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 advance 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 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 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, publication or other distribution 

of this information (including website posting) 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 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 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 responsibility 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 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, thereby allowing unrestricted introduction of the 

article. 

Monsanto Company is submitting this request to APHIS for a determination of nonregulated status for the new biotechnology-derived corn (Zea mays L.) product, 

MON 87460, any progeny derived from crosses between MON 87460 and conventional 

corn, and any progeny derived from crosses of MON 87460 with other biotechnologyderived corn which has been granted non-regulated status 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 source in the developed world. These demands 

are exceeding production, leading to diminished grain reserves worldwide. In addition, 

climate change may have variable impacts on crop yields, potentially creating further 

supply disruptions. The combination of these 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 and agronomic practices have 

made contributions to increasing corn drought tolerance and yield potential. Nearly all 

conventional corn hybrids currently on the market have been bred to exhibit some degree 

of drought tolerance. Biotechnology provides a significant new tool that can be used in 

combination with conventional breeding and agronomic practices to increase corn yield 

stability under water-limited conditions. 

Monsanto has developed MON 87460 that reduces yield loss under water-limited 

conditions compared to conventional corn. 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 water-limited conditions, particularly 

during flowering and grainfill periods when corn yield potential is most sensitive to stress 
Monsanto Company 07-CR-191U Page 5 of 544 

by disrupting kernel development. Under severe 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 stresses by binding cellular RNA and unfolding nontranslatable secondary structures affecting RNA stability and translation. As in bacteria, 

the CSPB protein in MON 87460 binds RNA and appears to help maintain plant cellular 

functions. Data suggest that MON 87460 reduces yield loss, primarily through increased 

kernel number per ear, under water-limited conditions by minimizing the effect of water 

limitation on photosynthesis, stomatal conductance, 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, embryo loss, and/or reduced grain filling in 

viable kernels. Studies on conventional germplasm with enhanced drought tolerance 

show that yield improvements are attained through improvements in all of these 

endpoints. Therefore, the enhanced yield 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 response 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 potential 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 addition, 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 variability 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 through conventional breeding. 

Field studies were established using three different water management regimes: (1) wellwatered, (2) both well-watered and water-limited treatments established in the same field, 

or (3) water managed according to typical or standard agronomic practices, which 

included typical amounts of supplemental irrigation at relevant 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 benefit. Finally, field studies managed according 

to typical agronomic practices allowed an evaluation of MON 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 levels of the inserted proteins in several 
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tissue types, (2) compositional analysis of forage and grain, (3) phenotypic and 

agronomic characteristics at several plant development stages, and (4) environmental 

interactions. The variation 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 from these field studies are relevant to the 

plant pest potential assessment 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-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. In the case of MON 87460, the data 

and analysis presented in this petition support the conclusion that MON 87460 is unlikely 

to be a plant pest. 

Data and Information Presented to Assess 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 potential 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 inserted 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 within the corn genome. The third is a 

detailed biochemical characterization of the CSPB and NPTII proteins produced in 

MON 87460, which shows very low levels of these proteins and demonstrates that the 

proteins have a history of safe use. The fourth line of evidence 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 and nutritional assessment which confirms that 

MON 87460 forage and grain are not compositionally different from conventional corn. 

The sixth line of evidence is the extensive evaluation of the MON 87460 phenotypic and 

agronomic characteristics and environmental interactions, which demonstrates that 

MON 87460 poses no increased plant pest potential, including weediness potential, and 

no adverse environmental impact compared to conventional corn. The seventh is an 

assessment on the potential impact on non-target organisms (NTO) and threatened or 

endangered species, which concludes that MON 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 confirming that the introduction of MON 87460 is no 

more likely to have an impact on land use, cultivation practices, or the management of 

weeds, diseases, and insects than the use of conventionally bred drought tolerant corn. 

Plant Pest Potential of Modern Corn

In the U.S., corn is not listed as a weed in the major weed references, 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 
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purposes during its evolution as a crop. During domestication of corn, traits often 

associated with weediness such as seed dormancy, 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 processing facilities, sustainable 

volunteer corn populations are not found growing in fence rows, ditches, and road sides. 

Although corn seed can overwinter and emerge as volunteer plants in rotational crops, the 

populations do not persist, and agronomic management 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 single transfer DNA (T-DNA). The T-DNA 

contains two expression cassettes. The first expression cassette produces CSPB and the 

second expression cassette produces NPTII, a selectable 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 present at a single locus 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 provided the complete DNA sequence of the 

insert and confirmed the organization of the elements within the insert. 

The stability of the integrated DNA is demonstrated by the fact that the Southern blot 

fingerprint of MON 87460 was maintained for seven generations tested in the breeding 

history. Furthermore, these generations have been shown not to contain any backbone 

sequence from plasmid PV-ZMAP595. The stability was further confirmed by the fact 

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

segregation. 

Characterization of the CSPB and NPTII Proteins

The expression levels of CSPB and NPTII proteins were determined in MON 87460 

tissues produced during two growing seasons from 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 NPTII protein levels. The results demonstrate 

that both CSPB and NPTII proteins were expressed in all tissues collected, including leaf, 

root, forage, silk, pollen, grain, and stover, the expression 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 forage, 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 in 
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stover. In tissues harvested throughout the growing season, mean CSPB protein levels 

(μg/g dwt) across all test sites 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 over 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 leaves 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 test sites under well-watered 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 leaves of plants at V2-V4 stage, 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 season, mean CSPB protein levels (μg/g dwt) 

across all test sites under well-watered and water-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-watered 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 level declined over the growing season, and there were no obvious 

differences in expression level in tissues collected from plants grown under well-watered 

or water-limited conditions. The level of NPTII 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 collected from plants grown under 

well-watered or water-limited conditions. 

Detailed safety assessments of CSPB and NPTII and their respective donor organisms 

establish that these proteins are safe for human consumption. CSPB has a history of safe 

consumption and NPTII is present in several biotechnology-derived crops that have 

undergone previous safety assessments. The history 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 history of safe consumption. Proteins containing cold shock 

domains are ubiquitous in nature, being present in many plants and common bacteria 

including species that are normally present in gastrointestinal 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 gastrointestinal 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 bacterial cultures. In addition, Bacillus, Lactobacillus, 

and Lactococcus species containing CSPs are involved in many food fermentation 

processes of milk, meats, cereals, and vegetables. 
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Allergenicity and Toxicity Potential of the CSPB and NPTII Proteins

CSPB protein does not share any amino acid sequence similarities with known allergens, 

gliadins, glutenins, or protein toxins which have adverse effects on mammals. This has 

been shown by extensive assessments with bioinformatic tools, such as FASTA sequence 

alignment search and an eight-amino acid sliding window search. Digestive fate 

experiments conducted with the CSPB protein demonstrate 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 small transiently stable CSPB protein 

fragment was very quickly degraded during short exposure to simulated intestinal fluid 

(SIF). Rapid digestion of the 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 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. Acute 

oral toxicity studies with mice demonstrate that CSPB protein is not acutely toxic and 

does not cause any adverse 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 establishes 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 exposure in animals will also be low with chickens, swine, 

and dairy cows consuming only nanogram quantities of each protein per kilogram of 

body weight. Taken together, these data indicate that food and feed derived from 

MON 87460, which contain the CSPB protein, are safe for consumption. 

The safety of NPTII has been extensively evaluated through several lines of experimental 

evidence, and several products containing NPTII have been approved by regulatory 

agencies on a global basis. NPTII 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). 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) affirmed its conclusion that the presence of 

nptII does not pose a threat to human health or the environment. Extensive bioinformatic 

assessments with tools such as FASTA sequence alignment search and an eight-amino 

acid sliding window search demonstrate that NPTII does not share any amino acid 

sequence similarities with known allergens, gliadins, glutenins, or protein toxins which 

have adverse effects on mammals. Numerous studies have suggested that the presence of 

this antibiotic-resistance gene in any crop or crop products will have no impact on food 

safety. Studies using purified NPTII protein revealed that NPTII degrades rapidly in 

simulated gastric and intestinal fluids suggesting that the protein is unlikely to cause an 

allergic response. Acute oral toxicity studies with mice demonstrate that NPTII is not 

acutely toxic and does not cause any adverse effects even at the highest dose levels 
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tested, 5,000 mg/kg body weight. A dietary safety assessment for MON 87460 based on 

the acute toxicity data and a corn product dietary consumption pattern established that the 

MOEs are very high for the overall U.S. population and for children aged 1-6 years old, 

an age group with the highest corn consumption on a body weight basis. NPTII is 

ubiquitous in E. coli, and, therefore, is normally present within the human gastrointestinal 

tract. Moreover, USDA previously evaluated the safety of NPTII in other biotechnologyderived products, including corn. Similar to these 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 reported that the amount of free 

kanamycin accumulating in soils, through the action of microorganisms or animal feces, 

is restricted by absorption to soil components so that no direct selection pressure for 

kanamycin resistant plants can occur. Also, enhanced physiological 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 NPTII 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 corn. The composition of MON 87460 was determined from 

forage and grain tissues produced during two growing seasons from multiple field sites in 

the major corn production regions of the 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 breeding 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. 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 included well-watered and water-limited treatments 

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

data there were no consistent differences between MON 87460 and the control. Within 

the U.S. 2006 combined-site analysis, there were no significant differences (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 test and the control. The 27 detected 

differences were not consistent across sites, 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 hybrids 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, there were no significant differences (p>0.05) 

for 59 (96.7%) of 61 comparisons. Furthermore, results from well-watered plots in the 

Chile study show that there were no significant differences (p>0.05) for 230 (94.3%) of 

the 244 total comparisons made between MON 87460 and the control. The 14 detected 

differences were not consistent across sites, were small in magnitude, and the mean 

component values of the test and control substances were within the 99% tolerance 
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interval established from the commercial reference hybrids that were produced at the 

same time and field sites as MON 87460 and the 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. Furthermore, 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 were not consistent across sites, were small in 

magnitude and the mean component values of the test and control substances 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, support the conclusion that MON 87460 is not different from 

conventional corn when grown under a broad range of environmental conditions. 

Phenotypic and Agronomic Characteristics and Environmental Interactions

The plant characterization and environmental 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, greenhouse, 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 assessments included evaluations among 14 plant 

growth and development characteristics, five seed germination parameters, two pollen 

characteristics, observations for plant-insect and plant-disease interactions, abiotic 

stressors, volunteer potential, and survival outside cultivation. Each of the measured 

characteristics is useful to assess familiarity and plant pest potential of MON 87460 

compared to conventional corn. In addition, certain characteristics can be used to assess 

weediness potential, including seed germination and dormancy (hard seed), pre-harvest 

seed loss characteristics (lodging and ear drop), and the potential to volunteer in 

cultivated areas or survive outside cultivation. 

The phenotypic field studies were conducted 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 practices 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 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 
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the control (5.6 vs. 1.5, respectively). For 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-limited 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 water-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 treatments (6.3 vs. 8.3), respectively. For 

the typical agronomic practices regime, one study 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 phenotypic data support the conclusion that MON 87460 

possesses no characteristics that would confer increased plant pest potential, including 

weediness potential, or result in adverse environmental impacts compared to 

conventional 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 potential 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 conclusion 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 Threatened or Endangered Species

The environmental assessment of MON 87460 demonstrates 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 familiarity 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 pest 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 conventional 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 proximity to each other, e.g., in areas of 
Monsanto 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 flowering time, geographical separation, and development factors make 

natural crosses in the U.S. highly unlikely. In contrast with corn and teosinte which 

easily hybridize under certain conditions, special techniques are required to hybridize 

corn and Tripsacum and the open literature indicates that the offspring of the cross show 

varying levels of sterility. 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 unlikely there would be any impact 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 more likely to impact land use, cultivation practices, or 

the management of weeds, diseases, and insects 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, disease, and insect management practices are not expected 

from the introduction of MON 87460. Conventionally 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 water-limited conditions, it is foreseeable 

that continued 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 the Great Plains. This trend 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, primarily 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 environmental impact. Therefore, Monsanto Company 

requests a determination from APHIS that 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 Determination 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 the 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 Environmental 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 ...................... 135
Monsanto Company 07-CR-191U Page 15 of 544 

V.F. Inheritance of the Genetic Insert in MON 87460 ............................................. 135

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

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

VI. Characterization of the Introduced 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 Assessment 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 .......... 151

VII.B. Chile 2006/2007 Composition Study under Well-Watered and WaterLimited Conditions ................................................................................. 159

VII.C. Additional Secondary Metabolites 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 Environmental 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 Phenotypic, 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 Agronomic 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...................................................................271
Monsanto Company 07-CR-191U Page 16 of 544 

Appendix A. Materials and Methods Used for Molecular Analyses of 

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

Appendix B. Materials, Methods, and Results for Characterization of CSPB 

Protein ......................................................................................................................276

Appendix C. Materials, Methods, and Results for Characterization of NPTII 

Protein ......................................................................................................................294

Appendix D. Materials and Methods for Expression Levels of CSPB and NPTII 

Proteins ....................................................................................................................300

Appendix E. Materials and Methods for Forage and Grain Compositional 

Analysis ....................................................................................................................310

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 Data 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, Methods and Individual Site Results for Seed Dormancy 

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

Appendix K. Materials and Methods for Pollen Morphology and Viability 

Analysis ....................................................................................................................508

Appendix L. Materials and Methods for Volunteer Potential Assessment ..............510

Appendix M. Materials and Methods for Survival Outside Cultivation 

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

Appendix N. USDA Notifications Approved for MON 87460 ...................................519

Appendix O. Petitioner’s Environmental Assessment ................................................524

 
Monsanto 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 Physiology Data from a Water-Limited Kansas 

Field Trial in 2003 ....................................................................................... 47

Table I-3. Yield Component Data from a Well-Watered and Water-Limited 

California Field Trial in 2007 ...................................................................... 48

Table I-4. Average Relative Water Content 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 Elements in Plasmid Vector PV-ZMAP595 ............ 100

Table V-1. Summary of Genetic Elements in MON 87460 ........................................... 108

Table V-2. Summary Chart of the Expected 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 Control 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 Control 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 ................................................................................... 171
Monsanto 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 Chile 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 Studies Established under Three Different 

Water Management Regimes for Phenotypic Evaluation of 

MON 87460 during 2006 and 2007 .......................................................... 189

Table VIII-3. Site Inclusion 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: Phenotypic 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 and Water-Limited Treatments .............. 195

Table VIII-7. Chile 2006/2007: Phenotypic 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 and Water-Limited Treatments .............. 196

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 Established with 

Well-Watered and Water-Limited Treatments .......................................... 199

Table VIII-9. U.S. 2007 Study-1: Phenotypic 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: 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 Established with 

Well-Watered and Water-Limited Treatments .......................................... 201

Table VIII-11. U.S. 2007 Study-2: Phenotypic 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 Agronomic Comparison of MON 87460 to the 

Control in the Combined-Site Analysis from a 2006 U.S. Field 

Study Conducted under Typical Agronomic Conditions .......................... 204

Table VIII-13. Seed Germination and Dormancy Parameters Evaluated ...................... 207
Monsanto Company 07-CR-191U Page 19 of 544 

Table VIII-14. Germination of MON 87460 and a Conventional 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 Production in the U.S. (1996 – 2008) ...................................... 242

Table IX-2. Field Corn Production by Regions and States in the U.S. (2008) .............. 243

Table IX-3. Comparison of Field Corn Production 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 Corn 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 Sequence 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 Methods 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 Ranges and Limits of Quantitation .......................... 315

Table E-4. Unit Conversions .......................................................................................... 320
Monsanto Company 07-CR-191U Page 20 of 544 

Table E-5. Comparison of Proximates, Fiber, and Mineral Content in Forage 

from MON 87460 and Conventional Control for Combined Sites in 

the U.S. during 2006 under Typical Agronomic Conditions .................... 328

Table E-6. Comparison of the Proximates 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 Combined 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-nutrient 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, Fiber, and Mineral Content in Forage 

from MON 87460 and Conventional Control for Combined Sites in 

Chile during 2006/2007 under Well-Watered Conditions ........................ 337

Table E-13. Comparison of the Proximates 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 Combined 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 Combined 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-nutrient and Secondary Metabolite Content 

in Grain from MON 87460 and Conventional Control for Combined 

Sites in Chile during 2006/2007 under Well-Watered Conditions ........... 345

Table E-19. Comparison of Proximates, Fiber, and Mineral Content in Forage 

from MON 87460 and Conventional Control for Combined Sites in 

Chile during 2006/2007 under Water-Limited Conditions ....................... 346
Monsanto Company 07-CR-191U Page 21 of 544 

Table E-20. Comparison of the Proximates 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 Combined 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 Combined 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-nutrient and Secondary Metabolite Content 

in Grain from MON 87460 and Conventional Control for Combined 

Sites in Chile during 2006/2007 under Water-Limited Conditions .......... 354

Table E-26. Comparison of the Additional Secondary Metabolite Composition of 

Forage 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 Additional Secondary Metabolite Composition of 

Grain from MON 87460 and Conventional Control for CombinedSites from the 2006/2007 Chile Production Conducted under WellWatered Conditions ................................................................................... 356

Table E-28. Comparison of the Additional Secondary Metabolite Composition of 

Forage from MON 87460 and Conventional Control for CombinedSites from the 2006/2007 Chile Production Conducted under WaterLimited Conditions .................................................................................... 357

Table E-29. Comparison of the Additional Secondary Metabolite Composition of 

Grain from MON 87460 and Conventional Control for CombinedSites from the 2006/2007 Chile Production 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 Temperature and Monthly Accumulated 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, Fiber, and Mineral Content in Forage 

from MON 87460 and Conventional Control from the QUI Site in 

Chile during 2006/2007 under Well-Watered Conditions ........................ 365
Monsanto Company 07-CR-191U Page 22 of 544 

Table E-34. Comparison of the Proximates 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-nutrient and Secondary Metabolite Content 

in Grain from MON 87460 and Conventional Control from the QUI 

Site in Chile during 2006/2007 under Well-Watered Conditions ............. 373

Table E-40. Comparison of Proximates, Fiber, and Mineral Content in Forage 

from MON 87460 and Conventional Control from the QUI Site in 

Chile during 2006/2007 under Water-Limited Conditions ....................... 374

Table E-41. Comparison of the Proximates 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-nutrient and Secondary Metabolite Content 

in Grain from MON 87460 and Conventional 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 Additional Secondary Metabolite Content in 

Forage from MON 87460 and Conventional Control from the QUI 

Site in Chile during 2006/2007 under Well-Watered Conditions ............. 384
Monsanto Company 07-CR-191U Page 23 of 544 

Table E-49. Comparison of the Additional Secondary Metabolite Content in 

Grain from MON 87460 and Conventional Control from the QUI 

Site in Chile during 2006/2007 under Well-Watered Conditions ............. 385

Table E-50. Comparison of the Additional 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 Additional Secondary Metabolite Content in 

Grain from MON 87460 and Conventional Control from the QUI 

Site in Chile during 2006/2007 under Water-Limited Conditions ............ 387

Table F-1. Field Phenotypic Evaluation 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 Reference Corns used in the 2006/2007 Chilean 

Field Study Established with Well-Watered and Water-Limited 

Treatments ................................................................................................. 397

Table F-5. Study-1, Test, Control, 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, Control, 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, Planting Information, Soil Description, and 

Cropping History in a 2006 Field Study under Well-Watered 

Conditions ................................................................................................. 400

Table F-9. Test Site Locations, Planting Information, Soil Description, and 

Cropping History in 2007 U.S. Field Studies under Well-Watered 

Conditions ................................................................................................. 401

Table F-10. Test Site Locations, Planting Information, Soil Description, and 

Cropping History in a 2006/2007 Chilean 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, Planting Information, Soil Description, and 

Cropping History in a 2006 U.S. Field Study Established with Water 

Managed According to Local Agronomic Practices ................................. 404

Table F-14. Phenotypic Comparison of MON 87460 to the Control at Each Site 

in the 2006 U.S. Field Study Under Well-Watered Conditions ................ 405
Monsanto Company 07-CR-191U Page 24 of 544 

Table F-15. Phenotypic Comparison 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 Comparison 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 Comparison 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 Comparison of MON 87460 to the Control at 

Each Site in 2007 Field Studies Established with Well-Watered and 

Water-Limited Treatments ........................................................................ 413

Table F-19. Study-1, Phenotypic Comparison of MON 87460 to the Control at 

Each Site in 2007 Field Studies Established with Well-Watered and 

Water-Limited Treatments ........................................................................ 414

Table F-20. Study-2, Phenotypic Comparison of MON 87460 to the Control at 

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

and Water-Limited Treatments ................................................................. 415

Table F-21. Phenotypic Comparison 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: Water-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 Control 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 Control 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 Control 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 at 

the IL1, KS, and NE Sites in a 2006 U.S. Field Study under WellWatered Conditions ................................................................................... 435
Monsanto 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, and NE Sites in a 2006 U.S. Field 

Study under Well-Watered Conditions ..................................................... 436

Table H-7. Beneficial Arthropod Abundance 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 Control 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 Control and References in a 2007 

U.S. Field Study under Well-Watered Conditions .................................... 443

Table H-10. Arthropod Stressor 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 Compared to the Control and 

References Combined across IA1, IL1, 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 Compared 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. Field 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. Field Study Established with Water 

Managed According to Local Agronomic Practices ................................. 454

Table H-17. Arthropod Stressor Incidence of MON 87460 Compared to the 

Control and References in a 2006 U.S. Field Study Established with 

Water Managed According to Local Agronomic Practices ...................... 455

Table H-18. Chile 2006/2007: Abiotic Stressor Incidence of MON 87460 

Compared to the Control and the References in a Field Study 

Established with Well-Watered and 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 and Water-Limited Treatments .............. 457
Monsanto 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 and 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 Field Studies Established with WellWatered and Water-Limited Treatments ................................................... 459

Table H-22. U.S. 2007 Study-1: Disease Damage Evaluations Using an 

Observational Severity Scale for MON 87460 Compared to the 

Control and References in Field Studies Established with WellWatered and Water-Limited Treatments ................................................... 460

Table H-23. U.S. 2007 Study-1: Arthropod Damage Evaluated using an 

Observational Severity Scale for MON 87460 Compared to the 

Control and References in Field Studies Established with WellWatered and Water-Limited Treatments ................................................... 461

Table H-24. U.S. 2007 Study-1: Quantitative 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: Arthropod Pest abundance on MON 87460 

Compared to the Control and References at TX in Field Studies 

Established with Well-Watered and Water-Limited Treatments .............. 463

Table H-26. U.S. 2007 Study-1: Arthropod 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 Field Studies Established with WellWatered and Water-Limited Treatments ................................................... 467

Table H-28. U.S. 2007 Study-2: Disease Damage Evaluations using an 

Observational Severity Scale for MON 87460 Compared to the 

Control and References in Field Studies Established with WellWatered and Water-Limited Treatments ................................................... 468

Table H-29. U.S. 2007 Study-2: Arthropod Damage Evaluations using an 

Observational Severity Scale for MON 87460 Compared to the 

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

Table I-1. U.S. 2006 Well-Watered Sites - Monthly Average Minimum and 

Maximum Temperatures and Temperature Range .................................... 499

Table I-2. U.S. 2007 Well-Watered Sites - 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 ............................. 501
Monsanto Company 07-CR-191U Page 27 of 544 

Table I-4. Chile 2006/2007 Well-Watered and Water-Limited Sites - Monthly 

Average Minimum and Maximum Temperatures and Temperature 

Range ......................................................................................................... 502

Table I-5. U.S. 2007 Study-1 Well-Watered and Water-Limited Sites - Monthly 

Average Minimum and Maximum Temperatures and Temperature 

Range ......................................................................................................... 502

Table I-6. U.S. 2007 Study-2 Well-Watered 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 Cropping History Information for Volunteer 

Potential Study .......................................................................................... 512

Table M-1. Test, Control, and Reference Starting Seed for Survival Outside 

Cultivation Study ....................................................................................... 514

Table M-2. Study Area Planting Information and Soil Description for Survival 

Outside Cultivation study .......................................................................... 516

Table M-3. Monthly Temperature and Rainfall Amounts 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 Approved for MON 87460 and Status of Trials 

Conducted Under these Notifications. ...................................................... 520

Table O-1. Comparison of Likely Environmental 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 ..................................................... 537

 
Monsanto Company 07-CR-191U Page 28 of 544 

List of Figures 

Figure I-1. Percent Yield Advantage for 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-Immunoprecipitation of RNA from MON 87460 using CSPB and 

the CSPB_F30A Variant ............................................................................. 50

Figure I-4. Gel Shift Analysis of Total RNA Isolated from MON 87460 ....................... 51

Figure I-5. Gel Shift Analysis of rRNA and Luciferase mRNA ...................................... 52

Figure I-6. Co-Immunoprecipitation of CSPB-FLAG:RNA Complexes from Leaf 

Tissue .......................................................................................................... 53

Figure I-7. Differential Accumulation of CSPB across Leaves of MON 87460 ............. 54

Figure I-8. Expression and Accumulation 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 Localization of CSPB in Plant Tissue ..................... 57

Figure I-11. Quantum Efficiency of Photosystem II-Mediated Electron Transfer, 

or ΦII, for MON 87460 and the Control over Time ................................... 58

Figure I-12. CO2 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-Drought (T0-3 to T0) and Post-Drought 

Initiation (T1-T4) ........................................................................................ 61

Figure I-15. Leaf 6 Daily LER Pre-Drought (T0-3 to T0) and Post-Drought 

Initiation (T1-T4) ........................................................................................ 62

Figure I-16. Leaf 7 Daily LER Pre-Drought (T0-3 to T0) and Post-Drought 

Initiation (T1-T4) ........................................................................................ 63

Figure I-17 Leaf 8 Daily LER Pre-Drought (T0-3 to T0) and Post-Drought 

Initiation (T1-T4) ........................................................................................ 64

Figure I-19. Automated LER Data and Plant Temperature for an Approximately 

48 Hour Growth Period during Well-Watered Conditions ......................... 66

Figure I-20. Automated LER Data and Plant 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 Transgenic CSPB Plants and the Control 

throughout the Duration of Stress Imposition ............................................. 69

Figure I-23. Osmotic Potential of Transgenic CSPB Plants and the Control 

throughout the Duration of Stress Imposition ............................................. 70

Figure I-24. Average Abscisic Acid Content 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 Content in Transgenic CSPB Plants and 

the Control ................................................................................................... 72
Monsanto Company 07-CR-191U Page 29 of 544 

Figure I-26. Leaf Raffinose and Inositol Content in Transgenic CSPB Plants and 

the Control ................................................................................................... 73

Figure I-27. Choline and Proline of Transgenic CSPB Plants and the Control 

throughout the Duration of Stress Imposition ............................................. 74

Figure I-28. Relative Glycine Betaine Levels of Transgenic CSPB Plants and the 

Control throughout the Duration of Stress Imposition ................................ 75

Figure II-1. Growth Stages of Corn from 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 Sequence of the Full Length CSPB Protein 

Present in MON 87460 .............................................................................. 102

Figure IV-2. Nucleotide Sequence Encoding the NPTII Protein in PV-ZMAP595 ...... 102

Figure IV-3. Deduced Amino Acid Sequence 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 Plasmid PV-ZMAP595 Showing Probes 1-6 .................. 111

Figure V-3. Circular Map of Plasmid PV-ZMAP595 Showing Probes 7-13 ................ 112

Figure V-4. Southern Blot Analysis of MON 87460: Insert 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: Insert 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 Interpretation of Detected Differences ............... 181

Figure VIII-2. Decision Diagram for Site Inclusion in Plant 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. Western 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 ........ 290
Monsanto 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 and 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 Accumulated Soil Water Values at OH in 2007 ............... 486

Figure I-17. Calculated and Accumulated Soil Water Values at PA in 2007 ................ 487

Figure I-18. Accumulated Water at IAE in 2006 ........................................................... 488

Figure I-19. Accumulated Water 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 Water at CL in 2006/2007 ................................................... 491

Figure I-24. Accumulated Water at CT in 2006/2007 ................................................... 492

Figure I-25. Accumulated Water at LUM in 2006/2007 ............................................... 493

Figure I-26. Accumulated water at QUI in 2006/2007 .................................................. 493

Figure I-27. Calculated and Accumulated Soil Water Values at CA Study-1 in 

2007 ........................................................................................................... 494

Figure I-28. Calculated and Accumulated Soil Water Values at TX Study-1 in 

2007 ........................................................................................................... 495

Figure I-29. Calculated and Accumulated Soil Water Values at KS Study-2 in 

2007 ........................................................................................................... 496

Figure I-30. Calculated and Accumulated Soil Water Values at NE Study-2 in 

2007 ........................................................................................................... 497

Figure I-31. Calculated and Accumulated Soil Water Values at TX Study-2 in 

2007 ........................................................................................................... 498

 
Monsanto 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-HCl, 5% (v/v) 2-mercaptoethanol, 

2% (w/v) sodium dodecyl sulfate, 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 Cauliflower 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-nucleotidyltransferase from the 

transposon Tn7 

AD8 Allergen, gliadin, and glutenin protein sequence database 

ADF Acid detergent fiber 

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

ALLERGENSEARCH Computer program for the search against known allergens 

ALLPEPTIDES Protein sequence database comprised of NRAA and SwissProt 

databases 

AOAC Association of Official Analytical Chemists 

AOCS American Oil Chemists Society 

AOSA Association of Official 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 acre 
Monsanto 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 and 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 Electrical conductivity 

E. coli Escherichia coli

EcoO109 I Restriction enzyme isolated from E. coli

EcoR V Restriction enzyme isolated from E. coli

ECL Enhanced chemiluminescence 

EDTA Ethylenediaminetetraacetic acid 

EFSA European Food Safety Authority 

ELISA Enzyme-linked immunosorbent assay 

EPA Environmental Protection Agency 
Monsanto 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 recognized as safe 

H29 Histidine 29 

HC Health Canada 

HI Harvest index 

HMM Health Ministry of Mexico 

Hind 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 Composition 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 Ionization - Time of Flight 

MH+ Protonated mass ion 
Monsanto 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 product, 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 product, 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 producing 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 phosphotransferase 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 pBR322 for maintenance of plasmid in 

E. coli 

ori V Origin of replication for Agrobacterium derived from the broad 

host range plasmid RK2 
Monsanto 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 saline 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 Statistical 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 diode 
Monsanto 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 Tris 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 termination 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 badrii
Monsanto Company 07-CR-191U Page 37 of 544 

I. Rationale for the Development of MON 87460 

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

Part 340.6 

The Animal and Plant Health Inspection Service (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 prevent 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, thereby allowing unrestricted introduction of the 

article. 

I.B. Product Concept and Benefits of MON 87460 

I.B.1. Product Concept 

Monsanto has developed MON 87460 that reduces yield loss under water-limited 

conditions compared to conventional corn. 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 water-limited conditions, particularly 

during flowering and grainfill periods when corn yield potential is most sensitive to 

stress, by disrupting kernel development (Claassen 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 scientists hypothesized that inserting a stress response protein 

into plants could impart a desirable 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 plants were tested for their ability 

to yield more grain than control plants under water-limited conditions. The CSPB protein 

expressed in MON 87460 exhibits key behaviors and properties that are similar to what is 

reported for bacterial cold shock proteins and cold shock domain-containing proteins in 

plants. 

I.B.2. Product Benefits 

MON 87460 is expected to provide 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 important factor that 

reduces global crop yields. In North America alone, it is estimated that 40% of annual 

crop losses are due to sub-optimal water availability (Boyer, 1982). In both temperate 

and tropical regions suitable for commercial corn production globally, the average annual 

corn yield loss attributable to moderate water 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 will 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 likely to be the Western Dryland region of the Corn Belt. 

This region has also been defined as the Great Plains and includes portions of ten states 

(Colorado, Kansas, Montana, Nebraska, New Mexico, North Dakota, Oklahoma, South 

Dakota, Texas and Wyoming) contributing 26% of the national corn production 

(Riebsame, 1990). Annual precipitation in this region averages 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 provide benefits due to reduced yield loss 

under water-limited conditions. While much progress has been made to improve corn 

yield in water-limited environments through breeding, selection, and agronomic 

management practices, there remains potential 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 impacts on yield and improved 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 conditions. 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 environmental 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 different experimental designs and water 

management regimes: (1) well-watered only, or (2) both well-watered and water-limited 

treatments established in the same field arranged in either a strip- or split-plot design. 

Field studies managed to impose only well-watered 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 expected between MON 87460 and the control. 

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 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-limited 
Monsanto 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 control. The well-watered 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 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 vegetative 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). Specifically, 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 approximately 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 indicators of moisture deficit 

stress in corn (Campos et al., 2006); therefore, these characteristics 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 the 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 the relative stress level imposed among the 

nine field sites (Figure I-1). Yield advantage 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 of the commercial references grown under waterlimited conditions compared to well-watered treatments. 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 presented 

data 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 deficit 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). Yield loss of the references at 

these three sites did not meet the required 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 grown under well-watered field 

conditions, MON 87460 did not provide a yield advantage compared 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 under water-limited conditions, and under severe 
Monsanto Company 07-CR-191U Page 40 of 544 

water deficit, yield can be reduced to zero. Intuitively, as stress levels (i.e., yield loss of 

the references) increase beyond the range presented in Figure I-1, the yield advantage of 

MON 87460 compared to the control should be expected to decrease and be eliminated as 

the grain yield for both MON 87460 and the control falls to zero. 

A comparison of the average percent yield 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 control, 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 strip- and split-plot experimental designs, yield 

potentials were 207 and 144 bu/ac for the well-watered and water-limited treatments, 

respectively. As expected, MON 87460 provided 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 MON 87460 in the water-limited treatment were 

highly variable, but are representative of the natural variation expected for corn grown 

under sub-optimal soil moisture conditions. 

It is known that corn hybrids are 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. The variation in 

yield response observed among MON 87460, the 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 relevant 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 yield 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 environmental conditions suited for commercial corn 

production, it is expected that MON 87460 will produce a range of responses based on 

the timing, severity, and duration of water-limited conditions experienced during the 

growing season. On average, under water-limited conditions, MON 87460 hybrids are 

expected to provide a 6% or greater yield advantage compared to commercial hybrids. 

Monsanto and other seed companies commonly 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). Commercial hybrids of MON 87460 will be evaluated 

for drought tolerance in a manner consistent with the breeding and selection of other 
Monsanto Company 07-CR-191U Page 41 of 544 

commercial corn hybrids. As per standard process for all commercial products, both 

conventional and biotechnology-derived, Monsanto 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 for 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 references was calculated as the average yield of the 

reference materials in the well-watered treatment compared to the average yield under the waterlimited treatment within each site. Data presented 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 WLsp

Percent 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: WWonly = 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 based on its yield advantage under waterlimited conditions compared to the control and absence of negative pleiotropic effects on 

plant performance. The insertion of the cspB gene in MON 87460 confers tolerance to 

water-limited conditions that would otherwise negatively impact yield. The nptII 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) 

179 207 144 

Yield advantage of MON 87460 vs. control 

(% difference)

Experimental design and treatment type 
Monsanto Company 07-CR-191U Page 43 of 544 

I.D. Mode of Action of the Drought Tolerance Trait 

Cold shock proteins confer environmental 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 belongs to the CSP family, which has been 

extensively studied in bacteria. Early investigations of bacterial responses to coldinduced stress led to the discovery of CSPs, a group of small proteins that contain a 

highly conserved RNA-binding sequence identified as a cold shock domain (CSD). 

In bacteria, a variety of environmental stresses are known to disrupt normal cell 

physiology, in part due to the production of RNA secondary structures which leads to a 

reduction in protein synthesis. Under environmental stress, CSD-containing proteins 

have been shown to bind many types of RNA (Cristofari and Darlix, 2002), leading to 

sustainable translation, maintenance of mRNA levels, and improved cellular function. 

While some members of the bacterial CSP family accumulate strictly in response to 

temperature shift (Etchegaray et al., 1996), others, including the B. subtilis CSPB protein, 

are also involved in maintaining normal cellular 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-localizing with 

the ribosomes (Mascarenhas et al., 2001; Weber et al., 2001). In stationary-phase cells 

CSPB is distributed throughout the cell, indicating 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 environmental stress tolerance in plants.

Similar to bacteria, CSD-containing proteins in plants also bind RNA, unfold RNA 

secondary structures caused by environmental stress, 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). Plant 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-containing 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 CSD proteins could be involved in a more 

general response to stress by binding RNAs and, therefore, helping cells to maintain 

cellular functions following the stress. CSD proteins from rice and Arabidopsis have 

been shown to be highly expressed in apical meristems, ovules, embryos, and seeds 

(Fusaro et al., 2007; Chaikam and Karlson, 2008) and, therefore, could potentially affect 

growth rate, flowering time, and seed development. The CSD proteins have been 

localized both in the 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 potentially be involved in multiple steps of RNA 

metabolism including localization, translation and stability. 

CSPB protein expressed in MON 87460 has a similar structure and function to other 

CSD-containing proteins.

As with bacterial and other plant CSD-containing proteins, the CSPB protein from B. 

subtilis, which is expressed in MON 87460, binds 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 effective in unfolding secondary RNA structures in 

vitro, while variants of the CSPB protein (CSPB_F30R and CSPB_F30A) with impaired 

RNA binding functions were unable to bind and unfold RNA (Table I-1). In vitro coimmunoprecipitation experiments with CSPB, CSPB_F30A and RNA from MON 87460 

further confirm that CSPB interacts with RNA while a variant lacking a functional RNA 

binding site will not interact with RNA in vitro (Figure I-3). Finally, gel shift 

experiments demonstrate that CSPB is capable 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-immunoprecipitated from leaf tissue, confirming that CSPB 

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

In MON 87460, the expression of the CSPB protein is under control of the rice actin 

promoter which enables constitutive expression of the protein and decouples expression 

of CSPB protein from the cold shock response in bacteria. Using a CSPB specificEnzyme-Linked Immunosorbent Assay (ELISA), the pattern of CSPB accumulation in 

MON 87460 leaf tissue, seedlings and developing reproductive tissues was evaluated 

(Figures I-7 – I-9). CSPB accumulation is highest in rapidly growing areas of the leaf 

and seedlings and tends to decline as the tissue matures. CSPB concentrations tend to 

increase over time in developing ears and decline over time in silks. Likewise, CSPB 

concentrations increase over time in immature tassels and either remain the same or 

decline in pollen. Sub-cellular localization of the CSPB protein was evaluated by 

immunohistochemistry. In MON 87460 coleoptiles, CSPB was distributed between 

cytoplasm and nucleus (Figure I-10). Similar sub-cellular localization of the CSPB 

protein was previously observed in corn protoplasts (Castiglioni et al., 2008). 

Taken together, the data on RNA binding, CSPB accumulation and CSPB localization in 

MON 87460 are consistent with the pattern of RNA binding, accumulation, localization, 

and functions described for plant CSD-containing proteins (Fusaro 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 corn yield potential is most 

sensitive to stress by disrupting fertilization 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 approach, Castiglioni et al. (2008) demonstrated 

that bacterial CSPs can confer improved stress adaptation to multiple plant species. 

CSPB-containing events were evaluated in water-limited field trials in environments that 

received no rainfall during the 10 to 14 days immediately prior 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 protein 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 yield (16.5%), kernels per 

ear (13.1%), and kernel weight (3.9%) (Table I-2). Similar results were observed in a 

subsequent study, where differences 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 component contributing to the improved yield of 

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

which is consistent with the current understanding of the effect of drought stress on corn 

yield potential (Westgate et al., 2004; Campos 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 growth rate relative to the control plants and 

overall better vegetative performance as measured by photosynthetic rate, carbon 

fixation, and stomatal conductance (Figures I-11 – I-13, respectively). The improved 

vegetative productivity is believed to be correlated to the high accumulation of CSPB in 

the rapidly growing tissues (Figures I-14 – I-21). Leaf growth 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 (Andrade 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 common drought tolerant responses in corn 

plants

Common mechanisms of plant response to drought stress are not altered 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 compared with controls. The plants containing 

CSPB protein show broadly the same relative water content (Table I-4), leaf water 

potential (Figure I-22), and leaf 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, fructose, glucose, choline, proline, glycine 
Monsanto 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 the CSPB protein in MON 87460 results in reduced yield loss 

under drought conditions when compared to conventional corn grown under identical 

conditions. The major component contributing 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 proteins moderate stress responses in bacteria and plants, 

primarily through stabilization of RNA. Like endogenous CSD proteins found in plants, 

the CSPB protein in MON 87460 interacts with 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 MON 87460 compared to the control, but 

accumulation of ABA and osmotically active solutes are not altered in transgenic CSPBcontaining corn plants. 

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

Protein 

Specific Activity 

(pmoles opened DLP1/μg CSPB)

CSPB 0.905 ± 0.033 

CSP_F30R <LOD2

CSPB_F30A <LOD 

1

DLP – Dual-Labeled Probe 

2

LOD – Limit of Detection 

The melting activities of CSPB and the different variants were measured for all 

proteins using 3 μg of protein for each replicate. The activity represents the 

average of three replicates. In this assay, a hairpin-shaped (stem-loop) molecular 

beacon is labeled with a fluorophore at the 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

Yield2 (bu/ac)3 80.0 (10.9) 68.7 (10.9) 11.3 16.4 0.153 

Kernels per ear4 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 plot6

 33.8 (1.3) 33.5 (1.3) 0.3 0.9 0.834 

Stomatal 

conductance

(mmol/m2

/s) 

262.7 (20.78) 235.8 (20.78) 26.9 11.4 0.064 

Photosynthetic rate 

(μmol CO2/m2

/s)7 37.2 (2.24) 34.1 (2.24) 3.1 9.1 0.066 

Transpiration rate 

(mmol/m2/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 kernel 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 replicates 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 measurements 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 from 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 ear4 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 measurements were collected from three plants in 

each of the ten water-limited replicates. 

The well-watered treatment contained five replicates. 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_M388351

 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 period 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 described 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% agarose 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 immunoprecipitate 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 B
Monsanto 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) purified 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 B
Monsanto 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 incubated 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 Bioanalyzer. 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 shows 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-immunoprecipitated 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 MON 87460 (i.e., rice actin promoter and Tr7 

terminator). The event used for the Co-IP experiments had a single copy transgene insertion and 

it expressed the expected protein as confirmed by western blot and MALDI-TOF Mass 

Spectrometry. Plants were kept well watered and 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 Marker 25nt Marker 25nt Marker

FLAG-CSPB Co-IP 

Negative Control Co -IP

FLAG-CSPB Co-IP

FLAG-CSPB Co-IP + RNase

FLAG-CSPB Co-IP

FLAG-CSPB Co-IP + DNase
Monsanto 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 contained 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 46

pg CSPB/ μg Total Protein

Basal to Distal Sampling

B
Monsanto Company 07-CR-191U Page 55 of 544 

Figure I-8. Expression and Accumulation 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 determined 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 11

ng/g fwt

Days after germination

Basal-Leaf Distal-Leaf Coleoptile

0

10

20

30

40

50

3 5 6 7 10 11

mg/g fwt

Days After Germination

Basal Leaf Distal Leaf Coleoptile

CSPB mRNA

28S rRNA

Level of CSPB (ng /g fwt) Total Protein (ng /g fwt)

MON 87460

d3 d5 d6 d7 d10 d11 

Days After Planting 

Days After Planting 
Monsanto Company 07-CR-191U Page 56 of 544 

Figure I-9. Expression of CSPB in Tissues 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 60

CSPB (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 60

CSPB (ppm)

Days After Planting (DAP)

Immature Ear Silk A

B
Monsanto Company 07-CR-191U Page 57 of 544 

Figure I-10. Immunohistochemical Localization of CSPB in Plant Tissue 

Shoot (coleoptiles) sections from 3-day post-germination plants were incubated with affinity 

purified goat anti-CSPB. The CSPB antibody was 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 D
Monsanto Company 07-CR-191U Page 58 of 544 

Figure I-11. Quantum Efficiency of Photosystem II-Mediated Electron Transfer, or 

ΦII, for MON 87460 and the Control over Time 

Measurements of the maximum fluorescence quantum yield of photosystem II (PS2) mediated 

electron transfer (ΦII) were obtained with an OS30P chlorophyll fluorometer as a measure of 

photosystem II activity. Readings were made on leaves that were at the top of the canopy and 

whose surface was visibly exposed to illumination. 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 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 T4

PS2Quantum Efficiency (ΦII; Fm'‐Fs/Fm')

Control

MON 87460
Monsanto Company 07-CR-191U Page 59 of 544 

Figure I-12. CO2 Assimilation over Time for MON 87460 and the Control 

Measurements of CO2 assimilation (AN) 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-state in the measurement chamber. Gas exchange 

parameters were obtained under steady-state illumination. 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 beginning 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 T4

CO2 assimilation(AN;μmol CO2m‐2s‐1)

Control

MON 87460
Monsanto Company 07-CR-191U Page 60 of 544 

Figure I-13. Stomatal Conductance over Time for MON 87460 and the Control 

Measurements of stomatal conductance (gs) 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-state in the measurement chamber. Gas exchange 

parameters were obtained under steady-state illumination. 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 beginning 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 T4

Stomatal Conductance (gs; mmol

H

O2 m‐2s‐1)

Control

MON 87460
Monsanto 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 based on three-day height difference between T1 and 

T0. T0-1, -2 & -3 are all well-watered measurements 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-limited 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 T4

LER (cm)

Control

MON 87460
Monsanto 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 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-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 T4

LER (cm)

Control

MON 87460
Monsanto 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 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-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 T4

LER (cm)

Control

MON 87460
Monsanto Company 07-CR-191U Page 64 of 544 

Figure I-17 Leaf 8 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 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 T4

LER (cm)

Control

MON 87460
Monsanto Company 07-CR-191U Page 65 of 544 

Figure I-18. Leaf 9 Daily LER during the Drought Progression (T1-T4) 

T1 LER is an average based on three-day height 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 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 T4

LER (cm)

Control

MON 87460
Monsanto Company 07-CR-191U Page 66 of 544 

TIMESTAMP

0

10

20

30

40

50

60

LER 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 made using a draw wire sensor potentiometer 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 

Temperature
Monsanto Company 07-CR-191U Page 67 of 544 LER 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 made using a draw wire sensor potentiometer 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 

Control
Monsanto 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 made using a draw wire sensor potentiometer 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 87460
Monsanto 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-containing events and a control. All events 

contained the same construct as MON 87460. Entries were planted in two row plots with three 

replications per experiment. Plots were irrigated 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 indicates a statistically significant difference at a p 

value of less than 0.100 using an analysis of variance. 

-16.0

-14.0

-12.0

-10.0

-8.0

-6.0

-4.0

-2.0

0.0

6:00 AM8:00 AM

10:00 AM

12:00 PM

2:00 PM4:00 PM6:00 PM

6:00 AM8:00 AM

10:00 AM

12:00 PM

2:00 PM4:00 PM6:00 PM

6:00 AM8:00 AM

10:00 AM

12:00 PM

2:00 PM4:00 PM6:00 PM

June 25 June 30 July 6

Total 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 Transgenic CSPB Plants and the Control 

throughout the Duration of Stress Imposition 

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

contained the same construct as MON 87460. Entries were planted in two row plots with three 

replications per experiment. Plots were irrigated 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 indicates a statistically significant difference at a p 

value of less than 0.100 using an analysis of variance. 

 

-20.0

-18.0

-16.0

-14.0

-12.0

-10.0

-8.0

-6.0

-4.0

-2.0

0.0

6:00 AM8:00 AM

10:00 AM

12:00 PM

2:00 PM4:00 PM6:00 PM

6:00 AM8:00 AM

10:00 AM

12:00 PM

2:00 PM4:00 PM6:00 PM

6:00 AM8:00 AM

10:00 AM

12:00 PM

2:00 PM4:00 PM6:00 PM

June 25 June 30 July 6

Osmotic Potential (bars)

Osmotic Potential: CSPB Events Osmotic Potential: Control

* *

*
Monsanto Company 07-CR-191U Page 71 of 544 

Figure I-24. Average Abscisic Acid Content 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-containing events (identified with codes beginning 

‘ZM’) and a control. All events contained the same construct as MON 87460. All plants were 

grown for approximately five weeks prior to the 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

7000

neg

ZM M38840ZM M39872

neg

ZM M38840ZM M39872

neg

ZM M38840ZM M39872

neg

ZM M38840ZM M39872

neg

ZM M38840ZM M39872

neg

ZM M38840ZM M39872

neg

ZM M38840ZM M39872

neg

ZM 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 16

Average ABA Content (ng/g DW)
Monsanto Company 07-CR-191U Page 72 of 544 

Figure I-25. Leaf Fructose and Glucose Content in Transgenic CSPB Plants and the 

Control 

The study was conducted using three CSPB-containing 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-limited

Sugar Content (mmoles/gram FW)

Fructose Content: Control Fructose Content: Test Glucose Content: Control Glucose Content: Test
Monsanto 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-containing 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-limited

Sugar Content (mmoles/gram FW)

Raffinose Content: Control Raffinose Content: Test Inositol Content: Control Inositol Content: Test
Monsanto Company 07-CR-191U Page 74 of 544 

Figure I-27. Choline and Proline of Transgenic CSPB Plants and the Control 

throughout the Duration of Stress Imposition 

The study was conducted using MON 87460, five other 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 extraction, deuterated homologues (d9 choline and d7 

proline) were added as internal standards to control 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 38862

Relative Metabolite Ratios 

(endogenous metabolite/internal standard)

Relative choline levels

Relative proline levels
Monsanto Company 07-CR-191U Page 75 of 544 

Figure I-28. Relative Glycine Betaine Levels of Transgenic CSPB Plants and the 

Control throughout the Duration of Stress Imposition 

The study was conducted using MON 87460, five other 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 for 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 38862

Relative Metabolite Ratios 

(endogenous metabolite/internal standard)

Relative glycine betaine levels
Monsanto Company 07-CR-191U Page 76 of 544 

I.E. Adoption, Estimated Use, and Global Trade Flows of MON 87460 

As described previously (Section I.B.2), 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 likely to be the Western Dryland region of the Corn 

Belt known as the Great Plains, which contributes 25% of the U.S. corn production 

(Riebsame, 1990). Other corn producing geographies 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 acre will help conserve the total number of 

acres needed to meet the needs for food, feed, 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 derived 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 nutritional 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 import and production approvals will 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 foreign government regulatory agencies, including 

Japan’s Ministry of Agriculture, Forestry and Fisheries (MAFF) and the Ministry of 

Health, Labor and Welfare (MHLW); the Canadian Food Inspection Agency (CFIA) and 

Health Canada; the Health Ministry of Mexico (HMM); the European Food Safety 

Authority (EFSA), and the regulatory authorities in many other countries. As 

appropriate, notifications of import will be made to importing countries that do not have a 

formal approval process. 
Monsanto Company 07-CR-191U Page 77 of 544 

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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 Regulatory Services of the United States 

Department of Agriculture-Animal and Plant Health Inspection Service (USDA-APHIS, 

2009); (3) information provided in the USDA petition for MON 89034 (Monsanto 

Company petition No. 06-CR-166U); and (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 source in the developed world. These demands 

are exceeding production, leading to diminished grain reserves worldwide. In addition, 

climate change may have variable impacts on crop yields, potentially creating further 

supply disruptions. The combination of these 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 (Boyer, 1982). 

Consequently, increasing drought tolerance in corn is a major goal of agricultural 

breeding and biotechnology. Advances in breeding and agronomic practices have made 

significant contributions to increasing corn yield potential and drought tolerance. 

Biotechnology provides additional tools that can be used in combination with breeding 

and agronomic practices to improve productivity. 

Monsanto has developed drought tolerant corn MON 87460 that provides reduced yield 

loss under water-limited conditions compared to conventional corn. Efficacy in 

MON 87460 is derived by expression of the inserted Bacillus subtilis cold shock protein 

B (CSPB). Understanding the applications, benefits, and function of MON 87460 in 

meeting the global demands on corn production requires an understanding of corn’s 

origins and history, its biology and cultivation, and its importance to global trade. A 

summary of the history, taxonomy, and biology of corn, including gene flow between 

cultivated corn and its wild relatives, and 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 that suit the needs of consumers and growers 

who wish to produce corn grain. Corn originated 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 crop 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 grain 
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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 20th 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 yield increases (Duvick et al., 2004). 

In the 1940s, when hybrid corn began to predominate, average U.S. yields were 30 – 40 

bushels/acre (bu/ac, 2.2 metric tons/hectare (MT/ha)) (Duvick et al., 2004). By 2000, 

average U.S. yields were over 130 bu/ac (8.6 MT/ha). Advances in breeding and the 

widespread availability of fertilizers and pesticides made significant contributions to 

these advancements (Troyer, 2004). Biotechnology 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 resistance traits were grown on 73% of U.S. corn 

acres. Combined trait products, with combinations of herbicide tolerance and insect 

resistance, were the largest category at 28% of U.S. corn acres (NCGA, 2008). As a 

complement to these existing traits, MON 87460 provides yield stability under waterlimited conditions that otherwise limit plant performance. Increasing demand for corn in 

the food, feed, and fuel sectors places a premium 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; teosinte 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 distinguishing 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 has 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 and its close relatives. 
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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 - Manisuris
Monsanto 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 subspecies. All species within 

the genus Zea, except corn, are different species of teosinte. 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 characterized of the higher plants. 

Because of the separation of male and female inflorescence, number of seeds produced 

on the female inflorescence, ease in handling (growing and hand pollinating), 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 laboratories to obtain a basic 

understanding of mitosis, meiosis, chromosome segregation, linkage and effects of 

crossing-over, and transposable elements. Because 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 trait loci (QTLs). QTLs are regions of 

chromosomes that are known to play a role in imparting physiological traits of interest 

and represent a significant area 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 QTLs, 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 stress tolerance is the 

difficulty in imposing targeted stresses under field conditions to select for desirable 

phenotypes (Takeda and Matsuoka, 2008). In addition, 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 visible 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 formation is complete and all leaves 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 through 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 number 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 (Westgate 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 index (HI) is the ratio of grain biomass to shoot 

biomass and is used to quantify the relationship between sink tissue and source tissue. In 

modern hybrids HI is approximately 0.5 (Westgate et al., 2004). The amount of shoot 

biomass available at the end of the season to support kernel development is a function of 

the amount of photosynthetically active radiation (PAR) the plant 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, adverse 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 geographical range, but its growth, development, and 

yield potential are still closely related to environmental conditions, including 

temperature, nutrients, and water. Soil temperatures 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 sufficient 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 temperatures will adversely effect corn growth and 

development. As canopy closure begins in the V6 growth stage, high plant density (more 
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than 30,000 plants per acre) can limit PAR interception, reducing growth rate (Westgate 

et al., 2004) and limiting the supply of photosynthate. Shading caused by high planting 

densities also reduces the amount of available photosynthate, reducing the ability of a 

plant to support developing kernels (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 availability (Boyer, 1982). As a C4 

plant, corn is inherently more water use efficient and transpires only half as much water 

as C3 plants (Taiz, 1991), e.g., rice, soybean, and cotton. Despite this key advantage, 

water limitation still adversely impacts corn growth, development, and yield, particularly 

during reproductive growth stages. In general, water limitation causes 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 manifests itself visually in corn as reduced 

plant height and yield. Limiting water during later reproductive growth stages results in 

scorching and leaf firing (Hsiao, 1973; Westgate et al., 2004). 

The flowering growth stages (VT – R1) are a particularly vulnerable period for corn plant 

development and the resulting yields. Four days 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 reduce 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) occurring 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 values (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 inability 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 also be a significant factor in yield loss 

under water limitation (Westgate et al., 2004). Water limitation during late vegetative 

stages through flowering will reduce kernel number and increase the number of plants 

without ears (barrenness). Water limitation during grain filling will 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 able to produce better 

yields than non-drought tolerant hybrids. The primary mechanism for improvement was 

by selection for synchronous flowering. Breeders 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 selecting for plants that were able to maintain 
Monsanto Company 07-CR-191U Page 86 of 544 

normal function under drought stress. A retrospective study of advances in drought 

tolerance over 50 years 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, although 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 Mexico 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 input with 50-60 cm of water needed under 

temperate conditions. Corn rapidly depletes nitrogen and phosphorous 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 inputs, the appropriate 

choice of hybrid is essential for optimal yield and will vary with planting location. 

Hybridization is a fundamental concept used in global corn breeding and production. 

Corn was originally developed as an open-pollinated (cross-fertilizing) crop species, and 

until the beginning of the 20th 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 individuals for multiple generations results in 

pure (inbred) lines within which every plant has similar traits. In these inbred lines, 

heterozygous loci are eliminated 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 (heterosis). Selecting 

for inbred lines and crosses that possess desirable traits forms the basis of modern corn 

breeding. Recent techniques such as marker-assisted breeding have reduced the time and 

number of generations required to produce 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 while decreasing inputs (Brookes and Barfoot, 

2008; Hicks and Thomison, 2004; Kaeppler, 2004). Globally, between 1996 and 2006, 

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

and insect resistant corn allowed a 5.0% reduction in the use of insecticide active 

ingredients. Notably, over this same period, an additional $1.1 billion and an additional 

$3.6 billion of global farm income can be attributed to the adoption of herbicide tolerant 

and insect resistant corn, respectively. Improved yields are responsible for 43% of these 

economic gains (Brookes and Barfoot, 2008). 

Rotation with other crops is typically 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 sorghum is also used. In the Western Dryland region, 

wheat, sorghum and soybean may be planted in rotation with corn. Wheat-fallow 
Monsanto Company 07-CR-191U Page 87 of 544 

rotation is also used to store rainfall during the fallow season. Recent research has shown 

that wheat-fallow rotations may actually reduce the amount of soil moisture available to 

wheat crops because fallow season management requires 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 wheat (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 genetic 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 hybrids combined with the types of selection 

operating in the teosinte populations. Wilkes (1967) suggested the presence of hybrids 

under natural conditions in Central Plateau and Valley of Mexico as an unequivocal 

indication of cross compatibility between corn and teosinte. Baltazar et al. (2005) and 

Ellstrand et al. (2007) presented further evidence of hybridization 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, which is approximately 1–

2% of the ovules normally produced on a hybrid corn ear (Baltazar et al., 2005). 

Hybridization in the other direction is difficult but teosinte ears can produce up to 0.2–0.3 

seeds per ear when pollinated with corn pollen (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 Cultivated Zea Varieties 

Gene flow in corn is closely associated with the biology of the staminate and pistillate 

inflorescences. Corn is a cross- and wind-pollinated crop that produces pollen in copious 

quantities. A hybrid tassel of normal size can produce up to 25 million pollen grains 

(Kiesselbach, 1999). Corn pollen has a mean 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 direction, turbulence and velocity are directly 

linked to pollen movement (Jones and Brooks, 1950; Di-Giovanni and Kevan, 1991). 

Other factors such as pollen density, air density and viscosity, pollen sedimentation 

velocity, and pollen radius seem to influence pollen transport and deposition (Paterniani 

and Stort, 1974; Di-Giovanni et al., 1995; Aylor, 2002). Once in the atmosphere, pollen 

grains must remain viable long enough to be able to reach a viable silk to complete the 

pollination process. On average, corn pollen loses 100% viability after two hours of 

atmospheric exposure (Luna et al., 2001; Aylor, 2003). 
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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 rows, 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 appearance 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 selection in the evolution of 

corn. In contrast with weedy plants, corn has a polystichous female inflorescence (or ear) 

on a stiff central spike (or cob) enclosed with 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 corn, 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 survive 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 habitat. Corn’s closest 

wild relative, teosinte, is native to Mexico and 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 biotechnology to increase drought tolerance, this basic water 

requirement has not been surpassed and will not allow corn to be grown or persist outside 

of current corn growing regions 

Corn is the target of a variety of microbial pathogens and insect pests. Rusts, smuts, leaf 

blights, and stalk rots are among the more common diseases of microbial origin. Their 

prevalence and economic importance vary from 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 can exacerbate fungal infections (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 (Diabrotica 
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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 conducted with LH59, an inbred corn line. A 

single transformed plant was then self-crossed to increase seed supplies. A homozygous 

inbred line was developed though self-crossing and selection, and then used to produce 

other lines used for product testing, regulatory studies, and commercial production. 

II.E. Global Use of Corn 

Corn is used globally for food, feed, and fuel, and in recent years the demand for corn has 

increased, leading to higher prices for raw grain and its derivatives (NCGA, 2008). 

OECD-FAO’s joint 2008-2017 Agricultural Outlook 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, changing 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 product supply and possibly result in price spikes (OECD-FAO, 

2008). 

Food uses include sweet corn, popcorn, and processed field corn, which are all hybrids of 

Zea mays. Of the corn used for food and industrial 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 industrial starches, corn gluten feed and corn 

gluten meal are also manufactured through 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 starch 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 also 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 feed, 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 grain solubles (DDGS) 

which are another source of animal feed (RFA, 2008). 

II.E.1. Historical Uses of Corn and 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 in 
Monsanto Company 07-CR-191U Page 90 of 544 

developing countries throughout Latin America and Africa. In industrialized countries 

and developing Asian countries, the primary use for corn grain is animal feed although 

ethanol production has been rising steadily (NCGA, 2008; OECD, 2003b; OECD-FAO, 

2008). 

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

Products 

From 2003 to 2007, worldwide corn grain production 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 production 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 available, 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 exporters were the United States, Argentina, 

Brazil, China, and Ukraine. Together, these countries accounted for 93% of average 

annual exports (USDA-FAS, 2008). The major 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 from approximately 0.7 MMT to just under 1.8 

MMT, second only to gluten feed in terms of processed corn product exports from the 

U.S. (ProExporter, 2008). Annual exports increased between 2003 and 2007 from 79 

MMT to 92 MMT. From 2003 to 2007, the top five grain importers were Japan, Korea, 

Mexico, EU and Egypt, accounting for 51% of average annual imports (FAOSTAT, 

2007). 

Between 1997 and 2007, global demand for cereals (coarse grains, wheat and rice) was 

equal to or greater than production (FAO, 2008). This has 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% between 2007 and 2008. Similarly, wheat and rice prices have 

doubled in some markets. For corn, the price change is largely the result of increased 

demand while for the other cereals, particularly 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 the 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 the 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 greater consumption of meat and dairy 

products (von Braun, 2007). 

