# Monsanto Company 12-CT-244U 1 of 620 

# Petition for the Determination of No nregulated Status for Dicamba and

Glufosinate-Tolerant Cotton MON 88701

The undersigned submits this petition under 7 CFR § 340.6 to request that the

Administrator make a determination that the article should not be regulated under

7 CFR Part 340.

July 2, 2012

(Revised September 10, 2012)

OECD Unique Identifier: MON-887Ø1-3

Monsanto Petition Number: 12-CT-244U

Submitted by:

Marianne Malven, M.S.

Monsanto Company

800 North Lindbergh Blvd.

St. Louis, MO 63167

Phone: (314) 694-2225

Fax: (314) 694-8414

E-mail: marianne.malven@monsanto.com

Prepared by:

S.M. Arackal, M.S., B.A. Comstock, B.S ., D. Chandu, Ph.D., A.E. Deffenbaugh, B.S.,

S.R. Eskelsen, Ph.D., D. Howard, B.S., M. Malven, M.S., J.K. Soteres, Ph.D.

Contributors and/or Pr incipal Investigators:

E. Bell, Ph.D., S.L. Bollman, Ph.D., P.L. Bo mmireddy, Ph.D., L. Burzio, Ph.D., B. Chen,

Ph.D., K.S. Crowley, Ph.D., R. Frazier, M.S ., A. Galadima, Ph.D., C.W. Garnaat, M.S.,

Xin Gu, Ph.D., J.M. Harrison, M.Sc., M.A., J.L. Hart, M.S., R.A. Hernan, Ph.D.,

R. Heeren, B.S., T. Kaempfe, B.S., H.T. Kang, M.S., M. Koch, Ph.D., K.R. Lawry, M.S.,

T.C. Lee, Ph.D., S.L. Lemke, Ph.D., K.J. Mathis, K.E. Niemeyer, M.S., L.F. Ralston,

Ph.D., A. Silvanovich, Ph.D., M.B. Spaur, B.S., Q. Tian, M.D., Ph.D., R. Wang, M.S.

# Monsanto Company 12-CT-244U 2 of 620 

# RELEASE OF INFORMATION

Monsanto is submitting the information in this petition for review by the USDA as part of

the regulatory process. By submitting this information, Monsanto does not authorize its

release to any third party. In the event the USDA receives a Freedom of Information Act

request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of this

information, Monsanto expects that, in adva nce of the release of the document(s), USDA

will provide Monsanto with a copy of the material proposed to be released and the

opportunity to object to the release of a ny information based on appropriate legal

grounds, e.g ., responsiveness, confiden tiality, and/or competitiv e concerns. Monsanto

understands that a copy of this information may be made available to the public in a

reading room and upon 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 (incl uding website posting) without Monsanto's

prior notice and consent.

©2012 Monsanto Company. All Rights Reserved.

This document is protected under copyright la w. This document is for use only by the

regulatory authority to which it has been submitted by Monsanto Company and only in

support of actions requested by Monsanto Co mpany. 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 to use or license the

information or intellectual propert y described in this document.

# CERTIFICATION

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

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

includes all relevant data and information known to the petitioner that are unfavorable to

the petition.

Marianne Malven, M.S.

Regulatory Affairs Manager

Address:

Monsanto Company

800 North Lindbergh Blvd., C3SD

St. Louis, MO 63167

Tel: (314) 694-2225

Fax: (314) 694-8414

Monsanto Company 12-CT-244U 30f620

# Monsanto Company 12-CT-244U 4 of 620 

# EXECUTIVE SUMMARY

The Animal and Plant Health Inspection Service (APHIS) of the United States

Department of Agriculture (USDA) has resp onsibility under the Plant Protection Act

(Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the

introduction and dissemination of plant pe sts 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 arti cle does not present a pl ant pest risk and no

longer should be regulated. If APHIS dete rmines that the regulated article does not

present a plant pest risk, the petition is gr anted, thereby allowing unr estricted introduction

of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of

nonregulated status for th e new biotechnology-derived co tton product, MON 88701, any

progeny derived from crosses between M ON 88701 and conventional cotton, and any

progeny derived from crosses of MON 88701 with biotechnology-derived cotton that

have previously been granted nonre gulated status under 7 CFR Part 340.

Product Description

Monsanto Company has developed dicam ba and glufosinate-tolerant cotton,

MON 88701, which will allow in-crop applicati ons of dicamba herbic ide for the control

of broadleaf weeds from preemergence to seven days preharvest and glufosinate

herbicide for broad spectrum weed control from emergence through early bloom growth

stage. MON 88701 provides a wider dicamba window of application beyond the current

preplant cotton uses and glufos inate application rates and timi ngs that are equivalent to

current commercial glufosinate-tolerant co tton. The combination of these two unique

herbicide modes-of-action provides an eff ective weed management system for cotton

production. Dicamba provides effective cont rol of over 95 annual and biennial weed

species, and suppression of over 100 perennial broadleaf and woody plant species.

Glufosinate, a broad-spectrum contact herb icide, provides nons elective control of

approximately 120 broadleaf and grass weeds. Additionally, dicamba and glufosinate

provide control of herbicide-resistant weed s, including glyphosate- resistant biotypes of

Palmer amaranth ( Amaranthus palmeri ), marestail ( Conyza canadensis ), common

ragweed ( Ambrosia artemisiifolia ), giant ragweed ( Ambrosia trifida ) and waterhemp

( Amaranthus tuberculatus ).

MON 88701 contains a de methylase gene from Stenotrophomonas maltophilia that

expresses a dicamba mono-oxygenase (DMO) pr otein to confer tolerance to dicamba

herbicide. DMO protein rapi dly demethylates dicamba to the herbicidally inactive

metabolite 3,6-dichlorosalicylic acid (DCSA). DCSA has been previously identified as a

metabolite of dicamba in cotton, soybean, livestock, and soil. Monsanto will request a

registration from U.S. EPA for the expande d use of dicamba on MON 88701, an increase

in the dicamba residue tolerance for cottonseed, the establishment of a tolerance for

cotton gin by-products, and the inclusion of DCSA in the residue definitions for both

cottonseed and gin by-products. No other revisions to th e dicamba pesticide residue

tolerances are necessary including animal products such as meat, eggs, and milk.

# Monsanto Company 12-CT-244U 5 of 620 

# Furthermore, the use of dicamba on MON 88701 does not present any new environmental

exposure scenarios not previously evalua ted and deemed acceptable by U.S. EPA.

MON 88701 also contains a bialaphos resistance ( bar ) gene from Streptomyces

hygroscopicus that expresses the phos phinothricin N-acetyltransf erase (PAT) protein to

confer tolerance to glufos inate herbicide. PAT ( bar ) 1 

# protein acetylates the free amino

group of glufosinate to produ ce non-herbicidal N-acetyl gl ufosinate, a well known

metabolite in glufosinate-tolerant plants. The use pattern and rate of glufosinate on

MON 88701 will follow the existing glufosinat e-tolerant cotton uses outlined on the

glufosinate herbicide label. The glufosinate residues in MON 88701 treated with

commercial glufosinate rates are below the es tablished pesticide residue tolerances for

both cottonseed and gin by-products. Therefore, Monsanto will not seek any changes in

the glufosinate label or the established tolerances for its use on MON 88701 cotton.

MON 88701 will be combined, through traditional breeding methods, with other

deregulated herbicide-tolerant ( e.g ., glyphosate-tolerant) even ts. The in-crop use of

dicamba and glufosinate herbicides, in a ddition to glyphosate he rbicide, provides

improved weed management options in cotton to control a broad spectrum of grass and

broadleaf weed species and effective contro l of weeds resistant to several herbicide

families. Successful integration of MON 88701 into glyphosate-tolerant cotton systems

will provide: 1) an opportunity for an effici ent, effective weed management system for

hard-to-control and herbicide-resistant weeds; 2) a flexible system for two additional

herbicide modes-of-action for in-crop application in current cotton production systems as

recommended by weed science experts to manage future weed resistance development;

3) an option to delay or prevent further re sistance to glyphosate and other critically

important cotton herbicides; in pa rticular, herbicides in the acetolactate synthase inhibitor

(ALS) and protoporphyrinogen oxidase inhibitor (PPO) class of chemistry; 4) crop safety

to dicamba, glufosinate, and glyphosate; and 5) additional weed management tools to

enhance weed management systems necessary to maintain yield and quality to meet the

growing needs of the food, f eed, and industrial markets.

Data and Information Presented Confirms th e Lack of Plant Pest Potential and the

Food and Feed Safety of MON 88701 Compared to Conventional Cotton

The data and information presented in this petition demonstrate MON 88701 is

agronomically, phenotypically, and compos itionally comparable to commercially

cultivated cotton, with the exception of its to lerances to both dicamba and glufosinate.

Moreover, the data presented demonstrate MON 88701 is unlikely to pose an increased

plant pest risk, including weediness, or a dverse environmental impact, compared to

commercially cultivated cotton. The food , feed, and environmental safety of

MON 88701 was confirmed based on multiple, well-established lines of evidence:

1 

# PAT ( bar ) indicates the PAT protein encoded by the bar gene isolated from S. hygroscopicus . The pat

gene from S. viridochromogenes also encodes a PAT protein that confers glufosinate tolerance.

# Monsanto Company 12-CT-244U 6 of 620 

#  Cotton is a familiar crop that does not possess any of the attributes commonly

associated with weeds, and has a history of safe usage and consumption.

 A detailed molecular characterization of the inserted DNA demonstrated a single,

intact copy of the T-DNA insert in a single locus within the cotton genome.

 Extensive evaluation of the proteins expressed in MON 88701, dicamba monooxygenase (MON 88701 DMO) and phosphinothr icin acetyltransferase [PAT

( bar )], confirmed they are unlikely to be to xins or allergens. In addition, PAT

proteins are in several other commerc ially-available crops that have been

reviewed and previously deregulated by USDA, includ ing those in cotton, corn,

soy, canola, sugarbeet, and rice.

 A compositional assessment of cottonseed confirmed that MON 88701 is

compositionally equivalent to co mmercially cultivated cotton.

 An extensive evaluation of phenotypi c, agronomic, and plant mapping

characteristics, as well as envir onmental interactions of MON 88701,

demonstrated no increased plant pest potential compared to commercially

cultivated cotton.

 An assessment of potential impact on non-ta rget organisms (NTOs) indicated that,

under anticipated agricultura l conditions, MON 88701 is unlikely to have adverse

effects on these organisms compared to commercially cultivated cotton.

 Evaluation of MON 88701 using current agronomic management practices for

cotton concluded that deregulation of MO N 88701 is not likely to impact cotton

agronomic practices or land use, with the exception of the expanded window of

dicamba application.

Cotton is a Familiar Crop La cking Weedy Characteristics

Cotton, as a commodity crop, has a longstanding history of cultivation; its by-products,

including processed fractions, also have a hi story of safe use and consumption. Cotton is

grown in 17 states across the southern U.S. and in over 80 countries world-wide. In

2011, U.S. growers planted approximate ly 14.7 million acres of cotton.

The commercial cotton species in the U.S. ( Gossypium hirsutum and Gossypium

barbadense L. Merr.) do not exhibit weedy characteristics as defined by USDA, and

neither invade established ecosystems, nor out cross to weedy relati ves. Cotton is not

listed as a weed in major weed references, nor is it present on the lists of noxious weed

species distributed by the fe deral government (7 CFR Part 360). Cotton does not possess

any of the attributes commonly associated with weeds, such as long persistence of the

seed in the soil, ability to disperse, invade , or become a dominant species in new or

diverse landscapes, or the abili ty to compete well with nativ e vegetation. It is recognized

that in some agricultural systems, cotton can volunteer in a subseque nt rotational crop.

However, volunteers are easily controlled through tillage or the use of appropriate

# Monsanto Company 12-CT-244U 7 of 620 

# herbicides with diverse modes-of-action ( e.g. , ALS inhibitor, chloroacetamide, EPSPS,

PPO inhibitor, PSI disruption, PSII inhibito r, synthetic auxin, and tubulin inhibitor

classes). Specificity studies using the aforem entioned herbicides as potential substrates

for MON 88701 DMO showed similar injury levels for MON 88701 compared to the

conventional control, indicating that these herbicides do not serve as a substrate for

MON 88701 DMO at commercial application rates. Additio nally, the specificity of

PAT ( bar ) has been established in the published scientific literature. Therefore,

herbicides effective for cont rol of volunteer conve ntional cotton can still be used to

control MON 88701 volunteers.

In the continental U.S ., wild populations of Gossypium species and some feral

populations of cultivated variants of G. hirsutum exist, but these species able to cross

with cultivated cotton are not known to exist in cotton growing areas. Importantly,

MON 88701 would not be expected to confer a selective advantage to, or enhance the

pest potential of, progeny resulting from such a cross if it were to occur, and could easily

be controlled through current agronomic pract ices used to control conventional cotton.

Thus, with environmental and biological lim itations and varying chemical and agronomic

practices available in the areas with wild and/or feral populations, there is limited

probability for MON 88701 or any Gossypium species to outcross with wild or feral

plants.

Conventional Cotton Coker 130 is an Appropriate Comparator to MON 88701

Cotton variety Coker 130 is the near isogenic line to MON 88701 and was used as the

conventional cotton comparat or to support the safety assessment of MON 88701.

MON 88701 and the near isogenic conventiona l cotton control Coker 130 have similar

genetic backgrounds with the exception of the dmo and bar expression cassettes; thus, the

effect of the dmo and bar expression cassettes and th e expressed MON 88701 DMO and

PAT ( bar ) proteins could be evaluated.

Molecular Characterization Verified the Integrity and Stability of the Inserted DNA

in MON 88701

MON 88701 was developed through Agrobacterium -mediated transformation of

hypocotyls from cotton variety Coke r 130 utilizing vector PV-GHHT6997.

PV-GHHT6997 contains one T-DNA that is delin eated by Left and Right Border regions.

The T-DNA contains the dmo and bar expression cassettes. The dmo expression cassette

is regulated by the PC1SV promoter, the TEV 5 ′ leader sequence, and the E6 3 ′

untranslated region. The chlo roplast transit peptide CTP2 directs transport of the

MON 88701 DMO protein to the chlo roplast and is derived from CTP2 target sequence

of the Arabidopsis thaliana shkG gene. The bar expression cassette is regulated by the

e35S promoter, the Hsp70 leader, and the nos 3 ′ untranslated region. After

transformation, self pollination and segregat ion were used to select those plants

containing a single homozygous copy of the T-DNA, including both the dmo and bar

expression cassettes, resulting in the selection of MON 88701.

# Monsanto Company 12-CT-244U 8 of 620 

# Molecular characterization de termined that MON 88701 contai ns one copy of the T-DNA

at a single integration locus and all genetic elements are present. These data also

demonstrated that MON 88701 does not contain detectable backbone sequences from the

plasmid vector. The complete DNA sequence of the insert and adjacent genomic DNA

sequences in MON 88701 confirmed the integrity of the inserted dmo and bar expression

cassettes and identified the 5 ′ and 3 ′ insert to flank DNA junctions. Molecular

characterization analysis also demonstrated that the insert in MON 88701 has been

maintained over five consecutive genera tions of breeding, th ereby confirming the

stability of the insert. Furthermore, result s from segregation analyses show inheritance

and stability of the insert were as e xpected across multiple generations, which

corroborates the molecular insert stability analysis determination that the MON 88701

T DNA resides at a single chromosomal locus within the cotton genome.

Data Confirms MON 88701 DMO and PAT ( bar) Protein Safety

A multistep approach was used to characteri ze and assess the safety of the MON 88701

DMO and PAT ( bar ) proteins expressed in MON 88701 resulting from the genetic

modification. The expression levels of the MON 88701 DMO and PAT ( bar ) proteins in

selected tissues of MON 88701 were determin ed. An assessment of the allergenic

potential of the MON 88701 DMO and PAT ( bar ) proteins supports th e conclusion that

neither protein poses a significant allergenic risk to humans or animals. In addition, the

donor organisms for the MON 88701 DMO and PAT ( bar ) protein coding sequences,

Stenotrophomonas maltophilia and Streptomyces hygroscopicus , respectively, are

ubiquitous in the environment and are not commonly known for human or animal

pathogenicity or allergenicit y. Bioinformatics analysis determined that the MON 88701

DMO and PAT ( bar ) proteins lack structural simila rity to known allergens, gliadins,

glutenins, or protein toxins . The MON 88701 DMO and PAT ( bar ) proteins are rapidly

digested in simulated gastrointestinal fluids and neither protein de monstrates acute oral

toxicity in mice at the levels tested. Hence, the consumption of the MON 88701 DMO

and PAT ( bar ) proteins from MON 88701 or its proge ny poses no meaningful risk to the

environment or human and animal health.

MON 88701 is Compositionally Equivalent to Conventional Commercial Cotton

Detailed compositional analyses were conducted in accordance with OECD guidelines to

assess whether levels of key nutrients and anti-nutrients in M ON 88701 cottonseed were

comparable to levels in the conventional control, Coker 130, and several commercial

reference cotton varieties. These compos itional comparisons were made by analyzing

cottonseed harvested from eight U.S. field sites in which MON 88701 was treated with

dicamba and glufosinate, with the conventio nal control, and a ra nge of commercial

reference varieties that were grown concurrent ly in the same field trial. Compositional

comparisons of MON 88701 not treated with dica mba or glufosinate herbicides were also

conducted to further support the assessmen t of MON 88701 traits. The commercial

reference varieties used to es tablish a range of na tural variability for key nutrients and

anti-nutrients have a history of safe consum ption. Nutrients assessed in this analysis

included proximates (ash, carbohydrates, and cal ories by calculation, moisture, protein,

and fat), fibers (ADF, crude fiber, NDF, and TDF), amino acids (18 components), fatty

# Monsanto Company 12-CT-244U 9 of 620 

# acids (C8-C22), minerals (calcium, coppe r, iron, magnesium, manganese, phosphorus,

potassium, sodium, and zinc) and vitamin E. The anti-nutrients assessed in this analysis

included gossypol and the cycl opropenoid fatty acids dihydr osterculic, malvalic, and

sterculic.

Combined-site analyses were conducted to de termine if there were any statisticallysignificant differences (5 % level of significance) between MON 88701 and the

conventional control cottonseed samples. Significant differences noted from the

combined-site statistical comparison were assessed using considerations relevant to the

safety and nutritional quality of MON 88701 when compared to the co nventional control.

Considerations used to assess the relevance of each combined-site statistically significant

difference included: 1) the relative magnitude of the difference in the mean values of

nutrient and anti-nutrient components betw een MON 88701 and the conventional control;

2) whether the MON 88701 component mean va lue is within the range of natural

variability of that component as represented by the 99% tolerance interval of the

commercial reference varieties gr own concurrently in the same trial; 3) evaluation of the

reproducibility of the statistical (p < 0.05) combined-site component differences at

individual sites; and 4) an assessment of th e differences within the context of natural

variability of commercial cotton composition p ublished in the scientific literature and in

the International Life Sciences Inst itute (ILSI) Crop Composition Database.

Based on these criteria, the observed differenc es were not meaningful to food and feed

safety or nutritional value, and led to the conclusion that MON 88701 is compositionally

equivalent to commercially cultivated cott on that has a history of safe consumption.

These results support the overall food and feed safety of MON 88701.

MON 88701 Does Not Change Cotton Plant Pest Potential or Environmental

Interactions

Plant pest potential of a biotechnology-de rived crop is assessed from the basis of

familiarity that the USDA recognizes as an important underlying concept in risk

assessment. The concept of familiarity is based on the fact that the biotechnologyderived plant is developed from a conventiona l plant hybrid or vari ety whose biological

properties and plant pest potential are well known. Familiarity considers the biology of

the plant, the introduced trait(s), the receivi ng environment, and the interactions among

these factors. This provides a basis fo r comparative risk assessment between a

biotechnology-derived plant and the convent ional control. Thus, the phenotypic,

agronomic, plant mapping, and environmental interaction assessment of MON 88701

included the parental conventi onal control as a comparator . This evaluation used a

weight-of-evidence approach and considered statistical differences between MON 88701

and the conventional control with resp ect to reproducibility, magnitude, and

directionality. The observat ions were taken on plants not treated with dicamba or

glufosinate, in order to evaluate the impact of the introduced traits in MON 88701. To

further support the trait as sessment, similar supplementa l observations were also

conducted on the agronomic system that in cludes MON 88701 treated with dicamba and

glufosinate herbicides. Comparison to a range of commercial reference varieties

established the range of natura l variability for cotton, and pr ovided a context from which

# Monsanto Company 12-CT-244U 10 of 620 

# to further evaluate any statistical differen ces. Characteristics assessed included: seed

dormancy and germination, pollen morphol ogy, plant phenotypic observations, plant

mapping, and environmental interaction eval uations conducted in the field. The

phenotypic, agronomic, and environmental in teraction assessment demonstrated that

MON 88701 is comparable to conventional cotton. Thus, MON 88701 is unlikely to

have increased weediness or plant pest poten tial compared to commercially cultivated

cotton.

Seed dormancy and germination characte rization demonstrated that MON 88701

cottonseed had germination characteristics similar to cottonseed of the conventional

control. In particular, the lack of hard s eed, a well-accepted charac teristic of weediness

affecting seed germination, supports a c onclusion of no increased weediness of

MON 88701 when compared to the conventiona l control. Additionally, there were no

statistically significant (5% level of si gnificance) differences observed between

MON 88701 and the conventional control for poll en viability and diameter, and no visual

differences in general pollen morphology were observed. Collectively, these results

support the conclusion that MON 88701 is not likely to exhibit increased plant pest

potential compared to commer cially cultivated cotton.

The field evaluation of phenotypic, agr onomic, plant mapping, and environmental

interaction characteristics of MON 88701 al so support the conclusion that MON 88701 is

not likely to have an increased plant pest potential compared to commercially cultivated

cotton. The evaluations were conducted at 26 replicated fiel d sites across the U.S. cotton

producing region. These assessments in cluded plant growth and development

characteristics, including cotton plant mapping evaluations at harvest, as well as

observations for plant responses to abiotic st ressors and plant-dis ease and plant-arthropod

interactions. The observed phenotypic char acteristics were similar between MON 88701

and the conventional control.

In a combined-site analysis of plant growth and development characte ristics, data showed

no statistically significant di fferences (5% level of sign ificance) between MON 88701

and the conventional control for stand count at 14 and 30 days after planting (DAP), final

stand count, number of nodes above white flow er at one of three observations, seed

cotton yield, immature seed per boll, weight per boll, micronaire, fiber elongation, fiber

uniformity, and fiber length. The mean values for MON 88701 were statistically

different from the conventiona l control for eight parameters in the combined-site

analysis. MON 88701 had shorter plants at 30 DAP and harvest, an increased number of

nodes above white flower at two observations , a lower seed index, increased seed per

boll, increased mature seeds per boll, and in creased fiber strength. However, the mean

values of MON 88701 were within the range of values observed for the commercial

reference varieties for each of the characteristics listed abov e. Therefore, none of these

differences were considered bi ologically meaningful in te rms of increased plant pest

potential of MON 88701 compared to commercially cultivated cotton.

Plant mapping is a process commonly used by cotton agronomists and breeders to

quantify growth and development parameters of a cotton plant, including boll retention.

Plant mapping parameters, which include deline ation of boll position and spatial retention

# Monsanto Company 12-CT-244U 11 of 620 

# of bolls, are used to measure crop productivity and are influenced by abiotic and biotic

stressors. In the combined-site analysis of plant mapping parameters, no statistically

significant differences were detected be tween MON 88701 and the conventional control

for number of mainstem nodes, number of nodes to first fruiting branch, total number of

bolls per plant, number of vegetative bolls pe r plant, percent retent ion of first-position

bolls, and percent first-position bolls. On e statistically signifi cant difference was

detected between MON 88701 and the conven tional control in the combined-site

analysis. The mean value for first-positio n bolls per plant wa s higher for MON 88701

than the conventional control. However, th e mean value of the number of first-position

MON 88701 bolls was within the range of th e commercial reference varieties. Thus,

MON 88701 is similar to commercially cultiv ated cotton varieties and unlikely to have

increased plant pest potential, increased weed iness, or an adverse environmental impact

compared to commercially cultivated cotton.

In an individual site assessment of abio tic stress response and disease damage, no

differences were observed between MON 88701 and the conventional control for any of

the 296 comparisons for the assessed abiotic st ressors or for any of the 299 comparisons

for the assessed diseases among all observations at the 26 sites. In an assessment of

arthropod-related damage, no differences were detected betw een MON 88701 and the

conventional control for any of the 288 comp arisons for the assessed arthropods. The

lack of significant biological differences in plant responses to ab iotic stress, disease

damage, and arthropod-related damage fo r MON 88701 support the conclusion that the

introduction of the dicamba and glufosinate tolerance traits are unlikely to result in

increased plant pest potential or an a ltered environmental impact from MON 88701

compared to commercially cultivated cotton.

In an assessment of pest- and beneficial-a rthropod abundance, no stat istically significant

differences (5% level of significance) were detected between MON 88701 and the

conventional control for 173 out of 178 comp arisons (including 89 arthropod-pest and 89

beneficial-arthropod comparisons) among the multiple collections conducted during the

season at five geographically diverse sites. Fo r the five detected di fferences in arthropod

abundance, two were arthropod pests (stink bugs and tarnished plant bugs) and three were

beneficial arthropods ( Nabis spp. and Orius spp.). The differences detected in pest- and

beneficial-arthropod abundance were small in magnitude and were not consistent with

other collections at the individua l sites or across the sites. Consequently, it is concluded

that the differences in pest- and benefici al-arthropod abundance are not indicative of a

consistent plant response associated with MON 88701 and are not biologically

meaningful in terms of increased plant pest potential or an altered environmental impact

from MON 88701 compared to commercially cultivated cotton.

Field evaluations of phenot ypic, agronomic, and plant mapping characteristics of

MON 88701 treated with dicamba and glufosin ate herbicides were also conducted to

further support the assessment of MON 88701 tr aits. Data were collected from field

trials conducted at eleven sites within the U.S. cotton-producing region. These

assessments included plant growth and develo pment characteristics, as well as plant

mapping evaluations at harvest. The phe notypic, agronomic, and plant mapping

assessments demonstrated that herbicide-tr eated MON 88701 is not different than the

# Monsanto Company 12-CT-244U 12 of 620 

# conventional control, which further suppor ts that MON 88701, whether treated or not

with dicamba and glufosinate, is unlikely to have an altered plant pest potential compared

to commercially cultivated cotton.

In summary, the phenotypic, agronomic, pl ant mapping and envir onmental interaction

data were evaluated to characterize MON 88701, and to assess whether the introduction

of the traits in MON 88701 a lters the plant pest potential compared to conventional

cotton. The evaluation, using a weight-o f-evidence approach, considered the

reproducibility, magnitude, and direction of detected differences between MON 88701

and the conventional control, and comparison to the range of the commercial reference

varieties. Results from the phenotypic, agronomic, plant mapping, and environmental

interactions assessment indicated that MON 88701 does not possess weedy

characteristics, increased susceptibility or tole rance to specific abiotic stress, diseases, or

arthropods, or charact eristics that would confer a plan t pest risk or a significant

environmental impact compared to commercially cultivated cotton.

MON 88701 Will Not Adversely Affect NTOs

Evaluation of the impacts of a biotechnol ogy-derived crop on non-target organisms

(NTOs) is a component of the plant pest risk assessment. Since MON 88701 does not

possess pesticidal activity, all organisms that interact with MON 88701 are considered to

be NTOs. The environmental assessment demo nstrated that the presence of the dicamba

and glufosinate-tolerance tra its in MON 88701 did not alter plant-arthropod interactions,

including beneficial arthropods, or alter disease susceptibility compared to the

conventional control. In addition, plant mapping data, which is utilized to determine crop

productivity in relation to abiotic and biotic stresses affecting yield, demonstrated that

both MON 88701 plots treated and not treated w ith dicamba and glufosinate herbicides

each had only a single significant difference fr om the conventional control, an increased

number of first-position bolls that was with in the range of the commercial reference

varieties. From these data it can be concluded that both MON 88701 plants treated and

not treated with dicamba and glufosinate re sponded to stressors in a similar manner.

The biochemical information and experime ntal data for eval uation of MON 88701

included molecular characteri zation, MON 88701 DMO and PAT ( bar ) safety

assessments, the history of environmental exposure to mono-oxygenases (the class of

enzymes to which MON 88701 DMO belongs) and the PAT proteins in several

commercial glufosinate-tolerant events, information from the environmental interaction

assessment, demonstration of compositional equivalence to conventional cotton, and

demonstration of agronomic and phenotypic equi valence to conventional cotton. Overall,

these data support the conclusion that MON 88701 has no reasonable mechanism for

harm to NTOs and does not pose any additiona l risk to NTOs compared to commercially

cultivated cotton.

The potential for outcrossing and gene in trogression from MO N 88701 to sexually

compatible species in the U.S. is unlikely, since th e only known wild Gossypium species

related to cultivated cotton do not grow in areas where cotton is cultivated, cotton pollen

movement by wind is limited due to it is large and sticky nature, and several studies have

# Monsanto Company 12-CT-244U 13 of 620 

# demonstrated that cross-pollination, even in the presence of high pollinator activity is

limited by distance. Furthermore, should cross-pollination occur, MON 88701 and its

progeny are not expected to exhibit a sign ificant environmental impact because, as

described above, evaluations have shown that the presence of the dicamba and

glufosinate-tolerance traits are not likely to enhance weediness or plant-pest potential.

Therefore, the environmental consequence of pollen transfer from MON 88701 to other

Gossypium species is considered negligible.

Deregulation of MON 88701 is Not Likely to Impact Cotton Agronomic Practices or

Land Use

Cotton fields are typically hi ghly managed agricultural areas that are dedicated to crop

production for many years. Cultivation of MON 88701 would not be expected to differ

from typical cotton cultivation, with the sole exception of an expanded window of

dicamba application, due to the presence of the dicamba-tolerance trait in MON 88701.

As glufosinate is already utilized within the U.S. cotton-growing areas, no change in

agronomic practices or land use would occur with the cultivation of MON 88701 and the

presence of the glufosinate- tolerance trait. MON 88701 likely would be used in common

rotations on land currently used for agricultu ral purposes. As demonstrated, MON 88701

is similar to commercially cultivated cotton in its agronomic, phenotypic, ecological, and

compositional characteristics, and has comparable levels of resistance to insects, diseases,

and abiotic stresses as comp ared to commercial cotton. Th erefore, the introduction of

MON 88701 into the existing cotton system is no t expected to have a significant impact

on current cultivation and pest management practices for cotton. The adoption of

MON 88701 into glyphosate-tolerant cotton systems will provide growers with two

additional herbicide modes-of-action and the means to control broadleaf weeds, including

hard-to-control and herbicide-resistant br oadleaf weeds, and will help preserve

conservation tillage practices by providing gr owers with an additional weed management

tool. Based on these considerations, M ON 88701 is not likely to impact agronomic

practices or land use, with th e exception of the expanded appli cation window of dicamba.

Conclusion

Based on the data and information presented in this petition, it is concluded that

MON 88701 is not likely to be a plant pest. Therefore, M onsanto Company requests a

determination from USDA-APHIS that MON 88701 and any progeny derived from

crosses between MON 88701 and conventional Gossypium cotton species or deregulated

biotechnology-derived cotton be granted nonregulated status under 7 CFR Part 340.

# Monsanto Company 12-CT-244U 14 of 620 

# TABLE OF CONTENTS

CERTIFICATION .............................................................................................................3

EXECUTIVE SUMMARY ...............................................................................................4

TABLE OF CONTENTS ................................................................................................14

LIST OF TABLES ...........................................................................................................19

LIST OF FIGURES .........................................................................................................27

ABBREVIATIONS AND DEFINITIONS .....................................................................29

I. RATIONALE FOR THE DEVELOPMENT OF MON 88701 ...............................33

I.A. Basis for the Request for a Determ ination of Nonregulated Status under

7 CFR § 340.6 ...............................................................................................................33

I.B. Rationale for the Development of Dicamba and Glufosinate-Tolerant

Cotton – MON 88701 ....................................................................................................33

I.C. Submissions to Other Regulatory Agencies ..........................................................36

I.C.1. Submission to FDA .....................................................................................36

I.C.2. Submission to EPA .....................................................................................36

I.C.3. Submissions to Fore ign Government Agencies ..........................................37

II. THE BIOLOGY OF COTTON ................................................................................38

II.A. Cotton as a Crop ..................................................................................................38

II.B. Characteristics of the Recipient Plant ..................................................................39

II.C. Cotton as a Test System in Product Safety Assessment ......................................39

III. DESCRIPTION OF TH E GENETIC MODIFICATION ....................................40

III.A. PV-GHHT6997 ...................................................................................................40

III.B. Description of the Transformation System .........................................................40

III.C. The dmo Coding Sequence and the MON 88701 DMO Protein ........................44

III.D. The bar Coding Sequence and PAT ( bar ) Protein .............................................44

III.E. Regulatory Sequences .........................................................................................44

III.F. T-DNA Borders ...................................................................................................44

III.G. Genetic Elements Outside of the T-DNA Borders .............................................45

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION .....................50

IV.A. Insert and Copy Number of T-DNA in MON 88701 .........................................57

IV.A.1. T-DNA Probes 1 and 5 ............................................................................57

IV.A.2. T-DNA Probes 2 and 4 ............................................................................58

IV.A.3. T-DNA Probe 3 ........................................................................................58

IV.B. Southern Blot Analysis to De termine the Presence or Absence of

PV-GHHT6997 Backbone Sequences in MON 88701 .................................................63

# Monsanto Company 12-CT-244U 15 of 620 

# IV.B.1. Backbone Probes 6, 7, and 8 ....................................................................63

IV.C. Organization and Sequence of th e Insert and Adjacent Genomic DNA in

MON 88701 ...................................................................................................................65

IV.D. PCR and DNA Sequence Analyses to Examine the MON 88701

Insertion Site ................................................................................................................ ..67

IV.E. Southern Blot Analysis to Examine Insert Stability in Multiple

Generations of MON 88701 ..........................................................................................69

IV.E.1. T-DNA Probe 2 and 4 ..............................................................................69

IV.F. Inheritance of the Ge netic Insert in MON 88701 ...............................................72

IV.G. Genetic Modification Ch aracterization Conclusion ...........................................76

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE

MON 88701 DMO AND PAT ( bar ) PROTEINS PRODUCED IN MON 88701 ........77

V.A. Identity and Function of the MON 88701 DMO and PAT ( bar ) Proteins

from MON 88701 ..........................................................................................................77

V.A.1. Mode-of-Action of DMO and MON 88701 DMO ...................................77

V.A.2. Mode-of-Action of PAT Proteins..............................................................81

V.B. Characterization and Equi valence of MON 88701 DMO and PAT ( bar )

Proteins from MON 88701 ............................................................................................82

V.C. Expression Levels of MON 88701 DMO and PAT ( bar ) Proteins in

MON 88701 ...................................................................................................................83

V.C.1. Expression Levels of MON 88701 DMO Protein .....................................84

V.C.2. Expression Levels of PAT ( bar ) Protein ...................................................86

V.D. Assessment of Potential A llergenicity of the MON 88701 DMO and

PAT ( bar ) Proteins ........................................................................................................88

V.D.1. Assessment of Potential Allergenicity of the MON 88701 DMO

Protein ....................................................................................................................88

V.D.2. Assessment of Potentia l Allergenicity of the PAT ( bar ) Protein ..............88

V.E. Safety Assessment Summ ary of MON 88701 DMO and PAT ( bar )

Proteins in MON 88701 ................................................................................................89

V.E.1. MON 88701 DMO Donor Organism , History of Safe Use, and

Specificity ..............................................................................................................89

V.E.2. PAT ( bar ) Donor Organism, History of Safe Use, and Specificity ...........92

V.E.3. MON 88701 DMO and PAT ( bar ) Proteins in MON 88701 are Not

Homologous to Known Allergens or Toxins .........................................................94

V.E.4. MON 88701 DMO and PAT ( bar ) Proteins in MON 88701 are

Labile in in vitro Digestion Assays ........................................................................94

V.E.5. MON 88701 DMO and PAT ( bar ) Proteins in MON 88701 are Not

Acutely Toxic .........................................................................................................95

# Monsanto Company 12-CT-244U 16 of 620 

# V.E.6. Human and Animal Exposure to the MON 88701 DMO and

PAT ( bar ) Proteins .................................................................................................95

V.F. MON 88701 DMO and PAT ( bar ) Protein Characterization and Safety

Conclusion .................................................................................................................... .96

VI. COMPOSITIONAL ASSESSMENT OF MON 88701 .........................................98

VI.A. Compositional Equivalence of MON 88701 Cottonseed to Conventional

Cotton ........................................................................................................................ ....99

VI.A.1 Nutrient Levels in Cottonseed ................................................................100

VI.A.2. Anti-Nutrient Levels in Cottonseed .......................................................105

VI.B. Compositional Assessment of MON 88701 Conclusion ..................................134

VII. PHENOTYPIC, AGRONO MIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT ...............................................................................136

VII.A. Characteristics Measured for Assessment ......................................................136

VII.B. Interpretation of Phenotypic and Environmental Interaction Data .................142

VII.B.1. Interpretation of Detected Differences Criteria ....................................142

VII.C. Comparative Assessments of the Phenotypic, Agronomic, Plant

Mapping, and Environmenta l Interaction Characte ristics of MON 88701 .................145

VII.C.1. Seed Dormancy and Germination Characteristics ................................145

VII.C.2. Field Phenotypic, Agronomic, Plant Mapping, and

Environmental Interactio ns Characteristics .........................................................148

VII.C.3. Pollen Characteristics ...........................................................................164

VII.D. Conclusions for Phenotypi c, Agronomic, Plant Mapping, and

Environmental Interact ions Evaluation .......................................................................166

VIII. U.S. AGRONOMIC PRACTICES.....................................................................167

VIII.A. Introduction ...................................................................................................167

VIII.B. Overview of U.S. Cotton Production .............................................................168

VIII.B.1. Cotton Production................................................................................168

VIII.B.2. Cotton Seed Production .......................................................................175

VIII.C. Production Management Considerations .......................................................176

VIII.C.1. Pre-Season ...........................................................................................176

VIII.C.2. Planting and Early Season ...................................................................177

VIII.C.3. Mid- to Late-Season ............................................................................178

VIII.C.4. Preharvest and Harvest ........................................................................179

VIII.D. Management of Insect and Other Pests .........................................................179

VIII.E. Management of Diseases ...............................................................................182

VIII.F. Weed Management .........................................................................................183

# Monsanto Company 12-CT-244U 17 of 620 

# VIII.F.1. Methods of Weed Control in Cotton....................................................187

VIII.F.2. Herbicide Resistant Weeds in Cotton ..................................................204

VIII.G. Introduction of Dicamba an d Glufosinate-Tolerant Cotton -

MON 88701 .................................................................................................................206

VIII.G.1. MON 88701 Product Concept .............................................................206

VIII.G.2. Dicamba and Glufosinate Usage in MON 88701 ...............................206

VIII.G.3. MON 88701 in Combination with Glyphosate-Tolerant Cotton

Systems ................................................................................................................209

VIII.G.4. MON 88701 as a Weed Resistance Management Tool ......................211

VIII.G.5. Introduction of Dicamba and Glufosinate-Tolerant Cotton -

MON 88701 - Conclusion ....................................................................................212

VIII.H. Crop Rotation Practices in Cotton .................................................................215

VIII.H.1. Cotton Volunteer Management ...........................................................228

VIII.I. Stewardship of MON 88701 ...........................................................................231

VIII.J. Impact of the Introduction of MON 88701 on Agricultural Practices ............232

IX. ENVIRONMENTAL ANALYSIS ........................................................................233

IX.A. Introduction ......................................................................................................233

IX.B. Plant Pest Assessment of MO N 88701 Insert and Expressed Proteins ............233

IX.B.1. Characteristics of the Gene tic Insert and Expressed Protein .................234

IX.B.2. Compositional Characteristics ...............................................................235

IX.B 3. Phenotypic, Agronomic, Plant Mapping, and Environmental

Interaction Characteristics ...................................................................................235

IX.C. Weed Potential of MON 88701 ........................................................................238

IX.D. Potential for Pollen Mediated Gene Flow and Introgression ...........................239

IX.D.1. Hybridization with Cultivated Cotton ....................................................239

IX.D.2. Hybridization with Wild and Feral Gossypium species .........................241

IX.D.3. Transfer of Genetic Inform ation to Species with which Cotton

Cannot Interbreed (Horizontal Gene Flow) .........................................................242

IX.E. Potential Impact on Cotton Agronomic Practices ............................................242

IX.F. Conventional Breeding with Other Biotechnology-derived or

Conventional Cotton ....................................................................................................243

IX.G. Summary of Plant Pest Assessments ................................................................244

X. ADVERSE CONSEQUENC ES OF INTRODUCTION .......................................245

REFERENCES ...............................................................................................................246

APPENDICES ................................................................................................................262

Appendix A: Notifications .............................................................................................263

# Monsanto Company 12-CT-244U 18 of 620 

# Appendix B: Materials, Methods, and Results for Molecular Analyses of

MON 88701 ..................................................................................................................... 266

Appendix C: Protein Reac tion Products, Materials, Methods, and Results

for Characterization of MON 88701 DMO and PAT ( bar ) Proteins Produced

in MON 88701, and Substrate Specificity ....................................................................273

Appendix D: Materials and Methods Used for the Analysis of the Levels of

MON 88701 DMO and PAT ( bar ) Proteins in MON 88701 .......................................332

Appendix E: Materials, Methods, and Individual Site Results for

Compositional Analysis of MON 88701 Cottonseed ...................................................337

Appendix F: Materials, Methods, a nd Individual Site Results for Seed

Dormancy and Germination Assessment of MON 88701 ..........................................526

Appendix G: Materials, Methods, Dicamb a and Glufosinate Treated Results,

and Individual Site Results from the Phenotypic, Agronomic, Plant Mapping,

and Environmental Interaction Assessment of MON 88701 under Field

Conditions .................................................................................................................... ...532

Appendix H: Materials and Methods for Pollen Morphology and Viability

Assessment .................................................................................................................... ..595

Appendix I: Herbicide Resistance ...............................................................................599

# Monsanto Company 12-CT-244U 19 of 620 

# LIST OF TABLES

Table III-1. Summary of Gene tic Elements in PV-GHHT6997 ..................................46

Table IV-1. Summary Chart of th e Expected DNA Segments Based on

Hybridizing Probes and Restriction Enzymes Used in MON 88701 Analysis ...........54

Table IV-2. Summary of Ge netic Elements in MON 88701 ........................................55

Table IV-3. Segregation of the T-DNA During the Development of

MON 88701: 1:1 Segregation ..........................................................................................75

Table IV-4. Segregation of the T-DNA During the Development of

MON 88701: 1:2:1 Segregation.......................................................................................75

Table V-1. Herbicides Applied to MON 88701 and Conventional Control ...............81

Table V-2. Summary of MON 88701 DMO Protein Levels in Tissues from

MON 88701 Grown in 2010 U.S. Field Trials ...............................................................85

Table V-3. Summary of PAT ( bar ) Protein Levels in Tissues from

MON 88701 Grown in 2010 U.S. Field Trials ...............................................................87

Table VI-1. Summary of Differences (p<0.05) for the Comparison of

Cottonseed Component Levels for MO N 88701 vs. Conventional Control ..............107

Table VI-2. Statistica l Summary of CombinedSite Cottonseed Nutrients for MON 88701 vs. Conventional Control ......................123

Table VI-3. Statistical Summary of Combined-Site Cottonseed Antinutrients for MON 88701 vs. Conventional Control ..................................................131

Table VI-4. Literature and ILSI Ranges for Components in Cottonseed ...............132

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction

Characteristics Evaluated in United States Field Trials and Laboratory

Studies ....................................................................................................................... ......138

Table VII-2. Combined-Site Comparison of MON 88701 to Conventional

Control for Germination Characteristics ....................................................................147

Table VII-3. Study 1 and Study 2 Data Location Summary .....................................149

Table VII-4. Field Phenotypic E valuation Sites for MON 88701 during 2010........151

Table VII-5. Study 1 Combined-Site Comparison of MON 88701 to

Conventional Control during 2010 for Phenotypic and Agronomic

Characteristics................................................................................................................ 153

Table VII-6. Study 2 Combined-Site Comparison of MON 88701 to

Conventional Control during 2010 for Phenotypic and Agronomic

Characteristics................................................................................................................ 155

Table VII-7. Study 2 Combined-Site Comparison of MON 88701 to

Conventional Control during 2010 for Plant Mapping Characteristics ...................157

# Monsanto Company 12-CT-244U 20 of 620 

# Table VII-8. Study 1 Summary of Qua litative Environmental Interactions

Assessments Including MON 88701 Response to Abiotic Stress, Disease, and

Arthropod Damage during 2010...................................................................................160

Table VII-9. Study 1 Combined-Site Comparison of MON 88701 to

Conventional Control during 2010 for Assessment of Thrips Damage ....................160

Table VII-10. Study 1 Combined-Site Comparison of MON 88701 to

Conventional Control during 2010 for Qu antitative Assessment of Heliothine

Damage......................................................................................................................... ...161

Table VII-11. Study 2 Summary of Qual itative Environmen tal Interactions

Assessments Including MON 88701 Respon se to Abiotic Stress and Disease

and Arthropod-related Damage during 2010 ..............................................................163

Table VII-12. MON 88701 Compared to the Conventional Control during

2010 for Pollen Characteristics .....................................................................................166

Table VIII-1. Cotton Production in the U.S., 2000-2010 1 

# ..........................................173

Table VIII-2. U.S. Cotton Production by Region and State in 2010 1 

# .......................174

Table VIII-3. Insect Losses in Cotton in U.S. in 2010 1 

# ..............................................181

Table VIII-4. Common and Scientific Na mes of Weeds Referred to in this

Petition ...................................................................................................................... ......184

Table VIII-5. Common Weeds in Cotton Production in the Southeast Region

of the U.S. 1,2 

# ....................................................................................................................185

Table VIII-6. Common Weeds in Cotton Production in the Midsouth Region

of the U.S. 1,2 

# ....................................................................................................................186

Table VIII-7. Common Weeds in Cotton Production in the Southwest

Region of the U.S. 1,2 

# .......................................................................................................186

Table VIII-8. Common Weeds in Cotton Production in the West Region of

the U.S. 1,2 

# .........................................................................................................................187

Table VIII-9. Herbicide Applications Registered for Use in Cotton in 2010 1 

# .........192

Table VIII-10. Grass Weed Species Cont rol Ratings to Preplant Burndown

Herbicides in Cotton ......................................................................................................194

Table VIII-11. Grass Weed Species Control Ratings to Preplant,

Preemergence and Postemergence Herbicides in Cotton ...........................................195

Table VIII-12. Broadleaf Weed Speci es Control Ratings to Preplant

Burndown Herbicides in Cotton ...................................................................................198

Table VIII-13. Broadleaf Weed Speci es Control Ratings to Preplant,

Preemergence and Postemergence Herb icides in Cotton - Part I .............................199

Table VIII-14. Broadleaf Weed Speci es Control Ratings to Preplant,

Preemergence and Postemergence Herbicides in Cotton – Part II ...........................201

Table VIII-15. Common Weeds in Cotton and Weed Resistance to Herbicide

Modes of Action in the U.S. 1 

# .........................................................................................205

# Monsanto Company 12-CT-244U 21 of 620 

# Table VIII-16. Anticipated Weed Management Recommendations for

MON 88701 Combined with Glyphosate- Tolerant Cotton Systems for MO,

AR, TN, AL, FL, GA, NC, SC, VA, LA, MS and eastern TX 1 

# ...................................208

Table VIII-17. Anticipated Weed Management Recommendations for

MON 88701 Combined with Glyphosat e-Tolerant Cotton Systems for

western TX, NM, KS, OK, CA, and AZ 1 

# .....................................................................209

Table VIII-18. Responses of Common Broadleaf Weeds to Dicamba and

Glufosinate Compared to Labeled Postemergence Herbicides in Cotton

Production – Part I ........................................................................................................213

Table VIII-19. Responses of Common Broadleaf Weeds to Dicamba and

Glufosinate Compared to Labeled Postemergence Herbicides in Cotton

Production – Part II .......................................................................................................214

Table VIII-20. Rotational Practices in the U.S. Following Cotton Production .......217

Table VIII-21. Rotational Practices Following Cotton Production in the

Southeast Region ............................................................................................................21 8

Table VIII-22. Rotational Practices Following Cotton Production in the

Midsouth Region ............................................................................................................221

Table VIII-23. Rotational Practices Following Cotton Production in the

Southwest Region ...........................................................................................................223

Table VIII-24. Rotational Practices Following Cotton Production in the

West Region ................................................................................................................... .226

Table VIII-25. Herbicides and Applicat ion Timing for Control of Volunteer

Cotton in Labeled Rotational Crops 1 

# ...........................................................................230

Table IX-1. Summary of Published Lit erature on Cotton Cross Pollination ..........241

Table A-1. USDA Notifications and Permits Approved for MON 88701 and

Status of Trials Conducted under These Notifications ...............................................264

Table B-1. Hybridization Conditions of Utilized Probes ...........................................269

Table C-1. Summary of MON 88701 DMO Protein Identity and Equivalence ......280

Table C-2. Summary of the Tryptic Masses 1 

# Identified for the MON 88701

DMO Protein Using MALDI-TOF MS ........................................................................284

Table C-3. Comparison of Immunoreactive Signals Between MON 88701

DMO and E. coli -produced MON 88701 DMO Proteins ...........................................289

Table C-4. Molecular Weight Comparison Between the MON 88701 DMO

and E. coli -produced MON 88701 DMO Proteins Based on SDS-PAGE .................291

Table C-5. MON 88701 DMO Functional Activity Assay .........................................297

Table C-6. Herbicides Tested in Exoge nous Specificity Herbicide Tolerance

Greenhouse Trials ..........................................................................................................297

Table C-7. Herbicide Tolerance Trials Injury Ratings .............................................299

Table C-8. Compounds Used in Specificity In Vitro Experiments ...........................300

# Monsanto Company 12-CT-244U 22 of 620 

# Table C-9. Summary of MON 88701-produced PAT ( bar ) Protein Identity

and Equivalence .............................................................................................................30 9

Table C-10. N-Terminal Sequence of the MON 88701-produced PAT ( bar )

Protein ....................................................................................................................... ......311

Table C-11. Summary of the Tryptic Masses 1 

# Identified for the MON 88701-

produced PAT ( bar ) Protein Using MALDI-TOF MS ...............................................314

Table C-12. Comparison of Immuno reactive Signals Between MON 88701-

and E. coli -produced PAT ( bar ) Proteins ....................................................................319

Table C-13. Molecular Weight Comparison Between the MON 88701- and

E. coli -produced PAT ( bar ) Proteins Based on SDS-PAGE .......................................321

Table C-14. PAT ( bar ) Functional Activity ................................................................327

Table D-1. MON 88701 DMO Protein Extraction Methods for Tissue

Samples ....................................................................................................................... ....333

Table D-2. PAT ( bar ) Protein Extraction Methods for Tissue Samples ..................333

Table E-1. Commercial Reference Varieties ..............................................................337

Table E-2. Re-expression Formulas for Statistical Analysis of Composition

Data .......................................................................................................................... .......346

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for

MON 88701 (Treated) vs. Conventional Control ........................................................348

Table E-4. Statistical Summary of Site ARTI Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................356

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for

MON 88701 (Treated) vs. Conventional Control ........................................................357

Table E-6. Statistical Summary of Site GACH Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................365

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for

MON 88701 (Treated) vs. Conventional Control ........................................................366

Table E-8. Statistical Summary of Site KSLA Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................374

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for

MON 88701 (Treated) vs. Conventional Control ........................................................375

Table E-10. Statistical Summary of Site LACH Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................383

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients

for MON 88701 (Treated) vs. Conventional Control ..................................................384

Table E-12. Statistical Summary of Site NCBD Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................392

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for

MON 88701 (Treated) vs. Conventional Control ........................................................393

# Monsanto Company 12-CT-244U 23 of 620 

# Table E-14. Statistical Summary of Site NMLC Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................401

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for

MON 88701 (Treated) vs. Conventional Control ........................................................402

Table E-16. Statistical Summary of Site SCEK Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................410

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for

MON 88701 (Treated) vs. Conventional Control ........................................................411

Table E-18. Statistical Summary ofSite TXPL Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................419

Table E-19. Summary of Differences (p<0.05) for the Comparison of

Cottonseed Component Levels for MON 88701 (Not Treated) vs.

Conventional Control ....................................................................................................426

Table E-20. Statistical Summary of Co mbined-Site Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................440

Table E-21. Statistical Summary ofCombined-Site Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................448

Table E-22 Literature and ILSI Rang es for Components in Cottonseed ................449

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................451

Table E-24. Statistical Summary of Site ARTI Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................459

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................460

Table E-26. Statistical Summary of Site GACH Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................468

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................469

Table E-28. Statistical Summary of Site KSLA Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................477

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................478

Table E-30. Statistical Summary of Site LACH Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................486

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................487

Table E-32. Statistical Summary of Site NCBD Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................495

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................496

# Monsanto Company 12-CT-244U 24 of 620 

# Table E-34. Statistical Summary of Site NMLC Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................504

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................505

Table E-36. Statistical Summary of Site SCEK Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................513

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for

MON 88701 (Not Treated) vs. Conventional Control.................................................514

Table E-38. Statistical Summary of Site TXPL Cottonseed Antinutrients for MON 88701 (Not Treate d) vs. Conventional Control ..........................522

Table F-1. Starting Seed of MO N 88701, Conventional Control and

Commercial Cotton Reference Varieties Used in Dormancy Assessment ................526

Table F-2. Comparison of MON 88701 to the Conventional Control for

Dormancy and Germination Characterist ics of Cottonseed Produced at Each

of Three Sites ................................................................................................................ ..529

Table G-1. Starting Seed for Study 1 ..........................................................................534

Table G-2. Starting Seed for Study 2 ..........................................................................536

Table G-3. Study 1 Field and Planting Information ..................................................538

Table G-4. Study 2 Field and Planting Information ..................................................539

Table G-5. Study 1 Data Missing or Excluded from Analysis ..................................554

Table G-6. Study 2 Data Missing or Excluded from Analysis ..................................555

Table G-7. Study 1 - Individual Site Phenotypic Comparison – Growth and

Development Characteristics - of MO N 88701 Not Treated with Dicamba or

Glufosinate Herbicides Compared to the Conventional Control ..............................556

Table G-8. Study 1 - Individual Site Phenotypic Comparison – Seed

Characteristics - of MON 88701 Not Trea ted with Dicamba or Glufosinate

Herbicides Compared to th e Conventional Control. ..................................................559

Table G-9. Study 1 - Individual Site Phenotypic Comparison – Boll and

Fiber Characteristics - of MON 88 701 Not Treated with Dicamba or

Glufosinate Herbicides Compared to the Conventional Control ..............................560

Table G-10. Study 2 - Individual Site Phenotypic Comparison – Growth and

Development Characteristics - of MON 88701 Not Treated with Dicamba or

Glufosinate Herbicides Compared to the Conventional Control ..............................561

Table G-11. Study 2 - Individual Site Phenotypic Comparison – Seed

Characteristics - of MON 88701 Not Trea ted with Dicamba or Glufosinate

Herbicides Compared to th e Conventional Control ...................................................564

Table G-12. Study 2 - Individual Site Phenotypic Comparison – Boll and

Fiber Characteristics - of MON 88 701 Not Treated with Dicamba or

Glufosinate Herbicides Compared to the Conventional Control ..............................565

# Monsanto Company 12-CT-244U 25 of 620 

# Table G-13. Study 2 - Combined-Site Phenotypic Comparison - Growth and

Development Characteristics - of MON 88701 Treated with Dicamba and

Glufosinate Herbicides Compared to the Conventional Control ..............................567

Table G-14. Study 2 - Individual Site Phenotypic Comparison – Growth and

Development Characteristics - of MON 88701 Treated with Dicamba and

Glufosinate Herbicides Compared to the Conventional Control ..............................568

Table G-15. Study 2 - Individual Site Phenotypic Comparison – Seed

Characteristics - of MON 88701 Treate d with Dicamba and Glufosinate

Herbicides Compared to th e Conventional Control ...................................................571

Table G-16. Study 2 - Individual Site Phenotypic Comparison – Boll and

Fiber Characteristics - of MON 88701 Treated with Dicamba and

Glufosinate Herbicides Compared to the Conventional Control ..............................572

Table G-17. Study 2 - Individual Si te Phenotypic Comparison – Plant

Mapping - of MON 88701 Not Treate d with Dicamba or Glufosinate

Herbicides Compared to th e Conventional Control ...................................................574

Table G-18. Study 2 – Combined-Sit e Phenotypic Comparison – Plant

Mapping - of MON 88701 Treated with Dicamba or Glufosinate Herbicides

Compared to the Conventional Control ......................................................................576

Table G-19. Study 2 - Individual Si te Phenotypic Comparison – Plant

Mapping - of MON 88701 Treated with Dicamba or Glufosinate Herbicides

Compared to the Conventional Control ......................................................................577

Table G-20 Study 1 – Qualitative Asse ssment of Plant Response to Abiotic

Stressors - MON 88701 Not Treated with Dicamba or Glufosinate Herbicides

Compared to the Conventional Control ......................................................................579

Table G-21. Study 1 – Qualitativ e Assessment of Disease Damage -

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared

to the Conventional Control ..........................................................................................580

Table G-22. Study 1 – Qualitative A ssessment of Arthropod-related Damage

- MON 88701 Not Treated with Dica mba or Glufosinate Herbicides

Compared to the Conventional Control ......................................................................581

Table G-23. Study 1 - Individual Site An alysis: Quantitative Assessment of

Thrips Damage - MON 88701 Not Treated with Dicamba or Glufosinate

Herbicides Compared to th e Conventional Control ...................................................582

Table G-24. Study 1 - Individual Site An alysis: Quantitative Assessment of

Heliothine Damage - MON 88701 Not Trea ted with Dicamba or Glufosinate

Herbicides Compared to th e Conventional Control ...................................................583

Table G-25. Study 1 - Abundance of Pest Arthropods - MON 88701 Not

Treated with Dicamba or Glufosinate Herbicides Compared to the

Conventional Control ....................................................................................................584

Table G-26. Study 1 - Abundance of Beneficial Arthropods - MON 88701

Not Treated with Dicamba or Glufosi nate Herbicides Compared to the

Conventional Control ....................................................................................................588

# Monsanto Company 12-CT-244U 26 of 620 

# Table G-27 Study 2 – Qualitative Asse ssment of Plant Response to Abiotic

Stressors - MON 88701 Not Treated with Dicamba or Glufosinate Herbicides

Compared to the Conventional Control ......................................................................591

Table G-28. Study 2 – Qualitative Assessment of Disease Damage of

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared

to the Conventional Control ..........................................................................................592

Table G-29. Study 2 – Qualitative A ssessment of Arthropod-related Damage

-MON 88701 Not Treated with Dica mba or Glufosinate Herbicides

Compared to the Conventional Control ......................................................................593

Table H-1. Starting Seed for Pollen Morphology and Viability Assessment ...........597

Table I-1. Management Recommenda tions for Control of Dicamba-,

Glufosinate- and Other Selected Sy nthetic Auxin-Resistant Weeds .........................609

# Monsanto Company 12-CT-244U 27 of 620 

# LIST OF FIGURES

Figure III-1. Circular Map of PV -GHHT6997 Showing Probes 1-8 ..........................42

Figure III-2. Schematic of the Development of MON 88701 ......................................43

Figure III-3. Deduced Amino Acid Sequence of the MON 88701 DMO

Protein ....................................................................................................................... ........49

Figure III-4. Deduced Amino Acid Sequence of the PAT ( bar ) Protein ....................49

Figure IV-1. Schematic Representation of the Insert and Flanking DNA in

MON 88701 ..................................................................................................................... ..53

Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA in MON 88701: Probes 1 and 5 ....................................................................60

Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA in MON 88701: Probes 2 and 4 ....................................................................61

Figure IV-4. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA in MON 88701: Probe 3 ................................................................................62

Figure IV-5. Southern Blot Analysis to Determine the Presence or Absence

of PV-GHHT6997 Backbone Sequences in MON 88701: Probes 6, 7, and 8 ............64

Figure IV-6. Overlapping PCR Analys is across the Insert in MON 88701 ...............66

Figure IV-7. PCR Amplification of the MON 88701 Insertion Site in

Conventional Control ......................................................................................................68

Figure IV-8. Breeding History of MON 88701 .............................................................70

Figure IV-9. Southern Blot Analysis to Examine Insert Stability in Multiple

Generations of MON 88701: Probes 2 and 4 .................................................................71

Figure IV-10. Breeding Path for Ge nerating Segregation Data for

MON 88701 ..................................................................................................................... ..74

Figure V-1. Three Components of the DMO Oxygenase System ...............................78

Figure V-2. Dicamba and Potential Endogenous Substrates Tested through

In Vitro Experiments with DMO ....................................................................................80

Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data

Interpretation Methods .................................................................................................143

Figure VIII-1. Planted Upland Cotton A cres by County in the U.S. in 2010 ...........171

Figure VIII-2. Planted Pima Cotton A cres by County in the U.S. in 2010 ...............172

Figure C-1. Forms of DMO Protein a nd Their Relation to the Wild-Type

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

Figure C-2. MALDI-TOF MS Coverage Map of the MON 88701 DMO

Protein ....................................................................................................................... ......285

Figure C-3. Western Blot Analysis of MON 88701 DMO and E. coli

produced MON 88701 DMO Proteins..........................................................................288

# Monsanto Company 12-CT-244U 28 of 620 

# Figure C-4. Molecular Weight and Pu rity Analysis of the MON 88701 DMO

Protein ....................................................................................................................... ......292

Figure C-5. Glycosylation Analysis of the MON 88701 DMO Protein ....................295

Figure C-6. UPLC Separation of Dicamba (DCB) and DCSA in Five

Substrate Analysis ..........................................................................................................30 3

Figure C-7. E. coli -produced DMO Conversion of Endogenous Substrates ...........304

Figure C-8. UPLC Separation of Di camba (DCB) and DCSA in Bridging

Analysis ...................................................................................................................... .....305

Figure C-9. E. coli -produced MON 88701 DMO Conversion of o- Anisic Acid ......306

Figure C-10. MALDI-TOF MS Cover age Map of the MON 88701-produced

PAT ( bar ) Protein ...........................................................................................................315

Figure C-11. Western Blot An alysis of the MON 88701- and E. coli

- produced PAT ( bar ) Proteins ........................................................................................318

Figure C-12. Molecular Weight and Purity Analysis of the MON 88701-

produced PAT ( bar ) Protein .........................................................................................322

Figure C-13. Glycosylation Analysis of the MON 88701-produced PAT ( bar )

Protein ....................................................................................................................... ......325

Figure I-1. Weed Resistance to Various Herbicide Families 1 

# ...................................604

# Monsanto Company 12-CT-244U 29 of 620 

# ABBREVIATIONS AND DEFINITIONS 2



# Symbol or Abbrev. Definition

~ Approximately

α -Cyano

2,4-D

2,4-DB α -Cyano-4-hydroxycinnamic acid

2,4-Dichlorophenoxyacetic acid

2,4-DB = 4-(2,4-dichlorophenoxy)butyric acid

AA Amino Acid

AAbA α -aminobutyric acid

ADF Acid Detergent Fiber

a.e.

a.i.

ALS acid equivalent

active ingredient

acetolactate synthase inhibitor

APHIS Animal and Plant Health Insp ection Service of the United States

Department of Agriculture

bar

BIO Bialaphos Resistance Gene from Streptomyces hygroscopicus

Biotechnology Industry Organization

BLOCKS A database of amino acid motifs found in protein families

BLOSUM Blo cks Su bstitution M atrix, used to score similarities between

pairs of distantly related prot ein or nucleotide sequences

BSA Bovine Serum Albumin

CFR Code of Federal Regulations

CHT Ceramic hydroxyapatite

CoA Coenzyme A

COA Certificate of Analysis

CTAB Hexadec ylt rimethyla mmonium bromide

DAP Days After Planting

Da Dalton

dCTP Deoxyc ytidine trip hosphate

DEAE- D ie thyla minoe thylDHB 2,5-d ih ydroxyb enzoic acid

DCSA 3,6-d ic hloros alicylic acid

DDI

DGA Daily Dietary Intake

Diglycolamine

dicamba 3,6-dichloro-2-methoxybenzoic acid

dmo Mono-oxygenase gene from Stenotrophomonas maltophilia

DMO Dicamba mono-oxygenase

DNA

DSMA Deoxyribon ucleic acid

Disodium methanearsonate

DTNB 5,5’-d it hio-bis (2-n itrob enzoic acid)

DTT Dit hiot hreitol

dw Dry weight

DWCF Dry weight conversion factor

2 

# Alred, G.J., C.T. Brusaw, and W.E. Oliu. 2003. Handbook of Technical Writing, 7th edn., pp. 2-7.

Bedford/St. Martin's, Boston, MA.

# Monsanto Company 12-CT-244U 30 of 620 

# ECL Enhanced Chemil uminescence

E. coli Escherichia coli

E.coli -produced

MON 88701 DMO DMO protein produced from E. coli with the same sequence as

MON 88701 DMO

ELISA Enzyme-linked Immunos orbent Assay

EPA Environmental Protection Agency

E -Score

ETS Expectation score

Excellence Through Stewardship SM



# FA Fatty Acid

FARRP Food Allergy Research and Resource Program

FASTA Algorithm used to find local high scoring alignments between a

pair of protein or nucleotide sequences

FDA Food and Drug Administration (U.S.)

FFDCA Federal Food, Drug and Cosmetic Act (U.S.)

FT Flow through

fw Fresh weight

glufosinate butanoic acid, 2-am ino-4-(hydroxymethylphosphinyl)

GLP Good Laboratory Practice

ha hectare

HPLC High Performance Liquid Chromatography

HRP Horser adish Peroxidase

HU Hemagglutinating Unit

ILSI

ISO International Life Sciences Institute

International Organizatio n for Standardization

kb Kilob ase

kDa Kiloda lton

kg Kilogram

LB Laemmli buffer

LOD Limit of Detection

LOQ Limit of Quantitation

MALDI-TOF-MS Matrix Assisted Laser Desorpti on Ionization - Time of Flight

Mass Spectrometry

μ g

μ l Microgram

Microliter

mg

mic Milligram

micronaire

MOE

MON 87708 Margin of Exposure

Dicamba-tolerant soybean developed by Monsanto Company

MON 88701 DMO

MRL

MSMA DMO protein produced in MON 88701

Maximum Residue Levels

Monos odium methanea rsonate

MW Molecular Weight

MWCO Molecular Weight Cuto ff

N-acetyl glufosinate 2-acetamido- 4-methylphosphinico-butanoic acid

NADH Nicotinamide adenine dinucleotide

# Monsanto Company 12-CT-244U 31 of 620 

# NCBI National Center for Biotechnology Information at the National

Institutes of Health, Bethesda, MD, USA

NDF Neutral Detergent Fiber

NFDM Non-fat Dried Milk

NOAEL No Observable Adverse Effect Level

OECD Organisation for Economic Co-operation and Development

ORF Open Reading Frame

OSL Overs eason Leaf

p Probability from PRESS

PAT Phosphinothricin N-a cetylt ransferase

PAT ( bar ) PAT protein produced by the bar gene

PBS Phosphate Buffered Saline

PBST Phosphate Buffered Sa line containing Tween-20

PCR Polymerase Chain Reaction

PI

PPA Prediction Interval

Plant Protection Act (7 U.S.C. § 7701-7772)

ppm

PPO parts per million

protoporphyrinogen oxidase inhibitor

PPT Phosp hinot hricin

PRESS Predicted Residual Sum of Squares

PRT_2011 GenBank protein database , 181.0 (Released December 18, 2010)

PTH Phenylt hioh ydantoin

PVDF Polyv inylidene dif luoride

PVP Polyv inyl pyrrolidone

RBD Refined, Bleached, and Deodorized

RED Reregistration Eligibility Decision

RT

SCST Room temperature

Society of Commercial Seed Technologists

SDS Sodium Dodecyl Sulfate

SDS-PAGE Sodium Dodecyl Sulfate-Polya crylamide Gel Electrophoresis

SE Standard Error

SGF Simulated Gastric Fluid

S. hygroscopicus Streptomyces hygroscopicus

SIF Simulated Intestinal Fluid

Sinapinic Acid 3,5-dimet hoxy-4-hydroxycinnamic acid

S. maltophilia Stenotrophomonas maltophilia

SOP Standard Operating Procedure

TBA Tris-borate buffer with L-ascorbic acid

TBS Tris Buffered Saline

TCEP Tris(2-c arboxye thyl)p hosphine

T-DNA Transfer DNA

TDF

tex Total Dietary Fiber

Grams of 1000 meters of fiber

TFA Trif luoroacetic Acid

TFE 2,2,2,-trif luoroe thanol

TIU Trypsin Inhibitor Unit

# Monsanto Company 12-CT-244U 32 of 620 

# Tm Melting temperature

TNB 5-thio-nitrob enzoate

TOX_2011 Toxin protein sequence database (Release date February 18,

2011)

V volts

v/v volume to volume ratio

w/v weight to volume ratio

# Monsanto Company 12-CT-244U 33 of 620 

# I. RATIONALE FOR THE DEVELOPMENT OF MON 88701

I.A. Basis for the Request for a Determ ination of Nonregulated Status under

7 CFR § 340.6

The Animal and Plant Health Inspection Service (APHIS) of the United States

Department of Agriculture (USDA) has res ponsibility, under the Plant Protection Act

(Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772), to prevent the

introduction and dissemination of plant pe sts 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 arti cle does not present a pl ant pest risk and no

longer should be regulated. If APHIS dete rmines that the regulated article does not

present a plant pest risk, the petition is gr anted, thereby allowing unr estricted introduction

of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of

nonregulated status for th e new biotechnology-derived co tton product, MON 88701, any

progeny derived from crosses between M ON 88701 and conventional cotton, and any

progeny derived from crosses of MON 88701 with biotechnology-derived cotton that

have previously been granted nonre gulated status under 7 CFR Part 340.

I.B. Rationale for the Development of Dicamba and Glufosinate-Tolerant Cotton –

MON 88701

Biotechnology derived cotton and the introduct ion of glyphosate-tolerant cotton systems

permit in-crop application of agricultural he rbicides containing the active ingredient

glyphosate for effective weed control. The value of glyphosate-tolerant cotton systems

has been demonstrated by the significant growth in the nu mber of glyphosate-tolerant

acres planted since introduction of this tec hnology in 1997. Today, more than 75% of all

cotton acres grown in the U.S. are gl yphosate-tolerant (USDA-NASS, 2010). The

glyphosate-tolerant systems deliver effectiv e broad spectrum weed control, provides

flexibility of application tim ing, increased adoption of reduc ed tillage practices, and has

resulted in increased grower income (Carpent er and Gianessi, 2001; Hu rley et al., 2009).

Additionally, the glyphosate-tolerant syst ems provide incremental environmental

benefits, including reduced overall herb icide usage (Brookes and Barfoot, 2012;

Carpenter and Gianessi, 2001). Furthermore, glyphosate, as concluded by the U.S. EPA

(1993), has a favorable safety profile. C ontinued use of glyphosate-tolerant cotton

systems will maintain effective and familiar weed control management practices that are

fully compatible with all current tillage and land management practices, including

conservation tillage practices. Growth of c onservation tillage in the U.S. was greatly

accelerated with the introduction of glyphosate-tolerant crops in large part because of the

broad spectrum postemergence control offe red by glyphosate (Pri ce et al., 2011). The

benefits associated with conservation tillag e, include reduced soil erosion, reduced fuel

and labor costs, and conservati on of soil moisture (CTIC, 2011).

As with all herbicides used in agriculture, there is potential for weeds to develop

resistance to frequent and continual use of the same herbicide over an extended time

# Monsanto Company 12-CT-244U 34 of 620 

# period (Powles, 2008). Plant populations can de velop resistance to a herbicide due to the

selection of individuals that carry altered ge netic codes producing alleles that can render

those individuals tolerant to the lethal eff ects of a herbicide. Weed populations with

confirmed herbicide-resistance are listed on the International Survey of Resistant Weeds

website ( www.weedscience.org ). Without effective weed management practices in

agricultural systems, herbicide resistance in weeds can become a limiting factor in crop

production. As with many agri cultural use herbicides, glyph osate has documented cases

of weed resistance. While there have been thirteen confirmed gl yphosate-resistant weeds

in the U.S. (Heap, 2012a), glyphosate still effectively controls more than 160 weed

species (Roundup WeatherMax ® 

# herbicide label, EPA Re g. No.524-537) and remains an

extremely valuable tool for U.S. cotton crop production. Studies have shown that

resistance can be postponed, containe d, and managed through good management

practices. One of the management practices most often recommended by

University/Cooperative Extension Service and industry is the use of multiple herbicide

modes-of-action. Simultaneously using mul tiple herbicides with different modesof-action significantly reduces the probability of weeds developing resistance to any or

all of the applied herbicides (Beckie and Reboud, 2009; Powles et al., 1996). Other weed

management recommendations include the use of multiple herbicide modes-of-action in

sequence and the inclus ion of mechanical or cultural weed management practices, in

addition to the use of a herbicide.

Monsanto Company has developed dicam ba and glufosinate-tolerant cotton,

MON 88701, which will allow in-crop appli cations of dicamba (3,6-dichloro-2-

methoxybenzoic acid) herbicide for the control of broadleaf weeds from preemergence to

seven days preharvest and glufosinate herbic ide for broad spectrum weed control from

emergence through early bloom growth stag e. MON 88701 provides dicamba tolerance

that allows for the in-crop application of dicamba beyond th e current preplant uses in

cotton and also provides glufosinate tole rance equivalent to current commercial

glufosinate-tolerant cotton events. The co mbination of the two herbicides’ distinct

modes-of-action provides an effective weed management system. Dicamba provides

effective control of over 95 annual and bie nnial weed species, and suppression of over

100 perennial broadleaf and woody plant species (BASF, 2008) (EPA Reg. No. 7969-

137) and glufosinate is a broad-spectrum c ontact herbicide that provides nonselective

control of about 120 broadleaf and grass w eeds (Bayer CropScience, 2011) (EPA Reg.

No. 264-829). Additionally, dicamba and gluf osinate each provide control of many

herbicide-resistant weeds, including glyphosat e-resistant biotypes of Palmer amaranth

( Amaranthus palmeri ), marestail ( Conyza Canadensis ), common ragweed ( Ambrosia

artemisiifolia ), giant ragweed ( Ambrosia trifida ) and waterhemp [ Amaranthus

tuberculatus ). Weeds that are hard-to- control using glyphosate (See Roundup

WeatherMax ® 

# label (U.S. EPA Reg. No. 524-537) fo r a listing], generally require a

higher rate and/or application at a smaller growth stage in order to consistently achieve

commercially acceptable control. To date, only four species with known dicambaresistant biotypes ( i.e. , common hempnettle, Galeopsis tetrahit ; kochia, Kochia scoparia ;

prickly lettuce, Lactuca serriola ; and wild mustard, Sinapis arvensis ) and one species

® 

# Roundup and WeatherMax are registered tr ademarks of Monsanto Technology, LLC.

# Monsanto Company 12-CT-244U 35 of 620 

# with a known glufosinate-resistant biotype ( i.e. , Italian ryegress, Lolium multiflorum )

have been identified in North America (Heap, 2012b; 2012c). Known resistant weed

populations to dicamba and glufosinate are pr imarily found in the western U.S. and, thus,

are not present in the major cotton geographi es. See Appendix I for additional details.

MON 88701 will be combined, through traditional breeding methods, with other

approved herbicide-tolerant ( e.g. , glyphosate-tolerant) events. The opportunity for incrop use of dicamba and glufosinate herbicid es, in addition to gl yphosate herbicide,

provides new weed management options in co tton to control a broad spectrum of grass

and broadleaf weed species and effective cont rol of weeds resistant to several herbicide

families. Successful integration of MON 88701 into glyphosate-tolerant cotton systems

will provide: 1) an opportunity for an effi cient, effective weed management system for

hard-to-control and herbicide-resistant weeds; 2) a flexible system for two additional incrop herbicide modes-of-action in current cotton production practices as recommended

by weed science experts to manage future w eed resistance development; 3) an option to

delay or prevent further resistance to gl yphosate and other critically important cotton

herbicides, in particular herbicides in the AL S and PPO class of chemistry; 4) crop safety

to dicamba, glufosinate and glyphosate; and 5) additional weed ma nagement tools to

enhance weed management systems necessary to maintain yield and quality to meet the

growing needs of fiber, food, and feed.

MON 88701 contains a de methylase gene from Stenotrophomonas maltophilia that

expresses a dicamba mono-oxygenase (DMO) pr otein to confer tolerance to dicamba

herbicide and a bialaphos resistance ( bar ) gene from Streptomyces hygroscopicus that

expresses the phosphinothricin N-acetyltransfera se (PAT) protein to confer tolerance to

glufosinate herbicide. DMO protein rapidly demethylates dicamba to the herbicidally

inactive metabolite 3,6-dichlorosalicylic acid (DCSA), a well known metabolite of

dicamba in conventional cotton, soybean, li vestock, and soil (FAO-WHO, 2011a; 2011b;

U.S. EPA, 2009). Monsanto will request a re gistration from U.S. EPA for the expanded

use of dicamba on MON 88701 cotton, an increas e in the dicamba residue tolerance for

cottonseed, the establishment of a tolerance for cotton gin by-products, and the inclusion

of DCSA in the residue definitions for co ttonseed and gin by-pr oducts. No other

revisions to the dicamba pesticide residue tolerances are necessary, including those for

animal products such as meat, eggs, and milk. Furthermore, the use of dicamba on

MON 88701 does not present any new environm ental exposure scenarios not previously

evaluated and deemed acceptable by EPA.

PAT ( bar ) protein acetylates the free amino group of glufosinat e to produce nonherbicidal N-acetyl glufosinat e, a well known metabolite in glufosinate-tolerant plants

(OECD, 2002a). The use pattern and rate of glufosinate on MON 88701 will follow the

existing glufosinate-tolerant cotton uses outlin ed on the glufosinate herbicide labels and

the glufosinate residues in MON 88701 treated with commercial glufosinate rates are

below the established pesticide residue to lerances established by U.S. EPA for both

cottonseed and gin by-products (40 CFR § 180.473). Theref ore, Monsanto will not

pursue any changes in the glufosinate labels or the established tolerances for its use on

MON 88701 cotton.

# Monsanto Company 12-CT-244U 36 of 620 

# I.C. Submissions to Other Regulatory Agencies

Under the Coordinated Framework for Regulation of Biotechnology (CFR) (USDAAPHIS, 1986), the responsibil ity for regulatory oversight of biotechnology-derived crops

falls primarily on three U.S. agencies: U. S. Food and Drug Admi nistration (FDA), the

United States Department of Agriculture (USDA), and in the case of herbicide-tolerant

products, the Environmental Protection Agency (EPA). A request for deregulation of

MON 88701 made to USDA constitutes only one component of the overall regulatory

oversight and review of this product. As a practical matter, MON 88701 cannot be

released and marketed until FDA and USDA have completed their reviews and

assessments under their respective jurisdicti ons. Additionally, EPA must complete its

review and assessments prior to approving th e use and allowable residues of dicamba on

MON 88701.

I.C.1. Submission to FDA

MON 88701 falls within the scope of th e 1992 FDA policy statement concerning

regulation of products derive d from new plant varieties, including those developed

through biotechnology (U.S. FDA, 1992). In co mpliance with this policy, Monsanto has

initiated a consultation with the FDA (BNF No. 135) on th e food and feed safety and

compositional assessment of MON 88701. Monsanto submitted a safety and nutritional

assessment summary document to the FDA in April 2012.

I.C.2. Submission to EPA

The safety of dicamba use on many crops, including cotton, was reviewed by the

Environmental Protection Agency (EPA) as part of the food, feed, and environmental

safety reassessment in 2006 (U.S. EPA, 2009) . Dicamba can currently be applied to

cotton in the U.S. as a pre- plant application, at least 21 days prior to planting. The

tolerance of MON 88701 to dicamba facilitate s a wider window of application on cotton,

allowing pre-emergence application of the he rbicide up to the day of crop emergence and

post-emergence in-crop applications through seven days pre-harvest. Monsanto will

request a registration from U.S. EPA for the expanded use of dicamba on MON 88701,

an increase in the dicamba residue tolera nce from 0.2 ppm to 3 ppm for cottonseed, the

establishment of a tolerance of 70 ppm for cotton gin by-pr oducts, and the inclusion of

DCSA in the residue definitions for cottons eed and gin by-products. No other revisions

to dicamba pesticide residue tolerances ar e needed including animal products such as

meat, eggs, or milk.

The existing 0.2 ppm pesticide residue tole rance for cottonseed supporting the current

registered uses of dicamba on cotton (40 CF R § 180.227) is for the combined residues of

parent dicamba and its metabolite 5-hydr oxy dicamba. Cotton gin by-products, a

ruminant feed supplement, have no establishe d dicamba tolerance. Studies have shown

that the proposed use of dicamba on MO N 88701 cotton results in total residue

concentrations of parent dicamba and its metabolites, including DCSA and 5-hydroxy

dicamba, are less than 3 ppm for cottonseed and less than 70 ppm for gin by-products.

# Monsanto Company 12-CT-244U 37 of 620 

# The safety of glufosinate use on many crops , including cotton, was reviewed by the

Environmental Protection Agency (EPA) as part of the food, feed, and environmental

safety reassessment in 2000 (U.S. EPA, 2003). In addition, glufosinate has been used

over-the-top of glufosinate-to lerant crops since 1995 with no significant adverse effects

reported. Glufosinate is currently labeled fo r in-crop application on glufosinate-tolerant

cotton from emergence through early bloom gr owth stage (Bayer CropScience, 2011).

The use pattern and rate of glufosinat e on MON 88701 will fo llow the existing

glufosinate-tolerant cotton uses outlined on the glufosinate herbicide label. Furthermore,

glufosinate residues in M ON 88701 treated with glufosinate are below the EPAestablished residue tolerances of 4.0 ppm and 15.0 ppm for cottonseed and gin byproducts, respectively (U.S. EPA, 2003) (40 CFR § 180.473). Both of these tolerances

include the combined residues of parent glufosinate and its metabolites N-acetyl

glufosinate and 3-methylphosphini co-propionic acid. Currently glufosinate is undergoing

reregistration at EPA with the Reregistrati on Eligibility Decision (R ED) expected by the

end of 2013 (U.S. EPA, 2008). It is likely that EPA will affirm the safety and efficacy of

glufosinate and approve its continued us e in the marketplace upon completion of the

reregistration process. Therefore, Monsanto will not pursue any changes in the

glufosinate label, use patter n, or the established tolera nces for its use on MON 88701

cotton.

I.C.3. Submissions to Forei gn Government Agencies

To support commercial introduction of MON 88701 in the U.S., re gulatory submissions

will be made to countries that import signi ficant quantities of cotton or its processed

fractions from the U.S. These will include submissions to a number of foreign

government regulatory authorities, including: Ja pan’s Ministry of Agriculture, Forestry,

and Fisheries and the Ministry of Healt h, Labour, and Welfare; the Canadian Food

Inspection Agency; Health Canada; the In tersectoral Commission for Biosafety of

Genetically Modified Organisms, Mexico; the Korea Food and Drug Administration; and

the Rural Development Administration of Korea, as well as to regulatory authorities in

other cotton importing countries with functi oning regulatory systems. As appropriate,

notifications will be made to countries that import signi ficant quantities of cotton and

cotton products that do not have a formal regulatory revi ew process for biotechnologyderived crops.

# Monsanto Company 12-CT-244U 38 of 620 

# II. THE BIOLOGY OF COTTON

The Organisation for Economic Co-operati on and Development Consensus Document

(OECD, 2008) on the biology of cotton ( Gossypium spp.) provides key information on:

- general description of cotton biology, including taxonomy and

morphology and use of cotton as a crop plant

- agronomic practices in cotton cultivation

- geographic centers of origin

- reproductive biology

- inter-species/genus introgression into relatives and interactions

with other organisms

- summary of the ecology of cotton

Additional information on the biology and grow th and development of cotton is available

in the literature (Kohel and Lewis, 1984; OGTR, 2008; Smith and Cothren, 1999).

To support the evaluation of the plant pe st potential of MON 88701 relative to

conventional cotton, additional information regarding several aspects of cotton biology

can be found elsewhere in this petition. This includes: agronomic practices for cotton in

Section V.III; volunteer management of cott on in Sections VIII.H and IX.C; and interspecies/genus introgression potential in Section IX.D.

II.A. Cotton as a Crop

Cotton belongs to the genus Gossypium that currently has a pproximately 50 species

which are widely cultivated in tropical and subtropical regions around the world (OECD,

2008; Percival et al., 1999). There are four cultivated species that were domesticated

independently, two of which account for grea ter than 95% of world cotton production.

Gossypium hirsutum (often called upland, American, Me xican, or Acala cotton) accounts

for 90% and Gossypium barbadense (often called extra long-s taple, Pima, and Egyptian

cotton) accounts for 5% of world cotton production. Due to the utility of the fibers for

the production of textiles, human selection pr essure on cotton has al tered the plant from

essentially perennial shrubs or trees with small impermeable seeds and sparse hairs to a

compact annual row crop, yielding large, ea sily germinating seeds with white, thick,

long, and strong fibers (B rubaker et al., 1999).

The four cultivated species, which are widely cultivated across the entire globe, are

comprised of two diploid species G. arboretum and G. herbaceum , which evolved from

Africa and the Middle East, and two allotetraploid species G. barbadense and G.

hirsutum , which evolved in the Americas (Brubaker et al., 1999).

Improved modern varieties of G. hirsutum and G. barbadense are currently cultivated in

the southern U.S., with G. barbadense grown primarily in the western states of Arizona,

California, New Mexico, and Texas; and G. hirsutum produced throughout the 17 states

comprising the U.S. cotton growing region, co mmonly referred to as the cotton belt.

G. hirsutum comprises the vast majority of U.S. cotton production with nearly 11 million

# Monsanto Company 12-CT-244U 39 of 620 

# acres planted and 18 million bales harvested, whereas G. barbadense varieties accounted

for approximately 200,000 acres and half a m illion bales in 2010 (U SDA-NASS, 2011e).

Commercial cotton, including G. hirsutum and G. barbadense , has a long history of

agricultural production (Lee, 1984; USDA -AMS, 2001; USDA-NASS, 2012c). Extralong staple lint from G. barbadense is segregated and classed separately from G.

hirsutum and is sold at a premium (USDA- AMS, 2001). However, cottonseed and

cottonseed by-products ( e.g. , oil and meal) are not genera lly distinguished by species

(OECD, 2008; USDA-FAS, 2005).

II.B. Characteristics of the Recipient Plant

The G. hirsutum cotton variety used as the recipient for the DNA insertion to create

MON 88701 was Coker 130, a non-transgenic, conventional, upland inbred variety

developed by Coker Pedigreed Seed Co., co mmercialized in 1990 in the U.S. (Bowman

et al., 2006).

II.C. Cotton as a Test System in Product Safety Assessment

Coker 130 was used as the near isogenic, conventional parental cotton comparator

(referred to in this petition as the conven tional control) in the safety assessment of

MON 88701. MON 88701 and the conventiona l control have similar genetic

backgrounds with the exception of the T-DNA, thus, the effect of the T-DNA and the

expressed MON 88701 DMO and PAT ( bar ) proteins could be assessed. In addition,

commercial cotton varieties (referred to in this consultation document as commercial

reference varieties) were used as reference materials to establish ranges of natural

variability representative of commercial co tton varieties. The commercial reference

varieties used at each field trial location we re selected based on their availability and

agronomic fit for the resp ective geographic region.

# Monsanto Company 12-CT-244U 40 of 620 

# III. DESCRIPTION OF TH E GENETIC MODIFICATION

MON 88701 was developed through Agrobacterium tumefaciens -mediated

transformation of cotton tissues from C oker 130 variety utilizing plasmid vector

PV-GHHT6997. This section describes the plasmid vector, the donor gene, and the

regulatory elements used in the development of MON 88701, as well as the deduced

amino acid sequence of the MON 88701 DMO protein and PAT ( bar ) protein produced

in MON 88701. In this section, transfer DNA (T-DNA) refers to DNA that is transferred

to the plant during transforma tion. An expression cassette is comprised of sequences to

be transcribed and the regulatory elements necessary for the expression of those

sequences.

III.A. PV-GHHT6997

PV–GHHT6997 was used in the transformation of cotton to produce MON 88701 and its

plasmid map is shown in Figure III-1. The elem ents included in this plasmid vector are

described in Table III-1. PV- GHHT6997 is approximately 9.4 kb and contains one

T-DNA that is delineated by Left Border and Right Border regions. The T-DNA contains

the dmo and bar expression cassettes. The dmo expression cassette is regulated by the

peanut chlorotic streak caulimovirus ( PC1SV ) promoter, the tobacco etch virus ( TEV ) 5 ′

leader sequence, and the 3 ′ untranslated sequence of the E6 gene from Gossypium

barbadense . The chloroplast transit peptide CTP2 directs transport of the DMO protein

to the chloroplast in MON 88701 and is derived from the CTP2 target sequence of the

Arabidopsis thaliana shkG gene (Herrmann, 1995; Klee et al., 1987). The bar expression

cassette is regulated by the e35S promoter from the 35S RNA of cauliflower mosaic virus

(CaMV), the heat shock protein 70 ( Hsp70) leader, and the nopaline synthase ( nos)

3 ′ untranslated region.

The backbone region of PV–GHHT6997, locate d outside of the T-DNA, contains two

origins of replication for maintenan ce of plasmid vector in bacteria ( oriV and

ori-pBR322 ), a bacterial selectable marker gene ( aadA ), and a coding sequence for

repressor of primer ( rop ) protein for maintenance of plasmid vector copy number in

Escherichia coli ( E. coli ). A description of the geneti c elements and their prefixes ( e.g.

, B-, P-, L-, TS-, CS-, T-, and OR-) in PV-GHHT6997 is provided in Table III-1.

III.B. Description of the Transformation System

MON 88701 was developed through Agrobacterium -mediated transformation of

PV-GHHT6997 (Figure III-1) into cotton hypocotyls, based on published methods

(Duncan, 2010; Duncan and Ye, 2011). In summary, hypocotyl segments were excised

from dark grown seedlings of germinated Coker 130 seed. After co-culturing with the

Agrobacterium 3 

# carrying the vector, the hypocotyl segments were placed on a sequence

of media for callus growth containing carbeni cillin and cefotaxime to inhibit the growth

of excess Agrobacterium and glufosinate to inhibit growth of untransformed cells. The

3 

# Agrobacterium strain used contained a disarmed Ti plasmid.

# Monsanto Company 12-CT-244U 41 of 620 

# somatic embryos developing on the culture medium were th en placed on medium that

contained plant growth regulators conducive to shoot regeneration, but no antibiotics or

glufosinate. Rooted plants (R 0 

# ) with normal phenotypic character istics were selected and

transferred to soil for grow th and further assessment.

The R 0 

# plants generated through the Agrobacterium -mediated transformation were selfpollinated to produce R 1 

# seed. R 0 

# and R 1 

# plants were evaluated for tolerance to dicamba

and glufosinate and screened for the presence of the T-DNA ( dmo and bar expression

cassettes) and absence of plasmid vector backbone (o riV ). Subsequently, the dmo and

bar homozygous positive R 1 

# plant was self-pollinated to give rise to R 2 

# plants.

Homozygous positive R 2 

# plants containing only a si ngle T-DNA insertion, were

identified by a combination of analytical techniques including di camba and glufosinate

sprays, polymerase chain reaction (PCR), an d Southern blot anal ysis, resulting in

production of dicamba and glufosinate- tolerant cotton MON 88701. MON 88701 was

selected as the lead event based on superi or phenotypic characteristics and its molecular

characteristics. Studies on M ON 88701 were initiated to furt her characterize the genetic

insertion and the expressed proteins, and to establish the food, feed, and environmental

safety relative to conventional cotton. The major steps involved in the development of

MON 88701 are depicted in Figure III-2.

# Monsanto Company 12-CT-244U 42 of 620 

# Probe Start Position (bp) End Position (bp) Total Le ngth (~kb)

1 1 1310 1.3

2 1223 2241 1.0

3 2142 3252 1.1

4 3153 3914 0.8

5 3832 4625 0.8

6 4626 6282 1.7

7 6204 7708 1.5

8 7630 9379 1.8



# Figure III-1. Circular Map of PV-GHHT6997 Showing Probes 1-8

A circular map of PV-GHHT6997 used to develop MON 88701 is shown.

PV-GHHT6997 contains a single T-DNA. Genetic elements and restriction sites (in

bold) used in Southern analyses (with positions relative to the first base pair of the

plasmid vector) are shown on the exterior of the map. The probes used in the Southern

analyses are shown on the interior of the map and listed in the table.

# Monsanto Company 12-CT-244U 43 of 620 

# Figure III-2. Schematic of the Development of MON 88701 Transformed hypocotyl tissue from Coker 130 with PV-GHHT6997 in

Agrobacterium tumefaciens containing a disarmed Ti plasmid

Selected transformants and generated rooted shoots from the

transformed hypocotyl tissue

Identified MON 88701 as lead candidate and further evaluated its

progeny in laboratory and field assessments for in sert integrity, dicamba

and glufosinate tolerances and agronomic performance Assembled Agrobacterium binary plasmid vector PV-GHHT6997 and

transferred to Agrobacterium tumefaciens strain ABI

Evaluated the transformed plants for tolerance to dicamba and glufosinate

and screened the transformed plants for the presence of the T-DNA ( dmo

and bar expression cassettes) and absence of backbone (o riV )

Selected homozygous plants were id entified by dicamba and glufosinate

sprays, polymerase chain reaction (PCR) , and Southern blot analysis

# Monsanto Company 12-CT-244U 44 of 620 

# III.C. The dmo Coding Sequence and the MON 88701 DMO Protein

The dmo expression cassette encodes a ~ 39 kDa MON 88701 DMO precursor protein

consisting of a single polypeptide of 416 amino acids (Figure III-3). The dmo coding

sequence is the codon optimized coding sequence from Stenotrophomonas maltophilia

that encodes the DMO protein (Herman et al ., 2005; Wang et al., 1997). The presence of

MON 88701 DMO protein confers dicamba tolerance.

III.D. The bar Coding Sequence and PAT ( bar ) Protein

The bar expression cassette encodes a ~21 kDa PAT ( bar ) protein consisting of a single

polypeptide of 183 amino acids (Thomps on et al., 1987) (Figure III-4). The bar coding

sequence is from Streptomyces hygroscopicus and encodes the phosphinothricin

N-acetyltransferase (PAT) protein (Thomps on et al., 1987). The presence of PAT ( bar )

protein confers glufosinate tolerance.

III.E. Regulatory Sequences

The dmo coding sequence in MON 88701 is under the regulation of the PC1SV promoter,

the TEV 5 ′ leader, and the E6 3 ′ untranslated region. The PC1SV promoter is the

promoter for the Full-Length Transcript (FLt ) of peanut chlorotic streak caulimovirus

(Maiti and Shepherd, 1998) that direct s transcription in plant cells. The TEV leader is the

5 ′ untranslated region from the tobacco etch virus (Niepel and Gallie, 1999) and is

involved in regulating gene ex pression. The chloroplast tr ansit peptide CTP2 directs

transport of the DMO protein to the chloro plast in MON 88701 and is derived from the

CTP2 target sequence of the Arabidopsis thaliana shkG gene (Herrmann, 1995; Klee et

al., 1987). The E6 3 ′ non-translated region is the 3 ′ untranslated region from the E6 gene

of Gossypium barbadense encoding a fiber protein, wh ich functions to direct

polyadenylation of the mRNA (John, 1996).

The bar coding sequence in MON 88701 is under the regulation of the e35S promoter,

the Hsp70 leader, and the nos 3 ′ untranslated region. The e35S promoter is the promoter

for the 35S RNA of cauliflower mosaic virus (CaMV) (Odell et al., 1985), containing the

duplicated enhancer region (Kay et al., 1987) that directs transc ription in plant cells. The

Hsp70 leader is the 5 ′ untranslated region from the DnaK gene from Petunia hybrida

(Rensing and Maier, 1994; Wi nter et al., 1988) and is involved in regulating gene

expression. The nos 3 ′ untranslated region is the 3 ′ untranslated region from the nopaline

synthase ( nos ) gene of Agrobacterium tumefaciens encoding NOS and directs

polyadenylation of the mR NA (Bevan et al., 1983; Fraley et al., 1983).

III.F. T-DNA Borders

PV-GHHT6997 contains Right Border and Le ft Border regions (Figure III-1 and

Table III-1), which were derived from Agrobacterium tumefaciens plasmids. The border

regions each contain a 24-25 bp nick site th at is the site of DNA exchange during

transformation (Barker et al., 1983; Depicker et al., 1982; Zambryski et al., 1982). The

border regions separate the T-DNA from the pl asmid backbone region and are involved

in the efficient transfer of T-DNA into the cotton genome.

# Monsanto Company 12-CT-244U 45 of 620 

# III.G. Genetic Elements Outside of the T-DNA Borders

Genetic elements that exis t outside of the T-DNA border regions are those that are

essential for the maintenance or selection of PV-GHHT6997 in bacteria. The origin of

replication, oriV, is required for the maintenance of the plasmid in Agrobacterium and is

derived from the broad host plasmid RK2 (Stalker et al., 1981). The orig in of replication,

ori-pBR322, is required for the maintenance of the plasmid in E. coli and is derived from

the plasmid vector pBR322 (Sutcliffe, 1979). Coding sequence rop encodes the repressor

of primer (ROP) protein which is necessary for the maintenance of plasmid copy number

in E. coli (Giza and Huang, 1989). The selectable marker aadA is a bacterial promoter

and coding sequence for an enzyme from transposon Tn7 that confers spectinomycin and

streptomycin resistance (Fling et al., 1985) in E. coli and Agrobacterium during

molecular cloning. Because these elements ar e outside the border re gions, they are not

expected to be transferred into the cotton ge nome. The absence of detectable backbone

sequence in MON 88701 has been confirmed by Southern blot analyses (See Section IV–

B).

# Monsanto Company 12-CT-244U 46 of 620 

# Table III-1. Summary of Ge netic Elements in PV-GHHT6997

Genetic

Element Location in

Plasmid

Vector (bp) Function (Reference)



# T-DNA

B 1 

# -Right

Border

Region 

# 1-331 DNA region from Agrobacterium tumefaciens containing

the Right Border sequence used for transfer of the T-DNA

(Depicker et al., 1982; Zambryski et al., 1982)

Intervening

Sequence 332-433 Sequence used in DNA cloning

P 2



# - PC1SV 

# 434-866 Promoter from the Full-Length Transcript (FLt) of peanut

chlorotic streak caulimovirus ( PC1SV ) that directs

transcription in plant cells (Maiti and Shepherd, 1998)

Intervening

Sequence 867-872 Sequence used in DNA cloning

L 3



# - TEV 

# 873-1004 5 ′ UTR leader sequence from the RNA of tobacco etch virus

(TEV) (Niepel and Gallie, 1999) that is involved in

regulating gene expression

Intervening

Sequence 1005 Sequence used in DNA cloning

TS 4



# - CTP2 

# 1006-1233 Targeting sequence of the ShkG gene from Arabidopsis

thaliana encoding the EPSPS transi t peptide region that

directs transport of the protei n to the chloroplast (Herrmann,

1995; Klee et al., 1987)

CS 5



# - dmo 

# 1234-2256 Codon optimized coding sequence for the dicamba

mono-oxygenase (DMO) protein of Stenotrophomonas

maltophilia that confers dicamba to lerance (Herman et al.,

2005; Wang et al., 1997)

Intervening

Sequence 2257-2310 Sequence used in DNA cloning

T 6



# - E6 

# 2311-2625 3 ′ UTR sequence of the E6 gene from Gossypium

barbadense (cotton) encoding a fibe r protein involved in

early fiber development (John, 1996) that directs

polyadenylation of mRNA

Intervening

Sequence 2626-2637 Sequence used in DNA cloning

P- e35S 

# 2638-3249 Promoter from the 35S RNA of cauliflower mosaic virus

(CaMV) (Odell et al., 1985) containing the duplicated

enhancer region (Kay et al., 1987) that directs transcription

in plant cells

Intervening

Sequence 3250-3252 Sequence used in DNA cloning

# Monsanto Company 12-CT-244U 47 of 620 

# Table III-1. Summary of Genetic Elements in PV-GHHT6997 (continued)

Genetic

Element Location in

Plasmid

Vector (bp) Function (Reference)

L- Hsp70 

# 3253-3348 5 ′ UTR leader sequence of the DnaK gene from Petunia

hybrida that encodes heat s hock protein 70 (HSP70)

(Rensing and Maier, 1994; Winter et al., 1988) that is

involved in regulati ng gene expression

Intervening

Sequence 3349-3354 Sequence used in DNA cloning

CS- bar 

# 3355-3906 Coding sequence for the phosphinothricin

N-acetyltransferase (PAT) protein of Streptomyces

hygroscopicus that confers glufosin ate tolerance (Thompson

et al., 1987)

Intervening

Sequence 3907-3911 Sequence used in DNA cloning

T- nos 

# 3912-4164 3 ′ UTR sequence of the nopaline synthase ( nos ) gene from

Agrobacterium tumefaciens pTi encoding NOS that directs

polyadenylation (Bevan et al ., 1983; Fraley et al., 1983)

Intervening

Sequence 4165-4183 Sequence used in DNA cloning

B-Left

Border

Region 

# 4184-4625 DNA region from Agrobacterium tumefaciens containing

the Left Border sequence used for transfer of the T-DNA

(Barker et al., 1983)

Plasmid Vector Backbone

Intervening

Sequence 4626-4711 Sequence used in DNA cloning

OR 7



# - oriV 

# 4712-5108 Origin of replication from the broad host range plasmid

RK2 for maintenance of plasmid in Agrobacterium (Stalker

et al., 1981)

Intervening

Sequence 5109-6616 Sequence used in DNA cloning

CS- rop 

# 6617-6808 Coding sequence for repressor of primer protein from the

ColE1 plasmid for maintenance of plasmid copy number in

E. coli (Giza and Huang, 1989)

Intervening

Sequence 6809-7235 Sequence used in DNA cloning

OR- oripBR322 

# 7236-7824 Origin of replication from plasmid pBR322 for maintenance

of plasmid in E. coli (Sutcliffe, 1979)

# Monsanto Company 12-CT-244U 48 of 620 

# Table III-1. Summary of Genetic Elements in PV-GHHT6997 (continued)

Genetic

Element Location in

Plasmid

Vector (bp) Function (Reference)



# Intervening

Sequence 7825-8354 Sequence used in DNA cloning

aadA 

# 8355-9243 Bacterial promoter, coding sequence, and 3 ′ UTR for an

aminoglycoside-modifying enzyme,

3'(9)- O -nucleotidyltransferase from the transposon Tn7

(Fling et al., 1985) that confers spectinomycin and

streptomycin resistance

Intervening

Sequence 9244-9379 Sequence used in DNA cloning

1 

# B, Border

2 

# P, Promoter

3 

# L, Leader

4 

# TS, Targeting Sequence

5 

# CS, Coding Sequence

6 

# T, Transcription Termination Sequence

7 

# OR, Origin of Replication

# Monsanto Company 12-CT-244U 49 of 620 1 MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG

51 LKKSGMTLIG SELRPLK VMS SVSTAC MLTF VRNAWYVAAL PEELSEKPLG

101 RTILDTPLAL YRQPDGVVAA LLDICPHRFA PLSDGILVNG HLQCPYHGLE

151 FDGGGQCVHN PHGNGARPAS LNVRSFPVVE RDALIWIWPG DPALADPGAI

201 PDFGCRVDPA YRTVGGYGHV DCNYKLLVDN LMDLGHAQYV HRANAQTDAF

251 DRLEREVIVG DGEIQALMKI PGGTPSVLMA KFLRGANTPV DAWNDIRWNK

301 VSAMLNFIAV APEGTPKEQS IHSRGTHILT PETEASCHYF FGSSRNFGID

351 DPEMDGVLRS WQAQALVKED KVVVEAIERR RAYVEANGIR PAMLSCDEAA

401 VRVSREIEKL EQLEAA



# Figure III-3. Deduced Amino Acid Sequence of the MON 88701 DMO Protein

The amino acid sequence of the MON 88701 DM O precursor protein was deduced from

the full-length coding nucleotide sequen ce present in PV-GHHT6997 (See Table III-1 for

more detail). The chloroplast transit peptid e (CTP2) and the first 76 amino acids of the

precursor protein are underlined. CTP2 targets MON 88701 DMO protein to the

chloroplast. The CTP2 is cl eaved in the chloroplast produc ing the mature 349 amino acid

MON 88701 DMO protein that begins with the valine at position 68 (See Appendix C.1).

The double underline shows the nine amino acids from CTP2 that are at the N-terminus

of the mature MON 88701 protein.

1 MSPERRPADI RRATEADMPA VCTIVNHYIE TSTVNFRTEP QEPQEWTDDL

51 VRLRERYPWL VAEVDGEVAG IAYAGPWKAR NAYDWTAEST VYVSPRHQRT

101 GLGSTLYTHL LKSLEAQGFK SVVAVIGLPN DPSVRMHEAL GYAPRGMLRA

151 AGFKHGNWHD VGFWQLDFSL PVPPRPVLPV TEI



# Figure III-4. Deduced Amino Acid Sequence of the PAT ( bar ) Protein

The amino acid sequence of the MON 88701-produced PAT ( bar ) protein was deduced

from the full-length coding nucleotide sequence present in PV-GHHT6997 (See

Table III-1 for more detail).

# Monsanto Company 12-CT-244U 50 of 620 

# IV. CHARACTERIZATION OF THE GENETIC MODIFICATION

Characterization of the DNA insert in MON 88701 was conducted by Southern blot,

PCR, and DNA sequence analyses. The results of this characterization demonstrate that

MON 88701 contains a single copy of the dmo and bar expression cassettes and lacks

plasmid backbone; the T-DNA is stably integr ated at a single locus and is inherited

according to Mendelian principles over multi ple generations. These conclusions were

based on several lines of evid ence: 1) Southern blot analys es assayed the entire cotton

genome for the presence of the T-DNA and ab sence of the plasmid backbone sequences

derived from PV-GHHT6997, and demonstrated that only a single copy of the T-DNA

was inserted at a single genomic site and that the insert is stably inherited; 2) DNA

sequence analyses to determine the exact sequence of the inserted DNA and the DNA

sequences flanking the 5 ′ and 3 ′ ends of the insert, allowing a comparison to the T-DNA

sequence in the plasmid vector to confir m that only the expected sequences were

integrated; 3) DNA sequences flanking the 5 ′ and 3 ′ ends of the insert were compared to

the sequence of the insertion site in convent ional cotton to identif y any rearrangements

that occurred at the insertion site dur ing transformation. Taken together, the

characterization of the genetic modification demonstrates that a single copy of the

T-DNA was stably integrated at a single locu s of the cotton genome and that no plasmid

backbone sequences are present in MON 88701.

Southern blot analyses were used to determine the copy numb er and insertion sites of the

integrated DNA as well as the presence or absence of plas mid vector backbone

sequences. The Southern blot strategy wa s designed to ensure that all potential

transgenic segments would be identified. The entire cotton genome was assayed with

probes that spanned the complete plasmid vector to detect the presen ce of the insert as

well as confirm the absence of any plasmi d vector backbone sequences. This was

accomplished by using probes that were not more than 2.5 kb in length to ensure a high

level of sensitivity. This high level of sensitivity was demonstrated for each blot by

detection of a positive control added at 0.1 copi es per genome equivalent. Two sets of

restriction enzymes were specifically chosen to fully characterize the T-DNA and detect

any potential fragments of the T-DNA and b ackbone sequences. The restriction enzyme

sets were chosen such that each enzyme set cleaves once within the inserted T-DNA and

at least once within the known DNA flanking the 5 ′ or 3 ′ end of the insert. As a

consequence, at least one segment containing a portion of the insert with the adjacent 5 ′

flanking DNA generated by one set of the enzy me(s) is of a predictable size and overlaps

with another predictable size segment containi ng a portion of the insert with the adjacent

3 ′ flanking DNA generated by another set of the enzyme(s). This two-set enzyme design

ensures that the entire insert is identified in a predictable hybridi zation pattern. This

strategy also maximizes the possi bility of detecting an insert ion elsewhere in the genome

that could be overlooked if that band co-m igrated on the gel with an expected band.

To determine the number of copies and insert ion sites of the T-DNA, and the presence or

absence of the plasmid vector backbone seque nces, duplicated sample s that consisted of

equal amounts of digested DNA were run on th e agarose gel. One set of samples was run

for a longer period of time (long run) than th e second set (short run). The long run allows

for greater resolution of large molecular we ight DNA, whereas the short run allows for

# Monsanto Company 12-CT-244U 51 of 620 

# retaining the small molecular weight DNA on th e gel. The molecular weight markers on

the left of the figures were used to estimate the sizes of the bands presen t in the long run

lanes of the Southern blots, and the molecula r weight markers on the right of the figures

were used to estimate the sizes of bands pres ent in the short run la nes of the Southern

blots (Figure IV-2 through Figure IV-5). Any minor discrepa ncies between the

molecular weight marker and the genomic DNA samples are lik ely due to differences in

the migration rate of DNA during agarose ge l electrophoresis caused by differences in

salt concentration, base composition, or seque nces of DNA (Elder and Southern, 1983;

Sambrook and Russell, 2001). Southern blot analyses determined that a single copy of

the T-DNA was inserted at a single locus of the cotton genome, and no additional genetic

elements, including backbone sequences, from PV-GHHT6997 were detected in

MON 88701.

The PCR and DNA sequence analyses compleme nt the Southern analyses. PCR and

DNA sequence analyses performed on MON 88701 determined the complete DNA

sequence of the insert and flanking genom ic DNA sequences in MON 88701, confirmed

the predicted organizati on of the genetic elements within the insert, and determined the

sequences flanking the insert. In addition, DNA sequence analyses confirmed that each

genetic element (except for the border regions) in the insert is intact and the sequence of

the insert is identical to the correspondi ng sequence in PV-GHHT6997 (Figures IV-6 and

IV-7). Furthermore, genomic organiza tion at the MON 88701 insertion site was

determined by comparing the sequence flanking the 5 ′ and 3 ′ ends of the insert to the

sequence of the insertion s ite in conventional cotton.

The stability of the T-DNA present in MON 88701 across multiple generations was

demonstrated by Southern blot fingerprint an alysis (Figure IV-9). Genomic DNA from

five generations of MON 88701 (Figure IV-8) wa s digested with one of the enzyme sets

used for the insert and copy number analyses and was hybridized w ith two probes that

detect restriction segments that encompass th e entire insert. This fingerprint strategy

consists of two insert segments each containing its adjacent genomic DNA that assesses

not only the stability of the in sert, but also the stability of the DNA directly adjacent to

the insert.

Segregation analysis was conducted to determ ine the inheritance and stability of the

T-DNA insert in MON 88701. Results from th is analysis demonstrated that the

inheritance and stability of the insert was as expected across multiple generations

(Figure IV-8, Table IV-3, and Table IV-4), which corroborates the molecular insert

stability analysis and establishes the ge netic behavior of th e T-DNA at a single

chromosomal locus.

The Southern blot analyses confirmed that the T-DNA reported in Figure IV-1 represents

the only detectable insert in MON 88701. A circular map of PV-GHHT6997 annotated

with the probes used in the Southern blot analysis is presented in Figure III-1 and the

genetic elements within the MON 88701 insert are summarized in Table IV-2. A linear

map depicting restriction sites within the insert as well as within the DNA immediately

flanking the insert in MON 88701 is shown in Figure IV-1. Based on the plasmid map

and the linear map of the in sert, a table summarizing the expected DNA segments for

# Monsanto Company 12-CT-244U 52 of 620 

# Southern analyses is presented in Table IV -1. The results from the Southern blot

analyses are presented in Figure IV-2 thr ough Figure IV-5. PCR amplification of the

MON 88701 insert and the insertion site in the conventional control for DNA sequence

analysis are shown in Figure IV -6 and Figure IV-7, respectively. The generations used in

the generational stability analysis are de picted in the breeding history shown in

Figure IV-8 and the results fr om the generational stability analysis are presented in

Figure IV-9. The breeding path for genera ting the segregation data is shown in

Figure IV-10 and the results for the segregati on analysis are presente d in Table IV-3 and

IV-4. Materials and methods used for the ch aracterization of the insert in MON 88701

are found in Appendix B.Monsanto Company 12-CT-244U 53 of 620Figure IV-1. Schematic Representation ofthe Insert and Flanking DNA in MON 88701A linear map of the insert and DNA flanking the insert in MON 88701is shown. Angled arrows indicate the ends of the integratedT-DNA and the beginning of the flanking DNA.Identified on the linear map are genetic elements within the insert, as well as the sitesof the restriction enzymes usedin the Southern analyseswith positions relative to the first base pair of the DNA sequence representedin this map. The relative sizes and locations of the T-DNA probes and the expected sizes of restriction fragments are indicated in thelower portion of the scheme. This schematicdiagram is not drawn to scale. Locationsof genetic elements and T-DNA probes areapproximate. Probes are alsoshown in Figure III-1.r1Superscript in Left Border Region indicates that the sequence in MON 88701was truncated compared to the sequences in PV–GHHT6997.Monsanto Company 12-CT-244U 54 of 620Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and Restriction Enzymes Used inMON 88701 AnalysisSouthern Blot Analysis T-DNABackboneFigureIV-2 IV-3IV-4IV-5Probe(s) Used1,5 2,4 3 6, 7, 8Probing TargetDigestionenzymeExpected Band Sizes on each Southern BlotPV-GHHT6997PciI~6.2 kb~3.2 kb~6.2 kb~6.2 kb~6.2 kb~3.2 kbProbe Templates1N/A~1.3 kb~0.8 kb~1.0 kb~0.8 kb~~2~1.5 kb~1.7 kb~1.8 kbMON 88701BclI≥3.1 kb~2.4 kb≥3.1 kb~2.4 kb≥3.1 kb~2.4 kbNoneSspI~3.4 kb~1.2 kb~3.4 kb~1.2 kb~3.4 kbNone1Probe template spikes were used as positive hybridization controlsin Southern blot analyses when multiple probes were hybridized to the blotsimultaneously.2’ ~~’ indicates that probe template was not used.

# Monsanto Company 12-CT-244U 55 of 620 

# Table IV-2. Summary of Ge netic Elements in MON 88701

Genetic Element Location in

Sequence (bp) Function (Reference)



# 5 ′ Flank 1-1126 Cotton genomic DNA

Intervening

Sequence 1127-1219 Sequence used in DNA cloning

P 1



# - PC1SV 

# 1220-1652 Promoter from the Full-Length Transcript

(FLt) of peanut chloro tic streak caulimovirus

( PC1SV ) that directs transcription in plant

cells (Maiti and Shepherd, 1998)

Intervening

Sequence 1653-1658 Sequence used in DNA cloning

L 2



# - TEV 

# 1659-1790 5 ′ UTR leader sequence from the RNA of

tobacco etch virus (TEV) (Niepel and Gallie,

1999) that is involved in regulating gene

expression

Intervening

Sequence 1791 Sequence used in DNA cloning

TS 3



# - CTP2 

# 1792-2019 Targeting sequence of the ShkG gene from

Arabidopsis thaliana encoding the EPSPS

transit peptide region that directs transport of

the protein to the ch loroplast (Herrmann,

1995; Klee et al., 1987)

CS 4



# - dmo 

# 2020-3042 Codon optimized coding sequence for the

dicamba mono-oxygenase (DMO) protein of

Stenotrophomonas maltophilia that confers

dicamba tolerance (Herman et al., 2005;

Wang et al., 1997)

Intervening

Sequence 3043-3096 Sequence used in DNA cloning

T 5



# - E6 

# 3097-3411 3 ′ UTR sequence of the E6 gene from

Gossypium barbadense (cotton) encoding a

fiber protein involve d in early fiber

development (John, 1996) that directs

polyadenylation of mRNA

Intervening

Sequence 3412-3423 Sequence used in DNA cloning

P- e35S 

# 3424-4035 Promoter from the 35S RNA of cauliflower

mosaic virus (CaMV) (Odell et al., 1985)

containing the duplicat ed enhancer region

(Kay et al., 1987) that di rects transcription in

plant cells

# Monsanto Company 12-CT-244U 56 of 620 

# Table IV-2. Summary of Genetic Elements in MON 88701 (continued)

Genetic Element 

# Location in

Sequence

(bp) Function (Reference)

Intervening

Sequence 4036-4038 Sequence used in DNA cloning

L- Hsp70 

# 4039-4134 5 ′ UTR leader sequence of the DnaK gene

from Petunia hybrida that encodes heat

shock protein 70 (HSP70) (Rensing and

Maier, 1994; Winter et al., 1988) that is

involved in regulati ng gene expression

Intervening

Sequence 4135-4140 Sequence used in DNA cloning

CS- bar 

# 4141-4692 Coding sequence for the phosphinothricin

N-acetyltransferase (PAT) protein of

Streptomyces hygroscopicus that confers

glufosinate tolerance (Thompson et al., 1987)

Intervening

Sequence 4693-4697 Sequence used in DNA cloning

T

- nos 

# 4698-4950 3 ′ UTR sequence of the nopaline synthase

( nos ) gene from Agrobacterium tumefaciens

pTi encoding NOS that directs

polyadenylation (Bevan et al., 1983; Fraley et

al., 1983)

Intervening

Sequence 4951-4969 Sequence used in DNA cloning

B 6 

# -Left Border

Region r1 

# 4970-5231 DNA region from Agrobacterium

tumefaciens containing the Left Border

sequence used for transfer of the T-DNA

(Barker et al., 1983)

3 ′ Flank 5232-6369 Cotton genomic DNA

1 

# P, Promoter

2 

# L, Leader

3 

# TS, Targeting Sequence

4 

# CS, Coding Sequence

5 

# T, Transcription Termination Sequence

6 

# B, Border

r1 

# Superscript in Left Border Region indicates that the sequence in MON 88701 was truncated

compared to the sequences in PV-GHHT6997.

# Monsanto Company 12-CT-244U 57 of 620 

# IV.A. Insert and Copy Number of T-DNA in MON 88701

The numbers of copies and insertion sites of the T-DNA sequences in the cotton genome

were evaluated by digesting MON 88701 and conventional control genomic DNA

samples with the restriction enzyme Bcl I or the restriction enzyme Ssp I and hybridizing

Southern blots with probes that span the T-DNA (Figure III-1). Each restriction digest is

expected to produce a specific banding pattern on the Southern blots (Table IV-1). Any

additional copies and/or integration sites w ould be detected as additional bands on the

blots.

The restriction enzyme Bcl I cleaves once within the in serted T-DNA and within the

known genomic DNA flanking the 3 ′ end of the insert (Figure IV-1). Therefore, if

T-DNA sequences were present as a single copy at a single integration site in

MON 88701, the digestion with Bcl I was expected to generate two border segments with

expected sizes of ≥ 3.1 kb and ~2.4 kb (Figure IV-1 and Table IV-1). The restriction

enzyme Ssp I cleaves once within the inserted T-DNA and within the known genomic

DNA flanking the 5 ′ and 3 ′ ends of the insert (Figure IV -1). If T-DNA sequences were

present as a single copy at a single integration site in MON 88701, the digestion with

Ssp I was expected to generate two border segments with ex pected sizes of ~3.4 kb and

~1.2 kb (Figure IV-1 and Table IV-1).

The Southern blots were hybridized with T-DN A probes that collectively span the entire

inserted DNA sequence (Figures III-1 and IV -1, Probe 1, Probe 2, Probe 3, Probe 4, and

Probe 5). Conventional cont rol genomic DNA digested w ith the restriction enzyme Bcl I

and spiked with either probe templates and/or digested PV-GHHT6997 DNA served as

positive hybridization controls. The positiv e hybridization control was spiked at

approximately 0.1 and 1.0 copies of genome equivalents to demonstrate sufficient

sensitivity of the Southern blot. Conventi onal control genomic D NA digested with the

appropriate restriction enzymes was used as a negative control. The results of these

analyses are shown in Figur e IV-2 through Figure IV-4.

IV.A.1. T-DNA Probes 1 and 5

Conventional control genomic DNA digested with Bcl I (Figure IV-2, Lane 1 and Lane 8)

or with Ssp I (Figure IV-2, Lane 3 and Lane 10) and simultaneously hybridized with

Probe 1 and Probe 5 (Figures III-1 and IV-1) produced no detectable hybridization bands

as expected for the negative co ntrol in the reported exposure shown in Figure IV-2. In a

longer exposure of the blot, faint endogenous hybridization bands we re present in both

the Bcl I digest and the Ssp I digest in the conventional control genomic DNA (data not

shown). Conventional control genomic DNA digested with Bcl I and spiked with probe

templates of Probe 1 and Probe 5 (Figure I II-1) produced the expected bands at ~1.3 kb

and ~0.8 kb (Figure IV-2, Lane 5 and Lane 6). Conventional control genomic DNA

digested with Bcl I and spiked with the PV-GHHT6997 DNA, previously digested with

the restriction enzyme Pci I (Figure III-1), produced two bands at ~6.2 kb and ~3.2 kb

(Figure IV-2, Lane 7), as exp ected. Detection of the positiv e controls indicates that the

probes hybridized to their target sequences.

# Monsanto Company 12-CT-244U 58 of 620 

# MON 88701 DNA digested with Bcl I and simultaneously hybridized with Probe 1 and

Probe 5 (Figures III-1 and IV-1) produced the expected bands at ~3.5 kb and ~2.4 kb

(Figure IV-2, Lane 2 and Lane 9) whic h is consistent with the expected ≥ 3.1 kb and

~2.4 kb bands (Figure IV-1 and Table IV-1 ), respectively. MON 88701 DNA digested

with the restriction enzyme Ssp I and hybridized with Probe 1 and Probe 5 (Figures III-1

and IV-1) produced two bands at ~3.4 kb and ~1.2 kb (Figure IV-2, Lane 4 and Lane 11),

as expected.

The results presented in Figur e IV-2 indicate that the se quences covered by Probe 1 and

Probe 5 reside at a single detectable locus of integration in MON 88701.

IV.A.2. T-DNA Probes 2 and 4

Conventional control genomic DNA digested with Bcl I (Figure IV-3, Lane 1 and Lane 8)

or with Ssp I (Figure IV-3, Lane 3 and Lane 10) and simultaneously hybridized with

Probe 2 and Probe 4 (Figures III-1 and IV-1) produced no detectable hybridization bands

as expected for the negative control. Conventional contro l genomic DNA digested with

Bcl I and spiked with probe templates of Probe 2 and Probe 4 (Figur e III-1) produced the

expected bands at ~1.0 kb and ~0.8 kb (Figure IV-3, Lane 5 and Lane 6). Conventional

control genomic DNA digested with Bcl I and spiked with the PV-GHHT6997 DNA,

previously digested with the restriction enzyme Pci I (Figure III-1), produced one band at

~6.2 kb (Figure IV-3, Lane 7), as expected. Detection of th e positive controls indicates

that the probes hybridized to their target sequences.

MON 88701 DNA digested with Bcl I and simultaneously hybridized with Probe 2 and

Probe 4 (Figures III-1 and IV-1) produced the expected bands at ~3.5 kb and ~2.4 kb

(Figure IV-3, Lane 2 and Lane 9), whic h is consistent with the expected ≥ 3.1 kb and

~2.4 kb bands (Figure IV-1 and Table IV-1 ), respectively. MON 88701 DNA digested

with the restriction enzyme Ssp I and hybridized with Probe 2 and Probe 4 (Figures III-1

and IV-1) produced two bands at ~3.4 kb and ~1.2 kb (Figure IV-3, Lane 4 and Lane 11),

as expected.

The results presented in Figur e IV-3 indicate that the se quences covered by Probe 2 and

Probe 4 reside at a single detectable locus of integration in MON 88701.

IV.A.3. T-DNA Probe 3

Conventional control DNA digested with Bcl I (Figure IV-4, Lane 1 and Lane 7) or with

Ssp I (Figure IV-4, Lane 3 and Lane 9) and hy bridized with Probe 3 (Figures III-1 and

IV-1) produced endogenous hybridization si gnals that were present in all lanes

(Figure IV-4, Lane 1 through Lane 10). Th e same hybridization band was produced in

conventional control and MON 88701 DNA lanes when digested with the same enzyme.

When digested with Bcl I and hybridized with Probe 3 hybr idization bands of ~1.9 kb and

~1.7 kb were produced with conventiona l control genomic D NA and MON 88701 DNA

(Figure IV-4, Lane 1, Lane 2, and Lane 5 through Lane 8). When digested with Ssp I and

hybridized with Probe 3, a hybridization band of ~2.5 kb was produced with conventional

control genomic DNA and MON 88701 DNA (Figure IV-4, Lane 3, Lane 4, Lane 9, and

# Monsanto Company 12-CT-244U 59 of 620 

# Lane 10). Since these bands are present in both control and test substances, these signals

are considered to be weak hybrid ization of probes to endogenous E6 sequences and are not

specific to the inserted DNA in MON 88701.

Conventional control genomic DNA digested with Bcl I and spiked with the

PV-GHHT6997 DNA, previously digested with the restriction enzyme Pci I

(Figure III-1), produced one ba nd at ~6.2 kb (Figure IV- 4, Lane 5 and Lane 6), as

expected. Detection of the spik ed controls indicates that the probe hybridized to its target

sequence.

MON 88701 DNA digested with Bcl I and hybridized with Pr obe 3 (Figures III-1 and

IV-1) produced two expected ba nds at ~3.5 kb and ~2.4 kb, whic h is consistent with the

expected ≥ 3.1 kb and ~2.4 kb bands (Figure IV-1 and Table IV-1), and is in addition to

the endogenous hybridization bands discussed a bove (Figure IV-4, Lane 2 and Lane 8).

The ~3.5 kb band is less intense than the ~ 2.4 kb band. The difference in band intensity

is likely due to hybridization of a smaller porti on of Probe 3 to the ~3.5 kb fragment. The

~3.5 kb band represents the 5 ′ end of the inserted DNA and the adjacent DNA flanking

the 5 ′ end of the insert; this correlates with the expected border fragment size of ≥ 3.1 kb.

The ~2.4 kb band represents the 3 ′ end of the inserted DNA and the adjacent DNA

flanking the 3 ′ end of the insert. MON 88701 DNA digested with Ssp I (Figure IV-4,

Lane 4 and Lane 10, Figure IV-1, and Table IV -1) and hybridized with Probe 3 produced

one expected band at ~3.4 kb in addition to the endog enous hybridization bands

discussed above. The ~3.4 kb band represents the 5 ′ end of the inserted DNA and the

adjacent DNA flanking the 5 ′ end of the insert.

The results presented in Figure IV-4 indi cate that the sequence covered by Probe 3

resides at a single detectable lo cus of integration in MON 88701.

# Monsanto Company 12-CT-244U 60 of 620 

# Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA in MON 88701: Probes 1 and 5

The blot was simultaneou sly hybridized with two 32 

# P-labeled probes that span a portion

of the T-DNA sequence (Figure III-1, Probe 1 and Probe 5). Each lane contains

approximately 10 μg of digested genomic DNA. Arrows denote th e size of the DNA, in

kilobase pairs, obtained fr om 1 Kb DNA Extension Ladder on the ethidium bromide

stained gel. Lane desi gnations are as follows:

Lane Description

1 Conventional Control ( Bcl I)

2 MON 88701 ( Bcl I)

3 Conventional Control ( Ssp I)

4 MON 88701 ( Ssp I)

5 Conventional Control ( Bcl I) spiked with Probe 1 and Probe 5 template [~1.0 genome

equivalent]

6 Conventional Control ( Bcl I) spiked with Probe 1 and Probe 5 template [~0.1 genome

equivalent]

7 Conventional Control ( Bcl I) spiked with PV-GHHT6997 ( Pci I) [~1.0 genome equivalent]

8 Conventional Control ( Bcl I)

9 MON 88701 ( Bcl I)

10 Conventional Control ( Ssp I)

11 MON 88701 ( Ssp I)

# Monsanto Company 12-CT-244U 61 of 620 

# Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA in MON 88701: Probes 2 and 4

The blot was simultaneou sly hybridized with two 32 

# P-labeled probes that span a portion

of the T-DNA sequence (Figure III-1, Probe 2 and Probe 4). Each lane contains

approximately 10 μg of digested genomic DNA. Arrows denote th e size of the DNA, in

kilobase pairs, obtained fr om 1 Kb DNA Extension Ladder on the ethidium bromide

stained gel. Lane desi gnations are as follows:

Lane Description

1 Conventional Control ( Bcl I)

2 MON 88701 ( Bcl I)

3 Conventional Control ( Ssp I)

4 MON 88701 ( Ssp I)

5 Conventional Control ( Bcl I) spiked with Probe 2 and Probe 4 template [~1.0 genome

equivalent]

6 Conventional Control ( Bcl I) spiked with Probe 2 and Probe 4 template [~0.1 genome

equivalent]

7 Conventional Control ( Bcl I) spiked with PV-GHHT6997 ( Pci I) [~1.0 genome equivalent]

8 Conventional Control ( Bcl I)

9 MON 88701 ( Bcl I)

10 Conventional Control ( Ssp I)

11 MON 88701 ( Ssp I)

# Monsanto Company 12-CT-244U 62 of 620 

# Figure IV-4. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA in MON 88701: Probe 3

The blot was hybridized with a 32 

# P-labeled probe that span s a portion of the T-DNA

sequence (Figure III-1, Probe 3). Each lane contains approx imately 10 μg of digested

genomic DNA. Arrows denote the size of th e DNA, in kilobase pa irs, obtained from

1 Kb DNA Extension Ladder on the ethidium bromide stained gel. Lane designations are

as follows:

Lane

1 Conventional Control ( Bcl I)

2 MON 88701 ( Bcl I)

3 Conventional Control ( Ssp I)

4 MON 88701 ( Ssp I)

5 Conventional Control ( Bcl I) spiked with PV–GHHT6997 ( Pci I) [~1.0 genome equivalent]

6 Conventional Control ( Bcl I) spiked with PV–GHHT6997 ( Pci I) [~0.1 genome equivalent]

7 Conventional Control ( Bcl I)

8 MON 88701 ( Bcl I)

9 Conventional Control ( Ssp I)

10 MON 88701 ( Ssp I)

# Monsanto Company 12-CT-244U 63 of 620 

# IV.B. Southern Blot Analysis to Determine the Presence or Absence of

PV-GHHT6997 Backbone Sequences in MON 88701

To determine the presence or absence of the PV-GHHT6997 backbone sequences,

MON 88701 and conventional control genomic DNA were digested with the restriction

enzyme Bcl I or restriction enzyme Ssp I, and hybridized with the three backbone probes

that collectively span the en tire backbone sequences (Figur e III-1, Probe 6, Probe 7, and

Probe 8). If backbone sequences are pres ent in MON 88701, then probing with backbone

probes should result in hybridizing bands. Conventional control genomic DNA digested

with the restriction enzyme Bcl I and spiked with probe templates or with digested

PV-GHHT6997 DNA served as positive hybr idization controls. The positive

hybridization control was spiked at appr oximately 0.1 and 1.0 copies of genome

equivalents to demonstrate sufficient sensit ivity of the Southern blot. Conventional

control genomic DNA digested with the appropriate restriction enzymes was used as a

negative control. The results of these analyses are shown in Figure IV-5.

IV.B.1. Backbone Probes 6, 7, and 8

Conventional control DNA digested with Bcl I (Figure IV-5, Lane 1 and Lane 10) or the

restriction enzyme Ssp I (Figure IV-5, Lane 3 and Lane 12) and hybridized with Probe 6,

Probe 7, and Probe 8 (Figure III-1) produced no detectable hybridization bands as

expected for the negative control.

Conventional control geno mic DNA digested with Bcl I and spiked with probe templates

of Probe 7 and Probe 8 (Figure III-1) produced the expected bands at ~1.5 kb and ~1.8 kb

(Figure IV-5, Lane 5 and Lane 6). Conven tional control genomic DNA digested with

Bcl I and spiked with probe template of Probe 6 (Figure III-1) produced the one expected

band at ~1.7 kb (Figure IV-5, Lane 7 and La ne 8). Conventional control DNA digested

with Bcl I and spiked with th e PV-GHHT6997 DNA, previous ly digested with the

restriction enzyme Pci I (Figure III-1), produced two bands at ~6.2 kb and ~3.2 kb

(Figure IV-5, Lane 9), as exp ected. Detection of the positiv e controls indicates that the

probe hybridized to its target sequence.

MON 88701 DNA digested with Bcl I (Figure IV-5, Lane 2 and Lane 11) or the

restriction enzyme Ssp I (Figure IV-5, Lane 4 and Lane 13) and hybrid ized with Probes 6,

7, and 8 produced no detectable bands.

The results presented in Figure IV-5 indi cate that MON 88701 cont ains no detectable

backbone sequences covered by Probes 6, 7, and 8.

# Monsanto Company 12-CT-244U 64 of 620 

# Figure IV-5. Southern Blot Analysis to Determine the Presen ce or Absence of

PV-GHHT6997 Backbone Sequences in MON 88701: Probes 6, 7, and 8

The blot was hybridized with three 32 

# P-labeled probes that span the plasmid vector

backbone sequences (Figure III-1, Probes 6, 7, a nd 8). Each lane contains approximately

10 μg of digested genomic DNA. Arrows denot e the size of the DNA, in kilobase pairs,

obtained from λ DNA/ Hind III fragments on the ethidium bromide stained gel. Lane

designations are as follows:

Lane Description

1 Conventional Control ( Bcl I)

2 MON 88701 ( Bcl I)

3 Conventional Control ( Ssp I)

4 MON 88701 ( Ssp I)

5 Conventional Control ( Bcl I) spiked with Probe 7 and Probe 8 template [~1.0 genome

il ] 6 Conventional Control ( Bcl I) spiked with Probe 7 and Probe 8 template [~0.1 genome

il ] 7 Conventional Control ( Bcl I) spiked with Probe 6 template [~1.0 genome equivalent]

8 Conventional Control ( Bcl I) spiked with Probe 6 template [~0.1 genome equivalent]

9 Conventional Control ( Bcl I) spiked with PV-GHHT6997 ( Pci I) [~1.0 genome equivalent]

10 Conventional Control ( Bcl I)

11 MON 88701 ( Bcl I)

12 Conventional Control ( Ssp I)

13 MON 88701 ( Ssp I)

# Monsanto Company 12-CT-244U 65 of 620 

# IV.C. Organization and Sequence of th e Insert and Adjacent Genomic DNA in

MON 88701

The organization and sequence of the el ements within the MON 88701 insert was

confirmed by DNA sequence analysis. PCR primers were designed with the intent to

amplify three overlapping DNA amplicons that sp an the entire length of the insert and the

associated DNA flanking the 5 ′ and 3 ′ ends of the insert (Figure IV-6). The amplified

PCR products were subjected to DNA sequence an alyses. This analysis determined that

the DNA sequence of the MON 88701 insert is 4105 bp long (Table IV-2) and is identical

to the corresponding T-DNA sequence of PV -GHHT6997 as described in Table III-1.

# Monsanto Company 12-CT-244U 66 of 620 

# Figure IV-6. Overlapping PCR Analys is across the Insert in MON 88701

PCR was performed on both conventional cont rol genomic DNA and MON 88701 genomic DNA

using three pairs of primers to generate overlapping PCR fragments from MON 88701 for

sequence analysis. Approximately five microlite rs of each of the PCR reactions was loaded on

the gel. The expected product size for each amplicon and an illustration of the insert in

MON 88701 is provided at the bottom of the figure. Arrows on the agarose gel photograph

denote the size of the DNA, in kilobase pairs, obtained from 1 Kb DNA ladder on the ethidium

bromide stained gel. Lane designations are as follows:

Lane Description



# 1 1 Kb DNA Ladder

2 Conventional Control

3 MON 88701

4 No template DNA control

5 Conventional Control

6 MON 88701

7 PV–GHHT6997

8 No template DNA control

9 Conventional Control

10 MON 88701

11 No template DNA control

12 1 Kb DNA Ladder

# Monsanto Company 12-CT-244U 67 of 620 

# IV.D. PCR and DNA Sequence Analyses to Examine the MON 88701 Insertion Site

PCR and sequence analyses were perf ormed on genomic DNA extracted from

MON 88701 and the conventional control to examine the M ON 88701 insertion site. The

PCR was performed with a forward primer specific to the genomic DNA sequence

flanking the 5 ′ end of the insert paired with a reverse primer specific to the genomic DNA

sequence flanking the 3 ′ end of the insert (Figure IV -7). The amplified PCR product

from the conventional contro l was subjected to DNA sequen ce analysis. Alignments

between the conventional contro l sequence obtained from this analysis and the sequences

immediately flanking the 5 ′ and 3 ′ end of the MON 88701 insert were separately

performed to determine the integrity and geno mic organization of the insertion site in

MON 88701. The alignment analyses indica ted a 123 base pair deletion from the

conventional genomic DNA occurred upon T-DNA insertion in MON 88701. Minor

deletions and/or insertions of DNA due to double-strand brea k repair mechanisms in the

plant during Agrobacterium- mediated transformation process are not uncommon

(Salomon and Puchta, 1998).

# Monsanto Company 12-CT-244U 68 of 620 

# Figure IV-7. PCR Amplification of the MON 88701 Insertion Site in Conventional

Control

PCR was performed on both conventiona l control genomic DNA and MON 88701

genomic DNA, using Primer A specific to the 5 ′ flanking sequence and Primer B specific

to the 3 ′ flanking sequence of the insert in MON 88701, to generate DNA fragments for

sequence analysis. The insertion site in the conventional contro l (top) and MON 88701

(bottom) are illustrated at the bottom of the figure. Approximately five microliters of

each of the PCR reactions were loaded on the gel. Arrows on the agarose gel photograph

denote the size of the DNA, in kilobase pa irs, obtained from 1 Kb DNA Ladder on the

ethidium bromide stained gel. La ne designations are as follows:

Lane Description



# 1 1 Kb DNA Ladder

2 Conventional Control

3 MON 88701

4 No template DNA control

5 1 Kb DNA Ladder 0.5 1.0 1.6 2.0

0.5 1.0 1.6 2.0 1 2 3 4 5

Primer A Primer B

Primer AB-Left Border Regionr1L-TEVCS-dmoT-E6P-PC1SV



# 5' Flank 3' FlankTS-CTP2P-e35SL-Hsp70CS-barT-nos 

# Primer B

# Monsanto Company 12-CT-244U 69 of 620 

# IV.E. Southern Blot Analysis to Examine Insert Stability in Multiple Generations of

MON 88701

In order to demonstrate the st ability of the insert in M ON 88701, Southern blot analysis

was performed using genomic DNA extracted from leaf tissues from five breeding

generations of MON 88701. For reference, the breeding history of MON 88701 is

presented in Figure IV-8. The specific genera tions tested are indicat ed in the legend of

Figure IV-8. The R 3 

# generation was used for the molecular characterization analyses

shown in Figure IV-2 through Figur e IV-5. To analyze insert stability, four samples from

four additional generations of MON 88701 were evaluated by Southern blot analysis and

compared to the R 3 

# generation. Genomic DNA, isolated from each of the selected

generations of MON 88701, was digested with the restriction enzyme Bcl I and

simultaneously hybridized with Probe 2 and Probe 4 (Figures III-1 and IV-1), which was

designed to detect both fragments generated by the Bcl I digest. Any instability

associated with the insert would be detected as extra bands within the fingerprint on the

Southern blot. The Southern blot has the sa me controls as described in Section IV.A.2.

IV.E.1. T-DNA Probe 2 and 4

Conventional control genomic DNA di gested with restriction enzyme Bcl I and

simultaneously hybridized with Probe 2 and Pr obe 4 (Figures III-1 and IV-1) produced

no hybridization signals (Figure IV-9, Lane 1) as expected for the negative control.

Conventional control genomic DNA digested with Bcl I and spiked with the

PV-GHHT6997 DNA, previously digested with the restriction enzyme Pci I (Figure III-1

and Table IV-1), produced one expected band at ~6.2 kb (Figure IV-9, Lane 2).

Conventional control geno mic DNA digested with Bcl I and spiked with probe templates

of Probe 2 and Probe 4 produced the expected bands at ~1.0 kb and ~0.8 kb (Figure IV-9,

Lane 3 and Lane 4). Detection of the pos itive controls indicates that the probes

hybridized to their target sequences.

MON 88701 genomic DNA digested with Bcl I and hybridized with Probe 2 and Probe 4

(Figures III-1 and IV-1) is expected to produ ce a Southern fingerprint with two bands at

~3.5 kb and ~2.4 kb (Figure IV-1 and Table IV-1 ). Southern fingerprints produced from

multiple generations (Figure IV-9, Lane 5, La nes 7-9) of MON 88701 are consistent with

the one produced from the fu lly characterized generation R 3 

# (Figure IV-3, Lane 2 and

Lane 9, and Figure IV-9, Lane 6), indicati ng that MON 88701 contains one copy of the

T-DNA insert that is stable across multiple generations.

# Monsanto Company 12-CT-244U 70 of 620 

# Figure IV-8. Breeding History of MON 88701

R 0 

# corresponds to the original transformed cotton plant.  designates self-pollination.

The R 3 

# generation was used for the molecu lar characterization and commercial

development of MON 88701. The R 2 

# , R 3 

# , R 4 

# , R 5 

# , and R 6 

# generations of MON 88701

were used to analyze the stability of the insert across generations. The R 5 

# generation was

used for protein expression in tissues other than seed a nd for agronomic, phenotypic, and

environmental interaction analyses. The R 6 

# generation was used for protein expression in

seed and for composition analysis.

# Monsanto Company 12-CT-244U 71 of 620 

# Figure IV-9. Southern Blot Analysis to Examine Insert Stability in Multiple

Generations of MON 88701: Probes 2 and 4

The blot was simultaneou sly hybridized with two 32 

# P-labeled probes that span a portion

of the T-DNA sequence (Figure III-1, Probe 2 and Probe 4). Each lane contains

approximately 10 μg of digested genomic DNA. Arrows denote th e size of the DNA, in

kilobase pairs, obtained from 1Kb DNA Extension Ladde r on the ethidium bromide

stained gel. Lane desi gnations are as follows:

Lane

1 Conventional control ( Bcl I)

2 Conventional control ( Bcl I) spiked with PV-GHHT6997 ( Pci I) [~1.0 genome equivalent]

3 Conventional control ( Bcl I) spiked with Probe 2 and Probe 4 template [~1.0 genome

equivalent]

4 Conventional control ( Bcl I) spiked with Probe 3 and Probe 4 template [~0.1 genome

equivalent]

5 MON 88701 (R 2 

# ) ( Bcl I)

6 MON 88701 (R 3 

# ) ( Bcl I)

7 MON 88701 (R 4 

# ) ( Bcl I)

8 MON 88701 (R 5 

# ) ( Bcl I))

9 MON 88701 (R 6 

# ) ( Bcl I)

# Monsanto Company 12-CT-244U 72 of 620 

# IV.F. Inheritance of the Genetic Insert in MON 88701

The MON 88701 T-DNA resides at a single locus within the cotton genome and is

inherited according to Mendelian principles of inheritanc e. During development of

MON 88701, phenotypic and genotypic segregatio n data were record ed to assess the

inheritance and stabil ity of the MON 88701 T-DNA using Chi-square ( χ 2 

# ) analysis over

several generations. The χ 2 

# analysis is based on comparing the observed segregation ratio

to the expected segregation ratio according to Mendelian principles.

The MON 88701 breeding path for generating pollin ated segregation data is described in

Figure IV-10. The transformed R 0 

# plant was self-polli nated to generate R 1 

# seed. The

segregating R 1 

# generation was assessed using Real-Time TaqMan analysis for the dmo

coding region. A single homozygous positive R 1 

# plant was selected a nd self-pollinated to

give rise to R 2 

# plants that were self -pollinated to produce R 3 

# seed. Phenotypic and

genotypic assays confirmed the lack of inse rt segregation in these self-pollinated

generations.

Homozygous positive R 3 

# plants were crossed to a Monsanto proprietary cotton inbred,

which does not contain the dmo or bar coding sequence, via traditional breeding

techniques to produce hemizygous F 1 

# seed. The F 1 

# plants, hemizygous for the dicamba

and glufosinate tolerant trait, were crossed with a M onsanto proprietary cotton inbred,

which does not contain the dmo or bar coding sequence, to produce BC1F 1 

# seed. The

BC1F 1 

# generation was assessed using a glufosinat e herbicide application to select for

plants containing the MON 88701 T-DNA. The plants that survived the herbicide

application were confirmed to be he mizygous for the MON 88701 T-DNA using an

event-specific End-Point TaqMan analysis. The hemizygous BC1F 1 

# plants were

self-pollinated to produce the BC1F 2 

# plants. For the BC1F 2 

# generation, the plants were

assessed using a glufosinate herbicide applicat ion and the surviving plants were assessed

using an event-specific End-Point Ta qMan analysis for the MON 88701 T-DNA.

The inheritance of the MON 88701 T-DNA was assessed in the R 1 

# , BC1F 1 

# , and BC1F 2 

# generations. At the BC1F 1 

# generation, the MON 88701 T- DNA was predicted to

segregate at a 1:1 ratio (hemizygous: homozygous negative) according to Mendelian

inheritance principles. At the R 1 

# and BC1F 2 

# generations, the MON 88701 T-DNA was

predicted to segregate at a 1:2:1 ratio

(homozygous positive: hemizygous: homozygous negative) according to Mendelian

inheritance principles.

A Chi-square ( χ 2 

# ) analysis was used to compare the observed segregation ratios of the

MON 88701 T-DNA to the expected ratios. The Chi-square ( χ 2 

# ) analysis used the

statistical program R Version 2.12.0 (2010-10-15).

The Chi-square was calculated as:

χ 2



# = ∑ [( | o – e | ) 2 

# / e]

# Monsanto Company 12-CT-244U 73 of 620 

# where o = observed frequency of the genotype or phenotype and e = expected frequency

of the genotype or phenotype. The level of st atistical significance was predetermined to

be 5% ( α = 0.05).

The results of the χ 2 

# analysis of the MON 88701 segreg ating progeny are presented in

Table IV-3 and Table IV-4. The χ 2 

# value in the BC1F 1 

# generation indicated no

statistically significant difference between the observed and expected 1:1 segregation

ratio (hemizygous: homozygous negative) of the MON 88701 T-DNA. The χ 2 

# value for

the R 1 

# and BC1F 2 

# generations indicated no statistically significant difference between the

observed and expected 1: 2:1 segregation ratio

(homozygous positive: hemizygous: homozygous negative) of MON 88701 T-DNA.

These results support the c onclusion that the MON 88701 T-DNA resides at a single

locus within the cotton genome and is inherited according to Mendelian principles of

inheritance. These results are also consiste nt with the molecular characterization data

indicating that MON 88701 contains a single intact copy of the dmo and bar expression

cassettes inserted at a single locus in the cotton genome.Monsanto Company12-CT-244U74 of 620Figure IV-10. Breeding Path for Generating Segregation Data for MON 88701*Chi-square analysis was conducted on segregation data from the R1, BC1F1, and BC1F2generations (bolded text).†The cotton line used in the cross that did not contain thedmoorbargenes is a Monsanto proprietary cotton inbred.=Self- PollinatedMonsanto Company 12-CT-244U 75 of 620Table IV-3. Segregation of the T-DNA Duringthe Development of MON 88701: 1:1 Segregation1:1 SegregationGeneration Total PlantsObserved #PlantsHemizygousObserved #PlantsHomozygousNegativeExpected #PlantsHemizygousExpected #PlantsHomozygousNegativeχ2Probability2BC1F11261 123 138 130.5 130.5 0.862 0.35321Segregation was evaluated using a glufosinate herbicide application followed by End-Point TaqMan analysis for the MON 88701 insert.2Chi-square analysis was performed to analyze the segregation ratios (p≤0.05).Table IV-4. Segregation of the T-DNA Duringthe Development of MON88701: 1:2:1 Segregation1:2:1 SegregationGenerationTotalPlantsObserved #PlantsHomozygousPositiveObserved #PlantsHemizygousObserved #PlantsHomozygousNegativeExpected #PlantsHomozygousPositiveExpected #PlantsHemizygousExpected #PlantsHomozygousNegativeχ2Probability3R11173 33 99 41 43.25 86.50 43.25 4.353 0.1135BC1F22118 36 56 26 29.50 59.00 29.50 2.000 0.36791Segregation was evaluated using Real-Time TaqMan analysis for thedmocoding region.2Segregation was evaluated using a glufosinate herbicide application followed by End-Point TaqMan analysis for the MON 88701 insert.3Chi-square analysis was performed to analyze the segregation ratios (p≤0.05).

# Monsanto Company 12-CT-244U 76 of 620 

# IV.G. Genetic Modification Characterization Conclusion

Molecular characterization of MON 88701 by Sout hern blot analyses confirmed that the

T-DNA was inserted into the co tton genome at a single locus containing one copy of the

dmo and bar expression cassettes. No backbone DNA sequences from PV-GHHT6997

were detected in MON 88701.

PCR and DNA sequence analyses perf ormed on MON 88701 and the conventional

control determined the following: the complete DNA sequence of the insert and the DNA

sequences flanking the 5 ′ and 3 ′ ends of the insert in MON 88701; the organization of the

genetic elements within the insert; and the 5 ′ and 3 ′ insert-to-genomic DNA junctions.

The PCR and DNA sequence analysis also dete rmined the DNA sequence at the insertion

site in the conventional control and identifi ed a rearrangement (123 base pair deletion)

that occurred at the insertion site in MON 88701. Minor deletions an d/or insertions of

DNA due to double-strand break repair mechanisms in the plant during

Agrobacterium- mediated transformation process are not uncommon (Salomon and

Puchta, 1998).

Southern blot analysis of multiple MON 88701 generations demonstrated that the

inserted DNA has been stably maintained thr ough five generations of breeding, thereby,

confirming the stability of the insert. Results from segregation analyses show inheritance

and stability of the insert was as expected across multiple generations, which

corroborates the molecular insert stability anal ysis and establishes th e genetic behavior of

the T-DNA in MON 88701 at a single chromosomal locus.

