON 87460 and the control for any of the 14 assessed phenotypic and agronomic characterist ics (Table VIII-5).

Summary of U.S. 2006 and 2007 Well-Watered The results from these studies support a conclu sion of no increased plant pest potential of MON 87460 compared to conventional co rn under well-watered conditions.Monsanto Company 07-CR-191U Page 192 of 544 Table VIII-4. U.S. 2006: Phenotypic and Agronomic Comparison of MON 87460 to

the Control in the Combined -Site Analysis from a 2006 U. S. Field Study Conducted

under Well-Watered Conditions

Phenotypic characteristic Units Mean ± S.E. Reference Range 1

MON 87460 Control Min Max

Seedling vigor 0-9 scale 2 7.2 ± 0.30 7.2 ± 0.25 6.3 8.3
Early stand count #/plot 84.8 ± 3.94 84.7 ± 4.20 71.5 115.0
Days to 50% pollen shed Days 60.8 ± 0.84 60.9 ± 0.82 55.3 63.8
Days to 50% silking Days 60.0 ± 0.65 59.8 ± 0.73 55.3 63.3
Stay green 0-9 scale 3 4.1 ± 0.25 4.1 ± 0.22 2.7 6.5
Ear height in 49.6 ± 1.15 49.2 ± 1.25 31.5 53.7
Plant height in 97.2 ± 1.84 98.8 ± 1.57 77.1 107.3
Dropped ears #/plot 0.4 ± 0.17 0.3 ± 0.11 0.0 2.0
Stalk lodged plants #/plot 9.3 ± 3.15 8.2 ± 2.36 0.3 46.0
Root lodged plants #/plot 5.6* ± 2.03 1.5 ± 0.45 0.0 30.3

Final stand count #/plot 71.8 ± 3.41 69.6 ± 3.14 52.8 96.3

Grain moisture % 16.9 ± 0.57 17.0 ± 0.60 13.3 21.7
Test weight lbs/bu 57.3 0.61 57.7 ± 0.63 54.1 60.4

Yield bu/a 163.9 ± 4.82 164.1 ± 5.96 151.2 209.6

S.E. = standard error * Indicates a statistically significant difference between the test and control at p< 0.05.

1 Reference range = Minimum and maximum mean values among the 19 reference corn hybrids.

2 Seedling vigor rating scale: 0 = dead and 9 = above average vigor.

3 Stay green rating scale: 0 = entire plan t is dried and 9 = entire plant is green.Monsanto Company 07-CR-191U Page 193 of 544 Table VIII-5. U.S. 2007: Phenotypic and Agronomic Comparison of MON 87460 to

the Control in the Combined -Site Analysis from a 2007 U. S. Field Study Conducted

under Well-Watered Conditions

Phenotypic

characteristic Units Mean ± S.E. Reference Range 1

MON 87460 Control Min Max
Seedling vigor 1-9 scale 2 3.0 ± 0.34 3.0 ± 0.33 1.5 3.9 Early stand count #/plot 87.1 ± 3.87 89.4 ± 3.77 74.4 95.8 Days to 50% pollen shed Days 63.6 ± 1.04 63.4 ± 1.05 60.0 68.2 Days to 50% silking Days 62.4 ± 1.03 62.3 ± 1.01 58.6 67.0 Stay green 1-9 scale 3 6.0 ± 0.33 5.4 ± 0.40 4.0 5.9 Ear height in 46.7 ± 1.47 45.2 ± 1.57 39.3 52.2 Plant height in 97.5 ± 2.55 96.8 ± 2.76 82.2 119.4 Dropped ears #/plot 0.6 ± 0.16 0.5 ± 0.19 0.0 0.9 Stalk lodged plants #/plot 3.5 ± 0.72 2.5 ± 0.62 0.8 5.6 Root lodged plants #/plot 0.5 ± 0.25 0.5 ± 0.23 0.0 1.1 Final stand count #/plot 71.7 ± 2.78 73.1 ± 2.80 61.0 78.7

Grain moisture % 17.3 ± 0.76 17.9 ± 0.76 15.0 23.3 Test weight lbs/bu 54.6 ± 0.72 55.1 ± 0.43 53.1 56.9

Yield bu/a 162.4 ± 5.82 160.6 ± 7.00 163.4 199.2

S.E. = standard error Note: No statistically significant differenc es between MON 87460 and the control ( α =0.05).

1 Reference range = minimum and maximum values observed among the 11 reference corn hybrids

2 Seedling vigor rating scale: 1 = above average vigor and 9 = poor.

3 Stay green rating scale: 1 = 90-100% green and 9 = 0-19% green.

VIII.C.2. Field Studies Established und er Both Well-Watered and Water-Limited

Conditions

These field studies were designed to evaluate MON 87460 under water-limitedconditions where MON 87460 was expected to reduce yield loss. In addition, the wellwatered treatment was included to evaluate whether any potential differences observed

between MON 87460 and the control under wate r-limited conditions (e.g., grain yield) could be attributed to the drought tolerance trait. Data from sites that did not meet the

inclusion criteria were not included in statistical analyses, but are provided in Appendix G. Three different studies totaling ni ne field sites were established in the U.S.and Chile during 2006 and 2007. Due to untimely rainfall during the imposed waterlimitation treatments and complications in wate r management techniques, only six of the

nine sites established met the inclusion crit eria of having both well-watered and waterlimited treatments for plant characterization assessments requiring both treatments.

VIII.C.2.1. Chile 2006/2007

Chile 2006/2007 Well-Watered and Water-Limited In 2006/2007, a field study with four sites was established in Chile with well-watered and water-limited treatments (site codes CL, CT, LUM, and QUI) (Table VIII-2).Monsanto Company 07-CR-191U Page 194 of 544 Comparative assessments of phenotypic and ag ronomic characteristics were conducted

on MON 87460 and a conventional control. In addition, 12 commercial corn hybrids

were included as references. Data from three sites met the inclusion criteria and results are presented in Tables VIII-6 and VIII-7. The QUI site was not established with the appropriate water stress treatments; thus, da ta for this site were not included in the statistical analysis. Data from the QUI site are presented in Appendix G. They appeared consistent with other phenotypic data for MO N 87460 and did not impact the conclusions of the plant pest risk assessment

Assessment In the combined-site analysis of the well-wa tered treatment in Chile, no differences were

detected between MON 87460 and the conventi onal control for any of the 14 assessed phenotypic and agronomic characteristics (Table VIII-6). In the combined-site analysis of the water-limited treatment in Chile, one significant difference was detected between

MON 87460 and the control out of the 14 a ssessed characteristics. As expected, MON 87460 exhibited higher yi eld (reduced yield loss) (p ≤ 0.05) than the conventional control (114.5 vs. 86.7 bushe ls/acre, respecti vely) under water-l imited conditions (Table VIII-7). Reduced yield loss is a desi rable agronomic characteristic and is not per

se considered to be associated with plant pest potential.

Summary of Chile 2006/2007 Well-Watered and Water-Limited The results from this study support the ex pectation of no statistically significant

differences between MON 87460 and the cont rol under well-watered conditions and a yield advantage for MON 87460 over the control under water-limited conditions.Monsanto Company 07-CR-191U Page 195 of 544 Table VIII-6. Chile 2006/2007: Phenotyp ic and Agronomic Comparison of

MON 87460 to the Control under Well-Watered Conditions in the Combined-Site Analysis in 2006/2007 Chilean Field Tria ls Established with Well-Watered and

Water-Limited Treatments

Well-Watered Treatment

Mean ± S.E. Reference Range 1

Phenotypic

characteristic Units MON 87460 Control Min Max

Seedling vigor 0-9 scale 2 4.9 ± 0.20 4.7 ± 0.17 4.3 6.0

Early stand count #/plot 76.1 ± 1.48 73.0 ± 2.53 71.0 80.0

Days to 50% pollen shed Days 66.8 ± 1.35 66.7 ± 1.17 65.0 74.3

Days to 50% silking Days 65.2 ± 1.04 65.3 ± 0.91 62.7 71.0

Stay green 0-9 scale 3 2.4 ± 0.50 2.9 ± 0.65 1.0 6.7

Ear height in 55.9 ± 2.81 52.8 ± 2.03 46.1 69.1

Plant height in 101.1 ± 3.18 99.0 ± 2.13 94.4 116.4

Dropped ears 4 #/plot 0.0 ± 0.0 0.0 ± 0.0 0.0 0.0

Stalk lodged plants 4 #/plot 0.0 ± 0.0 0.0 ± 0.0 0.0 0.0

Root lodged plants 4 #/plot 0.0 ± 0.0 0.0 ± 0.0 0.0 0.0

Final stand count #/plot 75.2 ± 1.40 74.0 ± 1.95 71.3 79.3

Grain moisture % 14.8 ± 1.00 15.2 ± 1.27 10.1 20.2

Test weight lbs/bu 56.4 ± 0.64 55.8 ± 0.84 54.0 61.2

Yield bu/a 220.7 ± 7.87 220.0 ± 10.19 166.7 248.4

S.E. = standard error Note: No statistically significant differenc es between MON 87460 and the control ( α =0.05).

1 Reference range was calculated from the 3 sites exhibiting a water treatment effect among the 12 reference corn hybrids.

2 Seedling vigor rating scale: 0 = dead and 9 = above average vigor.

3 Stay green rating scale: 0 = entire plan t is dried and 9 = entire plant is green.

4 No statistical comparisons were made due to lack of variability in the data. The test was considered

effectively not different from the control because the test and control mean values were identical.Monsanto Company 07-CR-191U Page 196 of 544 Table VIII-7. Chile 2006/2007: Phenotyp ic and Agronomic Comparison of

MON 87460 to the Control under Water-Limit ed Conditions in the Combined-Site Analysis in 2006/2007 Chilean Field Tria ls Established with Well-Watered and

Water-Limited Treatments

Water-Limited Treatment

Mean ± S.E. Reference Range 1

Phenotypic

characteristic Units MON 87460 Control Min Max

Seedling vigor 0-9 scale 2 5.0 ± 0.29 4.8 ± 0.22 4.0 6.0

Early stand count #/plot 76.8 ± 0.81 75.7 ± 1.35 67.3 80.7

Days to 50% pollen shed Days 67.4 ± 1.30 68.1 ± 1.47 65.7 75.0

Days to 50% silking Days 67.3 ± 1.70 66.8 ± 1.75 63.3 74.3

Stay green 0-9 scale 3 4.3 ± 0.78 4.7 ± 0.80 1.0 7.0

Ear height in 48.0 ± 4.86 45.1 ± 3.86 40.0 60.5

Plant height in 83.9 ± 5.94 78.1 ± 5.44 64.9 96.8

Dropped ears 4 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Stalk lodged plants 4 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Root lodged plants 4 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Final stand count #/plot 76.7 ± 0.91 75.1 ± 1.23 71.3 80.7

Grain moisture % 19.5 ± 2.53 21.3 ± 3.29 9.6 25.5

Test weight lbs/bu 56.7 ± 1.20 56.0 ± 1.33 51.3 62.2

Yield bu/a 114.5* ± 16.04 86.7 ± 14.17 56.4 167.6

S.E. = standard error

* Indicates statistical difference between the test and the control (p ≤ 0.05).

1 Reference range was calculated from the 3 sites exhibiting a water treatment effect among the 12 reference corn hybrids.

2 Seedling vigor rating scale: 0 = d ead and 9 = above average vigor.

3 Stay green rating scale: 0 = entire plan t is dried and 9 = entire plant is green.

4 No statistical comparisons were made due to lack of variability in the data. The test was considered

effectively not different from the control because the test and control mean values were identical.Monsanto Company 07-CR-191U Page 197 of 544 VIII.C.2.2. U.S. 2007

U.S. 2007 Well-Watered and Water-Limited In 2007, two separate field studies were conducted in the U.S. where sites were

established with well-watered and water-limited treatments (Table VIII-2). The two trials had different experimental designs whic h precluded a combined-study analysis.

U.S. 2007 – Study-1 In Study-1, two sites were established (site codes CA and TX) with well-watered and water-limited treatments (Table VIII-2). Comparative assessments of phenotypic and agronomic characteristics were conduc ted on MON 87460 and a conventional control. In addition, seven commercial corn hybrids were included as references. A total of 14 different phenotypic and agronomic characteristics were evaluated. In the combined-site analysis for the well-watered plots, no statistical differences were

detected between MON 87460 and the conventi onal control for any of the assessed phenotypic and agronomic characteristics (Table VIII-8). The results from this study support the expectation of no differences between MON 87460 and the control under

well-watered conditions. In the combin ed-site analysis of the water-limited treatment, no statistical differences we re detected between MON 87460 and the conventional control for any of the assesse d phenotypic and agronomic characteristics (Table VIII-9).

U.S. 2007 – Study-2 In Study-2, three sites were establis hed with well-watered and water-limited treatments (site codes KS, NE, and TX) (Tab le VIII-2). Comparative assessments of phenotypic and agronomic characteristic s were conducted on MON 87460 and a conventional control. In addition, 12 commercial corn hybrids were included as references in the well-watered plots (KS, NE, and TX), and four additional references hybrids were included from the TX water- limited plots. A total of 14 different phenotypic and agronomic charact eristics were evaluated. TX was the only site to

meet the inclusion criteria for both well-watered and water-limited treatments. Due to rainfall during the imposed water-limitation treatments at the KS and NE sites, the

well-watered treatments met the inclusion criteria but the water-limited treatments did not (Figure VIII-2, Step 2). Thus, the wa ter-limited treatment data from KS and NE were not included in the statistical analys is. The KS and NE data excluded from analyses are provided in Appendix G. They appeared consistent with other phenotypic data for MON 87460 and did not imp act the conclusions of the plant pest

risk assessment.

Assessment of U.S. 2007 Study-1

No statistical differences between MON 87460 and the control we re observed in the combined-site analysis.

Assessment of U.S. 2007 Study-2 In the combined-site analysis for the well-wa tered plots (KS, NE and TX), no statistical

differences were detected between MON 87460 and the conventional control for seedling vigor, early stand count, days to 50% pollen shed, days to 50% silking, ear height, plantMonsanto Company 07-CR-191U Page 198 of 544 height, dropped ears, stalk lodged plants, ro ot lodged plants, final stand count, grain moisture, test weight, and yield (Table VIII- 10). The only statistica l difference detected was a lower stay green rating (more green tis sue) compared to the control (5.8 vs. 6.7, respectively). Stay green is an indication of plant senescence and more green tissue at this late reproductive stage, just prior to ha rvest, is not a characte ristic that is likely associated with an increase in plant pest potential (Radosevic h et al., 1997). Although MON 87460 had more green tissue compared to the control, the mean value observed for MON 87460 was within the range of values obs erved in the commercial references. The difference in stay green rating was small in magnitude, does not represent a trend in the data across studies and years, and is not cons idered biologically meaningful in terms of increased plant pest potential (Figure VIII-1, Step 3).

In the single-site analysis of the water-limited treatment in TX, no statistical differences

were detected between MON 87460 and the co nventional control for seedling vigor, early stand count, days to 50% pollen shed, days to 50% silking, ear height, plant height, dropped ears, stalk lodged plants, root lodge d plants, final stand count, grain moisture, test weight, and yield (Table VIII-11). The only significant diffe rence detected was a lower stay green rating (more green tissu e) compared to the control (6.3 vs. 8.3, respectively). More green tissue in MON 87460 is not a characteristic that is interpreted as an increase in plant pest potential. Furt hermore, this difference in stay green was not consistently observed in the water-limited treatment at other sites. Thus, the difference detected in stay green at this single site was small in magnitude, does not represent a trend in the data, and is not considered to be biologically meaningful in terms of increased plant pest potential (Figure VIII-1, Step 5).

Summary of U.S. 2007 Studies 1 and 2 Well-Watered and Water-Limited

Results of the 2007 studies in the U.S. de monstrate that the observed phenotypic and agronomic characteristics for MON 87460 were within the range of responses expected

for corn under well-watered and water-limited conditions. Based on the measured phenotypic and agronomic char acteristics, the re sults support a conclusion of no increased plant pest potential of M ON 87460 compared to conventional corn.Monsanto Company 07-CR-191U Page 199 of 544 Table VIII-8. U.S. 2007 Study-1: Phenotypic and Agronomic Comparison of

MON 87460 to the Control under Well-Watered Conditions in the Combined-Site Analysis in 2007 U.S. Field Trials Esta blished with Well-Watered and WaterLimited Treatments

Well-Watered Treatment

Mean ± S.E. Reference Range 1

Phenotypic

characteristic Units MON 87460 Control Min Max

Seedling vigor 1-9 scale 2 3.0 ± 0.76 3.0 ± 0.76 1.0 5.0
Early stand count #/plot 90.4 ± 5.62 88.3 ± 7.28 74.5 98.5
Days to 50% pollen shed Days 60.8 ± 1.08 60.9 ± 1.22 57.8 63.5
Days to 50% silking Days 58.5 ± 0.57 58.4 ± 0.60 57.0 60.3
Stay green 1-9 scale 3 2.1 0.35 3.1 ± 0.30 2.5 4.5
Ear height in 42.3 ± 3.46 41.3 ± 2.66 32.2 52.7
Plant height in 85.5 ± 5.84 85.9 ± 4.23 72.2 101.7
Dropped ears #/plot 0.0 ± 0.00 0.1 ± 0.13 0.0 0.3
Stalk lodged plants #/plot 0.3 ± 0.16 0.1 ± 0.13 0.0 0.6
Root lodged plants #/plot 0.3 ± 0.16 0.6 ± 0.38 0.0 7.3
Final stand count #/plot 79.8 ± 6.15 78.9 ± 6.03 62.3 93.3

Grain moisture % 14.7 ± 0.14 14.6 ± 0.23 13.5 15.4
Test weight 4 lbs/bu 58.4 ±0.96 57.8 ± 0.63 57.6 58.8

Yield bu/a 227.5 ± 6.41 200.2 ± 11.65 205.0 220.3

S.E. = standard error Note: No statistically significant differenc es between MON 87460 and the control ( α =0.05).

1 Reference range = Minimum and maximum mean values among the seven references.

2 Seedling vigor rating scale: 1 = above average vigor and 9 = poor.

3 Stay green rating scale: 1 = 90-100% green and 9 = 0-19% green

4 Test weight values reported from the TX site only. Data not provided from the CA site.Monsanto Company 07-CR-191U Page 200 of 544 Table VIII-9. U.S. 2007 Study-1: Phenotypic and Agronomic Comparison of

MON 87460 to the Control under Water-Limit ed Conditions in the Combined-Site Analysis in 2007 U.S. Field Trials Esta blished with Well-Watered and WaterLimited Treatments

Water-Limited Treatment

Mean ± S.E. Reference Range 1

Phenotypic

Characteristic Units MON 87460 Control Min Max

Seedling vigor 1-9 scale 2 3.3 ± 0.81 3.0 ± 0.76 1.0 5.0
Early stand count #/plot 85.3 ± 5.77 90.8 ± 6.53 72.5 106.5
Days to 50% pollen shed Days 60.9 ± 1.26 61.4 ± 1.05 58.3 63.5
Days to 50% silking Days 59.1 ± 0.77 58.5 ± 0.73 57.0 60.5
Stay green 1-9 scale 3 3.0 ± 0.53 4.1 ± 0.35 2.9 5.5
Ear height in 41.7 ± 4.87 40.2 ± 4.06 28.7 54.1
Plant height in 82.5 ± 7.07 78.7 ± 6.30 61.7 103.4
Dropped ears #/plot 0.4 ± 0.30 0.8 ± 0.37 0.0 0.8
Stalk lodged plants #/plot 0.6 ± 0.43 0.8 ± 0.41 0.0 1.5
Root lodged plants #/plot 2.7 ± 2.09 0.8 ± 0.49 0.0 4.3
Final stand count #/plot 77.0 ± 6.73 79.6 ± 6.20 61.8 96.0

Grain moisture % 14.7 ± 0.35 14.5 ± 0.19 13.5 14.8
Test weight 4 lbs/bu 57.6 ± 0.74 56.7 ± 0.72 56.5 57.2

Yield bu/a 187.3 ± 16.88 160.6 ± 14.10 156.5 191.0

S.E. = standard error Note: No statistically significant differenc es between MON 87460 and the control ( α =0.05).

1 Reference range = Minimum and maximum mean values among the seven references.

2 Seedling vigor rating scale: 1 = above average vigor and 9 = poor.

3 Stay green rating scale: 1 = 90-100% green and 9 = 0-19% green

4 Test weight values reported from the TX site only. Data not provided from the CA site.Monsanto Company 07-CR-191U Page 201 of 544 Table VIII-10. U.S. 2007 Study-2: Phenotypic and Agronomic Comparison of

MON 87460 to the Control under Well-Watered Conditions in the Combined-Site Analysis in 2007 U.S. Field Trials Esta blished with Well-Watered and WaterLimited Treatments

Well-Watered Treatment

Mean ± S.E. Reference Range 1

Phenotypic characteristic Units MON 87460 Control Min Max Seedling vigor 1-9 scale 2 2.3 ± 0.29 2.2 ± 0.32 1.3 2.7 Early stand count #/plot 55.8 ± 4.32 59.2 ± 2.62 52.0 68.7 Days to 50% pollen shed 3 Days 63.6 ± 0.82 63.3 ± 0.87 61.0 69.0 Days to 50% silking 3 Days 62.2 ± 1.02 62.3 ± 1.03 59.0 69.0 Stay green 1-9 scale 4 5.8* ± 0.32 6.7 ± 0.67 2.0 9.0 Ear height in 37.9 ± 3.35 37.6 ± 2.55 26.0 45.8 Plant height in 84.2 ± 2.73 83.9 ± 2.59 72.8 100.1 Dropped ears #/plot 0.4 ± 0.24 0.3 ± 0.24 0.0 1.0 Stalk lodged plants 3 #/plot 1.2 ± 0.46 1.9 ± 0.77 0.0 14.3 Root lodged plants #/plot 0.9 ± 0.65 5.1 ± 4.99 0.0 9.7 Final stand count #/plot 52.7 ± 3.16 56.1 ± 1.93 52.7 63.7

Grain moisture % 13.9 ± 0.42 13.7 ± 0.53 12.3 17.3 Test weight lbs/bu 60.5 ± 0.27 60.7 ± 0.26 59.3 61.5

Yield bu/a 192.3 ± 17.07 189.6 ± 23.59 133.3 261.3

S.E. = standard error *Indicates significant differe nces detected between MON 87460 and the control (p< 0.05).

1 Reference range = minimum and maximum mean values among the 12 references at KS, NE, and TX.

2 Seedling vigor rating scale: 1 = above average vigor and 9 = below average vigor.

3 No comparisons were made for Days to 50% pollen shed, Days to 50% silking, and Stalk lodged plants

due to a lack of variability.

4 Stay green rating scale: 1 = 90-100% green and 9 = 0-19% green.Monsanto Company 07-CR-191U Page 202 of 544 Table VIII-11. U.S. 2007 Study-2: Phenotypic and Agronomic Comparison of

MON 87460 to the Control under Water-Limi ted Conditions in a 2007 Texas Field

Trial Established with Well-Wat ered and Water-Limited Treatments

Water-Limited Treatment

Mean ± S.E. Reference Range 1

Phenotypic

characteristic Units MON 87460 Control Min Max
Seedling vigor 1-9 scale 2 1.7 ± 0.33 1.7 ± 0.33 1.7 2.0 Early stand count #/plot 55.3 ± 3.33 44.0 ± 7.77 51.3 59.0 Days to 50% pollen shed 3 Days 61.0 ± 0.00 61.0 ± 0.00 61.0 61.0 Days to 50% silking 3 Days 59.0 ± 0.00 59.0 ± 0.00 59.0 59.0 Stay green 1-9 scale 4 6.3* ± 0.33 8.3 ± 0.33 8.3 9.0 Ear height in 25.5 ± 2.02 27.9 ± 2.51 26.6 30.3 Plant height in 73.5 ± 3.18 75.6 ± 1.63 74.1 83.2 Dropped ears #/plot 0.0 ± 0.00 0.7 ± 0.67 0.0 0.7 Stalk lodged plants 3 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0 Root lodged plants #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 0.3 Final stand count #/plot 54.7 ± 2.67 44.0 ± 7.77 49.3 58.0

Grain moisture % 13.0 ± 0.07 12.7 ± 0.18 12.3 12.8 Test weight lbs/bu 59.7 ± 0.26 59.7 ± 0.52 59.6 60.4

Yield bu/a 228.3 ± 11.53 168.8 ± 27.78 173.8 208.1

S.E. = standard error *Indicates significant differe nces detected between MON 87460 and the control (p< 0.05).

1 Reference range = minimum and maximum mean values among the four references at TX.

2 Seedling vigor rating scale: 1 = above average vigor and 9 = below average vigor.

3 No comparisons were made for Days to 50% pollen shed, Days to 50% silking, and Stalk lodged plants

due to a lack of variability.

4 Stay green rating scale: 1 = 90-100% green and 9 = 0-19% green.Monsanto Company 07-CR-191U Page 203 of 544 VIII.C.3. Field Studies Established und er Water Conditions Typical for Local Agronomic Practice

This field study was designed to eval uate MON 87460 under a broad range of

environmental conditions relevant to commer cial corn production. No differences were expected between MON 87460 and the control w ith the exception of reduced yield loss for MON 87460 if soil moisture became limiting.

Assessment In 2006, a field study with water managed according to local practices was established at five sites (Table VIII-2). Comparative assessments of phenot ypic and agronomic characteristics were conducted on MON 87460 and a conventional control. In addition, 15 commercial corn hybrids were included as references. In the combined-site analysis,

no statistical differences were detected be tween MON 87460 and the control for any of the 14 assessed phenotypic and agronomi c characteristics (Table VIII-12).

Summary of U.S. 2006 under Conditions Ty pical for Local Agronomic Practice The results from these studies support a conclu sion of no increased plant pest potential of MON 87460 compared to conventional corn und er conditions where water was managed according to typical local agronomic practices.Monsanto Company 07-CR-191U Page 204 of 544 Table VIII-12. Phenotypic and Agronomic Comparison of MON 87460 to the Control in the Combined-Site Analysis from a 2006 U.S. Fiel d Study Conducted under Typical Agronomic Conditions

Mean ± S.E. Reference Range 1

Phenotypic

characteristic Units MON 87460 Control Min Max

Seedling vigor 0-9 scale 2 7.6 ± 0.13 7.8 ± 0.14 7.0 8.0
Early stand count #/plot 64.5 ± 0.67 62.3 ± 1.21 58.7 72.3
Days to 50% pollen shed Days 63.1 ± 0.55 63.1 ± 0.53 59.0 65.0
Days to 50% silking Days 62.2 ± 0.71 61.7 ± 0.69 57.3 64.7
Stay green 0-9 scale 3 3.7 ± 0.52 3.7± 0.55 1.0 6.7
Ear height in 45.6 ± 1.54 44.9 ± 1.53 31.9 51.2
Plant height in 97.8 ± 1.74 97.2 ± 1.72 84.9 108.3
Dropped ears #/plot 0.1 ± 0.13 0.1 ± 0.09 0.0 0.7
Stalk lodged plants #/plot 5.5 ± 1.03 5.1 ± 1.55 0.3 7.7
Root lodged plants #/plot 2.1 ± 0.64 1.1 ± 0.51 0.0 5.7
Final stand count #/plot 57.9 ± 0.82 57.4 ± 0.96 53.5 58.7

Grain moisture % 17.5 ± 0.52 17.7 ± 0.63 15.5 22.6
Test weight lbs/bu 55.3 ± 0.73 54.8 ± 0.95 49.7 60.3

Yield bu/a 170.2 ± 6.26 165.3 ± 7.41 143.6 213.4

S.E. = standard error Note: No statistically significant differenc es between MON 87460 and the control ( α =0.05).

1 Reference range = Minimum and maximum mean values among the 15 reference corn hybrids.

2 Seedling vigor rating scale: 0 = dead and 9 = above average vigor.

3 Stay green rating scale: 0 = entire plan t is dried and 9 = entire plant is green.

VIII.D. Seed Germination and Dormancy Assessment

APHIS considers the potential for weedines s to constitute a plant pest factor. (Section IX). As mentioned in Section VIII.A, several weediness indicators (lodging, ear drop) were included in the testing described in Section VIII.C and no biologically meaningful differences were shown betw een MON 87460 and conventional corn. Other indicators, specifically seed germination and dormancy, are also relevant to weediness determinations, and these were tested separately as described below.

The purpose of this study was to assess seed germination and dormancy characteristics of MON 87460 compared to a conventional contro l, which had a genetic background similar to MON 87460 but did not possess the drought to lerance trait. In addition, seed from multiple conventional corn hybrids (references) we re included in the analysis to establish a range of natural variability for each characteristic. Seed germination and dormancy mechanisms vary with species and their genetic basis tends to be complex. Seed dormancy (e.g., hard seed) is an important char acteristic that is of ten associated with plants that are considered as weeds (A nderson, 1996; Lingenfelter and Hartwig, 2003). Information on germination and dormancy is therefore useful when assessing a plant forMonsanto Company 07-CR-191U Page 205 of 544 increased weediness potential. For corn, the potential for hard seed (dormant) is negligible or nonexistent. Standardized ge rmination assays are available and routinely used to measure the germination characteristics of corn seed. The A ssociation of Official Seed Analysts (AOSA), an internationall y recognized seed testing organization, recommends a temperature range of 20-30 ° C as optimal for germination of corn (AOSA, 2006). The results of this study, in partic ular the absence of hard seed, support a conclusion of no increased weediness potenti al of MON 87460 compared to conventional

corn.

Seed of MON 87460, the conventional contro l, and three commercial corn hybrids (references; nine total, thre e per location) were produced in 2006 at three locations (Greene County, IA; Stark County, IL; and Pawnee County, KS) with variable environmental conditions representative of corn producing regions in the U.S. The conventional control corn had a gene tic background similar to MON 87460.

The germination characteristics, temperature regimes and the evaluation descriptions are presented in Table VIII-13. The tests were conducted in temperature-controlled growth chambers using the rolled towel test method. Four replicates of the seed materials from each location were tested in seven growth chambers, each maintained in the dark under one of the following temperature regimes: cons tant target temperature of approximately 5, 10, 20, or 30°C; and alternating target te mperatures of approximately 10/20, 10/30, or 20/30°C (Table VIII-13). In the alternating temperature regimes, the lower temperature was maintained for 16 h and the higher temper ature for 8 h. Seeds were evaluated four and seven days after planting in the AOSA -recommended temperature regime (20/30°C) and at four, seven, and twelve days after planting in the additional temperature regimes.

A statistical comparison between MON 87460 and the control was conducted using SAS (SAS Release 9.1.3 (TS1M3). 2002-2003). St atistical significance was set at p< 0.05. A summary of the results is provided in Table VIII-14, while the details of the materials, methods, and results from individual sites analysis are provided in Appendix J.

In the initial analysis, no pr oduction site × seed substance in teractions were detected for any combination except percent germinated and pe rcent viable firm swo llen seed at 10°C.

These two characteristics were analyzed separately by site to account for the interaction. In the combined site analysis, no stat istical differences were detected ( α = 0.05) between MON 87460 and the control in any temperat ure regime for any characteristic (27 comparisons, Table VIII-14).

As noted above, significant pr oduction site × seed substan ce type interactions were detected in percent germinated and percent vi able firm swollen seed at 10°C. Analyses

were therefore conducted on an individual site basis for thes e variables at 10°C. No

differences were detected between MON 87460 and the control for seed from the IL or KS production sites. For the IA site, M ON 87460 had greater percent germination and lower percent viable firm swollen seed than the control (91.5 vs. 87.0; 6.5 vs. 11.2, respectively). However, no differences were de tected at other sites, and for the IA site, the values for MON 87460 and the control at 10°C were within the reference range forMonsanto Company 07-CR-191U Page 206 of 544 both percent germination and percent viable fi rm swollen seed. Thus, the difference is not expected to constitute a meaningful bi ological change for the crop in terms of weediness potential (Figure VIII-1, Step 3).

The biological characteristics evaluated in this study were used to characterize MON 87460 in the context of plant pest risk assessment. The result s of this study, in particular the absence of hard seed, s upport a conclusion of no increased weediness potential of MON 87460 compar ed to conventional corn.Monsanto Company 07-CR-191U Page 207 of 544 Table VIII-13. Seed Germination a nd Dormancy Parameters Evaluated

Germination

characteristic Temperature regime (°C) Evaluation description

Normally

Germinated 20/30 Seedlings that exhibited normal developmental

characteristics and possessed both a root and a

shoot.

Abnormally

Germinated 20/30 Germinated, but with

insufficient root or shoot development, may have possessed a hollow coleoptile, or exhibited mechanical damage.

Total Germinated 5, 10, 20, 30, 10/20, 10/30 Seedlings that had germinated.

Dead 5, 10, 20, 30, 10/20, 10/30,

20/30 Seeds that had visibly

deteriorated and had

become soft to the touch.

Viable Hard 5, 10, 20, 30, 10/20, 10/30,

20/30 Seeds that did not imbibe

water and remained hard to

the touch.

Viable Firm Swollen 5, 10, 20, 30, 10/20, 10/30,

20/30 Seeds that had visibly swollen (imbibed water)

and were firm to the touch but lacked any evidence of growth.

Note: Germination tests were c onducted in temperature-controlle d growth chambers using the

rolled towel test method to measure the describ ed characteristics. Four replicates of MON 87460, the control, and the nine references were tested in seven growth chambers, each

maintained in the dark under one of the follo wing temperature regimes: constant target temperature of approximately 5, 10, 20, or 30 ° C; and alternating target temperatures of approximately 10/20, 10/30, or 20/30 ° C. In these alternating temperature regimes, the lower temperature was maintained for 16 h and the higher temperature for 8 h. Counts for characteristics were made four and seven days after planting for seed placed in the AOSA– recommended temperature of 20/30°C; counts we re made on four, seven, and twelve days after planting for seed in the add itional temperature regimes.Monsanto Company 07-CR-191U Page 208 of 544 Table VIII-14. Germination of MON 87460 and a Conventional Control Corn

across Sites 1

Mean % ± S.E. Reference Ran g e
Tem p erature Characteristic MON 8746 Control Min Max

5 ° C Dea d 3.9 ± 0.90 4.5 ± 0.93 4.0 11.0

Germinate d 0.0 ± 0.00 0.0 ± 0.00 0.0 0.3

Viable firm swolle n 96.1 ± 0.90 95.5 ± 0.93 89.0 96.0

Viable har d 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

10°C Dea d 1.0 ± 0.30 1.0 ± 0.32 0.8 6.0

Germinated 3

- - - - Viable firm swollen 3

- - - - Viable hard 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

20°C Dea d 0.6 ± 0.23 0.3 ± 0.18 0.5 5.8

Germinate d 99.4 ± 0.23 99.8 ± 0.18 94.3 99.5

Viable firm swollen 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable har d 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

30°C Dea d 1.2 ± 0.55 1.7 ± 0.36 0.5 6.0

Germinate d 98.8 ± 0.55 98.3 ± 0.36 94.0 99.5

Viable firm swollen 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable har d 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

20 / 30°C (AOSA) Dea d 0.5 ± 0.19 0.9 ± 0.23 0.3 4.3

Normal g erminate d 98.7 ± 0.38 98.4 ± 0.26 93.3 98.0

Abnormal 0.8 ± 0.30 0.7 ± 0.22 1.0 4.3

Viable firm swollen 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable har d 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

10 / 20°C Dea d 1.0 ± 0.39 1.7 ± 0.34 0.3 5.0

Germinate d 99.0 ± 0.39 98.3 ± 0.34 95.0 99.8

Viable firm swollen 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable har d 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

10 / 30°C Dea d 1.5 ± 0.34 1.3 ± 0.37 0.3 5.0

Germinate d 98.5 ± 0.34 98.8 ± 0.37 95.0 99.8

Viable firm swollen 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable har d 2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0
S.E. = standard error; n = 12 for MON 87460 and control. Note: No statistically significant differe nces between MON 87460 and the control ( α =0.05).

1 Seed was produced at Greene County, IA, Stark County, IL, and Pawnee County, KS, in the

U.S.

2 Analysis could not be conducted due to lack of variability in the data. For these data the values were all zero, indicating no biological differences.

3 Across-site analyses could not be run due to site × substance interactions.

The data in this table are the combined-site resu lts of the seeds from the three production sites. The results for the individual site analysis wher e the site × substance interaction occurred are

included in Appendix J.Monsanto Company 07-CR-191U Page 209 of 544 VIII.E. Pollen Morphology and Viability Assessment

APHIS also considers the potential for gene flow to, and introgression of the biotechnology-derived trait into, other corn varieties to determine the potential of increased weedy or invasive characteristics in other plant species. Therefore, pollen morphology and viability of M ON 87460 were also assessed.

The purpose of this study was to assess whet her the introduct ion of the drought tolerance trait and the expression of the CSPB protein altered th e pollen characteristics of MON 87460 compared to a conventional contro l, which had a genetic background similar to MON 87460 but did not posse ss the drought tolerance trait. In addition, pollen from multiple conventional corn hybrids (references) was included in the analysis to establish a range of natural variab ility for each characteristic. Pollen was collected from four replications of MON 87460, the control, and four commercial corn references grown in California during 2007 under a split-plot de sign, under well-watered and water-limited conditions (Table VIII-2). Pollen samples from three plants per plot were fixed and stained with 1:5 diluted Alexander stain (A lexander, 1980) and evaluated for viability and general morphology. Data analysis comp ared the mean of MON 87460 to the mean of the control corn for average pollen diameter and percent viable pollen. Additional

materials and methods are provided in Appendix K.

No differences were detected ( α = 0.05) between MON 87460 and the control for pollen diameter or viability in either the we ll-watered or the water-limited treatment (Tables VIII-15 and VIII-16). No visual differences in general pollen morphology were

observed between MON 87460 and the control. These results demonstrate that the introduction of the drought tolerance trait and the expression of the CSPB protein did not alter the overall morphology or viability of pollen from MON 87460 compared to the

control under well-watered or water-limited cond itions. The lack of differences between the pollen collected from MON 87460 compar ed to the conventional control for the

assessed characteristics demonstrate that the observed values were within the range of responses expected for corn.

Table VIII-15. Viability and Diameter of Pollen Collected from MON 87460 and a

Conventional Control Corn under Well-Watered Conditions in 2007

Mean ± S.E. Reference Range 1

Characteristic MON 87460 Control Min Max

Viability (%) 98.4 ± 0.50 98.8 ± 0.44 97.4 98.6
Diameter ( μ m) 86.3 ± 1.35 86.5 ± 1.20 85.5 88.6

S.E. = standard error; n = 4 for MON 87460 and control. No statistically significant differences between MON 87460 and the control ( α =0.05).

1 Minimum and maximum mean values of reference substances. References were four commercial hybrids.Monsanto Company 07-CR-191U Page 210 of 544 Table VIII-16. Viability and Diameter of Pollen Collected from MON 87460 and a

Conventional Control Corn under Watered-Limited Conditions in 2007

Mean ± S.E. Reference Range 1

Characteristic MON 87460 Control Min Max

Viability (%) 96.9 ± 0.64 97.2 ± 0.67 96.3 98.5
Diameter ( μ m) 86.9 ± 1.01 85.6 ± 0.73 86.2 89.7

S.E. = standard error; n = 4 for MON 87460 and control. No statistically significant differences between MON 87460 and the control ( α =0.05).

1 Minimum and maximum mean values of reference substances. References were four commercial hybrids.

VIII.F. Environmental In teractions Assessment

Environmental interactions evaluations were conducted as part of the plant

characterization studies for M ON 87460. These data are used to evaluate plant pest potential for MON 87460 compared to a conven tional control. In addition, multiple conventional corn hybrids (references) were incl uded in the analysis to establish a range of natural variability for each characteristic . The environmental interactions evaluation included data collected in the phenotypic studie s (plant-insect, plant-disease, and plantenvironment interactions) and studies on abioti c stress tolerance, potential for the crop to volunteer, and survival outside of cultivation.

These environmental interactions characteristics are all useful to assess plant pest potential of MON 87460. In addition, cert ain characteristics, including volunteer potential and survival outside cultivation, can be used for an assessment of weediness, an element of plant pest potential. Abiotic stre ss tolerance was evaluated qualitatively from natural occurrences in the phenotypic fiel d studies and also quantitatively under controlled conditions in gree nhouse and growth chamber studi es. Studies to assess tolerance to drought, cold, heat, and salt stress at sensitive young growth stages under

controlled conditions are useful for characterizing the exte nt of stress tolerance imparted by the insertion of the cspB gene. Such a characterizatio n is useful to identify any potential changes in tolerance that warrant further investiga tion of plant pest risk under stress conditions. If no changes in toleran ce are observed, further investigation is not

warranted because the characteristics of the biotechnology-derived plant are not meaningfully different from the control under stress conditions.

Results of the environmental interactions a ssessment show the drought tolerance trait did not unexpectedly alter MON 87460 compared to conventional corn. The lack of differences in plant response to abiotic stressors (excludi ng drought stress), disease damage, arthropod damage, and arthropod pest and beneficial abundance indicate that the introduction of the drought tolerance trait is unlikely to be biologic ally meaningful in terms of increased plant pest potential. Finally, arthropod (pest and beneficial) abundance indicates no significant impact on non-target organisms for purposes of

NEPA.Monsanto Company 07-CR-191U Page 211 of 544 VIII.F.1. Insect, Disease, and Abiotic Stress Interactions Assessment in Field Studies

In the two years of field studies conducte d for evaluation of phenotypic and agronomic characteristics of MON 87460, observational da ta on the presence of and differential response to biotic (insects, diseases) and abiotic (e.g., drought, wind, nutrient deficiency)

stressors were also collected to examine th e environmental interactions of MON 87460 compared with those of the conventional co ntrol corn. The observed stressors were “natural” (i.e., no artificial infestation or in terference was used). Therefore, the same stressors were not necessarily observed at each field site.

In these studies, environmental interactions were assessed qualitatively in all 31 sites and quantitatively for insect interactions in si x selected sites. Observation of plant interactions with insect pests and diseases, and plant responses to abiotic stressors were collected from each of the 31 field sites in 2006 and 2007. The purpose of these evaluations was to assess plant-insect or pl ant-disease interactions , or plant response to abiotic stressors of MON 87460 compared to the control. For the plant-insect interactions, plant-disease interactions, a nd plant responses to abiotic stressors, the reported values represent the range of ra tings observed across the three or four replications at each site. MON 87460 and the control were considered qualitatively different in response to a st ressor if the ratings between MON 87460 and the control corn did not overlap across all repl ications for that particular stressor (e.g. “none” rating vs. “slight-moderate” rating). The ratings obser ved among the commercial reference hybrids provide qualitative assessment data common to the crop for each stressor assessed.

Qualitative Assessments Across all 31 sites, approximately 21 arthropods (species or group), 19 disease categories (species or group) and 15 abiotic stressors we re evaluated. No qualitative differences were observed in MON 87460 compared to the control for disease, damage or plant response to abiotic stressors (other than wate r stress) evaluated. Fo r pest and beneficial arthropod evaluations, two differences we re observed out of approximately 388 comparisons. Grasshopper damage was lowe r for MON 87460 compared to the control (none vs. slight) at the IAE site at Ob servation 3 (Appendix H, Table H-17). The difference in grasshopper damage was not c onsidered biologically meaningful because

the difference detected at th e IAE site was within the ra nge of the references (noneslight). Additionally, no differences were detected between MON 87460 and the control for grasshopper damage for Observations 2 a nd 4 at the IAE site, or at any observation time at the other sites (Appendix H, Table H-17). At the IA1 site, European corn borer damage was higher for MON 87460 compared to the control (moderate vs. slight; Appendix H, Table H-10). However, the observed incidence of European corn borer damage was not detected at other sites or in additional observation times at this site. Thus, the difference in European corn borer da mage does not represent a trend in the data

and is not considered bi ologically meaningful.Monsanto Company 07-CR-191U Page 212 of 544 Quantitative Assessments

European Corn Borer and Corn Earworm Damage Specific quantitative assessments for corn earworm and European corn borer damage

were conducted at three well-watered site s in 2006 (Appendix H, Tables H-4 and H5, at three well-watered s ites in 2007 (Appendix H, Tables H-11 and H-12), and a single site at TX in U. S. 2007 Study-1, with well-w atered and water-limited treatments (Appendix H, Table H-24). In combined-site analyses across all years and sites, and for the TX site in Study-1, no st atistical differences we re detected between MON 87460 and control for corn earworm damage or for European corn borer damage.

Arthropod Abundance Arthropod collections were conducted at th ree well-watered sites in 2006 (Appendix H, Tables H-6 and H-7), at three well-w atered sites in 2007 (Appendix H, Tables H13 and H-14) and a single site at TX in U.S. 2007 Study-1, with well-watered and water-limited treatments (Appendix H, Tables H-25 and H-26). In an assessment of pest and beneficial arthropod abundance across all sites, a total of eight differences were detected out of 326 comparisons (163 pest and 163 beneficial arthropod comparisons) between MON 87460 and the contro l. For three of the eight detected differences, the mean abundance values fr om MON 87460 were within the respective reference ranges. For the remaining di fferences, the mean abundance value from MON 87460 was higher (two comparisons) or lower (three comparisons) than the reference range. Furthermore, each detected difference was observed at a single observation time point. This suggests the dete cted differences were not indicative of a consistent response in the data associat ed with the trait and are unlikely to be biologically meaningful in te rms of plant pest potentia l of MON 87460 compared to

the control.

Summary of Insect, Disease, and Abiotic St ressor Interactions Assessment in Field

Studies

The results of the environm ental interactions evaluation for MON 87460 supports the conclusion that the introduction of the drought tolerance trait did not unexpectedly alter MON 87460 compared to conventional corn. The lack of differences in plant response to abiotic stressors (excluding moisture defi cit), disease damage, and arthropod damage

indicates that the introduction of the drought to lerance trait is unlikel y to be biologically meaningful in terms of increas ed pest potential. Finally, arthropod (pest and beneficial) abundance indicates no significant impact on non-target organisms for purposes of

NEPA.

VIII.F.2. Abiotic Stress Tolerance Assessmen t in Controlled Environment Studies

Abiotic stress tolerance was evaluated quali tatively from natural occurrences in the phenotypic field studies (Section VIII.F.1) and also quantitatively under controlled environment conditions in greenhouse and grow th chamber studies as presented in thisMonsanto Company 07-CR-191U Page 213 of 544 section. Studies to assess tolerance to drough t, cold, heat, and salt stress at sensitive young growth stages under controlled conditions are useful for charac terizing the extent of stress tolerance imparted by the inserted st ress tolerance protein (CSPB) (see mode of action Section I.D). Although drought, cold, h eat or salt tolerance could be associated with some plants that are weed s, a change in these factors in corn would not necessarily

be correlated with increased weediness. This is because of the inherent non-weedy nature of corn exemplified by a lack of dispersal mechanisms, the lack of ability to survive in highly competitive plant communities wit hout the intervention of man, a lack of dormancy mechanism that allows long term survival in the soil, and lack of other factors

that would be needed for corn to function as a weed. In all studies, MON 87460 was compared to a conventional control from planting through study completion, which in no cases lasted past the vegetative growth stages , so that yield was not an observed factor.

Results from these studies show that M ON 87460 is susceptible to drought, cold, heat, and salt stress and support the conclusion that the abiotic stress tolerance of MON 87460 during young plant growth stages is not meani ngfully different compared to conventional

corn.

VIII.F.2.1. Drought Tolerance Assessment

The purpose of this study was to assess the effe ct of various levels of drought stress on the growth and development of plants of MON 87460 compared to a conven tional control in a greenhouse environment. The test s ubstance, MON 87460, and a conventional corn control were established in pot s in a greenhouse in excess of what was needed for the study. At the V4 growth stage, 80 test plan ts and 80 control plants were selected for uniformity and arranged in a complete fact orial treatment structure (8 treatment combinations) in a randomized complete bloc k design with 20 replic ations. The factors were drought level (well-wate red, mild, moderate, or seve re) and plant substance type (test or control).

Drought stress treatments were initiated at the V4 growth stage and continued for 15 days. Pots in each treatment were irrigated to maintain target pot weights that were intended to provide a range of growth reductions as follows:

Treatment Pot weight target for irrigation (g)

Well-watered 4700 - 4800

Mild 3500 - 3900

Moderate 2700 - 2900

Severe 2160 - 2400

Plants were evaluated prior to drought initiation and at 7 and 15 days after drought stress treatment (DAT). Plants were evaluated for plant height, grow th stage, chlorophyll content, and leaf rolling score. Fresh a nd dry weights of above -ground biomass were measured at the conclusion of the experiment.Monsanto Company 07-CR-191U Page 214 of 544 Prior to the imposition of the drought stress tr eatments, no statistical differences were detected ( α = 0.05) between MON 87460 and the contro l for chlorophyll content. No

numeric differences were detected between MON 87460 and the control for growth stage, where statistical comparisons could not be made due to lack of variability (Table VIII17). MON 87460 exhibited shorter plant heig ht than the control (88.0 vs. 89.6 cm, respectively). This difference is small in ma gnitude, does not represent a consistent trend

across the other treatments in this study, and likely represen ts biological variation in

MON 87460 and the control.

Well-watered treatment

Plants in the well-watered treatment were included to provide plant characterization

data common to MON 87460 and the control in the absence of drought stress. In the well-watered treatment, no significant differences were detected between MON 87460 and control for plant height, chlorophyll content and leaf rolling. Three differences were observed in plants that received the well-watered treatment after drought treatments were imposed. MON 87460 had fewer leaves than the control (growth stage 9.6 vs. 9.9, resp ectively) and had lower fres h and dry weight (620.6 vs. 659.4 g and 65.5 vs. 70.5 g, respectively). (Table VIII-17).

Mild drought treatment In the mild drought treatment, no significan t differences were detected between MON 87460 and control for any of the meas ured characteristics (Table VIII-17).

Moderate drought treatment In the moderate drought treatment, no signifi cant differences were detected between MON 87460 and control for chlorophyll conten t and for biomass (fresh weight and dry weight). Four differe nces were detected between MON 87460 and the control in the moderate drought treatment. MON 87460 ha d less plant height than the control 14 DAT (127.0 vs. 130.2 cm, respectively), ha d fewer leaves at 7 DAT and 14 DAT (growth stage 5.4 vs. 5.8 and 7.3 vs. 7.7, re spectively) and MON 87460 had a higher leaf rolling score 7 DAT than the contro l (2.4 vs. 2.1, respectively, Table VIII-17).

Severe drought treatment In the severe drought treatment, no significa nt differences were detected between MON 87460 and control for plan t height, growth stage, chlorophyll content, or biomass (fresh weight and dry weight). One difference was detected between MON 87460 and the control for plants in the severe drought tr eatment. MON 87460 had a lower leaf rolling score than the c ontrol (2.6 vs. 3.0, respectively, Table VIII17).

Summary of Drought Tolerance Assessment The biological endpoints measured in this study were used to assess drought stress on the growth and development of the biotechnology- derived crop compared to a conventional control. Compared to test and control pl ants in the well-watered treatment, test and control plants in the mild, moderate, and se vere treatments exhibited a dose-dependent pattern of lower plant height, growth stage, fresh weight , and dry weight with increasingMonsanto Company 07-CR-191U Page 215 of 544 water stress. Furthermore, the differences ob served in plant height, growth stage, and leaf rolling were not consistent across treat ments, do not indicate a competitive advantage for MON 87460, and do not represent a meani ngful trend. Therefore, based on the characteristics measured to assess drought tole rance, the results s upport a conclusion that

MON 87460 is still sensitive to drought stress and does not differ from conventional corn after exposure to a range of stress levels im posed during early vegetative growth stages.Monsanto Company 07-CR-191U Page 216 of 544Table VIII-17. Comparison of MON 87460 to the Control Prior toDrought Treatment and at 7 and 15 Days after TreatmentWell-wateredMean ± S.E.MildMean ± S.E.ModerateMean ± S.E.SevereMean ± S.E.Evaluationtiming CharacteristicMON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlPrior to treatment1Plant height(cm)88.0*± 0.4189.6± 0.52- - - - - -Growth stage24.03± 0.0 4.03± 0.0 - - - - - -Chlorophyll451.4 ± 0.35 50.9 ± 0.46 - - - - - -Post-treatment Plant height(cm)126.3± 0.86127.9± 1.09119.1± 1.23119.8± 1.07112.2± 0.92114.3± 0.70112.3± 0.74112.4± 0.83(7 DAT)Growth stage6.2 ± 0.08 6.1 ± 0.1 5.9 ± 0.07 6.0 ± 0.05 5.4* ± 0.11 5.8 ± 0.1 5.4 ± 0.11 5.6 ± 0.11Chlorophyll54.4± 0.5353.7± 0.855.1± 0.7153.9± 0.5253.4± 1.0953.7± 0.4854.0± 0.6552.4± 0.71Leaf roll51.0± 0.01.0± 0.01.0± 0.01.0± 0.02.4*± 0.182.1± 0.142.6*± 0.183.0± 0.17Post-treatment Plant height(cm)175.3± 0.97178.0± 0.88152.1± 1.25151.0± 1.75127.0*± 0.93130.2± 1.0119.1± 0.68120.3± 0.97(15 DAT)Growth stage9.6* ± 0.11 9.9 ± 0.07 9.0 ± 0.0 8.9 ± 0.08 7.3* ± 0.1 7.7 ± 0.11 6.1 ± 0.07 6.3 ± 0.11Chlorophyll53.4± 0.4953.8± 0.4353.6± 0.6452.8± 0.556.0± 0.7656.2± 0.6455.6± 0.7254.7± 0.56Leaf roll1.0 ± 0.0 1.0 ± 0.0 1.1 ± 0.05 1.0 ± 0.0 2.2 ± 0.12 2.1 ± 0.11 3.8 ± 0.1 3.7 ± 0.11Fresh weight (g)620.6*± 14.73659.4± 9.24457.6± 14.64430.9± 14.60230.8± 5.5249.1± 6.22174.1± 4.53179.9± 4.05Dry weight (g)65.5*± 1.5170.5± 0.9351.4± 1.3249.1± 1.3632.4± 0.6535.1± 0.7226.5± 0.6726.8± 0.59S.E. = standard error; n=20 for test andcontrol plants in each treatment. *Indicates significant differences detected betweenMON 87460 and the control(p<0.05) provided by t-test comparisons.1Due to the randomization of the substances and drought treatments to the plants within a replication before droughttreatments were applied, the pre-treatment measurements for each substance consist of four sub-samples (n=20).2Growth stage was determined by the numberof leaves with a visible leaf collar.3Data could not be analyzed due to lack of variability.4Chlorophyll - relative chlorophyll content was an average ofreadings at the base, midpoint and tip of the fourth leaf measured with a Single Photon Avalanche Diode-containing (SPAD) meter.5Leaf roll was rated on ascale of 1 to 5 (1=no rolling, 2=slight rolling upper leaves (younger), 3=more rolling upper leaves and slight rolling lower leaves, 4=all leaves show signs ofrolling and 5=severe leaf rolling with little or no top leaf visible).Monsanto Company 07-CR-191U Page 217 of 544 VIII.F.2.2. Cold Tolerance Assessment

The purpose of this study was to assess the effect of cold temperature-induced stress on the growth and development of plants of MON 87460 compared to a conventional corn control. The test substance, MON 87460, and a conventional control were established in pots, in excess of what was needed for th e study, in growth chambers programmed for optimal conditions (30/22 °C). At the V3 grow th stage, uniform plants were selected and transferred to growth chambe rs with optimal (30/22° C), mild (20/15 °C), moderate (15/10 °C), and severe (4/4 °C) cold treatm ents for eight days. In each chamber, 20 replicates (one plant per pot ) each of MON 87460 and the control were arranged in a completely randomized design with a photop eriod of 16 hours. Each treatment was considered to be a separate experiment. All plants were watered as needed for the duration of the experiment.

Plants were evaluated for plant height, growth stage, chlorophyll content, and vigor three times during the experiment: prior to treatment and at 4 and 8 DAT. Fresh and dry weights of above-ground biomass were measur ed at the conclusion of the experiment. No significant differences we re observed in plants prior to temperature treatments (Table VIII-18).

Optimal temperature treatment (30/22° C) Plants in the optimal temperature treat ment were included to provide plant

characterization data for MON 87460 and the co ntrol in the absence of cold stress and to validate that the cold temperature treatmen ts affected corn growth as expected. In the optimal temperature treatment, no signifi cant differences were detected between MON 87460 and the control for plant height, chlorophyll content, vigor, or fresh weight. Three differences were detected for plants in the optimal chamber.

MON 87460 had more leaves than the cont rol 4 and 8 DAT (growth stage 3.6 vs. 3.3 and 4.5 vs. 4.1, respectively) and MON 87460 had greater dry weight than the control (2.8 g vs. 2.4 g, respectively). (Table VIII-18).

Mild temperature treatment (20/15° C) In the 20/15° C treatment, no differences were detected between MON 87460 and the control for any of the measured characteristics (Table VIII-18).

Moderate temperature treatment (15/10° C) In the 15/10° C treatment, no differences were detected between MON 87460 and the control for any of the measured characteristics (Table VIII-18).

Severe temperature treatment (4/4° C) In the 4/4° C treatment, no differences were detected between MON 87460 and the control for any of the measured characteristics (Table VIII-18).

Summary of Cold Tolerance Assessment The biological endpoints measured in this study were used to assess the effects of cold temperatures on the growth and devel opment of the biotechnology-derived crop compared to a conventional cont rol. Compared to test and control plants in the optimalMonsanto Company 07-CR-191U Page 218 of 544 temperature group, test and cont rol plants in the mild, mode rate and severe treatments exhibited a dose-dependent pattern of lower plant height, growth stage, vigor, fresh weight and dry weight. No significant di fferences were observed between MON 87460 and the control in the mild, moderate, or severe cold treatments. Based on the

characteristics measured to assess cold to lerance, the results s upport a conclusion that

MON 87460 is still sensitive to cold stress a nd any differences from conventional corn in response to cold stress are not re lated to the gene of interest.Monsanto Company 07-CR-191U Page 219 of 544Table VIII-18. Comparison of MON 87460 to the Control Priorto Cold Treatment and at 4 and 8 Days after TreatmentOptimal Mean ± S.E.Mild Mean ± S.E.Moderate Mean ± S.E.Severe Mean ± S.E.(30/22°C)(20/15°C)(15/10°C)(4/4°C)Evaluation Timing Characteristic MON 87460 Control MON 87460 ControlMON 87460 ControlMON 87460 ControlPrior to treatment Plant height (cm)43.1± 0.4742.3± 0.3343.2± 0.4542.4± 0.3943.9± 0.4242.8± 0.4444.3± 0.3544.1± 0.56Growth stage13.0± 0.03.0± 0.03.0± 0.03.0± 0.03.0± 0.03.0± 0.03.0± 0.03.0± 0.0Chlorophyll240.3± 0.7939.5± 0.6541.3± 0.5439.9± 0.8239.8± 0.7440.2± 0.8843.6± 0.6542.9± 0.75Vigor3,42 ± 0.0 2 ± 0.0 2 ± 0.0 2 ± 0.0 2 ± 0.0 2 ± 0.0 2 ± 0.0 2 ± 0.0Post-treatment Plant height (cm)62.6± 0.4162.7± 0.4654.5± 0.8353.4± 1.0148.8± 0.6647.6± 0.9344.4± 0.544.4± 0.75(4 DAT) Growth stage3.6*± 0.113.3± 0.13.04± 0.03.04± 0.03.04± 0.03.04± 0.03.04± 0.03.04± 0.0Chlorophyll45.1± 0.8344.1± 0.6743.9± 0.5743.3± 1.0339.2± 0.7740.2± 0.7544.8± 0.6644.4± 0.6Vigor42 ± 0.0 2 ± 0.0 3 ± 0.0 3 ± 0.0 3 ± 0.0 3 ± 0.0 4 ± 0.0 4 ± 0.0Post-treatment Plant height (cm)80.2± 0.7781.7± 0.9564.3± 0.7462.6± 0.7654.0± 0.6153.5± 0.7444.3± 0.5244.5± 0.8(8 DAT) Growth stage4.5*± 0.114.1± 0.073.9± 0.084.0± 0.053.04± 0.03.04± 0.03.04± 0.03.04± 0.0Chlorophyll41.5± 0.8241.5± 0.6845.0± 0.8443.2± 1.0238.4± 0.739.2± 0.7944.7± 0.4144.5± 0.84Vigor42 ± 0.0 2 ± 0.0 3 ± 0.0 3 ± 0.0 5 ± 0.0 5 ± 0.0 6 ± 0.0 6 ± 0.0Fresh weight (g)26.2± 1.3723.4± 1.0616.5± 0.6515.8± 0.899.4± 0.378.9± 0.44.5± 0.274.4± 0.28Dry weight (g)2.8*± 0.142.4± 0.091.3± 0.061.3± 0.071.3± 0.051.2± 0.050.5± 0.030.6± 0.04S.E. = standard error; n=20 for MON 87460 and control in each temperature treatment. *Indicates significant differences detected between MON 87460 and thecontrol (p<0.05) provided by t-test comparisons.1Number of leaves with a visible leaf collar.2Relative chlorophyll content was anaverage of readings at thebase, midpoint, and tip of the leaf measured with a Single Photon Avalanche Diode (SPAD)-containing meter.3Plant vigor was rated using a scale of 1 – 9,where 1 = good and 9 = poor.4Not statistically analyzed due to lack of variation.Monsanto Company 07-CR-191U Page 220 of 544 VIII.F.2.3. Heat Tolerance Assessment

The purpose of this study was to assess the e ffect of heat-induced stress on the growth and development of plants of MON 87460 compar ed to a conventional control. The test substance, MON 87460, and a conventional contro l were established in pots, in excess of what was needed for the study, in growth chambers programmed for optimal conditions (30/22 °C). At the V3 growth stage, uniform plants were selected and transferred to growth chambers with optimal (30/22°C), mild (40/35°C), moderate (43/35°C) and severe (47/35°C) heat treatments (day/night ) for five days. In each chamber, 20 replicates (one plant per pot ) each of MON 87460 and the control were arranged in a completely randomized design with a photop eriod of 16 hours. Each treatment was considered to be a separate experiment. All plants were watered as needed for the duration of the experiment.

Plants were evaluated for pl ant height, growth stage, ch lorophyll content, vigor and necrosis prior to heat treatment and at 3 and 5 DAT. Fresh and dry weights of aboveground biomass were measured at th e conclusion of the experiment.

Prior to imposition of the heat treatments, no numeric differences were detected between MON 87460 and the control for growth stage, vigor, and necrosis, where statistical comparisons could not be made due to a lack of variability. M ON 87460 was taller (44.8 vs. 42.7 cm) and had less chlorophyll cont ent (41.0 vs. 43.3 SPAD units [Single Photon Avalanche Diode-containing meter]) than the c ontrol in plants selected for the optimal

treatment. MON 87460 was shorter than the control (43.9 vs. 45.2 cm) in plants selected

for the severe treatment. These differences are small in magnitude, do not represent a

consistent trend across the other treatments in this study, and likely represent biological

variation in MON 87460 and th e control (Table VIII-19).

Optimal temperature treatment (30/22°C) Plants in the optimal temperature treat ment were included to provide plant

characterization data for MON 87460 and the co ntrol in the absence of heat stress and to validate that high temperat ures affected corn growth as expected. In the optimal temperature treatment no significant differe nces were detected between MON 87460 and the control for plant height, vigor and n ecrosis at 3 and 5 DAT. Five differences were observed in plants grown under op timal conditions. MON 87460 had less chlorophyll content compared to the c ontrol 3 and 5 DAT (45.9 vs. 47.7 and 45.6 vs. 47.0 SPAD units, respectively). MON 87460 ha d more leaves than the control 3 DAT (growth stage 3.6 vs. 3.2, respectively). At the conclusion of the experiment, MON 87460 had greater fresh and dry weight than the control (14.5 vs. 12.5 g and 1.1 vs. 1.0 g, respectively) (Table VIII-19).

Mild temperature treatment (40/35°C) In the 40/35 °C treatment, no differences were detected between MON 87460 and the control for plant height, growth stage, vigor, necrosis, and plant biomass (fresh weight and dry weight). One difference was detected between MON 87460 and the control for plants in the mild treatme nt. MON 87460 had lower chlorophyll content than the control 3 DAT (42.3 vs. 43.8, respectively, Table VIII-19).Monsanto Company 07-CR-191U Page 221 of 544 Moderate temperature treatment (43/35°C) In the 43/35 °C treatment, no significan t differences were detected between MON 87460 and the control for any of the m easured characteristics (Table VIII-19).

Severe temperature treatment (47/35°C) In the 47/35 °C treatment, no significan t differences were detected between MON 87460 and the control for any measured characteristics, with the exception of vigor. MON 87460 had less vigor than the control 5 DAT (8.9 vs. 8.6, respectively, Table VIII-19).

Summary of Heat Tolerance Assessment The biological endpoints measured in this st udy were used to asse ss heat stress on the growth and development of the biotechnology- derived crop compared to a conventional control. Compared to test and control plan ts in the optimal temperature group, test and control plants in the mild, moderate and severe treatment s exhibited a dose-dependent pattern of lower plant height, growth stage, vigor, fresh weight and dry weight with increasing temperature. Two diffe rences were observed for plants exposed to heat stress. A reduction in chlorophyll content in the mild treatment 3 DAT and reduced vigor

observed in the severe trea tment 5 DAT are not indicative of heat tolerance in

MON 87460. The differences in chlorophyll content and vigor were not observed across treatments, and therefore, did not represent consistent trends in the data. Based on the

characteristics measured to a ssess heat tolerance, the re sults support a conclusion that

MON 87460 is still sensitive to heat stress and any differences from conventional corn in response to heat stress are not re lated to the gene of interest.Monsanto Company 07-CR-191U Page 222 of 544Table VIII-19. Comparison of MON 87460 to the Control Priorto Heat Treatment and at 3 and 5 Days after TreatmentOptimal Mean ± S.E.Mild Mean ± S.E.Moderate Mean ± S.E.Severe Mean ± S.E.30/22°C40/35°C 43/35°C 47/35°CEvaluation timing CharacteristicMON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlPrior to treatmentPlant height (cm)44.8*± 0.4242.7± 0.443.6± 0.543.3± 0.5943.9± 0.3443.4± 0.5943.9*± 0.445.2± 0.42Growth stage13.05± 0.03.05± 0.03.05± 0.0 3.05± 0.0 3.05± 0.0 3.05± 0.0 3.05± 0.0 3.05± 0.0Chlorophyll241.0*± 0.7143.3± 0.4344.3± 0.4345.8± 0.6244.4± 0.5444.4± 0.5245.2± 0.3744.2± 0.64Vigor31.05± 0.01.05± 0.01.05± 0.0 1.05± 0.0 1.05± 0.0 1.05± 0.0 1.05± 0.0 1.05± 0.0Necrosis41.05± 0.01.05± 0.01.05± 0.0 1.05± 0.0 1.05± 0.0 1.05± 0.0 1.05± 0.0 1.05± 0.0Post-treatmentPlant height (cm)64.0± 0.3763.4± 0.5157.9± 0.556.8± 0.6952.1± 0.7150.3± 0.8944.7± 0.4845.8± 0.4(3 DAT)Growth stage3.6* ± 0.113.2 ± 0.083.05± 0.0 3.05± 0.0 3.05± 0.0 3.05± 0.0 3.05± 0.0 3.05± 0.0Chlorophyll45.9*± 0.4847.7± 0.4342.3*± 0.4843.8± 0.5838.6± 0.6237.6± 0.6220.0± 1.922.1± 2.19Vigor1.05± 0.01.05± 0.01.1 ± 0.07 1.1 ± 0.05 1.6 ± 0.25 1.5 ± 0.2 8.8 ± 0.09 8.7 ± 0.11Necrosis1.05± 0.01.05± 0.01.05± 0.0 1.05± 0.0 1.05± 0.0 1.05± 0.0 4.4 ± 0.17 4.2 ± 0.2Post-treatmentPlant height (cm)71.7± 0.6571.7± 0.760.1± 0.5158.8± 0.6554.8± 0.652.7± 0.9144.6± 0.4645.8± 0.42(5 DAT)Growth stage4.05± 0.04.05± 0.03.4 ± 0.11 3.5 ± 0.11 3.0 ± 0.0 3.0 ± 0.0 3.05± 0.0 3.05± 0.0Chlorophyll45.6*± 0.547.0± 0.4340.1± 0.5140.9± 0.5432.6± 0.9433.2± 0.888.9± 0.738.1± 0.82Vigor1.05± 0.01.05± 0.01.5 ± 0.11 1.8 ± 0.1 5.1 ± 0.1 5.0 ± 0.11 8.9* ± 0.07 8.6 ± 0.15Necrosis1.05± 0.01.05± 0.01.05±0.0 1.05± 0.0 1.1 ± 0.05 1.0 ± 0.0 4.8 ± 0.12 4.6 ± 0.15Fresh weight (g)14.5*± 0.3812.5± 0.39.8± 0.249.8± 0.297.2± 0.246.9± 0.291.2± 0.121.5± 0.19Dry weight (g)1.1*± 0.031.0± 0.031.0± 0.031.0± 0.030.7± 0.040.7± 0.030.4± 0.010.4± 0.01S.E. = standard error; n = 20 for MON 87460 and the control in each temperature treatment. *Indicatessignificant differences detected between MON 87460 andthe control (p<0.05) provided by t-test comparisons.1Number of leaves with a visible leaf collar.2Relative chlorophyll content wasan average of readings atthe base, midpoint and tip of the fourth leaf measured with a Single Photon Avalanche Diode-containing (SPAD) meter.3Plant vigor was rated using a scale of1-9, where 1=good and 9=poor.4Necrosis was rated using a scale of 1-5, where 1=good and 5=poor.5Not statistically analyzed due to lack of variation.Monsanto Company 07-CR-191U Page 223 of 544 VIII.F.2.4. Salt Tolerance Assessment

The purpose of this study was to assess the eff ect of various salt trea tments on the growth and development of plants of MON 87460 compar ed to a conventional control. The test substance, MON 87460, and a conventional corn control were established in pots in a greenhouse in excess of what was needed for the study. Fourteen days after planting, 80 test plants and 80 control plan ts were selected for uniformity and placed in a complete

factorial treatment structure (8 treatment combinations) in a randomized complete block design with 20 replications. The factors were salt level (no salt, mild, moderate, or severe) and plant substance t ype (test or control).

Beginning 17 days after planting and continui ng for 12 days, pots in each salt treatment were irrigated with either reverse osmosis water or varying concentrations of a NaClCaCl 2 solution (up to 600 mM) to achieve diffe rent soil electri cal conductivity (EC)

levels for each salt treatment as follows:

Treatment Target EC (dS/m) Average EC (dS/m) Total salt per pot (g)

No Salt < 1 0.5 0.0

Mild approx. 2 – 4 2.6 14.4

Moderate approx. 5 – 7 4.6 40.2

Severe approx. 8 – 10 7.6 58.4

Plants were evaluated for plant height, growth stage, chlorophyll content, and vigor three times during the experiment: prior to treat ment and at 9 and 12 DAT. Fresh and dry weights of above-ground biomass were measured at the conclusion of the experiment.

Prior to the imposition of the salt treatments, no statistical differences were detected ( α = 0.05) between MON 87460 and the control for pl ant height or vigor (Table VIII-20).

No numeric differences were detected be tween MON 87460 and the control for growth stage, where statistical compar isons could not be made due to lack of variability. MON 87460 exhibited greater chlorophy ll content than the control (50.2 vs. 48.4, respectively) but this difference diminished over time in untreated plants.

No Salt Treatment

Plants that did not receive a salt trea tment were included to provide plant

characterization data for MON 87460 and the co ntrol in the absence of salt stress and

to validate that the salt treatments affected corn growth as expected. Within the untreated group, no significant differences were detected between MON 87460 and the control for any measured character istic at 9 and 12 DAT (Table VIII-20).

Mild Salt Treatment In the mild salt treatment, no differences were detected between MON 87460 and the control for any measured characteristics, with the exception of dry weight. MON 87460 exhibited lower dry weight than th e control (11.3 vs. 11.7 g, respectively; Table VIII-20).Monsanto Company 07-CR-191U Page 224 of 544 Moderate Salt Treatment

In the moderate sa lt treatment, no differences we re detected between MON 87460 and the control for plant height, growth st age, and biomass (fresh weight and dry weight). Two differences were detect ed between MON 87460 and the control for plants in the moderate treatment. M ON 87460 had increased chlorophyll content 9 DAT (48.4 vs. 46.1 SPAD units, respectively) and had increased vigor over the control 12 DAT (6.2 vs. 6.6, respectively, Table VIII-20).

Severe Salt Treatment In the severe salt treatment, no differe nces were detected between MON 87460 and the control for growth stage, chlorophyll c ontent, and biomass (fresh weight and dry weight). Two differences were detect ed between MON 87460 and the control for plants in the severe treatment. MON 87460 was shorter than the control 9 DAT (73.2 vs. 75.6 cm, respectively) and had decrease d vigor 12 DAT (7.9 vs. 7.5 respectively, Table VIII-20).

Summary of Salt Tolerance Assessment The biological endpoints measured in this study were used to assess various salt levels on the growth and development of the bi otechnology-derived crop compared to a conventional control. Compared to test and control plants in the untreated group, test and control plants in the mild, moderate and severe salt treatments exhibited a dosedependent pattern of lower plant height, growth stage, vigor, fresh weight and dry weight. Across salt treatments, a tota l of five differences were detected between MON 87460 and the control. Reduced dry weight for MON 87460 compared to the control in the mild treatment was not indicative of salt tolerance. The increased chlorophyll content and improved vigor in the moderate salt treatment were not observed in the mild or severe

salt treatments. The differences detected between MON 87460 and the control were not consistent across treatments and did not repr esent a trend in the data. Based on the

characteristics measured to a ssess salt tolerance, the re sults support a conclusion that

MON 87460 is still sensitive to salt stress an d any differences from conventional corn in response to salt stress are not rela ted to the gene of interest.Monsanto Company 07-CR-191U Page 225 of 544Table VIII-20. Comparison of MON 87460 to the Control Priorto Salt Treatment and at 9 and 12 Days after TreatmentUntreated Mean ± S.E.Mild Mean ± S.E.Moderate Mean ± S.E.Severe Mean ± S.E.Evaluation timingCharacteristicMON 87460ControlMON 87460ControlMON 87460ControlMON 87460ControlPrior to treatment1Plant height (cm)54.2± 0.4154.8± 0.41------Growth stage24.03± 0.004.03± 0.00------Chlorophyll450.2*± 0.3548.4± 0.36------Vigor52.1 ±0.042.1± 0.06------Post-treatmentPlant height (cm)107.8± 1.01109.5± 1.2085.7± 0.5787.5± 0.7276.4± 0.6377.3± 0.973.2*± 0.7575.6± 0.66(9 DAT)Growth stage7.2± 0.117.1± 0.076.5± 0.116.5± 0.115.7± 0.135.8± 0.125.5± 0.115.6± 0.11Chlorophyll48.6± 0.6847.4± 1.0652.1± 0.5652.5± 0.7648.4*± 0.7646.1± 0.6732.4± 2.0234.9± 1.29Vigor2.0± 0.072.1± 0.053.7± 0.113.5± 0.145.6± 0.115.7± 0.116.6± 0.116.4± 0.11Post-treatmentPlant height (cm)125.2± 1.98127.4± 1.1394.4± 0.7096.2± 1.0477.1± 0.8277.4± 0.6773.4± 0.7775.9± 0.63(12 DAT)Growth stage8.0± 0.058.0± 0.006.9± 0.077.0± 0.075.8± 0.095.9± 0.075.5± 0.115.7± 0.11Chlorophyll43.5± 0.9942.9± 1.0147.8± 0.7748.3± 0.5642.8± 0.6342.9± 0.5424.1± 1.5525.9± 1.48Vigor2.0± 0.071.9± 0.084.0± 0.073.9± 0.146.2*± 0.096.6± 0.147.9*± 0.077.5± 0.11Fresh weight (g)217.9± 3.22218.8± 3.05108.8± 2.10111.1± 1.6253.0± 1.0153.8± 0.9242.7± 0.7946.1± 0.80Dry weight (g)19.4± 0.2319.8± 0.4011.3*± 0.1811.7± 0.136.8± 0.176.8± 0.145.6± 0.085.9± 0.10S.E. = standard error; n = 20 for MON 87460 and control for each treatment. *Indicates significant differences detected between MON 87460 and the control(p<0.05) provided by t-test comparisons.1Due to the randomization of the substances and treatments within a replication before the treatments were applied, thepre-treatment measurements consist of four sub-samples (n=20).2Number of leaves witha visible leaf collar.3Data could not be analyzed due to lack ofvariability.4Relative chlorophyll content was an averageof readings at the base, midpoint and tip of the third (pre-treatment) or fourth (post-treatment) leafmeasured with a Single Photon Avalanche Diode-containing (SPAD) meter.5Plant vigor was rated using a scale of 1 – 9, where 1 = good and 9 = poor.Monsanto Company 07-CR-191U Page 226 of 544 VIII.F.3. Volunteer Potential Assessment

Volunteer potential can also play a role in determining whether a regulated article has increased weediness potential. The purpose of this study was to assess the volunteer potential of MON 87460 compared to a conven tional control. In some crops, seed remaining in the field after harvest have the potential to over-winter and volunteer in the subsequent cropping season. In the fall of 2006, field trials were established at three

locations to assess vo lunteer potential. Comparative assessments were conducted on

MON 87460 and a conventional control. In ad dition, six commercial corn hybrids were included as references. Normal seed germ ination rates were confirmed for MON 87460, control, and reference starting seeds by standa rd viability testing. The trials were

established at each location as a randomi zed complete block design with three replications. Each plot was 20 ft long by 5 ft wide and was hand-seeded by uniformly scattering approximately 200 seed on the soil su rface. Seed were then incorporated with

a disk or field cultivator. Plots were asse ssed for volunteer plants in the fall of 2006 and spring of 2007. Additional materials and methods are provided in Appendix L.

Agronomic practices used to prepare and mainta in each study site were characteristic of each respective region. No irrigation was applied to the study areas and no plot management was required after the seed were in corporated into the soil. Volunteer plant counts were taken after planti ng in fall 2006 until so il temperatures dropped below 50°F and re-commenced in spring 2007 approximate ly a week prior to the average local planting date for each field site. Voluntee r plant counts were taken approximately every two weeks thereafter until mid-June, for a total of six to seven observations at each site.

No volunteer plants were observed at any site or observation time during the fall or spring (Table VIII-21). The fact that no plants of any study substance emerged and survived as volunteer plants supports a conc lusion that the introdu ction of the drought

tolerance trait did not alter the voluntee r potential of MON 87460 compared to conventional corn. Furthermore, these results demonstrate that the dr ought tolerance trait in MON 87460 confers no biologically meaningf ul change to the invasiveness or potential for corn to persist in the environment.Monsanto Company 07-CR-191U Page 227 of 544 Table VIII-21. Observed Volunteer Corn Plants of MON 87460 Compared to the

Control and References in a 2006/2007 U.S. Field Trial

Site 1 Season MON 87460 Control References 2

BG Fall 3

- - - Spring 0 0 0

RL Fall 0 0 0
Spring 0 0 0

RV Fall 0 0 0
Spring 0 0 0

N = 3 for MON 87460 and control at each site.

1 Site code: BG = Guthrie County, IA; RL = Jefferson County, IA; RV = Parke County, IN.

2 Minimum and maximum values for the reference range could not be calculated because no volunteer

plants were observed.

3 No fall observations were made at BG because so il temperature was <50°F at the first observation timepoint.

VIII.F.4. Survival Outside of Cultivation Assessment

Weediness or invasiveness may al so be indicated if corn exhi bited an increased rate of survival outside of cultivation. The purpose of this study was to assess the ability of MON 87460 to establish and pers ist in areas that are not cu ltivated for agricultural production. Four sites were established in 2007. Each site was unmanaged and received no agricultural inputs allowing MON 87460, the conventional control, and the reference corn hybrids (three per site) to compete with existing vegeta tion and abiotic and biotic stressors present in each environment. Add itional materials and met hods are provided in Appendix M.

Phenotypic and agronomic characteristics enco mpassing plant growth, development, and yield were assessed for MON 87460, the contro l, and the references in unmanaged environments. The experiment was established at each of four sites in a randomized complete block design with three replicatio ns. Early stand count , final stand count, vegetative plant height, plant height at ma turity, number of ears produced per plot, number of ears produced per plant, and seed produced per plot were evaluated (Table VIII-22).

Additionally, replacement values were calc ulated for each environment. Each replacement value is the ratio of the number of seeds produced to the number of seeds sown, represented by a number equal to or gr eater than zero. A replacement value less than one means that fewer seeds were produced than were sown. This is interpreted to mean that the population is not replacing itself and is declining. For corn, this would mean that it is not exhibiting a tr end towards increased weediness.

No differences were observed between MON 87460 and the control for vegetative plant height, plant height at maturity, number of ears produced per plot, number of ears produced per plant, and seed pr oduced per plot at any of the four sites (Table VIII-22).

Two differences were observed between MON 87460 and the control at the MO site.Monsanto Company 07-CR-191U Page 228 of 544 Early stand count was greater for MON 87460 wh en compared to the control (41.3 vs. 24.0 plants per plot, respectivel y) and final stand count wa s greater for MON 87460 when compared to the control (38.3 vs. 21.0 plants per plot, respective ly). It is noteworthy that MO was the only site of the four planted s ites where seeds were produced; however, the replacement values for MON 87460 and the cont rol were much less than one (0.15 and 0.03, respectively). This means that the populations for MON 87460 and the control are declining and that MON 87460 is not exhib iting increased weediness. Thus, MON 87460 is not likely to persist in any of the four unmanaged envir onments and did not demonstrate a competitive advantage in this study compared to conventional corn. As such, the drought tolerance trait in MON 87460 confers no biologically meaningful change to the fitness, invasiveness, or poten tial for corn to persist outside of managed agricultural environments.Monsanto Company 07-CR-191U Page 229 of 544Table VIII-22. Survival Outside of Cultivation Phenotypic Comparison of MON 87460 tothe Control in 2007 U.S. Field TrialsEarly Stand Count(plants/plot)Mean±S.E.Late Vegetative Plant Height(inches)Mean±S.E.Final Stand Count(plants/plot)Mean±S.E.Site1MON87460Control Reference MON87460Control Reference MON87460Control ReferenceIL 53.0 ± 2.08 57.3 ± 5.24 30.7-48.3 8.1 ± 1.53 8.8 ± 0.87 7.6-10.4 16.7 ± 1.45 22.0 ± 1.15 9.3-19.3MO 41.3* ± 1.76 24.0 ± 3.06 21.3-30.3 22.0 ± 1.60 23.6 ± 2.41 22.9-31.3 38.3* ± 1.67 21.0 ± 2.31 19.3-28.0NE 2.3 ± 2.33 0.3 ± 0.33 0.0-1.0 17.8 16.0 19.5-28.0 1.0 ± 1.00 0.0 ± 0.00 0.0-0.3TX - - - - - - - - -Plant Height at Maturity(inches)Mean±S.E.# Ears Produced(ears/plot)Mean±S.E.# Seed Produced(seed/plot)Mean±S.E.Site MON87460Control Reference MON87460Control Reference MON87460Control ReferenceIL 6.9 ± 1.00 8.1 ± 1.11 6.9-8.9 - - - - - -MO 27.9 ± 1.33 29.8 ± 3.31 29.1-38.9 8.7 ± 3.18 3.7 ± 1.45 3.0-10.7 7.7 ± 1.86 1.3 ± 0.88 2.3-28.3NE 20.7 - 30.0-30.0 - - - - - -TX - - - - - - - - -Average # Ears(ears/plant)Mean±S.E.Replacement Value2Site MON87460Control Reference MON87460Control ReferenceIL - - - 0.0 0.0 0.0-0.0MO 0.2 ± 0.08 0.2 ± 0.05 0.2-0.4 0.15 0.03 0.05-0.57NE - - - 0.0 0.0 0.0-0.0TX - - - 0.0 0.0 0.0-0.0S.E. = standard error; n = 3 for MON 87460 and controlat each site. *Indicates significantdifferences detected between MON 87460 and the control (p<0.05).Dash (-) indicates no data available. Mean values without S.E. values are from asample size of one and S.E. cannot be calculated.1Site codes: IL = Effingham County, IL; MO = Shelby County, MO; NE = York County, NE; TX = Carson County, TX2Due to seed counting errors, the exact numberof seed sown in each plot varied. Therefore, replacement values at the MO site were conservatively based on 50seeds sown per plot using the formula, Replacement Value = [Number of Seed Produced] /50. Replacement values were not statistically analyzed.Replacement values < 1.0 indicate the seed population is declining.Monsanto Company 07-CR-191U Page 230 of 544 VIII.G. Overall Conclusions of Phenotypic, Agronomic, and Environmental

Interactions Assessment of MON 87460

An extensive and robust set of information and data were used to assess whether the introduction of the drought tolerance trait and the expression of the CSPB and NPTII proteins altered the plant pe st potential of MON 87460 compar ed to a control, which had a genetic background similar to MON 87460 but did not possess the drought tolerance trait. The assessment was based on thorough phenotypic, agronomic, and environmental

interactions characterizati on, and comparison of MON 87460 to control and conventional reference corn hybrids. Data were coll ected for seed dormancy and germination parameters, phenotypic and agronomic char acteristics during plant growth and development, pollen characteristics, observations for plant-insect, plant disease and plantabiotic stressor interactions, abiotic stress to lerance, volunteer pote ntial, and survival

outside cultivation.

Results from the phenotypic and agronomic assessments indicate that MON 87460 does not possess characteristics that would confer a plant pest ri sk compared to conventional

corn. Data indicate that MON 87460 does not confer any increased susceptibility or tolerance to specific insect, di sease, or abiotic stressors, with the exception of drought. MON 87460 is expected to provide reduced yield loss under water-limited conditions compared to conventional corn. This yi eld response is a desirable agronomic

characteristic and is not considered to be associated with plant pest potential. Taken together, these data support a conclusion that MON 87460 poses no increased plant pest potential, including weediness potential, an d no adverse environmental impact compared

to conventional corn.

VIII.H. References

Alexander, M.P. 1980. A versatile stain for pollen fungi, yeast and bacteria. Stain Technology. 55:13-18.

Anderson, W.P. 1996. Weed Science Princi ples and Applicati ons, 3rd ed. West Publishing Company, St. Paul, MN. pp 27-37.

AOSA. 2006. Association of Official Seed Anal ysts. Rules for testing seeds. AOSA, Lincoln, NE.

Barker, T., H. Campos, M. Cooper, D. Dolan, G. Edmeades, J. Habben, J. Schlusser, D. Wright, and C. Zinselmeier. 2005. Improvi ng Drought Tolerance in Maize. Pages 173-253 in Plant Breeding Reviews. Vol 25, J. Janick, (ed.) John Wiley and Sons, Inc., Hoboken, New Jersey.

Black, R.D. and D.H. Rogers. 1989. Soil water measurements: An aid to irrigation water management. Cooperative Extension Service, Kansas State University. Manhattan, KS.

Boyer, J. S. and M. E. West gate. 2004. Grain yields with limited water. Journal of Experimental Botany. 55:2385-2394.Monsanto Company 07-CR-191U Page 231 of 544 Campos, H., M. Cooper, G.O. Edmeades, C. Loffler, J.R. Schussl er, and M. Ibanez. 2006. Changes in drought tolerance in ma ize associated with fifty years of breeding for yield in the U.S. Corn Belt. Maydica. 51:369-381.

Claassen, M. M. and R. H. Shaw. 1970. Wa ter deficit effects on corn. II. Grain components. Agronomy Journal. 62: 652-655.

Lingenfelter, D.D. and N.L. Hartwig. 2003. Introduction to Weeds and Herbicides. Agricultural Research and Cooperative Extension CAT UC175. The Pennsylvania State Universi ty, University Park, PA.

OECD. 1993. Safety considerations for biotechnology: Scale-up of crop plants. Organization for Economic Co-operati on and Development, Paris, France http://www.biosafety.be/CU/BSL_ Ressources/PDF/M00034525.pdf Accessed January 9, 2009.

Radosevich, S., J. Holt, and C. Gher sa. 1997. Weed Ecology: Implications for Management. Second ed. Pages 3-6. John Wiley & Sons, Inc., New York, NY.

Yuan, S. and Lin, H.H. 2008. Role of salicylic acid in plant abiotic stress. Zeitschrift Fur Naturforschung Section C-a Jour nal of Bioscience. 63:313-320.Monsanto Company 07-CR-191U Page 232 of 544 IX. Environmental Consequences a nd Impact on Agronomic Practices

This section provides a brief review and assessment of the plant pest potential of MON 87460 and its impact on agronomic pr actices. USDA-AHPIS has responsibility, under the Plant Protection Act (PPA) (7 U. S.C. § 7701-7772), to prevent the introduction and dissemination of plant pests into the U. S. APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to ev aluate submitted data to determine that a particular regulated article does not present a plant pe st risk and should no longer be regulated. If APHIS determines that the regu lated article does not present a plant pest risk, the petition is granted, thereby allowi ng unrestricted introduc tion of the article.

The definition of plant pest in the PPA is broad and include s living organisms that could directly or indirectly injure, damage, or cau se disease in any plant or plant product [7 U.S.C. § 7702(14)]. This section summari zes the plant pest risk for MON 87460 based on the information presented in the previous se ctions of the petition. Information related to plant pest risk characteristics include di sease and pest susceptibilities, expression of the gene product (CSPB, NPTII), changes to plant metabolism, weediness of the regulated article, any impacts on the weedines s of any other plant with which it can interbreed, and the transfer of genetic information to or ganisms with which it cannot interbreed. Issues related to agricultural or cultivation practices are considered in the Appendix O of this petition.

The regulatory end-point for “GE plants” is not zero risk but rather a determination that deregulation of the regulated arti cle is not likely to pose a plant pest risk. As part of its plant pest risk assessment, the genetic cons truct inserted into MON 87460 was evaluated to determine if those sequences cause plant disease. Morphologica l characteristics of MON 87460 were analyzed to determine if it will become weedy or invasive. The potential for gene flow and introgression of th e genetic construct into other plant varieties or wild relatives are also evaluated to determine the potential of increased weedy or

invasive characteristics in other plant species. Finall y, the propensity of MON 87460 to become a greater reservoir of plant pests (ins ects or pathogens) compared to conventional plants and the potential for horizontal gene transfer was evaluated. Using this risk assessment process, the data and analysis pres ented in this petition leads to a conclusion that MON 87460 is unlikely to be a plant pest and therefore should no longer be subject to regulation under 7 CFR § 340.