Within this context, the impacts of climate change take on a new urgency with respect to 

corn production. In a 2008 report, the U.S. Climate Change Science Program stated that 

climate change is already impacting agricultural productivity. The report concluded that 
Monsanto Company 07-CR-191U Page 91 of 544 

a combination of increased temperature variability, altered rainfall patterns and the 

resulting increases in drought frequency will magnify yield variability (CCSP, 2008; 

Hatfield et al., 2008). 

II.F. Conclusions 

Corn is a versatile crop that provides food, feed and fuel. This versatility, coupled with 

growing economies in the developing 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 disruptions. Climate change will have variable 

impacts on crop yields, potentially creating supply disruptions. The combination of these 

factors places a premium on corn yield stability in sub-optimal environments. 

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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, Class: Bacilli). The transformation was 

performed with the binary plasmid vector PV-ZMAP595 (Figure III-1). Agrobacterium 

tumefaciens strain ABI contains a modified Ti plasmid that is incapable of inducing 

tumor formation due to the deletion of the phytohormone genes originally present in the 

Agrobacterium plasmid (Koncz and Schell, 1986). The vector, PV-ZMAP595, contains 

both the left and right border sequences flanking 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 Phillips (Armstrong and Phillips, 1988). Briefly, 

freshly isolated immature corn embryos were used for callus initiation. After coculturing with Agrobacterium carrying the transformation vector, the calli were 

transferred from filter paper to callus initiation medium containing carbenicillin to 

eliminate Agrobacterium and paromomycin to eliminate cells that were not transformed, 

so that only cells containing the T-DNA survived. The resulting transformed cells were 

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

The R0 plants generated through the above transformation were self-pollinated, and the 

subsequent R1 plants were screened for the presence 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 kanamycin were advanced for 

development, and their progenies were subjected 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, phenotypic characteristics, and molecular 

profile. Additional studies were conducted with MON 87460 to further characterize the 

genetic insertion and the expressed protein, and to establish the food, feed, and 

environmental safety relative to conventional corn. The 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. Genetic and cytogenetic variation in plants 

regenerated from organogenic and friable, embryogenic tissue cultures 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 carried 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

T-tr7 CS-cspB

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

T-tr7 CS-cspB

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

T-tr7 CS-cspB

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

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

PV- ZMAP595

9379 bp
Monsanto Company 07-CR-191U Page 98 of 544 

Figure III-2. Schematic Representation of the Development of MON 87460 

 

Assembled Agrobacterium binary plasmid vector 

PV-ZMAP595 and transferred to Agrobacterium 

tumefaciens, strain ABI

Transformed LH59 (a conventional corn variety) callus 

tissue with the vector PV-ZMAP595 in Agrobacterium 

tumefaciens

Selected transformants and generated 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 studies 
Monsanto Company 07-CR-191U Page 99 of 544 

IV. Donor Genes and Regulatory Sequences 

This section describes the donor genes and regulatory sequences used in the development 

of MON 87460 and the deduced amino acid sequences 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 transformation of corn embryos to produce 

MON 87460 is shown in Figure III-1 and its elements 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 confers resistance to kanamycin. 

The T-DNA that is expected to incorporate into 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 contains 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 repressor 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 protein, an RNA chaperone protein from Bacillus 

subtilis, which is associated with reduced yield loss under water limitation through the 

unfolding of RNA secondary structure thereby, facilitating RNA translation (Phadtare et 

al., 2002). The amino acid sequence of the CSPB protein produced in MON 87460 is 

identical to the native CSPB protein (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 amino acid change was implemented to facilitate 

the assembly of the plasmid vector PV-ZMAP595 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 Elements in Plasmid Vector PV-ZMAP595 

Genetic Element 

Location in 

Plasmid Function (Reference) 

Vector Backbone 

Intervening Sequence 1 – 52 Sequence used in DNA cloning 

CS1

-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 

OR2

-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 an 

aminoglycoside-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 

B3-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 

P4

-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 

T6-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; 2OR – Origin of Replication; 3B – Border; 4P – Promoter; 5I – 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.) 

T6-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 transcriptional termination sequence and polyadenylation 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 Sequence 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 Encoding 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 Sequence of the Full Length NPTII Protein 

Present in MON 87460 

The amino acid sequence of the NPTII 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 polyadenylation (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 facilitate 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 shown 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 Virus (Odell et al., 1985). Following the nptII coding 

sequence is the 3' nontranslated 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 involved 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 the 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 for 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 (Fling et al., 1985) prior to plant 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 transcription 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. Seurinck, M. v.Montague and J. Schell. 1983. 

Identification of sequences involved in polyadenylation 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 Tn7

gene encoding an aminoglycoside-modifying enzyme, 3′(9)-Onucleotidyltransferase. Nucleic 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. Fry, 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 and R. Wu. 1991. Construction of expression 

vectors based on the rice actin 1(Act1) 5′ region for use in monocot 

transformation. Molecular and 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. Helinski. 1981. Nucleotide sequence of the region 

of the origin of replication of the broad 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 affecting 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 characterize the integrated DNA in MON 87460. 

Southern blot analyses were used to determine 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 the plant. The Southern strategy was used 

to insure that all detectable fragments would have been detected in the analysis. The corn 

genome was assayed with probes that spanned the entire transformation 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 1/10th

genome equivalent of the positive control. Two restriction enzyme sets were specifically 

chosen to independently confirm the presence of the insert and minimize the possibility 

that two DNA fragments could comigrate on the gel. In addition, 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 fragments 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 multiple generations was also demonstrated by 

Southern blot fingerprint analysis. Seven generations of MON 87460 were digested with 

one of the enzyme sets utilized for the copy number analysis and were hybridized with 

probes that would detect the entire insert (two hybridization bands). This fingerprint 

strategy consists of two border fragments that assay not only the stability of the insert, but 

also the stability of genomic DNA directly adjacent to the insert. 

The DNA sequencing analyses compliment the Southern 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 addition, 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 genome 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 characterize the T-DNA that was integrated into 

the corn genome. For each digest, there were duplicated samples which 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 potential 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 hybridized bands on Southern blots. Minor 
Monsanto Company 07-CR-191U Page 107 of 544 

differences in the sizes of the hybridization 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 information and results derived from the molecular 

analyses were used to construct a linear map 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 provides information on the expected banding patterns and sizes 

of the DNA fragments after restriction enzyme digestion. The linear map is shown in 

Figure V-1. Based on the insert linear map and the plasmid map, a table 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 Element1

Location in 

Sequence2 Function (Reference) 

Sequence flanking 5' 

end of the insert 

1-1121 

Corn genomic DNA 

P3-Ract187460 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 

CS5

-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 

T6

-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 intervening 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 transcriptional termination sequence and polyadenylation 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 

B7

-Left Border87460 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 – Border
Monsanto Company 07-CR-191U Page 110 of 544 

Figure V-1. Schematic Representation of the Insert and Genomic Flanking Sequences in MON 87460 A linear map of the insert and known genomic DNA flanking the insert in MON 87460 is shown. Identified on the m

within the insert, as well as restriction sites with positions relative to the size of the linear map for enzymes used in the So

on the lower portion of the map are the expected sizes of the DNA fragments after digestions with respective restriction enenzymes. The dotted line indicates the additional DNA fragment that would be present if partial digestion of the internsite occurs. Arrows with dotted lines indicate the end of the insert and the beginning of corn genomic flanking sequence.

EcoO109 I 64

EcoR

V

Hind III

EcoO109I/Not I

EcoO109 I 875 EcoR

V 2225

Not I 2553

>2.2 kb 2.7 kb

>4.5 kb

0.8 kb 1.7 kb >2.7 kb

P-Ract187460

I-Ract1

CS-cspB

T-tr7

loxP

loxP

P-35S

CS-nptII

T-nos

B-Left Border87460

5' Genomic Flank DNA

1 

EcoO109 I 64

EcoR

V

Hind III

EcoO109I/Not I

EcoO109 I 875 EcoR

V 2225

Not I 2553

>2.2 kb 2.7 kb

>4.5 kb

0.8 kb 1.7 kb >2.7 kb

P-Ract187460

I-Ract1

CS-cspB

T-tr7

loxP

loxP

P-35S

CS-nptII

T-nos

B-Left Border87460

5' Genomic Flank DNA

1 

EcoO109 I 64

EcoR

V

Hind III

EcoO109I/Not I

EcoO109 I 875 EcoR

V 2225

Not I 2553

>2.2 kb 2.7 kb

>4.5 kb

0.8 kb 1.7 kb >2.7 kb

P-Ract187460

I-Ract1

CSEcoO109 I 64

EcoR

V

Hind III

EcoO109I/Not I

EcoO109 I 875 EcoR

V 2225

Not I 2553

>2.2 kb 2.7 kb

>4.5 kb

0.8 kb 1.7 kb >2.7 kb

P-Ract187460

I-Ract1

CS-cspB

T-tr7

loxP

loxP

P-35S

CS-nptII

T-nos

B-Left Border87460

5' Genomic Flank DNA

1 

EcoO109 I 64

EcoR

V

Hind III

EcoO109I/Not I

EcoO109 I 875 EcoR

V 2225

Not I 2553

>2.2 kb 2.7 kb

>4.5 kb

0.8 kb 1.7 kb >2.7 kb

P-Ract187460

I-Ract1

CS-cspB

T-tr7

loxP

loxP

P-35S

CS-nptII

T-nos

B-Left Border87460

5' Genomic Flank DNA

1 
Monsanto 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 Plasmid 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 exterior 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 the map) are detailed in the accompanying table. 

 

PV- ZMAP595

9379 bp

Blp I 2140

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

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

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

Blp I 2140

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

CS-rop

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

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

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

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

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

Blp I 2140

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

CS-rop

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

T-DNA

1

3

2

4

5

6
Monsanto 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 Plasmid 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 exterior 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 probes 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

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

T-DNA

7

10 9 8

13

12 

11

PV- ZMAP595

9379 bp

Blp I 2140

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

Blp I 2140

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

T-DNA

7

10 9 8

13

12 

11

CS-rop

PV- ZMAP595

9379 bp

Blp I 2140

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

T-DNA

7

10 9 8

13

12 

11

PV- ZMAP595

9379 bp

Blp I 2140

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

Blp I 2140

EcoO109 I 3198

EcoO109 I 8956

EcoO109 I 8998

EcoR V 5041

Not I 5369

Xba I 5376

OR-ori.pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

T-tr7 CS-cspB

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

T-DNA

7

10 9 8

13

12 

11

CS-rop
Monsanto Company 07-CR-191U Page 113 of 544 

Table V-2. Summary Chart of the Expected 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 

BlpI + 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 -- -- -- -- -- -- -- -- 

EcoR 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 

EcoO109 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 probes were used in Southern blot analysis. 

2

 ‘--’ Indicates that the particular restriction enzyme 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 number) 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 detected indicates the number of inserts present in MON 87460. 

The number of copies of the T-DNA (copy number) 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 with the entire T-DNA will result in two 

bands, each representing a portion 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 determine 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 Hind III or EcoR V 

produced several hybridization signals. These hybridization signals result from the 
Monsanto Company 07-CR-191U Page 114 of 544 

probes hybridizing to endogenous sequences residing 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 considered to be endogenous background. 

To ensure that each of the T-DNA probes was able to hybridize to their 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 copy equivalence) with the control DNA pre-digested with 

EcoR 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 comparison 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 are hybridizing 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 EcoR V. The expected hybridization bands of approximately 3.2 and 

6.1 kb (Figure V-4, lane 7) were detected (refer to Figure V-2 and Figure V-3 plasmid 

map). 

MON 87460 DNA digested with Hind III (Figure V-4, lanes 2 and 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 greater than 4.5 kb (Figure V-1) and confirms 

that MON 87460 contains one insert located within a 6.8 kb Hind III restriction fragment. 

MON 87460 DNA digested with EcoR 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 inserted DNA along with the adjacent genomic 

DNA flanking the 5' end of the insert. 

The results of MON 87460 presented in Figure V-4 show that MON 87460 contains a 

single copy of the T-DNA that resides at a single locus of integration on an 

approximately 6.8 kb Hind III restriction fragment. Southern blot analyses described 

below using a combination of EcoO109 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 EcoO109 I and Not I or the restriction enzyme EcoR V to determine if 

PV-ZMAP595 sequences, other than the T-DNA region, were inserted in MON 87460. 

The samples were electrophoresed, blotted, and the blot was hybridized simultaneously 

with three overlapping probes (Figure V-2, probes 4-6) that spanned the backbone 

sequence of PV-ZMAP595. The results are shown in Figure V-5. Probe template spikes 

(Figure V-2, probes 4-6) generated from the plasmid PV-ZMAP595 were mixed with the 
Monsanto Company 07-CR-191U Page 115 of 544 

pre-digested control genomic DNA to serve as a positive hybridization control. 

Additionally, plasmid PV-ZMAP595 DNA previously digested with the combination of 

Blp I and Xba I was mixed with control genomic DNA digested with EcoR 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 were detected when probing with the 

backbone sequences, the blot contained conventional corn genomic DNA digested with a 

combination of EcoO109 I and Not I (Figure V-5, lanes 1 and 8) or EcoR V (Figure V-5, 

lanes 3 and 10). The conventional corn genomic DNA digested with EcoO109 I and 

Not I (Figure V-5, lanes 1 and 8) or EcoR 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 produced in both test and control lanes, and 

therefore are considered to be endogenous background. 

To ensure that each backbone probe was capable 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 concentrations with the control DNA pre-digested 

with EcoR V were included on the blot (Figure V-5, lanes 5 and 6). The expected 

hybridization bands at 1.4, 1.7, and 2.0 kb were detected. The results show that the three 

probes hybridized, as expected, to the target DNA. To ensure that the backbone probes 

hybridize to the plasmid used for transformation, plasmid PV-ZMAP595 digested with 

Blp I/Xba I was mixed with conventional control DNA digested with EcoR 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 the 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 EcoO109 I and Not I (Figure V-5, lanes 2 and 9) or EcoR V (Figure V-5, 

lanes 4 and 11) showed no detectable hybridization signal besides 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 32P-labeled T-DNA probes that 

span the insert (Figure V-2, probes 1-3). Each lane contains ~10 μg of digested genomic DNA 

isolated from seed. Lane designations are as follows: 

 1: Conventional (Hind III) 

 2: MON 87460 (Hind III) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with probe templates [~0.1 copy] 

 6: Conventional (EcoR V) spiked with probe templates [~1 copy] 

 7: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 8: Conventional (Hind III) 

 9: MON 87460 (Hind III) 

 10: Conventional (EcoR V) 

 11: MON 87460 (EcoR 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 RUN
Monsanto 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 32P-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 from seed. Lane designations are as follows: 

 1: Conventional (EcoO109 I/Not I) 

 2: MON 87460 (EcoO109 I/Not I) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with probe templates [~0.1 copy] 

 6: Conventional (EcoR V) spiked with probe templates [~1 copy] 

 7: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 8: Conventional (EcoO109 I/Not I) 

 9: MON 87460 (EcoO109 I/Not I) 

 10: Conventional (EcoR V) 

 11: MON 87460 (EcoR 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 RUN
Monsanto 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 assessed by digesting MON 87460 genomic DNA with 

restriction enzyme EcoR V or a combination of EcoO109 I and Not I and hybridizing 

Southern blots with probes covering the inserted cspB and nptII cassettes. The size of the 

genomic fragments and the T-DNA elements expected to be contained in each of those 

fragments is indicated below and summarized in Table V-2. 

Digestion of MON 87460 genomic DNA with the combination of EcoO109 I and Not I 

was expected to generate two border fragments with expected sizes of 1.7 kb and greater 

than 2.7 kb (Figure V-1). The 1.7 kb restriction fragment contains genomic DNA 

flanking the 5' end of the insert, Ract187460 promoter and Ract1 leader, Ract1 intron, cspB

coding sequence, and the tr7 3' nontranslated sequence. The 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 EcoR 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 contains genomic DNA flanking the 5' end of the 

insert, Ract187460 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) contained several controls. To determine if 

any endogenous background hybridization bands were detected when probing with probe 

7, the blot contained conventional corn genomic DNA digested with a combination of 

EcoO109 I and Not I (Figure V-6, lanes 1 and 7) or EcoR 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. These signals were produced in both test and 

control lanes, and therefore 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 control 
Monsanto 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 EcoO109 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 (Figure V-6, lane 8) appears slightly larger 

than the corresponding band in the short run (Figure V-6, lane 2), most likely due to 

better resolution of the band in the longer run. MON 87460 DNA digested with EcoR V 

(Figure V-6, lanes 4 and 10) produced a single unique band of approximately 7.5 kb. 

This is consistent with the expected band being greater than 2.2 kb (Figure V-1). This 

band in the long run (Figure V-6, lane 10) appears slightly larger than the corresponding 

band in the short run (Figure V-6, lane 4), most likely due to better resolution of the band 

in the longer run. There were no additional bands detected using the Right Border, 

promoter and leader sequence probe. Based on the results presented in Figure V-6, 

MON 87460 contains no additional, 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 digested with a combination of EcoO109 I and Not I 

(Figure V-7, lanes 1 and 7) or EcoR V (Figure V-7, lanes 3 and 9). The results of this 

analysis show no detectable 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 EcoR V pre-digested control DNA. 

Results of this experiment produced an expected band which migrated at approximately 

3.1 kb (Figure V-7, lanes 5 and 6). 

MON 87460 DNA digested with a combination of EcoO109 I and Not I (Figure V-7, 

lanes 2 and 8) that was electrophoresed, blotted, and hybridized with probe 8 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 likely 

due to better resolution of the band in the long run. MON 87460 DNA digested with

EcoR 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 hybridization bands detected using the Ract1 intron probe. 

Based on the results presented in Figure 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 digested with a combination of EcoO109 I and Not I 
Monsanto Company 07-CR-191U Page 120 of 544 

(Figure V-8, lanes 1 and 7) or EcoR V (Figure V-8, lanes 3 and 9). The results of this 

analysis show no detectable hybridization bands, 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 EcoO109 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 likely due to better resolution of the band in 

the long run. MON 87460 DNA digested with EcoR 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 expected 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, MON 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 detected when probing with probe 10, the blot contained 

conventional corn genomic DNA digested with a combination of EcoO109 I and Not I 

(Figure V-9, lanes 1 and 7) or EcoR V (Figure V-9, lanes 3 and 9). The results of this 

analysis show no detectable hybridization bands, 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 EcoO109 I and Not I (Figure V-9, 

lanes 2 and 8) and hybridized with probe 10 produced the expected 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 approximately 2.5 kb that is likely a result of 

partial digestion. The 2.5 kb band is consistent with partial digestion of genomic DNA 

because there is an EcoO109 I site at position 875 in the 5' flanking genomic DNA 

(Figure V-1). MON 87460 DNA digested with EcoR V (Figure V-9, lanes 4 and 10) and 

hybridized with probe 10 produced the expected 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 expected size for the 3 ́ border fragment 

(Figure V-1). The 2.7 kb band is less intense 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 bands detected using the tr7 3' nontranslated probe. Based on 

the results presented in Figure V-9, MON 87460 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) contained 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 EcoO109 I 

and Not I (Figure V-10, lanes 1 and 7) or EcoR 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 EcoO109 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 EcoR V (Figure V-10, lanes 4 and 10) 

and hybridized with probe 11 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-10 

show that MON 87460 contains no additional, detectable loxP sequence or 35S promoter 

elements other than those associated 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 detected when probing with probe 12, the blot contained 

conventional corn genomic DNA digested with a combination of EcoO109 I and Not I 

(Figure V-11, lanes 1 and 7) or EcoR V (Figure V-11, 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 

and hybridized with probe 12. Results of this 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 EcoO109 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 appears slightly larger than 

the corresponding band in the short run, most likely 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 EcoR V (Figure V-11, lanes 4 and 10) and 
Monsanto Company 07-CR-191U Page 122 of 544 

hybridized with probe 12 produced the expected single unique 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 Figure 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) contained several controls. To determine if 

any endogenous background hybridization bands were detected when probing with probe 

13, the blot contained conventional corn genomic DNA digested with a combination of 

EcoO109 I and Not I (Figure V-12, lanes 1 and 7) or EcoR 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 EcoO109 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 EcoR 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 32P-labeled probe that spanned the Right Border, Ract1 promoter 

and leader (Figure V-3, probe 7). Each lane contains approximately 10 μg of digested genomic 

DNA isolated from seed. Lane designations are as follows: 

 1: Conventional (EcoO109 I/Not I) 

 2: MON 87460 (EcoO109 I/Not I) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~0.1 copy] 

 6: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 7: Conventional (EcoO109 I/Not I) 

 8: MON 87460 (EcoO109 I/Not I) 

 9: Conventional (EcoR V) 

 10: MON 87460 (EcoR 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

15
Monsanto 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 32P-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 (EcoO109 I/Not I) 

 2: MON 87460 (EcoO109 I/Not I) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~0.1 copy] 

 6: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 7: Conventional (EcoO109 I/Not I) 

 8: MON 87460 (EcoO109 I/Not I) 

 9: Conventional (EcoR V) 

 10: MON 87460 (EcoR 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 RUN
Monsanto 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 32P-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 (EcoO109 I/Not I) 

 2: MON 87460 (EcoO109 I/Not I) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~0.1 copy] 

 6: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 7: Conventional (EcoO109 I/Not I) 

 8: MON 87460 (EcoO109 I/Not I) 

 9: Conventional (EcoR V) 

 10: MON 87460 (EcoR 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 RUN
Monsanto 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 32P-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 (EcoO109 I/Not I) 

 2: MON 87460 (EcoO109 I/Not I) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~0.1 copy] 

 6: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 7: Conventional (EcoO109 I/Not I) 

 8: MON 87460 (EcoO109 I/Not I) 

 9: Conventional (EcoR V) 

 10: MON 87460 (EcoR 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 RUN
Monsanto 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 32P-labeled probe that spanned the loxP sequence and 35S

promoter (Figure V-3, probe 11). Each lane contains approximately 10 μg of digested genomic 

DNA isolated from seed. Lane designations are as follows: 

 1: Conventional (EcoO109 I/Not I) 

 2: MON 87460 (EcoO109 I/Not I) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~0.1 copy] 

 6: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 7: Conventional (EcoO109 I/Not I) 

 8: MON 87460 (EcoO109 I/Not I) 

 9: Conventional (EcoR V) 

 10: MON 87460 (EcoR 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 RUN
Monsanto 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 32P-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 (EcoO109 I/Not I) 

 2: MON 87460 (EcoO109 I/Not I) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~0.1 copy] 

 6: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 7: Conventional (EcoO109 I/Not I) 

 8: MON 87460 (EcoO109 I/Not I) 

 9: Conventional (EcoR V) 

 10: MON 87460 (EcoR 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 RUN
Monsanto 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 32P-labeled probe that spanned the nos 3' nontranslated sequence, 

loxP sequence and Left Border sequence (Figure V-3, probe 13). Each lane contains 

approximately 10 μg of digested genomic DNA isolated from seed. Lane designations are as 

follows: 

 1: Conventional (EcoO109 I/Not I) 

 2: MON 87460 (EcoO109 I/Not I) 

 3: Conventional (EcoR V) 

 4: MON 87460 (EcoR V) 

 5: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~0.1 copy] 

 6: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 7: Conventional (EcoO109 I/Not I) 

 8: MON 87460 (EcoO109 I/Not I) 

 9: Conventional (EcoR V) 

 10: MON 87460 (EcoR 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 RUN
Monsanto Company 07-CR-191U Page 130 of 544 

V.D. Southern Blot Analyses of MON 87460 across Multiple Generations 

In order to assess the stability of the inserted DNA in MON 87460 across generations, 

Southern blot analyses were performed using 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 EcoR V (refer to generations indicated in bold in Figure V-13). Digestion 

of the test materials with EcoR V was expected to release two border fragments with 

expected sizes of 2.7 kb and >2.2 kb (Figure V-1). The blot was hybridized 

simultaneously with three radiolabeled probes 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)F1] 

generation to determine insert stability. 

The Southern blot used to confirm generational stability of the T-DNA (Figure V-14) 

contained several controls. To determine if any endogenous background hybridization 

bands were detected when hybridizing with the three radiolabeled probes that span the 

entire T-DNA, the blot contained conventional control DNA digested with EcoR V

(Figure V-14, lane 1). The results of this analysis 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 spikes (Figure V-2, probes 1-3) that were generated from 

plasmid PV-ZMAP595 and mixed at different concentrations with control DNA 

pre-digested with EcoR V (Figure V-14, lanes 2 and 3). When hybridized with three 

overlapping 32P-labeled probes that span the entire T-DNA (Figure V-2, probes 1-3), the 

expected hybridization bands at approximately 1.4, 1.6, and 2.0 kb were detected. The 

0.1 and 1 copies of the 1.4 kb band are faint in comparison 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 are 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 EcoR V (Figure V-14, lane 4). 

Hybridization with probes 1-3 produced the expected size bands at approximately 3.2 and 

6.1 kb in addition to the endogenous background. 

MON 87460 DNA isolated from multiple generations of MON 87460 (Figure V-13), 

digested with restriction enzyme EcoR V, and hybridized with three overlapping 32Plabeled probes that span the entire T-DNA (Figure V-2, probes 1-3) produced two 

hybridization bands at 2.7 kb and approximately 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 the 
Monsanto 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 F1 generation 

(LH59 R3 x LH244) shown in Figure V-4 (lanes 4 and 11). There were no additional 

unexpected bands detected, indicating that the single copy 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 stability of the insert were also tested 

for the presence of backbone sequence by Southern blot analysis. Test and control DNA 

samples were digested with EcoR V and the blot was hybridized simultaneously with 

three radiolabeled probes that span the entire backbone sequence of plasmid PVZMAP595 (Figure V-2, probes 4-6). 

The Southern blot used to confirm the absence of the PV-ZMAP595 backbone sequences 

(Figure V-15) contained several controls. To determine if any endogenous background 

hybridization bands were detected when hybridizing with the three radiolabeled backbone 

probes, the blot contained conventional control DNA digested with EcoR V (Figure V15, lane 1). Several hybridization bands were detected. These signals were produced in 

all lanes, including those lanes containing the conventional control DNA material, and 

therefore they are considered endogenous background. 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 of the backbone probes was capable of hybridizing to its respective 

target, the blot contained probe template spikes (Figure V-2, probes 4-6) that were 

generated from plasmid PV-ZMAP595 and mixed with the control DNA pre-digested 

with EcoR V (Figure V-15, lanes 2 and 3). The expected 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 weight markers are slightly higher than 

expected. 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 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 was digested with a combination of Blp I and Xba I 

and mixed with control DNA pre-digested with EcoR V (Figure V-15, lane 4). 

Hybridization with probes 4-6 produced the expected size bands at approximately 3.2 and 

6.1 kb, in addition to the endogenous background produced by the conventional control 

DNA. 

MON 87460 DNA isolated from multiple generations of MON 87460 (refer to Breeding 

History of MON 87460, Figure V-13), digested with restriction enzyme EcoR V, and 

hybridized with three overlapping 32P-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 tested 
Monsanto Company 07-CR-191U Page 132 of 544 

do not contain any detectable backbone sequence from the transformation vector 

PV-ZMAP595. 

Figure V-13. MON 87460 Breeding Diagram 

The (LH59 R3 × LH244)F1 generation was used for the molecular characterization of 

MON 87460. The (LH59 R2 × LH244) F1, (LH59 R3 × LH244) F1, (LH59 R3 × LH244) F2, 

LH59 R4, (LH59 R4 × HCL301) F1, (LH59 R4 × HCL301) F2, and (RP×BC2F1) BC3F1 generations 

were used for generational stability (indicated in bold). The (LH59 R3 × LH244) F2 and (LH59 

R4 × HCL301) F2 generations were used for expression and composition analyses. 

R0 = transformed plant; F(#) = filial generation; ⊗ = self-pollination; BC(#) = 

backcross generation; RP = recurrent parent for commercial seed 

development; TI = trait integration for commercial seed development. 

R0

TI: (RP × LH59 R4)BC0F1

TI: (RP × BC2F1)BC3F1

LH59 R0

LH59 R1

LH59 R2

LH59 

LH59 R3

LH59 R5

⊗

⊗

⊗

⊗

⊗

× LH59 

(LH59 R2 × LH244)F1 

(LH59 R3 × LH244)F1 

(LH59 R3 × LH244)F2

(LH59 R4 × HCL301)F1

(LH59 R4 × HCL301)F2

× 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 32P-labeled probes that spanned the entire 

backbone sequence (Figure V-2, probes 1-3). Each 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 (EcoR V) 

 2: Conventional (EcoR V) spiked with probe templates [~0.1 copy] 

 3: Conventional (EcoR V) spiked with probe templates [~1 copy] 

 4: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 5: MON 87460 [(LH59 R2 x LH244)F1, EcoR V] 

 6: MON 87460 [(LH59 R3 x LH244)F1, EcoR V] 

 7: MON 87460 [(LH59 R3 x LH244)F2, EcoR V] 

 8: MON 87460 (LH59 R4, EcoRV) 

 9: MON 87460 [(LH59 R4 x HCL301)F1, EcoR V] 

 10: MON 87460 [(LH59 R4 x HCL301)F2, EcoR V] 

 11: MON 87460 [(RP x BC2F1)BC3F1, EcoR 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 11
Monsanto 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 32P-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 MON 87460 is illustrated in Figure V-13. Lane 

designations are as follows: 

 1: Conventional (EcoR V) 

 2: Conventional (EcoR V) spiked with probe templates [~0.1 copy] 

 3: Conventional (EcoR V) spiked with probe templates [~1 copy] 

 4: Conventional (EcoR V) spiked with PV-ZMAP595 (Blp I/Xba I) [~1 copy] 

 5: MON 87460 [(LH59 R2 x LH244)F1, EcoR V] 

 6: MON 87460 [(LH59 R3 x LH244)F1, EcoR V] 

 7: MON 87460 [(LH59 R3 x LH244)F2, EcoR V] 

 8: MON 87460 (LH59 R4, EcoRV) 

 9: MON 87460 [(LH59 R4 x HCL301)F1, EcoR V] 

 10: MON 87460 [(LH59 R4 x HCL301)F2, EcoR V] 

 11: MON 87460 [(RP x BC2F1)BC3F1, EcoR 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 11
Monsanto Company 07-CR-191U Page 135 of 544 

V.E. Organization of the Inserted Genetic Elements of MON 87460 

The organization of the genetic elements within the insert of MON 87460 was confirmed 

by DNA sequence analyses. Several polymerase chain reaction (PCR) primers were 

designed to amplify overlapping DNA fragments spanning 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 base 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 MON 87460 insert and this molecular 

rearrangement presumably resulted from double-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 elements is as depicted in Table V-1. 

V.F. Inheritance of the Genetic Insert in MON 87460 

During the development of the MON 87460, trait segregation data were generated and 

analyzed. Chi-square analysis was performed over two generations to confirm the 

segregation and stability of the cspB gene in MON 87460. The Chi-square analysis is 

based on testing the observed segregation ratio to the expected segregation ratio 

according to Mendelian principles. The R0 plant was self-pollinated to produce R1 seed, 

which is expected to segregate 1:2:1 (1 homozygote: 2 hemizygous: 1 null segregant) for 

the gene. A homozygous selection (R1 plant) was identified from the segregating 

population by using an NPTII based Invader assay. The selected R1 plant was selfpollinated again to produce R2 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 BC3F1, BC3F2, BC3F3, BC4F1, and 

BC5F1 seed. These generations are derived from the R4 generation identified in 

Figure V-13. The BC3F1, BC4F1, and BC5F1 generations were expected to segregate 1:1 

(1 positive:1 negative). The BC3F2 generation was expected to segregate 1:2:1 (1 

homozygote:2 hemizygous:1 null segregant) for the gene. The BC3F3 generation was 

expected to be fixed for the trait. 

The Chi-square test was computed as: 

∑ − =

e

o e

x

( )

2

2

where o = observed frequency of the genotype and e = expected 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 R1, BC3F1, BC3F2, BC4F1, and BC5F1 generations indicated no 

significant differences between the observed and expected segregation ratios. The data 

for the R2 and BC3F3 generations confirmed that the populations 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 the expected Mendelian 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) 

 

R1 36 26 10 27 9 0.1481 NS 

 

R2 89 89 0 89 0 Fixed ⎯

 

BC3F1 178 84 94 89 89 0.562 NS

 

BC3F2 154 124 30 115.5 38.5 2.502 NS

 

BC3F3 474 474 0 474 0 Fixed ⎯

 

BC4F1 80 44 36 40 40 0.800 NS

 

BC5F1 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 elements from the transformation vector PVZMAP595, linked or unlinked to the intact DNA insert, were detected in the genome of 

MON 87460. Additionally, backbone sequence from PV-ZMAP595 was not detected. 

Generational stability analysis demonstrate that the expected Southern blot fingerprint of 

MON 87460 has been maintained across seven generations of breeding, thereby 

confirming the stability of the DNA insert over multiple generations. These generations 

were also shown not to contain any detectable backbone sequence from plasmid PVZMAP595. In addition, DNA sequence analyses confirmed the sequence identity 

between the MON 87460 insert and the portion of the T-DNA from PV-ZMAP595 that 

was integrated into the corn genome. These results also confirmed the organization of 

the genetic elements within the cspB and nptII expression cassettes of MON 87460, 

which was identical to that in plasmid PV-ZMAP595. Analysis of the T-DNA insertion 
Monsanto Company 07-CR-191U Page 137 of 544 

site indicates that there is a 22-bp deletion of genomic DNA at the insert-to-plant DNA 

junction. Segregation analyses show heritability and stability of the cspB and nptII genes 

occurred as expected across multiple generations, which corroborates the molecular insert 

stability analysis and establishes the genetic 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 transcription of the 

nopaline synthase gene region of T-DNA. Nucleic Acids Research. 11:369-385. 

Dhaese, P., H. d.Greve, J. Gielen, J. Seurinck, M. v.Montague and J. Schell. 1983. 

Identification of sequences involved in polyadenylation 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. Fry, 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 and R. Wu. 1991. Construction of expression 

vectors based on the rice actin 1(Act1) 5′ region for use in monocot 

transformation. Molecular and 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. 3rd edition. Cold 

Spring Harbor Laboratory Press, 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 affecting cell viability at low 

temperatures. Journal of Bacteriology. 174:6326-6335. 

 
Monsanto Company 07-CR-191U Page 138 of 544 

VI. Characterization of the Introduced CSPB and NPTII Proteins 

Characterization of the introduced proteins is important to establishing the basic 

components of the food, feed and environmental 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. coliproduced proteins used in protein safety studies, (b) the expression levels of the proteins 

determined in corn tissues, and (c) a summary 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 the cold-shock protein (CSP) family and is 

identical in amino acid sequence to the 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 purposes. 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 transition from exponential growth to stationary phase 

(Graumann et al., 1997; Graumann and Marahiel, 1999), indicating that CSPB 

accumulation in 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). 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 CSPB protein has a very low thermodynamic 

stability and is susceptible to rapid proteolytic degradation (Schindler 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 sites for single stranded nucleic 

acids called RNA-binding ribonucleoprotein (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-stranded polynucleotide unwinding 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 adopt 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 open (Phadtare et al., 

2002). It was suggested that CSPs bind to single stranded nucleic acids, RNA and 
Monsanto 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 confirmed by co-crystallization of the B. subtilis CSPB 

protein in a complex with single stranded polynucleotides (Bienert et al., 2004; Max et 

al., 2007). The crystal structure data revealed the stoichiometry and sequence 

determinants of the binding of single-stranded nucleic acids to a preformed site on CSPB. 

These findings together with the described mechanism 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 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. This assay has been broadly utilized to characterize the 

specificity of a variety of CSPs including cold-shock domain (CSD)-containing 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 different tissues of MON 87460 are relatively 

low. Therefore, it was necessary to produce the protein in a high-expressing, 

recombinant microorganism in order to obtain 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 characteristics and functional activity between the 

MON 87460-produced and E. coli-produced CSPB protein was confirmed by a panel of 

analytical techniques, including sodium dodecyl sulfate polyacrylamide gel 

electrophoresis (SDS-PAGE), western blot analysis, matrix assisted laser 

RNP1 RNP2

VEGKVKWFNSEKGFGFIEVEGQDDVFVHFSAIQGEGFKTLEEGQAVSFEIVEGNRGPQAANVTKEA 

β1 β2 β3 β4 β5
Monsanto Company 07-CR-191U Page 140 of 544 

desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), glycosylation 

analysis, and assay of biological 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 the identity of the MON 87460-produced CSPB 

protein and established the equivalence of the plant produced protein to the E. coliproduced CSPB reference protein standard. The molecular weight of the MON 87460- 

and E. coli-produced CSPB proteins was estimated 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 equivalent in their molecular weight. The 

electrophoretic mobility and immunoreactive 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 yielded 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 activities and the lack of glycosylation. 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 reference 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 transformation (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 cells 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 NPTII 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 previous safety assessment data performed on E. coliproduced 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 
Monsanto 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. coliproduced NPTII reference protein standard. The results of this analysis confirmed the 

identity of the MON 87460-produced 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 compare the immunoreactivity and apparent 

molecular weight of the MON 87460-produced NPTII protein to that of the previously 

characterized E. coli-produced NPTII reference protein standard. The MON 87460- and 

E. coli-produced NPTII proteins displayed similar immunoreactivity with NPTII-specific 

antibody and had identical electromobility on SDS-PAGE. Taken together, these data 

establish equivalence between 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 proteins in various tissues of MON 87460 were 

determined using enzyme-linked immunosorbent assays (ELISA). The materials and 

methods for the ELISA analysis, as well as a description 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. during 2006 under typical agronomic 

practices and water conditions. The second season was conducted at four sites in Chile 

during 2006/2007 using a strip-plot design to establish two water treatment levels (wellwatered and water-limited) to assess for any changes in CSPB and NPTII protein levels 

under different soil moisture conditions. The sites were located in the major corngrowing regions of the U.S. and Chile. Additional 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-season whole plant (OSWP) samples were 

collected four times (1-4) over the season corresponding to plant 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. 

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 expression 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 samples from both field studies. Consistent 

with the constitutive nature of the rice actin promoter, no obvious difference was 

observed in CSPB protein levels in tissues 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 levels. The NPTII protein was 

detected in three out of four analyzed tissue types, with the highest level determined in 
Monsanto 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), senescent root (0.041 μg/g dwt), and forage 

root (0.029μg/g dwt) (Table VI-1). In tissues harvested throughout the growing season, 

mean CSPB protein levels in MON 87460 across 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 represent 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 reference 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 established 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 three sites that were subjected to either wellwatered or water-limited conditions were highest 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 conditions), young root (OSR1, 1.3 μg/g dwt 

well-watered and 1.5 μg/g dwt water-limited conditions), silk (0.82 μg/g dwt wellwatered and 1.1 μg/g dwt μg/g dwt water-limited conditions), forage (0.11 μg/g dwt μg/g 

dwt well-watered and 0.15 μg/g dwt water-limited conditions), grain (0.048 μg/g dwt 

μg/g dwt well-watered and 0.038 μg/g dwt water-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 water-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 throughout 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 from 0.44 – 2.8 μg/g dwt in leaves harvested 

from plants grown under water-limited conditions. The mean CSPB protein levels in 

roots harvested from MON 87460 plants across 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 protein 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 mean NPTII protein levels across all sites 

under typical agronomic practices were highest 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 forage (0.12μg/g dwt) (Table VI-3). The 

levels of NPTII protein in grain were below the NPTII assay Limit of Quantitation 

(LOQ), which was 0.0047 μg/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 μg/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 (OSL1, 2.4 μg/g dwt well-watered 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). The levels of NPTII protein in grain were 

below the NPTII assay LOQ (0.0047 μg/g fwt for grain) for tissue collected from plants 

under both well-watered and water-limited conditions. The NPTII protein levels in 

MON 87460 were lower than NPTII levels determined for the equivalent tissue types in 

MON 863 that also relied on NPTII 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 μg/g 

fwt, respectively. The range in NPTII protein 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 μg/g 

fresh weight for well-watered and water-limited, respectively. 

 
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Table VI-1. CSPB Protein Levels in Tissues Collected from MON 87460 Produced 

in the U.S. during 2006 under Typical Agronomic Conditions 

Tissue Type1

Mean (SD)2

(μg/g fwt)3

Range4

(μg/g fwt) 

Mean (SD) 

(μg/g dwt)5

Range 

(μg/g dwt) 

LOQ / LOD 

(μg/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 the 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) were 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 (μg) of protein per gram (g) of tissue on a fresh weight (fwt) 

basis. 4

Minimum and maximum values were determined for each tissue type across all sites. 5

Protein levels are expressed as “μg/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. 

 
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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 

 Well-Watered Water-Limited

Tissue 

Type

1

Mean (SD)2 

Range3 

(μg/g fwt)4

Mean (SD) 

Range 

 (μg/g dwt)5 

Mean (SD) 

Range 

 (μg/g fwt) 

Mean (SD) 

Range 

 (μg/g dwt) 

LOQ / LOD

(μg/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 deviation (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 determined for each tissue type across all sites. 4

Protein levels are expressed as microgram (μg) of protein per gram (g) of tissue on a fresh weight (fwt) basis. 5

Protein levels are expressed as “μg/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. 
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Table VI-3. NPTII Protein Levels in Tissues Collected from MON 87460 Produced 

in the U.S. during 2006 under Typical Agronomic Conditions 

Tissue 

Type1

NPTII (μg/g fwt) 

Mean (SD)2

Range 

(μg/g fwt)3 

NPTII (μg/g dwt) 

Mean (SD)4

Range 

(μg/g dwt) 

LOQ / LOD 

(μg/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/A5 0.0047 / 0.0024

1

Over-season leaf (OSL-1) and over-season root (OSR-1) samples were collected at the V2-V4 plant 

growth stage. 2

Protein levels are expressed as microgram (μg) of protein per gram (g) of tissue on a fresh weight (fwt) 

basis. The arithmetic mean and standard deviation (SD) were calculated for each tissue type across all 

sites (n=18). 3

Minimum and maximum values were determined for each tissue type across sites. 4

Protein levels are expressed as μg/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 calculated 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-Watered and Water-Limited Conditions 

Well-Watered Water-Limited 

Tissue 

Type

1

Mean (SD)2 

Range3 

(μg/g fwt)4

Mean (SD) 

Range 

 (μg/g dwt)5 

Mean (SD) 

Range 

 (μg/g fwt) 

Mean (SD) 

Range 

 (μg/g dwt) 

LOQ / LOD 

(μg/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/A6

) 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 determined for each tissue type across sites. 4

Protein levels are expressed as microgram (μg) of protein per gram (g) of tissue on a fresh weight (fwt) 

basis. 5

Protein levels are expressed as μg/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 calculated if fresh weight values were less than the LOQ 

or LOD.
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VI.D. Food and Feed Safety Assessment Summary of the CSPB and NPTII Proteins 

Numerous factors have been considered in the safety assessment 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 designated enzyme preparations from this 

organism as GRAS (generally recognized as safe, FDA, 1999). Proteins 

containing cold shock domains are ubiquitous in nature, being present in many 

plants and common bacteria including species that are normally present in 

gastrointestinal flora. Cold shock proteins have no known toxicity and are not 

associated with pathogenicity. 

b) The B. subtilis CSPB shares a high percent of identity with CSPs present in other 

bacterial species broadly used by the food industry and with CSD-containing 

proteins in plant species used as a food. Many foods prepared 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 other naturally occurring CSD-containing proteins found in 

food and food products. The amino acid identity ranges from 35% to 98.5% 

across different plant and bacterial 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 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 bacterial 

cultures. These bacteria resist gastric acid, bile salts and pancreatic enzymes, and, 

thus, readily colonize the intestinal tract (Rolfe, 2000). In addition, Bacillus, 

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 Observable Effect Level (NOEL) from an acute 

mouse gavage study to estimates of the dietary 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 effects even at the highest 

tested dose levels, which are 4.7 and 5,000 mg/kg body weight for CSPB and 

NPTII proteins, respectively. The dietary 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 highest corn consumption on a body weight 

basis, the MOEs were greater than or equal to 11,400 and 208,000,000 for the 

CSPB and NPTII proteins, respectively. Dietary exposures in animals will also be 
Monsanto 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 consumption. A small transiently stable 

CSPB protein fragment was very quickly degraded during short exposure to SIF. 

Rapid digestion of the 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 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 protein toxins which have adverse effects 

to mammals. This has been shown by extensive assessments with bioinformatic 

tools, such as FASTA sequence alignment tool and an eight-amino acid sliding 

window search. 

f) The safety of the NPTII protein 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 made 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. 

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VII. Compositional Assessment 

Compositional analyses were conducted to assess the nutrient, anti-nutrient and key 

secondary metabolite levels in the forage and grain tissues derived 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 relevant to where commercial production would 

be expected. 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) 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 that 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. during 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). Results from these assessments were published 

by Harrigan et al. (2009). Samples from the Chile 2006/2007 study were also analyzed 

for 11 additional secondary metabolites that are potentially associated 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 intervals 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 selections 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 control corn were harvested from plants 

grown under typical agronomic practices with three replicates at each of six field sites in 

the U.S. within corn production regions. Two 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. Four sites were rainfed (IAE, IAW, IL, IN) and 

two (KS, NE) received supplemental irrigation. 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 from 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 commercial references grown at each site was 

collected from a single block. Forage was collected 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 statistical 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 all six field sites. Statistically 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 component 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 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 biological perspective. This comparative 

evaluation also considers natural 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-Nutrients, and Key Secondary Metabolites 

Forage and grain samples were harvested from all plots and analyzed for nutritional and 

anti-nutrient components. Compositional analyses of the forage samples included 

measurement of proximates (moisture, fat, protein, ash), carbohydrates 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, fatty acid composition (C8-C22), minerals 

(calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc), 

vitamins (vitamin B1 [thiamine], vitamin B2 [riboflavin], vitamin 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 and literature publications (Appendix E). 

In total, 77 different analytical 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, 15:0 
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pentadecanoic acid, 15:1 pentadecenoic acid, 17:0 heptadecanoic acid, 17:1 

heptadecenoic acid, 18:3 gamma linolenic 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) 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. There were a total of 434 comparisons 

made (seven sets of comparisons × 53 components 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 sites) analyses is presented in Table VII-2. 

Mean values, ranges, standard error values and statistical analyses 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 significant 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 control substances were within the 99% tolerance interval 

established from the commercial references. The individual site analysis show that there 

were no statistically significant differences (p>0.05) for 348 (93.5%) of the 372 

comparisons. Individual site differences were not reproducible across multiple sites and, 

where differences were observed, mean component values of the test and control 

substances were within the 99% tolerance interval (Table VII-2). Therefore, these 

differences were within the natural variability of corn for these components.

VII.A.2. Levels of Nutrients 

Corn forage and grain contain a variety of key nutrients that provide much of this crop’s 

value as a food and feed. The OECD consensus document on compositional 

considerations for corn describes the nutrients present in corn grain or processed corn 

products and includes proximates, fiber, minerals, 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 compositionally 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 combined-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 acid (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 the combined-site difference in the 18:0 stearic 

acid value was small (0.069% of total FA) and the mean component values for the test 
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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 small, and the mean values for these two 

components were within the 99% tolerance interval established from the commercial 

references grown at the same sites. Furthermore, 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 reflected the natural variability 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 protein, with each component difference being observed at 

only a single site. This lack of reproducibility 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 constituted no biological significance. For grain 

nutrients, individual site differences in components 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 linoleic acid, 18:3 linolenic acid, 22:0 behenic 

acid, ADF, TDF, thiamine, folic acid, and riboflavin (each at a single 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 interval 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 87460 and the control. The limited number of 

differences observed in this study reflected the natural variation of conventional corn and 

supported the compositional equivalence of MON 87460 to conventional corn. 

VII.A.3. Levels of Key Anti-Nutrients and Secondary Metabolites 

The OECD consensus document on compositional considerations for corn describes the 

anti-nutrients and secondary metabolites present in corn grain or processed corn products 

(OECD, 2002). The anti-nutrients assessed included phytic acid and raffinose. Phytic 

acid is widely distributed in plants and can limit the uptake of minerals such as calcium in 

higher animals (Lott et al., 2000; Novak and Haslberger, 2000). Raffinose is a 

nondigestible oligosaccharide that is considered to be an anti-nutrient due to gas 

production and the resulting flatulence caused 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 precursors for a large group of phenylpropanoid 

compounds. The non-starch polysaccharide pentosans are a major source of furfural 

(Adams et al., 1997). 
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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 recorded a significant difference for each of these 

components, these differences represented no meaningful trend and were considered to be 

not biologically meaningful. The limited number of differences recorded in this study 

reflected the natural variation 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 evaluation 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 relevant changes. The 

combined-site analysis show that there were no statistically significant 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 references. The individual site analysis show 

that there were no statistically significant differences (p>0.05) for 348 (93.5%) of the 372 

comparisons. Individual site differences were not reproducible across multiple sites and, 

where differences were observed, mean component values test and control substances 

were within the 99% tolerance interval. Furthermore, the limited number of component 

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, reflected no meaningful 

trends, and were of no biological significance.

Therefore, the corn grain and forage derived 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.
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Table VII-1. Monthly Temperature and Accumulated Water Data for the U.S. 2006 Study Site1 Measurement May June July August SepteIAE Accumulated water (in.) 2.5 2.3 2.3 6.2 2

Avg Max temp (°F) 72 81 86 86 7Avg Min temp (°F) 51 60 65 62 4Range2 (°F) 30 - 98 45 - 97 54 - 95 55 - 96 32 - 

IAW Accumulated water (in.) 1.8 0.4 2.8 4.9 8

Avg Max temp (°F) 72 84 88 83 7Avg Min temp (°F) 50 59 63 66 4Range2 (°F) 34 - 91 49 - 93 52 - 99 58 - 93 34 - 

IL Accumulated water (in.) 2.1 1.5 3.7 3.1 2

Avg Max temp (°F) 72 81 88 82 7Avg Min temp (°F) 51 57 64 62 4Range2 (°F) 36 - 94 46 - 92 46 - 97 51 - 96 34 - 

IN Accumulated water (in.) 4.8 5.5 3.4 5.6 2

Avg Max temp (°F) 71 80 86 83 7Avg Min temp (°F) 51 58 65 63 5Range2 (°F) 40 - 92 52 - 89 50 - 96 55 - 93 37 - 

KS Accumulated water (in.) 5.3 8.7 8 10.9 2 

Avg Max temp (°F) 81 92 96 92 8Avg Min temp (°F) 53 62 66 67 5Range2 (°F) 37 - 100 52 - 102 54 - 109 52 - 107 36 - 

NE Accumulated water (in.) 1.9 3.8 5.6 9.5 4

Avg Max temp (°F) 79 88 91 86 7Avg Min temp (°F) 51 61 65 63 5Range2 (°F) 37 - 96 52 - 101 55 - 103 52 - 100 36 - 

1 Site codes are as follows: IAE = Benton County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; KS = 

 NE = York County, NE. 

2 The range is the absolute maximum and minimum temperature in each month. 
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Table VII-2. Summary of Significant Differences (p<0.05) Comparing MON 87460 to the Conventionaduring 2006 under Typical Agronomic Conditions 

Tissue/Site/ 

Components (Units)1

Mean 

MON 87460 

Mean 

Control 

Mean Diff 

(% of Control) 

Signif. 

(p-value) MON 87460 (Range) 

ForageIL Carbohydrates (% DW) 87.31 85.75 1.81 0.007 (86.23 - 88.01) INMoisture (% FW) 66.23 67.80 -2.31 0.008 (64.70 - 67.40) 

Protein (% DW) 6.75 7.23 -6.60 0.031 (6.68 - 6.90) 

Grain

Combination of all sites 

Ash (% DW) 1.54 1.46 5.60 0.041 (1.33 - 1.83) 

18:0 Stearic (% Total FA) 2.05 1.98 3.50 0.024 (1.88 - 2.34) 20:1 Eicosenoic (% Total FA) 0.18 0.19 -4.05 0.007 (0.17 - 0.19) 

IAEMoisture (% FW) 9.78 9.22 6.08 0.049 (9.57 - 10.00) Histidine (% DW) 0.31 0.32 -2.68 0.032 (0.30 - 0.32) Methionine (% DW) 0.21 0.20 2.45 0.035 (0.20 - 0.21) 

Valine (% DW) 0.51 0.53 -3.71 0.028 (0.48 - 0.52) 

18:3 Linolenic (% Total FA) 1.22 1.25 -2.45 0.040 (1.17 - 1.25) Total Dietary Fiber (% DW) 12.23 11.60 5.40 0.029 (11.94 - 12.52) 

Raffinose (% DW) 0.20 0.15 27.28 0.009 (0.19 - 0.20) 

IAW

22:0 Behenic (% Total FA) 0.24 0.21 12.99 0.015 (0.20 - 0.25) Thiamine HCl (mg/kg DW) 2.85 2.48 14.81 0.011 (2.77 – 2.9) 

Ferulic Acid (μg/g DW) 1753.10 1847.90 -5.13 0.003 (1661.13 - 1914.78) 

Table VII-2 con
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Table VII-2 (cont.). Summary of Significant Differences (p<0.05) Comparing MON 87460 to the Conin the U.S. during 2006 under Typical Agronomic Conditions 

Tissue/Site/ 

Components (Units)1

Mean 

MON 87460 

Mean 

Control 

Mean Diff 

(% of Control) 

Signif. 

(p-value) MON 87460 (Range) 

ILMoisture (% FW) 9.94 10.43 -4.73 0.013 (9.71 - 10.30) 

Folic Acid (mg/kg DW) 0.28 0.27 4.19 0.033 (0.27 - 0.29) 

INCystine (% DW) 0.19 0.20 -5.55 0.039 (0.19 - 0.20) 

18:1 Oleic (% Total FA) 20.05 20.63 -2.83 0.025 (19.96 - 20.13) 18:2 Linoleic (% Total FA) 63.81 63.03 1.24 0.026 (63.67 - 63.95) 

Acid Detergent Fiber (% DW) 2.55 3.63 -29.68 0.036 (2.52 - 2.60) Riboflavin/Vitamin B2 (mg/kg DW)

1.52 1.09 39.64 0.039 (1.45 - 1.57) 

Phytic Acid (% DW) 0.63 0.77 -17.45 0.007 (0.60 - 0.66) KSLysine (% DW) 0.33 0.31 4.51 0.045 (0.31 - 0.34) 

20:1 Eicosenoic (% Total FA) 0.18 0.19 -7.81 0.043 (0.17 - 0.18) 

NE

Ash (% DW) 1.57 1.38 14.01 0.007 (1.49 - 1.62) 

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 we

Tables E-5 through E-11 in Appendix E present the full results of the combined site analysis for the compositional analyses from including means, standard error values and p-values. 
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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 were harvested from 

plants grown in three replicates at each of four field sites in Chile in 2006 - 2007. These field 

sites were located in commercial corn production regions of Chile. The sites were Calera de 

Tango (CT), Colina (CL), Lumbreras (LUM) and Quillota (QUI). These sites are well-suited 

for corn production, but as they typically do not receive any rainfall during the growing 

season, all water received at each site occurred through controlled irrigation. At each site, a 

strip-plot design was used allowing comparisons of the test and control substances 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 withholding irrigation during approximately the V10 – R2 growth 

stages, which represents the growth stages when corn grain yield potential is most susceptible 

to drought stress (Campos et al., 2006). Table VII-3 presents temperature data and applied 

water from the production period. In addition to MON 87460 and its conventional control, 

four different conventional commercial corn hybrids 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 water-limited conditions. Compositional analysis 

included components consistent with OECD guidelines. 

The experiment was arranged in a strip-plot design with three replicates per site, with 

irrigation treatment (well-watered or water-limited) 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 commercial 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 collected at physiological maturity. 

Within each treatment, the composition of forage 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-site 

data 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 the same reference hybrids planted in the wellwatered 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 established 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 the 
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replicated field sites and the fourth based on data 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 component 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 the compositional variability that occurs in corn currently grown 

commercially. It allows statistically significant differences between MON 87460 and the 

control to be placed in biological perspective. This comparative evaluation can also consider 

natural 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.B.1. Assessment of Nutrients, Anti-Nutrients, and Key Secondary Metabolites 

under Well-Watered Conditions 

The well-watered plots provide a compositional 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 under well-watered conditions. 

Forage and grain samples were harvested from all well-watered plots and analyzed for 

nutritional and anti-nutrient components as described in Section 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 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, 15:0 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 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, 61 components (9 in forage, 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. There were a total of 244 comparisons made (four 

sets of comparisons × 52 components from grain and four 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 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 significant differences observed, mean component 

values of the test and control substances were within the 99% tolerance interval 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 comparisons. 

Individual site differences were not reproducible across multiple sites and, where differences 

were observed, mean component values of the test 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 present in corn grain is provided in the OECD consensus document 

on compositional considerations for corn (OECD, 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 combined-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 significant difference (p<0.05) only at a single 

site. This lack of reproducibility across multiple sites established that the differences 

observed in the combined-site analysis in values for these two components were of no 

biological significance. The magnitude of the differences in total fat (0.17% DW) and 

magnesium (0.01% of DW) were extremely small, and the mean values for these two 

components were within the 99% tolerance interval established from the commercial 

references grown at the same sites. These findings confirmed that these minor differences 

reflected the natural variability of conventional corn. 

In the individual-site analysis of forage, 23 of the 27 comparisons were not significantly 

different (p>0.05). Differences included values for carbohydrates by calculation, moisture, 

ADF, and calcium, with each component difference being observed at only a single site. 

This lack of reproducibility across 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 grain nutrients, individual site differences in components not 

recorded in the combined-site analysis included values for serine, threonine, 18:0 stearic 

acid, 18:2 linoleic acid, 18:3 linolenic acid, and vitamin E. For each component, these 

differences were observed at a single 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 interval established from the 

commercial references, support a conclusion that the 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 of 
Monsanto Company 07-CR-191U Page 162 of 544 

differences recorded in this study reflected the natural variation of corn and supported the 

compositional equivalence of MON 87460 and conventional corn. 

VII.B.3. Levels of Anti-Nutrients and Key Secondary Metabolites under Well-Watered 

Conditions 

A description of the anti-nutrients and secondary metabolites present 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 metabolites 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 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 significant differences observed, mean component values of the 

test and control substances were within the 99% tolerance interval 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 comparisons. Individual site 

differences were not reproducible across multiple sites and, where differences were observed, 

mean component values test and control substances were within the 99% tolerance interval. 

Furthermore, the limited number of component 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, reflected no meaningful trends, 

and were of no biological significance.

Therefore, the corn grain and forage derived from MON 87460 grown under well-watered 

conditions, and consequently the 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 well-watered conditions is as safe as 

conventional corn with a history of safe consumption. 
Monsanto Company 07-CR-191U Page 163 of 544 

Table VII-3. Monthly Temperature and Accumulated Water Data for the Chile 2006/2007 Study Site1 Measurement December January February March ApCL 

Accumulated water (in.), well-watered 0.9 10.3 8.5 9.4 2Accumulated water (in.), water-limited2, 3 0.9 10.3 4.7 5.6 2

Avg Max temp (°F) NA5 88 85 82 7

Avg Min temp (°F) NA5 53 49 47 4Range4 (°F) NA5 46 - 97 42 - 94 41 - 94 33 - 

CT 

Accumulated water (in.), well-watered 2.8 9.4 8.5 10.3 2

Accumulated water (in.), water-limited2, 3 3.8 9.4 2.8 6.6 2

Avg Max temp (°F) NA5 84 79 79 7

Avg Min temp (°F) NA5 52 50 50 4Range4 (°F) NA5 46 - 91 42 - 90 42 - 88 31 - 

LUM 

Accumulated water (in.), well-watered 2.8 9.4 8.5 10.3 1

Accumulated water (in.), water-limited2, 3 2.8 9.4 2.8 6.6 2

Avg Max temp (°F) NA5 81 78 79 7

Avg Min temp (°F) NA5 52 50 49 4Range4 (°F) NA5 47 - 89 42 - 89 42 - 94 32 - 

1 Site 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 544 

Table VII-4. Summary of Significant Differences (p<0.05) Comparing MON 87460 to the Conventionduring 2006/2007 under Well-Watered Conditions 

Tissue/Site/ 

Components (Units)1

Mean 

MON 87460 

Mean 

Control 

Mean Diff 

(% of Control) 

Signif. 

(p-value) MON 87460 (Range) 

ForageCT

Moisture (% FW) 72.53 76.10 -4.69 <0.001 70.90 - 75.00 Carbohydrates (% DW) 87.22 86.14 1.26 0.048 86.98 - 87.46 Acid Detergent Fiber (% DW) 22.84 29.47 -22.51 0.002 17.95 - 31.28 

Calcium (% DW) 0.26 0.32 -18.99 0.047 0.25 - 0.28 Grain

Combination of all sites 

Total Fat (% DW) 3.89 3.72 4.52 0.029 3.45 - 4.23 

Magnesium (% DW) 0.12 0.11 8.64 0.012 0.10 - 0.14 CL

Vitamin E (mg/kg DW) 12.46 10.50 18.66 0.002 12.37 - 12.51 

CT

18:2 Linoleic (% Total FA) 64.64 63.82 1.28 0.048 64.11 - 65.10 

LUM

Total Fat (% DW) 3.96 3.61 9.54 0.010 3.80 - 4.23 Magnesium (% DW) 0.13 0.11 15.54 0.022 0.11 - 0.14 

Serine (% DW) 0.50 0.44 13.33 0.024 0.47 - 0.54 

Threonine (% DW) 0.35 0.32 10.08 0.047 0.33 - 0.37 18:0 Stearic (% Total FA) 1.83 1.71 6.83 0.024 1.74 - 1.97 18:3 Linolenic (% Total FA) 1.20 1.23 -2.41 0.042 1.17 - 1.23 

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 were set to 

Tables E-12 through E-18 in Appendix E present the full results of the combined site analysis for the compositional analyses f

production including means, standard error values and p-values.
Monsanto Company 07-CR-191U Page 165 of 544 

VII.B.5. Assessment of Nutrients, Anti-Nutrients, 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 background genetics but lacking the introduced trait. 

Both were grown under conditions intended to impose drought stress by withholding 

irrigation during approximately the V10 – R2 growth stages, which represents the growth 

stages when corn grain yield potential is most susceptible to drought 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-nutrient 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 individual 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 data are presented in Appendix E (Tables E-19 

through E-25). 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 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 significant differences observed, mean component 

values of the test and control substances were within the 99% tolerance interval 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 comparisons. 