# Monsanto Company 12-CT-244U 77 of 620 

# V. CHARACTERIZATION AND SA FETY ASSESSMENT OF THE

MON 88701 DMO AND PAT ( bar ) PROTEINS PRODUC ED IN MON 88701

Characterization of the introduced protein(s) in a biotechnology-derived crop is important

to establishing food, feed, and environmenta l safety. As descri bed in Section IV,

MON 88701 contains dmo and bar expression cassettes that, when transcribed and

translated, result in the expressi on of the MON 88701 DMO and PAT ( bar ) proteins,

respectively. This section summarizes: 1) the identity and function of the MON 88701

DMO and PAT ( bar ) proteins produced in MON 88701 ; 2) the demonstration of

equivalence between the plant-produced and E. coli -produced proteins, which were used

in various protein safety studies; 3) the expression levels of the MON 88701 DMO and

PAT ( bar ) proteins in MON 88701 plant tissues ; 4) the assessment of the potential

allergenicity of the MON 88701 DMO and PAT ( bar ) proteins produced in MON 88701;

and 5) the food, feed, and environmental safety assessment of the MON 88701 DMO and

PAT ( bar ) proteins produced in MON 88701. The data support a conclusion that these

two proteins produced in MON 88701 are safe for the environment and human or animal

consumption based on several lines of evidence summarized below. These data were

supplied to FDA for their evaluation in c onsultation BNF No. 135 on the food and feed

safety and compositional assessment of MON 88701.

V.A. Identity and Function of the MON 88701 DMO and PAT ( bar ) Proteins from

MON 88701

V.A.1. Mode-of-Action of DMO and MON 88701 DMO

Wild-type DMO was initially purified from Stenotrophomonas maltophilia ( S.

maltophilia ) strain DI-6, isolated from soil at a dicamba manufacturing plant (Krueger et

al., 1989). DMO is an enzyme that catalyzes the demethylation ofdicamba to the nonherbicidal compound DCSA and formaldehyde (Chakraborty et al., 2005). DMO is a

Rieske-type non-heme iron oxygenase, that is part of a three component system

comprised of a reductase, a ferredoxin, and a terminal oxygenase, in this case the DMO.

These three proteins work together in a re dox system similar to many other oxygenases to

transport electrons from nicotinamide ad enine dinucleotide (NADH) to oxygen and

catalyze the demethylation of an electron acceptor substrat e, in this case dicamba

(Behrens et al., 2007). This three component redox system is presented in Figure V-1.

# Monsanto Company 12-CT-244U 78 of 620 

# Figure V-1. Three Components of the DMO Oxygenase System

The crystal structure of a DMO has been solv ed (D'Ordine et al., 2009; Dumitru et al.,

2009) and shows that the DMO monomers cont ain a Rieske [2Fe-2S] cluster domain and

a non-heme iron center domain typical of a ll Rieske-type mono-oxygenases (Ferraro et

al., 2005). To catalyze the demethylation of dicamba, electrons transferred from NADH

are shuttled through an endogenous reductase and ferredoxin to the terminal DMO. The

electrons are received by the Rieske [2Fe -2S] cluster on one DMO monomer and

transferred to the non-heme iron center at the catalytic site of an adjacent monomer

(D'Ordine et al., 2009; Dumitru et al., 2009) , where it reductively activates oxygen to

catalyze the final demethylation of di camba. As a result of the reaction,

3

, 

# 6-dichlorosalicylic ac id (DCSA) and formaldehyde are formed. DCSA is a known

cotton, soybean, soil, and livestock metabolite whose safety has been evaluated by the

EPA (FAO-WHO, 2011a; 2011b; U.S. EPA, 2009). Formaldehyde is found naturally in

many plants at levels up to several hundr ed ppm (Adrian-Romero et al., 1999). An

assessment of the safety and potential effects of the DMO reaction products is provided

in Appendix C.1.

V.A.1.1. Description of MON 88701 DMO

DMO is targeted to chloroplasts for co-lo calization with the e ndogenous reductase and

ferredoxin enzymes that supply electrons for the DMO demethylation reaction as

described by Behrens et al. (2007). In the c onstruction of the plasmi d vector used in the

development of MON 88701, PV-GHHT6997, a transit peptide coding sequence ( CTP2,

Table IV-2) was joined to the dmo coding sequence; this codi ng sequence results in the

production of a precursor prot ein consisting of the DMO pr otein and an additional 76

amino acids at the N-terminus of the protei n. These additional amino acids correspond to

the chloroplast transi t peptide (CTP) from Arabidopsis thaliana EPSPS ( CTP2 ), which is

incorporated to improve the targeting of the precursor protein to the chloroplast

(Herrmann, 1995; Klee et al., 1987). Typically, transit peptides are precisely removed

from the precursor protein following delivery to the targeted plastid (Della-Cioppa et al.,

1986) resulting in the full-length protein. Howeve r, there are examples in the literature of

alternatively processed forms of a protein target ed to a plant’s chloroplast (Behrens et al.,

2007; Clark and Lamppa, 1992). Such alternative processing is observed with the

MON 88701 DMO protein produced in MON 88701. Reductase

(oxidized)

Flavin

Reductase

(reduced) Ferredoxin

(reduced)

[2Fe-2S]

Ferredoxin

(oxidized) NADH

+

H +



# NAD+ Oxygenase

(oxidized)

[2Fe-2S]

Fe+2

Oxygenase

(reduced) O 2 

# O OH

OCH 3



# Cl Cl O OH

OH

Cl Cl 3,6 DCSA

Dicamba + O

H H Dicamba

Mono-Oxygenase

Dicamba

Mono-Oxygenase Formaldehyde

# Monsanto Company 12-CT-244U 79 of 620 

# Analysis of cottonseed extracts from MON 88701 determined that the expressed protein

had an apparent molecular weight of 39.5 kDa and corres ponded to the DMO protein

with nine amino acids on the N-terminus or iginating from the EPSPS chloroplast transit

peptide. Except for the 9 amino acids derive d from the CTP2 and an additional leucine at

position two, the MON 88701 DMO protein has an identical sequence to the wild-type

DMO protein from the DI-6 strain of S. maltophilia (Herman et al., 2005). The

differences in the amino acid sequence between the wild-type DMO protein and

MON 88701 DMO protein are not e xpected to have an effect on structure, activity, or

specificity because the N-terminus and pos ition two are sterically distant from the

catalytic site (D'Ordine et al., 2009; Dumitr u et al., 2009). The DMO protein produced in

MON 88701 is hereinafter referred to as MON 88701 DMO protein. Accordingly, the

DMO protein produced from E. coli with the same sequence as MON 88701 DMO is

referred to as E

. coli -produced MON 88701 DMO protein.

As described previously the active form of DM O is a trimer (Chakrab orty et al., 2005;

Dumitru et al., 2009). For MON 88701 DMO to be functionally active and confer

dicamba tolerance to MON 88701 , a trimeric structure is required. The activity of

MON 88701 DMO was confirmed during characterization (Section V.B and

Appendix C).

V.A.1.2. Specificity of MON 88701 DMO

The substrate specificity of MON 88701 DM O was evaluated to understand potential

interactions DMO may have with potential substrates pr esent in MON 88701 cotton. The

literature indicates the specificity of DMO for dicamba is due to the specific interactions

that occur at the catalytic site (D'Ordine et al., 2009; Dumitru et al., 2009). Dicamba

interacts with amino acids in the catalytic site of DMO through both the carboxylate

moiety and the chlorine atoms of dicamba, wh ich are primarily involved in orienting the

substrate in the catalytic site. These chlori ne atoms are required for catalysis (D'Ordine

et al., 2009; Dumitru et al., 2009). Given the limited existence of chlorinated compounds

with structures similar to dicamba in plants and other eukaryotes (Wishart, 2010; Wishart

et al., 2009), it is unlikel y that MON 88701 DMO will catal yze the conversion of other

endogenous substrates.

The potential for MON 88701 DMO to metabolize endogenous plant substrates was

evaluated through in vitro experiments using a purified N-terminal histidine tagged DMO

that was identical to wild-type DMO, except for a histidine tag at the N-terminus added to

aid in protein purification. A comparison of DMO versions is shown in Appendix C,

Figure C-1. A set of potential endogenous s ubstrates was selected for evaluation based

on structural similarity of the compounds to dicamba and their presence in cotton, corn,

and soybean (Buchanan et al ., 2000; Janas et al., 2000; Lege et al., 1995; Schmelz et al.,

2003). The potential subs trates tested were o -anisic acid (2-methoxybenzoic acid),

vanillic acid (4-hydroxy-3-met hoxybenzoic acid), syringic acid

(3,5-dimethoxy-4-hydroxybenzoi c acid), ferulic acid [3 -(4-hydroxy-3-methoxy-phenyl)

prop-2-enoic acid] and sinapic acid [3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-enoic

acid] (Figure V-2). The assay mixture incl uded NADH, reductase, ferredoxin and DMO.

Dicamba was first used as a po sitive control to demonstrat e that the assay system was

# Monsanto Company 12-CT-244U 80 of 620 

# functional. The disappearance of potential substrates an d the formation of potential

oxidation products were monitored using LC- UV and LC-MS (Appendix C). None of the

tested substrates, except dicamba, were me tabolized by the histidine tagged DMO in

these in vitro experiments

. To assess whether MON 88701 DMO protein has the same

specificity as the histidine tagged DMO used in the in vitro experiments, the

E. coli -produced MON 88701 DMO protein ( i.e. , lacking a histidine), shown to be

equivalent to the plant produced MON 88701 DMO protein (Section V.B), was incubated

with o -anisic acid, the endogenous compound that has the greatest structural similarity to

dicamba. Again dicamba was us ed as a positive control to demonstrate the assay system

was functional. This analysis demonstrated that o -anisic acid was not metabolized by the

E. coli -produced MON 88701 DMO protein ( i.e. , lacking a histidine) , but dicamba was.

These results indicate that DMO, including the MON 88701 DMO protein, is specific for

dicamba as a substrate (See Section V.E.1.3 and Appendix C.3.2 for additional details).

Figure V-2. Dicamba and Potential E ndogenous Substrates Tested through In Vitro

Experiments with DMO

The arrow indicates methyl group removed by DMO.

The possibility that MON 88701 DMO can meta bolize exogenous substrates was tested

through in vivo greenhouse experiments. In addition to dicamba, nine other herbicides,

representing eight families with distinct m odes-of-action, some of which are approved for

use in cotton, were tested with MON 88701 and the conventi onal control (Table V-1).

Each herbicide was applied at two spray ra tes that are representative of potential

commercial rates needed to control broadleaf weeds. Herbicides were applied

preemergence or at the 2 to 5 leaf plant growth stage and plants were scored with a visual

rating based on the amount of injury observed. Across all of the herbicides tested,

MON 88701 and the conventional control were sim ilar in their level of injury, indicating

that these herbicides do not serve as a substrate for MON 88701 DMO (Appendix C.3.). Cl OCH 3 

# Cl COOH

Dicamba OCH 3 

# CH=CHCOOH

OH H 3 

# CO OCH 3 

# CH=CHCOOH

OH OCH 3 

# COOH



# 0-Anisic acid Vanillic acid Syringic acid ferulic acid si napic acid OCH 3 

# COOH

OH H 3 

# CO OCH 3 

# COOH

OH



# Dicamba o-Anisic Acid Vanillic Acid Syringic Acid Ferulic Acid Sinapic Acid

# Monsanto Company 12-CT-244U 81 of 620 

# Table V-1. Herbicides Applied to MON 88701 and Conventional Control

Herbicide Active

Ingredient Herbicide Chemical Family (Mode-of-Action) 1



# Dicamba Benzoic (Synthetic Auxin)

2,4-D Phenoxycarboxylic acid (Synthetic Auxin)

2,4-DB Phenoxycarboxylic acid (Synthetic Auxin)

Acetochlor Chloroacetamide (Inhibition of VLCFAs)

Atrazine Triazine (Inhibition of Photosynthesis at Photosytem II)

Oxyfluorfen Diphenylether (Inhibition of PPO)

Halosulfuron Sulfonylurea (Inhibition of ALS)

Trifluralin Dinitroaniline (Microtubule Assembly Inhibition)

Paraquat Bipyridilium (Photosys tem I electron diversion)

Glyphosate Glycine (Inhibition of EPSP synthase)

1 

# (HRAC, 2009)

2 

# 2,4-D = 2,4-Dichlorophenoxyacetic acid; 2,4-DB = 4-(2,4-dichlorophenoxy) butyric acid.



# V.A.2. Mode-of-Action of PAT Proteins

The mode-of-action for PAT protein has been extensively a ssessed, as numerous

glufosinate-tolerant products including thos e in cotton, corn, soy, canola, sugarbeet and

rice have been review ed by the FDA and several other regu latory agencies (ILSI-CERA,

2011; OECD, 1999a; 2002a). PAT, including the PAT ( bar ) protein produced in

MON 88701, is an enzyme classified as an ac etyltransferase which acetylates glufosinate

to produce non-herbicidal N-acetyl glufosinate. Glufosinate is a racemic mixture of the

D- and L- forms of phosphinothricin, though only the L-form has herbicidal activity. The

herbicidal activity of glufosinate results from the binding of L-phosphinothricin to

glutamine synthetase (OECD, 1999b; 2002a). Glutamine synthetase is responsible for

the assimilation of ammonia generated during photorespiration. The binding of

L-phosphinothricin to glutamine synthetase results in the inactiv ation of glutamine

synthetase and a subsequent toxic build-up of ammonia with in the plant, resulting in

death of the plant (Manderscheid and Wild, 1986; OECD, 1999b; 2002a; Wild and

Manderscheid, 1984).

The PAT ( bar ) protein produced in MON 88701 acet ylates the free amine group of Lphosphinothricin form of glufosinate to produ ce non-herbicidal N-acetyl glufosinate.

The acetylated glufosinate is una ble to bind to glutamine sy nthetase and therefore does

not disrupt photorespiration and avoids the build-up of ammonia. Therefore, the

production of PAT ( bar ) protein in MON 88701 confers glufosinate herbicide tolerance

through this mechanism.

V.A.2.1. Description of PAT ( bar )

Phosphinothricin N-acetyltransferase (PAT) prot eins conferring tolerance to glufosinate

herbicide (2-amino-4-(hydroxymet hylphosphinyl) butanoic acid) ha ve been isolated from

two separate species of Streptomyces

, S. hygroscopicus (Thompson et al., 1987) and S.

viridochromogenes (Wohlleben et al., 1988). The PAT protein isolated from

# Monsanto Company 12-CT-244U 82 of 620 

# S. hygroscopicus is encoded by the bar gene, and the PAT protein isolated from

S. viridochromogenes is encoded by the pat gene. These PAT proteins are made up of

183 amino acids with 85% identi ty at the amino aci d level. Based on previous studies

(Wehrmann et al., 1996) that have extensivel y characterized PAT pr oteins produced from

bar and pat genes, OECD recognizes both proteins to be equivalent with regard to

function and safety (OECD, 1999b). In addition, EPA has issued a tolerance exemption

for PAT protein regardless of the encoding gene (U.S. EPA, 1997). The safety of PAT

proteins present in biotec hnology-derived crops has been extensively as sessed (Hérouet

et al., 2005; ILSI-CERA, 2011).

The PAT protein produced in MON 88701 is from the bar gene, and for clarity, the PAT

protein produced in MON 88701 will be referred to as PAT ( bar ). Analysis of cottonseed

extracts from MON 88701 determined that the expressed protei n corresponded to the

183 amino acid polypeptide, resulting in a 24.1 kDa PAT ( bar ) protein. The activity of

the PAT ( bar ) protein purified from MON 88701 co ttonseed was confirmed during

characterization (Appendix C.4.).

V.A.2.2. PAT ( bar ) Specificity

The PAT proteins, including PAT ( bar ), are highly specific for glufosinate in the

presence of acetyl-CoA (Thompson et al., 1987; Wehrmann et al., 1996). While the

herbicidal activity of glufosinate comes from the L-amino acid form, other L-amino acids

are unable to be acetylated by PAT pr otein and competition assays containing

glufosinate, high concentrations of other amino acids and PAT showed no inhibition of

glufosinate acetylation (Wehrma nn et al., 1996). Furthermore, L-glutamate, an analogue

of glufosinate, also showed no inhibition of glufosinate acetylation in competition assays

(Wehrmann et al., 1996). In addition, the PAT ( bar ) protein has more than 30-fold

higher affinity towards L-phosphinothricin ove r other analogues (Thom pson et al., 1987).

Thus, the PAT ( bar ) protein has high substrate speci ficity for L-phosphinothricin, the

herbicidal component of glufosin ate, and it is unlikely to af fect the metabolic system of

MON 88701 cotton. Numerous glufosinate-to lerant products including those in cotton,

corn, soy, canola, sugarbeet, and rice have be en reviewed with no concerns identified

(ILSI-CERA, 2011).

V.B. Characterization and Eq uivalence of MON 88701 DMO and PAT ( bar )

Proteins from MON 88701

The safety assessment of crops derived th rough biotechnology incl udes characterization

of the physicochemical and functional propertie s of the protein(s) produced from the

inserted DNA, and confirmation of the safety of the protein(s). For the safety data

generated using E. coli -produced protein(s) to be applied to plant- produced protein(s), the

equivalence of the plant- and E. coli -produced proteins must be assessed. For

MON 88701 the physicochemical and functiona l characteristics of the MON 88701 DMO

and MON 88701-produced PAT ( bar ) proteins were determin ed and each was shown to

be equivalent to its respective E. coli -produced protein. A summary of the analytical

results for each protein are shown below and the details of the materials, methods, and

results are described in Appendix C.

# Monsanto Company 12-CT-244U 83 of 620 

# The MON 88701 DMO protein purified fr om cottonseed of MON 88701 was

characterized and the equivalence of the physicochemical and functional properties

between the MON 88701 DMO and the E. coli -produced MON 88701 DMO proteins was

established using a panel of analytical tests: 1) the identity could not be confirmed by

N-terminal sequence analysis; however, MALD I-TOF MS analysis of peptides derived

from tryptic digested MON 88701 DMO esta blished the N-terminal sequence of

MON 88701 DMO; 2) MALDI-TOF MS analysis yielded pep tide masses consistent with

the expected peptide masses from the theo retical trypsin digest of the MON 88701 DMO

sequence; 3) MON 88701 DMO protein was de tected on a western blot probed with

antibodies specific for DMO protein a nd the immunoreactive and physiochemical

properties of the MON 88701 DMO and E. coli -produced MON 88701 DMO proteins

were shown to be equivalent; 4) the elect rophoretic mobility and apparent molecular

weight of the MON 88701 DMO and E. coli -produced MON 88701 DMO proteins were

shown to be equivalent; 5) glycos ylation status of MON 88701 DMO and

E. coli -produced MON 88701 DMO proteins were determined to be equivalent; and

6) functional activity of the MON 88701 DMO and the E. coli -produced MON 88701

DMO proteins were demonstr ated to be equivalent.

The MON 88701-produced PAT ( bar ) protein purified from cottonseed of MON 88701

was characterized and the equivalence of the immunoreactive and physicochemical

characteristics and functional activ ity between the MON 88701- and the E. coli -produced

PAT ( bar ) proteins was established using a panel of analytical tests: 1) N-terminal

sequence analysis of th e MON 88701-produced PAT ( bar ) protein established identity;

2) MALDI-TOF MS analysis yielded pep tide masses consistent with the expected

peptide masses from the theoretical tr ypsin digest of the MON 88701-produced

PAT ( bar ) sequence; 3) MON 88701-produced PAT ( bar ) protein was detected on a

western blot probed with antibodies specific for PAT ( bar ) protein and the

immunoreactive properties of the MON 88701-produced and E. coli -produced PAT ( bar )

proteins were shown to be equivalent; 4) the electrophoretic mobility and apparent

molecular weight of the MON 88701-produced and E. coli -produced PAT ( bar ) proteins

were shown to be equivalent; 5) gl ycosylation status of MON 88701- and

E. coli -produced MON 88701 PAT ( bar ) proteins were determined to be equivalent; and

6) functional activity of the MON 88701- and E. coli -produced PAT ( bar ) proteins were

demonstrated to be equivalent.

Taken together, these data provide a de tailed characterizati on of the MON 88701 DMO

and PAT ( bar ) proteins and establish thei r respective equivalence to E. coli -produced

MON 88701 DMO protein and E. coli -produced PAT ( bar ) protein. This equivalence

justifies the use of the E. coli -produced proteins as test su btances in the protein safety

studies.

V.C. Expression Levels of MON 88701 DMO and PAT ( bar ) Proteins in

MON 88701

MON 88701 DMO and PAT ( bar ) protein levels in vari ous tissues of MON 88701

relevant to the risk assessment were determined by a validated enzyme-linked

immunosorbent assay (ELISA). Tissues of MON 88701 were co llected from four

# Monsanto Company 12-CT-244U 84 of 620 

# replicate plots planted in a randomized complete block field design during the 2010

growing season from the following eight fiel d sites in the U.S.: Arkansas (ARTI),

Georgia (GACH), Kansas (KSLA), Louisi ana (LACH), North Carolina (NCBD), New

Mexico (NMLC), South Carolina (SCEK), and Texas (TXPL). MON 88701 plots were

treated at the 3-5 leaf stage with glufosinate herbicide at the label rate (0.5 lbs active

ingredient [a.i.]/acre) and at the 6-10 leaf stage with dicamba herbicide at the proposed

label rate (0.5 lbs acid equivalent [a.e.]/acre) . The field sites were representative of

cotton-producing regions suitable for co mmercial production. Se ed, pollen, root, and

overseason leaf (OSL-1 through OSL-4) tissu e samples were collected from each

replicated plot at all field sites.

V.C.1. Expression Levels of MON 88701 DMO Protein

MON 88701 DMO protein levels were determined in all seven tissue types. The results

obtained from ELISA are summarized in Table V-2 and the details of the materials and

methods are described in Appendix D. Du e to a limited amount of tissue, moisture

content was not measured for pollen; therefore, pollen is reported on a fresh weight (fw)

basis only. MON 88701 DMO protein levels in MON 88701 across tissue types ranged

from <LOD to 410 μg/g dw. The mean MON 88701 DMO prot ein levels were

determined across eight sites, with the exceptio n of OSL-1 (7 sites) and OSL-4 (7 sites).

Samples <LOD were not included in mean determinations. The mean MON 88701 DMO

protein levels were highest in leaf (ra nging from OSL-2 and OSL-3 at 240 μg/g dw,

OSL-4 at 230 μg/g dw to OSL-1 at 180 μg/g dw ), followed by root at 43 μg/g dw, seed at

21 μg/g dw, and pollen at 14 μg/g fw.

# Monsanto Company 12-CT-244U 85 of 620 

# Table V-2. Summary of MON 88701 DMO Protein Levels in Tissues from

MON 88701 Grown in 2010 U.S. Field Trials

Tissue 1 

# Development

Stage 2 

# Days

After

Planting

(DAP) MON 88701

DMO

Mean (SD)

Range

(μg/g fw) 3 

# MON 88701

DMO

Mean (SD)

Range

(μg/g dw) 4 

# LOQ/LOD 5



# (μg/g fw)

OSL-1 2-4 leaf 14-25 27 (7.6) 180 (52) 0.168/0.313

13 – 42 110 – 280

OSL-2 4-7 leaf 25-37 41 (12) 240 (69) 0.168/0.313

19 – 65 110 – 380

OSL-3 9 leaf - Full flower 35-99 52 (17)

24 – 97 240 (75)

91 – 410 0.168/0.313

OSL-4 Cutout – Full

flower 70-121 57 (18)

0.70 – 91 230 (59)

2.8 – 310 0.168/0.313

Root 50% open flower –

Full flower 62-99 14 (3.7)

8.2 – 21 43 (12)

26 – 72 0.136/0.313

Pollen 50% open flower –

Full Flower 68-99 14 (28)

0.31 – 110 NA (NA)

NA 0.043/0.125

Seed Maturity 148-183 20 (4.6) 21 (5.0) 0.059/0.313

8.2 – 29 8.9 – 33

1 

# OSL= overseason leaf. Seed = black seed (ginned and delinted).

2 

# The crop development stag e each tissue was collected (Ritchie et al., 2007).

3 

# Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram ( μ g) of

protein per gram (g) of tissue on a fresh weight basis (fw). The means, SD, and ranges (minimum and

maximum values) were calculated for each tissue acro ss all sites (n=32, except OSL-3 n=31 due to one

sample <LOD, OSL-1 and OSL-4 n=28 due to missed sample collections, and pollen n=29 due to two

samples expressing <LOD and one being inconclusive).

4 

# Protein levels are expressed as μ g/g on a dry weight (dw) basis. The dry weight values were calculated by

dividing the μ g/g fw by the dry weight conversion factors obtained from moisture analysis data. NA= Not

Applicable.

5 

# LOQ=limit of quantitation; LOD=limit of detection.

# Monsanto Company 12-CT-244U 86 of 620 

# V.C.2. Expression Levels of PAT ( bar ) Protein

PAT ( bar ) protein levels were determined in all seven tissue types. The results obtained

from ELISA are summarized in Table V-3 a nd the details of the materials and methods

are described in Appendix D. Due to a lim ited amount of tissue, moisture content was

not measured for pollen; therefore, pollen is reported on a fresh wei ght (fw) basis only.

PAT ( bar ) protein levels in MON 88701 across tissue types range d from <LOQ to

10 μg/g dw. The mean PAT ( bar ) protein levels were determined across eight sites, with

the exception of OSL-1 (7 sites) and OSL-4 (7 sites). Samples <LOD were not included

in mean determinations. The mean PAT ( bar ) protein levels were highest in seed at

6.6 μg/g dw, followed by leaf (ranging from OSL-2 at 6.4 μg/g dw to OSL-4 at 3.2 μg/g

dw), root at 1.8 μg/g dw, and pollen at 0.56 μg/g fw.

# Monsanto Company 12-CT-244U 87 of 620 

# Table V-3. Summary of PAT ( bar ) Protein Levels in Tissues from MON 88701

Grown in 2010 U.S. Field Trials

Tissue 1 

# Development

Stage 2 

# Days

After

Planting

(DAP) PAT ( bar )

Mean (SD)

Range

(μg/g fw) 3 

# PAT ( bar )

Mean (SD)

Range

(μg/g dw) 4 

# LOD/LOQ 5



# (μg/g fw)

OSL-1 2-4 leaf 14-25 0.84 (0.21) 5.5 (1.5) 0.162/0.188

0.46 – 1.4 3.7 – 9.1

OSL-2 4-7 leaf 25-37 1.1 (0.26) 6.4 (1.4) 0.162/0.188

0.68 – 1.6 3.8 – 9.4

OSL-3 9 leaf – Full flower 35-99 1.0 (0.34) 4.8 (2.0) 0.162/0.188

0.34 – 1.7 1.3 – 10

OSL-4 Cutout – Full

flower 70-121 0.78 (0.29)

0.42 – 1.7 3.2 (1.2)

2.0 – 6.7 0.162/0.188

Root 50% open flowerFull flower 62-99 0.56 (0.18)

0.27 – 0.89 1.8 (0.75)

0.93 – 3.3 0.096/0.188

Pollen 50% open flower –

Full flower 68-99 0.56 (0.24)

0.27 – 0.90 NA (NA)

NA 0.021/0.188

Seed Maturity 148-183 6.1 (0.95) 6.6 (1.1) 0.032/0.188

4.8 – 8.8 5.2

– 9.6

1 

# OSL= overseason leaf. Seed = black seed (ginned and delinted).

2 

# The crop development stag e each tissue was collected (Ritchie et al., 2007).

3 

# Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram ( μ g) of

protein per gram (g) of tissue on a fresh weight basis (fw). The means, SD, and ranges (minimum and

maximum values) were calculated for each tissue across all sites (n=32, except OSL-1 n=28 due to missed

sample collections, OSL-4 n=27 due to missed samp le collections and one sample expressing <LOD,

OSL-3 n=31 due to one sample expressing <LOD, and pollen n=6 due to 26 samples expressing <LOQ).

4 

# Protein levels are expressed as μ g/g on a dry weight (dw) basis. The dry weight values were calculated by

dividing the μ g/g fw by the dry weight conversion factors obtained from moisture analysis data. NA= Not

Applicable.

5 

# LOQ=limit of quantitation; LOD=limit of detection.

# Monsanto Company 12-CT-244U 88 of 620 

# V.D. Assessment of Potential Allerg enicity of the MON 88701 DMO and PAT ( bar )

Proteins

Assessing the potential allergenicity of the expressed proteins is less relevant to

MON 88701 since only cottonseed oil and li nters from cotton are used in food

applications, which have undetectable or neg ligible amounts of tota l protein (Reeves and

Weihrauch, 1979; Sims et al., 1996). None theless, the allergenic potential of

MON 88701 DMO and PAT ( bar ) proteins was assessed by comparing the biochemical

characteristics of these introduced proteins to biochemical char acteristics of known

allergens (Codex Alimentarius, 2009). A protei n is not likely to be associated with

allergenicity if: 1) the protein is from a non- allergenic source; 2) th e protein represents a

very small portion of the total plant protein; 3) the protein does not share structural

similarities to known allergens based on the amino acid sequence; and 4) the protein is

rapidly digested in mammalian gastrointestinal systems.

V.D.1. Assessment of Potential All ergenicity of the MON 88701 DMO Protein

MON 88701 DMO has been assessed for its poten tial allergenicity accord ing to the safety

assessment guidelines described above, and conclusions were as follows.

1) MON 88701 DMO originates from S. maltophilia , an organism that has not

been reported to be a source of known allergens.

2) MON 88701 DMO represents no more th an 0. 008% of the total protein in the

cottonseed of MON 88701 4 

# . Therefore, the MON 88701 DMO protein represents

a very small portion of the total protei n in the cottonseed of MON 88701 and due

to the harsh conditions used in cottonseed processing is most likely absent in the

oil and linters that are used for food production.

3) Bioinformatics analyses demonstrated that the MON 88701 DMO does not

share amino acid sequence similarities with known allergens a nd, therefore, is

highly unlikely to contain immunologically cross-reactive allergenic epitopes.

4) In vitro digestive fate experiments conducted with the MON 88701 DMO

demonstrate that the proteins are rapidly digested in si mulated gastric fluid (SGF)

and in simulated intestinal fluid (SIF).

Taken together, these data support the c onclusion that MON 88701 DMO does not pose a

significant allerg enic risk.

V.D.2. Assessment of Potentia l Allergenicity of the PAT ( bar ) Protein

The non-allergenic nature of PAT ( bar ) protein is established in the scientific literature

(Hérouet et al., 2005) and by the tolerance exemption set by U.S. EPA (1997).

4 

# % protein = (Mean level of protein expression (μg/g)/ Mean dry weight of total protein in seed μg/g) x

100 %

# Monsanto Company 12-CT-244U 89 of 620 

# Furthermore, the safety of PA T proteins, including the PAT ( bar ) protein produced in

MON 88701, has been assessed extensively by regulatory agencies in 11 different

countries for more than 38 bi otechnology-derived events in eight different species (ILSICERA, 2011). In addition, poten tial allergenicity of PAT ( bar ) protein produced in

MON 88701 has been assessed acc ording to the safety assessment guidelines described

above, and conclusions were as follows.

1) PAT ( bar ) originates from S. hygroscopicus , an organism that has not been

reported to be a source of known allergens.

2) PAT ( bar ) represents no more than 0. 002% of the total protein in the

cottonseed of MON 88701. 5 

# Therefore, the PAT ( bar ) protein represents a very

small portion of the total protein in the cottonseed of MON 88701 and due to the

harsh conditions used in co ttonseed processing is most lik ely absent in the oil and

linters that are used for food production.

3) Bioinformatics analyses demonstrated that the PAT ( bar ) does not share amino

acid sequence similarities with known allerg ens and, therefore, is highly unlikely

to contain immunologically cross-re active allergenic epitopes.

4) In vitro digestive fate experiments conducted with the PAT ( bar ) demonstrate

that the proteins are rapidly digested in simulated gastric fluid (SGF) and in

simulated intestinal fluid (SIF).

Taken together, these data support the conclusion that PAT ( bar ) does not pose a

significant allerg enic risk.

V.E. Safety Assessment Summary of MON 88701 DMO and PAT ( bar ) Proteins in

MON 88701

Characterization of the introduced protein(s) in a biotechnology-deri ved crop product is

important to establishing its food, feed, and environmental safety. This section

summarizes: 1) the functionality of MON 88701 DMO and PAT ( bar ); 2) the

characterization of MON 88701 DMO and PAT ( bar ); 3) the levels of MON 88701 DMO

and PAT ( bar ) in plant tissues; 4) assessment of the potential allergenicity of

MON 88701 DMO and PAT ( bar ); and 5) the food, feed, and environmental safety

assessment of MON 88701 DMO and PAT ( bar ). The data support a conclusion that

MON 88701 is safe for the environment and human or animal consumption based on

several lines of evidence, all of which are summarized below.

V.E.1. MON 88701 DMO Donor Organism, History of Safe Use, and Specificity

Numerous factors have been considered in the safety assessment of MON 88701 DMO,

which include but are not limited to donor organism safety, the safety of mono-

5 

# % protein = (Mean level of protein expression (μg/g)/ Mean dry weight of total protein in seed μg/g) x

100 %

# Monsanto Company 12-CT-244U 90 of 620 

# oxygenases, and MON 88701 DMO protein specificity. A co mprehensive food, feed, and

environmental safety assessment of the MON 88701 DMO was conducted. The results

are summarized below, along with the conclusions reached from the assessment.

V.E.1.1. The dmo Donor Organism is Safe

The dmo gene is derived from the bacterium Stenotrophomonas maltophilia (Palleroni

and Bradbury, 1993). S. maltophilia is ubiquitous in the environment and is found

associated with the rhizosphere of plants. S. maltophilia can be found in a variety of

foods and feeds, and is wide spread in the home environm ent (Berg et al., 1999; Denton

and Kerr, 1998; Echemendia, 2010). Exposure to S. maltophilia is incidental to its

presence in food. It has been isolated from “ready to eat” salads, vegetables, frozen fish,

milk, and poultry (Qureshi et al., 2005; Ryan et al., 2009). S. maltophilia can be found in

healthy individuals without cau sing any harm to human heal th (Denton et al., 1998) and

infections caused by S. maltophilia are extremely uncommon (Cunha, 2010). Strains

have been found in the transient flora of hos pitalized patients as a commensal organism

(Echemendia, 2010) and, similar to the indigenous bacteria of the gast rointestinal tract,

S. maltophilia can be an opportunistic pa thogen (Berg, 1996). As such, S. maltophilia is

of low virulence in immuno-compromised patient s where a series of risk factors (severe

debilitation, the presence of indwelling device s such as ventilator tubes or catheters, for

prolonged periods of time and prolonged c ourses of antibiotics) must occur for

colonization by S. maltophilia in humans (Ryan et al., 2009). Therefore, infections by

S. maltophilia almost exclusively occur in hospital se ttings, in which case they are only

present in a minimal percentage of in fections (Ryan et al., 2009). Finally, S. maltophilia

has not been reported to be source of allergens.

The ubiquitous presence of S. maltophilia in the environment, the presence in healthy

individuals without causing in fections, the incidental pr esence in foods without any

adverse safety reports, and the lack of reporte d allergenicity establishes the safety of the

donor organism.

V.E.1.2. MON 88701 DMO Protein Belong s to a Common Class of MonoOxygenases

MON 88701 DMO is classified as an oxyge nase. Oxygenases are enzymes that

incorporate one or two oxygen atoms into substr ates and are widely distributed in many

universal metabolic pathways (Harayama et al ., 1992). Within this large enzymatic class

are mono-oxygenases that incorporate a singl e oxygen atom as a hydroxyl group with the

concomitant production of water and oxidatio n of NAD(P)H (Harayama et al., 1992).

Non-heme iron oxygenases, where iron is involve d in the catalytic si te, are an important

class of oxygenases. Within this class are Rieske oxygenases, which contain a Rieske

iron-sulfur [2Fe-2S] cluster. All Rieske non-heme iron oxygenases contain two catalytic

domains, a non-heme iron domain (nh-Fe) th at is a site of oxygen activation, and a

Rieske [2Fe-2S] domain (Ferraro et al., 2005). MON 88701 DMO bel ongs to this class

of oxygenases which are found in diverse phyla ra nging from bacteria to plants (Ferraro

et al., 2005; Schmidt and Shaw, 2001).

# Monsanto Company 12-CT-244U 91 of 620 

# As discussed previously, the crystal structure of a DMO has been solved (D'Ordine et al.,

2009; Dumitru et al., 2009). The crystallography results demonstrated that, similar to all

Rieske non-heme iron oxygenases, DMO contai ns two catalytically important and highly

conserved domains; a mononuclear non-heme iron domain (nh-Fe) that is a site of

oxygen activation, and a Rieske [2 Fe-2S] domain (D'Ordine et al., 2009; Dumitru et al.,

2009; Ferraro et al., 2005). The amino acids binding the non-heme iron and those that

constitute the Rieske [2Fe-2S] domain in th e DMO protein are also highly conserved in

these plant proteins, as is th eir spatial orientation (D'Ordin e et al., 2009; Ferraro et al.,

2005). Rieske domains are ubiquitous in nume rous bacterial and plant proteins like the

iron-sulfur protein of the cytochrome bc1 complex, chloroplast cytochrome b6/f complex,

and choline mono-oxygenases (Breyton, 2000; Darr ouzet et al., 2004; Gr ay et al., 2004;

Hibino et al., 2002; Rathinasabapathi et al ., 1997; Russell et al., 1998). The presence of

two conserved domains, a Rieske [2Fe-2 S] domain and a mononuclear iron domain,

suggests that all Rieske type non-heme iron oxygenases share the same reaction

mechanism, by which the Rieske domain transfers electrons from the ferredoxin to the

mononuclear iron to allow catalys is (Chakraborty et al., 2005; Dumitru et al., 2009;

Ferraro et al., 2005). The structure and mech anistic homologies are further evidence of

the evolutionary relatedness of all Rieske non-heme iron oxygenases to each other (Nam

et al., 2001; Rosche et al., 1997; Werlen et al., 1996). Additionally, a FASTA alignment

search of publicly availabl e databases using the MON 88701 DMO protein sequence as a

query yielded homologous sequences from many different species, predominantly

bacteria, with amino acid sequence ident ity ranging up to approximately 42%.

Alignments of MON 88701 DMO with plant proteins revealed homologous oxygenases

present in crops such as canola ( Brassica napus ), corn ( Zea mays ), pea ( Pisum sativum ),

rice ( Orysa sativa ), and soy ( Glycine max ), which were determined to have sequence

identities up to approximately 27%. The hi ghest homology was observed to proteins that

are involved in chlorophyll metabolism. Chlorophyllide A oxygenase (Accession

number: ACG42449) is Rieske-type oxygenase that is required for the formation of

chlorophyll b, which is present in all plants (Tanaka et al., 1998). Pheophorbide A

oxygenase (Accession number: ABD60316) is also a Rieske-type oxygenase that plays a

key role in the overall regulation of chlor ophyll degradation in plants (Rodoni et al.,

1997). Pheophorbide A oxygenase is constitutiv ely present in all green tissues and, at

slightly lower levels, in e tiolated and non-photosynthetic tiss ues including seeds (Yang et

al., 2004). As a Rieske-type oxygenase, Pheop horbide A oxygenase is expected to have

high degree of secondary and tertiary structur e homology to similar structural elements in

DMO as described above. The presence of th ese conserved structural domains in these

plant proteins is further evidence that exposure to a structural homolog of

MON 88701 DMO has occurred through consumption of these crops.

Therefore, MON 88701 DMO shares sequen ce identity and many catalytic domain

structural similarities with a wide variety of oxygenases present in bacteria and plants

currently widely prevalent in the environm ent and consumed, establishing that animals

and humans are extensively exposed to these types of enzymes.

# Monsanto Company 12-CT-244U 92 of 620 

# V.E.1.3. DMO Catalyzes a Specific Enzyme Reaction

DMO converts dicamba to DCSA. This demethyl ation is very specif ic to dicamba, where

both the carboxylate moiety and the chlorine atoms help position the substrate at the

active site of the enzyme (D'Ordine et al ., 2009; Dumitru et al., 2009). Crystallography

studies of the substrate in the active site de monstrated that these chlorines function as

steric “handles” that position the substrate in the proper orientation in the binding pocket

(Dumitru et al., 2009). Potent ial substrates abundant in co tton (o-anisic acid, vanillic

acid, syringic acid, ferulic acid and sinapic acid) that are structurally similar to dicamba,

were not metabolized by an E. coli -produced N-terminal histidine DMO. In addition, E.

coli -produced MON 88701 DMO did not meta bolize o-anisic acid, the endogenous

compound that has the greatest structural simila rity to dicamba. These laboratory tests

indicate that DMO, including MON 88701 DMO protein, is specific for dicamba (Section

V.A.1.2). Given the limited amount of chlorina ted metabolites with structures similar to

dicamba in plants and other eukaryotes (Wishart, 2010; Wishart et al., 2009), it is

unlikely that MON 88701 DMO will catalyze the conversion of other endogenous

substrates. Therefore, the activity of th e enzyme is specific for dicamba while it

maintains many structural pr operties common to oxygenases that are ubiquitous to all

organisms with a history of safe consumption.

V.E.2. PAT ( bar ) Donor Organism, History of Safe Use, and Specificity

The safety of PAT ( bar ) protein is established in the scientific literature (Hérouet et al.,

2005) and by the tolerance exemption set by th e EPA (U.S. EPA, 1997). In addition, the

safety of PAT proteins, including the PAT ( bar ) protein produced in MON 88701, has

been assessed extensively by regulatory agenci es in 11 different countries for more than

38 biotechnology-derived events in eight different spec ies (ILSI-CERA, 2011). The

PAT ( bar ) protein expressed in MON 88701 has the same functional activity as the PAT

proteins in all commercially available produc ts that provide glufosinate tolerance in

several crops, including cotton, corn, soybean, and canola. The lack of any documented

reports of adverse effects of glufosinate tole rant crops since their introduction in 1995

(Duke and Powles, 2009) further demonstrates the safety of PAT ( bar ) protein.

Numerous factors have been considered in the safety assessment of PAT ( bar ), which

include, but are not limited to, donor organism safety, the history of safe use, and PAT

protein specificity.

V.E.2.1. The bar Donor Organism is Safe

S. hygroscopicus is a saprophytic, soil-borne bacterium with no known safety issues.

Streptomyces species are widespread in the enviro nment and present no known allergenic

or toxicity issues (Kämpfer, 2006; Kutz ner, 1981) though human exposure is quite

common (Goodfellow and Williams, 1983). S. hygroscopicus is not considered

pathogenic to plants, humans or other an imals (Cross, 1989; Goodfellow and Williams,

1983; Locci, 1989). The history of safe use of S. hygroscopicus is discussed previously

(Hérouet et al., 2005), and this organism has been extensively reviewed during the

# Monsanto Company 12-CT-244U 93 of 620 

# deregulation of several glufosinate-tolerant events with no safety or allergenicity issues

identified.

The ubiquitous presence of S. hygroscopicus in the environment, the widespread human

exposure without any adverse safety or allerg enicity reports, and the successive reviews

resulting from the deregulation of several gluf osinate-tolerant events with no safety or

allergenicity issues identi fied establishes the safety of the donor organism.

V.E.2.2. PAT Protein has a History of Safe Use

The PAT ( bar ) protein expressed in MON 88701 is identical to the wild-type protein

produced in S. hygroscopicus and is analogous to the PA T proteins in commercially

available glufosinate-tolerant products in several crops including cotton, corn, soybean,

and canola. Based on studies characterizing the kinetic and chemical mechanisms of

PAT proteins (Wehrmann et al., 1996), OECD recognizes PAT proteins produced from

different genes to be equivalent with re gard to function and safety (OECD, 1999b).

The safety of PAT protein present in biot echnology-derived crops has been extensively

assessed (ILSI-CERA, 2011) and in 1997 a to lerance exemption was issued for PAT

proteins by U.S. EPA (U.S. EPA, 1997). This exemption was based on a safety

assessment that included rapid digestion in si mulated gastric fluids, lack of significant

homology to known toxins and known allergens, and lack of toxicity in an acute oral

mouse gavage study. Numerous glufosinate-to lerant products including those in corn,

soy, canola, sugarbeet and rice have been reviewed by the USDA and FDA with no

concerns identified. Furthe r, a comprehensive study on the safety of PAT proteins

present in biotechnology-derived crops (Hérou et et al., 2005) dem onstrated structural

similarity only with other acetyltransferases known to not cause adverse effects after

consumption, lack of sequence homology to know allergens and toxins, lack of

glycosylation sites, rapid degradation in ga stric and intestinal fluids, and no adverse

effects in mice treated with high doses of PA T proteins. Hérouet et al. concluded that

there is a reasonable certainty of no harm resulting from the inclusion of PAT proteins in

human food or animal feed (2005).

The history of safe use of PAT is supported by the lack of any documented reports of

adverse effects related to this protein since the introduction of glufosinate-tolerant crops

in 1995 (Duke and Powles, 2009). Since then, approvals have been issued by regulatory

agencies of 11 different countries for the environmental release of greater than 38

transformation events, including 8 different sp ecies of plants expressing the PAT protein

(ILSI-CERA, 2011).

V.E.2.3. PAT ( bar ) Catalyzes a Specific Enzyme Reaction

The mode-of-action for PAT protein has been extensively a ssessed, as numerous

glufosinate-tolerant products, including thos e in corn, soy, canola, sugarbeet, and rice,

have been reviewed by the FDA and severa l other regulatory agencies (ILSI-CERA,

2011; OECD, 1999b; 2002a). PAT, including the PAT ( bar ) protein produced in

MON 88701, is an enzyme classified as an ac etyltransferase which acetylates glufosinate

# Monsanto Company 12-CT-244U 94 of 620 

# to produce non-herbicidal N-acetyl glufosinate. Glufosinate is a racemic mixture of the

D- and L- forms of phosphinothricin. The herb icidal activity of gluf osinate results from

the binding of L-phosphinothricin to gl utamine synthetase (OECD, 1999b; 2002a).

Glutamine synthetase is responsible for th e assimilation of ammonia generated during

photorespiration. The binding of L-phosphinothric in to glutamine synthetase results in

the inactivation of glutamine synthetase a nd a subsequent toxic build-up of ammonia

within the plant, resulting in death of th e plant (Manderscheid and Wild, 1986; OECD,

1999b; 2002a; Wild and Manderscheid, 1984).

The PAT ( bar ) protein produced in MON 88701 acet ylates the free amine group of Lphosphinothricin form of glufosinate to produ ce non-herbicidal N-acetyl glufosinate.

The acetylated glufosinate is una ble to bind to glutamine sy nthetase and therefore does

not disrupt photorespiration and avoids the build-up of ammonia. Therefore, the

production of PAT ( bar ) protein in MON 88701 confers glufosinate herbicide tolerance

through this mechanism.

The PAT proteins, including PAT ( bar ), are highly specific for glufosinate in the

presence of acetyl-CoA (Thompson et al., 1987; Wehrmann et al., 1996). While the

herbicidal activity of glufosinate comes from the L-amino acid form, other L-amino acids

are unable to be acetylated by PAT pr otein and competition assays containing

glufosinate, high concentrations of other amino acids and PAT showed no inhibition of

glufosinate acetylation (Wehrma nn et al., 1996). Furthermore, L-glutamate, an analogue

of glufosinate, also showed no inhibition of glufosinate acetylation in competition assays

(Wehrmann et al., 1996). In addition, the PAT ( bar ) protein has more than 30-fold

higher affinity towards L-phosphinothricin ov er other plant analogue s (Thompson et al.,

1987). Thus, the PAT ( bar ) protein has high substrate sp ecificity for L-phosphinothricin,

the herbicidal component of glufosinate, and is unlikely to affect the metabolic system of

MON 88701 cotton. Numerous glufosinate-tolera nt products, including those in corn,

soy, canola, sugarbeet, and rice have been reviewed with no concerns identified (ILSICERA, 2011).

V.E.3. MON 88701 DMO and PAT ( bar ) Proteins in MON 88701 are Not

Homologous to Known Allergens or Toxins

Bioinformatics analyses were performed to assess the allergenic potential, toxicity, or

biological activity of MON 88701 DMO and PAT ( bar ). The analysis demonstrated that

neither protein shares amino acid sequence si milarities with known allergens, gliadins,

glutenins, or protein toxins which could have adve rse effects to human or animal health

(Section V.D).

V.E.4. MON 88701 DMO and PAT ( bar ) Proteins in MON 88701 are Labile in in

vitro Digestion Assays

MON 88701 DMO and PAT ( bar ) were readily digestible in simulated gastric fluid

(SGF) and simulated intestinal fluid (SIF ). Rapid degradation of the MON 88701 DMO

and PAT ( bar ) proteins in SGF and SIF makes it highl y unlikely that either protein would

# Monsanto Company 12-CT-244U 95 of 620 

# be absorbed in the small intestine and have any adverse effects on human or animal

health.

V.E.5. MON 88701 DMO and PAT ( bar ) Proteins in MON 88701 are Not Acutely

Toxic

Acute oral toxicology studies were conducted with MON 88701 DMO and PAT ( bar )

proteins individually. Results indicat e that neither MON 88701 DMO or PAT ( bar )

caused any adverse effects in mice, with No Observable Adverse Effect Levels

(NOAELs) for MON 88701 DMO at 283 mg/ kg bw and PAT ( bar ) at 1086 mg/kg bw,

respectively, the highest doses tested.

V.E.6. Human and Animal Exposu re to the MON 88701 DMO and PAT ( bar )

Proteins

Cottonseed is not consumed by humans because the majority of commercial cotton

varieties contain the anti-nut rients gossypol and cyclopropenoid fatty acids. The primary

human food currently produced from cottons eed is refined, bleached, and deodorized

(RBD) oil, and to a smaller extent, linters. RBD oil contains undetectable amounts of

protein (Reeves and Weihrauch, 1979); theref ore, oil produced from MON 88701 will

contain extremely low levels of MON 88701 DMO and PAT ( bar ) proteins. Linters are

an industrial by-product of ginning, and can be consumed as a highly processed product

composed of nearly pure ( i.e. , >99%) cellulose (NCPA, 2002; Nida et al., 1996).

Cottonseed RBD oil and linters are processe d fractions that cont ain undetectable or

negligible amounts of protei n there is minimal, if any, dietary exposure to MON 88701

DMO and PAT ( bar ) proteins from consumption of foods derive d from MON 88701.

Therefore, MOE values were not cal culated for the MON 88701 DMO or PAT ( bar )

proteins. Furthermore, the safety of PAT ( bar ) has been extensively assessed (Hérouet et

al., 2005), several glufosinate- tolerant crops that produce PAT proteins have been

reviewed by FDA and other regulatory ag encies (ILSI-CERA, 2011) and in 1997 a

tolerance exemption was issued fo r PAT proteins by U.S. EPA (1997).

Estimated exposure of MON 88701 DMO and PAT ( bar ) proteins in animal feed were

evaluated by calculating an estimate of da ily dietary intake ( DDI) for dairy cows.

Exposure was calculated for the worst-case scen ario, which assumes: 1) the source of

cottonseed in the diet is cottonseed meal; 2) cottonseed meal is only derived from

MON 88701 and contains no other cottonseed sources; 3) the pr otein expression level is

the maximum expression level measured for each protein; and 4) no loss of protein due to

heat. The maximum daily amou nt of MON 88701 DMO or PAT ( bar ) proteins

consumed from MON 88701 would be for the dairy cow and would be 0.00043 g/kg of

body weight for MON 88701 DMO and 0.000124 g/kg of body weight for PAT ( bar )

. These values represent 0.007 and 0.002% of prot ein consumed, respectively. These very

small levels of exposure of an imals to MON 88701 DMO and PAT ( bar ) in their feed, in

addition to the above mentioned safety data for both MON 88701 DMO and PAT ( bar ),

support the conclusion that there is no risk to animal health when MON 88701 DMO or

PAT ( bar ) are present in their diets.

# Monsanto Company 12-CT-244U 96 of 620 

# V.F. MON 88701 DMO and PAT ( bar ) Protein Characterization and Safety

Conclusion

MON 88701 DMO is a Rieske-t ype mono-oxygenase that cata lyzes the O-demethylation

of the herbicide dicamba and has homologs in bacteria and plants that share many of the

typical structural and functional characteri stics of these types of oxygenases, while

maintaining specificity for its substrate. The physicochemical characteristics of the

MON 88701 DMO protein were determined and equivalence between MON 88701 DMO

and E. coli -produced MON 88701 DMO proteins was demonstrated. This equivalence

justifies the use of the E. coli -produced MON 88701 DMO as a test substances in the

protein safety studies. E xpression studies using ELISA demonstrated that MON 88701

DMO was expressed at levels ranging from <LOD to 410 μg/g dw, representing a low

percentage of the total protein. An asse ssment of the allergenic potential of the

MON 88701 DMO protein supports the conc lusion that the M ON 88701 DMO protein

does not pose a significant allergenic risk. In addition, the donor organism for the

MON 88701 DMO coding sequence, S. maltophilia , is ubiquitous in the environment and

is not commonly known for human or animal pathogenicity or allergenicity. The

MON 88701 DMO protein lacks struct ural similarity to allergens, toxins or other proteins

known to have adverse effects on mammals. The MON 88701 DMO pr otein is rapidly

digested in simulated digestive fluids and dem onstrates no oral toxi city in mice at the

level tested. Based on the above information, the consumption of the MON 88701 DMO

protein from MON 88701 or its progeny is considered safe for humans and animals

PAT ( bar ) protein is an acetyltransferase that ca talyzes the acetylation of the herbicide

glufosinate. The PAT ( bar ) protein expressed in MON 88701 is analogous to the PAT

proteins in all commercially available produc ts that provide glufosinate tolerance in

several crops including cotton, corn, soybean, and canola. PAT proteins, including the

PAT ( bar ) protein isolated from MON 88701 have b een previously characterized, and the

safety of crops expressing th ese proteins has been well established. The data and

information provided in this section further confirms the food and feed safety of the

PAT ( bar ) protein in MON 88701. The physicochemi cal characteristics of the PAT ( bar )

protein were determined and equiva lence between MON 88701-produced and

E. coli -produced PAT ( bar ) proteins was demonstrated. This equivalence justifies the

use of the E. coli -produced PAT ( bar ) as a test substance in the protein safety studies.

Expression studies using ELISA demons trated that MON 88701-produced PAT ( bar ) was

expressed at levels ranging from <LOD to 10 μg/g dw, representing a low percentage of

the total protein. An assessment of the allergenic potential of the PAT ( bar ) protein

supports the conclusion that the PAT ( bar ) protein does not pose a significant allergenic

risk. In addition, the dono r organism for the PAT ( bar ) coding sequence,

S. hygroscopicus , is ubiquitous in the environm ent and is not commonly known for

human or animal pathogenicity, or allergenicity. The PAT ( bar ) protein lacks structural

similarity to allergens, toxins or other proteins known to have adverse effects on

mammals. The PAT ( bar ) protein is rapidly digested in simulated digestive fluids and

demonstrates no oral toxicity in mice at the level tested. Based on the above information,

the consumption of the PAT ( bar ) protein from MON 88701 or its progeny is considered

safe for humans and animals.

# Monsanto Company 12-CT-244U 97 of 620 

# The protein safety data presented herein support the conclusion that food and feed

products containing MON 88701 or derived from MON 88701 are as safe as cotton

products currently on the market fo r human and animal consumption.

# Monsanto Company 12-CT-244U 98 of 620 

# VI. COMPOSITIONAL ASSE SSMENT OF MON 88701

Safety assessments of biotechnology-derive d crops follow the comparative safety

assessment process (Codex Alimentarius, 2009) in which the composition of grain and/or

other raw agricultural commodities of the bi otechnology-derived crop is compared to the

appropriate conventional control that has a history of safe use. Compositional

assessments are performed using the princi ples and analytes outlined in the OECD

consensus document for cotton composition (OECD, 2009).

A recent review of compositional assessments conducted according to OECD guidelines

that encompassed a total of seven biotechnology-derived cr op varieties, nine countries

and eleven growing seasons concluded th at incorporation of biotechnology-derived

agronomic traits has had little impact on na tural variation in crop composition. Most

compositional variation is attributable to growing region, agr onomic practices and

genetic background (Harrigan et al., 2010). Co mpositional quality, therefore, implies a

very broad range of endogenous levels of indi vidual constituents. Numerous scientific

publications have further documented the extensive variability in the concentrations of

crop nutrients and anti-nut rients that reflect the influence of environmental and genetic

factors as well as extens ive conventional breeding efforts to improve nutrition,

agronomics and yield (Reynolds et al., 2005). This observation extends to publications

specific to cotton (Berberich et al., 1996; Hamilton et al., 2004; Nida et al., 1996).

Compositional equivalence between biotech nology-derived and conventional crops

supports an “equal or increased assurance of the safety of fo ods derived from genetically

modified plants” (OECD, 2002b). The OECD consensus document on considerations for

new varieties of cotton emphasi ze quantitative measurements of key nutrients and known

anti-nutrients (OECD, 2009). This is ba sed on the premise that such comprehensive and

detailed analyses will most effectively discern any compositional changes that imply

potential safety and nutritional concerns ( e.g ., anti-nutritional). Levels of the components

in the seed of the biotechnology-derived crop ar e compared to: 1) corresponding levels in

a conventional comparator, the genetically similar conventiona l line, grown concurrently,

under the same field conditions; and 2) natura l ranges generated from an evaluation of

commercial reference varieties grown concu rrently and from data published in the

scientific literature. The comparison to data published in the literature places any

potential differences between the assessed crop and its comparator in the context of the

well-documented variation in the concentratio ns of crop nutrients and anti-nutrients.

This section provides analyses of concentratio ns of key nutrients and anti-nutrients of

cottonseed from MON 88701 treated with both dicamba and glufosinate compared to the

conventional control grown and harvested under the same conditions, as appropriate. The

analyses of concentrations of ke y nutrients and anti-nutrients of cottonseed from

MON 88701 that was not treated with either dicamba or glufosinat e are presented in

Appendix E as supplemental information. In addition, conventional commercial cotton

reference varieties were included in the co mposition analyses to establish a range of

natural variability for each analyte, define d by a 99% tolerance interval. The production

of materials for the compositional analyses used field designs to allow accurate

assessments of compositional characteristics over a range of environmental conditions

# Monsanto Company 12-CT-244U 99 of 620 

# under which MON 88701 is expected to be gr own. The field trial design parameters

included a sufficient number of trial sites to allow adequate exposure to the variety of

conditions cotton plants typically encounter in na ture. Field sites were replicated with an

adequate number of plants sampled, and th e methods of analysis were sufficiently

sensitive and specific to detect variati ons in the components measured to allow

statistically rigorous analyses. The informa tion provided in this se ction also addresses

the relevant factors in Codex Plant Guide lines, Section 4, para graphs 44 and 45 for

compositional analyses (Codex Alimentarius, 2009).

VI.A. Compositional Equivalence of MON 88701 Cottonseed to Conventional

Cotton

Compositional analyses comparing MON 88701 treated with dicamba and glufosinate

herbicides to the conventi onal control variety (Coker 130) and conventional commercial

reference varieties demonstrated that MON 88701 is compositionally equivalent to

conventional cotton. Samples of acid-deli nted cottonseed were collected from

MON 88701 and the conventional control grow n in a 2010 U.S. field production. Nine

unique conventional cotton varieties, known as reference substances, were included

across all sites of the field pr oduction with four varieties per site to provide data on

natural variability of each compositional co mponent analyzed. The field production was

conducted at eight sites: Arkansas (ARTI), Georgia (GACH), Kansas (KSLA), Louisiana

(LACH), North Carolina (NCBD), New Mexico (NMLC), South Carolina (SCEK) and,

Texas (TXPL). The sites were planted in a randomized complete block design with four

blocks per site. All cott on plants, including MON 88701, the conventional control, and

the reference varieties, were grown under normal agronomic field conditions for their

respective geographic regions, including maintena nce pesticides as needed. In addition,

MON 88701 plots were treated at the 3-5 leaf st age with glufosinate herbicide at the label

rate (0.5 lbs a.i./acre), and at the 6-10 leaf stage with dicamb a herbicide at the label rate

(0.5 lbs a.e./acre).

Compositional analyses were conducted to assess whether levels of key nutrients and

anti-nutrients in MON 88701 were equivalent to levels in the conventional control and

comparable to the composition of conventio nal commercial reference varieties. A

description of nutrients and anti-nutrients present in cotton is provided in the OECD

consensus document on compositional consid erations for cottons eed (OECD, 2009).

Nutrients assessed in this an alysis included proximates (ash, calories and carbohydrates

by calculation, fat, moisture, and protein), aci d detergent fiber (ADF), neutral detergent

fiber (NDF), crude fiber (CF), total diet ary fiber (TDF), am ino acids (AA, 18

components), fatty acids (FA, C8-C22), mi nerals (calcium, copper, iron, magnesium,

manganese, phosphorus, potassium, sodium and zi nc), and vitamin E. Methods used in

the assessments of nutrients and anti-nutrients are found in Appendix E. In all, 65

different analytical components we re measured. Due to statistic al constraints, in order to

proceed with the statistical an alysis of any component in th is study, at least 50% of the

observed values for that analyte needed to be greater than the assay limit of quantitation

(LOQ). Of the 65 components measured, 13 had more than 50% of the observations

below the assay LOQ and were excluded fro m statistical analysis. Therefore, 52

components were statistically assessed using a mixed-model analysis of variance method.

# Monsanto Company 12-CT-244U 100 of 620 

# Values for all components were expressed on a dry weight basis with the exception of

moisture, expressed as percent fresh weight, a nd fatty acids, expressed as percent of total

FA.

For MON 88701, nine sets of statistical comp arisons to the conventional control were

conducted. One comparison was based on com positional data combined across all eight

field sites (the combined-site analysis) and eight separate comparisons to the

conventional control were conducted on data from each of the eight individual field sites.

Statistically significant differences were identifi ed at a 5% level of si gnificance (p<0.05).

Compositional data from the conventional commercial reference varieties, grown

concurrently in the same trial as MON 88701 and the conventiona l control, Coker 130,

were combined across all sites and used to calculate a 99% tolerance interval for each

component to define the natural variability in cotton varieties that ha ve a history of safe

consumption.

For the combined-site analysis, statistically significant differences (p < 0.05) in nutrient

and anti-nutrient components were evaluated fu rther using considerations relevant to the

safety and nutritional quality of MON 88701 when compared to the co nventional control.

The evaluation included: 1) the relative magn itude of the significa nt difference in the

mean values of nutrient and anti-nutrient components of MON 88701 compared to the

conventional control; 2) whether the MON 887 01 component mean values were within

the range of natural variabil ity of that component as represented by the 99% tolerance

interval of commercial conven tional reference varieties grow n concurrently in the same

trial; 3) analyses of the reproducibility of the significant combined-site component

differences at individual sites; and 4) assess ing the combined-site statistically significant

differences and reproducible indi vidual site significant differences within the context of

natural variability of commercial cottonseed composition published in the scientific

literature and/or in the International Life Sciences Institute Cr op Composition Database

(ILSI, 2011) (See Table VI-4). Statistical summaries of nutri ents and anti-nutrients for

individual sites are found in Appendix E.

This analysis provides a comprehensive comparative assessment of the levels of key

nutrients and anti-nutrients in cottons eed of MON 88701 and the conventional control

discussed in the context of natural variabi lity in composition of commercial cotton.

Results of the comparison indicate that the composition of the cottonseed of

MON 88701 is equivalent to th at of conventional cotton.

Compositional results from MON 88701 plots treated with dicam ba and glufosinate label

rates are summarized in the following subs ections. Similar results were obtained for

MON 88701 plots that were not treated with either dicamba or glufosinate, which are

provided as additional info rmation in Appendix E.

VI.A.1 Nutrient Levels in Cottonseed

In the combined-site analysis of nutrient levels in cott onseed, the following components

had no statistically significant differences (p<0.05) in mean values between MON 88701

and the conventional control: one proximate (pro tein), one type of fiber (crude fiber), 15

# Monsanto Company 12-CT-244U 101 of 620 

# amino acids (alanine, aspartic acid, cystine, gl utamic acid, glycine, histidine, isoleucine,

leucine, lysine, phenylalanine, serine, thre onine, tryptophan, tyrosine, and valine), seven

fatty acids (16:0 palmitic acid, 16:1 palmitole ic acid, 18:0 stearic ac id, 18:1 oleic acid,

18:3 linolenic acid, 20:0 arach idic acid, and 22:0 behenic acid), and four minerals

(copper, iron, phosphorus, and sodi um) (Table VI-1 and VI-2).

The components that had significant differences in mean values between MON 88701

and the conventional control in the combined -site analysis were: five proximates (ash,

calories, carbohydrates, moistu re, and total fat), three t ypes of fiber (ADF, NDF, and

TDF), three amino acids (arginine, methi onine and proline), two fatty acids (14:0

myristic acid and 18:2 linol eic acid), five minerals (c alcium, magnesium, manganese,

potassium, and zinc) and vitamin E (Table VI-1).

The statistically significant differences in nu trients were further ev aluated using the four

previously described considerations relevant to the safety and nutritional quality of

MON 88701 when compared to the conventional control:

1) All nutrient component differences obser ved in the combined -site statistical

analysis, whether reflecting increased or decreased MON 88701 mean values with

respect to the conventional control, were 14.09% or less. The relative magnitudes

of the differences were: 0.66 to 5.00% for proximates, 4.08 to 5.72% for fibers,

2.61 to 4.82% for amino acids, 0.69 to 2.69% for fatty acids, 4.94 to 14.09% for

minerals and 6.70% for vitamin E.

2) With the exception of methionine, mean values for all significantly different

nutrient components from the combined-site analysis of MON 88701 were within

the 99% tolerance interval establis hed from the conventional commercial

reference varieties grown concur rently in the same trial.

3) Assessment of the reproducibility of the combined-site differences at the eight

individual sites showed si gnificant differences for: NDF, methionine, proline and

18:2 linoleic acid at one site ; carbohydrates, total fat, ADF , manganese and zinc at

two sites; TDF, arginine, 14:0 myristic acid, potassium, and vitamin E at three

sites; magnesium at four sites, ash at six sites and calcium at seven sites.

Moisture and calories were not affected at any site. With the exception of

methionine, arginine, and zi nc, all individual site mean values of MON 88701 for

all nutrient components with signifi cant differences were within the 99%

tolerance interval established from the conventional commercial reference

varieties grown concurrently in the same trial.

4) All combined-site mean values and indi vidual mean values of MON 88701 for all

nutrient components, including those that we re significantly different, were within

the context of the natural variability of commercial cotton composition as

published in the scientific literature and/or available in the ILSI Crop

Composition Database (ILSI, 2011).

# Monsanto Company 12-CT-244U 102 of 620 

# Five of the 19 cottonseed nutrient statistically significant differences between

MON 88701 and the conventional control that were observed in the combined-site data

analysis were attributable to small differen ces in proximates (ash, carbohydrates, total fat

expressed as % dw, calories expressed as Kcal/100g dw, and moistu re expressed as %

fw). For ash, calories, and total fat the rela tive magnitude of the differences between the

mean value for MON 88701 and the conventional control were all small increases (5.00%

for ash, 0.66% for calories a nd 3.71% for total fat). The differences for carbohydrates

and moisture between the mean value for MON 88701 and the conventional control were

both small decreases (2.60% for carbohydrates and 4.51% for moisture). All of the

nutrient mean values for MON 88701 observed in the combined-site analysis for

proximates were within the 99% toleranc e interval established by conventional

commercial reference varieties grown concurrently in the same trial. Except for ash,

significant differences for most proximate mean values between MON 88701 and the

conventional control were not consistently obs erved among individual sites. There were

no significant differences at any of the individual sites for calories or moisture. Total fat

was increased at two sites ranging from 6.74 to 8.46% and carbohydrat es were decreased

at two sites, with decrease s ranging from 4.33 to 5.08%. Although ash was increased in

MON 88701 when compared to the conventional control at six sites, increases ranged

from 4.95 to 11.50%, which was less than the variability for the control samples (range

3.46 to 4.29, a relative difference of 24.0%, Tabl e VI-1). Overall, observed differences

in proximate values between MON 88701 a nd the conventional control were not

considered to be meaningful from a food and feed safety or nutritional perspective

because the magnitudes of combined-site differences ranged only from 0.66% to 5.00%,

most were not consistently reproduced across the individual s ites, and the mean

MON 88701 combined-site values were within the 99% tolerance inte rval established by

conventional commercial reference varieties gr own concurrently in the same trial, and

were within the context of the natural vari ability of commercial cotton composition as

published in the scientific literature and/ or available in the ILSI Crop Composition

Database (ILSI, 2011).

Three of the 19 cottonseed nutrient statis tically significant differences between

MON 88701 and the conventional control observed in the co mbined-site data analysis

were attributable to small differences in fiber (ADF, NDF, and TDF all expressed as %

dw). All relative magnitudes of the differe nces for fiber between the mean values for

MON 88701 and the conventional control we re small decreases (4.94% for ADF, 5.72%

for NDF and 4.08% for TDF). All of the nutrient mean values for MON 88701 observed

in the combined-site analysis for fiber were within the 99% tolerance interval established

by conventional commercial reference varieties grown concurrently in the same trial.

Significant differences for fiber mean va lues between MON 88701 and the conventional

control were not consistently observed am ong individual sites. TDF and ADF were

decreased at three and two sites, respec tively, with decreases ranging from 4.55 to 8.15%

for TDF and 9.27 to 9.86% for ADF. NDF was si gnificantly different at one site with a

small decrease of 7.40%. Overall, observe d differences in fiber values between

MON 88701 and the conventional control were not considered to be meaningful from a

food and feed safety or nutriti onal perspective because they we re small, not consistently

reproduced across the individual sites, a nd the mean MON 88701 combined-site values

# Monsanto Company 12-CT-244U 103 of 620 

# were within the 99% tolerance interval es tablished by conventiona l commercial reference

varieties grown concurrently in the same trial, and were within the context of the natural

variability of commercial cotton composition as published in the scientific literature

and/or available in the ILSI Cr op Composition Database (ILSI, 2011).

Three other combined-site nutrient statis tically significant differences between

MON 88701 and the conventional control observe d in the combined-site analysis were

attributed to small differences in amino acids (arginine, methionine, and proline;

expressed as % dw). For bot h arginine and proline, th e relative magnitude of the

differences between the mean values for MON 88701 and the conventional control were

small decreases (3.80% for arginine and 2.61% for proline). Methionine was increased

4.82% when MON 88701 was compared to the c onventional control. With the exception

of methionine, the nutrient mean values for MON 88701 observed in the combined-site

analysis for amino acids were within the 99% tolerance interval established by

conventional commercial referen ce varieties grown concurrently in the same trial. The

combined-site mean value for methionine was within the context of natural variation of

methionine found in commercial cotton as pub lished in the scientif ic literature or as

found in the ILSI Crop Composition Database (ILSI, 2011). Significant differences for

amino acid mean values between MON 88701 and the conventional control were not

consistently observed at all eight individual sites. Arginine and proline were decreased at

three sites and one site, re spectively, with decreases ranging from 6.10 to 8.35% for

arginine and 6.16% for proline. Methioni ne was increased 12.03% at only one site.

Overall, observed differences in amino acid values between MON 88701 and the

conventional control were not considered to be meaningful from a food and feed safety or

nutritional perspective because they were small in magnitude, not consistently

reproduced across the individual sites, and w ith the exception of methionine, the mean

MON 88701 combined-site values were within the 99% tolerance inte rval established by

conventional commercial reference varieties gr own concurrently in th e same trial. In

addition, all MON 88701 amino acid values we re within the cont ext of the natural

variability of commercial cotton composition as published in the scientific literature or

available in the ILSI Crop Co mposition Database (ILSI, 2011).

Two of the combined-site nutrient stat istically significant differences between

MON 88701 and the conventional control were a ttributed to the fatty acids 14:0 myristic

acid and 18:2 linoleic acid (expressed as % to tal FA). The relative magnitudes of the

differences between the mean fatty acid values for MON 88701 a nd the conventional

control in the combined-site an alysis were small decreases (2.69% for 14:0 myristic acid

and 0.69% for 18:2 linoleic acid). The nutri ent mean values for MON 88701 observed in

the combined-site analysis for both 14:0 myristic acid and 18:2 linoleic acid were within

the 99% tolerance interval established by conventional commercial reference varieties

grown concurrently in the same trial. Significant differences for fatty acid mean values

between MON 88701 and the conventional contro l were not consistently observed among

individual sites. 14:0 myrist ic acid was decreased at three sites while 18:2 linoleic acid

was decreased at one site with differences ranging from 4.43 to 8.36% for 14:0 myristic

acid and 1.93% for 18:2 linoleic acid. Overall, observed differences in fatty acid values

between MON 88701 and the conventional control were not considered to be meaningful

# Monsanto Company 12-CT-244U 104 of 620 

# from a food and feed safety and nutritional perspective be cause they were small, not

consistently reproduced acro ss the individual sites, a nd the mean MON 88701 values

were within the 99% tolerance interval es tablished by conventiona l commercial reference

varieties grown concurrently in the same trial and were within the context of the natural

variability of commercial cotton composition as published in the scientific literature

and/or available in the ILSI Cr op Composition Database (ILSI, 2011).

Five of the 19 cottonseed nutrient statistically significant differences between

MON 88701 and the conventional control observe d in the combined-site analysis were

attributed to small differences in minerals (calcium, magnesium, and potassium expressed

as % dw and manganese and zinc expressed as mg/kg dw). For calcium, magnesium,

potassium, and manganese, the relative magnit udes of the differences between the mean

values for MON 88701 and the conventional control were increases of 14.09% for

calcium, 5.63% for magnesium, 9.20% for ma nganese, and 4.94% for potassium. The

relative magnitude of the difference for zinc between the mean value for MON 88701 and

the conventional control was a decrease of 6.39% . All of the nutrient mean values for

MON 88701 observed in the combined-site analysis for minerals were within the 99%

tolerance interval es tablished by conventional commerci al reference varieties grown

concurrently in the same trial. Except fo r calcium, significant differences for mineral

mean values between MON 88701 and the conve ntional control were not consistently

observed among individual sites. Calcium was significantly diffe rent at seven sites, with

increases ranging from 6.92 to 22.7 0%; this was less than the variability observed for the

control samples (range 0.091 to 0.18, a relativ e difference of 97.8%, Table VI-1).

Magnesium, potassium, and manganese were si gnificantly different at four, three, and

two sites, respectively, w ith increases ranging from 5.54 to 9.36% for magnesium, 8.01 to

16.37% for potassium and from 16.52 to 20.59% fo r manganese. Zinc was significantly

different at two sites, with decreases ranging from 7.68 to 17.66%. Overall, observed

differences in mineral values between MON 88701 and the conventional control were not

considered to be meaningful from a food and feed safety or nutritional perspective

because they were small in magnitude, not consistently reproduced across the individual

sites (with the exception of calcium), a nd the mean MON 88701 combined-site values

were within the 99% tolerance interval es tablished by conventiona l commercial reference

varieties grown concurrently in the same trial and within the context of the natural

variability of commercial cotton composition as published in the scientific literature

and/or available in the ILSI Cr op Composition Database (ILSI, 2011).

One other nutrient difference observed in the combined-site analysis between

MON 88701 and the conventional control was attributed to vitamin E (expressed as

mg/kg dw). The relative magnitude of the difference between the mean vitamin E value

for MON 88701 and the conventional control in the combined-site analysis was a small

increase of 6.70%. The nutrient mean valu e for MON 88701 observed in the combinedsite analysis for vitamin E was within th e 99% tolerance interv al established by

conventional commercial reference varieties grown concurrently in the same trial.

Significant differences for vitamin E mean values between MON 88701 and the

conventional control were not consisten tly observed among indi vidual sites, with

# Monsanto Company 12-CT-244U 105 of 620 

# significant increases ranging from 7.78 to 13.28 % observed at three sites. Overall, the

observed difference in the vitamin E valu es between MON 88701 and the conventional

control in the combined-site an alysis were not considered to be meaningful from a food

and feed safety and nutritional perspect ive because they were 13.28% or less, not

consistently reproduced acro ss the individual sites, a nd the mean MON 88701 values

were within the 99% tolerance interval es tablished by conventiona l commercial reference

varieties grown concurrently in the same trial, and were within the context of the natural

variability of commercial cotton composition as published in the scientific literature

and/or available in the ILSI Cr op Composition Database (ILSI, 2011).

In summary, except for calcium and ash, statistical analyses found no consistent

differences between the levels of nutrien t components in cottonseed from MON 88701

and the conventional control. Differences were observed for calcium and ash in

combined-site analyses and most individual s ites, but the magnitudes of differences for

these nutrients were less than the variability for the control sample s, and values were

within the range of natura l variability for cottonseed. These findings support the

conclusion of compositional equivalence of MON 88701 to conventional cotton.

VI.A.2. Anti-Nutrient Levels in Cottonseed

Cottonseed was analyzed for five anti-nutrients and in the combined-site analysis the

following components had no significant differen ces (p<0.05) in mean values between

MON 88701 treated with dicam ba and glufosinate and th e conventional control: two

cyclopropenoid fatty acids (malvalic and ster culic) (Table VI-3). The components that

showed statistically significant differences in mean values between MON 88701 and the

conventional control were: one cyclopropenoid fatty acid (dihydrosterculic), free

gossypol, and total gossy pol (Table VI-1).

The statistically significant differences in an ti-nutrients were furt her evaluated using the

four previously described considerations rele vant to the safety a nd nutritional quality of

MON 88701 when compared to the conventional control:

1) All anti-nutrient component differences obs erved in the combin ed-site statistical

analysis, which reflected an increase in MON 88701 mean values with respect to

the conventional control, were small in ma gnitude. The relative magnitude of the

differences for dihydrosterculic acid, free gossypol, and total gossypol were

9.59%, 6.23%, and 6.75%, respectively.

2) Mean values for all significantly differe nt anti-nutrient co mponents from the

combined-site analysis of MON 88701 were within the 99% to lerance interval

established from the conventional co mmercial reference varieties grown

concurrently in the same trial.

3) Assessment of the reproducibility of the combined-site differences at the eight

individual sites showed signi ficant differences for: di hydrosterculic at one site;

free gossypol at two sites; and total gossypol at three sites. All individual site

mean values of MON 88701 for all anti-nutrient components with significant

# Monsanto Company 12-CT-244U 106 of 620 

# differences were within the 99% tolerance interval established from the

conventional commercial reference varieties grown concurrently in the same trial.

4) All combined-site mean values of M ON 88701 for all anti-nutrient components,

including those that were significantly di fferent, were within the context of the

natural variability of commercial cotton composition as published in the scientific

literature and/or availabl e in the ILSI Crop Compos ition Database (ILSI, 2011).

The three cottonseed anti-nutrient differences between MON 88701 and the conventional

control observed in the combined-site data analysis were attributed to small differences in

one cyclopropenoid fatty acid (d ihydrosterculic; expressed as % total fatty acid), free

gossypol, and total gossypol (expressed as % dw). For dihydrosterculic acid, free

gossypol, and total gossypol, the relative magnitude of the di fferences between the mean

values for MON 88701 and the conventiona l control were increases of 9.59% for

dihydrosterculic acid, 6.23% fo r free gossypol, and 6.75% for total gossypol. These antinutrient differences between MON 88701 and th e conventional control observed in the

combined-site analysis were within the 99% tolerance interval established by

conventional commercial reference varieties grown concurrently in the same trial.

Significant differences for the three anti- nutrient mean values between MON 88701 and

the conventional control were not consistently observed across all eight individual sites.

Dihydrosterculic acid, free gossypol, and tota l gossypol were significantly different at

one, two, and three sites respectively, with an increase of 28.35% for dihydrosterculic

acid, and increases ranging from 12.69 to 22.32% for free gossypol and 9.54 to 15.53%

for total gossypol. Overall, observed diff erences in anti-nutrient values between

MON 88701 and the conventional control were not considered to be meaningful from a

food and feed safety or nutri tional perspective because they were generally small, not

consistently reproduced acro ss the individual sites, a nd the mean MON 88701 values

were within the 99% tolerance interval es tablished by conventiona l commercial reference

varieties grown concurrently in the same trial and within the context of the natural

variability of commercial cotton composition as published in the scientific literature

and/or available in the ILSI Cr op Composition Database (ILSI, 2011).

In summary, statistical analyses found no cons istent statistically significant differences

between the levels of anti-nutrient com ponents in cottonseed from MON 88701 and the

conventional control and mean va lues for anti-nutrients were within the range of natural

variability for cottonseed. These findings supported the conclusi on of compositional

equivalence of MON 88701 to conventional cotton.Monsanto Company 12-CT-244U 107 of 620Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 vs.Conventional ControlMean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisCottonseed Proximate (% dw)Ash4.314.115.000.0013.77 - 4.743.42, 4.65Calories Kcal/100g498.50495.240.660.013482.46 - 517.46457.61, 527.56Carbohydrates44.6445.83-2.60<0.00141.40 - 48.8940.26, 56.45Moisture (% fw)7.157.48-4.510.0055.93 - 9.674.79, 9.92Total Fat23.1422.313.710.00119.79 - 26.7815.01, 28.51Cottonseed Fiber (% dw)Acid Detergent Fiber25.2726.58-4.940.00223.26 - 27.7422.24, 31.96Neutral Detergent Fiber30.7332.59-5.72<0.00125.13 - 34.4227.03, 42.49Monsanto Company 12-CT-244U 108 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisCottonseed Fiber (% dw)Total Dietary Fiber39.4441.12-4.08<0.00136.91 - 42.1334.52, 52.58Cottonseed Amino Acid (% dw)Arginine3.033.15-3.800.0022.33 - 3.602.38, 3.47Methionine0.400.384.820.0260.35 - 0.460.32, 0.38Proline1.001.03-2.610.0370.82 - 1.210.83, 1.08Cottonseed Fatty Acid (% Total FA)14:0 Myristic0.770.79-2.690.0090.66 - 0.950.16, 1.3718:2 Linoleic55.7756.15-0.690.02654.24 - 58.2247.49, 63.18Monsanto Company 12-CT-244U 109 of 620Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisCottonseed MineralCalcium (% dw)0.150.1314.09<0.0010.10 - 0.220.058, 0.21Magnesium (% dw)0.400.385.63<0.0010.35 - 0.440.28, 0.47Manganese (mg/kg dw)12.8111.739.200.00110.18 - 14.819.07, 17.33Potassium (% dw)1.121.074.940.0210.98 - 1.240.92, 1.21Zinc (mg/kg dw)37.5840.14-6.390.00527.31 - 46.7427.27, 44.95Cottonseed Vitamin (mg/kg dw)Vitamin E140.14131.336.70<0.00186.23 - 179.3441.91, 205.89Cottonseed CyclopropenoidFatty Acid (% Total FA)Dihydrosterculic Acid0.150.149.590.0030.11 - 0.190.078, 0.25Monsanto Company 12-CT-244U 110 of 620Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisCottonseed Gossypol (% dw)Free Gossypol0.940.896.230.0160.80 - 1.180.099, 1.57Total Gossypol1.040.976.75<0.0010.84 - 1.240.064, 1.76Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 7 SitesCalcium (% dw) Site ARTI0.150.1222.700.0100.14 - 0.160.058, 0.21Calcium (% dw) Site GACH0.130.1117.57<0.0010.13 - 0.130.058, 0.21Calcium (% dw) Site KSLA0.200.1814.740.0070.19 - 0.220.058, 0.21Calcium (% dw) Site NCBD0.150.146.920.0070.14 - 0.150.058, 0.21Calcium (% dw) Site NMLC0.150.1316.830.0030.14 - 0.150.058, 0.21Monsanto Company 12-CT-244U 111 of 620Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 7 SitesCalcium (% dw) Site SCEK0.110.09117.980.0270.10 - 0.110.058, 0.21Calcium (% dw) Site TXPL0.160.1415.31<0.0010.16 - 0.160.058, 0.21Cottonseed Proximate (% dw) - 6 SitesAsh Site GACH4.534.217.56<0.0014.45 - 4.573.42, 4.65Ash Site KSLA4.534.295.640.0274.25 - 4.663.42, 4.65Ash Site LACH4.354.125.560.0134.23 - 4.473.42, 4.65Ash Site NCBD4.344.144.950.0334.29 - 4.403.42, 4.65Ash Site SCEK4.113.749.950.0103.99 - 4.283.42, 4.65Monsanto Company 12-CT-244U 112 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701 vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Proximate (% dw) - 6 SitesAsh Site TXPL3.853.4611.500.0013.77 - 3.923.42, 4.65Cottonseed Fatty Acid (% Total FA) - 5 Sites18:0 Stearic Site ARTI2.682.516.700.0192.65 - 2.721.98, 2.9518:0 Stearic Site LACH2.682.526.040.0012.64 - 2.731.98, 2.9518:0 Stearic Site NCBD2.502.346.850.0362.39 - 2.641.98, 2.9518:0 Stearic Site NMLC2.512.64-5.13<0.0012.47 - 2.561.98, 2.9518:0 Stearic Site TXPL2.352.46-4.670.0062.30 - 2.431.98, 2.95Cottonseed Mineral - 4 SitesMagnesium (% dw) Site GACH0.410.386.92<0.0010.40 - 0.410.28, 0.47Monsanto Company 12-CT-244U 113 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 4 SitesMagnesium (% dw) Site KSLA0.430.406.850.0020.41 - 0.430.28, 0.47Magnesium (% dw) Site SCEK0.390.369.360.0050.37 - 0.410.28, 0.47Magnesium (% dw) Site TXPL0.350.345.540.0030.35 - 0.370.28, 0.47Cottonseed Fiber (% dw) - 3 SitesTotal Dietary Fiber Site KSLA38.3240.14-4.550.03437.62 - 38.7534.52, 52.58Total Dietary Fiber Site LACH39.8243.35-8.150.00239.02 - 40.8634.52, 52.58Total Dietary Fiber Site NMLC39.1641.10-4.730.01637.46 - 40.4434.52, 52.58Cottonseed Amino Acid (% dw) - 3 SitesArginine Site GACH2.953.21-8.350.0082.87 - 3.022.38, 3.47Monsanto Company 12-CT-244U 114 of 620Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Amino Acid (% dw) - 3 SitesArginine Site KSLA3.023.28-7.870.0132.95 - 3.102.38, 3.47Arginine Site NMLC3.483.71-6.100.0053.42 - 3.602.38, 3.47Cottonseed Fatty Acid (% Total FA) - 3 Sites14:0 Myristic Site KSLA0.680.72-5.330.0070.66 - 0.710.16, 1.3714:0 Myristic Site NCBD0.680.75-8.360.0020.66 - 0.700.16, 1.3714:0 Myristic Site NMLC0.930.98-4.430.0010.92 - 0.950.16, 1.37Cottonseed Mineral - 3 SitesPotassium (% dw) Site GACH1.211.128.01<0.0011.17 - 1.240.92, 1.21Potassium (% dw) Site SCEK1.131.0210.880.0421.11 - 1.170.92, 1.21Monsanto Company 12-CT-244U 115 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 3 SitesPotassium (% dw) Site TXPL1.010.8716.370.0040.98 - 1.060.92, 1.21Cottonseed Vitamin (mg/kg dw) - 3 SitesVitamin E Site GACH151.03140.127.780.025148.34 - 154.9541.91, 205.89Vitamin E Site LACH169.88149.9613.280.001163.34 - 175.3341.91, 205.89Vitamin E Site TXPL114.39103.6610.350.033107.81 - 118.3941.91, 205.89Cottonseed Gossypol (% dw) - 3 SitesTotal Gossypol Site KSLA1.131.0112.000.0491.00 - 1.240.064, 1.76Total Gossypol Site NMLC0.920.8015.530.0260.84 - 0.970.064, 1.76Total Gossypol Site SCEK1.171.079.540.0171.13 - 1.230.064, 1.76Monsanto Company 12-CT-244U 116 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Proximate (% dw) - 2 SitesCarbohydrates Site SCEK46.5648.67-4.330.03145.10 - 47.4840.26, 56.45Carbohydrates Site TXPL44.0346.39-5.080.01042.73 - 45.9940.26, 56.45Total Fat Site NCBD23.0421.596.740.02421.89 - 23.7615.01, 28.51Total Fat Site SCEK25.6523.658.460.01924.23 - 26.7815.01, 28.51Cottonseed Fiber (% dw) - 2 SitesAcid Detergent Fiber Site ARTI24.8127.53-9.860.00724.44 - 25.2022.24, 31.96Acid Detergent Fiber Site LACH25.7228.35-9.270.00524.16 - 27.0822.24, 31.96Cottonseed Amino Acid (% dw) - 2 SitesPhenylalanine Site GACH1.401.49-5.890.0391.37 - 1.431.12, 1.58Monsanto Company 12-CT-244U 117 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Amino Acid (% dw) - 2 SitesPhenylalanine Site KSLA1.441.53-5.880.0251.40 - 1.461.12, 1.58Cottonseed Fatty Acid (% Total FA) - 2 Sites16:0 Palmitic Site LACH24.4824.041.810.01424.37 - 24.5516.54, 30.5516:0 Palmitic Site SCEK24.7424.391.430.02924.59 - 24.9416.54, 30.5516:1 Palmitoleic Site NCBD0.460.48-3.880.0190.44 - 0.470.39, 0.7016:1 Palmitoleic Site NMLC0.530.54-2.270.0140.52 - 0.530.39, 0.7018:3 Linolenic Site ARTI0.140.1311.920.0120.14 - 0.150.060, 0.2418:3 Linolenic Site NMLC0.160.148.120.0090.15 - 0.160.060, 0.24Monsanto Company 12-CT-244U 118 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 2 SitesIron (mg/kg dw) Site NCBD43.2148.04-10.050.02541.96 - 44.4447.30, 97.12Iron (mg/kg dw) Site TXPL60.4779.02-23.470.03956.94 - 66.5047.30, 97.12Manganese (mg/kg dw) Site GACH13.4111.5116.520.00312.79 - 14.149.07, 17.33Manganese (mg/kg dw) Site TXPL10.919.0420.590.00710.18 - 11.379.07, 17.33Zinc (mg/kg dw) Site NCBD40.7949.54-17.660.00640.28 - 41.3727.27, 44.95Zinc (mg/kg dw) Site NMLC45.6349.43-7.680.00944.12 - 46.7427.27, 44.95Cottonseed Gossypol (% dw) - 2 SitesFree Gossypol Site KSLA1.070.9512.690.0141.03 - 1.100.099, 1.57Monsanto Company 12-CT-244U 119 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Gossypol (% dw) - 2 SitesFree Gossypol Site NMLC0.850.6922.320.0110.83 - 0.880.099, 1.57Statistical Differences Observed in One SiteCottonseed Proximate (% dw)Protein Site TXPL29.4328.483.330.01729.06 - 30.1422.30, 29.41Cottonseed Fiber (% dw)Crude Fiber Site KSLA16.4317.67-7.040.01916.06 - 17.2416.93, 22.68Neutral Detergent Fiber Site TXPL29.7532.12-7.400.00628.74 - 30.5627.03, 42.49Cottonseed Amino Acid (% dw)Alanine Site LACH1.071.033.730.0301.00 - 1.110.86, 1.11Aspartic Acid Site GACH2.312.45-6.030.0192.24 - 2.361.94, 2.57Monsanto Company 12-CT-244U 120 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in One SiteCottonseed Amino Acid (% dw)Glutamic Acid Site GACH4.574.96-7.950.0104.35 - 4.773.74, 5.28Isoleucine Site GACH0.900.94-4.210.0340.90 - 0.910.75, 0.96Leucine Site GACH1.511.58-4.320.0241.49 - 1.541.25, 1.62Lysine Site LACH1.261.187.010.0231.17 - 1.311.01, 1.30Methionine Site LACH0.420.3812.030.0130.37 - 0.440.32, 0.38Proline Site GACH0.981.05-6.160.0330.97 - 0.990.83, 1.08Threonine Site GACH0.850.90-5.140.0490.83 - 0.880.72, 0.89Tryptophan Site SCEK0.350.38-6.700.0230.33 - 0.380.34, 0.42Monsanto Company 12-CT-244U 121 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in One SiteCottonseed Amino Acid (% dw)Tyrosine Site GACH0.800.84-4.300.0370.79 - 0.820.67, 0.84Valine Site GACH1.211.26-4.190.0171.19 - 1.231.00, 1.28Cottonseed Fatty Acid (% Total FA)18:1 Oleic Site LACH14.7014.292.890.02114.48 - 15.0111.38, 20.6418:2 Linoleic Site LACH55.5356.63-1.930.00155.15 - 55.9947.49, 63.1820:0 Arachidic Site LACH0.310.296.780.0330.31 - 0.320.17, 0.3822:0 Behenic Site ARTI0.140.15-9.920.0080.13 - 0.140.070, 0.21Cottonseed MineralSodium (% dw) Site KSLA0.0220.0080178.300.0200.019 - 0.0250, 0.066Monsanto Company 12-CT-244U 122 of 620Table VI-1. Summary ofDifferences (p<0.05) for the Comparison ofCottonseed Component Levels for MON 88701vs.Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in One SiteCottonseed CyclopropenoidFatty Acid (% Total FA)Dihydrosterculic Acid Site GACH0.150.1228.350.0220.14 - 0.160.078, 0.251dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 was treated with dicamba and glufosinate.3Mean = least-square mean.4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 123 of 620Table VI-2. Statistical Summary ofCombined-Site Cottonseed Nutrients for MON 88701 vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.31 (0.11)4.11 (0.11)0.21 (0.052)0.094, 0.320.0013.42, 4.65(3.77 - 4.74)(3.34 - 5.00)(-0.49 - 0.61)(3.18 - 4.68)Calories (Kcal/100g)498.50 (1.65)495.24 (1.71)3.26 (1.29)0.70, 5.820.013457.61, 527.56(482.46 - 517.46)(487.70 - 512.65)(-14.30 - 18.37)(466.09 - 509.91)Carbohydrates44.64 (0.56)45.83 (0.57)-1.19 (0.32)-1.82, -0.56<0.00140.26, 56.45(41.40 - 48.89)(42.14 - 50.30)(-5.19 - 2.45)(43.28 - 54.90)Moisture (% fw)7.15 (0.26)7.48 (0.27)-0.34 (0.11)-0.56, -0.110.0054.79, 9.92(5.93 - 9.67)(6.15 - 9.19)(-1.82 - 0.79)(6.05 - 10.50)Protein27.91 (0.77)27.79 (0.77)0.13 (0.31)-0.53, 0.780.68522.30, 29.41(22.71 - 31.47)(23.53 - 31.27)(-1.99 - 3.73)(20.58 - 29.28)Total Fat23.14 (0.31)22.31 (0.33)0.83 (0.26)0.32, 1.340.00115.01, 28.51(19.79 - 26.78)(20.71 - 25.20)(-2.89 - 3.86)(16.58 - 25.25)Monsanto Company 12-CT-244U 124 of 620Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.27 (0.34)26.58 (0.35)-1.31 (0.35)-2.06, -0.570.00222.24, 31.96(23.26 - 27.74)(22.08 - 29.58)(-5.42 - 1.77)(23.42 - 31.62)Crude Fiber18.17 (0.37)18.54 (0.38)-0.38 (0.32)-1.02, 0.270.24616.93, 22.68(15.97 - 21.66)(16.06 - 21.70)(-3.36 - 4.75)(16.92 - 23.32)Neutral Detergent Fiber30.73 (0.51)32.59 (0.53)-1.86 (0.41)-2.68, -1.05<0.00127.03, 42.49(25.13 - 34.42)(28.87 - 35.89)(-6.95 - 1.16)(29.27 - 40.63)Total Dietary Fiber39.44 (0.39)41.12 (0.41)-1.68 (0.36)-2.45, -0.91<0.00134.52, 52.58(36.91 - 42.13)(39.05 - 44.37)(-5.34 - 1.09)(37.29 - 48.60)Amino Acid (% dw)Alanine1.06 (0.020)1.05 (0.020)0.0026 (0.0091)-0.017, 0.0220.7750.86, 1.11(0.91 - 1.14)(0.88 - 1.17)(-0.13 - 0.12)(0.83 - 1.22)Arginine3.03 (0.10)3.15 (0.10)-0.12 (0.033)-0.19, -0.0490.0022.38, 3.47(2.33 - 3.60)(2.41 - 3.77)(-0.47 - 0.39)(2.30 - 3.55)Monsanto Company 12-CT-244U 125 of 620Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.39 (0.062)2.41 (0.062)-0.015 (0.027)-0.072, 0.0420.5751.94, 2.57(1.94 - 2.64)(1.92 - 2.74)(-0.29 - 0.29)(1.79 - 2.72)Cystine0.41 (0.0091)0.40 (0.0094)0.0096 (0.0070)-0.0043, 0.0230.1740.31, 0.45(0.32 - 0.47)(0.31 - 0.46)(-0.063 - 0.082)(0.29 - 0.47)Glutamic Acid4.76 (0.13)4.84 (0.14)-0.079 (0.072)-0.23, 0.0770.2953.74, 5.28(3.80 - 5.38)(3.66 - 5.70)(-0.78 - 0.79)(3.39 - 5.45)Glycine1.10 (0.020)1.09 (0.020)0.0014 (0.011)-0.021, 0.0240.8960.90, 1.14(0.93 - 1.19)(0.91 - 1.20)(-0.13 - 0.14)(0.85 - 1.23)Histidine0.74 (0.019)0.75 (0.019)-0.0014 (0.0073)-0.017, 0.0140.8540.59, 0.81(0.58 - 0.85)(0.61 - 0.84)(-0.062 - 0.091)(0.57 - 0.84)Isoleucine0.91 (0.018)0.92 (0.018)-0.0066 (0.0079)-0.023, 0.0100.4210.75, 0.96(0.75 - 1.01)(0.77 - 1.03)(-0.077 - 0.096)(0.72 - 1.03)Monsanto Company 12-CT-244U 126 of 620Table VI-2. Statistical Summary ofCombined-Site Cottonseed Nutrients for MON 88701 vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.53 (0.032)1.54 (0.032)-0.0018 (0.013)-0.029, 0.0260.8921.25, 1.62(1.29 - 1.70)(1.28 - 1.69)(-0.14 - 0.16)(1.20 - 1.72)Lysine1.24 (0.025)1.23 (0.025)0.0069 (0.015)-0.026, 0.0390.6581.01, 1.30(1.05 - 1.38)(1.06 - 1.39)(-0.11 - 0.15)(0.99 - 1.44)Methionine0.40 (0.0079)0.38 (0.0084)0.018 (0.0081)0.0023, 0.0350.0260.32, 0.38(0.35 - 0.46)(0.32 - 0.46)(-0.066 - 0.12)(0.29 - 0.49)Phenylalanine1.43 (0.039)1.46 (0.039)-0.022 (0.014)-0.052, 0.00840.1441.12, 1.58(1.14 - 1.66)(1.15 - 1.66)(-0.18 - 0.19)(1.10 - 1.63)Proline1.00 (0.029)1.03 (0.029)-0.027 (0.012)-0.052, -0.00180.0370.83, 1.08(0.82 - 1.21)(0.81 - 1.25)(-0.12 - 0.10)(0.79 - 1.17)Serine1.08 (0.025)1.09 (0.026)-0.0036 (0.015)-0.035, 0.0280.8070.83, 1.21(0.90 - 1.23)(0.86 - 1.24)(-0.18 - 0.16)(0.81 - 1.24)Monsanto Company 12-CT-244U 127 of 620Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.87 (0.016)0.86 (0.016)0.0057 (0.0083)-0.012, 0.0230.5040.72, 0.89(0.74 - 0.94)(0.73 - 0.95)(-0.10 - 0.10)(0.67 - 0.96)Tryptophan0.41 (0.0092)0.42 (0.0095)-0.0061 (0.0066)-0.019, 0.00710.3610.34, 0.42(0.33 - 0.52)(0.37 - 0.52)(-0.081 - 0.078)(0.31 - 0.46)Tyrosine0.81 (0.017)0.81 (0.018)-0.0011 (0.0083)-0.019, 0.0170.8980.67, 0.84(0.67 - 0.92)(0.67 - 0.91)(-0.074 - 0.12)(0.63 - 0.91)Valine1.21 (0.027)1.23 (0.027)-0.012 (0.011)-0.036, 0.0120.2961.00, 1.28(1.00 - 1.40)(1.00 - 1.40)(-0.090 - 0.12)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.77 (0.030)0.79 (0.031)-0.021 (0.0071)-0.036, -0.00600.0090.16, 1.37(0.66 - 0.95)(0.71 - 0.98)(-0.077 - 0.047)(0.45 - 1.04)16:0 Palmitic23.95 (0.30)23.80 (0.30)0.15 (0.076)-0.016, 0.310.07316.54, 30.55(22.34 - 25.28)(22.69 - 25.05)(-0.68 - 0.76)(19.11 - 26.73)Monsanto Company 12-CT-244U 128 of 620Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.50 (0.0094)0.50 (0.0094)0.0022 (0.0038)-0.0060, 0.0100.5720.39, 0.70(0.44 - 0.54)(0.45 - 0.54)(-0.025 - 0.039)(0.44 - 0.67)18:0 Stearic2.54 (0.058)2.47 (0.058)0.068 (0.036)-0.0091, 0.140.0791.98, 2.95(2.29 - 2.85)(2.15 - 2.76)(-0.16 - 0.24)(1.98 - 2.97)18:1 Oleic15.10 (0.26)14.96 (0.26)0.14 (0.070)-0.0049, 0.290.05711.38, 20.64(14.15 - 16.45)(14.06 - 16.44)(-0.48 - 0.75)(13.71 - 18.39)18:2 Linoleic55.77 (0.39)56.15 (0.40)-0.39 (0.16)-0.72, -0.0530.02647.49, 63.18(54.24 - 58.22)(54.04 - 57.93)(-1.42 - 0.80)(49.78 - 59.61)18:3 Linolenic0.18 (0.022)0.17 (0.022)0.011 (0.0068)-0.0038, 0.0250.1360.060, 0.24(0.14 - 0.34)(0.12 - 0.30)(-0.0073 - 0.052)(0.10 - 0.29)20:0 Arachidic0.29 (0.0086)0.28 (0.0087)0.0044 (0.0047)-0.0057, 0.0150.3640.17, 0.38(0.23 - 0.32)(0.23 - 0.32)(-0.027 - 0.046)(0.20 - 0.36)Monsanto Company 12-CT-244U 129 of 620Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.15 (0.0051)0.15 (0.0051)-0.0035 (0.0029)-0.0098, 0.00290.2600.070, 0.21(0.12 - 0.19)(0.13 - 0.21)(-0.049 - 0.032)(0.051 - 0.19)MineralCalcium (% dw)0.15 (0.0093)0.13 (0.0093)0.018 (0.0022)0.013, 0.023<0.0010.058, 0.21(0.10 - 0.22)(0.081 - 0.19)(-0.012 - 0.038)(0.081 - 0.18)Copper (mg/kg dw)8.90 (0.70)8.93 (0.70)-0.025 (0.16)-0.34, 0.290.8752.97, 12.86(5.22 - 11.91)(5.40 - 11.92)(-2.59 - 1.29)(4.46 - 11.62)Iron (mg/kg dw)67.21 (4.40)71.33 (4.48)-4.12 (2.74)-9.96, 1.710.15347.30, 97.12(41.96 - 83.17)(45.03 - 95.10)(-38.15 - 12.79)(39.49 - 114.34)Magnesium (% dw)0.40 (0.0083)0.38 (0.0084)0.021 (0.0032)0.015, 0.028<0.0010.28, 0.47(0.35 - 0.44)(0.33 - 0.44)(-0.036 - 0.054)(0.31 - 0.46)Manganese (mg/kg dw)12.81 (0.47)11.73 (0.48)1.08 (0.28)0.48, 1.680.0019.07, 17.33(10.18 - 14.81)(8.61 - 14.11)(-1.95 - 2.54)(9.07 - 17.14)Monsanto Company 12-CT-244U 130 of 620Table VI-2. Statistical Summary ofCombined-Site Cottonseed Nutrients for MON 88701 vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.72 (0.031)0.72 (0.031)0.0081 (0.0067)-0.0053, 0.0210.2300.49, 0.87(0.56 - 0.84)(0.54 - 0.87)(-0.087 - 0.11)(0.48 - 0.87)Potassium (% dw)1.12 (0.028)1.07 (0.028)0.053 (0.020)0.0089, 0.0970.0210.92, 1.21(0.98 - 1.24)(0.79 - 1.27)(-0.12 - 0.27)(0.90 - 1.26)Sodium (% dw)0.034 (0.0095)0.029 (0.0096)0.0045 (0.0046)-0.0053, 0.0140.3460, 0.066(0.018 - 0.12)(0.0053 - 0.10)(-0.065 - 0.030)(0.0054 - 0.077)Zinc (mg/kg dw)37.58 (2.01)40.14 (2.02)-2.57 (0.77)-4.22, -0.910.00527.27, 44.95(27.31 - 46.74)(28.22 - 52.95)(-11.57 - 3.27)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E140.14 (9.87)131.33 (9.88)8.80 (2.07)4.39, 13.22<0.00141.91, 205.89(86.23 - 179.34)(91.78 - 162.98)(-6.54 - 26.36)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 was treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 131 of 620Table VI-3. Statistical Summary of Combined-Site Cottonseed Anti-nutrientsfor MON 88701 vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.0034)0.14 (0.0037)0.013 (0.0044)0.0044, 0.0220.0030.078, 0.25(0.11 - 0.19)(0.11 - 0.17)(-0.026 - 0.068)(0.038 - 0.23)Malvalic Acid0.39 (0.015)0.37 (0.016)0.013 (0.015)-0.016, 0.0430.3710.23, 0.54(0.20 - 0.55)(0.26 - 0.49)(-0.16 - 0.16)(0.11 - 0.59)Sterculic Acid0.22 (0.0067)0.21 (0.0072)0.0067 (0.0081)-0.0096, 0.0230.4120.17, 0.27(0.13 - 0.29)(0.17 - 0.27)(-0.085 - 0.078)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.94 (0.037)0.89 (0.037)0.055 (0.020)0.012, 0.0990.0160.099, 1.57(0.80 - 1.18)(0.68 - 1.20)(-0.086 - 0.20)(0.50 - 1.41)Total Gossypol1.04 (0.037)0.97 (0.037)0.066 (0.017)0.031, 0.10<0.0010.064, 1.76(0.84 - 1.24)(0.74 - 1.10)(-0.021 - 0.23)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 was treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.

# Monsanto Company 12-CT-244U 132 of 620 

# Table VI-4. Literature and ILSI Ranges for Components in Cottonseed

Cottonseed Tissue Components 1 

# Literature Range 2 

# ILSI Range 3



# Cottonseed Nutrients

Proximates (% dw)

Ash 3.87 – 5.29 a 

# ; 3.7 – 4.2 d 

# 3.761 – 5.342

Carbohydrates by calculation 45.28 – 53.62 a 

# 39.0 – 53.6

Calories by calculation

(Kcal/100g) 471.34 – 506.95 a 

# Not available

Moisture (% fw) 2.25 – 7.49 a 

# 2.3 – 9.9

Protein 24.54 – 30.83 a 

# ; 21.2 – 25.9 b 

# 21.48 – 32.97

Total Fat 17.37 – 25.16 a 

# ; 14.4 – 16.9 d 

# 17.201 – 27.292

Fiber (% dw)

Acid Detergent Fiber 21.10 – 34.8 a 

# ; 37.6 – 40.5 d 

# 19.74 – 38.95

Neutral Detergent Fiber 32.92 – 45.83 a 

# ; 50.0 – 53.6 d 

# 25.56 – 51.87

Crude Fiber 13.85 – 17.94 a 

# 13.86 – 23.10

Total Dietary Fiber not available 33.69 – 47.55

Amino Acids (% total AA) (% dw)

Alanine 4.16 – 4.41 a 

# ; 3.6 – 4.2 b 

# 0.80 – 1.22

Arginine 11.28 – 12.51 a 

# ; 10.9 – 12.3 b 

# 2.06 – 3.72

Aspartic acid 9.73 – 9.99 a 

# ; 8.8 – 9.5 b 

# 1.82 – 2.94

Cystine/Cysteine 1.60 – 1.92 a 

# ; 2.3 – 3.4 b 

# 0.35 – 0.56

Glutamic acid 20.76 – 21.61 a 

# ; 20.5 – 22.4 b 

# 3.91 – 6.72

Glycine 4.44 – 4.58 a 

# ; 3.8 – 4.5 b 

# 0.83 – 1.32

Histidine 3.00 – 3.12 a 

# ; 2.6 – 2.8 b 

# 0.57 – 0.91

Isoleucine 3.10 – 3.67 a 

# ; 3.0 – 3.4 b 

# 0.62 – 1.05

Leucine 6.27 – 6.65 a 

# ; 5.5 – 6.1 b 

# 1.14 – 1.86

Lysine 4.85 – 5.37 a 

# ; 4.2 – 4.6 b 

# 0.94 – 1.46

Methionine 1.46 – 1.88 a 

# ; 1.3 – 1.8 b 

# 0.30 – 0.47

Phenylalanine 5.56 – 5.77 a 

# ; 5.0 – 5.6 b 

# 1.02 – 1.72

Proline 4.06 – 4.28 a 

# ; 3.1 – 4.0 b 

# 0.75 – 1.23

Serine 4.45 – 4.86 a 

# ; 3.9 – 4.4 b 

# 0.91 – 1.35

Threonine 3.26 – 3.59 a 

# ; 2.8 – 3.2 b 

# 0.55 – 0.92

Tryptophan 0.97 – 1.21 a 

# ; 1.0 – 1.4 b 

# 0.194 – 0.319

Tyrosine 2.65 – 2.92 a 

# ; 2.8 – 3.3 b 

# 0.53 – 0.84

Valine 4.76 – 5.14 a 

# ; 4.3 – 4.7 b 

# 0.87 – 1.49

Fatty Acids (% total FA)

8:0 Caprylic not available not available

10:0 Capric not available not available

12:0 Lauric not available not available

14:0 Myristic 0.55 – 2.40 a 

# ; 0.6 – 1.5 b 

# 0.455 – 2.400

14:1 Myristoleic not available not available

15:0 Pentadecanoic 0.050 – 0.17 a 

# 0.103 – 0.481

15:1 Pentadecenoic not available not available

16:0 Palmitic 21.23 – 27.9 a 

# ; 17.6 – 24.8 b 

# 15.11 – 27.90

16:1 Palmitoleic 0.55 – 1.16 a 

# 0.464 – 1.190

17:0 Heptadecanoic not available 0.092 – 0.119

# Monsanto Company 12-CT-244U 133 of 620 

# Table VI-4. Literature and ILSI Ranges for Components in Cottonseed (continued)

Cottonseed Tissue Components 1 

# Literature Range 2 

# ILSI Range 3



# 17:1 Heptadecenoic not available not available

18:0 Stearic 1.99 – 3.11 a 

# ; 2.0 – 2.5 b 

# 0.20 – 3.11

18:1 Oleic 13.90 – 20.10 a 

# ; 15.0 – 20.7 b 

# 12.8 – 25.3

18:2 Linoleic 46.00 – 56.88 a 

# 46.0 – 59.4

18:3 Gamma Linolenic 0.050 – 0.25 a 

# 0.097 – 0.232

18:3 Linolenic 0.050 – 0.25 a 

# 0.11 – 0.35

20:0 Arachidic 0.25 – 0.33 a 

# 0.186 – 0.414

20:1 Eicosenoic not available 0.095 – 0.098

20:2 Eicosadienoic not available not available

20:3 Eicosatrienoic not available not available

20:4 Arachidonic not available not available

22:0 Behenic 0.13 – 0.17 a 

# 0.104 – 0.295

Vitamins (mg/kg fw) (mg/kg dw)

Vitamin E 99 – 224 c 

# 70.825 – 197.243

Minerals (% dw)

Calcium 0.10 – 0.33 a 

# 0.10323 – 0.32581

Copper (mg/kg dw) 3.54 – 11.14 a 

# 3.13 – 24.57

Iron (mg/kg dw) 40.58 – 56.54 a 

# 36.71 – 318.38

Magnesium 0.37 – 0.46 a 

# 0.34709 – 0.49312

Manganese (mg/kg dw) 11.06 – 18.31 a 

# 10.69 – 21.96

Phosphorus 0.60 – 0.84 a 

# 0.48254 – 0.99157

Potassium 0.98 – 1.24 a 

# 0.98345 – 1.44835

Sodium 0.0054 – 0.74 a 

# 0.01118 – 0.73548

Zinc (mg/kg dw) 30.21 – 47.75 a 

# 27.0 – 59.5

Cottonseed Anti-Nutrients

Gossypol, Total (% dw) 0.57 – 1.42 a 

# ; 0.55 – 0.77 d 

# 0.547 – 1.522

Gossypol, Free (% dw) 0.53 – 1.20 a 

# 0.454 – 1.399

Cyclopropenoid Fatty Acids

(% total FA)

Dihydrosterculic 0.13 – 0.24 a 

# 0.075 – 0.310

Malvalic 0.33 – 0.58 a 

# 0.229 – 0.759

Sterculic 0.21 – 0.56 a 

# 0.190 – 0.556

1 

# fw=fresh weight; dw=dry weight

2 

# Literature range references; a 

# (Hamilton et al., 2004); b 

# (Lawhon et al., 1977); c 

# (Smith and Creelman, 2001);

d 

# (Bertrand et al., 2005).

3 

# (ILSI, 2011).

# Monsanto Company 12-CT-244U 134 of 620 

# VI.B. Compositional Assessment of MON 88701 Conclusion

Detailed analyses were c onducted on nutrient and anti-nut rient levels in MON 88701

cottonseed from plants treated with dicamba and glufosinate, repor ted above, and plants

not treated with dicamba or glufosinat e (Appendix E). Component levels for

MON 88701 were compared to levels in the c onventional control. The analytes evaluated

are consistent with those id entified by the OECD as importa nt to understanding the safety

and nutrition of new varieties of biotechno logy-derived cotton (OECD, 2009). These

compositional comparisons were made by analyzing the acid-delinted cottonseed

harvested from plants grown at each of eight fiel d sites in the U.S. during the 2010 field

season. Composition analyses of all samples, conducted in accordance with OECD

guidelines, were performed for nutrients including proximates (ash, carbohydrates, and

calories by calculation, moistu re, protein, and fat), fibers (ADF, CF, NDF, and TDF),

amino acids, fatty acids (C8-C22), mine rals (calcium, copper, iron magnesium,

manganese, phosphorus, potassium, sodium, and zinc ), and vitamin E. The anti-nutrients

assessed in this analysis included total a nd free gossypol and cycl opropenoid fatty acids

(dihydrosterculic, malvalic, and sterculic). These analyses also included measurements

of the same nutrients and anti-nutrients in conventional commercial cotton varieties,

known as reference varieties, to provide data on natural variability of each compositional

component analyzed. All cotton plants in cluding MON 88701, the co nventional control,

and the conventional commercial reference va rieties were treated with maintenance

pesticides as necessary throughout the gr owing season. In addition, MON 88701 plots

were treated at the 3-5 leaf stage with glufosinate herbicid e at the label rate (0.5 lbs

a.i./acre) and at the 6-10 leaf stage with di camba herbicide at the label rate (0.5 lbs

a.e./acre).

For MON 88701 compared to the conventional control, the combined-site analysis of

cottonseed showed no statistic ally significant differences (p<0.05) between nutrient and

anti-nutrient components of MON 88701 and the control for 30 (57.7%) of the 52 mean

value comparisons. Cottonseed nutrient compon ent differences included mean values for

five proximates (ash, calories, carbohydrates, mo isture, and total fat), three types of fiber

(ADF, NDF, and TDF), three amino acids (arg inine, methionine, and proline), two fatty

acids (14:0 myristic acid a nd 18:2 linoleic acid), five minerals (calcium, magnesium,

manganese, potassium and zinc), and vitami n E. Cottonseed anti-nutrient component

differences included mean values for dihydroste rculic acid, free and total gossypol. All

nutrient and anti-nutrient component differences observed in the combined-site statistical

analysis, whether reflecting increased or d ecreased MON 88701 mean values with respect

to the conventional co ntrol, were 14.09% or less. Mean values for all significantly

different nutrient and anti- nutrient components from the combined-site analysis of

MON 88701, with the exception of methionine, were within the 99% tolerance interval

established from the conventional, commercial reference varieties grown concurrently in

the same trial. All combined-site mean valu es, including methionine, and individual site

mean values of MON 88701 for all nutrient a nd anti-nutrient compone nts were within the

context of the natural variability of comm ercial cotton composition as published in the

scientific literature and/or available in the ILSI Crop Composition Database (ILSI, 2011).

# Monsanto Company 12-CT-244U 135 of 620 

# Overall, for MON 88701 mean component values observed to be significantly different

from those of the conventional control, the differences with the control were generally

shown to be of small relative magnitudes. All MON 88701 mean component values in

the combined-site analysis, with the exception of methionine, were within the 99%

tolerance interval established from the c onventional commercial references varieties

grown concurrently and at the same field site s. All combined-site mean values including

methionine and individual site mean valu es of MON 88701 for all nutrient and antinutrient components were within the context of the natural variability of commercial

cotton composition as published in the scientif ic literature and/or av ailable in the ILSI

Crop Composition Database (ILSI, 2011).

For MON 88701 treated with dicamba and gluf osinate, compared to the conventional

control, most of the combined-site diffe rences were not reproducible among the

individual sites, with the exception of ash and calcium; however, all of the combined-site

component values were within the range of va lues reported in the sc ientific literature

and/or in the ILSI Crop Composition Database . Additionally, the concentrations of key

nutrients and anti-nutrients of cottonseed from MON 88701 that was not treated with

dicamba or glufosinate were also analyzed (See Appendix E). Results from this analysis

were similar to those of the dicamb a and glufosinate treated analysis. Based on the

results of this composition an alysis, it is concluded that cottonseed from MON 88701 is

compositionally equivalent to conventional cott on and therefore the food and feed safety

and nutritional quality of this product is comparable to that of the commercially

cultivated cotton.

Conventional cotton processing is described in Section II of this document. The

processing of MON 88701 is not expected to be any different from that of conventional

cotton. As described in this section, detail ed compositional analyses of key components

of MON 88701 have been performed and have demonstrated that MON 88701 is

compositionally equivalent to conventional co tton. Additionally, the mode of action of

the MON 88701 DMO and PAT ( bar ) proteins, as described in Section V.A., is well

understood, and there is no reason to expect interactions with important nutrients or

known anti-nutrients that are present in cotton. Therefore, when MON 88701 and its

progeny are used on a commercial scale as a source of food or fee d, these products are

not expected to be different from the e quivalent foods or feeds originating from

commercially cultivated cotton.

# Monsanto Company 12-CT-244U 136 of 620 

# VII. PHENOTYPIC, AGRO NOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides a comparative asse ssment of the phenotypic, agronomic, and

environmental interaction characteristics of MON 88701 compared to the conventional

control. The data support a conclusion th at MON 88701 is not meaningfully different

from the conventional control with the ex ception of the dicamba and glufosinatetolerance traits, and therefore is no more likely to pose a plant pest risk or have a

significant environmental impact compared to conventional cotton. These conclusions

are based on the results of multiple evaluations from laboratory and field experiments.

Phenotypic, agronomic, and environmental interaction characte ristics of MON 88701

were evaluated in a comparative manner to assess plant pest potential. These assessments

included evaluation of seed germination char acteristics, plant growth and development

characteristics, pollen characteristics, observa tions of plant responses to abiotic stress,

and plant-disease and plant- arthropod interactions. Resu lts from these assessments

demonstrate that MON 88701 does not possess: a) increased weediness ch aracteristics; b)

increased susceptibility or tolerance to specifi c abiotic stressors, diseases, or arthropods;

or c) characteristics that would confer a pl ant pest risk or a significant environmental

impact compared to the conventional control.

VII.A. Characteristic s Measured for Assessment

In the phenotypic, agronomic, and envi ronmental interactions assessment of

MON 88701, data were collected to evaluate altered plant pest po tential. A detailed

description of the regulated article phenotype is requested as part of the petition for

determination of nonregulated status in 7 CFR § 340.6 including differences from the

unmodified recipient organism that would “s ubstantiate that the regulated article is

unlikely to pose a greater plant pest risk th an unmodified organism from which it was

derived.” As part of the ch aracterization of MON 88701, data were collected to provide a

detailed description of the phenotypic, agronomic, and environmental interactions

characteristics of MON 88701.

The plant characterization of MON 88701 enco mpassed five general data categories:

1) seed germination, dormancy, and emergen ce; 2) vegetative growth; 3) reproductive

development (including pollen characteristics) ; 4) plant mapping; 5) plant response to

abiotic stress and interactions with diseases and arthropods

. An overview of the

characteristics assessed is presented in Table VII-1.

The phenotypic, agronomic, and environmental interactions data were evaluated from a

basis of familiarity (OECD, 1993) and were comprised of a combination of field and

laboratory studies conducted by scientists who are familiar with the production and

evaluation of cotton. In each of these assessments, MON 88701 was compared to a

conventional control, Coker 130, which has a genetic background similar to MON 88701,

but does not possess the dicamba and glufosinat e-tolerance traits. In addition, multiple

commercial reference varieties developed through conventional selection and breeding

(See Appendices F-H and Tables F-1, G-1, G-2, and H-1) were included to provide a

# Monsanto Company 12-CT-244U 137 of 620 

# range of comparative values that are representative of the variability in existing

commercial cotton varieties for each charact eristic. Data collected for the various

characteristics from the commercial reference varieties provides context for interpreting

experimental results.

# Monsanto Company 12-CT-244U 138 of 620 

# Table VII-1. Phenotypic, Agronomi c, and Environmental Interaction

Characteristics Evaluated in United Stat es Field Trials and Laboratory Studies

Data

Category Characteristics

measured

(associated

section where

discussed) 1 

# Evaluation timing (setting

of evaluation) 2 

# Evaluation description

(measurement endpoints)

Seed

germination,

dormancy, and

emergence Normal

germinated

(VII.C.1.) Day 4 and 12 (20/30oC)

(laboratory) Percentage of seed producing

seedlings exhibiting normal

developmental characteristics

Abnormal

germinated

(VII.C.1.) Day 12 (20/30°C)

(laboratory) Percentage of seed producing

seedlings that could not be

classified as normal germinated

Germinated

(VII.C.1.) Day 4, 12, and 18 (10, 20,

30, 10/20 and 10/30°C)

(laboratory) Percentage of seed that had

germinated normally and

abnormally

Dead

(VII.C.1.) Day 4 and 12 (10, 20, 30,

10/20, 10/30, and 20/30°C);

Day 18 (10, 20, 30, 10/20

and 10/30°C) (laboratory) Percentage of seed that had visibly

deteriorated and become soft to the

touch (also included non-viable

hard and nonviable firm-swollen

seed)

Viable hard

(VII.C.1.) Day 12 (20/30°C); Day 18

(10, 20, 30, 10/20 and

10/30°C) (laboratory) Percentage of seed that did not

imbibe water and remained hard to

the touch (viability determined by a

tetrazolium test 2 

# )

Viable firmswollen

(VII.C.1.) Day 12 (20/30°C); Day 18

(10, 20, 30, 10/20 and

10/30°C) (laboratory) Percentage of seed that imbibed

water and were firm to the touch but

did not germinate (viability

determined by a tetrazolium test 3 

# )

Stand count

(VII.C.2.1.) Approximately 14 and 30

DAP (Field) Number of emerged plants in two

rows, standardized to 20 ft rows

Final stand count

(VII.C.2.1.) Within approximately 7

days of harvest (Field) Number of plants in two rows,

standardized to 20 ft rows

Vegetative

Growth Plant vigor

(VII.C.2.1.) Approximately 14 and 30

DAP (Field) Rated on a 1-9 scale, where 1 =

excellent, 5 = average, and 9 = poor

vigor

Plant height (cm)

(VII.C.2.1.) Approximately 30 DAP and

within approximately 7 days

of harvest (Field) Distance from cotyledonary node (0

node) to the uppermost terminal bud

on 10 plants from two rows

Nodes above

white flower

(NAWF)

(VII.C.2.1.) Three weekly observations

starting approximately 7

days after first flower

(Field) Number of nodes from upper most

first-position white flower to the

terminal bud on 10 plants from two

rows

# Monsanto Company 12-CT-244U 139 of 620 

# Table VII-1. Phenotypic, Agronomi c, and Environmental Interaction

Characteristics Evaluated in United Stat es Field Trials and Laboratory Studies

(continued)

Data

Category Characteristics

Measured

(associated

section where

discussed) Evaluation timing (setting

of evaluation) Evaluation description

(measurement endpoints)

Reproductive

Development Seedcotton yield

(kg/ha)

(VII.C.2.1.) At harvest (Field) Hand harvested all seedcotton from

two rows

Seed index (g per

100 seed)

(VII.C.2.1.) Post harvest (Field) Mass of 100 ginned, fuzzy seed

Total seed per boll

(VII.C.2.1.) Post harvest (Field) Average number of seeds per boll

calculated from a 50-boll sample

Mature seed per

boll (VII.C.2.1.) Post harvest (Field) Average number of mature seed in a

boll calculated from a 50-boll

sample

Immature seed per

boll (VII.C.2.1.) Post harvest (Field) Average number of immature seed

in a boll calculated from a 50-boll

sample

Boll weight (g)

(VII.C.2.1.) Post harvest (Field) Average mass of a single boll

calculated from a 50-boll sample

Fiber micronaire

(mic units)

(VII.C.2.1.) Post harvest (Field) Measure of fiber fineness and

maturity (expressed in

dimensionless micronaire (mic)

units)

Fiber elongation

(%) (VII.C.2.1.) Post harvest (Field) Measure of the tensile-elastic

behavior of the fiber. It is a

measure of how much the fibers

stretch before they tear.

Fiber strength

(g/tex) (VII.C.2.1.) Post harvest (Field) Force in grams required to break a

bundle of fibers one tex unit in size.

One tex is the mass in grams of

1,000 meters of fiber.

Fiber length (cm)

(VII.C.2.1.) Post harvest (Field) Average length of the longer half of

combed fibers

Fiber uniformity

(%) (VII.C.2.1.) Post harvest (Field) Ratio between the mean length and

the longer half mean length of fibers

Pollen viability

(VII.C.3.) Flowering (laboratory) Percentage of viable pollen; viable

pollen stains purple due to the

presence of vital cytoplasmic

content

Pollen

morphology

(VII.C.3.) Flowering (laboratory) Diameter (μm) of viable pollen

grains and observations

# Monsanto Company 12-CT-244U 140 of 620 

# Table VII-1. Phenotypic, Agronomi c, and Environmental Interaction

Characteristics Evaluated in United Stat es Field Trials and Laboratory Studies

(continued)

Data

Category Characteristics

measured

(associated

section where

discussed) Evaluation timing (setting

of evaluation) Evaluation description

(measurement endpoints)

Plant Mapping

Characteristics Number of

mainstem nodes

per plant

(VII.C.2.2.) At harvest (Field) Number of mainstem nodes from

cotyledonary node (node 0) to

uppermost terminal meristem on 10

plants per plot

Number of nodes

to first fruiting

branch per plant

(VII.C.2.2.) At harvest (Field) Number of nodes from

cotyledonary node (node 0) up to

first fruiting branch on 10 plants per

plot

Number of firstposition bolls

(total, normal &

abnormal) per

plant

(VII.C.2.2.) At harvest (Field) Number of bolls at first position on

fruiting branches off of mainstem

on 10 plants per plot

Number of

vegetative bolls

per plant

(VII.C.2.2.) At harvest (Field) Number of vegetative bolls on 10

plants per plot

Total bolls per

plant (VII.C.2.2.) Post harvest (Field) Sum of first-position bolls, secondposition bolls, and vegetative bolls

per plant on 10 plants per plot

Retention of firstposition bolls (%)

(VII.C.2.2.) Post harvest (Field) Calculated first-position bolls

relative to number of fruiting

branches on the mainstem

First-position bolls

(%) (VII.C.2.2.) Post harvest (Field) Calculated first-position bolls per

plant relative to total bolls per plant

# Monsanto Company 12-CT-244U 141 of 620 

# Table VII-1. Phenotypic, Agronomi c, and Environmental Interaction

Characteristics Evaluated in United Stat es Field Trials and Laboratory Studies

(continued)

Data

Category Characteristics

measured

(associated

section where

discussed) Evaluation timing (setting

of evaluation) Evaluation description

(measurement endpoints)

Plantenvironmental

interactions Plant response to

abiotic stress

(VII.C.2.3.) 

# Four times per growing

season – approximately 30,

60, 90 and 120 DAP (Field) Qualitative assessment of each plot,

with rating on a 0- 9 scale, where 0

= no symptoms and 9 = severe

symptoms

Disease damage

(VII.C.2.3.) 

# Four times per growing

season – approximately 30,

60, 90 and 120 DAP (Field) Qualitative assessment of each plot,

with rating on a 0- 9 scale, where 0

= no symptoms and 9 = severe

symptoms

Arthropod-related

damage (VII.C.2.3.) 

# Four times per growing

season – approximately 30,

60, 90 and 120 DAP (Field) Qualitative assessment of each plot,

with rating on a 0- 9 scale, where 0

= no symptoms and 9 = severe

symptoms

Thrips damage

assessment

(VII.C.2.3.) 

# Three assessments at

approximately 14, 21 and 28

DAP (Field) Specific quantitative assessment of

thrips from 10 plants in each plot

using a 0-5 scale, where 0 = no

thrips or visible damage and 5 =

numerous thrips or severe damage

from thrips

Heliothine damage

assessment

(VII.C.2.3.) 

# Four assessments at

approximately 45, 60, 75

and 90 DAP (Field) Percent damage (number of

damaged fruiting bodies divided by

total number of fruiting bodies) and

number live larvae on the top 7

nodes of 10 plants in each plot

Arthropod

abundance

(VII.C.2.3.) 

# Four collections at

approximately 30, 60, 90

and 120 DAP (Field) Number of pest and beneficial

arthropods

1 

# All cottonseed was from mature open bolls.

2 

# Cotton plant growth stages were determined using descriptions and guidelines outlined in Cotton Growth

and Development (Ritchie et al., 2007).

3 

# Viability of hard and firm-swollen seed were determined by a tetrazolium test (AOSA, 2007).

# Monsanto Company 12-CT-244U 142 of 620 

# VII.B. Interpretation of Phenotypic and Environmental Interaction Data

Plant pest risk assessments for biotechnology-derived crops ar e comparative assessments.

Familiarity provides a basis from which th e potential environmental impact of a

biotechnology-derived plant can be evaluated. The concept of familiarity is based on the

fact that the biotechnology-derived plant is developed from a well-characterized

conventional plant variety. Familiarity considers the biology of the crop, the introduced

trait(s), the receiving environment and the in teraction of these factors, and provides a

basis for comparative environmental risk assessment between a biotechnology-derived

plant and its conventional counterpart.

Expert knowledge and experience with c onventionally bred cotton was the basis for

selecting appropriate endpoints and estimatin g the range of responses that would be

considered typical for cotton. As such, MON 88701 was compared to the conventional

control, Coker 130, in the asse ssment of measured characteri stics. An overview of the

characteristics assessed is presented in Table-VII-1. Evaluation of environmental

interaction characteristics ( e.g ., plant abiotic stress, plant- disease, and plant-arthropod

interactions) was also considered in the plan t pest assessment. Based on all of the data

collected, an assessment was made to dete rmine if MON 88701 is likely to pose an

increased plant pest risk compared to comme rcial cotton. Prior to analysis, the overall

dataset was evaluated for possible evidence of biologically releva nt changes and an

unexpected plant response. No unexpected observations or issues were identified.

VII.B.1. Interpretation of Detected Differences Criteria

Comparative plant characteriz ation data between a biotec hnology-derived crop and the

conventional control are interp reted in the contex t of contributions to increased plant

pest/weed potential as assessed by APHIS. Under the framew ork of familiarity,

characteristics for which no differences are de tected support a conclu sion of no increased

plant pest/weed potential of the biotechnology- derived crop compared to the conventional

crop. Characteristics for which differences ar e detected are considered in a step-wise

method (Figure VII-1) or in a similar fashion. All de tected differences for a

characteristic are considered in the cont ext of whether or not the difference would

increase the plant pest/weed potential of the biotechnology-derived crop. Ultimately, a

weight-of-evidence approach considering all characteristics and data is used for the

overall risk assessment of differences and their significance. In detail, Figure VII-1

illustrates the stepwise assessment process employed:

# Monsanto Company 12-CT-244U 143 of 620 

# Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data

Interpretation Methods

Note: A “no” answer at any step in dicates that the characteristic does not contribute to a biological or

environmental change for the crop in terms of plant pest/weed potential and subsequent steps are not

considered. If the answer is “yes” or “unc ertain” the subsequent step is considered.



# Steps 1 and 2 - Evaluate Detected St atistically Significant Differences

Data on each measured characteristic are sta tistically analyzed, where appropriate, within

each individual site and in a combined-site analysis, in which the data are pooled among

sites. All statistically significant differences are evaluated and considered in the context

of a change in plant pest/weed potential. Differences detected in individual site analyses

that are not detected when data across multip le environments are pooled in the combinedsite analysis are considered not biologically meaningful in terms of plant pest/weed

potential and, therefore, are not further consid ered in subsequent st eps. Any difference

detected in the combined-site analysis is further assessed.

Step 3 - Evaluate differences in the context of commercial reference varieties included

in the Study

If a difference for a characteristic is dete cted in the combined-site analysis across

multiple environments, then the mean value of the biotechnology-derived crop for the Hazard identification and

risk assessment on

difference Outside variation for crop? No

Yes

No

Yes 

# Adverse in terms of plant pest

potential? Yes 

# Outside variation of study references? No



# Not adverse; the direction

or magnitude of the

detected difference in the

measured characteristic

does not contribute to a

biological or

environmental change for

the crop in terms of plant

pest/weed potential Yes 

# Statistical differences detected

in combined-site analysis? No 

# Differences detected in the combined-site

and individual site analyses are evaluated

Step 2

Step 3

Step 4 

# The measured

characteristic does not

contribute to a biological

or environmental change

for the crop in terms of

plant pest/weed potential

Step 5

Step 6 Step 1

# Monsanto Company 12-CT-244U 144 of 620 

# characteristic is assessed relative to the ra nge of variation of th e commercial reference

varieties included in the study (e.g., reference range).

Step 4 - Evaluate Differences in the Context of the Crop

If the mean value of the ch aracteristics for a biotechnology-derived crop is outside the

variation of the commercial reference varie ties included in the study, the mean value of

the biotechnology-derived crop is assessed relative to known values common for the crop

( e.g. , published values).

Step 5 - Relevance of Differen ce to Plant Pest/Weed Potential

If the mean value of the ch aracteristics for a biotechnology-derived crop is outside the

range of values common for the crop, the detect ed difference for the characteristic is then

assessed for whether or not it is adverse in terms of plant pest/weed potential.

Step 6 - Conduct Risk Assessment on Identified Hazard

If an adverse effect (hazard) is identified, risk assessment on the difference is conducted.

The risk assessment considers contributions to enhanced plant pest/weed potential of the

crop itself, the impact of differences detect ed in other measured characteristics, and

potential for and effects of trait introgressi on into any populations growing outside of

cultivated environments or into a sexually-compatible species.

VII.B.1.1. Interpretation of Vigor and Environmental Interactions Data

For the qualitative assessments of vigor and abiotic stress response, disease damage, and

arthropod damage, the biotechnology-deri ved crop and conventional control were

considered different in plant re sponse ratings if the range of values or injury symptoms

did not overlap between the biotechnology-de rived crop and the conventional control

across all four replic ations. Any observed differences between the biotechnology-derived

crop and conventional control were assessed fo r biological significance in the context of

the range of the commercial reference varie ties, and consistency in other observation

times and sites. Differences that ar e not consistently observed at other

observations/collections and sites are consider ed not biologically meaningful in terms of

plant pest potential or an adverse environmental impact.

Quantitative assessments of arthropod damage we re analyzed within individual sites and

pooled across sites in a combined-site analys is. Statistically si gnificant differences

detected between the biot echnology-derived crop and c onventional control were

evaluated using the method outlined in Figure VII-1.

Quantitative assessments of arthropod abundan ce were only analyzed within each

individual site. Statistica lly significant differences be tween the biotechnology-derived

crop and conventional control were assessed fo r biological significance in the context of

the range of the commercial reference varieties, and for consistency with other collection

times and collection sites. Differences that are not consistently detected at other times

# Monsanto Company 12-CT-244U 145 of 620 

# and sites are considered not biol ogically meaningful in terms of plant pest potential or an

adverse environmental impact.

VII.C. Comparative Assessments of th e Phenotypic, Agronomic, Plant Mapping,

and Environmental Interaction Characteristics of MON 88701

This section provides the results of compar ative assessments condu cted in re plicated

laboratory and multi-site field experiments to provide a detailed phenotypic, agronomic,

plant mapping, and environmental inte raction description of MON 88701. The

MON 88701 characteristics evaluated in thes e assessments included: seed dormancy and

germination characteristics (Section VII.C .1.), plant phenotypic, plant mapping, and

environmental interaction obs ervations under field conditi ons (Section VII.C.2.), and

pollen characteristics (Section VII.C.3). Additional details for each assessment are

provided in Appendices F through H.

VII.C.1. Seed Dormancy and Germination Characteristics

USDA-APHIS considers the potential for weed iness to constitute a plant pest factor

(7 CFR § 340.6). Seed germination and dor mancy mechanisms vary among 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 weeds (Anderson,

1996; Lingenfelter and Hartwig, 20 07). Cotton does not exhibit significant levels of seed

dormancy as this characteristic has been removed through selection and conventional

breeding (Christiansen and Moore, 1959). To assess germination characteristics,

standardized germination assays are routinely used. The Association of Official Seed

Analysts (AOSA), an internationally recogni zed seed testing organization, recommends a

temperature range of alternating 20/30°C as optimal for testing the germination

characteristics of cottonseed (AOS A, 2007; 2010a; 2010b; AOSA/SCST, 2010).

Comparative assessments of seed dormancy and germination characteristics were

conducted on MON 88701 and the conventional co ntrol. In addition, nine unique

commercial reference varieties were included to provide a range of comparative values

that are representative of existing variability in commercial cotton varieties. The seed

lots for MON 88701, the conventional control, and the commercial reference varieties

were produced in three replicated field tr ials during 2010 located in Arkansas (ARPR),

North Carolina (NCME), and Texas (TXPL). These geographic areas represent

environmentally relevant conditions for co tton production. In a ddition to the AOSA

recommended temperature range of 20/30°C, seed was tested at five additional

temperature regimes of 10, 20, 30, alternati ng 10/20, and alternating 10/30°C to assess

seed germination properties. The details of the materials, experimental methods, and

germination data from all of the individual production sites are presented in Appendix F.

In the combined-site analysis, in which the data were pooled from the three individual

sites, no statistically significant differences (5% level of significance) were detected

between MON 88701 and the conventional control for any characteristic at the AOSA

temperature regime (alternating 20 o 

# C/30 o 

# C), or at the temperature regimes of 10 o 

# C, 20 o 

# C,

alternating 10 o 

# C/20 o 

# C, or alternating 10 o 

# C/30 o 

# C (Table VII-2). MON 88701 had a

# Monsanto Company 12-CT-244U 146 of 620 

# significantly higher percentage of germinated seed (96.7 vs. 94.4, respectively) and lower

percent dead seed (3.3 vs. 5.6, respective ly) than the conven tional control at 30 o 

# C. These

differences were small in magnitude, not obs erved at other temper atures and the mean

values of percent germinated and dead seed for MON 88701 were within the range of

commercial reference varieties. Therefore, the differences in percent germinated and

dead seed at 30 o 

# C are not considered to be biological ly meaningful in terms of altered

dormancy or germination characteristics (See Figure VII- 1 Step 3, answer “no”).

The dormancy and germination characteristics evaluated were used to assess MON 88701

in the context of plant pest risk. The results of this assessment, particularly the fact that

no hard seed were observed at any temperat ure, support the conclusion that there are no

seed germination characteristic differen ces between MON 88701 and the conventional

control. Thus, the introducti on of the dicamba and glufosinat e-tolerant traits into cotton

is not likely to result in increased plant pest potential, increased weediness, or an altered

environmental impact from MON 88701 compar ed to commercially cultivated cotton.

# Monsanto Company 12-CT-244U 147 of 620 

# Table VII-2. Combined-Site Comparison of MON 88701 to Conventional Control

for Germination Characteristics

Temperature

Regime Germination

Category Mean % (S.E.) 1



# MON 88701 2 

# Control Reference Range (%) 3



# 10 °C Germinated 32.8 (6.3) 34.8 (4.7) 14.2 - 58.0

Viable Hard 0.0 (0.0) 0.3 (0.2) 0.2 - 23.8

Dead 22.8 (3.8) 22.1 (3.3) 7.7 - 22.8

Viable Firm-Swollen 44.3 (7.3) 42.8 (5.9) 29.0 - 66.5

20 °C Germinated 95.7 (0.7) 95.3 (1.2) 88.5 - 97.8

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 - 4.0

Dead 4.3 (0.7) 4.7 (1.2) 2.0 - 9.0

Viable Firm-Swollen 0.0 (0.0) 0.0 (0.0) 0.0 - 2.0

30 °C Germinated 96.7 (0.8)* 94.4 (1.2) 90.5 - 97.8

Viable Hard † 

# 0.0 (0.0) 0.0 (0.0) 0.0 - 0.0

Dead 3.3 (0.8)* 5.6 (1.2) 2.3 - 9.5

Viable Firm-Swollen † 

# 0.0 (0.0) 0.0 (0.0) 0.0 - 0.0

10/20 °C Germinated 94.4 (1.3) 92.0 (1.4) 64.3 - 91.3

Viable Hard 0.0 (0.0) 0.1 (0.1) 0.1 - 17.5

Dead 4.3 (0.7) 6.6 (1.2) 5.0 - 10.3

Viable Firm-Swollen 1.3 (1.0) 1.3 (0.6) 0.0 - 21.3

10/30 °C Germinated 95.7 (1.0) 95.1 (1.5) 90.8 - 95.5

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 - 2.0

Dead 4.3 (1.0) 4.9 (1.5) 4.3 - 7.8

Viable Firm-Swollen 0.0 (0.0) 0.0 (0.0) 0.0 - 1.5

20/30 °C Normal Germinated 89.6 (1.9) 88.0 (2.9) 80.8 - 92.8

(AOSA) 4 

# Abnormal Germinated 4.8 (1.2) 6.0 (1.3) 2.0 - 6.3

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 - 4.8

Dead 5.6 (0.9) 6.0 (2.0) 4.0 - 10.8

Viable Firm-Swollen 0.1 (0.1) 0.0 (0.0) 0.0 - 3.8

Note: The experimental design was a split-plot with four replications (n = 12) and statistical analysis

consisted of an analysis of variance (ANOVA) model.

*Statistically significant differences detected ( α =0.05) between MON 88701 and the conventional control.

1 

# S.E. = Standard Error.

2 

# In some instances, the total percentage of MON 88701 did not equal 100% due to numerical rounding of

the means.

3 

# Minimum and maximum means determined from among the commercial reference varieties.

4 

# AOSA recommended.

†No statistical comparison could be made due to lack of variability in the data.

# Monsanto Company 12-CT-244U 148 of 620 

# VII.C.2. Field Phenotypic, Agronomi c, Plant Mapping, and Environmental

Interactions Characteristics

Phenotypic, agronomic, and plant mapping characteristics, and environmental

interactions were evaluated under field conditions as part of the plant characterization

assessment of MON 88701. These data were developed to provide USDA-APHIS with a

detailed description of MON 88701 relative to the conventional control and commercial

reference varieties. According to 7 CF R § 340.6, as part of th e petition to seek

deregulation, a petitioner must submit “a deta iled description of the phenotype of the

regulated article.” This information is being provided to assess whether there are

phenotypic differences between MON 88701 a nd the conventional control that may

impact its plant pest/weed poten tial. Environmental interactions were also assessed as an

indirect indicator of ph enotypic changes to MON 88701 compared to the same

comparators described above and are also c onsidered in the plant pest assessment.

The results of the asse ssment of agronomic, phenotypic, and plant mapping

characteristics demonstrated that the in troduction of the dicamba and glufosinatetolerance traits did no t meaningfully alter the weedine ss of MON 88701 compared to the

conventional control. Furtherm ore, the lack of meaningful differences in plant response

to abiotic stress, disease da mage, arthropod-related damage , and pest- and beneficialarthropod abundance also support the conclusion that the in troduction of the dicamba and

glufosinate-tolerance traits are not likely to result in increased plant pest potential,

increased weediness, or an adverse envir onmental impact from MON 88701 compared to

the conventional control.

VII.C.2.1. Field Phenotypic and Agronomic Characteristics

Two field studies were c onducted during 2010 to evalua te phenotypic and agronomic

characteristics of MON 88701 compared to the conventional control. One study was

designed to collect phenotypic and environmen tal interaction data, while the other was

designed for the collection of plant mapping data and tissue samples for expression and

compositional analyses. Field sites in both st udies were planted in randomized complete

block designs with four replicates per site. The sites were selected to provide a diverse

range of environmental and agronomic conditi ons representative of commercial cotton

production areas in North America (Table VII-3). All plots of MON 88701, the

conventional control, and the commercial refe rence varieties at each site were uniformly

managed in order to assess whether the in troduction of the dicamba and glufosinatetolerance traits altered th e phenotypic and agronomic characteristics of MON 88701

compared to the conventional control. Bo th studies included MON 88701 that was not

treated with dicamba or glufosinate herbicides to assess the effects of the traits on the

plant.

Study 1 was conducted at 15 sites in the U.S. (Table VII-3). MON 88701, the

conventional control, and four commercial reference varieties (three conventional

reference varieties and one glyphosate-tolerant reference variety) were evaluated at each

# Monsanto Company 12-CT-244U 149 of 620 

# site. Across sites, a total of 11 commerci al reference varieties (Table G-1) were

evaluated.

An additional study, Study 2, was conducted at 11 sites in the U.S. (Table VII-3).

MON 88701, the conventional control, and four conventional reference varieties were

evaluated at each site. Across sites a total of eight unique commercia l reference varieties

were evaluated. This study was designed for co llection of plant mapping data, as well as,

the production of tissues for the expression and compositiona l analyses discussed above

in Sections V.C. and VI, respectively. St udy 2 generated plant mapping information and

data across test locations treated and not treated with dicamba or glufosinate herbicides in

Study 2 (See section VII.C.2.2, C.2.3.2 and G.12.3), allowing for assessment of

MON 88701 under the agronomic system that it is expected to be used.

Results from Study 1 and Study 2 are pr esented in the following sections:

Table VII-3. Study 1 and Study 2 Data Location Summary

Data/Results 1 

# Study 1 Study 2 Petition Location

Not treated phenotypic characteristics   Section VII.C.2.1

Not treated plant mapping  Section VII.C.2.2

Not treated environmental interactions   Section VII.C.2.3

Treated phenotypic characteristics  Appendix G.12.3

Treated plant mapping  Appendix G.13.2

Treated environmental characteristics

through plant mapping  Section VII.C.2.3;

Appendix G.13.2

1 

# Not treated = not treated with dicamba or glufosin ate herbicides; treated = treated with dicamba and

glufosinate herbicides.



# All plant, seed, and fiber characteristic data , except for plant vigor (qualitative data),

were statistically analyzed within each site ( i.e ., individual site analysis) and in a

combined-site analysis in which the data were pooled across all sites within a study. The

reference range was determined from the mi nimum and maximum mean values from the

commercial reference varieties to provide pheno typic characteristic values representative

of commercial cotton varieties.

For the assessment of plant vigor MON 88701 and the conventional control were

considered different in plan t response rating if the range of values did not overlap

between the MON 88701 and the conventional cont rol across all four plot replications.

Any observed differences between MON 88701 and the conventional control were

assessed for biological significance in the context of the range of the commercial

reference varieties, and for consistency in ot her observations and sites. Differences that

# Monsanto Company 12-CT-244U 150 of 620 

# are not consistently observed at other observations and sites are considered not

biologically meaningful in te rms of plant pest potential or an adverse environmental

impact.

A description of the evaluated phenotypi c characteristics and the designated

developmental stages when evaluations occurr ed are listed in Table VII-1. The results

from Study 1 and 2 from combined-site anal yses of MON 88701 plot s not treated with

either dicamba or glufosinate compared to the conventional control are presented in the

following sub-sections. The re sults of these studies demons trate that the introduction of

the dicamba and glufosinate-tolerance tra its did not alter MON 88701 compared to the

conventional control in terms of weediness. The individual site data comparisons and

methods and detailed results of the suppl emental evaluations of MON 88701 plots that

were treated with dicamba and glufosinate in Study 2 are presente d and discussed in

Appendix G.

# Monsanto Company 12-CT-244U 151 of 620 

# Table VII-4. Field Phenotypic E valuation Sites for MON 88701 during 2010

Study 1 Locations 1 

# Study 2 Locations 2



# County, State Site Code County, State Site Code

Jackson, AR ARAU Crittenden, AR ARPR

Crittenden, AR ARPR Desha, AR ARTI

Desha, AR ARTI Tift, GA GACH

Tift, GA GACH Twiggs, GA GAJE

Twiggs, GA GAJE Pawnee, KS KSLA

Pawnee, KS KSLA Rapides, LA LACH

Rapides, LA LABU Perquimans, NC NCBD

Rapides, LA LACH Caswell, NC NCME

Perquimans, NC NCBD Dona Ana, NM NMLC

Caswell, NC NCME Barnwell, SC SCEK

Dona Ana, NM NMGA Hale, TX TXPL

Dona Ana, NM NMLC

Barnwell, SC SCEK

Hale, TX TXPL

San Patricio, TX TXPO

Note: Field trials at all sites were condu cted under USDA Notification number 10-071-101n.

1 

# MON 88701 was not treated with dicamba or glufosinate herbicides in Study 1.

2 

# Study 2 included plots not treated with dicamba or glufosinate herbicides and plots treated with

dicamba and glufosinate herbicides.



# VII.C.2.1.1. Field Phenotypic and Agro nomic Characteristics of MON 88701 –

Study 1

Vigor ratings were collected from each plot using a 1-9 scale, where 1 is outstanding

plant vigor and 9 is poorest plant vigor. Since vigor data are categoric al (qualitative), the

data were not statistically analyzed. There were no differences observed between

MON 88701 and the conventional c ontrol in plant vigor (Table G-7) at 14 and 30 days

after planting (DAP) for 29 of 30 comparisons fr om all sites. At ARPR at 30 days after

planting, MON 88701 had lower plant vigor than the conventional control (ranges of 4.0-

4.0 vs. 2.0-3.0, respectively), but was within the range of the commercial reference

varieties. Since only one difference (out of 30) was identified and it fell within the

reference range, MON 88701 is unlikely to have increased plant pest/w eed potential or an

adverse environmental impact compared to the conventional control (See Section

VII.B.1.1.).

