Petition for Determination of Nonregulat e d Statu s fo r Cor n Root w o r m Protected

and Glyphosate Tolerant MON 87411 Mai z e

The undersigned sub m its this petition under 7 CFR § 340.6 to request that the

Ad m inistrator m ake a deter m ination that the article should not be regulated under

7 CF R par t 340

October 4, 2013

OECD Unique Identifier: MON

- 87411

- 9

Monsanto P etition Number: CR240

- 13U1

Sub m itted b y:

John M. Cordts, M.S., M.B.A

Monsanto C o mpany

800 North Lindbergh Blvd.

St

. Louis

, M O 63167

Phone: (314) 694

- 4831

Fax: (314) 694

- 3080

E

- m ail: John.M.Cordt s @ m onsanto.com

Prepared b y :

A. Ah m ad, Ph.D., P.M. Bach m an, Ph.D., B.A. Co m stock, B.S.,

J.M

. Cordts

, M.S.

, M.B.A.

, S

. Dubel m an

, Ph.D.

, T.H

. Klu s meyer

, Ph.D.,

D.K. Kovalic, Ph.D., C.A. Lawrence, Ph.D., J.S. Petrick, Ph.D., Q. Tian, M.D., Ph.D.

Contributors and/or P rincipal Investigators:

K. Adu

- tutu, Ph.D., S. Arackal, M.S., D. Anstrom, M.S., A. Beyene, Ph.D.,

J. Bynu m

, Ph.D., S.M. Carleton, Ph.D., D.B

. Cars on

, Ph.D.

, M

. Chen

, Ph.D.,

K. Crowley

, Ph.D.

, S

. Drury

, B.A.

, J

. Fischer

, Ph.D.

, R

. Hile m a n

, Ph.D.,

K

. Howard

, Ph.D.

, T

. L e e

, Ph.D.

, S.L

. Levine, Ph.D., G. Mueller, M.A.,

M

. Paradise

, M.A.

, E

. Parks

, Ph.D.

, B

. S a m m on s

, Ph.D

. A

. S i lvanovich

, P h.D.,

Z. Song, M.S., J. Tan, Ph.D., J. Uff m an, B. S

., W. Urqhart, Ph.D., J.M. Ward, Ph.D.,

B.J. W arner, M.S., J. W arren, B.S., F. Zapata, M.S.

M ons a n t o Co m pany CR 2 4 0

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R E L E A S E O F INFORMATION

Monsanto is sub m itting the in f or m ation in this petition f or review by the USDA as part of

the regulatory process. Monsanto under stand s tha t th e USD A complie s wit h the

provisio n s o f the Freed o m of Info r m ation Act (FOIA). In t h e event t h e USDA receives a

FOI A request

, pursuan t t o 5 U.S.C.

, § 552

, an d 7 CF R Par t 1

, coverin g al l o r so m e o f the

infor m ation in this petition, Monsanto expec t s that, in advance of the release of the

docu m ent(s)

, USD A wi l l provid e Monsant o wit h a copy of the m aterial proposed to be

release d an d th e opportunit y t o objec t t o th e releas e of any infor m ation based on

appropriate legal grounds, e.g., responsiven e ss, confidentiality, and/or co mpetitive

concerns. Monsanto understands that a CBI

- d eleted copy of this infor m ation may be

m ade available to the public in a reading room and upon individual request as part of a

public co mm ent period. Monsanto also under s tands that w hen dee m ed co mplete, a copy

of the petition m ay be posted to the USDA

- APHI S BR S webs it e o r othe r U.S.

govern m ent websites (e.g., www. r egulations.gov). Except in accordance with the

foregoing, Monsanto does not a u thorize the release, publicatio n o r othe r distributio n of

this in f or m ation witho u t Monsa n to ' s prior notice and consent.

© 2013 Monsanto Company. All Rights Reserved.

This document is protected under national and international copyright law and treaties.

This docu m ent and a n y acco mpanying m aterial are for use only b y the reg u latory

autho r ity to which it h as been s u b m itted by Monsanto Co m pany and its a ff iliates,

collectively “Monsanto Co m pany”, and only in support of actions r e quested by Monsanto

Co mpany. Any other use, c opying, or trans m ission, includ i n g interne t posting

, o f this

docu m ent a nd the m aterials described in or acco mpanying this docu m ent, without prior

consent of Monsanto C o mpany, is strictly prohibited; except t h at Monsanto Co mp any

hereby grants such consent to th e regulator y authorit y wher e required under applicable

law or regulation. The intellectual property, in f or m ation and m aterials described in or

acco mpanying this doc um ent are owned by Monsanto Company, which has filed for or

been g r ant e d pate n ts o n those m a te r ials. By sub m itting this doc um ent and any

acco mpanying m aterial s , Monsanto Co mpany does not gra n t any party o r entity any right

or license to the infor m a tion, m aterial or i n tellectual property des cribe d o r containe d in

this sub m ission.

M ons a n t o Co m pany CR 2 4 0

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CERTIFICATION

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

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

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

the petition.

John M. Cordts, M.S., M.B.A.

Regulatory Affairs Manager

Monsanto Company

800 North Lindbergh Blvd., Mail Stop C3SD

St. Louis, MO 63167

Tel: (314)

- 694

- 4831

Fax: (314)

- 694

- 3080

Monsanto Company CR240

- 13Ul 3 of372CB I Delete d Copy

EXECUT I V E SUM M ARY

The Ani m al and Plant Health Ins p ection Ser v ic e (APHIS ) o f th e Unite d State s (U.S.)

Depart m ent of Agriculture (USDA) has res p o nsibility u n der the Pl a nt Prote c ti o n Act

(Titl e I V Pub

. L

. 106

- 224

, 11 4 Stat

. 438

, 7 U.S.C

. § 7701

- 7772 ) t o preven t the

introduction and disse m ination of plant pests into the U.S. APHIS regulation

7 CF R § 340. 6 provide s tha t a n applican t m ay petition APHIS to evaluate sub m itted data

to deter m ine that a particular regulated art i cle does not p resent a p l ant pest risk and no

longer should be regulated. If APHIS deter m ines that the regulated article does not

present a plant pest risk, the petition is g r anted, thereby allowing un r estri c ted introd u ction

of the article.

Monsanto Co mp any is sub m itti n g this req u est to APHIS f or a deter m ination of

nonregulated status for the new biote c hnology

- derived m aize p roduct

, MO N 87411

, any

progeny derived from c rosses bet w een MO N 8741 1 an d conventiona l m a ize

, an d any

progen y derive d fro m crosse s o f MO N 8741 1 with biotechnology

- derived m aize that

hav e previousl y bee n grante d nonre gulated status under 7 CFR part 340.

Product Description

Monsanto Co m pany has developed biot echnology

- derive d m a ize

, MO N 87411

, that

confers protection against corn rootwo r m (CR W ) ( Diabrotica sp p .) a n d tolera n ce to the

herbicid e glyphosate

. MO N 8741 1 contain s a suppressio n c a ssett e tha t expresse s an

inverted repeat sequence designed to m at c h the sequence of western corn rootwo r m

(WCR ; Diabrotica virgifera virgifer a ). The expression of the suppression cassette results

i n th e formatio n o f a double

- strande d R N A (dsRNA ) transcrip t containin g a 24 0 bp

frag m ent of the WCR Snf7 gen e (DvSnf7)

. Upo n con s umption

, th e plant

- produced

dsRNA in MON 87411 is rec o gnized by the CR W ’s RNA interference ( R NAi)

m achinery resulting in down

- regu la tion of the targ e ted D v Sn f 7 gene leading to C RW

mortality. MON 87411 also contains a cry3B b 1 gene that produces a modified Bacillus

thuringiensis (subsp. kumamotoensis ) Cry3Bb1 protein to protect against CRW larval

feeding. In addition, MO N 8741 1 contain s th e cp4 epsps gen e fro m Agrobacteriu m sp.

strai n CP 4 tha t encode s fo r th e 5

- enolpy ruvylshiki m ate

- 3

- phosphate synthase (EPSPS)

protein, which confers tolerance to glypho s ate, the active ingredien t i n Roundup ®

agric u lt u ral herbi c ides.

MO N 8741 1 build s upo n th e curren t B t protein

- base d m ode

- of

- actio n (MOA ) fo r CRW

control by the addition of a n e w R NA

- m e diate d MO A tha t o ffers enhanced control of

target insect pests and prolonged durability o f existing B t technologies designed to

contr o l CRW. MON 87411 will provide benefits to growers si m ilar to those obtained by

use of existing CRW

- protec te d m a iz e hybrids

, whic h includ e reduce d nee d for

inse c ti c ides and asso c iated i m prove m ents in worker sa fe t y

, incr e ased yield protection,

and water conservation. MON 87411 is also glyphosate t o lera n t and will contin u e to

® R o un d u p an d Ro u nd up Rea dy a re r e g istere d tr a d e m ark s o f Monsan t o T echnolog y LL C

M ons a n t o Co m pany CR 2 4 0

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provid e benefit s associate d wit h conservatio n tillage m ethods, including reduced soil

erosion, reduced fuel and labor costs, i mproved air quality and conservation of soil

moisture.

MON 87411 will not be offered for com m ercial u se as a stan d

- alone p ro d uct, but will be

co m bined, t hrough traditional breeding m e t hods, with other deregulated biotechnology

derived traits to provide protection against both above

- g round and below

- ground maize

pests as w e ll as t o le r an c e to m ultiple herbicide s

. These n e xt gene r ati o n co m bined

- tr a it

m aize products will offer broader grower choice, i m proved production efficiency,

increased pest control durability, and enhanced grower profit potentials.

Data and I n formation Presented Confirms t h e Lack of Plant Pest Potential and the

Food and Feed Safety of MON 87411 Compared to Conventional Maize

The data and infor m ation presented in t h i s petitio n demonstrat e tha t MO N 8741 1 is

agrono m ically, phenot y pically, a n d co m positionally c o mparable to commercially

cultivated maize. Moreover, the data and i n for m ation presented herein de m onstrate that

MO N 8741 1 i s no t expecte d t o pos e a n increase d plant pest risk, i n cludin g weediness,

co mpared to com m ercially c u lti v ated m aize. Th e food

, feed

, an d enviro nm enta l safet y of

MON 87411 was confir m ed based on m ultiple, well

- e s ta b li s hed lines of evidence:

• The CP4 EPSPS protein in MON 87411 is identical to the C P 4 EPSPS protein present

in several other com m ercially a v ailable c r ops t h at ha v e been reviewed b y USDA and

previously deregulated ( e.g

. , Roundu p Read y varietie s o f soybean, m aize, cotton,

sugarbeet, canola, and alfalfa). The safety an d mode

- of

- actio n o f CP 4 EPSP S proteins

i s wel l docu m ente d an d i s th e subjec t o f nu m erous publications. Si m ilarly, the safety

of the Cry3Bb1 protein has been previously assessed in two other corn rootwor m

protected products (M O N 863 and MON 88017) that have been grown on tens of

m illions of acres in the U.S. since t h eir intr o duction. T he m ode

- of

- action of Bt

protein s ha s als o bee n e xten s ively studied and is w e ll

- docu m ented in nu m erous

publications.

• Th e RNA

- base d suppressio n o f th e Snf7 gene i n w estern c orn ro o t w o r m that resu l ts

from expression of the DvSnf7 suppress i on cassette in MON 87411 is m ediated by

dsRN A m o lecules

. D ouble

- strande d RNA s are com m only used by eukaryotes,

inclu d ing plants, for e n dogenous g ene suppre s sion and as descri b ed in this p etiti o n,

pose no novel risks from a feed/food and env i ron m ent perspective. Nucleic acids, as

the co mponents of RNA, have a long history of safe consumption and are considered

GRA S b y t h e U.S

. FDA.

• A co m positional assess m ent supports t h e conclusion that MON 87411 grain and

forage are equivalent to grain a nd forage of conventional m aize.

• Evaluation of the agrono m ic and pheno typi c characteristic s o f MO N 87411

, using

current m aize cultivation and m a nage m ent practices, leads to the conclusion that

deregulation of MON 87411 would not have an effect on m aize agrono m ic practices.

M ons a n t o Co m pany CR 2 4 0

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Mai z e is a Familiar Crop Lacki n g Weedy Characteristics

Maize is grown extensively t h roughout the w orld, and is t h e largest cultivated crop

followed by wheat ( Triticu m sp.) and rice ( Ory z a sativa L.) in t o tal g lo b al prod u cti o n. I n

the U.S., maize is g rown in al m ost all stat e s a n d is the l a r g est crop gr o w n in ter m s of

acrea g e planted and net value. Maize has been studied exten s ively, and the

do m estication of m aize can be traced back to approxi m ately 10,00 0 year s ag o i n southern

Mexico.

Maize is not listed as a weed in the m ajor lite r ature re f e r en c es on weed s

, nor is it p r esent

on the lists of noxious weed species publish e d by the federal govern m ent (7 CFR Part

360). In a d dition, m aize has been g rown th r ou g hout the world witho u t any report that it

is a serious weed. Maize is poorly suited to survive witho u t hu m an assistance and is not

capable of surviving as a weed d u e to p ast s e lection in the do m esti c ation of m a ize.

During domestication of m aize, traits often a s sociated with weedines s

, such as s eed

dor m ancy, a dispersal m echani s m

, or the a b ility to establish reproducin g populations

outside of c ulti v ation, h ave not been selected. S i m ilarly, t h e history of h y brid b ree d i n g in

the U.S. does not indicate there are any changes in the charact e ristics of m aize that would

chang e th e weedines s profil e o f th e crop

. Although m aize seed can overwinte r int o a

rotation with soybeans and other crops, m echa n ical and che m ical m easures are ro u tinely

used to control m aize volunte e rs. So m e populations of wild annual and perennial species

that could hybridize with MON 87411 are kno w n to exist in the U.S., however key

differences in several factors such as flowering ti m e, geograph i cal separation, and

develop m ent ti m ings m a ke natural cr o sses in the U.S. highly unlikely.

Conventional Mai z e MPA640B and NL61 69 are Appropriate Comparators for

MON 87411

Based on s e ed av a ila b ility and the a ppropri a te f it for various studies, conventiona l control

m aterials w ere develo p ed for use as co m pa r ators in safety assess m ent stu d ies. The

conventiona l contro l m a terial s include d th e original transfor m at i on line (LH244) and two

hybrid conventional control lines (hybrids MPA640B and/or NL6169), both of wh i ch

have si m ilar genetic backgrounds to the hyb r id MON 87411 test m aterial (LH244 is one

parent of each of the control hybrids). Both MPA640B (LH244 × LH287) and NL6169

(LH244 × HCL645) were used as controls in m o lecular characterization studies. NL6169

was used as the conventional control in co mpositional ana lysi s studie s whil e MPA640B

was used as the conventional control in phenotypic, agrono m ic and environ m ental

interactions assess m ents. W here appropri a te, commercial reference m aize hybrids were

use d t o establis h a rang e o f varia b ility or resp o nses re p res e ntative of c o m m ercial m aize

(re f ere n ce h ybrids) in t h e U.S.

Molecul a r Characteri z ation Veri fi ed the Integrity and Stability o f the Inserted DNA

in MON 87411

MON 87411 was developed through Agrobacteriu m

- m ediated trans f ormation of m a ize

im m ature embryos from line LH244 u tilizing plas m i d vector PV

- ZMIR10871. PV

ZMIR1087 1 contain s on e transfe r DN A (T

- DNA ) that is delineated by Left and Right

M ons a n t o Co m pany CR 2 4 0

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Border regions. The T

- DNA co n tains the DvSnf7 suppression cassette, the cry3Bb1

expres s ion cassette, a n d the cp4 epsp s expre s sion cas s ette. The DvSnf7 suppression

cassette is regulated by the e35S promote r fro m th e 35 S RN A o f cauliflowe r m o sai c virus

(CaMV), the heat shock protein 70 ( Hsp70 ) intr o n f rom Zea may s , and the 3' untranslated

sequence of the E 9 gen e fro m P i sum sativ u m

. The c r y3Bb1 expression cassette is

regulated b y the pIIG pro m oter from Ze a m a y s

, the chlorophyll a/b binding protein

(CAB) lea d er from Triticum aestiv u m

, the Ract 1 intr o n f r o m Oryza sativ a

, and the heat

shock protein 17 ( Hsp1 7 ) 3′ untranslate d regio n fro m Triticum aestiv u m

. The cp4 epsps

expression cassette is regulated by the Tub A promoter from Oryza sativ a

, the Tub A leader

fr o m Oryza sativa

, the Tub A intron f rom Oryza sativa

, t h e CTP 2 chloropla s t

- tar g eting

sequence from Arabidopsi s thalian a

, and the Tub A 3′ untranslated region from Oryza

sativ a

.

Characterization of the DNA insert in MON 87411 was conducted using a co m bination

of sequencing, PCR, and bioinfor m atics. The results of this characterization d e m onstrate

tha t MO N 8741 1 contain s on e cop y o f th e intende d T

- DN A containin g th e DvSnf7

suppression cassette and the cry3B b 1 and cp 4 epsp s expression cassettes that is stably

inte g rated at a single locus and is inher i ted according to Mend e lian princi p les over

multiple g e n erations. T h ese con c lusions are ba se d on sever a l lines of evi d ence:

• Molecular characterization of MON 87411 by Next Generation Sequencing and

Junction S equence Analysis (NGS/ J SA) d e m onstrated that DNA from PV

ZMIR1087 1 DN A wa s integrate d at a single locus in M O N 87411.

• Directed sequencing (locus

- specific PCR

, DN A sequencin g an d analyses ) was

perfor m ed on MON 87411, which deter m ined the co m plete sequence of the single

PV

- ZMIR10871 T

- DNA insert, the adjacent fl ankin g DNA

, an d th e 5 ' a n d 3 ' insert

to

- f lank ju n ctions. T h is con f ir m ed that the sequence and organization of the T

- DNA

insert is identical to the correspondi n g regio n i n PV

- ZMIR10871

. Thi s sequencing

also confir m ed that no vector backbone or other unintended plas m id sequences are

present in MON 87411. Furthermore, the geno m ic organization at the insertion site

was assessed by c o mparing the sequences flanking the T

- DNA insert in MON 874 1 1

to the sequence of the insert i on site in conventional m aize. This analysis also assessed

potential rearrange m ents at the i n sertion site in MON 87411 upon T

- DNA

inte g ration.

• Generational stability analysis by NGS/JSA de m onstrated that the single T

- DNA

inser t i n MO N 8741 1 ha s bee n m a intaine d throug h fiv e breedin g generations

, thereby

confir m ing the stability of the intende d T

- DN A i n MO N 87 4 11.

• Segregation analyses showed expected heritabilit y that

, along with NGS/JSA,

de m onstrated stability of the T

- DNA insert across m ultiple generations.

Taken together, the characterization of the genetic modification in MON 87411

de m onstrates that a single copy of the int e nded T

- DNA was stably i n tegrated at a single

locus of the m aize geno m e and t hat no plas m id backbone sequences are present in

MO N 87411.

M ons a n t o Co m pany CR 2 4 0

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Data Confirms CP4 EPSPS and Cry3Bb1 P r otein Safety

A m ultistep approach was used to chara c te r ize and assess the sa f ety of the CP4 EPSPS

and Cry3Bb1 proteins expressed in M ON 87411. The expression levels of the

CP4 EPSPS and Cry3Bb1 proteins in selec t e d tissue s o f MO N 8741 1 wer e determined

and exposure to hu m ans and ani m als through diet was eval u ated. In a d dition, the d onor

organis m s fo r th e CP 4 EPSP S an d Cry3Bb 1 protei n codin g sequences

, Agrobacteriu m sp

. strain CP4 and Bacillus thurin g i ensis ssp kumamotoensi s

, are ubiquitous in the

environ m ent and are not commonly known f o r hu m an or ani m al pathogenicity or

allergenicity. Bioinfor m atics analysis d e ter m ined that the CP4 EPSPS and Cry3Bb1

proteins lack structural si m ilarity to known allergens or protein t o xins

. A s ha s been

previously shown in safety asses sm ents of other Roundup Ready and Cry3Bb1

-

containin g crops

, th e CP 4 EPSP S an d Cry3Bb 1 protein s ar e rapidl y digeste d i n si m u lated

digestiv e fluid s an d demonstrat e n o acut e ora l to x icity in m ice at t h e lev e ls te s ted.

Hence

, th e consu m ptio n o f th e CP 4 EPSP S an d Cry3Bb 1 protein s fro m M O N 8 7 41 1 or

its progeny poses no meaningful risk to hu m an and ani m al health or an increased plant

pes t risk.

Data Confirms DvSnf7 RNA Safety

DvSnf 7 RN A fro m M O N 8741 1 i s a dsRN A tha t upo n consu m ptio n b y wester n corn

rootworm c auses gene suppression of the targeted DvSnf7 gene. In MON 87411, the

predo m inant RNA transcript produced from the suppression cassette was identified as

being 968 nucleotides (nt) in lengt h

. Because of the extre m ely low expression of DvSnf7

RN A i n MO N 87411

, i t wa s necessar y t o produc e RN A throug h in v itro tra n sc ri ption

m ethods in order to obtain sufficient qu a ntities of DvSnf7_968 RNA for subsequent

safety studies. The molecular characte r istics of the MON 87411 DvSnf7 RNA were

deter m ined and equivalence b etwee n MO N 8741 1 DvSnf 7 RN A an d in vitro

- produced

DvSnf7_968 RNA was de m onstrated. This equivalenc e justifie s th e us e o f the

in vitro

- produce d DvSnf7_96 8 RN A a s a tes t substanc e i n studie s assessin g the

specificity and pote n tial i mpact o f DvSn f 7 RN A o n non

- targe t organis m s

. Tissue

specific expression studies de m onstrated tha t MO N 8741 1 DvSnf 7 RN A wa s expressed

at m ean levels ranging from 0.091 × 1 0

- 3 μg/g fw (in grain) to 14.4 × 10

- 3 μg/ g f w (in

ove r seaso n lea f a t growt h stage V14

- R1). Anticipated hu m an dietary exposure to

DvSnf 7 RN A i s als o ver y lo w ( ≤ 0.4 ng/kg body weight per day) relative to esti m ated

total daily RNA intake. Based on the ubiqui t ous nature of RNAi suppression utilizing

dsRN A i n a wid e variet y o f consume d plan t s p e cies, de m onstr a tion of the spe c i f icity of

DvSnf7 suppression in CRW, the long history o f saf e consumptio n o f RN A fro m a range

of sources, and the apparent lack of toxicity or allergenicity of dietary RNA; the DvSnf7

RNA i supp r essio n sequenc e use d i n MO N 87 4 11 poses no observed or theoretical risks

to hu m ans or ani m als. Therefore, t h e consu m ption of the DvSnf7 RNA fr o m

MO N 8741 1 o r it s progen y i s considered safe for hu m ans and ani m als and poses no

increase d plan t pes t risk.

MON 87411 is Compositionally E quivalent to Conventional Mai z e

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Co mpositi o nal anal y sis was conducted on grain and forage of MON 87411, a

conventiona l contro l an d 2 0 diff e ren t com m e rcial reference hybrids grown at eight

representative sites in a 2011/2012 field production in Argentina. Productio n i n th e U.S.

corn belt a n d Argenti n a m aize

- g r owing regions o ccurs a t rel a tiv e ly si m il a r l a titudes w ith

an approxi m ate 6 m o nth offset. The a v erage growing season temperatures and

preci p itation are co m parable and as a resul t

, maize hybrids deve l oped in the U.S. a r e

often used directly by far m ers i n th e souther n growin g regio n s of Argentina. As such,

co mpositional analyses from m aize grown in Ar g entina are a ppropri a te f or a co mparative

safety assess m ent and study results are relevant to the use of this m a ize grown in the U.S.

The co m positio n al anal y sis, b a sed on the OECD consensus d ocu m ent f or m aize, includ e d

m easur e m ent of nutrients, a n ti

- nutrie n ts and secondary m etabolites in con v e n tional

commercial reference hybrids to provide data on the natural v a riabilit y o f each

co mpositional co m ponent analyzed. A total of 78 co m ponents were assayed (nine in

forage and 69 in grain). Of the 78 co m ponents assayed, 18 had m o r e than 50% of

observations that were below the assay li m it of quantitation and were therefore exclu d ed

from statistical analysis. Of the 60 re m a ining co mponents statistically assessed, only 12

co mponents (protein, histidine, tyrosine, ole i c acid, neutral deterge n t fiber, copper, iron,

m anganese, zinc, niacin, vita m in B1 in grain, and ash in forage) showed a statistically

significant difference between MON 87411 and t h e conventiona l control

. Fo r thes e 12

co mponents, the m ean difference in co m pon e nt values between MON 87411 and the

conventional control, however, was less t h an the natural variation found within the

conventio n a l contr o l and reference hybrid values. Additionally, M ON 87411 m e a n

co mponent values were within the tolerance intervals of the reference hybrids, the values

f

or m aize o bserved in the lite r atu r e, and/or t h e I ntern a tion a l Li f e Scienc e s Instit u te C rop

Co mpositi o n Database ( ILSI

- CCDB) values. T h ese data indicated that t h e co m positional

co mponents with statistically signi f icant differences were not m eaningful from a food

and feed safety or n u tritional pers p e c tive.

These results support the overall conclusion that MON 87411 was not a m ajor contributor

to variation in co mponent lev e ls in m aize grain and f orage, and con f ir m e d the

co mpositional equivalence of grain and for a ge from M ON 87411 to conventional maize.

These results support the overall foo d an d fee d safet y an d lac k o f plan t pes t ris k of

MO N 87411.

MON 87411 Does Not Change Mai z e P l ant Pest P otential or Environmental

Interactio n s

Plant pest potential of a biotechnology

- d erive d cro p i s assesse d fro m th e basi s of

f

a m iliarity that the USDA recognizes as an i m portant underl y ing concept in risk

assess m ent. The concept of fa m iliarity is b a s ed on t h e f act that t h e biotechnology

derived plant is developed from a c onvention a l plan t hybri d o r var i ety whose biological

properties a nd plant pe s t pote n tial a re well kn o wn. Fa m iliarity con s i d ers the b iol o gy of

the plant, the introduced tra i t

, th e receivin g environ m ent

, and the interactions among

thes e factors

. Thi s provide s a basi s f or co mparative risk assess m ent between a

biotechnology

- derived plant and the conven t ional control. Thus, the phenotypic,

agrono m ic, and environ m ental interaction assess m ent of MON 87411 included a

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genetically si m ilar conventional control as a c o mparator

. Thi s evalu a tio n use d a weight

of evidence approach and considered st a tistical differences between MON 87411 a nd the

conventio n a l contr o l with respect to repro d ucibility, m agnitude, and directi o nality.

Co mparison to a range of com m er c i a l referenc e hybrid s gro wn concurrently established

the ran g e of natur a l va ri ability f or m aize, and provided a context from which to further

evaluate any observed statistical differences. Characteri s tics asse s sed include d : s eed

dor m ancy and ger m ination, pollen morpho l ogy, and plant phenotypic observation s and

environ m ental interaction evaluations conducte d i n th e fie l d

. T he phenotypic,

agrono m ic, and environ m ental interaction a s sess m ent d e m o nstrated that MON 87411 is

co mparable to the conventional control. Thus, MON 87411 is not expected to have

increased w eediness or plant pest r i sk co mpared to conventional m aize.

Seed dor m a ncy and ger m ination characterization indicated that MON 87411 seed had

dor m ancy and ger m ination characteristics si m ilar to seed of the conventional control. In

particular, the lack of hard seed, a well rec o g nized seed characteristic associated with

weediness, supports a conclusi o n o f n o increase d weedines s o f MO N 8741 1 co m p are d to

the conventional control. For pollen c h aracteristic assess m ents, there were no

statistically significant differences ( α =0.05 ) detecte d betwee n M O N 8741 1 an d the

conventional control for pollen viability a n d dia m eter

, an d n o visua l difference s in

general pollen m orphology were observed.

The field evaluation of phenotypic, agrono m i c

, and environ m ental characteristics also

supports the conclusion that MON 87411 is not likel y t o hav e increase d weedines s or

plan t pes t potentia l compare d t o conventiona l m a ize

. Evaluation s w e r e conducte d a t nine

replicate d fiel d s ite s acros s th e U.S

. cor n belt. These assess m ents included 13 plant

growth and develop m ent characteristics, as well as observations for plant responses to

abiotic stressors and plant

- disease and plant

- arthropod intera ctions

. Th e observed

phenotypi c characteristic s w e r e comparabl e betwee n M ON 87411 and the conventional

contr o l. Across sit e s, data sh o w no statisti c ally si g ni f icant d i ff erences b e tween

MON 87411 and the convention a l contro l fo r an y o f th e assesse d characteristics,

including early stand count, d a ys to 50% pollen shed and s i lking

, sta y green

, ea r height,

plant height, dropped ears, stalk and root lodg i ng, final stand count, grain m oisture, test

weight, and yield. Thus, the phenotypic cha racteristic s o f MO N 8741 1 wer e no t altered

in ter m s of pest/weed p o tential co m p ared to the convention a l control.

In an assess m ent of abiotic stress response and disease da m age, no differences were

observed between M O N 87411 and the con v entional control for any of the 100

co mparisons for the assessed abiotic stresso r s or for any of t he 119 comparisons for the

assessed di s eases a m ong all observations across the sites. In an assess m ent of arthro p od

related da m age, no differences were det e cted between M O N 87411and the conventional

contr o l for any of the 102 co mpa r isons for t h e asses s ed arthropo d s. Additionall y

, no

statistically significant differences were detecte d acros s site s betwee n MO N 8741 1 a nd

the conventional control for quantitative evalu a tions of corn earworm or European corn

borer da m age. The lack of differences in p l ant response to abiotic stress

, diseas e damage,

and arthropod

- related da m age support the conclusion that the intr oduce d trait s in

MON 87411 are not expected to pose an incre a sed plant pest/weed potential co m pared to

the conventional control.

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In an assess m ent of arth ropo d abundanc e collecte d usin g stick y traps

, n o statistically

significant differences were detected b e tween MON 87411 a nd the conventional control

plots for 104 out of 108 co mparisons a m ong the collections at the fou r site s wher e these

evaluations were m ade. The m ean art hropo d abundanc e value s fro m M O N 8741 1 were

within the respective range of reference hybrids for one of the four detected differences.

For the re m aining three differences, the m ean abundance values for MON 87411 were

outside of the reference range; ho w ever, the s e difference s wer e no t c o nsiste n t a c r oss

coll e ction ti m es or sit e s. These r esults a r e no t i ndi ca t i v e o f a co n siste n t r es p on se

associated with the traits and are not con s i d e red bi o logi c ally m eani n g f ul in t er m s of

increased pest/weed potenti a l of MON 87411 compared to th e conventiona l control.

In an assess m ent of arthropod abundance from visual counts, no statistically significant

differences were detected between MON 87411 and the conventional control for 60 out

of 61 co m p arisons a m ong the collections at t h e fou r site s wher e thes e evaluation s were

m ade. For t he single detected difference, the m ean abundance value for MON 87411 was

outside of the reference r ange ; however

, thi s differenc e wa s no t consisten t across

collections. Thus, this difference was not indicativ e o f a consisten t respons e associated

with the tr a its and is not considered biologically m eani n gful in ter m s of increased

pest/weed potential of MON 87411 co m pared to the conventional control.

Separate st u dies de m onstrated the efficacy of MON 87411 again s t two different CRW

species and low root feeding da m age ratin g s t o MO N 8741 1 hybri ds in CRW

- infested

fields.

I n sum m ary

, th e phenotypic

, agrono m ic

, an d environ m ental interaction data were

evaluated to characterize MON 87411, and to assess whether the t rait s introduce d in

MON 87411 alter the plant pest potential compared to conventional m aize. The

evaluation, using a w eight of evidence a p proach

, considere d th e reproducibility,

m agnitude, and direction of detected differences between MON 87411 and the

conventional control, and co mparison to the range of the commercial reference hybrids.

Results from the phenotypic, agrono m ic, a n d environ m e n ta l interaction s assess m e nt

indicat e tha t MO N 8741 1 doe s no t posses s enha nced weediness charact e ristics, increased

susceptibility or tolerance to specific abi o tic stressors, diseas es

, o r arthropods

, or

characteristics that would confer a plant p e st risk co mpared to conventional m aize.

MON 87411 Will Not Negative l y Affect Non

- target Organisms Including Those

Benefici a l to Agricult u re

An evaluation of the impacts of MON 87411 on non

- target organis m s (NTOs) is a

co mponent of the plant pest risk assess m ent. The NTO assess m ent has taken into

consideration a nu m ber of characteristics of the expressed products in MON 87411 to

evalu a te potenti a l haz ar ds to NTOs, includ i ng threatened and endangered species and

organis m s beneficial to agriculture. Cha r acteristics evaluated included MOA, spectrum

of insecticidal acti v it y an d exposur e level s t o th e C P4 EPSP S an d Cry3Bb 1 protein s and

DvSnf 7 RNA

. Bot h th e CP 4 E P SP S an d C r y3Bb1 proteins have been assessed in

multipl e pr o duct s b y U S DA

- APHIS

, U.S

. FDA

, and U.S. EPA in past y e ars. Cry3 B b1

protei n i s produce d i n bot h MO N 86 3 an d MO N 8801 7 tha t wer e grante d non

- regulated

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status and co m parable levels of this protein are produced i n MO N 87411

. Additionally,

starting in 1994 with Monsanto’s 40

- 3

- 2 soybean, a nu m ber of Roundup Ready crops

(canola, m a ize, sugarbeet, cotton, alfalfa) containing CP4 EPSPS pro t eins have been

granted non

- regulated status by USDA

- APHIS. After bot h extensiv e testin g and

widescale com m ercial cultivation, in no ins t ance have adverse i mp act s t o NTO s been

associated with exposure to Cry3Bb1 or CP4 E PSPS proteins from these biotechnology

derived crops.

As noted previously, the suppression cassett e i n MO N 8741 1 contain s th e CaM V e35S

pro m oter, maize hsp7 0 intron

, th e tw o DvSnf7 p sequences (240 nt e a ch) separated by a

149 bp intervening sequence, and a pea E 9 3’ untranslated region. W h e n the suppression

cassette is transcri b ed, the predo m inant RNA transcri p t ex p r essed is 968 nt in len g th and

fo r m s a hairpin loop, thereby allowing the for m ation of the 240 bp DvSnf7 dsRNA.

When consu m ed by CR W

, this 240 bp DvSnf7 dsRNA activates the RNAi process

leading to suppression of the targeted CRW Snf7 gene.

To address potential impacts to NTOs, specific laboratory bioassay studies using

DvSnf7_240 dsRNA, the active insecticidal product in MON 87411, were conducted on a

variety of NTOs including several Coleopteran, two Hy m enopteran, o n e He m ipteran and

fou r Lepidoptera n species

. No impacts to su r vival, gro w th, or develop m ent of these

species were noted when fed extre m ely high doses (relative to levels present in

MON 87411) of DvSnf7_240 dsRNA over m ulti

- day bioa s say periods.

Additional NTO assess m ents were conducted on a battery of organis m s based on

recom m endations published by the U.S. EPA. Organi s m s tested included earth w orm,

honeybee, parasitic wasp, ladybird beetle, carabid beetle and the insidious flower bug. In

these stu d ie s

, test conce n tr a tions we r e based on the m easured DvSnf7 RNA expression in

the tissue types that the NTO w ould most likely be exposed to in the environ m ent. Based

o n U.S

. EP A recom m e ndations , a targete d m a r gin of exposure (MOE) of greater than 10

-

ti m es the m axi m u m expected envir o n m ental c on cent r ation ( MEEC) was used to est a blish

test concentrations. MOEs that exceed 10 are considered as indicative of m in i m al risk in

worst

- case laboratory assays by U.S

. EPA

. I n al l case s w h er e MOE s coul d b e calculated,

they were > 10

- f old the predi c ted e x posure le v el for these s p ecies, indicating that DvSnf7

RNA is not likely to have effects on terrestrial beneficial invertebr a te species at field

exposur e levels.

Additional assess m ents for pote n tial exposure of aquatic o r ganis m s and threatened and

endangered species to DvSnf7 RNA contain e d i n MO N 8741 1 conclud e tha t du e t o the

lack of proxi m ity of t hese organis m s to m a ize cultivation, lack of relevant exposure

because of feeding ecology and the restricted acti v ity of the DvSnf7 RNA, that

cultivation o f MON 87411 will have no e ff ect on these sp e ci e s or th e ir h a bit a ts.

Deregulati o n of MON 87411 is Not Expected to Have Effects on Maize Agronomic

Practices

An assessment of curre n t m aize agr o no m ic p r a c tices was co n ducted to d e ter m ine wheth e r

th e cultivatio n o f MO N 8741 1 ha s th e potent i al to i m p a ct current m aize agrono m ic

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practices. Maize fields are typically highly m a naged areas that are dedicated to grain

and/or forage production.

MO N 8741 1 wa s develope d t o provid e tw o e ffective M O As against the target corn

rootworm pests of m aize in the U.S. corn bel t a s wel l a s glyphosat e toleranc e i n a single

product. As tolerance to glyphosate and pro t ection from c orn rootworm co mplex pests

are present in m any currently a v ailable m aize hybrids that have been widely grown in the

U.S. since 2003, the introduction of MON 87411 is expected to have no i mpact on

current agrono m ic or m anag e m ent practices for m aize. As phenotypic evaluations,

evaluations of stress responses, and pest/disease susce p tibility showed no difference

between MON 87411 a nd reference hybrids (ot h er than protection fro m CR W larval

feeding), no changes are anticipa t ed in crop r o t a tions, tilla g e practices, planting p ractices,

fertility m anage m ent, weed and disease m anage m ent, and volunteer manage m ent from

the introduction of MON 87411.

MON 87411 is si m ilar to conventional maize in its agronomic, phenotypic,

environ m ental, and co m positio n al c hara c te r isti c s and has n atur a lly o c c urring lev e ls of

protection against pests (other than CR W ) a nd diseases comparable to and typical of

conventional com m ercial m aize hybrids. Based on this assess m ent, the introduction of

MON 87411 is not expected to result in ch a nges or i m pacts to current maize agrono m ic

practices.

Conclusion

Based on the data and infor m ation presented i n thi s petition

, i t i s conclude d that

MON 87411 is not expected to be a plant pes t

. Therefore, Monsanto Company requests a

deter m ination from USDA

- APHIS that MON 87411 and any progeny derived from

crosse s betwee n MO N 8741 1 an d convention a l m aize or deregulated biotechnology

derived m aize be granted nonregula te d statu s unde r 7 CF R par t 340.

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TABL E O F CONTENTS

RELEAS E O F INFORMATION.

................................ ................................ .................... 2

CERTIFICATION

................................ ................................ ................................ ............ 3

EXECUT I V E SUM M ARY

................................ ................................ ................................ 4

TABLE OF CONTENTS

................................ ................................ ............................... 14

LIST OF TABLES

................................ ................................ ................................ ........... 20

LIST OF FIGURES

................................ ................................ ................................ ........ 23

ABBREV I ATION S AN D DEFINITIONS

................................ ................................ ..... 25

I. RATIONAL E FO R TH E DEVELOPMEN T O F MO N 87411

.............................. 28

I.A. Basis for the Requ e st for a Det e r m ination of Nonregulated Status under

7 CFR § 340.6

................................ ................................ ................................ .............. 28

I.B. Rationale for the Develop m e n t of Insect

- Protected and Glyphosate

Tolerant Maize MON 87411

................................ ................................ ........................ 28

I.B.1 Benefits of Insect

- Protecti o n an d Herbicid e To lerance Traits

...................... 29

I.B.2. Introduction of Insect

- P r otectio n Trait s i n th e U.S.

................................ .... 30

I.B.3. Develop m ent of CR W

- Protected and Glyphosate Tolerant Maize

MON 87411

................................ ................................ ................................ .......... 30

I.C. RN A I n terferenc e (RNAi)

................................ ................................ ..................... 31

I.C.1. Applications of RNAi in Plants

................................ ................................ .. 31

I.C.2. Application s o f RNA i i n Insects

................................ ................................ . 32

I.D. Modes

- of

- Action of the Inserte d Geneti c Components

................................ ........ 32

I.D.1. Mode

- of

- Action of the RNA i Co m ponen t o f MO N 87411

........................ 32

I.D.2. Modes

- of

- Action of the CP 4 EPSP S an d Cry3Bb 1 Proteins

...................... 35

I.E. Product Efficacy

................................ ................................ ................................ .... 35

I.F. Sub m issions to Other Regulatory Agencies

................................ .......................... 36

I.F.1. Submission to FDA

................................ ................................ ................... 36

I.F.2. Submission to EPA

................................ ................................ .................... 36

I.F.3. Submissions to For e ign Govern m ent Agencies

................................ .......... 37

II. THE BIOLOGY OF MA I Z E

................................ ................................ .................. 38

II.A. Maize as a Crop

................................ ................................ ................................ .. 38

II.B. Characteristics of the Recipient Plant

................................ ................................ .. 39

II.C. Maize as a Test System in Product Safety Assess m ent

................................ ....... 39

III. DESCRIPTIO N O F T H E GENETI C M O DIFICATION

................................ ... 41

III.A. The P las m id Vector PV

- ZMIR10871

................................ ................................ 41

III.B. Description of the Transfor m a tion System

................................ ........................ 41

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III.C. The cry3Bb 1 Coding Sequence and the Cry3Bb1 Protein

................................ .. 45

III.D. The cp 4 epsp s Codin g Sequenc e an d th e C P 4 EPSP S Protein

........................... 45

III.E. DvSnf 7 p sequence

................................ ................................ .............................. 45

III.F. Regulatory Sequences

................................ ................................ ........................ 45

III.G. T

- DNA Borders

................................ ................................ ................................ . 46

III.H. Genetic Ele m ents Outside of the T

- DNA Borders

................................ ............. 46

IV. CHARACTER I ZATIO N O F TH E GENETI C MODIFICAT I ON

..................... 52

IV.A. Deter m ining the N u m ber of DNA inserts in MON 87411

................................ 61

IV.A.1. N e x t Genera tio n Sequencin g (NGS ) fo r MO N 8741 1 and

Conventional Control Geno m ic DNA

................................ ................................ ... 62

IV.A. 2 Ch a r acterizatio n o f inser t numbe r i n MO N 8741 1 using

Bioinfor m a t ic Analysis

................................ ................................ ......................... 64

IV.B. Organizatio n an d Sequenc e o f th e Insert and Adjacent Flanking DNA in

MON 87411

................................ ................................ ................................ .................. 68

IV.C. Sequencing of the MO N 8741 1 Insertio n Site

................................ ................... 71

IV.D. Deter m ination of Insert Sta b ility over Multiple Generations of

MON 87411

................................ ................................ ................................ .................. 73

IV.D.1 Deter m ination of the Insert Nu m ber

................................ ........................ 73

IV.E. Inheritance of the Genetic Insert in MON 87411

................................ ............... 77

IV.F. Characterization of the Genetic Modificatio n Sum m a r y an d Conclusion

........... 81

V. CHARAC T ER I ZATIO N AN D SA F ET Y ASSE S SMEN T O F THE

Cry3Bb 1 an d CP 4 EPSP S PROTE I N S PR O DUCE D I N MO N 87411

....................... 82

V.A. Identity and Function of the Cry3Bb 1 an d CP 4 EPS PS Protein s from

MON 87411

................................ ................................ ................................ .................. 82

V.A.1. Identity and Function of the C ry3Bb1 Protein from MON 87411

............. 82

V.A.2. Identity and Function of the C P 4 EPSPS Protein from MON 87411

........ 83

V.B. Characterization and Equivale n ce of Cry3Bb1 and CP4 EPSPS P r oteins

from MON 87411

................................ ................................ ................................ ......... 84

V.C. Expressio n Level s o f Cry3Bb 1 an d CP 4 EPSP S Protein s i n MO N 87411

.......... 84

V.C.1. Exp r essio n Level s o f Cry3Bb 1 Protein

................................ ..................... 85

V.C.2. Exp r essio n Level s o f CP 4 EPSP S Protein

................................ ................ 87

V.D. Assess m ent of Potenti a l All e rgenicit y o f th e Cry3Bb 1 an d CP 4 EPSPS

Proteins

................................ ................................ ................................ ......................... 90

V.D.1. As s ess m ent of Potential All e rgenicit y o f th e Cry3Bb 1 Protein

................ 90

V.D.2. As s ess m ent of Potential Aller g enicit y o f th e CP 4 EPSP S Protein

........... 90

V.E. Safety Assess m ent Sum m ary of Cry3Bb 1 a n d CP 4 EPSP S Protein s in

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MON 87411

................................ ................................ ................................ .................. 91

V.E.1. Cry3Bb1 Donor Organis m

, History of Safe Use, and Specificity

............ 91

V.E. 2 CP 4 EPSP S Do n or Organis m

, Histo ry o f Saf e Use

, an d Specificity

......... 92

V.E.3. Cry 3 Bb 1 an d CP 4 EPSP S P r otein s i n MO N 8741 1 a re Not

Ho m o logous to Known Allergens or Toxins

................................ ........................ 93

V.E.4. Cry 3 Bb 1 an d CP 4 EPSP S P r o t ein s i n MO N 8741 1 a re Labil e i n in

vitro Digestio n Assays

................................ ................................ ........................... 93

V.E.5. Cry 3 Bb 1 an d CP 4 EPSP S P r otein s i n MO N 8741 1 a re Not

Acutel y Toxic

................................ ................................ ................................ ........ 93

V.E.6. H u man and Ani m al Exposure to the Cry3Bb1 and CP4 EPSPS

Proteins

................................ ................................ ................................ ................. 93

V.E.7. CP 4 EPSP S Activity

................................ ................................ ................. 95

V.E.8. Cry3Bb1 Activity

................................ ................................ ...................... 95

V.E.9. Non

- Target Assess m ent for CP4 EPSPS Protein

................................ ...... 95

V.E.10. Non

- Target Assess m ent for Cry3Bb1 Protein

................................ ......... 95

V.F. Cry3Bb 1 an d CP 4 EPSP S Prote ins Characterization and Safety

Conclusion

................................ ................................ ................................ .................... 97

VI. CHARAC T ER I ZATIO N AN D SA F ET Y ASSE S SMEN T O F THE

DvSnf 7 RN A PRODUCE D I N MO N 87411

................................ ................................ ... 98

VI.A. Histor y o f Saf e Us e o f RNA

- m ediated Gene Suppression in Plants

................. 98

VI.B. Characterization and Equival e nc e o f DvSnf 7 RN A fro m MO N 87411

........... 100

VI.C. Expressio n Level s o f DvSnf 7 RN A i n MO N 87411

................................ ........ 101

VI.D. Human and Ani m al Exposure to the Dv S nf7 RNA

................................ ......... 104

VI.E. Laboratory Tests to Characterize the Spectrum of Activity of DvSnf7

RNA 105

VI.E.1. Test Substances Used to A ssess the Activity Spectrum of DvSnf7

RNA

................................ ................................ ................................ .................... 105

VI.E.2. Results from A c tivity Spec t rum Bioassays for DvSnf7 RNA

............... 105

VI.E.3. Non

- Target Organism Assess m ent for DvSnf7

................................ ...... 108

VI.F. Characterization and Safety Conclusions

................................ ......................... 111

VII. COMPOSITIO NA L ASSESSMEN T O F MO N 87411

................................ .... 112

VII.A. Compositio n al E quivale n ce o f MON 87411 Grain a n d Forage to

Conventional Maize

................................ ................................ ................................ .... 113

VII.A.1. Nutrien t Level s i n Maiz e Grain

................................ ............................ 115

VII.A.2. Anti

- Nutrient L evels in Maize Grain

................................ .................. 120

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VII.A.3. Secondary Metabolites Level s i n Maiz e Grain

................................ .... 120

VII.A.4. Nutrient Lev e ls in Maize Forage

................................ ......................... 121

VII.B. Compositio n al A ssess m ent of MON 87411 Conclusi o n

................................ 138

VIII. PHENOTYPIC

, AGR O NOMIC

, AN D ENV I RONMENTAL

INTERACTION S ASSESSMENT

................................ ................................ ............... 139

VIII.A. Characteristics Measured f o r Assess m ent

................................ ..................... 139

VIII.B. Interpretation of Phenotypic and Enviro nm ental Interaction Data

................ 143

VIII.B.1. I n terpretation of D e tected Differences Criteria

................................ .. 144

VIII.B.2. I n terpretation of Env i ronmental Interactions Data

............................. 145

VIII.C. Comparative A ssess m ents of the Phenotypic, Agro n o m ic, and

Environ m ental Interaction Characteristics of M O N 87411

................................ ........ 146

VIII.C.1. Seed Dor m ancy and Ger m ination Characteristics

.............................. 146

VIII.C.2. Field Phenot ypic, Agron om ic, and Environ m ental Interactions

Characteristics

................................ ................................ ................................ ..... 149

VIII.C.3. Pollen Characteristics

................................ ................................ ......... 160

VIII.D. Conclusions for Phenotypic, Agrono m ic, and Enviro nm ental

Interactions Evaluation

................................ ................................ ............................... 161

IX. U.S

. AGRONOMI C PRACT I CES

................................ ................................ ....... 163

IX.A. Introduction

................................ ................................ ................................ ..... 163

IX.B. Overview of U.S. Maize Production

................................ ................................ 163

IX.B.1. Maize Production

................................ ................................ .................. 163

IX.C. Production Manage m ent Considerations

................................ ......................... 165

IX.D. Manage m ent of Insect Pests

................................ ................................ ........... 165

IX.D.1. Insect Resistance Manage m ent

................................ ............................. 169

IX.E. Manage m ent of D i seases and Other Pests

................................ ....................... 170

IX.F. W eed Manage m ent

................................ ................................ ........................... 170

IX.F.1. W e ed Resistance Manage m ent

................................ .............................. 171

IX.G. Cro p Rotatio n Practice s i n Maize

................................ ................................ .... 171

IX.H. Maize Volunteer Manage m ent

................................ ................................ ......... 172

IX.I. Stewardshi p o f MO N 87411

................................ ................................ .............. 172

IX.J. I m pa c t of the Introduction of MO N 8741 1 o n Agric u ltural Practices

.............. 173

X. PLANT PEST ASSESSMENT

................................ ................................ ............... 174

X.A. Characteristics of the Genetic Insert and E xpressed Products.

......................... 174

X.A.1. Genetic Insert

................................ ................................ ......................... 174

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 17 o f 3 72CB I Delete d Copy

X.B. Mode

- of

- Action

................................ ................................ ................................ 175

X.C. Expression and C haracterization of Gene Products

................................ .......... 175

X.C.1. Protein Safety and Expression Levels

................................ ..................... 175

X.C.2. RNA Safety and Expression Levels

................................ ........................ 176

X.D. Compositio n al C h aracteristics

................................ ................................ .......... 176

X.E. Phenotypic, Agrono m ic, and Environ m ental Interaction Characteristics

.......... 177

X.F. W eed i ness Potential of MON 87411

................................ ................................ . 178

X.F.1. Seed Do r m ancy and Ger m ination

................................ ........................... 178

X.F.2. Plant Growth and Develop m e nt

................................ .............................. 178

X.F.3. Pollen Morphology and Viability

................................ ........................... 179

X.G. I m pa c t to Non

- Target Organis m s, Including Those Beneficial to

Agriculture

................................ ................................ ................................ .................. 179

X.G.1. I m pact on Threatened and E ndangered S pecies

................................ ..... 179

X.H. Environ m ental Fate of C R W Products Expressing DvSnf7_968

...................... 180

X.I. Potential for Pollen Mediated Gene Flow and Introgression

.............................. 182

X.I.1. Hybridization with Cultivated Maize

................................ ....................... 182

X.I.2. Hybridizatio n wit h W ild Annual Species of Zea mays subsp.

mexicana

................................ ................................ ................................ ............. 190

X.I.3. Hybridization with the W ild Perennial Species of Subgenus

Tripsacum

................................ ................................ ................................ ........... 190

X.J. Transfer of Genetic Infor m at i on to Species with which Maize Cannot

Interbreed (Horizontal Gene Flow)

................................ ................................ ............ 190

X.K. Potential I m pact on Maize Agrono m ic Practices

................................ .............. 191

X.L. Conv e ntional Breeding with Othe r Biotechnology

- derive d or

Conventional Maize

................................ ................................ ................................ .... 191

X.M. Sum m ary of Plant Pest Assess m ents

................................ ................................ 192

XI. ADVERSE CONSEQUEN CE S O F INTRODUCTI O N

................................ ..... 194

REFERENCES

................................ ................................ ................................ .............. 195

APPEND I CES

................................ ................................ ................................ ............... 211

Appendix A: USDA Not i ficatio n s and Permits

................................ .......................... 212

Appendix B: Materials, Methods, and Suppl e mentary Results for Molecular

Analyses of MON 87411

................................ ................................ .............................. 216

Appendi x C : Materials

, Method s and Results for Characteri z ation of

Cry3Bb1 and CP4 EPSPS Proteins Produced in MON 87411

................................ .. 248

Appendi x D : Material s an d Met h ods Used for the Analysis of the Levels of

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 18 o f 3 72CB I Delete d Copy

Cry3Bb1 and CP4 EPSPS Proteins in MON 87411

................................ ................... 278

Appendi x E : Materials

, Method s an d Results for Characteri z ation of the

DvSnf7 RNA Produced in MON 87411

................................ ................................ ....... 283

Appendi x F : Material s an d Method s U s ed for the Analysis of Expression

Levels of D vSnf7 RNA in MON 87411

................................ ................................ ........ 292

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

Compositional Analysis of MON 87411 Mai z e Grain and Forage

............................ 298

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

Dormancy and Germination Assessment of MON 87411

................................ ........... 314

Appendi x I : Materials

, Methods

, and Individual Site Results from

Phenotypic, Agronomic, and Environmental I n teraction Assessment of

MON 87411 under Field Conditions

................................ ................................ ............ 321

Appendix J: Materials and Methods for Pollen Morphology and Viabilit y

Assessment

................................ ................................ ................................ ...................... 356

Appendi x K : Summar y o f Non

- targe t Organis m Studies

................................ .......... 360

Appendix L: Next

- Generation Se q u encing and Junction Sequence Analysis

for the Characteri z ation of DNA I n serted into Crop Plants.

................................ ..... 366

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 19 o f 3 72CB I Delete d Copy

LIS T O F T A BLES

Table III

- 1. Summary of Gen e tic Elements in PV

- Z M I R10871

................................ . 47

Table IV

- 1. Summary of G e netic Elements in MON 87411

................................ ....... 59

Tabl e IV

- 2

. Sequencin g (NGS ) Conducte d fo r MO N 8741 1 an d Con t rol

Genomic DNA.

................................ ................................ ................................ .................. 63

Table IV

- 3. Summary of NGS Data fo r th e Conventiona l Contro l DNA

Sample Spiked w ith PV

- Z MIR10871 DNA

................................ ................................ .. 63

Table IV

- 4. Unique Junction Sequence Class Results

................................ ................. 65

Table IV

- 5. Junction Sequence Classes Detected

................................ ......................... 73

Table IV

- 6. Segregation of the T

- DN A Durin g th e Developmen t of

MON 87411 1:1 Segregation

................................ ................................ .......................... 80

Table IV

- 7. Segregation of the T

- DN A Durin g th e Developmen t of

MON 87411 1:2:1 Segregation

................................ ................................ ....................... 80

Table V

- 1. Summary of Cry3Bb1 Protein Levels in Tiss u es from

MON 87411 Gro w n in 2011

– 2012 Argentina Field Trials

................................ ......... 86

Tabl e V

- 2

. Summar y o f CP 4 EPSP S Protein Levels in Tissues from

MON 87411 Gro w n in 2011

– 2012 Argentina Field Trials

................................ ......... 88

Tabl e VI

- 1

. Summar y o f DvSnf 7 RN A L e vels in Mai z e Tissues Collected

from MON 87411 Produced in Argentina Field Trials during 2011

- 2012

................ 102

Table VI

- 2. Susceptibility to DvSnf7_240 dsRNA in laboratory bioassays

.............. 107

Table VI

- 3. Maximum expected e n vir o nment a l concentr a t ions (MEECs), no

observed effect concentrations (NOECs) from non

- target organism s t udies

and estimated margins of exposure (MOEs) for DvSnf7 RNA

................................ .. 110

Table VII

- 1. Summary of Mai z e Grain Protein and Amino Acids for

MON 87411, Conventional Control, and Reference Hybrids

................................ .... 122

Table VII

- 2. Summary of Mai z e Grain Fat and Fatty Acids for MON 87411,

Conventional Control, and Reference Hybri d s

................................ ........................... 126

Table VII

- 3. Summary of Mai z e Grain Carbohydrates by Calculation and

Fiber for MON 87411, Conventional Control, and Reference Hybri d s.

................... 128

Table VII

- 4. Summary of Mai z e Gra i n As h an d Mineral s fo r MO N 87411,

Conventional Control, and Reference Hybri d s

................................ ........................... 129

Table VII

- 5. Summary of Mai z e Grain Vitamins for MON 87411,

Conventional Control, and Reference Hybri d s

................................ ........................... 131

Table VII

- 6. Summary of Mai z e Grain Anti

- n utrients a n d Secondary

Metabolites for MON 8 7411, Conventional C o ntrol

, an d Referenc e Hybrids

......... 133

Table VII

- 7. Summary of Mai z e Forage Proximates, Fiber and Minerals for

MON 87411, Conventional Control, and Reference Hybrids

................................ .... 134

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 20 o f 3 72CB I Delete d Copy

Table VII

- 8. Literature and ILSI Database Ranges for Components in Mai z e

Forage and Grain

................................ ................................ ................................ ......... 136

Table VIII

- 1. Phenotypic, Agronomic, and Environmental Interaction

Characteristics Evaluated in U.S. F iel d Trial s an d Laborator y Studies

................... 141

Tabl e VIII

- 2

. Germin a t io n Cha racteristics of MON 87411 and the

Conventional Control

................................ ................................ ................................ ... 148

Table VIII

- 3. Field Phenotypic Evaluation Sites for MON 87411 during 2012

...... 151

Table VIII

- 4. Combined

- Site C o mparison o f MON 87 4 11 to Conventional

Control for Phenotypic and Agron o mic Characteristics During 2012

..................... 152

Table VIII

- 5. Summary of Qualit a tive Envir o nmental Interactions

Assessments during 2012

................................ ................................ .............................. 1 55

Table VIII

- 6 Combined

- Site Comparison of Pest Dama g e to MON 8 7411

Compared to the Conventional Control duri n g 2012

................................ ................ 158

Tabl e VIII

- 7

. Summar y o f Arthr o po d Abundance Assessments and Detected

Differences during 2012

................................ ................................ ............................... 159

Table VIII

- 8. Pollen Characteristics of MON 87411 Compared to t h e

Conventional Control during 2012

................................ ................................ .............. 161

Table IX

- 1. Mai z e Production in the U.S., 2007

- 2012

................................ ............... 164

Table IX

- 2. Insect Pe st s in Mai z e in the North Central States and Typical

Time of Damage

................................ ................................ ................................ ............ 166

Table IX

- 3. Insecticide Applications in Mai z e in 2010 in the U.S.

........................... 167

Table X

- 1. Summary of Published Literature on Mai z e Cross Pollination

............ 183

Table A

- 1. USDA Notifications a n d Permits A pproved for MON 87411 and

Status of Trials Planted under These Notifications

................................ .................... 213

Table C

- 1. N

- Terminal Sequence of the MON 87411

- produced Cry3Bb1

............. 250

Table C

- 2. Summary o f the Tryptic Masses I d entified for the

MON 87411

- Produced Cry3Bb1 Using MALDI

- TOF MS

................................ ....... 252

Table C

- 3. Comparison of Immu n oreactive Signal Bet w e en MON 87411

-

an d E coli

- produced Cry3Bb1Proteins

................................ ................................ ...... 256

Table C

- 4. Molecular Weight Comparison Bet w een the MON 87411

- and

E. coli

- pro d uced Cry3Bb1 Proteins.

................................ ................................ ............ 256

Table C

- 5. Cry3Bb1 Funct i onal Activity Assay

................................ ........................ 262

Table C

- 6. N

- Terminal Sequence of the MON 87411

- produced CP4EPSPS

.......... 265

Table C

- 7. Summary o f the Tryptic Masses I d entified for the

MON 87411

- produced CP4 EPSPS Using MALDI

- TOF MS

................................ ... 267

Table C

- 8. Comparison of Immu n oreactive Signal Bet w e en MON 87411

-

an d E coli

- produced CP4 EPSPS Protein

................................ ................................ .. 271

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 21 o f 3 72CB I Delete d Copy

Table C

- 9. Molecular Weight Comparison Bet w een the MON 87411

- and

E. coli

- pro d uced CP4 EPSPS Proteins

................................ ................................ ......... 272

Tabl e C

- 10

. CP 4 EPSP S Fun ctional Activity Assay

................................ .................. 276

Table D

- 1. Cry3Bb1 Protein Extraction Method s 1 for Tissue Samples

................... 279

Table D

- 2. CP4 EPSPS Protein Extraction Method s 1 for T issue Samples

.............. 279

Table G

- 1. Conventional Commercial Reference Mai z e Hybrids

............................ 298

Table G

- 2. Re

- expression Formulas for Statistical Analysis of Composition

Data

................................ ................................ ................................ ................................ . 308

Table H

- 1. Starting Seed of MON 87411, Conventional C ontrol and

Commerci a l Mai z e Reference Hy b rids Used in Dormancy Assessment

................... 317

Tabl e H

- 2

. Germinatio n an d Do r m anc y Characteristic s o f MO N 8741 1 and

the Conventional Control Seed (Selfed F2 Grain) Produced at each of the

Three Field Sites

................................ ................................ ................................ ............. 318

Table I

- 1. Starting Seed for Pheno t ypic, Agronomic, a n d Environmental

Interaction Assessment

................................ ................................ ................................ . 323

Tabl e I

- 2

. Fiel d an d P l anting Information

................................ ................................ .. 324

Table I

- 3. Data Missing or Excluded from Analysis

................................ .................. 332

Tabl e I

- 4

. Individua l Sit e Phenoty pic Comparison of MON 87411 Compared

to the Conventional Control

................................ ................................ .......................... 334

Table I

- 5. Qualit a ti v e Assessment: A b iotic Stressor Evaluations Using a

Categorical Scale for MON 87411 and the Conventional Control

............................ 339

Table I

- 6. Qualit a ti v e Assessment: Disease D amage Evaluations Using a

Categorical Scale for MON 87411 and the Conventional Control

............................ 340

Table I

- 7. Qualit a ti v e Assessment: Ar thropo d Damag e Evaluation s Usin g a

Categorical Scale for MON 87411 and the Conventional Control

............................ 341

Table I

- 8. Individual

- S ite Analysis: Quantitative Assessment of Corn

Ea r w or m an d Europea n Cor n Bo r e r Damag e t o MO N 8741 1 Compare d to

the Conventional Control

................................ ................................ .............................. 342

Table I

- 9. Individual

- S ite Analysis: Arthropod Abundance in Sticky Trap

Samples Collected from MON 87411 Compared to the C onventional Control

....... 343

Table I

- 10. Individual

- Site Analysis: Ar thropo d Abundanc e i n Visua l Counts

from MON 87411 Compared to t h e Conventional Control

................................ ...... 349

Table J

- 1. Starting Seed for Pollen Morphology and Viability Asses s ment

............. 357

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 22 o f 3 72CB I Delete d Copy

LI S T O F FIG UR E S

Figure I

- 1. Diagram o f MO N 8741 1 dsRN A O r a l Deliver y t o Suppress

DvSnf 7 E x pressio n vi a th e RNA i Path w a y i n CRW

................................ .................... 33

Figur e I

- 2

. Mode l depictin g endos o mal

- auto p h agy pathway involved in

intracellul a r sorting a n d degradation of receptors along with other

macrom o lecules in a n ormal cell (left) and a D vSnf7 deficient cell (ri g ht).