Individual site differences were not reproducible across multiple sites and, where differences 

were observed, mean component values of the test 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 Nutrients under Water-Limited Conditions 

In the combined-site analysis of forage, eight 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 total 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 difference 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, indicating 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 only 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 significantly different (p>0.05). 

Individual site differences included only a single value for moisture. This lack of 

reproducibility across multiple sites established 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 grain nutrients, individual site differences in 

components not recorded in the combined-site analysis included values for iron, phosphorus, 

18:1 oleic acid, 22:0 behenic 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 sites were within the 99% tolerance interval 

established from the commercial references and 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 variability of corn for 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 the natural variation of conventional corn and 

supported the compositional equivalence of MON 87460 and conventional corn. 

VII.B.7. Levels of Anti-Nutrients and Key Secondary Metabolites 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 meaningful 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 comprehensive evaluation of anti-nutrient 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 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 significant differences observed, mean component values of the 

test and control substances were within the 99% tolerance interval 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 comparisons. Individual site 

differences were not reproducible across multiple sites and, where differences were observed, 

mean component values of the test and control 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, reflected no meaningful trends, 

and were of no biological significance.

Therefore, the corn grain and forage derived 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 availability is as safe as conventional 

corn with a history of safe consumption.
Monsanto Company 07-CR-191U Page 168 of 544 

Table VII-5. Summary of Significant Differences (p<0.05) Comparing MON 87460 to the Conventionduring 2006/2007 under Water-Limited Conditions 

Tissue/Site/ 

Components (Units)a

Mean 

MON 87460 

Mean 

Control 

Mean Diff 

(% of Control) 

Signif. 

(p-value) 

MON 87460 (Range) 

ForageCombination of all sites

Total Fat (% DW) 1.32 0.84 56.03 0.045 0.50 - 1.92 

CT

Moisture (% FW) 72.13 74.23 -2.83 0.005 72.00 - 72.30 Grain

Combination of all sites

20:1 Eicosenoic (% Total FA) 0.1763 0.1845 -4.43 0.042 0.1617 - 0.1916 CL

22:0 Behenic (% Total FA) 0.17 0.11 57.68 0.029 0.13 - 0.20 Vitamin E (mg/kg DW) 13.34 11.16 19.54 0.001 12.57 - 14.24 Phytic Acid (% DW) 0.87 0.69 25.47 0.012 0.84 - 0.89 CTIron (mg/kg DW) 17.61 18.81 -6.34 0.046 17.06 -18.24 

Phosphorus (% DW) 0.32 0.35 -8.35 0.027 0.32 - 0.32 

20:1 Eicosenoic (% Total FA) 0.17 0.18 -9.16 0.016 0.16 - 0.17 Folic Acid (mg/kg DW) 0.30 0.25 19.36 0.046 0.25 - 0.37 LUM

18:1 Oleic (% Total FA) 20.38 20.89 -2.46 0.030 20.20 - 20.48 

aDW= dry weight; FA=fatty acid. cWith 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were se

Tables E-19 through E-25 in Appendix E present the full results of the combined site analysis for the compositional analyses fproduction including means, standard error values and p-values.
Monsanto Company 07-CR-191U Page 169 of 544 

VII.C. Additional Secondary Metabolites 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 and 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 with stress responses in a 

range of plants and tissues, and thus possibly relevant in corn. There is no evidence in 

the literature for such components that are unique to corn. 

Eleven additional secondary metabolites were selected for comparison between 

MON 87460 and the control. These selected metabolites included: osmoprotectants, such 

as sugars and polyols (sucrose, glucose, fructose, sorbitol, mannitol, and glycerol), free 

proline, glycine betaine and choline (Yancey, 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 extremely minor fraction of corn biomass. 

Field design and sampling are described in Section VII.B. Samples from test, control, 

and reference substances from all four sites in Chile were subjected to additional 

compositional analysis. This section describes results of combined-site data from three 

(CL, CT, LUM) of the four sites established. 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 the same reference 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 established with the appropriate water 

stress treatments; therefore, data for the QUI site are presented in Appendix E. 

Statistical comparisons between the test and control substances were performed within 

each irrigation treatment. A range of component values and a statistical population were 

determined for the reference substances within each irrigation treatment. Thus, four sets 

of statistical analyses were made for each treatment, three based on the data from each of 

the replicated field sites and the fourth based on data from a combination of all three field 

sites. Statistically significant differences were determined at the 5% level of significance 

(α = 0.05) using established statistical methods. 

VII.C.1. Levels of Additional Secondary Metabolites under Well-Watered 

Conditions 

Two of the 11 metabolites (sorbitol, mannitol) had more than 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 were 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 (four sets of comparisons × nine components 

from grain and four 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 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 Appendix E (Tables E-26 and E-27). 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 E30. 

The combined-site analysis show that there were no statistically significant 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 abscisic 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 individual 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 multiple sites and, where differences were 

observed, with the exception of the highly variable forage abscisic acid, mean component 

values test and control substances were within the 99% tolerance 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 Additional Secondary Metabolites WellWatered Conditions 

In summary, statistical analysis highlighted no consistent differences across sites in the 

levels of the additional secondary metabolites from MON 87460 and the control. The 

limited number of differences observed in this study reflected 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 compositionally equivalent to 

conventional corn. 
Monsanto Company 07-CR-191U Page 171 of 544 

Table VII-6. Summary of Significant Differences in Additional Secondary Metabolite Composition 

MON 87460 to the Conventional Control in Chile during 2006/2007 under Well-Watered Conditions 

Tissue/Site/ 

Components (Units)1

Mean 

MON 87460 

Mean 

Control 

Mean Diff 

(% of Control) 

Signif. 

(p-value) 

MON 87460 (Range) 99% 

In

ForageCombination of all sitesAbscisic Acid (ppb FW) 37.03 15.66 136.54 0.040 (11.90 - 122.00) [1.CLAbscisic Acid (ppb FW) 75.23 15.63 381.24 0.003 (18.50 - 122.00) [1.CTFree Proline (% DW) 0.027 0.019 40.22 0.025 (0.023 - 0.031) [0Choline (ppm FW) 135.67 108.97 24.50 <0.001 [114.00 - 148.00] [76.1DW= dry weight; FW=fresh weight. 2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were s

Tables E-26 and E-27 in Appendix E present the full results of the combined site analysis for the compositional analyses from the Cincluding means, standard error values and p-values.
Monsanto Company 07-CR-191U Page 172 of 544 

VII.C.3. Levels of Additional Secondary 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, standard error values and 

statistical analyses for the combined-site data are presented in Appendix E (Tables E-28 

and E-29). 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 significant 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 single significant difference observed, mean 

component values of the test and control substances were within the 99% tolerance 

interval established from the commercial references. The individual site analysis show 

that there were no statistically significant differences (p>0.05) for 44 (81.5%) of the 54 

comparisons. Individual site differences were not reproducible across multiple sites and, 

where differences were observed, mean component values test and control substances 

were within the 99% tolerance interval (Table VII-7). Therefore, these differences were 

within the natural variability of corn for these components. 

VII.C.4. Conclusions for Chile 2006/2007 Additional 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 544 

Table VII-7. Summary of Significant Differences in Additional Secondary Metabolite Composition 

MON 87460 to the Conventional Control in Chile during 2006/2007 under Water-Limited Conditions 

Tissue/Site/ 

Components (Units)1

Mean 

MON 87460 

Mean 

Control 

Mean Diff 

(% of Control) 

Signif. 

(p-value) 

MON 87460 (Range) 99% 

ForageCL

Glycine Betaine ppm FW) 102.40 147.00 -30.34 0.016 [73.20 - 122.00] Salicylic Acid (ppm FW) 0.11 0.33 -68.43 0.002 [0.10 - 0.11] CTAbscisic Acid (ppb FW) 42.27 23.57 79.35 0.043 [32.90 - 54.30] 

Choline (ppm FW) 157.00 144.00 9.03 0.024 [153.00 - 160.00] [66LUM

Choline (ppm FW) 167.67 151.67 10.55 0.035 [153.00 - 181.00] [66Salicylic Acid (ppm FW) 0.41 0.26 57.57 0.016 [0.27 - 0.58] Grain

Combination of all sites

Sucrose (% DW) 1.63 1.86 -12.40 0.008 [1.33 - 1.86] [CL

Abscisic Acid (ppb FW) 10.03 16.59 -39.52 0.041 [8.78 - 11.90] Glycerol (% DW) 0.030 0.023 31.03 0.045 [0.025 - 0.034] CTSucrose (% DW) 1.71 2.03 -15.85 0.010 [1.44 - 1.86] [LUM

Sucrose (% DW) 1.41 1.58 -10.55 0.040 [1.33 - 1.54] [

1DW= dry weight; FW=fresh weight. 2With 95% confidence, the interval contains 99% of the values expressed in the population of commercial lines. Negative limits were sTables E-28 and E-29 in Appendix E present the full results of the combined site analysis for the compositional analyses from the Cincluding means, standard error values and p-values.
Monsanto Company 07-CR-191U Page 174 of 544 

VII.D. Overall Conclusions from Compositional 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 corn under a broad range of environmental 

conditions. 

Components evaluated in samples harvested from both studies included (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 key 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 values 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 metabolites associated with stress tolerance was 

conducted for samples from the Chile 2006/2007 study. Statistical comparisons between 

the test and control substances were performed 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 control were within the 

99% tolerance interval. Therefore, these differences were within the natural variability of 

corn for these components. The evaluation 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 maize 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 forage and grain of MON 87460, a droughttolerant corn hybrid, are compositionally equivalent 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. Substantial equivalence of antinutrients and 

inherent plant toxins in genetically modified novel foods. Food Chemical. 

Toxicology. 38: 473-483. 

OECD. 2002. Consensus document on compositional considerations for new varieties of 

maize (Zea mays): key food and feed nutrients, anti-nutrients and secondary plant 

metabolites. ENV/JM/MONO(2002)25. Organization for Economic 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. Science Progress. 87:1-24. 

Yancey, P.H. 2005. Organic osmolytes as compatible, metabolic and counteracting 

cytoprotectants in high osmolarity and other 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 Journal of Bioscience. 63:313-320. 
Monsanto Company 07-CR-191U Page 176 of 544 

VIII. Phenotypic, Agronomic, and Environmental 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 risk 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 interpret 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 comprise the comprehensive assessment of 

MON 87460. 

Phenotypic and agronomic characteristics of MON 87460 were evaluated in a 

comparative manner to assess plant pest potential (OECD, 1993). These assessments 

included 14 plant growth and development characteristics, five seed germination 

parameters, two pollen characteristics, and observations 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 demonstrate increased plant 

pest characteristics compared to conventional corn. Data on environmental interactions 

indicate that MON 87460 does not confer any increased susceptibility or tolerance to 

specific diseases, insects, or abiotic stressors, with the exception of the drought tolerance 

trait. 

VIII.A. Characteristics Measured for Assessment 

In the phenotypic, agronomic, and environmental 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 interactions 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 each of these assessments, 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 

commercial corn hybrids (references) were included 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 were adapted to the geographic region in which they were 

grown with selections 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. Data collected from the 
Monsanto Company 07-CR-191U Page 177 of 544 

commercial references reflect a range of selection and breeding for desirable 

characteristics including drought tolerance and can therefore 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 known that corn hybrids are 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 and other seed companies commonly 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). Approximately 

75% of the conventionally bred hybrids offered for planting in the dryer western areas of 

the Great Plains are rated as having very good to excellent drought tolerance and have 

been recommended for commercial production fields that regularly experience drought 

stress. The variation in yield response observed among MON 87460, the control, and 

commercial reference hybrids in the field studies discussed below is within normal levels 

expected for conventional corn grown under sub-optimal soil moisture environments. 

The characteristics measured among the field, greenhouse, and laboratory studies are 

listed in Table VIII-1. The standard phenotypic, agronomic, and environmental 

interactions characteristics are well known to experts familiar with corn breeding and 

agronomic performance. MON 87460 is expected to provide reduced 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. Additional plant characteristics assessed included germination and 

dormancy, pollen characteristics collected under well-watered and water-limited 

conditions, abiotic stress tolerance to multiple levels of drought, cold, heat, and salt 

conditions, volunteer potential in a subsequent season under cultivated soil 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 assess weediness potential, including seed 

germination and dormancy (hard seed), pre-harvest 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 MON 87460 across a broad 

range of soil moisture and environmental conditions relevant to where commercial 

production would be expected. Agronomic and phenotypic characteristics data were 

collected from plants grown under a combination 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 agronomic 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 Measured 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 Number 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 percentage 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. Measurements 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, phenotypic assessments from planting 

to harvest that includes early and final stand 

counts, vigor ratings, plant height, and number 

of ears and seed per plot 

*Rating scale changed in 2007 to be consistent with assessments used by corn breeders 
Monsanto 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 potential of a biotechnology-derived plant compared to the 

conventional crop. The concept of familiarity is based on the fact that the biotechnologyderived plant is developed from a conventional plant variety whose biological properties 

and plant pest potential are known to experts. Familiarity considers the biology of the 

crop, the introduced trait, the receiving environment, and the interaction among these 

factors, and provides a basis for comparative risk assessment between a biotechnologyderived plant and its conventional counterpart. 

Expert knowledge and experience with conventionally bred corn was the basis for 

selecting appropriate endpoints and estimating 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 conventional 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., dormancy, lodging or pre-harvest seed loss 

characteristics, volunteer potential and survival outside cultivation) was used to assess 

whether there is an increased weediness potential, an element of APHIS’s plant pest 

determination. Data on abiotic stress tolerance 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 potential changes in tolerance required 

additional evaluation as a component of the plant pest risk assessment. 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 conventional counterpart. 

Agronomists familiar with the experimental design, evaluation criteria, and expected 

outcomes were involved in all steps of data collection, summarization, and analyses. 

This supervision ensured that measurements were taken appropriately, data were 

consistent with expectations based on experience with the crop, and that the experimental 

and field sites were carefully monitored. These scientists were expected to report any 

unexpected observations or issues during the course of the studies. The overall dataset 

was evaluated for evidence of biologically relevant changes and for possible evidence of 

an unexpected plant response and the data were subsequently submitted for statistical 

analysis. 

VIII.B.1. Interpretation of Detected Differences Criteria 

Comparative plant characterization data between a biotechnology-derived crop and the 

control are interpreted in the context of contributions to increased plant pest potential as 

assessed by APHIS. Under the framework of familiarity, characteristics for which no 

differences are detected support a conclusion of no increased plant pest potential of the 

biotechnology-derived crop compared to the conventional crop. Characteristics for 

which differences are detected are considered in the step-wise method (Figure VIII-1). 

All detected differences for a characteristic are considered in the context of whether or 

not the difference would increase the plant pest potential of the biotechnology-derived 
Monsanto Company 07-CR-191U Page 180 of 544 

crop. Ultimately, a weight of evidence approach considering all characteristics and 

studies was used for the overall risk assessment of differences and their significance. In 

detail, Figure VIII-1 illustrates the stepwise 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 detected in the individual-site analysis 

must be observed in the combined-site analysis to be considered further for 

plant pest potential. A difference in the 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 reference 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 assessed 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 potential 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 the crop or a sexually 

compatible species. 
Monsanto Company 07-CR-191U Page 181 of 544 

Note: A “no” answer at any step indicates that the characteristic does not contribute to a 

biological or ecological change for the crop in terms 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 Interpretation 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 environmental consequences of 

MON 87460 across a broad range of soil moisture and environmental conditions relevant 

to where commercial production would be expected. Two 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 phenotypic 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

Yes 

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 analysis 
Monsanto Company 07-CR-191U Page 182 of 544 

The well-watered treatment allowed a comparison 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 V10 through R3) allowed a comparison of 

MON 87460 and the control under conditions where MON 87460 was expected to 

provide a yield benefit. Water treatment levels were managed by using a combination of 

either a water budgeting program (KanSched 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 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 

approximately 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 indicators of moisture deficit in corn (Campos et al., 2006); 

therefore, these characteristics 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 set criteria to determine 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 Differential Water Treatments Imposed. A review of the 

water management practices and rainfall data is conducted for each site. All 

rainfall events and irrigation amounts during the water stress period are 

evaluated for compliance with soil moisture 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 comparing the references in the well-watered 

treatment to the references in the water-limited treatment. Indicators of water 

limitation included yield reduction (minimum 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-watered 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 requirement for a 15% yield reduction in 

the reference substances due to the imposed water stress treatment must be met 

for 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 significant 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 assessments may be appropriate under the 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 

Yes 
Monsanto Company 07-CR-191U Page 185 of 544 

Note

The preceding two sections provide an introduction to Monsanto’s 

approach to the plant pest risk assessment 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 agronomic characteristics 

of MON 87460 compared to a 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) were included in the analysis to establish 

a range of natural variability for each characteristic. Because MON 87460 reduces yield 

loss under water-limited conditions, field studies were designed to evaluate the 

environmental consequences of MON 87460 across a broad range of soil moisture and 

environmental conditions relevant to where commercial production would be expected. 

Field studies were established using three different water management regimes: (1) wellwatered, (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. 

Well-Watered Treatment

Field studies established under well-watered conditions were intended to 

provide optimal grain yield and 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 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 supplemental 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 in 

the U.S. and Chile during 2006 and 2007 (Table VIII-2). The wellwatered 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 and provide optimal grain yield. The waterlimited 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 vegetative 
Monsanto Company 07-CR-191U Page 186 of 544 

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). See Section II.D.3 for additional information on the 

effects of drought stress on corn growth 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) and composition of forage and grain 

(Section VII). 

Typical Agronomic Practices Treatment

A field study established under water conditions typical of local 

agronomic practices allowed an evaluation of MON 87460 under a broad 

range of environmental conditions relevant 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 irrigation 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 determine CSPB and 

NPTII expression levels (Section VI.C) and composition of forage and 

grain (Section VII) as the staking error 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 collected from 31 field site locations over two 

years: 13 U.S. sites in 2006, 4 Chilean sites 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 study. These field site locations provided a 

broad range of environmental and agronomic conditions representative of U.S. corngrowing regions. The California and Chile locations selected for a sub-set of these sites 

provide environments that are well-suited for corn production but typically do not receive 

any rainfall during the growing season. These environments 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 replications or as strip- or split-plot designs 

with either three or four replications. 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 field study: seedling 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, grain moisture, test weight, and yield. 

The evaluations and timing of plant assessments 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 (referred to as combined-site analysis), in 

which data were pooled from all sites and analyzed statistically, or data are presented for 

an individual location (referred to as by-site analysis) where appropriate. For each 

characteristic, MON 87460 was compared to the control. Differences were considered 

significant at the 5% level (p≤0.05). Additional details on materials, 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 Appendices 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 reported based on a whole plot visual assessment. Early and 

final stand counts, dropped ears, stalk lodging 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 weight 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 disease damage were assessed from two rows on 

a whole plot basis. Stalk and ear/kernel 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 each plot. The sticky traps were left in place 

for seven days per deployment. European corn borer and corn earworm damage 

assessments were made on five non-systematically 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 Appendix 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 from the raw data. Thus, each 

test and control mean has its own individual standard deviation and hence a different 

standard error. 

Regardless of the water regime established, the following studies demonstrate that no 

phenotypic or agronomic differences were observed for MON 87460 that would lead to a 

conclusion of increased plant pest potential. MON 87460 is expected to provide reduced 
Monsanto 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 Studies Established under Three Different 

Water Management Regimes for Phenotypic 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 544 

Table VIII-3. Site Inclusion Assessment for U.S. and Chile Field Studies during 2006 and 2007 

Yield ± S.E. (bu/ac) 

Plant height ± S.E. 

(in) 

Ear height ± S.E. 

(in) 

Days t

Study, Location, and Year 

Site code1

WellWatered Water- Limited Reduction (%)2 Well- Watered Water- Limited Well- Watered Water- Limited Well

Water

Chile 2006/2007 CL 185.5 ± 10.72 82.3* ± 11.18 56 110.7 ± 2.74 79.7* ± 7.24 63.4 ± 2.38 50.9* ± 3.94 63.1 ± 0.1

CT 236.5 ± 4.95 152.3* ± 9.82 36 105.9 ± 2.91 92.1 ± 2.17 55.0 ± 2.08 46.0 ± 1.79 66.2 ± 0.4LUM 213.9 ± 4.28 94.4* ± 4.81 56 97.9 ± 1.96 75.0* ± 1.22 50.4 ± 1.63 41.8* ± 0.90 70.3 ± 0.3QUI 203.1 ± 5.27 196.3 ± 2.51 3 112.0 ± 1.72 112.8 ± 2.54 63.5 ± 2.12 63.4 ± 2.06 67.7 ± 0.3

U.S. 2007 CA 215.9 ±2.01 178.8 ±7.91 17 100.4 ±1.01 101.1 ±1.16 51.7 ±0.42 52.1 ±0.68 60.3 ±0.2

Study-1 TX 212.2 ±2.86 165.2* ±3.78 22 72.9 ±0.58 62.6* ±0.33 32.5 ±0.23 29.6* ±0.48 57.0 ±0.0

U.S. 2007 KS 141.0 ± 3.79 138.3 ± 7.55 2 88.1 ± 2.15 88.8 ± 2.15 42.1 ± 2.29 42.5 ± 1.91 63.2 ± 0.2

Study-2 NE 223.0 ± 1.59 214.1 ± 1.88 4 91.0 ± 3.16 87.7* ± 3.56 41.5 ± 1.71 42.1 ± 1.41 68.4 ± 0.2

TX 233.7 ± 10.50 191.8* ± 8.62 18 76.1 ± 1.72 77.1 ± 2.09 27.9 ± 0.67 28.2 ± 0.94 59.0 ± 0.0

Note: Phenotypic data are mean values for the commercial references 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 a

1CL = Colina; CT = Calera de Tango; LUM = Lumbreras; QUI = Quillota; CA = California, Study-1; TX = Texas, Study-1 and Stu

NE = Nebraska. 

2Percent yield reduction calculated as difference between reference mean under well-watered and water-limited conditions, divided 

under well-watered conditions. Sites QUI, KS, and NE did not meet the inclusion criteria of a minimum 15% yield reduction in referethe water-limited treatment. The lack of differences in plant height, ear height, and days to 50% silking between the water treatmenconfirmed that the plants did not experience meaningful water stress. Consequently, these three sites were not included in assessments 

limited conditions.
Monsanto Company 07-CR-191U Page 191 of 544 

VIII.C.1. Field Studies Established under Well-Watered Conditions 

Field studies established under well-watered conditions were intended to provide optimal 

grain yield and allowed an evaluation of MON 87460 in the absence 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 established at eight sites (Table VIII-2). 

Comparative assessments of phenotypic and agronomic characteristics were conducted 

on MON 87460 and the conventional control. In addition, 19 commercial corn hybrids 

were included as references. In the combined-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 moisture, test weight, or yield 

(Table VIII-4). The only significant difference detected was an increase in root lodged 

plants for MON 87460 compared to the control (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 number of root lodged plants was greater for 

MON 87460 compared to the control, the mean value observed for MON 87460 was 

within the range of values observed in the commercial 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 agronomic characteristics were conducted 

on MON 87460 and the conventional control. In addition, 11 commercial corn hybrids 

were included as references. In the combined-site analysis no differences were detected 

between MON 87460 and the control for any of the 14 assessed phenotypic and 

agronomic characteristics (Table VIII-5). 

Summary of U.S. 2006 and 2007 Well-Watered

The results from these studies support a conclusion of no increased plant pest potential of 

MON 87460 compared to conventional corn 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 Range1

MON 87460 Control Min Max

Seedling vigor 0-9 scale2 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 scale3 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 plant 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 

Range1

MON 87460 Control Min Max 

Seedling vigor 1-9 scale2 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 scale3

 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 differences 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 under Both Well-Watered and Water-Limited 

Conditions 

These field studies were designed to evaluate MON 87460 under water-limited 

conditions 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 water-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 nine 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 water management techniques, only six of the 

nine sites established met the inclusion criteria 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 agronomic 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, data 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 MON 87460 and did not impact the conclusions 

of the plant pest risk assessment 

Assessment

In the combined-site analysis of the well-watered treatment in Chile, no differences were 

detected between MON 87460 and the conventional 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 assessed characteristics. As expected, 

MON 87460 exhibited higher yield (reduced yield loss) (p≤0.05) than the conventional 

control (114.5 vs. 86.7 bushels/acre, respectively) under water-limited conditions 

(Table VIII-7). Reduced yield loss is a desirable 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 expectation of no statistically significant 

differences between MON 87460 and the control 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: Phenotypic 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 and 

Water-Limited Treatments 

Well-Watered Treatment 

 Mean ± S.E. 

Reference 

Range1

Phenotypic 

characteristic Units MON 87460 Control Min Max 

Seedling vigor 0-9 scale2 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 scale3

 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 ears4

 #/plot 0.0 ± 0.0 0.0 ± 0.0 0.0 0.0 

Stalk lodged plants4

 #/plot 0.0 ± 0.0 0.0 ± 0.0 0.0 0.0 

Root lodged plants4

 #/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 differences 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 plant 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: Phenotypic 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 and 

Water-Limited Treatments 

Water-Limited Treatment 

 Mean ± S.E. 

Reference 

Range1

 Phenotypic 

characteristic Units MON 87460 Control 

 

Min Max 

Seedling vigor 0-9 scale2 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 scale3

 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 ears4

 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0 

Stalk lodged plants4

 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0 

Root lodged plants4

 #/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 = dead and 9 = above average vigor. 3

 Stay green rating scale: 0 = entire plant 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 which 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 conducted 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 conventional 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 combined-site analysis of the water-limited 

treatment, no statistical differences were detected between MON 87460 and the 

conventional control for any of the assessed phenotypic and agronomic characteristics 

(Table VIII-9). 

U.S. 2007 – Study-2

In Study-2, three sites were established with well-watered and water-limited 

treatments (site codes KS, NE, and TX) (Table VIII-2). Comparative assessments of 

phenotypic and agronomic characteristics 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 characteristics 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 water-limited treatment data from KS and NE 

were not included in the statistical analysis. 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 impact the conclusions of the plant pest 

risk assessment. 

Assessment of U.S. 2007 Study-1

No statistical differences between MON 87460 and the control were observed in the 

combined-site analysis. 

Assessment of U.S. 2007 Study-2

In the combined-site analysis for the well-watered 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, plant 
Monsanto Company 07-CR-191U Page 198 of 544 

height, dropped ears, stalk lodged plants, root lodged plants, final stand count, grain 

moisture, test weight, and yield (Table VIII-10). The only statistical difference detected 

was a lower stay green rating (more green tissue) 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 harvest, is not a characteristic that is likely 

associated with an increase in plant pest potential (Radosevich 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 observed 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 considered 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 conventional 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 lodged plants, final stand count, grain moisture, 

test weight, and yield (Table VIII-11). The only significant difference detected was a 

lower stay green rating (more green tissue) 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. Furthermore, 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. demonstrate 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 characteristics, the results support a conclusion of no 

increased plant pest potential of MON 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 Established with Well-Watered and WaterLimited Treatments 

Well-Watered Treatment 

 Mean ± S.E. 

Reference 

Range1

Phenotypic 

characteristic Units MON 87460 Control Min Max 

Seedling vigor 1-9 scale2 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 scale3 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 weight4

 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 differences 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-Limited Conditions in the Combined-Site 

Analysis in 2007 U.S. Field Trials Established with Well-Watered and WaterLimited Treatments 

Water-Limited Treatment 

 Mean ± S.E. 

Reference 

Range1

Phenotypic 

Characteristic Units MON 87460 Control Min Max 

Seedling vigor 1-9 scale2 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 scale3 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 weight4

 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 differences 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 Established with Well-Watered and WaterLimited Treatments 

 

Well-Watered Treatment 

 Mean ± S.E. 

Reference 

Range1

Phenotypic characteristic Units MON 87460 Control Min Max 

Seedling vigor 1-9 scale2 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 shed3

 Days 63.6 ± 0.82 63.3 ± 0.87 61.0 69.0 

Days to 50% silking3

 Days 62.2 ± 1.02 62.3 ± 1.03 59.0 69.0 

Stay green 1-9 scale4

 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 plants3

 #/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 differences 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-Limited Conditions in a 2007 Texas Field 

Trial Established with Well-Watered and Water-Limited Treatments 

Water-Limited Treatment 

 Mean ± S.E. 

Reference 

Range1

Phenotypic 

characteristic Units MON 87460 Control Min Max 

Seedling vigor 1-9 scale2 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 shed3

 Days 61.0 ± 0.00 61.0 ± 0.00 61.0 61.0 

Days to 50% silking3 Days 59.0 ± 0.00 59.0 ± 0.00 59.0 59.0 

Stay green 1-9 scale4

 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 plants3

 #/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 differences 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 under Water Conditions Typical for Local 

Agronomic Practice 

This field study was designed to evaluate MON 87460 under a broad range of 

environmental conditions relevant to commercial corn production. No differences were 

expected between MON 87460 and the control with 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 phenotypic 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 between MON 87460 and the control for any of 

the 14 assessed phenotypic and agronomic characteristics (Table VIII-12). 

Summary of U.S. 2006 under Conditions Typical for Local Agronomic Practice

The results from these studies support a conclusion of no increased plant pest potential of 

MON 87460 compared to conventional corn under 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. Field Study Conducted 

under Typical Agronomic Conditions 

 

 

Mean ± S.E. 

Reference 

Range1

Phenotypic 

characteristic Units MON 87460 Control Min Max 

Seedling vigor 0-9 scale2 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 scale3 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 differences 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 plant is dried and 9 = entire plant is green. 

VIII.D. Seed Germination and Dormancy Assessment 

APHIS considers the potential for weediness 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 between 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 control, which had a genetic background similar 

to MON 87460 but did not possess the drought tolerance trait. In addition, seed from 

multiple conventional corn hybrids (references) were 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 characteristic that is often associated with 

plants that are considered as weeds (Anderson, 1996; Lingenfelter and Hartwig, 2003). 

Information on germination and dormancy is therefore useful when assessing a plant for 
Monsanto Company 07-CR-191U Page 205 of 544 

increased weediness potential. For corn, the potential for hard seed (dormant) is 

negligible or nonexistent. Standardized germination assays are available and routinely 

used to measure the germination characteristics of corn seed. The Association of Official 

Seed Analysts (AOSA), an internationally 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 particular the absence of hard seed, support a 

conclusion of no increased weediness potential of MON 87460 compared to conventional 

corn. 

Seed of MON 87460, the conventional control, and three commercial corn hybrids 

(references; nine total, three 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 genetic 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: 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 (Table VIII-13). In the alternating temperature regimes, the lower temperature 

was maintained for 16 h and the higher temperature 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). Statistical 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 production site × seed substance interactions were detected for 

any combination except percent germinated and percent viable firm swollen seed at 10°C. 

These two characteristics were analyzed separately by site to account for the interaction. 

In the combined site analysis, no statistical differences were detected (α = 0.05) between 

MON 87460 and the control in any temperature regime for any characteristic (27 

comparisons, Table VIII-14). 

As noted above, significant production site × seed substance type interactions were 

detected in percent germinated and percent viable firm swollen seed at 10°C. Analyses 

were therefore conducted on an individual site basis for these 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, 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 for the IA site, 

the values for MON 87460 and the control at 10°C were within the reference range for 
Monsanto Company 07-CR-191U Page 206 of 544 

both percent germination and percent viable firm swollen seed. Thus, the difference is 

not expected to constitute a meaningful biological 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 results of this study, in 

particular the absence of hard seed, support a conclusion of no increased weediness 

potential of MON 87460 compared to conventional corn. 

 
Monsanto Company 07-CR-191U Page 207 of 544 

Table VIII-13. Seed Germination and 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 conducted in temperature-controlled growth chambers using the 

rolled towel test method to measure the described 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 following 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 were made on four, seven, and twelve days 

after planting for seed in the additional temperature regimes. 
Monsanto Company 07-CR-191U Page 208 of 544 

Table VIII-14. Germination of MON 87460 and a Conventional Control Corn 

across Sites1

 Mean % ± S.E. Reference Range

Temperature Characteristic MON 8746 Control Min Max

5°C Dead 3.9 ± 0.90 4.5 ± 0.93 4.0 11.0

Germinated 0.0 ± 0.00 0.0 ± 0.00 0.0 0.3

Viable firm swollen 96.1 ± 0.90 95.5 ± 0.93 89.0 96.0

Viable hard2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

10°C Dead 1.0 ± 0.30 1.0 ± 0.32 0.8 6.0

Germinated3 - - - - 

Viable firm swollen3 - - - - 

Viable hard2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0 

20°C Dead 0.6 ± 0.23 0.3 ± 0.18 0.5 5.8

Germinated 99.4 ± 0.23 99.8 ± 0.18 94.3 99.5

Viable firm swollen2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable hard2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

30°C Dead 1.2 ± 0.55 1.7 ± 0.36 0.5 6.0

Germinated 98.8 ± 0.55 98.3 ± 0.36 94.0 99.5

Viable firm swollen2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable hard2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

20 / 30°C 

(AOSA) 

Dead 0.5 ± 0.19 0.9 ± 0.23 0.3 4.3

Normal germinated 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 swollen2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable hard2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

10 / 20°C Dead 1.0 ± 0.39 1.7 ± 0.34 0.3 5.0

Germinated 99.0 ± 0.39 98.3 ± 0.34 95.0 99.8

Viable firm swollen2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable hard2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

10 / 30°C Dead 1.5 ± 0.34 1.3 ± 0.37 0.3 5.0

Germinated 98.5 ± 0.34 98.8 ± 0.37 95.0 99.8

Viable firm swollen2 0.0 ± 0.00 0.0 ± 0.00 0.0 0.0

Viable hard2 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 differences 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 results of the seeds from the three production sites. 

The results for the individual site analysis where 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 MON 87460 were also assessed. 

The purpose of this study was to assess whether the introduction of the drought tolerance 

trait and the expression of the CSPB protein altered the pollen characteristics of 

MON 87460 compared to a conventional control, which had a genetic background similar 

to MON 87460 but did not possess the drought tolerance trait. In addition, pollen from 

multiple conventional corn hybrids (references) was included in the analysis to establish a 

range of natural variability 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 design, 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 (Alexander, 1980) and evaluated for viability 

and general morphology. Data analysis compared 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 well-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 conditions. The lack of differences between 

the pollen collected from MON 87460 compared 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 Range1

Characteristic MON 87460 Control Min Max 

Viability (%) 98.4 ± 0.50 98.8 ± 0.44 97.4 98.6 

Diameter (μm) 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 Range1

Characteristic MON 87460 Control Min Max 

Viability (%) 96.9 ± 0.64 97.2 ± 0.67 96.3 98.5 

Diameter (μm) 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 Interactions Assessment 

Environmental interactions evaluations were conducted as part of the plant 

characterization studies for MON 87460. These data are used to evaluate plant pest 

potential for MON 87460 compared to a conventional control. In addition, multiple 

conventional corn hybrids (references) were included in the analysis to establish a range 

of natural variability for each characteristic. The environmental interactions evaluation 

included data collected in the phenotypic studies (plant-insect, plant-disease, and plantenvironment interactions) and studies on abiotic 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, certain characteristics, including volunteer 

potential and survival outside cultivation, can be used for an assessment of weediness, an 

element of plant pest potential. Abiotic stress tolerance was evaluated qualitatively from 

natural occurrences in the phenotypic field studies and also quantitatively under 

controlled conditions in greenhouse and growth chamber studies. Studies to assess 

tolerance to drought, cold, heat, and salt stress at sensitive young growth stages under 

controlled conditions are useful for characterizing the extent of stress tolerance imparted 

by the insertion of the cspB gene. Such a characterization is useful to identify any 

potential changes in tolerance that warrant further investigation of plant pest risk under 

stress conditions. If no changes in tolerance 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 assessment 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 (excluding 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 biologically 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 conducted for evaluation of phenotypic and agronomic 

characteristics of MON 87460, observational data 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 the environmental interactions of MON 87460 

compared with those of the conventional control corn. The observed stressors were 

“natural” (i.e., no artificial infestation or interference 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 six 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 plant-disease interactions, or plant response to 

abiotic stressors of MON 87460 compared to the control. For the plant-insect 

interactions, plant-disease interactions, and plant responses to abiotic stressors, the 

reported values represent the range of ratings observed across the three or four 

replications at each site. MON 87460 and 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 replications for that particular stressor (e.g. “none” rating vs. 

“slight-moderate” rating). The ratings observed 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 were evaluated. No qualitative differences 

were observed in MON 87460 compared to the control for disease, damage or plant 

response to abiotic stressors (other than water stress) evaluated. For pest and beneficial 

arthropod evaluations, two differences were observed out of approximately 388 

comparisons. Grasshopper damage was lower for MON 87460 compared to the control 

(none vs. slight) at the IAE site at Observation 3 (Appendix H, Table H-17). The 

difference in grasshopper damage was not considered biologically meaningful because 

the difference detected at the IAE site was within the range of the references (noneslight). Additionally, no differences were detected between MON 87460 and the control 

for grasshopper damage for Observations 2 and 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 damage does not represent a trend in the data 

and is not considered biologically 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 sites in 2006 (Appendix H, Tables H-4 and H5, at three well-watered sites in 2007 (Appendix H, Tables H-11 and H-12), and a 

single site at TX in U.S. 2007 Study-1, with well-watered 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 statistical differences were detected between 

MON 87460 and control for corn earworm damage or for European corn borer 

damage. 

Arthropod Abundance

Arthropod collections were conducted at three well-watered sites in 2006 (Appendix 

H, Tables H-6 and H-7), at three well-watered 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 control. For three of the eight detected 

differences, the mean abundance values from MON 87460 were within the respective 

reference ranges. For the remaining differences, 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 detected differences were not indicative of 

a consistent response in the data associated with the trait and are unlikely to be 

biologically meaningful in terms of plant pest potential of MON 87460 compared to 

the control. 

Summary of Insect, Disease, and Abiotic Stressor Interactions Assessment in Field 

Studies

The results of the environmental 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 deficit), disease damage, and arthropod damage 

indicates that the introduction of the drought tolerance trait is unlikely to be biologically 

meaningful in terms of increased 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 Assessment in Controlled Environment Studies 

Abiotic stress tolerance was evaluated qualitatively from natural occurrences in the 

phenotypic field studies (Section VIII.F.1) and also quantitatively under controlled 

environment conditions in greenhouse and growth chamber studies as presented in this 
Monsanto Company 07-CR-191U Page 213 of 544 

section. Studies to assess tolerance to drought, cold, heat, and salt stress at sensitive 

young growth stages under controlled conditions are useful for characterizing the extent 

of stress tolerance imparted by the inserted stress tolerance protein (CSPB) (see mode of 

action Section I.D). Although drought, cold, heat or salt tolerance could be associated 

with some plants that are weeds, 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 without 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 MON 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 meaningfully different compared to conventional 

corn. 

VIII.F.2.1. Drought Tolerance Assessment 

The purpose of this study was to assess the effect of various levels of drought stress on 

the growth and development of plants of MON 87460 compared to a conventional control 

in a greenhouse environment. 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. At the V4 growth stage, 80 test plants and 80 control plants were selected for 

uniformity and arranged in a complete factorial treatment structure (8 treatment 

combinations) in a randomized complete block design with 20 replications. The factors 

were drought level (well-watered, mild, moderate, or severe) 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, growth stage, chlorophyll 

content, and leaf rolling score. Fresh and 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 treatments, no statistical differences were 

detected (α = 0.05) between MON 87460 and the control 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 height than the control (88.0 vs. 89.6 cm, 

respectively). This difference is small in magnitude, does not represent a consistent trend 

across the other treatments in this study, and likely represents 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, respectively) and had lower fresh 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 significant differences were detected between 

MON 87460 and control for any of the measured characteristics (Table VIII-17). 

Moderate drought treatment

In the moderate drought treatment, no significant differences were detected between 

MON 87460 and control for chlorophyll content and for biomass (fresh weight and 

dry weight). Four differences were detected between MON 87460 and the control in 

the moderate drought treatment. MON 87460 had less plant height than the control 

14 DAT (127.0 vs. 130.2 cm, respectively), had fewer leaves at 7 DAT and 14 DAT 

(growth stage 5.4 vs. 5.8 and 7.3 vs. 7.7, respectively) and MON 87460 had a higher 

leaf rolling score 7 DAT than the control (2.4 vs. 2.1, respectively, Table VIII-17). 

Severe drought treatment

In the severe drought treatment, no significant differences were detected between 

MON 87460 and control for plant 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 treatment. MON 87460 

had a lower leaf rolling score than the control (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 plants in the well-watered treatment, test and 

control plants in the mild, moderate, and severe treatments exhibited a dose-dependent 

pattern of lower plant height, growth stage, fresh weight, and dry weight with increasing 
Monsanto Company 07-CR-191U Page 215 of 544 

water stress. Furthermore, the differences observed in plant height, growth stage, and 

leaf rolling were not consistent across treatments, do not indicate a competitive advantage 

for MON 87460, and do not represent a meaningful trend. Therefore, based on the 

characteristics measured to assess drought tolerance, the results support 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 imposed during early vegetative growth stages. 
Monsanto Company 07-CR-191U Page 216 of 544 

Table VIII-17. Comparison of MON 87460 to the Control Prior to Drought Treatment and at 7 and 15 Da

Well-watered 

Mean ± S.E. 

Mild 

Mean ± S.E. 

Moderate 

Mean ± S.E. 

Evaluation timing Characteristic 

MON 

87460 Control 

MON 

87460 Control 

MON 

87460 Control 

Prior to treatment 1 Plant height 

(cm) 

88.0* 

± 0.41 

89.6 

± 0.52 - - - - 

Growth stage 2 4.03 ± 0.0 4.03 ± 0.0 - - - - 

Chlorophyll 4 51.4 ± 0.35 50.9 ± 0.46 - - - - 

Post-treatment Plant height 

(cm)

126.3 

± 0.86 

127.9 

± 1.09 

119.1 

± 1.23 

119.8 

± 1.07 

112.2 

± 0.92 

114.3 

± 0.70 

(7 DAT) Growth stage 6.2 ± 0.08 6.1 ± 0.1 5.9 ± 0.07 6.0 ± 0.05 5.4* ± 0.11 5.8 ± 0.1 

Chlorophyll 54.4 ± 0.53 53.7 ± 0.8 55.1 ± 0.71 53.9 ± 0.52 53.4 ± 1.09 53.7 ± 0.48 

Leaf roll 5 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 2.4* ± 0.18 2.1 ± 0.14 

Post-treatment Plant height 

(cm)

175.3 

± 0.97 

178.0 

± 0.88 

152.1 

± 1.25 

151.0 

± 1.75 

127.0* 

± 0.93 

130.2 

± 1.0 

(15 DAT) Growth stage 9.6* ± 0.11 9.9 ± 0.07 9.0 ± 0.0 8.9 ± 0.08 7.3* ± 0.1 7.7 ± 0.11 

Chlorophyll 53.4 ± 0.49 53.8 ± 0.43 53.6 ± 0.64 52.8 ± 0.5 56.0 ± 0.76 56.2 ± 0.64 

Leaf roll 1.0 ± 0.0 1.0 ± 0.0 1.1 ± 0.05 1.0 ± 0.0 2.2 ± 0.12 2.1 ± 0.11 

Fresh weight (g) 620.6* ± 14.73 659.4 ± 9.24 457.6 ± 14.64 430.9 ± 14.60 230.8 ± 5.5 249.1 ± 6.22 

Dry weight (g) 65.5* ± 1.51 70.5 ± 0.93 51.4 ± 1.32 49.1 ± 1.36 32.4 ± 0.65 35.1 ± 0.72 

S.E. = standard error; n=20 for test and control plants in each treatment. *Indicates significant differences detected between MO(p<0.05) provided by t-test comparisons. 1 Due to the randomization of the substances and drought treatments to the plants within a 

treatments were applied, the pre-treatment measurements for each substance consist of four sub-samples (n=20). 2 Growth stage was 

of leaves with a visible leaf collar. 3 Data could not be analyzed due to lack of variability. 4 Chlorophyll - relative chlorophyll c

readings at the base, midpoint and tip of the fourth leaf measured with a Single Photon Avalanche Diode-containing (SPAD) meter. 

scale of 1 to 5 (1=no rolling, 2=slight rolling upper leaves (younger), 3=more rolling upper leaves and slight rolling lower leaves, 4

rolling 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 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 (20/15 °C), moderate 

(15/10 °C), and severe (4/4 °C) cold treatments 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 photoperiod 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 measured at the conclusion of the experiment. 

No significant differences were observed in plants prior to temperature treatments 

(Table VIII-18). 

Optimal temperature treatment (30/22° C)

Plants in the optimal temperature treatment were included to provide plant 

characterization data for MON 87460 and the control in the absence of cold stress and 

to validate that the cold temperature treatments affected corn growth as expected. In 

the optimal temperature treatment, no significant 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 control 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 development of the biotechnology-derived crop 

compared to a conventional control. Compared to test and control plants in the optimal 
Monsanto Company 07-CR-191U Page 218 of 544 

temperature group, test and control plants in the mild, moderate and severe treatments 

exhibited a dose-dependent pattern of lower plant height, growth stage, vigor, fresh 

weight and dry weight. No significant differences were observed between MON 87460 

and the control in the mild, moderate, or severe cold treatments. Based on the 

characteristics measured to assess cold tolerance, the results support a conclusion that 

MON 87460 is still sensitive to cold stress and any differences from conventional corn in 

response to cold stress are not related to the gene of interest. 
Monsanto Company 07-CR-191U Page 219 of 544 

Table VIII-18. Comparison of MON 87460 to the Control Prior to Cold Treatment and at 4 and 8 Days af

Optimal Mean ± S.E. Mild Mean ± S.E. Moderate Mean ± S.E. 

(30/22°C) (20/15°C) (15/10°C) 

Evaluation Timing Characteristic MON 87460 Control MON 87460 Control MON 87460 Control M

Prior to treatment Plant height (cm) 

43.1 

± 0.47 

42.3 

± 0.33 

43.2 

± 0.45 

42.4 

± 0.39 

43.9 

± 0.42 

42.8 

± 0.44 

 Growth stage1

3.0 

± 0.0 

3.0 

± 0.0 

3.0 

± 0.0 

3.0 

± 0.0 

3.0 

± 0.0 

3.0 

± 0.0 

 Chlorophyll2

40.3 

± 0.79 

39.5 

± 0.65 

41.3 

± 0.54 

39.9 

± 0.82 

39.8 

± 0.74 

40.2 

± 0.88 

 Vigor3,4 2 ± 0.0 2 ± 0.0 2 ± 0.0 2 ± 0.0 2 ± 0.0 2 ± 0.0 

Post-treatment Plant height (cm) 

62.6 

± 0.41 

62.7 

± 0.46 

54.5 

± 0.83 

53.4 

± 1.01 

48.8 

± 0.66 

47.6 

± 0.93 

(4 DAT) Growth stage 

3.6* 

± 0.11 

3.3 

± 0.1 

3.04

± 0.0 

3.04

± 0.0 

3.04

± 0.0 

3.04

± 0.0 

 Chlorophyll 

45.1 

± 0.83 

44.1 

± 0.67 

43.9 

± 0.57 

43.3 

± 1.03 

39.2 

± 0.77 

40.2 

± 0.75 

 Vigor4 2 ± 0.0 2 ± 0.0 3 ± 0.0 3 ± 0.0 3 ± 0.0 3 ± 0.0 

Post-treatment Plant height (cm) 

80.2 

± 0.77 

81.7 

± 0.95 

64.3 

± 0.74 

62.6 

± 0.76 

54.0 

± 0.61 

53.5 

± 0.74 

(8 DAT) Growth stage 

4.5* 

± 0.11 

4.1 

± 0.07 

3.9 

± 0.08 

4.0 

± 0.05 

3.04

± 0.0 

3.04

± 0.0 

 Chlorophyll 

41.5 

± 0.82 

41.5 

± 0.68 

45.0 

± 0.84 

43.2 

± 1.02 

38.4 

± 0.7 

39.2 

± 0.79 

 Vigor4 2 ± 0.0 2 ± 0.0 3 ± 0.0 3 ± 0.0 5 ± 0.0 5 ± 0.0 

 Fresh weight (g) 

26.2 

± 1.37 

23.4 

± 1.06 

16.5 

± 0.65 

15.8 

± 0.89 

9.4 

± 0.37 

8.9 

± 0.4 

 Dry weight (g) 

2.8* 

± 0.14 

2.4 

± 0.09 

1.3 

± 0.06 

1.3 

± 0.07 

1.3 

± 0.05 

1.2 

± 0.05 

S.E. = standard error; n=20 for MON 87460 and control in each temperature treatment. *Indicates significant differences detected bet

control (p<0.05) provided by t-test comparisons. 1 Number of leaves with a visible leaf collar. 2 Relative chlorophyll content was an 

base, midpoint, and tip of the leaf measured with a Single Photon Avalanche Diode (SPAD)-containing meter. 3 Plant vigor was ra

where 1 = good and 9 = poor. 4 Not 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 effect of heat-induced stress on the growth 

and development of plants of MON 87460 compared to a conventional control. The test 

substance, MON 87460, and a conventional control 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 photoperiod 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, vigor and 

necrosis prior to heat treatment and at 3 and 5 DAT. Fresh and dry weights of aboveground biomass were measured at the 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. MON 87460 was taller (44.8 

vs. 42.7 cm) and had less chlorophyll content (41.0 vs. 43.3 SPAD units [Single Photon 

Avalanche Diode-containing meter]) than the control 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 the control (Table VIII-19). 

Optimal temperature treatment (30/22°C)

Plants in the optimal temperature treatment were included to provide plant 

characterization data for MON 87460 and the control in the absence of heat stress and 

to validate that high temperatures affected corn growth as expected. In the optimal 

temperature treatment no significant differences were detected between MON 87460 

and the control for plant height, vigor and necrosis at 3 and 5 DAT. Five differences 

were observed in plants grown under optimal conditions. MON 87460 had less 

chlorophyll content compared to the control 3 and 5 DAT (45.9 vs. 47.7 and 45.6 vs. 

47.0 SPAD units, respectively). MON 87460 had 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 treatment. 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 significant differences were detected between 

MON 87460 and the control for any of the measured characteristics (Table VIII-19). 

Severe temperature treatment (47/35°C)

In the 47/35 °C treatment, no significant 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 study were used to assess heat stress on the 

growth and development of the biotechnology-derived crop compared to a conventional 

control. Compared to test and control plants in the optimal temperature group, test and 

control plants in the mild, moderate and severe treatments exhibited a dose-dependent 

pattern of lower plant height, growth stage, vigor, fresh weight and dry weight with 

increasing temperature. Two differences 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 treatment 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 assess heat tolerance, the results 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 related to the gene of interest. 
Monsanto Company 07-CR-191U Page 222 of 544 

Table VIII-19. Comparison of MON 87460 to the Control Prior to Heat Treatment and at 3 and 5 Days af

Optimal Mean ± S.E. Mild Mean ± S.E. Moderate Mean ± S.E. 

30/22°C 40/35°C 43/35°C 

Evaluation timing Characteristic 

MON 

87460 Control 

MON 

87460 Control 

MON 

87460 Control 

Prior to treatment Plant height (cm) 44.8* ± 0.42 42.7 ± 0.4 43.6 ± 0.5 43.3 ± 0.59 43.9 ± 0.34 43.4 ± 0.59 

Growth stage 1 3.05 ± 0.0 3.05 ± 0.0 3.05 ± 0.0 3.05 ± 0.0 3.05 ± 0.0 3.05 ± 0.0 

Chlorophyll 2 41.0* ± 0.71 43.3 ± 0.43 44.3 ± 0.43 45.8 ± 0.62 44.4 ± 0.54 44.4 ± 0.52 

Vigor 3 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 

Necrosis4 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 

Post-treatment Plant height (cm) 64.0 ± 0.37 63.4 ± 0.51 57.9 ± 0.5 56.8 ± 0.69 52.1 ± 0.71 50.3 ± 0.89 

(3 DAT) Growth stage 3.6* ± 0.11 3.2 ± 0.08 3.05 ± 0.0 3.05 ± 0.0 3.05 ± 0.0 3.05 ± 0.0 

Chlorophyll 45.9* ± 0.48 47.7 ± 0.43 42.3* ± 0.48 43.8 ± 0.58 38.6 ± 0.62 37.6 ± 0.62 

Vigor 1.05 ± 0.0 1.05 ± 0.0 1.1 ± 0.07 1.1 ± 0.05 1.6 ± 0.25 1.5 ± 0.2 

Necrosis 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 1.05 ± 0.0 

Post-treatment Plant height (cm) 71.7 ± 0.65 71.7 ± 0.7 60.1 ± 0.51 58.8 ± 0.65 54.8 ± 0.6 52.7 ± 0.91 

(5 DAT) Growth stage 4.05 ± 0.0 4.05 ± 0.0 3.4 ± 0.11 3.5 ± 0.11 3.0 ± 0.0 3.0 ± 0.0 

Chlorophyll 45.6* ± 0.5 47.0 ± 0.43 40.1 ± 0.51 40.9 ± 0.54 32.6 ± 0.94 33.2 ± 0.88 

Vigor 1.05 ± 0.0 1.05 ± 0.0 1.5 ± 0.11 1.8 ± 0.1 5.1 ± 0.1 5.0 ± 0.11 

Necrosis 1.05 ± 0.0 1.05 ± 0.0 1.05 ±0.0 1.05 ± 0.0 1.1 ± 0.05 1.0 ± 0.0 

Fresh weight (g) 14.5* ± 0.38 12.5 ± 0.3 9.8 ± 0.24 9.8 ± 0.29 7.2 ± 0.24 6.9 ± 0.29 

Dry weight (g) 1.1* ± 0.03 1.0 ± 0.03 1.0 ± 0.03 1.0 ± 0.03 0.7 ± 0.04 0.7 ± 0.03 

S.E. = standard error; n = 20 for MON 87460 and the control in each temperature treatment. *Indicates significant differences detected 

the control (p<0.05) provided by t-test comparisons. 1 Number of leaves with a visible leaf collar. 2 Relative chlorophyll content was 

the base, midpoint and tip of the fourth leaf measured with a Single Photon Avalanche Diode-containing (SPAD) meter. 3 Plant vigor 

1-9, where 1=good and 9=poor. 4 Necrosis was rated using a scale of 1-5, where 1=good and 5=poor. 5 Not statistically analyzed due t
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 effect of various salt treatments on the growth 

and development of plants of MON 87460 compared 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 plants 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 type (test or control). 

Beginning 17 days after planting and continuing for 12 days, pots in each salt treatment 

were irrigated with either reverse osmosis water or varying concentrations of a NaClCaCl2 solution (up to 600 mM) to achieve different soil electrical 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 treatment 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 plant height or vigor (Table VIII-20). 

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. MON 

87460 exhibited greater chlorophyll 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 treatment were included to provide plant 

characterization data for MON 87460 and the control 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 characteristic 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 the 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 salt treatment, no differences were detected between MON 87460 

and the control for plant height, growth stage, and biomass (fresh weight and dry 

weight). Two differences were detected between MON 87460 and the control for 

plants in the moderate treatment. MON 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 differences were detected between MON 87460 and 

the control for growth stage, chlorophyll content, and biomass (fresh weight and dry 

weight). Two differences were detected 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 decreased 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 biotechnology-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 total 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 represent a trend in the data. Based on the 

characteristics measured to assess salt tolerance, the results support a conclusion that 

MON 87460 is still sensitive to salt stress and any differences from conventional corn in 

response to salt stress are not related to the gene of interest. 
Monsanto Company 07-CR-191U Page 225 of 544 

Table VIII-20. Comparison of MON 87460 to the Control Prior to Salt Treatment and at 9 and 12 Days a

Untreated Mean ± S.E. Mild Mean ± S.E. Moderate Mean ± S.E. 

Evaluation timing Characteristic MON 87460 Control MON 87460 Control MON 87460 Control MPrior to treatment1 Plant height (cm) 54.2 ± 0.41 54.8 ± 0.41 - - - - 

Growth stage2 4.03 ± 0.00 4.03 ± 0.00 - - - - Chlorophyll4 50.2* ± 0.35 48.4 ± 0.36 - - - - Vigor5 2.1 ± 0.04 2.1 ± 0.06 - - - - 

Post-treatment Plant height (cm) 107.8 ± 1.01 109.5 ± 1.20 85.7 ± 0.57 87.5 ± 0.72 76.4 ± 0.63 77.3 ± 0.9 (9 DAT) Growth stage 7.2 ± 0.11 7.1 ± 0.07 6.5 ± 0.11 6.5 ± 0.11 5.7 ± 0.13 5.8 ± 0.12 

Chlorophyll 48.6 ± 0.68 47.4 ± 1.06 52.1 ± 0.56 52.5 ± 0.76 48.4* ± 0.76 46.1 ± 0.67 Vigor 2.0 ± 0.07 2.1 ± 0.05 3.7 ± 0.11 3.5 ± 0.14 5.6 ± 0.11 5.7 ± 0.11 

Post-treatment Plant height (cm) 125.2 ± 1.98 127.4 ± 1.13 94.4 ± 0.70 96.2 ± 1.04 77.1 ± 0.82 77.4 ± 0.67 (12 DAT) Growth stage 8.0 ± 0.05 8.0 ± 0.00 6.9 ± 0.07 7.0 ± 0.07 5.8 ± 0.09 5.9 ± 0.07 

Chlorophyll 43.5 ± 0.99 42.9 ± 1.01 47.8 ± 0.77 48.3 ± 0.56 42.8 ± 0.63 42.9 ± 0.54 Vigor 2.0 ± 0.07 1.9 ± 0.08 4.0 ± 0.07 3.9 ± 0.14 6.2* ± 0.09 6.6 ± 0.14 Fresh weight (g) 217.9 ± 3.22 218.8 ± 3.05 108.8 ± 2.10 111.1 ± 1.62 53.0 ± 1.01 53.8 ± 0.92 Dry weight (g) 19.4 ± 0.23 19.8 ± 0.40 11.3* ± 0.18 11.7 ± 0.13 6.8 ± 0.17 6.8 ± 0.14 

S.E. = standard error; n = 20 for MON 87460 and control for each treatment. *Indicates significant differences detected between M(p<0.05) provided by t-test comparisons. 1 Due to the randomization of the substances and treatments within a replication before the tr

pre-treatment measurements consist of four sub-samples (n=20). 2 Number of leaves with a visible leaf collar. 3 Data could not b

variability. 4 Relative chlorophyll content was an average of readings at the base, midpoint and tip of the third (pre-treatment) or f

measured with a Single Photon Avalanche Diode-containing (SPAD) meter. 5 Plant vigor was rated using a scale of 1 – 9, where 1 = go
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 conventional 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 volunteer potential. Comparative assessments were conducted on 

MON 87460 and a conventional control. In addition, six commercial corn hybrids were 

included as references. Normal seed germination rates were confirmed for MON 87460, 

control, and reference starting seeds by standard viability testing. The trials were 

established at each location as a randomized 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 surface. Seed were then incorporated with 

a disk or field cultivator. Plots were assessed 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 maintain 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 incorporated into the soil. Volunteer plant 

counts were taken after planting in fall 2006 until soil temperatures dropped below 50°F 

and re-commenced in spring 2007 approximately a week prior to the average local 

planting date for each field 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. 

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 conclusion that the introduction of the drought 

tolerance trait did not alter the volunteer potential of MON 87460 compared to 

conventional corn. Furthermore, these results demonstrate that the drought tolerance trait 

in MON 87460 confers no biologically meaningful 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 

Site1

 Season MON 87460 Control References2

BG Fall3 - - - 

 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 soil temperature was <50°F at the first observation 

timepoint.

VIII.F.4. Survival Outside of Cultivation Assessment 

Weediness or invasiveness may also be indicated if corn exhibited an increased rate of 

survival outside of cultivation. The purpose of this study was to assess the ability of 

MON 87460 to establish and persist in areas that are not cultivated 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 vegetation and abiotic and biotic 

stressors present in each environment. Additional materials and methods are provided in 

Appendix M. 

Phenotypic and agronomic characteristics encompassing plant growth, development, and 

yield were assessed for MON 87460, the control, and the references in unmanaged 

environments. The experiment was established at each of four sites in a randomized 

complete block design with three replications. Early stand count, final stand count, 

vegetative plant height, plant height at maturity, 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 calculated 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 greater 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 trend 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 produced 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 when compared to the control (41.3 vs. 

24.0 plants per plot, respectively) and final stand count was greater for MON 87460 when 

compared to the control (38.3 vs. 21.0 plants per plot, respectively). It is noteworthy that 

MO was the only site of the four planted sites where seeds were produced; however, the 

replacement values for MON 87460 and the control 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 exhibiting increased weediness. Thus, MON 87460 

is not likely to persist in any of the four unmanaged environments 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 potential for corn to persist outside of managed 

agricultural environments. 
Monsanto Company 07-CR-191U Page 229 of 544 

Table VIII-22. Survival Outside of Cultivation Phenotypic Comparison of MON 87460 to the Control in 2

 Early Stand Count 

(plants/plot) 

Mean

± S.E. 

Late Vegetative Plant Height 

(inches) 

Mean

± S.E. 

Final Sta

(plant

Mean

Site1 MON 

87460 

Control Reference MON 

87460 

Control Reference MON 

87460 

Co

IL 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 

MO 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 

NE 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 TX - - - - - - - 

 Plant Height at Maturity 

(inches) 

Mean

± S.E. 

# Ears Produced 

(ears/plot) 

Mean

± S.E. 

# Seed P

(seed

Mean

Site MON 

87460 

Control Reference MON 

87460 

Control Reference MON 

87460 

Co

IL 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 

NE 20.7 - 30.0-30.0 - - - - TX - - - - - - - 

 Average # Ears 

(ears/plant) 

Mean

± S.E. 

Replacement Value2

Site MON 

87460 

Control Reference MON 

87460 

Control Reference 

IL - - - 0.0 0.0 0.0-0.0 

MO 0.2 ± 0.08 0.2 ± 0.05 0.2-0.4 0.15 0.03 0.05-0.57 

NE - - - 0.0 0.0 0.0-0.0 TX - - - 0.0 0.0 0.0-0.0 

S.E. = standard error; n = 3 for MON 87460 and control at each site. *Indicates significant differences detected between MON 87460 

Dash (-) indicates no data available. Mean values without S.E. values are from a sample size of one and S.E. cannot be calculated. 