The plant pest risk assessment of MON 87460 was based primarily on eight lines of evidence: (1) modern corn has inherently low plant pest potential, (2 ) insertion of a single functional copy of the inserted cspB and nptII expression cassettes, (3) characterization of the CSPB and NPTII proteins expressed in MON 87460, (4) lack of allergenicity and toxicity of the CSPB and NPTII proteins, (5) compositional and nut ritional equivalence of forage and grain as compared to conve ntional corn, (6) phenot ypic and agronomic characteristics demonstrating no increased plant pest potential, (7) negligible risk to NTOs and threatened or endangered species , and (8) no greater likelihood to impact agronomic practices, including land use, cu ltivation practices, or the management of weeds, diseases, and insects than conv entionally bred drought tolerant corn.Monsanto Company 07-CR-191U Page 233 of 544 APHIS has recently proposed to amend 7 CFR Part 340 to incl ude its noxious weed authority. Because the data show that MON 87460 has no potential to cause injury, damage or disease to any protected intere st, MON 87460 would also not be considered a “noxious weed” as defined by the Plant Protection Act.

IX.A. Characterization of MON 87460 and the expressed CSPB and NPTII Proteins

IX.A.1. Mode of Action

MON 87460 reduces yield loss under water-lim ited conditions compared to conventional corn. Under well-watered conditions, grai n yield for MON 87460 is not different from

conventional corn. Like conventional cor n, MON 87460 is still subject to yield loss under water-limited conditions, particularly dur ing flowering and grainfill periods when corn yield potential is most sensitive to stress by disr upting kernel development.

Efficacy in MON 87460 is derive d by expression of the inserted Bacillus subtilis cold shock protein B (CSPB). A detailed descript ion of the mode of act ion for CSPB and its drought tolerance trait is provide d in Section I.D. CSPB is an extensively studied, stressinducible protein known to faci litate adaptatio n to environmental stresses in bacteria. CSPB acts by interacting with RNA and unfolding secondary structures in RNA molecules, which is known to have an eff ect on RNA stability and on the ability of the cell to translate those RNA molecules, thus helping to preserve nor mal cellular functions (Schindler et al., 1999; Graumann et al., 1997). The CSPB protein in MON 87460 binds RNA and helps to maintain plant cellular f unctions in rapidly gr owing and reproductive organs under water-limited conditions. Our data suggest that CSPB in MON 87460 works by minimizing the effect of wate r limitation on photosynthesis, stomatal conductance, and carbon fixation, and ultimat ely improves corn grain yield, primarily through increased kernel number per ear. The nptII gene was inserted to facilitate selection of plants containing cspB during early product deve lopment and does not pose any safety concerns (EFSA, 2007; FDA 1994 and 1998; Flavell et al., 1992; Fuchs et al., 1993a and 1993b; and Nap et al., 1992).

Knowing that stress response proteins al low organisms to survive in adverse environments, it was hypothesized that inse rting a stress response protein into plants could impart a desirable phenotype. Usi ng a high through-put screening biotechnology approach, Castiglioni et al. ( 2008) demonstrated that bacteria l cold shock proteins (CSPs) can confer improved stress adaptation to mu ltiple plant species. Twenty-two events expressing the cold shock prot ein B (CSPB) were evaluated in water-limited field trials using commercial grade corn in environments th at received no rainfall during the 10 to 14 days immediately prior to flowering. The water-limited treatment resulted in an average reduction in growth rates of 50% of the we ll-watered rate. Analyzing data from all events, the CSPB containing events demonstrat ed a 3.6% - 24% increase in leaf extension rates relative to non-transgenic controls, improvements in chlorophyll content (2.5% - 4.4%), and photosynthetic rates (3.6% - 8.5 %). These measures of vegetative performance indicated that the CSPB prot ein has a positive impact on overall plant productivity and, therefore, yield potential. When plants were grown under well-watered conditions in both the greenhouse and field, no appreciable difference between CSPB-Monsanto Company 07-CR-191U Page 234 of 544 expressing lines and the control were de tected. MON 87460 was chosen for further development based on its yield performance under water-limited conditions compared to the control and its favorable agronomic performance.

As with bacterial and other plant cold s hock domain (CSD) cont aining proteins, the CSPB protein from B. subtilis , expressed in MON 87460 accumulates in actively growing

tissues where it binds to R NA and facilitates the unfolding of RNA secondary structures that are formed due to stress leading to mo re normal translation under stress conditions (Schindler et al., 1999; Graumann et al., 1997). Data from in vitro and in vivo experiments indicate that CSPB preferentially binds plant RNA. CSPB was also effective in unfolding secondary RNA structures in vitro, while variants of the CSPB protein with impaired RNA binding functions were unable to bind and unfold RNA. In

vitro co-immunoprecipitation experiments with CSPB, CSPB with an impaired binding

function and RNA from MON 87460 further confirm that CSPB interacts with RNA while the variant lacking a functional RNA binding site will not interact with RNA in

vitro

.

Water-limited conditions during the growing se ason can diminish corn productivity and yield, particularly dur ing flowering and grainfill period s when corn yiel d potential is most sensitive to stress by di srupting fertilizatio n and kernel development (Claassen and Shaw, 1970; Boyer and Westgate, 2004; Ca mpos et al., 2006). In field trials, improvements in MON 87460 yield and yield components under water-limited conditions were demonstrated in multiple years. Results from these studies demonstrate that the major component contributing to the impr oved yield of MON 87460 under water-limited

conditions is the increased number of kernels per ear, which is consistent with our current understanding of the effect of drought stress on corn yield potential (Westgate et al., 2004; Campos et al., 2006; Welcker et al., 2007).

IX.A.2. CSPB and NPTII Protein Safety

The safety assessment of the CSPB protein included extensive protein characterization demonstrating the lack of similarity to known allergens and toxins and the long history of safe consumption of similar proteins (Sectio n VI). CSPB protein does not share any amino acid sequence similarities with known alle rgens, gliadins, glutenins, or protein

toxins which have adverse effects on mammals. This has been shown by extensive assessments with bioinformatics tools, such as FASTA sequence alignment search and an eight-amino acid sliding window search (Section VI.D). Digestive fate experiments conducted with the CSPB protein demonstrated that the full-length protein is rapidly digested in simulated gastric fluid (SGF), a characteristic shared among many proteins with a history of safe consumption. A sma ll transiently stable CSPB protein fragment was very quickly degraded during short exposu re to simulated intestinal fluid (SIF). Rapid digestion of the full-le ngth CSPB protein in SGF a nd SIF, together with rapid degradation of the small transiently stable fr agment in SIF, indicates that it is highly unlikely that the CSPB protein and its frag ment will reach absorptive cells of the intestinal mucosa (Section VI.D). Proteins that are rapidly digestible in mammalian gastrointestinal systems are unlikely to be allergens when consumed. Finally, the CSPB protein represents no more than 0.00007% of the total protein in the grain of MON 87460Monsanto Company 07-CR-191U Page 235 of 544 (Section VI.D). Acute oral toxicity studies with mice demonstrated that CSPB protein is not acutely toxic and does not cause any adve rse effects even at the highest dose levels tested, which was 4.7 mg/kg body weight. The dietary safety assessment of CSPB based on the acute toxicity data and corn product dietary pattern esta blishes that the margin of exposure (MOE) for the overall U.S. population is 26,700. For children aged 1-6 years old, an age group with the highest corn consumption on a body weight basis, the MOE was greater than or equal to 11,400 for CSPB. Dietary exposu re in animals will also be low with chickens, swine, a nd dairy cows consuming only nanogram quantities of each protein per kilogram of body we ight (Section VI.D). Collec tively, these data establish the dietary safety of the CSPB protein. Because NTOs, including humans, will primarily come in contact with CSPB through dietary e xposure it can be concluded with reasonable certainty that the CSPB protein has no mean ingful toxic potential to exposed organisms

in the environment.

The dietary safety of NPTII has been exte nsively evaluated thr ough several lines of experimental evidence, and several products containing NPTII have been approved by regulatory agencies on a global basis. NPTI I is the most commonly used antibiotic resistance marker in several commercially grown biotechnology-derived crops including

YieldGard ® Rootworm corn (MON 863), Bollgard ® cotton (MON 531), Bollgard ® II cotton (MON 15985), and Roundup Ready cotton (MON 1445). The safety of NPTII has been addressed in multiple publications (Flave ll et al., 1992; Nap et al., 1992; Fuchs et al., 1993a and 1993b; EFSA, 2009). FDA eval uated NPTII as part of a petition for

FLAVR SAVR ® tomatoes and approved its use as a food additive. Additionally, EPA established an exemption from the requireme nt of a tolerance for NPTII for use as a selectable marker in raw agricultural co mmodities (40 CFR Part 180.1134). In 2007, the European Food Safety Authority (EFSA) affi rmed its conclusion th at the presence of nptII does not pose a threat to human health or the environment. Moreover, the USDA previously evaluated the safety of NPT II in other biotechnology-derived products, including corn. Similar to th ese products, there is negligible risk for the production of

NPTII in MON 87460 to result in a plant pest risk. A published assessment of the ecological impact of NPTII in crops repo rted that the amount of free kanamycin accumulating in soils, through the action of micr oorganisms or animal feces, is restricted by absorption to soil component s so that no direct select ion pressure for kanamycin resistant plants can occur (Nap et al., 1992). Also, enhanced physiological fitness resulting from potential pleiotropic effects of nptII gene expression is not likely to occur (Nap et al., 1992; EFSA, 2009). Thus, based on all the available ev idence, it can be concluded that the NPTII protei n is safe for use as a select able marker in biotechnologyderived plants and it has no meaningful toxi c potential to exposed organisms in the

environment.

IX.A.3. Composition and Nutrition of Forage and Grain

Compositional equivalence between corn impr oved through biotechnology-derived traits and conventional hybrids provides an “equal or in creased assurance of the safety of foods derived from genetically modified plants ” (OECD, 1998). Compositional analyses of forage and grain tissues from MON 87460 were conducted to assess the levels of key nutrients, anti-nutrient s, and key secondary metabolites for comparison to conventionalMonsanto Company 07-CR-191U Page 236 of 544 corn. These results, based on evaluation of 77 different components (9 in forage and 68 in grain) confirmed that th e corn grain and forage de rived from MON 87460, and the intended foods and feeds derived from MON 87460, can be considered compositionally and nutritionally equivalent to conventional corn hybrids that have a history of safe consumption that are currently in commerce (Section VII).

IX.A.4. Phenotypic and Agronomic Characteristics

Phenotypic and agronomic char acteristics of MON 87460 were evaluated in field studies conducted during 2006 and 2007 in the major co rn production regions of the U.S. and Chile (Section VIII). Because MON 87460 reduces yield loss under water-limited conditions, field studies were designed to ev aluate the relevant characteristics of MON 87460 across a broad range of soil moisture and environmental conditions relevant to where commercial production would be exp ected. Six field studies totaling 31 sites were established using three water management regimes: (1) well-watered (17 sites), (2) well-watered and water-limited treatments (9 sites), and (3) water managed according to typical local agronomic prac tices and water conditions (5 sites). Phenotypic and agronomic characteristics and environmenta l interactions were assessed under these water management regimes. Characteristics evaluated include seed ling vigor, early stand count, days to 50% pollen shed, days to 50% silking, stay green, ear height, plant height, dropped ears, stalk lodging, root lodging, final stand count, gr ain moisture, test weight and yield. For each characteristic, MON 87460 was compared to the control and means were calculated across sites (referred to as combined-site analysis, in which data were pooled from all sites and analyzed sta tistically).

Results from the combined-site analyses within each of the six studies detected only four

instances of a difference between MON 87460 and the control. Fo r the well-watered regime, two separate studies totaling 17 sites were established in the U.S. during 2006 (8 sites) and 2007 (9 sites). In the combined-site analyses of these data, no differences were

detected between MON 87460 and the control in the 2007 study. In the 2006 study, an increase in root lodged plants was detected for MON 87460 compared to the control (5.6 vs. 1.5, respectively). For th e well-watered and water-limite d regime, three different studies totaling nine field sites (six site s included in combined-site analyses) were established in Chile (1 study, 3 sites in 2006/ 2007) and the U.S. (2 studies, 3 sites in 2007). In the Chile 2006/2007 study, no differen ces were detected with the exception of reduced yield loss for MON 87460 compared to the control under water-limited conditions (114.5 vs. 86.7 bushels/acre, respec tively). In the U.S. 2007 Study-1, no

differences were detected in either the well-watered or water-limited treatments. In the U.S. 2007 Study-2, stay green rating was lo wer (more green tissue) for MON 87460 compared to the control in both the well -watered (5.8 vs. 6.7) and water-limited treatments (6.3 vs. 8.3), respectively. For th e typical agronomic practices regime, one study with five sites was established in th e U.S. during 2006, and no differences between

MON 87460 and the control were detected in th is study. In summary of the phenotypic data, the results support the conclusion that MON 87460 possesses no characteristics that would indicate an increased plant pest ri sk compared to conventional corn. The magnitude of the detected differences was sma ll, the differences did not represent a trend in the data across studies and years, and th e mean values of MON 87460 were within theMonsanto Company 07-CR-191U Page 237 of 544 ranges of values observed for the commercial references. In no instance did the field studies suggest that MON 87460 would cause damage, injury, or disease to plants.

Efficacy of MON 87460 can be demonstrated by directly comparing its grain yield to anon-drought tolerant, conventional control under water-limited conditions. The wellwatered and water-limited field trials descri bed above are relevant to demonstrate the efficacy of MON 87460. A comparison of th e average percent reduced yield loss of MON 87460 compared to the control under both field trial designs demonstrates the efficacy of MON 87460 under water-limited cond itions (Section I.B.2). As expected, MON 87460 provided reduced yield loss compar ed to the control under water-limited conditions and equivalent yield to the cont rol under well-watered conditions. Reduced yield loss values for MON 87460 in the water-li mited treatment were highly variable, but are representative of the natu ral variation expected for co rn grown under sub-optimal soil

moisture conditions.

IX.A.5. Potential for Risks to Non-target Organisms

Evaluation of the poten tial risks to non-target organism s (NTOs) may be considered a component of a plant pest risk assessment, or could be considered separately as part of a

NEPA environmental assessment. The na ture of MON 87460 as a product with no pesticidal activity can lead to a conclusion that a ll exposed organisms are considered to be NTOs. During the U.S. and Chile phenotypi c field studies at 31 locations in 2006 and 2007, each field site was rated at four time in tervals during the season for specific insects (pest and non-pests), and diseases (Section VI II.F.1). The purpose of these observations was to assess whether the plant-disease or plant-insect interac tions of MON 87460 were altered compared to commercial corn. Twenty-one pest a nd non-pest arthropod categories (species or group) and 19 disease categories were evaluated.

Of the more than 388 pest and non-pest arthr opod evaluations, only two differences were

observed between MON 87460 and the control. Grasshopper damage was lower for MON 87460 compared to the control (none vs. slight) and European corn borer damage was higher for MON 87460 compared to the control (moderate vs. slight) (Section VIII.F.1). The differences detected were either within the range of the references, or were isolated to a single st udy-site location. The fe w differences detected were small in magnitude, did not represent a tr end in the data, and are not considered to be biologically meaningful in terms of incr eased plant pest potential. Out of the more than 425 disease stressor observations, no di fferences were detected between MON 87460 and the control. These results support the conclusion that MON 87460 does not

have altered environmental interactions relativ e to other corn and expression of the CSPB protein show no apparent direct impact on arthropods or diseases of corn.

In addition, compositional analyses of forage and grain (Section VII) confirm that the levels of key nutrients, anti-nutrients, and secondary metabolites in MON 87460 are comparable to those in conve ntional corn and that the fo rage and grain derived from MON 87460 are compositionally equivalent to conventional corn. In total, these studies demonstrate no biologically meaningful inte ractions with the species exposed to

MON 87460.Monsanto Company 07-CR-191U Page 238 of 544 IX.B. Weediness Potential

In the U.S., corn is not listed as a weed in the major weed refere nces (Crockett, 1977; Holm et al., 1979; Muenscher, 1980), nor is it present on the lists of noxious weed species distributed by the federal government (7 CFR Part 360). Modern corn cannot survive as a weed due to intense selection for domestication purposes during the evolution of corn. During domestication of co rn, traits often associ ated with weediness such as seed dormancy, a dispersal mechan ism, or the ability to form reproducing populations outside of cultivation, have not been selected. For example, the corn ear is enclosed with husks. Consequently, seed disp ersal of individual kernels is limited. Even

if individual kernels of corn were distribute d within a field or along transportation routes from the fields to storage or processing f acilities, sustainable volunteer corn populations are not found growing in fence rows, ditches, and road sides. Corn is poorly suited to

survive without human assistan ce and is not capable of surv iving as a weed (Baker, 1965; Keeler, 1989; Galinat, 1988). Although corn seed can ove rwinter and emerge as volunteer plants in rotationa l crops, the populations do not persist, and agronomic management practices, including mechanical and chemical measures, can be used to control the volunteer plants.

In comparative studies conducted betw een MON 87460 and a non-drought-tolerant, conventional control, dormancy and germ ination, growth and development, and reproductive characteristics were evaluated fo r changes that would impact plant pest potential, and in particular, plant weediness potential. No meaningful differences from conventional corn were observed (Section VIII). The introduction of the drought tolerance trait did not unexpectedly alter th e assessed characteristics compared to the control. Thus, the results support a conclu sion of no increased w eediness potential of MON 87460 compared to conventional corn. Furthermore, extensive post-harvest monitoring of field trial plots planted with MON 87460 under USDA-APHIS notifications did not reveal any differences in survivability or persistence relative to other corn. A complete list of USDA notifica tions approved for MON 87460 is presented in Appendix N

. These data suggest that MON 87460 is no more likely to become a weed

than conventional corn.

IX.C. Gene Flow

Gene flow (often used synonymously with th e term “outcrossing” or “cross pollination”) is a natural biological process that occurs in most crop species, including corn. Pollenmediated gene flow is a term used to desc ribe the movement of plant genes from one plant to another via pollen. The rate of polle n-mediated gene flow depends on biotic and abiotic factors such as plant biology, po llen biology and volume, plant phenotype, overlap of flowering times, proximity of the pollen source and sink, ambient conditions such as temperature and humidity, and field architecture.Monsanto Company 07-CR-191U Page 239 of 544 IX.C.1. Vertical Gene Flow – Transfer of Genetic Information among Species with

which Corn Can Interbreed

Corn morphology fosters cross-pollination; ther efore, high levels of pollen-mediated gene flow can occur in this species. Researcher s also recognize that: (1) the amount of gene flow that occurs can be high because of ope n-pollination, (2) the percent gene flow will vary by population, hybrid or i nbred, (3) the level of gene flow decreases with greater distance between the source and recipient plan ts; (4) environmental factors affect the level of gene flow, (5) corn pollen is viable for a shor t period of time under field conditions, (6) corn produces ample pollen ove r an extended period of time, and (7) there are no purposeful insect pollinators of corn (pollinating insects, especially bees, are

occasional visitors to the tassels but rarely visit silks of corn).

For gene flow to occur by normal sexual tran smission, certain condit ions must exist: (1) the two parents must be sexually compatible ; (2) there must be overlapping phenology; and (3) a suitable factor (such as wind or insects) must be present and capable of transferring pollen betw een the two parents.

Several studies have been conduc ted on the extent of pollen-medi ated gene flow in corn. As expected, results were found to va ry depending on the experimental design,

environmental conditions and detection method. In general, percent gene flow was found to diminish with increasing distance from th e source field. It wa s previously reported that corn cross-pollination rate fell below 1% at distances >200 m (Jemison and Vayda, 2000; Luna et al., 2001). A more recent study showed that corn cross pollination rate decreased below 0.9% beyond 15 m (Bannert, 2006).

As discussed in Sections II.C and II.D.5, corn and annual teosinte are genetically compatible, wind-pollinated and may hybridize when in close proximity to each other e.g., in areas of Mexico and Guatemala. Corn easily crosses with teosinte; however, teosinte is not present in the U.S. other than as an occasional botanical garden specimen or small feral populations of Zea mexicana in Florida, Alabam a and Maryland and Zea perennis in South Carolina (http://plants.usda.go v). These specimens would only flower at the same time as corn if they were subjec t to artificial day length shortening for several weeks at a time (Wilkes, 1967). Differences in factors such as flowering time, geographical separation, and de velopment factors make natura l crosses in the U.S. highly unlikely. Additionally these states are not part of the targeted geographic area in which MON 87460 is expected to have the greatest benefit, the U. S. Great Plains where corn production is subject to fr equent drought stress.

In contrast with corn and teosinte, which easily hybridize under certain conditions, it is only with extreme difficulty and special techni ques that corn and another closely related species, Tripsacum (gamma grass) hybridize. Furthe rmore, the offspring of the cross show varying levels of sterility (Galinat , 1988; Mangelsdorf, 1974; Russell and Hallauer, 1980) (Section II.D.5). A single species, Tripsacum floridanum , found in the extreme southern Florida counties of Miami-Dade, Collier and Monroe has been categorized as a threatened species by the state of Florida and listed on the USDA’s Natural Resources Conservation Service Database (http://p lants.usda.gov/java/profile?symbol=TRFL4).Monsanto Company 07-CR-191U Page 240 of 544 However, given the level of difficulty fo r natural hybridization between species of Tripsacum and Zea , and the occurrence of T. floridanum primarily in both highly urbanized and non-agricultural, swampy areas of th e state, it is very unlikely there would be any impact on this species due to the intr oduction of MON 87460.

IX.C.2. Horizontal Gene Flow – Transfer of Genetic Information to Species with

which Corn Cannot Interbreed

Monsanto is aware of no reports of the transfer of genetic material from corn to other species with which corn cannot sexually inte rbreed. Southern blots examining multiple generations of MON 87460 demonstrate that the cspB and nptII expression cassettes are stably inherited with patterns typical of Mendelian genetics (Sectio n V.D.1). It is therefore not expected that these genes woul d have any altered poten tial to transfer to other organisms. The probability for horizont al gene flow to occur is judged to be exceedingly small. Even if it were to occur, the consequences would be negligible since the genes introduced into MON 87460 are of bacterial origin and the two proteins produced have no meaningful toxicity to hum ans and other NTOs under the conditions of use. In addition, the nptII gene is not expected to pose a ny additional risk of antibiotic

resistance when MON 87460 is cultivated. A study of corn expressing an antibiotic resistance marker gene demonstrated that the presence of such crops does not affect the frequency of antibiotic re sistance in soil bacteria (Demanèche et al., 2008).

IX.D. Corn Production, Current Agronomic Practices, and Land Use

This section provides a review of corn grai n and seed production, agronomic and land use practices for corn, and any anticipated environmental consequences from the

commercialization of MON 87460.

Areas such as the Midwestern U.S. Corn Be lt have sufficient precipitation to routinely support high levels of corn production. As shown in Sections VII and VIII, no phenotypic, compositional, or environm ental differences between MON 87460 and

conventional corn have been observed under well- watered conditions. Therefore, it is not anticipated that adoption of MON 87460 in Midw estern regions of the U.S. will have any significant impact on cultural practices, including tillage and pest management. In the Great Plains, there is often insufficient pr ecipitation for corn production resulting in

water-limited conditions. Under these conditio ns, some potential impacts to irrigation practices and dryland acreage are foreseeable, and are discussed in more detail below.

However the introduction of MON 87460 is no mo re likely to impact irrigation practices and dryland acreage in the Grea t Plains than the use of conventionally bred drought

tolerant corn.

IX.D.1. Overview of U.S. Corn Production

IX.D.1.1. Grain Production

Corn is the largest U.S. crop in terms of acreag e planted and net crop value. In the past 10 years (1997-2007), total annual corn acreage planted varied from approximately 76 to 93 million acres (http://www.nass.usda.gov). Total annual production ranged from 9 toMonsanto Company 07-CR-191U Page 241 of 544 13.1 billion bushels, and total annual value fluctuated from 17 to 52 billion dollars depending on production output and co mmodity prices (Table IX-1).

Corn is planted in almost every state in the continental U.S. The two largest corn producing regions are the Midwest, comprisi ng eight states (Illinoi s, Indiana, Iowa, Michigan, Minnesota, Missouri, Ohio and Wi sconsin) contributing 65% to the national corn production total, and the Great Plains , as defined by Riebsame (1990), including portions of ten states (Col orado, Kansas, Montana, Ne braska, New Mexico, North Dakota, Oklahoma, South Dakota, Texas and Wyoming) contributing 26% to the national corn production total (Table IX-2).

Yields vary considerably from region to regi on because of the dive rsity in rainfall and irrigation, climatic conditions, and soil produc tivity. A comparison of the Midwest and the Great Plains (Table IX-3) illustrates that while average bushel/acre yields are not very dissimilar, the acreage routinely devoted to corn production in the Great Plains is approximately 44% of that utili zed in the Midwest. This is due to a number of factors including significantly lower rainfall, with the Great Plains having approximately a 20 inch (50 cm) annual average while the Midw est has a 34 inch (86 cm) annual average (http://www.met.utah.edu/jhorel/h tml/wx/climate/normrain.html).Monsanto Company 07-CR-191U Page 242 of 544 Table IX-1. Field Corn Production in the U.S. (1996 – 2008)

Year Acres planted Acres

harvested Yield Production Price Value

(x 1000) (x 1000) (bu/acre) (x 1000 bu) ($/bu) ($ billion)

1996 79,229 72,644 127.1 9,232,557 2.71 25.15

1997 79,537 72,671 126.7 9,206,832 2.43 22.35

1998 80,165 72,589 134.4 9,758,685 1.94 18.92

1999 77,386 70,487 133.8 9,430,612 1.82 17.10

2000 79,551 72,440 136.9 9,915,051 1.85 18.50

2001 75,702 68,768 138.2 9,502,580 1.97 18.88

2002 78,894 69,330 129.3 8,966,787 2.32 20.88

2003 78,603 70,944 142.2 10,087,292 2.42 24.48

2004 80,929 73,631 160.3 11,805,581 2.06 24.38

2005 81,779 75,117 147.9 11,112,187 2.00 22.20

2006 78,327 70,638 149.1 10,531,123 3.04 32.09

2007 93,527 86,520 150.7 13,037,875 4.00 52.09

2008 85,982 78,640 153.9 12,101,238 NA* NA*

Source: http://www.nass.usda.gov *Data currently not availableMonsanto Company 07-CR-191U Page 243 of 544 Table IX-2. Field Corn Production by Regions and States in the U.S. (2008)

State Acres planted Acres

harvested Yield Production % to national

total
(x 1000) (x1000) (bu/acre) (x1000 bu) production

Midwest Region

Illinois 12,100 11,900 179 2,130,100

Indiana 5,700 5,460 160 873,600

Iowa 13,300 12,800 171 2,188,800

Michigan 2,400 2,140 138 295,320

Minnesota 7,700 7,200 164 1,180,800

Missouri 2,800 2,650 144 381,600

Ohio 3,300 3,120 135 421,200

Wisconsin 3,800 2,880 137 394,560

Regional total 51,100 48,150 154(avg) 7,865,980 65.00

Northeast Region

New York 1,090 640 144 92,160

Pennsylvania 1,350 880 133 117,040

Connecticut 27

Maine 29

Massachusetts 19

New Hampshire 15

Rhode Island 2

Vermont 94

Regional total 2,626 1,520 139(avg) 209,200 1.70

Mid-Atlantic Region

Delaware 160 152 125 19,000

Maryland 460 400 121 48,400

New Jersey 85 74 116 8,584

Virginia 470 340 108 36,720

West Virginia 43 26 130 3,380

Regional total 1,218 992 120(avg) 116,084 0.96

Southeast Region

Alabama 260 235 104 24,440

Arkansas 440 430 155 66,650Monsanto Company 07-CR-191U Page 244 of 544 Table IX-2 (cont.). Field Corn Production by Regions and States in the U.S. (2008)

State Acres planted Acres

harvested Yield Production % to national

total
(x 1000) (x1000) (bu/acre) (x1000 bu) production

Florida 70 35 105 3,675

Georgia 370 310 140 43,400

Louisiana 520 510 144 73,440

Kentucky 1,210 1,120 136 152,320

Mississippi 720 700 140 98,000

North Carolina 900 830 78 64,740

South Carolina 355 315 65 20,475

Tennessee 690 630 118 74,340

Regional total 5,524 5,115 119 (avg) 621,480 5.15

Great Plains Region

Colorado 1,250 1,080 137 147,960

Kansas 3,850 3,630 134 486,420

Montana 78 35 136 4,760

Nebraska 8,800 8,550 163 1,393,650

New Mexico 140 55 180 9,900

North Dakota 2,550 2,300 124 285,200

Oklahoma 370 320 115 36,800

South Dakota 4,750 4,400 133 585,200

Texas 2,300 2,030 125 253,750
Wyoming 95 52 134 6,968

Regional total 24,183 22,452 138 (avg) 3,210,608 26.53

Northwest Region

Washington 165 90 205 18,450

Oregon 60 33 200 6,600

Idaho 300 80 170 13,600

Regional total 525 203 192 (avg) 38,650 0.33

Southwest Region

Arizona 50 15 165 2,475

California 670 170 195 33,150

Nevada 5

Utah 70 23 157 3,611

Regional total 795 208 172 (avg) 39,236 0.33

United States 85,982 78,640 154 (avg) 12,101,238 Source: http://www.nass.usda.govMonsanto Company 07-CR-191U Page 245 of 544 Table IX-3. Comparison of Field Corn Pr oduction in the Midwest vs. the Great

Plains

2007 Corn Yield

Measurements and Average Precipitation Midwest Great Plains

Total Acres Planted 56,300,000 24,834,000 Avg. Bushels/Acre 150 132 Total Bushel Yield 8,515,450,000 3,315,645,000 Avg. Precipitation (inches)* 34.45 20.38
*Thirty year (1961-1990) annual average precipitation Source: http://www.met.utah.edu/jhorel/html/wx/climate/normrain.html

IX.D.1.2. Seed Production

Standardized seed production practices are re sponsible for maintaining high quality seed stocks, an essential basis for U.S. agriculture. By the early 20 th century, agronomists learned how to develop specific plant varieties with desirable traits. In the U.S., state agricultural experiment stations developed ma ny seed varieties which were distributed to farmers for use. As seeds were saved by farm ers and later sold to neighbors however, the

desirable traits of the varieties often we re lost through random genetic changes and contamination with other crop and weed s eeds (Sundstrom et al., 2002). The value of seed quality (including genetic purity, vigor, weed seed presen ce, seed borne diseases and inert materials, such as dirt) was quickly id entified as a major fact or in crop yields. States developed seed laws and certificati on agencies to ensure that purchasers who

received certified seed coul d be assured that the seed met established seed quality standards (Bradford, 2006). The federal governme nt passed the U.S. Federal Seed Act of 1939 to recognize seed cer tification and official certifying agencies. Regulations first adopted in 1969 under the Federal Seed Act re cognize land history, field isolation, and varietal purity standards for foundation, re gistered, and certified seed. Under international agreements such as the Or ganization for Economic Co-Operation and Development (OECD) scheme, the U.S. an d other countries mutually recognize minimum seed quality standards (Bradford, 2006). The Association of Official Seed Certifying Agencies (AOSCA) represents state and private seed certification in the U.S.,

and includes international member countries in North and South America, as well as

Australia and New Zealand.

Seed certification is based on varietal lineage, as well as quality production and processing standards. Seeds produced for sale to a crop grower (certified seeds) are a limited number of generations from a verified seed stock of the specified variety (Bradford, 2006). Breeder seed is generally produced under the strictest standards and under the supervision of the breeder. Breeder seed is used to produce foundation seed, which is used to produce registered seed, whic h is then used to produce certified seed that is sold for commercial planting (Bradfor d, 2006). In addition to documenting the pedigree of the seed, certification programs also monitor crop rotations, previous crops and weeds in the field, as well as isolation of the field fromother varieties of the sameMonsanto Company 07-CR-191U Page 246 of 544 genus or species (Bradford, 2006) . Inspectors walk the fiel ds to note the occurrence of off-type plants, other crop plants, weeds, or disease. After seed harvesting and cleaning,

the seed is later tested for germination capacity, and analyzed for the presence of seed of other varieties or other crops, weed seeds and inert matter (e.g., dirt or stones) to assure high quality before the seed bags are tagge d as “certified” (Bradford, 2006). Within a seed crop, the main sources of off-types, or seed from another plant, result from “volunteers,” or seed from crops grown in th e field at an earlier date, pollen transfer and mixing that occurs during harvesting and ha ndling (Bradford, 2006). Seed producers can and do take steps, such as cleaning equi pment, appropriate crop rotation and other stewardship measures, to control for these factors.

Seed producers also learned over the years to account for pollen flow between nearby fields, and seed certifying agencies adopted spatial isolation requi rements (codified in federal regulations for certain crops) that must be followed to produce certified seed that will guarantee the purchaser receives the intend ed seed variety. The isolation required for a particular crop depends on its floweri ng characteristics (including timing), sexual compatibility with neighboring crops, pollen quantity and viability, and the mode of pollen dissemination (Sundstrom et al., 2002).

The standards put in place in the first half of the 20 th century were based on physical appearance and performance determined by field inspections. Those standards are still in place today for the vast majority of crops pr oduced. These standards have never required 100% varietal purity. For example, AOSCA st ated that the maximum limits for seed of other varieties or off-types in foundation seed lots range from 0 to 0.2% among different species, while the limits for certified seed range from 0.1 to 2% by weight. This also can be demonstrated by stating that the maximum number of seeds of other varieties of the same crop permitted in one pound of certified seed is one seed for cotton and wheat, two for watermelons, four for rice, and six for sunflowers (Bradford, 2006; AOSCA, 2004; CCIA, 2005)

. The standards that have served societ y well for at least ha lf a century were adopted to reflect a balance between the leve l of purity required to meet market needs and prevent consumer fraud and the cost of ac hieving that purity standard. While it is possible to achieve higher levels of purity, this involves higher production costs, often prohibitively higher. A recent study found that relative to standard corn seed production practices, the costs to achieve higher levels of genetic purity (in this case, a 0.3% biotech threshold) would be approximately 35% higher (Bradford, 2006; Kalaitzandonakes and Magnier, 2004). A non-biotechnology-derived co rn seed standard has been established by AOSCA (http://www.identitypreserved.c om/handbook/aosca-nongmocorn.htm) requiring 660 feet of isolati on distance, field inspections and purity tests using AOSCA approved laboratories. This standard al lows up to a 1% presence of biotechnologyderived seed in corn seed sold as “non-GMO. ” This AOSCA standard has been in place for a number of years, coexistent with wide scale planting of biotec hnology-derived corn. MON 87460 will have no greater impact on this or any other seed purity standard than any other corn hybrid, conventional or biotechnology-derived.

Almost all seed corn currently utilized in the U.S. is produced th rough hybridization. In this process one variety of corn produces pol len to fertilize a second variety, which hasMonsanto Company 07-CR-191U Page 247 of 544 been de-tasseled (the pollen producing male or gans are removed) so it cannot fertilize itself. Because so much grain corn is grown in the continen tal U.S., hybrid seed production and bulk up is often done elsewhere (Sout h America and Hawaii are commonly used) and in the off-season. Overall, three types of isolation practices (i.e., spatial, temporal, and physical barriers) help reduce the frequency of unwanted gene flow into seed corn via pollen transport. Most se ed companies meet or exceed all national and international standards with regard to isolation and quality production practices.

In addition to instituting their own requireme nts, seed companies are working together regionally, nationally and intern ationally to establish betterguidelines for biotechnologyderived seed production. Several groups se t standards for production and monitor the sale and transportation of seed. Among thes e organizations, various standards act to guide the production of high-quality commerci al seed including the certifications specified below.

Certification by the OECD is applied to vari eties that meet established conditions of identity, uniformity, and stab ility. OECD certified variet ies have an added economic value and are published in official OECD lis ts. The OECD helps ensure the varietal identity and quality of seed by setting appr opriate requirements a nd controls throughout production, processing and labeling. Cer tified seeds are produced and officially controlled according to comm on harmonized procedures. OE CD certification provides official worldwide recognition of "quality-g uaranteed" seed, facilitating international trade and contributing to removal of technical trade barriers.

The AOSCA is dedicated to assisting comp anies in the producti on, identification, distribution and promotion of certified classes of seed. Established in 1919 in the United States, the organization has grown to incl ude members from around the world. AOSCA establishes minimum standards for quality an d identity. Its goal is to standardize certification regulations and pr ocedures internationally so companies compete under one set of standards. The association coopera tes with the OECD a nd other international organizations to develop standards, regula tions, procedures, and policies to expedite movement of seed and encourage intern ational commerce in improved varieties.

It is anticipated that MON 87460 seed will be produced and marketed in accordance with

OECD and AOSCA standards and the U.S. Fe deral Seed Act and will have no adverse impact on seed production practices.

IX.D.2. U.S. Corn Agronomic Practices

MON 87460 reduces yield loss under water-limited conditions. The targeted geographic area where MON 87460 is expected to have the greatest impact is th e Great Plains, an area of significant corn production that is prone to frequent drought stress. Approximately 24 M acres in the Great Plains were planted to co rn in 2008 resulting in 26% of the national corn producti on total. Key considerati ons for corn production in the Great Plains include climate and environment, soil quality and fertility, tillage and crop rotation practices, hybrid selec tion, nutrients, moisture, irri gation, and the management ofMonsanto Company 07-CR-191U Page 248 of 544 insects, weeds (including volunteer corn), and diseases. A summary of the corn cultivation and management practices in the Great Plains is provided below.

IX.D.2.1. Climate and Environment

The climate of the Great Plains is characte rized by two primary gradients which define the area and determine its environment; these are precipitation (decr easing east to west) and temperature (increasing nor th to south). Total annual precipitation ra nges from 30

inches in the east to less than 15 inches in the west (Hubbard, 1997, Parton et al., 1994). The growing season varies from 110 days in the north to approximately 300 days in the south (Skold, 1997). Lack of moisture has resulted in a steppe or semi-arid natural grassland ecosystem with plant growth limite d by precipitation and nut rient availability (Parton et al., 1994). Drought is a factor in this system with the degree and timing controlled by temperature, pr ecipitation and the ratio of precipitation to potential evapotranspiration (Parton et al., 1994). Cr ops grown vary according to the climatic gradients of rainfall and temperature. Rain fed corn and soybean are produced in the east, dryland wheat and sorghum in the west. However, irrigation pract ices in the

western sections of the Great Pl ains have supported significant corn production. It is in this type of climate that MON 87460 is exp ected to provide benefit and reduce grower risk due to drought.

IX.D.2.2. Soil Qualit y and Fertility

Corn is grown in a variety of soils in the U. S. ranging from the sand hills of Nebraska and Colorado to the clays of delta regions, from strongly acidic to strongly alkaline soils, and from shallow soils on residual material to deep soils in loess, till or alluvium. The primary types of soils found in the Great Plains are classified as Mollisols (http://soils.usda.gov/technical/classification /taxonomy) and, due to their fertility and distribution throughout the world, are cons idered agriculturally and economically important. Mollisols form in semi-arid to semi-humid areas, typically under a grassland cover. They have a high organic matter, nutrient-enriched surface soil, typically between 60-80 cm thick, clay subsurface, and a soft, gra nular soil structure. This fertile surface soil layer results from the long-term addition of organic materials derived from plants. The combination of high organic matter and clay subsurface soil, in conjunction with good structural properties, f acilitates water and nutrient storage and a degree of permeability favorable to water intake and ai r exchange. Corn hybrids perform well in

mollisols unless environmental conditions such as drought intervene, causing yield loss. It is anticipated that MON 87460, which has agronomic and phenotypic characteristics equivalent to conventional corn (Section VI II), will also grow well in mollisol soils.

IX.D.2.3. Tillage Practices

There are three main tillage practices employe d in all corn production areas: conventional tillage, reduced tillage, and conservation tillage. Conventi onal tillage practices leave <15% crop residue cover after planting and i nvolve the use of a moldboard plow or other intensive tillage procedure. Reduced tillag e practices leave between 15-30% crop residue cover after planting and exclude the use of a moldboard plow or other intensive tillage procedures. Conservation tillage is a system that covers 30 % or more of the soil surfaceMonsanto Company 07-CR-191U Page 249 of 544 with crop residue after planti ng to reduce soil er osion by water and consists of three subtypes: no-till, ridge-till and mulch-till. These subt ypes differ in the timing of

cultivation of the seedbed and type of equipment used. Over the last two decades there

has been a trend toward the increased use of conservation tillage practices with most of the growth coming from the expanded adoption of no-till. Studies in Great Plains states Nebraska, Montana and Texas (Smika and Wicks, 1968; Tanaka and Aase, 1987; Unger and Wiese, 1979) indicate that soil precipitation storage efficien cies increase when tillage is minimized or eliminated thus reducing the number of times moist soil is brought to the surface. Crop residues on the surface trap snow, absorb raindrop impact, slow runoff and minimize evaporation and wind velo cities as well as aid in w eed control. Considering the moisture retention benefits of minimal tillage, it is therefore not su rprising that in 2004, four of the top five states with significant in creases in no-till corn were from the Great Plains Table IX-4 (Peterson, 2005).

Table IX-4. Greatest Increases in No-Till Corn in 2004 Rank State Increases (acres) 1 South Dakota >253,000 2 Colorado >224,000 3 North Dakota >201,000
4 Kansas >191,000 5 Wisconsin >121,000

Source: Peterson, 2005

Coupled with public awareness of environm ental issues, the trend toward increased conservation tillage continues particularly in the Mississippi watershed (Figure IX-1) whose tributaries, the Missouri, the Platte, the Arkansas and Oklahoma Rivers, drain the

states of both the Midwest and the Great Plai ns. Given that, with the exception of yield under water-limited conditions, MON 87460 is agronomically and phenotypically equivalent to conventional cor n, it is not anticipated that the introduction of MON 87460 will have any impact on current tillage pr actices or on the trend towards increasing adoption of conservation tillage by growers.Monsanto Company 07-CR-191U Page 250 of 544 Figure IX-1. U.S. Conservation Tillage Acreage by Watershed

Source: http://agecon.okstate.edu /isct/labranza/walters/conservation.doc

IX.D.2.4. Corn Hybrids

In the U.S. hundreds of corn hybrids are market ed by seed companies. Their selection is primarily based on yield potential, climatic e nvironment, and disease/pest resistance for

that locale.

Due to the extensive north-south orientation of the Great Plains , one of the most variable factors is the length and warmth of the gr owing season. Corn planting can begin in March in the Southern areas of the Great Plains while significant planting in the cooler Northern Great Plains typically does not begin until May. Harvest may occur as early as August in Texas or as late as October in North Dakota. Planting date and hybrid selection are managed to avoid occurrence of the critical corn pollination stage during the hottest, most stressful times of midsummer. Additionally growers in the Southern Great Plains can grow hybrids with a much longer maturity cycle than is possible in the Northern Great Plains (Nielson and Wishart, 2004) . Planting densities in dryland areas is a subject of debate with ranges of 11,000 to 24,000 kernels of co rn per acre being claimed to produce optimal yield results (Blumenthal et al., 2003; Norwood, 2001).Monsanto Company 07-CR-191U Page 251 of 544 Hybrid characteristics that co ntribute to drought tolerance are complex and may not be readily apparent. The best indicator of dr ought tolerance is corn grain yield under

moisture stress. Over the la st several decades, breeding progr ams have been particularly successful at increasing the abil ity of corn to withstand drou ght occurring at flowering as demonstrated by decreases in anthesis-silking in terval, and increases in ears per plant and kernels per ear, all of which result in yi eld increases (Campos et al., 2006). These breeding efforts have resulted in hybrids that are characterized as having excellent to very good drought tolerance and are availabl e from major seed companies:

http://www.asgrowanddekalb.com/seedresourceguide/search/seeds http://www.pioneer.com/web/site/portal/menuitem.b98005ee4490c1a5d6c1f492d10093a0/

Seed companies provide recommendations to growers for seed selection based on geographic location. Approximately 25% of th e conventionally bred hybrids offered for planting in the dryer western areas of the Gr eat Plains were rated as having excellent drought tolerance and were recommended for fields that regularly experience drought stress; 50-65% of the hybrids o ffered for planting in these areas were rated as having very good performance in fields that regularly e xperience some drought stress, but were not recommended for fields routinely experiencing extreme drought stress.

If approved for use, it is highly likely th at biotechnology-derive d traits conferring drought tolerance will become part of breed ing programs. This additional tool for breeders has the potential to result in an expanded select ion of hybrids for growers to choose from as they consider their indivi dual growing needs and the likelihood of waterlimited conditions during the growing season. It is not anticipated that addition of the MON 87460 drought tolerance trait in future breeding programs will result in a reduction in the selection of hybrids currently availabl e to growers or in the introduction of an enhanced susceptibility to insects or dis ease, based on the mode of action and field observations (Sections I.D and VIII).

IX.D.2.5. Nutritional and Moisture (Includi ng Irrigation) Requirements of Corn

Corn, like all higher plants, requires at least thirteen elements from the soil for growth and development (Olson and Sander, 1988). Th e thirteen elements include the primary elements (nitrogen, phosphorus, and pota ssium), secondary elements (calcium, magnesium, sulfur), and micr onutrient elements (iron, manganese, zinc, copper, boron, molybdenum and chlorine). By far the most important are the primary elements, which are depleted in the soil as the corn plan t develops. Whereas nitrogen and phosphate uptake continues until maturity, potassium ab sorption is largely completed by the silking stage. This is why fertilization of corn fields is essential to ensure production and profitability. In 2005, nitrogen was applied to 96% of the planted corn acreage at an average usage rate of 138 pounds of nitroge n per acre per crop year. Phosphate was applied to 81% of the corn acreage at an av erage rate of 58 pounds pe r acre per crop year. Potassium, applied at 84 pounds per acre pe r crop year, was applied to 65% of the acreage planted to corn (USDA-NASS, 2006a).Monsanto Company 07-CR-191U Page 252 of 544 Corn is extremely sensitive to soil moisture deficits. As stated previously total annual precipitation in the Great Plains ranges from 30 inches in the east to less than 15 inches in the west (Hubbard, 1997; Parton et. al., 1994). The additional moisture requirement can be supplemented by irrigation, which is practiced on 10-11 million corn acres or approximately 15% of the total U.S. corn acreage (USDA-ERS, 2002). The impact of irrigation on corn yields for the area is dramatic; an average 157 bushels/acre from irrigated production vs. an average 75 bushels /acre, from dryland production (Nielson and Wishart, 2004).

Water supply sources in the Great Plains incl ude rain, surface water in rivers, streams, and lakes primarily from snowmelt, and groundwat er in aquifers. Agriculture is the main demand for water use (Skold, 1997) and depletion of existing water basins and aquifers is associated with the increased use of irriga tion. Irrigated land for all crops in the Great

Plains increased more than fourfold from 3.5M acres in 1950 to 15M acres in 1990 (Opie, 1996) while total water applied increased by only 11% (USDA-ERS, 2003). Adoption of more water conserving practices and produc tion shifts of some commodities to more humid and cooler areas is largely responsib le for more efficient water use. Most agricultural freshwater withdrawals from su rface and ground water for corn production occur in Nebraska, South Dakota, western Kansas, eastern Colorado and northern Texas where irrigated acres are c oncentrated (Figure IX-2).

Figure IX-2. U.S. Acres of Irrigated Corn Harvested for Grain

Source: http://www.nass.usda.gov/research/2002mapgallery/fieldcrops.htmlMonsanto Company 07-CR-191U Page 253 of 544 The shaded area of the Great Plains (Figure IX-2) correlates with the location of the Ogallala aquifer which was tapped for irri gation ground water in th e 1950s resulting in depletion of the aquifer (McGuire, 2007). With the exception of New Mexico and Colorado, total irrigated acres pl anted to corn in Great Plains states increased from 1992 to 2007 (Table IX-5). Of the states with more than 1M acres under irrigation, only Kansas experienced a significan t decline in the pe rcent total corn ac res under irrigation and a return to dryland production while Nebraska, South Dakota and Texas numbers remained relatively the same. According to Mapp (1988) the primar y historic causes for a decline in irrigated acres are increases in pumping cost s for pressurized groundwater irrigation systems and the low profitability of irrigated crops relative to dryland crops. Costs of supplying irrigation water vary widely reflecting different combinations of water sources, suppliers, distribution systems and other factors such as field proximity to water, topography, aquifer conditions and energy sour ces. Ground water is usually pumped onfarm with higher energy costs than surface water which is usually supplied from off-farm sources through storage and canal systems. In 2003, the average cost nationwide for ground water application was $40 per acre or ~$ 1.2B total. The aver age cost nationwide for surface water application was $26 per acr e or ~$0.9B total (USDA-ERS, 2003). However, in recent years the growing biofue ls industry and worldwide concern over food shortages have caused commodity prices to increase significantly. This may result in the continued high levels of irrigati on use despite the higher costs.

Given the above, it is foreseeable that growers may choose to determine whether MON 87460 can be used in dryl and production without benefit of irrigation. This is a decision driven by economic considerations, an d is not anticipated to have a significant impact on irrigation practices.Monsanto Company 07-CR-191U Page 254 of 544Table IX-5. Changes to IrrigatedCorn Acreage in the Great PlainsSource: http://www.agcensus.usda.gov/index.aspState 1992 1997 2002 Acres 2007 AcresTotalAcresIrrigatedAcres%Irg.TotalAcresIrrigatedAcres%Irg.TotalAcresIrrigatedAcres%Irg.Total Acres IrrigatedAcres%Irg.Colorado 891,720 808,351 92 919,784 769,567 84 708,197 634,015 90 1,060,000 700,000 66Kansas 1,748,802 1,154,370 66 2,497,516 1,465,920 59 2,494,179 1,346,807 54 3,700,000 1,569,000 42Montana 18,240 17,323 95 12,925 12,263 95 11,642 11,642 100 38,000 38,000 100Nebraska 7,235,528 4,703,268 65 8,279,499 4,966,816 60 7,344,715 4,505,579 61 9,200,000 5,725,000 62New Mexico 72,348 70,194 97 80,122 75,870 95 48,096 47,904 100 55,00 55,000 100North Dakota 595,347 53,836 9 578,953 46,654 8 991,390 54,445 5 2,350,000 106,900 5Oklahoma 123,567 69,104 56 150,404 85,678 57 182,777 99,457 54 270,000 140,000 52South Dakota 3,097,251 172,233 6 3,175,113 148,001 5 3,165,190 123,229 4 4,500,000 201,000 5Texas 1,549,680 740,431 48 1,656,229 871,364 53 1,815,560 658,177 36 2,000,000 975,000 49Wyoming 54,341 52,767 97 49,717 47,931 96 34,095 33,507 98 60,000 57,500 96Monsanto Company 07-CR-191U Page 255 of 544 IX.D.2.6. Crop Rotation

Growers make crop rotation decisions based on several factors, but a large consideration is the economic return resulting from differen ces in input costs, crop yield potential, and commodity prices. Crop rotations in the West ern Dryland region of th e Great Plains have relied on wheat and sorghum due to their abi lity to tolerate moderate water stress and produce acceptable yields. Changes in tillage management in the Great Plains cited previously (Section IX.D.2.3) have allowed fa rmers to change from the traditional wheatfallow rotation to more intensive rotations such as wheat-corn-fallow. Intensive cropping systems typically have higher precipitation use efficiency, thus incr easing crop yield per inch of rain. Dryland producers in Colorado have been adopting more intensive cropping systems, including dryland corn in rotation with wheat, at an increasi ng rate since 1990. The area planted to corn in northeastern Colorado, typically a wheat-fallow area, increased from 20,000 acres per year in year s previous to 1990 to 220,000 acres in 1999(Davis and Peterson, 2002). In addition to enhanced moisture conservation, a fourteenyear study of no-till in tensive dryland rotation manageme nt showed increases in soil carbon, nitrogen, and organic matter and aggreg ate stability (USDA- ARS, 2007). These increases in soil quality measurements were correlated with greater rotation intensity and with less fallow frequency.

Depending on commodity prices, growers may c ontinue to choose to increase the amount

of corn in their rotations. Given that MON 87460 is agronomically and phenotypically equivalent to conventional corn, it is anticipa ted that it will readily become part of any dryland crop rotation system that includes co rn. Corn hybrids with enhanced drought tolerance, either biotechnology-derived or conventional, may provide additional economic benefit in place of crops more co mmonly grown in these drier agricultural areas. If so, this decision w ill be driven largely by grower’s economic concerns. In this scenario, MON 87460 is no more likely to disp lace wheat or sorghum than conventional drought tolerant corn hybrids al ready available to the grower.

IX.D.2.7. Management of Diseases and Insects

MON 87460 was evaluated to determine whethe r the presence of th e drought tolerance trait would have an impact on disease and in sect management. Management of diseases and insects during corn growth and developmen t is essential for protecting the yield of the harvested grain. Estimates for annual yield losses becaus e of diseases have ranged from 7 to 17% (Shurtleff, 1980). Incidence of disease infestation is highly variable and depends on many factors such as location, climate, and ot her environmental factors. Most corn hybrids on the ma rket today have acceptable le vels of resistance to common

diseases. The diseases found to occur in co rn grown in the U.S. are summarized in Table IX-6. In addition, seve ral nematode species have been known to cause diseases in corn (Dicke and Guthrie, 1988). The use of fungicides in corn is limited because the incidence and severity of most diseases tends to be low and quite variable. The fungicides currently used on corn plants in the U.S. include azoxys trobin, chlorothalonil, propiconazole (USDA-NASS, 2006a).

The corn crop is subject to attack by a comp lex of insects from the time it is planted until

it is used as food and feed. The economically important in sect pests in North AmericaMonsanto Company 07-CR-191U Page 256 of 544 include wireworms, the black cutworm, Eur opean corn borer, Southwestern corn borer, the corn rootworms, grasshoppers, fall army worm, and corn earworm. Table IX-7 lists the insect pests in corn grown in the U.S. Approximately 27 active pesticidal ingredients are registered for use in corn for the control of insect pests. In its annual survey of agricultural chemical usage, USDA determined that 23% of the corn acreage was treated with insecticides in 2005 (USDA-NASS, 2006a). Tefluthrin, cyfluthrin, and tebupirimphos were the most wide ly applied insecticid es, at 7, 7, and 6% , respectively, to the corn acres. Chlorpyrifos was only applied to 2% of th e acres, but total quantity applied is more than 3 times greater than next highest insecticide at 2.0 million pounds.

The introduction of biotechnology- derived insect protected co rn has offered growers an

alternative and effective solution for the control of major insect pests in corn and in 2006 approximately 40% of the total corn acreage in the U.S. was planted with hybrids possessing insect protection traits (USDA-NASS, 2006b).

MON 87460 has no pesticidal activity. As de monstrated by environmental observations among numerous field studies (Section VIII.F .1), it has no apparent impact on arthropods and diseases of corn. Therefore, no change s to current disease and insect management practices, including pesticide use, conventional breeding selection for resistance, or adoption of biotechnology-derived traits ar e anticipated from the introduction of

MON 87460.

IX.D.2.8. Weed Management

MON 87460 was evaluated to determine whet her the presence of the drought tolerance trait would have an impact on weed manage ment. Weeds cause significant losses and require careful management by growers becaus e they interfere with corn plants by competing for available resources including water, nutrients and light. Economically damaging weeds in corn include annuals and perennials, grasses, broadleaf and sedge species. Some weeds can tolerate cold, wet conditions better than corn, and can get a head start prior to planting. Fields infe sted with perennial weeds present special problems for corn growers. Like annual w eeds, perennials can reproduce by seeds, but they also regrow and spread vegetatively. Their rhizomes propagate new shoots, usually soon after corn is planted. Unless effectiv ely controlled, perennial weeds can quickly gain a season-long advantage over the corn crop.

Corn yield loss is generally proportional to th e amount of weeds presen t. While the ratio is not always one-to-one, some studies sugge st that for every pound of weed dry matter, there is a reduction of approximately one pound of corn plant dry matte r (Gianessi et al., 2002). Competition for light, nutrients, and moisture resources by the crop and weeds can lead to proportional reductions in yield (Knake et al., 19 90). Numerous studies have shown that weed control early in the growing season is necessary to reduce yield losses in corn. Weed species such as giant foxtail, barnyardgrass and pigweed can reduce corn yields by up to 13, 35 and 50% respectively (Bosnic and Swanton, 1997; Fausay et al., 1997; Knake and Slife, 1965). In a study of mixed weed populations competing with corn, corn yields were reduced by up to 20% when the weed plants reached a height of eight inches (Carey and Kells, 1995).Monsanto Company 07-CR-191U Page 257 of 544 Corn is typically planted in wide rows (30 in ches) and has an upright leaf orientation. As a result, corn is not successful in compe ting with weeds early in the growing season. Corn is also usually planted early when soil temperature and w eather conditions favor weed over corn growth. A survey of Ex tension Service weed scientists solicited estimates of the percent of corn acreage infe sted with individual weed species by state or region, as well as the potential impact on corn yi elds if the species were left uncontrolled. In this survey, 12 annual broadleaf, nine a nnual grass, and seven pe rennial species were identified as troublesome weeds (Table IX-8) (Gianessi et al., 2002). Estimates of yield loss ranged from a low of 15% due to wire stem muhly and sandbur s to a high of 48%

from burcucumber.

Until the early 1950s, tillage and cultivation practices were primarily used for weed control in corn, but since then they have been largely replaced by the use of herbicides. Herbicide use in corn became widespread by the end of the 1970s. In 2005, herbicides were applied to 97% of the corn plante d acreage (USDA-NASS 2006a). Atrazine continues to be the most widely applied he rbicide with 66% of the planted acreage being treated. It was applied at an average rate of 1.133 pounds per acre. Glyphosate isopropylamine salt was applied to 31% of planted acres, up from 19% in 2003, at an average rate of 0.963 pounds per acre . In terms of area applied, that was followed closely by S-metolachlor and acetochlor, at 23% of the plan ted corn acreage tr eated. In 2001, the EPA identified glyphosate as the most widely us ed conventional agricu ltural pesticide in the U.S. (Kiely et al., 2004). The addition of herbicide-tolerant corn to an integrated weed management program offers multiple benefits to growers including a broader spectrum of weeds controlled, reduced crop injury, and less herbicide carry-over. Roundup Ready Corn 2 developed by Monsanto and Liberty Link corn developed by Bayer provide glyphosate-tolera nce and glufosinate-tolerance, respectively. These herbicide-tolerance technologies are easy to use and both herbicides are more environmentally friendly than other herb icide options (Knezevic and Cassman, 2003).

Volunteer corn commonly occurs in rota tional crops in the season following corn cultivation regardless of whether or not the corn was conventi onal or biotechnologyderived. When corn is grow n for silage, on approximately 9% of the U.S. corn acres, volunteer corn plants ty pically do not occur in rotationa l crops since corn harvested for silage does not produce viable seed. In the warmer clim ates of the Southeast and Southwest, the occurrence of volunteer corn is rare because any corn grain remaining after harvest is likely to germinate in the fall and the resulting plants can usually be controlled by tillage or by freezing temperatures in the winter. In the Northern corngrowing regions, volunteer corn does not alwa ys occur in the rota tional crop because of seed decomposition over the winter, efficien t harvest procedures, and tillage prior to planting rotational crops.

The first step to manage volunteer corn in ro tational crops is to mi nimize or reduce the potential for volunteers. Th e following practices are implemented to reduce volunteer corn in rotational crop s: (1) adjust harvest equipment to minimize the amount of corn grain lost in the field, (2) pl ant corn hybrids that reduce the extent of ear drop, (3) chooseMonsanto Company 07-CR-191U Page 258 of 544 corn hybrids with superior stalk strength and reduced lo dging, and (4) practice no-till production to significantly reduce the potential for volunteer growth in the rotational crop. If volunteer corn does occur in subse quent crops, pre-plan t tillage or in-crop cultivation are very effective management tools. Selectiv e herbicides labeled for the

control of volunteer corn in the particular rotational crop are available. Assure II ®

(quizalofop), Fusilade ® DX (fluazifop), Fusion ® (fluazifop + fenoxaprop), Poast ®

(sethoxydim), and Select ® 2EC (clethodim) provide effective postemergence control of volunteer corn in labeled crops. These products are labeled for use in eight field crops, including soybean, cotton, sugar beet and al falfa, and eleven ve getable rotation crops.

MON 87460 will likely be combined through conventional breeding with deregulated herbicide tolerance traits, and consequently, growers will be able to achieve the same high level of weed control as they have with other biotechnology-derived herbicide tolerant corn hybrids. Additionally, because MON 87460 is agronomically and phenotypically equivalent to c onventional corn, it is not anticipated that MON 87460 will respond differently to commonly used herbicid es, or that its introduction will have any impact on weed management practices in the U.S. compared to current production practices that include biotechnology-derived or conventional corn hybrids, including drought tolerant varieties.

® Assure II is a trademark of E.I. DuPont de Nemours, Inc.

® Fusilade and Fusion are trademar ks of Syngenta Group Company.

® Poast is a trademark of BASF Corporation.

® Select is a trademark of Valent U.S.A. Corporation.Monsanto Company 07-CR-191U Page 259 of 544 Table IX-6. Diseases of Corn

Common Name Causative Agent [transmittal agent]
Seed rots and seedling blights Fusarium moniliform, Pythium spp. Foliar Diseases Bacterial leaf blight and stalk rot Pseudomonas avenae Bacterial stripe Pseudomonas andropogonis

Stewart’s wilt Erwinia stewartii Chocolate spot Pseudomonas coronafaciens

Goss’s wilt Clavibacter michiganense Holcus spot Pseudomonas syringe

Anthracnose Colletotrichum graminicola Eyespot Kabatiella zeae Gray leaf spot Cercospora zeae-maydis Northern leaf spot Bipolaris zeicola Northern corn leaf blight Exserohilum turcicum Physoderma brown spot Physoderma maydis Southern corn leaf blight Bipolaris maydis Yellow leaf blight Phyllosticta maydis

Common rust Puccinia sorghi

Southern corn rust Puccinia polysora

Common corn smut Ustilago maydis Systemic Diseases

Head smut Sphacelotheca reiliana Crazy top Sclerophthora macrospora Sorghum downy mildew Peronosclerospora sorghi Maize dwarf mosaic virus [aphids] Maize chlorotic dwarf virus [leafhoppers] Corn lethal necrosis [chrysomelid beetles] Maize white line mosaic virus [not identified] Corn stunt [leafhoppers] Maize bushy stunt [leafhoppers]

Stalk and root rots

Gibberella stalk rot Gibberella zeae Diplodia stalk rot Stenocarpella maydis

Anthracnose stalk rot Colletotrichum graminicola

Charcoal rot Macrophomina phaseolina

Fusarium stalk rot Fusarium moniliforme Pythium stalk rot Pythium aphanidermatum

Bacterial stalk rot diseases Erwinia chrysanthemi

Root rots Pythium spp

. Ear rots and storage molds

Fusarium ear rot Fusarium moniliforme

Gibberella ear rot Gibberella zeae Diplodia ear rot Diplodia maydis Aspergillus ear and kernel rot Aspergillus flavus Storage molds Penicillium spp ., Aspergillus spp

. Source: Smith and White, 1988Monsanto Company 07-CR-191U Page 260 of 544 Table IX-7. Insect Pests of Corn

Common Name Latin name

Soil Insects

Northern corn rootworm Diabrotica barberi

Western corn rootworm Diabrotica virgifera virgifera

Southern corn rootworm Diabrotica undecimpunctata

Black cutworm Agrotis ipsilon

Wireworms A. mancus, Horistonotus uhlerii, Melanotus
cribulosus, others Billbugs Sphenophorus spp

. White grubs Phyllophaga spp

. Corn root aphid Anuraphis maidiradicis Seedcorn maggot Delia platura Grape colaspis Colaspis brunnea

Seed corn beetle Stenolophus lecontei

Insects attacking the leaf, stalk, and ear

Corn earworm Helicoverpa zea European corn borer Ostrinia nubilalis Corn leaf aphid Rhopalosiphum maidis Fall armyworm Spodoptera frugiperda

Stalk borers Diatraea spp. Armyworm Pseudaletia unipuncta

Lesser stalk borer Elasmopalpus lignosellus Chinch bug Blissus leucopterus leucopterus Grasshoppers Melanoplus differentialis

Corn flea beetle Chaetocnema pulicaria Japanese beetle Popillia japonica

Other insects Thrips Anaphothrips spp., Frankliniella spp.

Leafhoppers Trigonotylus brevipes , others

Western bean cutworm Striacosta albicosta

Corn blotch leaf miner Agromyza parvicornis Spider mites Oligonychus spp ., Tetranychus spp

. Pink scavenger caterpillar Pyroderces rileyi Garden symphlan Scuttigerella immaculata Hop-vine borer Hydraecia immanis Sod webworms Subfamily Cramdinae

Leaf rollers Stink bugs

Insect disease vectors Several

Sources: Dicke and Guthrie, 1988; and University of Missouri, 1998.Monsanto Company 07-CR-191U Page 261 of 544 Table IX-8. Troublesome Weeds in U.S. Corn Fields

Weed Species Area Infested 1 Acreage Infested (%) Potential Yield Loss (%)

Annuals

Broadleaves

Burcucumber PA/OH/TN/SE 5-10 48

Cocklebur MW/NP/SE 20-60 33

Jimsonweed MW/CO 5-20 17

Kochia NP/NW 10-70 33 Lambsquarters MW/SE/NE/CA 15-80 33 Morningglory MW/SE/SP 20-75 33 Nightshade MW/NP/CA 25-50 26 Pigweeds/Waterhemp US 30-90 36 Ragweed, Common MW/SE/NE 20-70 30 Ragweed, Giant MW/NP 10-45 28

Smartweeds MW/SD/NE/SE 30-70 22

Velvetleaf MW/NE/NP 25-70 28

Grasses Barnyardgrass SP/NW/CA 80-90 23 Bermudagrass MD/SE/UT/CA 10-20 47 Crabgrass spp. MW/SE/NE 20-80 29 Cupgrass, Woolly IA/WI 15-20 29 Foxtail spp. MW/NE/NP 50-90 31 Millet, Wild-Proso UT/WY/CO/ID 15-40 31 Panicum, Fall MW/SE/NE/NP 15-80 30

Sandburs NP/UT/WY 5-30 15

Shattercane MW/SP 5-40 33

Perennials Bindweed, Field ND/SW/CA 40-80 18 Dogbane, Hemp IL/MO 2-20 21 Johnsongrass MW/SE/SW/CA 20-60 45 Muhly, Wirestem PA 2 15 Nutsedge, Yellow MW/SE/NE/NP/CA 10-70 21 Quackgrass MW/NE/UT 10-70 27 Thistle, Canada NE/MW/NP/CO 5-25 26
Source: Gianessi et al., 2002.

1 Regions States

US: United States CA: California OH: Ohio MW:Midwest CO: Colorado PA: Pennsylvania

NE: Northeast ID: Idaho SD: South Dakota

NP: Northern Plains IA: Iowa TN: Tennessee NW: Northwest MD: Maryland UT: Utah

SE: Southeast MO: Missouri WI: Wisconsin SW: Southwest ND: North Dakota WY: Wyoming

SP: Southern PlainsMonsanto Company 07-CR-191U Page 262 of 544 IX.E. Corn Acreage, Land Use, and the Conservation Reserve Program

Total corn acreage in the U.S. has remain ed relatively steady from 1996 to 2006 with a yearly average of 78 million acres indicati ng that biotechnology-derived corn, which has been commercially available for over a decade, has had little to no impact on total corn acreage. In 2007, the total co rn acreage increased by 15% from 78 million to 92.9 million acres. This increase was attributed to increased demand from ethanol producers (http://www.ncga.com/node/83) and strong exports sales. The increase in total corn acres resulted primarily from fewer acres of soybean planted in the Corn Belt and Great Plains (USDA-NASS, 2007).

A small percentage of the increase in corn acreage came from additional corn plantings on land that had previously been enrolled in the Conservation Reserve Program (CRP). The CRP is a voluntary program offering annual rental payments over a 10-year contract period, as well as cost-share assistance, to pr oducers establishing specific types of plant cover on marginal farmland. Of the 36M acres enrolled in the CRP in 2006, 21.2M acres

or 59% were from Great Plains states with 7.9M acres or 22% from Midwest “corn-belt” states (USDA-FSA-CRP, 2007).

Re-enrollment for 15.7M eligible acres with CRP contracts due to expire in 2007 dropped by 2.6 M to 13.1M acres compared with 2006 (USDA-FSA-CRP, 2007). This drop could be attributed to the growing biofuels indus try and worldwide con cern over food shortages that have caused commodity prices to increa se, with the result that some growers are not renewing their enrollmen t in the CRP. This trend is already underway and may be accelerated by the introduction of a corn hybrid with enhanced drought tolerance characteristics, regardless of whether the hybrid has been developed through biotechnology or conventional breeding. In this scenario, introduction of MON 87460 is no more likely to impact changes in corn ac reage and land use than a conventionally bred drought tolerant hybrid. A decrease in commo dity prices would likely be accompanied by an increase in CRP enrollment, despite the availability of drought-tolerant corn.

IX.F. Mitigation and Remediation Measures Before and After Deregulation of

MON 87460 in the U.S.

Under the Coordinated Framework for Regul ation of Biotechnology, the responsibility for regulatory oversight of non-pesticidal biotechnology-derived crop s falls on two lead federal agencies: FDA and USDA (OSTP, 1986). Deregulation of MON 87460 by USDA constitutes only one component of the overall regulatory oversight and review of this product. As a practical matter, MON 87460 cannot be released and marketed until both FDA and USDA have completed thei r reviews and assessments under their respective jurisdictions.

Food and feed from biotechnology-derived cr ops are subject to regulatory review by FDA under the Federal Food, Drug, and Cosmetic Act [21 U.S.C. 346 a(d)]. Since 1992, FDA has used a voluntary consultation proces s to work together with biotechnologyderived product developers to identify and resolve any issu es regarding the safety and

nutritional content of food and feed from biotechnology-derive d crops. Using the currentMonsanto Company 07-CR-191U Page 263 of 544 process, Monsanto has initiated and will co mplete a consultation with FDA prior to

commercial distribution of MON 87460 (Section I.F.1).

The Animal and Plant Health Inspection Services (APHIS) of the United States Department of Agriculture (USDA) has res ponsibility under the Pl ant Protection Act (7 U.S.C. § 7701-7772) to prevent the introduction and disseminatio n of plant pests into the U.S. The APHIS regulation at 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and should no longer be regulated.

Monsanto has provided to both the FDA and the USDA a comprehensive data package including: (1) the intended technical effect of the modification of the corn plants; (2) a

molecular characterization of the modification, including the identities, sources, and functions of the introduced genetic materia l; (3) information on the expressed CSPB and NPTII proteins encoded by the introduced genes; (4) information on assessment of potential allergenicity and toxicity of the introduced proteins; (5) information on the compositional and nutritional characteristics of the food and feed derived form the regulated article and, in the cas e of the USDA, (6) informati on on the plant pest potential of the introduction of MON 87460, and (7) an assessment of the potential impact on NTOs and threatened or endangered species. Based on these data, there is no reason to expect that food and feed de rived from MON 87460 constitute a risk to human health.

Because these data demonstrate that MON 8746 0 does not pose a plant pest risk, it is not anticipated that mitigation or remediation m easures will be necessary for this product.

Monsanto employs a rigorous product stewards hip program that demonstrates respect for our customers, their markets and the envir onment. Our market stewardship program considers many factors to support global integr ation and assure increased transparency. In keeping with past practice, we will not launch MON 87460 without first gaining regulatory approval from the key corn import countries to assure global compliance and support the flow of internati onal trade. We commit to be st industry practices on seed quality assurance and control to prevent adventitious pr esence of unapproved traits. Before commercializing MON 87460, a detection method will be made available to grain producers, processors, and buyers. Our stewards hip policy is the shared responsibility of Monsanto, our licensees, and our customers to assure that our products are used properly. We are committed to our legal and ethical ob ligation to ensure th at our products and technologies are safe and environmentally responsible, and do not pose undue risks to human health or the environment during any stage of their life cycle. As such, Monsanto has policies in place to meet these comm itments as we research, develop, design, manufacture, market, and discontinue products through their product life cycle.

IX.G. Conclusions

MON 87460 has been shown to be no different from conventional corn in its phenotypic, environmental, or compositional characteri stics, with the exception of the drought tolerance trait (Sections VII and VIII). Thus, MON 87460 is similar to conventional corn in its agronomic characteristics and plant pest potential. Tillage, crop rotation, planting density, hybrid selection, and irrigation practi ces have historically been modified toMonsanto Company 07-CR-191U Page 264 of 544 optimize corn yields in the Great Plains wh ere precipitation use efficiency is a primary concern. As biotechnology-derive d agricultural products have offered safe and effective

alternatives to weed and insect control in the pa st, MON 87460 is another option for growers to help minimize production risk by providing increased yi eld stability under

water-limited conditions.

Monsanto is not aware of a ny study results or observati ons associated with MON 87460 that would suggest that an incr eased plant pest risk would be anticipated to result from its introduction. MON 87460 reduces yield loss under water-limited conditions. The greatest benefit is expected to occur in regi ons that are suitable for corn production but prone to frequent drought stress, such as th e Western Dryland region of the Great Plains. As demonstrated by field and laboratory st udy results, the only phenotypic difference

between MON 87460 and conventional corn is reduced yield loss under water-limited conditions, due to expressi on of the CSPB protein.

The plant pest assessment of MON 87460 wa s based on multiple lines of evidence developed from a detailed characterizati on of MON 87460 compared to conventional corn, followed by a risk assessment on detected differences. The risk assessment

considered various factors including: (1) the negligible risk for MON 87460 or its progeny to establish or persist in the envir onment without human assistance, (2) minimal availability of suitable hosts or habitats for MON 87460, and (3) the negligible risk for MON 87460 to cause damage to plants and plant products.

The assessment for potential impacts of MO N 87460 on agronomic practices was based on an evaluation of the current production, la nd use, and agronomic practices for corn, with particular reference to the Western Dryland region of the Great Plains. Those

assessments indicate that the introduction of MON 87460 is no more likely to impact agronomic and land use practices in the Great Plains than th e use of conven tionally bred drought tolerant corn. Finally, due to the inherently low plan t pest potential of modern corn and the lack of (a) weediness and horizon tal gene transfer poten tial, (b) any impact on disease or injury to plants or plant pest s in the field, or (c) any observed effects on non-target or beneficial or ganisms in the agro-ecosystem, it is concluded that MON 87460 is unlikely to pose a plant pest risk.

Based on the data and information presented in this petition, it is concluded that MON 87460 is unlikely to be a plant pest. The adoption of MON 87460 may increase economic and environmental benefits primar ily in the Western Dryland region of the Great Plains, due to the protection of corn yield under water-limited conditions, but is not expected to have a significant environmen tal impact. Therefore, Monsanto Company requests a determination from APHIS th at MON 87460 and any progeny derived from

crosses between MON 87460 and other commercial corn be granted non-regulated status

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USDA-NASS. 2006a. Agricultural chemical usage 2005 field crops summary. United States Department of Agri culture, National Agricultural Statistics Service. Ag Ch1 (06).

USDA-NASS. 2006b. USDA-NASS. 2006. National Agricultural Statistics Service, United States Department of Agriculture . Acreage Report, Cr Pr2-5 (6-06) a. Accessed June 30, 2006.

USDA-NASS. 2007 National Agricultural Statisti cs Service, United States Department of Agriculture. Acreage Report, Cr Pr 2-5 (6-07) Accessed June 30, 2008.

Welcker, C., B. Boussuge, C. Bencivenni, J-M. Ribaut and F. Tardieu. 2007. Are source and sink strengths genetically linked in mai ze plants subjected to water deficit? A QTL study of the responses of leaf growth and of anth esis-silking interval to

water deficit. Journal of E xperimental Botany. 58:339 - 349.Monsanto Company 07-CR-191U Page 270 of 544 Westgate, M.E., M.E. Otegui, and F.H. Andr ade. 2004. Physiology of the corn plant in Corn: Origin, History, Technology, and Production, C.W. Smith, J. Betran and E.C.A. Runge, (eds.) John Wiley and Sons, Hoboken, New Jersey.

Wilkes, H.G. 1967. Teosinte: the Closet Rela tive of Maize. Bussey Inst. Harvard Univ., Cambridge, Massachusetts, p. 69.Monsanto Company 07-CR-191U Page 271 of 544 X. Adverse Consequences of Introduction

Monsanto knows of no study results or observations associated with MON 87460 suggesting that adverse environmental cons equences would be anticipated from its introduction. MON 87460 reduces yield loss under water-limited conditions. Under well-watered conditions, grain yield for MON 87460 is not different from conventional corn. Like conventional corn, MON 87460 is still subject to yield loss under waterlimited conditions, particularly during flowering and grainfill periods when corn yield potential is most sensitive to stress by di srupting kernel development. Under severe water deficit, corn grain yield for MON 87460, as well as conventional corn, can be

reduced to zero.

The data and information presented in th is petition demonstrate that MON 87460 is unlikely to pose an increased plant pest pote ntial compared to conventional corn. This conclusion is reached based on multiple lines of evidence developed from a detailed characterization of the product compared to conventional corn, followed by risk

assessment on detected differences. Modern co rn has inherently low plant pest potential because it is poorly suited to survive wit hout human assistance and is not capable of surviving as a weed due to intense sel ection for domestication purposes during its evolution as a crop. The characterization studies for MON 87460 included molecular and protein analyses, which confirmed the inse rtion of a single f unctional copy of the cspB

and nptII expression cassettes at a single locus within the corn genome and that the protein was expressed in tissues at levels that are efficaci ous for reduced yield loss under

water-limited conditions. Alle rgenicity assessment conclude d that the CSPB and NPTII proteins are unlikely to be a llergens for humans. Toxicity tests, including acute oral gavage studies with mice for both CSPB and NPTII, showed no signs of adverse effects at high doses. Compositional analysis of key nutrients, anti-nutrients, and secondary metabolites showed that the forage and gr ain from MON 87460, and the foods and feeds derived from such, are compositionally equiva lent to those derived from conventional corn. Extensive characterization of the plan t phenotype also showed that MON 87460 is unchanged compared to conventional corn, with the exception of the intended modification. An endangered species risk assessment concluded that MON 87460 is unlikely to have adverse effects on these orga nisms. Finally, an assessment of agronomic practices confirmed that the introduction of MON 87460 is no mo re likely to impact land use, cultivation practices, or the management of weeds, diseases, a nd insects than the use of conventionally bred drought to lerant corn. Therefore, th e risks for humans, animals, and other non-target organisms from MON 87460 are negligible under the conditions of use. The introduction of MON 87460 may increa se economic and environmental benefits due to the protection of corn yields under water-limited conditions. Based on the results of these assessments, no adverse environmen tal consequences have been found to be

associated with the introduction of MON 87460.

Therefore, on the basis of the benefits that this product offers for reduced yield loss under water-limited conditions, Monsanto requests that MON 87460 and a ny progeny derived

from crosses between MON 87460 and other co mmercial corn be granted non-regulated

status under 7 CFR Part 340.Monsanto Company 07-CR-191U Page 272 of 544 Appendix A. Materials and Methods Used for Molecular Analyses of MON 87460

A.1. Materials

The DNA used in molecular analyses was isolated from MON 87460 seeds (seed lot number GLP-0604-17132-S). A dditional DNA extracted from seeds of various generations of MON 87 460 (seed lot numbers GLP-0704-18549-S; GLP-0604-17132-S (F 1 seed); GLP-0604-17132-S (F 2 grain); GLP-0704-18550-S; GLP-0609-17631-S; GLP-0609- 17631-S; GLP-0703-18435-S) was used in generation stab ility analyses. The control DNA

was isolated from the seed of a conventional corn with the same genetic background (seed lot number GLP-0604-17133-S). The refere nce substances included the PV-ZMAP595 plasmid, probe templates generated from this plasmid, and the size estimation molecular weight standards. As a positive control on Southern blots, PV-ZMAP595 plasmid DNA was digested with combination of enzymes to produce the banding patterns that were

most relevant to the assessment of the te st substance digested with appropriate enzyme(s). The plasmid DNA was digested first and then added to pre-digested conventional corn genomic DNA. The molecu lar weight standards include the 1 kb DNA Extension Ladder (Invitrogen) and λ DNA/ Hin d III fragments (Invitrogen) for size

estimations on Southern blots. The 100 bp and 500 bp DNA ladders (Invitrogen) were

used for size estimations for PCR analyses

.

A.2. Characterization of the Materials The quality of the source materials fr om MON 87460 and conventional corn were verified by PCR analysis to confirm the pr esence or absence of MON 87460 except the materials used in the generational stability an alyses where the identity of the materials was confirmed by the generation stability Southe rn blots themselves. The stability of the genomic DNA was confirmed in each Southern analysis by observation of the digested DNA sample on an ethidium bromide-stained agarose gel.

A.3. DNA Isolation for South ern Blot and PCR Analyses Genomic DNA samples from MON 87460 and conven tional corn used in the insert and copy number, copy number of each element, ba ckbone analysis, and PCR analyses were isolated from corn seeds that were pro cessed into a fine powder using a Harbil ® 5G highspeed paint shaker. DNA was extracted from the processed seeds using the method described by Rogers and Bendi ch (Rogers and Bendich 1985).

Genomic DNA was isolated using the fo llowing method. Place about 6 grams of processed seed tissue in a 50 mL centrifuge tube and add ~16 ml of cetyltriethylammonium brom ide (CTAB) extraction bu ffer [1.5% CTAB, 75 mM tris(hydroxymethyl)aminomethane (Tri s) pH 8.0, 100 mM ethylenediaminet etraacetic acid (EDTA), 1.05 M NaCl, 0.75% polyvinyl pyrrolidone (PVP) (40K)] and 8-10 μ L of 10 mg/mL RNase. Incubate the samples at 65 o C for 25-35 minutes and mix halfway through the incubation. Let the samp les cool to room temperature, and add 16 mL of 24:1 chloroform: isoamyl alcohol, mix for 5 minut es and centrifuge for 5 minutes at 16,000 × g and 20-25 ° C to separate the aqueous and organic phases. Transfer the upper aqueous phase to a clean 50 mL centrifuge tube, a dd 1.6 mL of 10% (w/v) CTAB (10% CTAB, 0.71M NaCl) solution, mix by inversion, and add 16 mL of 24:1 chloroform: isoamyl

alcohol. Mix the tubes for 5 minutes be fore centrifuge for 5 minutes at 16,000 × g and 20-Monsanto Company 07-CR-191U Page 273 of 544 25 ° C to separate the aqueous and organic phase s. Transfer the upper aqueous phase to a clean 50 mL centrifuge tube which contains 15 mL of CTAB precipitation buffer (1% CTAB, 50 mM Tris HCl, pH 8.0, 10 mM EDTA , pH 8.0). Mix the tubes gently by inversion, and let stand at RT for 50-70 minut es. Centrifuge for 9-11 minutes at 16,000 ×

g and 20-25 ° C to pellet the DNA. Discard the supe rnatant. Add 2 mL of high salt TE buffer (10 mM Tris HCl, pH 8.0, 1 mM ED TA, pH 8.0, 1M NaCl) and incubate at 35-60 ο C with gentle shaking until the pellet goes in to solution. Pr ecipitate the DNA by adding 1/10 the volume of 3 M sodium acetate, pH 5.2, an d two times the volume of 100% ethanol. Mix by inversion. Remove the DNA using a pipette tip, inoculat ion loop, or closed Pasteur pipette. Place the DNA in a clean 1.5 mL microcentrifuge tube containing 0.5-1.0 mL of 70% (v/v) ethanol, microcentrifuge fo r 5 minutes at maximum speed to pellet the DNA, and discard the supernatant. Dry the DNA pellet by vacuum drying for ≤ 10 minutes or by air drying for ≤ 2 hours. Resuspend the DNA pellet in 500-1000 μL of TE buffer (10 mM Tris HCl, pH 8.0, 1 mM EDTA, pH 8.0). To facilitate resusp ension of the DNA, additional TE buffer may be added and/or the solution may be heated up to 70 ° C for 1-4

hours. Store the DNA in a 4 ° C refrigerator or -20 ° C or -80 ° C freezer.

A.4. Quantification of Genomic DNA Quantification of DNA samples was perf ormed using a Hoefer DyNA Quant 200

Fluorometer with Roche molecular size ma rker IX as a DNA calibration standard.

A.5. Restriction Enzyme Di gestion of Genomic DNA Approximately 10 or 20 μ g of genomic DNA extracted from the test and control substances were used for restriction enzyme diges tions. When digesting genomic DNA with Hin d III (Roche) or Eco R V (Roche), 10X Buffer B (Roche) wa s used. When digesting genomic DNA with the enzyme combination Eco O109 I (New England BioLabs, Beverly, MA)

and Not I (Roche), NEbuffer 4 (New England BioLabs) was used. Finally, 100× BSA (New England BioLabs) was added to the Eco O109 I/ Not I digests to a final

concentration of 1×. All di gests were performed at 37 ° C in a total volume of approximately 500 μ l using ~100 units of the appropria te restriction enzyme(s).

A.6. DNA Probe Preparation for Southern Blot Analyses Probe template DNA containing sequences of plasmid PV-ZMAP595 was prepared by PCR amplification using a standard pr ocedure based on Sambrook and Russell (Sambrook and Russell 2001). Approximately 25 ng of each probe template were

radiolabeled with either 32 P-deoxycytidine triphosphate (dCTP) or 32 P-deoxyadenosine triphosphate (dATP) (6000 Ci/mmol) usi ng the random priming method (RadPrime DNA Labeling System, Invitrogen) or PCR method. Probe locations relati ve to the genetic elements in plasmid PV-ZMAP595 are depi cted in Figure IV-6 and Figure IV-7.

A.7. Southern Blot Analyses of Genomic DNA Digested DNA was separated us ing 0.8% (w/v) agarose gel electrophoresis. Except for generational stability analyses, DNA samples were loaded on th e gels for a long run and a short run in an effort to provide better reso lution of larger DNA fr agments while retaining smaller DNA fragments on the gel. Afte r transferring the DNA to the membrane, Southern blots were hybridized at 55 ° C, 60 ° C, or 65 ° C. The table below lists the temperature and radiolabeling conditions of the probes used in this study. MultipleMonsanto Company 07-CR-191U Page 274 of 544 exposures of each blot were then generated using Kodak Biomax MS film in conjunction

with one Kodak Biomax MS in tensifying screen in a -80 ° C freezer.

Probe DNA Probe Labeling

Method Probe labeled

with dNTP ( 32 P) Hybridization Temperature ( ° C)

1 T-DNA Probe 1 RadPrime dCTP 65

2 T-DNA Probe 2 RadPrime dCTP 65

3 T-DNA Probe 3 RadPrime dCTP 65

4 Backbone Probe 1 RadPrime dCTP 65

5 Backbone Probe 2 RadPrime dCTP 65

6 Backbone Probe 3 RadPrime dCTP 65

7 P- Ract1 Probe RadPrime dCTP 65

8 I- Ract1 Probe PCR dCTP 65

9 CS- cspB Probe PCR dATP 60

10 T- tr7 Probe PCR dATP 55

11 loxP + P- 35S Probe RadPrime dCTP 60

12 CS- nptII Probe RadPrime dCTP 65

13 T- nos + loxP + Left Border Probe RadPrime dATP 60

dNTP = deoxyribonucleotide triphosphate

A.8. DNA Sequence Analyses of the MON 87460 Insert Overlapping PCR products were generated th at span the insert in MON 87460. These products were sequenced to determine th e nucleotide sequence of the insert in MON 87460 as well as the nucleotide sequence of the genomic DNA flanking the 5' and

3' ends of the insert.

The PCR analyses were conducted using a pproximately 75 ng of genomic DNA template or approximately 10 ng of plasmid DNA in a 50 μ l reaction volume containing a final concentration of 2 mM MgCl 2 , 0.2 μ M of each primer, 0.2 mM each deoxyribonucleotide triphosphate (dNTP), and 1 unit of DNA polymerase mix. The DNA polymerase mix used to generate the products was Platinum Taq (Invitrogen) or Platinum High Fidelity Taq (Invitrogen).

Aliquots of each PCR reaction were sepa rated on a 1.0 % (w/v) agarose gel and visualized by ethidium bromide staining to ve rify that the products were of the expected size prior to sequencing. The PCR product wa s sequenced with the multiple primers used for PCR amplification. In addition, primers internal to the PCR primers were used to sequence other regions of the amplified produc t. All sequencing was performed by the Monsanto Genomics Sequencing Center using dye-terminator chemistry.

A.9. PCR and DNA Sequence Analyses of the Parental Corn Genome To demonstrate that the DNA sequences flanki ng the insert in MON 87460 are native to the corn genome, PCR analysis was pe rformed on genomic DNA from both MON 87460 and conventional corn. The primers used in this analysis were designed from the DNA sequences flanking the insert in MON 87460. One primer designed from the genomic DNA sequence flanking the 5' end of the insert was paired with a second primer located in the genomic DNA sequence flanking the 3' end of the insert.Monsanto Company 07-CR-191U Page 275 of 544 The PCR analyses were conducted using a pproximately 75 ng of genomic DNA template

in a 50 μ l reaction volume containing a fina l concentration of 2 mM MgCl 2 , 0.2 μ M of each primer, 0.1 mM each dNTP, and 1 unit of Platinum Taq DNA polymerase High Fidelty (Invitrogen).

Aliquots of each PCR reaction were separated on a 1.0% (w/v) agarose gel and visualized by ethidium bromide staining to verify that the product was of the expected size prior to sequencing. The PCR product was sequen ced with the primers used for PCR amplification. All sequencing was perfor med by the Monsanto Genomics Sequencing Center using dye-terminator chemistry.

A.10. References:

Rogers, S. O. and A. J. Bendich. 1985. Ex traction of DNA from milligram amounts of fresh, herbarium and mummified plant tissue. Plant Molecular Biology. 5:69-76.

Sambrook, J. and D. Russell. 2001. Chapter 5 Protocol 1: Agarose gel electrophoresis. Pages 5.4 to 5.13 in Molecular cl oning: a laboratory manual. 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Monsanto Company 07-CR-191U Page 276 of 544 Appendix B. Materials, Methods, and Result s for Characterization of CSPB Protein

The expression levels of CSPB protein in di fferent tissues of MON 87460 are relatively low. Therefore, it was necessary to produce the protein in a high-expressing, recombinant microorganism in order to obtai n sufficient quantities of the protein for safety studies. A recombinant CSPB protein was produced in Escherichia coli , the sequence of which was engineered to ma tch that of CSPB protein produced in MON 87460. The equivalence of the physicochemical char acteristics and functional activity between the MON 87460-produced and E. coli -produced CSPB protein was confirmed by a panel of analytical tech niques, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), glycosylation analysis, and a ssay of biological act ivity. The details of the materials, methods, and results are described below.