# Monsanto Company 12-CT-244U 152 of 620 

# In the combined-site analysis (Table VII-5), no statistically significant differences were

detected between MON 88701 and the conventiona l control for stand count at 14 and 30

DAP, final stand count, number of nodes above white flower at obs ervation 1, seedcotton

yield, number of immature seed per boll, weight per boll, fiber micronaire, fiber

elongation, fiber uniformity, and fiber length. The following statistically significant

differences were detected in the combined-s ite analysis. MON 88701 plants were shorter

at 30 DAP (18.3 vs. 19.7 cm) and at harves t (109.8 vs. 116.4 cm), had increased nodes

above white flower at observation 2 (6.0 vs. 5.7) and observation 3 (4.9 vs. 4.6), a

decreased seed index (9.8 vs. 10.5 g per 100 fu zzy seed), increased total seed per boll

(29.0 vs. 27.4), increased mature seeds pe r boll (22.6 vs. 19.7), and increased fiber

strength (31.8 vs. 31.0 g/tex) compared to th e conventional control. However, the mean

values of MON 88701 were within the range of the commercial reference varieties for the

eight characteristics listed above. Thus, MON 88701 is unli kely to have increased plant

pest/weed potential or an adverse environmental impact compared to the conventional

control. (See Figure VII-1, Step 3, answer “no”).Monsanto Company 12-CT-244U 153 of 620Table VII-5. Study 1 Combined-Site Comparison of MON 88701to Conventional Control during 2010 for Phenotypic andAgronomic CharacteristicsPhenotypic Characteristic (units)MON 88701ControlReference Range1Mean (SE)2Mean (SE)MinimumMaximumStand Count at 14 DAP3(# in 2 rows per plot)146.0 (4.3)152.4 (4.2)96.2143.5Stand Count at 30 DAP (# in 2 rows per plot)131.8 (5.5)137.7 (5.5)86.7140.8Final Stand Count at harvest (# in 2 rows per plot)125.2 (5.9)128.9 (6.0)88.2131.4Plant Height at 30 DAP (cm)18.3 (1.2)*19.7 (1.2)8.323.3Plant Height at harvest (cm)109.8 (3.8)*116.4 (4.2)84.4131.3Nodes Above White Flower: (# of nodes at observation 1)6.9 (0.2)6.7 (0.2)5.88.6(# of nodes at observation 2)6.0 (0.2)*5.7 (0.2)5.16.9(# of nodes at observation 3)4.9 (0.3)*4.6 (0.3)3.75.7Seedcotton Yield (kg/ha)2937.8 (153.7)2869.9 (156.0)2107.03636.5Seed Index (g per 100 fuzzy seed)9.8 (0.2)*10.5 (0.1)8.911.8Total Seed per Boll (# per boll)29.0 (0.4)*27.4 (0.3)26.430.6Mature Seed per Boll (# per boll)22.6 (0.7)*19.7 (0.6)11.827.2Immature Seed per Boll (# per boll)6.4 (0.5)7.7 (0.5)3.416.0Weight per Boll (g)4.8 (0.1)4.8 (0.1)4.26.0Fiber Micronaire (mic units)44.6 (0.1)4.5 (0.1)4.05.0Fiber Elongation (%)6.0 (0.1)6.0 (0.1)4.88.0Fiber Strength (g/tex)31.8 (0.2)*31.0 (0.1)30.734.5Fiber Uniformity (%)84.0 (0.1)83.7 (0.1)83.784.8Fiber Length (cm)2.8 (0.0)2.9 (0.0)2.83.1* Indicates a statisticallysignificant difference (α=0.05) between MON 88701 and the conventional control (n = 60).1Reference range = Minimum and maximum mean values across all 15sites and eleven referencesfrom the Study 1 field trial.2SE = standard error.3DAP = days after planting.4Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).

# Monsanto Company 12-CT-244U 154 of 620 

# VII.C.2.1.2. Field Phenotypic and Agro nomic Characteristics of MON 88701 –

Study 2

Vigor ratings were collected from each plot using a 1-9 scale, where 1 is outstanding

plant vigor and 9 is poorest plant vigor. Due to the non-specific nature of the scale used,

the data were not statistica lly analyzed. There were no differences between MON 88701

and the conventional control in plant vigor (Table G-10) at 14 and 30 DAP for 22 of 22

comparisons from all sites. Therefore, the lack of differences in plant vigor supports a

conclusion that MON 88701 is unlikely to have increased plant pest/w eed potential or an

adverse environmental impact compared to the conventional control (See Section

VII.B.1.1.).

In the combined-site analysis of MON 88701 (Table VII-6), no statistically significant

differences were detected between MON 88701 and the conventional control for stand

count at 14 and 30 DAP, final stand count at harvest, nodes above white flower

observations 1 and 3, seedcotton yield, immature seed per boll, weight per boll, fiber

micronaire, fiber elongation, fiber uniform ity, and fiber length. The following

statistically significant differences were detected in the combined-site analysis.

MON 88701 plants were shorter than the c onventional control at the 30 DAP (18.0 vs.

19.2 cm) and at harvest (96.1 vs. 105.0 cm), ha d increased nodes above white flower at

observation 2 (5.5 vs. 5.2), ha d a decreased seed index ( 9.4 vs. 10.7 g/100 seed), had

increased total seed per boll (29.1 vs. 27.0 seed ) and increased mature seed per boll (23.3

vs. 20.1), and had increased fiber strength as compared to the conventional control (30.9

vs. 30.2 g/tex). However, the mean values of MON 88701 were within the reference

range for the seven characteris tics listed above. Thus, MON 88701 is unlikely to have

increased plant pest/weed potential or an a dverse environmental impact compared to the

conventional control. (See Figur e VII-1, Step 3, answer “no”).

Results of the supplemental evaluation of MON 88701, described above, under the

agronomic system in which it is expected to be used ( i.e. , MON 88701 treated with

dicamba and glufosinate herbic ides) are provided in Appendi x G and further demonstrate

that MON 88701 is unlikely to have increased plant pest/weed potential or an adverse

environmental impact compared to the conventional c ontrol (See G.12.3).

VII.C.2.1.3. Field Phenotypic and Agro nomic Characteristics – Conclusion

The results of the agronomic and phenot ypic assessments on MON 88701 from Study 1

and Study 2 demonstrate that the introduction of the dicamba and glufosinate-tolerant

traits did not alter MON 88701 compared to the conventional control relating to plant

pest/weed potential. Additionally, ag ronomic and phenotypic assessments of

MON 88701 treated with dicamba and glufosinate herbicides we re also comparable to the

conventional control. Thus, the introduction of the dicamba and glufosinate-tolerant

traits into cotton is not likely to result in increased plant pest potential, increased

weediness or an altered environmental impact from MON 88701 compared to

commercially cultivated cotton.Monsanto Company 12-CT-244U 155 of 620Table VII-6. Study 2 Combined-Site Comparison of MON 88701to Conventional Control during 2010 for Phenotypic andAgronomic CharacteristicsPhenotypic Characteristic (units)MON 887011ControlReference Range2Mean (SE)3Mean (SE)MinimumMaximumStand Count at 14 DAP4(# in 2 rows per plot)150.5 (4.2)155.0 (4.4)108.4135.8Stand Count at 30 DAP (# in 2 rows per plot)149.4 (3.9)152.8 (4.0)105.8134.1Final Stand Count at harvest (# in 2 rows per plot)146.3 (4.0)150.5 (4.3)110.5137.7Plant Height at 30 DAP (cm)18.0 (1.1)*19.2 (1.1)11.420.7Plant Height at harvest (cm)96.1 (4.2)*105.0 (4.9)85.2121.9Nodes Above White Flower: (# of nodes at observation 1)6.6 (0.2)6.4 (0.2)6.07.3(# of nodes at observation 2)5.5 (0.3)*5.2 (0.3)4.85.7(# of nodes at observation 3)4.1 (0.2)3.8 (0.2)3.24.6Seedcotton Yield (kg/ha)3,334.1 (210.2)3,164.1 (210.8)2,181.73,970.8Seed Index (g per 100 fuzzy seed)9.4 (0.2)*10.7 (0.2)9.412.4Total Seed per Boll (# per boll)29.1 (0.4)*27.0 (0.4)26.130.7Mature Seed per Boll (# per boll)23.3 (0.7)*20.1 (0.8)14.627.0Immature Seed per Boll (# per boll)5.8 (0.6)6.9 (0.6)2.714.4Weight per Boll (g)4.9 (0.1)4.8 (0.1)4.55.9Fiber Micronaire (mic units)54.7 (0.1)4.6 (0.1)4.25.0Fiber Elongation (%)6.1 (0.1)6.2 (0.1)5.68.1Fiber Strength (g/tex)30.9 (0.2)*30.2 (0.2)30.734.0Fiber Uniformity (%)83.6 (0.2)83.4 (0.2)82.884.3Fiber Length (cm)2.8 (0.0)2.8 (0.0)2.73.1* Indicates a statisticallysignificant difference (α=0.05) between MON 88701 and the conventional control (n = 44).1MON 88701 plots were not treatedwith dicamba or glufosinate.2Reference range = Minimum and maximum mean values across all 11 sites and eight references from the Study 2 field trial.3SE = standard error.4DAP = days after planting.5Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).

# Monsanto Company 12-CT-244U 156 of 620 

# VII.C.2.2. Plant Mapping Characteristics

Plant mapping is a process commonly used by agronomists and breeders to quantify

growth and development parameters of a cott on plant, including boll retention (Kerby et

al., 2010; Plant and Kerby, 1995). Plant mappi ng parameters are used to measure crop

productivity and are influenced by abiotic and biotic stressors. Plant mapping

characteristics (Table VII-7) were evaluated under field conditions to provide USDAAPHIS with a detailed description of MON 88701 boll retention and distribution relative

to the conventional control and commercial reference varieties, and to consider

differences in context of pest/weed potential.

In addition to the methods discussed in S ection VII.C.2.1, 10 plants from each plot in

Study 2 were mapped at harvest for the numbe r of mainstem nodes, number of nodes to

the first fruiting branch, total number of bo lls (sum of first-position, second-position and

vegetative bolls), total number of first-pos ition bolls, and total number of vegetative

bolls. The percent of first-po sition bolls relative to total bolls and percent retention of

first-position bolls on mainstem fruiting bran ches were calculated from plant mapping

data. The combined-site statistical anal ysis comparing MON 88701 not treated with

either dicamba or glufosinate to the conven tional control is summarized below. Results

of the individual site data comparisons are presented in Appendix G.13.1. Also the

experimental methods and detailed results from the supplemental analyses comparing

MON 88701 treated with dicamba and glufosinat e herbicides to the conventional control

are presented and discussed in Appendix G.13.2.

In the combined-site analysis of plant mappi ng parameters (Table VII-7), no statistically

significant differences were detected be tween MON 88701 and the conventional control

for number of mainstem nodes per plant, num ber of nodes to first fruiting branch, total

number of bolls per plant, ve getative bolls per plant, percen t retention of first-position

bolls and percent first-position bolls (relative to total bolls). The mean value for firstposition bolls per plant was higher in MON 887 01 than the conventional control (5.2 vs.

4.6) (Table VII-7). However, the mean value for first-position bolls per plant was within

the reference range. Furthermore, similar results of the plant mapping evaluation of

dicamba and glufosinate-treated MON 88701, the agronomic system in which

MON 88701 is expected to be used, were observed (See Ap pendix G.13.2; Table G-18).

Thus, MON 88701 is unlikely to ha ve increased plant pest/weed potential or an adverse

environmental impact compared to the conventional control. (See Figure VII-1, Step 3,

answer “no”).Monsanto Company 12-CT-244U 157 of 620Table VII-7. Study 2 Combined-Site Comparisonof MON 88701 to Conventional Control during 2010forPlant MappingCharacteristicsPhenotypic Characteristic (units)MON 887011ControlReference Range2Mean (SE)3Mean (SE)MinimumMaximumMainstem Nodes (# per plant)18.1 (0.4)18.2 (0.4)16.021.6Nodes to First Fruiting Branch (# per plant)5.2 (0.2)5.5 (0.2)4.27.6Total Bolls4(# per plant)9.8 (0.6)9.0 (0.7)8.613.4Total First-Position Bolls (# per plant)5.2 (0.3)*4.6 (0.3)2.96.3Total Vegetative Bolls (# per plant)2.0 (0.6)1.9 (0.6)0.75.0% Retention of First-Position Bolls (per plant)42.1 (2.5)38.6 (2.6)21.253.5% First-Position Bolls relative to total bolls (per plant)57.5 (2.2)56.5 (2.1)36.059.6* Indicates a statisticallysignificant difference (α=0.05) between MON 88701 and the conventional control (n = 44).1MON 88701 plots were not treatedwith dicamba or glufosinate.2Reference range = Minimum and maximum mean values among eight conventional commercial reference varieties.3SE = standard error.4Total Bolls = number of first-position bolls + numberof second-position bolls + number of vegetative bolls.

# Monsanto Company 12-CT-244U 158 of 620 

# VII.C.2.3. Environmental Interaction Characteristics

USDA-APHIS considers the environmental interactions of the biotechnology-derived

crop compared to its conventional counterpart to determine the potential for increased

plant pest characteristics. Evaluations of environmental in teractions were conducted as

part of the plant characteri zation for MON 88701. In the 201 0, US field trials conducted

for evaluation of phenotypic and agronomic characteristics of MON 88701, data were

also collected on plant response to abiotic stress ( i.e. , drought, wind, nutrient deficiency,

etc.), disease damage, arthropod-related da mage, and arthropod abundance (Appendix G;

Tables G-20 through G-29). These data were us ed as part of the environmental analysis

(Section IX) to assess plant pest potential an d provide an indication of potential effects of

MON 88701 on non-target organisms (NTOs) comp ared to the conventio nal control. The

results of the field evaluations showed that the dicamba and glufosinate-tolerance traits

did not unexpectedly alter the assessed e nvironmental interactions of MON 88701

compared to the conventional control. The lack of significant biologically meaningful

differences in plant response to abiotic stre ss, disease damage, arthropod-related damage,

and pest- and beneficial-arthropod abunda nce supports the conclusion that the

introduction of the dicamba and glufosinate-tolerance traits are unlikely to result in

increased plant pest potential or an a ltered environmental impact from MON 88701

compared to commercial cotton.

VII.C.2.3.1. Study 1 Environmental Interactions of MON 88701

MON 88701 was compared to the conventional control for qualitative and quantitative

environmental interactions in Study 1 (See S ection VII.C.2.1.). Qualitative assessments

were conducted at 15 sites and included pl ant response to abiotic stressors, disease

damage, and arthropod damage. The assessme nts were conducted four times during the

growing season on all plots (4 time points x 4 plot replications = 16 data points per

assessment). The first assessment was made at approximately 30 days after planting

and the three subsequent assessments at approximately 30 day intervals thereafter.

Plant response to abiotic stressors, disease damage, and arthropod damage were assessed

at natural levels (no artificial infestation or imposed abiotic stress); therefore these levels

typically varied between observations at a site and among sites. Plant response to abiotic

stress, and disease damage a nd arthropod damage data were collected from each plot

using a 0 – 9 scale of increasi ng severity of observed damage for each stressor. This

scale was utilized to allow for the evaluatio n of the wide variety of potential abiotic

stressor, disease damage, and arthropod damage symptoms potentially occurring across

the season and across sites. Due to the non-sp ecific nature of the scale used, the data

were not statistically analyzed but rather were placed into one of the following

categories: none (0), slight (1-3), moderate (4-6), or severe (7-9). MON 88701 and

conventional control cotton were considered diff erent in plant response to stressors if the

range of injury symptoms across all four replications did not overlap between

MON 88701 and the conventional control. Any observed differe nces between the

MON 88701 and conventional control were as sessed for biological significance in the

context of the range of the commercial refere nce varieties, and for consistency in other

observations and sites.

# Monsanto Company 12-CT-244U 159 of 620 

# In addition to the qualitative assessment, quantitative arthropod assessments were

conducted at five sites and included thrips damage, heliothine damage, and pest- and

beneficial-arthropod abundance. Thrips dama ge was assessed three times (approximately

14, 21, and 28 DAP) during the growing season, heliothine damage was assessed four

times (approximately 45, 60, 75, and 90 DAP) du ring the growing season, and arthropod

abundance was assessed from collections performed four times (approximately 30, 60,

90, and 120 DAP) during the growing season.

Thrips damage was quantitativ ely assessed in each plot from 10 randomly selected plants

using the arthropod-specific 0–5 rating scale of increasing severity. Heliothine damage

was assessed quantitatively by recording the to tal number of fruiting bodies (flower buds,

flowers, and bolls), number of damaged fruiti ng bodies and number of live larvae on the

top 7 nodes from 10 randomly selected plants of each plot. These numerical data along

with the quantitative arthropod abundance data were subj ected to statistical analysis.

VII.C.2.3.1.1. Qualitative Asse ssment Results - Study 1

In an individual site assessment of qualita tive data (Tables V II-8, G-20 through G-22),

no differences were observed between MON 88701 and the conventional control for any

of the 169 comparisons for plant response to abiotic stressors, including compaction,

drought/dry, flood, hail, heat, nutrient defici ency, wet soil/ excess precipitation, and

wind damage. Also, no differences were observed between MON 88701 and the

conventional control for any of the 170 comp arisons for the assessed diseases, including

anthracnose, Ascochyta leafblight, bacterial b light, boll rot, cotton l eaf rust, damping off,

Fusarium wilt, leaf spots, Pythium , reniform nematode, Rhizoctonia , root-knot

nematode, Thielaviopsis , and Verticillium wilt. Finally, no diffe rences were observed

between MON 88701 and the conventional cont rol for any of the 159 comparisons for

the assessed arthropods, including aphids, b eet armyworms, cut worms, fall armyworms,

fleahoppers, grasshoppers, heliothines, sout hern corn rootworm beetles, soybean

loopers, spider mites, stink bugs, tarnished pl ant bugs, thrips, and whiteflies. Since no

differences were observed between MON 88701 and the conventional control for plant

response to abiotic stressors, disease dama ge, and arthropod-related damage in multiple

environments, the assessed results support the conclusion that M ON 88701 is unlikely to

have increased plant pest/weed potential or an adverse environmental impact compared

to the conventional contro l (See Section VII.B.1.1.).

# Monsanto Company 12-CT-244U 160 of 620 

# Table VII-8. Study 1 Summary of Qualitative Environmental Interactions

Assessments Including MON 88701 Response to Abiotic Stress, Disease, and

Arthropod Damage during 2010

Stressor Number of

Observations

Across All Sites Number of Observations

with No Observed

Differences Between

MON 88701 and the

Conventional Control

Abiotic stressors 169 169

Disease damage 170 170

Arthropod-related damage 159 159

Total 498 498

Note: The experimental design was a randomized comp lete block with four replications (n = 60).



# VII.C.2.3.1.2. Quantitative Assessment Results - Study 1

In the combined-site analysis of thrips damage data (Table VII-9), no statistically

significant differences were detected betw een MON 88701 and the conventional control.

There were no biological differe nces in thrips damage that would contribute to increased

pest potential of MON 88701 compared to the conventional control (See Section

VII.B.1.1.).

Table VII-9. Study 1 Combined-Site Comparison of MON 88701 to Conventional

Control during 2010 for Assessment of Thrips Damage

Observation MON 88701 (SE) 1 

# Control (SE) Reference range

1 0.5 (0.1) 0.4 (0.1) 0.0 – 1.2

2 0.1 (0.0) 0.1 (0.0) 0.0 – 0.2

3 0.0 (0.0) 0.1 (0.0) 0.0 – 0.5

Note: The experimental design was a randomized co mplete block design with four replications (n

= 20). No statistically significant differences ( α =0.05) were detected between MON 88701 and

the conventional control.

1 

# SE= standard error.



# In the combined-site analysis of heliothine damage data (Table VII-10), no statistically

significant differences were detected be tween MON 88701 and the conventional control

for percent damaged fruiting bodies and the nu mber of live larvae. Thus, there is no

biological difference in heliothine damage th at would contribute to increased pest

potential of MON 88701 compared to the c onventional control (S ee Section VII.B.1.1.).

# Monsanto Company 12-CT-244U 161 of 620 

# Table VII-10. Study 1 Combined-Site Co mparison of MON 88701 to Conventional

Control during 2010 for Quantitative A ssessment of Heliothine Damage

Percent Damaged Fruiting Bodies # of Live Larvae 1



# Observation MON 88701

(SE) 2 

# Control

(SE) Reference

Range MON 88701

(SE) Control

(SE) Reference

Range

1 2.8 (0.9) 1.8 (0.8) 0.0 – 8.7 0.1 (0.1) 0.0 (0.0) 0.0 – 0.2

2 5.3 (3.0) 6.3 (2.8) 1.2 – 28.1 0.2 (0.1) 0.1 (0.0) 0.0 – 0.5

3 3.7 (0.7) 2.6 (0.5) 1.2 – 5.2 0.1 (0.0) 0.1 (0.0) 0.0 – 0.1

4 6.3 (1.9) 6.9 (1.8) 2.6 – 12.1 0.1 (0.0) 0.1 (0.0) 0.0 – 0.3

Note: No statistically significant differences ( α =0.05) were detected between MON 88701 and

conventional control (n = 20).

1 

# Number of immature heliothines.

2 

# SE = standard error.



# For arthropod abundance, a total of 178 comparisons we re made between MON 88701

and the conventional control for the followi ng pest- and beneficial-arthropods: aphids,

cabbage loopers, fall armyworms, fleahoppers , heliothines, southern armyworms, stink

bugs, tarnished plant bugs, thrips, white fl ies, big eyed bugs, braconids, lacewings,

ladybird beetles, Damsel bugs, Orius spp., and spiders (Araneae) (Tables G-25 and G26). No statistically significant differenc es were detected between MON 88701 and the

conventional control for 173 out of 178 comp arisons, including 89 pest arthropod

comparisons and 89 beneficial arthropod comparisons.

The five differences detected between MON 88701 and the conventional control included

two differences for pest arthropods and th ree differences for beneficial arthropods

(Tables G-25 and G-26). In the pest arthropod assessment, MON 88701 had lower

abundance than the conventional control for stink bugs (0.3 vs. 1.8 per plot) and for

tarnished plant bugs (0.5 vs. 2.0 per plot) in Co llection 4 at the LABU site. For tarnished

plant bugs, the mean abundance value for M ON 88701 was within the reference range.

For stink bugs, the mean abundance value for MON 88701 was outside the reference

range. However, the statistical differences detected in stink bugs abundance were not

consistent across collections or sites (Table G-25).

In the beneficial arthropod assessment, MON 88701 had increased abundance compared

to the conventional control (Table G-26) fo r Damsel bugs in Collection 2 at the GACH

site (6.0 vs. 2.3). MON 88701 had lower abu ndance than the conventional control for

Orius spp. in Collection 2 (0.0 vs. 1.5 per plot ) and collection 3 (0.5 vs. 2.8 per plot) at

the ARAU site. The mean abundance value for MON 88701 was within the reference

ranges for the differences detected for Dams el bugs The mean abundance values for

Orius spp. in Collection 2 and collection 3 at th e ARAU site were out side their respective

reference range. However, the differences detected for Orius spp. were not consistently

detected across collections or sites (Table G-26).

# Monsanto Company 12-CT-244U 162 of 620 

# Since the arthropod differences detected we re not consistently observed at other

collections and sites, MON 88701 is unlikely to have increased plant pest/w eed potential

or an adverse environmental impact compared to the conventional control (See Section

VII.B.1.1.).

VII.C.2.3.2. Study 2 Environmental Interactions of MON 88701

MON 88701 was compared to the conventiona l control for qualitative environmental

interactions in Study 2 (See section VII.C.2.1.). Only qu alitative assessments were

conducted at all 11 sites and included plan t response to abioti c stressors, arthropod

damage, and disease damage. The observations of plant res ponse to abiotic stressors,

disease damage, and arthropod damage were performed four times during the growing

season at each site on all plots (4 replica tions). The first obse rvation was made at

approximately 30 days after planting and the three subsequent observations at

approximately 30 day intervals thereafter. (Section VII.C.2.3.1).

VII.C.2.3.2.1 Qualitative Asse ssment Results - Study 2

In an individual site assessment for Study 2 qualitative data (T able VII-11, G-27, G-28

and G-29), no differences were observe d between MON 88701 and the conventional

control for any of the 127 comparisons for pl ant response to abiotic stressors, including

compaction, drought (dry), flood, hail damage, h eat, nutrient deficien cy, wet soil (excess

precipitation), and wind damage. Also, no differences were observed between

MON 88701 and the conventional control fo r any of the 129 comparisons for the

assessed diseases, including anthracnose, ascoch yta leaf blight, bacter ial blight, boll rot,

cotton leaf rust, damping off, Fusarium wilt, leaf spots, Pythium , reniform nematode,

Rhizoctonia , root-knot nematode , thielaviopsis, and Verticillium wilt

. 

# Finally, no

differences were observed be tween MON 88701 and the conven tional control for any of

the 129 comparisons for the assessed arthropods, including aphids, beet armyworms,

cabbage loopers, cut worms, fall armywo rms, fleahoppers, grasshoppers, heliothines,

southern corn rootworm beetle, soybean lo opers, spider mites, stink bugs, tarnished

plant bugs, thrips and white flies. Sin ce no differences were observed between

MON 88701 and the conventional control for pl ant response to abiotic stressors, disease

damage, and arthropod-related damage in multip le environments, the assessed results are

similar to those in Study 1 and support th e conclusion that the biotechnology-derived

traits in MON 88701 are unlikel y to have increased plant pest/weed potential or an

adverse environmental impact compared to the conventional control (See Section

VII.B.1.1.).

# Monsanto Company 12-CT-244U 163 of 620 

# Table VII-11. Study 2 Summary of Qua litative Environmental Interactions

Assessments Including MON 88701 Response to Abiotic Stress and Disease and

Arthropod-related Damage during 2010

Stressor Number of

Observations

Across All Sites Number of Observations

with No Observed

Differences Between

MON 88701 and the

Conventional Control

Abiotic stressors 127 127

Disease damage 129 129

Arthropod-related damage 129 129

Total 385 385

Note: The experimental design was a randomized complete block with four replications (n =

60



# VII.C.2.3.2.2. Plant Mapping as an Indicato r of Plant Response to Environmental

Stress

Final boll retention and distri bution, as reflected in the plant mapping data, can provide

an indication of the effect that abiotic and biotic stressors had on a cotton plant because

squares and early bolls tend to abort if the plant experiences stress (Guinn, 1982; Kerby

et al., 2010; University of California, 1996). For example, if plants experienced severe

stress during early flowering, this could resu lt in fewer bolls on the lowermost fruiting

branches compared to unstressed plants. If plant map results are similar between two

cotton lines this usually indicates that plan ts responded to stress in a similar manner.

Within a study location and based on the proxi mity of plots within a location, it can be

concluded that all plots would be subjected to similar stressors.

As previously indicated, th ere were no differences in plant mapping parameters between

MON 88701 not treated with dicamba or gluf osinate herbicides and the conventional

control that would be indica tive of a differential plant re sponse to abiotic or biotic

stressors (Study 2, Section VII.C.2.2, Table VI I-7). Similar results were observed for

MON 87701 plots treated with dicamba and gl ufosinate (Table G-18). Thus, since all

Study 2 plots would be subjected to simila r stressors and since MON 88701 treated and

not treated with dicamba or glufosinate he rbicides had similar plant map results, each

compared to the conventional control (See Tables VII-7 and G-18); it can be concluded

that both responded to stressors in a similar manner. Results showed that only the mean

number of first-position bolls was signifi cantly different in both comparisons of

MON 88701 not treated compared to the conve ntional control (5.2 vs. 4.6, respectively)

and MON 88701 treated compared to the conventi onal control (5.2 vs. 4.6, respectively).

Both of the mean values of the number of first-position bolls in MON 88701 were within

the reference range. Therefor e, these data suppo rt the conclusion th at the biotechnologyderived traits in MON 88701 are unl ikely to have increased plant pest potential, increased

# Monsanto Company 12-CT-244U 164 of 620 

# weediness, or an adverse environmental imp act compared to commercially cultivated

cotton.

VII.C.2.3.3. Conclusions - Qualitative and Quantitative Environmental Interactions

The results of the qualitative a nd quantitative data of MON 88701 from Study 1 and

qualitative data from Study 2 showed that th e dicamba and glufosinate-tolerance traits did

not unexpectedly alter the assessed envir onmental interactions of MON 88701 compared

to the conventional control. The lack of significant biological differences in plant

responses to abiotic stress, disease damage, arthropod-related damage, thrips damage,

heliothine damage, and pest- and bene ficial-arthropod abundance for MON 88701

supports the conclusion that the introduction of the dicamba and glufosinate-tolerance

traits are unlikely to result in increased plan t pest potential, increased weediness or an

altered environmental impact from MON 88701 compared to commercially cultivated

cotton, irrespective of whether or not dicamba and glufosinate herbicide treatments were

applied.

VII.C.3. Pollen Characteristics

USDA-APHIS considers the potential for gene flow and introgression of the

biotechnology-derived trait(s) in to other cotton varieties and wild relatives to assess the

potential for increased weedy or invasive char acteristics of the rece iving species. Pollen

morphology and viability information are per tinent to this assessment and, therefore,

were assessed for MON 88701. In additi on, characterization of pollen produced by

MON 88701 and the conventional c ontrol is relevant to the pl ant pest risk assessment

because it adds to the detailed descripti on of the phenotype of MON 88701 compared to

the conventional control.

The purpose of this evaluation was to as sess the morphology and viability of pollen

collected from MON 88701 compared to that of the conventional control. Pollen was

collected from MON 88701, the conventional c ontrol, and four commercial reference

varieties grown under similar agronomic c onditions in Crittenden County, Arkansas

(ARPR). The field trial was arranged in a randomized complete bl ock design with four

replications. Five flowers (subsamples) were collected from each plot; pollen was

extracted from each flower and stained with Alexander’s stain (Alexander, 1980). Pollen

viability was evaluated for each subsample a nd pollen grain diameter was evaluated for

ten representative viable po llen grains per subsample. General morphology of the pollen

was observed for each subsample. MON 88701 was compared to the conventional

control for percentage viable pollen and pollen diameter. A reference range was

calculated from the minimum and maximum mean values of the commercial reference

varieties to provide pollen viability and pol len diameter values representative of

commercial cotton (See Appendix H).

No statistically significant differences ( α =0.05) were detected between MON 88701 and

the conventional control for percentage viable pollen or pollen grain diameter (Table VII12). Furthermore, no visual differences in general pollen morphology were observed

between MON 88701 and the conventional control. These results demonstrate that the

# Monsanto Company 12-CT-244U 165 of 620 

# introduction of the dicamba and glufosinate- tolerance traits did not alter the overall

morphology or pollen viability of MON 88701 comp ared to the convent ional control (See

Figure VII-1, Step 2, answer “no”). The pol len characterization da ta contribute to the

detailed phenotypic descripti on of MON 88701 compared to the conventional control.

The results support an overa ll conclusion that MON 88701 is not different than the

conventional control in terms of plant pest or weed characteristics and is no more likely

to pose a plant pest risk than commercially cultivated cotton.

# Monsanto Company 12-CT-244U 166 of 620 

# Table VII-12. MON 88701 Compared to the Conventional Control during 2010 for

Pollen Characteristics

MON 88701 Conventional

Control Reference Range 1



# Pollen Characteristic Mean (S.E.) 2 

# Mean (S.E.) Minimum Maximum

Viability (%) 97.8 (0.46) 97.9 (0.40) 96.0 98.2

Diameter (μm) 95.2 (1.49) 92.2 (1.47) 94.0 95.2

The experimental design was a randomized complete block design with four replications (n = 4).

No significant differences were detected between MON 88701 and the conventional control

( α =0.05) using analysis of variance (ANOVA).

1 

# Reference ranges = Minimum and maximum values of four commercial reference varieties.

2 

# S.E. = Standard Error.



# VII.D. Conclusions for Phenotypic, Agro nomic, Plant Mapping, and Environmental

Interactions Evaluation

Domesticated cotton lacks characteristics that are commonly associated with plants that

are considered weeds (e.g. seed dormancy, s eed dispersal mechanisms, ability to compete

with and displace native vegetation). An exte nsive and robust set of information and data

were used to assess whether the introduction of the dicamba and glufosinate-tolerant

traits altered the plant pe st potential of MON 88701 comp ared to the conventional

control. These assessments included five gene ral data categories: 1) seed dormancy and

germination characteristics; 2) agronomi c and phenotypic characteristics; 3) plant

mapping characteristics; 4) observations of abiotic stress response, disease damage,

arthropod related damage, and pest- and bene ficial-arthropod abundance; and 5) pollen

characteristics. Results from these assessments comparing MON 88701 and the

conventional control demonstrate that MON 88701 does not possess weedy

characteristics, increased susceptibility or tole rance to specific abio tic stressors, diseases,

or arthropods, or characteristics that would confer a plant pest risk or significant

environmental impact compared to conventi onal cotton. Therefore, based on the results

of multiple assessments discussed above, MON 88701 is comparable to commercially

cultivated cotton, and is no more likely to pose a plant pes t/weediness risk or have a

significant environmental impact.

# Monsanto Company 12-CT-244U 167 of 620 

# VIII. U.S. AGRO NOMIC PRACTICES

VIII.A. Introduction

As part of the plant pest assessment re quired by 7 CFR § 340.6(c)(4), impacts to

agricultural and cultivation practices must be considered. This section provides a

summary of current agronomic practices in the U.S. for producing cotton and is included

in this petition as a baseline to assess possi ble impacts to agricultural practices due to the

cultivation of MON 88701. Discussions include cotton production, seed production,

plant growth and development, general management practices during the season,

management of insects, diseases and weed s, cotton rotational cr ops, and volunteer cotton

management. Information presented in th e previous section demonstrated that

MON 88701 is no more susceptible to diseases or pests than commercially cultivated

cotton. Additionally, data presented in Sec tion VII show that, with the exception of

tolerances to both dicamba and glufosin ate herbicides, MON 88701 is phenotypically

equivalent to commercially cultivated cott on. Thus, there are no changes to the inputs

needed for MON 88701, and no likely impacts to the majority of th e agronomic practices

employed for the production of co tton. Agronomic practices that maybe influenced from

the deregulation of MON 88701 are discussed.

Cotton production in the U.S. is limited prim arily by climate. Cotton is a woody, warmseason perennial plant that is planted in 17 st ates across the southern U.S. Aside from

temperature, the most influential climatic f actor impacting cotton agronomic practices is

moisture. Rainfall requirements and patterns are a major determinant of the cotton

production practices adopted in both dryland and irrigate d cotton. The length of the

season may vary between cotton producti on regions, but the production cycle and

production practices used are fairly consiste nt among geographic regions and between the

upland and Pima cotton types. Proper seedbe d preparation, appropriate variety selection,

appropriate planting dates a nd plant population, and good inte grated pest management

practices are important for optimizing the yiel d potential and economic returns of cotton.

Annual and perennial weeds are a serious pr oblem and must be managed in order to

maximize cotton yield and quality. Weeds comp ete with cotton for water, nutrients, and

light, resulting in reduced cott on lint yields and lint quality wh en left uncontrolled. Weed

species in cotton vary from region to region and from state to state, but the economic

thresholds of cotton require some form of weed management pr actice on all cotton

acreage. Weed management practices include mechanical tillage, crop rotations, cultural

practices ( e.g. , planting clean seed, cleaning till age and harvesting equipment), and

herbicide application. Nume rous selective herbicides are utilized for preplant,

preemergence, and postemergence control of annual and perennial weeds in cotton.

Approximately 97% of the cotton acreage in th e U.S. receives a herbicide application.

Herbicide-tolerant cotton is currently grow n on 78% of U.S. cotton acres (Brookes and

Barfoot, 2012) and glyphosate-tolerant cotton weed control systems have become the

standard program for weed management in cotton since commerci al introduction of

glyphosate-tolerant cotton in 1997 (Brookes and Barfoot, 2012). Glyphosate-tolerant

cotton has facilitated the gr owth of conservation tillage systems in cotton production

which has resulted in reduced soil erosion, reduced fuel and labor costs, improved water

# Monsanto Company 12-CT-244U 168 of 620 

# quality, and conserved soil moisture. Herbicides can replace the need for preplant tillage

for weed control in no-tillage production systems. Insect pe sts, diseases, and nematodes

are also common and continuous threats to cotton production and integrated pest

management programs must be implemented to prevent yield losses due to these pests.

Volunteer cotton ( i.e ., cotton plants that have germinat ed and emerged unintentionally in

a subsequent crop) is not cons idered a significant problem in rotational crops primarily

because mechanical and chemical control meth ods are available to manage the occasional

volunteer cotton plant. Preplant tillage genera lly destroys volunteer cotton plants prior to

planting rotational crops. Volunteer cotton is generally more of a problem in no-till

cotton because of the lack of preplant tillage , but herbicides are av ailable for control of

volunteer cotton in rotational crops. Given that MON 88701 is agronomically,

phenotypically, and ecologically comparable to commercially cultivated cotton, the

introduction of MON 88701 in the cotton produc tion system is expect to have no impact

on the management of cotton volunteer plants in rotational crops such as corn, soybean,

sorghum, and wheat. The numerous control m easures that are effective on conventional

and glyphosate-tolerant volunteer plants will continue to be effective on volunteer

MON 88701 plants if they arise. See Sect ion VIII.H.1 for additional information on

control of MON 88701 volunteers.

As shown in Sections VI and VII, with th e exception of the tolerances to both dicamba

and glufosinate herbicides, no biologically meaningful differences were observed in

composition, phenotype, or environmental interactions between MON 88701 and

commercially cultivated cotton. Moreover, herbicide-tolerant cotton is currently grown

on 78% of U.S. cotton acres (Brookes and Ba rfoot, 2012). Therefore, it is anticipated

that commercialization of MON 88701 in the U. S. is not likely to impact current cotton

cultivation and/or agronomi c practices, beyond the intende d benefits of effective

management of common and troublesome weed s, including herbicide-resistant weeds.

VIII.B. Overview of U.S. Cotton Production

VIII.B.1. Cotton Production

The majority of the value of the producer’s cotton crop is based on the quality and

quantity of the lint produced, a nd with the exception of contracted acres for planting seed

production. Little consideration is given by gr owers to the disposi tion of the cottonseed

and its by-products. Most of the worl d’s cotton production (116.40 million bales

annually) is grown in China (30.5 million bale s), India (26.4 million bales), United States

(18.1 million bales), Pakistan (8.6 million bale s) and Brazil (9.0 million). Figures are

from the 2010/2011 cotton season (USDA-FAS, 2012). In 2010/2011, the U.S. supplied

over 14 million bales of the world’s cotton e xports, accounting for approximately 40% of

the total world export market for cott on (USDA-FAS, 2011). China, Bangladesh,

Indonesia, and Turkey are major importers of cotton. The largest customers for U.S.

cotton are Asian countries a nd Mexico, due to the preval ence of textile manufacturing

(NCCA, 2010). Cottonseed produ ction currently results in approximately 10% of the

world’s oilseed production (U SDA-FAS, 2010), and is exceeded by soybean (58%) and

rapeseed (13%).

# Monsanto Company 12-CT-244U 169 of 620 

# Gossypium hirsutum (upland cotton) cultiv ars account for more than 90% of the world’s

annual cotton crop and 97% of the U.S. co tton production (Smith and Cothren, 1999;

USDA-NASS, 2011e). G. barbadense , known as extra-long staple, Pima, or Egyptian

cotton, is also grown in the U.S, which accoun ts for approximately 3% of the acreage in

the U.S. (USDA-NASS, 2012c). The long, stro ng, fine fibers produced by Pima are ideal

for specialized uses, but due to the geogra phic limitation for optimum production it is

economically less viable than the G. hirsutum cultivars in the U.S. Pima cotton requires a

longer growing season than upland cotton, and production is limited to the Southwestern

states.

Cotton is a crop that produces two commodities: fiber and seed. The modern cotton gin

has enhanced the value of cotton commodities by separating the fiber from the seed and

by removing foreign matter, while preserving th e inherent qualities of the fiber and seed

(Smith and Cothren, 1999). The fiber is the more valuable product of the crop, normally

accounting for approximately 85% of the valu e. For every 100 pounds of fiber produced

by the cotton plant, it also produces about 162 pounds of cottonseed (NCCA, 2010).

Cottonseed is crushed for oil and meal used in both food products and in livestock feed.

Cotton ( Gossypium spp.) is grown in the U.S. across southern states where the climate is

warmer and the season is longer (Figures VIII-1 and VIII-2). The total U.S. cotton

acreage in the past 10 years has varied from approximately 9.15 to 15.77 million planted

acres, with the lowest acreag e recorded in 2009 and the high est in 2001 (Table VIII-1).

Average cotton yields have varied from 632 to 879 pounds per acre over this same time

period. Total annual cotton production ranged from 12.19 to 23.89 million bales (480

pounds/bale) over the past ten y ears. The variations obse rved in cotton acreage and

production is driven by current market condition s, rather than agronomic considerations.

According to data from USDA-NASS (U SDA-NASS, 2011b), co tton was planted on

approximately 11 million acres in the U.S. in 2010, producing approximately 18 million

bales of cotton (Table VIII-1 ). The value of cotton pr oduction reached $7.32 billion in

the U.S. in 2010 (USDA-NASS, 2011b).

U.S. cotton production is divided into the fo llowing four major cotton growing regions,

which span the southern and southwestern st ates: Southeast region (AL, FL, GA, NC, SC,

and VA), Midsouth region (AR, LA, MS, MO, a nd TN), Southwest region (KS, NM, OK,

and TX), and West region (AZ and CA) (Tab le VIII-2). Cotton planting and production

figures for these regions in 2010 are shown in Table VIII-2 and discussed below (USDANASS, 2011e). Approximately 5.6 million acr es of cotton were planted in Texas,

representing about 51% of the total U.S. cotton acres. Texas produced 8.1 million bales

(480 pounds/bale) of cotton, which represents approximately 44% of the U.S. cotton

production. The second largest productio n state for cotton was Georgia with

approximately 12% of U.S. cotton production. Average cotton yields across the four

cotton growing regions ranged from 727 to 1416 pounds cotton lint pe r acre, with the

highest yields in the West with full irriga tion, and the lowest yiel ds in areas such as

Alabama, Oklahoma, and Texas, where little to no irrigation is employe d (Table VIII-2).

The average cotton yield across all regions is 821 pounds cotton lint per acre. The value

of the cotton lint production among the four regions ranged from $0.86 billion in the

West region to $3.35 billion in the Southwes t region. The total value of the cottonseed

# Monsanto Company 12-CT-244U 170 of 620 

# production in the U.S. in 2010 was $1 billion with the value among the regions ranging

from $134 million in the West region to $461 million in the Southwest region.

# Monsanto Company 12-CT-244U 171 of 620 

# Figure VIII-1. Planted Upland Cotton Acres by County in the U.S. in 2010

(USDA-NASS, 2012a)

# Monsanto Company 12-CT-244U 172 of 620 

# Figure VIII-2. Planted Pima Cotton A cres by County in the U.S. in 2010

(USDA-NASS, 2012b)

# Monsanto Company 12-CT-244U 173 of 620 

# Table VIII-1. Cotton Production in the U.S., 2000-2010 1



# Year Acres

Planted

(×1000) Acres

Harvested

(×1000) Average

Yield

(lbs/acre) Total Production

(480 lb bales) Value

(billions $)

2010 10,973 10,707 821 18,314,500 7.318

2009 9,150 7,691 777 12,187,500 3.788

2008 9,471 7,569 813 12,815,300 3.021

2007 10,872 10,489 879 19,206,900 5.653

2006 15,274 12,732 814 21,587,800 5.013

2005 14,245 13,803 831 23,890,200 5.695

2004 13,659 13,057 855 23,250,700 4.853

2003 13,480 12,003 730 18,255,200 5.517

2002 13,958 12,417 665 17,208,600 3.777

2001 15,769 13,828 705 20,302,800 3.122

2000 15,517 13,053 632 17,188,300 4.260

1 

# (USDA-NASS, 2011b)Monsanto Company 12-CT-244U 174 of 620Table VIII-2. U.S. Cotton Production by Region and State in 20101Region/StateAcres Planted(thousands)Acres Harvested(thousands)Average Yield(pounds/acre)Total Production(thousand bales)Cotton Lint $Value (thousands)Cottonseed $Value (thousands)Southeast RegionAlabama340337684480199,06620,856Florida928980915054,7925,720Georgia1,3301,3208112,230926,96691,120North Carolina550545854970338,95744,992South Carolina202201872365136,65616,756Virginia838268511746,0516,300Region Totals2,5972,57480443121,702,488185,744Midsouth RegionArkansas5455401,0491,180395,91471,400Louisiana255250864450174,96024,024Mississippi420415983850308,85644,616Missouri3103081,068685226,21440,630Tennessee390387843680275,48242,180Region Totals1,9201,9009713,8451,381,426222,850Southwest RegionKansas51497848034,6753,712New Mexico50491,08411046,7217,215Oklahoma285270738415180,27620,727Texas5,5675,3677238,0823,083,472429,814Region Totals5,9535,7347278,6873,345,144461,468WestArizona1981961,46059524638446,200California3063031,38887661004287,599Region Totals5044991,4161,471856,426133,799U.S. Total10,97310,70782118,3157,317,7041,003,8611(USDA-NASS, 2011e; 2011c)

# Monsanto Company 12-CT-244U 175 of 620 

# VIII.B.2. Cotton Seed Production

Standardized seed production practices are responsible for maintaining high-quality seed

stocks, which is essential for U.S. agricult ure. The value of s eed quality (including

genetic purity, vigor, and absence of weed see d, seed-borne diseases, and inert materials,

such as dirt) are a major factor impacting cr op yield potential. St ates developed seed

laws and certification agencies to ensure that purchasers who received certified seed

could be assured that the seed met establis hed seed quality standards (Bradford, 2006).

The federal government passed the U.S. Fe deral Seed Act of 1939 to recognize seed

certification and the establishment of offici al certifying agencies. Regulations first

adopted in 1969 under the Federal Seed Act re cognize land history, field isolation, and

varietal purity standards for foundation, re gistered, and certified seed. Under

international agreements such as the Organisation for Economic Co-operation and

Development (OECD) system, 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 priv ate seed certification organizations in the

U.S., and includes international member countries in North and South America,

Australia, and New Zealand.

Cotton seed is separated into four seed classes: 1) breeder ; 2) foundation; 3) registered;

and 4) certified (AOSCA, 2012). Breeder seed is seed di rectly controlled by the

originating or sponsoring plan t breeding organization or firm . Foundation seed is firstgeneration seed increased from breeder seed and is handled in a manner to maintain

specific levels of varietal pu rity and identity. Register ed seed is the progeny of

foundation seed that is handled to maintain satisfactory varietal purity and identity.

Certified seed is the progeny of breeder, foundation or registered seed, and is typically

two generations removed from foundation see d. While not all cotton seed sold to

growers is officially certified, commercial co tton seed sold and planted for typical cotton

production is produced predominately to meet or exceed certified seed standards. This

section of the petition will provide a broad ov erview of the practices used in producing

certified seed.

The majority of the cotton seed is produced in Texas with significan t quantities produced

in Arizona, Arkansas, California, and Missi ssippi (McDonald and Copeland, 1997). The

entire seed production process at the majority of the seed companie s uses International

Organization for Standardization (ISO) certif ication standards and therefore include

internal and external audits (ISO, 2009). ISO standards ensu re desirable characteristics

of seeds and services, such as quality, safety, reliability, and efficiency. The ISO

standards represent an international cons ensus on good management practices with the

aim of ensuring that the organization can co nsistently deliver excellent products or

services. The standards must meet the cu stomer’s requirements, applicable seed

regulatory requirements, and continually improve the pr ocess and process control

systems (ISO, 2009). Agronomic practices for producing cotton seed are similar to

commercial cotton production. However, incr eased management is needed in certain

agronomic practices ( e.g. , fertility, water management, cu ltivation, use of plant growth

# Monsanto Company 12-CT-244U 176 of 620 

# regulators, etc.) to produce seed with high quality, high germination rates, and high

genetic purity.

After harvest and the ginning and delinting pr ocesses, commercially certified cotton seed

must meet state and federal seed standards and labeling requirements. AOSCA standards

for certified cotton seed are as follows: 98% pure seed (minimum), 2% inert matter

(maximum), 0.02% weed seed (maximum), 0.3% other crop seeds (maximum), and 70%

germination (minimum) (AOSCA, 2009). The co tton seed industry hi storically sets a

minimum of 80% germination for labeling purpos es. State seed certification standards

vary slightly from state to state and can be more restrictive than the seed standards of

AOSCA.

When deregulated, MON 88701 seed will be produced in the same manner as

commercially certified cotton seed, such that it will meet all state and federal seed

standards and labeling requirements.

VIII.C. Production Management Considerations

VIII.C.1. Pre-Season

Production decisions regarding crop rotation, tillage system, soil fertility, variety

selection, and row spacing need to be made we ll in advance of planting the cotton crop.

Many of the decisions in this area are made pr ior to or immediately after harvest of the

previous crop. The rotation of cotton with ot her crops should be an integral part of a

farm management program. Ideally, cotton sh ould be rotated with other crops on a

regular basis to maintain soil productivity and reduce the incidence of various weeds,

insect pests or diseases (Hak e et al., 1996d). However, produc tion costs, rela tive rate of

return, and the current market conditions will dictate which crops to rotate with cotton or

whether to grow continuous cotton. See Se ction VIII.H for additional details on crop

rotation practices in cotton.

Tillage has been an integral part of production agriculture and is synonymous with

seedbed preparation. The primary purposes of preplant tillage are to incorporate residue

from the previous crop, reduce wheel tra ffic compaction from the previous season,

improve water filtration and soil aeration, c ontrol weeds, loosen the soil for root

penetration, and provide a suitable environment for the planting and germination of

cottonseed (Hake et al., 1996d). Decreased profitab ility in cotton produ ction, as well as

soil erosion concerns, have increased intere st in conservation tillage systems. The

benefits of conservation tillage or no-ti ll systems are well documented and include

reduced soil erosion, reduced fuel and labor costs, and co nservation of soil moisture

(CTIC, 2011).

Maintaining optimum crop nutrition is critical in achieving high yields and quality in

cotton. Pre-season soil test results for nitrogen, phosp horus, and potassium plus

determination of pH, together with previous cropping and fertilization history determine

the fertilizer and liming needs for the upcom ing cotton crop. In the Southwest and West

regions, monitoring soil salinity is of additional importa nce because cotton is most

# Monsanto Company 12-CT-244U 177 of 620 

# sensitive to sodium and salts during the germination and se edling growth stage (Hake et

al., 1996d). Soil salinity will severely delay em ergence, which can make the plants more

vulnerable to seedling disease.

Yield potential has generally been the most important factor considered by growers in

variety selection (Smith and Cothren, 1999). Growers also need to consider fiber

properties ( e.g., length, strength, microna ire, etc.), cold tolerance, seedling vigor, heat

tolerance, leaf hairiness, insect and diseas e resistance, maturity, and a number of other

factors. Cotton varieties are classified into three maturity groups: short-, medium-, or

long-season varieties (Smith and Cothren, 1999). More determinate plants are planted in

the short season northern portions of the cottonbelt and longer-season or more

indeterminate varieties planted in the sout h. Growers in areas of western Texas and

Oklahoma have tended to select ‘stripper’ or ‘s tormproof’ varieties which produce a boll

that is more resistant against yield loss unde r storm and hail conditions. “Picker” cotton

varieties grown in the high plains of Texas produce large open bolls and are susceptible

to yield loss from seasonally strong thundersto rm activity. Growers are advised to plant

three or four varieties to reduce the risk of planting the entire farm to a poor-yielding

variety or using traits that do not add value to their cropping system (NCCA, 2007).

VIII.C.2. Planting and Early Season

The yield potential of a cotton crop is determin ed in the first 30 to 40 days after seed is

placed in the ground (Deterling and El-Zik, 1982). Planting date management is an

important element in achieving early fruit se t, and establishing a strong yield potential

(Smith and Cothren, 1999). Cotton should be planted into prepared seedbeds that are

firm, warm, and moist. Cotton specialis ts recommend planting cotton when soil

temperatures at seeding depth ar e at 64° F or higher at 8 a. m. for three consecutive days,

with a favorable five-day forecast (Det erling and El-Zik, 1982; Smith and Cothren,

1999). Under favorable conditions, emergence can occur anywhere from 5 to 15 days

following planting. Once emerged, the cotton pl ant goes through a period of slow growth

and development before entering phases of rapid vegetative and reproductive growth.

The growth of the cotton plant is temperat ure dependent and growth ceases when the

average daily temperature falls below 60 ° F (Hake et al., 1996b). It is during this early

period of slow development that cotton must be protected from damaging weed, insect,

and disease pests to prevent yield losses (H ake et al., 1996b; Smith and Cothren, 1999).

When planting is delayed significantly due to time constraints or weather conditions,

growers are advised to switch to more determin ate (short-season) type varieties. Planting

good quality seed with a germination of 85% or higher is also important for establishing a

good and uniform stand of cotton (Deterlin g and El-Zik, 1982). The single most

important practice for minimizing damage fromseedling diseases is selection of highquality planting seed (Smith and Cothren, 1999) . Most cottonseed sold commercially is

treated with a fungicide to protect the germ inating seed and seed lings from seed- and

soil-borne pathogens (Smith and Cothren, 1999)

.

Plant population management contributes toward early fruiting, good fr uit retention, and

improved earliness of crop maturity (Smith and Cothren, 1999). Seeding rates vary

# Monsanto Company 12-CT-244U 178 of 620 

# across the cotton growing region of the U.S. The seeding range will vary depending

upon row-spacing, soil classification, availabl e moisture, and overall environmental

conditions. Cotton has the ability to compensate in response to row spacing and plant

populations. However, higher plant densities te nd to cause cotton plants to grow taller,

develop more vegetative growth, and create more shading within the canopy (Smith and

Cothren, 1999). These characteristics ca n result in delayed fruiting, alter the

reproductive/vegetative balance, and decrea se fruit retention. Conversely, low

populations can delay overall plant maturity, allow sunlight to penetration through the

canopy contributing to more weeds, and resu lt in insufficient st ructure to produce

adequate fruit which can influen ce overall harvestable yields.

VIII.C.3. Mid- to Late-Season

After early development, the next critical st age in the development of a cotton crop is

rapid vegetative growth that includes the init iation of the first ‘squares.’ These floral

buds develop into the subsequent fruiting forms called bolls. Fruiting development

generally begins with the formation of fr uiting branches on nodes four through eight

(Deterling and El-Zik, 1982). After the accumulation of 40 to 60 days following

emergence, the first square becomes visible, which is normally five to eight weeks after

planting, depending on the area and temperature (Deterling and El-Zik, 1982; Hake et al.,

1996c). Approximately 85% of the total bolls that are harvested come from squares set

during the first four to five weeks of squaring (Deterling and El-Zik, 1982). Therefore, it

is critical to properly manage cotton during this period to maximize yields.

Management practices, such as water ma nagement, plant nutrition management, and

weed, disease, and insect control are critical during this reproductive growth phase. To

maximize yield fruiting square a nd resulting boll rete ntion is critical, especially the first

bolls set on the plant. The first three ‘ positions’ on each reproductive branch are the key

sites for fruiting and will account for the vast majority of the plant’s yield (Deterling and

El-Zik, 1982). Further, the first-position, or squares nearest the main stem, will account

for over 50% of the total lint produced per plant. The second-pos ition squares account

for another one-third or more of the ha rvest, while squares further out on each

reproductive branch produce 15% or less of the final number of mature bolls harvested

that contribute to yield (Deterling and El-Zik, 1982).

Most growers or crop consultants currently use a number of measurements during

midseason to monitor and manage cotton pl ant growth. Although each will not be

discussed in detail here, the grower may monitor any one or more of the following

parameters: 1) plant height; 2) number of mainstem nodes; 3) node number of first

fruiting branch; 4) total number of fruiting br anches; 5) height-to- node ratios; and 6)

square or fruit retention (Hake et al., 1996c). These parameters are commonly referred to

as plant mapping. In general, as cotton is a perennial grown as an annual the cotton

grower is seeking to favor reproductive growth at the expense of vegetative growth. This

transition from vegetative to reproductive gr owth influences crop maturity and season

length. Available options to influence cotton plant growth include the use of a plant

growth regulator such as mepiquat chloride, fertility management (primarily nitrogen and

potassium), and water management. Also, weed management and insect pest control are

# Monsanto Company 12-CT-244U 179 of 620 

# important at mid-season, both of which can dramatically decrease both square and

immature boll retention.

As the end of the growing season approaches , the yield is established and management

efforts shift to protecting the crop yield and quality. The stage in cotton when vegetative

growth ceases is generally referred to as “cu t-out” (Hake et al., 1996a). When the nodes

above the first-position white fl ower decline to four or five, cut-out has been reached.

This is also the point at which the last eff ective bloom, which could contribute to yield, is

on the plant. The timing of cu t-out is critical for both yield and quality of cotton. If cutout occurs too early, due to environment and management practices, the crop may not

take full advantage of the available season. Late cut-out is often associated with poor

early-season fruit retention and results in delayed maturity and harvest.

VIII.C.4. Preharvest and Harvest

The complete defoliation or desiccation of leaf tissue in preparation for harvest is a

necessity with harvesting (Hake et al., 1996a). Leaves not only interf ere with harvesting,

but contribute to trash and moisture content, which infl uences ginning, cleaning, and

overall quality of cotton lint. Effective defo liation is an essential step in the overall

process of harvesting high quality cotton lint with the grower seeking to accomplish a

complete, quick and efficient defoliation by chemical means. Defoliation attempts to

speed up and control the natural process of senescence. An add itional objective of

defoliation is to kill or desiccate weeds that can reduce harvest efficiency, contribute to

the weed seed bank, and reduce both the quality and value of the lint because of staining

by vegetation (University of Georgia, 2012). Successful defoliation in cotton depends on

a number of factors including: 1) plant-water status; 2) n itrogen fertility status; 3)

weather conditions; and 4) the chemical defoliant(s) (Smith and Cothren, 1999).

Defoliation should begin as early as possible by balancing the realistic yield potential of

the crop with the need to preserve quality, schedule harvesting equipment, and minimize

losses from weathering. Defoliation normally occurs when approximately 60% of the

bolls are open (Smith and Cothren, 1999). Co tton is then harvested mechanically using

pickers or strippers.

VIII.D. Management of Insect and Other Pests

Insect and mite pests are a common and c ontinuous threat to cotton production in all

regions of the U.S., leading to decreased yield and quality. Ge nerally, fewer than 25

insect pests are considered persistent probl ems causing economic losses in cotton (Smith

and Cothren, 1999). The susceptib ility of cotton plants to insect pests varies across and

within the various production regions. Ins ect and mite pests a ffect cotton production by

decreasing yield and reducing quality. Near ly every phenological stage of cotton is

susceptible to injury by one or more insect pests during the growi ng season. Therefore,

cotton fields must be monitored regularly to detect the presence of insect pests. The

susceptibility of cotton plants to economic yield losses from in sect pests is influenced by

pest population density, timing of infestat ions as related to plant phenology, local

environmental conditions, and agronomic pr actices (Smith and Cothren, 1999).

# Monsanto Company 12-CT-244U 180 of 620 

# Numerous insect species are observed in cott on fields across the U. S., but only a few are

considered of economic importance. Yield lo ss and treatment costs for the most common

insect pests in cotton in 2010 are shown in Table VIII-3. These data are estimates

collected from surveys of county agents, exte nsion specialists, private consultants, and

research entomologists. Inse ct damage resulted in yield losses of approximately 986

thousand bales of cotton in 2010 or a 3.9% yi eld loss which represented an average loss

of $22.56 per acre. The lepidopteran pests, bollworm/budworm, caused the greatest yield

reductions followed by stink bugs and lygus in sects, both of which are piercing and

sucking insects. Thrips infested more acr es in 2010 than any other insect in cotton.

However, this insect ranked sixth in yield reductions, due to the da mage occurring early

in the growing season before the deve lopment of fruiting structures.

Successful and economical management of ins ect pests in cotton is accomplished through

an integrated pest management approach of variety selection and implementation of

cultural, biological, and chemical strategies (University of Georgia, 2011). Preplant

tillage and crop rotation are impor tant agronomic or cultural practices utilized to reduce

insect populations prior to planting cotton. Other agronomic practices are utilized to

promote early maturity and reduce that period of time the crop is susceptible to insect and

mite pests, and to increase the probability that an acceptabl e yield can be produced before

insect pest densities exceed economic thre shold levels (Smith and Cothren, 1999).

Nematodes are another serious pest in cotton and have the potential to cause significant

loss of yield, reduction in fiber quality, and crop maturity. Yield losses in cotton from

nematodes exceed $400 million annually in the U.S (NCCA, 2007). Management

decisions for controlling nematodes must be made prior to or at planting since few

control options are avai lable during the season.Monsanto Company 12-CT-244U 181 of 620Table VIII-3. Insect Losses in Cotton in U.S. in 20101Insect Pest% YieldReductionCotton AcresInfestedCottonAcresTreatedTreatmentCost($/Acre)Cotton BalesLostBollworm/Budworm 1.186 8,148,844 2,113,842 2.54 263,902Stink bugs 0.724 6,712,988 2,782,462 3.45 162,397Lygus spp. 0.677 5,932,835 2,458,413 6.86 191,826Cotton Fleahopper 0.362 4,487,032 2,357,727 2.79 81,048Aphids 0.286 7,133,029 1,270,253 1.39 60,377Thrips 0.200 10,165,601 3,469,195 2.32 45,964Spider mites 0.199 3,522,479 885,684 1.75 57,189Fall Armyworm 0.199 2,762,701 203,109 0.20 41,256Clouded Plant Bugs 0.024 614,569 213,683 0.14 6,465Silverleaf Whitefly 0.020 508,430 111,902 0.81 4,935Cutworms 0.003 487,946 543,570 0.19 699Grasshoppers 0.001 1,293,128 53,300 0.02 373Beet Armyworm 0.001 967,552 52,222 0.06 146Loopers 0.001 735,998 9,160 0.01 197Saltmarsh caterpillar 0.001 689,127 3,400 0.00 140Banded winged whitefly 0.000 483,273 - 0.00 0Southern Armyworms 0.000 242,500 - 0.00 0Boll Weevil 0.000 115,470 25,920 0.01 0Pink Bollworm 0.000 97,725 - 0.00 0Cotton Leaf Perforator 0.000 14,988 14,988 0.00 0European cornborer 0.000 0 - 0.00 0Other Insects (1-4)0.023 649,594 26,648 0.01 68,906Total 3.906 22.56 985,8211(Williams, 2010).

# Monsanto Company 12-CT-244U 182 of 620 

# VIII.E. Management of Diseases

Disease management is essential in cotton production to achieve optimum yields and

economic returns. Plant pathologists estimate that diseases cause annual losses in cotton

production of 1.8 million bales or a yield redu ction of approximately 9.0 % in the U.S.

(Blasingame et al., 2008). Seed ling diseases, fungal wilts, r oot rots, and foliar diseases

constitute the major disease complex in cotton (Smith and Cothren, 1999). Yield losses

are often underestimated because most of the diseases are caused by soil-borne pathogens

that attack the roots and cause little reduc tion of the plant size or change to the crop

canopy (Smith and Cothren, 1999). These types of infestation can result in yield losses

of as much as 20% without any awareness of th e root infections by soil-borne pathogens.

The major seedling disease complex ( i.e. , Pythium spp., Rhizoctonia spp., Fusarium spp.,

and Thielaviopsis spp.) are cau sed by fungal pathogens and ar e generally classified as

seed-borne pathogens that occu r on or in seed prior to pl anting and soil-borne pathogens

that reside in soil (Smith and Cothren, 1999) . The soil-borne pathogens are the most

important causes of seedling disease and the most difficult to control. Verticillium wilt

and Fusarium wilt are the two major fungal wilt diseases causing losses in cotton

production. The pathogens penetr ate root tips and enter the xylem vessels of the cotton

plant and the plants subsequently develop the characteristics of wilt symptoms.

Phymatotrichum root rot, macrophomina root rot, agrobacterium root rot and root gall are

the primary diseases attacking roots of olde r cotton plants (Smith and Cothren, 1999).

The foliar diseases are caused by pathogens that infect leaves, stems, bolls, and

occasionally seedling roots. Bacterial blight, boll rot, fungal leaf spots, fungal boll rots,

viran and mycoplasmal make up the primary foliar diseases.

An integrated management system is the best means of controlling diseases in cotton.

This includes agronomic and cultural practices ( i.e., fertility, water management, crop

rotation), use of resistant va rieties, applications of f ungicides and bactericides, and

applications of biocontrol agents (Smith and Cothren, 1999). The single most important

practice for minimizing damage from seedling diseases is selection of high-quality

planting seed that has minimal seed coat da mage and has been assessed for germination

and vigor (Smith and Cothren, 1999). S eedbed conditions that encourage rapid

germination and emergence will minimize seedling disease losses (NCCA, 2007).

Selection of varieties with sati sfactory levels of resistance is also an important step in the

control of certain other diseases. Various cu ltural practices such as crop rotation, proper

fertility and water management, clean tillage systems, early planting, eliminating weeds

which are host plants to the pathogen, and practices that increase decomposition of crop

residues can reduce the severity of diseases (Smith and Cothren, 1999). Fungicides are

used to protect seeds and seedlings from seed - and soil-borne pathoge ns during their first

few weeks of growth. Commercial cottonseed is normally treated and planted with a

mixture of chemical fungicides applied to c ontrol these soil borne pathogens (Smith and

Cothren, 1999). Fungicides are also used to prevent epidemics of foliar diseases when

they approach economically damaging levels. An average of three fungicide treatments

were made to cotton in 2007 (USDA-ERS, 2 012a). Foliar fungici des are applied to

approximately 2% of the cotton acreage (USDA-NASS, 2008).

# Monsanto Company 12-CT-244U 183 of 620 

# VIII.F. Weed Management

Weed control in cotton is essential to maxi mize both cotton fiber yield and quality. In

contrast to other crops, including corn a nd soybean, cotton emergence and above ground

growth is relatively slow during the first few weeks after planting. Th e slow early growth

of cotton does not permit the crop to aggressively compete against weed species that

often grow more rapidly (Smith and Cothren, 1999). This is especially true under cool

weather or adverse growing c onditions which often prevail af ter cotton is planted. The

extent or degree to which weeds interfere w ith cotton growth and yield is dependent on

the species, densities, durati on, and environmental conditi ons. For example, a single

common cocklebur over 30 row-feet can redu ce cotton yields by 8.85%, while a single

prickly sida plant at the same density reduces cotton yields by only 0.26% (Smith and

Cothren, 1999). Weed-crop competition studies have demonstrated that the control of

weeds during the first four to eight weeks after cotton planting is critical as weeds

compete against the crop for water, nutrients , light, and other resources necessary for

growth (Smith and Cothren, 1999). Although la te-season infestations may not impact

yield, they reduce harvesting e fficiency, contribute to the weed seed bank, and lower the

lint grade (Vargas et al., 1996). Weeds can al so have an impact on cotton diseases and

insect management because certain weed species can be a host for Rhizoctonia and

Verticillium wilt and harbor insects such as lygus bugs.

The occurrence and frequency of individual we ed species in cotton vary greatly between

and within each state and geographical grow ing region. Cultural and chemical control

practices can cause shifts in the composition of weed populat ions (Smith and Cothren,

1999). Weed populations are affected over time by edaphic (soil-rated) factors,

reproductive ability, control methods, cropping sequences, herbicide regimes, herbicideresistance, climatic changes, and other environmental situations (Smith and Cothren,

1999). The most common weeds in cotton ar e not necessarily th e most troublesome

weeds. The degree of importance depends on th e interference to co tton growth and yield,

reduction in lint quality, and expense of c ontrol (Smith and Cothren, 1999). The proper

identification of the weed species , especially in the immature st ages of growth is essential

to the development of an e ffective weed management program. Table 4 lists the common

and species names of all weeds referred to in this petition. Tables VIII-5 through VIII-8

provide summaries of the most common weeds in cotton for each of the four major cotton

growing regions (Southeast, Midsouth, Southwes t, and West). Barnyardgrass, crabgrass,

pigweed spp. (including Palmer amaranth), morningglory spp., common cocklebur, and

common lambsquarters are common annual weed species in almost all cotton growing

regions. Johnsongrass, bermudagrass and nutse dge are common perennial weed species.

Weed species of the Solan aceae family, such as the nightshade spp. and groundcherry,

are more common in the Southwest and West regions. Palmer amaranth, morningglory

spp., and nutsedge spp. are not only common in cotton, but are freque ntly reported as

some of the most troublesome or serious weed species in cotton (Webster et al., 2009).

# Monsanto Company 12-CT-244U 184 of 620 

# Table VIII-4. Common and Scientific Names of Weeds Referred to in this Petition

Common Name Scientific Name Common Name Scientific Name

Annual bluegrass Poa annua Little barley Hordeum pusillum

Barnyardgrass Echinochloa crus-galli Jimsonweed Datura stramonium

Bermudagrass Cynodon dactylon Johnsongrass Sorghum halepense

Bindweed, field Convolvulus arvensis Junglerice Echinochloa colona

Black nightshade

(Eastern) Solanum ptychanthum Large Crabgrass Digitaria sanguinalis

Broadleaf signalgrass Urochloa platyphylla Morningglory spp Ipomoea spp.

Browntop millet Urochloa ramosa Mustard spp. Brassica spp.

Buttercup Ranunculus spp. Nightshade, hairy Solanum physalifolium

Carolina geranium Geranium carolinianum Nightshade,

silverleaf Solanum

elaeagnifolium

Chickweed Stellaria media Nutsedge spp. Cyperus spp.

Citronmelon Citrullus lanatus Palmer amaranth Amaranthus palmeri

Common cocklebur Xanthium strumarium Pigweed spp. Amaranthus spp

Common hempnettle Galeopsis tetrahit Prickly lettuce Lactuca serriola

Common lambsquarters Chenopodium album Prickly sida Sida spinosa

Common ragweed Ambrosia artemisiifolia Purple nutsedge Cyperus rotundus

Crabgrass spp. Digitaria spp. Purslane, common Portulaca oleracea

Crowfootgrass Dactyloctenium

aegyptium Purslane, horse Trianthema

portulacastrum

Cupgrass, southwestern Eriochloa acuminata Red rice Oryza punctata

Curly dock Rumex crispus Redweed Melochia corchorifolia

Cutleaf eveningprimrose Oenothera laciniata Russian thistle Salsola tragus

Devil's claw Proboscidea louisianica Sandbur Cenchrus spp.

Fall panicum Panicum dichotomiflorum Shepard’s purse Capsella bursa-pastoris

Florida beggarweed Desmodium tortuosum Sicklepod Senna obtusifolia

Florida pusley Richardia scabra Smartweed spp. Polygonum spp.

Foxtail spp. Setaria faberi Smellmellon Cucumis melo

Giant foxtail Setaria spp. Sprangletop, red Leptochloa panicea

Giant ragweed Ambrosia trifida Spreading dayflower Commelina diffusa

Goosegrass Eleusine indica Spurge spp. Euphorbia spp.

Groundcherry spp. Physalis spp. Spurred anoda Anoda cristata

Henbit Lamium amplexicaule Sunflower Helianthus annuus

Hemp sesbania Sesbania herbacea Texas blueweed Helianthus ciliaris

Horseweed (marestail) Conyza canadensis Texas millet Urochloa texana

Italian ryegrass Lolium multiflorum Texas panicum Urochloa reptans

Kochia Kochia scoparia Tropical spiderwort Tradescantia ohiensis

Velvetleaf Abutilon theophrasti

# Monsanto Company 12-CT-244U 185 of 620 

# Table VIII-4. Common and Scie ntific Names of Weeds Referred to in this Petition

(continued)

Common Name Scientific Name

Virginia pepperweed Lepidium virginicum

Volunteer corn Zea Mays

Volunteer peanut Arachis hypogaea

Common waterhemp Amaranthus rudis

Tall waterhemp Amaranthus tuberculatus

Wild lettuce Lactuca canadensis

Wild mustard Sinapis arvensis

Wild radish Raphanus raphanistrum

Woolyleaf bursage Ambrosia grayi

Yellow nutsedge Cyperus esculentus



# Table VIII-5. Common Weeds in Cotton Produc tion in the Southeast Region of the

U.S. 1,2



# Crabgrass spp.(6)

Morningglory spp.(6)

Prickly sida(5)

Florida pusley(4)

Nutsedge spp.(4)

Sicklepod(4)

Broadleaf signalgrass(3)

Goosegrass(3) Pigweed spp.(3)

Common cocklebur(2)

Common lambsquarters(2)

Common ragweed(2)

Florida beggarweed(2)

Palmer amaranth(2)

Texas millet(2)

Bermudagrass(1) Crowfootgrass(1)

Horseweed (marestail)(1)

Jimsonweed(1)

Johnsongrass(1)

Smartweed spp.(1)

Spurge spp.(1)

Volunteer peanut(1)

1 

# OK data (Webster et al., 2009).

2 

# Number provided in parenthesis is the number of stat es out of the six total states (AL, FL, GA, NC, SC,

and VA) in the Southeast region reporting each weed as one of the ten most common weeds.

# Monsanto Company 12-CT-244U 186 of 620 

# Table VIII-6. Common Weeds in Cotton Produc tion in the Midsouth Region of the

U.S. 1,2



# Morningglory spp.(5)

Broadleaf

signalgrass(4)

Crabgrass spp.(4)

Nutsedge spp.(4)

Prickly sida(4)

Spurge spp.(4)

Pigweed spp.(3) Velvetleaf(3)

Barnyardgrass(2)

Horseweed

(marestail)(2)

Johnsongrass(2)

Palmer amaranth(2)

Bermudagrass(1)

Browntop millet(1) Common cocklebur(1)

Cutleaf eveningprimrose(1)

Goosegrass(1)

Hemp sesbania(1)

Henbit(1)

Spurred anoda(1)

1 

# AR, LA & MO data (Webster et al., 2009); MS & TN data (Webster et al., 2005).

2 

# Number provided in parenthesis is the number of states out of the fi ve total states (AR, LA, MS, MO, &

TN) in the Midsouth region reporting each weed as one of the ten most common weeds.



# Table VIII-7. Common Weeds in Cotton Production in the Southwest Region of the

U.S. 1,2



# Johnsongrass(4)

Nutsedge spp.(4)

Cocklebur, common(3)

Palmer amaranth(3)

Silverleaf Nightshade

(3)

Common

lambsquarters(2)

Large Crabgrass(2)

Devil's claw(2)

Morningglory spp.(2) Mustard spp.(2)

Pigweed spp.(2)

Russian thistle(2)

Barnyardgrass(1)

Bermudagrass(1)

Bindweed, field (1)

Foxtail spp.(1)

Groundcherry spp.(1)

Kochia(1)

Horseweed

(marestail)(1) Shepard’s purse(1)

Smartweed(1)

Smellmelon(1)

Spurred anoda(1)

Sprangletop, red(1)

Sunflower(1)

Texas blueweed(1)

Texas millet(2)

Velvetleaf(1)

Woolyleaf bursage(1)

1 

# OK data (Webster et al., 2009); KS – Stewart Dunc an, Ph.D., Kansas State University – Personal

Communication November, 2010; NM –Jamshid Ashigh, Ph.D., New Mexico State University – Personal

Communication November, 2010; TX –Wayne Keeling, Ph.D. and Gaylon Morgan, Ph.D., Texas A&M

University - Personal Communications November, 2010.

2 

# Number provided in parenthesis is the number of states out of the four total states (OK, KS, TX, & NM) in

the Southwest region reporting each weed as one of the ten most common weeds.

# Monsanto Company 12-CT-244U 187 of 620 

# Table VIII-8. Common Weeds in Cotton Pro duction in the West Region of the

U.S. 1,2



# Barnyardgrass(2)

Morningglory spp.(2)

Sprangletop(2)

Bermudagrass(1)

Bindweed, field(1)

Cupgrass,

southwestern(1) Groundcherry spp.(1)

Lambsquarters,

common(1)

Johnsongrass(1)

Junglerice(1)

Nutsedge spp.(1)

Pigweed spp.(1) Nightshade, black(1)

Nightshade, hairy(1)

Nightshade, silverleaf(1)

Palmer amaranth(1)

Purslane, common(1)

Purslane, horse(1)

Volunteer corn(1)

1.

1 

# Source:AZ – Bill McCloskey, Ph.D., University of Arizona – Personal Communication, November, 2010;

CA – Steven Wright, Ph.D., University of California - Personal Communication November, 2010.

2 

# Number provided in parenthesis is th e number of states out of the two total states (AZ & CA) in the West

Region reporting each weed as one of the ten most common weeds.



# VIII.F.1. Methods of Weed Control in Cotton

Weeds in cotton are controlled through the inte grated use of various cultural, mechanical,

and chemical methods (Hake et al., 1996d). Crop rotation, or the lack of rotation, in

conjunction with other weed control methods, can play an important factor on the weed

spectrum and drastically impact weed populations (Smith and Cothren, 1999).

Historically, mechanical tilla ge and hand hoeing were the mo st important tools in cotton

weed control. Current weed management practices include as many as five tillage

operations in conventional tillage systems a nd two or three tillage operations in mulch

tillage, or no-till systems (USDA-ERS, 2012b) . Approximately, 38% of the total cotton

acres are post-plant cultivated and within c onventional tillage systems, over 50% cotton

acres are cultivated for w eed control (USDA-ERS, 2012b).

The use of chemical methods for weed contro l began to develop in cotton in the 1940s

and 1950s with the discovery and development of several selective herbicides (Buchanan,

1992). Dinoseb, chloropropham, dalapon, and diuron were developed and used in cotton.

Despite the increased use of he rbicides in the late 1950s, less than 10% of the total U.S.

cotton acreage received a herbicide treatm ent. However, herbicide use rapidly

accelerated in the 1960s as a series of more se lective herbicides were introduced into the

market. These herbicides pr ovided good weed control with le ss cotton injury than most

products used a decade earlier. These products included trifluralin, DSMA/MSMA,

prometryn, and fluometuron. These herbicides , representing different chemical families

and modes-of-action, are still widely used t oday. Additional herbicides were introduced

during the 1970s that were effi cient, effective, and relatively economical on a wide range

of weed species. Glyphosate was introduced in the early 1970s and quickly became one

of the most effective herbicides for nons elective spot treatme nts for control of

johnsongrass and other weeds (Buchanan, 199 2). Glyphosate was also an effective

burndown treatment within no-till cotton produ ction. The use of dinoseb for broadleaf

weed control was halted in 1987 with th e suspension of the registration by the

Environmental Protection Agency (McWhorte r and Bryson, 1992). Registrations were

also discontinued for dinitramine, flurach loralin, profluralin, dalapon, dipropetryn, and

# Monsanto Company 12-CT-244U 188 of 620 

# perfluidone. Numerous additional selective herbicides for grass and broadleaf weed

control were introduced in the 1980s in cotton including fluazifop, metolachlor,

oxyfluorfen, and sethoxydim. However, the use of these products does not equal the

acreage treated with the herbic ides which were discontinue d (Buchanan, 1992). By the

mid 1980s, there were 33 herbicides and he rbicide combinations applied in cotton

(Buchanan, 1992). The greatest us e of herbicides on a per-acre basis was in the Midsouth

which averaged 5.7 herbicide applications per acre each year. During the 1990s, the

herbicides lactofen, bromoxynil, clethodim, clomazone, quizalifop, and pyrithiobac were

introduced for use in cotton.

The first biotechnology-derived herbicide-to lerant cotton became available in 1995 and

provided tolerance to bromoxynil (Stalker et al., 1996). Approximately 50,000 acres of

bromoxynil-tolerant cotton were planted the year of introduction a nd approximately 2500

growers planted 200,000 acres of bromoxynil- tolerant cotton in 1996 (Smith and

Cothren, 1999). The second herbicide-tole rant cotton product, the first generation

glyphosate-tolerant cotton, was introduced in 1997. Glyphosate-tolerant cotton in

combination with glyphosate herbicide be came the standard program for weed

management in cotton. The first generation gl yphosate-tolerant weed control system in

cotton provided postemergence control of a broad spectrum of weeds with excellent

early-season crop safety (Wilcut et al., 2003) . Glyphosate-tolerant cotton expanded the

grower’s options for weed management and made the mechanics of weed control much

easier, more convenient, and less expensive (Car penter and Gianessi, 2001; Wilcut et al.,

2003). This system also provided a better f it into no-till and reduced-tillage systems,

resulting in an increase in conservation tilla ge systems in cotton (Baldwin and Baldwin,

2002; Carpenter and Gianessi, 2001). Glyphos ate could be applied postemergence to

glyphosate-tolerant cotton from emergence thro ugh the four-leaf stage. After the four

leaf stage and up to layby (canopy closure in th e row), glyphosate had to be applied as a

post-directed spray between the crop rows to minimize contact with the cotton plants to

prevent potential crop injury.

In 2003, glyphosate-tolerant cotton was plan ted on approximately 59% of the cotton

acres in the U.S. (USDA-NASS, 2003). Glyphosate was the most widely used herbicide

in cotton in terms acres treated (US DA-NASS, 2004). However, cotton growers

continued to use a variety of herbicides with various modes-of-action in glyphosatetolerant cotton. Triflura lin and pendimethalin were used on nearly half of the U.S. cotton

acreage for small seeded grass and broadleaf weed control. Various substituted urea

herbicides (diuron, prometryn, fluometuron an d linuron) were also used on 50% of the

U.S. cotton acreage (USDA-NASS, 2004). The so il residual activity of these herbicides

on a number of weed species provided additi onal season-long control of continuously

germinating weeds in glyphosate-tolerant cott on systems (Askew et al., 2002; Wilcut et

al., 2003). Other herbicide pr oducts representing additional modes-of-action, including

carfentrazone, MSMA, pyrithiobac and metolach lor, were also used on cotton ranging

from four to 11% of the acres (USDA-NASS, 2002).

In 2006, a second generation glyphosate-tole rant product was introduced providing

increased tolerance to glyphosat e in the reproductive stages of cotton. This allowed for

an expanded window for over-th e-top applications of glyp hosate in cotton. Glyphosate

# Monsanto Company 12-CT-244U 189 of 620 

# can be applied over-the top in second generation glyphos ate-tolerant cotton from

emergence up to 7 days prior to harvest. With this additional application flexibility,

growers were able to more effectively manage weeds in cotton using over-the-top

applications as opposed to pos t-directed or hooded sprayer applications with previous

glyphosate-tolerant varieties. In addition, fo liar insecticides coul d be combined with

glyphosate in a single application during the se ason for secondary pests such as thrips,

aphids, and plant bugs. Mepiquat chloride, a plant growth regulator commonly used in

cotton production to reduce vege tative growth and increase fr uit retention, could also be

applied with glyphosate in a single applic ation. In 2010, approxi mately 78% of the

cotton acreage was planted to herbicide-tole rant cotton, which was nearly all glyphosatetolerant (Brookes and Barfoot, 2012).

The third herbicide-tolerant cotton product, gl ufosinate-tolerant co tton, was introduced in

2003. Only 3% was planted to glufosinat e-tolerant cotton in 2010 (USDA-ERS-FAS,

2010). Approximately 50% of the acres plan ted to cotton variet ies containing both

herbicide- and insect-tolerant traits (USDA-NASS, 2011a).

Table VIII-9 provides a summary of the herbic ide applications registered for use in

cotton in 2010, the data are discussed below. Herbicides are used on essentially all

(99+%) cotton acres in the U.S. (Monsanto Company, 2011). A total of 32.8 million

pounds of herbicide active ingredient were applied in cotton in 2010. Glyphosate was the

predominate herbicide used in cotton with 19.6 million pounds active ingredient being

applied on 91% of the acres. The numberof glyphosate applications, on glyphosatetolerant cotton, average approxima tely 2.4 applications per year at an average rate of 2.0

pounds of glyphosate active ingredient per acre per crop year (Monsanto, 2011).

Dinitroanaline herbicides (pendimethalin an d trifluralin) were a pplied on 53% of the

cotton acres. Diuron (18%), flumioxazin (1 6%), metolachlor (16%), pyrithiobac (15%),

fomesafen (13%), and 2,4-D (13%) were also frequently used herbicides in cotton

(Monsanto Company, 2011).

According to USDA-ERS (2012a) statistics, gr owers make on average a total of four

herbicide applications in cotton during the growing season. Approximately 16-19% of

the growers utilizing the latest glyphosate-tole rant cotton varieties ap plied a fall herbicide

application to control weeds prior to pl anting cotton depending on their crop rotation

(Prince et al., 2011). Approxi mately 53-97% of the grower s applied spring burndown

treatments in glyphosate-tolerant cotton, wh ich consisted of predominately glyphosate

and/or synthetic auxi ns (2,4-D, dicamba).

Dicamba is currently labeled for use in cotton, although dicamba use is limited because

applications are restricted to early preplant only, due to cotton injury. Before planting

cotton, a minimum accumulation of one inch of ra infall or overhead irrigation must occur

and a waiting interval of 21 da ys is required per 0.25 lbs acid equivalent (a.e .) or less.

Dicamba-treated acres have increased in cott on primarily because it is a leading herbicide

recommendation for glyphosate-resistant marest ail (horseweed) in the Midsouth region

(McClelland et al., 2006).

# Monsanto Company 12-CT-244U 190 of 620 

# Glufosinate may be used for weed contro l in non-glufosinate-tolerant cotton when

applied with a hood sprayer in-crop to avoid c ontact with cotton plan ts. Glufosinate can

also be applied in glufosin ate-tolerant cotton from emer gence up to the early bloom

growth stage.

Approximately 15, 39, and 42% of growers ma de 1, 2, and 3 in-crop applications of

glyphosate in continuous cotton, respectively (Prince et al., 2011). Although glyphosate

is used extensively in glyphosate-tolera nt cotton, non-glyphosate herbicides with

different modes-of-action are al so utilized to provide residua l weed control, improve the

control of certain weed species, extend weed co ntrol, and/or control resistant weeds. The

use of herbicides with different modes-of-act ion is an effective practice to reduce the

potential risk of weeds deve loping resistance to glyphosate or other herbicide modes-ofaction. Approximately 49-76% of growers applied non-glyphosate he rbicides prior to

planting, at planting, or postemergence in gl yphosate-tolerant co tton in 2010 depending

on cropping system (Prince et al., 2011). The non-glyphosate herbicides were ALS

inhibitors (trifloxysulfur on, pyrithiobac), photosystem II inhibitors (prometryn,

fluometuron, diuron), mitosi s inhibitors (metolachlor), PPO inhibitors (flumioxazin,

fomesafen), and synthetic a uxins (2,4-D, dicamba).

Weed management in conventional cotton varietie s is very similar. The major difference

in the herbicide programs is that alternativ e postemergence herbicid es or herbicide tank

mixtures are applied in place of glyphosate as in-crop pos t applications. Glyphosate can

still be applied alone or in combinations w ith other herbicides in preplant burndown or

preharvest applications in conventional cotton. A herbicide or combination of herbicides

(trifluralin, pendimethalin, fl uometuron, fomesafen, flumioxazin) is generally applied at

planting for residual grass and broadleaf weed control. Ge nerally, at leas t two in-crop

post applications are made for control of emerged weeds during the growing season.

Pyrithiobac, trifloxysulfur on, prometryn, clethodim, and sethoxydim are some of the

more common herbicides used post in cotton. In addition, a layby a pplication of one or

more herbicides is applied such as di uron, MSMA, prometryn, or trifloxysulfuron.

Tables VIII-10 through VIII -14 provide a summary of th e control ratings of common

weed species to various herbic ides and herbicide combinations in cotton. These tables

list only the most commonly used herbicides or herbicide treatment s in cotton production

and control ratings are for non- glyphosate-resistant weeds. Seldom would one field or

farm have all weed species, but they generally have a mi xture of grass and broadleaf

weed species. These ratings are utilized to f acilitate the selection of a herbicide program

for the cotton crop, which offers the best overall control of the weed species.

Dinitroanalines (trifluralin and pendimethalin ) provide effective cont rol of most listed

annual grasses, but only certain broadlea f species. Postemergence treatments of

quizalofop, fluazifop, sethoxydim a nd clethodim are effective on the annual grasses and

perennial grasses listed such as johnsongrass and bermudagrass, but provide no control of

the broadleaf species. On the other hand, preem ergence or postemergence applications of

fluometuron or pyrithiobac provide good contro l of many broadleaf weeds and poor or no

control of most grasses. In-crop applica tions of glyphosate and glufosinate provide good

to excellent control of a broa d spectrum of annual grass and broadleaf weeds. However,

# Monsanto Company 12-CT-244U 191 of 620 

# glyphosate provides more effective control of perennial weeds such as bermudagrass,

johnsongrass, and nutsedge species as compared to glufosinate. In addition, glyphosate

combinations are the most effective herbicide treatments for silverleaf nightshade, Texas

blueweed, and woolyleaf bursage, which are problem weeds in the Southwest region.

Post-directed layby applications of MSMA in combination w ith diuron, flumioxazin, or

prometryn and the premix combination of prometryn/trifloxysulfuron provide broad

spectrum weed control. Due to the broad range of weed species present in cotton,

multiple treatments and/or combinations of herbicides are used to achieve effective

season-long weed c ontrol in cotton.Monsanto Company 12-CT-244U 192 of 620Table VIII-9. Herbicide ApplicationsRegistered for Use in Cotton in 20101Herbicide Chemical FamilyMode of Action(MOA)Cotton AcresTreated (%)Cotton AcresTreated perMOA (%)QuantityApplied (1000lbs a.i.2)Total QuantityApplied/MOA(1000 lbs a.i.2)Glyphosate Glycine EPSPS inhibitor 91 91 19,602 19,602Pendimethalin DinitroanalineMicrotubuleinhibitor18531,5845,138Trifluralin Dinitroanaline 35 3,554Diuron UreaPSII inhibitor18341,5272,827Prometyrn Triazine 8 650Fluometuron Urea 7 619Linuron Urea <1 31Carfentrazone TriazolinonePPO inhibitor1302465Flumioxazin N-phenylphthalimide 16 114Fomesafen Diphenylether 13 342Lactofen Diphenylether <1 1Oxyfluorfen Diphenylether <1 6Pyraflufen Phenylpyrazole <1 <12,4-D PhenoxySynthetic Auxin13218911,0842,4-DB Phenoxy <1 1Dicamba Benzoic acid 8 192Monsanto Company 12-CT-244U 193 of 620Table VIII-9. Herbicide ApplicationsRegistered for Use in Cotton in 20101(continued)Herbicide Chemical FamilyMode of Action(MOA)Percent ofCotton AcresTreatedPercent ofCotton AcresTreated perMOAQuantityApplied(1000 lbsa.i.2)Total QuantityApplied/MOA(1000 lbs a.i.2)Pyrithiobac BenzoateALS inhibitor15207275Thifensulfuron Sulfonylurea <1<1Tribenuron Sulfonylurea <1<1Trifloxysulfuron Sulfonylurea 43Acetochlor ChloroacetamideLong-chain fattyacid inhibitor<116471,813Metolachlor Chloroacetamide 16 1,766Paraquat BipyridyliumPhotosystem-I-electron diverter10 10 547 547GlufosinateammoniumPhosphinic acid Glutaminesynthesis inhibitor8 8 535 535MSMA OrganoarsenicalCell membranedisruption6 6 747 747Clethodim CyclohexanedioneACCase inhibitor<1167FluazifopAryloxyphenoxy-propionate<1 1Diflufenzopyr Semicarbazone Auxin transport <1 <1 <1 <1Clomazone IsoxazolidinoneDiterpenesynthesis inhibitor<1 <1 10 10Total 99.4 32,8561(Monsanto, 2011).2a.i.= active ingredient.Monsanto Company 12-CT-244U 194 of 620Table VIII-10. Grass Weed Species Control Ratings to Preplant Burndown Herbicides in CottonCommon Grass Weeds1Product AB2BG2CG2GFT4GG2IRG2JGs2LB2RR4SB2TP2VC22,4-DN N N 0 N N N N 0 N N NGlufosinate63- 83- 83839373- - - -Glufosinate + 2,4-D ordicamba64- 7484- 54- 7474- - -GlyphosateE F E 8 E G G-E E 8 E E EGlyphosate + 2,4-DE F G-E 8 G-E G G E 8 G-E G-E EGlyphosate + dicambaE F G-E 8 G-E G G E 8 G-E G-E EGlyphosate + carfentrazone orpyraflufenE F E - E G G-E E - E E EGlyphosate + diuronE F G - G F F-G E - G G EGlyphosate + thifensulfuron/tribenuronE F E - E G G-E E - E E EGlyphosate + flumioxazinE F E 8 E G G-E E 8 E E EParaquatG-E P F-G 8 F-G F P G 7 G G F-GParaquat + diuronE P G - G F-G P G-E - G G-E F-G1Weed Species: AB = annual bluegrass, BG = bermudagrass, CG = crabgrass, GFT = giant foxtail, GG = goosegrass, IRG = Italian ryegrass, JGs = seedlingjohnsongrass, LB = little barley, RR = red rice, SB = sandbur,TP = Texas millet (Texas panicum), VC = volunteer corn.2(University of Georgia, 2012). Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-80% control; P = 30% to 60% control;N = < 30% control.3(MSU, 2012).Rating scale: 0-3 = none to slight, 4-6 = fair, 7-8 = good, 9-10 = excellent control.4(University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control.Monsanto Company 12-CT-244U 195 of 620Table VIII-11. Grass Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbicides in CottonCommon Grass Weeds & Nutsedge1ProductBYG2BSG3CG3CFG3CPG4FT3GG3JR5JGs3ST4TP3BG3JGr3NSy3NSp3Preplant Incorporated OnlyPendimethalin9GEECEECE-GNPNNTrifluralin9GEECEECECGNPNNPreemergencePendimethalin-FGGCGGCG-FNPNNClomazone-EEG-EE-G-FP-FNNNFluometuron7PF-GF-G-F-GF-P-PNNNNDiuron7PF-GF-GC-FCPNPNNNNFomesafen-F-GF-G-------FN-G-E-Pyrithiobac6PP--PP-F-F-G-NNNFFPostemergence Residual ControlMetolachlor-F-GEECEECFCP-FNPFPPyrithiobac-PP--PP-F-F-NNNP-FFTrifloxsulfuron-PPP-PP-P-PNN--Monsanto Company 12-CT-244U 196 of 620Table VIII-11. Grass Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbicides in Cotton(continued)Common Grass Weeds & Nutsedge1ProductBYG2BSG3CG3CFG3CPG4FT3GG3JR4JGs3ST4TP3BG3JGr3NSy3NSp3Postemergence Over-The-TopQuizalofop8GGG-EG-E-GGENNFluazifop7G-EGFCEGCG-ECGGG-ENNSethoxydim8EG-EF-GCEG-ECG-ECEFGNNClethodim8EG-EGCEG-ECECEGG-ENNMSMA-PPPP-PNPNN-PNPPN-PFluometuron-PP-FP-F--P-F-P-NNNNNPyrithiobac2NNNNN-PN-PNPNNNN-PP-FP-FTrifloxsulfuron7NPNNN-PN-PNFNN-PNPGF-GGlyphosate9EEECEECECEFG-EFF-GGlyphosate +Pyrithiobac9EEE-EE-E-EFG-EF-GF-GGlyphosate +Trifloxsulfuron-EEE-EE-E-EFG-EG-EGGlufosinate8GGGCGPCGCGNFPPMonsanto Company 12-CT-244U 197 of 620Table VIII-11. Grass Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbicides in Cotton(continued)Common Grass Weeds & Nutsedge1ProductBYG2BSG3CG3CFG3CPG4FT3GG3JR4JGs3ST4TP3BG3JGr3NSy3NSp3Postemergence Directed – LaybyMSMA-FFFPFFNFNPNPF-GFDiuron + MSMA9GGF-G-F-GF-G-F-G-FNPGFPrometryn + MSMA9F-GF-GF-G-F-GF-G-F-G-FNPF-GFFlumioxazin + MSMA9FFF-FF-F-P-FNPGF-GPrometryn/trifloxysulfuron+MSMA9F-GF-GF-G-F-GF-G-F-G-FNPEE1Weed species: BYG = barnyardgrass, BSG = broadleaf signalgrass, CG = crabgrass, CFG = crowsfootgrass, CPG = cupgrass, FP = fallpanicum,GG = goosegrass, JR = junglerice, JGs = seedling johnsongrass, ST =sprangletop, TP = Texas panicum (Texas millet), BG = bermudagrass, JGr= rhizome johnsongrass, NSy = yellow nutsedge, and NSp = purple nutsedge.2(University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control.3(University of Georgia, 2012).Weed control ratings key: E = 90%or better control; G = 80% to 90% control; F = 60%-80% control;P = 30% to60% control; N = < 30% control.4(University of California, 2012). Ratings Key: C = control,P = partial control, N = no control, - = no information.Monsanto Company 12-CT-244U 198 of 620Table VIII-12. Broadleaf Weed Species Control Ratings to Preplant BurndownHerbicides in CottonCommon Broadleaf Weeds1Product CD2BC3CG3CW3CM3CLP3HB3MT3PA3SA3VP3WL3WR32,4-D9G F P F E P-F G-E F F-GG-E G GGlufosinate4- - 8 10 - 7 6 9 - -9 - -Glufosinate + 2,4-D ordicamba28 10 8 10 - 8 10 9 9 10 - -Glyphosate 7 G-E P-F E G-E P-F G-E G-E E G G G-E F-GGlyphosate + 2,4-D 9 E F-G E E E E E E E G-E EGlyphosate + dicamba 9 E G E E G E E E - G-E G-E G-EGlyphosate + carfentrazone orpyraflufen- G-E F-G E E F E G-E E G G G-E GGlyphosate + diuron - G-E G EG-E F-G E G-E E G G G-E GGlyphosate + thifensulfuron /tribenuron nuron- G-E G-E E G-E F E G-E E - G G-E EGlyphosate + flumioxazin 7 G-E G E E F-G E G-E E - G-E E GParaquat 5 E G-E E F F G-E P-F F-G F-G G P F-GParaquat + diuron - E E E GG-E E F-G G-E F-G G F G-E1Weed Species: CD = curly dock, BC = buttercup,CG = Carolina geranium, CW = chickweed,CM = citronmelon, CLP = cutleaf primrose, HB =henbit, MT = marestail, PA = Palmer amaranth, SA spurred anoda, VP = Virginia pepperweed, WL = wild lettuce, WR = wild radish.2(University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control.3(University of Georgia, 2012). Weed control ratings key: E =90% or better control; G = 80% to 90% control; F = 60%-80% control; P = 30% to60% control; N = < 30% control.4(MSU, 2012).Rating scale: 0-3 = none to slight, 4-6 = fair, 7-8 = good, 9-10 = excellent control.Monsanto Company 12-CT-244U 199 of 620Table VIII-13. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbicides in Cotton -Part ICommon Broadleaf Weeds1Product CB2DC3FB2FP2GC4HS2NS4JW2LQ2MG2PA2PW2PS2Preplant IncorporatedPendimethalin N N P E N N N N G-E P G G-E NTrifluralin N N P E N N N N G-E P G G-E NPreemergencePendimethalinN N P F-G N N N N G P P-F F-G NClomazone F F F-G F-G - F - G G P-F N-P P EFluometuron F-G F-G G-E F-G - P - G G-E G F G-E GDiuron F F G P-F C P C G G-E F F-G G-E FFomesafen G F-G P P - P - - E P-F E E -Pyrithiobac N-P F-G G F - P - F-G G F G-E E GPostemergence Residual ControlMetolachlor P N P-F G - P - - F P G G-E FPyrithiobac N-P G G F - P - F-G G F G-E G-E GTrifloxsulfuron - G F-G P-F - - - - - - P-F F -Monsanto Company 12-CT-244U 200 of 620Table VIII-13. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbicides in Cotton– Part I (continued)Common Broadleaf Weeds1Product CB2DC3FB2FP2GC4HS2NS3JW2LQ2MG2PA2PW2PS2Postemergence Over-The-TopQuizalofopNNNN-N-NNNNNNFluazifopNNNNNNNNNNNNNSethoxydimNNNNNNNNNNNNNClethodimNNNNNNNNNNNNNMSMAEP-FEN-PP-PPPP-FPPPFluometuronF-GF-GGP-F---GGGP-FFF-GPyrithiobacGGGN-PPG-ECENGFGFTrifloxsulfuronG-EGG-EP---NGGP-FF-GNGlyphosateEEEP-GCP-FCEGF-GEEF-GGlyphosate + PyrithiobacEEEP-G-G-E-EGG-EEEGGlyphosate + TrifloxsulfuronEEEP-G---EEEEEGGlufosinateEG-EGFC-CEEEF-GGFPostemergence Directed – LaybyMSMAEP-FEPPNPFP-FFPP-FPDiuron + MSMAEG-EEF-P-F-GGG-EGG-EG-EPrometryn + MSMAEG-EEF-P-F-GGG-EFGG-EFlumioxazin + MSMAEG-EEF-G---EF-GEF-GG-EG-EPrometryn/ trifloxysulfuron + MSMAEG-EEF---GG-EEGG-EG-E1Weed species: CB = common cocklebur, DC = devil’s claw, FB = Florida beggarweed, FP = Florida pusley, GC = ground cherry, HS =hemp sesbania, NS =nightshade, JW = jimsonweed, LG = Common lambsquarters, MG = morningglory species, PA = Palmeramaranth, PW = pigweed species,PS = Prickly sida.2(University of Georgia, 2012).Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-80% control; P = 30% to 60% control; N= < 30% control3Personal communications with Dr. Wayne Keeling, Texas A & M University - 2011, Weed control ratings key: E = 90% or better control; G = 80% to 90%control; F = 60%-80% control; P = 30% to 60% control; N = < 30% control.4(University of California, 2012).Ratings Key: C = control, P = partial control, N = no control, - = no information.Monsanto Company 12-CT-244U 201 of 620Table VIII-14. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbicides in Cotton– Part IICommon Broadleaf Weeds1Product PL2RW2RdW2SP2SG2SN3SW2TB3TSW2VL4WB3Preplant IncorporatedPendimethalin E N N N N N N N N 0 NTrifluralin E N N N N N N N N 0 NPreemergencePendimethalinG N N N N N N N N 0 NClomazone G-E G G-E P N N E N F 10 NFluometuron E E E G P-F N G N F 3 NDiuron E G G-E F F N G N P-F 7 NFomesafen G G - P - N - N N 1 GPyrithiobac G N-P G-E P-F G N G N P 85NPostemergence Residual ControlMetolachlor G P - P P-F N - N E - NPyrithiobac G N-P G-E P G N G N P - NTrifloxsulfuron - - - P-F - N - N - - NMonsanto Company 12-CT-244U 202 of 620Table VIII-14. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbicides in Cotton– Part II (continued)Common Broadleaf Weeds1ProductPL2RW2RdW2SP2SG2SN3SW2TB3TSW2VL4WB3Postemergence Over-The-TopQuizalofopNNNNNNNNN0NFluazifopNNNNNNNNN0NSethoxydimNNNNNNNNN0NClethodimNNNNNNNNN0NMSMAP-FP-FNP-FNP-FN-PNP-GFluometuronF-GGF-GF-GP-FNF-GNP-NPyrithiobacFP-P-FF-GPGNF9NTrifloxsulfuron-GGE-PGNP-F-NGlyphosateF-GEEEGEGGP-G7GGlyphosate + PyrithiobacGEEEGEEGG-GGlyphosate + TrifloxsulfuronGE-EGEEGP-G-GGlufosinateF-GE-EF-GF-GGF-GP-F105F-GMonsanto Company 12-CT-244U 203 of 620Table VIII-14. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbicides in Cotton– Part II (continued)Common Broadleaf Weeds1ProductPL2RW2RdW2SP2SG2SN3SW2TB3TSW2VL4WB3Postemergence Directed – LaybyMSMAP-FFNFNNPNF-GDiuron + MSMAGEG-EG-EGFFNG65GPrometryn + MSMAF-GEGG-EGFFNF-G65GFlumioxazin + MSMAGG-E-G-EGF-GGNG-E95GPrometryn/ trifloxysulfuron +MSMA-E-E-F-NF-G95N1Weed species: PL = purslane, RW = Common ragweed, RdW = redweed, SP= sicklepod, SG = Spurge, SN = silverleaf nightshade, SW =smartweed, TB = Texas blueweed, TSW = tropical spiderwort, VL = velvetleaf, WB = woolyleaf bursage.2(University of Georgia, 2012).Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-80% control; P = 30% to60% control; N = < 30% control.3Personal communications with Dr.Wayne Keeling, Texas A & M University - 2011, Weed control ratings key: E = 90% or better control; G =80% to 90% control; F = 60%-80% control; P = 30% to 60% control; N = < 30% control.4(MSU, 2012).Rating scale: 0-3 = none to slight, 4-6 = fair, 7-8 = good, 9-10 = excellent control.5(University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control.

# Monsanto Company 12-CT-244U 204 of 620 

# VIII.F.2. Herbicide Resistant Weeds in Cotton

Table VIII-15 provides a summary of the co mmon weeds in cotton that have biotypes

reported resistant to the various herbicide modes-of-action in the U.S. To date there are

only two species with biotypes confirmed to be resistant to dicamba in the U.S. after over

40 years of use – kochia and prickly lettuce (Heap, 2012c). Additionally, a population of

lambsquarters has been confirmed as resi stant to dicamba in New Zealand, and in

Canada, common hempnettle and wild mustard have been confirmed as resistant, for a

total of five species worldwide with confir med resistance to dicamba. Currently in the

U.S., six grass species and eight broadleaf speci es have been confirmed to have resistance

to glyphosate. Dicamba provides good to excelle nt control of all eight of these broadleaf

species. None of these broadleaf weed biot ypes have been shown to have populations

that are resistant to both glyphosate and dicamba . The first species in the U.S. with a

biotype resistant to glufosinate was recently confirmed in a glyphos ate-resistant Italian

Ryegrass population (Avila-G arcia and Mallory-Smith, 2011) . Additionally, a population

of goosegrass from Malaysia has been confir med resistant to glufosinate (Seng et al.,

2010). Thus, there are a tota l of two species worldwide with biotypes that have

resistance to glufosinate. A discussion regarding the useful ness of MON 88701 in

management of herbicide resistant weeds can be found in Section VIII.G., and the

potential for development of dicamba and gl ufosinate resistance in weeds following the

introduction of MON 88701 can be found in Appendix I.

# Monsanto Company 12-CT-244U 205 of 620 

# Table VIII-15. Common Weeds in Cotton an d Weed Resistance to Herbicide Modes

of Action in the U.S. 1



# Mode of Action

Weed SpeciesACCase InhibitorsALS InhibitorsChloroacetamidesDinitroanilinesGlycinesOrganoarsenicalsPhotosystem IIInhibitorsThiocarbamatesUreas & AmidesSynthetic AuxinsBipyridiliumsPPO InhibitorsGlutamineSynthase Inhibitors



# Annual Grasses

Barnyardgrass X X X X X

Crabgrass spp. (large,

smooth) X X

Foxtail spp. (giant, green) X X X X

Italian ryegrass X X X X X

Goosegrass X X X X X

Junglerice X

Annual Broadleaves

Black nightshade

(Eastern) X X

Common cocklebur X X

Common purslane X X

Common ragweed X X X X

Horseweed (marestail) X X X X X

Jimsonweed X

Lambsquarters X X

Palmer amaranth X X X X

Prickly sida X

Pigweed spp. (redroot,

smooth, Powell,

waterhemp) X X X X X

Russian thistle X

Smartweed spp.

(Pennsylvania,

ladysthumb) X

Sunflower X

Velvetleaf X

Perennial Grasses

Johnsongrass X X X X

Perennial Broadleaves

Field bindweed X

1 

# (Heap, 2012d)

# Monsanto Company 12-CT-244U 206 of 620 

# VIII.G. Introduction of Dicamba and Glufo sinate-Tolerant Cotton - MON 88701

VIII.G.1. MON 88701 Product Concept

Monsanto has developed he rbicide-tolerant cotton, MON 88701, which will offer

growers cotton varieties that are tolerant to both dicamba and glufosinate herbicides.

Herbicide tolerances to dicamba and glufos inate were developed due to the benefits

associated with these herbicides, including: th e ability to control glyphosate-resistant and

hard-to-control weeds with two unique modes-of-action and the familiarity growers have

with these herbicides. Since dicamba is currently labeled for only preplant applications

in cotton, MON 88701 will facilita te a wider window of applic ation for dicamba in cotton

by allowing preemergence applications of di camba up to the day of crop emergence, as

well as postemergence in-crop applications up to seven days preharvest. MON 88701

will provide the ability for in-crop postemergence applications of glufosinate from

emergence up to the early bloom growth stage, which is the same as the current

application timing for glufosinate-tolerant cotton. MON 88701 will be combined with

glyphosate-tolerant cotton utilizing traditiona l breeding techniques. This combination of

herbicide-tolerance traits w ill allow the use of dicamba, glufosinate, and glyphosate

herbicides in an integrated weed manageme nt program to control a broad spectrum of

grass and broadleaf weed species in cotton. Th ese herbicides will pr ovide three distinct

modes-of-action for use in conjunction w ith other herbicide active ingredients and

modes-of-action for an effective weed re sistance management program in cotton.

Dicamba will offer improved in-crop posteme rgence control of glyphosate’s difficult-tocontrol broadleaf weeds, including Florid a pusley, hemp sesbania, lambsquarters,

morningglory species, prickly sida, purslane, and Pennsylvania smartweed (Table VIII16). Glufosinate will offer improved contro l of certain broadleaf weeds, including

lambsquarters, morningglory species, and velvetleaf, as compared to glyphosate.

Dicamba and glufosinate will also offer effec tive control options for glyphosate-resistant

broadleaf weed biotypes, including glyphosat e-resistant biotypes of Palmer amaranth,

marestail, common ragweed, giant ragweed, and waterhemp. Additionally, dicamba and

glufosinate will offer an effective control op tion for broadleaf species resistant to other

herbicide classes ( e.g. , ALS and PPO chemistries).

VIII.G.2. Dicamba and Glufosinate Usage in MON 88701

The current labeled use of dicamba in cotton is limited to early preplant application.

Significant restrictions exist in cotton for pr eplant application of dicamba, including a

maximum application rate of 0.25 lbs a.e. per acre, a 21-day interval between application

and planting cotton, and a minimum of one inch of rainfall or overhead irrigation before

planting cotton to avoid cotton injury (B ASF, 2008). To support the introduction of

MON 88701, Monsanto will be submitting an application to U.S. EPA to amend

Registration Number 524-582, a DGA salt formul ation, to allow preemergence and incrop postemergence dicamba applications to MON 88701. If approved, growers would

be authorized to apply dicamba alone or in mixtures with glyphosate, glufosinate, or

# Monsanto Company 12-CT-244U 207 of 620 

# other herbicides for preplant or postemer gence in-crop applications on MON 88701.

Pending EPA registration, dicamba would be authorized to be applied up to 1.0 lb a.e. per

acre prior to planting, up to the emerge nce of cotton, and postemergence in-crop

applications, up to 0.5 lbs a.e. per acre each, could be applied up through seven days prior

to harvest. These application rates are well within the dicamba rates applied to other

crops, such as corn and sugarcane (BASF, 2008). Maximum application amounts for

dicamba will be established for total preplant/preemergence applications and in-crop

applications with the combined total not to exceed 2.0 lbs a.e. per acre of dicamba per

year for all applications. Based on the dicamba label requested by Monsanto, aerial

applications of dicamba will not be allowed on MON 88701.

Glufosinate is currently labele d for preplant and in-crop applications in cotton varieties

designated as glufosinate-to lerant (Bayer CropScience, 2011). No changes to the

glufosinate product labels will be necessary to permit broadcast in-c rop applications of

glufosinate to MON 88701. Glufosinate can also be applied as a burndown treatment

prior to planting or prior to emergence of any conventional or non-glufosinate herbicidetolerant cotton varieties. Directed postem ergence applications are also permitted in nonglufosinate-tolerant varieties, provided no he rbicide contacts the cotton foliage. Once

MON 88701 is available, growers will be able to apply glufosinate alone or tank-mixed

with dicamba for preplant or postemerg ence in-crop applications on MON 88701.

Application rates and timings for glufosinate al one will be the same as currently labeled

for glufosinate use in glufosinate-tolerant varieties ( i.e. , from emergence up to the early

bloom stage at 0.402 to 0.530 lbs a.i./acre, seasonal maximum of 1.59 lbs a.i. per acre)

(Bayer CropScience, 2011).

The expected use patterns for dicamba and glufosinate on MON 88701 will vary across

U.S. cotton growing regions. This variability is dictated by the environment and weed

spectrum variations across these regions. The recommendations for the Midsouth and

Southeast regions are sh own in (Table VIII-16 ) . In these regions, conventional tillage

planted acres are expected to receive a single in-crop application per season of dicamba at

0.5 lbs a.e. per acre and conser vation tillage or no-tillage ac res are expected to receive

two applications (one prepla nt application at 0.375 lbs a.e. per acre and one in-crop

application at 0.50 lbs a.e. per acre). All acres in this regi on where glyphosate-resistant

weeds are present, regardless of tillage, are expected to receive a single in-crop

application of glufosinate as 0.53 lbs a.i. per acre. For the remaining acres where

glyphosate-resistant weeds are not present, glyp hosate will likely be used for control of

late-emerging weeds. Dicamba and glufosinate use in eastern Texas, is expected to be

similar to that described for the Mi dsouth and Southeast regions.

# Monsanto Company 12-CT-244U 208 of 620 

# Table VIII-16. Anticipated Weed Man agement Recommendations for MON 88701

Combined with Glyphosate-Tolerant Cotton Systems for MO, AR, TN, AL, FL, GA,

NC, SC, VA, LA, MS and eastern TX 1



# Application

Timing Conventional Tillage Conservation Tillage

(No-till or reduced till)

No GR

Weeds 2 

# GR Weeds or Suspected

GR Weeds 2 

# No GR

Weeds 2 

# GR Weeds or Suspected

GR Weeds 2



# Preemergence

(burndown, at

planting) 3 

# Residual Residual Dicamba

+

Glyphosate

+

Residual Dicamba

+

Glyphosate

+

Residual

Postemergence 3 

# Dicamba

+

Glyphosate Dicamba

+

Glyphosate

+

Residual Dicamba

+

Glyphosate

+

Residual Dicamba

+

Glyphosate

+

Residual

Postemergence 3 

# Glyphosate Glufosinate Glyphosate Glufosinate

+

Residual

1 

# The anticipated use patterns repres ent a high-end estimate for predic ting dicamba use associated with

MON 88701 combined with glyphosate-tolerant cotton. Actual weed control practices by growers will

vary depending on the specific weed spectrum and ag ronomic situation of the individual cotton field,

specifically dicamba use could be lower especially for the preemergence a nd second postemergence

applications.

2 

# Recommendations for all fields in these regions will assume GR weeds are present.

3 

# Monsanto and academics recommend the use of soil resi duals as part of a comprehensive weed resistance

management program to ensure that two effective herbicide modes-of-action are used in cotton and to

provide protections against additional resistance development to existing cotton herbicides.



# In western Texas, New Mexico, Kansas, Ok lahoma, California and Arizona, dicamba is

expected to be utilized more extensively than glufosinate for management of hard-tocontrol and/or glyphosate-resi stant weeds in MON 88701. Gluf osinate is considered less

effective on the weed spectrum under th e high temperature and low humidity

environmental conditions in these regions (Bayer CropScience, 2011). The

recommendations for these cotton growing areas are shown in (Table VIII-17 ) . All acres

are expected to receive one pr eplant application of dicamb a (0.375 lbs a.e. per acre).

Areas with glyphosate-resistan t weeds are also expected to receive two in-crop

applications of dicamba (0. 50 lbs a.e. /acre) per season, wh ereas areas without

glyphosate-resistant weeds will only receive one in-crop application of dicamba (0.50 lbs

a.e./acre).

# Monsanto Company 12-CT-244U 209 of 620 

# Table VIII-17. Anticipated Weed Man agement Recommendations for MON 88701

Combined with Glyphosate-Tolerant Cotto n Systems for western TX, NM, KS, OK,

CA, and AZ 1



# Application

Timing Conventional Tillage Conservation Tillage

(No-till or reduced till)

No GR

Weeds GR Weeds or Suspected

GR Weeds No GR

Weeds GR Weeds or Suspected

GR Weeds

Preemergence

(burndown, at

planting) 2 

# Dicamba

+

Glyphosate

+

Residual Dicamba

+

Glyphosate

+

Residual Dicamba

+

Glyphosate

+

Residual Dicamba

+

Glyphosate

+

Residual

Postemergence 2 

# Dicamba

+

Glyphosate Dicamba

+

Glyphosate Dicamba

+

Glyphosate Dicamba

+

Glyphosate

Postemergence 2 

# Glyphosate Dicamba

+

Glyphosate Glyphosate Dicamba

+

Glyphosate

1 

# The anticipated use patterns represent a high-end es timate for predicting dicamba use associated with

MON 88701 combined with glyphosate-tolerant cotton. Actual weed control practices by growers will

vary depending on the specific weed spectrum and ag ronomic situation of the individual cotton field,

specifically dicamba use could be lower especially for the preemergence a nd second postemergence

applications.

2 

# Monsanto and academics recommend the use of soil resi duals as part of a comprehensive weed resistance

management program to ensure that two effective herbicide modes-of-action are used in cotton and to

provide protections against additional resistance development to existing cotton herbicides.



# VIII.G.3. MON 88701 in Combination with Glyphosate-Tolerant Cotton Systems

With the introduction of MON 88701 into gl yphosate-tolerant sy stems, growers will

continue to be able to use dicamba, glufos inate, and glyphosate herbicides for preplant

burndown, without the plant-back restrictions currently in place. Tables VIII-10 and

VIII-12 show weed control ratings for glyphosate, glufosinate, glyphosate tank-mixed

with dicamba, and glufosinate tank-mixed with dicamba compared to other herbicide

regimes when applied as a preplant burndown application to common broadleaf weed

species found in fields prio r to planting cotton. Glyphos ate alone provides excellent

control of many grass species and is superior to glufosin ate on many grass species.

Therefore, glufosinate a pplications in MON 88701 are not expected to provide

improvement in control of grass species inno-till systems, except where glyphosateresistant grass species may be present. Cert ain hard-to-control broadleaf weeds such as

curly dock, Carolina geranium, cutleaf primrose , and wild radish, are difficult to control

with glyphosate. Similarly, glufosinate provides unsatisfactory control of certain

# Monsanto Company 12-CT-244U 210 of 620 

# broadleaf weeds such as Carolina geranium, cutleaf primrose, and henbit. Tank-mixing

dicamba with glyphosate or glufosinate improve s the control of the difficult-to-control

weed species for both of these products. The dicamba tank mix combinations will also

provide excellent control of glyphosate-resist ant marestail and fair to good control of

glyphosate-resistant Palmer amaranth. Dicamba will be complementary to glyphosate or

glufosinate for preplant burndow n weed control in cotton and will offer growers equal or

superior weed control to other preplant herbicides or herbicide tank mixtures.

MON 88701 will provide additional application flexibility with dicamba allowing

applications up to the day of planting in cott on. In addition to complementing the weed

control of glyphosate, dicamba and glufosinate will provide additiona l modes-of-action in

the glyphosate-tolerant cotton systems to lo wer the potential risk of weed species

developing resistance to glyphosate. Furtherm ore, dicamba and glufosinate will provide

alternative modes-of-action fo r control of broadleaf weeds with populations known to be

resistant to glycine, ALS, and PPO cla sses of herbicides (see Table VIII-15).

Upon integration of MON 88701 into glyphos ate-tolerant cotton systems, in-crop

postemergence applications of dicamba, gluf osinate, and glyphosate herbicides will be

permitted in cotton production. Tables VIII-18 and VIII-19 illustrate common broadleaf

weed responses to dicamba, glyphosate, glufos inate, and several ot her labeled in-crop

over-the-top herbicide treatments in cotton. Si nce dicamba is not currently labeled for incrop applications in cotton, weed control ratin gs for dicamba were taken from labeled incrop applications of dicamba in corn for comparison purposes. Glyphosate provides

good to excellent control of all the listed annua l grasses and most of the annual broadleaf

weeds. When compared to glufosinate, glyph osate provides better co ntrol of some of the

annual grasses (broadleaf signa lgrass, crabgrass, crowsfoot grass, foxtail, goosegrass,

seedling johnsongrass, Texas panicum), perennial grasses (johnsongrass and

bermudagrass), and some of the broadleaf weeds (devil’s claw, Florida beggarweed,

Florida pusley, pigweed species, and silverleaf nightshade). Howeve r, glufosinate data

illustrates slightly better control of some of the broadleaf weeds compared to glyphosate,

namely lambsquarter, morningglory, and velv etleaf. Dicamba as a tank mixture will

complement the weed control of in-crop application(s) of either glyphosate or

glufosinate. The use of dicam ba will improve control of most of the broadleaf weeds

where either glyphosate or gluf osinate provide unsatisfactory control, with the exception

of spurge, silverleaf nightshade, Texas blue weed, tropical spiderwort, velvetleaf, and

woolyleaf bursage. Other he rbicide treatments are available when needed to provide

effective control of these weed species. In addition, an in-c rop application of dicamba in

combination with either glyphosate or glufos inate will assist in the management of

glyphosate-resistant weed biotypes.

Currently, many residual and non-residual he rbicides are used in combination with

glyphosate in preplant and in-crop postemergen ce applications in cott on. The addition of

dicamba and glufosinate to the system are e xpected to offer increased benefits over the

current alternative herbicides as supplemen ts to glyphosate for preplant and in-crop

applications on MON 88701, including increased flexibility and reduced crop injury.

# Monsanto Company 12-CT-244U 211 of 620 

# Since planting interval restrictions following preplant applications of dicamba in cotton

will be removed, dicamba will have greater fl exibility for preplant applications than

current preplant applications of 2,4- D and will potentially replace some 2,4-D

applications in cotton. The broadleaf weed control provided by dicam ba and glufosinate,

plus the crop tolerance when applied to MON 88701, will allow the potential replacement

of some other in-crop alternat ive herbicides used for broa dleaf weed control in cotton,

particularly diuron, fomesafen, fluometu ron, and paraquat. Considering the

characteristics of dicamba and glufosinate from the perspective of weed control and

compatibility with glyphosate, it is c oncluded that MON 88701 will complement the

established safety and efficacy of glyphosat e use in glyphosate-tolerant cotton systems.

VIII.G.4. MON 88701 as a Weed Resistance Management Tool

Although herbicide resistance may eventually oc cur in a weed species when an herbicide

is widely used, resistance can be delaye d, contained, and mana ged through research,

education, and good management practices. The addition of dicamba and glufosinate

tolerance to the glyphosate-tolerant cotton sy stems will provide an efficient method for

incorporation of additional m odes-of-action in the system, and reduce the potential for

further resistance development to glyphosate, dicamba, and glufosinate, as well as other

important cotton herbicides. Current rese arch, conducted by Monsan to, to define the

optimum weed management systems indicate the following: 1) in MO, AR, TN, AL, FL,

GA, NC, SC, VA, LA, MS, and eastern TX, th e recommendation will be to apply a soilactive residual herbicide followed by an in -crop early postemergence application of

dicamba tank-mixed with glyphosate, and a residual product, follo wed by a late postemergence application of glufosinate tank-mixe d with a residual pr oduct (Table VIII-16 ) ;

and 2) in western TX, KS, OK, NM, AZ, a nd CA, the recommendation will be to apply a

soil-active residual herbicide, followed by an in-crop postemergence application of

dicamba tank-mixed with glyphosate at early and late postemergence (Table VIII-17 )

. This will ensure the use of more than one mode -of-action against the targeted species. In

both areas, a preplant application of di camba tank-mixed with glyphosate may be

recommended, in addition to the in-crop app lications described ab ove. This is not

expected to increase selec tion pressure on either produc t since the preplant weed

spectrum is generally different from the in-crop spectrum.

Stewardship of dicamba and glufosinate to pr eserve their usefulne ss for growers is an

important aspect of Monsanto’s stewardship co mmitment, as is discussed in Appendix I.

Specifically, Monsanto has implemented and will continue to develop and proactively

provide weed resistance management practices 6 

# , and will utilize multiple methods to

distribute technical and stewardship inform ation to growers, academics, and grower

advisors through a variety of communication tools. Monsanto’s Technology Use Guide

6 

# Weed resistance management guidelines available at http://www.weedtool.com and

http://www.monsanto.com/weedma nagement/Pages/default.aspx

# Monsanto Company 12-CT-244U 212 of 620 

# (TUG) will set forth the requirements and best practices for the cultivation of

MON 88701 including recommendations on weed resistance management practices.

Growers purchasing products containing MON 88701 are required by the Monsanto

Technology Stewardship Agreement (MTSA) to read and follow the TUG. Furthermore,

Monsanto is committed to actively evaluate herbicide performance and weed efficacy on

a continuing basis, and develop additional mitigation plans as necessary to manage

resistance development for glyphosat e, dicamba, and glufosinate.

VIII.G.5. Introduction of Dicamba a nd Glufosinate-Tolerant Cotton - MON 88701

- Conclusion

Integration of MON 88701 into glyphosate-tole rant cotton systems will allow the use of

dicamba, glufosinate, and glyphosate herbicid es in an integrated weed management

program to control a broad sp ectrum of grass and broadleaf weed species in cotton.

These herbicides will also provide three distinct modes-of-action for an effective

proactive and reactive weed resistance management program in cotton. Due to the crop

safety of MON 88701 to dicamba and glufosinat e, growers will be afforded two effective

herbicide modes-of-action fo r in-crop control of glyphosate’s hard-to-control and

resistant broadleaf weeds that are present in U. S. cotton production.

Furthermore, the integration of MON 88701, along with the glyphosate-tolerant cotton

systems, will provide growers with the abil ity to continue use of established cotton

production practices including tillage syst ems; the same planting and harvesting

machinery; traditional management of insects, diseases, and other pe sts; and many of the

current herbicides used for weed control, including glyphosate with its established

environmental and grower benefits. Th erefore, it is anticipated that the

commercialization of MON 88701 in the U.S. is not likely to impact current cotton

agronomic practices, cultivation or seed production practices, beyond the intended

benefits of more effective and improve d management of common and troublesome

weeds, including herbicide-resistant weeds.Monsanto Company 12-CT-244U 213 of 620Table VIII-18. Responses of Common Broadleaf Weeds to Dicamba and Glufosinate Compared to Labeled PostemergenceHerbicides in Cotton Production – Part ICommon Broadleaf Weeds1Product CB2DC3FB2FP2GC4HS2NS3JW2LQ2MG2PA2PW2PS2Postemergence Over-The-TopDicambaE5G-EG5G5C4E5C4E5E5E5G-E5G-E5E5MSMA E P-F E N-P P - P P P P-F P P PFluometuron F-G F-G G P-F - - - G G G P-F F F-GPyrithiobac G G G N-P P G-E C E N G F G FTrifloxsulfuron G-E G G-E P - - - N G G P-F F-G NGlyphosateEEEP-GCP-FCEGF-GEEF-GGlyphosate +Pyrithiobac E E E P-G - G-E - E G G-E E E GGlyphosate +Trifloxsulfuron E E E P-G - - - E E E E E GGlufosinateEG-EGFC-CEEEF-GGF1Weed species: CB = common cocklebur, DC = devil’s claw, FB = Florida beggarweed, FP = Florida pusley, GC = ground cherry, HS =hempsesbania, NS = nightshade, JW = jimsonweed, LG = Common lambsquarters, MG = morningglory species, PA = Palmer amaranth, PW =pigweed species, PS = Prickly sida.2(University of Georgia, 2012). Weed control ratings key: E =90% or better control; G = 80% to 90% control; F = 60%-80% control; P = 30% to60% control; N = < 30% control.3Personal communications with Dr. Wayne Keeling, Texas A & M University - 2011, Weed control ratings key: E = 90% or better control; G =80% to 90% control; F = 60%-80% control; P = 30% to 60% control; N = < 30% control.4(University of California, 2012).Ratings Key: C = control, P = partial control, N = no control, - = no information.5(University of Georgia, 2010).Weed control ratings key: E = Excellent control, 90%or above; G = Good control, 80% or above; F = Faircontrol, less than 80% control; P = Poor control.Monsanto Company 12-CT-244U 214 of 620Table VIII-19. Responses of Common Broadleaf Weeds to Dicamba and Glufosinate Compared to Labeled PostemergenceHerbicides in Cotton Production – Part IICommon Broadleaf Weeds1Product PL2RW2RdW2SP2SG2SN3SW2TB3TSW2VL4WB3Postemergence Over-The-TopDicambaE5E5-E5P6F3E5F3P5F-G5F3MSMA P-F P-F N P-F N P-F N-P N P - GFluometuron F-G G F-G F-G P-F N F-G N P - NPyrithiobac F P G P-F F-G P G N F 9 NTrifloxsulfuron - G G E - P G N P-F - NGlyphosateF-GEEEGEGGP-G7GGlyphosate +Pyrithiobac G E E E G E E G G - GGlyphosate +Trifloxsulfuron G E - E G E E G P-G - GGlufosinateF-GE-EF-GF-GGF-GP-F107F-G1Weed species: PL = purslane, RW = Common ragweed, RdW = redweed, SP= sicklepod, SG = Spurge, SN = silverleaf nightshade, SW =smartweed, TB = Texas blueweed, TSW = tropical spiderwort, VL = velvetleaf, WB = woolyleaf bursage.2(University of Georgia, 2012). Weed control ratings key: E =90% or better control; G = 80% to 90% control; F = 60%-80% control; P = 30% to60% control; N = < 30% control.3Personal communications with Dr. Wayne Keeling, Texas A & M University, Weed control ratings key: E = 90% or better control; G= 80% to90% control; F = 60%-80% control; P = 30% to 60% control; N = < 30% control.4(MSU, 2012).Rating scale: 0-3 = none to slight, 4-6 = fair, 7-8 = good, 9-10 = excellent control.5(University of Georgia, 2010).Weed control ratings key: E = Excellent control, 90%or above; G = Good control, 80% or above; F = Faircontrol, less than 80% control; P = Poor control.6(University of California, 2012).Ratings Key: C = control, P = partial control, N = no control, - = no information.7(University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control.

# Monsanto Company 12-CT-244U 215 of 620 

# VIII.H. Crop Rotation Practices in Cotton

The rotation of cotton with othe r crops is an integral part of most farm management

programs across the southern United States cotton growing region. Ideally, cotton should

be rotated with other crops on a regular basis to maintain soil productivity and reduce the

incidence of various weeds, inse ct pests, or diseases (Hake et al., 1996d). Rotating cotton

with grass crops such as corn helps to re duce the soil inoculum level of the seedling

disease fungi Pythium and Rhizoctonia . These seedling diseases can increase in

continuous cotton cropping systems. Crop rotati ons or the lack of rotations, along with

weed control programs used in these crops, can play an important factor on the weed

spectrum (Smith and Cothren, 1999). In ad dition, the crop rotati on and weed control

programs can increase or decrease the populat ions of certain weed species. Production

costs, relative rate of return, and the current market conditions will dictate which crops to

rotate with cotton or whether to grow c ontinuous cotton. These economic factors may

outweigh the agronomic benefits of crop ro tation. According to Sandretto and Payne

(2006) statistics, cotton was grown in a continuous cropping system on 73% of the

acreage in the major cotton grow ing states in 2003. Cotton was rotated with other row

crops such as corn or soybean on about 20% of the acreage.

Crop rotations for cotton vary fr om region to region and state to state and often within a

state. This section provides a detailed desc ription and quantitative assessment by state of

the rotational cropping pract ices immediately following cotton production. This

assessment accounts for about 99% of the total cotton acreage. These data are presented

in Tables VIII-20 through VIII-24). Sevent een crops immediately follow cotton in the

crop rotation sequence according to this assess ment. In the U.S., approximately 54% of

the cotton acres are followed by cotton in the crop rotation sequence. Corn (16%),

soybean (8%), sorghum (8%), wheat (9%), a nd peanuts (4%) are th e other crops most

frequently following cotton. The other crops following cotton are 0.5% or less of the

cotton acres.

Grower survey data available to Monsan to (2011) for dicamba, glufosinate, and

glyphosate herbicide usage were utilized for this as sessment. For the purpose of this

assessment, a 50% adoption rate in U.S. cotton and soybean production was assumed for

both MON 88701 and MON 87708 (dicamba-tolerant soybean), respectively. In the

following data tables, columns F, H, and J provide the number of acres of dicamba,

glufosinate, glyphosate that follow cotton in th e rotation crops, respectively. Columns K,

L, and M provide the percentage of dicamba , glufosinate, and gl yphosate usage in the

total rotational crop acreage ( i.e. , the percentage of cott on acres where dicamba,

glufosinate and glyphosate, respec tively, are used in the subse quent crop). For the entire

U.S. (Table VIII-20), 33.1%, 11.9%, and 75.0% of the rotational crop acreage would be

treated with dicamba, glufosin ate, and glyphosate, respec tively. The percentage of

dicamba usage in the rotation would be the highest in the Southeast region (35.8%) and

lowest in the West region (17.9%). MO (45.6%) and OK (48.0%) would be the states

with the highest dicamba usage in the rotati on and NM (5.8%) and AZ (3.2%) the lowest.

The percentage of glufosinate usage in the ro tation would be the highe st in the Southeast

(25.7%) and Midsouth (21.4%) regions and lowest in the Southwest region (2.5%). MO

(43.0%) and OK (45.1%) would be the states w ith the highest glufos inate usage in the

# Monsanto Company 12-CT-244U 216 of 620 

# rotation and LA (1.8%), AZ (0%), and NM (0.2%) the lowest. The percentage of

glyphosate usage in the rotation would be th e highest in the Midsouth region (89.9%) and

lowest in the West region (37.8.0%). MO (95.3%) would have the highest glyphosate

usage in the rotation and AZ (19.7%) the lowest. In the S outhwest region where almost

55% of the cotton is grown, 33.1%, 2.5%, and 70.4% of the rotational crop acres

following cotton would be treated with dicamba, glufosinate, and glyphosate,

respectively.Monsanto Company 12-CT-244U 217 of 620Table VIII-20. Rotational Practices in the U.S. Following Cotton ProductionA B C D E F G H I J K L MState/TotalCottonAcres1RotationalCropsFollowingCottonRotationalCropAcres2%RotationalCrop ofTotalCotton3DicambaUsage inRotationalCrop4GlufosinateUsage inRotationalCrop5GlyphosateUsage inRotationalCrop6% Usage in Total Rotational CropAcres7% Acres % Acres % Acres Dicamba Glufosinate GlyphosateUnitedStates10,974CottonCornSoybeanSorghumWheatBarleyPeanutSunflowerAlfalfa8Vegetables9Dry BeansPeppersTomatoesOnionsTobacco585817368618361025404322247500.5824630Total:1010,97453.415.87.87.69.30.43.90.20.40.50.0050.10.20.060.3508.1508.35.65.0NLNLNLNLNLNLNLNLNL29301414316957Total:3,63021.62.11.0NLNLNLNLNLNLNLNLNLNLNLNL1264378Total:1,30990.288.095.934.714.127.521.172.750240.037.545.833.3NL528415278262901451191162410.23112Total:8,23126.71.33.90.60.50.0233.111.50.30.111.948.213.97.52.61.30.10.80.10.20.010.0020.030.10.0275.0This table was developed by compiling the data from all four regional summaries (Tables VIII-21 through VIII-24). All acreagesare expressed as 1000s of acres.NL indicates not labeled for use.1Cotton acreage based on 2010planting data (USDA-NASS, 2011e).2Column C is obtained by compiling the data from the four regional summaries.3Column D is obtained by dividing Column C by Column A.4Column E is obtained by dividing Column F by Column C; Column F is obtained by compiling the data from all four regional summaries.5Column G is obtained by dividing Column H by Column C; Column H is obtained by compiling the data from all four regional summaries.6Column I is obtained by dividing Column J by Column C; Column J is obtained by compiling the data from all four regional summaries7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Column CTotal; Column M isobtained by dividing Column J Total by Column C Total.8Newly seeded alfalfa.9Vegetables: Cauliflower (37k acres), lettuce (271 kacres), and broccoli (124k acres) (USDA-NASS, 2011d).10Totals may not be exact due to rounding.Monsanto Company 12-CT-244U 218 of 620Table VIII-21. Rotational Practices Following Cotton Production in the Southeast RegionA B C D E F G H I J K L MState/TotalCottonAcres1RotationalCropsFollowingCottonRotationalCropAcresFollowingCotton2%RotationalCropAcres3DicambaUsage inRotationalCrop4GlufosinateUsage inRotationalCrop5GlyphosateUsage inRotational Crop6% Usage in Total Rotational CropAcres7% Acres % Acres % Acres Dicamba Glufosinate GlyphosateRegion2597CottonCornSoybeanPeanutTobacco131134050741030Total:10259750.513.119.515.81.1506.250NLNL65621254Total:930502.30.9NLNL65684Total:66895.188.397.820.9NL124730049686Total:212825.20.89.835.825.20.30.225.748.011.519.13.381.9AL340CottonCornSoybeanPeanut1026811951Total:34030203515507850NL51560Total:115502818NL5111Total:54979710030996611915Total:29915.01.417.533.915.00.40.415.829.119.435.04.588.0FL92CottonCornSoybeanPeanut469532Total:925010535507850NL2312Total:26502818NL2310.05Total:24997889482746749Total:6625.00.72.528.225.01.10.126.149.57.84.79.571.5GA1330CottonCornSoybeanPeanut798133133266Total:133060101020507850NL399967Total:475502818NL39931Total:4039392991874212213248Total:104430.00.75.035.730.00.20.130.355.89.29.93.678.5Monsanto Company 12-CT-244U 219 of 620Table VIII-21. Rotational Practices Following CottonProduction in the Southeast Region (continued)A B C D E F G H I J K L MState/TotalCottonAcres1RotationalCropsFollowingCottonRotationalCropAcres2%RotationalCrop ofTotalCotton3DicambaUsage inRotationalCrop4GlufosinateUsage inRotationalCrop5GlyphosateUsage inRotationalCrop6% Usage in Total Rotational CropAcres7% Acres % Acres % Acres Dicamba Glufosinate GlyphosateNC550CottonCornSoybeanPeanutTobacco231831654428Total:55042153085507850NLNL116383Total:201502818NLNL11621Total:119997995238NL2296515710Total:46121.00.515.036.521.00.40.221.541.611.928.51.883.8SC202CottonCornSoybeanPeanutTobacco101306182Total:20250153041507850NLNL51230Total:83502818NLNL5111Total:52100869823810126592Total:18825.01.115.041.125.00.30.325.650.012.929.40.993.2VA83CottonCornSoybeanPeanut3317258Total:8340203010507850NL17112Total:30502818NL170.30.2Total:179179992383013252Total:7020.01.415.036.420.00.40.320.736.415.829.72.384.2The Southeast region summary was developed by compiling the data from all the states within the region. Column C, Column F, Column H, and Column J areobtained by compiling data from all the states within this region; Column D is obtained by dividing Column C by Column A, Column E is obtained by dividingColumn F by Column C, Column G is obtained by dividing Column H by Column C, Column I is obtained by dividing Column J by Column C. All acreages areexpressed as 1000s of acres. NLindicates not labeled for use.1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e).2Column C is obtained by multiplying Column A by Column D.Monsanto Company 12-CT-244U 220 of 6203The rotational crop percentages in Column D are based on estimates from individual state Extension Crop Production Specialist and Extension Weed ControlSpecialist in cotton (Dale Monks, Auburn University; David Wright,Ph.D., University of Florida; Jared Whitaker, Ph.D., University of Georgia; MichaelJones, Ph.D., Clemson University; and Henry Wilson, Ph.D., Virginia Tech University, Personal Communications, November, 2010).4Dicamba usage data in Column E except for cotton and soybean is based on 2010 data (Monsanto Company, 2011). Dicamba usage in cotton (50%) and soybean(50%) are future market adoption estimates.5Glufosinate usage data in Column G except for cotton is based on 2010 (Monsanto Company, 2011). Glufosinate usage in cotton (50%) is the future marketadoption estimate.6Glyphosate usage data in Column I is based on 2010 data (Monsanto Company, 2011).7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Column CTotal, and Column Mis obtained by dividing Column J Total by Column C Total.8Since no data was reported or the survey sample size was too small for the herbicide data in the state to be statistically reliable, the percent usage for theherbicide/crop in the U.S. was used.10Totals may not be exact due to rounding.Monsanto Company 12-CT-244U 221 of 620Table VIII-22. Rotational Practices FollowingCotton Production in the Midsouth RegionA B C D E F G H I J K L MState/TotalCottonAcres1RotationalCropsFollowingCottonRotationalCropAcres2%RotationalCrop ofTotalCotton3DicambaUsage inRotationalCrop4GlufosinateUsage inRotationalCrop5GlyphosateUsage inRotationalCrop6% Usage in Total Rotational CropAcres7% Acres % Acres % Acres Dicamba Glufosinate GlyphosateRegion1920CottonWheatCornSoybeanSorghum7865971133727Total:9192040.93.037.017.61.4506.010.150123934721693Total:64050NL2.11.0NL393154Total:4119915859445780960731712Total:172520.50.23.78.80.233.320.50.80.221.440.60.531.616.50.689.9AR545CottonCornSoybeanSorghum21816413627Total:545403025550785012810916683Total:196502818NL10931Total:1141007888745821812811912Total:47620.02.912.50.636.020.00.60.320.940.023.421.82.387.4LA255CottonCornSoybean019164Total:2550752550785001332Total:45502818041Total:410078100014964Total:2135.312.517.81.50.31.858.525.083.5MS420CottonCornSoybean16818963Total:420404515507850841332Total:1295028188441Total:8899989916618562Total:41420.03.27.530.720.00.90.221.139.644.114.998.6Monsanto Company 12-CT-244U 222 of 620Table VIII-22. Rotational Practices Following CottonProduction in the Midsouth Region (continued)A B C D E F G H I J K LState/TotalCottonAcres1RotationalCropsFollowingCottonRotationalCropAcres2%RotationalCrop ofTotalCotton3DicambaUsage inRotationalCrop4GlufosinateUsage inRotationalCrop5GlyphosateUsage inRotationalCrop6% Usage in Total Rotational Crop Acres7% Acres % Acres % Acres Dicamba Glufosinate GlyphosateMO310CottonCornSoybean2643116Total:3108510550785013228Total:142504213210.3Total:13310059882641814Total:29542.50.62.545.642.50.40.143.085.05.94.495.3TN390CottonWheatCornSoybean1375913759Total:39035153515506811506842729Total:12850NL28186831Total:729715893100132912759Total:32717.50.96.97.532.817.50.70.218.434.02.332.615.083.8The Midsouth region summary was developed by compiling the data from all the states within the region. Column C, Column F, Column H, and Column J areobtained by compiling data from all the states within this region; Column D is obtained by dividing Column C by Column A, Column E is obtained by dividingColumn F by Column C, Column G is obtained by dividing Column H by Column C, Column I is obtained by dividing Column J by Column C. All acreages areexpressed as 1000s of acres. NLindicates not labeled for use.1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e)2Column C is obtained by multiplying Column A by Column D.3The rotational crop percentages in Column D are based on estimates from individual state Extension Crop Production Specialist and Extension Weed ControlSpecialist in cotton (Tom Barber, Ph.D., University of Arkansas;John Kruse, Louisiana State University; Darrin Dobbs, Ph.D.,Mississippi State University;Andrea Jones, Ph.D., University of Missouri; and Larry Steckel, Ph.D., University of Tennessee, Personal Communications November, 2010).4Dicamba usage data in Column E except for cotton and soybean is based on 2010 data (Monsanto Company, 2011). Dicamba usage in cotton (50%) and soybean(50%) are future market adoption estimates.5Glufosinate usage data in Column G except for cotton is based on 2010 (Monsanto Company, 2011). Glufosinate usage in cotton (50%) is the future marketadoption estimate.6Glyphosate usage data in Column I is based on 2010 data (Monsanto Company, 2011).7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Column CTotal, and Column Mis obtained by dividing Column J Total by Column C Total.8Since no data was reported or the survey sample size was too small for the herbicide data in the state to be statistically reliable, the percent usage for theherbicide/crop in the U.S. was used.9Totals may not be exact due to rounding.Monsanto Company 12-CT-244U 223 of 620Table VIII-23. Rotational Practices FollowingCotton Production in the Southwest RegionA B C D E F G H I J K L MState/TotalCottonAcres1RotationalCropsFollowingCottonRotationalCropAcres2%RotationalCrop TotalCotton3DicambaUsage inRotationalCrop4GlufosinateUsage inRotational Crop5GlyphosateUsage inRotationalCrop6% Usage in Total Rotational Crop Acres7% Acres % Acres % Acres Dicamba Glufosinate GlyphosateRegion5953CottonWheatCornSoybeanSorghumDry BeanAlfalfa8PeanutsSunflowerPeppers3607765685178080.51822228Total:10595360.612.911.50.313.60.010.30.40.40.1505.37.0508.2NLNLNLNLNL18044148866Total:19673.8NL2.01.0NLNLNLNLNLNL138140.2Total:15286.913.990.578.234.435.050.023.074.043.03136106620132780.295163Total:418830.30.70.80.11.133.12.30.20.0032.552.71.810.40.24.70.0030.10.10.30.170.4KS51CottonWheatCornSoybeanSorghum31320313Total:515.025.040.05.025.0501311501912212Total:950NL2919NL10.40.03Total:21008889657311827Total:312.53.34.22.54.717.22.50.80.13.45.02.035.24.814.361.3Monsanto Company 12-CT-244U 224 of 620Table VIII-23. Rotational Practices Following CottonProduction in the Southwest Region (continued)A B C D E F G H I J K L MState/TotalCottonAcres1RotationalCropsFollowingCottonRotationalCropAcres2%RotationalCrop ofTotalCotton3DicambaUsage inRotationalCrop4GlufosinateUsage inRotationalCrop5GlyphosateUsage inRotationalCrop6% Usage in Total Rotational Crop Acres7% Acres % Acres % Acres Dicamba Glufosinate GlyphosateNM50CottonWheatCornSorghumDry BeansAlfalfa8Peppers312550.5188Total:505.024.010.010.01.035.015.0506979129NLNLNL10.70.41Total:30NL29NLNLNLNL00.1Total:0.19191597894593595043922420.293Total:222.51.40.71.25.80.20.24.63.67.84.50.417.56.544.8OK285CottonCornSoybeanSorghum2579146Total:285903525079501291280.670.7Total:137502919NL1280.20.1Total:129996975352546112Total:27345.00.22.50.248.045.00.10.145.189.12.13.80.795.6TX5567CottonWheatCornSorghumPeanutsSunflower33467406517852222Total:556760.113.311.714.10.40.450578NLNL1673394563Total:18190.25NL29NLNLNL813Total:21861491342397492877104593267516Total:386230.10.70.81.132.70.20.20.451.71.910.64.80.10.369.4The Southwest region summary was developed by compiling the data from all the states within the region. Column C, Column F, Column H, and Column J areobtained by compiling data from all the states within this region; Column D is obtained by dividing Column C by Column A, Column E is obtained by dividingColumn F by Column C, Column G is obtained by dividing Column H by Column C, Column I is obtained by dividing Column J by Column C. All acreages areexpressed as 1000s of acres. NLindicates not labeled for use.1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e).Monsanto Company 12-CT-244U 225 of 6202Column C is obtained by multiplying Column A by Column D.3The rotational crop percentages in Column D are based on estimates from individual state Extension Crop Production Specialist and Extension Weed ControlSpecialist in cotton (Stewart Duncan, Ph.D., Kansas State University; Jamshid Ashigh, Ph.D., New Mexico State University; DonMurray, Ph.D., OklahomaState University; Wayne Keeling, Ph.D., Texas A & M University;and Gaylon Morgan, Ph.D., Texas A & M University, Personal CommunicationsNovember, 2010).).4Dicamba usage data in Column E except for cotton and soybean is based on 2010 data (Monsanto Company, 2011). Dicamba usage in cotton (50%) and soybean(50%) are future market adoption estimates.5Glufosinate usage data in Column G except for cotton is based on 2010 (Monsanto Company, 2011). Glufosinate usage in cotton (50%) is the future marketadoption estimate for most of the Southwest region. Noglufosinate usage is expected in NM and West TX.6Glyphosate usage data in Column I is based on 2010 data (Monsanto Company, 2011).7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Column CTotal, and Column Mis obtained by dividing Column J Total by Column C Total.8Newly seeded alfalfa; Glyphosate usage in alfalfa (50%) is future market adoption estimate.9Since no data was reported or the survey sample size was too small for the herbicide data in the state to be statistically reliable, the percent usage for theherbicide/crop in the U.S. was used.10Totals may not be exact due to rounding.Monsanto Company 12-CT-244U 226 of 620Table VIII-24. Rotational Practices FollowingCotton Production in the West RegionA B C D E F G H I J K L MState/TotalCottonAcres1RotationalCropsFollowingCottonRotationalCropAcres2%RotationalCrop ofTotalCotton3DicambaUsage inRotationalCrop4GlufosinateUsage inRotationalCrop Option5GlyphosateUsage inRotationalCrop6% Usage in Total Rotational Crop Acres7% Acres % Acres % Acres Dicamba Glufosinate GlyphosateRegion504CottonWheatBarleyAlfalfa8TomatoesOnionsVegetables9153202403024650Total:1150430.440.07.95.94.91.29.850.06.04.0NLNLNLNL77122Total:9050.0NLNLNLNLNLNL77Total:7779.015.027.050.043.027.03.01213011151121Total:19015.22.40.317.915.215.224.06.02.12.92.10.30.337.8AZ198WheatBarleyAlfalfa8Vegetables979403050Total:19840201525610410NLNL52Total:6NLNLNLNLTotal:015102710503101211151Total:391.60.53.20.06.05.47.50.819.7CA306CottonWheatTomatoesOnions153122246Total:30650408250610NLNL777Total:8450NLNLNL77Total:77791510432712118112Total:15125.02.427.425.025.039.56.03.40.549.5The West region summary was developed by compiling the data from all the states within the region. Column C, Column F, Column H, and Column J areobtained by compiling data from all the states within this region; Column D is obtained by dividing Column C by Column A, Column E is obtained by dividingColumn F by Column C, Column G is obtained by dividing Column H by Column C, Column I is obtained by dividing Column J by Column C. All acreages areexpressed as 1000s of acres. NLindicates not labeled for use.1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e).2Column C is obtained by multiplying Column A by Column D.Monsanto Company 12-CT-244U 227 of 6203The rotational crop percentages in Column D are based on estimates from individual state Extension Crop Production Specialist and Extension Weed ControlSpecialist in cotton (Bill McCloskey, Ph.D.,University of Arizona; and Steve Wright,Ph.D., University of California, PersonalCommunications, November,2010).4Dicamba usage data in Column E except for cotton and soybean is based on 2010 data (Monsanto Company, 2011). Dicamba usage in cotton (50%) and soybean(50%) are future market adoption estimates.5Glufosinate usage data in Column G except for cotton is based on 2010 (Monsanto Company, 2011). Glufosinate usage in cotton (50%) is the future marketadoption estimate.6Glyphosate usage data in Column I is based on 2010 data (Monsanto Company, 2011).7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Column CTotal, and Column Mis obtained by dividing Column J Total by Column C Total.8Newly seeded alfalfa; Glyphosate usage in alfalfa (50%) is future market adoption estimate.9AZ acreage: cauliflower (3), lettuce (62), broccoli (7); U.S. acreage: cauliflower(35), lettuce (199), broccoli (125).10Since no data was reported or the survey sample size was too small for the herbicide data in the state to be statistically reliable, the percent usage for theherbicide/crop in the U.S. was used.11Totals may not be exact due to rounding.