1 Site codes: IL = Effingham County, IL; MO = Shelby County, MO; NE = York County, NE; TX = Carson County, TX 2 Due to seed counting errors, the exact number of seed sown in each plot varied. Therefore, replacement values at the MO site were c

seeds sown per plot using the formula, Replacement Value = [Number of Seed Produced] / 50. Replacement values were 

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 pest potential of MON 87460 compared 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 characterization, and comparison of MON 87460 to control and conventional 

reference corn hybrids. Data were collected for seed dormancy and germination 

parameters, phenotypic and agronomic characteristics during plant growth and 

development, pollen characteristics, observations for plant-insect, plant disease and plantabiotic stressor interactions, abiotic stress tolerance, volunteer potential, 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 risk compared to conventional 

corn. Data indicate that MON 87460 does not confer any increased susceptibility or 

tolerance to specific insect, disease, 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 yield 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, and 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 Principles and Applications, 3rd ed. West 

Publishing Company, St. Paul, MN. pp 27-37. 

AOSA. 2006. Association of Official Seed Analysts. 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. Improving 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. Westgate. 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. Schussler, and M. Ibanez. 

2006. Changes in drought tolerance in maize 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. Water 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 University, University Park, PA. 

OECD. 1993. Safety considerations for biotechnology: Scale-up of crop plants. 

Organization for Economic Co-operation and Development, Paris, France 

http://www.biosafety.be/CU/BSL_Ressources/PDF/M00034525.pdf Accessed 

January 9, 2009. 

Radosevich, S., J. Holt, and C. Ghersa. 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 Journal of Bioscience. 63:313-320. 
Monsanto Company 07-CR-191U Page 232 of 544 

IX. Environmental Consequences and 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 practices. 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 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, thereby allowing unrestricted introduction of the article. 

The definition of plant pest in the PPA is broad and includes living organisms that could 

directly or indirectly injure, damage, or cause disease in any plant or plant product [7 

U.S.C. § 7702(14)]. This section summarizes the plant pest risk for MON 87460 based 

on the information presented in the previous sections of the petition. Information related 

to plant pest risk characteristics include disease and pest susceptibilities, expression of 

the gene product (CSPB, NPTII), changes to plant metabolism, weediness of the 

regulated article, any impacts on the weediness of any other plant with which it can 

interbreed, and the transfer of genetic information to organisms 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 article is not likely to pose a plant pest risk. As part of its 

plant pest risk assessment, the genetic construct inserted into MON 87460 was evaluated 

to determine if those sequences cause plant disease. Morphological characteristics of 

MON 87460 were analyzed to determine if it will become weedy or invasive. The 

potential for gene flow and introgression of the 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. Finally, the propensity of MON 87460 to 

become a greater reservoir of plant pests (insects or pathogens) compared to conventional 

plants and the potential for horizontal gene transfer was evaluated. Using this risk 

assessment process, the data and analysis presented 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 nutritional equivalence 

of forage and grain as compared to conventional corn, (6) phenotypic 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, cultivation practices, or the management of 

weeds, diseases, and insects than conventionally bred drought tolerant corn. 
Monsanto Company 07-CR-191U Page 233 of 544 

APHIS has recently proposed to amend 7 CFR Part 340 to include its noxious weed 

authority. Because the data show that MON 87460 has no potential to cause injury, 

damage or disease to any protected interest, 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-limited conditions compared to conventional 

corn. 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 water-limited conditions, particularly during flowering and grainfill periods when 

corn yield potential is most sensitive to stress by disrupting kernel development. 

Efficacy in MON 87460 is derived by expression of the inserted Bacillus subtilis cold 

shock protein B (CSPB). A detailed description of the mode of action for CSPB and its 

drought tolerance trait is provided in Section I.D. CSPB is an extensively studied, stressinducible protein known to facilitate adaptation to environmental stresses in bacteria. 

CSPB acts by interacting with RNA and unfolding secondary structures in RNA 

molecules, which is known to have an effect on RNA stability and on the ability of the 

cell to translate those RNA molecules, thus helping to preserve normal cellular functions 

(Schindler et al., 1999; Graumann et al., 1997). The CSPB protein in MON 87460 binds 

RNA and helps to maintain plant cellular functions in rapidly growing and reproductive 

organs under water-limited conditions. Our data suggest that CSPB in MON 87460 

works by minimizing the effect of water limitation on photosynthesis, stomatal 

conductance, and carbon fixation, and ultimately 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 development 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 allow organisms to survive in adverse 

environments, it was hypothesized that inserting a stress response protein into plants 

could impart a desirable phenotype. Using a high through-put screening biotechnology 

approach, Castiglioni et al. (2008) demonstrated that bacterial cold shock proteins (CSPs) 

can confer improved stress adaptation to multiple plant species. Twenty-two events 

expressing the cold shock protein B (CSPB) were evaluated in water-limited field trials 

using commercial grade corn in environments that 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 well-watered rate. Analyzing data from all 

events, the CSPB containing events demonstrated 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 protein 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 detected. 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 shock domain (CSD) containing proteins, the 

CSPB protein from B. subtilis, expressed in MON 87460 accumulates in actively growing 

tissues where it binds to RNA and facilitates the unfolding of RNA secondary structures 

that are formed due to stress leading to more 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 season can diminish corn productivity and 

yield, particularly during flowering and grainfill periods when corn yield potential is 

most sensitive to stress by disrupting fertilization and kernel development (Claassen and 

Shaw, 1970; Boyer and Westgate, 2004; Campos 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 improved 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 (Section VI). CSPB protein does not share any 

amino acid sequence similarities with known allergens, 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 small transiently stable CSPB protein fragment 

was very quickly degraded during short exposure to simulated intestinal fluid (SIF). 

Rapid digestion of the 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 fragment 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 87460 
Monsanto 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 adverse 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 establishes 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 exposure in animals will also be 

low with chickens, swine, and dairy cows consuming only nanogram quantities of each 

protein per kilogram of body weight (Section VI.D). Collectively, these data establish 

the dietary safety of the CSPB protein. Because NTOs, including humans, will primarily 

come in contact with CSPB through dietary exposure it can be concluded with reasonable 

certainty that the CSPB protein has no meaningful toxic potential to exposed organisms 

in the environment. 

The dietary safety of NPTII has been extensively evaluated through several lines of 

experimental evidence, and several products containing NPTII have been approved by 

regulatory agencies on a global basis. NPTII 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 (Flavell et al., 1992; Nap et al., 1992; Fuchs et 

al., 1993a and 1993b; EFSA, 2009). 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 (40 CFR Part 180.1134). In 2007, the 

European Food Safety Authority (EFSA) affirmed its conclusion that the presence of 

nptII does not pose a threat to human health or the environment. Moreover, the USDA 

previously evaluated the safety of NPTII in other biotechnology-derived products, 

including corn. Similar to these 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 reported that the amount of free kanamycin 

accumulating in soils, through the action of microorganisms or animal feces, is restricted 

by absorption to soil components so that no direct selection 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 evidence, it can be 

concluded that the NPTII protein is safe for use as a selectable marker in biotechnologyderived plants and it has no meaningful toxic potential to exposed organisms in the 

environment. 

IX.A.3. Composition and Nutrition of Forage and Grain 

Compositional equivalence between corn improved through biotechnology-derived traits 

and conventional hybrids provides an “equal or increased 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-nutrients, and key secondary metabolites for comparison to conventional 
Monsanto 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 the corn grain and forage derived 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 characteristics of MON 87460 were evaluated in field studies 

conducted during 2006 and 2007 in the major corn 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 evaluate the relevant characteristics of 

MON 87460 across a broad range of soil moisture and environmental conditions relevant 

to where commercial production would be expected. 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 practices and water conditions (5 sites). Phenotypic and 

agronomic characteristics and environmental interactions were assessed under these 

water management regimes. Characteristics evaluated include seedling 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, grain 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 statistically). 

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. For 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 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 (1 study, 3 sites in 2006/2007) and the U.S. (2 studies, 3 sites in 

2007). In the Chile 2006/2007 study, no differences 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, respectively). 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 lower (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 the typical agronomic practices regime, one 

study 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 of the phenotypic 

data, the results support the conclusion that MON 87460 possesses no characteristics that 

would indicate an increased plant pest risk compared to conventional corn. 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 
Monsanto 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 a 

non-drought tolerant, conventional control under water-limited conditions. The wellwatered and water-limited field trials described above are relevant to demonstrate the 

efficacy of MON 87460. A comparison of the 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 conditions (Section I.B.2). As expected, 

MON 87460 provided reduced yield loss compared to the control under water-limited 

conditions and equivalent yield to the control under well-watered conditions. Reduced 

yield loss values for MON 87460 in the water-limited treatment were highly variable, but 

are representative of the natural variation expected for corn grown under sub-optimal soil 

moisture conditions. 

IX.A.5. Potential for Risks to Non-target Organisms 

Evaluation of the potential risks to non-target organisms (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 nature of MON 87460 as a product with no 

pesticidal activity can lead to a conclusion that all exposed organisms are considered to 

be NTOs. During the U.S. and Chile phenotypic field studies at 31 locations in 2006 and 

2007, each field site was rated at four time intervals during the season for specific insects 

(pest and non-pests), and diseases (Section VIII.F.1). The purpose of these observations 

was to assess whether the plant-disease or plant-insect interactions of MON 87460 were 

altered compared to commercial corn. Twenty-one pest and non-pest arthropod 

categories (species or group) and 19 disease categories were evaluated. 

Of the more than 388 pest and non-pest arthropod 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 study-site location. The few differences detected 

were small in magnitude, did not represent a trend in the data, and are not considered to 

be biologically meaningful in terms of increased plant pest potential. Out of the more 

than 425 disease stressor observations, no differences were detected between MON 

87460 and the control. These results support the conclusion that MON 87460 does not 

have altered environmental interactions relative 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 conventional corn and that the forage and grain derived from 

MON 87460 are compositionally equivalent to conventional corn. In total, these studies 

demonstrate no biologically meaningful interactions 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 references (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 corn, traits often associated with weediness 

such as seed dormancy, 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 processing facilities, sustainable volunteer corn populations 

are not found growing in fence rows, ditches, and road sides. Corn is poorly suited to 

survive without human assistance and is not capable of surviving as a weed (Baker, 1965; 

Keeler, 1989; Galinat, 1988). Although corn seed can overwinter and emerge as 

volunteer plants in rotational 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 between MON 87460 and a non-drought-tolerant, 

conventional control, dormancy and germination, growth and development, and 

reproductive characteristics were evaluated for 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 the assessed characteristics compared to the 

control. Thus, the results support a conclusion of no increased weediness 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 notifications 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 the 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 describe the movement of plant genes from one 

plant to another via pollen. The rate of pollen-mediated gene flow depends on biotic and 

abiotic factors such as plant biology, pollen 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; therefore, high levels of pollen-mediated gene 

flow can occur in this species. Researchers also recognize that: (1) the amount of gene 

flow that occurs can be high because of open-pollination, (2) the percent gene flow will 

vary by population, hybrid or inbred, (3) the level of gene flow decreases with greater 

distance between the source and recipient plants; (4) environmental factors affect the 

level of gene flow, (5) corn pollen is viable for a short period of time under field 

conditions, (6) corn produces ample pollen over 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 transmission, certain conditions 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 between the two parents. 

Several studies have been conducted on the extent of pollen-mediated gene flow in corn. 

As expected, results were found to vary depending on the experimental design, 

environmental conditions and detection method. In general, percent gene flow was found 

to diminish with increasing distance from the source field. It was 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, Alabama and Maryland and Zea 

perennis in South Carolina (http://plants.usda.gov). These specimens would only flower 

at the same time as corn if they were subject to artificial day length shortening for several 

weeks at a time (Wilkes, 1967). Differences in factors such as flowering time, 

geographical separation, and development factors make natural 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 frequent drought stress. 

In contrast with corn and teosinte, which easily hybridize under certain conditions, it is 

only with extreme difficulty and special techniques that corn and another closely related 

species, Tripsacum (gamma grass) hybridize. Furthermore, 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://plants.usda.gov/java/profile?symbol=TRFL4). 
Monsanto Company 07-CR-191U Page 240 of 544 

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 unlikely there would 

be any impact on this species due to the introduction 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 interbreed. 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 (Section V.D.1). It is 

therefore not expected that these genes would have any altered potential to transfer to 

other organisms. The probability for horizontal 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 humans and other NTOs under the conditions of 

use. In addition, the nptII gene is not expected to pose any 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 resistance in soil bacteria (Demanèche et al., 2008). 

IX.D. Corn Production, Current Agronomic Practices, and Land Use 

This section provides a review of corn grain 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 Belt have sufficient precipitation to routinely 

support high levels of corn production. As shown in Sections VII and VIII, no 

phenotypic, compositional, or environmental 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 Midwestern 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 precipitation for corn production resulting in 

water-limited conditions. Under these conditions, 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 more likely to impact irrigation practices 

and dryland acreage in the Great 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 acreage 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 to 
Monsanto 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 commodity 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, comprising eight states (Illinois, Indiana, Iowa, 

Michigan, Minnesota, Missouri, Ohio and Wisconsin) contributing 65% to the national 

corn production total, and the Great Plains, as defined by Riebsame (1990), including 

portions of ten states (Colorado, Kansas, Montana, Nebraska, 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 region because of the diversity in rainfall and 

irrigation, climatic conditions, and soil productivity. 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 utilized 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 Midwest has a 34 inch (86 cm) annual average 

(http://www.met.utah.edu/jhorel/html/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 available 
Monsanto 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 66,650 

 
Monsanto 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.gov 

 
Monsanto Company 07-CR-191U Page 245 of 544 

Table IX-3. Comparison of Field Corn Production 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 responsible for maintaining high quality seed 

stocks, an essential basis for U.S. agriculture. By the early 20th century, agronomists 

learned how to develop specific plant varieties with desirable traits. In the U.S., state 

agricultural experiment stations developed many seed varieties which were distributed to 

farmers for use. As seeds were saved by farmers and later sold to neighbors however, the 

desirable traits of the varieties often were lost through random genetic changes and 

contamination with other crop and weed seeds (Sundstrom et al., 2002). The value of 

seed quality (including genetic purity, vigor, weed seed presence, seed borne diseases and 

inert materials, such as dirt) was quickly identified as a major factor in crop yields. 

States developed seed laws and certification agencies to ensure that purchasers who 

received certified seed could be assured that the seed met established seed quality 

standards (Bradford, 2006). The federal government passed the U.S. Federal Seed Act of 

1939 to recognize seed certification and official certifying agencies. Regulations first 

adopted in 1969 under the Federal Seed Act recognize land history, field isolation, and 

varietal purity standards for foundation, registered, and certified seed. Under 

international agreements such as the Organization for Economic Co-Operation and 

Development (OECD) scheme, the U.S. and 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, which is then used to produce certified seed that 

is sold for commercial planting (Bradford, 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 from other varieties of the same 
Monsanto Company 07-CR-191U Page 246 of 544 

genus or species (Bradford, 2006). Inspectors walk the fields 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 tagged 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 the field at an earlier date, pollen transfer and 

mixing that occurs during harvesting and handling (Bradford, 2006). Seed producers can 

and do take steps, such as cleaning equipment, 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 requirements (codified in 

federal regulations for certain crops) that must be followed to produce certified seed that 

will guarantee the purchaser receives the intended seed variety. The isolation required 

for a particular crop depends on its flowering 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 20th 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 produced. These standards have never required 

100% varietal purity. For example, AOSCA stated 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 society well for at least half a century were 

adopted to reflect a balance between the level of purity required to meet market needs 

and prevent consumer fraud and the cost of achieving 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 corn seed standard has been established 

by AOSCA (http://www.identitypreserved.com/handbook/aosca-nongmocorn.htm) 

requiring 660 feet of isolation distance, field inspections and purity tests using AOSCA 

approved laboratories. This standard allows 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 widescale planting of biotechnology-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 through hybridization. In 

this process one variety of corn produces pollen to fertilize a second variety, which has 
Monsanto Company 07-CR-191U Page 247 of 544 

been de-tasseled (the pollen producing male organs are removed) so it cannot fertilize 

itself. Because so much grain corn is grown in the continental U.S., hybrid seed 

production and bulk up is often done elsewhere (South 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 seed companies meet or exceed all national and 

international standards with regard to isolation and quality production practices. 

In addition to instituting their own requirements, seed companies are working together 

regionally, nationally and internationally to establish better guidelines for biotechnologyderived seed production. Several groups set standards for production and monitor the 

sale and transportation of seed. Among these organizations, various standards act to 

guide the production of high-quality commercial seed including the certifications 

specified below. 

Certification by the OECD is applied to varieties that meet established conditions of 

identity, uniformity, and stability. OECD certified varieties have an added economic 

value and are published in official OECD lists. The OECD helps ensure the varietal 

identity and quality of seed by setting appropriate requirements and controls throughout 

production, processing and labeling. Certified seeds are produced and officially 

controlled according to common harmonized procedures. OECD certification provides 

official worldwide recognition of "quality-guaranteed" seed, facilitating international 

trade and contributing to removal of technical trade barriers. 

The AOSCA is dedicated to assisting companies in the production, identification, 

distribution and promotion of certified classes of seed. Established in 1919 in the United 

States, the organization has grown to include members from around the world. AOSCA 

establishes minimum standards for quality and identity. Its goal is to standardize 

certification regulations and procedures internationally so companies compete under one 

set of standards. The association cooperates with the OECD and other international 

organizations to develop standards, regulations, procedures, and policies to expedite 

movement of seed and encourage international 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. Federal 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 the 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 corn in 2008 resulting in 

26% of the national corn production total. Key considerations for corn production in the 

Great Plains include climate and environment, soil quality and fertility, tillage and crop 

rotation practices, hybrid selection, nutrients, moisture, irrigation, and the management of 
Monsanto 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 characterized by two primary gradients which define 

the area and determine its environment; these are precipitation (decreasing east to west) 

and temperature (increasing north to south). Total annual precipitation ranges 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 limited by precipitation and nutrient availability 

(Parton et al., 1994). Drought is a factor in this system with the degree and timing 

controlled by temperature, precipitation and the ratio of precipitation to potential 

evapotranspiration (Parton et al., 1994). Crops 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 practices in the 

western sections of the Great Plains have supported significant corn production. It is in 

this type of climate that MON 87460 is expected to provide benefit and reduce grower 

risk due to drought. 

IX.D.2.2. Soil Quality 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 considered 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, granular 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, facilitates water and nutrient storage and a degree of 

permeability favorable to water intake and air 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 VIII), will also grow well in mollisol soils. 

IX.D.2.3. Tillage Practices 

There are three main tillage practices employed in all corn production areas: conventional 

tillage, reduced tillage, and conservation tillage. Conventional tillage practices leave 

<15% crop residue cover after planting and involve the use of a moldboard plow or other 

intensive tillage procedure. Reduced tillage 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 surface 
Monsanto Company 07-CR-191U Page 249 of 544 

with crop residue after planting to reduce soil erosion by water and consists of three 

subtypes: no-till, ridge-till and mulch-till. These subtypes 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 efficiencies 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 velocities as well as aid in weed control. Considering the 

moisture retention benefits of minimal tillage, it is therefore not surprising that in 2004, 

four of the top five states with significant increases 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 environmental 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 Plains. Given that, with the exception of yield 

under water-limited conditions, MON 87460 is agronomically and phenotypically 

equivalent to conventional corn, it is not anticipated that the introduction of MON 87460 

will have any impact on current tillage practices 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 marketed by seed companies. Their selection is 

primarily based on yield potential, climatic environment, 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 growing 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 corn 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 contribute to drought tolerance are complex and may not be 

readily apparent. The best indicator of drought tolerance is corn grain yield under 

moisture stress. Over the last several decades, breeding programs have been particularly 

successful at increasing the ability of corn to withstand drought occurring at flowering as 

demonstrated by decreases in anthesis-silking interval, and increases in ears per plant and 

kernels per ear, all of which result in yield 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 available 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 the conventionally bred hybrids offered for 

planting in the dryer western areas of the Great Plains were rated as having excellent 

drought tolerance and were recommended for fields that regularly experience drought 

stress; 50-65% of the hybrids offered for planting in these areas were rated as having very 

good performance in fields that regularly experience some drought stress, but were not 

recommended for fields routinely experiencing extreme drought stress. 

If approved for use, it is highly likely that biotechnology-derived traits conferring 

drought tolerance will become part of breeding programs. This additional tool for 

breeders has the potential to result in an expanded selection of hybrids for growers to 

choose from as they consider their individual 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 available to growers or in the introduction of an 

enhanced susceptibility to insects or disease, based on the mode of action and field 

observations (Sections I.D and VIII). 

IX.D.2.5. Nutritional and Moisture (Including 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). The thirteen elements include the primary 

elements (nitrogen, phosphorus, and potassium), secondary elements (calcium, 

magnesium, sulfur), and micronutrient 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 plant develops. Whereas nitrogen and phosphate 

uptake continues until maturity, potassium absorption 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 nitrogen per acre per crop year. Phosphate was 

applied to 81% of the corn acreage at an average rate of 58 pounds per acre per crop year. 

Potassium, applied at 84 pounds per acre per 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 include rain, surface water in rivers, streams, 

and lakes primarily from snowmelt, and groundwater 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 irrigation. 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 production shifts of some commodities to more 

humid and cooler areas is largely responsible for more efficient water use. Most 

agricultural freshwater withdrawals from surface and ground water for corn production 

occur in Nebraska, South Dakota, western Kansas, eastern Colorado and northern Texas 

where irrigated acres are concentrated (Figure IX-2). 

Figure IX-2. U.S. Acres of Irrigated Corn Harvested for Grain 

Source: http://www.nass.usda.gov/research/2002mapgallery/fieldcrops.html 

 
Monsanto 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 irrigation ground water in the 1950s resulting in 

depletion of the aquifer (McGuire, 2007). With the exception of New Mexico and 

Colorado, total irrigated acres planted 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 significant decline in the percent total corn acres under irrigation 

and a return to dryland production while Nebraska, South Dakota and Texas numbers 

remained relatively the same. According to Mapp (1988) the primary historic causes for 

a decline in irrigated acres are increases in pumping costs 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 sources. 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 average cost nationwide 

for surface water application was $26 per acre or ~$0.9B total (USDA-ERS, 2003). 

However, in recent years the growing biofuels industry and worldwide concern over food 

shortages have caused commodity prices to increase significantly. This may result in the 

continued high levels of irrigation use despite the higher costs. 

Given the above, it is foreseeable that growers may choose to determine whether 

MON 87460 can be used in dryland production without benefit of irrigation. This is a 

decision driven by economic considerations, and is not anticipated to have a significant 

impact on irrigation practices. 
Monsanto Company 07-CR-191U Page 254 of 544 

Table IX-5. Changes to Irrigated Corn Acreage in the Great Plains Source: http://www.agcensus.usda.gov/index.asp

State 1992 1997 2002 Acres 

Total 

Acres 

Irrigated 

Acres 

% 

Irg. 

Total 

Acres 

Irrigated 

Acres 

% 

Irg. 

Total 

Acres 

Irrigated 

Acres 

% 

Irg. 

Total Ac

Colorado 891,720 808,351 92 919,784 769,567 84 708,197 634,015 90 1,060,0

Kansas 1,748,802 1,154,370 66 2,497,516 1,465,920 59 2,494,179 1,346,807 54 3,700,0

Montana 18,240 17,323 95 12,925 12,263 95 11,642 11,642 100 38,00

Nebraska 7,235,528 4,703,268 65 8,279,499 4,966,816 60 7,344,715 4,505,579 61 9,200,0

New Mexico 72,348 70,194 97 80,122 75,870 95 48,096 47,904 100 55,00 

North Dakota 595,347 53,836 9 578,953 46,654 8 991,390 54,445 5 2,350,0

Oklahoma 123,567 69,104 56 150,404 85,678 57 182,777 99,457 54 270,00

South Dakota 3,097,251 172,233 6 3,175,113 148,001 5 3,165,190 123,229 4 4,500,0

Texas 1,549,680 740,431 48 1,656,229 871,364 53 1,815,560 658,177 36 2,000,0

Wyoming 54,341 52,767 97 49,717 47,931 96 34,095 33,507 98 60,00
Monsanto 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 differences in input costs, crop yield potential, and 

commodity prices. Crop rotations in the Western Dryland region of the Great Plains have 

relied on wheat and sorghum due to their ability 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 farmers 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 increasing 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 increasing rate since 1990. 

The area planted to corn in northeastern Colorado, typically a wheat-fallow area, 

increased from 20,000 acres per year in years 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 intensive dryland rotation management showed increases in soil 

carbon, nitrogen, and organic matter and aggregate 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 continue 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 anticipated that it will readily become part of any 

dryland crop rotation system that includes corn. Corn hybrids with enhanced drought 

tolerance, either biotechnology-derived or conventional, may provide additional 

economic benefit in place of crops more commonly grown in these drier agricultural 

areas. If so, this decision will be driven largely by grower’s economic concerns. In this 

scenario, MON 87460 is no more likely to displace wheat or sorghum than conventional 

drought tolerant corn hybrids already available to the grower. 

IX.D.2.7. Management of Diseases and Insects 

MON 87460 was evaluated to determine whether the presence of the drought tolerance 

trait would have an impact on disease and insect management. Management of diseases 

and insects during corn growth and development is essential for protecting the yield of 

the harvested grain. Estimates for annual yield losses because 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 other environmental factors. 

Most corn hybrids on the market today have acceptable levels of resistance to common 

diseases. The diseases found to occur in corn grown in the U.S. are summarized in 

Table IX-6. In addition, several 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 azoxystrobin, chlorothalonil, 

propiconazole (USDA-NASS, 2006a). 

The corn crop is subject to attack by a complex of insects from the time it is planted until 

it is used as food and feed. The economically important insect pests in North America 
Monsanto Company 07-CR-191U Page 256 of 544 

include wireworms, the black cutworm, European corn borer, Southwestern corn borer, 

the corn rootworms, grasshoppers, fall armyworm, 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 widely applied insecticides, at 7, 7, and 6%, respectively, to 

the corn acres. Chlorpyrifos was only applied to 2% of the 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 corn 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 demonstrated by environmental observations 

among numerous field studies (Section VIII.F.1), it has no apparent impact on arthropods 

and diseases of corn. Therefore, no changes to current disease and insect management 

practices, including pesticide use, conventional breeding selection for resistance, or 

adoption of biotechnology-derived traits are anticipated from the introduction of 

MON 87460. 

IX.D.2.8. Weed Management 

MON 87460 was evaluated to determine whether the presence of the drought tolerance 

trait would have an impact on weed management. Weeds cause significant losses and 

require careful management by growers because 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 infested with perennial weeds present special 

problems for corn growers. Like annual weeds, perennials can reproduce by seeds, but 

they also regrow and spread vegetatively. Their rhizomes propagate new shoots, usually 

soon after corn is planted. Unless effectively controlled, perennial weeds can quickly 

gain a season-long advantage over the corn crop. 

Corn yield loss is generally proportional to the amount of weeds present. While the ratio 

is not always one-to-one, some studies suggest that for every pound of weed dry matter, 

there is a reduction of approximately one pound of corn plant dry matter (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., 1990). 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 inches) and has an upright leaf orientation. As 

a result, corn is not successful in competing with weeds early in the growing season. 

Corn is also usually planted early when soil temperature and weather conditions favor 

weed over corn growth. A survey of Extension Service weed scientists solicited 

estimates of the percent of corn acreage infested with individual weed species by state or 

region, as well as the potential impact on corn yields if the species were left uncontrolled. 

In this survey, 12 annual broadleaf, nine annual grass, and seven perennial 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 wirestem muhly and sandburs 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 planted acreage (USDA-NASS 2006a). Atrazine 

continues to be the most widely applied herbicide 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 planted corn acreage treated. In 2001, the 

EPA identified glyphosate as the most widely used conventional agricultural 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-tolerance and glufosinate-tolerance, respectively. These 

herbicide-tolerance technologies are easy to use and both herbicides are more 

environmentally friendly than other herbicide options (Knezevic and Cassman, 2003). 

Volunteer corn commonly occurs in rotational crops in the season following corn 

cultivation regardless of whether or not the corn was conventional or biotechnologyderived. When corn is grown for silage, on approximately 9% of the U.S. corn acres, 

volunteer corn plants typically do not occur in rotational crops since corn harvested for 

silage does not produce viable seed. In the warmer climates 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 always occur in the rotational crop because of 

seed decomposition over the winter, efficient harvest procedures, and tillage prior to 

planting rotational crops. 

The first step to manage volunteer corn in rotational crops is to minimize or reduce the 

potential for volunteers. The following practices are implemented to reduce volunteer 

corn in rotational crops: (1) adjust harvest equipment to minimize the amount of corn 

grain lost in the field, (2) plant corn hybrids that reduce the extent of ear drop, (3) choose 
Monsanto Company 07-CR-191U Page 258 of 544 

corn hybrids with superior stalk strength and reduced lodging, and (4) practice no-till 

production to significantly reduce the potential for volunteer growth in the rotational 

crop. If volunteer corn does occur in subsequent crops, pre-plant tillage or in-crop 

cultivation are very effective management tools. Selective 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 alfalfa, and eleven vegetable 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 conventional corn, it is not anticipated that MON 87460 will 

respond differently to commonly used herbicides, 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 trademarks 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, 1988 
Monsanto 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 Plains 

 
Monsanto 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 remained relatively steady from 1996 to 2006 with a 

yearly average of 78 million acres indicating 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 corn 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 producers 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 industry and worldwide concern over food shortages 

that have caused commodity prices to increase, with the result that some growers are not 

renewing their enrollment 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 acreage and land use than a conventionally bred 

drought tolerant hybrid. A decrease in commodity 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 Regulation of Biotechnology, the responsibility 

for regulatory oversight of non-pesticidal biotechnology-derived crops 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 their reviews and assessments under their 

respective jurisdictions. 

Food and feed from biotechnology-derived crops 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 process to work together with biotechnologyderived product developers to identify and resolve any issues regarding the safety and 

nutritional content of food and feed from biotechnology-derived crops. Using the current 
Monsanto Company 07-CR-191U Page 263 of 544 

process, Monsanto has initiated and will complete 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 responsibility under the Plant Protection Act (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 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 material; (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 case of the USDA, (6) information 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 derived from MON 87460 constitute a risk to human health. 

Because these data demonstrate that MON 87460 does not pose a plant pest risk, it is not 

anticipated that mitigation or remediation measures will be necessary for this product. 

Monsanto employs a rigorous product stewardship program that demonstrates respect for 

our customers, their markets and the environment. Our market stewardship program 

considers many factors to support global integration 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 international trade. We commit to best industry practices on seed 

quality assurance and control to prevent adventitious presence of unapproved traits. 

Before commercializing MON 87460, a detection method will be made available to grain 

producers, processors, and buyers. Our stewardship 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 obligation 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 stage of their life cycle. As such, Monsanto 

has policies in place to meet these commitments 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 characteristics, 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 practices have historically been modified to 
Monsanto Company 07-CR-191U Page 264 of 544 

optimize corn yields in the Great Plains where precipitation use efficiency is a primary 

concern. As biotechnology-derived agricultural products have offered safe and effective 

alternatives to weed and insect control in the past, MON 87460 is another option for 

growers to help minimize production risk by providing increased yield stability under 

water-limited conditions. 

Monsanto is not aware of any study results or observations associated with MON 87460 

that would suggest that an increased 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 regions that are suitable for corn production but 

prone to frequent drought stress, such as the Western Dryland region of the Great Plains. 

As demonstrated by field and laboratory study results, the only phenotypic difference 

between MON 87460 and conventional corn is reduced yield loss under water-limited 

conditions, due to expression of the CSPB protein. 

The plant pest assessment of MON 87460 was based on multiple lines of evidence 

developed from a detailed characterization 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 environment 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 MON 87460 on agronomic practices was based 

on an evaluation of the current production, land 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 the use of conventionally bred 

drought tolerant corn. Finally, due to the inherently low plant pest potential of modern 

corn and the lack of (a) weediness and horizontal gene transfer potential, (b) any impact 

on disease or injury to plants or plant pests in the field, or (c) any observed effects on 

non-target or beneficial organisms 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 primarily 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 environmental impact. Therefore, Monsanto Company 

requests a determination from APHIS that 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 265 of 544 

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X. Adverse Consequences of Introduction 

Monsanto knows of no study results or observations associated with MON 87460 

suggesting that adverse environmental consequences 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 disrupting 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 this petition demonstrate that MON 87460 is 

unlikely to pose an increased plant pest potential 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 corn has inherently low plant pest potential 

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 characterization studies for MON 87460 included molecular and 

protein analyses, which confirmed the insertion of a single functional 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 efficacious for reduced yield loss under 

water-limited conditions. Allergenicity assessment concluded that the CSPB and NPTII 

proteins are unlikely to be allergens 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 grain from MON 87460, and the foods and feeds 

derived from such, are compositionally equivalent to those derived from conventional 

corn. Extensive characterization of the plant 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 organisms. Finally, an assessment of agronomic 

practices confirmed that the introduction of MON 87460 is no more likely to impact land 

use, cultivation practices, or the management of weeds, diseases, and insects than the use 

of conventionally bred drought tolerant corn. Therefore, the 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 increase economic and environmental benefits 

due to the protection of corn yields under water-limited conditions. Based on the results 

of these assessments, no adverse environmental 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 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 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). Additional DNA extracted from seeds of various 

generations of MON 87460 (seed lot numbers GLP-0704-18549-S; GLP-0604-17132-S (F1

seed); GLP-0604-17132-S (F2 grain); GLP-0704-18550-S; GLP-0609-17631-S; GLP-0609-

17631-S; GLP-0703-18435-S) was used in generation stability 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 reference 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 test substance digested with appropriate 

enzyme(s). The plasmid DNA was digested first and then added to pre-digested 

conventional corn genomic DNA. The molecular weight standards include the 1 kb DNA 

Extension Ladder (Invitrogen) and λ DNA/Hind 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 from MON 87460 and conventional corn were 

verified by PCR analysis to confirm the presence or absence of MON 87460 except the 

materials used in the generational stability analyses where the identity of the materials 

was confirmed by the generation stability Southern 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 Southern Blot and PCR Analyses 

Genomic DNA samples from MON 87460 and conventional corn used in the insert and 

copy number, copy number of each element, backbone analysis, and PCR analyses were 

isolated from corn seeds that were processed 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 Bendich (Rogers and Bendich 1985). 

Genomic DNA was isolated using the following method. Place about 6 grams of 

processed seed tissue in a 50 mL centrifuge tube and add ~16 ml of 

cetyltriethylammonium bromide (CTAB) extraction buffer [1.5% CTAB, 75 mM 

tris(hydroxymethyl)aminomethane (Tris) pH 8.0, 100 mM ethylenediaminetetraacetic acid 

(EDTA), 1.05 M NaCl, 0.75% polyvinyl pyrrolidone (PVP) (40K)] and 8-10 μL of 10 

mg/mL RNase. Incubate the samples at 65oC for 25-35 minutes and mix halfway through 

the incubation. Let the samples cool to room temperature, and add 16 mL of 24:1 

chloroform: isoamyl alcohol, mix for 5 minutes 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, add 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 before 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 phases. 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 minutes. Centrifuge for 9-11 minutes at 16,000 ×

g and 20-25°C to pellet the DNA. Discard the supernatant. Add 2 mL of high salt TE 

buffer (10 mM Tris HCl, pH 8.0, 1 mM EDTA, pH 8.0, 1M NaCl) and incubate at 35-60οC 

with gentle shaking until the pellet goes into solution. Precipitate the DNA by adding 1/10 

the volume of 3 M sodium acetate, pH 5.2, and two times the volume of 100% ethanol. 

Mix by inversion. Remove the DNA using a pipette tip, inoculation 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 for 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 resuspension 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 performed using a Hoefer DyNA Quant 200 

Fluorometer with Roche molecular size marker IX as a DNA calibration standard. 

A.5. Restriction Enzyme Digestion of Genomic DNA 

Approximately 10 or 20 μg of genomic DNA extracted from the test and control substances 

were used for restriction enzyme digestions. When digesting genomic DNA with Hind III 

(Roche) or EcoR V (Roche), 10X Buffer B (Roche) was used. When digesting genomic 

DNA with the enzyme combination EcoO109 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 EcoO109 I/Not I digests to a final 

concentration of 1×. All digests were performed at 37°C in a total volume of 

approximately 500 μl using ~100 units of the appropriate 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 procedure based on Sambrook and Russell 

(Sambrook and Russell 2001). Approximately 25 ng of each probe template were 

radiolabeled with either 32P-deoxycytidine triphosphate (dCTP) or 32P-deoxyadenosine 

triphosphate (dATP) (6000 Ci/mmol) using the random priming method (RadPrime DNA 

Labeling System, Invitrogen) or PCR method. Probe locations relative to the genetic 

elements in plasmid PV-ZMAP595 are depicted in Figure IV-6 and Figure IV-7. 

A.7. Southern Blot Analyses of Genomic DNA 

Digested DNA was separated using 0.8% (w/v) agarose gel electrophoresis. Except for 

generational stability analyses, DNA samples were loaded on the gels for a long run and a 

short run in an effort to provide better resolution of larger DNA fragments while retaining 

smaller DNA fragments on the gel. After 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. Multiple 
Monsanto 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 intensifying screen in a -80°C freezer. 

Probe DNA Probe Labeling 

Method 

Probe labeled 

with 

dNTP (32P) 

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 that span the insert in MON 87460. These 

products were sequenced to determine the 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 approximately 75 ng of genomic DNA template 

or approximately 10 ng of plasmid DNA in a 50 μl reaction volume containing a final 

concentration of 2 mM MgCl2, 0.2 μM 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 separated on a 1.0 % (w/v) agarose gel and 

visualized by ethidium bromide staining to verify that the products were of the expected 

size prior to sequencing. The PCR product was 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 product. 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 flanking the insert in MON 87460 are native to 

the corn genome, PCR analysis was performed 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 approximately 75 ng of genomic DNA template 

in a 50 μl reaction volume containing a final concentration of 2 mM MgCl2, 0.2 μM 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 sequenced with the primers used for PCR 

amplification. All sequencing was performed by the Monsanto Genomics Sequencing 

Center using dye-terminator chemistry. 

A.10. References: 

Rogers, S. O. and A. J. Bendich. 1985. Extraction 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 cloning: 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 Results for Characterization of CSPB Protein 

The expression levels of CSPB protein in different tissues of MON 87460 are relatively 

low. Therefore, it was necessary to produce the protein in a high-expressing, 

recombinant microorganism in order to obtain sufficient quantities of the protein for 

safety studies. A recombinant CSPB protein was produced in Escherichia coli, the 

sequence of which was engineered to match that of CSPB protein produced in 

MON 87460. The equivalence of the physicochemical characteristics and functional 

activity between the MON 87460-produced and E. coli-produced CSPB protein was 

confirmed by a panel of analytical techniques, 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 assay of biological activity. 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 purified 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 fractionation, anion exchange chromatography, 

immunoaffinity chromatography, and size exclusion chromatography. Protein 

purification records are archived at Monsanto Company under Orion lot 10000842, and 

the purification methods are described below. 

The ground grain (10 kg) was mixed with a Tris-borate extraction buffer (89 mM TrisBorate, 2 mM EDTA, pH 8.3) for 17 h at approximately 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 utilizing a polysulfone hollow fiber cartridge 

with a 3 kDa Molecular Weight Cut Off (MWCO) (surface area: 3.25 m2

, GE Healthcare, 

Piscataway, NJ). An ammonium sulfate precipitate 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. After centrifugation, the ammonium 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 Healthcare, 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 sodium 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 fractions were pooled and concentrated using 

diafiltration to a final 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-CSPB antibody (Leinco Technologies Inc, St 

Louis, MO) had been conjugated. Bound CSPB protein was eluted using 100 mM 

triethylamine buffer and neutralized with 1/20th volume of 1 M sodium phosphate, pH 

6.8. The process was repeated a total of 19 times to capture and elute most of the CSPB 

protein present in the concentrated 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 approximately 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 Healthcare, Piscataway, NJ) equilibrated in 

20 mM Tris-HCL pH 7.0. The fractions containing CSPB protein were identified by 

western blot and a final pool of ~105 ml was concentrated by diafiltration 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 dialysis 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 submitted 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 from 10 to 60 ng) was used to create a standard curve. 

Aliquots of the MON 87460-produced CSPB protein and reference standard 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 gradient 18-well gel. A 

50-fold diluted MON 87460-produced protein solution was mixed with 5× LB and three 

different amounts were loaded in duplicate on the gel. Electrophoresis 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 Staining Kit Protocol (Owl Separation Systems, 

Portsmouth, NH). The following steps were performed during the gel staining procedure: 

1. Fixing for 10 min in 150 ml of fixing solution (60 ml deionized water, 75 ml 

methanol, and 15 ml acetic acid);

2. 15 min incubation in 150 ml of a second fixing solution (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 silver staining solution (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 bands occurred in 150 ml of developer 

solution (7.5 ml Concentrated Developer 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 coliproduced 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 were 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-well gel. The three amounts of each protein 

were loaded in duplicate on the gel. Electrophoresis was performed at a constant voltage 

of 170 V for 70 min. Pre-stained 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 immunoreactive bands observed. 

Electrotransfer to a 0.45 μm nitrocellulose membrane (Invitrogen, 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 dry 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 incubated 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 SRX-101A automated film processor (Tokyo, 

Japan). 

The immunoreactive bands of the MON 87460-produced CSPB protein in each lane 

migrating to the same level as the reference standard protein were quantitated and 

compared to the signals corresponding to the CSPB reference 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) file 
Monsanto Company 07-CR-191U Page 279 of 544 

for the pair wise comparison of all the loads. 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 was 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-produced 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-stained 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 μm 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 was performed for 15 cycles using automated 

Edman degradation chemistry (Hunkapiller and Hood, 1983). An Applied Biosystems 

494 Procise Sequencing System with 140C Microgradient system and 785 Programmable 

Absorbance Detector and ProciseTM Control Software (version 1.1a) were used. 

Chromatographic data were collected using Atlas99 software (version 3.59a, LabSystems, 

Altrincham, Cheshire, England). A phenylthiohydantoin (PTH)-amino acid standard 

mixture (Applied Biosystems, Foster City, 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 the analysis of the two CSPB protein bands 

that were analyzed as a single sample, to verify that the sequencer 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 was not deemed necessary to separate the protein by SDS-PAGE. 

An aliquot of the MON 87460-produced CSPB protein was diluted ten-fold to a final 

concentration of ~12 μg/ml. Mass spectral analyses were performed as follows. Mass 

calibration of the instrument was performed using an external peptide mixture from a 

Sequazyme Peptide Mass Standards kit (Applied Biosystems). Samples (0.3 μl) from 

each of the tryptic samples were co-crystallized with 0.75 μl 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-less MALDI-TOF instrument specific value). 
Monsanto Company 07-CR-191U Page 280 of 544 

Protonated (MH+) peptide masses were observed monoisotopically in linear mode 

(Aebersold, 1993; Billeci and Stults, 1993). GPMAW32 software (Applied Biosystems, 

version 4.23) was used to generate a theoretical mass of the expected CSPB (plant) 

protein sequence based upon the nucleotide sequence. Peaks were not assessed if the 

peak heights were less than 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. 

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 protein 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 concentrator. The sample was digested for 16 hr at 

37 °C with 660 ng of trypsin (Promega, Madison, 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 peptides using the ProteoMassTM 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 NH4OH) 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 peptide mixture from a Sequazyme Peptide Mass 

Standards kit (Applied Biosystems). Samples (0.3 μL) from each of the trypsin digested 

samples were co-crystallized with 0.75 μL 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 were observed monoisotopically in reflector 

mode (Aebersold, 1993; Billeci and Stults, 1993). GPMAW32 software (Applied 

Biosystems, version 4.23) was used to generate a theoretical trypsin digest of the 

expected MON 87460-produced CSPB protein 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 more isotopically resolved peaks, 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 than 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 fragments from trypsin digestion were identified in 

the raw data. The tryptic mass map coverage was considered acceptable if ≥ 40 % of the 

protein sequence was identified by matching experimental masses observed for the 

tryptic peptide fragments to the expected masses for the fragments. 

 
Monsanto Company 07-CR-191U Page 281 of 544 

B.1.6. Molecular Weight and Purity 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/μl. The MON 87460-produced protein was analyzed in 

duplicate at 50, 100, and 150 ng of total 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-well mini-gel (Invitrogen, Carlsbad, CA). 

Pre-stained molecular weight markers (Invitrogen SeeBlue Plus2, Carlsbad, CA) were 

loaded in parallel. Electrophoresis was performed at a constant voltage of 170 V for 70 

min. 

The gel was stained using the Owl Silver Staining Kit Protocol (Owl Separation Systems, 

Portsmouth, NH). The same procedure described 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 apparent molecular weight of the MON 87460-

produced CSPB protein. For the purity evaluation, all visible bands within each lane 

were quantified. The purity and estimated molecular weight of the MON 87460-

produced CSPB protein were reported as the average of the six 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 with covalently bound carbohydrate moieties. 

Aliquots of the MON 87460-produced CSPB protein, the E. coli-produced CSPB 

reference standard protein, and the positive controls, transferrin (~ 76 – 81kDa, SigmaAldrich, St. Louis, MO), and horseradish peroxidase (~ 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, and 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 molecular 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. Electrotransfer to a 0.45 μm PVDF 

membrane (Invitrogen) was performed for 90 min at a constant voltage of 35 V. 

Carbohydrate detection was performed directly 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 performed 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 acid 
Monsanto Company 07-CR-191U Page 282 of 544 

(wash solution) were followed by a 20 min oxidation in 25 ml of an oxidizing solution 

containing periodic acid (Component C from kit). Membrane was washed three times, 10 

min each, in 50 ml of wash solution. The blot was then incubated in 25 ml of Pro-Q 

Emerald Staining Solution that was prepared using the kit reagents. 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 visualize the fluorescentlylabeled glycosylated proteins. 

After glycosylation analysis the blot was stained 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 manufacturer’s instructions were followed and all 

steps were performed at room temperature and 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 Imager 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 functional activity of the MON 87460-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 their ability to unfold polynucleotide hairpin 

structures. Activity is expressed as the amount 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 separate 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 calibration 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 (Molecular Devices, Sunnyvale, 

CA). The fluorescence was determined with an excitation wavelength 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-produced CSPB protein during storage in a 

freezer set to maintain -80°C was evaluated by comparing the purity and molecular 

weight values obtained on day 0 to the purity and molecular weight values obtained on 

day 14 of storage. Day 0 stability analysis 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/μl, 

heated at 100 °C for 3 min, and loaded in duplicate (50, 100, and 150 ng per lane) onto a 

tricine 10 - 20% gradient polyacrylamide 10-well gel. Staining and densitometric 

analysis 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 comprising 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 removal of the N-terminal 

methionine, catalyzed by methionine aminopeptidase, is a common modification that 

occurs co-translationally before completion of the nascent protein chain and has no effect 

on protein structure or activity (Arfin and Bradshaw, 1988; Polevoda and Sherman, 

2000). The N-terminal sequence information, therefore, confirms the identity of the 

CSPB protein isolated from 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 sequence 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 without consideration for potential 

natural amino acid modifications such as glycosylation. The protein sample was heatdenatured, chemically reduced, alkylated, digested with trypsin, guanidinated, and the 

masses of the tryptic peptides were measured. 

CSPB is a small protein with a limited number 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-digested peptides. The 

identified masses were used to assemble a coverage map indicating the matched peptide 

sequences for the entire CSPB protein (Figure 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-CSPB serum was conducted to determine the 

relative immunoreactivity of the MON 87460-produced CSP protein and the E. coliproduced CSPB reference standard. The results demonstrated that the anti-CSPB 

antibody recognized the MON 87460-produced CSPB that migrated identically to the E. 

coli-produced reference standard protein (Figure 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 equivalent 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 acid1

residue # from the 

N-terminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Predicted CSPB 

Sequence2 →

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 protein was deduced from the coding region of 

the full length cspB gene present in MON 87460. 
Monsanto Company 07-CR-191U Page 285 of 544 

Table B-2. Summary of the Tryptic Masses Identified for the MON 87460-Produced CSPB Protein Using Spectrometry Only experimental masses that matched expected masses are listed in the table. 

Observed Mass (Da) Expected 

Mass 

(Da) 

Mass 

Difference 

(Da) 

Crude AA Position Fragment Sequen

Sample 

Guanidinated 

Sample 

810.42 - 810.38 0.04 7-12 WFNSEK 

- 852.51 852.38 0.13 7-12G WFNSEK 

885.54 - 885.48 0.06 56-64 GPQAANVTK 

- 927.64 927.48 0.16 56-64G GPQAANVTK 

1878.11 - 1877.92 0.19 39-55 TLEEGQAVSFEIVEG

- 2903.32 2903.36 0.04 13-38G GFGFIEVEGQDDVFVHFSA
Monsanto 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-produced 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 total amino acids) of the expected protein 

sequence were identified. 

 
Monsanto Company 07-CR-191U Page 287 of 544 

Figure B-2. Western Blot Analysis 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 polyvinylidene 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-Stained 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 protein 
Monsanto Company 07-CR-191U Page 288 of 544 

B.2.4. CSPB Protein Molecular Weight Equivalence 

The equivalence in apparent molecular weight of the purified MON 87460- and the E. 

coli-produced CSPB proteins was demonstrated using SDS-PAGE (Figure B-3). The 

MON 87460-produced CSPB protein migrated with a molecular weight indistinguishable 

to that of the E. coli-produced protein standard analyzed 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 obtained for CSPB was 7220 Da. This 

experimentally obtained mass differs from the theoretical mass calculated for the CSPB 

reference standard protein by 131 Da. The difference between the expected and the 

observed mass for MON 87460-produced CSPB corresponds 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 structures, simple oligosaccharides or 

monosaccharides. In contrast, prokaryotic organisms such as non-virulent E. coli strains 

used for cloning and expression purposes, lack the necessary biochemical synthetic 

capacity required for protein glycosylation. An investigation of glycosylation status 

therefore is necessary to confirm that the MON 87460-produced 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 post-translational glycosylation of the MON 87460-produced 

CSPB protein occurred, the purified protein sample was subjected 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 covalently-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 performed 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 concentration-dependent manner (Figure 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 were present for carbohydrate 

detection and glycosylation analysis, the membrane was stained with SYPRO® Ruby 

stain to detect proteins (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. Approximate molecular weights (kDa) of markers 

loaded in Lanes 1 and 18 are shown on the left side of the gel. 

 Lane Sample Amount Loaded 

 (ng) (μl)

 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 10 11 12 13 14 15 16 17 18

210 

105 

78 

55 

45

34 

17 

16 

7

4 

CSPB protein 
Monsanto 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 peroxidase (positive control) and transferrin 

(positive control) were separated by SDS-PAGE (10-20% gradient) and electrotransferred 

to a PVDF membrane. (A) Where present, periodate-oxidized protein-bound 

carbohydrate moieties reacted with Pro-Q Emerald 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-Stained 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 molecular weight standards ⎯

 

1 2 3 4 5 6 7 8 9 10

180

82

42

18

kDa kDa 1 2 3 4 5 6 7 8 9 10 kDa

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 B
Monsanto 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 unfolds or “melts” a DNA-hairpin structure. 

Results confirm the two proteins exhibit similar functional activity. The DNA-hairpin 

structure is labeled with a fluorophore at the 5’- 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. This assay has been broadly utilized to characterize the specificity of a 

variety of CSPs including CSD-containing proteins identified in bacteria and plants 

(Karlson et al., 2002; Kim et al., 2007; Phadtare et al., 2002). 

In this assay protein specific activity is expressed as the amount (pmol) of open Dual 

Labeled Probe (DLP) that is induced by a microgram (μg) of CSPB. The E. coli- and 

MON 87460-produced CSPB proteins 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-carboxyfluorescein (6-FAM) 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 the complementary bases 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 binding to the loop will separate the double 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/μg protein 

and the E. coli-produced reference standard had a specific activity of 0.757 pmole open 

DLP/μg protein. The difference in specific activities was 12.8% (Table B-3). These 

results clearly demonstrate that the CSPB 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 (pmol) of open Dual Labeled Probe (DLP) that 

is induced by a microgram (μg) of 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. Fluorescence was determined with an excitation 

wavelength of 485 nm and an emission wavelength 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.) 

% Difference1

(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 ActivityEcoli

ActivityEcoli ActivityPlant

×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 the identity of the MON 87460-produced CSPB 

protein and established the equivalence of the plant produced protein to the E. coliproduced CSPB reference protein standard. The molecular weight of the MON 87460- 

and E. coli-produced CSPB proteins was estimated by SDS-PAGE. SDS-PAGE 

demonstrated that the proteins migrated at the same molecular weight indicating that the 

CSPB proteins from both sources are equivalent in their molecular weight. The 

electrophoretic mobility and immunoreactive properties of the MON 87460-produced 

CSPB protein were 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 yielded peptide masses consistent with the expected peptide 

masses from the translated cspB coding sequence. The MON 87460- and the E. coliproduced 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 isolated 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. Cotranslational 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. Podtelejnikov and M. Mann. 1997. Identification of the components 

of simple protein mixtures by high-accuracy 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 shares 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. Three amino acids in 

Escherichia coli CspE surface-exposed aromatic patch are critical for nucleic acid 

melting activity leading to transcription antitermination and cold acclimation of 

cells. Journal of Biological Chemistry. 277:46706-46711. 

Polevoda, B. and F. Sherman. 2000. N-terminal 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 Results 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 previous safety assessment 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 extracted 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 control) were place in a polypropylene mesh 

bag from a Plant Protein Extraction kit (Pierce, 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 centrifuged 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 from 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% gradient polyacrylamide 

SDS 12-well gel (Invitrogen, Carlsbad, CA) was 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 Protein 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 antibodies (Sigma. St. Louis, MO, 

Cat No N6412) in 1% (w/v) NFDM in PBST for 60 min. Excess antibodies were 

removed using three 5 min washes with PBST. 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, Hercules, 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 dilution was made in the incubation solution) to visualize 

the position of the WesternC protein molecular weight markers. 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 Hyperfilm ECL high performance chemiluminescence 

film (GE Healthcare, GE healthcare, Piscataway, NJ). Films 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 the 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 their apparent molecular weight and 

immunoreactivity with NPTII specific antibodies. The criteria for these tests were preestablished during developmental work taking into 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 same mobility as the NPTII 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 initial laboratory stages of plant 

cell selection following transformation (Horsch at al., 1984; DeBlock et al., 1984). 

NPTII uses adenosine triphosphate (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 cells after transformation. In general, the 

frequency of plant cells that are transformed 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 establishment of the equivalence between 

MON 87460- and E. coli-produced proteins included: 

1. immunoblot analysis to establish protein identity through immunoreactivity 

with NPTII–specific antibody and demonstrate immuno-equivalence between 

MON 87460 and E. coli-produced NPTII proteins. 

2. SDS-PAGE to assess the apparent molecular 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 properties. The expression levels of NPTII 

protein in MON 87460 leaf tissue allowed detection of the protein with an NPTII-specific 

antibody directly in leaf extracts without additional enrichment. An extract was also 

prepared from a leaf sample of conventional 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 altered as a result of 

matrix effects, the reference substance was spiked into the leaf extract from conventional 

corn and analyzed alongside the leaf extract from MON 87460. The 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 using an anti-NPTII antibody. A co-migrating 

immunoreactive band was observed in the leaf extract from MON 87460 (Figure C-1, 

Lanes 4-6), leaf NPTII-spiked conventional corn leaf extract (Figure C-1, Lanes 7-9), and 
Monsanto Company 07-CR-191U Page 297 of 544 

pure E. coli-produced NPTII protein (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 extract from conventional corn spiked with 

the E. coli-produced NPTII protein. No immunoreactive 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 both 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 blot (Figure C-1). The electromobility of the 

MON 87460-produced protein was indistinguishable from the electromobility of the 

E. coli-produced NPTII protein. The estimated molecular weight of the MON 87460-

produced NPTII protein was 27.4 kDa, which was similar to the previously determined 

molecular weight of the E. coli-produced NPTII reference standard (27.1 kDa). Based on 

the identical electrophoretic mobility and apparent molecular masses, the MON 87460- 

and E. coli-produced NPTII proteins have equivalent molecular weights. 

C.2.5. Conclusions of the NPTII Protein Characterization 

MON 87460-produced and E. coli-produced NPTII proteins have equivalent 

immunoreactivities and apparent molecular weights. The results of this analysis 

confirmed the identity of the MON 87460-produced 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 compare the immunoreactivity and 

apparent molecular weight of the MON 87460-produced NPTII protein to that of the 

previously characterized E. coli-produced NPTII reference protein standard. The 

MON 87460- and E. coli-produced NPTII proteins displayed similar immunoreactivity 

with NPTII-specific antibody and had identical electromobility on SDS-PAGE. Taken 

together, these data establish equivalence between the MON 87460-produced and E. coliproduced 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. coliproduced NPTII spiked into leaf extract of conventional 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 antibody 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 exposure is shown and is representative of the bands 

observed in the other exposures. 

Lane Sample Amount Loaded 

 (ng) (μl) 

 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 12 
Monsanto 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. Montagu, J. Schell and P. Zambryski. 1984. 

Expression of foreign genes in regenerated 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. Fry, 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 foreign genes in plants. Science. 223:496-498. 
Monsanto Company 07-CR-191U Page 300 of 544 

Appendix D. Materials and Methods for Expression Levels of CSPB and NPTII 

Proteins 

The levels of the CSPB and NPTII proteins in various tissues of MON 87460 were 

determined using enzyme-linked immunosorbent assays (ELISA). The materials and 

methods for the ELISA analysis, as well as a description of the tissue types, are provided 

below. To produce the tissues for analysis, MON 87460 and conventional corn were 

each planted in field trials conducted during 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 strip-plot design to establish two water treatment levels (wellwatered and water-limited) to assess for any changes in CSPB and NPTII protein levels 

under different soil moisture conditions. The sites were located in the major corngrowing regions of the U.S. and Chile. Forage, 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 plant (OSWP) samples were collected four 

times (1-4) over the season corresponding to plant 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 established 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 conducted in the U.S. during 2006. 

The trials were located in Iowa, Illinois, Indiana, 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 commercial production of corn. At each site, 

three replicated plots of MON 87460 and a conventional control hybrid were planted 

using a randomized complete block field design. Over-season leaf (OSL), over-season 

whole plant (OSWP), over-season root (OSR), pollen, silk, forage, forage root, grain, 

stover, and senescent root tissues were collected from each replicated plot at all field 

sites. 

D.1.2. Description of Collected Tissues 

Leaf. Leaves were randomly collected from plants in each plot at each site. Twenty 

leaves were combined to form the leaf sample for each plot. There were 18 leaf samples 

across all sites for OSL-1, OSL-3, and OSL-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 development 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 collected from four plants in each plot 

at each site at the V2-V4 stage and combined 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 combined. 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 planting from five plants, except for the Indiana 

and Iowa sites, where silks were collected from ten plants. Silks were only collected 

from ears of plants that were covered with shoot 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 approximately 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 ice for processing and 

analysis. Grain was stored and shipped at room 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 for NPTII, the number of tissues evaluated for 

NPTII expression was fewer than those evaluated for CSPB protein. The NPTII levels 

were evaluated in four of the 19 tissue types 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 types. Protein levels for all tissue types are 

provided in μg/g fresh weight tissue (fwt) and μg/g dry weight tissue (dwt) basis. 

D.1.3. Tissue Processing and Protein Extraction 

All samples produced at the field sites were shipped to Monsanto’s processing 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 into capped tubes and stored in a -80°C freezer until 

use. 

CSPB and NPTII were extracted from all tissues by shaking tubes mounted in a Harbil 

mixer for a specified period of time. Each extraction tube contained 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 

Ratio1

Shake Time 

(minutes) 

Sample 

Clarification 

Method 

CSPB/NPTII Leaf 2

 PBST/BSA3 1:100 7.0 Serum filter 

CSPB/NPTII Root4

 PBST/BSA 1:20 7.0 Serum filter 

CSPB/NPTII Forage5

 TB6 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 Na2B4O7 · 10H2O, 0.01 M MgCl2, 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 clarified extracts were aliquot, 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-specific IgG was purified by Protein 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 spectrophotometric methods. The purified antibody was 

stored in 137 mM NaCl, 10 mM Na2HPO4 .

 7 H2O, 1 mM KH2PO4, and 2.7 mM KCl, pH 

7.4 (1X phosphate buffered saline (PBS)). CSPB protein affinity purified IgG was used 

as the well coating antibody. 

Protein G agarose affinity purified goat polyclonal anti-CSPB was coupled with biotin 

(Sigma, St. Louis, MO) according to the manufacturer’s instructions and assigned lot G806080-2. The detection reagent was NeutrAvidin (Pierce, Rockford, IL) conjugated to 

HRP. 

D.1.5. NPTII Antibodies 

Rabbit polyclonal antibodies (lot G-805224) specific for the NPTII protein were purified 

using Protein-A agarose affinity chromatography 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 preserved with 0.01% (w/v) sodium azide.

The purified NPTII antibodies were coupled 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 antibodies were diluted in coating buffer 

(15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and immobilized onto 96-well microtiter 

plates at 2.0 μg/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 well of 1X PBST with 

1% BSA for 60 to 70 min at 37°C. CSPB protein standard or sample extract was added 

at 100 μl per well and incubated for 1 h at 37°C. The captured CSPB protein was 

detected by the addition of 100 μl per well of biotinylated goat anti-CSPB antibodies and 

NeutrAvidin-HRP (Pierce). Plates were developed by adding 100 μl per well of HRP 

substrate, 3,3',5,5'-tetramethylbenzidine (TMB; Kirkegaard & Perry, Gaithersburg, MD). 

The enzymatic reaction was terminated by the addition of 100 μl per well of 3 M H3PO4. 

Quantitation of the CSPB protein was accomplished 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 antibodies were diluted in coating buffer (15 mM Na2CO3 and 

35 mM NaHCO3, pH 9.6) and immobilized onto 96-well microtiter plates at 5.0 μg/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 μl per well and incubated for 1 h at 37°C. The captured NPTII protein was 

detected by the addition of 100 μl per well of biotinylated rabbit anti-NPTII antibodies 
Monsanto Company 07-CR-191U Page 304 of 544 

and NeutrAvidin-HRP. Plates were developed by adding 100 μl per well of TMB. The 

enzymatic reaction was terminated by the addition of 100 μl per well of 6 M H3PO4. 