B.1. Materials and Methods

B.1.1. Protein Purification

The MON 87460-produced CSPB protein was pur ified from the grain of MON 87460. The CSPB protein was purified at ~4 °C from an extract of ground grain using a

combination of ammonium sulfate fract ionation, anion exchange chromatography, immunoaffinity chromatography, and size exclusion chromatography. Protein purification records are ar chived at Monsanto Comp any under Orion lot 10000842, and the purification methods are described below.

The ground grain (10 kg) was mixed with a Tr is-borate extraction buffer (89 mM TrisBorate, 2 mM EDTA, pH 8.3) for 17 h at appr oximately a 1:10 sample weight to buffer volume ratio. To remove lipids, diatomaceous earth (Advanced Minerals Corp, Goleta, CA) was added to a final concentration of 7.5% (w/v) and mixed for 3 hours. The final slurry was filtered using an Ertel Alsop filter press (Kingston, NY) with Die 42 micro media filter pads and a Cuno filter (45115-12- 90S, Hagedorn & Gannon Co., Inc). The filtrate was concentrated by diafiltration ut ilizing a polysulfone hollow fiber cartridge with a 3 kDa Molecular Weight Cu t Off (MWCO) (surface area: 3.25 m 2 , GE Healthcare, Piscataway, NJ). An ammoni um sulfate precipit ate was prepared by the addition of

ammonium sulfate salt to the clarified extract to a final saturation of 40% and was allowed to dissolve overnight at 4 °C. Afte r centrifugation, the am monium sulfate pellet was discarded and the supernatant collected and diafiltered against 20 mM Tris-HCl pH 7.0, resulting in a final volume of 14 L.

The sample was loaded onto a 4.4 L (14 cm x 20 cm) Q Sepharose Fast Flow anion exchange resin column (GE H ealthcare, Piscataway, NJ ), which was equilibrated with AEC buffer A (20 mM Tris-HCl , pH 7.0). The bound CSPB was eluted with a linear salt gradient that increased from 0 M to 0.5 M sodi um chloride (in AEC buffer A) over 44 L. Fractions containing MON 87460-produced CSPB protein were identified by western blot analysis and totaled 9.2 L. These fr actions were pooled and concentrated using

diafiltration to a fina l volume of ~0.6 L.Monsanto Company 07-CR-191U Page 277 of 544 The concentrated sample containing MON 87460-produced CSPB protein was recirculated over two AminoLink (Pierce, Rockford, IL) columns (2.4 ml: 1.6x1.2 cm; 4.4 ml: 1.6x2.2 cm) to which a monoclonal anti-C SPB antibody (Leinco Technologies Inc, St Louis, MO) had been conjugated. Bound CSPB protein was el uted using 100 mM triethylamine buffer and neutralized with 1/20 th volume of 1 M sodium phosphate, pH 6.8. The process was repeated a total of 19 tim es to capture and elute most of the CSPB protein present in the concentr ated AEC pool. After analysis of fractions by western blot, those containing CSPB protein were combined to a final volume of 205 ml. The pool was concentrated by diafiltration to approximate ly 27 ml and then divided into three 9 ml samples that were further purified by size exclusion chromatography on a 320 ml, 60 cm bed height, Sephacryl S-100 HR column (GE H ealthcare, Piscataway, NJ) equilibrated in 20 mM Tris-HCL pH 7.0. The fractions co ntaining CSPB protein were identified by western blot and a final pool of ~105 ml was concentrated by diafil tration with a mini cartridge to approximately 12 ml. Further concentration to 0.5 ml was accomplished by placing the solution into a slide-A-lyzer dial ysis cassette (MWCO: 3.5 kDa, size: 0.5-3 ml, Pierce, Rockford, IL) and removing the excess of solvent (~11.5 ml) by exposure to a water absorbing polymer powder (Aquacide I, EMD, Gibbstown, NJ). The concentrated pool of MON 87460-produced protein was submitte d to the Analytical Protein Standard (APS) program as 23 aliquots that were assigned APS lot 1000842.

B.1.2. Protein Concentration

The concentration of the MON 87460-produced CSPB protein was estimated using quantitative densitometric analysis of silver stained SDS-PAGE. The E. coli -produced CSPB protein (amounts ranging fr om 10 to 60 ng) was used to create a standard curve. Aliquots of the MON 87460-produced CSPB protei n and reference sta ndard were diluted in 20 mM Tris-HCl pH 7.0 and 5 × Laemmli buffer (5 × LB), heated at 100.3 °C for 3 min, and applied to a pre-cast tricine 10 - 20% polyacrylamide grad ient 18-well gel. A 50-fold diluted MON 87460-produced pr otein solution was mixed with 5 × LB and three different amounts were loaded in duplicate on the gel. Electrophores is was performed at a constant voltage of 200 V for 45 min. Pre-stained molecular weight markers (Invitrogen SeeBlue Plus2, Carlsbad, CA) were loaded in parallel.

The gel was stained using the Owl Silver Stai ning Kit Protocol (Owl Separation Systems, Portsmouth, NH). The following steps were pe rformed during the gel staining procedure:

1. Fixing for 10 min in 150 ml of fixing so lution (60 ml deionized water, 75 ml methanol, and 15 ml acetic acid);

2. 15 min incubation in 150 ml of a second fixing solu tion (82.5 ml deionized water, 45 ml methanol, 15 ml acetic acid and 7.5 ml Reagent Bottle 1);

3. 10 min incubation in 150 ml of a pretreatment solution (75 ml methanol, 7.5 ml Pretreatment Reagent, and 67.5 ml deionized water);

4. Washing for 5 min with 150 ml of deionized water;

5. 15 min staining in 150 ml of the silv er staining soluti on (7.5 ml Staining Solution A, 7.5 ml Staining Solution B, and 135 ml deionized water);

6. Washing three times for 2 min each with 150 ml of deionized water;Monsanto Company 07-CR-191U Page 278 of 544 7. Development of stained protein band s occurred in 150 ml of developer solution (7.5 ml Concentrated Devel oper and 142.5 ml deionized water) for 8 min, and was stopped by addition of 7.5 ml stopping solution for 10 min;

8. The gel was washed three times for 2 min each with 150 ml of deionized water and the gel was then changed to 50 ml of a 20% ethanol solution.

Analysis of the gel was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA) using the lane finding and contour tool. The raw data were exported to a Microsoft Excel (version 2002, SP3) file

for the construction of the calibration curve and the final concentration determination of the MON 87460-produced CSPB concentration.

B.1.3. Immunoblot Analysis

Immunoblot analysis was performed to confirm the identity of the CSPB protein purified from MON 87460 and to compare the immunoreactivity of the MON 87460- and E coli

produced CSPB proteins.

The MON 87460- and E. coli -produced CSPB proteins were loaded onto the same gel at equal amounts of 3, 6, and 9 ng. Aliquots of each protein we re diluted in 20 mM TrisHCl pH 7.0 and mixed with 5 × LB, heated at 100 °C for 3 min, and applied to a pre-cast tricine 10 - 20% polyacrylamide gradient 15-we ll gel. The three amounts of each protein were loaded in duplicate on the gel. Electr ophoresis was performed at a constant voltage

of 170 V for 70 min. Pre-stained molecular weight markers (SeeBlue Plus2 Prestained, Invitrogen, Carlsbad, CA) were lo aded in parallel to verify electrotransfer of protein to

the membrane and estimate the size of the immunoreactive bands observed.

Electrotransfer to a 0.45 μ m nitrocellulose membrane (In vitrogen, Carlsbad, CA) was performed for 90 min at a constant voltage of 35 V.

The membrane was blocked for 1 hour with 5% (w/v) non-fat dr y milk (NFDM) in

1 × phosphate-buffered saline containing Tween -20 (PBST). The membrane was probed

with a 1:1000 dilution of goat anti-CSPB antibody (Orion lot 10000798, aliquot # 101) in 5% (w/v) NFDM in PBST for 14 hr. Excess antibody was removed using three 10 min washes with PBST. Finally, the membrane was probed with horseradish peroxidaseconjugated rabbit anti-goat IgG (Sigma, St. Louis, MO) at a dilution of 1:10000 in 5% (w/v) NFDM in PBST for 60 min. Excess HRP-conjugate was removed using three 10

min washes with PBST. All incubations were performed at room temperature, except for the primary antibody which was incubate d at 4°C. Immunoreactive bands were visualized using the ECL detection system (Amersham Biosciences, Piscataway, NJ) and exposed (10, 30, and 60 s) to BioMax XAR film (Eastman Kodak, Rochester, NY). Films were developed using a Konica SR X-101A automated film processor (Tokyo, Japan).

The immunoreactive bands of the MON 8746 0-produced CSPB protein in each lane migrating to the same level as the refere nce standard protein were quantitated and compared to the signals co rresponding to the CSPB refe rence standard protein. Quantitation of the blot was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA) using the lane finding and contour tool. The raw data were exported to a Microsoft Excel (version 2002, SP3) fileMonsanto Company 07-CR-191U Page 279 of 544 for the pair wise comparison of all the lo ads. An average percent difference was calculated for each comparison to determine the immunoreactivity equivalence.

B.1.4. N-Terminal Sequencing

N-terminal sequencing by Edman degradation wa s used to confirm the identity of the MON 87460-produced CSPB and to determine if the N-terminal methionine was present in the protein.

Protein Blot for N-Terminal Analysis An aliquot of MON 87460-produc ed CSPB was removed from storage, diluted with 20 mM Tris-HCl pH 7.0 and mixed with 5 × LB to a final concentration of 10 ng/μl, heated

at 100.3 ° C for 3 min, and loaded in duplicate at 250 ng per lane onto a tricine 10-20% gradient polyacrylamide 10-well gel. Pre-st ained molecular weight markers (SeeBlue Plus2 Prestained, Invitrogen, Carlsbad, CA ) were loaded in parallel to verify electrotransfer of protein to the membrane and estimate the size of the stained bands observed. Electrophoresis was performed at a constant voltage of 170 V for 70 min.

Electrotransfer to a 0.45 μ m PVDF membrane (Invitrogen, Carlsbad, CA) was performed for 90 min at a constant voltage of 25 V. The blot was stained with Ponceau S (Sigma, St. Louis, MO) to visualize the markers and the CSPB protein.

N-Terminal Sequencing The bands corresponding to MON 87460-produced CSPB protein were excised from the blot and N-terminal sequence analysis wa s performed for 15 cycl es using automated Edman degradation chemistry (Hunkapiller and Hood, 1983). An Applied Biosystems 494 Procise Sequencing System with 140C Mi crogradient system and 785 Programmable

Absorbance Detector and Procise TM Control Software (ver sion 1.1a) were used. Chromatographic data were collected using Atlas 99 software (version 3.59a, LabSystems, Altrincham, Cheshire, England). A pheny lthiohydantoin (PTH)-ami no acid standard mixture (Applied Biosystems, Foster Cit y, CA) was used to chromatographically calibrate the instrument for each analysis. This mixture served to verify system suitability criteria such as percent peak resolution and relative amino acid chromatographic retention times. A control protein (10 pmole β -lactoglobulin, Applied Biosystems) was analyzed before and after th e analysis of the two CSPB protein bands that were analyzed as a single sample, to verify that the seque ncer met performance criteria for repetitive yield and sequence identity.

B.1.5. MALDI-TOF Mass Analysis

MALDI-TOF mass spectrometry was used to confirm the molecular weight of the MON 87460-produced CSPB. Since the protein was determined to be very pure (97%) prior to this analysis, it wa s not deemed necessary to separate the protein by SDS-PAGE.

An aliquot of the MON 87460-produced CSPB pr otein was diluted ten-fold to a final concentration of ~12 μg/ml. Mass spectral an alyses were performed as follows. Mass

calibration of the instrument was performed using an extern al peptide mixture from a Sequazyme Peptide Mass Standards kit (Applied Biosystems). Samples (0.3 μ l) from each of the tryptic samples we re co-crystallized with 0.75 μ l sinapinic acid on the analysis plate. The sample was analyzed in the 1000 to 25000 Da range using 200 shots at a laser intensity setting of 2603 (a unit-le ss MALDI-TOF instrument specific value).Monsanto Company 07-CR-191U Page 280 of 544 Protonated (MH+) peptide masses were obs erved monoisotopically in linear mode (Aebersold, 1993; Billeci and Stults, 1993). GPMAW32 software (Applied Biosystems, version 4.23) was used to generate a theo retical mass of the expected CSPB (plant) protein sequence based upon the nucleotide sequ ence. Peaks were not assessed if the peak heights were less than approximately tw ice the baseline noise, or when a mass could not be assigned due to overlap with a stronger signal ± 2 Da from the mass analyzed.

MALDI-TOF Tryptic Mass Map Analysis MALDI-TOF mass spectrometry was used to confirm the identity of the MON 87460- produced CSPB protein. Since the protein was determined to be very pure (97%) prior to this analysis, it was not deemed necessary to separate the protein by SDS-PAGE.

An aliquot of the MON 87460-produced CSPB pr otein was diluted ten-fold to a final concentration of ~12 μg/ml. A 30 μl sample was transferred to a micro vial tube and evaporated to dryness in a Speed-Vac concentrat or. The sample was digested for 16 hr at 37 °C with 660 ng of trypsin (Promega, Madi son, WI) in 20 μl of a 25 mM ammonium

bicarbonate buffer.

Ten μl of the trypsin digested sample was transferred to a separate micro vial for guanidination of the peptid es using the ProteoMass TM guanidination kit (Sigma, St. Louis, MO). To the tube, 10 μl of guanidination reagent (O-methylisourea hemisulfate) solution and 10 μl of base (2.85 M NH 4 OH) were added and the tube was vortexed. The tube was incubated at 65 °C for 30 min, then 10 μl of stop solution (10% trifluoroacetic acid (TFA)) was added.

Mass spectral analyses were performed as follows. Mass calibration of the instrument was performed using an external peptid e mixture from a Sequazyme Peptide Mass Standards kit (Applied Biosystems). Samples (0.3 μ L) from each of the trypsin digested samples were co-crystallized with 0.75 μ L α -cyano-4-hydroxycinnamic acid on the analysis plate. The sample was analyzed in the 500 to 5000 Da range using 100 shots at a laser intensity setting of 1783 and 2175 (a unit-less MALDI-TOF instrument specific value). Protonated (MH+) peptide masses we re observed monoisotopically in reflector mode (Aebersold, 1993; Billeci and St ults, 1993). GPMAW32 software (Applied Biosystems, version 4.23) was used to gene rate a theoretical trypsin digest of the expected MON 87460-produced CSPB protei n sequence based upon the nucleotide sequence. Masses were calculated for each theoretical peptide and compared to the raw mass data. Experimental masses (MH+) were assigned to peaks in the 500 to 1000 Da range if there were two or mo re isotopically resolved peak s, and in the 1000 to 5000 Da range if there were three or more isotopically resolved peaks in the spectra. Peaks were not assessed if the peak heights were less th an approximately twice the baseline noise, or when a mass could not be assigned due to overlap with a stronger signal ± 2 Da from the mass analyzed. Known autocatalytic fragment s from trypsin digestion were identified in the raw data. The tryptic mass map c overage was considered acceptable if ≥ 40 % of the protein sequence was identified by matc hing experimental masses observed for the tryptic peptide fragments to the e xpected masses for the fragments.Monsanto Company 07-CR-191U Page 281 of 544 B.1.6. Molecular Weight and Puri ty Estimation – SDS-PAGE

Aliquots of the E. coli -produced reference standard and MON 87460-produced CSPB proteins were diluted with 20 mM Tris-HCl, pH 7.0 and mixed with 5 × LB to a final protein concentration of 10 ng/ μ l. The MON 87460-produced protein was analyzed in duplicate at 50, 100, and 150 ng of to tal protein per lane. The E. coli -produced CSPB protein reference standard was analyzed at 100 ng of purity corrected full-length protein. All samples were heated in a thermo-block at 100.3 ° C for 3 min and applied to a pre-cast tricine 10-20% polyacrylamide gradient 10-we ll mini-gel (Invitrogen, Carlsbad, CA). Pre-stained molecular weight markers (Invi trogen SeeBlue Plus2, Carlsbad, CA) were loaded in parallel. Electrophor esis was performed at a cons tant voltage of 170 V for 70

min.

The gel was stained using the Owl Silver Stai ning Kit Protocol (Owl Separation Systems, Portsmouth, NH). The same procedure desc ribed previously in this appendix was followed, though the solutions were proportionately adjusted to a final volume of 50 ml. Also, the development of protein bands occurred during incubation of the gel in the developer solution.

Analysis of the gel was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). The molecular weight markers were used to estimate the appa rent molecular wei ght of the MON 87460- produced CSPB protein. For the purity evalua tion, all visible bands within each lane were quantified. The purity and estimat ed molecular weight of the MON 87460- produced CSPB protein were reported as th e average of the si x values obtained by densitometric analysis.

B.1.7. Glycosylation Analysis

Glycosylation analysis was used to determine whether the MON 87460-produced CSPB protein was post-translationally modified w ith covalently bound carbohydrate moieties. Aliquots of the MON 87460-produced CSPB protein, the E. coli -produced CSPBreference standard protein, and the positive controls, transferrin (~ 76 – 81kDa, SigmaAldrich, St. Louis, MO), and horseradish pe roxidase (~ 40kDa, Pierce, Rockford, IL) were each diluted with 20 mM Tris-HCl pH 7.0 and mixed with 5 × LB. These samples were heated at 100 °C for 3 min, cooled, an d loaded on a tricine 10-20% polyacrylamide gradient 10-well mini-gel. Each sample was loaded at 25 and 50 ng per lane. SeeBlue Plus2 pre-stained protein mol ecular weight markers (Invitrogen) were loaded to verify electrotransfer of the proteins to the membrane and the CandyCaneTM Glycoprotein Molecular Weight Standards (Molecular Probes, Eugene, OR) were loaded as positive/negative controls and markers for molecular weight. Electrophoresis was performed at a constant voltage of 170 V for 60 min. Elect rotransfer to a 0.45 μ m PVDF membrane (Invitrogen) was performed for 90 min at a constant voltage of 35 V.

Carbohydrate detection was performed direct ly on the PVDF membrane using the ProQ® Emerald 488 Glycoprotein Gel and Blot Stain Kit (Molecular Probes). The manufacturer’s protocol was followed. All steps were pe rformed at room temperature.

The PVDF membrane was fixed in 25 ml of a solution containing 50% methanol and 5% glacial acetic acid for 45 min, the solution was then changed and the membrane was incubated overnight. Two, 10 min washes (50 ml each) with 3% (v/v) glacial acetic acidMonsanto Company 07-CR-191U Page 282 of 544 (wash solution) were followed by a 20 min oxida tion in 25 ml of an oxidizing solution containing periodic acid (Component C from k it). Membrane was washed three times, 10 min each, in 50 ml of wash solution. The bl ot was then incubated in 25 ml of Pro-Q Emerald Staining Solution that wa s prepared using the kit reagen ts. After 1 hr of staining in the dark, two 20 min, 50 ml wash cycles were followed by two 30 min, 50 ml wash cycles. The final wash cycles included two 50 ml, 1 min deionized water washes followed by three 5 min methanol washes(EMD , San Diego, CA). The blot was then scanned using the BioRad Molecular Imager FX using the Alexa 488 illumination setting (Quantity One software; version 4.6, build 036) in order to visual ize the fluorescentlylabeled glycosylated proteins.

After glycosylation analysis the blot was stai ned to visualize the proteins present on the membrane. Proteins were stained using the SYPRO ® Ruby Protein Blot Stain (Molecular Probes). Sections 2.4 to 2.6 of the manufactur er’s instructions were followed and all steps were performed at room temperature a nd incubations were done on a shaking table. The blot used for glycosylation was stained in 10 ml of the SYPRO staining solution for

15 min. The solution was discarded and the blot was washed twice for 5 min in 50 ml of

deionized water. The blot was stored in 25 ml of deionized water. The blot was then scanned using the BioRad Molecular Imag er FX using the SYPRO Ruby illumination setting (Quantity One software; version 4.6, build 036) in order to visualize the fluorescently-labeled proteins.

B.1.8. Functional Activity Assay

In order to assess the functi onal activity of the MON 8746 0-produced CSPB protein and to compare its activity to the E. coli -produced CSPB reference standard protein, aliquots of the MON 87460-produced CSPB protein and E. coli -produced CSPB reference standard protein were analyzed for thei r ability to unfold polynucleotide hairpin structures. Activity is expressed as the am ount of DLP that is unfolded by CSPB. The probe consists of a custom synthesized 35- base oligonucleotide DNA fragment with a 6-

FAM fluorescent label at the 5’ end and a black hole quencher at the 3’ end. The oligonucleotide probe forms a double strand stem of six base pairs due to complementary

bases located at the 5’ and 3’ ends. The 23 nucleotides (dT) in the middle form a loop. and the binding of CSPB to the loop will se parate the double strands of the probe, thus separating the fluorophore from the quencher, allowing fluorescence to be emitted and

measured.

The assay was carried out on a micro titer plate. A calibr ation curve using the 6-FAM

was constructed from serial dilutions of a 100 nM stock solution of the 6-FAM. The dilutions were done in Assay buffer (25 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.5) and the final concentrations of the 6-FAM were 0.234, 0.468, 0.938, 1.875, 3.75, 7.50, and 15.00 pmoles/well. The sample wells were prepared by adding 175 μl of a reagent solution containing 0.34 μM DLP in the assay buffer. The plate was incubated at 30.1 °C for 30 min. Then, 25 μl of dilutions of each MON 87460- and E.coli -produced CSPB protein (3 μg total CSPB), in triplicate, were added to the test wells and the plate was incubated at 30 °C in a SpectraMax M2 plate reader (Molecula r Devices, Sunnyvale, CA). The fluorescence was dete rmined with an excitation wa velength of 485 nm and an emission wavelength of 520 nm using a template created within the SpectraMax Pro GxP software (version 5.0.1).Monsanto Company 07-CR-191U Page 283 of 544 B.1.9. Storage Stability

The short-term stability of the MON 87460-pr oduced CSPB protein during storage in a

freezer set to maintain -80 ° C was evaluated by compari ng the purity and molecular weight values obtained on day 0 to the purit y and molecular weight values obtained on day 14 of storage. Day 0 stability analys is corresponds to the purity and molecular weight determination. On day 14, an aliquot was removed from a -80 ° C freezer, diluted with 20 mM Tris-HCl pH 7.0 and mixed with 5 × LB to a final concentration of 10 ng/ μ l,

heated at 100 ° C for 3 min, and loaded in duplicat e (50, 100, and 150 ng per lane) onto a tricine 10 - 20% gradient polyacrylamide 10-well gel. Staining and densitometricanalysis were performed as described for Molecular Weight and Purity Estimation-SDSPAGE. The protein samples were considered to have undergone degradation if a > 10% decrease in purity and/or molecular weight was observed relative to the value determined on Day 0.

B.2. Results

B.2.1. CSPB Protein N-terminal Sequence Analysis

Sequencing of the first 15 amino acids co mprising the N-terminal of MON 87460- produced CSPB protein produced the expected result (Table B-1). The N-terminal

methionine was not detected. This result is expected as remova l of the N-terminal methionine, catalyzed by methionine aminope ptidase, is a common modification that occurs co-translationally before completion of the nascen t protein chain and has no effect on protein structure or activity (Arfin and Bradshaw, 1988; Polevoda and Sherman, 2000). The N-terminal sequence information, th erefore, confirms th e identity of the CSPB protein isolated fro m MON 87460 and the intactness of its N-terminus.

B.2.2. CSPB Protein MALDI-TOF Mass Spectrometry Analysis

The identity of the MON 87460-produced CSPB protein was further confirmed by tryptic peptide mass mapping analysis using MALDI-TOF MS. Protein identification made by peptide mapping is reliable if the measured coverage of the sequenc e is 15% or higher with a minimum of five matched peptides (Jensen et al., 1997). Observed tryptic peptides were considered a match to the expected tryptic mass when differences in molecular weight of less than one Dalton (Da) were found between the observed and predicted fragment masses. Such matches were made wit hout consideration for potential natural amino acid modifications such as gl ycosylation. The prot ein sample was heatdenatured, chemically reduced, alkylated, di gested with trypsin, guanidinated, and the masses of the tryptic peptides were measured.

CSPB is a small protein with a limited numbe r of the trypsin-digested peptides that are amenable to identification by MALDI-TOF. There were four unique peptide fragments identified that matched expected masses of the CSPB trypsin-dige sted peptides. The

identified masses were used to assemble a coverage map indicating the matched peptide sequences for the entire CSPB protein (Fig ure B-1), resulting in an 88% (58 out of 66 amino acids) coverage of the total protein. This analysis confirmed the identity of the MON 87460-produced CSPB protein. Table B- 2 presents the tryptic masses of CSPB.Monsanto Company 07-CR-191U Page 284 of 544 B.2.3. CSPB Protein Immunoreactivity

A western blot analysis using goat anti-C SPB serum was conducted to determine the relative immunoreactivity of the M ON 87460-produced CSP protein and the E. coli

produced CSPB reference standard. The results demonstrated that the anti-CSPB antibody recognized the MON 8746 0-produced CSPB that migr ated identically to the E.

coli -produced reference standard protein (Fi gure B-2). Furthermore, the immunoreactive signal increased with increasing levels of CSPB loading. Immunoreactivities between the

MON 87460- and E. coli -produced proteins were similar based on densitometric analysis

of the western blot. Based on the analysis, the MON 87460- and E. coli -produced CSPB proteins demonstrated e quivalent immunoreactive properties, which confirmed the identity and equivalence of the two proteins.

Table B-1. N-terminal Amino Acid Sequence Analysis of the CSPB Protein Purified

from Grain Tissue of MON 87460

Amino acid 1

residue # from the

N-terminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Predicted CSPB Sequence 2 → M V E G K V K W F N S E K G F G

⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐ ⏐

Observed Sequence → - V E G K V K W F N S E K G F G

1 The single letter amino acid code is: E, Glutamic acid; F, Phenylalanine; G, Glycine; K, Lysine; M, Methionine; N, Asparagine, S, Serine; V valine and W, Tryptophan.

2 The predicted amino acid sequence of the CSPB pr otein was deduced from the coding region of the full length cspB gene present in MON 87460.Monsanto Company 07-CR-191U Page 285 of 544Table B-2. Summary of the Tryptic Masses Identified forthe MON 87460-Produced CSPBProtein Using MALDI-TOF MassSpectrometryOnly experimental masses that matched expected masses are listed in the table.Observed Mass (Da)ExpectedMass(Da)MassDifference(Da)AA Position Fragment SequenceCrudeSampleGuanidinatedSample810.42 - 810.38 0.04 7-12WFNSEK- 852.51 852.38 0.13 7-12GWFNSEK885.54 - 885.48 0.06 56-64GPQAANVTK- 927.64 927.48 0.16 56-64GGPQAANVTK1878.11 - 1877.92 0.19 39-55TLEEGQAVSFEIVEGNR- 2903.32 2903.36 0.04 13-38GGFGFIEVEGQDDVFVHFSAIQGEGFKMonsanto Company 07-CR-191U Page 286 of 544 0001 VEGKVKWFNS EKGFGFIEVE GQDDVFVHFS AIQGEGFKTL EEGQAVSFEI

0051 VEGNRGPQAA NVTKEA

Figure B-1. MALDI-TOF MS Coverage Map of the CSPB Protein Isolated from

MON 87460

The amino acid sequence of the plant-produ ced CSPB protein was deduced from the coding region of the full-length cspB gene present in MON 87460. Boxed regions correspond to tryptic peptide masses that were identified from the protein sample using MALDI-TOF MS. In total, 88% (58 of 66 to tal amino acids) of the expected protein sequence were identified.Monsanto Company 07-CR-191U Page 287 of 544 Figure B-2. Western Blot An alysis of MON 87460- and E. coli -produced CSPB

Proteins

Aliquots of the purified, MON 87460- and E. coli -produced CSPB proteins were separated by SDS-PAGE, and electrotransferred to a polyv inylidene difluoride (PVDF) membrane. The membrane was probed with goat anti-CSPB serum and developed using an enhanced chemiluminescense (ECL) system (GE Healthcare). Approximate molecular weights (kDa) of markers loaded in Lane 1 are shown on the left side of the blot.

Lane Sample Amount Loaded (ng)

1 See Blue® Plus2 Pre-Staine d molecular weight markers

⎯ 2 E. coli -produced CSPB reference standard 3

3 E. coli -produced CSPB reference standard 3

4 E. coli -produced CSPB reference standard 6

5 E. coli -produced CSPB reference standard 6

6 E. coli -produced CSPB reference standard 9

7 E. coli -produced CSPB reference standard 9 8 MON 87460-produced CSPB protein 3 9 MON 87460-produced CSPB protein 3 10 MON 87460-produced CSPB protein 6 11 MON 87460-produced CSPB protein 6 12 MON 87460-produced CSPB protein 9 13 MON 87460-produced CSPB protein 9 210

105

78

55

45

34

17
16

7

4 1 2 3 4 5 6 7 8 9 10 11 12 13

CSPB proteinMonsanto Company 07-CR-191U Page 288 of 544 B.2.4. CSPB Protein Molecu lar Weight Equivalence

The equivalence in apparent molecular we ight of the purified MON 87460- and the E.

coli -produced CSPB proteins was demonstrat ed using SDS-PAGE (Figure B-3). The MON 87460-produced CSPB protein migrated wit h a molecular weight indistinguishable

to that of the E. coli -produced protein standard analyz ed concurrently (Figure B-3). Based on comparable electrophoretic mobilities, the MON 87460- and E. coli -produced CSPB proteins were determined to have equivalent apparent molecular weights.

The predicted mass of the MON 87460-produced CSPB protein was also confirmed by MALDI-TOF MS. The average mass obt ained for CSPB was 7220 Da. This experimentally obtained mass differs from th e theoretical mass calculated for the CSPB reference standard protein by 131 Da. The difference be tween the expected and the observed mass for MON 87460-produced CSPB co rresponds to the mass of methionine (131 Da). The absence of the N-terminal methionine was confirmed by N-terminal sequencing (Section B.2.1).

B.2.5. CSPB Protein Glycosylation Equivalence

Some eukaryotic proteins are post-translationally modified by the addition of carbohydrate moieties (Rademacher et al., 1988). These carbohydrate moieties may be complex, branched polysaccharide struct ures, simple oligosaccharides or monosaccharides. In contrast, prokaryot ic organisms such as non-virulent E. coli strains used for cloning and expression purposes, la ck the necessary biochemical synthetic capacity required for protein glycosylation. An investigation of glycosylation status therefore is necessary to confirm that the MON 87460-p roduced CSPB protein is equivalent to the E. coli -produced CSPB protein. Results of this analysis confirm that the proteins are equivalent in this respect.

To assess whether potential pos t-translational glycosylati on of the MON 87460-produced CSPB protein occurred, the purified protei n sample was subjecte d to glycosylation analysis. The E. coli -produced CSPB reference standard represented a negative control. The positive controls were the transferrin and horseradish peroxidase (HRP) proteins which are known to have multiple covalen tly-linked carbohydrate modifications. The transferrin protein and HRP, as well as the purified CSPB protein isolated from

MON 87460 and E. coli were separated on SDS-PAGE, transferred to a PVDF membrane, and glycosylation analysis was perf ormed to detect carbohydrate moieties on the proteins. The results of this analysis are shown in Figure B-4. The positive controls, transferrin and HRP, were detected at the expected molecular weights of ~75 and ~50 kDa, respectively, in a con centration-dependent manner (Fi gure B-4, Panel A, Lanes 4-5 and 2-3). No detectable signal was observed for the MON 87460- and E. coli -produced CSPB proteins (Figure B-4, Panel A, Lanes 6-7 and 8-9). To confirm that sufficient

MON 87460- and E. coli -produced CSPB proteins we re present for carbohydrate detection and glycosylati on analysis, the membrane was stained with SYPRO ® Ruby stain to detect protei ns (Figure B-4, Panel B). Both MON 87460- and E. coli -produced CSPB were clearly detected on the membrane (Figure B-4, Panel B, Lanes 6-9). These

results demonstrate that the MON 87460-produced CSPB protein is not glycosylated and, thus is equivalent to the E. coli -produced CSPB reference standard.Monsanto Company 07-CR-191U Page 289 of 544 Figure B-3. SDS-PAGE of E. coli - and MON 87460-produced CSPB Proteins

Aliquots of the MON 87460-produced CSPB and the E. coli -produced CSPB reference standard were separated by a tricine 10-20% polyacrylamide gradient gel and stained with an Owl Silver Staining kit. Approxi mate molecular weights (kDa) of markers

loaded in Lanes 1 and 18 are show n on the left side of the gel.

Lane Sample Amount Loaded (ng) ( μ l)
1 See Blue® Plus2 Pre-Stained molecular weight markers

⎯ 15

2 E. coli- produced CSPB standard 10

⎯ 3 E. coli- produced CSPB standard 10

⎯ 4 E. coli- produced CSPB standard 20

⎯ 5 E. coli- produced CSPB standard 20

⎯ 6 E. coli- produced CSPB standard 30

⎯ 7 E. coli- produced CSPB standard 30

⎯ 8 E. coli- produced CSPB standard 40

⎯ 9 E. coli- produced CSPB standard 40

⎯ 10 E. coli- produced CSPB standard 60

⎯ 11 E. coli- produced CSPB standard 60

⎯ 12 MON 87460-produced CSPB protein

⎯ 10 13 MON 87460-produced CSPB protein

⎯ 10 14 MON 87460-produced CSPB protein

⎯ 20 15 MON 87460-produced CSPB protein

⎯ 20 16 MON 87460-produced CSPB protein

⎯ 30 17 MON 87460-produced CSPB protein

⎯ 30 18 See Blue® Plus2 Pre-Stained molecular weight markers

⎯ 15 kDa 1 2 3 4 5 6 7 8 9 1 0 11 12 1 3 14 1 5 1 6 1 7 1 8

210

105

78

55

45

34

17

16

7

4 CSPB proteinMonsanto Company 07-CR-191U Page 290 of 544 Figure B.4. Glycosylation Analysis of the MON 87460-produced CSPB Protein

Aliquots of the MON 87460-produced CSPB protein, E. coli -produced CSPB reference standard (negative control), horseradish pero xidase (positive cont rol) and transferrin (positive control) were separated by SDS-PAGE (10

- 20% gradient) and electrotransferred to a PVDF membrane. ( A ) Where present, periodat e-oxidized protein-bound carbohydrate moieties reacted with Pro-Q Em erald 488 glycoprotein stain and emitted a fluorescent signal at 488 nm (Lanes 1-5). ( B ) The same blot was stained with SYPRO Ruby. The signal was captured using a Bio- Rad Molecular Imager FX. Approximate molecular weights (kDa) correspond to the See Blue® Plus2 pre-stained dual color molecular weight marker loaded in Lane 1 and CandyCane glycosylated markers loaded

in Lane 10.

Lane Sample Amount (ng)

1 See Blue® Plus2 Pre-Staine d molecular weight markers

⎯ 2 Horseradish Peroxidase (positive control) 25 3 Horseradish Peroxidase (positive control) 50 4 Transferrin (positive control) 25 5 Transferrin (positive control) 50 6 MON 87460-produced CSPB 25 7 MON 87460-produced CSPB 50

8 E. coli -produced CSPB (negative control) 25

9 E. coli -produced CSPB (negative control) 50 10 CandyCane Glycoprotein mo lecular weight standards

⎯ 1 2 3 4 5 6 7 8 9 10

180

82

42

18 k

Da k Da 1 2 3 4 5 6 7 8 9 10 k Da

210

105

78 55
45
34

17 16

7

4 210

105

78 55
45
34

17 16

7

4 180

82

42

18 66

29

14
CSPB protein 

### A BMonsanto Company 07-CR-191U Page 291 of 544 B.2.6. CSPB Protein Functional Activity Equivalence

The functional activities of the E. coli - and MON 87460-produced CSPB proteins were measured using an assay where protein unfol ds or “melts” a DNA-hairpin structure. Results confirm the two proteins exhibit similar functional activity. The DNA-hairpin

structure is labeled with a fl uorophore at the 5’- and quencher at the 3’-terminus. Due to the close proximity of the fluorescent tag an d quencher in the hair pin conformation, the fluorescence is efficiently quenched. Wh en a CSPB protein “melts” the hairpin conformation, the fluorescent tag and quencher are spatiall y separated which permits fluorescence. This assay has been broadly ut ilized to characterize the specificity of a variety of CSPs including CSD- containing proteins identifie d in bacteria and plants (Karlson et al., 2002; Kim et al., 2007; Phadtare et al., 2002).

In this assay protein specifi c activity is expressed as th e amount (pmol) of open Dual Labeled Probe (DLP) that is induced by a microgram ( μ g) of CSPB. The E. coli - and MON 87460-produced CSPB protei ns were considered functionally equivalent if the specific activity of one protein was within 25% of the other.

The DLP consists of a custom synthesized 35-base oligonucleotide DNA fragment with a fluorescein amidite derived from 6-carboxyfluor escein (6-FAM) label at the 5’ end and a black hole quencher at the 3’ end. The oligonucleotide pr obe forms a double strand stem of six base pairs due to the complementary ba ses located at the 5’ and 3’ ends. The 23 nucleotides (dT) in the middle form a loop. CSPB has been shown to have a high affinity for poly dT sequences and its bi nding to the loop w ill separate the dou ble strands of the probe, which separates the fluorophore from the quencher, allowing fluorescence to be

emitted and measured.

MON 87460-produced CSPB had a specific activity of 0.660 pmole open DLP/ μ g protein

and the E. coli -produced reference standard had a sp ecific activity of 0.757 pmole open

DLP/ μ g protein. The difference in specific activities was 12.8% (Table B-3). These results clearly demonstrate that the C SPB proteins derived from MON 87460 and E. coli have equivalent functional activities.Monsanto Company 07-CR-191U Page 292 of 544 Table B-3. CSPB Functional Assay Results Assay activity is expressed as the amount (pmo l) of open Dual Labeled Probe (DLP) that is induced by a microgram ( μ g) of CSPB. The probe consis ts of a custom synthesized 35-base oligonucleotide DNA frag ment with a 6-FAM fluorescen t label at the 5’ end and a black hole quencher at the 3’ end. Fluor escence was determined with an excitation wavelength of 485 nm and an emission wa velength of 520 nm. The amount of open probe was determined relative to the standard curve prepared from serial dilutions of 6-FAM. The opening of the DLP was measured for both proteins, using 3 μg of CSPB for each replicate. The activity represents the average of three replicates.

Specific Activity (pmoles opened DLP/μg CSPB ± S.E.) % Difference 1

(MON 87460 vs. E. coli )

CSPB

MON 87460 – CSPB E. coli – CSPB

12.8

0.660 ± 0.029 0.757 ± 0.032

1 Percent difference was calculated as follows:

Difference oli ActivityEc ant ActivityPl oli ActivityEc % 100 = × −

Note: S.E. = ܵܦ/ √ 3

B.2.7. Conclusions of the CSPB Protein Characterization

A comparison of the MON 87460-produced CSPB to the E. coli -produced CSPB reference protein standard confirmed th e identity of the MON 87460-produced CSPB protein and established the equivalence of the plant produced protein to the E. coli

produced CSPB reference protein standard. The molecular weight of the MON 87460-

and E. coli -produced CSPB proteins was estim ated by SDS-PAGE. SDS-PAGE demonstrated that the proteins migrated at the same molecular weight indicating that the CSPB proteins from both sources are equi valent in their molecular weight. The electrophoretic mobility and immunoreactiv e properties of the MON 87460-produced CSPB protein were equiva lent to those of the E. coli -produced CSPB reference standard. The N-terminus of the MON 87460-produced C SPB was consistent with the predicted amino acid sequence translated from the cspB coding sequence, and the MALDI-TOF mass spectrometry analysis yielded peptide ma sses consistent with the expected peptide

masses from the translated cspB coding sequence. Th e MON 87460- and the E. coli

produced CSPB reference standard were also equivalent based on the functional activities and the lack of glycosylation. Taken together, these data provide a detailed characterization of the CSPB protein is olated from MON 87460 and establish its equivalence to the E. coli -produced CSPB reference protein standard.Monsanto Company 07-CR-191U Page 293 of 544 B.3. References:

Aebersold, R. 1993. Mass spectrometry of proteins and peptides in biotechnology. Current Opinion in Biotechnology. 4:412-419.

Arfin, S.M. and R.A. Bradshaw. 1988. Cotransl ational processing and protein turnover in eukaryotic cells. Biochemistry-US. 27:7979-7984.

Billeci, T.M. and J.T. Stults. 1993. Tryptic mapping of recombinant proteins by matrixassisted laser desorption/ionization mass spectrometry. Analytical Chemistry.

85:1709-1716.

Hunkapiller, M.W. and L.E. Hood. 1983. Protein sequence analysis: automated microsequencing. Science. 219:650-659.

Jensen, O.L., A.V. Podtelej nikov and M. Mann. 1997. Iden tification of the components of simple protein mixtures by high-accu racy peptide mass mapping and database searching. Analytical Chemistry. 69:4741-4750.

Karlson, D., K. Nakaminami, T. Toyomasu and R. Imai. 2002. A cold-regulated nucleic acid-binding protein of winter wheat shar es a domain with bacterial cold shock proteins. Journal of Biological Chemistry. 277:35248-35256.

Kim, J.S., S.J. Park, K.J. Kwak, Y.O. Kim, J.Y. Kim, J. Song, B. Jang, C.-H. Jung and H. Kang. 2007. Cold shock domain proteins and glycine-rich RNA-binding proteins

from Arabidopsis thaliana can promote the cold adaptation process in Escherichia

coli . Nucleic Acids Research. 35:506-516.

Phadtare, S., S. Tyagi, M. Inouye and K. Severinov. 2002. Th ree amino acids in

Escherichia coli CspE surface-exposed aromatic pa tch are critical fo r nucleic acid melting activity leading to transcription an titermination and cold acclimation of cells. Journal of Biological Chemistry. 277:46706-46711.

Polevoda, B. and F. Sherman. 2000. N-term inal acetylation of eukaryotic proteins. Journal of Biological Chemistry. 275:36479-36482.

Rademacher, T.W., R.B. Parekh, and R.A. Dwek. 1988. Glycobiology. Annual Review of Biochemistry. 57:785-838.Monsanto Company 07-CR-191U Page 294 of 544 Appendix C. Materials, Methods, and Re sults for Characterization of NPTII

Protein

The NPTII protein produced in MON 87460 was characterized and its equivalence to a previously characterized E. coli -produced NPTII reference substance was demonstrated. Demonstration of the equivalence between E. coli - and MON 87460-produced NPTII proteins allows utilization of previ ous safety assessmen t data performed on E. coli produced NPTII to confirm the safety of the NPTII protein in MON 87460. The analyses employed for the characterization of MON 87460-produced NPTII protein and establishment of the equivalence between MON 87460- and E. coli -produced proteins included western blot and SDS-PAGE analyses . The details of the materials, methods,

and results are described below.

C.1. Materials and Methods

C.1.1. Preparation of Protein Extracts from Leaf Samples

Frozen leaf samples from MON 87460 and conventional corn with a genetic background

similar to that of the test material were ex tracted using phosphate saline buffer containing 0.1 % Triton X-100 (PBST) and Complete Mini (protease inhibitor) EDTA-free protein inhibitor (Roche, Indianapolis, IN) as the extraction buffer. Samples of leaf tissues (0.22 g of MON 87460 and 0.21 g of conventional co ntrol) were place in a polypropylene mesh bag from a Plant Protein Extraction kit (Pie rce, Rockford, IL) and the extraction buffer was added to the bag at a tissue to buffer ratio of 1:5 (w/v). With the open end of the bag upright and held closed, the lower portion of the bag was placed on a hard flat surface, and pressed and rubbed with the backend of a marker pen 15 times. The extracts were

transferred to 1.5 ml labeled tubes and cen trifuged for 5 min at approximately 15,000 × g at room temperature. Each supernatant was transferred to a clean, labeled 1.5 ml tube,

stored on ice and used within the day for the analysis.

To produce a spiked assay control, 5 μl of a 0.5 mg/ml NPTII protein reference standard solution was mixed with 35 μl leaf extract fr om conventional corn. The resulting final

concentration of NPTII protein was 0.0625 mg/ml.

C.1.2. SDS-PAGE and Immunoblotting

Aliquots of 40 μl of each sample were mixed with 10 μl of 5 × loading buffer (5 × LB) and heated for three min at 96.2 oC. A pre-cast tris-glycine 4-20% gr adient polyacrylamide SDS 12-well gel (Invitrogen, Carlsbad, CA) wa s loaded with the following samples:

5, 10, and 15 μl of leaf extract from MON 87460,

5, 10, and 15 μl of the spiked assay control,

10 μl of NPTII reference standard,

10 μl of leaf extract from conventional corn,

5 μl of Precision Plus Prot ein WesternC molecular weight markers in triplicate (Bio-Rad, Hercules, CA).Monsanto Company 07-CR-191U Page 295 of 544 Electrophoresis was performed at a constant voltage of 125 V for 90 min. Proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane (0.45 μm pore size, Invitrogen, Carlsbad, CA) at a constant voltage of 25 V for 90 min.

The membrane was blocked for 18 h at ~ 4 oC with 5% (w/v) NFDM in PBST. From this point on, all incubations were performed at room temperature. The membrane was probed with a 1:2000 dilution of a rabbit anti-NPTII antibodie s (Sigma. St. Louis, MO, Cat No N6412) in 1% (w/v) NFDM in PB ST for 60 min. Excess antibodies were removed using three 5 min washes with PB ST. The membrane was probed with HRPconjugated goat anti-rabbit IgG (Vector lab, Burlingame, CA) secondary antibody at a dilution of 1:5000 in 1% (w/v) NFDM in PBST for 60 min. Precision Protein StrepTactin-HRP conjugates (Bio-Rad, Herc ules, CA) were added to the secondary antibody incubation solution at a dilution of 1:50000 (a 10-fold dilution was made first with PBST, then a 1 to 5000 d ilution was made in the incuba tion solution) to visualize the position of the WesternC protein molecular weight mark ers. Excess HRP-conjugates were removed using three 5 min washes with PBST. Immunoreactive bands were visualized using the ECL detection system (GE healthcare, Piscataway, NJ) and films were exposed for 5, 10 and 20 s to Hyperf ilm ECL high performance chemiluminescence film (GE Healthcare, GE heal thcare, Piscataway, NJ). Fi lms were developed using a

Konica SRX-101A automated film processor (Tokyo, Japan).

C.1.3. Immunoblot Analysis

The 10 s exposure film was scanned using a Bio-Rad GS-800 densitometer (Hercules, CA) and used for the image analysis. The image analysis was performed using Quantity One software (Version 4.6, Bio-Rad, Hercules , CA). The apparent molecular weights of the MON 87460-produced NPTII protein and the NPTII reference standard in the spiked assay control were determined relative to th e known values of the Precision Plus Protein WesternC molecular weight markers loaded on the gel. The apparent molecular weight was calculated as the average value for all loads of each sample and the average values were compared.

C.1.4. Equivalence criteria

The equivalence of the MON 87460- and E. coli -produced NPTII proteins was established by direct comparison of th eir apparent molecular weight and immunoreactivity with NPTII sp ecific antibodies. The criteriafor these tests were preestablished during developmental work taking in to consideration the inherent variability of each analytical method. These criteria were as follows:

The immunoreactive band corresponding to the NPTII protein from the leaf extract of MON 87460 should migrate to the same position as the NPTII protein in the spiked assay

control. No immunoreactive band with the sa me mobility as the NP TII protein reference

standard should be observed in leaf extract from conventional corn.

The apparent molecular weight of the MON 87460-produced NPTII protein should be

within ± 10% of the E. coli -produced NPTII protein in the spiked assay control.Monsanto Company 07-CR-191U Page 296 of 544 C.2. Results

C.2.1. Identity and Function of the NPTII Protein

The NPTII protein functions as a selectable marker in the initia l laboratory stages of plant cell selection following tran sformation (Horsch at al., 19 84; DeBlock et al., 1984). NPTII uses adenosine triphosphate (ATP ) to phosphorylate neomycin and related aminoglycoside antibiotics, th ereby inactivating them. Ce lls that prod uce the NPTII enzyme selectively survive exposur e to these aminoglycosides. The nptII coding sequence is derived fr om the prokaryotic E. coli transposon Tn5 (Beck et al., 1982). The purpose of inserting the gene encoding the NPT II protein into corn cells along with CSPB was to have an effective method for selecting cells after transformati on. In general, the frequency of plant cells that are tran sformed is often low, ranging from 1x10 -5 to lx10 –4 of cells treated (Fraley et al., 1983) . Therefore, the selectable marker, NPTII, facilitates the screening process.

C.2.2. Characterization of the NPTII Protein

The NPTII protein produced in MON 87460 was characterized and its equivalence to a previously characterized E. coli -produced NPTII reference substance was demonstrated. Demonstration of the equivalence between E. coli - and MON 87460-produced NPTII proteins allows utilization of previous safety assessment data to confirm the safety of the NPTII protein in MON 87460. The analyses employed for the characterization of MON 87460-produced NPTII protein and esta blishment of the equivalence between

MON 87460- and E. coli -produced proteins included:

1. immunoblot analysis to establish prot ein identity through immunoreactivity
with NPTII–specific antibody and dem onstrate immuno-equivalence between

MON 87460 and E. coli -produced NPTII proteins.

2. SDS-PAGE to assess the apparent mo lecular weight of the protein and establish equivalence of the apparent molecular weight between MON 87460-

and E. coli -produced proteins.

C.2.3. NPTII Protein Immunoreactivity

Immunoblot analysis established that MON 87460-produced NPTII and E. coli -produced NPTII have equivalent immunoreactive proper ties. The expression levels of NPTII protein in MON 87460 leaf tissue allowed detect ion of the protein with an NPTII-specific antibody directly in leaf extr acts without additional enrichme nt. An extract was also prepared from a leaf sample of conventiona l corn with a similar genetic background as MON 87460 to serve as a negative control for the presence of the NPTII protein. To ensure that the electromobility of the NPTII protein had not been a ltered as a result of matrix effects, the reference substance was sp iked into the leaf extract from conventional corn and analyzed alongside the leaf extr act from MON 87460. Th e leaf extract from MON 87460, E. coli -produced NPTII protein, and NPTII-spiked conventional corn leaf extract were subjected to a reducing and denaturing SDS-PAGE and then transferred to a

nitrocellulose membrane for detection us ing an anti-NPTII antibody. A co-migrating

immunoreactive band was observed in the l eaf extract from MON 87460 (Figure C-1, Lanes 4-6), leaf NPTII-spiked conventional corn leaf extract (Figure C-1, Lanes 7-9), andMonsanto Company 07-CR-191U Page 297 of 544 pure E. coli -produced NPTII protei n (Figure C-1, Lane10) . As expected, the immunoreactive signal increased with increased loading levels of the leaf extract from

MON 87460 and increased amount of the leaf ex tract from conventional corn spiked with

the E. coli -produced NPTII protein. No immunor eactive bands were observed in the leaf extract from conventional corn (Figure C-1, Lane 3). Based on this analysis, the

MON 87460- and E. coli -produced NPTII proteins demonstrated equivalent immunoreactive properties, which confirmed bot h the identity and equivalence of the two proteins.

C.2.4. NPTII Protein Molecular Weight Equivalence

The molecular weight of the MON 87460-produced NPTII protein and its equivalence to the molecular weight of the E. coli -produced NPTII reference standard was confirmed using densitometric analysis of the western bl ot (Figure C-1). The electromobility of the MON 87460-produced protein wa s indistinguishable from the electromobility of the

E. coli -produced NPTII protein. The estimat ed molecular weight of the MON 87460- produced NPTII protein was 27.4 kDa, which wa s similar to the previously determined molecular weight of the E. coli -produced NPTII reference sta ndard (27.1 kDa). Based on the identical electrophoretic mobility and a pparent molecular masses, the MON 87460-

and E. coli -produced NPTII proteins have equivalent molecular weights.

C.2.5. Conclusions of the NPT II Protein Characterization

MON 87460-produced and E. coli -produced NPTII proteins have equivalent immunoreactivities and apparent molecular we ights. The results of this analysis confirmed the identity of the MON 87460-pr oduced NPTII protein and established the equivalence of the plant produced protein to the E. coli -produced NPTII reference protein

standard. A western blot analysis was utilized to co mpare the immunoreactivity and apparent molecular weight of the MON 87460-produced NPT II protein to that of the previously characterized E. coli -produced NPTII reference protein standard. The

MON 87460- and E. coli -produced NPTII proteins disp layed similar immunoreactivity with NPTII-specific antibody and had identi cal electromobility on SDS-PAGE. Taken together, these data establish equiva lence between the MON 87460-produced and E. coli

produced NPTII reference protein standard.Monsanto Company 07-CR-191U Page 298 of 544 Figure C-1. Western Blot Analysis of the MON 87460- and E. coli -produced NPTII

Protein

Corn leaf extracts from MON 87460 and conventional corn. E. coli -produced NPTII and E. coli

produced NPTII spiked into leaf extract of c onventional corn were separated by SDS-PAGE and

electrotransferred to a nitrocellulose membrane . The membrane was probed with rabbit antiNPTII antibody and an HPR-conjugated secondary an tibody and visualized using an ECL system. Approximate molecular weights (kDa) are shown on the left and correspond to the protein marker loaded in Lanes 2, 11 and 12. The 10 s expos ure is shown and is representative of the bands observed in the other exposures. Lane Sample Amount Loaded (ng) ( μ l)
1 Empty

⎯ ⎯ 2 Precision Plus Protein WesternC markers

⎯ 5

3 Leaf extract from conventional corn

⎯ 10

4 Leaf extract from corn MON 87460

⎯ 5

5 Leaf extract from corn MON 87460

⎯ 10

6 Leaf Extract from corn MON 87460

⎯ 15

7 E. coli- produced NPTII spiked* 0.25 5

8 E. coli- produced NPTII spiked* 0.5 10

9 E. coli- produced NPTII spiked* 0.75 15

10 E. coli- produced NPTII 0.5 10

11 Precision Plus Protein WesternC markers

⎯ 5

12 Precision Plus Protein WesternC markers

⎯ 5

* E. coli- produced NPTII spiked in leaf extract from conventional corn kDa

250

150

100

75

50

37.5

25

20

15

10 1 2 3 4 5 6 7 8 9 10 11 12Monsanto Company 07-CR-191U Page 299 of 544 C.3. References:

Beck, E., G. Ludwig, E.A. Auerswald, B. Reiss and H. Schaller. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from

transposon Tn5 . Gene. 19:327-336.

DeBlock, M., L. Herrera-Estrella, M.V. M ontagu, J. Schell and P. Zambryski. 1984. Expression of foreign genes in regenera ted plants and in their progeny. EMBO

Journal. 3:1681-1689.

Fraley, R.T., S.G. Rogers, R.B. Horsch, P.R. Sanders, J.S. Flick, S.P. Adams, M.L. Bittner, L.A. Brand, C.L. Fink, J.S. Fr y, G.R. Galluppi, S.B. Goldberg, N.L. Hoffmann and S.C. Woo. 1983. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences USA. 80:4803-4807.

Horsch, R.B., R.T. Fraley, S.G. Rogers, P. R. Sanders, A. Lloyd and N. Hoffmann. 1984.

Inheritance of functional fore ign genes in plants. Science. 223:496-498.Monsanto Company 07-CR-191U Page 300 of 544 Appendix D. Materials and Methods for Ex pression Levels of CSPB and NPTII

Proteins

The levels of the CSPB and NPTII protei ns in various tissues of MON 87460 were determined using enzyme-linked immunosorbe nt assays (ELISA). The materials and methods for the ELISA analysis, as well as a de scription of the tissue types, are provided below. To produce the tissues for anal ysis, MON 87460 and conventional corn were each planted in field trials conducted duri ng two different growing seasons. The first

season was conducted at six sites in the U.S. during 2006 under typical agronomic practices and water conditions. The second season was conducted at four sites in Chile during 2006/2007 using a st rip-plot design to establish tw o water treatment levels (wellwatered and water-limited) to assess for any changes in CSPB and NPTII protein levels

under different soil moisture conditions. Th e sites were located in the major corngrowing regions of the U.S. and Chile. Fora ge, stover, silk, pollen, and grain samples were collected at appropriate times of plant development. Over-season leaf (OSL), overseason root (OSR), and over-season whole pl ant (OSWP) samples were collected four times (1-4) over the season corresponding to pl ant growth stages V2-V4, V6-V8, V10- V12, and pre-VT (pre-tasseling) , respectively. The expression levels of CSPB and NPTII proteins in these tissues are shown in Tables VI-1 to VI-4. As discussed previously (Section I.B.5, and in more detail in Section VIII.C and Table VIII-3), the QUI site in the Chile 2006/2007 study was not estab lished with the appropriate water stress treatments; therefore, data and analysis for the QUI site are presented separately in Section D.3.

D.1. U.S. 2006 Study

D.1.1. Field Design

Tissue samples were collected from six field trials conducte d in the U.S. during 2006. The trials were located in Iowa, Illinois, I ndiana, Kansas, and Nebraska which represent the major corn-growing regions of the U. S. and provide a range of environmental

conditions that would be encountered in the co mmercial production of co rn. At each site, three replicated plots of MON 87460 and a conventional c ontrol hybrid were planted using a randomized complete block field de sign. Over-season l eaf (OSL), over-season whole plant (OSWP), over-season root (OSR), pollen, silk, forage, forage root, grain, stover, and senescent root tissues were collect ed from each replicated plot at all field

sites.

D.1.2. Description of Collected Tissues

Leaf. Leaves were randomly collected from plan ts in each plot at each site. Twenty leaves were combined to form the leaf samp le for each plot. There were 18 leaf samples across all sites for OSL-1, OSL-3, and OS L-4 and 17 leaf samples for OSL-2. OSL samples were collected as follows:Monsanto Company 07-CR-191U Page 301 of 544 Over-season leaf (OSL) Corn developme nt stage Days after planting (DAP)

OSL-1 V2-V4 15-22

OSL-2 V6-V8 27-38

OSL-3 V10-V12 41-56 OSL-4 pre-VT (pre-tasseling) 49-63

Whole Plant . The aerial portion of the plant was colle cted from four plants in each plot at each site at the V2-V4 stage and combin ed to form the whole plant sample. Two plants were collected and combined to form the whole plant samples for the later growth stages. OSWP samples were collected as follows:

Overseason whole plant (OSWP) Corn development stage DAP

OSWP-1 V2-V4 15-22

OSWP-2 V6-V8 27-38

OSWP-3 V10-V12 41-56 OSWP-4 pre-VT (pre-tasseling) 49-63

Root . Roots remaining after collection of whole plants from each plot were combined to form the root sample. Therefore, roots from four plants at the V2-V4 stage and roots from two plants at later stages were combin ed. OSR samples were collected as follows.

Overseason root (OSR) Corn development stage DAP

OSR-1 V2-V4 15-22

OSR-2 V6-V8 27-38

OSR-3 V10-V12 41-56 OSR-4 pre-VT (pre-tasseling) 49-63 Forage root early dent stage (R4-R6) 90-103

Senescent root after harvest 135-151

Pollen and Silk . Approximately 10 ml of pollen was collected from multiple tassels in each plot at each site at pollination, approximately 59-68 days after planting. Silks were collected approximately 58-74 days after planti ng from five plants, except for the Indiana and Iowa sites, where silks were collected fr om ten plants. Silks were only collected from ears of plants that were covered with s hoot bags to preserve their genetic identity.

Forage and Forage Root . Two whole plants in each plot at each site were cut above the soil surface at an early dent stage, at appr oximately 96-109 days after planting, and then combined to form the forage sample. The roots of these plants were combined to form the forage root sample.

Grain, Stover, and Senescent Root . Grain was harvested at maturity from all plants in each plot at each site and dried to a moisture content of 11-17%. Following harvest, approximately 136-158 days after planting, two whole plants in each plot at each site

were cut above the soil surface and combined to form the stover sample. The roots of these plants were also removed, washed and combined to form a senescent root sample.Monsanto Company 07-CR-191U Page 302 of 544 All tissue samples, except grain, were stored and shipped on dry ic e for processing and analysis. Grain was stored and shipped at ro om temperature. All tissue samples were stored in a –80oC freezer upon receipt.

CSPB expression levels were determined in all 19 tissue types described above. Because of the extensive historical safety data fo r NPTII, the number of tissues evaluated for NPTII expression was fewer than those evalua ted for CSPB protein. The NPTII levels

were evaluated in four of the 19 tissue t ypes including OSL-1 (V2- V4), OSR-1 (V2-V4), forage and grain. These four tissues were selected to span the life cycle of corn. Moisture content was measured for all tissue ty pes. Protein levels for all tissue types are provided in μg/g fresh weight tissue (fwt) and μg/g dry we ight tissue (dwt) basis.

D.1.3. Tissue Processing and Protein Extraction

All samples produced at the field sites were sh ipped to Monsanto’s pr ocessing facility in Creve Coeur, MO. During the processing step, dry ice was combined with the individual samples, and vertical cutters or mixers were used to thoroughly grind and mix the tissues. Processed samples were transferred in to capped tubes and stored in a -80 ° C freezer until

use.

CSPB and NPTII were extracted from all tissu es by shaking tubes mounted in a Harbil mixer for a specified period of time. Each extraction tube containe d eight 1⁄4” diameter Chrome-steel beads, buffer and a tissue to buffer ratio as specified below.

Table D-1. Protein Extraction Methods for U.S. 2006 Tissue Samples

Protein Tissue Extraction Buffer T:B

Ratio 1 Shake Time (minutes) Sample Clarification

Method

CSPB/NPTII Leaf 2 PBST/BSA 3 1:100 7.0 Serum filter

CSPB/NPTII Root 4 PBST/BSA 1:20 7.0 Serum filter

CSPB/NPTII Forage 5 TB 6 1:30 7.0 Serum filter

CSPB/NPTII Grain TB 1:25 10.5 Serum filter

CSPB Silk PBST/BSA 1:50 7.0 Serum filter

CSPB Pollen TB 1:50 10.5 Serum filter

1 T:B Ratio – Tissue to buffer ratio

2 Overseason leaf (OSL1, OSL2, OSL3, and OSL4)

3 1x Phosphate Buffered Saline + 0.05% (w/v)Tween + 0.1%(w/v) Bovine Serum Albumin

4 Overseason root (OSR1, OSR2, OSR3, and OSR4), forage root, and senescent root

5 Forage, overseason whole plant (OSWP1, OSWP2, OSWP3, and OSWP4), and stover

6 1x Tris borate buffer (0.1 M Tris, 0.1 M Na 2 B 4 O 7 · 10H 2 O, 0.01 M MgCl 2 , 0.05% (v/v) Tween-20, pH 7.8)

.

Following shaking, insoluble material was removed from the extracts using a serum filter (Fisher Scientific, Pittsburgh, PA). The clarif ied extracts were aliquo t, and stored frozen

in a -80 ° C freezer until ELISA analysis.Monsanto Company 07-CR-191U Page 303 of 544 D.1.4. CSPB Antibodies

Goat polyclonal CSPB-specifi c IgG was purified by Pr otein G-agarose affinity chromatography followed by affinity chromatography on AminoLink immobilized CSPB protein (lot G-812159). The concentration of the CSPB protein affinity purified IgG was determined to be 0.8 mg/ml by spectrophot ometric methods. The purified antibody was stored in 137 mM NaCl, 10 mM Na 2 HPO 4 . 7 H 2 O, 1 mM KH 2 PO 4 , and 2.7 mM KCl, pH 7.4 (1X phosphate buffered saline (PBS)). CSPB protein affinity pu rified IgG was used as the well coating antibody.

Protein G agarose affinity pur ified goat polyclonal anti-CSP B was coupled with biotin (Sigma, St. Louis, MO) according to the manufacturer’s instructions and assigned lot G806080-2. The detection reagent was NeutrAvi din (Pierce, Rockford, IL) conjugated to

HRP.

D.1.5. NPTII Antibodies

Rabbit polyclonal antibodies (l ot G-805224) specific for the NP TII protein were purified using Protein-A agarose affinity chromatogr aphy by TechServ Associates (St. Louis, MO). The concentration of the purified IgG was determined to be 5.6 mg/ml by spectrophotometric methods. The purified antibody was stored in 20 mM potassium phosphate, 150 mM NaCl, pH 7.3 and pres erved with 0.01% (w/v) sodium azide.

The purified NPTII antibodies were couple d with biotin (Sigma, St. Louis, MO) according to the manufacturer’s instructions and assigned lot G-814147. The detection reagent was NeutrAvidin (Pierce, Rockford, IL) conjugated to HRP.

D.1.6. CSPB ELISA Method

Affinity-purified goat anti-CSPB capture an tibodies were diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and immobilized onto 96-well microtiter plates at 2.0 μ g/ml followed by incubation in a 4 ° C refrigerator for >8 h. Prior to each step in the assay, plates were washed with 1X PBS containing 0.05% (w/v) Tween-20 (1X PBST). Plates were blocked with the addition of 150 μl per we ll of 1X PBST with

1% BSA for 60 to 70 min at 37 ° C. CSPB protein standard or sample extract was added

at 100 μ l per well and incubated for 1 h at 37 ° C. The captured CSPB protein was detected by the addition of 100 μ l per well of biotinylated goat anti-CSPB antibodies and NeutrAvidin-HRP (Pierce). Plat es were developed by adding 100 μ l per well of HRP substrate, 3,3',5,5'-tetramethylbenzidine (T MB; Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction was term inated by the addition of 100 μ l per well of 3 M H3PO4. Quantitation of the CSPB protein was acco mplished by interpolation from a CSPB protein standard curve that ranged from 0.05 – 1.6 ng/ml.

D.1.7. NPTII ELISA Method

Rabbit anti-NPTII capture an tibodies were diluted in coating buffer (15 mM Na 2 CO 3 and

35 mM NaHCO 3, pH 9.6) and immobilized onto 96- well microtiter plates at 5.0 μ g/ml followed by incubation in a 4 ° C refrigerator for > 8 h. Prior to each step in the assay, plates were washed with 1X PBST. NPTII protein standard or sample extract was added

at 100 μ l per well and incubated for 1 h at 37 ° C. The captured NPTII protein was detected by the addition of 100 μ l per well of biotinylated rabbit anti-NPT II antibodiesMonsanto Company 07-CR-191U Page 304 of 544 and NeutrAvidin-HRP. Plates were developed by adding 100 μ l per well of TMB. The enzymatic reaction was terminated by the addition of 100 μ l per well of 6 M H 3 PO 4

. Quantitation of the NPTII protein was acco mplished by interpolation from a NPTII protein standard curve that ranged from 0.094 – 3.0 ng/ml.

D.1.8. Moisture Analysis

All tissues were analyzed fo r moisture content using an IR-200 Moisture Analyzer (Denver Instrument Company, Arvada, CO). A homogeneous tissue-specific site pool (TSSP) was prepared using the test and contro l samples of a given tissue type grown at a given site. These pools were prepared for all tissues in this study. The average percent moisture for each TSSP was calculated from triplicate analyses. A TSSP Dry Weight Conversion Factor (DWCF) was calculated as follows:

DWCF = 1 – [Mean % TSSP Moisture / 100]

The DWCF was used to convert protein levels assessed on a μg/g fres h weight (fwt) basis into levels reported on a μg/g dry weight (dwt) basis using the following calculation:

The protein levels that were re ported to be less than or equal to the limit of detection (LOD) or less than the limit of quantitati on (LOQ) on a fresh wei ght basis were not reported on a dry weight basis.

D.1.9. Data Analyses

All CSPB and NPTII ELISA plates were an alyzed on a SPECTRAmax Plus (Molecular Devices, Sunnyvale, CA) or a SPECTRAFluor Pl us (Tecan, Research Triangle Park, NC) microplate spectrophotometer, using a dual wa velength detection method. All protein concentrations were determined by optical absorbance at a wavele ngth of 450 nm with a simultaneous reference reading of 620-650 nm. Data reduction analyses were performed using Molecular Devices SOFTmax PRO vers ion 4.7.1 or SOFTmax Pro GxP version 5.0.1. Absorbance readings and protein standa rd concentrations were fitted with a fourparameter logistic curve fit. Following the interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was reported on a μ g/g fwt basis. For all proteins, this conversion utilized a samp le dilution factor and a tissue- to-buffer ratio. The protein

values in μ g/g fwt were also converted to μ g/g dwt by applying the DWCF. Microsoft Excel 2002 (Version 10.6834.6830 SP3, Microsoft, Redmond, WA) was used to calculate the CSPB and NPTII protein levels in corn tissues.

D.2. Chile 2006/2007 Study

D.2.1. Field Design

The second season field trial was designed to assess protein levels in MON 87460 under a range of typical environmenta l conditions relevant to its commercial produc tion. This trial was conducted in Chile during 2006/2007 us ing two water treatment levels (well- 

### ( ) ( )

DWCF Weight Fresh Level otein Pr Weight Dry in Level otein Pr =Monsanto Company 07-CR-191U Page 305 of 544 watered and water-limited) to assess the impact of different soil moisture conditions on protein expression. The levels of the CSPB and NPTII proteins in various tissues of

MON 87460 that are relevant to the risk a ssessment were assessed by a validated ELISA. OSL, OSWP, OSR, pollen, silk, forage, forage root, grain, stover, and senescent root

tissues were collected from four field sites. The trial locations were Calera de Tango (CT), Colina (CL), Lumbreras (LUM) and Quillota (QUI), covering a range of environmental conditions representative of commercial corn production areas for MON 87460. At each site, three replicat ed plots of MON 87460, as well as the conventional control, were plante d using a strip-plot design with three re plicates per site. The whole-plot for each replicate was an irrigation treatment (well-watered or waterlimited). The sub-plot for each irrigation trea tment was substance type (test and control substances), which was randomized in strips ac ross the irrigation treatments to assess for any changes in protein leve ls under different soil moistu re conditions. Well-watered plots were irrigated to achieve optimal yiel d, whereas water-limited plots were managed to impose a drought stress by withholding irri gation during the late vegetative through early grain fill growth stages (i.e., approximately V10 thr ough R2). For a site to be included in the combined-site analysis, co mmercial reference hybrids had to exhibit phenotypic responses indicative of a treatment effect. Speci fically, reference hybrids in the water-limited plots had to exhibit a mini mum 15% reduction in yield compared to the same reference hybrids planted in the well-watered plots. Mode rate water deficits result in approximately a 15% yield lo ss annually for corn grown in both temperate and tropical regions (Barker et al., 2005). Assessments fo r plant height, ear hei ght and days to 50% silking were also made as reduced height and a delay in silking are indicators of moisture deficit in corn (Campos et al., 2006). Refe rence hybrids at CT, CL and LUM exhibited the expected phenotypic response. Results from the Chile 2006/2007 represent combined-site data from three (CL, CT, LUM) of the four sites established. As discussed previously (Section VIII.C, Table VIII-3), the QUI site in the Chile 2006/2007 study was not established with the appropriate water stre ss treatments; therefore, data and analysis for the QUI site are presented below in Section D.3.

D.2.2. Description of Collected Tissues

Leaf. The youngest leaves were rand omly collected from plants in each plot at each site. Forty leaves were combined to form the l eaf sample for each plot. Over-season leaf samples were collected as follows:

Over-season leaf (OSL) Corn developm ent stage Days after planting (DAP)

OSL-1 V2-V4 13-20

OSL-2 V6-V8 32-38

OSL-3 V10-V12 48-52

OSL-4 ~VT 62-65Monsanto Company 07-CR-191U Page 306 of 544 Whole Plant . The aerial portion of the plant was colle cted from four plants in each plot at each site at the V2-V4 stage and combined to form the whole plant sample. Overseason whole plant samples were collected as follows:

Overseason whole plant (OSWP) Corn development stage DAP

OSWP-1 V2-V4 13-20

OSWP-2 V6-V8 32-38

OSWP-3 V10-V12 48-52

OSWP-4 ~VT 62-65

Root . Roots remaining after collection of whole plants from each plot were combined to form the root sample. Overseason root samples were collected as follows:

Overseason root (OSR) Corn development stage DAP

OSR-1 V2-V4 13-20

OSR-2 V6-V8 32-38

OSR-3 V10-V12 48-52

OSR-4 ~VT 62-65 Forage root early dent stage 106-113

Senescent root after harvest 151-153

Pollen and Silk . Quantities of pollen ranging from 0.5-40 ml and averaging approximately 10 ml were collected from multip le tassels in each plot at each site at pollination, approximately 67-74 days after planting. Silks were collected approximately 64-72 days after planting from five plants. Silk s were only collected from ears of plants that were covered with shoot bags to preserve the genetic identity.

Forage and Forage Root . Four whole plants in each plot at each site were cut above the soil surface at an early dent stage, approxi mately 106-113 days after planting, and then combined to form the forage sample. The roots of these plants were also removed, washed and combined to form the forage root sample.

Grain, Stover, and Senescent Root . Grain was harvested at maturity from all plants in each plot at each site and dried to a moisture content of 11-17%. Following harvest, approximately 151-153 days after planting, four whole plants in each plot at each site

were cut above the soil surface and combined to form the stover sample. The roots of these plants were also removed, washed and combined to form a senescent root sample.

All tissue samples, except grain, were stored and shipped on dry ic e for processing and analysis. Grain was stored and shipped at ro om temperature. All tissue samples were stored in a –80 oC freezer upon receipt. Ti ssue samples were extracted and analyzed by a validated ELISA according to applicable standard operating procedures (SOPs).

CSPB expression levels were determined in all 19 tissue types described above. Given the extensive historical safety data for NPTII, the number of tissues evaluated for NPTII expression was fewer than those evaluated fo r CSPB protein. The NPTII levels wereMonsanto Company 07-CR-191U Page 307 of 544 evaluated in four of the 19 tissue types including OSL1 (V2-V4), OSR1 (V2-V4), forage, and grain. These four tissues were select ed to span the life cycle of corn.

Moisture content was measured for all tissue ty pes. Protein levels for all tissue types are provided in μg/g fwt and μg/g dwt basis.

D.2.3. Tissue Processing and Protein Extraction

Same as described for U.S. 2006 study.

D.2.4. CSPB Antibodies

Same as described for U.S. 2006 study.

D.2.5. NPTII Antibodies

Same as described for U.S. 2006 study.

D.2.6. CSBP ELISA Method

Same as described for U.S. 2006 study.

D.2.7. NPTII ELISA Method

Same as described for U.S. 2006 study.

D.2.8. Moisture Analysis

Same as described for U.S. 2006 study.

D.2.9. Data Analysis

Same as described for U.S. 2006 study.

D.2.10. References

Barker, T., H. Campos, M. Cooper, D. Dolan, G. Edmeades, J. Habben, J. Schlusser, D. Wright and C. Zinselmeier. 2005. Impr oving drought tolerance in maize. Pages 173-253 in Plant Breeding Reviews. Vol 25. J. Janick, (ed.). John Wiley and Sons, Inc., Hoboken, NJ.

Campos, H., M. Cooper, G.O. Edmeades, C. Loffler, J.R. Schussler, and M. Ibanez. 2006. Changes in drought tolerance in ma ize associated with fifty years of breeding for yield in the U.S. Corn Belt. Maydica. 51:369-381.

D.3. Supplementary Protein Level Data for QUI Site in Chile 2006/2007 Study

This section presents protein expression da ta for MON 87460 grown at an individual site (QUI) of the Chile 2006/2007 study. As discussed previously (Section VIII.C, Table VIII-3), the QUI site in the Chile 2006/2007 study was not established with the appropriate water stress treatments; therefor e, data and analysis for the QUI site are presented in this appendix. Table D-2 presen ts results for CSPB and Table D-3 presents

results for NPTII. Evaluation of the overall da ta set confirmed expres sion levels were as expected. No unexpected values for either C SPB or NPTII were observed. The QUI site results reported below do not impact the co nclusion of protein expression studies of MON 87460 presented in Section VI.C.Monsanto Company 07-CR-191U Page 308 of 544 Table D-2. CSPB Protein Levels in Tissu es Collected from MON 87460 Produced at the QUI site in Chile during 2006/2007 under Well-Watered and Water-Limited

Conditions

Well-Watered Water-Limited

Tissue Type Mean (SD) 1

Range 2

( μ g/g fwt.) 3 Mean (SD) Range ( μ g/g dwt.) 4 Mean (SD) Range ( μ g/g fwt.) Mean (SD) Range ( μ g/g dwt.) LOQ / LOD ( μ g/g fwt)

OSL-1 0.46 (0.10) 3.1 (0.68) 0.47 (0.076) 2.5 (0.40) 0.015 / 0.0069 0.36 - 0.56 2.4 - 3.7 0.39 - 0.53 2.0 - 2.8

OSL-2 0.28 (0.037) 1.5 (0.19) 0.31 (0.015) 1.6 (0.077) 0.015 / 0.0069 0.24 - 0.31 1.3 - 1.6 0.29 - 0.32 1.5 - 1.6

OSL-3 0.11 (0.036) 0.50 (0.16) 0.11 (0.049) 0.46 (0.21) 0.015 / 0.0069 0.084 - 0.15 0.37 - 0.67 0.061 - 0.16 0.26 - 0.68

OSL-4 0.093 (0.0015) 0.44 (0.0073) 0.10 (0.031) 0.48 (0.16) 0.015 / 0.0069 0.092 - 0.095 0.44 - 0.45 0.059 - 0.12 0.30 - 0.58

OSR-1 0.12 (0.023) 1.2 (0.23) 0.10 (0.0063) 1.0 (0.063) 0.0020 / 0.0018 0.10 - 0.14 0.95 - 1.4 0.10 - 0.11 0.99 - 1.1

OSR-2 0.13 (0.044) 1.3 (0.44) 0.10 (0.022) 0.92 (0.20) 0.0020 / 0.0018 0.10 - 0.18 1.0 - 1.8 0.081 - 0.12 0.74 - 1.1

OSR-3 0.046 (0.010) 0.46 (0.10) 0.052 (0.023) 0.52 (0.23) 0.0020 / 0.0018 0.036 - 0.056 0.36 - 0.56 0.033 - 0.077 0.33 - 0.77

OSR-4 0.048 (0.014) 0.40 (0.11) 0.059 (0.0029) 0.45 (0.022) 0.0020 / 0.0018 0.035 - 0.062 0.29 - 0.52 0.056 - 0.062 0.43 - 0.47

OSWP-1 0.34 (0.0032) 3.1 (0.029) 0.31 (0.039) 2.8 (0.35) 0.0045 / 0.0043 0.34 - 0.35 3.1 - 3.1 0.29 - 0.36 2.6 - 3.2

OSWP-2 0.20 (0.023) 2.2 (0.26) 0.18 (0.019) 2.5 (0.28) 0.0045 / 0.0043 0.18 - 0.23 2.0 - 2.5 0.16 - 0.20 2.3 - 2.8

OSWP-3 0.082 (0.0079) 0.68 (0.066) 0.077 (0.0054) 0.70 (0.049) 0.0045 / 0.0043 0.073 - 0.087 0.61 - 0.72 0.071 - 0.081 0.64 - 0.74

OSWP-4 0.11 (0.017) 0.74 (0.11) 0.12 (0.0058) 0.79 (0.039) 0.0045 / 0.0043 0.092 - 0.12 0.61 - 0.81 0.11 - 0.13 0.76 - 0.84

Forage Root 0.0090 (0.0013) 0.056 (0.0082) 0.0062 (0.0027) 0.041 (0.018) 0.0020 / 0.0018 0.0080 - 0.010 0.050 - 0.066 0.0038 - 0.0092 0.025 - 0.061

Senescent

Root 0.0051 (0.0041) 0.032 (0.025) 0.0028 (N/A 5 ) 0.018 (N/A) 0.0020 / 0.0018 0.0021 - 0.010 0.013 - 0.061 N/A N/A

Forage 0.023 (0.0030) 0.10 (0.013) 0.021 (0.0040) 0.091 (0.017) 0.0045 / 0.0043 0.021 - 0.027 0.091 - 0.12 0.017 - 0.025 0.073 - 0.11

Stover 0.014 (0.00059) 0.048 (0.0020) 0.012 (0.0041) 0.039 (0.013) 0.0045 / 0.0043 0.013 - 0.015 0.046 - 0.050 0.0082 - 0.016 0.026 - 0.053

Silk 0.053 (0.014) 0.66 (0.17) 0.040 (0.012) 0.40 (0.12) 0.0075 / 0.0047 0.036 - 0.066 0.45 - 0.82 0.032 - 0.054 0.32 - 0.54

Pollen 12 (1.1) 18 (1.7) 11 (0.84) 17 (1.2) 0.050 / 0.045 12 - 14 17 - 20 10 - 12 16 - 18

Grain 0.043 (0.010) 0.050 (0.012) 0.036 (0.0091) 0.042 (0.011) 0.0038 / 0.0017 0.031 - 0.051 0.036 - 0.059 0.029 - 0.046 0.034 - 0.054

1 The mean and standard deviation were calculated across sites (n=3 for well-watered and n=3 for waterlimited, except silk where n=4 for well-watered and senescent root where n=1 under water-limited).

2 Minimum and maximum values were determ ined for each tissue type across sites.

3 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a fresh weight basis.

4 Protein levels are expressed as μ g/g on a dry weight basis. The dry weight values were calculated by dividing the fresh weight by the dry weight conversion factors obtained from moisture analysis data.

5 N/A – not applicableMonsanto Company 07-CR-191U Page 309 of 544 Table D-3. NPTII Protein Levels in Ti ssues Collected from MON 87460 Produced at the QUI site in Chil e during 2006/2007 under Well-W atered and Water-Limited

Conditions

Well-Watered Water-Limited

Tissue Type Mean (SD) 1

Range 2

( μ g/g fwt.) 3 Mean (SD) Range ( μ g/g dwt.) 4 Mean (SD) Range ( μ g/g fwt.) Mean (SD) Range ( μ g/g dwt.) LOQ / LOD ( μ g/g fwt)

OSL-1 0.42 (0.039) 2.8 (0.26) 0.50 (0.0093) 2.6 (0.049) 0.047/ 0.0090 0.38 - 0.45 2.5 - 3.0 0.49 - 0.50 2.6 - 2.7

OSR-1 0.044 (0.016) 0.44 (0.16) 0.036 (0.0094) 0.36 (0.094) 0.0075/ 0.0043 0.025 - 0.055 0.25 - 0.55 0.028 - 0.046 0.28 - 0.46

Forage 0.034 (0.0020) 0.15 (0.0088) 0.031 (0.0023) 0.13 (0.010) 0.0056/ 0.0024 0.032 - 0.036 0.14 - 0.15 0.028 - 0.033 0.12 - 0.14

Grain <LOQ (N/A 5 ) N/A (N/A) <LOQ (N/A) N/A (N/A) 0.0047 / 0.0024 N/A N/A <LOD-<LOQ N/A

1 The mean and standard deviation were calculated across sites (n=3 for well-watered and n=3 for waterlimited, except silk where n=4 for well-watered and senescent root where n=1 under water-limited).

2 Minimum and maximum values were determ ined for each tissue type across sites.

3 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a fresh weight basis.

4 Protein levels are expressed as μ g/g on a dry weight basis. The dry weight values were calculated by dividing the fresh weight by the dry weight conversion factors obtained from moisture analysis data.

5 N/A – not applicableMonsanto Company 07-CR-191U Page 310 of 544 Appendix E. Materials and Methods for Fora ge and Grain Compositional Analysis

Compositional comparisons between MON 87460 and a conventional control were performed using the principles and analytes outlined in the OECD consensus documents for corn composition (OECD, 2002; 2006). Th ese principles are a ccepted globally and have been employed previously in asse ssments of corn products derived through biotechnology. The material s and methods for component analysis, as well as a description of th e tissue types, ar e provided below.

The compositional assessment was conducted on forage and grain samples harvested from two different growing seasons. The fi rst season was conducted in the U.S. during 2006 under typical agronomic pr actices and water conditions . The second season was conducted in Chile during 2006/2007 using a st rip-plot design to establish two water treatment levels (well-watered and water- limited) to assess for any changes in compositional equivalence under different soil moisture conditions. Samples from the Chile 2006/2007 study were also analyzed for 11 additional secondary metabolites that are potentially associated with drought stress. Square brackets in the tables presented in this section denote ranges for the test and control or 99% tolerance intervals for the reference materials. As discussed previously (Section VIII.C, Table VIII-3), the QUI site in the Chile 2006/2007 study was not establis hed with the approp riate water stress treatments; therefore, data and analysis for the QUI site are also presented below in

Section E.8.

E.1. Test, Control and Reference Substances

E.1.1. Test Substance

The test substance was MON 87460. Forage and grain tissues of corn MON 87460 were evaluated in this study.

E.1.2. Control Substance

The control substance was conve ntional corn hybrid with genetic background similar to MON 87460. The forage and grain tissues of th e control substance were evaluated in this study.

E.1.3. Reference Substances

The reference substances were 15 conventional commercial corn hybrids. A single replicate of the forage and grain tissues fr om each reference substance was evaluated in this study. The following conventi onal corn hybrids were analyzed:Monsanto Company 07-CR-191U Page 311 of 544 Table E-1. Reference Substances for U.S. 2006 Composition Study

Material Name Seed Lot No. Field Code

DKC 61-42 GLP-0603-16998-S IAE

DKC 60-15 GLP-0604-17072-S IAE

DKC 63-78 GLP-0604-17073-S IAE

H8991 GLP-0603-16996-S IAW

DKC 61-50 GLP-0603-16999-S IAW

33N29 GLP-0604-17088-S IAW

33K39 GLP-0604-17076-S IL

M-3744 GLP-0604-17077-S IL

M-3765 GLP-0604-17078-S IL

BT-6512 GLP-0604-17079-S IN

B-625 GLP-0604-17083-S IN

B-645 GLP-0604-17084-S IN

S-2721 GLP-0604-17146-S KS

32B33 GLP-0604-17147-S KS

33H25 GLP-0604-17071-S KS

G-8424 GLP-0604-17089-S NE

NC+4822 GLP-0604-17090-S NE

34N43 GLP-0604-17091-S NE

Table E-2. Reference Substances for Chile 2006/2007 Composition Study

Material Name Seed Lot No. Field Code

33D11 GLP-0604-17075-S CL

BT 6011 GLP-0610-17684-S CL

Garst 8424 GLP-0610-17687-S CL

DKC62-30 GLP-0609-17618-S CL

33N09 GLP-0610-17691-S CT

33K39 GLP-0604-17076-S CT

BT 6613 GLP-0610-17683-S CT

DKC63-78 GLP-0609-17613-S CT

33N29 GLP-0604-17088-S LUM

Garst 8445 GLP-0610-17688-S LUM

DKC61-50 GLP-0609-17612-S LUM

RX 715 GLP-0609-17615-S LUM

34N43 1 GLP-0604-17091-S QUI 1

BT 6610 1 GLP-0610-17685-S QUI 1

Garst 8545 1 GLP-0609-17689-S QUI 1

DKC60-15 1 GLP-0609-17610-S QUI 1Monsanto Company 07-CR-191U Page 312 of 544 E.2. Test, Control and Reference Substance Characterization

The identities of the forage and grain samp les from each test, control, and reference substance were verified by the Study Director by confirming the chain-of-custody documentation supplied with the forage and grai n collected from the plots. The grain of the test, control, and reference substances we re also characterized, by event-specific PCR analysis, for the presence of the cspB coding region.

E.3. Field Trial Description

U.S. 2006 Seed was planted in the spring of 2006 at six sites (IAE, IAW, IL, IN , KS, and NE) in the

United States. Locations of the field sites are as follows: IAE, Benton County, Iowa; IAW, Greene County, Iowa; IL, Stark County, Illinois; IN, Parke C ounty, Indiana; KS, Pawnee County, Kansas, and NE, York County, Nebraska. At each field site, the T/C/R seed starting substances were planted in a randomized comp lete block design with three replicates per block. Each block (replicate) c onsisted of five plots with one plot for each test, control, and reference substance. Production was managed according to normal agronomic field practices. Grain and forage samples were harvested from all plots at ambient temperature and forwarded to Mons anto Company (St. Louis, MO). A subsample for compositional analysis was obtained from each tissue sample harvested. These sub-samples were then ground and stored in a freezer set to maintain a temperature of -

20 o C until their shipment on dry ice to Cova nce Laboratories Inc. (Madison, WI) for analysis.

Chile 2006/2007 Seed was planted in the winter of 2006 at four replicated fi eld sites (CL, CT, LUM, and QUI) in Chile. Locations of the field sites are as follows: CL, Colina, Region Metropolitana; CT, Calera de Tango, Region Metropolitana; LUM, Lumbreras, Region Metropolitana, and QUI, Quillota, “V.” The test and control substances were grown at all

field sites. Four different c onventional reference s ubstances were also grown at each of the field sites. The field design incorporated a strip-plot design. The whole plot factor was irrigation treatment. Well-watered was irrigation management for optimal yield. Water-limited was irrigation management to target replacement of 55-65% of water evapotranspiration starting at plant growth stage ~V10 and continuing through ~R2. The design for the whole plot factor was a randomi zed complete block design. The strip-plot factor consisted of the test, c ontrol, and reference substances.

Grain and forage samples were harvested fr om all plots at ambient temperature and forwarded to Monsanto Company (St. Loui s, MO). A sub-sample for compositional analysis was obtained from each tissue sample collected. These sub-samples were then ground and stored in a freezer set to maintain a temperature of -20 o C until their shipment on dry ice to Covance Laboratories, Inc. (Madison, WI) for analysis.

E.4. Analytical Methods

Components assessed in forage samples included proximates (protein, fat, ash, and moisture), carbohydrates by calc ulation, acid deterg ent fiber (ADF), neutral detergentMonsanto Company 07-CR-191U Page 313 of 544 fiber (NDF), calcium, and phosphorus. Compon ents assessed in gr ain samples included proximates (protein, fat, ash, and moistu re), carbohydrates by ca lculation, ADF, NDF, total detergent fiber (TDF), total amino acid composition, fatty acid composition, minerals (calcium, copper, iron, magnesium , manganese, phosphorus , potassium, sodium, zinc), vitamins (vitamin B1 [thiamine], vita min B2 [riboflavin], vitamin B6 [pyridoxine], vitamin E, niacin, folic acid), furfural, raffinose, phytic acid, p -coumaric acid, and ferulic

acid. All compositional analyses were performed at Covance Laboratories, Inc. (Madison, Wisconsin). Methods for analysis were ba sed on internationally-r ecognized procedures and literature publications. Brie f descriptions of the methods utilized for the analyses are

described below.