# Monsanto Company 12-CT-244U 228 of 620 

# VIII.H.1. Cotton Volunteer Management

Volunteer cotton refers to plants that have germinated, emerged and established

unintentionally from the previous year’s co tton crop (Roberts et al., 2002). Volunteer

cotton plants generally come from seed that falls to the ground as a result of preharvest

losses due to adverse weather condition or losses during the harvesting operation.

Volunteer cotton will compete with the rota tional crop and potentially cause yield loss

and act as early host plants for pests such as spider mites and aphids (Roberts et al.,

2002). Although volunteer cotton in soybean fields can impact yield, recent studies

indicate that other common grasses or broadl eaf weeds are more problematic in soybean

(Lee et al., 2009). The occurrence of volun teer cotton depends on the tillage after

harvesting the crop and the severity of wint ers. Cotton volunteers are more frequently

observed in conservation tillage systems where tillage is not used prior to planting. An

integrated weed management system of tillage and herbicides has traditionally been the

most common method of volunteer co tton control (Roberts et al., 2002).

Mechanical tillage prior to planting is an effective and efficient method for controlling

seedling volunteer cotton plants. This is accomplished in most soil conditions because

the root and hypocotyls of seedling cotton are easily destroyed by the tillage process

(Roberts et al., 2002). Any damage occurri ng below the cotyledons will kill the plant

because there are no growing points from which the plant can recover. The

disadvantages of tillage are moisture loss under arid conditions and the possibility of

increased soil erosion. In-cr op cultivation is a highly eff ective option for satisfactory

control of volunteer seedlings. Where cultiva tion is not appropriate, the use of herbicides

is effective in controlling volunteers.

University weed specialists have identified numerous effective and economical herbicide

treatments for control of volunteer cotton in the various rotational crops including cotton

(Table VIII-25). University studies have shown that the timing of the herbicide

application can greatly impact the effectiveness of many herbicides. Newly emerged

cotton (up to 2- 3-leaf stage) as a volunteer is much easier to control with herbicides than

more mature cotton (Thompson and Steckel, 2010) . If the volunteer cotton plants contain

the glyphosate-tolerance trait, the use of glyphosate alone in subsequent rotational crops

will not control these seedlings. Simila rly, volunteer cotton plants containing the

dicamba and glufosinate-tolerant traits from MON 88701 would not be controlled with

either dicamba or glufosinate herbicides, and alternative herbicides would be required for

control.

Currently both dicamba and glufosinate are labe led for use in crops rotated with cotton.

Dicamba herbicide is labeled for weed cont rol in soybean, corn, cotton, sorghum, wheat,

barley, oats, millet, pasture, rangeland, aspara gus, sugarcane, turf, grass grown for seed,

conservation reserve programs, and fallow croplands. Glufosinate is used for

postemergence weed control in canola, cor n, cotton, and soybean varieties containing

glufosinate-tolerance. It may also be a pplied as a preplant burndown application in

conventional or herbicid e-tolerant varieties of canola, corn, cotton, soybean, or sugarbeet.

The herbicide control options available in rotational crops will continue to result in the

ability to manage cotton volunteers.

# Monsanto Company 12-CT-244U 229 of 620 

# Preplant burndown applications of carfent razone, paraquat, or flumioxazin will

effectively control emerged vol unteer cotton prior to plantin g rotational crops (Murdock

et al., 2002; Roberts et al., 2002). In most situations, these preplant measures are

sufficient and no additional control measur es specifically for cotton volunteers are

required the remainder of the season. In the event these measures are not sufficient, the

preplant treatment will generally reduce infestation levels, allowing for more effective incrop management of the remaining vol unteer cotton in rotational crops.

In emerged cotton, in-crop cu ltivation has been traditionally used in the subsequent

cotton crop or other crops to effectivel y remove weeds and volunteer cotton plants

between the crop rows. In redu ced tillage situations, special high-residue cultivators with

sweeps may be used to effectively lift w eeds out of the soil to leave the ground cover

undisturbed. Cotton emerging within the ro w can negatively impact cotton growth and

management decisions due to increased plant population and disease susceptibility

(Roberts et al., 2002). However, plants remaining at the end of the season can generally

be harvested with the planted population by mechanical picking or stripping. Several

herbicides are also available for control of volunteer cotton plants after the emergence of

the rotational crop. In emerged cotton, applications of carfentrazone or paraquat with

hooded sprayers or other selectiv e equipment will effectively control volunteer plants and

other weeds in row middles (Alford et al., 2002; Murdock et al., 2002). However, special

precautions must be taken to ensure that thes e non-selective herbicid es do not contact the

cotton crop (Gray et al., 2002). Chlorimuron and imazaquin provide control of volunteer

cotton in soybean (Clemmer et al., 2001; York et al., 2004) (Table VIII-25). Volunteer

cotton in corn generally is not an issue because of the sensitivity of cotton to a number of

commonly used corn herbicides ( e.g. , atrazine).

# Monsanto Company 12-CT-244U 230 of 620 

# Table VIII-25. Herbicides and Applicatio n Timing for Control of Volunteer Cotton

in Labeled Rotational Crops 1



# Rotation Crop/ Herbicide Product Rate

Product/Acre Preplant /

Preemergent InCrop

Cotton

Gramoxone (paraquat)

C 32 oz X X*

Aim (carfentrazone) 1 oz X X*

ET (pyraflufen) 1.5 oz X X*

Layby Pro (linuron/diuron) 32 oz X*

Sharpen (saflufenacil) 1.5-2.0 oz X

Corn

Atrazine 32 oz X X

Callisto (mesotrione) 3 fl oz X X

Sharpen (saflufenacil) 1.5-2.0 oz X

Status (diflufenzopyr/dicamba) 0.75 oz X X

2,4-D 32 oz X X

Soybean

Resource (flumiclorac) 8 oz X

Gramoxone Inteon (paraquat) 32 oz X

Sencor + Classic (metribuzin +

chlorimuron) X

Classic (chlorimuron) 2/3 oz X

2,4-D 32 oz X

Peanuts

Gramoxone Inteon (paraquat) 32 oz X

Classic (chlorimuron) 2/3 oz X

Sunflower

Paraquat 32 oz X

Sharpen (saflufenacil/) 1.5-2.0 oz X

Buctril 1 pt X

Sorghum

Gramoxone Inteon (paraquat) 32 oz X

Sharpen (saflufenacil) 1.5-2.0 oz X

Atrazine 32 oz X X

Wheat

CleanWave (aminopyralid/fluroxypyr) 14 oz X

Buctril (bromoxynil) 1 pt X

2,4-D 32 oz X

Starane (fluroxypyr) 16 oz X

Sharpen (saflufenacil) 1.5-2.0 oz X

1 

# (Grichar et al., 2010; Keeling et al., 2009; Roberts et al., 2002; Thompson and Steckel, 2010; York et al.,

2004).

* Hooded or selective equipment only

# Monsanto Company 12-CT-244U 231 of 620 

# VIII.I. Stewardship of MON 88701

Monsanto develops effective products and tech nologies and is committed to assuring that

its products and technologies are safe and environmentally responsible. Monsanto

demonstrates this commitment by implem enting product stew ardship processes

throughout the lifecycle of a product and by pa rticipation in the Excellence Through

Stewardship SM 

# (ETS) Program (BIO, 2010). Thes e policies and practices include

rigorous field compliance and quality management systems and verification through

auditing. Monsanto’s Stewards hip Principles are also ar ticulated in Technology Use

Guides (Monsanto Company, 2012) and Monsanto Technology Stewardship Agreements

that are signed by growers who utilize Monsan to branded traits, to ensure stewardship

compliance.

As an integral action of fulfilling this st ewardship commitment, Monsanto will seek

biotechnology regulatory appr ovals for MON 88701 in all ke y cotton import countries

with a functioning regulatory sy stem to assure global compliance and support the flow of

international trade. These actions will be consistent with the Biotechnology Industry

Organization (BIO) Policy on Product Launc h (BIO, 2010). Monsanto continues to

monitor other countries that are key impor ters of cotton from the U.S., for the

development of formal biotechnology approval processes. If new functioning regulatory

processes are developed, Monsanto will make appropriate and timely regulatory

submissions. In addition, Monsanto actively interacts with and participates in cotton

industry groups, such as the National Cotton Council, state grower boards, Farm Bureau,

Cotton Inc., and trade affiliates, to obtain input on market trends to ensure awareness of

the current key markets for whole cottonseed and cottonseed by-products.

Monsanto also commits to industry best pract ices on seed quality assurance and control

to ensure the purity and integrity of MON 8870 1 cottonseed. As with all of Monsanto’s

products, before commercializing MON 88701 in any country, a MON 88701 detection

method will be made available to cotton producers, processors, and buyers.

The dicamba and glufosinate-tolerant cotton system, which is applying dicamba and/or

glufosinate herbicide to MON 88701 integrated into the gly phosate cotton systems, will

enable expanded use of dicamba herbicide in cotton production. Monsanto is seeking

regulatory approvals with the U.S. EPA fo r the expanded application of dicamba

herbicide as a weed control tool in cott on. Furthermore, Monsanto will establish

appropriate dicamba Maximum Residue Leve ls (MRLs) for key cotton import countries

. No additional regulatory approvals with U.S. EPA will be required for glufosinate

products for use in MON 88701.

As with all U.S. EPA registered herbicides fo r agricultural use, it is possible that offsite

movement during and/or following application can occur such that non-target plants may

be exposed to direct spray or to spray drift. Research has demonstrated that herbicide

formulation, application equipment, and a pplication procedures can be optimized to

significantly reduce spray drif t potential in most circumstances (Jordan et al., 2009;

SDTF, 1997). Monsanto is addressing potenti al offsite movement of dicamba by seeking

U.S. EPA registration of a low volatility dicamba formulation (DGA salt) for ground

# Monsanto Company 12-CT-244U 232 of 620 

# application only. Additionally Monsanto wi ll implement a robust stewardship program

that will include a st rong emphasis on grower and appli cator training. Furthermore,

Monsanto will consult with U.S. EPA to id entify what additional measures, if any are

necessary, to address any potential impact of off-site movement of these herbicides.

Stewardship of dicamba and glufosinate, to pr eserve their usefulness for growers, is also

an important aspect of Monsanto’s stewardship commitment. Detailed information

regarding dicamba and glufos inate weed resistance and the usefulness of dicamba and

glufosinate-tolerant cotton in combination with glyphosate-tolerant cotton to address

herbicide-resistance issues is presen ted in Section VIII.G and Appendix I.

VIII.J. Impact of the Introduction of MON 88701 on Agricult ural Practices

Introduction of MON 88701 is expected to have no impact on current agronomic,

cultivation and management practices for co tton, with the exception of expanded dicamba

application timings and more options for eff ective weed management. Dicamba has been

used in corn, soybean, and small grain cropping systems sinc e 1967. MON 88701 with

its excellent crop tolerance to dicamba allo ws preemergence applications through crop

emergence and in-crop postemergence applicati ons up to seven days prior to harvest.

MON 88701 will be combined with glyphos ate-tolerant cotton systems utilizing

traditional breeding techniques. Cotton containing both MON 88701 and glyphosatetolerance will allow the use of glyphosate, di camba, and glufosinate herbicides in an

integrated weed management program to control a broad spectrum of grasses and

broadleaf weed species, and to sustain and complement the benefits and value of the

glyphosate use in glyphosate-tol erant cotton systems.

MON 88701 has been shown to be comparable to commercially cultivated cotton in its

agronomic, phenotypic, and compositional characte ristics (refer to Se ctions VI, VII, and

VIII), and has the same levels of susceptibility to insect pests and diseases as commercial

cotton. Like other herbicide-to lerant cotton, such as glyphosat e-tolerant cotton that have

been cultivated and consumed in the U.S. since 1996, dicamba and glufosinate tolerant

cotton (MON 88701) will improve the current agricultural practices for U.S. cotton

growers by providing two additional in-crop herbicide modes-of-action for the control of

glyphosate’s hard-to-control and resistant broadl eaf weeds, as well as weeds resistant to

other herbicide families, thereby improving th e efficiency in the U.S. cotton production

system to maximize or maintain cotton yield potential, and help m eet growing needs for

fiber, food, and feed.

# Monsanto Company 12-CT-244U 233 of 620 

# IX. ENVIRONMENTAL ANALYSIS

IX.A. Introduction

This section provides a brief review and assessment of the plant pest potential of

MON 88701 and its impact on current ag ronomic practices. USDA-APHIS has

responsibility, under th e Plant Protection Act (7 U.S. C. § 7701-7772), to prevent the

introduction and dissemination of plant pe sts 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 arti cle does not present a pl ant pest risk and no

longer should be regulated. If APHIS dete rmines that the regulated article does not

present a plant pest risk, the petition must be granted, thereby allowing unrestricted

introduction of the article.

The definition of “plant pest” in the Plant Protection Act (PPA) in cludes living organisms

that could directly or indirectly injure, da mage, or cause disease in any plant or plant

product (7 U.S.C. § 7702[14]).

The regulatory endpoint under the PPA for bi otechnology-derived crop products is not

zero risk, but rather a determination that dere gulation of the regulated article is unlikely

to pose a plant pest risk. The approach us ed to assess the plant pest potential of

MON 88701 is a weight-of-evidence approach ba sed primarily on eight lines of evidence:

1) insertion of a single functional copy of the dmo and bar expression cassettes;

2) characterization of MON 88701 DMO and PAT ( bar ) expressed in MON 88701;

3) protein safety of MON 88701 DMO and PAT ( bar ); 4) compositional equivalence of

harvested MON 88701 seed to conventiona l commercial cotton; 5) phenotypic,

agronomic, plant mapping, and environmental in teraction characteristics demonstrating

no increased plant pest potential compared to commercially cultiv ated cotton, including

disease and pest susceptibilities; 6) negligible risk to NTOs; 7) familiarity with cotton as

a cultivated crop and the inherently low plant pest potential of cott on; and 8) no greater

likelihood to impact agronomic practices, incl uding land use, cultivation practices, or the

management of weeds (other than the intende d benefits of dicamba and glufosinate for

weed control), diseases, and insects than commercially cultivated cotton.

Using the aforementioned assessment, the data a nd analysis presented in this petition lead

to a conclusion that MON 88701 is unlikely to be a plant pest, and therefore should no

longer be subject to regulati on under 7 CFR Part 340.

IX.B. Plant Pest Assessment of MO N 88701 Insert and Expressed Proteins

This section summarizes the details of the genetic insert, characteristics of the genetic

modification, and safety and expr ession of the MON 88701 DMO and PAT ( bar ) proteins

used to evaluate the food, feed, and environmental safety of MON 88701.

# Monsanto Company 12-CT-244U 234 of 620 

# IX.B.1. Characteristics of the Gene tic Insert and Expressed Protein

IX.B.1.1. Genetic Insert

As described in Section IV, molecular anal yses demonstrated that MON 88701 contains a

single copy of the inserted T-DNA at a single integration locus. No backbone sequences

from the PV–GHHT6997 were detected in th e genome of MON 88701. In addition, data

confirmed the organization and sequence of the insert and the stability of the insert over

several breeding generations.

IX.B.1.2. Mode-of-Action

MON 88701 exhibits tolerance to the herbicide dicamba through the insertion of a

demethylase gene from Stenotrophomonas maltophilia that encodes DMO and the

herbicide glufosinate through the inserti on of a N-acetyltransferase gene from

Streptomyces hygroscopicus that encodes PAT. The DMO protein is a Rieske-type nonheme iron oxygenase that catalyzes the demet hylation of dicamba to the non-herbicidal

compound DCSA (Section V.A.1.). As shown in section V.A. a nd by Dumitru et. al.

(2009), DMO is specific for dicamba.

The PAT protein has been extensively asse ssed, as numerous glufosinate-tolerant

products including those in cotton, corn, so y, canola, sugarbeet and rice have been

reviewed by the USDA and several other re gulatory agencies (ILSI-CERA, 2011; OECD,

1999b; 2002a). The PAT ( bar ) protein produced in MON 88701 acetylates the free

amine group of L-phosphinothricin form of glufosinate to produce non-herbicidal

N-acetyl glufosinate (Section V.A.2.). PAT is specific for glufosinate (Thompson et al.,

1987; Wehrmann et al., 1996).

IX.B.1.3. Protein Safety and Expression

The safety of the expressed proteins is detail ed in Section V. A history of safe use has

been established for both MON 88701 DMO and PAT ( bar ) proteins (Section V.E).

MON 88701 DMO and PAT ( bar ) lack structural similarity to known allergens, gliadins,

glutenins, or protein toxins (S ection V.D). MON 88701 DMO and PAT ( bar ) are present

at very low levels in MON 88701 cottonseed (Section V.C.) and will constitute a small

portion of the total protein present in feed derived from MON 88701 (Section V.E.). No

consumption of the MON 88701 DMO or PAT ( bar ) proteins derived from MON 88701

is expected for the U.S. gene ral population at the present time given th at the only foods

produced from cottonseed are RBD oil and linters, which contain undectable and

neligible amounts of total protein, respectively. As shown in Section V.E,

MON 88701 DMO and PAT ( bar ) are readily digestible in simulated gastric and

simulated intestinal fluids and show no oral toxicity in mice (Section V.E.). In addition,

PAT proteins have been evaluated in several previous safety assessments with no safety

concerns identified.

# Monsanto Company 12-CT-244U 235 of 620 

# IX.B.2. Compositional Characteristics

Detailed compositional analys es in accordance with OECD guidelines were conducted to

assess whether levels of key nutrients and anti-nutrients in M ON 88701, both treated and

not treated with dicamba or gl ufosinate herbicides, were comparable to levels present in

the near isogenic conventional cotton cont rol Coker 130 and se veral conventional,

commercial reference varieties (Section VI). Seed were harvested from eight individual

sites in which MON 88701, the conventional control, and a range of commercial

reference varieties were grown concurrently in the same field trial. The commercial

reference varieties were used to establish a range of natural variability for the key

nutrients and anti-nutrients in commercial co tton varieties that have a history of safe

consumption.

The combined-site analysis was conducted to determine statis tically significant

differences (5% level of significance) be tween MON 88701 treated with dicamba and

glufosinate and the conventiona l control. The biological si gnificance of difference from

the combined-site data were reviewed using considerations releva nt to food and feed

safety and nutritional quality. These consider ations included: 1) the relative magnitude

of differences in the mean values of nutrient and anti-nutrient components of

MON 88701 and the conventional control; 2) whether the MON 88701 component mean

value was within the range of natural variabili ty of commercial cotton as represented by

the 99% tolerance interval of the commercial reference varieties grown concurrently in

the same field trial; 3) whether the MON 88701 component mean value was within the

context of the natural variability of comm ercial cotton composition as published in the

scientific literature and/or available in the ILSI Crop Composition Database; and

4) analyses of the reproducib ility of the statistic ally significant comb ined-site component

differences at in dividual sites.

Assessment of the analytical results confir med that the differences observed in the

combined-site analysis were not meaningful to food and f eed safety or the nutritional

quality of MON 88701 cotton. To furthe r support the safety assessment, similar

compositional analyses were also conducted on cottonseed from MON 88701 not treated

with dicamba or glufosinate herbicides. Ba sed on the analyzed nutrient and anti-nutrient

levels of both dicamba and glufosinate- treated and not trea ted MON 88701, MON 88701

is compositionally equivalent to conventional commercial cotton and therefore the food

and feed safety and nutritional quality of this product is comparable to that of

commercially cultivated cotton. These re sults support the overa ll conclusion that

MON 88701 is unlikely to be a plant pest.

IX.B 3. Phenotypic, Agronomic, Plant Mapping, and Environmental Interaction

Characteristics

An extensive set of comparative plant charac terization data were used to assess whether

the introduction of the dicamba and glufosinat e tolerance traits a ltered the plant pest

potential of MON 88701 compared to th e conventional control (Section VII).

Phenotypic, agronomic, plant mapping, and envi ronmental interaction characteristics of

MON 88701 were evaluated and compared to those of the conventio nal control (Section

# Monsanto Company 12-CT-244U 236 of 620 

# VII.B). As described below, these assessmen ts included: seed dormancy and germination

characteristics; agronomic and phenotypic char acteristics; plant mapping characteristics;

observations for abiotic stress response, di sease damage, arthropod-related damage,

arthropod abundance, and pollen ch aracteristics. To support th e trait assessment, similar

observations were also conduc ted on MON 88701 treated with dicamba and glufosinate

herbicides. Results from all phenotypic, agronomic, plant mappi ng, and environmental

interaction assessments demonstrated that MON 88701 does not possess weedy

characteristics, or increased susceptibility or tolerance to specific diseases, insects, or

abiotic stressors compared to the conventional control. Taken together, the results of the

analysis support a determination that MON 88701 is no more likely to pose a plant pest

risk or have a biologically meaningful change in environm ental impact than

commercially cultivated cotton.

IX.B.3.1. Seed Dormancy and Germination

Seed dormancy and germination characteri zation demonstrated that MON 88701 seed

had germination characteristics similar to those of the conventi onal control (Section

VII.C.1). In particular, the lack of ha rd seed, a well-accepte d characteristic often

associated with plants that are weeds, suppor ts a conclusion of no increased weediness or

plant pest potential of MON 88701 compared to commercially cultivated cotton.

IX.B.3.2. Plant Growth and Development

Of the growth and development characte ristics assessed between MON 88701 and the

conventional control, eight si gnificant differences were detected in a combined-site

analysis (Section VII.C.2.1). MON 88701 obser ved a reduction in plant height at 30

DAP and at harvest, increased number of nodes above white flower at observations 2 and

3, a lower seed index, increased total and immature seed per boll, and increased fiber

strength. The differences in mean values of MON 88701 were within the range of the

commercial reference varieties for these charact eristics. Thus, the differences in these

parameters are not considered to be biol ogically meaningful in terms of increased

weediness or plant pest potential of MON 88701 compared to commercially cultivated

cotton.

Plant mapping is a process commonly used to quantify growth and development

parameters of the cotton plant, includi ng boll retention and distribution (Section

VII.C.2.2). Of the plant mapping characte ristics assessed between MON 88701 and the

conventional control, one significant diffe rence was detected where MON 88701 had

increased first-position bolls per plant compared to the conventional control. However,

the mean value of the number of first-posit ion bolls for MON 88701 was within the range

of values observed for the commercial referenc e varieties. Thus, it is unlikely that the

difference is biologically meaningful in the context of increased weediness of

MON 88701 compared to commercially cultivated cotton.

# Monsanto Company 12-CT-244U 237 of 620 

# IX.B.3.3. Response to Abiotic Stressors

No biologically meaningful differences were observed during comparative field

observations between MON 88701 and the conventional cont rol in their response to

abiotic stressors, such as compaction, dr ought, high winds, nutrien t deficiency, etc.

(Section VII.C.2.3). The lack of significant biologically meaningful differences in the

MON 88701 response to abiotic stress support th e conclusion that the introduction of the

dicamba and glufosinate-tolerance traits are unlikely to result in increased weediness or

plant pest potential compared to commercially cultivated cotton.

IX.B.3.4. Pollen Morphology and Viability

Evaluations of pollen morphology and viabili ty from field-grown plants provide

information useful in a plant pest assessment as it relates to the potential for gene flow

and introgression of the biotechno logy-derived trait(s) into ot her cotton varie ties and wild

relatives (Section VII.C.3). Pollen morphol ogy and viability evaluations demonstrated

no statistically signifi cant differences between MON 88701 a nd the conventional control.

Taken together, these comparative assessments indicate that MON 88701 is not likely to

have increased weediness or plant pest poten tial compared to commercially cultivated

cotton.

IX.B.3.5. Interactions with Non-target Organisms

Evaluation of MON 88701 for potential adverse impacts on NTOs is a component of the

plant pest risk assessment. Since MON 88701 is not intended to have pesticidal activity,

all organisms that interact with MON 88701 can be considered to be NTOs. In 2010 U.S.

field trials, observational da ta on environmental interactions were collected for

MON 88701 and the conventional control. In addition, multiple commercial reference

varieties were included in the analysis to establish a range of natural variability for each

characteristic among commercial cotton vari eties. The environmental interactions

assessment (Section VII.C.2.3) included data collected on plant-arth ropod, plant-disease

interactions, and plant mapping. The result s of this assessment indicated that the

presence of the dicamba and glufosinate-to lerance traits did not alter plant-arthropod

interactions, including benefi cial arthropods and arthropod pe sts, nor did it alter disease

susceptibility of MON 88701 compared to c onventional cotton. In addition, there were

no differences in plant mapping paramete rs between MON 88701, not treated with

dicamba or glufosinate herbicides, and the c onventional control that would be indicative

of a differential plant response to abiotic or biotic stressors. Thus, since all plots

evaluated for plant mapping ch aracteristics were at the same sites they would be

subjected to similar stressors. Given that MON 88701 plants treated and not treated with

dicamba and glufosinate herbicides had similar plant map results, it can be concluded that

both responded to stressors in a similar ma nner. Therefore, these data support the

conclusion that the biotechnol ogy-derived traits in MON 8 8701, treated or not treated

with dicamba and glufosinate herbicides, ar e unlikely to have increased plant pest

potential, weediness, or an adverse environmental impact compared to commercially

cultivated cotton. The lack of biologically meaningful di fferences in disease damage,

arthropod-related damage, pest- and benefi cial-arthropod abundance, and plant mapping

# Monsanto Company 12-CT-244U 238 of 620 

# data demonstrate that the introduction of the dicamba and glufosinate-tolerance traits are

unlikely to be biologically meaningful in terms of increased plant pest potential as

compared to commercially cultivated cotton.

The potential for MON 88701 to influence NTOs was evaluated using a combination of

biochemical information and experimental data. The biochemical information and

experimental data included molecular characterization, the MON 88701 DMO and

PAT ( bar ) safety assessments, the history of environmental exposure to

mono-oxygenases (the class of enzymes to which DMO belongs) and PAT proteins,

results from the environmental interac tions assessment described above, and the

demonstration of compositional, agronomic and phenotypic equivalence to conventional

cotton. Taken together, these data support the conclusion that M ON 88701 is unlikely to

adversely affect NTOs, including those bene ficial to agricultur e. Any effects on nontarget organisms that could potentially result from proposed changes in herbicide labels

will be evaluated by the EPA.

IX.C. Weed Potential of MON 88701

Cotton is not listed as a weed in the major w eed references (Crockett, 1977; Holm et al.,

1997), nor is it present on the lists of noxious weed species distributed by the federal

government (7 CFR Part 360). United States Department of Agriculture has previously

determined that “cotton is not considered to be a serious, principal or common weed pest

in the U.S.” (USDA-APHIS, 1995). Commercial Gossypium species in the U.S. are not

considered weeds and are not effective in invading establis hed ecosystems. Cotton is not

considered to have weedy characteristics in the U.S. and does not possess attributes

commonly associated with weeds, such as long soil persistence, the ab ility to invade and

become a dominant species in new or divers e landscapes, or the ability to compete well

with native vegetation. It is recognized th at in some agricultural systems, cotton can

volunteer in a subsequent rotational crop. However, volunteers are easily controlled

through tillage or use of appropriate herbicides (Alford et al., 2002; Murdock et al., 2002;

Roberts et al., 2002).

In comparative studies between MON 88701 a nd the conventional control, phenotypic,

agronomic, plant mapping, and environmental in teraction data were evaluated (Section

VII) for changes that would impact the plant pe st potential and in pa rticular, plant weed

potential. Results of these ev aluations show that there is no biologically meaningful

difference between MON 88701 and the conve ntional control for characteristics

potentially associated with weediness. Furthermore, comparative field observations

between MON 88701 and its conven tional control in their res ponse to abiotic stressors,

such as drought, heat stre ss, and high winds, i ndicated no biologically meaningful

differences and, therefore, no increased weed potential. Data on environmental

interactions also indicate that MON 88701 does not confer a ny biologically meaningful

increased susceptibility or tolerance to speci fic diseases or insect pests. Collectively,

these findings support the conclusion th at MON 88701 has no increased weediness

compared to commercially cultivated cotton.

# Monsanto Company 12-CT-244U 239 of 620 

# Volunteer MON 88701, like volunteer commer cial cotton, would compete poorly with

any succeeding crops and soon die, making it extremely unlikely to have any prolonged

negative effects. Volunteer MON 88701 woul d also not be “extremely difficult to

manage” because it can be controlled easil y with numerous altern ative herbicides and

other mechanical means (Alford et al., 2002; Mu rdock et al., 2002; Robe rts et al., 2002).

IX.D. Potential for Pollen Mediated Gene Flow and Introgression

Pollen mediated gene flow (often referred to as cross pollination) occurs when pollen of

one plant fertilizes ovules of a second plant. Pollen mediated gene flow is affected by

both biotic and abiotic factors such as plant biology, pollen bi ology/volume, plant

phenology, overlap of flowering times, proximity of the pollen source and sink, ambient

conditions such as temperature and humidit y, and field architecture. Pollen mediated

gene flow is a natural biological proce ss, and therefore does not constitute an

environmental risk in and of itself.

Introgression is a process wher eby one or more genes successfully incorporate into the

genome of a recipient plant. Pollen mediated gene flow and gene introgression must be

considered in the context of the transgenes inserted into the biot echnology-derived plant,

and the likelihood that the presen ce of the transgenes and th eir subsequent transfer to

recipient plants and plant populations will resu lt in increased plant pest potential. The

potential for gene flow and introgression from deregulatio n of MON 88701 is discussed

in greater detail below.

IX.D.1. Hybridization with Cultivated Cotton

Although natural crossing can occur, cotton is normally considered to be a selfpollinating crop (Niles and Feaster, 1984). There are no morphological barriers to crosspollination based on flower structure. Howe ver, the pollen is heavy and sticky and

transfer by wind is limited. Pollen is transferred instead by insects, in particular by

various wild bees, bumble bees ( Bombus sp.), and honeybees ( Apis mellifera ) (Van

Deynze et al., 2005). Numerous studies on cotto n cross-pollination have been conducted,

and the published results, with and without supplemental pollinators, are summarized in

Table IX-1. Recent cotton lit erature shows that the fre quency of cross-pollination

decreases with distance from the pollen so urce. McGregor (1976) traced movement of

pollen by means of fluorescent particles and found that, even among fl owers located only

150 to 200 feet from a cotton field that was surrounded by a large numbe r of bee colonies

to ensure ample opportunity for transfer of pollen, fluorescent particles were detected on

only 1.6% of the flowers. In a 1996 study with various fiel d designs, Llewellyn and Fitt

(1996) also found low levels of cross-pollination in cotton. At one meter from the source

they observed cross-pollination frequencies of 0.15 to 0.4%, decreasi ng to below 0.3% at

16 meters from the source. Umbeck et al. ( 1991) used a selectable marker to examine

cross-pollination from a 30 x 136 meter source of biotechnology-deri ved cotton. Crosspollination decreased from five to less than one percent from one to seven meters,

respectively, away from the source plot. A low level of cross-pollination (less than one

percent) was sporadically detected at the fu rthest sampling distance of 25 meters. Berkey

et al. (2002) reported that cro ss pollination between fields separated by a 13 foot road

# Monsanto Company 12-CT-244U 240 of 620 

# decreased from 1.89% in the row nearest the so urce to zero percent in the 24th row. Van

Deynze et al. (2005) conducted a two year study on pollen-mediated gene flow with high

and low pollinator activity. In the presen ce of high pollinator activity the pollination

frequency was 7.65% at 0.3 meters and less th an 1% at greater than nine meters.

Whereas, the pollination frequency in the presence of low pollinator activity was below

1% at just over a meter. In a 2008 study, pollination frequencies of 5.00% and 0.00%

were demonstrated at 1 and 8 meters, respectively (Kairichi et al., 2008).

The potential for outcrossing and gene in trogression from MON 88701 to cultivated

cotton in the U.S. is low since cotton polle n movement by wind is limited due to it is

large and sticky nature, and several studies have demonstrated that cross-pollination,

even in the presence of high pollinator activity, is limited by distance. Therefore, the

environmental consequences of pollen tr ansfer from MON 88701 to other cotton or

related Gossypium species is considered to be negligible.

# Monsanto Company 12-CT-244U 241 of 620 

# Table IX-1. Summary of Published Li terature on Cotton Cross Pollination

Distance

from Pollen

Source

(meters) CrossPollination

(%) Comments Reference

45-61 1.60% Used fluorescent particles to follow

pollinator movement in cotton fields

over one season. (McGregor, 1976)

1 0.15-0.4% Used a selectable marker to examine

cross-pollination in the progeny of

buffer row plants over one season. (Llewellyn and Fitt,

1996) 4 <0.08%

16 <0.03%

1 5% Used a selectable marker to examine

cross-pollination from a 20 x 136

meter source of biotechnologyderived cotton over one season. (Umbeck et al., 1991)

1-25

<1%

5 1.89% Used herbicide bioefficacy to

examine pollen flow between fields

separated by a 13 foot road over one

season. (Berkey et al., 2002) 10.5 0.77%

17 0.13%

25 0.00%

0.3 7.65% * Used herbicide bioefficacy confirmed

by DNA testing to measured pollenmediated gene flowing in four

directions over 2 years. (Van Deynze et al.,

2005) >9 < 1% *

>1 < 1% **

1625 0.04% **

1 5.00% Used ELISA strips to examine

pollen-mediated gene flow in four

directions from Bt source over a

period of one season. (Kairichi et al., 2008) 2-7 2.00%

8 0.00%

* High pollinator activity

** Low pollinator activity



# IX.D.2. Hybridization with Wild and Feral Gossypium species

Based on cytological evidence, seven genomic types, A through G, many with subtypes,

have been identified for the genus Gossypium (Endrizzi et al., 1984) . The domesticated

species G. hirsutum and G. barbadense are allotetraploid (AADD, 2n=4x=52), while G.

thurberi is a diploid (DD, 2n=2x=26), and G. tomentosum is an allotetraploid (AADD,

2n=4x=52). G. tomentosum is capable of crossing with domesticated cotton to produce

fertile offspring (Waghmare et al., 2005). However, Hawaii is the only U.S. region

where G. tomentosum is found and domesticated cotton is not grown commercially in

Hawaii, with the exception of potentia l counter-season breeding nurseries where

appropriate isolation distances and practices are required (Wagner et al., 1990). Thus, the

potential for gene flow to these wild rela tives is limited. Importantly, MON 88701 would

not be expected to confer a selective adva ntage to, or enhance th e pest potential of,

progeny resulting from such a cross if it were to occur. Any potential gene exchange

between G. thurberi and domesticated cotton, if it were to occur, would result in triploid

# Monsanto Company 12-CT-244U 242 of 620 

# (ADD, 3x=39), sterile plants because G. hirsutum and G. barbadense are allotetraploids

(AADD, 2n=4x=52) and G. thurberi is a diploid (DD, 2n=2x=26). Such sterile hybrids

have not been observed to persist in the wil d. Fertile allohexaploi ds (6x=78) have not

been reported in the wild.

Only two ‘wild’ Gossypium species related to cultivated cotton are known to be present

in the U.S., G. thurberi Todaro, which is known in Arizona (Fryxell, 1984), and feral

populations of cultivated G. hirsutum and ‘wild’ populations of G. hirsutum are known to

occur in South Florida and Puerto Rico (Bruba ker et al., 1999). Bo th of these species

would be capable of crossing with cultivated cotton, but they are not known to exist in

cotton growing areas. Importantly, MON 88701 would not be expected to confer a

selective advantage to, or e nhance the pest potential of , progeny resulting from such

crosses if they were to occur.

Importantly, the environmental consequences of pollen transfer from MON 88701 to

other cotton or related Gossypium species is considered to be negligible due to the plant

biology and limited movement of cotton pollen, the safety of the introduced protein, and

the lack of any selective advantage by the di camba and glufosinate tr aits that might be

conferred on a recipient pl ant of feral or wild co tton, or a wild relative.

IX.D.3. Transfer of Genetic Informatio n to Species with which Cotton Cannot

Interbreed (Horizontal Gene Flow)

Monsanto is unaware of any reports regardi ng the unaided transfer of genetic material

from cotton species to other sexually-incompatible plant species. The likelihood for

horizontal gene flow to occur is exceedingly small. Therefore, potential ecological risk

associated with horizontal gene flow fr om MON 88701 due to the presence of the

dicamba and glufosinate-toleran ce traits are not expected. The consequence of horizontal

gene flow of the dicamba and glufosinate-to lerance traits into other plants that are

sexually-incompatible is negligible since, as data presented in this petition confirm, the

genes and traits confer no increased plant pe st potential to cotton. Thus, in the highly

unlikely event that horizontal gene transfer we re to occur, the pres ence of the dicamba

and glufosinate-toleranc e traits would not be expected to increase pest potential in the

recipient species.

IX.E. Potential Impact on Cotton Agronomic Practices

An assessment of current cotton agronomic practices was conducted to determine

whether the cultivation of MON 88701 has th e potential to impact current cotton and

weed management practices (Section VIII). Cotton fields are typically highly managed

agricultural areas that are dedicated to crop production. MON 88701 is likely to be used

in common rotations on land pr eviously used for agricultura l purposes. Certified seed

production will continue to use well-established industry practices to deliver high quality

seed containing MON 88701 to growers. Cu ltivation of MON 88701 is not expected to

differ from typical cotton cultivation, with the exception of an expanded window of

dicamba applications. As gl ufosinate is alrea dy utilized within U.S. cotton growing

areas, no change in agronomic practices or land use would occur with the cultivation of

# Monsanto Company 12-CT-244U 243 of 620 

# MON 88701 and the presence of the glufosinate-to lerance trait. Due to the crop safety of

MON 88701 to dicamba and glufosinate, gr owers will have two herbicide modes-ofaction for in-crop control of glyphosate’s ha rd-to-control and resi stant broadleaf weeds

that are present in U.S. cotton production. As a result of cultivation of MON 88701

integrated into the glyphosat e-tolerant cotton systems, th e number of dicamba-treated

cotton acres will likely increase, whereas the number of glufosinate-treated cotton acres

is expected to remain relatively static with minimal increase in use as cotton varieties

utilizing the biotechnology-deri ved glufosinate-tolerance trai t are currently commercially

available and being utilized across the U.S. cottonbelt. Additionally, due to the expanded

timing of in-crop applications to cotton, dica mba treatments will be later in the growing

season than most current labeled dicamba uses.

MON 88701 is similar to commercially cultivated cotton in its agronomic, phenotypic,

ecological, and compositional characteristics, and has levels of resistance to insect pests

and diseases comparable to commercially cultivated cotton. Based on this assessment,

the introduction of MON 88701 is not likely to impact current U.S. cotton agronomic or

cultivation practices, or weed management practices, other than the intended weed

control benefits.

IX.F. Conventional Breeding with Oth er Biotechnology-derived or Conventional

Cotton

Several biotechnology-derived cotton products have been deregulated or are under

consideration for deregulation. Once deregul ated, MON 88701 may be bred with these

deregulated biotechnology-deri ved cotton products, as well as with conventional cotton,

creating new improved varieties. APHIS ha s determined that none of the individual

biotechnology-derived cotton prod ucts it has previously dere gulated displays increased

plant pest characteristics. APHIS has al so concluded that a ny progeny derived from

crosses of these deregulated biotechnology-de rived cotton products with conventional or

previously deregulated biot echnology-derived cotton are unlikely to exhibit new plant

pest properties. This presumption, that combined-trait biot echnology products are

unlikely to exhibit new characteristics that would pose new plant pest risks or potential

environmental impacts not observed in the single event biotech product, is based upon

several facts. Namely: 1) st ability of the genetic inserts is confirmed in each approved

biotech-derived cotton product across multiple generations (See Section IV.E for

MON 88701 data); 2) stabilit y of each of the introduced traits is continually and

repeatedly assessed as new combined-trait varieties are created by plant breeders and

tested over multiple seasons prior to commerci alization; 3) combined-trait products are

developed using the well established proce ss of conventional breed ing that has been

safely used for thousands of years to generate new varietie s (Cellini et al ., 2004; NRC,

2004; WHO, 1995); 4) worldwide organizations , such as World Health Organization,

Food and Agriculture Organization/World H ealth Organization, International Seed

Federation, CropLife International and U.S. FDA, conclude that the safety of the

combined-trait product can be based on the safe ty of the parental GE events (CLI, 2005;

FAO-WHO, 1996; ISF, 2005; U.S. FDA, 2001; WHO, 1995); and 5) practical

applications in the field have shown that two unrelated biotechnol ogy traits combined

together by conventional breeding do not di splay new characteri stics or properties

# Monsanto Company 12-CT-244U 244 of 620 

# distinct from those present in the single ev ent biotech products (Brookes and Barfoot,

2012; James, 2010; Lemaux, 2008; Pilaci nski et al., 2011; Sankula, 2006)

.

Therefore, based on the considerations a bove and the conclusion that MON 88701 is no

more likely to pose a plant pest risk than commercially cultivated cotton it can be

concluded that any progeny derived from crosses between MON 88701 and conventional

cotton or deregulated biotechnol ogy-derived cotton are no more likely to pose a plant risk

than commercially cultivated cotton.

IX.G. Summary of Plant Pest Assessments

Plant pests are defined in the PPA as certa in living organisms th at can directly or

indirectly injure, cause damage to, or cause disease to an y plant or plant product (7

U.S.C. § 7702[14]). Characte rization data presented in S ections III through VII of this

petition confirm that although MON 88701 contains the dicamba and glufosinate-tolerant

traits, it is not different from commercially cu ltivated cotton in terms of pest potential in

its phenotypic, agronomic, plant mapping, and e nvironmental interactio n characteristics.

Monsanto is not aware of a ny study results or observati ons associated with MON 88701

that would suggest an increas ed plant pest risk would re sult from its introduction.

The plant pest assessment was based on multiple lines of evidence developed from a

detailed characterization of MON 88701 comp ared to commercially cultivated cotton,

followed by a risk assessment on detected di fferences. The risk assessment considered

various factors, including: 1) inser tion of a single functional copy of the dmo and bar

expression cassettes; 2) characterizati on and safety of th e MON 88701 DMO and

PAT ( bar ) proteins; 3) compositional equivale nce of harvested MON 88701 cottonseed

as compared to commercially cultivated cotton; 4) phenotypic, agronomic, and

environmental interaction characteristics demonstrating no increased plant pest potential

compared to commercially cultivated cotton; 5) negligible risk to NTOs; 6) familiarity

with cotton as a cultivated crop and the inherent ly low plant pest potential of cotton; and

7) no greater likelihood to im pact agronomic practices, incl uding land use, cultivation

practices, or the management of weeds, diseases, and insects than commercially

cultivated cotton, with the exception of the expanded window of dicamba application.

Based on the data and information presented in this petition, it is concluded that, like

conventional cotton and currently dere gulated biotechnology-derived cotton,

MON 88701 is highly unlikely to be a plant pest. Therefore, Monsanto Company

requests a determination from APHIS th at MON 88701 and any progeny derived from

crosses between MON 88701 and other commercial cotton be granted nonregulated status

under 7 CFR Part 340.

# Monsanto Company 12-CT-244U 245 of 620 

# X. ADVERSE CONSEQUENCES OF INTRODUCTION

Monsanto does not know of any results or observations associated with MON 88701 or

the MON 88701 DMO and PAT ( bar ) proteins indicating that there would be an adverse

environmental consequence from the in troduction of MON 88701. MON 88701 contains

DMO and PAT that confers dicamba and gluf osinate tolerance to the cotton plant,

respectively. As demonstrated by field re sults and laboratory tests, the only phenotypic

differences between MON 88701 and conventiona l cotton are the tolerances to dicamba

and glufosinat e herbicides.

The data and information presented in th is petition demonstrate that MON 88701 is

unlikely to pose an increased plant pest risk or to have an adverse environmental

consequence compared commercially cultivated cotton. This conclusion is reached based

on multiple lines of evidence developed from a detailed characterization of the product

compared to commercially cultivated cott on, followed by a risk assessment on detected

differences. The characterization evalua tion included molecular analyses, which

confirmed the insertion of a single functional copy of the dmo and bar expression

cassettes at a single locus within the cott on genome. The amino acid sequence of the

MON 88701 DMO and PAT ( bar ) proteins expressed in MON 88701 are identical to the

amino acid sequences of the respective E. coli -produced proteins utilized in the protein

safety studies supporting the safety of the pr oteins. Analyses of key nutrients and, antinutrients of MON 88701 seed demonstrate that MON 88701 is compositionally

equivalent to commercially cultivated cotton. The phenotypic evaluations of

MON 88701, including an assessment of seed germination and dormancy characteristics,

plant growth and development characteri stics, plant mapping parameters, pollen

characteristics, and environmental interacti ons also indicated that MON 88701 is no more

weedy than commercially cultivated cott on. There is no indication that MON 88701

would have an adverse impact on beneficial or non-target or ganisms. Therefore, based

on the lack of increased plant pest potenti al or adverse environmental consequences

compared to commercially cultivated cott on, the risks for humans, animals, and other

NTOs from introducing MON 88701 are neg ligible under the conditions of use.

Successful integration of MON 88701 into glyphosate-tolerant cotton systems will

provide growers with an opportunity for an ef ficient, effective weed management system

for the management of glyphosate’s hard-to- control and resistant broadleaf weeds;

provide a flexible system for inclusion of a second and third herbicide mode-of-action in

cotton production practices as recommended by weed science experts to manage weed

resistance development; and continue to pr ovide cotton growers with effective weed

control systems necessary for production yiel ds to meet the growing needs of the food,

feed, and industrial markets.

# Monsanto Company 12-CT-244U 246 of 620 

# REFERENCES

Adrian-Romero, M., G. Blunden, B.G. Carpenter and E. Tyihák. 1999. HPLC quantification of

formaldehyde, as formaldemethone, in plants and plant-like organisms. Chromatographia 50:160-166.

Alexander, M.P. 1980. A versatile stain for po llen fungi, yeast and bacteria. Stain Technology 55:13-18.

Alford, J.L., R.M. Hayes, T.C. Muelle r and G.N. Rhodes. 2002. Roundup Ready ® 

# soybean ( Glycine max )

and cotton ( Gossypium hirsutum L.) control in Roundup Ready ® 

# cotton ( Gossypium hirsutum L.). Pages 2-

3 in 2002 Proceedings of the Southern Weed Science Society, Atlanta, Georgia.

Anderson, W.P. 1996. Weed ecology. Pages 27-38 in Weed Science: Principles and Applications. Third

Edition. West Publishing Company, St. Paul, Minnesota.

AOSA. 2007. Tetrazolium testing handbook. Contribution No. 29. Association of Official Seed Analysts,

Lincoln, Nebraska.

AOSA. 2010a. AOSA Rules for testing seeds. Volume 1: Principles and procedures. Volume 1: Principles

and procedures, Association of Offici al Seed Analysts, Ithaca, New York.

AOSA. 2010b. AOSA Rules for testing seeds. Volume 4: Seedling evaluation. Volume 4: Seedling

evaluation, Association of Official Seed Analysts, Ithaca, New York.

AOSA/SCST. 2010. Tetrazolium testing handbook. Associ ation of Official Seed Analysts and the Society

of Commercial Seed Techno logists, Ithaca, New York.

AOSCA. 2009. Cotton ( Gossypium ) certification standards. Associa tion of Official Seed Certifying

Agencies, Moline, Illinois.

AOSCA. 2012. Seed certification. Association of Of ficial Seed Certifying Agencies, Moline, Illinois.

http://www.aosca.org/seed %20certification.htm [Accessed April 23, 2012].

Askew, S.D., W.A. Bailey, G.H. Scott and J.W. Wilcut. 2002. Economic assessment of weed management

for transgenic and nontransgenic cotton in tilled and nontilled systems. Weed Science 50:512-520.

Avila-Garcia, W.V. and C. Mallory-Smith. 201 1. Glyphosate-resistant Italian ryegrass ( Lolium perenne )

populations also exhibit resistance to glufosinate. Weed Science 59:305-309.

Baldwin, F.L. and T.L. Baldwin. 2002. Impact of herbicide resistant crops in the Americas - A southern

prospective. Pages 650-654 in Thirteenth Australia n Weeds Conference, Council of Australian Weed

Societies Inc., Perth, Western Australia.

Barker, R.F., K.B. Idler, D.V. Thompson and J.D. Kemp. 1983. Nucleotide sequ ence of the T-DNA region

from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Molecular Biology 2:335-350.

BASF. 2008. Clarity herbicide label. BASF Corpor ation, Research Triangle Park, North Carolina.

Bayer CropScience. 2011. Liberty herbicide label. Bayer CropScience LP, Research Triangle Park, North

Carolina.

Beckie, H.J. and X. Reboud. 2009. Selecting for weed resistance: Herbicide ro tation and mixture. Weed

Technology 23:363-370.

# Monsanto Company 12-CT-244U 247 of 620 Behrens, M.R., N. Mutlu, S. Chakraborty, R. Dumitr u, W.Z. Jiang, B.J. LaVallee, P.L. Herman, T.E.

Clemente and D.P. Weeks. 2007. Dicamba resistance: Enlarging and preserving biotechnology-based weed

management strategies. Science 316:1185-1188.

Berberich, S.A., J.E. Ream, T.L. Jackson, R. Wood, R. Stipanovic, P. Harvey, S. Patzer and R.L. Fuchs.

1996. The composition of insect-protected cottonseed is equivalent to that of conventional cottonseed.

Journal of Agricultural and Food Chemistry 44:365-371.

Berg, G., N. Roskot and K. Smalla. 1999. Genotypic and phenotypic relationships between clinical and

environmental isolates of Stenotrophomonas maltophilia . Journal of Clinical Microbiology 37:3594-3600.

Berg, R.D. 1996. The indigenous gastrointestinal microflora. Trends in Microbiology 4:430-435.

Berkey, D.A., B.R. Savoy, S.R. M iller and P.G. Johnson. 2002. Pollen dissemination from adjacent fields

of genetically enhanced cotton in the Mississippi Delta. 2002 Pr oceedings of th e Beltwide Cotton

Conference, Atlanta, Georgia.

Bertrand, J.A., T.Q. Sudduth, A. Condon, T.C. Jenkin s and M.C. Calhoun. 2005. Nutrient content of whole

cottonseed. Journal of Dairy Science 88:1470-1477.

Bevan, M., W.M. Barnes and M.-D. Chilton. 1983. St ructure and transcription of the nopaline synthase

gene region of T-DNA. Nucleic Acids Research 11:369-385.

BIO. 2010. Product launch stewardship policy. Biotechnology Industry Organization, Washington, D.C.

http://www.bio.org/articles/prod uct-launch-stewardship-policy [Accessed May 31, 2012].

Blasingame, D., M.V. Patel, W. Gazaway, M. Olsen, T. Kirkpatrick, M. Davis, R.K. Sprenkel, B.

Kemerait, P. Colyer, A. Wrather, N. Goldberg, S. Koenning, J.C. Banks, J. Muller, M. Newman, J.

Woodward and P. Phipps. 2008. Cotton disease lo ss estimate committee report: Table 1. Estimated

reduction in 2007 cotton yield resulting from diseases. National Cotton Council of America, Cordova,

Tennessee.

Bowman, D.T., O.A. Gutierrez, R.G. Percy, D.S. Ca lhoun and O.L. May. 2006. Pedigrees of upland and

pima cotton cultivars released between 1970 and 2005 . Mississippi State University, Mississippi State,

Mississippi.

Bradford, K.J. 2006. Methods to maintain genetic purity of seed stocks. Publication 8189. University of

California, Oakland, California.

Breyton, C. 2000. The cytochrome b 6 

# f

complex: Structural studies and comparison with the bc 1 

# complex.

Biochimica et Biophysica Acta 1459:467-474.

Brookes, G. and P. Barfoot. 2012. GM crops: Global socio-economic and environmental impacts 1996-

2010. PG Economics, Ltd, Dorchester, United Kingdom.

Brubaker, C.L., F.M. Bourland and J.F. Wendel. 1999. The origin and domestication of cotton. Pages 3-31

in Cotton: Origin, History, Technology, and Production C.W. Smith and J.T. Cothren (eds.). John Wiley &

Sons, Inc., New York, New York.

Buchanan, B.B., W. Gruissem and R.L. Jones. 2000. Phenylpropanoid and phenylpropanoid-acetate

pathway metabolites. Pages 1286-1289 in Biochemistry and Molecular Biology of Plants. American

Society of Plant Biologists, Rockville, Maryland.

Buchanan, G.A. 1992. Trends in weed control methods. Pages 47-72 in Weeds of Cotton: Characterization

and Control. C.G. McWhorter and J.R. Abernathy (eds.). The Cotton Foundation, Memphis, Tennessee.

# Monsanto Company 12-CT-244U 248 of 620 Carpenter, J. and L. Gianessi. 2001. Why US farmers have adopted genetically modified crops and the

impact on US agricu lture. AgBiotechNet 3:1-4.

Cellini, F., A. Chesson, I. Colquhoun, A. Constable, H.V. Davies, K.H. Engel, A.M.R. Gatehouse, S.

Kärenlampi, E.J. Kok, J.-J. Leguay, S. Lehesranta, H.P.J.M. Noteborn, J. Pedersen and M. Smith. 2004.

Unintended effects and their detection in genetically modified crops. Food and Chemical Toxicology

42:1089-1125.

Chakraborty, S., M. Behrens, P.L. Herman, A.F. Arendsen, W.R. Hagen, D.L. Carlson, X.-Z. Wang and

D.P. Weeks. 2005. A three-component dicamba  -demethylase from Pseudomonas maltophilia , strain DI6: Purification and characterization. Ar chives of Biochemistry and Biophysics 437:20-28.

Christiansen, M.N. and R.P. Moore. 1959. Seed coat st ructural differences that influence water uptake and

seed quality in hard seed cotton. Agronomy Journal 51:582-584.

Clark, S.E. and G.K. Lamppa. 1992. Processing of the precursors for the light-harvesting chlorophyllbinding proteins of photosystem II and photosystem I during import and in an organelle-free assay. Plant

Physiology 98:595-601.

Clemmer, K.C., A.C. York and A.S. Culpepper. 2001. Control of volunteer glyphosate-resistant cotton in

glyphosate-resistant soybean. Pages 53-54 in 2001 Proceedings, Southern Weed Science Society, Biloxi,

Mississippi.

CLI. 2005. Position paper: Regulation of plant biotechnology products containing two or more traits

combined by conventional plant breeding. CropLife International, Brussels, Belgium.

Codex Alimentarius. 2009. Foods derived from modern biotechnology. Codex Alimentarius Commission,

Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations,

Rome, Italy.

Crockett, L. 1977. Contents. Pages vii-ix in Wildly Successful Plants: A Handbook of North American

Weeds. Macmillan Publishing Co ., Inc., New York, New York.

Cross, T. 1989. Other genera. Pages 2586-2615 in Bergey's Manual of Systematic Bacteriology. Volume 4.

S.T. Williams and M.E. Sharpe (eds.). Williams & Wilkins, Balitmore, Maryland.

CTIC. 2011. Top 10 conservation tillage benefits: C onservation for agriculture's future. Conservation

Technology Information Center, West Lafayette, Indiana.

http://www.ctic.purdue.edu/Core4/ CT/CTSurvey/10Benefits.html [Accessed September 23, 2011].

Cunha, B.A. 2010. Stenotrophomonas maltophilia . WebMD, LLC, New York, New York.

http://www.emedicine.com/med/topic3457.htm [Accessed January 2, 2010].

D'Ordine, R.L., T.J. Rydel, M.J. Storek, E.J. Sturman, F. Moshiri, R.K. Bartlett, G. R. Brown, R.J. Eilers, C.

Dart, Y. Qi, S. Flasinski and S.J. Franklin. 2009. Dicamba monooxygenase: Structural insights into a

dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation. Journal of Molecular Biology

392:481-497.

Darrouzet, E., J.W. Cooley and F. Daldal. 2004. The cytochrome bc 1 

# complex and its homologue the b 6 

# f

complex: Similarities and differences. Photosynthesis Research 79:25-44.

Della-Cioppa, G., S.C. Bauer, B.K. Klein, D.M. Shah , R.T. Fraley and G.M. Ki shore. 1986. Translocation

of the precursor of 5- enol pyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro

. Proceedings of the National Academy of Sc iences of the United States of America 83:6873-6877.

# Monsanto Company 12-CT-244U 249 of 620 Denton, M. and K.G. Kerr. 1998. Microbiological a nd clinical aspects of infection associated with

Stenotrophomonas maltophilia . Clinical Microbiology Reviews 11:57-80.

Denton, M., N.J. Todd, K.G. Kerr, P.M. Hawkey a nd J.M. Littlewood. 1998. Molecular epidemiology of

Stenotrophomonas maltophilia isolated from clinical specimens from patients with cystic fibrosis and

associated environmental samples. Journal of Clinical Microbiology 36:1953-1958.

Depicker, A., S. Stachel, P. Dhaese, P. Zambry ski and H.M. Goodman. 1982. Nopaline synthase:

Transcript mapping and DNA sequence. Journal of Molecular and Applied Genetics 1:561-573.

Deterling, D. and K.M. El-Zik. 1982. How a cotton plant grows. Progressive Farmer, Birmingham,

Alabama.

Duke, S.O. and S.B. Powles. 2009. Glyphosate-resistant crops and weeds: Now and in the future.

AgBioForum 12:346-357.

Dumitru, R., W.Z. Jiang, D.P. Weeks and M.A. Wilson. 2009. Crystal structure of dicamba

monooxygenase: A Rieske nonheme oxygenase that catalyzes oxidative demethylation. Journal of

Molecular Biology 392:498-510.

Duncan, D.R. 2010. Cotton transformation. Pages 65-77 in Cotton: Biotechnological Advances. Volume

65. U.B. Zehr (ed.). Springer-Verlag, Berlin, Germany.

Duncan, D.R. and G. Ye. 2011. Methods for inducing cotton embryogenic callus. Patent 7,947,869, U.S.

Patent Office, Washington, D.C.

Echemendia, Y. 2010. Microorganism of the month: Stenotrophomonas maltophilia . Environmental

Microbiology Laboratory, Inc., Cherry Hill, New Jersey. http://www.emlab.com/s/sampling/env-report-07-

2007.html [Accessed August 10, 2010].

Elder, J.K. and E.M. Southern. 1983. Measurement of DNA length by gel electrophoresis II. Comparison of

methods for relating mobility to fragme nt length. Analytical Biochemistry 128:227-231.

Endrizzi, J.E., E.I. Turcotte and R. J. Kohel. 1984. Qualitative genetics, cytology, cytogenetics. Pages 81-

129 in Cotton. R.J. Kohel and C.F. Lewis (eds.). American Society of Agronomy, Inc., Crop Science

Society of America, Inc., and Soil Society of America, Madison, Wisconsin.

FAO-WHO. 1996. Joint FAO/WHO expert consultation on biotechnology and food safety. Paper 61. Food

and Agriculture Organization, World Health Organization, Rome, Italy.

FAO-WHO. 2011a. Pesticide residues in food 2010: Joint FAO/WHO meeting on pesticide residues. FAO

Plant Production and Protection Paper 200. Food and Agriculture Organization of the United Nations,

World Health Organization, Rome, Italy.

FAO-WHO. 2011b. Summary report: Acceptable daily in takes, acute reference doses, short-term and longterm dietary intakes, recommended maximum residue limits and supervised trials median residue values

recorded by the 2011 meeting. Food and Agriculture Organization of the United Nations, World Health

Organization, Geneva, Switzerland.

Ferraro, D.J., L. Gakhar and S. Ramaswamy. 2005. Rieske business: Structure-function of Rieske nonheme oxygenases. Biochemical and Biophysical Research and Communications 338:175-190.

Fling, M.E., J. Kopf and C. Richards. 198 5. Nucleotide sequence of the transposon Tn 7 gene encoding an

aminoglycoside-modifying enzyme, 3"(9)- O -nucleotidyltransferase. Nucleic Acids Research 13:7095-

7106.

# Monsanto Company 12-CT-244U 250 of 620 Fraley, R.T., S.G. Rogers, R.B. Hors ch, 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. Pro ceedings of the National Academy of Sciences of the United States of

America 80:4803-4807.

Fryxell, P.A. 1984. Taxonomy and germplasm resources. Pages 27-58 in Cotton. R.J. Kohel and C.F. Lewis

(eds.). American Society of Agrono my, Inc., Crop Science Society of America, Inc., Soil Science Society

of America, Inc., Madison, Wisconsin.

Giza, P.E. and R.C.C. Hu ang. 1989. A self-inducing runaway-replication plasmid expression system

utilizing the Rop protein. Gene 78:73-84.

Goodfellow, M. and S.T. Williams. 1983. Ecology of actinomycetes. Annual Review of Microbiology

37:189-216.

Gray, C.J., D.R. Shaw and M.L. Tagert. 2002. Control of volunteer Roundup Ready crops in soybean and

cotton systems. 2002 Proceedings of the Southern Weed Science Society, Southern Weed Science Society,

Atlanta, Georgia.

Gray, J., E. Wardzala, M. Yang, S. Reinbothe, S. Haller and F. Pauli. 2004. A small family of LLS1-related

non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers. Plant Molecular

Biology 54:39-54.

Grichar, W.J., D.D. Fro mme, P.A. Dotray and J.W. Keeling. 2010. Controlling volunteer cotton with

postmergence herbicides. Page 1635 in 2010 Beltwide Cotton Conferences, New Orleans, Louisiana.

Guinn, G. 1982. Causes of square and boll shedding in cotton. Technical Bulletin Number 1672. United

States Department of Agriculture, Agricu ltural Research Services, Washington, D.C.

Hake, S.J., K.D. Hake and T.A. Kerby. 1996a. Mid- to late-bloom decisions. Pages 64-79 in Cotton

Production Manual. S.J. Hake, T.A. Kerby, and K.D. Hake (eds.). University of California Division of

Agriculture and Natural Resources, Davis, California.

Hake, S.J., K.D. Hake and T.A. Kerby. 1996b. Planting and stand establishment. Pages 21-22 in Cotton

Production Manual. S.J. Hake, T.A. Kerby, and K.D. Hake (eds.). University of California Division of

Agriculture and Natural Resources, Davis, California.

Hake, S.J., K.D. Hake and T.A. Kerby. 1996c. Prebloom decisions. Pages 29-32 in Cotton Production

Manual. S.J. Hake, T.A. Kerby, and K.D. Hake (eds.). University of California Division of Agriculture and

Natural Resources, Davis, California.

Hake, S.J., T.A. Kerby and K.D. Ha ke. 1996d. Preparation for the new cr op season - Fall/winter. Pages 6-

14 in Cotton Production Manual. S.J. Hake, T.A. Kerby, and K.D. Hake (eds.). University of California

Division of Agriculture and Natura l Resources, Davis, California.

Hamilton, K.A., P.D. Pyla, M. Breeze, T. Olson, M. Li, E. Robinson, S.P. Gallagher, R. Sorbet and Y.

Chen. 2004. Bollgard II cotton: Compositional analysis and feeding studies of cottonseed from insectprotected cotton ( Gossypium hirsutum L.) producing the Cry1Ac and Cry2Ab2 proteins. Journal of

Agricultural and Food Chemistry 52:6969-6976.

Harayama, S., M. Kok and E.L. Neidle. 1992. Func tional and evolutionary relationships among diverse

oxygenases. Annual Review of Microbiology 46:565-601.

Harrigan, G.G., D. Lundry, S. Drury, K. Berman, S.G. Riordan, M.A. Nemeth, W.P. Ridley and K.C.

Glenn. 2010. Natural variation in crop composition and the impact of transgenesis. Nature Biotechnology

28:402-404.

# Monsanto Company 12-CT-244U 251 of 620 Heap, I. 2012a. Herbicide resistan t weeds - Glycines (G/9) resistant weeds by species and country.

WeedScience.org, International Survey of Herbicide Resistant Weeds, Corvallis, Oregon.

http://www.weedscience.org/summary/MOASummary.asp [Accessed June 11, 2012].

Heap, I. 2012b. Herbicide resistant weeds - Glutamine sy nthase inbibitors (H/10) resistant weeds by species

and country. WeedScience.org, International Survey of Herbicide Resistant Weeds, Corvallis, Oregon.

http://www.weedscience.org/summary/MOASummary.asp [Accessed June 11, 2012].

Heap, I. 2012c. Herbicide resistant weeds - Synthetic auxins (O/4) re sistant weeds. WeedScience.org,

International Survey of Herbicide Resistant Weeds, Corvallis, Oregon.

http://www.weedscience.org/summary/MOASummary.asp [Accessed June 11, 2012].

Heap, I. 2012d. Herbicide resistan t weeds summary table. WeedScien ce.org, International Survey of

Herbicide Resistant Weeds, Corvallis, Oregon. http://www.weedscience.or g/summary/MOASummary.asp

[Accessed June 15, 2012].

Herman, P.L., M. Behrens, S. Chak raborty, B.M. Chrastil, J. Barycki and D.P. Weeks. 2005. A threecomponent dicamba  -demethylase from Pseudomonas maltophilia , strain DI-6: Gene isolation,

characterization, and heterologous expression. Journal of Biological Chemistry 280:24759-24767.

Hérouet, C., D.J. Esdaile, B.A. Mallyon, E. Debruyne, A. Schulz, T. Currier, K. Hendrickx, R.-J. van der

Klis and D. Rouan. 2005. Safety evaluation of the phosphinothricin acetyltransfer ase proteins encoded by

the pat and bar sequences that confer tolerance to glufosin ate-ammonium herbicide in transgenic plants.

Regulatory Toxicology and Pharmacology 41:134-149.

Herrmann, K.M. 1995. The shikimate pathway: Early st eps in the biosynthesis of aromatic compounds. The

Plant Cell 7:907-919.

Hibino, T., R. Waditee, E. Araki, H. Ishikawa, K. Aoki, Y. Tanaka and T. Takabe. 2002. Functional

characterization of choline monooxygenase, an enzyme for betaine synthesis in plants. Journal of

Biological Chemistry 277:41352-41360.

Holm, L., J. Doll, E. Holm, J. Pancho and J. Herberger. 1997. Chenopodium murale L. Pages 178-182 in

World Weeds: Natural Histories and Distribution. John Wiley & Sons, Inc., New York, New York.

HRAC. 2009. Classification of herb icides according to mode of ac tion. Herbicide Resistance Action

Committee, Corvallis, Oregon.

http://www.hracglobal.com/Publications/Classificatio nofHerbicideModeofAction/tabid/222/Default.aspx

[Accessed June 29, 2009].

Hurley, T.M., P.D. Mitchell and G.B. Frisvold. 2009. Weed management costs, weed best management

practices, and the Roundup Ready we ed management program. AgBioForum 12:281-290.

ILSI-CERA. 2011. A review of the environmental safety of the PAT protein. International Life Sciences

Institute, Center for Environmental Risk Assessment, Washington, D.C. http://ceragmc.org/docs/cera_publications/pub_05_2011.pdf

.

ILSI. 2011. Crop Composition Database, Version 4.2. In ternational Life Sciences Institute, Washington,

D.C. http://www.cropcomposition.org/

.

ISF. 2005. Genetically modified crops and plant breeding. International Seed Foundation, Nyon,

Switzerland.

ISO. 2009. Selection and use of the ISO 9000 family of standards. International Standards Organization,

Geneva, Switzerland.

# Monsanto Company 12-CT-244U 252 of 620 James, C. 2010. Global status of commercialized biotech/GM crops: 20 10. Brief 42. International Service

for the Acquisition of Agri-Biotech Applications, It haca, New York.

Janas, K.M., M. Cvikrová, A. Pal ą giewicz and J. Eder. 2000. Alterations in phenylpropanoid content in

soybean roots during low temperature acclimation. Plant Physiology and Biochemistry 38:587-593.

John, M.E. 1996. Structural char acterization of genes corresponding to cotton fiber mRNA, E6: Reduced

E6 protein in transgenic plants by antisense gene. Plant Molecular Biology 30:297-306.

Jordan, T., G. Nice, B. Johnson and T. Bauman. 2009. Reducing spray drift from glyphosate and growth

regulator herbicide drift caution. Purdue Extensio n, Purdue University, West Lafayette, Indiana.

http://www.ag.purdue.edu/btny/weedscience/Documents/ReducingDrift09.pdf [Accessed December 18,

2009].

Kairichi, M.N., B.W. Waswa, C.N. Waturu, W. Wiesel, R.G. Ngigi, G.K. Njenga and S.M. Njinju. 2008.

Assessment of pollen-mediated gene flow of Bt -cotton to local commerical variety, Hart 89m in KariMwea Station. Page 257 in Proceedings of the 1 st 

# All African Congress on Biotechnology, Nairobi, Kenya.

Kämpfer, P. 2006. The family Streptomycetaceae , Part I: Taxonomy. Pages 538-604 in The Prokaryotes. A

Handbook on the Biology of Bacter ia: Archaea. Bacteria: Firmicutes, Actinomycetes. Volume 3.

M.Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleife r, and E. Stackebrandt (eds.). Springer+ Business

Media, LLC., New York, New York.

Kay, R., A. Chan, M. Daly and J. McPherson. 1987. Duplication of CaMV 35 S promoter sequences creates

a strong enhancer for plant genes. Science 236:1299-1302.

Keeling, J.W., J.D. Reed, D.M. Petty and B.A. Brow n. 2009. Control of volunteer Roundup Ready Flex

cotton with postemergence herbicid es. Page 1333 in 2009 Beltwide Cotton Conferences, San Antonio,

Texas.

Kerby, T.A., F.M. Bourla nd and K.D. Hake. 2010. Physiologyical rationales in plant monitoring and

mapping. Pages 304-317 in Physiology of Cotton. J.M. Stewart, D.M. Oosterhuis, J.J. Heitholt, and J.R.

Mauney (eds.). Springer Science+Busine ss Media, Dordrecht, Netherlands.

Klee, H.J., Y.M. Muskopf and C.S. Gasser. 1987. Cloning of an Arabidopsis thaliana gene encoding 5-

enolpyruvylshikimate-3-phosphate synthase: Sequence analysis and manipulation to obtain glyphosatetolerant plants. Molecular and General Genetics 210:437-442.

Kohel, R.J. and C.F. Lewis. 1984. Cotton. American Society of Agronomy, Inc., Crop Science Society of

America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.

Krueger, J.P., R.G. Butz, Y.H. Atallah and D.J. Cork. 1989. Isolation and identification of microorganisms

for the degradation of dicamba. Journal of Agricultural and Food Chemistry 37:534-538.

Kutzner, H.J. 1981. The family streptomycetaceae. Pages 2028-209 0 in The Prokaryo tes: A Handbook on

Habitats, Isolation, and Identification of Bacteria. Volume 2. M.P. Starr, H. Stolp, H.G. Trüper, A. Balows,

and H.G. Schlegel (eds.). Springer-Verlag, Berlin, Germany.

Lawhon, J.T., C.M. Cater and K.F. Mattil. 1977. Evaluati on of the food use potential of sixteen varieties of

cottonseed. Journal of the Amer ican Oil Chemists' Society 54:75-80.

Lee, D.R., D.K. Miller and D.C. Blouin. 2009. Gl yphosate-resistant cotton interference in glyphosateresistant soybean. Journal of Cotton Science 13:174-177.

# Monsanto Company 12-CT-244U 253 of 620 Lee, J.A. 1984. Cotton as a world crop. Pages 1-25 in Cotton. R.J. Kohel and C.F. Lewis (eds.). American

Society of Agronomy, Inc., Crop Science Society of Am erica, Inc., Soil Science Society of America, Inc.,

Madison, Wisconsin.

Lege, K.E., J.T. Cothren and C.W. Smith. 1995. Phenolic acid and condensed tannin concentrations of six

cotton genotypes. Environmental and Experimental Botany 35:241-249.

Lemaux, P.G. 2008. Genetically engineered plants and fo ods: A scientist's analysis of the issues (Part I).

Annual Review of Plant Biology 59:771-812.

Lingenfelter, D.D. and N.L. Hartwig. 2007. Introduction to weeds and herbicides. Penn State University

Agricultural Research and Cooperative Exte nsion, University Park, Pennsylvania.

Llewellyn, D. and G. Fitt. 1996. Pollen dispersal from two field trials of transgenic cotton in the Namoi

Valley, Australia. Molecular Breeding 2:157-166.

Locci, R. 1989. Streptomycetes and related genera. Pages 2451-2508 in Bergey's Manual of Systematic

Bacteriology. Volume 4. S.T. Williams and M.E. Shar pe (eds.). Williams & Wilkins, Baltimore, Maryland.

Maiti, I.B. and R.J. Shepherd. 1998. Isolation and expression analysis of peanut chlorotic streak

caulimovirus (PCISV) full-length transcript (FLt) promoter in transgenic plants. Biochemical and

Biophysical Research Communications 244:440-444.

Manderscheid, R. and A. Wild. 1986. Studies on the mechanism of inhibition by phosphinothricin of

glutamine synthetase isolated from Triticum aestivum L. Journal of Plant Physiology 123:135-142.

McClelland, M.R., K.L. Smith and J.K. Norsworthy. 2006. Managing glyphosate-resistant horseweed in

conservation-tillage cotton production: Final summary and recommendations. AAES Research Series 552.

University of Arkansas Agricultural Experiment Station, Fayetteville, Arkansas.

McDonald, M.B. and L.O. Copeland. 1997. Cotton ( Gossypium hirsutum L.). Pages 264-273 in Seed

Production: Principles and Practices. Chapman & Hall, New York, New York.

McGregor, S.E. 1976. Insect pollin ation of cultivated crop plants. Agricultural Handbook No. 496. U.S.

Department of Agriculture, Agricultural Research Service, Washington, D.C.

McWhorter, C.G. and C.T. Bryson. 1992. Use trends in the 1960s. Pages 241-254 in Weeds of Cotton:

Characterization and Control. C.G. McWhorter and J.R. Abernathy (eds.). The Cotton Foundation,

Memphis, Tennessee.

Monsanto Company. 2011. Unpublished grower survey data performed by a contracting agency. St. Louis,

Missouri.

Monsanto Company. 2012. U.S. Technology use guide. Monsanto Company, St. Louis, Missouri.

http://www.monsanto.com/ourcommitments/Pages/technology-use-guides.aspx [Accessed January 19,

2012].

MSU. 2012. 2012 weed control guidelines for Mississippi. Mississippi State University Extension Service,

Mississippi State, Mississippi. http://msucares.com/pubs /publications/p1532.pdf [Accessed June 15, 2012].

Murdock, E.C., M.A. Jones and R.F. Graham. 2002. Control of volunteer glyphosa te (Roundup)-tolerant

cotton and soybean in Roundup R eady cotton. 2002 Proceedings of the Beltwide Cotton Conference,

National Cotton Council, Atlanta, Georgia.

# Monsanto Company 12-CT-244U 254 of 620 Nam, J.-W., H. Nojiri, T. Yoshida, H. Habe, H. Yama ne and T. Omori. 2001. New classification system for

oxygenase components involved in ring-hydroxylating oxygenations. Bioscience, Biotechnology, and

Biochemistry 65:254-263.

NCCA. 2007. The first 40 days: The most critical period in cotton production. National Cotton Council of

America, The Cotto n Foundation, Atlanta, Georgia.

NCCA. 2010. Cotton: From field to fabric. Nationa l Cotton Council of Ameri ca, Atlanta, Georgia.

NCPA. 2002. Cottonseed and its products. National Cottonseed Products Association, Cordova, Tennessee.

http://www.cottonseed.com/publicatio ns/cottonseedanditsproducts.asp [Accessed October 17, 2011].

Nida, D.L., S. Patzer, P. Harvey, R. Stipanovic, R. Wood and R.L. Fuchs. 1996. Glyphosate-tolerant

cotton: The composition of the cottonseed is equivalent to that of conventional cottonseed. Journal of

Agricultural and Food Chemistry 44:1967-1974.

Niepel, M. and D.R. Gallie. 1999. Identification and ch aracterization of the functional elements within the

tobacco etch virus 5' leader required for cap-independent translation. Journal of Virology 73:9080-9088.

Niles, G.A. and C.V. Feaster. 1984. Breeding. Pages 201-231 in Cotton. R.J. Kohel and C.F. Lewis (eds.).

American Society of Agronomy, Inc. , Crop Science Society of Ameri ca, Inc., Soil Scie nce Society of

America, Inc., Madison, Wisconsin

NRC. 2004. New approaches for identifying unintended changes in food composition. Pages 73-102 in

Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects. Institute of

Medicine and National Research Council, Na tional Academies Press, Washington, D.C.

Odell, J.T., F. Nagy and N.-H. Chua. 1985. Identifi cation of DNA sequences required for activity of the

cauliflower mosaic virus 35S promoter. Nature 313:810-812.

OECD. 1993. Safety considerations for biotechnology: Scale-up of crop plants. Organisation for Economic

Co-operation and Development, Paris, France.

www.biosafety.be/CU/BSL_Ressources/PDF/M00034525.pdf [Accessed January 9, 2009].

OECD. 1999a. Consensus document on general information concerning the genes and their enzymes that

confer tolerance to phosphinothricin herbicid e. ENV/JM/MONO(99)13. Series on Harmonization of

Regulatory Oversight in Biotechnology No.11. Organisation for Economic Co-operation and Development,

Paris, France.

OECD. 1999b. Consensus document on general information concerning the genes and their enzymes that

confer tolerance to glyphosate herbicide. ENV/JM /MONO(99)9. Series on Harmonization of Regulatory

Oversight in Biotechnology No.10. Organisation for Economic Co-operation and Development, Paris,

France.

OECD. 2002a. Module II: Herbicide biochemistry, herbicide metabolism and the residues in glufosinateammonium (Phosphinothricin)-tolentant transgen ic plants. ENV/JM/MONO(2002)14. Series on

Harmonization of Regulatory Oversight in Biotechnology No. 25. Organisation for Economic Co-operation

and Development, Paris, France.

OECD. 2002b. Report of the OECD workshop on the toxicological and nutritional testing of novel foods.

SG/ICGB(1998)1/FINAL. Organisation for Economic Co-operation and Development, Paris, France.

OECD. 2008. Consensus document on the biology of cotton ( Gossypium spp.). ENV/JM/MONO(2008)33.

Organisation for Economic Co-operation and Development, Paris, France.

# Monsanto Company 12-CT-244U 255 of 620 OECD. 2009. Consensus document on the compositional considerations for new varities of cotton

( Gossypium hirsutum and Gossypium barbadense ): Key food and feed nutrients and anti-nutrients.

ENV/JM/MONO(2004)16. Organisation for Economic Co-operation and Development, Paris, France.

OGTR. 2008. The biology of Gossypium hirsutum L. and Gossypium barbadense L. (cotton). Australian

Government, Department of Health and Ageing, Office of the Gene Technology Regulator, Canberra,

Australia.

Palleroni, N.J. and J.F. Bradbury. 1993. Stenotrophomonas , a new bacterial genus for Xanthomonas

maltophilia (Hugh 1980) Swings et al. 1983. Interna tional Journal of Systematic Bacteriology 43:606-609.

Percival, A.E., J.F. Wendel and J.M. Stewart. 1999 . Taxonomy and germplasm resources. Pages 33-63 in

Cotton: Origin, History, Technology, and Production. W.C. Smith and J.T. Cothren (eds.). John Wiley &

Sons, Inc., New York, New York.

Pilacinski, W., A. Crawford, R. Downey, B. Harvey, S. Huber, P. Hunst, L.K. Lahman, S. MacIntosh, M.

Pohl, C. Rickard, L. Tagliani and N. Weber. 2011. Plants with genetically modified events combined by

conventional breeding: An assessment of the need for additional regulatory data. Food and Chemical

Toxicology 49:1-7.

Plant, R.E. and T.A. Kerby. 1995. CPM: Software for cotton final plant mapping. Agronomy Journal

87:1143-1147.

Powles, S.B. 2008. Evolved glyphosate-resistant weeds around the world: Lessons to be learnt. Pest

Management Science 64:360-365.

Powles, S.B., C. Preston, I.B. Bryan and A.R. Jutsum. 1996. Herbicide resistance: Impact and management.

Pages 57-93 in Advances in Agronomy. Volume 58. D.L. Sparks (ed.). Academic Press, San Diego,

California.

Price, A.J., K.S. Balkcom, S.A. Culpepper, J.A. Kelton, R.L. Nichols and H. Schomberg. 2011.

Glyphosate-resistant Palmer amaranth: A threat to conservation tillage. Journal of Soil and Water

Conservation 66:265-275.

Prince, J.M., D.R. Shaw, W.A. Givens, M.E. Newman, M.D.K. Owen, S.C. Weller, B.G. Young, R.G.

Wilson and D.L. Jordan. 2011. Benchmark study: III. Survey on changing herbicide use patterns in

glyphosate-resistant cropping systems. Weed Sc ience Society of America, Champaign, Illinois.

Qureshi, A., L. Mooney, M. Denton and K.G. Kerr. 2005. Stenotrophomonas maltophilia in salad.

Emerging Infectious Diseases 11:1157-1158.

Rathinasabapathi, B., M. Burnet, B.L. Russell, D.A. Gage, P.-C. Liao, G.J. Nye, P. Scott, J.H. Golbeck and

A.D. Hanson. 1997. Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of

Glycine betaine synthesis in plants: Prosthetic grou p characterization and cDNA cloning. Proceedings of

the National Academy of Sciences of the United States of America 94:3454-3458.

Reeves, J.B. and J.L. Weihrauch. 1979. Composition of foods. Agriculture Handbook 8-4. U.S. Department

of Agriculture, Washington, D.C.

Rensing, S.A. and U.-G. Maier. 1994. Phylogenetic analysis of the stress-70 protein family. Journal of

Molecular Evolution 39:80-86.

Reynolds, T.L., M.A. Nemeth, K.C. Glenn, W.P. Ridl ey and J.D. Astwood. 2005. Natural variability of

metabolites in maize grain: Differences due to genetic background. Journal of Agricultural and Food

Chemistry 53:10061-10067.

# Monsanto Company 12-CT-244U 256 of 620 Ritchie, G.L., C.W. Bednarz, P.H. Jost and S.M. Brown. 2007. Cotton growth and development. University

of Georgia Cooperative Extension, Athens, Georgia.

Roberts, G., S. Kerlin and M. Hickman. 2002. Controlling volunteer cotton. Page F4 in WEEDpak - A

Guide for Integrated Management of Weeds in Cotton. Cotton Catchment Communities CRC, Australian

Cotton Research Institute, Narrabri, New South Wales.

Rodoni, S., W. Mühlecker, M. Anderl, B. Kräutler, D. Moser, H. Thomas, P. Matile and S. Hörtensteiner.

1997. Chlorophyll breakdown in senescent chloroplasts (cleavage of pheophorbide a in two enzymic steps).

Plant Physiology 115:669-676.

Rosche, B., B. Tshisuaka, B. Hauer, F. Lingens and S. Fetzner. 1997. 2-oxo-1,2-dihydroquinoline 8-

monooxygenase: Phylogenetic relati onship to other multicomponent nonheme iron oxygenases. Journal of

Bacteriology 179:3549-3554.

Russell, B.L., B. Rathinasabapathi and A.D. Hanson . 1998. Osmotic stress induces expression of choline

monooxygenase in sugar beet and amaranth. Plant Physiology 116:859-865.

Ryan, R.P., S. Monchy, M. Cardinale, S. Taghavi, L. Crossman, M.B. Avison, G. Berg, D. van der Lelie

and J.M. Dow. 2009. The versatility and adaptation of bacteria from the genus Stenotrophomonas . Nature

Reviews Microbiology 7:514-525.

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. Protocol 1: Agar ose gel electrophoresis. Pages 5.4-5.13 in Molecular

Cloning. Third Edition. Cold Springs Laborato ry Press, Cold Springs Harbor, Maine.

Sandretto, C. and J. Payne. 2006. Soil management and conservation. Pages 96-106 in Agricultural

Resources and Environmental Indicators. K. Wiebe and N. Gollehon (eds.). U.S. Department of

Agriculture, Economic Research Service, Washington, D.C.

Sankula, S. 2006. Quantification of the impacts on US agriculture of biotechnology-derived crops planted

in 2005. National Center for Food and Agricultural Policy, Washington, D.C.

Schmelz, E.A., J. Engelberth, H.T. Alborn, P. O'Donnell, M. Sammons, H. Toshima and J.H. Tumlinson.

2003. Simultaneous analysis of ph ytohormones, phytotoxins, and vola tile organic compounds in plants.

Proceedings of the National Academy of Sc iences of the United States of America 100:10552-10557.

Schmidt, C.L. and L. Shaw. 2001. A comprehensive phylogenetic analysis of Rieske and Rieske-type ironsulfur proteins. Journal of Bioenergetics and Biomembranes 33:9-26.

SDTF. 1997. A summary of ground application studies. Spray Drift Task Force, St. Louis, Missouri.

http://www.agdrift.com/PDF_FILES/Ground.pdf [Accessed August 10, 2010].

Seng, C.T., L. Van Lun, C.T. San and I. Sahid. 2010 . Initial report of glufosinate and paraquat multiple

resistance that evolved in a biotype of goosegrass ( Eleusine indica ) in Malaysia. Weed Biology and

Management 10:229-233.

Sims, S.R., S.A. Berberich, D.L. Nida, L.L. Segalini, J.N. Leach, C.C. Ebert and R.L. Fuchs. 1996.

Analysis of expressed proteins in fiber fractions from insect-protected and glyphosate-tolerant cotton

varieties. Crop Science 36:1212-1216.

Smith, C.W. and J.T. Cothren. 1999. Cotton: Origin, history, technology, and production. John Wiley and

Sons, Inc., New York, New York.

# Monsanto Company 12-CT-244U 257 of 620 Smith, C.W. and R.A. Creelman. 2001. Vitamin E concentration in upland cotton seeds. Crop Science

41:577-579.

Stalker, D.M., J.A. Kiser, G. Baldwin, B. Coulombe and C.M. Houck. 1996. Cotton weed control using the

BXN TM 

# system. Pages 93-105 in Herbic ide-Resistant Crops: Agricultural, Environmental, Economic,

Regulatory, and Technical Aspects. S.O. Duke (ed.). CRC Press, Inc., Boca Raton, Florida.

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. Pages 77-90

in Cold Spring Harbor Symposia on Quantitative Biology, Cold Spring Harbor, New York.

Tanaka, A., H. Ito, R. Tanaka, N.K. Tanaka , K. Yoshida and K. Ok ada. 1998. Chlorophyll a oxygenase

( CAO ) is involved in chlorophyll b formation from chlorophyll a . Proceedings of the National Academy of

Sciences of the United States of America 95:12719-12723.

Thompson, A. and L. Steckel. 2010. Controlling volunteer cotton in soybeans. W168. University of

Tennessee Extension, Memphis, Tennessee.

Thompson, C.J., N.R. Movva, R. Tizard, R. Crameri, J.E. Davies, M. Lauwereys and J. Botterman. 1987.

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus . EMBO Journal

6:2519-2523.

U.S. EPA. 1993. RED facts: Glyphosate. U.S. Envi ronmental Protection Agency , Office of Prevention,

Pesticides, and Toxic Substances, Washington, D.C.

http://www.epa.gov/oppfead1/endanger/lits tatus/effects/glyphosate-red.pdf [Accessed August 16, 2010].

U.S. EPA. 1997. Phosphinot hricin acetyltransferase and the genetic ma terial necessary for its production in

all plants; Exemption from the requirement of a tolerance on all raw agricultural commodities. Federal

Register 62:17717-17720.

U.S. EPA. 2003. Memorandum: Glufosinate ammonium (PC code 128850). Section 3 registrations for

transgenic cotton and cotton (ID# - 0F06140), transgenic rice (ID# - 0F06210), and bushberry (ID# -

2E06404). Human health risk assessment. DP barcode: D290086. Case number: 293386. Submission:

S635308. 40 CFR 180.473. U.S. Environmental Protection Agency, Office of Pevention, Pesticides, and

Toxic Substances, Washington, D.C.

U.S. EPA. 2008. Glufosinate final work plan registration review: August 2008. Docket Number: EPA-HQOPP-2008-0190. U.S. Environmental Protection Agency, Washington, D.C.

U.S. EPA. 2009. Reregistration eligibility decision fo r dicamba and associated salts. U.S. Environmental

Protection Agency, Office of Prevention, Pesticides, and Toxic Substances, Washington, D.C.

U.S. FDA. 1992. Statement of policy: Foods derived from new plant varieties. Federal Register 57: 22984-

23005.

U.S. FDA. 2001. Premarket notice concerning bioengineered foods. Federal Register 66:4706-4738.

Umbeck, P.F., K.A. Barton, E.V. Nordheim, J.C. McCarty, W.L. Parrott and J.N. Jenkins. 1991. Degree of

pollen dispersal by insects from a field test of genetically engineered cotton. Journal of Economic

Entomology 84:1943-1950.

University of Arkansas. 2011. Recommended chemicals for weed and brush control. University of

Arkansas, Cooperative Extension Service, Divi sion of Agriculture, Fayetteville, Arkansas.

# Monsanto Company 12-CT-244U 258 of 620 University of California. 1996. Development and growth requirements of the cotton plant. Pages 11-23 in

Integrated Pest Management for Cotton in the Western Region of the United States. Publication 3305.

Second Edition. University of Ca lifornia, Oakland, California.

University of California. 2012. Cotton: Susceptibility of weeds to herbicide control. University of

California Agriculture & Natural Resources Statewide Integrated Pest Management Program, Davis,

California. http://ipm.ucdavis.edu/PMG/r114700411.html [Accessed May 1, 2012].

University of Georgia. 2010. Corn production guide. University of Georgia, College of Agricultural &

Environmental Sciences, Athens, Georgia.

http://www.caes.uga.edu/commodities/fieldcro ps/gagrains/2008ComProductionGuide.html [Accessed

December 9, 2010].

University of Georgia. 2011. 2011 Georgia cotton production guide. University of Georgia College of

Agricultural and Environmental Sciences, Athens, Georgia.

University of Georgia. 2012. 2012 Georgia cotton production guide. University of Georgia College of

Agricultural and Environmental Sciences, Athens, Georgia.

USDA-AMS. 2001. The classification of cotton. Agricultural Handbook 566. U.S. Department of

Agriculture, Agricultural Marketing Service, Washington, D.C.

USDA-APHIS. 1986. Coordinated framework for regulation of biotechnology. Federal Register 51:23302.

USDA-APHIS. 1995. USDA/APHIS determination on a petition 94-308-01p of Monsanto Agricultural

Company seeking nonregulated status of lepidopteran-resistant cotton lines 531, 757, 1076: Environmental

assessment and finding of no significant impact. U.S. Department of Agriculture, Animal and Plant Health

Inspection Service, Washington, D.C.

USDA-ERS-FAS. 2010. Outlook for U.S. agricultural trad e: FY 2010 exports expected to rise to $104.5

billion; Imports drop to $76.5 billion. U.S. Departme nt of Agriculture, Economic Research Service and

Foreign Agricultural Service, Washington, D.C. http://www.fas.usda.gov/cmp/outlook/2010/May10/May2010_fullreport.pdf [Accessed July 11, 2011].

USDA-ERS. 2012a. 2007 crop production practices for cotton: All survey states. U.S. Department of

Agriculture, Economic Research Service, Washington, D.C. http://ers.usda.gov/data-products/arms-farmfinancial-and-crop-production- practices/tailored-reports.aspx [Accessed June 18, 2012].

USDA-ERS. 2012b. Crop production practices for cotton: National. U.S. Department of Agriculture,

Economic Research Service, Washington, D.C.

USDA-FAS. 2005. Oilseeds: World markets and trade. Circular Series FOP 7 - 05. U.S. Department of

Agriculture, Foreign Agricultural Service, Washington, D.C.

USDA-FAS. 2010. Record early-season soybean sales brighten U.S. export outlook. U.S. Department of

Agriculture, Foreign Agricultural Service, Washington, D.C.

USDA-FAS. 2011. Cotton: World markets and trade. Ci rcular Series FOP 12-11. U.S. Department of

Agriculture, Foreign Agricultural Service, Washington, D.C.

USDA-FAS. 2012. Cotton: World markets and trade. U. S. Department of Agriculture, Foreign Agricultural

Service, Washington, D.C.

USDA-NASS. 2002. 2002 census of agriculture. Geographic Area Series Part 51. U.S. Department of

Agriculture, National Agricultural Statistics Service, Washington, D.C.

# Monsanto Company 12-CT-244U 259 of 620 USDA-NASS. 2003. Acreage - June 2003. U.S. Departme nt of Agriculture, National Agricultural Statistics

Service, Washington, D.C.

USDA-NASS. 2004. Agricultural chemical usage - 2003 field crops summary. U.S. Department of

Agriculture, National Agricultural Statistics Service, Washington, D.C.

http://usda.mannlib.cornell.edu/MannUsda /viewDocumentInfo.do?documentID=1560 [Accessed May 12,

2010].

USDA-NASS. 2008. Agricultural chemical usage - 2007 field crops summary. U.S. Department of

Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2010. Acreage - June 2010. U.S. Departme nt of Agriculture, National Agricultural Statistics

Service, Washington, D.C.

http://usda.mannlib.cornell.edu/MannUsda /viewDocumentInfo.do?documentID=1000 [Accessed July 31,

2010].

USDA-NASS. 2011a. Acreage - June 2011. U.S. Department of Agriculture, National Agricultural

Statistics Service, Washington, D.C.

http://usda.mannlib.cornell.edu/MannUsda /viewDocumentInfo.do?documentID=1000 [Accessed June 22,

2012].

USDA-NASS. 2011b. Crop production: Historical track records, April 2011. U.S. Department of

Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2011c. Crop values: 2010 summary, February 2011. U.S. Department of Agriculture,

National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2011d. Vegetables: 2010 summary, January 2011. Vg 1-2 (11). U.S. Department of

Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2011e. Crop production: 2010 summary, January 2011. U.S. Department of Agriculture,

National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2012a. Upland cotton 2010 planted acres by county for selected stat es. U.S. Department of

Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2012b. Pima cotton 2012 planted acres by county for selected stat es. U.S. Department of

Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2012c. Quick stats: Cotton, acres planted, Pima - acres planted, Pima - price received,

Upland acres. U.S. Department of Agriculture, Nationa l Agricultural Statistics Service, Washington, D.C.

http://quickstats.nass.usda.gov/data/printable/2550EF99-0495-3D73-97B4-C71FFC453437 [Accessed

April 4, 2012].

Van Deynze, A.E., F.J. Sundstrom and K.J. Bradford. 2005. Pollen-mediated gene flow in California cotton

depends on pollinator activity. Crop Science 45:1565-1570.

Vargas, R.N., W.B. Fischer, H.M. Kempen and S.D. Wright. 1996. Cotton weed management. Pages 187-

188 in Cotton Production Manual. S.J. Hake, T.A. Kerby, and K.D. Hake (eds.). University of California

Division of Agriculture and Natura l Resources, Davis, California.

Waghmare, V.N., J. Rong, C.J. Rogers, G.J. Pierce, J.F. Wendel and A.H. Paterson. 2005. Genetic mapping

of a cross between Gossypium hirsutum (cotton) and the Hawaiian endemic, Gossypium tomentosum

. Theoretical and Applied Genetics 111:665-676.

# Monsanto Company 12-CT-244U 260 of 620 Wagner, W.L., D.R. Herbst and S.H. Sohmer. 1990. Manua l of the Flowering Plants of Hawai'i. University

of Hawaii Press, Honolulu, Hawaii.

Wang, X.-Z., B. Li, P.L. Herman and D.P. Weeks. 1 997. A three-component enzyme system catalyzes the

O demethylation of the herbicide dicamba in Pseudomonas maltophilia DI-6. Applied and Environmental

Microbiology 63:1623-1626.

Webster, T.M., M. Patterson, J. Everest, J. Ferrell, B. Brecke, A.S. Culpepper, E.P. Prostko, J.D. Green,

J.R. Martin, E.Webster, S. Kelly, J. Griffin, D. Sanders, J. Byrd, A. Kendig, A. York, D. Jordan, L. Fisher,

C. Medlin, D. Murray, J. Norsworthy , J. Chapin, L. Nelson and L. Steck el. 2005. Weed survey - Southern

states 2005: Broadleaf crops subsection (cotton, peanut, soybean, tobacco, and forestry). Pages 291-306 in

2005 Proceedings of the Southern Weed Science Society, Memphis, Tennessee.

Webster, T.M., M. Patterson, J. Everes t, K. Smith, D. Oliver, J. Norswort hy, N. Burgos, B. Scott, J. Byrd,

C. Fristoe, J. Ferrell, B. Brecke, P. Minogue, A.S. Culpepper, E.P. Prostko, D. Moorhead, J.M. Moore, J.D.

Green, J.R. Martin, B. Williams, D. Stephenson, D. Mille r, D. Sanders, J. Griffin, K. Bradley, A. York, D.

Jordan, L. Fisher, D. Murray, M. Marshall, S. Hagood and C. Johnson. 2009. Weed survey

 Southern

states 2009: Broadleaf crops subsection (cotton, peanut, soybean, tobacco, and forestry). Pages 509-524 in

2009 Proceedings of the Southern Weed Science Society, Las Cruces, New Mexico.

Wehrmann, A., A.V. Vliet, C. Opsomer, J. Botte rman and A. Schulz. 1996. The similarities of bar and pat

gene products make them equally applicable for plant engineers. Nature Biotechnology 14:1274-1278.

Werlen, C., H.-P.E. Kohler and J.R. van der Meer. 1996. The broad substrate chlorobenzene dioxygenase

and cis -chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily

to the enzymes for benzene and toluene degr adation. Journal of Biological Chemistry 271:4009-4016.

WHO. 1995. Application of the principles of substan tial equivalence to the safety evaluation of foods or

food components from plants derived by modern biotechnology. World Health Organization, Geneva,

Switzerland.

Wilcut, J.W., R.M. Hayes, R.L. Ni chols, S.B. Clewis, J. Summerlin, D.K. Miller, A. Kendig, J.M.

Chandler, D.C. Bridges, B. Brecke, C.E. Snipes and S.M. Brown. 2003 . Weed management in transgenic

cotton. North Carolina State Univer sity Agricultural Research Service, Raleigh, North Carolina.

Wild, A. and R. Manderscheid. 1984. The effect of phosphinothricin on the assimilation of ammonia in

plants. Zeitschrift für Naturforschung C 39:500-504.

Williams, M.R. 2010. Cotton insect losses 2010. Mississippi State University, Mississippi State,

Mississippi. http://www.entomology.msstate.edu/reso urces/tips/cotton-losses/data/2010/ [Accessed May

22, 2012].

Winter, J., R. Wright, N. Duck, C. Gasser, R. Fraley and D. Shah. 1988. The inhibition of petunia hsp70

mRNA processing during CdCl 2 

# stress. Molecular and General Genetics 211:315-319.

Wishart, D.S. 2010. Human metabolome database. www.hmdb.ca [Accessed June 2, 2010].

Wishart, D.S., C. Knox, A.C. Guo, R. Eisner, N. Youn g, B. Gautam, D.D. Hau, N. Psychogios, E. Dong, S.

Bouatra, R. Mandal, I. Sinelnikov, J. Xia, L. Jia, J.A. Cruz, E. Lim, C.A. Sobsey, S. Shrivastava, P. Huang,

P. Liu, L. Fang, J. Peng, R. Fradette, D. Cheng, D. Tzur, M. Clements, A. Lewis, A. De Souza, A. Zuniga,

M. Dawe, Y. Xiong, D. Clive, R. Greiner, A. Nazyrova, R. Shaykhutdinov, L. Li, H.J. Vogel and I.

Forsythe. 2009. HMDB: A knowledgebase for th e human metabolome. Nucleic Acids Research 37:D603-

D610.

# Monsanto Company 12-CT-244U 261 of 620 Wohlleben, W., W. Arnold, I. Broer, D. Hillemann, E. Strauch and A. Pühler. 1988. Nucleotide sequence

of the phosphinothricin N -acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its

expression in Nicotiana tabacum . Gene 70:25-37.

Yang, M., E. Wardzala, G.S. Johal and J. Gray. 2004. The wound-inducible Lls1 gene from maize is an

orthologue of the Arabidopsis Acd1 gene, and the LLS1 protein is present in non-photosynthetic tissues.

Plant Molecular Biology 54:175-191.

York, A.C., A.M. Stewart, P.R. Vidrine and A.S. Culpepper. 2004. Control of volunteer glyphosateresistant cotton in glyphosate-resistant soybean. Weed Technology 18:532-539.

Zambryski, P., A. Depicker, K. Kruger and H.M. Goodman. 1982. Tumor induction by Agrobacterium

tumefaciens : Analysis of the boundaries of T-DNA. Journal of Molecular and Applied Genetics 1:361-370.

# Monsanto Company 12-CT-244U 262 of 620 

# APPENDICES

# Monsanto Company 12-CT-244U 263 of 620 

# Appendix A: Notifications

Field trials of MON 88701 have been conducted in the U.S. since 2007. The protocols

for these trials include fiel d performance, breeding and observation, agronomics, and

generation of field materials and data necessary for this petition. In addition to the

MON 88701 phenotypic assessment data, observati onal 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 2011-

2012 seasons, are still in prep aration. A list of trials conducted under USDA notifications

and the status of the final reports for th ese trials are provide d in Table A-1.

# Monsanto Company 12-CT-244U 264 of 620 

# Table A-1. USDA Notifications and Perm its Approved for MON 88701 and Status

of Trials Conducted under These Notifications

USDA # Effective Date 

# # Release sites per state Trial Status

2007

07-241-107n 9/28/2007 PR-2 Submitted to USDA

2008

08-042-109n 3/12/2008 TX-2, TN-1, NC-2, MS-3, GA-4 Submitted to USDA

08-056-112n 3/26/2008 NM-2 Submitted to USDA

08-056-117n 3/26/2008 TX-3, SC-2, NC-2, MS-2, LA-1, GA-4,

AR-1 Submitted to USDA

08-266-130n 10/19/2008 PR-3 Submitted to USDA

2009

09-058-104n 3/29/2009 CA-1 Submitted to USDA

09-065-111n 4/5/2009 AZ-5, GA-1, MS-3, SC-2, TX-4 Submitted to USDA

09-068-108n 4/8/2009 AL-1, AR-2, AZ-1, GA-1, IL-1, LA-1,

MS-1, NC-4, NM-2, TX-1 Submitted to USDA

09-072-103n 4/8/2009 AR-1, MS-2, SC-5, TN-2, TX-5 Submitted to USDA

09-224-101n 9/21/2009 PR-2 Submitted to USDA

2010

10-054-134n 3/20/2010 TX-4 Submitted to USDA

10-059-109n 3/28/2010 GA-2, NC-9, SC-3 Submitted to USDA

10-061-102n 7/10/2010 MS-1, PR-7 Submitted to USDA

10-064-101n 4/3/2010 CA-2, GA-1, LA-1, MO-1, OK-3, SC-1,

AR-1 Submitted to USDA

10-067-104n 4/7/2010 AZ-5, IL-1, MS-4, NM-2, PR-2, TX-10 Submitted to USDA

10-071-101n 4/9/2010 AR-4, AZ-2, GA-2, KS-1, LA-1, NC-2,

NM-1, SC-1, TX-2 Submitted to USDA

10-071-102n 4/10/2010 AR-1, GA-1, LA-1, MS-1, NC-1, SC-1,

TN-1, TX-2 Submitted to USDA

10-242-102n 9/29/2010 PR-2 Submitted to USDA

10-285-105n 11/11/2010 AR-1, GA-1, LA-1, NM-1 Submitted to USDA

2011

11-045-101n 3/16/2011 MS-1, PR-2 Pending

11-052-105n 3/23/2011 AL-1, FL-2, GA-9, MS-1, NC-6, SC-4 Pending

11-053-105n 3/25/2011 AR-3, LA-2, MO-2, MS-8, TN-5, TX-4 Pending

11-075-107n 4/15/2011 AL-1, AR-1, AZ-4, IL-1, LA-1, MO-1,

MS-4, NC-1, SC-1, TX-9 Pending

11-068-103n 4/8/2011 AL-2, AR-2, AZ-1, CA-2. GA-2, LA-1,

NC-1, NM-1, SC-1, TX-5 Pending

11-083-104n 4/23/2011 AL-1,MS-1 Pending

11-084-107n 4/24/2011 NC-1 Pending

11-091-102n 5/1/2011 TX-1 Pending

# Monsanto Company 12-CT-244U 265 of 620 

# Table A-1. USDA Notifications and Perm its Approved for MON 88701 and Status

of Trials Conducted under Thes e Notifications (continued)

USDA # Effective Date # Release sites per state Trial Status

2011 cont.

11-094-101n 5/4/2011 AZ-1 Pending

11-111-104n 5/21/2011 FL-1 Pending

11-133-103n 6/12/2011 IL-1 Pending

11-153-101n 7/2/2011 MS-1, PR-2 Pending

11-152-101n 7/1/2011 GA-1 Pending

11-199-102n 8/17/2011 PR-1 Pending

11-290-101n 11/16/2011 MS-1, PR-3 Pending

2012

12-018-101n 2/17/2012 AL-1, TX-2 Pending

12-053-110n 3/23/2012 AR-3, CA-1, GA-2, LA-2, MS-11, NC1, TN-1, TX-2 Pending

12-046-104n 3/16/2012 AL-1, AR-4, FL-1, GA-2, LA-1, NC-3,

SC-1, TN-4, TX-2 Pending

12-051-106n 3/21/2012 AL-3, AR-3, FL-1, GA-3, MS-7, SC-1,

TN-1, TX-5 Pending

12-051-105n 3/21/2012 GA-5, MS-4, NC-6, SC-2, TN-1, TX-5 Pending

12-046-109n 3/16/2012 AR-1, MO-5, TN-13, TX-2 Pending

12-055-101n 3/25/2012 AR-1, CA-1, SC-1, TX-1 Pending

12-068-101n 4/7/2012 CA-4 Pending

12-053-109n 3/23/2012 AL-1, NC-1, SC-1, TX-4 Pending

12-069-101n 4/8/2012 GA-9, TX-2 Pending

12-075-102n 4/14/2012 AL-1, AR-2, MS-1, NC-1, SC-1, TX-4 Pending

12-074-107n 4/13/2012 TX-1 Pending

12-081-101n 4/20/2012 AL-1, TX-2 Pending

# Monsanto Company 12-CT-244U 266 of 620 

# Appendix B: Materials, Methods, and Results for Molecular Analyses of

MON 88701

B.1. Materials

The genomic DNA used in molecular analyses was isolated from leaf tissue of the R 3 

# generation of MON 88701 and the conventional control (Coker 130). The leaf tissue was

harvested from a greenhouse production in 20 10. For generational stability analysis,

genomic DNA was extracted fr om leaf tissue of the R 2 

# , R 3 

# , R 4 

# , R 5 

# and R 6 

# generations of

MON 88701. The leaf tissue was harv ested from production plan PPN-10-113. The

reference substance, PV-GHHT6997 (Figure II I-1), was used as a positive hybridization

control in Southern blot analyses. Probe templates generated from PV-GHHT6997 were

used as additional positive hybrid ization controls. As additio nal reference standards, the

1 Kb DNA Extension Ladder and λ DNA/ Hind III Fragments from Invitrogen (Carlsbad,

CA) were used for size estimations on agarose gels and subsequent So uthern blots. The

1 Kb DNA Ladder from Invitrogen was used for size estimations on agarose gels for PCR

analyses.

B.2. Characterization of the Materials

The identity of the source materials was verified by methods used in molecular

characterization to confirm the presence or absence of MON 88701. The stability of the

genomic DNA was confirmed by observation of interpretable signals from digested DNA

samples on ethidium bromide stained agarose gels and/or specific PCR products, and the

samples did not appear visibly degraded on the ethidium bromide stained gels.

B.3. DNA Isolation for South ern Blot and PCR Analyses

Genomic DNA was isolated from MON 88701 leaf tissue using a

hexadecyltrimethylammonium bromide (CTAB) based method. Briefly, 20 ml of CTAB

buffer (1.5% w/v CTAB, 75 mM Tris HCl, 100 mM EDTA, 1.05 M NaCl, and

0.75% w/v PVP) and 10 mg RNase A were adde d to approximately 4 ml of ground leaf

tissue and incubated at 60-70 °C for 40-50 min with intermittent mixing. Twenty

milliliters of chloroform was added to the samples and mixed by hand for 2-3 min, then

centrifuged at 10,300 × g for 8-10 min. The u pper aqueous phase was put into a clean

tube and the chloroform step was repeated tw ice. After the last chloroform step, the

aqueous phase was put into a clean tube a nd the DNA was precipitat ed with 20 ml of

100% v/v ethanol. The sample was centrifuge d for one minute to condense the pellet,

and then the precipitated DNA was hooked out and put into a tube with 4-6 ml of

70% v/v ethanol to wash th e DNA pellet. The samples we re centrifuged at 5,100 × g for

5 min to pellet the DNA. DNA pellets were air dried, then resuspended in 250 μl of TE

buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8. 0). All extracted DNA was stored in a

-20 C freezer.

B.4. Quantification of DNA

Genomic DNA was quantified using a QubitTM Fluorometer (Invitrogen, Carlsbad, CA).

# Monsanto Company 12-CT-244U 267 of 620 

# B.5. Restriction Enzyme Digestion of DNA

Approximately 10 μg of genomic DNA ex tracted from MON 88701 and conventional

control were digested with restriction enzyme Bcl I (New England Biolabs, Inc., Beverly,

MA) or with restriction enzyme Ssp I- HF (New England Bi olabs, Inc.). All Bcl I digests

were conducted in 10X NEBuffer 3 buffer at 50 °C in a total volume of ~500 μl with

~50 units of restriction enzyme. All Ssp I-HF digests were conduc ted in 10X NEBuffer 4

at 37 °C in a total volume of ~500 μl with ~100 units of restriction enzyme. For the

purpose of running positive hybrid ization controls, ~10 μg of genomic DNA extracted

from the conventional control was dige sted with the restriction enzyme Bcl I and the

appropriate positive hybridization control(s) were added to these digests prior to loading

the agarose gel.

B.6. Agarose Gel Electrophoresis

Digested DNA was resolved on ~0.8% (w /v) agarose gels. For T-DNA insert/copy

number and plasmid vector backbone analyses , individual digests containing ~10 μg each

of MON 88701 and conventional control genomic DNA were loaded on the same gel in a

long run/short run format. Th e long run allows for greater resolution of large molecular

weight DNA, whereas the short run allows fo r retaining the small molecular weight DNA

on the gel. The positive hybridization controls were only run in the short run format. For

the insert stability analysis, individual digests of ~10 μg each of genomic DNA extracted

from five leaf samples from multiple gene rations of MON 88701 and the conventional

control along with the positive hybridization co ntrols were loaded on the agarose gel in a

single run format.

B.7. DNA Probe Preparation fo r Southern Blot Analyses

Probe templates were prepared by PCR am plification using th e PV-GHHT6997 DNA as

template. The PCR products were separate d on an agarose gel by electrophoresis and

purified from the gel using QIAquick Ge l Extraction Kit (Qiagen, Valencia, CA)

according to manufacturer’s instruction. The probe templates were designed based on the

nucleotide composition (%GC) of the sequence in order to optimize the detection of

DNA sequences during hybridization. When po ssible, probes possessing similar melting

temperature (Tm) were combined in th e same Southern blot hybridization.

Approximately 25 ng of each probe templa te were radiolabeled with either [ α

32 

# P]

deoxycytidine triphosphate (d CTP) (6000 Ci/mmol) or [ α

32 

# P] deoxyadenosine

triphosphate (dATP) (6000 Ci/mmol) us ing RadPrime DNA Labeling System

(Invitrogen, Carlsbad, CA) according to manufacturer’s instruction.

B.8. Southern Blot Analyses of DNA

Genomic DNA isolated from MON 88701 and th e conventional control was digested and

evaluated using Southern blot analys es (Southern, 1975). The PV-GHHT6997 DNA,

previously digested with the restriction enzyme Pci I was added to conventional control

genomic DNA digested with Bcl I to serve as positive hybridization control on each

Southern blot. When multiple probes were hybridized simultaneously to one Southern

# Monsanto Company 12-CT-244U 268 of 620 

# blot, the probe templates were spiked in the digested conventional control genomic DNA

to serve as additional positive hybridization c ontrols on the Southern blot. The DNA was

then separated by agarose gel electrophoresis and transferred onto a nylon membrane.

Southern blots were hybridized and washed at 55 °C, 60 °C, or 65 °C, depending on the

calculated melting temperature (Tm) of the pr obes that were used. Table B-1 lists the

radiolabeling conditions and hybrid ization temperatures of the probes used in this study.

Multiple exposures of each blot were then generated using Kodak Biomax MS film

(Eastman Kodak, Rochester, NY) in conjunction with one Kodak Biomax MS

intensifying screen in a -80 °C freezer.

# Monsanto Company 12-CT-244U 269 of 620 

# Table B-1. Hybridization Conditions of Utilized Probes

Probe Labeling

Method Element Sequence Spanned by

DNA Probe Probe labeled

with dNTP

(32P) Hybridization/

Wash

Temperature (  C)

1 RadPrime B-Right Border, P- PC1SV, LTEV, TS- CTP2, CS- dmo

(portion) dATP 55

2 RadPrime TS- CTP2 (portion), CS- dmo

(portion) dCTP 65

3 RadPrime CS- dmo (portion), T- E6 , P- e35S dATP 55

4 RadPrime P- e35S (portion), L- Hsp70 , CSbar , T- nos (portion) dCTP 65

5 RadPrime CS- bar(portion) , T-nos, BLeft Border dATP 55

6 RadPrime OR- ori V dCTP 60

7 RadPrime CS- rop, OR -ori-pBR322

(portion) dCTP 60

8 RadPrime OR -ori-pBR322 (portion) , aadA dCTP 60

# Monsanto Company 12-CT-244U 270 of 620 

# B.9. DNA Sequence Analyses of the Insert

Overlapping PCR products, denoted as Pr oduct A, Product B, and Product C, were

generated that span the insert and adjacent 5 ′ and 3 ′ flanking DNA sequences in

MON 88701. These products were analyzed to determine the nucleotide sequence of the

insert in MON 88701, as well as that of the DNA flanking the 5 ′ and 3 ′ ends of the insert.

The PCR analysis for Product A was conducte d using ~100 ng of genomic DNA template

in a 50  l reaction volume. The reaction volume cont ained either a final concentration of

2 mM MgSO 4 

# , 0.2  M of each primer, 0.2 mM of each dNTP, and 0.02 units/μl of

Accuprime Taq DNA Polymerase High Fidelity (Invitroge n) or a final concentration of,

1.5 mM MgCl 2, 

# 0.2  M of each primer, 0.2 mM of each dNTP, and 0.02 units/μl of

Phusion Hot Start II High Fidelity DNA Po lymerase (Finnzymes, Espoo, Finland). The

Phusion Hot Start II High Fidelity DNA Polymerase was used to enhance the

amplification and sequencing of a sma ll A-rich region located in Product A.

The PCR analyses for Product B and Product C were each conducted using ~100 ng of

genomic DNA template in a 50  l reaction volume containing a final concentration of

2 mM MgSO 4 

# , 0.2  M of each primer, 0.2 mM of eac h dNTP, 2.5% (v/v) DMSO, and

0.02 units/μl of Accuprime Taq DNA Polymerase High Fidelity (Invitrogen).

The amplification of Product A using Accuprime Taq was performed under the following

cycling conditions: 1 cycle at 94  C for 2 minutes; 35 cycles at 94  C for 15 seconds,

59  C for 30 seconds, 68  C for 2.25 minutes; 1 cycle at 68  C for 5 minutes. The

amplification of Product A using Phusion Hot Start II DNA Polymerase was performed

under the following cycling conditions: 1 cycle at 98  C for 30 seconds, 35 cycles at

98  C for 10 seconds, 64  C for 15 seconds, and 72  C for 1.25 minutes. The

amplification of Product B was performe d under the following cycling conditions:

1 cycle at 94  C for 2 minutes; 35 cycles at 94  C for 15 seconds, 50  C for 30 seconds,

68  C for 3 minutes; 1 cycle at 68  C for 5 minutes. The amplification of Product C was

performed under the following cyc ling conditions: 1 cycle at 94  C for 2 minutes;

35 cycles at 94  C for 15 seconds, 62  C for 30 seconds, 68  C for 2 minutes; 1 cycle at

68  C for 5 minutes.

A small aliquot of each PCR product was se parated on a 1.0% (w/v) agarose gel and

visualized by ethidium bromide staining to verify that the products were the expected

size. Prior to sequencing, each verified PCR product was purified using the QIAquick

PCR Purification Kit (Qiagen, Inc., Vale ncia, CA) and quantified using a Qubit

fluorometer. The purified PCR products we re sequenced using multiple primers,

including primers used for PCR amplifi cation. All sequencing was performed by

Monsanto TGAC (The Genome Analysis Ce nter) using BigDye terminator chemistry

(Applied Biosystems, Foster City, CA).

A consensus sequence was generated by compiling multiple sequencing reactions

performed on the overlapping PCR products. Th is consensus sequence was aligned to the

# Monsanto Company 12-CT-244U 271 of 620 

# PV-GHHT6997 sequence to determine the integr ity and organization of the integrated

DNA and the 5 ′ and 3 ′ insert-to-flank DNA junctions in MON 88701.

B.10. PCR and DNA Sequence Analysis to Examine the MON 88701 Insertion Site

To examine the MON 88701 insertion site in conventional cotton, PCR and sequence

analyses were performed on genomic DNA from both MON 88701 and conventional

cotton. The primers used in this analys is were designed from the DNA sequences

flanking the insert in MON 88701. A forwar d primer specific to the DNA sequence

flanking the 5 ′ end of the insert was paired with a reverse primer specific to the DNA

sequence flanking the 3 ′ end of the insert.

The PCR reactions were conducted using ~ 100 ng of genomic DNA template in a 50  l

reaction volume containing a fina l concentration of 2 mM MgSO 4 

# , 0.2  M of each

primer, 0.2 mM of each dNTP, and 0.02 units/μl of Accuprime Taq DNA Polymerase

High Fidelity (Invitrogen). The amplifica tion was performed under the following cycling

conditions: 1 cycle at 94  C for 2 minutes; 35 cycles at 94  C for 15 seconds, 58  C for

30 seconds, 68  C for 30 seconds; 1 cycle at 68  C for 5 minutes.

A small aliquot of each PCR product was se parated on a 1.2% (w/v) agarose gel and

visualized by ethidium bromide staining to verify that the PCR products were the

expected size prior to seque ncing. Only the verified PCR product from the parental

conventional control was purified with the QI Aquick PCR Purification Kit (Qiagen) and

quantified using a Qubit Fluorometer. Th e purified PCR product was sequenced using

multiple primers, including primers used fo r PCR amplification. All sequencing was

performed by the Monsanto TGAC (The Ge nome Analysis Cent er) using BigDye

terminator chemistry (Applied Biosystems).

A consensus sequence was generated by compiling multiple sequencing reactions

performed on the verified PCR product. Th is consensus sequence was aligned to the 5 ′

and 3 ′ sequences flanking the MON 88701 inse rt to determine the integrity and

organization of the insertion site.

# Monsanto Company 12-CT-244U 272 of 620 

# References for Appendix B

Southern, E.M. 1975. Detection of specifi c sequences among DNA fragments separated

by gel electrophoresis. Jour nal of Molecular Biology 98:503-517.

# Monsanto Company 12-CT-244U 273 of 620 

# Appendix C: Protein Reaction Products , Materials, Methods, and Results for

Characterization of MON 88701 DMO and PAT ( bar ) Proteins Produced in

MON 88701, and Substrate Specificity

C.1. DMO Reaction Products

MON 88701 when treated with dicamba herbic ides will yield th e reaction products 3,6-

dichlorosalicylic acid (DCSA) and formaldehyde during demethylation of the herbicide.

These products, as you will see in the text be low, have either been previously deemed

safe (DCSA) or are commonly pr oduced in nature and at sufficiently low levels in this

MON 88701 cropping system (formaldehyde) so as to not raise concerns with regard to

the plant pest risk assessment for MON 88701.

C.1.1. DCSA in MON 88701

DCSA is a metabolite generated when di camba herbicide is sprayed on MON 88701

cotton and soybean and is also produced by livestock and soil whose safety has been

evaluated by the Environmental Protection Agency (U.S. EPA, 2009; FAO-WHO, 2011).

DCSA residue levels were measured in dicamba-treated MON 88701 to support

Monsanto’s registration request for the incl usion of DCSA in the cottonseed and gin byproduct dicamba residue definitions . DCSA is structurally sim ilar to salicylic acid (SA).

Numerous studies have reported on the st ress defense activities of SA, although most

studies have looked at the pr otective effects of exogenously applied SA (Janda et al,

2007).

C.1.2. Formaldehyde in the Environment

Formaldehyde is ubiquitous in the environm ent; plants and animals are constantly

exposed to low levels alrea dy present in the e nvironment and the atmosphere from a

variety of biogenic ( e.g ., plant and animal) and anthropogenic ( e.g ., automotive or

industrial emissions) sources. In water, fo rmaldehyde dissipates through biodegradation

to low levels in a few days (USHHS-ATSDR , 1999). Aerobic biodegr adation half-lives

are estimated to be 1-7 days for surface wate r and 2-14 days for ground water (U.S. EPA,

2008). The half-life of formaldehyde in air is dependent on a number of factors (light

intensity, temperature, and lo cation). Through reaction withhydroxyl radical, the halflife of formaldehyde in air varies from 7 to 70 hours (U.S. EPA, 2008). The photolytic

half-life of formaldehyde in air ( e.g ., in the presence of sunli ght) is estimated to be 1.6-6

hours (U.S. EPA, 2008; USHHS-ATSDR, 1999). Formaldehyde is rapidly consumed in

the atmosphere through direct photolysis or by oxidation with hydroxyl or nitrate radicals

(USHHS-ATSDR, 1999).

Humans are constantly exposed to low le vels of formaldehyde. Human exposure to

formaldehyde is primarily due to indoor air exposures (USHHS-ATSDR, 1999).

Formaldehyde is found in a variety of consum er products such as cosmetics and paints,

often as an antimicrobial ag ent, and is used extensivel y in urea-formaldehyde “slowrelease” fertilizer formulations and adhesives (USHHS-ATSDR, 1999). Indoor

formaldehyde air concentrations are gene rally significantly hi gher than outdoor air

# Monsanto Company 12-CT-244U 274 of 620 

# concentrations (USHHS-ATSDR, 1999) as a result of combustion (cooking, heating,

tobacco use) and the emission of formaldehyde from a variety of construction materials

( e.g ., particle board, plywood or foam insulation) as well as permanent press fabrics ( e.g

., clothing or draperies) (U.S. CPSC, 1997). Formaldehyde present in outdoor air results

from a number of sources, and levels of fo rmaldehyde are generally higher in urban areas

than in rural areas (WHO-IPCS, 1989). Dire ct contributions of formaldehyde to the

atmosphere ( i.e ., those in the form of formaldehyde itself) from man-made sources are

present, but are generally considered to be small relative to natural sources or indirect

production of formaldehyde in th e atmosphere (WHO, 2002).

C.1.3. Formaldehyde in MON 88701

Formaldehyde is a metabolite when dicamba is sprayed on MON 88701 cotton.

However, formaldehyde is not considered a relevant metabolite in the demethylation of

dicamba by U.S. EPA. According to the guidelines published by Of fice of Prevention,

Pesticides and Toxic Substances, United St ates Environmental Protection Agency (US

EPA OPPTS 860.1300), the methoxy side chain that is cleaved from dicamba to form

formaldehyde would specifically not be chosen to be labeled in a metabolism study (U.S.

EPA, 1996). This is because it is not metabolically stable and would not be considered a

significant moiety as it would be readily meta bolized and incorporated into the 1-carbon

pool of the plant through known pathways. Therefore, formaldehyde was not measured

in the residue study when dicamba was applied to MON 88701.

The maximum theoretical production of form aldehyde produced from dicamba-treated

MON 88701 is estimated to be 6.3 mg/kg and 33 mg/kg 7 

# . This is well within the range of

formaldehyde concentrations measured for a variety of agricultural commodities,

including up to 60 mg/kg in fruits and vegetables (WHO-IPCS, 1989). Plants have a

large capacity to metabolize formaldehyde natu rally produced from in ternal processes (A.

Hanson (2011), C.V. Griffin, Sr. Eminent Schola r, Horticulture Department, University

of Florida, Personal Communication), and an y additional amount of formaldehyde that

could be theoretically produced in the pl ant by dicamba treatment in MON 88701 would

be metabolized very quickly. Thus the in cremental increase in formaldehyde over and

above the levels already presum ed to be present in the cott on plant would be small and

transient and associated with an outdoor application of dicamba herbicide. Further, since

current literature supports that formaldehyde is only emitted from foliage under certain

7 

# Calculation based an assumption that the entire 0.56 kg/ha (0.5 lb/acre a.e.) application of dicamba that is

intercepted by the MON 88701 cotton plant at the 6-leaf or first bloom plus 15 day growth stage is

instantaneously and completely absorbed, and then instantaneously metabolized by the DMO enzyme

(Complete demethylation of 560 g (2.5 mol)/ ha dicamba would yield 2.5 mol/ha formaldehyde). Canopy

closure, and thus spray interception, is estimated at 30 % at the 6-leaf stage (Krutz et al., 2012), resulting in

production of 23 g/ha formaldehyde. Canopy closure is near complete at the first bloom plus 15 day

growth stage (Reddy et al., 2009), so no adjustment is applied. Above-ground biomass of 6-leaf plants is

estimated to be 0.7 metric tons/ha (Ducamp et al., 2012), and the estimated maximum theoretical

concentration is 33 mg/kg formaldehyde in planta . For dicamba applications at first bloom plus 15 day

growth stage, the crop biomass is estimated to be 12 metric tons/ha (Boquet and Breitenbeck, 2000), and

the estimated maximum theoretical fo rmaldehyde concentration produced in planta is 6.3 mg/kg.

# Monsanto Company 12-CT-244U 275 of 620 

# conditions (Nemecek-Marshall et al., 1995; Cojo cariu et al., 2004; Cojo cariu et al., 2005)

and that emission rates are low (Nemecek-Marsha ll et al., 1995), little opportunity exists

for formaldehyde to be released from MON 88701 after dicamba treatment. Therefore

human safety concerns of formaldehyde re leased from dicamba-treated MON 88701 are

considered to be negligible and the most re levant route of exposure is from repeated

inhalation of concentrated levels associated with indoor or occupati onal environments.

USHHS-NTP (2011) has already st ated that there is no evid ence to suggest that dietary

intake of formaldehyde is important, despite NTP’s 12 th 

# Report on Carcinogens

reclassifying formaldehyde as a known hu man carcinogen by (USHHS-NTP, 2011). In

addition, the only human food currently produc ed from cottonseed is refined, bleached,

and deodorized (RBD) oil, and to a smaller exte nt, linters. Therefore, the potential for

human exposure to any formaldehyde in dicamba-treated MON 88701 cottonseed is

highly unlikely.

C.1.4. Conclusion

Data from both dicamba and glufosinate-tr eated and not treated MON 88701 compared to

a conventional control are available from multiple sites across the U.S., where

agronomic, phenotypic and environmental interac tion data were collected. The results of

this assessment demonstrate no biological ly meaningful difference between MON 88701

treated with and without dicamba and gluf osinate and the conventional control, and

support a conclusion that the formation of DCSA and formaldehyde does not alter the

weedy characteristics or increase susceptibility or tolerance to diseases, insect pests or

abiotic stresses. Therefore, MON 88701, as cul tivated, is no more likely to be a plant

pest risk or have a biologically meaningf ul change in environmental impact than

conventional cotton.

C.2. Characterization of MO N 88701 DMO Protein in MON 88701

C.2.1 Forms of DMO

Various forms of the DMO protei n (Figure C-1) were used to establish enzyme structure,

activity, substrate specificity and safety of the proteins in MON 88701. The wild-type

DMO was first isolated and characterized from Stenotrophomonas maltophilia (Herman

et al., 2005). The MON 88701 DMO protein pres ent in MON 88701 is identical to the

wild-type DMO, except for an additional leuc ine at position two and an additional nine

amino acids at the N-terminus from the chloro plast transit peptide, CTP2 (Figure C-1).

The E. coli -produced form of DMO is identical to the wild-type DMO, but with a

histidine-tag on the N-terminus (Figure C-1), was used for specificity experiments. The

differences in the amino acid sequence or the addition of N-terminal histidine tag did not

appear to have an effect on m ode-of-action, structure, functio nal activity, or specificity of

DMO, as these changes are sterically distant from the catalytic domain centers involved

in electron transport (Rieske and non-heme iron centers) and the catalytic centers for the

dicamba substrate (D’Ordine et al ., 2009; Dumitru et al., 2009).

# Monsanto Company 12-CT-244U 276 of 620 

# Figure C-1. Forms of DMO Protein and Their Relation to the Wild-Type DMO

Protein

The diagram represents the various DMO forms described in this pe tition. The wild-type

DMO form isolated from S. maltophilia was the first form sequenced (Herman et al.,

2005). The MON 88701 DMO protei n has an insertion of a leucine at position 2, and the

addition of 9 amino acids fr om CTP2 at the N-terminus . MON 88701 DMO was purified

from cottonseed of MON 88701. E. coli -produced MON 88701 DMO has the same

sequence as plant-produced MON 88701 DMO; equivalence between the two proteins

has been demonstrated (Section V.B and Append ix C). The N-terminal histidine-tagged

DMO was produced in E. coli and was used for in vitro specificity studies (Section

V.A.1.2).

C.2.2. Materials

The MON 88701 DMO protein (lot 11299151) was purified from cottonseed of

MON 88701 (lot 11287350). The MON 88701 DM O protein was stored in a -80 oC

freezer in a buffer solution containing 50 mM potassium phosphate, pH 8.0, 1 mM DTT,

1 mM benzamidine HCl, 0.1 M sodi um chloride and 10% glycerol.

The E. coli -produced MON 88701 DMO protein (lot 11300031) was used as the

reference substance. The DMO protein refe rence substance was generated from cell

paste produced by large- scale fermentation of E. coli containing the pMON136400

expression plasmid. The coding sequence for dmo contained on the expression plasmid

(pMON136400) was confirmed prior to and after fermentation. The E. coli -produced

MON 88701 DMO protein was pr eviously characterized.

C.2.3. Description of Assay Control

Protein MW standards (Precision Plus Protei n Standards Dual color; Bio-Rad, Hercules,

CA) were used to calibrate some SDS-PAGE gels and verify protein transfer to

polyvinylidene difluoride (PVDF) and nitro cellulose membranes. Broad Range

SDS-PAGE MW standards (Bio-Rad, Hercules, CA) were used to generate a standard

curve for the apparent MW estimation. Bovine serum albumin (BSA) and

 -aminobutyric acid (AAbA) were used as hydrolysis control and internal calibration

standard for amino acid analysis. The E. coli -produced MON 88701 DMO reference

standard was used to constr uct a standard curve for the estimation of total protein

concentration using a Bio-Rad protein assa y. A phenylthiohydantoi n (PTH) amino acid

standard mixture (Applied Biosystems, Fost er City, CA) was used to calibrate the

# Monsanto Company 12-CT-244U 277 of 620 

# Applied Biosystems 494 Procise Sequencing System for each analysis. A peptide

mixture (Sequazyme Peptide Mass Standards ki t, Applied Biosystems, Foster City, CA)

was used to calibrate the MALDI-TOF mass spectrometer fo r tryptic mass analysis.

Transferrin (Sigma-Aldrich, St. Louis, MO) was used as positive control for

glycosylation analysis.

C.2.4. Protein Purification

The MON 88701 DMO was purified from cottonseed of MON 88701. The purification

procedure was not performed under a GL P plan; however, all procedures were

documented on worksheets and, where applicable, SOPs were followed. The

MON 88701 DMO protein was purified from an extract of ground cottonseed using a

combination of ammonium sulfate precipitation, hydrophobic interaction

chromatography, anion exchange chroma tography, mixed mode ion exchange

chromatography and size exclusion chromat ography. The purification procedure is

briefly described below.

Approximately 1 kg of MON 88701 cottonseed expressing the DMO protein was mixed

with ~1 kg of dry ice and ground to fine powder using a laboratory mill (model 3100,

Perten Instruments). The ground powder was suspended in two liters of hexane (EMD

Chemicals Inc., Gibbstown, NJ ) and filtered. This process was repeated four times in

order to completely defat the powder. Af ter drying overnight, the powder was ready for

further processing. All grinding and defatti ng steps were done in a fume hood at room

temperature.

The ground powder was mixed with extr action buffer (50 mM Tris, pH 8.0, 2.0 M

deionized urea, 0.2 M boric acid, 1.0 mM dithiothreitol (DTT), 1.0 mM

benzamidine-HCl, 1.0 μm bestatin, 1.0 μM E-64 and Complete EDTA free protease

inhibitor cocktail (Roche Applied Science, I ndianapolis, IN)) to a final volume of 8 liters

and incubated for 2 h at room temperature. The slurry was cen trifuged at 15000 × g for

30 min at 4 °C. The supernatant was collec ted and brought to 0.05% polyethyleneimine

(PEI). The solution was stirred at ~4 °C for 30 min and then centrifuged at 15000 × g for

30 min. The supernatant was collected and ~2.2 kg of ammonium sulfate was slowly

added to bring the solution to 50% w/v amm onium sulfate saturati on. This solution was

stirred at ~4 °C for 2 h and the pellet wa s collected by centrifugation at 15000 × g for

30 min. The pellet was resuspended in 10 liters of the resuspension buffer (50 mM

Tris-HCl, pH 8.0, 0.35 M ammonium sulfate, 10 mM DTT, 1.0 mM benzamidine-HCl,

1.0 μM bestatin, 1.0 μM E-64 and Complete EDTA free protease inhibitor cocktail). The

solution was stirred in the cold room overnight and then centrifuged at 30,000  g for 1 h.

Supernatant was collected and loaded ont o a 1 liter butyl sepharose column (GE

Healthcare) equilibrated with butyl sepha rose equilibration (BSE) buffer (50 mM

Tris-HCl, pH 8.0, 0.35 M ammonium sulfate, 1 mM DTT, 1.0 mM benzamidine-HCl,

1.0 μM bestatin, 1.0 μM E-64 and Complete EDTA -free protease inhibito r cocktail). All

column steps were run at room temperature. The column was washed with 5 liters BSE

buffer. Proteins were eluted with 1 lite r of buffer containing 25 mM Triethanolamine,

pH 8.0, 100 μM dicamba, 1.0 mM DTT, 1.0 mM benzamidine-HCl, 1.0 μM bestatin,

1.0 μM E-64 and Complete EDTA free protease inhibitor cocktail (B SEL buffer). After

# Monsanto Company 12-CT-244U 278 of 620 

# eluting the proteins with 1 liter of BSEL buffer, the flow was stopped for one hour and

then elution was continued with additional 1 l iter of BSEL buffer. Both elutions were

pooled and loaded onto a 25 ml DEAE macropr ep column (Bio Rad) equilibrated with

DEAE macroprep equilibration (DME) bu ffer (50 mM Tris-HCl, pH 8.0, 100 μM

dicamba, 1.0 mM DTT, 1.0 mM benzamidin e-HCl, 1.0 μM bestatin, 1.0 μM E-64 and

Complete EDTA free protease inhibitor cocktail). All steps associated with DEAE

macroprep were performed at ~4 °C. The column was washed with 125 ml DME buffer

and proteins were eluted with 75 ml DME buf fer containing 70 mM NaCl and then with a

linear gradient that increased from 70 mM to 350 mM NaCl over 500 ml. Fractions

containing MON 88701 DMO we re pooled and loaded onto a 2.5 ml ceramic

hydroxyapatite (CHT) column (Bio-Rad) e quilibrated with CHT equilibration buffer

(50 mM Tris-HCl, pH 8.0, 100 μM dicamba, 1.0 mM DTT, 1.0 mM benzamidine-HCl,

1.0 μM bestatin, 1.0 μM E-64 and Complete EDTA free protease inhibitor cocktail ). All

steps associated with CHT were performed at ~4 °C. Most of the MON 88701 DMO was

found in the flow through and wash fractions . Flow through and wash fractions from

CHT were pooled separately (P ooled FT and Pooled Wash, re spectively) and reloaded on

two separate CHT columns as follows.

The Pooled FT was loaded onto a ~10 ml CHT column (CHT3). The column was

washed with 50 ml of CHT equilibration buffer and step eluted using the CHT

equilibration buffer containing 1 mM, 2 mM and 3 mM potassium phosphate, pH 8.0.

The Pooled Wash was loaded onto a ~3 ml CHT column (CHT2). CHT2 was washed

with ~45 ml of CHT equilibration buffer and step eluted using the CHT equilibration

buffer containing 1 mM, 2 mM and 3 mM potassium phosphate, pH 8.0.

Wash fractions from both the CHT2 and CHT3 chromatography runs that contained

MON 88701 DMO were pooled and loaded ont o a ~1 ml DEAE macroprep column

equilibrated with DME buffer for concentrati on. The column was washed with 10 ml of

the DME buffer and eluted with DME buffer containing 500 mM NaCl. The

MON 88701 DMO containing fractions were pooled and loaded onto a Hi-Prep

Sephacryl S 100 size exclusion column equilib rated at ~4 °C with 50 mM potassium

phosphate, pH 8.0, 1 mM DTT, 1 mM benzamidine-HCl, 0.1 M NaCl and 10% (v/v)

glycerol. Fractions containing MON 88701 DM O were pooled and concentrated with

aquacide (EMD Bioscienes, Inc., La Jolla, CA) at ~4 °C to a final volume of 750 μl.

Elution fractions (1-3 mM potassium phospha te, pH 8.0 fractions) from both the CHT2

and CHT3 that contained MON 88701 DMO were pooled a nd concentrated using a

Amicon ultra spin concentrator (Millipor e, Bedford, MA) with a 10K MWCO. The

centriprep concentrated pool was then load ed onto a Hi Prep Sephacryl S 100 size

exclusion column equilibrated at ~4 °C with 50 mM potassium phosphate, pH 8.0, 1 mM

DTT, 1 mM benzamidine-HCl, 0.1 mM NaCl and 10% (v/v) glycerol. Fractions

containing MON 88701 DMO were pooled and concentrated w ith aquacide at ~4 °C to a

final volume of 750 μl.

Both aquacide concentrated samples were pool ed to a final volume of 1.5 ml. The final

buffer composition of the purified MON 88701 DMO protein was 50 mM potassium

phosphate, pH 8.0, 1 mM DTT, 1 mM benzamidine-HCl, 100 mM sodium chloride and

# Monsanto Company 12-CT-244U 279 of 620 

# 10% (v/v) glycerol. This MON 88701 DM O purified from the cottonseed of

MON 88701 was aliquoted and stored in a -80 °C freezer.

# Monsanto Company 12-CT-244U 280 of 620 

# C.2.5. Summary of DMO Protein Identity and Equivalence

Table C-1. Summary of MON 88701 DMO Protein Identity and Equivalence

Analytical Test Assessment Analytical Test Outcome

1. N-terminal sequence analysis of the

MON 88701 DMO protein to assess

identity  The identity could not be confirmed by

N-terminal sequence analysis

 MALDI-TOF MS 1 

# analysis of peptides

derived from tryptic digested MON 88701

DMO established the N-terminal sequence of

MON 88701 DMO

2. MALDI-TOF MS 1 

# analysis of peptides

derived from tryptic digested

MON 88701 DMO protein to assess

identity  MALDI-TOF MS 1 

# analysis yielded peptide

masses consistent with the expected peptide

masses from the theoretical trypsin digest of

the MON 88701 DMO sequence

3. Western blot analysis using anti-DMO

polyclonal antibodies to assess identity

and immunoreactive equivalence

between MON 88701 DMO and the

E. coli -produced MON 88701 DMO

proteins  MON 88701 DMO protein identity was

confirmed using a western blot probed with

antibodies specific for DMO protein

 Immunoreactive properties of the

MON 88701 DMO and the E. coli -produced

MON 88701 DMO proteins were shown to be

equivalent

4. SDS-PAGE 2 

# to assess equivalence of

the apparent molecular weight between

MON 88701 DMO and the

E. coli -produced MON 88701 DMO

proteins  Electrophoretic mobility and apparent

molecular weight of the MON 88701 DMO

and the E. coli -produced MON 88701 DMO

proteins were shown to be equivalent

5. Glycosylation analysis of the

MON 88701 DMO protein to assess

equivalence between the MON 88701

DMO and E. coli -produced

MON 88701 DMO proteins  Glycosylation status of MON 88701 DMO

and E. coli -produced MON 88701 DMO

proteins were shown to be equivalent

6. DMO enzymatic activity analysis to

assess functional equivalence between

MON 88701 DMO and the

E. coli -produced MON 88701 DMO

proteins  Functional activity of the MON 88701 DMO

and the E. coli -produced MON 88701 DMO

proteins were shown to be equivalent

1 

# MALDI-TOF MS = Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

2 

# SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis



# C.2.6. N-Terminal Sequencing

C.2.6.1. Methods

N-terminal sequencing by automated Edman de gradation chemistry was carried out in an

attempt to confirm the identity of MON 88701 DMO.

# Monsanto Company 12-CT-244U 281 of 620 

# MON 88701 DMO was separated by SDS-PAGE and transferred to PVDF membrane.

The blot was stained using Coomassie Blue R-250. The major band at ~39 kDa

containing MON 88701 DM O was excised from the blot and was used for N-terminal

sequence analysis. The analysis was perf ormed for 15 cycles using automated Edman

degradation chemistry (Hunkapiller et al ., 1983) using an Applied Biosystems 494

Procise Sequencing System equipped with 140 C Microgradient system a Perkin Elmer

Series 200 UV/VIS Absorbance Detector with Procise  

# Control Software (version 2.1)

for amino acid detection after each cycle. Chromatographic data were collected using

SequencePro (version 2.1) software. A control protein,  -lactoglobulin, (Applied

Biosystems, Foster City, CA) was analyzed be fore and after the sequence analysis of the

MON 88701 DMO protein to verify that the sequencer met performance criteria for

repetitive yield and sequence identi ty. Identity was established if ≥ 8 amino acids,

consistent with the predicted sequence of the N-terminus of the MON 88701 DMO, were

observed during analysis.

C.2.6.2. Results of the N-t erminal Sequence Analysis

N-terminal sequencing reaction was pe rformed on MON 88701 DMO protein. The

reaction did not yield any observable sequen ce presumably because the N-terminus was

blocked. Although this analysis did not yield N-terminal se quence data, the N-terminus

of the MON 88701 DMO protein was determin ed using MALDI-TOF tryptic mass map

analysis (see Section C.2.6).

C.2.7. MALDI-TOF Tryptic Mass Map Analysis

C.2.7.1. Methods

MALDI-TOF tryptic mass fingerprint analysis was used to confirm the identity of the

MON 88701 DMO protein. MON 88701 DMO protein (~15 μg) was chilled in a -20 °C

freezer for at least 10 min. The chilled protein was precipitated with 200 μl of

95% acetone in a -20 °C freezer overnight. Pr ecipitated protein sample was pelleted in a

refrigerated centrifuge for at least 45 min at more than 13,000  g. The supernatant was

carefully removed and discarded. The protei n pellet was washed twice with 200 μl of

chilled ethanol to remove residual supernatant. The pellet was dried to completion using

a Speed Vac concentrator and resuspended in 30 μl of 40% 2,2,2,-trifluoroethanol (TFE)

in 25 mM ammonium bicarbonate. The re suspended protein was vortexed vigorously

and then sonicated for 5 min in a water bath. The sample was incubated at ~37 °C for 1 h

to denature the proteins. Denatured pr otein sample was reduced with ~5 mM

tris(2-carboxyethyl)pho sphine (TCEP) for 1 h at ~37 °C . Reduced protein sample was

then alkylated in the dark for 30 min at room temperature with ~10 mM iodoacetic acid.

Additional TCEP was added to ~5 mM and the sample was incubated for 10 min at room

temperature. The reduced and denatured test substance was mixed with 67 μl of 25 mM

ammonium bicarbonate and 2.5 μl of trypsin solution (0.2 μg/μl in 25 mM ammonium

bicarbonate). The tryptic diges tion was allowed to proceed for 15 h at 37 °C followed by

quenching with 1 μl of formic acid. Proteolytic peptides we re dried to completion using

Speed Vac concentrator. To solubilize the dried peptides, a solution of 50% acetonitrile,

0.1% TFA was added and sonicated for 5 min. Aliquots from the digest were spotted to

# Monsanto Company 12-CT-244U 282 of 620 

# three wells on an analysis plate. For each spot, 0.75 μl of 2, 5 dihydroxybenzoic acid

(DHB), α -cyano-4-hydroxycinnamic acid ( α -Cyano), or

3, 5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (Thermo Fisher Scientific Inc.)

was added to one of the spots. The sample in DHB matrix was analyzed in the 300 to

5000 Da range. Samples in α -Cyano and Sinapinic acid were analyzed in the 500 to

5000 Da and 500 to 7000 Da range, respectivel y. The analysis was performed using a

Voyager TM 

# DE Pro Biospectrometry TM 

# workstation (Applied Biosystems) using Voyager

Instrument Control Panel software (version 5.10.2) and Data Explorer data analysis

software (version 4.0.0.0). Protonated peptide masses were monoisotopically resolved in

reflector mode (Aebersold, 1993; Billeci and Stults, 1993). CalMix 2 was used as the

external calibrant (Sequazyme Peptide Mass Standards kit, Applied Biosystems, Foster

City, CA) for the analysis. GPMAW32 softwa re (Lighthouse Data, Odense M, Denmark)

was used to generate a theoretical tryps in digest of the MON 88701 DMO protein

sequence. Masses within 1 Da of a m onosiotopic mass were matched against the

theoretical digest of the MON 88701 DMO se quence. All matching masses were tallied

and a coverage map was generated for the ma ss fingerprint. The tr yptic mass fingerprint

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 (Biron et al., 2006, Krause et al., 1999).

C.2.7.2. Results of MALDI-TOF Tryptic Mass Map Analysis

The identity of the MON 88701 DMO prot ein was confirmed by MALDI-TOF MS

analysis of peptide fragments produced fr om tryptic digestion of the MON 88701 DMO

protein. The ability to iden tify a protein using this met hod is dependent upon matching a

sufficient number of observed tryptic pept ide fragment masses w ith predicted tryptic

peptide fragment masses. In general, prot ein identification made by peptide mapping is

considered to be reliable if  40% of the protein sequenc e was identified by matching

experimental masses observed for the tryptic peptide fragments to the expected masses

for the fragments (Biron et al ., 2006, Krause et al., 1999).

There were 19 unique peptides identified that corresponded to the masses expected to be

produced by tryptic digestion of the MON 88701 DMO protein (Table C-2). The

identified masses were used to assemble a coverage map of the entire MON 88701 DMO

protein (Figure C-2). The experimentally determined mass coverage of the MON 88701

DMO protein was 66.5% (232 out of 349 amino acids). This anal ysis serves as identity

confirmation for the MON 88701 DMO protein.

To identify the N-terminus, the experiment ally determined masses of the peptides

produced from tryptic digestion of the M ON 88701 DMO protein were examined for the

presence of a mass that matched the theoretical mass expected from the MON 88701

DMO protein deduced from the dmo gene present in MON 88701. A mass was identified

that corresponded to the predicted mass of an acetylated peptide with nine amino acids

from CTP2 followed by the MON 88701 DMO protein deduced from the dmo gene

present in MON 88701. The additional nine amino acids of CTP2 resulted from the

alternative processing of CTP 2. Alternative processing of DMO precursor proteins has

been observed in other dicamba-t olerant plants containing the dmo gene (Behrens et al.,

# Monsanto Company 12-CT-244U 283 of 620 

# 2007). Hence, the MON 88701 DMO protein was de signated to have an N-terminal end

as shown in Figure C-2.Monsanto Company 12-CT-244U 284 of 620Table C-2. Summary of the Tryptic Masses1Identified for the MON 88701 DMO Protein Using MALDI-TOF MS-cyanoDHB Sinapinic acid Expected Mass Diff.2Fragment3Sequence720.40 720.37 0.03 140-145 VDPAYR833.51 833.45 833.45 0.06 108-114 SFPVVER856.49 856.43 0.06 251-257 EQSIHSR914.60 914.53 0.07 305-312 VVVEAIER1030.58 1030.57 0.01 293-301 SWQAQALVK1108.61 1108.59 1108.50 0.11 176-185 ANAQTDAFDR1275.87 1275.83 1275.73 0.14 35-45 TILDTPLALYR1286.83 1286.70 0.13 302-312 EDKVVVEAIER1428.84 1428.83 1428.69 0.15 218-230 GANTPVDAWNDIR1470.74 1470.63 0.11 146-158 TVGGYGHVDCNYK1501.91 1501.79 0.12 189-202 EVIVGDGEIQAALMK1506.86 1506.73 0.13 176-188 ANAQTDAFDRLER1577.89 1577.80 1577.73 0.16 279-292 NFGIDDPEMDGVLR1731.92 1731.80 0.12 1-15 VMSSVSTACMLTFVR +42 Da (N-acetylation)1745.09 1744.99 1744.93 0.16234-250 VSAMLNFIAVAPEGTPK1994.30 1994.23 1994.03 0.27 159-175 LLVDNLMDLGHAQYVHR2143.35 2143.12 0.23 16-34 NAWYVAALPEELSEKPLGR2398.37 2398.35 2398.09 0.28258-278 GTHILTPETEASCHYFFGSSR2724.72 2724.31 0.41 115-139 DALIWIWPGDPALADPGAIPGCR1Only experimental masses that matched expected masses are listed in the table.2The difference between the expected mass and the first column mass.Other masses shown within a row are also within 1 Da of the expected mass.3Position refers to amino acid residues within the predicted MON 88701 DMO sequence as depicted in Figure C-2.DHB = 5-dihydroxybenzoic acid matrix,α-cyano =α-cyano-4-hydroxycinnamic acid matrix, Sinapinic acid = 3, 5-dimethoxy-4-hydroxycinnamic acid matrix.