Quantitation of the NPTII protein was accomplished 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 for 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 control 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 fresh weight (fwt) basis 

into levels reported on a μg/g dry weight (dwt) basis using the following calculation: 

 

The protein levels that were reported to be less than or equal to the limit of detection 

(LOD) or less than the limit of quantitation (LOQ) on a fresh weight basis were not 

reported on a dry weight basis. 

D.1.9. Data Analyses 

All CSPB and NPTII ELISA plates were analyzed on a SPECTRAmax Plus (Molecular 

Devices, Sunnyvale, CA) or a SPECTRAFluor Plus (Tecan, Research Triangle Park, NC) 

microplate spectrophotometer, using a dual wavelength detection method. All protein 

concentrations were determined by optical absorbance at a wavelength of 450 nm with a 

simultaneous reference reading of 620-650 nm. Data reduction analyses were performed 

using Molecular Devices SOFTmax PRO version 4.7.1 or SOFTmax Pro GxP version 

5.0.1. Absorbance readings and protein standard 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 μg/g fwt basis. For all proteins, 

this conversion utilized a sample dilution factor and a tissue-to-buffer ratio. The protein 

values in μg/g fwt were also converted to μg/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 environmental conditions relevant to its commercial production. This 

trial was conducted in Chile during 2006/2007 using two water treatment levels (well-

( )

( ) DWCF

Protein Level FreshWeight Protein Level in Dry Weight =
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 assessment 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 replicated plots of MON 87460, as well as the 

conventional control, were planted using a strip-plot design with three replicates per site. 

The whole-plot for each replicate was an irrigation treatment (well-watered or waterlimited). The sub-plot for each irrigation treatment was substance type (test and control 

substances), which was randomized in strips across the irrigation treatments to assess for 

any changes in protein levels under different soil moisture conditions. Well-watered 

plots were irrigated to achieve optimal yield, whereas water-limited plots were managed 

to impose a drought stress by withholding irrigation during the late vegetative through 

early grain fill growth stages (i.e., approximately V10 through R2). For a site to be 

included in the combined-site analysis, commercial reference hybrids had to exhibit 

phenotypic responses indicative of a treatment effect. Specifically, 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. Moderate water deficits result 

in approximately a 15% yield loss annually for corn grown in both temperate and tropical 

regions (Barker et al., 2005). Assessments for plant height, ear height 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). Reference 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 stress 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 randomly collected from plants in each plot at each site. 

Forty leaves were combined to form the leaf sample for each plot. Over-season leaf 

samples were collected as follows: 

Over-season leaf (OSL) Corn development 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 62-65 

 
Monsanto Company 07-CR-191U Page 306 of 544 

Whole Plant. The aerial portion of the plant was collected 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 multiple 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. Silks 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, approximately 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 ice for processing and 

analysis. Grain was stored and shipped at room temperature. All tissue samples were 

stored in a –80 oC freezer upon receipt. Tissue 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 for CSPB protein. The NPTII levels were 
Monsanto 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 selected to span the life cycle of corn. 

Moisture content was measured for all tissue types. 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. Improving 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 maize 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 data 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; therefore, data and analysis for the QUI site are 

presented in this appendix. Table D-2 presents results for CSPB and Table D-3 presents 

results for NPTII. Evaluation of the overall data set confirmed expression levels were as 

expected. No unexpected values for either CSPB or NPTII were observed. The QUI site 

results reported below do not impact the conclusion 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 Tissues 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 

Range2 

(μg/g fwt.)3

Mean (SD) 

Range 

(μg/g dwt.)4

 Mean (SD) 

Range 

(μg/g fwt.) 

Mean (SD) 

Range 

(μg/g dwt.) 

LOQ / LOD 

(μg/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/A5

) 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 determined for each tissue type across sites. 3

Protein levels are expressed as microgram (μg) of protein per gram (g) of tissue on a fresh weight basis. 4

Protein levels are expressed as μg/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 applicable 
Monsanto Company 07-CR-191U Page 309 of 544 

Table D-3. NPTII Protein Levels in Tissues 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 

Range2 

(μg/g fwt.)3

Mean (SD) 

Range 

(μg/g dwt.)4

Mean (SD) 

Range 

(μg/g fwt.) 

Mean (SD) 

Range 

(μg/g dwt.) 

LOQ / LOD 

(μg/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/A5

) 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 determined for each tissue type across sites. 3

Protein levels are expressed as microgram (μg) of protein per gram (g) of tissue on a fresh weight basis. 4

Protein levels are expressed as μg/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 applicable 

 
Monsanto Company 07-CR-191U Page 310 of 544 

Appendix E. Materials and Methods for Forage 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). These principles are accepted globally and 

have been employed previously in assessments of corn products derived through 

biotechnology. The materials and methods for component analysis, as well as a 

description of the tissue types, are provided below. 

The compositional assessment was conducted on forage and grain samples harvested 

from two different growing seasons. The first season was conducted in the U.S. during 

2006 under typical agronomic practices and water conditions. 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. 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 established with the appropriate 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 conventional corn hybrid with genetic background similar to 

MON 87460. The forage and grain tissues of the 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 from each reference substance was evaluated in 

this study. The following conventional 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 

34N431 GLP-0604-17091-S QUI1

BT 66101 GLP-0610-17685-S QUI1

Garst 85451 GLP-0609-17689-S QUI1

DKC60-151 GLP-0609-17610-S QUI1

 
Monsanto Company 07-CR-191U Page 312 of 544 

E.2. Test, Control and Reference Substance Characterization 

The identities of the forage and grain samples 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 grain collected from the plots. The grain of 

the test, control, and reference substances were 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 County, 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 complete block design with three 

replicates per block. Each block (replicate) consisted 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 Monsanto 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 -

20oC until their shipment on dry ice to Covance Laboratories Inc. (Madison, WI) for 

analysis. 

Chile 2006/2007

Seed was planted in the winter of 2006 at four replicated field 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 conventional reference substances 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 randomized complete block design. The strip-plot 

factor consisted of the test, control, and reference substances. 

Grain and forage samples were harvested from all plots at ambient temperature and 

forwarded to Monsanto Company (St. Louis, 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 -20o

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 calculation, acid detergent fiber (ADF), neutral detergent 
Monsanto Company 07-CR-191U Page 313 of 544 

fiber (NDF), calcium, and phosphorus. Components assessed in grain samples included 

proximates (protein, fat, ash, and moisture), carbohydrates by calculation, 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], vitamin 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 based on internationally-recognized procedures 

and literature publications. Brief descriptions of the methods utilized for the analyses are 

described below. 

E.4.1 Moisture

Sample was dried in a vacuum 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, 18th 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 nonvolatile matter remaining was quantitated gravimetrically and 

calculated to determine percent ash. The limit of quantitation was 0.100%. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method 923.03, 

AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005). 

E.4.3 Protein

Nitrogenous compounds in the sample were reduced 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 and converted to equivalent protein using the 

factor 6.25. The limit of quantitation was 0.100%. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th 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. The 

fat was extracted with ether and hexane. The extract was evaporated on a steam bath, redissolved in hexane and filtered through a sodium sulfate column. The hexane extract 
Monsanto Company 07-CR-191U Page 314 of 544 

was then evaporated again on a steam bath under nitrogen, dried, and weighed. The limit 

of quantitation was 0.100%. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th 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 thimble 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, 18th 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 and washed with an acidic boiling detergent 

solution that dissolved the protein, carbohydrate, and ash. An acetone wash removed the 

fats and pigments. The lignocellulose fraction 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 fritted vessel and washed with a neutral boiling detergent solution 

that dissolved the protein, carbohydrate, enzyme, 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 products and moisture. Protein content was 

determined for one of the duplicates; ash content was determined for the other. The total 

dietary fiber in the sample was calculated using the protein and ash values. The limit of 

quantitation was 1.0%. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method 985.29, 

AOAC INTERNATIONAL: Gaithersburg, 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 acid, treated with hydrochloric acid, taken to 

dryness, and put into a solution of 5% hydrochloric acid. The amount of each element 

was determined at appropriate wavelengths by comparing the emission of the unknown 

sample, measured on the inductively coupled plasma spectrometer, with the emission of 

reference standards. 

Table E-3. Reference Calibration Ranges and Limits of Quantitation 

Mineral Reference Calibration Range (μg/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, 18th 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. The sulfur-containing amino acids required an 

oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the 

samples for the remaining amino acids was accomplished through direct acid hydrolysis 

with hydrochloric acid. Once hydrolyzed, the individual amino acids were then 

quantitated using an automated amino acid analyzer. The limit of quantitation was 0.100 

mg/g. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method 982.30, 

AOAC INTERNATIONAL: Gaithersburg, 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 methylated with 14% boron trifluoride in methanol. The 

resulting methyl esters were extracted with heptane containing an internal standard. The 

methyl esters of the fatty acids were analyzed by gas chromatography using external 

standards for quantitation. The limit of quantitation was 0.00400%. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method 996.06, 

AOAC INTERNATIONAL, Gaithersburg, Maryland, (2005). 

Official Methods and Recommended 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 phosphate buffer with the addition of ascorbic 

acid to protect the folic acid during autoclaving. Following hydrolysis by autoclaving, 

the sample was treated with a chicken-pancreas 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 acid standard. This response was measured 

turbidimetrically. The limit of quantitation was 0.060 μg/g. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th 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 acid 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. The limit of quantitation 

was 0.300 μg/g. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method 944.13, 

AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005). 

E.4.15 Thiamine Hydrochloride 

Sample was autoclaved under weak acid conditions to extract the thiamine. 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 fluorometer, 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, 18th Ed., Methods 942.23, 

953.17, and 957.17, AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005). 

E.4.16 Vitamin B2 (Riboflavin) 

Sample was hydrolyzed with dilute hydrochloric 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 standards. The growth response was measured turbidimetrically. 

The limit of quantitation was 0.200 μg/g. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th 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 sulfuric 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 response of a pyridoxine standard. The response was 

measured turbidimetrically. The limit of quantitation was 0.070 μg/g. 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Method 961.15, 

AOAC INTERNATIONAL: Gaithersburg, 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 break down any fat and release vitamin E. The saponified 

mixture was extracted with ethyl ether and then quantitated by high-performance liquid 

chromatography using a silica column. The 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 Composition of Some 

Seed Oils as Determined by High-Performance Liquid Chromatography with 

Fluorometric Quantitation,” Journal of Food Science, 50(1):121-124, (1985). 

McMurray, C. H., Blanchflower, W. J., and Rice, D. A., “Influence 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 ultrasonication, hydrolyzed using 4N sodium 

hydroxide, buffered using acetic acid/sodium hydroxide, acidified with 3N hydrochloric 

acid, and filtered. The levels of p-coumaric and ferulic acids in the extract were 

determined by reverse phase high-performance liquid chromatography with ultraviolet 

detection. The limit of quantitation 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 with 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, 

5μm (150 x 4.1mm) with a refractive index detector. The limit of quantitation was 

approximately 0.100%. 

Lehrfeld, Jacob, “HPLC Separation and Quantitation 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-Performance Liquid Chromatography Analysis of Phytic Acid 

on a pH-Stable, Macroporous 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 hydroxylamine 

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 Chromatography 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 Agricultural 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 limit 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 Furfural Compounds In Infant Milk Formulas 
Monsanto Company 07-CR-191U Page 319 of 544 

By High-Performance Liquid 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 from the sample with water. Aliquots were 

dried under inert gas and reconstituted with a hydroxylamine hydrochloride solution in 

pyridine containing phenyl- β -D-glucoside as the internal standard. The resulting 

oximes were converted to silyl derivatives with hexamethyldisilazane (HMDS) and 

trifluoracetic acid (TFA) treatment and analyzed by gas chromatography using a flame 

ionization detector. The limit of quantitation 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 Derivations," Methods in 

Carbohydrate Chemistry, 6:3-8, Academic Press, New York, NY. 1972. 

E.4.24 Free Proline 

The sample was extracted in acid. Determination was by high-performance liquid 

chromatography (HPLC) with fluorescence or diode array detection. Primary amino 

acids were derivatized with o-phthalaldehyde and the secondary amino acids were 

derivatized with fluorenylmethyl chloroformate before injection. The limit of 

quantitation for this study was 0.0100 mg/g. 

R. Schuster, "Determination of Amino Acids in Biological, Pharmaceutical, Plant and 

Food Samples by Automated Precolumn Derivatization and HPLC," Journal of 

Chromatography, 1988, 431, 271-284 

Henderson, J. W., Ricker, R. D., Bidlingmeyer, B. A., Woodward, C., "Rapid 

Accurate, Sensitive, and Reproducible HPLC Analysis of Amino Acids, Amino Acid 

Analysis Using Zorbax Eclipse-AAA columns 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., "Determination of Carbohydrates, Sugar Alcohols, and 

Glycols in Cell Cultures and Fermentation 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, Salicylic 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 specific precursor-fragment response. 

E.5. Control of Bias 

The test, control, and reference substances from 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 specified by a computer-generated randomized 

sample list. The Study Director generated the randomized sample list 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 statistical 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 103 (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)Xj/ΣX, for 

each FAj where 

ΣX is over all 

the FA 

‘X’ is the individual sample value; ‘d’ is the fraction of the sample that is dry matter. 

E.6.1.1. U.S. 2006 Data Processing 

In order to complete a statistical analysis for a compositional constituent 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 analytes with greater than 50% of observations 
Monsanto Company 07-CR-191U Page 321 of 544 

below the assay LOQ were excluded from statistical analysis: 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, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 

gamma linolenic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, and 20:4 

arachidonic acid, sodium, and furfural. These components naturally 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 outliers using a studentized PRESS residuals 

calculation. A predicted residual sums of squares (PRESS) residual is the difference 

between any value and its predicted value from a statistical model that excludes the 

datum point. The studentized version scales these 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 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 had a PRESS residual value outside of the ± 6 

studentized PRESS residual range. 

E.6.1.2. Chile 2006/2007 Data Processing 

In order to complete a statistical analysis for a compositional constituent 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 below the assay LOQ were excluded from 

summaries and analysis. The following 16 analytes with more than 50% of observations 

below the assay LOQ were excluded from statistical analysis: 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 eicosadienoic acid, 20:3 

eicosatrienoic acid, 20:4 arachidonic acid, sodium, 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 represented in Listing 2 of the Statistical Subreport. 

PRESS residuals were used to identify outliers. 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 these 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 that are also outside of the ± 6 

studentized PRESS residual range are considered for exclusion, as outliers, from the final 

analyses. The following result had 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 and 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 analysis. 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 reference 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: 

 Yij = U + Ti + Bj + eij , 

where Yij = unique individual observation, U = overall mean, Ti = hybrid effect, Bj = 

random block effect, and eij = residual error. 

Combined site analyses used the model: 
Monsanto Company 07-CR-191U Page 323 of 544 

Yijk = U + Ti + Lj + B(L)jk + LTij + eijk , 

where Yijk = unique individual observation, U = overall mean, Ti = hybrid effect, Lj = 

random location effect, B(L)jk = random block within location effect, LTij = random 

location by hybrid interaction effect, and eijk = residual error. For each compositional 

component, the forage and grain from the test substance was compared to the 

conventional control. 

A range of observed values from the reference substances was determined for each 

analytical component. Additionally, the reference substances data were used to develop 

population tolerance intervals. 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 compositional component, 

99% tolerance intervals were calculated that are expected to contain, with 95% 

confidence, 99% of the quantities expressed in the population of commercial references 

(George et al., 2004; Ridley et al., 2002). Each tolerance interval estimate was based 

upon one observation per unique reference substance. Individual substances with 

multiple observations were summarized within sites to obtain a single estimate for 

inclusion in tolerance interval calculations. Because negative quantities 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 present p-values from SAS® as either <0.001 or the actual 

value truncated to three decimal places. 

E.6.3. Statistical Methodology for Chile 2006/2007 

All T/C/R substances were grown in single plots randomly assigned within each of three 

replication blocks. All corn compositional analysis components were statistically 

analyzed using a mixed model analysis of variance. The three replicated sites were 

analyzed both separately and combined. Individual replicated site analyses used model 

(1). 

(1) Yijk = U + Bi + Tj + BTij + Sk + BSik + TSjk + eijk, 

where Yijk = unique individual observation, U = overall mean, Bi = random block effect, 

Tj = irrigation treatment effect, BTij = random block by treatment interaction effect, Sk = 

substance effect, BSik = random block by substance effect, TSjk = treatment by substance 

interaction effect and eijk = residual error. 

 
Monsanto Company 07-CR-191U Page 324 of 544 

Combined site analyses used model (2). 

(2) Yijkl = U + Li + B(L)ij + Tk + LTik + TB(L)ijk + Sl + SB(L)ijl + TSkl + LSil + LTSikl

+ eijkl, 

where Yijkl = unique individual observation, U = overall mean, Li = random location 

effect, B(L)ij = random block within location effect, Tk = irrigation treatment effect, LTik

= random location by treatment interaction effect, 

TB(L)ijk = random treatment by block within location interaction effect, 

Sl = substance effect, SB(L)ijl = random substance by block within location interaction 

effect, TSkl = treatment by substance interaction effect, 

LSil = random location by substance interaction effect, LTSikl = random location by 

treatment by substance interaction effect and eijkl = 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 compositional component within each irrigation 

treatment, 99% tolerance intervals were calculated that are expected to contain, with 95% 

confidence, 99% of the quantities expressed in the population of commercial 

conventional substances. Each tolerance interval estimate was based upon one 

observation per unique reference substance within each treatment. For each treatment, 

data were first summarized by substance within site and then by substance across sites. 

Because negative quantities are not possible, negative calculated lower tolerance bounds 

were set to zero. 

SAS® programming was used to generate all summary 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 value 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 furfural 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 Methods of Analysis, 

18th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 923.03. 2005. Official Methods of Analysis, 18th Ed. 

Association of Official Analytical 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 Methods of Analysis, 

18th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 940.33 and 960.46. 2005. Official Methods of Analysis, 

18th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 942.23, 953.17 and 957.17. 2005. Official Methods of 

Analysis, 18th Ed. Association of Official Analytical Chemists, Gaithersburg, 

MD. 

AOAC. International Methods 944.13. 2005. Official Methods of Analysis, 18th Ed. 

Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC International Methods 955.04 and 979.09. 2005. Official Methods of Analysis, 

18th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 960.39 and 948.22. 2005. Official Methods of Analysis, 

18th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 960.46 and 992.05. 2005. Official Methods of Analysis, 

18th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 961.15. 2005. Official Methods of Analysis, 18th Ed. 

Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 982.30. 2005. Official Methods of Analysis, 18th Ed. 

Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 984.27 and 985.01. 2005. Official Methods of Analysis, 

18th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 985.29. 2005. Official Methods of Analysis, 18th Ed. 

Association of Official Analytical Chemists, Gaithersburg, MD. 

AOAC. International Methods 996.06. 2005. Official Methods of Analysis, 18th Ed. 

Association of Official Analytical Chemists, Gaithersburg, MD. 

AOCS. Method Ce 1-62. 1997. Official Methods and Recommended Practices of the 

American Oil Chemists Society, 5th Ed. American Oil Chemists Society, 

Champaign, IL. 

Atkins, L., A.S. Schultz, W.L. Williams, and 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 The Kjeldahl Method for 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 and B.D. Williams. 1983. Vitamin E content of 

feedstuffs determined by high-performance 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 that 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. Determination of carbohydrates, sugar alcohols, and 

glycols in cell cultures and fermentation broths using high-performance anionexchange chromatography with pulsed amperometric detection. Analytical 

Biochemistry. 283:192-199. 

Henderson, J.W., R.D. Ricker, B.A., Bidlingmeyer, C. Woodward. 2000. Rapid 

Accurate, Sensitive, and Reproducible HPLC 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 chromatography analysis of phytic acid on a 

pH-stable, macroporous polymer column. Cereal Chemistry. 66:510-515. 

Lehrfeld, J. 1994. HPLC separation and quantitation of phytic acid and some inositol 

phosphates in foods: problem and solutions Journal of Agricultural and Food 

Chemistry. 42:2726-2731. 

Mason, B.S. and H.T. Slover. 1971. A gas chromatographic method for the determination 

of sugars in foods. Journal of Agricultural 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 Analytical Chemists. 63:1258-1261. 

Methods of Analysis for Infant Formulas. Section C-2. 1985. Infant Formula Council, 

Atlanta, GA. 

OECD. 2002. Consensus document on compositional considerations for new varieties of 

maize (Zea mays): key food and feed nutrients, antinutrients and secondary plant 

metabolites. Organisation for Economic Co-operation and Development. Paris, 

France. 

OECD, 2006. An Introduction to the Food/Feed 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 profile of glyphosate-tolerant 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 derivatization 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. Schreurs. 1985. Vitamin E composition of some 

seed oils as determined by high-performance liquid chromatography with 

fluorometric detection. Journal 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. Agriculture 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 Agriculture, Washington, D.C. 
Monsanto Company 07-CR-191U Page 328 of 544 

Table E-5. Comparison of Proximates, Fiber, and Mineral Content in Forage from MON 87460 and ConvCombined Sites in the U.S. during 2006 under Typical Agronomic Conditions 1DW = dry weight; FW = fresh 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 were set t

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Fiber Acid Detergent Fiber (% DW) 24.10 (0.96) 24.64 (0.96) -0.54 (0.92) -2.49, 1.41 0.567 

 [17.78 - 34.43] [19.11 - 29.21] [-6.50 - 8.60] 

Neutral Detergent Fiber (% DW) 38.69 (1.99) 38.75 (1.99) -0.056 (1.08) -2.33, 2.22 0.959 

 [31.10 - 49.44] [27.73 - 48.35] [-11.60 - 6.51] 

Mineral Calcium (% DW) 0.21 (0.018) 0.22 (0.018) -0.018 (0.012) -0.048, 0.013 0.189 

 [0.14 - 0.30] [0.13 - 0.33] [-0.075 - 0.089] 

Phosphorus (% DW) 0.18 (0.0089) 0.19 (0.0089) -0.0095 (0.0055) -0.021, 0.0021 0.101 

 [0.14 - 0.22] [0.14 - 0.23] [-0.069 - 0.022] 

Proximate Ash (% DW) 3.76 (0.35) 4.21 (0.35) -0.44 (0.37) -1.39, 0.50 0.281 

 [2.17 - 5.34] [2.94 - 8.01] [-3.73 - 1.22] 

Carbohydrates (% DW) 86.45 (0.54) 85.77 (0.54) 0.68 (0.49) -0.57, 1.93 0.220 

 [83.78 - 88.75] [81.88 - 89.26] [-2.08 - 2.89] 

Moisture (% FW) 70.94 (1.25) 71.46 (1.25) -0.52 (0.37) -1.30, 0.25 0.174 

 [64.70 - 77.90] [66.50 - 75.70] [-2.50 - 2.40] 

Protein (% DW) 7.56 (0.25) 7.85 (0.25) -0.30 (0.20) -0.71, 0.12 0.146 

 [6.65 - 8.57] [6.45 - 10.24] [-2.63 - 1.14] 

Total Fat (% DW) 2.23 (0.20) 2.17 (0.20) 0.059 (0.13) -0.22, 0.34 0.659 

 [1.07 - 3.24] [1.28 - 2.88] [-0.88 - 0.90] 
Monsanto Company 07-CR-191U Page 329 of 544 

Table E-6. Comparison of the Proximates and Fiber Content in Grain from MON 87460 and ConvCombined Sites in the U.S. during 2006 under Typical Agronomic Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E.

[Range] 

Control Mean ± 

S.E. 1

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Proximate 

Ash (% DW) 1.54 (0.039) 1.46 (0.039) 0.082 (0.038) 0.0033, 0.16 0.041 

 [1.33 - 1.83] [1.32 - 1.79] [-0.22 - 0.38] 

Carbohydrates (% DW) 84.22 (0.56) 84.10 (0.56) 0.13 (0.20) -0.30, 0.56 0.539 

 [81.40 - 87.04] [81.31 - 86.05] [-1.57 - 1.98] 

Moisture (% FW) 9.94 (0.18) 10.09 (0.18) -0.15 (0.15) -0.54, 0.25 0.377 

 [9.12 - 11.00] [9.17 - 11.20] [-1.36 - 0.83] 

Protein (% DW) 10.50 (0.54) 10.74 (0.54) -0.24 (0.18) -0.61, 0.13 0.195 

 [8.19 - 13.21] [8.77 - 13.33] [-2.05 - 1.35] 

Total Fat (% DW) 3.74 (0.051) 3.71 (0.051) 0.029 (0.067) -0.14, 0.20 0.678 

 [3.44 - 4.06] [3.57 - 3.96] [-0.52 - 0.32] 

Fiber Acid Detergent Fiber (% DW) 3.03 (0.25) 3.02 (0.25) 0.0095 (0.36) -0.79, 0.81 0.979 

 [1.57 - 4.94] [1.94 - 4.08] [-2.51 - 3.00] 

Neutral Detergent Fiber (% DW) 8.97 (0.32) 8.95 (0.32) 0.019 (0.46) -1.00, 1.03 0.967 

 [6.45 - 11.63] [7.82 - 12.22] [-4.07 - 3.32] 

Total Dietary Fiber (% DW) 12.59 (0.34) 12.15 (0.34) 0.44 (0.35) -0.28, 1.16 0.216 

 [10.42 - 14.57] [10.76 - 14.87] [-3.32 - 3.67] 

1DW = dry weight; FW = fresh 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 were set t
Monsanto Company 07-CR-191U Page 330 of 544 

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

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Calcium (% DW) 0.0054 (0.00019) 0.0054 (0.00019) -0.00006 (0.00007) -0.00020, 0.00009 0.431 

 [0.0047 - 0.0061] [0.0048 - 0.0063] [-0.00059 - 0.00056] 

Copper (mg/kg DW) 1.89 (0.14) 1.86 (0.14) 0.022 (0.16) -0.32, 0.37 0.892 

 [1.47 - 4.61] [1.54 - 3.43] [-1.31 - 2.11] 

Iron (mg/kg DW) 18.24 (0.62) 18.30 (0.62) -0.067 (0.50) -1.34, 1.21 0.898 

 [15.02 - 24.88] [14.17 - 20.58] [-2.34 - 7.02] 

Magnesium (% DW) 0.11 (0.0042) 0.12 (0.0042) -0.0013 (0.0016) -0.0047, 0.0020 0.418 

 [0.095 - 0.13] [0.095 - 0.13] [-0.010 - 0.013] 

Manganese (mg/kg DW) 6.79 (0.43) 6.89 (0.43) -0.097 (0.12) -0.41, 0.22 0.462 

 [5.02 - 8.64] [5.50 - 8.34] [-0.97 - 0.68] 

Phosphorus (% DW) 0.31 (0.011) 0.32 (0.011) -0.0085 (0.0047) -0.018, 0.0014 0.089 

 [0.27 - 0.35] [0.27 - 0.37] [-0.030 - 0.034] 

Potassium (% DW) 0.38 (0.0030) 0.38 (0.0030) 0.0019 (0.0037) -0.0060, 0.0097 0.624 

 [0.36 - 0.39] [0.35 - 0.39] [-0.025 - 0.038] 

Zinc (mg/kg DW) 20.86 (0.95) 21.24 (0.95) -0.38 (0.32) -1.03, 0.27 0.238 

 [18.24 - 24.75] [17.41 - 25.20] [-3.02 - 1.85] 

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 were set t
Monsanto Company 07-CR-191U Page 331 of 544 

Table E-8. Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Controin the U.S. during 2006 under Typical Agronomic Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Alanine (% DW) 0.80 (0.047) 0.82 (0.047) -0.011 (0.013) -0.037, 0.016 0.410 

 [0.60 - 1.04] [0.64 - 1.04] [-0.10 - 0.10] 

Arginine (% DW) 0.45 (0.019) 0.44 (0.019) 0.0067 (0.011) -0.022, 0.035 0.577 

 [0.33 - 0.54] [0.38 - 0.52] [-0.071 - 0.087] 

Aspartic acid (% DW) 0.65 (0.028) 0.66 (0.028) -0.0062 (0.0075) -0.022, 0.0096 0.419 

 [0.52 - 0.79] [0.54 - 0.78] [-0.065 - 0.060] 

Cystine (% DW) 0.23 (0.0085) 0.23 (0.0085) -0.0040 (0.0018) -0.0087, 0.00069 0.079 

 [0.19 - 0.27] [0.20 - 0.26] [-0.016 - 0.012] 

Glutamic acid (% DW) 2.07 (0.12) 2.09 (0.12) -0.025 (0.034) -0.097, 0.046 0.462 

 [1.52 - 2.66] [1.64 - 2.67] [-0.26 - 0.28] 

Glycine (% DW) 0.39 (0.013) 0.39 (0.013) 0.0019 (0.0041) -0.0085, 0.012 0.656 

 [0.33 - 0.45] [0.34 - 0.43] [-0.024 - 0.035] 

Histidine (% DW) 0.32 (0.012) 0.32 (0.012) -0.00085 (0.0049) -0.013, 0.012 0.868 

 [0.25 - 0.38] [0.27 - 0.37] [-0.029 - 0.040] 

Isoleucine (% DW) 0.38 (0.021) 0.38 (0.021) -0.0022 (0.0088) -0.025, 0.020 0.810 

 [0.28 - 0.50] [0.31 - 0.48] [-0.042 - 0.070] 
Monsanto Company 07-CR-191U Page 332 of 544 

Table E-8 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conve

Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Leucine (% DW) 1.41 (0.088) 1.43 (0.088) -0.020 (0.026) -0.075, 0.035 0.453 

 [1.01 - 1.85] [1.11 - 1.87] [-0.20 - 0.22] 

Lysine (% DW) 0.30 (0.0076) 0.30 (0.0076) 0.0024 (0.0040) -0.0080, 0.013 0.579 

 [0.26 - 0.34] [0.26 - 0.33] [-0.023 - 0.027] 

Methionine (% DW) 0.22 (0.013) 0.22 (0.013) -0.00089 (0.0030) -0.0086, 0.0068 0.777 

 [0.16 - 0.28] [0.17 - 0.26] [-0.019 - 0.019] 

Phenylalanine (% DW) 0.56 (0.031) 0.56 (0.031) -0.0059 (0.0090) -0.025, 0.013 0.518 

 [0.41 - 0.72] [0.45 - 0.72] [-0.067 - 0.074] 

Proline (% DW) 1.01 (0.047) 1.02 (0.047) -0.0048 (0.017) -0.050, 0.040 0.793 

 [0.78 - 1.23] [0.83 - 1.21] [-0.082 - 0.17] 

Serine (% DW) 0.53 (0.028) 0.53 (0.028) -0.0070 (0.0086) -0.025, 0.011 0.430 

 [0.40 - 0.64] [0.43 - 0.67] [-0.089 - 0.046] 

Threonine (% DW) 0.37 (0.016) 0.37 (0.016) -0.00059 (0.0050) -0.011, 0.010 0.908 

 [0.30 - 0.45] [0.31 - 0.45] [-0.036 - 0.037] 

Tryptophan (% DW) 0.066 (0.0027) 0.068 (0.0027) -0.0015 (0.0017) -0.0050, 0.0021 0.394 

 [0.054 - 0.088] [0.055 - 0.085] [-0.014 - 0.016] 
Monsanto Company 07-CR-191U Page 333 of 544 

Table E-8 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conve

Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions 

Difference (Test minus Control) 

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Tyrosine (% DW) 0.32 (0.024) 0.30 (0.024) 0.014 (0.022) -0.044, 0.071 0.565 

 [0.16 - 0.43] [0.15 - 0.43] [-0.12 - 0.21] 

Valine (% DW) 0.52 (0.024) 0.52 (0.024) -0.0019 (0.011) -0.029, 0.026 0.866 

 [0.40 - 0.64] [0.43 - 0.62] [-0.053 - 0.079] 

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 were set t
Monsanto Company 07-CR-191U Page 334 of 544 

Table E-9. Comparison of the Fatty Acid Content in Grain from MON 87460 and Conventional Controin the U.S. during 2006 under Typical Agronomic Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

16:0 Palmitic (% Total FA) 12.12 (0.20) 11.94 (0.20) 0.18 (0.19) -0.31, 0.66 0.394 

 [11.60 - 15.21] [11.45 - 12.38] [-0.28 - 2.84] 

16:1 Palmitoleic (% Total FA) 0.17 (0.0073) 0.17 (0.0073) -0.0020 (0.0036) -0.0095, 0.0055 0.576 

 [0.15 - 0.20] [0.15 - 0.23] [-0.042 - 0.015] 

18:0 Stearic (% Total FA) 2.05 (0.033) 1.98 (0.033) 0.069 (0.022) 0.013, 0.13 0.024 

 [1.88 - 2.34] [1.80 - 2.10] [-0.041 - 0.33] 

18:1 Oleic (% Total FA) 20.26 (0.18) 20.49 (0.18) -0.23 (0.12) -0.48, 0.015 0.064 

 [19.32 - 21.08] [19.50 - 21.77] [-1.13 - 0.85] 

18:2 Linoleic (% Total FA) 63.34 (0.35) 63.34 (0.35) -0.0079 (0.26) -0.67, 0.65 0.976 

 [59.90 - 65.07] [61.88 - 64.70] [-3.07 - 1.05] 

18:3 Linolenic (% Total FA) 1.28 (0.012) 1.27 (0.012) 0.0066 (0.015) -0.029, 0.042 0.673 

 [1.17 - 1.46] [1.22 - 1.33] [-0.046 - 0.20] 

20:0 Arachidic (% Total FA) 0.41 (0.0078) 0.41 (0.0078) 0.0037 (0.0032) -0.0031, 0.010 0.263 

 [0.39 - 0.44] [0.37 - 0.45] [-0.017 - 0.024] 

20:1 Eicosenoic (% Total FA) 0.18 (0.0024) 0.19 (0.0024) -0.0078 (0.0027) -0.013, -0.0022 0.007 

 [0.17 - 0.19] [0.17 - 0.22] [-0.035 - 0.0086] 

22:0 Behenic (% Total FA) 0.20 (0.012) 0.20 (0.012) -0.0044 (0.013) -0.037, 0.028 0.742 

 [0.14 - 0.27] [0.14 - 0.27] [-0.099 - 0.078] 

1FA = 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 were set t
Monsanto Company 07-CR-191U Page 335 of 544 

Table E-10. Comparison of the Vitamin Content in Grain from MON 87460 and Conventional Control fo

the U.S. during 2006 under Typical Agronomic Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Folic Acid (mg/kg DW) 0.30 (0.012) 0.30 (0.012) 0.0058 (0.0059) -0.0094, 0.021 0.371 

 [0.25 - 0.36] [0.24 - 0.35] [-0.033 - 0.041] 

Niacin (mg/kg DW) 18.59 (0.77) 18.52 (0.77) 0.069 (0.53) -1.29, 1.42 0.901 

 [15.53 - 22.23] [15.26 - 21.85] [-3.73 - 4.43] 

Thiamine HCl Vitamin B1 (mg/kg DW) 

3.31 (0.16) 3.21 (0.16) 0.094 (0.066) -0.077, 0.26 0.216 

 [2.67 - 3.89] [2.33 - 3.89] [-0.44 - 0.54] 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 - 2.04] [0.94 - 1.94] [-0.88 - 0.60] 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 

 [5.03 - 7.49] [5.21 - 7.41] [-1.55 - 1.66] Vitamin E (mg/kg DW) 14.73 (0.80) 14.69 (0.80) 0.045 (0.53) -1.31, 1.40 0.935 

 [11.09 - 20.02] [9.47 - 18.44] [-3.95 - 4.77] 

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 were set t
Monsanto Company 07-CR-191U Page 336 of 544 

Table E-11. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain froConventional Control for Combined Sites in the U.S. during 2006 under Typical Agronomic Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Anti-nutrient 

Phytic Acid (% DW) 0.83 (0.038) 0.84 (0.038) -0.0087 (0.028) -0.080, 0.063 0.767  [0.60 - 1.00] [0.69 - 1.09] [-0.15 - 0.19] Raffinose (% DW) 0.19 (0.0081) 0.18 (0.0081) 0.014 (0.0082) -0.0074, 0.035 0.155  [0.15 - 0.22] [0.15 - 0.22] [-0.036 - 0.050] Secondary Metabolite Ferulic Acid (μg/g DW) 1772.22 (47.57) 1693.18 (47.57) 79.04 (62.05) -80.47, 238.55 0.258  [1561.63 - 1966.67] [1245.83 - 1997.77] [-210.94 - 533.93] p-Coumaric Acid (μg/g DW) 115.95 (4.15) 126.55 (4.15) -10.61 (4.60) -22.44, 1.22 0.069  [99.45 - 136.67] [94.77 - 156.25] [-38.32 - 27.59] 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 were set to 
Monsanto Company 07-CR-191U Page 337 of 544 

Table E-12. Comparison of Proximates, Fiber, and Mineral Content in Forage from MON 87460 and Cfor Combined Sites in Chile during 2006/2007 under Well-Watered Conditions 1DW = dry weight; FW = fresh 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 were set to 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Fiber Acid Detergent Fiber (% DW) 28.37 (1.45) 30.43 (1.45) -2.07 (1.79) -5.85, 1.72 0.264 

 [17.95 - 34.70] [24.98 - 35.12] [-10.93 - 5.34] 

Neutral Detergent Fiber (% DW) 42.02 (1.52) 44.51 (1.52) -2.49 (1.81) -6.10, 1.11 0.171 

 [36.08 - 50.00] [39.08 - 47.24] [-8.10 - 8.70] 

Mineral Calcium (% DW) 0.26 (0.020) 0.27 (0.020) -0.0091 (0.019) -0.048, 0.029 0.628 

 [0.24 - 0.28] [0.22 - 0.39] [-0.11 - 0.051] 

Phosphorus (% DW) 0.16 (0.0077) 0.16 (0.0077) 0.0011 (0.0061) -0.011, 0.013 0.852 

 [0.12 - 0.19] [0.13 - 0.20] [-0.030 - 0.033] 

Proximate Ash (% DW) 4.71 (0.22) 4.89 (0.22) -0.18 (0.20) -0.59, 0.22 0.366 

 [4.25 - 5.35] [3.88 - 6.05] [-1.33 - 0.73] 

Carbohydrates (% DW) 87.61 (0.42) 87.11 (0.42) 0.50 (0.40) -0.29, 1.29 0.208 

 [86.51 - 89.58] [85.87 - 88.50] [-0.52 - 2.34] 

Moisture (% FW) 74.02 (0.73) 75.19 (0.73) -1.17 (0.70) -2.65, 0.31 0.113 

 [70.90 - 75.90] [74.20 - 78.00] [-4.40 - 1.70] 

Protein (% DW) 6.53 (0.40) 6.71 (0.40) -0.18 (0.22) -0.62, 0.25 0.407 

 [5.29 - 7.10] [6.01 - 7.44] [-1.20 - 1.09] 

Total Fat (% DW) 1.16 (0.16) 1.30 (0.16) -0.14 (0.23) -0.60, 0.33 0.557 

 [0.57 - 1.96] [0.51 - 2.33] [-0.71 - 0.68] 
Monsanto Company 07-CR-191U Page 338 of 544 

Table E-13. Comparison of the Proximates and Fiber Content in Grain from MON 87460 and ConvCombined Sites in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± 

S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Proximate 

Ash (% DW) 1.44 (0.038) 1.42 (0.038) 0.015 (0.048) -0.083, 0.11 0.751 

 [1.35 - 1.53] [1.26 - 1.60] [-0.22 - 0.20] 

Carbohydrates (% DW) 85.17 (0.27) 85.53 (0.27) -0.37 (0.36) -1.08, 0.35 0.310 

 [82.98 - 87.63] [84.91 - 86.31] [-2.41 - 2.56] 

Moisture (% FW) 12.09 (0.15) 12.09 (0.15) 0 (0.21) -0.44, 0.44 1.000 

 [11.80 - 12.50] [11.30 - 12.80] [-0.70 - 0.70] 

Protein (% DW) 9.50 (0.23) 9.32 (0.23) 0.18 (0.29) -0.40, 0.76 0.533 

 [7.57 - 11.32] [8.55 - 9.77] [-1.93 - 1.61] 

Total Fat (% DW) 3.89 (0.082) 3.72 (0.082) 0.17 (0.071) 0.018, 0.32 0.029 

 [3.45 - 4.23] [3.60 - 3.90] [-0.42 - 0.61] 

Fiber Acid Detergent Fiber (% DW) 2.57 (0.21) 2.47 (0.21) 0.11 (0.27) -0.44, 0.65 0.696 

 [2.08 - 3.18] [1.41 - 4.41] [-1.43 - 0.96] 

Neutral Detergent Fiber (% DW) 8.66 (0.34) 8.60 (0.34) 0.063 (0.44) -0.86, 0.99 0.887 

 [8.19 - 9.45] [7.74 - 9.70] [-1.41 - 1.62] 

Total Dietary Fiber (% DW) 12.70 (0.44) 12.53 (0.44) 0.17 (0.50) -0.90, 1.24 0.737 

 [11.59 - 16.00] [11.20 - 13.98] [-1.15 - 2.97] 

1DW = dry weight; FW = fresh 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 were set to 
Monsanto Company 07-CR-191U Page 339 of 544 

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

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Calcium (% DW) 0.0047 (0.00045) 0.0045 (0.00045) 0.00014 (0.00029) -0.00044, 0.00072 0.630 

 [0.0035 - 0.0058] [0.0036 - 0.0059] [-0.00060 - 0.0012] 

Copper (mg/kg DW) 1.86 (0.24) 1.87 (0.24) -0.011 (0.27) -0.60, 0.58 0.966 

 [1.58 - 2.17] [1.47 - 2.81] [-0.89 - 0.37] 

Iron (mg/kg DW) 16.60 (0.68) 16.95 (0.68) -0.36 (0.61) -1.58, 0.86 0.561 

 [14.53 - 20.30] [15.22 - 19.95] [-2.07 - 1.58] 

Magnesium (% DW) 0.12 (0.0034) 0.11 (0.0034) 0.0094 (0.0037) 0.0021, 0.017 0.012 

 [0.10 - 0.14] [0.10 - 0.11] [-0.0076 - 0.028] 

Manganese (mg/kg DW) 6.27 (0.40) 6.03 (0.40) 0.24 (0.29) -0.59, 0.87 0.434 

 [5.25 - 7.08] [4.64 - 7.58] [-1.51 - 1.61] 

Phosphorus (% DW) 0.32 (0.0099) 0.30 (0.0099) 0.019 (0.0098) -0.00060, 0.038 0.057 

 [0.29 - 0.34] [0.26 - 0.33] [-0.034 - 0.086] 

Potassium (% DW) 0.40 (0.011) 0.40 (0.011) -0.0039 (0.0084) -0.021, 0.013 0.643 

 [0.37 - 0.41] [0.36 - 0.45] [-0.078 - 0.047] 

Zinc (mg/kg DW) 22.00 (1.00) 21.02 (1.00) 0.99 (0.80) -0.60, 2.57 0.219 

 [19.20 - 25.09] [18.36 - 25.34] [-3.91 - 5.47] 

1DW = dry weight; FW = fresh 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 were set to 
Monsanto Company 07-CR-191U Page 340 of 544 

Table E-15. Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional CoSites in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Alanine (% DW) 0.71 (0.018) 0.70 (0.018) 0.017 (0.025) -0.032, 0.066 0.500 

 [0.63 - 0.86] [0.63 - 0.76] [-0.077 - 0.14] 

Arginine (% DW) 0.39 (0.012) 0.38 (0.012) 0.0079 (0.017) -0.026, 0.042 0.639 

 [0.32 - 0.47] [0.32 - 0.43] [-0.030 - 0.14] 

Aspartic Acid (% DW) 0.61 (0.013) 0.60 (0.013) 0.0088 (0.017) -0.024, 0.042 0.598 

 [0.54 - 0.70] [0.56 - 0.63] [-0.057 - 0.073] 

Cystine (% DW) 0.22 (0.0043) 0.21 (0.0043) 0.0030 (0.0047) -0.0064, 0.012 0.527 

 [0.20 - 0.23] [0.20 - 0.22] [-0.0082 - 0.017] 

Glutamic Acid (% DW) 1.84 (0.047) 1.80 (0.047) 0.043 (0.064) -0.085, 0.17 0.503 

 [1.63 - 2.21] [1.62 - 1.97] [-0.22 - 0.35] 

Glycine (% DW) 0.35 (0.0063) 0.34 (0.0063) 0.0063 (0.0077) -0.0090, 0.022 0.414 

 [0.32 - 0.39] [0.31 - 0.35] [-0.029 - 0.041] 

Histidine (% DW) 0.29 (0.0056) 0.29 (0.0056) 0.0034 (0.0074) -0.011, 0.018 0.645 

 [0.26 - 0.34] [0.26 - 0.30] [-0.031 - 0.037] 

Isoleucine (% DW) 0.33 (0.0092) 0.33 (0.0092) 0.0014 (0.013) -0.024, 0.026 0.908 

 [0.29 - 0.42] [0.30 - 0.36] [-0.044 - 0.074] 
Monsanto Company 07-CR-191U Page 341 of 544 

Table E-15 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conve

Combined Sites in Chile during 2006/2007 under Well-Watered Conditions

Difference (Test minus Control)

Analytical 

Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Leucine (% DW) 1.23 (0.034) 1.19 (0.034) 0.036 (0.046) -0.057, 0.13 0.441 

 [1.08 - 1.52] [1.08 - 1.30] [-0.13 - 0.28] 

Lysine (% DW) 0.28 (0.0045) 0.28 (0.0045) 0.00075 (0.0055) -0.010, 0.012 0.892 

 [0.26 - 0.31] [0.26 - 0.29] [-0.027 - 0.018] 

Methionine (% DW) 0.18 (0.0073) 0.18 (0.0073) 0.0032 (0.0065) -0.0098, 0.016 0.625 

 [0.16 - 0.21] [0.16 - 0.19] [-0.021 - 0.027] 

Phenylalanine (% DW) 0.49 (0.012) 0.48 (0.012) 0.011 (0.017) -0.023, 0.044 0.535 

 [0.43 - 0.60] [0.43 - 0.52] [-0.056 - 0.10] 

Proline (% DW) 0.88 (0.027) 0.87 (0.027) 0.012 (0.030) -0.049, 0.072 0.705 

 [0.77 - 1.07] [0.82 - 0.95] [-0.092 - 0.16] 

Serine (% DW) 0.47 (0.012) 0.45 (0.012) 0.019 (0.016) -0.013, 0.051 0.224 

 [0.43 - 0.54] [0.40 - 0.50] [-0.041 - 0.071] 

Threonine (% DW) 0.32 (0.0069) 0.32 (0.0069) 0.0043 (0.0090) -0.014, 0.022 0.634 

 [0.28 - 0.37] [0.29 - 0.33] [-0.033 - 0.040] 

Tryptophan (% DW) 0.051 (0.0020) 0.051 (0.0020) 0.00095 (0.0025) -0.0039, 0.0058 0.701 

 [0.039 - 0.063] [0.046 - 0.054] [-0.013 - 0.012] 
Monsanto Company 07-CR-191U Page 342 of 544 

Table E-15 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conve

Combined Sites in Chile during 2006/2007 under Well-Watered Conditions

Difference (Test minus Control) 

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Tyrosine (% DW) 0.23 (0.023) 0.25 (0.023) -0.022 (0.032) -0.089, 0.044 0.496 

 [0.12 - 0.35] [0.13 - 0.32] [-0.11 - 0.079] 

Valine (% DW) 0.46 (0.011) 0.46 (0.011) 0.0017 (0.014) -0.027, 0.031 0.909 

 [0.42 - 0.56] [0.41 - 0.49] [-0.054 - 0.077] 

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 were set to 
Monsanto Company 07-CR-191U Page 343 of 544 

Table E-16. Comparison of the Fatty Acid Content in Grain from MON 87460 and Conventional Contro

in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

16:0 Palmitic (% Total FA) 10.93 (0.13) 11.15 (0.13) -0.22 (0.16) -0.54, 0.10 0.173 

[10.63 - 11.60] [10.96 - 11.32] [-0.69 - 0.33] 

18:0 Stearic (% Total FA) 1.80 (0.045) 1.78 (0.045) 0.020 (0.047) -0.078, 0.12 0.675 

[1.74 - 1.97] [1.66 - 1.91] [-0.11 - 0.22] 

18:1 Oleic (% Total FA) 20.93 (0.37) 21.01 (0.37) -0.079 (0.29) -0.69, 0.53 0.786 

[20.29 - 21.28] [19.78 - 21.93] [-1.20 - 0.51] 

18:2 Linoleic (% Total FA) 64.51 (0.45) 64.21 (0.45) 0.30 (0.42) -0.59, 1.19 0.485 

[63.92 - 65.27] [63.22 - 65.48] [-0.81 - 1.59] 

18:3 Linolenic (% Total FA) 1.18 (0.016) 1.21 (0.016) -0.027 (0.014) -0.056, 0.00085 0.057 

[1.15 - 1.23] [1.19 - 1.25] [-0.049 - -0.013] 

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 - 0.34] [0.29 - 0.34] [-0.026 - 0.0090] 

20:1 Eicosenoic (% Total FA) 0.18 (0.0039) 0.19 (0.0039) -0.0041 (0.0039) -0.012, 0.0038 0.301 

[0.17 - 0.20] [0.17 - 0.20] [-0.016 - 0.0089] 

22:0 Behenic (% Total FA) 0.15 (0.020) 0.13 (0.020) 0.015 (0.026) -0.039, 0.070 0.559 

[0.062 - 0.26] [0.063 - 0.17] [-0.075 - 0.097] 

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 were set to 
Monsanto Company 07-CR-191U Page 344 of 544 

Table E-17. Comparison of the Vitamin Content in Grain from MON 87460 and Conventional Control fo

Chile during 2006/2007 under Well-Watered Conditions

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Folic Acid (mg/kg DW) 0.26 (0.018) 0.27 (0.018) -0.0094 (0.016) -0.044, 0.025 0.569 

 [0.23 - 0.29] [0.22 - 0.33] [-0.085 - 0.029] Niacin (mg/kg DW) 19.17 (1.77) 19.23 (1.77) -0.054 (1.81) -3.66, 3.56 0.976  [16.42 - 21.50] [17.42 - 21.17] [-1.62 - 2.69] Thiamine HCl/Vitamin B1 (mg/kg DW) 

2.86 (0.084) 2.86 (0.084) 0.00065 (0.088) -0.19, 0.19 0.994 

[2.61 - 3.19] [2.74 - 3.06] [-0.22 - 0.44] 

Riboflavin/Vitamin B2 (mg/kg DW) 

2.01 (0.14) 1.97 (0.14) 0.040 (0.14) -0.24, 0.32 0.781 

[1.61 - 2.54] [1.46 - 2.63] [-1.02 - 0.61] 

Pyridoxine HCl/Vitamin B6 (mg/kg DW) 

6.32 (0.27) 6.83 (0.27) -0.51 (0.33) -1.21, 0.19 0.143 

[5.49 - 7.39] [6.17 - 7.37] [-1.32 - 0.21] 

Vitamin E (mg/kg DW) 11.90 (0.40) 10.99 (0.40) 0.91 (0.54) -0.23, 2.05 0.110 

 [10.64 - 13.57] [9.30 - 12.78] [-0.81 - 2.32] 

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 were set to 
Monsanto Company 07-CR-191U Page 345 of 544 

Table E-18. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain froConventional Control for Combined Sites in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Anti-nutrient 

Phytic Acid (% DW) 0.76 (0.035) 0.76 (0.035) 0.0069 (0.043) -0.083, 0.096 0.873 

 [0.58 - 0.93] [0.63 - 0.90] [-0.21 - 0.31] 

Raffinose (% DW) 0.11 (0.013) 0.11 (0.013) -0.0015 (0.0050) -0.012, 0.0089 0.767 

 [0.075 - 0.12] [0.077 - 0.14] [-0.022 - 0.013] 

Secondary Metabolite Ferulic Acid (μg/g DW) 1849.20 (114.60) 1753.19 (114.60) 96.01 (160.59) -224.84, 416.86 0.552 

 [1265.68 - 2240.00] [820.14 - 2128.15] [-660.93 - 1232.32] 

p-Coumaric Acid (μg/g DW) 137.39 (15.51) 149.37 (15.51) -11.98 (18.53) -50.95, 26.99 0.526 

 [68.64 - 188.64] [64.03 - 204.06] [-88.07 - 87.50] 

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 were set to 
Monsanto Company 07-CR-191U Page 346 of 544 

Table E-19. Comparison of Proximates, Fiber, and Mineral Content in Forage from MON 87460 and Cfor Combined Sites in Chile during 2006/2007 under Water-Limited Conditions 1DW = dry weight; FW = fresh 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 were set to 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Fiber Acid Detergent Fiber (% DW) 27.15 (1.45) 26.73 (1.45) 0.42 (1.79) -3.36, 4.21 0.816 

 [23.03 - 32.00] [23.10 - 30.79] [-6.30 - 8.90] 

Neutral Detergent Fiber (% DW) 39.06 (1.52) 40.10 (1.52) -1.04 (1.81) -4.65, 2.56 0.565 

 [33.29 - 44.10] [31.81 - 50.61] [-9.65 - 8.94] 

Mineral Calcium (% DW) 0.32 (0.020) 0.34 (0.020) -0.023 (0.019) 0.002, 0.015 0.219 

 [0.20 - 0.44] [0.26 - 0.41] [-0.14 - 0.11] 

Phosphorus (% DW) 0.16 (0.0077) 0.17 (0.0077) -0.0078 (0.0061) -0.020, 0.0044 0.204 

 [0.14 - 0.18] [0.15 - 0.21] [-0.033 - 0.014] 

Proximate Ash (% DW) 5.29 (0.22) 5.49 (0.22) -0.20 (0.20) -0.60, 0.21 0.332 

 [4.51 - 6.29] [4.59 - 6.90] [-1.02 - 0.94] 

Carbohydrates (% DW) 85.92 (0.42) 86.02 (0.42) -0.10 (0.40) -0.89, 0.69 0.798 

 [84.14 - 88.81] [84.51 - 87.52] [-1.84 - 1.29] 

Moisture (% FW) 74.98 (0.73) 75.42 (0.75) -0.44 (0.70) -1.92, 1.04 0.552 

 [72.00 - 77.40] [73.00 - 77.60] [-3.20 - 4.10] 

Protein (% DW) 7.47 (0.40) 7.67 (0.40) -0.20 (0.22) -0.63, 0.24 0.373 

 [5.49 - 8.76] [6.52 - 9.14] [-1.02 - 0.36] 

Total Fat (% DW) 1.32 (0.16) 0.84 (0.16) 0.47 (0.23) 0.010, 0.94 0.045 

 [0.50 - 1.92] [0.20 - 1.66] [-0.42 - 0.92] 
Monsanto Company 07-CR-191U Page 347 of 544 

Table E-20. Comparison of the Proximates and Fiber Content in Grain from MON 87460 and ConvCombined Sites in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± 

S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Proximates 

Ash (% DW) 1.47 (0.038) 1.50 (0.038) -0.032 (0.048) -0.13, 0.066 0.505 

[1.24 - 1.75] [1.39 - 1.63] [-0.31 - 0.35] 

Carbohydrates (% DW) 84.21 (0.27) 84.10 (0.27) 0.11 (0.36) -0.60, 0.82 0.754 

[82.64 - 85.64] [82.95 - 85.98] [-2.06 - 1.84] 

Moisture (% FW) 12.10 (0.15) 11.98 (0.15) 0.12 (0.21) -0.31, 0.56 0.563 

[11.60 - 12.50] [11.30 - 12.50] [-0.70 - 1.20] 

Protein (% DW) 10.30 (0.23) 10.44 (0.23) -0.13 (0.29) -0.71, 0.45 0.645 

[9.41 - 11.45] [9.17 - 11.50] [-1.61 - 1.41] 

Total Fat (% DW) 4.02 (0.082) 3.96 (0.082) 0.054 (0.071) -0.096, 0.20 0.459 

 [3.71 - 4.28] [3.47 - 4.23] [-0.19 - 0.54] 

Fiber Acid Detergent Fiber (% DW) 2.59 (0.21) 2.33 (0.21) 0.26 (0.27) -0.28, 0.80 0.342 

[1.85 - 3.58] [1.83 - 3.05] [-0.53 - 0.99] 

Neutral Detergent Fiber (% DW) 8.87 (0.34) 8.22 (0.34) 0.64 (0.44) -0.28, 1.57 0.161 

[7.33 - 11.31] [7.91 - 8.66] [-1.25 - 3.07] 

Total Dietary Fiber (% DW) 12.48 (0.44) 12.15 (0.44) 0.33 (0.50) -0.73, 1.40 0.515 

[10.78 - 14.43] [11.06 - 13.70] [-1.99 - 2.19] 

1DW = dry weight; FW = fresh 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 were s
Monsanto Company 07-CR-191U Page 348 of 544 

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

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Calcium (% DW) 0.0052 (0.00045) 0.0050 (0.00045) 0.00017 (0.00029) -0.00041, 0.00075 0.563 

 [0.0046 - 0.0065] [0.0041 - 0.0063] [-0.0014 - 0.0015] 

Copper (mg/kg DW) 2.19 (0.24) 2.16 (0.24) 0.031 (0.27) -0.56, 0.62 0.910 

 [1.88 - 2.49] [1.87 - 2.30] [-0.40 - 0.62] 

Iron (mg/kg DW) 17.67 (0.68) 18.60 (0.68) -0.93 (0.61) -2.14, 0.29 0.131 

 [16.38 - 19.27] [16.12 - 22.21] [-3.53 - 1.02] 

Magnesium (% DW) 0.13 (0.0034) 0.13 (0.0034) 0.00044 (0.0037) -0.0069, 0.0077 0.905 

 [0.11 - 0.14] [0.10 - 0.14] [-0.022 - 0.024] 

Manganese (mg/kg DW) 6.71 (0.40) 6.54 (0.40) 0.18 (0.30) -0.45, 0.80 0.565 

 [5.28 - 8.66] [5.25 - 7.77] [-1.13 - 2.28] 

Phosphorus (% DW) 0.32 (0.0099) 0.33 (0.0099) -0.0095 (0.0098) -0.029, 0.010 0.334 

 [0.25 - 0.36] [0.27 - 0.38] [-0.074 - 0.075] 

Potassium (% DW) 0.40 (0.011) 0.40 (0.011) -0.0024 (0.0082) -0.019, 0.015 0.777 

 [0.37 - 0.43] [0.37 - 0.43] [-0.038 - 0.038] 

Zinc (mg/kg DW) 23.30 (1.00) 24.37 (1.00) -1.07 (0.80) -2.66, 0.52 0.183 

 [18.36 - 26.77] [21.29 - 27.79] [-3.62 - 2.49] 

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 were set to 
Monsanto Company 07-CR-191U Page 349 of 544 

Table E-22. Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional CoSites in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Alanine (% DW) 0.78 (0.018) 0.79 (0.018) -0.010 (0.025) -0.059, 0.039 0.682 

 [0.67 - 0.85] [0.68 - 0.89] [-0.12 - 0.12] 

Arginine (% DW) 0.43 (0.012) 0.42 (0.012) 0.013 (0.017) -0.022, 0.047 0.457 

 [0.41 - 0.44] [0.34 - 0.47] [-0.058 - 0.10] 

Aspartic Acid (% DW) 0.65 (0.013) 0.65 (0.013) -0.0072 (0.017) -0.040, 0.026 0.665 

 [0.59 - 0.71] [0.59 - 0.73] [-0.090 - 0.098] 

Cystine (% DW) 0.23 (0.0043) 0.23 (0.0043) -0.00090 (0.0047) -0.010, 0.0085 0.848 

 [0.22 - 0.25] [0.20 - 0.24] [-0.021 - 0.027] 

Glutamic Acid (% DW) 2.01 (0.047) 2.03 (0.047) -0.019 (0.064) -0.15, 0.11 0.772 

 [1.74 - 2.21] [1.71 - 2.29] [-0.31 - 0.32] 

Glycine (% DW) 0.36 (0.0063) 0.36 (0.0063) 0.0018 (0.0077) -0.014, 0.017 0.818 

 [0.34 - 0.39] [0.33 - 0.39] [-0.033 - 0.037] 

Histidine (% DW) 0.31 (0.0056) 0.31 (0.0056) -0.0022 (0.0074) -0.017, 0.013 0.768 

 [0.28 - 0.32] [0.27 - 0.34] [-0.031 - 0.034] 

Isoleucine (% DW) 0.37 (0.0092) 0.37 (0.0092) -0.0065 (0.013) -0.031, 0.018 0.605 

 [0.32 - 0.38] [0.32 - 0.41] [-0.052 - 0.048] 
Monsanto Company 07-CR-191U Page 350 of 544 

Table E-22 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conve

Combined Sites in Chile during 2006/2007 under Water-Limited Conditions

Difference (Test minus Control)

Analytical 

Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Leucine (% DW) 1.36 (0.034) 1.37 (0.034) -0.011 (0.046) -0.10, 0.081 0.806 

 [1.16 - 1.47] [1.13 - 1.56] [-0.22 - 0.24] 

Lysine (% DW) 0.29 (0.0045) 0.29 (0.0045) -0.0044 (0.0055) -0.016, 0.0067 0.428 

 [0.27 - 0.31] [0.28 - 0.31] [-0.023 - 0.029] 

Methionine (% DW) 0.20 (0.0073) 0.20 (0.0073) 0.0010 (0.0065) -0.012, 0.014 0.873 

 [0.18 - 0.22] [0.16 - 0.22] [-0.023 - 0.038] 

Phenylalanine (% DW) 0.53 (0.012) 0.54 (0.012) -0.0044 (0.017) -0.038, 0.029 0.797 

 [0.46 - 0.58] [0.45 - 0.61] [-0.079 - 0.086] 

Proline (% DW) 0.96 (0.027) 0.97 (0.027) -0.011 (0.030) -0.072, 0.050 0.722 

 [0.85 - 1.04] [0.84 - 1.11] [-0.15 - 0.16] 

Serine (% DW) 0.51 (0.012) 0.51 (0.012) -0.0023 (0.016) -0.034, 0.030 0.886 

 [0.45 - 0.58] [0.43 - 0.59] [-0.087 - 0.11] 

Threonine (% DW) 0.35 (0.0069) 0.35 (0.0069) 0.0015 (0.0090) -0.016, 0.019 0.868 

 [0.32 - 0.39] [0.31 - 0.39] [-0.042 - 0.067] 

Tryptophan (% DW) 0.053 (0.0020) 0.052 (0.0020) 0.0012 (0.0025) -0.0037, 0.0060 0.639 

 [0.046 - 0.059] [0.042 - 0.063] [-0.0044 - 0.0095] 
Monsanto Company 07-CR-191U Page 351 of 544 

Table E-22 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and Conve

Combined Sites in Chile during 2006/2007 under Water-Limited Conditions

Difference (Test minus Control) 

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Tyrosine (% DW) 0.29 (0.023) 0.24 (0.023) 0.050 (0.032) -0.017, 0.12 0.133 

 [0.18 - 0.33] [0.12 - 0.35] [-0.11 - 0.16] 

Valine (% DW) 0.50 (0.011) 0.51 (0.011) -0.0078 (0.014) -0.037, 0.021 0.590 

 [0.44 - 0.51] [0.45 - 0.55] [-0.068 - 0.050] 

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 were set to 
Monsanto Company 07-CR-191U Page 352 of 544 

Table E-23. Comparison of the Fatty Acid Content in Grain from MON 87460 and Conventional Contro

in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

16:0 Palmitic (% Total FA) 11.06 (0.13) 11.18 (0.13) -0.12 (0.16) -0.45, 0.20 0.447 

 [10.54 - 11.33] [10.75 - 11.45] [-0.39 - 0.32] 

18:0 Stearic (% Total FA) 1.86 (0.045) 1.86 (0.045) -0.0062 (0.047) -0.10, 0.092 0.896 

 [1.73 - 1.95] [1.68 - 2.08] [-0.15 - 0.25] 

18:1 Oleic (% Total FA) 20.99 (0.37) 20.83 (0.37) 0.16 (0.29) -0.45, 0.77 0.589 

 [20.20 - 21.60] [19.59 - 21.98] [-1.03 - 1.33] 

18:2 Linoleic (% Total FA) 64.29 (0.45) 64.30 (0.45) -0.0089 (0.42) -0.90, 0.88 0.983 

 [63.27 - 65.10] [62.75 - 65.65] [-1.32 - 0.94] 

18:3 Linolenic (% Total FA) 1.19 (0.016) 1.21 (0.016) -0.013 (0.014) -0.041, 0.015 0.354 

 [1.13 - 1.25] [1.12 - 1.26] [-0.079 - 0.049] 

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 - 0.34] [0.30 - 0.33] [-0.025 - 0.014] 

20:1 Eicosenoic (% Total FA) 0.1763 (0.0039) 0.1845 (0.0039) -0.0082 (0.0039) -0.016, -0.00030 0.042 

 [0.16 - 0.19] [0.17 - 0.20] [-0.025 - 0.014] 

22.0 Behenic (% Total FA) 0.12 (0.020) 0.12 (0.020) 0.0022 (0.026) -0.052, 0.056 0.933 

 [0.058 - 0.20] [0.059 - 0.15] [-0.092 - 0.13] 

1FA = 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 were set to 
Monsanto Company 07-CR-191U Page 353 of 544 

Table E-24. Comparison of the Vitamin Content in Grain from MON 87460 and Conventional Control fo

Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± S.E.