E.4.1 Moisture Sample was dried in a vac uum oven at approximately 100 ° C to a constant weight. The moisture weight loss was determined and converted to percent moisture. The limit of quantitation was 0.100%.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 926.08 and 925.09, AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005).

E.4.2 Ash Sample was placed in an electric furnace at 550 ° C and ignited to drive off all volatile organic matter. The nonvolat ile matter remaining was quan titated gravimetrically and calculated to determine pe rcent ash. The limit of quantitation was 0.100%.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Method 923.03,

AOAC INTERNATIONAL: Gaither sburg, Maryland, (2005).

E.4.3 Protein Nitrogenous compounds in the sample were re duced in the presence of boiling sulfuric acid and a mercury catalyst mixture to form ammonia. The acid digest was made

alkaline. The ammonia was distilled and then titrated with a previously standardized acid. The percent nitrogen was calculated a nd converted to equivale nt protein using the factor 6.25. The limit of quantitation was 0.100%.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 955.04 and 979.09, AOAC INTERNATIONAL, Gaithersburg, Maryland, (2005).

Bradstreet, R. B., The Kjeldahl Method for Organic Nitrogen , Academic Press: New York, New York, (1965).

E.4.4 Fat by Acid Hydrolysis (Forage Analysis) Forage sample was hydrolyzed with hydrochloric acid at an elevated temperature. Thefat was extracted with ether and hexane. The extract was evaporated on a steam bath, redissolved in hexane and filtered through a s odium sulfate column. The hexane extractMonsanto Company 07-CR-191U Page 314 of 544 was then evaporated again on a steam bath unde r nitrogen, dried, and weighed. The limit of quantitation was 0.100%.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 922.06 and 954.02, AOAC INTERNATIONAL, Gaithersburg, Maryland, (2005).

E.4.5 Fat by Soxhlet Extraction (Grain Analysis) The sample was weighed into a cellulose thim ble containing sodium sulfate and dried to remove excess moisture. Pentane was dripped through the sample to remove the fat. The extract was then evaporated, dried, and weighed. The limit of quantitation was 0.100%.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Method 960.39 and 948.22, AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005)

E.4.6 Carbohydrate (CHO) The total carbohydrate level was calculated by difference using the fresh weight-derived data and the following equation:

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

The limit of quantitation was 0.100%.

United States Department of Agriculture

, “Energy Value of Foods,” Agriculture
Handbook No. 74, pp. 2-11, (1973).

E.4.7 Acid Detergent Fiber Sample was placed in a fritted vessel an d washed with an acidic boiling detergent solution that dissolved the protein, carbohydrat e, and ash. An acetone wash removed the fats and pigments. The lignocellulose fracti on was collected on the frit and determined gravimetrically. The limit of quantitation was 0.100%.

Forage and Fiber Analyses , Agriculture Handbook No.379, United States Department of Agriculture, Washington, D.C. (1970).

E.4.8 Neutral Detergent Fiber Sample was placed in a fritte d vessel and washed with a ne utral boiling detergent solution that dissolved the protein, carbohydrate, enzy me, and ash. An acetone wash removed the fats and pigments. Hemicellulose, cellulose , and lignin fractions were collected on the frit and determined gravimetrically. The limit of quantitation was 0.100%.

Approved Methods of the American Association of Cereal Chemists , 9th Ed.,

Method 32.20, (1998).

Forage and Fiber Analyses , Agriculture Handbook No. 379, United States Department of Agriculture, (1970).Monsanto Company 07-CR-191U Page 315 of 544 E.4.9 Total Dietary Fiber Duplicate samples were gelatinized with α -amylase and digested with enzymes to break down starch and protein. Ethanol was added to each sample to precipitate the soluble fiber. The samples were filtered, and the residue was rinsed with ethanol and acetone to remove starch and protein degradation pr oducts and moisture. Protein content was determined for one of the duplicates; ash conten t was determined for the other. The total dietary fiber in the sample was calculated us ing the protein and ash values. The limit of quantitation was 1.0%.

Official Methods of Analysis of AOAC INTERNATIONAL

, 18 th Ed., Method 985.29,

AOAC INTERNATIONAL: Gaither sburg, Maryland, (2005).

E.4.10 Mineral Analysis by ICP Emission Spectrometry The sample was dried, precharred, and ashed overnight in a muffle set to maintain 500 ° C. The ashed sample was re-ashed with nitric aci d, treated with hydrochloric acid, taken to dryness, and put into a solution of 5% hydroc hloric acid. The amount of each element was determined at appropriate wavelengt hs by comparing the emission of the unknown sample, measured on the inducti vely coupled plasma spectrom eter, with the emission of

reference standards.

Table E-3. Reference Calibration Rang es and Limits of Quantitation

Mineral Reference Calibration Range (μ g/ml) Limit of Quantitation (ppm) Calcium 200, 1000 20.0 Copper 2, 10 0.50 Iron 10, 50 2.00 Magnesium 50, 250 20.0 Manganese 2, 10 0.30 Phosphorus 200, 1000 20.0 Potassium 200, 1000 100 Sodium 200, 1000 100 Zinc 10, 50 0.40

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 984.27 and 985.01, AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005).

E.4.11 Amino Acid Composition Samples were assayed by three methods to obtain the full profile. Tryptophan required a base hydrolysis with sodium hydroxide. Th e sulfur-containing ami no acids required an oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the samples for the remaining amino acids wa s accomplished through direct acid hydrolysis with hydrochloric acid. Once hydrolyzed, the individual amino acids were then quantitated using an automate d amino acid analyzer. The limit of quantitation was 0.100 mg/g.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Method 982.30,

AOAC INTERNATIONAL: Gaither sburg, Maryland, (2005).Monsanto Company 07-CR-191U Page 316 of 544 E.4.12 Fatty Acid Composition The lipid was extracted and saponified with 0.5N sodium hydroxide in methanol. The saponification mixture was met hylated with 14% boron trifl uoride in methanol. The resulting methyl esters were extracted with heptane containing an in ternal standard. The methyl esters of the fatty acids were an alyzed by gas chromatography using external standards for quantitation. Th e limit of quantitation was 0.00400%.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Method 996.06, AOAC INTERNATIONAL, Gaither sburg, Maryland, (2005).

Official Methods and Recommende d Practices of the AOCS , 5th Ed., Method Ce 1-62, American Oil Chemists’ Society: Champaign, Illinois, (1997).

E.4.13 Folic Acid Sample was hydrolyzed in a potassium phosph ate buffer with the ad dition of ascorbic acid to protect the folic acid during autoclav ing. Following hydrolysis by autoclaving, the sample was treated with a chicken-panc reas enzyme and incubated approximately 18

hours to liberate the bound folic acid. The amount of folic acid was determined by comparing the growth response of the sample, using the bacteria Lactobacillus casei

, with the growth response of a folic aci d standard. This response was measured turbidimetrically. The li mit of quantitation was 0.060 μ g/g.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 960.46 and 992.05, AOAC INTERNATIONAL, Gaithersburg, Maryland, (2005).

Methods of Analysis for Infant Formulas , Infant Formula Council, Atlanta, Georgia, Section C-2, (1985).

E.4.14 Niacin Sample was hydrolyzed with sulfuric aci d and the pH was adjusted to remove

interferences. The amount of niacin was determined by comparing the growth response of the sample, using the bacteria Lactobacillus plantarum, with the growth response of a niacin standard. This response was measured turbidimetrically. Th e limit of quantitation

was 0.300 μ g/g.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Method 944.13,

AOAC INTERNATIONAL: Gaither sburg, Maryland, (2005).

E.4.15 Thiamine Hydrochloride Sample was autoclaved under weak acid conditio ns to extract the thia mine. The resulting solution was incubated with a buffered enzyme solution to release any bound thiamine. The solution was purified on a cation-exchange column. An aliquot was reacted with potassium ferricyanide to convert thiamine to thiochrome. The thiochrome was extracted into isobutyl alcohol, measured on a fluorom eter, and quantitated by comparison to a known standard. The limit of quantitation was 0.01 mg/100g.Monsanto Company 07-CR-191U Page 317 of 544 Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 942.23, 953.17, and 957.17, AOAC INTERNATIONAL : Gaithersburg, Maryland, (2005).

E.4.16 Vitamin B 2 (Riboflavin) Sample was hydrolyzed with dilute hydrochlor ic acid and the pH was adjusted to remove

interferences. The amount of riboflavin was determined by comparing the growth response of the sample, using the bacteria Lactobacillus casei , with the growth response of multipoint riboflavin standard s. The growth response was measured turbidimetrically. The limit of quantitation was 0.200 μ g/g.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 940.33 and 960.46, AOAC INTERNATIONAL, Gaithersburg, Maryland, (2005).

The United States Pharmacopeia, Twenty-Ninth Revision, p. 1913, United States Pharmacopeial Convention, Inc.: Rockville, Maryland, (2005).

E.4.17 Pyridoxine Hydrochloride The sample was hydrolyzed with dilute sulf uric acid in the autoclave and the pH was adjusted to remove interferences. The amount of pyridoxine was determined by comparing the growth response of the sample, using the yeast Saccharomyces carlsbergensis, with the growth res ponse of a pyridoxine sta ndard. The response was measured turbidimetrically. The limit of quantitation was 0.070 μ g/g.

Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Method 961.15,

AOAC INTERNATIONAL: Gaither sburg, Maryland, (2005).

Atkins, L., Schultz, A. S., Williams, W. L ., and Frey, C. N., “Yeast Microbiological Methods for Determination of Vitamins,” Industrial and Engineering Chemistry, Analytical Edition, 15:141-144, (1943).

E.4.18 Vitamin E The product was saponified to br eak down any fat and release vitamin E. The saponified mixture was extracted with ethyl ether and then quantitated by hi gh-performance liquid chromatography using a silica column. Th e limit of quantitation for this study was approximately 0.500 mg/100g.

Cort, W. M., Vincente, T. S., Waysek, E. H., and Williams, B. D., “Vitamin E Content of Feedstuffs Determined by High- Performance Liquid Chromatographic Fluorescence,” Journal of Agricultural Food Chemistry, 31 :1330-1333, (1983).

Speek, A. J., Schrijver, J., and Schreurs, W. H. P., “Vitamin E Co mposition of Some Seed Oils as Determined by High-Pe rformance Liquid Ch romatography with Fluorometric Quantitation,” Journal of Food Science, 50 (1):121-124, (1985).

McMurray, C. H., Blanchflower, W. J., an d Rice, D. A., “Influ ence of Extraction Techniques on Determination of α -Tocopherol in Animal Feedstuffs,” Journal of the Association of Official Analytical Chemists, 63(6):1258-1261, (1980).Monsanto Company 07-CR-191U Page 318 of 544 E.4.19 p-Coumaric Acid and Ferulic Acid Sample was extracted with methanol using ul trasonication, hydroly zed using 4N sodium hydroxide, buffered using acetic acid/sodium hydroxide, acidified wit h 3N hydrochloric acid, and filtered. The leve ls of p-coumaric and ferulic acids in the extract were determined by reverse phase high-performan ce liquid chromatogra phy with ultraviolet detection. The limit of quantitat ion was approximately 50.0 ppm.

Hagerman, A. E. and Nicholson, R. L., “High-Performance Liquid Chromatographic Determination of Hydroxycinnamic Acids in Maize Mesocotyl,” Journal of Agricultural and Food Chemistry , 30 (No. 6):1098-1102, (1982).

E.4.20 Phytic Acid Sample was extracted using 0.5M HCl w ith ultrasonication. Purification and concentration were accomplished on a silica- based anion-exchange column. The sample was analyzed on a polymer high-performance liquid chromatography column PRP-1,

5 μ m (150 x 4.1mm) with a refractive index detector. The limit of quantitation was approximately 0.100%.

Lehrfeld, Jacob, “HPLC Separation and Quantit ation of Phytic Acid and Some Inositol Phosphates in Foods: Problem and Solutions,” Journal of Agricultural and Food Chemistry , 42:2726-2731, (1994).

Lehrfeld, Jacob, “High-Perfor mance Liquid Chromatography Analysis of Phytic Acid on a pH-Stable, Macropor ous Polymer Column,” Cereal Chemistry , 66(6):510-515, (1989).

E.4.21 Raffinose Sample was extracted with deionized water and the extract treated with a hydr oxylamine hydrochloride solution in pyridine, containing phenyl- β -D-glucoside as an internal standard. The resulting oximes were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization detector. The limit of quantitation was 0.0500%.

Brobst, K. M., "Gas-Liquid Chromatogr aphy of Trimethylsilyl Derivatives,” Methods in Carbohydrate Chemistry , Volume 6, Academic Press: New York, New York, (1972).

Mason, B. S., and Slover, H. T., "A Gas Chromatographic Method for the Determination of Sugars in Foods," Journal of Agricultu ral and Food Chemistry

, 19(3):551-554, (1971).

E.4.22 2-Furaldehyde (Furfural) Ground sample was extracted with 4% trichloroacetic acid and injected directly on a high-performance liquid chromatography system for quantitation of free furfurals by

ultraviolet detection. The lim it of quantitation was 0.500 ppm.

Albala-Hurtado S., Veciana-Nogues, M. T., Izquierdo-Pulido, M., and Vidal-Carou, M. C., “Determination of Free and Total Furf ural Compounds In Infant Milk FormulasMonsanto Company 07-CR-191U Page 319 of 544 By High-Performance Li quid Chromatography,” Journal of Agricultural and Food Chemistry , 45:2128-2133, (1997).

E.4.23 Sugar and Sugar Alcohols (SGAL) Sugars and sugar alcohols were extracted fr om the sample with wa ter. Aliquots were dried under inert gas and reconstituted with a hydroxylamine hydrochloride solution in pyridine containing phenyl- β -D-glucoside as the intern al standard. The resulting oximes were converted to silyl derivatives with hexamethyldisilazane (HMDS) and trifluoracetic acid (TFA) trea tment and analyzed by gas chromatography using a flame ionization detector. The limit of qua ntitation for this study was 0.0500%.

Mason, B. S. and Slover, H. T., "A Gas Chromatographic Method for the Determination of Sugars in Foods," Journal of Agricultural and Food Chemistry, 1971.

Brosbt, K., "Gas Liquid Chromatography of Trimethylsilyl Deriva tions," Methods in Carbohydrate Chemistry, 6:3-8, Acad emic Press, New York, NY. 1972.

E.4.24 Free Proline The sample was extracted in acid. De termination was by high-performance liquid chromatography (HPLC) with fluorescence or diode array detection. Primary amino acids were derivatized with o-phthalalde hyde and the secondary amino acids were

derivatized with fluo renylmethyl chloroformate before injection. The limit of quantitation for this study was 0.0100 mg/g.

R. Schuster, "Determination of Amino Acid s in Biological, Pharmaceutical, Plant and Food Samples by Automated Precolumn De rivatization and HP LC," Journal of Chromatography, 1988, 431, 271-284

Henderson, J. W., Ricker, R. D., Bidli ngmeyer, B. A., Woodward, C., "Rapid Accurate, Sensitive, and Reproducible HPLC Analysis of Amino Acids, Amino Acid Analysis Using Zorbax Eclipse-AAA colu mns and the Agilent 1100 HPLC," Agilent Publication, 2000.

E.4.25 Glycerol Glycerol was extracted from the sample with water. A portion of the extract was passed through glass microfiber filter paper and an appropriate dilution was made. The sample was injected onto a high performance anion exchange chromatograph (HPAEC) equipped with a Pulsed Amperometric Detector (PAD) . The amount of glycerol present was quantitated relative to an external standard curve using regression analysis. The limit of quantitation for this study was 20 ppm.

Hanko, V. P. and Rohrer, J. S., "Determi nation of Carbohydrates, Sugar Alcohols, and Glycols in Cell Cultures and Fermentati on Broths Using High-Performance AnionExchange Chromatography with Pulsed Amperometric Detection", Analytical Biochemistry, 283:192-199, (2000).Monsanto Company 07-CR-191U Page 320 of 544 E.4.26 Glycine Betaine, Choline, Sa licylic acid and Abscisic Acid Internal standard and extraction solvent (0.1 % formic acid in 50:50 methanol:water) were added to the sample. After centrifugation and filtration samples were analyzed by Liquid chromatography using MS/MS for detection. Specific precursor-fragment transitions were monitored for each analyte using the multiple reaction monitoring (MRM) technique. The analytes were identified by comparison to reference standards using the retention time of the specifi c precursor-fragment response.

E.5. Control of Bias The test, control, and reference substances fr om each respective plot within the field sites were produced under similar agronomic conditions . To control and/or minimize bias, the samples were analyzed in the order spec ified by a computer-generated randomized sample list. The Study Director generated th e randomized sample lis t and forwarded it to Covance Laboratories, Inc. prior to analysis.

E.6. Statistical Analysis

E.6.1. Data Processing

After compositional analyses were performed at Covance Laboratories, Inc., data spreadsheets were sent to Monsanto Company. The data were reviewed, formatted, and sent to Certus International, Inc. for stat istical analysis. A statistical sub-report was generated by Certus and sent to Monsanto Company. The following formulas were used for re-expression of the data for statistical analysis:

Table E-4. Unit Conversions
Component From (X) To Formula Proximates (excluding moisture), Fiber,

Anti-nutrients % FW % DW X/d

Calcium, Phosphorus, Magnesium, Potassium, Sodium ppm FW % DW (X/d) X 10 -4

Copper, Iron, Manganese, Zinc ppm FW mg/kg DW X/d Secondary Metabolites ppm FW μg/g DW X/d Thiamine HCl mg/100g FW mg/kg DW 10 (X/d) Vitamin E mg/g FW mg/kg DW 10 3 (X/d) Folic Acid, Niacin, Riboflavin, Pyridoxine HCl/Vitamin B6 μg/g FW mg/kg DW X/d

Amino Acids (AA) mg/g FW % DW X/(10*d)

Fatty Acids (FA) % FW % Total FA (100)X j / Σ X, for

each FA j where

Σ X is over all

the FA ‘X’ is the individual sample value; ‘d’ is the fr action of the sample that is dry matter.

E.6.1.1. U.S. 2006 Data Processing

In order to complete a statistical analysis fo r a compositional constitu ent in this study, at least 50% of the values for an analyte had to be greater than the assay LOQ. Analytes with greater than 50% of observations below the assay LOQ were excluded from summaries and analysis. The following 15 analyt es with greater than 50% of observationsMonsanto Company 07-CR-191U Page 321 of 544 below the assay LOQ were excluded from st atistical analysis: 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic ac id, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 17:0 heptadecan oic acid, 17:1 heptadecenoic acid, 18:3 gamma linolenic acid, 20:2 ei cosadienoic acid, 20:3 eico satrienoic acid, and 20:4 arachidonic acid, sodium, and furfural. These components na turally occur at very low

levels in corn.

For individual measurements below the assay’s LOQ, where fewer than 50% of the total values were below the LOQ, results were assigned a value equal to half the quantitation limit. The following analytes were assigned values:

Obs. Below LOQ

Component Units N (%) Total

N LOQ Value Assigned
Grain Fatty Acid

16:1 Palmitoleic % FW 13 24.1 54 0.0040 0.0020

22:0 Behenic % FW 1 1.9 54 0.0040 0.0020

The data were assessed for potential outli ers using a studentized PRESS residuals calculation. A predicted resi dual sums of squares (PRESS) residual is the difference between any value and its predicted value fr om a statistical model that excludes the datum point. The studentized version scales th ese residuals so that the values tend to

have a standard normal distribution when out liers are absent. Thus , most values are expected to be between ± 3. Extreme data points that are also outside of the ± 6 studentized PRESS residual range are considered for exclusion, as outliers, from the final analyses. For this study, no results ha d a PRESS residual va lue outside of the ± 6 studentized PRESS residual range.

E.6.1.2. Chile 2006/2007 Data Processing

In order to complete a statistical analysis fo r a compositional constitu ent in this study, at least 50% of the values for an analyte had to be greater than the assay LOQ. Analytes

with more than 50% of observations be low the assay LOQ were excluded from summaries and analysis. The following 16 anal ytes with more than 50% of observations below the assay LOQ were excluded from sta tistical analysis: 8: 0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic ac id, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic ac id, 16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gamma linolen ic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, 20 :4 arachidonic acid, so dium, and furfural.

Otherwise, results below the LOQ were assigned a value equal to half the quantitation limit. The following analytes were assigned values:Monsanto Company 07-CR-191U Page 322 of 544 Obs. Below LOQ

Component Units N (%) Total

N LOQ Value Assigned
Forage Proximate

Total Fat % FW 9 7.9 114 0.10 0.050 Grain Fatty Acid

22:0 Behenic % FW 30 26.5 113 0.0040 0.0020

Grain Vitamin

Vitamin E mg/g FW 5 4.4 113 0.0050 0.0025

Grain Anti-nutrients

Raffinose % FW 2 1.8 113 0.050 0.025

Individual samples assigned a value are repres ented in Listing 2 of the Statistical Subreport.

PRESS residuals were used to identify ou tliers. A PRESS residual is the difference between any value and its value predicted from a statistical model that excludes the datum point. The studentized version scales th ese residuals so that the values tend to have a standard normal distribution when outliers are absent. Thus, most values are expected to be between ± 3. Extreme datum points th at are also outside of the ± 6 studentized PRESS residual range are considered for exclusion, as outliers, from the final analyses. The following result ha d a PRESS residual value outside of ± 6 range:

Site Rep Description Analyte ID Sent

Value Value PRESS Std

Residual

Grain Mineral

CL 1 DM1718 Copper 0645B302-00804 12 13.5287 17.1470

The copper value was considered an outlier a nd was removed from further analysis. The outlier test procedure was reapplied to all remaining copper data to detect potential outliers that were masked in the first analys is. No further PRESS residuals were outside

of ± 6 range.

E.6.2. Statistical Methodology for U.S. 2006 At the field sites, the test, control, and re ference substances were grown in single plots randomly assigned within each of three replication blocks. The compositional components for the test and control substances were statistically analyzed using a mixed model analysis of variance. The data from the six replicated sites were analyzed separately and as a combined data set.

Individual replicated site analyses used the model:

Y ij = U + T i + B j + e ij ,

where Y ij = unique individual observa tion, U = overall mean, T i = hybrid effect, B j = random block effect, and e ij = residual error.

Combined site analyses used the model:Monsanto Company 07-CR-191U Page 323 of 544 Y ijk = U + T i + L j + B(L) jk

+ LT ij + e ijk ,

where Y ijk = unique individual observa tion, U = overall mean, T i = hybrid effect, L j = random location effect, B(L) jk

= random block within location effect, LT ij = random location by hybrid interaction effect, and e ijk = residual error. For each compositional component, the forage and grain from th e test substance was compared to the

conventional control.

A range of observed values from the refere nce substances was determined for each analytical component. Additionally, the refere nce substances data were used to develop population tolerance intervals. A tolerance interval is an interval that one can claim, with a specified degree of co nfidence, contains at least a specified proportio n, p, of an entire sampled population for the parameter measured. For each compositional component,

99% tolerance intervals were calculated th at are expected to contain, with 95% confidence, 99% of the quantities expressed in the population of co mmercial references (George et al., 2004; Ri dley et al., 2002). Each tolera nce interval estimate was based upon one observation per unique reference s ubstance. Individual substances with multiple observations were summarized with in sites to obtain a single estimate for

inclusion in tolerance interval calculations. Because negative quantiti es are not possible, calculated negative lower tolerance bounds were set to zero. SAS ® software was used to generate all summary statistics and perform all analyses (SAS ® Software Release 9.1, 2002-2003). Report tables pres ent p-values from SAS ® as either <0.001 or the actual value truncated to three decimal places.

E.6.3. Statistical Meth odology for Chile 2006/2007 All T/C/R substances were grown in single plots randomly assigned within each of three replication blocks. All co rn compositional analysis components were statistically analyzed using a mixed model analysis of va riance. The three replicated sites were analyzed both separately and combined. Indi vidual replicated site analyses used model (1).

(1) Y ijk = U + B i + T j + BT ij + S k + BS ik + TS jk + e ijk ,

where Y ijk = unique individual observa tion, U = overall mean, B i = random block effect,

T

j = irrigation treatment effect, BT ij = random block by treatmen t interaction effect, S k = substance effect, BS ik = random block by substance effect, TS jk

= treatment by substance

interaction effect and e ijk = residual error.Monsanto Company 07-CR-191U Page 324 of 544 Combined site analyses used model (2).

(2) Y ijkl = U + L i + B(L) ij + T k + LT ik + TB(L) ijk + S l + SB(L) ijl + TS kl + LS il + LTS ikl
+ e ijkl ,

where Y ijkl = unique individual observa tion, U = overall mean, L i = random location effect, B(L) ij = random block within location effect, T k = irrigation treat ment effect, LT ik = random location by treatment interaction effect, TB(L) ijk = random treatment by block within location interaction effect,

S l = substance effect, SB(L) ijl = random substance by block within location interaction effect, TS kl = treatment by substance interaction effect,

LS il = random location by substance interaction effect, LTS ikl = random location by treatment by substance in teraction effect and e ijkl = residual error.

For each component analysis, mean comparison tests of each test substance versus the conventional control substance within each irrigation treatment were conducted.

A tolerance interval is an interval that one can claim, with a specified degree of confidence, contains at least a specified proportion, p, of an entire sampled population for the parameter measured. For each compos itional component with in each irrigation treatment, 99% toleranc e intervals were calcula ted that are expected to contain, with 95% confidence, 99% of the quantities expr essed in the population of commercial

conventional substances. Each toleran ce interval estimate was based upon one observation per unique referen ce substance within each treatment. For each treatment, data were first summarized by substance withi n site and then by subs tance across sites. Because negative quantities are not possible, negative calculated lower tolerance bounds

were set to zero.

SAS ® programming was used to generate all su mmary statistics and perform all analyses (Version 9.1.3, SAS Institute , Inc. 2002-2003). Report tables present p-values from SAS

as either <0.001 or the actual valu e truncated to three decimal places.

E.7. References

AACC. Method 32.20. 1998. Approved Methods of the American Association of Cereal Chemists, 9th Ed.

Albala-Hurtado S., M.T. Veciana-Nogues, M. Izquierdo-Pulido and M.C. Vidal-Carou.

1997. Determination of free and total furfur al compounds in infant milk formulas by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry. 45:2128-2133.

AOAC. International Methods 922.06 and 954.02. 2005. Official Meth ods of Analysis,

18 th Ed. Association of Official Anal ytical Chemists, Gaithersburg, MD.

AOAC. International Methods 923.03. 2005. Official Methods of Analysis, 18 th Ed. Association of Official Analytic al Chemists, Gaithersburg, MD.

® SAS is a registered trademark of SAS Institute Inc.Monsanto Company 07-CR-191U Page 325 of 544 AOAC. International Methods 926.08 and 925.09. 2005. Official Meth ods of Analysis,

18 th Ed. Association of Official Anal ytical Chemists, Gaithersburg, MD.

AOAC. International Methods 940.33 and 960.46. 2005. Official Meth ods of Analysis,

18 th Ed. Association of Official Anal ytical Chemists, Gaithersburg, MD.

AOAC. International Methods 942.23, 953.17 and 957.17. 2005. Official Methods of Analysis, 18 th Ed. Association of Official An alytical Chemists, Gaithersburg,

MD.

AOAC. International Methods 944.13. 2005. Official Methods of Analysis, 18 th Ed. Association of Official Analytic al Chemists, Gaithersburg, MD.

AOAC International Methods 955.04 and 979.09 . 2005. Official Methods of Analysis,

18 th Ed. Association of Official Anal ytical Chemists, Gaithersburg, MD.

AOAC. International Methods 960.39 and 948.22. 2005. Official Meth ods of Analysis,

18 th Ed. Association of Official Anal ytical Chemists, Gaithersburg, MD.

AOAC. International Methods 960.46 and 992.05. 2005. Official Meth ods of Analysis,

18 th Ed. Association of Official Anal ytical Chemists, Gaithersburg, MD.

AOAC. International Methods 961.15. 2005. Official Methods of Analysis, 18 th Ed. Association of Official Analytic al Chemists, Gaithersburg, MD.

AOAC. International Methods 982.30. 2005. Official Methods of Analysis, 18 th Ed. Association of Official Analytic al Chemists, Gaithersburg, MD.

AOAC. International Methods 984.27 and 985.01. 2005. Official Meth ods of Analysis,

18 th Ed. Association of Official Anal ytical Chemists, Gaithersburg, MD.

AOAC. International Methods 985.29. 2005. Official Methods of Analysis, 18 th Ed. Association of Official Analytic al Chemists, Gaithersburg, MD.

AOAC. International Methods 996.06. 2005. Official Methods of Analysis, 18 th Ed. Association of Official Analytic al Chemists, Gaithersburg, MD.

AOCS. Method Ce 1-62. 1997. Official Meth ods and Recommended Practices of the American Oil Chemists Society, 5 th Ed. American Oil Chemists Society, Champaign, IL.

Atkins, L., A.S. Schultz, W.L. Williams, a nd C.N. Frey. 1943. “Yeast Microbiological Methods for Determination of Vitamins,” Industrial and Engineering Chemistry, Analytical Edition. 15:141-144.

Bradstreet, R. B. 1965. Pages 97-100 in Th e Kjeldahl Method fo r Organic Nitrogen. Academic Press, New York, NY.

Brobst, K. M. 1972. Gas-liquid chromatography of trimethylsilyl derivatives. Analysis of corn syrup. Pages 3-8 in Methods in Carbohydrate Chemistry. Vol. 6. Academic Press, New York, NY.

Cort, W.M., T.S. Vincente, E.H. Waysek a nd B.D. Williams. 1983. Vitamin E content of feedstuffs determined by high-performa nce liquid chromatographic fluorescence. Journal of Agricultural and Food Chemistry. 31:1330-1333.Monsanto Company 07-CR-191U Page 326 of 544 George, C., W.P. Ridley, J.C. Obert, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 2004. Composition of grain and forage from corn rootworm-protected corn event MON 863 is equivalent to th at of conventional corn ( Zea mays L.). Journal of Agricultural and Food Chemistry. 52:4149-4158.

Hagerman, A.E. and R. L. Nicholson. 1982. High-performance liquid chromatographic determination of hydroxycinnamic acids in the maize mesocotyl. Journal of Agricultural and Food Chemistry. 30:1098-1102.

Hanko, V.P. and J.A. Rohrer. 2000. Determin ation of carbohydrates, sugar alcohols, and glycols in cell cultures and fermentati on broths using high-performance anionexchange chromatography with pulsed amperometric detection. Analytical Biochemistry. 283:192-199.

Henderson, J.W., R.D. Ricker, B.A., Bi dlingmeyer, C. Woodward. 2000. Rapid Accurate, Sensitive, and Reproducible HP LC Analysis of Amino Acids, Amino Acid Analysis Using Zorbax Eclipse- AAA columns and the Agilent 1100 HPLC. Agilent Publication.

Lehrfeld, J. 1989. High-performance liquid ch romatography analysis of phytic acid on a pH-stable, macroporous polymer colu mn. Cereal Chemistry. 66:510-515.

Lehrfeld, J. 1994. HPLC separation and qua ntitation of phytic acid and some inositol phosphates in foods: problem and solutio ns Journal of Agricultural and Food Chemistry. 42:2726-2731.

Mason, B.S. and H.T. Slover. 1971. A gas ch romatographic method for the determination of sugars in foods. Journal of Agricu ltural and Food Chemistry. 19:551-554.

McMurray, C.H., W.J. Blanchflower and D. A. Rice. 1980. Influence of extraction techniques on determination of α -tocopherol in animal feedstuffs. JournalAssociation of Official An alytical Chemists. 63:1258-1261.

Methods of Analysis for Infant Formulas. S ection C-2. 1985. Infant Formula Council, Atlanta, GA.

OECD. 2002. Consensus document on composi tional considerations for new varieties of maize ( Zea mays ): key food and feed nutrients, an tinutrients and secondary plant metabolites. Organisation for Economic Co-operation and Development. Paris,

France.

OECD, 2006. An Introduction to the Food/Fe ed Safety Consensus Documents of the Task Force. Organisation for Economic Co-operation and Development. Paris,

France.

Ridley, W.P., R.S. Sidhu, P.D. Pyla, M. A. Nemeth, M.L. Breeze, and J.D. Astwood. 2002. Comparison of the nutritional prof ile of glyphosate-tole rant corn event
NK603 with that of conventional corn ( Zea mays L.). Journal of Agricultural and Food Chemistry. 50:7235-7243.

Schuster, R. 1988. Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn de rivatization and HPLC. Journal of Chromatography. 431:271-284.Monsanto Company 07-CR-191U Page 327 of 544 Speek, A.J., J. Schrijver, and W.H.P. Schr eurs. 1985. Vitamin E composition of some seed oils as determined by high-perf ormance liquid chromatography with

fluorometric detection. Journa l of Food Science. 50:121-124.

The United States Pharmacopeia. 2005. Twenty -Ninth Revision, p. 1913, United States Pharmacopeial Convention, Inc.: Rockville, Maryland.

USDA. 1970. Forage and Fiber Analysis. Ag riculture Handbook No. 379. United States Department of Agriculture, Washington, D.C.

USDA. 1973. Energy value of foods. Pages 2-11 in Agriculture Handbook No. 74. United States Department of Agri culture, Washington, D.C.Monsanto Company 07-CR-191U Page 328 of 544Table E-5. Comparison of Proximates, Fiber, and Mineral Content in Forage from MON 87460and Conventional Control forCombined Sites in the U.S. during2006 under Typical Agronomic Conditions1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Difference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]FiberAcid Detergent Fiber (% DW) 24.10 (0.96) 24.64 (0.96) -0.54 (0.92) -2.49, 1.41 0.567 (19.44 - 30.49)[17.78 - 34.43] [19.11 - 29.21] [-6.50 - 8.60] [13.04, 35.77]Neutral Detergent Fiber (% DW) 38.69 (1.99) 38.75 (1.99) -0.056 (1.08) -2.33, 2.22 0.959 (32.12 - 49.62)[31.10 - 49.44] [27.73 - 48.35] [-11.60 - 6.51] [24.23, 56.48]MineralCalcium (% DW) 0.21 (0.018) 0.22 (0.018) -0.018 (0.012) -0.048, 0.013 0.189 (0.12 - 0.25)[0.14 - 0.30] [0.13 - 0.33] [-0.075 - 0.089] [0.044, 0.35]Phosphorus (% DW) 0.18 (0.0089) 0.19 (0.0089) -0.0095 (0.0055) -0.021, 0.0021 0.101 (0.090 - 0.26)[0.14 - 0.22] [0.14 - 0.23] [-0.069 - 0.022] [0.074, 0.32]ProximateAsh (% DW) 3.76 (0.35) 4.21 (0.35) -0.44 (0.37) -1.39, 0.50 0.281 (2.67 - 4.43)[2.17 - 5.34] [2.94 - 8.01] [-3.73 - 1.22] [1.52, 5.75]Carbohydrates (% DW) 86.45 (0.54) 85.77 (0.54) 0.68 (0.49) -0.57, 1.93 0.220 (84.97 - 88.89)[83.78 - 88.75] [81.88 - 89.26] [-2.08 - 2.89] [82.09, 90.80]Moisture (% FW) 70.94 (1.25) 71.46 (1.25) -0.52 (0.37) -1.30, 0.25 0.174 (64.20 - 75.50)[64.70 - 77.90] [66.50 - 75.70] [-2.50 - 2.40] [59.32, 81.14]Protein (% DW) 7.56 (0.25) 7.85 (0.25) -0.30 (0.20) -0.71, 0.12 0.146 (5.80 - 8.63)[6.65 - 8.57] [6.45 - 10.24] [-2.63 - 1.14] [4.92, 10.30]Total Fat (% DW) 2.23 (0.20) 2.17 (0.20) 0.059 (0.13) -0.22, 0.34 0.659 (1.60 - 3.62)[1.07 - 3.24] [1.28 - 2.88] [-0.88 - 0.90] [0, 4.67]Monsanto Company 07-CR-191U Page 329 of 544Table E-6. Comparison of the Proximates and Fiber Content in Grain from MON 87460 and Conventional Control forCombined Sites in the U.S. during2006 under Typical Agronomic ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.[Range]Control Mean ±S.E.1[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]ProximateAsh (% DW) 1.54 (0.039) 1.46 (0.039) 0.082 (0.038) 0.0033, 0.16 0.041 (1.17 - 2.01)[1.33 - 1.83] [1.32 - 1.79] [-0.22 - 0.38] [0.55, 2.30]Carbohydrates (% DW) 84.22 (0.56) 84.10 (0.56) 0.13 (0.20) -0.30, 0.56 0.539 (82.11 - 87.06)[81.40 - 87.04] [81.31 - 86.05] [-1.57 - 1.98] [80.32, 89.92]Moisture (% FW) 9.94 (0.18) 10.09 (0.18) -0.15 (0.15) -0.54, 0.25 0.377 (8.74 - 11.30)[9.12 - 11.00] [9.17 - 11.20] [-1.36 - 0.83] [7.58, 12.13]Protein (% DW) 10.50 (0.54) 10.74 (0.54) -0.24 (0.18) -0.61, 0.13 0.195 (8.27 - 11.50)[8.19 - 13.21] [8.77 - 13.33] [-2.05 - 1.35] [6.26, 13.45]Total Fat (% DW) 3.74 (0.051) 3.71 (0.051) 0.029 (0.067) -0.14, 0.20 0.678 (2.95 - 4.40)[3.44 - 4.06] [3.57 - 3.96] [-0.52 - 0.32] [2.08, 5.12]FiberAcid Detergent Fiber (% DW) 3.03 (0.25) 3.02 (0.25) 0.0095 (0.36) -0.79, 0.81 0.979 (1.82 - 4.48)[1.57 - 4.94] [1.94 - 4.08] [-2.51 - 3.00] [0.62, 5.72]Neutral Detergent Fiber (% DW) 8.97 (0.32) 8.95 (0.32) 0.019 (0.46) -1.00, 1.03 0.967 (6.51 - 12.28)[6.45 - 11.63] [7.82 - 12.22] [-4.07 - 3.32] [3.45, 15.08]Total Dietary Fiber (% DW) 12.59 (0.34) 12.15 (0.34) 0.44 (0.35) -0.28, 1.16 0.216 (10.65 - 16.26)[10.42 - 14.57] [10.76 - 14.87] [-3.32 - 3.67] [8.11, 17.95]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 330 of 544Table E-7. Comparison of the MineralContent in Grain from MON 87460 and Conventional Control for Combined Sites inthe U.S. during 2006 under Typical Agronomic ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Calcium (% DW) 0.0054 (0.00019) 0.0054 (0.00019) -0.00006 (0.00007) -0.00020, 0.00009 0.431 (0.0036 - 0.0068)[0.0047 - 0.0061] [0.0048 - 0.0063] [-0.00059 - 0.00056] [0.0019, 0.0076]Copper (mg/kg DW) 1.89 (0.14) 1.86 (0.14) 0.022 (0.16) -0.32, 0.37 0.892 (1.14 - 2.56)[1.47 - 4.61] [1.54 - 3.43] [-1.31 - 2.11] [0.39, 3.21]Iron (mg/kg DW) 18.24 (0.62) 18.30 (0.62) -0.067 (0.50) -1.34, 1.21 0.898 (16.89 - 23.40)[15.02 - 24.88] [14.17 - 20.58] [-2.34 - 7.02] [13.28, 26.47]Magnesium (% DW) 0.11 (0.0042) 0.12 (0.0042) -0.0013 (0.0016) -0.0047, 0.0020 0.418 (0.091 - 0.14)[0.095 - 0.13] [0.095 - 0.13] [-0.010 - 0.013] [0.059, 0.16]Manganese (mg/kg DW) 6.79 (0.43) 6.89 (0.43) -0.097 (0.12) -0.41, 0.22 0.462 (4.83 - 8.05)[5.02 - 8.64] [5.50 - 8.34] [-0.97 - 0.68] [2.27, 9.92]Phosphorus (% DW) 0.31 (0.011) 0.32 (0.011) -0.0085 (0.0047) -0.018, 0.0014 0.089 (0.24 - 0.36)[0.27 - 0.35] [0.27 - 0.37] [-0.030 - 0.034] [0.20, 0.40]Potassium (% DW) 0.38 (0.0030) 0.38 (0.0030) 0.0019 (0.0037) -0.0060, 0.0097 0.624 (0.29 - 0.37)[0.36 - 0.39] [0.35 - 0.39] [-0.025 - 0.038] [0.26, 0.42]Zinc (mg/kg DW) 20.86 (0.95) 21.24 (0.95) -0.38 (0.32) -1.03, 0.27 0.238 (16.78 - 28.17)[18.24 - 24.75] [17.41 - 25.20] [-3.02 - 1.85] [11.61, 32.63]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 331 of 544Table E-8. Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Control for Combined Sitesin the U.S. during 2006 under Typical Agronomic ConditionsDifference (Test minus Control)AnalyticalComponent1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Alanine (% DW) 0.80 (0.047) 0.82 (0.047) -0.011 (0.013) -0.037, 0.016 0.410 (0.60 - 0.91)[0.60 - 1.04] [0.64 - 1.04] [-0.10 - 0.10] [0.43, 1.08]Arginine (% DW) 0.45 (0.019) 0.44 (0.019) 0.0067 (0.011) -0.022, 0.035 0.577 (0.34 - 0.51)[0.33 - 0.54] [0.38 - 0.52] [-0.071 - 0.087] [0.24, 0.60]Aspartic acid (% DW) 0.65 (0.028) 0.66 (0.028) -0.0062 (0.0075) -0.022, 0.0096 0.419 (0.52 - 0.72)[0.52 - 0.79] [0.54 - 0.78] [-0.065 - 0.060] [0.39, 0.84]Cystine (% DW) 0.23 (0.0085) 0.23 (0.0085) -0.0040 (0.0018) -0.0087, 0.00069 0.079 (0.19 - 0.24)[0.19 - 0.27] [0.20 - 0.26] [-0.016 - 0.012] [0.15, 0.27]Glutamic acid (% DW) 2.07 (0.12) 2.09 (0.12) -0.025 (0.034) -0.097, 0.046 0.462 (1.54 - 2.32)[1.52 - 2.66] [1.64 - 2.67] [-0.26 - 0.28] [1.06, 2.76]Glycine (% DW) 0.39 (0.013) 0.39 (0.013) 0.0019 (0.0041) -0.0085, 0.012 0.656 (0.33 - 0.42)[0.33 - 0.45] [0.34 - 0.43] [-0.024 - 0.035] [0.26, 0.47]Histidine (% DW) 0.32 (0.012) 0.32 (0.012) -0.00085 (0.0049) -0.013, 0.012 0.868 (0.25 - 0.33)[0.25 - 0.38] [0.27 - 0.37] [-0.029 - 0.040] [0.20, 0.36]Isoleucine (% DW) 0.38 (0.021) 0.38 (0.021) -0.0022 (0.0088) -0.025, 0.020 0.810 (0.30 - 0.41)[0.28 - 0.50] [0.31 - 0.48] [-0.042 - 0.070] [0.22, 0.49]Monsanto Company 07-CR-191U Page 332 of 544Table E-8 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Control forCombined Sites in the U.S. during2006 under Typical Agronomic ConditionsDifference (Test minus Control)AnalyticalComponent1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Leucine (% DW) 1.41 (0.088) 1.43 (0.088) -0.020 (0.026) -0.075, 0.035 0.453 (1.02 - 1.55)[1.01 - 1.85] [1.11 - 1.87] [-0.20 - 0.22] [0.68, 1.90]Lysine (% DW) 0.30 (0.0076) 0.30 (0.0076) 0.0024 (0.0040) -0.0080, 0.013 0.579 (0.27 - 0.32)[0.26 - 0.34] [0.26 - 0.33] [-0.023 - 0.027] [0.22, 0.36]Methionine (% DW) 0.22 (0.013) 0.22 (0.013) -0.00089 (0.0030) -0.0086, 0.0068 0.777 (0.17 - 0.24)[0.16 - 0.28] [0.17 - 0.26] [-0.019 - 0.019] [0.14, 0.28]Phenylalanine (% DW) 0.56 (0.031) 0.56 (0.031) -0.0059 (0.0090) -0.025, 0.013 0.518 (0.43 - 0.61)[0.41 - 0.72] [0.45 - 0.72] [-0.067 - 0.074] [0.30, 0.74]Proline (% DW) 1.01 (0.047) 1.02 (0.047) -0.0048 (0.017) -0.050, 0.040 0.793 (0.74 - 1.01)[0.78 - 1.23] [0.83 - 1.21] [-0.082 - 0.17] [0.56, 1.19]Serine (% DW) 0.53 (0.028) 0.53 (0.028) -0.0070 (0.0086) -0.025, 0.011 0.430 (0.39 - 0.60)[0.40 - 0.64] [0.43 - 0.67] [-0.089 - 0.046] [0.27, 0.70]Threonine (% DW) 0.37 (0.016) 0.37 (0.016) -0.00059 (0.0050) -0.011, 0.010 0.908 (0.29 - 0.40)[0.30 - 0.45] [0.31 - 0.45] [-0.036 - 0.037] [0.22, 0.46]Tryptophan (% DW) 0.066 (0.0027) 0.068 (0.0027) -0.0015 (0.0017) -0.0050, 0.0021 0.394 (0.047 - 0.070)[0.054 - 0.088] [0.055 - 0.085] [-0.014 - 0.016] [0.037, 0.081]Monsanto Company 07-CR-191U Page 333 of 544Table E-8 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Control forCombined Sites in the U.S. during2006 under Typical Agronomic ConditionsDifference (Testminus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Tyrosine (% DW) 0.32 (0.024) 0.30 (0.024) 0.014 (0.022) -0.044, 0.071 0.565 (0.13 - 0.37)[0.16 - 0.43] [0.15 - 0.43] [-0.12 - 0.21] [0.0046, 0.54]Valine (% DW) 0.52 (0.024) 0.52 (0.024) -0.0019 (0.011) -0.029, 0.026 0.866 (0.42 - 0.54)[0.40 - 0.64] [0.43 - 0.62] [-0.053 - 0.079] [0.33, 0.62]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 334 of 544Table E-9. Comparison of the Fatty Acid Content in Grain from MON 87460 and Conventional Control for Combined Sitesin the U.S. during 2006 under Typical Agronomic ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]16:0 Palmitic (% Total FA) 12.12 (0.20) 11.94 (0.20) 0.18 (0.19) -0.31, 0.66 0.394 (8.80 - 13.33)[11.60 - 15.21] [11.45 - 12.38] [-0.28 - 2.84] [6.35, 16.03]16:1 Palmitoleic (% Total FA) 0.17 (0.0073) 0.17 (0.0073) -0.0020 (0.0036) -0.0095,0.0055 0.576 (0.059 - 0.15)[0.15 - 0.20] [0.15 - 0.23] [-0.042 - 0.015] [0, 0.21]18:0 Stearic (% Total FA) 2.05 (0.033) 1.98 (0.033) 0.069 (0.022) 0.013, 0.13 0.024 (1.36 - 2.14)[1.88 - 2.34] [1.80 - 2.10] [-0.041 - 0.33] [1.00, 2.51]18:1 Oleic (% Total FA) 20.26 (0.18) 20.49 (0.18) -0.23 (0.12) -0.48, 0.015 0.064 (21.17 - 33.71)[19.32 - 21.08] [19.50 - 21.77] [-1.13 - 0.85] [11.92, 39.78]18:2 Linoleic (% Total FA) 63.34 (0.35) 63.34 (0.35) -0.0079 (0.26) -0.67, 0.65 0.976 (49.31 - 62.94)[59.90 - 65.07] [61.88 - 64.70] [-3.07 - 1.05] [45.91, 72.47]18:3 Linolenic (% Total FA) 1.28 (0.012) 1.27 (0.012) 0.0066 (0.015) -0.029, 0.042 0.673 (0.89 - 1.56)[1.17 - 1.46] [1.22 - 1.33] [-0.046 - 0.20] [0.39, 1.85]20:0 Arachidic (% Total FA) 0.41 (0.0078) 0.41 (0.0078) 0.0037 (0.0032) -0.0031, 0.010 0.263 (0.30 - 0.49)[0.39 - 0.44] [0.37 - 0.45] [-0.017 - 0.024] [0.23, 0.56]20:1 Eicosenoic (% Total FA) 0.18 (0.0024) 0.19 (0.0024) -0.0078 (0.0027) -0.013, -0.0022 0.007 (0.20 - 0.29)[0.17 - 0.19] [0.17 - 0.22] [-0.035 - 0.0086] [0.15, 0.33]22:0 Behenic (% Total FA) 0.20 (0.012) 0.20 (0.012) -0.0044 (0.013) -0.037, 0.028 0.742 (0.069 - 0.28)[0.14 - 0.27] [0.14 - 0.27] [-0.099 - 0.078] [0, 0.37]1FA = fatty acid S.E. = standarderror; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 335 of 544Table E-10. Comparison of the VitaminContent in Grain from MON 87460 and Conventional Control for Combined Sites inthe U.S. during 2006 under Typical Agronomic ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Folic Acid (mg/kg DW) 0.30 (0.012) 0.30 (0.012) 0.0058 (0.0059) -0.0094, 0.021 0.371 (0.19 - 0.31)[0.25 - 0.36] [0.24 - 0.35] [-0.033 - 0.041] [0.13, 0.38]Niacin (mg/kg DW) 18.59 (0.77) 18.52 (0.77) 0.069 (0.53) -1.29, 1.42 0.901 (15.07 - 32.38)[15.53 - 22.23] [15.26 - 21.85] [-3.73 - 4.43] [4.67, 36.68]Thiamine HCl VitaminB1 (mg/kg DW)3.31 (0.16) 3.21 (0.16) 0.094 (0.066) -0.077, 0.26 0.216 (2.43 - 4.17)[2.67 - 3.89] [2.33 - 3.89] [-0.44 - 0.54] [1.84, 4.94]Riboflavin/Vitamin B2(mg/kg DW)1.54 (0.084) 1.44 (0.084) 0.10 (0.097) -0.15, 0.36 0.331 (0.95 - 2.42)[0.95 - 2.04] [0.94 - 1.94] [-0.88 - 0.60] [0.047, 2.91]Pyridoxine HCl/Vitamin B6 (mg/kg DW)6.10 (0.25) 6.24 (0.25) -0.13 (0.17) -0.48, 0.21 0.436 (4.93 - 7.53)[5.03 - 7.49] [5.21 - 7.41] [-1.55 - 1.66] [3.12, 8.09]Vitamin E (mg/kg DW) 14.73 (0.80) 14.69 (0.80) 0.045 (0.53) -1.31, 1.40 0.935 (5.96 - 17.70)[11.09 - 20.02] [9.47 - 18.44] [-3.95 - 4.77] [0, 26.07]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 336 of 544Table E-11. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain from MON 87460 andConventional Control for Combined Sites in the U.S. during 2006 under Typical Agronomic ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Anti-nutrientPhytic Acid (% DW) 0.83 (0.038) 0.84 (0.038) -0.0087 (0.028) -0.080, 0.063 0.767 (0.69 - 0.98)[0.60 - 1.00] [0.69 - 1.09] [-0.15 - 0.19] [0.50, 1.11]Raffinose (% DW) 0.19 (0.0081) 0.18 (0.0081) 0.014 (0.0082) -0.0074, 0.035 0.155 (0.079 - 0.19)[0.15 - 0.22] [0.15 - 0.22] [-0.036 - 0.050] [0.039, 0.26]Secondary MetaboliteFerulic Acid (μg/g DW) 1772.22 (47.57) 1693.18 (47.57) 79.04 (62.05) -80.47, 238.55 0.258 (1205.75 - 2873.05)[1561.63 - 1966.67] [1245.83 - 1997.77] [-210.94 - 533.93] [395.96, 3485.38]p-Coumaric Acid (μg/g DW) 115.95 (4.15) 126.55 (4.15) -10.61 (4.60) -22.44, 1.22 0.069 (128.21 - 327.39)[99.45 - 136.67] [94.77 - 156.25] [-38.32 - 27.59] [7.61, 408.53]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 337 of 544Table E-12. Comparison of Proximates, Fiber, and MineralContent in Forage from MON 87460 and Conventional Controlfor Combined Sites in Chile during 2006/2007 under Well-Watered Conditions1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Difference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]FiberAcid Detergent Fiber (% DW) 28.37 (1.45) 30.43 (1.45) -2.07 (1.79) -5.85, 1.72 0.264 (25.07 - 37.22)[17.95 - 34.70] [24.98 - 35.12] [-10.93 - 5.34] [16.01, 45.98]Neutral Detergent Fiber (% DW) 42.02 (1.52) 44.51 (1.52) -2.49 (1.81) -6.10, 1.11 0.171 (37.84 - 49.16)[36.08 - 50.00] [39.08 - 47.24] [-8.10 - 8.70] [27.28, 58.88]MineralCalcium (% DW) 0.26 (0.020) 0.27 (0.020) -0.0091 (0.019) -0.048, 0.029 0.628 (0.17 - 0.36)[0.24 - 0.28] [0.22 - 0.39] [-0.11 - 0.051] [0.043, 0.46]Phosphorus (% DW) 0.16 (0.0077) 0.16 (0.0077) 0.0011 (0.0061) -0.011, 0.013 0.852 (0.13 - 0.18)[0.12 - 0.19] [0.13 - 0.20] [-0.030 - 0.033] [0.086, 0.22]ProximateAsh (% DW) 4.71 (0.22) 4.89 (0.22) -0.18 (0.20) -0.59, 0.22 0.366 (4.12 - 6.12)[4.25 - 5.35] [3.88 - 6.05] [-1.33 - 0.73] [2.42, 8.00]Carbohydrates (% DW) 87.61 (0.42) 87.11 (0.42) 0.50 (0.40) -0.29, 1.29 0.208 (85.54 - 89.52)[86.51 - 89.58] [85.87 - 88.50] [-0.52 - 2.34] [82.51, 92.09]Moisture (% FW) 74.02 (0.73) 75.19 (0.73) -1.17 (0.70) -2.65, 0.31 0.113 (71.40 - 76.80)[70.90 - 75.90] [74.20 - 78.00] [-4.40 - 1.70] [69.22, 81.25]Protein (% DW) 6.53 (0.40) 6.71 (0.40) -0.18 (0.22) -0.62, 0.25 0.407 (5.56 - 7.39)[5.29 - 7.10] [6.01 - 7.44] [-1.20 - 1.09] [4.12, 8.77]Total Fat (% DW) 1.16 (0.16) 1.30 (0.16) -0.14 (0.23) -0.60, 0.33 0.557 (0.20 - 2.26)[0.57 - 1.96] [0.51 - 2.33] [-0.71 - 0.68] [0, 3.59]Monsanto Company 07-CR-191U Page 338 of 544Table E-13. Comparison of the Proximates and Fiber Content in Grain from MON 87460 and Conventional Control forCombined Sites in Chile during2006/2007 under Well-Watered ConditionsDifference (Test minus Control)Analytical Component1Test Mean ±S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]ProximateAsh (% DW) 1.44 (0.038) 1.42 (0.038) 0.015 (0.048) -0.083, 0.11 0.751 (1.14 - 1.47)[1.35 - 1.53] [1.26 - 1.60] [-0.22 - 0.20] [0.90, 1.76]Carbohydrates (% DW) 85.17 (0.27) 85.53 (0.27) -0.37 (0.36) -1.08, 0.35 0.310 (83.60 - 86.65)[82.98 - 87.63] [84.91 - 86.31] [-2.41 - 2.56] [81.08, 89.71]Moisture (% FW) 12.09 (0.15) 12.09 (0.15) 0 (0.21) -0.44, 0.44 1.000 (11.00 - 12.20)[11.80 - 12.50] [11.30 - 12.80] [-0.70 - 0.70] [10.10, 13.35]Protein (% DW) 9.50 (0.23) 9.32 (0.23) 0.18 (0.29) -0.40, 0.76 0.533 (8.69 - 11.33)[7.57 - 11.32] [8.55 - 9.77] [-1.93 - 1.61] [5.83, 13.57]Total Fat (% DW) 3.89 (0.082) 3.72 (0.082) 0.17 (0.071) 0.018, 0.32 0.029 (3.16 - 4.07)[3.45 - 4.23] [3.60 - 3.90] [-0.42 - 0.61] [2.47, 4.68]FiberAcid Detergent Fiber (% DW) 2.57 (0.21) 2.47 (0.21) 0.11 (0.27) -0.44, 0.65 0.696 (1.95 - 3.76)[2.08 - 3.18] [1.41 - 4.41] [-1.43 - 0.96] [0.29, 5.01]Neutral Detergent Fiber (% DW) 8.66 (0.34) 8.60 (0.34) 0.063 (0.44) -0.86, 0.99 0.887 (7.15 - 9.41)[8.19 - 9.45] [7.74 - 9.70] [-1.41 - 1.62] [5.23, 10.90]Total Dietary Fiber (% DW) 12.70 (0.44) 12.53 (0.44) 0.17 (0.50) -0.90, 1.24 0.737 (10.24 - 13.51)[11.59 - 16.00] [11.20 - 13.98] [-1.15 - 2.97] [6.72, 16.07]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 339 of 544Table E-14. Comparison of the Mineral Content in Grain from MON 87460 and Conventional Control for Combined Sites inChile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Calcium (% DW) 0.0047 (0.00045) 0.0045 (0.00045) 0.00014 (0.00029) -0.00044, 0.00072 0.630 (0.0032 - 0.0057)[0.0035 - 0.0058] [0.0036 - 0.0059] [-0.00060 - 0.0012] [0.00076, 0.0080]Copper (mg/kg DW) 1.86 (0.24) 1.87 (0.24) -0.011 (0.27) -0.60, 0.58 0.966 (1.29 - 4.16)[1.58 - 2.17] [1.47 - 2.81] [-0.89 - 0.37] [0, 5.74]Iron (mg/kg DW) 16.60 (0.68) 16.95 (0.68) -0.36 (0.61) -1.58, 0.86 0.561 (14.37 - 19.48)[14.53 - 20.30] [15.22 - 19.95] [-2.07 - 1.58] [10.40, 25.42]Magnesium (% DW) 0.12 (0.0034) 0.11 (0.0034) 0.0094 (0.0037) 0.0021, 0.017 0.012 (0.095 - 0.13)[0.10 - 0.14] [0.10 - 0.11] [-0.0076 - 0.028] [0.064, 0.16]Manganese (mg/kg DW) 6.27 (0.40) 6.03 (0.40) 0.24 (0.29) -0.59, 0.87 0.434 (4.55 - 9.02)[5.25 - 7.08] [4.64 - 7.58] [-1.51 - 1.61] [0.69, 10.70]Phosphorus (% DW) 0.32 (0.0099) 0.30 (0.0099) 0.019 (0.0098) -0.00060, 0.038 0.057 (0.27 - 0.36)[0.29 - 0.34] [0.26 - 0.33] [-0.034 - 0.086] [0.21, 0.40]Potassium (% DW) 0.40 (0.011) 0.40 (0.011) -0.0039 (0.0084) -0.021, 0.013 0.643 (0.32 - 0.42)[0.37 - 0.41] [0.36 - 0.45] [-0.078 - 0.047] [0.25, 0.47]Zinc (mg/kg DW) 22.00 (1.00) 21.02 (1.00) 0.99 (0.80) -0.60, 2.57 0.219 (18.12 - 29.69)[19.20 - 25.09] [18.36 - 25.34] [-3.91 - 5.47] [7.39, 38.63]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 340 of 544Table E-15. Comparison of the AminoAcid Content in Grain from MON 87460 and Conventional Control for CombinedSites in Chile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Alanine (% DW) 0.71 (0.018) 0.70 (0.018) 0.017 (0.025) -0.032, 0.066 0.500 (0.66 - 0.89)[0.63 - 0.86] [0.63 - 0.76] [-0.077 - 0.14] [0.44, 1.06]Arginine (% DW) 0.39 (0.012) 0.38 (0.012) 0.0079 (0.017) -0.026, 0.042 0.639 (0.34 - 0.46)[0.32 - 0.47] [0.32 - 0.43] [-0.030 - 0.14] [0.23, 0.55]Aspartic Acid (% DW) 0.61 (0.013) 0.60 (0.013) 0.0088 (0.017) -0.024, 0.042 0.598 (0.58 - 0.77)[0.54 - 0.70] [0.56 - 0.63] [-0.057 - 0.073] [0.39, 0.88]Cystine (% DW) 0.22 (0.0043) 0.21 (0.0043) 0.0030 (0.0047) -0.0064, 0.012 0.527 (0.20 - 0.24)[0.20 - 0.23] [0.20 - 0.22] [-0.0082 - 0.017] [0.16, 0.27]Glutamic Acid (% DW) 1.84 (0.047) 1.80 (0.047) 0.043 (0.064) -0.085, 0.17 0.503 (1.64 - 2.26)[1.63 - 2.21] [1.62 - 1.97] [-0.22 - 0.35] [1.09, 2.72]Glycine (% DW) 0.35 (0.0063) 0.34 (0.0063) 0.0063 (0.0077) -0.0090, 0.022 0.414 (0.31 - 0.38)[0.32 - 0.39] [0.31 - 0.35] [-0.029 - 0.041] [0.26, 0.42]Histidine (% DW) 0.29 (0.0056) 0.29 (0.0056) 0.0034 (0.0074) -0.011, 0.018 0.645 (0.24 - 0.30)[0.26 - 0.34] [0.26 - 0.30] [-0.031 - 0.037] [0.20, 0.34]Isoleucine (% DW) 0.33 (0.0092) 0.33 (0.0092) 0.0014 (0.013) -0.024, 0.026 0.908 (0.30 - 0.41)[0.29 - 0.42] [0.30 - 0.36] [-0.044 - 0.074] [0.19, 0.49]Monsanto Company 07-CR-191U Page 341 of 544Table E-15 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Control forCombined Sites in Chile during2006/2007 under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Leucine (% DW) 1.23 (0.034) 1.19 (0.034) 0.036 (0.046) -0.057, 0.13 0.441 (1.06 - 1.53)[1.08 - 1.52] [1.08 - 1.30] [-0.13 - 0.28] [0.66, 1.87]Lysine (% DW) 0.28 (0.0045) 0.28 (0.0045) 0.00075 (0.0055) -0.010, 0.012 0.892 (0.25 - 0.31)[0.26 - 0.31] [0.26 - 0.29] [-0.027 - 0.018] [0.19, 0.35]Methionine (% DW) 0.18 (0.0073) 0.18 (0.0073) 0.0032 (0.0065) -0.0098, 0.016 0.625 (0.18 - 0.23)[0.16 - 0.21] [0.16 - 0.19] [-0.021 - 0.027] [0.14, 0.26]Phenylalanine (% DW) 0.49 (0.012) 0.48 (0.012) 0.011 (0.017) -0.023, 0.044 0.535 (0.44 - 0.60)[0.43 - 0.60] [0.43 - 0.52] [-0.056 - 0.10] [0.28, 0.72]Proline (% DW) 0.88 (0.027) 0.87 (0.027) 0.012 (0.030) -0.049, 0.072 0.705 (0.72 - 0.99)[0.77 - 1.07] [0.82 - 0.95] [-0.092 - 0.16] [0.48, 1.18]Serine (% DW) 0.47 (0.012) 0.45 (0.012) 0.019 (0.016) -0.013, 0.051 0.224 (0.43 - 0.55)[0.43 - 0.54] [0.40 - 0.50] [-0.041 - 0.071] [0.32, 0.65]Threonine (% DW) 0.32 (0.0069) 0.32 (0.0069) 0.0043 (0.0090) -0.014, 0.022 0.634 (0.30 - 0.37)[0.28 - 0.37] [0.29 - 0.33] [-0.033 - 0.040] [0.23, 0.42]Tryptophan (% DW) 0.051 (0.0020) 0.051 (0.0020) 0.00095 (0.0025) -0.0039, 0.0058 0.701 (0.040 - 0.059)[0.039 - 0.063] [0.046 - 0.054] [-0.013 - 0.012] [0.022, 0.078]Monsanto Company 07-CR-191U Page 342 of 544Table E-15 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Control forCombined Sites in Chile during2006/2007 under Well-Watered ConditionsDifference (Testminus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Tyrosine (% DW) 0.23 (0.023) 0.25 (0.023) -0.022 (0.032) -0.089, 0.044 0.496 (0.14 - 0.32)[0.12 - 0.35] [0.13 - 0.32] [-0.11 - 0.079] [0, 0.53]Valine (% DW) 0.46 (0.011) 0.46 (0.011) 0.0017 (0.014) -0.027, 0.031 0.909 (0.41 - 0.54)[0.42 - 0.56] [0.41 - 0.49] [-0.054 - 0.077] [0.29, 0.62]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 343 of 544Table E-16. Comparison of the Fatty Acid Content in Grain from MON 87460 and ConventionalControl for Combined Sitesin Chile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]16:0 Palmitic (% Total FA)10.93 (0.13) 11.15 (0.13) -0.22 (0.16) -0.54, 0.10 0.173 (9.53 - 12.33)[10.63 - 11.60] [10.96 - 11.32] [-0.69 - 0.33] [7.43, 14.09]18:0 Stearic (% Total FA)1.80 (0.045) 1.78 (0.045) 0.020 (0.047) -0.078, 0.12 0.675 (1.28 - 2.13)[1.74 - 1.97] [1.66 - 1.91] [-0.11 - 0.22] [0.60, 2.58]18:1 Oleic (% Total FA)20.93 (0.37) 21.01 (0.37) -0.079 (0.29) -0.69, 0.53 0.786 (22.13 - 31.09)[20.29 - 21.28] [19.78 - 21.93] [-1.20 - 0.51] [12.40, 36.28]18:2 Linoleic (% Total FA)64.51 (0.45) 64.21 (0.45) 0.30 (0.42) -0.59, 1.19 0.485 (55.17 - 64.97)[63.92 - 65.27] [63.22 - 65.48] [-0.81 - 1.59] [49.61, 73.18]18:3 Linolenic (% Total FA)1.18 (0.016) 1.21 (0.016) -0.027 (0.014) -0.056, 0.00085 0.057 (1.00 - 1.32)[1.15 - 1.23] [1.19 - 1.25] [-0.049 - -0.013] [0.72, 1.66]20:0 Arachidic (% Total FA)0.31 (0.011) 0.32 (0.011) -0.0054 (0.0058) -0.018, 0.0067 0.367 (0.29 - 0.42)[0.29 - 0.34] [0.29 - 0.34] [-0.026 - 0.0090] [0.19, 0.52]20:1 Eicosenoic (% Total FA)0.18 (0.0039) 0.19 (0.0039) -0.0041 (0.0039) -0.012, 0.0038 0.301 (0.20 - 0.31)[0.17 - 0.20] [0.17 - 0.20] [-0.016 - 0.0089] [0.10, 0.36]22:0 Behenic (% Total FA)0.15 (0.020) 0.13 (0.020) 0.015 (0.026) -0.039, 0.070 0.559 (0.061 - 0.33)[0.062 - 0.26] [0.063 - 0.17] [-0.075 - 0.097] [0, 0.48]1DW = dry weight; FA = fatty acid; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 344 of 544Table E-17. Comparison of the VitaminContent in Grain from MON 87460 and Conventional Control for Combined Sites inChile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Folic Acid (mg/kg DW) 0.26 (0.018) 0.27 (0.018) -0.0094 (0.016) -0.044, 0.025 0.569 (0.26 - 0.41)[0.23 - 0.29] [0.22 - 0.33] [-0.085 - 0.029] [0.11, 0.55]Niacin (mg/kg DW) 19.17 (1.77) 19.23 (1.77) -0.054 (1.81) -3.66, 3.56 0.976 (14.92 - 26.80)[16.42 - 21.50] [17.42 - 21.17] [-1.62 - 2.69] [5.96, 38.50]Thiamine HCl/VitaminB1 (mg/kg DW)2.86 (0.084) 2.86 (0.084) 0.00065 (0.088) -0.19, 0.19 0.994 (2.94 - 4.78)[2.61 - 3.19] [2.74 - 3.06] [-0.22 - 0.44] [1.01, 6.00]Riboflavin/Vitamin B2(mg/kg DW)2.01 (0.14) 1.97 (0.14) 0.040 (0.14) -0.24, 0.32 0.781 (1.62 - 2.62)[1.61 - 2.54] [1.46 - 2.63] [-1.02 - 0.61] [0.87, 3.38]Pyridoxine HCl/VitaminB6 (mg/kg DW)6.32 (0.27) 6.83 (0.27) -0.51 (0.33) -1.21, 0.19 0.143 (4.01 - 6.70)[5.49 - 7.39] [6.17 - 7.37] [-1.32 - 0.21] [1.86, 8.29]Vitamin E (mg/kg DW) 11.90 (0.40) 10.99 (0.40) 0.91 (0.54) -0.23, 2.05 0.110 (2.83 - 11.69)[10.64 - 13.57] [9.30 - 12.78] [-0.81 - 2.32] [0, 19.32]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 345 of 544Table E-18. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain from MON 87460 andConventional Control for Combined Sites inChile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Anti-nutrientPhytic Acid (% DW) 0.76 (0.035) 0.76 (0.035) 0.0069 (0.043) -0.083, 0.096 0.873 (0.58 - 0.97)[0.58 - 0.93] [0.63 - 0.90] [-0.21 - 0.31] [0.28, 1.15]Raffinose (% DW) 0.11 (0.013) 0.11 (0.013) -0.0015 (0.0050) -0.012, 0.0089 0.767 (0.028 - 0.15)[0.075 - 0.12] [0.077 - 0.14] [-0.022 - 0.013] [0, 0.21]Secondary MetaboliteFerulic Acid (μg/g DW) 1849.20 (114.60) 1753.19 (114.60) 96.01 (160.59) -224.84, 416.86 0.552 (1504.52 - 2224.72)[1265.68 - 2240.00] [820.14 - 2128.15] [-660.93 - 1232.32] [1019.70, 2703.40]p-Coumaric Acid (μg/g DW) 137.39 (15.51) 149.37 (15.51) -11.98 (18.53) -50.95, 26.99 0.526 (84.79 - 239.33)[68.64 - 188.64] [64.03 - 204.06] [-88.07 - 87.50] [0, 378.84]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 346 of 544Table E-19. Comparison of Proximates, Fiber, and MineralContent in Forage from MON 87460 and Conventional Controlfor Combined Sites in Chile during 2006/2007 under Water-Limited Conditions1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Difference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]FiberAcid Detergent Fiber (% DW) 27.15 (1.45) 26.73 (1.45) 0.42 (1.79) -3.36, 4.21 0.816 (20.73 - 33.39)[23.03 - 32.00] [23.10 - 30.79] [-6.30 - 8.90] [11.54, 42.87]Neutral Detergent Fiber (% DW) 39.06 (1.52) 40.10 (1.52) -1.04 (1.81) -4.65, 2.56 0.565 (36.08 - 49.33)[33.29 - 44.10] [31.81 - 50.61] [-9.65 - 8.94] [25.58, 58.01]MineralCalcium (% DW) 0.32 (0.020) 0.34 (0.020) -0.023 (0.019) 0.002, 0.015 0.219 (0.21 - 0.37)[0.20 - 0.44] [0.26 - 0.41] [-0.14 - 0.11] [0.085, 0.50]Phosphorus (% DW) 0.16 (0.0077) 0.17 (0.0077) -0.0078 (0.0061) -0.020, 0.0044 0.204 (0.13 - 0.19)[0.14 - 0.18] [0.15 - 0.21] [-0.033 - 0.014] [0.077, 0.23]ProximateAsh (% DW) 5.29 (0.22) 5.49 (0.22) -0.20 (0.20) -0.60, 0.21 0.332 (4.80 - 6.62)[4.51 - 6.29] [4.59 - 6.90] [-1.02 - 0.94] [3.59, 7.93]Carbohydrates (% DW) 85.92 (0.42) 86.02 (0.42) -0.10 (0.40) -0.89, 0.69 0.798 (84.11 - 87.54)[84.14 - 88.81] [84.51 - 87.52] [-1.84 - 1.29] [81.74, 90.41]Moisture (% FW) 74.98 (0.73) 75.42 (0.75) -0.44 (0.70) -1.92, 1.04 0.552 (73.40 - 77.50)[72.00 - 77.40] [73.00 - 77.60] [-3.20 - 4.10] [70.85, 80.94]Protein (% DW) 7.47 (0.40) 7.67 (0.40) -0.20 (0.22) -0.63, 0.24 0.373 (5.56 - 8.59)[5.49 - 8.76] [6.52 - 9.14] [-1.02 - 0.36] [2.94, 11.20]Total Fat (% DW) 1.32 (0.16) 0.84 (0.16) 0.47 (0.23) 0.010, 0.94 0.045 (0.20 - 1.76)[0.50 - 1.92] [0.20 - 1.66] [-0.42 - 0.92] [0, 3.25]Monsanto Company 07-CR-191U Page 347 of 544Table E-20. Comparison of the Proximates and Fiber Content in Grain from MON 87460 and Conventional Control forCombined Sites in Chile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)Analytical Component1Test Mean ±S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]ProximatesAsh (% DW)1.47 (0.038)1.50 (0.038)-0.032 (0.048)-0.13, 0.0660.505(1.27 - 1.63)[1.24 - 1.75][1.39 - 1.63][-0.31 - 0.35][1.06, 1.93]Carbohydrates (% DW)84.21 (0.27)84.10 (0.27)0.11 (0.36)-0.60, 0.820.754(82.10 - 85.17)[82.64 - 85.64][82.95 - 85.98][-2.06 - 1.84][80.40, 87.76]Moisture (% FW)12.10 (0.15)11.98 (0.15)0.12 (0.21)-0.31, 0.560.563(11.70 - 13.20)[11.60 - 12.50][11.30 - 12.50][-0.70 - 1.20][10.50, 14.11]Protein (% DW)10.30 (0.23)10.44 (0.23)-0.13 (0.29)-0.71, 0.450.645(9.99 - 12.19)[9.41 - 11.45][9.17 - 11.50][-1.61 - 1.41][8.12, 13.56]Total Fat (% DW) 4.02 (0.082) 3.96 (0.082) 0.054 (0.071) -0.096, 0.20 0.459 (3.18 - 4.22)[3.71 - 4.28] [3.47 - 4.23] [-0.19 - 0.54] [2.07, 5.10]FiberAcid Detergent Fiber (% DW)2.59 (0.21)2.33 (0.21)0.26 (0.27)-0.28, 0.800.342(1.83 - 3.39)[1.85 - 3.58][1.83 - 3.05][-0.53 - 0.99][0.88, 4.63]Neutral Detergent Fiber (% DW)8.87 (0.34)8.22 (0.34)0.64 (0.44)-0.28, 1.570.161(6.08 - 10.36)[7.33 - 11.31][7.91 - 8.66][-1.25 - 3.07][2.87, 13.22]Total Dietary Fiber (% DW)12.48 (0.44)12.15 (0.44)0.33 (0.50)-0.73, 1.400.515(10.57 - 14.56)[10.78 - 14.43][11.06 - 13.70][-1.99 - 2.19][6.50, 17.54]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 348 of 544Table E-21. Comparison of the Mineral Content in Grain from MON 87460 and Conventional Control for Combined Sites inChile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Calcium (% DW) 0.0052 (0.00045) 0.0050 (0.00045) 0.00017 (0.00029) -0.00041, 0.00075 0.563 (0.0035 - 0.0070)[0.0046 - 0.0065] [0.0041 - 0.0063] [-0.0014 - 0.0015] [0, 0.010]Copper (mg/kg DW) 2.19 (0.24) 2.16 (0.24) 0.031 (0.27) -0.56, 0.62 0.910 (1.39 - 2.76)[1.88 - 2.49] [1.87 - 2.30] [-0.40 - 0.62] [0.22, 3.82]Iron (mg/kg DW) 17.67 (0.68) 18.60 (0.68) -0.93 (0.61) -2.14, 0.29 0.131 (15.90 - 24.66)[16.38 - 19.27] [16.12 - 22.21] [-3.53 - 1.02] [7.05, 30.38]Magnesium (% DW) 0.13 (0.0034) 0.13 (0.0034) 0.00044 (0.0037) -0.0069, 0.0077 0.905 (0.11 - 0.14)[0.11 - 0.14] [0.10 - 0.14] [-0.022 - 0.024] [0.083, 0.16]Manganese (mg/kg DW) 6.71 (0.40) 6.54 (0.40) 0.18 (0.30) -0.45, 0.80 0.565 (4.78 - 9.35)[5.28 - 8.66] [5.25 - 7.77] [-1.13 - 2.28] [0.72, 11.82]Phosphorus (% DW) 0.32 (0.0099) 0.33 (0.0099) -0.0095 (0.0098) -0.029, 0.010 0.334 (0.30 - 0.38)[0.25 - 0.36] [0.27 - 0.38] [-0.074 - 0.075] [0.25, 0.42]Potassium (% DW) 0.40 (0.011) 0.40 (0.011) -0.0024 (0.0082) -0.019, 0.015 0.777 (0.36 - 0.43)[0.37 - 0.43] [0.37 - 0.43] [-0.038 - 0.038] [0.29, 0.49]Zinc (mg/kg DW) 23.30 (1.00) 24.37 (1.00) -1.07 (0.80) -2.66, 0.52 0.183 (18.25 - 30.44)[18.36 - 26.77] [21.29 - 27.79] [-3.62 - 2.49] [6.01, 42.60]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 349 of 544Table E-22. Comparison of the AminoAcid Content in Grain from MON 87460 and Conventional Control for CombinedSites in Chile during 2006/2007under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Alanine (% DW) 0.78 (0.018) 0.79 (0.018) -0.010 (0.025) -0.059, 0.039 0.682 (0.77 - 0.96)[0.67 - 0.85] [0.68 - 0.89] [-0.12 - 0.12] [0.59, 1.09]Arginine (% DW) 0.43 (0.012) 0.42 (0.012) 0.013 (0.017) -0.022, 0.047 0.457 (0.41 - 0.50)[0.41 - 0.44] [0.34 - 0.47] [-0.058 - 0.10] [0.32, 0.56]Aspartic Acid (% DW) 0.65 (0.013) 0.65 (0.013) -0.0072 (0.017) -0.040, 0.026 0.665 (0.63 - 0.76)[0.59 - 0.71] [0.59 - 0.73] [-0.090 - 0.098] [0.52, 0.88]Cystine (% DW) 0.23 (0.0043) 0.23 (0.0043) -0.00090 (0.0047) -0.010, 0.0085 0.848 (0.20 - 0.26)[0.22 - 0.25] [0.20 - 0.24] [-0.021 - 0.027] [0.15, 0.30]Glutamic Acid (% DW) 2.01 (0.047) 2.03 (0.047) -0.019 (0.064) -0.15, 0.11 0.772 (1.94 - 2.44)[1.74 - 2.21] [1.71 - 2.29] [-0.31 - 0.32] [1.51, 2.80]Glycine (% DW) 0.36 (0.0063) 0.36 (0.0063) 0.0018 (0.0077) -0.014, 0.017 0.818 (0.35 - 0.42)[0.34 - 0.39] [0.33 - 0.39] [-0.033 - 0.037] [0.30, 0.45]Histidine (% DW) 0.31 (0.0056) 0.31 (0.0056) -0.0022 (0.0074) -0.017, 0.013 0.768 (0.27 - 0.33)[0.28 - 0.32] [0.27 - 0.34] [-0.031 - 0.034] [0.23, 0.36]Isoleucine (% DW) 0.37 (0.0092) 0.37 (0.0092) -0.0065 (0.013) -0.031, 0.018 0.605 (0.34 - 0.44)[0.32 - 0.38] [0.32 - 0.41] [-0.052 - 0.048] [0.27, 0.50]Monsanto Company 07-CR-191U Page 350 of 544Table E-22 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Control forCombined Sites in Chile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Leucine (% DW) 1.36 (0.034) 1.37 (0.034) -0.011 (0.046) -0.10, 0.081 0.806 (1.29 - 1.65)[1.16 - 1.47] [1.13 - 1.56] [-0.22 - 0.24] [0.98, 1.91]Lysine (% DW) 0.29 (0.0045) 0.29 (0.0045) -0.0044 (0.0055) -0.016, 0.0067 0.428 (0.28 - 0.31)[0.27 - 0.31] [0.28 - 0.31] [-0.023 - 0.029] [0.25, 0.34]Methionine (% DW) 0.20 (0.0073) 0.20 (0.0073) 0.0010 (0.0065) -0.012, 0.014 0.873 (0.19 - 0.30)[0.18 - 0.22] [0.16 - 0.22] [-0.023 - 0.038] [0.095, 0.35]Phenylalanine (% DW) 0.53 (0.012) 0.54 (0.012) -0.0044 (0.017) -0.038, 0.029 0.797 (0.51 - 0.63)[0.46 - 0.58] [0.45 - 0.61] [-0.079 - 0.086] [0.41, 0.72]Proline (% DW) 0.96 (0.027) 0.97 (0.027) -0.011 (0.030) -0.072, 0.050 0.722 (0.78 - 1.03)[0.85 - 1.04] [0.84 - 1.11] [-0.15 - 0.16] [0.64, 1.23]Serine (% DW) 0.51 (0.012) 0.51 (0.012) -0.0023 (0.016) -0.034, 0.030 0.886 (0.48 - 0.60)[0.45 - 0.58] [0.43 - 0.59] [-0.087 - 0.11] [0.36, 0.71]Threonine (% DW) 0.35 (0.0069) 0.35 (0.0069) 0.0015 (0.0090) -0.016, 0.019 0.868 (0.33 - 0.39)[0.32 - 0.39] [0.31 - 0.39] [-0.042 - 0.067] [0.28, 0.44]Tryptophan (% DW) 0.053 (0.0020) 0.052 (0.0020) 0.0012 (0.0025) -0.0037, 0.0060 0.639 (0.043 - 0.063)[0.046 - 0.059] [0.042 - 0.063] [-0.0044 - 0.0095] [0.031, 0.082]Monsanto Company 07-CR-191U Page 351 of 544Table E-22 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Control forCombined Sites in Chile during 2006/2007 under Water-Limited ConditionsDifference (Testminus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Tyrosine (% DW) 0.29 (0.023) 0.24 (0.023) 0.050 (0.032) -0.017, 0.12 0.133 (0.25 - 0.41)[0.18 - 0.33] [0.12 - 0.35] [-0.11 - 0.16] [0.12, 0.52]Valine (% DW) 0.50 (0.011) 0.51 (0.011) -0.0078 (0.014) -0.037, 0.021 0.590 (0.47 - 0.58)[0.44 - 0.51] [0.45 - 0.55] [-0.068 - 0.050] [0.39, 0.64]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 352 of 544Table E-23. Comparison of the Fatty Acid Content in Grain from MON 87460 and ConventionalControl for Combined Sitesin Chile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]16:0 Palmitic (% Total FA) 11.06 (0.13) 11.18 (0.13)-0.12 (0.16) -0.45, 0.20 0.447 (9.84 - 12.33)[10.54 - 11.33] [10.75 - 11.45] [-0.39 - 0.32] [7.71, 14.14]18:0 Stearic (% Total FA) 1.86 (0.045) 1.86 (0.045) -0.0062 (0.047) -0.10, 0.092 0.896 (1.30 - 2.10)[1.73 - 1.95] [1.68 - 2.08] [-0.15 - 0.25] [0.71, 2.57]18:1 Oleic (% Total FA) 20.99 (0.37) 20.83 (0.37) 0.16 (0.29) -0.45, 0.77 0.589 (20.78 - 29.13)[20.20 - 21.60] [19.59 - 21.98] [-1.03 - 1.33] [12.15, 35.55]18:2 Linoleic (% Total FA) 64.29 (0.45) 64.30 (0.45) -0.0089 (0.42) -0.90, 0.88 0.983 (56.51 - 64.46)[63.27 - 65.10] [62.75 - 65.65] [-1.32 - 0.94] [50.63, 72.71]18:3 Linolenic (% Total FA) 1.19 (0.016) 1.21 (0.016) -0.013 (0.014) -0.041, 0.015 0.354 (1.03 - 1.38)[1.13 - 1.25] [1.12 - 1.26] [-0.079 - 0.049] [0.67, 1.76]20:0 Arachidic (% Total FA) 0.31 (0.011) 0.32 (0.011) -0.0034 (0.0058) -0.016, 0.0087 0.561 (0.30 - 0.41)[0.30 - 0.34] [0.30 - 0.33] [-0.025 - 0.014] [0.18, 0.52]20:1 Eicosenoic (% Total FA) 0.1763 (0.0039) 0.1845 (0.0039) -0.0082 (0.0039) -0.016, -0.00030 0.042 (0.18 - 0.27)[0.16 - 0.19] [0.17 - 0.20] [-0.025 - 0.014] [0.11, 0.34]22.0 Behenic (% Total FA) 0.12 (0.020) 0.12 (0.020) 0.0022 (0.026) -0.052, 0.056 0.933 (0.062 - 0.18)[0.058 - 0.20] [0.059 - 0.15] [-0.092 - 0.13] [0, 0.32]1FA = fatty acid; S.E. = standarderror; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 353 of 544Table E-24. Comparison of the VitaminContent in Grain from MON 87460 and Conventional Control for Combined Sites inChile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Folic Acid (mg/kg DW) 0.29 (0.018) 0.28 (0.018) 0.011 (0.016) -0.023, 0.046 0.497 (0.26 - 0.42)[0.25 - 0.37] [0.23 - 0.35] [-0.062 - 0.13] [0.098, 0.58]Niacin (mg/kg DW) 18.54 (1.77) 21.73 (1.77) -3.18 (1.81) -6.79, 0.43 0.083 (13.64 - 27.42)[16.23 - 25.00] [16.36 - 42.06] [-24.26 - 2.78] [2.23, 41.53]Thiamine HCl/VitaminB1 (mg/kg DW)3.10 (0.084) 2.98 (0.084) 0.12 (0.088) -0.070, 0.30 0.203 (2.87 - 4.33)[2.84 - 3.42] [2.71 - 3.19] [-0.11 - 0.45] [1.55, 5.85]Riboflavin/Vitamin B2(mg/kg DW)2.12 (0.14) 2.29 (0.14) -0.16 (0.14) -0.45, 0.12 0.255 (1.81 - 2.78)[1.43 - 2.89] [1.64 - 2.81] [-1.30 - 0.50] [0.88, 3.61]Pyridoxine HCl/VitaminB6 (mg/kg DW)6.17 (0.27) 6.15 (0.27) 0.013 (0.33) -0.69, 0.71 0.969 (5.30 - 8.22)[5.43 - 6.57] [4.97 - 8.27] [-1.96 - 1.20] [2.06, 9.98]Vitamin E (mg/kg DW) 13.01 (0.40) 12.16 (0.40) 0.84 (0.54) -0.30, 1.99 0.135 (2.84 - 15.53)[12.16 - 14.24] [10.15 - 13.64] [-0.45 - 2.42] [0, 22.61]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 354 of 544Table E-25. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain from MON 87460 andConventional Control for Combined Sites inChile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)Analytical Component1Test Mean ± S.E.1[Range]Control Mean ±S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Anti-nutrientPhytic Acid (% DW) 0.79 (0.035) 0.77 (0.035) 0.022 (0.043) -0.067, 0.11 0.612 (0.67 - 0.94)[0.63 - 0.89] [0.60 - 0.89] [-0.16 - 0.27] [0.40, 1.12]Raffinose (% DW) 0.11 (0.013) 0.12 (0.013) -0.0087 (0.0050) -0.019, 0.0017 0.095 (0.061 - 0.15)[0.087 - 0.14] [0.097 - 0.15] [-0.018 - 0.0025] [0, 0.21]Secondary MetaboliteFerulic Acid (μg/g DW) 1923.79 (114.60) 1852.11 (114.60) 71.68 (160.59) -249.18, 392.53 0.656 (1011.40 - 2539.86)[1208.67 - 2352.27] [1088.34 - 2301.59] [-852.84 - 788.80] [0, 4071.51]p-Coumaric Acid (μg/g DW) 137.29 (15.51) 149.45 (15.51) -12.16 (18.53) -51.13, 26.81 0.519 (84.15 - 259.68)[85.52 - 168.18] [66.48 - 208.43] [-122.91 - 65.49] [0, 378.67]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 355 of 544Table E-26. Comparison of the Additional Secondary Metabolite Composition of Forage from MON 87460 and ConventionalControl for Combined-Sites from the 2006/2007 ChileProduction Conducted under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Free Proline (% DW)0.023 (0.0025) 0.019 (0.0025) 0.0035 (0.0018) -0.00026, 0.0073 0.066 (0.0094 - 0.030)[0.014 - 0.031] [0.012 - 0.024] [-0.0077 - 0.014] [0, 0.042]Abscisic Acid (ppb FW)37.03 (8.19) 15.66 (7.97) 21.38 (9.64) 1.01, 41.75 0.040 (12.70 - 23.80)[11.90 - 122.00] [10.30 - 21.70] [-5.50 - 106.90] [1.22, 33.02]Choline (ppm FW)137.00 (6.68) 128.77 (6.68) 8.23 (6.28) -4.99, 21.46 0.207 (111.00 - 154.00)[114.00 - 159.00] [94.90 - 145.00] [-10.00 - 36.00] [76.96, 179.64]Glycerol (% DW)0.16 (0.0085) 0.14 (0.0085) 0.019 (0.0097) -0.00090, 0.039 0.060 (0.097 - 0.18)[0.11 - 0.21] [0.12 - 0.18] [-0.023 - 0.048] [0.024, 0.25]Glycine Betaine (ppm FW)84.24 (9.15) 76.27 (9.15) 7.98 (11.23) -15.64, 31.59 0.486 (4.46 - 147.00)[66.40 - 104.00] [55.60 - 91.00] [-17.40 - 24.90] [0, 271.19]Salicylic Acid (ppm DW)0.14 (0.049) 0.19 (0.049) -0.046 (0.045) -0.14, 0.051 0.327 (0.11 - 0.34)[0.072 - 0.21] [0.060 - 0.30] [-0.15 - 0.060] [0, 0.51]Fructose (% DW)8.56 (1.15) 9.06 (1.15) -0.50 (0.89) -2.38, 1.38 0.581 (4.32 - 10.04)[6.74 - 10.29] [7.63 - 9.80] [-1.86 - 0.77] [1.20, 14.57]Glucose (% DW)9.22 (1.07) 9.68 (1.07) -0.45 (0.96) -2.48, 1.58 0.642 (4.19 - 11.67)[7.31 - 10.43] [7.63 - 11.04] [-2.26 - 0.87] [1.01, 16.70]Sucrose (% DW)0.46 (1.07) 0.86 (1.07) -0.40 (1.11) -2.61, 1.82 0.722 (0.076 - 5.36)[0.10 - 1.03] [0.094 - 2.35] [-1.58 - 0.46] [0, 9.76]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 356 of 544Table E-27. Comparison of the Additional Secondary Metabolite Composition of Grain from MON 87460 and ConventionalControl for Combined-Sites from the 2006/2007 ChileProduction Conducted under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Free Proline (% DW)0.058 (0.011) 0.055 (0.011) 0.0026 (0.0035) -0.0048, 0.0099 0.476 (0.0093 - 0.076)[0.029 - 0.090] [0.030 - 0.083] [-0.0036 - 0.010] [0, 0.12]Abscisic Acid (ppb FW)9.73 (2.00) 11.49 (2.00) -1.76 (2.71) -7.16, 3.63 0.517 (9.48 - 116.00)[3.61 - 20.70] [7.24 - 21.90] [-10.40 - 8.40] [0, 162.21]Choline (ppm FW)219.89 (13.09) 235.78 (13.09) -15.89 (10.18) -37.49, 5.71 0.138 (174.00 - 264.00)[181.00 - 255.00] [203.00 - 265.00] [-48.00 - 33.00] [129.07, 327.26]Glycerol (% DW)0.023 (0.0035) 0.022 (0.0035) 0.0013 (0.0024) -0.0035, 0.0061 0.595 (0.015 - 0.037)[0.020 - 0.029] [0.017 - 0.030] [-0.0017 - 0.0051] [0, 0.048]Glycine Betaine (ppm FW)2.27 (0.33) 2.41 (0.33) -0.14 (0.26) -0.70, 0.41 0.588 (0.50 - 7.67)[1.31 - 3.11] [1.32 - 4.19] [-1.08 - 0.36] [0, 12.03]Salicylic Acid (ppm FW)0.088 (0.018) 0.094 (0.018) -0.0060 (0.016) -0.039, 0.027 0.705 (0.061 - 0.71)[0.065 - 0.12] [0.069 - 0.11] [-0.030 - 0.030] [0, 0.95]Fructose (% DW)0.44 (0.029) 0.44 (0.029) -0.0074 (0.034) -0.078, 0.063 0.830 (0.21 - 0.57)[0.34 - 0.50] [0.25 - 0.53] [-0.19 - 0.24] [0, 0.87]Glucose (% DW)0.46 (0.029) 0.48 (0.029) -0.018 (0.035) -0.091, 0.054 0.607 (0.23 - 0.54)[0.35 - 0.55] [0.34 - 0.56] [-0.20 - 0.069] [0.038, 0.81]Sucrose (% DW)1.77 (0.17) 1.82 (0.17) -0.053 (0.076) -0.21, 0.11 0.490 (1.47 - 2.86)[1.47 - 2.49] [1.40 - 2.47] [-0.33 - 0.085] [0.41, 3.46]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 357 of 544Table E-28. Comparison of the Additional Secondary Metabolite Composition of Forage from MON 87460 and ConventionalControl for Combined-Sites from the 2006/2007 Chile Production Conducted under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Free Proline (% DW) 0.018 (0.0025) 0.018 (0.0025) -0.00028 (0.0018) -0.0041, 0.0035 0.876 (0.011 - 0.025)[0.013 - 0.027] [0.011 - 0.025] [-0.0092 - 0.0069] [0, 0.036]Abscisic Acid (ppb FW) 31.60 (7.97) 29.42 (7.97)2.18 (9.46) -17.91, 22.27 0.820 (16.00 - 58.50)[20.40 - 54.30] [15.80 - 56.60] [-31.10 - 31.80] [0, 94.59]Choline (ppm FW) 155.11 (6.68) 145.89 (6.68) 9.22 (6.28) -4.00, 22.45 0.159 (118.00 - 166.00)[134.00 - 181.00] [136.00 - 163.00] [-13.00 - 34.00] [66.54, 217.46]Glycerol (% DW) 0.14 (0.0085) 0.14 (0.0085) -0.0038 (0.0097) -0.024, 0.016 0.697 (0.10 - 0.19)[0.11 - 0.17] [0.12 - 0.17] [-0.029 - 0.042] [0.025, 0.24]Glycine Betaine (ppm FW) 116.80 (9.15) 119.79 (9.15)-2.99 (11.23) -26.61, 20.63 0.793 (7.19 - 189.00)[73.20 - 138.00] [89.60 - 176.00] [-64.00 - 32.50] [0, 357.15]Salicylic Acid (ppm FW) 0.21 (0.049) 0.24 (0.049)-0.024 (0.045) -0.12, 0.073 0.607 (0.12 - 0.47)[0.10 - 0.58] [0.12 - 0.49] [-0.39 - 0.34] [0, 0.82]Fructose (% DW) 11.12 (1.15) 10.91 (1.15) 0.21 (0.89) -1.67, 2.09 0.813 (7.53 - 14.83)[7.77 - 20.17] [7.52 - 14.95] [-2.41 - 7.53] [0.69, 18.60]Glucose (% DW) 12.31 (1.07) 11.87 (1.07) 0.43(0.96) -1.59, 2.46 0.655 (8.11 - 15.87)[8.60 - 20.87] [8.60 - 15.05] [-2.82 - 8.81] [1.24, 20.22]Sucrose (% DW) 2.27 (1.07) 1.88 (1.07) 0.39(1.11) -1.82, 2.60 0.724 (0.12 - 4.68)[0.10 - 6.36] [0.11 - 4.07] [-3.62 - 4.83] [0, 8.87]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 358 of 544Table E-29. Comparison of the Additional Secondary Metabolite Composition of Grain from MON 87460 and ConventionalControl for Combined-Sites from the 2006/2007 Chile Production Conducted under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Free Proline (% DW) 0.051 (0.011) 0.058 (0.011) -0.0063 (0.0035) -0.014, 0.0011 0.089 (0.013 - 0.056)[0.029 - 0.076] [0.029 - 0.079] [-0.027 - 0.016] [0, 0.11]Abscisic Acid (ppb FW) 11.43 (2.00) 13.54 (2.00)-2.11 (2.71) -7.50, 3.29 0.438 (7.37 - 120.00)[8.78 - 17.70] [6.79 - 23.90] [-15.12 - 7.01] [0, 176.41]Choline (ppm FW) 238.11 (13.09) 241.56 (13.09) -3.44 (10.18) -25.04, 18.16 0.739 (202.00 - 306.00)[191.00 - 308.00] [209.00 - 284.00] [-45.00 - 76.00] [104.72, 381.48]Glycerol (% DW) 0.030 (0.0035) 0.029 (0.0035) 0.00069(0.0024) -0.0041, 0.0055 0.776 (0.019 - 0.045)[0.023 - 0.049] [0.018 - 0.043] [-0.020 - 0.017] [0, 0.060]Glycine Betaine (ppm FW) 2.21 (0.33) 1.99 (0.33)0.21 (0.26) -0.34, 0.77 0.421 (0.50 - 11.40)[1.52 - 3.24] [1.18 - 3.98] [-2.22 - 1.68] [0, 21.14]Salicylic Acid (ppm FW) 0.11 (0.018) 0.12 (0.018)-0.0026 (0.016) -0.036, 0.031 0.871 (0.057 - 0.60)[0.073 - 0.19] [0.084 - 0.15] [-0.074 - 0.060] [0, 1.00]Fructose (% DW) 0.47 (0.029) 0.48 (0.029) -0.011 (0.034) -0.082, 0.059 0.739 (0.29 - 0.74)[0.37 - 0.60] [0.38 - 0.63] [-0.26 - 0.19] [0, 1.12]Glucose (% DW) 0.48 (0.029) 0.50 (0.029) -0.015 (0.035) -0.087, 0.058 0.671 (0.32 - 0.77)[0.38 - 0.59] [0.39 - 0.64] [-0.26 - 0.17] [0, 1.17]Sucrose (% DW) 1.63 (0.17) 1.86 (0.17) -0.23(0.076) -0.39, -0.069 0.008 (1.41 - 2.19)[1.33 - 1.86] [1.37 - 2.27] [-0.72 - 0.17] [0.61, 2.84]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereset to zero.Monsanto Company 07-CR-191U Page 359 of 544 Table E-30. Literature and ILSI Database Ranges of Components of Corn Forage

and Grain

Tissue/ Component 1 Literature Range 2 ILSI Range 3

Forage Proximates (% DW)