................ 34

Figure III

- 1 . Circular Map of PV

- Z MIR10871

................................ ........................... 43

Figure III

- 2

. Schematic of the De v elopment o f MON 87411

................................ ....... 44

Figure III

- 3

. Deduced Amino Acid Sequence of the Cry 3 Bb1 Protein

...................... 51

Figure III

- 4

. Deduced Amino Acid Sequence of the CTP2 Targeting

Sequence and CP4 EPSPS Protein

................................ ................................ ................. 51

Figure IV

- 1

. Molecul a r Charact e ri z ation using Sequencing and

Bioinformatics

................................ ................................ ................................ .................. 53

Figure IV

- 2. Junctions and Junction Sequences

................................ .......................... 54

Figure IV

- 3. T w o Unique Junction Sequence Classes are Produced by the

Insertio n o f a Singl e Plasmi d Region

................................ ................................ ............. 54

Figure IV

- 4

. Breeding Hist o ry of MON 87411

................................ ............................. 57

Figure IV

- 5

. Schematic Representation of the Insert and Flanking Sequences

in MON 87411

................................ ................................ ................................ .................. 58

Figure IV

- 6. Junction Sequences Detected by NGS/JSA

................................ ............. 66

Figur e IV

- 7

. Analysi s o f Overlappin g PC R P r oduct s Acros s th e MO N 87411

Insert

................................ ................................ ................................ ................................ . 69

Figur e IV

- 8

. PC R A m plificatio n of the MON 87411 Insertion Site

............................ 72

Figure IV

- 9. Junction sequences detected by JSA. Junction Sequence Class

A Alignment (All Generations Tested)

................................ ................................ .......... 75

Figure IV

- 10. Junction sequences detected by JSA. Junction Sequence Class

B Alignment (All Generations Tested)

................................ ................................ ........... 76

Figure IV

- 1 1. Breeding Path for G e nerating Segregation Data for

MON 87411

................................ ................................ ................................ ....................... 79

Figure VIII

- 1. Schematic Diagram of Agronomic and Phenotypic Data

Interpretation Methods

................................ ................................ ................................ . 144

Figure IX

- 1. Planted Mai z e Acr e s by County in the U.S. in 2012

............................ 164

Figure IX

- 2

. Mai z e acres in the U

. S. planted to hybrids containing Bt

proteins for corn rootworm protection

................................ ................................ ........ 169

Figure C

- 1. MALDI

- TOF MS Cove r age Map of the MON 87411

- produced

Cry3Bb1

................................ ................................ ................................ .......................... 253

Figure C

- 2. Western B lot Analysis of MON 87411

- and E. coli

- produced

Cry3Bb1 Proteins

................................ ................................ ................................ ........... 255

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 23 o f 3 72CB I Delete d Copy

Figur e C

- 3

. Molecula r Weigh t a n d Purit y Analysi s o f the

MON 87411

- produced Cry3Bb1 Protein

................................ ................................ .... 257

Figur e C

- 4

. Glycosylatio n Analysi s o f th e MO N 87411

- produce d Cry3Bb1

Protein

................................ ................................ ................................ ............................ 260

Figure C

- 5. MALDI

- TOF MS Cove r age Map of the MON 87411

- Produced

CP4 EPSPS Protein

................................ ................................ ................................ ....... 268

Figure C

- 6. Western B lot Analysis of MON 87411

- and E. col i

- Produced

CP4 EPSPS Proteins

................................ ................................ ................................ ..... 270

Figur e C

- 7

. Molecula r Weigh t a n d Purit y Analysi s o f the

MON 87411

- produced CP4EPSPS Protein

................................ ................................ . 272

Figure C

- 8. Glycosylation Analysis o f th e MO N 87411

- produce d CP 4 EPSPS

Protein

................................ ................................ ................................ ............................ 274

Figure E

- 1. PCR Amp l ification o f MO N 8741 1 DvSnf 7 cDNA

................................ 287

Figure E

- 2. Sequence Alignment bet w ee n M ON 8741 1 DvSnf 7 cDN A and

DvSnf7_968 cDNA

................................ ................................ ................................ ........ 288

Figure E

- 3. Northern Blot Analysis to Confirm the Equivalence bet w een the

dsRN A i n DvSnf7_96 8 RN A an d MO N 8741 1 DvSnf 7 RNA

................................ .... 290

Figure J

- 1. General Morphology of Pollen from MON 87411, the

Conventional Control, and the Reference Hy b rids under 200X Magnification

...... 358

Figure L

- 1. Sequencing and Sequ e nce Selection

................................ ........................ 368

Figure L

- 2. Junctions and Junction Sequences

................................ ......................... 369

Figure L

- 3. T w o Unique Junction S e quence Classes are Produced by the

Insertio n o f a Singl e Plasmi d Region

................................ ................................ ........... 370

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 24 o f 3 72CB I Delete d Copy

ABBREV I ATION S AN D DEFINITION S 1

~ approxi m a tely

ADF acid detergent fiber

ANOVA anal y s is of v a riance

AOSA Association of Official Seed Anal y s ts

APHIS An i m al and Plant Health Inspection Service

APS anal y tical protein standards

bp base pairs

BSA bovine serum albu m in

Bt Bacillus thur i ngiensis

bu/A bushels per acre

bw bo d y weight

cD NA c o m p l e ment a ry deo x y r i b o nucleic acid

CEW corn earwo r m

CFR Code of Federal Regulations

CHT ceramic h y d r ox y a patite

CP4 EPSPS 5

- enolp y r uvylshiki m a t e

- 3

- phosphate s y n t hase protein from

Agrobacterium tumefacie n s strain CP4

CRW corn rootworm

CTAB hexadec y ltr im eth y lam m o nium br o m ide

CV coefficient of variation

DAP da y s after pl a nting

dATP deo x y a denos i ne triphos p hate

DDI daily dieta r y i ntake

DEEM

- FCID Dietary Exp o sure Evaluation M o del

- F o od Com m odity Intake Database

DHB 2,5

- d ih y dr ox ybenzoic acid

dNTP deo x y ri b onu c leotide

dsRNA double stran d ed RNA

DTT dithio t hreitol

DvSnf7 Snf7 gene fr o m Diabrotica virgifera vi rg ifer a encod i ng the SNF7 subunit

of the ESCRT

- III c o m plex

DvSnf7 RNA RNA expressed fr o m the suppression casset t e that contains an inverted

repeat sequence derived fr o m the western corn rootworm (WCR;

Diabrotica virgifera virgifer a ) DvSnf7 gene

DvSnf7_ 2 40 the active insecticidal RNA in MON 87411

DvSnf7_ 9 68 an in vitro transcribed DvSnf7 single stranded RNA

DvSnf7 p partial coding sequence of the Snf7 gene fr o m Diabrotica virgifera

virgifer a encoding the Sn f 7 subunit of the ESCRT

- III c o m plex

dw d r y weight

DWCF d r y weight c o nversion factor

ECB European corn borer

1 A l r e d, G

. J., C .T. Br us a w, and W.E. O li u. 2 0 0 3. H a nd b oo k of Tec h n i cal Wr iti ng, 7 t h e d n

. , p p

. 2

- 7

. Be d f o r d / S t

. M a r ti n ' s, B os to n, M A.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 25 o f 3 72CB I Delete d Copy

EDV extended diapause variant

E. coli Escherichia coli

ELISA enz y me

- linked im m unoso r bent assay

EPA Environ mental Protection Agency

ESCRT Endoso m al Sorting Co m plex Required for Transport

EUP experi m e ntal use per m it

ETS Excellence T hrough Stewardship

FA fatty acid

FDA U.S. Food a n d Drug Ad m i nistration

FIFRA Federal Insecticide, F ungi c ide and Rod e nticide Act

FMOC fluoren y l m e t h y l chloroformate

FSE farm scale e v aluation

fw fresh weight

GC gas chr o m a t o graphy

Gb gigabases

ha hectare

HPLC high

- perfor m ance liquid chro m a togra p h y

HRP horseradish peroxidase

HT herbicide tolerance

ILSI CCDB International Life Scien c es Institute

- Crop Co m position Database

IPM integrated pest management

IRM insect resis t ance m a nag e ment

JSC junction sequence cl a s s

kDa kilodalt o n

kg/hl kilogra m s per hectoliter

LOD li m it of detection

LOQ li m it of quantitation

MEEC maxi m u m expected environmental concentration

MESA 4

- Morpholin e pranesulfonic acid

- eth y lenedia m in e tetraa c etic a c id

-

sodium a c eta t e

MFI median fluorescence intens i ty

Mg/ha megagr a m s/ h ectare

m iRNA m i c ro RNA

MMT million me tr i c tons

MOA m ode

- of

- act i on

MOE margin of exposure

MVB m ulti

- vesicular bodies

n nu m ber of sa m ples

NCR northern corn rootworm

NDF neutral detergent fiber

NFDM nonfat d r y m i lk

NGS/JSA Next Generation Sequen ci ng/Juncti o n Se quence Anal y s is

NHANES National Hea l th and Nutrit i on Exa m inat i on Su r vey

NOAEL no observable adverse eff e ct level

NOEC no observable effect conc e ntration

nt nucleotide

NTO non

- target o r ganism

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 26 o f 3 72CB I Delete d Copy

OECD Organisation for Econo m ic Co

- operation and Develo pm ent

OM organic matter

OPA o

- phthalalde h yde

OSL over season leaf

OSR over season root

OSWP over season whole plant

PBST phosphate bu ffered saline containing 0

. 05% (v/v) Tw een

PCR pol y mer a se chain reaction

PIP plant incorporated protect a nt

Pol y (A) m ultiple ade n osine m onophosphates

PPA Plant Protection Act

PTH

- AA phen y lthiohydantoin

- a m i n o acid

QC

- negative qua l ity control

QC+ positive qual i ty control

RDR root da m a ge rating

RH relative hu m i di t y

RISC RNA

- induced silencing c o m plexes

RNA ribonucleic acid

RNAi RN A interfe r ence

RNase ribonuclease

RT room te m p erature

SAP Scientific Adviso r y Panel

SBV soybean variant

SCR southern corn rootworm

SD standard deviation

SDS sodium dodec y l sulfate

S.E. standard error

SGF si m ulated gastric fluid

SIF s i m ulated intestinal fluid

siRNA s m all interfering RNA

sp. species

TDF total dietary fiber

T

- DNA transfer DNA

TFA trifluoroacetic acid

TSSP tissue

- specific site pool

TTC threshold of t oxicolo g ical concern

Tz tetrazoli u m

USDA United States Depar t ment of Agriculture

UTR untranslated region

UV ultraviolet

v/v volu me to vo l ume

WCR western corn rootworm

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 27 o f 3 72CB I Delete d Copy

I. RATI O NAL E F O R T H E DEVELOP M EN T O F M O N 87411

I.A. Basis for the Request for a Deter minatio n o f Nonregulate d Statu s under

7 CF R § 340.6

The Ani m al and Plant Health Ins p ection Ser v ic e (APHIS ) o f th e Unite d State s (U.S.)

Depart m ent of Agriculture (USDA) has re s ponsibility, under the Pla n t Protection Act

(Titl e I V Pub

. L

. 106

- 224

, 11 4 S t at

. 438

, 7 U.S.C

. § 7701

- 7772)

, t o preven t the

introduction and disse m ination of plant pests into the U.S. APHIS regulation

7 CF R § 340. 6 provide s tha t a n applican t m ay petition APHIS to evaluate sub m itted data

to deter m ine that a particular regulated art i cle does not p resent a p l ant pest risk and no

longer should be regulated. If APHIS deter m ines that the regulated article does not

present a plant pest risk, the petition is g r anted, thereby allowing un r estri c ted introd u ction

of the article.

Monsanto Co mpany is sub m itti n g this req u est to APHIS f or a deter m ination of

nonregulated status for the new biote c hnology

- derived m aize p roduct

, MO N 87411

, any

progeny derived from c rosses bet w een MO N 8741 1 an d conventiona l m a ize

, an d any

progen y derive d fro m crosse s o f MO N 8741 1 with biotechnology

- derived m aize that

hav e previousl y bee n grante d nonre gulated status under 7 CFR Part 340.

I.B. Rationale for t h e Development of I n sect

- Pr o tected and Glyphosate Tolerant

Mai z e MON 87411

Maiz e ( Zea mays L. ) i s th e larges t cro p grow n i n th e U.S. in ter m s of acreage pla n ted and

net val u e. In 2012, m aize was pla n ted on over 9 7 m illion acres and grain harvested fr o m

87.4 m illion acres (U S DA

- NASS, 2013a). A v erage y ields in t h e pre v ious five y e a r s

range d fro m 14 7 bushel s pe r acr e (bu/A ) ( 2011 ) t o 16 5 bu/ A (2009 ) an d wer e valued

between $46.7 billion (2009) and $76.9 billion (2011) (USDA

- NASS, 2013d); however,

in 2012, a widespread drought in the U.S. resulted in an average yield of only 123 bu/A,

with a total production of about 10.8 billion b u shels (USDA

- NASS, 2013a), valued at

approxi m ately $77 billion (USDA

- NASS, 2013c).

In 2012, a p proxi m ately 85 m illion acres in t h e U.S. (or 88% of the total U.S. m aize

acreage ) wer e plante d wit h biotechnology

- derive d m a iz e hybr ids, and approxi m ately 64

m illion acres (or 67% of the total m aize acreage) were planted with m aize hybrids

contai n ing insecticidal crystal (Cry ) protein s derive d fro m Bacillus thuringiensis ( B t )

(USDA

- ERS, 2013). Of those 64 m illion acres, over 50 million acres were pla n ted with

co m bined

- trait hybrids containing B t and her b icide t o l e r a nce (HT) t r aits (USDA

- ERS,

2013). S ince the early to m id

- 2000’s, ma ny of these co m bined

- trait hybrids have

contai n ed multi p le B t genes with m u lti p le m odes

- of

- action (MOA) for robust and d urable

efficacy against a broad range of a bove

- ground le p i dopteran and below

- g round

coleopteran m aize pests. Esti m ates are t h at a pproxi m ately 50 m illi o n acres of corn

rootworm (CR W )

- protected hybrids were pla n ted in the U.S. in 2011 (Marra, et al.,

2012).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 28 o f 3 72CB I Delete d Copy

I.B.1 Benefits of Insect

- Protecti o n and Herbicide Tolerance Traits

The introduction of i nsect

- protecte d an d h erbicide tolerant (HT) biotechnology

- derived

m aize hybrids has been valuable to growers for two pri m ary reasons: 1) they per m it the

in

- crop application of broad

- spectrum agricult u ral herbicides for effective weed control,

whic h promote s th e adoptio n o f conservatio n t i llag e practices ; an d 2 ) the y provid e highly

effective targeted pest control to m a nage so m e of a grower’s most da m aging m aize pests.

The value of HT m aize hybrids to growers ha s bee n d e m o nstrate d b y th e significant

growth in acres planted to HT m aize. In 2000, just 7% of m a ize a c res were planted w ith

hybrids containing a trait con ferrin g herbicid e tolerance

, while that percentage had

increased to 73% by 2012 (USDA

- ERS, 2013). Co m petition for light, nutrie n ts, and

moisture re s ources by weeds can lead to proportional and signifi can t reduction s i n crop

yiel d (Knake

, e t al.

, 1990)

. Nu m e rou s studie s hav e show n t hat weed c o ntrol early in the

growing season is necessary to reduce yield losses in corn.

Insect

- protecte d m a iz e hybrid s hav e als o d e livere d significan t valu e t o grower s as

de m onstrated by significant growth in insect

- protected m aize acres planted. In 2000,

19 % o f plante d m a iz e acre s ha d insect

- pro tection t r ait s

, while t h at perce n tage had

increased to 67% by 2012 (USDA

- ERS, 2013). Inc l uded in that total a r e insect

- protected

products for above

- ground lepidopteran contr ol

, below

- groun d coleoptera n contro l and

hybrids containing traits for con t rol of both types of pests.

Of the several insect species t h at can cause da m a ge to m aize plants, the most da m aging in

m ajor U.S. m aize growing re g ions are larvae o f the CRW co m plex 2 ( D iabrotica spp.,

Coleoptera: Chryso m e l idae) (Chandler, et al., 2008). The corn rootworm complex

includes Diabrotica species that are significa n t pests of m aize including western C R W

( D. virgife r a vir g ifer a )

, norther n CR W ( D. barber i )

, an d souther n CR W ( D.

undecimpunctata howardi ). These insect lar v ae da m age m a ize by fee d i ng on the roots,

reducing the ability of t h e pla n t to a b sorb wat e r a nd nut r ients f rom the soil, and cau s ing

harvesting difficulties because of plant l odgi n g (Riedell, 1990; Spike and Tolle f son,

1991). CRW has long been descri b ed as the “ b illio n

- dollar pest” co mplex, based on costs

associ a ted with the a p plic a tion of soil in s ecticides and c rop losses f rom pest da m age

(Mitchell

, e t al.

, 2004)

. A s the adoption of CR W

- protection t rait s ha s increase d fro m its

first pla n ti n gs in 2003 to approxi m a tely 50 m illion acres in 2011 (Marra et al., 2012), the

use of these traits has led to the decreas e d use of conventional insecticides for CRW

control by more than 75% (U.S. EPA, 2011).

2 The c o rn rootworm c o m plex inc l ud e s D i a b r o tic a s p ecie s tha t ar e significa nt pes t s o f m ai ze i nc l u di ng

weste r n C R W ( D

. vir g ifer a vir g ifera )

, n o r t h e rn CR W ( D

. ba r ber i ), a nd s ou t he r n C R W ( D.

u n dec imp unc t at

a h o w ard i ).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 29 o f 3 72CB I Delete d Copy

I.B.2. Introduction of Insect

- Protectio n Trait s i n th e U.S.

Biotechnology traits providing protection against CRW were firs t introduce d i n the

United States in 2003 when YieldGar d ® Rootworm (MON 863 that expresses Cry3Bb1

protein) m aize hybrids were co mm ercialize d

. Thi s trai t provid e d a highl y effective

solution for CRW con t rol. Shortly after YieldGard ® Rootworm was launched, other

product s providin g CR W protectio n f r om Dow AgroSciences (Hercule x ® /DA S 59122

that expresses Cry34/35Ab1 proteins) and Syngenta (Agrisur e ® /MI R 60 4 tha t expresse s a

modified Cry3A protein (mCry3A)) were intro duce d (Marr a e t al.

, 2012)

. Followin g the

develop m ent and introduction of YieldGar d ® Rootwo r m m aize hybrids, Monsanto

introduced YieldGard VT Rootwor m /RR 2 ® ( MON 88017 that expresses Cry3Bb1 and

CP4 EPSPS proteins) with genes for both CRW protection and Roundu p ® herbici d e

toleranc e i n a singl e produ c t

. Tha t introductio n wa s followe d shortl y thereafte r by

co m bined

- trait hybrids containing B t proteins for both abov e

- ground lepidopteran and

below

- grou n d coleopt e r a n (CR W ) pest contr o l, a s well as he r bicide tol e r a nce (Yield G ard

VT Tripl e ®

, Genuit y ® V T T rip l e PRO ®

, and Genuit y ® S m artsta x ® ). Mon s a nto’s

YieldGard VT Triple (MON 88017 × MON 810) contains single B t protein s t o control

targeted lepidopteran (Cry1Ab) and CRW (Cry3Bb1) m aize pests while Monsanto’s

Genuit y V T Tripl e PR O (MO N 8801 7 × MO N 89034 ) provide s tw o B t protein s wit h two

MOAs (Cry1A.105 and Cry2Ab2) for l epidoptera n pest s an d a singl e B t protein

(Cry3Bb1) for CRW c ontrol. Th e join t introductio n o f S m artStax m aize hybrids by

Monsanto and Dow Ag r oSciences introd u ced the first m aize hybrids with six B t proteins

(Cry1A.105, Cry2Ab2, Cry1F, C r y34/35Ab1, and Cry3Bb1), three effective MOA

against the pri m ary lepidopteran pest s o f m a iz e i n th e U.S

. corn belt, and two effective

MOA against the pri m a ry CRW pests of m aize in t h e U.S. corn b e lt. These two MOA

provide d i n MO N 8741 1 (fro m C r y3Bb 1 protei n an d DvSnf 7 R NA) can be expected to

i m prove the durability of CR W

- protection traits and extend the use f ul li f eti m e of these

products ( B ates, et al., 2005; Roush, 1998). All of these noted CR W

- protected products

hav e provide d highl y effectiv e contro l o f CR W across wide growing regions in the U.S.

I.B.3. Development of CRW

- Protected a n d Glyphosat e Toleran t Maiz e MO N 87411

In its continuing efforts to provid e highl y effective

, du r a ble co n tr o l of CRW f o r its

custo m ers, Monsanto C o m pany has devel ope d biotechnology

- derive d m a iz e MO N 87411

tha t confer s protectio n agains t CR W ( Diabrotica spp. ) an d toleranc e to t h e h e rbicide

glyphosate. MON 87411 builds upon current B t protein

- base d CR W control technology

b y introducin g a ne w MO A base d o n RNA

- m e dia t ed gene suppression (RNAi) that offe r s

increased c o ntrol of tar g et insect pe s ts and will p rolong the d urability of existing CRW

controlling B t technologies.

® Y i e l dG a rd a nd Y i e ld Ga r d VT Ro o two r m /RR 2 are r e g i s t ered t r a de m arks o f Mo ns a n t o Te c h n o lo gy LL C

. Her c ulex is a r e gistered tr a de m a r k o f D o w A gr oScien c es LLC. A gri s ure i s a regis t ere d trade m a r k of

S y ng e n t a P a r tici pa ti ons A G.

® Yiel dG ar d V T Tr i p le

, Ge nu it y V T Tri p l e Pro

, Ge n u it y S m ar t S t a x, a n d Ro u nd up are reg i s t e r e d

t rade m arks of M ons a n t o Te c h n o lo gy LL C

.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 30 o f 3 72CB I Delete d Copy

MON 87411 contains a suppress i on cassette that expresses an inverted repeat sequence

designed to m atch the sequence in wester n cor n rootwor m (WCR ; Diabrotica virgifera

virgifer a ). The expression of the suppression cassette results in the fo r m ation of a

double

- strande d RN A (dsRNA ) transcript containing a 240 bp frag m ent of the W CR Snf7

gene (DvSnf7). Upon consu m ption, the plant

- produce d dsRN A i n MO N 8741 1 is

recognized by the CR W ’s RNA interference (RNAi) machinery (Hammond, 2005;

Ketting and Plasterk, 2 0 04; To m ari and Za m ore, 2005) res u lting in the down

- regulation

of the tar g eted DvSnf7 gene leading to CRW m ortali t y (Bolognesi, et al., 2012).

MON 87411 also produces a m odified Bacillu s thuringiensi s (subsp

. kumamotoensi s )

Cry3Bb1 protein to protect against CRW l a rva l feeding

. I n addition

, MO N 87411

contain s th e cp4 epsps gene from Agrobacteriu m sp

. strai n CP 4 tha t encode s fo r the

5

- enolpyruvylshiki m ate

- 3

- phosphate synthase (EPSPS) protein, whi c h confer s tolerance

to glyphosate, the active ingredient i n Roundu p agricultur al herbicides.

MON 87411 will provi d e benefits to growers s i m ilar to those obtained by use of existing

CR W

- protected m aize hybrids, which includes reduced use of insecticides, increased

yield protection, water conservation, and i n creased worker safety (Rice, 2004).

MON 87411 is also glyphosate tolerant and will continue to provide benefit s associated

with conservation tillage m ethods, including r educe d soi l erosion

, reduce d fue l an d labor

costs, i m proved air quality and conservation of soil m oisture (CTIC, 2011; Hurley, et al.,

2009; Towery and W erblow, 2010).

MO N 8741 1 wil l no t b e offere d fo r com m ercia l us e a s a stand

- alon e product, but w ill be

co m bined, t hrough traditional breeding m e t hods, with other deregulated biotechnology

derived traits to provide protection against both above

- g round and below

- ground maize

pests as w e ll as t o le r an c e to m ultiple herbicide s

. These n e xt gene r ati o n co m bined

- tr a it

m a iz e product s wil l offe r th e abilit y t o m axi m ize grower choice, improve production

efficiency, increa s e pest contr o l durabili t y, and improve grower profit potentials.

I.C. RN A I nterferenc e (RNAi)

I.C.1. App l ication s o f RNA i i n Plants

Naturally occurring RNA

- m ediated gene suppression (RNAi) in plants has been

previously docu m ented and includes selection f o r soybean seed coat c o l o r (Tuteja, e t al.,

2004) and m aize stalk color (Della Vedova, et al., 2005). In both of these instances,

production of chalcone synthase was suppressed leading to significantly decreased

pig m entation in soybean seed coats and m aize stalks, respectively. In addition, a low

glutelin rice variety has been studied and has been deter m ined to result from production

o f a dsRN A an d conco m itan t suppressio n o f g l uteli n gene s (Kusaba

, e t al.

, 2003)

. R N A

m ediated gene suppression has also been u s ed in a nu m ber of b i otechnology

- derive d food

crops that have previously been deregulated by USDA or other regulatory authorities

including virus resistant papaya, squash, pot ato

, commo n bean

, an d plu m a s wel l a s a

delayed ri p e ning to m ato and a soybean with altered oil co mpositi o n (Parrott, et al., 2 0 10).

Safety assess m ents have been conducted (Parrott et al., 2010; Petrick, e t al.

, 2013 ) and

global regulatory approvals have been obtained for products e m ploying RNAi gene

suppression.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 31 o f 3 72CB I Delete d Copy

I.C.2. App l ication s o f RNA i i n In s ects

RNAi can also achieve gene silencing in s u sceptible i n sects follo w ing ingestion of

dsRNAs (Bau m

, et al., 2007b; Terenius, et al.

, 2011 ; W h yard

, e t al.

, 2009)

. Insect

control products can be dev e lo p ed utilizing RNAi seque n ce

- sp e ci f ic gene s ilen c ing to

suppress g e nes critical for insect survival. Because of this se quence

- specifi c gene

silencing, these products have t h e potential to se l ectively target a na rro w grou p o f closely

relate d pes t specie s an d g reatly reduce the likelihood of advers e effect s o n non

- target

organis m s (NTOs), including those beneficial to agric u lt u re. The spectr u m of activity for

DvSnf 7 dsRN A ha s bee n show n t o b e narro w and acti v ity is only evi d e nt in a su bs et of

beetles wit h in the Gal er ucinae sub f a m ily of Chryso m elidae within the Order Cole o ptera

(Bach m a n

, e t al.

, 2013)

, a s describe d i n m o re detail in Section VI.E below.

I.D. Modes

- of

- Action o f the Inserted Genetic Components

I.D.1. Mode

- of

- Action of the RNAi Component of MON 87411

MON 87411 contains a DvSnf7 suppression cassette that expresses an inverted repeat

sequence designed to m atch the sequence in W C R an d thereb y utilize s th e RNA i pathw a y

to control C RW ( Diabrotica spp.). The expression of the s uppression cassette results in

the fo r m ation of a dsRNA transcript containin g a 24 0 b p fra g m ent of the WCR Snf7 gene

(DvSnf7)

. Upo n consumptio n o f MO N 8741 1 b y WCR

, DvSnf 7 dsRN A i s recognized

by the pest’s RNAi m a chinery, resulting in the down

- regulation of the targeted DvSnf7

gene lea d ing to WCR mortality (Bol o gnesi et al., 2012) (Figure I

- 1).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 32 o f 3 72CB I Delete d Copy

F ig u r e I

- 1. D iag r a m of M ON 87411 d s RN A O r al De liv er y t o S upp re ss D v Snf 7

Ex p re ssion via t h e RN A i P a t h w ay in CR W

The R NA i p a th w a y is a n a tu ra l p r o ce ss in e uk a r y otic o r g a nisms f or the re g ul a tion of

e ndog e nous g e ne e x p re s sion ( D y k x hoo r n, e t a l., 2003; P a rr ott e t a l., 2 0 1 0)

. The dsR N A

mol ec ule th a t a c tiv a t e s t h e m ec h a nism is f i r st p r o ce ss e d b y a c l a ss of R N a se II I e n z y m e s

ca ll e d D i c er s into sm a ll int erfe r i n g R N A s ( siR NA s

, ~21

- 24 nu c l e ot i d e s ) (Ha mmond,

2005; Z a mo r e

, e t a l., 2 0 0 0)

. The re sulting siR N A mol ec ul e s ar e th e n i n c o rpo ra t e d into

multip r ot e in R NA

- indu c e d sil e n c i n g c ompl e x e s ( R I S C )

, w hi c h f a c ilit a t e c ompl e m e nt a r y

s e qu e n c e rec o g nition a n d mR N A c l ea v a g e th a t l ea ds to sp ec i f ic supp re s s ion of the t ar g e t

mR N A (Ha mmond, 200 5 ; Tom ar i a nd Z a mo re

, 2 0 05 )

. I n the ca se of C RW th a t c onsume

M O N 87411, the D v S n f 7 g e n e in CRW is supp re ss e d.

D v S n f 7 wa s s e l ec t e d a s the t ar g e t mR N A in C R W d ue to i ts vit a l ce l l u l a r f un c tion th a t

ca n be sup p re ss e d a t r e l a tiv e l y low c o n ce nt r a tions w h e n t a r g e t e d b y ds R N A ( B a um e t a l.,

2007b )

. S n f 7 is a c l a ss E v a c uol a r so r ti n g p r ot e i n a nd b e lon g s to the E ndosom a l S o r ting

Compl e x R e qui re d f or T ra nspo r t ( E S CRT )

– II I c ompl e x

, w hi c h h a s b ee n sho w n to be

involv e d in so r ting of t r a nsm e mb ra ne p r ot e ins e n r oute to l y sosom a l d e g r a d a tion th r ou g h

the e ndosom a l

- a utoph a g i c p a th w a y in a numb e r o f o r g a nisms ( K im, e t a l., 2011; L e e a nd

Ga o, 2008; Rust e n, e t a l., 200 8 ; T e is, e t a l., 2 0 08; Vac c ar i, e t a l., 2 0 0 9 ) ( F i g u r e I

- 2 )

. E S CRT

- I I I c ompon e nts pl a y c r iti ca l r ol e s in distin c t st e ps of th i s p a th w a y (He nn e

, e t a l.,

2011; R o x r ud, e t a l., 201 0)

. Da ta h a ve sho w n th a t supp re ssion of D v S n f 7 in W CR l ea ds

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 33 o f 3 72CB I Delete d Copy

to accu m ulation of ubiquitinated protein s 3 desti n ed f or lysoso m a l degrad a tion

(Ra m aseshadri, et al., 2013). Sorting of tra n s m e m brane proteins is critical to regulate

signa l transductio n i n cell s an d a s such

, s uppressio n o f thi s so r tin g throug h th e E S CRT

III co mplex i m pairs cell ho m eostasis and fun c tioning, lea d i n g to cell u lar death and CRW

mortality. We have also shown syste m ic spread of the RNAi effect to ti s sues di s tal to t h e

WCR m idgut (Bolog n esi et al., 2012). S im ilar lethal phenotypes res u lting from

knockdow n o f Snf7 have been shown in Drosophila (Sweeney, et al., 2006) and C.

elegans (Michelet

, e t al.

, 2010).

Figure I

- 2. Model depicting endos o mal

- autophagy path w ay involved in

intracellul a r sorting and degradation of receptors along w ith other macrom o lecules

in a normal cell (left) and a DvSnf7 defi c ient c ell (right).

In the nor m al cell, internalization and ubiqu i tin a ti o n of cargo p r oteins (1) d e

- ubiqui t inati o n of

cargo proteins (2), biogenesis of m ulti

- v esicul a r bodies (MVB) (3), formation of autophagosomes

engulfing m acr o m olecules (4), and fus i on of late en d osomes, autophagoso m e and l y soso me s into

auto l y soso mes for degradation of cargo proteins and macr o m ol e cules (5) are depicted. In the

3 U b iq u iti n ati on i s a p o st

- tra n s lati on a l m od ificati on of a p rotei n i n w h ic h o ne or m o r e u b i qu iti n m o lec u les

ar e ad d e d t o t he pr o tei n (Pic k art

, 2 00 1 )

. U b iq u iti ns ar e smal l re gu lat o r y pro tein s f o u nd i n al l e uk ary o tic

cell s a nd t h ei r a dd itio n t o a pr o tei n of te n lea ds t o d e gr a d at i on o f t h a t p r o tei n. T h i s p r o ces s o f u b i qu itinati on

and pr o t e i n d e gr a da tio n a llo ws th e ce l l t o m odu l a t e t he c onc e n t ra tio n o f essen tia l pr o t e i ns w i t h i n t hat ce ll

.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 34 o f 3 72CB I Delete d Copy

DvSnf7 deficient cell, the i m p a i r ment of de

- ubiquitination, acc u mulation of autophagoso m es, and

failure of fusion of endo s omes, autophagosomes a n d l y soso mes and auto l y s o s o m a l activity are

highlighted (Ra m aseshadri et al., 2013).

Induction of RNAi

- m ediated gene suppression in insects via an oral route of exposure

requires efficient uptake of d s RNAs by m idgut cells followed b y suppressio n o f th e target

mRNA leading to significant effects on growth, develop m ent and survival. In the case of

WCR

, onl y th e relativel y lon g ds R N A ( e.g

., DvSnf7 240

- m er) is taken up by the insect

and significant biological activity w a s only observed with dsRNA sequences ≥ 6 0 bp

(Bolognesi et al., 2012). Finally, sever a l key points have been identified in

de m onstrating efficacy of MON 87411 against WCR: 1) oral deliver y / u ptake of dsRNA

into W CR gut cells, 2) suppression of the targeted DvSnf7 mRNA expression followed

by suppression of the production of the DvSnf7 protein, 3) syste m ic spread of

suppression of DvSnf7 e xpr e ssion beyond W CR m idgut tissues, and 4) growth inhibition

and W CR mortality (Bo l ognesi et al., 2012).

I.D.2. Modes

- of

- Action of the CP 4 EPSP S an d Cry3Bb 1 Proteins

MON 87411 contains the identi c al CP4 EPSPS protein that i s expresse d i n MO N 88017

m aize (USDA

- APHIS Petition N o. 04

- 125

- 01p) and nu m erous other Roundup Ready

crops ( m aize, cotton, soybean, canola, alfalfa, sugar beet). The CP4 EPSPS protein is

structurally si m ilar and functionally identical to endogenous pla n t EPSPS enzy m e s

, but

has a m uch reduced affinity for glypho s ate relative to endogenous plant EPSPS

(Padgette

, e t al.

, 1996)

. I n MO N 87411

, a s i n othe r Roundu p Read y plants

, aro m atic

a m ino acids and other m etabolites necessary for plant growth and develop m ent are

produced by the continued action of the CP4 EPSPS e nzy m e in t he presence of

glyphosate (Padgette et al., 1996).

MON 87411 also contains an expressio n cassett e tha t co des for the sa m e Cry3Bb1

protein as the expression cassette that is present in MON 88017 m aize (USDA

- APH I S

Petition No. 04

- 125

- 01p) that was granted non

- regulated status by USDA

- APHIS in 2006

(USDA

- APHIS, 2013). The a m ino acid sequ e nce deduced from the Cry3Bb1 expression

cassettes of MON 87411 and MON 88017 is also 99.8% identical to the deduced a m ino

acid seque n ce for Cry3Bb1 protein in MO N 86 3 (USDA

- APHI S Petitio n No

. 01

- 137

- 01p) that w as granted non

- regulated status b y USDA

- APHI S i n 200 2 (USDA

- APHIS,

2013). The use of B t

- containing crops in U.S. agricult u re has been widespread a n d the

mode

- of

- action ( i.e

., solubilization of Cry protein, processing to the active fo r m

, binding

to m idgut receptors and insertion of the toxin into cellular me m branes) and specificity of

B t protei n s has been studied e x tensively and is well understood ( G ill, et al., 1 992;

Whalon and W ingerd, 2003).

I.E. Product Efficacy

Monsanto conducted field trials in 2011 a nd 2012 to assess the efficacy of MON 87411

in reducing root da m age caused by CRW l a rvae. In both years, MON 87411 was

co mpared to genetically si m ilar control hyb r ids (one pare n t o f each hybrid was LH244)

which also contained biot echnology

- derive d MO N 8903 4 exp r essin g tw o Cr y proteins

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(Cry1A.105 and Cry2Ab2) for protection aga i nst above

- ground lepidopteran pests.

Cry1A.10 5 an d Cry2Ab 2 ar e no t act i v e agains t Coleoptera n ins e ct s an d therefor e d o not

i m pact root feeding by CRW larvae. Data were collected from replicated blocks at nine

trial locations in 2011 and five locations in 2012 from m a i ze production regions in Iowa,

Illinois, and Indiana.

In both years, when plants reached the V2 growth stage, five plants per plot were infested

with corn rootworm eggs at a rate of over 3200 eggs per plant. At the V10 growth stage,

these five plants were dug and the roots were washed and evaluated for feeding da m age.

The evaluations were based on a root da m age rating (RDR) (Oleson, et al., 2005) scale of

0 to 3, where 0 is no root da m age detected and a 3 is where a ll three below

- ground nodes

are co mpletely m issing or totally da m aged.

In 2011 trials, the average RDR ac r oss all 9 locations for control hybrids not containing

Cry3Bb 1 o r DvSnf 7 d s RN A wa s 1.5

. D a m a g e ratings across these location s range d from

0.9 to 2.4. These ratings are ind i cative of the relatively high rootworm pressure overall.

The average RDR for M ON 87411 hybrids was 0.13, d e m onstrating significant efficacy

against larval CRW fe e d ing. In 2012 trials, the average R D R across the 5 locations for

control hybrids was 1.06, confir m ing significa n t rootworm pressure. Averaged RDR’s

across these sites for MON 87411 hybrids in these trials was 0.07, again de m onstrating

significa n t efficacy against larval C RW feeding.

I.F. Submissions to Other Regulatory Agencies

Under the Coordinated Fra m ework for Regulation of Biotechnology (CFR) (USDA

APHIS, 1986), the responsibility for regulatory oversight of biotechnology

- derive d crops

falls p ri m arily on three U.S. agencies: U .S

. Foo d an d Dru g Ad m inist r ation (FDA), t h e

United States Depart m e nt of Agriculture (USDA), and in the case of plant inc o rp o rated

protectants (PIPs), the Environ m e ntal Pro t ectio n Agenc y (EPA)

. Deregulatio n of

MON 87411 by USDA constitutes only one co mponent of the overall regulatory

oversight and review of this product. A s a practica l matter

, MO N 8741 1 canno t be

rel e ased and m arketed until FDA, EPA, and USDA have completed t h eir re v iews a nd

assess m ents under their res p ective jurisdictions.

I.F.1. Submission to FDA

MO N 8741 1 fall s withi n th e scop e o f th e 199 2 FD A polic y state m en t concerning

regulatio n o f product s deriv e d fro m ne w plan t varieties

, including those develop e d

through bi o t echnology (U.S. FDA, 1992). In compliance with this policy, Monsanto will

be sub m itti n g a food/feed safety and nut r itio n al assess m ent summary docu m ent to FDA

in the n e ar f uture.

I.F.2. Submission to EPA

Substances that are pesticides, as defined under the Federal Insecticide, Fungicide and

Rodenticid e Ac t (FIFRA ) [ 7 U.S.C

. §136(u)]

, are subject to regulation by the EPA.

Pesti c ides p roduced in planta

, referred to as PIPs, are also subject to regulation by the

EP A unde r FIFRA.

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Pursuant to §408(d) of the Federal Food Drug and Cos m etic Ac t [2 1 U.S.C

. 34 6 a(d)],

Monsanto Co mpany petitioned E P A for an exe mption from the require m ent of a

tol e rance f o r the PIP B acillus thuringiensis Cry3Bb 1 protei n i n o r o n al l foo d an d fe e d

commodities of field corn, sweet c o rn, and popcorn and t h e genetic m aterial nece s sary

fo r it s productio n i n thes e product s i n 1997

. O n Marc h 31

, 2004

, th e EP A establishe d a

per m anent exe mption from the require m ent of a tolerance for the PIP Bacillus

thuringiensis Cry3Bb1 protein and the genetic m ater i al necessary for its production in

foo d an d fee d com m odi t i e s o f fiel d corn

, swee t cor n an d popcor n (4 0 CF R § 180.1214).

Additionally, and applicable to M O N 87411, is EPA’s establish m ent of an ex e mption

f rom the re q uire m ent of a tol e rance f or re s idues of nucleic a cids t h at a re part of a plant

incorporated protect an t (4 0 CF R § 174.475).

On May 4, 2012, Monsanto filed an experi m ental use per m it (EUP) application for

MON 87411 and the genetic m aterial necessary for its prod u ction with the U.S. EPA to

facilitate MON 87411 field te s ting and safety evaluations. EP A grante d th e EU P (524

-

EUP

- 104 ) o n Marc h 1

, 2013

. Monsant o wil l m a k e a n applicatio n t o th e EP A fo r a

Breeding Registration for MON 87411 and the genetic m aterial ( P V

- ZMIR10871)

necess a ry f or its prod u ction in m aize in the n e ar f uture. Addition a ll y

, Monsanto will

m ake the a ppropriate Section 3 registration application(s) when final decisions about

specific stacked m aize products (breeding stacks) are m ade.

I.F.3. Submissions to Fore i gn Government A gencies

Consistent with our com m i t m ents to t h e Biotechnology Industry Organization’s

Excellence Through Stewardshi p ® (ETS) Progra m 4

, Monsant o intend s t o obtai n the

appropriate approvals from all key m aize i m port m arkets with functionin g regulatory

syste m s prior to com m e r cial planting of M O N 87411. As appropriate, notifications will

be m ade to countries that i m port significa n t quantities of m aize and m aize products and

do not have fo r m al regulatory review p r ocesse s fo r biotechnology

- derive d crops.

® Exce lle nce T h r o u gh S t e wa r d s h i p i s a r e g i s t ered trade m ark of Excelle n ce T h r ou gh S t ew a r dsh i p,

Wa s hington, DC

. 4 h tt p ://www.excelle n ce t h r o ug h stewards h i p .or g /

.

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II. TH E BIOLOG Y O F MAIZE

The Organisation for Econo m ic Co

- Operati o n and Develop m ent (OECD) Consensus

Docu m ent on the biology of m aize (OECD, 2003) provides key infor m a tion on:

- genera l descriptio n o f maiz e biology

, including taxono m y and m orphology and use

of m aize as a crop plant

- agrono m ic practices in maize cultivation

- geographic centers o f origin

- reproductiv e biology

- cultivated m aize as a volunteer weed

- int e r

- sp e cie s /genus i n t r ogression into r e lati v es and i n tera c tions with oth e r

organis m s

- a sum m ary of the ecol o gy of m aize

Additional info r m ation on the biology and u s e s o f m a iz e ca n als o b e foun d o n the

Australian Govern m ent Depart m ent of Health an d Agein g (Offic e o f th e Gene

Technology Regulator) web site (OGTR, 2008), and in the USDA

- ARS GRI N database

(USDA

- ARS, 2013).

To support the evaluation of the plant p e st potential of MON 87411 relative to

conventio n a l m aize, additio n al inf o r m ation regarding se v eral aspects o f m aize biology

can be found elsewhere in this petition. This includes: agr o no m ic practices for m aize in

Section IX; volunteer m anag e m ent of maize in Section IX .H; and inter

- spe c ies/ g enus

introgression potential i n Sectio n X.I.

II.A. Mai z e as a Crop

Maize is g r own in nea r ly all a reas of the w o rld and is the largest culti v ated crop in the

world followed by wh e at ( Triticum sp.) and rice ( Oryza sativa L.) in t o tal glo b al met r ic

ton production. In 2012, m aize w a s planted glo b ally on ~174 m illion hectares (ha) with a

tota l grai n productio n o f a n estimate d 85 4 millio n m e tri c ton s (MMT ) (USDA

- FAS,

2013). The top five production regions in 201 2 were : U S A (27 4 MMT)

, Chin a (208

MMT), Brazil (73 MMT), EU

- 27 (55 MMT), and Argentina (27 MMT) (USDA

- FAS,

2013). In the U.S., m ai z e is grown in al m o st every state and in 2012, its production value

of over $77 billion was the highest o f an y cro p (USDA

- NASS

, 2013c).

In industrialized countries m a ize has two m ajor use s : (1) as ani m al feed in the form of

grain, f ora g e or silag e ; and (2) as a raw m ateri a l f or wet

- or dr y

- m illed processed

products such as high fructose m aize syrup, o i l, starch, glucose, dextrose and ethanol.

By

- products of the wet

- and dry

- m ill proce s s es are also used as ani m al feed. These

processed products are used as ingredients in m a ny industrial applications and in hu m an

food products. Most m aize produced in industriali z ed countries is used as ani m al f e ed or

for industrial purposes, but m aize re m ains an i m portant food staple in many developing

regions

, especiall y sub

- Sahara n Afric a an d Cen tral A m erica, where it is frequently the

m ainstay of hu m an diets (Morris, 1998).

Maize is a v ery f a m iliar plant t h at h a s been r i g o r ously stu d i e d due to its use as a st a ple

food/feed and the econo m ic opportunity it brings to growers. The do m estication of maize

M ons a n t o Co m pany CR 2 4 0

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likel y occurre d i n souther n Mexic o between 7,000 and 10,000 y ears ago (Good m an,

1988). W h ile the putative pro g enito r specie s o f m a iz e hav e no t bee n recovered

, i t is

lik e ly th a t t e osinte p lay e d an i mportant r o le in contributing to t h e geneti c backgroun d of

m aize. Although grown extensively throughout the world, m aize is not considered a

persistent weed or a plant that is difficult to control. Maize, as we know it today, cannot

survive in the wild be c ause the f e m ale i n florescence (the ear) is co v ered by a h usk

thereby restricting seed disper s al, it h as no seed dor m ancy, and is a poor co m petitor in an

un m anaged ecosyste m . The transfor m ation from a wild

, weed y specie s t o on e dependent

on hu m ans for its survival m ost likely e volved over a long period of time through plant

breeding by the indigenous i nhabitants of the Western He m i sphere. Today, virtually all

the m aize grown in the U.S. is a hybrid, a pr oductio n practic e tha t starte d i n th e 1930’s

( W ych

, 1988)

. Maiz e hybrid s ar e develop e d an d use d base d o n the positive yield

increases and plant vigor associated with heterosis

, als o know n a s hybri d vigor.

Conventional plant breeding res u lts in desirable characteri s tics in a plant through the

unique combination of genes al r ea d y prese n t in the pla n t. However, there is a li m it to

genetic diversity with conventional plant br eeding

. Biotechnology

, a s a n additiona l tool

to conventional breeding, offers access to g r eater g enetic diversity than conventional

breeding alone, resulting in expression of highly desirable tr a its that ar e profitabl e to

growers.

II.B. Characteristics o f the Recipient Plant

The transfor m ation for MON 87411 was conducted with inbred m aize line LH244, a

patented m a ize line assigned to Holden’s Foundatio n Seed s LL C i n 200 1 (U.S

. patent

#6,252,148). LH244 is a m edium season yellow den t m a iz e lin e wit h a Stif f Stalk

background that is best adapted to the central regions of the U.S. corn belt. LH244 was

initiate d fro m a singl e cros s o f LH19 7 × LH19 9 followe d b y a backcros s t o LH197

. The

F 2 co m bination ((LH19 7 × LH197 ) × LH199 ) wa s the n selfe d an d use d i n the

develop m ent of LH244.

Following transfor m ation of immature LH244 embryos, a single transfor m ed plant w a s

selected and self

- crossed to increase seed supplies. A ho m ozygous inbred line was

developed though further self

- crossing and sel e ction and was then used to produce other

lines which were used for product testing, safety assess m e nt studies, and commercial

production.

II.C. Mai z e as a Test System in Product Safety Assessment

Based on s eed av a ilability and ap p ropri a te f it f or vario u s studies, hy b rid m aize lines

MPA640 B (LH24 4 × LH287 ) an d NL616 9 (LH24 4 × HCL645 ) wer e use d a s near

isogenic, conventional c ontrols for this sub m ission (hereafter referred to as conventional

controls). A s noted, one parent of each of these control m aize lines is LH244, the inbred

from which MON 87411 is derived, while the other parents ( LH28 7 an d HCL645 ) are

other m aize inbreds. As such, both of these m aize lines co n stit u te r ele v ant co mparato r s

for MON 87411. In ad d ition, o ther commercial m aize hybrids (hereafter referred to as

reference hybrids) were used to e s t a blish r anges of natural varia b ility r eprese n tati v e of

commercial m aize hybrids. Reference hybrids used at each field trial location were

sele c ted ba s ed on their availa b ility and a g rono m ic fit for the respective geographic

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regions. Both MPA640B and NL6169 were used in m olecular characterization studies.

NL6169 was used as the conventio n al co n tr o l in co m positi o nal a n aly s is while MP A 640B

was used in phenotypic, agrono m ic and environ m ental interactions assess m ents. Where

appropriate, reference hybrids were used to establish a range of variability or responses

repre s ent a tive of commercial m aize in the U.S.

In developing the data to support this p e tition, appropriate MON 87411 test m aterials

were generated for t h e m olecular charact e rization (Sections III and IV), protein

characterization and expression analysis (Section V), RNA characterization and

expres s ion (Section V I), co mposition a l an al ysis (Section VII), a nd phenot ypic,

agrono m ic and environmental i n teractions a ss es sm ent (Section VIII). The full m olecular

characterization studies (NGS/ J SA) were conduct e d with the R 4 generation (Figure I V

- 4).

Initiation of commercial breeding efforts was conducted with the R 5 generatio n (Figure

IV

- 4). Pr o t ein and RNA chara c te ri zation and e xpression a n alysis, co mp osition an al ysis,

and phenotypic, agrono m ic and environ m ent a l interactions assess m ent were conducted

wit h variou s MO N 8741 1 breedin g generat i on s a s note d i n th e Breedin g Tree

(Figure IV

- 4 ).

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III. DESCRIPTI O N O F T H E G ENETI C MODIFICATION

MON 87411 was developed through Agrobacterium tumefaciens

- m ediated

transfor m ation of m aize im m a ture embryos from line LH 2 44 utilizing PV

- ZMIR10871.

This section describes the plas m id vector, the donor gene, and the regulatory ele m en t s

used in the develop m ent of MON 87411 as well as the deduced a m ino acid sequence of

th e Cry3Bb 1 protei n an d CP 4 EPSP S protei n produce d i n MO N 87411

. In this section,

transfe r DN A (T

- DNA ) refer s t o DN A tha t is trans f erred to th e plan t during

transfor m ation. An expression (or suppression) cassette is c o mprised of sequences to be

transcri b ed and the reg u lat o ry ele m ents nece s sary for the ex p ression of t h ose sequen c es.

III.A. The Plasmid Vector PV

- Z MIR10871

PV

- ZMIR10871 was used in the transfo r m ation of m aize to produce M O N 87411 and its

plas m id m a p is shown in Figure III

- 1. The ele m ents incl u ded in this plas m id ve c tor a re

described in Table III

- 1. PV

- ZMIR10871 is approxi m ately 16.5 kb and contains one

T

- DN A tha t i s delineate d b y Lef t an d Righ t B o rde r regions

. Th e T

- DN A contain s the

DvSnf7 suppression cassette, t h e cry3Bb1 expres s ion cassette, a n d the cp4 epsps

expressio n cassette

. Th e DvSnf 7 suppression ca s sette is reg u lated by the e35S pro m oter

f rom the 35S RNA of cauli f lower m osaic virus (CaMV), the heat shock protein 70

( Hsp70 ) intron from Zea may s

, and the 3' untranslated sequence of the E 9 gene f r om

Pisu m sativu m

. The cry3Bb1 expression cassette is regulated by the pII G pro m oter fr o m

Zea may s

, the chlorophyll a/b binding protein (CAB) leader from Triti c um aestivu m

, the

Ract1 intron from Oryza sativa

, an d th e hea t shoc k protei n 1 7 ( Hsp17 ) 3 ′ untranslated

region from Triticum a estivu m

. The cp4 epsps expres s ion cassette is regulated b y the

Tub A promoter from Oryza sativa

, the Tub A leader from Oryza sativa

, the Tub A intr o n

fr o m Oryza sativa

, the CTP 2 targeting sequence from Arabidopsis thaliana

, and the TubA

3 ′ untranslated region from Oryza sativa

.

The backbone region of PV

- ZMIR10871, located outside of the T

- DNA, contains two

origins of replication for m aintenance of the plas m id vector in bacteria ( o ri V

, ori

pBR322 ), a bacterial selecta b le m arker gene ( aad A ), and a coding sequence for

repressor of pri m er ( R OP) protein for m aintenance of plas m id vector copy nu m ber in

Escheric h ia coli ( E

. coli ). A description of the genet i c ele m ents and their prefixes ( e

. g

. , B

- , P

- , L

- , I

- , TS

- , CS

- , T

- , and OR

- ) in PV

- ZMIR10871 is provided in Table III

- 1.

III.B. Description of the Transformation System

MON 87411 was developed through Agrobacteriu m

- m ediated transfor m ation of

im m ature maize e m bryos based on the m e t hod described by Sidorov and Duncan (2009),

utilizing P V

- ZMIR10871. I m m ature e m bryos were excised from a po s t

- pollinated m aize

ea r o f LH244

. Afte r co

- culturin g the excised im m ature e m bryos with Agrobacterium

carr y ing the plas m id vector, the i mm ature e m bryos were p laced on selection m edi u m

contai n ing g lyphosate and carbe n icillin diso d i u m salt in order to inhi b it t h e growth of

untransformed plant cells and excess Agrobacterium

. Onc e transfo r m ed callus

developed, the callus was placed o n m edia c onducive to shoot and root develo pm ent.

The rooted plants ( R 0 ) with nor m al phenotypic charact e ristics were selected and

transferred to soil for growth and further ass e ss m ent. As de m onstrated in this p e tition,

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the use of disar m ed Agrobacterium tumefaciens strain ABI, a designat e d plan t pest

, a s the

transfor m ation vector has not i m parted plant pest characteristics to MON 87411.

The R 0 plant s generate d throug h th e transfor m a tion process describ e d abov e ha d already

been exposed to glyphosate in the selection m edium a nd de m onstrated glyphosate

tolerance. T he R 0 plants self

- pollinated to produce R 1 seed and R 1 plants were evaluated

for the presence of the T

- DNA via q uantitative poly m erase chain rea c tion (PCR) analysis.

The R 1 plants ho m ozy g ous for the T

- DNA we r e selecte d fo r furthe r develop m en t and

their proge n ies were s u bjected to f urther m olecular and phenotypic assess m ents. As is

typica l o f a com m ercia l even t productio n a n d selectio n process

, hundred s o f different

transfor m ation events (regenerants) were generated in the laboratory using PV

ZMIR10871. After m any m o nths of care f u l selectio n an d evaluatio n o f these

hundreds of events in the l aboratory

, greenhous e an d field

, MO N 8741 1 wa s selecte d as

the lead event based on superior agrono m ic, phenotypic, and m olecular characteristics.

Studies on MON 87411 were initi ate d t o furthe r characteriz e th e geneti c insertio n an d the

expressed products, and to establish the food, f e ed, and environ m ental safety relative to

commercial m aize. The m ajor steps involv e d in the develop m ent of MON 87411 are

depicted in Figure III

- 2.

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Figure III

- 1

. Circular Map of PV

- Z MIR10871

A circular map of the plas m id vector PV

- Z MIR10871 used to develop MON 87411 is shown.

PV

- ZMIR10871 contains a single T

- DNA. Gene t ic elements are shown on the exterior of the

map. P Superscript in DvSnf7 indicates partial sequence.

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Asse m bled Agrobacteriu m binary plas m id vector PV

- ZMIR10871 and

transferred to Agrobacterium tumefaciens s tr a in ABI

Transfor m e d LH244 (a m aize line for m ore eff i cient transfor m ation)

im m ature embryos with PV

- ZMIR10871 in Agrobacterium tumefaciens

Selected transfo r m ants and generated rooted shoots from the

trans f or m ed call u s tissu e s

Evaluated the transfor m ed plants for the prese n ce of the T

- DNA and

selecte d homozygou s plant s b y qua ntitative poly m erase chain reaction

(PCR) anal y ses

Evaluated p l ants f or ins e rt i n teg rit y b y Souther n blo t analysis

Identified MON 87411 a s lead candidate and further evaluated its

progeny in laboratory and field a ssess m ents for insert inte g rity,

glyphosate tolera n ce, efficacy a g ainst CRW larval da m age and superi o r

phenotypic characteristics

Figure III

- 2

. Schematic of the De v elopment o f MON 87411

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III.C. Th e 

### cry3Bb 1 C o din g Sequenc e an d th e Cry3Bb 1 P r otein

The cry3B b 1 expression cassette encodes a 74.5 kD a Cry3Bb 1 protei n consistin g o f a

single polypeptide of 653 a m ino acids (Figure III

- 3). The cry3Bb 1 codin g sequenc e is

the codon opti m ized coding sequence from Bacillus thuringiensis t h at encodes the

Cry3Bb1 protein (English, et al., 2000). The presence of Cry3Bb 1 protei n provides

protection from corn rootworm feeding.

III.D. Th e 

### cp 4 epsp s Codin g Sequenc e an d th e CP 4 EPSP S Protein

The cp 4 epsp s expressio n cassette

, encode s a 47. 6 kD a CP 4 EPSP S protei n consistin g of

a single polypeptide of 455 a m ino acids ( F igure III

- 4) ( P adgette et al., 1996). The

cp4 epsps coding sequence is the codon o p ti m ized coding sequence of the aroA gene

fr o m Agrobacte r ium sp. strain CP4 encoding CP4 EPSPS (Barry, et al., 2001; Padgette et

al., 1996). The CP4 EPSPS protein is si m ilar and functionally i dentica l t o endogenous

plant EPSPS enz y m es, but has a m uch red u ced affinity for glyphosate, the active

ingredient in Roundup a g ricultural herbicides, relative to endogenous plant EPSPS (Barry

et al., 2001; Padgette et al., 1996). The presence of this protein renders the plant tolerant

to Roundup.

III.E. DvSnf 7 p sequence

Th e DvSnf 7 p sequenc e i s th e partia l codin g sequenc e o f th e Snf7 gen e f r o m Diabrotica

virgifera vi rg ifera (Baum et al., 2007b; Bau m

, et al., 2007a) encoding the SNF7 subunit

of the ESCRT

- III co m plex (Bab s t, et a l., 20 0 2). The DvSnf7 suppression cas s ette

contain s tw o 24 0 b p DvSnf 7 p s e quences in an inverted orientation. There is an

intervening sequence of 149 nucl eotide s betwee n th e tw o D v Snf7 p sequences (noted on

Tables III

- 1 and IV

- 1 ). W hen the s u ppression c a ssette is tra n scribe d

, th e RN A expressed

fo r m s a hairpi n loo p thereb y allowin g th e formatio n o f doubl e strande d DvSnf 7 RNA.

Th e DvS n f 7 p sequences in the suppression cassette produce a 240 bp d s RNA that upon

trans c ri p tion trig g ers the RNAi m echanis m

.

III.F. Regulatory Seq u ences

The cry3B b 1 coding sequence in MON 87411 is unde r th e regulatio n o f th e pIIG

pro m oter, the chlorophyll a/b binding protein (CAB) leader, the Ract1 intron

, an d the

heat shock protein 17 ( Hsp17) 3 ′ untranslate d region

. Th e pIIG pro m oter, which directs

trans c ri p tion in pla n t cells, is f rom the pIIG gene f a m ily encoding the physical i mpedance

induced protein from Zea mays (Huang, et al., 1998). The CA B leade r is t h e 5'

untranslated region from the chloro phyl l a/b

- bindin g (CAB ) protei n o f Triticu m aestivum

and is involved in regulating gene expression (La m ppa, et al., 1985). The Ract1 int r on is

the intron fr o m the act1 gene from Oryza sati v a (McElroy, et al., 1990). The Hsp1 7 3 ′

non

- translated region is the 3 ′ untranslated region from the heat shock protein, Hsp17, of

Triticum a e stivum (McElwai n an d Spiker

, 1989 ) tha t directs polyadenylation of the

mR N A

.

The cp4 epsps coding sequence in MON 87411 is under the regulation of the T u bA

pro m oter, the Tub A leader, the Tub A intron, the CTP 2 targeting sequence, and the TubA

3 ′ untranslated region. The Tub A pro m oter, which directs trans cr iption in pla n t c e lls, is

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 45 o f 3 72CB I Delete d Copy

fr o m the OsTub A gene fa m ily from Oryz a sativ a (rice) encoding α

- tubulin (Jeon, et al.,

2000)

. Th e Tub A intr o n is t h e i n t r on f rom the OsTub A gene fa m ily f r om Oryza sati v a

(ri c e) enco d i ng α

- tubulin (Jeon et al., 2000). The chl o roplast transit peptide CTP2 directs

transport of the CP4 EPSPS protein to the c h loroplast in MON 87411 and is derived fr o m

CTP 2 target sequence of the Arabidopsi s thalian a ShkG gene (Herr m ann, 1995; Klee, et

al., 1987). The Tub A 3 ′ non

- translated region is the 3 ′ untranslated region from the

OsTub A ge n e f a m ily fr o m Oryza sativa (rice) e n coding α

- tubulin (Jeon et al., 2000) that

directs polyadenyl atio n o f mRNA.

Th e DvSnf 7 p sequenc e i n MO N 8741 1 i s unde r the regulation of the e3 5 S pro m oter, the

heat shock protein 70 ( Hsp70) intron

, an d th e E 9 3 ′ untranslated region. The e35S

pro m oter

, whic h direct s transcriptio n i n p l ant cells, contains t h e duplicated enhancer

region (Kay, et al., 1987) from the cauliflower mosaic virus (CaMV) 35S RN A promoter

(Odell, et al., 1985). As de m onstrated i n thi s petition

, th e us e o f th e CaM V 35S promoter

containing the duplicated enha n cer region, derived from a d e signated plant pest, has not

i m parted plant pest characteristics to MON 87411. The hsp7 0 intron is the f irst i n tron

from the m a ize heat shock protein 70 gene (Brow n an d Santino

, 1997 ; Rochester

, e t al.,

1986). The E 9 3 ′ non

- translated region is the 3 ′ untranslated region from the rbcS gene of

Pisu m sativu m (pea) encoding the s m all subunit of ribulose bisphosphate carboxylase

protein (Coruzzi, et al., 1984) that directs polyadenyla t ion of the mRNA.

III.G. T

- D NA Borde r s

PV

- ZMIR10871 contains Right Border and L eft Border regions (Figure III

- 1 and

Table III

- 1 ) that were d erived from Agrobacterium tumefaciens plas m ids. The border

regions each contain a nick s i te that is the site of DNA exchange during transfor m ation

(Barker, et al., 1983; Depicker, et al., 1982; Zambryski, et al., 1982). The border regions

separate the T

- DNA from the plasmid backbone region and are involved in the efficient

transfer of T

- DNA into the m aize geno m e. As de m onstrated in this petition

, th e us e of

Right Border and Left Border regions derived from Agrobacterium tumefaciens

, a

designated plant pest, has not i mparted p l ant pest characteristics to MON 87411.

III.H. Genetic Eleme n ts Outside of the T

- DNA Borders

Genetic el e m ents that exist out s ide of the T

- DNA borders are those that are esse n tial for

the m aintenance or selection of PV

- ZMIR10871 in bacteria. The origin of replication

ori V is re q uired f or t h e m aintena n ce of the plas m id in Agrobacterium an d i s derived

from the broad host plas m id R K 2 (Stalker, et al., 1981)

. Th e origi n o f replication

ori

- pBR322 is required for the m aintenance of the plas m id in E. coli and is derived from

the plas m id vector pBR322 (Sutcliffe, 1979). Coding s equence r o p is the coding

sequence of the repressor of pri m er (ROP) p r otein and is necessary for the m aintenance

of plas m id copy nu m ber in E. c o li (Giza and H uang, 1989)

. The selectable m arker aadA

is a bacterial pro m oter and coding sequence for an enzyme from transposon T n 7 that

confers spectino m ycin and streptomycin re s istance (Fling, et al.

, 1985 ) i n E

. col i and

Agrobacteriu m during molecular cloning. Because these ele m ents are outside t h e border

regions, they are not e x pected to b e tra n sf e rred into the m aize geno m e. The abse n c e of

the backbone and other unintended plas m id sequence in MON 87411 has been confir m ed

by sequencing and bioinfo r m atic analyses (see Section IV.A).

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Table III

- 1. Summary of Gen e tic Elements in PV

- Z M I R10871

Genetic Element Location in

Plasmid

Vector Functio n (Reference)

T

- DNA

B 1

- Left Bo rd er

Region 1

- 442 DN A regio n fr o m Agro b a cterium

tumefaciens containing the left border

sequence used for trans f er of the T

- DNA

(Barker et al., 1983)

Intervening Sequence 443

- 485 Sequence used in DNA cloning

T 2

- E9 486

- 1118 3′ UTR of the rbcS gene f a m ily from

Pisu m sativu m (pea) encoding the s m all

subunit of ribulose bisphosphate

carboxylase protein (Coruzzi et al., 1984)

that directs polyadenylation of the mRNA

Intervening Sequence 1119

- 1147 Sequence used in DNA cloning

DvSnf 7 p 1148

- 1387 Partial coding sequence of the Snf7 gene

fr o m Diabrotica virgifera virgifera (Ba u m

et al., 2007b; Baum et al., 2007a)

encoding the SNF7 subunit of the

ESCRT

- III co m plex (Babst et al., 2 002)

that for m s part of the s u ppression cassette

Intervening Sequence 1388

- 1537 Sequence used in DNA cloning

DvSnf 7 p 1538

- 1777 Partial coding sequence of the Snf7 gene

fr o m Diabrotica virgifera virgifera (Ba u m

et al., 2007b; Baum et al., 2007a)

encoding the SNF7 subunit of the ESCRT

III co mplex (Babst et al., 2 002) that for m s

part of the s u ppression cassette

Intervening Sequence 1778

- 1813 Sequence used in DNA cloning

I 3

- Hsp70 1814

- 2617 Intron and flanking exon sequence of the

hsp70 gene from Ze a m a y s ( m aize)

encoding the heat shock protein 70

(HSP70) (Rochester et al., 1986) that is

involved in regulati n g gen e expression

(Brown and Santino, 1997)

P 4

- e35S 2618

- 3238 Pro m oter f r om the 35S RNA of

cauli f lower mosaic vir u s (CaMV) (Odell

et al., 1985) containing the duplicated

enhancer region (Kay et al., 1987) that

dire c ts tran s cri p tion in p lant c ells

Intervening Sequence 3239

- 3264 Sequence used in DNA cloning

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Table III

- 1 (continued). Summary of Genetic Elements in PV

- Z M I R10871

Genetic Element Location in

Plasmid

Vector Functio n (Reference)

P

-

pIIG 3265

- 4213 Pro m oter sequence of the pIIG gene

encoding the physical impedance induced

protei n fro m Zea mays (Huang et al., 1998)

( m aize) that dire c ts tran s cri p tion in p lant

cells

Intervening Sequence 4214

- 4219 Sequence used in DNA cloning

L 5

- Cab 4220

- 4280 5' UTR leader sequence from chlorophyll

a/b

- binding (CAB) protein of Tritic u m

aestivum (wheat) that is involved in

regulating gene expression (La mppa et al.,

1985)

Intervening Sequence 4281

- 4296 Sequence used in DNA cloning

I

- Ract1 4297

- 4776 Intron and flanking UTR sequence of the

act1 gene from Oryza sativa (rice)

encoding rice Actin 1 protein is involved in

regulating gene expression (McElroy et al.,

1990)

Intervening Sequence 4777

- 4785 Sequence used in DNA cloning

CS 6

- cry3Bb1 4786

- 6747 Codon opti m ized coding sequence f rom Cry3Bb1 protein of Ba c illus thu r in g iensis

that provides insect r e sistance (English et

al., 2000)

Intervening Sequence 6748

- 6766 Sequence used in DNA cloning

T

- Hsp17 6767

- 6976 3' UTR sequence from a heat shock

protein

, Hsp17

, o f Triti c um aestivum

(wheat) (McElwain and Spiker, 1989) that

directs polyadenylation of the mRNA

Intervening Sequence 6977

- 7024 Sequence used in DNA cloning

P

- TubA 7025

- 9205 Pro m oter, 5′UTR leader and intron

sequences of the OsTub A gene f a m ily from

Oryza sativa (ri c e) e n co d ing α

- tubulin

(Jeon et al., 2000) that directs transcription

in pla n t cells

Intervening Sequence 9206

- 9209 Sequence used in DNA cloning

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Table III

- 1 (continued). Summary of Genetic Elements in PV

- Z M I R10871

Genetic Element Location in

Plasmid

Vector Functio n (Reference)

TS 7

- CTP2 9210

- 9437 Targeting sequence of the ShkG gene from

Arabidopsis thaliana encodin g th e E PSPS

transit peptide region t h at directs transport

of the protein to the c h l o ropla s t

(Herr m ann, 1995; Klee et al., 1987)

CS

- cp4 epsps 9438

- 10805 Codon opti m ized coding sequence of the

aroA gene from Agrobacterium sp

. strain

CP 4 encodin g th e nativ e CP 4 EPSPS

protein that provides herbicide tolerance

(Barry et al., 2001; Padgette et al., 1996)

Intervening Sequence 10806

- 10812 Sequence used in DNA cloning

T

- TubA 10813

- 11394 3' UTR sequence of the OsTub A gene

f

a m ily fr o m Oryza sativa (ri c e) e n co d ing α

tubulin (Jeon et al., 2000) that directs

polyadenylatio n o f mRNA

Intervening Sequence 11395

- 11412 Sequence used in DNA cloning

B

- Right Border

Region 11413

- 11743 DN A regio n fr o m Agro b a cterium

tumefaciens containing the right border

sequence used for trans f er of the T

- DNA

(Depicker et al., 1982; Z a m bryski et al.,

1982)

Vecto r Backbone

Intervening Sequence 11744

- 11879 Sequence used in DNA cloning

aadA 11880

- 12768 Bacterial pro m oter, coding sequence, and

3' UTR for an a m inoglycoside

- m odifying

enzy m e, 3' ' ( 9)

- O

- nucle o tidyltra n sferase

from the transposon T n 7 (Fling et al.,

1985) that confers spectino m ycin and

strepto m ycin resistance

Intervening Sequence 12769

- 13298 Sequence used in DNA cloning

O R 8

- or i

- pBR322 13299

- 13887 Origin of replication from plas m id pBR322

for m aintenance of plasmid in E

. coli

(Sutcliffe, 1979)

Intervening Sequence 13888

- 14314 Sequence used in DNA cloning

CS

- rop 14315

- 14506 Coding sequence for repressor of pri m er protein from the ColE1 plas m id for

m aintenance of plas m id copy nu m b e r in E.

coli (Giz a an d Huang

, 1989)

Intervening Sequence 14507

- 16014 Sequence used in DNA cloning

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Table III

- 1 (continued). Summary of Genetic Elements in PV

- Z M I R10871

Genetic Element Location in

Plasmid

Vector Functio n (Reference)

OR

- ori V 16015

- 16411 Origin of replication from the broad host

range plasmid RK2 for m aintenance of

plas m id in Agrobacteriu m (Stalker et al.,

1981)

Intervening Sequence 16412

- 16497 Sequence used in DNA cloning

1 B

, B o r d er

2 T

, Tra n scri p ti on Ter m i n ati on Seq u e n ce

3 I, I nt ro n

4 P, P ro m o t er

5 L

, Le a d er

6 C S, Co d in g S e qu e nce

7 TS

, Ta rg etin g Seq u e n ce

8 OR, Ori g i n o f Re p licati on

p S up e rscri pt i n D v S n f 7 i nd icate s t he p artia l se qu e n ce

. Withi n t he D v S n f 7 cassette , b ase s 1 14 8

- 1 3 8 7 are

rev e rse c o m p le m e nt t o b a ses 1 53 8

- 1 7 7 7.