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Folic Acid (mg/kg DW) 0.29 (0.018) 0.28 (0.018) 0.011 (0.016) -0.023, 0.046 0.497 

 [0.25 - 0.37] [0.23 - 0.35] [-0.062 - 0.13] Niacin (mg/kg DW) 18.54 (1.77) 21.73 (1.77) -3.18 (1.81) -6.79, 0.43 0.083  [16.23 - 25.00] [16.36 - 42.06] [-24.26 - 2.78] Thiamine HCl/Vitamin B1 (mg/kg DW) 

3.10 (0.084) 2.98 (0.084) 0.12 (0.088) -0.070, 0.30 0.203 

[2.84 - 3.42] [2.71 - 3.19] [-0.11 - 0.45] 

Riboflavin/Vitamin B2 (mg/kg DW) 

2.12 (0.14) 2.29 (0.14) -0.16 (0.14) -0.45, 0.12 0.255 

[1.43 - 2.89] [1.64 - 2.81] [-1.30 - 0.50] 

Pyridoxine HCl/Vitamin B6 (mg/kg DW) 

6.17 (0.27) 6.15 (0.27) 0.013 (0.33) -0.69, 0.71 0.969 

[5.43 - 6.57] [4.97 - 8.27] [-1.96 - 1.20] 

Vitamin E (mg/kg DW) 13.01 (0.40) 12.16 (0.40) 0.84 (0.54) -0.30, 1.99 0.135  [12.16 - 14.24] [10.15 - 13.64] [-0.45 - 2.42] 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 were set to 
Monsanto Company 07-CR-191U Page 354 of 544 

Table E-25. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain froConventional Control for Combined Sites in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical Component1

Test Mean ± S.E. 1

[Range] 

Control Mean ± 

S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Anti-nutrient 

Phytic Acid (% DW) 0.79 (0.035) 0.77 (0.035) 0.022 (0.043) -0.067, 0.11 0.612 

 [0.63 - 0.89] [0.60 - 0.89] [-0.16 - 0.27] 

Raffinose (% DW) 0.11 (0.013) 0.12 (0.013) -0.0087 (0.0050) -0.019, 0.0017 0.095 

 [0.087 - 0.14] [0.097 - 0.15] [-0.018 - 0.0025] 

Secondary Metabolite Ferulic Acid (μg/g DW) 1923.79 (114.60) 1852.11 (114.60) 71.68 (160.59) -249.18, 392.53 0.656 

 [1208.67 - 2352.27] [1088.34 - 2301.59] [-852.84 - 788.80] 

p-Coumaric Acid (μg/g DW) 137.29 (15.51) 149.45 (15.51) -12.16 (18.53) -51.13, 26.81 0.519 

 [85.52 - 168.18] [66.48 - 208.43] [-122.91 - 65.49] 

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 were set to 
Monsanto Company 07-CR-191U Page 355 of 544 

Table E-26. Comparison of the Additional Secondary Metabolite Composition of Forage from MON 874

Control for Combined-Sites from the 2006/2007 Chile Production Conducted under Well-Watered Condit

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

[9

Free Proline (% DW) 0.023 (0.0025) 0.019 (0.0025) 0.0035 (0.0018) -0.00026, 0.0073 0.066 

[0.014 - 0.031] [0.012 - 0.024] [-0.0077 - 0.014] 

Abscisic Acid (ppb FW) 37.03 (8.19) 15.66 (7.97) 21.38 (9.64) 1.01, 41.75 0.040 

[11.90 - 122.00] [10.30 - 21.70] [-5.50 - 106.90] 

Choline (ppm FW) 137.00 (6.68) 128.77 (6.68) 8.23 (6.28) -4.99, 21.46 0.207 

[114.00 - 159.00] [94.90 - 145.00] [-10.00 - 36.00] 

Glycerol (% DW) 0.16 (0.0085) 0.14 (0.0085) 0.019 (0.0097) -0.00090, 0.039 0.060 

[0.11 - 0.21] [0.12 - 0.18] [-0.023 - 0.048] 

Glycine Betaine (ppm FW) 84.24 (9.15) 76.27 (9.15) 7.98 (11.23) -15.64, 31.59 0.486 

[66.40 - 104.00] [55.60 - 91.00] [-17.40 - 24.90] 

Salicylic Acid (ppm DW) 0.14 (0.049) 0.19 (0.049) -0.046 (0.045) -0.14, 0.051 0.327 

[0.072 - 0.21] [0.060 - 0.30] [-0.15 - 0.060] 

Fructose (% DW) 8.56 (1.15) 9.06 (1.15) -0.50 (0.89) -2.38, 1.38 0.581 

[6.74 - 10.29] [7.63 - 9.80] [-1.86 - 0.77] 

Glucose (% DW) 9.22 (1.07) 9.68 (1.07) -0.45 (0.96) -2.48, 1.58 0.642 

[7.31 - 10.43] [7.63 - 11.04] [-2.26 - 0.87] 

Sucrose (% DW) 0.46 (1.07) 0.86 (1.07) -0.40 (1.11) -2.61, 1.82 0.722 

[0.10 - 1.03] [0.094 - 2.35] [-1.58 - 0.46] 

1DW = dry weight; FW = fresh 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 were 
Monsanto Company 07-CR-191U Page 356 of 544 

Table E-27. Comparison of the Additional Secondary Metabolite Composition of Grain from MON 874

Control for Combined-Sites from the 2006/2007 Chile Production Conducted under Well-Watered Condit

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

C

[99% 

Free Proline (% DW) 0.058 (0.011) 0.055 (0.011) 0.0026 (0.0035) -0.0048, 0.0099 0.476 (0.

[0.029 - 0.090] [0.030 - 0.083] [-0.0036 - 0.010] 

Abscisic Acid (ppb FW) 9.73 (2.00) 11.49 (2.00) -1.76 (2.71) -7.16, 3.63 0.517 (9

[3.61 - 20.70] [7.24 - 21.90] [-10.40 - 8.40] 

Choline (ppm FW) 219.89 (13.09) 235.78 (13.09) -15.89 (10.18) -37.49, 5.71 0.138 (17

[181.00 - 255.00] [203.00 - 265.00] [-48.00 - 33.00] [12

Glycerol (% DW) 0.023 (0.0035) 0.022 (0.0035) 0.0013 (0.0024) -0.0035, 0.0061 0.595 (0

[0.020 - 0.029] [0.017 - 0.030] [-0.0017 - 0.0051] 

Glycine Betaine (ppm FW) 2.27 (0.33) 2.41 (0.33) -0.14 (0.26) -0.70, 0.41 0.588 (

[1.31 - 3.11] [1.32 - 4.19] [-1.08 - 0.36] 

Salicylic Acid (ppm FW) 0.088 (0.018) 0.094 (0.018) -0.0060 (0.016) -0.039, 0.027 0.705 (0

[0.065 - 0.12] [0.069 - 0.11] [-0.030 - 0.030] 

Fructose (% DW) 0.44 (0.029) 0.44 (0.029) -0.0074 (0.034) -0.078, 0.063 0.830 (

[0.34 - 0.50] [0.25 - 0.53] [-0.19 - 0.24] 

Glucose (% DW) 0.46 (0.029) 0.48 (0.029) -0.018 (0.035) -0.091, 0.054 0.607 (

[0.35 - 0.55] [0.34 - 0.56] [-0.20 - 0.069] [0

Sucrose (% DW) 1.77 (0.17) 1.82 (0.17) -0.053 (0.076) -0.21, 0.11 0.490 (

[1.47 - 2.49] [1.40 - 2.47] [-0.33 - 0.085] [

1DW = dry weight; FW = fresh 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 were set t
Monsanto Company 07-CR-191U Page 357 of 544 

Table E-28. Comparison of the Additional Secondary Metabolite Composition of Forage from MON 874

Control for Combined-Sites from the 2006/2007 Chile Production Conducted under Water-Limited Condi

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

C

[99% 

Free Proline (% DW) 0.018 (0.0025) 0.018 (0.0025) -0.00028 (0.0018) -0.0041, 0.0035 0.876 (0

 [0.013 - 0.027] [0.011 - 0.025] [-0.0092 - 0.0069] 

Abscisic Acid (ppb FW) 31.60 (7.97) 29.42 (7.97) 2.18 (9.46) -17.91, 22.27 0.820 (1

 [20.40 - 54.30] [15.80 - 56.60] [-31.10 - 31.80] 

Choline (ppm FW) 155.11 (6.68) 145.89 (6.68) 9.22 (6.28) -4.00, 22.45 0.159 (11

 [134.00 - 181.00] [136.00 - 163.00] [-13.00 - 34.00] [6

Glycerol (% DW) 0.14 (0.0085) 0.14 (0.0085) -0.0038 (0.0097) -0.024, 0.016 0.697 (

 [0.11 - 0.17] [0.12 - 0.17] [-0.029 - 0.042] [

Glycine Betaine (ppm FW) 116.80 (9.15) 119.79 (9.15) -2.99 (11.23) -26.61, 20.63 0.793 (7

 [73.20 - 138.00] [89.60 - 176.00] [-64.00 - 32.50] 

Salicylic Acid (ppm FW) 0.21 (0.049) 0.24 (0.049) -0.024 (0.045) -0.12, 0.073 0.607 (

 [0.10 - 0.58] [0.12 - 0.49] [-0.39 - 0.34] 

Fructose (% DW) 11.12 (1.15) 10.91 (1.15) 0.21 (0.89) -1.67, 2.09 0.813 (

 [7.77 - 20.17] [7.52 - 14.95] [-2.41 - 7.53] [

Glucose (% DW) 12.31 (1.07) 11.87 (1.07) 0.43 (0.96) -1.59, 2.46 0.655 (

 [8.60 - 20.87] [8.60 - 15.05] [-2.82 - 8.81] [

Sucrose (% DW) 2.27 (1.07) 1.88 (1.07) 0.39 (1.11) -1.82, 2.60 0.724 (

 [0.10 - 6.36] [0.11 - 4.07] [-3.62 - 4.83] 

1DW = dry weight; FW = fresh 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 were set t
Monsanto Company 07-CR-191U Page 358 of 544 

Table E-29. Comparison of the Additional Secondary Metabolite Composition of Grain from MON 874

Control for Combined-Sites from the 2006/2007 Chile Production Conducted under Water-Limited Condi

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

C

[99% 

Free Proline (% DW) 0.051 (0.011) 0.058 (0.011) -0.0063 (0.0035) -0.014, 0.0011 0.089 (0

 [0.029 - 0.076] [0.029 - 0.079] [-0.027 - 0.016] 

Abscisic Acid (ppb FW) 11.43 (2.00) 13.54 (2.00) -2.11 (2.71) -7.50, 3.29 0.438 (7

 [8.78 - 17.70] [6.79 - 23.90] [-15.12 - 7.01] 

Choline (ppm FW) 238.11 (13.09) 241.56 (13.09) -3.44 (10.18) -25.04, 18.16 0.739 (20

 [191.00 - 308.00] [209.00 - 284.00] [-45.00 - 76.00] [10

Glycerol (% DW) 0.030 (0.0035) 0.029 (0.0035) 0.00069 (0.0024) -0.0041, 0.0055 0.776 (0

 [0.023 - 0.049] [0.018 - 0.043] [-0.020 - 0.017] 

Glycine Betaine (ppm FW) 2.21 (0.33) 1.99 (0.33) 0.21 (0.26) -0.34, 0.77 0.421 (

 [1.52 - 3.24] [1.18 - 3.98] [-2.22 - 1.68] 

Salicylic Acid (ppm FW) 0.11 (0.018) 0.12 (0.018) -0.0026 (0.016) -0.036, 0.031 0.871 (

 [0.073 - 0.19] [0.084 - 0.15] [-0.074 - 0.060] 

Fructose (% DW) 0.47 (0.029) 0.48 (0.029) -0.011 (0.034) -0.082, 0.059 0.739 (

 [0.37 - 0.60] [0.38 - 0.63] [-0.26 - 0.19] 

Glucose (% DW) 0.48 (0.029) 0.50 (0.029) -0.015 (0.035) -0.087, 0.058 0.671 (

 [0.38 - 0.59] [0.39 - 0.64] [-0.26 - 0.17] 

Sucrose (% DW) 1.63 (0.17) 1.86 (0.17) -0.23 (0.076) -0.39, -0.069 0.008 (

 [1.33 - 1.86] [1.37 - 2.27] [-0.72 - 0.17] 

1DW = dry weight; FW = fresh 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 were set t
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/ 

Component1

Literature 

Range2 

ILSI 

Range3 

Forage 

Proximates (% DW) 

Ash 2.43-9.64a; 2-6.6b 1.527 – 9.638 

Carbohydrates 83.2-91.6b; 76.5-87.3a 76.4 – 92.1 

Fat, total 0.35-3.62b; 1.42-4.57a 0.296 – 4.570 

Moisture (% fw) 56.5-80.4a;55.3-75.3b 49.1 – 81.3 

Protein 4.98-11.56a 3.14 – 11.57 

 

Fiber (% DW) 

Acid detergent fiber (ADF) 18.3-41.0b; 17.5-38.3a 16.13 – 47.39 

Neutral detergent fiber (NDF) 26.4-54.5b; 27.9-54.8a 20.29 – 63.71 

 

Minerals (% DW) 

Calcium 0.0969-0.3184b 0.0714 – 0.5768 

Phosphorous 0.1367-0.2914b 0.0936 – 0.3704 

 

Grain

Proximates (% DW) 

Ash 1.1-3.9d; 0.89-6.28b 0.616 – 6.282 

Carbohydrates 77.4-87.2b; 82.2-88.1a 77.4 – 89.5 

Fat, total 3.1-5.7d; 2.48-4.81b 1.742 – 5.823 

Moisture (% FW) 7-23d; 8.18-26.2b 6.1 – 40.5 

Protein 6-12d; 9.7-16.1c 6.15 – 17.26 

 

Fiber (% dw) 

Acid detergent fiber (ADF) 3.3-4.3d; 2.46-11.34a,b 1.82 – 11.34 

Neutral detergent fiber (NDF) 8.3-11.9d; 7.58-15.91b 5.59 – 22.64 

Total dietary fiber (TDF) 10.99-11.41h 8.82 – 35.31 

 

Minerals 

Calcium (% DW) 0.01-0.1d 0.00127 – 0.02084 

Copper (mg/kg DW) 0.9-10d 0.73 – 18.50 

Iron (mg/kg DW) 1-100d 10.42 – 49.07 

Magnesium (% DW) 0.09-1d 0.0594 – 0.194 

Manganese (mg/kg DW) 0.7-54d 1.69 – 14.30 

Phosphorus (% DW) 0.26-0.75d 0.147 – 0.533 

Potassium (% DW) 0.32-0.72d 0.181 – 0.603 

Zinc (mg/kg DW) 12-30d 6.5 – 37.2 

 
Monsanto 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/ 

Component1

Literature 

Range2 

ILSI 

Range3 

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 7-19e 7.94 – 20.71 

16:1 Palmitoleic 1e 0.095 – 0.447 

18:0 Stearic 1-3e 1.02 – 3.40 

18:1 Oleic 20-46e 17.4 – 40.2 

18:2 Linoleic 35-70e 36.2 – 66.5 

18:3 Linolenic 0.8-2e 0.57 – 2.25 

20:0 Arachidic 0.1-2e 0.279 – 0.965 

20:1 Eicosenoic - 0.170 – 1.917 

22:0 Behenic - 0.110 – 0.349 

 

Vitamins (mg/kg DW) 

Folic acid 0.3d 0.147 – 1.464 

Niacin 9.3-70d 10.37 – 46.94 

Vitamin B1 3-8.6e 1.26 – 40.00 

Vitamin B2 0.25-5.6e 0.50 – 2.36 

Vitamin B6 5.3d; 9.6e 3.68 – 11.32 

Vitamin E 3-12.1e; 17-47d 1.5 – 68.7 
Monsanto Company 07-CR-191U Page 361 of 544 

Table E-30 (cont.). Literature and Historical Ranges of Components of Corn 

Forage and Grain

Tissue/ 

Component1

Literature 

Range2 

ILSI 

Range3 

Grain 

Anti-nutrients (% DW) 

Phytic acid 0.48-1.12a 0.111 – 1.570 

Raffinose 0.08-0.30e 0.020 – 0.320 

 

Secondary Metabolites 

 (μg/g dw) 

 

Ferulic acid 113-1194f; 3000g 291.9 – 3885.8 

p-Coumaric acid 22-75f 53.4 – 576.2 

1

FW=fresh weight; DW=dry weight; Niacin =Vitamin B3; Vitamin B1 =Thiamine; Vitamin B2

=Riboflavin; Vitamin B6 =Pyridoxine ; N/A = not available as percent dry weight. 

2

Literature range references: aRidley et al., 2002. bSidhu et al., 2000. cJugenheimer, 1976. 

d

Watson, 1987. eWatson, 1982. fClassen et al., 1990. gDowd and Vega, 1996. hChoi et al., 1999. 

3

ILSI range is from ILSI CCD, 2006. 

Conversions: % DW x 104 = μg/g dw; mg/g dw x 103 = mg/kg DW; mg/100g dw x 10 = mg/kg 

DW
Monsanto 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 collected from MON 87460 

grown at an individual site (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 analyte results were as expected from the 

respective assays and were similar to reference and published ranges for conventional 

corn. No unexpected compositional values for any components 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, ranges, and statistical 

analyses data are presented in (Tables E-33 through E-39) for the well-watered treatment 

and Tables E-40 through E-46) for the water-limited treatment. 

A summary of significant differences (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 544 

Table E-31. Monthly Temperature and Monthly Accumulated Water Data for the QUI Site in the Chile 2Site1 Measurement December January February March ApQUI 

Accumulated water (in.), well-watered 1.9 10.3 8.5 9.4 1Accumulated water (in.), water-limited1, 2 1.9 10.3 1.9 5.6 1

Avg Max temp (°F) NA4 83 79 77 7

Avg Min temp (°F) NA4 51 51 49 4Range3 (°F) NA4 48 - 92 44 - 90 41 - 93 34 - 

1 Water limitation began at the V10 growth stage which occurred at approximately February 7. 2 Water limitation ended at the R2 growth stage which occurred at approximately March 13. 3 The range is the absolute maximum and minimum temperature in each month. 4 Temperature 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 364 of 544 

Table E-32. Summary of Significant Differences (p<0.05) Comparing MON 87460 to the Conventional CSite in Chile during 2006/2007 under Well-Watered and Water-Limited Conditions 

Tissue/Site/ 

Components (Units)1

Mean 

MON 87460 

Mean 

Control 

Mean Diff 

(% of Control) 

Signif. 

(p-value) 

MON 87460 

(Range) 

ForageWater-LimitedMoisture (% FW) 73.53 76.57 -3.96 0.019 (72.40 - 75.30) 

Protein (% DW) 5.78 6.69 -13.68 0.013 (5.25 - 6.57) 

Total Fat (% DW) 1.44 0.82 75.70 0.049 (0.58 - 2.34) Grain

Well-watered

Moisture (% FW) 13.63 8.48 60.83 <0.001 (13.10 - 14.2) 

Magnesium (% DW) 0.12 0.11 7.86 0.043 (0.12 - 0.13) 

Phosphorus (% DW) 0.32 0.30 7.63 0.047 (0.31 - 0.34) 16:0 Palmitic (% Total FA) 11.06 11.38 -2.77 0.017 (10.92 - 11.29) 

Vitamin E (mg/kg DW) 11.63 9.85 18.09 0.023 (11.06 - 12.47) 

Water-LimitedCarbohydrates (% DW) 85.95 85.21 0.87 0.038 (85.69 - 86.33) Moisture (% FW) 13.60 8.72 55.96 <0.001 (12.70 - 14.10) Protein (% DW) 8.69 9.24 -5.90 0.043 (8.24 - 8.99) Neutral Detergent Fiber (% DW 9.65 10.89 -11.36 0.042 (8.85 - 10.54) 

Copper (mg/kg DW) 1.61 4.22 -61.82 0.032 (1.53 - 1.65) 

Iron (mg/kg DW) 14.35 15.48 -7.29 0.037 (13.75 - 14.77) Alanine (% DW) 0.62 0.70 -11.03 0.006 (0.59 - 0.65) Aspartic Acid (% DW) 0.56 0.60 -7.42 0.018 (0.54 - 0.58) 

Glutamic Acid (% DW) 1.60 1.76 -9.04 0.020 (1.50 - 1.66) Isoleucine (% DW) 0.29 0.33 -10.09 0.007 (0.28 - 0.31) 

Leucine (% DW) 1.04 1.16 -10.50 0.015 (0.97 - 1.09) Lysine (% DW) 0.27 0.29 -5.44 0.037 (0.27 - 0.27) 

Phenylalanine (% DW) 0.43 0.47 -8.79 0.022 (0.40 - 0.44) Valine (% DW) 0.43 0.46 -7.50 0.018 (0.41 - 0.44) 

1DW= dry weight; FW=fresh weight, FA= fatty acid. 
Monsanto Company 07-CR-191U Page 365 of 544 

Table E-33. Comparison of Proximates, Fiber, and Mineral Content in Forage from MON 87460 and Cfrom the QUI Site in Chile during 2006/2007 under Well-Watered Conditions 1DW = dry weight; FW = fresh 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 s

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range]

95% CI1

(Lower,Upper) p-Value 

Fiber Acid 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] 

Neutral Detergent Fiber (% 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] 

Mineral Calcium (% 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] 

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] 

Proximate Ash (% 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] 

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] 

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] 

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] 

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] 
Monsanto Company 07-CR-191U Page 366 of 544 

Table E-34. Comparison of the Proximates and Fiber Content in Grain from MON 87460 and Conventio

QUI Site in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Proximates Ash (% 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] 

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] 

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] 

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] 

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] 

Fiber Acid 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] 

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] 

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] 

1DW = dry weight; FW = fresh 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 s
Monsanto Company 07-CR-191U Page 367 of 544 

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

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Calcium (% DW) 0.0047 (0.00021) 0.0047 (0.00021) 0 (0.00022) -0.00049, 0.00047 0.966 

 [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 

 [1.54 - 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 

 [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 

 [0.12 - 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 

 [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 

 [0.31 - 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 

 [0.38 - 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 

 [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 s
Monsanto Company 07-CR-191U Page 368 of 544 

Table E-36. Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Contrin Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

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] 
Monsanto Company 07-CR-191U Page 369 of 544 

Table E-36 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and ConventionQUI Site in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

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] 
Monsanto Company 07-CR-191U Page 370 of 544 

Table E-36 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and ConventionQUI Site in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control) 

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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 
Monsanto Company 07-CR-191U Page 371 of 544 

Table E-37. Comparison of the Fatty Acid Content in Grain from MON 87460 and Conventional Control 

Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

1FA = fatty acid; 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 s
Monsanto Company 07-CR-191U Page 372 of 544 

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

Difference (Test minus Control)

Analytical Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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 s
Monsanto Company 07-CR-191U Page 373 of 544 

Table E-39. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain froConventional Control from the QUI Site in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Anti-nutrient Phytic 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] 

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] 

Secondary Metabolite Ferulic 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] 

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] 

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 s
Monsanto Company 07-CR-191U Page 374 of 544 

Table E-40. Comparison of Proximates, Fiber, and Mineral Content in Forage from MON 87460 and Cfrom the QUI Site in Chile during 2006/2007 under Water-Limited Conditions 1DW = dry weight; FW = fresh 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 s

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Fiber Acid 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] 

Neutral Detergent Fiber (% 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] 

Mineral Calcium (% 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] 

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] 

Proximate Ash (% 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] 

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] 

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] 

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] 

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] 
Monsanto Company 07-CR-191U Page 375 of 544 

Table E-41. Comparison of the Proximates and Fiber Content in Grain from MON 87460 and Conventio

QUI Site in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Proximates Ash (% 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] 

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] 

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] 

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] 

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] 

Fiber Acid 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] 

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] 

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] 

1DW = dry weight; FW = fresh 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 s
Monsanto Company 07-CR-191U Page 376 of 544 

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

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

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 s
Monsanto Company 07-CR-191U Page 377 of 544 

Table E-43. Comparison of the Amino Acid Content in Grain from MON 87460 and Conventional Contrin Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

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] 
Monsanto Company 07-CR-191U Page 378 of 544 

Table E-43 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and ConventionQUI Site in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

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] 
Monsanto Company 07-CR-191U Page 379 of 544 

Table E-43 (cont). Comparison of the Amino Acid Content in Grain from MON 87460 and ConventionQUI Site in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control) 

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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 s
Monsanto Company 07-CR-191U Page 380 of 544 

Table E-44. Comparison of the Fatty Acid Content in Grain from MON 87460 and Conventional Control 

Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

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] 

1FA – fatty acid; 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 s
Monsanto Company 07-CR-191U Page 381 of 544 

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

Difference (Test minus Control)

Analytical Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

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] 

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] 

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] 

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] 

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] 

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] 

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 s
Monsanto Company 07-CR-191U Page 382 of 544 

Table E-46. Comparison of the Anti-nutrient and Secondary Metabolite Content in Grain froConventional Control from the QUI Site in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Anti-nutrient Phytic 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] 

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] 

Secondary Metabolite Ferulic 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] 

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] 

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 s
Monsanto Company 07-CR-191U Page 383 of 544 

Table E-47. Summary of Significant Differences in Additional Secondary Metabolite Composition MON 87460 to the Conventional Control from the QUI Site in Chile during 2006/2007 under Well-WLimited Conditions 

Tissue/Site/ 

Components (Units)1

Mean 

MON 87460 

Mean 

Control 

Mean Diff 

(% of Control) 

Signif. 

(p-value) 

MON 87460 

(Range) 

ForageWell-wateredFructose (% DW) 5.30 6.95 -23.77 0.005 (4.40 - 6.31) Glucose (% DW) 6.09 7.73 -21.18 0.005 (5.22 - 6.96) Water-Limited

Glucose (% DW) 6.10 7.53 -18.94 0.012 (5.55 - 6.81) 

GrainWell-wateredAbscisic Acid (ppb FW) 10.80 18.93 -42.94 0.001 (9.41 - 12.60) Glucose (% DW) 0.47 0.57 -17.65 0.021 (0.46 - 0.48) Sucrose (% DW) 1.81 2.22 -18.67 0.002 (1.76 - 1.85) Water-LimitedAbscisic Acid (ppb FW) 8.04 23.13 -65.24 <0.001 (7.02 - 8.66) Sucrose (% DW) 1.84 2.10 -12.24 0.040 (1.67 - 2.08) 

1DW= dry weight; FW=fresh weight. 
Monsanto Company 07-CR-191U Page 384 of 544 

Table E-48. Comparison of the Additional Secondary Metabolite Content in Forage from MON 8746Control from the QUI Site in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Free Proline (% DW) 0.020 (0.0017) 0.018 (0.0017) 0.0019 (0.0023) -0.0030, 0.0069 0.422 

 [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 

 [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 

 [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 

 [0.14 - 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 

 [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 

 [0.088 - 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 

 [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 

 [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 

 [2.32 - 4.74] [2.08 - 2.93] [-0.25 - 2.25] 1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval. 
Monsanto Company 07-CR-191U Page 385 of 544 

Table E-49. Comparison of the Additional Secondary Metabolite Content in Grain from MON 8746Control from the QUI Site in Chile during 2006/2007 under Well-Watered Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Free Proline (% DW) 0.072 (0.0030) 0.076 (0.0030) -0.0041 (0.0039) -0.012, 0.0042 0.313 

 [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 

 [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 

 [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 - 0.022] [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 

 [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 

 [0.076 - 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 

 [0.44 - 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 

 [0.46 - 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 

 [1.76 - 1.85] [2.10 - 2.38] [-0.53 - -0.30] 1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval. 
Monsanto Company 07-CR-191U Page 386 of 544 

Table E-50. Comparison of the Additional Secondary Metabolite Content in Forage from MON 8746Control from the QUI Site in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Free Proline (% DW) 0.024 (0.0017) 0.020 (0.0017) 0.0040 (0.0023) -0.00095, 0.0089 0.106 

 [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 

 [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 

 [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 

 [0.15 - 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 

 [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 

 [0.073 - 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 

 [5.00 - 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 

 [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 

 [1.14 - 4.26] [1.49 - 5.56] [-3.96 - 2.14] 1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval. 
Monsanto Company 07-CR-191U Page 387 of 544 

Table E-51. Comparison of the Additional Secondary Metabolite Content in Grain from MON 8746Control from the QUI Site in Chile during 2006/2007 under Water-Limited Conditions 

Difference (Test minus Control)

Analytical 

Component1

Test 

Mean ± S.E. 1

[Range] 

Control 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower,Upper) p-Value 

Free Proline (% DW) 0.068 (0.0030) 0.073 (0.0030) -0.0045 (0.0039) -0.013, 0.0038 0.268 

 [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 

 [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 

 [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 

 [0.018 - 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 

 [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 

 [0.087 - 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 

 [0.41 - 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 

 [0.45 - 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 

 [1.67 - 2.08] [1.96 - 2.18] [-0.51 - -0.086] 1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval. 
Monsanto Company 07-CR-191U Page 388 of 544 

Appendix F. Phenotypic and Agronomic Assessment: Individual Site Results from 

2006 and 2007 Field Studies 

F.1. Materials 

The materials for phenotypic, agronomic, and 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 were 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 County, 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 established at four sites: Calera de Tango, 

Region Metropolitana de Santiago (CT); Colina, 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 studies 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); York 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 mean values, was determined from the 

references among the sites. The means of the test and control substance and the results 

of the statistical analyses are reported. Standard 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 lodged plants, root lodged plants, final stand 

count, grain moisture percentage, test weight, insect abundance, 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 characteristics evaluated at eight wellwatered sites in 2006 are presented in Table F-14. In the individual site analyses, no 

differences between MON 87460 and the control were detected for seedling vigor, 

days to 50% pollen shed, days to 50% silking, 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 early stand count than the control at IL1 

(104.3 vs. 86.0) and a lower early stand count than the control 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 lodged plants than the control at NE (9.0 

vs. 5.0) and more root lodged plants than the control at IL1 (23.7 vs. 2.7). 

MON 87460 had a higher final stand than the 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 early 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 control (Figure VIII-1, Step 2). The results 

of this study support a conclusion that the introduction of the drought tolerance trait 

did not unexpectedly alter the assessed phenotypic characteristics of MON 87460 

compared to conventional corn. Thus, the results support a conclusion of no 

increased pest potential or significant environmental 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 control were detected for seedling vigor, 

early stand count, days to 50% pollen shed, days to 50% silking, ear height, plant 

height, root lodged plants, final stand count, test weight, and yield. Eight differences 

were detected in the individual site analysis 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 than the control at IA2 (18.0 

vs. 17.1, respectively), but was lower in grain moisture than the control at IL3 (13.0 

vs. 14.7, respectively). Differences in stay 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 response associated with the trait and are unlikely to be 

biologically meaningful in terms of increased pest potential of MON 87460 compared 

to the control (Figure VIII-1, Step 2). The results of this study support a conclusion 

that the introduction of the drought tolerance trait did not unexpectedly alter the 

assessed phenotypic characteristics of MON 87460 compared to conventional corn 

and a conclusion of no increased pest potential or significant environmental impact 

of MON 87460 compared to conventional corn under well watered conditions. 

F.4.3. 2006/2007 Chilean Field Study Established 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-watered 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, root lodged plants, final 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 all three sites (Table VIII-7). Thus, the 
Monsanto Company 07-CR-191U Page 391 of 544 

differences detected for these phenotypic characteristics are not indicative of a 

consistent response associated with the trait and are unlikely to be biologically 

meaningful in terms of increased 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 benefit of this trait and was also detected in 

the combined-site analysis (Table VIII-7). Furthermore, statistical differences in ear 

height and plant height are expected differences between MON 87460 and the control 

under water-limited conditions (Figure VIII-2). 

The results of this study support a conclusion 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 Established with Well-Watered and Water-Limited 

Treatments 

The results of the 14 phenotypic or agronomic characteristics 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 differences between MON 87460 and control were 

detected for seedling vigor, days to 50% pollen shed, days to 50% silking, ear height, 

dropped ears, root lodged plants, final stand count, grain moisture, and test weight 

(TX site only) in the well-watered treatment. Five comparisons between MON 87460 

and the control were significantly different in the individual-site analysis under wellwatered conditions. At the CA site, MON 87460 had higher 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 MON 87460 had more yield than the control 

(239.7 vs. 181.4 bu/acre). At the TX site, 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 plants) 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 Table 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 yield, stay green and plant height were not 

detected in the combined-site analysis for the well-watered treatment (Table VIII-8). 

Therefore, the differences detected in the individual site analysis are unlikely to be 

adverse and do not contribute to a biological or environmental change for 

MON 87460 in terms of pest potential. 

The results of the 14 phenotypic or agronomic characteristics 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 differences between MON 87460 and the control were 

detected 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 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 significantly different in 

the individual-site analysis under water-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 difference detected in stay green is not 

interpreted as a characteristic that would confer 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 analysis 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 Established 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 differences between MON 87460 and control were 

detected 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 lodged plants, 

final stand count, test weight, and yield in the well-watered treatment. Three 

comparisons between MON 87460 and the control were significantly different in the 

individual-site analysis under well-watered 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 reference 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 (Figure 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 met 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 conclusion that MON 87460 was not unexpectedly 

altered for the assessed phenotypic characteristics compared to conventional corn 

under water-limited conditions. Thus, the results support a conclusion of no 

increased pest potential or significant environmental impact of MON 87460 

compared to a conventional control under well-watered or water-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 phenotypic or agronomic characteristics evaluated at five sites 

where water was managed by local agronomic practices in 2006 are presented in 

Table F-21. In the individual site analysis, no differences were detected between 

MON 87460 and the control at any site for early stand count, days to 50% pollen 

shed, stay green rating, ear height, plant height, dropped ears, final 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 exhibited 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 stalk 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, stalk lodging and root lodging were not 

detected when the data were pooled across all five sites (Table VIII-12). Thus, the 

differences detected for these 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 544 

Table F-1. Field Phenotypic Evaluation Sites for MON 87460 during 2006 and 2007 Water Management Study Year Location 

Site Code 

Wellwatered 2006 Jefferson Co., Iowa IA1 (randomized complete block desi

g

n) 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 desi

g

n) 2007 Jefferson Co., Iowa IA1 

 Van Horne Co., Iowa IA2 

 Star

k Co., Illinois IL1 

 Warren Co., Illinois IL2 

 Clinton Co., Illinois IL3  Boone Co., Indiana IN 

 York Co., Nebraska

NE 

 Fa

yette Co., Ohio OH 

 Berks Co., Penns

ylvania PA 

Well-watered and water-limite

d 2006/2007 Calera de Tan

go, Chile1 CT 

(stri

p-plot desi

g

n) Colina, Chile1 CL 

 Lumbreras, Chile1 LUM 

Quillota, Chile1 QUI 

(split-plot desi

g

n) 2007 Stud

y-1 Sutter Co., California CA 

 Carson Co., Texas TX 

(stri

p-plot desi

g

n) 2007 Stud

y-2 Pawnee Co., Kansas CA 

 York Co., Nebraska

NE 

 Carson Co., Texas TX 

Typical a

gronomic 

practices, 2006 Benton Co., Iowa IAE 

(rainfed and normal irri

gation) Greene Co., Iowa IAW 

(randomized complete block desi

g

n) Stark Co., Illinois IL 

 Parke Co., Indiana IN 

 York Co., Nebraska

NE 
Monsanto 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 Names1 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, and Reference Substances in the 

2007 U.S. Field Study under Well-Watered Conditions 

Corn Hybrid Names1

 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 Water-Limited Treatments 

Corn Hybrid Names1 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 commercial 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 Water-Limited Treatments 

Corn Hybrid Names1 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 are 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 Established with Well-Watered and WaterLimited Treatments 

Corn Hybrid Names1 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 designations

 
Monsanto 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 

Names1 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 544 

Table F-8. Test Site Locations, Planting Information, Soil Description, and Cropping History in a 2006 F

Well-Watered Conditions 

Study site1

Planting 

date

(m/dd/yy)

Planting 

rate

(seeds/row)2

Planting 

depth

(in)

Plot size 

(ft) Reps3 Soil series; organic matter; soil pH

IA1 5/26/06 40 1.5 10 x 20 3 Silty clay loam; 3.6%; 7.7 IA2 5/19/06 40 2.0 10 x 20 3 Silty clay loam; 3-4%; 6.2 IL1 5/25/06 60 1.5 20 x 30 3 Plano silt loam; 3.5%; 6.2 IL2 5/23/06 40 2.0 10 x 20 3 Muscatine silt loam; 3.6%; 6.1 IN1 5/23/06 40 1.5-1.8 10 x 20 3 Silt loam; 2.1%; 6.9 

IN2 6/06/06 40 1.5 10 x 20 3 Reesville silt loam; 2.0%; 6.9 

KS 5/17/06 60 1.5 20 x 30 3 Lubbock silt Loam; 1.2%; 7.9 

NE 5/13/06 60 1.5 20 x 30 3 Hastings silt loam; 3.0%; 6.6 

1 Study sites: IA1 = Jefferson County, IA; IA2 = Benton, County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IN1 = Boon

County, IN; KS = Pawnee County, KS, NE = York County, NE. 

2 After seedling vigor and early stand count data were collected at each site, all plots were thinned to a uniform density. 3 Reps = replications. 
Monsanto Company 07-CR-191U Page 401 of 544 

Table F-9. Test Site Locations, Planting Information, Soil Description, and Cropping History in 2007 Uunder Well-Watered Conditions Study Site1

Planting 

date 

(m/dd/yy)

Planting 

rate2

(seed/row)

Planting

depth 

(in)

Plot size

(ft) Reps3 Soil series; organic matter (%); and pH

2006 crop

IA1 5/11/07 60 1.8 10 x 30 3 Tainter-Mahska silty clay loam; 3.9%; 6.5 SoybIA2 5/15/07 40 2.0 10 x 20 3 Tama silty clay loam; 2.8%; 6.3 Soyb

IL1 5/16/07 60 1.8 10-13 x 30 3 Sawmill silt loam; 3.4%; 6.3 Corn 

IL2 5/17/07 40 1.8 10 x 20 3 Sable silty clay loam; 6%; 5.6-7.3 Fallo

IL3 5/17/07 40 1.5 10 x 20 3 Cisne silt loam; 1.5-2.0%; 6.5-7.0 Corn IN 5/16/07 60 1.5 10 x 30 3 Crosby silt loam; 2.1%; 6.9 Corn NE 5/11/07 40 2.0 10 x 20 3 Hastings silt loam; 3.0%; 6.1 SoybOH 5/21/07 40 1.5 10 x 20 3 Millsdale silt loam; 2.0%; 7.1 Corn PA 5/22/07 40 1.5 10 x 25 3 Philo Atkin silt loam; 2.4%; 5.8 Fallo

1 Study sites: IA1 = Jefferson County, IA; IA2 = Van Horne County, IA; IL1 = Wyoming County, IL; IL2 = Monmouth County, IL; 

IN = Sheridan County, IN; NE = York County, NE; OH = Fayette County, OH; PA = Hamburg County, PA. 2 After seedling vigor and early stand count data were collected at each site, all plots were thinned to a uniform density. 

3 Reps = replications. 
Monsanto Company 07-CR-191U Page 402 of 544 

Table F-10. Test Site Locations, Planting Information, Soil Description, and Cropping History in a 200Study Established with Well-Watered and Water-Limited Treatments Study site 1

Planting 

date 

(mm/dd/yy)

Planting 

rate 

(seeds/row)2

Planting 

depth 

(in)

Plot size 

(ft) Reps3 Soil series; organic matter; pH

20

CL 12/19/06 42

2 16.4 x 19.7 3 Clay; 3 – 5%; 5.5 – 6.5 Ta

gr

CT 12/15/06 42

2 16.4 x 19.7 3 Clay; 4 – 5%; 7 – 9 O

LUM 12/15/06 42

2 16.4 x 19.7 3 Sandy clay; 3 – 5%; 5.5 – 6.5 Pu

QUI† 12/16/06 42

2 16.4 x 19.7 3 Clay; 3 – 5% ; 5.5 – 6.5

Swchcibe

1 Study sites: CL = Colina; CT = Calera de Tango; LUM = Lumbreras; QUI = Quillota. All sites were managed as well-watered and w†Site QUI did not meet defined non-stress / stress criteria. Data are reported in Appendix G. 2 After seedling vigor and early stand count data were collected, all plots were thinned to approximately 40 plants per row. 3 Reps = replications. 
Monsanto Company 07-CR-191U Page 403 of 544 

Table F-11. Study-1, Test Site Locations, Planting Information, Soil Description, and Cropping Histo

Studies Established with Well-Watered and Water-Limited Treatments 

Study Site1

Planting

date

(m/dd/yy)

Planting 

rate

(seeds/row)2

Planting 

depth

(in)

Plot size 

(ft) Reps3 Soil series; organic matter; pH

2c

CA 5/22/07 40 1.0-2.0 10 x 20

4 Subaco clay loam; 1.9%; 7.0 F

TX 5/31/07 60 0.5-0.8 20 x 30

4 Pullman loam; 2.3%; 7.2 S

1 Study sites: CA = Sutter County, CA; TX = Carson, County, TX 2 After seedling vigor and early stand count data were collected at each site, all plots were thinned to a uniform density. 3 Reps = replications. Table F-12. Study-2, Test Site Locations, Planting Information, Soil Description, and Cropping Histor

Studies Established with Well-Watered and Water-Limited Treatments 

Study 

Site1

Planting date 

(m/dd/yy) 

Planting rate 

(seeds/row)2

Planting depth 

(in) 

Plot size 

(ft) Reps3 Soil Series; organic matter; pH 

KS 5/12/07 35 1.5 20 × 20 3 Farnum silt loam; 3.1%; 7.8 

NE 5/03/07 35 2 15 × 20 3 Hastings silt loam; 3%; 6.3 TX 5/30/07 35 0.5 20 × 20 3 Pullman silty clay loam; 2.2%: 7.1 

1Study 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 = replications 
Monsanto Company 07-CR-191U Page 404 of 544 

Table F-13. Test Site Locations, Planting Information, Soil Description, and Cropping History in a 20

Established with Water Managed According to Local Agronomic Practices 

Study site1

Planting 

date 

(mm/dd/yy)

Planting 

rate 

(seeds/row)2

Planting 

depth 

(in)

Plot size 

(ft) Reps3 Soil series; organic matter; pH

20cr

IAE 5/19/06 34 2.0 20 x 15 3 Silty clay loam; 3.5%; 6.2 FaIAW 5/16/06 34 2.0 20 x 15 3 Loam; 3 – 4%; 5.6 – 7.3 So

IL 5/23/06 34 1.8 20 x 15 3 Plano silt loam; 3.5%; 6.2 FieIN 6/06/06 34 1.4 20 x 15 3 Silt loam; 2.0%; 6.9 CoNE 5/12/06 34 1.5 20 x 15 3 Silt loam; 3.0%; 6.6 So

1 All sites were managed according to local agronomic practices. Site codes are as follows: IAE = Benton County, IA; IAW = Gree

County, IL; IN = Parke County, IN; NE = York County, NE. 

2 After seedling vigor and early stand count data were collected, all plots were thinned to a uniform density. 3 Reps = replications. 
Monsanto Company 07-CR-191U Page 405 of 544 

Table F-14. Phenotypic Comparison of MON 87460 to the Control at Each Site in the 2006 U.S. Field Well-Watered Conditions 

Phenotypic Characteristic, (units), Mean ± S.E.

Seedling vigor 

(0=9 scale)

Early stand count 

(#/plot)1

Days to 50% pollen 

shed Days to 50% silking

Site

MON 

87460 Control

MON 

87460 Control

MON 

87460 Control

MON 

87460 Control

IA1 8.0 ± 0.00 8.0 ± 0.00 74.7 ± 1.45 72.0 ± 2.89 55.7 ± 0.67 57.0 ± 0.58 56.0 ± 0.58 55.3 ± 1.20 IA2 7.0 ± 0.00 7.0 ± 0.00 68.7 ± 1.33 70.7 ± 2.03 65.0 ± 0.00 65.0 ± 0.00 63.0 ± 0.00 63.0 ± 0.00 IL1 9.0 ± 0.00 9.0 ± 0.00 104.3* ± 0.88 86.0 ± 7.51 65.7 ± 0.33 66.0 ± 0.58 63.0 ± 0.00 63.3 ± 0.33 IL2 7.7 ± 0.33 7.3 ± 0.33 - - 57.3 ± 0.33 57.7 ± 0.67 58.0 ± 0.00 58.0 ± 0.58 IN1 4.3 ± 0.33 5.0 ± 0.00 62.7 ± 9.21 65.3 ± 4.98 65.0 ± 0.00 65.0 ± 0.00 63.0 ± 0.00 63.0 ± 0.00 IN2 8.3 ± 0.33 8.0 ± 0.00 77.3 ± 1.33 75.7 ± 0.88 57.3 ± 0.33 57.0 ± 0.00 56.3 ± 0.33 56.0 ± 0.58 KS 5.7 ± 0.33 6.0 ± 0.00 104.0 ± 4.04 108.7 ± 1.20 57.0 ± 0.58 56.7 ± 0.33 57.3 ± 0.67 56.7 ± 0.33 NE 7.3 ± 0.33 7.3 ± 0.33 102.0* ± 0.58 114.3 ± 1.33 63.0 ± 0.00 62.7 ± 0.33 63.0 ± 0.00 63.3 ± 0.33 S.E. = standard error *Indicates significant differences detected between MON 87460 and the control (p<0.05). 1 Dashes indicate that data are missing or excluded from statistical analysis. 
Monsanto Company 07-CR-191U Page 406 of 544 

Table F-14 (continued). Phenotypic Comparison of MON 87460 to the Control at Each Site in the 2006 UWell-Watered Conditions 

Phenotypic Characteristic, (units), Mean ± S.E.

Ear height (in) Plant height (in)

Dropped ears 

(#/plot)

Stalk lodged plants 

(#/plot)

Site

MON 

87460 Control

MON 

87460 Control

MON 

87460 Control

MON 

87460 Control

IA1 50.1 ± 0.52 49.7 ± 1.29 

96.5 ± 

0.47 

97.9 ± 

1.44 0.0 ± 0.00 0.0 ± 0.00 7.0 ± 0.58 8.3 ± 3.18 

IA2 53.1 ± 0.35 54.2 ± 0.53 

107.1 ± 

1.33 

106.4 ± 

0.60 0.7 ± 0.33 0.0 ± 0.00 3.7 ± 0.33 3.7 ± 0.67 

IL1 51.7 ± 0.64 53.3 ± 0.07 

99.1 ± 

0.99 

98.1 ± 

1.46 0.0 ± 0.00 0.0 ± 0.00 1.0 ± 0.58 3.3 ± 1.86 

IL2 52.3 ± 2.60 49.6 ± 2.75 

100.8 ± 

1.29 

100.5 ± 

2.95 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 

IN1 49.9 ± 2.23 49.9 ± 2.49 

94.9 ± 

6.06 

102.5 ± 

2.31 1.0 ± 1.00 0.7 ± 0.33 2.0 ± 1.00 2.0 ± 0.58 

IN2 46.8 ± 2.71 46.3 ± 1.22 

95.8 ± 

2.25 

97.7 ± 

2.79 0.7 ± 0.67 0.3 ± 0.33 5.0 ± 0.58 6.7 ± 0.88 

KS 37.8 ± 0.83 36.4 ± 2.21 

78.7 ± 

 0.97 

82.5 ± 

3.16 1.0 ± 0.58 1.0 ± 0.58 46.7 ± 8.82 36.7 ±3.18 

NE 55.1 ± 0.82 54.4 ± 0.92 

104.7 ± 

1.64 

104.7 ± 

1.57 0.0 ± 0.00 0.3 ± 0.33 9.0* ± 1.15 5.0 ± 1.53 

S.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 544 

Table F-14 (continued). Phenotypic Comparison of MON 87460 to the Control at Each Site in the 2006 U.Well-Watered Conditions 

Phenotypic Characteristic, (units), Mean ± S.E.

Final stand count (#/plot) Grain moisture (%) Test weight (lbs/bu) 

Site MON 87460 Control

MON 

87460 Control MON 87460 Control MON 8

IA1 71.3 ± 1.76 67.0 ± 5.00 14.0 ± 0.21 14.5 ± 0.50 60.6 ± 3.27 62.3 ± 3.35 172.7 ± IA2 62.0 ± 0.58 62.3 ± 0.33 13.5 ± 0.03 13.5 ± 0.21 57.7 ± 0.88 57.7 ± 0.67 173.9 ± 1IL1 94.0* ± 1.00 77.3 ± 0.67 18.9 ± 0.23 18.9 ± 0.18 52.7 ± 1.20 53.3 ± 0.33 19.04 ± IL2 62.0 ± 1.53 62.0 ± 1.15 17.9 ± 0.21 17.6 ± 0.15 57.5 ± 0.17 57.7 ± 0.12 184.0 ± IN1 52.3 ± 3.28 55.3 ± 3.18 19.3 ± 0.23 20.3 ± 0.52 58.9 ± 0.67 59.3 ± 0.25 127.1 ± IN2 53.0 ± 2.65 51.3 ± 2.91 20.4 ± 0.45 20.7 ± 0.61 56.03 ± 0.00 56.03 ± 0.00 161.2 ± 1KS 87.3 ± 2.91 86.0 ± 2.00 13.1 ± 0.59 13.0 ± 0.62 56.4 ± 0.26 56.2 ± 0.37 159.4* ± NE 92.0 ± 1.73 94.3 ± 0.67 17.8 ± 0.38 17.4 ± 0.31 59.0 ± 0.06 59.1 ± 0.20 168.8 ± S.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 544 

Table F-15. Phenotypic Comparison of MON 87460 to the Control at Each Site in the 2007 U.S. Field 

Watered Conditions 

 Phenotypic Characteristic, (units), Mean ± S.E. 

Study 

Seedling vigor 

(1-9 scale) 

 Early stand count 

(#/plot) 

 Days to 50% 

pollen shed 

 Days to 50% 

silking 

 

Site1 MON 87460 Control MON 87460 Control MON 87460 Control MON 87460 Control IA1 1.0 ± 0.00 1.0 ± 0.00 118.0 ± 1.15 117.0 ± 1.53 61.3 ± 0.33 61.0 ± 0.58 59.7 ± 0.67 60.0 ± 1.00 IA2 1.02 ± 0.00 1.0 ± 0.00 78.3 ± 1.45 79.7 ± 0.33 59.7 ± 0.33 59.3 ± 0.33 57.0 ± 0.00 57.0 ± 0.00 IL1 2.02 ± 0.00 2.0 ± 0.00 113.3 ± 1.45 112.3 ± 1.33 64.7 ± 0.33 65.0 ± 0.00 63.02 ± 0.00 63.0 ± 0.00 IL2 4.3 ± 0.67 4.0 ± 1.00 77.7 ± 1.76 78.0 ± 2.31 61.0 ± 0.58 60.7 ± 0.33 60.3 ± 0.33 60.3 ± 0.33 IL3 3.7 ± 0.33 4.0 ± 0.00 78.3 ± 1.20 78.3 ± 1.20 58.0 ± 0.58 58.0 ± 0.00 56.7 ± 0.33 57.0 ± 0.00 IN 5.3 ± 0.33 4.7 ± 0.33 111.0 ± 3.51 118.7 ± 4.06 77.02 ± 0.00 77.0 ± 0.00 75.02 ± 0.00 75.0 ± 0.00 NE 4.3 ± 0.33 5.3 ± 0.33 68.0 ± 2.52 72.0 ± 1.00 61.7 ± 0.33 61.3 ± 0.33 61.7 ± 0.33 61.3 ± 0.33 OH 1.0 ± 0.00 1.3 ± 0.33 70.0 ± 1.15 77.0 ± 0.58 63.3 ± 0.88 63.3 ± 1.20 63.0 ± 1.15 63.0 ± 1.15 PA 4.7 ± 0.33 4.0 ± 0.00 69.7 ± 3.18 72.0 ± 3.06 65.3 ± 0.67 64.7 ± 0.33 65.3 ± 0.67 64.0 ± 0.00 S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05). 1 Dashes (-) indicate that data are missing or excluded from statistical analysis. 2 Not statistically analyzed due to lack of variation. 
Monsanto Company 07-CR-191U Page 409 of 544 

Table F-15 (continued). Phenotypic Comparison of MON 87460 to the Control at Each Site in the 2007 U.Well-Watered Conditions 

 Phenotypic Characteristic, (units), Mean ± S.E. 

Study 

Ear height 

(in) 

Plant height 

(in) Dropped ears (#/plot) 

Stalk lodged plants 

(#/plot) 

Site1 MON 87460 Control MON 87460 Control MON 87460 Control MON 87460 Control IA1 42.1 ± 0.53 42.5 ± 0.64 93.9 ± 1.14 93.5 ± 2.21 0.0 ± 0.00 0.0 ± 0.00 1.0 ± 0.58 0.7 ± 0.33 IA2 45.0 ± 0.53 44.1 ± 0.55 93.5 ± 0.35 94.7 ± 0.87 0.3 ± 0.33 0.0 ± 0.00 0.3 ± 0.33 1.0 ± 1.00 IL1 60.1 ± 1.39 59.0 ± 0.62 121.8 ± 1.65 124.3 ± 1.97 0.02 ± 0.00 0.0 ± 0.00 6.3 ± 1.76 6.0 ± 2.52 IL2 - - - - - - - - IL3 50.3 ± 1.23 47.1 ± 0.59 100.9 ± 0.55 100.1 ± 0.55 1.7 ± 0.33 2.7 ± 0.33 2.3 ± 0.67 3.0 ± 0.00 IN 41.3 ± 1.18 37.4 ± 1.30 94.2 ± 1.33 85.9 ± 0.75 0.3 ± 0.33 0.7 ± 0.33 10.7 ± 1.20 7.3 ± 1.45 0NE 54.2 ± 1.45 54.0 ± 0.31 108.2 ± 2.47 106.6 ± 0.76 0.3 ± 0.33 0.0 ± 0.00 1.3 ± 0.67 1.0 ± 0.58 0OH 39.7 ± 0.64 35.8 ± 0.87 81.1 ± 2.68 78.7 ± 2.66 0.7 ± 0.67 0.3 ± 0.33 3.7* ± 0.88 1.0 ± 0.58 0PA 41.3 ± 0.57 41.5 ± 0.71 86.4 ± 2.40 90.9 ± 1.57 1.3* ± 0.33 0.0 ± 0.00 2.3* ± 0.33 0.3 ± 0.33 0S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05). 1 Dashes (-) indicate that data are missing or excluded from statistical analysis. 2 Not statistically analyzed due to lack of variation. 
Monsanto Company 07-CR-191U Page 410 of 544 

Table F-15 (continued). Phenotypic Comparison of MON 87460 to the Control at each Site in the 2007 U.Well-Watered Conditions 

 Phenotypic Characteristic, (units), Mean ± S.E. 

Study 

Final stand count 

 (#/plot) 

 Grain moisture 

 (%) 

 Test weight 

(lbs/bu) 

 

Site1 MON 87460 Control MON 87460 Control MON 87460 Control MON IA1 81.7 ± 4.10 83.3 ± 2.73 11.8 ± 0.23 12.4 ± 0.10 53.9 ± 0.35 54.5 ± 0.48 200.2 IA2 64.7 ± 0.88 66.0 ± 1.15 18.0* ± 0.23 17.1 ± 0.25 57.3 ± 0.44 57.0 ± 0.58 206.1 IL1 96.0 ± 0.00 95.7 ± 0.33 21.1 ± 1.12 22.4 ± 1.32 57.3 ± 0.48 56.1 ± 0.98 155.4 IL2 - - - - - - IL3 61.7 ± 1.86 64.3 ± 2.03 13.0* ± 0.50 14.7 ± 0.95 56.1 ± 0.24 56.4 ± 0.42 144.7 IN 85.3 ± 1.86 89.7 ± 3.33 18.5 ± 0.98 18.6 ± 0.38 48.0 ± 2.11 53.3 ± 0.57 136.9 NE 61.7 ± 0.88 63.0 ± 0.58 18.5 ± 0.40 19.4 ± 0.95 58.2 ± 0.20 58.0 ± 0.35 156.4 OH 62.0 ± 1.00 62.0 ± 1.53 15.1 ± 0.63 15.3 ± 0.64 51.5 ± 0.59 51.9 ± 0.05 137.0 PA 60.7 ± 3.53 61.0 ± 1.00 22.6 ± 0.29 23.1 ± 0.29 54.5 ± 0.29 54.0 ± 0.00 162.2 S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05). 1 Dashes (-) indicate that data are missing or excluded from statistical analysis. 2 Not statistically analyzed due to lack of variation. 
Monsanto Company 07-CR-191U Page 411 of 544 

Table F-16. Phenotypic Comparison of MON 87460 to the Control at Each Site in a 2006/2007 ChilEstablished with Well-Watered and Water-Limited Treatments in 2006/2007 Well-Watered Treatment 

CL 

Mean ± S.E. 

CT 

Mean ± S.E. 

Me

Phenotypic characteristic Units MON 87460 Control MON 87460 Control MON 874Seedling vigor 0-9 scale3 5.3 ± 0.33 4.3 ± 0.33 5.0 ± 0.00 5.0 ± 0.00 4.3 ± 0.33 Early stand count #/plot 74.0 ± 3.51 66.0 ± 3.06 75.7 ± 2.60 77.7 ± 2.33 78.7 ± 1.2

Days to 50% pollen shed Days 65.0 ± 2.31 64.0 ± 1.15 64.3 ± 0.88 65.0 ± 0.58 71.0 ± 1.5Days to 50% silking Days 64.0 ± 1.53 64.0 ± 1.00 63.0 ± 1.00 63.3 ± 0.33 68.7 ± 0.6Stay green 0-9 scale4 0.7 ± 0.33 1.0 ± 0.00 3.7 ± 0.33 4.3 ± 1.33 3.0 ± 0.5

Ear height in 64.7 ± 3.52 59.1 ± 1.67 53.9 ± 2.00 53.5 ± 0.57 49.2 ± 3.9

Plant height in 110.3 ± 4.47 104.3 ± 2.61 99.5 ± 2.38 100.8 ± 1.81 93.4 ± 4.7Dropped ears2 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 

Stalk lodged plants2 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 Root lodged plants2 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 Final stand count #/plot 74.0 ± 3.46 68.7 ± 2.33 74.7 ± 2.96 77.3 ± 2.03 77.0 ± 0.5

Grain moisture % moisture 12.1 ± 0.33 11.9 ± 0.47 14.0 ± 1.21 13.9 ± 1.07 18.3 ± 0.6Test weight lbs/bu 56.2 ± 0.17 54.8 ± 0.44 58.7 ± 0.44 58.7 ± 0.83 54.5 ± 0.5Yield bu/a 213.0 ± 16.59 195.6 ± 4.80 240.7 ± 8.68 258.8 ± 7.85 208.5 ± 9.