Ash 2.43-9.64 a ; 2-6.6 b 1.527 – 9.638 Carbohydrates 83.2-91.6 b ; 76.5-87.3 a 76.4 – 92.1 Fat, total 0.35-3.62 b ; 1.42-4.57 a 0.296 – 4.570 Moisture (% fw) 56.5-80.4 a ;55.3-75.3 b 49.1 – 81.3

Protein 4.98-11.56 a 3.14 – 11.57

Fiber (% DW) Acid detergent fiber (ADF) 18.3-41.0 b ; 17.5-38.3 a 16.13 – 47.39 Neutral detergent fiber (NDF) 26.4-54.5 b ; 27.9-54.8 a 20.29 – 63.71

Minerals (% DW)

Calcium 0.0969-0.3184 b 0.0714 – 0.5768 Phosphorous 0.1367-0.2914 b 0.0936 – 0.3704

Grain Proximates (% DW)

Ash 1.1-3.9 d ; 0.89-6.28 b 0.616 – 6.282 Carbohydrates 77.4-87.2 b ; 82.2-88.1 a 77.4 – 89.5 Fat, total 3.1-5.7 d ; 2.48-4.81 b 1.742 – 5.823 Moisture (% FW) 7-23 d ; 8.18-26.2 b 6.1 – 40.5

Protein 6-12 d ; 9.7-16.1 c 6.15 – 17.26

Fiber (% dw) Acid detergent fiber (ADF) 3.3-4.3 d ; 2.46-11.34 a, b 1.82 – 11.34 Neutral detergent fiber (NDF) 8.3-11.9 d ; 7.58-15.91 b 5.59 – 22.64 Total dietary fiber (TDF) 10.99-11.41 h 8.82 – 35.31

Minerals Calcium (% DW) 0.01-0.1 d 0.00127 – 0.02084 Copper (mg/kg DW) 0.9-10 d 0.73 – 18.50 Iron (mg/kg DW) 1-100 d 10.42 – 49.07 Magnesium (% DW) 0.09-1 d 0.0594 – 0.194 Manganese (mg/kg DW) 0.7-54 d 1.69 – 14.30 Phosphorus (% DW) 0.26-0.75 d 0.147 – 0.533 Potassium (% DW) 0.32-0.72 d 0.181 – 0.603 Zinc (mg/kg DW) 12-30 d 6.5 – 37.2Monsanto Company 07-CR-191U Page 360 of 544 Table E-30 (cont.). Literature and ILSI Database Ranges of Components of Corn Forage and Grain

Tissue/ Component 1 Literature Range 2 ILSI Range 3

Grain Amino Acids (% DW)

Alanine N/A 0.439 – 1.393 Arginine N/A 0.119 – 0.639 Aspartic acid N/A 0.335 – 1.208 Cystine N/A 0.125 – 0.514

Glutamic acid N/A 0.965 – 3.536 Glycine N/A 0.184 – 0.539

Histidine N/A 0.137 – 0.434

Isoleucine N/A 0.179 – 0.692

Leucine N/A 0.642 – 2.492 Lysine N/A 0.172 – 0.668

Methionine N/A 0.124 – 0.468 Phenylalanine N/A 0.244 – 0.930

Proline N/A 0.462 – 1.632

Serine N/A 0.235 – 0.769

Threonine N/A 0.224 – 0.666 Tryptophan N/A 0.0271 – 0.215 Tyrosine N/A 0.103 – 0.642

Valine N/A 0.266 – 0.855

Fatty Acids (% total fat) (% total fatty acid)

16:0 Palmitic 7-19 e 7.94 – 20.71

16:1 Palmitoleic 1 e 0.095 – 0.447

18:0 Stearic 1-3 e 1.02 – 3.40

18:1 Oleic 20-46 e 17.4 – 40.2

18:2 Linoleic 35-70 e 36.2 – 66.5

18:3 Linolenic 0.8-2 e 0.57 – 2.25

20:0 Arachidic 0.1-2 e 0.279 – 0.965

20:1 Eicosenoic - 0.170 – 1.917

22:0 Behenic - 0.110 – 0.349

Vitamins (mg/kg DW)

Folic acid 0.3 d 0.147 – 1.464

Niacin 9.3-70 d 10.37 – 46.94

Vitamin B 1 3-8.6 e 1.26 – 40.00

Vitamin B 2 0.25-5.6 e 0.50 – 2.36

Vitamin B 6 5.3 d ; 9.6 e 3.68 – 11.32

Vitamin E 3-12.1 e ; 17-47 d 1.5 – 68.7Monsanto Company 07-CR-191U Page 361 of 544 Table E-30 (cont.). Literature and Histo rical Ranges of Components of Corn Forage and Grain

Tissue/ Component 1 Literature Range 2 ILSI Range 3

Grain Anti-nutrients (% DW) Phytic acid 0.48-1.12 a 0.111 – 1.570

Raffinose 0.08-0.30 e 0.020 – 0.320

Secondary Metabolites ( μ g/g dw)

Ferulic acid 113-1194 f ; 3000 g 291.9 – 3885.8 p-Coumaric acid 22-75 f 53.4 – 576.2

1 FW=fresh weight; DW=dry weight; Niacin =Vitamin B 3 ; Vitamin B 1 =Thiamine; Vitamin B 2 =Riboflavin; Vitamin B 6 =Pyridoxine ; N/A = not available as percent dry weight.

2 Literature range references: a Ridley et al., 2002. b Sidhu et al., 2000. c Jugenheimer, 1976.

d Watson, 1987. e Watson, 1982. f Classen et al

. , 1990. g Dowd and Vega, 1996. h Choi et al

. , 1999.

3 ILSI range is from ILSI CCD, 2006.

Conversions: % DW x 10 4 = μ g/g dw; mg/g dw x 10 3 = mg/kg DW; mg/100g dw x 10 = mg/kg

DWMonsanto Company 07-CR-191U Page 362 of 544 E.8. Compositional Analysis for the QUI Site in the Chile 2006/2007 Study

This section presents composition data on forage and grain colle cted from MON 87460 grown at an individual si te (QUI) of the Chile 2006 /2007 study. As noted in Section VII.2, this site was excluded from the combined-site data analysis because it did not meet the requirements specified by the intended water-limited conditions. Table E-31 presents data on applied water and temperatures from the production period for the QUI site.

Evaluation of the overall data set confirmed an alyte results were as expected from the respective assays and were similar to re ference and published ranges for conventional corn. No unexpected com positional values for any co mponents were observed.

A summary of significant differences (p<0 .05) between test and control for the compositional data is presented in Table E- 32. Mean values, ra nges, and statistical analyses data are presented in (Tables E-33 through E-39) fo r the well-watered treatment and Tables E-40 through E-46) fo r the water-limited treatment.

A summary of significant differe nces (p<0.05) between test and control for the additional secondary metabolites is presented in Table E- 47. Mean values, ranges, and statistical analyses data are presented in (Tables E-48 and E-49) for the well-watered treatment and Tables E-50 and E-51) for the water-limited treatment.

The QUI site results reported below do not impact the conclusion of compositional equivalence of MON 87460 to conventional corn as established in Section VII.Monsanto Company 07-CR-191U Page 363 of 544Table E-31. Monthly Temperature and Monthly AccumulatedWater Data for the QUI Sitein the Chile 2006/2007 StudySite1Measurement December January February March April MayQUIAccumulated water (in.),well-watered 1.9 10.3 8.5 9.4 1.9 0.0Accumulated water (in.),water-limited1, 21.9 10.3 1.9 5.6 1.9 0.0Avg Max temp (°F) NA48379777468Avg Min temp (°F) NA45151494437Range3(°F) NA448 - 9244 - 9041 - 9334 - 9229 – 771Water limitation began at the V10 growth stage which occurred at approximately February 7.2Water limitation ended at the R2 growth stage which occurred at approximately March 13.3The range is the absolute maximum and minimum temperature in each month.4Temperature data are available from January 6 through May 25; planting occurredin late December and early January.Rainfall did not occur during the production period.Monsanto Company 07-CR-191U Page 364 of 544Table E-32. Summary of Significant Differences (p<0.05) Comparing MON 87460 to the Conventional Control from the QUISite in Chile during 2006/2007 under Well-Watered and Water-Limited ConditionsTissue/Site/Components (Units)1MeanMON 87460MeanControlMean Diff(% of Control)Signif.(p-value)MON 87460(Range)Commercial(Range)ForageWater-LimitedMoisture (% FW)73.53 76.57 -3.96 0.019 (72.40 - 75.30) (74.30 - 77.30)Protein (% DW)5.78 6.69 -13.68 0.013 (5.25 - 6.57) (5.44 - 6.38)Total Fat (% DW)1.44 0.82 75.70 0.049 (0.58 - 2.34) (0.58 - 1.42)GrainWell-wateredMoisture (% FW)13.63 8.48 60.83 <0.001 (13.10 - 14.2) (9.41 - 13.70)Magnesium (% DW)0.12 0.11 7.86 0.043 (0.12 - 0.13) (0.10 - 0.12)Phosphorus (% DW)0.32 0.30 7.63 0.047 (0.31 - 0.34) (0.27 - 0.33)16:0 Palmitic (% Total FA)11.06 11.38 -2.77 0.017 (10.92 - 11.29) (9.56 - 12.93)Vitamin E (mg/kg DW)11.63 9.85 18.09 0.023 (11.06 - 12.47) (2.88 - 8.47)Water-LimitedCarbohydrates (% DW) 85.95 85.21 0.870.038 (85.69 - 86.33) (85.53 - 86.50)Moisture (% FW) 13.60 8.72 55.96<0.001 (12.70 - 14.10) (9.55 - 13.40)Protein (% DW) 8.69 9.24 -5.900.043 (8.24 - 8.99) (8.32 - 9.67)Neutral Detergent Fiber (% DW9.65 10.89 -11.36 0.042 (8.85 - 10.54) (7.75 - 10.73)Copper (mg/kg DW)1.61 4.22 -61.82 0.032 (1.53 - 1.65) (1.37 - 1.87)Iron (mg/kg DW)14.35 15.48 -7.29 0.037 (13.75 - 14.77) (14.04 - 19.16)Alanine (% DW)0.62 0.70 -11.03 0.006 (0.59 - 0.65) (0.62 - 0.70)Aspartic Acid (% DW)0.56 0.60 -7.42 0.018 (0.54 - 0.58) (0.53 - 0.60)Glutamic Acid (% DW)1.60 1.76 -9.04 0.020 (1.50 - 1.66) (1.53 - 1.76)Isoleucine (% DW)0.29 0.33 -10.09 0.007 (0.28 - 0.31) (0.29 - 0.32)Leucine (% DW)1.04 1.16 -10.50 0.015 (0.97 - 1.09) (0.97 - 1.14)Lysine (% DW)0.27 0.29 -5.44 0.037 (0.27 - 0.27) (0.26 - 0.29)Phenylalanine (% DW)0.43 0.47 -8.79 0.022 (0.40 - 0.44) (0.40 - 0.46)Valine (% DW)0.43 0.46 -7.50 0.018 (0.41 - 0.44) (0.40 - 0.45)1DW= dry weight; FW=fresh weight, FA= fatty acid.Monsanto Company 07-CR-191U Page 365 of 544Table E-33. Comparison of Proximates, Fiber, and MineralContent in Forage from MON 87460 and Conventional Controlfrom the QUI Site in Chile during 2006/2007 under Well-Watered Conditions1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Difference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)FiberAcid Detergent Fiber (% DW) 34.70 (1.57) 32.92(1.57) 1.78 (2.21) -2.91, 6.47 0.433[31.50 - 39.52] [31.44 - 34.55] [-3.05 - 8.08] (32.07 - 39.64)Neutral DetergentFiber (% DW) 46.04 (1.90) 48.39 (1.90)-2.35 (2.45) -7.57, 2.87 0.353[42.13 - 50.79] [43.78 - 52.67] [-7.46 - 2.06] (46.84 - 50.22)MineralCalcium (% DW) 0.31 (0.021) 0.33 (0.021)-0.020 (0.26) -0.075, 0.034 0.443[0.29 - 0.34] [0.31 - 0.35] [-0.033 - - 0.0051] (0.26 - 0.33)Phosphorus (% DW) 0.16 (0.010) 0.16 (0.010)0.0075 (0.014) -0.022, 0.037 0.594[0.14 - 0.17] [0.13 - 0.17] [-0.021- 0.041] (0.13 - 0.17)ProximateAsh (% DW) 4.13 (0.18) 4.55 (0.18) -0.42 (0.25) -0.96, 0.13 0.123[4.02 - 4.33] [4.51 - 4.62] [-0.50 - -0.29] (4.73 - 6.65)Carbohydrates (% DW) 88.78 (0.34) 88.57 (0.34) 0.21 (0.47) -0.78, 1.20 0.665[88.57 - 89.20] [88.12 - 88.91] [-0.34 – 0.52] (86.88 - 88.82)Moisture (% FW) 74.43 (0.96) 76.27 (0.96)-1.83 (1.17) -4.30, 0.64 0.135[73.90 - 74.80] [75.70 - 76.70] [-2.10 - -1.60] (75.10 - 77.90)Protein (% DW) 5.89 (0.24) 6.12 (0.24)-0.23 (0.34) -0.93, 0.47 0.504[5.63 - 6.10] [5.92 - 6.30] [-0.51 – 0.18] (5.78 - 6.47)Total Fat (% DW) 1.20 (0.22) 0.76 (0.22)0.44 (0.29) -0.18, 1.06 0.154[0.84 - 1.47] [0.56 - 1.06] [0.29 - 0.61] (0.23 - 0.86)Monsanto Company 07-CR-191U Page 366 of 544Table E-34. Comparison of the Proximates and Fiber Contentin Grain from MON 87460 and Conventional Control from theQUI Site in Chile during 2006/2007under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)ProximatesAsh (% DW) 1.48 (0.033) 1.44 (0.033)0.036 (0.046) -0.061, 0.13 0.448[1.39 - 1.54] [1.34 - 1.57] [-0.18 - 0.20] (1.30 - 1.36)Carbohydrates (% DW) 85.22 (0.24) 85.41 (0.24) -0.19 (0.33) -0.89, 0.51 0.571[85.00 - 85.65] [84.63 - 86.01] [-1.01 - 1.02] (86.05 - 86.57)Moisture (% FW) 13.63 (0.24) 8.48 (0.24) 5.16 (0.34) 4.42, 5.89 <0.001[13.10 - 14.20] [8.15 - 8.82] [4.95 - 5.38] (9.41 - 13.70)Protein (% DW) 9.23 (0.19) 9.20 (0.19)0.029 (0.25) -0.50, 0.56 0.909[9.16 - 9.30] [8.87 - 9.45] [-0.29 - 0.37] (8.35 - 9.23)Total Fat (% DW) 4.07 (0.14) 3.94 (0.14)0.13 (0.19) -0.28, 0.54 0.518[3.81 - 4.25] [3.71 - 4.35] [-0.54 - 0.55] (3.15 - 4.16)FiberAcid Detergent Fiber (% DW) 3.58 (0.37) 3.34(0.37) 0.24 (0.52) -0.84, 1.32 0.652[2.45 - 4.15] [2.48 - 3.78] [-1.33 - 1.67] (2.83 - 4.02)Neutral Detergent Fiber (% DW) 10.25 (0.40) 9.12 (0.40) 1.13 (0.57) -0.057, 2.32 0.060[10.01 - 10.61] [8.63 - 9.54] [0.94 - 1.38] (8.91 - 10.10)Total Dietary Fiber (% DW) 14.32 (0.58) 13.55(0.58) 0.77 (0.70) -0.71, 2.25 0.286[13.89 - 14.92] [12.96 - 14.26] [0.45 - 1.20] (11.76 - 13.56)1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 367 of 544Table E-35. Comparison of the Mineral Content in Grain from MON 87460 and Conventional Control from the QUI Site inChile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Calcium (% DW) 0.0047 (0.00021) 0.0047 (0.00021) 0(0.00022) -0.00049, 0.00047 0.966 (0.0041 - 0.0054)[0.0044 - 0.0051] [0.0046 - 0.0051] [-0.00068 - 0.00059]Copper (mg/kg DW) 1.70 (0.80) 3.78 (0.80) -2.08 (1.14) -4.46, 0.29 0.082 (1.33 - 1.72)[1.54 - 1.79] [1.93 - 7.38] [-5.61 - -0.25]Iron (mg/kg DW) 16.87 (0.37) 16.20 (0.37) 0.66(0.50) -0.39, 1.71 0.202 (13.79 - 18.40)[16.32 - 17.59] [14.81 - 17.26] [0.14 - 1.51]Magnesium (% DW) 0.12 (0.0030) 0.11 (0.0030) 0.0090 (0.0042) 0.00028, 0.018 0.043 (0.10 - 0.12)[0.12 - 0.13] [0.11 - 0.12] [-0.0088 - 0.020]Manganese (mg/kg DW) 6.46 (0.19)6.68 (0.19) -0.22 (0.26) -0.76, 0.33 0.421 (4.92 - 5.28)[6.01 - 7.10] [5.91 - 7.08] [-1.07 - 1.19]Phosphorus (% DW) 0.32 (0.0076) 0.30 (0.0076) 0.023 (0.011) 0.00032, 0.046 0.047 (0.27 - 0.33)[0.31 - 0.34] [0.28 - 0.33] [-0.013 - 0.046]Potassium (% DW) 0.39 (0.0084) 0.38 (0.0084) 0.014 (0.011) -0.011,0.038 0.256 (0.32 - 0.40)[0.38 - 0.40] [0.36 - 0.39] [-0.015 - 0.033]Zinc (mg/kg DW) 18.18 (0.58) 19.09 (0.58) -0.91 (0.78) -2.54, 0.73 0.260 (16.12 - 18.18)[17.01 - 19.35] [17.96 - 19.96] [-2.32 - 0.22]1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 368 of 544Table E-36. Comparison of the Amino Acid Content in Grain from MON 87460 and ConventionalControl from the QUI Sitein Chile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)Analytical Component1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Alanine (% DW) 0.67 (0.018) 0.68 (0.018)-0.0098 (0.025) -0.062, 0.043 0.697[0.67 - 0.68] [0.64 - 0.71] [-0.032 - 0.032] (0.62 - 0.72)Arginine (% DW) 0.43 (0.011) 0.40 (0.011) 0.023 (0.015) -0.0096, 0.055 0.158[0.42 - 0.43] [0.39 - 0.42] [0.016 - 0.034] (0.38 - 0.42)Aspartic Acid (% DW) 0.60 (0.012) 0.58 (0.012) 0.011 (0.017) -0.025, 0.047 0.530[0.59 - 0.60] [0.55 - 0.61] [-0.014 - 0.042] (0.52 - 0.63)Cystine (% DW) 0.21 (0.0050) 0.21 (0.0050)0.0028 (0.0058) -0.0096, 0.015 0.639[0.21 - 0.22] [0.20 - 0.21] [-0.0043 - 0.0089] (0.19 - 0.21)Glutamic Acid (% DW) 1.73 (0.045) 1.71 (0.045) 0.014 (0.063) -0.12, 0.14 0.830[1.71 - 1.74] [1.61 - 1.76] [-0.034 - 0.10] (1.52 - 1.81)Glycine (% DW) 0.34 (0.0050) 0.33 (0.0050)0.0097 (0.0068) -0.0048, 0.024 0.175[0.34 - 0.34] [0.31 - 0.34] [0.00028 - 0.026] (0.32 - 0.35)Histidine (% DW) 0.29 (0.0053) 0.28 (0.0053)0.0078 (0.0072) -0.0073, 0.023 0.292[0.29 - 0.29] [0.26 - 0.29] [-0.0014 - 0.022] (0.24 - 0.28)Isoleucine (% DW) 0.32 (0.0079) 0.32 (0.0079) 0.0027 (0.011) -0.021, 0.026 0.813[0.32 - 0.33] [0.31 - 0.33] [-0.0077 - 0.020] (0.28 - 0.32)Monsanto Company 07-CR-191U Page 369 of 544Table E-36 (cont). Comparison of the Amino Acid Contentin Grain from MON 87460 and Conventional Control from theQUI Site in Chile during 2006/2007under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Leucine (% DW) 1.15 (0.033) 1.14 (0.033)0.0086 (0.045) -0.087, 0.10 0.851[1.14 - 1.15] [1.07 - 1.18] [-0.025 - 0.068] (0.99 - 1.17)Lysine (% DW) 0.29 (0.0050) 0.28 (0.0050)0.0059 (0.0071) -0.0089, 0.021 0.413[0.28 - 0.29] [0.27 - 0.29] [-0.0043 - 0.020] (0.27 - 0.30)Methionine (% DW) 0.17 (0.0045) 0.16 (0.0045) 0.0047 (0.0060) -0.0081, 0.017 0.447[0.16 - 0.17] [0.16 - 0.17] [-0.0072 - 0.011] (0.16 - 0.18)Phenylalanine (% DW) 0.46 (0.012) 0.46 (0.012) 0.0050 (0.017) -0.030, 0.040 0.767[0.46 - 0.46] [0.43 - 0.48] [-0.012 - 0.028] (0.41 - 0.48)Proline (% DW) 0.88 (0.022) 0.86 (0.022)0.025 (0.032) -0.041, 0.090 0.445[0.86 - 0.90] [0.80 - 0.91] [-0.048 - 0.084] (0.74 - 0.85)Serine (% DW) 0.43 (0.013) 0.43 (0.013)-0.0020 (0.018) -0.039, 0.035 0.912[0.42 - 0.44] [0.41 - 0.46] [-0.019 - 0.010] (0.39 - 0.46)Threonine (% DW) 0.31 (0.0071) 0.30 (0.0071) 0.0074 (0.010) -0.013, 0.028 0.468[0.31 - 0.32] [0.29 - 0.32] [-0.0015 - 0.015] (0.28 - 0.33)Tryptophan (% DW) 0.051 (0.0013) 0.049 (0.0013)0.0019 (0.0018) -0.0020, 0.0058 0.318[0.049 - 0.052] [0.048 - 0.050] [0.00056 - 0.0031] (0.045 - 0.054)Monsanto Company 07-CR-191U Page 370 of 544Table E-36 (cont). Comparison of the Amino Acid Contentin Grain from MON 87460 and Conventional Control from theQUI Site in Chile during 2006/2007under Well-Watered ConditionsDifference (Testminus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Tyrosine (% DW) 0.29 (0.021) 0.28 (0.021) 0.0038 (0.030) -0.060, 0.067 0.901[0.27 - 0.30] [0.27 - 0.32] [-0.026 - 0.029] (0.24 - 0.30)Valine (% DW) 0.46 (0.0095) 0.45 (0.0095)0.0049 (0.013) -0.023, 0.033 0.716[0.45 - 0.46] [0.42 - 0.47] [-0.014 - 0.035] (0.39 - 0.46)1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 371 of 544Table E-37. Comparison of the Fatty Acid Content in Grainfrom MON 87460 and Conventional Control from the QUI Site inChile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)16:0 Palmitic (% Total FA) 11.06 (0.099) 11.38 (0.099) -0.31 (0.12) -0.57, -0.063 0.017[10.92 - 11.29] [11.21 - 11.53] [-0.41 - -0.24] (9.56 - 12.93)18:0 Stearic (% Total FA) 1.83(0.038) 1.84 (0.038) -0.0084 (0.053) -0.12, 0.11 0.877[1.74 - 1.89] [1.75 - 1.90] [-0.13 - 0.12] (1.32 - 2.11)18:1 Oleic (% Total FA) 20.91 (0.22) 20.88 (0.22) 0.030 (0.27) -0.56, 0.62 0.913[20.27 - 21.55] [20.41 - 21.29] [-0.67 - 0.50] (20.59 - 31.56)18:2 Linoleic (% Total FA) 64.43 (0.26) 64.06(0.26) 0.36 (0.31) -0.29, 1.02 0.257[63.86 - 64.94] [63.58 - 64.86] [-0.39 - 1.20] (55.08 - 64.79)18:3 Linolenic (% Total FA) 1.21 (0.017) 1.26(0.017) -0.043 (0.023) -0.091, 0.0058 0.080[1.19 - 1.23] [1.22 - 1.28] [-0.055 - -0.030] (1.10 - 1.61)20:0 Arachidic (% Total FA) 0.30 (0.0063) 0.30(0.0063) -0.0010 (0.0081) -0.018, 0.016 0.901[0.29 - 0.30] [0.28 - 0.31] [-0.010 - 0.017] (0.32 - 0.36)20:1 Eicosenoic (% Total FA) 0.18 (0.0024) 0.18 (0.0024) -0.0020 (0.0033) -0.0089, 0.0050 0.560[0.17 - 0.18] [0.17 - 0.18] [-0.0097 - 0.0047] (0.20 - 0.26)22:0 Behenic (% Total FA) 0.087 (0.021) 0.11 (0.021) -0.026 (0.029) -0.086, 0.035 0.386[0.060 - 0.14] [0.058 - 0.15] [-0.086 - 0.081] (0.060 - 0.17)1FA = fatty acid; S.E. = standarderror; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 372 of 544Table E-38. Comparison of the Vitamin Content in Grain from MON 87460 and Conventional Control from the QUI Site inChile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)Analytical Component1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Folic Acid (mg/kg DW) 0.29 (0.024) 0.24 (0.024) 0.055 (0.026) -0.0020, 0.11 0.057[0.26 - 0.35] [0.23 - 0.25] [0.027 - 0.099] (0.28 - 0.30)Niacin (mg/kg DW) 21.92 (1.34) 23.52 (1.34)-1.60 (1.73) -5.31, 2.10 0.369[20.49 - 23.01] [22.48 - 24.71] [-2.89 - -0.22] (24.16 - 29.08)Thiamine HCl (mg/kg DW) 2.89 (0.12) 2.88 (0.12) 0.017 (0.17) -0.33, 0.37 0.917[2.80 - 3.01] [2.74 - 2.95] [-0.063 - 0.060] (2.19 - 3.59)Vitamin B2 (mg/kg DW) 2.17 (0.22) 2.14 (0.22) 0.034 (0.31) -0.62, 0.69 0.913[1.77 - 2.87] [1.86 - 2.57] [-0.69 - 1.01] (1.96 - 2.40)Vitamin B6 (mg/kg DW) 5.64 (0.21) 5.40 (0.21) 0.24 (0.30) -0.39, 0.87 0.430[5.32 - 6.05] [5.36 - 5.44] [-0.073 - 0.61] (5.37 - 5.80)Vitamin E (mg/kg DW) 11.63 (0.53) 9.85 (0.53) 1.78 (0.71) 0.27, 3.29 0.023[11.06 - 12.47] [8.30 - 10.97] [1.08 - 2.76] (2.88 - 8.47)1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 373 of 544Table E-39. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain from MON 87460 andConventional Control from the QUI Site inChile during 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Anti-nutrientPhytic Acid (% DW) 0.76 (0.042) 0.71 (0.042) 0.046 (0.060) -0.079, 0.17 0.450[0.65 - 0.83] [0.68 - 0.73] [-0.034 - 0.11] (0.57 - 0.71)Raffinose (% DW) 0.096 (0.0064) 0.093 (0.0064)0.0031 (0.0090) -0.016, 0.022 0.738[0.089 - 0.11] [0.082 - 0.10] [-0.010 - 0.024] (0.029 - 0.095)Secondary MetaboliteFerulic Acid (μg/g DW) 1988.05 (116.42) 1904.98 (116.42) 83.07 (158.06) -250.94, 417.09 0.606[1910.24 - 2097.90] [1704.17 - 2072.82] [-27.70 - 251.85] (1263.58 - 2704.49)p-Coumaric Acid (μg/g DW) 201.09 (10.65) 176.29(10.65) 24.81 (14.34) -5.32, 54.93 0.100[180.56 - 213.29] [159.49 - 188.64] [21.06 - 28.71] (119.71 - 286.21)1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 374 of 544Table E-40. Comparison of Proximates, Fiber, and MineralContent in Forage from MON 87460 and Conventional Controlfrom the QUI Site in Chile during 2006/2007 under Water-Limited Conditions1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Difference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)FiberAcid Detergent Fiber (% DW) 29.22 (1.57) 30.72(1.57) -1.50 (2.21) -6.19, 3.20 0.508[26.90 - 32.72] [28.45 - 33.32] [-3.49 - -0.40] (28.33 - 32.37)Neutral DetergentFiber (% DW) 41.55 (1.90) 45.54 (1.90)-4.00 (2.45) -9.22, 1.22 0.123[38.01 - 44.93] [42.49 - 48.44] [-10.43 - -0.77] (37.80 - 47.14)MineralCalcium (% DW) 0.27 (0.021) 0.30 (0.021)-0.028 (0.027) -0.085, 0.030 0.316[0.24 - 0.31] [0.29 - 0.30] [-0.051 - 0.013] (0.23 - 0.32)Phosphorus (% DW) 0.15 (0.010) 0.14 (0.010)0.0082 (0.014) -0.021, 0.037 0.561[0.13 - 0.16] [0.13 - 0.15] [-0.0045 - 0.025] (0.14 - 0.17)ProximateAsh (% DW) 3.82 (0.18) 4.10 (0.18) -0.28 (0.25) -0.82, 0.27 0.291[3.61 - 4.09] [3.86 - 4.53] [-0.92 - 0.18] (4.80 - 5.14)Carbohydrates (% DW) 88.97 (0.34) 88.38 (0.34) 0.58 (0.47) -0.41, 1.57 0.232[88.28 - 89.83] [87.74 - 89.09] [-0.81 - 2.08] (87.43 - 89.00)Moisture (% FW) 73.53 (0.96) 76.57 (0.96)-3.03 (1.17) -5.50, -0.56 0.019[72.40 - 75.30] [74.40 - 78.60] [-6.20 - -1.40] (74.30 - 77.30)Protein (% DW) 5.78 (0.24) 6.69 (0.24)-0.91 (0.34) -1.62, -0.21 0.013[5.25 - 6.57] [6.25 - 7.47] [-1.96 - 0.32] (5.44 - 6.38)Total Fat (% DW) 1.44 (0.22) 0.82 (0.22) 0.62 (0.29) 0.00006, 1.24 0.049[0.58 - 2.34] [0.77 - 0.88] [-0.31 - 1.58] (0.58 - 1.42)Monsanto Company 07-CR-191U Page 375 of 544Table E-41. Comparison of the Proximates and Fiber Contentin Grain from MON 87460 and Conventional Control from theQUI Site in Chile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)ProximatesAsh (% DW) 1.39 (0.033) 1.43 (0.033) -0.039 (0.046) -0.14, 0.058 0.409[1.38 - 1.40] [1.39 - 1.48] [-0.085 - -0.0024] (1.21 - 1.50)Carbohydrates (% DW) 85.95 (0.24) 85.21 (0.24) 0.74 (0.33) 0.042, 1.44 0.038[85.69 - 86.33] [84.92 - 85.65] [0.18 - 1.41] (85.53 - 86.50)Moisture (% FW) 13.60 (0.24) 8.72 (0.24) 4.88 (0.34) 4.15, 5.61 <0.001[12.70 - 14.10] [8.53 - 8.89] [4.17 - 5.26] (9.55 - 13.40)Protein (% DW) 8.69 (0.19) 9.24 (0.19)-0.54 (0.25) -1.07, -0.018 0.043[8.24 - 8.99] [9.20 - 9.28] [-1.05 - -0.24] (8.32 - 9.67)Total Fat (% DW) 3.97 (0.14) 4.13 (0.14)-0.16 (0.19) -0.56, 0.25 0.429[3.80 - 4.06] [3.71 - 4.41] [-0.36 - 0.088] (3.46 - 3.97)FiberAcid Detergent Fiber (% DW) 3.14 (0.37) 3.58(0.37) -0.43 (0.52) -1.51, 0.65 0.412[2.91 - 3.57] [3.10 - 3.93] [-0.98 - 0.47] (2.85 - 4.42)Neutral Detergent Fiber (% DW) 9.65 (0.40) 10.89 (0.40) -1.24 (0.57) -2.42, -0.049 0.042[8.85 - 10.54] [10.32 - 11.70] [-1.47 - -1.08] (7.75 - 10.73)Total Dietary Fiber (% DW) 13.81 (0.58) 14.17(0.58) -0.36 (0.70) -1.84, 1.12 0.612[13.02 - 14.43] [13.26 - 15.15] [-1.18 - 0.33] (11.74 - 14.40)1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 376 of 544Table E-42. Comparison of the Mineral Content in Grain from MON 87460 and Conventional Control from the QUI Site inChile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Calcium (% DW) 0.0045 (0.00021) 0.0044 (0.00021) 0.00012 (0.00022) -0.00036, 0.00060 0.595[0.0041 - 0.0049] [0.0042 - 0.0046] [-0.00022 - 0.00030] (0.0038 - 0.0055)Copper (mg/kg DW) 1.61 (0.80) 4.22 (0.80)-2.61 (1.14) -4.99, -0.24 0.032[1.53 - 1.65] [2.17 - 7.76] [-6.23 - -0.52] (1.37 - 1.87)Iron (mg/kg DW) 14.35 (0.37) 15.48 (0.37)-1.13 (0.50) -2.18, -0.075 0.037[13.75 - 14.77] [15.20 - 15.70] [-1.45 - -0.79] (14.04 - 19.16)Magnesium (% DW) 0.11 (0.0030) 0.11 (0.0030)-0.0013 (0.0042) -0.010, 0.0075 0.767[0.11 - 0.11] [0.11 - 0.12] [-0.010 - 0.0052] (0.11 - 0.11)Manganese (mg/kg DW) 5.84 (0.19) 6.07 (0.19) -0.23 (0.26) -0.77, 0.32 0.397[5.66 - 6.07] [5.94 - 6.17] [-0.51 - -0.037] (4.59 - 5.18)Phosphorus (% DW) 0.30 (0.0076) 0.29 (0.0076)0.00040 (0.011) -0.022, 0.023 0.970[0.29 - 0.30] [0.28 - 0.32] [-0.025 - 0.019] (0.26 - 0.31)Potassium (% DW) 0.38 (0.0084) 0.38 (0.0084)0.0033 (0.011) -0.021, 0.028 0.774[0.38 - 0.39] [0.38 - 0.38] [-0.0023 - 0.0089] (0.31 - 0.38)Zinc (mg/kg DW) 18.17 (0.58) 19.13 (0.58)-0.96 (0.78) -2.60, 0.67 0.231[17.33 - 18.67] [17.86 - 20.77] [-2.10 - -0.26] (15.65 - 19.27)1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 377 of 544Table E-43. Comparison of the Amino Acid Content in Grain from MON 87460 and ConventionalControl from the QUI Sitein Chile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)Analytical Component1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Alanine (% DW) 0.62 (0.018) 0.70 (0.018)-0.077 (0.025) -0.13, -0.025 0.006[0.59 - 0.65] [0.68 - 0.72] [-0.093 - -0.049] (0.62 - 0.70)Arginine (% DW) 0.40 (0.011) 0.41 (0.011)-0.015 (0.015) -0.047, 0.018 0.352[0.38 - 0.40] [0.38 - 0.45] [-0.063 - 0.027] (0.34 - 0.42)Aspartic Acid (% DW) 0.56 (0.012) 0.60 (0.012) -0.045 (0.017) -0.081, -0.0086 0.018[0.54 - 0.58] [0.59 - 0.61] [-0.058 - -0.027] (0.53 - 0.60)Cystine (% DW) 0.20 (0.0050) 0.21 (0.0050)-0.0097 (0.0058) -0.022, 0.0026 0.112[0.19 - 0.21] [0.20 - 0.22] [-0.029 - 0.0015] (0.19 - 0.22)Glutamic Acid (% DW) 1.60 (0.045) 1.76 (0.045) -0.16 (0.063) -0.29, -0.027 0.020[1.50 - 1.66] [1.72 - 1.79] [-0.22 - -0.10] (1.53 - 1.76)Glycine (% DW) 0.33 (0.0050) 0.34 (0.0050)-0.0088 (0.0068) -0.023, 0.0057 0.218[0.33 - 0.34] [0.33 - 0.35] [-0.018 - -0.0014] (0.31 - 0.33)Histidine (% DW) 0.27 (0.0053) 0.29 (0.0053)-0.013 (0.0072) -0.028, 0.0020 0.084[0.26 - 0.28] [0.28 - 0.29] [-0.018 - -0.0080] (0.23 - 0.27)Isoleucine (% DW) 0.29 (0.0079) 0.33 (0.0079)-0.033 (0.011) -0.056, -0.0099 0.007[0.28 - 0.31] [0.33 - 0.33] [-0.047 - -0.020] (0.29 - 0.32)Monsanto Company 07-CR-191U Page 378 of 544Table E-43 (cont). Comparison of the Amino Acid Contentin Grain from MON 87460 and Conventional Control from theQUI Site in Chile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Leucine (% DW) 1.04 (0.033) 1.16 (0.033)-0.12 (0.045) -0.22, -0.026 0.015[0.97 - 1.09] [1.15 - 1.17] [-0.18 - -0.074] (0.97 - 1.14)Lysine (% DW) 0.27 (0.0050) 0.29 (0.0050)-0.016 (0.0071) -0.030, -0.00096 0.037[0.27 - 0.27] [0.28 - 0.30] [-0.033 - -0.0028] (0.26 - 0.29)Methionine (% DW) 0.17 (0.0045) 0.17 (0.0045) -0.0027 (0.0060) -0.016, 0.010 0.658[0.16 - 0.17] [0.16 - 0.18] [-0.022 - 0.016] (0.17 - 0.19)Phenylalanine (% DW) 0.43 (0.012) 0.47 (0.012) -0.041 (0.017) -0.076, -0.0063 0.022[0.40 - 0.44] [0.46 - 0.48] [-0.057 - -0.022] (0.40 - 0.46)Proline (% DW) 0.81 (0.022) 0.87 (0.022)-0.062 (0.032) -0.13, 0.0039 0.063[0.76 - 0.84] [0.84 - 0.91] [-0.11 - -0.0054] (0.71 - 0.86)Serine (% DW) 0.41 (0.013) 0.44 (0.013)-0.030 (0.018) -0.067, 0.0074 0.109[0.39 - 0.43] [0.42 - 0.46] [-0.044 - -0.020] (0.37 - 0.43)Threonine (% DW) 0.30 (0.0071) 0.31 (0.0071)-0.017 (0.010) -0.038, 0.0035 0.098[0.29 - 0.30] [0.30 - 0.33] [-0.028 - -0.011] (0.27 - 0.31)Tryptophan (% DW) 0.052 (0.0013) 0.050 (0.0013)0.0019 (0.0018) -0.0019, 0.0058 0.304[0.048 - 0.055] [0.048 - 0.051] [-0.00035 - 0.0038] (0.048 - 0.052)Monsanto Company 07-CR-191U Page 379 of 544Table E-43 (cont). Comparison of the Amino Acid Contentin Grain from MON 87460 and Conventional Control from theQUI Site in Chile during 2006/2007 under Water-Limited ConditionsDifference (Testminus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Tyrosine (% DW) 0.26 (0.021) 0.27 (0.021)-0.0032 (0.030) -0.067, 0.060 0.915[0.22 - 0.30] [0.20 - 0.32] [-0.10 - 0.097] (0.13- 0.28)Valine (% DW) 0.43 (0.0095) 0.46 (0.0095) -0.034 (0.013) -0.062, -0.0065 0.018[0.41 - 0.44] [0.46 - 0.47] [-0.049 - -0.020] (0.40 - 0.45)1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 380 of 544Table E-44. Comparison of the Fatty Acid Content in Grainfrom MON 87460 and Conventional Control from the QUI Site inChile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)16:0 Palmitic (% Total FA) 11.35 (0.099) 11.60 (0.099) -0.25 (0.12) -0.50, 0.0021 0.051[11.21 - 11.45] [11.48 - 11.72] [-0.51 - -0.080] (9.61 - 12.95)18:0 Stearic (% Total FA) 1.93 (0.038) 1.89 (0.038) 0.035 (0.053) -0.079, 0.15 0.527[1.84 - 2.01] [1.84 - 1.92] [-0.0075 - 0.10] (1.39 - 2.21)18:1 Oleic (% Total FA) 21.26 (0.22) 21.03 (0.22) 0.23 (0.27) -0.36, 0.82 0.412[20.92 - 21.89] [20.83 - 21.29] [-0.33 - 1.06] (21.04 - 31.63)18:2 Linoleic (% Total FA) 63.60 (0.26) 63.64 (0.26) -0.043 (0.31) -0.70, 0.61 0.890[62.77 - 64.18] [63.46 - 63.85] [-1.07 - 0.57] (54.81 - 65.11)18:3 Linolenic (% Total FA) 1.23 (0.017) 1.21(0.017) 0.019 (0.023) -0.029, 0.067 0.414[1.22 - 1.24] [1.18 - 1.23] [0.0040 - 0.041] (1.14 - 1.60)20:0 Arachidic (% Total FA) 0.32 (0.0063) 0.31(0.0063) 0.012 (0.0081) -0.0054, 0.029 0.168[0.30 - 0.33] [0.29 - 0.31] [-0.0081 - 0.034] (0.32 - 0.35)20:1 Eicosenoic (% Total FA) 0.18 (0.0024) 0.19 (0.0024) -0.0057 (0.0033) -0.013, 0.0013 0.103[0.18 - 0.18] [0.18 - 0.19] [-0.015 - 0.0041] (0.20 - 0.25)22:0 Behenic (% Total FA) 0.14 (0.021) 0.14(0.021) 0.0034 (0.029) -0.057, 0.064 0.906[0.13 - 0.15] [0.12 - 0.18] [-0.035 - 0.032] (0.063 - 0.16)1FA – fatty acid; S.E. = standarderror; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 381 of 544Table E-45. Comparison of the Vitamin Content in Grain from MON 87460 and Conventional Control from the QUI Site inChile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)Analytical Component1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Folic Acid (mg/kg DW) 0.27 (0.024) 0.31 (0.024) -0.032 (0.026) -0.089, 0.026 0.251[0.23 - 0.31] [0.24 - 0.39] [-0.11 - 0.025] (0.27 - 0.36)Niacin (mg/kg DW) 24.28 (1.34) 23.37 (1.34)0.91 (1.73) -2.79, 4.62 0.605[20.00 - 28.64] [21.92 - 24.71] [-1.92 - 3.93] (24.60 - 28.88)Thiamine HCl (mg/kg DW) 2.97 (0.12) 2.85 (0.12) 0.12 (0.17) -0.23, 0.47 0.468[2.86 - 3.14] [2.74 - 2.95] [-0.088 - 0.29] (2.88 - 3.66)Vitamin B2 (mg/kg DW) 2.15 (0.22) 2.11 (0.22) 0.036 (0.31) -0.62, 0.69 0.908[1.79 - 2.42] [1.96 - 2.27] [-0.49 - 0.46] (1.91 - 2.75)Vitamin B6 (mg/kg DW) 5.43 (0.21) 5.51 (0.21) -0.075 (0.30) -0.71, 0.56 0.804[5.41 - 5.46] [5.25 - 5.71] [-0.29 - 0.22] (4.19 - 6.07)Vitamin E (mg/kg DW) 11.52 (0.53) 10.57 (0.53) 0.95 (0.71) -0.56, 2.46 0.201[10.34 - 12.37] [10.05 - 10.94] [-0.40 - 2.32] (5.20 - 9.95)1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 382 of 544Table E-46. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain from MON 87460 andConventional Control from the QUI Site in Chile during 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Anti-nutrientPhytic Acid (% DW) 0.78 (0.042) 0.69 (0.042) 0.093 (0.060) -0.032, 0.22 0.136[0.75 - 0.80] [0.63 - 0.74] [0.052 - 0.17] (0.55 - 0.73)Raffinose (% DW) 0.10 (0.0064) 0.091 (0.0064)0.0094 (0.0090) -0.0094, 0.028 0.308[0.092 - 0.11] [0.081 - 0.11] [-0.010 - 0.028] (0.029 - 0.069)Secondary MetaboliteFerulic Acid (μg/g DW) 2079.28 (116.42) 1986.45 (116.42) 92.82 (158.06) -241.19, 426.84 0.564[2034.88 - 2119.13] [1898.80 - 2066.25] [40.58 - 185.01] (1503.59 - 2078.52)p-Coumaric Acid (μg/g DW) 192.51 (10.65) 189.88(10.65) 2.62 (14.34) -27.50, 32.75 0.856[181.40 - 201.40] [175.61 - 202.72] [-21.32 - 25.79] (127.14 - 277.33)1DW = dry weight; S.E. = standard error; CI = confidence interval.2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.Monsanto Company 07-CR-191U Page 383 of 544Table E-47. Summary of Significant Differences in Additional Secondary Metabolite Composition (p<0.05) ComparingMON 87460 to the Conventional Control from the QUI Site in Chile during 2006/2007under Well-Watered and WaterLimited ConditionsTissue/Site/Components (Units)1MeanMON 87460MeanControlMean Diff(% of Control)Signif.(p-value)MON 87460(Range)Commercial(Range)ForageWell-wateredFructose (% DW) 5.30 6.95 -23.770.005 (4.40 - 6.31) (6.50 - 8.09)Glucose (% DW) 6.09 7.73 -21.180.005 (5.22 - 6.96) (7.93 - 8.97)Water-LimitedGlucose (% DW) 6.10 7.53 -18.940.012 (5.55 - 6.81) (6.72 - 7.58)GrainWell-wateredAbscisic Acid (ppb FW) 10.80 18.93 -42.94 0.001 (9.41 - 12.60) (23.00 - 35.80)Glucose (% DW) 0.47 0.57 -17.650.021 (0.46 - 0.48) (0.41 - 0.64)Sucrose (% DW) 1.81 2.22 -18.670.002 (1.76 - 1.85) (2.07 - 2.74)Water-LimitedAbscisic Acid (ppb FW) 8.04 23.13 -65.24 <0.001 (7.02 - 8.66) [(9.30 - 30.500Sucrose (% DW) 1.84 2.10 -12.240.040 (1.67 - 2.08) (2.12 - 2.59)1DW= dry weight; FW=fresh weight.Monsanto Company 07-CR-191U Page 384 of 544Table E-48. Comparison of the Additional Secondary Metabolite Content in Forage from MON 87460 and ConventionalControl from the QUI Site in Chileduring 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Free Proline (% DW) 0.020 (0.0017) 0.018 (0.0017) 0.0019 (0.0023) -0.0030, 0.0069 0.422 (0.014 - 0.015)[0.017 - 0.022] [0.017 - 0.019] [-0.0015 - 0.0042]Abscisic Acid (ppb FW) 15.70 (4.35) 12.53 (4.35)3.17 (5.90) -9.39, 15.72 0.599 (13.00 - 19.80)[14.60 - 16.90] [10.50 - 14.00] [1.50 - 6.40]Choline (ppm FW) 127.67 (6.53) 120.00 (6.53) 7.67 (8.71) -10.78, 26.11 0.391 (111.00 - 115.00)[121.00 - 135.00] [111.00 - 133.00] [-6.00 - 24.00]Glycerol (% DW) 0.14 (0.0084) 0.14 (0.0084) 0.0019(0.011) -0.021, 0.025 0.864 (0.12 - 0.18)[0.14 - 0.14] [0.12 - 0.16] [-0.026 - 0.028]Glycine Betaine (ppm FW) 63.53 (5.24) 57.83 (5.24)5.70 (6.24) -7.73, 19.13 0.376 (49.80 - 64.20)[58.40 - 69.80] [51.60 - 62.80] [-4.40 - 10.80]Salicylic Acid (ppm FW) 0.098 (0.040) 0.16 (0.040)-0.058 (0.057) -0.18, 0.061 0.321 (0.075 - 0.15)[0.088 - 0.11] [0.091 - 0.21] [-0.12 - 0.020]Fructose (% DW) 5.30 (0.46) 6.95 (0.46) -1.65 (0.51) -2.74, -0.57 0.005 (6.50 - 8.09)[4.40 - 6.31] [6.22 - 7.44] [-2.26 - -0.88]Glucose (% DW) 6.09 (0.52) 7.73 (0.52) -1.64 (0.50) -2.71, -0.57 0.005 (7.93 - 8.97)[5.22 - 6.96] [6.99 - 8.15] [-2.05 - -1.10]Sucrose (% DW) 3.80 (1.31) 2.53 (1.31) 1.27(1.74) -2.41, 4.95 0.475 (0.11 - 3.77)[2.32 - 4.74] [2.08 - 2.93] [-0.25 - 2.25]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.Monsanto Company 07-CR-191U Page 385 of 544Table E-49. Comparison of the Additional Secondary Metabolite Content inGrain from MON 87460 and ConventionalControl from the QUI Site in Chileduring 2006/2007 under Well-Watered ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Free Proline (% DW) 0.072 (0.0030) 0.076 (0.0030) -0.0041 (0.0039) -0.012, 0.0042 0.313 (0.048 - 0.059)[0.068 - 0.076] [0.075 - 0.078] [-0.0091 - 0.0016]Abscisic Acid (ppb FW) 10.80 (1.71) 18.93 (1.71)-8.13 (2.05) -12.48, -3.78 0.001 (23.00 - 35.80)[9.41 - 12.60] [17.60 - 20.30] [-8.50 - -7.70]Choline (ppm FW) 210.67 (12.86) 227.33 (12.86) -16.67 (17.84) -54.25, 20.92 0.363 (194.00 - 265.00)[206.00 - 219.00] [213.00 - 248.00] [-29.00 - -6.00]Glycerol (% DW) 0.019 (0.0019) 0.019 (0.0019) 0.00018(0.0025) -0.0051, 0.0054 0.944 (0.018 - 0.025)[0.018 - 0.022] [0.016 - 0.024] [-0.0057 - 0.0061]Glycine Betaine (ppm FW) 1.89 (0.17) 1.75 (0.17)0.14 (0.23) -0.35, 0.64 0.548 (0.50 - 3.34)[1.69 - 2.08] [1.61 - 1.82] [0.080 - 0.26]Salicylic Acid (ppm FW) 0.092 (0.026) 0.12 (0.026)-0.032 (0.030) -0.099, 0.035 0.317 (0.069 - 0.32)[0.076 - 0.12] [0.095 - 0.15] [-0.043 - -0.019]Fructose (% DW) 0.46 (0.031) 0.54 (0.031) -0.081 (0.040) -0.17, 0.0034 0.058 (0.41 - 0.65)[0.44 - 0.48] [0.54 - 0.54] [-0.10 - -0.067]Glucose (% DW) 0.47 (0.031) 0.57 (0.031) -0.10(0.039) -0.18, -0.017 0.021 (0.41 - 0.64)[0.46 - 0.48] [0.53 - 0.60] [-0.12 - -0.071]Sucrose (% DW) 1.81 (0.083) 2.22 (0.083) -0.41(0.12) -0.66, -0.17 0.002 (2.07 - 2.74)[1.76 - 1.85] [2.10 - 2.38] [-0.53 - -0.30]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.Monsanto Company 07-CR-191U Page 386 of 544Table E-50. Comparison of the Additional Secondary Metabolite Content in Forage from MON 87460 and ConventionalControl from the QUI Site in Chileduring 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Free Proline (% DW) 0.024 (0.0017) 0.020 (0.0017) 0.0040 (0.0023) -0.00095, 0.0089 0.106 (0.012 - 0.027)[0.019 - 0.027] [0.018 - 0.021] [-0.0020 - 0.0076]Abscisic Acid (ppb FW) 17.10 (4.35) 11.56 (4.35)5.54 (5.90) -7.01, 18.09 0.362 (14.10 - 28.70)[13.80 - 21.40] [9.98 - 14.70] [3.82 - 6.70]Choline (ppm FW) 127.67 (6.53) 117.00 (6.53) 10.67 (8.71) -7.78, 29.11 0.238 (115.00 - 119.00)[116.00 - 139.00] [101.00 - 134.00] [0 - 27.00]Glycerol (% DW) 0.17 (0.0084) 0.15 (0.0084) 0.022(0.011) -0.0017, 0.045 0.067 (0.14 - 0.17)[0.15 - 0.20] [0.14 - 0.16] [0.0014 - 0.052]Glycine Betaine (ppm FW) 51.17 (5.24) 52.97 (5.24)-1.80 (6.24) -15.23, 11.63 0.777 (46.60 - 64.00)[42.20 - 61.10] [41.90 - 66.60] [-5.50 - 0.30]Salicylic Acid (ppm FW) 0.098 (0.040) 0.11 (0.040)-0.017 (0.057) -0.14, 0.10 0.769 (0.077 - 0.41)[0.073 - 0.15] [0.078 - 0.18] [-0.035 - -0.0042]Fructose (% DW) 5.48 (0.46) 6.53 (0.46) -1.05 (0.51) -2.14, 0.030 0.055 (5.68 - 6.42)[5.00 - 5.97] [6.33 - 6.68] [-1.68 - -0.61]Glucose (% DW) 6.10 (0.52) 7.53 (0.52) -1.43 (0.50) -2.49, -0.36 0.012 (6.72 - 7.58)[5.55 - 6.81] [7.51 - 7.56] [-1.96 - -0.70]Sucrose (% DW) 2.33 (1.31) 3.06 (1.31) -0.72(1.74) -4.40, 2.96 0.682 (0.095 - 4.24)[1.14 - 4.26] [1.49 - 5.56] [-3.96 - 2.14]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.Monsanto Company 07-CR-191U Page 387 of 544Table E-51. Comparison of the Additional Secondary Metabolite Content inGrain from MON 87460 and ConventionalControl from the QUI Site in Chileduring 2006/2007 under Water-Limited ConditionsDifference (Test minus Control)AnalyticalComponent1TestMean ± S.E.1[Range]ControlMean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower,Upper)p-ValueCommercial(Range)Free Proline (% DW) 0.068 (0.0030) 0.073 (0.0030) -0.0045 (0.0039) -0.013, 0.0038 0.268 (0.044 - 0.067)[0.064 - 0.075] [0.067 - 0.078] [-0.012 - 0.0076]Abscisic Acid (ppb FW) 8.04 (1.71) 23.13 (1.71) -15.09 (2.05) -19.44, -10.74 <0.001 (19.30 - 30.50)[7.02 - 8.66] [22.50 - 23.90] [-16.88 - -13.84]Choline (ppm FW) 231.00 (12.86) 234.00 (12.86) -3.00 (17.84) -40.59, 34.59 0.868 (175.00 - 235.00)[209.00 - 265.00] [212.00 - 262.00] [-53.00 - 53.00]Glycerol (% DW) 0.019 (0.0019) 0.018 (0.0019) 0.0016(0.0025) -0.0036, 0.0069 0.520 (0.017 - 0.021)[0.018 - 0.020] [0.014 - 0.024] [-0.0032 - 0.0042]Glycine Betaine (ppm FW) 2.09 (0.17) 2.10 (0.17)-0.010 (0.23) -0.50, 0.48 0.966 (0.50 - 2.91)[1.99 - 2.18] [1.63 - 2.44] [-0.34 - 0.36]Salicylic Acid (ppm FW) 0.094 (0.026) 0.14 (0.026)-0.048 (0.030) -0.11, 0.019 0.143 (0.055 - 0.38)[0.087 - 0.10] [0.11 - 0.18] [-0.080 - -0.019]Fructose (% DW) 0.50 (0.031) 0.48 (0.031) 0.022 (0.040) -0.063, 0.11 0.586 (0.38 - 0.66)[0.41 - 0.57] [0.45 - 0.52] [-0.038 - 0.054]Glucose (% DW) 0.52 (0.031) 0.48 (0.031) 0.031(0.039) -0.053, 0.11 0.449 (0.38 - 0.66)[0.45 - 0.57] [0.47 - 0.52] [-0.021 - 0.063]Sucrose (% DW) 1.84 (0.083) 2.10 (0.083) -0.26(0.12) -0.50, -0.013 0.040 (2.12 - 2.59)[1.67 - 2.08] [1.96 - 2.18] [-0.51 - -0.086]1DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.Monsanto Company 07-CR-191U Page 388 of 544 Appendix F. Phenotypic and Agronomic A ssessment: Individual Site Results from

2006 and 2007 Field Studies

F.1. Materials

The materials for phenotypic, agronomic, a nd plant pest assessment studies are included in Tables F-2 – F-7, and planting information for each site is reported in Tables F-8 – F-13. The presence or absence of the MON 87460 insert in the test and control materials was confirmed by event-specific PCR analyses.

F.2. Test Sites

F.2.1. 2006 and 2007 U.S. Field Studies under Well-Watered Conditions

Field trials included eight test sites that we re established as well watered locations in 2006: (site codes in parenthesis): Jefferson Co., IA (IA1), Benton, Co., IA (IA2), Stark Co., IL (IL1), Warren Co., IL (IL2), Boone Co ., IN (IN1), Parke Co., IN (IN2), Pawnee Co., KS (KS) and York Co., NE (Tables F-1, F-2, and F-8); and nine sites in 2007 (site codes in parenthesis): Jefferson County, IA (IA1); Van Horne County, IA ( IA2); Stark County, IL ( IL1); Warren County, IL (IL2); Clinton County, IL (IL3); Boone County, IN (IN); York County, NE (NE); Fayette Count y, OH (OH); and Berks County, PA (Tables F-1, F-3, and F-9).

F.2.2. 2006 and 2007 U.S. and Chilean Field Studies Established with Well-Watered

and Water-Limited Treatments

In 2006/2007, a Chile field study was establis hed at four sites: Calera de Tango, Region Metropolitana de Santiago (CT); Co lina, Region Metropolitana de Santiago (CL); Lumbreras, Region Metropolitana de Santiago (LUM); and Quillota, Region de Valparaiso (QUI) (Tables F-1, F-4, and F-10)

In 2007, two U.S. field studie s were established. Study-1 had two sites: Sutter County, CA (CA) and Carson, County, TX (Tables F-1, F-5, and F-11). Study-2 had three sites: Pawnee County, KS (KS); Yo rk County Nebraska (NE); and Carson County, TX (Tables F-1, F-6, and F-12).

F.2.3. 2006 U.S. Field Study Established with Water Managed According to Local Agronomic Practices

In 2006, five sites where water was managed according to local agronomic practices were established (site codes in parenthesis): Benton County, IA (IAE) ; Greene County, IA (IAW); Stark County, IL (IL); Parke County, IN (IN); and York County, NE (Tables F-1, F-7, and F-13).Monsanto Company 07-CR-191U Page 389 of 544 F.3. Statistical Analyses

Statistical analyses were performed by the Monsanto Statistics Technology Center. SAS was used for analysis and contrast statements in SAS were used to compare the test to the control. The level of statistical significance was 5% ( α = 0.05). No statistical comparisons were made between the test and reference substances. The reference range,

defined as the minimum and maximum me an values, was determined from the references among the sites. The means of th e test and control subs tance and the results of the statistical analyses are reported. Sta ndard error was calculated from the raw data.

Each test and control mean has its own individual standard deviation and a different standard error. The characteristics reported for each substance type include: seedling vigor, early stand count, days to 50% pollen shed, days to 50 % silking, stay green rating, ear height, plant height, dropped ears, stalk l odged plants, root lodged plants, final stand count, grain moisture percenta ge, test weight, insect abun dance, European corn borer and corn earworm damage, and yield.

F.4. Individual Field Study Results

F.4.1. 2006 U.S. Field Study under Well-Watered Conditions

The results of the 14 phenotypic or agronomic characteri stics evaluated at eight wellwatered sites in 2006 ar e presented in Table F-14. In the individual site analyses, no

differences between MON 87460 and the contro l were detected for seedling vigor, days to 50% pollen shed, days to 50% silk ing, ear height, plant height, dropped ears, grain moisture, or test weight at any location. A total of seven out of 96 site × characteristic comparisons were significantly different between MON 87460

and the control. The differences were distributed among six of the 14 phenotypic characteristics. MON 87460 had a higher earl y stand count than the control at IL1 (104.3 vs. 86.0) and a lower early stand c ount than the contro l at NE (102.0 vs. 114.3). MON 87460 had lower stay green (less green tissue) than the control at IA2 (4.0 vs. 5.0). MON 87460 had more stalk lodg ed plants than the control at NE (9.0 vs. 5.0) and more root lodged plants th an the control at IL1 (23.7 vs. 2.7). MON 87460 had a higher final stand than th e control at IL1 (94. 0 vs. 77.3). Yield was lower for MON 87460 compared to the control at KS (159.4 vs. 182.3).

The differences detected in the individual site analyses for ear ly stand count, stay green, stalk lodging, final stand, and yield were not detected in the combined-site analysis (Table VIII-4). Thus, the differences detected for these phenotypic characteristics in the by-site analysis were not indicative of a consistent trend in the data and are unlikely to be biologically meaningful in terms of increased weed potential of MON 87460 compared to the cont rol (Figure VIII-1, Step 2). The results of this study support a conclu sion that the introduction of the drought tolerance trait did not unexpectedly alte r the assessed phenotypic ch aracteristics of MON 87460 compared to conventional corn. Thus, the results support a conclusion of no increased pest potential or significant environmen tal impact of MON 87460 compared to conventional corn.Monsanto Company 07-CR-191U Page 390 of 544 F.4.2. 2007 U.S. Field Study under Well-Watered Conditions

The results of the 14 phenotypic or agronomic characteristics evaluated at nine wellwatered sites in 2007 are presented in Table F-15. In the individual analysis, no

differences between MON 87460 and the contro l were detected for seedling vigor, early stand count, days to 50% pollen shed, days to 50% silking, ear height, plant height, root lodged plan ts, final stand count, test weight , and yield. Eight differences

were detected in the individual site an alysis in the 2007 trials spanning four assessments. Stay green values were higher for MON 87460 vs. the control (less green tissue) than the control at IA2 (5.7 vs. 5.0), at IL1 (6.7 vs. 5.3), and at IL3 (4.0 vs. 3.0, respectively at each site). Dropped ears were greater for MON 87460 compared to the control at PA (1.3 vs. 0.0 respectively). MON 87460 had more stalk lodged plants than the control at OH (3.7 vs. 1.0) and at PA (2.3 vs. 0.3, respectively at each site). MON 87460 was higher in grain moisture th an the control at IA2 (18.0 vs. 17.1, respectively), but was lower in grai n moisture than the control at IL3 (13.0 vs. 14.7, respectively). Differences in st ay green, dropped ears, stalk lodged plants, and grain moisture were not observed in the combined-site analysis reported in Table VIII-5). Thus, the differences detected for these phenotypic characteristics are not

indicative of a consistent re sponse associated with the tr ait and are unlikely to be biologically meaningful in te rms of increased pest potential of MON 87460 compared to the control (Figure VIII-1, Step 2). Th e results of this study support a conclusion

that the introduction of the drought tolerance tr ait did not unexpectedly alter the assessed phenotypic characteristics of MO N 87460 compared to conventional corn and a conclusion of no increased pest poten tial or significant environmental impact of MON 87460 compared to conventiona l corn under well watered conditions.

F.4.3. 2006/2007 Chilean Field Study Estab lished with Well-Watered and WaterLimited Treatments

The results of the 14 phenotypic or agronomic characteristics evaluated at three sites

in Chile for the well-watered treatment in 2006/ 2007 are presented in Table F-16. In the individual site analysis of the well-w atered treatment, no significant differences were detected at the CL, CT, and LUM sites for any measured characteristic supporting the hypothesis of no statistical differences between MON 87460 and the

control under well-watered conditions.

The results of the 14 phenotypic or agronomic characteristics evaluated at three sites

in Chile for the water-limited treatment in 2006/2007 are presented in Table F-17. Data for a fourth site, QUI, are reported in Appendix G. In the individual site analysis of the water-limited treatment, no significant differences were detected between MON 87460 and the control at the CL, CT, and LUM sites for seedling vigor, early stand count, days to 50% pollen shed, days to 50% silking, stay green rating, dropped ears, stalk lodged plants, ro ot lodged plants, fina l stand count, grain moisture, and test weight. Ear height and plant height were higher for MON 87460 compared to the control at the CL site (61. 0 vs. 51.4 in; 97.2 vs. 79.3 in, respectively). Yield was higher for MON 87460 than the control for the CT site (160.9 vs. 123.7 bu/A, respectively). The differences detected in ear height and plant height were not detected when the data were pooled across al l three sites (Table VIII-7). Thus, theMonsanto Company 07-CR-191U Page 391 of 544 differences detected for th ese phenotypic characteristics are not indicative of a consistent response associated with the trait and are unlikely to be biologically meaningful in terms of in creased pest potential of MON 87460 compared to the control. (Figure VIII-1, Step 2). However, the difference in yield detected at the CT site is consistent with the expected yield bene fit of this trait and was also detected in the combined-site analysis (Table VIII-7). Furthermore, statistical differences in ear height and plant height ar e expected differences betw een MON 87460 and the control under water-limited conditions (Figure VIII-2).

The results of this study support a conclu sion of no increased pest potential or significant environmental impact for MON 87460 when grown under well-watered or

water-limited conditions in the field.

F.4.4. 2007 U.S. Field Study-1 Establishe d with Well-Watered and Water-Limited

Treatments

The results of the 14 phenotypic or agronomic characte ristics evaluated at two sites with the well-watered treatment in Study-1 in 2007 are presented in Table F-18. In the individual-site analysis, no differe nces between MON 87460 and control were detected for seedling vigor, days to 50% pol len shed, days to 50% silking, ear height, dropped ears, root lodged plan ts, final stand count, grain moisture, and test weight (TX site only) in the well-watered treatm ent. Five comparisons between MON 87460 and the control were significantly different in the individual-si te analysis under wellwatered conditions. At th e CA site, MON 87460 had highe r early stand count than the control at (75.8 vs. 70.0 plants), MON 87460 had more stalked lodged plants than the control (0.5 vs. 0.0 plants) and M ON 87460 had more yield than the control (239.7 vs. 181.4 bu/acre). At the TX s ite, MON 87460 had a lower stay green value (more green tissue) than the control (1.8 vs. 3.5), and MON 87460 was shorter than the control (70.4 vs. 75.1 in.). At the CA site, mean values for MON 87460 for early stand count (75.8 plants) and stalk lodged plants (0.5 plan ts) are within the range of reference hybrid values (Table VIII-8). Therefore, the differences in early stand count and stalk lodged plants are unlikely to be biologically meaningful in terms of increased weed potential of MON 87460 compared to the control (Figure VIII-1, Step 3). The differences reported in Tabl e F-18 at CA for yield of MON 87460 (239.7 bushel/acre), and at TX for stay green (1.8 rating) and for plant height (70.4 inches) are not interpreted as characteristics that would confer an increase in weediness potential. The differences detected for yiel d, stay green and plant height were not detected in the combined-site analysis for th e well-watered treatment (Table VIII-8). Therefore, the differences detected in the individual site analysis are unlikely to be

adverse and do not contribut e to a biological or environmental change for MON 87460 in terms of pest potential.

The results of the 14 phenotypic or agronomic characte ristics evaluated at two sites with the water-limited treatment in Study-1 in 2007 are presented in Table F-19. In the individual-site analysis, no differen ces between MON 87460 and the control were detected for seedling vigor, early stand c ount, days to 50% polle n shed, days to 50% silking, ear height, plant height, dropped ear s, stalk lodged plants, root lodged plants,Monsanto Company 07-CR-191U Page 392 of 544 final stand count, grain moisture, and test weight in the water-limited treatment). Two comparisons between MON 87460 and the control were si gnificantly different in the individual-site analysis under wate r-limited conditions. At the CA site, MON 87460 had more yield than the control (188.3 vs. 148. 1 bu/acre, respectively). At the TX site, MON 87460 had a lower stay green value (more green tissue) than the control (1.7 vs. 4.3 respectively). The di fference detected in stay green is not interpreted as a characteristic that would c onfer an increase in weediness potential. Furthermore, the difference in stay green was not detected in the combined-site analysis for plants in the water-limited treatment (Table VIII-9). Therefore, the

difference detected in the individual site an alysis for stay green is likely not adverse

and does not contribute to a biological or environmental change for MON 87460 in terms of pest potential.

F.4.5. 2007 U.S. Field Study-2 Establishe d with Well-Watered and Water-Limited

Treatments

The results of the 14 phenotypic or agronomic characteristics evaluated at three sites with the well-watered treatment in Study-2 in 2007 are presented in Table F-20. In the individual-site analysis, no differe nces between MON 87460 and control were detected for seedling vigor, early stand c ount, days to 50% polle n shed, days to 50% silking, ear height, plant height, dropped ear s, stalk lodged plants, root lodged plants, final stand count, test wei ght, and yield in the well-w atered treatment. Three comparisons between MON 87460 and the contro l were significantly different in the individual-site analysis under well-wa tered conditions. MON 87460 had a higher stay green rating at NE (4.7 vs. 4.0) and a lower stay green rating at TX (6.3 vs. 8.0, respectively) than the control. MON 87460 was higher in grain moisture than the control at KS (13.7 vs. 12.4, respectively). The differences detected in stay green and grain moisture are within the range of re ference hybrid values (Table VIII-12). Therefore, the differences in stay green and grain moisture are unlikely to be biologically meaningful in terms of increased weed potential of MON 87460 compared to the control (F igure VIII-1, Step 3).

Results from the water-limited treatment are presented in Section VIII.C.2 and Appendix G. TX was the only site that me t the inclusion criteria (Section VIII.C, Table VIII-3). Data for the water-limited treatment at TX are presented in Section VIII.C.2.2, Table VIII-11. Data for the water-limited treatment at KS and NE are presented in Appendix G, Table G-3.

The results of this study support a conc lusion that MON 87460 wa s not unexpectedly altered for the assessed phenotypic characte ristics compared to conventional corn under water-limited conditions. Thus, the results support a conclusion of no increased pest potential or significant environmen tal impact of MON 87460 compared to a conventional control under well-watered or wa ter-limited conditions.Monsanto Company 07-CR-191U Page 393 of 544 F.4.6. 2006 U.S. Field Study Established with Water Managed According to Local Agronomic Practices

The results of the 14 phenotypi c or agronomic characteristic s evaluated at five sites where water was managed by local agronom ic practices in 2006 are presented in Table F-21. In the individual site analys is, no differences were detected between MON 87460 and the control at any site fo r early stand count, days to 50% pollen shed, stay green rating, ear height, plant height, dropped ears, fi nal stand count, grain moisture, test weight, and yield. A total of four differences were detected across four

different characteristics. At the NE site, MON 87460 exhi bited lower vigor than the control (7.0 vs. 8.0) and greater days to 50% silking (65.0 vs. 64.0). At the IN site

MON 87460 exhibited more st alk lodged plants and root lodged plants than the control (8.3 vs. 4.7 plants, and 3.3 vs. 0. 3 plants, respectively). The differences detected in vigor, days to 50% silking, stal k lodging and root lodging were not detected when the data were pooled across a ll five sites (Table VIII-12). Thus, the

differences detected for th ese phenotypic characteristics are not indicative of a consistent response associated with the trait and are unlikely to be biologically meaningful in terms of increased weed potential under well-watered conditions.Monsanto Company 07-CR-191U Page 394 of 544Table F-1. Field Phenotypic Evaluation Sites for MON 87460 during 2006 and 2007Water Management Study Year LocationSiteCodeWellwatered2006JeffersonCo.,Iowa IA1(randomized complete block design)Benton Co.,Iowa IA2Stark Co.,Illinois IL1Warren Co.,Illinois IL2Boone Co.,Indiana IN1Parke Co.,Indiana IN2Pawnee Co.,Kansas KSYork Co.,NebraskaNE(randomized complete block design)2007 Jefferson Co.,Iowa IA1Van Horne Co.,Iowa IA2StarkCo.,Illinois IL1Warren Co.,Illinois IL2Clinton Co.,Illinois IL3Boone Co.,Indiana INYork Co.,NebraskaNEFayette Co.,Ohio OHBerks Co.,Pennsylvania PAWell-watered and water-limited2006/2007 Calera de Tango,Chile1CT(strip-plotdesign)Colina,Chile1CLLumbreras,Chile1LUMQuillota,Chile1QUI(split-plotdesign)2007 Study-1 Sutter Co.,California CACarson Co.,Texas TX(strip-plotdesign)2007 Study-2 Pawnee Co.,Kansas CAYork Co.,NebraskaNECarson Co.,Texas TXTypical agronomicpractices,2006 Benton Co.,Iowa IAE(rainfed and normal irrigation)Greene Co.,Iowa IAW(randomized complete block design)Stark Co.,Illinois ILParke Co.,Indiana INYork Co.,NebraskaNEMonsanto Company 07-CR-191U Page 395 of 544 Table F-2. Test, Control, and Reference Substances in the 2006 U.S. Field Study

under Well-Watered Conditions

Corn Hybrid Names 1 Seed Types Site

MON 87460 Test All

H1548126 Control All

DKC 61-42 Reference IA1, IA2

DKC 60-15 Reference IA1, IA2

DKC 63-78 Reference IA1, IA2

33N29 Reference IA1, IA2

33K39 Reference IL1, IL2

M-3744 Reference IL1, IL2

M-3765 Reference IL1, IL2

RX715AR Reference IL1, IL2

631 MF-B17 Reference IN1, IN2

33N56 Reference IN1, IN2

H8920 Reference IN1, IN2

RX715AR Reference IN1, IN2

S-2721 Reference KS

32B33 Reference KS

33H25 Reference KS

H8991 Reference KS

G-8424 Reference NE

NC+4822 Reference NE

34N43 Reference NE

DKC 61-50 Reference NE

1 Test and control substance names are a Monsanto Regulatory ID. Reference substance names are commercial corn hybrid designations.Monsanto Company 07-CR-191U Page 396 of 544 Table F-3. Test, Control, a nd Reference Substances in the 2007 U.S. Field Study under Well-Watered Conditions

Corn Hybrid Names 1 Seed Types Site

MON 87460 Test All

DM1718 Control All

DKC 57-83 Reference IL2, IL3, OH, PA

DKC 60-15 Reference IA1, IL1, IL3, OH

G-8424 Reference IA1, IA2, IL2, IN

RX 715 Reference IA1, IA2, IL2, IL3

DKC 61-42 Reference IA2, IN, NE, PA

DKC 61-50 Reference IA2, IL1

H-8920 Reference IL1,NE

RX754RR Reference OH, PA

DKC 63-78 Reference IL1, IL3, IN, PA

Pioneer 33H25 Reference IA1, IN, NE

DKC 64-27 Reference IL2, NE, OH

1 Test and control substance names are a Monsanto Regulatory ID. Reference

substance names are commercial corn hybrid designations.Monsanto Company 07-CR-191U Page 397 of 544 Table F-4. Test, Control, and Reference Corns used in the 2006/2007 Chilean Field Study Established with

Well-Watered and Wat er-Limited Treatments

Corn Hybrid Names 1 Seed Types Site

MON 87460 Test All Sites

DM1718 Control All Sites

33N09 Reference CT

33K39 Reference CT

BT 6613 Reference CT

DKC63-78 Reference CT

33D11 Reference CL

BT 6011 Reference CL

Garst 8424 Reference CL

DKC62-30 Reference CL

33N29 Reference LUM

Garst 8445 Reference LUM

DKC61-50 Reference LUM

RX 715 Reference LUM

34N43 Reference QUI

BT 6610 Reference QUI

Garst 8545 Reference QUI

DKC60-15 Reference QUI

1 Test and control substance names are a Monsanto Regulatory ID.

Reference substance names are comm ercial corn hybrid designations.