M ons a n t o Co m pany CR 2 4 0

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1 MANPNNRSEH DTIKVTPNSE LQTNHNQYPL ADNPNSTLEE LNYKEFLRMT EDSSTEVLDN

61 STVKDAVGTG ISVVGQILGV VGVPFAGALT SFYQSFLNTI WPSDADPWKA FMAQVEVLID

121 KKIEEYAKSK ALAELQGLQN NFEDYVNALN SWKKTPLSLR SKRSQDRIRE LFSQAESHFR

181 NSMPSFAVSK FEVLFLPTYA QAANTHLLLL KDAQVFGEEW GYSSEDVAEF YRRQLKLTQQ

241 YTDHCVNWYN VGLNGLRGST YDAWVKFNRF RREMTLTVLD LIVLFPFYDI RLYSKGVKTE

301 LTRDIFTDPI FLLTTLQKYG PTFLSIENSI RKPHLFDYLQ GIEFHTRLRP GYFGKDSFNY

361 WSGNYVETRP SIGSSKTITS PFYGDKSTEP VQKLSFDGQK VYRTIANTDV AAWPNGKVYL

421 GVTKVDFSQY DDQKNETSTQ TYDSKRNNGH VSAQDSIDQL PPETTDEPLE KAYSHQLNYA

481 ECFLMQDRRG TIPFFTWTHR SVDFFNTIDA EKITQLPVVK AYALSSGASI IEGPGFTGGN

541 LLFLKESSNS IAKFKVTLNS AALLQRYRVR IRYASTTNLR LFVQNSNNDF LVIYINKTMN

601 KDDDLTYQTF DLATTNSNMG FSGDKNELII GAESFVSNEK IYIDKIEFIP VQL

Figure III

- 3

. Deduced Amino Acid Sequence of the Cry 3 Bb1 Protein

The a m ino acid sequence of the Cr y 3Bb1 p r otein was d e duced fr o m the full

- length co d ing

nucleotide sequence present in PV

- ZM I R1087 1

.

1 MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG LKKSGMTLIG

6 1 SELRPLKVMS SVSTA C MLH G ASSRPATAR K SSGLSGTVR I PGDKSISHR S FMFGGLASGE

121 TRITGLLEGE DVINTGKAMQ AMGARIRKEG DTWIIDGVGN GGLLAPEAPL DFGNAATGCR

181 LTMGLVGVYD FDSTFIGDAS LTKRPMGRVL NPLREMGVQV KSEDGDRLPV TLRGPKTPTP

241 ITYRVPMASA QVKSAVLLAG LNTPGITTVI EPIMTRDHTE KMLQGFGANL TVETDADGVR

301 TIRLEGRGKL TGQVIDVPGD PSSTAFPLVA ALLVPGSDVT ILNVLMNPTR TGLILTLQEM

361 GADIEVINPR LAGGEDVADL RVRSSTLKGV TVPEDRAPSM IDEYPILAVA AAFAEGATVM

421 NGLEELRVKE SDRLSAVANG LKLNGVDCDE GETSLVVRGR PDGKGLGNAS GAAVATHLDH

481 RIAMSFLVMG LVSENPVTVD DATMIATSFP EFMDLMAGLG AKIELSDTKA A

Figure III

- 4

. Deduced Amino Acid Seque n ce of the CTP2 Targeting Sequence and

CP4 EPSPS Protein

The transit peptide CTP2 for t he CP4 EPSPS p r otein is underlined. Acc u m ulation of the

CP4 EPSPS protein is targeted to the chloroplasts u s ing cleavable CTP2, the transit peptide of the

Arabidopsis t halia n a EP S PS protein. The a m ino acid sequence of the CP4 EPSPS protein was

deduced from the full

- leng t h coding nuc le otide sequence present in PV

- ZMIR10871.

M ons a n t o Co m pany CR 2 4 0

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IV. CHARACT E R I ZATIO N O F TH E G E NET I C M O DIF I CAT I ON

Characterization of the DNA insert in MON 87411 was conducted using a co m bination

of sequencing, PCR, and bioinfor m atics. The results of this characterization d e m onstrate

that MON 87411 contains one copy of the i n tended transfer DNA (T

- DNA) containing

th e DvSnf 7 suppressio n cassett e an d th e cry3B b 1 and cp 4 epsp s expres s ion ca s settes that

is stably i n tegrated at a single loc u s and is in h erited a c co r ding to Me n delian p rin c iples

over m ultiple gener a tio n s. These c o nclusions a r e based on s ever a l li n es of evidence:

• Molecular characterization of MON 87411 by Next Gen e ration Sequencing and

Junction Sequence Analysis (NGS/JSA) de m on s trated that MO N 8741 1 containe d a

single DNA insert. These whole

- geno m e sequence analyses provided a

co mprehensive assess m ent of MON 87411 to deter m ine the presence of sequences

derive d fro m PV

- ZMIR1087 1 (DuBose

, et al., 2013; Kovalic, et al., 2012),

de m onstrated that MON 87411 c ontaine d a singl e DN A insert.

• Directed sequencing (locus

- specific P CR, DNA sequencing and analyses) of

MON 87411 was used to deter m ine the co mp lete sequence of the single DNA insert

from PV

- ZMIR10871, the a d jace n t flanking D NA, and the 5' and 3' insert

- to

- flank

junctions. This analysis confir m ed that th e sequenc e an d organizatio n o f th e DN A i s

identical to the corresponding region in the PV

- ZMIR10871 T

- DNA. This also

confir m ed t hat no vector backbone or other unintended plas m id sequences are

presen t i n MO N 87411

. Furth e r m ore, the geno m ic organiza tio n a t th e insertio n site

was assessed by c o mparing the sequences flanking the T

- DNA insert in MON 874 1 1

to the sequence of the insert i on site in conventional m aize. This analysis deter m ined

that no m ajor DNA rearrange m ent o c curred at the insertion site in MON 87411 upon

DNA integration.

• Generation a l sta b ility a n alysis by NGS/JSA d e m onstrated th a t the s ingle PV

ZMIR10871 T

- DNA insert in MON 87411 has been m aintained through five

breeding generations, thereby confir m ing the stability of the T

- DNA in MON 8741 1

.

• Segregation analysis corroborates the insert stability de m onstrated by N G S/JSA and

independently establishes the nature of t h e T

- DNA as a single chro m oso m al locus.

Taken together, the characterization of the genetic modification in MON 87411

de m onstrates that a single copy of the int e nded T

- DNA was stably i n tegrated at a single

locus of the m aize geno m e and t hat no plas m id backbone sequences are present in

MO N 87411.

A sch e m atic representation of the NGS/JSA m ethodology and the basis of the

chara c te r iz a tion using NGS/JSA and PCR sequencing a r e illu s tr a ted in Figure IV

- 1

below. These techniques and their value in DNA characterization i n cro p plant s are

further described in Appendices B and L.

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NGS/JSA

Directed

Sequencing

Figure IV

- 1. Molecular Characteri z ation using Sequencing and Bioinformatics

Gen o m ic DN A fr o m MO N 8741 1 an d th e conv entional control was sequenced using

NG S technolog y tha t produce s a se t o f short

, rando m ly distributed sequenc e read s (each

approxi m ately 100 bp long) that comprehensively cove r th e geno m e s (Ste p 1)

. Utilizing

these geno m ic sequences, bioinfor m atics search to o ls w e re u s ed to s ele c t a ll se q uence

reads (100

- m ers) that were significantly si m ilar to the t ransfo r m a tio n plas m id (Ste p 2)

and Junction Sequence Analysis (JSA) bioin f or m atics was used to d e ter m ine the insert

nu m ber (Step 3). Overlapping PCR products are produced which span any insert(s) and

their wild type loci ( S tep 4 and Step 5, r e spectively). These P CR products are sequenced

to provi d e a detailed characterization of the inse rt ion site( s ).

The NGS/JSA m ethod characterized t h e geno m ic DNA from MON 87411 and the

conventiona l contro l usin g shor t (~10 0 bp ) rando m ly distributed sequence frag m ents

(sequencing reads) generated in su ff icie n t nu m ber to e n sure co mprehensive coverage of

the sa mple geno m es. Bioinfor m a t ics analysis was then used to select sequencing reads

that contai n ed sequenc e s si m ilar to the tra n sfor m ation plas m id, and these were analysed

to deter m ine the nu m ber of DNA inserts. NGS/JSA was run on all MON 87411 s a mples

and the conventional controls; results of NGS/JSA are shown i n Sectio n IV. A an d I V.D

below.

The nu m ber of DNA inserts was deter m ined by analyzing sequences for novel junctions.

The junctions of the DNA insert and the flanking DNA are unique for each insertion; an

exa mple is shown in Figure IV

- 2 below (Kovalic e t al.

, 2012)

. The r efore

, insertio n sites

can be recognized by analyzing for s e quence reads containing such junctions.

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J u n c t i o n

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C G

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C GT G T

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C GT G TCG

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C GT G TC G GG

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C GT G TC G GG A T

P l a s m i d D N A F l a n k in g D N A

Figure IV

- 2. Junctions and Junction Sequences

Depicted above are five exa m ple junction s e qu e nces fo r m at t e d and labeled to indicate the

plas m id/flanking DNA portions of the sequences and with the junction point indicated (plas m id

DNA is shown in bold, underlined text and flank DNA is shown in plain t e xt). Junctions are

detected by ex a m ining the NGS d a ta f or seque n ces having port i ons of plasmid sequences that

span less than the full read. Detected junctions are typically characterist i c of plas m id insert i ons

in the geno me.

Each ins e rtion will pr o duce two unique jun c tion sequence class e s ch a racteristic o f the

geno m ic locus, one at the 5' end of t he insert (illustr a ted in s ee Figure IV

- 3 below, and

na m ed junction sequence class A, or JSC

- A, in this case) and one at the 3' end of the

insert, na m ed junction sequence class B, or JSC

- B

, i n thi s cas e (Kovali c e t al.

, 2012).

J un c t i o n S e q u e n c e s : C l a s s A I ns e r t J u n c t i o n Re g i on s



## 5 ’ F la n k D N A in s e r t 3 ’ F l a n k

J un c t i o n S e qu e n c e s : Cl a s s B

Figur e IV

- 3

. T w o Uniqu e Junctio n Seque nce Classes are Produced b y th e Insertion

of a Single Plasmid Region

A sche m at i c representati o n of a single DNA insert i on within the genome showing the inserted

DNA, the 5 ' and 3' flanks (depicted as areas bound e d by dotted lin e s ), and the t wo distinct regions

spanning t he junctions be tw een inserted DNA and flanking DNA (shaded boxes). The group of

~100

- me r sequences in which each read contains sequences fr o m both the DNA insert and the

adjacent fla n king DNA at a given junction is called a Junction Sequence Cl a ss. In this ex a m ple,

two distinct junction sequence cla s ses (in this case: Clas s A a t th e 5 ' en d an d Clas s B a t th e 3'

end) are repr e sented.

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B y evaluatin g th e numbe r o f uniqu e junctio n c lasses detected, the n u mbe r o f insertion

sites of the plas m id sequence can be deter m i n ed. A longer description of the m olecular

m ethods us e d to characterize MON 87411 can be found in Appendices B and L. For a

single i n sert two juncti o n sequence classes are expected, one each origi n ating from either

en d o f th e insert

, bot h contain i n g portion s o f plas m id DN A inser t an d flankin g sequence.

I n th e cas e o f MO N 87411

, tw o uniqu e jun c tion sequence classes, both containing

portions of T

- DNA a nd flanking sequence, were detected indicating MON 87411

contai n s a single D N A insert (results a r e described in Section IV.A, m ethods and

supple m entary data are presented in Append i x B). The identity of the DNA insert ( i.e

. , T

- DNA or b ackbone) is deter m ined by d i rec t sequencin g describe d below.

The NGS/JSA strategy to deter m ine inse r t number of the integrated plas m id DNA was

designed to ensure th a t all trans g enic s e g m ents would be identified. The d e pth of

coverage (the average nu m ber of ti m es each base of t h e geno m e is inde p endently

sequenced) was >7 5 x for each s a mple geno m e. It has pre v iou s ly been demonstrated t h at

75 x coverage of the soybean geno m e is adequ a te to provide comprehensive coverage and

ensure detection of inserted DNA ( K ovalic et al., 2012) and si m ilarly 7 5 x coverage

provide s comprehensiv e coverag e o f th e m aize geno m e (Clarke and Carbon, 1976). The

level of se n siti v ity of this m ethod was d e m onstrated by detection of a positi v e c o ntrol

spike d a t 1/1 0 th copy

- per

- geno m e equivalent.

Directed sequencing (locus

- specific PCR and DNA sequencing analyses, Figure IV

- 1,

step 4) comple m ents the NGS/JSA analyses. A s indicate d above

, NGS/JS A r e sults

deter m ined that MON 87411 contains a sin g le DNA insertion site. Sequencing of the

insert and flanking geno m ic DNA deter m ined the co m plete sequence o f the insert and

flanks; it deter m ined that the sequence and o r ganization of the DNA insert is identical to

thos e i n th e T

- DN A o f PV

- ZM IR10871, and that each genetic ele m ent (except for the

border regions) in the insert is intact, and also that no vector backbone, or other

unintended plas m id sequences were inserted in MON 87411. Further m ore, the geno m ic

organization at the insertion site was assessed by co m paring the insert and MON 87411

flanking sequence to the sequence of the ins e rtion site in conventional m aize. This

assess m ent indicated that the integration sit e i n th e MO N 8741 1 geno m e include d a

118 bp deletion of geno m ic DNA but is other w ise identical to t h e native sequence.

Result s ar e describe d i n Sectio n IV. B and Section IV.C; m ethods are presented in

Appendi x B.

The stability of the T

- DNA present in MON 87411 across m ultiple generations was

evaluated by NGS/JSA analyses. Gen o m ic DN A fr o m fiv e generation s o f MO N 87411

(Figure IV

- 4) was assayed for all unique j unctio n sequenc e classe s a s describe d above.

This in f ormation was u s ed to det e rmine the number and ide n tity of inse rt ion sit e s. For a

single insert, two junction sequence classes are expecte d ; each one originates from either

end of the insert, both conta inin g portion s o f DN A inser t and flanking sequence. In the

case of MON 87411, two identical junction sequence classes were detected in all the

generations tested, con f ir m ing t h at the sin g le inse r t is s t a bly in h erit e d over m ultiple

generations.

Segregation analysis of the T

- DNA was co n ducted to deter m ine the inheritance and

stability of the insert in MON 87411. Results from this analysis de m on s trate inheritance

accor d ing to Mendelian princi p les and the stabil i ty of the insert is as expected across

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 55 o f 3 72CB I Delete d Copy

multiple g e nerations (F igure IV

- 1 1, Table I V

- 4, and T a ble IV

- 5). The segr e gation

analysis corroborates th e inser t stabil i ty de m onstrated by NGS/JSA a nd independently

establishes the genetic behavior of t h e T

- DNA as a single chr o m os o m al locus.

Th e result s o f thes e analyse s o f MO N 8741 1 de m onstrate that a single copy of the

intended T

- DNA derived from PV

- ZMIR10871 was inserte d a t a singl e locu s o f the

MON 87411 geno m e, t h at the sequence and organization of the T

- DNA insert is identical

to the corresponding region in PV

- ZMIR10871 and that no additional genetic ele m ents,

including backbone sequence s

, were detected in MON 87411. Generational stability

analysis demonstrated that t h e single insert in MON 87411 was m aintained through five

generations of the breeding history, ther e by confir m ing the sta b ility of T

- DNA in

MON 87411. In addition, results from segregation analyses confir m ed the genetic

behavior of the T

- DNA a s a single chro m os o m al locus.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 56 o f 3 72CB I Delete d Copy

LH24 4 R 0



LH24 4 R 1



LH24 4 R 2



LH24 4 R 3



LH244 R 4 1

, 2 × HCL645

R 4 F 1 (LH244 × HCL645) 2

, 4



LH244 R 5 2

, 3 × LH287

R 5 F 1 (LH24 4 × LH287) 2

, 5



LH244 R 6 2

Figure IV

- 4

. Breeding Hist o ry of MON 87411

R 0 corresponds to the transformed plant, F # is the filial generat i on, R # is the regenerant and

subsequent ge nerations,  designates self

- pollination.

1 Gen e ratio n u s e d fo r ful l m o lecula r c h a r acterization.

2 Ge n e rati o n s use d t o c o nfir m i n ser t sta b ility.

3 G e n e r ati on use d f o r c o mme r cia l d e v e l op m e nt o f M ON 87 4 1 1.

4 Gen e ra ti on u s ed f o r c o m pos iti onal a n a ly s i s and RN A e x p r ess i on s tu d ie s.

5 G e nera tio n u sed f o r a g ro n o m i c /

pheno t yp i c s tu d ie s

M ons a n t o Co m pany CR 2 4 0

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Figure IV

- 5. Schematic Representation of t h e Insert and Flanking Sequences in MON 87411

Linear m a p showing DNA derived from the T

- DNA of PV

- ZMIR10871 int e grated into MON 87411. Right

- angl e d arrows ind i cate the

ends of the integrated T

- DNA and the beginn i ng of the flanking sequence. Identified on the m a p are genetic el e m ents within the inser t

. This sche m at i c diagr a m is drawn to scale; the exact c oordinates of every element is shown in Table IV

- 1.

r1 Sup e rs c r i pt i n L e ft a n d R i g ht Bo rd e r R e g i ons i n d i ca t e th at t he se qu e n c e i n MO N 8 7 41 1 w a s tru n cate d c o m pared t o the s e qu e nces i n P V

- ZM I R10 8 7 1.

P Su p erscri pt i n DvSnf 7 in d icate s t he p artia l seq u e n ce.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 58 o f 3 72CB I Delete d Copy

Table IV

- 1. Summary of G e netic Elements in MON 87411

Genetic Element 1 Location in

Sequenc e 2 Functio n (Reference)

5′ Flank 1

- 1460 Sequence flanking the 5 ′ end of the insert

B 3

- Left Bo rd er Regio n r1 1461

- 1723 DN A regio n fr o m Agrobacte r ium

tumefaciens containing the left border

sequence used for trans f er of the

T

- DN A (B a r ke r e t al.

, 1983)

Intervening Sequence 1724

- 1766 Sequence used in DNA cloning

T 4

- E9 1767

- 2399 3' UTR sequence from Pisum sativ u m rbcS

gene fa m ily encoding the s m all subunit of

ribulose bisphosphate carboxylase protein

(Coruzzi et al., 1984 ) tha t directs

polyadenylation of the mRNA

Intervening Sequence 2400

- 2428 Sequence used in DNA cloning

DvSnf 7 P 2429

- 2668 Partial coding sequence of the Snf7 gene

fr o m Diabrotica virgifera virgifera (Baum et

al., 2007b; Baum et al., 2007a) encoding the

Snf7 subunit of the ESCRT

- III co m plex

(Babst et al., 2002) that for m s part of the

suppression cassette

Intervening Sequence 2669

- 2818 Sequence used in DNA cloning

DvSnf 7 P 2819

- 3058 Partial coding sequence of the Snf7 gene

fr o m Diabrotica virgifera virgifera (Ba u m

et al., 2007b; Baum et al., 2007a) encoding

the Snf7 subunit of the E S CRT

- III c o mplex

(Babst et al., 2002) that for m s part of the

suppression cassette

Intervening Sequence 3059

- 3094 Sequence used in DNA cloning

I 5

- Hsp70 3095

- 3898 Intron and flanking exon sequence of the

hsp70 gene from Ze a m a y s ( m aize) encoding

th e hea t shoc k protei n 7 0 (HSP70)

(Rochester et al., 1986)i s involve d in

regulating gene expression (Brown and

Santino, 1997)

P 6

- e35S 3899

- 4519 Pro m oter f r om the 35S RNA of cauliflower

mosaic virus (CaMV) (Odell et al., 1985)

containing the duplicated enhancer region

(Kay et al., 1987) that d i rects transcription in

plant c ells

Intervening Sequence 4520

- 4545 Sequence used in DNA cloning

P

-

pIIG 4546

- 5494 Pro m oter sequence from the physical

i m pedance induced protein of Zea mays

( m aize) (Huang et al., 1998) that directs

trans c ri p tion in pla n t cells

M ons a n t o Co m pany CR 2 4 0

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Table IV

- 1 (continued). Summary of Genetic Elements in MON 87411

Genetic Element 1 Location in

Sequenc e 2 Functio n (Reference)

Intervening Sequence 5495

- 5500 Sequence used in DNA cloning

L 7

- Cab 5501

- 5561 5 ' UT R leade r sequenc e fro m chloroph y l l a/b

bindin g (CAB ) protei n o f Triticu m aestivum

(wheat ) tha t i s involve d i n regulatin g gene

expressio n (La m pp a e t al.

, 1985)

Intervening Sequence 5562

- 5577 Sequence used in DNA cloning

I

- Ract1 5578

- 6057 Intron and flanking UTR sequence of the

act1 gene from Oryza sativa (rice) encoding

rice Actin 1 protein is involve d i n regulating

gene expression (Mc E lroy et al., 1990)

Intervening Sequence 6058

- 6066 Sequence used in DNA cloning

CS 8

- cry3Bb1 6067

- 8028 Codon opti m ized coding sequence f or

Cry3Bb1 protein of Ba c illus thu r in g iensis

that provides insect res i stance (English et al.,

2000)

Intervening Sequence 8029

- 8047 Sequence used in DNA cloning

T

- Hsp17 8048

- 8257 3' UTR sequence from a heat shock protein,

HSP17

, o f Triticum aestivum (wheat)

(McElwain and Spiker, 1989) that directs

polyadenylation of the mRNA

Intervening Sequence 8258

- 8305 Sequence used in DNA cloning

P

- TubA 8306

- 10486 Pro m oter

, 5 ′ UT R leade r an d intro n s e quences

o f th e O s Tu b A gen e fa m il y fro m Oryz a sativa

(rice ) encodin g α

- tubuli n (Jeo n e t al.

, 2000)

tha t direct s transcriptio n i n plan t cells

Intervening Sequence 10487

- 10490 Sequence used in DNA cloning

TS 9

- CTP2 10491

- 10718 Targeting sequence of the ShkG gene from

Arabidopsis thaliana encodin g th e EPSPS

transit peptide region that directs transport of

the protein to the c h loroplast (Herr m ann,

1995 ; Kle e e t al.

, 1987)

CS

- cp4 epsps 10719

- 12086 Coding sequence of the aroA gene from

Agrobacteriu m sp. strain CP4 encoding the

native CP4 EPSPS protein that provides

herbi c ide t o lerance ( B arry et al., 2001;

Padgette et al., 1996)

Intervening Sequence 12087

- 12093 Sequence used in DNA cloning

T

- TubA 12094

- 12675 3' UTR sequence of the OsTub A gene from

Oryza sativa (ri c e) e n co d ing α

- tubulin (Jeon

et al., 2000) that dir e cts polyadenylation of

mR NA

M ons a n t o Co m pany CR 2 4 0

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Table IV

- 1 (continued). Summary of Genetic Elements in MON 87411

Genetic Element 1 Location in

Sequenc e 2 Functio n (Reference)

Intervening Sequence 12676

- 12693 Sequence used in DNA cloning

B

- Right Border Regi on r1 12694

- 12708 DN A regio n fr o m Agrobacte r ium

tumefaciens containing the right border

sequence used for trans f er of the T

- DNA

(Depicker et al., 1982; Z a m bryski et al.,

1982)

3′ Flank 12709

- 14511 Sequence flanking the 3 ′ end of the insert

1 A lth o ug h f l a nk i ng s e qu e n c es a n d i n te rv e ni ng se q u e nce a re not f u nc tiona l gene ti c e l e m en t s, t hey co m pr i se

a p o rtio n of t he seq u e n ce.

2 Nu m bering refers to t he s e quence of th e inse rt i n M O N 8 7 41 1 a nd ad j ace nt DN A.

3 B

, Bor d er

4 T, T ranscript i on Ter m ination S e quence

5 I, I n t r on

6 P, P r o m o t er

7 L

, Le a d er

8 C S, Co d i ng S e qu e nce

9 TS

, Tar g etin g Seq u e n ce

r1 S u pers c r i pt i n L e ft a n d R i g ht Bo rd e r R e g i ons in d i ca t e th at t he s e qu e nce i n MO N 8 7 41 1 was tru nca t ed

co m p are d t o th e seq u e n ce s i n P V

- ZM I R10 8 71

P Su p erscri pt i n DvSnf 7 in d icate s t he p artia l seq u e n ce

. W it h i n t he D vSnf 7 cassette , b a se s 2 4 29

- 26 68 a re

rev e rse c o m p le m e nt t o b a ses 2 81 9

- 3 0 5 8.

IV.A. Determinin g th e Numbe r o f DN A insert s i n MO N 87411

Th e nu m be r o f insertio n site s o f PV

- ZMIR1087 1 DN A i n th e MO N 8741 1 wa s assessed

by perfor m i ng NGS/JSA on MON 87411 geno m i c DNA. A single geno m ic DNA

insertion is expected to produce two junctio n sequenc e classe s an d an y additional

integratio n site s woul d produc e additional junction sequence cl asses

. A plas m id m a p of

PV

- ZMIR10871 is shown in Figure III

- 1. A s c he m atic representation of the insert and

flanking sequences in MON 87411 is shown in Figur e IV

- 5

. Th e generation s studie d are

depicted in the breeding history diagram show n i n Figur e IV

- 4

. Th e NG S conducte d for

all sa mples and its adequate depth of c overage in each case is summarized in

Section IV.A.1, Section IV.A.2 a nd Table IV

- 2. The sensitivity of the m e thod is

de m onstrated in Section IV.A.1 with data sho w n in Table IV

- 3. The JSA analysis of the

R 4 generatio n i s sho w n i n Sectio n IV.A. 2 w ith data presented in F igure IV

- 6 and

supple m ental data sho w n in Appendix B; the othe r generation s tha t wer e use d i n the

generational stability analysis are shown in Figure IV

- 4 with the results of JSA analysis

described in Section I V .D, with JSA res u lts shown in Figure I V

- 9, Figure IV

- 10 and

supple m ental data included in Appendix B. For full details on m aterials and m ethods see

Appendi x B.

M ons a n t o Co m pany CR 2 4 0

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IV.A.1. Nex t Generatio n Sequencin g (NGS ) fo r MO N 8741 1 an d Conventional

Control Genomic DNA

Gen o m ic DN A fr o m fiv e generation s o f MON 87411 and the conventional controls

(inbred L H 244 and hybrids NL6169 and MPA640 B ) w e re isolated and prepared for

sequencing accor d ing to the m anufacturer’s p ro t ocol (Illu m ina, TruSeq library prot o col.

For m aterial and m ethod details see Appendix B). These geno m ic DNA libraries we r e

used to generate short (~100 bp) rando m ly distributed sequence frag m e nts (sequencing

reads) in su f f icient nu m bers to ensu r e co m p rehensive coverage of the m aize geno m e (see

Figur e IV

- 1

, Ste p 1).

To confirm sufficient sequence coverage i n al l generation s o f MO N 8741 1 an d the

conventional controls, the 100

- m er sequence r eads from all sa mples were analyzed to

deter m ine the effective depth of coverage ( i.e

. , th e averag e nu m b e r o f ti m es an y bas e of

the geno m e is ex p ected to be independentl y sequenced ) b y m a ppin g al l read s t o a known

single

- copy endoge n ous gene (Pyruvate decarb o xylase ( pd c 3 ), Ge n B ank

accession.version: AF370006.2). The analysis showed that pdc3 w as covere d by 100

-

m ers at >10 7 x for each sa mple (Table IV

- 2). It has p r e viously b een de m onstrated that

7 5 x coverage of the soybean geno m e is adequate to provide co mprehensive coverag e

an d ensur e detectio n o f inser ted DNA (Kovalic et al., 2012) and si m ilarly 75 x coverage

provides co m prehen s ive coverage of the m aize geno m e (Clarke and Carbon, 1976)

. A

summar y o f NG S an d eff e ctiv e dept h o f coverag e ar e show n i n Tabl e IV

- 2.

In order to confi r m the m e t hod’s ability to detect any sequences derived from the PV

ZMIR10871 transfor m ation plas m id, a s a mple of conventional control m aize DNA

spike d wit h PV

- ZMIR1087 1 DN A a t 1 an d 1/1 0 th geno m e equivalent was analyzed by

NGS and bioinfor m atics. At 1 geno m e equ i valent, 100% nucleotide identity was

observed over 100% of PV

- ZMIR10871 (Table IV

- 3). This result de m onstrates that all

nucleotides of the transfor m ation pla sm id ar e observe d b y th e sequencin g and

bioinfor m atic assess m ents perfor m e d. Also, observed coverage was adequate (Clarke

and Carbon, 1976) at a level of at most 1/1 0 th geno m ic equivalent (99.64% coverage at

100% identity for the 1/1 0 th geno m e equivalent spiked c o ntrol sa mple, Table IV

- 3) and,

hence, a detection le v el of at l e ast 1 / 1 0 th geno m e equivalent was achieved for the plas m i d

DN A sequ e nc e assessment.

M ons a n t o Co m pany CR 2 4 0

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Sa mple

LH244 Total Nucl e otides (Gb)

346.9

LH244 × HCL645 309.5

LH244 × LH287 342.3

R 4 334.9

R 4 F 1 338.9

R 5 F 1 346.6

R 5 352.3

R 6 363.9 Tabl e IV

- 2

. Sequencin g (NGS ) Condu c te d fo r MO N 8741 1 an d Contro l Genomic

DNA

Effective Median Depth of

Coverage (x

- fold)

126x

107x

118x

125x

113x

126x

140x

134x

For each sa m p l e the r aw data produced are presented in te r m s of total nucleotide nu m ber.

Effective depth of coverage is deter m ined by m a pping and alignment of all r a w data to a well

known single copy locus ( pdc3 : p y ruvate dec a rbox y l ase) within the maize genome. The m e dian

effective depths of coverage are shown for all sa m ples.

Tabl e IV

- 3

. Summar y o f NG S Dat a fo r th e Conventiona l Contro l DN A S a mple

Spiked w ith PV

- Z M I R10871 DNA

1/1 0 th copy Spike 1 copy Spike

Extent of co ve rag e 1 of PV

- ZMIR10871 99. 6 4% 100%

Percent identity of coverag e 2 of PV

- ZMIR10871 100% 100%

1 Extent of coverage is calculated as the perce n t of all PV

- ZMIR10871 bases observed in the se q uencing of the s pike

in sa m ples:

e x te nt o f c o v e r ag e numbe r o f spi k e i n base s detected

t otal length ( bp ) of spi k e in plasmid x 100

2 Percent i d entity of co v era g e is calculated as t h e pe r ce n t of all PV

- ZMIR10871 bases observed in t h e seque n ci n g of

the spi k e

- in sa m ples:

P e r c ent identity of c o v e r a ge numbe r o f identica l base s ( spi k e i n v s

. plasmi d sequence ) detec t ed

t otal length ( bp ) of spi k e i n plasmi d detected x 100

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 63 o f 3 72CB I Delete d Copy

IV.A.2 Characteri z a tio n o f inser t number in MON 87411 usi n g Bioinformatic

Analysis

Th e nu m be r o f insertio n site s o f DN A fr o m PV

- ZMIR10871 in the MON 87411 was

assesse d b y perfor m i n g NGS/JS A o n MO N 8741 1 geno m i c DN A usin g th e R 4 generation. A single geno m ic DNA inserti o n is expected to produce two junction

sequence classes and any additio n a l inte g ra t ion sites would produce a d ditio n al ju n ction

sequence classes.

IV.A.2.1 S e lectio n o f S e quenc e Read s C ontaining Sequence of the P V

- Z MIR10871

PV

- ZMIR1087 1 wa s transfo r m ed int o th e p a renta l lin e LH24 4 t o produc e MO N 87411.

Consequently, any DNA inserted into MON 87411 will consist of sequences t h at are

si m ilar to the PV

- ZMIR10871 DNA sequence. T o full y characteriz e th e DN A from

PV

- ZMIR1087 1 inserte d i n MO N 87411

, i t i s suff i cient to co mpletely analyze only the

sequence r e ads that ha v e si m ilarity to the t r ansfor m ation plas m id (Figure IV

- 1, Step 2).

In order to analyze the sequence data for insert nu m ber, all sequences that have

significant sequence similarity t o PV

- ZMIR1087 1 wer e selec t e d fro m th e ful l sequencing

datasets (Kovalic et al., 2012). Due to the d e pth of sequence coverage demonstrated with

this m ethodology (see Section IV

. A .1), on a v erage, any area of the geno m e will be

covere d b y mor e tha n 10 7 o f the 100

- m er sequences; this en sure s tha t sequence s from

PV

- ZMIR10871 inserted into t h e ge n o m e will be detected by the analy s is.

Using established criteria (which are d e scribed in the m aterials and m ethods,

Appendix B), reads si m ilar to the trans f or m ation plas m id were selected from

MON 87411 and the conventional control sequence datasets and were then used as input

dat a fo r bioinfo r m a ti c junctio n sequenc e analysis.

IV.A.2.2 Determinatio n o f th e Inser t Number

The NGS/JSA m ethod described above used the entire P V

- ZMIR10871 plas m id as a

query to d e ter m ine the DNA inse r tion site n u mber. Any DNA inse r ts, reg a rdl e s s of

whether the sequence was fr o m b ackbone o r T

- DNA

, c an b e detecte d b y junction

sequences. Therefore, unlike the tra d itional Southern blot analy s is th a t se p a rat e ly

hybridizes T

- DNA or backbone probes, in N G A/JSA the deter m ination of the T

- DNA

insert nu m ber and of the absence of backbone or unintended sequences are si mply

represented by the deter m ination of the over a ll insert nu m ber in the geno m e follow e d by

deter m inati o n of the exact ide n tity of a ny DNA insert using directed sequencing and

sequence analysis.

By evaluating the nu m ber of unique junction cl a sses, the n u mber of DNA insertion sit e s

can be deter m ined (Figure IV

- 1, Step 3). For a single insert, at a s i ngle geno m ic locus, a

single pair of junction sequences classes, e ach one originating from either end of the

insert

, i s expected

. I f MO N 8741 1 contain s a singl e T

- DN A insert two junction sequence

classes (JS C s) each containing portions of T

- DN A sequenc e an d flankin g sequenc e will

b e detected.

M ons a n t o Co m pany CR 2 4 0

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To deter m ine the insert nu m ber in MON 87411, the selected sequence reads described

above were analyzed using J S A (Kovali c e t al.

, 2012)

. JS A uses bioin f or m atic analysis

to find and classify partially m atched reads characteri s tic of the ends of insertions. The

nu m ber of resultant unique JSCs were deter m ined by this analysis and are shown in the

table b elow.

Table IV

- 4. Unique Juncti o n Sequenc e Clas s Results

Sa mple JSCs Detected

MO N 87411 2

LH244 0

Detailed s e quence in f o r m ation of the jun c t io n sequence s detecte d b y JS A i s show n in

Figure IV

- 6 (Panel B) a n d Figu r e B

- 1 in Appendix B. The location and orientation of the

junctio n sequence s relativ e t o th e T

- DN A inser t deter m ine d fo r MO N 8741 1 (as

described in Section IV.B) is shown in Figure IV

- 6

, Pane l A

. A s show n i n th e figure,

there are two junction sequence classes identifie d i n MO N 87411

. Junctio n Sequence

Clas s A an d Clas s B (JSC

- A an d JSC

- B ) bo t h contai n th e T

- DN A bo r de r sequenc e joined

to flanking sequence, indicating t h at they repre s ent the seq u ences at t h e junctions o f the

intende d T

- DN A inser t an d flankin g sequence.

The presence of two, and only two, junct i on sequence classes (joining T

- DNA border and

f lanking se q uences) indicate this si n gle p a ir of J S Cs likely a rises f rom the ins e rtion o f the

intended P V

- ZMIR10871 T

- DNA at a single l o cu s i n th e gen o m e o f M O N 87411

. JSC

A represents the junction of the T

- DNA Left Border seque n ce to the 5' flank and JSC

- B

represents the junction of t h e T

- DNA Right Bo r der sequence to the 3' f l ank. As shown

by exact a n d co mplete align m ent of the JSCs to the f ull f l a nk/ins e rt s e quence ( d e s cribed

in Section IV.B and shown in Figure IV

- 6, Panel B) both of these JSCs o rigi n ate fr o m the

sa m e locus of the M ON 87411 geno m e and are linked by contiguous, known and

expected D NA that m a kes up the single insert.

Based on this co mprehensive NGS/JSA study it is concluded that MON 87411 contains

one DNA inserted into a single loc us

, as s hown in Figure IV

- 5. The identity of the DNA

inser t wa s deter m ine d b y th e sequencin g an d analysi s o f overlap p in g P C R product s from

this loc u s a s descri b ed below in Section I V .B. Additionally, the lack of detectable

junction sequences attributable to plas m id backbone sequences leads to the conclusion

that no backbone sequences from PV

- ZMIR10871 are present in MON 87411.

M ons a n t o Co m pany CR 2 4 0

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Panel A.

Figur e IV

- 6

. Junctio n Sequence s Detecte d b y NGS/JSA

Panel A: Linear map of MON 87411 illustrating the relationship of the detected junction s e quences to the insert locus. The in d i vidual

junction sequences detected by JSA are illus t rated as stacked bars; each detec t ed juncti o n sequence read is shown trimmed to in clud e only

30 bases of plas m id seque n ce. The sca l e of the identified junction s e quences relat i ve to the ins e rt map is depicted by the bra ces.

r1 Superscript in Left and Right Border Regions indicate that the sequence in MON 87411 was trunca t ed c o m pared t o the sequences in

PV

- ZMIR10871.

P Superscript in DvSnf7 in dicates the partial sequence.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 66 o f 3 72CB I Delete d Copy

**JSC**

**- A**  **Alignment:**

**JSC_A: 1**  **60 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||** 

**Directed Seq.:**  **1554 1495** 

**JSC_A: 61**  **120**

**|||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||** 

**Directed Seq.:**  **1494 1435** 

**JSC_A: 121**  **164 ||||||||||||||||||||||||||||||||||||||||||||** 

**Directed Seq.:**  **1434 1391** 

**JSC**

**- B**  **Alignment:**

**JSC**

**- B:**  **1 60** 

**||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||**

**Directed Seq.:**  **12616 12675** 

**JSC**

**- B:**  **61 120** 

**||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||** 

**Directed Seq.:**  **12676 12735** 

**JSC**

**- B:**  **121 163** 

**|||||||||||||||||||||||||||||||||||||||||||**

**Directed Seq.:**  **12736 12778** 

Panel B.

Figure IV

- 6 (continue d ). Junction Sequence s Detecte d b y NGS/JSA

Panel B: F u ll consensus sequence for jun c tion s e quence Cl a s ses A and B (JSC

- A and JSC

- B) sh o w ing exact alignment to the

independent l y deter m ined in planta sequence at t h e insert loc u s (labeled “ D irected S e q.”). The n u m b ers f l anking the sequence text

represent the base pair nu m b e ring of t he JSA consensus sequence or the insert sequence, respectivel y

. Double unde r lined text indica te s

plas m id DNA sequence, single underl i ned text i nd i cates plant g e nome sequence, and the carat character “^” indicates the juncti o n po i nt

between the MON 87411 insert and the flank.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 67 o f 3 72CB I Delete d Copy

IV.B. Organ i z atio n an d Sequenc e o f th e Inser t an d Adjacen t Flan ki n g DN A in

MO N 87411

The orga n i z ation of the ele m ents within t h e D N A inse r t and the adj a ce n t geno m ic DNA

wa s assesse d usin g directe d DN A sequenc e analysi s (refe r t o Figure IV

- 1, Step 4). PCR

pri m ers were designed to a m plify eight overlapping regions of the MON 87411 geno m ic

DNA that span the entire length of the insert (Fi g ure IV

- 7). The a mplified PCR products

were subjected to DNA s equencing analyses. The results of this analysis confirm that the

MO N 8741 1 inser t i s 11,24 8 b p an d tha t eac h gen e tic ele m ent in the ins e r t i s intact

, with

the exception of the Right and Left bord e r regions. The border region s bot h contain

s m all ter m inal d e letions with the r e m ainder of the ins e rted border regi o ns being id e nti ca l

to the sequence in PV

- ZMIR10871. The sequence and organization of the insert was also

shown to be identical to the corresp o nding T

- DNA of PV

- ZMIR10871, confir m ing that a

single copy of T

- DNA was inserted as inten d ed. This analysis also shows that only

T

- DNA ele m ents (described in Table I V

- 1 ) wer e presen t an d n o PV

- ZMIR10871

backbone sequences were present in MON 87411.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 68 o f 3 72CB I Delete d Copy

P r o d u c t A P ro du c t B

1 2 3 4 5 6 7 8 P r o d u c t C P ro du c t D

9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 17 P r o d u c t E P ro du c t F

1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 26 P r odu c t G P r odu c t H

2 7 2 8 2 9 3 0 3 1 3 2 3 3 34

40

40

4 0

3 0

4 0 2 0

3 0 1 6

2 0 1 0

1 6

1 0

P r od u c t A : ~ 2.1 kb

P r od u c t B : ~ 1.1 k b P r od uc t D : ~ 2.2 kb

P r o d u c t E : ~ 3.2 kb P r o d u c t F : ~ 2 .1 kb

P r od u c t H : ~ 2.8 kb

P r o d u c t C : ~ 2.1 kb P r o d u c t G : ~ 2.5 kb

Figur e IV

- 7

. Analysi s o f Overlappin g PC R P r oduct s Acros s th e MO N 8741 1 Insert

PCR was performed on both conven t ional control geno m i c DNA and geno m ic DNA of the R 4 generation of MON 8 74 11 usi n g ei gh t pairs

of pri mers to generate overlapping PCR fra g ments fr o m MON 87411 f o r seque nc ing anal y s is. To veri f y t h e production of PCR pro d u cts,

5 μ l of each of the PCR reactions was loaded on t h e gel, except where noted below. The expected product size f o r each a m plicon is

provided in t he illustration.

r1 Sup e rs c r i pt i n L e ft a n d R i g ht Bo rd e r R e g i ons in d i ca t e th at t he se qu e n c e i n MO N 8 7 41 1 w a s tru n cate d c o m pared t o the s e qu e nces i n P V

- ZM I R10 8 7 1.

P Su p erscri pt i n DvSnf 7 in d icate s t he p artia l seq u e n ce.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 69 o f 3 72CB I Delete d Copy

Lane Sample Lane Sample

1 1 kb DNA Extension Ladder 18 1 kb DNA Extension Ladder

2 Conventi o nal Control LH 2 44 19 Conventi o nal Control LH 2 44

3 MON 87411 20 MON 87411

4 No te m plate control 21 PV

- ZMIR10871

5 Conventi o nal Control LH 2 44 22 No te m plate control

6 MON 87411 23 Conventi o nal Control LH 2 44

7 PV

- ZMIR10871 (2 μ l) 24 MON 87411

8 No te m plate control 25 PV

- ZMIR10871

9 Conventi o nal Control LH 2 44 26 No te m plate control

10 MON 87411 27 Conventi o nal Control LH 2 44

11 PV

- ZMIR10871 (2 μ l) 28 MON 87411

12 No te m plate control 29 PV

- ZMIR10871

13 Conventi o nal Control LH 2 44 30 No te m plate control

14 MON 87411 (10 μ l) 31 Conventi o nal Control LH 2 44

15 PV

- ZMIR10871 32 MON 87411

16 No te m plate control 33 No te m plate control

17 1 kb DNA Extension Ladder 34 1 kb DNA Extension Ladder Lane designations are as follows:

Figure IV

- 7 (continue d ). Analysis of Overla p p ing PCR Products Across the MON 87411 Insert

Arrows next to the agarose gel photog r a ph denote the size of t h e DNA, in kilobase pairs, obtained from t he 1 kb DNA Extension L adder

(Invitrogen, Grand Island, NY) on t h e ethidium bromide stained gel.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 70 o f 3 72CB I Delete d Copy

IV.C. Sequencing of t h e MON 87411 Insertion Site

PC R an d s e quenc e analysi s wer e perfo r m ed on gen o mic DNA e x tracted from the

conventional control to exa m ine t he inse r tion site in conventional m aize (refer to

Figure IV

- 1, Step 5). The PCR was perfor m ed with one pri m er specific to the geno m ic

DN A sequenc e flankin g th e 5 ' en d o f th e MO N 8741 1 inser t paire d with a second pri m er

specific to the geno m ic DNA sequ e nce flanking t h e 3' end of the insert (Figure IV

- 8). A

sequence comparison between the PCR product generated from the conventional control

an d th e sequenc e generate d fro m th e 5 ' an d 3 ' flankin g sequence s o f MO N 87411

indicates there was a 118 base pair deletion that occurred during integration of the

T

- DNA, with the remainder of the flanks in MON 8 7 411 being identical to the

conventional control. Such changes are common during plant transfor m ation and these

changes presu m ably resulted from double

- stran d ed break repair m echanis m s in the plant

during the Agrobacteriu m

- m ediated transfor m ation process (Salo m on and Puchta, 1998).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 71 o f 3 72CB I Delete d Copy

1 2 3 4

4 0 40

5

. 0

4

. 0

3

. 0

2

. 0

1

. 6 5

. 0

4

. 0

3

. 0

2

. 0

1

. 6

1

. 0 1

. 0

0

. 5 0

. 5

P r i

m e r A P r im e r B

P r im e r A P r i m e r B

Figur e IV

- 8

. PC R A m plificatio n of the MON 87411 Insertion Site

PCR Anal y s is was performed to eval u a te the MON 874 1 1 insertion site. PCR was performed on

conventional control DNA using Pri mer A, specific to the 5 ′ fla n king sequen c e, and Pri mer B,

specific to the 3 ′ flanki n g sequence of the MON 8 7411 i nsert. The DNA generated fr o m the

conventional control PCR was used for sequen c ing anal y s is. This illustration depicts the

MON 87411 insertion site in the conventional con t rol (upper pa nel) and a schematic of the

MON 87411 insert (lower panel). Approxi m a tely 5 μ l of each of the PCR reactions was l o aded

on the gel. La ne designations are as follows:

Lane Sample

1 1 kb DNA Extension Ladder

2 Conventi o nal Control LH 2 44

3 No t e m plate DNA control

4 1 kb DNA Extension Ladder

Arrows on the agarose gel phot o graph denote the size of the DNA, in kiloba s e pairs, obtained

from the 1 kb DNA Extension Ladder (Invitrogen, Grand Island, NY) on the ethidium bromide

stained gel.

r1 Su p erscri pt i n Lef t an d Rig ht B o r d e r Regi ons in d icat e t h a t t he seq u enc e i n MO N 87 4 1 1 wa s tru n cated

co m pared t o the sequ e nce s i n P V

- ZM I R10 8 7 1.

P Su p erscri pt i n DvSnf 7 in d icate s t he p artia l seq u e n ce.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 72 o f 3 72CB I Delete d Copy

IV.D. Determination of Insert S t a b ili t y o v er Multiple Generations of MON 8 7 411

In order to de m onstrate th e stabilit y o f th e T

- DN A present in MON 87411 through

multiple generations, N G S/JSA analysis was perfor m ed using DNA obtained from five

breeding generations of MON 87411. The br eedin g histor y o f M O N 8741 1 i s presented

in Figure I V

- 4, and the specific generations tes t ed are indicated in t h e figure legend. The

MON 87411 ( R 4 ) generation was used for t h e m o lecular characterization a n al y ses

discussed in Sections IV.A

- IV.C and s how n i n Figur e IV

- 6 an d Figur e B

- 1 in

Appendix B. To assess s tability, f our additio n al g ener a tions w ere ev a luat e d by NGS/JSA

analy s is as p reviou s ly descr i bed in Section IV.A, and co mp ared to the fully characterized

MON 87411 ( R 4 ) generation

. Th e conventiona l con t rols used for the generational

stabilit y analysi s include d LH244

, whic h has a genetic background si m ilar to the

MON 87411 ( R 4 )

, MO N 8741 1 (R 5 ) an d MO N 8741 1 ( R 6 ) generation s an d represent s the

original transfo r m ation lin e

. The conventional contr o l hybri d LH24 4 × HCL64 5 ha s a

genetic background si m i la r t o th e MO N 8741 1 R 4 F 1 hybrid. In addit i on

, th e conventional

control hybrid LH244 × LH287, has a g e netic background si m ilar to the

MO N 8741 1 R 5 F 1 hybri d

. Geno m ic DNA isolated from each of the selected generations

of MON 87411 and conventional controls were used for NGS/JSA analysis. The results

are shown in Figure IV

- 9, Figure I V

- 10 and Figure B

- 1 in Appendix B.

IV.D.1 Determination of the Insert Number

To deter m ine the insert nu m ber in the MON 87411 generations, the sequences selected as

described in Section IV.A.2.1 were analyzed using JSA (Kovalic et al., 2012), where the

nu m ber of r esultant JSCs containing P V

- ZMIR10871 DNA sequence deter m ined by this

analysis is shown in the table b elow.

Table IV

- 5. Junction Sequence Classes Detected

Sa mple JSCs Detected

MON 87411 ( R 4 ) 2

MON 87411 ( R 4 F 1 ) 2

MON 87411 ( R 5 ) 2

MON 87411 ( R 5 F 1 ) 2

MON 87411 ( R 6 ) 2

LH244 0

LH244 × HCL645 0

LH244 × LH287 0

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 73 o f 3 72CB I Delete d Copy

Figure IV

- 9 and Figure IV

- 10, below, ide n ti f y the presence of two, and only two,

identical ju n ction seque n ce clas s es in each of the five assessed MON 87411 generati o ns

( R 4

, R 5

, R 6

, R 4 F 1 and R 5 F 1 ) as expe c ted f or a st a bly m aintained single i ns ert. This si n gle

identica l pai r o f JSC s i s observe d du e to the insertion of PV

- ZMIR1087 1 T

- DN A a t a

single locus in the geno m e of MON 87411. The consistency of these JSC data across all

generations tested de m o nstrates that this single locu s i s stabl y m a intaine d throughou t the

MO N 8741 1 breedin g process.

These results, therefore, de m onstrate that the MON 87411 s ingle locus of integration has

been m aintained through s e veral generations of the b r eeding of M O N 87411; thereby

con f ir m ing the sta b ility of the inse rt

. B a sed on this co m prehensive sequence data and

bioinfor m atic analysis (NGS/JSA), it is conclude d tha t MO N 8741 1 contain s a singl e and

stabl e T

- DN A insertion.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 74 o f 3 72CB I Delete d Copy

R 4

Directe d Seq.

R 6

R 4 F 1

R 5

R 5 F 1

R 4

Directe d Seq.

R 6

R 4 F 1

R 5

R 5 F 1

R 4

Directe d Seq.

R 6

R 4 F

1 R 5

R 5 F

1

Figur e IV

- 9

. Junctio n sequence s detecte d b y JSA

. J unction Sequence Class A Alignment (All Generations Tested)

Full consensus sequence f o r JSC

- A showing exact align ment to the independen t ly deter m ined in planta locus specific sequence (labeled

“Direc t ed Seq.” in the figure); individual consensus sequences for each of the five generations are labeled according to their generation

( R 4 , R 5 , R 6 , R 4 F 1 and R 5 F 1 ). Double underlined text indicates plasmid DNA s e quence, single underlined text indicates plant geno me

sequence, a n d the carat character “^” i n dicates the junction p oint be tween the MON 87411 in sert and the flank. The asterisk ch aracte r “*”

indicates identical nucleotide in eve r y sequ e nce at that position in th e alignment. Dash charac t e rs indicate positions past th e end of the

consensus s e quence for a particular generation.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 75 o f 3 72CB I Delete d Copy

R 5 F 1

Directe d Seq.

R 5

R 4 F 1

R 4

R 6

R 5 F 1

Directe d Seq.

R 5

R 4 F 1

R 4

R 6

R 5 F 1

Directe d Seq.

R 5

R 4 F 1

R 4

R 6

Figur e IV

- 10

. Junctio n sequence s detecte d b y JSA

. Junction Sequence Class B Align m ent (All Gener a tions Teste d )

Full consen s us sequence for JSC

- B showing exact align ment to the independently deter m ined in planta locus specific s e quence (labeled

“Direc t ed Seq.” in the figure); individual consensus sequences for each of the five generations are label e d according to their gene ra tion

( R 4 , R 5 , R 6 , R 4 F 1 and R 5 F 1 ). Do u ble underlined t e xt indicates plas m id DNA sequence, single under l ined text i n d i cates plant genome

sequence, a n d the carat c h aracter “^” i n dicates the j u nction p o int be tween the MON 87411 i n sert and the flank. The asterisk char a c t e r “*”

indicates id e ntical nucleotide in every sequence at t hat position in the alignment. Dash charact e rs indi c a te positions past t h e end of the

consensus s e quence for a particular generation.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 76 o f 3 72CB I Delete d Copy

IV.E. Inh e ritance of the Genetic Insert in MON 87411

Th e MO N 8741 1 T

- DN A reside s a t a singl e locu s within the m aize geno m e and therefo r e

should be inherited accor d ing to Mendelian principles of inheritance. During

develop m ent of MON 87411, phenotypic and genotypic segregation data were recorded

t o asses s th e inheritanc e an d stabili ty o f th e MO N 8741 1 T

- DN A usin g Chi

- squar e ( χ 2 )

analysis over sever a l generations

. Th e χ 2 analysis is based on co mparing the observed

segregation ratio to t h e expected s egreg a tion ratio accor d ing to Mendelian principles.

The MON 87411 breeding path for generating segregation data i s describe d in

Figure IV

- 11. The transfo r m ed R 0 plan t wa s self

- pollinate d t o generat e R 1 seed

. An

individ u al h o m ozygous positi v e plant was identified in the R1 segregating population via

a Real

- Ti m e TaqMa n  PC R assay.

The ho m o z ygous positive R 1 pla n t was sel f

- pollin a ted to give ri s e to R 2 seed

. Th e R 2 plants were self

- po l linated to produce R 3 see d

. The R 3 plant s wer e self

- pollinate d to

produce R 4 seed. Homozygous positi v e R 4 pl a nts were cr o ssed via tr a ditio n al br ee ding

techniques to a recurrent parent (H C L645 ) tha t doe s no t contai n th e DvSnf 7 p

, cp4 epsps

,

or cry3Bb1 coding sequences to produce he m izygous R 4 F 1 seed. The R 4 F 1 plants were

crossed with the recurrent pare n t to produce B C 1 F 1 seed. The B C 1 F 1 generation was

teste d fo r th e presenc e o f th e T

- DN A b y E nd

- Poin t TaqMa n PC R t o selec t for

he m i zygou s MO N 8741 1 plants

. B C 1 F 1 plants he m izygous for MON 87411 T

- DNA

were crossed with the recurrent parent to pr o duce the B C 2 F 1 plants. BC 2 F 1 plants

he m izygous for MON 87411 T

- DNA were self

- pollinated to produce the B C 2 F 2 p l ants.

BC 2 F 1 plant s he m i zygo u s fo r MO N 8741 1 T

- DN A wer e crossed with the recurrent parent

to produce the B C 3 F 1 plants.

The inheritance of the MON 87411 T

- DNA was assessed in the B C 2 F 1

, BC 2 F 2

, and

BC3F 1 generations. At the B C 2 F 1 an d BC 3 F 1 generations

, th e MO N 8741 1 T

- DN A was

predicted to segregate at a 1:1 rat i o (he m izygous po s iti v e: ho m ozygous negative)

accor d ing to Mendelian inheritance p rinciples. A t the B C 2 F 2 generation, the MON 87411

T

- DNA was predicted to segregate at a 1 : 2:1 ratio (ho m o z ygous positive: he m izygous

positi v e: h o mozygous negati v e) according to M endelian in h eritance p ri n ciples.

A Pearson’s Chi

- square ( χ 2 ) analy s is was used to co mpare the observed s egregation ratios

of the MON 87411 T

- DNA to the expected ratios. The Chi

- square ( χ 2 ) analy s is used the

statistical program R V e rsion 2.15.2 (2012

- 10

- 26).

The Chi

- sq u are was calculated as:

χ 2

= ∑ [ ( | o

– e | ) 2 / e]

 TaqMa n i s a r e g i s te r e d t r a d ema rk of R o c he M o lec u la r S yst e m s, In c

.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 77 o f 3 72CB I Delete d Copy

where o = observed frequency of the genotype or phenotype and e = expected frequency

of the genotype or phenotype. The level of s t atistical sig n ificance was predeter m ined to

be 5% ( α = 0.05).

The res u lts of the χ 2 analysi s o f th e MO N 8741 1 segre g atin g progen y ar e presente d in

Table IV

- 6 and Table IV

- 7. The χ 2 values in t h e B C 2 F 1 an d BC 3 F 1 generation s indicated

no stati s tically significa n t difference between the observed and expected 1:1 segregation

ratio (he m izygous positive: ho m oz y gous negative) of the M ON 87411 T

- DNA. T he χ 2

value f or t h e B C 2 F 2 generation indicated no statistic a lly significant d ifference between

the observed and expected 1:2:1 segreg a tion ratio (ho m o z ygous positive: he m izygous

positive : homozygou s negative ) o f MO N 87411 T

- DNA. These results support the

conclusion that the MON 87411 T

- DNA resides at a single locus within the m ai z e

geno m e and is inherited accordi n g to Mendelian pri n ci p les. These results are also

consistent w ith the m olecular characteriza t ion data indicating that MON 87411 contains a

single intact copy of the T

- DNA inserted at a single locus in the m aize gen o m e.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 78 o f 3 72CB I Delete d Copy

Tra n s f or m e d L H 24 4 R 0 Pla nt



L H 2 4 4 R 1



L H 2 4 4 R 2



L H 2 4 4 R 3



L H 2 4 4 R 4

B reed in g pa t h

con tin u e d × RP R 4 F 1 (L H 2 4 4 x H CL 6 45)

× RP

TI:BC 1 F 1

× RP

x R P (E xpected seg r egation 1:1) TI:B C 2 F 1 (H e m iz y gous positive: H o m oz y gous negative)



TI:B C 3 F 1 (E xpected seg re gation 1:1)

( H e m i z y gous positive: H o m oz y gous negative) TI:B C 2 F 2

(E xpected seg re gation 1:2:1)

(H o m o z y gous pos i tive: H e m i z y gous positive: H o m o z y gous negative)

Figure IV

- 1 1. Breeding Path for Ge n erating Segregation Data for MON 87411

*Chi

- square anal y s is was conduct e d on s e gregation data from the B C 2 F 1 , B C 2 F 2 , and B C 3 F 1 generations (bo l ded text).

TI: T rait I ntegration: Rep l acement of genetic background of M O N 87411 b y r ecurrent backgrou n d except inserted gene.

RP: Recurring parent.

 =Self

- Poll i nated

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 79 o f 3 72CB I Delete d Copy

Tabl e IV

- 6

. Segregatio n o f th e T

- DN A Durin g the Development of MON 87411 1:1 Segregation

1 : 1 Seg r e g ation

Expected # Expected #

Obs e r ved # Obs e r ved # Plants Plants

Ge n e ration T o ta l Pla n ts Plants

P o siti ve Plants

Ne g ati ve He m i z y go u s

(Positi v e) Ho m oz y g o us

Ne g ati ve χ 2 Pro b a b ilit y 2

BC 2 F 1 3 5 1

1 1 7 2 1 7 9 1 7 5 .5 0 1 7 5 .5 0 0

. 14 0

. 7 0 9

BC 3 F 1 2 2 3

1 1 0 4 1 1 9 1 1 1 .5 0 1 1 1 .5 0 1

. 01 0

. 3 1 5

1 Segregation was evaluated using an E n d

- Point Ta q Man anal y s is for the MON 874 1 1 insert.

2 Chi

- square anal y s is was performed to anal y ze the segregation ratios (p ≤ 0.05 )

.

Tabl e IV

- 7

. Segregatio n o f th e T

- DN A Durin g the Development of MON 87411 1:2:1 Segregation

Obs e r ved # Obs e r ved # Expected # 1 : 2 : 1 S e gr e ga ti on Expected #

Ge n e ration

1 T o tal

Plants Plants

Ho m oz y g o us

P o siti ve Obs e r ved #

Plants

He m i z y go u s Plants

Ho m oz y g o us

Ne g ati ve Plants

Ho m oz y g o us

P o siti ve Expected #

Plants

He m i z y go u s Plants

Ho m oz y g o us

Ne g ati ve χ 2 Pro b a b ilit y 2

BC 2 F 2 6 2 3 1 5 2 3 1 4 1 5 7 1 5 5 .7 5 3 1 1 .5 0 1 5 5 .7 5 0

. 12 0

. 9 42

1 Segregation was evaluated using Real

- Ti me TaqMan ana l y s is for the MON 87411 i nsert.

2 Chi

- square anal y s is was performed to anal y ze the segregation ratios (p ≤ 0.05 )

.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 80 o f 3 72CB I Delete d Copy

IV.F. Cha r acteri z ation of the Ge n etic Mo d ification Summary and Conclusion

Molecular characterization of MON 87411 by NGS/JSA and directed sequencing

de m onstrated that a single copy of the i ntende d transfe r DN A (T

- DNA) containing the

DvSnf7 suppression ca s sette and t h e cry3Bb1 an d cp4 eps p s expression cassettes from

PV

- ZMIR10871 was integrated into the m aize geno m e at a single locus. These analyses

als o showe d n o PV

- ZMIR1087 1 backbon e DN A ha d bee n inserted.

Directed sequence analyses perfor m ed on MON 87411 confir m ed the organization and

intactness of the full T

- DNA and all expected ele m ents within the insert, with the

exception of inco mp lete Right and Left Bo r der sequences that do not affect the

functionality of the DvSnf7 suppression or cry 3 Bb1 and cp4 epsps expression cassettes.

Analysis of the T

- DNA insertion site in m a iz e show s th e flank s i n MO N 8741 1 are

identical to the conventional control, exce p tin g a 11 8 b p deletio n o f geno m ic DN A a t the

insertion site in MON 87411. This deletion is not expected to affect food or feed safety.

Generational stability ana lysi s b y NGS/JS A de m ons t r ate d tha t th e T

- DN A in

MON 87411 was m aintained through five br eedin g generations

, thereb y confir m i n g the

stability of the insert. Results fro m segregatio n analyse s sho w heritability and stability of

the insert o ccurred as expected across m u ltiple generati o ns, which corroborates the

molecular insert stability analy s is and e s tablishes the p resen c e of the T

- DNA in

MON 87411 at a single chro m oso m al locus.

M ons a n t o Co m pany CR 2 4 0

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V. CHARACT E R I ZATIO N AN D SAF ET Y AS S ES S MEN T O F TH E Cry 3 Bb1

an d CP 4 EPSP S PROTE I N S PRODUCE D I N MO N 87411

Characterization of the introduced protein(s) in a biotechnology

- derived crop is i mportant

to establishing food, f e ed, and environ m ental safety. As described in Section IV,

MON 87411 contains c r y3Bb1 and cp4 epsps e x pression ca s settes that, when transcribed

and t r ansl at ed, res u lt in t h e expression of the Cry3Bb1 an d CP 4 EPSP S proteins

. The

characterization and safety assess m ent for the DvSnf7 suppression cassette is described

in Section VI.

This se c tion summariz e s: 1) the i d entity and f unction of the Cry3Bb1 and CP4 EPSPS

proteins produced in MON 87411; 2) assess m ent of equivalence between the plant

produced and E

. coli

- produce d proteins ; 3 ) th e leve l o f th e Cry3Bb 1 and CP4

EPSPS proteins in plant tissues from MON 87411; 4) assess m ent of the potential

allergenicit y o f th e Cry3Bb 1 an d CP 4 EPSPS proteins produced in MON 87411; and

5) the food and feed safety assess m ent o f th e Cry3Bb 1 an d CP 4 EPSP S pr o t eins

produced in MON 87411. The data are consistent wit h prio r safet y assess m ent s o f th e se

two proteins and support a conclusion that the proteins produced in MON 87411 are safe

for hu m an or ani m al consu mption and safe for the environ m ent based on several lines of

evidence s u mmarized below.

V.A. Identity and Function o f the Cry3Bb 1 an d CP 4 EPSP S Protein s from

MO N 87411

V.A.1. Identity and Function of t h e Cry3Bb 1 Protei n fro m MO N 87411

Cry3Bb1 protein originates from Bacillus thuringiensis ( B t ), a u b iquit o us gra m

- positi v e

soil bacteri u m that acc u mulates crystal pr o te i ns during spor u lation. Cry3Bb1 protein is a

m e mber of the 3D

- Cry fa m ily of insecticidal proteins (Crickmore, 2012). Proteins within

the 3D

- Cry proteins are subdi vide d int o differen t group s base d o n th e hig h specificity

they have for their target categ o r y of in s ects. Because of their narrow spectr u m of

activity, they lack an impact on broader i nsec t population s o r o ther organis m s. For

exa mple, C r y3 proteins have insecticidal acti v ity specifically against c o leopteran i n sect s

, while Cry1A proteins have i nsecticida l activit y specifical l y against lepidopteran insects

(Höfte and Whiteley, 1989).

Th e generalize d MO A fo r Cr y p r otein s wa s describe d b y E ngli sh an d Slati n (1992)

. It

includes ingestion of the crystals by insects and s olubiliz a tion of the cry s tals in the in s ect

m idgut, followed by activation through proteolytic processin g o f th e solubl e Cr y protein

by digestive enzy m es in the m idguts. The activ a ted pr o tein t h en binds to spe c i f ic

rece p tors on the surface of the m idgut epit h e lium of target insects a n d inserts into the

m e mbrane, leading to pore for m ation and g e neralized disruption of the trans m e m brane

gradie n ts a n d, therefore, cell i n tegrity. Wh ile alternate mechani s m s hav e als o been

proposed, a review of the availa bl e data h as recently been published and the a u thors

concluded that the original m odel, pore for ma tion, is the m ost valid m odel for Cry protein

mode of action (Vachon, et al., 2012).