S.E. = standard error Note: No statistically significant differences between MON 87460 and the control (

α=0.05). 1 Study sites: CL = Colina; CT = Calera de Tango; LUM = Lumbreras. 

2 No statistical comparisons were made for this rating due to lack of variability in the data. The test was considered effectively not 

because the test and control mean values were identical. 3 Seedling vigor rating scale: 0 = dead and 9 = above average vigor. 

4 Stay green rating scale: 0 = entire plant is dried and 9 = entire plant is green.
Monsanto Company 07-CR-191U Page 412 of 544 

Table F-17 Phenotypic Comparison of MON 87460 to the Control at Each Site in a 2006/2007 ChilEstablished with Well-Watered and Water-Limited Treatments Water-Limited Treatment 

CL 

Mean ± S.E. 

CT 

Mean ± S.E. 

M

Phenotypic characteristic Units MON 87460 Control MON 87460 Control MON 874Seedling vigor 0-9 scale3 5.0 ± 0.58 5.0 ± 0.00 5.3 ± 0.33 4.7 ± 0.33 4.7 ± 0.6Early stand count #/plot 75.7 ± 1.33 73.0 ± 3.79 77.7 ± 0.88 76.0 ± 1.00 77.0 ± 2.0

Days to 50% pollen shed Days 65.0 ± 1.53 65.3 ± 0.67 65.3 ± 0.33 65.3 ± 0.33 72.0 ± 1.5Days to 50% silking Days 64.3 ± 0.88 62.7 ± 0.33 63.7 ± 0.33 64.0 ± 0.58 74.0 ± 0.5Stay green 0-9 scale4 1.3 ± 0.33 1.7 ± 0.67 5.7 ± 0.33 5.7 ± 0.33 6.0 ± 0.5

Ear height in 61.0* ± 3.56 51.4 ± 4.55 44.9 ± 11.41 43.2 ± 10.76 38.1 ± 1.3

Plant height in 97.2* ± 3.19 79.3 ± 10.73 87.5 ± 13.21 84.5 ± 12.94 67.0 ± 0.5Dropped ears2 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.0

Stalk lodged plants2 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.0Root lodged plants2 #/plot 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.0Final stand count #/plot 74.3 ± 1.20 71.3 ± 1.20 78.0 ± 0.58 76.3 ± 2.03 77.7 ± 2.0

Grain moisture % moisture 14.6 ± 0.75 16.3 ± 0.68 15.5 ± 0.30 15.6 ± 1.40 28.5 ± 3.9Test weight lbs/bu 57.0 ± 0.00 55.7 ± 0.67 60.5 ± 0.50 60.5 ± 1.26 52.5 ± 1.0Yield bu/a 96.9 ± 32.09 65.6 ± 30.99 160.9* ± 18.60 123.7 ± 18.05 85.8 ± 8.3

S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05). 1 Study sites: CL = Colina; CT = Calera de Tango; LUM = Lumbreras. 2 No statistical comparisons were made for this rating due to lack of variability in the data. The test was considered effectively not 

because the test and control mean values were identical. 3 Seedling vigor rating scale: 0 = dead and 9 = above average vigor. 

4 Stay green rating scale: 0 = entire plant is dried and 9 = entire plant is green.
Monsanto Company 07-CR-191U Page 413 of 544 

Table F-18. Study-1, Phenotypic Comparison of MON 87460 to the Control at Each Site in 2007 Field with Well-Watered and Water-Limited Treatments Well-Watered Treatment  Phenotypic Characteristic, (units), Means ± S.E.  Seedling vigor 

(1-9 scale) 

 Early stand count 

(#/plot)§

 Days to 50% 

pollen shed 

 Days to 50% 

silking 

Site 

MON 

87460 Control MON 87460 Control 

 MON 

87460 Control 

 MON 

87460 Control 

CA 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 TX 1.0 ± 0.00 1.0 ± 0.00 105.0 ± 2.12 106.5± 4.35 63.5 ± 0.50 64.0 ±0.58 57.0 ± 0.00 57.0± 0.00  Phenotypic Characteristic, (units), Means ± S.E.  Ear height 

(in) 

 Plant height 

(in) Dropped ears (#/plot) 

 Stalk lodged plants 

(#/plot) 

Site 

MON 

87460 Control MON 87460 Control 

 MON 

87460 Control 

 MON 

87460 Control 

CA 51.4 ± 1.01 48.1± 0.89 100.5 ± 2.50 96.8 ±1.34 0.01 ± 0.00 0.0 ± 0.00 0.5* ± 0.29 0.0 ± 0.00 TX 

33.3 ± 

0.63 34.6 ± 1.39 70.4* ± 1.51 75.1± 1.77 0.0 ± 0.00 0.3 ± 0.25 0.0 ± 0.00 0.3 ± 0.25 

 Phenotypic 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 CA 63.5 ± 0.50 63.0 ± 0.41 14.6 ± 0.28 14.2 ± 0.31 - - 239.7* ± 7.34 TX 96.0 ± 0.00 94.8 ± 1.25 14.8 ± 0.07 15.0 ± 0.20 58.4 ± 0.96 57.8 ± 0.63 215.4 ± 6.24 S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05). § Note: For CA, the r

TX, rows were 30 feet 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 544 

Table F-19. Study-1, Phenotypic Comparison of MON 87460 to the Control at Each Site in 2007 Field with Well-Watered and Water-Limited Treatments Water-Limited Treatment 

 Phenotypic Characteristic, (units), Mean ± S.E.  Seedling vigor 

(1-9 scale) 

Early stand count 

(#/plot)§

Days to 50% pollen 

shed 

Days to 50% 

silking 

Site 

MON 

87460 Control 

MON 

87460 Control 

MON 

87460 Control 

MON 

87460 Control 

CA 5.01 ± 0.00 5.0 ± 0.00 73.5 ± 2.53 74.0 ± 2.65 58.3 ± 0.25 58.8 ± 0.48 60.8 ± 0.25 60.0 ± 1.00 TX 1.0 ± 0.00 1.0 ± 0.00 101.0 ± 2.08 107.5 ± 2.18 64.3 ±0.67 64.0 ± 0.58 57.0 ± 0.00 57.0 ± 0.00  Phenotypic Characteristic, (units), Mean ± S.E.  Ear height 

(in) 

Plant height 

 (in) 

Dropped ears 

(#/plot) 

Stalk lodged 

plants (#/plot) 

Site 

MON 

87460 Control 

MON 

87460 Control 

MON 

87460 Control 

MON 

87460 Control 

CA 52.0 ± 0.57 50.7 ± 1.91 97.5 ± 0.87 95.3 ± 1.34 0.01 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 4TX 27.9 ± 0.69 29.7 ± 0.26 62.6 ± 0.55 62.2 ± 0.73 1.0 ± 0.58 1.5 ± 0.5 1.3 ± 0.88 1.5 ± 0.65 0 Phenotypic Characteristic, (units), Mean ± 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 Con

CA 62.8 ± 0.75 63.3 ± 0.63 15.2 ± 0.49 14.7 ± 0.40 - - 188.3* ± 31.49 148.1 ± TX 96.0 ± 0.00 96.0 ± 0.00 14.0 ± 0.10 14.4 ± 0.07 57.6 ± 0.74 56.7 ± 0.72 186.0 ± 2.98 173.1 ± S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05). §Note: For CA, the r

TX, rows were 30 feet 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 544 

Table F-20. Study-2, Phenotypic Comparison of MON 87460 to the Control at Each Site in 2007 Established with Well-Watered and Water-Limited Treatments Well-Watered Treatment1

 Phenotypic Characteristic, (units), Mean ± S.E. 

Seedling vigor 

(0-9 scale) 

 Early stand count 

(#/plot) 

 Days to 50% 

pollen shed 

 Days to 50% 

silking 

 

Site Test Control Test Control Test Control Test Control 

KS 2.7 ± 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 

NE 2.7 ± 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 TX 1.7 ± 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 

 Phenotypic Characteristic, (units), Mean ± S.E. 

Ear height 

(in) 

 Plant height 

(in) 

 Dropped ears 

(#/plot) 

 Stalk lodged plants 

(#/plot) 

 

Site Test Control Test Control Test Control Test Control 

KS 45.7 ± 1.83 44.2 ± 1.21 90.6 ± 1.94 88.8 ± 2.43 1.0 ± 0.58 0.7 ± 0.67 3.0 ± 0.00 4.7 ± 0.88 

NE 42.9 ± 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 TX 25.1 ± 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 

 Phenotypic Characteristic, (units), Mean ± S.E. 

Final stand count 

(#/plot) 

 Grain moisture 

(%) 

 Test weight 

(lbs/bu) 

 

Site Test Control Test Control Test Control T

KS 59.3 ± 2.73 61.3 ± 0.33 13.7* ± 0.03 12.4 ± 0.09 61.4 ± 0.04 61.7 ± 0.04 137.2 

NE 47.7 ± 2.03 50.3 ± 1.76 15.4 ± 0.25 15.7 ± 0.19 60.5 ± 0.15 60.3 ± 0.12 189.4 TX 51.0 ± 8.5 56.7 ± 3.33 12.7 ± 0.34 12.9 ± 0.12 59.6 ± 0.25 60.0 ± 0.19 250.3 

S.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 NE site, and Days to 50% pollen shed, Days to 50% silking, and Stalk lodged plants variability.

1Water-limited data for the TX site that met the inclusion criteria are presented in Section VIII.C.2, Table VIII-13. Data for KS and NE are reported in 
Monsanto Company 07-CR-191U Page 416 of 544 

Table F-21. Phenotypic Comparison of MON 87460 to the Control at Each Site in a 2006 U.S. Field StuWater Managed According to Local Agronomic Practices 

Phenotypic Characteristic, (units), Mean ± S.E. 

Seedling vigor 2

Early stand count 

(#/plot) Days to 50% pollen shed Days to 50% silking 3 

Site1

MON 

87460 Control 

MON 

87460 Control 

MON 

87460 Control 

MON 

87460 Control 

M

87

IAE 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 ± IAW 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 ± IL 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 ± IN 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 ± NE 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 ± 

Phenotypic Characteristic, (units), Mean ± S.E. 

Ear height (in) Plant height (in) Dropped ears (#/plot) 4

Stalk lodged plants 

(#/plot) 

Site1

MON 

87460 Control MON 87460 Control 

MON 

87460 Control 

MON 

87460 Control 

IAE 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 5IAW 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 0IL 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 0IN 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 3.3NE 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

S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05). 1 Site codes are as follows: IAE = Benton County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; NE = 2 Lack of variance prevented statistical analysis of seedling vigor at the IAE, IL, and IN sites. 3 Lack of variance prevented statistical analysis of days to 50% silking at the IAE and IN sites. 4 Lack of variance prevented statistical analysis of dropped ears at the IAE, IAW, and IL sites. 5 Lack of variance prevented statistical analysis of root lodged plants at the IL site.
Monsanto Company 07-CR-191U Page 417 of 544 

Table F-21 (continued). Phenotypic Comparison of MON 87460 to the Control at Each Site in a 2006 Field Study Established with Water Managed According to Local Agronomic Practices

Phenotypic Characteristic, (units), Mean ± S.E. 

Final stand count (#/plot) Grain moisture (%) Test weight (lbs/bu) Yield (bu/a) 

Site1 MON 87460 Control MON 87460 Control MON 87460 Control MON 87460 ContrIAE 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 ± 5IAW 53.3 ± 2.33 52.7 ± 3.53 15.9 ± 0.64 16.4 ± 0.23 50.7 ± 0.00 48.8 ± 0.94 154.9 ± 5.32 138.5 ± 1IL 58.7 ± 1.45 59.3 ± 0.33 20.6 ± 0.91 20.0 ± 2.40 56.2 ± 0.45 56.1 ± 0.99 52.0 ± 3.69 35.8 ± 18IN 59.0 ± 0.58 57.0 ± 1.73 18.3 ± 0.31 19.4 ± 0.36 54.2 ± 0.32 53.6 ± 0.12 146.8 ± 5.20 161.9 ± 1NE 58.0 ± 1.00 59.3 ± 0.33 16.8 ± 0.45 17.1 ± 0.61 58.6 ± 0.36 58.7 ± 0.09 191.3 ± 3.27 179.8 ± 1

S.E. = standard error. Note: No statistically significant differences between MON 87460 and the control (

α=0.05). 1 Site codes are as follows: IAE = Benton County, IA; IAW = Greene County, IA; IL = Stark County, IL; IN = Parke County, IN; 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 that were excluded from the combined-site analyses discussed 

in Section VIII.C. All data reported here are for sites that did not meet the well-watered 

and water-limited inclusion criteria described 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 differences 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 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 the 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 differences 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 not different from the control because the test 

and control mean values were identical. 

 
Monsanto Company 07-CR-191U Page 420 of 544 

Site Codes, Test Location, and YearKS (Pawnee Co.) and NE (York Co.), 2007 Table G-3. U.S. 2007 Study-2: Water-Limited Treatment – Phenotypic Comparison of MON 87460 to 

Site 

 Phenotypic Characteristic, (units), Mean ± S.E. 

Seedling vigor 

(0-9 scale) 

Early stand count 

(#/20 ft plot) 

Days to 50% 

pollen shed 

Days to 50% 

silking 

Site Test Control Test Control Test Control Test Control KS 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 NE 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 Phenotypic Characteristic, (units), Mean ± S.E. 

Ear height 

(in) 

Plant height 

(in) 

Dropped ears 

(#/plot) 

Stalk lodged plants 

(#/plot) 

Site Test Control Test Control Test Control Test Control KS 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 NE 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  Phenotypic Characteristic, (units), Mean ± S.E. 

Final stand count 

(#/plot) 

Grain moisture 

(%) 

Test weight 

(lbs/bu) 

Site Test Control Test Control Test Control Test KS 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 ± 6NE 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 ± 5S.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 NE site, and days to 50% pollen shed, days to 50% silking, and stalk lodue to a lack of variability. 
Monsanto Company 07-CR-191U Page 421 of 544 

Appendix H. Environmental Interactions 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 beneficial 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 occurring environmental stressors during the growing season. 

During each observation, each plot was evaluated for the severity of symptoms caused by 

three arthropod, three disease, and three abiotic stressors that commonly occur at the 

study sites. With a few exceptions, these stressors were predetermined by the individual 

site Principal Investigators (PIs) based on their experience. The environmental stressors 

evaluated were not artificially induced and could vary between sites. Plots were rated on 

the 0 – 9 rating scale of increasing symptomology described below but the results were 

reported as categorical (none, slight, 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 following 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 (intermediate 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 representative 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 (intermediate 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 damage ratings were performed at three wellwatered sites in 2006, at three well-watered sites in 2007, and a single site in TX with 

well-watered and water-limited treatments (Study-2) in 2007. 

European corn borer damage was evaluated 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 the 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 during the growing season at the following 

intervals: 

 Collection 1: V2 – V4 

 Collection 2: V10 – V15 

 Collection 3: VT – R3 

 Collection 4: R6 
Monsanto 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 deployed at the approximate corn ear level for 

the remainder of the arthropod collections. The sticky traps were deployed for 

approximately seven days. The sticky traps were sent to the Department of Entomology 

at the University of Arkansas in Fayetteville, AR for arthropod identification and 

enumeration. Up to six of the most abundant 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 per plot) from a given 

collection at each site. The arthropods assessed 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 for evaluation of phenotypic and agronomic 

characteristics of MON 87460, observational data on the presence of and differential 

response to biotic (insects, diseases) and abiotic (drought, wind, nutrient deficiency etc.) 

stressors were also collected to examine the environmental interactions of MON 87460 

compared with those of the conventional control corn. The observed stressors were 

“natural” (i.e., no artificial infestation or interference was used). Therefore, the same 

stressors were not necessarily observed at each field site. A summary of environmental 

interactions data are presented in Section VIII. Individual site data are reported in this 

appendix. 

Environmental interactions were assessed qualitatively, and for selected sites, insect 

interactions data were collected quantitatively. Observation of plant interactions with 

insect pests and diseases, and plant responses to abiotic stressors were collected from 

each of the 33 field site locations in 2006 and 2007. The purpose of these evaluations 

was to assess whether plant-insect or plant-disease interactions, or plant response to 

abiotic stressors of MON 87460 were altered compared to the conventional control corn. 

For the plant-insect interactions, plant-disease interactions, and plant responses to abiotic 

stressors, the reported values represent the range of ratings observed across the three or 

four replications at each site. MON 87460 and 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). The ratings observed among the commercial 

reference hybrids provide qualitative 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 according 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 corn borer damage, and arthropod abundance. 

Additionally, the corn earworm damage and European corn borer damage data were 

pooled across sites (combined-site analysis) for a statistical comparison of the test and 

control substances. The test substance was not statistically compared to the reference 

substances, and the reference range was calculated 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 according to a split-plot design with four 

replications using SAS®. The whole plot factor was the irrigation treatment and the 

subplot factor was substance with the experimental unit consisting of one plot. The level 

of statistical significance was 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 reference 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 assessments across both years. For quantitative 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 abiotic stressors were detected between 

MON 87460 and the control for cold, compaction, drought, flood, frost, green snap, 

hail, heat, mineral toxicity, nutrient deficiency, 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, southern leaf blight, stalk rot, 

Stewarts wilt, or yellow leaf blight evaluated 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 detected between MON 87460 and the control for 

aphids, armyworms, corn earworm, corn rootworms, cutworms, European corn 
Monsanto Company 07-CR-191U Page 425 of 544 

borers, flea beetles, grape colaspis, grasshoppers, Japanese beetles, leafhoppers, 

seedcorn maggots, southwestern corn borers, spider mites, white grubs, or wireworms 

evaluated using the observational severity scale 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/ exit 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 IL1, KS, and NE sites (Tables H-4 and H-5). 

Arthropod Abundance

In the individual site analysis, no statistical differences were detected between 

MON 87460 and the control for the following pest and beneficial arthropods at the 

IL1, KS, and NE sites: delphacid planthoppers, grasshoppers, leafhoppers, northern 

corn rootworm beetles, sap beetles, southern corn rootworm beetles, western corn 

rootworm beetles, brown lacewings, green lacewings, lady bird beetles, macroparasitic hymenopterans (parasitoids), nabids, minute pirate bugs, and spiders (Tables 

H-6 and H-7). Aphid abundance was statistically lower at the NE site at collection 1 

(0.0 vs. 3.3 per trap) and statistically higher at the IL1 site at collection 3 (70.7 vs. 

63.3 per trap) for MON 87460 compared to the control out of 12 collections 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 beetles per trap) for MON 87460 compared to the 

control out of nine collections (Table H-6). Micro-parasitic hymenopteran abundance 

was statistically lower at the KS site for collections 2 and 3 (30.0 vs. 42.3 per trap and 

13.0 vs. 23.3 per trap, respectively) for MON 87460 compared to the control out of 

12 collections (Table H-7). However, no consistent statistical differences for aphids, 

corn flea beetles, or micro-parasitic hymenopterans 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 treatments for cold, 

compaction, drought, flood, frost, hail, heat, mineral toxicity, nutrient deficiency, or 

wind evaluated using the observational 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 anthracnose, 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 aphids, leafhoppers, thrips, grasshoppers, 

armyworms, corn earworm larvae, cutworms, billbugs, flea beetles, grape colaspis 

adults, Japanese beetles, northern corn rootworm, 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 87460 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 were detected between MON 87460 and the 

control for corn earworm damage using the adapted Widstrom (1967) scale in the 

combined-site analysis (Table H-11). No statistical differences were detected 

between MON 87460 and the control for European corn borer damage including: 

number of live larvae; number of entry/ exit holes; number of galleries; and length of 

galleries (in.) in the stalk in the combined-site analysis (Table H-11). However, 

number of live larvae and the 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 detected in the individual site analysis were 

not significant in the combined-site analysis 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, grasshoppers, leafhoppers, corn flea beetles, Northern corn 

rootworms, sap beetles, southern corn rootworm 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 control for micro-parasitic hymenopterans 

(parasitoids) abundance were detected across sites or arthropod collections. 

2006 U.S. Field Study Established with Water Managed According to Local Agronomic 

Practices 

Abiotic Stressors

No differences in plant response to abiotic stressors were detected between 

MON 87460 and the control for cold, compaction, flood, frost, hail, heat, nutrient 

deficiency, or wind using the observational severity 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 were detected between MON 87460 and the 

control for aphids, armyworms, billbugs, corn earworms, corn rootworm beetles, 

cutworms, European corn borers, flea beetles, 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 site at Observation 

3. However, the detected difference was within the range of the references. 

Additionally, no differences were detected between MON 87460 and the control for 

grasshopper damage using the observational severity 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 drought tolerance trait did not 

unexpectedly alter MON 87460 compared to conventional corn based on the assessed 

environmental interactions. 

 
Monsanto Company 07-CR-191U Page 428 of 544 

2006/2007 Chilean Field Study Established with Well-Watered and Water-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 abiotic 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, northern 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 were detected between MON 87460 and the 

control for aphids, seed corn maggots, thrips, 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 Well-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 were established (KS, NE, TX), but only one 

site (TX) met the inclusion criteria. The two trials had different experimental designs 

which precluded a combined-study analysis. 

Study-1 – Abiotic Stress Response

In assessment of abiotic stress response in the well-watered and water-limited 

treatments, no differences in plant response to abiotic stressors were observed 

between MON 87460 and the control treatments for drought, hail, heat, heavy 

thunderstorm, nutrient deficiency, 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 mosaic 
Monsanto Company 07-CR-191U Page 429 of 544 

virus, rust, seedling blight, smut, stalk rot, or southern leaf blight evaluated using the 

observational severity scale at the 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 observed between MON 87460 and the control for 

aphids, armyworms, western corn rootworm 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 were detected between MON 87460 and the 

control for corn earworm damage using the adapted Widstrom (1967) scale at the TX 

site under well-watered and water-limited treatments (Table H-24). No statistical 

differences were detected between MON 87460 and the control for European corn 

borer damage including: number of live larvae, number of entry/ exit holes; number 

of galleries; and length 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 under 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 rootworm 

beetles, western corn rootworm beetles, green lacewings, lady bird beetles, macroparasitic hymenopterans (parasitoids), micro-parasitic hymenopterans (parasitoids), 

Orius, big-eyed bugs, and spiders (Tables H-25 and H-26). Brown lacewing 

abundance was statistically higher at collection 3 (0.5 vs. 0.0 per trap) for 

MON 87460 compared to the control, but was 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 beetles, macro-parasitic hymenopterans 

(parasitoids), micro-parasitic hymenopterans (parasitoids), Orius, big-eyed bugs, and 

spiders (Tables H-25 and H-26). Southern corn rootworm beetle abundance was 

statistically higher at collection 4 (2.5 vs. 0.8 per trap) for MON 87460 compared to 

the control and was outside of the reference range (Table H-25). However, 

differences between MON 87460 and the control for southern corn rootworm 

abundance were not detected for 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 well-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 virus, northern corn leaf blight, rust, seedling blight, smut, 

stalk rot assessment by splitting the 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 blight, stalk rot assessment by splitting the stalk, or southern 

corn leaf blight evaluated using the observational severity 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 between MON 87460 and the control for aphids, 

leafhoppers, grasshoppers, armyworms, corn earworms, cutworms, European corn 

borers, southwestern corn borers, corn rootworms, 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 between MON 87460 and the control for aphids, 

grasshoppers, armyworms, corn earworms, cutworms, European corn borers, 

southwestern corn borers, corn rootworms using the observational severity scale at 

the TX site (Table H-29) 

H.4. Summary 

The results of the environmental 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 beneficial 
Monsanto Company 07-CR-191U Page 431 of 544 

abundance indicate that the introduction of the drought tolerance trait is unlikely to be 

biologically meaningful in terms of increased pest potential. 

H.5. References 

Widstrom, N.W. 1967. An evaluation for measuring corn earworm injury. Journal of 

Economic Entomology. 60:791-794. 

Table H-1. Abiotic Stressor Evaluations using 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 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 = Jefferson 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 plots 
Monsanto Company 07-CR-191U Page 432 of 544 

Table H-2. Disease Damage Evaluations using 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 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 = Jefferson 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 rust, and common/southern rust; 6 Includes charcoal stalk rot ; 7 Stalk 

rot assessed externally by observing the outside of the stalk only ; 8

 Stalk rot assessed by splitting the stalks of 5 nonsystematically selected plants and evaluating the disease 
Monsanto 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 and 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 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 = Jefferson 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 rootworms, and southern corn rootworms 
Monsanto Company 07-CR-191U Page 434 of 544 

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 

Pest Damage Assessment MON 

87460 

Control Reference 

Range1

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 differences 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 544 

Table H-5. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 874Control and References at the IL1, KS, and NE Sites in a 2006 U.S. Field Study under Well-Watered Cond

Pest Arthropod Damage Assessment Site MON 874Corn earworm Mean ± SE of 5 ears (0 – 9 rating scale) IL1 0.0 ± 0.0

Mean ± SE of 5 ears (0 – 9 rating scale) KS 5.0 ± 0.3

 Mean ± SE of 5 ears (0 – 9 rating scale) NE 0.0 ± 0.0European corn borer Mean ± SE # of ear shank entry/exit holes of 5 plants IL1 0.0 ± 0.0

Mean ± SE # of ear shank entry/exit holes of 5 plants KS 1.0 ± 0.2

 Mean ± SE # of ear shank entry/exit holes of 5 plants NE 0.0 ± 0.0European corn borer Mean ± SE # of ear shank galleries per plant of 5 plants IL1 0.0 ± 0.0

Mean ± SE # of ear shank galleries per plant of 5 plants KS 0.5 ± 0.0

 Mean ± SE # of ear shank galleries per plant of 5 plants NE 0.0 ± 0.0European corn borer Mean ± SE ear shank gallery length per plant of 5 plants (in.) IL1 0.0 ± 0.0

Mean ± SE ear shank gallery length per plant of 5 plants (in.) KS 0.6 ± 0.

 Mean ± SE ear shank gallery length per plant of 5 plants (in.) NE 0.0 ± 0.0European corn borer Mean ± SE # of stalk entry/exit holes of 5 plants IL1 0.5 ± 0.

Mean ± SE # of stalk entry/exit holes of 5 plants KS 4.9 ± 1.7

 Mean ± SE # of stalk entry/exit holes of 5 plants NE 0.3 ± 0.European corn borer Mean ± SE # of stalk galleries per plant of 5 plants IL1 0.5 ± 0.

Mean ± SE # of stalk galleries per plant of 5 plants KS 2.4 ± 0.7

 Mean ± SE # of stalk galleries per plant of 5 plants NE 0.3 ± 0.European corn borer Mean ± SE stalk gallery length per plant from 5 plants (in.) IL1 0.6 ± 0.3

Mean ± SE stalk gallery length per plant from 5 plants (in.) KS 14.0 ± 2.

 Mean ± SE stalk gallery length per plant from 5 plants (in.) NE 0.2 ± 0.0

S.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). 1 Quantitative insect locations.
Monsanto Company 07-CR-191U Page 436 of 544 

Table H-6. Arthropod Pest Abundance on MON 87460 Compared to the Control and References at the ILin a 2006 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Aphids IL1 7.3 ± 1.67 9.3 ± 0.67 7.7 – 10.3 25.7 ± 1.76 23.0 ± 4.51 18.0 – 27.3 70.7 ± 8.35 63.3 ± 13.84 42.7 – 69.7 9.3 

0.

 KS 9.0 ± 1.53 12.3 ± 2.33 13.7– 19.3 4.0 ± 2.08 5.0 ± 1.53 4.0 – 7.3 5.3* ± 2.19 13.0 ± 1.00 1.7 – 5.7 3.7 

1.

 NE 0.0* ± 0.00 3.3 ± 0.33 0.7 – 2.7 3.0 ± 1.00 3.7 ± 0.88 2.7 – 6.3 76.3 ± 36.68 40.0 ± 14.22 33.0 – 62.7 1.0 

0.

Corn flea beetles IL1 – – – 23.3 ± 5.81 16.7 ± 3.18 15.0 – 25.0 6.0 ± 2.00 4.3 ± 1.33 4.7 – 7.0 – 

 KS 5.3 ± 2.33 7.3 ± 0.88 2.0 – 6.7 10.3 ± 3.28 15.0 ± 2.08 12.3 – 17.7 1.7 ± 0.33 1.3 ± 0.33 0.7 – 1.7 0.3 

0.

 NE – – – 0.0 ± 0.00* 1.3 ± 0.33 0.0 – 1.0 0.7 ± 0.67 1.3 ± 0.88 0.0 – 1.0 0.0 

0.

Delphacid planthoppers IL1 0.0† ± 0.00 0.0 ± 0.00 0.0 – 0.0 1.0 ± 0.58 1.0 ± 0.58 0.3 – 2.0 – – – – 

Grasshoppers KS 0.3 ± 0.33 0.0 ± 0.00 0.0 – 0.3 – – – – – – – 

 NE 0.0 ± 0.00 0.0 ± 0.00 0.0 – 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 enumerated at this observation and si

† p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical. 
Monsanto Company 07-CR-191U Page 437 of 544 

Table H-6 (Continued). Arthropod Pest Abundance on MON 87460 Compared to the Control and Referand NE Sites in a 2006 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Leafhoppers KS 0.0 ± 0.00 0.3 ± 0.33 0.0 – 1.0 0.3 ± 0.33 1.0 ± 0.00 0.3 – 1.7 0.3 ± 0.33 1.0 ± 0.58 0.0 – 1.0 1.0 

1.

 NE – – – 0.0 ± 0.00† 0.0 ± 0.00 0.0 – 0.0 – – – 3.7 

0.

Northern corn rootworms IL1 – – – 2.0 ± 1.15 2.3 ± 0.33 2.3 – 3.7 2.7 ± 0.67 2.3 ± 2.33 1.3 – 2.3 – 

Sap beetles IL1 0.7 ± 0.67 0.7 ± 0.33 0.3 – 1.7 – – – 0.0 ± 0.00 0.3 ± 0.33 0.3 – 1.0 – 

 KS 1.0 ± 1.00 0.3 ± 0.33 0.0 – 0.7 – – – 0.0 ± 0.00 0.3 ± 0.33 0.0 – 0.3 – 

 NE – – – 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 0.3 

0.

Southern corn rootworms IL1 0.0† ± 0.00 0.0 ± 0.00 0.0 – 0.00 – – – – – – – 

 KS – – – – – – 0.0 ± 0.00 0.3 ± 0.33 0.0 – 0.3 – 

 NE – – – – – – 0.3 ± 0.33 0.0 ± 0.00 0.0 – 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 enumerated at this observation and si

† p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical. 
Monsanto Company 07-CR-191U Page 438 of 544 

Table H-6 (Continued). Arthropod Pest Abundance on MON 87460 Compared to the Control and Referand NE Sites in a 2006 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Western corn rootworms IL1 – – – 14.0 ± 4.04 16.3 ± 3.84 10.0 – 16.3 21.7 ± 5.24 19.7 ± 2.91 18.7 – 28.0 –  KS – – – 0.7 ± 0.33 0.7 ± 0.33 0.3 – 1.0 – – – – 

NE – – – 0.3 ± 0.33 0.7 ± 0.33 0.0 – 1.0 9.0 ± 1.00* 3.0 ± 2.08 5.0 – 9.0 0.0 

0.

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 enumerated at this observation and si
Monsanto Company 07-CR-191U Page 439 of 544 

Table H-7. Beneficial Arthropod Abundance on MON 87460 Compared to the Control and References at 

Sites in a 2006 U.S. Field Study under Well-Watered Conditions 

 Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Brown lacewings IL1 – – – – – – – – – 0.0 

0.

 KS – – – – – – – – – 0.0 

0.

Green lacewings IL1 – – – 0.7 ± 0.33 0.0 ± 0.00 0.0 – 0.7 0.0 ± 0.00 1.0 ± 1.00 0.3 – 1.3 –  KS 0.3 ± 0.33 0.0 ± 0.00 0.0 – 0.7 – – – 0.7 ± 0.33 2.0 ± 0.58 0.0 – 1.7 – 

 NE 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 0.3 ± 0.33 0.0 ± 0.00 0.0 – 2.0 4.7 ± 0.88 2.3 ± 1.20 0.7 – 4.3 0.0 

0.

Ladybird beetles IL1 2.3 ± 0.33 2.7 ± 0.67 3.7 – 5.7 4.7 ± 0.88 4.0 ± 1.73 2.3 – 3.7 3.0 ± 1.00 2.3 ± 1.20 2.3 – 3.0 – 

 KS 4.7 ± 1.76 7.0 ± 0.58 4.0 – 7.0 0.7 ± 0.67 1.7 ± 0.33 1.7 – 2.7 0.3 ± 0.33 0.3 ± 0.33 0.3 – 1.0 0.3 

0.

 NE 0.7 ± 0.33 0.0 ± 0.00 0.7 – 2.7 2.7 ± 0.88 1.0 ± 0.58 1.7 – 3.3 4.3 ± 1.76 3.7 ± 1.86 1.0 – 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 at this observation and site
Monsanto Company 07-CR-191U Page 440 of 544 

Table H-7 (Continued). Beneficial Arthropod Abundance on MON 87460 Compared to the Control and RKS, and NE Sites in a 2006 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Macro-parasitic hymenopterans1 IL1 0.0 ± 0.00 0.0 ± 0.00 0.3 – 0.7 – – – – – – 2.7 

0.

 KS 0.0 ± 0.00† 0.0 ± 0.00 0.0 – 0.0 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 0.0 ± 0.00 0.7 ± 0.67 0.0 – 0.7 – 

 NE – – – – – – – – – 0.3 

0.

Micro-parasitic hymenopterans2 IL1 15.0 ± 2.52 12.3 ± 3.67 12.0 – 16.3 53.7 ± 9.94 42.0 ± 5.51 44.0 – 58.3 20.0 ± 4.73 17.3 ± 4.70 21.0 – 38.7 9.3 

2.

 KS 11.3 ± 2.96 13.3 ± 4.91 15.3 – 25.0 30.0* ± 3.51 42.3 ± 1.45 32.0 – 54.0 13.0* ± 0.58 23.3 ± 6.33 10.7 – 16.7 22

4.

 NE 2.0 ± 0.58 3.0 ± 1.15 0.0 – 3.3 23.3 ± 4.67 19.7 ± 6.33 11.7 – 36.7 18.3 ± 5.55 17.0 ± 4.93 16.0 – 30.3 18

7.

Minute pirate bugs IL1 0.0 ± 0.00 0.3 ± 0.33 0.7 – 1.7 5.7 ± 0.88 4.7 ± 1.45 7.7 – 10.0 11.7 ± 3.71 5.3 ± 0.88 6.0 – 8.3 0.3 

0.

 KS 1.3 ± 0.67 0.3 ± 0.33 0.7 – 1.7 2.3 ± 1.20 5.0 ± 1.53 2.7 – 5.0 3.3 ± 1.20 4.3 ± 1.20 2.7 – 5.3 1.3 

0.

 NE – – – 0.3 ± 0.33 0.3 ± 0.33 0.0 – 0.7 7.3 ± 0.88 8.7 ± 2.60 7.7 – 9.7 1.0 

0.

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 at this observation and site

1 Greater than 5mm in length ; 2 Less than 5mm in length. † p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because mean values were identical. 
Monsanto Company 07-CR-191U Page 441 of 544 

Table H-7 (Continued). Beneficial Arthropod Abundance on MON 87460 Compared to the Control and RKS, and NE Sites in a 2006 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Nabids IL1 – – – 0.0 ± 0.00† 0.0 ± 0.00 0.0 – 0.0 – – – – Spiders IL1 1.3 ± 0.88 2.7 ± 0.67 0.3 – 2.3 – – – 1.0 ± 0.00 2.0 ± 1.00 0.3 – 2.0 1.3 

0.6

 KS – – – 0.0 ± 0.00 0.7 ± 0.33 0.7 – 1.7 – – – 2.7 

1.2

 NE 1.0 ± 0.58 1.0 ± 0.00 0.3 – 1.0 0.0 ± 0.00 1.0 ± 0.58 0.0 – 0.3 1.7 ± 0.88 0.7 ± 0.67 1.0 – 2.0 3.7 

0.6

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 at this observation and site

† p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical. 
Monsanto Company 07-CR-191U Page 442 of 544 

Table H-8. Abiotic Stressor 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 

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 differences between MON 87460 and the control (α=0.05). The experimental design 

was a randomized complete block with three replications; Data were 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 = Jefferson County, IA; IA2 = Van Horne County, IA; IL1 = Stark County, IL; IL2 = 

Warren County, IL; IL3 = Clinton County, IL; IN = Boone County, IN; NE = York County, NE; OH = Fayette 

County, OH; PA = Berks County, PA 
Monsanto Company 07-CR-191U Page 443 of 544 

Table H-9. Disease Stressor 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 

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 differences between MON 87460 and the control (α=0.05). The experimental design 

was a randomized complete block with three replications; Data were 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 = Jefferson County, IA; IA2 = Van Horne County, IA; IL1 = Stark County, IL; IL2 = 

Warren County, IL; IL3 = Clinton County, 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 plants 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 observing the outside of the stalk only; 6

 Stalk rot assessed by splitting the stalks of 5 non-systematically selected plants and evaluating the disease 
Monsanto 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 and 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 MON 87460 and the control for European corn borer at the IA1 site during the 

fourth observation (moderate vs. slight). The difference detected was outside the range of the reference (no-sl) 

Note: The experimental design was a randomized complete block with three replications; Data were 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 = Jefferson County, IA; IA2 = Van Horne County, IA; IL1 = Stark County, IL; IL2 = 

Warren County, IL; IL3 = Clinton County, IL; IN = Boone County, IN; NE = York County, NE; OH = Fayette 

County, OH; PA = Berks County, PA 

1

 External observation, ear husks were not pulled back for evaluation; 2 External observation; 3 Includes western corn 

rootworms and northern corn rootworms 

 
Monsanto Company 07-CR-191U Page 445 of 544 

Table H-11. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87Control and References Combined across IA1, IL1, and IN Sites in a 2007 U.S. Field Study under Well-WPest Arthropod Damage Assessment MON 87460 Control Corn earworm Mean ± SE of 10 ears (0 – 9 rating scale) 1.8 ± 0.95 2.6 ± 1.20 European corn borer Mean ± SE # of stalk entry/exit holes of 10 plants 1.6 ± 0.35 1.4 ± 0.43 European corn borer Mean ± SE # of stalk galleries per plant of 10 plants 1.4 ± 0.26 1.3 ± 0.35 European corn borer Mean ± SE stalk gallery length per plant of 10 plants (in.) 2.1 ± 0.49 2.0 ± 0.80 European corn borer Mean ± SE live larvae per plant from 10 plants 0.2 ± 0.09 0.2 ± 0.09 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). 1 Reference range (the minimum and maximum values of the reference means). 
Monsanto Company 07-CR-191U Page 446 of 544 

Table H-12. Quantitative Assessment for Corn Earworm and European Corn Borer Damage to MON 87Control, and References at the IA1, IL1, and IN sites in a 2007 U.S. Field Study under Well-Watered Cond

Pest Arthropod Damage Assessment Site MON 874Corn earworm Mean ± SE of 10 ears (0 – 9 rating scale) IA1 0.2 ± 0.0 Mean ± SE of 10 ears (0 – 9 rating scale) IL1 0.0 ± 0.0 Mean ± SE of 10 ears (0 – 9 rating scale) IN 5.1 ± 1.5European corn borer Mean ± SE # of stalk entry/exit holes of 10 plants IA1 0.9 ± 0.1 Mean ± SE # of stalk entry/exit holes of 10 plants IL1 1.2 ± 0.2 Mean ± SE # of stalk entry/exit holes of 10 plants IN 2.7 ± 0.7European corn borer Mean ± SE # of stalk galleries per plant of 10 plants IA1 0.8 ± 0.2 Mean ± SE # of stalk galleries per plant of 10 plants IL1 1.2 ± 0.2 Mean ± SE # of stalk galleries per plant of 10 plants IN 2.2 ± 0.4European corn borer Mean ± SE stalk gallery length per plant from 10 plants (in.) IA1 0.8 ± 0.2 Mean ± SE stalk gallery length per plant from 10 plants (in.) IL1 1.8* ± 0.3 Mean ± SE stalk gallery length per plant from 10 plants (in.) IN 3.7 ± 0.7European corn borer Mean ± SE live larvae per plant from 10 plants IA1 0.0 ± 0.0 Mean ± SE live larvae per plant from 10 plants IL1 0.4* ± 0. Mean ± SE live larvae per plant from 10 plants IN 0.3 ± 0.1

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).
Monsanto Company 07-CR-191U Page 447 of 544 

Table H-13 Arthropod Pest Abundance on MON 87460 Compared to the Control and References at the IAin a 2007 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Aphids IA1 5.7 ± 1.86 3.0 ± 1.73 3.7 – 6.7 4.7 ± 1.45 4.0 ± 1.73 1.7 – 6.3 32.0 ± 11.02 45.3 ± 9.96 18.0 – 44.3 25

3.

 IL1 45.7 ± 27.81 39.0 ± 23.25 7.0 – 94.7 2.0 ± 1.00 0.3 ± 0.33 1.0 – 2.7 49.0 ± 24.58 42.7 ± 9.35 15.7 – 126.7 4.0 

1.

 IN 8.3 ± 0.88 6.3 ± 1.33 8.7 – 18.3 11.3 ± 3.53 12.3 ± 5.84 4.7 – 27.0 7.7 ± 1.45 40.7 ± 29.34 3.0 – 118.3 6.7 

1.

Delphacid planthoppers IA1 –

– – 

0.3 ± 

0.33 

1.7 ± 

1.67 

0.0 – 

0.7 

18.3 ± 

4.84 

23.0 ± 

8.33 

9.3 – 

21.0 

0.0 

0.

 IL1 67.0 ± 15.50 80.0 ± 32.75 53.0 – 137.7 6.7 ± 3.18 13.7 ± 2.96 3.0 – 12.3 4.3 ± 1.86 4.7 ± 0.88 2.3 – 7.3 1.0 

0.

 IN –

– – 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.3 – 

1.7 

100.0 ± 

18.68 

81.3 ± 

17.70 

47.7 – 

92.0 

0.3 

0.

Grasshoppers IN 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 0.0 ± 0.00† 0.0 ± 0.00 0.0 – 0.0 

–

–

–

–

Leafhoppers IA1 0.0† ± 0.00 0.0 ± 0.00 0.0 – 0.00 

–

–

–

–

–

–

–

 IL1 12.7 ± 6.64 9.7 ± 3.84 7.7 – 15.7 

–

–

–

–

–

–

0.3 

0.

 IN 3.3 ± 1.45 2.7 ± 1.45 1.3 – 3.3 20.3 ± 13.45 15.0 ± 6.03 13.3 – 66.7 6.3 ± 4.37 12.3 ± 5.67 3.0 – 17.3 0.3 

0.

S.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 at this observation and site

† p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical.
Monsanto Company 07-CR-191U Page 448 of 544 

Table H-13 (Continued). Arthropod Pest Abundance on MON 87460 Compared to the Control and ReIL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Corn flea beetle IA1 0.3 ± 0.33 0.3 ± 0.33 0.0 – 0.7 0.3 ± 0.33 0.7 ± 0.33 0.0 – 0.7 0.0 ± 0.00 0.3 ± 0.33 0.3 – 1.0 0.0 

0.

 IL1 23.0 ± 11.14 18.0 ± 6.51 10.3 – 26.7 7.7 ± 0.88 6.7 ± 0.88 3.7 – 10.0 1.0 ± 1.00 0.7 ± 0.33 0.0 – 2.0 0.3 

0.

 IN 0.7 ± 0.67 0.0 ± 0.00 0.0 – 0.7 15.0 ± 11.53 9.0 ± 3.06 8.7 – 19.3 9.7 ± 1.86 23.3 ± 8.82 4.3 – 23.7 7.7 

0.

Northern corn rootworms IA1 – – – – – – 0.7 ± 0.33 1.7 ± 0.88 1.7 – 2.3 0.3 

0.

 IL1 – – – 0.0 ± 0.00 0.7 ± 0.67 0.0 – 1.0 3.7 ± 0.33 3.0 ± 1.53 4.3 – 17.3 – 

Sap beetles IA1 0.7 ± 0.67 0.3 ± 0.33 0.0 – 0.3 0.3 ± 0.33 0.7 ± 0.67 0.3 – 1.7 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 0.0 

0.

 IL1 0.3 ± 0.33 0.0 ± 0.00 0.0 – 0.3 – – – 1.0 ± 0.58 0.0 ± 0.00 0.0 – 0.7 0.3 

0.

 IN – – – – – – 0.3 ± 0.33 0.0 ± 0.00 0.0 – 0.0 0.0 

0.

Southern corn rootworms IA1 –

– – 

0.0 ± 

0.00

0.0 ± 

0.00

0.0 – 

0.7 – – – – 

 IL1 –

– – 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.0 – 

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 at this observation and site
Monsanto Company 07-CR-191U Page 449 of 544 

Table H-13 (Continued). Arthropod Pest Abundance on MON 87460 Compared to the Control and ReIL1, and IN sites in a 2007 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

874

Western corn rootworms IA1 – – – 0.3 ± 0.33 0.0 ± 0.00 0.0 – 0.3 0.7 ± 0.67 0.0 ± 0.00 0.0 – 0.3 0.0 

0.0

 IL1 26.0 ± 4.16 16.0 ± 5.20 10.0 – 44.7 6.3 ± 2.33 5.3 ± 1.86 2.3 – 6.0 16.7 ± 3.76 28.3 ± 9.40 24.3 – 37.0 0.3 

0.

 IN –

– – 

32.0 ± 

11.53

31.7 ± 

5.78

33.0 – 

39.3 

25.0 ± 

11.72 

25.3 ± 

7.62 

15.3 – 

41.3 

0.0 0.0

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 at this observation and site

† p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical.
Monsanto Company 07-CR-191U Page 450 of 544 

Table H-14. Beneficial Arthropod Abundance on MON 87460 Compared to the Control and References IN Sites in a 2007 U.S. Field Study under Well-Watered Conditions  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

87

Araneae (spiders) IA1 0.7 ± 0.33 0.7 ± 0.33 0.3 – 1.0 1.3 ± 0.88 0.3 ± 0.33 1.3 – 2.0 0.7 ± 0.67 2.3 ± 0.88 1.0 – 2.3 2.

0

 IL1 1.0 ± 1.00 0.3 ± 0.33 0.0 – 1.0 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 2.3 ± 0.67 0.7 ± 0.67 0.0 – 2.0 3.

0

 IN 4.0 ± 1.73 3.7 ± 1.20 3.7 – 6.7 0.3 ± 0.33 0.3 ± 0.33 0.3 – 1.7 0.0 ± 0.00 0.0 ± 0.00 0.0 – 2.3 1.

0

Macro-parasitic hymenoptera1 IA1 –

– – –

– – –

– – 

0.

0

 IN –

– – –

– – 

0.0 ± 

0.00 

0.7 ± 

0.67 

0.7 – 

0.7 

0.

0

Micro-parasitic hymenoptera2 IA1 8.7 ± 1.20 6.7 ± 2.40 7.7 – 13.7 23.7 ± 4.48 27.3 ± 3.67 24.0 – 35.0 28.0 ± 10.21 21.0 ± 5.51 20.3 – 27.3 22

2

 IL1 85.3 ± 22.70 63.0 ± 17.06 36.3 – 87.7 72.7 ± 13.42 77.7 ± 11.14 64.3 – 90.7 15.3 ± 1.76 11.7 ± 1.67 12.3 – 16.3 75

5

 IN 12.7 ± 3.38 9.3 ± 2.40 9.0 – 14.3 59.0 ± 10.44 72.0 ± 35.47 54.0 – 74.7 48.7* ± 19.70 118.3 ± 22.67 53.7 – 112.0 307

9

Nabids IN 0.0 ± 0.00† 0.0 ± 0.00 0.0 – 0.0 0.7 ± 0.67 0.3 ± 0.33 0.3 – 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 at this observation and site

1 Greater than 5mm in length. 2 Less than 5mm in length. † p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical.
Monsanto Company 07-CR-191U Page 451 of 544 

Table H-14 (Continued). Beneficial Arthropod Abundance on MON 87460 Compared to the Control a

IA1, IL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered Conditions 

 Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

87

Orius IA1 0.0† ± 0.00 0.0 ± 0.00 0.0 – 0.0 3.7 ± 1.76 1.0 ± 0.58 3.7 – 7.0 15.3 ± 0.67 18.7 ± 3.76 5.7 – 12.7 2.

1

 IL1 12.7 ± 4.48 5.3 ± 2.67 3.7 – 17.3 0.7 ± 0.33 0.7 ± 0.33 0.3 – 1.0 9.3 ± 1.45 15.3 ± 0.67 6.0 – 15.3 7

 IN 0.0 ± 0.00 0.3 ± 0.33 0.0 – 0.0 4.3 ± 1.76 1.0 ± 0.00 1.3 – 3.7 16.0 ± 9.54 13.3 ± 4.26 5.0 – 16.0 0.

0

Brown lacewings IA1 –

– – –

– – 

0.3 ± 

0.33 

0.7 ± 

0.67 

0.0 – 

0.3 

 IL1 –

– – –

– – 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.0 – 

0.3 

Green lacewings IA1 –

– – 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.0 – 

0.3 

–

– – 

 IL1 –

– – –

– – 

3.7 ± 

0.33 

5.7 ± 

1.86 

1.3 – 

7.3 

 IN –

– – 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.0 – 

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 at this observation and site

† p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical.
Monsanto Company 07-CR-191U Page 452 of 544 

Table H-14 (Continued). Beneficial Arthropod Abundance on MON 87460 Compared to the Control a

IA1, IL1, and IN Sites in a 2007 U.S. Field Study under Well-Watered Conditions 

 Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod Site MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MO

87

Ladybird beetles IA1 0.3 ± 0.33 1.3 ± 0.88 0.3 – 1.3 3.0 ± 0.58 3.0 ± 0.58 0.7 – 3.3 10.0 ± 2.31 15.3 ± 0.88 7.7 – 14.0 8.

2

 IL1 6.3 ± 2.03 8.7 ± 1.45 1.7 – 7.3 13.0 ± 1.53 6.3 ± 3.38 3.0 – 14.7 1.0 ± 0.00 1.3 ± 0.33 0.7 – 2.3 13

1

 IN 0.3 ± 0.33 0.0 ± 0.00 0.3 – 1.0 8.0 ± 0.58 6.3 ± 1.45 4.3 – 11.3 8.7 ± 2.73 4.3 ± 2.85 2.3 – 5.7 9.

1

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 at this 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 Study 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 data 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 = 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 454 of 544 

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

and References in a 2006 U.S. Field Study 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 data 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 = Benton 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-systematically 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/plot 
Monsanto Company 07-CR-191U Page 455 of 544 

Table H-17. Arthropod Stressor Incidence of MON 87460 Compared to the Control 

and References in a 2006 U.S. Field Study 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 stages, Observation 1 = appx. V2-V4 growth 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 = Benton 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 rootworms 

 
Monsanto Company 07-CR-191U Page 456 of 544 

Table H-18. Chile 2006/2007: Abiotic Stressor Incidence of MON 87460 Compared 

to the Control and the References in a Field 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 and the control. The experimental design was a split plot 

design with three replications; Observational data were collected at four crop developmental stages, Observation 1 = 

appx. V2-V4 growth 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: 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 and the control. The experimental design was a split plot 

design with three replications; Observational data were collected at four crop developmental stages, Observation 1 = 

appx. V2-V4 growth 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: CL =Colina; CT Calera de Tango; LUM = Lumbreras 

 
Monsanto Company 07-CR-191U Page 457 of 544 

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 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 and the control. The experimental design was a split plot 

design with three replications; Observational data were collected at four crop developmental stages, Observation 1 = 

appx. V2-V4 growth 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: 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 and the control. The experimental design was a split plot 

design with three replications; Observational data were collected at four crop developmental stages, Observation 1 = 

appx. V2-V4 growth 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: CL =Colina; CT Calera de Tango; LUM = Lumbreras 
Monsanto Company 07-CR-191U Page 458 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 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 and the control. The experimental design was a split plot 

design with three replications; Observational data were collected at four crop developmental stages, Observation 1 = 

appx. V2-V4 growth 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: 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 and the control. The experimental design was a split plot 

design with three replications; Observational data were collected at four crop developmental stages, Observation 1 = 

appx. V2-V4 growth 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: CL =Colina; CT Calera de Tango; LUM = Lumbreras 

 
Monsanto 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 MON 87460 Compared to the Control and 

References in Field Studies Established 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 and the control. The experimental design was a split plot 

design with three replications; Observational data were collected at four crop developmental stages, Observation 1 = 

appx. V2-V4 growth 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: 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 and the control. The experimental design was a split plot 

design with three replications; Observational data were collected at four crop developmental stages, Observation 1 = 

appx. V2-V4 growth 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: CA =Sutter County, CA; TX = Carson County, TX 

 
Monsanto Company 07-CR-191U Page 460 of 544 

Table H-22. U.S. 2007 Study-1: Disease Damage Evaluations Using an 

Observational Severity Scale for MON 87460 Compared to the Control and 

References in Field Studies Established 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 and the control. The experimental design was a split plot 

design with four replications; Observational data were 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: CA =Sutter County, CA; TX = Carson County, TX 1 ear rot assessed by pulling husk back from ears of 5 non-systematically selected plants and evaluating the disease 2

 stalk rot assessed by splitting the stalks of 5 non-systematically selected 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 and the control. The experimental design was a split plot 

design with four replications; Observational data were 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: CA =Sutter County, CA; TX = Carson County, TX 1 ear rot assessed by pulling husk back from ears of 5 non-systematically selected plants and evaluating the disease 2

 stalk rot assessed by splitting the stalks of 5 non-systematically selected plants and evaluating the disease
Monsanto Company 07-CR-191U Page 461 of 544 

Table H-23. U.S. 2007 Study-1: Arthropod Damage Evaluated using an 

Observational Severity Scale for MON 87460 Compared to the Control and 

References in Field Studies Established 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 and the control. The experimental design was a split plot 

design with four replications; Observational data were 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: 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 and the control. The experimental design was a split plot 

design with four replications; Observational data were 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: CA =Sutter County, CA; TX = Carson County, TX 
Monsanto Company 07-CR-191U Page 462 of 544 

Table H-24. U.S. 2007 Study-1: Quantitative Assessment for Corn Earworm and European Corn MON 87460 Compared to the Control and References at TX in Field Studies Established with Well-WLimited Treatments Well-Watered Treatment Pest Damage Assessment MON 87460 Control Corn earworm Mean ± SE of 5 ears (0 – 9 rating scale) 3.2 ± 0.32 3.4 ± 0.29 European corn borer Mean ± SE larvae/5 plants 0.0† ± 0.00 0.0 ± 0.00 

European corn borer Mean ± SE # of stalk entry/exit holes of 5 plants 0.0 ± 0.00 0.0 ± 0.00 European corn borer Mean ± SE # of stalk galleries per plant of 5 plants 0.0 ± 0.00 0.0 ± 0.00 European corn borer Mean ± SE stalk gallery length per plant of 5 plants (in.) 0.0 ± 0.00 0.0 ± 0.00 

Water-Limited TreatmentPest Damage Assessment MON 87460 Control Corn earworm Mean ± SE of 5 ears (0 – 9 rating scale) 2.5 ± 0.12 2.7 ± 0.09 European corn borer Mean ± SE larvae/5 plants 0.0† ± 0.00 0.0 ± 0.00 

European corn borer Mean ± SE # of stalk entry/exit holes of 5 plants 0.0† ± 0.00 0.0 ± 0.00 European corn borer Mean ± SE # of stalk galleries per plant of 5 plants 0.0† ± 0.00 0.0 ± 0.00 European corn borer Mean ± SE stalk gallery length per plant of 5 plants (in.) 0.0† ± 0.00 0.0 ± 0.00 

S.E. = standard error; the experimental design was a split plot design with four replications. 

No statistically significant differences between MON 87460 and the control (α=0.05). 

1 Reference range (the minimum and maximum values of the reference means). † p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical.
Monsanto Company 07-CR-191U Page 463 of 544 

Table H-25. U.S. 2007 Study-1: Arthropod Pest abundance on MON 87460 Compared to the Control and Field Studies Established with Well-Watered and Water-Limited Treatments Well-Watered Treatment  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 8746Aphids 5.5 ± 1.32 5.0 ± 1.08 7.5 – 10.0 67.0 ± 17.92 62.5 ± 13.86 50.3 – 79.3 6.0 ± 1.41 6.5 ± 2.22 4.3 – 6.0 17.8 

3.0

Delphacid planthoppers 

0.0 ± 

0.00 

0.3 ± 

0.25 

0.3 – 

0.3 

1.3 ± 

0.48 

1.3 ± 

0.75 

0.5 – 

1.8 

5.8 ± 

2.46 

5.3 ± 

2.10 

4.3 – 

11.8 

3.8 

0.8

Leafhoppers 17.8 ± 2.69 11.8 ± 3.22 9.0 – 15.8 1.3 ± 0.75 1.3 ± 0.75 0.3 – 2.3 11.5 ± 2.99 13.8 ± 4.50 9.0 – 12.5 163.8 

45.4

Grasshoppers 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 – – – – – – – 

Corn flea beetles 1.8 ± 0.48 1.3 ± 0.25 0.3 – 1.5 13.0 ± 4.02 11.0 ± 4.92 9.8 – 20.5 26.8 ± 6.69 23.8 ± 7.30 15.8 – 23.5 13.3 

1.3

Sap beetles – – – – – – 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 – 

Southern corn rootworms 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.0 – 

0.3 

 1.0 ± 

0.41 

1.3 ± 

1.25 

0.8 – 

1.8 – – – 

0.3 

0.2

Western corn rootworms – – – 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.0 – 

0.5 

0.0† ± 

0.00 

0.0 ± 

0.00 

0.0 – 

0.0 

0.0† 

0.0

S.E. = standard error; the experimental design was a split plot design 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 enumerated at this observation and si

† p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical.
Monsanto Company 07-CR-191U Page 464 of 544 

Table H-25 (Continued). U.S. 2007 Study-1: Arthropod Pest Abundance on MON 87460 Compared References at TX in Field Studies Established with Well-Watered and Water-Limited Treatments Water-Limited Treatment Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 8746Aphids 7.3 ± 3.28 5.5 ± 1.04 7.0 – 11.8 80.8 ± 23.26 80.0 ± 8.67 69.3 – 87.5 4.8 ± 1.11 8.3 ± 1.93 3.5 – 7.8 16.3 

2.02 

Delphacid planthoppers – – – 

1.8 ± 

0.75 

1.3 ± 

0.48 

0.8 – 

2.0 

4.8 ± 

1.38 

7.3 ± 

2.93 

2.8 – 

10.0 

5.3 

2.6

Leafhoppers 9.3 ± 1.31 17.0 ± 6.82 8.5 – 20.5 1.8 ± 1.18 0.8 ± 0.25 0.5 – 1.8 10.8 ± 2.81 10.5 ± 4.29 8.8 – 12.5 137.5 

43.0

Grasshoppers 0.3 ± 0.25 0.0 ± 0.00 0.0 – 0.3 – – – – – – – 

Corn flea beetles 1.3 ± 0.25 1.5 ± 0.50 0.3 – 1.3 21.5 ± 6.25 13.3 ± 2.21 9.3 – 18.3 18.0 ± 4.88 23.5 ± 9.21 12.5 – 23.8 16.5 

3.7

Sap beetles – – – – – – – – – – Southern corn rootworms – – – 

 0.5 ± 

0.29 

0.8 ± 

0.48 

0.3 – 

0.8 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.0 – 

0.3 

 2.5* 

0.6

Western corn 

rootworms – – – 

0.3 ± 

0.25 

0.0 ± 

0.00 

0.0 – 

0.3 

0.3 ± 

0.25 

0.8 ± 

0.75 

0.0 – 

0.0 

0.0 0.00

S.E. = standard error; the experimental design was a split plot design 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 enumerated at this observation and si

† p-values could not be generated due to lack of variability in the data. MON 87460 was considered effectively not different from the control because 

mean values were identical.
Monsanto Company 07-CR-191U Page 465 of 544 

Table H-26. U.S. 2007 Study-1: Arthropod Beneficial Abundance on MON 87460 Compared to the Contr

TX in Field Studies Established with Well-Watered and Water-Limited Treatments 

Well-Watered Treatment  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 8746Araneae (spiders) 17.5 ± 5.85 21.0 ± 3.85 15.0 – 24.5 4.0 ± 1.29 5.0 ± 1.00 2.5 – 2.8 2.8 ± 1.11 2.0 ± 1.08 1.8 – 2.8 10.8 

2.32 

Brown lacewings – – – – – – 0.5* ± 0.29 0.0 ± 0.00 0.0 – 0.5 – 

Green lacewings – – – 2.0 ± 0.41 0.5 ± 0.29 0.5 – 1.5 2.0 ± 0.91 3.5 ± 1.76 2.5 – 4.3 2.5 

1.5

Big-eyed bug 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 – – – – – – – 

Orius 0.3 ± 0.25 0.8 ± 0.75 0.3 – 1.0 4.3 ± 0.63 4.8 ± 1.84 2.3 – 6.8 3.0 ± 0.71 3.0 ± 1.41 2.0 – 3.3 3.0 

1.0

Ladybird beetle 1.3 ± 0.75 0.5 ± 0.29 0.5 – 0.8 5.0 ± 1.08 6.0 ± 1.35 5.0 – 6.8 8.8 ± 2.29 5.0 ± 1.73 5.3 – 12.0 3.0 

0.5

Macro-parasitic  hymenoptera – – – 

0.5 ± 

0.29 

1.0 ± 

0.41 

0.8 – 

4.3 – – – 

0.8 

0.4

Micro-parasitic  hymenoptera 

35.0 ± 

5.57 

37.3 ± 

7.42 

21.3 – 

32.8 

118.5 ± 

14.77 

98.8 ± 

18.43 

101.5 – 

128.5 

59.3 ± 

1.93 

51.0 ± 

5.43 

39.3 – 

49.5 

217.3 

23.2

S.E. = standard error; the experimental design was a split plot design 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 enumerated at this observation and si
Monsanto Company 07-CR-191U Page 466 of 544 

Table H-26 (Continued). U.S. 2007 Study-1: Arthropod Beneficial Abundance on MON 87460 Compare

References at TX in Field Studies Established with Well-Watered and Water-Limited Treatments 

Water-Limited Treatment  Mean Arthropod abundance ± S.E.  Collection 1 Collection 2 Collection 3 Arthropod MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 87460 Contr Ref Range MON 8746Araneae (spiders) 21.0 ± 3.08 19.3 ± 3.42 19.3 – 22.0 2.3 ± 0.48 2.5 ± 0.65 1.5 – 3.0 4.8 ± 2.17 1.8 ± 0.48 1.5 – 4.3 10.3 

1.9

Brown lacewings – – – – – – 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 – Green lacewings – – – 0.5 ± 0.50 0.5 ± 0.29 0.8 – 1.5 2.3 ± 0.63 2.0 ± 0.71 2.0 – 3.8 2.3 

0.8

Big-eyed bug 0.0 ± 0.00 0.0 ± 0.00 0.0 – 0.3 – – – – – – – 

Orius 0.3 ± 0.25 0.0 ± 0.00 0.0 – 0.8 5.0 ± 1.22 7.0 ± 1.58 3.8 – 7.8 3.3 ± 1.97 2.8 ± 1.25 1.5 – 3.5 3.3 

1.4

Ladybird beetle 0.3 ± 0.25 0.0 ± 0.00 0.3 – 1.8 6.5 ± 1.26 5.8 ± 0.25 4.8 – 7.5 2.8 ± 0.85 6.0 ± 0.91 6.0 – 9.0 2.8 

0.6

Macro-parasitic  hymenoptera 

0.0 ± 

0.00 

0.0 ± 

0.00 

0.0 – 

0.8 – – – – – – 

1.3 

0.4

Micro-parasitic 

 hymenoptera 

24.8 ± 

2.90 

27.3 ± 

6.02 

27.5 – 

43.0 

111.5 ± 

7.80 

101.8 ± 

14.08 

90.0 – 

112.8 

48.0 ± 

4.02 

51.8 ± 

7.19 

46.5 – 

51.5 

240.3 

53.0

S.E. = standard error; the experimental design was a split plot design 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 enumerated at this observation and si
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 MON 87460 Compared to the Control and 

References in Field Studies Established 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 and the control. The experimental design was a split plot 

design with three replications; Data were 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: NE = York County, NE; KS = Pawnee County, 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 and the control. The experimental design was a split plot 

design with three replications; Data were 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 code is as follows: TX = Carson County, TX 
Monsanto Company 07-CR-191U Page 468 of 544 

Table H-28. U.S. 2007 Study-2: Disease Damage Evaluations using an 

Observational Severity Scale for MON 87460 Compared to the Control and 

References in Field Studies Established 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 and the control. The experimental design was a split plot 

design with three replications; Data were 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: NE = York County, NE; KS = Pawnee County, 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-systematically 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 and the control. The experimental design was a split plot 

design with three replications; Data were 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 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 plants and evaluating the disease 

 
Monsanto Company 07-CR-191U Page 469 of 544 

Table H-29. U.S. 2007 Study-2: Arthropod Damage Evaluations using an 

Observational Severity Scale for MON 87460 Compared to the Control and 

References in Field Studies Established 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 and the control. The experimental design was a split plot 

design with three replications; Data were 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: NE = York County, NE; KS = Pawnee County, 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 and the control. The experimental design was a split plot 

design with three replications; Data were 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 code is as follows: TX = Carson County, TX 1

 Includes western corn rootworm and southern corn rootworm 
Monsanto 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 presented in Appendices F, G, and H. 