Table F-5. Study-1, Test, Control, and Reference Corns

used in the 2007 U.S. Field Studies Established with

Well-Watered and Wat er-Limited Treatments

Corn Hybrid Names 1 Seed Types Site Code

MON 87460 Test CA, TX

DM1718 Control CA, TX

DKC63-78 Reference CA, TX

RX715 Reference CA

RX754RR2 Reference CA

DKC61-50 Reference CA

33H25 Reference TX

DKC61-42 Reference TX

DKC57-83 Reference TX

1 Test and control substance names are a Monsanto Regulatory ID.

Reference substance names ar e commercial corn hybrid designations.Monsanto Company 07-CR-191U Page 398 of 544 Table F-6. Study-2, Test, Control, and Reference Corns used in the

2007 U.S. Field Studies Establishe d with Well-Watered and WaterLimited Treatments

Corn Hybrid Names 1 Substance Type Phenotype Sites

MON 87460 Test drought tolerant All

DM 1718 Control conventional All

Burrus 645 Reference conventional KS

DKC63-78 Reference conventional KS

33H25 Reference conventional KS

RX 772 Reference conventional KS

33D11 Reference conventional NE

DKC62-30 Reference conventional NE

BT 6613 Reference conventional NE

Garst 8445 Reference conventional NE

SC 1085 Reference conventional TX

DKC57-01 Reference conventional TX

RX 708 Reference conventional TX

Garst 8545 Reference conventional TX

1 Test and control substance names are a Monsanto Regulatory ID. Reference substance names are commercial corn hybrid designationsMonsanto Company 07-CR-191U Page 399 of 544 Table F-7. Test, Control, and Reference Substances in the 2006 U.S. Field Study under Typical Agronomic Conditions

Corn Hybrid

Names 1 Seed Types Site

MON 87460 Test All Sites

H1548126 Control All Sites

DKC 61-42 Reference IAE

DKC 60-15 Reference IAE

DKC 63-78 Reference IAE

H8991 Reference IAW

DKC 61-50 Reference IAW

33N29 Reference IAW

33K39 Reference IL

M-3744 Reference IL

M-3765 Reference IL

BT-6512 Reference IN

B-625 Reference IN

B-645 Reference IN

G-8424 Reference NE

NC+4822 Reference NE

34N43 Reference NE

1 Test and control substance names are a Monsanto Regulatory ID. Reference substance names are commercial corn hybrid designations.Monsanto Company 07-CR-191U Page 400 of 544Table F-8. Test Site Locations, Planting Information, Soil Description, and Cropping History in a 2006 Field Study underWell-Watered ConditionsStudysite1Plantingdate(m/dd/yy)Plantingrate(seeds/row)2Plantingdepth(in)Plot size(ft)Reps3Soil series; organic matter; soil pH2005crop2004cropIA15/26/06401.510 x 203Silty clay loam; 3.6%; 7.7SoybeanCornIA25/19/06402.010 x 203Silty clay loam; 3-4%; 6.2FallowCornIL15/25/06601.520 x 303Plano silt loam; 3.5%; 6.2CornCornIL25/23/06402.010 x 203Muscatine silt loam; 3.6%; 6.1SoybeanCornIN15/23/06401.5-1.810 x 203Silt loam; 2.1%; 6.9CornSoybeanIN26/06/06401.510 x 203Reesville silt loam; 2.0%; 6.9CornCornKS5/17/06601.520 x 303Lubbock silt Loam; 1.2%; 7.9SorghumSoybeanNE5/13/06601.520 x 303Hastings silt loam; 3.0%; 6.6SoybeanSoybean1Study sites: IA1 = Jefferson County, IA; IA2 = Benton, County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IN1 = Boone County, IN; IN2 = ParkeCounty, IN; KS = Pawnee County, KS, NE = York County, NE.2After seedling vigor and early stand count data were collected at each site, all plots were thinned to a uniform density.3Reps = replications.Monsanto Company 07-CR-191U Page 401 of 544Table F-9. Test Site Locations, Planting Information, SoilDescription, and Cropping History in 2007 U.S. Field Studiesunder Well-Watered ConditionsStudySite1Plantingdate(m/dd/yy)Plantingrate2(seed/row)Plantingdepth(in)Plot size(ft)Reps3Soil series; organic matter (%); and pH2006crop2005cropIA15/11/07601.810 x 303Tainter-Mahska silty clay loam; 3.9%; 6.5SoybeanCornIA25/15/07402.010 x 203Tama silty clay loam; 2.8%; 6.3SoybeanCornIL15/16/07601.810-13 x 303Sawmill silt loam; 3.4%; 6.3CornCornIL25/17/07401.810 x 203Sable silty clay loam; 6%; 5.6-7.3FallowSoybeanIL35/17/07401.510 x 203Cisne silt loam; 1.5-2.0%; 6.5-7.0CornWheat/SoybeanIN5/16/07601.510 x 303Crosby silt loam; 2.1%; 6.9CornSoybeanNE5/11/07402.010 x 203Hastings silt loam; 3.0%; 6.1SoybeanCornOH5/21/07401.510 x 203Millsdale silt loam; 2.0%; 7.1CornSoybeanPA5/22/07401.510 x 253Philo Atkin silt loam; 2.4%; 5.8FallowTomato1Study sites: IA1 = Jefferson County, IA; IA2 = Van Horne County, IA; IL1 = Wyoming County, IL; IL2 = Monmouth County, IL; IL3= Carlyle County, IL;IN = Sheridan County, IN; NE = York County, NE;OH = Fayette County, OH; PA = Hamburg County, PA.2After seedling vigor and early stand count data were collected at each site, all plots were thinned to a uniform density.3Reps = replications.Monsanto Company 07-CR-191U Page 402 of 544Table F-10. Test Site Locations, Planting Information, Soil Description, and Cropping History in a2006/2007 Chilean FieldStudy Established with Well-Watered and Water-Limited TreatmentsStudysite1Plantingdate(mm/dd/yy)Plantingrate(seeds/row)2Plantingdepth(in)Plot size(ft)Reps3Soil series; organic matter; pH2005-2006crop2004-2005cropCL12/19/0642216.4 x 19.73Clay; 3 – 5%; 5.5 – 6.5TablegrapesTablegrapesCT12/15/0642216.4 x 19.73Clay; 4 – 5%; 7 – 9OatsTablegrapesLUM12/15/0642216.4 x 19.73Sandy clay; 3 – 5%; 5.5 – 6.5PumpkinsCornQUI†12/16/0642216.4 x 19.73Clay; 3 – 5% ; 5.5 – 6.5Swisschard,cilantro,beetsSwisschard,cilantro,beets1Study sites: CL = Colina; CT = Calera de Tango; LUM = Lumbreras; QUI = Quillota. All sites were managed as well-watered and water-limited locations.†Site QUI did not meet defined non-stress / stress criteria. Data are reported inAppendix G.2After seedling vigor and early stand count data were collected, all plots were thinned to approximately 40 plants per row.3Reps = replications.Monsanto Company 07-CR-191U Page 403 of 544Table F-11. Study-1, Test Site Locations,Planting Information, Soil Description,and Cropping History in 2007 U.S. FieldStudies Established with Well-Watered and Water-Limited TreatmentsStudySite1Plantingdate(m/dd/yy)Plantingrate(seeds/row)2Plantingdepth(in)Plot size(ft)Reps3Soil series; organic matter; pH2006crop2005cropCA5/22/07401.0-2.010 x 204Subaco clay loam; 1.9%; 7.0FallowFallowTX5/31/07600.5-0.820 x 304Pullman loam; 2.3%; 7.2SorghumWheat1Study sites: CA = Sutter County, CA; TX = Carson, County, TX2After seedling vigor and early stand count data were collected at each site, all plots were thinned to a uniform density.3Reps = replications.Table F-12. Study-2, Test Site Locations,Planting Information, Soil Description,and Cropping History in 2007 U.S. FieldStudies Established with Well-Watered and Water-Limited TreatmentsStudySite1Planting date(m/dd/yy)Planting rate(seeds/row)2Planting depth(in)Plot size(ft) Reps3Soil Series; organic matter; pH2006crop2005cropKS 5/12/07 35 1.5 20 × 20 3 Farnum silt loam; 3.1%; 7.8 wheat alfalfaNE 5/03/07 35 2 15 × 20 3 Hastingssilt loam; 3%; 6.3 soybean soybeanTX 5/30/07 35 0.5 20 × 20 3 Pullman silty clay loam; 2.2%: 7.1 cotton wheat1Study sites: KS – Pawnee County, KS; NE = York County, NE; and TX = Carson County, TX.2After seedling vigor and early stand count data were collected,all plots at each site were thinned to a uniform density.3Reps = replicationsMonsanto Company 07-CR-191U Page 404 of 544Table F-13. Test Site Locations, Planting Information, SoilDescription, and Cropping History in a 2006 U.S. Field StudyEstablished with Water Managed According to Local Agronomic PracticesStudysite1Plantingdate(mm/dd/yy)Plantingrate(seeds/row)2Plantingdepth(in)Plot size(ft)Reps3Soil series; organic matter; pH2005crop2004cropIAE5/19/06342.020 x 153Silty clay loam; 3.5%; 6.2FallowCornIAW5/16/06342.020 x 153Loam; 3 – 4%; 5.6 – 7.3SoybeansCornIL5/23/06341.820 x 153Plano silt loam; 3.5%; 6.2Field cornField cornIN6/06/06341.420 x 153Silt loam; 2.0%; 6.9CornCornNE5/12/06341.520 x 153Silt loam; 3.0%; 6.6SoybeansSoybeans1All sites were managed according to local agronomic practices.Site codes are as follows: IAE = Benton County, IA; IAW = Greene County, IA; IL = StarkCounty, IL; IN = Parke County, IN; NE = York County, NE.2After seedling vigor and early stand count data were collected, all plots were thinned to a uniform density.3Reps = replications.Monsanto Company 07-CR-191U Page 405 of 544Table F-14. Phenotypic Comparison of MON 87460 to the Control at Each Site in the 2006 U.S. Field Study UnderWell-Watered ConditionsPhenotypic Characteristic, (units), Mean ± S.E.Seedling vigor(0=9 scale)Early stand count(#/plot)1Days to 50% pollenshedDays to 50% silkingStay green(0-9 scale)SiteMON87460ControlMON87460ControlMON87460ControlMON87460ControlMON87460ControlIA18.0 ± 0.00 8.0 ± 0.00 74.7 ± 1.45 72.0 ± 2.8955.7 ±0.6757.0 ±0.5856.0 ± 0.58 55.3 ± 1.20 3.3 ± 0.67 2.7 ± 0.33IA27.0 ± 0.007.0 ± 0.0068.7 ± 1.33 70.7 ± 2.0365.0 ±0.0065.0 ±0.0063.0 ± 0.00 63.0 ± 0.00 4.0* ± 0.00 5.0 ± 0.00IL19.0 ± 0.009.0 ± 0.00104.3* ±0.8886.0 ± 7.5165.7 ±0.3366.0 ±0.5863.0 ± 0.00 63.3 ± 0.33 4.0 ± 0.00 4.0 ± 0.00IL27.7 ± 0.337.3 ± 0.33- -57.3 ±0.3357.7 ±0.6758.0 ± 0.00 58.0 ± 0.58 4.3 ± 0.33 4.0 ± 0.00IN14.3 ± 0.335.0 ± 0.0062.7 ± 9.21 65.3 ± 4.9865.0 ±0.0065.0 ±0.0063.0 ± 0.00 63.0 ± 0.00 2.0 ± 0.58 2.3 ± 0.33IN28.3 ± 0.338.0 ± 0.0077.3 ± 1.33 75.7 ± 0.8857.3 ±0.3357.0 ±0.0056.3 ± 0.33 56.0 ± 0.58 4.7 ± 0.33 4.7 ± 0.33KS5.7 ± 0.336.0 ± 0.00104.0 ± 4.04 108.7 ± 1.2057.0 ±0.5856.7 ±0.3357.3 ± 0.67 56.7 ± 0.33 4.7 ± 0.33 5.0 ± 0.00NE7.3 ± 0.337.3 ± 0.33102.0* ±0.58114.3 ± 1.3363.0 ±0.0062.7 ±0.3363.0 ± 0.00 63.3 ± 0.33 6.0 ± 0.00 5.0 ± 0.58S.E. = standard error*Indicates significant differences detected between MON 87460 and the control (p<0.05).1Dashes indicate that data are missingor excluded from statistical analysis.Monsanto Company 07-CR-191U Page 406 of 544Table F-14 (continued). Phenotypic Comparison of MON 87460 to the Control at EachSite in the 2006 U.S. Field study underWell-Watered ConditionsPhenotypic Characteristic, (units), Mean ± S.E.Ear height (in)Plant height (in)Dropped ears(#/plot)Stalk lodged plants(#/plot)Root lodged plants(#/plot)SiteMON87460ControlMON87460ControlMON87460ControlMON87460ControlMON87460ControlIA150.1 ± 0.52 49.7 ± 1.2996.5 ±0.4797.9 ±1.44 0.0 ± 0.00 0.0 ± 0.00 7.0 ± 0.58 8.3 ± 3.18 0.02± 0.000.0 ± 0.00IA253.1 ± 0.35 54.2 ± 0.53107.1 ±1.33106.4 ±0.60 0.7 ± 0.33 0.0 ± 0.00 3.7 ± 0.33 3.7 ± 0.67 3.7 ± 1.33 4.7 ± 1.67IL151.7 ± 0.64 53.3 ± 0.0799.1 ±0.9998.1 ±1.46 0.0 ± 0.00 0.0 ± 0.00 1.0 ± 0.58 3.3 ± 1.86 23.7* ± 5.21 2.7 ± 1.45IL252.3 ± 2.60 49.6 ± 2.75100.8 ±1.29100.5 ±2.95 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.02± 0.000.0 ± 0.00IN149.9 ± 2.23 49.9 ± 2.4994.9 ±6.06102.5 ±2.31 1.0 ± 1.00 0.7 ± 0.33 2.0 ± 1.00 2.0 ± 0.58 0.02± 0.000.0 ± 0.00IN246.8 ± 2.71 46.3 ± 1.2295.8 ±2.2597.7 ±2.79 0.7 ± 0.67 0.3 ± 0.33 5.0 ± 0.58 6.7 ± 0.88 2.3 ± 1.20 3.7 ± 0.67KS37.8 ± 0.83 36.4 ± 2.2178.7 ±0.9782.5 ±3.16 1.0 ± 0.58 1.0 ± 0.58 46.7 ± 8.82 36.7 ±3.18 0.02± 0.000.0 ± 0.00NE55.1 ± 0.82 54.4 ± 0.92104.7 ±1.64104.7 ±1.57 0.0 ± 0.00 0.3 ± 0.33 9.0* ± 1.15 5.0 ± 1.53 15.0 ± 8.39 1.3 ± 0.88S.E. = standard error*Indicates significant differences detected between MON 87460 and the control (p<0.05).2Not statistically analyzed due to lack of variation.Monsanto Company 07-CR-191U Page 407 of 544Table F-14 (continued). Phenotypic Comparison of MON 87460 to the Control at EachSite in the 2006 U.S. Field Study underWell-Watered ConditionsPhenotypic Characteristic, (units), Mean ± S.E.Final stand count (#/plot)Grain moisture (%)Test weight (lbs/bu)Yield (bu/a)SiteMON 87460ControlMON87460ControlMON 87460ControlMON 87460ControlIA171.3 ± 1.76 67.0 ± 5.00 14.0 ± 0.21 14.5 ± 0.50 60.6 ± 3.27 62.3 ± 3.35 172.7 ± 8.13 182.9 ± 7.02IA262.0 ± 0.58 62.3 ± 0.33 13.5 ± 0.03 13.5 ± 0.21 57.7 ± 0.88 57.7 ± 0.67 173.9 ± 10.39 174.9 ± 6.43IL194.0* ± 1.00 77.3 ± 0.67 18.9 ± 0.23 18.9 ± 0.18 52.7 ± 1.20 53.3 ± 0.33 19.04± 5.4922.34± 4.50IL262.0 ± 1.53 62.0 ± 1.15 17.9 ± 0.21 17.6 ± 0.15 57.5 ± 0.17 57.7 ± 0.12 184.0 ± 9.98 181.0 ± 15.59IN152.3 ± 3.28 55.3 ± 3.18 19.3 ± 0.23 20.3 ± 0.52 58.9 ± 0.67 59.3 ± 0.25 127.1 ± 3.31 113.6 ± 6.71IN253.0 ± 2.65 51.3 ± 2.91 20.4 ± 0.45 20.7 ± 0.61 56.03± 0.00 56.03± 0.00 161.2 ± 17.33 144.5 ± 6.96KS87.3 ± 2.91 86.0 ± 2.00 13.1 ± 0.59 13.0 ± 0.62 56.4 ± 0.26 56.2 ± 0.37 159.4* ± 6.78 182.3 ± 3.98NE92.0 ± 1.73 94.3 ± 0.67 17.8 ± 0.38 17.4 ± 0.31 59.0 ± 0.06 59.1 ± 0.20 168.8 ± 2.33 169.5 ± 4.47S.E. = standard error.*Indicates significant differences detected between MON 87460 and the control (p<0.05).3Values are used for IN2 due to equipment malfunction.4Yield response at IL1 negatively impacted by heat stress during pollination.Monsanto Company 07-CR-191U Page 408 of 544Table F-15. Phenotypic Comparison ofMON 87460 to the Control at Each Site inthe 2007 U.S. Field Study under WellWatered ConditionsPhenotypic Characteristic, (units), Mean ± S.E.StudySeedling vigor(1-9 scale)Early stand count(#/plot)Days to 50%pollen shedDays to 50%silkingStay green(1-9 scale)Site1MON87460Control MON87460Control MON87460Control MON87460Control MON87460ControlIA11.0 ± 0.00 1.0 ± 0.00118.0 ±1.15117.0 ±1.5361.3 ±0.3361.0 ±0.5859.7 ± 0.6760.0 ±1.005.7 ± 0.33 5.0 ± 0.00IA21.02±0.001.0 ± 0.0078.3 ±1.4579.7 ± 0.3359.7 ±0.3359.3 ±0.3357.0 ± 0.0057.0 ±0.005.7* ± 0.33 5.0 ± 0.00IL12.02±0.002.0 ± 0.00113.3 ±1.45112.3 ±1.3364.7 ±0.3365.0 ±0.0063.02±0.0063.0 ±0.006.7* ± 0.33 5.3 ± 0.33IL24.3 ± 0.674.0 ± 1.0077.7 ±1.7678.0 ± 2.3161.0 ±0.5860.7 ±0.3360.3 ± 0.3360.3 ±0.33- -IL33.7 ± 0.334.0 ± 0.0078.3 ±1.2078.3 ± 1.2058.0 ±0.5858.0 ±0.0056.7 ± 0.3357.0 ±0.004.0* ± 0.00 3.0 ± 0.58IN5.3 ± 0.334.7 ± 0.33111.0 ±3.51118.7 ±4.0677.02±0.0077.0 ±0.0075.02±0.0075.0 ±0.009.0 ± 0.00 9.0 ± 0.00NE4.3 ± 0.335.3 ± 0.3368.0 ±2.5272.0 ± 1.0061.7 ±0.3361.3 ±0.3361.7 ± 0.3361.3 ±0.336.7 ± 0.33 6.3 ± 0.33OH1.0 ± 0.001.3 ± 0.3370.0 ±1.1577.0 ± 0.5863.3 ±0.8863.3 ±1.2063.0 ± 1.1563.0 ±1.155.0 ± 1.15 5.7 ± 1.67PA4.7 ± 0.334.0 ± 0.0069.7 ±3.1872.0 ± 3.0665.3 ±0.6764.7 ±0.3365.3 ± 0.6764.0 ±0.005.0 ± 0.58 3.7 ± 0.33S.E. = standard error.*Indicates significant differences detected between MON 87460 and the control (p<0.05).1Dashes (-)indicate that data are missing or excluded from statistical analysis.2Not statistically analyzed due tolack of variation.Monsanto Company 07-CR-191U Page 409 of 544Table F-15 (continued). Phenotypic Comparison of MON 87460 to the Control at EachSite in the 2007 U.S. Field Study underWell-Watered ConditionsPhenotypic Characteristic, (units), Mean ± S.E.StudyEar height(in)Plant height(in) Dropped ears (#/plot)Stalk lodged plants(#/plot)Root lodged plants(#/plot)Site1MON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlIA142.1 ±0.5342.5 ±0.6493.9 ±1.1493.5 ± 2.21 0.0 ± 0.00 0.0 ± 0.00 1.0 ± 0.58 0.7 ± 0.33 0.7 ± 0.67 0.0 ± 0.00IA245.0 ±0.5344.1 ±0.5593.5 ±0.3594.7 ± 0.87 0.3 ± 0.33 0.0 ± 0.00 0.3 ± 0.33 1.0 ± 1.00 0.7 ± 0.67 0.3 ± 0.33IL160.1 ±1.3959.0 ±0.62121.8 ±1.65124.3 ±1.970.02± 0.00 0.0 ± 0.00 6.3 ± 1.76 6.0 ± 2.52 0.0 ± 0.00 0.7 ± 0.33IL2--- - - - - - - -IL350.3 ±1.2347.1 ±0.59100.9 ±0.55100.1 ±0.551.7 ± 0.33 2.7 ± 0.33 2.3 ± 0.67 3.0 ± 0.00 3.0 ± 1.00 3.3 ± 0.33IN41.3 ±1.1837.4 ±1.3094.2 ±1.3385.9 ± 0.75 0.3 ± 0.33 0.7 ± 0.33 10.7 ± 1.20 7.3 ± 1.45 0.02± 0.00 0.0 ± 0.00NE54.2 ±1.4554.0 ±0.31108.2 ±2.47106.6 ±0.760.3 ± 0.33 0.0 ± 0.00 1.3 ± 0.67 1.0 ± 0.58 0.02± 0.00 0.0 ± 0.00OH39.7 ±0.6435.8 ±0.8781.1 ±2.6878.7 ± 2.66 0.7 ± 0.67 0.3 ± 0.33 3.7* ± 0.88 1.0 ± 0.58 0.02± 0.00 0.0 ± 0.00PA41.3 ±0.5741.5 ±0.7186.4 ±2.4090.9 ± 1.57 1.3* ± 0.33 0.0 ± 0.00 2.3* ± 0.33 0.3 ± 0.33 0.02± 0.00 0.0 ± 0.00S.E. = standard error.*Indicates significant differences detected between MON 87460 and the control (p<0.05).1Dashes (-)indicate that data are missing or excluded from statistical analysis.2Not statistically analyzed due to lack of variation.Monsanto Company 07-CR-191U Page 410 of 544Table F-15 (continued). Phenotypic Comparison of MON 87460 tothe Control at each Site in the 2007 U.S. Field Study underWell-Watered ConditionsPhenotypic Characteristic, (units), Mean ± S.E.StudyFinal stand count(#/plot)Grain moisture(%)Test weight(lbs/bu)Yield(bu/a)Site1MON 87460 Control MON 87460 Control MON 87460 Control MON 87460 ControlIA181.7 ± 4.10 83.3 ± 2.7311.8 ± 0.23 12.4 ± 0.1053.9 ± 0.35 54.5 ± 0.48200.2 ± 3.67 202.4 ± 6.33IA264.7 ± 0.88 66.0 ± 1.1518.0* ± 0.23 17.1 ± 0.2557.3 ± 0.44 57.0 ± 0.58206.1 ± 6.91 209.6 ± 4.04IL196.0 ± 0.00 95.7 ± 0.3321.1 ± 1.12 22.4 ± 1.3257.3 ± 0.48 56.1 ± 0.98155.4 ± 15.93 136.5 ± 8.01IL2- -- -- -- -IL361.7 ± 1.86 64.3 ± 2.0313.0* ± 0.50 14.7 ± 0.9556.1 ± 0.24 56.4 ± 0.42144.7 ± 6.75 148.1 ± 9.40IN85.3 ± 1.86 89.7 ± 3.3318.5 ± 0.98 18.6 ± 0.3848.0 ± 2.11 53.3 ± 0.57136.9 ± 5.24 120.9 ± 17.65NE61.7 ± 0.88 63.0 ± 0.5818.5 ± 0.40 19.4 ± 0.9558.2 ± 0.20 58.0 ± 0.35156.4 ± 4.00 156.5 ± 5.77OH62.0 ± 1.00 62.0 ± 1.5315.1 ± 0.63 15.3 ± 0.6451.5 ± 0.59 51.9 ± 0.05137.0 ± 11.72 131.9 ± 7.02PA60.7 ± 3.53 61.0 ± 1.0022.6 ± 0.29 23.1 ± 0.2954.5 ± 0.29 54.0 ± 0.00162.2 ± 7.96 179.2 ± 9.21S.E. = standard error.*Indicates significant differences detected between MON 87460 and the control (p<0.05).1Dashes (-)indicate that data are missing or excluded from statistical analysis.2Not statistically analyzed due to lack of variation.Monsanto Company 07-CR-191U Page 411 of 544Table F-16. Phenotypic Comparison of MON 87460 to theControl at Each Site ina 2006/2007 Chilean Field StudyEstablished with Well-Watered and Water-Limited Treatments in 2006/2007Well-Watered TreatmentCLMean ± S.E.CTMean ± S.E.LUMMean ± S.E.Phenotypic characteristicUnitsMON 87460ControlMON 87460ControlMON 87460ControlSeedling vigor0-9 scale35.3 ± 0.334.3 ± 0.335.0 ± 0.005.0 ± 0.004.3 ± 0.334.7 ± 0.33Early stand count#/plot74.0 ± 3.5166.0 ± 3.0675.7 ± 2.6077.7 ± 2.3378.7 ± 1.2075.3 ± 4.91Days to 50% pollen shedDays65.0 ± 2.3164.0 ± 1.1564.3 ± 0.8865.0 ± 0.5871.0 ± 1.5371.0 ± 0.58Days to 50% silkingDays64.0 ± 1.5364.0 ± 1.0063.0 ± 1.0063.3 ± 0.3368.7 ± 0.6768.7 ± 0.67Stay green0-9 scale40.7 ± 0.331.0 ± 0.003.7 ± 0.334.3 ± 1.333.0 ± 0.583.3 ± 0.67Ear heightin64.7 ± 3.5259.1 ± 1.6753.9 ± 2.0053.5 ± 0.5749.2 ± 3.9545.9 ± 1.57Plant heightin110.3 ± 4.47104.3 ± 2.6199.5 ± 2.38100.8 ± 1.8193.4 ± 4.7692.0 ± 2.04Dropped ears2#/plot0.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.00Stalk lodged plants2#/plot0.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.00Root lodged plants2#/plot0.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.00Final stand count#/plot74.0 ± 3.4668.7 ± 2.3374.7 ± 2.9677.3 ± 2.0377.0 ± 0.5876.0 ± 3.79Grain moisture% moisture12.1 ± 0.3311.9 ± 0.4714.0 ± 1.2113.9 ± 1.0718.3 ± 0.6419.8 ± 0.87Test weightlbs/bu56.2 ± 0.1754.8 ± 0.4458.7 ± 0.4458.7 ± 0.8354.5 ± 0.5854.0 ± 1.15Yield bu/a 213.0 ± 16.59 195.6 ± 4.80 240.7 ± 8.68 258.8 ± 7.85 208.5 ± 9.39 205.7 ± 2.82S.E. = standard errorNote: No statistically significant differences between MON 87460 and the control (α=0.05).1Study sites: CL = Colina; CT = Calera de Tango; LUM = Lumbreras.2No statistical comparisons were made for this rating due to lack of variability in the data. The test was considered effectively not different from the controlbecause the test and controlmean values were identical.3Seedling vigor rating scale: 0 = dead and 9 = above average vigor.4Stay green rating scale: 0 = entire plant is dried and 9 = entire plant is green.Monsanto Company 07-CR-191U Page 412 of 544Table F-17 Phenotypic Comparison of MON 87460 to the Control at Each Site in a 2006/2007 Chilean Field StudyEstablished with Well-Watered and Water-Limited TreatmentsWater-Limited TreatmentCLMean ± S.E.CTMean ± S.E.LUMMean ± S.E.PhenotypiccharacteristicUnitsMON 87460ControlMON 87460ControlMON 87460ControlSeedling vigor0-9 scale35.0 ± 0.585.0 ± 0.005.3 ± 0.334.7 ± 0.334.7 ± 0.674.7 ± 0.67Early stand count#/plot75.7 ± 1.3373.0 ± 3.7977.7 ± 0.8876.0 ± 1.0077.0 ± 2.0878.0 ± 0.58Days to 50% pollen shedDays65.0 ± 1.5365.3 ± 0.6765.3 ± 0.3365.3 ± 0.3372.0 ± 1.5373.7 ± 1.45Days to 50% silkingDays64.3 ± 0.8862.7 ± 0.3363.7 ± 0.3364.0 ± 0.5874.0 ± 0.5873.7 ± 0.33Stay green0-9 scale41.3 ± 0.331.7 ± 0.675.7 ± 0.335.7 ± 0.336.0 ± 0.586.7 ± 0.33Ear heightin61.0* ± 3.5651.4 ± 4.5544.9 ± 11.4143.2 ± 10.7638.1 ± 1.3940.9 ± 3.41Plant heightin97.2* ± 3.1979.3 ± 10.7387.5 ± 13.2184.5 ± 12.9467.0 ± 0.5670.4 ± 4.64Dropped ears2#/plot0.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.00Stalk lodged plants2#/plot0.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.00Root lodged plants2#/plot0.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.000.0 ± 0.00Final stand count#/plot74.3 ± 1.2071.3 ± 1.2078.0 ± 0.5876.3 ± 2.0377.7 ± 2.0377.7 ± 1.20Grain moisture% moisture14.6 ± 0.7516.3 ± 0.6815.5 ± 0.3015.6 ± 1.4028.5 ± 3.9331.9 ± 6.51Test weightlbs/bu57.0 ± 0.0055.7 ± 0.6760.5 ± 0.5060.5 ± 1.2652.5 ± 1.0451.8 ± 0.44Yield bu/a 96.9 ± 32.09 65.6 ± 30.99 160.9* ± 18.60 123.7 ± 18.05 85.8 ± 8.36 70.8 ± 9.55S.E. = standard error.*Indicates significant differences detected between MON 87460 and the control (p<0.05).1Study sites: CL = Colina; CT = Calera de Tango; LUM = Lumbreras.2No statistical comparisons were made for this rating due to lack of variability in the data. The test was considered effectively not different from the controlbecause the test and controlmean values were identical.3Seedling vigor rating scale: 0 = dead and 9 = above average vigor.4Stay green rating scale: 0 = entire plant is dried and 9 = entire plant is green.Monsanto Company 07-CR-191U Page 413 of 544Table F-18. Study-1, Phenotypic Comparison of MON 87460 to the Control at Each Site in 2007 Field Studies Establishedwith Well-Watered and Water-Limited TreatmentsWell-Watered TreatmentPhenotypic Characteristic, (units), Means ± S.E.Seedling vigor(1-9 scale)Early stand count(#/plot)§Days to 50%pollen shedDays to 50%silkingStay green(1-9 scale)SiteMON87460 ControlMON 87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlCA 5.01± 0.00 5.0 ±0.00 75.8* ± 0.25 70.0 ± 2.48 58.0 ± 0.41 57.8 ± 0.25 60.0 ± 0.00 59.8± 0.63 2.5 ± 0.65 2.8± 0.48TX1.0 ± 0.001.0 ± 0.00105.0 ± 2.12 106.5± 4.35 63.5 ± 0.50 64.0 ±0.58 57.0 ± 0.00 57.0± 0.001.8* ±0.253.5± 0.29Phenotypic Characteristic, (units), Means ± S.E.Ear height(in)Plant height(in)Dropped ears (#/plot)Stalk lodged plants(#/plot)Root lodged plants(#/plot)SiteMON87460 ControlMON 87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlCA51.4 ±1.0148.1± 0.89 100.5 ± 2.50 96.8 ±1.340.01±0.000.0 ± 0.000.5* ±0.290.0 ± 0.000.5 ±0.291.3 ± 0.63TX33.3 ±0.6334.6 ± 1.39 70.4* ± 1.51 75.1± 1.77 0.0 ± 0.00 0.3 ± 0.25 0.0 ± 0.00 0.3 ± 0.250.01±0.000.0 ± 0.00Phenotypic Characteristic, (units), Means ± S.E.Final stand count (#/plot)§Grain moisture(%)Test weight(lbs/bu)Yield(bu/a)Site MON 87460 Control MON 87460 Control MON 87460 Control MON 87460 ControlCA 63.5 ± 0.50 63.0 ± 0.41 14.6 ± 0.28 14.2 ± 0.31- -239.7* ± 7.34 181.4 ± 19.21TX 96.0 ± 0.00 94.8 ± 1.25 14.8 ± 0.07 15.0 ± 0.2058.4 ± 0.96 57.8 ± 0.63 215.4 ± 6.24 219.1 ± 5.22S.E. = standard error. *Indicates significant differences detected between MON87460 and the control (p<0.05).§Note: For CA, the row length was 20 feet; forTX, rows were 30feet in length.1Not statistically analyzed due to lack of variation. Test weight data not provided from CA site.Monsanto Company 07-CR-191U Page 414 of 544Table F-19. Study-1, Phenotypic Comparison of MON 87460 to the Control at Each Site in 2007 Field Studies Establishedwith Well-Watered and Water-Limited TreatmentsWater-Limited TreatmentPhenotypic Characteristic, (units), Mean ± S.E.Seedling vigor(1-9 scale)Early stand count(#/plot)§Days to 50% pollenshedDays to 50%silkingStay green(1-9 scale)SiteMON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlCA5.01±0.005.0 ± 0.00 73.5 ± 2.53 74.0 ± 2.65 58.3 ± 0.25 58.8 ± 0.4860.8 ±0.2560.0 ± 1.00 4.0 ± 0.41 4.0 ± 0.00TX1.0 ±0.001.0 ± 0.00101.0 ±2.08107.5 ±2.1864.3 ±0.67 64.0 ± 0.5857.0 ±0.0057.0 ± 0.001.7* ±0.334.3 ± 0.75Phenotypic Characteristic, (units), Mean ± S.E.Ear height(in)Plant height(in)Dropped ears(#/plot)Stalk lodgedplants (#/plot)Root lodged plants(#/plot)SiteMON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlCA52.0 ±0.5750.7 ±1.9197.5 ± 0.87 95.3 ± 1.34 0.01± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 4.8 ± 3.47 1.5 ± 0.87TX27.9 ±0.6929.7 ±0.2662.6 ± 0.55 62.2 ± 0.73 1.0 ± 0.58 1.5 ± 0.5 1.3 ± 0.88 1.5 ± 0.65 0.01± 0.00 0.0 ± 0.00Phenotypic Characteristic, (units), Mean ± S.E.Final stand count (#/plot)§Grain moisture(%)Test weight(lbs/bu)Yield(bu/a)Site MON 87460 ControlMON87460 ControlMON87460 Control MON 87460 ControlCA 62.8 ± 0.75 63.3 ± 0.63 15.2 ± 0.49 14.7 ± 0.40--188.3* ± 31.49 148.1 ± 28.69TX 96.0 ± 0.00 96.0 ± 0.00 14.0 ± 0.10 14.4 ± 0.0757.6 ± 0.74 56.7 ± 0.72 186.0 ± 2.98 173.1 ± 0.70S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05).§Note: For CA, the row length was 20 feet; forTX, rows were 30feet in length.1Not statistically analyzed due to lack of variation.-Test weight data not provided from CA site.Monsanto Company 07-CR-191U Page 415 of 544Table F-20. Study-2, Phenotypic Comparison of MON 87460 tothe Control at Each Site in 2007 U.S. Field StudiesEstablished with Well-Watered and Water-Limited TreatmentsWell-Watered Treatment1Phenotypic Characteristic, (units), Mean ± S.E.Seedling vigor(0-9 scale)Early stand count(#/plot)Days to 50%pollen shedDays to 50%silkingStay green(0-9 scale)Site Test Control Test Control TestControl Test Control Test ControlKS2.7 ± 0.33 3.0 ± 0.58 68.7 ± 1.86 65.3 ± 1.33 63.3 ± 0.88 62.3 ± 0.33 62.0 ± 1.15 62.0 ± 0.00 6.3 ± 0.33 8.0 ± 0.00NE2.7 ± 0.33 2.3 ± 0.33 46.3 ± 0.88 52.3 ± 3.18 66.3 ± 0.33 66.7 ± 0.33 65.7 ± 0.33 66.0 ± 0.58 4.7* ± 0.33 4.0 ± 0.00TX1.7 ± 0.67 1.3 ± 0.33 52.3 ± 9.28 60.0 ± 5.29 61.0 ± 0.00 61.0 ± 0.00 59.0 ± 0.00 59.0 ± 0.00 6.3* ± 0.33 8.0 ± 0.00Phenotypic Characteristic, (units), Mean ± S.E.Ear height(in)Plant height(in)Dropped ears(#/plot)Stalk lodged plants(#/plot)Root lodged plants(#/plot)Site Test Control Test Control TestControl Test Control Test ControlKS45.7 ± 1.83 44.2 ± 1.2190.6 ± 1.94 88.8 ± 2.431.0 ± 0.58 0.7 ± 0.673.0 ± 0.00 4.7 ± 0.882.7 ± 1.6715.0 ±15.00NE42.9 ± 0.13 40.7 ± 0.66 87.8 ± 1.33 88.6 ± 1.06 0.3 ± 0.33 0.0 ± 0.00 0.7 ± 0.33 1.0 ± 0.58 0.0 ± 0.00 0.0 ± 0.00TX25.1 ± 2.45 27.8 ± 1.51 74.1 ± 2.53 74.2 ± 1.92 0.0 ± 0.00 0.3 ± 0.33 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.3 ± 0.33Phenotypic Characteristic, (units), Mean ± S.E.Final stand count(#/plot)Grain moisture(%)Test weight(lbs/bu)Yield(bu/a)Site Test Control Test Control Test Control Test ControlKS59.3 ± 2.73 61.3 ± 0.33 13.7* ± 0.03 12.4 ± 0.09 61.4 ± 0.04 61.7 ± 0.04 137.2 ± 2.17 110.8 ± 7.20NE47.7 ± 2.03 50.3 ± 1.76 15.4 ± 0.25 15.7 ± 0.1960.5 ± 0.15 60.3 ± 0.12 189.4 ± 6.89 202.8 ± 7.54TX51.0 ± 8.5 56.7 ± 3.33 12.7 ± 0.34 12.9 ± 0.12 59.6 ± 0.25 60.0 ± 0.19 250.3 ± 15.58 255.1 ± 35.19S.E. = standard error.*Indicates significant differences detected between MON 87460 and the control (p<0.05).Note: No comparisons were made for Root lodged plants at the NEsite, and Days to 50% pollen shed, Days to 50% silking, and Stalk lodged plants at the TX site due to a lack ofvariability.1Water-limited data for the TX site that met the inclusion criteria are presented in SectionVIII.C.2, Table VIII-13. Data for KS and NE are reported in Appendix G,Table G-3.Monsanto Company 07-CR-191U Page 416 of 544Table F-21. Phenotypic Comparison ofMON 87460 to the Control at Each Site ina 2006 U.S. Field Study Established withWater Managed According to Local Agronomic PracticesPhenotypic Characteristic, (units), Mean ± S.E.Seedling vigor2Early stand count(#/plot) Days to 50% pollen shed Days to 50% silking3Stay greenSite1MON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlIAE 8.0 ± 0.00 8.0 ± 0.00 62.3 ± 0.33 63.0 ± 1.15 64.3 ± 0.33 63.7 ± 0.33 63.0 ± 0.00 61.0 ± 0.00 4.3 ± 0.33 5.0 ± 0.58IAW 7.0 ± 0.00 7.0 ± 0.58 63.0 ± 1.00 59.0 ± 5.51 64.0 ± 1.00 64.3 ± 0.67 63.7 ± 0.67 64.3 ± 0.67 0.7 ± 0.33 0.3 ± 0.33IL 8.0 ± 0.00 8.0 ± 0.00 65.0 ± 2.00 62.0 ± 1.15 63.3 ± 0.33 64.0 ± 0.58 62.0 ± 0.00 62.0 ± 0.00 5.0 ± 0.00 5.0 ± 0.00IN 8.0 ± 0.00 8.0 ± 0.00 64.7 ± 1.45 60.7 ± 0.67 59.3 ± 0.33 59.3 ± 0.33 57.3 ± 0.33 57.3 ± 0.33 2.7 ± 0.33 2.7 ± 0.33NE 7.0* ± 0.00 8.0 ± 0.00 67.3 ± 0.88 66.7 ± 0.88 64.3 ± 0.33 64.0 ± 0.00 65.0* ± 0.00 64.0 ± 0.00 6.0 ± 0.58 5.7 ± 0.33Phenotypic Characteristic, (units), Mean ± S.E.Ear height (in) Plant height(in) Dropped ears (#/plot)4Stalk lodged plants(#/plot)Root lodged plants(#/plot)5Site1MON87460 Control MON 87460 ControlMON87460 ControlMON87460 ControlMON87460 ControlIAE 54.1 ± 1.41 52.7 ± 1.46 108.3 ± 0.44 108.1 ± 0.18 0.0 ± 0.00 0.0 ± 0.00 2.3 ± 0.67 2.3 ± 0.67 5.7 ± 1.33 4.7 ± 0.67IAW 38.6 ± 0.81 37.7 ± 1.87 97.5 ± 0.74 97.9 ± 1.05 0.0 ± 0.00 0.0 ± 0.00 11.3 ± 1.20 13.7 ± 5.24 0.7 ± 0.33 0.7 ± 0.67IL 40.5 ± 1.05 40.1 ± 0.70 88.9 ± 1.43 89.2 ± 0.52 0.0 ± 0.00 0.0 ± 0.00 2.7 ± 0.88 2.0 ± 1.53 0.0 ± 0.00 0.0 ± 0.00IN 46.1 ± 0.47 46.4 ± 1.29 94.7 ± 0.35 94.4 ± 0.76 0.0 ± 0.00 0.3 ± 0.33 8.3* ± 0.67 4.7 ± 1.86 3.3* ± 0.88 0.3 ± 0.33NE 48.6 ± 1.03 47.4 ± 1.62 99.5 ± 1.27 96.6 ± 2.50 0.7 ± 0.67 0.3 ± 0.33 2.7 ± 0.33 3.0 ± 1.53 0.7 ± 0.33 0.0 ± 0.00S.E. = standard error. *Indicates significant differences detected between MON87460 and the control (p<0.05).1Site codes are as follows: IAE = Benton County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; NE= York County, NE.2Lack of variance prevented statistical analysisof seedling vigor at the IAE, IL, and IN sites.3Lack of variance prevented statistical analysis ofdays to 50% silking at the IAE and IN sites.4Lack of variance prevented statistical analysisof dropped ears at the IAE, IAW, and IL sites.5Lack of variance prevented statistical analysis of root lodged plants at the IL site.Monsanto Company 07-CR-191U Page 417 of 544Table F-21 (continued).Phenotypic Comparison of MON 87460 to theControl at Each Site in a 2006 U.S.Field Study Established with Water ManagedAccording to Local Agronomic PracticesPhenotypic Characteristic, (units), Mean±S.E.Final stand count (#/plot) Grain moisture(%) Test weight (lbs/bu) Yield (bu/a)Site1MON 87460 Control MON 87460 Control MON 87460 Control MON 87460 ControlIAE 60.3 ± 0.67 58.7 ± 0.88 16.1 ± 0.50 15.6 ± 0.19 56.7 ± 0.33 57.0 ± 0.58 187.8 ± 5.34 180.9 ± 5.72IAW 53.3 ± 2.33 52.7 ± 3.53 15.9 ± 0.64 16.4 ± 0.2350.7 ± 0.00 48.8 ± 0.94 154.9 ± 5.32 138.5 ± 15.26IL 58.7 ± 1.45 59.3 ± 0.33 20.6 ± 0.91 20.0 ± 2.4056.2 ± 0.45 56.1 ± 0.99 52.0 ± 3.69 35.8 ± 18.00IN 59.0 ± 0.58 57.0 ± 1.73 18.3 ± 0.31 19.4 ± 0.3654.2 ± 0.32 53.6 ± 0.12 146.8 ± 5.20 161.9 ± 10.18NE 58.0 ± 1.00 59.3 ± 0.33 16.8 ± 0.45 17.1 ± 0.6158.6 ± 0.36 58.7 ± 0.09 191.3 ± 3.27 179.8 ± 15.64S.E. = standard error.Note: No statistically significant differences between MON 87460 and the control (α=0.05).1Site codes are as follows: IAE = Benton County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; NE= York County, NE.Monsanto Company 07-CR-191U Page 418 of 544 Appendix G. Phenotypic Data from U.S. and Chile Field Sites Excluded from Combined-Site Analyses

This section contains data th at were excluded from the comb ined-site analyses discussed in Section VIII.C. All data reported here ar e for sites that did not meet the well-watered

and water-limited inclusion criteria desc ribed in Section VIII.C (Table VIII-3).

Site Code, Test Location, and Year Site Code QUI, Chile, 2006/2007

Table G-1. Individual Site Analysis of Phenotypic Characteristics of MON 87460 to the Control for the QUI Site in Chile during 2006/2007 under Well-Watered Conditions

Mean ± S.E.

Phenotypic Characteristic MON 87460 Control

Seedling vigor 5.7 ± 0.33 5.0 ± 0.00
Early stand count (#/plot) 73.0 ± 4.04 73.7 ± 4.06
Days to 50% pollen shed 69.3 ± 0.33 70.0 ± 1.00
Days to 50% silking 69.0 ± 0.00 68.3 ± 1.20
Stay green 3.0* ± 0.00 4.7 ± 0.88
Ear height (in) 68.3 ± 1.98 66.6 ± 3.99
Plant height (in) 110.8 ± 1.35 109.8 ± 4.03
Dropped ears (#/plot) 1 0.0 ± 0.00 0.0 ± 0.00
Stalk lodged plants (#/plot) 1 0.0 ± 0.00 0.0 ± 0.00
Root lodged plants (#/plot) 1 0.0 ± 0.00 0.0 ± 0.00
Final stand count (#/plot) 76.7 ± 2.03 75.0 ± 2.65
Grain moisture (%) 13.6 ± 0.84 14.1 ± 0.50
Test weight (lbs/bu) 57.3 ± 0.33 58.2 ± 1.01

Yield (bu/ac) 200.4 ± 5.75 217.8 ± 5.10

S.E. = standard error. *Indicates significant differe nces detected between MON 87460 and the control (p< 0.05).

1 No statistical comparisons were made for this rating due to lack of variability in

the data. The test was considered effectiv ely not different from the control because

the test and control mean values were identical.Monsanto Company 07-CR-191U Page 419 of 544 Table G-2. Individual Site Analysis of Phenotypic Characteristics of

MON 87460 to the Control for th e QUI site in Chile During

2006/2007 under Water-Limited Conditions

Mean ± S.E.

Phenotypic characteristic MON 87460 Control

Seedling vigor 6.0* ± 0.00 4.7 ± 0.33

Early stand count (#/plot) 81.0 ± 1.53 78.7 ± 1.67

Days to 50% pollen shed 66.0* ± 0.58 70.7 ± 0.67

Days to 50% silking 64.7* ± 0.33 68.3 ± 1.20

Stay green 2.3 ± 0.33 3.7 ± 0.33

Ear height (in) 72.1 ± 2.46 67.1 ± 0.39

Plant height (in) 116.9 ± 4.97 112.4 ± 2.79

Dropped ears (#/plot) 1 0.0 ± 0.00 0.0 ± 0.00

Stalk lodged plants (#/plot) 1 0.0 ± 0.00 0.0 ± 0.00

Root lodged plants (#/plot) 1 0.0 ± 0.00 0.0 ± 0.00

Final stand count (#/plot) 81.0 ± 1.53 78.7 ± 1.76

Grain moisture (%) 14.5* ± 0.82 12.5 ± 0.90

Test weight (lbs/bu) 57.2 ± 0.83 57.5 ± 0.29

Yield (bu/ac) 199.0 ± 14.60 202.0 ± 3.06

S.E. = standard error. *Indicates significant differe nces detected between MON 87460 and the control (p< 0.05).

1 No statistical comparisons were made for this rating due to lack of variability in the data. The test was considered effectively no t different from the control because the test

and control mean valu es were identical.Monsanto Company 07-CR-191U Page 420 of 544Site Codes, Test Location, and YearKS (Pawnee Co.) and NE (York Co.), 2007Table G-3. U.S. 2007 Study-2: Water-Limited Treatment – Phenotypic Comparisonof MON 87460 to theControl at EachSitePhenotypic Characteristic, (units), Mean±S.E.Seedling vigor(0-9 scale)Early stand count(#/20 ft plot)Days to 50%pollen shedDays to 50%silkingStay green(0-9 scale)SiteTest Control Test Control Test Control Test Control Test ControlKS 2.3 ± 0.33 2.7 ± 0.33 68.3 ± 1.33 66.3 ± 2.73 62.3 ± 0.33 62.0 ± 0.00 62.0 ± 0.00 62.0 ± 0.00 6.0 ± 1.00 6.7 ± 0.67NE 2.0 ± 0.00 2.3 ± 0.33 50.3 ± 3.18 47.7 ± 4.26 66.3 ± 0.33 66.7 ± 0.67 65.3 ± 0.33 66.3 ± 0.88 5.0* ± 0.00 4.0 ± 0.00Phenotypic Characteristic, (units), Mean±S.E.Ear height(in)Plant height(in)Dropped ears(#/plot)Stalk lodged plants(#/plot)Root lodged plants(#/plot)SiteTest Control Test Control Test Control Test Control Test ControlKS 43.3 ± 0.93 44.3 ± 1.39 88.7 ± 1.73 89.7 ± 1.51 1.3 ± 0.88 0.3 ± 0.33 2.3 ± 0.88 6.0 ± 2.00 0.7 ± 0.67 2.7 ± 1.76NE 41.6 ± 1.10 41.1 ± 0.79 87.4 ± 1.25 86.5 ± 1.16 0.0 ± 0.00 0.0 ± 0.00 0.3 ± 0.33 0.3 ± 0.33 0.0 ± 0.00 0.0 ± 0.00Phenotypic Characteristic, (units), Mean±S.E.Final stand count(#/plot)Grain moisture(%)Test weight(lbs/bu)Yield(bu/a)SiteTest Control Test ControlTest Control Test ControlKS 61.0 ± 2.52 60.0 ± 4.00 13.7 ± 0.06 12.6 ± 0.09 61.4 ± 0.04 61.5 ± 0.13 111.9 ± 6.25 121.1 ± 1.99NE 45.0 ± 1.15 45.3 ± 3.53 15.4 ± 0.12 15.9 ± 0.35 60.6 ± 0.06 60.3 ± 0.23 183.3 ± 5.48 183.0 ± 8.44S.E. = standard error.*Indicates significant differences detected between MON 87460 and the control (p<0.05).Note: No comparisons were made for root lodged plants at the NEsite, and days to 50% pollen shed, days to 50% silking, and stalk lodged plants at the TX sitedue to a lack of variability.Monsanto Company 07-CR-191U Page 421 of 544 Appendix H. Environmental Inte ractions Data for Individual Field Sites in U.S. and Chile Field Studies during 2006 and 2007

Environmental interaction evaluations were conducted as part of the plant characterization studies for MON 87460, and will be used as part of the plant pest risk

assessment. The environmental interactions evaluation included the collection and comparison of abiotic stressor data, disease damage data, arthropod damage data, and arthropod pest and be neficial data.

H.1. Materials and Methods for Disease and Insect Assessments

The plots at all sites were qualitatively evaluated at least four times for differential response to naturally occurr ing environmental stressors during the growing season. During each observation, each plot was evaluate d for the severity of symptoms caused by three arthropod, three disease, and three ab iotic stressors that commonly occur at the study sites. With a few exceptions, these st ressors were predetermined by the individual site Principal Investigators (P Is) based on their experience. The environmental stressors evaluated were not artificially induced and coul d vary between sites. Plots were rated on the 0 – 9 rating scale of incr easing symptomology described below but the results were reported as categorical (none, s light, moderate, or severe).

0 = none (no symptoms observed) 1 – 3 = slight (symptoms observed, not detrimental to plant growth and development) 4 – 6 = moderate (intermediate between slight and severe) 7 – 9 = severe (symptoms observed, detrimental to plant growth and development)

H.1.1. Stalk and Ear/Kernel Rot Assessment

Stalk Rot Disease

At harvest, incidence of stalk rot disease from 5 representative plants per plot was assessed. The stalk of each plant was cut longitudinally and examined for shredded and discolored pith tissue. The followi ng 0 – 9 rating scale was be used:

0 = none (no symptoms observed) 1 – 3 = slight (symptoms observed not detrimental to stalk quality) 4 – 6 = moderate (intermediat e between slight and severe) 7 – 9 = severe (symptoms observed, detrimental to stalk quality)Monsanto Company 07-CR-191U Page 422 of 544 Ear and Kernel Rot Disease

At harvest, incidence of ear and kernel rot disease from 5 represen tative ears (one per plant) per plot was assessed. The husks were pulled back so the ear and kernels could be examined for infection. The following 0 – 9 rating scale was used: 0 = none (no symptoms observed) 1 – 3 = slight (symptoms observed, not detrimental to grain quality) 4 – 6 = moderate (intermediat e between slight and severe) 7 – 9 = severe (symptoms observed, detrimental to grain quality)

H.1.2. Quantitative Assessment: European Corn Borer and Corn Earworm Damage

European corn borer, and corn earworm dama ge ratings were performed at three wellwatered sites in 2006, at three well-watered si tes in 2007, and a singl e site in TX with well-watered and water-limited treatments (Study-2) in 2007.

European corn borer damage was evalua ted at harvest by examining five nonsystematically selected plants. If damage was present, the number of live larvae, number of galleries, number of entry/ exit holes were counted, and th e length of galleries in each

stalk was measured.

Corn earworm damage was evaluated at harvest by examining ears from ten nonsystematically selected plants. For each ear, the following damage rating scale was used:

0 = No visible corn earworm damage 1 = Silk shows evidence of feeding, feeding on the ear is < 0.5 inches 2 = Corn earworm feeding to 0.5 in. beyond the ear tip 3 = Corn earworm feeding to 1.0 in. beyond the ear tip 4 = Corn earworm feeding to 1.5 in. beyond the ear tip 5 = Corn earworm feeding to 2.0 in. beyond the ear tip 6 = Corn earworm feeding to 2.5 in. beyond the ear tip 7 = Corn earworm feeding to 3.0 in. beyond the ear tip 8 = Corn earworm feeding to 3.5 in. beyond the ear tip 9 = Corn earworm feeding to 4.0 in. or greater beyond the ear tip

H.1.3. Quantitative Assessment: Arthropod Collection

Arthropod collections were performed at three well-watered sites in 2006, at three wellwatered sites in 2007, and a single site in TX with well-watered and water-limited treatments (Study-2) in 2007. Arthropods were collected four times dur ing the growing season at the following

intervals:

Collection 1: V2 – V4

Collection 2: V10 – V15

Collection 3: VT – R3

Collection 4: R6Monsanto Company 07-CR-191U Page 423 of 544 Arthropods were collected using yellow sticky traps. The sticky traps were placed at the approximate midpoint between the ground level and the top of the plant canopy. Once the main ear was visible, the sticky traps were de ployed at the approximate corn ear level for the remainder of the arthropod collectio ns. The sticky traps were deployed for approximately seven days. The sticky traps we re sent to the Department of Entomology at the University of Arkans as in Fayetteville, AR fo r arthropod identification and enumeration. Up to six of the most abunda nt pest and up to six of the most abundant beneficial arthropods were determined for each collection at each site. These arthropods were then enumerated across all plots (i.e ., one sticky trap pe r plot) from a given collection at each site. The arthropods assess ed often varied between collections due to differences in seasonal activity.

H.1.4. Abiotic, Disease, and Arthropod Assessments

In the two years of field studies fo r evaluation of phenotypic and agronomic characteristics of MON 87460, observational da ta on the presence of and differential response to biotic (insects, di seases) and abiotic (drought, wi nd, nutrient deficiency etc.)

stressors were also collected to examine th e environmental interactions of MON 87460 compared with those of the conventional control corn. The observed stressors were “natural” (i.e., no artificial infestation or in terference was used). Therefore, the same stressors were not necessarily observed at e ach field site. A summ ary of environmental interactions data are presented in Section VIII. Individual site data are reported in this appendix.

Environmental interactions were assessed qualitativ ely, and for selected sites, insect interactions data were collected quantitativel y. Observation of plant interactions with insect pests and diseases, and plant responses to abiotic stressors were collected from

each of the 33 field site loca tions in 2006 and 2007. The purpose of these evaluations was to assess whether plant-in sect or plant-disease intera ctions, or plant response to

abiotic stressors of MON 87460 were altered co mpared to the conventional control corn. For the plant-insect interactions, plant-diseas e interactions, and plant responses to abiotic stressors, the reported values represent the range of ratings observe d across the three or four replications at each site. MON 87460 a nd the control were considered qualitatively different in response to a stressor if the ratings between MON 87460 and the control corn did not overlap across all three or four replications for that particular stressor (e.g. “none” rating vs. “slight-moderate” rating). Th e ratings observed among the commercial reference hybrids provide qua litative assessment data common to the crop for each

stressor assessed.

H.2. Statistical Analysis

2006 and 2007 U.S. Field Studies under Well-Watered Conditions An analysis of variance was conducted accord ing to a randomized complete block design with three replications using SAS ® (SAS Version 9.2, SAS Institute, Inc. 2008) for the corn earworm damage, European corn borer damage, and the arthropod abundance. The level of statistical significance was predetermined to be 5% ( α = 0.05). The test substance was compared to the control substance at each site (individual-site analysis)Monsanto Company 07-CR-191U Page 424 of 544 for the corn earworm damage, European co rn borer damage, a nd arthropod abundance. Additionally, the corn earwor m damage and European corn borer damage data were pooled across sites (combined-si te analysis) for a statistica l comparison of the test and

control substances. The test substance was not statistically compared to the reference substances, and the reference range was ca lculated from the minimum and maximum

mean values observed in the references.

U.S. 2007 Field Study (Study- 1) Established with Well-Watered and Water-Limited

Treatments – TX Site An analysis of variance was conducted ac cording to a split-plo t design with four replications using SAS ® . The whole plot factor was th e irrigation treatment and the subplot factor was substance with the experime ntal unit consisting of one plot. The level of statistical significance wa s predetermined to be 5% ( α = 0.05). The test substance was compared to the control substance within irrigation treatments for the corn earworm damage, European corn borer damage, and arthropod abundance. The test substance was not statistically compared to the referen ce substances, and the reference range was

calculated from the minimum and maximum mean values observed in the references.

H.3. Results

2006 U.S. Field Study under Well-Watered Conditions

Environmental interaction data were collected from eight field trials in 2006 and nine

in 2007 that were established as well- watered locations. Six sites provided quantitative insect assessment s across both years. For quant itative insect sites, sticky traps were deployed for insect identification and enumeration. Quantitative insect damage assessments were made at all locations.

Plant Response to Abiotic Stressors No differences in plant response to ab iotic stressors were detected between MON 87460 and the control for cold, compact ion, drought, flood, frost, green snap, hail, heat, mineral toxicity, nutrient de ficiency, or wind evaluated using the observational severity scale at the IA1, IA2, IL1, IL2, IN1, IN2, KS, or NE sites (Table H-1).

Disease Damage No differences in disease damage were detected between MON 87460 and the control for anthracnose, an external assessment for ear rot, ear and kernel rot assessment after husk removal, eyespot, Fusarium , gray leaf spot, leaf blight, northern corn leaf blight, northern leaf spot, Pythium , rust, seedling blight, smut, sout hern leaf blig ht, stalk rot, Stewarts wilt, or yellow leaf blight evalua ted using the observational severity scale at the IA1, IA2, IL1, IL2, IN1, IN2, KS, or NE sites (Table H-2).

Arthropod Damage No differences in damage were detect ed between MON 87460 and the control for aphids, armyworms, corn earworm, corn rootworms, cutworms, European cornMonsanto Company 07-CR-191U Page 425 of 544 borers, flea beetles, grape colaspis, gr asshoppers, Japanese beetles, leafhoppers, seedcorn maggots, southwestern corn borers, spider mites, white grubs, or wireworms evaluated using the observational severity scal e at the IA1, IA2, IL1, IL2, IN1, IN2, KS, or NE sites (Table H-3).

Corn Earworm and European Corn Borer Damage

No statistical differences were detected between MON 87460 and the control for corn earworm damage using the adapted Widstrom (1967) scale in the combined-site or individual-site analyses at the IL1, KS, and NE sites (Tables H-4 and H-5). No

statistical differences were detected between MON 87460 and the control for European corn borer damage including: number of entry/ ex it holes; number of galleries; and length of galleries (in.) in the ear shank and the stalk, in the combinedsite or individual-site analyses at the IL 1, KS, and NE sites (Tables H-4 and H-5).

Arthropod Abundance In the individual site analysis, no statis tical differences were detected between MON 87460 and the control for the following pest and beneficial arthropods at the IL1, KS, and NE sites: delphacid plan thoppers, grasshoppers, leafhoppers, northern corn rootworm beetles, sap beetles, southe rn corn rootworm beetles, western corn rootworm beetles, brown lacewings, gr een lacewings, lady bird beetles, macroparasitic hymenopterans (parasitoids), nabids , minute pirate bugs, and spiders (Tables H-6 and H-7). Aphid abundance was statistica lly lower at the NE site at collection 1 (0.0 vs. 3.3 per trap) and sta tistically higher at the IL1 si te at collection 3 (70.7 vs. 63.3 per trap) for MON 87460 compared to the control out of 12 co llections at three sites (Table H-6). Corn flea beetle abundance was statistically lower at the NE site at collection 2 (0.0 vs. 1.3 corn flea beetle s per trap) for MON 87460 compared to the

control out of nine collecti ons (Table H-6). Micro-pa rasitic hymenopteran abundance was statistically lower at the KS site for co llections 2 and 3 (30.0 vs. 42.3 per trap and 13.0 vs. 23.3 per trap, respectively) for M ON 87460 compared to the control out of 12 collections (Table H-7). However, no cons istent statistical differences for aphids, corn flea beetles, or micro-parasitic hy menopterans were detected across sites or arthropod collections.Monsanto Company 07-CR-191U Page 426 of 544 2007 U.S. Field Study under Well-Watered Conditions

Plant Response to Abiotic Stressors

In an assessment of abiotic stress response, no differences in plant response to abiotic

stressors were observed between MON 87460 and the control trea tments for cold, compaction, drought, flood, frost, hail, heat, mineral toxicity, nutri ent deficiency, or wind evaluated using the observa tional severity scale at the IA1, IA2, IL1, IL2, IL3, IN, NE, OH or PA sites (Table H-8).

Disease Damage In an assessment of disease damage, no differences in disease damage were observed

between MON 87460 and the control for anthrac nose, an external assessment for ear rot, ear rot assessment after husk removal, an external assessment for stalk rot, stalk rot assessment by splitting the stalks, eyespot, Fusarium , gray leaf spot, leaf blight, northern corn leaf blight, Pythium , seedling blight, root rot, rust, smut, or Stewart’s wilt evaluated using the observational severity scale at the at the IA1, IA2, IL1, IL2, IL3, IN, NE, OH, or PA sites (Table H-9).

Arthropod Damage In an assessment of arthropod damage, no differences in damage were observed

between MON 87460 and the control for a phids, leafhoppers, thrips, grasshoppers, armyworms, corn earworm larvae, cutworms , billbugs, flea beetles, grape colaspis adults, Japanese beetles, northern corn r ootworm, western corn rootworm beetles, white grubs, wireworms, or seedcorn maggots evaluated using the observational severity scale at the IA1, IA2, IL1, IL2, IL3, IN, NE, OH, or PA sites (Table H-10).

One difference was observed between MON 874 60 and the control for European corn borer at the IA1 site during the fourth observation (moderate vs. slight). However, the observed incidence of European corn borer damage was not detected at other sites

or observations.

Additionally, no statistical differences we re detected between MON 87460 and the

control for corn earworm da mage using the adapted Wids trom (1967) scale in the combined-site analysis (Table H-11). No statistical differences were detected

between MON 87460 and the control for Eur opean corn borer damage including: number of live larvae; number of entry/ exit holes; numbe r of galleries; and length of galleries (in.) in the stalk in the combined-site analysis (Table H-11). However,

number of live larvae and th e length of galleries (in) in the stalk were significantly

different between MON 87460 and the control at the IL1 site in the individual site analysis (Table H-12). The differences dete cted in the individual site analysis were not significant in the combined-site analys is and; therefore, not indicative of a

consistent trend in the data.

Arthropod Abundance

No statistical differences were detected between MON 87460 and the control for the following pest and beneficial arthropods at the IA1, IL1 and IN sites: aphids,Monsanto Company 07-CR-191U Page 427 of 544 delphacid planthoppers, grass hoppers, leafhoppers, corn flea beetles, Northern corn rootworms, sap beetles, southern corn r ootworm beetles, western corn rootworm beetles, brown lacewings, green lacewings , lady bird beetles, macro-parasitic hymenopterans (parasitoids), Orius , and spiders (Tables H-13 and H-14). Microparasitic hymenopterans (parasitoids) abundance was statistically lower at the IN site for collection 3 (48.7 vs. 118.3 per trap) for MON 87460 compared to the control and was outside of the reference range (Table H-14). However, no consistent statistical

differences between MON 87460 and the cont rol for micro-parasitic hymenopterans (parasitoids) abundance were detected across sites or arthropod collections.

2006 U.S. Field Study Established with Wate r Managed According to Local Agronomic

Practices

Abiotic Stressors No differences in plant response to ab iotic stressors were detected between MON 87460 and the control for cold, compac tion, flood, frost, hail, heat, nutrient deficiency, or wind using the observational se verity scale at the IAE, IAW, IL, IN, and NE sites (Table H-15).

Disease Damage No differences in disease damage were detected between MON 87460 and the control for anthracnose, eyespot, ear rot, Fusarium , gray leaf spot, leaf blight, northern corn leaf blight, northern leaf spot, Pythium , root rot, rust, seedling blight, southern leaf blight, smut, stalk rot, or Stewarts wilt evaluated using the observational severity scale at the IAE, IAW, IL, IN , or NE sites (Table H-16).

Arthropod Damage No differences in arthropod damage we re detected between MON 87460 and the control for aphids, armyworms, billbugs, corn earworms, corn rootworm beetles, cutworms, European corn borers, flea beetle s, seedcorn maggots, southwestern corn borers, western bean cutworms, white grubs , and wireworms using the observational severity scale at the IAW, IL, IN, and NE sites. Grasshopper damage was lower for MON 87460 compared to the control (none vs. slight) at the IAE si te at Observation 3. However, the detected difference wa s within the range of the references. Additionally, no differences were detect ed between MON 87460 and the control for grasshopper damage using the observational seve rity scale for Observations 2 and 4 at the IAE site or at any observation time at the other sites (Table H-17). These results support the conclusion that the introduction of the drough t tolerance trait did not unexpectedly alter MON 87460 compared to c onventional corn based on the assessed

environmental interactions.Monsanto Company 07-CR-191U Page 428 of 544 2006/2007 Chilean Field Study Es tablished with Well-Wat ered and Wa ter-Limited

Treatments

Three locations were included in the combined-site based on site inclusion criteria (Section VIII.C, Figure VIII-3).

Abiotic Stressors No differences in plant response to ab iotic stressors were detected between MON 87460 and the control for cold, frost, hail, heat, nitrogen deficiency, or wind damage in the well-watered or water-limited treatments using the observational severity scale at the CL, CT, and LUM sites (Table H-18).

Disease Damage No differences in disease damage were detected between MON 87460 and the control for ear rot, Fusarium , gray leaf spot, leaf blight, north ern corn leaf blight, root rot, rust, seedling blight, smut, or stalk rot in the well-watered or water-limited treatments using the observational severity scale at the CL, CT, and LUM sites (Table H-19).

Arthropod Damage No differences in arthropod damage we re detected between MON 87460 and the control for aphids, seed corn maggots, thri ps, and wireworms in the well-watered or water-limited treatments using the observational severity scale at the CL, CT, and LUM sites (Table H-20).

2007 U.S. Field Studies Established with We ll-Watered and Water-Limited Treatments

In 2007, two separate field studies were conducted in the U.S. where sites were

established with well-watered and water- limited treatments. In Study-1, two sites were established (CA, TX) and both met the inclusion criteria (Section VIII.C, Figure VIII-3). In Study-2, three sites we re established (KS, NE, TX), but only one site (TX) met the inclusion criteria. The tw o trials had different experimental designs which precluded a combined-study analysis.

Study-1 – Abiotic Stress Response In assessment of abiotic stress respons e in the well-watered and water-limited treatments, no differences in plant res ponse to abiotic stressors were observed

between MON 87460 and the co ntrol treatments for dr ought, hail, heat, heavy thunderstorm, nutrient deficien cy, or wind evaluated using the observational severity scale at the CA and TX sites (Table H-21).

Study-1 – Disease Damage In assessment of disease damage in the well-watered and water-limited treatments, no differences in disease damage were observed between MON 87460 and the control for ear rot assessment after husk removal, Fusarium , gray mold, maize dwarf mosaicMonsanto Company 07-CR-191U Page 429 of 544 virus, rust, seedling blight, sm ut, stalk rot, or southern l eaf blight evaluated using the observational severity scale at th e CA and TX sites (Table H-22).

Study-1 – Arthropod Damage In assessment of arthropod damage in the well-watered and water-limited treatments, no differences in damage were observe d between MON 87460 a nd the control for aphids, armyworms, western corn rootwo rm beetles, cutworms, European corn borers, grasshoppers, leafhoppers, southwestern corn borers, or spider mites evaluated using the observational severity scale at the CA and TX sites (Table H-23).

Additionally, no statistical differences we re detected between MON 87460 and the

control for corn earworm dama ge using the adapted Widstr om (1967) scale at the TX

site under well-watered and water-limited tr eatments (Table H-24). No statistical

differences were detected between MON 87460 and the control for European corn borer damage including: number of live larv ae, number of entry/ exit holes; number of galleries; and le ngth of galleries (in.) in the stalk at the TX site under well-watered and water-limited treatments (Table H-24).

Study-1 – Arthropod Abundance In an assessment of arthropod damage unde r the well-watered treatment, no statistical

differences were detected between MON 87460 and the control for the following pest and beneficial arthropods at the TX site: aphids , delphacid planthoppers, grasshoppers, leafhoppers, corn flea beetles, sap beetles, southern corn rootwormbeetles, western corn rootworm beetles, green lacewings, lady bird beetles, macroparasitic hymenopterans (parasitoids), mi cro-parasitic hymenopt erans (parasitoids),

Orius , big-eyed bugs, and spiders (Table s H-25 and H-26). Brown lacewing abundance was statistically higher at co llection 3 (0.5 vs. 0.0 per trap) for MON 87460 compared to the control, but wa s within the range of the references (Table H-26).

In an assessment of arthropod damage under the water-limited treatment, no statistical

differences were detected between MON 87460 and the control for the following pest and beneficial arthropods at the TX site: aphids , delphacid planthoppers, grasshoppers, leafhoppers, corn flea beetles, sap beetles, western corn rootworm beetles, green lacewings, lady bird b eetles, macro-parasitic hymenopterans (parasitoids), micro- parasitic hymenopterans (parasitoids), Orius , big-eyed bugs, and spiders (Tables H-25 and H- 26). Southern corn root worm beetle abundance was statistically higher at collec tion 4 (2.5 vs. 0.8 per trap) for MON 87460 compared to

the control and was outside of the refe rence range (Table H-25). However,

differences between MON 87460 and the co ntrol for southern corn rootworm

abundance were not detected fo r other arthropod collections.Monsanto Company 07-CR-191U Page 430 of 544 Study-2 – Abiotic Stress Response In assessment of abiotic stress response in the well-watered treatment, no differences in plant response to abiotic stressors were observed between MON 87460 and the control treatments for drought, hail, heat, water logging, or wind evaluated using the observational severity scale at the NE, KS and TX sites (Table H-27).

In assessment of abiotic stress response under water-limited treatment, no differences in plant response to abiotic stressors were observed between MON 87460 and the control treatments for drought, hail, heat, or wind evaluated using the observational severity scale at the TX site (Table H-27).

Study-2 – Disease Damage In assessment of disease damage in the we ll-watered treatment, no differences in disease damage were observed between MON 87460 and the control for crazy top, ear rot assessment after husk removal, Goss’s wilt, grey leaf spot, grey mold, leaf blight, maize dwarf mosaic viru s, northern corn leaf blight , rust, seedling blight, smut, stalk rot assessment by splitting th e stalk, or southern corn leaf blight evaluated using the observational severity scale at NE, KS and TX sites (Table H-28).

In assessment of disease damage in the water-limited treatment, no differences in disease damage were observed between MON 87460 and the control for ear rot assessment after husk removal, grey mold, maize dwarf mosaic virus, northern corn leaf blight, rust, seedling bli ght, stalk rot assessment by spli tting the stalk, or southern corn leaf blight evaluated using the observational severi ty scale at the TX site (Table H-28)

Study-2 – Arthropod Damage In an assessment of arthropod damage in the well-watered treatment, no differences in arthropod damage were observed betw een MON 87460 and the control for aphids, leafhoppers, grasshoppers, armyworms, corn earworms, cutworms, European corn borers, southwestern corn borers, corn r ootworms, wireworms, leafrollers or mites using the observational severity scale at the NE, KS, and TX sites (Table H-29)

In an assessment of arthropod damage in the water-limited treatment, no differences in arthropod damage were observed betw een MON 87460 and the control for aphids, grasshoppers, armyworms, corn earworms, cutworms, European corn borers, southwestern corn borers, co rn rootworms using the obser vational severity scale at the TX site (Table H-29)

H.4. Summary

The results of the environm ental interactions evaluation for MON 87460 supports the conclusion that the introduction of the drought tolerance trait did not unexpectedly alter MON 87460 compared to conventional corn. The lack of differences in plant response to abiotic stressors, disease damage, arthropod damage, and arthropod pest and beneficialMonsanto Company 07-CR-191U Page 431 of 544 abundance indicate that the in troduction of the drought tolera nce trait is unlikely to be biologically meaningful in term s of increased pest potential.

H.5. References

Widstrom, N.W. 1967. An evaluation for meas uring corn earworm injury. Journal of Economic Entomology. 60:791-794.

Table H-1. Abiotic Stressor Evaluations us ing an Observational Severity Scale for MON 87460 Compared to the Control and References in a 2006 U.S. Field Study

under Well-Watered Conditions

Abiotic stressor Number of observations

across all sites ∞ (IA1, IA2, IL1, IL2, IN1, IN2, KS, NE) Number of observations

where no differences were

detected across all sites

Total 70 70

Cold 3 3 Compaction (includes soil compaction) 4 4
Drought 3 3
Flood (includes excessive water) 6 6

Frost 2 2 Green Snap 1 1 1

Hail 13 13

Heat 6 6 Mineral toxicity 1 1 Nutrient deficiency 6 6

Wind 25 25

Note: No differences were detected between MON 87460 and the control. The experimental design was a randomized complete block with three replications; Data were collected at four crop developmental st ages, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site codes are as follows: IA1 = Jeffe rson County, IA; IA2 = Benton County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IN1 = Boone County, IN; NE = York County, NE; KS = Pawnee County, KS

1 Observations occurred between the V15 – VT growth stages across all plotsMonsanto Company 07-CR-191U Page 432 of 544 Table H-2. Disease Damage Evaluations us ing an Observational Severity Scale for MON 87460 Compared to the Control and References in a 2006 U.S. Field Study

under Well-Watered Conditions

Disease Number of observations

across all sites ∞ (IA1, IA2, IL1, IL2, IN1, IN2, KS, NE) Number of observations

where no differences were

detected across all sites

Total 112 112

Anthracnose 3 3

Ear rot 1 1 1

Ear and kernel rot 2 8 8
Eyespot 4 4

Fusarium 3 3 3 Gray leaf spot 22 22 Leaf blight 1 1 Northern corn leaf blight 4 12 12 Northern leaf spot 2 2 Pythium 4 4

Rust 5 18 18 Seedling blight 3 3

Smut 4 4 Southern leaf blight 5 5

Stalk rot 6,7 8 8

Stalk rot 8 8 8

Stewarts wilt 4 4 Yellow leaf blight 2 2

Note: No differences were detected between MON 87460 and the control. The experimental design was a randomized complete block with three replications; Data were collected at four crop developmental st ages, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site codes are as follows: IA1 = Jeffe rson County, IA; IA2 = Benton County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IN1 = Boone County, IN; NE = York County, NE; KS = Pawnee County, KS

1 Ear rot assessed externally by observing the outside of the ear only; 2 Ear and kernel rot assessed by peeling back the husk and evaluating the ear and kernels; 3 Fusarium was rated as Gibberella / Fusarium at the IN1 site; 4 Includes northern leaf blight; 5 Includes leaf rust, common ru st, and common/southern rust; 6 Includes charcoal stalk rot ; 7 Stalk rot assessed externally by observi ng the outside of the stalk only ; 8 Stalk rot assessed by splitting the stalks of 5 nonsystematically selected plants and evaluating the diseaseMonsanto Company 07-CR-191U Page 433 of 544 Table H-3. Arthropod Damage Evaluations using an Observational Severity Scale for MON 87460 Compared to the Control an d References in a 2006 U.S. Field Study

under Well-Watered Conditions

Arthropod Number of observations

across all sites ∞ (IA1, IA2, IL1, IL2, IN1, IN2, KS, NE) Number of observations

where no differences were

detected across all sites

Total 98 98

Aphids (includes corn leaf aphids) 12 12 Leafhoppers 2 2 Armyworms (includes fall armyworms) 4 4

Corn earworms 1 6 6
Cutworms (includes black cutworms) 5 5
European corn borers 2 25 25

Southwestern corn borers 1 1 Grasshoppers 6 6

Corn rootworms 3 18 18
Flea beetles (includes corn flea beetles) 3 3
Japanese beetles 4 4 Grape colaspis 4 4

Wireworms 3 3 White grubs 1 1 Spider mites (include mites) 2 2 Seedcorn maggots 2 2

Note: No differences were detected between MON 87460 and the control. The experimental design was a randomized complete block with three replications; Data were collected at four crop developmental st ages, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site codes are as follows: IA1 = Jeffe rson County, IA; IA2 = Benton County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IN1 = Boone County, IN; NE = York County, NE; KS = Pawnee County, KS

1 External observation, ear husks were not pulled back for evaluation ; 2 External observation; 3 Includes western corn rootworms, northern corn rootworm s, and southern corn rootwormsMonsanto Company 07-CR-191U Page 434 of 544 Table H-4. Quantitative A ssessment for Corn Earworm and European Corn Borer Damage to MON 87460 Compared to the Control and References in a 2006 U.S. Field Study under Well-Watered Conditions

Pest Damage Assessment MON

87460 Control Reference Range 1

Corn earworm Mean ± SE of 5 ears (0 – 9 rating scale) 1.7 ±

0.84 1.8 ±

0.93 0.0 – 5.20

European corn

borer Mean ± SE # of ear shank entry/exit holes of 5 plants 0.3 ±

0.18 0.3 ±

0.16 0.0 – 2.27

European corn

borer Mean ± SE # of ear shank galleries per plant of 5 plants 0.2 ±

0.09 0.2 ±

0.10 0.0 – 0.93

European corn

borer Mean ± SE ear shank gallery length per plant of 5 plants (in.) 0.2 ±

0.11 0.2 ±

0.11 0.0 – 0.93

European corn

borer Mean ± SE # of stalk entry/exit holes of 5 plants 1.9 ±

0.91 1.7 ±

0.65 0.07 – 6.53

European corn

borer Mean ± SE # of stalk galleries per plant of 5 plants 1.1 ±

0.40 1.0 ±

0.28 0.07 – 2.53

European corn

borer Mean ± SE stalk gallery length per plant of 5 plants (in.) 4.9 ±

2.37 3.3 ±

1.67 0.03 – 15.33

S.E. = standard error; the experimental design was a randomized complete block with three replicates. Note: No statistically significant differenc es between MON 87460 and the control ( α =0.05).

1 Reference range (the minimum and maximum values of the reference means).Monsanto Company 07-CR-191U Page 435 of 544Table H-5. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87460 Compared to theControl and References at the IL1, KS, and NE Sitesin a 2006 U.S. Field Study under Well-Watered ConditionsPest Arthropod Damage Assessment Site MON 87460 ControlCorn earworm Mean ± SE of 5 ears (0 – 9 rating scale) IL1 0.0 ± 0.00 0.0 ± 0.00Mean ± SE of 5 ears (0 – 9 rating scale) KS 5.0 ± 0.31 5.3 ± 0.96Mean ± SE of 5 ears (0 – 9 rating scale) NE 0.0 ± 0.00 0.0 ± 0.00European corn borer Mean ± SE # of ear shank entry/exit holes of 5 plants IL1 0.0 ± 0.00 0.0 ± 0.00Mean ± SE # of ear shank entry/exit holes of 5 plants KS 1.0 ± 0.20 0.8 ± 0.31Mean ± SE # of ear shank entry/exit holes of 5 plants NE 0.0 ± 0.00 0.0 ± 0.00European corn borer Mean ± SE # of ear shank galleries per plant of 5 plants IL1 0.0 ± 0.00 0.0 ± 0.00Mean ± SE # of ear shank galleries per plant of 5 plants KS 0.5 ± 0.07 0.5 ± 0.13Mean ± SE # of ear shank galleries per plant of 5 plants NE 0.0 ± 0.00 0.0 ± 0.00European corn borer Mean ± SE ear shank gallery length per plant of 5 plants (in.) IL1 0.0 ± 0.00 0.0 ± 0.00Mean ± SE ear shank gallery length per plant of 5 plants (in.) KS 0.6 ± 0.12 0.7 ± 0.10Mean ± SE ear shank gallery length per plant of 5 plants (in.) NE 0.0 ± 0.00 0.0 ± 0.00European corn borer Mean ± SE # of stalk entry/exit holes of 5 plants IL1 0.5 ± 0.18 1.0 ± 0.64Mean ± SE # of stalk entry/exit holes of 5 plants KS 4.9 ± 1.75 3.8 ± 1.15Mean ± SE # of stalk entry/exit holes of 5 plants NE 0.3 ± 0.13 0.3 ± 0.13European corn borer Mean ± SE # of stalk galleries per plant of 5 plants IL1 0.5 ± 0.18 0.7 ± 0.37Mean ± SE # of stalk galleries per plant of 5 plants KS 2.4 ± 0.70 1.9 ± 0.37Mean ± SE # of stalk galleries per plant of 5 plants NE 0.3 ± 0.13 0.3 ± 0.07European corn borer Mean ± SE stalk gallery length per plant from 5 plants (in.) IL1 0.6 ± 0.31 0.8 ± 0.45Mean ± SE stalk gallery length per plant from 5 plants (in.) KS 14.0 ± 2.45 9.0 ± 3.00Mean ± SE stalk gallery length per plant from 5 plants (in.) NE 0.2 ± 0.08 0.2 ± 0.05S.E. = standard error; the experimental design was a randomized complete block with three replicates.Note: No statistically significant differences between MON 87460 and the control (α=0.05).1Quantitative insect locations.Monsanto Company 07-CR-191U Page 436 of 544Table H-6. Arthropod Pest Abundance on MON 87460 Compared tothe Control and References at the IL1, KS, and NE Sitesin a 2006 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeAphids IL17.3 ±1.679.3 ±0.677.7 –10.325.7 ±1.7623.0 ±4.5118.0 –27.370.7 ±8.3563.3 ±13.8442.7 –69.79.3 ±0.333.0 ±0.585.0 –23.7KS9.0 ±1.5312.3 ±2.3313.7–19.34.0 ±2.085.0 ±1.534.0 –7.35.3* ±2.1913.0 ±1.001.7 –5.73.7 ±1.673.0 ±0.581.7 –2.0NE0.0* ±0.003.3 ±0.330.7 –2.73.0 ±1.003.7 ±0.882.7 –6.376.3 ±36.6840.0 ±14.2233.0 –62.71.0 ±0.001.3 ±0.881.0 –2.7Corn flea beetles IL1 – – –23.3 ±5.8116.7 ±3.1815.0 –25.06.0 ±2.004.3 ±1.334.7 –7.0– – –KS5.3 ±2.337.3 ±0.882.0 –6.710.3 ±3.2815.0 ±2.0812.3 –17.71.7 ±0.331.3 ±0.330.7 –1.70.3 ±0.330.0 ±0.000.0 –1.0NE – – –0.0 ±0.00*1.3 ±0.330.0 –1.00.7 ±0.671.3 ±0.880.0 –1.00.0 ±0.000.0 ±0.000.0 –1.0DelphacidplanthoppersIL10.0†±0.000.0 ±0.000.0 –0.01.0 ±0.581.0 ±0.580.3 –2.0– – – – – –Grasshoppers KS0.3 ±0.330.0 ±0.000.0 –0.3– – – – – – – – –NE0.0 ±0.000.0 ±0.000.0 –0.3– – – – – – – – –S.E. = standard error; the experimental design was a randomized complete block with three replicates.Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not enumeratedat this observation and site.†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 437 of 544Table H-6 (Continued). Arthropod PestAbundance on MON 87460 Compared to the Control and References at the IL1, KS,and NE Sites in a 2006 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeLeafhoppers KS0.0 ±0.000.3 ±0.330.0 –1.00.3 ±0.331.0 ±0.000.3 –1.70.3 ±0.331.0 ±0.580.0 –1.01.0 ±1.000.7 ±0.330.3 –1.7NE – – –0.0 ±0.00†0.0 ±0.000.0 –0.0– – –3.7 ±0.333.0 ±1.533.0 –5.3Northern cornrootwormsIL1 – – –2.0 ±1.152.3 ±0.332.3 –3.72.7 ±0.672.3 ±2.331.3 –2.3– – –Sap beetles IL10.7 ±0.670.7 ±0.330.3 –1.7– – –0.0 ±0.000.3 ±0.330.3 –1.0– – –KS1.0 ±1.000.3 ±0.330.0 –0.7– – –0.0 ±0.000.3 ±0.330.0 –0.3– – –NE – – –0.0 ±0.000.0 ±0.000.0 –0.30.0 ±0.000.0 ±0.000.0 –0.30.3 ±0.330.7 ±0.670.3 –1.0Southern cornrootwormsIL10.0†±0.000.0 ±0.000.0 –0.00– – – – – – – – –KS – – – – – –0.0 ±0.000.3 ±0.330.0 –0.3– – –NE – – – – – –0.3 ±0.330.0 ±0.000.0 –0.3– – –S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not enumeratedat this observation and site.†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 438 of 544Table H-6 (Continued). Arthropod PestAbundance on MON 87460 Compared to the Control and References at the IL1, KS,and NE Sites in a 2006 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeWestern cornrootwormsIL1 – – –14.0 ±4.0416.3 ±3.8410.0 –16.321.7 ±5.2419.7 ±2.9118.7 –28.0– – –KS – – –0.7 ±0.330.7 ±0.330.3 –1.0– – – – – –NE – – –0.3 ±0.330.7 ±0.330.0 –1.09.0 ±1.00*3.0 ±2.085.0 –9.00.0 ±0.000.0 ±0.000.0 –0.3S.E. = standard error; the experimental design was a randomized complete block with three replicates.*Indicates significant differences detected between MON 87460 and the control (p<0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not enumeratedat this observation and site.Monsanto Company 07-CR-191U Page 439 of 544Table H-7. Beneficial Arthropod Abundance on MON 87460 Compared to the Control and References at the IL1, KS, and NESites in a 2006 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeBrown lacewings IL1 – – – – – – – – –0.0 ±0.000.0 ±0.000.0 –1.0KS – – – – – – – – –0.0 ±0.000.0 ±0.000.0 –0.3Green lacewings IL1 – – –0.7 ±0.330.0 ±0.000.0 –0.70.0 ±0.001.0 ±1.000.3 –1.3– – –KS0.3 ±0.330.0 ±0.000.0 –0.7– – –0.7 ±0.332.0 ±0.580.0 –1.7– – –NE0.0 ±0.000.0 ±0.000.0 –0.30.3 ±0.330.0 ±0.000.0 –2.04.7 ±0.882.3 ±1.200.7 –4.30.0 ±0.000.3 ±0.330.0 –1.3Ladybird beetles IL12.3 ±0.332.7 ±0.673.7 –5.74.7 ±0.884.0 ±1.732.3 –3.73.0 ±1.002.3 ±1.202.3 –3.0– – –KS4.7 ±1.767.0 ±0.584.0 –7.00.7 ±0.671.7 ±0.331.7 –2.70.3 ±0.330.3 ±0.330.3 –1.00.3 ±0.330.0 ±0.000.0 –1.0NE0.7 ±0.330.0 ±0.000.7 –2.72.7 ±0.881.0 ±0.581.7 –3.34.3 ±1.763.7 ±1.861.0 –9.3– – –S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.Monsanto Company 07-CR-191U Page 440 of 544Table H-7 (Continued). Beneficial Arthropod Abundance on MON 87460 Comparedto the Control and References at the IL1,KS, and NE Sites in a 2006 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMacro-parasitichymenopterans1IL10.0 ±0.000.0 ±0.000.3 –0.7– – – – – –2.7 ±0.333.3 ±0.331.7 –4.0KS0.0 ±0.00†0.0 ±0.000.0 –0.00.0 ±0.000.0 ±0.000.0 –0.30.0 ±0.000.7 ±0.670.0 –0.7– – –NE – – – – – – – – –0.3 ±0.330.7 ±0.330.7 –2.0Micro-parasitichymenopterans2IL115.0 ±2.5212.3 ±3.6712.0 –16.353.7 ±9.9442.0 ±5.5144.0 –58.320.0 ±4.7317.3 ±4.7021.0 –38.79.3 ±2.199.0 ±2.657.3 –12.3KS11.3 ±2.9613.3 ±4.9115.3 –25.030.0* ±3.5142.3 ±1.4532.0 –54.013.0* ±0.5823.3 ±6.3310.7 –16.722.7 ±4.6322.0 ±3.069.7 –18.0NE2.0 ±0.583.0 ±1.150.0 –3.323.3 ±4.6719.7 ±6.3311.7 –36.718.3 ±5.5517.0 ±4.9316.0 –30.318.0 ±7.5121.0 ±6.5617.0 –42.0Minute pirate bugs IL10.0 ±0.000.3 ±0.330.7 –1.75.7 ±0.884.7 ±1.457.7 –10.011.7 ±3.715.3 ±0.886.0 –8.30.3 ±0.330.3 ±0.330.0 –0.3KS1.3 ±0.670.3 ±0.330.7 –1.72.3 ±1.205.0 ±1.532.7 –5.03.3 ±1.204.3 ±1.202.7 –5.31.3 ±0.671.3 ±0.330.0 –1.0NE – – –0.3 ±0.330.3 ±0.330.0 –0.77.3 ±0.888.7 ±2.607.7 –9.71.0 ±0.580.7 ±0.331.3 –3.3S.E. = standard error; the experimental design was a randomized complete block with three replicates.*Indicates significant differences detected between MON 87460 and the control (p<0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.1Greater than 5mm in length ;2Less than 5mm in length.†p-values could not be generated due tolack of variability in the data. MON 87460was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 441 of 544Table H-7 (Continued). Beneficial Arthropod Abundance on MON 87460 Comparedto the Control and References at the IL1,KS, and NE Sites in a 2006 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeNabids IL1 – – –0.0 ±0.00†0.0 ±0.000.0 –0.0– – – – – –Spiders IL11.3 ±0.882.7 ±0.670.3 –2.3– – –1.0 ±0.002.0 ±1.000.3 –2.01.3 ±0.671.0 ±0.580.7 –2.0KS – – –0.0 ±0.000.7 ±0.330.7 –1.7– – –2.7 ±1.201.0 ±0.000.3 –1.7NE1.0 ±0.581.0 ±0.000.3 –1.00.0 ±0.001.0 ±0.580.0 –0.31.7 ±0.880.7 ±0.671.0 –2.03.7 ±0.674.0 ±3.002.7 –5.0S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.†p-values could not be generated due tolack of variability in the data. MON 87460was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 442 of 544 Table H-8. Abiotic Stressor Evaluations us ing an Observational Severity Scale for MON 87460 Compared to the Control and References in a 2007 U.S. Field Study

under Well-Watered Conditions

Abiotic stressor Number of observations

across all sites ∞ (IA1, IA2, IL1, IL2, IL3, IN, NE, OH, PA) Number of observations

where no differences were

detected across all sites

Total 90 90

Cold 3 3 Compaction (includes soil compaction) 7 7
Drought 2 2
Flood (includes excessive water) 5 5

Frost 2 2

Hail 14 14

Heat 18 18 Mineral toxicity 1 1 Nutrient deficiency 14 14

Wind 24 24

Note: No statistically significant diffe rences between MON 87460 and the control ( α =0.05). The experimental design was a randomized complete block with th ree replications; Data we re collected at four crop developmental stages, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of

corn.

∞ Site codes are as follows: IA1 = Jeffe rson County, IA; IA2 = Van Horne County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IL3 = Clinton Count y, IL; IN = Boone County, IN; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PAMonsanto Company 07-CR-191U Page 443 of 544 Table H-9. Disease Stressor Evaluations us ing an Observational Severity Scale for MON 87460 Compared to the Control and References in a 2007 U.S. Field Study

under Well-Watered Conditions

Disease Number of observations

across all sites ∞ (IA1, IA2, IL1, IL2, IL3, IN, NE, OH, PA) Number of observations

where no differences were

detected across all sites

Total 120 120

Anthracnose 7 7

Ear rot 1 1 1

Ear rot 2 8 8
Eyespot 8 8

Fusarium 3 3 Gray leaf spot 21 21 Leaf blight 3 3 3 Northern corn leaf blight 14 14 Pythium 4 4 Seedling blight 6 6

Root rot 4 4

Rust 4 21 21

Smut 6 6

Stalk rot 5 1 1

Stalk rot 6 8 8

Stewarts wilt 5 5

Note: No statistically significant diffe rences between MON 87460 and the control ( α =0.05). The experimental design was a randomized complete block with th ree replications; Data we re collected at four crop developmental stages, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of

corn.

∞ Site codes are as follows: IA1 = Jeffe rson County, IA; IA2 = Van Horne County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IL3 = Clinton Count y, IL; IN = Boone County, IN; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PA

1 Ear rot assessed externally by observing the outside of the ear only; 2 Ear rot assessed by pulling husk back from ears of 5 non-systematically selected pl ants and evaluating the disease; 3 Includes southern leaf blight; 4 Includes leaf rust, common rust, and common/southern rust; 5 Stalk rot assessed externally by obs erving the outside of the stalk only;

6 Stalk rot assessed by splitting the stalks of 5 non-sys tematically selected plants and evaluating the diseaseMonsanto Company 07-CR-191U Page 444 of 544 Table H-10. Arthropod Stressor Evaluations using an Observational Severity Scale for MON 87460 Compared to the Control an d References in a 2007 U.S. Field Study

under Well-Watered Conditions

Arthropod Number of observations

across all sites ∞ (IA1, IA2, IL1, IL2, IL3, IN, NE, OH, PA) Number of observations

where no differences were

detected across all sites

Total 99 98

Aphids (includes corn leaf aphids) 10 10 Leafhoppers 2 2 Thrips 2 2 Grasshoppers 6 6 Armyworms (includes fall armyworms) 10 10

Corn earworms 1 6 6
Cutworms (includes black cutworms) 9 9

European corn borers 2, * 22 21

Corn rootworms 3 11 11
Flea beetles (includes corn flea beetles) 7 7 Grape colaspis 3 3
Japanese beetles 8 8 Billbugs 2 2 White grubs 1 1

* Differences were detected between M ON 87460 and the control for European corn borer at the IA1 site during the fourth observation (moderate vs . slight). The difference detected was out side the range of the reference (no-sl) Note: The experimental design was a randomized complete bloc k with three replications; Da ta were collected at four crop developmental stages, observation 1 = V2-V4, observati on 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn.