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Cry3Bb1 protein in MON 87411 is a protei n consistin g o f a singl e polypeptid e o f 652

a m ino acids. Like other Cry proteins, it is synthesized as a prototoxin and is likely

cleaved by digestive enzy m es in the m idgut of target organis m s to an approxi m ately

60 kDa activated protein (Bravo, et a l

., 2007). Cry3Bb1 is also expressed in

commerciall y availabl e YieldGar d V T Rootwo r m /RR2 (MON 88017) m aize and

S m artSta x ® m aize. The a m ino acid sequence deduced from the Cry3Bb1 expression

cassette present in YieldGard VT Rootwo r m /RR2 is identic a l to th a t deduced f rom the

Cry3Bb1 expression cassette present in MON 87411. A related Cry3Bb1 protein, w hich

has over 99% a m ino acid iden t ity to the Cry3B b 1 in Yi eldGar d V T Rootwor m /RR 2 and

MON 87411, is expressed in YieldGard Rootworm m aize (MON 863). Each of these

products were previously reviewed by U SDA

- APHI S an d foun d t o no t hav e an y unique

plant pest risks relative to conventional m aize and were subsequently deregulated.

V.A.2. Identity and Function of t h e CP4 EPSPS Protein from MON 87411

The enzy m e, 5

- enolpyruvylshiki m ate

- 3

- phosph a te synthase (EPSPS), catalyzes one of

the enzy m atic steps of the shiki m ic acid pathw a y, and is the target f or t h e broad spe c trum

herbicide glyphosate ( H asla m

, 1993; Herr m ann and W eaver, 1999; Kishore, et al., 1988;

Steinrücken and A mrh e in, 1980). The shi k i m ic acid pathway and EPSPS enz y mes are

ubiquitous to plants and m icroorganis m s, but a b sent in m a m m als, fi s h, birds, reptiles, and

insects (Alibhai and Stalling s

, 2001)

. EPSP S protein s hav e b een isolated f rom both plant

and m icrobial sources and their properties have been extensively stud i ed (Harrison, et al.,

1996; Haslam, 1993; Schönbrunn, et al., 2001; Steinrücken and A mrhein, 1984). The

plant and m icrobial enzy m es are m o no

- funct i onal with a m o l ecular weight of 44

- 51 kDa

(Franz

, e t al.

, 1997 ; Kishor e e t al., 1988). EPSPS enzy m es catalyze the transfer of the

enolpyruvyl group from phosphoenolpyruva t e (PEP) to the 5

- hydroxyl of shiki m ate

- 3

- phosphate (S3P), there b y yieldin g inorgani c phosphat e and 5

- enolpyruvylshiki m ate

- 3

- phosphate (EPSP) ( A libhai and Stallings, 2001)

. Shiki m ic acid is a s ubstrate for the

b iosy n thesis of the aro m atic a m ino acids (phenylalanine, tryptophan and tyrosine) and

other aro m atic m olecules nece s sar y fo r plan t growth.

Th e EPSP S transgen e i n MO N 87 4 1 1 i s derive d fr o m Ag r obacterium sp. strain C P 4

( cp4 epsp s ). The cp4 epsps coding sequence encodes an EPSP S protei n consistin g o f a

single polypeptide of 455 a m ino acids (Padgette e t al.

, 1996)

. Th e CP 4 EPSP S protei n is

si m ilar and functionally identical to endogenous plant EPSPS enzy m es, but has a much

reduced affinity for glyphosate, the active ingredient in Roundup agricultural herbicides,

relativ e t o endogenou s p l an t EPSP S (Padgett e e t al.

, 1 9 96). In conventional plants,

including w eeds, glyphosate blocks the biosynt h esi s o f EPSP

, thereb y deprivin g plant s of

essential a m ino acids (Hasla m

, 1993; Ste i nrücken and Am rhein, 1980). In Roundup

Read y plants

, whic h ar e toleran t t o Roundu p agricultural herbicides, require m ents for

aro m atic a m ino acids and other m etabolites are m et by the conti n ued action of the

CP4 EPSPS enzy m e in the presence of glyphosate (Padgette et al., 1996). The

CP4 EPSPS protein expressed in MON 87411 i s identica l t o th e CP 4 EPSP S p r otein

expresse d i n Roundu p Read y product s acros s s e veral crops, including soybeans, corn,

canola, cotton, sugar b eet

, an d alfalfa.

M ons a n t o Co m pany CR 2 4 0

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V.B. Cha r acteri z ation and Equivalence of Cry3Bb1 and CP4 EPSPS Proteins from

MO N 87411

The safety assess m ent of crops derived t h rough biotechnology includes characterization

of the physicoche m ical and functional properti e s of the protein(s) produced from the

inserted DNA, and confir m ation of the safety of the protein(s). For safet y dat a generated

using proteins produced from a heterologous source ( e.g

., E

. col i

- produced protein) to be

applie d t o plant

- produce d protein(s ), th e equivalenc e o f th e plan t an d E

. coli

- produced

protein s m u s t b e assessed

. Th e physicoch e m ical and functional c h aracteristics of the

MON 87411

- produced CP4 EPSPS and MON 87411

- produced Cry3Bb1 proteins were

deter m ined and each was shown to be equivalent to its respective E. coli

- produced

protein. A summary of the anal y tical results f o r each pr o tein are sho w n below and the

details of the m aterials, m ethods, and results are described in Appendix C.

Th e Cry3Bb 1 an d CP 4 EPSP S p r otein s pu r ifie d fro m grai n o f MO N 8741 1 were

characterized and the equiv a lence of the physicoche m ical characteristic s betwee n the

MON 87411

- produced and the E. coli

- produced proteins was est a blished using a panel of

analytical tests: 1) N

- t e r m inal sequence analysis of the M O N 87411

- produced proteins

esta b lished identity; 2) MAL D I

- TOF MS analysis yielded p e ptide m asses consistent with

the expected peptide m asses from the theor e tic a l try p sin digest o f the

MON 87411

- produced sequences; 3) MON 87411

- produced Cry3Bb1 and CP4 EPSPS

protein s wer e detecte d o n w estern blots probed with the i r re spec ti v e p r o t e i n

- spec i f ic

antibo d ies and the immunoreactive proper t ies of the MON 87411

- produced and

E. coli

- produced proteins were sho w n to be equ i valent; 4) the apparent molecular weights

of the MON 87411

- produced and E. coli

- produce d proteins

, assesse d b y SDS

- PAGE,

were shown to be equival e nt ; 5 ) MO N 87411

- produce d an d E. coli

- produced

MON 87411 proteins were both deter m ined to be non

- glycosylate d ; and 6) functional

(biological ) activit y o f the MON 87411

- produced and E

. col i

- produce d protein s were

de m onstrated to be equivalent for both Cry3Bb1 and CP4 EPSPS.

Taken together, these data provide a det a iled char a ct er ization of the

MON 87411

- produced C ry3Bb1 and CP4 EPSPS p r oteins and establish their respective

equivalenc e t o E. coli

- produced Cry3Bb1 and CP4 EPSPS p r oteins. This equivalence

justifies the use of previously conducted protein studies using E. coli

- produce d Cry3Bb1

and CP4 EPSPS proteins to establish the safety of the Cry 3 Bb1 and CP4 EPSPS p r oteins

expresse d i n MO N 87411

, summ a rized in section V.E.

V.C. Expr e ssio n Level s o f Cry3Bb 1 an d CP 4 EPSP S Protein s i n MO N 87411

Cry3Bb 1 an d CP 4 EPSP S protei n levels in various tissues o f MO N 8741 1 relevan t t o the

risk assess m ent were deter m ined by a vali date d enzy m e

- linke d immunosorben t assay

(ELISA)

. Tissue s o f MO N 8741 1 wer e collecte d from four replicate plots planted in a

rando m ized co mplete block field design du r in g th e 201 1

- 201 2 growin g seaso n fro m the

following five field sites in Argentina: Perga m ino, Buenos Aires ( S ite Code BAFO);

Hunter, Buenos Aires (Site Code BAHT); Perga m ino, Bu e nos Aires (Site Code BAPE);

Sarasa, Buenos Aires ( S ite Code BASS) and Salto

, Bueno s Aire s (Sit e Cod e BATC)

. The

field sites were representative of m aize

- producin g region s suitabl e fo r commercia l maize

production. Maize production in the U.S. corn belt and Argentina growing regions

M ons a n t o Co m pany CR 2 4 0

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occurs at r e lativ e ly s i m ilar la titude s wit h a n approxi m a t e 6 m ont h offse t (Schnepf

, e t al.,

2001). The average growing season te m pera t ures and precipitation are co m parable

(Schnep f e t al.

, 2001 ) and

, a s a result

, m a iz e hybrid s develope d i n th e U.S

. ar e ofte n used

directly by fa r m ers in the southern growing regions of A r gentina. As such, protein

expression analyses from m aize grown at t h e s e sites are appropriate for a co mparative

assess m ent. Nineteen total tissue s a mples were coll e cted from each replicated plot at

each site, many over several ti m e points/ g rowth stages, t h r o ughout t h e season. Sa mples

include d ove r seaso n lea f (OSL 1 throug h O S L4)

, ove r season root (OSR1 through

OSR4), ov e r season w hole plant (OS W P1 through OSWP4), stover, senescent root,

forage root, forage, grain, pollen and s ilk. MON 87411 plots were treated with

glyphosate to generate sa m ples under cond i tions of the intended use (0.95 lbs active

ingredient/ hectare) o f th e product.

V.C.1. Expressio n Level s o f Cry3Bb 1 Protein

Cry3Bb1 protein levels were deter m ined in 19 tissue types. The ELISA results o b tained

for each s a mple were averaged across the fi v e sites and are sum m arized in Table V

- 1.

The details of the m aterials and m ethods are described in A ppendix D. The individual

Cry3Bb1 protein levels in MON 87411 across all sa mples analyzed from all sites ranged

from 3.0 to 460 μg/g dw. The m e an Cry3 B b1 protein level a m ong al l tissu e type s was

highest in OS W P 1 at 340 μg/g dw a nd lowest in grain at 4.0 μg/g dw.

M ons a n t o Co m pany CR 2 4 0

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Table V

- 1. Summary of Cry3Bb1 Protein Levels in Tissues from MON 87411

Gro w n in 2011

– 2012 Argentina Field Trials

Develo p m ent Da y s After

Planting Cr y 3 Bb1

Mean (SD)

Range Cr y 3 Bb1

Mean (SD)

Range LOQ/LOD

Tissu e 1 Stag e 2 (DAP ) 3 ( μ g/g fw ) 4 ( μ g/g dw) 5 ( μ g/g fw ) 6

OSL1 V3

- V4 21

- 22 45 (9

. 0) 270 ( 6 5) 0.0 3 5/0. 0 06

31

– 64 160

- 390

OSL2 V6

- V8 35

- 44 40 (7

. 8) 210 ( 4 0) 0.0 3 5/0. 0 06

26

– 56 120

– 2 7 0

OSL3 V10

- V13 50

- 55 40 (7

. 9) 170 ( 3 5) 0.0 3 5/0. 0 06

21

– 52 92

– 220

OSL4 V14

- R1 59

- 78 56 (19) 220 ( 6 3) 0.0 3 5/0. 0 06

31

– 89 130

– 3 4 0

OSR1 V3

- V4 21

- 22 25 (4

. 6) 180 ( 4 3) 0.0 3 5/0. 0 28

16

– 32 130

– 2 8 0

OSR2 V6

- V8 35

- 44 16 (4

. 0) 120 ( 2 4) 0.0 3 5/0. 0 28

9.4

– 25 67

– 170

OSR3 V10

- V13 50

- 55 15 (4

. 0) 84 (21) 0.0 3 5/0. 0 28

9.6

– 24 54

– 130

OSR4 V14

- R1 59

- 78 14 (3

. 3) 75 (19) 0.0 3 5/0. 0 28

9.0

– 21 43

– 120

OSWP1 V3

- V4 21

- 22 44 (4

. 9) 340 ( 4 9) 0.0 3 5/0. 0 08

33

– 53 250

– 4 6 0

OSWP2 V6

- V8 35

- 44 30 (5

. 3) 190 ( 3 0) 0.0 3 5/0. 0 08

21

– 40 130

– 2 7 0

OSWP3 V10

- V13 50

- 55 20 (6

. 8) 140 ( 3 9) 0.0 3 5/0. 0 08

9.2

– 33 59

– 210

OSWP4 V14

- R1 59

- 78 20 (4

. 8) 120 ( 2 8) 0.0 3 5/0. 0 08

12

– 29 71

– 170

Stover R6 136

- 1 55 10 (6

. 2) 21 (13) 0.0 3 5/0. 0 08

1.9

– 19 4.7

– 44

Senescent R o ot R6 136

- 155 4.8 (3.1) 19 (13) 0.035/0.028

0.76

– 1 2 3.0

– 50

M ons a n t o Co m pany CR 2 4 0

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Table V

- 1. (continued) Su mm ary of Cry3Bb1 Pr otein Levels in Tissues from MON 874 1 1

Grown in 2011

– 20 1 2 Argentina Field Trials

Develo p m ent Da y s After

Planting Cr y 3 Bb1

Mean (SD)

Range Cr y 3 Bb1

Mean (SD)

Range LOQ/LOD

Tissu e 1 Stag e 2 (DAP ) 3 ( μ g/g fw ) 4 ( μ g/g dw) 5 ( μ g/g fw ) 6

Forage Root R5 101

- 1 11 7.9 (3

. 5) 36 (16) 0.0 3 5/0. 0 28

2.6

– 15 13

– 66

Forage R5 101

- 1 11 12 (4

. 9) 39 (17) 0.0 3 5/0. 0 08

5.5

– 23 18

– 75

Grain R6 139

- 1 54 3.5 (0

. 45) 4.0 (0

. 56) 0.0 3 5/0. 0 07

2.7

– 4.4 3.1

– 5.1

Pollen VT

- R1 65

- 80 29 (3

. 0) 36 (4

. 0) 0.0 3 5/0. 0 18

23

– 34 30

– 42

Silk R1 65

- 81 16 (3

. 8) 160 ( 3 7) 0.0 3 5/0. 0 10

8.5

– 23 89

– 220

1 OSL

= ove r s e aso n leaf ; OS R = ove r seas on r oo t ; O S WP= o ver sea s on wh o l e p l a nt

2 The c r op d e velop m ent stage each tis s ue w a s collected.

3 The n u m ber of da y s a f te r p l an tin g tha t ea c h tissu e w a s c ollected.

4 Protei n l e vel s ar e expre s s e d a s th e arithmeti c m ean and s t a n da r d de vi a ti on ( S D) as m i crogram ( μ g) o f

p r o t e i n per gr a m (g) o f ti s s ue o n a fr e sh w e i ght bas i s ( f w )

. T he m eans, SD, a n d r a ng e s ( m i ni m u m a n d

maxi m u m values) were calcu l a ted for ea c h tissu e a c ros s al l site s (n

= 20).

5 Protei n l e vel s ar e expre s s e d a s th e arithmeti c m ean and s t a n da r d de vi a ti on ( S D) as m i crogram ( μ g) o f

p r o t e i n per gr a m (g) of ti s s u e on a d ry w e i ght b a s i s ( d w ). T he dry w e i ght va l ues w e re ca l cu late d b y

d i v i d i ng t he μg/ g f w by t he dr y w eig ht con v e r sio n f act or o b t ai n e d f r om m o ist ure a n alysi s d ata.

6 LOQ=l i m i t of qu a n titati on; LOD=li m i t of d etecti o n.

V.C.2. Expression Levels of CP4 EPSPS Protein

CP4 EPSPS protein levels were deter m ined i n al l 1 9 tissu e types

. Th e ELIS A results

obtained for each sa mple were avera g ed across t h e five sites and are sum m arized in Table

V

- 2. The details of the m aterials and m ethods are described in Appendix D. The

individua l C P 4 EPSP S p r otei n level s i n MO N 87411 across all sa mples analyzed from all

sites ranged f rom less than the li m it of qua n tit a tion (<LOQ) to 76 μg/g dw. The mean

CP4 EPSPS protein level a m ong all tissue typ e s was highest in OS W P1 at 63 μg/g dw

an d lowes t i n grai n a t 1. 9 μg/ g dw.

M ons a n t o Co m pany CR 2 4 0

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Table V

- 2. Summary of CP4 EPSPS Protein Levels in Tissues from MON 87411

Gro w n in 2011

– 2012 Argentina Field Trials

Develo p m ent Da y s After

Planting CP4 EPSPS

Mean (SD)

Range CP4 EPSPS

Mean (SD)

Range LOQ/LOD

Tissu e 1 Stag e 2 (DAP ) 3 ( μ g/g fw ) 4 ( μ g/g dw) 5 ( μ g/g fw ) 6

OSL1 V3

- V4 21

- 22 7.1 (0

. 83) 42 (5

. 9) 0.1 3 7/0. 0 71

5.8

– 9.0 33

– 55

OSL2 V6

- V8 35

- 44 7.0 (0

. 64) 36 (3

. 1) 0.1 3 7/0. 0 71

6.0

– 7.9 29

– 39

OSL3 V10

- V13 50

- 55 7.4 (0

. 78) 32 (3

. 8) 0.1 3 7/0. 0 71

6.4

– 8.9 27

– 42

OSL4 V14

- R1 59

- 78 7.8 (0

. 85) 31 (3

. 5) 0.1 3 7/0. 0 71

6.6

– 9.5 24

– 37

OSR1 V3

- V4 21

- 22 6.5 (0

. 86) 48 (6

. 6) 0.0 6 8/0. 0 33

4.4

– 8.0 38

– 63

OSR2 V6

- V8 35

- 44 5.2 (1

. 0) 37 (7

. 0) 0.0 6 8/0. 0 33

3.8

– 7.1 23

– 48

OSR3 V10

- V13 50

- 55 5.6 (0

. 84) 31 (4

. 7) 0.0 6 8/0. 0 33

4.0

– 7.1 24

– 37

OSR4 V14

- R1 59

- 78 5.7 (0

. 80) 30 (4

. 8) 0.0 6 8/0. 0 33

4.2

– 7.1 20

– 38

OSWP1 V3

- V4 21

- 22 8.1 (0

. 90) 63 (6

. 7) 0.1 3 7/0. 0 70

6.6

– 9.8 54

– 76

OSWP2 V6

- V8 35

- 44 5.6 (0

. 94) 36 (5

. 8) 0.1 3 7/0. 0 70

3.4

– 7.4 21

– 46

OSWP3 V10

- V13 50

- 55 4.6 (1

. 1) 33 (6

. 2) 0.1 3 7/0. 0 70

2.3

– 6.6 21

– 45

OSWP4 V14

- R1 59

- 78 4.3 (0

. 87) 25 (5

. 0) 0.1 3 7/0. 0 70

2.9

– 5.5 17

– 32

Stover R6 136

- 1 55 1.0 (0

. 60) 2.2 (1

. 2) 0.1 3 7/0. 0 70

0.30

– 2

. 1 0.59

– 4

. 9

Senescent R o ot R6 136

- 155 1.4 (0.69) 5.4 (2.9) 0.068/0.033

0.49

– 2

. 6 1.8

– 11

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Table V

- 2. (continued) Summary of CP4 E PSPS Protein Levels i n Tissues fr o m

MON 87411 Grown in 2011

– 20 1 2 Argentina Field Trials

Develo p m ent Da y s After

Planting CP4 EPSPS

Mean (SD)

Range CP4 EPSPS

Mean (SD)

Range LOQ/LOD

Tissu e 1 Stag e 2 (DAP ) 3 ( μ g/g fw ) 4 ( μ g/g dw) 5 ( μ g/g fw ) 6

Forage Root R5 101

- 1 11 2.2 (0

. 81) 10 (3

. 7) 0.0 6 8/0. 0 33

1.1

– 4.1 5.1

– 19

Forage R5 101

- 1 11 2.4 (0

. 71) 8.0 (2

. 3) 0.1 3 7/0. 0 70

1.5

– 3.8 5.2

– 13

Grain R6 139

- 1 54 1.7 (0

. 27) 1.9 (0

. 31) 0.2 2 8/0. 1 52

1.4

– 2.7 1.6

– 3.1

Pollen VT

- R1 65

- 80 15 (1

. 9) 19 (2

. 8) 0.1 3 7/0. 0 99

12

– 19 16

– 24

Silk R1 65

- 81 4.0 (0

. 69) 40 (5

. 0) 0.1 3 7/0. 1 21

3.1

– 5.1 32

– 49

1 OSL= ov e r s e ason leaf; OS R = over sea s on r oo t ; O S WP= o ver sea s on wh o l e p l a nt

2 The c r op d e velop m ent stage each tis s ue w a s collected.

3 The n u m ber of da y s a f te r p l an tin g tha t ea c h tissu e w a s c ollected.

4 Protei n l e vel s ar e expre s s e d a s th e arithmeti c m ean and s t a n da r d de vi a ti on ( S D) as m i crogram ( μ g) o f

p r o t e i n per gr a m (g) o f ti s s ue o n a fr e sh w e i ght bas i s ( f w )

. T he m eans, SD, a n d r a ng e s ( m i ni m u m a n d

maxi m u m values) were cal c u lated for ea c h tissue acr o s s al l site s ( n =2 0, e x ce pt for stover wh e re n = 19

due t o o ne sa m p l e exp r essin g <L O Q ).

5 Protei n l e vel s ar e expre s s e d a s th e arithmeti c m ean and s t a n da r d de vi a ti on ( S D) as m i crogram ( μ g) o f

p r o t e i n per gr a m (g) of ti s s u e on a d ry w e i ght b a s i s ( d w ). T he dry w e i ght va l ues w e re ca l cu late d b y

d i v i d i ng t he μg/ g f w by t he dr y w eig ht con v e r sio n f act or o b t ai n e d f r om m o ist ure a n alysi s d ata.

6 LOQ=li m it of q u a n titati on; LOD=li m i t of d etecti o n.

.

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V.D. Assessment of Potential Aller genicit y o f th e Cry3Bb 1 an d CP 4 EPSPS

Proteins

The aller g e nic p o tential of an introduced protein is assessed b y co mp arin g the

physioche m ical characteristics of th e introduce d protei n t o ph ysioche m ical characteristics

of known allergens (Codex Ali m entarius, 2009). Using a we i ght of evidence approach, a

protein is not likely to be associated with a l lergenicity if: 1) the protein is from a non

allergenic s ource; 2) t h e protein represents a s m all porti o n of the total plant p r o tei n ;

3) the protein does not share structural si m ilarities to known allergens based on the a m ino

acid sequence; and 4) the pro t ein does not show resistance to pepsi n digestion

. The

Cry3Bb1 and CP4 EPSPS proteins have been assessed f or their pote n ti a l aller g e n icity

accor d ing to these safety assess m ent guidelines.

V.D.1. As s e ssmen t o f Potentia l All ergenicity of the Cry3Bb1 Protein

The Cry3Bb1 protein has been a s sessed for i t s potenti a l allergenicity accor d ing to the

Codex safety assess m ent guidelines describ e d above

, an d conclusion s wer e a s follows.

1) The Cry3Bb1 protein originates from B t

, an organism that has not been reported

t o b e a sourc e o f know n allergens.

2) The Cry3Bb1 protein represents no more than 0.004% of the total protein in the

grai n o f MO N 87411.

3) Bioinfor m a t ics analyses de m onstrated that the Cry3Bb1 protein does not share

a m ino acid sequence s i m ilarities with known allergens an d

, the r efore, is hig h ly

unlikely to contain immuno l ogically cross

- reacti v e a llergeni c epitopes.

4) Finally, in v itro digestive fate experi m ents c onducted with the Cry3Bb1 protein

de m onstrate that the protein is rapidly digested by pepsin in a si m ulated gastric

fluid (SGF) assay.

Taken together, these data support the concl u sio n tha t th e Cry3Bb1protei n doe s no t pose

a significant allergenic risk to hu m ans or ani m als.

V.D.2. As s e ssmen t o f Potentia l All ergenicity of the CP4 EPSPS Protein

The CP4 EPSPS protein has been assesse d fo r it s potentia l all e rgenicit y accordin g t o the

Codex safety assess m ent guidelines describ e d above, and conclusions are as follows.

1) Th e CP 4 E PSP S protei n originate s fr o m Ag r obacterium sp

. strain CP4, an

organism that has not been reported to be a source of known allergens.

2) The CP4 EPSPS protein represents no m ore than 0.002% of the total protein in

the seed of MON 87411.

3) Bioinfor m a t ics analyses de m onstrated tha t th e CP 4 EPSP S p r otei n doe s no t share

a m ino acid sequence s i m ilarities with known allergens an d

, the r efore, is hig h ly

unlikely to contain immuno l ogically cross

- reacti v e a llergeni c epitopes.

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4) Finally, in vitro digestive fate experi m ents cond u cte d wit h th e CP 4 EPSP S protein

de m onstrate that the protein i s rapidl y digeste d b y p e psi n i n a SG F assay.

Taken together, these data support the con c lusion that the CP4 EPSPS protein does not

pose a sig n ificant allergenic risk to hu m ans or ani m als.

V.E. Safet y Assessmen t Summar y o f Cry3Bb 1 an d CP 4 EPSP S Protei n s in

MO N 87411

A co mprehensive set of factors have been considered and assessed in the s afety

assess m ent of the Cry3Bb1 and CP4 EPSPS proteins in food and feed or the

environ m ent. The results are su mm arized below along w ith the conclusions reached

from each assess m ent.

V.E.1. Cry3Bb1 Donor Organism, Hist o r y o f Saf e Use

, an d Specificity

V.E.1.1 The cry3Bb1 Donor Organism is Safe

The donor organism for cry3Bb1

, B a cillus th u ri n giensis, has been used com m ercially in

th e Unite d State s sinc e 195 8 t o produc e m i cr obial

- derived products wit h insecticidal

acti v ity. T he extre m ely low m a m m alian toxicity of Bt

- base d insecticid e product s has

been de m onstrated in nu m erous safety s t udies, and there are no con f ir m ed cases of

all e rgic re ac tions to Cry prot e ins in applicators of m icrobial

- deri v ed B t products during

50 years of use.

Applications of sporulated B t hav e a lon g histor y o f saf e us e fo r pes t contro l in

agriculture, especially in organic far m ing (Cannon, 1993; IPCS, 1999; U.S. EPA, 1988).

Micro b ial p esticides c o ntai n ing Bt Cry3 proteins have been used for more than 30 years

and subjected to extensive toxicity testing sho w ing no adverse effects to hu m an h e alth

(Bau m

, et al., 1999; Betz, et al., 2000; Mc C lintock, et al., 1995; Mendelsohn, et al., 2003;

U.S

. EPA

, 2001b ; 2005).

V.E.1.2. C r y3Bb 1 Protei n ha s a Histor y o f Saf e Use

A histor y o f saf e us e o f Cry3Bb1 protein has been established (Cannon, 1993; IPCS,

1999; U.S. EPA, 1988). Micro b ial pesticides containing B t Cry proteins have been used

for m ore than 50 years and subjected to extensive toxicity testing showing no adverse

effects to hu m an health (Baum et al., 1999; B e tz et al., 2000; McC lintoc k e t al.

, 1995;

Mendelsohn et al., 2003; U.S. EPA, 2001b; 20 0 5). Cry3Bb1, specifically, is one of the

acti v e in g r e dients in t h e m icrobial p esticide m ixture Rave n ® Oil Flowa b le Bioin s ecticide

that was c o mmercializ e d in the United States to contr o l Col o rado Pot a to Beetle (Ba u m et

al., 1999). Cry3Bb1 is also expressed in com m ercially avail a ble YieldGard VT

Rootwo r m /RR2 (MON 88017) m aize and S m artStax ® m aize. The a m ino acid sequence

deduced from the Cry3Bb1 expression casset t e present in Y ieldGard VT Rootwor m / RR2

is identical to that deduced from the Cry3Bb1 expression cassett e presen t i n MO N 87411.

A related Cry3Bb1 protein, which h as over 9 9 % a m ino acid ide n tity to the Cry3Bb1 in

YieldGard VT Rootwor m /RR2 and MON 87411, is expressed in YieldGard Rootworm

m aize (MON 863). The United States EPA has approved com m e rcial use of the

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Cry3Bb1 as expressed in m aize and has estab lishe d a n exemptio n fro m th e require m e nt

of a tolera n ce for resi d ues of the Cry3Bb1 protein and the genetic m aterial for its

production in m aize (U.S. EPA, 2004b). The history of large scale c u lti v ation of these

Cry3Bb1

- expressing crops without any indicati o n of ha r m ful i m pact on t he environ m ent,

non

- tar g et o rganis m s, or m a m m als provi d es a dditio n al e v idence for the safety of the

Cry3Bb1 protein. Taken togethe r

, thes e dat a demonstrat e tha t th e Cry3Bb 1 protei n ha s a

history of safe use and does not pose any unexpected effects to hu m an and ani m al health

or the en v ir o n m ent.

V.E.1.3. C r y3Bb 1 Protei n D e monstrate s Specificity

Cry3Bb1 protein, like other Cry3 p r ot e ins, has inse c ti c idal acti v ity s p e ci f ically ag a in s t

coleopteran insects (Höfte and W hiteley, 1989) and is covered in m ore detail in Section

X.C.1.

V.E. 2 CP 4 EPSP S Dono r Organism

, Histo ry of Safe Use, and Specificity

V.E.2.1 The cp4 epsps Donor Organism is S a f e

The donor organism for cp4 epsps

, Agrobacterium sp. strain CP4, is not known for

hu m an or ani m al pathogenicity, and is not commonly allergenic (F A O

- W HO, 1991).

The history of safe use of Agrobacterium sp

. strai n CP 4 ha s bee n previousl y reviewe d as

a part of the safety assess m ent of this donor organism for U S DA

- APHIS deregulations,

as well as co mpleted Monsanto consult a tions w ith the FDA regarding Roundup Ready

varieties of soybean ( 1 995 and 2007), canola (1995, 2002, and 2012), m aize (1998 and

2000), sugar beet (1998 and 2004), alfalfa (2004), and cotton (1995 and 2005).

V.E.2.2. CP4 EPSPS Protein has a History of Safe Use

The CP4 EPSPS protein present in MON 87411 is si m ilar to EPSP S protein s consu m ed

i n a variet y o f foo d an d fee d sources

. CP 4 EPSP S protei n i s ho m ol o gou s t o EPSPS

proteins naturally present in pla n ts, including f ood and feed crops ( e. g

., soybean and

m aize) and fungal and m icrobial food sources such as baker’s yeast ( Saccharomyces

cerevisia e ), all of which have a history of safe consumption (Harrison et al., 1996;

Padgette et al., 1996). The si m ilarity of t h e CP4 EPSPS p r otein to E P SPS proteins in a

variety of foods and feeds supports extensive safe consu mp t ion of the fa m ily of EPSPS

proteins and the lack of hu m an or ani m al health concerns. The ubiquitous presence of

h o m ologous EPSPS enzy m es in c rops and c o m m on microorganis m s establishes that

EPSPS proteins, and their enzy m atic activity, p o se no hazards to hu m ans, ani m als, or the

environ m ent. In addition, the CP4 EPSPS protein in MON 87411 is identical to the

CP4 EPSPS protein in nu m erous other Roundup Ready varieties of soybean

, m a ize,

canola, su g ar beet, cotton and al f alfa. F urther

, th e U.S

. EP A ha s establishe d an

exe mption from the require m ent of a tolera nc e fo r residue s o f CP 4 EPSPS protein and the

genetic m aterial necessary for its pr o du c tion in all pla n ts (U.S. EPA, 1996).

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V.E.2.3. CP4 EPSPS Cataly z es a Specific Enzyme Reaction

CP 4 EPSPS

, lik e othe r EPSP S enz y m e s

, funct i on s i n th e shiki m a te pathway that is

integral to aro m atic a m i no acid biosynthes i s in plants and m icroorganis m s (Levin and

Sprinson, 1964; Steinrücken and Amrhein, 1980 )

. Therefore, EPSPS and its activity are

found widely in food a nd feed derived from plant and m icrobial sources. Genes for

n u m erous EPSPS proteins have been cloned (Padgette et al., 1996) an d th e catalytic

d o m a in s o f thi s grou p o f protein s ar e conse r ved

. Bacteria l EPSP S protein s hav e been

well cha r a c te r ized with respe c t to their t h ree di m ensional X

- ray crystal structures

(Stallings

, e t al.

, 1991 ) an d d e taile d kineti c an d che m ica l m echani s m s (Anderson and

Johnson, 1990).

V.E.3. Cry3Bb1 and CP4 EPSPS Proteins in MON 87411 are Not Homologous to

Kno w n Allergens or Toxins

Bioinfor m a t ics analyses were perfor m ed to a ssess the p o tential f or a ll e r g enicity, to x i city,

o r biologica l activit y o f Cry3Bb 1 an d CP 4 EPSPS. The analyses demonstrated that

neither protein shares a m ino acid sequence s i milaritie s wit h know n allergens

, gliadins,

glutenins, or protein toxins tha t coul d hav e advers e effec t s to hu m an or ani m al health.

V.E.4. C r y3Bb1 and CP4 EPSPS Proteins in MON 87411 are Labile in in v itro

Digestion A ssays

A s ha s bee n describe d i n previou s regu latory sub m issions for a number of Roundup

Ready crops and MON 88017 (APHIS Petition No. 04

- 125

- 01), both the Cry3Bb1 and

CP4 EPSPS proteins are readily digestible i n S G F (USDA

- APHIS

, 2013)

. Digestio n in

si m ulated i n testinal fl u id (SIF) was also ass e sse d

. CP4 EPSPS was rea d ily di g estible in

SIF. As e x pected, Cr y 3Bb1 subjected to SIF alone was processed to a tryp s in

- resistant

core of ~66 kDa which was stable throughout t h e SIF digestion period. This result is

consistent w ith observations for this and other C ry proteins subjected to SIF digestion.

Rapid degradation of the Cry3Bb1 and C P 4 EPSPS proteins in SGF m akes it highly

unlikely that either protein would be absorbed in a fo r m other than as co mponent

nutrition a l a m ino acids in t h e s m all intestine o r hav e an y advers e effect s o n hu m a n or

ani m al health.

V.E.5. Cry3Bb 1 an d CP 4 EPSP S Prote ins in MON 87411 are Not Acutely Toxic

Acute oral toxicology stud ie s wer e conducte d wit h Cry3 B b 1 an d CP 4 EPSP S proteins

individually

. Result s indicat e tha t neithe r Cry3Bb 1 no r CP 4 EPSP S cause d an y adverse

effects in m ice, with No Observable Adver s e Effec t Level s (NOA E Ls ) fo r CP 4 EP S P S at

572 m g/kg (Harrison et al., 1996) and for C r y3Bb1 at 1930 m g/kg bw ( F SANZ, 2006),

the highest doses tested. As described below, the highest dose levels tested are m any fold

higher than levels of Cry3Bb1 or CP4 EPSPS t ha t ar e presen t i n tissue s o f MO N 874 1 1.

V.E.6. Human and Animal Exposure to the Cry3Bb1 and CP4 EPSPS Proteins

A com m on approach u s ed to ass e ss potential health risks for potentially toxic m aterials is

to calculate a Margin of Exposure (MOE) between the lowest NOAEL fr o m a n

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appropriate ani m al toxicity study and an esti m ate of hu m an exposure. Since no evidence

o f m a mma l i a n toxicit y ha s bee n reporte d fo r Cry3Bb 1 o r CP 4 EP SPS, dietary ri s k

assess m ents would nor m ally not be considered necessary

. Nevertheless

, a dietar y risk

assess m ent was still co n ducted for these protei n s in order to provide further assurances of

safety by calculating a MOE between t h e NOAELs for the Cry3Bb1 and CP4 EPSPS

proteins in m ouse acute oral tox icit y studie s (Sectio n V.E.5 ) an d 9 5 th per c entile

consu mption esti m ates of acute dietary exposure deter m ined using the Dietary Exposure

Evaluation Model

- Food Com m odity Intake Database (DEEM

- FCID) (U.S

. EPA

, 2013).

DEEM

- FCID utilizes food consumption data from the National Health and Nutrition

Exa m ination Survey (NHANES) conducted in 2003

- 2008 (Included: vegetable m a ize,

popcorn, and field m aize (flour, m e al, bran a n d starch))

. B a se d o n level s o f expressed

protei n o n a fres h weigh t basi s ( μ g/g) discussed above (Se c tion s V.C. 1 an d V.C.2)

, 95 th

percentile exposures to Cry3Bb1 and CP4 E PSPS for the general U.S. population were

esti m ated to be 7.2 and 3.5 μ g/kg body weight (bw), res p ectively

. Fo r non

- nursing

infants in the U.S., the most highl y expose d sub

- population

, 9 5 th percentil e exposure s to

Cry3Bb1 and CP4 EPSPS were esti m ated to be 17.1 and 8.3 μ g/kg, respectively. For the

Cry3Bb1 protein, MOEs for acute dietary intake were esti m ated to be 270,000 (2.7 × 1 0 5 )

and 110,000 (1.1 × 10 5 ) for the general population and non

- nu r sing infants, respectively.

For the CP4 EPSPS protein, MOEs for acute dietary intake were esti m ated to be 160,000

(1.6 × 1 0 5 ) and 69,000 (6.9 × 1 0 4 ) fo r th e genera l populatio n and non

- nursing infants,

respe c tiv e ly. Actual MOEs will li k ely be m uch higher be c au s e: 1) the e x p osure esti m ates

utilized are conservative (9 5 th percentile, assu m e 100% MON 87411) and 2) as described

in section V.E.4, Cry 3 Bb1 and CP4 EPSPS are rapidly digested, further m in i m izing

exposures. These very large MOE s 5 indic a te t h a t th e re is no m eaningful risk to hu m an

healt h fro m dietar y expos ure to the Cry3Bb1 or CP4 EPSP S protein s produce d by

MO N 87411.

There are a nu m ber of steps in the proces sin g o f m a iz e t o m a ke food ingredients,

including high te m perature treat m e nts, hydrolyse s

, soakin g i n slightl y acidi c water

, and

drying that can denature a protein. Changes in te mperature, pH, and physical disruptions

associated w ith food processing and cooking/preparation gener all y lea d t o los s o f protein

structure and functionality ( H am m o nd and Jez, 2011). Like othe r proteins

, th e Cry3Bb1

and CP4 EPSPS proteins in MON 87411 are expected to be si m ilarly susceptible to

denaturatio n whe n expose d t o hig h te mperatures, pH extre m es, and di g e stive

environ m ents encountered during proces sin g an d cookin g o f food s containing

MON 87411. Thus, there are likely to be significantl y lowe r exposure s t o the

functionally active forms of these proteins through consu m ption of MON 87411 than the

levels estimated above.

The potential Cry3Bb1 and CP4 EPSPS protein exposure to ani m als from consu m ption of

MO N 8741 1 i n feed s wa s evaluate d b y calculat i ng an esti m ate of daily dietary intake

(DDI). The highest percentage of Cry3Bb1 and CP4 EPSPS proteins (g/kg bw) per total

5 These M OEs reflect that a hu m an would have to eat th ou s a n ds of k i l o g ra m s of m a ize i n a s h o rt t i m e

per i od t o a c h i eve e x p o s ures t o t he e x p res s e d pr o t e i ns i n M ON 8 7 41 1 t h a t w e re n ot t ox i c t o m ice

, wh ich

wo u l d be a ph y sica l i m po ssi b i lity.

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protein consu m ed was i n the lactating d a iry cow, 0.014% ( g/g) and 0.0034% (g/g) of the

tota l dietar y protei n intak e (0.00087 6 g Cry3Bb1/k g b w divid ed b y 6. 2 g dietar y protein,

and 0.000210 g CP4 EPSPS/kg bw divided by 6.2 g dietary protein w hich is the total

dietary protein intake for the cow), respecti v ely. The chicken and pig percentages of the

Cry3Bb1 and CP4 EPSPS proteins consu m ed as part of the d aily pr o t e in intake are m u ch

less than for the lactating dairy cow. In th e wors t cas e scenario

, pe r k g bod y weight,

poultry, swine and lactating dair y cattl e woul d b e con s u m in g 0.02 % (g/g ) an d 0.004%

(g/g ) o r les s o f thei r tota l protein as Cry3Bb1 and CP4 EPSPS proteins, respectively,

from MON 87411 m aize.

In summary, there is n o signi f ica n t risk to h um an and ani m al health associated with

dietar y exposur e t o th e Cry3Bb 1 an d CP 4 E PSP S protein s i n foo d an d fee d products

derive d fro m MO N 87411.

V.E.7. CP 4 EPSP S Activity

The CP4 EPSPS protein produced in MON 87411 is si m ilar to native EP S PS proteins that

are ubiquitous in plant and m icrobial tissues in the environ m ent and is not known to be

toxic to other organis m s (ILSI

- C ERA

, 2010 ; 2011 ; USDA

- APHIS

, 2013).

V.E.8. Cry3Bb1 Activity

The spectr u m of activity of the m o dified Cry3Bb1 protein has previously been reviewed

by USDA

- A PHIS in s ub m issions for both MON 863 and MON 88017 (USDA

- APHIS,

2013 ) an d U.S

. EP A (U.S

. EPA

, 2010)

. Signif i cant insecticidal act i vit y ha s onl y been

seen in the f a m ily Chryso m elidae within t h e Order Coleoptera (U.S. EPA, 2010). The

Cry3Bb 1 gene s presen t i n MO N 86 3 an d MO N 8801 7 code for Cry3Bb1 proteins that are

>99.8% identical and which have been shown to be functionally equivalent in insect

bioassays w ith two species sensitive to the Cry3Bb1 protein (U.S. EPA, 2010). The

Cry3Bb 1 gene present in MON 87411 codes for Cry3 B b1 p r otein that is identical to that

in MON 88017. Con s equently, the Cry3Bb1 in MON 87411 is predicted to have

equivalent functional activity and spectrum of activity as in those two previously

registered and deregulated m aize product s

.

V.E.9. Non

- Targe t Assessmen t for CP4 EPSPS Protein

The USDA

- APHIS has previously deter m i n ed that the gene i mparting glyphosate

tol e rance, cp4 epsps, an d th e CP 4 EPSP S protei n tha t i t encodes, poses no significant risk

to non

- target organis m s due to its long histo r y of use and no known toxicity to non

- target

organis m s (Harrison et al., 1996; IL S I

- CERA

, 2010 ; USDA

- APHIS

, 2013).

V.E.10. Non

- Target Assessment for Cry3Bb1 Protein

USDA

- APHI S an d EP A hav e conducte d plan t pest and environ m ental assess m ents of

MO N 86 3 an d MO N 8801 7 tha t prod uce the Cry3Bb1 protein. The Cry3Bb 1 gene

present in MON 87411 has the sa m e coding sequence as the Cry3Bb 1 gene present in

MON 88017 and the tissue expression of the Cry3Bb1 protein is comparable to the

previousl y registere d maiz e product s MO N 8801 7 an d MO N 863

. Fo r MO N 863

, a

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series of NTOs were evaluated to assess for potential negative i m pacts of the Cry3Bb1

protei n (U. S

. EPA

, 2010)

. Exposur e concent r ations for the MON 863 studies was based

upon ≥ 10X the m ax i m u m observed Cry3Bb1 expression in tissue and no unacceptable

negative impacts were observed in the N T O t esting battery. Because the m aximum

Cry3Bb 1 protei n level s ar e highe r i n MO N 86 3 co mp are d t o MO N 8741 1 previous

studies per f or m ed for MON 863 are sufficient to inform and provide a protective NTO

assess m ent for MON 87411. In the environ m ental assess m ent for MON 863 (USDA

APHIS, 2013)(USDA

- APHIS Petition No. 01

- 137

- 01p), USDA

- APHIS considered the

potential i m pact of the Cry3Bb1 protein on NTOs, including beneficial organis m s and

threatened or endanger e d species concluding:

“It [MON 86 3 ] has no potential to have a greater damaging, harmful, or

toxic effect on organisms beneficial to agriculture than does other

cultivated c orn. In ad d ition to our finding o f n o plant pe s t risk, t h ere w ill

be no effect on threatened or endangered species resulting from a

determination of non

- regulated status for MON 863 c orn and its

progeny.” (USDA

, 2002)

Additionally, USDA

- APHIS concluded in their assess m ent of MON 88017 that;

“Th e Cry3Bb 1 protei n expresse d i n MO N 8801 7 cor n ha s activit y only

against select beetle (Order Coleop t era) species within the family

Chrysomelidae, namely CRW [corn rootworm] and Colorado potato

beetle

. Fiel d observations

, compositiona l analyses

, an d dat a o n th e safety

o f th e engineere d EPSP S an d Cry3Bb 1 protein s al l indicat e that

MON 88017 should not have greater pote n tial than other cultivated corn

to damage or harm organisms beneficial to agriculture. In addition to our

finding of n o plant pest risk, there will be no e ffe c t on the threatened or

endangered species res u lting from a determin a ti o n of non

- regulated st a tus

for MON 88017 and its progeny.” (USDA

, 2005)

The EPA also conducted an extensive env i ron m ental hazard assess m e nt of Cry3Bb1

- producing m aize based on studies conducted with si m ilar Cry3Bb1 proteins on

repre s entative species of b i rd, fish, and terrestrial non

- target insects, including

endangered species, to support the re gistratio n o f MO N 86 3 an d MO N 88017

concluding:

“ Due to a demonstrated lack of toxicity and/or exposure, no effects from

Cry3Bb1 protein are anticipated for any non

- target species, including

federally listed threatened and endangered (“listed”) lepidopteran and

coleopteran species and their designate d critica l habitats. ” (U.S. EPA,

2010)

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 96 o f 3 7 2CB I Delete d Copy

V.F

. Cry3Bb 1 an d CP 4 EPSP S Protein s C haracteri z ation and Safety Conclusion

The data and info r m ation provided in this section address the questions i mportant to the

food, feed and environ m ental safety of the Cry3Bb1 and CP4 EPSPS proteins i n

MON 87411, including their pote n tial allergenicity an d toxicity

. T o summarize

, the

physicoche m i ca l characteristic s o f th e C r y3Bb 1 an d th e CP 4 EPS P S proteins from

MON 87411 were deter m ined and shown to be equivalent to those of their

E. coli

- produce d counterparts

. Give n th e ver y large MOEs for Cry3Bb1 and CP4 EPSPS

proteins for hu m ans and the m in i m a l exposu r e to ani m als, both support a conclusion of

no significant risk to hu m an or ani m al health associated with dietary exposure to these

proteins in food and feed product s derive d fro m MO N 8 7 411

. A n assess m en t o f the

allergenic potential of the proteins support s th e conclusio n tha t th e Cry3Bb 1 and

CP4 EPSPS protein do not pose a significant aller g e n ic risk to hu m ans or ani m als. The

donor organis m s for the Cry3Bb1 a nd CP4 EPSPS coding sequences, Bacillus

thuringiensis and Agrobacterium sp. strain CP4, respectively, are ubiquitous in the

environ m ent and not commonly known for hu m an or ani m al pathogenicity, or

allergenicity. The Cry3Bb1 and CP4 EPSPS p r otei n s lack str u ctural si m ilarity to

allergens, toxins or other proteins known to have adverse effects on m a mmals. The

Cry3Bb1 and CP4 EPSPS proteins are rapidly d i gested in simulated digestive fluid and

de m onstrate no oral toxicity in m ice at the level tested. Based on the above infor m ation,

the consu m ption of the Cry3Bb1 and C P 4 EPSPS proteins fr o m MON 87411 or its

progen y i s considere d saf e f o r hu m a ns and ani m als.

The protein safety data presented herein support the conclusion that food and feed

product s containin g MO N 8741 1 or derived from MON 87411 are as safe as m aize

currently on the m arket for hu m an a nd ani m al c onsu mption. Given the identical nature

of these proteins to proteins contained in other products that have been registered by EPA

and deregulated by U S DA

- APHIS, as well as previous safety ass e ssments, the proteins

contained in MON 87411 are also considered as safe for the environ m ent as other maize

products.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 97 o f 3 7 2CB I Delete d Copy

VI. CHARACT E R I ZATIO N AN D SA FET Y ASS E SSM E N T O F T H E Dv Snf7

RN A PR O DUCE D I N MO N 87411

VI.A. History of Safe U se of RNA

- me diate d Gen e Suppressio n i n Plants

RNA

- m e di a t e d gen e suppressio n (RNAi ) i s a naturally occurring, ub i quitou s proces s in

eukaryotes, including plants and ani m als consu m ed as food and feed. Endogenous RNA

m ediated gene m odulation is responsible for certai n characteristic s of conventional crops

(Della Vedova et al., 2005; Kusaba et al., 2003 ; Tutej a e t al.

, 2004 ) an d ha s als o been

utilized in so m e biotechnology

- derive d crop s approve d fo r cu ltivation and use as f ood

and feed (Ivashuta, et al., 2009; Parrott et al., 2010; Petrick et al., 201 3 ). Therefore, there

is a history of safe con s u mption of the R NA molecules m e diating gen e suppressio n in

plants, including those with ho m ology to genes in hu m ans a nd other ani m als (Ivashuta et

al., 2009; Jensen, et al., 2013). Ad d itio n ally, there is no e v i d ence to sug g est that d ietary

consu mption of nucleic acids is associated w ith toxicity (Petrick et al., 2013; U.S. FDA,

1992) and U.S. EPA has an established toleran c e exe m ption for nucleic acids that are part

o f PI P product s (U.S

. EPA

, 2001a)

. FD A re cognizes that all food a l lergens are proteins

FDA, 1992; 2001) and there is also no e v idence of allergenicity of dietary RNA in

the pe e r r e v iewed s c ienti f ic lit e rat u r e. This lack of toxicity or aller g eni c ity f or inge s ted

RNA also extends to R NA m olecules associa t ed with dsRNA

- m ediated gene regulation.

Therefore an exten s ive history of safe consu mp t io n fo r dietar y RNAs

, includin g dsRNAs,

has been established, as reviewed (Petrick et al., 2013). The reason for this history of

safe consumption of dietary RNAs is that extensive sequence

- independent physiological

and bioche m ical barriers are k n own to exi s t in hu m ans and other a n i m als that li m it the

potential for uptake or acti v ity of ingested nucleic acids (J u l iano, et al., 2009; O'Neill, et

al., 2011; Petrick et al., 2013).

A recent publication reported that ingestion of large doses of a particular s m all ( m icro)

RNA ( m iRNA) fr o m rice led to so m e absorpt i on of the m iRNA, detection of the miRNA

in serum and liver, and an apparent i mpact on a target protein and plas m a LDL in m i c e

(Zhang, et al., 2012a). The authors suggest that a “cross

- kingdo m ” effe c t

– a plant gene

product ( m iR168a) regulating ani m al gene expression

– m ay be a common pheno m e non;

and that m iRNAs in food m a y regulate spe c ific genes in ani m als based upon sequence

identity between plant m iRNAs and m a mmalian genes. A second publication

(Heine m ann, et al., 2013), a review article relying al m ost exclusively on the Zhang

(2012a) study, suggests that the current safet y /risk assess m ent approach is not sufficient

fo r RNA

- base d biotechnology

- derive d products

. As stated in the preceding paragraph,

there are no safety concerns related to the consumptio n o f RN A an d RNAi in plants and

therefore t h e current s afety asse s s m ent approach for dsRNA

- containing products,

including MON 87411, is appropriate for a s sessin g produc t safety

. Th e following

evidence supports t h i s conclusion:

• Hu m ans regularly con s u m e plants that co n tain s m all RNAs. Recent research

de m onstrates that m any existing plant RNAs share sequences with human genes

(Ivashuta et al., 2009). This work has also been followed up by studies

de m onstrating that not only s m all RNAs but als o lon g dsRNA s i n plant s share

sequence i d entity to h um an transc r ipts (Jense n e t al.

, 2013)

. Further

, hu m a ns

regularly consu m e an i m al

- derived foods t h at are likely to contain m ore ani m al

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 98 o f 3 7 2CB I Delete d Copy

m i RNAs that have 100% identity to hu m an g e nes than plant m iRNAs. Despite

this routine ingestion of plant and ani m al s m all RNAs, no i mpacts on hu m an

health have been reported

• A follow up study to Zhang et al. (2012a) involving Monsanto scientists (Zhang,

et al., 2012b) revealed confounding factors which likely explain, in part, the

unexpected findings in the 2012 study (Zhang et al., 2012a). In this follow up

study, plant m iRNAs (including m iR168a) were shown to be over

- represented

relative to their dietary abundance in so m e public ani m al s m all RNA datasets.

This in d icates t h at their appare n t presence in m ouse tissues likely resulted, at least

in p a rt, f r o m cross

- co n ta m ination during the s equencing p rocedu r e, t h us c a lling

into que s ti o n the pote n ti a l f or si g ni f icant uptake of ingested pla n t m i RNAs

(Zhang et al., 2012b).

• Differences in diet co mposition, rather than cross kingdom gene regulation by

plant m iRNAs, were likely responsible for alteration s i n plas m a LD L c holesterol

when Zhang et al., (2012a) f e d a n all

- ric e die t to m ice (Petrick et al., 2013). These

results are consistent with the kno w n chall e nges to oral delivery of nucleic acids

(O ' N eill et al., 2011).

• In a rece n t study, Monsanto and m iRagen T h era p eutics scientists collaborated o n

a rice feeding study in m ice to evaluate clai m s of dietary m iRNA uptake and

physiolo g ical i mpact (Dickinson, et al., 2013. Accepted for publicatio n ). In this

study, m iR168a uptake was not reproduce d

, LDLRAP 1 protei n level s were

unaffected, and LDL was only m odulate d wit h a hig h ric e die t an d no t wit h a

nutritionally balanced rice diet. These results support the conclusion that

previously reported observations ( Z hang et al., 2012a) were due to variability in

gene expression and protein expression data and nutritional differences in ani m al

diets

, rathe r tha n die t ary exposure to m i R168a.

• A recent re p ort (Snow, et al., 2013) notes the presence of endogenous miRNAs at

substantial levels in diets of hu m an s

, m ice and honey bees. The authors provide

e mpirical data to de m onstrate that des p ite consu m ption of m i RNAs, horizontal

deliver y vi a ora l ingestio n fro m a typica l die t i s neithe r frequen t no r prevalent

across these consu m ing organis m s.

• A recent report ( W itwer, et al., 2 0 13) conducted a feeding study in nonhu m an

pri m ates with an m iRNA rich food source and based on their results, concluded

that, “ the r e is little e v i d ence f or pr e sence of th e se pla n t m i RNA in nonhu m an

pri m ate blood prior to or following die t ary intake of a plant m iRNA

- rich

substance

. ”

• Nu m erous barriers to the syste m ic and ce llula r uptak e o f exogenou s nuclei c acids

exist (salivary enzy m es, sto m ach and int e stinal acids and enzy m es, nucleases in

seru m

, etc.) (Akhtar, 2009; Haupenthal, e t al.

, 2006 ; Jain

, 2008; O ' Neill et al.,

2011; Petrick et al., 2013).

• Other authors, organizations, and regula t ory bodies have looked specifically at

biotechnology

- derived RNA

- based products and support current risk assess m ent

approaches (FSANZ, 2013; ILSI

- CE R A, 2011; Parrott et al., 2010).

• Data prese n ted below in this p e tition regar d i n g speci f i c ity an d lac k o f adverse

i mpacts on NTOs (S e ction VI.E.) supports the safety of the consumption of

MO N 8741 1 an d th e expresse d constru c t

- derive d RN A molecule s contained

within it.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 99 o f 3 7 2CB I Delete d Copy

Finally, these nu m erous points regarding the ubiquitous nature of RNA in foods, the

known barriers to syste m ic and cellular upta k e o f exogenou s nuclei c acids, the known

sequence si m ilarity between so m e pla n t and hu m an RNAs, and e m pirical data

de m onstratin g th e lac k o f advers e impact s o n non

- target organis m s l ea d u s t o conclude

that unique risk issues rel ate d t o expressio n o f DvSnf 7 RN A i n MO N 8741 1 d o no t exist

and the current risk/safety as s ess m ent appr o ac h use d b y USDA

- A P HI S i s appropriat e for

assessing this product.

VI.B. Cha r acteri z a tio n an d Equ iv a lenc e o f D v Snf 7 RN A fro m MO N 87411

Despite the long history of exposure to an d consumptio n o f nuclei c acids

, a

characterization and safety assess m ent of the DvSnf7 RNA produced in MON 87411 was

conducted. The safety assess m ent of the DvSnf7 transcript required large a m ounts of

DvSnf7 RNA as the test substance. Because of the extre m ely low expression of DvSnf7

RNA in MON 87411 (Table VI

- 1), it was necessary to produce RNA through in vitro

transcription m ethods in order to obtain sufficient quantities of DvSnf7 RNA

(DvSnf7_968 RNA 6 ) for subsequent safety studies. In order to de m o nstrate that the

in vitro

- p ro d uced RNA was the a ppropri a te test m ateri a l f or the s af ety asses sm e nt

studies, the molecular characteristics of the RNA produced in MON 87411 (MON 87411

DvSnf7 RNA) were deter m ined and were shown to be equivalent to those of in vitro

produce d R NA

, DvSnf7_96 8 RNA

. A sum m a ry o f th e result s i s sho w n belo w an d the

details of the m aterials, m ethods, and results are described in Appendix E.

Regarding the overall potential for toxicity an d allergenicity

, RN A i s d i fferen t fr o m

proteins. While certain rare protein s ar e capabl e o f de m onstrating toxi c ity ( u sually

occurring acutely) (Pariza and Johnson, 2001; S j oblad, et al., 1992), t h er e i s n o evidence

that inge s t e d dsRNA is capable of eli c iting to x icity in h um ans r e gardless of sequence

(Petrick et al., 2013). Additionall y

, all kno w n food allergens are proteins (U.S. FDA,

1992; 2001) and there is no evidence to suggest that ingested RNA c a n be allergenic.

Therefore, experi m ental endpoints intended to address toxicity and/or allergenicity of

protein s ar e no t necessar y o r usefu l fo r eva l uatin g th e foo d an d fee d safet y o f ingested

RNA.

The characterization and equivalence between MON 87411 DvSnf7 RNA and

DvSnf7_96 8 RN A wa s establishe d b y tw o m o l ecular analyses: 1) DNA sequencing o f

the reverse transcribed RNA deter m ined that the sequence of the MON 87411 DvSnf7

an d DvSnf7_96 8 RNA s wa s identical

, includ ing the sequence within the 240 bp inverted

7 repeat regions; and 2) RNase I f digestion follo w ed by northern blot analysis detected the

24 0 b p dsRN A i n MO N 8741 1 DvSnf 7 RN A an d d e m on s trated its equivalence to the

in vitro produce d DvSnf7_96 8 RNA.

6 D v Snf7_ 9 6 8 i s t he pr ed o m i n a nt RN A tra n scri pt prod u ce d fr om t he DvS n f 7 s u p p ressio n cassett e an d, in

a dd itio n t o t he D v S n f 7 p s e que nces, c o n t a i ns p or tio ns o f th e CaM V 3 5 S p ro m o t er, m aiz e h s p 70 i n tro n, and

pea E 9 3’ u n t r a n slate d r e g i o n f r om P V

- Z MIR 1 08 7 1.

7 Endonucleas e RNas e I f s pec ifi ca ll y d i ges t s s i ng l e s t r a nd e d RNA

, leav i ng dsRN A i n tact

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 100 o f 3 7 2CB I Delete d Copy

Take n together

, thes e dat a provide a detailed charact e rizatio n o f MO N 8741 1 DvSnf7

RNA and establish its equivalence to the in v itr o

- produced DvSnf7_968 RNA. The

equivalenc e justifie s th e us e o f th e DvSnf7_9 68 RNA as a test subtance in the safety

assess m ent studies (Section VI.E.3.) ( e.g

. , studie s o n earthwo r m s, hone y bees

, Lady

beetle, environ m ental fate of D v Snf7 RNA, etc.) of MON 87411.

VI.C. Exp r essio n Level s o f DvSnf 7 RN A i n MO N 87411

DvSnf7 RNA levels in various tissues of MON 87411 w e re deter m ined by a validated

QuantiGen e ® Plex 2.0 Assay. Plant m aterial from MON 87411 was collected in 2011

-

2012 from the sa m e replicated field sites in Argentina (BAFO, BAHT, BAPE, BASS,

and BATC) using the sa m e 19 tissue types as described for protein expression analysis

(Section V.C.).

DvSn f 7 RNA levels we r e deter m in e d in all 19 tissue types. The r e sults obtained for each

sa mple by the QuantiGene ® Plex 2

. 0 Assay were averaged across the five sites and are

summarize d i n Tabl e VI

- 1

. Th e detail s o f the m aterials and m ethods are described in

Appendi x F

. Th e individua l DvSnf 7 R N A level s i n M O N 8741 1 acros s al l samples

analyzed fr o m all sit e s ranged fr o m lower than the li m it of detection (<LOD) to

213 × 1 0

- 3 μ g/g dw. The m ean DvSnf7 RNA level for each tissue type was highest in

OSWP1 at 84.8 × 1 0

- 3 μg/g dw and lowest in grain at 0.104 × 1 0

- 3 μg/g dw. Samples

with an ex p ression level <LOD or l o wer than the li m it of q u antitation ( < LOQ) were not

included in the m ean deter m inations.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 101 o f 3 7 2CB I Delete d Copy

Tissue Develo p m ent Mean

(SD) Mean

(SD)

Typ e 1 Stag e 2 Range

( μ g/g fw ) 3 Range

( μ g/g dw ) 4

OSL1 V3

- V4 12.6 × 1 0

- 3

(2.11 × 1 0

- 3 ) 73.9 × 1 0

- 3

(14.5 × 1 0

- 3 )

8.53 × 1 0

- 3

- 16.6 × 1 0

- 3 43.3 × 1 0

- 3

- 103 × 1 0

- 3

OSL2 V6

- V8 13.2 × 1 0

- 3

(4.08 × 1 0

- 3 )

7.77 × 1 0

- 3

- 20.1 × 1 0

- 3 67.3 × 1 0

- 3

(19.4 × 1 0

- 3 )

37.1 × 1 0

- 3

- 98.9 × 1 0

- 3

OSL3 V10

- V13 10.3 × 1 0

- 3

(1.89 × 1 0

- 3 )

6.19 × 1 0

- 3

- 12.8 × 1 0

- 3 44.6 × 1 0

- 3

(8.51 × 1 0

- 3 )

27.5 × 1 0

- 3

- 58.8 × 1 0

- 3

OSL4 V14

- R1 14.4 × 1 0

- 3

(6.71 × 1 0

- 3 )

5.40 × 1 0

- 3

- 33.8 × 1 0

- 3 56.9 × 1 0

- 3

(28.5 × 1 0

- 3 )

22.1 × 1 0

- 3

- 153 × 1 0

- 3

OSR1 V3

- V4 3.15 × 1 0

- 3

(1.79 × 1 0

- 3 )

1.74 × 1 0

- 3

- 8.00 × 1 0

- 3 23.9 × 1 0

- 3

(15.1 × 1 0

- 3 )

12.5 × 1 0

- 3

- 67.0 × 1 0

- 3

OSR2 V6

- V8 2.32 × 1 0

- 3

(0.758 × 10

- 3 )

0.9 2 8 × 10

- 3

- 3.76 × 10

- 3 16.3 × 1 0

- 3

(4.84 × 1 0

- 3 )

6.62 × 1 0

- 3

- 25.7 × 1 0

- 3

OSR3 V10

- V13 1.81× 1 0

- 3

(0.749× 10

- 3 ) 10.2× 1 0

- 3

(4.77× 1 0

- 3 )

0.9 4 2 × 10

- 3

- 4.00× 1 0

- 3 5.13 × 1 0

- 3

- 24.3 × 1 0

- 3

OSR4 V14

- R1 1.28× 1 0

- 3

(0.471× 10

- 3 )

0.5 3 0 × 10

- 3

- 2.40× 1 0

- 3 6.84× 1 0

- 3

(2.67× 1 0

- 3 )

2.66 × 1 0

- 3

- 13.0× 1 0

- 3

OSWP1 V3

- V4 10.5 × 1 0

- 3 84.8 × 1 0

- 3

(4.25 × 1 0

- 3 )

6.78 × 1 0

- 3

- 23.1 × 1 0

- 3 (43.8 × 1 0

- 3 )

51.1 × 1 0

- 3

- 213 × 1 0

- 3

OSWP2 V6

- V8 8.54 × 1 0

- 3

(3.54 × 1 0

- 3 )

5.01 × 1 0

- 3

- 16.0 × 1 0

- 3 55.1 × 1 0

- 3

(23.1 × 1 0

- 3 )

33.0 × 1 0

- 3

- 106 × 1 0

- 3

OSWP3 V10

- V13 3.53 × 1 0

- 3

(1.17 × 1 0

- 3 )

2.03 × 1 0

- 3

- 5.89 × 1 0

- 3 25.5 × 1 0

- 3

(9.53 × 1 0

- 3 )

13.0 × 1 0

- 3

- 45.9 × 1 0

- 3 Tabl e VI

- 1

. Summar y o f DvSnf 7 RN A L e v els in Maize Tissues Collected from

MON 87411 Produced in Argenti n a Field Trials during 2011

- 2012

LOD/

LO Q 5

( μ g/g fw)

0.1 2 9 × 10

- 3 /

0.5 6 6 × 10

- 3

0.1 1 4 × 10

- 3 /

0.5 0 2 × 10

- 3

0.1 0 6 × 10

- 3 /

0.4 6 8 × 10

- 3

0.1 1 0 × 10

- 3 /

0.4 8 2 × 10

- 3

0.0 2 9 × 10

- 3 /

0.1 2 8 × 10

- 3

0.0 2 1 × 10

- 3 /

0.0 9 3 × 10

- 3

0.0 2 0 × 10

- 3 /

0.0 8 8× 10

- 3

0.0 1 5× 10

- 3 /

0.0 6 7× 10

- 3

0.0 7 8 × 10

- 3 /

0.3 4 5 × 10

- 3

0.0 5 4 × 10

- 3 /

0.2 3 9 × 10

- 3

0.0 2 7 × 10

- 3 /

0.1 1 9 × 10

- 3

M ons a n t o Co m pany CR 2 4 0

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Table VI

- 1. (continued). Summary of DvSnf7 RNA Levels in Mai z e Tis s ues

Collected from MON 87411 Produced in Argentina Field Trials during 2011

- 2012

Tissue

Typ e 1 D eve l op m ent

Stag e 2 Mean

(SD)

Range

( μ g/g fw ) 3

, 6 Mean

(SD)

Range

( μ g/g dw ) 4 LOD/

LO Q 5

( μ g/g fw)

OSWP4 V14

- R1 3.16 × 1 0

- 3 18.5 × 1 0

- 3 0.0 2 8 × 10

- 3 /

(1.03 × 1 0

- 3 ) (6.27 × 1 0

- 3 ) 0.1 2 3 × 10

- 3

1.89 × 1 0

- 3

- 5.37 × 1 0

- 3 10.3 × 1 0

- 3

- 32.2 × 1 0

- 3

Forage

Root R5 0.5 3 6 × 10

- 3 2.37 × 1 0

- 3 0.0 1 3 × 10

- 3 /

(0.295 × 10

- 3 ) (1.29 × 1 0

- 3 ) 0.0 5 9 × 10

- 3

0.0 8 6 × 10

- 3

- 1.07 × 10

- 3 0.4 2 5 × 10

- 3

- 4.61 × 10

- 3

Forage R5 1.28 × 1 0

- 3 4.26 × 1 0

- 3 0.0 3 6 × 10

- 3 /

(0.361 × 10

- 3 ) (1.26 × 1 0

- 3 ) 0.1 5 7 × 10

- 3

0.6 0 1 × 10

- 3

- 2.31 × 10

- 3 2.00 × 1 0

- 3

- 7.72 × 1 0

- 3

Senescent

Root R6 0.3 5 3 × 10

- 3 1.39 × 1 0

- 3 0.0 1 5 × 10

- 3 /

(0.203 × 10

- 3 ) (0.815 × 10

- 3 ) 0.0 6 5 × 10

- 3

0.1 2 7 × 10

- 3

- 0.9 4 7 × 10

- 3 0.4 7 8 × 10

- 3

- 3.68 × 10

- 3

Stover R6 0.3 1 0 × 10

- 3 0.6 7 7 × 10

- 3 0.0 4 7 × 10

- 3 /

(0.077 × 10

- 3 ) (0.201 × 10

- 3 ) 0.2 0 7 × 10

- 3

0.1 9 0 × 10

- 3

- 0.4 4 9 × 10

- 3 0.4 0 1 × 10

- 3

- 1.04 × 10

- 3

Pollen VT

- R1 0.1 0 3 × 1 0

- 3 0.1 3 4 × 10

- 3 0.0 1 3 × 10

- 3 /

(0.069 × 10

- 3 ) (0.090 × 10

- 3 ) 0.0 5 7 × 10

- 3

0.0 5 6 × 10

- 3

- 0.2 2 4 × 10

- 3 0.0 7 3 × 10

- 3

- 0.2 9 2 × 10

- 3

Silk R1 0.5 3 0 × 10

- 3 5.42 × 1 0

- 3 0.0 0 4 × 10

- 3 /

(0.190 × 10

- 3 ) (2.05 × 1 0

- 3 ) 0.0 1 9 × 10

- 3

0.2 1 5 × 10

- 3

- 0.8 9 3 × 10

- 3 1.99 × 1 0

- 3

- 9.03 × 1 0

- 3

Grain R6 0.0 9 1 × 1 0

- 3 0.1 0 4 × 10

- 3 0.0 0 8 × 10

- 3 /

(0.028 × 10

- 3 ) (0.033 × 10

- 3 ) 0.0 3 6 × 10

- 3

0.0 4 9 × 10

- 3

- 0.1 5 3 × 10

- 3 0.0 5 6 × 10

- 3

- 0.1 7 5 × 10

- 3

1 OSL

= ove r s e aso n leaf

, OS R = ove r sea s o n r oo t

, a n d O S W P= o v e r sea s o n w ho l e p l ant

2 The c rop d e velop m ent stag e s at whic h eac h tissu e wa s c o llected.

3 Th e DvSnf 7 RN A l e vel s a r e calculate d a s microgra m (μ g) o f D v S n f7 RNA (≥ 20 0 nt ) p e r gram ( g ) o f

ti ssue o n a fre s h we i ght ( f w ) bas i s. T he s a m p l e m eans, SDs, a nd ra n ges ( m i ni m u m and m a xi m u m va l ues)

wer e cal c u late d fo r eac h tis s ue typ e a c ro s s al l 5 site s (n

= 2 0

, e x cep t fo r senesc e nt roo t n=19

, stove r n=16,

po lle n n= 5

, a n d gra i n n= 1 8 due t o e x pr e s s i ons f rom t wo p o lle n sa m p le s <L O D a n d f rom t he rest o f t he

sa m p le s fo r s e nesc e nt root

, s t ov e r

, pollen

, a nd grai n <LOQ)

4 Th e DvSnf 7 RN A l e vel s ar e calculated as m i crogram (μ g) of D v S n f 7 R NA per g ram of ti s s ue o n a dry

weigh t (dw ) basis

. T he sa m p l e m eans

, SDs

, an d ranges (mini m u m and maxi m u m values) were cal c ulated

for each tis s ue type across a l l 5 sites (n

= 20, exc e pt f o r s e nesc e nt root n=19, stover n = 16, pollen n=5, and

gra i n n= 1 8 du e t o e x press i o ns f r om t wo po lle n sa m p le s <L O D a n d f r om t he rest of t he sa m p l e s f or

senesc e nt ro o t

, s to ver, p o lle n, a n d gra i n < L O Q ).