# Monsanto Company 12-CT-244U 285 of 620 

# Figure C-2. MALDI-TOF MS Coverage Map of the MON 88701 DMO Protein

The amino acid sequence of the MON 88701 DMO protein was deduced from the dmo

gene present in MON 88701. Boxed regions correspond to regions covered by tryptic

peptides that were identified from the MON 88701 DMO protein sample using

MALDI-TOF MS. Underlined region correspo nds to the nine amino acids from CTP2

retained at the N-terminus of the MON 88701 DMO. In total, 66.5% (232 of 349 total

amino acids) of the expected protein sequen ce was covered by the identified peptides.

# Monsanto Company 12-CT-244U 286 of 620 

# C.2.8. Western Blot Analysis-Immunoreactivity

C.2.8.1. Methods

Western blot analysis was performed to confirm the identity of the MON 88701 DMO

protein purified from cottonseed of MON 88701 and to compare the immunoreactivity of

the MON 88701 DMO and E. coli -produced MON 88701 DMO proteins.

The MON 88701 DMO and E. coli -produced MON 88701 DMO prot eins were analyzed

concurrently on the same gel using three lo adings of 0.5, 2, and 6 ng. Loadings of the

three concentrations were made in duplicate on the gel. Aliquots of each protein were

diluted in water and 5X Laemmli buffer (LB) containing 312 mM Tris-HCl, 25% (v/v)

2-mercaptoethanol, 10% (w/v) SDS, 0.025% (w/v) bromophenol blue, 50% (v/v)

glycerol, pH 6.8, heated at 101 °C for 3 min, and applied to a 15 well pre-cast

Tris-glycine 4-20% polyacrylamide gradient gel (Invitrogen, Carlsbad, CA). Pre-stained

molecular weight markers (Precision Plus Protein Standards Dual color; Bio-Rad,

Hercules, CA) were loaded in parallel to veri fy electrotransfer of the proteins to the

membrane and to estimate the size of the immunoreactive bands observed.

Electrophoresis was performed at a constant voltage of 150 V for 90 min. Electrotransfer

to a 0.45 μm nitrocellulose membrane (Invi trogen, Carlsbad, CA) was performed for

105 min at a constant voltage of 25 V. Af ter electrotransfer, the membrane was stored

overnight with 1  phosphate buffered saline containi ng 0.05% (v/v) Tween-20 (PBST) at

4 °C. The membrane was blocked for 1 h with 5% (w/v) NFDM in PBST at room

temperature. The membrane was then probe d with a 1:5000 dilution of goat anti-DMO

antibody (lot 11223358) in 2% NFDM in PBST for 1 h at room temperature. Excess

antibody was removed using two 1 min washes followed by three 5 min washes with

PBST. Finally, the membrane was probe d with horseradish peroxidase (HRP)-

conjugated horse anti-goat IgG (Thermo, Ro ckford, IL) at a dilution of 1:10,000 in

2% NFDM in PBST for 1 h at room temper ature. Excess HRP-conjugate was removed

using two 1 min washes and three 5 min washes with PBST. All washes were performed

at room temperature. Immunoreactive bands were visualized using the ECL detection

system (GE, Healthcare, Pi scataway, NJ) with exposure to Amersham Hyperfilm ECL

(GE, Healthcare, Piscataway, NJ). The f ilm was developed using a Konica SRX-101A

automated film processor (Konica Minolta Medical & Graphic, Inc., Tokyo, Japan).

Quantification of the bands on the blot was performed using a Bio-Rad GS-800

densitometer with the supplied Quantity On e software (version 4.4.0, Hercules, CA)

using the band location and volume tool. Th e signal intensities of the immunoreactive

bands observed for the MON 88701 DMO and E. coli -produced MON 88701 DMO

proteins migrating at the expected position on the blot film were quantified as “adjusted

volume” values. The raw data was exported to a Microsoft Excel (2007) file. The

immunoreactivity of the MON 88701 DMO and E. coli -produced MON 88701 DMO

proteins were reported as the mean signal in tensity at each amount of protein analyzed.

The immunoreactivity of the MON 88701 DMO and E. coli -produced MON 88701 DMO

proteins were considered equivalent if th e overall mean of the immunoreactive signal of

the MON 88701 DMO protein was within ± 35% of the overall mean of immunoreactive

signal of the E. coli -produced MON 88701 DMO protein.

# Monsanto Company 12-CT-244U 287 of 620 

# C.2.8.2. Results of MON 88701 DMO Protei n Immunoreactivity Equivalence

Western blot analysis was conducted using goat anti-DMO polyclonal antibodies to

1) assess the identity of the MON 88701 DMO protein isolated from the cottonseed of

MON 88701 and 2) determine the relative immunoreactivity of the MON 88701 DMO

and the E. coli -produced MON 88701 DMO proteins. Th e results demonstrated that the

anti-DMO antibodies recognized the MON 88701 DMO protein that migrated to the same

position on the blot as the E. coli -produced MON 88701 DMO pr otein (Figure C-3).

Furthermore, the immunoreactive signal increased with increasing amounts of

MON 88701 DMO protein loaded. Two other bands, one migrating at ~75 kDa and the

other at ~17 kDa were also observed. These bands were prominent in lanes with higher

load amounts (Figure C-3, Lanes 3-6), and may represent products of aggregation and

degradation of DM O, respectively.

Densitometric analysis was conducted to compare the immunoreactivity of MON 88701

DMO and E. coli -produced MON 88701 DMO proteins. The mean signal intensity

(OD  mm 2 

# ) from the MON 88701 DMO bands and from the E. coli -produced

MON 88701 DMO bands at each amount of prot ein analyzed was calculated and then

overall mean signal intensity was calculate d (Table C-3). Th e immunoreactivity was

considered equivalent if the overall m ean signal intensity of all MON 88701 DMO

protein bands was within ±35% of the overall mean signal intensity of E. coli -produced

MON 88701 DMO protein bands across all loading levels.

The overall mean signal intensity of the E. coli -produced MON 88701 DMO bands was

6.500 OD × mm 2 

# , and the overall mean signal inte nsity of the MON 88701 DMO bands

was 4.440 OD × mm 2 

# . Because overall mean signa l intensity of the MON 88701 DMO

protein bands was between 4.225 and 8.775 (between -35% and +35% of the

E. coli -produced MON 88701 DMO bands), the MON 88701 DMO and E. coli -produced

MON 88701 DMO proteins were determined to have equivalent immunoreactivity.

# Monsanto Company 12-CT-244U 288 of 620 

# Figure C-3. Western Blot Analysis of MON 88701 DMO and E. coli -produced

MON 88701 DMO Proteins

Aliquots of the MON 88701 DMO protein and the E. coli -produced MON 88701 DMO

protein were subjected to SDS-PAGE and electrotransfe rred to a nitrocellulose

membrane. The membrane was incubated with anti-DMO antibodies and

immunoreactive bands were visualized using an ECL system (GE Healthcare,

Piscataway, NJ). Approximate molecular we ights (kDa) are shown on the left. Lanes

loaded with molecular weight markers were cropped, and lanes were renumbered relative

to the original gel loading. The 6 min e xposure is shown. Lane designations are as

follows:

Lane Sample Amount (ng)

1 E. coli -produced MON 88701 DMO protein 0.5

2 E. coli -produced MON 88701 DMO protein 0.5

3 E. coli -produced MON 88701 DMO protein 2

4 E. coli -produced MON 88701 DMO protein 2

5 E. coli -produced MON 88701 DMO protein 6

6 E. coli -produced MON 88701 DMO protein 6

7 MON 88701 DMO protein 0.5

8 MON 88701 DMO protein 0.5

9 MON 88701 DMO protein 2

10 MON 88701 DMO protein 2

11 MON 88701 DMO protein 6

12 MON 88701 DMO protein 6

# Monsanto Company 12-CT-244U 289 of 620 

# Table C-3. Comparison of Immunoreactive Signals Between MON 88701 DMO and

E. coli -produced MON 88701 DMO Proteins

Mean Signal intensity from

MON 88701 DMO

(OD × mm 2 

# ) Mean Signal intensity from

E. coli -produced

MON 88701 DMO

(OD × mm 2 

# ) Preset Acceptance limits for

MON 88701 DMO 1



# (OD × mm 2 

# )

4.440 6.500 4.225 – 8.775

1 

# The acceptance limits for MO N 88701 DMO are based on the interval between +35% (6.500 × 1.35) and

-35% (6.500 × 0.65) of the overall mean of the E. coli -produced MON 88701 DMO signal intensity across

six loads.

# Monsanto Company 12-CT-244U 290 of 620 

# C.2.9. Molecular Weight and Purity Estimation using SDS-PAGE

C.2.9.1. Methods

MON 88701 DMO and E. coli -produced MON 88701 DMO proteins were mixed with

5X LB and diluted with wate r to a final total protein concentration of 0.1 μg/μl.

Molecular Weight Standards, Bi o-Rad broad range (Hercules, CA) were diluted to a final

total protein concentration of 0.9  g/  l. The MON 88701 DMO was analyzed in

duplicate at 0.5, 1, and 1.5 μg protein per lane. The E. coli -produced MON 88701 DMO

reference standard was analyzed at 0.5 μg to tal protein in a single lane. The samples

were loaded onto a 10-well pre-cast Tris gl ycine 4-20% (w/v) polyacrylamide gradient

mini-gel (Invitrogen, Carlsbad, CA) and el ectrophoresis was performed at a constant

voltage of 125 V for 90 min. Proteins were fixed by placing the gel in a solution of

40% (v/v) methanol and 7% (v /v) acetic acid for 25 min, stai ned for ~16 h with Brilliant

Blue G-Colloidal stain (Sigma-Aldrich, St. L ouis, MO). Gels were destained once for

30 sec with a solution containi ng 10% (v/v) acetic acid and 25 % (v/v) methanol, and with

25% (v/v) methanol for a total of 6 h. Analysis of the gel was performed using a Bio-Rad

GS-800 densitometer with the supplied Quan tity One software (version 4.4.0, Hercules,

CA). The apparent MW of each observed band was estimated from a standard curve

generated by the Quantity One software wh ich was based on the MWs of the markers and

their migration distance on the gel. To determine purity, al l visible bands within each

lane were quantified using Quantity One software. Apparent MW and purity were

reported as an average of all si x lanes containing the MON 88701 DMO.

C.2.9.2. Results of MON 88701 DMO Protei n Molecular Weight Equivalence

The molecular weight and purity of the M ON 88701 DMO protein were determined to be

39.5 kDa and 97%, respectively. To assess molecular weight (MW) and purity, the

MON 88701 DMO protein was su bjected to SDS-PAGE. Th e gel was stained with

Brilliant Blue G Colloidal stain and an alyzed by densitometry (Figure C-4).

E. coli -produced MON 88701 DMO protein was loaded in a single lane for reference

(Figure C-4, Lane 2). The MON 88701 DMO protein (Figure C-4, Lanes 3-8) had an

apparent MW of 39.5 kDa (Table C- 4). The apparent MW of the E. coli -produced MON

88701 DMO protein as reported on its Certificat e of Analysis was 38.7 kDa (Table C-4).

Because the apparent MW of MON 88701 DMO protein was within the preset

acceptance limits for equivalence (Table C-4), the MON 88701 DMO and

E. coli -produced MON 88701 DMO proteins were determined to have equivalent

apparent MWs.

The purity of the MON 88701 DMO protein was calculated based on the six loads on the

gel (Figure C-4, Lanes 3 to 8). The aver age purity was determined to be 97%.

# Monsanto Company 12-CT-244U 291 of 620 

# Table C-4. Molecular Weight Comp arison Between the MON 88701 DMO and

E. coli -produced MON 88701 DMO Proteins Based on SDS-PAGE

Apparent MW of MON 88701

DMO Protein 1



# (kDa) Apparent MW of

E. coli -Produced MON 88701

DMO Protein 2



# (kDa) Preset Acceptance

Limits for

MON 88701 DMO 3



# (kDa)

39.5 38.7 38.5-39.7

1 

# The reported value is the mean mol ecular weight across all six loads.

2 

# The molecular weight of the E

. coli -produced MON 88701 DMO protein as reported on its Certificate of

Analysis.

3 

# See Section C.6.

# Monsanto Company 12-CT-244U 292 of 620 

# Figure C-4. Molecular Weight and Purity Analysis of the MON 88701 DMO

Protein

Aliquots of the MON 88701 DMO and the E. coli -produced MON 88701 DMO proteins

were separated by SDS-PAGE and then stained with Brilliant Blue G-Colloidal stain.

Approximate molecular weights are shown on the left and correspond to the markers

loaded in Lanes 1 and 9. Empty lane was pa rtially cropped. Lane designations are as

follows:

Lane Sample Amount (μg)

1 Broad Range Molecular Weight Markers 4.5

2 E. coli -produced MON 88701 DMO protein 0.5

3 MON 88701 DMO protein 0.5

4 MON 88701 DMO protein 0.5

5 MON 88701 DMO protein 1

6 MON 88701 DMO protein 1

7 MON 88701 DMO protein 1.5

8 MON 88701 DMO protein 1.5

9 Broad Range Molecular Weight markers 4.5

# Monsanto Company 12-CT-244U 293 of 620 

# C.2.10. Glycosylation Analysis

C.2.10.1. Methods

Glycosylation analysis was used to de termine whether the MON 88701 DMO was posttranslationally modified with covalently bound carbohydrate moieties. Aliquots of the

MON 88701 DMO protein, the E. coli -produced MON 88701 DMO (negative control)

and the positive control, tran sferrin (Sigma-Aldrich, St L ouis, MO), were each diluted

with water and brought to 1X LB. These samp les were heated at ~101 °C for 3 min. The

MON 88701 DMO, the E. coli - produced MON 88701 DMO and transferrin were loaded

at approximately 50 and 100 ng per lane on a Tris-glycine 10 well 4-20% polyacrylamide

gradient mini-gel (Invitrogen, Carlsbad, CA ). Precision Plus Protein Dual color

Standards (Bio-Rad, Hercules, CA) were also loaded to verify electrotransfer of the

proteins to the membrane and as markers for molecular weight. Electrophoresis was

performed at a constant voltage of 150 V fo r 90 min. Electrotransfer to a 0.45 μm PVDF

membrane (Invitrogen, Carlsbad, CA) was perfor med for 90 min at a constant voltage of

25 V.

Carbohydrate detection was performed dir ectly on the PVDF me mbrane at room

temperature using the Amersham ECL glycopr otein Detection Module (GE, Healthcare,

Piscataway, NJ). With this module, carbohydrate moieties of pr oteins were ox idized with

sodium metaperiodate and then biotinylated with biotin-X-hydrazide. The biotinylated

proteins can be detected on the blot by addi tion of streptavidin conjugated to HRP for

luminol-based detection using ECL reagents (GE, Healthcare, Pis cataway, NJ) and with

subsequent exposure to Amersham Hyperfilm (G E, Healthcare). The film was developed

using a Konica SRX-101A automated film processor (Konica Minolta Medical &

Graphic, Inc., Tokyo, Japan).

An identical blot run in parall el to that used for the glycos ylation analysis was stained to

visualize the proteins present on the membrane. Proteins were stained for 30 sec to 2 min

using Coomassie Brilliant Blue R-250 stai ning solution (Bio-Rad, Hercules, CA) and

then destained with 1X Coomassie Brilliant Blue R-250 destaining solution (Bio-Rad) for

5 min. After washing with water, the blot was dried and scanne d using Bio-Rad GS-800

densitometer with the supplied Quantity One software (version 4.4.0).

C.2.10.2. Results of Glycosylation Analysis

Some eukaryotic proteins are post-translationally modified by the addition of

carbohydrate moieties (Rademacher et al., 1988) . To test whether DMO protein was

glycosylated when expressed in the cottonseed of MON 88701, the MON 88701 DMO

protein was analyzed using an ECL Glycopr otein Detection Modul e (GE, Healthcare,

Piscataway, NJ). Transferrin, a glycosylated protein, was used as a positive control in the

assay. To assess equivalence of the MON 88701 DMO and E. coli -produced

MON 88701 DMO proteins, the E. coli -produced MON 88701 DMO protein was also

analyzed. The positive control was clearly detected at expected molecular weight

(~80 kDa) and the band intens ity increased with increasing concentration (Figure C-5,

Panel A, Lanes 1-2). In cont rast, signals were not observed in the lanes containing the

# Monsanto Company 12-CT-244U 294 of 620 

# MON 88701 DMO or E. coli -produced MON 88701 DMO prot eins at the expected

molecular weight for the M ON 88701 DMO protein (Figure C- 5 Panel A, Lanes 7-8 and

Lanes 4-5, respectively). To asse ss that sufficient MON 88701 DMO and

E. coli -produced MON 88701 DMO proteins were pr esent for glycosylation analysis, a

second membrane (with identical loadings and transfer times ) was stained with

Coomassie Blue R250 for protein detection (Figure C-5 Panel B). Both the MON 88701

DMO and E. coli -produced MON 88701 DMO proteins we re clearly detected (Figure C5 Panel B, Lanes 7-8 and Lanes 4-5, respec tively). These data indicate that the

glycosylation status of MON 88701 DMO and E. coli -produced MON 88701 DMO

proteins are equivalent and th at neither is glycosylated.

# Monsanto Company 12-CT-244U 295 of 620 

# Figure C-5. Glycosylation Analysis of the MON 88701 DMO Protein

Aliquots of the transferrin (positive control), E. coli -produced MON 88701 DMO protein

and MON 88701 DMO prot ein were subjected to SDS-PA GE and electrotransferred to

PVDF membranes. Panel A corresponds to detection of the labeled carbohydrate

moieties, where present, using the ECL-base d system with exposure to Hyperfilm. A

6 min exposure is shown. Panel B corres ponds to Coomassie Brilliant Blue R250

staining of an equivalent bl ot to confirm the presence of proteins. The signal was

captured using a Bio-Rad GS-800 with Qu antity One software (version 4.4.0).

Approximate molecular weights (kDa) co rrespond to the Precision Plus, dual color

markers (used to verify transfer and MW). Lanes loaded with molecular weight markers

were partially cropped, and lanes were renumbere d relative to the original gel loading.

Arrows indicate the expected migration MON 88701 DMO protein. Lane designations

are as follows:

Lane Sample Amount (ng)

1 Transferrin (positive control) 50

2 Transferrin (positive control) 100

3 Empty

-

4 E. coli -produced MON 88701 DMO (negative control) 50

5 E. coli -produced MON 88701 DMO (negative control) 100

6 Empty

-

7 MON 88701 DMO 50

8 MON 88701 DMO 100

# Monsanto Company 12-CT-244U 296 of 620 

# C.2.11. Functional Activity Analysis

C.2.11.1. Methods

The specific activity of MON 88701 DMO and E. coli -produced MON 88701 DMO was

determined by quantifying the conversio n of 3,6-dichloro-2-methoxybenzoic acid

(dicamba) to 3,6-dichlorosalicylic acid (D CSA) via HPLC (Agilent Technologies 1100

series, Santa Clara, CA) separation and fluo rescence detection (Agi lent Technologies

1200 series, G1321A). Each assay reaction contained 25 mM potassium phosphate,

pH 7.2, 3.4 μ g ferredoxin, 3.4 μ g reductase, 0.5 mM FeSO 4 

# , 10 mM MgCl 2 

# , 0.7 mM

NADH, 0.3 mM dicamba, 2 μ l (42.48 U/ml) of formaldehyde dehydrogenase and either

2.9

 μ g MON 88701 DMO or 3 μ g his-DMO as an assay positive control. The reactions

were performed in PCR tubes (Sorenson, Salt Lake City, UT) and incubated at 30 °C for

15 min. Reactions (200 μl) were initiated by the addition of dicamba and quenched with

the addition of 50 μ l of 5% H 2 

# SO 4 

# . Reactions were then filtered using Whatman

Anotop 10 filters (0.2 μ m, GE healthcare), and 40 μ l was transferred to a HPLC sample

vial (200 μ l, Agilent) for analysis. Twenty-five microliters of the filtered reaction was

injected onto a Phenomenex® Synergi 4 μ m C18/ODS Hydro-RP column (150 × 4.6 mm

ID, Torrance, CA). The mobile phase cons isted of solvent A (21.5 mM phosphoric acid)

and solvent B (100% acetonitr ile) running at 1.5 ml/min. DC SA was eluted from the

column using a linear gradient from 90% to 40% solvent A for the first 14 min, followed

by a step to 10% solvent A for 1 min and th en re-equilibration at 90% solvent A for

10 min before the next inject ion. DCSA was monitored by the detection of fluorescent

emission at 424 nm (excitation 306 nm) and qu antified relative to a standard curve of

DCSA generated using 0.1, 0.3, 0.6, 0.9, 1.2, 2.4, and 4.8 nmol/250 μ l. Chromatographic

data were collected using Atlas TM 

# 2003 software (Thermo Fisher Scientific Inc). The

specific activity was calculated based on the amount of purity corrected MON 88701

DMO protein added to the reaction mixture and expressed as nmol of DCSA produced

per minute per mg of MON 88701 DMO protein (nmol  min -1 

# × mg -1 

# ).

C.2.11.2. Results of Functional Activity

The functional activities of the MON 88701 DMO and E. coli -produced MON 88701

DMO proteins were determined by quantifyi ng the conversion of dicamba to DCSA

using HPLC separation and fluorescence detectio n. In this assay, protein-specific activity

is expressed as nmol DCSA × minute -1 

# × mg -1 

# of DMO.

The experimentally-determined specifi c activities for the MON 88701 DMO and

E. coli -produced MON 88701 DMO proteins are pr esented in Table C-5. The specific

activities of MON 88701 DMO and E. coli -produced MON 88701 DMO proteins were

5.48 and 7.23 nmol DCSA × minute -1 

# × mg -1 

# of DMO, respectively. Because the mean

specific activities of the MON 88701-produced and E. coli -produced MON 88701 DMO

proteins fall within the pr eset acceptance criterion (T able C-5), the MON 88701 DMO

and E. coli -produced MON 88701 DMO proteins were determined to have equivalent

functional activity.

# Monsanto Company 12-CT-244U 297 of 620 

# Table C-5. MON 88701 DMO Functional Activity Assay

MON 88701 DMO 1



# (nmol DCSA × minute -1 

# × mg -1 

# ) 

# E. coli -produced

MON 88701 DMO 1



# (nmol DCSA × minute -1 

# × mg -1 

# ) 

# Preset Acceptance Limits

for MON 88701 DMO 2



# (nmol DCSA × minute -1 

# × mg -1 

# )



# 5.48 ± 1.3 7.23 ± 2.1 1.69-20.74

1 

# Value refers to mean and standard deviation calculated based on n = 5.

2 

# See Section C.6.



# C.3. Substrate Specificity of MON 88701 DMO Protein

C.3.1. Exogenous Specificity Herbicid e Tolerance - Greenhouse Analysis

C.3.1.1. Materials

MON 88701 (lots IG2000000439645080059904 and IG2000000371459002138624) and

the near isogenic conventional control, Coker 130, (lots IG2000000025726392598528,

IG2000000025726407540736, and IG2000000025726372937728) were grown in

greenhouses during 2010 and 2011. At the 2-5 leaf growth stage or pre-emergent,

MON 88701 and the near isogenic conventiona l control, Coker 130, were sprayed with

different herbicides. The herbicid es tested are listed in Table C-6.

Table C-6. Herbicides Tested in Exoge nous Specificity Herbicide Tolerance

Greenhouse Trials

 

# Harness and Roundup WeatherMax are registered tr ademarks of Monsanto Technology LLC. All other

trademarks are the property of their respective owners.



# Manufacturer/

Retailer Herbicide Herbicide

Formulation Lot Number

Albaugh 2,4-DB Butyrac  

# 200 HPR-0404-14987-F

BASF dicamba Clarity ® 

# KIH-0702-18134-F

Dow atrazine Atrazine AGT-0804-19336-F

Dow trifluralin Treflan ® 

# MB231656T7

Dow oxyfluorfen Goal ® 

# 2XL EWP-0107-11628-F

Helena 2,4-D 2,4-D Amine 4 RUD-0502-15805-F

Monsanto acetochlor Harness ® 

# MUS-0704-18520-F

Monsanto halosulfuron Permit ® 

# MUS-0405-15154-F

Monsanto glyphosate Roundup WeatherMax ® 

# MUS-0905-19887-F

Syngenta paraquat Gramoxone ® 

# GTA-0606-17421

# Monsanto Company 12-CT-244U 298 of 620 

# C.3.1.2. Exogenous Specificity Herbicide Tolerance Greenhouse Method

MON 88701 and the near isogenic conventional control, Coker 130, were planted in pots

containing Redi-earth® and Os mocote® 14-14-14 slow releas e fertilizer or Peters® 20-

20-20 fertilizer. There were 10 replicate pot s and one plant per re plicate of MON 88701

and the conventional control for each herbicid e and rate tested. Th e pots were randomly

placed in a greenhouse and grown under normal agronomic conditions for cotton (relative

humidity 10-70%, temperature 21-34  ̊ C, 14 hour photoperiod, and watering as needed).

At pre-emergence or when the plants were at the 2-5 leaf growth stage, the replicates

were treated with a single herbicide and rate (Table 1). Two different application rates of

each herbicide were applied to different repl icate sets (Table 2). Based on the U.S.

herbicide labeled rates, the rates for the expe riments were chosen and then adjusted for

use on cotton and for the optimal growing c onditions in the greenhouse in order to

achieve approximately 40 to 80% injury. Twen ty to 22 days after a pplication, all plants

were rated for percent injury. Ratings were based on visual assessment of chlorosis,

necrosis, malformation, stunting, and biomass reduction with 0 being no visible injury

and 100 being completely dead. All 10 replicate ratings were averaged.

C.3.1.3. Results of Herbicide Tolerance Greenhouse Trials

MON 88701 demonstrated reduced injury rating s for dicamba, but similar injury ratings,

and therefore similar levels of susceptibility as the near isogenic conventional control,

Coker 130, for the remaining 9 herb icides tested (Table C-7).Monsanto Company 12-CT-244U 299 of 620Table C-7. Herbicide Tolerance Trials Injury RatingsInjury Observations(days afterapplication)Injury ratings (%)3Formulation Manufacturer Herbicide1Labeled Rate Range(g/ha)2Rates Applied(g/ha)2Control4Average (Range)MON 887015Average (Range)Clarity®BASF dicamba 140-2242 (a.e.)561 (a.e.)1120 (a.e.) 2287 (80-90)92 (85-95)05 (3-8)2,4-D Amine 4 Helena 2,4-D 140-2242 (a.e.)280 (a.e.)561 (a.e.) 2286 (80-90)96 (90-99)88 (85-95)98 (95-99)Butyrac®200 Albaugh 2,4-DB 130-1682 (a.e.)280 (a.e.)561 (a.e.) 2279 (70-85)96 (90-99)84 (75-95)96 (90-99)Gramoxone®Syngenta paraquat 280-1120 (a.e.)561 (a.e.)841 (a.e.) 2088 (85-95)95 (90-98)84 (80-90)95 (90-100)Harness®Monsanto acetochlor 930-4485 (a.i.)4485 (a.i.)6732 (a.i.) 2170 (30-100)84 (40-100)68 (50-100)93 (80-100)Atrazine Dow atrazine 1100-3800 (a.i.)1682 (a.i.)3364 (a.i.) 2128 (20-40)48 (20-95)29 (20-40)62 (20-100)Treflan®Dow trifluralin 560-2242 (a.i.)4485 (a.i.)6732 (a.i.) 212 (0-5)4 (0-10)1 (0-5)5 (0-10)Roundup WeatherMax®Monsanto glyphosate 280-4162 (a.e.)75 (a.e.)240 (a.e.) 219 (5-20)46 (35-60)12 (5-25)50 (40-60)Goal®2XL Dow oxyfluorfen 280-2242 (a.i.)561 (a.i.)841 (a.i.) 2137 (25-25)46 (30-80)41 (30-50)46 (35-80)Permit®Monsanto halosulfuron 36-140 (a.i.)75 (a.i.)200 (a.i.) 2148 (40-55)59 (50-65)50 (45-55)59 (55-65)1Herbicides applied pre-emergent were acetochlor, atrazine, and trifluralin. All other herbicides were applied when the plantswere at the 2-5 leaf growth stage.2a.e. = acid equivalent; a.i. = active ingredient. Each herbicidecontains the active ingredient directly or the salt form of the active ingredient. Whendetermining the rate ofapplication, the salt form is calculated back to the acid that is the active ingredient and therefore called acid equivalent.Each labeled rate is for cereal or/and broad acre row cropssince these herbicides are not labeled tobe sprayed on cotton or are labeled for cotton only as a pre-plant treatment. Basedon the labeled rates, the rates for the experiments werechosen and then adjusted for use in-crop on cotton and for the optimal growing conditions in the greenhouse.3Injury ratings were determined by visual inspection of each plant. Ratings were based on visual assessment of chlorosis, necrosis, malformation, stunting,and biomass reduction.0 percent = no visual adverse effects and 100 percent = completely dead.4Control plants were near isogenic conventional cotton control Coker 130. Reported average and range of 10 replicate plants.5Reported average and range of 10 replicate plants.

# Monsanto Company 12-CT-244U 300 of 620 

# C.3.2. In Vitro Endogenous Specificity Experiments

C.3.2.1 Materials

The DMO protein used in the endogenous specificity in vitro experiments was generated

in Escherichia coli with a histidine-tag at the N-term inus and has an identical amino acid

sequence to MON 88701 DMO with the exception of the lack of leuc ine at the second

position and the nine amino acids from CTP2 (Figure C-1). The compounds tested and

standards used in the in vitro experiments are listed in Table C-8.

Table C-8. Compounds Used in Specificity In Vitro Experiments

C.3.2.2. In Vitro Specificity Experiments Enzymatic Reaction Mixture Method

The reaction of E. coli -produced DMO with different compounds evaluated as potential

substrates was carried out using similar reaction co nditions described in the

characterization portion of this a ppendix (Appendix C.2.10.). The compounds

(Table C-8) were combined with E. coli -produced DMO at 0.2 a nd/or 0.012 mM. The

concentrations tested ensured adequate reac tion conditions in terms of the substrate for

the detection of product formation or disappearance of substrate.

C.3.2.3. In Vitro Experiments Liquid Chromat ography Separation Method

The reaction mixture was separated by U ltra Performance Liquid Chromatography

(UPLC) using an ACQUITY UPLC BEH C18 Column containing 1.7 μm Bridged Ethyl

Hybrid (BEH) particles and an ACQUITY BEH C18 VanGuard Pre-column. The Manufacturer/

Retailer Compound Common

Name Lot/Product

Number

Compounds Tested:

Aldrich 2-methoxybenzoic acid o-anisic acid A0230443

Chem Service 3,6-dichloro-2-methoxybenzoic acid dicamba 341-9143

Sigma 2,4-dichlorophenoxyacetic acid 2,4-D D7299-100G

Sigma 3-(4-hydroxy-3,5-

dimethoxyphenyl)prop-2-enoic acid sinapic acid D7927-1G

Fluka 3,5-dimethoxy-4-hydroxybenzoic acid syringic acid 86230

Fluka 4-hydroxy-3-methoxybenzoic acid vanillic acid 94770

Fluka 3-(4-hydroxy-3-methoxy-phenyl)prop2-enoic acid ferulic acid 46278

Compounds Used as Standards:

Monsanto 3,6-dichlorosalicylic acid DCSA GLP-0603-16959-T

Riedel-de Haen 2,4-dichlorophenol 2,4-DCP 35811

# Monsanto Company 12-CT-244U 301 of 620 

# column was heated to 40°C. The tested s ubstrates and potential oxidative by-products

were monitored by ACQUITY UPLC photodiode array (PDA) with wavelength range

from 200nm to 320nm with 1.2nm resolution. The chromatography was performed at

0.25ml/min and directed to th e mass spectrometer following the separation. Both mobile

phase A (water) and solvent B (acetonitrile) contained 0.1% v/v formic acid. Gradients

used were substrate-specific:

 The gradient for dicamba was run from 40 to 50% solvent B in 3min, 50 to 100%

solvent B in 0.1 min and then kept at 100% solvent B for 1min before returning to

40% solvent B in 0.1 min.

 The gradient for 2,4-D was run from 40 to 45% solvent B in 6min, held at 45%

solvent B for 1min, 45 to 100% solvent B in 0.1 min, and then held at 100%

solvent B for 0.5 min before returning to 40% solvent B in 0.1 min.

 The gradient for ferulic acid, o-anisic aci d, sinapic acid, syringic acid, and vanillic

acid were run from 0 to 100% solvent B in 4 min and then held at 100% solvent B

for 1 min before returning to 0% solvent B in 0.1 min.

Five microliters injection of each sample was used for UPLC analysis where the

disappearance of the potential s ubstrate was monitored, and a 50 μ l injection was used for

UPLC analysis where formation of potent ial oxidative by-products was monitored.

C.3.2.4. In Vitro Experiments Mass Spectrometry Detection Method

Elution from the UPLC column (C.3.2.3) flowed directly to a Waters Micro Q-TOF mass

spectrometer. The parameters used for th e mass determination were: negative mode,

capillary voltage of 2800 V, sample cone vol tage of 26 V for all analytes with the

exception of 2,4-D and 2,4-DCP, which was 10 V. The extraction cone was 1.5 V. The

source temperature was 150 °C and the de solvation temperature was 390 °C. The

desolvation gas flow was 500 L/hour. Scan time was 0.76 seconds and inter-scan delay

was 0.1 seconds. The m/z range used was specific to each substrate and product. The

m/z range for dicamba and DCSA was from 16 0 to 225 from 0 to 4 minutes. The m/z at

175, which is the fragment ion of dicamba, wa s used as a detection method for dicamba.

This fragment ion of dicamba gave better sensiti vity than the parent ion. The m/z at 205

or 207 was used to detect DCSA. The m/ z range for 2,4-D and 2,4-DCP was from 160-

164 or 160-225, dependent on the specific experiment from 0 to 6 minutes. The m/z

range for all other acids is fr om 120 to 230 within 4 minutes.

C.3.2.5. Results of In Vitro Experiments with Endogenous Cotton Compounds

The reaction of dicamba with E. coli -produced DMO has been well characterized

utilizing an in vitro enzymatic assay that monitors the formation of DCSA by LC-MS,

which allows for the detection of the product with high sensitivity. Both the substrate

and reaction products can be detected by LC-UV and LC-MS after separation by UPLC

(Figure C-6).

# Monsanto Company 12-CT-244U 302 of 620 

# Compounds structurally similar to dicamba and found in cotton, soybean and corn were

used as potential substrates to determine if these com pounds could be metabolized by

DMO (Table C-8). The compounds tested were syringic acid, o-anisic acid, vanillic acid,

ferulic acid, and sinapic acid. Mass spectrome try scans were taken from 120 m/z to 250

m/z to cover the range of all potential oxi dation products formed by DMO. Standard

reaction conditions of dicamba with a histidine tagged E. coli -produced DMO were used

as a positive control. LC-MS data demonstrated that there are no additional peaks

formed when reactions of each com pound incubated with histidine tagged E. coli

produced DMO and without histidine tagged E. coli -produced DMO are compared

(Figure C-7) (dicamba m/z 205, 2, 4-D m/z 163, ferulic acid m/z 175, o-anisic acid m/z

137, sinapic acid m/z 209, syringic acid m/z 183, and vanillic acid m/z 153). There were

no peaks observed at the resp ective masses for the predicte d reaction products of each

compound incubated with histidine tagged E. coli -produced DMO, indicating these

compounds are not catabolized by DMO.

To assess whether MON 88701 DMO protein has the same specificity as the histidine

tagged DMO used in the in vitro experiments, the E. coli -produced MON 88701 DMO

protein ( i.e. , lacking a histidine tag), shown to be equivalent to th e plant produced

MON 88701 DMO protein (Secti on V.B), was incubated with o -anisic acid, the

endogenous compound that has the greatest st ructural similarity to dicamba. Again

dicamba was used as a positive control to demonstrate the assay system was functional

(Figure C-8). This analysis demonstrated that o -anisic acid was not metabolized by the

E. coli -produced MON 88701 DMO protein ( i.e. , lacking a histidin e tag), but dicamba

was (Figures C-8 and C-9). These results indicate that DMO, including the MON 88701

DMO protein, is specific for dicamba as a substrate.

# Monsanto Company 12-CT-244U 303 of 620 

# Figure C-6. UPLC Separation of Dica mba (DCB) and DCSA in Five Substrate

Analysis

Dicamba and DCSA were separated by UPLC and detected by UV absorbance using a Photo

Diode Array (PDA) and mass spectrometry (MS).Monsanto Company 12-CT-244U 304 of 620Figure C-7.E. coli-produced DMO Conversion of Endogenous SubstratesEndogenous substrates, as well as dicamba and 2,4-D, were incubated withE. coli-produced DMO and the formation of products and disappearance of substratewas monitored by LC-MS (top two chromatograms) and LC-UV (bottom two chromatograms) for a positive control (dicamba (a)), (2,4-D (b)) and eachendogenous compound:, sinapic acid(c), ferulicacid (d), anisic acid (e), syringicacid (f), and vanillic acid (g). For eachexperiment the reaction mixture wasmade with (+E. coli-produced DMO, upper) and without (-E. coli-produced DMO, lower). The red line indicatesthe migration of the substrates (and DCSA inthe case of dicamba) in each chromatogram.8,107,108 G4 Vanillic2.50 2.75 3.00 3.25 3.50 3.75

%01002.50 2.75 3.00 3.25 3.50 3.75

%0100Dicamba-02-12-09-11261: TOF MS ES1533.14e33.733.572.772.483.443.283.172.96Dicamba-02-12-09-11301: TOF MS ES1533.66e32.483.783.613.492.772.99Time2.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+22.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+2Dicamba-02-12-09-11263: Diode ArrayRange: 2.491e+22.68Dicamba-02-12-09-11303: Diode ArrayRange: 2.586e+22.682.38+DMO+DMO-DMO-DMO8,107,108 G5 Syringic2.50 2.75 3.00 3.25 3.50 3.75

%01002.50 2.75 3.00 3.25 3.50 3.75

%0100Dicamba-02-12-09-11751: TOF MS ES1831.24e32.762.512.683.033.47Dicamba-02-12-09-11791: TOF MS ES1831.05e32.772.512.302.683.013.52Time2.50 2.75 3.00 3.253.50 3.75

AU0.02.0e+22.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+2Dicamba-02-12-09-11753: Diode ArrayRange: 2.417e+22.672.78Dicamba-02-12-09-11793: Diode ArrayRange: 2.419e+22.672.422.78+DMO+DMO-DMO-DMO8,107,108 G8 Anistic2.50 2.75 3.00 3.25 3.50 3.75

%01002.50 2.75 3.00 3.25 3.50 3.75

%0100Dicamba-02-12-09-11481: TOF MS ES1371.54e33.453.192.783.333.71Dicamba-02-12-09-11521: TOF MS ES1372.12e33.453.192.653.32Time2.50 2.75 3.00 3.25 3.50 3.75

AU0.01.0e+22.0e+22.50 2.75 3.00 3.25 3.50 3.75

AU0.01.0e+2Dicamba-02-12-09-11483: Diode ArrayRange: 1.661e+23.092.442.94Dicamba-02-12-09-11523: Diode ArrayRange: 2.097e+23.092.44+DMO+DMO-DMO-DMO8,107,108 G7 Ferrulic2.50 2.75 3.00 3.25 3.50 3.75

%01002.50 2.75 3.00 3.25 3.50 3.75

%0100Dicamba-02-12-09-11031: TOF MS ES1791.89e32.703.022.86Dicamba-02-12-09-11071: TOF MS ES1792.20e32.703.00Di b 02 12 09 11033 Di d ATime2.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+22.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+2Dicamba-02-12-09-11033: Diode ArrayRange: 2.866e+2.912.99Dicamba-02-12-09-11073: Diode ArrayRange: 2.978e+2.912.392.99+DMO+DMO-DMO-DMO8,107,108 B2 DCB2.00 2.50 3.00

%01002.00 2.50 3.00

%0100D icam ba-02-12-09-10231: TO F MS ES2054.56e32.54D icam ba-02-12-09-10271: TO F MS ES2059 92.642.042.382.812.873.383.09Tim e2.00 2.50 3.00

AU0.01.0e+12.00 2.50 3.00

AU0.01.0e+12.0e+1D icam ba-02-12-09-10233: D iode ArrayR ange: 2.288e+12.662.44D icam ba-02-12-09-10273: D iode ArrayR ange: 1.837e+12.65+DMO-DMO+DMO-DMO3,6‐DCSA3,5‐DCSADCBT im e3.00 4.00 5.00

AU0.05.0e+ 13.00 4.00 5.00

AU0.05.0e+ 1Range:9.014e +1x1 63.7 33.384.33D ica m b a-0 2 -1 2 -09 -10 7 03 : D io d e A rrayR ange: 8.997e+ 1x163.733.384.338,107,108 C 3 2,4-D3.00 4.00 5.00

%01003.00 4.00 5.00

%0100D ica m b a-0 2 -1 2 -09 -10 7 01: T O F M S E S -1639 43.833.553.494.433.965.264.51D ica m b a-0 2 -1 2 -09 -10 7 41: T O F M S E S -1638 93.863.073.303.904.435.235.12+DMO+DMO-DMO-DMO2,4‐DCP8,107,108 H6 Sinapic2.00 2.50 3.00 3.50

%01002.00 2.50 3.00 3.50

%0100Dicamba-02-12-09-11971: TOF MS ES209472.382.192.131.932.492.713.173.123.54Dicamba-02-12-09-12021: TOF MS ES209432.522.362.292.261.941.832.783.253.063.45Time2.00 2.50 3.00 3.50

AU0.02.0e+22.00 2.50 3.00 3.50

AU0.02.0e+2Dicamba-02-12-09-11973: Diode ArrayRange: 2.36e+22.151.692.822.32Dicamba-02-12-09-12023: Diode ArrayRange: 2.501e+22.151.782.822.332.66 3.103.20+DMO+DMO-DMO-DMOea. Dicamba (DCB)b. 2,4-Dc. Sinapic Acidd. Ferulic Acide. Anisic Acidf. Syringic Acidg. Vanillic Acid

# Monsanto Company 12-CT-244U 305 of 620 

# Figure C-8. UPLC Separation of Dicamb a (DCB) and DCSA in Bridging Analysis

Dicamba and DCSA were separated by UPLC and detected by UV absorbance using a Photo

Diode Array (PDA) and mass spectrometry (MS).

# Monsanto Company 12-CT-244U 306 of 620 

# Figure C-9. E. coli -produced MON 88701 DMO Conversion of o- Anisic Acid

o- anisic acid was incubated with E. coli- produced MON 88701 DMO and the formation

of predicted oxidative produc t and the disappearance of o- anisic acid was monitored by

LC-UV (A chromatograms) and LC -MS (B and C chromatograms). o- Anisic acid was

included in a reaction mixture made with (+DMO, upper) and w ithout (-DMO, lower)

MON 88701 DMO. The dotted li ne indicates the solvent de lay between the UV and MS

detectors as they are connected in series.

# Monsanto Company 12-CT-244U 307 of 620 

# C.4. Characterization of PAT ( bar ) Protein in MON 88701

C.4.1. Materials

The MON 88701-produced PAT ( bar ) protein (lot 11295997) was purified from

cottonseed of MON 88701 (lot 1 1287350). The MON 88701-produced PAT ( bar )

protein was stored in a -80 oC freezer in a buffer soluti on containing 50 mM Tris-HCl,

pH 7.5, 0.16 M sodium chlori de and 20% glycerol.

The E. coli -produced PAT ( bar ) protein (lot 11270310) was used as the reference

substance. The PAT ( bar ) protein reference substance was generated from cell paste

produced by large-scale fermentation of E. coli containing the pMON106653 expression

plasmid. The coding sequence for bar contained on the expression plasmid

(pMON106653) was confirmed prior to and after fermentation. The E. coli -produced

PAT ( bar ) protein was previously characterized.

C.4.2. Description of Assay Control

Protein MW standards (Precision Plus Protei n Standards Dual color; Bio-Rad, Hercules,

CA) were used to calibrate some SDS-PAGE gels and verify protein transfer to

polyvinylidene difluoride (PVDF) and nitro cellulose membranes. Broad Range

SDS-PAGE molecular weight st andards (Bio-Rad, Hercules, CA) were used to generate a

standard curve for the apparent MW estim ation. Bovine serum albumin (BSA) and

 -aminobutyric acid (AAbA) were used as hydrolysis control and internal calibration

standard for amino acid analysis. A phe nylthiohydantoin (PTH) amino acid standard

mixture (Applied Biosystems, Foster City, CA) was used to calibrate the Applied

Biosystems 494 Procise Sequencing System for each analysis. A peptide mixture

(Sequazyme Peptide Mass Standards kit, App lied Biosystems, Foster City, CA) was used

to calibrate the MALDI-TOF mass spectromete r for tryptic mass analysis. Transferrin

(Sigma-Aldrich, St. Louis, MO) was used as positive control for glycosylation analysis.

C.4.3. PAT ( bar ) Protein Purification

The plant-produced PAT ( bar ) protein was purified from cottonseed of MON 88701.

The purification procedure was not performe d under a GLP plan; however, all procedures

were documented on worksheets and, where applicable, SOPs were followed. The plantproduced PAT ( bar ) was purified from an extract of ground cottonseed using a

combination of dye affinity chromatography and anionic exchange chromatography. The

purification procedure is briefly described below.

Approximately 1 kg of cottonseed of M ON 88701 was ground to fine powder using a

laboratory mill (model 3100, Perten Instrument s). The ground powder was suspended in

4 liters of hexane (EMD Chemicals Inc., Gibbstown, NJ ) and filtered 3 times in order to

defat the powder. After drying overnight, th e powder was ready for further processing.

All grinding and defatting steps were done in a fume hood at room temperature.

# Monsanto Company 12-CT-244U 308 of 620 

# A portion (200 g) of the defatted powder was extracted with 2 liters of 20 mM Tris-HCl,

pH 7.5, and the solids were re moved by centrifugation at 25,000  g for 20 min. The

decanted solution was treated with 15 ml of 1 M CaCl2 solution to precipitate some

proteins and centrifuged at 25,000  g for 20 min to remove th e precipitated proteins.

The soluble portion (~1450 ml), containing the PAT ( bar ) protein, was batch absorbed

onto 20 ml of reactive brown 10 agarose (Sig ma-Aldrich, St. Louis, MO) equilibrated

with 200 ml of 20 mM Tris-HCl, pH 7.5. Th e reactive brown 10 agarose was centrifuged

at 1000  g for 2 min and the resin, after decanting the supernatant, was transferred to a

column. To remove unbound proteins, reactive brown 10 agarose was washed with 80 ml

of 20 mM Tris-HCl buffer, pH 7.5, follo wed by 120 ml of 20 mM Tris-HCl buffer,

pH 7.5, 1.5 M NaCl. Finally, the column was rinsed with 120 ml of 20 mM Tris-HCl

buffer, pH 7.5. The PAT ( bar ) protein was then eluted, wi th 80 ml of 1 mM acetyl CoA

in 20 mM Tris-HCl, pH 7.5. The eluted PAT ( bar ) protein was loaded onto a 1 ml Q

Sepharose Fast Flow (GE Healthcare) colu mn, equilibrated with 10 ml of 20 mM

Tris-HCl, pH 7.5, using an automated chromat ography system (AKTA, GE Healthcare).

The Q Sepharose Fast Flow column was wash ed with 20 ml of 20 mM Tris-HCl, pH 7.5,

0.1 M NaCl and consecutive step wise el ution using 0.2 M and 0.5 M NaCl in 20 mM

Tris-HCl, pH 7.5 to a total volume of 23 ml was conducted. Fractions containing

PAT ( bar ) protein were pooled (8 ml) and concen trated to a volume of 1170 μl using a

centrifugal filter (Ultracel 10K; Millipore, Billerica, MA; Molecular Weight Cutoff

(MWCO) of 10 kDa). Buffer was added to th e concentrated sample to bring the final

volume to 2 ml and the final buffer composition to 50 mM Tris-HCl, pH 7.5, 0.16 M

NaCl, and 20% (v/v) glycerol. This PAT ( bar ) protein purified from the cottonseed of

MON 88701 was aliquoted and stored in a -80 °C freezer.

# Monsanto Company 12-CT-244U 309 of 620 

# C.4.4. Summary of PAT ( bar ) Protein Identity and Equivalence

Table C-9. Summary of MON 88701-produced PAT ( bar ) Protein Identity and

Equivalence

Analytical Test Assessment Section

Cross

Reference Analytical Test Outcome

1. N-terminal sequence analysis of

the MON 88701-produced

PAT ( bar ) protein to assess

identity VI.C.3.1.  The identity was confirmed by

N-terminal sequence analysis

2. MALDI-TOF MS 1 

# analysis of

peptides derived from tryptic

digested MON 88701-produced

PAT ( bar ) protein to assess

identity VI.C.3.2.  MALDI-TOF MS 1 

# analysis yielded

peptide masses consistent with the

expected peptide masses from the

theoretical trypsin digest of the

MON 88701 PAT ( bar ) sequence

3. Western blot analysis using antiPAT ( bar ) polyclonal

antibodies to assess identity and

immunoreactive equivalence

between MON 88701- and the

E. coli -produced PAT ( bar )

proteins VI.C.3.3.  MON 88701-produced PAT ( bar )

protein identity was confirmed using a

western blot probed with antibodies

specific for PAT protein

 Immunoreactive properties of the

MON 88701- and the E. coli -produced

PAT ( bar ) proteins were shown to be

equivalent

4. SDS-PAGE 2 

# to assess

equivalence of the apparent

molecular weight between

MON 88701- and the

E. coli -produced PAT ( bar )

proteins VI.C.3.4.  Electrophoretic mobility and apparent

molecular weight of the MON 88701-

and the E. coli -produced PAT ( bar )

proteins were shown to be equivalent

5. Glycosylation analysis of the

PAT ( bar ) protein to assess

equivalence between the

MON 88701- and the

E. coli -produced PAT ( bar )

proteins VI.C.3.5.  Glycosylation status of MON 88701-

and the E. coli -produced PAT ( bar )

proteins were shown to be equivalent

6. PAT ( bar ) enzymatic activity

analysis to assess functional

equivalence between

MON 88701- and the

E. coli -produced PAT ( bar )

proteins VI.C.3.6.  Functional activity of the MON 88701-

and the E. coli -produced PAT ( bar )

proteins were shown to be equivalent

1 

# MALDI-TOF MS = Matrix-assisted laser desorption/ionization time-of-flight mass

spectrometry

2 

# SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis

# Monsanto Company 12-CT-244U 310 of 620 

# C.4.5. N-Terminal Sequencing

C.4.5.1. Methods

N-terminal sequencing, carried out by automa ted Edman degradation chemistry, was used

to confirm the identity of the MON 88701-produced PAT ( bar ).

One aliquot of MON 88701-produced PAT ( bar ) was used for N-terminal sequence

analysis. The analysis was performed for 15 cycles. using automated Edman degradation

chemistry (Hunkapiller et al., 1983). An A pplied Biosystems 494 Procise Sequencing

System equipped with 140C Microgradient system a Perkin Elmer Series 200 UV/VIS

Absorbance Detector with Procise  

# Control Software (version 2.1) was used for amino

acid detection after each cycle. Chromatographic data were collected using SequencePro

(version 2.1) software. A control protein,  -lactoglobulin, (Applied Biosystems, Foster

City, CA) was analyzed before and afte r the sequence analysis of the MON 88701-

produced PAT ( bar ) protein to verify that the seque ncer met performance criteria for

repetitive yield and sequence identity. Identity was established if ≥ 8 amino acids,

consistent with the predic ted sequence of the N-termi nus of the MON 88701-produced

PAT ( bar ), were observed during analysis.

C.4.5.2. Results of the N-t erminal Sequence Analysis

N-terminal sequencing of the first 15 amino acids was performed on

MON 88701-produced PAT ( bar ). The expected sequence for the PAT ( bar ) protein

deduced from the bar gene present in MON 88701 was observed. The data obtained

correspond to the deduced PAT ( bar ) protein beginning at amino acid positions 2 and 3

(Table C-10, Experimental Sequence 1 and 2, respectively). The N-terminal methionine

residue in the PAT ( bar ) protein was not observed. This result is expected as removal of

the N-terminal methionine, ca talyzed by methionine aminope ptidase, is common in many

organisms and has no effect on protein structure or activity (Arfin and Bradshaw, 1988;

Bradshaw et al., 1998; Polevoda and Sherma n, 2000). Hence, the sequence information

confirms the identity of the PAT ( bar ) protein isolated from the cottonseed of

MON 88701.

# Monsanto Company 12-CT-244U 311 of 620 

# Amino acid

residue # from

the N-terminus → 

# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Expected

Sequence → 

# M S P E R R P A D I R R A T E A

│ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Experimental

Sequence 1 →



# - S P E R R P A D I R R A T E A

│ │ │ │ │ │ │ │ │ │ │ │ │

Experimental

Sequence 2 →



# - - P E R X X A D I X X X T E

-



# Table C-10. N-Terminal Sequence of the MON 88701-produced PAT ( bar ) Protein

The expected amino acid sequence of the N-terminus of PAT ( bar ) protein was deduced

from the bar coding region present in MON 88701. The experimental sequences

obtained from the MON 88701-produced PAT ( bar ) protein were compared to the

expected sequence. The sing le letter IUPAC-IU B amino acid code is M, methionine;

S, serine; P, proline; E, glutamic acid; R, arginine; A, alanine; D, aspatic acid;

I, isoleucine; and T, threonine. X indicat es that the residue was not identifiable;

(-) indicates the residue was not observed.

# Monsanto Company 12-CT-244U 312 of 620 

# C.4.6. MALDI-TOF Tryptic Mass Map Analysis

C.4.6.1. Methods

MALDI-TOF tryptic mass fingerprint analysis was used to confirm the identity of the

MON 88701-produced PAT ( bar ) protein. MON 88701-produced PAT ( bar ) protein was

subjected to SDS-PAGE and the gel was staine d using Brilliant Blue G Colloidal stain.

Each ~25 kDa band was excised and transferred to a microcentrifuge tu be. The gel slices

were destained with 40% (v/v) methanol/ 10 % (v/v) acetic acid and washed in 100 mM

ammonium bicarbonate and then, to reduce the protein in each, gel slices were incubated

in 100 μl of 10 mM DTT at ~37 °C for 1 h. The protein was then al kylated in the dark

for 20 min with 100 μl of 20 mM iodoacetic acid and washed three times for 15-20 min

each with 200 μl of 25 mM ammonium bicar bonate. Gel slices were dried with a

Speed-Vac ® 

# concentrator (Thermo Fisher Scient ific, Waltham, MA) and then rehydrated

with 20 μl of trypsin solution (20 μg/ml) . After 1.25 h, excess liquid was removed and

the gel was incubated overnight at ~37.5 °C in 40 μl of 10% acetonitrile in 25 mM

ammonium bicarbonate. Gel slices were soni cated for 5 min to further elute proteolytic

fragments. The resulting extracts were transferred to new microcentrifuge tubes labeled

Extract 1 and dried using Speed-V ac concentrator. The gel slic es were re-extracted twice

with 30 μl of a 60% acetonitrile, 0.1% trifluoroacetic acid, 0.1% β -octylglucopyranoside

solution and sonicated for 5 min. Both extr acts were pooled into a new tube labeled

Extract 2 and dried with a Speed-Vac conc entrator. A solution (20 μl) of 0.1%

trifluoroacetic acid (TFA) was added to all Extract 1 and 2 tubes and the samples were

dried to completion via vacuum centrifugation. To solubilize the extracts, 5 μl of 50%

acetonitrile, 0.1% trifluoroacetic acid was added to Extract 1 tube and 10 μl of 50%

acetonitrile, 0.1% trifluoroacetic acid was added to Extract 2 tube and all were sonicated

for 5 min. Each extract was spotted to three we lls on an analysis plate. For each extract,

0.75 μl of 2, 5 dihydroxybenzoic acid (DHB), α -cyano-4-hydroxycinnamic acid

( α -Cyano), or 3, 5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (Thermo Fisher

Scientific Inc.) was added to one of the spots. The samples in DHB matrix were analyzed

in the 300 to 5000 Da range. Samples in α -Cyano and sinapinic acid were analyzed in

the 500 to 5000 Da and 500 to 7500 Da range, re spectively. The anal ysis was performed

using a Voyager TM 

# DE Pro Biospectrometry TM 

# workstation (Applied Biosystems) using

Voyager Instrument Control Panel software (version 5.10.2) and Data Explorer data

analysis software (version 4.0.0.0). Protona ted peptide masses were monoisotopically

resolved in reflector mode (Aebersold, 1993; Billeci and St ults, 1993). CalMix 2 was

used as the external calibrant (Sequazy me Peptide Mass Standards kit, Applied

Biosystems, Foster City, CA) for the anal ysis. GPMAW32 software (Lighthouse Data,

Odense M, Denmark) was used to generate a theoretical trypsin digest of the PAT ( bar )

protein sequence. Masses within 1 Da of a monosiotopic mass were matched against the

theoretical digest of the PAT ( bar ) sequence. All matching masses were tallied and a

coverage map was generated for the mass fi ngerprint. The tryp tic mass fingerprint

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 (Biron et al., 2006, Krause et al., 1999).

# Monsanto Company 12-CT-244U 313 of 620 

# C.4.6.2. Results of MALDI-TOF Tryptic Mass Map Analysis

The identity of the MON 88701-produced PAT ( bar ) protein was also confirmed by

MALDI-TOF MS analysis of peptide fragment s produced from tryptic digestion of the

MON 88701-produced PAT ( bar ) protein. The ability to identify a protein using this

method is dependent upon matching a suffi cient number of observed tryptic peptide

fragment masses with predicted tryptic peptid e fragment masses. In general, protein

identification made by peptide mapping is considered to be reliable if  40% of the

protein sequence was identified by matc hing experimental masses observed for the

tryptic peptide fragments to the expected masses for the fragments (Biron et al., 2006,

Krause et al., 1999).

There were 10 unique peptides identified that corresponded to the masses expected to be

produced by tryptic digestion of the PAT ( bar ) protein (Table C-11) . The identified

masses were used to assemble a covera ge map of the entire MON 88701-produced

PAT ( bar ) protein (Figure C-10). The experimental ly determined mass coverage of the

MON 88701-produced PAT ( bar ) protein was 84.7% (155 out of 183 amino acids). This

analysis serves as additional identity confirmation for the MON 88701-produced

PAT ( bar ) protein.Monsanto Company 12-CT-244U 314 of 620Table C-11. Summary of the Tryptic Masses1Identified for the MON 88701-produced PAT (bar) Protein Using MALDI-TOFMS-cyanoDHB Sinapinic acid ExpectedMassDiff.2Fragment3SequenceExtract 1 Extract 2 Extract 1 Extract 2 Extract 1 Extract 2879.65879.460.19113-120SLEAQGFK1144.651144.751144.841144.560.09136-145MHEALGYAPR1403.931404.031404.121404.181403.790.14100-112TGLGSTLYTHLLK1523.021523.131523.141523.191522.931522.860.16121-135SVVAVIGLPNDPSVR1843.071843.181843.271842.981843.191842.850.2238-52TEPQEPQEWTDDLVR1859.061859.221859.221858.981859.181858.860.2081-96NAYDWTAESTVYVSPR2391.452391.642391.200.2557-78YPWLVAEVDGEVAGIAYAGPWK2676.672676.642676.882676.350.3255-78ERYPWLVAEVDGEVAGIAYAGPWK2840.622840.320.3013-37ATEADMPAVCTIVNHYIETSTVNFR3353.143353.363353.173353.483352.730.41155-183HGNWHDVGFWQLDFSLPVPPRPVLPVTEI1Only experimental masses that matched expected masses are listed in the table.2The difference between the expected mass and the first column mass.Other masses shown within a row are also within 1 Da of the expected mass.3Position refers to amino acid residues within the predicted PAT (bar) sequence as depicted in Figure C-10.DHB = 5-dihydroxybenzoic acid matrix,α-cyano =α-cyano-4-hydroxycinnamic acid matrix, Sinapinic acid = 3, 5-dimethoxy-4-hydroxycinnamic acid matrix

# Monsanto Company 12-CT-244U 315 of 620 

# Figure C-10. MALDI-TOF MS Coverage Map of the MON 88701-produced

PAT ( bar ) Protein

The amino acid sequence of the PAT ( bar ) protein was deduced from the bar gene

present in MON 88701. Boxed regions correspon d to regions covered by tryptic peptides

that were identified from the MON 88701-produced PAT ( bar ) protein sample using

MALDI-TOF MS. In total, 84.7% (155 out of 183 amino acids) of the expected protein

sequence was covered by the identified peptides.

# Monsanto Company 12-CT-244U 316 of 620 

# C.4.7. Western Blot Analysis-Immunoreactivity

C.4.7.1. Methods

Western blot analysis was performed to confirm the identity of the PAT ( bar ) protein

purified from cottonseed of MON 88701 and to compare the immunoreactivity of the

MON 88701- and E. coli -produced proteins.

The MON 88701- and E. coli -produced PAT ( bar ) proteins were analyzed concurrently

on the same gel using three loadings of 2, 4, and 6 ng. Loadings of the three

concentrations were made in duplicate on the ge l. Aliquots of each protein were diluted

in water and 5X Laemmli buffer (LB) containing 312 mM Tris-HCl, 25% (v/v)

2-mercaptoethanol, 10% (w/v) SDS, 0.025% (w/v) bromophenol blue, 50% (v/v)

glycerol, pH 6.8, heated at ~96 °C for 4 min, and applied to a 15 well pre-cast

Tris-glycine 4-20% polyacrylamide gradient gel (Invitrogen, Carlsbad, CA). Pre-stained

moelcular weight markers (Precision Plus Protein Standards Dual color; Bio-Rad,

Hercules, CA) were loaded in parallel to veri fy electrotransfer of the proteins to the

membrane and to estimate the size of the immunoreactive bands observed.

Electrophoresis was performed at a constant voltage of 150 V for 85 min. Electrotransfer

to a 0.45 μm PVDF membrane (Invitrogen, Carl sbad, CA) was performed for 60 min at a

constant current of 200 mA. After electrot ransfer, the membrane was blocked for 1 h

with 10% (w/v) non-fat dried milk (NFDM) in 1X phosphate buffered saline containing

0.05% (v/v) Tween-20 (PBST). The membrane was then probed with a 1:2000 dilution

of goat anti-PAT ( bar ) antibody (lot G863803) in 5% NFDM in PBST for 1 h at room

temperature. Excess antibody was removed using three 10 min washes with PBST.

Finally, the membrane was probed with horse radish peroxidase (HRP)-conjugated horse

anti-goat IgG (Thermo, Rockford, IL) at a d ilution of 1:10,000 in 5% NFDM in PBST for

1 h at room temperature. Excess HRP-conj ugate was removed using three 10 min washes

with PBST. All washes were performed at room temperature. Immunoreactive bands

were visualized using the ECL detection syst em (GE, Healthcare, Piscataway, NJ) with

exposure to Amersham Hyperfilm ECL (GE, Healthcare, Piscataway, NJ). The film was

developed using a Konica SRX-101A automate d film processor (Konica Minolta Medical

& Graphic, Inc., Tokyo, Japan).

Quantification of the bands on the blot was performed using a Bio-Rad GS-800

densitometer with the supplied Quantity On e software (version 4.4.0, Hercules, CA)

using the lane selection and contour tool. The signal intensities of the immunoreactive

bands observed for the MON 88701- and E. coli -produced proteins migrating at the

expected position on the blot film were quant ified as “contour quantity” values. The raw

data was exported to a Microsoft Excel (2 007) file. The imm unoreactivity of the

MON 88701- and E. coli -produced PAT ( bar ) proteins were reporte d as the mean signal

intensity at each amount of protein analy zed. The immunoreactivity of the MON 88701-

and E. coli -produced PAT ( bar ) proteins were considered e quivalent if the overall mean

of the immunoreactive signal of the MON 88701 -produced protein was within ± 35% of

the overall mean of imm unoreactive signal of the E. coli -produced PAT ( bar ) protein.

# Monsanto Company 12-CT-244U 317 of 620 

# C.4.7.2. Results of PAT ( bar ) Protein Immunoreactivity Equivalence

Western blot analysis was conducted using goat anti- PAT ( bar ) polyclonal antibodies to

1) assess the identity of the PAT ( bar ) protein isolated from the cottonseed of

MON 88701 and 2) to determine the relati ve immunoreactivity of the MON 88701- and

the E. coli -produced PAT ( bar ) proteins. The results demonstrated that the

anti-PAT ( bar ) antibodies recognized the MON 88701-produced PAT ( bar ) protein that

migrated to an identical pos ition on the blot as the E. coli -produced PAT ( bar ) protein

(Figure C-11). Furthermore, the immunor eactive signal increased with increasing

amounts of PAT ( bar ) protein loaded.

Densitometric analysis was conducte d to compare the immunoreactivity of

MON 88701- and the E. coli -produced PAT ( bar ) proteins. The mean signal intensity

(OD  mm 2 

# ) from the MON 88701-produced PAT ( bar ) bands and from the

E. coli -produced PAT ( bar ) bands at each amount of protein analyzed was calculated and

then overall mean signal intensity was cal culated (Table C-12). The immunoreactivity

was considered equivalent if the ove rall mean signal intensity of all

MON 88701-produced PAT ( bar ) protein bands was within ±35% of the overall mean

signal intensity of all E. coli -produced PAT ( bar ) protein bands.

The overall mean signal intensity of the E. coli -produced PAT ( bar ) bands was 4.669

OD × mm 2 

# , and the overall mean signal inte nsity of the MON 88701-produced PAT ( bar )

bands was 4.167 OD × mm 2 

# . Because overall mean signal intensity of the

MON 88701-produced PAT ( bar ) protein bands was be tween 3.035 and 6.303 OD × mm 2



# (between -35% and +35% of the E. coli -produced PAT ( bar ) bands), the

MON 88701-produced and E. coli -produced PAT ( bar ) proteins were determined to have

equivalent immunoreactivity.

# Monsanto Company 12-CT-244U 318 of 620 

# Figure C-11. Western Blot An alysis of the MON 88701- and E. coli -produced

PAT ( bar ) Proteins

Aliquots of the MON 88701-produced PAT ( bar ) protein and the E. coli -produced

PAT ( bar ) protein were subjected to SDS-PAGE and electrotransferred to a PVDF

membrane. The membrane was incubated with anti-PAT ( bar ) antibodies and

immunoreactive bands were visualized using an ECL system (GE Healthcare,

Piscataway, NJ). Approximate molecular we ights (kDa) are shown on the left. Lanes

loaded with molecular weight markers were cropped, and lanes were renumbered relative

to the original gel loading. The 1 min e xposure is shown. Lane designations are as

follows:

Lane Sample Amount (ng)

1 E. coli -produced PAT ( bar ) protein 2

2 E. coli -produced PAT ( bar ) protein 2

3 E. coli -produced PAT ( bar ) protein 4

4 E. coli -produced PAT ( bar ) protein 4

5 E. coli -produced PAT ( bar ) protein 6

6 E. coli -produced PAT ( bar ) protein 6

7 Empty -

8 MON 88701-produced PAT ( bar ) protein 2

9 MON 88701-produced PAT ( bar ) protein 2

10 MON 88701-produced PAT ( bar ) protein 4

11 MON 88701-produced PAT ( bar ) protein 4

12 MON 88701-produced PAT ( bar ) protein 6

13 MON 88701-produced PAT ( bar ) protein 6

# Monsanto Company 12-CT-244U 319 of 620 

# Table C-12. Comparison of Immunorea ctive Signals Between MON 88701- and

E. coli -produced PAT ( bar ) Proteins

Mean Signal intensity from

MON 88701-produced

PAT ( bar )

(OD × mm 2 

# ) Mean Signal intensity from

E. coli -produced

PAT ( bar )

(OD × mm 2 

# ) Preset Acceptance limits

for MON 88701-produced

PAT ( bar ) 1



# (OD × mm 2 

# )

4.167 4.669 3.035 – 6.303

1 

# The acceptance limits for th e MON 88701-pr oduced PAT ( bar ) are based on the interval between +35%

(4.669 × 1.35) and -35% (4.669 × 0.65) of the overall mean of the E. coli -produced PAT ( bar ) signal

intensity across all loads.

# Monsanto Company 12-CT-244U 320 of 620 

# C.4.8. Molecular Weight and Purity Estimation using SDS-PAGE

C.4.8.1. Methods

MON 88701- and E. coli -produced PAT ( bar ) proteins were mixed with 5X LB and

diluted with water to a final total protein c oncentration of 0.136 μg/μl. Molecular Weight

Standards, Bio-Rad broad rang e (Hercules, CA) were diluted to a final total protein

concentration of 0.9  g/  l. The MON 88701-produced PAT ( bar ) was analyzed in

duplicate at 1, 2, and 3 μg protein per lane. The E. coli -produced PAT ( bar ) reference

standard was analyzed at 1 μg total protein in a single lane. The samples were loaded

onto a 10-well pre-cast Tris gl ycine 4-20% polyacrylamide gr adient mini-gel (Invitrogen,

Carlsbad, CA) and electrophoresis was perfor med at a constant voltage of 150 V for

95 min. Proteins were fixed by placing the gel in a solution of 40% (v/v) methanol and

7% (v/v) acetic acid for 30 min, stained for 16.25 h with Brilliant Blue G-Colloidal stain

(Sigma-Aldrich, St. Louis, MO). Gels were destained once for 30 to 45 sec with a

solution containing 10% (v/v) acetic acid and 25 % (v/v) methanol, and four times for 2 h

each (for a total of 8 h) with 25% (v/v) methanol. An alysis of the gel was performed

using a Bio-Rad GS-800 densitometer with th e supplied Quantity One software (version

4.4.0, Hercules, CA). The apparent MW of each observed band was estimated from a

standard curve generated by the Quantity On e software which was based on the MWs of

the markers and their migrati on distance on the gel. To dete rmine purity, all visible bands

within each lane were quantified using Quantity One software. Apparent MW and purity

were reported as an average of all si x lanes containing the MON 88701-produced

PAT ( bar ).

C.4.8.2. Results of PAT ( bar ) Protein Molecular Weight Equivalence

The molecular weight and purity of the PAT ( bar ) protein was determined to be 24.1 kDa

and 99%, respectively. To assess appare nt molecular weight (MW) and purity, the

MON 88701-produced PAT ( bar ) protein was subjected to SDS-PAGE. The gel was

stained with Brilliant Blue G Colloidal stain and analyzed by densitometry (Figure C-12).

E. coli -produced PAT ( bar ) protein was loaded in a single lane for reference (Figure C12, Lane 2). The MON 88701-produced PAT ( bar ) protein (Figure C-1 2, Lanes 3-8) had

an apparent MW of 24.1 kDa (Table C-13). The apparent MW of the E. coli -produced

PAT ( bar ) protein as reported on its Certificate of Analysis was 25.0 kDa (Table C-8).

Because the apparent MW of MON 88701-produced PAT ( bar ) protein was within the

preset acceptance limits (Table C-13), the MON 88701-produced and E. coli -produced

PAT ( bar ) proteins were determined to have equivalent apparent MWs.

The purity of the MON 88701-produced PAT ( bar ) protein was calculated based on the

six loads on the gel (F igure C-12, Lanes 3-8). The averag e purity was determined to be

more than 99%.

# Monsanto Company 12-CT-244U 321 of 620 

# Table C-13. Molecular Weight Comparison Between the MON 88701- and

E. coli -produced PAT ( bar ) Proteins Based on SDS-PAGE

Apparent Molecular Weight

of MON 88701-Produced

PAT ( bar ) Protein 1



# (kDa) Apparent Molecular Weight

of E. coli -Produced

PAT ( bar ) Protein 2



# (kDa) Preset Acceptance Limits

for MON 88701-

produced PAT ( bar ) 3



# (kDa)

24.1 25.0 23.9-25.4

1 

# The reported value is the mean mol ecular weight across all six loads.

2 

# The molecular weight of the E

. coli -produced PAT ( bar ) protein as reported on its Certificate of Analysis.

3 

# See Section C.6.

# Monsanto Company 12-CT-244U 322 of 620 

# Figure C-12. Molecular Weight and Puri ty Analysis of the MON 88701-produced

PAT ( bar ) Protein

Aliquots of the MON 88701-produced and the E. coli -produced PAT ( bar ) proteins were

subjected to SDS-PAGE and then stained with Brilliant Blue G-Colloidal stain.

Approximate molecular weights are shown on the left and correspond to the markers

loaded in Lanes 1 and 9. Empty lane was pa rtially cropped. Lane designations are as

follows:

Lane Sample Amount (μg)

1 Broad Range Molecular Weight Markers 4.5

2 E. coli -produced PAT ( bar ) protein 1

3 MON 88701-produced PAT ( bar ) protein 1

4 MON 88701-produced PAT ( bar ) protein 1

5 MON 88701-produced PAT ( bar ) protein 2

6 MON 88701-produced PAT ( bar ) protein 2

7 MON 88701-produced PAT ( bar ) protein 3

8 MON 88701-produced PAT ( bar ) protein 3

9 Broad Range Molecular Weight markers 4.5

# Monsanto Company 12-CT-244U 323 of 620 

# C.4.9. Glycosylation Analysis

C.4.9.1. Methods

Glycosylation analysis was used to determine whether the MON 88701-produced

PAT ( bar ) was post-translationally modified with covalently bound carbohydrate

moieties. Aliquots of the MON 88701-produced PAT ( bar ) protein, the E. coli -produced

PAT ( bar ) (negative control) and the positive control, transferrin (Sigma-Aldrich,

St. Louis, MO), were each diluted with wate r and brought to 1X LB. These samples were

heated at ~102 °C for 4 mi n. The MON 88701-produced PAT ( bar ), the E. coli -produced

PAT ( bar ) and transferrin were lo aded at approximately 50 and 100 ng per lane on a

Tris-glycine 10 well 4-20% polyacrylamide grad ient mini-gel (Invitrogen, Carlsbad, CA).

Precision Plus Protein Dual color Standards (B io-Rad, Hercules, CA) were also loaded to

verify electrotransfer of the proteins to the membrane and as markers for molecular

weight. Electrophoresis was performed at a constant voltage of 150 V for 90 min.

Electrotransfer to a 0.45 μm PVDF membrane (Invitrogen, Carlsbad, CA) was performed

for 60 min at a constant current of 200 mA.

Carbohydrate detection was performed dir ectly on the PVDF me mbrane at room

temperature using the Amersham ECL glycopr otein Detection Module (GE, Healthcare,

Piscataway, NJ). With this module, carbohydrate moieties of pr oteins were ox idized with

sodium metaperiodate and then biotinylated with biotin-X-hydrazide. The biotinylated

proteins can be detected on the blot by addi tion of streptavidin conjugated to HRP for

luminol-based detection using ECL reagents (GE, Healthcare, Pis cataway, NJ) and with

subsequent exposure to Amersham Hyperfilm (G E, Healthcare). The film was developed

using a Konica SRX-101A automated film processor (Konica Minolta Medical &

Graphic, Inc., Tokyo, Japan).

An identical blot run in parall el to that used for the glycos ylation analysis was stained to

visualize the proteins present on the membrane. Proteins were stained for 30 sec to 2 min

using Coomassie Brilliant Blue R-250 stai ning solution (Bio-Rad, Hercules, CA) and

then destained with 1X Coomassie Brilliant Blue R-250 destaining solution (Bio-Rad) for

more than 5 min. After washing with water, the blot was dried and scanned using BioRad GS-800 densitometer with the supplied Quantity One software (version 4.4.0).

C.4.9.2. Results of Glycosylation Analysis

Some eukaryotic proteins are post-translationally modified by the addition of

carbohydrate moieties (Rademacher et al., 1988). To test whether PAT ( bar ) protein was

glycosylated when expressed in the cottonseed of MON 88701, the

MON 88701-produced PAT ( bar ) protein was analyzed us ing an ECL Glycoprotein

Detection Module (GE, H ealthcare, Piscataway, NJ). Transf errin, a glycosylated protein,

was used as a positive control in the assay. To assess equivalence of the MON 88701-

and E. coli -produced PAT ( bar ) proteins, the E. coli -produced PAT ( bar ) protein was

also analyzed. The positive control was clearly detected at the expected molecular

weight (~80 kDa) and the band intensity increased with increasing concentration

(Figure C-13, Panel A, Lanes 1- 2). In contrast, signals were not observed in the lanes

# Monsanto Company 12-CT-244U 324 of 620 

# containing the MON 88701- or E. coli -produced protein at the e xpected molecular weight

for the PAT ( bar ) protein (Figure C-13 Panel A, Lane s 7-8 and Lanes 4-5, respectively).

To assess whether the MON 88701- and E. coli -produced PAT ( bar ) proteins were

loaded appropriately for glycosylation anal ysis, a second membrane (with identical

loadings and transfer times) was stained w ith Coomassie Blue R-250 for protein detection

(Figure C-13 Panel B). Both the MON 88701- and E. coli -produced PAT ( bar ) proteins

were clearly detected (Figure C-13 Panel B, Lanes 7-8 and Lane s 4-5, respectively).

These data indicate that the glycosyl ation status of MON 88701-produced PAT ( bar )

protein and E. coli -produced PAT ( bar ) protein are equivalent and that neither is

glycosylated.

# Monsanto Company 12-CT-244U 325 of 620 

# Figure C-13. Glycosylation Analysis of the MON 88701-produced PAT ( bar )

Protein

Aliquots of the transfe rrin (positive control), E. coli -produced PAT ( bar ) protein and

MON 88701-produced PAT ( bar ) protein were subjec ted to SDS-PAGE and

electrotransferred to PVDF membranes. Panel A corresponds to detection of labeled

carbohydrate moieties, where present, using th e ECL-based system with exposure to

Hyperfilm. A 7 min exposure is shown. Pa nel B corresponds to Coomassie Blue R-250

staining of an equivalent bl ot to confirm the presence of proteins. The signal was

captured using a Bio-Rad GS-800 with Quantity One software (version 4.4.0).

Approximate molecular weights (kDa) co rrespond to the Precision Plus, dual color

markers (used to verify transfer and MW). Lanes loaded with molecular weight markers

were cropped, and lanes were renumbered rela tive to the original gel loading. Arrows

indicate the expected migration of PAT ( bar ) protein. Lane designa tions are as follows:

Lane Sample Amount (ng)

1 Transferrin (positive control) 50

2 Transferrin (positive control) 100

3 Empty

-

4 E. coli -produced PAT ( bar ) (negative control) 50

5 E. coli -produced PAT ( bar ) (negative control) 100

6 Empty

- 7 MON 88701-produced PAT ( bar ) 50

8 MON 88701-produced PAT ( bar ) 100

# Monsanto Company 12-CT-244U 326 of 620 

# C.4.10. Functional Activity Analysis

C.4.10.1. Methods

PAT ( bar ) catalyzes the reaction of phosphinothricin (PPT) with acetyl CoA to form

acetyl PPT and free CoA. To assess functional activity of PAT ( bar ), the amount of CoA

released during the reaction can be monito red using the reduction of 5,5’-dithio-bis

(2-nitrobenzoic acid) (DTNB) by CoA to form the colorimetric reagent

5-thio-nitrobenzoate (TNB) (Wehrmann et al., 1996).

Prior to functional activity analysis, both MON 88701- and E. coli -produced PAT ( bar )

proteins were diluted to a purity corrected concentration of 1 ng/μl with a 50 mM Tris,

pH 7.5, and 0.5 mM EDTA buffer. Assays for both proteins were c onducted using five

replicates. The reaction mixtures contai ning 2 mM acetyl CoA, 1 mM DTNB, 50 mM

Tris, pH 7.8, and 0.5 mM EDTA with or without 1 mM phosphinothricin were pre

incubated at ~30 °C for 10-60 min. The reactio ns were then initiated by the addition of

10 ng of PAT ( bar ) enzyme. The reaction rate was monitored in each well at 412 nm and

~30 °C using a plate reader in one minute intervals for 30 min. A response curve was

prepared using 3.9 μM to 250 μM β -mercaptoethanol in 1 mM DTNB, 50 mM Tris,

pH 7.8, and 0.5 mM EDTA. The response curve was generated only to verify assay

conditions and instrument performance. The in itial assay results are reported as the mean

velocity of the reaction of PAT ( bar ) (generated by the KC4 software, Power Wave Xi,

Bio Tek, Richmond, VA) and expressed as min -1 

# . The specific activities of the

MON 88701- and E. coli -produced PAT ( bar ) proteins were then calculated using the

molar absorptivity of product releas ed during the assay, TNB (13,600 M -1 

#  cm -1 

# or

13.6 μmol- 1 

#  ml). Specific activity is expresse d as μmol of TNB released per minute

per mg of PAT ( bar ) (μmol  min -1 

# × mg -1 

# ). Calculations of the specific activities were

performed using Microsoft Excel (2007).

C.4.10.2. Results of Functional Activity

The functional activitie s of the MON 88701- and E. coli -produced PAT ( bar ) proteins

were assessed using a colorimetric assay that measures PAT ( bar ) catalyzed release of

coenzyme A (CoA) from acetyl-CoA upon transfer of an acetyl-group to

phosphinothricin. In this assay, protei n-specific activity is expressed as

μmol × minute -1 

# × mg -1 

# of PAT enzyme.

The experimentally determined speci fic activities for the MON 88701- and

E. coli -produced PAT ( bar ) proteins are presented in Tabl e C-14. The specific activities

of MON 88701- and E. coli -produced PAT ( bar ) proteins were 36.4 and

46.2 μmol × minute -1 

# × mg -1 

# of PAT ( bar ), respectively. Because the specific activities

of the MON 88701-produced and E. coli -produced PAT ( bar ) proteins fall within the

preset acceptance criterion (T able C-14), the MON 88701- and E. coli -produced

PAT ( bar ) proteins were determined to have equivalent functional activity.

# Monsanto Company 12-CT-244U 327 of 620 

# Table C-14. PAT ( bar ) Functional Activity

MON 88701-produced

PAT ( bar ) 1



# (μmol × minute -1 

# × mg -1 

# ) E. coli -produced

PAT ( bar ) 1



# (μmol × minute -1 

# × mg -1 

# ) Preset Acceptance Limits

for

MON 88701-produced

PAT ( bar ) 2



# (μmol × minute -1 

# × mg -1 

# )

36.4 ± 1.3 46.2 ± 2.1 30.17 - 51.70

1 

# Value refers to mean and standard deviation calculated based on n = 5.

2 

# See Section C.6.



# C.5. Substrate Specificity of PAT ( bar ) Protein Produced in MON 88701

The PAT proteins, including PAT ( bar ) protein, were demonstrated to be highly specific

for glufosinate in the presence of acetyl-CoA. Si nce the specificity of PAT proteins has

been well established in literat ure, and due to the lack of any documented reports of nonspecific effects of PAT proteins since the in troduction of glufosinat e tolerant crops in

1995, in-house experiments were not conduct ed to further demonstrate substrate

specificity of PAT ( bar ) protein isolated from MON 88701.

C.6. Prediction Intervals as Acceptance criteria

Acceptance criteria (acceptan ce limits) based on prediction in tervals were used to assess

the equivalence of the MON 88701-produced and E. coli -produced proteins for apparent

MW and functional activity. A prediction interval is an estimate of an interval in which a

randomly selected future observation from a pop ulation will fall, with a certain degree of

confidence, given what has already b een observed (Hahn and Meeker, 1991a; b); i.e

., prediction intervals are generated based on the statistical analysis of the existing data.

Data obtained from multiple assays of E. coli -produced protein conducted under GLP

guidelines were used for this purpose.

To generate the 95% prediction interval (PI), the mean and standard deviation of the data

from several assays were calculated. The num ber of assays used to calculate the mean

and the number of future assays (one for equi valence studies) were used in the following

formula to generate the PI:



# X ഥ േ ݎሺ1 െ ߙ ; ݉, ݊ሻ ሺݏሻ

r (1-  ; m, n) 

# is estimated using the formula given below:

ݎ

ሺଵିఈ;௠,௡ሻ 

# ؆ ݐ ሺଵି.଴ହ/ሺଶ௠ሻ; ௡ିଵሻ 

# ඨ

1൅ 1

݊

# Monsanto Company 12-CT-244U 328 of 620 

# Where X ҧ is mean of the replicate assays; s is standard devi ation of the replicates;  is

the level of confidence; n is th e number of assays used to ge nerate the mean; and m is the

number of future assays (one for e quivalence studies). The t-value is the

100(1-.05/(2m)) th 

# percentile from Student’s t-distribu tion with n-1 degrees of freedom.

With 95% confidence, all m future values of the assay will fall within this interval (Hahn

and Meeker, 1991a; b). If the assay means do not appear to have been derived from a

normal distribution, but the logarithms of th e raw values do follow a normal distribution,

then prediction intervals may be applied to the logarithms of the raw values (Hahn and

Meeker, 1991a; b).

# Monsanto Company 12-CT-244U 329 of 620 

# References for Appendix C

Aebersold, R. 1993. Mass spectr ometry of proteins and peptides in biotechnology.

Current Opinion in Biotechnology 4:412-419.

Arfin, S.M. and R.A. Bradshaw. 1988. Cotransla tional processing and pr otein turnover in

eukaryotic cells. Biochemistry 27:7979-7984.

Behrens, M.R., N. Mutlu, S. Chakraborty, R. Dumitru, W.Z. Jiang, B.J. LaVallee, P.L.

Herman, T.E. Clemente and D.P. Weeks. 2007. Dicamba resistance: Enlarging and

preserving biotechnology-based weed management strategies. Science 316:1185-1188.

Billeci, T.M. and J.T. Stults. 1993. Tryptic mapping of recombinant proteins by matrixassisted laser desorption/ionization mass spectrometry. Analytical Chemistry 65:1709-

1716.

Biron, D. G.; Brun, C.; Lefevre, T.; Lebarbenchon, C.; Loxdale, H. D.; Chevenet, F.;

Brizard, J. P.; Thomas, F., 2006 The pitfalls of proteomics experiments without the

correct use of bioinformatics tools. Proteomics 6:5577

− 5596.

Boquet, D.J. and G.A. Breitenbeck. 2000. Nitrogen rate effect on partitioning of nitrogen

and dry matter by cotton. Crop Science 40:1685-1693.

Bradshaw, R.A., W.W. Bric key and K.W. Walker. 1998. N- terminal processing: The

methionine aminopeptidase and N a 

# -acetyl transferase families. Trends in Biochemical

Sciences 23:263-267.

Cojocariu, C., P. Escher, K.-H. Häberle, R. Matyssek, H. Rennenberg and J. Kreuszeiser.

2005. The effect of ozone on the emission of carbonyls from leaves of adult Fagus

sylvatica . Plant, Cell and Environment 28:603-611.

Cojocariu, C., J. Kreuzwieser and H. Re nnenberg. 2004. Correlation of short-chained

carbonyls emitted from Picea abies with physiological and e nvironmental parameters.

New Phytologist 162:717-727.

D'Ordine, R.L., T.J. Rydel, M.J. Storek, E.J. Sturman, F. Moshiri, R.K. Bartlett, G.R.

Brown, R.J. Eilers, C. Dart, Y. Qi, S. Flasinski and S.J. Franklin. 2009. Dicamba

monooxygenase: Structural insigh ts into a dynamic Rieske oxygenase that catalyzes an

exocyclic monooxygenation. Journal of Molecular Biology 392:481-497.

Ducamp, F., F.J. Arriaga, K.S. Balkcom, S.A. Prior, E. van Santen and C.C. Mitchell.

2012. Cover crop biomass harvest influences co tton nitrogen utiliza tion and productivity.

International Journal of Agronomy 2012:1-12.

Dumitru, R., W.Z. Jiang, D.P. Weeks and M.A. Wilson. 2009. Crystal structure of

dicamba monooxygenase: A Rieske nonheme ox ygenase that catalyzes oxidative

demethylation. Journal of Molecular Biology 392:498-510.

# Monsanto Company 12-CT-244U 330 of 620 

# FAO-WHO. 2011. Pesticide residues in food 2010: Joint FAO/WHO meeting on

pesticide residues. FAO Plant Production a nd Protection Paper 200. Food and Agriculture

Organization of the United Nations, Wo rld Health Organization, Rome, Italy.

Hahn, G.J. and W.Q. Meeker. 1991a. Methods fo r calculating statistical intervals for a

normal distribution. Pages 53-74 in Statistical Intervals: A Guide for Practitioners. John

Wiley and Sons, Inc., Hoboken, New Jersey.

Hahn, G.J. and W.Q. Meeker. 1991b. Overview of different types of st atistical intervals.

Pages 27-40 in Statistical Intervals: A Guid e for Practitioners. John Wiley and Sons, Inc.,

Hoboken, New Jersey.

Herman, P.L., M. Behrens, S. Chakraborty, B. M. Chrastil, J. Barycki and D.P. Weeks.

2005. A three-component dicamba  -demethylase from Pseudomonas maltophilia , strain

DI-6: Gene isolation, characterization, and heterologous expression. Journal of Biological

Chemistry 280:24759-24767.

Hunkapiller, M.W., R.M. Hewick, W.J. Dr eyer, and L.E. Hood. 1983. High-sensitivity

sequencing with gas-phase sequenato r. Methods in Enzymology 91:399-413.

Janda, T., E. Horvath, G. Szalai and E. Paldi. 2007. Role of salicylic acid in the induction

of abiotic stress tolerance. Pages 91-150 in Salicylic Acid: A Plant Hormone. S. Hayat

and A. Ahmad (eds.). Springer, Dordrecht, The Netherlands.

Krause, E., Wenschuh, H., Jungblut, P. R., 1999. The dominance of arginine-containing

peptides in MALDI-derived tryptic mass finge rprints of proteins. Analytical Chemistry

71:4160-4165

Krutz, L.J., M.A. Locke, R.W. Steinriede, K.N. Reddy, L. Libous-Bailey and I.C. Burke.

2012. Water, sediment, and metolachlor trans port differences between wide- and narrowrow cotton production systems. Journa l of Soil and Water Conservation 67:8-15.

Nemecek-Marshall, M., R.C. Macdonald, J.J. Franzen, C.L. Wojciechowski and R. Fall.

1995. Methanol emission from leaves: Enzymatic detection of gas-phase methanol and

relation of methanol fluxes to stomatal conductance and leaf development. Plant

Physiology 108:1359-1368.

Polevoda, B. and F. Sherman. 2000. N α 

# -terminal acetylation of eukaryotic proteins.

Journal of Biological Chemistry 275:36479-36482.

Rademacher, T.W., R.B. Pa rekh and R.A. Dwek. 1988. Glycobiology. Annual Review of

Biochemistry 57:785-838.

# Monsanto Company 12-CT-244U 331 of 620 

# Reddy, K.N., I.C. Burke, J.C. Boykin and J.R. Williford. 2009. Narrow-row cotton

production under irrigated and non-irrigated environment: Plant popul ation and lint yield.

Journal of Cotton Science 13:48-55.

U.S. CPSC. 1997. An update on formaldehyde. U.S. Consumer Product Safety

Commission, Wash ington, D.C.

U.S. EPA. 1996. Residue chemistry test guid elines - Nature of the residue - Plants,

livestock. OPPTS 860.1300. U.S. Environm ental Protection Agency, Office of

Prevention, Pesticides and Toxic Substances, Washington, D.C.

U.S. EPA. 2008. Reregistration eligib ility decision for formaldehyde and

paraformaldehyde. EPA 739-R-08-004. U.S. Envi ronmental Protection Agency, Office of

Pesticide Programs Washington, D.C.

U.S. EPA. 2009. Reregistration eligibility decision fo r dicamba and associated salts. U.S.

Environmental Protection Agency, Office of Pr evention, Pesticides, and Toxic Substances,

Washington, D.C.

USHHS-ATSDR. 1999. Toxicological profile for formaldehyde. U.S. Department of

Health and Human Services, Agency for Toxic Substances and Disease Registry,

Washington, D.C.

USHHS-NTP. 2011. Report on carcinogens. U.S. Department of Health and Human

Services, National Toxicology Program, Washington, D.C.

Wehrmann, A., A.V. Vliet, C. Opsomer, J. Botterman and A. Schulz. 1996. The

similarities of bar and pat gene products make them equally applicable for plant

engineers. Nature Biotechnology 14: 1274-1278.

WHO. 2002. Concise international chemi cal assessment document 40: Formaldehyde.

World Health Organization, Geneva, Switzerland.

WHO-IPCS. 1989. Environmental health crit eria 89: Formaldehyde. World Health

Organization, Internat ional Programme on Chemical Safety, Geneva, Switzerland.

http://www.inchem.org/documents/ehc/ehc/e hc89.htm [Accessed February 2, 2011].

# Monsanto Company 12-CT-244U 332 of 620 

# Appendix D: Materials and Methods Used for the Analysis of the Levels of

MON 88701 DMO and PAT ( bar ) Proteins in MON 88701

D.1. Materials

Seed, over season leaf (OSL-1-4), root, a nd pollen tissue samples from dicamba and

glufosinate-treated MON 88701 were harvested fr om eight field sites in the U.S. during

the 2010 growing season from starting seed lot 11268129, with the exception of OSL-1

(7 sites) and OSL-4 (7 sites). MON 88701 plot s were treated at the 3-5 leaf stage with

glufosinate herbicide at the labe l rate (0.5 lbs a.i./acre) and at the 6-10 l eaf stage with

dicamba herbicide at the labe l rate (0.5 lbs a.e./acre). E. coli -produced MON 88701

DMO (lot 11293429) and PAT ( bar ) protein (lot 11270310) were used as the analytical

reference standards.

D.2. Characterization of the Materials

The identity of MON 88701 was confirme d by conducting MON 88701 event specific

polymerase chain reaction (PCR) analyses on the harvested seed from each site. Any

seed sample and its associated tissues for wh ich three or more pools out of four tested

unexpectedly during PCR verification we re not analyzed in this study.

D.3. Field Design and Tissue Collection

Field trials were initiate d during the 2010 planting season to generate MON 88701 seed,

OSL-1-4, root, and pollen sample s at various cotton growing lo cations in the U.S. The

tissue samples from the following field sites were analyzed: Arkansas (ARTI), Georgia

(GACH), Kansas (KSLA), Louisiana (LAC H), North Carolina (NCBD), New Mexico

(NMLC), South Carolina (SCEK) and Te xas (TXPL). These field sites were

representative of cotton producing regions su itable for commercial production. At each

site, four replicated plots of plants containing MON 88701 were planted using a

randomized complete-block field design. Seed , over season leaf (OSL-1-4), root, and

pollen samples were collected from each replicat ed plot at all field sites, except OSL-1 at

site TXPL and OSL-4 at site LACH. See Tabl es V-4 and V-5 for deta iled descriptions of

when the samples were collected.

D.4. Tissue Processing and Protein Extraction

Tissue samples were shipped to Monsanto Company (St. Louis, Missouri), and were

prepared by the Monsanto Sample Management Team. The prepared tissue samples were

stored in a -80° C freezer until transferre d on dry ice to the analytical facility.

D.4.1. MON 88701 DMO Protein

MON 88701 DMO protein was extracted from tissu e samples as described in Table D-1.

MON 88701 DMO was extracted from over s eason leaf (OSL-1-4) and root tissues

samples with the appropriate amount of Tris borate buffer with 0.5% (w/v) bovine serum

albumin (1 × TB + 0.5% BSA) [0.1 M Tris, 0.1 M Na 2 

# B 4 

# O 7 

# , 0.05 M MgCl 2 

# , 0.05% (v/v)

# Monsanto Company 12-CT-244U 333 of 620 

# Tween 20 at pH 7.8, 0.5% (w/v) BSA]. MON 88701 DMO was extracted from pollen

and seed tissues with the a ppropriate amount of phosphate buffered saline (PBS) with

Tween 20 (1 × PBST). Extractions were done using 8 1/4 ′′ chrome-steel beads, and

shaking in a Harbil mixer (Fluid Management, Wheeling, Illinois). Insoluble material

was removed from all tissue extracts using a serum filter (Fisher Scientific, Pittsburgh,

PA) The extracts were aliquoted and stor ed frozen in a -80 °C freezer until ELISA

analysis.

Table D-1. MON 88701 DMO Protein Extrac tion Methods for Tissue Samples

Sample Type Tissue-to-Buffer Ratio Extraction Buffer

Leaf 1 

# 1:100 1 × TB + 0.5% BSA



# Root 1:100 1 × TB + 0.5% BSA



# Pollen 1:100 1 × PBST



# Seed 1:100 1 × PBST

1 

# Over- season leaf (OSL-1, OSL-2, OSL-3, and OSL-4).



# D.4.2. PAT ( bar ) Protein

PAT ( bar ) protein was extracted from tissue sa mples as described in Table D-2.

PAT ( bar ) was extracted from over season leaf (O SL-1-4) and root tissues samples with

the appropriate amount of Tris borate buffer with L-ascorbic acid (1× TBA) [0.1 M Tris,

0.1 M Na 2 

# B 4 

# O 7 

# , 0.05 M MgCl 2 

# , 0.05% (v/v) Tween 20 at pH 7.8, 0.2% (w/v) L-ascorbic

acid]. PAT ( bar ) was extracted from pollen and seed tissues with the appropriate amount

of 1 × PBST. Extractions were done using 8 1/4 ′′ chrome-steel beads, and shaking in a

Harbil mixer. Insoluble material was rem oved from all tissue extracts using a serum

filter. The extracts were aliquoted and st ored frozen in a - 80 °C freezer until ELISA

analysis.

Table D-2. PAT ( bar ) Protein Extraction Methods for Tissue Samples

Sample Type Tissue-to-Buffer Ratio Extraction Buffer

Leaf 1 

# 1:100 1× TBA



# Root 1:100 1× TBA



# Pollen 1:100 1× PBST



# Seed 1:100 1× PBST

1 

# Over- season leaf (OSL-1, OSL-2, OSL-3, and OSL-4)

# Monsanto Company 12-CT-244U 334 of 620 

# D.5. Protein Antibodies

D.5.1. MON 88701 DMO Protein

Goat polyclonal antibodies specific for the DMO protein were purif ied using Protein G

affinity chromatography. The concentration of the purified IgG was determined to be

8.1 mg/ml by spectrophotometric methods. The purified antibody was stored in 1× PBS.

Protein G-affinity purified goat polyclonal an ti-DMO antibodies were coupled with biotin

(Thermo Fisher Scientific, Rockford, IL) accord ing to the manufacturer’s instructions.

The detection reagent was NeutrAvidin (T hermo Fisher Scientific, Rockford, IL)

conjugated to horseradish peroxidase (HRP).

D.5.2. PAT ( bar ) Protein

Goat polyclonal PAT ( bar )-specific IgG was purified by Protein G-affinity

chromatography followed by PAT ( bar ) antigen affinity chromatography. The

concentration of the purified IgG was determ ined to be 3.6 mg/ml by spectrophotometric

methods. The purified antibody was stored in 1× PBS.

Protein G-affinity purified goat polyclonal anti-PAT ( bar ) antibodies were coupled with

biotin (Thermo Fisher Scientific, Rockfo rd, IL) according to the manufacturer’s

instructions. The detection reagent was NeutrAvidin (Thermo Fisher Scientific,

Rockford, IL) conjugated to horseradish peroxidase (HRP).

D.6. Protein ELISA Method

D.6.1. MON 88701 DMO Protein

Goat anti-DMO antibodies were diluted in coating buffer (15 mM Na 2 

# CO 3 

# , 35 mM

NaHCO 3 

# , and 150 mM NaCl) to a final concentr ation of 5 μg/ml and immobilized onto

96 well microtiter plates followed by incubati on in a 4° C refrigerator for > 8 hours.

Prior to each step in the assay, plates were washed with 1× PBST. Plates were blocked

with the addition of 200 μ l per well of blocking buffer, Blocker Casein (Thermo Fisher

Scientific, Rockford, IL) in Tris Buffered Saline (TBS) for 60 to 70 minutes at room

temperature (RT). DMO protei n standard or sample extrac t was added at 100 μl per well

and incubated for 60 to 65 minutes at 37° C. Biotinylated goat anti-DMO antibodies

prepared in 1× Tris-borate buffer with 10% Blocker Casein in TBS were added at 100 μl

per well and incubated for 60 to 65 minutes at 37° C. NeutrAvi din HRP conjugate was

added at 100 μl per well and incubated for 30 to 35 minutes at 37° C. Plates were

developed by adding 100 μl per well of s ubstrate, 3,3',5,5' tetramethyl benzidine (TMB;

Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction was terminated by the

addition of 100 μl per well of 3 M H 3 

# PO 4 

# . Quantification of the DMO protein was

accomplished by interpolation from a DMO pr otein standard curve that ranged from

0.313 – 10 ng/ml.

# Monsanto Company 12-CT-244U 335 of 620 

# D.6.2. PAT ( bar ) Protein

Affinity purified goat anti PAT ( bar ) antibodies were diluted in coating buffer (15 mM

Na 2 

# CO 3 

# , 35 mM NaHCO 3 

# , and 150 mM sodium chloride) to a final concentration of

4 μg/ml and immobilized onto 96 well microtiter plates, followed by incubation in a 4° C

refrigerator for >12 h. Prior to each step in the assay, plates were washed with 1× PBST.

Plates were blocked w ith the addition of 200 μ l per well of blocking buffer

(1× PBST+1% BSA) for 60 to 70 minutes at 37° C. PAT ( bar ) protein standard or

sample extract was added at 100 μl per well an d incubated for 60 to 70 minutes at 37° C.

Biotinylated goat anti-PAT ( bar ) antibodies diluted in 1 × PBST + 0.1% BSA were added

at 100 μl per well and incubated for 60 to 70 minutes at 37° C. NeutrAvidin HRP

conjugate was added at 100 μl per well and incubated for 60 to 70 minutes at 37° C.

Plates were developed by a dding 100 μl per well of TMB substrate. The enzymatic

reaction was terminated by the addi tion of 100 μl per well of 3 M H 3 

# PO 4 

# . Quantification

of the PAT ( bar ) protein was accomplished by interpolation from a PAT ( bar ) protein

standard curve that ranged from 0.625 – 20 ng/ml.

D.7. Moisture Analysis

Tissue moisture content was determined us ing an IR-200 Moisture Analyzer (Denver

Instrument Company, Arvada, CO). A homogeneous tissue -specific site pool (TSSP)

was prepared consisting of samples of a give n tissue type grown at a given site. The

average percent moisture for each TSSP was cal culated from triplicate analyses. A TSSP

Dry Weight Conversion Factor (D WCF) was calculated as follows:

DWCF  ൌ 1 െ ൬ Mean% TSSP Moisture

100 ൰

The DWCF was used to convert protein levels assessed on a μg/g fres h weight (fw) basis

into levels reported on a μg/g dry weight (dw) basis usi ng the following calculation:

Protein Level in Dry Weight  ൌ Protein Level Fresh Weight

DWCF

Due to a limited amount of tissue, pollen wa s not analyzed for moisture content.

Therefore, no dry weight calculation was performed and pollen was reported on a μg/g

fresh weight (fw) basis only.

The protein levels (ng/ml) 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.8. Data Analyses

All MON 88701 DMO and PAT ( bar ) ELISA plates were analyzed on a SPECTRAmax

Plus 384 (Molecular Devices, Sunnyvale, CA) microplate spectrophotometer, using a

dual wavelength detection method. All protei n concentrations were determined by

optical absorbance at a wavelength of 450 nm with a simultaneous reference reading of

# Monsanto Company 12-CT-244U 336 of 620 

# 620-650 nm. Data reduction analyses were performed using Molecular Devices

SOFTmax PRO GxP version 5.0.1 software. Ab sorbance readings and protein standard

concentrations were fitted with a four-par ameter logistic curve fit. Following the

interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was

converted to a μg/g fw basis for data that we re greater than or equa l to the LOQ. This

conversion utilized a sample dilution factor, and tissue-to-buffer ratio. The protein values

in μg/g fw were also converted to μg/g dw by applying the DWCF (except pollen).

Microsoft Excel 2007 (Microsoft, Redmond, WA) wa s used to calculate the protein levels

in all cotton tissues. The sample means, standard deviations, and ranges were also

calculated by Microsoft Excel 2007. All protein expression levels were rounded to two

significant figures.

Any MON 88701 sample extracts that resulted in an unexpectedly negative result by

ELISA analysis was re extracted twice for th e protein of interest and re analyzed by

ELISA to confirm the results. Samples with confirmed unexpected results were omitted

from all calculations.

# Monsanto Company 12-CT-244U 337 of 620 

# Appendix E: Materials, Methods, and Indi vidual Site Results for Compositional

Analysis of MON 88701 Cottonseed

E.1. Materials

Cottonseed from MON 88701 (Seed Lot Numb er 11268129) treated with dicamba and

glufosinate (T) and MON 88701 not treated with dicamba or glufosinate (NT) and the

conventional control (Seed Lot Number 11268128) was evaluated. The conventional

control has background genetics similar to that of MON 88701 but does not contain either

the dicamba mono-oxygenase (DMO) or phos phinothricin N-acetyltransferase (PAT)

proteins. The commercial re ference varieties were nine conventional cotton varieties

(Table E-1).

Table E-1. Commercial Reference Varieties

Material Name Seed Lot No. Field Sites 1



# SG 125 11266155 ARTI, SCEK, NCBD, TXPL

DP 435 11266762 ARTI, NCBD, TXPL

DP 5415 11266157 ARTI, LACH, KSLA

FM 989 10001810 ARTI, LACH, GACH

Delta Opal 11266158 SCEK, GACH, NCBD

Atlas 11266765 SCEK, TXPL, KSLA, NMLC

ST 474 11266156 GACH, LACH, NCBD, NMLC, SCEK

DP 565 11266764 GACH, LA CH, KSLA, NMLC

NM 1517-99 11268233 TXPL, KSLA

1 

# Field sites described in Section VII.A.



# E.2. Characterization of the Materials

The identities of MON 88701(T) and MON 88701( NT), the conventio nal control, and

commercial reference varieties were conf irmed by verifying the chain of custody

documentation prior to analysis. To furthe r confirm the identities of MON 88701(T) and

MON 88701(NT), the conventional control, and commercial reference varieties, eventspecific polymerase chain reaction (PCR) an alyses were conducted on the harvested,

acid-delinted cottonseed from each site to confirm the presence or absence of the

MON 88701 event.

E.3. Field Production of the Samples

Cottonseed samples were collected from MON 88701(T) and M ON 88701(NT) and the

conventional control Coker 130 grown in a 201 0 U.S. field production. Four different

conventional cotton varieties, known as refere nce substances, were included at each site

of the field production to provide data on natural variability of each compositional

# Monsanto Company 12-CT-244U 338 of 620 

# component analyzed. The field production was conducted at eight sites: Arkansas

(ARTI), Georgia (GACH), Kansas (KSL A), Louisiana (LACH), North Carolina

(NCBD), New Mexico (NMLC), South Carolina (SCEK) and Texas (TXPL). The sites

were planted in a randomized complete bl ock design with four blocks per site.

MON 88701 plots were treated at the 3-5 leaf stage with glufos inate herbicide at the label

rate (0.5 lbs a.i. /acre) and at the 6-10 leaf stage with dicam ba herbicide at the label rate

(0.5 lbs a.e./acre). T/C/R substances were grown under normal agronomic field

conditions for their resp ective geographic regions. Cottonseed samples were harvested

and ginned from all plots and shipped at ambi ent temperature to Monsanto Company (St.

Louis, Missouri). The samples were acid-de linted and a subsample was obtained from

each for compositional analyses. These subsam ples were ground and stored in a freezer

set to maintain

 20°C until their shipment on dry i ce to Covance Laboratories Inc.

(Madison, Wisconsin) for analysis. The label on the samples shipped listed the protocol

(study) number, tissue type, material name, storage conditions, and a unique sample ID

number.

E.4. Summary of Analytical Methods

Harvested, acid-delinted cottonseed samples were analyzed by Covance Laboratories Inc.

Upon receipt, the samples were stored in a freezer set to ma intain -20 °C until their use.

Nutrients assessed in this analysis included proximates (ash, fat, moisture, protein, and

carbohydrates and calories by cal culation), acid detergent fibe r (ADF), neutral detergent

fiber (NDF), crude fiber (CF) , total dietary fiber (TDF), amino acids (AA), fatty acids

(C8-C22), minerals (Ca, Cu, Fe, Mg, Mn, P, K, Na, Zn) and vitamin E ( α -tocopherol).

Anti-nutrients analyzed included gossypol a nd cyclopropenoid fatty acids (CPFA).

E.4.1. Acid Detergent Fiber

The ANKOM2000 Fiber Analyzer automated th e process of removal of proteins,

carbohydrates, and ash. Fats and pigments were removed with an ace tone wash prior to

analysis. The fibrous residue that is prim arily cellulose, lignin, and insoluble protein

complexes remained in the Ankom filter bag, and were determined gravimetrically.

(Komarek, et al., 1993; USDA, 1970). The resu lts were reported on fresh weight basis.

The limit of quantitation was 0.100%.

# Monsanto Company 12-CT-244U 339 of 620 

# E.4.2. Amino Acid Composition

The following 18 amino acids were analyzed:

Total threonine Total aspartic acid (including asparagine)

Total serine Total tyrosine

Total phenylalanine Total glut amic acid (including glutamine)

Total proline Total histidine

Total glycine Total lysine

Total alanine Total arginine

Total valine Total tryptophan

Total isoleucine Total methionine

Total leucine Total cystine (including cysteine)

The samples were hydrolyzed in 6N hydrochloric acid for approximately 24 hours at

approximately 106-110oC. Phenol was added to the 6N hydrochloric acid to prevent

halogenation of tyrosine. Cystine an d cysteine are converted to S-2-

carboxyethylthiocysteine by the addition of dithiodipropionic acid. Tryptophan was

hydrolyzed from proteins by heating at approximately 110oC in 4.2N sodium

hydroxide for 20 hours. The samples were analyzed by HPLC after pre-injection

derivatization. The primary am ino acids were derivatized with o-phthalaldehyde (OPA)

and the secondary amino acids are derivati zed with fluorenylmethyl chloroformate

(FMOC) before injection. (AOAC, 2011a ; Barkholt and Jensen, 1989; Henderson and

Brooks, 2010; Henderson, et al., 2000; Schuster, 1988). The results were reported on

fresh weight basis. The lim it of quantitation was 0.100 mg/g.

Reference Standards:

Component Manufacturer Lot No. Purity (%)

L-Alanine Sigma-Aldrich 1440397 99.9

L-Arginine Monohydrochloride Sigma-Aldrich 1361811 100

L-Aspartic Acid Sigma- Aldrich BCBB9274 100.6

L-Cystine Sigma-Aldrich 1418036 99.9

L-Glutamic Acid Sigma-Aldrich 1423805 100.2

Glycine Sigma-Aldrich 1119375 100

L-Histidine Monohydrochloride Mono hydrate Sigma-Aldrich BCBB1348 99.9

L-Isoleucine Sigma-Aldrich 1423806 100

L-Leucine Sigma-Aldrich BCBB1733 98.6

L-Lysine Monohydrochloride Sigma-Aldrich 1362380 100.2

L-Methionine Sigma-Aldrich 1423807 99.9

L-Phenylalanine Sigma-Aldrich BCBB9200 100

L-Proline Sigma-Aldrich 1414414 99.7

L-Serine Sigma-Aldrich 1336081 99.9

L-Threonine Sigma-Aldrich 1402329 100

L-Tryptophan Sigma-Aldrich BCBB1284 99.8

L-Tyrosine Sigma-Aldrich BCBB5393 99.5

L-Valine Sigma-Aldrich 1352709 100

# Monsanto Company 12-CT-244U 340 of 620 

# E.4.3. Ash

The sample was placed in an electric fu rnace at 550 °C and i gnited. The nonvolatile

matter remaining was quantified gravimetrically and calculated to determine percent ash

(AOAC, 2011b). The limit of quantitation was 0.100%.

E.4.4. Calories

Calories were calculated using the Atwater factors with the fresh weight-derived data and

the following equation:

calories (Kcal/100g) = (4 × % protein) + (9 × % fat) + (4 × % carbohydrates)

The limit of quantitation was calculated as 2.00 Kcalories/ 100g on a fresh weight basis

(Code of Federal Regulation, Title 21, Part 101.9, pp. 24-25).

E.4.5. Carbohydrates

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 results were reported on fresh weight basis (USDA, 1973). The limit of quantitation

was 0.100%.

E.4.6. Crude Fiber

Crude fiber was quantitated as the loss on ignition of dried residue remaining after

digestion of the sample with 1.25% sulfuric acid and 1.25% sodium hydroxide solutions

under specific conditions (AOAC, 2011c). Th e results were reported on fresh weight

basis. The limit of quantitation was 0.100%.

E.4.7. Fat by Soxhlet Extraction

The sample was weighed into a cellulose thim ble containing sodium sulfate and dried to

remove excess moisture. Pentane was dripped through the sample to re move the fat. The

extract was then evaporated, dried, and we ighed (AOAC, 2011d; e). The results were

reported on fresh weight basis. The limit of quantitation was 0.100%.

# Monsanto Company 12-CT-244U 341 of 620 

# E.4.8. Cyclopropenoid Fatty Acids

The total lipid fraction was extracted from the sample using chloroform and methanol. A

portion of the lipid fraction was then saponified with a mild alkaline hydrolysis. The free

fatty acids were extracted with ethyl ether and hexane. The free fatty acids were then

converted to their phenacyl derivatives with 2-bromoacetophenone. The derivatives were

quantitated on a high-performance liquid ch romatography system equipped with an

ultraviolet detector. The amount of malvalic , sterculic and dihydrosterculic acids were

determined by comparison to an external ca libration curves of si milarly derivatized

reference standards (Wood, 1986). The results we re expressed on a fresh weight basis.

The limit of quantitation was 50.0 μg/g.

Reference Standards:

 Monsanto, Malvalic Acid, 100%, Lot Number GLP-0208-12964-A

 Monsanto, Sterculic Acid, 99% , Lot Number GLP-0208-12963-A

 Monsanto, Dihydrosterculic, 98 %, Lot Number GLP-0311-14467-A

E.4.9. Fatty Acids

The lipid was extracted and saponified w ith 0.5 N methanolic sodium hydroxide, and

methylated with 14% boron trifluoride in meth anol. The resulting methyl esters of the

fatty acids were extracted with heptane cont aining an internal standard. The methyl

esters of the fatty acids we re analyzed by gas chromatogr aphy using external standards

for quantitation (AOCS, 1997; 2001; 2009a; c). The results were reported on fresh

weight basis. The limit of quantitation was 0.0200%.

# Monsanto Company 12-CT-244U 342 of 620 

# Reference Standards:

Component Lot Number Component Weight

(%) Purity

(%)

Nu-Chek Prep GLC

Reference Standard

Hazelton No. 1 JY20-U Methyl Octanoate 16.66 99.6

Methyl Decanoate 16.66 99.6

Methyl Laurate 16.66 99.8

Methyl Myristate 16.66 99.8

Methyl Palmitoleate 16.66 99.7

Methyl Linolenate 16.66 99.5

Nu-Chek Prep GLC

Reference Standard

Hazelton No. 2 AU16-U Methyl Arachidate 33.33 99.6

Methyl 11-Eicosenoate 33.33 99.5

Methyl Arachidonate 33.33 99.6

Nu-Chek Prep GLC

Reference Standard

Hazelton No. 3 J28-U Methyl Myristoleate 12.5 99.5

Methyl Pentadecanoate 12.5 99.6

Methyl 10-Pentadecenoate 12.5 99.5

Methyl Heptadecanoate 12.5 99.6

Methyl 10-Heptadecenoate 12.5 99.5

Methyl 11-14 Eicosadienoate 12.5 99.6

Methyl Behenate 12.5 99.8

Methyl 11-14-17

Eicosatrienoate 12.5 99.5

Nu-Chek Prep GLC

Reference Standard

Hazelton No. 3 F15-V Methyl Myristoleate 12.5 99.5

Methyl Pentadecanoate 12.5 99.6

Methyl 10-Pentadecenoate 12.5 99.5

Methyl Heptadecanoate 12.5 99.6

Methyl 10-Heptadecenoate 12.5 99.5

Methyl 11-14 Eicosadienoate 12.5 99.6

Methyl Behenate 12.5 99.8

Methyl 11-14-17

Eicosatrienoate 12.5 99.5

Nu-Chek Prep GLC

Reference Standard

Hazelton No. 4 MA30-U Methyl Palmitate 27.0 99.6

Methyl Stearate 19.0 99.5

Methyl Oleate 27.0 99.8

Methyl Linoleate 27.0 99.8

Nu-Chek Prep GLC

Reference Standard

Hazelton No. 4 JA31-V Methyl Palmitate 27.0 99.7

Methyl Stearate 19.0 99.7

Methyl Oleate 27.0 99.8

Methyl Linoleate 27.0 99.8

Nu-Chek Prep Methyl

Gamma Linolenate U-63M-M18-U Not applicable Not applicable >99

Nu-Chek Prep Methyl

Gamma Linolenate U-63M-N2-U Not applicable Not applicable >99

Nu-Chek Prep

Methyl Tridecanoate N-13M-F16-V Not applicable Not applicable >99

Nu-Chek Prep

Methyl Tridecanoate N-13M-MA25-T Not applicable Not applicable >99

# Monsanto Company 12-CT-244U 343 of 620 

# E.4.10. Free and Total Gossypol

For free gossypol, the sample was extracted w ith aqueous acetone. The solution was then

filtered and the free gossypol wa s reacted with aniline. Fo r total gossypol analysis, the

sample was extracted using a complexing reagent containing acetic acid,

3-amino-1-propanol, and dimethylformamide. The solution was then filtered and the

total gossypol was reacted with aniline. For both analys es, the dianilinogossypol was

quantitated spectrophotometrica lly using a standard curve ( AOCS, 2011a; b) The results

were reported on fresh weight basis. The limit of quantitation was 0.00200%.

Reference Standard:

 Sigma-Aldrich, Gossypol, 97.7%, Lot Number 059K4046

E.4.11. ICP Emission Spectrometry

The sample was dried, precharred, and ashed overnight in a muffle furnace set to

maintain 500  C. The ashed sample was re-ashed with nitric acid, treated with

hydrochloric acid, taken to dryne ss, 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, measur ed on the inductively coupled plasma

spectrometer, with the emission of the standa rd solutions (AOAC, 2011f; g). The results

were reported on fresh weight basis.

Inorganic Ventures Reference Sta ndards and Limits of Quantitation

Mineral Lot Numbers Concentration

(μg/mL) Limit of

Quantitation

(ppm)



# Calcium E2-MEB360079MCA, E2-MEB360081 200, 1000 20.0

Copper E2-MEB360079MCA, E2-MEB360080MCA 2.00, 10.0 0.500

Iron E2-MEB360079MCA, E2-MEB360082 10.0, 50.0 2.00

Magnesium E2-MEB360079MCA, E2-MEB360080MCA 50.0, 250 20.0

Manganese E2-MEB360079MCA, E2-MEB360080MCA 2.00, 10.0 0.300

Phosphorus E2-MEB360079MCA, E2-MEB360081 200, 1000 20.0

Potassium E2-MEB360079MCA, E2-MEB360081 200, 1000 100

Sodium E2-MEB360079MCA, E2-MEB360081 200, 1000 100

Zinc E2-MEB360079MCA, E2-MEB360080MCA 10.0, 50.0 0.400

# Monsanto Company 12-CT-244U 344 of 620 

# E.4.12. Moisture

The 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 (AOAC,

2011h; i). The results were reported on fresh weight basis. The limit of quantitation was

0.100%.

E.4.13. Neutral Detergent Fiber, Enzyme Method

The ANKOM2000 Fiber Analyzer automated the process of the removal of proteins,

carbohydrates, and ash. The fats and pigments were removed with an acetone wash prior

to analysis. Hemicellulose, cellulose, lignin, and insoluble protein fr action were left in

the filter bag and determined gravimetri cally (AACC, 1999; Ko marek et al., 1994;

USDA, 1970). The results were reported on fr esh weight basis. The limit of quantitation

was 0.100%.

E.4.14. Protein

The protein and other organic nitrogen in the sample were converted to ammonia by

digesting the sample with sulfur ic acid containing a catalyst mixture. The acid digest was

made alkaline. The ammonia was distille d and then titrated with a previously

standardized acid. The per cent nitrogen was calculated a nd converted to equivalent

protein using the factor 6.25 (AOAC, 2011j; k; AOCS, 2009a) The results were reported

on fresh weight basis. The limit of quantitation was 0.100%.

E.4.15. Total Dietary Fiber

Duplicate samples were gelatinized with  -amylase and digested with enzymes to break

down starch and protein. Ethanol was added to each sample to precipitate the soluble

fiber. The sample was filtered, and the residue was rinsed with ethanol and acetone to

remove starch and protein degradation pr oducts and moisture. Protein content was

determined for one of the duplicates; ash conten t was determined for the other. The total

dietary fiber in the sample was calculated us ing protein and ash valu es (AOAC, 2011j).

The results were reported on fresh weight basis. The limit of quantitation was 1.00%.

E.4.16. Vitamin E

The sample was saponified to break down any fat and release vitamin E. The saponified

mixture was extracted with ethyl ether a nd then quantified by high-performance liquid

chromatography using a silica column (Cort, et al., 1983; McMurray, et al., 1980; Speek,

et al., 1985). The results were reported on fresh weight basis. The limit of quantitation

was 0.500 mg/100g.

Note: Alpha tocopherol is part of a mixed sta ndard which also includes beta, delta, and

gamma isomers. The reference standard ma terial for those isomers may contain small

amounts of alpha tocopherol. All reference standards that contri buted to the alpha

tocopherol concentration are listed below.

# Monsanto Company 12-CT-244U 345 of 620 

# Reference Standards:

 USP, Alpha Tocopherol, 98.9%, Lot Number N0F068

 Acros Organics, D-gamma-Tocopherol, 99.4%, A0083534

 Sigma-Aldrich, (+)-delta- Tocopherol, 92%, 090M1916V

E.5. Data Processing and Statistical Analysis

After compositional analyses were performed, data spreadsheets containing individual

values for each analysis were sent to M onsanto Company (St. Louis, Missouri) for

review. Data were then transferred to Certus International (Chesterfield, MO) where they

were converted into the appropr iate units and statis tically analyzed. The formulas that

were used for re-expression of composition da ta for statistical analysis are listed in

Table E-2.

# Monsanto Company 12-CT-244U 346 of 620 

# Table E-2. Re-expression Formulas for St atistical Analysis of Composition Data

Component From (X) To Formula 1



# Proximates (excluding Moisture

and Calories), Fiber, Gossypol % fw % dw X/d

Calories Kcal/100g fw Kcal/100g dw X/d

Copper, Iron, Manganese, Zinc ppm fw mg/kg dw X/d

Calcium, Magnesium,

Phosphorus, Potassium, Sodium ppm fw % dw X/(10 4 

# d)

Vitamin E mg/100g fw mg/kg dw 10(X/d)

Amino Acids (AA) mg/g fw % dw X/(10d)

Sterculic, Malvalic, and

Dihydrosterculic Acids 2 

# μ g/g fw % fw X/10 4



# Fatty Acids (FA) % fw % Total FA (100)X j 

# /  X,

for each FA j



# where  X is

over all the

FA

1 

# d is the fraction of the sample that is dry matter.

2 

# Sterculic Acid, Malvalic Acid and Dihydroste rculic Acid were first converted to % fw

as an intermediate step for final re-expression as % Total FA.



# In order to complete a statistical analysis for a compositional component in this study, at

least 50% of the values for a component had to be grea ter than the assay limit of

quantitation (LOQ). Components with more than 50% of ob servations below the assay

LOQ were excluded from summar ies and analysis. The following 13 components with

more than 50% of the observations below the assay LOQ were excluded: 8:0 caprylic

acid, 10:0 capric acid, 12:0 lauric acid, 14:1 myristolei c acid, 15:0 pentadecanoic acid,

15:1 pentadecenoic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gammalinolenic acid, 20:1 eicosenoic acid; 20:2 eicosa dienoic acid, 20:3 eicosatrienoic acid, and

20:4 arachidonic acid.

If less than 50% of the observations for a component we re below the LOQ, individual

analyses that were below the LOQ were assign ed a value equal to one-half the LOQ. In

this study 187 values for 22:0 behenic acid were assigned a value of 0.010% fw and 187

values for sodium were a ssigned a value of 50.00 ppm fw.

The data were assessed for potential outli ers using a studentized PRESS residuals

calculation. A PRESS residual is the di fference between any value and its value

predicted from a statistical model that excludes the data point. The studentized version

scales these residuals so that the values te nd to have a standard normal distribution when

outliers are absent. Thus, most va lues 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. One sodium value from the

commercial control at the ARTI site and one sodium value from a commercial reference

# Monsanto Company 12-CT-244U 347 of 620 

# at the ARTI site were extreme data points that were outside the  6 studentized PRESS

residual range and were removed fr om the statistical analysis.

All cottonseed components were statistically analyzed using a mixed model analysis of

variance. The eight replicated field sites we re analyzed individually and as a combined

data set. Individual site analysis mean co mparison tests were not conducted on site ARTI

sodium content because only one Coker 130 re plicate was available at that site.

Analyses of the combined replicated sites were performed using model (1).

(1) Y ijk 

# = U + T i 

# + L j 

# + B(L) jk



# + LT ij 

# + e ijk,



# where Y ijk 

# = unique individua l observation, U = overall mean, T i 

# = substance effect,

L

j 

# = random site effect, B(L) jk



# = random block within site effect,

LT ij 

# = random site by substance interaction effect, and e ijk 

# = residual error.

Individual sites were also analyzed separate ly. Individual site analyses were performed

using model (2).

(2) Y ij 

# = U + T i 

# + B j 

# + e ij 

# ,

where Y ij 

# = unique individual observa tion, U = overall mean, T i 

# = substance effect,

B j 

# = random block effect, and e ij 

# = residual error.

Pairwise comparisons between the test and control materials were defined within the

ANOVA and tested using t-tests. The variab ility from the ANOVA wa s used to compute

the standard error of the di fference and to conduct the t-tests for the comparisons.

For each compositional component, a range of observed values and a 99% tolerance

interval were calculated. A tolerance interval is an interval that one can claim, with a

specified degree of confidence, contains at least a specifi ed proportion, p, of an entire

sampled population for the parameter measure d. The calculated tole rance intervals are

expected to contain, with 95% confidence, 99% of the quantitie s expressed in the

population of conventional cotton. Each tolerance interval estimate was based upon the

average observation for each unique reference material. Because negative quantities are

not possible, negative calculated lower tolerance bounds were set to zero.

SAS ® 

# (Version 9.2) software was used to genera te all summary statistics and perform all

analyses.

Report tables present p-values from SAS as either <0.001 or the actual value truncated to