∞ Site codes are as follows: IA1 = Jeffe rson County, IA; IA2 = Van Horne County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IL3 = Clinton Count y, IL; IN = Boone County, IN; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PA

1 External observation, ear husks we re not pulled back for evaluation; 2 External observation; 3 Includes western corn

rootworms and northern corn rootwormsMonsanto Company 07-CR-191U Page 445 of 544Table H-11. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87460 Compared to theControl and References Combined across IA1, IL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered ConditionsPest ArthropodDamage AssessmentMON 87460ControlReference Range1Corn earwormMean ± SE of 10 ears (0 – 9 rating scale) 1.8 ± 0.95 2.6 ± 1.20 0.00 – 6.37European corn borerMean ± SE # of stalk entry/exit holes of 10 plants 1.6 ± 0.35 1.4 ± 0.43 0.50 – 2.70European corn borerMean ± SE # of stalk galleries per plant of 10 plants 1.4 ± 0.26 1.3 ± 0.35 0.48 – 2.28European corn borerMean ± SE stalk gallery length per plant of 10 plants (in.) 2.1 ± 0.49 2.0 ± 0.80 0.62 – 4.50European corn borerMean ± SE live larvae per plant from 10 plants 0.2 ± 0.09 0.2 ± 0.09 0.07 – 0.55S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant differences between MON 87460 and the control (α=0.05).1Reference range (the minimum and maximum values of the reference means).Monsanto Company 07-CR-191U Page 446 of 544Table H-12. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87460 Compared to theControl, and References at the IA1, IL1, and IN sitesin a 2007 U.S. Field Study under Well-Watered ConditionsPest Arthropod Damage Assessment Site MON 87460 ControlCorn earworm Mean ± SE of 10 ears (0 – 9 rating scale) IA1 0.2 ± 0.09 0.4 ± 0.09Mean ± SE of 10 ears (0 – 9 rating scale) IL1 0.0 ± 0.00 0.0 ± 0.00Mean ± SE of 10 ears (0 – 9 rating scale) IN 5.1 ± 1.57 7.2 ± 0.93European corn borer Mean ± SE # of stalk entry/exit holes of 10 plants IA1 0.9 ± 0.12 0.8 ± 0.19Mean ± SE # of stalk entry/exit holes of 10 plants IL1 1.2 ± 0.24 0.8 ± 0.20Mean ± SE # of stalk entry/exit holes of 10 plants IN 2.7 ± 0.70 2.8 ± 0.85European corn borer Mean ± SE # of stalk galleries per plant of 10 plants IA1 0.8 ± 0.20 0.7 ± 0.15Mean ± SE # of stalk galleries per plant of 10 plants IL1 1.2 ± 0.24 0.7 ± 0.18Mean ± SE # of stalk galleries per plant of 10 plants IN 2.2 ± 0.47 2.5 ± 0.61European corn borer Mean ± SE stalk gallery length per plant from 10 plants (in.) IA1 0.8 ± 0.25 0.8 ± 0.25Mean ± SE stalk gallery length per plant from 10 plants (in.) IL1 1.8* ± 0.39 0.7 ± 0.26Mean ± SE stalk gallery length per plant from 10 plants (in.) IN 3.7 ± 0.71 4.6 ± 1.60European corn borer Mean ± SE live larvae per plant from 10 plants IA1 0.0 ± 0.03 0.0 ± 0.00Mean ± SE live larvae per plant from 10 plants IL1 0.4* ± 0.17 0.1 ± 0.03Mean ± SE live larvae per plant from 10 plants IN 0.3 ± 0.18 0.5 ± 0.18S.E. = standard error; the experimental design was a randomized complete block with three replicates.*Indicates significant differences detected between MON 87460 and the control (p<0.05).Monsanto Company 07-CR-191U Page 447 of 544Table H-13 Arthropod Pest Abundance on MON 87460 Compared tothe Control and References at the IA1, IL1, and IN Sitesin a 2007 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeAphids IA15.7 ±1.863.0 ±1.733.7 –6.74.7 ±1.454.0 ±1.731.7 –6.332.0 ±11.0245.3 ±9.9618.0 –44.325.3 ±3.7623.7 ±14.6610.3 –51.0IL145.7 ±27.8139.0 ±23.257.0 –94.72.0 ±1.000.3 ±0.331.0 –2.749.0 ±24.5842.7 ±9.3515.7 –126.74.0 ±1.534.3 ±1.453.3 –6.3IN8.3 ±0.886.3 ±1.338.7 –18.311.3 ±3.5312.3 ±5.844.7 –27.07.7 ±1.4540.7 ±29.343.0 –118.36.7 ±1.205.7 ±0.881.0 –3.3DelphacidplanthoppersIA1 –––0.3 ±0.331.7 ±1.670.0 –0.718.3 ±4.8423.0 ±8.339.3 –21.00.0 ±0.000.3 ±0.330.0 –0.7IL167.0 ±15.5080.0 ±32.7553.0 –137.76.7 ±3.1813.7 ±2.963.0 –12.34.3 ±1.864.7 ±0.882.3 –7.31.0 ±0.581.3 ±0.330.3 –1.3IN –––0.0 ±0.000.0 ±0.000.3 –1.7100.0 ±18.6881.3 ±17.7047.7 –92.00.3 ±0.330.7 ±0.330.3 –1.7Grasshoppers IN0.0 ±0.000.0 ±0.000.0 –0.30.0 ±0.00†0.0 ±0.000.0 –0.0––––––Leafhoppers IA10.0†±0.000.0 ±0.000.0 –0.00–––––––––IL112.7 ±6.649.7 ±3.847.7 –15.7––––––0.3 ±0.330.0 ±0.000.0 –0.7IN3.3 ±1.452.7 ±1.451.3 –3.320.3 ±13.4515.0 ±6.0313.3 –66.76.3 ±4.3712.3 ±5.673.0 –17.30.3 ±0.330.7 ±0.330.0 –1.0S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant difference between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 448 of 544Table H-13 (Continued). Arthropod Pest Abundance on MON87460 Compared to the Control and References at the IA1,IL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeCorn flea beetle IA10.3 ±0.330.3 ±0.330.0 –0.70.3 ±0.330.7 ±0.330.0 –0.70.0 ±0.000.3 ±0.330.3 –1.00.0 ±0.000.7 ±0.670.0 –0.0IL123.0 ±11.1418.0 ±6.5110.3 –26.77.7 ±0.886.7 ±0.883.7 –10.01.0 ±1.000.7 ±0.330.0 –2.00.3 ±0.330.7 ±0.670.3 –0.7IN0.7 ±0.670.0 ±0.000.0 –0.715.0 ±11.539.0 ±3.068.7 –19.39.7 ±1.8623.3 ±8.824.3 –23.77.7 ±0.677.3 ±2.851.7 –5.7Northern cornrootwormsIA1 – – – – – –0.7 ±0.331.7 ±0.881.7 –2.30.3 ±0.330.7 ±0.330.0 –1.0IL1 – – –0.0 ±0.000.7 ±0.670.0 –1.03.7 ±0.333.0 ±1.534.3 –17.3– – –Sap beetles IA10.7 ±0.670.3 ±0.330.0 –0.30.3 ±0.330.7 ±0.670.3 –1.70.0 ±0.000.0 ±0.000.0 –0.30.0 ±0.000.0 ±0.000.0 –0.3IL10.3 ±0.330.0 ±0.000.0 –0.3– – –1.0 ±0.580.0 ±0.000.0 –0.70.3 ±0.330.0 ±0.000.0 –0.7IN – – – – – –0.3 ±0.330.0 ±0.000.0 –0.00.0 ±0.000.7 ±0.330.7 –1.0Southern cornrootwormsIA1 –––0.0 ±0.000.0 ±0.000.0 –0.7– – – – – –IL1 –––0.0 ±0.000.0 ±0.000.0 –0.3– – – –––S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.Monsanto Company 07-CR-191U Page 449 of 544Table H-13 (Continued). Arthropod Pest Abundance on MON87460 Compared to the Control and References at the IA1,IL1, and IN sites in a 2007 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeWestern cornrootwormsIA1 – – –0.3 ±0.330.0 ±0.000.0 –0.30.7 ±0.670.0 ±0.000.0 –0.30.0 ±0.00†0.0 ±0.000.0 –0.0IL126.0 ±4.1616.0 ±5.2010.0 –44.76.3 ±2.335.3 ±1.862.3 –6.016.7 ±3.7628.3 ±9.4024.3 –37.00.3 ±0.330.7 ±0.670.3 –1.3IN –––32.0 ±11.5331.7 ±5.7833.0 –39.325.0 ±11.7225.3 ±7.6215.3 –41.30.0 ±0.00†0.0 ±0.000.0 –0.0S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 450 of 544Table H-14. Beneficial Arthropod Abundance on MON 87460 Compared to the Control and References at the IA1, IL1, andIN Sites in a 2007 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeAraneae (spiders) IA10.7 ±0.330.7 ±0.330.3 –1.01.3 ±0.880.3 ±0.331.3 –2.00.7 ±0.672.3 ±0.881.0 –2.32.7 ±0.881.0 ±0.581.3 –4.3IL11.0 ±1.000.3 ±0.330.0 –1.00.0 ±0.000.0 ±0.000.0 –0.32.3 ±0.670.7 ±0.670.0 –2.03.3 ±0.883.0 ±1.001.3 –3.3IN4.0 ±1.733.7 ±1.203.7 –6.70.3 ±0.330.3 ±0.330.3 –1.70.0 ±0.000.0 ±0.000.0 –2.31.3 ±0.880.7 ±0.330.3 –2.0Macro-parasitichymenoptera1IA1 ––– ––– –––0.0 ±0.000.0 ±0.000.0 –0.7IN ––– –––0.0 ±0.000.7 ±0.670.7 –0.70.3 ±0.330.0 ±0.000.0 –0.3Micro-parasitichymenoptera2IA18.7 ±1.206.7 ±2.407.7 –13.723.7 ±4.4827.3 ±3.6724.0 –35.028.0 ±10.2121.0 ±5.5120.3 –27.322.7 ±2.4036.7 ±5.7820.3 –37.3IL185.3 ±22.7063.0 ±17.0636.3 –87.772.7 ±13.4277.7 ±11.1464.3 –90.715.3 ±1.7611.7 ±1.6712.3 –16.375.7 ±5.4692.7 ±11.7278.0 –168.7IN12.7 ±3.389.3 ±2.409.0 –14.359.0 ±10.4472.0 ±35.4754.0 –74.748.7* ±19.70118.3 ±22.6753.7 –112.0307.0 ±9.71256.0 ±73.00189.3 –656.0Nabids IN0.0 ±0.00†0.0 ±0.000.0 –0.00.7 ±0.670.3 ±0.330.3 –1.3––– –––S.E. = standard error; the experimental design was a randomized complete block with three replicates.*Indicates significant differences detected between MON 87460 and the control (p<0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.1Greater than 5mm in length.2Less than 5mm in length.†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 451 of 544Table H-14 (Continued). Beneficial Arthropod Abundance on MON 87460 Compared tothe Control and References at theIA1, IL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeOrius IA10.0†±0.000.0 ±0.000.0 –0.03.7 ±1.761.0 ±0.583.7 –7.015.3 ±0.6718.7 ±3.765.7 –12.72.3 ±1.330.7 ±0.330.0 –2.3IL112.7 ±4.485.3 ±2.673.7 –17.30.7 ±0.330.7 ±0.330.3 –1.09.3 ±1.4515.3 ±0.676.0 –15.37.0 ±4.516.0 ±1.157.3 –21.3IN0.0 ±0.000.3 ±0.330.0 –0.04.3 ±1.761.0 ±0.001.3 –3.716.0 ±9.5413.3 ±4.265.0 –16.00.7 ±0.671.3 ±0.670.0 –2.3Brown lacewings IA1 ––– –––0.3 ±0.330.7 ±0.670.0 –0.3–––IL1 ––– –––0.0 ±0.000.0 ±0.000.0 –0.3–––Green lacewings IA1 –––0.0 ±0.000.0 ±0.000.0 –0.3––– –––IL1 ––– –––3.7 ±0.335.7 ±1.861.3 –7.3–––IN –––0.0 ±0.000.0 ±0.000.0 –0.3––– –––S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 452 of 544Table H-14 (Continued). Beneficial Arthropod Abundance on MON 87460 Compared tothe Control and References at theIA1, IL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered ConditionsMean Arthropod abundance ± S.E.Collection 1 Collection2 Collection 3 Collection 4Arthropod SiteMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeLadybird beetles IA10.3 ±0.331.3 ±0.880.3 –1.33.0 ±0.583.0 ±0.580.7 –3.310.0 ±2.3115.3 ±0.887.7 –14.08.0 ±2.6510.3 ±2.609.7 –25.7IL16.3 ±2.038.7 ±1.451.7 –7.313.0 ±1.536.3 ±3.383.0 –14.71.0 ±0.001.3 ±0.330.7 –2.313.7 ±1.8616.7 ±2.0314.3 –29.3IN0.3 ±0.330.0 ±0.000.3 –1.08.0 ±0.586.3 ±1.454.3 –11.38.7 ±2.734.3 ±2.852.3 –5.79.3 ±1.207.3 ±2.966.0 –18.7S.E. = standard error; the experimental design was a randomized complete block with three replicates.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not evaluated atthis observation and site.Monsanto Company 07-CR-191U Page 453 of 544 Table H-15. Abiotic Stressor Incidence of MON 87460 Compared to the Control

and References in a 2006 U.S. Field St udy Established with Water Managed According to Local Agronomic Practices

Abiotic stressor Number of observations

across all sites ∞ (IAE, IAW, IL, IN, NE) Number of observations

where no differences were

detected across all sites

Total 47 47

Cold 2 2 Compaction 5 5

Flood 1 1

Frost 4 4

Hail 6 6

Heat 8 8 Nutrient deficiency 5 5

Wind 16 16

Note: No differences were detected between MON 87460 and the control. The experimental design was a randomized complete block with three replications; Observational da ta were collected at four crop developmental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 growth stage; Observation 3 = appx. VTR3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: IAE = Bent on County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; NE = York County, NE.Monsanto Company 07-CR-191U Page 454 of 544 Table H-16. Disease Stressor Inciden ce of MON 87460 Compared to the Control

and References in a 2006 U.S. Field St udy Established with Water Managed According to Local Agronomic Practices

Disease Number of observations

across all sites ∞ (IAE, IAW, IL, IN, NE) Number of observations

where no differences were

detected across all sites

Total 67 67

Anthracnose 4 4

Ear rot 1 5 5
Eyespot 3 3

Fusarium 1 1 Gray leaf spot 13 13 Leaf blight 1 1 Northern corn leaf blight 2 2 Northern leaf spot 5 5 Pythium 4 4 Seedling blight 5 5

Root rot 1 1

Rust 4 4 Southern leaf blight 2 2

Smut 4 4

Stalk rot 2 6 6

Stewarts wilt 7 7

Note: No differences were detected between MON 87460 and the control. The experimental design was a randomized complete block with three replications; Observational da ta were collected at four crop developmental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 growth stage; Observation 3 = appx. VTR3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: IAE = Bent on County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; NE = York County, NE.

1 Ear rot assessed by pulling husk back from ears of 5 non-syst ematically selected plants and evaluating the disease;

2 Stalk rot assessed by splitting the stalks of 5 non-systematically selected plants and evaluating the disease. At IL and IN, stalk rot data were collected both on a per plot basis and on 5 plants/plotMonsanto Company 07-CR-191U Page 455 of 544 Table H-17. Arthropod Stressor Inciden ce of MON 87460 Compared to the Control

and References in a 2006 U.S. Field St udy Established with Water Managed According to Local Agronomic Practices

Arthropod Number of observations

across all sites ∞ (IAE, IAW, IL, IN, NE) Number of observations

where no differences were

detected across all sites

Total 61 60

Aphids (includes corn leaf aphids) 5 5 Grasshoppers 12 11 Armyworms(includes fall armyworms) 2 2

Corn earworms 4 4

Cutworms 3 3
European corn borers 1 13 13

Southwestern corn borers 1 1

Western bean cutworms 2 2

Corn rootworms 2 7 7 Flea beetles (includes corn flea beetles) 2 2 Billbugs 1 1

Wireworms 3 3 White grubs 4 4 Seedcorn maggots 2 2

* Differences were detected between MON 87460 and the control for grasshopper at the IAE site during the third observation (none vs. slight). The difference detected was within the range of the reference (no-sl) Note: The experimental design was a randomized complete block with three replications ; Observational data were collected at four crop developmental st ages, Observation 1 = appx. V2-V4 grow th stage; Observation 2 = appx. V10- V15 growth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: IAE = Bent on County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; NE = York County, NE.

1 Includes corn borers; 2 Includes western corn rootworms and southern corn rootwormsMonsanto Company 07-CR-191U Page 456 of 544 Table H-18. Chile 2006/2007: Abiotic Stre ssor Incidence of MON 87460 Compared

to the Control and the References in a Fi eld Study Established with Well-Watered

and Water-Limited Treatments

Well-Watered Treatment

Abiotic stressor Number of observations

across all sites ∞ (CL, CT, LUM) Number of observations

where no differences were

detected across all sites

Total 29 29

Cold 1 1

Frost 3 3

Hail 1 1

Heat 9 9 Nitrogen deficiency 3 3

Wind 12 12

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Observational data were co llected at four crop developm ental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 gr owth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: CL =Colina; CT Calera de Tango; LUM = Lumbreras

Water-Limited Treatment

Abiotic stressor Number of observations

across all sites ∞ (CL, CT, LUM) Number of observations

where no differences were

detected across all sites

Total 29 29

Cold 1 1

Frost 3 3

Hail 1 1

Heat 9 9 Nitrogen deficiency 3 3

Wind 12 12

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Observational data were co llected at four crop developm ental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 gr owth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: CL =Colina; CT Calera de Tango; LUM = LumbrerasMonsanto Company 07-CR-191U Page 457 of 544 Table H-19. Chile 2006/2007: Disease Stre ssor Incidence of MON 87460 Compared

to the Control and References in a Field Study Established with Well-Watered and

Water-Limited Treatments

Well-Watered Treatment

Disease Number of observations

across all sites ∞ (CL, CT, LUM) Number of observations

where no differences were

detected across all sites

Total 35 35

Ear rot 2 2

Fusarium 3 3 Gray leaf spot 6 6 Leaf blight 5 5 Northern corn leaf blight 1 1

Root rot 3 3

Rust 9 9 Seedling blight 3 3

Smut 2 2

Stalk rot 1 1

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Observational data were co llected at four crop developm ental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 gr owth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: CL =Colina; CT Calera de Tango; LUM = Lumbreras

Water-Limited Treatment

Disease Number of observations

across all sites ∞ (CL, CT, LUM) Number of observations

where no differences were

detected across all sites

Total 35 35

Ear rot 2 2

Fusarium 3 3 Gray leaf spot 6 6 Leaf blight 5 5 Northern corn leaf blight 1 1

Root rot 3 3

Rust 9 9 Seedling blight 3 3

Smut 2 2

Stalk rot 1 1

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Observational data were co llected at four crop developm ental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 gr owth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: CL =Colina; CT Calera de Tango; LUM = LumbrerasMonsanto Company 07-CR-191U Page 458 of 544 Table H-20. Chile 2006/2007: Arthropo d Stressor Incidence of MON 87460 Compared to the Control and References in a Field Study Established with WellWatered and Water-Limited Treatments

Well-Watered Treatment

Arthropod Number of observations

across all sites ∞ (CL, CT, LUM) Number of observations

where no differences were

detected across all sites

Total 23 23

Aphids 9 9 Seedcorn maggots 3 3 Thrips 8 8

Wireworms 3 3

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Observational data were co llected at four crop developm ental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 gr owth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: CL =Colina; CT Calera de Tango; LUM = Lumbreras

Water-Limited Treatment

Arthropod Number of observations

across all sites ∞ (CL, CT, LUM) Number of observations

where no differences were

detected across all sites

Total 23 23

Aphids 9 9 Seedcorn maggots 3 3 Thrips 8 8

Wireworms 3 3

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Observational data were co llected at four crop developm ental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 gr owth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: CL =Colina; CT Calera de Tango; LUM = LumbrerasMonsanto Company 07-CR-191U Page 459 of 544 Table H-21. U.S. 2007 Study-1: Abiotic Stressor Evaluations using an Observational Severity Scale for MO N 87460 Compared to the Control and

References in Field Studies Establishe d with Well-Watered and Water-Limited

Treatments

Well-Watered Treatment

Abiotic stressor Number of observations

across all sites ∞ (CA, TX) Number of observations

where no differences were

detected across all sites

Total 16 16

Drought 3 3

Hail 4 4

Heat 2 2
Heavy thunderstorm 1 1 Nitrogen deficiency 2 2

Wind 4 4

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Observational data were co llected at four crop developm ental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 gr owth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: CA =Sutter County, CA; TX = Carson County, TX

Water-Limited Treatment

Abiotic stressor Number of observations

across all sites @

(CA, TX) Number of observations

where no differences were

detected across all sites

Total 16 16

Drought 3 3

Hail 4 4

Heat 2 2
Heavy thunderstorm 1 1 Nitrogen deficiency 2 2

Wind 4 4

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Observational data were co llected at four crop developm ental stages, Observation 1 = appx. V2-V4 growth stage; Observation 2 = appx. V10-V15 gr owth stage; Observation 3 = appx. VT-R3 growth stage; Observation 4 = appx. R6 growth stage.

∞ Site codes are as follows: CA =Sutter County, CA; TX = Carson County, TXMonsanto Company 07-CR-191U Page 460 of 544 Table H-22. U.S. 2007 Stud y-1: Disease Damage Evaluations Using an Observational Severity Scale for MO N 87460 Compared to the Control and

References in Field Studies Establishe d with Well-Watered and Water-Limited

Treatments

Well-Watered Treatment

Disease Number of observations

across all sites ∞ (CA, TX) Number of observations

where no differences were

detected across all sites

Total 18 18

Ear rot 1 2 2

Fusarium 1 1 Gray mold 2 2

Maize dwarf mosaic virus 1 1

Rust 1 1 Seedling blight 1 1
Smut (head and ear) 4 4

Stalk rot 2 2 2 Southern leaf blight 4 4

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with four replications; Observa tional data were collected at four cr op developmental stages, Observation 1 = V2-V4, Observation 2 = V10-V15, Observation 3 = VT-R 3, and Observation 4 = R6 growth stages of corn.

∞ Site codes are as follows: CA =Sutter County, CA; TX = Carson County, TX

1 ear rot assessed by pulling husk back from ears of 5 non-system atically selected plants and evaluating the disease

2 stalk rot assessed by splitting the stalks of 5 non-systematically select ed plants and evaluating the disease

Water-Limited Treatment

Disease Number of observations

across all sites ∞ (CA, TX) Number of observations

where no differences were

detected across all sites

Total 18 18

Ear rot 1 2 2

Fusarium 1 1 Gray mold 2 2

Maize dwarf mosaic virus 1 1

Rust 1 1 Seedling blight 1 1
Smut (head and ear) 4 4

Stalk rot 2 2 2 Southern leaf blight 4 4

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with four replications; Observa tional data were collected at four cr op developmental stages, Observation 1 = V2-V4, Observation 2 = V10-V15, Observation 3 = VT-R 3, and Observation 4 = R6 growth stages of corn.

∞ Site codes are as follows: CA =Sutter County, CA; TX = Carson County, TX

1 ear rot assessed by pulling husk back from ears of 5 non-system atically selected plants and evaluating the disease

2 stalk rot assessed by splitting the stalks of 5 non-systematically select ed plants and evaluating the diseaseMonsanto Company 07-CR-191U Page 461 of 544 Table H-23. U.S. 2007 St udy-1: Arthropod Damage Evaluated using an Observational Severity Scale for MO N 87460 Compared to the Control and

References in Field Studies Establishe d with Well-Watered and Water-Limited

Treatments

Well-Watered Treatment

Arthropod Number of observations

across all sites ∞ (CA, TX) Number of observations

where no differences were

detected across all sites

Total 18 18

Aphids 4 4 Leafhoppers 2 2 Grasshoppers 1 1 Armyworms (includes fall armyworms) 2 2

Cutworms 1 1 European Corn Borers 2 2

Southwestern Corn Borers 2 2

Western Corn Rootworms 1 1

Mites (includes spider mites) 3 3

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with four replications; Observa tional data were collected at four cr op developmental stages, Observation 1 = V2-V4, Observation 2 = V10-V15, Observation 3 = VT-R 3, and Observation 4 = R6 growth stages of corn.

∞ Site codes are as follows: CA =Sutter County, CA; TX = Carson County, TX

Water-Limited Treatment

Arthropod Number of observations

across all sites ∞ (CA, TX) Number of observations

where no differences were

detected across all sites

Total 18 18

Aphids 4 4 Leafhoppers 2 2 Grasshoppers 1 1 Armyworms (includes fall armyworms) 2 2

Cutworms 1 1 European Corn Borers 2 2

Southwestern Corn Borers 2 2

Western Corn Rootworms 1 1

Mites (includes spider mites) 3 3

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with four replications; Observa tional data were collected at four cr op developmental stages, Observation 1 = V2-V4, Observation 2 = V10-V15, Observation 3 = VT-R 3, and Observation 4 = R6 growth stages of corn.

∞ Site codes are as follows: CA =Sutter County, CA; TX = Carson County, TXMonsanto Company 07-CR-191U Page 462 of 544Table H-24. U.S. 2007 Study-1: Quantitative Assessment for Corn Earwormand European Corn Borer Damage toMON 87460 Compared to the Control and References at TX inField Studies Established with Well-Watered and WaterLimited TreatmentsWell-Watered TreatmentPest Damage Assessment MON 87460 Control Reference Range1Corn earworm Mean ± SE of 5 ears (0 – 9 rating scale) 3.2 ± 0.32 3.4 ± 0.29 2.70 – 3.00European corn borer Mean± SE larvae/5 plants 0.0†± 0.00 0.0 ± 0.00 0.00 – 0.00European corn borer Mean ± SE # of stalk entry/exit holes of 5 plants 0.0 ± 0.00 0.0 ± 0.00 0.00 – 0.05European corn borer Mean ± SE # of stalk galleries per plant of 5 plants 0.0 ± 0.00 0.0 ± 0.00 0.00 – 0.03European corn borer Mean ± SE stalk gallery length per plant of 5 plants (in.) 0.0 ± 0.00 0.0 ± 0.00 0.00 – 0.01Water-Limited TreatmentPest Damage Assessment MON 87460 Control Reference Range1Corn earworm Mean ± SE of 5 ears (0 – 9 rating scale) 2.5 ± 0.12 2.7 ± 0.09 2.60 – 2.80European corn borer Mean± SE larvae/5 plants 0.0†± 0.00 0.0 ± 0.00 0.00 – 0.00European corn borer Mean ± SE # ofstalk entry/exit holes of 5 plants 0.0†± 0.00 0.0 ± 0.00 0.00 – 0.00European corn borer Mean ± SE # of stalk galleries per plant of 5 plants 0.0†± 0.00 0.0 ± 0.00 0.00 – 0.00European corn borer Mean ± SE stalk gallery length per plant of 5 plants (in.) 0.0†± 0.00 0.0 ± 0.00 0.00 – 0.00S.E. = standard error; the experimental design was a split plotdesign with four replications.No statistically significant differences between MON 87460 and the control (α=0.05).1Reference range (the minimum and maximum values of the reference means).†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 463 of 544Table H-25. U.S. 2007 Study-1: Arthropod Pest abundance onMON 87460 Compared to the Control and References at TX inField Studies Established with Well-Watered and Water-Limited TreatmentsWell-Watered TreatmentMean Arthropod abundance ± S.E.Collection 1 Collection 2Collection 3 Collection 4ArthropodMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeAphids5.5 ±1.325.0 ±1.087.5 –10.067.0 ±17.9262.5 ±13.8650.3 –79.36.0 ±1.416.5 ±2.224.3 –6.017.8 ±3.0711.5 ±3.529.8 –17.5Delphacidplanthoppers0.0 ±0.000.3 ±0.250.3 –0.31.3 ±0.481.3 ±0.750.5 –1.85.8 ±2.465.3 ±2.104.3 –11.83.8 ±0.854.5 ±1.444.5 –7.3Leafhoppers17.8 ±2.6911.8 ±3.229.0 –15.81.3 ±0.751.3 ±0.750.3 –2.311.5 ±2.9913.8 ±4.509.0 –12.5163.8 ±45.48159.3 ±38.52142.5 –190.8Grasshoppers0.0 ±0.000.0 ±0.000.0 –0.3– – – – – – – – –Corn flea beetles1.8 ±0.481.3 ±0.250.3 –1.513.0 ±4.0211.0 ±4.929.8 –20.526.8 ±6.6923.8 ±7.3015.8 –23.513.3 ±1.3115.0 ±2.7413.0 –16.0Sap beetles – – – – – –0.0 ±0.000.0 ±0.000.0 –0.3– – –Southern cornrootworms0.0 ±0.000.0 ±0.000.0 –0.31.0 ±0.411.3 ±1.250.8 –1.8– – –0.3 ±0.250.5 ±0.290.3 –1.3Western cornrootworms– – –0.0 ±0.000.0 ±0.000.0 –0.50.0†±0.000.0 ±0.000.0 –0.00.0†±0.000.0 ±0.000.0 –0.0S.E. = standard error; the experimental design was a split plotdesign with four replications.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not enumeratedat this observation and site.†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 464 of 544Table H-25 (Continued). U.S. 2007 Study-1: ArthropodPest Abundance on MON 87460 Compared to the Control andReferences at TX in Field Studies Establishedwith Well-Watered and Water-Limited TreatmentsWater-Limited TreatmentMean Arthropod abundance ± S.E.Collection 1 Collection 2Collection 3 Collection 4ArthropodMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeAphids7.3 ±3.285.5 ±1.047.0 –11.880.8 ±23.2680.0 ±8.6769.3 –87.54.8 ±1.118.3 ±1.933.5 –7.816.3 ±2.0216.8 ±2.6611.5 –16.5Delphacidplanthoppers– – –1.8 ±0.751.3 ±0.480.8 –2.04.8 ±1.387.3 ±2.932.8 –10.05.3 ±2.634.3 ±1.935.0 –8.8Leafhoppers9.3 ±1.3117.0 ±6.828.5 –20.51.8 ±1.180.8 ±0.250.5 –1.810.8 ±2.8110.5 ±4.298.8 –12.5137.5 ±43.06179.8 ±32.5396.8 –171.0Grasshoppers0.3 ±0.250.0 ±0.000.0 –0.3– – – – – – – – –Corn flea beetles1.3 ±0.251.5 ±0.500.3 –1.321.5 ±6.2513.3 ±2.219.3 –18.318.0 ±4.8823.5 ±9.2112.5 –23.816.5 ±3.7515.5 ±2.537.8 –15.8Sap beetles – – – – – – – – – – – –Southern cornrootworms– – –0.5 ±0.290.8 ±0.480.3 –0.80.0 ±0.000.0 ±0.000.0 –0.32.5* ±0.650.8 ±0.480.8 –1.5Western cornrootworms– – –0.3 ±0.250.0 ±0.000.0 –0.30.3 ±0.250.8 ±0.750.0 –0.00.0 ±0.00†0.0 ±0.000.0 –0.0S.E. = standard error; the experimental design was a split plotdesign with four replications.*Indicates significant differences detected between MON 87460 and the control (p<0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not enumeratedat this observation and site.†p-values could not be generated due to lack of variabilityin the data. MON 87460 was considered effectively not different from the control because the MON 87460 and controlmean values were identical.Monsanto Company 07-CR-191U Page 465 of 544Table H-26. U.S. 2007 Study-1: Arthropod Beneficial Abundance on MON 87460 Compared to the Control and References atTX in Field Studies Established withWell-Watered and Water-Limited TreatmentsWell-Watered TreatmentMean Arthropod abundance ± S.E.Collection 1 Collection 2Collection 3 Collection 4ArthropodMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeAraneae (spiders)17.5 ±5.8521.0 ±3.8515.0 –24.54.0 ±1.295.0 ±1.002.5 –2.82.8 ±1.112.0 ±1.081.8 –2.810.8 ±2.328.3 ±2.327.8 –9.0Brown lacewings – – – – – –0.5* ±0.290.0 ±0.000.0 –0.5– – –Green lacewings – – –2.0 ±0.410.5 ±0.290.5 –1.52.0 ±0.913.5 ±1.762.5 –4.32.5 ±1.552.5 ±0.960.0 –3.3Big-eyed bug0.0 ±0.000.0 ±0.000.0 –0.3– – – – – – – – –Orius0.3 ±0.250.8 ±0.750.3 –1.04.3 ±0.634.8 ±1.842.3 –6.83.0 ±0.713.0 ±1.412.0 –3.33.0 ±1.082.8 ±0.851.5 –2.5Ladybird beetle1.3 ±0.750.5 ±0.290.5 –0.85.0 ±1.086.0 ±1.355.0 –6.88.8 ±2.295.0 ±1.735.3 –12.03.0 ±0.583.3 ±0.481.3 –3.3Macro-parasitichymenoptera– – –0.5 ±0.291.0 ±0.410.8 –4.3– – –0.8 ±0.481.5 ±0.650.0 –1.3Micro-parasitichymenoptera35.0 ±5.5737.3 ±7.4221.3 –32.8118.5 ±14.7798.8 ±18.43101.5 –128.559.3 ±1.9351.0 ±5.4339.3 –49.5217.3 ±23.25248.0 ±13.35189.0 –222.8S.E. = standard error; the experimental design was a split plotdesign with four replications.*Indicates significant differences detected between MON 87460 and the control (p<0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not enumeratedat this observation and site.Monsanto Company 07-CR-191U Page 466 of 544Table H-26 (Continued). U.S. 2007 Study-1: Arthropod Beneficial Abundance on MON 87460 Compared to the Control andReferences at TX in Field Studies Established with Well-Watered and Water-Limited TreatmentsWater-Limited TreatmentMean Arthropod abundance ± S.E.Collection 1 Collection 2Collection 3 Collection 4ArthropodMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeMON87460ContrRefRangeAraneae (spiders)21.0 ±3.0819.3 ±3.4219.3 –22.02.3 ±0.482.5 ±0.651.5 –3.04.8 ±2.171.8 ±0.481.5 –4.310.3 ±1.936.0 ±1.877.5 –11.3Brown lacewings – – – – – –0.0 ±0.000.0 ±0.000.0 –0.3– – –Green lacewings – – –0.5 ±0.500.5 ±0.290.8 –1.52.3 ±0.632.0 ±0.712.0 –3.82.3 ±0.851.5 ±0.500.8 –2.0Big-eyed bug0.0 ±0.000.0 ±0.000.0 –0.3– – – – – – – – –Orius0.3 ±0.250.0 ±0.000.0 –0.85.0 ±1.227.0 ±1.583.8 –7.83.3 ±1.972.8 ±1.251.5 –3.53.3 ±1.491.3 ±0.631.5 –4.3Ladybird beetle0.3 ±0.250.0 ±0.000.3 –1.86.5 ±1.265.8 ±0.254.8 –7.52.8 ±0.856.0 ±0.916.0 –9.02.8 ±0.632.3 ±1.111.3 –2.3Macro-parasitichymenoptera0.0 ±0.000.0 ±0.000.0 –0.8– – – – – –1.3 ±0.481.5 ±0.650.5 –2.3Micro-parasitichymenoptera24.8 ±2.9027.3 ±6.0227.5 –43.0111.5 ±7.80101.8 ±14.0890.0 –112.848.0 ±4.0251.8 ±7.1946.5 –51.5240.3 ±53.00199.8 ±28.54172.8 –266.8S.E. = standard error; the experimental design was a split plotdesign with four replications.No statistically significant differences between MON 87460 and the control (α=0.05).Contr = Control; Ref Range = minimum and maximum mean values of the references; dash (–) = arthropods not enumeratedat this observation and site.Monsanto Company 07-CR-191U Page 467 of 544 Table H-27. U.S. 2007 Study-2: Abiotic Stressor Evaluations using an Observational Severity Scale for MO N 87460 Compared to the Control and

References in Field Studies Establishe d with Well-Watered and Water-Limited

Treatments

Well-Watered Treatment

Abiotic stressor Number of observations

across all sites ∞

(KS, NE, TX) Number of observations

where no differences were

detected across all sites

Total 29 29

Drought 1 1

Hail 8 8

Heat 5 5 Water logging 3 3

Wind 12 12

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Data were collected at four crop developmental stag es, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site codes are as follows: NE = York County, NE; KS = Pawnee C ounty, KS; TX = Carson County, TX

Water-Limited Treatment

Abiotic stressor Number of observations

across all sites ∞ (TX) Number of observations

where no differences were

detected across all sites

Total 12 12

Drought 1 1

Hail 4 4

Heat 3 3

Wind 4 4

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Data were collected at four crop developmental stag es, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site code is as follows : TX = Carson County, TXMonsanto Company 07-CR-191U Page 468 of 544 Table H-28. U.S. 2007 Study-2: Dise ase Damage Evaluations using an Observational Severity Scale for MO N 87460 Compared to the Control and

References in Field Studies Establishe d with Well-Watered and Water-Limited

Treatments

Well-Watered Treatment

Disease Number of observations

across all sites ∞

(KS, NE, TX) Number of observations

where no differences were

detected across all sites

Total 42 42

Crazy top 1 1

Ear rot 1 3 3 Gray leaf spot 7 7 Gray mold 3 3

Maize dwarf mosaic 1 1 Northern corn leaf blight 7 7

Rust 2 8 8 Seedling blight 2 2

Smut 3 3

Stalk rot 3 3 3 Southern corn leaf blight 4 4

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Data were collected at four crop developmental stag es, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site codes are as follows: NE = York County, NE; KS = Pawnee C ounty, KS; TX = Carson County, TX

1 Ear rot assessed externally by observing the outside of the ear only; 2 Includes common rust and leaf rust; 3 Stalk rot assessed by splitting the stalks of 5 non-systemati cally selected plants and evaluating the disease

Water-Limited Treatment

Disease Number of observations

across all sites ∞ (TX) Number of observations

where no differences were

detected across all sites

Total 14 14

Ear rot 1 1 1 Gray mold 3 3

Maize dwarf mosaic 1 1 Northern corn leaf blight 3 3 Seedling blight 1 1

Stalk rot 2 1 1 Southern corn leaf blight 4 4

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Data were collected at four crop developmental stag es, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site code is TX = Carson County, TX

1 Ear rot assessed externally by observing the outside of the ear only; 2 Stalk rot assessed by splitting the stalks of 5 non-systematically selected plan ts and evaluating the diseaseMonsanto Company 07-CR-191U Page 469 of 544 Table H-29. U.S. 2007 Study-2: Arthro pod Damage Evaluations using an Observational Severity Scale for MO N 87460 Compared to the Control and

References in Field Studies Establishe d with Well-Watered and Water-Limited

Treatments

Well-Watered Treatment

Arthropod Number of observations

across all sites ∞

(KS, NE, TX) Number of observations

where no differences were

detected across all sites

Total 36 36

Aphids 1 1 Leafhoppers 1 1 Grasshoppers 4 4 Armyworms 5 5

Corn earworms 4 4
Cutworms (includes black cutworms) 2 2
European corn borers 8 8

Southwestern corn borers 3 3

Corn rootworm 1 6 6

Wireworms 1 1

Leafrollers 1 1

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Data were collected at four crop developmental stag es, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site codes are as follows: NE = York County, NE; KS = Pawnee C ounty, KS; TX = Carson County, TX

1 Includes western corn rootworm and southern corn rootworm

Water-Limited Treatment

Arthropod Number of observations

across all sites ∞ (TX) Number of observations

where no differences were

detected across all sites

Total 12 12

Aphids 1 1 Grasshoppers 1 1 Armyworms 1 1

Corn earworms 3 3
Cutworms (includes black cutworms) 1 1
European corn borers 2 2

Southwestern corn borers 2 2

Corn rootworm 1 1 1

Note: No differences were detected between MON 87460 a nd the control. The experimental design was a split plot design with three replications; Data were collected at four crop developmental stag es, observation 1 = V2-V4, observation 2 = V10-V15, observation 3 = VT-R3, and observation 4 = R6 growth stages of corn

∞ Site code is as follows : TX = Carson County, TX

1 Includes western corn rootworm and southern corn rootwormMonsanto Company 07-CR-191U Page 470 of 544 Appendix I. Water Management and Environmental Data for U.S. and Chile Field Studies during 2006 and 2007

This section contains water management and temperature data for the phenotypic characterization field sites presen ted in Appendices F, G, and H.

Contents

Section I.1. Calculated and accumulated wa ter data for U.S. and Chile field studies during 2006 and 2007

• U.S. 2006 and 2007 well-watered sites

o Section I.1.1; Figures I-1 – I-17

• U.S. 2006 typical local practice sites

o Section I.1.2; Figures I-18 – I-22

• Chile 2006/2007 well-watered and water-limited sites

o Section I.1.3; Figures I-23 - I-26

• U.S. 2007 Study-1 well-watered and water-limited sites

o Section I.1.4; Figures I-27 and I-28

• U.S. 2007 Study-2 well-watered and water-limited sites

o Section I.1.5; Figures I-29 - I-31

Section I.2. Temperature data (monthly minimum and maximum) for U.S. and Chile field studies during 2006 and 2007

• U.S. 2006 and 2007 well-watered sites

o Section I.2.1; Tables I-1 and I-2

• U.S. 2006 typical local practice sites

o Section I.2.2; Table I-3

• Chile 2006/2007 well-watered and water-limited sites

o Section I.2.3; Table I-4.

• U.S. 2007 Study-1 well-watered and water-limited sites

o Section I.2.4; Table I-5.

• U.S. 2007 Study-2 well-watered and water-limited sites

o Section I.2.5; Table I-6.Monsanto Company 07-CR-191U Page 471 of 544 I-1. Calculated and Accumulated Water Da ta for U.S. and Chile Field Studies during 2006 and 2007

I.1.1. U.S. 2006 and 2007 Well-Watered Sites
Site Code, Test Location, and Year Site Code IA1, Jefferson County, Iowa, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-1. Calculated and Accumula ted Soil Water Values at IA1 in 2006

(Note: moisture levels below 50% of fi eld capacity represen t stress conditions)Monsanto Company 07-CR-191U Page 472 of 544 Site Code, Test Location, and Year Site Code IA2, Benton County, Iowa, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-2. Calculated and Accumula ted Soil Water Values at IA2 in 2006

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 473 of 544 Site Code, Test Location, and Year Site Code IL1, Stark County, Illinois, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-3. Calculated and Accumula ted Soil Water Values at IL1 in 2006

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 474 of 544 Site Code, Test Location, and Year Site Code IL2, Warren County, Illinois, 2006.

. Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date 6/1/06 7/1/06 8/1/06 9/1/06Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-4. Calculated and Accumula ted Soil Water Values at IL2 in 2006

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 475 of 544 Site Code, Test Location, and Year Site Code IN1, Boone County, Indiana, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25 30

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-5. Calculated and Accumula ted Soil Water Values at IN1 in 2006

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 476 of 544 Site Code, Test Location, and Year Site Code IN2, Parke County, Indiana, 2006.

Date 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date 7/1/06 8/1/06 9/1/06Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-6. Calculated and Accumula ted Soil Water Values at IN2 in 2006

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 477 of 544 Site Code, Test Location, and Year Site Code KS, Pawnee County, Kansas, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 10 20 30 Accumualted Water

Date

6/1/06 7/1/06 8/1/06 9/1/06Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-7. Calculated and Accumula ted Soil Water Values at KS in 2006

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 478 of 544 Site Code, Test Location, and Year Site Code NE, York County, Nebraska, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-8. Calculated and Accumula ted Soil Water Values at NE in 2006

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 479 of 544 Site Code, Test Location, and Year Site Code IA1, Jefferson County, Iowa, 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25 30

Accumulated Water

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-9. Calculated and Accumula ted Soil Water Values at IA1 in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 480 of 544 Site Code, Test Location, and Year Site Code IA2, Van Horne County, Iowa, 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-10. Calculated and Accumu lated Soil Water Values at IA2 in 2007

(Note: moisture levels below 50% of fi eld capacity represen t stress conditions)Monsanto Company 07-CR-191U Page 481 of 544 Site Code, Test Location, and Year Site Code IL1, Stark County, Illinois, 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-11. Calculated and Accumu lated Soil Water Values at IL1 in 2007

(Note: moisture levels below 50% of fi eld capacity represen t stress conditions)Monsanto Company 07-CR-191U Page 482 of 544 Site Code, Test Location, and Year Site Code IL2, Warren County, Illinois, 2007.

Water data not available for this site.

Figure I-12. Calculated and Accumu lated Soil Water Values at IL2 in 2007Monsanto Company 07-CR-191U Page 483 of 544 Site Code, Test Location, and Year Site Code IL3, Clinton County, Illinois, 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-13. Calculated and Accumu lated Soil Water Values at IL3 in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 484 of 544 Site Code, Test Location, and Year Site Code IN, Boone County, Indiana, 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-14. Calculated and Accumula ted Soil Water Values at IN in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 485 of 544 Site Code, Test Location, and Year Site Code NE, York County, Nebraska, 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-15. Calculated and Accumu lated Soil Water Values at NE in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 486 of 544 Site Code, Test Location, and Year Site Code OH, Fayette County, Ohio, 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-16. Calculated and Accumu lated Soil Water Values at OH in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 487 of 544 Site Code, Test Location, and Year Site Code PA, Berks County, Pennsylvania, 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25 30

Accumulated Water

Date 06/01 07/01 08/01 09/01Calculated Soil Water Availability (%)

0 20 40 60 80 100

Calculated Soil Water Availability

50% Reference Line

Figure I-17. Calculated and Accumu lated Soil Water Values at PA in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 488 of 544 I.1.2. U.S. 2006 Typical Local Practice Sites, Accumulated Water Data Only

Site Code, Test Location, and Year Site Code IAE, Benton County, Iowa, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Figure I-18. Accumulated Water at IAE in 2006

Site Code, Test Location, and Year Site Code IAW, Greene County, Iowa, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Figure I-19. Accumulated Water at IAW in 2006Monsanto Company 07-CR-191U Page 489 of 544 Site Code, Test Location, and Year Site Code IL, Stark County, Illinois, 2006.

Figure I-20. Accumulated Water at IL in 2006

Site Code, Test Location, and Year Site Code IN, Parke County, Indiana, 2006.

Date 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Figure I-21. Accumulated Water at IN in 2006 Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated WaterMonsanto Company 07-CR-191U Page 490 of 544 Site Code, Test Location, and Year Site Code NE, York County, Nebraska, 2006.

Date 6/1/06 7/1/06 8/1/06 9/1/06Accumulated Water (in.)

0 5 10 15 20 25

Accumulated Water

Figure I-22. Accumulated Water at NE in 2006Monsanto Company 07-CR-191U Page 491 of 544 I.1.3. Chile 2006/2007 Well-Watered and Wat er-Limited Sites, Accumulated Water Data Only

Site Code, Test Location, and Year Site Code CL, Colina, Chile, 2006/2007.

Date 1/1/07 2/1/07 3/1/07 4/1/07Accumulated Water (in.)

0 10 20 30 Water-Limited

Well-Watered

Figure I-23. Accumulated Water at CL in 2006/2007Monsanto Company 07-CR-191U Page 492 of 544 Site Code, Test Location, and Year Site Code CT, Calera de Tango, Chile, 2006/2007.

Date 1/1/07 2/1/07 3/1/07 4/1/07Accumulated Water (in.)

0 10 20 30 Water-Limited

Well-Watered

Figure I-24. Accumulated Water at CT in 2006/2007

Site Code, Test Location, and Year Site Code LUM, Lumbreras, Chile, 2006/2007.

Date 1/1/07 2/1/07 3/1/07 4/1/07Accumulated Water (in.)

0 10 20 30 Water-Limited

Well-WateredMonsanto Company 07-CR-191U Page 493 of 544 Figure I-25. Accumulated Water at LUM in 2006/2007

Site Code, Test Location, and Year Site Code QUI, Quillota, Chile, 2006/2007.

Date 1/1/07 2/1/07 3/1/07 4/1/07Accumulated Water (in.)

0 10 20 30 Water-Limited

Well-Watered

Figure I-26. Accumulated water at QUI in 2006/2007Monsanto Company 07-CR-191U Page 494 of 544 I.1.4. U.S. 2007 Study-1 Well-Watered and Water-Limited Sites

Site Code, Test Location, and Year Site Code CA, Sutter County, California, Study-1 in 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Water-Limited

Well-Watered

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Water-Limited

50% Reference Line

Well-Watered

Figure I-27. Calculated and Accumulate d Soil Water Values at CA Study-1 in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 495 of 544 Site Code, Test Location, and Year Site Code TX, Carson County, Texas, Study-1 in 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25 30

Water-Limited

Well-Watered

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Water-Limited

50% Reference Line

Well-Watered

Figure I-28. Calculated and Accumulate d Soil Water Values at TX Study-1 in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 496 of 544 I.1.5. U.S. 2007 Study-2 Well-Watered and Water-Limited Sites

Site Code, Test Location, and Year Site Code KS, Pawnee County, Kansas, Study-2 in 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Water-Limited

Well-Watered

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Water-Limited

50% Reference Line

Well-Watered

Figure I-29. Calculated and Accumulate d Soil Water Values at KS Study-2 in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 497 of 544 Site Code, Test Location, and Year Site Code NE, York County, Nebraska, Study-2 in 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25

Water-Limited

Well-Watered

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Water-Limited

50% Reference Line

Well-Watered

Figure I-30. Calculated and Accumulate d Soil Water Values at NE Study-2 in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 498 of 544 Site Code, Test Location, and Year Site Code TX, Carson County, Texas, Study-2 in 2007.

Date 6/1/07 7/1/07 8/1/07 9/1/07Accumulated Water (in.)

0 5 10 15 20 25 30

Water-Limited

Well-Watered

Date 6/1/07 7/1/07 8/1/07 9/1/07Calculated Soil Water Availability (%)

0 20 40 60 80 100

Water-Limited

50% Reference Line

Well-Watered

Figure I-31. Calculated and Accumulate d Soil Water Values at TX Study-2 in 2007

(Note: moisture levels below 50% of field capacity repres ent stress conditions)Monsanto Company 07-CR-191U Page 499 of 544 I.2. Temperature Data for U.S. and Chile Field Studies during 2006 and 2007

I.2.1. U.S. 2006 and 2007 Well-Watered Sites Temperature Data

Table I-1. U.S. 2006 Well-Watered Si tes - Monthly Average Minimum and Maximum Temperatures and Temperature Range Study

Site 1 May June July

Min 2 Max Range 3 Min Max Range Min Max Range

IA 1 52.2 71.9 33-91 60.9 81.1 51-90 66.8 88.1 53-96

IA 2 51.1 72.5 30-98 59.5 81.1 45-97 64.9 85.6 54-95

IL 1 50.5 71.7 36-94 57.1 80.9 46-92 64.2 87.6 46-97

IL 2 50.5 71.1 33-94 59.5 80.4 48-90 65.1 87.2 54-96

IN 1 51.2 70.4 37-92 58.8 79.7 49-92 65.5 85.0 50-96

IN 2 51.1 70.6 40-92 58.1 80.1 52-89 64.8 86.3 50-96

KS 53.3 78.8 39-99 61.7 88.6 53-99 66.5 92.8 55-104

NE 51.4 79.1 37-96 61.3 88.1 52-101 65.5 91.3 55-103

Study

Site August September October Min Max Range Min Max Range Min Max Range

IA 1 64.6 82.1 55-92 51.0 72.9 33-85 39.2 59.2 21-92

IA 2 62.0 86.5 55-96 48.9 75.6 32-94 36.8 58.8 19-94

IL 1 61.8 82.2 51-96 48.2 74.4 34-88 35.8 60.0 19-92

IL 2 64.2 82.5 55-97 51.1 72.9 35-84 38.0 60.9 22-94

IN 1 62.4 81.8 52-92 52.0 72.6 37-85 41.9 61.4 27-85

IN 2 63.2 82.7 55-93 52.8 74.5 37-87 40.6 63.1 25-90

KS 65.8 88.8 52-104 51.2 77.7 37-93 41.9 67.7 22-96

NE 63.0 86.1 52-100 50.0 75.8 36-93 37.2 63.0 18-96

1 Study sites: IA1 = Jefferson Co., IA; IA2 = Benton, Co., IA; IL1 = Stark Co., IL;

IL2 = Warren Co., IL; IN1 = Boone Co., IN; IN2 = Parke Co., IN; KS = Pawnee Co., KS, NE = York Co., NE

2 Average daily minimum and maximum temperature (°F)

3 Range of daily minimum and maximum temperatures (°F)Monsanto Company 07-CR-191U Page 500 of 544 Table I-2. U.S. 2007 Well-Watered Si tes - Monthly Average Minimum and Maximum Temperatures and Temperature Range

1 Study sites: IA1 = Jefferson Co., IA; IA2 = Van Horne Co., IA; IL1 = Wyoming Co., IL; IL2 = Monmouth Co., IL; IL3 = Carlyle Co., IL; IN = Sheridan Co., IN; MO = Bethel Co., MO; NE = York Co., NE; OH = New Holland Co., OH; PA = Hamburg Co., PA

2 Average daily minimum and maximum temperature (°F)

3 Range of daily minimum and maximum temperatures (°F)

4 The IL2 site was destroyed by a storm on August 28, 2007 and subsequent weather data was not collected

Study May June July

Site 1 Min 2 Max 2 Range 3 Min Max Range Min Max Range

IA1 56.1 76.7 43-87 62.3 81.5 52-93 63.5 84.9 54-91

IA2 54.0 76.6 39-89 61.6 80.4 48-92 61.5 84.2 52-91

IL1 53.8 79.4 32-91 58.3 82.4 42-94 58.1 83.2 49-91

IL2 57.3 78.1 44-87 61.6 82.2 50-91 60.7 81.9 53-90

IL3 59.7 79.8 40-88 64.2 85.4 53-92 64.7 85.7 55-91

IN 53.7 81.0 30-90 58.0 84.5 44-95 56.8 84.3 45-93

MO 57.1 78.3 42-89 62.8 82.6 51-92 65.0 86.4 54-92

NE 55.5 76.0 45-91 60.2 82.4 48-90 65.9 86.7 56-96

OH 51.4 80.1 38-92 59.7 84.6 48-96 59.5 83.1 48-93

PA 52.1 86.0 38-93 58.4 82.2 47-93 59.7 84.1 46-94

Study August September October

Site1 Min Max Range Min Max Range Min Max Range

IA1 67.2 85.9 53-93 54.2 79.4 34-90 47.4 67.4 31-87

IA2 65.6 84.0 49-90 50.3 78.1 30-89 44.6 65.8 25-88

IL1 64.1 85.5 48-93 50.9 82.7 29-93 43.4 70.1 25-90

IL2 4 67.3 86.0 58-93 - - - - - -

IL3 69.6 95.0 55-104 60.0 84.4 40-96 50.6 72.0 31-92

IN 63.5 87.0 49-95 53.7 82.0 36-93 49.4 70.7 32-91

MO 69.6 90.8 60-99 57.8 83.3 45-93 47.9 69.9 32-87

NE 66.7 86.3 56-95 53.5 79.0 40-90 44.9 67.1 29-87

OH 64.3 88.8 51-98 53.2 83.9 35-95 48.3 71.7 28-93

PA 62.1 84.8 52-96 52.6 80.9 39-91 47.7 72.3 29-90Monsanto Company 07-CR-191U Page 501 of 544 I.2.2. U.S. 2006 Typical Local Practice Sites Temperature Data

Table I-3. U.S. 2006 Typical Local Practi ce Sites - Monthly Average Minimum and Maximum Temperatures and Temperature Range

Study

Site 1 May June July

Min 2 Max 2 Range 3 Min Max Range Min Max Range

IAE 51 72 30 - 98 60 81 45 - 97 65 86 54 - 95

IAW 50 72 34 - 91 59 84 49 - 93 63 88 52 - 99

IL 51 72 36 - 94 57 81 46 - 92 64 88 46 - 97

IN 51 71 40 - 92 58 80 52 - 89 65 86 50 - 96

NE 51 79 37 - 96 61 88 52 - 101 65 91 55 - 103

Study

Site August September October

Min Max Range Min Max Range Min Max Range

IAE 62 86 55 - 96 49 76 32 - 94 37 59 19 - 94

IAW 66 83 58 - 93 48 71 34 - 86 38 60 23 - 90

IL 62 82 51 - 96 48 75 34 - 88 36 60 19 - 92

IN 63 83 55 - 93 53 75 37 - 87 41 63 25 - 90

NE 63 86 52 - 100 50 76 36 - 93 37 63 18 - 96

1 Site codes are as follows: IAE = Benton County, IA ; IAW = Greene County, IA; IL = Stark County, IL;

IN = Parke County, IN; NE = York County, NE.

2 Average daily minimum and maximum temperature (°F)

3 The range is the absolute maximum a nd minimum temperature in each month.Monsanto Company 07-CR-191U Page 502 of 544 I.2.3. Chile 2006/2007 Well-Watered and Water-Limited Sites Temperature Data

Table I-4. Chile 2006/2007 Well-Watered and Water-Limited Sites - Monthly Average Minimum and Maximum Temperatures and Temperature Range

Study January February March

Site 1 Min 2 Max 2 Range 3 Min Max Range Min Max Range

CL 52.9 87.6 45.9-97.2 49.3 84.6 41.5-94.1 47.3 82.4 40.8-94.1

CT 51.6 83.7 45.9-91.0 49.5 79.3 42.0-89.6 49.5 78.8 41.5-88.2

LUM 52.0 81.1 46.8-88.9 50.4 77.5 41.5-88.9 48.9 78.8 41.5-94.1
QUI 51.4 83.3 47.5-91.8 50.9 78.6 43.7-89.6 48.6 77.4 40.8-93.4

Study April May

Site 1 Min Max Range Min Max Range

CL 40.8 74.3 33.1 - 88.9 33.1 66.6 25.5 - 78.1

CT 41.0 71.4 30.6 - 86.7 36.7 65.7 28.9 - 77.4

LUM 41.9 72.5 32.2 - 87.4 36.5 67.1 28.0 - 76.6

QUI 43.9 73.6 33.8 - 91.8 37.4 68.0 28.9 - 76.6

1 Study sites: CL = Colina; CT = Calera de Tango; LUM = Lumbreras; QUI = Quillota.

2 Average daily minimum and maximum temperature (°F) 3The range is the absolute maximum and minimum temperature in each month.

I.2.4. U.S. Study-1 Well-Watered and Water-Limited Sites Temperature Data

Table I-5. U.S. 2007 Study-1 Well-Water ed and Water-Limited Sites - Monthly Average Minimum and Maximum Temperatures and Temperature Range

Study May June July

Site 1 Min 2 Max 2 Range 3 Min Max Range Min Max Range

CA 50.7 84.5 39.0-94.0 55.1 91.5 47.0-107.0 58.2 95.8 52.0-109.0

TX 50.8 75.9 38.0-86.0 57.3 86.0 43.0-96.0 2 61.0 94.8 57.0-100.0

Study August September October

Site 1 Min Max Range Min Ma x Range Min Max Range

CA 57.4 95.4 49.0-107.0 52.0 86.0 41.0-105.0 45.7 73.3 40.0-87.0

TX 63.7 95.9 59.0-105.0 56.2 86.9 46.0-105.0 43.9 80.0 32.0-98.0 2

1 Source for weather data: University of California, Agriculture and Natural Resources. IPM Online for Yuba City, CA, and Record of Climatological Observations, Panhandle Station for Carson Count, TX.

2 Data missing or not reported for June 10, June 26-30, and for October 30-31

3 The range is the absolute maximum a nd minimum temperature in each month.Monsanto Company 07-CR-191U Page 503 of 544 I.2.5. U.S. Study-2 Well-Watered and Water-Limited Sites Temperature Data

Table I-6. U.S. 2007 Study-2 Well-Water ed and Water-Limited Sites - Monthly Average Minimum and Maximum Temperatures and Temperature Range

Study May June July

Site 1 Min 2 Max 2 Range 3 Min Max Range Min Max Range

KS 55.3 78.3 42-84 61.3 85.4 48-91 66.0 91.2 60-95

NE 55.0 76.0 42-91 60.0 82.0 46-91 66.0 87.0 55-98

TX 53.0 76.0 44-86 60.0 84.0 44-89 64.0 90.0 59-96

Study August September October

Site 1 Min Max Range Min Ma x Range Min Max Range

KS 69.5 94.6 60-102 58.1 85.4 44-96 44.5 75.8 28-91

NE 67.0 86.0 55-96 53.0 79.0 41-91 45.0 67.0 30-89

TX 66.0 93.0 62-98 60.0 85.0 46-98 47.0 76.0 32-91

1 Note: Average Daily Minimum and Average Da ily Maximum values were provided by the principle investigator at each site. The Range values were downloaded from the internet source: http://www.wunderground.com using the nearest airport weather data for each site.

2 Average daily minimum and maximum temperature (°F) 3The range is the absolute maximum and minimum temperature in each month.Monsanto Company 07-CR-191U Page 504 of 544 Appendix J. Materials, Metho ds and Individual Site Results for Seed Dormancy and

Germination

J.1. Materials

Seed of MON 87460, the conventional control co rn, and reference corn were produced in Greene County, IA, Stark County, IL, and Pawnee County, KS in 2006.

Material Material Type Phenotype Production 1

Site
MON 87460 Test Drought tolerant IA

H1548126 Control Conventional IA

H8991 Reference Conventional IA

DKC 61-50 Reference Conventional IA

33N29 Reference Conventional IA

MON 87460 Test Drought tolerant IL

H1548126 Control Conventional IL

33K39 Reference Conventional IL

M-3744 Reference Conventional IL

M-3765 Reference Conventional IL

MON 87460 Test Drought tolerant KS

H1548126 Control Conventional KS

S-2721 Reference Conventional KS

32B33 Reference Conventional KS

33H25 Reference Conventional KS

J.2. Characterization of the Materials

The presence or absence of MON 87460 insert was verified by event-specific PCR analysis for MON 87460, control, and reference corn. The results of these ve rifications were as expected with the following exceptions: anal ysis of the H1548126 seed from the IA production site reve aled the presence of MON 87460 at ≤ 1.84%. In addition, the analysis

of seed of three reference substances from the IA production si te and two reference substances from the KS production site revealed the presence of MON 87460 at ≤ 1.84%. This result was not unexpected as the seed of the test, control and reference substances were produced in a common nursery. The le vel of MON 87460 was low and was deemed to have no negative effect on the quality of the study or interpreta tion of the results.

J.3. Performing Facility and Experimental Methods

Dormancy and germination evaluations were conducted at BioDiagnostics, Inc. in River Falls, WI. The principal investigator wa s certified to conduc t seed dormancy and germination testing consistent with the standards established by the Association of Official Seed Analysts (AOSA), a seed tr ade association (AOSA, 2000; AOSA, 2006).Monsanto Company 07-CR-191U Page 505 of 544 Seven germination chambers were used in the study and each chamber was maintained dark under one of the following seven temperat ure regimes: constant temperature of approximately 5, 10, 20, or 30 o C or alternating temperatur es of approximately 10/20, 10/30, or 20/30 o C. The alternating temperature regimes were maintained at the lower temperature for 16 h and the higher temperatur e for 8 h. The temperature inside each germination chamber was monitored and record ed throughout the duration of the study.

Germination towels for MON 87460, control, an d reference materials were prepared per the facility SOPs. Each germination towel repr esented one replication. The types of data collected depended on the temperature regime. Each rolled germination towel in the AOSA-recommended temperature regime (i.e ., 20/30°C) was assessed periodically during the study for normally germinated, abnormally germinated, hard (viable and nonviable), dead, and firm swollen (viabl e and nonviable) seed as defined by AOSA guidelines (AOSA, 2006). Each rolled germin ation towel in the additional temperature regimes (i.e., 5, 10, 20, 30, 10/20 and 10/30 o C) was assessed periodically during the study for germinated, hard (viable and nonviab le), dead, and firm swollen (viable and nonviable) seed.

J.4. Statistical Analysis

Statistical analyses were performed by Mons anto Statistics Technology Center. The data were analyzed according to a split-plot design, with production site as the whole plot and seed substance type as the sub-plot. SAS ® (SAS Version 9.1.3, 2002-2003) was used to compare MON 87460 and the control material for each characteristic ac ross all sites with

a level of statistica l significance of 5% ( α = 0.05). A combined-site analysis was conducted when no significant production site-by- seed substance type interactions were

detected. An individual-s ite analysis was conducted wh en production site-by-seed substance type interactions we re detected. Summary statisti cs were provided for each production site and temperatur e regime combination. Th e test substance was not statistically compared to the reference subs tances, and no comparisons were made across temperature regimes. The reference ra nge was calculated from the minimum and

maximum mean values observed in the references.

For the AOSA recommended temperature ra nge, the data reported for each seed substance included the percentage of norma l germinated, abnormal germinated, dead, viable firm swollen, and viable hard seed. For non-AOSA temperatures, the data reported for each seed substance included the pe rcentage of germinated, dead, viable firm swollen, and viable hard seed.

J.5. Individual Site Seed Dorman cy and Germination Results

MON 87460, the control, and reference seed ma terials were produced at three sites to assess germination characteristic s of seed grown under various environmental conditions.

® SAS is a registered trademark of SAS Institute, Inc.Monsanto Company 07-CR-191U Page 506 of 544 No production site × seed substance interact ions were detected for any combination except percent germinated and percent viab le firm swollen at 10°C. These two characteristics were analyzed separately by site to account for the inte raction. In percent germinated and percent viable firm swollen seed at 10°C, significant production site × seed substance type interactions were dete cted. Analyses were therefore conducted for these two variables at this temperature on an individual site basis. No differences were

detected between MON 87460 and the control at the IL or KS producti on sites. For the IA site, MON 87460 had greater percent germination and lower percent viable firm swollen seed than the control (91.5 vs. 87.0; 6.5 vs. 11.2, respectively). However, no

differences were detected at other sites, and the values for MON 87460 and the control at 10°C were within the reference range for bot h percent germination and percent viable firm swollen seed (Table J-1). Furthermor e, of the 15 comparisons over the germination characteristics from the across-site analysis , no statistically significant differences were

detected between MON 87460 and the control (Table VIII-3).

The biological characteristics evaluated in th is study are used to characterize MON 87460 in the context of plant pest risk assessment. The results of this st udy, in particular the absence of hard seed, support a conclusion that seed of MON 87460 does not exhibit altered dormancy and germination characteristics compared to the conventional control. These results support a conc lusion of no increased pe st potential of MON 87460 compared to conventional corn.

J.6. References

AOSA. 2000. Tetrazolium Testing Handbook. Asso ciation of Official Seed Analysts. Lincoln, NE. AOSA. 2006. Rules for Testing Seeds. Associa tion of Official Seed Analysts. Lincoln,

NE.Monsanto Company 07-CR-191U Page 507 of 544Table J-1. Individual Site Analysis for Germination of MON 87460 and the Control in 2006IA2,3Mean ± S.E.IL2,3Mean ± S.E.KS2,3Mean ± S.E.ReferenceRangeTemperatureCharacteristicMON87460ControlMON87460ControlMON87460ControlMinMax10°CGerminated91.5 ± 1.5287.0* ± 1.7486.0 ± 1.5889.0 ± 0.4192.0 ± 1.7894.0 ± 0.4187.799.0Viable firm swollen6.5 ± 1.8611.2* ± 1.4013.3 ± 1.1810.5 ± 0.657.8 ± 1.655.3 ± 0.480.08.3S.E. = standard error.*Indicates significant differences detected between MON 87460 and the control (p<0.05).1Analysis run only on variables where production site × seed substance interaction prevented combined-site analysis.2IA = Greene County, IA; IL = Stark County, IL; KS = Pawnee County, KS.3Totals are a mean percent.Monsanto Company 07-CR-191U Page 508 of 544 Appendix K. Materials and Methods for Po llen Morphology and Viability Analysis

K.1. Test, Control, and Reference Substances

Assessment for pollen morphology and viabilit y was conducted in a U.S. field study in CA during 2007 on pollen collected from plants that received either the well-watered or water-limited treatment (Tables F-1 and F-6).

K.1.1. Test Substance

The test substance was MON 87460.

K.1.2. Control Substance

The control substance was conventional corn DM1718, which has a genetic background

similar to the test substance but did not possess the drought tolerance trait.

K.1.3. Reference Substances

The reference substances were co mmercially available corn hybrids: DKC63-78, RX715, RX754RR2, and DKC61-50.

K.2. Experimental Methods

Plant Production MON 87460, a conventional control, and four re ferences were grown in California under similar agronomic conditions, with the excepti on of irrigation treatment. The study was arranged in a split-plot design with four re plications, where irrigation treatment was the whole-plot and plant substance type was th e sub-plot. A pollen sample was collected from three plants per plot an d was fixed and staine d with 1:5 diluted Alexander stain. Pollen viability and pollen diameter were evaluated and general pollen morphology was observed. The test substance was compared to the control substance for mean pollen diameter and percenta ge viable pollen.

Pollen Collection Tassel bags were placed on three to five, non- systematically select ed plants per plot during pollen shed. The following morni ng, pollen was collected from each of the bagged plants and transferred to a uniquely labeled tube. Within approximately 30 minutes of collection, Alexander’s stain solu tion (Alexander, 1980) in a 1:5 dilution was added to each tube (at least 2: 1 (v/v) stain to pollen) to fi x and stain the pollen, rendering the pollen non-viable; the tubes were closed and the contents sh aken until thoroughly mixed. Subsamples were placed on wet ic e within 30 minutes of pollen collection and

maintained under those conditions until receipt at the perf orming laboratory. Pollen collected from each plant in a plot represente d a subsample, and three to five subsamples made up one pollen sample.Monsanto Company 07-CR-191U Page 509 of 544 Data collection Pollen samples were assessed for viability, diameter and general morphology. Slides were prepared by aliquoting 15 μ l of suspended pollen / stai n solution onto a slide. Pollen samples were viewed under an Olympus Provis AX70 light/fluorescence microscope with an Olympus DP70 digital co lor camera. The associated PC computer [run on Microsoft Windows 2000 Professional (© 1981-1999, Microsoft Corp.)] had microscope and camera software (DP Cont roller v1.2.1.108 and DP Manager v1.2.1.107, respectively) (© 2001-2003, Olympus Optical Co., Ltd.).

To assess pollen viability, one hundred pollen gr ains were evaluated under the 10X ocular lens (100X magnification) for each of the three to five subsam ples per plot, and the mean of each whole sample (calculated from the subsamples) was analyzed. When exposed to the staining solution, viable pollen grains stained purple due to the presence of vital cytoplasmic content, while dead pollen grains stained clear to li ght blue-green. In addition, viable pollen grains appeared round and turgid, whereas non-viable pollen grains may have appeared flaccid, depe nding on the degree of hydration.

Pollen diameter was measured using Im age-Pro Plus v4.5.1.27 (© 1993-2002, Media Cybernetics, Inc.) software to view digita l images. For a single subsample per pollen sample, pollen diameter was measured along tw o perpendicular axes for ten viable pollen grains. The mean of the 20 diameter measurements per sample was analyzed.

Pollen morphology was observed from digital imag es for one of the three to five samples for each test, control, or reference substance.

K.3. Statistical Analysis

Monsanto Statistics Technology Center perfor med the statistical analysis. The design was a split plot with four replications, wher e treatment was the whole plot and substance type was the sub-plot. SAS ® was used to compare the test substance to the control substance within each treatment for pollen vi ability and diameter, with a significance level of 5% ( α = 0.05). A reference range consis ting of minimum and maximum mean

values of the reference substances in each treatment was reported for each characteristic.

K.4. Reference

Alexander, M.P. 1980. A versatile stain fo r pollen fungi, yeast and bacteria. Stain Technology. 55:13-18.

® SAS is a registered trademark of SAS Institute, Inc.Monsanto Company 07-CR-191U Page 510 of 544 Appendix L. Materials and Methods fo r Volunteer Potential Assessment

In some crops, seed remaining in the field after harvest have the potential to over-winter and volunteer in the subsequent cropping s eason. The purpose of this study was to evaluate volunteer pote ntial of MON 87460 compared to co nventional corn. These data are used to assess pest potential, and ultimately, the environmental risk of the biotechnology-derived crop as comp ared to the conventional crop.

L.1. Study Sites

The experiment was established at Jefferson County, Iowa (RL); Gu thrie County, Iowa (BG), and Parke County, Indiana (RV). Th ese three locations provided a range of environmental and agronomic conditions repres entative of major corn growing regions where commercial production of MON 87460 is expected. The Principal Investigator at each site was familiar with corn production a nd the occurrence and identification of corn

volunteers.

L.2. Test, Control, and Reference Substances

Starting seed for the test, control and refere nce substances were produced during 2006 in Jefferson County, IA (Tables F-1 and F-2).

The viability of the starting seed was determin ed in the laboratory (BioDiagnostics Inc.) by conducting warm germination testing of each test, control, and reference starting seed lot. A sub-report that includes details of th e germination testing is included in the study file. The seed germination was ≥ 90% for all test, control, and reference lots.

In this study, emerged plants of MON 87460, the control, or references were evaluated.

L.2.1. Test Substance

The test substance was MON 87460.

L.2.2. Control Substance

The control substance was conventional co rn H1548126, which has a genetic background

similar to the test substance but did not possess the drought tolerance trait.

L.2.3. Reference Substances

The reference substances were conventional, comme rcially available co rn hybrids: C63- 78, 33H25, H8991, C60-15, H8920, and 33N29.Monsanto Company 07-CR-191U Page 511 of 544 L.3. Experimental Methods

At each site, the study areas used for volunteer potential assessment had not been used for corn production during the 2006 growing season nor were co rn seed observed to be inadvertently present.

Planting, soil, and cropping hist ory information for each site ar e presented in Table K-1. In November 2006, plots were established at each of the th ree study sites in a randomized complete block design with three replications . Each plot was 20 ft long by 5 ft wide. Alleyways between blocks were 3 to 10 ft in length. To avoid mixing of substances between adjacent plots during seed incorporation, a confined seeding area of 18 ft by 3 ft (1 ft from each edge of the plot) was es tablished. Each plot was hand-planted by uniformly scattering approximately 200 seed on the soil surface within the confined plot area. Seed were then incorporated with a disk or field cultivator.

Agronomic practices used to prepare and mainta in each study site were characteristic of each respective region. No irrigation was applied to the study areas and no plot management was required after the se ed were scattered in the plots.

Volunteer plant counts were taken after planting in Fall 2006 until soil temperatures dropped below 50°F and re-commenced in Sp ring 2007 approximately a week prior to the average local planting date for each fiel d site. Volunteer plant counts were taken approximately every two weeks thereafter until mid-June, for a total of six to seven

observations at each site.

L.4. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data co llection and summariza tion. Study personnel ensured that assessments were taken properly, data were consiste nt with expectations based on experience with the crop, and the ex periment was carefully monitored. Any unexpected observations or issues during the study that would impact the study objectives were noted by the St udy Director.

L.5. Statistical Analysis

Statistical analysis of cumu lative plant count data was to be conducted according to a randomized complete block design with three replications. However, because no volunteer plants emerged at any s ites, no analysis was warranted.Monsanto Company 07-CR-191U Page 512 of 544 Table L-1. Planting, Soil type, and Cro pping History Information for Volunteer Potential Study

Site 1 Planting

Date Planting Depth (in) Soil Series Description, Organic Matter (%), and pH 2006 Crop 2005 Crop

BG 11/16/06 1 - 2 Loam, 3-4%, 6.5 soybean soybean

RL 11/22/06 1.5 Taintor silty clay loam, 2.4%, 7.3 none soybean

RV 11/25/06 1 - 2.5 Reesville silt loam, 2.0%, 7.5 soybean corn

1 BG = Guthrie County, IA; RL =Jefferson County, IA; RV = Parke County, IN.Monsanto Company 07-CR-191U Page 513 of 544 Appendix M. Materials and Methods for Survival Outside Cultivation Assessment

Monsanto Company has developed drought tolerant corn MON 87460 that provides a yield benefit when yield is limited by water av ailability. The purpose of this study was to assess the ability of plan ts of MON 87460 to survive in unmanaged, competitive environments compared to a conventional corn control.

M.1. Study Site Descriptions

Field trials were established in 2007 at four locations to evaluate whether MON 87460 could establish and persist in unmanaged environments. The study sites listed below were located within major corn growing region s of the U.S. where seed or grain may be incidentally returned to the environment dur ing harvest, handling, or transport.

• Effingham County, Illinois (site code: IL)

• Shelby County, Missour i (site code: MO)

• York County, Nebraska (site code: NE)

• Carson County, Texas (site code: TX)

M.2. Test, Control, and Reference Substances

The starting seed of the test, control, and reference substances are summarized in Table M-1. Since seed loss is more likely dur ing harvest operations, starting seed of the F2 generation, produced by self-pollination, were used. The test and control starting seed were produced in Stark County, IL, in 2006. The reference starti ng seed were produced in Greene County, IA, Stark County, IL, or Pawnee County, KS. Within the production site, the study substances were grown under similar agronomic conditions. Plant phenotypic and agronomic characteristics of the test, control, and reference substances were evaluated in this study.

M.2.1. Test Substance

The test substance was MON 87460.

M.2.2. Control Substance

The control substance was H1548126, which has background genetics si milar to the test substance with the exception of the drought tolerance trait.

M.2.3. Reference Substances

The reference substances were commercially-a vailable corn hybrids th at did not express the drought tolerance trait.Monsanto Company 07-CR-191U Page 514 of 544 Table M-1. Test, Control, and Reference Starting Seed for Survival Outside Cultivation Study

Site

Code 1 Substance

Name Substance Type Phenotype/ Genotype

All MON 87460 Test Droughttolerant

All H1548126 Control Conventional

IL, TX DKC 61-50 Reference Conventional

NE, MO 33N29 Reference Conventional

NE 33K39 Reference Conventional

MO M-3744 Reference Conventional

NE M-3765 Reference Conventional

MO, IL, TX 33H25 Reference Conventional

IL, TX 32B33 Reference Conventional

1 Study sites: IL = Effingham County., IL; MO = Shelby County., MO; NE = York County., NE; and TX = Carson County., TX.

M.3. Experimental Methods

M.3.1. Plot Design

The experiment was established at each of th e four sites in a randomized complete block design with three repli cations. Each plot was approximately 100 ft 2 in size, consisting of four rows, approximately 10 ft. in length, with an inter-row spacing of approximately 30 inches. A target of 100 seeds per plot were to be planted; however, due to a seed packaging error, several plots were plante d with less than 100 seeds. The minimum number of seed planted in any plot was 53 at the IL site an d 76 at the TX site. Planting

occurred at the MO and NE sites before a recount could be reque sted. The Principal Investigator at the MO site, however, confirmed that he plan ted a minimum of 50 seeds. Therefore, the minimum number of seed plante d in any plot was c onservatively estimated

to be 50 based on the information received.

M.3.2. Planting and Study Area Description

Within each study area, the study substances were grown under similar conditions. The study areas consisted of unmanaged areas and no plot preparation was performed. No fertilizers, tillage, or herbicides were applied to the study area prior to or after planting. Seed in each plot was incorporated into the soil using methods appropriate to the site (e.g., no-till or drill planti ng equipment or by hand). Planting information and soil description of each study site are provided in Table M-2.Monsanto Company 07-CR-191U Page 515 of 544 The IL study area was typical of Conservati on Reserve Program (CRP) acres, with a mix of native grasses, forbs, and weeds. The ground cover was estimated to be approximately

60%. The area had last been used for corn and soybean rotation in 2003. The MO study area was in an area adjacent to a lake and consisted of a nnual grasses, broadleaf weeds,

and volunteer wheat. The ground cover was estimated to be approximately 98%. The area had last been used for soybean in 2006 and winter wheat in 2005-2006. The NE study area was adjacent to a field watered by a center pivot irrigation system and contained a mixture of weeds. The ground cover was estimated to be approximately 25%. The area was usually not used for crop production. The TX study area was a pasture of native grasses. The ground cove r was estimated to be approximately 85%.

The area had last been cultivated in 1983.

Weather data collected at each site throughout the growi ng season is presented in

Table M-3. Weather conditions at the sites during 2007 were typical for their respective regions and there were no unusual weather events.

M.3.3. Data Collection and Assessment

The characteristics evaluate d and evaluation timing are presented in Table M-4. Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data collection, summariza tion, and analysis. Study personnel assessed that measurements were ta ken properly, data were consistent with expectations based on experi ence with the crop, and the experiment was carefully monitored. Prior to analysis , the overall dataset was eval uated by the Study Director for evidence of biologically relevant changes and for possible evidence of an unexpected plant response. Any unexpected observations or issues during the study that would impact the study objectives were noted by the Study Director. Data were then subjected to statistical analysis as indicated below.

M.3.4. Statistical Analysis

Analysis of variance was conducted according to a randomized complete block design using SAS ® (Version 9.2). The level of significance was α =0.05. For each statistically analyzed characteristic, MON 87460 was compared to the contro l at each site . There was no intention to analyze the data across all s ites since the sites were selected for their varying attributes. The reference range was determined for each characteristic from the

minimum and maximum mean values of the references.

® SAS is a registered trad emark of the SAS Institute.Monsanto Company 07-CR-191U Page 516 of 544Table M-2. Study Area PlantingInformation and Soil Descriptionfor Survival Outside CultivationstudyStudySite1Planting Date(mm/dd/yy)Planting Rate(seeds/plot)2PlantingDepth (in)Plot Size (ft) Reps Soil Series; Organic Matter; pHIL 06/11/07 53-100 1.2-1.5 10 x 103 Bluford silt loam; 1.7%; 6.7MO 05/29/07 53-100 1.3 10 x 10 3 Putnam loam; 2.0%; 5.7NE 06/01/07 53-100 0.5 10 x 10 3Hastings silt loam; 3.0%; 6.6TX 06/09/07 76-100 0.5-0.8 10 x 10 3 Pullman silty clay loam; 2.9%; 6.91Study sites: IL = Effingham County., IL; MO = Shelby County., MO; NE = York County., NE; and TX = Carson County., TX.2Planting rate was documented for each plotat the IL and TX sites. Planting rate atthe MO and NE sites were estimated basedoncounts from the IL and TX sites.Monsanto Company 07-CR-191U Page 517 of 544Table M-3. Monthly Temperature and Rainfall Amounts for Survival Outside Cultivation StudyStudySite1May June JulyLow2High3Range4Rain5Low High Range Rain Low High Range RainIL 55.8 80.3 38-89 3.0 61.8 85.9 53-97 3.4 61.6 85.1 54-94 3.2MO 57.1 78.3 42-89 4.3 62.8 82.6 51-92 3.6 65.0 86.4 54-92 0.8NE 55.5 76.0 45-91 5.8 60.2 82.4 48-90 2.2 65.9 86.7 56-98 6.9TX 50.8 75.9 38-86 2.4 57.3 86.0 43-96 0.5 61.0 94.8 57-100 0.5StudySiteAugust September OctoberLow High Range Rain Low High Range Rain Low High Range RainIL 66.6 93.5 57-100 1.0 56.5 84.7 40-98 2.4 47.2 72.6 29-93 3.7MO 69.6 91.1 60-99 6.3 57.8 83.3 45-93 1.6 47.9 69.2 32-87 4.4NE 66.7 86.3 56-95 4.8 53.5 79.0 40-90 2.9 44.9 67.2 29-87 5.2TX 63.7 95.9 59-105 0.9 56.2 86.9 46-105 2.3 43.9 80.0 32-98 0.71Study sites: IL = Effingham County., IL; MO = Shelby County., MO; NE = York County., NE; and TX = Carson County., TX.2Average daily low temperature (°F)3Average daily high temperature (°F)4Range of daily high and low temperatures (°F)5Total rainfall (inches)Monsanto Company 07-CR-191U Page 518 of 544Table M-4. Characteristics Evaluated at EachSite for Survival Outside Cultivation StudyCharacteristic Timing of Evaluation Evaluation DescriptionEarly stand count V2-V4 growth stage (at least oneplant) Number of emerged plants per plotGrowth stage monitoring V2-V4 growth stage (at least one plant) andevery two to four weeks thereafterAverage growth stage of emerged corn plantsusing Iowa State University guidance(Herman, 1997)Vigor monitoring V2-V4 growth stage (at least one plant) andevery two to four weeks thereafterVigor rating on a 1-9 scale, where 1-3 = aboveaverage, 4-6 = average, 7-9 = below average,based on PI experience with cornLate vegetative plant height V10growth stage (approximately 50% ofplants)The average height of up to five plantsPollen shed interval Pollen shed Datewhen the earliest plant begins sheddingpollen and the date when the latest plant stopsshedding pollenSilking interval Silking Date when the earliest plant begins silking andthe date when the latest plant stops silkingFinal stand count Within one week of typical harvest time Number of standing plants per plotPlant height at maturity R6 growth stage (approximately 50% ofplants)The average height of up to five plantsNumber of ears produced Harvest Number of ears produced per plotNumber of seed produced Harvest Number of seed produced per plotAverage number of ears per plant Derived from other data Number of ears produced divided by the finalstand count per plotReplacement value Derived from other data Ratio of the number of seed produced to thenumber of seed plantedMonsanto Company 07-CR-191U Page 519 of 544 Appendix N. USDA Notifica tions Approved for MON 87460

Field trials of MON 87460 we re conducted in the U.S. since 2002. The protocols for these trials include field performance, agr onomics, and generation of field materials and data necessary for this petition. In addition to the phenotypic assessment data provided for MON 87460, observational data on pest a nd disease stressors were collected from these product development trials. The majority of the final reports have been submitted to the USDA. However, some final reports, mainly from the 2007 and 2008 seasons, are still in preparation. A list of trials conducted under USDA notification and the status of the final reports for these trials are provided in Table N-1.Monsanto Company 07-CR-191U Page 520 of 544 Table N-1. USDA Notificati ons Approved for MON 87460 and Status of Trials

Conducted Under these Notifications.

USDA No. Effective Date (m/d/y) Re lease State (sites) Trial Status

2002 Field Trials
02-256-06n 11/19/02 HI (3) Submitted to USDA
02-256-07n 10/13/02 PR (2) Submitted to USDA

2003 Field Trials

03-059-04n 03/30/03 CA, KS Submitted to USDA
03-218-02n 10/08/03 HI (5) Submitted to USDA
03-276-09n 11/02/03 PR (2) Submitted to USDA
03-276-10n 11/02/03 PR (2) Submitted to USDA

03-317-01r 04/07/04 CA (5),CT (2), IA (10), IL (11), KS (7), MO (1) Submitted to USDA

03-317-01r * 03/24/05 CA(7), IA (8), IL (20), KS (8) Submitted to USDA

*Amendment 1 For Permit 03-317-01r

2004 Field Trials

04-022-03n 03/25/04 CT (2), IA (2), IL (2), KS, MI (2), MN Submitted to USDA

04-036-04n 03/22/04 CT (2), IA (2), IL (2), KS, MI (2), MN, WI (2) Submitted to USDA

04-042-12n 03/12/04 CA (3), KS (7), NE Submitted to USDA
04-042-13n 03/12/04 IA (5), IL (10) Submitted to USDA

04-058-06n 03/28/04 CT Submitted to USDA
04-237-03n 09/30/04 PR (2) Submitted to USDA
04-238-01n 09/30/04 HI (4), PR (2) Submitted to USDA
04-238-03n 09/27/04 HI (3) Submitted to USDA

2005 Field Trials
05-045-07n 03/22/05 IA (6), IL (5) Submitted to USDA
05-045-08n 03/22/05 CA (6), KS (5), NE, TX Submitted to USDA
05-053-25n 03/17/05 CT (2) Submitted to USDA
05-088-01r 08/12/05 HI (3), PR Submitted to USDA
05-171-02n 08/15/05 HI (6), PR (2) Submitted to USDA
05-213-06n 09/06/05 HI (5), PR (2) Submitted to USDA

05-230-01n 10/03/05 HI (5), PR (2) Submitted to USDA

05-290-01r 02/23/06 CA(8), CT (2), IA (15), IL (27), IN (7), KS (9) Submitted to USDA

2006 Field Trials
06-039-06n 05/18/06 PR (3) Submitted to USDA
06-039-07n 05/18/06 HI (12) Submitted to USDA
06-045-23n 04/17/06 IA (7), IL (2), IN (4), KS, NE Submitted to USDA
06-052-08n 04/24/06 IL (2), KS(8), NE (3), TX Submitted to USDA
06-052-10n 03/23/06 CA (6), KS (5), NE Submitted to USDA

Table N-1 continues on next page.Monsanto Company 07-CR-191U Page 521 of 544 Table N-1 (continued). USDA Notifications Approved for MON 87460 and Status of

Trials Conducted under these Notifications.

USDA No. Effective Date (m/d/y) Re lease State (sites) Trial Status
06-054-04n 03/20/06 IL (7) Submitted to USDA
06-054-05n 04/24/06 IA (7), KS Submitted to USDA
06-054-06n 03/23/06 CA (6) Submitted to USDA
06-054-07n 03/23/06 IL (7), IN (4), NE Submitted to USDA
06/089-01r 07/01/06 HI (3), PR Submitted to USDA
06-089-05n 05/18/06 HI (5), PR (2) Submitted to USDA

06-118-01n 05/09/06 IA Submitted to USDA
06-142-103n 08/15/06 HI (14) Submitted to USDA
06-142-106n 08/15/06 PR (3) Submitted to USDA
06-153-102n 08/13/06 HI (2) Submitted to USDA
06-233-101n 09/20/06 IA (2), IN Submitted to USDA
06-272-105n 11/14/06 HI (7) Submitted to USDA
06-275-105n 11/08/06 HI (10) Submitted to USDA

06-347-107n 01/12/07 KS Cancelled
06-347-109n 01/12/07 NE (2) Cancelled

06-348-103n 01/13/07 OK Submitted to USDA
06-353-101n 01/29/07 HI (7) Submitted to USDA
06-353-102n 01/29/07 HI (10) Submitted to USDA
06-353-103n 01/18/07 IN (4) Submitted to USDA
06-354-104n 01/19/07 KS (5) Submitted to USDA
06-355-102n 01/20/07 SD (15) Submitted to USDA
06-355-103n 01/20/07 SD (10) Submitted to USDA
06-355-105n 01/20/07 NE (13) Submitted to USDA
06-355-106n 01/20/07 NE (15) Submitted to USDA
06-355-107n 01/20/07 KS (5), NE (10) Submitted to USDA

06-355-108n 01/20/07 OK Submitted to USDA

2007 Field Trials

07-084-102r 04/12/07 CA (8), CT (2), IA (19), IL (31), IN (6), KS (18) Submitted to USDA

07-010-106n 02/09/07 OK Cancelled

07-012-102n 03/18/07 NM Submitted to USDA

07-024-103n 02/23/07 TX Submitted to USDA

07-024-107n 02/23/07 KS Submitted to USDA

07-024-109n 02/23/07 NE (2) Submitted to USDA

07-024-111n 02/23/07 CO Submitted to USDA

07-028-120n 03/17/07 CO Submitted to USDA

07-033-107n 03/21/07 IL(3), KS (3), MS Submitted to USDA

07-033-108n 03/19/07 TX (2) Submitted to USDA

07-036-109n 03/21/07 KS (7), TX Submitted to USDA

07-036-110n 03/19/07 CA (8), CT (2) Submitted to USDA

07-037-120n 03/22/07 IL Submitted to USDA

07-037-121n 03/18/07 IA, KS Submitted to USDA

Table N-1 continues on next page.Monsanto Company 07-CR-191U Page 522 of 544 Table N-1 (continued). USDA Notifications Approved for MON 87460 and Status of

Trials Conducted under these Notifications.