5 LOD=li m it of d etectio n; LOQ=l i m i t of q u a n titati on.

6 C o n v ertin g μ g / g t o part s p e r b illi on ( p p b) show s DvS n f 7 R N A ex p ressi on ra ng i ng fr om 0. 0 91 p pb i n

gr a i n t o 1 4

. 4 p pb i n o v e r seas on lea f a t t h es e growt h stage s

.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 103 o f 3 7 2CB I Delete d Copy

VI.D. Huma n an d An i m a l Exposur e t o th e DvSnf 7 RNA

The potential dietary intake of DvSnf7 RNA from the consumption of food derived from

MON 87411 can b e e s ti m ated by multipl y ing m aize co n su mption (total of all m aize

fractions consu m ed) by the level of DvSnf7 RNA in MON 87411. For the purposes of

this ass e ssment, the co n cent r ation o f the DvSnf 7 RN A i n th e include d m a iz e fractions

(Deter m ined using DEEM

- FCI D 8 as in Section V.E.6.; flour, m eal, bran, starch, sweet

corn and popcorn) was assu m ed t o be equal to the m e a n expression level in whole

MO N 8741 1 grain

. Base d o n D EEM

- FCI D exposur e esti m a te s an d level s o f expressed

DvSnf 7 RN A i n grai n fro m MO N 87411 on a fresh weight basis ( μ g/g ) discusse d above

(Table VI

- 1 ), 9 5 th percentile exposure esti m ates for t h e genera l U.S

. populatio n an d for

non

- nursing infants (the m ost highly exposed sub

- population) were esti m ated to be 0.2

an d 0. 4 ng/k g bod y weigh t pe r day

, respectively

. These exposure esti m ates are likely to

be overesti m ates because t h ey a s su m e that ther e i s n o los s o f DvSnf 7 RN A during

storage, processing and/or cooking o f th e grai n o r food.

Total nucleic acid consu m ption in the d i e t fo r DN A an d RN A (base d o n purin e and

pyri m idine intake) is esti m ated to be 1

- 2 gra m s per day ( S uchner, et al., 2000) which

would equate to a m axi m al intake of 0.2 g / kg/day (2 g in a 10 kg non

- nursing infant;

assu m ing all of the intake is RNA). In co m p a rison, anticipated exposures to DvSnf7

RNA from consu mption of M ON 87411 are extre m ely low ( ≤ 0.4 ng/kg/day) relative to

this esti m ate of tot a l d aily RNA intake (a 5 0 0 m illion f old di ff ere n ce between daily

consu mptio n o f DvSnf 7 RN A fro m M O N 8 7 411 and total RNA). Esti m ated DvSnf7

exposure values are m o re than 3,300 fold lower than the 1.5 μ g/kg/da y intak e value

defined as the threshold of toxicological concern (TTC) for co mpounds not triggering

structural alerts for to x icity (Kroe s

, et al., 2 0 05). The TTC concept esta b lis h es an

exposure level for che m icals (with or without toxicit y data ) belo w whic h ther e woul d be

negligible risk to hu m an health. Further m o r e, it is notewo r t h y to poi n t o ut th a t t h e T T C

value reflects chronic ( m ean per c apita) int a ke values and the exposures herein are

conservative, as they reflect ac u te ( s hort

- ter m

, 95th percentile) intake values. Based on

this infor m ation, the exposure assess m ent des c ribed above, rather than a dietary risk

assess m ent is con s ide re d m ost appropri a te f or evalu a tion o f the DvSnf7 RNA and the

deter m inati o n of sa f ety is ba s ed on the w e ight

- o f

- the

- evi d ence f or R NA sa f ety ( e.g.

, general RNA history of safe use, v e ry low exposure to the specific DvSnf7 RNA, a nd the

lack of oral toxicity to n u cleic aci d s).

The potential DvSnf7 RNA exposure to a n imals from consu m ption of MON 87411 in

feeds was evaluated by calculating an esti m ate of daily dietary intake. Th e highest

dietary exposure of DvSnf7 RNA is 81.0 ng/kg bw/day in the lactating dairy cow. The

dietar y exposur e o f DvSnf 7 RN A fo r broile r chicke n an d finishin g pi g is

8 D ietar y E xp o su re E va l ua t i on M o d el

– F o o d C o m m odi t y Intake D a t abase

. Thi s i s a softw a re

- based

ex p o s ure a na ly s i s s y s t em for pe r f o r m i ng ch r on i c a nd a c u t e d i e t ary e x po s ure as s e ss m en t s de velo ped by t he

U. S . E P A. D E E M

- F CI D c a n be us e d t o es ti m a t e d i e t ary i n ta ke o f a ny c o m ponent of fo od o r wa te r.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 104 o f 3 7 2CB I Delete d Copy

10.5 ng/kg bw/day and 4.6 ng/kg bw/day, respectively. Therefore, there is m ini m al

dietary exposure of animals to DvSnf 7 RN A fro m MO N 87411.

Negligible exposures and lack o f ora l toxicit y o f RN A i n h i gher organis m s indicates that

ther e woul d b e n o significan t ris k t o hu m a n and a n i m al health associated with

consu mption of DvSnf7 RNA in food and feed products derived from MON 87411.

VI.E. Laboratory Tests to Characteri z e the Spectrum of Activity o f DvSnf7 RNA

VI.E.1. Te s t Substance s Use d t o Asses s th e Activit y Spectru m o f DvSnf 7 RNA

As noted earlier, MON 87411 maize, expr esse s a 96 8 nucleotid e (nt ) DvSnf 7 RNA

pri m ary tra n scri p t. The DvSnf7 target sequence or “active” region is a 2 40 bp stretch of

dsRNA included within the larger 968 nt RNA m olecule. The DvSnf7 240 bp dsRNA

was confir m ed to be equipotent (in ter m s of m ortality to W CR and southern corn

rootworm ( S CR, Diabrotica undecimpunctata howardi )) to the DvSnf7 968 nt RNA in

diet bioassays with both the W CR a nd SCR, justifying the use of the 240 bp dsRNA. The

DvSnf7 240 bp dsRNA, herein referred to a s DvSnf7_24 0 dsRNA

, wa s synthesize d in

vitro f or use in i n itial in s ect d iet bio a ssays to characterize the spectr u m of activity (T a ble

VI

- 2). De fi niti v e st u di e s used f or t h e quantit a ti v e ecolo g ic a l risk as s es sm ent utili ze d an

in vitro synthesize d 96 8 n t DvSnf 7 (know n a s DvSnf7_96 8 RNA ) a s expresse d in

MON 87411 m aize and are descr i bed below (Table VI

- 3).

VI.E.2. Re s u lts from Activity Spectru m Bioassay s fo r DvSnf 7 RNA

An assessment of the s pectrum of acti v ity f o r a pesticide is desig n ed to characterize

activity against a range of insect taxa that includes t h e target organism as well as non

target organis m s (Raybould, 2006; Ro m eis, et al., 2013; U .S. EPA, 2007). Efficacy of

the DvSnf7 dsRNA against the target organ i sm WCR, and the closely related SCR, was

characterized with larvae in 12

- da y continuous

- feedin g d i et

- incorporatio n bioassay s to

9 characterize the conce n t r ation

- effect relationsh i p and to e s ti m ate the L C 50 va lue for

DvSnf7_240 dsRNA (Table VI

- 2) (Bach m an et al.

, 2013 ; Bolognes i e t al.

, 2012)

. Both

WCR and SCR d e m onstrated co mp a rable concentration

- dep e ndent effects with m ean 12

-

da y LC 50 value s o f 4. 4 n g DvSnf7_24 0 dsRNA / m l diet and 1.2 ng DvSnf7_240

dsRNA / m l diet, respe c tively (Table VI

- 2). The rel a tiv e ly sm all di ff erence in sen s itivities

between S C R and W CR species to DvSnf7_240 dsRN A m ay b e relate d to each species’

feeding physiology, how well they perform on an artificial diet in the laboratory

bioassays and SCR typically having higher gro w th rates in laboratory s e ttings than W CR.

Result s fro m feedin g DvSnf7_24 0 dsRN A t o these two species de monstrate s efficacy

against target pests in these assays.

Characterization of t h e spectrum of inse c ticidal a c tivity, MOA, as well as an

understanding of environ m ental exposure l e vels and pathways provides i m portant

9 The L C 50 va lu e i s t he m easure o f t ox i c it y of a s ubs t a nce ( L etha l C o n ce n tratio n), t h a t w h e n fe d t o a test

o r g a n i sm over a g i v e n ti m e per io d, re s u lt s i n dea t h o f 50% o f th ose o rg a n i s m s.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 105 o f 3 7 2CB I Delete d Copy

infor m ation that can narrow the sc o pe of N T O testing for an ecolo g ical risk asse ssm ent

(Ro m eis, et al., 2008; Ro m eis et al., 2013). NTO testing is typically perfor m ed in a

sequential sche m e ( e.g. Tier 1, Tier 2, etc.) and builds upon characterization of the

acti v ity sp e ctru m

. Results f rom the NTO batt e ry co m ple m ents sp e ci f icity d a ta by

evaluating organis m s that m ay be phylogenetically related and/or provide i mportant

functional roles ( e.g.

, det r itiv o res, predators, parasitoi d s, polli n ato r s) in rele v ant and

reliable laboratory studies. In 2011, a tripartit e grou p (govern m e nt

, industr y and

acade m ia) evaluated the enviro nm ental risk asse s s m ent ap p roach for genetically

engineered plants ( e.g. B t

- expressing plants) and conclud e d tha t th e curren t ecological

risk assessment fra m ework and effects tests for NTOs are ap p lica b le to plants expr e ssing

an RNAi trait (ILSI

- C E R A, 2011).

The spe c tr u m of acti v ity f or DvSn f 7 RNA wa s characterize d b y s e l e cting and t e sting

insects based upon their taxono m ic relatedness to the W CR ( T able VI

- 2) (Bach m an et al.,

2013). In total, 14 i ns ect species were te s ted, repre s enti n g 10 f a m ilies and 4 orders.

Representative inse c ts f rom the foll o wing orders were tested: He m iptera ( Orius

insidiosus ), Hy m enoptera ( Nasonia vitripennis and Pediobius foveolatu s )

, Lepidoptera

( Spodopter a frugiperda

, Helicoverp a ze a

, Ostrinia nubilalis

, and Bomby x mori ) and

Coleoptera (WCR, SCR, Leptinotar s a decemlineata, Triboli u m castaneum, Coleomegilla

maculata, Epilachna varivestis, and Poecilus c h alcites )

. A representative insect from the

Order Diptera was n o t exa m ined because a previou s stud y o n a Dipteran s p ecies

( Drosophila spp. ) ha d show n i t t o b e insensitiv e t o dietar y dsRN A withou t th e us e o f a

transfection agent ( W hyard et al

. , 2009)

. Give n tha t th e targ et species are in the Order

Coleoptera, a strong focus was placed on this order and in total, 7 species across four

f

a m ilies in this Order w ere tested w ith t h e goal to m ore f ully chara c te ri ze the r ange of

acti v ity of DvSn f 7 dsRNA within the Order Col e optera.

Diet bioassays were conducted utilizing in vitro synthesized DvSnf7_240 dsRNA, at test

concentrations ranging from 500 to 5000 ng DvSnf7_24 0 dsRNA / m l die t an d exceed

DvSnf 7 d s RN A expressio n value s in p l an ta (Sectio n VI.B.)

. Bioassay s fo r activity

spectrum studies were designed to: 1) pro v ide continuous exposure of the DvSnf7_240

dsRNA to e ach test species, and 2) provide a sufficient duration of exposure to evaluate

th e potentia l effect s o f DvSn f7_240 dsRNA on growth, deve lop m en t an d survival

. At

exposure concentrations that will greatly exceed fie l d exposur e levels

, n o negative

i m pacts attributable to DvSnf7_240 were detected against the Chryso m elidae species,

Leptinot a rsa decemline a ta (phylogenetically the c l osest to the target species), or in

representative species that are even less phylogeneticall y relate d fro m th e othe r three

f

a m ilies (C o ccin e lli d ae, Tenebrio n i d ae, and Ca r abida e ) te st ed in the o r der Coleoptera.

Of the Coleoptera tested, two species L. decemline a ta and Tribolium castaneum have,

however, been shown to be sensitive to ingeste d dsRN A whe n fe d thei r conspecific

(species

- specific) dsR N A (Bach m a n et al., 2013; Baum e t al., 2007b; W hyard et al.,

2009). Additio n ally, no negati v e impacts were detected in repre s entative s p ecies from

the orders H e m iptera, Hy m enopt era

, o r Lepidoptera

. Result s fro m thes e bioassays

de m onstrate that the spectrum of activity o f D v Snf7_24 0 dsRN A i s restricte d t o the

sub f a m ily Galer u cinae in the f a m ily Chryso m elidae within the order Coleopt e ra (T a ble

VI

- 2).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 106 o f 3 7 2(da y s ) ( n g / m l or g d i e t )

C o leo p tera C h rys o m eli d ae Galer u ci n a e D ia bro tic a vir g ifer a vir g ifera Wes t e r n c o r n r oo t w o rm 12 S 4

. 4 2

C h rys o m eli d ae Galer u ci n a e D ia bro ti c a u nd ec i m p unc t a t

a h o w a r di S o u t h e rn c o rn r o o tw orm 12 S 1

. 2 2

C h rys o m eli d ae C h rys o m eli n ae L eptinotar s a decemlinea t a C o l or a d o po t a t o bee tl e 12 S, G 5

, 0 00 3

Ten e br io n ida e Ten e br io n ina e Tr i bo l i um cas t an e u m Re d f l o u r bee tl e 30 S, G 5

, 0 00 3

C o cci n elli d ae C o cci n elli n ae C o leome g ill a m a c u l a t a P i n k

- s po tte d la dy bee tl e 24 S, G, D 3

, 0 0 0 3

C o cci n elli d ae E p ilach n i n a e Ep il a c h n a v a r i vestis Me x i can be a n bee tl e 28 S, G, D 3

, 0 00 3

Ca rab i dae Ha r pa li nae Po ecil us ch a lcites Ca rab i d bee tl e 35 S, G, D 5

, 0 00 3

He m i ptera An t ho c oridae An t ho c o r i nae Ori us i n s i d i o s us In s i d io us Flowe r b ug 9 S

, D 5

, 0 0 0 3

H y m e nop t era Eu lop h i dae En tedo n i nae Ped io b i us f ov e o l a tu s Eu lop h i d wa s p 21 S 3

, 0 0 0 3

Ptero m alidae Ptero m alinae N a so n i a vitripe nn i s Jewel wasp 20 S 5

, 0 0 0 3

Lep ido p te ra Noc t u i dae Noc t u i nae S po d o

p ter a fru g i p erda Fal l ar m yworm 8 S

, G 50 0 3

N o c t u i d a e Heli o t h i n ae He lic ove r pa z e a C o r n e a r w orm 12 S, G 5

, 0 0 0 3

Cra m bidae Pyraustinae Ostri n i a nu b ilalis Eu ro pean c or n b o rer 12 S, G 5

, 0 0 0 3

Bo m b ycidae Bo m b ycinae Bombyx mori S il k w orm 14 S, G 5

, 0 0 0 3 CB I Delete d Copy

Table VI

- 2. Susceptibility to DvSnf7_240 dsRNA in laboratory bioassays

Bioassay LC 50 2 or N o

O r der Famil y Su bf a m il y Specie s Com m o n Name Duration E n d p o i n ts 1 Obs e rved Eff e ct

3 C on ce n tration

1 S : Sur v i v al ; G : Growt h; D : De v e l op m e nt

2 LC 50 s u r v i v a l d at a rep o rte d i n (Bach m a n e t al.

, 2013)

3 N o obs e rve d effec t c o ncentratio n ( N OEC ) i s eq u a l t o t he ma xi m u m c o nce n tratio n teste d i n t h es e b i o a ssays.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 1 0 7 o f 3 7 2CB I Delete d Copy

VI.E.3. Non

- Targe t Organis m Assessmen t fo r DvSnf7

Evaluation of the pote n tial risks to NTOs is an i m port a nt co mponent of APHIS’s plant

pest risk assess m ent of a biotechnology

- der i ved crop. Assess m ent of the potential risks

to NTOs associated with t h e introduction of a biotechnolog y

- derive d cro p producin g an

insecticidal trait is b ase d o n th e characteristic s o f th e c r o p an d th e introduce d trait

. S i nce

risk is a function of hazard and exposure, it is c ritic a l to d e ter m ine the potential h a zard

and exposure scenarios that are most li k ely and that require evaluation through

experi m ental studies. Selec t ion of the test organis m s and te s t m ate r ial are i mporta n t

decision s tha t ar e base d o n th e characteristic s o f th e trai t a n d the product (Ro m eis et al.,

2008). In the U.S., regulatory guidelines for NTO testing and risk assess m ent of insect

protected crops have been developed by the EPA and testing is conducted according to a

tier

- based system (U.S. EPA, 2010). A dditionally

, th e EP A ha s convene d several

Scienti f ic Advisory P anel (SAP) m eet i ngs to m ake recommendations and provide

guidance for NTO testing and risk assess m e n t for agricultural products produced by

m ethods of biotechn o logy (U.S. EPA, 2001c; 2002; 2004a; 2010).

Based on the results from the activity s p ectrum (Section VI.E.2.) and known expression

o f DvSnf 7 RN A i n MO N 8741 1 (Se c tio n VI.B.)

, a n evaluatio n o f the potential toxicity to

selecte d N T O s a t fiel d expo s ure le v els was co n ducted. The hazard assess m ent incl u ded

toxicity testing against a soil deco mposer [earthworm ( Eisenia andre i )]

, an d five

beneficial insect species [honeybee ( Apis mellifer a ), parasitic wasp ( Pediobius

foveol a tu s ), ladybird beetle ( Coleomegilla maculat a ), c a rabid beetle ( Poec ilus cha l c i te s ),

an d Insidiou s Flowe r bu g ( Orius insidiosu s ) ] (Appendi x K)

. Al l studie s wer e conducted

using in vit r o produced DvSnf7_968 RNA as the test substance. W ith the exception of

the earthworm study, all studies utilizing the in vitr o

- produced test substance included a

diet analy s is using a s ensitive in s ect ( Diabrotica undecimpunctata howardi; Southern

corn rootwor m

, SCR) to confirm that th e DvSnf7_96 8 RN A containe d i n th e test

substance d i et was biol o g ically acti v e and h a d the expected level of biological activity.

Additionally

, wher e appropriat e base d upo n th e diet m atrix, the ho m ogeneity of the test

m a teria l an d stabilit y (o f th e DvSnf7_96 8 RN A test substance) over the period of storage

was also confir m ed. A dose confir m ation was not performed for the earthworm study

due to the rapid degradation of RNA in the soil mat r ix (disc u s sed in Section X.H.). The

no observed effect concentrations (NOECs) det e r m ined for each of the tests used in the

NTO risk assess m ent for MON 87411 a r e sum m a rized in Table VI.3.

Exposure info r m ation was developed to det e r m i ne the m aximum expected environmental

concentration (MEEC) for the Dv S nf7 RNA produced in MON 87411 to NTOs. Test

concentrations were based on the m easured DvSnf7 RNA expression in the tissue(s)

types that the NTO would m ost likely be exposed to in t h e environ m ent. A tar g eted

MOE of greater than 1 0

- ti m es the MEEC was u s ed to set test conce n trations (U.S. EPA,

2010). The NTO studies were perfor m ed at a no m inal concentration of 1,000 ng

DvSnf7_96 8 RN A / m l o r g diet

. I n additio n t o thi s concentr ation providing an adequate

MOE, it also was high enough to allow for the assess m ent of the functional activity of the

RNA in 12

- day dose confir m ation diet bioassa y s wit h SCR

. Fo r predator y beetle s that

consu m e h e rbivorous prey and have an ind i rect exposure to m a i ze expressed DvSnf7

RNA, the maxi m u m expression value from the leaf develo pm ent stage with the hi g hest

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 108 o f 3 7 2CB I Delete d Copy

expression (V14

- R1) was used to represent worst

- case s ce n ario to calc u late the M O E f or

these s p ecies. The most ecol o gically rel e vant ro u te of exposure for soil

- d welling

organis m s, such earthwor m s, was considered to be from de co mposing late season plant

tissue that enters the soil en v iro nm ent. Con s equently, for soil

- dwelling NTOs, the

MEECs were based on the level of esti m ated DvSnf7 RNA i n senescent m aize roots.

For the non

- target organism studies, MOEs w e re calculated based on the ratio of the

NOECs to the MEECs. MOEs calc u lated for ea c h speci e s were >1 0

- fold of the pre d icted

exposure level for NTOs (Table VI

- 3). MOEs that exceed 10 are considered as indicative

o f m i n i m al ris k i n worst

- cas e laborator y assay s b y (U.S

. EPA

, 2010)

. Therefore, as with

the previously assessed Cry3Bb1 protein, DvSnf7 RNA is not likely to produce adverse

effects on terrestrial benefic i al invertebrate species at fiel d exposur e levels

. T h is

conclu s ion is in ag r eement with p rior publi s hed literature which reported that DvSnf7

acti v ity is very spe c i f ic and is r e stricted to the Gal e ru c inae sub fa m ily within the

Chryso m elidae fa m ily in the Order Coleoptera (Bach m an et al., 2013).

Th e Augus t 200 2 EP A SA P repor t (U.S

. E P A

, 2002 ) recommende d tha t non

- target

testing should be focused on species exposed to the crop being evaluated ( i.e

. , for

MON 87411 beneficial organis m s found in m aize fields). Though aquatic habitats m ay

be located near agricult u ral areas, t h e EPA concluded that e x posure of aquatic o rganis m s

to biotech crops is li m ited te mporally and spatially and that the potential exposure of

aquatic organis m s is therefore low to negli g ible (U.S. EPA, 2010). Sinc e ther e i s no

m eaningful ecologically

- relevant exposur e t o aquati c organis m s from maize, other than

through purposeful feeding of processed m a i ze products, e f fects tes t s on aquatic species

were not c o nducted for MON 87411. Additionally, based u pon the narrow spectr u m of

activity for DvSnf7 RNA the likelihood of a d verse effects to aquatic organis m s from

DvSnf7 is extre m ely low (Bachman et al., 2 013). Further m ore, successful RNAi in

organis m s such as fish, reptiles, and birds h a s o nly been achieved with cell lin e s a n d/or

e m bryos and has required the use of transfecti o n agents, direct injection, electroporation,

or other invasive RNA deli v ery techniques (Schyth, 2008; Sifuentes

- Ro m ero, et al.,

2011; Ubuka, et al., 2012). Based on m in i m a l exposure and the narrow spectrum of

activity of DvSnf7, the likelihood of negati v e i m pacts to non

- target aquatic organisms

from cultivation of MON 87411 is concluded to be extre m ely low.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 109 o f 3 7 2CB I Delete d Copy

Table VI

- 3. Maximum expected e n vironment a l concentr a t ions ( M EECs), no observed effect concentrations (NOECs) from non

target o rg a nism studies and estimated margins of expo s u re (MOE s ) for DvSnf7 RNA

Test o rg a n i sm O r der M EEC 1 NOEC M OE 2

C o leome g ill a m a c u l a t a C o leo p te ra 0

. 2 2 4 n g / g f w po lle n 1 00 0 ng / g ≥ 44 64

P o ecil us ch a lcites C o leo p tera 3 3. 8 n g / g f w lea f 3 1 00 0 ng / g ≥ 30

A

p i s mellifer a larvae H y m e nop t era 0

. 0 00 4 4 8 n g 4 11. 3 ng/lar v a e 5 ≥ 2 5 ,2 23

A

p i s mellifer a adu l t H y m e nop t era 0

. 2 2 4 n g / g f w po lle n 1 00 0 ng / g ≥ 44 64

Ped io b i us f ov e o l a tu s H y m e nop t era 0

. 2 2 4 n g / g f w po lle n 1 00 0 ng / g ≥ 44 64

Ori us i n s i d i o s us

E i sen i a a n d rei He m i p t era

Hap l o t ax i da 0

. 2 2 4 n g / g f w po lle n

3

. 68 n g / g d w s e nesc e nt r oot 1 00 0 ng / g

5 00 0 ng / g dry so i l ≥ 44 64

≥ 13 59

1 Ma x i m u m express i o n le ve l s de te r m i ned f r om M ON 87 4 1 1.

2 M OE va l ues were ca l cu late d bas e d on t he ra ti o of t he N O EC t o M EE C

. The M OE was de te r m i ned b as e d on t he m a x i m u m express io n leve l o f the D v S n f7 R N A

i n t he tiss ue fr om MO N 87 4 1 1d ee m e d m o s t rele v a nt t o t he NT O e x p o s u re.

3 The m a x i m u m express io n val ue f rom t he l eaf deve l op m ent s t age w it h th e h ig hest e x pr e ss i on ( V 14

- R1 ) wa s used t o re pres e nt w o rs t

- case

- sc e n ari o fo r a pr e d ator

co n su m i ng a n he r b iv o ro us p r e y

. 4 M EEC bas e d u po n m ean a m o u nt of D v S n f 7 RN A e x pr e s s e d i n 2 mg of M ON 8 7 41 1 p o lle n ( f w )

. T he av e rage c o n s u m pt i on of p o lle n by ho n e y b e e larva e i s 2 mg

d u r in g d e v elopme nt ( Ba b en d reie r, e t al.

, 200 4 )

. T he MEE C w a s calc u late d a s f o ll o ws : (2 m g po lle n × ( 0

. 2 2 4 n g Dv S n f 7 R NA / 1 00 0 μ g)).

5 The NOEC r e pres e nts t he co n ce n t r ati on o f t he tes t s o l u tio n us e d fo r d o sin g in d i v i du a l lar v a l cells

. A sin g l e d o s e o f 10 μ l of 10 00 n g / g s o l u tio n was add e d t o ea c h

lar v a l cel l f or a t o ta l m as s of 1 1

. 3 ng D v S n f 7 / cell

. T he co n ce n tratio n of 1 0 0 0 ng / g DvSnf7

_ 96 8 RN A i n t he d ie t s o l u tio n i s cal c u l a t ed bas e d on t he de ns it y of t he

3 0 % s uc r ose /wate r ( w /v ) s o l u t i on o f 1

. 1 27 0 g / m l

.

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VI.F. Cha r acteri z ation and Safety Conclusions

DvSnf 7 RN A fro m M ON 8741 1 i s a dsRN A that upon consu mption by CRW species

causes gene suppression of the targeted DvS n f7 gene. The molecular characteristics of

the MON 87411 DvSnf7 RNA were deter m ined and equivalence between MON 87411

DvSnf 7 RNA an d in vitr o

- produce d DvSnf7_96 8 RNA s w a s de m o nstrated. This

equivalence justifies the use of the in vitr o

- produce d D v Snf7_96 8 RN A a s a test

substanc e i n th e RN A safet y studie s (desc r ibed in m ore detail in Section VI.E.3.).

Expression studies using a Quantigene Plex 2.0 assay de m onstrated that MON 87411

DvSnf7 was expressed at very l o w m ean levels ranging from 0.104 × 10

- 3 μg/g dw to

84.8 × 1 0

- 3 μg/g dw, representing a low percentage of the t o tal RNA. Anti c ipated d i etary

exposures to DvSnf7 in hu m ans are very low ( ≤ 0. 4 ng/k g b w pe r day)

. Anticipated

dietary exp o sures to D v Snf7 in ani m als are a l s o very low, the h ighest anti c ipated levels

being in lactating dairy cows (81 ng/kg bw per day).

Additionally, activity spectrum testing on 14 d i fferent species showed that activity of

DvSn f 7 RNA is restri ct ed to the s ub f a m ily Galer u cinae in the f a m ily Chryso m e lidae

within the order Coleoptera. Specific testing on a battery of NTOs (honeybee,

earthwor m

, ladybird beetle, and others) f u rther supports a conclusion that negative

i m pacts resulting from e xposure to DvSnf7 RNA from MON 87411 are unlikely. Based

on the ubiquitous nature of the RNA

- bas e d suppression m echani s m utilizing dsRNA,

de m onstration of specificity of DvSnf7 suppression in CR W , lack of i m pact to NTOs, the

history of safe consu m p t ion of RNA, and the a pparent lack of toxicity or allergenicity of

dietar y RNA ; th e RNA

- m e diate d gen e suppressio n use d i n MO N 8741 1 pose s no

observed or theoretical risks to hu m ans or a n i m als. Therefore, the consu mption of the

DvSnf 7 RN A fro m M O N 8741 1 o r it s progen y is considered safe for hu m ans and

ani m als.

The safety data presented h e rein support the conclusion t ha t foo d an d fee d products

containing MON 87411 or derived from MON 87411 are as safe as m aize products

curre n tly on the m arket for hu m an and a n i m al consu mption and the environ m ent.

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VII. COMPOSITIONA L A S SESSMEN T O F MO N 87411

Safety assess m ents of biotechnology

- deriv e d crops follow the comparative safety

assess m ent process (C o dex Ali m entarius, 200 9 ) in which the co mposition of grain and/or

other raw agric u ltural com m odities of the biotechnology

- derived crop are co mpared to

the appropriate conventional c ontrol that has a history of safe use. Compositional

assess m ents are perfor m ed using the princ i ples and analytes outlined in the OECD

consensus docu m ent for m aize co m p osition (OECD, 2002a).

A recent re v iew of co m p ositio n al as s ess m ents conducted according to OECD guidelines,

which encompassed a total of seven biotechno l ogy

- derive d cro p varieti e s, nine countries

and eleven growing seasons concluded t h a t incorporatio n o f biotechnology

- derived

agrono m ic traits has h a d little i mpact on n a tural variation in crop composition. Most

co mpositional variation is attributable to growin g region

, agronomi c practices

, and

genetic background (Harrigan, et al., 2010). Nu m erous scientific publications have

f urther doc um ented the exten s ive v a ria b ility in the concentrations of crop nutrients, anti

nutrients, and secondary m etabolites that r eflec t th e influenc e o f environ m enta l and

genetic factors as well as extensi v e conventio n al bree d ing efforts to improve nutritio n

,

agrono m ics, and yield (Harrigan et al., 2010; Harrigan, et al., 2009; Ridley, et al., 2011;

Zhou, et al., 2011).

Co mpositi o nal equivalence between biotec hn ology

- derived and conventional crops

supports an “equal or increased assurance of the safety of foods derived from genetically

modifie d plants ” (OECD

, 2002b)

. OEC D c onsensus d ocu m ents on co m positional

considerations for new crop varieties e m phas i ze quantitati v e m easure m ents of ess e nti a l

nutrients and known anti

- nutrie n ts. These quantitative m easu r e m ents effectively discern

any co mpositional changes that i mply potential nutritional or safety ( e.g., anti

- nutriti o nal)

concerns. Levels of the co mponents in grain and/or other raw agricult u ral commodities

of the biotechnology

- derived crop product are co m pared to: 1) corresponding levels in a

conventional co mparator, a genetically si m ilar conventional line, grown concurrently

under s i m ilar field co n ditions, and 2) natural ranges g enerated from an eval u ati o n of

commercial reference hybrids grown concurrently and from data published in the

scie n ti f ic literat u re. The co m parison to data published in the lit e r a ture places any

potential differences between the a s sessed crop and its co m parat o r in t h e conte x t of the

well

- docu m ented variation in the concentrati o ns of crop nutrients, anti

- nutrients, and

secondary m etabolites.

Thi s sectio n provide s analyse s o f concentra tions of key nutrient s

, anti

- nutrients, and

secondary m etabolites of MON 87411 co m pared to that of a conventi o nal contr o l m aize

hybrid g r o w n and harvested un d er si m ilar c onditions, as appropriate. In addition,

conventional commercial reference m aize hyb r ids (herea f ter refered to as reference

hybrids) were i n cluded in the co mposition analy s es to p rovi d e additional infor m ation on

the range o f natural varia b ility for each co mp onent. The production of m aterials for

co mpositional analyses used a sufficient variety of f ield trial s ites, robus t fiel d designs

(rando m ized co m plete block design with four blocks), and sensitive analytical m ethods

that allow accurate a s sess m ents of co mpos i tional characteristics o ver a ran g e of

environ m ental conditions under which MON 87411 is expected to be grown.

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The in f ormation pro v ided in this section addresses relevant factors in Codex Plant

Guidelin e s, Section 4, paragraphs 44 and 45 f or compositio n al a nalyses (C o dex

Ali m entarius, 2009).

VII.A. Compositional Equivalence of MON 87411 Grain and Forage to

Conventional Mai z e

Grain and forage sa m ples were collected from M ON 87411, t he conventional control, and

a total of 20 different reference hybri d s grown in Argentina during a 2011/2012 field

production. The refere n ce hybri d s were inclu d ed in t h e c o mposition analyses to p rovide

data on the natural variability fo r eac h co m pon e nt

. Th e fiel d productio n wa s conducted

a t eigh t site s (Appendi x G)

. T he field sites were planted in a rando m ized co mp lete block

design with four blocks per site. MON 87411, conventional control, and reference

hybrids were grown under nor m a l agrono m ic field conditions for their respective

geographic regions, in areas that were t ypical for m aize production in Argentina.

Production in the U.S. corn belt and Argentina m aize

- growing regions o c curs at relatively

si m ilar latitudes with an approxi m ate 6 m onth offset (Sch nep f e t al.

, 2001)

. Th e average

growing season te m peratures and precipitation ar e co mp arabl e (Schnep f e t al.

, 2001 ) and

m aize hybrids developed in the U.S. are often u s ed directly by fa r m ers in Argentina. As

such, co mp ositio n al a n alyses f rom m aize grown in Argentina are approriate f or a

co mparative safety assess m ent. MON 87411 plots were treated with glyphosate

(0.95 lbs a.i./ha) to generate sa mples under conditions of the intended use of the product.

Th e evaluatio n o f MO N 8741 1 fo l l owe d consid erations relevant to t h e co mpositional

quality of maize as defined by the OECD con s ensus docu m ent (OECD, 2002a). Grain

sa mples w e re analyzed for levels of nu t rients including proxi m ates, carbohydrates by

calculation, fiber, a m ino acids, fatty acids, m inerals, and vita m ins. The anti

- nutrients

analyzed in grain inclu d ed phytic acid and r a ffi n ose. Seco n dary m etabolites analyz e d in

grain included furfural, ferulic acid, and p

- c oumaric acid. Forage sa m ples were analyzed

for levels of proxi m ates, ca rbohydrate s b y calculation

, fiber, and m ineral s

. In total, 78

different components were assayed (ni n e in forage and 69 in grain).

Of those 78 co m ponen t s, 14 fatty acids (capr y lic, capric, lauric, m yristic, m yristoleic,

pentadecan o ic, penta d ecenoic, pal m itolei c

, heptadecanoic, heptadecenoic, gamma

linolenic, eicosadienoic, eic o satrienoic, and arachidonic acid s ), sodiu m

, and furfural had

more than 50% of observations below the a s say li m it of q u antiti a tion (L OQ) and were

excluded fr o m statistical analysis. Moist u re on two co m ponents (grain and forage) w a s

m easure d fo r conversio n o f co m pon e nt s t o dr y weight, but w as not st atisticall y analyzed.

Therefore, 60 co mponents were statistically analyzed.

The statistical co m parison of MON 87411 and the conventional control was based on

co mpositional data c o mbined ac r oss all f ie l d sites (t h e co m bined

- site analysis).

Stati s tically significa n t differences were identified at t h e 5% level ( α = 0.05). The

co mpositional data from the reference hybrids were combined a cro s s all f i e ld s ites to

calc u late a 99% tolera n ce interval f or each c o mponent to esti m ate the natural varia b ility

of each component in m aize.

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A statistically significant differenc e betwee n MO N 8741 1 a n d th e conventiona l control

does not i m ply biological relevance from a food and feed safety perspective. Therefore,

statistically significa n t differences observ e d in the combined

- s ite a nalysis between

MON 87411 and the conventional control were evaluated further to deter m ine whether

the detected difference indicated a biologically relevant co m positional change or

supported a conclu s ion o f co m positi o nal equi v alence, as foll o ws:

1) Deter m ination of the m ean diffe r ence between MON 87411 and the conventional

contr o l t h at was used in steps two and t h ree

, below

. Fo r p r otei n an d a m in o acids

only 10

, the relative m agnitude of the differ e nce (percent change r e l a tive to t h e

control ) betwee n MO N 8741 1 an d th e conventi onal control was deter m ined to allow

an assess m ent of the difference in amino aci d s in rel a tion to a di fferenc e i n protein;

2) Assess m ent of the relative i mpact of MON 87411 co m pared to variati o n within the

conventio n a l contr o l ger mplasm g r own across multiple s ites ( i.e

., variation due to

environ m ental influence). This assess m e n t compares the m ean difference between

MON 87411 and the conventional control to t h e range of values for the conventional

control ( m axi m um value m inus the m in i m u m value) derived from the co m bined

- site

analy s is. When a mean difference is l e ss than ran g es seen d u e to nat u ral

environ m ental variation within e v en a single, closely related germplas m

, the

difference is typically not a food o r fee d safet y concern ; and

3) Assess m ent of the relative i mpact of MON 87411 co m pared to natural variation due

to m ultiple s ources ( e.g. , environ m ental and germplasm influences). This assess m ent

co mpares the m ean difference between M ON 87411 and the conventional control to

variation in conventional m aize as esti m ated by in

- study re f erence hybrid values and

assessing w hether the m ean value of MON 87411 was within the 99% tolerance

int e rval, t h e lit e rat u re v alues, and/ o r the ILSI Crop Co mp osition Dat a base values

(ILSI

- CCDB) (ILSI, 2011).

These evaluations of natural variation within the context of the conventional control and

conventio n a l m aize references are i mport a nt as crop c o mposition is known to be

influenced by environ m ent and ger mpla s m

. Although used as the first step in the

co mparative assess m ent process, d etection of st a tistic a lly s i g ni f ica n t d ifferences between

MON 87411 and conventional control m ean values does not i m ply a m eaningful

contribution by MON 87411 to compositional v a ria b ility. Only if m ean differences

between MON 87411 a nd the conventional control are large relative to natural variation

inherent to conventional m aize would fur the r assess m ent s b e require d t o establish

10 S i nce t o ta l a mi no ac i ds m easur e d i n a g ra i n ana ly s i s a re p re d o m i nan tl y der i ved f ro m h y dro ly s i s of

p r o tei n, change s i n pro tei n le v el s wil l l i k el y res u l t i n c o rres p on d in g c h an g e s i n ami no aci ds le v els.

Howe v er

, t he mea n d iffere n c e f or i n d i v i du a l a m i no aci d le v el s wil l be l es s t h a n t he mea n d iffere n c e for

protein bec ause each a m ino acid constitu t e s only a part of the pro t e i n. For this reason, the re l a tive

m a gn it ud e s o f d i ff e rence ( p ercent ch a n ge re lati ve t o t he c o n tr o l ) fo r a mi no ac i ds and p r o t e i n were

d eter m i n e d t o all ow a n assess m e nt of t he differe n c e i n am i no ac i ds i n re lati on t o a d i ff e rence i n prot e i n.

W h e n t he relati ve m a gn itude s of d ifferenc e fo r a m i no aci ds wer e re l ate d t o t he relati ve m a gn itu de of

d i ff e r e nce fo r p r o t e i n, t h e n s t eps 2 a n d 3 are n ot d i s c uss e d f o r a m i no ac id s.

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whether the change in co mposition would have an i mpact from a food and feed safety

perspective. The ste p s reviewed in t h is as s e ss m ent therefore de s cribe whether the

differences between MON 87411 a nd the conventional control are m e aningful from a

food/feed perspective o r whether t h ey suppo r t a conclu s ion o f co m positi o nal equi v alence.

The co mpositio n al an al ysis provi d e d a co mp rehensive c o mparative a ssess m ent of the

levels of key nutrients, anti

- nutrients, and secondary m etabolites in m aize grain and

forage of MON 87411 and the conventional contr o l. Of the 60 co mponents statistically

assesse d ther e wer e n o statist i cally significant differences in 48 co mponents. Only 12

co mponents (protein, histidine, tyrosine, ole i c acid, neutral deterge n t fiber, copper, iron,

m anganese, zinc, niacin, vita m in B1 in gra i n and ash in forage) showed a significant

difference between M O N 87411 and the conve n tional control in the co m bined site

analysis. F or these 12 co mponents, the m ean differences in co mponent values between

MON 87411 and the conventional contro l wer e les s tha n th e observe d rang e o f the

conventional control values (Step 2 above) and the reference hybrid values (Step 3

above) (Ta b les VII

- 1,

- 3,

- 4,

- 5, and

- 7 below). The MON 87411 m ean component

values of the 12 components that were st a tistically different were within the 99%

tol e rance intervals, the values ob s e r ved in the lit e rat u re, a n d/or t h e IL S I

- CCDB values

(Step 3 above). These results support the overall conclusion that MON 87411 w a s not a

m ajor contributor to variation in co m ponent levels in maize grain and forage and

confir m ed the equivalence of MON 87411 to t h e conventional control in levels of all

m easured components. A detailed description of the assessment of statistically significant

differences observed between MON 87411 and t h e conventional control is provided in

the following section. These data con f irme d tha t th e co mponen t s wit h observed

signi f ica n t d i ff erences were not compositio n ally m eaning f ul f rom a f ood and f eed s af ety

perspective.

VII.A.1. Nutrient Levels in Mai z e Grain

Grain sa mp les were a n alyzed for levels of nutrients including a sh

, protein

, tota l fat,

carbohydrates by calculation, fiber (three co m ponents), a m ino acids (18 co mponents),

fatty acids (22 co m p onents), m inerals ( n ine co mponents), and vita m ins (seven

co mponents). Moisture was m easured for c onversion of components from fresh to dry

weight

, bu t wa s no t sta t istically analyzed.

VII.A.1.1. Proteins a n d Amino Acids

Maize grain is typically co mposed of approxi m a tel y 10 % protei n an d th e level s o f protein

an d associate d a m in o acid s ca n var y dependin g o n loca l growin g c onditions (Harrigan et

al., 2009; Ridley et al., 2011; Z hou et al., 2011). This naturally occurrin g variabilit y is

i m portant in assessing the bio l o g ic a l r e leva n ce of statistically sig n i f ica n t di ff erenc e s in

co mposition.

A statisticall y significan t differenc e (p<0.05 ) betwee n MO N 8741 1 an d th e conventional

contr o l was observed for protein (Table VII

- 1 ). The m ean protein value was 10.71% dw

for MON 87411 and 10.28% dw for the conve n tional control, a m e an difference of

0.43% dw. This difference was evaluated in t h e contex t o f th e rang e o f th e conventional

control values, 4.54% dw, ca l culated from the m in i m u m (8.06% dw) and m ax i m u m

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(12.60% d w ) protein values. The mean diff e renc e i n protei n va lues bet w een MON 87411

an d th e conventiona l contro l w a s les s tha n th e rang e o f th e conventiona l contro l values,

indicating that MON 87411 does not i mpact levels m ore than natural variation within the

conventio n a l control g r o w n at m ultiple locatio n s. The m e a n difference in protein v alues

between MON 87411 a nd the conventional control was also less than the variation seen

in the reference hybrid val u es (ranged 7.21 to 14.59% dw, a m a gnitud e o f 7.38 % dw),

and the MON 87411 m e an value for protein was within the 99% tol e rance interval, the

values observed in the published lit e ratu r e and/ o r the ILS I

- CCDB values (Table VII

- 8 ).

Since t o tal a m ino acids m easured in a seed analysis are predo m inantly derived from

hydrolysis of protein, differences in a m ino acid levels between MON 87411 and the

conventional control w ere ass esse d relativ e t o th e differen ce in protein levels. The

relative m agnitude of the difference in m ean protein values for MON 87411 and the

conventional control, when expressed as a percent of the conventional control, was

4.19 % (Tabl e VII

- 1)

. Corr espondingly, relative m agnitudes o f differenc e fo r th e 18

a m ino acids m easured were ≤ 4.77%. These differences were significant for two of the

a m ino acids (histidine, tyrosine) (Table VII

- 1), and reflected s m all relative m agnitudes of

differences between MON 87411 a nd the conve n tional control, as would be expected

based on the s m all relative m agnitude of difference in protein.

The data demonstrated that MON 87411 was not a m ajor contributor to variation in

protein and a m ino acid levels in m aize grain and confir m ed the co mpositional

equivalenc e o f MO N 8741 1 t o th e conventiona l control in levels of these co mponents.

Also

, th e mea n value s o f thes e component s wer e withi n th e 99 % toleranc e intervals

, the

values obse r ved in the liter a ture, an d /or t h e ILSI

- CCDB values. These data confir m ed

that the s ig n i f icant d i ff erences (p<0

. 05) in m ean values of protein and two a m ino acids

were not compositionally m ea n ingful from a food and f eed safety perspective.

VII.A.1.2. Total Fat and Fatty Acids

Maize g rain is ty p ically co mposed of approxi m a tely 4 % f at and the lev e ls of tot a l f at and

fatty acid levels can vary depending on local growin g condition s (Harriga n e t al.

, 2009;

Ridle y e t al.

, 2011 ; Zho u e t a l

. , 2011)

. Thi s naturall y occurring variability is i mportant in

assessing the biological relev a nce of statistically significant differences in co m position.

No stati s ti c ally s ignifi c ant differe n ce was obs e rved for total fat (Ta b le VII

- 2). There

were also n o stati s tically significant differences for seven of the eight fatty a c ids (FA)

assessed. One statistically significant difference (p<0.0 5 ) wa s observe d fo r olei c acid

(Table VII

- 2). The m e an oleic acid value was 21.89% total FA for MON 87411 and

21.70% total FA for the conventional control, a difference of 0.19% total FA. This

observed s t a tistic a lly si g ni f icant d i ff erence was evalu a ted in the co n te x t of the ra ng e of

th e conventiona l contro l values

, 2.67 % tot al F A

, calculated from the m i ni m u m (20.81%

total FA) and m ax i m u m (23.49% total FA) ole i c acid values. The m e a n difference in

oleic acid values between MON 87411 and t h e conventional control was less than the

range of the conventional control value s

, indicating that M O N 8741 1 doe s no t impact

levels m ore than nat u ral variation within the conventio n al control g rown at m u lti p le

locations. The m ean difference in ole i c aci d value s betwee n MO N 8741 1 an d the

conventional control was also less than the v a riation seen in the reference hybrid values

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(ranged from 20.52 to 42.88% total FA, a m a gnitud e o f 22.36 % tota l FA)

, an d the

MON 87411 m ean value was within the 99% t o lerance interval, the values observed in

the lit e rat u r e

, and/or the ILSI

- CCDB values ( T able VII

- 8).

Th e dat a demonstrate d tha t MO N 8741 1 wa s no t a m a jo r contributo r t o variatio n i n total

f

at and f atty acid lev e ls in m aize g r ain and confir m ed the co mpositional equivalence of

MON 87411 to the conventional control in lev e ls of these co m ponents. Also, the m ean

values of these co mponents were w ithin the 99 % toleranc e interva l

, the values observed

in the lite r ature, and/ o r the ILSI

- CCDB value s

. These data confir m e d that the sig n i f icant

difference in the m ean value of oleic acid was not co m pos i tionally m eaningful from a

foo d an d fee d safet y perspective.

VII.A.1.3. Carbohyd r a te s b y Calculatio n a n d Fiber

I n additio n t o protei n an d fat

, m a jo r b i omass co m ponents as s essed in m aize grain

include d carbohydrate s b y calculatio n an d fib e r [aci d detergen t fibe r (ADF)

, neutral

detergent fiber (NDF), and total dietary fiber ( T DF)]. Maize grain is t y pically co mposed

of approximately 85% carbohydrates by calc u lation, which includes fiber (ADF, NDF,

an d TDF)

, an d th e level s o f thes e co m ponen t s can vary widely depending on local

growin g condition s (Harriga n e t al., 2009; Ridley et al., 20 1 1; Zhou et al., 2011). T his

natur a lly o c curring v a riability is important in assessing the biological relevance of

statisticall y significan t diff e rences in co mposition.

No statistically significant difference s wer e observe d f or carbohydrates by calculation,

ADF, or TDF (Table V I I

- 3). A statisti c ally significant difference (p<0.05) was observed

for NDF. The m ean NDF value was 8.26% dw for MON 87411 and 8.74% dw for the

conventional control, a m ean diffe r ence of

- 0.48 % dw

. Thi s differenc e wa s evaluate d in

th e contex t o f th e rang e o f the conventional control val u es, 2.94% dw, ca lculate d from

the m ini m u m (7.36% dw) and m aximum (10.30% dw) NDF values. The m ean diff e rence

in NDF values between MON 87411 and the conventional control was l es s tha n th e range

of the conventional control values, indic a ting that MON 87411 does not i mpact levels

more than natural variation within the conv e ntional contr o l grown at multi p le locations.

The m ean d i fference in NDF values was also less than the variation seen in the reference

hybri d value s (range d f r o m 7.4 1 t o 14.80 % dw

, a m a gnitud e o f 7.39 % dw)

, an d the

MON 87411 m ean value for NDF was within the 99% tolerance interval, the values

observed in the lit e rat u r e

, and/or the ILSI

- CCDB values ( T able VII

- 8).

The data demonstrated that MON 87411 was not a m ajor contributor to variation in

carbohydrates by calculation and fiber levels i n m a iz e grai n an d confir m ed the

co mpositional equivalence of MON 87411 to the conventional control in levels of these

co mponents. Also, the m ean values of the s e components were wit h in the 99% tolerance

int e rval, the values obs e rved in the lit e rat u r e

, and/or the IL S I

- CCDB va l ues. These data

confir m ed that the significant differe n ce in the m ean value of NDF was not

co mpositionally m eaningful from a food and feed safety perspective.

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VII.A.1. 4 As h an d Minerals

Ash and m inerals were also assessed in m aize grain. Mineral co m ponents (calcium,

copper, iron, m agnesium, m angan e se, phosphorus, potassium and zinc), constituents of

ash, are also discussed in this section. The levels of these co mponents can vary widely

depending on local gro w ing conditions (Harrig a n et al., 2009; Ridley et al., 2011; Zhou et

al., 2011). This naturally occurring variabili t y is i m portant in assessing the biological

relevance of statistically signif i cant differences in co mposition.

Ash levels were not statistically sign i ficantly different between MON 87411 and the

conventiona l contro l (Tabl e VII

- 4)

. The re wer e als o n o stati s ticall y significant

differences observed for four of the eight m inerals assessed, including calciu m

,

m agnesiu m

, phosphorus, or potassiu m

. Statistically significant difference s (p<0.05 ) were

observed between MON 87411 and the conventional control for copper, iron, m anganese,

and zinc (T a ble VII

- 4 ).

Fo r copper

, th e m ea n valu e wa s 1.3 3 m g /k g d w fo r MO N 8741 1 an d 1.4 1 m g /k g d w for

the conventional control, a difference of

- 0.07 7 mg/k g dw

. Thi s differenc e wa s evaluated

in the context of the range of the convent i onal control values, 0.65 m g/kg dw, calculated

from the m i ni m u m (1.13 m g/kg dw) and m aximum (1.78 mg/kg dw) copper values. The

m ean d i fference in copper values between MON 87411 and the conventional control was

less than the range of the conventiona l contro l values

, indicatin g tha t MO N 8741 1 does

not i mpact levels m ore than nat u ral vari a tion within the c o nventi ona l contro l grow n at

multiple l o cations. The m ean differ e nce in c opper values was also less t h an the variation

seen in the reference hybrid value s (range d 1.1 0 t o 3.2 3 m g/kg dw, a m agnitude of 2.13

mg/k g dw)

, an d th e MO N 8741 1 m ea n valu e wa s within the 99% tol eranc e interval

, the

values observed in the literature, and/or the ILSI

- CCDB values (Table VII

- 8).

Fo r iron

, th e m ea n valu e wa s 16.8 4 mg/k g dw for MON 87411 and 16.33 m g/kg dw for

the conventional control, a difference of 0.51 mg/kg dw. The m ean difference in iron

values between MON 87411 and the conventional control was less than the range of the

conventiona l contro l value s (4.6 7 mg/k g d w ; 13.7 2 t o 18.3 9 m g /k g d w ), indicating that

MON 87411 does not impact levels m ore than natural variation within the conventional

contr o l gr o w n at m ultiple lo c atio n s. The m ean di ff erence in iron v alues was al s o less

tha n th e variatio n see n i n th e referenc e hybrid values (ranged 13.80 to 24.48 m g/kg dw, a

m agnitude of 10.68 mg/kg dw), and the M ON 87411 m ean value was within the 99%

tol e rance i n terv a l, the v alues obs er ved in t h e lit e rat u re, a n d/or the I L SI

- CCDB values

(Table VII

- 8 ).

For m anganese, the m ean value was 6.16 m g/kg dw for M ON 87411 and 5.99 m g/kg dw

for the co n ventional c o ntrol, a d ifference of 0.17 m g/kg dw. The m ean difference in

m anganese values bet w een MON 87411 and the conventional control was less than the

range of the conventional control values (2.68 m g/kg dw; 4.63 to 7.32 m g/kg dw),

indicating that MON 87411 does not i mpact levels m ore than natural variation within the

conventio n a l co n trol grown at m ultiple loc a ti o ns. The mean differe n ce in m anganese

values was a lso l e ss t h an the v a ri a ti o n seen in the re f eren c e h ybrid value s (range d 4.9 3 to

10.42 m g/kg dw, a m agnitude of 5.49 m g/kg dw), and the MON 87411 m ean value was

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within the 9 9% tol e ran c e int e rval, the values observed in the literature, and/or the ILSI

CCDB values (Table VII

- 8).

For zinc, the m ean value was 21.44 m g/kg dw for MON 87411 and 20.93 m g/kg dw for

the conventional control, a difference of 0.50 mg/kg dw. The m ean difference in zinc

values between MON 87411 and the conventional control was less than the range of the

conventiona l contro l value s (6.4 5 mg/k g d w ; 17.7 6 t o 24.2 1 m g /k g d w ), indicating that

MON 87411 does not impact levels m ore than natural variation within the conventional

contr o l gr o w n at m ultiple locatio n s. The m ean difference in zinc values was al s o less

tha n th e variatio n see n i n th e referenc e hybrid values (ranged 16.40 to 33.92 m g/kg dw, a

m agnitude of 17.52 mg/kg dw), and the M ON 87411 m ean value was within the 99%

tol e rance i n terv a l, the v alues obs er ved in t h e lit e rat u re, a n d/or the I L SI

- CCDB values

(Table VII

- 8 ).

The data demonstrated that MON 87411 was not a m ajor contributor to variatio n i n ash

and m ineral levels in m aize grain and confir m ed the compositional equivalence of

MON 87411 to the conventional control in lev e ls of these co m ponents. Also, the m ean

values of these co mponents were w ithin the 99 % toleranc e interva l

, the values observed

in the lite r ature, and/ o r the ILSI

- CCDB value s

. These data confir m e d that the sig n i f icant

differences in m ean values of these s e veral m inerals were not co m positionally

m eaningful from a food and feed safety perspective.

VII.A.1. 5 Vitamins

Maize grain contains both wat e r

- soluble vita m ins (folic acid, niacin, B1, B2, and B6) and

f

at

- solu b le v ita m ins [ vit a m ins A ( β

- carotene) and E]. The levels of these co m ponents can

vary widely depending on local growing conditions (Harrigan et al., 2009; Ridley et al.,

2011 ; Safawo

, e t al.

, 2010)

. Thi s naturall y occurrin g variabilit y is i mportant in assessing

the biological relevance of statistically significant diff e rences in co mposition.

No statistically significant differences were observed for folic acid, vita m in A, vita m in

B2

, vita m in B6

, an d vita m in E (Tabl e VII

- 5 ). St a ti s ti c ally signi f i can t d i fferences

(p<0.05 ) wer e observe d betwee n MO N 8741 1 an d th e conventiona l contro l fo r niaci n and

vita m in B1.

For niacin, the m ean value was 17.33 m g /kg dw for MON 87411 and 18.78 m g/kg dw for

th e conventiona l control

, a differenc e o f

- 1.4 5 m g /k g dw

. Thi s d i fferenc e wa s evaluated

in the context of the range of the convent i onal control values, 20.69 m g / kg dw, calculated

from the m i ni m u m (13.73 m g/kg dw) and m ax i mu m (34.4 1 m g /k g dw ) values. The m ean

difference in niacin values between MON 87411 and the conventional control was less

than the range of the conventional contr o l values, indicating that MON 87411 does not

i m pact levels m ore than natur a l vari a tion within the co n vention a l c ontrol gro w n at

multiple l o c ations. The m ean di ff erence in nia c in values was also less than the variation

seen in the reference hybrid values (r a nged 14.90 to 38.07 m g/kg dw, a m agnitude of

23.17 m g/kg dw), and the MON 87411 m ean value was within t h e 99% tolerance

int e rval, the values obs e rved in the lit e rat u re, a n d/or t h e IL S I

- CCDB values (Table VII

8).

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For vita m in B1, the m e a n value was 3.44 m g/kg dw for MON 87411 and 3.56 m g/kg dw

for the con v entional co n trol, a difference of

- 0.12 m g/kg dw. The m ean difference in

vita m in B1 values between MON 87411 and t h e conventional control was less than the

range of the conventional control values (0.85 m g/kg dw; 3.10 to 3.94 m g/kg dw),

indicating that MON 87411 does not i mpact levels m ore than natural variation within the

conventio n a l contr o l grown at m ultiple locatio n s. The m e a n difference in vita m in B1

values was a lso l e ss t h an the v a ri a ti o n seen in the re f eren c e h ybrid value s (range d 2.7 9 to

4.96 m g/kg dw, a m agnitude of 2.17 m g/kg dw), and the MO N 8741 1 m ea n valu e was

within the 9 9% tol e ran c e int e rval, the values observed in the literature, and/or the ILSI

CCDB values (Table VII

- 8).

The data demonstrated that MON 87411 was not a m ajor contributor to variation in

vita m in levels in m aize grain and con f irmed the compositional equivalence of

MON 87411 to the conventional control in lev e ls of these co m ponents. Also, the m ean

values of these co mponents were w ithin the 99 % toleranc e interva l

, the values observed

in the lite r ature, and/ o r the ILSI

- CCDB value s

. These data confir m e d that the sig n i f icant

di ff erences in m ean val u es of these v ita m ins were not co m positio n ally meaning f ul fr o m a

foo d an d fee d safet y perspective.

VII.A.2. Anti

- Nutrient Levels in Mai z e Grain

Anti

- nutrients assessed included phytic acid and raffinose. Phytic acid, the m ajor storage

fo r m of phosphorus in m aize grain, is considered an anti

- nutrie n t du e t o it s m i neral

chelating properties and the sequestration of phosphorus in phytic acid, reducing

phosphoru s bioavailabity

. Raffinos e i s a low m olecular weight non

- digestible

carbohydrate that is consi d ere d t o b e a n ant i

- nutrien t du e t o th e enteri c ga s production

and resulting flatule n ce caused by its consu m ption (Lien er

, 2000)

. Th e level s o f these

co mponents can vary widely depending on l o cal growing conditions (Harrigan et al.,

2009 ; Ridle y e t al.

, 2011)

. Thi s naturall y occurrin g variabilit y is i mportant in assessing

the biological relevance of statistically significant diff e rences in co mposition.

N o statisticall y significan t difference s wer e observe d fo r phyti c aci d an d raffinose

(Table VII

- 6). The data de m onstrated t h a t MO N 8741 1 w a s no t a m a jo r contributo r to

variation in phytic acid and raffinose lev e ls in m aize grain and confir m ed the

co mpositional equivalence of MON 87411 to the conventional control in levels of these

co mponents.

VII.A.3. Secondary Metabolites L evels in Maize Grain

Secondary m etabolites m easured in MON 87411 g r ain i n cl u ded furfural, ferulic acid, and

p

- cou m aric acid accor d ing to the OECD consensus docu m e nt (OECD, 2002a). Furfural

was not detected in the grain of MON 8741 1

, the conventional control, or reference

hybrids. Ferulic acid and p

- cou m a ric acid are derived from phenylalanine and tyrosine

(Buchanan

, e t al.

, 2000 ) an d s e rv e a s precursor s fo r a larg e grou p o f phenylpropanoid

co mpounds and fiber. The levels of the s e secondary m etabolites can vary widely

depending o n local g rowing con d iti o ns (Harrig a n et al., 200 9 ; Ridley et al., 2011). T his

natur a lly o c curring v a riability is important in assessing the biological relevance of

statisticall y significan t diff e rences in co mposition.

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No statistically significant d i fferences were observed for fer u lic acid or p

- cou m aric acid

(Table VII

- 6). The data de m onstrated t h a t MO N 8741 1 w a s no t a m a j or contributo r to

variation in ferulic acid and p

- cou m aric acid levels in m aize grain and confir m ed the

co mpositional equivalence of MON 87411 to the conventional control in levels of these

co mponents.

VII.A.4. Nutrient Levels in Mai z e Forage

Forage sa mples were assessed for levels of ash, protein, total fat

, car bohydrates by

calculation, fiber (ADF and NDF), and m inerals (calcium and phosphorus). The levels of

these co mponents can vary widely depending on local growing conditions (Harrigan et

al., 2009; Ridley et al., 2011). This naturally occurring v a riability is i m portant in

assessing the biological relev a nce of statistically significant differences in co m position.

W ith the exception of ash, there were no stati s tically s igni fi cant di ff er e nces in lev e ls of

proxi m ates, fiber, or m inerals (Table VII

- 7 ). T he m ean ash value was 5.57% dw for

MON 87411 and 5.95% dw for the conventiona l control

, a diffe rence of

- 0.39% dw. This

difference was evaluated in th e context of the r a nge of the conventional control values,

5.04% dw, calculated from the m inimum (4.51% dw) and m ax i m u m (9.55% dw) values.

The m ean difference in ash values bet w ee n MO N 8741 1 an d th e conventiona l control

was less than the range of the convention a l control values, indicating that MON 87411

does not impact levels more t ha n natura l variatio n withi n the conventional contro l grown

at m ultiple locatio n s. T he m ean di ff erence was also le s s t h an the va r i a tion s een in the

reference values (ranged 3.53 to 8.33% d w

, a m agnitude of 4.80% dw), and the

MON 87411 m ean value was within the 99% t o lerance interval, the values observed in

the lit e rat u r e

, and/or the ILSI

- CCDB values ( T able VII

- 8).

The data demonstrated that MON 87411 was not a m ajor contributor to variatio n i n ash

levels in m a ize forage and confir m e d the co mp ositio n al e q uivale n ce of MON 87411 t o

the conventional control in levels of these components. A lso, the m e an value of this

co mponent was within the 99% tolerance interval, th e value s observe d i n th e literature,

and/or the ILSI

- CCDB values. T hese data confir m ed tha t th e observe d significant

di ff erence in m ean value of ash w as not compositio n ally m eaning f ul f rom a f ood and

feed safety perspective.

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Table VII

- 1. Summary of Ma i z e Grain Protein and Amino Acids for MON 87411, Conventional Control, and Reference

Hybrids

Difference (Test m inus Control)

MON 87411 Conventi o nal

Control Refere n c e H y brids Conventi o nal

C o m ponent ( % dw)1 Mean (S.E.)2

Range Mean (S.E.)