three decimal places.Monsanto Company 12-CT-244U 348 of 620Table E-3. Statistical Summary of Site ARTI CottonseedNutrients for MON 88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Unis)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.62 (0.087)4.74 (0.12)-0.13 (0.15)-0.55, 0.290.4403.42, 4.65(4.51 - 4.74)(4.49 - 5.00)(-0.49 - 0.047)(3.18 - 4.68)Calories (Kcal/100g)495.41 (3.00)488.30 (3.88)7.11 (3.90)-3.73, 17.940.142457.61, 527.56(487.88 - 504.08)(487.70 - 494.60)(4.92 - 16.38)(466.09 - 509.91)Carbohydrates45.08 (0.58)46.35 (0.81)-1.27 (0.96)-3.94, 1.390.25440.26, 56.45(43.42 - 46.31)(45.03 - 47.37)(-3.95 - -0.33)(43.28 - 54.90)Moisture (% fw)7.10 (0.27)7.63 (0.35)-0.53 (0.35)-1.49, 0.430.1974.79, 9.92(6.71 - 7.58)(7.32 - 7.40)(-0.69 - -0.36)(6.05 - 10.50)Protein27.53 (0.24)27.04 (0.33)0.49 (0.41)-0.65, 1.630.29722.30, 29.41(27.16 - 28.11)(26.97 - 27.11)(0.12 - 0.65)(20.58 - 29.28)Total Fat22.76 (0.59)21.50 (0.78)1.26 (0.81)-0.98, 3.500.19315.01, 28.51(21.32 - 24.40)(21.15 - 22.89)(0.69 - 3.25)(16.58 - 25.25)Monsanto Company 12-CT-244U 349 of 620Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber24.81 (0.32)27.53 (0.45)-2.71 (0.55)-4.23, -1.200.00722.24, 31.96(24.44 - 25.20)(26.57 - 28.49)(-3.98 - -2.13)(23.42 - 31.62)Crude Fiber18.33 (0.90)19.47 (1.20)-1.14 (1.26)-4.64, 2.360.41716.93, 22.68(15.97 - 20.56)(19.33 - 19.85)(-3.36 - -0.40)(16.92 - 23.32)Neutral Detergent Fiber31.27 (0.79)32.89 (1.06)-1.61 (1.13)-4.75, 1.530.22727.03, 42.49(29.99 - 32.89)(30.67 - 34.42)(-4.43 - 0.095)(29.27 - 40.63)Total Dietary Fiber40.85 (1.06)41.67 (1.50)-0.82 (1.83)-5.91, 4.270.67834.52, 52.58(39.82 - 42.13)(40.50 - 42.84)(-0.70 - 1.09)(37.29 - 48.60)Amino Acid (% dw)Alanine1.05 (0.015)1.02 (0.018)0.029 (0.017)-0.018, 0.0770.1610.86, 1.11(1.01 - 1.09)(0.99 - 1.05)(0.011 - 0.036)(0.83 - 1.22)Arginine3.00 (0.052)3.02 (0.073)-0.018 (0.084)-0.25, 0.210.8402.38, 3.47(2.86 - 3.07)(2.89 - 3.13)(-0.098 - -0.032)(2.30 - 3.55)Monsanto Company 12-CT-244U 350 of 620Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.41 (0.045)2.32 (0.060)0.087 (0.065)-0.093, 0.270.2521.94, 2.57(2.29 - 2.48)(2.19 - 2.42)(-0.031 - 0.10)(1.79 - 2.72)Cystine0.40 (0.010)0.37 (0.014)0.034 (0.017)-0.015, 0.0820.1240.31, 0.45(0.38 - 0.42)(0.35 - 0.39)(0.014 - 0.074)(0.29 - 0.47)Glutamic Acid4.82 (0.099)4.51 (0.14)0.31 (0.17)-0.15, 0.780.1343.74, 5.28(4.61 - 5.07)(4.34 - 4.67)(0.11 - 0.27)(3.39 - 5.45)Glycine1.10 (0.020)1.08 (0.026)0.027 (0.028)-0.052, 0.110.3970.90, 1.14(1.05 - 1.14)(1.03 - 1.11)(-0.015 - 0.021)(0.85 - 1.23)Histidine0.74 (0.016)0.74 (0.023)0.0028 (0.026)-0.071, 0.0760.9190.59, 0.81(0.71 - 0.76)(0.71 - 0.77)(-0.0019 - -0.00095)(0.57 - 0.84)Isoleucine0.89 (0.018)0.91 (0.024)-0.010 (0.025)-0.079, 0.0580.6960.75, 0.96(0.87 - 0.92)(0.88 - 0.93)(-0.027 - -0.0076)(0.72 - 1.03)Monsanto Company 12-CT-244U 351 of 620Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.53 (0.027)1.50 (0.037)0.029 (0.042)-0.086, 0.140.5241.25, 1.62(1.47 - 1.55)(1.44 - 1.55)(-0.0060 - 0.032)(1.20 - 1.72)Lysine1.22 (0.044)1.23 (0.058)-0.0029 (0.062)-0.18, 0.170.9651.01, 1.30(1.15 - 1.27)(1.19 - 1.26)(-0.041 - -0.016)(0.99 - 1.44)Methionine0.39 (0.015)0.35 (0.021)0.041 (0.025)-0.029, 0.110.1810.32, 0.38(0.35 - 0.43)(0.35 - 0.36)(-0.014 - 0.087)(0.29 - 0.49)Phenylalanine1.43 (0.027)1.41 (0.038)0.016 (0.045)-0.11, 0.140.7371.12, 1.58(1.36 - 1.45)(1.34 - 1.48)(-0.027 - 0.022)(1.10 - 1.63)Proline0.97 (0.021)1.00 (0.028)-0.027 (0.030)-0.11, 0.0570.4170.83, 1.08(0.95 - 1.00)(0.98 - 1.02)(-0.027 - -0.024)(0.79 - 1.17)Serine1.11 (0.022)1.03 (0.031)0.089 (0.038)-0.016, 0.190.0790.83, 1.21(1.07 - 1.19)(0.99 - 1.06)(0.011 - 0.096)(0.81 - 1.24)Monsanto Company 12-CT-244U 352 of 620Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.88 (0.012)0.85 (0.017)0.029 (0.021)-0.028, 0.0870.2300.72, 0.89(0.85 - 0.90)(0.82 - 0.88)(-0.0045 - 0.033)(0.67 - 0.96)Tryptophan0.41 (0.0062)0.42 (0.0087)-0.012 (0.011)-0.042, 0.0170.3060.34, 0.42(0.40 - 0.42)(0.40 - 0.44)(-0.041 - 0.0060)(0.31 - 0.46)Tyrosine0.82 (0.015)0.79 (0.021)0.030 (0.026)-0.042, 0.100.3130.67, 0.84(0.79 - 0.84)(0.76 - 0.82)(0.0011 - 0.028)(0.63 - 0.91)Valine1.19 (0.021)1.21 (0.027)-0.018 (0.026)-0.090, 0.0550.5371.00, 1.28(1.14 - 1.23)(1.17 - 1.24)(-0.030 - -0.016)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.77 (0.0085)0.78 (0.012)-0.0049 (0.013)-0.041, 0.0310.7230.16, 1.37(0.76 - 0.79)(0.77 - 0.78)(-0.029 - 0.0018)(0.45 - 1.04)16:0 Palmitic25.16 (0.10)24.98 (0.14)0.18 (0.18)-0.31, 0.670.36016.54, 30.55(25.01 - 25.28)(24.92 - 25.05)(0.028 - 0.094)(19.11 - 26.73)Monsanto Company 12-CT-244U 353 of 620Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.53 (0.0052)0.52 (0.0073)0.017 (0.0089)-0.0082, 0.0410.1370.39, 0.70(0.52 - 0.54)(0.52 - 0.52)(-0.00060 - 0.017)(0.44 - 0.67)18:0 Stearic2.68 (0.026)2.51 (0.036)0.17 (0.045)0.044, 0.290.0191.98, 2.95(2.65 - 2.72)(2.45 - 2.57)(0.083 - 0.22)(1.98 - 2.97)18:1 Oleic14.81 (0.11)14.68 (0.15)0.12 (0.17)-0.34, 0.590.50111.38, 20.64(14.46 - 15.08)(14.58 - 14.70)(-0.24 - 0.18)(13.71 - 18.39)18:2 Linoleic54.73 (0.22)55.31 (0.31)-0.59 (0.37)-1.62, 0.450.18947.49, 63.18(54.24 - 55.29)(55.26 - 55.38)(-0.45 - 0.037)(49.78 - 59.61)18:3 Linolenic0.14 (0.0021)0.13 (0.0030)0.015 (0.0035)0.0056, 0.0250.0120.060, 0.24(0.14 - 0.15)(0.12 - 0.14)(0.0064 - 0.021)(0.10 - 0.29)20:0 Arachidic0.31 (0.0079)0.29 (0.011)0.023 (0.011)-0.0087, 0.0540.1150.17, 0.38(0.31 - 0.32)(0.27 - 0.31)(0.0032 - 0.046)(0.20 - 0.36)Monsanto Company 12-CT-244U 354 of 620Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.14 (0.0026)0.15 (0.0033)-0.015 (0.0031)-0.024, -0.00650.0080.070, 0.21(0.13 - 0.14)(0.15 - 0.16)(-0.019 - -0.012)(0.051 - 0.19)MineralCalcium (% dw)0.15 (0.0035)0.12 (0.0050)0.028 (0.0061)0.011, 0.0450.0100.058, 0.21(0.14 - 0.16)(0.12 - 0.13)(0.024 - 0.035)(0.081 - 0.18)Copper (mg/kg dw)9.66 (0.34)9.64 (0.41)0.018 (0.38)-1.03, 1.060.9632.97, 12.86(9.23 - 10.15)(8.79 - 9.79)(-0.57 - 0.58)(4.46 - 11.62)Iron (mg/kg dw)75.27 (5.63)80.76 (7.78)-5.49 (8.79)-29.91, 18.930.56647.30, 97.12(72.55 - 77.65)(72.89 - 87.72)(-15.17 - 2.25)(39.49 - 114.34)Magnesium (% dw)0.41 (0.0076)0.40 (0.010)0.0099 (0.011)-0.020, 0.0400.4130.28, 0.47(0.40 - 0.42)(0.38 - 0.41)(0.0077 - 0.016)(0.31 - 0.46)Manganese (mg/kg dw)13.27 (0.63)11.50 (0.89)1.77 (1.06)-1.18, 4.730.1719.07, 17.33(12.58 - 13.63)(11.34 - 11.55)(1.03 - 1.95)(9.07 - 17.14)Monsanto Company 12-CT-244U 355 of 620Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.83 (0.012)0.84 (0.016)-0.015 (0.018)-0.066, 0.0360.4500.49, 0.87(0.82 - 0.84)(0.82 - 0.87)(-0.027 - 0.0054)(0.48 - 0.87)Potassium (% dw)1.18 (0.029)1.11 (0.040)0.071 (0.047)-0.059, 0.200.2040.92, 1.21(1.16 - 1.20)(1.06 - 1.15)(0.028 - 0.14)(0.90 - 1.26)Sodium (% dw)0.027 (0.0038)ND60, 0.066(0.023 - 0.029)(0.013 - 0.013)(0.0054 - 0.077)Zinc (mg/kg dw)37.08 (1.65)40.81 (2.03)-3.72 (1.87)-8.92, 1.470.11727.27, 44.95(35.15 - 38.88)(38.63 - 40.71)(-3.48 - -3.41)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E147.20 (2.44)136.55 (3.46)10.65 (4.23)-1.10, 22.400.06541.91, 205.89(144.99 - 149.40)(133.79 - 139.31)(6.47 - 15.60)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.6Not determined due to insufficient number of observations for the control.Monsanto Company 12-CT-244U 356 of 620Table E-4. Statistical Summary of SiteARTI Cottonseed Anti-nutrients for MON88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.0073)0.15 (0.010)0.0022 (0.013)-0.033, 0.0370.8670.078, 0.25(0.14 - 0.16)(0.14 - 0.15)(-0.0021 - -0.00026)(0.038 - 0.23)Malvalic Acid0.37 (0.016)0.36 (0.023)0.0088 (0.025)-0.061, 0.0780.7420.23, 0.54(0.34 - 0.39)(0.33 - 0.37)(-0.031 - 0.032)(0.11 - 0.59)Sterculic Acid0.21 (0.014)0.20 (0.020)0.013 (0.023)-0.052, 0.0780.6050.17, 0.27(0.19 - 0.22)(0.19 - 0.20)(-0.0076 - 0.032)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.91 (0.035)0.82 (0.049)0.090 (0.060)-0.076, 0.260.2070.099, 1.57(0.80 - 1.02)(0.80 - 0.84)(-0.035 - 0.10)(0.50 - 1.41)Total Gossypol0.97 (0.034)0.93 (0.044)0.042 (0.044)-0.081, 0.160.3990.064, 1.76(0.89 - 1.04)(0.94 - 0.98)(0.0013 - 0.10)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 357 of 620Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.53 (0.044)4.21 (0.047)0.32 (0.034)0.23, 0.41<0.0013.42, 4.65(4.45 - 4.57)(4.12 - 4.23)(0.31 - 0.34)(3.18 - 4.68)Calories (Kcal/100g)497.72 (2.28)496.55 (2.63)1.16 (3.48)-7.79, 10.120.751457.61, 527.56(489.91 - 504.20)(494.57 - 498.27)(-6.91 - 8.49)(466.09 - 509.91)Carbohydrates44.91 (0.59)45.84 (0.68)-0.94 (0.91)-3.27, 1.390.34840.26, 56.45(43.42 - 46.94)(44.64 - 47.09)(-3.23 - 1.14)(43.28 - 54.90)Moisture (% fw)6.98 (0.15)7.23 (0.18)-0.25 (0.23)-0.85, 0.350.3284.79, 9.92(6.42 - 7.33)(6.99 - 7.48)(-1.06 - 0.12)(6.05 - 10.50)Protein27.41 (0.33)27.30 (0.37)0.11 (0.37)-0.83, 1.060.77022.30, 29.41(26.87 - 27.78)(26.45 - 28.21)(-0.43 - 0.96)(20.58 - 29.28)Total Fat23.14 (0.46)22.67 (0.53)0.47 (0.70)-1.34, 2.280.53615.01, 28.51(21.58 - 24.46)(22.26 - 23.02)(-1.16 - 1.93)(16.58 - 25.25)Monsanto Company 12-CT-244U 358 of 620Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.69 (0.52)27.52 (0.60)-1.83 (0.79)-3.86, 0.200.06822.24, 31.96(25.04 - 26.77)(26.81 - 28.13)(-3.09 - -0.87)(23.42 - 31.62)Crude Fiber19.27 (0.68)19.92 (0.78)-0.64 (1.00)-3.21, 1.920.54716.93, 22.68(17.10 - 20.64)(18.70 - 21.18)(-1.60 - -0.54)(16.92 - 23.32)Neutral Detergent Fiber31.47 (0.90)33.92 (1.04)-2.44 (1.38)-5.98, 1.090.13527.03, 42.49(29.71 - 34.04)(32.79 - 35.89)(-5.13 - -1.40)(29.27 - 40.63)Total Dietary Fiber39.64 (0.86)41.11 (1.00)-1.46 (1.32)-4.85, 1.930.31834.52, 52.58(37.72 - 41.91)(39.89 - 42.04)(-4.32 - -0.44)(37.29 - 48.60)Amino Acid (% dw)Alanine1.04 (0.019)1.10 (0.022)-0.059 (0.025)-0.12, 0.00500.0640.86, 1.11(1.02 - 1.05)(1.06 - 1.17)(-0.13 - -0.016)(0.83 - 1.22)Arginine2.95 (0.061)3.21 (0.067)-0.27 (0.064)-0.43, -0.100.0082.38, 3.47(2.87 - 3.02)(3.07 - 3.46)(-0.47 - -0.14)(2.30 - 3.55)Monsanto Company 12-CT-244U 359 of 620Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.31 (0.043)2.45 (0.047)-0.15 (0.044)-0.26, -0.0350.0191.94, 2.57(2.24 - 2.36)(2.36 - 2.60)(-0.29 - -0.054)(1.79 - 2.72)Cystine0.40 (0.012)0.40 (0.013)-0.0089 (0.018)-0.054, 0.0360.6360.31, 0.45(0.38 - 0.42)(0.38 - 0.43)(-0.052 - 0.011)(0.29 - 0.47)Glutamic Acid4.57 (0.098)4.96 (0.11)-0.39 (0.099)-0.65, -0.140.0103.74, 5.28(4.35 - 4.77)(4.77 - 5.21)(-0.63 - -0.17)(3.39 - 5.45)Glycine1.08 (0.020)1.13 (0.023)-0.052 (0.026)-0.12, 0.0140.0990.90, 1.14(1.06 - 1.12)(1.09 - 1.20)(-0.13 - 0.011)(0.85 - 1.23)Histidine0.73 (0.013)0.76 (0.014)-0.029 (0.012)-0.061, 0.00300.0670.59, 0.81(0.68 - 0.76)(0.75 - 0.78)(-0.022 - -0.020)(0.57 - 0.84)Isoleucine0.90 (0.010)0.94 (0.012)-0.040 (0.014)-0.075, -0.00420.0340.75, 0.96(0.90 - 0.91)(0.92 - 0.97)(-0.074 - -0.012)(0.72 - 1.03)Monsanto Company 12-CT-244U 360 of 620Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.51 (0.022)1.58 (0.024)-0.068 (0.021)-0.12, -0.0130.0241.25, 1.62(1.49 - 1.54)(1.52 - 1.65)(-0.14 - -0.030)(1.20 - 1.72)Lysine1.24 (0.018)1.23 (0.020)0.0073 (0.024)-0.055, 0.0690.7751.01, 1.30(1.21 - 1.28)(1.22 - 1.25)(0.0016 - 0.032)(0.99 - 1.44)Methionine0.40 (0.017)0.42 (0.019)-0.025 (0.024)-0.086, 0.0360.3370.32, 0.38(0.36 - 0.43)(0.37 - 0.46)(-0.057 - -0.011)(0.29 - 0.49)Phenylalanine1.40 (0.030)1.49 (0.033)-0.088 (0.032)-0.17, -0.00640.0391.12, 1.58(1.37 - 1.43)(1.41 - 1.61)(-0.18 - -0.033)(1.10 - 1.63)Proline0.98 (0.020)1.05 (0.022)-0.065 (0.022)-0.12, -0.00750.0330.83, 1.08(0.97 - 0.99)(1.03 - 1.09)(-0.097 - -0.032)(0.79 - 1.17)Serine1.03 (0.031)1.12 (0.035)-0.090 (0.039)-0.19, 0.0100.0690.83, 1.21(0.96 - 1.11)(1.08 - 1.20)(-0.18 - 0.011)(0.81 - 1.24)Monsanto Company 12-CT-244U 361 of 620Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.85 (0.015)0.90 (0.017)-0.046 (0.018)-0.092, -0.000060.0490.72, 0.89(0.83 - 0.88)(0.87 - 0.95)(-0.10 - -0.0034)(0.67 - 0.96)Tryptophan0.42 (0.011)0.42 (0.013)0.0013 (0.017)-0.043, 0.0460.9420.34, 0.42(0.39 - 0.45)(0.39 - 0.44)(-0.044 - 0.041)(0.31 - 0.46)Tyrosine0.80 (0.011)0.84 (0.013)-0.036 (0.013)-0.069, -0.00300.0370.67, 0.84(0.79 - 0.82)(0.81 - 0.89)(-0.068 - -0.015)(0.63 - 0.91)Valine1.21 (0.018)1.26 (0.019)-0.053 (0.015)-0.091, -0.0140.0171.00, 1.28(1.19 - 1.23)(1.23 - 1.32)(-0.090 - -0.022)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.78 (0.0068)0.77 (0.0079)0.0029 (0.010)-0.024, 0.0300.7930.16, 1.37(0.76 - 0.79)(0.76 - 0.78)(-0.026 - 0.018)(0.45 - 1.04)16:0 Palmitic24.40 (0.14)24.12 (0.16)0.28 (0.20)-0.24, 0.800.22716.54, 30.55(24.27 - 24.66)(23.78 - 24.45)(-0.11 - 0.56)(19.11 - 26.73)Monsanto Company 12-CT-244U 362 of 620Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.52 (0.0058)0.51 (0.0067)0.011 (0.0089)-0.012, 0.0340.2680.39, 0.70(0.51 - 0.54)(0.50 - 0.52)(-0.012 - 0.025)(0.44 - 0.67)18:0 Stearic2.54 (0.032)2.43 (0.037)0.11 (0.046)-0.0057, 0.230.0581.98, 2.95(2.45 - 2.67)(2.37 - 2.46)(0.066 - 0.21)(1.98 - 2.97)18:1 Oleic14.64 (0.098)14.39 (0.11)0.25 (0.13)-0.078, 0.580.10711.38, 20.64(14.39 - 14.84)(14.06 - 14.61)(-0.0098 - 0.34)(13.71 - 18.39)18:2 Linoleic55.81 (0.24)56.59 (0.28)-0.78 (0.35)-1.67, 0.120.07547.49, 63.18(55.04 - 56.24)(56.02 - 57.32)(-1.39 - 0.12)(49.78 - 59.61)18:3 Linolenic0.15 (0.0066)0.15 (0.0076)0.0076 (0.010)-0.018, 0.0330.4810.060, 0.24(0.15 - 0.16)(0.14 - 0.15)(0.00091 - 0.013)(0.10 - 0.29)20:0 Arachidic0.29 (0.0098)0.28 (0.011)0.013 (0.013)-0.021, 0.0470.3760.17, 0.38(0.26 - 0.31)(0.26 - 0.29)(0.0016 - 0.015)(0.20 - 0.36)Monsanto Company 12-CT-244U 363 of 620Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.14 (0.0026)0.14 (0.0030)-0.00014 (0.0037)-0.0097, 0.00950.9710.070, 0.21(0.14 - 0.15)(0.14 - 0.15)(-0.0017 - 0.0028)(0.051 - 0.19)MineralCalcium (% dw)0.13 (0.0015)0.11 (0.0018)0.019 (0.0023)0.013, 0.025<0.0010.058, 0.21(0.13 - 0.13)(0.11 - 0.11)(0.014 - 0.024)(0.081 - 0.18)Copper (mg/kg dw)8.51 (0.26)8.21 (0.30)0.30 (0.39)-0.70, 1.310.4732.97, 12.86(8.02 - 9.13)(7.48 - 8.64)(-0.49 - 1.13)(4.46 - 11.62)Iron (mg/kg dw)75.42 (3.80)78.00 (4.39)-2.58 (5.81)-17.51, 12.350.67547.30, 97.12(70.35 - 79.72)(75.01 - 80.40)(-8.31 - 4.71)(39.49 - 114.34)Magnesium (% dw)0.41 (0.0052)0.38 (0.0054)0.026 (0.0034)0.018, 0.035<0.0010.28, 0.47(0.40 - 0.41)(0.37 - 0.39)(0.021 - 0.031)(0.31 - 0.46)Manganese (mg/kg dw)13.41 (0.31)11.51 (0.34)1.90 (0.37)0.95, 2.850.0039.07, 17.33(12.79 - 14.14)(10.81 - 11.75)(1.18 - 2.39)(9.07 - 17.14)Monsanto Company 12-CT-244U 364 of 620Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.78 (0.011)0.76 (0.012)0.018 (0.0082)-0.0028, 0.0400.0760.49, 0.87(0.75 - 0.81)(0.75 - 0.79)(0.0052 - 0.029)(0.48 - 0.87)Potassium (% dw)1.21 (0.012)1.12 (0.013)0.090 (0.010)0.064, 0.12<0.0010.92, 1.21(1.17 - 1.24)(1.10 - 1.13)(0.075 - 0.11)(0.90 - 1.26)Sodium (% dw)0.022 (0.0045)0.017 (0.0052)0.0049 (0.0069)-0.013, 0.0230.5150, 0.066(0.019 - 0.027)(0.013 - 0.022)(-0.0020 - 0.014)(0.0054 - 0.077)Zinc (mg/kg dw)39.10 (0.67)39.55 (0.75)-0.45 (0.83)-2.58, 1.680.61027.27, 44.95(37.41 - 40.18)(38.49 - 40.84)(-1.35 - 0.51)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E151.03 (2.27)140.12 (2.63)10.90 (3.47)1.97, 19.830.02541.91, 205.89(148.34 - 154.95)(133.64 - 145.15)(3.19 - 18.52)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 365 of 620Table E-6. Statistical Summary of SiteGACH Cottonseed Anti-nutrients for MON 88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.0067)0.12 (0.0077)0.033 (0.010)0.0069, 0.0590.0220.078, 0.25(0.14 - 0.16)(0.11 - 0.13)(0.021 - 0.050)(0.038 - 0.23)Malvalic Acid0.37 (0.027)0.32 (0.031)0.044 (0.038)-0.055, 0.140.3040.23, 0.54(0.26 - 0.45)(0.31 - 0.34)(-0.052 - 0.12)(0.11 - 0.59)Sterculic Acid0.21 (0.011)0.18 (0.013)0.030 (0.015)-0.0072, 0.0680.0920.17, 0.27(0.18 - 0.25)(0.18 - 0.20)(-0.0018 - 0.050)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.85 (0.017)0.86 (0.019)-0.016 (0.020)-0.068, 0.0370.4740.099, 1.57(0.83 - 0.88)(0.85 - 0.90)(-0.055 - 0.028)(0.50 - 1.41)Total Gossypol0.97 (0.019)0.96 (0.020)0.019 (0.017)-0.026, 0.0630.3240.064, 1.76(0.93 - 1.01)(0.90 - 0.99)(-0.012 - 0.033)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 366 of 620Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.53 (0.063)4.29 (0.071)0.24 (0.078)0.040, 0.440.0273.42, 4.65(4.25 - 4.66)(4.21 - 4.33)(0.042 - 0.34)(3.18 - 4.68)Calories (Kcal/100g)496.63 (2.75)495.83 (3.16)0.80 (3.87)-9.15, 10.750.844457.61, 527.56(492.91 - 499.03)(492.30 - 504.10)(-8.23 - 6.50)(466.09 - 509.91)Carbohydrates44.10 (0.71)44.23 (0.82)-0.14 (1.09)-2.94, 2.670.90540.26, 56.45(42.20 - 44.98)(42.53 - 45.14)(-0.77 - 2.45)(43.28 - 54.90)Moisture (% fw)7.02 (0.17)7.36 (0.19)-0.34 (0.20)-0.86, 0.180.1534.79, 9.92(6.63 - 7.36)(7.17 - 7.62)(-0.73 - 0.19)(6.05 - 10.50)Protein28.42 (0.62)28.82 (0.72)-0.40 (0.93)-2.80, 1.990.68122.30, 29.41(26.95 - 30.82)(28.58 - 29.04)(-1.63 - -0.87)(20.58 - 29.28)Total Fat22.96 (0.55)22.62 (0.64)0.34 (0.81)-1.75, 2.430.69215.01, 28.51(22.34 - 23.50)(21.87 - 24.18)(-1.58 - 1.56)(16.58 - 25.25)Monsanto Company 12-CT-244U 367 of 620Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber24.04 (0.55)24.01 (0.61)0.027 (0.65)-1.64, 1.700.96822.24, 31.96(23.86 - 24.16)(22.08 - 25.22)(-1.08 - 1.77)(23.42 - 31.62)Crude Fiber16.43 (0.24)17.67 (0.28)-1.24 (0.37)-2.19, -0.290.01916.93, 22.68(16.06 - 17.24)(17.49 - 17.88)(-1.69 - -0.24)(16.92 - 23.32)Neutral Detergent Fiber28.04 (0.88)30.20 (1.01)-2.15 (1.21)-5.27, 0.970.13627.03, 42.49(25.13 - 30.18)(28.87 - 32.60)(-2.93 - 0.63)(29.27 - 40.63)Total Dietary Fiber38.32 (0.42)40.14 (0.49)-1.83 (0.63)-3.46, -0.190.03434.52, 52.58(37.62 - 38.75)(39.32 - 41.35)(-3.74 - -0.57)(37.29 - 48.60)Amino Acid (% dw)Alanine1.04 (0.011)1.06 (0.012)-0.022 (0.014)-0.058, 0.0140.1750.86, 1.11(1.02 - 1.05)(1.02 - 1.10)(-0.048 - -0.00010)(0.83 - 1.22)Arginine3.02 (0.053)3.28 (0.060)-0.26 (0.069)-0.43, -0.0820.0132.38, 3.47(2.95 - 3.10)(3.10 - 3.43)(-0.34 - -0.14)(2.30 - 3.55)Monsanto Company 12-CT-244U 368 of 620Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.38 (0.037)2.50 (0.043)-0.11 (0.053)-0.25, 0.0220.0831.94, 2.57(2.32 - 2.46)(2.39 - 2.64)(-0.26 - -0.073)(1.79 - 2.72)Cystine0.43 (0.010)0.42 (0.012)0.0076 (0.016)-0.033, 0.0480.6510.31, 0.45(0.41 - 0.44)(0.41 - 0.43)(-0.017 - 0.015)(0.29 - 0.47)Glutamic Acid4.82 (0.12)5.07 (0.14)-0.25 (0.18)-0.71, 0.210.2193.74, 5.28(4.58 - 5.22)(4.86 - 5.43)(-0.78 - -0.017)(3.39 - 5.45)Glycine1.08 (0.018)1.11 (0.021)-0.029 (0.027)-0.099, 0.0400.3300.90, 1.14(1.06 - 1.12)(1.07 - 1.18)(-0.11 - -0.0024)(0.85 - 1.23)Histidine0.75 (0.013)0.77 (0.014)-0.024 (0.011)-0.052, 0.00350.0740.59, 0.81(0.73 - 0.77)(0.74 - 0.80)(-0.045 - 0.0022)(0.57 - 0.84)Isoleucine0.92 (0.014)0.95 (0.015)-0.027 (0.014)-0.063, 0.00910.1120.75, 0.96(0.91 - 0.95)(0.91 - 0.98)(-0.062 - 0.0079)(0.72 - 1.03)Monsanto Company 12-CT-244U 369 of 620Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.54 (0.020)1.58 (0.022)-0.043 (0.021)-0.097, 0.0110.0981.25, 1.62(1.51 - 1.57)(1.52 - 1.65)(-0.083 - -0.0065)(1.20 - 1.72)Lysine1.22 (0.017)1.25 (0.019)-0.033 (0.021)-0.087, 0.0210.1781.01, 1.30(1.21 - 1.23)(1.19 - 1.30)(-0.073 - 0.027)(0.99 - 1.44)Methionine0.39 (0.015)0.39 (0.017)0.0021 (0.019)-0.046, 0.0500.9150.32, 0.38(0.37 - 0.42)(0.34 - 0.44)(-0.017 - 0.027)(0.29 - 0.49)Phenylalanine1.44 (0.022)1.53 (0.025)-0.090 (0.029)-0.16, -0.0160.0251.12, 1.58(1.40 - 1.46)(1.45 - 1.58)(-0.13 - -0.049)(1.10 - 1.63)Proline1.01 (0.018)1.07 (0.020)-0.060 (0.027)-0.13, 0.00900.0750.83, 1.08(0.98 - 1.03)(1.03 - 1.12)(-0.12 - -0.042)(0.79 - 1.17)Serine1.08 (0.029)1.11 (0.034)-0.031 (0.045)-0.15, 0.0830.5130.83, 1.21(1.03 - 1.18)(1.06 - 1.20)(-0.17 - 0.0023)(0.81 - 1.24)Monsanto Company 12-CT-244U 370 of 620Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.86 (0.013)0.88 (0.015)-0.022 (0.019)-0.071, 0.0280.3170.72, 0.89(0.84 - 0.88)(0.83 - 0.92)(-0.053 - 0.0050)(0.67 - 0.96)Tryptophan0.42 (0.0061)0.42 (0.0070)-0.0028 (0.0090)-0.026, 0.0200.7710.34, 0.42(0.42 - 0.43)(0.42 - 0.43)(-0.016 - 0.011)(0.31 - 0.46)Tyrosine0.80 (0.014)0.84 (0.016)-0.039 (0.019)-0.087, 0.00960.0940.67, 0.84(0.78 - 0.83)(0.79 - 0.87)(-0.074 - -0.015)(0.63 - 0.91)Valine1.21 (0.017)1.26 (0.019)-0.048 (0.023)-0.11, 0.0100.0871.00, 1.28(1.19 - 1.23)(1.21 - 1.30)(-0.085 - -0.027)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.68 (0.0087)0.72 (0.0096)-0.038 (0.0090)-0.061, -0.0150.0070.16, 1.37(0.66 - 0.71)(0.71 - 0.73)(-0.049 - -0.016)(0.45 - 1.04)16:0 Palmitic22.61 (0.089)22.73 (0.10)-0.12 (0.13)-0.46, 0.210.39416.54, 30.55(22.34 - 22.84)(22.69 - 22.78)(-0.11 - 0.093)(19.11 - 26.73)Monsanto Company 12-CT-244U 371 of 620Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.50 (0.0057)0.49 (0.0062)0.0067 (0.0059)-0.0086, 0.0220.3120.39, 0.70(0.48 - 0.52)(0.49 - 0.50)(-0.0046 - 0.021)(0.44 - 0.67)18:0 Stearic2.31 (0.031)2.20 (0.036)0.11 (0.047)-0.016, 0.230.0751.98, 2.95(2.29 - 2.32)(2.15 - 2.28)(0.039 - 0.17)(1.98 - 2.97)18:1 Oleic14.69 (0.11)14.83 (0.13)-0.14 (0.17)-0.59, 0.310.45411.38, 20.64(14.51 - 14.88)(14.74 - 14.99)(-0.48 - 0.14)(13.71 - 18.39)18:2 Linoleic57.84 (0.19)57.78 (0.22)0.059 (0.29)-0.67, 0.790.84347.49, 63.18(57.49 - 58.22)(57.65 - 57.93)(-0.44 - 0.19)(49.78 - 59.61)18:3 Linolenic0.18 (0.0027)0.17 (0.0030)0.0066 (0.0033)-0.0018, 0.0150.1000.060, 0.24(0.18 - 0.19)(0.17 - 0.18)(0.0051 - 0.010)(0.10 - 0.29)20:0 Arachidic0.23 (0.0029)0.24 (0.0034)-0.0021 (0.0045)-0.014, 0.00940.6510.17, 0.38(0.23 - 0.24)(0.23 - 0.24)(-0.0063 - 0.0087)(0.20 - 0.36)Monsanto Company 12-CT-244U 372 of 620Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.13 (0.0030)0.13 (0.0035)-0.0051 (0.0046)-0.017, 0.00670.3180.070, 0.21(0.12 - 0.14)(0.13 - 0.14)(-0.0084 - 0.0036)(0.051 - 0.19)MineralCalcium (% dw)0.20 (0.0060)0.18 (0.0065)0.026 (0.0061)0.010, 0.0420.0070.058, 0.21(0.19 - 0.22)(0.17 - 0.19)(0.013 - 0.038)(0.081 - 0.18)Copper (mg/kg dw)10.08 (0.54)11.01 (0.61)-0.93 (0.71)-2.75, 0.890.2462.97, 12.86(8.74 - 11.06)(10.09 - 11.33)(-2.59 - 0.69)(4.46 - 11.62)Iron (mg/kg dw)78.87 (5.50)74.39 (6.35)4.48 (8.40)-17.11, 26.070.61647.30, 97.12(74.42 - 82.99)(72.65 - 76.27)(3.93 - 8.73)(39.49 - 114.34)Magnesium (% dw)0.43 (0.0033)0.40 (0.0038)0.027 (0.0046)0.015, 0.0390.0020.28, 0.47(0.41 - 0.43)(0.39 - 0.40)(0.015 - 0.041)(0.31 - 0.46)Manganese (mg/kg dw)13.34 (0.29)12.56 (0.33)0.78 (0.44)-0.35, 1.900.1359.07, 17.33(12.74 - 13.60)(11.96 - 13.28)(-0.53 - 1.64)(9.07 - 17.14)Monsanto Company 12-CT-244U 373 of 620Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional Control (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.79 (0.0090)0.78 (0.010)0.0090 (0.011)-0.019, 0.0370.4400.49, 0.87(0.75 - 0.82)(0.77 - 0.79)(-0.024 - 0.029)(0.48 - 0.87)Potassium (% dw)1.09 (0.010)1.08 (0.012)0.018 (0.015)-0.020, 0.0560.2810.92, 1.21(1.08 - 1.11)(1.05 - 1.10)(-0.0048 - 0.063)(0.90 - 1.26)Sodium (% dw)0.022 (0.0028)0.0080 (0.0032)0.014 (0.0042)0.0033, 0.0250.0200, 0.066(0.019 - 0.025)(0.0054 - 0.013)(0.0098 - 0.016)(0.0054 - 0.077)Zinc (mg/kg dw)40.79 (1.18)42.00 (1.37)-1.21 (1.81)-5.85, 3.440.53327.27, 44.95(37.59 - 43.87)(40.59 - 43.50)(-5.91 - 3.27)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E93.11 (2.67)92.34 (2.91)0.76 (2.71)-6.20, 7.720.78941.91, 205.89(86.23 - 100.03)(91.78 - 95.85)(-6.54 - 4.18)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 374 of 620Table E-8. Statistical Summary of SiteKSLA Cottonseed Anti-nutrients for MON 88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.010)0.12 (0.012)0.026 (0.014)-0.011, 0.0630.1290.078, 0.25(0.11 - 0.18)(0.12 - 0.13)(-0.0087 - 0.054)(0.038 - 0.23)Malvalic Acid0.45 (0.030)0.37 (0.034)0.083 (0.039)-0.018, 0.180.0880.23, 0.54(0.34 - 0.55)(0.33 - 0.39)(-0.024 - 0.15)(0.11 - 0.59)Sterculic Acid0.23 (0.014)0.20 (0.016)0.031 (0.019)-0.017, 0.0790.1590.17, 0.27(0.18 - 0.28)(0.19 - 0.21)(-0.023 - 0.078)(0.061 - 0.34)Gossypol (% dw)Free Gossypol1.07 (0.027)0.95 (0.030)0.12 (0.033)0.036, 0.200.0140.099, 1.57(1.03 - 1.10)(0.86 - 1.05)(0.051 - 0.20)(0.50 - 1.41)Total Gossypol1.13 (0.033)1.01 (0.038)0.12 (0.047)0.00016, 0.240.0490.064, 1.76(1.00 - 1.24)(1.00 - 1.02)(0.0061 - 0.23)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 375 of 620Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.35 (0.047)4.12 (0.047)0.23 (0.066)0.066, 0.390.0133.42, 4.65(4.23 - 4.47)(4.06 - 4.15)(0.11 - 0.41)(3.18 - 4.68)Calories (Kcal/100g)494.11 (3.01)494.75 (3.01)-0.64 (4.19)-10.89, 9.610.883457.61, 527.56(482.46 - 501.83)(490.27 - 498.67)(-14.30 - 4.88)(466.09 - 509.91)Carbohydrates45.92 (0.79)47.84 (0.79)-1.91 (1.11)-4.64, 0.810.13640.26, 56.45(44.17 - 48.89)(46.77 - 50.30)(-5.19 - 1.91)(43.28 - 54.90)Moisture (% fw)6.70 (0.26)6.98 (0.26)-0.28 (0.36)-1.17, 0.620.4784.79, 9.92(6.46 - 6.94)(6.15 - 7.40)(-0.90 - 0.79)(6.05 - 10.50)Protein27.44 (0.56)25.80 (0.56)1.64 (0.80)-0.31, 3.590.08422.30, 29.41(27.06 - 27.92)(23.53 - 27.85)(0.074 - 3.73)(20.58 - 29.28)Total Fat22.27 (0.64)22.25 (0.64)0.018 (0.90)-2.18, 2.220.98415.01, 28.51(19.79 - 23.86)(21.29 - 23.02)(-2.89 - 1.18)(16.58 - 25.25)Monsanto Company 12-CT-244U 376 of 620Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.72 (0.45)28.35 (0.45)-2.63 (0.63)-4.18, -1.080.00522.24, 31.96(24.16 - 27.08)(27.81 - 29.58)(-5.42 - -0.73)(23.42 - 31.62)Crude Fiber18.73 (0.66)19.62 (0.66)-0.89 (0.93)-3.15, 1.380.37616.93, 22.68(17.75 - 19.77)(18.46 - 20.54)(-1.79 - 0.17)(16.92 - 23.32)Neutral Detergent Fiber33.12 (0.65)34.05 (0.65)-0.93 (0.42)-1.97, 0.100.07027.03, 42.49(32.24 - 34.42)(32.61 - 35.84)(-1.65 - 0.23)(29.27 - 40.63)Total Dietary Fiber39.82 (0.49)43.35 (0.49)-3.53 (0.69)-5.21, -1.850.00234.52, 52.58(39.02 - 40.86)(42.33 - 44.37)(-5.34 - -1.47)(37.29 - 48.60)Amino Acid (% dw)Alanine1.07 (0.018)1.03 (0.018)0.038 (0.014)0.0050, 0.0720.0300.86, 1.11(1.00 - 1.11)(1.00 - 1.06)(0.00025 - 0.082)(0.83 - 1.22)Arginine2.96 (0.073)2.98 (0.073)-0.017 (0.084)-0.22, 0.190.8492.38, 3.47(2.64 - 3.13)(2.89 - 3.13)(-0.25 - 0.15)(2.30 - 3.55)Monsanto Company 12-CT-244U 377 of 620Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.37 (0.050)2.30 (0.050)0.074 (0.062)-0.077, 0.220.2781.94, 2.57(2.17 - 2.49)(2.25 - 2.40)(-0.12 - 0.22)(1.79 - 2.72)Cystine0.41 (0.016)0.38 (0.016)0.026 (0.019)-0.021, 0.0740.2220.31, 0.45(0.38 - 0.46)(0.36 - 0.44)(-0.024 - 0.082)(0.29 - 0.47)Glutamic Acid4.78 (0.13)4.52 (0.13)0.27 (0.16)-0.13, 0.670.1513.74, 5.28(4.32 - 5.26)(4.37 - 4.75)(-0.15 - 0.79)(3.39 - 5.45)Glycine1.11 (0.020)1.06 (0.020)0.057 (0.025)-0.0034, 0.120.0600.90, 1.14(1.02 - 1.18)(1.04 - 1.09)(-0.024 - 0.14)(0.85 - 1.23)Histidine0.74 (0.020)0.72 (0.020)0.019 (0.014)-0.014, 0.0520.2060.59, 0.81(0.68 - 0.78)(0.67 - 0.76)(0.013 - 0.029)(0.57 - 0.84)Isoleucine0.90 (0.018)0.88 (0.018)0.020 (0.020)-0.028, 0.0680.3550.75, 0.96(0.84 - 0.97)(0.87 - 0.90)(-0.028 - 0.096)(0.72 - 1.03)Monsanto Company 12-CT-244U 378 of 620Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.51 (0.026)1.48 (0.026)0.039 (0.028)-0.030, 0.110.2171.25, 1.62(1.40 - 1.58)(1.44 - 1.52)(-0.035 - 0.12)(1.20 - 1.72)Lysine1.26 (0.024)1.18 (0.024)0.083 (0.027)0.016, 0.150.0231.01, 1.30(1.17 - 1.31)(1.12 - 1.23)(0.021 - 0.15)(0.99 - 1.44)Methionine0.42 (0.017)0.38 (0.017)0.045 (0.013)0.013, 0.0770.0130.32, 0.38(0.37 - 0.44)(0.32 - 0.42)(0.020 - 0.075)(0.29 - 0.49)Phenylalanine1.41 (0.027)1.39 (0.027)0.015 (0.033)-0.066, 0.0950.6681.12, 1.58(1.28 - 1.47)(1.36 - 1.43)(-0.077 - 0.094)(1.10 - 1.63)Proline1.00 (0.015)0.98 (0.015)0.017 (0.022)-0.036, 0.0710.4590.83, 1.08(0.94 - 1.04)(0.95 - 1.02)(-0.039 - 0.093)(0.79 - 1.17)Serine1.07 (0.022)1.03 (0.022)0.038 (0.030)-0.036, 0.110.2570.83, 1.21(0.99 - 1.15)(1.01 - 1.06)(-0.047 - 0.12)(0.81 - 1.24)Monsanto Company 12-CT-244U 379 of 620Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.87 (0.016)0.84 (0.016)0.027 (0.012)-0.0036, 0.0570.0740.72, 0.89(0.80 - 0.90)(0.82 - 0.86)(-0.021 - 0.060)(0.67 - 0.96)Tryptophan0.41 (0.014)0.39 (0.014)0.019 (0.018)-0.026, 0.0630.3470.34, 0.42(0.36 - 0.46)(0.38 - 0.43)(-0.024 - 0.078)(0.31 - 0.46)Tyrosine0.80 (0.018)0.79 (0.018)0.010 (0.021)-0.042, 0.0630.6430.67, 0.84(0.72 - 0.84)(0.76 - 0.81)(-0.045 - 0.058)(0.63 - 0.91)Valine1.21 (0.024)1.18 (0.024)0.026 (0.029)-0.044, 0.0960.3971.00, 1.28(1.11 - 1.29)(1.17 - 1.19)(-0.054 - 0.12)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.74 (0.013)0.75 (0.013)-0.012 (0.018)-0.057, 0.0320.5230.16, 1.37(0.71 - 0.76)(0.73 - 0.78)(-0.032 - 0.0064)(0.45 - 1.04)16:0 Palmitic24.48 (0.091)24.04 (0.091)0.44 (0.13)0.12, 0.750.01416.54, 30.55(24.37 - 24.55)(23.92 - 24.16)(0.21 - 0.61)(19.11 - 26.73)Monsanto Company 12-CT-244U 380 of 620Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.51 (0.0035)0.50 (0.0035)0.0090 (0.0049)-0.0030, 0.0210.1160.39, 0.70(0.50 - 0.52)(0.49 - 0.51)(-0.0051 - 0.020)(0.44 - 0.67)18:0 Stearic2.68 (0.018)2.52 (0.018)0.15 (0.026)0.089, 0.220.0011.98, 2.95(2.64 - 2.73)(2.49 - 2.57)(0.11 - 0.24)(1.98 - 2.97)18:1 Oleic14.70 (0.095)14.29 (0.095)0.41 (0.13)0.084, 0.740.02111.38, 20.64(14.48 - 15.01)(14.13 - 14.53)(0.16 - 0.72)(13.71 - 18.39)18:2 Linoleic55.53 (0.14)56.63 (0.14)-1.09 (0.20)-1.59, -0.600.00147.49, 63.18(55.15 - 55.99)(56.52 - 56.72)(-1.42 - -0.63)(49.78 - 59.61)18:3 Linolenic0.15 (0.0042)0.15 (0.0042)0.0063 (0.0059)-0.0082, 0.0210.3270.060, 0.24(0.14 - 0.17)(0.13 - 0.15)(-0.0073 - 0.019)(0.10 - 0.29)20:0 Arachidic0.31 (0.0054)0.29 (0.0054)0.020 (0.0071)0.0022, 0.0370.0330.17, 0.38(0.31 - 0.32)(0.29 - 0.30)(0.016 - 0.023)(0.20 - 0.36)Monsanto Company 12-CT-244U 381 of 620Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.14 (0.0039)0.14 (0.0039)0.00029 (0.0055)-0.013, 0.0140.9590.070, 0.21(0.14 - 0.15)(0.14 - 0.15)(-0.0046 - 0.0027)(0.051 - 0.19)MineralCalcium (% dw)0.12 (0.0039)0.12 (0.0039)0.0047 (0.0050)-0.0075, 0.0170.3810.058, 0.21(0.11 - 0.13)(0.12 - 0.12)(-0.012 - 0.015)(0.081 - 0.18)Copper (mg/kg dw)8.48 (0.30)8.70 (0.30)-0.22 (0.42)-1.26, 0.820.6212.97, 12.86(7.86 - 9.49)(8.11 - 9.14)(-1.27 - 0.99)(4.46 - 11.62)Iron (mg/kg dw)76.74 (4.19)68.59 (4.19)8.15 (4.72)-3.40, 19.690.13447.30, 97.12(73.99 - 83.17)(66.28 - 70.38)(3.68 - 12.79)(39.49 - 114.34)Magnesium (% dw)0.41 (0.0065)0.39 (0.0065)0.021 (0.0093)-0.0018, 0.0430.0650.28, 0.47(0.39 - 0.44)(0.38 - 0.41)(-0.014 - 0.054)(0.31 - 0.46)Manganese (mg/kg dw)13.13 (0.37)12.87 (0.37)0.26 (0.52)-1.03, 1.540.6429.07, 17.33(11.92 - 13.79)(12.31 - 13.87)(-1.95 - 1.16)(9.07 - 17.14)Monsanto Company 12-CT-244U 382 of 620Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.75 (0.014)0.71 (0.014)0.036 (0.019)-0.012, 0.0830.1130.49, 0.87(0.71 - 0.80)(0.69 - 0.73)(-0.023 - 0.11)(0.48 - 0.87)Potassium (% dw)1.18 (0.022)1.17 (0.022)0.0099 (0.029)-0.061, 0.0810.7420.92, 1.21(1.16 - 1.22)(1.12 - 1.27)(-0.087 - 0.064)(0.90 - 1.26)Sodium (% dw)0.023 (0.0043)0.015 (0.0043)0.0078 (0.0060)-0.0069, 0.0220.2420, 0.066(0.021 - 0.024)(0.0053 - 0.027)(-0.0031 - 0.017)(0.0054 - 0.077)Zinc (mg/kg dw)33.97 (0.93)35.74 (0.93)-1.77 (1.32)-4.99, 1.450.22727.27, 44.95(31.68 - 37.84)(35.10 - 37.09)(-5.41 - 2.55)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E169.88 (2.48)149.96 (2.48)19.92 (3.48)11.40, 28.430.00141.91, 205.89(163.34 - 175.33)(148.96 - 152.67)(14.16 - 26.36)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 383 of 620Table E-10. Statistical Summary of SiteLACH Cottonseed Anti-nutrients for MON 88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.011)0.13 (0.011)0.019 (0.016)-0.019, 0.0570.2710.078, 0.25(0.13 - 0.19)(0.12 - 0.14)(-0.0082 - 0.068)(0.038 - 0.23)Malvalic Acid0.39 (0.035)0.35 (0.035)0.032 (0.049)-0.088, 0.150.5350.23, 0.54(0.33 - 0.53)(0.31 - 0.38)(-0.047 - 0.16)(0.11 - 0.59)Sterculic Acid0.22 (0.016)0.20 (0.016)0.020 (0.023)-0.037, 0.0760.4320.17, 0.27(0.19 - 0.29)(0.17 - 0.21)(-0.011 - 0.076)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.86 (0.026)0.81 (0.026)0.056 (0.036)-0.033, 0.140.1750.099, 1.57(0.81 - 0.92)(0.78 - 0.84)(0.0028 - 0.13)(0.50 - 1.41)Total Gossypol0.93 (0.025)0.90 (0.025)0.033 (0.036)-0.055, 0.120.3950.064, 1.76(0.90 - 1.00)(0.82 - 0.94)(-0.015 - 0.086)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 384 of 620Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.34 (0.057)4.14 (0.064)0.20 (0.070)0.024, 0.390.0333.42, 4.65(4.29 - 4.40)(3.94 - 4.26)(0.13 - 0.35)(3.18 - 4.68)Calories (Kcal/100g)497.98 (1.61)491.80 (1.86)6.18 (2.46)-0.15, 12.510.053457.61, 527.56(491.46 - 501.77)(488.93 - 494.48)(-3.01 - 10.35)(466.09 - 509.91)Carbohydrates44.40 (0.59)44.36 (0.68)0.044 (0.87)-2.20, 2.290.96140.26, 56.45(43.84 - 45.31)(43.65 - 45.15)(-1.31 - 0.79)(43.28 - 54.90)Moisture (% fw)9.18 (0.22)8.96 (0.23)0.22 (0.19)-0.26, 0.700.2874.79, 9.92(8.64 - 9.67)(8.59 - 9.19)(-0.050 - 0.48)(6.05 - 10.50)Protein28.24 (0.61)29.84 (0.70)-1.60 (0.85)-3.78, 0.590.11922.30, 29.41(26.53 - 29.33)(29.62 - 30.42)(-1.99 - -0.53)(20.58 - 29.28)Total Fat23.04 (0.30)21.59 (0.34)1.45 (0.46)0.28, 2.630.02415.01, 28.51(21.89 - 23.76)(21.03 - 22.21)(-0.32 - 2.22)(16.58 - 25.25)Monsanto Company 12-CT-244U 385 of 620Table E-11. Statistical Summary of Site NCBD CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.91 (1.12)26.31 (1.29)-0.40 (1.71)-4.80, 3.990.82222.24, 31.96(24.26 - 27.74)(24.72 - 28.08)(-1.88 - -0.096)(23.42 - 31.62)Crude Fiber17.01 (0.29)16.93 (0.33)0.086 (0.38)-0.90, 1.080.83116.93, 22.68(16.31 - 17.78)(16.30 - 17.90)(-0.12 - 0.31)(16.92 - 23.32)Neutral Detergent Fiber31.08 (1.03)31.14 (1.19)-0.057 (1.58)-4.11, 4.000.97227.03, 42.49(29.23 - 32.66)(30.85 - 31.49)(-1.62 - 1.16)(29.27 - 40.63)Total Dietary Fiber38.51 (0.55)39.52 (0.64)-1.01 (0.85)-3.19, 1.160.28534.52, 52.58(36.91 - 39.40)(39.05 - 39.86)(-0.90 - -0.088)(37.29 - 48.60)Amino Acid (% dw)Alanine1.11 (0.020)1.10 (0.023)0.011 (0.031)-0.068, 0.0910.7270.86, 1.11(1.07 - 1.14)(1.05 - 1.14)(-0.0051 - 0.026)(0.83 - 1.22)Arginine3.08 (0.052)3.20 (0.060)-0.12 (0.080)-0.33, 0.0800.1782.38, 3.47(2.97 - 3.20)(3.11 - 3.27)(-0.19 - -0.031)(2.30 - 3.55)Monsanto Company 12-CT-244U 386 of 620Table E-11. Statistical Summary of Site NCBD CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.50 (0.053)2.55 (0.061)-0.045 (0.081)-0.25, 0.160.6041.94, 2.57(2.43 - 2.58)(2.46 - 2.63)(-0.042 - 0.0096)(1.79 - 2.72)Cystine0.43 (0.018)0.42 (0.021)0.016 (0.028)-0.056, 0.0880.5940.31, 0.45(0.40 - 0.46)(0.39 - 0.46)(-0.051 - 0.055)(0.29 - 0.47)Glutamic Acid4.71 (0.13)5.08 (0.15)-0.37 (0.20)-0.88, 0.140.1203.74, 5.28(4.64 - 4.79)(4.85 - 5.40)(-0.66 - -0.19)(3.39 - 5.45)Glycine1.11 (0.019)1.11 (0.022)0.0013 (0.029)-0.072, 0.0750.9640.90, 1.14(1.06 - 1.16)(1.08 - 1.14)(-0.015 - 0.023)(0.85 - 1.23)Histidine0.76 (0.013)0.76 (0.015)0 (0.019)-0.049, 0.0490.9990.59, 0.81(0.73 - 0.79)(0.74 - 0.79)(-0.032 - 0.052)(0.57 - 0.84)Isoleucine0.96 (0.018)0.96 (0.020)-0.00058 (0.020)-0.053, 0.0510.9780.75, 0.96(0.90 - 1.00)(0.93 - 0.97)(-0.030 - 0.040)(0.72 - 1.03)Monsanto Company 12-CT-244U 387 of 620Table E-11. Statistical Summary of Site NCBD CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.60 (0.023)1.60 (0.027)-0.0061 (0.035)-0.097, 0.0850.8691.25, 1.62(1.55 - 1.65)(1.55 - 1.63)(-0.025 - 0.023)(1.20 - 1.72)Lysine1.25 (0.029)1.26 (0.033)-0.010 (0.043)-0.12, 0.100.8191.01, 1.30(1.17 - 1.36)(1.22 - 1.29)(-0.060 - 0.077)(0.99 - 1.44)Methionine0.40 (0.0093)0.41 (0.011)-0.011 (0.014)-0.048, 0.0260.4770.32, 0.38(0.38 - 0.42)(0.40 - 0.42)(-0.046 - 0.016)(0.29 - 0.49)Phenylalanine1.48 (0.027)1.52 (0.031)-0.043 (0.041)-0.15, 0.0630.3421.12, 1.58(1.44 - 1.54)(1.45 - 1.56)(-0.058 - -0.012)(1.10 - 1.63)Proline1.03 (0.022)1.09 (0.025)-0.065 (0.029)-0.14, 0.00980.0750.83, 1.08(0.98 - 1.11)(1.07 - 1.12)(-0.094 - -0.010)(0.79 - 1.17)Serine1.11 (0.027)1.13 (0.032)-0.017 (0.042)-0.12, 0.0900.6960.83, 1.21(1.11 - 1.13)(1.09 - 1.19)(-0.087 - 0.028)(0.81 - 1.24)Monsanto Company 12-CT-244U 388 of 620Table E-11. Statistical Summary of Site NCBD CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.90 (0.014)0.89 (0.016)0.0096 (0.021)-0.045, 0.0640.6690.72, 0.89(0.88 - 0.92)(0.86 - 0.92)(-0.0050 - 0.015)(0.67 - 0.96)Tryptophan0.41 (0.021)0.45 (0.024)-0.039 (0.032)-0.12, 0.0450.2850.34, 0.42(0.40 - 0.44)(0.39 - 0.52)(-0.081 - 0.024)(0.31 - 0.46)Tyrosine0.84 (0.012)0.84 (0.014)0.0015 (0.018)-0.045, 0.0470.9370.67, 0.84(0.81 - 0.87)(0.82 - 0.87)(-0.026 - 0.039)(0.63 - 0.91)Valine1.26 (0.025)1.30 (0.027)-0.038 (0.021)-0.091, 0.0160.1301.00, 1.28(1.17 - 1.31)(1.24 - 1.32)(-0.067 - -0.010)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.68 (0.0074)0.75 (0.0086)-0.063 (0.011)-0.091, -0.0340.0020.16, 1.37(0.66 - 0.70)(0.74 - 0.76)(-0.077 - -0.044)(0.45 - 1.04)16:0 Palmitic22.89 (0.12)23.10 (0.14)-0.21 (0.18)-0.68, 0.250.28616.54, 30.55(22.47 - 23.15)(23.07 - 23.15)(-0.68 - 0.079)(19.11 - 26.73)Monsanto Company 12-CT-244U 389 of 620Table E-11. Statistical Summary of Site NCBD CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.46 (0.0047)0.48 (0.0053)-0.018 (0.0055)-0.033, -0.00440.0190.39, 0.70(0.44 - 0.47)(0.47 - 0.49)(-0.025 - -0.0092)(0.44 - 0.67)18:0 Stearic2.50 (0.037)2.34 (0.043)0.16 (0.057)0.015, 0.310.0361.98, 2.95(2.39 - 2.64)(2.32 - 2.38)(0.011 - 0.22)(1.98 - 2.97)18:1 Oleic15.04 (0.12)14.70 (0.14)0.35 (0.19)-0.14, 0.840.12711.38, 20.64(14.58 - 15.26)(14.51 - 14.83)(-0.17 - 0.75)(13.71 - 18.39)18:2 Linoleic56.95 (0.23)57.19 (0.26)-0.24 (0.35)-1.13, 0.660.52847.49, 63.18(56.35 - 57.88)(57.01 - 57.46)(-1.12 - 0.80)(49.78 - 59.61)18:3 Linolenic0.31 (0.012)0.29 (0.014)0.028 (0.018)-0.018, 0.0740.1780.060, 0.24(0.27 - 0.34)(0.27 - 0.30)(-0.0012 - 0.052)(0.10 - 0.29)20:0 Arachidic0.28 (0.0065)0.28 (0.0075)0.0046 (0.0099)-0.021, 0.0300.6630.17, 0.38(0.26 - 0.30)(0.27 - 0.28)(-0.024 - 0.024)(0.20 - 0.36)Monsanto Company 12-CT-244U 390 of 620Table E-11. Statistical Summary of Site NCBD CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.16 (0.0056)0.15 (0.0065)0.011 (0.0086)-0.011, 0.0330.2580.070, 0.21(0.15 - 0.19)(0.15 - 0.16)(-0.0044 - 0.032)(0.051 - 0.19)MineralCalcium (% dw)0.15 (0.0014)0.14 (0.0017)0.0095 (0.0022)0.0039, 0.0150.0070.058, 0.21(0.14 - 0.15)(0.14 - 0.14)(0.0056 - 0.013)(0.081 - 0.18)Copper (mg/kg dw)6.82 (0.36)6.91 (0.41)-0.084 (0.54)-1.48, 1.310.8832.97, 12.86(5.81 - 7.58)(6.64 - 7.19)(-1.38 - 0.53)(4.46 - 11.62)Iron (mg/kg dw)43.21 (1.00)48.04 (1.15)-4.83 (1.53)-8.75, -0.900.02547.30, 97.12(41.96 - 44.44)(45.03 - 50.87)(-6.43 - -2.22)(39.49 - 114.34)Magnesium (% dw)0.41 (0.010)0.40 (0.012)0.011 (0.016)-0.030, 0.0520.5290.28, 0.47(0.40 - 0.43)(0.37 - 0.44)(-0.036 - 0.049)(0.31 - 0.46)Manganese (mg/kg dw)14.12 (0.36)13.83 (0.42)0.29 (0.56)-1.14, 1.720.6229.07, 17.33(13.57 - 14.81)(13.65 - 14.11)(-0.54 - 0.52)(9.07 - 17.14)Monsanto Company 12-CT-244U 391 of 620Table E-11. Statistical Summary of Site NCBD CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.64 (0.020)0.66 (0.023)-0.020 (0.031)-0.099, 0.0590.5480.49, 0.87(0.61 - 0.67)(0.59 - 0.71)(-0.087 - 0.068)(0.48 - 0.87)Potassium (% dw)1.06 (0.019)1.08 (0.022)-0.022 (0.029)-0.097, 0.0520.4710.92, 1.21(1.04 - 1.09)(1.03 - 1.16)(-0.12 - 0.032)(0.90 - 1.26)Sodium (% dw)0.11 (0.0084)0.099 (0.0096)0.0074 (0.011)-0.022, 0.0360.5390, 0.066(0.068 - 0.12)(0.094 - 0.10)(-0.031 - 0.030)(0.0054 - 0.077)Zinc (mg/kg dw)40.79 (1.30)49.54 (1.50)-8.75 (1.98)-13.84, -3.660.00627.27, 44.95(40.28 - 41.37)(44.04 - 52.95)(-11.57 - -3.76)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E169.03 (3.66)156.99 (4.23)12.04 (5.60)-2.34, 26.430.08441.91, 205.89(163.57 - 179.34)(151.55 - 162.98)(8.84 - 16.38)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 392 of 620Table E-12. Statistical Summary of SiteNCBD Cottonseed Anti-nutrients for MON88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.14 (0.0093)0.13 (0.011)0.012 (0.014)-0.024, 0.0480.4220.078, 0.25(0.13 - 0.15)(0.12 - 0.14)(0.0062 - 0.024)(0.038 - 0.23)Malvalic Acid0.36 (0.040)0.37 (0.046)-0.017 (0.056)-0.16, 0.130.7730.23, 0.54(0.20 - 0.43)(0.36 - 0.41)(0.0058 - 0.056)(0.11 - 0.59)Sterculic Acid0.22 (0.024)0.22 (0.028)-0.0024 (0.037)-0.098, 0.0930.9510.17, 0.27(0.13 - 0.27)(0.21 - 0.23)(0.0078 - 0.040)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.94 (0.026)0.90 (0.030)0.038 (0.039)-0.063, 0.140.3740.099, 1.57(0.91 - 0.97)(0.81 - 0.95)(-0.024 - 0.097)(0.50 - 1.41)Total Gossypol1.12 (0.067)1.09 (0.077)0.037 (0.10)-0.22, 0.300.7310.064, 1.76(1.07 - 1.18)(1.08 - 1.10)(-0.0038 - 0.050)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 393 of 620Table E-13. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.16 (0.087)4.27 (0.10)-0.11 (0.13)-0.45, 0.220.4343.42, 4.65(3.77 - 4.38)(4.20 - 4.39)(-0.093 - 0.13)(3.18 - 4.68)Calories (Kcal/100g)496.46 (2.64)492.81 (3.05)3.66 (4.04)-6.73, 14.040.406457.61, 527.56(486.87 - 500.48)(490.52 - 494.31)(-7.44 - 9.96)(466.09 - 509.91)Carbohydrates42.09 (0.46)42.60 (0.53)-0.50 (0.70)-2.31, 1.300.50440.26, 56.45(41.40 - 43.69)(42.14 - 43.05)(-1.65 - 1.09)(43.28 - 54.90)Moisture (% fw)6.59 (0.25)7.28 (0.28)-0.70 (0.32)-1.53, 0.130.0824.79, 9.92(5.93 - 7.28)(6.63 - 7.75)(-1.82 - -0.34)(6.05 - 10.50)Protein31.15 (0.13)31.18 (0.15)-0.025 (0.19)-0.52, 0.470.90222.30, 29.41(30.63 - 31.47)(31.00 - 31.27)(-0.65 - 0.46)(20.58 - 29.28)Total Fat22.59 (0.54)21.95 (0.62)0.64 (0.83)-1.48, 2.770.47115.01, 28.51(20.62 - 23.58)(21.42 - 22.22)(-1.60 - 2.16)(16.58 - 25.25)Monsanto Company 12-CT-244U 394 of 620Table E-13. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber24.46 (0.51)26.40 (0.59)-1.94 (0.79)-3.96, 0.0800.05622.24, 31.96(23.26 - 25.45)(25.76 - 27.10)(-3.08 - -1.80)(23.42 - 31.62)Crude Fiber17.90 (0.74)17.71 (0.84)0.20 (0.94)-2.23, 2.630.84116.93, 22.68(17.33 - 18.57)(16.06 - 20.78)(-2.21 - 1.44)(16.92 - 23.32)Neutral Detergent Fiber29.73 (0.87)32.83 (1.00)-3.09 (1.32)-6.49, 0.310.06627.03, 42.49(27.53 - 32.00)(31.58 - 34.49)(-6.95 - -0.41)(29.27 - 40.63)Total Dietary Fiber39.16 (0.71)41.10 (0.75)-1.94 (0.55)-3.36, -0.530.01634.52, 52.58(37.46 - 40.44)(39.09 - 43.00)(-3.61 - -0.88)(37.29 - 48.60)Amino Acid (% dw)Alanine1.11 (0.011)1.13 (0.013)-0.019 (0.014)-0.054, 0.0150.2120.86, 1.11(1.10 - 1.13)(1.09 - 1.17)(-0.044 - 0.0067)(0.83 - 1.22)Arginine3.48 (0.049)3.71 (0.053)-0.23 (0.048)-0.35, -0.100.0052.38, 3.47(3.42 - 3.60)(3.67 - 3.77)(-0.34 - -0.17)(2.30 - 3.55)Monsanto Company 12-CT-244U 395 of 620Table E-13. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.60 (0.033)2.66 (0.037)-0.062 (0.043)-0.17, 0.0480.2061.94, 2.57(2.55 - 2.64)(2.58 - 2.74)(-0.11 - -0.031)(1.79 - 2.72)Cystine0.43 (0.013)0.44 (0.015)-0.010 (0.020)-0.061, 0.0400.6200.31, 0.45(0.39 - 0.47)(0.43 - 0.45)(-0.063 - 0.038)(0.29 - 0.47)Glutamic Acid5.30 (0.087)5.46 (0.10)-0.16 (0.13)-0.50, 0.180.2853.74, 5.28(5.24 - 5.38)(5.29 - 5.70)(-0.46 - -0.0085)(3.39 - 5.45)Glycine1.16 (0.014)1.18 (0.015)-0.021 (0.016)-0.063, 0.0200.2470.90, 1.14(1.14 - 1.19)(1.17 - 1.20)(-0.037 - -0.013)(0.85 - 1.23)Histidine0.82 (0.010)0.83 (0.012)-0.0065 (0.015)-0.044, 0.0310.6750.59, 0.81(0.80 - 0.85)(0.83 - 0.84)(-0.026 - 0.024)(0.57 - 0.84)Isoleucine0.97 (0.017)0.98 (0.018)-0.011 (0.012)-0.041, 0.0190.3970.75, 0.96(0.94 - 1.01)(0.94 - 1.03)(-0.038 - 0.021)(0.72 - 1.03)Monsanto Company 12-CT-244U 396 of 620Table E-13. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.65 (0.021)1.66 (0.023)-0.0069 (0.022)-0.063, 0.0490.7631.25, 1.62(1.62 - 1.70)(1.63 - 1.69)(-0.039 - 0.0098)(1.20 - 1.72)Lysine1.33 (0.019)1.36 (0.022)-0.030 (0.022)-0.087, 0.0270.2321.01, 1.30(1.26 - 1.38)(1.32 - 1.39)(-0.058 - -0.0056)(0.99 - 1.44)Methionine0.43 (0.012)0.40 (0.014)0.033 (0.018)-0.014, 0.0810.1290.32, 0.38(0.40 - 0.46)(0.38 - 0.41)(-0.013 - 0.071)(0.29 - 0.49)Phenylalanine1.61 (0.026)1.61 (0.028)-0.0044 (0.022)-0.061, 0.0520.8501.12, 1.58(1.56 - 1.66)(1.60 - 1.66)(-0.038 - 0.015)(1.10 - 1.63)Proline1.14 (0.027)1.18 (0.031)-0.040 (0.039)-0.14, 0.0600.3530.83, 1.08(1.09 - 1.21)(1.10 - 1.25)(-0.080 - -0.015)(0.79 - 1.17)Serine1.17 (0.026)1.20 (0.030)-0.028 (0.040)-0.13, 0.0750.5120.83, 1.21(1.15 - 1.23)(1.16 - 1.24)(-0.069 - -0.0042)(0.81 - 1.24)Monsanto Company 12-CT-244U 397 of 620Table E-13. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.92 (0.015)0.93 (0.017)-0.0072 (0.021)-0.062, 0.0480.7470.72, 0.89(0.89 - 0.94)(0.91 - 0.94)(-0.024 - 0.0027)(0.67 - 0.96)Tryptophan0.46 (0.014)0.44 (0.016)0.019 (0.020)-0.032, 0.0710.3820.34, 0.42(0.43 - 0.52)(0.43 - 0.45)(-0.019 - 0.076)(0.31 - 0.46)Tyrosine0.87 (0.014)0.89 (0.015)-0.012 (0.014)-0.048, 0.0240.4400.67, 0.84(0.85 - 0.92)(0.86 - 0.91)(-0.039 - 0.0089)(0.63 - 0.91)Valine1.32 (0.024)1.34 (0.026)-0.021 (0.019)-0.070, 0.0290.3291.00, 1.28(1.26 - 1.40)(1.31 - 1.40)(-0.048 - 0.0048)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.93 (0.0046)0.98 (0.0054)-0.043 (0.0071)-0.062, -0.0250.0010.16, 1.37(0.92 - 0.95)(0.97 - 0.98)(-0.060 - -0.037)(0.45 - 1.04)16:0 Palmitic24.19 (0.088)24.11 (0.10)0.083 (0.13)-0.26, 0.430.56216.54, 30.55(24.02 - 24.42)(23.89 - 24.34)(-0.32 - 0.33)(19.11 - 26.73)Monsanto Company 12-CT-244U 398 of 620Table E-13. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.53 (0.0022)0.54 (0.0025)-0.012 (0.0033)-0.021, -0.00370.0140.39, 0.70(0.52 - 0.53)(0.53 - 0.54)(-0.019 - -0.0082)(0.44 - 0.67)18:0 Stearic2.51 (0.020)2.64 (0.021)-0.14 (0.016)-0.18, -0.094<0.0011.98, 2.95(2.47 - 2.56)(2.61 - 2.70)(-0.15 - -0.095)(1.98 - 2.97)18:1 Oleic16.21 (0.067)16.21 (0.076)0.0024 (0.088)-0.22, 0.230.97911.38, 20.64(16.03 - 16.40)(16.10 - 16.35)(-0.11 - 0.24)(13.71 - 18.39)18:2 Linoleic54.32 (0.084)54.29 (0.097)0.029 (0.13)-0.30, 0.360.83347.49, 63.18(54.30 - 54.33)(54.04 - 54.50)(-0.18 - 0.30)(49.78 - 59.61)18:3 Linolenic0.16 (0.0019)0.14 (0.0022)0.012 (0.0029)0.0043, 0.0190.0090.060, 0.24(0.15 - 0.16)(0.14 - 0.15)(0.0078 - 0.014)(0.10 - 0.29)20:0 Arachidic0.30 (0.0052)0.31 (0.0060)-0.011 (0.0080)-0.031, 0.00950.2250.17, 0.38(0.29 - 0.30)(0.28 - 0.32)(-0.025 - 0.014)(0.20 - 0.36)Monsanto Company 12-CT-244U 399 of 620Table E-13. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.16 (0.0065)0.19 (0.0075)-0.023 (0.0099)-0.049, 0.00210.0650.070, 0.21(0.16 - 0.17)(0.17 - 0.21)(-0.049 - -0.0086)(0.051 - 0.19)MineralCalcium (% dw)0.15 (0.0029)0.13 (0.0034)0.021 (0.0042)0.011, 0.0320.0030.058, 0.21(0.14 - 0.15)(0.12 - 0.13)(0.0081 - 0.031)(0.081 - 0.18)Copper (mg/kg dw)11.35 (0.15)11.75 (0.17)-0.40 (0.22)-0.97, 0.180.1342.97, 12.86(11.11 - 11.91)(11.46 - 11.92)(-0.76 - 0.060)(4.46 - 11.62)Iron (mg/kg dw)63.88 (3.33)64.62 (3.84)-0.74 (5.08)-13.80, 12.320.89047.30, 97.12(60.27 - 66.59)(63.58 - 66.45)(-6.18 - 2.76)(39.49 - 114.34)Magnesium (% dw)0.39 (0.0053)0.37 (0.0061)0.019 (0.0081)-0.0015, 0.0400.0620.28, 0.47(0.38 - 0.41)(0.36 - 0.38)(0.0045 - 0.036)(0.31 - 0.46)Manganese (mg/kg dw)12.93 (0.23)12.90 (0.26)0.029 (0.34)-0.86, 0.920.9369.07, 17.33(12.73 - 13.13)(12.00 - 13.47)(-0.47 - 1.13)(9.07 - 17.14)Monsanto Company 12-CT-244U 400 of 620Table E-13. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.77 (0.010)0.79 (0.012)-0.020 (0.016)-0.060, 0.0210.2640.49, 0.87(0.74 - 0.80)(0.78 - 0.80)(-0.035 - 0.019)(0.48 - 0.87)Potassium (% dw)1.10 (0.016)1.11 (0.018)-0.0086 (0.017)-0.052, 0.0350.6360.92, 1.21(1.05 - 1.14)(1.07 - 1.14)(-0.020 - -0.0045)(0.90 - 1.26)Sodium (% dw)0.021 (0.0034)0.013 (0.0039)0.0075 (0.0052)-0.0057, 0.0210.2030, 0.066(0.019 - 0.022)(0.0054 - 0.023)(-0.0044 - 0.016)(0.0054 - 0.077)Zinc (mg/kg dw)45.63 (0.60)49.43 (0.69)-3.80 (0.92)-6.16, -1.440.00927.27, 44.95(44.12 - 46.74)(47.66 - 50.87)(-5.64 - -0.92)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E114.29 (2.04)112.18 (2.36)2.11 (3.12)-5.90, 10.120.52841.91, 205.89(107.78 - 119.15)(107.02 - 115.99)(-5.75 - 12.13)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 401 of 620Table E-14. Statistical Summary of Site NMLC CottonseedAnti-nutrients for MON 88701 (Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.16 (0.0079)0.14 (0.0092)0.019 (0.012)-0.012, 0.0500.1780.078, 0.25(0.14 - 0.18)(0.12 - 0.15)(-0.0065 - 0.041)(0.038 - 0.23)Malvalic Acid0.34 (0.023)0.29 (0.026)0.054 (0.035)-0.035, 0.140.1770.23, 0.54(0.30 - 0.38)(0.26 - 0.31)(-0.0093 - 0.074)(0.11 - 0.59)Sterculic Acid0.20 (0.018)0.18 (0.020)0.018 (0.027)-0.051, 0.0870.5310.17, 0.27(0.18 - 0.23)(0.17 - 0.19)(-0.012 - 0.032)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.85 (0.026)0.69 (0.030)0.15 (0.040)0.052, 0.260.0110.099, 1.57(0.83 - 0.88)(0.68 - 0.70)(0.14 - 0.18)(0.50 - 1.41)Total Gossypol0.92 (0.026)0.80 (0.030)0.12 (0.040)0.022, 0.230.0260.064, 1.76(0.84 - 0.97)(0.74 - 0.87)(0.060 - 0.18)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 402 of 620Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.11 (0.095)3.74 (0.095)0.37 (0.10)0.12, 0.620.0103.42, 4.65(3.99 - 4.28)(3.38 - 3.98)(0.25 - 0.61)(3.18 - 4.68)Calories (Kcal/100g)511.69 (2.44)503.38 (2.44)8.31 (3.45)-0.13, 16.750.052457.61, 527.56(505.01 - 517.46)(499.09 - 512.65)(-7.65 - 18.37)(466.09 - 509.91)Carbohydrates46.56 (0.54)48.67 (0.54)-2.11 (0.75)-3.95, -0.270.03140.26, 56.45(45.10 - 47.48)(47.50 - 49.59)(-3.20 - -0.23)(43.28 - 54.90)Moisture (% fw)6.73 (0.17)7.08 (0.17)-0.35 (0.19)-0.81, 0.120.1194.79, 9.92(6.27 - 7.13)(6.63 - 7.37)(-0.89 - 0.030)(6.05 - 10.50)Protein23.70 (0.42)23.92 (0.42)-0.22 (0.49)-1.43, 0.980.66922.30, 29.41(22.71 - 24.70)(23.56 - 24.61)(-0.85 - 0.64)(20.58 - 29.28)Total Fat25.65 (0.44)23.65 (0.44)2.00 (0.63)0.46, 3.540.01915.01, 28.51(24.23 - 26.78)(22.92 - 25.20)(-0.97 - 3.86)(16.58 - 25.25)Monsanto Company 12-CT-244U 403 of 620Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.95 (0.44)27.04 (0.44)-1.09 (0.60)-2.56, 0.370.11822.24, 31.96(24.75 - 26.52)(26.24 - 27.74)(-1.75 - -0.099)(23.42 - 31.62)Crude Fiber19.72 (0.80)19.13 (0.80)0.59 (1.13)-2.17, 3.360.61716.93, 22.68(17.98 - 21.66)(16.91 - 21.70)(-2.59 - 4.75)(16.92 - 23.32)Neutral Detergent Fiber31.34 (0.67)33.60 (0.67)-2.27 (0.95)-4.59, 0.0490.05327.03, 42.49(29.42 - 32.89)(32.74 - 35.52)(-6.10 - -0.44)(29.27 - 40.63)Total Dietary Fiber39.72 (0.62)41.87 (0.62)-2.14 (0.88)-4.29, 0.00150.05034.52, 52.58(38.66 - 40.44)(40.16 - 43.29)(-3.56 - -0.63)(37.29 - 48.60)Amino Acid (% dw)Alanine0.96 (0.022)0.94 (0.022)0.017 (0.029)-0.055, 0.0890.5830.86, 1.11(0.91 - 1.00)(0.88 - 0.97)(-0.020 - 0.033)(0.83 - 1.22)Arginine2.52 (0.088)2.59 (0.088)-0.063 (0.10)-0.31, 0.180.5562.38, 3.47(2.33 - 2.74)(2.41 - 2.71)(-0.18 - 0.021)(2.30 - 3.55)Monsanto Company 12-CT-244U 404 of 620Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.08 (0.054)2.07 (0.054)0.0087 (0.070)-0.16, 0.180.9041.94, 2.57(1.94 - 2.20)(1.92 - 2.18)(-0.085 - 0.096)(1.79 - 2.72)Cystine0.36 (0.017)0.35 (0.017)0.0082 (0.018)-0.036, 0.0520.6660.31, 0.45(0.32 - 0.41)(0.31 - 0.39)(-0.018 - 0.024)(0.29 - 0.47)Glutamic Acid4.14 (0.13)4.10 (0.13)0.039 (0.18)-0.40, 0.470.8333.74, 5.28(3.80 - 4.40)(3.66 - 4.40)(-0.11 - 0.14)(3.39 - 5.45)Glycine0.99 (0.022)0.98 (0.022)0.016 (0.031)-0.061, 0.0930.6270.90, 1.14(0.93 - 1.04)(0.91 - 1.02)(-0.0040 - 0.033)(0.85 - 1.23)Histidine0.64 (0.021)0.64 (0.021)0.0012 (0.025)-0.060, 0.0620.9610.59, 0.81(0.58 - 0.70)(0.61 - 0.66)(-0.053 - 0.033)(0.57 - 0.84)Isoleucine0.81 (0.023)0.82 (0.023)-0.0053 (0.030)-0.078, 0.0680.8650.75, 0.96(0.75 - 0.88)(0.77 - 0.83)(-0.077 - 0.054)(0.72 - 1.03)Monsanto Company 12-CT-244U 405 of 620Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.36 (0.034)1.36 (0.034)0.0086 (0.046)-0.10, 0.120.8581.25, 1.62(1.29 - 1.46)(1.28 - 1.40)(-0.053 - 0.064)(1.20 - 1.72)Lysine1.12 (0.027)1.11 (0.027)0.0024 (0.025)-0.059, 0.0640.9251.01, 1.30(1.05 - 1.18)(1.06 - 1.17)(-0.018 - 0.026)(0.99 - 1.44)Methionine0.38 (0.016)0.33 (0.016)0.041 (0.020)-0.0093, 0.0910.0930.32, 0.38(0.35 - 0.42)(0.32 - 0.35)(0.017 - 0.077)(0.29 - 0.49)Phenylalanine1.22 (0.033)1.23 (0.033)-0.0097 (0.039)-0.11, 0.0860.8111.12, 1.58(1.14 - 1.31)(1.15 - 1.27)(-0.075 - 0.043)(1.10 - 1.63)Proline0.87 (0.026)0.87 (0.026)0.0028 (0.026)-0.060, 0.0660.9160.83, 1.08(0.82 - 0.92)(0.81 - 0.93)(-0.035 - 0.034)(0.79 - 1.17)Serine0.98 (0.028)0.96 (0.028)0.019 (0.033)-0.062, 0.100.5870.83, 1.21(0.90 - 1.04)(0.86 - 1.03)(-0.0099 - 0.042)(0.81 - 1.24)Monsanto Company 12-CT-244U 406 of 620Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.79 (0.019)0.77 (0.019)0.016 (0.023)-0.041, 0.0730.5180.72, 0.89(0.74 - 0.83)(0.73 - 0.81)(0.0053 - 0.039)(0.67 - 0.96)Tryptophan0.35 (0.0093)0.38 (0.0093)-0.025 (0.0085)-0.046, -0.00480.0230.34, 0.42(0.33 - 0.38)(0.37 - 0.40)(-0.040 - 0.0010)(0.31 - 0.46)Tyrosine0.72 (0.020)0.71 (0.020)0.0042 (0.025)-0.058, 0.0660.8730.67, 0.84(0.67 - 0.78)(0.67 - 0.74)(-0.033 - 0.040)(0.63 - 0.91)Valine1.07 (0.029)1.07 (0.029)0.0056 (0.039)-0.090, 0.100.8891.00, 1.28(1.00 - 1.14)(1.00 - 1.10)(-0.084 - 0.049)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.70 (0.011)0.73 (0.011)-0.028 (0.012)-0.057, 0.000460.0520.16, 1.37(0.67 - 0.72)(0.72 - 0.75)(-0.049 - 0.0062)(0.45 - 1.04)16:0 Palmitic24.74 (0.086)24.39 (0.086)0.35 (0.12)0.049, 0.650.02916.54, 30.55(24.59 - 24.94)(24.07 - 24.59)(0.17 - 0.61)(19.11 - 26.73)Monsanto Company 12-CT-244U 407 of 620Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.48 (0.0056)0.48 (0.0056)-0.0075 (0.0076)-0.026, 0.0110.3610.39, 0.70(0.47 - 0.49)(0.47 - 0.49)(-0.020 - 0.018)(0.44 - 0.67)18:0 Stearic2.78 (0.036)2.67 (0.036)0.11 (0.050)-0.015, 0.230.0761.98, 2.95(2.75 - 2.85)(2.58 - 2.76)(0.0076 - 0.18)(1.98 - 2.97)18:1 Oleic14.40 (0.11)14.46 (0.11)-0.059 (0.15)-0.43, 0.310.70611.38, 20.64(14.15 - 14.68)(14.42 - 14.49)(-0.33 - 0.19)(13.71 - 18.39)18:2 Linoleic55.54 (0.18)55.87 (0.18)-0.33 (0.25)-0.94, 0.290.24247.49, 63.18(55.18 - 55.96)(55.61 - 56.29)(-1.11 - 0.13)(49.78 - 59.61)18:3 Linolenic0.15 (0.0017)0.15 (0.0017)0.0044 (0.0023)-0.0012, 0.00990.1030.060, 0.24(0.15 - 0.16)(0.14 - 0.15)(-0.00022 - 0.011)(0.10 - 0.29)20:0 Arachidic0.29 (0.0070)0.30 (0.0070)-0.0046 (0.0099)-0.029, 0.0200.6560.17, 0.38(0.27 - 0.31)(0.29 - 0.30)(-0.027 - 0.019)(0.20 - 0.36)Monsanto Company 12-CT-244U 408 of 620Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.14 (0.0033)0.14 (0.0033)-0.0014 (0.0046)-0.013, 0.00990.7650.070, 0.21(0.13 - 0.14)(0.13 - 0.14)(-0.0030 - 0.00006)(0.051 - 0.19)MineralCalcium (% dw)0.11 (0.0040)0.091 (0.0040)0.016 (0.0056)0.0025, 0.0300.0270.058, 0.21(0.10 - 0.11)(0.081 - 0.095)(0.0059 - 0.023)(0.081 - 0.18)Copper (mg/kg dw)5.82 (0.24)5.64 (0.24)0.18 (0.32)-0.61, 0.970.5932.97, 12.86(5.22 - 6.30)(5.40 - 5.85)(-0.28 - 0.55)(4.46 - 11.62)Iron (mg/kg dw)63.78 (4.68)73.46 (4.68)-9.68 (4.30)-20.21, 0.840.06547.30, 97.12(59.75 - 67.62)(63.01 - 89.93)(-22.31 - -1.16)(39.49 - 114.34)Magnesium (% dw)0.39 (0.0090)0.36 (0.0090)0.033 (0.0080)0.014, 0.0530.0050.28, 0.47(0.37 - 0.41)(0.34 - 0.37)(0.014 - 0.044)(0.31 - 0.46)Manganese (mg/kg dw)11.39 (0.51)9.72 (0.51)1.67 (0.72)-0.080, 3.420.0589.07, 17.33(10.88 - 11.68)(8.61 - 11.03)(0.65 - 2.27)(9.07 - 17.14)Monsanto Company 12-CT-244U 409 of 620Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.67 (0.022)0.63 (0.022)0.035 (0.018)-0.0093, 0.0800.1000.49, 0.87(0.64 - 0.71)(0.58 - 0.68)(0.0036 - 0.072)(0.48 - 0.87)Potassium (% dw)1.13 (0.031)1.02 (0.031)0.11 (0.043)0.0051, 0.220.0420.92, 1.21(1.11 - 1.17)(0.88 - 1.08)(0.046 - 0.23)(0.90 - 1.26)Sodium (% dw)0.022 (0.0033)0.015 (0.0033)0.0070 (0.0046)-0.0043, 0.0180.1800, 0.066(0.018 - 0.027)(0.012 - 0.023)(0.0039 - 0.013)(0.0054 - 0.077)Zinc (mg/kg dw)29.14 (0.82)30.08 (0.82)-0.94 (0.82)-2.96, 1.080.29727.27, 44.95(27.31 - 31.57)(28.22 - 31.74)(-2.85 - 1.07)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E162.17 (1.77)158.20 (1.77)3.97 (2.50)-2.16, 10.100.16441.91, 205.89(158.92 - 165.82)(153.15 - 162.63)(2.38 - 7.55)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 410 of 620Table E-16. Statistical Summary of SiteSCEK Cottonseed Anti-nutrients for MON 88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.0087)0.15 (0.0087)0.0014 (0.012)-0.029, 0.0310.9140.078, 0.25(0.12 - 0.18)(0.14 - 0.15)(-0.023 - 0.043)(0.038 - 0.23)Malvalic Acid0.41 (0.029)0.43 (0.029)-0.022 (0.041)-0.12, 0.0780.6070.23, 0.54(0.33 - 0.52)(0.39 - 0.46)(-0.11 - 0.13)(0.11 - 0.59)Sterculic Acid0.23 (0.014)0.24 (0.014)-0.011 (0.020)-0.060, 0.0380.6070.17, 0.27(0.19 - 0.28)(0.22 - 0.25)(-0.060 - 0.060)(0.061 - 0.34)Gossypol (% dw)Free Gossypol1.10 (0.029)1.13 (0.029)-0.030 (0.027)-0.096, 0.0350.3030.099, 1.57(1.05 - 1.18)(1.06 - 1.20)(-0.086 - 0.00085)(0.50 - 1.41)Total Gossypol1.17 (0.025)1.07 (0.025)0.10 (0.031)0.026, 0.180.0170.064, 1.76(1.13 - 1.23)(1.05 - 1.10)(0.074 - 0.13)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 411 of 620Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash3.85 (0.051)3.46 (0.051)0.40 (0.072)0.22, 0.570.0013.42, 4.65(3.77 - 3.92)(3.34 - 3.61)(0.24 - 0.58)(3.18 - 4.68)Calories (Kcal/100g)498.01 (2.54)494.42 (2.54)3.59 (3.47)-4.90, 12.070.340457.61, 527.56(489.04 - 502.78)(489.10 - 500.98)(-1.24 - 13.67)(466.09 - 509.91)Carbohydrates44.03 (0.48)46.39 (0.48)-2.36 (0.64)-3.92, -0.790.01040.26, 56.45(42.73 - 45.99)(45.65 - 47.07)(-3.73 - -0.88)(43.28 - 54.90)Moisture (% fw)6.88 (0.21)7.47 (0.21)-0.59 (0.29)-1.30, 0.130.0904.79, 9.92(6.32 - 7.37)(7.11 - 7.79)(-1.47 - 0.18)(6.05 - 10.50)Protein29.43 (0.24)28.48 (0.24)0.95 (0.29)0.24, 1.660.01722.30, 29.41(29.06 - 30.14)(28.09 - 28.77)(0.38 - 1.82)(20.58 - 29.28)Total Fat22.71 (0.48)21.70 (0.48)1.01 (0.65)-0.58, 2.610.16915.01, 28.51(20.94 - 23.59)(20.71 - 22.88)(0.15 - 2.88)(16.58 - 25.25)Monsanto Company 12-CT-244U 412 of 620Table E-17. Statistical Summary of Site TXPL CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.53 (0.37)25.53 (0.37)0.0023 (0.41)-1.01, 1.010.99522.24, 31.96(25.31 - 25.83)(24.51 - 26.91)(-1.08 - 0.91)(23.42 - 31.62)Crude Fiber17.93 (0.38)18.10 (0.38)-0.17 (0.45)-1.26, 0.920.71616.93, 22.68(17.17 - 18.84)(17.35 - 19.63)(-1.48 - 1.07)(16.92 - 23.32)Neutral Detergent Fiber29.75 (0.41)32.12 (0.41)-2.38 (0.58)-3.80, -0.960.00627.03, 42.49(28.74 - 30.56)(30.49 - 33.05)(-4.31 - -0.26)(29.27 - 40.63)Total Dietary Fiber39.54 (0.62)40.47 (0.62)-0.93 (0.72)-2.69, 0.830.24534.52, 52.58(38.76 - 40.86)(39.15 - 42.09)(-3.24 - 0.56)(37.29 - 48.60)Amino Acid (% dw)Alanine1.07 (0.019)1.05 (0.019)0.022 (0.026)-0.042, 0.0860.4380.86, 1.11(1.05 - 1.10)(0.97 - 1.10)(-0.040 - 0.12)(0.83 - 1.22)Arginine3.25 (0.074)3.25 (0.074)-0.0020 (0.10)-0.26, 0.250.9852.38, 3.47(3.15 - 3.33)(2.94 - 3.49)(-0.34 - 0.39)(2.30 - 3.55)Monsanto Company 12-CT-244U 413 of 620Table E-17. Statistical Summary of Site TXPL CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.51 (0.047)2.45 (0.047)0.060 (0.067)-0.10, 0.220.4071.94, 2.57(2.43 - 2.55)(2.26 - 2.62)(-0.092 - 0.29)(1.79 - 2.72)Cystine0.41 (0.015)0.40 (0.015)0.0068 (0.017)-0.034, 0.0470.6970.31, 0.45(0.39 - 0.43)(0.36 - 0.45)(-0.030 - 0.073)(0.29 - 0.47)Glutamic Acid4.94 (0.13)5.02 (0.13)-0.072 (0.19)-0.53, 0.390.7143.74, 5.28(4.73 - 5.14)(4.41 - 5.32)(-0.44 - 0.73)(3.39 - 5.45)Glycine1.12 (0.022)1.11 (0.022)0.0062 (0.031)-0.070, 0.0820.8490.90, 1.14(1.07 - 1.15)(1.03 - 1.19)(-0.073 - 0.12)(0.85 - 1.23)Histidine0.77 (0.018)0.76 (0.018)0.017 (0.026)-0.046, 0.0790.5380.59, 0.81(0.73 - 0.81)(0.71 - 0.82)(-0.062 - 0.091)(0.57 - 0.84)Isoleucine0.95 (0.013)0.93 (0.013)0.013 (0.017)-0.027, 0.0540.4570.75, 0.96(0.93 - 0.97)(0.89 - 0.97)(-0.023 - 0.051)(0.72 - 1.03)Monsanto Company 12-CT-244U 414 of 620Table E-17. Statistical Summary of Site TXPL CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.58 (0.026)1.55 (0.026)0.028 (0.037)-0.064, 0.120.4861.25, 1.62(1.52 - 1.61)(1.45 - 1.64)(-0.072 - 0.16)(1.20 - 1.72)Lysine1.26 (0.026)1.24 (0.026)0.024 (0.037)-0.067, 0.120.5371.01, 1.30(1.21 - 1.33)(1.19 - 1.33)(-0.11 - 0.13)(0.99 - 1.44)Methionine0.40 (0.021)0.39 (0.021)0.016 (0.029)-0.056, 0.0880.6050.32, 0.38(0.37 - 0.44)(0.32 - 0.44)(-0.066 - 0.12)(0.29 - 0.49)Phenylalanine1.51 (0.034)1.48 (0.034)0.026 (0.048)-0.092, 0.140.6141.12, 1.58(1.46 - 1.55)(1.36 - 1.61)(-0.14 - 0.19)(1.10 - 1.63)Proline1.04 (0.024)1.04 (0.024)0.0028 (0.033)-0.079, 0.0840.9350.83, 1.08(0.99 - 1.11)(1.01 - 1.11)(-0.12 - 0.10)(0.79 - 1.17)Serine1.11 (0.031)1.11 (0.031)0.00043 (0.044)-0.11, 0.110.9920.83, 1.21(1.06 - 1.13)(0.97 - 1.17)(-0.087 - 0.16)(0.81 - 1.24)Monsanto Company 12-CT-244U 415 of 620Table E-17. Statistical Summary of Site TXPL CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.89 (0.016)0.86 (0.016)0.032 (0.022)-0.022, 0.0860.1970.72, 0.89(0.86 - 0.92)(0.80 - 0.91)(-0.026 - 0.10)(0.67 - 0.96)Tryptophan0.42 (0.013)0.43 (0.013)-0.0094 (0.018)-0.054, 0.0350.6260.34, 0.42(0.40 - 0.44)(0.38 - 0.47)(-0.070 - 0.030)(0.31 - 0.46)Tyrosine0.84 (0.017)0.81 (0.017)0.031 (0.024)-0.028, 0.0890.2450.67, 0.84(0.82 - 0.86)(0.74 - 0.87)(-0.048 - 0.12)(0.63 - 0.91)Valine1.26 (0.030)1.23 (0.030)0.024 (0.042)-0.079, 0.130.5861.00, 1.28(1.21 - 1.29)(1.16 - 1.29)(-0.046 - 0.12)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.86 (0.010)0.84 (0.010)0.017 (0.013)-0.016, 0.0500.2460.16, 1.37(0.84 - 0.88)(0.81 - 0.87)(-0.010 - 0.047)(0.45 - 1.04)16:0 Palmitic23.15 (0.14)23.01 (0.14)0.13 (0.16)-0.27, 0.530.44316.54, 30.55(22.72 - 23.62)(22.86 - 23.25)(-0.27 - 0.76)(19.11 - 26.73)Monsanto Company 12-CT-244U 416 of 620Table E-17. Statistical Summary of Site TXPL CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.48 (0.0058)0.46 (0.0058)0.016 (0.0072)-0.0018, 0.0340.0700.39, 0.70(0.47 - 0.49)(0.45 - 0.47)(0.0040 - 0.039)(0.44 - 0.67)18:0 Stearic2.35 (0.025)2.46 (0.025)-0.11 (0.028)-0.18, -0.0470.0061.98, 2.95(2.30 - 2.43)(2.40 - 2.52)(-0.16 - -0.097)(1.98 - 2.97)18:1 Oleic16.34 (0.078)16.16 (0.078)0.18 (0.11)-0.083, 0.450.14311.38, 20.64(16.22 - 16.45)(15.86 - 16.44)(-0.14 - 0.53)(13.71 - 18.39)18:2 Linoleic55.42 (0.21)55.58 (0.21)-0.15 (0.29)-0.87, 0.560.61647.49, 63.18(54.97 - 55.95)(55.18 - 56.13)(-1.16 - 0.77)(49.78 - 59.61)18:3 Linolenic0.17 (0.0056)0.17 (0.0056)0.0057 (0.0079)-0.014, 0.0250.4970.060, 0.24(0.17 - 0.18)(0.16 - 0.17)(0.00091 - 0.011)(0.10 - 0.29)20:0 Arachidic0.28 (0.0055)0.28 (0.0055)-0.0041 (0.0078)-0.023, 0.0150.6180.17, 0.38(0.27 - 0.28)(0.28 - 0.29)(-0.0061 - -0.0022)(0.20 - 0.36)Monsanto Company 12-CT-244U 417 of 620Table E-17. Statistical Summary of Site TXPL CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.16 (0.0015)0.16 (0.0015)0.0020 (0.0020)-0.0029, 0.00680.3620.070, 0.21(0.16 - 0.16)(0.16 - 0.16)(-0.00054 - 0.0042)(0.051 - 0.19)MineralCalcium (% dw)0.16 (0.0016)0.14 (0.0016)0.021 (0.0023)0.015, 0.027<0.0010.058, 0.21(0.16 - 0.16)(0.13 - 0.14)(0.018 - 0.023)(0.081 - 0.18)Copper (mg/kg dw)10.49 (0.15)9.98 (0.15)0.51 (0.22)-0.023, 1.040.0572.97, 12.86(10.09 - 10.87)(9.58 - 10.41)(0.12 - 1.29)(4.46 - 11.62)Iron (mg/kg dw)60.47 (5.67)79.02 (5.67)-18.55 (7.06)-35.82, -1.280.03947.30, 97.12(56.94 - 66.50)(67.45 - 95.10)(-38.15 - -0.95)(39.49 - 114.34)Magnesium (% dw)0.35 (0.0045)0.34 (0.0045)0.019 (0.0040)0.0087, 0.0280.0030.28, 0.47(0.35 - 0.37)(0.33 - 0.34)(0.0082 - 0.024)(0.31 - 0.46)Manganese (mg/kg dw)10.91 (0.34)9.04 (0.34)1.86 (0.48)0.70, 3.030.0079.07, 17.33(10.18 - 11.37)(8.83 - 9.54)(0.64 - 2.54)(9.07 - 17.14)Monsanto Company 12-CT-244U 418 of 620Table E-17. Statistical Summary of Site TXPL CottonseedNutrients for MON 88701 (Treated) vs. Conventional Control(continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.58 (0.0099)0.57 (0.0099)0.0036 (0.011)-0.022, 0.0300.7420.49, 0.87(0.56 - 0.61)(0.54 - 0.60)(-0.0098 - 0.018)(0.48 - 0.87)Potassium (% dw)1.01 (0.023)0.87 (0.023)0.14 (0.033)0.062, 0.220.0040.92, 1.21(0.98 - 1.06)(0.79 - 0.93)(0.073 - 0.27)(0.90 - 1.26)Sodium (% dw)0.024 (0.010)0.047 (0.010)-0.023 (0.014)-0.058, 0.0120.1610, 0.066(0.019 - 0.027)(0.019 - 0.090)(-0.065 - 0.0062)(0.0054 - 0.077)Zinc (mg/kg dw)34.10 (0.45)34.96 (0.45)-0.86 (0.64)-2.44, 0.710.22727.27, 44.95(33.36 - 35.30)(33.70 - 35.89)(-2.31 - 1.61)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E114.39 (2.75)103.66 (2.75)10.73 (3.90)1.20, 20.260.03341.91, 205.89(107.81 - 118.39)(93.92 - 109.90)(6.69 - 14.40)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 419 of 620Table E-18. Statistical Summary of SiteTXPL Cottonseed Anti-nutrients for MON88701 (Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.16 (0.0047)0.16 (0.0047)-0.0044 (0.0066)-0.021, 0.0120.5330.078, 0.25(0.14 - 0.17)(0.15 - 0.17)(-0.026 - 0.021)(0.038 - 0.23)Malvalic Acid0.41 (0.020)0.47 (0.020)-0.054 (0.029)-0.12, 0.0170.1120.23, 0.54(0.33 - 0.47)(0.44 - 0.49)(-0.16 - 0.0068)(0.11 - 0.59)Sterculic Acid0.23 (0.013)0.26 (0.013)-0.026 (0.018)-0.070, 0.0170.1890.17, 0.27(0.18 - 0.27)(0.25 - 0.27)(-0.085 - 0.024)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.98 (0.024)0.93 (0.024)0.054 (0.033)-0.028, 0.140.1570.099, 1.57(0.91 - 1.06)(0.91 - 0.95)(-0.025 - 0.13)(0.50 - 1.41)Total Gossypol1.06 (0.024)1.01 (0.024)0.053 (0.022)-0.00013, 0.110.0500.064, 1.76(1.03 - 1.11)(0.97 - 1.05)(-0.021 - 0.092)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2MON 88701 plants were treated with dicamba and glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.

# Monsanto Company 12-CT-244U 420 of 620 

# E.5. Composition of Cottonseed Not T reated with Dicamba or Glufosinate

E.5.1. Nutrient Levels in Cottonseed Not Treated with Dicamba or Glufosinate

In the combined-site an alysis of nutrient leve ls in cottonseed, the following components had no

significant differences (p<0.05) in mean valu es between MON 88701 not treated with dicamba

or glufosinate and the conventi onal control: one proximate (prote in), 17 amino acids (alanine,

aspartic acid, cystine, glutamic acid, glycine, hi stidine, isoleucine, leuc ine, lysine, methionine,

phenylalanine, proline, serine, th reonine, tryptophan, tyrosine, and valine), eight fatty acids (16:0

palmitic acid, 16:1 palmitoleic acid, 18:0 stearic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3

linolenic acid, 20:0 arachidic acid, and 22:0 behe nic acid), and four minerals (copper, iron,

phosphorus, and sodium) (Tables E-19 and E-20).

The nutrient components that had significant diffe rences in mean values between MON 88701

and the conventional control in the combined-site analysis were: five pr oximates (ash, calories,

carbohydrates, moisture, and total fat), four type s of fiber (ADF, crude fiber, NDF, and TDF),

one amino acid (arginine), one fatty acid (14:0 myristic acid), five minerals (calcium,

magnesium, manganese, potassium, and zinc) and vitamin E (Table E-19).

The significant differences in nutrients were furt her evaluated using consid erations relevant to

the safety and nutritional quality of MON 88701 when compared to the conventional control:

All nutrient component differences observed in th e combined-site statistical analysis, whether

reflecting increased or decrea sed MON 88701 mean values, with respect to the conventional

control, were 16.14% or less. Th e relative magnitude of the diffe rences were as follows: 1.04 to

6.45% for proximates; 3.35 to 4.12% for fibers; 3.82% for arginine; 2.39% for 14:0 myristic

acid; 5.48 to 16.14% for minerals; and 5.84% for vitamin E.

1) All mean values for all signi ficantly different nutrient co mponents from the combinedsite analysis of MON 88701 were within the 99% to lerance interval established from the

conventional commercial reference varieties grown concurrently in the same trial.

2) Assessment of the reproducibility of the comb ined-site differences at the eight individual

sites showed statistically significant differenc es for: calories, carbohydrates, total fat,

crude fiber, and NDF at one site; moisture, ADF , TDF, arginine and zinc at two sites;

14:0 myristic, potassium, and vitamin E at thre e sites; ash and magnesium at four sites,

manganese at five sites and calcium at 7 site s. With the exception of potassium, arginine

and zinc, each at a single site, all individual site mean values of MON 88701 for all

nutrient components with signifi cant differences were within the 99% tolerance interval

established from the conventional commercial reference varieties gr own concurrently in

the same trial. The control values for argini ne and zinc were also outside the tolerance

interval at the individual sites where the MON 88701 value was outside the tolerance

interval

.

3) With the exception of calories, combined-site mean values and individual site mean

values of MON 88701 for all nutrient components including those that were significantly

different were within the context of the natural variability of commercial cotton

# Monsanto Company 12-CT-244U 421 of 620 

# composition as published in the scientific literature and/or available in the ILSI Crop

Composition Database (ILSI, 2011).

Five of the 17 cottonseed nutri ent statistically significant di fferences between MON 88701 and

the conventional control observed in the combined-s ite data analysis were attributable to small

differences in proximates (ash, carbohydrates expressed as % dw, calories expressed as Kcal/100g

% dw, 

# moisture expressed as % fw and total fat expr essed as % dw). For ash, calories, and total

fat, the relative magnitudes of the differences between the mean values for MON 88701 and the

conventional control were all small increases (4.77% for ash, 1.04% for calories and 5.37% for

total fat). The relative magnitude s of differences for mean values of carbohydrates and moisture

between MON 88701 and the conventional cont rol were both small decreases; 2.96% for

carbohydrates and 6.45% for moisture. All of the nutrient mean values for MON 88701

observed in the combined-site analysis for proximates were within the 99% tolerance interval

established by conventional commercial reference vari eties grown concurrently in the same trial.

Significant differences for proximate mean values between MON 88701 and the conventional

control were not consistently observed among sites. Ash was si gnificantly different at four

individual sites, with relative magnitudes of differences rangi ng from 4.34 to 11.0%. Moisture

was decreased at two sites, with relative magn itude of differences ranging from 8.51 to 11.58%,

carbohydrates had a relative decrease of 4.71% at one site. Both calorie s and total fat had small

increases at one site with 1.30% for calories and 6.90% for to tal fat. Overall, observed

differences in proximate valu es between MON 88701 and the c onventional control were not

considered to be meaningful from a food and feed safety or nutritional perspective because they

were small, not consistently reproduced acro ss the individual sites, and the mean MON 88701

combined-site values were w ithin the 99% tolera nce interval establ ished by conventional

commercial reference varieties grown concurrently in the same trial and within the context of the

natural variability of commerci al cotton composition as published in the scientific literature

and/or available in the ILSI Cr op Composition Database (ILSI, 2011).

Four of the 17 cottonseed nutrient differences between MON 88701 and the conventional control

observed in the combined-site data analysis were attributable to small differences in fiber (ADF,

crude fiber, NDF, and TDF, all ex pressed as % dw). All relativ e magnitudes of the differences

between the mean value for MON 88701 and the conventional control were small decreases

(3.93% for ADF, 4.12% for crude fiber, 3.56% for NDF, and 3.35% for TDF). All of the

nutrient mean values for MON 88701 observed in the combined-site analysis for fiber were

within the 99% toleran ce interval established by conventiona l commercial reference varieties

grown concurrently in the same trial. Signifi cant differences for fiber mean values between

MON 88701 and the conventional control were not consistently observed am ong individual sites.

ADF and TDF were significantly di fferent at two sites, with sm all decreases ranging from 8.50

to 8.91% for ADF and 5.21 to 6.83% for TDF. Cr ude fiber and NDF were significantly different

at one site, with a small decrease of 5.46% for crude fiber and 5.66% for NDF. Overall,

observed differences in fiber values between MON 88701 and the conventional control were not

considered to be meaningful from a food and feed safety or nutritional perspective because they

were small, not consistently reproduced acro ss the individual sites, and the mean MON 88701

combined-site values were w ithin the 99% tolera nce interval establ ished by conventional

commercial reference varieties grown concurrently in the same trial and within the context of the

# Monsanto Company 12-CT-244U 422 of 620 

# natural variability of commerci al cotton composition as published in the scientific literature

and/or available in the ILSI Cr op Composition Database (ILSI, 2011).

One of the cottonseed nutrient differences between MON 88701 and the conventional control

observed in the combined-site analysis was attr ibuted to a small difference in one amino acid

(arginine, expressed as % dw). The relative ma gnitude of the difference between the mean value

for MON 88701 and the conventional control was a small decrease of 3.82%. The mean arginine

value for MON 88701 observed in the combined-sit e analysis was within the 99% tolerance

interval established by conventi onal commercial reference varietie s grown concurrently in the

same trial. Significant differences for argi nine mean values between MON 88701 and the

conventional control were not c onsistently observed among individua l sites, with small decreases

ranging from 5.36 to 8.48% observed at two sites. Overall, observed differences in arginine

values between MON 88701 and the conventional cont rol were not considered to be meaningful

from a food and feed safety or nutritional perspective because they were small, not consistently

reproduced across the individua l sites, and the mean MON 88701 combined-site values was

within the 99% toleran ce interval established by conventiona l commercial reference varieties

grown concurrently in the same trial or with in the context of the natural variability of

commercial cotton composition as published in the sc ientific literature and/or available in the

ILSI Crop Composition Data base (ILSI, 2011).

One of the cottonseed nutrient differences between MON 88701 and the conventional control

observed in the combined-site analysis was attributed to the fatty acid 14:0 myristic acid

(expressed as % total FA). The relative magnitude of the difference between the mean fatty acid

value for MON 88701 and the conventional control in the combined-site analysis was a small

decrease of 2.39%. The mean 14:0 myris tic acid value for MON 88701 observed in the

combined-site analysis was within the 99% to lerance interval established by conventional

commercial reference varieties grown concurrently in the same trial. Significant differences for

14:0 myristic acid mean values between MO N 88701 and the conventional control were not

consistently observed among individual sites, with three sites with sma ll decreases ranging from

3.08 to 4.79%. Overall, observed differences in 14:0 myristic acid va lues between MON 88701

and the conventional control were not considered to be meaningful from a food and feed safety

and nutritional perspective because they were small, not consistently reproduced across the

individual sites, and the mean MON 88701 combin ed-site value was within the 99% tolerance

interval established by conventi onal commercial reference varietie s grown concurrently in the

same trial and within the context of the natura l variability of commercial cotton composition as

published in the scientific literature and/or av ailable in the ILSI Crop Composition Database

(ILSI, 2011).

Five of the 17 cottonseed nutri ent differences between MON 88701 and the conventional control

observed in the combined-site data analysis were attributable to small differences in minerals

(calcium, magnesium, and potassium expressed as % dw and manga nese and zinc expressed as

mg/kg dw). For calcium, magnesium, manganese and potassium, the relative magnitude of the

differences between the mean values for MO N 88701 and the conventional control were all

increases (14.72% for calcium, 5.81% for ma gnesium, 16.14% for manganese and 5.48% for

potassium). The difference for zinc betw een the mean value for MON 88701 and the

conventional control was a decreas e of 5.81%. All of the nutrient mean values for MON 88701

# Monsanto Company 12-CT-244U 423 of 620 

# observed in the combined-site analysis for mine rals were within the 99% tolerance interval

established by conventional commercial reference vari eties grown concurrently in the same trial.

With the exception of calcium and manganese, significant differences for mineral mean values

between MON 88701 and the conve ntional control were not consistently observed among

individual sites. Alth ough calcium was significan tly different at seven sites, with increases

ranging from 9.33 to 24.41%, the variability obser ved for the test (mean range 0.10 to 0.21%

dw) and control samples (mean range 0.081 to 0.19% dw) were very similar (Table E-20).

Manganese was significantly differe nt at five sites with increased differences ranging from 12.88

to 29.26%. Magnesium was significantly different at four sites with relative increases in

MON 88701, ranging from 5.69 to 8.36%, and potassium was significantly increased at three

sites, with relative increases ranging from 8.76 to 18.36%. Zinc was significantly different at

two sites with small decreases in relative magnitude of differe nces ranging from 8.98 to 16.23%.

Overall, observed differences in mineral values between MON 88701 and the conventional

control were not considered to be meaningful from a food and feed safety or nutritional

perspective because they were generally small, not consistently reproduced across individual

sites (with the exception of calcium), and the mean MON 88701 combined-site values were

within the 99% tolerance interval established by conventional, commerci al reference varieties

grown concurrently in the same trial or with in the context of the natural variability of

commercial cotton composition as published in the sc ientific literature and/or available in the

ILSI Crop Composition Da tabase (ILSI, 2011).

The relative magnitude of the difference between the mean vitamin E value for MON 88701

and the conventional control in the combined-site analysis was a small increase of 5.84%. The

mean vitamin E value for MON 88701 observed in the combined-site anal ysis was within the

99% tolerance interval established by conven tional commercial reference varieties grown

concurrently in the same trial. The significant difference for the vitamin E mean value between

MON 88701 and the conventional control was not consistently observed among individual sites,

with significant increases ranging from 6.54 to 11.36% observed at three si tes. Overall, the

observed difference in the vitamin E value between MON 88701 and the conventional control

was not considered to be meaningful from a food and feed safety and nutritional perspective

because it was small, not consistently reprodu ced across the individual sites, and the mean

MON 88701 combined-site value was within th e 99% tolerance interval established by

conventional commercial reference varieties grown concurrently in the same trial and was within

the context of the natural variability of co mmercial cotton composition as published in the

scientific literature and/or available in the ILSI Crop Composition Database (ILSI, 2011).

In summary, with the exception of calcium a nd manganese, statistical analyses found no

consistent differences between the levels of nutrient components in cottonseed from MON 88701

and the conventional control. Differences were observed for calcium and manganese in

combined analyses and most individual sites, but the magnitudes of differences for this nutrient

was less than the variability for the control samples, and values were within the range of natural

variability for cottonseed.

These findings support the conclusion of compositional equivalence of MON 88701 to

conventional cotton.

# Monsanto Company 12-CT-244U 424 of 620 

# E.5.2. Anti-Nutrient Levels in Cottonseed Not Treated with Dicamba or Glufosinate

Cottonseed was analyzed for five anti-nutrients, namely: dihydrosterculic acid, malvalic acid,

sterculic acid, free gossypol, and total gossypol. Out of these five anti-nutrients, in the

combined-site analysis of MON 88701 not trea ted with dicamba or glufosinate and the

conventional control, malvalic and sterculic acids, as well as free gossypol, did not show any

significant differences (p<0.05) in their mean values (Table E-21). In the combined-site analysis

dihydrosterculic acid and total gossypol we re significantly diffe rent (Table E-2).

The significant differences in anti-nutrients were further evaluated using considerations relevant

to the safety and nutritional quality of MON 88701 when compared to the conventional control:

1) All anti-nutrient component differences observe d in the combined-site statistical analysis,

which reflected an increase in MON 88701 mean values with respect to the conventional

control, were 12.64% or less. The relative ma gnitudes of the differences for dihydrosterculic

acid and total gossypol were 12.64% and 6.26%, respectively.

2) Mean values for all significantly different an ti-nutrient components fr om the combined-site

analysis of MON 88701 were within the 99% tolerance interval established from the

conventional commercial reference varieties grown concurrently in the same trial.

3) Assessment of the reproducibility of the combined-site differe nces at the eight individual

sites showed significant differences for: dihydrosterculic and tota l gossypol at only one site.

All individual site mean values of MON 88701 for both anti-nutrient components with

significant differences were within the 99% tolerance interval established from the

conventional commercial reference varieties grown concurrently in the same trial.

4) All combined-site mean values and individual site mean va lues of MON 88701 for all antinutrient components including those that were significantly differe nt were within the context

of the natural variability of commercial cotton composition as published in the scientific

literature and/or availabl e in the ILSI Crop Compos ition Database (ILSI, 2011).

The two cottonseed anti-nutrient differences between MON 88701 and the conventional control

observed in the combined-site data analysis were attributed to small differences in one

cyclopropenoid fatty acid (dihydrosterculic; expr essed as % total fatty acid) and total gossypol

(expressed as % dw). The relative magnitude of the differences between the mean values for

MON 88701 and the conventional control were in creases of 12.64% for di hydrosterculic acid

and 6.26% for total gossypol. These anti-nutri ent mean values for MON 88701 observed in the

combined-site analysis were within the 99% to lerance interval establ ished by conventional

commercial reference varieties grown concurrently in the same trial. Significant differences for

the two anti-nutrient mean values between MO N 88701 and the conventional control were not

consistently observed among individual sites. Both dihydrosterculic acid and total gossypol

were significantly different at only one site with an increase of 33.11% for dihydrosterculic acid

and an increase of 7.99% for total gossypol. Overal l, observed differences in anti-nutrient values

between MON 88701 and the conventional control were not considered to be meaningful from a

food and feed safety or nutritional perspectiv e because they were generally small, not

# Monsanto Company 12-CT-244U 425 of 620 

# consistently reproduced across the individual sites, and th e mean MON 88701 values were

within the 99% toleran ce interval established by conventiona l commercial reference varieties

grown concurrently in the same trial and with in the context of the natural variability of

commercial cotton composition as published in the sc ientific literature and/or available in the

ILSI Crop Composition Da tabase (ILSI, 2011).

In summary, statistical analyses found no consiste nt statistically significa nt differences between

the levels of anti-nutrient components in cottonseed from MON 88701 and the conventional

control and mean values for anti-nutrients were within the natural variability found for

cottonseed. These findings supported the conclusion of compositional equivalence of

MON 88701 to conventional cotton.Monsanto Company 12-CT-244U 426 of 620Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701(Not Treated) vs. Conventional ControlMean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisCottonseed Proximate (% dw)Ash4.304.114.770.0023.76 - 4.883.42, 4.65Calories (Kcal/100g)500.37495.241.04<0.001487.62 - 511.92457.61, 527.56Carbohydrates44.4745.83-2.96<0.00141.07 - 48.8140.26, 56.45Moisture (% fw)7.007.48-6.45<0.0015.81 - 9.074.79, 9.92Total Fat23.5122.315.37<0.00120.99 - 25.5415.01, 28.51Cottonseed Fiber (% dw)Acid Detergent Fiber25.5326.58-3.930.00923.30 - 30.4322.24, 31.96Crude Fiber17.7818.54-4.120.02014.54 - 20.7316.93, 22.68Monsanto Company 12-CT-244U 427 of 620Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701(Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisCottonseed Fiber (% dw)Neutral Detergent Fiber31.4332.59-3.560.00528.05 - 37.2727.03, 42.49Total Dietary Fiber39.7541.12-3.350.00136.22 - 43.2234.52, 52.58Cottonseed Amino Acid (% dw)Arginine3.033.15-3.820.0022.31 - 3.622.38, 3.47Cottonseed Fatty Acid (% Total FA)14:0 Myristic0.770.79-2.390.0180.66 - 0.950.16, 1.37Cottonseed MineralCalcium (% dw)0.150.1314.72<0.0010.10 - 0.210.058, 0.21Magnesium (% dw)0.400.385.81<0.0010.35 - 0.450.28, 0.47Monsanto Company 12-CT-244U 428 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisCottonseed MineralManganese (mg/kg dw)13.6311.7316.14<0.00110.59 - 17.479.07, 17.33Potassium (% dw)1.131.075.480.0120.99 - 1.320.92, 1.21Zinc (mg/kg dw)37.8140.14-5.810.00927.60 - 46.0427.27, 44.95Cottonseed Vitamin (mg/kg dw)Vitamin E139.01131.335.840.00287.22 - 184.4741.91, 205.89Cottonseed CyclopropenoidFatty Acid (% Total FA)Dihydrosterculic Acid0.150.1412.64<0.0010.12 - 0.190.078, 0.25Cottonseed Gossypol (% dw)Total Gossypol1.030.976.26<0.0010.84 - 1.520.064, 1.76Monsanto Company 12-CT-244U 429 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 7 SitesCalcium (% dw) Site ARTI0.150.1224.410.0070.14 - 0.160.058, 0.21Calcium (% dw) Site GACH0.130.1117.80<0.0010.13 - 0.130.058, 0.21Calcium (% dw) Site KSLA0.200.1810.110.0320.18 - 0.210.058, 0.21Calcium (% dw) Site NCBD0.150.149.330.0020.15 - 0.150.058, 0.21Calcium (% dw) Site NMLC0.150.1315.030.0060.14 - 0.150.058, 0.21Calcium (% dw) Site SCEK0.110.09121.620.0130.10 - 0.120.058, 0.21Calcium (% dw) Site TXPL0.160.1417.83<0.0010.16 - 0.170.058, 0.21Monsanto Company 12-CT-244U 430 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Fatty Acid (% Total FA) - 5 Sites18:0 Stearic Site ARTI2.652.515.540.0352.59 - 2.711.98, 2.9518:0 Stearic Site LACH2.612.523.540.0142.58 - 2.641.98, 2.9518:0 Stearic Site NCBD2.532.347.940.0212.49 - 2.571.98, 2.9518:0 Stearic Site NMLC2.522.64-4.70<0.0012.49 - 2.561.98, 2.9518:0 Stearic Site TXPL2.312.46-6.140.0012.26 - 2.341.98, 2.95Cottonseed Mineral - 5 SitesManganese (mg/kg dw) Site ARTI14.8611.5029.260.03413.28 - 17.479.07, 17.33Manganese (mg/kg dw) Site GACH14.2611.5123.89<0.00113.82 - 15.049.07, 17.33Monsanto Company 12-CT-244U 431 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 5 SitesManganese (mg/kg dw) Site KSLA14.1812.5612.880.01313.48 - 14.849.07, 17.33Manganese (mg/kg dw) Site SCEK12.449.7228.040.00810.59 - 13.879.07, 17.33Manganese (mg/kg dw) Site TXPL11.389.0425.840.00210.73 - 12.839.07, 17.33Cottonseed Proximate (% dw) - 4 SitesAsh Site GACH4.394.214.340.0034.24 - 4.503.42, 4.65Ash Site NCBD4.334.144.560.0434.20 - 4.483.42, 4.65Ash Site SCEK4.103.749.720.0113.91 - 4.233.42, 4.65Ash Site TXPL3.843.4611.000.0013.76 - 3.983.42, 4.65Monsanto Company 12-CT-244U 432 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Fatty Acid (% Total FA) - 4 Sites18:3 Linolenic Site ARTI0.140.139.880.0220.14 - 0.140.060, 0.2418:3 Linolenic Site NCBD0.360.2925.900.0080.34 - 0.380.060, 0.2418:3 Linolenic Site NMLC0.160.149.670.0040.15 - 0.160.060, 0.2418:3 Linolenic Site SCEK0.160.156.700.0050.15 - 0.160.060, 0.24Cottonseed Mineral - 4 SitesMagnesium (% dw) Site GACH0.410.388.36<0.0010.40 - 0.430.28, 0.47Magnesium (% dw) Site KSLA0.420.405.690.0040.41 - 0.430.28, 0.47Magnesium (% dw) Site SCEK0.380.367.250.0170.36 - 0.410.28, 0.47Monsanto Company 12-CT-244U 433 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 4 SitesMagnesium (% dw) Site TXPL0.360.347.43<0.0010.35 - 0.370.28, 0.47Cottonseed Fatty Acid (% Total FA) - 3 Sites14:0 Myristic Site KSLA0.690.72-4.640.0130.66 - 0.700.16, 1.3714:0 Myristic Site NCBD0.710.75-4.790.0240.69 - 0.730.16, 1.3714:0 Myristic Site NMLC0.950.98-3.080.0080.94 - 0.950.16, 1.37Cottonseed Mineral - 3 SitesPotassium (% dw) Site GACH1.221.128.76<0.0011.18 - 1.240.92, 1.21Potassium (% dw) Site SCEK1.141.0211.890.0311.11 - 1.190.92, 1.21Potassium (% dw) Site TXPL1.030.8718.360.0020.99 - 1.090.92, 1.21Monsanto Company 12-CT-244U 434 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Vitamin (mg/kg dw) - 3 SitesVitamin E Site GACH149.29140.126.540.046142.84 - 153.5541.91, 205.89Vitamin E Site LACH167.00149.9611.360.002158.95 - 173.3041.91, 205.89Vitamin E Site NCBD174.64156.9911.240.025165.41 - 184.4741.91, 205.89Cottonseed Proximate (% dw) - 2 SitesMoisture (% fw) Site GACH6.397.23-11.580.0156.19 - 6.644.79, 9.92Moisture (% fw) Site SCEK6.487.08-8.510.0196.16 - 6.984.79, 9.92Cottonseed Fiber (% dw) - 2 SitesAcid Detergent Fiber Site ARTI25.0727.53-8.910.01024.53 - 25.4822.24, 31.96Acid Detergent Fiber Site LACH25.9428.35-8.500.00825.56 - 26.3322.24, 31.96Monsanto Company 12-CT-244U 435 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Fiber (% dw) - 2 SitesTotal Dietary Fiber Site LACH41.0943.35-5.210.01639.95 - 42.2634.52, 52.58Total Dietary Fiber Site NMLC38.3041.10-6.830.00337.12 - 39.3234.52, 52.58Cottonseed Amino Acid (% dw) - 2 SitesArginine Site KSLA3.003.28-8.480.0092.88 - 3.052.38, 3.47Arginine Site NMLC3.513.71-5.360.0083.34 - 3.622.38, 3.47Lysine Site KSLA1.191.25-5.160.0281.16 - 1.221.01, 1.30Lysine Site LACH1.251.186.250.0351.23 - 1.271.01, 1.30Cottonseed Fatty Acid (% Total FA) - 2 Sites18:2 Linoleic Site LACH56.0456.63-1.030.02755.71 - 56.3547.49, 63.18Monsanto Company 12-CT-244U 436 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteCottonseed Fatty Acid (% Total FA) - 2 Sites18:2 Linoleic Site NCBD56.0457.19-2.010.02155.76 - 56.3147.49, 63.18Cottonseed Mineral - 2 SitesZinc (mg/kg dw) Site NCBD41.5049.54-16.230.00939.45 - 43.0527.27, 44.95Zinc (mg/kg dw) Site NMLC44.9949.43-8.980.00444.56 - 46.0427.27, 44.95Statistical Differences Observed in One SiteCottonseed Proximate (% dw)Calories (Kcal/100g) Site NCBD498.21491.801.300.048496.22 - 499.62457.61, 527.56Carbohydrates Site TXPL44.2046.39-4.710.01443.36 - 44.5440.26, 56.45Total Fat Site NCBD23.0821.596.900.02222.78 - 23.3915.01, 28.51Monsanto Company 12-CT-244U 437 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in One SiteCottonseed Fiber (% dw)Crude Fiber Site KSLA16.7117.67-5.460.04716.10 - 17.3716.93, 22.68Neutral Detergent Fiber Site LACH32.1234.05-5.660.00330.20 - 33.9427.03, 42.49Cottonseed Amino Acid (% dw)Aspartic Acid Site KSLA2.352.50-5.910.0382.32 - 2.371.94, 2.57Glutamic Acid Site GACH4.694.96-5.450.0414.51 - 4.983.74, 5.28Histidine Site KSLA0.730.77-6.410.0060.69 - 0.760.59, 0.81Isoleucine Site KSLA0.900.95-5.130.0170.88 - 0.940.75, 0.96Leucine Site KSLA1.511.58-4.680.0171.47 - 1.551.25, 1.62Monsanto Company 12-CT-244U 438 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in One SiteCottonseed Amino Acid (% dw)Phenylalanine Site KSLA1.421.53-6.880.0141.37 - 1.451.12, 1.58Tyrosine Site KSLA0.790.84-5.940.0450.76 - 0.800.67, 0.84Cottonseed Fatty Acid (% Total FA)18:1 Oleic Site NCBD15.2914.704.040.02615.01 - 15.5011.38, 20.6422:0 Behenic Site ARTI0.140.15-6.750.0290.14 - 0.150.070, 0.21Cottonseed CyclopropenoidFatty Acid (% Total FA)Dihydrosterculic Acid Site GACH0.160.1233.110.0120.14 - 0.170.078, 0.25Sterculic Acid Site GACH0.230.1824.510.0280.20 - 0.240.17, 0.27Monsanto Company 12-CT-244U 439 of 620Table E-19. Summary of Differences (p<0.05) for theComparison of Cottonseed Component Levels forMON 88701 (Not Treated) vs. Conventional Control (continued)Mean Difference(MON 88701 minus Control)Analytical Component (Units)1MON 887012Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88701RangeCommercialTolerance Interval5Statistical Differences Observed in One SiteCottonseed Gossypol (% dw)Free Gossypol Site NMLC0.860.6923.910.0080.76 - 0.950.099, 1.57Total Gossypol Site TXPL1.091.017.990.0091.03 - 1.160.064, 1.761dw = dry weight; fw = fresh weight; FA = fatty acid.2Test refers to MON 88701 (Not Treated). These plantswere not treated with dicamba or glufosinate.3Mean = least-square mean.4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 440 of 620Table E-20.Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701(Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.30 (0.11)4.11 (0.11)0.20 (0.052)0.084, 0.310.0023.42, 4.65(3.76 - 4.88)(3.34 - 5.00)(-0.42 - 0.79)(3.18 - 4.68)Calories (Kcal/100g)500.37 (1.65)495.24 (1.71)5.13 (1.29)2.57, 7.69<0.001457.61, 527.56(487.62 - 511.92)(487.70 - 512.65)(-5.97 - 15.41)(466.09 - 509.91)Carbohydrates44.47 (0.56)45.83 (0.57)-1.36 (0.32)-1.99, -0.73<0.00140.26, 56.45(41.07 - 48.81)(42.14 - 50.30)(-5.09 - 1.56)(43.28 - 54.90)Moisture (% fw)7.00 (0.26)7.48 (0.27)-0.48 (0.11)-0.71, -0.26<0.0014.79, 9.92(5.81 - 9.07)(6.15 - 9.19)(-1.45 - 0.15)(6.05 - 10.50)Protein27.71 (0.77)27.79 (0.77)-0.075 (0.31)-0.73, 0.580.81022.30, 29.41(22.49 - 31.29)(23.53 - 31.27)(-2.53 - 4.39)(20.58 - 29.28)Total Fat23.51 (0.31)22.31 (0.33)1.20 (0.26)0.69, 1.71<0.00115.01, 28.51(20.99 - 25.54)(20.71 - 25.20)(-1.21 - 3.21)(16.58 - 25.25)Monsanto Company 12-CT-244U 441 of 620Table E-20.Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.53 (0.34)26.58 (0.35)-1.05 (0.35)-1.79, -0.300.00922.24, 31.96(23.30 - 30.43)(22.08 - 29.58)(-4.02 - 5.70)(23.42 - 31.62)Crude Fiber17.78 (0.37)18.54 (0.38)-0.76 (0.32)-1.41, -0.120.02016.93, 22.68(14.54 - 20.73)(16.06 - 21.70)(-5.57 - 3.82)(16.92 - 23.32)Neutral Detergent Fiber31.43 (0.51)32.59 (0.53)-1.16 (0.41)-1.97, -0.350.00527.03, 42.49(28.05 - 37.27)(28.87 - 35.89)(-4.66 - 6.42)(29.27 - 40.63)Total Dietary Fiber39.75 (0.39)41.12 (0.41)-1.38 (0.36)-2.15, -0.610.00134.52, 52.58(36.22 - 43.22)(39.05 - 44.37)(-4.41 - 2.04)(37.29 - 48.60)Amino Acid (% dw)Alanine1.05 (0.020)1.05 (0.020)-0.0034 (0.0091)-0.023, 0.0160.7140.86, 1.11(0.88 - 1.15)(0.88 - 1.17)(-0.076 - 0.11)(0.83 - 1.22)Arginine3.03 (0.10)3.15 (0.10)-0.12 (0.033)-0.19, -0.0500.0022.38, 3.47(2.31 - 3.62)(2.41 - 3.77)(-0.37 - 0.26)(2.30 - 3.55)Monsanto Company 12-CT-244U 442 of 620Table E-20.Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.39 (0.062)2.41 (0.062)-0.022 (0.027)-0.079, 0.0350.4221.94, 2.57(1.95 - 2.69)(1.92 - 2.74)(-0.28 - 0.20)(1.79 - 2.72)Cystine0.40 (0.0091)0.40 (0.0094)-0.0018 (0.0070)-0.016, 0.0120.7930.31, 0.45(0.33 - 0.49)(0.31 - 0.46)(-0.074 - 0.086)(0.29 - 0.47)Glutamic Acid4.71 (0.13)4.84 (0.14)-0.13 (0.072)-0.29, 0.0250.0933.74, 5.28(3.79 - 5.57)(3.66 - 5.70)(-1.01 - 0.57)(3.39 - 5.45)Glycine1.09 (0.020)1.09 (0.020)-0.0061 (0.011)-0.029, 0.0170.5770.90, 1.14(0.92 - 1.19)(0.91 - 1.20)(-0.11 - 0.088)(0.85 - 1.23)Histidine0.74 (0.019)0.75 (0.019)-0.0076 (0.0073)-0.023, 0.00790.3120.59, 0.81(0.58 - 0.84)(0.61 - 0.84)(-0.069 - 0.064)(0.57 - 0.84)Isoleucine0.91 (0.018)0.92 (0.018)-0.0087 (0.0079)-0.026, 0.00820.2910.75, 0.96(0.76 - 1.01)(0.77 - 1.03)(-0.074 - 0.074)(0.72 - 1.03)Monsanto Company 12-CT-244U 443 of 620Table E-20.Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.53 (0.032)1.54 (0.032)-0.0080 (0.013)-0.036, 0.0200.5441.25, 1.62(1.28 - 1.68)(1.28 - 1.69)(-0.095 - 0.14)(1.20 - 1.72)Lysine1.23 (0.025)1.23 (0.025)-0.0019 (0.015)-0.034, 0.0310.9041.01, 1.30(1.03 - 1.37)(1.06 - 1.39)(-0.084 - 0.14)(0.99 - 1.44)Methionine0.39 (0.0079)0.38 (0.0084)0.011 (0.0081)-0.0049, 0.0270.1670.32, 0.38(0.33 - 0.44)(0.32 - 0.46)(-0.081 - 0.088)(0.29 - 0.49)Phenylalanine1.43 (0.039)1.46 (0.039)-0.026 (0.014)-0.056, 0.00440.0881.12, 1.58(1.13 - 1.63)(1.15 - 1.66)(-0.14 - 0.11)(1.10 - 1.63)Proline1.02 (0.029)1.03 (0.029)-0.014 (0.012)-0.039, 0.0110.2460.83, 1.08(0.78 - 1.16)(0.81 - 1.25)(-0.13 - 0.10)(0.79 - 1.17)Serine1.08 (0.025)1.09 (0.026)-0.0031 (0.015)-0.035, 0.0280.8340.83, 1.21(0.93 - 1.28)(0.86 - 1.24)(-0.14 - 0.14)(0.81 - 1.24)Monsanto Company 12-CT-244U 444 of 620Table E-20.Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.87 (0.016)0.86 (0.016)0.0033 (0.0083)-0.014, 0.0210.6940.72, 0.89(0.73 - 0.95)(0.73 - 0.95)(-0.062 - 0.086)(0.67 - 0.96)Tryptophan0.41 (0.0092)0.42 (0.0095)-0.0092 (0.0066)-0.022, 0.00410.1710.34, 0.42(0.34 - 0.50)(0.37 - 0.52)(-0.099 - 0.11)(0.31 - 0.46)Tyrosine0.81 (0.017)0.81 (0.018)-0.0030 (0.0083)-0.021, 0.0150.7180.67, 0.84(0.68 - 0.88)(0.67 - 0.91)(-0.076 - 0.074)(0.63 - 0.91)Valine1.21 (0.027)1.23 (0.027)-0.013 (0.011)-0.037, 0.0110.2571.00, 1.28(0.98 - 1.38)(1.00 - 1.40)(-0.10 - 0.15)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.77 (0.030)0.79 (0.031)-0.019 (0.0071)-0.034, -0.00360.0180.16, 1.37(0.66 - 0.95)(0.71 - 0.98)(-0.087 - 0.041)(0.45 - 1.04)16:0 Palmitic23.93 (0.30)23.80 (0.30)0.13 (0.076)-0.035, 0.290.11616.54, 30.55(22.30 - 25.45)(22.69 - 25.05)(-0.48 - 0.63)(19.11 - 26.73)Monsanto Company 12-CT-244U 445 of 620Table E-20.Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.50 (0.0094)0.50 (0.0094)-0.00052 (0.0038)-0.0087, 0.00770.8940.39, 0.70(0.45 - 0.55)(0.45 - 0.54)(-0.024 - 0.024)(0.44 - 0.67)18:0 Stearic2.52 (0.058)2.47 (0.058)0.045 (0.036)-0.032, 0.120.2281.98, 2.95(2.16 - 2.93)(2.15 - 2.76)(-0.24 - 0.26)(1.98 - 2.97)18:1 Oleic15.05 (0.26)14.96 (0.26)0.094 (0.070)-0.055, 0.240.19611.38, 20.64(14.05 - 16.29)(14.06 - 16.44)(-0.54 - 1.00)(13.71 - 18.39)18:2 Linoleic55.84 (0.39)56.15 (0.40)-0.31 (0.16)-0.64, 0.0230.06547.49, 63.18(54.22 - 58.48)(54.04 - 57.93)(-1.65 - 0.73)(49.78 - 59.61)18:3 Linolenic0.18 (0.022)0.17 (0.022)0.013 (0.0068)-0.0017, 0.0270.0780.060, 0.24(0.11 - 0.38)(0.12 - 0.30)(-0.032 - 0.11)(0.10 - 0.29)20:0 Arachidic0.29 (0.0086)0.28 (0.0087)0.0044 (0.0047)-0.0057, 0.0150.3650.17, 0.38(0.23 - 0.32)(0.23 - 0.32)(-0.049 - 0.042)(0.20 - 0.36)Monsanto Company 12-CT-244U 446 of 620Table E-20.Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.15 (0.0051)0.15 (0.0051)-0.0010 (0.0029)-0.0074, 0.00530.7300.070, 0.21(0.12 - 0.17)(0.13 - 0.21)(-0.057 - 0.027)(0.051 - 0.19)MineralCalcium (% dw)0.15 (0.0093)0.13 (0.0093)0.019 (0.0022)0.014, 0.024<0.0010.058, 0.21(0.10 - 0.21)(0.081 - 0.19)(0.0037 - 0.039)(0.081 - 0.18)Copper (mg/kg dw)8.94 (0.70)8.93 (0.70)0.015 (0.16)-0.30, 0.330.9252.97, 12.86(5.02 - 12.15)(5.40 - 11.92)(-2.19 - 1.72)(4.46 - 11.62)Iron (mg/kg dw)72.43 (4.40)71.33 (4.48)1.10 (2.74)-4.74, 6.940.69347.30, 97.12(41.73 - 109.70)(45.03 - 95.10)(-20.63 - 27.89)(39.49 - 114.34)Magnesium (% dw)0.40 (0.0083)0.38 (0.0084)0.022 (0.0032)0.016, 0.028<0.0010.28, 0.47(0.35 - 0.45)(0.33 - 0.44)(-0.015 - 0.055)(0.31 - 0.46)Manganese (mg/kg dw)13.63 (0.47)11.73 (0.48)1.89 (0.28)1.29, 2.50<0.0019.07, 17.33(10.59 - 17.47)(8.61 - 14.11)(-0.84 - 5.26)(9.07 - 17.14)Monsanto Company 12-CT-244U 447 of 620Table E-20.Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.71 (0.031)0.72 (0.031)-0.0035 (0.0067)-0.017, 0.00990.6050.49, 0.87(0.58 - 0.87)(0.54 - 0.87)(-0.078 - 0.072)(0.48 - 0.87)Potassium (% dw)1.13 (0.028)1.07 (0.028)0.059 (0.020)0.015, 0.100.0120.92, 1.21(0.99 - 1.32)(0.79 - 1.27)(-0.13 - 0.31)(0.90 - 1.26)Sodium (% dw)0.026 (0.0095)0.029 (0.0096)-0.0035 (0.0046)-0.013, 0.00640.4660, 0.066(0.0053 - 0.082)(0.0053 - 0.10)(-0.085 - 0.026)(0.0054 - 0.077)Zinc (mg/kg dw)37.81 (2.01)40.14 (2.02)-2.33 (0.77)-3.99, -0.670.00927.27, 44.95(27.60 - 46.04)(28.22 - 52.95)(-13.50 - 1.99)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E139.01 (9.87)131.33 (9.88)7.68 (2.07)3.26, 12.090.00241.91, 205.89(87.22 - 184.47)(91.78 - 162.98)(-7.82 - 32.93)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2Test refers to MON 88701 (Not Treated). These plantswere not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 448 of 620Table E-21.Statistical Summary of Combined-Site Cottonseed Anti-nutrients for MON 88701 (Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.0034)0.14 (0.0037)0.017 (0.0044)0.0086, 0.026<0.0010.078, 0.25(0.12 - 0.19)(0.11 - 0.17)(-0.011 - 0.058)(0.038 - 0.23)Malvalic Acid0.39 (0.015)0.37 (0.016)0.019 (0.015)-0.011, 0.0480.2100.23, 0.54(0.24 - 0.50)(0.26 - 0.49)(-0.11 - 0.19)(0.11 - 0.59)Sterculic Acid0.22 (0.0067)0.21 (0.0072)0.0099 (0.0081)-0.0064, 0.0260.2290.17, 0.27(0.12 - 0.27)(0.17 - 0.27)(-0.075 - 0.093)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.93 (0.037)0.89 (0.037)0.041 (0.020)-0.0031, 0.0840.0650.099, 1.57(0.76 - 1.10)(0.68 - 1.20)(-0.14 - 0.27)(0.50 - 1.41)Total Gossypol1.03 (0.037)0.97 (0.037)0.061 (0.017)0.026, 0.096<0.0010.064, 1.76(0.84 - 1.52)(0.74 - 1.10)(-0.028 - 0.44)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2Test refers to MON 88701 (Not Treated). These plantswere not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.

# Monsanto Company 12-CT-244U 449 of 620 

# Table E-22 Literature and ILSI Ra nges for Components in Cottonseed

Cottonseed Tissue Components 1 

# Literature Range 2 

# ILSI Range 3



# Cottonseed Nutrients

Proximates (% dw)

Ash 3.87 – 5.29 a 

# ; 3.7 – 4.2 d 

# 3.761 – 5.342

Carbohydrates by calculation 45.28 – 53.62 a 

# 39.0 – 53.6

Calories by calculation

(Kcal/100g) 471.34 – 506.95 a 

# Not available

Moisture (% fw) 2.25 – 7.49 a 

# 2.3 – 9.9

Protein 24.54 – 30.83 a 

# ; 21.2 – 25.9 b 

# 21.48 – 32.97

Total Fat 17.37 – 25.16 a 

# ; 14.4 – 16.9 d 

# 17.201 – 27.292

Fiber (% dw)

Acid Detergent Fiber 21.10 – 34.8 a 

# ; 37.6 – 40.5 d 

# 19.74 – 38.95

Neutral Detergent Fiber 32.92 – 45.83 a 

# ; 50.0 – 53.6 d 

# 25.56 – 51.87

Crude Fiber 13.85 – 17.94 a 

# 13.86 – 23.10

Total Dietary Fiber not available 33.69 – 47.55

Amino Acids (% total AA) (% dw)

Alanine 4.16 – 4.41 a 

# ; 3.6 – 4.2 b 

# 0.80 – 1.22

Arginine 11.28 – 12.51 a 

# ; 10.9 – 12.3 b 

# 2.06 – 3.72

Aspartic acid 9.73 – 9.99 a 

# ; 8.8 – 9.5 b 

# 1.82 – 2.94

Cystine/Cysteine 1.60 – 1.92 a 

# ; 2.3 – 3.4 b 

# 0.35 – 0.56

Glutamic acid 20.76 – 21.61 a 

# ; 20.5 – 22.4 b 

# 3.91 – 6.72

Glycine 4.44 – 4.58 a 

# ; 3.8 – 4.5 b 

# 0.83 – 1.32

Histidine 3.00 – 3.12 a 

# ; 2.6 – 2.8 b 

# 0.57 – 0.91

Isoleucine 3.10 – 3.67 a 

# ; 3.0 – 3.4 b 

# 0.62 – 1.05

Leucine 6.27 – 6.65 a 

# ; 5.5 – 6.1 b 

# 1.14 – 1.86

Lysine 4.85 – 5.37 a 

# ; 4.2 – 4.6 b 

# 0.94 – 1.46

Methionine 1.46 – 1.88 a 

# ; 1.3 – 1.8 b 

# 0.30 – 0.47

Phenylalanine 5.56 – 5.77 a 

# ; 5.0 – 5.6 b 

# 1.02 – 1.72

Proline 4.06 – 4.28 a 

# ; 3.1 – 4.0 b 

# 0.75 – 1.23

Serine 4.45 – 4.86 a 

# ; 3.9 – 4.4 b 

# 0.91 – 1.35

Threonine 3.26 – 3.59 a 

# ; 2.8 – 3.2 b 

# 0.55 – 0.92

Tryptophan 0.97 – 1.21 a 

# ; 1.0 – 1.4 b 

# 0.194 – 0.319

Tyrosine 2.65 – 2.92 a 

# ; 2.8 – 3.3 b 

# 0.53 – 0.84

Valine 4.76 – 5.14 a 

# ; 4.3 – 4.7 b 

# 0.87 – 1.49

Fatty Acids (% total FA)

8:0 Caprylic not available not available

10:0 Capric not available not available

12:0 Lauric not available not available

14:0 Myristic 0.55 – 2.40 a 

# ; 0.6 – 1.5 b 

# 0.455 – 2.400

14:1 Myristoleic not available not available

15:0 Pentadecanoic 0.050 – 0.17 a 

# 0.103 – 0.481

15:1 Pentadecenoic not available not available

16:0 Palmitic 21.23 – 27.9 a 

# ; 17.6 – 24.8 b 

# 15.11 – 27.90

16:1 Palmitoleic 0.55 – 1.16 a 

# 0.464 – 1.190

17:0 Heptadecanoic not available 0.092 – 0.119

# Monsanto Company 12-CT-244U 450 of 620 

# Table E-22. Literature and ILSI Ranges fo r Components in Cottonseed (continued)

Cottonseed Tissue Components 1 

# Literature Range 2 

# ILSI Range 3



# 17:1 Heptadecenoic not available not available

18:0 Stearic 1.99 – 3.11 a 

# ; 2.0 – 2.5 b 

# 0.20 – 3.11

18:1 Oleic 13.90 – 20.10 a 

# ; 15.0 – 20.7 b 

# 12.8 – 25.3

18:2 Linoleic 46.00 – 56.88 a 

# 46.0 – 59.4

18:3 Gamma Linolenic 0.050 – 0.25 a 

# 0.097 – 0.232

18:3 Linolenic 0.050 – 0.25 a 

# 0.11 – 0.35

20:0 Arachidic 0.25 – 0.33 a 

# 0.186 – 0.414

20:1 Eicosenoic not available 0.095 – 0.098

20:2 Eicosadienoic not available not available

20:3 Eicosatrienoic not available not available

20:4 Arachidonic not available not available

22:0 Behenic 0.13 – 0.17 a 

# 0.104 – 0.295

Vitamins (mg/kg fw) (mg/kg dw)

Vitamin E 99 – 224 c 

# 70.825 – 197.243

Minerals (% dw)

Calcium 0.10 – 0.33 a 

# 0.10323 – 0.32581

Copper (mg/kg dw) 3.54 – 11.14 a 

# 3.13 – 24.57

Iron (mg/kg dw) 40.58 – 56.54 a 

# 36.71 – 318.38

Magnesium 0.37 – 0.46 a 

# 0.34709 – 0.49312

Manganese (mg/kg dw) 11.06 – 18.31 a 

# 10.69 – 21.96

Phosphorus 0.60 – 0.84 a 

# 0.48254 – 0.99157

Potassium 0.98 – 1.24 a 

# 0.98345 – 1.44835

Sodium 0.0054 – 0.74 a 

# 0.01118 – 0.73548

Zinc (mg/kg dw) 30.21 – 47.75 a 

# 27.0 – 59.5

Cottonseed Anti-Nutrients

Gossypol, Total (% dw) 0.57 – 1.42 a 

# ; 0.55 – 0.77 d 

# 0.547 – 1.522

Gossypol, Free (% dw) 0.53 – 1.20 a 

# 0.454 – 1.399

Cyclopropenoid Fatty Acids

(% total FA)