USDA No. Effective Date (m/d/y) Re lease State (sites) Trial Status

07-040-104n 03/26/07 IA (2), IL (3), KS, NE Submitted to USDA

07-044-108n 03/22/07 CA, TX, KS (2) Submitted to USDA

07-045-108n 04/13/07 IL (2) Submitted to USDA

07-052-106n 03/23/07 IN, PA Submitted to USDA

07-052-113n 03/23/07 IL (2) Submitted to USDA

07-053-103n 03/24/07 IA, MO, OH Submitted to USDA

07-053-105n 04/03/07 IA, IL Submitted to USDA

07-054-104n 03/25/07 OK Submitted to USDA

07-054-110n 03/25/07 NE, TX Submitted to USDA

07-058-107n 03/29/07 HI (5), PR (2) Submitted to USDA

07-061-102rm 06/08/07 HI (5), PR Submitted to USDA

07-065-114n 04/06/07 HI (4) Submitted to USDA

07-065-115n 04/09/07 PR (3) Submitted to USDA

07-065-116n 04/05/07 MO Submitted to USDA

07-066-111n 04/06/07 IL (3), NE (2) Submitted to USDA

07-084-102r 04/12/07 CA (6), CT (2), IA (8), IL (21), IN (5), KS (9) Submitted to USDA

07-215-101n 09/02/07 PR (4) Submitted to USDA

07-215-102n 09/01/07 HI (6) In Process

07-243-104n 09/30/07 HI (5), PR (2) In Process

07-257-108rm 02/14/08 HI (5), PR In Process

07-292-104n 11/20/07 HI In Process

07-292-105n 11/20/07 PR In Process

07-295-101n 12/06/07 HI In Process

07-295-105n 12/07/07 PR In Process

07-295-109n 11/21/07 HI In Process

07-295-110n 12/06/07 PR In Process

07-295-113n 12/06/07 HI, PR In Process

07-295-114n 12/06/07 HI, PR In Process

07-324-101rm 04/11/08 CA (5), CT (2), IA (10), IL (13), IN (3), KS (11), MD,

OH In Process

07-351-101n 01/18/08 HI (5), PR (2) In Process

2008 Field Trials
08-016-102n 02/15/08 IL (2) In Process
08-016-104n 02/15/08 KS (11) In Process
08-016-106n 02/15/08 KS (15) In Process

08-016-113n 02/15/08 MO In Process
08-016-114n 02/15/08 NE (12) In Process

Table N-1 continues on next page.Monsanto Company 07-CR-191U Page 523 of 544 Table N-1 (continued). USDA Notifications Approved for MON 87460 and Status of

Trials Conducted under these Notifications.

USDA No. Effective Date (m/d/y) Re lease State (sites) Trial Status
08-016-115n 02/15/08 NE (9) In Process
08-016-116n 02/23/08 IN (2) In Process
08-016-117n 02/23/08 MI (2) In Process

08-016-118n 02/15/08 OH In Process

08-016-121n 02/22/08 OK In Process
08-017-103n 02/25/08 SD (12) In Process
08-017-104n 02/16/08 SD (14) In Process
08-017-105n 02/25/08 TN (2) In Process
08-017-106n 02/16/08 WI (2) In Process
08-017-107n 02/16/08 IA (15) In Process
08-017-108n 02/16/08 IL (14) In Process
08-017-110n 02/20/08 MN (8) In Process
08-017-111n 02/16/08 NE (15) In Process
08-043-127n 03/13/08 TX (2) In Process

08-056-110n 03/26/08 TX In Process
08-056-111n 03/26/08 CA (4) In Process
08-056-113n 03/26/08 KS (5) In Process

08-058-108n 03/28/08 NE In Process

08-058-110n 03/28/08 IL, IN, LA, NE In Process

08-058-112n 03/28/08 CO In Process
08-058-116n 03/28/08 KS (3), SD (3) In Process

08-058-117n 03/28/08 TX In Process

08-058-118n 03/28/08 KS In Process
08-063-105n 04/02/08 AR, CA (2) In Process

08-063-109n 04/02/08 KS In Process

08-072-102n 04/11/08 CA In Process

08-079-104n 04/18/08 TX In Process

08-080-105n 04/19/08 OK In Process

08-080-107n 04/24/08 CA In Process
08-080-110n 04/19/08 TX (2) In Process

08-084-105n 04/25/08 IA, NE In Process

08-091-108n 04/30/08 TX In Process
08-106-111rm 06/30/08 HI (5), PR In Process

08-108-103n 05/17/08 IL, MS In Process

08-113-101n 05/22/08 TX In Process
08-185-103n 08/02/08 HI (2) In Process
08-200-102n 08/17/08 HI (4), PR (2) In Process
08-242-103n 09/26/08 HI (2) In Process
08-297-105n 11/22/08 HI, PR (2) In ProcessMonsanto Company 07-CR-191U Page 524 of 544 Appendix O. Petitioner’s Environmental Assessment

This section provides a brie f summary of three key areas to be covered in an environmental assessment prepared by APHIS for MON 87460 under NEPA: (1) Alternatives, (2) the Affected Environm ent, and (3) Potential Environmental Consequences. The significance of the pot ential environmental impact of MON 87460

takes into consideration bot h the context and intensity of the proposed action of deregulation in whole for MON 87460. This as sessment provides data and analysis that appropriately addresses and evaluates potentia l elements of each as described in the implementing NEPA regulations (40 CFR Part 1508.27). MON 87460 has been the subject of numerous field trials conducte d in the U.S. under AP HIS notification since 2002. Information has been developed from these field trials, other tests and the literature to specifically assess whether the drought tolerance trait or the plant transformation process altered MON 87460 corn in any way that would impart plant pest characteristics or cause significant environm ental impacts, including cumulative impacts.

O.A. Alternatives to Consider

The action of deregulation is governed by 7 CF R 340.6 (d)(3)(i) which states that APHIS may approve the petition in whole or in part, resulting in one of three possible outcomes from Monsanto’s petition:

• No action

o MON 87460 would remain a regulated article

• Approval in part

o MON 87460 would be granted deregulated status with some restrictions
(e.g., geographic)

• Approval in whole

o MON 87460 would be granted full deregulated status

Rejection of the “no action” and “approval in part” options is dependent upon a finding of no plant pest risk for MON 87460. M ON 87460 has been thoroughly characterized and extensive information presented in Sections I through IX of this petition demonstrates that MON 87460 does not present a plant pest risk. On the basis of this analysis, Monsanto is requesting an “approva l in whole” or full deregulated status for MON 87460. Information and arguments pr esented in this section will further demonstrate that MON 87460 does not present a significant environmental impact; thus, the requirements of NEPA ar e satisfied by an Environmen tal Assessment and Finding of No Significant Impact (FONSI). A comparison of likely environmental impacts between the “no action” and “approval in whole” alternatives to be considered under 7 CFR 340.6 (d)(3)(i) is presented in Table O-1.Monsanto Company 07-CR-191U Page 525 of 544 Table O-1. Comparison of Likely En vironmental Impacts Between “No Action” and “Approval in Whole” Alternatives

Attribute/Measure No Action Alternative Approval in Whole Alternative Meets Purpose and Need Objectives No Yes

Unlikely to Pose Plant Pest Risk Satisfied through use of regulated field trials Satisfied through use of regulated field trials and safety assessment

Commercial Use Unchanged Unchanged due to well established Stewardship policies and lack of plant pest potential Management Practices

Corn Seed Production Unchanged Unchanged; seed production will be in

accordance with OECD and AOSCA

standards Cropping Practices Unchanged Unchanged due to agronomic and phenotypic equivalency to conventional

corn Specialty Corn Production Unchanged Unchanged due to well established processes developed to protect seed purity

Pesticide Use Unchanged Unchanged due to agronomic and phenotypic equivalency to conventional

corn
Human and Animal Health

Risk to Human and Animal

Health Unchanged Unchanged; supported by low oral toxicity, protein safety, and compositional equivalence Risk to Worker Safety Unchanged Unchanged since pesticide use practices will not change

Environment

Risk to Plants Unchanged Unchanged due to no increase in plant pest potential, including weediness, and low potential for gene introgression

Risk to Animals Unchanged Unchanged; supported by low oral toxicity, protein safety, and compositional equivalence Land Use Including CRP Unchanged Unchanged; no more likely to survive on marginal farmland or displace other crops in Western Dryland than conventionally bred drought tolerant corn

Water Use Unchanged Unchanged or may result in some decrease in irrigation Cumulative Impacts Unchanged Unchanged for land use, agronomic practices, specialty corn production, biodiversity, risk to threatened or endangered species and non-target organisms due to agronomic and phenotypic equivalence to conventional corn, well-established seed production practices, lack of weediness, food and feed safety; possible positive economic and environmental benefits due to yield stability under water-limiting conditions and potential for decrease in irrigationMonsanto Company 07-CR-191U Page 526 of 544 O.B. Affected Environment

Corn is an intensively cultivated row crop that is planted in almost every state in the U.S. The two largest corn producing regions are th e Midwest contributing 65% of U.S. corn production and the Great Plains contributing 25% of U.S. production. Over the last several decades breeding programs have been successful at increasing the ability of

conventional corn to withsta nd drought, resulting in hybrids that are characterized as having very good to excellent drought tolera nce. These hybrids are currently planted widely throughout the Great Plains and th e Midwest. MON 87460 would provide an additional tool for growers faced with uncer tain weather conditions throughout the Corn Belt, particularly in the Western Dr yland region of the Great Plains.

The affected environment is described in deta il in Section IX of this document. The environment includes corn, its uses as human food and animal feed, areas where corn is currently grown commercially, land where co rn may be grown given the characteristics of the trait, and animal and plant communities. The affected environment also includes related agricultural practices such as tillage, crop rotation, pesticide use, weed management, irrigation practices, and non-agri cultural lands. Finally, the affected environment includes related corn production an d marketing practices such as specialty corn production, including organic corn produc tion, seed production, and related human activities associated with marketing of corn grain.

O.C. Potential Environmental Consequences

O.C.1. Corn Production

O.C.1.1. Acreage and Areas of Corn Production

MON 87460, with the exception of reduced yiel d loss under water limited conditions, is agronomically and phenotypically equivalent to conventional corn. Therefore it is not anticipated that the introduction of MON 87460 will have any signifi cant impact on total current U.S. corn acreage or the areas in which corn is produced. Between 1996 and 2006, a period during which improved conventiona lly bred drought tolerant corn hybrids (Campos, 2006) and new biotech nology-derived corn produc ts were introduced, total corn acreage remained relatively st eady, averaging 78 M acres per year (http://www.nass.usda.gov). In 2007, the total corn acreage increased by 15% from 78 M

to 92.9 M acres. The increase was attributed to increased demand from ethanol producers (http://www.ncga.com/node/83) and strong expo rts sales. The increased corn acreage was obtained primarily by plan ting fewer acres of soybean in the Corn Belt and Great Plains (USDA-NASS, 2007).

A small portion of the increase in 2007 corn ac reage came from land that had previously

been enrolled in the Conservation Reserve Program (CRP). The CRP is a voluntary federal program offering annual rental payments over a 10-year contract period, as well as cost-share assistance, to producers esta blishing specific type s of plant cover on marginal farmland. According to the Farm Service Agency: “CRP plantings and practices offer our nation vast environmenta l benefits, including reducing soil erosion, improving surface and ground water quality, creati ng wildlife habitat, restoring wetlands, sequestering carbon, preserving soil produc tivity and reducing offsite wind erosionMonsanto Company 07-CR-191U Page 527 of 544 damage.” Of the 36 M acres enrolled in th e CRP in 2006, 21.2 M acres or 59% were

from Great Plains states with 7.9 M acres or 22% from Midwes t “corn-belt” states (USDA-FSA-CRP, 2007).

Corn prices are a significant driver of decisi ons to withdraw land from the CRP. In 2007, re-enrollment for 15.7 M eligible acres with CRP contracts due to expire dropped by 2.6 M acres to 13.1 M acres compared wit h 2006 (USDA-FSA-CRP, 2007) Corn commodity prices were at an all-time high at $4/bushel in 2007 (http://www.nass.usda.gov). Consequently, so me growers did not re new their enrollment in the CRP and instead planted their land to crops, a decision driv en by market demands and corn prices (Hart, 2006). It is highly likely much of the corn planted on these marginal farmland acres were conven tional hybrids with good drought tolerance characteristics. In this scenario, it is important to recall that MON 87460 requires the same agronomic management practices as c onventionally bred drough t tolerant corn in order to produce acceptable yiel ds. Under severe water defi cit, corn grain yield for MON 87460, as well as for conventionally bred drought tolerant corn, can be reduced to zero. Therefore MON 87460 is no more likely to impact changes in corn acreage and land use including marginal land use, than a conventional drought tolerant hybrid.

O.C.1.2. Cropping Practices

Given that, with the exception of enhan ced yield under water-limited conditions, MON 87460 is agronomically and phenotypicall y equivalent to c onventional corn and that numerous drought tolerant conventional co rn hybrids are already planted in the U.S., it is not anticipated that the introduction of MON 87460 will have any impact on current cropping practices including seed production, crop rotation, tilla ge, or disease, insect, and weed management (Section IX).

In the event this product is commercialized, it is likely that the drought tolerance trait expressed in MON 87460 will become part of breeding programs. However it is not anticipated that the availabil ity of the MON 87460 drought tolera nce trait will result in a

reduction in the selection of hybrids currently available to growers or, based on the mechanism of action and field observations, in troduction of an enhanc ed susceptibility to

insects or diseases.

Corn is extremely sensitive to soil moisture de ficits. Due to the arid climate there is a significant amount of acreage unde r irrigation in the Great Pl ains. The growing market

for biofuels and worldwide concern over f ood shortages that have caused commodity prices to increase significantly may result in changes to cropping a nd irrigation patterns in the Great Plains as growers plant more of their acres to corn. Given the above, it is foreseeable that growers in the future may evaluate whether MON 87460 and other drought tolerant hybrids can be used in drylan d production without bene fit of irrigation. While the introduction of MON 87460 may facilita te an increase in dryland productivity,

it will be the result of the market forces noted above, not to the availability of a new drought tolerant hybrid per se.Monsanto Company 07-CR-191U Page 528 of 544 O.C.1.3. Specialty Corn Production

When considering impacts to the human e nvironment, specialty and organic grower concerns with regard to the possibility of a decrease in the value of their crops due to potential gene flow should be addressed. For several decades the corn industry has created and adopted systems to maintain a nd preserve the purity of corn germplasm developed for various uses, including specialty and organic uses. To maintain the genetic purity of inbred and hybrid corn populati ons, production activities for each type are

isolated from one another and from co mmercial grain production (Westgate et al

. , 2004; Wych, 1988). Isolation is achieved thr ough various means but may include physical separation to prevent cross pollination, temporal isolation by planting at different times to stagger pollination times of different material s, detasseling, and the use of cytoplasmic male sterility. Specialty corn producers use isolation measures to attain commercially acceptable purity standards for their crops. Organic corn producers use process-based standards outlined in the USDA’s National Organic Program to ensure that there is no opportunity for gene flow from biotechnol ogy-derived corn to non-biotechnology derived corn. Consequently, the advent of biotechno logy-derived crops does not appear to have had a negative impact on specialty or orga nic crop production. Indeed, organic production has seen its fastest growth in the decade since biotechnology-derived crops came on the market. From 1995 to 2005, orga nic corn acreage increased by 300% in the U.S. During the same timeframe, the acreag e of biotechnology-deri ved corn increased from none to 43% of total U.S. acreage for all crops in 2006 (USDA-NASS 2006).

The U.S. acreage devoted to corn hybrid s with biotechnology traits has grown dramatically since the first such hybrid was pl anted in 1996. Those tr aits include insect resistance and herbicide tolerance. In 2006, approximately 61% of U. S. corn acres were planted with a hybrid that contains a biotechnology trait (James, 2006; http:// www.ers.usda.gov/Data/BiotechCrops/ExtentofA doptionTable1.htm). Despite this large acreage of biotechnology-derived corn, about 5% of the total corn production acres are devoted to production of non-bi otechnology, specialty corn hybrids, including popcorn, waxy corn, high oil corn, white corn, blue co rn, Indian corn, and high- and modifiedprotein corn hybrids (USGC, 2007).

Organic farming is a small but rapidly growin g sector of the U.S. agriculture industry with about 0.5% of all U.S. cropland and 0.5% of all U.S. pasture being certified organic in 2005 (USDA-ERS, 2007). Only a small per centage of the most widely planted U.S. field crops (0.16% of corn, 0.17% of s oybean, and 0.48% wheat) were grown under certified organic farming systems (Table O-2) . Driven by a desire to have a single,

consistent national standard that could resu lt in greater public conf idence in foods that were labeled as “organic,” Congress pa ssed The Organic Foods Production Act of 1990 (OFPA). The OFPA required USDA to estab lish a National Organic Program (NOP) to develop uniform standards and a certifica tion process for those producing and handling food products offered for sale as organically produced (USDA-ARS, 2000). The OFPA requires the NOP to be process-based, speci fically describing the NOP as “an organic certification program for producer s and handlers of agricultura l products that have beenMonsanto Company 07-CR-191U Page 529 of 544 produced using organic methods.” 1 . No claims or assumptions regarding the quality or content of organic products may be made be yond the fact that the product was produced under these standards. Impor tantly, NOP prohibits the use of biotechnology: growers selling crops as organic may not intentionally plant seed produced through biotechnology. USDA repeatedly stated in the finalized NOP that the mere presence of plant materials produced thr ough biotechnology in a crop will not jeopardize the integrity of products labeled as organic:

“We have not accepted the comments that requested adding the products of excluded methods to the definition [of excluded methods]. The emphasis and basis of these standards is on process, not product. We have specifically structured the prov isions relating to excluded methods

to refer to the use of methods. Including the products of excluded

methods in the definition would not be consistent with th is approach to organic standards as a process- based system” (USDA-ARS, 2000).

Although small, organic production has been one of the fastest growing segments of U.S. agriculture for the past decade. The U.S. had less than one million acres of certified organic farmland when Congress passed th e OFPA. By the time USDA implemented national organic standards in 2002, certified organic farm land had doubled, and doubled again between 2002 and 2005 (USDA-ERS, 2007). For example, from 1995 to 2005 the organic corn acreage increased by 300% in th e U.S. (Table O-2), while during the same time frame the biotechnology-derived corn acrea ge increased from none to 52% of total U.S. acreage for corn in 2005 (USDA-NASS, 2006).

These national statistics do not suggest that the increase in orga nic corn production and the increase in biotechnology-derived corn pr oduction are correlated, but they do indicate that the adoption of biotechnology-derived co rn did not adversely affect organic corn production. Accordingly, there is no reason to expect that Monsanto’s drought tolerant corn product, MON 87460, will have any negative impact on the production of organic corn or other specialty corn.

1 7 U.S.C. § 6503(a).Monsanto Company 07-CR-191U Page 530 of 544Table O-2. Certified Organic and Total U.S.Acreage of Selected Crops (USDA-ERS, 2007)ItemTotal certified organic (acres)U.S.Cropland2005 (acres)Certifiedorganic tototal (%)1995 1997 2000 2001 2002 2003 2004 2005Total cropland:638,500 850,1731,218,9051,302,3921,299,6321,451,6011,452,3531,723,271340,650,083 0.51%Grains--Corn32,65042,70377,91293,55196,270105,57499,111130,67281,759,000 0.16%Wheat96,100125,687181,262194,640217,611234,221214,244277,48757,229,000 0.48%Oats13,25029,74829,77133,25453,45946,07442,61646,4654,246,000 1.09%Barley17,15029,82941,90431,47834,03130,26526,62939,2713,875,000 1.01%Sorghum--3,0751,6029383,0434,1524,4536,0426,454,000 0.09%Rice8,40011,04326,87029,02222,38120,15222,17326,4283,384,000 0.78%Spelt12,3501,70412,6067,6396,9399,7196,2038,169 -- --Millet18,55012,28515,10323,36617,57526,93521,03614,175565,000 2.51%Buckwheat13,2507,61610,59914,3118,3888,0867,9606,364 -- --Rye2,9004,3657,4887,0569,64411,61610,2898,5971,433,000 0.60%Beans--Soybean47,20082,143136,071174,467126,540122,403114,239122,21772,142,000 0.17%Dry beans--4,64114,01015,0802,4309,8367,64210,5611,659,300 0.64%Dry peas & lentils5,9005,18710,1449,3627,47616,18815,89317,757571,000 3.11%Oilseeds--Flax5,8508,05325,07620,67220,48414,94035,10430,843983,000 3.14%Sunflowers14,20010,89419,34215,2957,6247,1219,7426,0872,709,000 0.22%Monsanto Company 07-CR-191U Page 531 of 544 O.C.1.4. Commercial Use

Corn is a globally traded commodity and the U.S. is the single largest exporter. The

commercial use of corn in the U.S. would not be feasible without approvals from key trading partners. While there have been occasional market disruptions in the past involving agricultural biotechnol ogy products, these have not occurred when the product has successfully completed the determinati on process in key import markets such as Canada, Mexico, Japan, (Section I.F.2) and in no instance was any market disruption due to plant death, damage, or disease from th e biotechnology-derived product. Monsanto does not intend to commercially release M ON 87460 until all key corn import markets with functioning regulatory systems have also granted approval of MON 87460. The lack of significant adverse effects on non-target organisms, a lack of increased fitness or weediness characteristics, and no effect on the health of other plants indicates that there is no apparent potential for significant impact on commercial use. MON 87460 has been shown to be compositionally and phenotypicall y equivalent to conve ntional corn and is expected to enter commerce as part of the norma l trade flow of yellow corn to be utilized in the same manner as any other yellow corn.

Monsanto employs a rigorous product stewards hip program that demonstrates respect for our customers, their markets, and the envi ronment. Our market stewardship program considers many factors to ensu re global integration and increased transparency. In keeping with past practice, MON 87460 will not be sold commercia lly without regulatory approvals from key corn import countries to ensure global compliance and the flow of

international trade. Monsanto is committed to the industry practices on seed quality and control to prevent adventitious presence of unapproved traits. Before commercializing MON 87460, a detection method will be made av ailable to grain producers, processors, and buyers. Our stewardship policy is the shared responsibility of Monsanto, our licensees, and our customers to insure that our produc ts are used properl y. Monsanto is also committed to legal and ethical obligat ions to ensure that our products and technologies are safe and environmentally responsible, and do not pose undue risks to human health or the environment during any stag e of their life cycle. As such, Monsanto has policies in place to monitor its procedur es as products are re searched, developed, designed, manufactured, marketed, and discontinued through their life cycle.

O.C.2. Health and Safety

O.C.2.1. Food and Feed Safety

Data presented in this petition demonstrate th e dietary safety of th e drought tolerance trait expressed in MON 87460. The safety asse ssment of the CSPB and NPTII proteins included extensive protein characterization demo nstrating the lack of similarity to known allergens and toxins and the long history of safe consumption of similar proteins (Section VI). In addition, data confirmed the CSPB and NPTII proteins digestibility in

vitro , and the lack of acute oral toxicity in mice. From these data, it can be concluded with reasonable certainty that the CSPB a nd NPTII proteins have no meaningful toxic potential to humans, livestock , or other farm animals.Monsanto Company 07-CR-191U Page 532 of 544 Compositional equivalence between corn impr oved through biotechnology-derived traits and conventional hybrids provides an “equal or in creased assurance of the safety of foods derived from genetically modified plants ” (OECD, 1998). Compositional analyses of forage and grain from MON 87460 were conducted to assess the levels of key nutrients, anti-nutrients, and secondary metabolites for comparison to conventional corn. These results, based on evaluation of 77 different components (9 in forage and 68 in grain) confirmed that the forage and grain deri ved from MON 87460, and the intended foods

and feeds derived from MON 87460 can be co nsidered compositionally and nutritionally equivalent to those derived from conventiona l corn with a history of safe consumption (Section VII). As such, it can be conclude d with reasonable certainty that dietary exposure to MON 87460 poses no m eaningful risk to humans, livestock, or other farm

animals.

O.C.2.2. Worker Safety

As discussed in Sections IX.D.2.7 and IX.D.2.8, since it is agronomically and phenotypically equivalent to conventiona l corn, introduction of MON 87460 is not anticipated to have any impact on the mana gement of diseases, insects and weeds. Therefore, worker safety issues related to the use of pesticides during agricultural production would not be affected.

O.C.3. Gene Movement from MON 87460 in to Sexually Compatible Relatives

In assessing potential risks associated with gene introgression from MON 87460 with its sexually compatible relatives, teosinte and ga mma grass, there are two primary concerns: the potential for gene flow and introgression and the potential impact of the introgression.

As discussed in Sections II.C and II.D.5, corn and annual teosin te are genetically compatible, wind-pollinated and hybridize when in close proximity to each other, e.g., in areas of Mexico and Guatemala. However, in the U.S., hybridization in nature is extremely unlikely because geographical distri butions of teosinte, found only as a small feral population in southern Florida, and corn do not overlap and because of differences in developmental morphology and reproduc tive timing between the two species (Section II.D.5). First genera tion corn-teosinte hybrids are ge nerally less fit for survival and dissemination, and they show significan tly reduced reproductive capacity. Therefore it is highly unlikely that gene introgression from MON 87460 into teosinte will occur or, if it did, that it would result in robust wild weed populations.

In contrast with corn and teosinte, whic h easily hybridize, it is only with extreme difficulty and special techniques that corn and gamma grass hybridize. Furthermore, the offspring of the cross show varying levels of sterility (Galinat, 1988; Mangelsdorf, 1974; Russell and Hallauer, 1980) (Sec tion II.D.5). As no cases of gene flow between corn and

its wild relatives are known to occur naturally in the U.S., th ere is no oppor tunity for an environmental impact. Therefore it is not anticipated that the introduction of MON 87460 will result in any increased potenti al for harm to the environment due to gene flow to sexually compatible wild relatives.Monsanto Company 07-CR-191U Page 533 of 544 O.C.4. Plant and Animal Communities including Threatened or Endangered Species

O.C.4.1. Plants

Modern corn cannot survive as a weed due to intense selection duri ng domestication, in

which traits often associated with weedin ess such as seed dormancy, dispersal mechanisms, or the ability to form reproduc ing populations outside of cultivation have not been selected. Conseque ntly corn is not capable of surviving without human assistance (Baker, 1965; Keeler , 1989; Galinat, 1988) and all corn hybrids, including MON 87460, have extremely low potential for w eediness. Even when individual kernels

of corn are distributed within a field or al ong transportation routes from the fields to storage or processing faciliti es, sustainable, volunteer corn populations are not found growing in fence rows, d itches, or road sides.

In comparative studies conducted between MON 87460 and a conventional control, dormancy and germination, growth and deve lopment, and reproductive characteristics were evaluated for changes that would impact plant pest potential, and in particular, plant weediness potential. No meaningful diffe rences were detected between MON 87460 and control corn (Section VIII). Therefore, the introduction of the drought tolerance trait did not unexpectedly alter the assessed characteris tics compared to the control. These data

indicate that MON 87460 exhibits no charact eristics that would im prove the ability of

this corn to survive without human interv ention, and that its cultivation should not interfere with the cultivation of other corn hybrid s or result in its unc ontrolled spread into non-agricultural environments. Thus, the re sults support a conclusion of no increased weediness potential of MON 87460 compared to conventional corn. Additionally, APHIS has proposed to amend 7 CFR Part 340 to include its noxious weed authority.

MON 87460 would not be considered a “n oxious weed” as defined by the Plant

Protection Act because the data show that it has no potential to cause direct injury or damage (physical harm) to any protected interest.

MON 87460 will be grown on agricultural acres managed by growers in a manner similar to conventionally bred drought tolerant co rn. As discussed above, the weediness potential of MON 87460 has not been altered by the presen ce of the CSPB and NPTII proteins. Therefore it is highly unlikely that MON 87460 pos es any more of a risk to threatened or endangered plant species than conventionally bred dr ought tolerant corn. Additionally the potential for gene flow from MON 87460 is limited to sexually compatible relatives (teosinte and Tripsacum (gamma grass) species. As discussed in Section IX.C.1, differences in flower ing times, geographical separation, and developmental factors result in negligible opportunity for natural crosses in the U.S.

O.C.4.2. Animals

MON 87460 is a product beneficial to agriculture with no pesticid al activity. As such all exposed organisms, including threatened or endangered species are considered to be NTOs. During the U.S. phenotypic field tria ls at multiple locations in 2006 and 2007, multiple observations for plant disease inciden ce as well as effects on several insect and arthropod species were made to assess wh ether the plant-diseas e or plant-insectMonsanto Company 07-CR-191U Page 534 of 544 interactions of MON 87460 were altere d compared to commercial corn (Section VIII.F.1). Of the more than 375 pest and non-pest arth ropod evaluations, only

two differences were observed between M ON 87460 and the control. The differences detected were either within the range of the commercial reference hybrids or were isolated to a single st udy-site location. Because of the small number of differences that were not consistently observed, they are not co nsidered to have biol ogical significance.

Out of the more than 425 dis ease stressor observations, no differences were detected

between MON 87460 and the control. Thes e results support the conclusion that

MON 87460 does not have altered environm ental interactions relative to other conventional corn, and plants that express th e CSPB protein show no apparent impact on

NTOs.

The potential for MON 87460 to pose a meaningf ul risk to threatened or endangered species was assessed in two ways. First, the likelihood of MON 87460 to be a plant pest impacting threatened or endangered spec ies was considered. As described in Section VIII, evidence is presented dem onstrating that MON 87460 has no increased weediness potential compared to conventio nal corn. Second, the possibility that threatened and endangered species were at ri sk through hazard and level of exposure was

examined. As discussed in Sections VI a nd VIII.F.1, the CSPB and NPTII proteins are unlikely to pose a significant hazard to arthro pods and to non-insect animals. Based on the nature of the proteins, their known activity, and the characteristics of the donor organisms, the CSPB and NPTII proteins have a history of safety to organisms exposed at levels found in MON 87460. In addition, co mpositional analyses of forage and grain tissues (Section VII) that might be consumed by NTOs, confirm that the levels of key

nutrients and anti-nutrients in MON 87460 are comparable to those in conventional corn, and that the forage and grain derived from MON 87460 are compositionally equivalent to conventional corn. Thus, the potential for MON 87460 to have any effect on threatened and endangered species is neglig ible, regardless of exposure.

O.C.4.3. Biodiversity

As noted above, no potentially adverse effects on non-target organisms or threatened and endangered species were det ected based on extensive char acterization of MON 87460, which included phenotypic evaluation conducte d in field studies over a broad range of environmental conditions. Furthermore, environmental interaction field studies, including evaluations of pest and non-pest arthropods and interacti on with other biotic factor testing on MON 87460 reve aled no potential to adversel y impact NTOs. As such, the potential consequences to biodiversity are the same as with conventional corn. This

conclusion is based on the hi story of safe use of the hos t plant, corn, the extensive characterization of MON 87460 compared to a conventional cont rol, including: phenotypic assessments, compositional and nutritional equivalence, and the extensive characterization and history of safe use of the expressed CSPB and NPTII proteins.

The introduced drought tolerance trait confers a selective ad vantage only under specific conditions in the field, i. e., under water-limited conditions , which are predictable and spatially limited. This selectiv e advantage is consistent w ith that shown by conventional drought tolerant corn hybrids cu rrently planted in the U.S. This trait was shown not toMonsanto Company 07-CR-191U Page 535 of 544 provide a meaningful selectiv e advantage or disadvantage th at altered the survival of MON 87460 volunteer plants in cult ivated fields (Section VIII.F .3) or in areas that were not cultivated for agricultural production (Sectio n VIII.F.4). Thus, it is concluded that the likelihood of MON 87460 spreading in the en vironment to non-agricultural lands and causing harm to ecosystem biodiversity is negligible, as corn is neither persistent nor invasive and these parameters are unaltered in MON 87460 compared to conventional corn.

O.C.5. Cumulative Impacts

O.C.5.1. Potential for Cumulative Impa cts from MON 87460 in Combined Trait Products Resulting from Conventional Breeding with Other Biotechnology-Derived

Products

Corn hybrids produced by conventional plant breeding that combine multiple biotechnology-derived traits , have been commercialized since 2000 and have a demonstrated history of safety. For biot echnology-derived products , once single events

are assessed and determined to be safe for hu man and animal consumption and safe to the environment, then combining the unrelated single events through conventional breeding should not pose any new characteristics whic h would change the safety assessment conclusions. Further, the use of conventi onal breeding to produce combined trait or combined event products would identify o ff-types and non-perfor ming germplasm during development of new inbreds and hybrids and be removed from further development. Breeders use standard testing and assessment procedures to further examine and confirm the equivalence of the combined trait products to the single event products in terms of phenotypes, agronomic characteristics, and the efficacy of the traits.

Corn hybrids with various combinations of herb icide tolerance and inse ct protection traits have been grown commercially in the U.S. since 2000 with no reported incidence of increased weediness potential. MON 87460, which provides reduc ed yield loss under water-limited conditions, will likely be combined via conventional breeding techniques with one or more of these herbicide tolerance and insect protection traits. Similarly, MON 87460 presents negligible risk for increas ed pest potential or significant impact to

the human environment or thre atened and endangered species. In comparative studies

conducted between MON 87460 and a conventiona l control, dormancy and germination, growth and development, and reproductive ch aracteristics were evaluated for changes and none were observed (Section VIII). Additi onally, abiotic stress studies for tolerance to cold, heat and salt were conducted with MON 87460 to evaluate the potential for

increased tolerance to these adverse condi tions and no differences from conventional corn were observed (Section VIII.F.2). Ther efore, cumulative impacts are not anticipated from combining MON 87460 with one or more de regulated herbicide to lerance or insect protected traits.

O.C.5.2. Potential for Cumula tive Impacts on Biodiversity, Preservation of Corn Germplasm Purity, and Specialty Corn Production

The petition has discussed, in great detail, the outcrossing poten tial of contemporary

domestic corn varieties and their wild relatives . Sections VII and V III presented data on the phenotypic, agronomic, environmental in teractions, and compositional assessment ofMonsanto Company 07-CR-191U Page 536 of 544 MON 87460. Sections VII and II.D.5 examined the potential of weediness and gene flow of MON 87460 corn. These analyses c oncluded that (1) MON 87460 exhibits no

characteristics that would cause increased weediness and is not likely to become invasive or form self-sustaining populations outside of agricultural fi elds; (2) unconfined

cultivation should not lead to increased weed iness of sexually compatible relatives (of which there are none in the U.S.); and (3 ) use of MON 87460 in breeding for improved corn hybrids will not reduce or limit the genetic diversity available to corn breeders in the future. In addition, the assessment in Sectio n VIII.F.1 concluded that it is unlikely that MON 87460 would have long-term direct or indirect effects on non-target organisms common to agricultural production areas or on threatened an d endangered species. Based on these analyses, there is negligible poten tial for adverse effect s on biodiversity from MON 87460 compared to conventional and previously approved biotechnology-derived corn already under cultivation.

Since the development of hybrid corn systems in the 1930’s, the corn industry has created and adopted systems to maintain and preser ve the purity of corn germplasm developed

for various uses includ ing food, feed, processing, biofue ls, and specialty uses such as popcorn, sweet corn, high oil corn, white corn , blue corn, and high- protein corn (Wych, 1988). These systems have been developed and implemented over several decades to maintain and meet industry standards on ge netic purity and seed quality. Biotechnologyderived corn traits have been incorporated into these systems while achieving accepted purity standards for the corn industry and taki ng into consideration the cumulative effects of biotechnology-deri ved corn products.

To maintain the genetic purity of inbr ed and hybrid corn populations, production activities for each type are isolated from one another and from commercial grain production (Westgate et al

. , 2004; Wych, 1988). Isolation is achieved through various means but may include physical separation to prevent cross pollination, temporal isolation by planting at different times to sta gger pollination times of different materials, detasseling, and the use of cytoplasmic male sterility. Additionally, production guidelines and operating proced ures are used to ensure genetic purity and quality throughout the entire production process from planting and growth of the crop, through harvesting, transport, conditioning, packagi ng, storage and sale. With these operating procedures being widely used in the corn indus try, the industry has been able to introduce new inbreds and hybrids, incl uding biotechnology-derived co rn, into the marketplace while still maintaining the desired levels of pur ity and quality of individual types. Thus, over the last 70 years, systems have been developed to mainta in the purity and quality of the diverse corn types needed to meet th e needs and desires of corn farmers and consumers. These systems have allowed th e introduction of biotec hnology-derived traits into the desired inbred lines while at the same time allowing for the maintenance of desired conventional inbred lines. Although 52% of the total U.S. corn acreage was planted to biotechnology-derived hybrids in 2005, organic and conventional corn production remains an option for growers as evidenced by the ex istence of numerous seed suppliers, a small subset of which is listed below (Table O-3). Therefore the introduction of MON 87460 is not expected to have any cumulative impact on the processes and practices that en sure the genetic purity of c onventional and or ganic seeds.Monsanto Company 07-CR-191U Page 537 of 544 Table O-3. Organic and Conventional Seed Sources Organic Corn Seed Sources 1 Conventional Corn Seed Sources

Albert Lea Seed House Garst Seed 2

Blue River Hybrids Heirloom Seed 3

Golden Grains Kruger Seed 4

Great Harvest Organics Monsanto (Asgrow/DeKalb) 5

Merit Seeds Pioneer 6

Prairie Hybrids Seeds Welter Seed and Honey Company

1 http://www.organicgrains.ncsu.edu/

2 http://www.garstseed.com/GarstClient/Products/Corn/

3 http://www.heirloomseeds.com/corn.htm

4 http://www.krugerseed.com/index.php

5 http://www.asgrowanddekalb.com/ seedresourceguide/search/seeds

6 http://www.pioneer.com/web/site/portal/

There has been a long history of preserving, protecting and enhancing corn germplasm. The introduction of biotechnology-derived corn has had little impact on genetic diversity and germplasm resources of corn, even cons idering the potential cumulative effect of multiple biotechnology-derived traits bei ng introduced to corn. Plant breeders, institutions, government and non-government agenci es take great stride s to maintain corn germplasm resources. Corn genetic divers ity is maintained through public and private plant breeding efforts, seed conservation in gene bank s, germplasm collections maintained by the International Maize and Wheat Improvement Center (CIMMYT) and other governmental agencies, on-farm conser vation and enhancement activities in Latin America, and conservation of co rn wild relatives in Mexico and Central America. These efforts maintain and preserve corn geneti c diversity necessary for improving the base genetics of corn, separate from, or in comb ination with, the breeding practices used to introduce biotechnology-der ived traits into new corn i nbreds and hybrids. Thus, the genetic diversity of corn will continue to be maintained.

The introduction and large scale cultivation of biotechnology-derived corn in the past decade did not have any negative impact on organic corn production or other specialty corn production in the U.S. In 2005, 0.16% of U.S. corn acreage was devoted to organic production. It is worthwhile to note that states with the great est percentage of organic corn production often have above-average pene tration of biotechnology -derived crops. For example, the leading organic corn-g rowing states are Iowa, Minnesota, and Wisconsin. Of these, Iowa and Minneso ta have above-average penetration of biotechnology-derived crop plantin gs (32% and 36%, respectively, of total corn plantings relative to the U.S. average of 26% in 2001) (Brookes and Barfoot, 2005).

Seed manufacturers and growers of specialty use corn, such as organic corn, sweet corn and popcorn have been using the same establis hed processes and methods (e.g., isolation, detasseling, and use of cytoplas m male sterility) as described above to ensure the genetic purity of their corn products. Organic produc ers use process-based standards outlined inMonsanto Company 07-CR-191U Page 538 of 544 the USDA’s National Organic Program to assure that products produced meet a standard set by the industry or the purch asers of the products. No cl aims or assumptions regarding the quality or content of organic products may be made beyond the fact that the product was produced under these standards. Due to well-recognized segreg ation practices, seed producers have numerous organic corn seed hybrids available to them and AOSCA has developed protocols by whic h a seed developer can pr oduce 99% “non-GMO” seed. Therefore, it is not anticipated that th e introduction of MON 87460 will have a negative impact on specialty and organic farming.

In conclusion, measures of preservation of co rn germplasm purity including temporal and spatial isolation, detasseling, and the use of cytoplasmic ma le sterility have been well

established and have a track record of eff ectiveness over many years. The adoption and large scale cultivation of biotechnology-derived corn in the past decade did not have any negative impact on biodivers ity, genetic purity of corn, or the production of specialty corn including organic corn in the U.S. There is no ev idence to suggest that the

introduction of MON 87460 corn will adversel y impact biodiversity, genetic purity of corn germplasm, or small scale specialty use corn production ev en considering the potential cumulative effect of multiple biotechn ology-derived traits. It is also concluded based on past experience and standards that the introduction of M ON 87460 will not have an impact on the production of specialty corn products, incl uding organic corn.

O.C.5.3. Potential for Cumula tive Impacts on Land Use and Agronomic Practices

The introduction and rapid adoption of biot echnology-derived corn pr oducts in the past decade have had no significant impact on cr opland acreage in the U.S. The total crop area in the U.S. has remained relatively steady for decades, as is the case for field corn acreage. From 1996 to 2006, the total annua l field corn acres fluctuated around 78 million acres (Table IX-3), while in the same time frame the adoption rate for biotechnology-derived corn pr oducts increased from zero to 61%. In 2007, a significant increase (16%) in corn acreage to 93M acr es occurred with a corr esponding increase of 73% in the adoption rate for biotechnology- derived corn products (USDA-NASS, 2007). This increase in total corn acreage resulted primarily from fewer acres of soybean planted in the Corn Belt and the Great Plains (U SDA-NASS, 2007). Farmers rotate crops and adjust planting acreages of different crops based on market demand and commodity prices. Currently U.S. and global corn ma rket demand remains relatively high for food, feed, ethanol production, a nd other industrial uses.

From the information above it is clear th at starting with firs t biotechnology-derived product, MON 810 originally cultivated in 19 97, the addition of he rbicide tolerant and coleopteran resistant events have not significantly altered the total number of acres dedicated to agriculture or corn production in the U.S The cultivation of biotechnologyderived products has substantially reduced chemical insecticid e and herbicide use and has supported the increase in no-till corn acreage re sulting in less soil erosion and less runoff of pesticides and water (Brookes and Ba rfoot, 2005; Carpenter and Gianessi, 2001; Fawcett and Towery, 2002; Carpenter et al., 2004; Sankula, 2006; NCGA, 2007).

However since MON 87460 does not contain a pesticide tra it, its introduc tion is not anticipated to provide additional benefits in terms of decreased pesticide use.Monsanto Company 07-CR-191U Page 539 of 544 Cumulative changes to seed production, cr op rotation and tillage practices, or weed, disease, and insect management practices in the Great Plains are not expected from the introduction of MON 87460. Conventionally br ed, drought tolerant hybrids have been planted in the U.S. for decades without a ny documentation of such effects. MON 87460 is not phenotypically and agronomically di fferent from conventional corn, with the exception of reduced yield loss under water- limited conditions (Section VIII). It is foreseeable that increased drought tolerance in corn from the expressed trait in MON 87460 and continued improvements in drought tolerance from conventional breeding may reduce yield loss with or withou t the added benefits of irrigation. Over time, this may result in a cumulative benef it by decreasing the amount of water used for agriculture in the Great Plains.

O.C.5.4. Potential for Cumula tive Impacts on Non-agricultural Environments and Threatened and Endangered Species

Basic to all evaluati ons of biotechnology-de rived corn’s potential for cumulative impact to the environment or threatened and endange red species is the fact that corn cannot persist as a weed. It is an annual, wind-pollinated crop which lacks sexually compatible wild relatives (including threatened or enda ngered plant species) in the U.S., except for an occasional botanical garden spec imen or small feral populations of Zea mexicana in Florida, Alabama and Maryland or Zea perennis in South Carolina (http://plants.usda.gov). Corn exhibits ex tremely limited seed dormancy, has no weedy characteristics, and volunteers ar e easily controlled. It is not capable of establishing persistent populations in unmanaged envir onments. As demonstrated previously, the presence of the CSPB protein in no way a lters the weediness potential or gene flow potential of MON 87460.

Lack of toxicity of the CSPB protein has also been demonstrated (Section VI). The safety assessment of the CSPB protein in cluded extensive prot ein characterization demonstrating the lack of similarity to known allergens and toxins and the long history of safe consumption of similar proteins. In addition, data confir med the CSPB protein digestibility in vitro , and the lack of acute oral toxicity in mice. Collectively, these data establish the safety of the CSPB protein and indicate that the CSPB protein has no meaningful toxic potential to expo sed organisms in the environment.

Compositional equivalence between corn impr oved through biotechnology-derived traits, and conventional hybrids provides an “equal or in creased assurance of the safety of foods derived from genetically modified plants ” (OECD, 1998). Compositional analyses of forage and grain from MON 87460 were conducted to assess the levels of key nutrients, anti-nutrients, and secondary metabolites for comparison to conventional corn. These results confirmed that the forage and gr ain derived from MON 87460, and the intended foods and feeds derived from MON 87460, can be considered compositionally equivalent

to those derived from conventi onal corn with a history of sa fe consumption (Section VII). As such, it can be concluded with reasonabl e certainty that environmental exposure to MON 87460 poses no meaningful risk to or ganisms in the environment including

humans.Monsanto Company 07-CR-191U Page 540 of 544 Given these attributes and th e fact that MON 87460 has a me chanism of action different from previously deregulated biotechnology-de rived products, it is hi ghly unlikely that MON 87460 when combined through traditiona l breeding methods wit h insect protected

and/or herbicide tolerant traits will ha ve a cumulative impact on threatened and endangered species or non-agricultural environments.

O.C.5.5. Potential for Cumulative Impa cts on Economic and Environmental

Interests

The introduction and adoption of biotechnology-d erived corn products in the past decade has had a negligible impact on cropland use as evidenced by the fact that from 1996 to 2006, the total annual field corn acre s fluctuated around 78 million acres ( Table IX-3 ), while during the same period, the adoption rate for biotechnology-derived corn products

increased from zero to 61%. Economics factors largely influence total crop acreage, as in 2007, when corn acreage increased to 93 m illion acres in response to increased demand

which resulted in subs tantially higher prices for corn. However in 2008, as corn prices declined, total corn acreage also fell to 86 million acres.

Biotechnology-derived products have contributed to increas ed corn yields, reduced mycotoxin levels in corn grain, enhanced simplicity and flexibility of insect pest control and weed management, reduced chemical insect icide and herbicide use, increased no-till corn acreage which resulted in less soil er osion and less runoff of pesticides and water (Koziel et al., 1993; Martin and Hyde, 2001; Ca rpenter and Gianessi, 2001; Gianessi et al., 2002; Shelton et al., 2002; Fawcett and Towe ry, 2002; Hyde et al., 2003; Carpenter et al., 2004; Sankula and Blumenthal, 2004; Sankul a et al., 2005; Sankula, 2006; Wu, 2006; NCGA, 2007).

In a recent study, economist s Brookes and Barfoot (2008) quantified the cumulative economic and environmental impacts of biotech crops grown during the past eleven years (1996-2006). The authors report that biotech cr ops have resulted in substantial global

economic and environmental benefits. Over the past 11 year s biotechnology-derived crop adoption has positively impacted th e environment by reducing greenhouse gas emissions from agriculture and reducing pest icide spraying. This technology has also contributed to higher yields for many farmers contributing to increased grower incomes. A new study issued by the National Center for Food Agricultural Policy reported an increased net return to U.S. growers of $2.6B (http://www.ncfap.org) for 2006 alone. The estimated farm income benefit to gr owers worldwide for a ll biotechnology-derived crops is $30B (Brookes and Barfoot, 2008). Additionally, there is speculation that, without wide adoption of this technology, world prices for corn and soybean could be even higher than the current prices. In 2004, the United Nations Food and Agriculture Organization noted that agricu ltural biotechnology is a comple mentary tool to traditional farming methods that can help poor farmer s and consumers and improve food security (UN-FAO, 2004).

These benefits are from biot echnology crops that were designe d to be tolerant of or

resistant to biotic environmental stressors su ch as weeds (via novel herbicide tolerances)Monsanto Company 07-CR-191U Page 541 of 544 and insects. The introduction of MON 87460 marks the first attempt to turn biotechnology towards the amelioration of ab iotic environmental stressors such as drought. Globally, drought is the most impor tant limiting abiotic f actor of corn yield (Barker et al., 2005; Campos et al., 2006). Sign ificant progress has been made to reduce corn yield loss in water-limited environmen ts through breeding and cultural practices, but there remains potential for additional improvement. Positive impacts on yield and reduced yield loss in water-lim ited environments will con tinue to provide value to producers, consumers, and the environment.

Given the above, Monsanto fully anticipates that drought tolera nt corn MON 87460, particularly when combined with other biotechnology-derived traits, will add to the positive cumulative impact of biotechnology products on the U.S. economy and the

environment.

O.D. Highly Uncertain, Unique, or Unknown Risks

None of the effects on the human environmen t previously discussed are highly uncertain or involve unique or unknown ri sks. Any impacts would be similar to those already observed for current biotechnology- derived and conventional corn.

O.E. Conclusions

MON 87460 has been shown to be no different from conventional corn in its phenotypic, environmental, and compositional characteri stics, with the ex ception of the drought tolerance trait. As to the drought tolerance tr ait in MON 87460, it is similar to that found in conventionally bred, drought tolerant corn hybr ids currently planted in the U.S., both in terms of functional characteristics and lack of potential environmental impact. Thus, MON 87460 is expected to be similar in its agronomic characteristics and environmental impact to conventional corn.

No adverse impacts to threatened or enda ngered species, biodiversity, specialty and organic farming, and non-agricultural enviro nments is envisione d since corn cannot establish and persist outs ide an agricultural envir onment and MON 87460 has been demonstrated to be phenotypically and compos itionally equivalent to conventional corn. Any potential changes in agricultural practi ces would be consiste nt with the commercial production of conventional hybrid s with good drought tolerance, thus these agricultural practices would not be significantly im pacted by the intr oduction of MON 87460.

The drought tolerance trait of MON 87460 has a mechanism of action unique from other deregulated biotechnology-derive d traits. Therefore, there are no anticipated cumulative impacts from combining MON 87460 with de regulated herbicide tolerant, insect protected, or nutritionally e nhanced biotechnology-derived pr oducts, when viewed in the context of pre-existing agricultural practices. For the same reason, cumulative impacts on biodiversity, threatened and endangered spec ies, specialty and organic corn production are also not anticipated. Because MON 87460 reduces yield loss under water-limited conditions, it is foreseeable that continue d improvements in drought tolerance for corn, including the introduction of MON 87460, may result in the cumulative benefit of decreasing water demands for irrigation in th e Great Plains. This trend is alreadyMonsanto Company 07-CR-191U Page 542 of 544 underway due to the continued improvements in conventionally bred drought tolerant corn, and the introduction of MON 87460 is not anticipated to have a significant impact.

MON 87460 has been thoroughly characterized and the extensive information presented in Sections I through X of this petition de monstrate that MON 87460 does not present a plant pest risk. On the basis of this anal ysis, the requested acti on of deregulation in whole does not present a signifi cant environmental impact and should lead to a Finding of No Significant Impact.

O.F. References

Baker, H.G. 1965. Characteristics and modes of origin of weeds. Page 147-172 in The Genetics of Colonizing Species. Baker, H. G. and G.L. Stebbins (eds.). Academic Press, New York.

Barker, T., H. Campos, M. Cooper, D. Dolan, G. Edmeades, J. Habben, J. Schlusser, D. Wright, and C. Zinselmeier. 2005. Impr oving Drought Tolerance in Maize. Pages 173-253 in Plant Breeding Reviews. Vol 25, J. Janick, (ed.) John Wiley and Sons, Inc., Hoboken, New Jersey.

Brookes, G. and P. Barfoot. 2008. GM Crops: The global socio-economic and environmental impacts 1996–2006. PG Economics, Ltd., Dorchester, UK.

Campos, H., M. Cooper, G.O. Edmeades, C. Loffler, J.R. Schussler, and M. Ibanez. 2006. Changes in drought tolerance in ma ize associated with fifty years of breeding for yield in the U.S. Corn Belt. Maydica. 51:369-381.

Carpenter, J.E., and L.P. Gianessi. 2001. Agricultural biotechnol ogy: Updated benefits

estimates. In Report published by the National Ce nter for Food and Agricultural Policy, Washington, D.C.

Carpenter, J.E., S. Sankula, C.E. Silvers, and L.P. Gianessi. 2004. Insecticidal Bacillus thuringiensis plants versus chemical insectic ides. ACS Symposium Series 866 - Agricultural Biotechnology. Chapter 3:37-51.

Fawcett, R., and D. Towery. 2002. Conser vation tillage and pl ant biotechnology: How new technologies can impr ove the environment by reducing the need to plow. Report of Conservation Technology Informa tion Center, Purdue University, West Lafayette, IN.

Galinat, W.C. 1988. The origin of corn. Pages 1-31 in Corn and Corn Improvement. 3 rd
ed., Sprague, G.F. and J.W. Dudley, (eds.). American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison,

WI.

Gianessi, L.P., C.S. Silvers, S. Sankula, a nd J.E. Carpenter. 2002. Plant biotechnology: current and potential impact for improving pest management in U.S. agriculture. an analysis of 40 case studies. National Center for Food & Agricultural Policy, Washington, D.C.

Hart, C. E. 2006. Feeding the ethanol boom : where will the corn come from? Iowa Agriculture Review. 12:3-5.Monsanto Company 07-CR-191U Page 543 of 544 Hyde, J., M.A. Martin, P.V. Preckel, L.L. Buschman, C.R. Edwards, P.E. Sloderbeck, and R.A. Higgins. 2003. The value of Bt corn in southwest Kansas: A Monte Carlo simulation approach. Journal of Agricultural and Resource Economics.

28:15-33.

James, C. 2006. Global Status of Commercialized Transgen ic Crops. ISAAA Brief No. 35. International Service for the Acqui sition of Agri-Biotech Applications, Ithaca, New York.

Keeler, K. 1989. Can genetically engineered crops become weeds? BioTechnology.

7:1134-1139.

Koziel, M.G., G.L. Beland, C. Bowman, N.B. Carozzi, R. Crenshaw, L. Crossland, J. Dawson, N. Desai, M. Hill, S. Kadwell, K. Lewis, D. Maddox, K. McPherson, M.R. Meghji, E. Merlink, R. Rhodes, G.W. Warren, M. Wright, and S.V. Evola. 1993. Field performance of elite tran sgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis . BioTechnology. 11:194-

200.

Mangelsdorf, P.C. 1974. Corn: its Origin , Evolution, and Improvement. Harvard University. Press. Cambridge, Massachusetts.

Martin, M.A., and J. Hyde. 2001. Econo mic considerations for the adoption of transgenic crops: The case of Bt corn . Journal of Nematology. 33:173-177.

NCGA. 2007. No-till Corn on the Rise, Natio nal Corn Growers Association Notes (3-26- 07). http://www.ncga.com/news/notd/2007/march/032607.asp

OECD. 1998. Report of the OECD Workshop on the Toxicological and Nutritional Testing of Novel Foods. Organization for Economic Co-operation and Development. Paris, France.

Russell, W.A. and A.R. Hallauer. 1980. Co rn. Pages 299-312 in Hybr idization of Crop Plants. Fehr, W.R. and H.H. Hadley (eds.). American Society of Agronomy, and Crop Science Society of America, Publishers, Madison, Wisconsin.

Sankula, S. 2006. Quantification of the imp acts on US agricultu re of biotechnologyderived crops planted in 2005. National Ce nter for Food and Agriculture Policy. http://www.ncfap.org/whatwedo/pdf/ 2005biotechimpacts-finalversion.pdf

Sankula, S., and E. Blumenthal. 2004. Impacts on US agriculture of biotechnologyderived crops planted in 2003: an update of eleven case studies. www.ncfap.org/whatwedo/biotech-us.php

Sankula, S., G. Marmon, and E. Blumenth al. 2005. Biotechnology-derived crops planted in 2004 – impacts on US agriculture. www.ncfap.org/whatwedo/biotech-us.php

Shelton, A.M., J.Z. Zhao, and R.T. Roush. 2002. Economic, ecologi cal, food safety, and social consequences of the deployment of Bt transgenic plants . Annual Review of Entomology. 47:845-81.

UN-FAO. 2004. The state of food and agri culture 2003-2004: Agricu ltural Biotechnology – Meeting the Needs of the Poor, Part 1. 39 pp.Monsanto Company 07-CR-191U Page 544 of 544 USDA-ARS. 2000. National Organic Program. Final Rule. 7 CFR Part 205. 65 Federal Registration. 80548.

USDA-ERS. 2007. Organic Production. United States Department of Agriculture,

Economic Research Service http://www.ers.usda.gov/Data/Organic

USDA-FSA-CRP. 2007. News Release: USDA A nnounces Results of Intentions to Reenroll and Extend CRP Contracts from Un ited States Department of Agriculture, Farm Service Agency, Conservation Reserve Program (USDA-FSA-CRP).

USDA-NAAS. 2006. USDA-NASS. 2006. Nationa l Agricultural Statistics Service, United States Department of Agriculture . Acreage Report, Cr Pr2-5 (6-06) a. Accessed June 30, 2006.

USDA-NASS. 2007. National Agricultural Statis tics Service, United States Department of Agriculture. Acreage Report, Cr Pr 2-5 (6-07) Accessed June 30, 2008.

USGC. 2007. Value Enhanced Corn Re port 2005/2006. United States Grain Council, Washington, D.C., http://www.grains.org /galleries/technica l_publications/

USGC%20Value%20Enhanced%20Cor n%20Report%202006%20%20(English). pdf

Westgate, M.E., M.E. Otegui, and F.H. Andrade. 2004. Physio logy of the Corn Plant. in Corn: Origin, History, Technology, and Production, C.W. Smith, J. Betran and E.C.A. Runge, (eds.) John Wile y and Sons, Hoboken, New Jersey.

Wu, F. 2006. Mycotoxin reduction in Bt corn: potential economic, health, and regulatory impacts. Transg enic Research. 15:277-289.

Wych, R.D. 1988. Production of hybrid s eed corn. Pages 565-608 in Corn and Corn Improvement. 3 rd ed., Sprague, G.F., and J. W. D udley, (eds.). American Society of Agronomy, Crop Science Society of America and Soil Science Society of America, Madison, WI.Monsanto Company 07-CR-191U Page 2 of 15 Release of Information

Monsanto is submitting the information in this ad dendum for review by the USDA as part of the regulatory process. By submitting this information, Monsanto does not authorize its release to any third party. In the event the USDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552 and 7 CFR Part 1, covering all or some of this information, Monsanto expects that, in advance of the release of the document(s), USDA will provide Monsanto with a copy of the material proposed to be released and th e opportunity to object to the release of any information based on appropriate legal grounds , e.g., responsiveness, confidentiality, and/or competitive concerns. Monsanto expects that no information that has been identified as CBI (confidential business information), will be prov ided to any third party. Monsanto understands that a CBI-deleted copy of this information ma y be made available to the public in a reading room and by individual request, as part of a public comment period. Except in accordance with the foregoing, Monsanto does not authorize the rel ease, publication or other distribution of this information (including website posting) wit hout Monsanto's prior notice and consent.

 2010 Monsanto Company. All Rights Reserved.

This document is protected under copyright law. This document is for use only by the regulatory authority to which this has been submitted by Monsanto Company, and only in support of actions requested by Monsanto Company. Any other use of this material, without prior written consent of Monsanto, is strictly prohibited. By submitting this document, Monsanto does not grant any party or entity any right or license to the information or intellectual property

described in this document.Monsanto Company 07-CR-191U Page 3 of 15 Addendum to USDA Petition No. 09- 055-01p for Determination of Non-regulated Status for MON 87460 June 18, 2010

Section 1 Supplementary Text on Impacts of MON 87460 on Water Relations Section I.D of Petition 09-055-01p presents data indicating that cold shock protein B (CSPB) does not alter water relations (i.e., relative water content, leaf water potential, leaf osmotic potential) compared to conventiona l corn. Subsequent st udies of soil moisture depletion and root

biomass confirm the water relations data and demonstrate that MON 87460 is not significantly

different from conventional corn in terms of soil moisture depletion over the course of the growing season or in terms of root biomass and root:shoot ratio. Taken together, these results further support the conclusion in Appe ndix O of Petition 09-055-01p that MON 87460 is expected to be similar to conventional corn with respect to its overall water utilization and potential environmental impact.

1.1. Data from Petition 09-055-01p

Table I-4 of Petition 09-055-01p (p. 49) presents re lative water conten t data for MON 87460, other CSPB-containing events and a conventiona l control grown under water-limited conditions in the greenhouse. The data show that the average relative water content of MON 87460 (75%) is numerically similar that of the control (78%) and that other corn events containing the same genetic construct as MON 87460 exhi bit a range of average relative water content values (75 – 82%) that bracket the value in the control plants.

Figure I-22 of Petition 09-055-01p (p. 69) presents total water potential data from three events containing the same construct as MON 87460 and a control grow n under water-limited

conditions in the field. The data show that the total water potent ial of corn transformation events containing the same genetic construct as M ON 87460 is not significantly different from the control across multiple time points throughout the day.

Figure I-23 of Petition 09-055-01p (p. 70) presents osmotic potential data from three events containing the same construct as MON 87460 and control grown under water-limited conditions

in the field. The data show that the osmotic potential of corn transfor mation events expressing the same genetic construct as MON 87460 is not significantly different from the control across multiple time points throughout the day.

Collectively, these data presented in Petiti on 09-055-01p support a conclusion that expression of CSPB in corn does not alter plant water relations after the imposition of water-limited

conditions.

1.2 Soil Moisture Depletion Analysis

Subsequent to the submission of Petition 09- 055-01p, data on the potential for MON 87460 to deplete soil moisture were assembled and analy zed. The two years of data collected on plants grown under well-watered and water-limited fiel d conditions demonstr ate that MON 87460 does not deplete soil moisture differently from conventi onal corn. This observation is consistent with the conclusion in Appendix O of Petition 09-0 55-01p (Petitioner’s Environmental Assessment) that MON 87460 is expected to be similar to conventional corn with respect to its agronomicAddendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 4 of 15 characteristics and environmental impact. Additi onally, these data indicate that the yield benefit observed in MON 87460 plants under water- limited conditions is not accompanied by a

withdrawal of more water from the soil than occurs with conven tional corn. Likewise, the data also demonstrate that MON 87460 is unlikely to withdraw more water from the soil than

conventional corn under conditions where soil moistu re is sufficient to pr oduce adequate yields.

Data for this analysis were collected from fi eld trials conducted in California during 2007 and

2008. The 2007 data are from the same field trial in which the yield data presented in Table I-3 (p. 48 of Petition 09-055-01p) were generated. Under well-watered conditions, yields for MON 87460 and the control were not signif icantly different (297 bushels/acre and 295 bushels/acre, respectively). Under water-limited conditions, MON 87460 exhibited a 9.6% yield advantage over the control (206 bushels/acre vs. 188 bushels/a cre) that was statistically significant (p<0.05). Similarly in 2008, yiel ds for MON 87460 and the control were not significantly different under well-watered cond itions (242 bushels/acre vs. 243 bushels/acre, respectively). Under water- limited conditions in 2008, MON 87460 exhibited a 7.6% yield advantage over the control (156 bushels/acre vs. 145 bushels/a cre) that was statistically significant (p<0.05).

Materials and methods

In the 2007 study, ten replicates each of M ON 87460 and the control were grown under waterlimited conditions from the V7 to R2 growth stag es. Five replicates each of MON 87460 and the control were grown under well-wat ered conditions. Each replicat e consisted of four six-row plots with 20.5 foot rows plante d with 50 kernels per row. Bo rder rows were grown between treatments to ensure water did not migrate from the well-watered to the water-limited plots. The plots in each irrigation treatment was planted using a separate randomized complete block design with test and control positioned adjacent one another (paired) to minimize variability.

In the 2008 study, twenty replicates each of MON 87460 and the control were grown under water-limited conditions from the V10 to V14 grow th stages. The same design was used for the well-watered treatment. Each replicate consiste d of four eight-row plot s with 20.5 foot rows planted with 50 kernels per row. Border rows were grown between treatments to ensure water did not migrate from the well-watered to the water-limited plots. Each irrigation treatment was planted using a separate randomized complete block design.

In both 2007 and 2008, capacitance soil mo isture probes (SenTek, EnviroSmart TM ) were utilized to characterize the depletion of moisture from the soil profile during the growing season. The probes have five sensors at soil depths of 20, 30, 50, 70 and 100 cm that monitor the available

soil moisture within a 10 cm radius from each sensor. Within the water-limited block the probes were installed within the plots from ten repl icates of MON87460 and ten replicates of the control. MON 87460 and the control were matched within each of the irrigation runs. Within the well-watered block, probes we re installed within five re plicates each for MON 87460 and

control.

Prior to installation of the soil moisture probes, each sensor wa s normalized against air and water to set relative readings for moisture content. Probes were installed within the plant rows – in between two adjacent corn plants at the middle of the plot. At installation, each probe was outfitted with radio telemetry devices that allow data to be transmitted wirelessly. As the moisture in the soil is depleted, the electric al output signals from the sensors decline inAddendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 5 of 15 proportion. The data are generated in scale freq uency units and converted to mm soil moisture using a soil type-specific calibration equation ob tained from the manufacturer. Soil moisture depletion was calculated by subtracting late r readings from earlier readings.

Cumulative soil moisture depletion calculations were made by adding th e daily soil moisture depletion values over the course of the growing season. Statistical analyses were made between MON 87460 and the control across the growing seas on using analysis of variance. The paired design assumes equal variance at each pair (replicat e) and standard error w ill therefore be equal across entries (test and control). The standard e rrors are calculated across all of the replicates within each of the two irrigation treatments, lead ing to a single standard error value for both the

test and control.

Results

Table 1-1 summarizes the cumulative soil mois ture depletion across th e entire 2007 growing

season under well-watered and water-limited conditions . The data demonstrate that soil moisture depletion for MON 87460 and the control was not significantly different under well watered (319.81 mm vs. 377.28 mm, respectiv ely) or water-limited conditions (272.29 mm vs. 284.19 mm, respectively).

Table 1-2 summarizes the cumulative soil mois ture depletion across th e entire 2008 growing

season under well-watered and water-limited conditions . The data demonstrate that soil moisture depletion for MON 87460 and the control was not significantly different under well-watered (193.84 mm vs. 178.15 mm, respectiv ely) or water-limited conditions (182.87 mm vs. 195.52 mm, respectively).

Collectively, these data presented show th at MON 87460 does not deplete soil moisture differently from conventional corn when grown under well-watered or water-limited conditions. They further demonstrate that the yield a dvantage of MON 87460 observed under water-limited conditions is not accompanied by a withdrawal of more water from the soil than occurs with

conventional corn.

1.3 Root Biomass Comparisons

Under both well-watered and water-limited conditi ons, key measures of root biomass do not

differ between MON 87460 and conventional corn. The results of this st udy further support data presented in Section VIII of Petition 09-055-01p demonstrati ng that MON 87460 does not differ phenotypically from conventional corn. This lack of differences also supports the conclusion that MON 87460 is unlikely to significantly alter agronomic practices including water use. Comparisons of root biomass provide an unde rstanding of whether roots from MON 87460 are likely to have extended deeper into the soil prof ile beyond the range of th e soil moisture probes

used in the field st udies described above.

Direct measures of root biomass collected under field conditions are hampered by difficulties in separating intact roots from the soil matrix. To reduce these difficulties, MON 87460 and the

conventional control were estab lished in pots in a greenhouse and grown under well-watered or

water-limited conditions. Measuremen ts were taken of gr owth stage, plant height, root and shoot biomass and water consumption. Water-limited c onditions were imposed from the V7 to VT/R1 growth stages.Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 6 of 15 Materials and methods

The test substance, MON 87460, and a conventional isogenic control were es tablished in 3-foot tall, 8-inch diameter pots fille d with Turface Field and Fairway TM , an inert, clay-based medium which provided good capillary action to distribute water evenly th roughout the pots and washed off roots easily. The study was planted in a greenhouse in a complete factorial treatment structure (2×2) in a randomized complete block design with 12 replications. The factors were irrigation treatment (well-watere d or water-limited) and plant substance type (MON 87460 test or control).

Irrigation treatments were managed according to volumetric water content (VWC). The wellwatered treatment was maintained at 0.56 VW C from planting through the duration of the experiment. The water-limited treatment maintained 0.56 VWC from planting through approximately the V7 growth stage, at which point the treatment was maintained at 0.3 – 0.4 VWC for the remainder of the experiment. Plan t fertility was maintained through the use of Hoagland’s nutrient solution for every irrigation ranging from0.5X concentration for wellwatered plants up to 2X for water-limited plants. Plants were harvested when plants in the wellwatered treatment reached the VT – R1 growth st age, 25 days after im position of drought stress

on the water-limited treatment.

Plants were evaluated within each treatment for plant height and growth stage three times duringthe experiment: prior to treatment, 13 days after treatment (DAT) when plants in the waterlimited treatment exhibited leaf rolling (an indicato r of drought stress), and at the conclusion of the experiment 25 DAT. Growth stage was dete rmined using methods described by Iowa State University (2008). Fresh and dry weights of shoot s and roots were measured at the conclusion of the experiment, root:shoot ratio, and total water used were calculated. Growth stage data were categorical and were not statistically analyzed.

Results

Table 1-3 presents the comparisons between MON 87460 and the conventional control. No statistically significant differe nces were detected between MON 87460 and the control for any endpoint, including root weight and ratios of root:shoot bioma ss. Root:shoot ratio was calculated to assess the part itioning of biomass into above- and below-ground biomass in response to drought stress. MON 87460 did not di ffer from the control in its partitioning of

biomass into roots and shoots under well-water ed or water-limited conditions. In addition, MON 87460 was not significantly different from the control in the total amount of water

consumed under well-watered or water-limited c onditions. The results of these measurements used to assess root biomass and developmen t and water use support a conclusion that

MON 87460 does not differ from conventional corn in total water use, root biomass, or the proportion of total biomass partitioned into th e roots under both well-watered and water-limited

conditions.

1.4 Summary

The data presented in this addendum to Pe tition 09-055-01p indicate that MON 87460 does not exhibit altered soil moisture depl etion patterns or altered root biomass compared to conventional corn. Across two years of field studies, MON 87460 depleted soil moisture to a similar extent as

TM Turface Field and Fairway is a register ed trademark of Profile Products LLC.Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 7 of 15 the conventional control under both well-watered and water-lim ited conditions. Within the water-limited treatments, MON 87460 exhibited stat istically significant yiel d advantages in 2007 and 2008 that exceeded the 6% target increase described in Section I of Petition 09-055-01p. Additionally, key measures of root biomass were not significantly different between

MON 87460 and conventional corn. The results of these studies further support data presented in Section VIII of Petition 09-055-01p de monstrating that MON 87460 does not differ agronomically or phenotypically from conventiona l corn and are consiste nt with the conclusion in Appendix O of Petition 09-055-01p that MON 87460 is expected to be si milar to conventional corn with respect to its wate r use characteristics and potenti al environmental impact.

References Iowa State University. 2008. How a Corn Plan t Develops, Special Report No. 48. Ames, IA.Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 8 of 15 Table 1-1. Full Season Cumulative Soil Moisture Depletion from MON 87460 and Control

Plots in a 2007 California Field Trial

Treatment Entry Cumulative Soil Moisture Depletion (mm water ± standard error) p-value

Well-watered MON 87460 319.81 ± 61.75 0.546 Control 377.28 ± 61.75

Water-limited MON 87460 272.29 ± 12.71 0.525 Control 284.19 ± 12.71

Note: No statistically significant differences ( α =0.05) were detected within treatment between MON 87460 and the control using analysis of variance.

Table 1-2. Full Season Cumulative Soil Moisture Depletion from MON 87460 and Control

Plots in a 2008 California Field Trial

Treatment Entry Cumulative Soil Moisture Depletion (mm water ± standard error) p-value

Well-watered MON 87460 193.84 ± 21.94 0.640 Control 178.15 ± 21.94

Water-limited MON 87460 182.87 ± 9.02 0.347 Control 195.52 ± 9.02

Note: No statistically significant differences ( α =0.05) were detected within treatment between MON 87460 and the control using analysis of variance.Addendum to USDA Petition No. 09-055-01pMonsanto Company 07-CR-191U Page 9 of 15Table 1-3. Plant Growth Parameters of MON 87460 and the Control Prior to Irrigation Treatment and at 13 and 25 DaysAfter Treatment (DAT)Well-Watered Water-LimitedCharacteristic MON 87460 Control MON 87460 ControlPrior toHeight (cm)104.8103.3105.3105.8treatmentGrowth stage1V7-V8V6-V8V7V7-V8Post-treatmentHeight (cm)166.2166.4133.2134.8(13 DAT)Growth stageV12-VTV12-VTV10-V12V9-V12Post-treatmentHeight (cm)198.1197.6178.9178.8(25 DAT)Growth stageVT-R2VT-R2VTVT-R1Shoot fresh weight (g)557.1537.5279.1263.2Root fresh weight (g)405.4411.9241.1234.6Shoot dry weight (g)104.5102.358.155.8Root dry weight (g)38.237.425.925.8Root:shoot ratio20.360.370.450.47Total water consumed3(L)35.034.019.619.1Note: No statistically significant differences (α=0.05) were detected within treatmentbetween MON 87460 and the control usinganalysis of variance.1Growth stage was determined using methodsdescribed by Iowa State University (2008).2Root: shoot ratio was calculated by root biomass divided by shoot biomass.3Total water consumed was calculated by subtracting the water remaining in the pot at the conclusion of the experiment from thetotalamount of water appliedthroughout the experiment.Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 10 of 15 Section 2 Supplementary Text on Impa cts of MON 87460 on Land Use As described in Section IX.D (pages 240-261), and Appendix O (Sections O.C.5.2 and O.C.5.3, pages 535-539) of Petition 09-055-01p, MON 87460 is not expected to have a significant impact on land use throughout the U.S. Information summarized in this addendum further supports this conclusion specifically for the Western Great Plains, the region where MON 87460 is expected to provide the greatest benefit. The statements in Section IX.D and Appendix O of the Petition are based on data presented in Section VIII of the Petition demonstr ating that MON 87460 does not differ agronomically or phenotypically from conventional corn and is no di fferent from conventional corn in its plant pest potential. This lack of differences combined w ith the information provided here also supports the conclusion that MON 87460 is unlikely to significan tly alter agronomic pr actices, crop rotation patterns and their impacts, or the number of acres planted with biot echnology-derived corn varieties in

the Western Great Plains.

2.1 Agronomic Practices

Agronomic practices are an important determinant of crop productivity and profitability. As described below, agronomic practices such as hybrid selection, reduc ed tillage and altered planting strategies have commonly been employed to optimize corn yiel ds in areas where water use efficiency is a primary concern. MON 87460 is expected to be in corporated into these practices similarly to conventionally bred drought tolerant varieties and is e xpected to comprise one part of a risk management strategy that emphasizes conventi onal breeding and agronomics in addition to biotechnology. For this reason, MON 87460 is not expected to have any significant impact on individual growers’ choices to implem ent their preferred agronomic practices.

Drought tolerance imparted through conventional breeding allows corn production to occur throughout areas of the Western Great Plains that typicall y experience dry conditions during portions of the growing season. Approximately 25% of the hybrids offered by DEKALB are rated as having excellent drought tolerance and are recommended for fields that regularly experi ence drought stress; an additional 50-65% of the hybrids offered for planti ng in these areas were ra ted as having very good performance in fields that regularly experien ce some drought stress (Monsanto Company, 2010). Other seed companies also breed corn for drought tolerance and provide drought tolerance ratings for their hybrids (DAS, 2010; PHII, 2010).

Reduced tillage and no-till practic es are well-known for improving wa ter management in dryland crop production and improving yield pote ntial (Croissant et al., 2008). Research conducted by the University of Nebraska demonstrated that plots managed under no-till cond itions over the long term had better soil structure, better rainfall infiltration rates and less runoff compared to conventionally tilled plots (Pryor, 2006). Accordingly, yields in the no-till plots were greater than those in conventionally tilled plots. The combination of reduced labor and increased yield also produced a corresponding increase in projec ted profits per unit area. MON 87460 is not expected to have a significant impact on the number of acr es being managed using reduced t ill or no-till pr actices because such practices are expected to comple ment the drought tolerance trait.

Plant density also impacts yield potential with op timum planting densities in dryland cropping systems determined by the amount of avai lable soil moisture at planting. Recommended planting densities for dryland corn in Nebraska, for example, range from 8,000 to 16,000 plants per acre. By altering plant density to reflect the amount of ava ilable soil moisture at planting, it is possible to maximize profits per unit area while also mitigating the risk of crop failure (Klein and Lyon, 2003). As MON 87460 provides a yield benefit compared to conventional corn when water is limiting during later growthAddendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 11 of 15 stages, its introduction is not expected to have a significant impact on plant de nsity decisions based on soil moisture at planting.

Skip row planting is a specific method for altering pl ant density and improving corn drought tolerance. As its name implies, skip row planting involves ski pping one or two rows of co rn for every one or two rows planted. The underlying idea is to prevent deve loping plants from exhaus ting soil moisture early in the season leaving a reserve of soil moisture to support repr oductive development (Klein, 2006). Accordingly, no-till management is recommende d for growers employing skip row practices. Research on skip row planting indi cates that the greatest benefit occu rs when yields obtainable through standard planting methods are less than 100 bushels/acr e (Lyon et al., 2009). For example, in dry years when standard planting me thods produced 16-21 bush els/acre, skip row planting produced 25-50 bushels/acre (Klein, 2006). Skip row planting, th erefore, can allow corn growers to maintain profitability in locations wher e soil moisture could otherwise preclude a profitable crop.

2.2 Crop Rotation

MON 87460 is not expected to significantly alter cr op rotation patterns because recent trends in crop rotations already favor corn. Farmer s rotate crops and adjust planting acreages of different crops based on market demand and commodity prices. From 1996 to 2006, the total number of field corn acres in each year was approximately 78 million acres (see Ta ble IX-1 in Petition 09-055-01p). In 2007, the number of corn acres increased significantly to 93 million acres as corn grain prices increased. This increase in total corn acreage resulted primarily from growers choosing to plant corn rather than soybean in the Corn Belt and the Great Plains (US DA NASS, 2007).

Rotation with other crops is advantageous for co rn yield. Rotation benefits corn production by allowing alternate weed, insect and disease manageme nt strategies and improving soil structure (Hicks and Thomison, 2004). Croissant et al. (2008) also advise that dryland growers avoid following a particular crop with itself to reduce problems with in sects, disease and volunteer plants. In the U.S. Corn Belt, soybean is a common ro tational crop although sorghum is also used. In the Western Great Plains, winter wheat, sorghum and soybean may be planted in rotation w ith corn. Wheat-fallow rotations have been employed to store soil moisture by eliminating the water use that would normally occur if a crop was grown during the fallow season. Recent researchhas shown, however, that wheatfallow rotations may actually reduce the amount of soil moisture available to winter wheat crops because fallow season management may require tillage to prevent we ed growth (Croissant et al., 2008). In no-till systems, the cost of herbicides to control weeds during fallow periods can exceed profits from the subsequent winter wheat crop. A ccording to Croissant et al. (2008), in order to maximize the return on any stored water, crops such as corn, sorghum or millet should be grown in

rotation with winter wheat. Rotation choices in the Western Great Plains are therefore expected to be made primarily in the context of crop profitability, water management and the need to control weeds, diseases and pests. While MON 87460 is expected to provide increased yield under water-limited conditions, its use is not expected to change the cu rrent rotational practices of Western Great Plains growers because current crop rotation trends already favor corn and crop rotation provides benefits in terms of weed, disease and pest management.

Input recommendations for various crops span broad and overlapping ranges as a result of crop type, expected yields, soil type, rainfa ll patterns, existing levels of nitrogen and phosphorous, soil organic matter and rotation history. The impact of crop rota tion choices in the Wester n Great Plains therefore will vary according to these factors. Table 2-1 summarizes recommended nitrogen and phosphorous inputs for corn, sorghum, soybean and winter wh eat, four crops commonly used in Western Great Plains rotations. The table also presents transpirati on ratios, the weight of water transpired to produce an equal weight of above ground dr y matter. Depending on the specif ic conditions of a given field,Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 12 of 15 corn and sorghum may potentially require more nitrogen and phosphor ous than soybean and winter wheat. Conversely, the amount of water needed to produce a pound of a bove-ground dry matter is lower for corn and sorghum than for soybean and winter wheat. Overall, the data in Table 2-1 illustrate the wide range of inputs that may be re quired in the Western Great Plains to produce a profitable crop. MON 87460 is not expected to ha ve a significant impact on the impact of crop rotation decisions because MON 87460 is not expected to change current rotation patterns and input requirements vary by crop and by location.

2.3 Adoption of Biotechnology-derived Corn Varieties

Appendix O of Petition 09-055-01p examines data for the U.S. as a whole and concludes that MON 87460 is not likely to alter the total number of acre s planted with biotechnology- derived corn varieties or impact the ability of organic and specialty co rn growers to maintain their current practices. Appendix O also notes that states with the greatest number of acr es planted with organic corn also have the greatest number of acres planted with biotechnology-derive d corn. Additional information presented here, specific to the Western Great Plains states, demonstrates that these states already have a significant percentage of their corn acres planted with biotechnology-deri ved crops and the conclusions for the U.S. presented in the Petition also apply to the Western Great Plains specifically. Table 2-2 presents the percent of total corn acres in states comprising Western Great Plains planted with biotechnology-derived varieties in 2009 (USDA ER S, 2009). For the Wester n Great Plains states where state-specific data are avai lable, the percentage of acres planted with biotechnology-derived corn varieties is near or above the national average ( 85%). For all other states where state-specific data are not available, 78% of corn acres were planted with biotechnology-derived varieties. The percent of total U.S. corn acres planted wi th biotechnology-derived varietie s has been increasing since the beginning of the last decade when 25% of corn acres were planted with biotechnology-derived varieties. MON 87460 is expected to be cultivated as a combin ed-trait product with herbicide

tolerance and insect resistance tr aits and is therefore the acres planted with MON 87460 are expected to be the same acres already planted w ith biotechnology-derived corn varieties.

Table 2-2 also presents the number of acres in each Western Great Plains state planted with organic corn for grain or seed. As observed for the U.S. as a whole, the states in the Western Great Plains that plant the greatest number of organic corn acres also have the highest percentages of their total corn acres planted with biotechnology-de rived varieties. For example, 143,074 acres of organic corn for grain or seed were planted in the U.S. in 2008 and the Western Great Plains state with the greatest number of organic corn acres was Nebraska with 8,991 acres (USDA NASS, 2010). Ninety-one percent of the total corn acres Nebraska are plan ted with biotechnology-derive d varieties (USDA ERS, 2009) indicating that the cultivation of biotechnol ogy-derived corn varietie s is not necessarily incompatible with the cultivation of organic corn.

2.4 Summary

This document augments the information presen ted in Petition 09-055-01p by focusing on land use considerations that are specific to the Western Great Plains, the area of the U.S. where MON 87460 is expected to provide the greatest benefit. As desc ribed in Section IX of the Petition, MON 87460 is unlikely to significantly alter agronomic practices , crop rotation patterns or the number of acres planted with biotechnology-derived co rn varieties. Growers in the We stern Great Plains have selected conventionally-bred drought tolerant hybrids, minimized tillage and al tered planting density for many years to manage soil moisture and maximize yi elds under conditions th at would otherwise be unfavorable for corn production. MON 87460 is expect ed to be incorporated into these practices similarly to conventionally bred drought tolerant varieties. Additionally, given the importance ofAddendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 13 of 15 economic factors and local condit ions in determining crop choice s, MON 87460 is not expected to have a significant impact on crop ro tation patterns or their impacts. Crop rotation provides significant benefits and decisions about which crops to plant in a given rotation are made in the context of crop profitability, water management and the need to control pests rather than the availability of a single trait. Finally, it is unl ikely that MON 87460 will significantly alte r the number of acres planted with biotechnology-derived corn varie ties in the Western Great Plains , given the current significant percentage of acres in the West ern Great Plains already planted with corn containing biotechnologyderived traits. As a result, MON 87460 is not expect ed to have a significant impact on land use within the Western Great Plains specifica lly or the U.S. as a whole.

References

Croissant, R.L., G.A. Peterson, and D.G. Westfall. 2008. Dryland cropping systems. Publication 0.544. Colorado State University Extension. www.ext.colo state.edu/pubs/crops/00516.html. Accessed May 7,

2010.

DAS. 2010. Dow AgroSciences LLC. Mycoge n Grain Corn Maturity 111-112. http://www.dowagro.com/mycogen/grain/111- 112.htm. Accessed June 10, 2010.

Davis, J.G. and D.G. Westfall, 2009. Fertilizing winter wheat. Publication 0.544. Colorado State University Extension. www.ext.colostate.edu/pubs/crops/00544.html. Accessed May 7, 2010.

Ferguson, R.B., C.A. Shapiro, A.R. Dobermann, C.S. Wortmann. 2006. Fertilizer recommendations for soybean. Publication G859. University of Nebraska – Lincol n Extension, Institute of Agriculture and Natural Resources. http://www.ianrpubs.unl.edu/ sendIt/g859.pdf. Access ed May 7, 2010.

Hicks, D.R., and P.R. Thomison. 2004. Corn Management. in Corn: Origin, History, Technology, and Production, C.W. Smith, J. Betran and E.C.A. Runge , (eds.) John Wiley and Sons, Hoboken, New Jersey.

Klein, R.N. 2006. Skip row corn for improved drought tolerance in rain fed corn. Publication EC196-12. University of Nebraska – Lincoln Extension, In stitute of Agriculture and Natural Resources. http://www.ianrpubs.unl.edu/sendIt/ ec196.pdf. Accessed May 7, 2010.

Klein R.N. and D.J. Lyon. 2003. Recommended seeding rates and hybrid selection for rainfed (dryland) corn in Nebraska. Publication G03-1528-A. University of Nebr aska – Lincoln Extension, Institute of Agriculture

and Natural Resources.

Lyon, D. J., Pavlista, A. D., Hergert, G. W., Klein, R. N., Shapiro C. A., Knezevic, S., Mason, S. C., Nelson, L. A., Baltensperger, D. D., Elmore, R. W., Vigil, M. F., Schlegel, A.J., Olson, B. L., and Aiken, R. M. 2009. Skip-row planting patterns stabilize corn grain yields in the central Great Plains. Crop Management. http://www.agronext.iastate.edu/corn/contact/roger_ elmore/docs/skip.pdf. Accessed May 25, 2009.

Monsanto Company. 2010. Asgrow and DEKALB seed resource guide. http://www.asgrowanddekalb.com/seedresourceguide /search/seeds. Accessed May 7, 2010.

PHII. 2010. Pioneer Hi-Bred International. Character izing Pioneer® brand Hybrids for Drought Tolerance. http://www.pioneer.com/web/site/portal/menuitem. b98005ee4490c1a5d6c1f492d100 93a0/. Accessed June 9, 2010.

Pryor, R. 2006. No-till farming in dryland cropping syst ems. Publication EC196-2. University of Nebraska – Lincoln Extension, Institute of Ag riculture and Natural Resources. http://www.ianrpubs.unl.edu/sendIt/ ec196.pdf. Accessed May 7, 2010.

Shapiro, C.A., R.B. Ferguson, G.W. Hergert, C.S. Wort man, D.T. Waters. 2008. Fertilizer suggestions for corn. Publication EC117. University of Nebraska – Lincol n Extension, Institute of Agriculture and Natural Resources. http://www.ianrpubs.unl.edu/se ndIt/ec117.pdf. Accessed May 7, 2010.

USDA ERS, 2009. Adoption of genetically engineered crops in the U.S.: corn varieties. http://www.ers.usda.gov/Data/BiotechCrops/Extento fAdoptionTable1.htm. Accessed May 7, 2010.Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 14 of 15 USDA NASS, 2007. Online databases for corn and so y at the United States Department of Agriculture, National Agricultural Statistics Service (USDA NASS). http://www.nass.usda.gov Accessed June 30, 2008.

USDA NASS, 2010. 2008 Organic Production Survey. Un ited States Department of Agriculture, National Agricultural Statistics Service (USDA NASS). http://www.agcensus.usda.gov/Publications/2007/Online _Highlights/Organics/index.asp. Accessed June 9,

2010.

Wortmann, C.S., A.R. Dobermann, R.B. Ferguson, G.W. Hergert, C.A. Shapiro, D. Tarlakson. 2006. Fertilizer Suggestions for Grain Sorghum. Publication G1669. Univ ersity of Nebraska – Lincoln Extension, Institute of Agriculture and Natural Resources. http://www. ianrpubs.unl.edu/sendIt/g1669.pdf. Accessed May 7,

2010.Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 15 of 15 Table 2-1. Inputs for corn, sorghum, soybea n and winter wheat under dryland conditions

Crop Nitrogen (lb N/acre) Phosphorous (lb P 2 O 5 /acre) Transpiration ratio 1,2

Corn 3 0 – 280 0 – 80 349

Sorghum 4 0 – 200 0 – 80 304

Soybean 5 0 – 100 0 – 65 646

Winter wheat 6 0 – 75 0 – 40 528

1 Pounds of water transpired/pound above-ground dry matter produced

2 Croissant et al., 2008

3 Shapiro et al., 2008

4 Wortmann et al., 2006

5 Ferguson et al., 2006

6 Davis and Westfall, 2009

Table 2-2. Percent of total corn acres plante d with biotechnology-derived varieties and the number of acres planted with organic corn in st ates comprising the U.S. Western Great Plains

State Percent of total acres within eachstate planted with biotechnologyderived varieties 1 Acres of organic corn planted for grain or seed in each state 2

Colorado 78 3 1,117

Kansas 91 3,746

Montana 78 3 NR

Nebraska 91 8,991

New Mexico 78 3 NR

North Dakota 93 1,189

Oklahoma 78 3 NA

South Dakota 96 2,694

Texas 84 2,985

Wyoming 78 3 331

1 Data are for 2009 and were obtained from USDA ERS, 2009.

2 Data are for 2008 and were obtai ned from USDA NASS, 2010, Table 26. The total reported for the U.S. was 143,074 acres of organic co rn planted for grain or seed.

3 Values not available for these st ates. The value presented in th e table (78%) is the aggregate percentage for all other states included in USDA’s corn estimating program. The average for the U.S.

as a whole is 85%.

NR – Values not reported for Montana and New Mexico in USDA NASS, 2010 to avoid disclosing

data for individual farms.

NA – No data provided for Oklahoma in USDA NASS, 2010.