Range (Range)3

Tolerance Interval 4 Control Ran g e

Valu e 5 Mean

(S.E.) p

- Value % Relativ e 6

Protein 10. 7 1 (0. 4 9) 10. 2 8 (0. 4 9) (7.21

- 14

. 59) 4.54 0.43 ( 0.17) 0.0 2 3 4.19

8.03

- 13

. 10 8.06

- 12

. 60 5.66, 15

. 13

Alanine 0.84 ( 0.0 4 4) 0.81 ( 0.0 4 4) (0.51

- 1. 2 0) 0.42 0.0 2 7 (0. 0 19) 0.1 6 6 3.34

0.62

- 1. 0 3 0.61

- 1. 0 3 0.37, 1. 2 4

Arginine 0.48 ( 0.0 1 3) 0.47 ( 0.0 1 3) (0.40

- 0. 6 6) 0.15 0.0 0 88 (0

. 0 0 55) 0.1 3 3 1.86

0.41

- 0. 5 4 0.40

- 0. 5 4 0.35, 0. 6 6

Aspartic Acid 0.68 ( 0.0 2 8) 0.66 ( 0.0 2 8) (0.49

- 0. 9 1) 0.28 0.0 2 2 (0. 0 11) 0.0 6 8 3.41

0.54

- 0. 8 0 0.52

- 0. 8 0 0.42, 0. 9 1

C y stine/C y steine 0.21 ( 0.0 0 61) 0.21 ( 0.0 0 61) (0.16

- 0. 3 0) 0.07 0.0 0 10 (0

. 0 0 34) 0.7 6 8 0.50

0.17

- 0. 2 5 0.17

- 0. 2 4 0.12, 0. 3 2

Glut a m ic A c i d 2.03 ( 0.11) 1.95 ( 0.11) (1.23

- 2. 8 7) 1.06 0.0 7 8 (0. 0 45) 0.1 0 8 3.98

1.44

- 2. 5 1 1.43

- 2. 4 9 0.87, 3. 0 2

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Table VII

- 1 (continued). Summary of Mai z e Grain Protein an d Amin o Acid s fo r MO N 87411

, Conventiona l Control

, and

Reference Hybrids

Difference (Test m inus Control)

MON 87411 Conventi o nal

Control Refere n c e H y brids Conventi o nal

C o m ponent ( % dw)1 Mean (S.E.)2

Range Mean (S.E.)

Range (Range)3

Tolerance Interval 4 Control Ran g e

Valu e 5 Mean

(S.E.) p

- Value % Relativ e 6

Gl y c ine 0.38 ( 0.0 0 99) 0.38 ( 0.0 0 99) (0.31

- 0. 5 1) 0.12 0.0 0 24 (0

. 0 0 44) 0.5 9 1 0.64

0.34

- 0. 4 3 0.32

- 0. 4 4 0.27, 0. 5 2

Histidine 0.28 ( 0.0 1 0) 0.27 ( 0.0 1 0) (0.20

- 0. 4 6) 0.12 0.0 0 96 (0

. 0 0 41) 0.0 3 3 3.54

0.23

- 0. 3 2 0.21

- 0. 3 4 0.13, 0. 4 5

Isoleucine 0.40 ( 0.0 2 0) 0.38 ( 0.0 2 0) (0.25

- 0. 5 4) 0.21 0.0 1 8 (0. 0 0 8 4) 0.0 5 0 4.77

0.29

- 0. 4 9 0.27

- 0. 4 8 0.19, 0. 5 6

Leucine 1.41 ( 0.0 8 6) 1.35 ( 0.0 8 6) (0.79

- 2. 0 1) 0.81 0.0 6 1 (0. 0 34) 0.1 0 0 4.50

0.96

- 1. 7 9 0.95

- 1. 7 6 0.51, 2. 1 4

L y sine 0.28 ( 0.0 0 57) 0.27 ( 0.0 0 57) (0.24

- 0. 3 4) 0.09 0.0 0 57 (0

. 0 0 42) 0.1 9 7 2.12

0.24

- 0. 3 2 0.22

- 0. 3 1 0.22, 0. 3 5

Methionine 0.21 ( 0.0 0 74) 0.21 ( 0.0 0 74) (0.15

- 0. 2 7) 0.08

- 0.00 0 24 ( 0. 0 040) 0.9 5 2

- 0.11

0.18

- 0. 2 5 0.17

- 0. 2 5 0.13, 0. 2 8

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Table VII

- 1 (continued). Summary of Mai z e Grain Protein an d Amin o Acid s fo r MO N 87411

, Conventiona l Control

, and

Reference Hybrids

Difference (Test m inus Control)

MON 87411 Conventi o nal

Control Refere n c e H y brids Conventi o nal

C o m ponent ( % dw)1 Mean (S.E.)2

Range Mean (S.E.)

Range (Range)3

Tolerance Interval 4 Control Ran g e

Valu e 5 Mean

(S.E.) p

- Value % Relativ e 6

Phen y l alanine 0.56 ( 0.0 3 2) 0.54 ( 0.0 3 2) (0.34

- 0. 7 8) 0.31 0.0 2 3 (0. 0 14) 0.1 3 0 4.20

0.41

- 0. 7 0 0.40

- 0. 7 0 0.24, 0. 8 3

Proline 0.97 ( 0.0 4 5) 0.95 ( 0.0 4 5) (0.64

- 1. 3 9) 0.41 0.0 1 9 (0. 0 18) 0.3 2 1 1.98

0.74

- 1. 1 8 0.74

- 1. 1 5 0.44, 1. 4 7

Serine 0.48 ( 0.0 2 3) 0.47 ( 0.0 2 3) (0.33

- 0. 6 7) 0.26 0.0 1 7 (0. 0 0 9 3) 0.0 9 5 3.53

0.35

- 0. 6 0 0.33

- 0. 5 9 0.25, 0. 6 9

Threonine 0.37 ( 0.0 1 5) 0.36 ( 0.0 1 5) (0.27

- 0. 5 0) 0.14 0.0 1 0 (0. 0 0 6 2) 0.1 3 1 2.74

0.30

- 0. 4 4 0.29

- 0. 4 4 0.22, 0. 5 1

Trypto p han 0.0 7 1 (0. 0 0 1 6) 0.0 7 1 (0. 0 0 1 6) (0.053

- 0

. 0 8 6) 0.02 0.0 0 018 ( 0. 0 0070) 0.8 0 5 0.25

0.0 6 4

- 0

. 080 0.0 5 9

- 0

. 082 0.0 5 3, 0

. 091

T y r osine 0.42 ( 0.0 2 0) 0.40 ( 0.0 2 0) (0.27

- 0. 5 7) 0.22 0.0 1 8 (0. 0 0 8 2) 0.0 4 6 4.42

0.31

- 0. 5 2 0.30

- 0. 5 2 0.22, 0. 5 8

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Table VII

- 1 (continued). Summary of Mai z e Grain Protein an d Amin o Acid s fo r MO N 87411

, Conventiona l Control

, and

Reference Hybrids

Difference (Test m inus Control)

Conventi o nal

MON 87411 Control Refere n c e H y brids Conventi o nal

C o m ponent ( % dw)1 Mean (S.E.)2

Range Mean (S.E.)

Range (Range)3

Tolerance Interval 4 Control Ran g e

Valu e 5 Mean

(S.E.) p

- Value % Relativ e 6

Valine 0.49 ( 0.0 2 1) 0.48 ( 0.0 2 1) (0.34

- 0. 7 0) 0.22 0.0 1 5 (0. 0 10) 0.1 6 7 3.04

0.39

- 0. 5 8 0.37

- 0. 5 9 0.27, 0. 7 0

1dw

= d r y weight.

2Mean (S.E.)

= least

- s quare mean (standard error).

3Range is the mini m u m a n d m a xi m u m raw values for the convent i onal reference m a ize h y br i ds.

4 With 95% confidence, t h e tolerance interval contains 99% of t h e values expressed in the population of reference h y brids. Negat i ve limits were

set to zero.

5 Maxi m u m v a lue m inus mini m u m value for the conv e ntional cont r o l maize h y br i d.

6 The relative magnitude of the difference in mean values between MON 87411 and the conven t ional control, expressed as a percent of the

conventional control.

M ons a n t o Co m pany CR 2 4 0

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Table VII

- 2. Summary of Mai z e Grain Fat and Fatty Acids for MON 87411, Conventional Control, and Reference Hybrids

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

C o m ponent Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

Total Fat (% dw ) 1 3.79 ( 0.0 4 7) 3.83 ( 0.0 4 7) (2.65

- 5. 6 0) 0.68

- 0.036 (0. 0 6 3 ) 0.5 7 3

3.42

- 4. 1 5 3.43

- 4. 1 0 1.55, 6. 6 9

16:0 Pal m itic 6 13. 6 1 (0. 0 45) 13. 6 2 (0. 0 45) (9.98

- 12

. 71) 0.73

- 0.00 6 3 (0

. 0 3 2) 0.8 4 2

13. 3 1

- 1 3 .97 13. 3 2

- 1 4 .05 8.50, 14

. 14

18:0 Stearic 1.68 ( 0.0 3 2) 1.70 ( 0.0 3 2) (1.56

- 2. 5 8) 0.43

- 0.021 (0. 0 1 8 ) 0.2 4 9

1.57

- 1. 8 8 1.53

- 1. 9 6 0.93, 2. 9 8

18:1 Oleic 21. 8 9 (0. 1 5) 21. 7 0 (0. 1 5) (20.52

- 4 2

. 8 8) 2.67 0.19 ( 0.0 9 1) 0.0 4 0

20. 8 6

- 2 2 .96 20. 8 1

- 2 3 .49 7.74, 50

. 71

18:2 L i noleic 60. 9 0 (0. 2 2) 61. 0 6 (0. 2 2) (42.82

- 6 4

. 1 0) 3.30

- 0.17 ( 0.0 9 9) 0.0 9 5

59. 3 8

- 6 1 .96 59. 0 8

- 6 2 .38 33. 6 3, 7 7

. 43

18:3 L i nolen i c 1.09 ( 0.0 0 93) 1.09 ( 0.0 0 93) (0.85

- 1. 4 1) 0.14 0.0 0 35 (0

. 0 0 59) 0.5 5 2

1.04

- 1. 1 7 1.02

- 1. 1 6 0.57, 1. 6 5

M ons a n t o Co m pany CR 2 4 0

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Table VII

- 2 (continue d ). Summary of Mai z e Grain Fat and F a tty Acids for MON 87411, Conventional Control, and Reference

Hybrids

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

C o m ponent Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

20:0 Arachid i c 0.41 ( 0.0 0 68) 0.42 ( 0.0 0 68) (0.33

- 0. 6 3) 0.09

- 0.00 2 4 (0

. 0 0 42) 0.5 7 1

0.39

- 0. 4 4 0.37

- 0. 4 7 0.21, 0. 7 0

20:1 Eicose n oic 0.26 ( 0.0 0 18) 0.26 ( 0.0 0 18) (0.19

- 0. 3 4) 0.03 0.0 0 38 (0

. 0 0 22) 0.1 0 1

0.24

- 0. 2 8 0.24

- 0. 2 7 0.12, 0. 3 8

22:0 Behenic 0.16 ( 0.0 0 16) 0.16 ( 0.0 0 16) (0.055

- 0

. 25) 0.03

- 0.00 1 6 (0

. 0 0 20) 0.4 3 4

0.14

- 0. 1 7 0.15

- 0. 1 8 0.0 0 65, 0.31

1dw

= d r y weight.

2Mean (S.E.)

= least

- s quare mean (standard error).

3Range is the mini m u m a n d m a xi m u m raw values for the convent i onal reference m a ize h y br i ds.

4 With 95% confidence, interval contains 99% of the values expres s ed in the population of convent i onal reference maize h ybrids. N egative li m its

set to zero.

5 Maxi m u m v a lue m inus mini m u m value for the conv e ntional cont r ol m a ize h y b r id.

6 Fatty acid means and ranges are expressed as a % of t otal fatty ac id

. Prefix nu m bers refer to nu m b e r of car b on ato m s and nu m ber of carbon

carbon do u b l e bonds in the fatty acid m olecule; 16:0 means sixteen carbon at om s and zero double b onds.

N u m bers a r e not included in text discussion for r easons of clarit y

. The following fatty acids with

m o r e than 50% of observations below the assay LOQ were excluded from stat i s tical anal y s is: capr y lic acid, capric acid, lauric a cid, m y ristic acid,

my ristoleic a c id, pentadecanoic acid, pentadecenoic a c id, pal m it o l e ic acid, heptadecanoic aci d

, heptadecenoic acid, gamma lino l enic acid,

eicosadienoic acid, eicosatrienoic acid, and arachidon i c acid.

M ons a n t o Co m pany CR 2 4 0

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C o m ponent ( % dw)1 Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

Carbo h y drates by 84. 1 3 (0. 5 1) 84. 5 3 (0. 5 1) (78.76

- 8 7

. 5 6) 4.77

- 0.40 ( 0.20) 0.0 6 8

Calculation 81. 8 4

- 8 6 .85 82. 1 8

- 8 6 .95 77. 7 2, 9 0

. 40

Acid Detergent Fiber 3.06 ( 0.0 8 3) 3.26 ( 0.0 8 3) (1.89

- 5. 1 6) 1.26

- 0.20 ( 0.10) 0.0 7 4

2.50

- 3. 6 2 2.73

- 4. 0 0 2.18, 4. 9 8

Neutral De t e r gent Fiber 8.26 ( 0.17) 8.74 ( 0.17) (7.41

- 14

. 80) 2.94

- 0.48 ( 0.20) 0.0 1 8

7.12

- 12

. 14 7.36

- 10

. 30 6.04, 13

. 44

Total Dietary Fiber 11. 5 0 (0. 2 0) 11. 8 2 (0. 2 0) (10.33

- 1 7

. 1 1) 4.60

- 0.33 ( 0.27) 0.2 4 7

10. 1 9

- 1 2 .90 10. 4 3

- 1 5 .03 9.83, 16

. 84 Table VII

- 3. Summary of Mai z e Grain Carbohydrates by Cal c ulation and Fiber for MON 87411, Conventional C ontrol, and

Reference Hybrids

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

1dw

= d r y weight.

2Mean (S.E.)

= least

- s quare mean (standard error).

3Range is the mini m u m a n d m a xi m u m raw values for the convent i onal reference m a ize h y br i ds.

4 With 95% confidence, interval contains 99% of the values expres s ed in the population of convent i onal reference maize h ybrids. N egative li m its

set to zero.

5 Maxi m u m v a lue m inus mini m u m value for the conv e ntional cont r ol m a ize h y b r id.

M ons a n t o Co m pany CR 2 4 0

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Table VII

- 4. Summary of Mai z e Grain Ash and Minerals for MON 8 7411, Conventional C o n t rol, and Reference Hybrids

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

C o m ponent Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

Ash (% dw ) 1 1.36 ( 0.0 2 6) 1.35 ( 0.0 2 6) (1.07

- 1. 7 5) 0.55 0.0 0 98 (0

. 0 3 0) 0.7 4 6

1.13

- 1. 5 1 1.05

- 1. 6 0 1.07, 1. 8 0

Calci u m (% dw) 0.0 0 31 (0

. 0 0 007) 0.0 0 30 (0

. 0 0 007) (0.00 2 6

- 0

. 0 056) 0.00 0.0 0 009 ( 0. 0 0005) 0.0 6 3

0.0 0 24

- 0 .0 0 37 0.0 0 26

- 0 .0 0 33 0.0 0 19, 0.0 06 2

Copper ( m g/kg dw) 1.33 ( 0.0 5 6) 1.41 ( 0.0 5 6) (1.10

- 3. 2 3) 0.65

- 0.077 (0. 0 3 0 ) 0.0 2 1

1.09

- 1. 8 8 1.13

- 1. 7 8 0.28, 3. 7 5

Iron ( m g/kg dw ) 16. 8 4 (0. 4 1) 16. 3 3 (0. 4 1) (13.80

- 2 4

. 4 8) 4.67 0.51 ( 0.18) 0.0 1 3

14. 5 9

- 1 9 .70 13. 7 2

- 1 8 .39 10. 7 1, 2 8

. 62

Magnesi u m (% dw) 0.11 ( 0.0 0 31) 0.12 ( 0.0 0 31) (0.097

- 0

. 15) 0.03

- 0.00 0 50 ( 0. 0 011) 0.6 5 7

0.0 9 4

- 0

. 13 0.0 9 4

- 0

. 13 0.0 8 6, 0

. 16

Manganese ( m g/kg dw) 6.16 ( 0.26) 5.99 ( 0.26) (4.93

- 10

. 42) 2.68 0.17 ( 0.0 7 0) 0.0 3 3

4.64

- 7. 1 3 4.63

- 7. 3 2 2.28, 12

. 14

M ons a n t o Co m pany CR 2 4 0

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C o m ponent Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

Phospho r us (% dw) 0.31 ( 0.0 0 71) 0.31 ( 0.0 0 71) (0.24

- 0. 3 9) 0.11

- 0.00 1 8 (0

. 0 0 33) 0.5 9 1

0.25

- 0. 3 3 0.23

- 0. 3 4 0.22, 0. 4 3

Potassi u m (% dw) 0.34 ( 0.0 0 61) 0.35 ( 0.0 0 61) (0.30

- 0. 4 3) 0.09

- 0.00 5 5 (0

. 0 0 34) 0.1 2 7

0.32

- 0. 3 9 0.31

- 0. 4 0 0.28, 0. 4 6

Zinc ( m g/kg dw) 21. 4 4 (0. 7 2) 20. 9 3 (0. 7 2) (16.40

- 3 3

. 9 2) 6.45 0.50 ( 0.22) 0.0 3 8

18. 5 4

- 2 6 .54 17. 7 6

- 2 4 .21 11. 6 3, 3 6

. 32 Table VII

- 4 (continued). Summary of Mai z e Grain Ash and Minerals for MON 87411, Conventional C ontrol, and Reference

Hybrids

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

1dw

= d r y weight.

2Mean (S.E.)

= least

- s quare mean (standard error).

3Range is the mini m u m a n d m a xi m u m raw values for the convent i onal reference hybrids.

4 With 95% confidence, t h e tolerance interval contains 99% of t h e values expressed in the population of c on ventional reference hybr i ds. Negative

li m i t s were set to zero.

5 Maxi m u m v a lue m inus mini m u m value for the conv e ntional cont r ol m a ize h y b r id.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 130 o f 3 7 2CB I Delete d Copy

Table VII

- 5. Summary of Mai z e Grain Vitamins for M O N 87411, Conventional Control, and Reference Hybrids

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

C o m ponent (m g/kg dw)1 Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

Folic Acid 0.28 ( 0.0 0 71) 0.28 ( 0.0 0 71) (0.19

- 0. 5 2) 0.17 0.0 0 41 (0

. 0 0 74) 0.5 8 0

0.23

- 0. 3 9 0.21

- 0. 3 7 0.0 8 4, 0

. 56

Niacin 17. 3 3 (0. 7 5) 18. 7 8 (0. 7 5) (14.90

- 3 8

. 0 7) 20. 6 9

- 1.45 ( 0.61) 0.0 2 1

14. 8 6

- 2 0 .70 13. 7 3

- 3 4 .41 4.69, 42

. 03

Vita m in A 1.29 ( 0.0 5 8) 1.38 ( 0.0 5 8) (0.32

- 4. 7 6) 2.94

- 0.095 (0. 0 6 4 ) 0.1 4 5

0.75

- 1. 5 7 0.65

- 3. 5 9 0, 4

. 91

Vita m in B1 3.44 ( 0.0 6 5) 3.56 ( 0.0 6 5) (2.79

- 4. 9 6) 0.85

- 0.12 ( 0.0 4 7) 0.0 2 1

3.04

- 3. 8 5 3.10

- 3. 9 4 1.86, 5. 0 7

Vita m in B2 1.53 ( 0.0 4 8) 1.64 ( 0.0 4 8) (1.15

- 2. 5 4) 1.01

- 0.11 ( 0.0 5 2) 0.0 5 8

1.22

- 2. 0 3 1.28

- 2. 3 0 0.94, 2. 3 7

Vita m in B6 6.16 ( 0.11) 6.10 ( 0.11) (5.09

- 12

. 13) 3.49 0.0 6 0 (0. 1 3) 0.6 4 8

5.15

- 7. 4 4 5.07

- 8. 5 6 3.84, 10

. 03

M ons a n t o Co m pany CR 2 4 0

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Table VII

- 5 (continue d ). Summary of Mai z e Grain Vitamins for MON 87411, Conventional Control, and Reference Hybrids

Difference

(Test m inus Control)

MON 87411 Conventi o nal

Control Refere n c e H y brids Conventi o nal

C o m ponent (m g/kg dw)1 Mean (S.E.)2

Range Mean (S.E.)

Range (Range)3

Tolerance Interval 4 Control Ran g e

Valu e 5 Mean

(S.E.) p

- Value

Vita m in E 10. 5 3 (0. 2 4) 10. 2 8 (0. 2 4) (6.37

- 31

. 91) 3.78 0.25 ( 0.29) 0.4 0 0

6.99

- 15

. 33 8.94

- 12

. 72 0, 3 0 .69

1dw

= d r y weight.

2Mean (S.E.)

= least

- s quare mean (standard error).

3Range is the mini m u m a n d m a xi m u m raw values for the convent i onal reference m a ize h y br i ds.

4 With 95% confidence, t h e tolerance interval contains 99% of t h e values expressed in the population of c on ventional reference hybr i ds. Negative

li m i t s were set to zero.

5 Maxi m u m v a lue m inus mini m u m value for the conv e ntional cont r ol m a ize h y b r id.

M ons a n t o Co m pany CR 2 4 0

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C o m ponent Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

Anti

- nutrie n t (% dw1)

Ph y tic Acid 0.99 ( 0.0 2 9) 0.98 ( 0.0 2 9) (0.60

- 1. 2 5) 0.52 0.0 1 2 (0. 0 22) 0.5 8 4

0.75

- 1. 2 7 0.67

- 1. 1 9 0.56, 1. 4 1

Raffinose 0.25 ( 0.0 0 81) 0.24 ( 0.0 0 81) (0.093

- 0

. 40) 0.19 0.0 0 66 (0

. 0 0 58) 0.2 5 6

0.14

- 0. 3 1 0.10

- 0. 2 9 0, 0

. 45

Secondary Metabolite (μg/g dw)

Ferulic Acid 184 6 .74 ( 28

. 24) 189 6 .61 ( 28

. 24) (1337

. 21

- 3 2 86. 5 5) 392

. 34

- 49.87 (29

. 3 0 ) 0.1 1 0

152 8 .09

- 2 0 31. 9 6 170 0 .68

- 2 0 93. 0 2 749

. 39, 34 2 1

. 84

p

- Cou m aric Acid 148

. 56 (2

. 91) 148

. 27 (2

. 91) (62.24

- 3 8 7

. 51) 33. 3 3 0.28 ( 3.31) 0.9 3 2

132

. 88

- 1 97

. 75 131

. 03

- 1 64

. 37 0, 4 6 1. 0 5 Table VII

- 6. Summary of Mai z e Grain A n ti

- n utrients a n d Secondary Metabolites for MON 87411, Conventional C ontrol, and

Reference Hybrids

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

1dw

= d r y weight.

2Mean (S.E.)

= least

- s quare mean (standard error).

3Range is the mini m u m a n d m a xi m u m raw values for the convent i onal reference hybrids.

4 With 95% confidence, t h e tolerance interval contains 99% of t h e values expressed in the population of c on ventional reference hybr i ds. Negative

li m i t s were set to zero.

5 Maxi m u m v a lue m inus mini m u m value for the conv e ntional cont r ol m a ize h y b r id.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 133 o f 3 7 2CB I Delete d Copy

Table VII

- 7. Summary of Mai z e Forage P roximates, Fiber an d Mineral s fo r MO N 87411

, Conventiona l C o ntrol

, and

Reference Hybrids

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

C o m ponent ( % dw)1 Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

Ash 5.57 ( 0.22) 5.95 ( 0.22) (3.53

- 8. 3 3) 5.04

- 0.39 ( 0.16) 0.0 1 8

3.95

- 7. 0 1 4.51

- 9. 5 5 3.38, 7. 5 9

Carbo h y drates by 84. 9 6 (0. 5 6) 84. 5 7 (0. 5 6) (79.74

- 9 0

. 3 7) 7.09 0.38 ( 0.25) 0.1 2 8

Calculation 81. 6 5

- 8 8 .55 80. 9 1

- 8 8 .00 80. 3 2, 8 9

. 79

Protein 7.58 ( 0.46) 7.42 ( 0.46) (4.19

- 11

. 38) 5.03 0.17 ( 0.16) 0.3 2 1

5.27

- 10

. 25 4.63

- 9. 6 6 3.12, 11

. 55

Total Fat 1.91 ( 0.17) 2.07 ( 0.17) (0.66

- 3. 6 0) 2.40

- 0.16 ( 0.15) 0.2 9 6

0.49

- 2. 9 3 0.99

- 3. 3 9 0.67, 3. 5 8

Acid Detergent Fiber 25. 4 5 (0. 9 4) 25. 0 7 (0. 9 4) (18.64

- 3 7

. 6 8) 12. 6 5 0.39 ( 0.66) 0.5 5 8

16. 5 8

- 3 4 .25 17. 8 9

- 3 0 .55 14. 7 5, 3 8

. 41

Neutral De t e r gent Fiber 44. 6 8 (1. 4 9) 42. 8 4 (1. 4 9) (34.97

- 6 7

. 3 9) 19. 1 7 1.84 ( 1.19) 0.1 4 5

28. 3 2

- 5 5 .35 32. 1 6

- 5 1 .33 28. 7 4, 6 2

. 39

M ons a n t o Co m pany CR 2 4 0

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C o m ponent ( % dw)1 Range Range Tolerance Interval 4 Valu e 5 (S.E.) p

- Value

Calci u m 0.17 ( 0.0 1 6) 0.18 ( 0.0 1 6) (0.074

- 0

. 37) 0.34

- 0.013 (0. 0 0 7 8) 0.1 1 2

0.0 9 3

- 0

. 26 0.0 8 4

- 0

. 42 0.0 1 5, 0

. 32

Phospho r us 0.19 ( 0.0 1 1) 0.19 ( 0.0 1 1) (0.11

- 0. 2 6) 0.13

- 0.00 0 02 ( 0. 0 058) 0.9 9 7

0.0 9 5

- 0

. 24 0.13

- 0. 2 5 0.0 8 2, 0

. 26 Table VII

- 7 (continued). Summa r y of Mai z e For a ge Proximates, Fiber and Minerals for MON 87411, Conventional Control,

and Reference Hybri d s

MON 87411

Mean (S.E.)2 Conventi o nal

Control

Mean (S.E.) Refere n c e H y brids

(Range)3 Conventi o nal

Control Ran g e Difference

(Test m inus Control)

Mean

1dw

= d r y weight.

2Mean (S.E.)

= least

- s quare mean (standard error).

3Range is the mini m u m a n d m a xi m u m raw values for the convent i onal reference m a ize h y br i ds.

4 With 95% confidence, t h e tolerance interval contains 99% of t h e values expressed in the population of c on ventional reference hybr i ds. Negative

li m i t s were set to zero.

5 Maxi m u m v a lue m inus mini m u m value for the conv e ntional cont r ol m a ize h y b r id.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 135 o f 3 7 2CB I Delete d Copy

Tabl e VII

- 8

. Literatur e an d ILS I Databa se Ranges for Components in Mai z e

Forage and Grain

Grain Tis s ue Co m pone n t s 1 Literatur e Range 2 ILS I Ra n g e 3

Grain N u trie n ts

Pro x ima t e s ( % dw)

Ash 1

. 17

– 2.0 1 a ; 1

. 27

– 1.6 3 b 0

. 6 1 6

– 6 .2 82

Carboh y drate s b y calculation

Fat

, t o tal

Prote i n 8 1

. 3 1

– 8 7

. 06 a ; 8 2

. 10

– 8 5 .9 8 b

2

. 95

– 4.4 0 a ; 3

. 18

– 4.2 3 b

8

. 27

– 1 3 .3 3 a ; 9

. 17

– 1 2 .1 9 b 7 7. 4

– 8 9 .5

1

. 7 4 2

– 5 .9 00

6

. 15

– 1 7 .26

Fibe r ( % dw)

Aci d d eter g e nt fi b e r 1

. 82

– 4.4 8 a ; 1

. 83

– 3.3 9 b 1

. 82

– 1 1 .34

Ne u t ra l d eterge nt f i b e r

T o ta l d ietar y fi b e r 6

. 51

– 1 2

. 28 a ; 6.0 8

– 1 0.3 6 b

1 0

. 6 5

– 1 6

. 26 a ; 1 0

. 57

– 1 4 .5 6 b 5

. 59

– 2 2 .64

9

. 01

– 3 5 .31

Amino Ac i ds ( % d w )

Alanine 0

. 60

– 1.0 4 a ; 0

. 68

– 0.9 6 b 0

. 44

- 1.39

A r g i n i ne

As p a rti c acid

Cysti ne

Gluta m ic acid

Glycine

Histi d i ne

Iso l e uc i ne

Leucine

Lysine

Met h i on i ne

Ph e n ylalanine

Prol i ne

Serine

Thr e onine

T r y p top h an

Tyros i ne

Vali ne 0

. 34

– 0.5 2 a ; 0

. 34

– 0.5 0 b

0

. 52

– 0.7 8 a ; 0

. 59

– 0.7 6 b

0

. 19

– 0.2 6 a ; 0

. 20

– 0.2 6 b

1

. 54

– 2.6 7 a ; 1

. 71

– 2.4 4 b

0

. 33

– 0.4 3 a ; 0

. 33

– 0

. 42 b

0

. 25

– 0.3 7 a ; 0

. 27

– 0.3 4 b

0

. 30

– 0.4 8 a ; 0

. 32

– 0.4 4 b

1

. 02

– 1.8 7 a ; 1

. 13

– 1.6 5 b

0

. 26

– 0.3 3 a ; 0

. 28

– 0.3 1 b

0

. 17

– 0.2 6 a ; 0

. 16

– 0.3 0 b

0

. 43

– 0.7 2 a ; 0

. 45

– 0.6 3 b

0

. 74

– 1.2 1 a ; 0

. 78

– 1.1 1 b

0

. 39

– 0.6 7 a ; 0

. 43

– 0.6 0 b

0

. 29

– 0.4 5 a ; 0

. 31

– 0.3 9 b

0

. 0 4 7

– 0 .0 85 a ; 0 .0 42

– 0.07 0 b

0

. 13

– 0.4 3 a ; 0

. 12

– 0.4 1 b

0

. 42

– 0.6 2 a ; 0

. 45

– 0.5 8 b 0

. 12

- 0.64

0

. 33

– 1.21

0

. 13

– 0.51

0

. 97

– 3.54

0

. 18

– 0.54

0

. 14

– 0.43

0

. 18

– 0.69

0

. 64

– 2.49

0

. 17

– 0.67

0

. 12

– 0.47

0

. 24

– 0.93

0

. 46

– 1.63

0

. 24

– 0.77

0

. 22

– 0.67

0

. 0 2 7

– 0 .2 15

0

. 10

– 0.64

0

. 27

– 0.86

F a tt y A cid s ( % T otal FA)

16 : 0 Pal m itic 8

. 80

– 1 3 .3 3 a ; 9

. 84

– 1 2 .3 3 b 7

. 94

– 2 0 .71

18 : 0 S t e a r i c

18 : 1 O l e i c

18 : 2 L i no l e i c

18 : 3 L i no l e n i c

20 : 0 Ara c h i d i c

20 : 1 E i c ose n o i c

22 : 0 B e hen i c 1

. 36

– 2.1 4 a ; 1

. 3 0

– 2

. 10 b

1 9

. 5 0

– 3 3

. 71 a ; 1 9

. 59

– 2 9 .1 3 b

4 9

. 3 1

– 6 4

. 70 a ; 5 6

. 51

– 6 5 .6 5 b

0

. 89

– 1.5 6 a ; 1

. 03

– 1.3 8 b

0

. 30

– 0.4 9 a ; 0

. 30

– 0.4 1 b

0

. 17

– 0.2 9 a ; 0

. 17

– 0.2 7 b

0

. 0 6 9

– 0 .2 8 a ; 0

. 0 5 9

– 0 .1 8 b 1

. 02

– 3.40

1 7. 4

– 4 0 .2

3 6. 2

– 6 6 .5

0

. 57

– 2.25

0

. 2 7 9

– 0 .9 65

0

. 1 7 0

– 1 .9 17

0

. 1 1 0

– 0 .3 49

Minerals

Calci u m ( % dw) 0

. 0 03 6

– 0

. 0 06 8 a ; 0.00 35

– 0. 0 07 0 b 0

. 0 01 27

– 0.02 0 84

C o p per ( m g /

kg d w) 1

. 14

– 3.4 3 a ; 1

. 39

– 2.7 6 b 0

. 73

– 1 8 .50

I r on ( m g /

kg d w) 1 4

. 1 7

– 2 3

. 40 a ; 1 5

. 90

– 2 4 .6 6 b 1 0

. 4 2

– 4 9

. 07

Ma gnes i um (% d w) 0

. 0 9 1

– 0 .1 4 a ; 0

. 10

– 0.1 4 b 0

. 0 59 4

– 0

. 1 9 4

Ma ng a nese ( m g /k g d w ) 4

. 83

– 8.3 4 a ; 4

. 78

– 9.3 5 b 1

. 69

– 1 4 .30

Ph os ph o ro us ( % d w) 0

. 24

– 0.3 7 a ; 0

. 27

– 0.3 8 b 0

. 1 4 7

– 0 .5 33

Po ta ss i um (% d w ) 0

. 29

– 0.3 9 a ; 0

. 36

– 0.4 3 b 0

. 1 8 1

– 0 .6 03

Z i nc ( m g /k g d w) 1 6

. 7 8

– 2 8

. 17 a ; 1 8

. 25

– 3 0 .4 4 b 6

. 5

– 3 7 .2

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Table VII

- 8 (continued). Literature and I LS I Databas e Range s fo r Component s in

Mai z e Forage and Grain

Grain Tis s ue Co m pone n t s 1 Literatur e Range 2 ILS I Ra n g e 3

Vitamin s ( m g/k g dw)

F o li c acid 0

. 19

– 0.3 5 a ; 0

. 23

– 0.4 2 b 0

. 1 4 7

– 1 .4 64

Vit a m i n A [ β

– Carotene]

Vit a m i n B 1 [ T hiamine]

Vit a m i n B 2 [Ri b ofla v i n]

Vit a m i n B 3 [N i acin]

Vit a m i n B 6 [P y r id ox i ne]

Vit a m i n E [ α

– To c o p he r o l ] 1 2 2

– 4 7 4 0 c

2

. 33

– 4.1 7 a ; 2

. 71

– 4.3 3 b

0

. 94

– 2.4 2 a ; 1

. 64

– 2.8 1 b

1 5

. 0 7

– 3 2

. 38 a ; 1 3

. 64

– 4 2 .0 6 b

4

. 93

– 7.5 3 a ; 4

. 97

– 8.2 7 b

5

. 96

– 1 8 .4 4 a ; 2

. 84

– 1 5 .5 3 b 0

. 19

– 4 6 .81

1

. 26

– 4 0 .00

0

. 50

– 2.36

1 0

. 3 7

– 4 6

. 94

3

. 68

– 1 1 .32

1

. 5 3 7

– 6 8

. 6 7 2

Grain A n ti

– Nutrients (% d w )

P h yti c acid 0

. 69

– 1.0 9 a ; 0

. 60

– 0.9 4 b 0

. 1 1 1

– 1 .5 70

Raffin o se 0

. 0 7 9

– 0 .2 2 a ; 0

. 0 6 1

– 0 .1 5 b 0

. 0 2 0

– 0 .3 20

Grain Secon d ary M e ta b o lites ( μ g / g d w )

Fer u li c acid p

–

C ou m a ric acid 1 20 5

. 75

– 287 3

. 05 a ; 1 0 1 1 .4 0

– 2 53 9

. 86 b

9 4

. 7 7

– 3 2 7

. 39 a ; 6 6

. 4 8

– 2 5 9. 68 b 2 9 1

. 9

– 3 8 8 5

. 8

5 3. 4

– 57 6 .2

Fo r age Ti ssue C om p one n ts 1 Literatur e Range 2 ILS I Ra n g e 3

Fo r age N utr i e nts

Pro x ima t e s ( % dw)

Ash 2

. 67

– 8.0 1 a ; 4

. 59

– 6.9 0 b 1

. 5 2 7

– 9 .6 38

Carboh y drate s b y calculation

Fat

, t o tal

Prote i n 8 1

. 8 8

– 8 9

. 26 a ; 8 4

. 11

– 8 7 .5 4 b

1

. 28

– 3.6 2 a ; 0

. 20

– 1.7 6 b

5

. 80

– 1 0 .2 4 a ; 5

. 56

– 9.1 4 b 7 6. 4

– 9 2 .1

0

. 2 9 6

– 4 .5 70

3

. 14

– 1 1 .57

Fibe r ( % dw)

Aci d d eter g e nt fi b e r 1 9

. 1 1

– 3 0

. 49 a ; 2 0

. 73

– 3 3 .3 9 b 1 6

. 1 3

– 4 7

. 39

Neu t ral det e r g ent f i ber 2 7

. 7 3

– 4 9

. 62 a ; 3 1

. 81

– 5 0 .6 1 b 2 0

. 2 9

– 6 3

. 71

Minerals ( % d w )

Calci u m 0

. 12

– 0.3 3 a ; 0

. 21

– 0.4 1 b 0

. 0 71 39

– 0.57 6 79

P h os ph o ro us 0

. 0 9 0

– 0 .2 6 a ; 0

. 13

– 0.2 1 b 0

. 0 93 62

– 0.37 0 41

1 dw

= d r y weight ; F A = fatt y aci ds.

2 Literature ra n ge ref e rences: a US a nd b C h il e (Harrig a n e t al.

, 2009)

, c (S a f a wo e t al.

, 2 0 10 ).

3 ILSI r a nge is from ILSI Crop Co m position D a tabas e

, 201 1 [Acce s se d 9 Januar y 2013 ] (I L SI,

2 01 1 ).

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VII.B. Compositional Assessme n t of MON 87411 Conclusion

Co mpositi o nal analy s is was conducted on gra i n and forage of MON 87411 treated with

glyphosate grown at eight sit e s in a 2011/2012 field product i on in Argentina that are

representative of commer c ial agricultural regions fo r m a iz e production

. The

co mpositional analysis, based on the OECD consensus docu m ent for m aize, incl u ded

m easur e m ent of nutrients, a n ti

- nutrie n ts and secondary m etabolites in con v e n tional

commercial reference hybrids to provide data on the natural v a riabilit y o f each

co mpositional co mponent analyzed.

Of the 60 co m ponents statistically ass esse d fo r MO N 87411

, onl y 1 2 co m p onents

(protein, histidine, t y rosine, oleic acid, neutral deter g ent fiber, copper, iron, m anganese,

zinc, niacin, and vita m in B1 in grain and a s h in forage) showed a signi f icant difference

between MON 87411 a nd the conventional contr o l in the co m bined

- site analysis. For

these 12 components, the m ean difference in component values between MON 87411 and

the conventional control was less than the r a nge of the conventional control values and

the reference hybrid values. For all co m ponents analyzed, the MON 87411 m ean

co mponent values were within the tolerance intervals, the val u es observed in the

literature, and/or the IL S I

- CCDB values.

These results support the overall conclusion that MON 87411 is not a m ajor contributor

to vari a tion in co m ponent lev e ls in m aize grain and f orage and con f ir m e d the

co mpositional equivalence of MON 87411 to the conventional control in levels of these

co mponents. These data confir m ed that t h e components with a s t atistically significant

differences (p<0.05) between MON 87411 a n d the conventional control were not

co mpositionally m eaningful from a food and feed safety perspective.

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VIII. P H ENOTYPIC

, AGRON O MIC

, AN D ENVIRONMENTAL

INTERACTION S ASSESSMENT

This section provides a co mparative ass e ss m ent of the phenotypic, agrono m ic, and

environ m ental interaction characteristics o f MO N 8741 1 compare d t o th e conventional

control. The data support a conclusion t h a t MO N 8741 1 i s no t m eaningfull y different

from the conventional control with the exception of the i n sect

- protected and glyphosate

tolerant traits, and therefore, is not expec t ed to pose a plant pest ri s k co m pared to

conventional m aize. These conclusions are based on the results of m ultiple evaluations

from laboratory and field assess m ents.

Phenotypic, agrono m ic, and environ m ental interaction charact eristic s o f MO N 87411

were evaluated in a comparative m a nner to as s e s s plant pe s t potential. T hese asse ssm en t s

included evaluation of seed ger m ination cha racteristics

, plan t growt h an d develop m ent

characteristics, observations of plant respon se s t o abioti c stress o rs

, plant

- diseas e and

plant

- arthropo d interactions

, an d polle n charac t eristics

. Result s f r om these assess m ents

de m onstrate that MON 87411 does not possess a) increased weed ines s characteristics ; b)

incr e ased s u sceptibility or t o le r ance to speci f i c abiotic s tre s sors, d isea s es, or arthro p ods;

or c) characteristics that w ould confer a plant pest risk compared to the conventional

control.

VIII.A. Characteristics Measured for Assessment

In the phenotypic, agrono m ic, and environ m ental interactions assess m ent of

MO N 87411

, dat a wer e collecte d t o evaluat e altered plant pest p o tential

. A detailed

descriptio n o f th e regulate d article phenotype is requested as p a rt of the p e tition f or

deter m ination of nonregulated status in 7 CFR § 340.6 including differences from the

un m odified recipient organism that would “ s ubstantiate that the re g ulated arti cl e is

unlikely to pose a greater plant pest risk than the un m odified organism f r om which it was

derived”. A s part of the c h aracterization of MON 87411, data were collected to provide a

detaile d descriptio n o f th e phenotypic

, agrono m i c

, an d environ m e n ta l interaction

characteristics of MON 87411. A s ubset of these data were included in an evaluation of

specific characteristics related to alt e red p la n t pest pot e nti a l ( e.g. seed dor m ancy,

lodging, ear drop, and environ m e n tal interactions data).

Th e plan t characterizatio n o f MO N 8741 1 encompassed five general data categories: 1)

seed ger m ination, dor m ancy, and e m ergence; 2 ) vegetativ e growth ; 3 ) reproductive

develop m ent (including pollen characteristic s) ; 4 ) lodgin g an d see d retentio n o n the

plant; 5) plant response to a b iotic stress and inter action s wit h disease s and arthropods.

An overview of the characteristics a ssessed is presented in T able VIII

- 1.

The phenotypic, agrono m ic, and environ m ental interactions data were evaluated from a

basis of fa m iliarity ( O ECD, 1993) and were co mp rised of a co m b ination of field and

laboratory s tudies con d ucted by scientists who are fa m iliar with the production and

evaluation o f m aize. In each of these assess m ents, MON 87411 was co m pared to a n

appropriate conventional control ( i. e

., MPA640B) that had a genetic background si m ilar

t o MO N 8741 1 bu t di d no t posses s insect

- protec t e d o r glyphosate

- toleranc e traits

. In

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addition, multi p le com m ercial conventional maize refere n ce hybrids developed through

conventional breeding and selection (see Appendix H, I, and J, and Tables H

- 1, I

- 1, and

J

- 1) were i n cluded to provide a ra n ge of co m p arative val u es for each characteristic that

are representative of the var i ability in existing com m ercial m aize hybrids. Data collected

for the various characteristics from the com m er c ial reference hybrids provid e contex t for

interpreting experi m ental results.

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Table VIII

- 1. Phenotypic, Agronom i c, and Environmental Inter a ction

Characteristics Evaluated in U.S. F iel d Trial s an d Laborator y Studies

Characteristics

m easured

(associate d se c tion E v al u atio n timi ng Eva l ua tio n de s c r i p tio n

Dat a category wh e re discuss e d) (Settin g of e v al u ati o n) 1 ( m easure m en t endpoints)

Ger m i n ati on, Nor m al ger m i n ated D a y 4 an d 7: Perc e n ta ge of s eed p ro duc i ng

dor m ancy

, and ( V I I I.C

. 1) 20 / 3 0 °C ( La bo ra t or y ) seed lin gs e xh i b iti ng n or m al

e m ergence deve l op m ental charact e ristics

A bn or m al Day 7: Perc e n ta ge of s eed p ro duc i ng

ger m inated 20 / 3 0 °C (L a b ora t or y ) seed lin gs t hat cou l d not be c l ass i f i ed

( V I I I.C

. 1) a s nor m a l germinated

Ger m inated D a y 4

, D a y 7

, a nd Perc e n ta ge of s eed t hat h a d

( V I I I.C

. 1) D a y 1 2: g e r m i n ate d (bot h no r m a l l y or

5, 1 0, 2 0, 3 0, 1 0 /2 0 a nd a b no r m a l ly)

10 / 3 0 °C ( La bo ra t or y )

Dead D a y 4 an d 7: Perc e n ta ge of s eed t hat h a d v i s i b l y

( V I I I.C

. 1) 5, 1 0, 2 0, 3 0, 1 0 /2 0, deteriorated and beco m e soft t o the

10 / 3 0

, a nd 2 0 / 3 0°C. t ou c h (a l so i n c l ud e d n on

- v i a bl e ha r d

Day 12: a nd no n

- v ia b l e f i r m

- swo lle n see d)

5, 1 0, 2 0, 3 0, 1 0 /2 0 a nd

10 / 3 0 °C (L a b ora t or y )

Viable ha r d Day 7: Perc e n ta ge of s eed t hat d i d n o t

( V I I I.C

. 1) 2 0 /3 0°C i m b i be wate r a nd re m ai n e d har d to

D a y 1 2:

5, 1 0, 2 0, 3 0, 1 0 /2 0 a nd t he to u c h ( v iabili ty d eter m i n e d by a

tetraz o li um te s t 2 )

10 / 3 0 °C ( La bo ra t or y )

Viable fir m

- swollen Day 7: Perc e n ta ge of s eed t hat i m b ibe d

( V I I I.C

. 1) 20 / 3 0 °C water a n d were firm to the t o uc h but

D a y 1 2:

5, 1 0, 2 0, 3 0, 1 0 /2 0 a nd d i d n ot g e r m i n at e ( v ia b ility

d eter m i n e d by a tetraz o li um t es t 2 )

10 / 3 0 °C ( La bo ra t or y )

Ear l y s t a n d c o unt V2

– V 5 g ro w t h sta ge Nu m ber of e m erg e d p l an t s i n t wo

( V I I I.C

. 2

. 1) (Fiel d) r o ws

F i nal s t a n d c ou nt Pre

- h a rvest (F i e ld) N u m b e r of p la n t s i n tw o rows

( V I I I.C

. 2

. 1)

Ve g etati ve P l ant v i g o r V2

– V 4 g ro w t h sta ge Rate d o n a 1

- 9 scale

, w h e re 1

=

g r ow t h ( V I I I.C

. 2

. 1) (Fiel d) exce lle nt v ig or a n d 9 = p o o r vi g o r

Sta y gre e n Mat u rit y (Field) Rate d o n a 1

- 9 scale , 1 = 90 to

( V I I I.C

. 2

. 1) 1 0 0%; 2 = 8 0

- 8 9% ; 3

= 7 0

- 7 9%; 4 =

6 0

- 69%; 5 = 5 0

- 5 9%; 6

= 4 0

- 4 9% ;

7

= 3 0

- 3 9 % ; 8

= 2 0

- 2 9 % ; an d 9

= 0

– 1 9 % gre e n ti ssue

Ear he ig ht R 1

– Maturit y (Fiel d) Distanc e fro m th e s o i l surfac e a t the

( V I I I.C

. 2

. 1) base of t he p l a nt t o t he e a r

a tt ach m e nt no d e o n f i ve p l an t s p e r

p l ot

P l ant he i ght R 1

– Maturit y (Fiel d) Distanc e fro m th e s o i l surfac e a t the

( V I I I.C

. 2

. 1) b as e of t he p la nt t o t he fla g leaf

co lla r on f iv e pl an t s p e r p l ot

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Tabl e VIII

- 1 (continued)

. Phenotypic

, A gronomic and Environmental Inter a ction

Characteristics Evaluated in U.S. F iel d Trial s an d Laborator y Studies

Dat a category Characteristics

measured E v al u atio n timi ng

(Setti ng o f e v al u ati o n ) 1 Eva l ua tio n de s c r i p tio n

Re p r o d uc ti ve

g r ow t h D ay s t o 5 0% p o llen

sh e d

( V I I I.C

. 2

. 1)

Day s t o 5 0% sil k i ng

( V I I I.C

. 2

. 1)

P o lle n v ia b ility

( V I I I.C

. 3)

Po lle n m o r p hol ogy

( V I I I.C

. 3)

Gra i n m o ist u re

( V I I I.C

. 2

. 1) P o lle n s h e d (F i el d)

Sil k i ng (Fiel d)

Tasse li ng (L a b ora t or y )

Tasse li ng (L a b ora t or y )

Har v es t (Fiel d) Day s fr om p la n ti ng u n ti l 50 % of

t he p l a n t s have b e g u n t o s h e d

po llen

Day s fr om p la n ti ng u n ti l 50 % of

t he p la n t s h a ve m u l t i p l e sil ks

e x p o sed

Perc e n ta ge of v i ab l e po ll e n

bas e d on p o lle n gra i n s t a i n i n g

charact e r istics

Dia m e t er of v i able pollen gr a i ns

a nd o b se rv atio ns

Perce n ta ge m o ist u r e of h a r v ested

she lle d gra i n

Tes t weight

( V I I I.C

. 2

. 1)

Yield

( V I I I.C

. 2

. 1) Har v es t (Fiel d)

Har v es t (Fiel d) Test we ig ht (k i l o g ra m / hec t o

liter ) o f h ar v este d s h elle d gr a i n

Calc u late d i n M g / h a

, adjuste d to

15.5 % grai n moistur e cont e nt

L o dg i ng a n d

See d rete n tion S t a l k lo dg e d p l an t s

( V I I I.C

. 2

. 1)

R oot l o d g e d p l an t s

( V I I I.C

. 2

. 1)

D r o p p e d e a rs

( V I I I.C

. 2

. 1) Pre

- h a rvest (F i e ld)

Pre

- h a rvest (F i e ld)

Pre

- h a rvest (F i e ld) Nu m ber of p l a n t s per p l ot

broke n be l ow th e e a r

Nu m ber of p l a n t s per p l ot

leanin g a t th e s o i l surfac e at

>30 ° fro m th e vertical

Nu m b e r of m a ture ea rs d r opped

f r om p l an t s

E n v i r o n m en ta l

i n teracti o ns Ab i otic stress

r espo n se

( V I I I.C

. 2

. 2

. 1)

Disease da m a ge

( V I I I.C

. 2

. 2

. 1)

Ar t h r o po d da ma ge

( V I I I.C

. 2

. 2

. 1)

Stal k ro t disea s e

( V I I I.C

. 2

. 2

. 1)

Ear/k e rnel r ot

disease

( V I I I.C

. 2

. 2

. 1) F o ur ti m es d uri ng g r o w in g

seaso n (Fiel d)

F o ur ti m es d uri ng g r o w in g

seaso n (Fiel d)

F o ur ti m es d uri ng g r o w in g

seaso n (Fiel d)

Har v es t (Fiel d)

Har v es t (Fiel d) Q u alitati ve assess m e nt of each

plot

, wit h c ategorica l scal e of

i n creasin g se v e rit y (n on e

, slig h t

,

m oderate, se v e re)

Q u alitati ve assess m e nt of each

plot

, wit h c ategorica l scal e of

i n creasin g se v e rit y (n on e

, slig h t

,

m oderate, se v e re)

Q u alitati ve assess m e nt of each

plot

, wit h c ategorica l scal e of

i n creasin g se v e rit y (n on e

, slig h t

,

m oderate, se v e re)

Q u alitati ve assess m e nt of each

plot

, wit h c ategorica l scal e of

i n creasin g se v e rit y (n on e

, slig h t

,

m oderate, se v e re)

Q u alitati ve assess m e nt of each

plot

, wit h c ategorica l scal e of

i n creasin g se v e rit y (n on e

, slig h t

,

m oderate, se v e re)

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Tabl e VIII

- 1 (continued)

. Phenotypic

, A gronomic and Environmental Inter a ction

Characteristics Evaluated in U.S. F iel d Trial s an d Laborator y Studies

Dat a category

E n v i r o n m en ta l

i n teracti o ns Characteristics

measured

C o r n earw orm

da m a ge

( V I I I.C

. 2

. 2

. 2)

E u ro p ea n cor n b o r e r

da m a ge

( V I I I.C

. 2

. 2

. 2) E v al u atio n timi ng

(Setti ng o f e v al u ati o n ) 1 Eva l ua tio n de s c r i p tio n

R 5 g r ow t h s t a g e (F ield ) Q u a n titati ve assess m e nt on 10

p l an t s per p l ot by exa m i n i ng ears

f o r da m a ged a r ea us i ng a p l as ti c

fi lm gr i d (eac h gri d cel l = 0

. 5

c m 2 )

R 6 g r ow t h s t a g e (F ield ) Q u a n titati ve assess m e nt on 10

p l an t s per p l ot by c ou n ti ng

nu m ber of fe e di ng ga lle r i es a n d

t o ta l le ng t h of fee d i ng g aller i es

i n eac h stalk

Ar t h r o pod

abundance

( V I I I.C

. 2

. 2

. 2) Fi ve c o llecti on t i m e s dur i ng

g r ow i n g sea s o n (F iel d) Q u a n titati ve assess m e nt of

ar t h ro p o d a b u n dance v i a s tick y

trap s c o llection s an d visual

counts

1 Pla nt g rowt h sta g e s wer e d eter m i n e d us i ng d escr i p ti o n s a nd g u i d el i ne s ou tli n e d i n C o r n Gr ow t h a nd

Deve l op m en t (Ritchie

, e t al.

, 1997).

2 Via b ilit y of har d an d fir m

- sw o lle n see d wer e d eter m i n e d by a tetraz o liu m tes t (AOSA / SCST

, 2 0 10).

VIII.B. Interpretation of Phenotypic and Environment a l Interaction Data

Plant pest risk assess m e nts for biotechnology

- derived crops are co m p arative assessments,

and are con s idered f rom a basis of f a m iliarity. The concept o f f a m iliarity is based on the

fact that the biotechnology

- derived plant is developed from a well

- characterized

conventional crop whose biological properties and plant pest potential are well

- known.

F a m iliarity considers the biology of the crop, the introduced traits, the receiving

environ m ent and the interaction of these factors, and provides a basis for co m parative

environ m ental risk a s sess m ent between a biotechnology

- der i v ed pla n t a n d its

conventional counterpart.

Exper t knowledg e an d experienc e wit h conventionally bred m aize was the basis for

selecting appropriate endpoints and esti m ati n g the range of responses that would be

considered typical for maize. As such, MON 87411 was co mpared to the conventional

control in the assess m ent of phenotypic, a g rono m ic, and environ m ental interaction

characteristics. An overview of the characteri stic s assesse d i s presente d i n Table

- VIII

- 1.

A subset of the data relating to well

- understood weedy characteristics ( e.g

. , seed

dor m ancy, ear drop, and lodging) was used to asses s whethe r ther e wa s a n increas e in

weediness potential of MON 87411 co mpared to a conventional m aize. Evaluation of

environ m ental int e ra c ti o n characteristics ( e.g

., plant abi o tic stress, plant

- d isea s e, and

plant

- arthropod interactions) was also considered in the plant pest assess m ent. Prior to

analysis, the overall dataset was evaluated for possible evidence of biologically

- relevant

change s an d unexpecte d plan t r esp o nses. No u nexpected o b servations or issues were

identi f ied. Based on all of the data coll e cted, a n assess m ent was m ade to det e r m ine if

MON 87411 could be expected to pose an i n creased plant pest risk co mpared to

conventional m aize.

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VIII.B.1. Interpret a tion of Detected Differences Criteria

C o mparative plant characteri z ation data between a biotechnology

- derived crop and the

conventional control are inter p reted in the conte x t of contributions to increased plant

pest/weed p otential. U nder the f r a m ework of f a m iliarity, chara c te r istics f or which no

differences are detected support a conclusion of no increased plant pest/weed potential.

Characteristics for which differences are det e cted are considered in a step

- wise m ethod

(Figure VIII

- 1) or in a si m ilar fashion. All detected differences for a characteristic are

considered in the context of whether or not the d ifference would increase the cro p ’s plant

pest/weed potential. Ulti m atel y

, a weight of evide n ce appro ac h consid e ring all

characteristics and data is used for the over a ll risk assess m ent of differences and t h eir

significance. In detail, Figure VIII

- 1 i l lustrates t h e stepwise assess m ent process

e mployed:

Figure VIII

- 1. Schematic D iagram of Agronomic and Phenotypic Data

Interpret a tion Methods

Note: A “ n o” answer at a ny step indicates that the c h a racteristic do e s not c o n t r ib u t e t o a b i o l og i cal c h a nge

f o r t he c r o p i n t er m s of p l ant pes t/ we e d p o t e n ti al and s u bs e qu e nt s t eps are not c o ns i d e r e d. If t he an s w er i s

“yes” or “unc e rtain”, t he s u b s equ e nt s t e p i s c ons i der e d.

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Steps 1 and 2

– Evaluate Detected Stati s tically Significant D iff e rences

Dat a o n eac h m easure d characteristi c ar e st ati s tically anal y z ed, where a ppropri a te, within

each indi v i d ual site and in a co m bined

- site analysis, in which the data are pooled among

sites. All stati s ti c ally signi f icant d i ff erences a re evalu a ted a nd consid er ed in the c o nte x t

of a change in plant pest/weed pote n tial. Differences detected in indivi d ual

- s ite a n alyses

that are not detected when data acr o ss m ulti p le environ m ents are pooled in the co m bined

site a n aly s is are consi d ered not biologically m eaningful in ter m s of plant pe s t/weed

potential a n d, therefore, are not further consi d ered in subsequent ste p s. Any difference

detected in the co m bined

- site analy s is is furt h er assessed.

Step 3

– Evaluate differences in the c o ntext of commercial reference materials

included in the Study

I f a differenc e fo r a characteristi c i s d e t e cted in the c o mbined

- site anal y sis across

multiple e n viron m ents, then the mean value of the biotechnology

- derived crop for t h e

chara c te r istic is asses s e d relative to the r a nge of variation of t h e com m ercial reference

m aterials included in the study ( e.g

. , reference range).

Step 4

– Evaluate Differences in t h e Context of the Crop

If the m ean value of t h e c h aracteristics for a b iotechn o lo g y

- deri v ed crop is o u tsi d e the

variation of the com m e r cial reference m ater i als included in the study, the m ean value of

the biotechnology

- derived crop is assessed relative to known values co m m on for the crop

( e.g.

, publishe d values).

Step 5

– Relevance of D iffere n ce to Plant Pest/Weed Pote n tial

If the m ean value of t h e c h aracteristics for a b iotechn o lo g y

- deri v ed crop is o u tsi d e the

rang e o f value s com m o n fo r th e crop

, th e diffe rence is then assessed for whether o r n ot it

is m eaningful in ter m s of plant pest/weed potential.

Step 6

– Conduct Risk Ass e ssment on Identi f

i e d Hazard

If an adverse effect (hazard) is ide n tified, risk assess m ent on the difference is conducted.

The risk assess m ent considers contributions to enhanced plant pest/ w eed potential of the

crop it s elf, the i m pact of differences detected in other measured c h aracteristics, and

potential for and effects of trait i n trogression into any populations growing outside of

cultivated e nviron m ents or into a s e xuall y

- co mpati b le spe c i e s.

VIII.B.2. Interpret a tion of Environmental Interactions Data

For the qualitative assess m ents of abiotic stress response, disease da m age, and arthropod

da m age, the biotechnology

- derived crop and conventiona l contro l ar e considered

different in susceptibility or tolera n ce if t h e range of injury sy m pto m s of each did not

overlap across all four repli c ations. Any observed differe n ces are assessed for biological

significa n ce in the context of the range of the commercial reference m aterials, and for

consistency in other observation ti m es and sit e s. Differences that are not consi s te n tly

observed in multiple en v iron m ents a r e consi d e r ed not b iologi c ally m eani n g f ul in terms of

plant pest potential.

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Quantit a tive assess m ents of corn ea r worm and European c o r n borer d amage a r e an al yzed

within individual sites and pooled across sites in a co m bined s ite analysis. Statistically

significant differences detected bet w een t h e biotechnology

- derived crop and conventional

contr o ls are evaluated u s ing the m ethod outli n ed in Figure VIII

- 1.

Quantitative assess m ents of arthropod abu n dance are only analyzed within each

individual site. Statistic a lly significant differences b e tween the biotechnology

- derived

crop and conventional control are assessed for biological significance in the context of

the range of the comm e rcial reference hybrids, and for consistency in other collection

ti m es and colle c tion s ites and in t h e conte x t of pest potential. Differences that are not

consistently detected in m ultiple environ m ents are c o nsidered n ot biolo g i c ally

m eaningful in ter m s of plant pest potential.

VIII.C. Comparative Assessments of the Phenotypic, Agronomic, and

Environmental Interaction Characteristics of MON 87411

Thi s sectio n provide s th e result s o f co m p a rative assess m ents cond u cted in r eplicated

labor a tory and/or m ulti

- s ite f ield expe r i m ents to provide a d e tailed phenot ypi c

,

agrono m ic, and environ m ental interac t ion s descriptio n o f MO N 87411

. The

characteristic s fo r MO N 8741 1 evaluate d i n these assess m ents included: seed

ger m ination and dor m ancy characteristics (Section VIII.C.1), plant p h e n otypic,

agrono m ic, and enviro nm ental interaction o b servations un d er field co n ditions (Secti o n

VIII.C.2), a nd pollen ch ara c te r isti c s (Se c ti o n VIII.C.3). Addition a l details f or each

assess m ent are provided in Appendices G, H, and I.

VIII.C.1. Seed Dormancy and Germination Characteristics

USDA

- APHIS conside r s the potential f or weediness to con s tit u te a pla n t pest f actor (7

CFR § 340.6). Seed ger m ination and dor m an c y m echanis m s vary with species and their

genetic basis tends to be co mplex. Seed dormancy ( e.g

., hard seed) is an i m portant

characteristic that is often associated with plants that are considered w eeds (Anderson,

1996 ; Lingenfelte r an d Hartw i g

, 2007)

. Infor m atio n o n ger m ination and dor m a ncy

characteristics is t h ere f ore useful when asse s sing a pla n t for increased wee d iness

potential. To assess g er m ination characteristics, standardized ger m ination assays are

available a n d routinely used. The Associati o n of Official Seed Analysts (AOSA), an

internationally recognized s eed testing organization, recommends a te m p erature range of

20/30 °C as opti m al for testing the ger m in a tion and dor m a ncy characteristics of m aize

seed (AOSA, 2012b; a). Additional te mp erature regi m es were also evaluated.

A co mparative assessment of seed ger m ination and dor m ancy characteristics was

conducted on MON 87411 and the conventional contr o l

. Th e see d lot s t o b e use d fo r the

ger m ination testing ( sel f ed F 2 grain) of MON 87411, the conventional control, and the

reference hybrids (4 per site, 11 unique across all locations) were produced in replicated

field trials during 2012 in Iowa (IA), Illinois (I L ), and Kansas (KS). These geographic

areas represent a broad range of environ m ental conditions for m aize production for this

product. T he plots were not treated with glyphosate. The ger m ination and dor m an c y

chara c teristics (as not e d in Table VIII

- 1 ) w e re assessed in seven s e parate split

- plot

experi m ents with four replications, o ne for each te m perature regi m e.

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Descriptions of the evaluated ger m ination a nd d o r m ancy characteri s tics a nd the ti m ing of

the evalu a tions f or all te m perature regi m es are listed in Table VIII

- 1. Addition a l details

on the m a t erials and experi m ental m ethods u s ed in this evaluation are presented in

Appendi x H.

In the co m bined

- site analysis, in which data were pooled across t h e three seed production

sites, no st at istically sig n i f icant d i ff erences ( α =0.05) were detected between MON 87411

and the co n ventional control for any chara c t e ristic a t the AOSA temperature r egime

(20/30 °C), or at the temperature regi m es of 5, 10, 20, 30, 10/20, 10/30 °C (Table VIII

- 2).

In addition, no hard seed were observed at any temperature.

The ger m ination and dor m ancy characteristics evaluated were u s ed to assess MON 87411

in the context of plant pest risk. The results of this asses sm ent, particula r ly the l a ck of

increase d har d seed

, an d n o change s i n oth er ger m ination and dor m ancy characteristics,

support the conclusion that the introductio n o f insect

- protec t e d an d glyphosate

- tolerant

tr a its is not expected to result in inc r eased plant pe s t/weed pote n tial co mpared to

conventional m aize.

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Table VIII

- 2. Germi n ation Characteris t ics of MON 87411 and the Conventional

Control

Te m p erature Assess m en t Mea n % (S.E. ) 1 Refer e nce R a n g e 2

(° C) Cate gory M ON 8 7 411 C on tr ol

5 Ger m inated 0

. 3 ( 0

. 1 4) 0

. 2 ( 0

. 1 1) 0

. 0

- 0

. 3

Viable Ha r d † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

Dead 3

. 0 ( 0

. 6 2) 2

. 2 ( 0

. 4 7) 1

. 3

- 5

. 3

Viable F i r m

- Swollen 9 6 .7 ( 0 .6 8) 9 7 .7 ( 0 .4 7) 9 4 .5

- 9 8

. 6

10 Ger m inated 8 2 .0 ( 2 .4 0) 8 3 .1 ( 2 .3 0) 8 6 .3

- 9 7

. 5

Viable Ha r d † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

Dead 0

. 9 ( 0

. 3 6) 1

. 0 ( 0

. 2 8) 0

. 5

- 3

. 8

Viable F i r m

- Swollen 1 7 .1 ( 2 .1 4) 1 5 .9 ( 2 .1 4) 0

. 0

- 1 1 .3

20 Ger m inated 9 8 .6 ( 0 .3 8) 9 9 .0 ( 0 .2 5) 9 5 .5

- 9 9

. 5

Viable Ha r d † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

Dead 1

. 3 ( 0

. 3 6) 1

. 0 ( 0

. 2 5) 0

. 5

- 4

. 5

Viable F i r m

- Swollen 0

. 1 ( 0

. 0 8) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

30 Ger m inated 9 9 .1 ( 0 .2 3) 9 8 .6 ( 0 .4 2) 9 2 .5

- 9 9

. 3

Viable Ha r d † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

Dead 0

. 9 ( 0

. 2 3) 1

. 4 ( 0

. 4 2) 0

. 8

- 7

. 5

Viable F i r m

- Swolle n † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

10 / 20 Ger m i na t ed 9 8 .7 ( 0 .2 6) 9 8 .3 ( 0 .4 7) 9 4 .8

- 9 9

. 3

Viable Ha r d † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

Dead 0

. 7 ( 0

. 2 2) 0

. 9 ( 0

. 2 9) 0

. 8

- 4

. 5

Viable F i r m

- Swollen 0

. 7 ( 0

. 2 8) 0

. 8 ( 0

. 2 8) 0

. 0

- 0

. 8

10 / 30 Ger m i na t ed 9 8 .8 ( 0 .3 7) 9 9 .3 ( 0 .2 5) 9 6 .3

- 9 9

. 5

Viable Ha r d † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

Dead 1

. 1 ( 0

. 3 1) 0

. 8 ( 0

. 2 5) 0

. 5

- 3

. 8

Viable F i r m

- Swollen 0

. 1 ( 0

. 0 8) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 3

20 / 30 N o r m al Germinated 9 8 .2 ( 0 .3 7) 9 8 .3 ( 0 .3 3) 9 3 .8

- 9 9

. 8

( A O S A) A bn or m al Ge r m inated 0

. 8 ( 0

. 2 4) 0

. 3 ( 0

. 1 4) 0

. 0

- 2

. 8

Viable Ha r d † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 0

Dead 1

. 0 ( 0

. 2 8) 1

. 3 ( 0

. 3 6) 0

. 3

- 4

. 3

Viable F i r m

- Swollen 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0

- 0

. 1

N o te : T he experi m e n ta l d esi gn wa s a s p lit

- p l ot wit h fo ur re p licati ons.