Dihydrosterculic 0.13 – 0.24 a 

# 0.075 – 0.310

Malvalic 0.33 – 0.58 a 

# 0.229 – 0.759

Sterculic 0.21 – 0.56 a 

# 0.190 – 0.556

1 

# fw=fresh weight; dw=dry weight

2 

# Literature range references; a 

# (Hamilton, et al., 2004); b 

# (Lawhon, et al., 1977); c 

# (Smith and Creelman, 2001);

d 

# (Bertrand, et al., 2005).

3 

# (ILSI, 2011).Monsanto Company 12-CT-244U 451 of 620Table E-23. Statistical Summary of SiteARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.71 (0.087)4.74 (0.12)-0.030 (0.15)-0.45, 0.390.8503.42, 4.65(4.58 - 4.88)(4.49 - 5.00)(-0.42 - 0.39)(3.18 - 4.68)Calories (Kcal/100g)499.07 (3.00)488.30 (3.88)10.76 (3.90)-0.069, 21.590.050457.61, 527.56(493.81 - 501.81)(487.70 - 494.60)(7.21 - 13.11)(466.09 - 509.91)Carbohydrates44.29 (0.58)46.35 (0.81)-2.06 (0.96)-4.72, 0.600.09740.26, 56.45(43.36 - 45.15)(45.03 - 47.37)(-2.98 - -1.67)(43.28 - 54.90)Moisture (% fw)7.08 (0.27)7.63 (0.35)-0.55 (0.35)-1.51, 0.410.1844.79, 9.92(6.14 - 7.86)(7.32 - 7.40)(-1.26 - -0.37)(6.05 - 10.50)Protein27.39 (0.24)27.04 (0.33)0.35 (0.41)-0.79, 1.480.44222.30, 29.41(26.65 - 28.02)(26.97 - 27.11)(-0.32 - 0.91)(20.58 - 29.28)Total Fat23.62 (0.59)21.50 (0.78)2.12 (0.81)-0.12, 4.360.05815.01, 28.51(22.57 - 24.08)(21.15 - 22.89)(1.18 - 2.93)(16.58 - 25.25)Monsanto Company 12-CT-244U 452 of 620Table E-23. Statistical Summary of Site ARTI CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.07 (0.32)27.53 (0.45)-2.45 (0.55)-3.97, -0.940.01022.24, 31.96(24.53 - 25.48)(26.57 - 28.49)(-3.34 - -1.42)(23.42 - 31.62)Crude Fiber17.00 (0.90)19.47 (1.20)-2.47 (1.26)-5.97, 1.030.12116.93, 22.68(14.54 - 18.91)(19.33 - 19.85)(-2.18 - -0.94)(16.92 - 23.32)Neutral Detergent Fiber31.02 (0.79)32.89 (1.06)-1.86 (1.13)-5.01, 1.280.17527.03, 42.49(29.94 - 32.82)(30.67 - 34.42)(-3.04 - -0.73)(29.27 - 40.63)Total Dietary Fiber39.70 (1.06)41.67 (1.50)-1.97 (1.83)-7.06, 3.120.34334.52, 52.58(36.22 - 42.86)(40.50 - 42.84)(-4.27 - -4.04)(37.29 - 48.60)Amino Acid (% dw)Alanine1.03 (0.015)1.02 (0.018)0.0099 (0.017)-0.037, 0.0570.5910.86, 1.11(1.00 - 1.05)(0.99 - 1.05)(0.0033 - 0.028)(0.83 - 1.22)Arginine2.93 (0.052)3.02 (0.073)-0.086 (0.084)-0.32, 0.150.3622.38, 3.47(2.82 - 3.03)(2.89 - 3.13)(-0.21 - 0.068)(2.30 - 3.55)Monsanto Company 12-CT-244U 453 of 620Table E-23. Statistical Summary of Site ARTI CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.30 (0.045)2.32 (0.060)-0.024 (0.065)-0.20, 0.160.7291.94, 2.57(2.23 - 2.36)(2.19 - 2.42)(-0.085 - 0.077)(1.79 - 2.72)Cystine0.38 (0.010)0.37 (0.014)0.016 (0.017)-0.032, 0.0640.4060.31, 0.45(0.36 - 0.40)(0.35 - 0.39)(0.0017 - 0.039)(0.29 - 0.47)Glutamic Acid4.52 (0.099)4.51 (0.14)0.0076 (0.17)-0.46, 0.470.9653.74, 5.28(4.29 - 4.73)(4.34 - 4.67)(0.057 - 0.11)(3.39 - 5.45)Glycine1.07 (0.020)1.08 (0.026)-0.0049 (0.028)-0.084, 0.0740.8720.90, 1.14(1.04 - 1.10)(1.03 - 1.11)(-0.015 - 0.029)(0.85 - 1.23)Histidine0.71 (0.016)0.74 (0.023)-0.021 (0.026)-0.094, 0.0530.4740.59, 0.81(0.66 - 0.74)(0.71 - 0.77)(-0.042 - 0.034)(0.57 - 0.84)Isoleucine0.88 (0.018)0.91 (0.024)-0.029 (0.025)-0.098, 0.0400.3040.75, 0.96(0.82 - 0.93)(0.88 - 0.93)(-0.054 - 0.0021)(0.72 - 1.03)Monsanto Company 12-CT-244U 454 of 620Table E-23. Statistical Summary of Site ARTI CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.48 (0.027)1.50 (0.037)-0.017 (0.042)-0.13, 0.0980.7001.25, 1.62(1.41 - 1.55)(1.44 - 1.55)(-0.071 - 0.045)(1.20 - 1.72)Lysine1.20 (0.044)1.23 (0.058)-0.032 (0.062)-0.21, 0.140.6331.01, 1.30(1.04 - 1.31)(1.19 - 1.26)(-0.016 - -0.0053)(0.99 - 1.44)Methionine0.38 (0.015)0.35 (0.021)0.027 (0.025)-0.043, 0.0970.3470.32, 0.38(0.36 - 0.40)(0.35 - 0.36)(0.036 - 0.050)(0.29 - 0.49)Phenylalanine1.39 (0.027)1.41 (0.038)-0.022 (0.045)-0.15, 0.100.6491.12, 1.58(1.34 - 1.43)(1.34 - 1.48)(-0.10 - 0.067)(1.10 - 1.63)Proline0.97 (0.021)1.00 (0.028)-0.023 (0.030)-0.11, 0.0610.4900.83, 1.08(0.89 - 1.03)(0.98 - 1.02)(-0.044 - 0.0082)(0.79 - 1.17)Serine1.05 (0.022)1.03 (0.031)0.022 (0.038)-0.083, 0.130.5900.83, 1.21(1.02 - 1.07)(0.99 - 1.06)(0.011 - 0.078)(0.81 - 1.24)Monsanto Company 12-CT-244U 455 of 620Table E-23. Statistical Summary of Site ARTI CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.85 (0.012)0.85 (0.017)-0.00041 (0.021)-0.058, 0.0570.9850.72, 0.89(0.82 - 0.86)(0.82 - 0.88)(-0.028 - 0.040)(0.67 - 0.96)Tryptophan0.41 (0.0062)0.42 (0.0087)-0.0085 (0.011)-0.038, 0.0210.4680.34, 0.42(0.41 - 0.42)(0.40 - 0.44)(-0.033 - 0.012)(0.31 - 0.46)Tyrosine0.80 (0.015)0.79 (0.021)0.0015 (0.026)-0.071, 0.0740.9570.67, 0.84(0.76 - 0.83)(0.76 - 0.82)(-0.034 - 0.043)(0.63 - 0.91)Valine1.17 (0.021)1.21 (0.027)-0.034 (0.026)-0.11, 0.0390.2631.00, 1.28(1.12 - 1.23)(1.17 - 1.24)(-0.059 - -0.0050)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.78 (0.0085)0.78 (0.012)-0.0013 (0.013)-0.037, 0.0350.9270.16, 1.37(0.76 - 0.80)(0.77 - 0.78)(-0.0043 - 0.0026)(0.45 - 1.04)16:0 Palmitic25.06 (0.10)24.98 (0.14)0.075 (0.18)-0.41, 0.560.69216.54, 30.55(24.85 - 25.45)(24.92 - 25.05)(0.012 - 0.41)(19.11 - 26.73)Monsanto Company 12-CT-244U 456 of 620Table E-23. Statistical Summary of Site ARTI CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.54 (0.0052)0.52 (0.0073)0.018 (0.0089)-0.0064, 0.0430.1070.39, 0.70(0.52 - 0.55)(0.52 - 0.52)(0.016 - 0.024)(0.44 - 0.67)18:0 Stearic2.65 (0.026)2.51 (0.036)0.14 (0.045)0.015, 0.260.0351.98, 2.95(2.59 - 2.71)(2.45 - 2.57)(0.10 - 0.26)(1.98 - 2.97)18:1 Oleic14.82 (0.11)14.68 (0.15)0.13 (0.17)-0.33, 0.600.47511.38, 20.64(14.65 - 14.99)(14.58 - 14.70)(-0.050 - 0.37)(13.71 - 18.39)18:2 Linoleic54.83 (0.22)55.31 (0.31)-0.49 (0.37)-1.52, 0.550.26347.49, 63.18(54.28 - 55.22)(55.26 - 55.38)(-1.09 - -0.074)(49.78 - 59.61)18:3 Linolenic0.14 (0.0021)0.13 (0.0030)0.013 (0.0035)0.0030, 0.0230.0220.060, 0.24(0.14 - 0.14)(0.12 - 0.14)(0.0051 - 0.019)(0.10 - 0.29)20:0 Arachidic0.31 (0.0079)0.29 (0.011)0.015 (0.011)-0.017, 0.0460.2660.17, 0.38(0.28 - 0.32)(0.27 - 0.31)(0.0037 - 0.012)(0.20 - 0.36)Monsanto Company 12-CT-244U 457 of 620Table E-23. Statistical Summary of Site ARTI CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.14 (0.0026)0.15 (0.0033)-0.010 (0.0031)-0.019, -0.00160.0290.070, 0.21(0.14 - 0.15)(0.15 - 0.16)(-0.018 - -0.0043)(0.051 - 0.19)MineralCalcium (% dw)0.15 (0.0035)0.12 (0.0050)0.030 (0.0061)0.013, 0.0470.0070.058, 0.21(0.14 - 0.16)(0.12 - 0.13)(0.024 - 0.039)(0.081 - 0.18)Copper (mg/kg dw)9.81 (0.34)9.64 (0.41)0.17 (0.38)-0.88, 1.210.6802.97, 12.86(9.20 - 10.96)(8.79 - 9.79)(-0.45 - 0.41)(4.46 - 11.62)Iron (mg/kg dw)86.87 (5.63)80.76 (7.78)6.11 (8.79)-18.31, 30.530.52547.30, 97.12(72.55 - 103.10)(72.89 - 87.72)(0.83 - 10.40)(39.49 - 114.34)Magnesium (% dw)0.43 (0.0076)0.40 (0.010)0.028 (0.011)-0.0019, 0.0580.0590.28, 0.47(0.41 - 0.45)(0.38 - 0.41)(0.013 - 0.037)(0.31 - 0.46)Manganese (mg/kg dw)14.86 (0.63)11.50 (0.89)3.36 (1.06)0.41, 6.320.0349.07, 17.33(13.28 - 17.47)(11.34 - 11.55)(2.51 - 3.29)(9.07 - 17.14)Monsanto Company 12-CT-244U 458 of 620Table E-23. Statistical Summary of Site ARTI CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.84 (0.012)0.84 (0.016)-0.0071 (0.018)-0.058, 0.0440.7170.49, 0.87(0.80 - 0.87)(0.82 - 0.87)(-0.027 - 0.012)(0.48 - 0.87)Potassium (% dw)1.22 (0.029)1.11 (0.040)0.12 (0.047)-0.013, 0.250.0670.92, 1.21(1.16 - 1.32)(1.06 - 1.15)(0.092 - 0.11)(0.90 - 1.26)Sodium (% dw)0.017 (0.0038)ND60, 0.066(0.0054 - 0.030)(0.013 - 0.013)(0.0054 - 0.077)Zinc (mg/kg dw)39.24 (1.65)40.81 (2.03)-1.56 (1.87)-6.76, 3.630.44927.27, 44.95(35.79 - 45.91)(38.63 - 40.71)(-2.84 - -2.25)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E141.82 (2.44)136.55 (3.46)5.27 (4.23)-6.49, 17.020.28141.91, 205.89(132.11 - 146.82)(133.79 - 139.31)(-7.20 - 12.36)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.6Not determined due to insufficient number of observations for the control.Monsanto Company 12-CT-244U 459 of 620Table E-24. Statistical Summary of Site ARTI Cottonseed Anti-nutrients for MON 88701 (NotTreated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.0073)0.15 (0.010)0.0079 (0.013)-0.027, 0.0430.5670.078, 0.25(0.14 - 0.18)(0.14 - 0.15)(-0.011 - 0.0068)(0.038 - 0.23)Malvalic Acid0.37 (0.016)0.36 (0.023)0.011 (0.025)-0.058, 0.0810.6740.23, 0.54(0.33 - 0.43)(0.33 - 0.37)(-0.0096 - 0.012)(0.11 - 0.59)Sterculic Acid0.23 (0.014)0.20 (0.020)0.030 (0.023)-0.035, 0.0940.2720.17, 0.27(0.17 - 0.27)(0.19 - 0.20)(-0.013 - 0.022)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.84 (0.035)0.82 (0.049)0.020 (0.060)-0.15, 0.190.7490.099, 1.57(0.78 - 0.92)(0.80 - 0.84)(-0.021 - -0.0027)(0.50 - 1.41)Total Gossypol0.93 (0.034)0.93 (0.044)0.0038 (0.044)-0.12, 0.130.9340.064, 1.76(0.87 - 1.04)(0.94 - 0.98)(-0.0059 - 0.063)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 460 of 620Table E-25. Statistical Summary of SiteGACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.39 (0.044)4.21 (0.047)0.18 (0.034)0.095, 0.270.0033.42, 4.65(4.24 - 4.50)(4.12 - 4.23)(0.12 - 0.25)(3.18 - 4.68)Calories (Kcal/100g)501.28 (2.28)496.55 (2.63)4.73 (3.48)-4.23, 13.690.232457.61, 527.56(498.88 - 503.43)(494.57 - 498.27)(2.32 - 6.61)(466.09 - 509.91)Carbohydrates44.81 (0.59)45.84 (0.68)-1.03 (0.91)-3.36, 1.300.30640.26, 56.45(44.28 - 45.20)(44.64 - 47.09)(-1.89 - -0.36)(43.28 - 54.90)Moisture (% fw)6.39 (0.15)7.23 (0.18)-0.84 (0.23)-1.43, -0.240.0154.79, 9.92(6.19 - 6.64)(6.99 - 7.48)(-0.97 - -0.57)(6.05 - 10.50)Protein27.03 (0.33)27.30 (0.37)-0.27 (0.37)-1.21, 0.670.49322.30, 29.41(26.24 - 28.02)(26.45 - 28.21)(-1.02 - 0.63)(20.58 - 29.28)Total Fat23.77 (0.46)22.67 (0.53)1.10 (0.70)-0.71, 2.910.18015.01, 28.51(23.35 - 24.21)(22.26 - 23.02)(0.40 - 1.47)(16.58 - 25.25)Monsanto Company 12-CT-244U 461 of 620Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber26.12 (0.52)27.52 (0.60)-1.40 (0.79)-3.43, 0.630.13622.24, 31.96(24.62 - 27.38)(26.81 - 28.13)(-3.01 - 0.58)(23.42 - 31.62)Crude Fiber18.88 (0.68)19.92 (0.78)-1.04 (1.00)-3.60, 1.530.34616.93, 22.68(17.57 - 19.90)(18.70 - 21.18)(-2.85 - 1.20)(16.92 - 23.32)Neutral Detergent Fiber32.42 (0.90)33.92 (1.04)-1.49 (1.38)-5.03, 2.050.32727.03, 42.49(30.60 - 34.01)(32.79 - 35.89)(-1.93 - 0.94)(29.27 - 40.63)Total Dietary Fiber39.71 (0.86)41.11 (1.00)-1.39 (1.32)-4.79, 2.000.33934.52, 52.58(37.96 - 42.04)(39.89 - 42.04)(-3.36 - 0.94)(37.29 - 48.60)Amino Acid (% dw)Alanine1.06 (0.019)1.10 (0.022)-0.034 (0.025)-0.098, 0.0300.2340.86, 1.11(1.02 - 1.10)(1.06 - 1.17)(-0.076 - 0.029)(0.83 - 1.22)Arginine3.06 (0.061)3.21 (0.067)-0.16 (0.064)-0.32, 0.00980.0602.38, 3.47(2.96 - 3.18)(3.07 - 3.46)(-0.28 - -0.062)(2.30 - 3.55)Monsanto Company 12-CT-244U 462 of 620Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.39 (0.043)2.45 (0.047)-0.066 (0.044)-0.18, 0.0470.1921.94, 2.57(2.30 - 2.49)(2.36 - 2.60)(-0.11 - 0.00069)(1.79 - 2.72)Cystine0.41 (0.012)0.40 (0.013)0.0020 (0.018)-0.043, 0.0470.9130.31, 0.45(0.38 - 0.43)(0.38 - 0.43)(-0.032 - 0.050)(0.29 - 0.47)Glutamic Acid4.69 (0.098)4.96 (0.11)-0.27 (0.099)-0.52, -0.0160.0413.74, 5.28(4.51 - 4.98)(4.77 - 5.21)(-0.26 - -0.23)(3.39 - 5.45)Glycine1.11 (0.020)1.13 (0.023)-0.016 (0.026)-0.083, 0.0500.5590.90, 1.14(1.06 - 1.14)(1.09 - 1.20)(-0.055 - 0.033)(0.85 - 1.23)Histidine0.76 (0.013)0.76 (0.014)-0.00082 (0.012)-0.033, 0.0310.9490.59, 0.81(0.73 - 0.78)(0.75 - 0.78)(-0.018 - 0.0081)(0.57 - 0.84)Isoleucine0.95 (0.010)0.94 (0.012)0.00070 (0.014)-0.035, 0.0360.9610.75, 0.96(0.91 - 0.97)(0.92 - 0.97)(-0.012 - 0.011)(0.72 - 1.03)Monsanto Company 12-CT-244U 463 of 620Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.56 (0.022)1.58 (0.024)-0.019 (0.021)-0.074, 0.0370.4251.25, 1.62(1.51 - 1.60)(1.52 - 1.65)(-0.049 - 0.0079)(1.20 - 1.72)Lysine1.26 (0.018)1.23 (0.020)0.029 (0.024)-0.033, 0.0910.2861.01, 1.30(1.19 - 1.29)(1.22 - 1.25)(-0.040 - 0.062)(0.99 - 1.44)Methionine0.40 (0.017)0.42 (0.019)-0.019 (0.024)-0.080, 0.0420.4640.32, 0.38(0.38 - 0.42)(0.37 - 0.46)(-0.081 - 0.035)(0.29 - 0.49)Phenylalanine1.45 (0.030)1.49 (0.033)-0.040 (0.032)-0.12, 0.0410.2591.12, 1.58(1.41 - 1.49)(1.41 - 1.61)(-0.12 - 0.0089)(1.10 - 1.63)Proline1.03 (0.020)1.05 (0.022)-0.021 (0.022)-0.078, 0.0360.3930.83, 1.08(0.97 - 1.10)(1.03 - 1.09)(-0.065 - 0.010)(0.79 - 1.17)Serine1.08 (0.031)1.12 (0.035)-0.040 (0.039)-0.14, 0.0610.3570.83, 1.21(1.02 - 1.14)(1.08 - 1.20)(-0.055 - 0.023)(0.81 - 1.24)Monsanto Company 12-CT-244U 464 of 620Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.88 (0.015)0.90 (0.017)-0.017 (0.018)-0.063, 0.0290.3950.72, 0.89(0.85 - 0.91)(0.87 - 0.95)(-0.040 - 0.016)(0.67 - 0.96)Tryptophan0.41 (0.011)0.42 (0.013)-0.016 (0.017)-0.061, 0.0280.3890.34, 0.42(0.39 - 0.42)(0.39 - 0.44)(-0.042 - 0.030)(0.31 - 0.46)Tyrosine0.84 (0.011)0.84 (0.013)-0.0011 (0.013)-0.034, 0.0320.9360.67, 0.84(0.82 - 0.85)(0.81 - 0.89)(-0.036 - 0.027)(0.63 - 0.91)Valine1.24 (0.018)1.26 (0.019)-0.020 (0.015)-0.059, 0.0180.2351.00, 1.28(1.19 - 1.28)(1.23 - 1.32)(-0.040 - 0.011)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.77 (0.0068)0.77 (0.0079)-0.0036 (0.010)-0.030, 0.0230.7450.16, 1.37(0.76 - 0.79)(0.76 - 0.78)(-0.026 - 0.022)(0.45 - 1.04)16:0 Palmitic24.27 (0.14)24.12 (0.16)0.15 (0.20)-0.37, 0.670.49416.54, 30.55(24.04 - 24.70)(23.78 - 24.45)(-0.20 - 0.26)(19.11 - 26.73)Monsanto Company 12-CT-244U 465 of 620Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.51 (0.0058)0.51 (0.0067)-0.0013 (0.0089)-0.024, 0.0220.8860.39, 0.70(0.50 - 0.52)(0.50 - 0.52)(-0.0059 - 0.013)(0.44 - 0.67)18:0 Stearic2.50 (0.032)2.43 (0.037)0.070 (0.046)-0.050, 0.190.1931.98, 2.95(2.45 - 2.54)(2.37 - 2.46)(0.039 - 0.16)(1.98 - 2.97)18:1 Oleic14.51 (0.098)14.39 (0.11)0.12 (0.13)-0.20, 0.450.37411.38, 20.64(14.39 - 14.63)(14.06 - 14.61)(0.00041 - 0.47)(13.71 - 18.39)18:2 Linoleic56.07 (0.24)56.59 (0.28)-0.52 (0.35)-1.41, 0.380.19747.49, 63.18(55.90 - 56.24)(56.02 - 57.32)(-1.16 - -0.12)(49.78 - 59.61)18:3 Linolenic0.14 (0.0066)0.15 (0.0076)-0.0076 (0.010)-0.033, 0.0180.4850.060, 0.24(0.11 - 0.15)(0.14 - 0.15)(-0.0064 - 0.0089)(0.10 - 0.29)20:0 Arachidic0.29 (0.0098)0.28 (0.011)0.015 (0.013)-0.019, 0.0490.3090.17, 0.38(0.26 - 0.30)(0.26 - 0.29)(0.0032 - 0.042)(0.20 - 0.36)Monsanto Company 12-CT-244U 466 of 620Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.15 (0.0026)0.14 (0.0030)0.0075 (0.0037)-0.0021, 0.0170.1000.070, 0.21(0.14 - 0.16)(0.14 - 0.15)(0.0012 - 0.013)(0.051 - 0.19)MineralCalcium (% dw)0.13 (0.0015)0.11 (0.0018)0.020 (0.0023)0.014, 0.026<0.0010.058, 0.21(0.13 - 0.13)(0.11 - 0.11)(0.016 - 0.024)(0.081 - 0.18)Copper (mg/kg dw)8.67 (0.26)8.21 (0.30)0.46 (0.39)-0.54, 1.470.2882.97, 12.86(8.13 - 9.20)(7.48 - 8.64)(0.055 - 1.72)(4.46 - 11.62)Iron (mg/kg dw)74.17 (3.80)78.00 (4.39)-3.83 (5.81)-18.76, 11.100.53947.30, 97.12(59.77 - 87.01)(75.01 - 80.40)(-20.63 - -0.99)(39.49 - 114.34)Magnesium (% dw)0.41 (0.0052)0.38 (0.0054)0.032 (0.0034)0.023, 0.040<0.0010.28, 0.47(0.40 - 0.43)(0.37 - 0.39)(0.026 - 0.034)(0.31 - 0.46)Manganese (mg/kg dw)14.26 (0.31)11.51 (0.34)2.75 (0.37)1.80, 3.70<0.0019.07, 17.33(13.82 - 15.04)(10.81 - 11.75)(2.07 - 3.10)(9.07 - 17.14)Monsanto Company 12-CT-244U 467 of 620Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.78 (0.011)0.76 (0.012)0.012 (0.0082)-0.0088, 0.0340.1920.49, 0.87(0.75 - 0.81)(0.75 - 0.79)(0.00079 - 0.021)(0.48 - 0.87)Potassium (% dw)1.22 (0.012)1.12 (0.013)0.098 (0.010)0.072, 0.12<0.0010.92, 1.21(1.18 - 1.24)(1.10 - 1.13)(0.087 - 0.11)(0.90 - 1.26)Sodium (% dw)0.022 (0.0045)0.017 (0.0052)0.0051 (0.0069)-0.013, 0.0230.4970, 0.066(0.0053 - 0.039)(0.013 - 0.022)(-0.0019 - 0.026)(0.0054 - 0.077)Zinc (mg/kg dw)38.62 (0.67)39.55 (0.75)-0.93 (0.83)-3.06, 1.200.31127.27, 44.95(37.11 - 40.86)(38.49 - 40.84)(-2.50 - 1.19)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E149.29 (2.27)140.12 (2.63)9.17 (3.47)0.24, 18.100.04641.91, 205.89(142.84 - 153.55)(133.64 - 145.15)(-2.30 - 15.25)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 468 of 620Table E-26. Statistical Summary of SiteGACH Cottonseed Anti-nutrients for MON 88701 (Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.16 (0.0067)0.12 (0.0077)0.039 (0.010)0.012, 0.0650.0120.078, 0.25(0.14 - 0.17)(0.11 - 0.13)(0.020 - 0.058)(0.038 - 0.23)Malvalic Acid0.41 (0.027)0.32 (0.031)0.087 (0.038)-0.011, 0.190.0720.23, 0.54(0.36 - 0.43)(0.31 - 0.34)(0.085 - 0.11)(0.11 - 0.59)Sterculic Acid0.23 (0.011)0.18 (0.013)0.045 (0.015)0.0071, 0.0820.0280.17, 0.27(0.20 - 0.24)(0.18 - 0.20)(0.039 - 0.059)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.91 (0.017)0.86 (0.019)0.044 (0.020)-0.0089, 0.0960.0850.099, 1.57(0.85 - 0.95)(0.85 - 0.90)(0.036 - 0.091)(0.50 - 1.41)Total Gossypol0.98 (0.019)0.96 (0.020)0.024 (0.017)-0.020, 0.0680.2210.064, 1.76(0.93 - 1.03)(0.90 - 0.99)(-0.010 - 0.061)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 469 of 620Table E-27. Statistical Summary of SiteKSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.46 (0.063)4.29 (0.071)0.16 (0.078)-0.038, 0.370.0913.42, 4.65(4.40 - 4.55)(4.21 - 4.33)(0.071 - 0.23)(3.18 - 4.68)Calories (Kcal/100g)500.94 (2.75)495.83 (3.16)5.11 (3.87)-4.84, 15.060.244457.61, 527.56(494.73 - 507.94)(492.30 - 504.10)(0.76 - 15.41)(466.09 - 509.91)Carbohydrates44.42 (0.71)44.23 (0.82)0.19 (1.09)-2.62, 2.990.87040.26, 56.45(42.57 - 46.25)(42.53 - 45.14)(-0.94 - 0.041)(43.28 - 54.90)Moisture (% fw)7.08 (0.17)7.36 (0.19)-0.29 (0.20)-0.81, 0.230.2134.79, 9.92(6.51 - 7.47)(7.17 - 7.62)(-0.85 - 0.13)(6.05 - 10.50)Protein27.36 (0.62)28.82 (0.72)-1.47 (0.93)-3.86, 0.930.17622.30, 29.41(26.05 - 28.45)(28.58 - 29.04)(-2.53 - -0.58)(20.58 - 29.28)Total Fat23.76 (0.55)22.62 (0.64)1.13 (0.81)-0.96, 3.220.22215.01, 28.51(22.48 - 25.18)(21.87 - 24.18)(0.42 - 3.21)(16.58 - 25.25)Monsanto Company 12-CT-244U 470 of 620Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber24.37 (0.55)24.01 (0.61)0.36 (0.65)-1.31, 2.030.60122.24, 31.96(23.30 - 25.67)(22.08 - 25.22)(-0.26 - 1.22)(23.42 - 31.62)Crude Fiber16.71 (0.24)17.67 (0.28)-0.96 (0.37)-1.91, -0.0150.04716.93, 22.68(16.10 - 17.37)(17.49 - 17.88)(-1.54 - -0.51)(16.92 - 23.32)Neutral Detergent Fiber28.65 (0.88)30.20 (1.01)-1.54 (1.21)-4.66, 1.570.25927.03, 42.49(28.05 - 29.04)(28.87 - 32.60)(-3.72 - -0.82)(29.27 - 40.63)Total Dietary Fiber38.55 (0.42)40.14 (0.49)-1.59 (0.63)-3.22, 0.0440.05434.52, 52.58(37.44 - 39.47)(39.32 - 41.35)(-2.29 - -1.13)(37.29 - 48.60)Amino Acid (% dw)Alanine1.03 (0.011)1.06 (0.012)-0.035 (0.014)-0.071, 0.000960.0540.86, 1.11(1.02 - 1.04)(1.02 - 1.10)(-0.061 - 0.0081)(0.83 - 1.22)Arginine3.00 (0.053)3.28 (0.060)-0.28 (0.069)-0.45, -0.100.0092.38, 3.47(2.88 - 3.05)(3.10 - 3.43)(-0.37 - -0.059)(2.30 - 3.55)Monsanto Company 12-CT-244U 471 of 620Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.35 (0.037)2.50 (0.043)-0.15 (0.053)-0.28, -0.0120.0381.94, 2.57(2.32 - 2.37)(2.39 - 2.64)(-0.27 - -0.047)(1.79 - 2.72)Cystine0.39 (0.010)0.42 (0.012)-0.032 (0.016)-0.073, 0.00830.0960.31, 0.45(0.36 - 0.42)(0.41 - 0.43)(-0.074 - 0.011)(0.29 - 0.47)Glutamic Acid4.68 (0.12)5.07 (0.14)-0.40 (0.18)-0.86, 0.0620.0763.74, 5.28(4.63 - 4.71)(4.86 - 5.43)(-0.72 - -0.18)(3.39 - 5.45)Glycine1.07 (0.018)1.11 (0.021)-0.047 (0.027)-0.12, 0.0230.1440.90, 1.14(1.05 - 1.08)(1.07 - 1.18)(-0.11 - 0.016)(0.85 - 1.23)Histidine0.73 (0.013)0.77 (0.014)-0.050 (0.011)-0.078, -0.0220.0060.59, 0.81(0.69 - 0.76)(0.74 - 0.80)(-0.069 - -0.031)(0.57 - 0.84)Isoleucine0.90 (0.014)0.95 (0.015)-0.049 (0.014)-0.085, -0.0130.0170.75, 0.96(0.88 - 0.94)(0.91 - 0.98)(-0.074 - -0.017)(0.72 - 1.03)Monsanto Company 12-CT-244U 472 of 620Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.51 (0.020)1.58 (0.022)-0.074 (0.021)-0.13, -0.0200.0171.25, 1.62(1.47 - 1.55)(1.52 - 1.65)(-0.095 - -0.013)(1.20 - 1.72)Lysine1.19 (0.017)1.25 (0.019)-0.065 (0.021)-0.12, -0.0100.0281.01, 1.30(1.16 - 1.22)(1.19 - 1.30)(-0.084 - -0.024)(0.99 - 1.44)Methionine0.39 (0.015)0.39 (0.017)0.0063 (0.019)-0.041, 0.0540.7470.32, 0.38(0.38 - 0.41)(0.34 - 0.44)(-0.053 - 0.049)(0.29 - 0.49)Phenylalanine1.42 (0.022)1.53 (0.025)-0.11 (0.029)-0.18, -0.0310.0141.12, 1.58(1.37 - 1.45)(1.45 - 1.58)(-0.14 - -0.013)(1.10 - 1.63)Proline1.00 (0.018)1.07 (0.020)-0.065 (0.027)-0.13, 0.00390.0590.83, 1.08(0.96 - 1.03)(1.03 - 1.12)(-0.094 - -0.00058)(0.79 - 1.17)Serine1.07 (0.029)1.11 (0.034)-0.044 (0.045)-0.16, 0.0710.3730.83, 1.21(1.05 - 1.07)(1.06 - 1.20)(-0.14 - 0.017)(0.81 - 1.24)Monsanto Company 12-CT-244U 473 of 620Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.84 (0.013)0.88 (0.015)-0.036 (0.019)-0.086, 0.0140.1210.72, 0.89(0.82 - 0.86)(0.83 - 0.92)(-0.062 - 0.021)(0.67 - 0.96)Tryptophan0.42 (0.0061)0.42 (0.0070)-0.0058 (0.0090)-0.029, 0.0170.5510.34, 0.42(0.40 - 0.44)(0.42 - 0.43)(-0.024 - 0.012)(0.31 - 0.46)Tyrosine0.79 (0.014)0.84 (0.016)-0.050 (0.019)-0.098, -0.00130.0450.67, 0.84(0.76 - 0.80)(0.79 - 0.87)(-0.076 - -0.0034)(0.63 - 0.91)Valine1.20 (0.017)1.26 (0.019)-0.058 (0.023)-0.12, 0.000070.0501.00, 1.28(1.15 - 1.23)(1.21 - 1.30)(-0.076 - -0.0020)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.69 (0.0087)0.72 (0.0096)-0.033 (0.0090)-0.056, -0.0100.0130.16, 1.37(0.66 - 0.70)(0.71 - 0.73)(-0.050 - -0.020)(0.45 - 1.04)16:0 Palmitic22.54 (0.089)22.73 (0.10)-0.19 (0.13)-0.52, 0.150.20816.54, 30.55(22.30 - 22.75)(22.69 - 22.78)(-0.48 - 0.057)(19.11 - 26.73)Monsanto Company 12-CT-244U 474 of 620Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.48 (0.0057)0.49 (0.0062)-0.0079 (0.0059)-0.023, 0.00730.2390.39, 0.70(0.47 - 0.50)(0.49 - 0.50)(-0.015 - 0.0026)(0.44 - 0.67)18:0 Stearic2.25 (0.031)2.20 (0.036)0.048 (0.047)-0.074, 0.170.3561.98, 2.95(2.16 - 2.34)(2.15 - 2.28)(-0.066 - 0.16)(1.98 - 2.97)18:1 Oleic14.65 (0.11)14.83 (0.13)-0.18 (0.17)-0.62, 0.270.35611.38, 20.64(14.23 - 14.99)(14.74 - 14.99)(-0.54 - 0.0051)(13.71 - 18.39)18:2 Linoleic58.08 (0.19)57.78 (0.22)0.30 (0.29)-0.43, 1.040.33547.49, 63.18(57.39 - 58.48)(57.65 - 57.93)(-0.26 - 0.73)(49.78 - 59.61)18:3 Linolenic0.18 (0.0027)0.17 (0.0030)0.0014 (0.0033)-0.0070, 0.00990.6840.060, 0.24(0.17 - 0.18)(0.17 - 0.18)(-0.0031 - 0.0059)(0.10 - 0.29)20:0 Arachidic0.23 (0.0029)0.24 (0.0034)-0.0038 (0.0045)-0.015, 0.00770.4350.17, 0.38(0.23 - 0.24)(0.23 - 0.24)(-0.011 - 0.0067)(0.20 - 0.36)Monsanto Company 12-CT-244U 475 of 620Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.13 (0.0030)0.13 (0.0035)-0.0068 (0.0046)-0.019, 0.00490.1960.070, 0.21(0.12 - 0.14)(0.13 - 0.14)(-0.019 - 0.0086)(0.051 - 0.19)MineralCalcium (% dw)0.20 (0.0060)0.18 (0.0065)0.018 (0.0061)0.0022, 0.0340.0320.058, 0.21(0.18 - 0.21)(0.17 - 0.19)(0.0037 - 0.021)(0.081 - 0.18)Copper (mg/kg dw)10.86 (0.54)11.01 (0.61)-0.15 (0.71)-1.97, 1.670.8422.97, 12.86(9.15 - 12.15)(10.09 - 11.33)(-2.19 - 1.26)(4.46 - 11.62)Iron (mg/kg dw)90.85 (5.50)74.39 (6.35)16.46 (8.40)-5.13, 38.050.10747.30, 97.12(74.66 - 109.70)(72.65 - 76.27)(1.19 - 27.33)(39.49 - 114.34)Magnesium (% dw)0.42 (0.0033)0.40 (0.0038)0.023 (0.0046)0.011, 0.0350.0040.28, 0.47(0.41 - 0.43)(0.39 - 0.40)(0.015 - 0.026)(0.31 - 0.46)Manganese (mg/kg dw)14.18 (0.29)12.56 (0.33)1.62 (0.44)0.50, 2.740.0139.07, 17.33(13.48 - 14.84)(11.96 - 13.28)(0.20 - 2.14)(9.07 - 17.14)Monsanto Company 12-CT-244U 476 of 620Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.76 (0.0090)0.78 (0.010)-0.014 (0.011)-0.042, 0.0140.2510.49, 0.87(0.75 - 0.78)(0.77 - 0.79)(-0.020 - -0.0075)(0.48 - 0.87)Potassium (% dw)1.11 (0.010)1.08 (0.012)0.029 (0.015)-0.0087, 0.0670.1040.92, 1.21(1.08 - 1.12)(1.05 - 1.10)(0.020 - 0.030)(0.90 - 1.26)Sodium (% dw)0.015 (0.0028)0.0080 (0.0032)0.0070 (0.0042)-0.0039, 0.0180.1590, 0.066(0.0054 - 0.022)(0.0054 - 0.013)(0.0062 - 0.016)(0.0054 - 0.077)Zinc (mg/kg dw)40.63 (1.18)42.00 (1.37)-1.37 (1.81)-6.01, 3.270.48227.27, 44.95(36.90 - 42.39)(40.59 - 43.50)(-6.60 - 0.49)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E94.05 (2.67)92.34 (2.91)1.71 (2.71)-5.26, 8.670.55641.91, 205.89(87.22 - 99.35)(91.78 - 95.85)(-2.10 - 7.57)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 477 of 620Table E-28. Statistical Summary of SiteKSLA Cottonseed Anti-nutrients for MON 88701 (Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.14 (0.010)0.12 (0.012)0.017 (0.014)-0.020, 0.0540.2960.078, 0.25(0.13 - 0.15)(0.12 - 0.13)(0.00032 - 0.031)(0.038 - 0.23)Malvalic Acid0.41 (0.030)0.37 (0.034)0.044 (0.039)-0.056, 0.150.3080.23, 0.54(0.35 - 0.45)(0.33 - 0.39)(-0.012 - 0.094)(0.11 - 0.59)Sterculic Acid0.21 (0.014)0.20 (0.016)0.012 (0.019)-0.036, 0.0600.5520.17, 0.27(0.19 - 0.22)(0.19 - 0.21)(-0.019 - 0.030)(0.061 - 0.34)Gossypol (% dw)Free Gossypol1.00 (0.027)0.95 (0.030)0.055 (0.033)-0.029, 0.140.1510.099, 1.57(0.96 - 1.03)(0.86 - 1.05)(-0.030 - 0.13)(0.50 - 1.41)Total Gossypol1.09 (0.033)1.01 (0.038)0.075 (0.047)-0.046, 0.200.1720.064, 1.76(1.05 - 1.12)(1.00 - 1.02)(0.073 - 0.11)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 478 of 620Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.27 (0.047)4.12 (0.047)0.16 (0.066)-0.0069, 0.320.0573.42, 4.65(4.14 - 4.40)(4.06 - 4.15)(0.081 - 0.25)(3.18 - 4.68)Calories (Kcal/100g dw)498.08 (3.01)494.75 (3.01)3.33 (4.19)-6.92, 13.580.457457.61, 527.56(493.35 - 504.84)(490.27 - 498.67)(-3.41 - 14.57)(466.09 - 509.91)Carbohydrates45.34 (0.79)47.84 (0.79)-2.50 (1.11)-5.22, 0.230.06640.26, 56.45(44.46 - 46.08)(46.77 - 50.30)(-5.09 - -1.23)(43.28 - 54.90)Moisture (% fw)6.44 (0.26)6.98 (0.26)-0.54 (0.36)-1.43, 0.350.1904.79, 9.92(5.81 - 7.10)(6.15 - 7.40)(-1.45 - 0.11)(6.05 - 10.50)Protein27.41 (0.56)25.80 (0.56)1.62 (0.80)-0.33, 3.560.08822.30, 29.41(26.70 - 28.07)(23.53 - 27.85)(-1.15 - 4.39)(20.58 - 29.28)Total Fat22.96 (0.64)22.25 (0.64)0.71 (0.90)-1.49, 2.900.46215.01, 28.51(22.01 - 24.43)(21.29 - 23.02)(-0.67 - 3.15)(16.58 - 25.25)Monsanto Company 12-CT-244U 479 of 620Table E-29. Statistical Summary of Site LACH CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.94 (0.45)28.35 (0.45)-2.41 (0.63)-3.96, -0.860.00822.24, 31.96(25.56 - 26.33)(27.81 - 29.58)(-4.02 - -1.48)(23.42 - 31.62)Crude Fiber17.47 (0.66)19.62 (0.66)-2.15 (0.93)-4.42, 0.120.05916.93, 22.68(14.96 - 19.14)(18.46 - 20.54)(-5.57 - 0.12)(16.92 - 23.32)Neutral Detergent Fiber32.12 (0.65)34.05 (0.65)-1.93 (0.42)-2.96, -0.890.00327.03, 42.49(30.20 - 33.94)(32.61 - 35.84)(-2.42 - -1.25)(29.27 - 40.63)Total Dietary Fiber41.09 (0.49)43.35 (0.49)-2.26 (0.69)-3.94, -0.580.01634.52, 52.58(39.95 - 42.26)(42.33 - 44.37)(-4.41 - -0.26)(37.29 - 48.60)Amino Acid (% dw)Alanine1.06 (0.018)1.03 (0.018)0.028 (0.014)-0.0052, 0.0620.0840.86, 1.11(1.02 - 1.07)(1.00 - 1.06)(0.012 - 0.047)(0.83 - 1.22)Arginine3.00 (0.073)2.98 (0.073)0.018 (0.084)-0.19, 0.220.8362.38, 3.47(2.92 - 3.09)(2.89 - 3.13)(-0.13 - 0.16)(2.30 - 3.55)Monsanto Company 12-CT-244U 480 of 620Table E-29. Statistical Summary of Site LACH CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.38 (0.050)2.30 (0.050)0.075 (0.062)-0.076, 0.230.2691.94, 2.57(2.35 - 2.45)(2.25 - 2.40)(-0.040 - 0.18)(1.79 - 2.72)Cystine0.38 (0.016)0.38 (0.016)-0.0067 (0.019)-0.054, 0.0410.7410.31, 0.45(0.35 - 0.40)(0.36 - 0.44)(-0.038 - 0.031)(0.29 - 0.47)Glutamic Acid4.67 (0.13)4.52 (0.13)0.15 (0.16)-0.25, 0.550.3843.74, 5.28(4.57 - 4.83)(4.37 - 4.75)(-0.18 - 0.36)(3.39 - 5.45)Glycine1.10 (0.020)1.06 (0.020)0.039 (0.025)-0.022, 0.0990.1680.90, 1.14(1.07 - 1.12)(1.04 - 1.09)(0.012 - 0.076)(0.85 - 1.23)Histidine0.73 (0.020)0.72 (0.020)0.010 (0.014)-0.023, 0.0440.4770.59, 0.81(0.68 - 0.77)(0.67 - 0.76)(-0.015 - 0.062)(0.57 - 0.84)Isoleucine0.91 (0.018)0.88 (0.018)0.029 (0.020)-0.019, 0.0770.1860.75, 0.96(0.88 - 0.93)(0.87 - 0.90)(0.017 - 0.056)(0.72 - 1.03)Monsanto Company 12-CT-244U 481 of 620Table E-29. Statistical Summary of Site LACH CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.52 (0.026)1.48 (0.026)0.045 (0.028)-0.024, 0.110.1581.25, 1.62(1.50 - 1.54)(1.44 - 1.52)(0.013 - 0.079)(1.20 - 1.72)Lysine1.25 (0.024)1.18 (0.024)0.074 (0.027)0.0069, 0.140.0351.01, 1.30(1.23 - 1.27)(1.12 - 1.23)(0.023 - 0.14)(0.99 - 1.44)Methionine0.40 (0.017)0.38 (0.017)0.028 (0.013)-0.0036, 0.0600.0730.32, 0.38(0.38 - 0.42)(0.32 - 0.42)(-0.0027 - 0.064)(0.29 - 0.49)Phenylalanine1.43 (0.027)1.39 (0.027)0.038 (0.033)-0.043, 0.120.2961.12, 1.58(1.41 - 1.46)(1.36 - 1.43)(-0.0091 - 0.090)(1.10 - 1.63)Proline1.03 (0.015)0.98 (0.015)0.045 (0.022)-0.0079, 0.0990.0820.83, 1.08(1.02 - 1.05)(0.95 - 1.02)(0.0012 - 0.10)(0.79 - 1.17)Serine1.06 (0.022)1.03 (0.022)0.025 (0.030)-0.049, 0.0990.4450.83, 1.21(1.04 - 1.08)(1.01 - 1.06)(0.0023 - 0.049)(0.81 - 1.24)Monsanto Company 12-CT-244U 482 of 620Table E-29. Statistical Summary of Site LACH CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.87 (0.016)0.84 (0.016)0.023 (0.012)-0.0073, 0.0530.1120.72, 0.89(0.83 - 0.89)(0.82 - 0.86)(0.0022 - 0.034)(0.67 - 0.96)Tryptophan0.40 (0.014)0.39 (0.014)0.0098 (0.018)-0.035, 0.0540.6100.34, 0.42(0.39 - 0.43)(0.38 - 0.43)(-0.033 - 0.041)(0.31 - 0.46)Tyrosine0.81 (0.018)0.79 (0.018)0.023 (0.021)-0.029, 0.0750.3200.67, 0.84(0.81 - 0.82)(0.76 - 0.81)(-0.0051 - 0.050)(0.63 - 0.91)Valine1.23 (0.024)1.18 (0.024)0.047 (0.029)-0.023, 0.120.1521.00, 1.28(1.20 - 1.26)(1.17 - 1.19)(0.0063 - 0.080)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.73 (0.013)0.75 (0.013)-0.020 (0.018)-0.065, 0.0240.3120.16, 1.37(0.69 - 0.77)(0.73 - 0.78)(-0.087 - 0.041)(0.45 - 1.04)16:0 Palmitic24.26 (0.091)24.04 (0.091)0.21 (0.13)-0.099, 0.530.14516.54, 30.55(23.95 - 24.55)(23.92 - 24.16)(-0.13 - 0.63)(19.11 - 26.73)Monsanto Company 12-CT-244U 483 of 620Table E-29. Statistical Summary of Site LACH CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.50 (0.0035)0.50 (0.0035)0.00039 (0.0049)-0.012, 0.0120.9390.39, 0.70(0.50 - 0.51)(0.49 - 0.51)(-0.011 - 0.012)(0.44 - 0.67)18:0 Stearic2.61 (0.018)2.52 (0.018)0.089 (0.026)0.025, 0.150.0141.98, 2.95(2.58 - 2.64)(2.49 - 2.57)(0.026 - 0.15)(1.98 - 2.97)18:1 Oleic14.46 (0.095)14.29 (0.095)0.17 (0.13)-0.16, 0.500.25211.38, 20.64(14.26 - 14.65)(14.13 - 14.53)(-0.27 - 0.52)(13.71 - 18.39)18:2 Linoleic56.04 (0.14)56.63 (0.14)-0.58 (0.20)-1.08, -0.0920.02747.49, 63.18(55.71 - 56.35)(56.52 - 56.72)(-0.91 - -0.30)(49.78 - 59.61)18:3 Linolenic0.15 (0.0042)0.15 (0.0042)0.0015 (0.0059)-0.013, 0.0160.8020.060, 0.24(0.14 - 0.16)(0.13 - 0.15)(-0.012 - 0.014)(0.10 - 0.29)20:0 Arachidic0.29 (0.0054)0.29 (0.0054)0.0028 (0.0071)-0.015, 0.0200.7100.17, 0.38(0.27 - 0.31)(0.29 - 0.30)(-0.022 - 0.024)(0.20 - 0.36)Monsanto Company 12-CT-244U 484 of 620Table E-29. Statistical Summary of Site LACH CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.15 (0.0039)0.14 (0.0039)0.0070 (0.0055)-0.0064, 0.0200.2470.070, 0.21(0.13 - 0.16)(0.14 - 0.15)(-0.0068 - 0.027)(0.051 - 0.19)MineralCalcium (% dw)0.13 (0.0039)0.12 (0.0039)0.0092 (0.0050)-0.0030, 0.0210.1150.058, 0.21(0.12 - 0.14)(0.12 - 0.12)(0.0045 - 0.017)(0.081 - 0.18)Copper (mg/kg dw)8.24 (0.30)8.70 (0.30)-0.47 (0.42)-1.51, 0.570.3112.97, 12.86(7.69 - 8.79)(8.11 - 9.14)(-1.27 - 0.086)(4.46 - 11.62)Iron (mg/kg dw)77.97 (4.19)68.59 (4.19)9.38 (4.72)-2.16, 20.930.09347.30, 97.12(69.22 - 98.27)(66.28 - 70.38)(-0.024 - 27.89)(39.49 - 114.34)Magnesium (% dw)0.41 (0.0065)0.39 (0.0065)0.020 (0.0093)-0.0022, 0.0430.0680.28, 0.47(0.41 - 0.42)(0.38 - 0.41)(0.0091 - 0.029)(0.31 - 0.46)Manganese (mg/kg dw)13.87 (0.37)12.87 (0.37)0.99 (0.52)-0.29, 2.280.1069.07, 17.33(13.02 - 14.71)(12.31 - 13.87)(-0.84 - 2.08)(9.07 - 17.14)Monsanto Company 12-CT-244U 485 of 620Table E-29. Statistical Summary of Site LACH CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.72 (0.014)0.71 (0.014)0.0034 (0.019)-0.044, 0.0510.8670.49, 0.87(0.68 - 0.73)(0.69 - 0.73)(-0.011 - 0.021)(0.48 - 0.87)Potassium (% dw)1.15 (0.022)1.17 (0.022)-0.023 (0.029)-0.093, 0.0480.4610.92, 1.21(1.13 - 1.18)(1.12 - 1.27)(-0.13 - 0.026)(0.90 - 1.26)Sodium (% dw)0.023 (0.0043)0.015 (0.0043)0.0074 (0.0060)-0.0072, 0.0220.2610, 0.066(0.0054 - 0.029)(0.0053 - 0.027)(-0.0081 - 0.024)(0.0054 - 0.077)Zinc (mg/kg dw)34.21 (0.93)35.74 (0.93)-1.54 (1.32)-4.75, 1.680.28727.27, 44.95(32.91 - 35.63)(35.10 - 37.09)(-2.57 - -1.04)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E167.00 (2.48)149.96 (2.48)17.04 (3.48)8.52, 25.550.00241.91, 205.89(158.95 - 173.30)(148.96 - 152.67)(9.99 - 20.70)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2Test refers to MON 88701 (Not Treated). These plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 486 of 620Table E-30. Statistical Summary of SiteLACH Cottonseed Anti-nutrients for MON 88701 (Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.16 (0.011)0.13 (0.011)0.030 (0.016)-0.0078, 0.0690.0990.078, 0.25(0.14 - 0.19)(0.12 - 0.14)(-0.0041 - 0.055)(0.038 - 0.23)Malvalic Acid0.41 (0.035)0.35 (0.035)0.059 (0.049)-0.062, 0.180.2780.23, 0.54(0.36 - 0.50)(0.31 - 0.38)(0.011 - 0.19)(0.11 - 0.59)Sterculic Acid0.23 (0.016)0.20 (0.016)0.029 (0.023)-0.028, 0.0850.2600.17, 0.27(0.21 - 0.27)(0.17 - 0.21)(-0.0031 - 0.093)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.84 (0.026)0.81 (0.026)0.031 (0.036)-0.057, 0.120.4200.099, 1.57(0.76 - 0.90)(0.78 - 0.84)(-0.046 - 0.12)(0.50 - 1.41)Total Gossypol0.93 (0.025)0.90 (0.025)0.035 (0.036)-0.052, 0.120.3630.064, 1.76(0.89 - 1.01)(0.82 - 0.94)(-0.028 - 0.18)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 487 of 620Table E-31. Statistical Summary of SiteNCBD Cottonseed Nutrients for MON 88701 (NotTreated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.33 (0.057)4.14 (0.064)0.19 (0.070)0.0083, 0.370.0433.42, 4.65(4.20 - 4.48)(3.94 - 4.26)(0.038 - 0.36)(3.18 - 4.68)Calories (Kcal/100g)498.21 (1.61)491.80 (1.86)6.41 (2.46)0.078, 12.750.048457.61, 527.56(496.22 - 499.62)(488.93 - 494.48)(3.24 - 10.35)(466.09 - 509.91)Carbohydrates43.25 (0.59)44.36 (0.68)-1.11 (0.87)-3.36, 1.130.25940.26, 56.45(41.07 - 44.88)(43.65 - 45.15)(-3.08 - 1.23)(43.28 - 54.90)Moisture (% fw)8.67 (0.22)8.96 (0.23)-0.29 (0.19)-0.76, 0.190.1844.79, 9.92(7.98 - 9.07)(8.59 - 9.19)(-0.61 - -0.010)(6.05 - 10.50)Protein29.35 (0.61)29.84 (0.70)-0.49 (0.85)-2.68, 1.700.59222.30, 29.41(27.82 - 31.29)(29.62 - 30.42)(-2.05 - 0.88)(20.58 - 29.28)Total Fat23.08 (0.30)21.59 (0.34)1.49 (0.46)0.32, 2.660.02215.01, 28.51(22.78 - 23.39)(21.03 - 22.21)(0.72 - 2.17)(16.58 - 25.25)Monsanto Company 12-CT-244U 488 of 620Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber26.21 (1.12)26.31 (1.29)-0.10 (1.71)-4.50, 4.300.95522.24, 31.96(23.72 - 30.43)(24.72 - 28.08)(-2.57 - 5.70)(23.42 - 31.62)Crude Fiber17.82 (0.29)16.93 (0.33)0.90 (0.38)-0.094, 1.880.06716.93, 22.68(17.57 - 18.04)(16.30 - 17.90)(-0.33 - 1.52)(16.92 - 23.32)Neutral Detergent Fiber32.76 (1.03)31.14 (1.19)1.61 (1.58)-2.44, 5.670.35327.03, 42.49(31.18 - 37.27)(30.85 - 31.49)(-0.26 - 6.42)(29.27 - 40.63)Total Dietary Fiber38.49 (0.55)39.52 (0.64)-1.03 (0.85)-3.21, 1.140.27734.52, 52.58(37.06 - 40.31)(39.05 - 39.86)(-2.58 - -1.10)(37.29 - 48.60)Amino Acid (% dw)Alanine1.13 (0.020)1.10 (0.023)0.027 (0.031)-0.052, 0.110.4180.86, 1.11(1.06 - 1.15)(1.05 - 1.14)(-0.075 - 0.11)(0.83 - 1.22)Arginine3.10 (0.052)3.20 (0.060)-0.10 (0.080)-0.31, 0.100.2532.38, 3.47(2.92 - 3.22)(3.11 - 3.27)(-0.30 - 0.011)(2.30 - 3.55)Monsanto Company 12-CT-244U 489 of 620Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.56 (0.053)2.55 (0.061)0.013 (0.081)-0.19, 0.220.8761.94, 2.57(2.35 - 2.67)(2.46 - 2.63)(-0.28 - 0.14)(1.79 - 2.72)Cystine0.42 (0.018)0.42 (0.021)0.0075 (0.028)-0.064, 0.0790.7980.31, 0.45(0.38 - 0.49)(0.39 - 0.46)(-0.073 - 0.086)(0.29 - 0.47)Glutamic Acid4.90 (0.13)5.08 (0.15)-0.18 (0.20)-0.69, 0.330.4053.74, 5.28(4.39 - 5.18)(4.85 - 5.40)(-1.01 - 0.18)(3.39 - 5.45)Glycine1.13 (0.019)1.11 (0.022)0.020 (0.029)-0.054, 0.0930.5220.90, 1.14(1.07 - 1.17)(1.08 - 1.14)(-0.063 - 0.062)(0.85 - 1.23)Histidine0.77 (0.013)0.76 (0.015)0.015 (0.019)-0.034, 0.0640.4660.59, 0.81(0.75 - 0.80)(0.74 - 0.79)(0.0060 - 0.024)(0.57 - 0.84)Isoleucine0.95 (0.018)0.96 (0.020)-0.0094 (0.020)-0.061, 0.0430.6610.75, 0.96(0.91 - 1.00)(0.93 - 0.97)(-0.028 - 0.022)(0.72 - 1.03)Monsanto Company 12-CT-244U 490 of 620Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.63 (0.023)1.60 (0.027)0.024 (0.035)-0.067, 0.110.5331.25, 1.62(1.56 - 1.68)(1.55 - 1.63)(-0.065 - 0.088)(1.20 - 1.72)Lysine1.32 (0.029)1.26 (0.033)0.057 (0.043)-0.054, 0.170.2431.01, 1.30(1.27 - 1.37)(1.22 - 1.29)(-0.0085 - 0.086)(0.99 - 1.44)Methionine0.42 (0.0093)0.41 (0.011)0.0089 (0.014)-0.028, 0.0460.5570.32, 0.38(0.39 - 0.44)(0.40 - 0.42)(-0.0049 - 0.019)(0.29 - 0.49)Phenylalanine1.49 (0.027)1.52 (0.031)-0.026 (0.041)-0.13, 0.0790.5481.12, 1.58(1.42 - 1.55)(1.45 - 1.56)(-0.14 - 0.055)(1.10 - 1.63)Proline1.08 (0.022)1.09 (0.025)-0.013 (0.029)-0.088, 0.0630.6840.83, 1.08(1.06 - 1.13)(1.07 - 1.12)(-0.057 - 0.032)(0.79 - 1.17)Serine1.16 (0.027)1.13 (0.032)0.032 (0.042)-0.076, 0.140.4830.83, 1.21(1.05 - 1.22)(1.09 - 1.19)(-0.14 - 0.13)(0.81 - 1.24)Monsanto Company 12-CT-244U 491 of 620Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.92 (0.014)0.89 (0.016)0.029 (0.021)-0.026, 0.0830.2370.72, 0.89(0.87 - 0.95)(0.86 - 0.92)(-0.050 - 0.079)(0.67 - 0.96)Tryptophan0.44 (0.021)0.45 (0.024)-0.016 (0.032)-0.099, 0.0670.6420.34, 0.42(0.41 - 0.50)(0.39 - 0.52)(-0.099 - 0.11)(0.31 - 0.46)Tyrosine0.85 (0.012)0.84 (0.014)0.0095 (0.018)-0.036, 0.0550.6180.67, 0.84(0.83 - 0.87)(0.82 - 0.87)(-0.0099 - 0.043)(0.63 - 0.91)Valine1.26 (0.025)1.30 (0.027)-0.036 (0.021)-0.090, 0.0170.1401.00, 1.28(1.21 - 1.32)(1.24 - 1.32)(-0.074 - -0.0017)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.71 (0.0074)0.75 (0.0086)-0.036 (0.011)-0.065, -0.00690.0240.16, 1.37(0.69 - 0.73)(0.74 - 0.76)(-0.065 - -0.013)(0.45 - 1.04)16:0 Palmitic23.40 (0.12)23.10 (0.14)0.30 (0.18)-0.16, 0.760.15216.54, 30.55(23.11 - 23.69)(23.07 - 23.15)(0.26 - 0.62)(19.11 - 26.73)Monsanto Company 12-CT-244U 492 of 620Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.47 (0.0047)0.48 (0.0053)-0.0094 (0.0055)-0.023, 0.00470.1470.39, 0.70(0.46 - 0.48)(0.47 - 0.49)(-0.024 - 0.0016)(0.44 - 0.67)18:0 Stearic2.53 (0.037)2.34 (0.043)0.19 (0.057)0.041, 0.330.0211.98, 2.95(2.49 - 2.57)(2.32 - 2.38)(0.11 - 0.26)(1.98 - 2.97)18:1 Oleic15.29 (0.12)14.70 (0.14)0.59 (0.19)0.11, 1.080.02611.38, 20.64(15.01 - 15.50)(14.51 - 14.83)(0.18 - 1.00)(13.71 - 18.39)18:2 Linoleic56.04 (0.23)57.19 (0.26)-1.15 (0.35)-2.04, -0.250.02147.49, 63.18(55.76 - 56.31)(57.01 - 57.46)(-1.65 - -0.71)(49.78 - 59.61)18:3 Linolenic0.36 (0.012)0.29 (0.014)0.074 (0.018)0.028, 0.120.0080.060, 0.24(0.34 - 0.38)(0.27 - 0.30)(0.048 - 0.11)(0.10 - 0.29)20:0 Arachidic0.30 (0.0065)0.28 (0.0075)0.023 (0.0099)-0.0021, 0.0490.0650.17, 0.38(0.29 - 0.30)(0.27 - 0.28)(0.013 - 0.031)(0.20 - 0.36)Monsanto Company 12-CT-244U 493 of 620Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.16 (0.0056)0.15 (0.0065)0.0093 (0.0086)-0.013, 0.0310.3270.070, 0.21(0.16 - 0.17)(0.15 - 0.16)(-0.00007 - 0.015)(0.051 - 0.19)MineralCalcium (% dw)0.15 (0.0014)0.14 (0.0017)0.013 (0.0022)0.0072, 0.0180.0020.058, 0.21(0.15 - 0.15)(0.14 - 0.14)(0.0067 - 0.018)(0.081 - 0.18)Copper (mg/kg dw)6.59 (0.36)6.91 (0.41)-0.31 (0.54)-1.71, 1.080.5872.97, 12.86(5.37 - 7.35)(6.64 - 7.19)(-0.067 - 0.19)(4.46 - 11.62)Iron (mg/kg dw)44.35 (1.00)48.04 (1.15)-3.69 (1.53)-7.62, 0.230.06047.30, 97.12(41.73 - 46.45)(45.03 - 50.87)(-9.14 - -0.44)(39.49 - 114.34)Magnesium (% dw)0.41 (0.010)0.40 (0.012)0.010 (0.016)-0.031, 0.0510.5450.28, 0.47(0.40 - 0.42)(0.37 - 0.44)(-0.015 - 0.055)(0.31 - 0.46)Manganese (mg/kg dw)14.78 (0.36)13.83 (0.42)0.95 (0.56)-0.48, 2.380.1489.07, 17.33(13.36 - 15.70)(13.65 - 14.11)(0.97 - 1.97)(9.07 - 17.14)Monsanto Company 12-CT-244U 494 of 620Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.64 (0.020)0.66 (0.023)-0.021 (0.031)-0.10, 0.0580.5210.49, 0.87(0.60 - 0.66)(0.59 - 0.71)(-0.078 - 0.072)(0.48 - 0.87)Potassium (% dw)1.04 (0.019)1.08 (0.022)-0.045 (0.029)-0.12, 0.0300.1830.92, 1.21(1.03 - 1.06)(1.03 - 1.16)(-0.13 - -0.0056)(0.90 - 1.26)Sodium (% dw)0.074 (0.0084)0.099 (0.0096)-0.026 (0.011)-0.055, 0.00310.0690, 0.066(0.061 - 0.082)(0.094 - 0.10)(-0.038 - -0.020)(0.0054 - 0.077)Zinc (mg/kg dw)41.50 (1.30)49.54 (1.50)-8.04 (1.98)-13.13, -2.950.00927.27, 44.95(39.45 - 43.05)(44.04 - 52.95)(-13.50 - -2.09)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E174.64 (3.66)156.99 (4.23)17.65 (5.60)3.27, 32.040.02541.91, 205.89(165.41 - 184.47)(151.55 - 162.98)(7.48 - 32.93)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 495 of 620Table E-32. Statistical Summary of SiteNCBD Cottonseed Anti-nutrients for MON 88701 (Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.0093)0.13 (0.011)0.023 (0.014)-0.013, 0.0590.1670.078, 0.25(0.12 - 0.18)(0.12 - 0.14)(-0.0060 - 0.032)(0.038 - 0.23)Malvalic Acid0.36 (0.040)0.37 (0.046)-0.018 (0.056)-0.16, 0.130.7600.23, 0.54(0.26 - 0.45)(0.36 - 0.41)(-0.11 - 0.039)(0.11 - 0.59)Sterculic Acid0.23 (0.024)0.22 (0.028)0.010 (0.037)-0.085, 0.110.7910.17, 0.27(0.16 - 0.27)(0.21 - 0.23)(-0.055 - 0.044)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.94 (0.026)0.90 (0.030)0.044 (0.039)-0.057, 0.140.3140.099, 1.57(0.88 - 0.99)(0.81 - 0.95)(-0.00010 - 0.18)(0.50 - 1.41)Total Gossypol1.21 (0.067)1.09 (0.077)0.12 (0.10)-0.14, 0.380.2870.064, 1.76(1.05 - 1.52)(1.08 - 1.10)(0.021 - 0.44)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 496 of 620Table E-33. Statistical Summary of SiteNMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.31 (0.087)4.27 (0.10)0.042 (0.13)-0.29, 0.380.7613.42, 4.65(4.25 - 4.38)(4.20 - 4.39)(-0.0052 - 0.087)(3.18 - 4.68)Calories (Kcal/100g)495.87 (2.64)492.81 (3.05)3.06 (4.04)-7.32, 13.450.482457.61, 527.56(487.62 - 499.09)(490.52 - 494.31)(-5.97 - 7.23)(466.09 - 509.91)Carbohydrates42.30 (0.46)42.60 (0.53)-0.30 (0.70)-2.10, 1.500.68840.26, 56.45(41.59 - 43.70)(42.14 - 43.05)(-0.90 - 1.56)(43.28 - 54.90)Moisture (% fw)7.03 (0.25)7.28 (0.28)-0.25 (0.32)-1.08, 0.580.4714.79, 9.92(6.78 - 7.42)(6.63 - 7.75)(-0.92 - 0.15)(6.05 - 10.50)Protein30.79 (0.13)31.18 (0.15)-0.39 (0.19)-0.89, 0.110.10322.30, 29.41(30.68 - 30.89)(31.00 - 31.27)(-0.59 - -0.31)(20.58 - 29.28)Total Fat22.62 (0.54)21.95 (0.62)0.67 (0.83)-1.46, 2.790.45515.01, 28.51(20.99 - 23.29)(21.42 - 22.22)(-1.21 - 1.54)(16.58 - 25.25)Monsanto Company 12-CT-244U 497 of 620Table E-33. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber26.43 (0.51)26.40 (0.59)0.030 (0.79)-1.99, 2.050.97122.24, 31.96(25.17 - 27.78)(25.76 - 27.10)(-0.16 - 1.44)(23.42 - 31.62)Crude Fiber17.75 (0.74)17.71 (0.84)0.040 (0.94)-2.39, 2.470.96716.93, 22.68(16.63 - 18.67)(16.06 - 20.78)(-2.11 - 2.09)(16.92 - 23.32)Neutral Detergent Fiber30.87 (0.87)32.83 (1.00)-1.96 (1.32)-5.36, 1.440.19927.03, 42.49(29.16 - 32.29)(31.58 - 34.49)(-4.66 - 0.72)(29.27 - 40.63)Total Dietary Fiber38.30 (0.71)41.10 (0.75)-2.81 (0.55)-4.22, -1.390.00334.52, 52.58(37.12 - 39.32)(39.09 - 43.00)(-4.03 - -1.98)(37.29 - 48.60)Amino Acid (% dw)Alanine1.11 (0.011)1.13 (0.013)-0.019 (0.014)-0.054, 0.0150.2110.86, 1.11(1.09 - 1.13)(1.09 - 1.17)(-0.044 - 0.023)(0.83 - 1.22)Arginine3.51 (0.049)3.71 (0.053)-0.20 (0.048)-0.32, -0.0750.0082.38, 3.47(3.34 - 3.62)(3.67 - 3.77)(-0.23 - -0.12)(2.30 - 3.55)Monsanto Company 12-CT-244U 498 of 620Table E-33. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.63 (0.033)2.66 (0.037)-0.033 (0.043)-0.14, 0.0760.4691.94, 2.57(2.52 - 2.69)(2.58 - 2.74)(-0.091 - 0.069)(1.79 - 2.72)Cystine0.43 (0.013)0.44 (0.015)-0.0077 (0.020)-0.058, 0.0430.7130.31, 0.45(0.41 - 0.46)(0.43 - 0.45)(-0.022 - 0.0095)(0.29 - 0.47)Glutamic Acid5.31 (0.087)5.46 (0.10)-0.15 (0.13)-0.49, 0.190.3093.74, 5.28(5.08 - 5.57)(5.29 - 5.70)(-0.50 - 0.18)(3.39 - 5.45)Glycine1.16 (0.014)1.18 (0.015)-0.018 (0.016)-0.060, 0.0230.3090.90, 1.14(1.11 - 1.19)(1.17 - 1.20)(-0.023 - 0.0094)(0.85 - 1.23)Histidine0.82 (0.010)0.83 (0.012)-0.010 (0.015)-0.048, 0.0270.5100.59, 0.81(0.79 - 0.84)(0.83 - 0.84)(-0.026 - 0.014)(0.57 - 0.84)Isoleucine0.97 (0.017)0.98 (0.018)-0.0018 (0.012)-0.032, 0.0280.8850.75, 0.96(0.94 - 1.01)(0.94 - 1.03)(-0.014 - 0.017)(0.72 - 1.03)Monsanto Company 12-CT-244U 499 of 620Table E-33. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.64 (0.021)1.66 (0.023)-0.015 (0.022)-0.071, 0.0410.5191.25, 1.62(1.57 - 1.67)(1.63 - 1.69)(-0.024 - 0.035)(1.20 - 1.72)Lysine1.31 (0.019)1.36 (0.022)-0.045 (0.022)-0.10, 0.0120.0971.01, 1.30(1.29 - 1.33)(1.32 - 1.39)(-0.057 - -0.0086)(0.99 - 1.44)Methionine0.42 (0.012)0.40 (0.014)0.021 (0.018)-0.026, 0.0690.2950.32, 0.38(0.39 - 0.44)(0.38 - 0.41)(-0.016 - 0.048)(0.29 - 0.49)Phenylalanine1.58 (0.026)1.61 (0.028)-0.026 (0.022)-0.083, 0.0300.2871.12, 1.58(1.50 - 1.63)(1.60 - 1.66)(-0.034 - 0.013)(1.10 - 1.63)Proline1.12 (0.027)1.18 (0.031)-0.057 (0.039)-0.16, 0.0430.2000.83, 1.08(1.07 - 1.16)(1.10 - 1.25)(-0.13 - 0.055)(0.79 - 1.17)Serine1.20 (0.026)1.20 (0.030)-0.0068 (0.040)-0.11, 0.0970.8720.83, 1.21(1.11 - 1.28)(1.16 - 1.24)(-0.055 - 0.063)(0.81 - 1.24)Monsanto Company 12-CT-244U 500 of 620Table E-33. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.92 (0.015)0.93 (0.017)-0.0029 (0.021)-0.058, 0.0520.8960.72, 0.89(0.86 - 0.95)(0.91 - 0.94)(-0.00072 - 0.037)(0.67 - 0.96)Tryptophan0.44 (0.014)0.44 (0.016)-0.0052 (0.020)-0.057, 0.0460.8030.34, 0.42(0.43 - 0.44)(0.43 - 0.45)(-0.020 - 0.0087)(0.31 - 0.46)Tyrosine0.87 (0.014)0.89 (0.015)-0.018 (0.014)-0.054, 0.0180.2500.67, 0.84(0.84 - 0.88)(0.86 - 0.91)(-0.029 - 0.011)(0.63 - 0.91)Valine1.29 (0.024)1.34 (0.026)-0.044 (0.019)-0.093, 0.00560.0711.00, 1.28(1.25 - 1.32)(1.31 - 1.40)(-0.078 - -0.019)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.95 (0.0046)0.98 (0.0054)-0.030 (0.0071)-0.048, -0.0120.0080.16, 1.37(0.94 - 0.95)(0.97 - 0.98)(-0.032 - -0.026)(0.45 - 1.04)16:0 Palmitic24.15 (0.088)24.11 (0.10)0.044 (0.13)-0.30, 0.390.75716.54, 30.55(24.06 - 24.37)(23.89 - 24.34)(-0.23 - 0.47)(19.11 - 26.73)Monsanto Company 12-CT-244U 501 of 620Table E-33. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.53 (0.0022)0.54 (0.0025)-0.0080 (0.0033)-0.017, 0.000540.0600.39, 0.70(0.53 - 0.53)(0.53 - 0.54)(-0.014 - -0.0036)(0.44 - 0.67)18:0 Stearic2.52 (0.020)2.64 (0.021)-0.12 (0.016)-0.17, -0.082<0.0011.98, 2.95(2.49 - 2.56)(2.61 - 2.70)(-0.14 - -0.12)(1.98 - 2.97)18:1 Oleic16.13 (0.067)16.21 (0.076)-0.085 (0.088)-0.31, 0.140.38211.38, 20.64(16.01 - 16.29)(16.10 - 16.35)(-0.23 - -0.072)(13.71 - 18.39)18:2 Linoleic54.43 (0.084)54.29 (0.097)0.14 (0.13)-0.19, 0.470.32647.49, 63.18(54.22 - 54.63)(54.04 - 54.50)(-0.11 - 0.59)(49.78 - 59.61)18:3 Linolenic0.16 (0.0019)0.14 (0.0022)0.014 (0.0029)0.0065, 0.0210.0040.060, 0.24(0.15 - 0.16)(0.14 - 0.15)(0.0032 - 0.020)(0.10 - 0.29)20:0 Arachidic0.30 (0.0052)0.31 (0.0060)-0.0075 (0.0080)-0.028, 0.0130.3890.17, 0.38(0.29 - 0.31)(0.28 - 0.32)(-0.026 - 0.022)(0.20 - 0.36)Monsanto Company 12-CT-244U 502 of 620Table E-33. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.17 (0.0065)0.19 (0.0075)-0.020 (0.0099)-0.046, 0.00530.0970.070, 0.21(0.16 - 0.17)(0.17 - 0.21)(-0.057 - -0.00078)(0.051 - 0.19)MineralCalcium (% dw)0.15 (0.0029)0.13 (0.0034)0.019 (0.0042)0.0082, 0.0300.0060.058, 0.21(0.14 - 0.15)(0.12 - 0.13)(0.015 - 0.024)(0.081 - 0.18)Copper (mg/kg dw)11.38 (0.15)11.75 (0.17)-0.37 (0.22)-0.95, 0.200.1572.97, 12.86(11.09 - 11.56)(11.46 - 11.92)(-0.77 - 0.018)(4.46 - 11.62)Iron (mg/kg dw)67.95 (3.33)64.62 (3.84)3.33 (5.08)-9.74, 16.390.54147.30, 97.12(61.15 - 83.28)(63.58 - 66.45)(-2.69 - -1.19)(39.49 - 114.34)Magnesium (% dw)0.38 (0.0053)0.37 (0.0061)0.0076 (0.0081)-0.013, 0.0280.3860.28, 0.47(0.37 - 0.39)(0.36 - 0.38)(-0.0069 - 0.021)(0.31 - 0.46)Manganese (mg/kg dw)13.23 (0.23)12.90 (0.26)0.33 (0.34)-0.55, 1.220.3779.07, 17.33(12.92 - 13.61)(12.00 - 13.47)(-0.55 - 1.20)(9.07 - 17.14)Monsanto Company 12-CT-244U 503 of 620Table E-33. Statistical Summary of Site NMLC CottonseedNutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.76 (0.010)0.79 (0.012)-0.031 (0.016)-0.071, 0.00990.1090.49, 0.87(0.75 - 0.78)(0.78 - 0.80)(-0.046 - -0.0099)(0.48 - 0.87)Potassium (% dw)1.11 (0.016)1.11 (0.018)-0.00071 (0.017)-0.045, 0.0430.9680.92, 1.21(1.09 - 1.12)(1.07 - 1.14)(-0.044 - 0.029)(0.90 - 1.26)Sodium (% dw)0.017 (0.0034)0.013 (0.0039)0.0041 (0.0052)-0.0092, 0.0170.4650, 0.066(0.0054 - 0.023)(0.0054 - 0.023)(-0.0019 - 0.018)(0.0054 - 0.077)Zinc (mg/kg dw)44.99 (0.60)49.43 (0.69)-4.44 (0.92)-6.80, -2.080.00427.27, 44.95(44.56 - 46.04)(47.66 - 50.87)(-6.30 - -3.03)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E115.90 (2.04)112.18 (2.36)3.72 (3.12)-4.29, 11.730.28541.91, 205.89(113.71 - 119.14)(107.02 - 115.99)(0.18 - 7.09)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 504 of 620Table E-34. Statistical Summary of SiteNMLC Cottonseed Anti-nutrients for MON 88701 (Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.16 (0.0079)0.14 (0.0092)0.022 (0.012)-0.0097, 0.0530.1360.078, 0.25(0.14 - 0.18)(0.12 - 0.15)(-0.0016 - 0.056)(0.038 - 0.23)Malvalic Acid0.33 (0.023)0.29 (0.026)0.039 (0.035)-0.050, 0.130.3080.23, 0.54(0.24 - 0.38)(0.26 - 0.31)(-0.070 - 0.13)(0.11 - 0.59)Sterculic Acid0.19 (0.018)0.18 (0.020)0.0079 (0.027)-0.061, 0.0770.7800.17, 0.27(0.12 - 0.24)(0.17 - 0.19)(-0.075 - 0.079)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.86 (0.026)0.69 (0.030)0.17 (0.040)0.063, 0.270.0080.099, 1.57(0.76 - 0.95)(0.68 - 0.70)(0.12 - 0.27)(0.50 - 1.41)Total Gossypol0.88 (0.026)0.80 (0.030)0.083 (0.040)-0.019, 0.190.0910.064, 1.76(0.84 - 0.92)(0.74 - 0.87)(-0.028 - 0.18)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 505 of 620Table E-35. Statistical Summary of SiteSCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.10 (0.095)3.74 (0.095)0.36 (0.10)0.12, 0.610.0113.42, 4.65(3.91 - 4.23)(3.38 - 3.98)(0.20 - 0.79)(3.18 - 4.68)Calories (Kcal/100g)508.96 (2.44)503.38 (2.44)5.58 (3.45)-2.87, 14.020.157457.61, 527.56(506.18 - 511.92)(499.09 - 512.65)(-4.16 - 9.98)(466.09 - 509.91)Carbohydrates47.15 (0.54)48.67 (0.54)-1.52 (0.75)-3.36, 0.320.09040.26, 56.45(46.17 - 48.81)(47.50 - 49.59)(-3.27 - -0.78)(43.28 - 54.90)Moisture (% fw)6.48 (0.17)7.08 (0.17)-0.60 (0.19)-1.07, -0.140.0194.79, 9.92(6.16 - 6.98)(6.63 - 7.37)(-1.04 - -0.17)(6.05 - 10.50)Protein23.61 (0.42)23.92 (0.42)-0.31 (0.49)-1.52, 0.890.54722.30, 29.41(22.49 - 24.37)(23.56 - 24.61)(-1.23 - 0.78)(20.58 - 29.28)Total Fat25.13 (0.44)23.65 (0.44)1.48 (0.63)-0.061, 3.020.05715.01, 28.51(24.62 - 25.54)(22.92 - 25.20)(-0.15 - 2.28)(16.58 - 25.25)Monsanto Company 12-CT-244U 506 of 620Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber25.58 (0.44)27.04 (0.44)-1.46 (0.60)-2.93, 0.00440.05022.24, 31.96(24.19 - 26.37)(26.24 - 27.74)(-3.49 - 0.082)(23.42 - 31.62)Crude Fiber19.49 (0.80)19.13 (0.80)0.36 (1.13)-2.41, 3.120.76316.93, 22.68(18.47 - 20.73)(16.91 - 21.70)(-3.23 - 3.82)(16.92 - 23.32)Neutral Detergent Fiber32.88 (0.67)33.60 (0.67)-0.73 (0.95)-3.04, 1.590.47227.03, 42.49(31.85 - 34.31)(32.74 - 35.52)(-3.28 - 0.90)(29.27 - 40.63)Total Dietary Fiber41.46 (0.62)41.87 (0.62)-0.40 (0.88)-2.55, 1.740.66134.52, 52.58(40.03 - 43.22)(40.16 - 43.29)(-3.26 - 2.04)(37.29 - 48.60)Amino Acid (% dw)Alanine0.93 (0.022)0.94 (0.022)-0.011 (0.029)-0.083, 0.0610.7240.86, 1.11(0.88 - 0.98)(0.88 - 0.97)(-0.059 - 0.10)(0.83 - 1.22)Arginine2.51 (0.088)2.59 (0.088)-0.075 (0.10)-0.32, 0.170.4822.38, 3.47(2.31 - 2.67)(2.41 - 2.71)(-0.20 - 0.26)(2.30 - 3.55)Monsanto Company 12-CT-244U 507 of 620Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.03 (0.054)2.07 (0.054)-0.037 (0.070)-0.21, 0.130.6101.94, 2.57(1.95 - 2.12)(1.92 - 2.18)(-0.17 - 0.20)(1.79 - 2.72)Cystine0.36 (0.017)0.35 (0.017)0.0031 (0.018)-0.041, 0.0470.8680.31, 0.45(0.33 - 0.38)(0.31 - 0.39)(-0.026 - 0.066)(0.29 - 0.47)Glutamic Acid3.96 (0.13)4.10 (0.13)-0.14 (0.18)-0.57, 0.300.4733.74, 5.28(3.79 - 4.14)(3.66 - 4.40)(-0.61 - 0.47)(3.39 - 5.45)Glycine0.96 (0.022)0.98 (0.022)-0.023 (0.031)-0.10, 0.0540.4940.90, 1.14(0.92 - 1.00)(0.91 - 1.02)(-0.087 - 0.088)(0.85 - 1.23)Histidine0.64 (0.021)0.64 (0.021)-0.00060 (0.025)-0.062, 0.0610.9810.59, 0.81(0.58 - 0.68)(0.61 - 0.66)(-0.058 - 0.064)(0.57 - 0.84)Isoleucine0.81 (0.023)0.82 (0.023)-0.0090 (0.030)-0.082, 0.0640.7740.75, 0.96(0.76 - 0.85)(0.77 - 0.83)(-0.067 - 0.074)(0.72 - 1.03)Monsanto Company 12-CT-244U 508 of 620Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.34 (0.034)1.36 (0.034)-0.014 (0.046)-0.13, 0.0990.7701.25, 1.62(1.28 - 1.42)(1.28 - 1.40)(-0.071 - 0.14)(1.20 - 1.72)Lysine1.08 (0.027)1.11 (0.027)-0.031 (0.025)-0.092, 0.0310.2691.01, 1.30(1.03 - 1.12)(1.06 - 1.17)(-0.066 - 0.049)(0.99 - 1.44)Methionine0.36 (0.016)0.33 (0.016)0.030 (0.020)-0.020, 0.0800.1880.32, 0.38(0.33 - 0.41)(0.32 - 0.35)(-0.012 - 0.088)(0.29 - 0.49)Phenylalanine1.20 (0.033)1.23 (0.033)-0.029 (0.039)-0.12, 0.0660.4801.12, 1.58(1.13 - 1.25)(1.15 - 1.27)(-0.10 - 0.098)(1.10 - 1.63)Proline0.86 (0.026)0.87 (0.026)-0.012 (0.026)-0.074, 0.0510.6650.83, 1.08(0.78 - 0.89)(0.81 - 0.93)(-0.080 - 0.077)(0.79 - 1.17)Serine0.94 (0.028)0.96 (0.028)-0.013 (0.033)-0.094, 0.0680.7100.83, 1.21(0.93 - 0.96)(0.86 - 1.03)(-0.11 - 0.098)(0.81 - 1.24)Monsanto Company 12-CT-244U 509 of 620Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.78 (0.019)0.77 (0.019)0.0049 (0.023)-0.052, 0.0620.8400.72, 0.89(0.73 - 0.82)(0.73 - 0.81)(-0.028 - 0.086)(0.67 - 0.96)Tryptophan0.36 (0.0093)0.38 (0.0093)-0.019 (0.0085)-0.040, 0.00150.0630.34, 0.42(0.34 - 0.38)(0.37 - 0.40)(-0.038 - 0.010)(0.31 - 0.46)Tyrosine0.71 (0.020)0.71 (0.020)-0.0059 (0.025)-0.068, 0.0560.8220.67, 0.84(0.68 - 0.74)(0.67 - 0.74)(-0.035 - 0.073)(0.63 - 0.91)Valine1.05 (0.029)1.07 (0.029)-0.013 (0.039)-0.11, 0.0820.7511.00, 1.28(0.98 - 1.11)(1.00 - 1.10)(-0.10 - 0.11)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.71 (0.011)0.73 (0.011)-0.013 (0.012)-0.042, 0.0150.3050.16, 1.37(0.69 - 0.74)(0.72 - 0.75)(-0.033 - 0.013)(0.45 - 1.04)16:0 Palmitic24.54 (0.086)24.39 (0.086)0.15 (0.12)-0.15, 0.440.27816.54, 30.55(24.37 - 24.64)(24.07 - 24.59)(-0.12 - 0.56)(19.11 - 26.73)Monsanto Company 12-CT-244U 510 of 620Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.49 (0.0056)0.48 (0.0056)0.0028 (0.0076)-0.016, 0.0210.7260.39, 0.70(0.47 - 0.50)(0.47 - 0.49)(-0.016 - 0.024)(0.44 - 0.67)18:0 Stearic2.79 (0.036)2.67 (0.036)0.12 (0.050)-0.0034, 0.240.0541.98, 2.95(2.72 - 2.93)(2.58 - 2.76)(0.033 - 0.24)(1.98 - 2.97)18:1 Oleic14.44 (0.11)14.46 (0.11)-0.019 (0.15)-0.39, 0.350.90111.38, 20.64(14.05 - 14.68)(14.42 - 14.49)(-0.40 - 0.20)(13.71 - 18.39)18:2 Linoleic55.63 (0.18)55.87 (0.18)-0.25 (0.25)-0.86, 0.370.36647.49, 63.18(55.18 - 56.00)(55.61 - 56.29)(-0.66 - 0.24)(49.78 - 59.61)18:3 Linolenic0.16 (0.0017)0.15 (0.0017)0.0098 (0.0023)0.0042, 0.0150.0050.060, 0.24(0.15 - 0.16)(0.14 - 0.15)(0.0036 - 0.014)(0.10 - 0.29)20:0 Arachidic0.31 (0.0070)0.30 (0.0070)0.012 (0.0099)-0.013, 0.0360.2810.17, 0.38(0.30 - 0.31)(0.29 - 0.30)(0.00039 - 0.024)(0.20 - 0.36)Monsanto Company 12-CT-244U 511 of 620Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.14 (0.0033)0.14 (0.0033)-0.0010 (0.0046)-0.012, 0.0100.8360.070, 0.21(0.13 - 0.15)(0.13 - 0.14)(-0.015 - 0.015)(0.051 - 0.19)MineralCalcium (% dw)0.11 (0.0040)0.091 (0.0040)0.020 (0.0056)0.0058, 0.0330.0130.058, 0.21(0.10 - 0.12)(0.081 - 0.095)(0.0064 - 0.030)(0.081 - 0.18)Copper (mg/kg dw)5.65 (0.24)5.64 (0.24)0.015 (0.32)-0.78, 0.800.9652.97, 12.86(5.02 - 6.58)(5.40 - 5.85)(-0.43 - 0.73)(4.46 - 11.62)Iron (mg/kg dw)67.74 (4.68)73.46 (4.68)-5.73 (4.30)-16.25, 4.800.23147.30, 97.12(60.53 - 81.81)(63.01 - 89.93)(-14.73 - 3.89)(39.49 - 114.34)Magnesium (% dw)0.38 (0.0090)0.36 (0.0090)0.026 (0.0080)0.0063, 0.0450.0170.28, 0.47(0.36 - 0.41)(0.34 - 0.37)(-0.00067 - 0.046)(0.31 - 0.46)Manganese (mg/kg dw)12.44 (0.51)9.72 (0.51)2.73 (0.72)0.97, 4.480.0089.07, 17.33(10.59 - 13.87)(8.61 - 11.03)(-0.44 - 5.26)(9.07 - 17.14)Monsanto Company 12-CT-244U 512 of 620Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.64 (0.022)0.63 (0.022)0.0061 (0.018)-0.039, 0.0510.7510.49, 0.87(0.59 - 0.72)(0.58 - 0.68)(-0.040 - 0.054)(0.48 - 0.87)Potassium (% dw)1.14 (0.031)1.02 (0.031)0.12 (0.043)0.015, 0.230.0310.92, 1.21(1.11 - 1.19)(0.88 - 1.08)(0.027 - 0.31)(0.90 - 1.26)Sodium (% dw)0.018 (0.0033)0.015 (0.0033)0.0028 (0.0046)-0.0085, 0.0140.5620, 0.066(0.0054 - 0.025)(0.012 - 0.023)(-0.018 - 0.013)(0.0054 - 0.077)Zinc (mg/kg dw)29.14 (0.82)30.08 (0.82)-0.94 (0.82)-2.96, 1.080.29727.27, 44.95(27.60 - 30.85)(28.22 - 31.74)(-2.60 - 1.99)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E159.04 (1.77)158.20 (1.77)0.84 (2.50)-5.29, 6.970.74941.91, 205.89(154.81 - 162.27)(153.15 - 162.63)(-7.82 - 7.76)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 513 of 620Table E-36. Statistical Summary of SiteSCEK Cottonseed Anti-nutrients for MON 88701 (Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.15 (0.0087)0.15 (0.0087)0.0062 (0.012)-0.024, 0.0360.6320.078, 0.25(0.15 - 0.16)(0.14 - 0.15)(-0.0031 - 0.012)(0.038 - 0.23)Malvalic Acid0.43 (0.029)0.43 (0.029)-0.0044 (0.041)-0.10, 0.0960.9180.23, 0.54(0.40 - 0.48)(0.39 - 0.46)(-0.026 - 0.015)(0.11 - 0.59)Sterculic Acid0.23 (0.014)0.24 (0.014)-0.011 (0.020)-0.060, 0.0380.5960.17, 0.27(0.21 - 0.25)(0.22 - 0.25)(-0.020 - -0.0016)(0.061 - 0.34)Gossypol (% dw)Free Gossypol1.06 (0.029)1.13 (0.029)-0.064 (0.027)-0.13, 0.00100.0520.099, 1.57(1.03 - 1.10)(1.06 - 1.20)(-0.14 - 0.012)(0.50 - 1.41)Total Gossypol1.14 (0.025)1.07 (0.025)0.069 (0.031)-0.0076, 0.150.0690.064, 1.76(1.06 - 1.23)(1.05 - 1.10)(-0.00076 - 0.18)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 514 of 620Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. Conventional ControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash3.84 (0.051)3.46 (0.051)0.38 (0.072)0.20, 0.560.0013.42, 4.65(3.76 - 3.98)(3.34 - 3.61)(0.20 - 0.63)(3.18 - 4.68)Calories (Kcal/100g)500.56 (2.54)494.42 (2.54)6.14 (3.47)-2.35, 14.630.127457.61, 527.56(499.03 - 501.60)(489.10 - 500.98)(-0.76 - 12.43)(466.09 - 509.91)Carbohydrates44.20 (0.48)46.39 (0.48)-2.18 (0.64)-3.75, -0.620.01440.26, 56.45(43.36 - 44.54)(45.65 - 47.07)(-3.71 - -1.24)(43.28 - 54.90)Moisture (% fw)6.86 (0.21)7.47 (0.21)-0.61 (0.29)-1.32, 0.0990.0794.79, 9.92(6.30 - 7.24)(7.11 - 7.79)(-0.89 - -0.29)(6.05 - 10.50)Protein28.77 (0.24)28.48 (0.24)0.30 (0.29)-0.42, 1.010.34822.30, 29.41(28.28 - 29.48)(28.09 - 28.77)(-0.49 - 0.74)(20.58 - 29.28)Total Fat23.19 (0.48)21.70 (0.48)1.49 (0.65)-0.097, 3.090.06115.01, 28.51(22.75 - 23.37)(20.71 - 22.88)(0.36 - 2.63)(16.58 - 25.25)Monsanto Company 12-CT-244U 515 of 620Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Acid Detergent Fiber24.53 (0.37)25.53 (0.37)-1.00 (0.41)-2.01, 0.00860.05122.24, 31.96(23.82 - 25.07)(24.51 - 26.91)(-2.23 - -0.13)(23.42 - 31.62)Crude Fiber17.12 (0.38)18.10 (0.38)-0.98 (0.45)-2.07, 0.110.07016.93, 22.68(16.76 - 17.54)(17.35 - 19.63)(-2.09 - -0.10)(16.92 - 23.32)Neutral Detergent Fiber30.70 (0.41)32.12 (0.41)-1.42 (0.58)-2.84, 0.00150.05027.03, 42.49(30.40 - 31.02)(30.49 - 33.05)(-2.03 - 0.031)(29.27 - 40.63)Total Dietary Fiber40.66 (0.62)40.47 (0.62)0.19 (0.72)-1.57, 1.950.79734.52, 52.58(38.95 - 42.48)(39.15 - 42.09)(-1.42 - 1.70)(37.29 - 48.60)Amino Acid (% dw)Alanine1.05 (0.019)1.05 (0.019)0.0027 (0.026)-0.061, 0.0670.9200.86, 1.11(1.04 - 1.09)(0.97 - 1.10)(-0.061 - 0.069)(0.83 - 1.22)Arginine3.15 (0.074)3.25 (0.074)-0.10 (0.10)-0.36, 0.150.3552.38, 3.47(3.10 - 3.24)(2.94 - 3.49)(-0.37 - 0.18)(2.30 - 3.55)Monsanto Company 12-CT-244U 516 of 620Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Aspartic Acid2.47 (0.047)2.45 (0.047)0.027 (0.067)-0.14, 0.190.7021.94, 2.57(2.42 - 2.55)(2.26 - 2.62)(-0.20 - 0.19)(1.79 - 2.72)Cystine0.41 (0.015)0.40 (0.015)0.0059 (0.017)-0.035, 0.0460.7340.31, 0.45(0.39 - 0.44)(0.36 - 0.45)(-0.019 - 0.050)(0.29 - 0.47)Glutamic Acid4.95 (0.13)5.02 (0.13)-0.068 (0.19)-0.53, 0.390.7283.74, 5.28(4.85 - 5.11)(4.41 - 5.32)(-0.48 - 0.57)(3.39 - 5.45)Glycine1.11 (0.022)1.11 (0.022)-0.0046 (0.031)-0.081, 0.0710.8860.90, 1.14(1.08 - 1.14)(1.03 - 1.19)(-0.086 - 0.050)(0.85 - 1.23)Histidine0.74 (0.018)0.76 (0.018)-0.013 (0.026)-0.075, 0.0500.6350.59, 0.81(0.70 - 0.77)(0.71 - 0.82)(-0.047 - 0.034)(0.57 - 0.84)Isoleucine0.92 (0.013)0.93 (0.013)-0.010 (0.017)-0.051, 0.0310.5690.75, 0.96(0.89 - 0.94)(0.89 - 0.97)(-0.058 - 0.022)(0.72 - 1.03)Monsanto Company 12-CT-244U 517 of 620Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Leucine1.55 (0.026)1.55 (0.026)0.00049 (0.037)-0.091, 0.0920.9891.25, 1.62(1.52 - 1.58)(1.45 - 1.64)(-0.090 - 0.10)(1.20 - 1.72)Lysine1.22 (0.026)1.24 (0.026)-0.016 (0.037)-0.11, 0.0750.6801.01, 1.30(1.17 - 1.26)(1.19 - 1.33)(-0.066 - 0.018)(0.99 - 1.44)Methionine0.37 (0.021)0.39 (0.021)-0.018 (0.029)-0.090, 0.0530.5530.32, 0.38(0.33 - 0.40)(0.32 - 0.44)(-0.068 - 0.051)(0.29 - 0.49)Phenylalanine1.48 (0.034)1.48 (0.034)0.00099 (0.048)-0.12, 0.120.9841.12, 1.58(1.47 - 1.52)(1.36 - 1.61)(-0.13 - 0.11)(1.10 - 1.63)Proline1.05 (0.024)1.04 (0.024)0.012 (0.033)-0.069, 0.0940.7240.83, 1.08(1.02 - 1.08)(1.01 - 1.11)(-0.074 - 0.076)(0.79 - 1.17)Serine1.12 (0.031)1.11 (0.031)0.0077 (0.044)-0.10, 0.120.8670.83, 1.21(1.07 - 1.17)(0.97 - 1.17)(-0.11 - 0.14)(0.81 - 1.24)Monsanto Company 12-CT-244U 518 of 620Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Threonine0.88 (0.016)0.86 (0.016)0.020 (0.022)-0.034, 0.0730.4070.72, 0.89(0.87 - 0.90)(0.80 - 0.91)(-0.042 - 0.075)(0.67 - 0.96)Tryptophan0.41 (0.013)0.43 (0.013)-0.013 (0.018)-0.058, 0.0320.5020.34, 0.42(0.39 - 0.43)(0.38 - 0.47)(-0.079 - 0.051)(0.31 - 0.46)Tyrosine0.82 (0.017)0.81 (0.017)0.015 (0.024)-0.043, 0.0740.5460.67, 0.84(0.81 - 0.85)(0.74 - 0.87)(-0.042 - 0.074)(0.63 - 0.91)Valine1.26 (0.030)1.23 (0.030)0.029 (0.042)-0.074, 0.130.5131.00, 1.28(1.19 - 1.38)(1.16 - 1.29)(-0.072 - 0.15)(0.97 - 1.36)Fatty Acid (% Total FA)14:0 Myristic0.83 (0.010)0.84 (0.010)-0.013 (0.013)-0.046, 0.0200.3580.16, 1.37(0.81 - 0.85)(0.81 - 0.87)(-0.063 - 0.019)(0.45 - 1.04)16:0 Palmitic23.24 (0.14)23.01 (0.14)0.22 (0.16)-0.18, 0.620.21916.54, 30.55(22.99 - 23.40)(22.86 - 23.25)(0.045 - 0.54)(19.11 - 26.73)Monsanto Company 12-CT-244U 519 of 620Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)16:1 Palmitoleic0.47 (0.0058)0.46 (0.0058)0.0043 (0.0072)-0.013, 0.0220.5750.39, 0.70(0.45 - 0.48)(0.45 - 0.47)(-0.017 - 0.019)(0.44 - 0.67)18:0 Stearic2.31 (0.025)2.46 (0.025)-0.15 (0.028)-0.22, -0.0830.0011.98, 2.95(2.26 - 2.34)(2.40 - 2.52)(-0.24 - -0.090)(1.98 - 2.97)18:1 Oleic16.14 (0.078)16.16 (0.078)-0.021 (0.11)-0.29, 0.250.85211.38, 20.64(16.04 - 16.22)(15.86 - 16.44)(-0.22 - 0.31)(13.71 - 18.39)18:2 Linoleic55.63 (0.21)55.58 (0.21)0.050 (0.29)-0.67, 0.770.86847.49, 63.18(55.38 - 55.91)(55.18 - 56.13)(-0.74 - 0.44)(49.78 - 59.61)18:3 Linolenic0.16 (0.0056)0.17 (0.0056)-0.0030 (0.0079)-0.022, 0.0160.7140.060, 0.24(0.14 - 0.18)(0.16 - 0.17)(-0.032 - 0.012)(0.10 - 0.29)20:0 Arachidic0.26 (0.0055)0.28 (0.0055)-0.018 (0.0078)-0.037, 0.000610.0550.17, 0.38(0.24 - 0.28)(0.28 - 0.29)(-0.049 - -0.0036)(0.20 - 0.36)Monsanto Company 12-CT-244U 520 of 620Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)22:0 Behenic0.16 (0.0015)0.16 (0.0015)0.0019 (0.0020)-0.0030, 0.00670.3850.070, 0.21(0.15 - 0.16)(0.16 - 0.16)(-0.0028 - 0.0060)(0.051 - 0.19)MineralCalcium (% dw)0.16 (0.0016)0.14 (0.0016)0.024 (0.0023)0.019, 0.030<0.0010.058, 0.21(0.16 - 0.17)(0.13 - 0.14)(0.018 - 0.033)(0.081 - 0.18)Copper (mg/kg dw)10.33 (0.15)9.98 (0.15)0.35 (0.22)-0.18, 0.880.1562.97, 12.86(9.96 - 10.53)(9.58 - 10.41)(-0.45 - 0.83)(4.46 - 11.62)Iron (mg/kg dw)69.52 (5.67)79.02 (5.67)-9.50 (7.06)-26.76, 7.770.22747.30, 97.12(59.77 - 92.17)(67.45 - 95.10)(-14.73 - -2.93)(39.49 - 114.34)Magnesium (% dw)0.36 (0.0045)0.34 (0.0045)0.025 (0.0040)0.015, 0.035<0.0010.28, 0.47(0.35 - 0.37)(0.33 - 0.34)(0.015 - 0.032)(0.31 - 0.46)Manganese (mg/kg dw)11.38 (0.34)9.04 (0.34)2.34 (0.48)1.17, 3.510.0029.07, 17.33(10.73 - 12.83)(8.83 - 9.54)(1.35 - 4.00)(9.07 - 17.14)Monsanto Company 12-CT-244U 521 of 620Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. ConventionalControl (continued)Difference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (% dw)0.58 (0.0099)0.57 (0.0099)0.0062 (0.011)-0.020, 0.0320.5820.49, 0.87(0.58 - 0.59)(0.54 - 0.60)(-0.020 - 0.032)(0.48 - 0.87)Potassium (% dw)1.03 (0.023)0.87 (0.023)0.16 (0.033)0.079, 0.240.0020.92, 1.21(0.99 - 1.09)(0.79 - 0.93)(0.061 - 0.30)(0.90 - 1.26)Sodium (% dw)0.018 (0.010)0.047 (0.010)-0.029 (0.014)-0.063, 0.00620.0910, 0.066(0.0054 - 0.024)(0.019 - 0.090)(-0.085 - 0.0040)(0.0054 - 0.077)Zinc (mg/kg dw)34.14 (0.45)34.96 (0.45)-0.82 (0.64)-2.39, 0.760.25027.27, 44.95(33.08 - 35.14)(33.70 - 35.89)(-2.58 - 0.54)(25.07 - 48.49)Vitamin (mg/kg dw)Vitamin E110.33 (2.75)103.66 (2.75)6.67 (3.90)-2.86, 16.200.13741.91, 205.89(103.52 - 114.05)(93.92 - 109.90)(-6.38 - 20.14)(84.07 - 162.76)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 12-CT-244U 522 of 620Table E-38. Statistical Summary of SiteTXPL Cottonseed Anti-nutrients for MON 88701 (Not Treated) vs. ConventionalControlDifference (MON 88701 minus Control)Analytical Component(Units)1MON 887012Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid0.16 (0.0047)0.16 (0.0047)-0.0020 (0.0066)-0.018, 0.0140.7770.078, 0.25(0.16 - 0.17)(0.15 - 0.17)(-0.0083 - 0.0048)(0.038 - 0.23)Malvalic Acid0.42 (0.020)0.47 (0.020)-0.046 (0.029)-0.12, 0.0250.1610.23, 0.54(0.40 - 0.44)(0.44 - 0.49)(-0.072 - -0.018)(0.11 - 0.59)Sterculic Acid0.23 (0.013)0.26 (0.013)-0.025 (0.018)-0.068, 0.0190.2160.17, 0.27(0.21 - 0.25)(0.25 - 0.27)(-0.043 - 0.0078)(0.061 - 0.34)Gossypol (% dw)Free Gossypol0.98 (0.024)0.93 (0.024)0.052 (0.033)-0.030, 0.130.1700.099, 1.57(0.94 - 1.05)(0.91 - 0.95)(0.0089 - 0.14)(0.50 - 1.41)Total Gossypol1.09 (0.024)1.01 (0.024)0.081 (0.022)0.028, 0.130.0090.064, 1.76(1.03 - 1.16)(0.97 - 1.05)(0.053 - 0.11)(0.56 - 1.61)1dw = dry weight; FA = fatty acid.2 MON 88701 plants were not treated with dicamba or glufosinate.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (Coker 130).5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.

# Monsanto Company 12-CT-244U 523 of 620 

# References for Appendix E

AACC. 1999. Official method 32-20. American Association of Ce real Chemists, St. Paul,

Minnesota.

AOAC. 2011a. AOAC official method 988.15: Tr yptophan in foods and food and feed

ingredients. Official Methods of Analysis of AOAC Inte rnational, Association of

Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011b. AOAC official method 923.03: Ash of flour . Official Methods of

Analysis of AOAC International, Associati on of Analytical Communities International,

Gaithersburg, Maryland.

AOAC. 2011c. AOAC official me thod 962.09: Fiber (crude) in an imal feed and pet food.

Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011d. AOAC official method 948.22: Fat (crude) in nuts and nut products.

Official Methods of Analysis of AOAC In ternational, Associa tion of Analytical

Communities International, Gaithersburg, Maryland.

AOAC. 2011e. AOAC offi cial method 960.39: Fat (crude) or ether extract in meat.

Official Methods of Analysis of AOAC In ternational, Associa tion of Analytical

Communities International, Gaithersburg, Maryland.

AOAC. 2011f. AOAC official method 984.27: Calcium, copper, iron, magnesium,

manganese, phosphorus, potassium, sodium, and zinc in infant formula. Official Methods

of Analysis of AOAC International, Association of Analytical Communities

International, Gaithersburg, Maryland.

AOAC. 2011g. AOAC offici al method 985.01: Metals and other elements in plants and

pet foods. Association of Analytical Comm unities International, Gaithersburg, Maryland.

AOAC. 2011h. AOAC official method 925.09: Solids (tot al) and loss on drying

(moisture) in flour. Official Methods of Analysis of AOAC In ternational, Association of

Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011i. AOAC official method 926.08: Loss on drying (moisture) in cheese.

Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011j. AOAC offi cial method 955.04: Nitrogen (total) in fertilizers. Official

Methods of Analysis of AOAC International, Association of Analytical Communities

International, Gaithersburg, Maryland.

AOAC. 2011k. AOAC offici al method 979.09: Protein in gr ains. Official Methods of

Analysis of AOAC International, Associati on of Analytical Communities International,

Gaithersburg, Maryland.

# Monsanto Company 12-CT-244U 524 of 620 

# AOCS. 1997. Preparation of meth yl esters of fatty acids. Method Ce 2-66, American Oil

Chemists' Society, Champaign, Illinois.

AOCS. 2001. Determination of fatty acids in edible oils and fats by capillary GLC.

Method Ce 1e-91, American Oil Chem ists' Society, Champaign, Illinois.

AOCS. 2009a. Official Met hods and Recommended Practices of the American Oil

Chemists' Society. Method Ce 1i-07, Amer ican Oil Chemists' Society, Champaign,

Illinois.

AOCS. 2009b. Nitrogen-ammonia- protein modified Kjeldahl method titanium dioxide +

copper sulfate catalyst. Offici al Methods and Recommended Practices of the American

Oil Chemists' Society, 5th Edition. Method Ac 4-91, American Oil Chemists' Society,

Champaign, Illinois.

AOCS. 2009c. Direct methylatio n of lipids in foods for the determination of total fat,

saturated, cis -monounsaturated, cis -polyunsaturated, and trans fatty acids by gas

chromatography. Method Ce 1k-09, American Oil Chemists' Society, Champaign,

Illinois.

AOCS. 2011a. Total gossypol. Method Ba 8-78, American Oil Chemists' Society,

Champaign, Illinois.

AOCS. 2011b. Free gossypol. Method Ba 7- 58, American Oil Chemists' Society,

Champaign, Illinois.

Barkholt, V. and A.L. Jensen. 1989. Amino acid analysis determination of cysteine plus

half-cystine in proteins afte r hydrochloric acid hydrolysis with a disulfide compound as

additive. Analytical Biochemistry 177:318-322.

Bertrand, J.A., T.Q. Sudduth, A. Condon, T.C. Jenkins and M.C. Calhoun. 2005. Nutrient

content of whole cottonseed. Journal of Dairy Science 88:1470-1477.

Cort, W., T.S. Vicente, E.H. Waysek a nd B.D. Williams. 1983. Vitamin E content of

feedstuffs determined by high-performance liquid chromatographic fluorescence. Journal

of Agriculture and Food Chemistry 31:1330-1333.

Hamilton, K.A., P.D. Pyla, M. Breeze, T. Ol son, M. Li, E. Robinson, S.P. Gallagher, R.

Sorbet and Y. Chen. 2004. Bollgard II cotton: Compositional analysis and feeding studies

of cottonseed from insect-protected cotton ( Gossypium hirsutum L.) producing the

Cry1Ac and Cry2Ab2 proteins. Journal of Agricultural and Food Chemistry 52: 6969-

6976.

Henderson, J.W. and A. Brooks. 2010. Improv ed amino acid methods using Agilent

ZORBAX Eclipse Plus C18 columns for a va riety of Agilent LC instrumentation and

separation goals. Agilent Technologies, Inc., Wilmington, Delaware.

# Monsanto Company 12-CT-244U 525 of 620 

# Henderson, J.W., R.D. Ricker, B.A. Bid lingmeyer and C. Woodward. 2000. Rapid,

accurate, sensitive, and reproducible HPLC analysis of amino acids. Agilent

Technologies, Inc., Wilmington, Delaware.

ILSI. 2011. Crop Composition Database, Vers ion 4.2. International Life Sciences

Institute, Washington, D.C. http://www.cropcomposition.org/

.

Komarek, A.R., J.B. Robertson and P.J. Va n Soest. 1993. A comparison of methods for

determining ADF using the Filter Bag Techni que versus conventional filtration. Journal

of Animal Science 72:114.

Komarek, A.R., J.B. Roberts on and P.J. Van Soest. 1994. Comparison of the filter bag

technique to conventional filt ration in the Van Soest NDF an alysis of 21 foods. National

Conference on Forage Quality, Evaluation, and Utilization, American Society of

Agronomy, Inc., Crop Science Society of Amer ica, Inc., Soil Society of America, Inc.,

Madison, Wisonsin.

Lawhon, J.T., C.M. Cater and K.F. Mattil. 1977 . Evaluation of the f ood use potential of

sixteen varieties of cottonseed. Journal of the American Oil Chemists' Society 54:75-80.

McMurray, C.H., W.J. Blanchflower and D.A. Rice. 1980. Infl uence of extraction

techniques on determination of  Tocopherol in animal feedstuffs. Journal of the

Association of Official Analytical Chemists 63:1258-1261.

Schuster, R. 1988. Determination of amino acids in biological pharmaceutical plant and

food samples by automated precolumn de rivatization and high-performance liquid

chromatography. Journal of Chromatography 431:271-284.

Smith, C.W. and R.A. Creelman. 2001. Vitamin E concentration in upland cotton seeds.

Crop Science 41:577-579.

Speek, A.J., J. de Schrijver and W.H.P. Schreurs. 1985. Vitamin E composition of some

seed oils as determined by high-performa nce liquid chromatography with fluorometric

detection. Journal of Food Science 50:121-124.

USDA. 1970. Forage fiber analyses (appara tus, reagents, procedures, and some

applications). Agricultur al Handbook No. 379. U.S. Department of Agriculture,

Washington, DC.

USDA. 1973. Energy value of foods: Basis an d derivation. Agricultural Handbook No.

74. US Department of Agriculture, Agricultu ral Research Service, Washington, D.C.

Wood, R. 1986. High-performance liquid chromat ography analysis of cyclopropene fattyacids. Biochemical Archives 2:63-72.

# Monsanto Company 12-CT-244U 526 of 620 

# Appendix F: Materials, Methods, and Individual Site Results for Seed

Dormancy and Germination Assessment of MON 88701

F.1. Materials

Seed dormancy and germination characterist ics were assessed on seed from MON 88701,

the conventional control, and commercial refe rence varieties produced in 2010 field trials

at the following sites: Crittenden County, Arkansas (ARPR); Caswell County, North

Carolina (NCME); and Hale County, Texas (T XPL) in 2010 (Table VII-3). The field

trial at each site was established in a ra ndomized complete block design with four

replications. The seed from MON 88701, the conventional control, and the commercial

reference varieties were harveste d from all four replicated plot s at each of the three field

sites.

Table F-1. Starting Seed of MON 88701, Conventional Control and Commercial

Cotton Reference Varieties Used in Dormancy Assessment

Site 1 

# Material Material Type Phenotype Sample ID

ARPR Coker 130 Control Conventional 11268128

ARPR MON 88701 Test DGT Cotton 1 

# 11268129

ARPR SG 125 Reference Conventional 11266155

ARPR DP 565 Reference Conventional 11266764

ARPR ST 474 Reference Conventional 11266156

ARPR DP 5415 Reference Conventional 11266157

NCME Coker 130 Control Conventional 11268128

NCME MON 88701 Test DGT Cotton 11268129

NCME DP 435 Reference Conventional 11266762

NCME Delta Opal Reference Conventional 11266158

NCME SG 125 Reference Conventional 11266155

NCME FM 989 Reference Conventional 10001810

TXPL Coker 130 Control Conventional 11268128

TXPL MON 88701 Test DGT Cotton 11268129

TXPL Atlas Reference Conventional 11266765

TXPL DP 435 Reference Conventional 11266762

TXPL SG 125 Reference Conventional 11266155

TXPL NM 1517-99 Reference Conventional 11268233

1 

# DGT Cotton = Dicamba and glufosinate-tolerant cotton.

# Monsanto Company 12-CT-244U 527 of 620 

# F.2. Characterization of the Materials

For the MON 88701, the parental conventional control, and the commercial reference

varieties starting seed lots, the pres ence or absence of the MON 88701 insert was

confirmed by event-specific pol ymerase chain reaction analyses.

F.3. Germination Testing Facili ty and Experimental Methods

Seed dormancy and germination evaluations we re conducted at BioDiagnostics, Inc. in

River Falls, WI. The principal investigat or was qualified to conduct seed dormancy and

germination testing consistent with the st andards established by the Association of

Official Seed Analysts (AOSA), a s eed trade association (AOSA, 2010a; b;

AOSA/SCST, 2010).

Seed lots of MON 88701, the conventional c ontrol, and four commercial reference

varieties were produced from each of three field sites and tested under six different

temperature regimes. Each temperature re gime constituted a different experiment ( i.e

., no comparisons were made between temperat ure regimes). Six germination chambers

were maintained dark under one of the following temperature regimes: constant

temperature of approximately 10, 20, or 30 °C, or alternating temperatures of

approximately 10/20, 10/30, or 20/30 °C. The alternating temperature regimes were

maintained at the lower temperature for 16 hours and the higher temperature for eight

hours. The temperature inside each germin ation chamber was monitored and recorded

every 15 minutes throughout the duration of the assessment. Prior to the study, the

starting seed were treated uniformly with the commercial seed treatment fungicides

mefenoxam and fludioxonil at labeled rates. Approximately 100 cottonseeds were placed

on the germination towels using a vacuum planting system. Two additional premoistened germination towels were placed on top of the cottonseed. The set was rolled

up and secured with a rubber ba nd. All rolled germination to wels were labeled and then

placed into an appropriately labeled bucket. One germination towel was prepared for

each of the six starting cottons eed entries from each individu al site, all of which were

placed into a single bucket. A bucket was prep ared for each site and replicaton for a total

of 12 buckets per temperature regime. Buckets were placed in the appropriate

germination chambers.

A description of each germination characterist ic evaluated and the timing of evaluations

are presented in Table VII-1. 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 evaluated periodically during the study for normal germinated,

abnormal germinated, viable hard, dead, an d viable firm-swollen seed as defined by

AOSA guidelines (AOSA, 2010a; b; AOS A/SCST, 2010). AOSA only provides

guidelines (AOSA, 2010a) for testing seed under optimal temperatures (20/30°C);

however, additional temperature regimes were included to test a range of temperature

conditions. Each rolled germination towel in the additional temperature regimes ( i.e. , 10,

20, 30, 10/20, and 10/30°C) was evaluated periodi cally for germinated, viable hard, dead,

and viable firm-swollen seed. Emergence and/ or development of essential structures of

seedlings that otherwise would be categor ized as “normal germinated” under optimal

# Monsanto Company 12-CT-244U 528 of 620 

# temperature conditions may not be so at non- optimal temperatures. Therefore, for the

additional temperature regimes, no distinct ion was made between normal and abnormal

germinated seed.

F.4. Statistical Analysis

An analysis of variance was conducted using SAS  

# Version 9.2 (SAS, 2008) according to

a split-plot design with four replications. MON 88701 was co mpared to the conventional

control for dormancy and germination characte ristics of cottonseed produced within each

site ( i.e ., individual site analysis) an d in a combined-site analysis in which the data were

pooled across all three s ites. The seed germination char acteristics analyzed included

percent germinated seed, percent viable hard seed, percent dead see d, and percent viable

firm-swollen seed. The percent germinated seed were categorized as either normal

germinated or abnormal germinated for the AOSA temperature regime. The level of

statistical significance was pr edetermined to be 5% (  =0.05). MON 88701 was not

statistically compared to th e reference varieties, nor we re comparisons made across

temperature regimes. The minimum and maxi mum mean values were determined from

the reference materials across all sites to provide a range of values ( i.e ., reference range)

representative of commercial cotton varieties. Results from the combined-site analysis

are presented in Table VII-2.

F.5. Individual Site Seed Dormancy and Germination Analyses

In the individual site analyses, no statisti cally significant differe nces were detected

between MON 88701 and the parental conventi onal control for any of the measured

characteristics ( i.e. , percent germinated, viab le hard, dead, or viable firm-swollen seed) in

any temperature regime for seed produced at the ARPR and NCME sites. Three

statistically significant diffe rences in total were detected between MON 88701 and the

conventional control for cottonseed produced at the TXPL site (Tab le F-2). At TXPL,

MON 88701 had fewer dead seed than the co nventional control at 10/20°C (4.5% vs.

9.5%); MON 88701 had more germinated seed th an the conventional control at 20/30°C

(92.5% vs. 84.0%); and, MON 88701 had fewer abnormal germinated seed than the

conventional control at 20/30°C (2.5% vs. 7.3%). Statistically sign ificant differences

between MON 88701 and the conventional control for germination characteristics in the

individual site analyses were not consistently detected acro ss temperature regimes or the

individual sites. While some statistically significant differences were detected in the

combined-site analysis, these statistical differences were within the range of values

expected for the commercial reference variet ies. Thus, MON 88701 is unlikely to have

increased plant pest/weed potential or an a dverse environmental impact compared to the

conventional control. (See Figure VII-1, Schematic Diagram of Agronomic and

Phenotypic Data Interpretation Methods , step 3, answer “no”).

 

# SAS is a registered trademark of SAS Institute, Inc.Monsanto Company 12-CT-244U 529 of 620Table F-2. Comparison of MON 88701 to the Conventional Control for Dormancy and Germination Characteristics ofCottonseed Produced at Each of Three SitesARPR1NCME1TXPL1Temperature Germination Mean % (S.E.)2Mean % (S.E.)2Mean % (S.E.)2Regime Category MON 88701 Control MON 88701 Control3MON 88701 Control10 °C Germinated 49.5 (13.5) 41.8 (11.2) 22.0 (7.2) 24.7 (4.2) 27.0 (7.6) 35.5 (5.5)Viable Hard 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.7 (0.7) 0.0 (0.0) 0.0 (0.0)Dead 16.0 (5.0) 15.0 (3.6) 32.0 (6.5) 26.7 (3.9) 20.5 (6.9) 25.8 (7.3)Viable Firm Swollen 34.5 (15.7) 43.0 (13.7) 46.0 (12.2) 48.0 (5.8) 52.5 (11.8) 38.8 (10.3)20 °C Germinated 96.0 (0.7) 96.8 (0.5) 94.5 (1.6) 97.3 (0.9) 96.5 (1.5) 92.3 (3.0)Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 4.0 (0.7) 3.3 (0.5) 5.5 (1.6) 2.7 (0.9) 3.5 (1.5) 7.8 (3.0)Viable Firm Swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)30 °C Germinated 99.0 (0.7) 97.0 (0.9) 95.8 (1.7) 93.7 (1.9) 95.2 (1.2) 92.3 (2.5)Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 1.0 (0.7) 3.0 (0.9) 4.3 (1.7) 6.3 (1.9) 4.8 (1.2) 7.8 (2.5)Viable Firm Swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Monsanto Company 12-CT-244U 530 of 620Table F-2. Comparison of MON 88701 to the Conventional Control for Dormancy and Germination Characteristics ofCottonseed Produced at Eachof Three Sites (continued)ARPR1NCME1TXPL1Temperature Germination Mean % (S.E.)2Mean % (S.E.)2Mean % (S.E.)2Regime Category MON 88701 Control MON 88701 Control3MON 88701 Control10/20 °C Germinated 98.0 (0.7) 95.3 (1.1) 90.3 (2.5) 90.3 (3.9) 95.0 (1.2) 90.0 (2.1)Viable Hard0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 2.0 (0.7) 4.3 (0.9) 6.5 (0.6) 6.0 (2.5) 4.5 (0.9)* 9.5 (2.1)Viable Firm Swollen0.0 (0.0) 0.3 (0.3) 3.3 (2.9) 3.7 (1.5) 0.5 (0.5) 0.5 (0.3)10/30 °C Germinated 97.3 (0.8) 97.8 (0.9) 93.8 (2.7) 96.7 (0.9) 96.0 (1.4) 91.3 (3.5)Viable Hard0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 2.8 (0.8) 2.3 (0.9) 6.3 (2.7) 3.3 (0.9) 4.0 (1.4) 8.8 (3.5)Viable Firm Swollen0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)20/30 °C Normal Germinated 92.0 (1.9) 94.5 (0.3) 84.2 (4.6) 84.7 (3.1) 92.5 (1.3)* 84.0 (7.2)(AOSA) Abnormal Germinated 3.8 (1.4) 2.5 (0.3) 8.0 (2.6) 9.0 (3.0) 2.5 (0.9)* 7.3 (2.2)Viable Hard0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 4.3 (1.5) 3.0 (0.6) 7.5 (2.0) 6.3 (0.9) 5.0 (0.7) 8.8 (5.5)Viable Firm Swollen0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Note: The experimental design for the germination test was a split-plot with four replications and statistical analysis consisted of an analysis of variance(ANOVA) model.*Statistically significant differences detected (α= 0.05) between MON 88701 and the conventional control (n = 11).1In some instances, the total percentage of MON 88701 or the control did not equal 100% due to numerical rounding of the means.2SE = Standard Error.3Control – The NCME site only had 3 reps across all temperature regimes compared to 4 reps for other sites and treatments

# Monsanto Company 12-CT-244U 531 of 620 

# References for Appendix F

AOSA. 2010. AOSA Rules for testing seeds Vo l. 1: Principles and procedures.

Association of Official Seed Analysts, Ithaca, New York.

AOSA. 2010. AOSA Rules for testing seeds Vol. 4: Seedling evaluatio n. Association of

Official Seed Analys ts, Ithaca, New York.

AOSA/SCST. 2010. Tetrazolium testing handbook. A ssociation of Official Seed Analysts

and the Society of Commercial Seed Technologists, Ithaca, New York.

# Monsanto Company 12-CT-244U 532 of 620 

# Appendix G: Materials, Methods, Dicamb a and Glufosinate Treated Results,

and Individual Site Results from the Phenotypic, Agronomic, Plant Mapping,

and Environmental Interaction A ssessment of MON 88701 under Field

Conditions

G.1. Materials

Data were collected from two different st udies during 2010 to ev aluate phenotypic,

agronomic, and environmental interaction ch aracteristics. In Study 1, MON 88701 not

treated with dicamba or glufosinate herbic ides was evaluated against the conventional

control (Table G-1). In Study 2, MON 88701 not tr eated with dicamba or glufosinate

herbicides and MON 88701 treated with dicamba- and glufosin ate herbicides were both

evaluated against the conventional control (Table G-2) . Assessments were conducted on

the agronomic system that includes MON 88701 treated with dicamba and glufosinate

herbicides to support the assessment of MON 88701. In Study 1, a total of 11 cotton

reference varieties were evaluated among the s ites (Table G-1). Seven of the commercial

varieties were conventional and four of th e commercial varieties were tolerant to

glyphosate herbicide. Three conventional refe rence varieties and one glyphosate-tolerant

reference variety were grown at each location. In Study 2, a total of 8 commercial

conventional reference varie ties were evaluated among the sites. Four commercial

conventional reference vari eties were planted per site (Table G-2).

G.2. Characterization of the Materials

The presence or absence of the MON 88701 in sert in the starting seed of MON 88701

and the conventional control were confirmed by event-specific polymerase chain reaction

analyses.

G.3. Field Sites and Plot Design

Field sites in both studies (T able VII-3) are representative of commercial cotton growing

areas and are distributed acros s a geographical area to incl ude a variety of agronomic

practices, soils and climatic factors. The res earchers at each field site were familiar with

the growth, production, and evaluation of cotton ch aracteristics. The starting seed in each

study were planted at each site in a rando mized complete-block design with four

replications.

Data from Study 1 were collected at fifteen field sites in the U.S. during 2010 (Table VII3). Each plot (Table G-3) at the ARAU (Arkansas), ARPR (Arkansas), GACH

(Georgia), LABU (Louisiana), and SCEK (S outh Carolina) consisted of 12 rows on 0.91

to 1.02 meter centers and rows were 9.1 meters long. In general, rows 2 and 3 were

designated for the collection of following da ta: phenotypic, plant response to abiotic

stress, disease damage, and arthropod damage . Rows 5 and 7 were designated for the

collection of arthropod samples. Rows 9 a nd 10 were designated for the collection of

damage data from thrips and heliothine spp. Rows 1, 4, 6, 8, 11, and 12 were used as

buffer rows.

# Monsanto Company 12-CT-244U 533 of 620 

# Each plot (Table G-3) at the ARTI (Arkan sas), GAJE (Georgia), KSLA (Kansas), LACH

(Louisiana), NCBD (North Carolina), NCME (North Carolina), NMGA (New Mexico),

NMLC (New Mexico), TXPL (Texas), and TXPO (Texas) was 4 rows, row spacing of

0.76 to 1.02 meters and rows were 6.1 meters long. In general, rows 2 and 3 were

designated for the collection of the following data: phenotypic, plant response to abiotic

stress, disease damage, and arthr opod damage. Rows 1 and 4 were used as buffer rows.

Data from Study 2 were collected at eleven field sites in the U.S. during 2010 (Section

VII, Table VII-3). Each plot (Table G-4) was eight rows, row spacing was 0.76 to 1.02

meters and rows were 6 meters long. Rows 2 and 3 were designated for the collection of

phenotypic, plant mapping and enviro nmental interaction data.

G.4. Planting and Field Operations

Field and planting information for Study 1 and Study 2 are listed in Tables G-3 and G-4,

respectively. Prior to planting, the Field Coope rator at each field site prepared the plot

area with a proper seed bed according to lo cal agronomic practices , including tillage,

fertilization and pH adjustment, and pest management. During the growing season, all

plots were assessed for agronomic conditi ons and pest populations, including pest

arthropods, diseases, and weed s. Fertilizer, irrigation, agri cultural chemicals and other

management treatments were applied as necessary. All maintenance operations

mentioned above were performed unif ormly across all plots at a site.

# Monsanto Company 12-CT-244U 534 of 620 

# Table G-1. Starting Seed for Study 1

Site 1 

# Material Name Phenotype 2 

# Regulatory

Lot Number

All MON 88701 DGT 11268129

All Coker 130 Control 11268128

ARTI Phytogen Phy315RF Glyphosate-Tolerant 11266967

ARTI Delta Opal Conventional 11266158

ARTI DP435 Conventional 11266762

ARTI ST474 Conventional 11266156

LABU Bayer FM9058F Glyphosate-Tolerant 11266968

LABU DP565 Conventional 11266764

LABU SG125 Conventional 11266155

LABU ST474 Conventional 11266156

NCME Nex Gen NG3410RF Glyphosate-Tolerant 11266969

NCME DP5415 Conventional 11266157

NCME Delta Opal Conventional 11266158

NCME DP435 Conventional 11266762

GAJE Bayer FM9058F Glyphosate-Tolerant 11266968

GAJE DP493 Conventional 11266763

GAJE DP565 Conventional 11266764

GAJE SG125 Conventional 11266155

NCBD Phytogen Phy315RF Glyphosate-Tolerant 11266967

NCBD ST474 Conventional 11266156

NCBD DP5415 Conventional 11266157

NCBD Delta Opal Conventional 11266158

TXPO Bayer FM9058F Glyphosate-Tolerant 11266968

TXPO DP435 Conventional 11266762

TXPO DP493 Conventional 11266763

TXPO DP565 Conventional 11266764

TXPL All tex patriotRF Glyphosate-Tolerant 11266966

TXPL SG125 Conventional 11266155

TXPL ST474 Conventional 11266156

TXPL DP5415 Conventional 11266157

ARPR Nex Gen NG3410RF Glyphosate-Tolerant 11266969

ARPR ST474 Conventional 11266156

ARPR DP493 Conventional 11266763

ARPR SG125 Conventional 11266155

ARAU Phytogen Phy315RF Glyphosate-Tolerant 11266967

ARAU Delta opal Conventional 11266158

ARAU DP435 Conventional 11266762

ARAU ST474 Conventional 11266156

SCEK Bayer FM9058F Glyphosate-Tolerant 11266968

SCEK SG125 Conventional 11266155

SCEK ST474 Conventional 11266156

SCEK DP5415 Conventional 11266157

# Monsanto Company 12-CT-244U 535 of 620 

# Table G-1. Starting Seed for Study 1 (continued)

Site 1 

# Material Name Phenotype 2 

# Regulatory

Lot Number

GACH Nex Gen NG3410RF Glyphosate-Tolerant 11266969

GACH Delta opal Conventional 11266158

GACH DP435 Conventional 11266762

GACH DP493 Conventional 11266763

LACH Phytogen Phy315RF Glyphosate-Tolerant 11266967

LACH DP5415 Conventional 11266157

LACH ST474 Conventional 11266156

LACH DP435 Conventional 11266762

KSLA All tex patriotRF Glyphosate-Tolerant 11266966

KSLA Delta opal Conventional 11266158

KSLA DP435 Conventional 11266762

KSLA DP493 Conventional 11266763

NMLC Nex Gen NG3410RF Glyphosate-Tolerant 11266969

NMLC DP565 Conventional 11266764

NMLC SG125 Conventional 11266155

NMLC ST474 Conventional 11266156

NMGA All tex patriotRF Glyphosate-Tolerant 11266966

NMGA DP5415 Conventional 11266157

NMGA Delta opal Conventional 11266158

NMGA DP435 Conventional 11266762

1 

# Sites - ARTI = Desha County, Arkansas; LABU = Rapides County, Louisiana; NCME = Caswell County,

North Carolina; GAJE = Twiggs County, Georgia; NCBD = Perquimans County, North Carolina; TXPO =

San Patricio County, Texas; TXPL = Hale County, Texas; ARPR = Crittenden County, Arkansas; ARAU =

Jackson County, Arkansas; SCEK = Barnwell County , South Carolina; GACH = Tift County, Georgia;

LACH = Rapides County, Louisiana; KSLA = Pawnee County, Kansas; NMLC = Dona Ana County, New

Mexico; NMGA = Dona Ana County, New Mexico.

2 

# Phenotype abbreviations: DGT = dicamba and glufosinate-tolerant.

# Monsanto Company 12-CT-244U 536 of 620 

# Table G-2. Starting Seed for Study 2

Site 1 

# Material Name 2 

# Phenotype 3 

# Regulatory Lot

Number

All Coker 130 Conventional 11268128

ARPR SG125 Conventional 11266155

ARPR DP 565 Conventional 11266764

ARPR ST 474 Conventional 11266156

ARPR DP 5415 Conventional 11266157

ARTI SG125 Conventional 11266155

ARTI DP 5415 Conventional 11266157

ARTI DP 435 Conventional 11266762

ARTI FM 989 Conventional 10001810

GACH DP 565 Conventional 11266764

GACH ST 474 Conventional 11266156

GACH FM 989 Conventional 10001810

GACH Delta Opal Conventional 11266158

GAJE SG125 Conventional 11266155

GAJE ST 474 Conventional 11266156

GAJE DP 5415 Conventional 11266157

GAJE DP 435 Conventional 11266762

KSLA DP 565 Conventional 11242914

KSLA DP 5415 Conventional 11266157

KSLA Atlas Conventional 11266765

KSLA NM 1517-99 Conventional 11268233

LACH DP 565 Conventional 11266764

LACH ST 474 Conventional 11266156

LACH DP 5415 Conventional 11266157

LACH FM 989 Conventional 10001810

NCBD SG125 Conventional 11266155

NCBD ST 474 Conventional 11266156

NCBD DP 435 Conventional 11266762

NCBD Delta Opal Conventional 11266158

NCME SG125 Conventional 11266155

NCME DP 435 Conventional 11266762

NCME FM 989 Conventional 10001810

NCME Delta Opal Conventional 11266158

# Monsanto Company 12-CT-244U 537 of 620 

# Table G-2. Starting Seed for Study 2 (continued)

Site 1 

# Material Name 2 

# Phenotype 3 

# Regulatory

Lot Number

NMLC DP 565 Conventional 11266764

NMLC ST 474 Conventional 11266156

NMLC Atlas Conventional 11266765

NMLC NM 1517-99 Conventional 11268233

SCEK SG125 Conventional 11266155

SCEK ST 474 Conventional 11266156

SCEK Delta Opal Conventional 11266158

SCEK Atlas Conventional 11266765

TXPL SG125 Conventional 11266155

TXPL DP 435 Conventional 11266762

TXPL Atlas Conventional 11266765

TXPL NM 1517-99 Conventional 11268233

All MON 88701 (U) DGT 11268129

All MON 88701 (S) DGT 11268129

1 

# ARPR= Crittenden County, Arkansas; ARTI = Desha County, Arkansas; GACH = Tift County, Georgia;

GAJE = Twiggs County, Georgia; KSLA = Pawnee County, Kansas; LACH = Rapides County, Louisiana;

NCBD = Perquimans County, North Carolina; NCME = Caswell County, North Carolina; NMLC = Dona

Ana County, New Mexico; SCEK = Barnwell County, South Carolina; TXPL = Hale County, Texas.

2 

# U = unsprayed, S = sprayed

3 

# DGT = dicamba and glufosinate-tolerant.Monsanto Company 12-CT-244U 538 of 620Table G-3. Study 1 Fieldand Planting InformationSite Planting Date1Planting Rate(seeds/m)Plot Size (m × m)2Rows/plot% Organic MatterSoil Texture 2009 Cropping HistoryARAU 5/25/2010 16 9.1 × 0.97 12 sandy loam 1.0 SoybeanARPR 5/19/2010 16 9.1 × 0.91 12 silt loam 0.9 CornARTI 5/24/2010 16 6.1 × 0.97 4 silt loam 2.2 CottonGACH 6/03/2010 16 9.1 × 0.91 12 loamy sand 1.0 CornGAJE 6/25/2010 16 6.1 × 0.91 4 loamy sand 0.8 FallowKSLA 6/02/2010 16 6.1 × 0.76 4 loam 2.1 CornLABU 5/20/2010 16 9.1 × 1.02 12 silt loam 0.6 CottonLACH 5/22/2010 16 6.1 × 1.02 4 very fine sandy 0.8 SoybeanNCBD 5/21/2010 16 6.1 × 0.97 4 loamy sand 1.6 CottonNCME 6/15/2010 16 6.1 × 0.76 4 loamy sand 0.9 SoybeanNMGA 5/20/2010 16 6.1 × 0.97 4 sandy loam 1.0 CottonNMLC 5/19/2010 16 6.1 × 0.97 4 loam 1.0 CottonSCEK 5/22/2010 16 9.1 × 1.02 12 loamy sand 1.5 CornTXPL 5/25/2010 16 6.1 × 1.02 4 clay loam 0.5 CornTXPO 5/21/2010 16 6.1 × 0.76 4 sandy clay loam 1.1 Corn1Month-day-year.2Length × width.Monsanto Company 12-CT-244U 539 of 620Table G-4. Study 2 Fieldand Planting InformationSite Code Planting Date1Planting Rate(seeds/m)Plot Size (m × m)2Rows/plot% Organic MatterSoil Texture 2009 Cropping HistoryARPR5/25/2010166 × 0.918Clay 1.3 MiloARTI5/24/2010166 × 0.978Silt loam 1.6 CottonGACH5/26/2010166 × 0.918Loamy sand 1.0 SoybeanGAJE6/25/2010166 × 0.918Loamy sand 0.8 FallowKSLA6/02/2010166 × 0.768Loam 3.1 SoybeanLACH5/21/2010166 x 1.028Silt loam 0.6 CottonNCBD5/27/2010166 x 0.978Loam 2.2 CottonNCME6/11/2010166 x 0.768Loamy sand 1.0 SoybeanNMLC5/17/2010166 x 0.978Sandy loam 1.0 CottonSCEK5/19/2010166 x 1.028Loamy sand 1.3 CornTXPL5/25/2010166 x 1.028Clay loam 0.5 Corn1Month-day-year.2Length × width.

# Monsanto Company 12-CT-244U 540 of 620 

# G.5. Phenotypic Observations

In both Study 1 and Study 2, the description of the characteristics measured and the

designated developmental stages when observati ons occurred are listed in Table VII-1.

G.6. Environmental Observations

In both Study 1 and Study 2, e nvironmental interactions ( i.e. , interactions between crop

plants and their receiving environment) were used to characterize MON 88701 by

evaluating plant response to abiotic stress, disease damage, and arthropod-related damage

using qualitative methods described in section G.7. In addition, pest damage and pestand beneficial-arthropod abundance were ev aluated in Study 1 using the quantitative

methods described in the following sections (G.7 and G.8).

G.7. Plant Response to Abiotic Stress, Disease Damage, and Arthropod-Related

Damage

In Study 1 and Study 2, plot s containing MON 88701 not treated with dicamba and

glufosinate herbicides and th e conventional control were evaluated qualitatively at all

sites for differences in plant response to abiotic stress, disease damage, and arthropodrelated damage. Three abiotic stressors, th ree diseases, and three arthropod pests were

evaluated four times during the growi ng season at the following intervals:

Observation 1: approximately 30 days after planting (DAP)

Observation 2: approximately 60 DAP

Observation 3: approximately 90 DAP

Observation 4: approximately 120 DAP

Method used for selecting st ressors at each field site:

1. Prior to each data collection, cotton was surv eyed in proximity to the study area or the

border rows of the study for abiotic stressors ( e.g. , drought), diseases ( e.g. , Alternaria

black spot), and arthropod damage ( e.g. , thrips).

2. Cooperators chose three abiotic stressors, three diseases, and three arthropod species

that are actively causing damage for subs equent evaluation in the study plots.

Cooperators were requested to select additional stressors if present.

3. If fewer than three abiotic stressors, diseas es, or arthropod species were present, the

cooperator chose additional abiotic stressors , diseases, and arthropod species that are

known to commonly occur in that geographi cal region and cause damage at the study

site at that time.

4. All plots at a site were rated for the same abiotic stressors, diseases, and arthropod

pests at a given observation, even if that se lected stressor was not present in some or

all of the plots.

5. If a selected stressor was not present, the cooperator reco rded the rating as “0” (=

none).

# Monsanto Company 12-CT-244U 541 of 620 

# As indicated above, the researcher at each fiel d site chose abiotic stressors, diseases, and

arthropod pests that were eith er actively causing pl ant injury in the study area or were

likely to occur in cotton during the given obser vation period. Therefore, abiotic stressors,

diseases, and arthropod pests assessed often varied between observations at a site and

between sites. Qualitative plant response to abiotic stre ss and disease damage and

arthropod-related damage observations were co llected from each plot using a continuous

0 – 9 scale of increasing severity (in Study 2, qualitative abiotic and biotic stressor were

not evaluated on plots with MON 88701 tr eated with dicamba and glufosinate

herbicides). Data were collected numerically and then placed into one of the following

categories for reporting purposes:

Rating Severity of plant damage

0 none (no symptoms observed)

1 – 3 slight (symptoms not damaging to plant development)

4 – 6 moderate (intermediate between slight and severe)

7 – 9 severe (symptoms damaging to plant development)

In Study 1, a quantitativ e assessment for differences in thrips and heliothine damage on

MON 88701 and the conventional control plants was conducted at ARAU, ARPR,

GACH, LABU, and SCEK.

Thrips damage was assessed quantitatively fr om rows 9 and 10 of each plot from 10

randomly selected plants using the arthropod -specific 0 – 5 rating scales of increasing

severity listed below. Damage was rated at approximately 14, 21, and 28 DAP.

Heliothine damage was assessed from rows 9 and 10 of each plot. Visual observations

were conducted at 45, 60, 75 and 90 DAP to r ecord total number of fruiting bodies

(flower buds, flowers and bolls), number of damaged fruiting bodies and number live

larvae on the top 7 nodes from 10 randomly selected plants. Rating Severity of plant damage

0 No Thrips or damage visible

1 Few thrips present; no brownish tinge along the edges of le aves and silvering

on the underside of leaves

2 Numerous thrips present; newest leaves show only a slight brownish tinge

along the edges of leaves and some silv ering on the underside of some leaves

3 Numerous thrips present; newest leav es show considerable browning along

the edges of leaves and some silver ing on the underside of most leaves

4 Numerous thrips present; extensive silver ing of leaves with some curling of

leaves

5 Numerous thrips present; extensive silvering of leaves, leaves often curl

upwards and the plant is gene rally ragged in appearance

# Monsanto Company 12-CT-244U 542 of 620 

# G.8. Arthropod Abundance

Pest and beneficial arthropods were colle cted at the ARAU, ARPR, GACH, LABU, and

SCEK sites four times during the growi ng season at the following intervals:

Collection 1: 30 DAP

Collection 2: 60 DAP

Collection 3: 90 DAP

Collection 4: 120 DAP

Arthropods were collected usi ng a vertical beat sheet samp ling method (Drees and Rice,

1985). The beat sheet was approximately 0.91 × 0.91 m, constructed of a stiff material

and had a collection trough at the bottom. The sheet was placed in a designated row and

the collecting trough was positioned near the base of the plants. Plants were shaken

vigorously along the length of the beat sheet to dislodge arthropods from the plants. This

sample constituted a subsample. Two subsamples were collected form both rows 5 and 7

for a total of 4 subsamples per plot. The s ubsamples collected with in the same row were

at least 1.5 m apart. The four sub-samples we re combined into one pre-labeled container

and placed on freezer ice packs and sent to a laboratory to be enumerated.

A maximum of the five pest and five benefi cial arthropods were enumerated for each

collection. For each individual collection ( e.g ., Collection 1, ARPR site), four randomly

selected samples were examined to determ ine presence and rela tive abundance of up to

five pest- and beneficial-arthropods to be e numerated for that particular collection and

site. Thus, the suite of pe st- and beneficial-arthropods as sessed often varied between

collections from a site and between sites due to differences in temporal activity and

geographical distributio n of arthropod taxa.

G.9. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were

involved in all components of data collection, summariza tion, and analysis. Study

personnel assessed that measurements were ta ken properly, data were consistent with

expectations based on experi ence with the crop, and the experiment was carefully

monitored. Prior to analysis, the overall dataset was evaluated for evidence of

biologically relevant changes and for possible evidence of an unexpected plant response.

Any unexpected observations or issues durin g the trials that would impact the trial

objectives were noted. Data were then subjected to categorical or statistical analysis as

indicated G.10 (categorical analysis ) and G.11 (statistical analysis).

G.10. Environmental Interactions Evalua tion Criteria for Qualitative Data

The following data were categorical and not st atistically analyzed: plant vigor at 14 DAP,

plant vigor at 30 DAP, plant response to ab iotic stress, disease damage and arthropod

damage. MON 88701 and the conventional contro l were considered different in plant

response rating if the range of vigor or st ressor values did not overlap between the

# Monsanto Company 12-CT-244U 543 of 620 

# MON 88701 and the conventional control across all four replications. Any observed

differences between the MON 88701 and the conventional control were assessed for

biological significance in the context of the ra nge of the commercial reference materials,

and for consistency in other observation times and sites. Differences that are not

consistently observed at other times and site s are considered not bi ologically meaningful

in terms of plant pest potential or an adverse environmental impact.

G.11. Statistical Analysis

Analysis of variance was conducted on the qu antitative data according to a randomized

complete block design using SAS  

# Version 9.2 (SAS, 2008). The level of significance

was predetermined to be  =0.05. In separate analysis , MON 88701 not treated with

dicamba or glufosinate herbicides from St udy 1, MON 88701 not treated with dicamba or

glufosinate herbicides from Study 2, and MON 88701 treated with dicamba and

glufosinate herbicides were each compared to the conventional contro l within each site

(individual site analysis) and in a combined -site analysis. In both Study 1 and Study 2,

no statistical comparisons were made between MON 88701 and the reference varieties.

The reference range for each characteristic an alyzed across sites was determined from the

minimum and maximum mean values from th e reference cotton vari eties planted among

the sites, within a study.

MON 88701 not treated with dicamba or gl ufosinate herbicides from Study 1 was

statistically compared to the conventional control within each site (individual site

analysis) and in a combined-sit e analysis, in which the follo wing data were pooled across

sites: stand count at 14 DAP, stand count at 30 DAP, final stand count at harvest, plant

height at 30 DAP, plant height at harvest, nodes above white flower (3 observations),

seed cotton yield, seed index, total seed pe r boll, total mature seed per boll, total

immature seed per boll, boll weight, fiber micronaire, fiber elonga tion, fiber strength,

fiber uniformity, fiber length, thrips damage and percent heliothine damaged fruiting

bodies and number of live he liothine larvae. Pest and be neficial arthropod abundance

data were statistically analyzed only within individual observations/ collections and sites

due to the variation in temporal activity a nd geographical distributi on of the taxa. The

reference range for pest- and beneficial -arthropod abundance and damage of each

arthropod evaluated from a given collection/ob servation and site was determined from

the minimum and maximum mean abundance or damage values collected from the

reference varieties at the site . Data excluded from Study 1 (Table G-5) and the reasons

for their exclusion are listed in the study file. Exclusion of these data did not adversely

affect the quality of the study.

MON 88701 not treated with dicamba or glufosinate herbicide and MON 88701 treated

with dicamba and glufosinate herbicides from Study 2 were each compared to the

conventional control within each site (indivi dual site analysis) a nd in a combined-site

analysis, in which the following data were poo led across sites: st and count at 14 DAP,

stand count at 30 DAP, final sta nd count, plant height at 30 DAP , plant height at harvest,

 

# SAS is a registered trad emark of the SAS Institute.

# Monsanto Company 12-CT-244U 544 of 620 

# nodes above white flower (3 observations), s eed cotton yield, number of mainstem nodes

per plant, number of nodes to first fruiting br anch, total number of bolls per plant, total

number of first-position bolls per plant, total number of vege tative bolls per plant, percent

retention of first-position bolls , percent first-position bolls of total bolls per plant, seed

index, total seed per boll, total mature seed per boll, total immature seed per boll, weight

per boll, fiber micronaire, fi ber elongation, fiber strength , fiber uniformity, and fiber

length. Data excluded from Study 2 (Table G- 6) and the reasons for their exclusion are

listed in the study file. Exclusi on of these data did not advers ely affect the quality of the

study.

G.12. Phenotypic Results from 2010 - Results and Discussion

The individual site data will be reported in three separate comparisons: Study 1 -

MON 88701 not treated with di camba or glufosinate herbic ides vs. the conventional

control; Study 2 - MON 88701 not tr eated with dicamba or gluf osinate herbicides vs. the

conventional control; and, Study 2 - MON 8870 1 treated with dicamba and glufosinate

herbicides vs. the conventional control. The agronomic system that includes MON 88701

treated with dicamba and glufosinate herbicid es was assessed to s upport the assessment

of MON 88701.

G.12.1. Individual Phenotypic Characteristic s - Results and Discussion for Study 1

- MON 88701 Not Treated with Dica mba or Glufosinate Herbicides

In the individual site analysis of MON 88701 da ta, a total of 59 stat istically si gnificant

differences were detected out of a total of 285 comparisons made between MON 88701

and the conventional control (Tables G-7, G-8 and G-9). At LACH (Table G-7),

MON 88701 had a lower stand count (plants per plot) than the conven tional control at 14

DAP (100.8 vs. 127.8), 30 DAP (101.8 vs. 125.0) and at harvest (63.3 vs. 73.5). At

GAJE, the final stand count was high er for MON 88701 than the control (167.5 vs. 156.3

plants/plot). Plants of MON 88701 were shorter (cm) than the conventional control at 30

DAP at LACH (12.4 vs. 14.5), NCBD (20.0 vs. 24.5) and NMLC (6.7 vs. 8.3). Plants of

MON 88701 were shorter (cm) than the cont rol at harvest at GAJE (87.1 vs. 98.6), KSLA

(129.9 vs. 142.0), LACH (120.5 vs. 140.5), NCBD (104.8 vs. 111.0), and TXPL (44.9 vs.

48.4). Plants of MON 88701 had more nodes a bove white flower at observation 1 at

KSLA (5.1 vs. 4.4), NMLC (8.4 vs. 7.4) and TXPL (5.0 vs. 4.5). Plants of MON 88701

had more nodes above white flower at obser vation 2 at ARPR (7.1 vs. 6.6) and KSLA

(4.2 vs. 3.2). Plants of MON 88701 had more nodes above white flower at observation 3

at KSLA (4.6 vs. 3.5), SCEK (5.2 vs. 4.6) a nd TXPO (6.4 vs. 5.7). Plants of MON 88701

a higher seedcotton yield (Kg/ha) than th e conventional control at ARAU (2912.9 vs.

2166.8) and at GAJE (2320.4 vs. 1843.1). Plants of MON 88 701 (Table G-8) had a lower

seed index (grams of 100 fuzzy seed) than the conventiona l control ARPR (9.4 vs. 10.3),

ARTI (10.1 vs. 10.9), GAJE (8.3 vs. 9.8), KSLA (11.8 vs. 12.5), LABU (9.4 vs. 10.5),

LACH (8.8 vs. 9.8), NCBD (9.0 vs. 9.8), NCME (9.7 vs. 10.6), and NMGA (10.6 vs.

11.4). Plants of MON 88701 had more total seed per boll than the c onventional control at

ARAU (31.6 vs. 29.6), ARPR (29.4 vs. 27.3) , KSLA (31.2 vs. 28.1), NCME (31.7 vs.

27.5), NMGA (34.4 vs. 30.3) and NMLC (31.0 vs. 25.9). Plants of MON 88701 had

more mature seed per boll th an the control at ARPR (27.3 vs. 21.7), ARTI (20.2 vs. 17.6),

# Monsanto Company 12-CT-244U 545 of 620 

# KSLA (29.9 vs. 26.6), NCME (28.4 vs. 22.1), NMGA (32.9 vs. 28.8), and NMLC (26.5

vs. 21.6). Plants of MON 88701 had fewer imma ture seed per boll th an the conventional

control at ARPR (2.1 vs. 5.7), ARTI (5.7 vs. 9.0), and SCEK (6.5 vs. 11.1). Plants of

MON 88701 (Table G-9) had lower boll weight (grams per boll) than the conventional

control at ARTI (4.1 vs. 4.8) and GACH (4.2 vs. 4.5) and a higher bol l weight at NMLC

(5.1 vs. 4.6). Plants of MON 88701 had hi gher fiber micronaire (mic units) than the

conventional control at NMGA (5.1 vs. 4.9) and TXPL (4.9 vs. 4.5). Plants of

MON 88701 had greater fiber el ongation (%) than the conventional control at ARAU (6.8

vs. 6.2) and lower fiber elonga tion at NCBD (5.7 vs. 6.6). Plants of MON 88701 had

greater fiber strength (g/tex) than the conventional control at KSLA (31.5 vs. 30.3),

NCBD (32.6 vs. 30.9), NMLC (31.2 vs. 30.0), and TXPL (34.1 vs. 32.2). Plants of

MON 88701 had greater fiber uni formity than the conventional control at TXPL (85.4 vs.

84.1%) and shorter cotton fiber at the NCME (2.8 vs. 2.9 cm).

The statistical differences det ected in the individua l site analyses for stand count at 14

DAP, stand count at 30 DAP, fi nal stand count at harvest, nodes above white flower at

observation 1, seedcotton yield, immature seed per boll, boll weight, micronaire, fiber

elongation, fiber uniformity, and fiber length were not detected in the combined-site

analysis. Therefore, the differences be tween MON 88701 and the conventional control

were not indicative of a cons istent plant response and MON 88701 is unlikely to have

increased plant pest/weed potential or an a dverse environmental impact compared to the

conventional control (See Figure VII-1, Schematic Diagram of Agronomic and

Phenotypic Data Interpretation Methods , step 2, answer “no”). While statistical

differences were detected in the combined-site analysis for plant height at 30 DAP at

three sites, plant height at ha rvest at five sites, nodes above white flower observation 2 at

two sites, and nodes above white flower observa tion 3 at three sites, seed index at nine

sites, total seed per boll at si x sites, and mature seed per bol l at six sites and fiber strength

at four sites, the assessed phenotypic value of MON 88701 for these phenotypic

characteristics were within their respective reference range. Therefore, the differences

between MON 88701 and the conventional contro l were not indicative of a consistent

plant response and MON 88701 is unlikely to ha ve increased plant pest/weed potential or

an adverse environmental impact compared to the conventional control (See Figure VII1, Schematic Diagram of Agronomic and Ph enotypic Data In terpretation Methods , step

3, answer “no”).

G.12.2. Individual Phenotypic Characteristics - Results and Discussion for Study 2

- MON 88701 Not Treated with Dica mba or Glufosinate Herbicides

In the individual site analysis for MON 88701 da ta, 43 statistic ally significan t differences

were detected out of 209 comparisons betw een MON 88701 and the conventional control

(Table G-10, G-11 and G-12). At 30 DAP, pl ants of MON 88701 were shorter (cm) than

the conventional control at KSLA (15.7 vs. 18.3), LACH (13.5 vs. 15.9), and NCME

(17.2 vs. 19.7 cm). At harvest, plants of MON 88701 were shorter (cm) than the

conventional control at GAJE (77.4 vs. 92.6), KSLA (113.5 vs. 127.0 cm), LACH (154.4

vs. 180.0), and NCME (71.4 vs. 85.0). Plan ts of MON 88701 had more nodes above

white flower than the conven tional control at observati on 1 at GACH (5.9 vs. 5.3), at

# Monsanto Company 12-CT-244U 546 of 620 

# observation 2 at NCBD (4.8 vs . 3.9) and TXPL (5.5 vs. 5.1) and at observation 3 at

GACH (4.1 vs. 3.6) and KSLA (3.7 vs. 2.5) . Plants of MON 88701 had higher

seedcotton yield than the conventional control at KSLA (4,487.0 vs. 3,726.5 kg/ha) and

NMLC (1,938.4 vs. 1,479.3 kg/ha). Plants of MON 88701 had a lower seed index (gram

of 100 seed) than the conven tional control at ARPR (8.8 vs. 10.0), ARTI (9.9 vs. 12.0),

GAJE (8.5 vs. 10.4), KSLA (11.7 vs. 12.8), LACH (9.4 vs. 10.3), NCBD (8.8 vs. 10.5),

NCME (8.8 vs. 10.2), SCEK (8.5 vs. 9.8), a nd TXPL (9.9 vs. 11.0). Plants of

MON 88701 had more total seed per boll than the conventional control at ARPR (26.8 vs.

23.9), KSLA (28.4 vs. 24.9), and NMLC (33.8 vs . 30.7). Plants of MON 88701 had more

mature seed per boll than the conventional control at ARPR (24.2 vs. 15.9), ARTI (23.7

vs. 18.0), GAJE (15.0 vs. 11.6), and KSLA (25.9 vs. 22.7). Plants of MON 88701 had

fewer immature seed per boll than the conve ntional control at ARPR (2.6 vs. 8.0) and

ARTI (4.6 vs. 8.9). Plants of MON 88701 had greater weight per boll than the

conventional control at KSLA (5.8 vs. 5.3 g/bol l) and NMLC (5.7 vs. 5.1 g/boll). Plants

of MON 88701 had higher fiber micronaire than the conventional control at NMLC (4.9

vs. 4.7 mic units), lower fiber elongation at ARPR (5.0 vs. 5.7 %), higher fiber strength

(g/tex) at KSLA (30.4 vs. 29.4), NMLC (30.1 vs. 27.3), and TXPL (32.0 vs. 31.0). Plants

of MON 88701 had higher fiber uniformity (%) than the conventional control at KSLA

(84.2 vs. 83.3) and NMLC (83.4 vs. 81.1 %) and s horter fiber length (cm) at ARPR (2.8

vs. 2.9) and NCBD (2.8 vs. 2.9).

The statistical differences dete cted in the individua l site analysis fo r nodes above white

flower (observations 1 and 3), seedcotton yiel d, immature seed per bo ll, weight per boll,

fiber micronaire, fiber elongation, fiber uniform ity, and fiber length were not detected in

the combined-site analysis. Therefore, the differences between MON 88701 and the

conventional control were not indicative of a consistent plant response and MON 88701

is unlikely to have increased plant pest/w eed potential or an adverse environmental

impact compared to the conventional control (See Figure VII-1, Schematic Diagram of

Agronomic and Phenotypic Data Interpretation Methods , step 2, answer “no”). Although

statistical differences were detected for plant height at 30 DAP at three sites, plant height

at harvest at four sites, node s above white flower observation 2 at two sites, seed index at

nine sites, total seed per boll at three sites, and mature seed per boll at four sites and fiber

strength at three sites, the assessed values of MON 88701 for the combined-site analysis

were within their respective reference ra nge. Therefore, the differences between

MON 88701 and the conventional control were not indicativ e of a consistent plant

response and MON 88701 is unlikely to have in creased plant pest/wee d potential or an

adverse environmental impact compared to the conventional control (See Figure VII-1,

Schematic Diagram of Agronomic and Ph enotypic Data Interpretation Methods , step 3,

answer “no”).

G.12.3. Combined-site and Individual Site Phen otypic Characteristics - Results and

Discussion for Study 2 - MON 88701 Trea ted with Dicamba and Glufosinate

Herbicides

To support the assessment of MON 88701, these assessments were also conducted on the

agronomic system that includes MON 88701 treated with dicamba and glufosinate

# Monsanto Company 12-CT-244U 547 of 620 

# herbicides. In the combined-site analysis (Table G-13), no statistically significant

differences were detected between MON 8870 1 treated with dicamba and glufosinate

herbicides and the conventional control for stand count at 14 DAP, stand count at 30

DAP, stand count at harvest, number of nodes above white flower observation 3,

seedcotton yield, immature seed per boll, weight per boll, fiber micronaire, fiber

elongation, fiber uniformity and fi ber length. Therefore, the lack of differences in the

above characteristics supports a conclusion th at MON 88701 is unlikel y to have increased

plant pest/weed potential or an adverse environmental impact compared to the

conventional control (See Figure VII-1, Schematic Diagram of Agronomic and

Phenotypic Data Interpretation Methods , step 2, answer “no”). The following

statistically significant differences were detected in the combined-site analysis. Plants of

MON 88701 were shorter (cm) than the conve ntional control at 30 DAP (18.1 vs. 19.2)

and at harvest (98.4 vs. 105.0). Plants of MON 88701 had a higher number of nodes

above white flower at observation 1 (6.7 vs. 6.4) and at observation 2 (5.6 vs. 5.2), lower

seed index (9.5 vs. 10.7 g per 100 fuzzy seed), more seed pe r boll (28.5 vs . 27.0), more

mature seed per boll (22.8 vs. 20.1), and, incr eased fiber strength (31.2 vs. 30.2 g/tex).

However, the mean values for the above charac teristics were within the reference range.

Thus, MON 88701 is unlikely to ha ve increased plant pest/weed potential or an adverse

environmental impact compared to the conventio nal control.

In the individual site analysis for MON 88701 plant growth and development data (Table

G-14 – G-16), a total of 41 statistically significant differences were detected out of a total

of 209 comparisons made between treated MON 88701 and the conventional control.

Plants of MON 88701 were shorter than the conventional control at 30 DAP at GAJE

(23.2 vs. 26.0 cm). Plants of MON 88701 we re shorter (cm) than the conventional

control at harvest at GAJE (81.7 vs . 92.6), KSLA (113.6 vs. 127.0), LACH (164.8

vs.180.0) and NCME (75.7 vs. 85.0). Plants of MON 88701 had more nodes above white

flower at observation 1 than the conventi onal control at LACH (8.6 vs. 7.6) and TXPL

(6.2 vs. 5.7), Plants of MON 88701 had more nodes above white flower at observation 2

than the conventional control at GAJE (6.1 vs. 5.7), NCBD (5.7 vs. 3.9) and TXPL (5.7

vs. 5.1). Plants of MON 88701 had more nodes a bove white flower at observation 3 than

the conventional control at ARTI (4.9 vs. 4.7), KSLA (3.4 vs. 2.5) , and NCBD (3.8 vs.

3.0). Plants of MON 88701 had lower seedcott on yield than the c onventional control at

GACH (4,107.9 vs. 4,471.5 kg/ha) a nd higher at NMLC (2,048.0 vs. 1,479.3 kg/ha).

Plants of MON 88701 had a lower seed index (g/100 fuzzy seed) th an the conventional

control at ARPR (9.1 vs. 10.0), ARTI (9.6 vs. 12.0), GACH (9.0 vs. 9.7), GAJE (8.1 vs.

10.4), LACH (9.5 vs. 10.3), NCBD (9.2 vs. 10. 5), NCME (8.9 vs. 10.2), SCEK (8.5 vs.

9.8), and TXPL (9.9 vs. 11.0). Plants of MO N 88701 had more total s eed per boll than

the conventional control at the ARPR ( 26.1 vs. 23.9) and GACH (29.7 vs. 27.4). Plants

of MON 88701 had more mature seed per bol l than the conventional control at ARPR

(23.2 vs. 15.9), ARTI (22.5 vs. 18.0) and GACH (27.2 vs. 23.4). Plants of MON 88701

had fewer immature seed per boll than the conventional control at ARPR (2.9 vs. 8.0) and

ARTI (5.3 vs. 8.9). Plants of MON 88701 had higher fiber micronaire than the control at

NMLC (4.9 vs. 4.7 mic units) and higher fiber strength (g/tex) at GACH (31.0 vs. 29.5),

KSLA (30.7 vs. 29.4), LACH (31.3 vs. 29.9), NMLC (29.3 vs. 27.3), SCEK (30.7 vs.

30.0) and the TXPL (33.0 vs. 31.0). Plants of MON 88701 had higher fiber uniformity

# Monsanto Company 12-CT-244U 548 of 620 

# (%) than the conventional control at KSLA (84.3 vs. 83.3) and lower at SCEK (82.5 vs.

83.9). Plants of MON 88701 had shorter fibe r length (cm) at SCEK (2.7 vs. 2.8).

The statistical differences dete cted in the individua l site analysis fo r nodes above white

flower observations 3 at three locations, seedcotton yield at two locations, immature seed

per boll at two locations, fiber micronaire at one location, fiber uniformity at two

locations and fiber length at one location were not detected in the combined-site analysis

(Table G-13). Therefore, the differe nces between MON 88701 and the conventional

control were not indicative of a consistent plant response and MON 88701 is unlikely to

have increased plant pest/weed potential or an adve rse environmental impact compared to

the conventional control. (See Figure VII-1, Schematic Diagram of Agronomic and

Phenotypic Data Interpretation Methods , step 3, answer “no”). Although statistical

differences were detected for plant height at 30 DAP at one site, plant height at harvest at

four sites, nodes above white flower observati on 1 at two sites, nodes above white flower

observation 2 at three sites, seed index at nine sites, total seed per boll at two sites, and

mature seed per boll at three sites and fiber strength index at 6 sites, the assessed values

of MON 88701 for the combined-site analysis were within their respective reference

range. Therefore, the differences betw een MON 88701 and the c onventional control

were not indicative of a cons istent plant response and MON 88701 is unlikely to have

increased plant pest/weed potential or an a dverse environmental impact compared to the

conventional control (See Figure VII-1, Schematic Diagram of Agronomic and

Phenotypic Data Interpretation Methods , step 3, answer “no”).

G.13. Plant Mapping Results and Discussion

G.13.1. Individual Site Plant Mapping Resu lts for MON 88701 Not Treated with

Dicamba or Glufosinate Herbicides - Results and Discussion.

In the individual site analysis, 14 statistically significant differences were detected out of

77 comparisons between MON 88701 and the control (Table G-17). Plants of

MON 88701 had more mainstem nodes compared to the conventional control at ARTI

(20.9 vs. 19.8) and a fewer number of nodes to th e first fruiting branch at GACH (6.5 vs.

7.9) and GAJE (5.0 vs. 5.8). Plants of MON 88701 had fewer total bolls per plant

compared to the conventional control at KSLA (19.1 vs. 20.6) and more total bolls at

NCME (6.8 vs. 4.2) and NMLC (6.7 vs. 4.5). Plants ofMON 88701 had more firstposition bolls per plant compared to the conventional control at NCME (3.5 vs. 2.5),

NMLC (4.4 vs. 3.0), and TXPL (7.8 vs. 7.1) and more vegetative bolls per plant at

NCME (1.0 vs. 0.1). Plants of MON 88701 retain ed a higher percenta ge (%) of firstposition bolls per plant compared to the c onventional control at the NCME (35.6 vs.

23.9) and NMLC (30.1 vs. 21.3) and a higher pe rcent of first-position bolls per plant

relative to total bo lls per plant at GAJE (67.4 vs. 61.8) and SCEK (73.2 vs. 65.7).

The statistical differences (Table G-17) de tected between MON 88701 and the control in

the individual site analysis for total mainstem nodes per plan t at one location, the number

of nodes per plant to th e first fruiting branch at two site s, total bolls pe r plant at three

sites, total vegetative bolls per plant at one site, percent first-position boll retention per

plant at two sites and percent first-position bolls per plant relative to total bolls per plant

# Monsanto Company 12-CT-244U 549 of 620 

# at two sites were not detected in the combined -site analysis (Table VII-6). Therefore, the

differences between MON 88701 and the conven tional control were not indicative of a

consistent plant response and MON 88701 is un likely to have increased plant pest/weed

potential or an adverse environmental impact compared to the conventional control (See

Figure VII-1, Schematic Diagram of Agronomic and Phenotypic Data Interpretation

Methods , step 2, answer “no”). Although statistic al differences were detected between

the MON 88701 and the conventiona l control for total first-pos ition bolls at three sites

(Table G-17), the assessed values of M ON 88701 for the combined-site analysis were

within their respective refere nce range. Therefore, the differences between MON 88701

and the conventional control were not indica tive of a consistent plant response and

MON 88701 is unlikely to have increased plant pest/weed poten tial or an adverse

environmental impact compared to th e conventional control (See Figure VII-1, Schematic

Diagram of Agronomic and Phenotypic Data Interpretation Methods , step 3, answer

“no”).

G.13.2. Combined-site and Individual Site Pl ant Mapping Results for MON 88701

Treated with Dicamba and Glufosinate Herbicides - Results and Discussion.

To support the assessment of MON 88701, plant mapping assessments were also

conducted on the agronomic system that in cludes MON 88701 treated with dicamba and

glufosinate herbicides. In the combined-sit e analysis (Table G-18) of plant mapping

parameters, no statistically significant di fferences were detected between MON 88701

and the conventional control fo r total mainstem nodes per pl ant, the number of nodes per

plant to the first fruiting branch, total bolls per plant, total vegetative bolls per plant,

percent first-position boll rete ntion per plant and percent first-positi on bolls per plant

relative to total bolls per plant. Therefor e, the lack of diffe rences in the above

characteristics supports a conclusion that MON 88701 is unlikely to have increased plant

pest/weed potential or an adverse environmental impact compared to the conventional

control (See Figure VII-1, Schematic Diagram of Agronomic and Phenotypic Data

Interpretation Methods , step 2, answer “no”). Plants of MON 88701 had a significantly

higher number of first-position bolls per plant compared to the conventional control (5.2

vs. 4.6). However, the mean values of MON 88701 were within the reference range.

Thus, MON 88701 is unlikely to ha ve increased plant pest/weed potential or an adverse

environmental impact compared to the conventional control. (See Figure VII-1,

Schematic Diagram of Agronomic and Ph enotypic Data Interpretation Methods , step 3,

answer “no”).

In the individual site analysis, 15 statistically significant differences were detected out of

77 comparisons between MON 88701 treated w ith dicamba and glufosinate herbicides

compared to the conventional (Table G-19) . Plants of MON 88701 had more mainstem

nodes per plant compared to the conventional control at NCBD (16.5 vs. 14.8) and a

fewer number of nodes to the first fruiting branch at GACH (6.5 vs. 7.9) and GAJE (5.1

vs. 5.8). Plants of MON 88701 had a higher numb er of total bolls pe r plant compared to

the conventional control at NCME (7.1 vs. 4.2), NMLC (7.7 vs. 4.5) and TXPL (14.3 vs.

11.3) and a higher number of first-position bolls per plant at NCBD (6.5 vs. 5.5), NCME

(3.4 vs. 2.5), NMLC (4.7 vs. 3.0) and TXPL (8.1 vs. 7.1). Plants of MON 88701 had

# Monsanto Company 12-CT-244U 550 of 620 

# more vegetative bolls per plan t compared to the convention control at NCME (1.1 vs.

0.1) and less at SCEK (0.3 vs. 0.7). Plants of MON 88701 retained a higher percent of

first-position bolls compared to the control at NCME ( 33.7 vs. 23.9) and NMLC (32.2 vs.

21.3) and had a higher percentage of first-position bolls per pl ant relative to total bolls

per plant at the GACH (45.4 vs. 36.6).

The statistical differences detected in the i ndividual site analysis (Table G-19) for total

mainstem nodes per plant, the number of nodes pe r plant to the first fruiting branch, total

bolls per plant, total vegetati ve bolls per plant, percent first-positi on boll retention per

plant and percent first-position bolls per plant relative to total bolls per plant were not

detected in the combined-site analysis (Table G-18). Therefore, the differences between

MON 88701 and the conventional control were not indicativ e of a consistent plant

response and MON 88701 is unlikely to have in creased plant pest/wee d potential or an

adverse environmental impact compared to the conventional control (See Figure VII-1,

Schematic Diagram of Agronomic and Ph enotypic Data Interpretation Methods , step 2,

answer “no”). Although statistical differences were detect ed for the total number of firstposition bolls at four sites (Table G-19), the assessed values of MON 88701 for the

combined-site analysis were within their respective reference range. Therefore, the

differences between MON 88701 and the conven tional control were not indicative of a

consistent plant response and MON 88701 is un likely to have increased plant pest/weed

potential or an adverse environmental impact compared to the conventional control (See

Figure VII-1, Schematic Diagram of Agronomic and Phenotypic Data Interpretation

Methods , step 3, answer “no”).

G.14. Individual Site Environmental In teractions - Results and Discussion

G.14.1. Individual Site Environmental Intera ctions - Results and Discussion –

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides – Study 1

(Qualitative Data Assessment)

In an individual site assessment, no diffe rences were observed between MON 88701 and

the conventional control for any of the 169 comparisons for the assessed abiotic

stressors, including compacti on, drought/dry, flood, hail, he at, nutrient deficiency, wet

soil/excess precipitation, and wi nd damage (Table G-20).

In an individual site assessment, no diffe rences were observed between MON 88701 and

the control for any of the 170 comparisons for the assessed diseases, including

anthracnose, Ascochyta leaf blight, bacterial blight, boll rot, cotton l eaf rust, damping off,

Fusarium wilt, leaf spots, Pythium , reniform nematode, Rhizoctonia , root-knot nematode,

Thielaviopsis , and Verticillium wilt (Table G-21)

In an individual site assessment, no diffe rences were observed between MON 88701 and

the control for any of the 159 comparisons for the assessed arthropod stressors,

including aphids, beet armyworms, cu t worms, fall armyworms, fleahoppers,

# Monsanto Company 12-CT-244U 551 of 620 

# grasshoppers, heliothines, sout hern corn rootworm beetles, soybean loopers, spider

mites, stink bugs, tarnished plant bugs, th rips, and white flies (Table G-22).

( Quantitative Data Assessment)

Thrips Damage

A total of 15 thrips damage comparisons (thr ee observation events × 5 sites) were made

between MON 88701 and the conventional control in the individual site analysis (Table

G-23). Of these comparisons, no numerical differences were observed for 10

comparisons for which p-values could not be ge nerated due to lack of variability in the

data. Four of the remaining five compar isons were not signifi cantly different.

MON 88701 had significantly less damage from thrips in obs ervation 3 at the ARPR site

(0.1 vs. 0.3). However, there was no si gnificant difference between MON 88701 and the

conventional control for Observation 3 in th e combined-site analysis (Table VII-8).

Therefore, the lack of difference for 14 comparisons and the one site/observation

difference between MON 88701 and the conventi onal control were no t indicative of a

consistent plant response and MON 88701 is un likely to have increased plant pest/weed

potential or an adverse environmental impact compared to the conventional control (See

Figure VII-1, Schematic Diagram of Agronomic and Phenotypic Data Interpretation

Methods , step 2, answer “no”).

Heliothines Damage

A total of 40 heliothine damage comparisons (4 observations × 5 sites) were made

between MON 88701 and the conventional control in the individual site analysis (Table

G-24). Of these comparisons, no numeri cal differences were observed for three

comparisons for which p-values could not be ge nerated due to lack of variability in the

data. For the remaining 37 comparisons, no statistically significant differences were

detected between MON 88701 and the control for 35 out of 37 comparisons (Table G24). Two statistically significant differences were detected between MON 88701 and the

parental control. Plants of MON88701 had fewer helio thine damage fruiting bodies

compared to the conventional control in Observation 4 at ARAU (8.7 vs. 15.1%), and

more live larvae Observation 1 at GACH ( 0.5 vs. 0.1). Although the above statistical

differences were detected, the assessed va lues of MON 88701 were not significantly

different than the control in the combined-site analysis (Table VII-9). Therefore, the lack

of difference for 38 comparisons and the two site/observation differences between

MON 88701 and the conventional control were not indicativ e of a consistent plant

response, and MON 88701 is unlikely to have in creased plant pest/weed potential or an

adverse environmental impact compared to the conventional control (See Figure VII-1,

Schematic Diagram of Agronomic and Ph enotypic Data Interpretation Methods , step 2,

answer “no”).