Contents

Section I.1. Calculated and accumulated water 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 Data 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-1. Calculated and Accumulated Soil Water Values at IA1 in 2006 

(Note: moisture levels below 50% of field capacity represent 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-2. Calculated and Accumulated Soil Water Values at IA2 in 2006 

(Note: moisture levels below 50% of field capacity represent 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-3. Calculated and Accumulated Soil Water Values at IL1 in 2006 

(Note: moisture levels below 50% of field capacity represent 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-4. Calculated and Accumulated Soil Water Values at IL2 in 2006 

(Note: moisture levels below 50% of field capacity represent 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

30

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-5. Calculated and Accumulated Soil Water Values at IN1 in 2006 

(Note: moisture levels below 50% of field capacity represent 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

7/1/06 8/1/06 9/1/06 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-6. Calculated and Accumulated Soil Water Values at IN2 in 2006 

(Note: moisture levels below 50% of field capacity represent 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 9/1/06 

Accumulated Water (in.)

0

10

20

30 Accumualted Water

Date

6/1/06 7/1/06 8/1/06 9/1/06 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-7. Calculated and Accumulated Soil Water Values at KS in 2006 

(Note: moisture levels below 50% of field capacity represent 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/06 7/1/06 8/1/06 9/1/06 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-8. Calculated and Accumulated Soil Water Values at NE in 2006 

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

30

Accumulated Water

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-9. Calculated and Accumulated Soil Water Values at IA1 in 2007 

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-10. Calculated and Accumulated Soil Water Values at IA2 in 2007 

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-11. Calculated and Accumulated Soil Water Values at IL1 in 2007 

(Note: moisture levels below 50% of field capacity represent 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 Accumulated Soil Water Values at IL2 in 2007 
Monsanto 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-13. Calculated and Accumulated Soil Water Values at IL3 in 2007 

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-14. Calculated and Accumulated Soil Water Values at IN in 2007 

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-15. Calculated and Accumulated Soil Water Values at NE in 2007 

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-16. Calculated and Accumulated Soil Water Values at OH in 2007 

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

30

Accumulated Water

Date

06/01 07/01 08/01 09/01 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Calculated Soil Water Availability

50% Reference Line

Figure I-17. Calculated and Accumulated Soil Water Values at PA in 2007 

(Note: moisture levels below 50% of field capacity represent 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 9/1/06 

Accumulated 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Figure I-19. Accumulated Water at IAW in 2006 
Monsanto 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 9/1/06 

Accumulated 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water
Monsanto 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 9/1/06 

Accumulated Water (in.)

0

5

10

15

20

25

Accumulated Water

Figure I-22. Accumulated Water at NE in 2006 

 
Monsanto Company 07-CR-191U Page 491 of 544 

I.1.3. Chile 2006/2007 Well-Watered and Water-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 4/1/07 

Accumulated Water (in.)

0

10

20

30

Water-Limited

Well-Watered

Figure I-23. Accumulated Water at CL in 2006/2007 
Monsanto 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 4/1/07 

Accumulated 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 4/1/07 

Accumulated Water (in.)

0

10

20

30 Water-Limited

Well-Watered
Monsanto 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 4/1/07 

Accumulated Water (in.)

0

10

20

30 Water-Limited

Well-Watered

Figure I-26. Accumulated water at QUI in 2006/2007 

 
Monsanto 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Water-Limited

Well-Watered

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Water-Limited

50% Reference Line

Well-Watered

Figure I-27. Calculated and Accumulated Soil Water Values at CA Study-1 in 2007 

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

30

Water-Limited

Well-Watered

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Water-Limited

50% Reference Line

Well-Watered

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

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Water-Limited

Well-Watered

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Water-Limited

50% Reference Line

Well-Watered

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

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

Water-Limited

Well-Watered

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Water-Limited

50% Reference Line

Well-Watered

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

(Note: moisture levels below 50% of field capacity represent 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 9/1/07 

Accumulated Water (in.)

0

5

10

15

20

25

30

Water-Limited

Well-Watered

Date

6/1/07 7/1/07 8/1/07 9/1/07 

Calculated Soil Water Availability (%)

0

20

40

60

80

100

Water-Limited

50% Reference Line

Well-Watered

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

(Note: moisture levels below 50% of field capacity represent 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 Sites - Monthly Average Minimum and 

Maximum Temperatures and Temperature Range 

Study 

Site1

May June July 

Min2 Max Range3 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 Sites - 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 

Site1 Min2 Max2 Range3 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 

IL24 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 29-90 
Monsanto 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 Practice Sites - Monthly Average Minimum and 

Maximum Temperatures and Temperature Range 

Study 

Site 1

May June July 

Min2 Max2 Range3 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 and 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 

Site1 Min2 Max2 Range3 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 

Site1 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-Watered and Water-Limited Sites - Monthly 

Average Minimum and Maximum Temperatures and Temperature Range 

Study May June July 

Site1 Min2 Max2 Range3 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 43.0-96.02 61.0 94.8 57.0-100.0 

 

Study August September October 

Site1 Min Max Range Min Max 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 32.0-98.02

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 and 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-Watered and Water-Limited Sites - Monthly 

Average Minimum and Maximum Temperatures and Temperature Range 

Study May June July 

Site1 Min2 Max2 Range3 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 

Site1 Min Max Range Min Max 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 Daily 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, Methods and Individual Site Results for Seed Dormancy and 

Germination 

J.1. Materials 

Seed of MON 87460, the conventional control corn, and reference corn were produced in 

Greene County, IA, Stark County, IL, and Pawnee County, KS in 2006. 

Material Material Type Phenotype

Production1

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 verifications were as 

expected with the following exceptions: analysis of the H1548126 seed from the IA 

production site revealed the presence of MON 87460 at ≤1.84%. In addition, the analysis 

of seed of three reference substances from the IA production site 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 level of MON 87460 was low and was deemed 

to have no negative effect on the quality of the study or interpretation 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 was certified to conduct seed dormancy and 

germination testing consistent with the standards established by the Association of 

Official Seed Analysts (AOSA), a seed trade 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 temperature regimes: constant temperature of 

approximately 5, 10, 20, or 30o

C or alternating temperatures of approximately 10/20, 

10/30, or 20/30o

C. The alternating temperature regimes were maintained at the lower 

temperature for 16 h and the higher temperature for 8 h. The temperature inside each 

germination chamber was monitored and recorded throughout the duration of the study. 

Germination towels for MON 87460, control, and reference materials were prepared per 

the facility SOPs. Each germination towel represented 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 (viable and nonviable) seed as defined by AOSA 

guidelines (AOSA, 2006). Each rolled germination towel in the additional temperature 

regimes (i.e., 5, 10, 20, 30, 10/20 and 10/30o

C) was assessed periodically during the 

study for germinated, hard (viable and nonviable), dead, and firm swollen (viable and 

nonviable) seed. 

J.4. Statistical Analysis 

Statistical analyses were performed by Monsanto 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 across all sites with 

a level of statistical 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-site analysis was conducted when production site-by-seed 

substance type interactions were detected. Summary statistics were provided for each 

production site and temperature regime combination. The test substance was not 

statistically compared to the reference substances, and no comparisons were made across 

temperature regimes. The reference range was calculated from the minimum and 

maximum mean values observed in the references. 

For the AOSA recommended temperature range, the data reported for each seed 

substance included the percentage of normal germinated, abnormal germinated, dead, 

viable firm swollen, and viable hard seed. For non-AOSA temperatures, the data 

reported for each seed substance included the percentage of germinated, dead, viable firm 

swollen, and viable hard seed. 

J.5. Individual Site Seed Dormancy and Germination Results 

MON 87460, the control, and reference seed materials were produced at three sites to 

assess germination characteristics 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 interactions were detected for any combination 

except percent germinated and percent viable firm swollen at 10°C. These two 

characteristics were analyzed separately by site to account for the interaction. In percent 

germinated and percent viable firm swollen seed at 10°C, significant production site × 

seed substance type interactions were detected. 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 production 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 both percent germination and percent viable 

firm swollen seed (Table J-1). Furthermore, 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 this study are used to characterize MON 87460 

in the context of plant pest risk assessment. The results of this study, 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 conclusion of no increased pest potential of MON 87460 

compared to conventional corn. 

J.6. References 

AOSA. 2000. Tetrazolium Testing Handbook. Association of Official Seed Analysts. 

Lincoln, NE. 

AOSA. 2006. Rules for Testing Seeds. Association of Official Seed Analysts. Lincoln, 

NE. 
Monsanto Company 07-CR-191U Page 507 of 544 

Table J-1. Individual Site Analysis for Germination of MON 87460 and the Control in 2006 

IA2,3 

Mean ± S.E.

IL2,3 

Mean ± S.E.

KS2,3 

Mean ± S.E.

Temperature Characteristic

MON 

87460 Control

MON 

87460 Control

MON 

87460 Cont

10°C Germinated 91.5 ± 1.52 87.0* ± 1.74 86.0 ± 1.58 89.0 ± 0.41 92.0 ± 1.78 94.0 ± 

Viable firm swollen 6.5 ± 1.86 11.2* ± 1.40 13.3 ± 1.18 10.5 ± 0.65 7.8 ± 1.65 5.3 ± 

S.E. = standard error. *Indicates significant differences detected between MON 87460 and the control (p<0.05). 1 Analysis run only on variables where production site × seed substance interaction prevented combined-site analysis. 2 IA = Greene County, IA; IL = Stark County, IL; KS = Pawnee County, KS. 3 Totals are a mean percent. 
Monsanto Company 07-CR-191U Page 508 of 544 

Appendix K. Materials and Methods for Pollen Morphology and Viability Analysis 

K.1. Test, Control, and Reference Substances 

Assessment for pollen morphology and viability 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 commercially available corn hybrids: DKC63-78, RX715, 

RX754RR2, and DKC61-50.

K.2. Experimental Methods 

Plant Production

MON 87460, a conventional control, and four references were grown in California under 

similar agronomic conditions, with the exception of irrigation treatment. The study was 

arranged in a split-plot design with four replications, where irrigation treatment was the 

whole-plot and plant substance type was the sub-plot. A pollen sample was collected 

from three plants per plot and was fixed and stained 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 percentage viable pollen. 

Pollen Collection

Tassel bags were placed on three to five, non-systematically selected plants per plot 

during pollen shed. The following morning, 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 solution (Alexander, 1980) in a 1:5 dilution was 

added to each tube (at least 2:1 (v/v) stain to pollen) to fix and stain the pollen, rendering 

the pollen non-viable; the tubes were closed and the contents shaken until thoroughly 

mixed. Subsamples were placed on wet ice within 30 minutes of pollen collection and 

maintained under those conditions until receipt at the performing laboratory. Pollen 

collected from each plant in a plot represented 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 μl of suspended pollen / stain solution onto a slide. 

Pollen samples were viewed under an Olympus Provis AX70 light/fluorescence 

microscope with an Olympus DP70 digital color camera. The associated PC computer 

[run on Microsoft Windows 2000 Professional (© 1981-1999, Microsoft Corp.)] had 

microscope and camera software (DP Controller 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 grains were evaluated under the 10X ocular 

lens (100X magnification) for each of the three to five subsamples 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 light blue-green. In 

addition, viable pollen grains appeared round and turgid, whereas non-viable pollen grains 

may have appeared flaccid, depending on the degree of hydration. 

Pollen diameter was measured using Image-Pro Plus v4.5.1.27 (© 1993-2002, Media 

Cybernetics, Inc.) software to view digital images. For a single subsample per pollen 

sample, pollen diameter was measured along two perpendicular axes for ten viable pollen 

grains. The mean of the 20 diameter measurements per sample was analyzed. 

Pollen morphology was observed from digital images for one of the three to five samples 

for each test, control, or reference substance. 

K.3. Statistical Analysis 

Monsanto Statistics Technology Center performed the statistical analysis. The design 

was a split plot with four replications, where 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 viability and diameter, with a significance 

level of 5% (α = 0.05). A reference range consisting 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 for pollen fungi, yeast and bacteria. Stain 

Technology. 55:13-18. 

 

 

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Monsanto Company 07-CR-191U Page 510 of 544 

Appendix L. Materials and Methods for 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 season. The purpose of this study was to 

evaluate volunteer potential of MON 87460 compared to conventional corn. These data 

are used to assess pest potential, and ultimately, the environmental risk of the 

biotechnology-derived crop as compared to the conventional crop. 

L.1. Study Sites 

The experiment was established at Jefferson County, Iowa (RL); Guthrie County, Iowa 

(BG), and Parke County, Indiana (RV). These three locations provided a range of 

environmental and agronomic conditions representative of major corn growing regions 

where commercial production of MON 87460 is expected. The Principal Investigator at 

each site was familiar with corn production and the occurrence and identification of corn 

volunteers. 

L.2. Test, Control, and Reference Substances 

Starting seed for the test, control and reference substances were produced during 2006 in 

Jefferson County, IA (Tables F-1 and F-2). 

The viability of the starting seed was determined 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 the 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 corn 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, commercially available corn 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 corn seed observed to be 

inadvertently present. 

Planting, soil, and cropping history information for each site are presented in Table K-1. 

In November 2006, plots were established at each of the three 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 established. 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 maintain 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 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 Spring 2007 approximately a week prior to 

the average local planting date for each field 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 experimental design and evaluation criteria were 

involved in all components of data collection and summarization. Study personnel 

ensured that assessments were taken properly, data were consistent with expectations 

based on experience with the crop, and the experiment was carefully monitored. Any 

unexpected observations or issues during the study that would impact the study objectives 

were noted by the Study Director. 

L.5. Statistical Analysis 

Statistical analysis of cumulative 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 sites, no analysis was warranted. 
Monsanto Company 07-CR-191U Page 512 of 544 

Table L-1. Planting, Soil type, and Cropping History Information for Volunteer 

Potential Study 

Site1 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 availability. The purpose of this study was to 

assess the ability of plants 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 regions of the U.S. where seed or grain may be 

incidentally returned to the environment during harvest, handling, or transport. 

• Effingham County, Illinois (site code: IL) 

• Shelby County, Missouri (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 during 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 starting 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 similar to the test 

substance with the exception of the drought tolerance trait. 

M.2.3. Reference Substances 

The reference substances were commercially-available corn hybrids that 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 

Code1

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 the four sites in a randomized complete block 

design with three replications. Each plot was approximately 100 ft2

 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 planted with less than 100 seeds. The minimum 

number of seed planted in any plot was 53 at the IL site and 76 at the TX site. Planting 

occurred at the MO and NE sites before a recount could be requested. The Principal 

Investigator at the MO site, however, confirmed that he planted a minimum of 50 seeds. 

Therefore, the minimum number of seed planted in any plot was conservatively 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 planting 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 Conservation 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 annual 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 cover was estimated to be approximately 85%. 

The area had last been cultivated in 1983. 

Weather data collected at each site throughout the growing 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 evaluated and evaluation timing are presented in Table M-4. 

Experienced scientists familiar with the experimental design and evaluation criteria were 

involved in all components of data collection, summarization, and analysis. Study 

personnel assessed that measurements were taken properly, data were consistent with 

expectations based on experience with the crop, and the experiment was carefully 

monitored. Prior to analysis, the overall dataset was evaluated 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 control at each site. There was 

no intention to analyze the data across all sites 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 trademark of the SAS Institute. 
Monsanto Company 07-CR-191U Page 516 of 544 

Table M-2. Study Area Planting Information and Soil Description for Survival Outside Cultivation study Study Site1

Planting Date 

(mm/dd/yy) 

Planting Rate 

(seeds/plot)2

Planting 

Depth (in) Plot Size (ft) Reps Soil Series; O

IL 06/11/07 53-100 1.2-1.5 10 x 10 3 Bluford silt loam; 1MO 05/29/07 53-100 1.3 10 x 10 3 Putnam loam; 2.0%NE 06/01/07 53-100 0.5 10 x 10 3 Hastings silt loam; TX 06/09/07 76-100 0.5-0.8 10 x 10 3 Pullman silty clay l1 Study sites: IL = Effingham County., IL; MO = Shelby County., MO; NE = York County., NE; and TX = Carson County., TX. 2 Planting rate was documented for each plot at the IL and TX sites. Planting rate at the MO and NE sites were estimated based on 

 counts from the IL and TX sites.
Monsanto Company 07-CR-191U Page 517 of 544 

Table M-3. Monthly Temperature and Rainfall Amounts for Survival Outside Cultivation Study Study Site1

May June 

Low2 High3 Range4 Rain5 Low High Range Rain Low High 

IL 55.8 80.3 38-89 3.0 61.8 85.9 53-97 3.4 61.6 85.1 MO 57.1 78.3 42-89 4.3 62.8 82.6 51-92 3.6 65.0 86.4 NE 55.5 76.0 45-91 5.8 60.2 82.4 48-90 2.2 65.9 86.7 TX 50.8 75.9 38-86 2.4 57.3 86.0 43-96 0.5 61.0 94.8 Study Site 

August September O

Low High Range Rain Low High Range Rain Low High 

IL 66.6 93.5 57-100 1.0 56.5 84.7 40-98 2.4 47.2 72.6 MO 69.6 91.1 60-99 6.3 57.8 83.3 45-93 1.6 47.9 69.2 NE 66.7 86.3 56-95 4.8 53.5 79.0 40-90 2.9 44.9 67.2 TX 63.7 95.9 59-105 0.9 56.2 86.9 46-105 2.3 43.9 80.0 1 Study sites: IL = Effingham County., IL; MO = Shelby County., MO; NE = York County., NE; and TX = Carson County., TX. 2 Average daily low temperature (°F) 3 Average daily high temperature (°F) 4 Range of daily high and low temperatures (°F) 5 Total rainfall (inches)
Monsanto Company 07-CR-191U Page 518 of 544 

Table M-4. Characteristics Evaluated at Each Site for Survival Outside Cultivation Study Characteristic Timing of Evaluation Evaluation DescriptioEarly stand count V2-V4 growth stage (at least one plant) Number of emerged plaGrowth stage monitoring V2-V4 growth stage (at least one plant) and 

every two to four weeks thereafter 

Average growth stage using Iowa State (Herman, 1997) 

Vigor monitoring V2-V4 growth stage (at least one plant) and 

every two to four weeks thereafter 

Vigor rating on a 1-9 saverage, 4-6 = averagebased on PI experience 

Late vegetative plant height V10 growth stage (approximately 50% of 

plants) 

The average height of u

Pollen shed interval Pollen shed Date when the earlies

pollen and the date whshedding pollen 

Silking interval Silking Date when the earliest 

the date when the latest 

Final stand count Within one week of typical harvest time Number of standing plaPlant height at maturity R6 growth stage (approximately 50% of 

plants) 

The average height of u

Number of ears produced Harvest Number of ears producNumber of seed produced Harvest Number of seed producAverage number of ears per plant Derived from other data Number of ears produc

stand count per plot 

Replacement value Derived from other data Ratio of the number o

number of seed planted 
Monsanto Company 07-CR-191U Page 519 of 544 

Appendix N. USDA Notifications Approved for MON 87460 

Field trials of MON 87460 were conducted in the U.S. since 2002. The protocols for 

these trials include field performance, agronomics, 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 and 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 Notifications Approved for MON 87460 and Status of Trials 

Conducted Under these Notifications. 

USDA No. Effective Date (m/d/y) Release 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) Release 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) Release 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) Release 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 Process 

 
Monsanto Company 07-CR-191U Page 524 of 544 

Appendix O. Petitioner’s Environmental Assessment 

This section provides a brief 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 Environment, and (3) Potential Environmental 

Consequences. The significance of the potential environmental impact of MON 87460 

takes into consideration both the context and intensity of the proposed action of 

deregulation in whole for MON 87460. This assessment provides data and analysis that 

appropriately addresses and evaluates potential 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 conducted in the U.S. under APHIS 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 environmental impacts, including cumulative impacts. 

O.A. Alternatives to Consider 

The action of deregulation is governed by 7 CFR 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. MON 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 “approval in whole” or full deregulated status for 

MON 87460. Information and arguments presented in this section will further 

demonstrate that MON 87460 does not present a significant environmental impact; thus, 

the requirements of NEPA are satisfied by an Environmental 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 Environmental 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 irrigation
Monsanto 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 the 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 withstand drought, resulting in hybrids that are characterized as 

having very good to excellent drought tolerance. These hybrids are currently planted 

widely throughout the Great Plains and the Midwest. MON 87460 would provide an 

additional tool for growers faced with uncertain weather conditions throughout the Corn 

Belt, particularly in the Western Dryland region of the Great Plains. 

The affected environment is described in detail 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 corn 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-agricultural lands. Finally, the affected 

environment includes related corn production and marketing practices such as specialty 

corn production, including organic corn production, 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 yield 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 significant 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 conventionally bred drought tolerant corn hybrids 

(Campos, 2006) and new biotechnology-derived corn products were introduced, total 

corn acreage remained relatively steady, 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 exports sales. The increased corn acreage 

was obtained primarily by planting fewer acres of soybean in the Corn Belt and Great 

Plains (USDA-NASS, 2007). 

A small portion of the increase in 2007 corn acreage 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 establishing specific types of plant cover on 

marginal farmland. According to the Farm Service Agency: “CRP plantings and 

practices offer our nation vast environmental benefits, including reducing soil erosion, 

improving surface and ground water quality, creating wildlife habitat, restoring wetlands, 

sequestering carbon, preserving soil productivity and reducing offsite wind erosion 
Monsanto Company 07-CR-191U Page 527 of 544 

damage.” Of the 36 M acres enrolled in the CRP in 2006, 21.2 M acres or 59% were 

from Great Plains states with 7.9 M acres or 22% from Midwest “corn-belt” states 

(USDA-FSA-CRP, 2007). 

Corn prices are a significant driver of decisions 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 with 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, some growers did not renew their enrollment 

in the CRP and instead planted their land to crops, a decision driven by market demands 

and corn prices (Hart, 2006). It is highly likely much of the corn planted on these 

marginal farmland acres were conventional hybrids with good drought tolerance 

characteristics. In this scenario, it is important to recall that MON 87460 requires the 

same agronomic management practices as conventionally bred drought tolerant corn in 

order to produce acceptable yields. Under severe water deficit, 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 enhanced yield under water-limited conditions, 

MON 87460 is agronomically and phenotypically equivalent to conventional corn and 

that numerous drought tolerant conventional corn 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, tillage, 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 availability of the MON 87460 drought tolerance 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, introduction of an enhanced susceptibility to 

insects or diseases. 

Corn is extremely sensitive to soil moisture deficits. Due to the arid climate there is a 

significant amount of acreage under irrigation in the Great Plains. The growing market 

for biofuels and worldwide concern over food shortages that have caused commodity 

prices to increase significantly may result in changes to cropping and 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 dryland production without benefit of irrigation. 

While the introduction of MON 87460 may facilitate 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 environment, 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 and 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 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 

stagger pollination times of different materials, 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 biotechnology-derived corn to non-biotechnology derived 

corn. Consequently, the advent of biotechnology-derived crops does not appear to have 

had a negative impact on specialty or organic 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, organic corn acreage increased by 300% in the 

U.S. During the same timeframe, the acreage of biotechnology-derived 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 hybrids with biotechnology traits has grown 

dramatically since the first such hybrid was planted in 1996. Those traits 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/ExtentofAdoptionTable1.htm). Despite this large 

acreage of biotechnology-derived corn, about 5% of the total corn production acres are 

devoted to production of non-biotechnology, specialty corn hybrids, including popcorn, 

waxy corn, high oil corn, white corn, blue corn, Indian corn, and high- and modifiedprotein corn hybrids (USGC, 2007).

Organic farming is a small but rapidly growing 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 percentage of the most widely planted U.S. 

field crops (0.16% of corn, 0.17% of soybean, 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 result in greater public confidence in foods that 

were labeled as “organic,” Congress passed The Organic Foods Production Act of 1990 

(OFPA). The OFPA required USDA to establish a National Organic Program (NOP) to 

develop uniform standards and a certification 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, specifically describing the NOP as “an organic 

certification program for producers and handlers of agricultural products that have been 
Monsanto 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 beyond the fact that the product was produced 

under these standards. Importantly, 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 through 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 provisions 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 this 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 the OFPA. By the time USDA implemented 

national organic standards in 2002, certified organic farmland 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 the U.S. (Table O-2), while during the same 

time frame the biotechnology-derived corn acreage 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 organic corn production and 

the increase in biotechnology-derived corn production are correlated, but they do indicate 

that the adoption of biotechnology-derived corn 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 544 

Table O-2. Certified Organic and Total U.S. Acreage of Selected Crops (USDA-ERS, 2007) 

Item Total certified organic (acres) 

U.S. 

Cropland 

2005 (acres) 

Certif

organi

total (

1995 1997 2000 2001 2002 2003 2004 2005 

Total cropland: 638,500 850,173 1,218,905 1,302,392 1,299,632 1,451,601 1,452,353 1,723,271 340,650,083 0.51% Grains--  Corn 32,650 42,703 77,912 93,551 96,270 105,574 99,111 130,672 81,759,000 0.16%  Wheat 96,100 125,687 181,262 194,640 217,611 234,221 214,244 277,487 57,229,000 0.48%  Oats 13,250 29,748 29,771 33,254 53,459 46,074 42,616 46,465 4,246,000 1.09%  Barley 17,150 29,829 41,904 31,478 34,031 30,265 26,629 39,271 3,875,000 1.01%  Sorghum 

-- 3,075 1,602 938 3,043 4,152 4,453 6,042 6,454,000 0.09% 

 Rice 8,400 11,043 26,870 29,022 22,381 20,152 22,173 26,428 3,384,000 0.78%  Spelt 12,350 1,704 12,606 7,639 6,939 9,719 6,203 8,169 -- --  Millet 18,550 12,285 15,103 23,366 17,575 26,935 21,036 14,175 565,000 2.51%  Buckwheat 13,250 7,616 10,599 14,311 8,388 8,086 7,960 6,364 -- --  Rye 2,900 4,365 7,488 7,056 9,644 11,616 10,289 8,597 1,433,000 0.60% Beans--  Soybean 47,200 82,143 136,071 174,467 126,540 122,403 114,239 122,217 72,142,000 0.17%  Dry beans 

-- 4,641 14,010 15,080 2,430 9,836 7,642 10,561 1,659,300 0.64% 

 Dry peas & lentils 5,900 5,187 10,144 9,362 7,476 16,188 15,893 17,757 571,000 3.11% Oilseeds--  Flax 5,850 8,053 25,076 20,672 20,484 14,940 35,104 30,843 983,000 3.14%  Sunflowers 14,200 10,894 19,342 15,295 7,624 7,121 9,742 6,087 2,709,000 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 biotechnology products, these have not occurred when the product 

has successfully completed the determination 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 the biotechnology-derived product. Monsanto 

does not intend to commercially release MON 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 phenotypically equivalent to conventional corn and is 

expected to enter commerce as part of the normal trade flow of yellow corn to be utilized 

in the same manner as any other yellow corn. 

Monsanto employs a rigorous product stewardship program that demonstrates respect for 

our customers, their markets, and the environment. Our market stewardship program 

considers many factors to ensure global integration and increased transparency. In 

keeping with past practice, MON 87460 will not be sold commercially 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 available to grain producers, processors, 

and buyers. Our stewardship policy is the shared responsibility of Monsanto, our 

licensees, and our customers to insure that our products are used properly. Monsanto is 

also committed to legal and ethical obligations 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 stage of their life cycle. As such, Monsanto 

has policies in place to monitor its procedures as products are researched, 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 the dietary safety of the drought tolerance trait 

expressed in MON 87460. The safety assessment of the CSPB and NPTII proteins 

included extensive protein characterization demonstrating 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 and 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 improved through biotechnology-derived traits 

and conventional hybrids provides an “equal or increased 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 derived from MON 87460, and the intended foods 

and feeds derived from MON 87460 can be considered compositionally and nutritionally 

equivalent to those derived from conventional corn with a history of safe consumption 

(Section VII). As such, it can be concluded with reasonable certainty that dietary 

exposure to MON 87460 poses no meaningful 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 conventional corn, introduction of MON 87460 is not 

anticipated to have any impact on the management 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 into Sexually Compatible Relatives 

In assessing potential risks associated with gene introgression from MON 87460 with its 

sexually compatible relatives, teosinte and gamma 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 teosinte 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 distributions 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 reproductive timing between the two species 

(Section II.D.5). First generation corn-teosinte hybrids are generally less fit for survival 

and dissemination, and they show significantly 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, which 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) (Section II.D.5). As no cases of gene flow between corn and 

its wild relatives are known to occur naturally in the U.S., there is no opportunity for an 

environmental impact. Therefore it is not anticipated that the introduction of 

MON 87460 will result in any increased potential 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 during domestication, in 

which traits often associated with weediness such as seed dormancy, dispersal 

mechanisms, or the ability to form reproducing populations outside of cultivation have 

not been selected. Consequently 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 weediness. Even when individual kernels 

of corn are distributed within a field or along transportation routes from the fields to 

storage or processing facilities, sustainable, volunteer corn populations are not found 

growing in fence rows, ditches, or road sides. 

In comparative studies conducted between MON 87460 and a conventional control, 

dormancy and germination, growth and development, and reproductive characteristics 

were evaluated for changes that would impact plant pest potential, and in particular, plant 

weediness potential. No meaningful differences were detected between MON 87460 and 

control corn (Section VIII). Therefore, the introduction of the drought tolerance trait did 

not unexpectedly alter the assessed characteristics compared to the control. These data 

indicate that MON 87460 exhibits no characteristics that would improve the ability of 

this corn to survive without human intervention, and that its cultivation should not 

interfere with the cultivation of other corn hybrids or result in its uncontrolled spread into 

non-agricultural environments. Thus, the results 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 “noxious 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 corn. As discussed above, the weediness 

potential of MON 87460 has not been altered by the presence of the CSPB and NPTII 

proteins. Therefore it is highly unlikely that MON 87460 poses any more of a risk to 

threatened or endangered plant species than conventionally bred drought 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 flowering 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 pesticidal activity. As such all 

exposed organisms, including threatened or endangered species are considered to be 

NTOs. During the U.S. phenotypic field trials at multiple locations in 2006 and 2007, 

multiple observations for plant disease incidence as well as effects on several insect and 

arthropod species were made to assess whether the plant-disease or plant-insect 
Monsanto Company 07-CR-191U Page 534 of 544 

interactions of MON 87460 were altered compared to commercial corn 

(Section VIII.F.1). Of the more than 375 pest and non-pest arthropod evaluations, only 

two differences were observed between MON 87460 and the control. The differences 

detected were either within the range of the commercial reference hybrids or were 

isolated to a single study-site location. Because of the small number of differences that 

were not consistently observed, they are not considered to have biological significance. 

Out of the more than 425 disease stressor observations, no differences were detected 

between MON 87460 and the control. These results support the conclusion that 

MON 87460 does not have altered environmental interactions relative to other 

conventional corn, and plants that express the CSPB protein show no apparent impact on 

NTOs. 

The potential for MON 87460 to pose a meaningful 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 species was considered. As described in 

Section VIII, evidence is presented demonstrating that MON 87460 has no increased 

weediness potential compared to conventional corn. Second, the possibility that 

threatened and endangered species were at risk through hazard and level of exposure was 

examined. As discussed in Sections VI and VIII.F.1, the CSPB and NPTII proteins are 

unlikely to pose a significant hazard to arthropods 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, compositional 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 negligible, 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 detected based on extensive characterization of MON 87460, 

which included phenotypic evaluation conducted in field studies over a broad range of 

environmental conditions. Furthermore, environmental interaction field studies, 

including evaluations of pest and non-pest arthropods and interaction with other biotic 

factor testing on MON 87460 revealed no potential to adversely impact NTOs. As such, 

the potential consequences to biodiversity are the same as with conventional corn. This 

conclusion is based on the history of safe use of the host plant, corn, the extensive 

characterization of MON 87460 compared to a conventional control, 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 advantage only under specific 

conditions in the field, i.e., under water-limited conditions, which are predictable and 

spatially limited. This selective advantage is consistent with that shown by conventional 

drought tolerant corn hybrids currently planted in the U.S. This trait was shown not to 
Monsanto Company 07-CR-191U Page 535 of 544 

provide a meaningful selective advantage or disadvantage that altered the survival of 

MON 87460 volunteer plants in cultivated fields (Section VIII.F.3) or in areas that were 

not cultivated for agricultural production (Section VIII.F.4). Thus, it is concluded that the 

likelihood of MON 87460 spreading in the environment 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 Impacts 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 biotechnology-derived products, once single events 

are assessed and determined to be safe for human and animal consumption and safe to the 

environment, then combining the unrelated single events through conventional breeding 

should not pose any new characteristics which would change the safety assessment 

conclusions. Further, the use of conventional breeding to produce combined trait or 

combined event products would identify off-types and non-performing 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 herbicide tolerance and insect protection traits 

have been grown commercially in the U.S. since 2000 with no reported incidence of 

increased weediness potential. MON 87460, which provides reduced 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 increased pest potential or significant impact to 

the human environment or threatened and endangered species. In comparative studies 

conducted between MON 87460 and a conventional control, dormancy and germination, 

growth and development, and reproductive characteristics were evaluated for changes 

and none were observed (Section VIII). Additionally, 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 conditions and no differences from conventional 

corn were observed (Section VIII.F.2). Therefore, cumulative impacts are not anticipated 

from combining MON 87460 with one or more deregulated herbicide tolerance or insect 

protected traits. 

O.C.5.2. Potential for Cumulative Impacts on Biodiversity, Preservation of Corn 

Germplasm Purity, and Specialty Corn Production 

The petition has discussed, in great detail, the outcrossing potential of contemporary 

domestic corn varieties and their wild relatives. Sections VII and VIII presented data on 

the phenotypic, agronomic, environmental interactions, and compositional assessment of 
Monsanto 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 concluded 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 fields; (2) unconfined 

cultivation should not lead to increased weediness 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 Section 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 and endangered species. Based 

on these analyses, there is negligible potential for adverse effects 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 preserve the purity of corn germplasm developed 

for various uses including food, feed, processing, biofuels, 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 genetic purity and seed quality. Biotechnologyderived corn traits have been incorporated into these systems while achieving accepted 

purity standards for the corn industry and taking into consideration the cumulative effects 

of biotechnology-derived corn products. 

To maintain the genetic purity of inbred 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 stagger pollination times of different materials, 

detasseling, and the use of cytoplasmic male sterility. Additionally, production 

guidelines and operating procedures are used to ensure genetic purity and quality 

throughout the entire production process from planting and growth of the crop, through 

harvesting, transport, conditioning, packaging, storage and sale. With these operating 

procedures being widely used in the corn industry, the industry has been able to introduce 

new inbreds and hybrids, including biotechnology-derived corn, into the marketplace 

while still maintaining the desired levels of purity and quality of individual types. Thus, 

over the last 70 years, systems have been developed to maintain the purity and quality of 

the diverse corn types needed to meet the needs and desires of corn farmers and 

consumers. These systems have allowed the introduction of biotechnology-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 existence 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 ensure the genetic purity of conventional and organic seeds. 
Monsanto Company 07-CR-191U Page 537 of 544 

Table O-3. Organic and Conventional Seed Sources 

Organic Corn Seed Sources1 Conventional Corn Seed Sources 

Albert Lea Seed House Garst Seed2

Blue River Hybrids Heirloom Seed3

Golden Grains Kruger Seed4

Great Harvest Organics Monsanto (Asgrow/DeKalb)5

Merit Seeds Pioneer6

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 considering the potential cumulative effect of 

multiple biotechnology-derived traits being introduced to corn. Plant breeders, 

institutions, government and non-government agencies take great strides to maintain corn 

germplasm resources. Corn genetic diversity is maintained through public and private 

plant breeding efforts, seed conservation in gene banks, germplasm collections 

maintained by the International Maize and Wheat Improvement Center (CIMMYT) and 

other governmental agencies, on-farm conservation and enhancement activities in Latin 

America, and conservation of corn wild relatives in Mexico and Central America. These 

efforts maintain and preserve corn genetic diversity necessary for improving the base 

genetics of corn, separate from, or in combination with, the breeding practices used to 

introduce biotechnology-derived traits into new corn inbreds 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 greatest percentage of organic 

corn production often have above-average penetration of biotechnology-derived crops. 

For example, the leading organic corn-growing states are Iowa, Minnesota, and 

Wisconsin. Of these, Iowa and Minnesota have above-average penetration of 

biotechnology-derived crop plantings (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 established processes and methods (e.g., isolation, 

detasseling, and use of cytoplasm male sterility) as described above to ensure the genetic 

purity of their corn products. Organic producers use process-based standards outlined in 
Monsanto 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 purchasers of the products. No claims 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 segregation practices, seed 

producers have numerous organic corn seed hybrids available to them and AOSCA has 

developed protocols by which a seed developer can produce 99% “non-GMO” seed. 

Therefore, it is not anticipated that the introduction of MON 87460 will have a negative 

impact on specialty and organic farming. 

In conclusion, measures of preservation of corn germplasm purity including temporal and 

spatial isolation, detasseling, and the use of cytoplasmic male sterility have been well 

established and have a track record of effectiveness over many years. The adoption and 

large scale cultivation of biotechnology-derived corn in the past decade did not have any 

negative impact on biodiversity, genetic purity of corn, or the production of specialty 

corn including organic corn in the U.S. There is no evidence to suggest that the 

introduction of MON 87460 corn will adversely impact biodiversity, genetic purity of 

corn germplasm, or small scale specialty use corn production even considering the 

potential cumulative effect of multiple biotechnology-derived traits. It is also concluded 

based on past experience and standards that the introduction of MON 87460 will not have 

an impact on the production of specialty corn products, including organic corn. 

O.C.5.3. Potential for Cumulative Impacts on Land Use and Agronomic Practices 

The introduction and rapid adoption of biotechnology-derived corn products in the past 

decade have had no significant impact on cropland 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 annual field corn acres fluctuated around 78 

million acres (Table IX-3), while in the same time frame the adoption rate for 

biotechnology-derived corn products increased from zero to 61%. In 2007, a significant 

increase (16%) in corn acreage to 93M acres occurred with a corresponding 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 (USDA-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 market demand remains relatively high for food, 

feed, ethanol production, and other industrial uses. 

From the information above it is clear that starting with first biotechnology-derived 

product, MON 810 originally cultivated in 1997, the addition of herbicide 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 insecticide and herbicide use and has 

supported the increase in no-till corn acreage resulting in less soil erosion and less runoff 

of pesticides and water (Brookes and Barfoot, 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 trait, its introduction 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, crop 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 bred, drought tolerant hybrids have been 

planted in the U.S. for decades without any documentation of such effects. MON 87460 

is not phenotypically and agronomically different 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 without the added benefits of irrigation. Over 

time, this may result in a cumulative benefit by decreasing the amount of water used for 

agriculture in the Great Plains. 

O.C.5.4. Potential for Cumulative Impacts on Non-agricultural Environments and 

Threatened and Endangered Species 

Basic to all evaluations of biotechnology-derived corn’s potential for cumulative impact 

to the environment or threatened and endangered 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 endangered plant species) in the U.S., except for 

an occasional botanical garden specimen or small feral populations of Zea mexicana in 

Florida, Alabama and Maryland or Zea perennis in South Carolina 

(http://plants.usda.gov). Corn exhibits extremely limited seed dormancy, has no weedy 

characteristics, and volunteers are easily controlled. It is not capable of establishing 

persistent populations in unmanaged environments. As demonstrated previously, the 

presence of the CSPB protein in no way alters 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 included extensive protein characterization 

demonstrating the lack of similarity to known allergens and toxins and the long history of 

safe consumption of similar proteins. In addition, data confirmed 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 exposed organisms in the environment. 

Compositional equivalence between corn improved through biotechnology-derived traits, 

and conventional hybrids provides an “equal or increased 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 grain derived from MON 87460, and the intended 

foods and feeds derived from MON 87460, can be considered compositionally equivalent 

to those derived from conventional corn with a history of safe consumption (Section VII). 

As such, it can be concluded with reasonable certainty that environmental exposure to 

MON 87460 poses no meaningful risk to organisms in the environment including 

humans. 
Monsanto Company 07-CR-191U Page 540 of 544 

Given these attributes and the fact that MON 87460 has a mechanism of action different 

from previously deregulated biotechnology-derived products, it is highly unlikely that 

MON 87460 when combined through traditional breeding methods with insect protected 

and/or herbicide tolerant traits will have a cumulative impact on threatened and 

endangered species or non-agricultural environments. 

O.C.5.5. Potential for Cumulative Impacts on Economic and Environmental 

Interests 

The introduction and adoption of biotechnology-derived 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 acres 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 million acres in response to increased demand 

which resulted in substantially 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 increased corn yields, reduced 

mycotoxin levels in corn grain, enhanced simplicity and flexibility of insect pest control 

and weed management, reduced chemical insecticide and herbicide use, increased no-till 

corn acreage which resulted in less soil erosion and less runoff of pesticides and water 

(Koziel et al., 1993; Martin and Hyde, 2001; Carpenter and Gianessi, 2001; Gianessi et 

al., 2002; Shelton et al., 2002; Fawcett and Towery, 2002; Hyde et al., 2003; Carpenter et 

al., 2004; Sankula and Blumenthal, 2004; Sankula et al., 2005; Sankula, 2006; Wu, 2006; 

NCGA, 2007). 

In a recent study, economists 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 crops have resulted in substantial global 

economic and environmental benefits. Over the past 11 years biotechnology-derived 

crop adoption has positively impacted the environment by reducing greenhouse gas 

emissions from agriculture and reducing pesticide 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 growers worldwide for all 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 agricultural biotechnology is a complementary tool to traditional 

farming methods that can help poor farmers and consumers and improve food security 

(UN-FAO, 2004). 

These benefits are from biotechnology crops that were designed to be tolerant of or 

resistant to biotic environmental stressors such 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 abiotic environmental stressors such as 

drought. Globally, drought is the most important limiting abiotic factor of corn yield 

(Barker et al., 2005; Campos et al., 2006). Significant progress has been made to reduce 

corn yield loss in water-limited environments through breeding and cultural practices, but 

there remains potential for additional improvement. Positive impacts on yield and 

reduced yield loss in water-limited environments will continue to provide value to 

producers, consumers, and the environment. 

Given the above, Monsanto fully anticipates that drought tolerant 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 environment previously discussed are highly uncertain 

or involve unique or unknown risks. 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 characteristics, with the exception of the drought 

tolerance trait. As to the drought tolerance trait in MON 87460, it is similar to that found 

in conventionally bred, drought tolerant corn hybrids 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 endangered species, biodiversity, specialty and 

organic farming, and non-agricultural environments is envisioned since corn cannot 

establish and persist outside an agricultural environment and MON 87460 has been 

demonstrated to be phenotypically and compositionally equivalent to conventional corn. 

Any potential changes in agricultural practices would be consistent with the commercial 

production of conventional hybrids with good drought tolerance, thus these agricultural 

practices would not be significantly impacted by the introduction of MON 87460. 

The drought tolerance trait of MON 87460 has a mechanism of action unique from other 

deregulated biotechnology-derived traits. Therefore, there are no anticipated cumulative 

impacts from combining MON 87460 with deregulated herbicide tolerant, insect 

protected, or nutritionally enhanced biotechnology-derived products, when viewed in the 

context of pre-existing agricultural practices. For the same reason, cumulative impacts 

on biodiversity, threatened and endangered species, specialty and organic corn production 

are also not anticipated. Because MON 87460 reduces yield loss under water-limited 

conditions, it is foreseeable that continued 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 the Great Plains. This trend is already 
Monsanto 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 demonstrate that MON 87460 does not present a 

plant pest risk. On the basis of this analysis, the requested action of deregulation in 

whole does not present a significant environmental impact and should lead to a Finding 

of No Significant Impact. 

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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 impacts on US agriculture of biotechnologyderived crops planted in 2005. National Center 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. Blumenthal. 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, ecological, 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 agriculture 2003-2004: Agricultural Biotechnology 

– Meeting the Needs of the Poor, Part 1. 39 pp. 
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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 

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Farm Service Agency, Conservation Reserve Program (USDA-FSA-CRP). 

USDA-NAAS. 2006. 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 Statistics Service, United States Department 

of Agriculture. Acreage Report, Cr Pr 2-5 (6-07) Accessed June 30, 2008. 

USGC. 2007. Value Enhanced Corn Report 2005/2006. United States Grain Council, 

Washington, D.C., http://www.grains.org/galleries/technical_publications/ 

USGC%20Value%20Enhanced%20Corn%20Report%202006%20%20(English).

pdf 

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. 

Wu, F. 2006. Mycotoxin reduction in Bt corn: potential economic, health, and 

regulatory impacts. Transgenic Research. 15:277-289. 

Wych, R.D. 1988. Production of hybrid seed corn. Pages 565-608 in Corn and Corn 

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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 addendum 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 the 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 provided to any third party. Monsanto understands 

that a CBI-deleted 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, publication or other distribution of this 

information (including website posting) without 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 conventional corn. Subsequent studies 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 Appendix 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 relative water content data for MON 87460, 

other CSPB-containing events and a conventional 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 exhibit 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 grown under water-limited 

conditions in the field. The data show that the total water potential of corn transformation events 

containing the same genetic construct as MON 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 transformation 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 Petition 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 analyzed. The two years of data collected on plants 

grown under well-watered and water-limited field conditions demonstrate that MON 87460 does 

not deplete soil moisture differently from conventional corn. This observation is consistent with 

the conclusion in Appendix O of Petition 09-055-01p (Petitioner’s Environmental Assessment) 

that MON 87460 is expected to be similar to conventional corn with respect to its agronomic 
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Monsanto Company 07-CR-191U Page 4 of 15 

characteristics and environmental impact. Additionally, 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 conventional 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 moisture is sufficient to produce adequate yields. 

Data for this analysis were collected from field 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 significantly 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/acre) that was statistically 

significant (p<0.05). Similarly in 2008, yields for MON 87460 and the control were not 

significantly different under well-watered conditions (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/acre) that was statistically 

significant (p<0.05). 

Materials and methods 

In the 2007 study, ten replicates each of MON 87460 and the control were grown under waterlimited conditions from the V7 to R2 growth stages. Five replicates each of MON 87460 and the 

control were grown under well-watered conditions. Each replicate consisted of four six-row 

plots 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. 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 growth stages. The same design was used for the 

well-watered treatment. Each replicate consisted of four eight-row plots 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 moisture probes (SenTek, EnviroSmartTM) 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 replicates 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 were installed within five replicates each for MON 87460 and 

control. 

Prior to installation of the soil moisture probes, each sensor was 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 electrical output signals from the sensors decline in 
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Monsanto Company 07-CR-191U Page 5 of 15 

proportion. The data are generated in scale frequency units and converted to mm soil moisture 

using a soil type-specific calibration equation obtained from the manufacturer. Soil moisture 

depletion was calculated by subtracting later readings from earlier readings. 

Cumulative soil moisture depletion calculations were made by adding the 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 season using analysis of variance. The paired 

design assumes equal variance at each pair (replicate) and standard error will therefore be equal 

across entries (test and control). The standard errors are calculated across all of the replicates 

within each of the two irrigation treatments, leading to a single standard error value for both the 

test and control. 

Results 

Table 1-1 summarizes the cumulative soil moisture depletion across the 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, respectively) or water-limited conditions (272.29 mm vs. 284.19 

mm, respectively). 

Table 1-2 summarizes the cumulative soil moisture depletion across the 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, respectively) or water-limited conditions (182.87 mm vs. 195.52 

mm, respectively). 

Collectively, these data presented show that 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 advantage 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 conditions, key measures of root biomass do not 

differ between MON 87460 and conventional corn. The results of this study further support data 

presented in Section VIII of Petition 09-055-01p demonstrating 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 understanding of whether roots from MON 87460 are 

likely to have extended deeper into the soil profile beyond the range of the soil moisture probes 

used in the field studies 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 established in pots in a greenhouse and grown under well-watered or 

water-limited conditions. Measurements were taken of growth stage, plant height, root and shoot 

biomass and water consumption. Water-limited conditions were imposed from the V7 to VT/R1 

growth stages. 
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Materials and methods 

The test substance, MON 87460, and a conventional isogenic control were established in 3-foot 

tall, 8-inch diameter pots filled with Turface Field and FairwayTM, an inert, clay-based medium 

which provided good capillary action to distribute water evenly throughout 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-watered 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 VWC 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. Plant fertility was maintained through the use of 

Hoagland’s nutrient solution for every irrigation ranging from 0.5X 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 stage, 25 days after imposition of drought stress 

on the water-limited treatment. 

Plants were evaluated within each treatment for plant height and growth stage three times during 

the experiment: prior to treatment, 13 days after treatment (DAT) when plants in the waterlimited treatment exhibited leaf rolling (an indicator of drought stress), and at the conclusion of 

the experiment 25 DAT. Growth stage was determined using methods described by Iowa State 

University (2008). Fresh and dry weights of shoots 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 differences were detected between MON 87460 and the control for any 

endpoint, including root weight and ratios of root:shoot biomass. Root:shoot ratio was 

calculated to assess the partitioning of biomass into above- and below-ground biomass in 

response to drought stress. MON 87460 did not differ from the control in its partitioning of 

biomass into roots and shoots under well-watered 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 conditions. The results of these measurements 

used to assess root biomass and development 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 the roots under both well-watered and water-limited 

conditions. 

1.4 Summary 

The data presented in this addendum to Petition 09-055-01p indicate that MON 87460 does not 

exhibit altered soil moisture depletion 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 registered 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-limited conditions. Within the 

water-limited treatments, MON 87460 exhibited statistically significant yield 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 demonstrating that MON 87460 does not differ 

agronomically or phenotypically from conventional corn and are consistent with the conclusion 

in Appendix O of Petition 09-055-01p that MON 87460 is expected to be similar to conventional 

corn with respect to its water use characteristics and potential environmental impact. 

References 

Iowa State University. 2008. How a Corn Plant 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. 09-055-01p

Monsanto Company 07-CR-191U Page 9 o

Table 1-3. Plant Growth Parameters of MON 87460 and the Control Prior to Irrigation Treatment anAfter Treatment (DAT) 

 Well-Watered Water-Lim

Characteristic MON 87460 Control MON 87460 

Prior to Height (cm) 104.8 103.3 105.3 treatment Growth stage1 V7-V8 V6-V8 V7 

Post-treatment Height (cm) 166.2 166.4 133.2 

 (13 DAT) Growth stage V12-VT V12-VT V10-V12 

Post-treatment Height (cm) 198.1 197.6 178.9 

(25 DAT) Growth stage VT-R2 VT-R2 VT 

Shoot fresh weight (g) 557.1 537.5 279.1 Root fresh weight (g) 405.4 411.9 241.1 Shoot dry weight (g) 104.5 102.3 58.1 Root dry weight (g) 38.2 37.4 25.9 Root:shoot ratio2 0.36 0.37 0.45 Total water consumed3 (L) 35.0 34.0 19.6 

Note: No statistically significant differences (α=0.05) were detected within treatment between MON 87460 

analysis of variance. 

1 Growth stage was determined using methods described by Iowa State University (2008). 2 Root: shoot ratio was calculated by root biomass divided by shoot biomass. 3 Total water consumed was calculated by subtracting the water remaining in the pot at the conclusion of the exp

amount of water applied throughout the experiment. 
Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 10 of 15 

Section 2 

Supplementary Text on Impacts 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 demonstrating that MON 87460 does not differ agronomically 

or phenotypically from conventional corn and is no different from conventional corn in its plant pest 

potential. This lack of differences combined with the information provided here also supports the 

conclusion that MON 87460 is unlikely to significantly alter agronomic practices, crop rotation 

patterns and their impacts, or the number of acres planted with biotechnology-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, reduced tillage and altered planting strategies 

have commonly been employed to optimize corn yields in areas where water use efficiency is a 

primary concern. MON 87460 is expected to be incorporated into these practices similarly to 

conventionally bred drought tolerant varieties and is expected to comprise one part of a risk 

management strategy that emphasizes conventional 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 implement their preferred agronomic practices. 

Drought tolerance imparted through conventional breeding allows corn production to occur throughout 

areas of the Western Great Plains that typically 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 experience drought stress; an 

additional 50-65% of the hybrids offered for planting in these areas were rated as having very good 

performance in fields that regularly experience 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 practices are well-known for improving water management in dryland crop 

production and improving yield potential (Croissant et al., 2008). Research conducted by the 

University of Nebraska demonstrated that plots managed under no-till conditions 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 projected profits per unit area. MON 87460 is not expected to have a 

significant impact on the number of acres being managed using reduced till or no-till practices because 

such practices are expected to complement the drought tolerance trait. 

Plant density also impacts yield potential with optimum planting densities in dryland cropping systems 

determined by the amount of available 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 available 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 growth 
Addendum 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 density decisions based on 

soil moisture at planting. 

Skip row planting is a specific method for altering plant density and improving corn drought tolerance. 

As its name implies, skip row planting involves skipping one or two rows of corn for every one or two 

rows planted. The underlying idea is to prevent developing plants from exhausting soil moisture early 

in the season leaving a reserve of soil moisture to support reproductive development (Klein, 2006). 

Accordingly, no-till management is recommended for growers employing skip row practices. 

Research on skip row planting indicates that the greatest benefit occurs when yields obtainable through 

standard planting methods are less than 100 bushels/acre (Lyon et al., 2009). For example, in dry 

years when standard planting methods produced 16-21 bushels/acre, skip row planting produced 25-50 

bushels/acre (Klein, 2006). Skip row planting, therefore, can allow corn growers to maintain 

profitability in locations where soil moisture could otherwise preclude a profitable crop. 

2.2 Crop Rotation 

MON 87460 is not expected to significantly alter crop rotation patterns because recent trends in crop 

rotations already favor corn. Farmers 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 Table 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 (USDA NASS, 2007). 

Rotation with other crops is 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). Croissant et al. (2008) also advise that dryland growers avoid following a 

particular crop with itself to reduce problems with insects, disease and volunteer plants. In the U.S. 

Corn Belt, soybean is a common rotational crop although sorghum is also used. In the Western Great 

Plains, winter wheat, sorghum and soybean may be planted in rotation with 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 research has 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 weed 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. According 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 current 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, rainfall patterns, existing levels of nitrogen and phosphorous, soil organic 

matter and rotation history. The impact of crop rotation choices in the Western Great Plains therefore 

will vary according to these factors. Table 2-1 summarizes recommended nitrogen and phosphorous 

inputs for corn, sorghum, soybean and winter wheat, four crops commonly used in Western Great 

Plains rotations. The table also presents transpiration ratios, the weight of water transpired to produce 

an equal weight of above ground dry matter. Depending on the specific 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 phosphorous than soybean and winter 

wheat. Conversely, the amount of water needed to produce a pound of above-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 required in the Western Great Plains to produce a 

profitable crop. MON 87460 is not expected to have 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 acres planted with biotechnology-derived corn varieties 

or impact the ability of organic and specialty corn growers to maintain their current practices. 

Appendix O also notes that states with the greatest number of acres planted with organic corn also 

have the greatest number of acres planted with biotechnology-derived 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-derived 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 ERS, 2009). For the Western Great Plains states 

where state-specific data are available, 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 with biotechnology-derived varieties 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 combined-trait product with herbicide 

tolerance and insect resistance traits and is therefore the acres planted with MON 87460 are expected 

to be the same acres already planted with 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-derived 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 planted with biotechnology-derived varieties (USDA ERS, 

2009) indicating that the cultivation of biotechnology-derived corn varieties is not necessarily 

incompatible with the cultivation of organic corn. 

2.4 Summary 

This document augments the information presented 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 described 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 corn varieties. Growers in the Western Great Plains have selected 

conventionally-bred drought tolerant hybrids, minimized tillage and altered planting density for many 

years to manage soil moisture and maximize yields under conditions that would otherwise be 

unfavorable for corn production. MON 87460 is expected to be incorporated into these practices 

similarly to conventionally bred drought tolerant varieties. Additionally, given the importance of 
Addendum to USDA Petition No. 09-055-01p

Monsanto Company 07-CR-191U Page 13 of 15 

economic factors and local conditions in determining crop choices, MON 87460 is not expected to 

have a significant impact on crop rotation 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 unlikely that MON 87460 will significantly alter the number of acres planted with 

biotechnology-derived corn varieties in the Western Great Plains, given the current significant 

percentage of acres in the Western Great Plains already planted with corn containing biotechnologyderived traits. As a result, MON 87460 is not expected to have a significant impact on land use within 

the Western Great Plains specifically 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.colostate.edu/pubs/crops/00516.html. Accessed May 7, 

2010. 

DAS. 2010. Dow AgroSciences LLC. Mycogen 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 – Lincoln Extension, Institute of Agriculture and Natural 

Resources. http://www.ianrpubs.unl.edu/sendIt/g859.pdf. Accessed 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, Institute 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 Nebraska – 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. Characterizing Pioneer® brand Hybrids for Drought Tolerance. 

http://www.pioneer.com/web/site/portal/menuitem.b98005ee4490c1a5d6c1f492d10093a0/. Accessed June 

9, 2010. 

Pryor, R. 2006. No-till farming in dryland cropping systems. Publication EC196-2. University of Nebraska – 

Lincoln Extension, Institute of Agriculture 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. Wortman, D.T. Waters. 2008. Fertilizer suggestions for corn.

Publication EC117. University of Nebraska – Lincoln Extension, Institute of Agriculture and Natural 

Resources. http://www.ianrpubs.unl.edu/sendIt/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/ExtentofAdoptionTable1.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 soy 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. United 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. University of Nebraska – Lincoln Extension, Institute 

of Agriculture and Natural Resources. http://www.ianrpubs.unl.edu/sendIt/g1669.pdf. Accessed May 7, 

2010.

 
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Monsanto Company 07-CR-191U Page 15 of 15 

Table 2-1. Inputs for corn, sorghum, soybean and winter wheat under dryland conditions 

Crop 

Nitrogen 

(lb N/acre)

Phosphorous 

(lb P2O5/acre) Transpiration ratio1,2

Corn3 0 – 280 0 – 80 349 

Sorghum4 0 – 200 0 – 80 304 

Soybean5

 0 – 100 0 – 65 646 

Winter wheat6

 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 planted with biotechnology-derived varieties and the 

number of acres planted with organic corn in states comprising the U.S. Western Great Plains 

State 

Percent of total acres within each 

state planted with biotechnologyderived varieties1Acres of organic corn planted for 

grain or seed in each state2

Colorado 783 1,117 

Kansas 91 3,746 

Montana 783 NR 

Nebraska 91 8,991 

New Mexico 783 NR 

North Dakota 93 1,189 

Oklahoma 783 NA 

South Dakota 96 2,694 

Texas 84 2,985 

Wyoming 783

 331 

1

 Data are for 2009 and were obtained from USDA ERS, 2009. 

2 Data are for 2008 and were obtained from USDA NASS, 2010, Table 26. The total reported for the 

U.S. was 143,074 acres of organic corn planted for grain or seed. 

3 Values not available for these states. The value presented in the 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.