N o statisticall y sig n ifica nt d iffere n c e b etwee n MO N 8741 1 an d th e c onv e ntiona l contro l wer e detected

( α= 0 .0 5) us i ng A N OV A.

† N o statistica l c o m p aris on co u l d be m a de d u e t o lac k o f v a ria b ilit y i n t he data.

1 M ON 8 7 41 1 a nd t he c o nv e n t i onal c on t rol val ues r e p resent m eans w it h s t and a r d e rr or ( S .E.) i n

par e n t hes e s. n = 1 2. I n s o m e i ns t ances, t he t o ta l p e rcen t a ge o f bo t h MO N 8 74 11 a n d t he co n v e n ti onal

con t rol d i d n ot equal 1 0 0 % d u e t o nu m er i cal r ou nd i ng o f th e m eans.

2 Re ference r a n ge i s t he m i ni m u m and m a xi m u m m ean va l ues o bse r v e d a m ong t he 1 1 u n iq ue ref e ren c e

h yb r i d s.

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VIII.C.2. Field Phenotypic, Agronomic, and Environmental Interactions

Characteristics

Phenotypic and agron om ic characteristics, and e nviron m ental inter a ctio n s were ev a l u at e d

under field conditions as part of the plant characterization assess m ent of MON 87411.

These data were developed to provide U SDA

- APHI S wi t h a detaile d descriptio n of

MON 87411 relative to the conventional contr o l an d referenc e hybrids

. Accordin g t o 7

CF R § 340.6

, a s par t o f th e petitio n t o see k deregulation, a petitioner m ust submit “a

detailed description of the ph e noty p e of the re g ulated a r ti c le.” This in f or m ation is being

provided to assess whether there are pheno t ypic differences between MON 87411 a nd the

conventional control that m ay impact i t s plant pest/weed potential. Specific

characteristics that are related to weediness ( e

. g

. , lodging and ear drop) were used to

assess whether there is a potential increase in weediness of MON 87411 co mpa r ed to

conventional m aize. Environ m ental interactions including plant response to abiotic

stress, disease da m age, specific arthropod

- r elate d da m a ge

, an d a rthropod abundance were

also assessed as an indicat o r o f change s t o M O N 8741 1 an d ar e als o considere d i n the

plant pest assess m ent.

The results of the assess m ents of agrono m i c and phenotypic characteristics de m onstrated

that the introduction of the g l yphosate tolerant and insect protected traits did not

m eaningfully alter the p l ant pest/weed potential of MON 87411 co m pared to

conventional m aize. Further m ore, the lack of m e aningful differences in plant response to

abiotic stress, disease da m age, arthropod

- r e late d da m a ge

, an d a rthropod abundance also

support the conclusion that the introducti o n of the glyphosate tolerant and insect

prote c ted t r aits is not expected to result in increa s ed plant pest/weed potential for

MON 87411 co mpared to conventional m aize.

VIII.C.2.1. Field Phenotypic and Agronomic C h aracteristics

Data were collected at nine sites in t h e U.S. during 2012 to evaluate phenotypic and

agrono m ic characteristics of MON 87411 co m p a r e d t o th e convent ional contr o l (Table

VIII

- 3 ). These nine sites provi d e d a diverse range of environ m ental and agro n o m ic

conditions representative of comm e rcial m a i ze production areas in North A m erica. The

experi m ental design at each site was a rando m i z ed co mplete block with four replications.

At all sites, MON 87411, the conventional c o ntrol, and f our reference hybrids were

evaluated. A total of 22 unique reference hybrids were evaluated a m ong the nine sites

(Appendix I, Table I

- 1). The p l anted plot di m ensions var i ed b e tw e en sit e s, d u e to

varia b ility in available p l anting equip m ent (Appendix I, Table I

- 2). All plots of

MON 87411, the conventional control, and t h e reference hybrids at each site w ere

unifor m ly m anaged in order to assess wheth e r the intro d uction of i n sect

- p rotect e d and

glyphosate

- tolerant traits altered the pheno typi c an d agrono m i c characteristic s of

MON 87411 co mpared to the conventional control.

Description s o f th e evaluate d phenotypi c cha ra c teri s ti c s and the ti m in g o f th e evaluations

are listed in Table VIII

- 1. The materials, m ethods, details concerning the ti m ing of

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phenotypic assess m ents, and detailed results of the in d ivid u al

- s ite data co m parisons a r e

presented and discussed in Appendix I (Table I

- 4). The results of t h e co m bined

- site

analyses are summarized below.

In a co m bined

- site analysis in which the data were pooled a m ong the sites, no statistically

significa n t differences were detected ( α =0.05) between MON 87411 and the conventional

control for any of the assessed characteristi c s, including early stand count, 50% pollen

shed

, day s t o 50 % silking

, sta y gree n rat i ng, ear height, plant height, dropped ear count,

stal k lodge d plants

, roo t lodged plants, final stand count, grain m oisture, test weight, and

yield (Ta b le VIII

- 4). T h us, the phe n otypic and agrono m ic charact e ristics of MON 87411

were not altered in ter m s of pest/weed pote n tial co mpared to conv e ntional m aize (Figure

VIII

- 1, s tep 2, answer “ n o”).

Plant vigor data were summar i zed as ranges within individual sites. MON 87411 and the

conventional control w ere considered differ e n t i f th e rang e o f vigo r value s di d not

overlap across all four rep l ications. There were no d i fference s observe d between

MON 87411 and the conventional c o ntro l i n plan t vigo r acros s sites (Appendix I, Table I

4).

The phenotypic and agrono m ic characteristics evaluated in this study were used to

provide a detailed descripti o n o f MO N 8741 1 co m p are d t o t h e conventional control. A

subset of these characteristics was used to assess the weediness of MON 87411. The

results of the agronomic and phenotypic a s sess m ent d e m o nstrate that there were no

unexpected changes in the phenotyp e o f MO N 8741 1 compare d t o th e conventional

control when m anaged under the agrono m ic practices for m aize production. Thus, the

introduction of insect

- protected and glyphosate

- tolerant traits is no t expecte d t o resul t in

increased plant pest/weed potential f rom M O N 87411 co m p a red to conventional m aize.

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Table VIII

- 3. Field Phenotypic Evaluation Sites for MON 87411 during 2012

Site Code County, State

IABG Greene, Io w a

IARL Jefferson, Iowa

ILMN W arren, Illinois

INSH Boone, Indiana

KSL A Pawnee

, Kansas

NCBD Perqui m ans, North Carolina

NED C Butler

, Nebraska

NEYO York, Nebraska

PAH M Berks

, Pennsylvania

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Early stand count (#/plot) 82. 1 (1.59) 83. 4 (1.20) 72. 0

– 92.5

Days to 50% pollen shed 63.3 (0.64) 63.1 (0.59) 60.0

– 67.0

Day s t o 50 % silking 63. 4 (0.62) 63. 3 (0.54) 59. 3

– 66.5

Stay green rating (1

- 9 scale) 5. 6 (0.42) 5. 6 (0.40) 2. 8

– 9.0

Ear height (c m ) 104.1 (1.99) 103.6 (2.07) 87.4

– 132.2

Plant height (c m ) 238.3 (4.13) 239.1 (4.32) 204.4

– 262.6

Dropped ears (#/plot) 0.7 (0.29) 1.0 (0.42) 0.0

– 10.9

Stal k lodge d plant s (#/pl o t) 1. 2 (0.29) 1. 1 (0.39) 0. 0

– 5.7

Roo t lodge d plant s (#/plot) 0. 1 (0.06) 0. 2 (0.13) 0. 0

– 1.3

Final stand count (#/plot ) 3 65. 0 (0.96) 64. 3 (0.81) 60. 5

– 70.8

Grain m oisture ( % ) 4 17. 6 (0.38) 17. 9 (0.42) 12. 6

– 22.4

Test W eight (kg/hl) 5 72. 8 (0.57) 73. 2 (0.64) 68. 2

– 77.7

Yield (Mg/ha) 6 11. 1 (0.44) 10. 9 (0.44) 7. 4

– 15.8 Table VIII

- 4. Combi n ed

- Site Comparison o f MON 87 4 11 to Conventional C o ntrol

for Phenotypic and Agronomic Characteristics During 2012

Phenotypic Characteri s tic Mean (S.E. ) 1 Reference

Rang e 2

(units) MON 87411 Control

Note: The experi me ntal design was a rando m ized c o m plete block with four rep l icates per site.

No statistica l ly significant differences were detected between MON 87411 and the conven t ional

control (α=0.05) using ANOVA.

1 MON 87411 and the co n ventional control values represent mea n s with s t andard error (S.E.) in

parentheses. n = 36, except where noted.

2 Reference range is calculated from the mini m u m a n d m a xi m u m mean values from among the 2 2

unique reference hybrids.

3 Final stand count was excluded from a single re p lication of MON 87411 at the NEDC site

because it was identified as an outlier. n = 35 for MON 87411; n = 36 f or the convent io nal

control.

4 Grain m oist u re was excluded from a single replic a tion of the co nve ntional cont r ol at the IARL

site because it was collec t ed incorrect l y

. n = 36 f or MON 87411; n = 35 for the c onventional

control.

5 Test weight (kg/hl

= ki logra m /hectoliter) w as exclu d ed from a single replication of the

conventional control at two sites (IARL and PAHM). n = 36 f or M ON 87411; n = 34 for the

conventional control.

6 Yield (Mg/ h a = me gagram/hectare) data w as dropped from a single replication of the

conventional control at the IARL site because the shelled plot wei g ht was collected incorrectly.

n = 36 for M ON 87411; n = 35 for the c onventi o nal control.

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VIII.C.2.2. Environmental Inter a ction Characteristics

USDA

- APHIS consid e rs the enviro nm ental i n teraction of the biotechnology

- derived crop

co mpared to its conventional control to deter m ine the potential for increased plant pest

characteristics. Evaluations of environ m ent a l interactions were conducted as part of the

plant characterization for MON 87411. In the 2012 U.S. field trials conducted to

evaluat e th e phenotypi c an d agrono m ic characte ristic s o f MO N 87411

, data were also

collected on plant response to abi o tic stre s s ( e.g.

, drought, wind, nutrient deficiency, etc.),

disease da m age, arthropod

- related da m age, and arthropod abundance ( T ables VIII

- 5 and

VIII

- 6 ; Appendix I; Ta b les I

- 5, I

- 6, I

- 7, I

- 8, I

- 9, and I

- 10, re s pecti v ely). These data were

used as part of the environ m ental analysis (Se c tion IX) to assess plan t pes t potential

co mpared to the conventional control and prov i de confi m atory data for the conclusion of

laboratory NTO testing data (Section VI.E.). T he results of the field evaluations showed

that insect

- protected and glyphos ate

- toleran t trait s di d no t u n expectedly alt e r th e assessed

environ m ental interactions of MON 87411 co mp ared to the conventional control. The

lac k o f significan t biologica l difference s i n plan t response s t o a b ioti c stress

, disease

da m age, arthropod

- related da m age, and pest

- an d beneficial

- arthropo d abundanc e support

th e conclusio n tha t th e introductio n o f th e ins ect

- protected and glypho sate

- toleran t traits

is not expected to result in increased pla n t pest potential from MON 87411 co m pa r ed to

commercial m aize.

VIII.C.2.2.1. Qualit a ti v e Environme n tal Inter a ctions Assessment

Plant responses to a b iotic stressors, disease damage, a n d arthropo d damag e wer e assessed

at nat u r a l l e vels, i

. e

., n o artificial infestation o r i mposed abiotic stres s ; therefore the s e

levels typically varied between observations at a site and a m ong sites. Plant responses to

abioti c stress

, diseas e da m a ge

, an d arthropo d da m age data were coll e cted from each plot

using a categorical scale (none, slight, m oder a te, and severe) of increasing severity of

observed da m age for each stressor. This scale w as utilized to allow for the eval u ati o n of

the wide variety of potenti a l abiotic stressor, disease damage

, an d arthropo d damage

sy mpto m s potentially occurring across the s ea s on and across sites. These data were

categ o rical and therefore were s u mmarized and not subjected to A NOVA. For a

particular stressor, all comparisons of the range of responses for MON 87411 to the range

of responses for the conventional control across all observation ti m es and sites are

reported.

Descriptions of the ev a l u ated en v iro nm ental inte r actio n s ch ar act e ri s tics a nd the ti m ing of

th e evaluation s ar e liste d i n Tabl e VIII

- 1

. T he m aterials, m ethods, additional details

concerning the qualitative environ m ental inter a ctions assessments, and detailed results of

the qualitative data comparisons are present e d and discussed in Appendix I (Tables I

- 5

throug h I

- 7).

In the qualitative assess m ent, no differences in the range of responses were observed

betwee n MO N 8741 1 a n d th e conventiona l con trol for any of the 100 co m parisons of

plant response to abiotic stressors, inclu d in g cold

, drought

, flood

, frost, hail, heat,

nutrient deficiency, soil co m paction, sunsca l d, and wind (Table VIII

- 5 and Appendix I;

Table I

- 5). Additionally, no differences in t h e range of responses were observed between

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MON 87411 and the convention a l control for any of the 119 co m parisons for plant

da m age caused by diseases, including anthracno s e, bacterial leaf spot, ear rot, eyespot,

Fusarium sp., Goss’s bacterial wilt, gray leaf s p ot, leaf blight, m aize rough dwarf virus,

Pythium sp.

, Rhizocto n ia sp.

, rust

, seedlin g blight

, s m ut

, stal k rot

, an d Stewart’ s bacterial

wilt (Table VIII

- 5 and Appendix I; Table I

- 6 ). Finally, n o di ff erenc e s in the ra n ge of

responses w ere observed between MON 87411 and the conventional control for any of

the 102 comparisons for plant da m age caus e d by arthropods, inc l uding aphid, ar m yworm,

billbug, cutwo r m

, corn earwor m

, corn flea bee t le, rootworm beetle, European corn borer,

grasshopper

, Japanes e beetl e

, sa p beetle

, spide r m ite

, stink bug, and wireworm adult

(Table VIII

- 5 and Appendix I; Table I

- 7).

The lack of differences observed between MON 87411 and the conventional control for

plant responses to abiotic s t ressors, disease da m age, and arthropod

- related da m age in

multiple en v iron m ents across t h e U.S. supports the conclu s i o n that the i n troduction of the

insect

- protected and glyphosate

- tolerant traits is not expec te d t o caus e a biologically

m eaningful change in ter m s of plant pest po t e n tial co m pared to the con v entional co n trol

(See Section VIII.B.2.).

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Table VIII

- 5. Summary of Qualitative E n vironment a l Interacti o ns Assessments

during 2012

Stressor N u m ber of

observations

acros s al l si t es N u m ber of observations with

no differences between

MON 87411 and the

conventional control across

all site s 1

Abiotic stressors 100 100

Disease dam a ge 119 119

Arthropod

- re la ted da m a ge 102 102

Total 321 321

No te : T he e xpe r i m e n t al des i gn was a r a n do m i zed co m p le te b l o c k w i t h fo ur r e p li ca t es p e r s ite

. No d i ff e rences w e re obs e r ved be t we e n M O N 8 74 11 a n d t he c o n v e n tio n a l con t rol d ur i n g a ny o bs e rva t i on

for da m a ge caus e d by any of the assess e d stressors.

1 MO N 8 7 41 1 a nd t he co nv e n t i on a l c on t r ol wer e c ons id ere d d iffere nt i n s u sce p ti b ilit y or t o lera n c e if

t he r a n ge of i nj ury s y m p t o m s acro s s fo ur r e pli ca ti ons d i d n ot o v e r la p be t ween MO N 8 7 4 1 1 a n d t he

co n v e n ti onal c on t ro l

.

VIII.C.2.2.2. Quantit a tive Enviro n me n tal I n teractions Assessme n t

Quantitative arthropod assess m ents on corn earwo r m (CE W : Helicoverpa ze a ) d a m age,

European corn borer ( E CB: Ostrinia nubilali s ) da m age, and arthropod abundance were

conducted at four sites (IABG, NEYO, N C BD, and PAH M ). CEW and ECB d a mage

was assessed once during the growing sea s on at each site. Arthropod abundance was

assessed f r o m colle c tio n s per f or m ed f ive ti m es during the growing sea s on at each s ite

using sticky traps and visual counts.

D a m age data were collected for CEW and ECB at the four sites in 2012. Both individual

and co m bined

- site (the four site pooled data) analyses were conducted. Descriptions of

the evaluated environ m ental interactions cha r acteristics and the ti m ing of the evaluations

ar e liste d i n Tabl e VIII

- 1

. Th e materials

, m e thods

, additiona l detail s concernin g the

specific arthropod da m a ge assess m e nts, and d e tailed results of the individual

- sit e data

co mparisons are presented and discussed in Appendix I ( T able I

- 8). The results of the

co m bined

- site analysis are sum m arized below.

In the combined

- s ite analysis, no statistically significa n t differences ( α =0.05) were

detected between MON 87411 and the conventional control for plant da m age caus e d by

corn earw o rm or European corn borer (Ta b les VI I I

- 6). Considering this l a ck of

significa n t differences, the results i n dicate no changes in su s cepti b ility o r resi s tan c e of

MO N 8741 1 t o thes e commo n m a i z e pests. (See Figure VIII

- 1, Step 2, answer “no”).

Arthropod abundance w as assessed from collec tion s perfor m ed usin g stick y trap s and

visual counts at the four sit e s in 2012. Variations in te m poral activity and geographical

distribution of arthropod taxa occurred bet w een sites, therefore, only individual

- site

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analyses were conducted for arthropod abu n dance data. Statistical analyses and

significance testing of differ e nces were only perfor m ed f o r the arthropods present in

sufficient nu m bers to allow m ore robust statistical analysis. An i n clusion criterion was

established where a given art h ropod m ust have an average coun t pe r plo t pe r collection

ti m e (acro s s all m aterials) of ≥ 1. Additional details of the arthropod abundance

assess m ents and detailed results of the in d ivid u al

- s ite d ata co mparisons are provided in

Appendix I (Tables I

- 9 and I

- 10). The results of these analyses are s u mmarized b elow

and in Table VIII

- 7.

In an assess m ent of arthropod abundance from sticky traps, a total of 108 statistical

co mparisons were m ade between MON 87411 a nd the conventional control for arthropod

abundance involving the following arthropods: aphid, corn flea beetle, delphacid

planthopper, green lacewing, l a dybird beetle, leafhopper, m i cro

- parasitic hy m enoptera,

m acro

- parasitic hy m enoptera, sap beetle, m inute pirate bug, and spider. Lack of

sufficient arthropod abundance precluded s t atistical co mparisons betwee n MO N 87411

and the conventional control for 79 addit i onal co mparisons; ho w ever, descriptive

statistics were provided for these comparison s (Append ix I ; Tabl e I

- 9).

No statistically significant differences ( α =0.05) were detected between MON 87411 and

th e conventiona l contro l fo r 10 4 ou t o f 10 8 comparisons

. T he fou r difference s detected

betwee n MO N 8741 1 an d th e conventiona l contro l include d aphid

, delphacid

planthopper, m inute pirate bug, and spider (Table VIII

- 7 and Appendix I; Table I

- 9). The

abundance of aphids was higher in MON 87411 than the conventional control in

Collection 3 (1.8 vs. 0.0 per plot) at the NEYO site. The m e a n value for aphid abundance

on MON 87411 was within the range of the referenc e hybrids

. Th e abundanc e of

delphaci d planthopper s wa s lowe r i n M O N 8741 1 tha n th e conventiona l contro l in

Collection 1 at the IABG site (0.0 vs. 1.8 per plot). MON 87411 had higher abundance

of spiders than the convent i onal control (3.3 vs. 0.3 per plot) in Collection 5 and lower

abundance than the conventional control for m inute pirate bugs (1.0 vs. 3.0 per plot) in

Collection 1 at the PAHM site. The m ean values for delphacid planthopper, m inute

pirat e bug

, an d spide r abundanc e o n MO N 8741 1 were outside the r espectiv e range s of

referenc e hybrids

. However

, thes e differen ces were not consistently detected across

coll e ctio n s or sites (T a ble V III

- 7 and Appendix I; Table I

- 9). Thus, these differences in

aphid, delphacid planthopper, m inute pir a t e bug

, an d spide r abundanc e w e r e not

indicativ e o f a consisten t re spo n se associated with t h e trait and are not con s i d ered

biologically m eaningful in t e r m s of i ncreased pest potential of MON 87411 co mpared to

the conve n tional control (See Section VIII.B.2.).

I n a n assess m en t o f arthropo d abundanc e fro m visua l counts

, a tota l o f 6 1 statistical

co mparisons were m ade between MON 87411 a nd the conventional control for arthropod

abundance involving the following pest and beneficial arthr o pods : ant

- lik e flowe r beetle,

click beetle, corn flea beetle, ladybir d beetl e adult

, ladybir d beetle larvae, m inute pirate

bug, sap beetle, shining flower beetle, a n d spider. Lack of sufficient arthropod

abundance precluded statistical co m pari s on s betwee n MO N 8741 1 an d th e conventional

control for 152 additional co m parisons; howeve r

, the des c ri p tive st atistic s wer e provided

for these comparisons (Appendix I, Table I

- 10).

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No statistically significant differences ( α =0.05) were detected between MON 87411 and

the conventional control for 60 out of 61 c o mparisons. One difference was detected

betwee n MO N 8741 1 an d th e conv entional control for ant

- li k e flower beetle (Table

VIII

- 7 and Appendix I; Table I

- 10). The abunda n ce of ant

- like flower beetles was lower

on MON 87411 than the conventional control in Collection 2 at the IABG site (0.8 vs. 4.3

per plot). The m ean value for ant

- like flower beetle abundance on MON 87411 was

lowe r tha n th e rang e o f referenc e hybrid s (1. 8

– 3. 3 pe r plot)

. However

, th e differen c e

for ant

- like flower beetle was not c onsiste n tly dete c ted a cr oss collecti o ns at the IABG

site (Ta b le VIII

- 7 and Appendix I; Table I

- 1 0 )

. Thus, this difference in ant

- li k e flower

beetle abundance was not indicati v e of a consistent response associated w ith the trait and

is not considered biologically meaningful in ter m s of increased pest pot e nti a l

of MON 87411 co m pa r ed to the conve n tional control (See Section VIII.B.2.).

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Table VIII

- 6 Combined

- Site Comparison of Pest Dama g e to MON 8 7411 Compared to the Conventional Control during 2012

Mean (S.E. ) 1 Reference

Pest Arthro p od D a m age Assess m ent MO N 87411 Control range 2

Corn earworm

( H. zea ) D a m age area (c m 2 ) of 10 plants per plot 1.9 (0.47) 1.3 (0.30) 0.2

– 3.2

European corn borer Nu m ber of s talk galleries of 10 pla nt s pe r plot 0. 4 (0.16) 0. 5 (0.21) 0. 0

– 1.8

( O. nubilali s )

European corn borer Stalk gallery length (c m ) o f 1 0 plant s pe r plot 1. 6 (0.72) 2. 0 (0.94) 0. 0

– 8.3

( O. nubilali s )

Note: The experi me ntal design was a rando m ized c o m plete block with four rep l icates per site.

No statistical l y significant differences were detected between MON 87411 and t h e convention a l control (α=0.05) using ANOVA.

1 MON 87411 and the conventional control values represent means with standard error (S.E.) in parenthese s

. n = 16.

2 Reference range is calculated from the mini m u m a n d m a xi m u m mean values from a m ong 15 un i que reference h y br i ds across four sites (IABG,

NCBD, N E Y O

, and PAHM).

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Table VIII

- 7. Summary of Arthr o pod Abundance A ssessments and Detected Differences d u ring 2012

Summary of Statistical C o m p arison s 1 Summar y o f Detec t e d Dif f erenc e s 2

N u m ber of Consistently

Arthropod N u m ber of c o m parisons where Within detecte d acr o ss

Abundance N u m b er c o m parisons no differences were Collection reference collections or

Ass e s s ment of sites across si t e s detected Arthropod Site N u m b er range? sites?

Sticky Traps 4 108 104 Aphids NEYO 3 Yes No

Delphacid

plantho p per

Minute pirate b

Spiders IABG 1 No No

ug PAHM 1 No No

PAHM 5 No No

Visual Counts 4 61 60 Ant

- like flower

beetle IABG 2 No No

1 Q u a n titati ve art h ro p o d a b u nd a n c e assessme n t s wer e statistica l l y a n a l yze d a t α =0. 0 5 us i ng A NO V A

. Lack of su f f i cie nt ar t h ro p o d a bu n da nce p rec lud e d

statistica l c o m p aris o n s b etwee n MO N 87 4 1 1 an d t he con v e n tio n a l co n t r ol f or a dd itio n a l 79 c o m p aris ons (stic ky tra p s ) a nd 1 5 2 c o m p aris o n s ( v is u a l cou n ts);

h o w e ve r

, desc ri p ti ve s t a ti s tic s w e re p r ov i ded f or t hese c o m par i s o n s i n Ap pe n d i x I (T a b l e s I

- 9 a n d I

- 10 ).

2 Fi ve statisticall y si gn ifica nt d iffere n ce s w e re de t ec t e d. T hese d i ff e r e n c es are f u r t h e r d i scus s e d i n S ec ti on V I II. C

. 2

. 2

. 2 us i n g t he ap p r o ac h o u tli n e d i n Section

V I II. B .2 .

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VIII.C.3. Polle n Characteristics

USDA

- APHIS consid er s the pot e nti a l f or gene flow and introgression of the

biotechnology

- derived trait(s) i n to sexually c o m patible p lants and wild r e lati v es to

deter m ine the potential for increa s ed weedy or inva s ive c hara c te r isti c s of the re c eivi n g

species. Pollen m orphology and viability infor m ation are pertinent to this assess m ent

and, there f ore, were assessed for MON 87411. In addition, m orphological

characterization of pollen produced by M ON 87411 and the conventional control is

rele v ant to the plant pest risk asses sm ent beca u se it adds to the detailed descri p tion of the

phenotype of MON 87411 co mpared to the conventional control.

The viability and m orphology of pollen collec t ed from M ON 87411 co mpared to that of

the conventional control was also assessed. Polle n wa s collecte d fro m MO N 87411

, the

conventional control, and four commercial references grown under si m ilar agrono m ic

conditions in a field trial in Iowa, a geographic area that repre s ents environ m entally

relevant conditions for m aize production for t h is product. The tri a l wa s arrange d i n a

rando m ized co mplete block design with four replications

. Onc e al l plant s acros s the

replications reached the flowering s t age, pollen was collected from three non

syste m atically selected plants per p lot and s tained for a s sess m ent. Description s o f the

evaluated pollen viability and morphology characteristics and the ti m ing of the

evaluations are listed in Table VIII

- 1. The d e tails of the m aterials and experi m ental

m ethods used in this evaluat i on are presented in Appendix J.

No statistically significant differences ( α =0.05) were detected between MON 87411 and

the con v entional co n tr o l f or perce n t viable poll e n o r polle n grai n dia m ete r (Tabl e VIII

- 8).

Further m ore, no visual differences in gen e ral pollen m orphology were observed bet w een

MON 87411 and the conventional control (Appendix J, Figure J

- 1).

The pollen characterization data c o ntrib u te to the det a i l e d phenotypi c descriptio n of

MON 87411 co mpared to the convention a l control. Based on the assessed

characteristics, the results support a conclusion that neither pollen via b ilit y nor

morphology of MON 87411 were altered c o mpared to conventional m aize.

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Table VIII

- 8. Pollen Characteristics of MON 87411 Compared to t h e Conventional

Control during 2012

Pollen

Characteri s tic Mean (S.E. ) 1

Reference Rang e 2

(unit) MON 87411 Control

Viabilit y 3 (%) 98. 7 (0.43) 98. 9 (0.21) 99.0

– 99.2

Dia m eter 4 (μ m ) 87. 0 (1.02) 88. 9 (1.29) 80.4

– 88.9

Note: The experi me ntal design was a rando m ized c o m plete block design with f our replicati o ns.

No significant differences were detected bet w een the MON 87411 and the conv e ntional cont r o l

(α=0.05) using ANOVA.

1 MON 87411 and the conventional control values represent means with standard error (S.E.) in

parentheses. n=4

2 Reference range is the m i ni m u m and maxi m u m mean value observed a m ong the four reference

h y br i d s.

3 Evaluated f r om three sub

- sa m ples per replication at 100X m a gnification.

4 Evaluated f r om 10 representative viable pollen grai n s per replication at 2 0 0X magnification.

VIII.D. Conclusions for Phenotypic, Agr o nomic, and Environmental Interactio n s

Evaluation

Co mp arative plant characteri z ation data between a biote c hnology

- derived crop and the

conventional control are interpr e ted in the context of contributions to increased plant pest

potentia l a s assesse d b y U S DA

- APHIS

. Unde r th e fr a m ewo r k o f f a m i l i arity,

characteristics for which no differences are d e tected support a concl u sion of no increased

plant pest potential of the biotechnology

- der i ved crop co m p ared to t h e conventional crop.

Ulti m ately, a weight

- o f

- evidence a p proach th a t conside r s all chara c te r i s tics and data is

used for the overall risk assess m ent of differences and their significance.

An extensive and robust set of agrono m ic, phenotypic, and environ m ental interactions

data, including specific weedy and plant pest pote n ti a l ch a ra c te r istics, we r e used to as s ess

whether the introduction of insect

- protected and glyphosate

- tolerant trait s altere d the

plant pest potential of MON 87411 co m pared to the conventional control, considered

within the context of the variation a m ong co m m ercial r e ference hybrids. These

assess m ents included five general data cat e gories: 1) seed ger m ination, dor m an c y, and

e m ergence; 2) vegetative growth; 3) r e productive develop m ent (including pollen

characteristics); 4) lodging a nd seed retention on the plant; 5) plant response to abiotic

stres s an d interaction s wit h diseases and arthropods. W ithin thes e dat a categories

, data

relevant to understanding specific characteristics associated with weediness were also

assessed to deter m ine whether there was a po t ential increase in w eediness of MON 87411

co mpared to conventional m aize.

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Results from these assess m ents comparing MON 87411 a nd the conventional control

de m onstrate that MON 87411 does not possess: 1 ) increase d weedines s characteristics;

2) in c rea s ed susceptibility or tol e ra n ce to s p ecific abi o tic stress, di s ease s

, or arthro p ods;

or 3) characteristics that would confer a pla n t p e st risk co mpared to con v ention a l m aize.

Therefore, b ased on the results of multi p le a s s e s s m ents discussed above and prese n ted in

th e appendices

, th e weight

- of

- evidenc e ind icates that MON 87411 is not m eaningfully

different from conventional m aize with the exception of insect

- p rotected and glyphosate

tolerant traits and is not expected to pose a plant pest risk co mpared to conventional

m aize.

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IX. U.S

. AGRONOMI C P RAC T IC E S

IX.A. Introduction

As part of the plant pest assessment r e quired by 7 CFR § 340.6(c)(4), i mpacts of

deregulation on agricultural and cultivation pr a cti c es m ust be conside r ed. This secti o n

provides a sum m ary of current a g r o no m ic practices in t h e U.S. and North A m erica for

producing m aize, and is included in this petiti o n a s a baselin e t o a sses s possibl e i mpacts

to agricultural practices due to the culti v ation of MON 87411. Discussions include m aize

production, plant growth and develop m ent, general m anage m ent practices during the

season, m anage m ent of weeds, insects and dis e ases, m aize rotational crops, and volunteer

m anag e m en t

. Infor m ation presented in t h e previou s sectio n demonstrate d that

MON 87411 is no m ore susceptible to diseases or pests than com m ercially cultivated

m aize. Additio n ally, data presented in Section VIII s h ow that M ON 87411 is not

expected to pose a plant pest risk compared to conventional m aize. Current CR W

- control

products available in the U.S. wer e grow n o n ove r hal f o f the m aize acres in 2011 (Marra

et al., 2012). MON 87411 is protected from CRW feeding using two different m od e s of

action derived from Cry3Bb1 protei n an d DvSnf 7 dsRNA

. Thus, there are no expected

changes to the inputs needed for MON 87411, and no expected i mpacts to agrono m ic

practices employed for production of m aize co mp ared to the current situation.

IX.B. Overview of U.S. Mai z e Production

IX.B.1. Mai z e Produc t ion

The U.S., China, Brazil, Argentina, and M e x ico are the top five countries producing

m aize globally (USDA

- FAS, 2013). As noted in Section I.B., m aize ( Ze a may s L.) is the

large s t crop grown in the U.S. in ter m s of acreage pla n ted (97.2 m illion acres in 2012)

(USDA

- NASS, 2013d), exceeding soybean a n d wheat with acrea g es of 77.2 and 55.7

m illion acres, respecti v ely (USDA

- NASS, 2013a). The value of m aize reached $ 77.4

billion in t h e United States in 2012, exceed i ng soybeans and wheat with values of $43.2

and $17.9 billion, respectively (U S DA

- NASS, 2013c )

. Th e principa l use s o f m a iz e are

fee d an d residual

, ethano l fuel

, export

, an d high

- fructos e c orn s yrup (Capehart, et al.,

2012).

Th e plantin g o f 97. 2 millio n acres of m aize in 2012 was t h e largest acrea g e since 1936

(Table IX

- 1 ) (USDA

- NASS, 2013d) and was up over 5.2 m illion acres from 2011. Much

of that production occurs in upper Midwest states ( F igure IX

- 1)

. O f th e 201 2 acreage,

approxi m ately 87.4 m illion acres were harvested for grain and 7.4 m illi(Gass m ann, et al

. , 2012)on acres were harvested for silage (USDA

- NASS

, 2013d)

. Tota l productio n was

approxi m ately 10.8 billion bushels with an a verag e yiel d o f 12 3 bushel s pe r acr e (Table

IX

- 1). The value of m a ize grain product i on in the U.S. has ranged from $46.7 to $77.4

billio n i n th e pas t 6 year s ( T able IX

- 1) (USDA

- NASS, 2013d).

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Table IX

- 1. Mai z e P r oduction in the U.S., 2007

- 2012

Year Acres

Planted

(×1000) Acres

Harvested

(×1000) Average

Yield

(bushels/acre) Total

Production

(×100 0 bushels) Value

(billion s $)

2012 97,155 87,375 123.4 10,780,296 77.35

2011 91,936 83,989 147.2 12,359,612 76.94

2010 88,192 81,446 152.8 12,446,865 64.64

2009 86,382 79,490 164.7 13,091,862 46.73

2008 85,982 78,570 153.9 12,091,648 49.31

2007 93,527 86,520 150.7 13,037,875 54.67

Source: ( U SDA

- NASS, 2013d)

Figure IX

- 1. Planted Mai z e Acr e s by County in the U.S. in 2012

Source: ( U SDA

- NASS, 2012)

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IX.C. Production Ma n agement Consideratio n s

Other than the specific insertion of a parti a l coding sequence of t h e DvSnf 7 gen e that

provides another MOA for protection again s t CRW, MON 87411 is si m ilar to several

other eve n ts present in CR W

- protected ma ize hybrids being grown in the U.S. ( e.g.

, Genuity SmartStax, Genuity VT Triple PRO, etc). Herbi c ide tol e rant m aize has been in

use commercially in the U.S. since 1997 and constit u ted 7 3 % of the total m aize crop in

2012 (USDA

- ERS, 2013). CR W

- protected m aize has been planted commercially since

2003 and in 2010 constituted 53% of the tot a l U.S. crop (Brookes and Barfoot, 2012).

Give n th e widesprea d us e o f these HT and i n sect

- p rotected m aize h y brids, Mon s anto

anticipates no specific production m anag em ent changes from introduction of

MON 87411 above and beyond those in current u s e on a m ajority of U.S. m aize acres.

IX.D. Management o f Ins ect P e s ts

Maize is subject to attack by nu m erous insects and global production losses from

arthropod pests are m ini m ally esti m ated at 12 %

, wit h potentia l losse s bein g m u c h higher

(Culy, 2000; Vaadia, 1985). Approxi m ately 100 s p ecies of insects cause most of the pest

da m age to m aize production in the U.S. (O'D a y

, e t al.

, 1998)

. Cert ain insect pests are

abundan t almos t ever y yea r an d ten d t o b e c h roni c problem s whil e othe r pes t infestations

are sporadic and occur only every 5 to 10 y e ars with a severity great enough to reach

econo m ic thresholds (O ' D ay et al., 1998). For m igratory pests, weather events m ust

favor the move m ent into m aize

- producing r e gions. In addition, m aize or other suitable

host plants must be a v aila b le and in the pre f erred growth stage when the inse c ts a rrive

and local weather conditions m u st also be favorable for t h e i n sects to survi v e a n d

reproduce. So m e of the m ost econo m ically i m portant m igratory m aize pests in the U.S.

include black cutwor m

, fall ar m yworm and corn earworm ( O 'Day et al., 1998). Other

econo m ically i m portant pests such as European corn borer and corn rootwo r m overwinter

in the field (O'Day et al., 1998). Table IX

- 4 lists the m ost important insect pests in m aize

in the North Central states of the U.S.

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Table IX

- 2. Insect Pests in Mai z e in the North Central States and Typical Time of

Damage

Typical Time of Da m a ge Growth Stage C o m m on Name of Insect Pest

Planting to F ull E mergence Planting to V2 Seed corn maggot

Seed corn beetles

Wirewor m s

E mergence to Knee

- High VE to V8 Wirewo r m s

White grubs

Grape colasp i s larva

Cinch bugs

Black cutwo r m

Stalk borer

Thrips

Corn leaf beetle

Sod webworms

Southern corn leaf beetle

Billbugs

A r my worms

Knee

- High to Tasseling V8 to VT Fall a r m y worm

Grasshoppers

European corn borer

Southwestern corn borer

Corn rootwo r m

Tasseling to Maturity VT to R6 Corn leaf aphid

Corn earworm

Source: (Dicke and Guthrie, 1988; O'Day et al., 1998)

Insect pests are best m anaged through the u s e of integrated pest m anage m ent (IPM)

progra m s where all v ia b le contr o l strate g ies are considered an d appropriat e strategie s are

selecte d fo r us e agains t specifi c insec t pes t s ( B radley

, e t al.

, 2013)

. Contro l strategies

e mployed in IPM progra m s m ay include che m i cal, c u ltural, m echanical, biological and

genetic options ( i.e

. , use of biotechnology

- derived or naturally occurring resistant or

tol e rant c rop vari e tie s ). Al m ost all m aize seed includes a treat m ent of an insectici d e such

as thia m ethoxam or clothianidin for contr o l or suppression of several seedling pests

including white grubs, wirewor m s, seed c o rn maggots, cinch bugs, black cutwor m

, and

billbugs ( B radley et al., 2013; University of Tennessee, 2013). Applications of

inse c ti c ide f or contr o l o f various maize i n se c t pests has ranged from 12% to 31% of the

planted acreage over the past 20 years (USDA

- NASS, 2013b) In 2010, however, only

12 % o f plante d acre s wer e t r eate d wit h a n insecticid e (USDA

- NASS

, 2013b)

. Nu m e r ous

inse c ti c idal acti v e ing re dients a re r egistered for use in m aize for the contr o l of insect

pests. However, only seven insecticide acti v e ingredie n ts ( bi f enthrin ( 2 %), chl o rp yri f os

(1%), cyfluthrin (2%), la m bda

- cyhalothrin ( 2 %), per m ethrin (1%), tebupiri m phos (2),

and te f luth r in (3% ) ) r e present a lmost all of the total tr e ated m aize acres in t h e U.S.

(USDA

- NASS, 2013b). These insecticides are effective in m anaging insect pests that

m ay cause da m age to maize from e m ergence through m aturity (Table IX

- 5) (Bradley et

al., 2013). Other IPM practices t ha t ar e use d includ e rotatio n t o non

- hos t crop s t o break

pest life cycles, soil tillage to m anage we e d, arthropod and disease pests, and r e m oval of

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crop residues that can harbor both arthropod and disease organis m s (Vasileiadis, et al.,

2011). Additionally, s om e researchers have e v aluated and found effective the u s e of

specific be n eficial n e m a todes for biological co n t rol of spe c i f ic m aize pest s (Kurtz

, e t al.,

2007).

Table IX

- 3. Insecticide Applicati o ns in Mai z e in 2010 in the U.S.

Herbi c ide Chemical F amily Mode of Action

(MOA) Percent of

Mai z e Acres

Treated Quantity

Applied

(1000 lbs ai)

Bifenthrin P y reth r oids

Sodium channel

m odulators 2.0 68

β

- C y fluthr i n P y reth r oids 2.0 15

La m bda

C y halothrin P y reth r oids 2.0 24

Per methrin P y reth r oids 1.0 72

Tefluthrin P y reth r oids 3.0 242

Z

- C y per m et h rin P y reth r oids 2

Chlorp y rifos Organophos p hates

Acet y l cholin

ester a se inhibitors 1.0 478

Tebupiri m phos Organophos p hates 2.0 195

Tebufos Organophos p hates 137

Di m e thoate Organophos p hates 52

Propargite Propargite Inhibitors of

mitochondrial ATP

s y nthase 109

Spiro mesifen Tetronic & tetra m ic

acid derivatives Inhibitors of acet y l

CoA carboxylase 59

Total 12 1631

Source: ( U SDA

- NASS, 2013b).

The m ost da m aging root

- feeding pests of m a i ze in the m ajor U.S. m aize growing regions

are larvae of the corn rootw o rm co m plex (CR W : Diabrotica ssp.

, Coleoptera;

Chryso m elidae) (Chandler et al., 2008). There are three pri m ary species of CRW in the

U.S.

, n a m e l y wester n cor n rootwor m ( WCR : Diabrotica virgifera virgifer a )

, northern

corn ro o tworm (NCR: Diabrotica barber i ) an d souther n cor n rootwor m (SCR:

Diabrotic a undecimpunctat a howard i ) and all are con s idered ma jor ins e ct pe s ts of m aize

(Culy, 2000). In the Midwest region, the W CR is the do m inant species while the S CR is

not considered an econo m ic pest there (O ' D ay et al., 1998). Corn rootwor m s can c a use

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seriou s injur y an d yiel d loss es in the following ways: 1) roo t prunin g an d tunneling

disrupting the transport of nutri ent s an d wate r fro m th e roo t syste m

, 2) lack of root

support causing gooseneck lodging, which can co m p licat e harvesting

, an d 3 ) roo t feeding

pro m otes invasion by secondary pathogen s suc h a s bacte r i a an d fungi

, increasin g the

incidence of root rots ( O' D ay et al., 1998). Yield losses depend on the nu m ber of larvae

per plant and on plant m atu r ity, soil fertility, and a m ount of m oisture following peak

injury, as well as the a b ility of the plant to regenerate root ti s sue (O ' Day et al.

, 1998).

Historically

, population s o f th e NC R an d W C R had been esti m ated to result in annual

yield losses and contr o l costs t h at e x ceeded $1 billion (Metcalf, 1986). Researchers h ave

reported yield losses of 0.8 to 2.5 % pe r larv a pe r plan t (Pet ty, et al., 1969; S m ith, 1979).

In addition, adult rootworm beetles can cause stripping of the upper layer of tissue from

the leaves and silk clipping, a l though this rarely has an i m p act on m aize yields (O ' D a y et

al.

, 1998).

In past years, various manage m ent practices hav e bee n e mploye d t o manag e CR W pests

inclu d ing c r op rot a tion, inse c ti c ide appli c at i on at planting to m anage larvae, and foliar

insecticide application to m anage adult beetles

. I n 2003

, m aize hybrids containing Bt

proteins to p rotect again s t CRW feeding beca m e availa b le and have been widely ado p ted

on broad acreages in the U.S. since then (Figure IX

- 2). Prior to introduction of these

corn ro o twor m

- protect e d B t hybrids

, tw o variant s o f W CR a n d NC R wer e identifie d that

m ade crop rotation as a m eans to m anage r ootworm populations less effective. The

soybean variant (SBV) of the WCR adapted its egg

- laying behavior to la y egg s i n crops

other than m aize, namely soybea n (Levin e a n d Olou m i

- Sadeghi

, 1996)

. Th e SB V has

been reported to occur in Illinois, Indiana, Michigan, Ohio, Minnesota, Iowa, and

Missouri (Gray, et al., 2009; O nstad

, e t al.

, 1999)

. Th e extende d diapaus e varian t ( E DV)

of the NCR has ada p ted to c r op r o tations as w ell. Al t hough m ost NCR eggs hatch the

following spring, so m e of the eggs of EDV hat c h after two winters a nd the larval stages

are able to feed on m aize roots even in r o tated m aize (Krysan, et al., 1984). The EDV

varia n t is m ost p revale n t in po r tions of Sout h D a kota

, Nebraska

, Iowa, and Minnesota

(Alston, et al., 2002). While these variant CRW populations do exist, corn root w or m

protected B t m aize products (both s i ngl e an d dua l MOA ) av a ilable to growers have

provide d an d continu e t o prov i d e e x cellent control of corn rootwor m

- complex pests on a

vast m ajority of acres.

The introduction of biotechnology

- derived i n sect

- protecte d m a iz e has offered growers

alternative and highly effective sol u tions f or control of m ajor inse c t pests in m aize. As

noted in S e ction I (Pr o duct Ratio n ale), in 20 1 2, approxi m ately 85 m illion a c res in the

U.S. (or 88% of the total U.S. m aize ac r eage ) wer e plante d wit h biotechnology

- derived

m aize hybrids, and ap p r oxi m ately 6 4 m illion acres ( o r 6 7 % of the t o tal m aize acreage)

were planted with m aize hybrids containing i n secticidal crystal (Cry) proteins derived

fr o m B t (USDA

- ERS, 2013). Of the 97.2 m illion acres of m aize planted in the U

. S. in

2012, approxi m ately 50 m illion contained B t proteins for protection against corn

rootworm (Figure IX

- 2 ).

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50

40

30

20

10

0

2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Year

Figure IX

- 2. Ma i z e acres in the U.S. planted to hybrids containi n g B t proteins for

corn rootworm protection

Source: (Marra et al., 2012) (updated with 2012 i nterna l Monsant o data)

MON 87411 was developed to build upon current B t protein

- base d CR W control

technology by introd u cing a new MOA based on R NAi that will co n ti n ue to effectively

control corn rootwor m

- co mplex pests in m a i ze fields and is expected to pr o lo n g the

durability of existing B t technologies

. B y incorporat i n g tw o distinc t MOAs

, MO N 87411

is expected to offer increased co n tr o l of target insect p ests and will continue to provide

benefits to growers similar to those obtained with the use of existing CR W

- protected

m aize hybrids. Thus, no changes to agrono m ic practices are expected related to grower

use of MON 87411.

IX.D.1. In s ect Resista n ce Management

MON 87411 will n o t b e offered for com m e r cial use as a stan d

- alone single

- event

product. Thus, no specific insect resis t ance m anag e m ent (IRM) program is being

developed for MON 87411 alone. Instead M O N 87411 is intended to be commercialized

as co m bin e d

- trait breeding stacks with previously deregulated biotechnology

- derived

traits. As these selected insect

- protecte d combined

- tr ait products are identified and

developed, Monsanto (or other developers) wil l develo p appropria te I R M progra m (s) for

these p rod u cts and su bm it them to U.S. EPA as part of its registr a tion package(s). EPA

has req u ired IRM pr o posals for each of t h e insect

- p rotected corn products it has

previously registered. I R M plans, which are included as conditions of U.S. EPA

registration, are designed to d e lay the develo pm ent of insect r esi s tan c e to spe c i f ic PIPs

and prolong the useful life of these products.

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IX.E. Management of D i sease s a n d Othe r Pests

Manage m ent of diseases during m aize growth and develop m ent is i mportant for

protecting the yield of harvested grain. Diseases can cause stand loss, r e duce

photosynthesis, increase lodging, a n d lower yields and grain quality ( M ueller and Pope,

2009). The incidence of diseases varies from year to y e a r becaus e the y ar e highly

influenced b y cli m ate a n d other en v i ron m ent a l f actors. E s ti m ates fo r annual yi e ld l o sses

because of d isease have ranged from 7 to 17 % (Shurtleff

, 1980)

. Mor e tha n 6 0 diseases

occur in m a ize in the U.S., but m any occur infrequently or are not prevalent enough to

cause m easurable loss (Stuc key

, e t al.

, 1993)

. Maiz e dise ases can be grouped into six

categ o rie s : 1) seed and seedling d i seases, 2) l e af diseases, 3) stalk rots, 4) ear rots, 5)

viral diseases, and 6) ne m atode diseases. M o st m aize seed is tre a ted with fungicide for

the prevention of seed and seedling diseases ( S tucke y e t al.

, 1993)

. O t he r disease s ca n be

m anaged to varying degrees with use of r e sistant hybrids, crop r otation

, tillage

, foliar

fungicides, and various cultur a l practices (Mueller and Pop e

, 2009; Stuckey et al., 1993).

In addition, several ne m atode species have b een known to cause dis e ase in m aize (S m i t h

and W hite, 1988). The best m ethods for m anage m ent of pest ne m atodes are primarily

crop rotation and stress protection (Stuckey et al., 1993). Other than use as part of a seed

treat m ents, fungicides were applie d o n jus t 8 % o f U.S

. m a i z e acreag e i n 201 0 (USDA

NASS

, 2013b)

. Fungicide s currentl y use d on m aize plants in the U.S. i n clude

azoxystrobin, propiconazole, pyraclostrobin, and trifloxystrobin ( U SDA

- NASS, 2013b).

Environ m ental observations in field studies have de m onstra t ed no apparent i mpact of

MON 87411 on diseases of m aize (Section VIII)

. Therefore

, n o change s i n curre n t

disease m anage m ent practices are antici p ated from the introduction of MON 87411.

IX.F. Weed Management

W eed cont r ol in m aize is e ssenti a l to o p ti m izing yield b e cause wee d s co mpete with

m aize for light, nutrients, and m oisture and can lead to red u ctions in yiel d (Knak e e t al.,

1990). W e ed control the first several weeks after m aize e m ergence is the m ost critical

period to avoid yield losses in m aize (Bo s ni c an d Swanton

, 1997 ; Care y an d Kells

, 1995;

Hall, et al., 1992). Some weeds can tolerate cold, wet conditions better than m aize, and

can gain an advantage prior to planting. Annual weed species such as giant foxtail

( Setaria spp.), barnyardgrass ( Echinochlo a crus

- gall i (L.) B eauv .) and Pal m er pigweed

( Amaranthus spp.) can reduce m a ize yields by up to 13, 35 and 74%, respectively

(Bosnic and Swanton, 1997; Fausey, et al., 199 7 ; Gianessi, et al., 2002; Knake and Slife,

1965). In a study of m ixed weed populations c o mpeting with m aize, yields were reduced

b y u p t o 20 % whe n th e weed s reache d a heigh t o f eigh t inche s (Care y an d Kells

, 1995;

Gianessi et al., 2002).

A survey of Extension Servi c e weed sci e ntists s oli c ited esti m ates of the per c ent of m aize

acreage infested with individu a l weed species by state or region, as well as the potential

i m pact on m aize yiel d s if the sp e cies w e re lef t uncontrolled

. I n thi s survey

, twelve

annual broadleaf, nine annual grass, and sev e n perennial species were identified as

troubleso m e weeds (Table IX

- 6) (Gianessi et al., 2002). Esti m ates of yield loss ranged

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from a low of 15% due to wirestem m uhly and sandburs to a high of 48% from

burcucu m ber.

Until the early 1950s, tillage and culti v ation p ractices were pri m arily used for weed

control in maize, but they have been largely re p laced by the u se of herbicides. Herbi c ide

use in m aize beca m e widespread by the e nd of the 1970s. In 2010, herbicides were

applied to 98% of the planted maize acreag e (USDA

- NASS

, 2013b)

. Currently,

glyphosate is the m ost widely applied herbici d e in m aize followed by atrazine at 78% and

61%, respectively, of p l anted acreage. The c h loraceta m ide herbicides (long chain fatty

acid inhi b itors such as acetochl o r, s

- m et o lachlor, di m ethena m id, and flufenacet) were

applied to 56% of the planted m a i ze acreage in 2010 (USDA

- NASS, 2013b).

The introduction of biotechnology

- derived HT m aize (like MON 87411) continues to

offer growers an effective in

- crop solution f o r control of weeds. In 2012, approxi m ately

73% of the total m aize acreage in the U.S. was planted with hybrids possessing HT traits.

Althoug h glyphosat e provide s effectiv e broad

- spectrum control of nu m e rous annual and

perennial w eed specie s

, pre

- e m ergence re s id u al her b ici d es are also recom m ended

co mponents of weed control progra m s f o r Roundup Ready m aize (Monsanto Company,

2013). P re

- e m ergence residual herbicides p rovid e earl y seaso n contro l t o reduc e early

weed co mp etition, i m prove control of certain har d t o contro l b r oadlea f wee d species

( e.g

., m orning glory spp.), and can provide co ntro l o f glyphosate

- resistan t weeds.

IX.F.1. Weed Resista n ce Management

As MON 87411 uses the sa m e gene ( cp4 epsps ) as other Roundup Ready m aize products,

no separate or additional recom m endations f o r m anaging glyphosate

- r e sistant weeds have

been developed for MON 87411. As describ e d in its Technology Use Guide (Monsanto

Co mpany, 2013), Monsanto continues to rec o mmend that growers i m ple m ent diversified

weed m anage m ent progra m s. Re co m m endations include s tarting with clean weed

- free

fields, use of a diverse set of w e ed contr o l tools, including res i dual herbicides and

multiple M OA, use of other c u lt u r al pr a ctice s

, as needed, and contr o l/re m oval of weed

escapes. G uidelines have been developed by a wide variety of stakeholders to help

facilitate the effective manage m ent of herbicide resistance (HRAC, 2011 )

.

IX.G. Crop Rotation Practices in Mai z e

Crop rot a ti o n is a w e ll

- esta b lished f ar m ing p r actice and a useful m an a ge m ent tool for

m aize production. Crop rotations are used to diversify farm inco m e, spread labor

require m ents throughout the year, and spread t h e crop loss risk associated with weather

and pest da m age across two or more crops. In ter m s of soil and pest m anag e ment,

rotation s ar e use d t o 1 ) m a n a g e weed

, insect

, an d diseas e pests

, 2 ) reduc e soi l erosio n by

wind and w ater, 3) m aintain or inc r ease s o il organic m atter, 4) pro v id e biologicall y fixed

nitrogen when legu m es are used in the rot a tion, and 5) m anage excess nutrients (Singer

and Bauer, 2009). Studies in U.S. corn belt s t ates indicate m aize yield is about 10

- 15%

higher in m a ize grown following soybean than m aize grown following m aize ( S inger and

Bauer, 2009). Despite the m any b e nefits of crop rotations, crop pr ic e fluctuations

, input

costs, rental agree m ents, govern m ent price s upports, weather, choice of fa r m ing syst e m

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and on

- farm resources, and o t he r factor s al l contribut e t o decision s regardin g crop

rotations. Market conditions such as U.S. govern m ent

- m a ndated ethanol use and record

high commodity m aize prices h ave increa s ed th e de m a n d fo r m a iz e g r ai n an d resulte d in

increases in m aize acreage in recent y ears ( S inger and Bauer, 2009; USDA

- NASS,

2013d). Introduction of MON 87411 is not, howe v er, expected to i mpact crop rotation

practices any m ore so than current C R W

- protected and HT p r oducts available to growers.

IX.H. Maize Volunteer Management

Volunteer maize com m only occ u rs in rotat i on a l crops in the season f oll o wing cultiv a tion

of conventional or biotechnology

- derived m aize. Viable grain is not produced on the

approxi m ately 8% of U.S. m aize acres that are culti v ated for the production of silage, and

volunteer maize plants typically do not occur in the rot a tio n al crops t h a t f ollow m aize

harvested as sila g e. In the war m er cli m ates o f th e Southeas t an d Southwest

, volunteer

m aize is rare becau s e maize g rain re m aining aft e r harvest is likely to ger m inate in t h e fall

and the res u lting pla n ts can usu a lly be control l e d by tilla g e or by f ree z ing te m peratures in

the winter. In northern m aize

- growing regions, volunteer maize does not always occur in

rot a tion a l c rops becau s e of seed deco mposition over the winter, e fficient harvest

procedures, and tillage prior t o plantin g rotation al crops. None of the agrono m ic or

phenotypic data collected for MON 87411 (Sect i o n VIII.C.)

, includin g dat a regarding

ger m ination and seed dor m ancy, i ndicates that MON 87411 is any more likely to

volunteer than existing com m ercial m aize hybrids.

Manage m ent of volunteer m aize in rotational crop s involve s m i n i m i zin g o r reducin g the

potential for volunteers through p r actices that include: 1) a d justing harvest equip m ent to

m in i m ize the a m ount of grain lost in the fie l d; 2) planting hybrids th a t reduc e th e extent

of ear drop; 3) choosing hybrids with superior stalk strength and reduced lodging; and 4)

practicing no

- till production to significantl y reduc e th e pote n tial for volunteer growth in

rotational crops. If volunteer m a iz e doe s occu r i n subsequen t crops

, pre

- plan t tillage

, in

crop c u lti v ation and t h e use of sele ct ive he r bi c i d es are very e ff ective m a nage m ent tools.

Introduction of M ON 87411 is not expected to i mpact maize volunteer m anag e m ent

practices any m ore so than current C R W

- protected and HT produ c ts available to growers.

IX.I. Stewardshi p o f MO N 87411

Monsanto develops effective products and technologies that deliver valu e t o grower s and

conserve resources that agriculture depends on, and is com m itted to assuring t h at its

products and technologies are safe and environ m entally responsible. Monsanto

de m onstrates this commi t m ent by i mpl e m entin g produc t stewardshi p processes

throughout the lifecycle of a product and by p a rticipation in the Excellence Through

Stewardshi p ® (ETS) Program (BIO, 2010)

. ETS policies and prac t ice s includ e rigorous

field co mpliance and quality m an a ge m ent syste m s and v e rification through auditing.

Monsanto’ s Stewardshi p Principle s ar e als o articulated in Technology Use Guides

(Monsanto Co mpany, 2013) and Monsanto Technology Stewardship Agree m ents that are

signed by growers who utilize Monsanto branded traits, to ensure stewardship

co mplian ce.

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As an i n te g ral action o f f ul f illing this s t ewar d ship com m it m ent, Monsanto will s eek

biotechnology regulatory approvals for MON 87411 in all key m aize i mporting countries

with functioning regulatory sys t e m s to assure global co mpliance and support the flow of

international trade. These actions will b e co n siste n t with the Biotechnology Industry

Organization Policy on Product Launch (BIO, 2010). Monsa n t o continue s t o m o nitor

other countries that are key i mporters of m a ize from the U.S., for the develop m ent of

fo r m a l biotechnolog y approva l p r ocesses

. I f ne w functionin g regulator y processe s are

developed, Monsanto w ill m ake appropri a te and ti m ely regulatory sub m issions.

Monsanto also com m its to industry best prac tice s o n see d qualit y assuranc e an d control

t o ensur e th e purit y an d integrit y o f MO N 87411 seed. As with all of Monsanto’s

products, before comm e rcializing MON 87411 p r oducts in any country, the appropriate

detection methods will be m ade available to m aize producers, processors, and buyers.

IX.J. Impact of the Introduction of MON 87411 on Agricultural Practices

MON 87411 has been developed to prov i d e tw o MO A agains t th e pri m ar y CR W

co mplex p e sts of m aize in the corn belt as well as glyphosate t o lerance in a single

product. As tolerance to glyphosate and pro t ection from c orn rootworm co m plex pests

are p rese n t in m any currently available m aize hybrids and are widely grow n i n th e U.S.,

th e introductio n o f MO N 8741 1 i s no t expec ted to have m ajor i mpacts on current

agrono m ic, cultivation and m anag e m ent practices fo r m a ize

. N o change s ar e anticipated

in crop r o t a tions, tilla g e pra c tic e s, planting p r actices, f ertility m anage m ent, weed and

disease m anage m ent, and volunteer m anag e m e nt from the introduction of MON 87411.

MON 87411 has been shown to be co mp arable to conventional m aize in its

co mpositional, phenot y pic, and agrono m ic c haracteristics (Secti o n s VII and VIII).

CR W

- protected and glyphosate tolerant m aize products have been widely grown in the

U.S. since 2 003 and the introducti o n of MON 87411, with its new M OA, will i m prove

the durability of CRW

- protection traits and extend the useful lifeti m e of these products.

Biotechnology

- derived CR W

- prot ecte d m a iz e ha s bee n cultivate d an d consu m e d i n the

U.S

. sinc e 200 3 an d ha s reduce d insecticid e ap plications direc te d a t thi s pe st (U.S. EPA,

2011)

. M O N 8741 1 introduce s a ne w MO A base d o n RNA i tha t offer s increase d control

of CRW pests and is expected to prolong the durability of existing CR W

- c ontrol

technolo g ie s

. As such, it will continue to p r ovide benefits to growers si m ilar to those

obtaine d b y us e o f existin g CR W

- p rotecte d m a iz e hybrids

, inc l udin g reduce d us e of

insecticides, increased yield protection, water conservation, and increased worker safety.

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X. PL AN T PE S T A SS ESS M ENT

Thi s sectio n provide s a brie f revie w an d assess m ent of the plant pest potential of

MON 87411 and its i mpact on agrono m ic practices and the environ m ent

. USDA

- APHIS

has responsibility, under the Plant Protection Act (PPA) (7 U.S.C. § 7701

- 7772), to

prevent the introduction and disse m ination of plant p e sts into t h e U.S. Regulati o n

7 CFR § 340.6 provides that an ap p l icant m ay petition APHIS to eval u a te sub m itted data

to deter m ine that a p artic u lar regulated a r ticle does not present a p la n t pest risk and

shoul d n o longe r b e regulated

. If APHIS deter m ines that t h e regulate d articl e doe s not

present a plant pest risk, the petition is g r anted, thereby allowing un r estri c ted introd u ction

of the article.

According to the PPA, the definition of “plant pest” inclu d es the li v ing stage of any of

the f ollowi n g that can d ire c tly o r i n dire c tly inj u re, cause d a m age to, or cause di s ea s e in

any plant or plant product: ( A ) a protozoan; (B) a nonhu m an a n i m al; (C) a parasitic plant;

(D ) a bacteriu m ; (E ) a fungus ; (F ) a viru s o r v i roid ; o r (G ) a n infectiou s agen t o r other

pathogen; (H) Any article si m ilar to or allied with any of the articles specified in the

prece d ing s u bparagraphs (7 U.S.C. § 7702[14]).

The regulatory endpoint under the PPA for b i otechnology

- derived crop products is not

zero risk, but rather a deter m ination that d e regulation of the article in question is not

expected to pose a greater plant pest risk than its un m odified co m parator. Info r m ation in

this petition related to plant pe s t risk c h aracteristics includes: 1) m ode

- of

- action and

changes to plant m etabolis m ; 2) co mposition; 3 ) expressio n an d characteristic s o f the

gene product; 4) potential for weediness of the regulated article; 5) i mpacts to NTOs; 6)

disease and pest susceptibilities; 7) i mpac t s on agrono m ic practice s ; and 8) i m pa c ts on

the weedin e ss of any other plant with which it can inter b re e d, as well as the potenti a l f or

gene flow. Using the assess m ent above, the data an d analysi s present ed i n thi s petition

lead to a conclusion that MON 87411 is not e x pected to be a plant pest, and there f ore

should no longer be subject to regulatio n unde r 7 CF R § 340.

X.A. Cha r acteristics of the Genetic Insert and Expressed Products

X.A.1. Genetic Insert

As described in Section III, MON 87411 was developed by Agrobacterium tumefaciens

m ediated transfor m ation of m aize e m b r yos using plas m id vector PV

- ZMIR 10871.

Characterization of the DNA insert in MON 87411 was conducted using a co m bination

of sequencing, PCR, a nd bioin f or m atics m ethods. The r esults of this characterization

de m onstrate that MON 87411 contains one c opy of the intended T

- DNA containing the

DvSnf7 suppression ca s sette and t h e cry3Bb1 an d cp 4 epsp s expre s sion cassettes t h at is

stably integrated at a s i n gle loc u s a n d is in h erited accor d ing to M e ndelian principles over

multiple generations. These m ethods also c o nfir m ed that no vector backbone o r other

unintended plas m id sequences are present in MON 87411. Additionally, t h e ge n o m ic

organi z ation at the ins er tion s ite w a s asse s s ed b y co m paring the s equences flanki n g the

T

- DNA insert in MON 87411 to the sequence of the insertio n sit e i n conventional m aize.

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This analysis deter m ined that no m ajor DNA r e arrange m ent occurred at the insertion site

i n MO N 8741 1 upo n DN A integration.

X.B. Mode

- of

- Action

A s note d i n Sectio n I.D.2.

, MO N 8741 1 con t ain s th e sam e CP 4 EPSP S an d Cry3Bb1

proteins as those found in MON 88017 m aize (APHIS petiti o n No. 04

- 125

- 01p) that was

grante d non

- regulate d statu s b y U SDA

- A P HI S i n 200 6 (USDA

- APHIS

, 2013)

. The

MOA of the CP4 EPSPS protein, w hich is found in nu m erous other approved Roundup

Ready crops, is well understood (Padg e tte et al., 1996). Si m ilarly, the MOA of Bt

proteins such as Cry3Bb1 has been extensi v ely studied and is well under s tood (Gill et al.,

1992; W ha l on and W ingerd, 2003).

As previously described in Section I.C.1., the R NAi m echanism is a natural p roces s in

eukaryotic organis m s for the regulation of gene expression. Double

- stranded RNA

molecules that activate the m echanism are f i rst processed into siRNAs which are then

incorporated into m ulti

- p rotein RNA

- induced silencing co mplexes (RISC). These RISCs

f

acilitate t a rget sequ e nce r e cog n ition a nd mRNA cleavage, lea d ing to sp e ci f ic

suppressio n o f th e targete d m R NA

. Th e targete d DvSnf 7 i s a vacuola r sortin g protei n in

the ESCRT

- III co m plex and is in v olved in s o rting trans m e m brane proteins enro u te to

lyso m so m al degradation. Snf7 is an esse n ti a l gene in CRW with signi f icant roles in

intracellular trafficking of protei n s and because of its key cell u lar function, its

suppression resulting from the expression of specific DvSnf7 dsRNAs, results in CRW

mortality.

Characterization of the insec t icida l mod e o f actio n o f D vSnf7 dsRNA has de m onstrated

that Snf7 is an essential gene in W CR that pla y s a significa n t role in i n tracellular pr o tein

trafficking and its function is consistent with its known function i n othe r eukaryotes

(Bolognesi et al., 2012; R a m ases h adri et al., 2013). The spectrum of activity for DvSnf7

dsRN A ha s bee n show n t o b e narro w an d activi t y is only evident in a subset of beetles

within the G aler u cinae s ub f a m ily of Chrys om elidae wit h in the Order Coleopt e ra (S e ction

VI.E.) (Bach m an et al., 2013). For the NTO assess m ent of MON 87411, in addition to

the a cti v ity spectrum testing (Se c tions VI.E.1. and VI.E.2.), an additiona l batter y of

beneficial organis m s was tested that included r epresentativ e pollinator s (honeybee)

, a

det r itiv o re ( earthwor m ) and inse c t predat o rs/ p a r asit o ids ( la d ybird be e tle, carabid b ee tle,

Orius

, parasitic wasp). The testing and as s ess m ent for DvSnf7 RNA lead to the

conclusion that no negative i m pacts to NTOs, including those benefic i al to agriculture,

will result from the cultivation of M ON 87411.