Arthropod Abundance

# Monsanto Company 12-CT-244U 552 of 620 

# A total of 178 comparisons were made between MON 88701 and the conventional

control for arthropod abundance involving the following pest- and beneficial-arthropods:

aphids, cabbage loopers, fall armyworms, flea hoppers, heliothines, southern armyworms,

stink bugs, tarnished plant bugs , thrips, white flies, big eyed bugs, braconids, Damsel

bugs, lacewings, ladybird beetles, Orius spp and Araneae (spiders) (Tables G-25 and G26). No statistically significant differenc es were detected between MON 88701 and the

conventional control for 173 out of 178 comp arisons, including 89 pest arthropod

comparisons and 89 beneficial arthropod comp arisons. The five differences detected

between MON 88701 and the conv entional control included two differences for pest

arthropods and three differences for beneficial arthropods.

At collection 4 at LABU, th e abundance of stink bugs pe r plot in MON 88701 was lower

compared to the conventional control (0.3 vs . 1.8 per plot) and lower for tarnished plant

bugs (0.5 vs. 2.0). For tarnished plant bugs , the mean abundance value for MON 88701

was within the reference ranges for the diffe rences detected. For stink bugs, the mean

abundance value for MON 88701 was outside the reference range for the difference

detected. Since the two differences mentione d above were not consis tently detected in

multiple environments, these data support a conclusion that the differences are considered

not biologically meaningful in terms of plan t pest potential or an adverse environmental

impact (See Section VII.B.1.1., Interpretation of Environmental In teractions Data).

The abundance of Damsel bugs per plot MON 88701 was higher compared to the

conventional control in Collection 2 at GACH (6.0 vs, 2.3) and lower for Orius spp. in

Collection 2 (0.0 vs. 1.5 per plot) and collection 3 (0.5 vs. 2.8 per plot) at the ARAU site.

The mean abundance value for MON 88701 wa s within the refere nce range for the

difference detected for Damsel bugs. The mean abundance values for Orius spp. in

Collection 2 and collection 3 at the ARAU site were outside their respective reference

range. However, the differences detected for Orius spp. were not consistently detected

across collections or sites (Table G-26). T hus, the detected differences in beneficial

arthropod abundance were not i ndicative of a consistent response associated with

MON 88701 and are not considered biological ly meaningful in terms of an adverse

environmental impact of MON 88701 compared to conventional co tton (See Section

VII.B.1.1., Interpretation of Envi ronmental Interactions Data).

G.14.2. Individual Site Environmental Intera ctions - Results and Discussion –

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides – Study 2

In an individual site assessment, no diffe rences were observed between MON 88701 and

the control for any of the 127 comparisons fo r the assessed abiotic stressors, including

compaction, drought, dry, flood, hail damage, heat , nutrient deficiency, wet soil, excess

precipitation, and wind damage (Table G-27).

In an individual site assessment, no diffe rences were observed between MON 88701 and

the control for any of the 129 comparisons for the assessed diseases, including

anthracnose, Ascochyta leaf blight, bacterial blight, bol l rot, cotton leaf rust, damping

# Monsanto Company 12-CT-244U 553 of 620 

# off, Fusarium wilt, leaf spots, Pythium , reniform nematode, Rhizoctonia , root-knot

nematode, Thielaviopsis , and Verticillium wilt (Table G-28).

In an individual site assessment, no diffe rences were observed between MON 88701 and

the control for any of the 129 comparisons for the assessed arthropod stressors,

including aphids, beet armyworms, cabba ge loopers, cut worms, fall armyworms,

fleahoppers, grasshoppers, heliothines, southern corn rootworm beetle, soybean loopers,

spider mites, stink bugs, tarnished plant bugs , thrips, and white flies (Table G-29).

These results support a conclusi on that MON 88701 w ould not confer a pl ant pest risk or

significant environmental impact compar ed to conventional cotton (See Section

VII.B.1.1., Interpretation of Envir onmental Interactions Data).Monsanto Company 12-CT-244U 554 of 620Table G-5. Study 1 Data Missingor Excluded from AnalysisSiteMaterial nameMaterial type Plots Characteristics Reason for exclusionAZME All All All AllSite was dropped due to low stand count in the plots, poor trialmaintenance, and non-reliable data collection by thecooperator.All All All AllNodes above crackedboll observations 1,2,and 3Nodes above white flower data were sufficient in providinggrowth and development trend relevant for showing cropadvancement towards cutout.ARAU andLABUAll All All Arthropod collection 1Neonate soybean looper identification was questionablebecause of the size.LABU All All All Arthropod collection 3Beneficial species from collection 2 were recorded forcollection 3 as there were no beneficial insects in collection 3KSLA All All All 14 D stand countsStand counts taken on 6/16 were dropped due to low count andwere taken again on 6/23 from all four rows of each plot. Thenrows with good count were chosen for data collection.KSLA All All All 14 D plant vigorVigor was taken at 6/16 and 6/23 but 6/23 data were droppedbecause it was taken 21 days after planting.NCBD Coker 130 Control 403 Final stand countStand count was poor in these plots as couple feet in the plotwere destroyed by the lightning strike.NCBD Coker 130 Control 403 Seed cotton yield A couple feet in the plot were destroyed by a lightening strike.NCMEDP 435 andDP 5415Reference 206, 305, 406Stand count at 30 DAPand final stand countStand count was poor in these plots because of poorgermplasm.NCME DP 5415 Reference 406 Seedcotton yield Yield was very low due to poor stand count.TXPL All All AllNodes above crackedboll observations 4This was extra collection and not needed.Monsanto Company 12-CT-244U 555 of 620Table G-6. Study 2 Data Missingor Excluded from AnalysisSiteMaterial nameMaterial type Plots Characteristics Reason for exclusionARTI SG125 Reference 106 Plant mapping Plant mappingdata sheet was misplaced after collection.TXPO All All All AllPlot area was destroyed by lightning.ALL All All AllNodes above cracked bolland days of planning tofirst cracked boll dateNodes above white flower data were sufficient in providinggrowth and development trends relevant for showing cropadvancement towards cutout. Repetition of summary wouldnot have added any value.ALL All All AllDays of planting to firstflower dateData on three observations on nodes above white flower wassufficient in proving data on crop growth and development.Repetition of summary would not have added any value.ARTIGACHLACHNCMEFM 989 Reference All ALLDue to poor germination and stand establishment, referenceFM 989 will be excluded from all phenotypic data analysisand reporting. Germination ranged from 9.5 to 40.5 % of thetarget stand counts.Monsanto Company 12-CT-244U 556 of 620Table G-7. Study 1 - Individual Site Phenotypic Comparison – Growth and Development Characteristics - of MON 88701 NotTreated with Dicamba or Glufosinate Herbicides Compared to the Conventional ControlPhenotypic CharacteristicsSiteStand count at 14 DAP(# per ~6 m)Stand count at 30 DAP(# per ~6 m) Final stand count Plant vigor at 14 DAP1Plant vigor at 30 DAP2MON 88701(SE)3Control (SE)MON 88701(SE) Control (SE)MON 88701(SE) Control (SE) MON 88701 Control MON 88701 ControlARAU 165.7 (10.0) 182.2 (12.0) 159.3 (7.3) 174.0 (11.1) 154.2 (9.0) 181.0 (4.9) 3.0-4.0 2.0-3.0 2.0-3.0 2.0-3.0ARPR 179.8 (3.2) 185.3 (4.1) 179.7 (3.2) 183.5 (4.7) 178.3 (3.5) 181.8 (4.7) 5.0-6.0 6.0-6.0 4.0-4.0 2.0-3.0ARTI 173.0 (6.5) 175.5 (5.0) 171.5 (6.4) 173.5 (5.3) 171.0 (6.4) 172.5 (4.9) 1.0-1.0 1.0-1.0 1.0-1.0 1.0-1.0GACH 170.7 (2.7) 171.7 (4.0) 168.8 (2.3) 167.7 (3.9) 166.8 (2.9) 168.5 (3.6) 3.0-4.0 3.0-4.0 2.0-5.0 2.0-4.0GAJE 178.8 (3.3) 182.8 (5.8) 168.5 (4.9) 160.8 (3.3) 167.5 (1.8)* 156.3 (4.9) 1.0-2.0 1.0-1.0 2.0-2.0 2.0-2.0KSLA 127.8 (2.8) 141.5 (3.2) 74.5 (2.5) 74.0 (0.7) 70.3 (2.3) 70.3 (0.8) 2.0-2.0 2.0-2.0 2.0-2.0 2.0-2.0LABU 147.5 (1.0) 155.7 (3.3) 146.2 (1.3) 160.0 (4.1) 94.3 (1.8) 91.8 (0.7) 1.0-3.0 2.0-3.0 1.0-2.0 1.0-2.0LACH 100.8 (3.0)* 127.8 (1.1) 101.8 (2.6)* 125.0 (2.6) 63.3 (0.9)* 73.5 (4.7) 2.0-3.0 2.0-3.0 2.0-2.0 1.0-2.0NCBD 117.3 (2.3) 125.3 (6.8) 106.8 (5.0) 116.5 (7.5) 106.5 (2.7) 115.3 (8.8) 3.0-3.0 2.0-3.0 3.0-4.0 2.0-3.0NCME 95.8 (22.8) 105.0 (11.8) 90.5 (23.0) 99.8 (12.3) 90.5 (21.6) 97.0 (12.2) 3.0-6.0 3.0-6.0 3.0-5.0 3.0-5.0NMGA 116.5 (17.3) 116.0 (12.8) 70.0 (0.0) 70.0 (0.0) 70.3 (0.5) 70.8 (0.6) 1.0-2.0 2.0-2.0 1.0-1.0 1.0-1.0NMLC 122.5 (6.7) 116.0 (18.8) 70.0 (0.0) 70.0 (0.0) 70.8 (0.9) 71.8 (1.0) 2.0-2.0 1.0-2.0 1.0-1.0 1.0-1.0SCEK 182.7 (4.3) 188.2 (3.2) 183.3 (3.4) 189.3 (3.1) 179.2 (2.6) 181.3 (3.1) 2.0-3.0 2.0-3.0 2.0-4.0 2.0-3.0TXPL 163.0 (3.8) 151.0 (15.9) 142.8 (8.3) 155.5 (9.5) 157.8 (1.9) 146.8 (13.9) 1.0-1.0 1.0-1.0 3.0-3.0 3.0-3.0TXPO 147.8 (13.3) 162.3 (12.1) 143.3 (9.1) 145.5 (13.0) 137.0 (6.1) 151.0 (5.4) 1.0-1.0 1.0-1.0 1.0-1.0 1.0-1.0Monsanto Company 12-CT-244U 557 of 620Table G-7. Study 1 - Individual Site Phenotypic Comparison – Growth and Development Characteristics - of MON 88701 NotTreated with Dicamba or Glufosinate Herbicides Compared to the Conventional Control (continued)Phenotypic CharacteristicsSitePlant height at 30 DAP(cm)Plant height before harvest(cm)Nodes above white flower(obs. 1)Nodes above white flower(obs. 2)Nodes above white flower(obs. 3)MON 88701(SE)Control(SE)MON 88701(SE) Control (SE)MON 88701(SE) Control (SE)MON 88701(SE) Control (SE)MON 88701(SE) Control (SE)ARAU 30.9 (0.5) 33.7 (1.2) 159.3 (2.9) 168.8 (8.8) 7.7 (0.1) 7.6 (0.2) 7.1 (0.3) 7.1 (0.1) 6.7 (0.5) 6.5 (0.2)ARPR 29.1 (1.5) 31.8 (2.6) 126.9 (3.6) 134.4 (3.1) 7.7 (0.2) 7.2 (0.2) 7.1 (0.1)* 6.6 (0.2) 6.1 (0.3) 5.5 (0.2)ARTI 29.5 (0.6) 29.4 (0.3) 131.9 (2.9) 139.2 (4.8) 8.5 (0.1) 8.4 (0.1) 7.5 (0.2) 7.1 (0.2) 4.7 (0.1) 4.8 (0.1)GACH 28.2 (0.4) 29.7 (1.4) 84.6 (2.6) 88.1 (2.0) 5.2 (0.1) 5.0 (0.2) 3.4 (0.2) 3.1 (0.3) 1.7 (0.1) 1.3 (0.2)GAJE 27.0 (0.8) 27.9 (0.7) 87.1 (3.7)* 98.6 (5.3) 7.3 (0.6) 7.7 (0.5) 6.0 (0.2) 5.9 (0.1) 3.9 (0.0) 4.0 (0.1)KSLA 17.5 (1.4) 19.4 (0.8) 129.9 (3.5)* 142.0 (5.5) 5.1 (0.2)* 4.4 (0.2) 4.2 (0.5)* 3.2 (0.2) 4.6 (0.5)* 3.5 (0.2)LABU 15.8 (0.8) 17.2 (0.7) 150.2 (3.7) 161.2 (6.4) 7.5 (0.2) 7.6 (0.4) 7.0 (0.2) 6.6 (0.2) 7.5 (0.1) 7.5 (0.2)LACH 12.4 (0.6)* 14.5 (0.6) 120.5 (3.2)* 140.5 (9.8) 8.2 (0.2) 8.5 (0.1) 7.8 (0.1) 7.6 (0.1) 6.9 (0.1) 7.0 (0.1)NCBD 20.0 (1.8)* 24.5 (1.0) 104.8 (9.5)* 111.0 (10.5) 6.2 (0.5) 5.6 (0.5) 4.0 (0.4) 3.7 (0.6) 2.9 (0.5) 2.4 (0.6)NCME 16.2 (1.1) 15.7 (0.3) 90.3 (4.4) 94.7 (2.1) 4.8 (0.1) 4.8 (0.1) 3.1 (0.1) 2.9 (0.1) 2.7 (0.3) 2.2 (0.3)NMGA 5.0 (0.2) 5.2 (0.4) 92.3 (3.0) 96.6 (3.8) 8.6 (0.4) 8.4 (0.5) 8.7 (0.2) 8.5 (0.3) 5.9 (0.9) 6.1 (0.9)NMLC 6.7 (0.7)* 8.3 (0.6) 96.0 (4.6) 94.6 (2.1) 8.4 (0.5)* 7.4 (0.3) 8.2 (0.3) 8.0 (0.2) 6.5 (0.4) 6.2 (0.3)SCEK 8.0 (0.9) 8.4 (1.5) 98.9 (8.0) 97.8 (5.0) 5.3 (0.2) 5.0 (0.3) 5.4 (0.4) 4.9 (0.3) 5.2 (0.3)* 4.6 (0.1)TXPL 8.0 (0.4) 8.1 (0.3) 44.9 (0.7)* 48.4 (0.8) 5.0 (0.1)* 4.5 (0.2) 4.4 (0.2) 3.9 (0.2) 2.0 (0.1) 1.8 (0.1)TXPO 19.9 (0.8) 22.6 (0.8) 129.7 (1.2) 129.9 (0.9) 8.8 (0.1) 8.5 (0.3) 7.0 (0.2) 6.6 (0.1) 6.4 (0.3)* 5.7 (0.2)Monsanto Company 12-CT-244U 558 of 620Table G-7. Study 1 - Individual Site Phenotypic Comparison – Growth and Development Characteristics - of MON 88701 NotTreated with Dicamba or Glufosinate Herbicides Compared to the Conventional Control (continued)Phenotypic CharacteristicsSiteSeed cotton yield(Kg/ha)MON 88701 (SE) Control (SE)ARAU 2912.9 (231.8)* 2166.8 (250.2)ARPR 3452.4 (202.0) 3309.7 (155.9)ARTI 3444.8 (90.4) 3488.7 (87.3)GACH 2880.8 (97.8) 2875.6 (101.1)GAJE 2320.4 (42.3)* 1843.1 (256.8)KSLA 4330.4 (184.1) 4341.9(365.1)LABU 1534.1 (153.7) 1719.4 (67.8)LACH 1464.1 (245.0) 1736.0 (87.4)NCBD 3990.5 (375.5) 4112.1 (299.2)NCME 1324.7 (110.5) 1296.3 (140.9)NMGA 3459.6 (211.1) 3498.9 (165.2)NMLC 4771.7 (199.9) 4522.5 (158.1)SCEK 3625.0 (125.2) 3901.9 (143.5)TXPL 3638.4 (114.1) 3689.3 (29.6)TXPO 916.5 (115.9) 856.8 (41.5)* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and conventionalcontrol (n = 4).1Plant vigor score range (minimum-maximum). The range of plant vigor score at 14 DAP for the references is as follows: ARAU 3-5; ARPR 3-6; ARTI 1-2;GACH 3-7; GAJE 1-3; KSLA 2-4; LABU 2-5; LACH 2-5; NCBD 3-4; NCME 3-6; NMGA 1-3; NMLC 2-3; SCEK 2-4; TXPL 1-1; TXPO 1-1. Plantvigor wasrated per plot using a rating scale of 1-9 where: 1-3 is excellent vigor, 4-6 is average vigor, and 7-9 is poor vigor.2Plant vigor score range (minimum-maximum). The range of plant vigor score at 30 DAP for the references is as follows: ARAU 3-5; ARPR 4-7; ARTI 1-1;GACH 3-7; GAJE 2-2; KSLA 2-3; LABU 2-3; LACH 1-4; NCBD 2-4; NCME 3-7; NMGA 1-5; NMLC 1-5; SCEK 3-4; TXPL 3-3; TXPO 1-1.3SE = Standard errorMonsanto Company 12-CT-244U 559 of 620Table G-8. Study 1 - Individual SitePhenotypic Comparison – Seed Characteristics - of MON 88701 Not Treated withDicamba or Glufosinate Herbicides Compared to the Conventional Control.SiteSeed index(g per 100 fuzzy seed)Total seed per boll(# per boll)Mature seed per boll(# per boll)Immature seed per boll(# per boll)MON 88701 (SE)1Control (SE) MON 88701 (SE) Control (SE) MON 88701 (SE) Control (SE) MON 88701 (SE) Control (SE)ARAU 10.6 (0.4) 10.6 (0.2) 31.6 (0.3)* 29.6 (1.0) 20.5 (1.2) 19.2 (0.9) 11.1 (1.4) 10.4 (0.8)ARPR 9.4 (0.2)* 10.3 (0.1) 29.4 (0.3)* 27.3 (0.9) 27.3 (0.6)* 21.7 (0.7) 2.1 (0.3)* 5.7 (1.0)ARTI 10.1 (0.2)* 10.9 (0.1) 26.0 (0.7) 26.6 (0.5) 20.2 (1.0)* 17.6 (0.4) 5.7 (1.2)* 9.0 (0.5)GACH 10.3 (0.6) 10.4 (0.5) 28.0 (0.9) 27.6 (0.7) 17.4 (0.5) 17.0 (0.8) 10.6 (1.3) 10.6 (1.3)GAJE 8.3 (0.4)* 9.8 (0.2) 25.8 (0.9) 26.3 (1.3) 15.6 (0.8) 15.2 (2.0) 10.2 (0.2) 11.1 (0.7)KSLA 11.8 (0.2)* 12.5 (0.2) 31.2 (1.1)* 28.1 (0.4) 29.9 (1.1)* 26.6 (0.4) 1.3 (0.1) 1.5 (0.1)LABU 9.4 (0.2)* 10.5 (0.1) 29.5 (0.4) 27.9 (0.7) 21.2 (0.8) 18.9 (1.1) 8.4 (1.0) 9.0 (1.4)LACH 8.8 (0.3)* 9.8 (0.3) 28.4 (0.8) 28.3 (0.7) 15.5 (1.2) 16.0 (0.7) 12.9 (0.8) 12.4 (0.5)NCBD 9.0 (0.1)* 9.8 (0.3) 30.1 (1.5) 27.8 (1.7) 21.1 (3.5) 16.2 (2.1) 9.0 (2.5) 11.6 (1.0)NCME 9.7 (0.2)* 10.6 (0.1) 31.7 (2.0)* 27.5 (0.8) 28.4 (1.0)* 22.1 (0.7) 3.4 (1.0) 5.4 (1.0)NMGA 10.6 (0.2)* 11.4 (0.2) 34.4 (0.4)* 30.3 (0.8) 32.9 (0.6)* 28.8 (0.6) 1.6 (0.3) 1.5 (0.3)NMLC 10.5 (1.0) 10.5 (0.6) 31.0 (0.7)* 25.9 (1.5) 26.5 (1.0)* 21.6 (1.3) 4.5 (0.6) 4.3 (0.5)SCEK 8.5 (0.2) 9.3 (0.2) 27.1 (0.9) 27.1 (0.8) 20.5 (0.8) 16.0 (2.1) 6.5 (0.5)* 11.1 (2.1)TXPL 11.5 (0.3) 11.5 (0.6) 28.3 (0.6) 28.0 (0.8) 25.4 (0.7) 24.5 (0.8) 2.9 (0.6) 3.5 (0.8)TXPO 9.0 (0.2) 9.6 (0.4) 23.3 (0.2) 22.7 (0.7) 16.8 (0.8) 14.7 (0.9) 6.6 (0.7) 8.0 (0.6)* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and conventional control (n = 4).1SE = Standard errorMonsanto Company 12-CT-244U 560 of 620Table G-9. Study 1 - Individual Site Phenotypic Comparison – Boll and FiberCharacteristics - of MON 88701 Not Treatedwith Dicamba or Glufosinate HerbicidesCompared to the Conventional ControlSiteBoll weight (g/boll) Micronaire (mic units)1Elongation (%) Strength (g/tex) Uniformity (%) Length (cm)MON 88701(SE)2Control(SE)MON 88701(SE)Control(SE)MON 88701(SE)Control(SE)MON 88701(SE)Control(SE)MON 88701(SE)Control(SE)MON 88701(SE)Control(SE)ARAU 4.9 (0.1) 4.9 (0.2) 3.8 (0.0) 3.6 (0.1) 6.8 (0.2)* 6.2 (0.1) 33.2 (0.5) 32.3 (0.2) 85.3 (0.1) 84.4 (0.3) 3.0 (0.0) 3.0 (0.0)ARPR 4.8 (0.1) 4.6 (0.2) 5.2 (0.1) 5.0 (0.2) 5.4 (0.2) 5.2 (0.4) 29.8 (0.3) 29.8 (0.6) 83.0 (0.4) 82.4 (0.4) 2.7 (0.0) 2.7 (0.1)ARTI 4.1 (0.1)* 4.8 (0.1) 4.5 (0.1) 4.7 (0.0) 5.2 (0.2) 5.5 (0.3) 32.1 (0.1) 31.0 (0.5) 84.4 (0.4) 83.6 (0.1) 2.9 (0.0) 2.8 (0.0)GACH 4.2 (0.1)* 4.5 (0.1) 4.6 (0.0) 4.6 (0.1) 7.1 (0.1) 6.8 (0.3) 30.8 (0.5) 30.6 (0.5) 83.8 (0.2) 84.1 (0.5) 2.8 (0.0) 2.9 (0.0)GAJE 3.6 (0.1) 4.0 (0.3) 3.9 (0.1) 3.7 (0.1) 7.1 (0.5) 6.7 (0.3) 32.2 (0.7) 32.1 (0.2) 84.0 (0.2) 83.8 (0.2) 2.8 (0.0) 2.9 (0.0)KSLA 6.7 (0.2) 6.3 (0.1) 4.5 (0.0) 4.5 (0.1) 5.8 (0.3) 5.6 (0.4) 31.5 (0.4)* 30.3 (0.3) 84.1 (0.5) 83.4 (0.3) 3.0 (0.0) 3.0 (0.0)LABU 4.5 (0.1) 4.7 (0.1) 4.7 (0.1) 4.7 (0.1) 5.0 (0.3) 5.4 (0.3) 32.3 (0.6) 31.2 (0.3) 84.1 (0.1) 84.1 (0.2) 2.8 (0.1) 2.9 (0.0)LACH 4.1 (0.2) 4.5 (0.2) 4.4 (0.0) 4.5 (0.0) 5.5 (0.2) 6.0 (0.4) 30.5 (0.2) 29.9 (0.2) 83.7 (0.3) 83.3 (0.4) 2.8 (0.0) 2.8 (0.0)NCBD 5.4 (0.2) 5.1 (0.4) 4.7 (0.0) 4.5 (0.5) 5.7 (0.3)* 6.6 (0.4) 32.6 (1.1)* 30.9 (0.8) 85.1 (0.4) 84.7 (0.4) 2.9 (0.1) 2.9 (0.1)NCME 5.7 (0.3) 5.3 (0.1) 5.0 (0.0) 4.8 (0.1) 6.2 (0.1) 6.4 (0.3) 32.6 (0.2) 31.8 (0.3) 84.1 (0.4) 84.2 (0.3) 2.8 (0.0)* 2.9 (0.0)NMGA 5.9 (0.1) 5.8 (0.1) 5.1 (0.1)* 4.9 (0.0) 5.9 (0.2) 6.0 (0.2) 30.9 (0.2) 31.2 (0.4) 82.3 (0.8) 83.7 (0.2) 2.8 (0.0) 2.9 (0.0)NMLC 5.1 (0.1)* 4.6 (0.3) 4.7 (0.1) 4.6 (0.1) 7.1 (0.2) 6.8 (0.2) 31.2 (0.3)* 30.0 (0.2) 83.5 (0.3) 83.0 (0.4) 2.9 (0.0) 2.9 (0.0)SCEK 4.1 (0.2) 4.4 (0.2) 4.7 (0.1) 4.7 (0.1) 5.1 (0.1) 5.2 (0.1) 32.1 (0.5) 31.2 (0.3) 83.3 (0.2) 83.7 (0.1) 2.8 (0.0) 2.8 (0.0)TXPL 5.8 (0.1) 6.0 (0.1) 4.9 (0.1)* 4.5 (0.1) 7.2 (0.1) 7.3 (0.3) 34.1(0.1)* 32.2 (0.5) 85.4 (0.4)* 84.1 (0.4) 2.9 (0.0) 2.9 (0.0)TXPO 3.3 (0.1) 3.2 (0.2) 4.6 (0.1) 4.5 (0.1) 5.0 (0.4) 4.8 (0.1) 31.0 (0.6) 31.2 (0.2) 83.8 (0.6) 83.2 (0.2) 2.7 (0.0) 2.8 (0.0)* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and conventional control (n = 4).1Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).2SE = Standard error.Monsanto Company 12-CT-244U 561 of 620Table G-10. Study 2 - Individual Site Phenotypic Comparison – Growth and Development Characteristics - of MON 88701Not Treated with Dicamba or Glufosinate Herbicides Compared to the Conventional ControlPhenotypic Characteristic (units)Stand count 14 DAP1(# per plot)Stand count at 30 DAP(# per plot)Final Stand Count atharvestPlant Height at 30 DAP(cm)Plant Heightbefore harvest (cm)MON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlSiteMean (SE)2Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR185.5 (1.9)179.8 (2.3)180.3 (0.9)179.5 (2.9)178.0 (0.4)177.5 (3.5)20.5 (0.6)21.1 (0.5)71.7 (5.0)73.5 (5.1)ARTI155.3 (5.9)164.8 (7.2)153.8 (6.3)164.3 (7.1)153.3 (6.5)163.8 (7.2)33.0 (0.2)32.8 (0.2)128.1 (3.0)126.9 (3.1)GACH131.3 (8.4)132.8 (8.0)129.0 (8.0)127.8 (10.3)128.5 (9.4)129.5 (9.1)25.1 (1.0)25.8 (0.8)100.2 (4.5)110.2 (8.7)GAJE179.8 (4.4)181.3 (2.7)166.0 (2.3)164.0 (3.0)162.3 (6.0)157.3 (8.4)23.6 (1.1)26.0 (0.7)77.4 (4.4)*92.6 (7.9)KSLA156.5 (7.4)162.5 (5.9)154.8 (7.4)158.5 (5.3)150.5 (7.0)150.0 (6.7)15.7 (0.6)*18.3 (1.2)113.5 (3.0)*127.0 (3.3)LACH98.8 (3.1)110.3 (5.4)101.0 (3.1)112.5 (7.6)92.3 (1.1)94.8 (2.9)13.5 (0.1)*15.9 (0.8)154.4 (5.7)*180.0 (3.1)NCBD141.8 (2.3)140.3 (3.2)140.8 (2.2)140.3 (3.6)132.5 (2.5)134.3 (2.3)17.8 (0.9)19.4 (0.7)80.3 (2.1)84.7 (4.8)NCME132.0 (8.2)148.0 (19.5)158.5 (6.3)160.3 (9.7)146.3 (9.0)166.3 (8.3)17.2 (0.7)*19.7 (0.9)71.4 (5.2)*85.0 (3.4)NMLC157.5 (7.5)168.8 (11.9)154.5 (7.3)164.0 (2.4)153.3 (6.2)163.3 (5.8)7.0 (0.2)7.2 (0.2)96.7 (2.4)102.7 (1.9)SCEK186.3 (3.7)191.5 (4.6)182.5 (3.6)190.3 (3.9)181.5 (3.0)188.8 (3.8)16.7 (1.1)17.9 (1.3)104.7 (3.6)111.7 (1.4)TXPL130.8 (3.3) 125.0 (10.4) 122.3 (7.4) 119.0 (8.8) 130.5 (3.2)130.0 (11.3) 7.8 (0.6) 7.4 (0.5) 58.6 (1.0) 60.9 (1.3)Monsanto Company 12-CT-244U 562 of 620Table G-10. Study 2 - Individual Site Phenotypic Comparison – Growth and DevelopmentCharacteristics - of MON 88701Not Treated with Dicamba or Glufosinate HerbicidesCompared to the Conventional Control (continued)1st Vigor3,4rating2ndVigor3,5ratingNodes above white flower(obs. 1)Nodes above white flower(obs. 2)MON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlSiteRangeRangeRangeRangeMean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR4 – 63 - 44 - 53 - 55.5 (0.1)5.7 (0.2)3.7 (0.2)3.6 (0.2)ARTI2 – 32 - 32 - 22 - 28.9 (0.3)8.5 (0.2)6.9 (0.3)6.5 (0.2)GACH3 – 42 - 53 - 54 - 65.9 (0.2)*5.3 (0.2)4.7 (0.1)4.1 (0.4)GAJE1 – 21 - 12 - 22 - 26.3 (0.3)6.6 (0.5)5.9 (0.1)5.7 (0.2)KSLA2 – 52 - 32 - 32 - 24.8 (0.3)4.6 (0.2)4.4 (0.7)4.2 (0.6)LACH4 – 73 - 52 - 31 - 27.9 (0.2)7.6 (0.1)8.2 (0.2)8.4 (0.1)NCBD2 – 22 - 33 - 33 - 37.2 (0.1)7.1 (0.5)4.8 (0.2)*3.9 (0.2)NCME2 – 51 - 52 - 32 - 35.1 (0.3)5.2 (0.2)3.2 (0.1)3.3 (0.2)NMLC1 – 32 - 21 - 21 - 17.4 (0.3)7.1 (0.2)8.1 (0.1)8.1 (0.3)SCEK2 – 43 - 62 - 33 - 37.5 (0.2)7.4 (0.2)4.8 (0.1)4.5 (0.1)TXPL1 -11 - 13 - 33 - 35.9 (0.2)5.7 (0.1)5.5 (0.2)*5.1 (0.1)Phenotypic Characteristic (units)Monsanto Company 12-CT-244U 563 of 620Table G-10. Study 2 - Individual Site Phenotypic Comparison – Growth and Development Characteristics - of MON 88701Not Treated with Dicamba or Glufosinate HerbicidesCompared to the Conventional Control (continued)Phenotypic characteristics (units)Nodes above white flower(obs. 3)Seedcotton yield(kg/ha)MON 88701ControlMON 88701ControlSiteMean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR2.3 (0.2)2.4 (0.4)2,561.6 (191.4)2,301.7 (173.4)ARTI 4.8 (0.1)4.7 (0.1)3,864.4 (250.8)3,869.0 (112.9)GACH4.1 (0.1)*3.6 (0.3)4,424.4 (74.0)4,471.5 (99.5)GAJE3.9 (0.1)3.8 (0.0)1,750.1 (261.1)1,548.4 (342.4)KSLA3.7 (0.4)*2.5 (0.4)4,487.0 (294.6)*3,726.5 (81.7)LACH5.8 (0.1)5.7 (0.1)1,956.0 (173.0)2,046.9 (210.4)NCBD3.4 (0.3)3.0 (0.3)4,224.0 (326.0)3,792.7 (283.0)NCME 2.0 (0.2)1.9 (0.1)1,508.4 (112.7)1,610.6 (206.9)NMLC7.5 (0.2)7.1 (0.2)1,938.4 (22.1)*1,479.3 (65.6)SCEK3.8 (0.3)3.5 (0.1)5,219.8 (236.4)5,424.3 (148.4)TXPL3.6 (0.1)3.5 (0.3)4,741.3 (165.8)4,534.5 (215.6)* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and control (n = 4).1DAP = Days after planting.2SE = Standard error.3Plant vigor was rated per plot using a rating scale of 1-9 where: 1-3 is excellent vigor, 4-6 is average vigor, and 7-9 is poorvigor.4First plant vigor score range (minimum-maximum). The range of plant vigor score at 14 DAP for the references is as follows: ARPR 3-6; ARTI 2-3; GACH 4-6; GAJE 1-3; KSLA 2-5; LACH 4-7; NCBD 3-3; NCME 1-6; NMLC 2-5;SCEK 4-6; TXPL 1-1.5Plant vigor score range (minimum-maximum). The range of plant vigor score at 30 DAP for the references is as follows: ARPR 4-6; ARTI 2-2; GACH 3-6;GAJE 2-2; KSLA 2-3; LACH 2-3; NCBD 3-3; NCME 2-3; NMLC 1-3; SCEK 2-3; TXPL 3-3.Monsanto Company 12-CT-244U 564 of 620Table G-11. Study 2 - Individual Site Phenotypic Comparison – Seed Characteristics - of MON 88701 Not Treated withDicamba or Glufosinate Herbicides Compared to the Conventional ControlSeed CharacteristicsSeed index(g per 100 seed)Total seed per boll(# per boll)Mature seed per boll(# per boll)Immature seed per boll(# per boll)MON 88701 Control MON 88701 ControlMON 88701 Control MON 88701 ControlSiteMean (SE)1Mean (SE) Mean (SE) Mean (SE) Mean(SE) Mean (SE) Mean (SE) Mean (SE)ARPR8.8 (0.2)* 10.0 (0.4) 26.8 (0.4)* 23.9 (0.7)24.2 (0.4)* 15.9 (0.6) 2.6 (0.2)* 8.0 (0.6)ARTI9.9 (0.3)* 12.0 (1.0) 28.4 (0.3) 26.8 (0.7)23.7 (0.7)* 18.0 (0.6) 4.6 (0.6)* 8.9 (1.2)GACH9.1 (0.3) 9.7 (0.3) 29.4 (0.7) 27.4 (1.1)24.9 (1.0) 23.4 (1.1) 4.5 (1.4) 4.0 (0.6)GAJE8.5 (0.4)* 10.4 (0.5) 26.6 (1.1) 24.6 (0.7)15.0 (0.5)* 11.6 (0.1) 11.6 (0.8) 13.0 (0.8)KSLA11.7 (0.2)* 12.8 (0.3) 28.4 (1.7)* 24.9 (0.4)25.9 (1.9)* 22.7 (1.0) 2.5 (0.6) 2.2 (0.6)LACH9.4 (0.2)* 10.3 (0.2) 31.4 (0.4) 28.7 (1.2) 20.7 (1.6) 17.5 (1.9) 10.7 (1.8) 11.2 (0.8)NCBD8.8 (0.2)* 10.5 (0.2) 27.0 (0.8) 25.7 (1.3) 18.4 (1.2) 18.2 (0.9) 8.6 (0.6) 7.5 (0.5)NCME8.8 (0.1)* 10.2 (0.1) 28.3 (0.5) 27.6 (1.7) 25.8 (0.6) 23.0 (1.7) 2.5 (0.7) 4.6 (0.2)NMLC10.3 (0.2) 10.9 (0.2) 33.8 (2.0)* 30.7 (1.1) 30.7 (2.2) 28.1 (1.4) 3.2 (1.0) 2.6 (0.4)SCEK8.5 (0.2)* 9.8 (0.2) 28.9 (2.0) 27.3 (1.3) 20.8 (1.4) 19.1 (1.4) 8.1 (2.0) 8.2 (1.2)TXPL9.9 (0.3)* 11.0 (0.3) 31.4 (0.6) 30.1 (0.9) 26.7 (0.8) 25.7 (0.7) 4.7 (0.8) 4.4 (1.1)* Indicates a statisticallysignificant difference (α= 0.05).between MON 88701 and control (n = 0.4).1SE = Standard error.Monsanto Company 12-CT-244U 565 of 620Table G-12. Study 2 - Individual SitePhenotypic Comparison – Boll and FiberCharacteristics - of MON 88701 Not Treatedwith Dicamba or Glufosinate HerbicidesCompared to the Conventional ControlWeight per Boll(g/boll)Fiber Micronaire(mic units)2Fiber Elongation (%)Fiber Strength (g/tex)MON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlSiteMean (SE)1Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR4.3 (0.1)4.1 (0.2)5.0 (0.1)4.9 (0.1)5.0 (0.3)*5.7 (0.2)30.8 (0.4)30.5 (0.4)ARTI4.5 (0.0)4.4 (0.1)5.0 (0.2)4.8 (0.1)5.4 (0.2)5.4 (0.2)29.7 (0.3)28.8 (0.4)GACH5.0 (0.2)4.9 (0.1)4.8 (0.1)4.7 (0.1)5.9 (0.1)6.3 (0.3)30.4 (0.4)29.5 (0.5)GAJE3.6 (0.2)3.7 (0.1)3.9 (0.1)3.6 (0.1)7.5 (0.5)7.0 (0.3)31.1 (0.6)31.5 (0.5)KSLA5.8 (0.3) *5.3 (0.1)4.3 (0.0)4.4 (0.1)6.3 (0.2)6.2 (0.3)30.4 (0.1)*29.4 (0.3)LACH5.4 (0.1)5.4 (0.3)4.7 (0.1)4.6 (0.1)5.5 (0.3)5.9 (0.3)30.7 (0.6)29.9 (0.2)NCBD4.6 (0.3)4.7 (0.2)4.4 (0.1)4.3 (0.1)5.9 (0.1)6.0 (0.2)32.6 (0.9)32.7 (0.4)NCME4.9 (0.1)5.0 (0.3)4.9 (0.1)4.9 (0.1)6.0 (0.3)5.9 (0.4)31.8 (0.2)31.2 (0.3)NMLC5.7 (0.3)*5.1 (0.3)4.9 (0.0)*4.7 (0.1)6.2 (0.1)6.4 (0.2)30.1 (0.3)*27.3 (0.5)SCEK4.6 (0.3)4.4 (0.4)4.9 (0.0)4.9 (0.0)7.0 (0.2)6.9 (0.2)29.9 (0.4)30.0 (0.4)TXPL5.6 (0.1)5.7 (0.1)4.6 (0.1)4.6 (0.1)6.6 (0.2)6.5 (0.2)32.0 (0.3)*31.0 (0.3)Monsanto Company 12-CT-244U 566 of 620Table G-12. Study 2 - Individual SitePhenotypic Comparison – Boll and FiberCharacteristics - of MON 88701 Not Treatedwith Dicamba or Glufosinate Herbicides Compared to the Conventional Control (continued)Fiber Uniformity (%)Fiber Length (cm)MON 88701ControlMON 88701ControlSiteMean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR83.9 (0.2)84.6 (0.1)2.8 (0.0)*2.9 (0.0)ARTI83.6 (0.4)83.6 (0.1)2.7 (0.0)2.8 (0.0)GACH83.6 (0.4)83.4 (0.4)2.8 (0.0)2.8 (0.0)GAJE82.6 (0.9)82.5 (0.6)2.8 (0.1)2.8 (0.1)KSLA 84.2 (0.2)*83.3 (0.1)3.1 (0.0)3.0 (0.0)LACH84.1 (0.3)83.4 (0.4)2.8 (0.0)2.8 (0.0)NCBD84.8 (0.2)84.8 (0.6)2.8 (0.0)*2.9 (0.0)NCME82.6 (0.5)83.2 (0.6)2.7 (0.0)2.8 (0.1)NMLC83.4 (0.6)*81.1 (0.7)2.8 (0.0)2.7 (0.1)SCEK83.1 (0.2)83.9 (0.1)2.7 (0.0)2.8 (0.0)TXPL83.5 (0.3)83.9 (0.3)2.8 (0.0)2.8 (0.0)* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and control (n = 4).1SE = Standard error.2Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).Monsanto Company 12-CT-244U 567 of 620Table G-13. Study 2 - Combined-Site Phenotypic Comparison - Growth and Development Characteristics - of MON 88701Treated with Dicamba and Glufosinate Herbicides Compared to the Conventional ControlPhenotypic Characteristic (units)MON 88701ControlReference Range1Mean (SE)2Mean (SE)MinimumMaximumStand Count at 14 DAP3(# per plot)152.5 (4.9)155.0 (4.4)108.4135.8Stand Count at 30 DAP (# per plot)151.1 (4.8)152.8 (4.0)105.8134.1Final Stand Count at harvest (# per plot)147.7 (5.0)150.5 (4.3)110.5137.7Plant Height at 30 DAP (cm)18.1 (1.1)*19.2 (1.1)11.420.7Plant Height at harvest (cm)98.4 (4.4)*105.0 (4.9)85.2121.9Nodes Above White Flower: (# of nodes at observation 1)6.7 (0.2)*6.4 (0.2)6.07.3(# of nodes at observation 2)5.6 (0.3)*5.2 (0.3)4.85.7(# of nodes at observation 3)4.0 (0.2)3.8 (0.2)3.24.6Seedcotton Yield (kg/ha)3,295.5 (191.0)3,164.1 (210.8)2,181.73,970.8Seed Index (g per 100 seed)9.5 (0.2)*10.7 (0.2)9.412.4Total Seed per Boll (# per boll)28.5 (0.4)*27.0 (0.4)26.130.7Mature Seed per Boll (# per boll)22.8 (0.7)*20.1 (0.8)14.627.0Immature Seed per Boll (# per boll)5.7 (0.6)6.9 (0.6)2.714.4Weight per Boll (g)4.7 (0.1)4.8 (0.1)4.55.9Fiber Micronaire (mic units)44.6 (0.1)4.6 (0.1)4.25.0Fiber Elongation (%)6.0 (0.1)6.2 (0.1)5.68.1Fiber Strength (g/tex)31.2 (0.2)*30.2 (0.2)30.734.0Fiber Uniformity (%)83.5 (0.2)83.4 (0.2)82.884.3Fiber Length (cm)2.8 (0.0)2.8 (0.0)2.73.1* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and the conventional control (n = 44).1Reference range = Minimum and maximum mean values across all 11 sites and nine references from the Study 2 field trial.2SE = Standard error.3DAP = days after planting.4Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).Monsanto Company 12-CT-244U 568 of 620Table G-14. Study 2 - Individual Site Phenotypic Comparison – Growth and Development Characteristics - of MON 88701Treated with Dicamba and Glufosinate Herbicides Compared to the Conventional ControlPhenotypic Characteristic (units)Stand count at 14 DAP1(# per plot)Stand count at 30 DAP(# per plot)Final Stand Count atHarvestPlant Height at 30 DAP(cm)Plant Height beforeharvestMON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlSiteMean (SE)2Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR182.0 (4.4)179.8 (2.3)178.8 (4.8)179.5 (2.9)177.0 (5.0)177.5 (3.5)20.7 (0.7)21.1 (0.5)66.8 (2.0)73.5 (5.1)ARTI169.3 (8.3)164.8 (7.2)167.5 (8.0)164.3 (7.1)166.5 (8.0)163.8 (7.2)33.2 (0.2)32.8 (0.2)122.2 (2.2)126.9 (3.1)GACH128.0 (15.7)132.8 (8.0)126.0 (16.4)127.8 (10.3)123.5 (16.9)129.5 (9.1)23.9 (0.6)25.8 (0.8)103.5 (6.5)110.2 (8.7)GAJE186.0 (2.3)181.3 (2.7)170.0 (2.1)164.0 (3.0)171.0 (2.8)157.3 (8.4)23.2 (1.0)*26.0 (0.7)81.7 (2.4)*92.6 (7.9)KSLA170.0 (1.1)162.5 (5.9)167.5 (3.4)158.5 (5.3)162.0 (2.8)150.0 (6.7)16.6 (0.81)18.3 (1.2)113.6 (1.5)*127.0 (3.3)LACH109.0 (6.4)110.3 (5.4)108.5 (5.7)112.5 (7.6)91.3 (2.7)94.8 (2.9)14.7 (0.4)15.9 (0.8)164.8 (3.6)*180.0 (3.1)NCBD141.8 (2.5)140.3 (3.2)141.8 (2.2)140.3 (3.6)136.8 (3.6)134.3 (2.3)17.8 (0.5)19.4 (0.7)87.4 (3.6)84.7 (4.8)NCME133.5 (8.3)148.0 (19.5)152.5 (3.4)160.3 (9.7)155.0 (7.0)166.3 (8.3)18.6 (0.6)19.7 (0.9)75.7 (2.0)*85.0 (3.4)NMLC166.8 (4.6)168.8 (11.9)168.8 (3.4)164.0 (2.4)160.0 (3.1)163.3 (5.8)7.4 (0.1)7.2 (0.2)98.3 (2.2)102.7 (1.9)SCEK183.5 (3.3)191.5 (4.6)182.8 (3.1)190.3 (3.9)180.3 (3.3)188.8 (3.8)15.6 (1.2)17.9 (1.3)107.0 (2.0)111.7 (1.4)TXPL107.8 (19.8)125.0 (10.4)98.0 (19.1)119.0 (8.8)101.8 (20.0)130.0 (11.3)7.8 (0.4)7.4 (0.5)60.9 (1.8)60.9 (1.3)Monsanto Company 12-CT-244U 569 of 620Table G-14. Study 2 - Individual Site Phenotypic Comparison – Growth and Development Characteristics - of MON 88701Treated with Dicamba and Glufosinate Herbicides Compared to the Conventional Control (continued)Phenotypic Characteristic (units)1st Vigor3,4rating2ndVigor3,5ratingNodes above white flower(obs. 1)Nodes above white flower(obs. 2)MON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlSiteRangeRangeRangeRangeMean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR3 – 53 - 44-53 - 55.6 (0.0)5.7 (0.2)3.7 (0.2)3.6 (0.2)ARTI 2 – 32 - 32-42 - 28.8 (0 .1)8.5 (0.2)6.6 (0.2)6.5 (0.2)GACH 4 – 52 - 54-64 - 65.8 (0.2)5.3 (0.2)4.7 (0.3)4.1 (0.4)GAJE1 – 11 - 12-32 - 26.3 (0.4)6.6 (0.5)6.1 (0.2)*5.7 (0.2)KSLA 1 – 32 - 32-32 - 24.4 (0.3)4.6 (0.2)4.1 (0.4)4.2 (0.6)LACH 3 – 63 - 51-21 - 28.6 (0.2)*7.6 (0.1)8.2 (0.0)8.4 (0.1)NCBD2 – 22 - 33-33 - 37.5 (0.2)7.1 (0.5)5.7 (0.3)*3.9 (0.2)NCME 3 – 51 - 52-32 - 34.9 (0.2)5.2 (0.2)3.6 (0.3)3.3 (0.2)NMLC 1 - 22 - 21-11 - 17.9 (0.1)7.1 (0.2)8.0 (0.2)8.1 (0.3)SCEK3 - 43 - 62-33 - 37.7 (0.3)7.4 (0.2)5.1 (0.4)4.5 (0.1)TXPL 1 - 11 - 13-33 - 36.2 (0.3)*5.7 (0.1)5.7 (0.0)*5.1 (0.1)Monsanto Company 12-CT-244U 570 of 620Table G-14. Study 2 - Individual Site Phenotypic Comparison – Growth and Development Characteristics - of MON 88701Treated with Dicamba and Glufosinate Herbicides Compared to the Conventional Control (continued)Phenotypic characteristics (units)Nodes above white flower(obs. 3)Seedcotton yield(kg/ha)MON 88701ControlMON 88701ControlSiteMean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR 2.3 (0.2)2.4 (0.4)2,705.9 (124.4)2,301.7 (173.4)ARTI4.9 (0.0)*4.7 (0.1)4,109.3 (266.9)3,869.0 (112.9)GACH4.0 (0.1)3.6 (0.3)4,107.9 (42.0)*4,471.5 (99.5)GAJE3.8 (0.0)3.8 (0.0)1,977.6 (341.5)1,548.4 (342.4)KSLA3.4 (0.1)*2.5 (0.4)4,182.0 (329.6)3,726.5 (81.7)LACH5.7 (0.1)5.7 (0.1)1,940.4 (182.3)2,046.9 (210.4)NCBD 3.8 (0.3)*3.0 (0.3)4,025.4 (189.5)3,792.7 (283.0)NCME2.1 (0.2)1.9 (0.1)1,785.4 (181.0)1,610.6 (206.9)NMLC7.0 (0.7)7.1 (0.2)2,048.0 (74.4)*1,479.3 (65.6)SCEK3.8 (0.3)3.5 (0.1)5,264.2 (106.1)5,424.3 (148.4)TXPL3.7 (0.2)3.5 (0.3)4,307.3 (109.1)4,534.5 (215.6)* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and control (n = 44).1DAP = Days after planting.2SE = Standard error.3Plant vigor was rated per plot using a rating scale of 1-9 where: 1-3 is excellent vigor, 4-6 is average vigor, and 7-9 is poorvigor.4First plant vigor score range (minimum-maximum). The range of plant vigor score at 14 DAP for the references is as follows: ARPR 3-6; ARTI 2-3; GACH 4-6; GAJE 1-3; KSLA 2-5; LACH 4-7; NCBD 3-3; NCME 1-6; NMLC 2-5;SCEK 4-6; TXPL 1-1.5Plant vigor score range (minimum-maximum). The range of plant vigor score at 30 DAP for the references is as follows: ARPR 4-6; ARTI 2-2; GACH 3-6;GAJE 2-2; KSLA 2-3; LACH 2-3; NCBD 3-3; NCME 2-3; NMLC 1-3; SCEK 2-3; TXPL 3-3.Monsanto Company 12-CT-244U 571 of 620Table G-15. Study 2 - Individual Site Phenotypic Comparison – Seed Characteristics - of MON 88701 Treated with Dicambaand Glufosinate Herbicides Compared to the Conventional ControlSeed CharacteristicsSeed index(g per 100 seed)Total seed per boll(# per boll)Mature seed per boll(# per boll)Immature seed per boll(# per boll)MON 88701 Control MON 88701 ControlMON 88701 Control MON 88701 ControlSiteMean (SE) Mean (SE) Mean (SE) Mean (SE)Mean (SE) Mean (SE) Mean (SE) Mean (SE)ARPR9.1 (0.2)* 10.0 (0.4) 26.1 (0.8)* 23.9 (0.7)23.2 (1.5)* 15.9 (0.6) 2.9 (0.9)* 8.0 (0.6)ARTI9.6 (0.1)* 12.0 (1.0) 27.7 (0.3) 26.8 (0.7)22.5 (1.0)* 18.0 (0.6) 5.3 (1.0)* 8.9 (1.2)GACH9.0 (0.3)* 9.7 (0.3) 29.7 (0.7)* 27.4 (1.1)27.2 (1.4)* 23.4 (1.1) 2.5 (0.7) 4.0 (0.6)GAJE8.1 (0.4)* 10.4 (0.5) 25.4 (1.8) 24.6 (0.7) 12.6 (0.7) 11.6 (0.1) 12.8 (1.7) 13.0 (0.8)KSLA12.3 (0.3) 12.8 (0.3) 25.8 (0.5) 24.9 (0.4)23.7 (0.3) 22.7 (1.0) 2.1 (0.8) 2.2 (0.6)LACH9.5 (0.3)* 10.3 (0.2) 29.8 (1.0) 28.7 (1.2) 21.6 (2.2) 17.5 (1.9) 8.3 (1.8) 11.2 (0.8)NCBD9.2 (0.2)* 10.5 (0.2) 28.0 (0.7) 25.7 (1.3) 19.7 (0.6) 18.2 (0.9) 8.3 (1.2) 7.5 (0.5)NCME8.9 (0.2)* 10.2 (0.1) 29.0 (0.4) 27.6 (1.7) 23.1 (1.4) 23.0 (1.7) 5.9 (1.4) 4.6 (0.2)NMLC10.2 (0.1) 10.9 (0.2) 31.8 (0.8) 30.7 (1.1)29.6 (1.4) 28.1 (1.4) 2.2 (0.7) 2.6 (0.4)SCEK8.5 (0.2)* 9.8 (0.2) 27.9 (1.3) 27.3 (1.3) 21.4 (1.5) 19.1 (1.4) 6.5 (1.9) 8.2 (1.2)TXPL9.9 (0.4)* 11.0 (0.3) 32.2 (0.9) 30.1 (0.9) 26.5 (0.8) 25.7 (0.7) 5.8 (1.1) 4.4 (1.1)* Indicates a statistically significant difference between MON 88701 and control (p≤0.05).Monsanto Company 12-CT-244U 572 of 620Table G-16. Study 2 - Individual Site Phenotypic Comparison – Boll and Fiber Characteristics - of MON 88701 Treated withDicamba and Glufosinate Herbicides Compared to the Conventional ControlFiber Boll weight(g/boll)Fiber Micronaire(mic units)1Fiber Elongation (%)Fiber Strength (g/tex)MON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlSiteMean (SE2)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR4.1 (0.2) 4.1 (0.2) 5.0 (0.0) 4.9 (0.1) 5.3 (0.1) 5.7 (0.2) 30.3 (0.7) 30.5 (0.4)ARTI4.4 (0.1) 4.4 (0.1) 4.8 (0.0) 4.8 (0.1) 5.1 (0.1) 5.4 (0.2) 29.2 (0.4) 28.8 (0.4)GACH4.8 (0.1) 4.9 (0.1) 4.8 (0.1) 4.7 (0.1) 5.8 (0.3) 6.3 (0.3) 31.0 (0.4)* 29.5 (0.5)GAJE3.3 (0.2) 3.7 (0.1) 3.7 (0.2) 3.6 (0.1) 7.5 (0.1) 7.0 (0.3) 31.5 (0.2) 31.5 (0.5)KSLA5.4 (0.2) 5.3 (0.1) 4.4 (0.0) 4.4 (0.1) 6.1 (0.1) 6.2 (0.3) 30.7 (0.3)* 29.4 (0.3)LACH5.0 (0.2) 5.4 (0.3) 4.7 (0.2) 4.6 (0.1) 5.4 (0.1) 5.9 (0.3) 31.3 (0.3)* 29.9 (0.2)NCBD4.7 (0.1) 4.7 (0.2) 4.3 (0.1) 4.3 (0.1) 6.2 (0.2) 6.0 (0.2) 33.9 (0.2) 32.7 (0.4)NCME4.8 (0.1) 5.0 (0.3) 4.8 (0.1) 4.9 (0.1) 5.9 (0.2) 5.9 (0.4) 32.2 (0.7) 31.2 (0.3)NMLC5.3 (0.2) 5.1 (0.3) 4.9 (0.1)* 4.7 (0.1) 6.1 (0.2) 6.4 (0.2) 29.3 (0.3)* 27.3 (0.5)SCEK4.3 (0.2) 4.4 (0.4) 4.9 (0.0) 4.9 (0.0) 6.7 (0.2) 6.9 (0.2) 30.7 (0.3)* 30.0 (0.4)TXPL5.8 (0.1)5.7 (0.1)4.8 (0.1)4.6 (0.1) 6.4 (0.3) 6.5 (0.2)33.0 (0.4)*31.0 (0.3)Monsanto Company 12-CT-244U 573 of 620Table G-16. Study 2 - Individual Site Phenotypic Comparison – Boll and Fiber Characteristics - of MON 88701 Treated withDicamba and Glufosinate Herbicides Comparedto the Conventional Control (continued)Fiber Uniformity (%)Fiber Length (cm)MON 88701ControlMON 88701ControlSiteMean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR83.9 (0.5) 84.6 (0.1) 2.8 (0.0) 2.9 (0.0)ARTI83.5 (0.2) 83.6 (0.1) 2.8 (0.0) 2.8 (0.0)GACH84.3 (0.6) 83.4 (0.4) 2.8 (0.1) 2.8 (0.0)GAJE82.6 (1.1) 82.5 (0.6) 2.8 (0.0) 2.8 (0.1)KSLA84.3 (0.3)* 83.3 (0.1) 3.1 (0.0) 3.0 (0.0)LACH83.7 (0.3) 83.4 (0.4) 2.8 (0.0) 2.8 (0.0)NCBD84.4 (0.4) 84.8 (0.6) 2.8 (0.0) 2.9 (0.0)NCME83.6 (0.2) 83.2 (0.6) 2.7 (0.0) 2.8 (0.1)NMLC81.4 (0.6) 81.1 (0.7) 2.7 (0.0) 2.7 (0.1)SCEK82.5 (0.5)* 83.9 (0.1) 2.7 (0.0)* 2.8 (0.0)TXPL84.2 (0.4)83.9 (0.3)2.8 (0.1)2.8 (0.0)* Indicates a statisticallysignificant difference (α= 0.05).between MON 88701 and control (n = 4).1Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).2SE = Standard error.Monsanto Company 12-CT-244U 574 of 620Table G-17. Study 2 - Individual Site Phenotypic Comparison– Plant Mapping - of MON 88701Not Treated with Dicamba orGlufosinate Herbicides Comparedto the Conventional ControlPhenotypic Characteristic (units)Total Mainstem Nodes(#)Nodes to first fruitingbranch (#)Total bolls1(# per plant)Total first-position bolls(# per plant)Vegetative bolls(# per plant)MON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlSiteMean (SE)2Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR16.4 (0.4)16.9 (0.5)6.1 (0.1)6.3 (0.2)6.2 (0.4)5.9 (0.6)3.3 (0.1)3.1 (0.3)1.1 (0.3)1.0 (0.3)ARTI20.9 (0.7)*19.8 (0.2)5.4 (0.1)5.4 (0.2)11.3 (1.2)9.7 (0.9)7.2 (1.0)6.1 (0.4)0.0 (0.0)0.1 (0.1)GACH21.6 (0.6)20.9 (0.4)6.5 (0.5)*7.9 (0.5)9.9 (0.4)9.4 (1.3)4.1 (0.5)3.2 (0.5)2.4 (0.5)3.5 (0.9)GAJE17.7 (0.4)17.5 (0.7)5.0 (0.2)*5.8 (0.2)9.0 (0.6)10.1 (0.6)5.8 (0.2)5.9 (0.3)0.2 (0.1)0.3 (0.2)KSLA18.9 (0.4)19.4 (0.6)3.8 (0.1)4.0 (0.3)19.1 (0.2)*20.6 (0.3)4.7 (0.4)5.2 (0.3)13.5 (0.4)13.9 (0.6)LACH20.9 (0.4)22.8 (1.4)7.1 (0.6)6.9 (0.4)7.1 (0.8)4.9 (1.2)2.9 (0.5)2.8 (0.6)1.8 (0.5)0.4 (0.3)NCBD15.0 (0.4)14.8 (0.6)4.2 (0.1)4.3 (0.2)9.8 (0.4)8.9 (0.5)6.3 (0.5)5.5 (0.3)0.6 (0.2)0.3 (0.1)NCME14.5 (0.5)15.2 (0.2)4.5 (0.1)4.6 (0.1)6.8 (0.7)*4.2 (0.2)3.5 (0.3)*2.5 (0.1)1.0 (0.2)*0.1 (0.1)NMLC19.6 (0.2)18.9 (0.3)4.8 (0.5)5.0 (0.4)6.7 (0.7)*4.5 (0.5)4.4 (0.3)*3.0 (0.4)0.4 (0.1)0.0 (0.0)SCEK15.8 (0.2)15.8 (0.2)4.6 (0.2)4.6 (0.1)9.7 (0.4)10.0 (0.6)6.9 (0.3)6.3 (0.3)0.3 (0.1)0.7 (0.2)TXPL18.3 (0.2)18.2 (0.3)5.7 (0.1)5.9 (0.2)12.4 (0.5)11.3 (0.4)7.8 (0.2)*7.1 (0.1)1.0 (0.3)0.8 (0.2)Monsanto Company 12-CT-244U 575 of 620Table G-17. Study 2 - Individual SitePhenotypic Comparison – Plant Mapping -of MON 88701 Not Treated with Dicamba orGlufosinate Herbicides Compared to the Conventional Control (continued)Phenotypic Characteristic (units)% First-position bollretention% First-position bolls overtotal bollsMON 88701ControlMON 88701ControlSiteMean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR33.0 (1.9)30.3 (3.7)56.6 (3.6)56.0 (4.4)ARTI45.4 (4.9)42.3 (2.8)63.9 (2.1)66.8 (2.9)GACH26.8 (2.0)23.0 (2.6)42.7 (5.0)36.6 (3.2)GAJE45.1 (1.5)52.6 (4.9)67.4 (2.3)*61.8 (1.9)KSLA32.2 (2.0)34.0 (2.4)24.8 (1.9)24.9 (1.3)LACH20.9 (2.8)18.4 (4.8)48.8 (1.0)55.8 (1.9)NCBD58.8 (4.3)52.5 (0.9)66.5 (4.5)64.5 (4.0)NCME35.6 (3.3)*23.9 (0.8)53.9 (2.7)58.4 (1.7)NMLC30.1 (2.1)*21.3 (3.3)69.5 (3.0)65.8 (2.8)SCEK62.2 (2.6)57.0 (2.5)73.2 (3.5)*65.7 (1.9)TXPL72.5 (1.4)69.6 (1.7)65.8 (2.0)64.9 (2.2)* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and conventional control (n = 4).1Total bolls = number of first-position bolls + numberof second-position bolls + number of vegetative bolls.2SE = Standard error.Monsanto Company 12-CT-244U 576 of 620Table G-18. Study 2 – Combined-Site Phenotypic Comparison – Plant Mapping - of MON 88701 Treated with Dicamba orGlufosinate Herbicides Comparedto the Conventional ControlPhenotypic Characteristic (units)MON 88701ControlReference Range1Mean (SE)2Mean (SE)MinimumMaximumMainstem Nodes (# per plant)18.3 (0.3)18.2 (0.4)16.021.6Nodes to First Fruiting Branch (# per plant)5.2 (0.1)5.5 (1.1)4.27.6Total Bolls (# per plant)310.1 (0.6)9.0 (0.7)8.613.4Total First-Position Bolls (# per plant)5.2 (0.3)*4.6 (0.3)2.96.3Total Vegetative Bolls (# per plant)2.1 (0.6)1.9 (0.6)0.75.0% Retention First-Position Bolls (per plant)41.7 (2.3)38.6 (2.6)21.253.5% First-Position Bolls of Total Bolls (per plant)56.7 (2.1)56.5 (2.1)36.059.6*Indicates a statisticallysignificant difference (α= 0.05).between the testand control (n = 44).1Reference range = Minimum and maximum mean values among the commercial conventional reference varieties.2SE = Standard error.3Total Bolls = number of first-position bolls + numberof second-position bolls + number of vegetative bolls.Monsanto Company 12-CT-244U 577 of 620Table G-19. Study 2 - Individual Site Phenotypic Comparison – Plant Mapping - of MON 88701 Treated with Dicamba orGlufosinate Herbicides Comparedto the Conventional ControlPhenotypic Characteristic (units)Total Mainstem Nodes(#)Nodes to first fruitingbranch (#)Total bolls1(# per plant)Total first-position bolls(# per plant)Vegetative bolls(# per plant)MON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlMON 88701ControlSiteMean (SE)2Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR16.3 (0.4)16.9 (0.5)6.0 (0.0)6.3 (0.2)5.7 (0.3)5.9 (0.6)3.4 (0.4)3.1 (0.3)0.7 (0.1)1.0 (0.3)ARTI19.7 (0.1)19.8 (0.2)5.2 (0.1)5.4 (0.2)11.0 (0.3)9.7 (0.9)6.8 (0.3)6.1 (0.4)0.3 (0.1)0.1 (0.1)GACH21.2 (0.9)20.9 (0.4)6.5 (0.5)*7.9 (0.5)8.7 (1.6 )9.4 (1.3)3.8 (0.8)3.2 (0.5)2.0 (0.6)3.5 (0.9)GAJE17.0 (0.2)17.5 (0.7)5.1 (0.1)*5.8 (0.2)9.5 (1.1)10.1 (0.6)5.5 (0.4)5.9 (0.3)0.7 (0.3)0.3 (0.2)KSLA 19.6 (0.7)19.4 (0.6)3.7 (0.1)4.0 (0.3)20.1 (0.7)20.6 (0.3)4.8 (0.3)5.2 (0.3)14.3 (0.7)13.9 (0.6)LACH21.4 (0.8)22.8 (1.4)6.4 (0.4)6.9 (0.4)7.3 (1.1)4.9 (1.2)3.8 (0.5)2.8 (0.6)1.0 (0.4)0.4 (0.3)NCBD16.5 (0.7)*14.8 (0.6)4.5 (0.2)4.3 (0.2)10.2 (1.0)8.9 (0.5)6.5 (0.4)*5.5 (0.3)0.4 (0.3)0.3 (0.1)NCME15.1 (0.3)15.2 (0.2)4.8 (0.1)4.6 (0.1)7.1 (0.4)*4.2 (0.2)3.4 (0.3)*2.5 (0.1)1.1 (0.1)*0.1 (0.1)NMLC19.4 (0.2)18.9 (0.3)4.8 (0.4)5.0 (0.4)7.7 (0.6)*4.5 (0.5)4.7 (0.2)*3.0 (0.4)0.3 (0.2)0.0 (0.0)SCEK16.1 (0.4)15.8 (0.2)4.6 (0.1)4.6 (0.1)9.4 (0.3)10.0 (0.6)6.5 (0.3)6.3 (0.3)0.3 (0.0)*0.7 (0.2)TXPL 18.5 (0.7)18.2 (0.3)5.3 (0.2)5.9 (0.2)14.3 (1.8)*11.3 (0.4)8.1 (0.2)*7.1 (0.1)1.8 (0.9)0.8 (0.2)Monsanto Company 12-CT-244U 578 of 620Table G-19. Study 2 - Individual Site Phenotypic Comparison – Plant Mapping - of MON 88701 Treated with Dicamba orGlufosinate Herbicides Compared tothe Conventional Control (continued)Phenotypic Characteristic (units)% Retention firstposition bolls (per plant)% First-position bolls oftotal bolls (per plant)2MON 88701ControlMON 88701ControlSiteMean (SE)Mean (SE)Mean (SE)Mean (SE)ARPR33.5 (3.7)30.3 (3.7)61.4 (2.6)56.0 (4.4)ARTI 46.7 (2.0)42.3 (2.8)61.6 (1.5)66.8 (2.9)GACH25.2 (4.2)23.0 (2.6)45.4 (3.7)*36.6 (3.2)GAJE45.6 (2.8)52.6 (4.9)61.4 (2.0)61.8 (1.9)KSLA32.0 (2.8)34.0 (2.4)24.5 (1.6)24.9 (1.3)LACH25.5 (2.0)18.4 (4.8)57.5 (5.2)55.8 (1.9)NCBD54.5 (1.4)52.5 (0.9)65.2 (3.1)64.5 (4.0)NCME 33.7 (2.4)*23.9 (0.8)50.5 (3.3)58.4 (1.7)NMLC32.2 (1.5)*21.3 (3.3)64.8 (4.6)65.8 (2.8)SCEK57.5 (3.0)57.0 (2.5)70.0 (2.7)65.7 (1.9)TXPL72.1 (2.3)69.6 (1.7)61.0 (6.3)64.9 (2.2)* Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and conventional control (n = 4).1Total bolls = number of first-position bolls + numberof second-position bolls + number of vegetative bolls.2SE = Standard error.

# Monsanto Company 12-CT-244U 579 of 620 

# Table G-20 Study 1 – Qualitative Assessment of Plant Response to Abiotic Stressors

- MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared to

the Conventional Control

Note 1: The experimental design was a randomized complete block with four replications. Data were

not subjected to statistical analysis. No diff erences were observed between MON 88701 and the

conventional control during any observation of plant response to abiotic stressors.



# Abiotic Stressor Number of observations

across all sites Number of observations where

no differences were observed

between MON88701 and the

conventional control

Total 169 169

Compaction 4 4

Drought/ Dry 40 40

Flood 1 1

Hail 6 6

Heat 46 46

Nutrient deficiency 22 22

Wet soil/excess precipitation 17 17

Wind damage 33 33

# Monsanto Company 12-CT-244U 580 of 620 

# Table G-21. Study 1 – Qualitative Asse ssment of Disease Damage - MON 88701 Not

Treated with Dicamba or Glufosinate Herb icides Compared to the Conventional

Control

Disease Number of

observations

across all sites Number observations where no

differences were observed between

MON 88701 and the conventional control

Total 170 170

Anthracnose 2 2

Ascochyta leaf blight 2 2

Bacterial blight 23 23

Boll rot 26 26

Cotton leaf rust 13 13

Damping off 1 1

Fusarium wilt 14 14

Leaf spots 1 

# 43 43

Pythium 11 11

Reniform nematode 1 1

Rhizoctonia 16 16

Root-knot nematode 6 6

Thielaviopsis 1 1

Verticillium wilt 11 11

Note 1: The experimental design was a randomized complete block with four replications. Data were

not subjected to statistical analysis. No diff erences were observed between MON 88701 and the

conventional control during any observation of plant response to abiotic stressors.

1 

# includes Septoria

# Monsanto Company 12-CT-244U 581 of 620 

# Table G-22. Study 1 – Qualitative Asse ssment of Arthropod-related Damage -

MON 88701 Not Treated with Dicamba or Glufo sinate Herbicides Compared to the

Conventional Control

Arthropod Number of

observations

across all sites Number observations

where no differences

were observed between

MON 88701 and the

conventional control

Total 159 159

A p hids ( A p hididae ) 31 31

Beet armyworms ( Spodoptera exigua) 2 2

Cut worms (Noctuidae) 1 1

Fall armyworms ( Spodoptera frugiperda ) 4 4

Fleahoppers ( Pseudatomoscelis seriatus) 4 4

Grasshoppers (Acrididae) 8 8

Heliothines ( Helicoverpa zea and

Heliothis virescens ) 25 25

Southern corn rootworm beetles

( Diabrotica undecimpunctata howardi ) 3 3

Soybean loopers ( Pseudoplusia inclunes ) 2 2

Spider mites ( Tetranychus spp.) 9 9

Stink bugs (Pentatomidae) 28 28

Tarnished plant bugs ( Lygus lineolaris ) 21 21

Thrips (Thripidae) 16 16

White flies ( B emisia spp. ) 5 5

Note 1: The experimental design was a randomized complete block with four replications. Data were

not subjected to statistical analysis. No diff erences were observed between MON 88701 and the

conventional control during any observation of plant response to abiotic stressors.

# Monsanto Company 12-CT-244U 582 of 620 

# Table G-23. Study 1 - Individual Site Analys is: Quantitative Assessment of Thrips

Damage - MON 88701 Not Treated with Dicamba or Glufosinate Herbicides

Compared to the Conventional Control

Rating 1 

# Site MON 88701 (SE) 2 

# Control (SE)

1 ARAU 0.0 (0.0) † 

# 0.0 (0.0)

ARPR 0.9 (0.1) 0.8 (0.1)

GACH 0.0 (0.0) † 

# 0.0 (0.0)

LABU 0.0 (0.0) † 

# 0.0 (0.0)

SCEK 1.5 (0.2) 1.4 (0.2)

2 ARAU 0.1 (0.0) 0.1 (0.0)

ARPR 0.3 (0.1) 0.2 (0.1)

GACH 0.0 (0.0) † 

# 0.0 (0.0)

LABU 0.0 (0.0) † 

# 0.0 (0.0)

SCEK 0.0 (0.0) † 

# 0.0 (0.0)

3 ARAU 0.0 (0.0) † 

# 0.0 (0.0)

ARPR 0.1 (0.0)* 0.3 (0.1)

GACH 0.0 (0.0) † 

# 0.0 (0.0)

LABU 0.0 (0.0) † 

# 0.0 (0.0)

SCEK 0.0 (0.0) † 

# 0.0 (0.0)

* Indicates a statistically significant difference ( α = 0.05) between MON 88701 and conventional control

(n = 4).

† 

# No p-values were generated due to lack of variability in the data.

1 

# Thrips damage observation # 1 was made at approximat ely 14 DAP and the two subsequent observations at

approximately seven day intervals thereafter.

2 

# SE = Standard error.Monsanto Company 12-CT-244U 583 of 620Table G-24. Study 1 - Individual Site Analysis: Quantitative Assessmentof Heliothine Damage - MON 88701 Not Treatedwith Dicamba or Glufosinate HerbicidesCompared to the Conventional ControlPercent Damaged Fruiting Bodies # of Live LarvaeObservation1Site MON 88701 (SE)2Control (SE) MON 88701 (SE) Control (SE)1ARAU 3.8 (1.0) 2.0 (0.7) 0.0 (0.0) 0.0 (0.0)ARPR 0.0 (0.0) 0.1 (0.1) 0.0 (0.0) 0.0 (0.0)GACH 9.2 (2.0) 6.8 (2.5) 0.5 (0.2)* 0.1 (0.0)LABU 1.0 (0.6) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0)SCEK 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)2ARAU 0.2 (0.2) 0.6 (0.6) 0.0†(0.0) 0.0 (0.0)ARPR 1.6 (0.3) 2.2 (0.4) 0.1 (0.1) 0.2 (0.1)GACH 0.8 (0.5) 0.4 (0.4) 0.0 (0.0) 0.1 (0.1)LABU 2.9 (1.7) 2.6 (0.9) 0.1 (0.1) 0.0 (0.0)SCEK 21.2 (13.3) 26.0 (9.2) 0.6 (0.4) 0.3 (0.1)3ARAU 2.5 (1.0) 1.4 (0.6) 0.0 (0.0) 0.1 (0.1)ARPR 4.9 (0.6) 4.9 (1.4) 0.3 (0.1) 0.2 (0.1)GACH 4.1 (2.0) 3.3 (1.4) 0.1 (0.0) 0.1 (0.0)LABU 4.7 (1.1) 2.9 (0.9) 0.1 (0.1) 0.2 (0.1)SCEK 2.3 (2.3) 0.6 (0.6) 0.0 (0.0) 0.0 (0.0)4ARAU 8.7 (1.5)* 15.1 (2.4) 0.0 (0.0) 0.1 (0.1)ARPR 0.0†(0.0) 0.0 (0.0) 0.0†(0.0) 0.0 (0.0)GACH 1.5 (0.5) 2.8 (1.3) 0.0 (0.0) 0.0 (0.0)LABU 20.0 (3.8) 16.4 (2.4) 0.4 (0.1) 0.3 (0.1)SCEK 1.2 (0.5) 0.4 (0.4) 0.0 (0.0) 0.0 (0.0)*Indicates a statistically significant difference (α= 0.05) between MON 88701 and the conventionalcontrol (n = 4).1Heliothine damage observation 1 was made at approximately 45 DAP and the two subsequentobservationsat approximately 15 day intervals thereafter.2SE = Standard Error.Monsanto Company 12-CT-244U 584 of 620Table G-25. Study 1 - Abundance of Pest Arthropods - MON 88701 Not Treated with Dicamba or Glufosinate HerbicidesCompared to the Conventional ControlPest ArthropodAphids (Aphididae) Cabbage loopers (Trichoplusia ni)Fall armyworms (Spodoptera frugiperda)Coll.1SiteMON 88701(SE)2Control(SE)Reference rangeMON 88701(SE)Control(SE)ReferencerangeMON 88701(SE)Control(SE)Referencerange1ARAU−−−2.0 (0.9) 2.3 (0.5) 0.8 – 1.8−−−ARPR 0.0 (0.0) 0.0 (0.0) 0.0 – 0.5−−−−−−GACH 3358.0 (656.5) 1971.8 (419.3) 1193.5 – 5796.0−−−−−−LABU 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 1.3 (0.6) 2.0 (0.4) 0.5 – 1.8−−−SCEK 27.3 (8.2) 20.0 (6.4) 10.3 – 31.3−−−−−−2ARAU 36.8 (8.4) 30.0 (4.6) 30.0 – 144.3−−−−−−ARPR 24.3 (10.0) 17.3 (5.6) 16.0 – 30.3 1.3 (0.6) 1.0 (0.4) 0.5 – 1.5−−−GACH 6.8 (1.6) 2.5 (0.9) 2.5 – 4.5−−−−−−LABU 54.3 (12.1) 51.0 (20.1) 70.0 – 156.0−−−−−−SCEK 4713.3 (1424.1) 7440.0 (1117.1) 994.0 – 6840.0−−−−−−3ARAU 6.0 (1.7) 10.3 (3.7) 8.0 – 23.0 5.3 (2.3) 3.0 (1.1) 1.8 – 6.0−−−ARPR 15.8 (8.8) 19.0 (10.5) 12.3 – 15.3 0.0 (0.0) 0.3 (0.3) 0.0 – 0.0−−−GACH 19.3 (6.5) 19.3 (6.6) 8.3 – 10.3 3.8 (1.6) 1.3 (1.0) 1.3 – 3.8 1.5 (1.5) 1.0 (0.6) 0.3 – 0.8LABU 4.8 (1.1) 10.0 (3.2) 3.8 – 18.0 0.3 (0.3) 0.3 (0.3) 0.0 – 0.8−−−SCEK 6.3 (3.0) 8.5 (1.9) 2.0 – 10.5 0.8 (0.5) 2.0 (1.2) 0.5 – 2.5−−−4ARAU 4.8 (1.3) 5.8 (1.5) 1.5 – 15.8 0.8 (0.3) 0.8 (0.5) 0.0 – 0.5−−−ARPR 3.5 (0.9) 5.5 (1.2) 1.3 – 5.0−−−−−−GACH 2.0 (1.3) 4.3 (2.3) 0.8 – 4.5−−−−−−LABU 1959.3 (303.3) 1993.8 (492.9) 795.3 – 2218.5−−−−−−SCEK 130.0 (15.9) 145.5 (38.0) 70.0 – 183.8−−−−−−Monsanto Company 12-CT-244U 585 of 620Table G-25. Study 1 - Abundance of Pest Arthropods - MON 88701 Not Treated with Dicamba or Glufosinate HerbicidesCompared to the Conventional Control (continued)Pest ArthropodFleahoppers (Pseudatomoscelis seriatus)Heliothines (Helicoverpa zeaandHeliothis virescens)Southern Armyworms (Spodopteraeridania)Coll.SiteMON 88701(SE)Control(SE)Reference rangeMON 88701(SE)Control(SE)ReferencerangeMON 88701(SE)Control(SE)Referencerange1ARAU−−−−−−−−−ARPR−−−−−−−−−GACH 0.5 (0.3) 0.5 (0.5) 0.0 – 0.5 0.3 (0.3) 0.8 (0.5) 0.0 – 0.3−−−LABU−−−−−−0.3 (0.3) 0.5 (0.3) 0.8 – 2.0SCEK−−−−−−−−−2ARAU−−−0.8 (0.5) 0.5 (0.3) 0.3 – 1.0−−−ARPR−−−2.5 (1.0) 2.0 (0.9) 1.3 – 3.5−−−GACH−−−0.3 (0.3) 0.3 (0.3) 0.0 – 1.3−−−LABU 0.5 (0.5) 0.0 (0.0) 0.3 – 0.8 1.3 (0.6) 0.8 (0.5) 0.3 – 1.5−−−SCEK 3.3 (1.0) 4.8 (3.0) 1.5 – 4.5 11.8 (4.6) 6.5 (2.7) 9.5 – 18.8−−−3ARAU−−−0.8 (0.5) 2.0 (0.9) 0.3 – 2.3−−−ARPR−−−0.3 (0.3) 0.8 (0.5) 0.3 – 2.5−−−GACH−−−0.5 (0.5) 1.3 (1.3) 0.0 – 0.5−−−LABU−−−1.5 (0.5) 0.8 (0.5) 0.5 – 1.8−−−SCEK 6.0 (1.8) 6.0 (1.6) 2.8 – 9.5 1.3 (0.5) 0.8 (0.3) 0.5 – 1.5−−−4ARAU−−−1.0 (0.7) 0.5 (0.3) 0.0 – 0.5−−−ARPR−−−−−−−−−GACH 0.3 (0.3) 0.0 (0.0) 0.0 – 0.0−−−−−−LABU−−−0.0 (0.0) 0.3 (0.3) 0.0 – 0.8−−−SCEK 2.5 (0.9) 2.3 (1.0)1.0 – 4.8 0.3 (0.3) 0.0 (0.0) 0.0 – 0.3−−−Monsanto Company 12-CT-244U 586 of 620Table G-25. Study 1 - Abundance of Pest Arthropods - MON 88701 Not Treated with Dicamba or Glufosinate HerbicidesCompared to the Conventional Control (continued)Pest ArthropodStink bugs (Pentatomidae) Tarnished plant bugs (Lygus lineolaris) Thrips (Thripidae)Coll. SiteMON 88701(SE)Control(SE)ReferencerangeMON 88701(SE)Control(SE)ReferencerangeMON 88701(SE)Control(SE)Referencerange1ARAU−−−−−−4.3 (1.7) 3.8 (2.3) 0.8 – 2.5ARPR−−−2.5 (0.9) 0.5 (0.3) 0.0 – 1.0 96.8 (31.9) 56.8 (17.6) 51.3 – 78.5GACH−−−−−−5.0 (1.6) 3.5 (1.3) 2.3 – 4.8LABU−−−−−−0.3 (0.3) 0.3 (0.3) 0.0 – 0.3SCEK−−−−−−−−−2ARAU 0.8 (0.5) 0.3 (0.3) 0.0 – 1.5 1.0 (0.4) 0.8 (0.5) 0.0 – 1.5−−−ARPR−−−1.8 (0.8) 3.3 (1.1) 2.0 – 4.0 24.0 (6.4) 25.3 (14.0) 17.5 – 32.5GACH−−−0.3 (0.3) 1.5 (0.7) 0.5 – 1.0 1.3 (0.5) 1.3 (0.3) 0.8 – 2.8LABU−−−1.3 (0.6) 0.8 (0.3) 0.5 – 0.8 1.0 (0.7) 1.5 (1.2) 0.5 – 1.5SCEK−−−−−−13.8 (2.8) 16.5 (5.3) 15.0 – 30.53ARAU−−−0.5 (0.5) 1.8 (0.9) 0.3 – 0.5−−−ARPR−−−0.0 (0.0) 0.3 (0.3) 0.3 – 2.0 2.8 (2.1) 1.0 (0.7) 1.3 – 2.3GACH−−−−−−24.0 (8.8) 19.5 (5.6) 10.5 – 25.0LABU−−−−−−1.0 (0.4) 0.8 (0.3) 0.3 – 2.8SCEK−−−−−−3.5 (1.8) 2.5 (0.9) 2.3 – 5.34ARAU 1.8 (0.9) 1.3 (0.6) 0.3 – 1.5−−−2.0 (1.4) 0.8 (0.3) 0.8 – 1.8ARPR 0.5 (0.3) 0.3 (0.3) 0.5 – 1.3 1.3 (1.3) 0.3 (0.3) 0.8 – 1.5 8.3 (1.7) 9.0 (2.7) 9.3 – 18.5GACH−−−−−−1.3 (1.0) 0.5 (0.3) 0.0 – 0.8LABU 0.3 (0.3)* 1.8 (0.5) 0.5 – 1.0 0.5 (0.5)* 2.0 (0.6) 0.5 – 2.0 1.5 (1.2) 1.3 (0.8) 1.5 – 10.0SCEK−−−−−−11.0 (3.5) 6.0 (1.8) 11.3 – 20.0Monsanto Company 12-CT-244U 587 of 620Table G-25 Study 1 - Abundance of Pest Arthropods - MON 88701 Not Treated with Dicambaor Glufosinate HerbicidesCompared to the Conventional Control (continued)White flies (Bemisiaspp.)Coll. SiteMON 88701 (SE) Control (SE) Reference range1ARAU−−−ARPR 0.0 (0.0) 0.3 (0.3) 0.0 – 0.5GACH 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3LABU−−−SCEK−−−2ARAU 0.5 (0.3) 0.3 (0.3) 0.5 – 1.5ARPR−−−GACH 1.3 (0.3) 0.5 (0.3) 1.3 – 2.3LABU−−−SCEK 0.5 (0.5) 0.5 (0.5) 0.0 – 0.53ARAU 0.0 (0.0) 0.3 (0.3) 0.0 – 0.5ARPR−−−GACH−−−LABU−−−SCEK−−−4ARAU−−−ARPR 2.0 (0.7) 3.0 (1.4) 0.8 – 3.0GACH 1.5 (0.7) 0.3 (0.3) 0.3 – 1.0LABU−−−SCEK 1.3 (1.0) 0.5 (0.3) 0.0 – 0.8Note: A dash (-) indicates arthropod not evaluated.*Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and the conventionalcontrol (n = 4).1Arthropod collection 1 was made at approximately 30 DAP and the three subsequent collections at approximately 30 day intervalsthereafter2SE = Standard error.Monsanto Company 12-CT-244U 588 of 620Table G-26. Study 1 - Abundance of Beneficial Arthropods - MON 88701 Not Treated with Dicamba or GlufosinateHerbicides Compared to the Conventional ControlBeneficial ArthropodBig eyed bugs (Geocorisspp.) Braconids (Braconidae) Damsel bugs (Nabisspp.)Coll.1SiteMON 88701(SE)2Control(SE)ReferencerangeMON 88701(SE)Control (SE)ReferencerangeMON 88701(SE)Control (SE)Referencerange1ARAU−−−−−−1.3 (0.5) 1.0 (0.7) 0.0 – 1.8ARPR 2.3 (0.6) 0.5 (0.5) 0.8 – 2.3−−−0.5 (0.5) 1.3 (0.6) 0.3 – 0.8GACH−−−−−−0.8 (0.5) 0.5 (0.3) 0.0 – 0.5LABU−−−0.0 (0.0) 0.0 (0.0) 0.0 – 0.3−−−SCEK 0.3 (0.3) 0.0 (0.0) 0.3 – 1.0−−−−−−2ARAU 0.3 (0.3) 0.0 (0.0) 0.0 – 1.3−−−−−−ARPR 1.0 (0.4) 1.5 (0.5) 0.3 – 0.8−−−−−−GACH 10.5 (1.3) 11.8 (1.9) 6.8 – 16.5−−−6.0 (2.0)* 2.3 (0.8) 2.5– 7.3LABU 1.5 (0.7) 2.3 (1.3) 0.5 – 3.3−−−0.5 (0.3) 0.0 (0.0) 0.0 – 0.5SCEK 0.8 (0.5) 1.3 (0.5) 1.0 – 3.3−−−−−−3ARAU 0.0 (0.0) 0.3 (0.3) 0.0 – 1.3−−−−−−ARPR−−−−−−−−−GACH 1.0 (0.4) 2.5 (0.9) 1.5 – 4.5−−−3.3 (1.0) 1.5 (0.9) 1.5 – 3.0LABU−−−−−−−−−SCEK 7.0 (0.7) 6.0 (2.0) 5.0 – 13.0−−−−−−4ARAU 1.5 (0.5) 2.5 (0.7) 1.5 – 3.5−−−1.8 (0.5) 3.0 (0.7) 0.8 – 2.5ARPR 7.5 (3.0) 4.0 (0.7) 3.0 – 7.5−−−−−−GACH 5.0 (1.6) 5.5 (3.0) 1.3 – 8.5−−−3.3 (0.8) 4.8 (1.9) 1.3 – 4.0LABU−−−0.5 (0.5) 2.0 (1.4) 3.0 – 8.8−−−SCEK 27.8 (9.4) 35.0 (4.8) 16.5 – 44.3−−−−−−Monsanto Company 12-CT-244U 589 of 620Table G-26. Study 1 - Abundance of Beneficial Arthropods - MON 88701 Not Treated with Dicamba or GlufosinateHerbicides Compared to the Conventional Control (continued)Beneficial ArthropodLacewings (Chrysopaspp.andHemerobiusspp.) Ladybird Beetles (Coccinellidae)Oriusspp.Coll.SiteMON 88701(SE)Control (SE)ReferencerangeMON 88701(SE)Control(SE)ReferencerangeMON 88701(SE)Control (SE)Referencerange1ARAU−−−0.5 (0.3) 0.5 (0.3) 0.3 – 0.8 0.0 (0.0) 0.3 (0.3) 0.0 – 0.3ARPR−−−2.3 (0.5) 3.3 (1.1) 1.0 – 2.5 1.8 (1.1) 0.5 (0.3) 0.0 – 2.8GACH 0.8 (0.8) 0.3 (0.3) 0.3 – 0.5 14.3 (4.6) 12.3 (3.9) 6.0 – 43.8 0.8 (0.5) 0.0 (0.0) 0.3 – 0.8LABU−−−0.5 (0.5) 0.3 (0.3) 0.0 – 0.8−−−SCEK−−−0.5 (0.3) 0.8 (0.3) 0.3 – 1.3−−−2ARAU 3.0 (0.4) 3.5 (0.9) 2.3 – 5.0 1.0 (0.4) 1.0 (0.7) 1.0 – 2.0 0.0 (0.0)* 1.5 (0.7) 0.3 – 0.8ARPR 2.5 (0.7) 3.0 (1.3) 1.8 – 4.0 9.0 (2.5) 4.8 (0.3) 5.5 – 8.5 1.0 (0.6) 1.0 (0.4) 0.5 – 1.8GACH 0.3 (0.3) 1.0 (0.4) 1.0 – 2.0 10.8 (2.3) 10.0 (1.4) 3.3 – 12.3−−−LABU−−−8.5 (1.4) 9.5 (2.4) 8.0 – 12.5 0.3 (0.3) 0.0 (0.0) 0.0 – 0.5SCEK 3.3 (0.8) 2.5 (1.0) 1.8 – 3.5 34.8 (6.5) 46.3 (10.8) 18.3 – 36.3 8.5 (3.1) 6.3 (1.9) 5.3 – 9.53ARAU 2.8 (1.3) 4.0 (0.7) 1.8 – 3.5 7.3 (3.2) 10.3 (2.7) 9.3 – 18.3 0.5 (0.3)* 2.8 (1.2) 0.8 – 3.3ARPR 1.0 (0.4) 1.5 (0.9) 0.5 – 1.5 6.5 (1.0) 6.0 (1.8) 5.5 – 8.0 0.3 (0.3) 1.0 (0.6) 0.0 – 0.5GACH 3.0 (1.8) 4.0 (1.2) 2.0 – 5.5 12.0 (3.2) 11.0 (2.4) 4.8 – 10.3−−−LABU−−−−−−−−−SCEK 0.3 (0.3) 1.0 (0.4) 0.3 – 0.8 7.8 (0.9) 5.0 (0.7) 6.3 – 8.3 2.0 (1.7) 3.5 (0.9) 1.5 – 5.04ARAU 4.3 (1.6) 2.5 (0.5) 1.8 – 3.5 10.8 (2.5) 12.0 (1.7) 9.3 – 13.0−−−ARPR 3.3 (1.1) 3.8 (1.3) 0.5 – 4.3 6.5 (1.6) 5.3 (1.0) 2.0 – 4.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.8GACH 0.0 (0.0) 0.0 (0.0) 0.3 – 0.5 1.3 (0.6) 2.0 (1.1) 3.3 – 7.0−−−LABU 0.3 (0.3) 0.3 (0.3) 0.5 – 2.3 6.3 (3.3) 3.5 (1.3) 4.0 – 8.0 3.8 (3.1) 3.3 (2.0) 2.8 – 10.3SCEK 1.0 (0.4) 0.3 (0.3) 0.5 – 1.8 7.5 (0.3) 5.0 (2.4) 5.0 – 10.8 3.8 (0.5) 4.0 (0.8) 3.0 – 10.3Monsanto Company 12-CT-244U 590 of 620Table G-26. Study 1 - Abundance of Beneficial Arthropods - MON 88701 Not Treated with Dicamba or GlufosinateHerbicides Compared to the Conventional Control (continued)Spiders (Araneae)Coll. SiteMON 88701 (SE) Control (SE) Reference range1ARAU 1.0 (0.4) 1.0 (0.4) 0.3 – 1.3ARPR 1.5 (0.5) 1.0 (0.7) 0.8 – 1.8GACH 1.5 (0.9) 2.5 (0.7) 0.3 – 2.0LABU 0.3 (0.3) 1.0 (1.0) 0.3 – 1.3SCEK 0.5 (0.5) 0.8 (0.3) 0.5 – 1.02ARAU 1.0 (0.4) 1.3 (0.5) 0.3 – 2.0ARPR 6.0 (1.3) 4.0 (0.4) 2.5 – 5.8GACH 4.5 (0.3) 6.0 (1.2) 4.3 – 6.8LABU 5.3 (1.5) 3.0 (1.3) 2.8 – 5.3SCEK 2.8 (0.5) 4.3 (1.3) 1.8 – 4.33ARAU 2.0 (0.4) 2.0 (1.1) 0.8 – 1.8ARPR 0.3 (0.3) 0.0 (0.0) 0.0 – 0.5GACH 4.0 (0.4) 2.8 (0.5) 3.8 – 4.3LABU−−−SCEK 4.3 (1.0) 2.8 (0.9) 3.5 – 7.84ARAU 3.3 (1.0) 3.5 (0.9) 3.5 – 7.3ARPR 1.5 (0.3) 2.0 (1.0) 0.3 – 1.8GACH 3.0 (0.8) 5.8 (1.8) 3.5 – 6.0LABU 0.8 (0.5) 0.8 (0.5) 0.3 – 1.5SCEK 6.5 (1.3) 6.0 (0.8) 8.0 – 12.5Note: A dash (-) indicates arthropod not evaluated.*Indicates a statisticallysignificant difference (α= 0.05) between MON 88701 and the conventionalcontrol (n = 4).1Arthropod collection 1 was made at approximately 30 DAP and the three subsequent collections at approximately 30 day intervalsthereafter2SE = Standard error.

# Monsanto Company 12-CT-244U 591 of 620 

# Table G-27 Study 2 – Qualitative Assessment of Plant Response to Abiotic Stressors

- MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared to

the Conventional Control

Abiotic Stressor Number of

observations

across all sites Number of observations where no

differences were observed between

MON 88701 and the control

Total 127 127

Compaction 4 4

Drought/ Dry 30 30

Flood 1 1

Hail Damage 6 6

Heat 30 30

Nutrient deficiency 10 10

Wet soil/excess precipitation 17 17

Wind damage 29 29

Note 1: The experimental design was a randomized complete block with four replications. Data were

not subjected to statistical analysis. No diff erences were observed between MON 88701 and the

conventional control during any observation of plant response to abiotic stressors.

# Monsanto Company 12-CT-244U 592 of 620 

# Table G-28. Study 2 – Qualitative A ssessment of Disease Damage of MON 88701

Not Treated with Dicamba or Glufosi nate Herbicides Compared to the

Conventional Control

Disease Number of observations

across all sites Number of observations where no

differences were observed between

MON 88701 and the control

Total 129 129

Anthracnose 3 3

Ascochyta leaf blight 3 3

Bacterial blight 14 14

Boll rot 15 15

Cotton leaf rust 7 7

Damping off 1 1

Fusarium wilt 11 11

Leaf spots 1 

# 36 36

Pythium 9 9

Reniform nematode 1 1

Rhizoctonia 12 12

Root-knot nematode 6 6

Thielaviopsis 1 1

Verticillium wilt 10 10

Note 1: The experimental design was a randomized complete block with four replications. Data were

not subjected to statistical analysis. No diff erences were observed between MON 88701 and the

conventional control during any observation of plant response to abiotic stressors

1 

# Includes Alternaria and Septoria

# Monsanto Company 12-CT-244U 593 of 620 

# Table G-29. Study 2 – Qualitative Asse ssment of Arthropod-related Damage -

MON 88701 Not Treated with Dicamba or Glufo sinate Herbicides Compared to the

Conventional Control

Arthropod Number of

observations

across all sites Number of

observations where no

differences were

observed between

MON 88701 and the

control

Total 129 129

A p hids ( A p hididae ) 24 24

Beet armyworms ( Spodoptera exigua) 1 1

Cabbage loopers ( Trichoplusia ni ) 1 1

Cut worms (Noctuidae) 3 3

Fall armyworms ( Spodoptera frugiperda ) 4 4

Fleahoppers ( Pseudatomoscelis seriatus) 2 2

Grasshoppers (Acrididae) 6 6

Heliothines ( Helicoverpa zea and

Heliothis virescens ) 23 23

Southern corn rootworm beetles

( Diabrotica undecimpunctata howardi ) 2 2

Soybean loopers ( Pseudoplusia inclunes ) 1 1

Spider mites ( Tetranychus spp.) 9 9

Stink bugs (Pentatomidae) 21 21

Tarnished plant bugs ( Lygus lineolaris ) 14 14

Thrips (Thripidae) 16 17

White flies ( B emisia spp. ) 2 2

Note 1: The experimental design was a randomized complete block with four replications. Data were

not subjected to statistical analysis. No diff erences were observed between MON 88701 and the

conventional control during any observation of plant response to abiotic stressors.

# Monsanto Company 12-CT-244U 594 of 620 

# References for Appendix G

Drees, B.M. and M.E. Rice. 1985. The vertical beat sheet: A new device for sampling

soybeans insects. Journal of Economic Entomology 78:1507-1510.

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.

# Monsanto Company 12-CT-244U 595 of 620 

# Appendix H: Materials and Method s for Pollen Morphology and Viability

Assessment

H.1. Plant Production

MON 88701, the conventional control, and f our commercial reference varieties were

grown under similar agronomic conditions in a field trial in Crittenden County, Arkansas

(Table G-1; ARPR site). The trial was arra nged in a randomized complete-block design

with four replications. Each plot consisted of eight rows approximately 6 m in length.

H.2. Flower Collection and Sample Preparation

Five flowers, each open less than 24 hours at the time of collection, were collected from

each plot. The pollen obtained from an indi vidual flower comprised a subsample of the

plot and was placed in a uniquely labele d, clean container. Six hundred μl of

Alexander’s stain (Alexander, 1980) diluted 1:5 with distille d water was added to each

container, and the container c ontents were thoroughly mixed. Containers were placed on

wet (water) ice within 10 minutes of pollen collection. After trans port to the performing

laboratory, the pollen in the containers was allowed to stain at ambient temperatures for

at least 20 hours.

H.3. Data Collection

Pollen subsamples were assessed for pollen viability, diameter, and general morphology.

Slides were prepared by aliquoting 30 μ l of suspended pollen/stain solution onto a slide.

The slides were viewed under an Olympus BX51TRF light/fl uorescence microscope with

an Olympus DP70 digital color camera. Th e associated PC computer had imaging

software for diameter measurement (I -Pro Plus version 6.2.1.491© 1993-2007, Media

Cybernetics, Inc.) and camera softwa re (DP Controller 1. 2. 1.108 © 2001-2003,

Olympus Optical Co., Ltd. and DP Manage r version 1, 2, 1, 107 © 2001-2003, Olympus

Optical Co., Ltd.).

H.3.1. Pollen Viability

To assess pollen viability, 77 or more pollen grains were evaluated under the 40X ocular

lens (400X total magnification) for each s ubsample. When exposed to the staining

solution, viable pollen grains stained purple because of the pr esence of vital cytoplasmic

content, while dead pollen grains stained clear to light blue-green. In addition, viable

pollen grains appeared round, whereas non-vi able pollen grains appeared round to

collapsed depending on the degree of hydration.

H.3.2. Pollen Diameter

Pollen diameter was measured under the 40X ocular lens (400X total magnification)

using software (Image-Pro Plus versi on 6.2.1.491© 1993-2007 Media Cybernetics, Inc.)

# Monsanto Company 12-CT-244U 596 of 620 

# to view digital images. For each replica tion, pollen diameter was measured along two

perpendicular axes for ten representative viable pollen grains.

H.3.3. General Pollen Morphology

General morphology of the pollen was observed for each subsample of MON 88701, the

conventional control, and the commercial re ference varieties duri ng determination of

pollen viability.

H.4. Statistical Analysis

Monsanto Statistics Technology Cent er performed the statistical analysis. An analysis of

variance was conducted according to a randomized complete block design using SAS 



# Version 9.2 (SAS, 2008) with a significance level of 5% (p ≤ 0.05). MON 88701 was

compared to the conventional control for percent viable pollen and pollen grain diameter.

MON 88701 was not statistically compared to the reference va rieties. A reference range

for each measured characteristic was determ ined from the minimum and maximum mean

values from among the four commercial refe rence varieties. General pollen morphology

was qualitative; therefore, no statistical analysis was conducted on these observations.

 

# SAS is a registered tradem ark of SAS Institute, Inc.

# Monsanto Company 12-CT-244U 597 of 620 

# Table H-1. Starting Seed for Pollen Morphology and Viability Assessment

Material Phenotype Monsanto ID

Coker 130 Conventional 11268128

Nex Gen NG3410RF Glyphosate-tolerant 11266969

ST474 Conventional 11266156

DP 493 Conventional 11266763

SG125 Conventional 11266155

MON 88701 Dicamba and glufosinate-tolerant 11268129

# Monsanto Company 12-CT-244U 598 of 620 

# References for Appendix H

Alexander, M.P. 1980. A versat ile stain for pollen fungi, yeast and bacteria. Stain

Technology 55:13-18.

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.

# Monsanto Company 12-CT-244U 599 of 620 

# Appendix I: Herbicide Resistance

I.1. Introduction

Based upon theory of natural selection, plant populations can devel op resistance to an

herbicide due to the selection of individuals that carry specif ic genes that can render those

individuals unaffected by the typical lethal effect s of an herbicide. The application of an

herbicide to the plant does not, itself, cause a mutation in subsequent generations.

Rather, over time, those few plant biotypes co ntaining resistant gene (s) become dominant

in the population with repeat ed use of the herbicide in the absence of other control

methods, such as use of other herbicides a nd/or use of cultural control methods. The

development of resistant populations is a possi bility for all herbicid es. The probability

for resistance to develop is a functio n of: frequency of resistant allele(s) 8 

# , mechanism of

resistance, dominance or recessive nature of th e resistant allele(s), re lative fitness of the

resistant biotype, and frequency or duration of herbicide use in the absence of other

control methods (Beckie, 2006; Jasieniuk, et al.,1996; Sammons et al., 2007). The

probability of resistance is not the same fo r all herbicides, with some herbicides ( e.g.

, ALS and ACCase classes) exhibiting resistan ce more quickly than other herbicides ( e.g. auxin class, glyphosate, dinitroanilines class).

Herbicide resistance can become a limiting factor in crop production if the resistant weed

population cannot be controlled with other herb icides or cultural practices. In general,

this has not been the case for any herbicide. In most crops, there are multiple herbicide

options for growers to use. However, good management practices to delay the

development of herbicide resistance have been identified and are be ing actively promoted

by the public and private sectors (HRAC, 2010) and are being implemented by growers.

Monsanto considers product st ewardship to be a fundament al component of customer

service and business practices. Stewardship of the dicamba and glufosinate herbicides to

preserve their usefulness for growers is an important aspect of Monsanto’s stewardship

commitment. Although herbicide resistance may eventually occur in weed species when

any herbicide is widely used, resistance can be postponed, contained, and managed

through research, education, and good manage ment practices. These are the key

elements of Monsanto’s approach to providi ng stewardship of di camba and glufosinate

used on MON 88701 integrated into the glyphos ate-tolerant cotton systems. Monsanto

will invest in research, and grower/retaile r education and training programs to provide

information on best practices to manage di camba and glufosinate weed resistance in

cotton production. This appendix provides an overview of Monsanto’s approach to the

development of best management practices to mitigate dicamba and glufosinate weed

resistance. Monsanto works closely with we ed scientists in academia and with other

companies to research and develop best management practices and to uniformly

communicate such practices to growers. Eviden ce of this cooperative effort is the recent

development and posting of herbicide-resist ant training modules on the WSSA website

8 

# An allele is any of several forms of a gene, usually arising through mutation, that are responsible for

hereditary variation.

# Monsanto Company 12-CT-244U 600 of 620 

# ( www.wssa.net ) and the publication of guidelines by the Herbicide Resistance Action

Committee (HRAC) on their website ( www.hracglobal.com ).

I.2. The Herbicide Dicamba

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is classified as a benzoic acid herbicide

belonging to the synthetic auxin group of herb icides (HRAC, 2010). The herbicides in

this group act as growth regulators similar to endogenous indole acetic acid (IAA) but are

structurally diverse. The synthetic auxin gr oup includes five chemical families (benzoic

acid, pyridine-carboxylic acid, quinoline car boxylic acid, phenoxy-car boxylic acid and a

separate class which includes one herbicide, be nazolin ethyl). The specific site of action

among the different synthetic auxin chemical families may be different. In addition to

dicamba, specific herbicides in this group include 2,4-D, 2,4-DB, mecoprop, MCPA,

clopyralid, picloram, quinclorac and several ot her active ingredients. Dicamba and other

synthetic auxin herbicides are classified in Herbicide Group 4 by the Weed Science

Society of America (HRAC, 2009). Most herb icides in this group are active on broadleaf

weeds only, but a few have si gnificant activity on grasses, e.g. , quinclorac. Dicamba

provides preemergence and postemergence c ontrol of over 95 annual and biennial

broadleaf weed species and control or s uppression of over 100 pere nnial broadleaf and

woody species (BASF, 2008). Dicamba is not ac tive on grass weeds and is often used in

combination with other herbicides to provide broad spectrum weed control.

Dicamba herbicide was commercialized in the U.S. for agricultural use in 1967 and is

currently labeled for preemergence and/or pos temergence weed control in corn, soybean,

cotton, sorghum, small grains (wheat, barley and oats), millet, pasture, rangeland,

asparagus, sugarcane, turf, grass grown for s eed, conservation reserve program land, and

fallow cropland, and for non-crop uses (U.S. EPA, 2009). Dicamba is sold as standalone

formulation which can be tank-mixed with one or more active ingredients depending

upon the crop and the weed spectrum. Dicamba is also sold as a premix formulation with

other herbicides.

Dicamba acts in plants by mimicking natura lly-occurring plant growth hormones called

auxins, thereby destroying tissue through uncont rolled cell division a nd growth (Ahrens,

1994). Ahrens (1994) further states that dicamba has been found to affect cell wall

integrity and nucleic acid metabolism whereas in other cases it has been found to increase

cell wall permeability, leading to cell enlargem ent. At low concentrations, dicamba has

been found to increase synthesis of DNA, RN A, and proteins, resulting in altered cell

division and growth. At high c oncentrations, inhibition of cell division and growth occur.

In general, dicamba and other synthetic au xin herbicides have been found to affect

multiple plant physiological systems. Grossmann (2010), in a review of auxin

herbicides, outlined a proposed mechanism a nd mode-of-action for auxin herbicides and

IAA at supraoptimal endogenous concentrations in dicot plan t species. The proposal was

based upon recent identification of receptors for auxins and hormone interaction in

signaling between auxin, ethyle ne, and the upregulations of abscisic acid biosynthesis

which would account for a large part of the various auxin-herbicide-mediated responses

that are seen in sensitive dico ts. In addition, research has indicated that there is a high

# Monsanto Company 12-CT-244U 601 of 620 

# level of redundancy in auxin receptors which may account for the lack of development of

widespread resistance to this he rbicide group (Walsh et al., 2006).

Dicamba is taken up by plants through the root s, stems, and foliage (Ahrens, 1994; NPIC,

2002). Dicamba translocates to all plant tissu es but accumulates in growing tissues.

Translocation of dicamba is typically slower in tolerant plants such as grasses compared

to broadleaf plants.

I.3. The Herbicide Glufosinate

Glufosinate [2-Amino-4-(hydroxymethylphosphinyl ) butanoic acid] is classified as a

phosphinic acid herbicide belonging to the gl utamine synthetase inhibitor group of

herbicides (HRAC, 2010). Bialaphos is the only other active ingred ient belonging to the

phosphinic acid chemical family. Glufosinate and bialaphos are classified in Herbicide

Group 10 by the Weed Science Society of Amer ica (HRAC, 2010). Glufosinate provides

postemergence control of over 90 annual gr ass and broadleaf weed species and 25

biennial and perennial grass and broadleaf weed species.

Glufosinate was first approved for use in the U.S. in 1994 (U.S. EPA, 2008) and is

currently labeled for non-crop uses, preplant burndown to gl ufosinate-tolerant and nontolerant crops and/or in-crop postemergence w eed control in glufosin ate-tolerant canola,

corn, cotton, and soybean, (Bayer CropScience, 2011). Glufosinate is sold as standalone

formulation which can be tank mixed with one or more active ingredients depending

upon the crop and the weed spectrum.

Glufosinate acts in plants by inhibiting the enzyme glutamine synthase, causing a toxic

buildup of ammonia within the treated plant (Bayer, 2010). Glufosinate is a nonselective

herbicide and has no residual activity. This herbicide has a different mode-of-action than

the other major herbicides used in cotton.

I.4. Herbicide-Resistant Weeds and Resistance Management Strategies

The development of herbicide-resistant weeds is not a new phenomenon and resistance is

not limited to certain select he rbicides. In 1957, the first U.S. herbicide-resistant weed, a

spreading dayflower biotype resistant to 2,4-D , was identified in Hawaii (Heap, 2012a).

See Table VIII-4 for scientif ic names of weeds mentioned in Appendix I. Through

November 2011, there are approximately 80 individual weed species with known

herbicide-resistant biotypes to one or more he rbicides in the U.S. For example, there are

45 weed species resistant to ALS herbicides, 16 to ACCase inhibitors , 24 to photosystem

II inhibitors, and 13 to glycine herbicides (Heap, 2012b). Growers have been managing

herbicide-resistant weeds for decades with the use of alternative herbicides and/or

cultural methods such as til lage or crop rotation.

The occurrence of an herbicide- resistant weed biotype does no t end the useful lifespan or

preclude the effective use of the herbicide as part of an overall diversified weed

management system. The three herbicide cl asses with the highest number of resistant

species, ALS, ACCase and triazine herbicides, ar e still effectively used by growers today.

# Monsanto Company 12-CT-244U 602 of 620 

# It is important to distinguish herbicide resistance from herbicide tolerance. A herbicide

resistant weed is one in which there is an inherited ability of a plant to survive and

reproduce following exposure to a dose of herbicide normally lethal to the wild type

(WSSA, 2012). A herbicide-tolerant weed species is one that is naturally tolerant to a

herbicide, for example a grass species is not killed by the appli cation of a broadleaf

herbicide (WSSA, 2012). Furthermore, certain weed species, while neither resistant nor

tolerant, are inherently difficult to control with a particular herb icide, requiring more

careful herbicide use and weed management practices.

Since the first confirmed cases of herbicide resistance, research has been directed at

determining which practices are best for ma naging existing resistan ce situations and how

best to reduce the development of herbicide resistance. Resistance management practices

most often recommended by University/C ooperative Extension Service (CES) and

industry are: 1) use of multiple herbicide m odes-of-action in mixture, sequence, or in

rotation; 2) crop rotation; 3) use of cultural control measures such as tillage and time of

planting; and 4) use of the labeled herb icide rate at the recommended timing of

application (Gressel and Sege l, 1990; Beckie, 2006). Recent research by Beckie and

Reboud (2009) indicates that in some cases he rbicide mixtures offer a better management

option than rotating herbicides. Simultaneous ly using two herbicides with different

modes-of-action, each effective on the same weed species, significantly reduce the

probability of weeds developing resistance to either or both herbicides (Beckie and

Reboud, 2009). Crop rotation is also an effec tive method for resistance management due

to the fact that it fosters the use of add itional herbicide modes-of -action and, potentially,

use of additional cultural practices to ma nage weeds over time. The use of multiple

methods of weed control in a single locati on is the technical basis for management

programs to delay the development of resistance. This general concept has been referred

to as applying “diversity” within a crop or across a crop rotation (Beckie, 2006; Powles,

2008).

It is generally accepted that conservation tillage practices (minimum-till and no-till)

create environments where herbicide resistan ce is more likely to develop (Beckie, 2006).

This is primarily due to selection pressure put on weeds by herbicide use due to the

absence of tillage as a cultural weed manageme nt practice to supplement herbicide use.

However, this is not always the case. Legere et al. (2000) found that an increase in the

use of ACCase inhibitors in a conservation tillage system ( e.g. , aryloxyphenoxy

propionates and phenylpyrazolines herbicide families) did not result in an increased

incidence of wild oat populations resistan t to ACCase inhibitors. In conclusion,

conservation tillage practices should not be co nsidered a primary contributing factor to

the development of re sistance in all cases.

I.5. Characteristics of H erbicides and Herbicide Us e Influencing Resistance

While the incidence of weed re sistance is often associated with repeated applications of

an herbicide, the actual probability for the deve lopment of resistant populations is related,

in part, to the specific herb icide active ingredient, chemical family and the herbicide

group. Some herbicides are more prone to the development of re sistance than others

(Heap, 2012c). The graph in Figure I-1 illustra tes the global instances of weed resistance

# Monsanto Company 12-CT-244U 603 of 620 

# to various herbicide groups. The different slopes of observed resist ance are largely due

to the factors described above, which relate to the specific herbicide active ingredient as

well as to the group and herbic ide family and its function.

# Monsanto Company 12-CT-244U 604 of 620 

# Figure I-1. Weed Resistance to Various Herbicide Families 1



# As can be seen in Figure I-1, weed resistance to the synthetic auxi n group of herbicides

has been slower to develop than for other herbicide groups even though these were the

first synthetic herbicides discovered and used commercially. Possible reasons for this are

discussed below.

1 

# Global number of resistant biotypes



# I.6. Mechanisms of Resistance and Inheritance of Resistance

To date, the three known basic mechanisms by which weed species develop resistance to

a herbicide have been identified: 1) target site alteration (targe t site), 2) enhanced

metabolism of the herbicides (metabolis m), and 3) reduced absorption and/or

translocation of the herbicide such that the herbicide does not get to the site of action

within the plant cell (exclu sion) (Sammons et al., 2007).

Herbicide resistance via target site alteration is the mostcommon resistance mechanism

among the various herbicide groups and chemical families. It has been found that a target

site mechanism is the most common mechanism for ALS inhibitors, ACCase inhibitors,

# Monsanto Company 12-CT-244U 605 of 620 

# and triazines, but is less common for other herbicide groups , such as glyphosate (Powles

and Yu, 2010). The most common type of target site alteration is one where amino acid

substitution(s) occur in the protein that is the target of the herbicide such that the

alteration prevents the binding of the herbicide to the protein and as a result the activity

of the targeted protein is not alte red and the plant grows normally.

In the case of synthetic auxin herbicides, resistance has been speculated to be due to

mutation(s) in genes encoding an auxin-bi nding protein causing reduced herbicide

binding (Zheng and Hall, 2001; Goss and Dyer, 2003). In several studies, differential

herbicide absorption, translocation, and metabolism were ruled out as possible

mechanisms of resistance in kochia (Cranston et al., 2001) and in wild mustard (Zheng

and Hall, 2001). However, cu rrent research has not presen ted convincing evidence for a

single mechanism of resistance and this inability to elucidate the mech anism of resistance

may be due to a lack of thorough understand ing of the mechanism (mode) of action of

auxin herbicides (Jasieniuk et al., 1996). Wals h et al. (2006) identified seven alleles at

two distinct genetic loci that conferred significant resistance to picolinate auxins

(picloram) in Arabidopsis , yet had minimal cross-resistance to 2,4-D and IAA, a naturally

occurring plant growth regulator..

Multiple mechanisms for inheritance of dicam ba resistance have been reported in the

literature. Jasieniuk et al. (1995) reported results indicating that inheritance of dicamba

resistance in wild mustard is determined by a single, completely dominant nuclear allele.

However, Cranston et al. (2001) reported results indicating th at dicamba resistance in

kochia is determined by a quan titative trait (two or more genes). The slow development

of weed resistance to synthetic auxin herbic ides may in part be due to their proposed

multiple sites of physiological action in plan ts (Jasieniuk et al., 1996) and to the

possibility that inheritance, at least in some species, is determined by a quantitative trait

(Cranston et al., 2001).

Little is known about the resistance mechanis ms in glufosinate-resistant biotypes. AvilaGarcia and Mallory-Smith (2011 ) conducted an initial set of experiments to understand

the mechanism of resistance in the ryegrass population th at was also resistant to

glyphosate. They found that resistance was not du e to an insensitive or altered target site

and hypothesized that reduced translocation is responsible for the resistance to both

glyphosate and glufosinate in these populations.

I.7. Weeds Resistant to Dicamba and Glufosinate

As noted earlier, like other he rbicides, the use of dicamba ma y lead to the development of

dicamba-resistant weed species. To date, th ere are four species with known resistant

biotypes to dicamba in the U.S./Canada afte r over 40 years of us e: common hempnettle,

kochia, prickly lettuce, and wild mustard (Heap, 2012a). Additi onally, a population of

common lambsquarters has been confirmed to be resistant in New Zealand, for a total of

five species worldwide with confirmed resistant biotypes to dicamba. For the synthetic

auxin group of herbicides ther e exist a total of 29 species globally with biotypes having

confirmed resistance to at l east one member of this group, but only nine species in the

U.S. and four species in Canada (Heap, 2012a). All of these populations are, except for

# Monsanto Company 12-CT-244U 606 of 620 

# two (wild carrot in OH and MI, and waterhem p in NE), found in western states or

western Canadian provinces. In some weed species, cross-resistance between different

herbicides within the auxin group has been confirmed (pla nt cross-resistance to another

herbicide as a result of exposure to a si milarly acting herbicide). Therefore,

consideration has to be given to the possibility that dicambaresistance could extend to

some of the other broadleaf species listed as resistant to other synt hetic auxin herbicides

(Cranston et al., 2001; Jasieni uk et al., 1995; Miller et al., 2001). However, because of

differences in sites of action among the chemistry families within this group ( i.e. , benzoic

acids compared to pyridine-carboxylic acids) cross resistance between the herbicide

groups is not a certaint y (Monaco et al., 2002).

With the introduction of MON 88701 into gl yphosate-tolerant cotton systems, where

dicamba will be applied in combination with gl yphosate and glufosinate, it is important to

note that kochia is the only br oadleaf species with resistant biotypes to either synthetic

auxins or glyphosate. However, there are no known kochia biotypes resistant to both of

these herbicides or resistant to glufosinate. In additi on, the evolution of a dicambaglyphosate resistant biotype is unlikely because dicamba, glyphosate, and/or glufosinate,

each with a distinct mode-of-action, will li kely be applied in the same season to

MON 88701 in the glyphosate-tole rant cotton systems. If popul ations with resistance to

both glyphosate and dicamba herbicides were to occur, there are other herbicide options

for managing the weed in cotton ( e.g. , glufosinate, clomazone and flumioxazin) and in its

rotational crops ( e.g. , atrazine and isoxaflutole in corn) (Table I-1). The glyphosateresistant kochia biotype may be found in western cotton growing areas of Texas and

Oklahoma.

To date there are two weed species with confirmed resist ance to glufosinate: goosegrass

in Malaysia and Italian ry egress in Oregon, U.S. (H eap, 2012d). In the case of

goosegrass, the resistant populations evolved due to use of glufosinate in a rubber

plantation (Seng et al, 2010). In the case of Italian ryegrass, the resistance was actually

discovered in populations exposed to glyphosat e that evolved resistance to glyphosate

and which had not been exposed to glufosinat e; exemplifying a case of cross-resistance

(Avila-Garcia and Mallory-Smith, 2011). No re sistance in a broadlea f species has been

found to date.

Italian ryegrass may require special c onsideration when de signing appropriate

management programs because of the potenti al for cross resistance between glyphosate

and glufosinate to exist. Avila-Garcia and Mallory-Smith (2011) demonstrated the only

case of glufosinate cross resistance, whic h developed when the populations evolved

resistance to glyphosate. It is not known if the reverse is true, t hough it is possible.

Where there are known glyphosate resistan t ryegrass populations Monsanto will

recommend not to use glufosinate to control these populations. Likewise, dicamba will

not be an option, since it does not control grasses such as ryegrass. Other herbicides such

as those in the ACCase or ALS classes will be recommended. It is important to note that

ryegrass is generally a weed target in pr eplant burndown applications and not in the

cotton crop itself because of the biology of the species.

# Monsanto Company 12-CT-244U 607 of 620 

# I.8. Sustainable Use of Dicamba and Glufosi nate as a Weed Management Option in

Cotton

MON 88701 will be sold only in cotton varieties that also cont ain other herbicide-tolerant

traits, including glyphos ate-tolerance. Cotton varie ties containing both MON 88701 and

a glyphosate-tolerant system will enable dica mba and glufosinate to be applied with

glyphosate and/or other cotton herbicides in an integrated weed management program.

Dicamba primarily will be used in mixtures with either glyphosate or glufosinate or in

sequence with glyphosate or glufosinate to control a broad spectrum of grass and

broadleaf weed species. Glyphosate and glufosin ate will not be used in mixtures due to

antagonism ( i.e. , glufosinate damages the leaf tissue before glyphosate gets into the plant

and/or can be translocated to growing pa rts of the plant) and reduced efficacy of

glyphosate on susceptible weed species. Di camba and glufosinate applications on

MON 88701 will provide effective control of glyphosate-resistant broadleaf weeds and

improve the control of annual and perennial broadleaf weed species, some of which are

difficult to control with glyphosate. Dicam ba and glufosinate will also help delay

development and/or combat existing weed resi stance issues that can limit the use of the

PPO- and ALS-inhibiting herbicide groups by providing additional modes-of-action for

management of certain broadleaf species known to be prone to resistance to many of the

current herbicide options for weed management ( i.e.

, Amarathus spp.). Likewise,

dicamba will help to mediate potential evoluti on of resistance to glufosinate in broadleaf

species and glufosinate will do the same for th e potential evolution of resistant broadleaf

species to dicamba. Cultivation of a co mbined MON 88701 and glyphosate-tolerance

trait product will foster the a doption of Integrated Pest Ma nagement (IPM) practices in

cotton by allowing growers to continue to primarily focus on postemergence in-crop

weed control, as they have practiced with the glyphosate-tole rant cotton systems. This

will allow growers to delay some herbicide treatments until field scouting indicates a

need for additional postemergence weed control wh ich is consistent with the principles of

IPM, and also herbicide resi stance management practices. Increasing postemergence

herbicide options in cotton is important, esp ecially in conservation tillage situations,

where consistency of postemergence herbicides has generally been greater than that of

soil active residual products, which have greate r degree of inconsistent weed control, and

thus has been a factor in the adoption of c onservation tillage systems in the U.S.

Upon the integration of MON 88701 into th e glyphosate-tolerant cotton systems and

pending approval of the use of dicam ba on MON 88701 by the U.S. EPA,

preplant/preemergence applications of dicam ba can be made up to 1.0 lb a.e./acre up

through crop emergence (cracking) and in-c rop postemergence applications up to 0.5 lb

a.e./acre could be applied through 7 days preharvest, with the combined total not to

exceed 2.0 lbs a.e. dicamba per year for all appl ications. Residual herbicides also will be

recommended for use, to provide early seas on weed control and to supplement dicamba

and glufosinate activity on certain hard -to-control and glyphosate-resistant weed

biotypes, such as glyphosate- resistant Palmer amaranth where weed populations can be

very substantial. See section I.8.1 for specific weed management recommendations.

Dicamba and glufosinate, as complementary herbicides to glyphosate, will provide new

weed control options in cotton that strengthen the utility and sustainability of glyphosate

# Monsanto Company 12-CT-244U 608 of 620 

# as a weed control tool in th e glyphosate-tolerant cotton system s. Likewise, glyphosate, as

a complementary herbicide to dicamba and gluf osinate, would streng then the utility and

sustainability of dicamba and glufosinate as weed control tools for the combined

MON 88701 glyphosate-tolera nce trait product.

In the event there is known or suspected pres ence of a dicamba-resi stant or glufosinateresistant weed biotype, other options for managing the resistant biotypes are available to

the grower. There are multiple preemergence (including soil residuals) and postemergent

herbicide options for managing weed populations that are resistant or may potentially

develop resistance to dicamba or glufosinate in cotton, as well for crops grown in rotation

with cotton. These options are noted in Table I-1.Monsanto Company 12-CT-244U 609 of 620Table I-1. Management Recommendations for Control of Dicamba-, Glufosinate- and Other Selected Synthetic AuxinResistant WeedsWeed Species1Herbicide ResistantBiotypesPrimaryCrop CottonRotational CropsCorn Sorghum Soybeans WheatKochiadicamba, fluroxpyr(populations alsoresistant toglyphosate)AtrazineaAtrazineaSaflufenacilaSaflufenacilaClomazoneaSaflufenacilaSaflufenacilaClomazoneaGlyphosateaFlumioxaziniIsoxaflutoleaIsoxaflutoleaFlumioxazinaBromoxynil/MCPAaGlyphosateiMesotrioneaMesotrioneaGlyphosateaParaquatiGlyphosateaGlyphosateaParaquataPrickly LettuceDicamba, 2,4 D,MCPAGlyphosateiSaflufenacilaSaflufenacilaSaflufenacilaSaflufenacilaParaquatiAtrazineaAtrazineaChlorimuron/metribuzinaTriasulfuronaFlumioxaziniCarfentrazone+ atrazineaCarfentrazone +atrazineaGlyphosate +imazethapyraMetsulfuron +thifensulfuronaIsoxaflutole +atrazineaIsoxaflutole +atrazineaWild mustardDicamba , 2,4D,MCPA, picloram,dichlorprop,mecopropGlyphosateiGlyphosatecGlyphosatecGlyphosatecParaquatiAtrazinecAtrazinecChlorimuroncPrimisulfuroncPrimisulfuroncChlorimuron/metribuzincNicosulfurondNicosulfurondHalosulfurondHalosulfurondFieldBindweed2,4 DGlyphosateiGlyphosateaGlyphosateaGlyphosateaGlyphosateaParaquatiGlyphosate +imazethapyraGlyphosate +imazethapyraFlumioxaziniGlyphosate +ImazamoxaGlyphosate +ImazamoxaYellowStarthistleePicloramMonsanto Company 12-CT-244U 610 of 620Table I-1. Management Recommendationsfor Control of Dicamba-, Glufosinate-and Other Selected Synthetic AuxinResistant Weeds (continued)Weed Species1HerbicideResistant BiotypesPrimaryCrop CottonRotational CropsCorn Sorghum Soybeans WheatSpreadingDayflower2,4 DBentazon halosulfuronpenoxsulam bispyribacfLambsquartersgDicambaIsoxaflutoleaMetribuzinbBromoxynilaParaquatiAtrazineaCloransulambChlorsulfuron/MetsulfuronaFlumioxaziniSaflufenacilaSaflufenacilaGlyphosateaGlyphosatebMesotrioneaImazamoxbSaflufenacilaBromoxynilbGlyphosatebGoosegrassGlufosinateClethodimhClethodimhGlyphosatehGlyphosatehGlyphosatehGlyphosatehGlyphosatehpendimethalinhpendimethalinhpendimethalinhtrifluralinhtrifluralinhItalian ryegrassGlufosinate(populations alsoresistant toglyphosate)Metolachlor (fallapplied)hMetolachlor(fall applied)hMetolachlor (fallapplied)hMetolachlor (fallapplied)hClethodimhClethodimhGlyphosatehGlyphosatehGlyphosatehGlyphosatehGlyphosatehParaquatiMonsanto Company 12-CT-244U 611 of 6201Scientific names for each weed species can be found in Table VIII-4.aBernards et al., 2010.bLoux et al., 2010.cMSU, 2010.dKells and Stachler, 1997.ePNWE, 2010.fUniversity of Arkansas CES, 2010.gResistance to lambsquarters has onlybeen confirmed in New Zealand.hSteckel et al., 2011iSmith et al., 2012

# Monsanto Company 12-CT-244U 612 of 620 

# I.9. Stewardship of Dicamba and Glufosinate Use on MON 88701

In order to steward the use of agricultural herbicides and herbicide-tolerant cropping

systems such as the combined trait MON 88701 and glyphosate-tolerant cotton product,

Monsanto has conducted investigations and wo rked extensively with academics and other

herbicide manufacturers to understand and recommend best practices to manage

herbicide resistance. These investigations have demonstrated that one of the major

factors contributing to the development of resistant weed biotypes has been poor weed

control management practices. The primary reasons for lack of adequate management

includes: 1) application of he rbicides at rates below those indicated on the product label

for the weed species, and 2) sole reliance on a particular herbicide for weed control

without the use of other herbic ides or cultural control met hods (Beckie, 2006; Peterson et

al., 2007).

I.9.1. Weed Control Recommendations

The proposed label for dicamba use on M ON 88701 is based on the maximum allowable

use rates and patterns. Pr ior to launch of MON 88701 in glyphosate-tolerant cotton

systems, Monsanto, in cooperation with acad emics, will conduct trials to confirm the

optimum rate and timing for dicamba, glufosinate and glyphosate, alone and in

combination, and other herbicides. Recomme ndations to growers will be developed from

this information and will be provided in herbicide product labels, Monsanto’s

Technology Use Guide (TUG), and in other e ducation and training materials to be

broadly distributed. Specifically, current re search conducted by Monsanto to define the

optimum weed management systems support use recommendations that include the

application of dicamba and glyphosate for preemergence on conservation tillage acres

and early postemergence in-crop applicati ons. In some situations, a second in-crop

application of either dicamba tank-mixed with glyphosate or glufosinate, with or without

a soil residual will be recommended (see Section VIII.G.4 for additional details)

These recommendations will ensure more than one mechanism of action against the

targeted species, which is a fundamental component of a good weed resistance

management program. These management syst ems, which include the use of multiple

effective herbicide modes-of-action, will reduce the potential for further resistance

development to glyphosate, dicamba, and glufos inate, as well as other critical cotton

herbicides. Furthermore, the preplant weed spectrum is generally different from the incrop weed spectrum therefore multiple applic ations of glyphosate and dicamba are not

expected to increase selection pr essure on either herbicide.

I.9.2. Dispersal of Technical and Stewardship Information

Monsanto will use multiple methods to dist ribute technical and stewardship information

to growers, academics and grower advisors. Monsanto’s TUG will set forth the

requirements and best practices for the cultivation of MON 88701 including

recommendations on weed resistance manage ment practices. Growers who purchase

# Monsanto Company 12-CT-244U 613 of 620 

# varieties containing MON 88701 will be required to enter into a limited use license with

Monsanto and must sign and comply with the Monsanto Tec hnology Stewardship

Agreement (MTSA), which requires th e grower to follow the TUG.

The weed resistance management practices that will be articulated in the TUG will also

be broadly communicated to growers and retail ers in order to minimize the potential for

the development of resistant weeds. Thes e practices will be communicated through a

variety of means, including direct mailings to each grower purchasing a cotton variety

containing MON 88701, a public website 9 

# , and reports in farm media publications. The

overall weed resistance management program will be reinforced through collaborations

with U.S. academics, who will provide their recommendations for appropriate

stewardship of dicamba and gluf osinate in cotton production, as well as by collaboration

with crop commodity groups who have launche d web-based weed resistance educational

modules. Finally, Monsanto will urge grow ers to report any inci dence of repeated nonperformance of dicamba or glufosinate on weeds in fields planted with MON 88701, and

Monsanto will investigate cases of unsatisfact ory weed control to determine the cause as

defined in I.9.

The EPA is the U.S. federal regulatory agen cy that administers th e federal law governing

pesticide sale and use under the Federal In secticide, Fungicide and Rodenticide Act

(FIFRA). EPA encourages pesticide manufact urers to provide grow ers with information

regarding an herbicide’s mode-of-action to aid growers in planning herbicide use

practices and to foster the a doption of effective weed resist ance management practices as

specified by EPA in Pesticide Registration (PR) Notice 2001-5 (U.S. EPA, 2001). In that

document EPA states that “thi s approach to resistance ma nagement is sound and would

be highly beneficial to pest icide manufacturers and pesticid e users.” EPA approves all

pesticide label use instructions based on its evaluation of supporting data supplied by the

pesticide registrant or manuf acturer. By approving a label, EPA has concluded that the

product will not cause unreasonable adverse eff ects to the environment when used in

accordance with the label’s dire ctions. After EPA approves a pesticide label, it is a

violation of federal law to use the pesticid e for a use or in a manner not in accordance

with the label directions. Monsanto in corporates EPA’s guidelines for pesticide

resistance management labeling on its agricultural herbicide labels, and will continue to

do so in the future. Monsanto will adopt a similar approach to pesticide resistance

management guidance on its dicamba product labels.

In summary, Monsanto will require weed re sistance management practices through the

MTSA and TUG for its biotechnology-derive d herbicide-tolerant products, such as

MON 88701 integrated into the glyphosate-tole rant cotton systems, and to promote these

practices through product labeling and educationa l outreach efforts as an effective means

to manage weed resistance development for both dicamba, glufosinate, and glyphosate.

9 

# http://www.monsanto.com/weedmanagement/Pages/default.aspx

# Monsanto Company 12-CT-244U 614 of 620 

# I.9.3. Weed Resistance Management Practices

Monsanto will provide information to grower s and grower advisors on best management

practices to delay the development of resistan ce to dicamba and glufosinate. The weed

resistance management reco mmendations for the use of dicamba and glufosinate in

conjunction with cotton varieties containi ng MON 88701 will be consistent with the

Herbicide Resistance Action Committee’s guid elines for prevention and management of

herbicide resist ance (HRAC, 2010) 10 

# . These guidelines reco mmend an integrated

approach to weed resistance mana gement, including crop management ( i.e. , cover crops,

crop rotation, etc.), cultivation techniques, and the use of multiple herbicide modes-ofaction to manage a weed population.

In cases where resistance is confirmed fo r dicamba or glufosinate in cotton producing

areas, Monsanto and University/Cooperative Extension Service (CES) personnel will

provide recommendations for alternative herb icide control methods to growers. These

recommendations would be made availabl e through Monsanto supplemental labels,

Monsanto and university publications, and internet sites to growers, consultants, retailers

and distributors. For all ex isting cases of dicamba-resist ant and glufosinate-resistant

weeds in the U.S. and globally today, altern ative herbicides and cultural methods are

available to growers to eff ectively control these biotypes. Examples of recommended

alternative herbicides from University/CES pe rsonnel that are applicable to weed species

known to be resistant to glufosinate, dicamba and other synthetic auxin herbicides are

found in Table I-1. However, th ese examples in Table I-1 ar e only a subset of product

combinations of available cotton herbicides.

I.10. Monsanto Weed Performance Evalua tion and Weed Resistance Management

Plan

An important part of a weed resistance mana gement plan is the timely acquisition of

information regarding product performance. M onsanto has an extens ive technical, sales

and marketing presence in the cotton markets where MON 88701 will be grown.

Through our relationships with farm advi sors, key University/CES personnel, and

growers using our seeds and tr aits products, Monsanto will acquire important and timely

information regarding product performance. This will allow the timely recognition of

performance issues that could arise related to weed resistance or other means. Field

employees and hired consultants are traine d and provided processes for responding to

product performance inquiries. Individual perf ormance issues that could be related to

potential resistance are promptly handled. In addition performance inquiries are

periodically reviewed by Mons anto for trends that could indicate the need for follow up

action on a broad scale.

If dicamba or glufosinate resi stance is confirmed, the scientific and grower communities

will be notified and a weed resistance mitigation plan will be implemented by Monsanto

10 

# The Herbicide Resistance Action Committee (HRAC) is an international body founded by the

agrochemical industry for the purpose of suppor ting a cooperative approach to the management of

herbicide resistance and the establishment of a worldwide herbicide resistance database.

# Monsanto Company 12-CT-244U 615 of 620 

# in cooperation with the Univer sity/CES and/or the appropria te herbicide producer . The

mitigation plan will be designed to manage the resistant biotype through effective and

economical weed management recommendations implemented by the grower. The scope

and level of intensity of the mitigation plan may vary depending on a combination of the

following factors: 1) biology a nd field characteristics of the weed (seed shed, seed

dormancy, etc.), 2) importance of the weed in the agricultural system, 3) resistance status

of the weed to other herbicides with altern ate modes-of-action, and 4) availability of

alternative control options. These factors ar e analyzed by Monsanto and University/CES

personnel in combination with economic and practical management considerations to

develop a tailored mitigation strategy. The plan considers what is technically appropriate

for the particular weed and incorporates pr actical management strategies that can be

implemented by the grower.

After a mitigation plan is developed, Monsan to communicates the plan to the grower

community through the use of supplemental herbicide labeling (labeling which includes

newly approved use directi ons, or other instructions) 11 

# , informational fact sheets, retailer

training programs, agriculture media a nd/or other means, as appropriate.

In addition to the grower inquiry initiated pr ocess, Monsanto, alone and/or in cooperation

with University/CES, will conduct field st udies to understand the potential for weed

resistance and weed shifts as the resu lt of various weed management programs

implemented for MON 88701 integrated into gl yphosate-tolerant cotton systems. These

studies will allow researchers to better tr ack specific factors that can influence the

development of resistance to specific weeds.

I.11. Summary

Development of weed resistance is a complex process that can be difficult to accurately

predict. Multiple methods for managing we ed resistance are available and no single

option is best for all farming situations. No single agronomic practice will mitigate

resistance for all herbicides or all weeds. As a result, weed resistance needs to be

managed on a case-by-case basis, tailored for the particular herbicide and weed species,

and utilize an integrated system approach to meet grower needs. Using good weed

management principles, built upon achievi ng high levels of c ontrol through proper

application rate, choice of cultural practic es, and appropriate companion weed control

products will allow dicamba and glufosinate herbic ides to continue to be used effectively.

In cases where weed populations have evolved or developed re sistance to dicamba and/or

glufosinate, effective management options are available and experience has shown that

growers will continue to find value in us ing dicamba and glufosinate in their weed

control programs.

The key principles for effective stewardship of dicamba and glufosinate use, including

the integration of MON 88701 in the glyphosa te-tolerant cotton systems, comprise:

11 

# Monsanto will communicate information broadly so re gistrants are aware of when Monsanto is not the

registrant or provider of the chemistry,.

# Monsanto Company 12-CT-244U 616 of 620 

# 1) basing weed management and weed resi stance management practices on local needs

and using the tools necessary to optimize cr op yield, 2) using proper rate and timing of

application, 3) not relying solely on one he rbicide weed control option across a cropping

system, 4) responding rapidly to instances of unsatisfactory weed control, and

5) providing up-to-date weed management and weed resistance management training.

Overall, there is a low potential for dicamba-re sistant broadleaf weed populations to arise

from the use of dicamba applied to MON 88701 integrated into glyphosate-tolerant

cotton systems. The reasons are as follows:

 Dicamba will be used in combination w ith glyphosate and/or glufosinate in a

majority of cropping situations, and weed recommendations will also include the

concurrent use of residual herbicides for complementary weed control and

different modes-of-action. These use patterns mean that there will be multiple

modes-of-action against the major broadleaf species present in cotton production.

This is a primary way to delay the development of resistance.

 The development of resistance to auxin he rbicides has been f ound to be relatively

slow. This observation is hypothesized to be due to multiple site s of action within

plants and evidence suggesting that resistance is determined by multiple genes

(quantitative traits), at least in some species.

 Only four broadleaf weed species have b een confirmed to be resistant to dicamba

in the U.S., and relatively low numbers of broadleaf species have been confirmed

to be resistant to synthetic auxin herbic ides even though dicamba has been widely

in use for over 40 years.

 Known resistant broadleaf populations to dicamba and other auxin herbicides are

primarily found in the western U.S. and, thus, are not present in the major cotton

geographies. In addition, the known dicamba-resistant biotypes are not major

weed species present in the U.S. cotton crop.

Likewise, the probability for weed species to evolve resistance to gl ufosinate as a result

of glufosinate use in the MON88701 system is considered to be low because:

 Two species have been confirmed to be resistant to glufosinate worldwide and

one (ryegrass) in the US. This suggests that the frequency for resistant alleles in

native weed populations is fairly low.

 Known resistant populations to glufosinat e herbicide within the U.S. are only

found in Oregon, and thus, are not presen t in the major cotton geographies.

 In the MON 88701 system, glufosinate will likely be used in combination with

dicamba and in sequence with glyphosate. Residual herbicides will also be

recommended and likely used in this cropp ing system. As noted above, these use

patterns mean that there will be multip le modes-of-action against the major

broadleaf species present in soybean produc tion. This is a primary way to delay

the development of resistance.

# Monsanto Company 12-CT-244U 617 of 620 

# References for Appendix I

Ahrens, W.H. 1994. Dicamba. 3,6-dichloro-2-methoxybenzoic acid. Pages 91-94 in

Herbicide Handbook. Seventh Edition. Weed Sc ience Society of America, Champaign,

Illinois.

Avila-Garcia, W.V. and C. Mallory-Smith. 2011. Glyphosate-re sistant Italian ryegrass

( Lolium perenne ) populations also exhibit resistan ce to glufosinate. Weed Science

59:305-309.

BASF. 2008. Clarity herbicide label. BASF Corporation, Res earch Triangle Park, North

Carolina.

Bayer CropScience. 2011. Liberty herbicide label. Bayer CropScience LP, Research

Triangle Park, North Carolina.

Beckie, H.J., and X. Reboud. 2009. Selecting for weed resistance: He rbicide rotation and

mixture. Weed Technology 23:363-370.

Beckie, H.J. 2006. Herbicide-resistant weeds: Management tactics and practices. Weed

Technology 20:793-814.

Bernards, M.L., R. E.Gaussoin, R.N. Hliei n, S.Z. Knezevic, D.J. Lyon, L.D. Sandell and

R.G Wilson. 2010. Guide for weed management in Nebraska (EC130). University of

Nebraska Extension, Lincoln, Nebraska.

Cranston, H. J., A. J. Kern, J. L. Hackett, E. D. Maxwell, B. D. Maxwell, and W. E.

Dyer. 2001. Dicamba resistance in ko chia. Weed Science 49:164-170.

Goss, G.A., and W. E. Dyer. 2003. Physiologi cal characterization ofauxinic herbicideresistant biotypes of Kochia ( Kochia scoparia ). Weed Science 51:839-844.

Gressel, J. and L. A. Segel. 1990. Modelling the effectiveness of herbicide rotations and

mixtures as strategies to delay or preclude resistan ce. Weed Technology 4:186-198.

Grossmann, K. 2010. Auxin herbicid es: current status of mech anism and mode of action.

Pest Management Science 66:113-120.

Heap, I. 2010. International survey of herb icide resistant weeds. WeedScience.com,

International Survey of Herbicide Re sistant Weeds, Corvallis, Oregon.

http://www.weedscience.org/in.asp [Accessed July 1, 2010]

Heap, I. 2011. Herbicide resistant weeds summar y table. WeedScience.com, International

Survey of Herbicide Resistan t Weeds, Corvallis, Oregon.

http://www.weedscience.org/summary/ MOASummary.asp [Accessed May 26, 2011].

# Monsanto Company 12-CT-244U 618 of 620 

# Heap, I. 2012a. Herbicide resi stant weeds - Synthetic auxi ns (O/4) resistant weeds.

WeedScience.org, International Survey of Herbicide Resist ant Weeds, Corvallis, Oregon.

http://www.weedscience.org/summary/M OASummary.asp [Accessed June 11, 2012].

Heap, I. 2012b. U.S. Herbicide resistant weeds summary table. WeedScience.org,

International Survey of Herbicide Resistant Weeds, Corvallis, Oregon.

http://www.weedscience.org/summary/M OASummary.asp [Accessed June 25, 2012].

Heap, I. 2012c. Herbicide resist ant weeds summary table. WeedScience.org, International

Survey of Herbicide Resistan t Weeds, Corvallis, Oregon.

http://www.weedscience.org/summary/M OASummary.asp [Accessed June 15, 2012].

Heap, I. 2012d. Herbicide resist ant weeds - Glutamine synt hase inbibitors (H/10)

resistant weeds by species and country. W eedScience.org, International Survey of

Herbicide Resistant Weeds, Corvallis, Oregon.

http://www.weedscience.org/summary/M OASummary.asp [Accessed June 11, 2012].

HRAC. 2009. Classification of herbicides according to mode of action. Herbicide

Resistance Action Committee, Corvallis, Oregon.

http://www.hracglobal.com/Publications/Cla ssificationofHerbicideModeofAction/tabid/2

22/Default.aspx Accessed June 29, 2009

.

HRAC. 2010. Guideline to the management of herbicide resistance. Herbicide Resistance

Action Committee, Calgary, Alberta. http://www.hracglobal.com Accessed April 1,

2010

.

Jasieniuk, M., I.N. Morrison, and A.L. Br ûlé-Babel. 1995. Inhe ritance of dicamba

resistance in wild mustard ( Brassica kaber ). Weed Science 43:192-195.

Jasieniuk, M., A.L. Brûlé-Babel, and I.N. Morrison. 1996. The evolution and genetics of

herbicide resistance in weeds. Weed Science 44:176-193.

Kells, J.J. and J.M. Stachler. 1997. Controlli ng wild carrot. Michig an State University

Extension, East Lansing, Michigan. http ://web1.msue.msu.edu/imp/mods1/fact9707.html

[Accessed May 19, 2010].

Legere, A., H.J. Beckie, F.C. Stevenson, a nd A.G. Thomas. 2000. Survey of management

practices affecting the o ccurrence of wild oat ( Avena fatua ) resistant to acetyl-coA

carboxylase inhibitors. Weed Technology 14:366-376.

Loux, M.M., D. Doohan, A.F. Dobbels, W.G. Johnson, G.R.W. Nice, T.N. Jordan and

T.T. Bauman. 2010. Weed control guide for Ohio and Indiana. Bulletin 789. Purdue

University and Ohio State University Extension, West Lafayette, Indiana.

# Monsanto Company 12-CT-244U 619 of 620 

# MSU. 2010. Controlling wild car rot. Mississippi State Univer sity Extension Service,

Mississippi State, Mississippi.

http://fieldcrop.msu.edu/documents/contro lling%20wild%20carrot.pdf [Accessed May

19, 2010].

Miller, T.W., S.L. Shinn, and D.C. Thill. 2001. Cross-resistance in and chemical control

of auxinic herbicide-resistant yellow starthistle ( Centaurea solstitialis ). Weed

Technology 15:293-299.

Monaco, T.J., S.C. Weller and F.M. Asht on. 2002. Growth regulator herbicides. Pages

291-310 in Weed Science: Principles and Pr actices. Fourth Edition. John Wiley & Sons,

Inc., New York, New York.

NPIC. 2002. Dicamba technical fact sheet. National Pesticide Information Center,

Corvallis, Oregon. http://npic.orst.edu/fact sheets/dicamba_tech.pdf [Accessed April 30,

2010]

PNWE. 2010. Control of problem weeds - Starthistle. Pacific Northwest weed

management handbook. Pacific Northwest Extension Service, Portland, Oregon.

http://uspest.org/pnw/wee ds [Accessed May 25, 2010]

Peterson, D., B. Olson, K. Al-Khatib, R. Currie, J.A. Dille, J. Falk, P. Geier, D. Regehr,

P. Stahlman and C. Thompson. 2007. Glyphos ate stewardship. MF-2767. Kansas State

University Agricultural Experiment St ation and Cooperative Extension Service,

Manhattan, Kansas.

Powles, S.B. 2008. Evolved glyphosate-resist ant weeds around the world: Lessons to be

learnt. Pest Manageme nt Science 64:360-365.

Powles, S.B. and Qin Yu. 2010. Evolution in action: Plants resistant to herbicides.

Annual Review of Plant Biology 61:317-347.

Sammons, R.D., D.C. Heeri ng, N. DiNicola, H. Glick, and G.A. Elmore. 2007.

Sustainability and stewardship of glyphos ate and glyphosate-resistant crops. Weed

Technology 21:347-354.

Seng, C.T., L. Van Lun, C.T. San and I. Bi n Sahid. 2010. Initial repor t of glufosinate and

paraquat multiple resistance that evolved in a biotype of goosegrass ( Eleusine indica ) in

Malaysia. Weed Biology and Management 10:229-233.

Smith, K. 2012 Recommended chemicals for w eed and brush control. University of

Arkansas, Cooperative Extension Service, Division of Agriculture, Fayetteville,

Arkansas. http://www.uaex.edu/Other_Areas /publications/HTML/MP-44.asp [Accessed

June 22, 2012]

# Monsanto Company 12-CT-244U 620 of 620 

# Steckel, L.E. 2011. Weed control manual fo r Tennessee. University of Tennessee

Institute of Agriculture, Knoxville, Tennessee. http://weeds.utk.edu/ [Accessed June 22,

2012]

U.S. EPA. 2001. Pesticide Registration (PR) Notice 2001-5, Guidance for Pesticide

Registrants on Pesticide Resist ance Management Labeling.

http://www.epa.gov/opppmsd1/ PR_Notices/pr2001-5.pdf.

U.S. EPA. 2008. Glufosinate summary docum ent registration review: Initial docket,

March, 2008. U.S. Environmental Protection Agency, Washington, D.C.

U.S. EPA. 2009. Reregistration Eligibility Decision (RED) for Dicamba and Associated

Salts. Case No. 0065. U.S. Environmental Protection Agency Office of Prevention,

Pesticides and Toxic Substances, Speci al Review and Reregistration Division,

Washington, D.C.

University of Arkansas. 2011. Recommended chemicals for weed and brush control.

University of Arkansas, Cooperative Exte nsion Service, Division of Agriculture,

Fayetteville, Arkansas.

Walsh, T.A., R. Neal, A.O. Merlo, M. Honma, G.R. Hicks, K. Wolff, W. Matsumura, and

J.P. Davies. 2006. Mutations in an auxin re ceptor homolog AFB5 and in SGT1b confer

resistance to synthetic pico linate auxins and not to 2,4- dichlorophenoxyacetic acid or

indole-3-acetic acid in Arabidopsis . Plant Physiology 142:542-552.

WSSA. 2012. WSSA lesson module: Herbicide re sistant weeds. Weed Science Society of

America, Champaign, Illinois. http://www. wssa.net/LessonModules/h erbicide-resistantweeds/index.htm [Acce ssed June 23, 2012].

Zheng, H. and J.C. Hall. 2001. Understanding auxinic herbicide re sistance in wild

mustard: physiological, biochemical, and mo lecular genetic approaches. Weed Science

49:276-281