X.C. Expr e ssion and Characteri z ation of Gene Products

X.C.1. Protein Safety and Expression Levels

The safety and expression of the Cry3Bb1 an d CP 4 EPSP S protein s ar e detaile d in

Section V. Expression levels were deter m i n e d fro m 1 9 tissu e type s f r om trials conducted

in Argenti n a and are p resented in Section V

. C. The expression in the various tissues

ranged from 3.0 to 460 μg/g dw for Cry3Bb1 an d <LO Q t o 7 6 μg/ g d w fo r CP 4 EPSPS.

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These pr o teins are p resent at a very s m all p e rc e ntage of the tot a l pr o tein in m aize s eed

(no m ore than 0.004% and 0.002% for Cry3B b 1 and CP4 EPSPS, re s pectively). Both

proteins also have established histories of safe use, havi n g bee n assesse d b y USDA

, FDA

and U.S. EPA on multiple occasio n s (Section V.E.). Neither protein ori g inat e s from an

organism known to be a source of allergens, a bi o infor m atic assess m ent of each shows no

shared a m ino acid seq u ence si m ilarities to kn o wn allerge n s, and each protein is rapidly

digested in a si m ulated gastric fluid assay (Section s V.D. 1 an d V.D.2)

. Take n together,

the results of these analyses support a det e r m ination that MON 87411 is no m ore likely to

pose a plant pest risk than conventional m aize.

X.C.2. RNA Safety and Expression Levels

The safety and expression of the DvSnf7 R NA expressed in MON 87411 is detailed in

Section VI. Expression levels were dete r m ined f rom 19 tissue types f rom tri a ls

conducted in Argentina and are presente d i n Sectio n VI.C . The expression in the various

tissue types ranged from <LOQ to 213 × 1 0

- 3 μg/g dw. As RNAi is a naturally occurring,

ubituitous process in eukaryote s

, there is a long h i story of safe consumption of the RNA

molecules m ediating this process. Further, there is no evidence sug g esting t h at d iet a ry

consu m ption of nucleic acids, like RNA, is associ a ted with toxi c ity or all e rg e nicity

(Petric k e t al.

, 2013 ; U.S

. FDA

, 1992)

. Tota l nucleic acid consu mption in the hu m an diet

for DNA a n d RNA is esti m ated to be 1

- 2 gram s pe r da y (Suchne r e t al.

, 2000 ) which

equates to an approxi m ate m ax i m al intake of 0.2 g/kg/day for a non

- nursing infant (the

highest consu m er per body weight). Given these para m eters, consu mption of DvSnf7

RN A fro m MO N 8741 1 wou ld be extre m ely low ( ≤ 0.4 ng/kg/day) relative to the total

esti m ate d dietar y RN A intake

. Take n toget h er

, th e result s o f t hese assess m ents, along

with the d e monstrated speci f i c ity of DvSnf7 RNA to CRW and the history of safe

consu mption of RNA, s upport a deter m inat i on that MON 87411 is no more likely to pose

a plant p est risk t h an conventional m aize.

X.D. Compositional Characteristics

Co mpositi o nal co m parisons based on OECD gui d ance w er e prese n ted in Section VII to

assess whether levels of nutrie nts

, anti

- nutrients

, an d second ary m etabolites in grain and

forage derived from MON 87411 are co mparable t o level s i n th e conventional control

and several reference hybrids for which the r e is an established history of safe

consu mption. Nutrients assessed in this analysi s include d prox i m ates, carbohydrates by

calculation, acid detergent fiber, neutral det e rgent fiber, total dietary fiber, m inerals,

a m ino acids, and vita m ins. Anti

- nutrients a s sessed in grain included phytic acid and

raffinose. Secondary m etabolites assessed in grai n include d furfural

, feruli c acid

, an d p

cou m aric acid. Forage sa m ples were assessed for levels of proxi m ates, fiber, m inerals

(calcium and phosphorus), and carbohydrates by calculation. In all, 78 different

co mponents were assayed (9 in forage a nd 69 in grain). While 78 co mponents were

assayed, 16 of these had m ore than 50% of observation s belo w th e assa y LO Q an d were

excluded fr o m stati s tical analy s is.

A co m bined

- site analysis of the data was c onducted to deter m ine sta tisticall y significant

differences (at 5%) between MON 87411 and the conventional control. The biological

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significance of differences from the data was reviewed using considerations relevant to

foo d an d fee d safet y an d nutritiona l quality

. Thes e con s ideration s included : 1 ) a

deter m ination of the mean differences of nutrient and anti

- nut r ient co m ponents of

MO N 8741 1 an d th e conventiona l control ; 2 ) consideration of the m ean differences

between MON 87411 a nd the conventional control in the context of the range of values

for the conventional control, 3) co mparison of the m ean diff e rences bet w een MON 87411

and the co n vention a l c o ntrol to variation in co n vention a l maize as e sti m ated by in

- stu d y

referenc e hybri d value s an d assessin g w hether the m ean values for MON 87411 were

within 99% tol e rance i n tervals, liter a ture v a lues, and/or ILSI C r op Co m p osition Database

values. These evaluations of variation wit h in the conventional c ontrol and conventional

m aize, incl u ding refere n ce hybrids, are i m portant as crop co m position is known to be

influenced by environ m ent and ger mplas m

.

The co mpositio n al an al ysis provi d e d a co mprehensive c o mparative a ssess m ent of the

levels of key nutrients, anti

- nutrients, and secondary m etabolites in m aize grain and

forage of MON 87411 and the conventional contr o l. Of the 60 co mponents statistically

assesse d ther e wer e n o statist i cally significant differences in 48 co mponents. Only 12

co mponents (protein, histidine, tyrosine, ole i c acid, neutral deterge n t fiber, copper, iron,

m anganese, zinc, niacin, vita m in B1 in g rai n an d as h i n forage ) showe d significant

difference s betwee n MO N 8741 1 an d convention al control

. Fo r thes e 1 2 co m ponents,

the m ean difference in co mponent values bet w een MON 87411 and the conventional

contro l wa s les s tha n th e rang e o f th e conventiona l contro l value s an d th e reference

hybrid values. Overall, MON 87411 m ean co mp onent values were within 99% tolerance

int e rvals, v alues ob s e r ved in the liter a tur e

, and/o r IL S I

- CCD B values

. Thes e data

indicate t h at the co m ponents with signif i ca n t differences were not co mpositionally

m eaningful from a food and f eed s af ety or nutrition a l pers p ecti v e. Th e se res u lts f urth e r

support the overall conclusion that MON 87411 was not a m ajor contributor to variation

in co mpon e nt levels in m aize grain and forag e an d confir m ed th e equivalenc e of

MON 87411 to the conventional control in levels of these components.

X.E. Phenotypic, Agr o nomic, and Environmental Inter a ction Characteri s tics

An extensi v e set of comparative plant chara c terization data were used to assess whether

the introduction of the insect

- p rotection and glyphosate toler a nce traits altered the plant

pest potential of MON 87411 compared to t h e conventi o nal control (Section VIII).

Phenotypic, agrono m ic, and environ m ental interaction charact eristic s o f MO N 87411

were evaluated and compared to th o se of the conventional control. A s describe d below,

these asse ssm ents incl u ded: seed d o r m ancy and ger m ination charact e ristics; agro n o m ic

an d plan t phenotypi c character i stics ; observation s fo r abioti c stres s response

, disease

da m age, arthropod

- related da m age, arthropod abundance, and pollen characteristics.

Results from all phenotypic, agrono m ic, and environ m ental interaction assessments

de m onstrated that M O N 87411 does not possess weedy characteristics, or increased

susceptibility or tolera n ce to specific diseases, insects, o r abiotic stres s ors co mpared to

th e conventiona l control

. Take n togethe r, the results of the analysis support a

deter m ination that MON 87411 is no m ore li kel y t o pos e a pla n t pes t ris k than

conventional m aize.

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X.F. Weediness Potential of MON 87411

Maize is not li s ted as a weed in the m ajor weed referenc e s ( C rockett, 1977; University of

Montana

, 2011)

, no r i s i t presen t o n th e list s o f noxiou s wee d specie s distribute d b y the

federal govern m ent (7 CFR § 360). In addi tion

, m a iz e ha s bee n grow n throughou t the

world with o ut any rep o r t that it is a serious we e d. During do m estic a tion of m aize, t r aits

often associated with weediness, s u ch as, s eed dor m ancy, a seed d ispersal m echanis m

, or

the ability to fo r m repro d ucing populations o u t s i d e of culti v ation, have not been selected.

Even if individual kernels of m aize were distributed within a field or along transportation

route s fro m th e field s t o storag e o r proc e ssing facilities, s ustai nabl e voluntee r m a ize

populations are not found growin g i n fenc e ro w s, ditches

, o r roa d sides

. Maiz e i s poorly

suited to s u rvive with o ut hu m an assistance a n d is not ca p able of sur v iving as a weed

(Galinat, 1988; Keeler, 1989).

In co m parative studies between MON 87411 a nd a conventional control, phenotypic,

agrono m ic and enviro nm ental interaction data were evaluated (Section VIII) for changes

that would i m pact the plant p est potential, in particular, plant we e diness p o t e nti a l.

Results of these ev a l u ations show that th e re is no f unda m ental di ff erence between

MON 87411 and the conventional con t rol for traits potentially a ssociated with weediness.

Further m ore, co m parative field observati on s betwee n MO N 8741 1 an d it s conventional

control and their response to abiotic stressors indi cate d n o difference s and

, therefore

, no

increased w eediness potential. Collectively, these findings support the conclusion that

MON 87411 has no increased weed potential co m p ared to conventional maize and it is no

more likely to beco m e a weed than conventional m aize.

X.F.1. Seed Dormancy and Germination

A co m parative assess m ent of seed ger m ination and dor m ancy cha r acteri s tics was

conducte d o n MO N 8741 1 an d th e conventiona l con t rol

. Th e result s o f this assess m ent,

particul a rly the lack of incr e ased h ard seed

, an d n o change s i n othe r ger m inatio n and

dor m ancy characteristics, support the concl u sio n tha t th e introdu c tio n o f MO N 8741 1 is

not expected to result in increased plant pest/ w eed potential co mp ared to conventional

m aize.

X.F.2. Plant Gro w th and Development

Evaluations of plant growth and develop m ent characteristics in the field are useful for

assessing potential weediness c h aracteristics such as stalk an d roo t lodging

. Phenotypic

characteristics such as early stan d count

, day s t o 50 % polle n she d an d silking

, sta y green,

ear height, plant height, dropped ears, stalk l odged plants, root lodged plants, final stand

count, grain moisture, test weight, and yield were assessed. In the co m b i ned

- site analysis

in which the data were pooled among the sit e s, no statistically si gnifican t differences

were detected between MON 87411 and the con v entiona l contro l fo r an y o f th e assessed

characteristics. Thus, the phenotypic and a g rono m ic characteristics of MON 87411 were

not altered in ter m s of pest/weed potenti a l co m p ared to con v ention a l m aize.

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X.F.3. Pollen Morphology and Viability

Evaluations of pollen m o rphology and viability from field

- gr ow n plant s provid e useful

in f or m ation in a plant p est asse s s m e nt as it r e lates to the potenti a l f or gene f low to, and

possibl e introgressio n o f a b i otechnology

- derive d trai t int o sexually

- compatibl e plants

and wild r elatives. No statisti c ally s ignificant differe n ces were d ete c ted between

MO N 8741 1 an d th e conventiona l contro l fo r percen t viabl e polle n o r polle n grain

dia m eter. Further m ore, no visual differences in general pollen m o rphology were

observed between MON 87411 and the conventional control. Based on the assessed

characteristics, the results support a conclusion that neither pollen via b ilit y nor

morphology of MON 87411 were altered c o m pared to conventional m aize.

X.G. Impact to Non

- Target Organisms, Including Those Benefici a l to Agriculture

Infor m ation presented previously i n Section s V.E

. an d VI.E

. i s relevan t t o th e plan t pest

risk assessment for MON 87411 b e cause it descr i bes the spectrum of activity for the CP4

EPSPS protein, the Cry3Bb1 protein, and the D vSnf 7 RNA

. Accordin g t o 7 CF R Part

340.6

, thi s info r m atio n i s par t o f th e require d infor m ation needed for evaluation of plant

pest potential. Given that the safety of CP 4 EPSP S an d Cry3Bb 1 pro teins as well as the

CR W

- resistance and glyphosate

- t o lerance traits have previously been evaluated by both

USDA

- APHI S i n regulator y sub m ission s fo r MO N 86 3 an d MO N 8801 7 (USDA

APHIS, 2013) and EPA (U.S. EPA, 2010), the pri m ary focus of the NTO section (Section

VI.E. ) wa s o n DvSnf 7 R NA.

The NTO a ssess m ent for MON 87411 has taken into consideration the MOAs, the

spectrum of insecticidal activity, and expo s ure levels to the CP4 EPSPS and Cry3Bb1

proteins and the DvSnf7 RNA produced by MON 87411. The data, analyses and

assess m ents presented s upport a c o nclus i on that MON 87411 will not negati v ely impact

NTOs, including those beneficial to agricult u re, and does not prese n t an incr e ased pla n t

pest risk compared to conventional m aize.

X.G.1. Impac t o n Threatene d an d Endangere d Species

Threatened and endangered species risk assess m ents were previously conducted by

USD A (USDA

- APHIS

, 2013 ) an d EP A (U.S

. EPA

, 2010 ) fo r Cry3B b1

- containing m ai z e

product s MO N 86 3 an d MO N 88017

, an d indicate d no direct or indirect effects to

threatened or endangered species.

Becaus e o f th e specificit y o f th e D v Snf 7 R NA for c o leopteran speci e s within the f a m ily

Chryso m elidae (Bach m an et al., 2013), endange re d specie s concern s fo r thi s trai t are

properly focused on the order Coleoptera. C u rrently

, ther e ar e 1 8 liste d Coleopter a on

the threatened and endangered species list, however none of these species are m e m be r s of

the f a m ily Chryso m elidae (USFWS, 2013b). Additionally, the listed threatened or

endangered coleopteran species are not expected to b e in or near m aize f ields as m any of

the endangered and threatened c o leopteran s p ecies occur in caves o r aquatic habitats

(U.S. EPA, 2010).

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Threatened and endangered coleopteran speci e s in the U.S. have re s tricti v e habitat

ranges, which generally do not include m aize fields (U.S. E P A, 2010). However, so m e

endangered and threate n ed col eoptera n habitat s m ay occu r n ear a g ricultural prod u c tion

areas. An analysis of the county level distribu t ions of threatened or endangered terrestrial

coleopteran species in d icates tha t th e potentia l concer n r e garding range overlap with

m aize production was restricted to the A m erican burying beetle ( Nicrophorus

americanus)

. The A m erican burying beetle is t h e l a rge s t ca r rion beetle in North A m erica

(USF W S

, 1991) and is only found in li m ited areas encompassing parts of 9 11 states,

including Arkansas, Kansas, Massachusett s

, Nebraska, Ohio, Oklahoma, Rhode Island,

South Dakota, and Texas (USF W S, 2013a). Adults feed on carrion and occasionally

othe r insect s (USFWS

, 1991)

, w hile larvae feed exclusive l y on buried carrion or carrion

regurgitations provided by th ei r parent s (U.S

. EPA

, 2 010 ; USFWS

, 2008))

. The

A m erican burying beetle’s habitat is variable a nd inclu d es m ature forests, shru b

- c o vered

areas, and so m e grassland habitats and the be et le’s preferred habitat h a s been correlated

wit h a n abundanc e o f s m al l vertebrat e b i o m ass (USF W S

, 2008). Considering that both

larvae and adult beetles are carrion f eeders, exposur e t o th e DvSnf 7 RN A i n MO N 87411

is highly unlikely due to thei r feedin g ecology.

There are s everal threatened and endangered coleopteran s pecies that occur in a q uatic

habitats. As discussed above, however, expo s ure of aquatic organis m s to biotechnology

derived crops has been shown to be li m ited t e mporally a n d spati a lly and the U.S. EPA

has previo u sly conclu d ed that potenti a l e xposure of aquatic organis m s is low to

negligibl e (U.S

. EPA

, 2010).

Due to t h e lack of proxi m ity to m aize c u lti v ati o n, lack of r e levant exp o sure b e cau s e of

feeding ecology and the restric te d activit y o f th e DvSnf 7 RNA, it is concluded that

cultivation of MON 87411 will have no effect on threate n ed and en d angered species,

inclu d ing c o leopteran s p ecies.

X.H. Environmental F ate of CRW Products Expressing DvSnf7_968

The two components of the i n secticida l activit y o f MO N 87411 are the Cry3Bb1 protein

and the DvSnf7_968 RNA. The environ m ental safety of the Cry3Bb1 protein has been

de m onstrat e d in m ultiple environ m ental studies and ass e ssed exten s ively as p a rt of m any

previous EPA product registrations, e.g.

, MON 863, EPA Reg. # 524

- 528; MON 863 x

MON 810, EPA Reg. # 524

- 545; MON 88017, EP A Reg

. # 524

- 551

, MO N 8801 7 x

MO N 810

, EP A Reg

. # 524

- 55 2 an d MO N 89034 x MON 88017, EPA Reg. # 524

- 576

a m ong others (U.S. EPA, 2010). Thus, the current environ m ental assess m ent pertains

exclusively to the RNA co m ponent of the insecticidal activity, DvSnf7_968.

Soil organis m s m ay be exposed to DvSnf7_968 by contact with roots and with above

groun d plan t residue s o r polle n reachin g th e s o il. Soil m icroorganis m s m ay be exposed

to DvSnf7_968 by degrading li v ing or decaying m aize bio m ass or by ingestion or

11 A n e xp e r i m e n t al p o pu l a tio n t hat i s li s te d as n on

- esse n tia l e x ist s i n Missou ri ( U SF W S

, 2 0 1 3b ).

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absorption of the dsRNA after its relea s e into the soil. As s ess m ent of t he environ m ental

fate of DvSnf7_968 and any other nucleic aci d (DN A o r RNA ) fro m biotechnology

derived crops should consider the a m ount of nucleic acids already i n th e environ m ent

from conventional sources. Thousands of tons o f nuclei c acid s a r e release d int o the

environ m ent every year from conv e ntional pla n t bio m ass (roots, leaves, pollen, etc.), in

additio n t o th e nuclei c aci d contributio n o f other decaying ani m al and m icrobial m atter

(Dale, et al., 2002). It is unlikely that nucleic acids originating from biotechnology

derived p lants will persist in s o il or inter a ct differently than nucleic acids from non

engineered organis m s, or persi s t b y being in c orporated i n t o m i crobe s vi a soi l uptake

(Dale, et al., 2001).

There are currently no published laboratory or f ield m onito r ing stu d ies that d ir e ctly

address the persistence or accu m ulation of RNA in m aize f ields. However, a laboratory

soil study of DNA that encodes rec o mbinant ne o m ycin phosphotransferase II (rNPT

- II), a

m arker used in early p l ant genetic engineer i ng, showed rapid dissi p ation of the target

DNA ( W i d m er, et al., 1996). The study showed that after the rNPT

- II DNA was ad d ed to

soil by incorporating ground t i ssue of transgenic tobacco containing the rN P T

- II

sequenc e an d b y additio n t o s o i l o f th e purifie d plas m id DNA

, bot h th e tissue

- derived

rNPT

- II se q uence and the plas m id DNA degraded rapidly. Only a small prop o rti o n of

th e DN A wa s detectabl e fo r longe r period s

. U p t o 0.08 % o f th e plas m id DN A was

detectabl e a t 4 0 days

, an d u p t o 0.14 % o f tissue

- derive d DN A sequenc e wa s detectable

for 120 days.

A laboratory soil degradation study conducted by Monsanto (unpublished, 2013) showed

that the dsRNA expressed in MON 87411 dissip a ted rapidly in different representative

agricultural soils (MO, IL an d ND ) tha t range d i n p H f r om 5.9 to 7.1, and contained

significant proportions of clay (u p t o 36% ) o r organi c m a tte r (up to 5.1%). Purified

DvSnf7_968 RNA and a m ixture of m aize root an d shoo t tissue s expressin g DvSnf7_968

RNA were added to ea ch soil in e x cess r el a tive to the m aximum concentr a tion po s sible

under m aize growing conditions. Elevated level s o f DvSnf7_96 8 RN A wer e use d to

i m prove the accuracy o f the analytical qu a ntifications, and to ensure that the D T 50 and

DT 90 (ti m e to 50% and 90% dissipation of the DvSnf7_968 ) coul d b e calculated from

m easurable values ( i.e

. , above the li m it of detection )

. Th e exces s DvSnf7_96 8 also

provided an increased m argin of environ m ental sa f ety to inclu d e any curr ently

unforeseen routes of RNA introduction into s o il. Soil extracts were analyzed using

QuantiGene, a m olecular analysis m e thod for nucleic acids, and by insect bioassay (corn

rootworm feeding), a functional measure of t h e loss of inse c ti c idal acti v ity in soil.

Results in d i cated a m axi m u m D T 50 of 29 hours, and a m aximum D T 90 of 35 hours for

DvSnf7_96 8 RNA

. N o DvSnf7_96 8 RN A wa s detecte d i n an y s o i l afte r 4 8 hour s of

incubation.

Since DvSnf7_968 dissipates co m pletely in less than 2 days in soil, negligible exposure

to NTOs is expected from the use of MON 87411.

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X.I. Potential for P o ll e n Mediated Gene Flow and Introgression

Pollen m ediated gene flow is a process where b y on e o r m o r e gene s successfull y integrate

into t h e ge n o m e of a recipie n t pla n t. Intro g ression is affected by both biotic and abiotic

factors such as plant biology, pollen biology/volu m e, plant phenology, overlap of

flowering ti m es, prox i m ity of the pollen s ource and sink, a m bient conditions such as

te mperature and relati v e hu m idi t y, a n d field arc h itect u re. Because gene introgression is a

natural biological process, it does not constitute an environ m ental risk in and of itself.

Gene introgression m ust be considered in the c o ntext of the transge n es inserted into the

biotechnology derived plant, and the likeliho o d that the p resence of the tra n sge n es and

their subse q uent transfer to reci p ie n t pla n ts w i ll result in i n crea s ed pla n t pest p o te n t ial.

The potential for gene introgression f r om MON 87411 is discussed belo w

.

X.I.1. Hybridi z ation w ith Cultivated Mai z e

Maize m orphology fosters cross p o lli n ation, t h erefore, h i g h levels of pollen m ediated

gene flow can occur in this species. In addition, researchers recognize that (1) the

a m ount of gene flow that occurs can be h i gh because of open polli n ati o n; (2) the perce n t

gene flow can vary by population, hybrid or inb r ed; (3) the level of gene flow decreases

with greater dista n ce between the s ource and r ecipient p l a nts; (4) en v i ron m ental f actors

affect the level of gene flo w ; (5) maize p o llen is viable f o r a short period of ti m e under

field conditions; (6) m aize produces a mple p o llen over an extended period of ti m e; and,

(7) m aize is al m ost excl u sively wind polli n ated.

Based on several studies conducted on the extent of pollen m ediated gene flow be t w een

m aize fields, results were found to v a ry depending on the experi m ental design,

environ m ental conditions, and detection m ethod, as expected. In general, the percent of

gene flow di m inished with increa s i n g di s tance from the so u rce field, g e nerally falling

below 1% at distances >200 m (~660 feet) (Table X

- 1). This infor m ation is useful for

m anaging gene flow during m aize breeding, s e ed production, identity preservation or

other applications; in addit i on, it f or m s the basis for the USDA

- APHIS perfor m ance

standards for m aize. All testing and production of regulated MON 87411 seed or grain

have been conducted u n der USDA notification accor d ing to these stan d ards. Gene flow

from fields planted with MON 87411 to other maize would not be of concern beca u se of

the lack of potential to cause harm to hu m ans and to the environ m ent.

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Table X

- 1. Summary of Published Literatu r e on Mai z e Cross Pollin a tion

Pollinator

Distance

(m) Reported

Outcros s ing

(%) Comments Country Referen c e

0 28.6 Frequencies by distance investigated. Three y ear stud y

. USA (Jones and Brooks, 1 950)

25

75 14.2

5.8 Single m a le and female. Pollen source was a y e llow

dent and the fe m a l e was a white swe e t corn..

125 2.3

200 1.2

300 0.5

400 0.2

500 0.2

1

- Frequencies by distance investigated. Single y e llow UK (Haskell and Dow, 1951)

3.6

4.8 95

- sweet corn hybrid was used as a pollen source and as pollen recipient. To measu r e levels of outcrossing, pollen

recipient plants were det a sseled.

6

-

7.3

-

8.5

-

9.8

-

11

-

12

-

13.4

-

18 10

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Table X

- 1 (continued). Summary of publish e d lit e rature on mai z e outcros s ing

Pollinat o r

Distance

(m) Reported

Outcros s i n g

( % ) Comments Country Reference

1 2.25 Dispersal of m aize pollen investigated. Single hybrid. Brazil (Pater n ia n i and

10 0.02 Gene flow decreased with grea t er di s tance from the source. Stort, 1974)

20 0.008 Closer correlation of number of plants with gene flow than

30 0.005 physica l distance

. Dat a repo r ted in this table represent

34 0.003 m eans from two of four fields

2

- 4 0.01 Gene flow in isolated and crossing blocks was evaluated.

Two year study. Single m ale and f e m ale. B t f e m ale hybrid

was detasseled. Mexico (Garcia, et al.,

1998)

30 1.04 Frequencies by distance i nvestigated. Two year study. USA (Je m ison and

40 0.03 Single RR m ale and non

- RR f e m a l e. Data reported in this Vayda, 2001)

350 0 table represent one of two years.

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Table X

- 1 (continued). Summary of publish e d lit e rature on mai z e outcros s ing

Pollinat o r

Distance

(m) Reported

Outcros s i n g

( % ) Comments Country Reference

100 0.01 Frequencies by dista n ce and pollen v iability investigated. Mexico (Luna, et al.,

150

- Two year study. Single m ale and f e m ale. A pu rple gene 2001)

200 0.01 m arker was utilized to measure p o ll e n m obility. Pollen

300

- viability la st ed one hour in t h e d rie s t

- hotte s t year and two

400

- hours in the most hu m id, less hot year.

1 30

- 40 Frequencies by distance investi g ated. Two sites/ o ne year. USA (Chilc u tt a n d

3 18

- 22 Six hybrid pairs. Six B t an d si x nea r isogeni c non

- Bt Tabashnik, 2004)

8 9

- 12 hybrids. H ybridization was assessed by m easuring the

16 3

- 5 expressio n o f Bt gene in kernels collected from

24 0

- 2 neighboring plants. Alternatively, sampled kernels were

32 2

- 4 grown and seedlings tested for expression of B t gene. Data

reported in this table represent estimates from a graph.

1 9.7

- 19.0 Frequency by distance investigated. Three year, three sites. Canada (Ma, et al., 2004)

5 1.3

- 2.6 Single m ale and f e m ale/location.

10 0.7

- 2.0

14 0.3

- 0.6

19 0.4

24 0

- 0.3

28 0.1

- 0.5

33 0

- 0.3

36 0

- 0.1

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Table X

- 1 (continued). Summary of publish e d lit e rature on mai z e outcros s ing

Pollinat o r

Distance

(m) Reported

Outcros s i n g

( % ) Comments Country Reference

200

300 0.03

0.02 Detasseling efficiency on p o llen contain m ent investigated.

Four inbreds were used as a source of pollen; yellow

inbred, two GM inbreds ( Bt an d RR ) an d a n IT

(i m idazolinone tolerant) inbred. The reci p ient p o llen traps

were two white inbreds and a m ale

- ste r ile hy b ri d

. Two

year/three locations study. USA (Stevens, et al.,

2004)

24

- 32 0.01

- 0.7 Frequencies by distance i nvestigated. Single m ale and 7 USA (Halsey, et al.,

60

- 62 0.01

- 0.2 f e m ales with different RM use d

. Th e m a l e paren t sourc e of 2005)

123

- 125 0.001

- 0.08 pollen co n t a ins the g enetic m arkers P1

- rr and R1

- nj. W hen

244

- 254 0

- 0.02 m ale pollen polli n ated fe m ale yellow plants a pu r ple

486

- 500 0

- 0.005 coloration o ccurred in t h e fertilized yellow kernels. Two

743

- 745 0

- 0.002 year/two site study. Data repo r ted in this table represent

results from one site.

1.8 1.0

- 2.5 Isolation distance investiga t ed. The objectives w ere (i) to USA (Ireland, et al.,

9.4 1.2

- 2.5 evaluate current industry is olatio n practice s t o produce 2006)

20.6 1.0

- 2.2 hybrid seed that m eets higher levels of genetic purity and

35.8 0.5

- 2.3 (ii) to identify practices t h at will i mprove reproductive

200 0.6

- 1.4 isolation in hybrid seed f i elds. Three year/315 fields.

Multi p le h y brids f rom 24 seed co m p anies tested. Data

reported in this table represent estimates from a graph.

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Table X

- 1 (continued). Summary of publish e d lit e rature on mai z e outcros s ing

Pollinat o r

Distance

(m) Reported

Outcros s i n g

( % ) Comments Country Reference

1 17.0

- 29.9 Frequencies of cross

- pollination by distance investigated.

Pollin a tion was quanti f ied by m easuring out

- cr o ssing f rom USA (Goggi

, e t al.,

2006)

10 1.5

- 2.5 a transgenic hybrid plot into a conventional grain

35 0.4 production field. A combination of three m arker genes was

100 0.03

- 0.05 utilized to d ete c t out c ro s ses: y1 (seed color gene), B t and

150 0.01

- 0.03 RR. Two y e ars/two sites. Single m ale and fe m al e

.

200 0.007

- 0.03

250 0.002

- 0.03

0

4.6

18.3 < 0.9% at

dista n ces < 20

m Efficiency of border rows a nd isolation distance on cross

polli n ation investi g ated. Avail a ble d atas e ts were utilized

to m ake predictions for reduc in g out

- crossin g t o levels

belo w 0.9%. USA (Gustafson, et al.,

2006)

0 3

- 13 Frequencies of cross

- polli natio n wit h a PC R base d m e thod Spain (Pla, et al., 2006)

2 0.2

- 10 investigated. The m ain objective of the study was to

5 0.1

- 2.3 co mpare a PCR based method to real cross

- fertilization

10 0.2

- 3.7 rates as deter m ined by phenotypic analysis. Four Bt

20 0.1

- 0.8 hybrids and a single non

- B t hybri d wer e use d a s a m a l e and

40 0

- 0.7 f e m a l e respectively

. On e year/on e site.

80 0.1

- 0.2

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Table X

- 1 (continued). Summary of publish e d lit e rature on mai z e outcros s ing

Pollinat o r

Distance

(m) Reported

Outcros s i n g

( % ) Comments Country Reference

0 10.5 Frequenc y o f cros s polli nation (expressed as %GM DNA) UK ( W eekes, et al.,

2 34.9 by distance investigated. T h e study was conducted in large 2007)

5 9.9 fa r m scale evaluation ( F SE) across the UK. Data reported

10 12.2 here are m a xi m u m raw values

15 0.5

20 8.2

25 4

40 3.7

50 5.9

70 0.13

75 0.28

80 0.12

100 2.3

120 0.16

142 0.06

147 0

150 5.4

160 0

200 0.24

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Table X

- 1 (continued). Summary of publish e d lit e rature on mai z e outcros s ing

Pollinat o r

Distance

(m) Reported

Outcros s i n g

( % ) Comments Country Reference

52 0.009 Cross

- pollination investigated using occurrence of yellow Switzerland (Banner t and

85 0.015 kernels in 1 3 white m aize f i elds

. I n n o case

, th e cross

- Sta mp, 2007)

105 0.003 polli n ation o f the whole field was > than 0.02%. In every

125 0.01 f ield so m e cross

- p o lli n ation with a l o w rate, on a n average

149 0.016 of 1.8% of the sa mpled ears, could be found. These

150

200 0.007

0.009 polli n atio n s were m ostly single cros s

- pollinations on the

ear.

287 0.005

371 0.008

402 0.005

458 0.0002

4125 0.006

4440 0.0005

1 42.2 Frequencies of cross

- pollination by distance investigated. USA (Goggi, et al.,

10 6.3 The pollen s ource was a stacked RR / B t yellow hybrid. The 2007)

35 1.3 recipient was a nontransgen i c white hybrid. Higher

100 0.1 outcrossing detected when white hybri d use d detasseled.

12 4.2 Frequency of cross pollination and coexistence by distance EU (Langhof, et al.,

12 11.7 investigated

. Tw o crop s wer e used as barriers to d eter m ine 2008)

12 3.8 their usefulness as buffer crops in m aize. Three genetic

m arkers to m easure outcrossing were used: GM B t m aize, a

kernel color m aize and a molecular m arker te s t

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X.I.2. Hybridi z ation w ith Wild Annual Sp e cies of Z ea mays subsp

. mexicana

For gene flow to occur by nor m al sexual trans m ission, the following conditions must

exist: (1) the two parents m ust be sexually compatibl e ; ( 2 ) there m ust be overlapping

flowerin g ti m es ; an d (3 ) a suitabl e facto r (suc h a s win d o r insects ) m u s t b e presen t and

capable of transferring p oll e n betwee n th e tw o parents.

Maize and annual teosinte ( Zea mays subsp. mexicana ), are g enetically c o mpatible, wind

polli n ated a nd teosi n te pollen c an p olli n ate m a i ze silks wh e n in clo s e p roxi m ity to each

other, e.g. in areas of Mexico and Guat e m ala ( W ilkes, 1972). Maize crosses with

teosi n te; h o wever, te o sinte is not p r esent in the U.S. other than as a n occasiona l botanical

garden speci m en or s m all feral populations in Florida, A laba m a and Maryland. In

experi m ental studies where m aize and teosinte species were planted together, very low

hybridization rates were observed for m aize and Zea mays subsp. mexic a na (Baltazar, et

al., 2005; Ellstrand, et al., 2007). Differences in factors such as flowering ti m e

, geographical distribution, and develop m ent f actors m ake natural crosses in the U.S.

highl y unlikely.

X.I.3. Hybridi z ation w ith the Wild Perennial Species of Subgenus Tripsac u m

In contra s t with m aize and teo s inte, whi c h hy b ridize under certain co n ditions, it is only

with extre m e difficulty and special techniq u es that m aize and the related pere n nial

species, Tripsacu m da c tyl o ides (gamma grass) hybridize. Further m o r e, hybrids of the

cross are m ale sterile, even after s everal bac k crosses to m aize (Russell and Hallauer,

1980).

A single species, Tripsacu m floridanu m (Florida g a m m a grass), found in the extre m e

southern F l o rida cou n ti e s of Mia m i

- Dade, Colli e r and Mo n r o e has been c atego r ized a s a

threatened s pecies by the state of Flori d a an d liste d o n th e USD A Natura l Resources

Conservation Service (USDA

- NRCS) datab as e (USDA

- NRCS

, 2012)

. Another species,

Tripsacum dactyl o ides (Easter n gamm a grass)

, foun d p r i m arily throughout the eastern

U.S., has been categorized as endangered in Massachusetts and Pe nnsylvania

, an d as

threatene d i n Ne w Yor k (U S DA

- NRCS, 2013). However, given the level of difficulty for

natura l hybridizatio n betwee n specie s o f Tripsacu m and Z ea a s m e ntione d above

, the

occurrence of T. floridanum pr i m arily in both highly u r banized and non

- agricultural,

swa mpy areas of the state where c o mmercial m ai z e i s no t typicall y g r o w n, as well as t h e

preference o f T. dactyloides for wet habitats where hybrid m aize production would not

occur, it is very unli k ely there would be any i m pact on this species due to the

introductio n o f MO N 87411.

X.J. Transfer of Genetic Information to Species with w h ich Mai z e Cannot

Interbreed (Hori z ontal Gene Flow)

Monsanto is aware of no reports confir m ing the transfer of genetic m aterial from maize to

other species with which m a i ze cannot interbreed. The prob abilit y fo r horizonta l gene

flow to occur is jud g ed to b e exceedin g ly s m all. Even if it w e re to occ u r, the

consequences would be negligible since th e CP 4 EPSP S an d Cry3Bb 1 protein s produced

M ons a n t o Co m pany CR 2 4 0

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i n MO N 8741 1 ar e th e sa m e protein s a s i n com m ercial M O N 863 and MON 88017 m aize

products and have been shown to have no m eaningful toxicity to hu m ans and NTOs.

Si m ilar to the situ a tion with tra n s f er of the p r otein ge n es, the li k elih o od of horizontal

transfer of the partial DvSnf7 gene from MON 87411 is also exceedin g ly s m all. The

consequenc e o f suc h transfer

, give n th e know n lack of toxicity to all but a very s m all

subset of organis m s (CRW species), would also b e expecte d t o b e inconsequential

. In

either case, the prese n ce of these genes wo u ld not be expected to incr e ase the pest

potential of the recipie n t species.

X.K. Potential I mpact on Mai z e Agronomic P ractices

An assessment of curre n t m aize agr o no m ic p r a c tices was co n ducted to d e ter m ine wheth e r

th e cultivatio n o f MO N 8741 1 h as the potential to i m pact current m aize m anage m ent

practices ( S ection IX). Maize fields are typicall y highl y m a nage d agricultura l area s that

are dedicated to crop production. O ther than t h e specific insertion of the partial Dv S nf7

gene that provides another MOA for protec t ion against CRW, MON 87411 is si m ilar to

m any other m aize hybrids being grown in the U.S. ( e.g.

, Ge n uity S m artStax, Genuity VT

Triple PRO, etc). Her b icide t o l e r a nt m aize has been in use commercially in the U.S.

since 1997 and constituted 73% of the tot a l maize crop in 2012 (USDA

- ERS, 2013).

CR W

- protected m aize has been planted com m e r c i all y sinc e 200 3 an d i n 201 0 constituted

53 % o f th e tota l U.S

. cro p (Brooke s an d B a rfoot

, 2012)

. C R W

- protecte d m a iz e hybrids

have been g rown on tens of m illions of acres in the U.S. since their i n troduction. Given

the widespread use of these HT and insect

- p rotected m aize hybrids, Monsanto anticipates

no specific changes to agrono m ic practices from introduction of MON 87411 above and

beyond those in current use.

MON 87411 is si m ilar to com m ercially cultivated m aize in its agrono m ic, pheno t ypic,

ecological, and co mpositio n al c h ar a cte r istics, a n d has le v els of resi s tan c e to i n sect p ests

and dise a s e s co mparable to o ther commerci a lly c u lti v ated m aize. Based on this

assess m ent, the introduction of MON 87411 is not likely to i m pact current U.S. m aize

agrono m ic or cultivation practices or lead to an increased plant pest potential co mpared

to other m aize hybrids w idely availabl e t o growers.

X.L. Conventional Breeding w i t h Ot h er Biotechnolog y

- derived or Conventional

Mai z e

Nu m erous biotechnology

- deriv e d m aize products have been deregulate d o r ar e under

consideration for deregulation by APHIS. Once deregulated, M ON 87411 m ay be bred

with these deregulated m aize products, as well as with convention a l maize, creating n e w

i m proved hybrids. APHIS has deter m ined that none of the individual biotechnology

derive d m a iz e product s i t ha s previousl y d e regulated displays i n creased plant pest

characteristics. APHIS has also c o ncluded tha t progen y derive d f r o m crosse s o f these

deregulated m aize products with convention a l or previously deregulated m aize are

unlikely to exhibit new plant pest properti e s. This presumption, that co m bined

- trait

biotechnology products are unli kel y t o exhibi t ne w charact e ristic s tha t woul d pos e new

plant pest risks not observed in the single even t biotec h product

, i s base d upo n several

facts. Namely: 1) s tability of the g enetic ins e rts is confirmed in each a pproved bi o tech

M ons a n t o Co m pany CR 2 4 0

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derived m aize product across m ulti p le generati o ns; 2) stability of each of the introd u ced

traits is continually and re p eatedly assessed as new co m bine d

- trai t hybrid s ar e create d by

plant b reed e rs and te s ted over m ulti p le s e asons p rior to co mm ercializ a tion; 3) co m b ined

trait products are developed u s ing conventional breeding that has been safely used for

thousands of years to generate new varie t ies (Steiner, et al., 2013; WHO, 1995); 4)

worldwide organizations, such as World Health Organization, Foo d an d Agriculture

Organization/ W orld Health Organization, International Seed Federation, CropLife

International and U.S. FDA, conclude that the safety of the co m bined

- trait product can be

base d o n th e safet y o f th e parenta l G E event s (CLI

, 2005 ; F AO

- WHO

, 1996 ; ISF

, 2005;

WHO, 1995); and 5) practical applications in the field have shown that two unrelated

biotechnology traits combined together by conventional breeding do not display new

characteristics or properties distinct from those present in the single event bi o tech

products (Pilacinski, et al., 2011 )

.

Therefore, based on the considerations abov e an d th e conclusio n tha t MO N 8741 1 i s no

more likely to pose a plant pe s t risk than c o mmercially cultivated m aize, it can be

concluded that any progeny derived from crosses between MON 87411 a nd conventional

m aize or deregulated biotechnology

- derived m a i ze are no more likely to pose a plant risk

than com m ercially c u lti v ated m aize.

X.M. Summary of Pl a n t Pest Assessments

A plant pest, as defined in the PPA, is t h e living stage of any of t he following, or a

si m ilar a r ticle, th a t can d ire c tly o r i n dire c tly injure, da m age, or cause disease in any plant

or plant product: (A) a protozoan; (B) a nonhu m an an i m al; ( C ) a parasiti c plant ; (D ) a

bacteriu m ; (E) a fungus; (F) a virus or viroi d ; (G) an infectious agent or other pathogen,

or (H) any article si m ilar to o r allied with any of the articles specified in the p recedi n g

subparagraphs (7 U.S.C. § 7702[14]). Charac t erization data presented in Sections IV

through VIII and Section X of this petiti o n confir m tha t MO N 87411

, with th e exception

of protection from CRW larval feeding and glyphosate tolerance, is not fund a m e ntally

different from conventional m aize, in ter m s of plant pest potential. Monsanto is not

aware of any study r e sult s o r observation s assoc i ate d wit h MO N 8741 1 tha t would

suggest an increased plant pest risk would result from its introduction.

The plant p est a s sess m ent was ba s ed on m ultiple lines of evidence d eveloped fr o m a

detaile d characterizatio n o f MON 87411 co mpared to conventional m aize, followed by a

risk assessment on detected differences. The p l ant pest risk assess m ent in this petition

was based on the following lines of evidence: 1) insertion of a single functional copy of

th e Cry3Bb 1 an d CP 4 E PSP S gene s an d DvSnf 7 suppression cassette; 2) characterization

and safety of the expressed prod u cts; 3 ) compositio n al equivale n ce of MON 87411

forage and grain co mpared to a convent i onal control; 4) phenotypic, agrono m ic, and

environ m ental ch a ra c te ri stics de m onstr a ting no increa s ed pl a nt pest p o t e nti a l co mpared

to conventional m aize; 5) ne g ligible risk to NTOs

, includin g org a nis m s beneficial to

agric u lt u re; 6) f a m ilia r ity with m aize as a culti v ated crop a n d 7) no greater likeliho o d to

i m pact agrono m ic practices, cultivation pract i ces

, o r th e m a nage m e n t o f weeds

, diseases

and insects, than conventional m aize.

M ons a n t o Co m pany CR 2 4 0

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Based on the data and info r m ation presented in this petition, it is concluded that, like

conventional m aize and previously d e regulated biotechnology

- derived m aize,

MON 87411 is not expected to be a plant pes t

. Results also support a conclusion of no

increased weediness potential of MON 87 4 11 co m pared to conventional m aize.

Therefore, Monsanto Co mpany r equests a deter m ination fr o m USDA

- APHIS that

MON 87411 and any progeny derived from c r osse s betwee n MO N 8741 1 an d other

commercial m aize be granted nonregula te d statu s unde r 7 CF R par t 340.

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XI. ADV E R S E C O NSEQUENCE S O F I N T RODUCT I ON

Monsanto knows of no study results or observations associated with MON 87411

indicating that there would be adverse consequences from its introduction. MON 87411

produces the Cry3Bb1 and CP4 EPSP S protein s an d th e DvSnf 7 RNA

. Th e Cry3Bb1

and CP4 EPSPS proteins produced in MON 87411 are identical to the sa m e proteins

presen t i n MO N 8801 7 tha t w a s previousl y grante d a det er m ination of nonregulated

status by USDA

- APHIS. The unique DvSnf7 RNA in MON 87411 has been thoroughly

characterized and its safety ha s bee n thoroughl y assesse d in this sub m ission. As

de m onstrated by field results and labora t ory tests, the only phenotypic differences

between MON 87411 a nd conventional m aize a r e protection from CRW larval feeding

and glyphosate tolerance.

The data and infor m ation presented in t h i s petitio n demonstrat e tha t MO N 8741 1 is

unlikely to pose an increased plant pest risk co mp ared to conventional m aize. This

conclu s ion is reached b ased on m u lti p le lines of evidence developed from a detailed

characterizatio n o f th e produc t compare d t o conventional m aize, followed by risk

assess m ent on detected differences. The cha r acterization e v aluations i n cluded m olecular

analyses, which confir m ed the insertion of on e cop y o f th e intende d DN A containin g the

DvSnf7 suppression ca s sette and t h e cry3Bb1 an d cp 4 epsp s expre s sion cassettes t h at is

stably integrated at a s i n gle loc u s a n d is in h erited accor d ing to M e ndelian principles over

multiple g e n erations.

Analysis of key nutrients, anti

- nutrie n t s

, and secondary m etabolites of MON 87411

de m onstrate that MON 87411 is compositionally equivale n t to conventional m aize. The

phenotypi c evaluations

, includin g an as s essment of seed ger m ination and dormancy

chara c te r istics, pla n t g r owth and develop m ent ch a ra c te ri stics, poll e n char a ct e ristics,

ecological interaction charact er isti c s, and environ m ental inter a ctio n s also in d icated

MON 87411 is unchanged co mpared to conventional m aize. There is no indication that

MON 87411 would have an adverse i m pact on beneficial or non

- target organis m s,

inclu d ing t h reatened or endangered species. T h erefore, based on the lack of increased

pest potential co mpared to conventional m a i ze, the risks for hu m ans, ani m als, and other

NTO s fro m MO N 8741 1 ar e negligible.

The introd u ction of MON 87411 will not ad v ersely i mpact cultivati o n practices or the

m a nag e m e n t o f weeds

, diseases

, an d insec ts in m aize production syste m s. Fa r m ers

f

a m iliar with CRW

- protected m ai z e products ( e.g

., Genuity VT Triple Pro or Genuity

S m artStax) curre n tly a v aila b le will be a dv ised to co n tin u e to e mploy the sa m e crop

rotational practices, weed contr o l practices an d / or volunteer contr o l m easures c u rre n tly in

place for t h ese products.

M ons a n t o Co m pany CR 2 4 0

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. W a n g

, T

. Zh a n g

, D. Z hu

, D

. Z h a n g

, J. X u

, Q. C h e n, Y. B a

, J. Liu

, Q. W a n g

, J

. C h en

, J

. W an g, M

. W a n g, Q. Z h a n g,

J. Zh a n g

, K. Zen a n d C

. - Y. Zha n g. 2 01 2 a

. E x og e n ous pl ant M I R 1 6 8a spec i f i ca ll y targe t s m a mm a lia n

LDL R A P 1: E vi dence o f c r os s

- k i n gd om reg ulati on by m i cr oR NA. Cel l Re search 2 2: 1 0 7

- 1 2 6.

Zh a n g

, Y.

, B.E

. W iggins

, C

. Lawrence

, J

. Pet r ic k, S

. Ivash u t a a nd G. H eck

. 2 0 12 b. An alysi s o f pla n t

d er i v e d m iR N A s i n a n i m a l smal l RN A d atasets

. BM C Gen o m ic s 13: 38 1.

Zh o u

, J.

, G.G

. Harri g a n, K.H

. Ber m a n, E.G

. W eb b, T.H

. Kl us m eye r an d M.A

. Ne m e th. 2 01 1. Sta b ilit y in

t he co m pos iti on e qu i va le n c e of g ra i n f ro m i nsec t

- p r otected m a ize a n d seed from glyphosate

- t o l erant

so y be a n t o c o nv e n tio nal co u n ter par t s o ver m ul ti p l e seasons, l oca tio ns, a nd bre e d in g g e r m p l as m s. Journal

of A g ric u lt u ra l an d Fo od C h emistr y 59: 882 2

- 8 8 2 8.

M ons a n t o Co m pany CR 2 4 0

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AP P END I C E S

M ons a n t o Co m pany CR 2 4 0

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App e ndi x A : USD A Not ification s an d Permits

Field trials of MON 87411 have been conducted in the U.S. since 2010

. Th e protocols

for these trials include field perfor m ance, breeding and observation, agrono m ics, and

generation of field m a t erials an d dat a necessar y fo r thi s petition. In addition to the

MON 87411 phenotypic assess m ent data, observational data on pest and disease stressors

were collected from these product develop m ent trials. The m ajority of the final reports

have been s ub m itted to the USDA. However, so m e f inal reports, m ainly f rom the 2012

-

2013 seasons, are still in pre p aration. A list of trials conducte d unde r USD A notifications

or per m its and the status of t h e f inal reports f or these t r i a ls are provided in Table A

- 1.

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Tabl e A

- 1

. USD A No t i fication s an d Per m it s Approve d fo r MO N 8741 1 an d Status

of Trials P l anted under These Notifications

USDA No. Effective date Release S t ate (# of site s ) Trial S tatus

2010 Field T r ials

10

- 0 5 0

- 1 0 1n 21

- Mar

- 2010 IL (4) Sub m itted

10

- 0 5 0

- 1 0 5n 23

- Mar

- 2010 IA (4) Sub m itted

10

- 0 5 4

- 1 4 1n 25

- Mar

- 2010 OH (1) Sub m itted

10

- 0 8 1

- 1 0 1n 16

- Apr

- 20 1 0 HI (1) Sub m itted

2011 Field T r ials

11

- 0 1 4

- 1 0 4n 13

- Feb

- 2 0 11 HI (2) Sub m itted

10

- 3 5 1

- 1 1 3 r m 15

- Feb

- 2 0 11 HI (1) Sub m itted

11

- 0 3 4

- 1 0 4n 02

- Mar

- 2011 IA (10) Sub m itted

11

- 0 3 4

- 1 0 2n 02

- Mar

- 2011 IL (12) Sub m itted

11

- 0 3 4

- 1 0 7n 02

- Mar

- 2011 OH (6) Sub m itted

11

- 0 3 4

- 1 0 5n 02

- Mar

- 2011 KS (6) Sub mitted

11

- 0 3 9

- 1 0 1n 09

- Mar

- 2011 IA (2), IL (4), NE (1) Sub mitted

11

- 0 4 1

- 1 0 7n 11

- Mar

- 2011 KS (5) Sub m itted

11

- 0 4 5

- 1 0 6n 13

- Mar

- 2011 IA (7) Sub m itted

10

- 3 5 1

- 1 1 7 r m 15

- Mar

- 2011 IL (1), IN (1) Sub m itted

11

- 0 4 5

- 1 0 3n 16

- Mar

- 2011 IL (8) Sub m itted

11

- 0 4 5

- 1 0 9n 16

- Mar

- 2011 NE (1) Sub m itted

11

- 0 4 7

- 1 0 2n 17

- Mar

- 2011 OH (4) Sub mitted

11

- 0 5 6

- 1 0 2n 27

- Mar

- 2011 IA (2), IL (2), NE (1) Sub mitted

11

- 0 6 2

- 1 0 1n 01

- Apr

- 20 1 1 IN (1) Sub m itted

11

- 1 5 2

- 1 0 4n 01

- Jul

- 2 0 11 PR (1) Sub m itted

11

- 1 2 3

- 1 0 4 r m 01

- Sep

- 2 0 11 HI (2) Sub m itted

11

- 2 6 5

- 1 0 4n 24

- Oct

- 2011 HI (2) Sub m itted

11

- 2 9 2

- 1 0 1n 18

- Nov

- 2 011 HI (1) Sub m itted

11

- 3 2 2

- 1 0 2n 18

- Dec

- 2011 HI (1) Sub m itted

11

- 3 2 2

- 1 0 1n 18

- Dec

- 2011 HI (1) Sub m itted

2012 Field T r ials

12

- 0 0 6

- 1 0 1n 05

- Feb

- 2 0 12 GA (1) Sub mitted

11

- 2 9 1

- 1 1 1 r m 15

- Feb

- 2 0 12 IA (4), IL (1), IN (1 ), KS (3) Sub mitted

11

- 3 0 5

- 1 0 5 r m 01

- Mar

- 2012 HI (3) Sub m itted

12

- 0 3 8

- 1 0 3n 08

- Mar

- 2012 IA (1) Sub mitted

11

- 3 2 0

- 1 0 5 r m 15

- Mar

- 2012 IA (5), IL (3), NE (4), SD (1) Sub mitted

12

- 51

- 101 20

- Mar

- 2012 IA (2), NE (3) Sub m itted

12

- 51

- 102 20

- Mar

- 2012 IA (1), IL (9) Sub m itted

12

- 0 5 1

- 1 0 3n 20

- Mar

- 2012 IA (4), IL (4) Sub m itted

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Table A

- 1 (continued). USDA No t ific a tions and Permits Approved for MON 87411

an d Statu s o f Trial s Plante d unde r Thes e Notifications

USDA No. Effective date Release S t ate (# of site s ) Trial S tatus

12

- 0 5 1

- 1 0 4n

11

- 3 2 6

- 1 0 8 r m

12

- 0 5 9

- 1 2 0n

12

- 0 5 9

- 1 0 9n

12

- 0 6 0

- 1 1 0n 20

- Mar

- 2012

22

- Mar

- 2012

28

- Mar

- 2012

28

- Mar

- 2012

30

- Mar

- 2012 IA (4), KS (2)

IA (11), IL (20)

IL (1), NE (1)

HI (1)

IA (1), IL (1), IN (1 ) Sub mitted

Sub mitted

Sub mitted

Sub mitted

Sub m itted

12

- 0 6 1

- 1 0 5n 31

- Mar

- 2012 IA (1), IL ( 1), NC (1), NE

(1), PA (1) Sub m itted

12

- 0 6 2

- 1 1 1n 01

- Apr

- 20 1 2 IA (1), IL (1), IN (1 ), KS (1) Sub m itted

12

- 0 6 5

- 1 0 9n 04

- Apr

- 20 1 2 IL (2), NE (1) Sub m itted

12

- 0 7 4

- 1 1 0n 13

- Apr

- 20 1 2 IA (1) Sub m itted

12

- 0 7 5

- 1 1 4n

12

- 1 1 6

- 1 0 5n 14

- Apr

- 20 1 2

07

- M a y

- 2 0 12 IA (1), IL (1), NE (1), SD (1)

CO (2), IA ( 2 ), IL (1), KS (5),

NE (1), SD (1) Sub m itted

Sub m itted

12

- 1 4 3

- 1 0 4n 20

- Jun

- 2 012 HI (4), PR (1) Sub m itted

12

- 1 2 5

- 1 0 6 r m

12

- 2 5 1

- 1 0 1n 01

- Sep

- 2 0 12

07

- Oct

- 2012 HI (3)

AR (1), IL ( 1), KS (1), NE

(1) In Progress

In Progress

12

- 3 1 2

- 1 0 3n 07

- Dec

- 2012 HI (2) In Progress

2013 Field T r ials

12

- 3 2 0

- 1 2 5 r m 07

- Mar

- 2013 KS (32), NE (20) In Progress

13

- 0 4 4

- 1 0 1 r m 07

- Mar

- 2013 HI (2), PR (2) In Progress

IA (32), NE ( 7), SD (10), TN

12

- 3 2 0

- 1 0 9 r m 08

- Mar

- 2013 (3) In Progress

12

- 3 1 2

- 1 0 9 r m 15

- Mar

- 2013 HI (8), PR(2) In Progress

IA (1), IL (37), IN (3), KS

12

- 3 2 0

- 1 1 4 r m 15

- Mar

- 2013 (10), MS (1) In Progress

13

- 0 3 9

- 1 0 2n 15

- Mar

- 2013 HI (11), PR ( 3) In Progress

IA (8), IL (23), KS (12), NE

13

- 0 5 1

- 1 0 1n 22

- Mar

- 2013

13

- 0 5 2

- 1 0 5n 23

- Mar

- 2013

13

- 53

- 109 24

- Mar

- 2013

13

- 53

- 110 24

- Mar

- 2013 (7) In Progress

IA (2), IL (1), MN (12), SD

(1) In Progress

IA (8), IL ( 8), MS (1 )

, NE

(4), SD (4) In Progress

IA (8), IL ( 8), MS (1 )

, NE

(4), SD (4) In Progress

13

- 0 5 9

- 1 0 3n 30

- Mar

- 2013 IA (5), IL (5), NE (4) In Progress

13

- 0 6 4

- 1 2 3n 04

- Apr

- 20 1 3 IA (3) In Progress

AR (1), CA (1), IA (5), IL

(10), IN (3), KS (3), LA ( 1),

MI (1), MO (3), MS (3), NC

(2), NE (5), OH (1), P A (2),

13

- 0 6 6

- 1 0 5n 06

- Apr

- 20 1 3 TX (2), WI (1) In Progress

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Table A

- 1 (continued). USDA No t ific a tions and Permits Approved for MON 87411

an d Statu s o f Trial s Plante d unde r Thes e Notifications

USDA No. Effective date Release S t ate (# of site s ) Trial S tatus

13

- 0 6 6

- 1 0 9n 06

- Apr

- 20 1 3 IA (11), IL (6), NE (4), SD

(5) In Progress

13

- 1 1 9

- 1 0 3n 29

- M a y

- 2 0 13 HI (11), PR ( 3) In Progress

13

- 1 9 1

- 1 0 2n 09

- Aug

- 2 013 GA (1) In Progress

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App e ndi x B : Materials

, Methods

, an d Supplementar y Result s fo r Molecular

Analyse s o f MO N 87411

B.1. Materials and Methods

B.1.1 Test Substance

Th e tes t substanc e i n thi s stud y wa s MO N 874 1 1

. Gen o m ic DN A fo r us e i n thi s study

was extr a ct e d f rom seed tissue li s ted in the table below.

Generation Seed Lot Number

R 4 113 0 88 1 7

R 4 F 1 113 2 01 7 3

R 5 113 2 63 1 3

R 5 F 1 113 3 31 7 6

R 6 113 3 88 5 8

B.1.2 Con t rol Substance

The control substances were the convention a l m aize lines which have si m ilar genetic

background as the generations as shown in t h e table below. Geno m ic DNA for use in

this study was extracted from seed tissue listed in the table below.

Control S u bstance Generations Seed Lot Nu m b er

LH244 R 4 , R 5 , R 6 112 6 47 4 7

LH244 × HCL645 R 4 F 1 ( h y br i d) 113 2 00 3 1

LH244 × LH287 R 5 F 1 ( h y br i d) 113 3 31 7 0

B.1.3 Reference Substance

The reference substance was plas m id vector PV

- ZMIR10871, which was used to develop

MON 87411. W hole plas m id served as a positi v e c o ntrol for s equencing and

bioin f or m atic analy s es. The ide n tity of t h e re f erence plas m id was con f ir m ed by

restriction enzy m e digestion p r ior to the study. Docu m entat i on of the confir m ation of the

plas m id vector ide n tity was archiv e d with the raw data. Appropriate m olecular size

m arkers from commercial sources were used f or siz e esti m a tion s o n agaros e gels

. The

unique identity of the molecular weight m arkers was docu m e nted in the raw data.

B.1.4 Cha r acteri z ation of Test, Control, and Reference S ubstances

The seed for the test and control substan c e s use d i n thi s stud y wer e obtaine d from

Monsanto T rait Develo pm ent. The synthesis re c ords f or the s e m ateri a ls a re loc a ted in the

ORION syste m

. The identities of the test s ubstance a n d the conventiona l control

substance were confir m ed by the sequencing in the study. No certificates of analysis

(COA) or v eri f ic a tion o f identity ( VOI) ce r ti f i cates were generated f o r t h ese m ateri a ls.

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The Study Director reviewed the chain of c u stody docu m entation to confirm the identity

of the test and control substances prior to the use of these materials in the study.

Test, control and reference DNA su b stance were considered stable dur i ng storage if they

yielded interpretable signals in sequencing experi m ents and/or did not appear visibly

degraded on the stained gels.

B.1.5 Genomic DNA Isolation

For sequencing library const r uction and PCR reactions, gen o m ic DNA was isolated from

seed tissues of the test and control substances. First the seeds were d e conta m inat e d by

vigorously agitating them by hand for 30 s econds with 0.05% (v/v) Tween

- 20, followed

by a tap w a ter rinse. T he seeds w e re then v ig o rously a g it a ted with 0. 5 % (w/v) N a OCl,

allowed to stand for one m inute at room t e m p e rature, and rinsed with tap water. The

seeds were then vigorously agitated with 1% (v/v ) HCl

, allowe d t o stan d fo r on e m i nute

at room temperat u re, a n d rinsed with tap water. The 1% (v/v) HCl rin s e was repe a ted

one ti m e, a n d then the s eeds were rinsed with d i stilled w a ter and p l ace d i n a dryin g oven

at 75°C

- 80°C to dry. The dri e d seed s wer e groun d t o a fin e powder in a Harbil paint

shaker. Geno m ic DNA was extracted using a hexadecyltri m ethylammonium bromide

(CTAB) extraction protocol. Briefly, 16 m l CTAB buffer (1.5% (w/v) CTAB, 75 m M

Tri s HC l (p H 8.0)

, 10 0 m M EDT A (p H 8.0)

, 1.05 M NaCl, and 0.75% (w/v) PVP) and

RNas e A wa s adde d t o groun d see d tissue

. Th e sa m p le s wer e incubate d a t ~6 5 ° fo r 60

m inutes with inter m ittent m ixing. The sa m ples were cooled to room te mperature and

subjecte d t o thre e round s o f chloroform :iso a m yl alcohol (24:1) extraction.

Approxi m ately 1.6 m l of 10% CTAB solution (10% (w/v) CTAB and 0.7 M NaCl) was

added to the sa m ples, m ixed by inversion and extra c ted once with chloro f or m :isoa m yl

alcohol (24:1). Approxi m ately 15 m l of CTAB precipitation buffer (1% (w/v) C T AB,

50 m M Tris HCl (pH 8.0), 10 m M EDTA (pH 8.0)) was added to the sa mples, m ixed by

inversion, and allowed to stand at room t e mp erature for 50

- 70 m inutes. Following

centrifugation to precipitate the DNA, the sa m p les were dissolved in high salt TE buffer

(10 m M Tr i s HCl (pH 8.0), 1 mM EDTA (pH 8.0), 1 M NaCl). The DNA was

preci p itated with 3 M sodium acetate (pH 5.2) and 100% (v/ v ) ethan o l. The DNA pellets

wer e washe d wit h 70 % ethanol

, ai r drie d an d resuspende d i n T E buffe r (1 0 m M Tris

HCl

, 1 m M EDTA

, p H 8.0).A ll extracted DNA was stored i n a 4° C refrigerator.

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B.1.6 DN A Quantification

PV

- ZMIR1087 1 DN A a n d extracte d geno m i c DN A wer e quantifie d usin g a QubitTM

Fluoro m eter (Invitrogen, Carlsbad, CA). Fo r d i recte d sequencing

, gen o m ic DN A was

quantified using a NanodropTM Spectrophoto m eter (Ther m o Scientific, W i l m ington, DE)

accor d ing to m anufacturer’s instructions.

B.1.7 Aga r ose Gel Electrophoresis

After quantification, approxi m ately 0.5

- 1 m i crogram (μg) of the extracted DNA for

NGS/JSA s e quencing library construction was r u n on a 1% (w/v) agarose gel to check the

quality.

B.1.8 She a r in g o f DNA

Approxi m a tel y 1 μ g o f DN A fr o m th e test

, contro l an d referenc e substance s wer e sheared

usin g a Covari s S

- 22 0 ultrasonicator

. Th e DN A wa s dilute d t o ~ 18

- 2 0 ng/μ l i n Buffer

EB (Qiagen Inc., Valencia, CA) and frag m en t ed using the f ollowing settings to cr e ate

approxi m ately 325 bp frag m ents with 3' or 5' overhangs : dut y cycl e o f 10 ; pea k incident

power of 175; intensity of 5.0, 200 bursts per c y cle, in the frequency s w eeping m ode at ~

6° C fo r 8 0 second s fo r tes t an d contr ol DN A o r 6 0 second s fo r referenc e DNA.

B.1.9 Bioanaly z er Analysis

On e m i crolite r o f sheare d geno m i c DN A wa s dilute d 1:1 0 i n Buffe r E B an d ru n o n a

DN A Hig h Sensitivit y chi p o n a n Agilen t 210 0 B ioanalyzer to check the quality of the

shearing. After pre p ari n g the c h ip according to the m anufa c turer’s instructions, 1 μ l of

each dil u ted DNA sa m ple or water was added to individual wells and the chip was r u n on

th e Bioanalyze r usin g th e dsDNA

, Hig h Sensitivit y Assa y reagents.

B.1.10 Paired End Library Preparation

Paired end geno m ic DNA libraries were pre p ared for the test, co n tr o l, and reference

substance s usin g th e Illu m i n a TruSe q DN A S a mple Preparation Kit (Illu m ina, San Diego,

CA) according to the m anufacturer’s instructi on s for the l o w

- through p ut procedure with

the following exception: a Sage Science P ippi n Pre p DN A Siz e Selectio n syste m (Sage

Science Inc., Beverly, MA) was used to size selec t th e DN A f r ag m e nt s instea d o f agarose

gel electro p horesis.

First, the 3' and 5' overhangs of the DNA frag m ents generated by the shearing process

were converted into blunt ends by adding 10 μl of Illu m ina Resuspension Buffer and

40 μl of Ill um ina End Repair m ix to each sa mple and m ixing thoroughly by pipette.

Then the libraries were incubated f or 30 m inutes at 30°C. The end

- repaired samples

were purified using AMPure XP beads (Beck m an Coult e r, Inc., Brea, CA) a n d

resuspended in 17.5 μl of Illu m ina Resuspension Buffer.

M ons a n t o Co m pany CR 2 4 0

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Fifteen m icroliters of each library w as tra n sferred to a new tube for adenylation, which

adds a single adenosine nucleotide to the 3' e n ds of the blunt frag m ents. Then 2.5 μl of

Illu m ina Resuspension Buffer and 12.5 μl of I llu m ina A

- Tailing Mix were added to each

library and m ixed thoroughly by pipetting. T h e libraries w ere incubated for 30 m inutes

at 37°C. After incubation, 2.5 μl each of ind i vidual adapter index, Illu m ina Resuspension

Buffer, and Illu m ina DNA Ligase Mix was i mm ediately added to each tube, and m ixed

thoroughly by pipetting to begin li g ation of each library. The libraries were incubated for

10 m inutes at 30°C. Then 5 μl of Illu m ina Stop Ligase Buffer was added to each tube

and m ixed thoroughly by pipetting to stop the ligation reaction. Next

, another

AMPure XP bead clea n up was perfor m ed on the libr a ri e s which were then resus p e nded

in 32.5 μl of Illu m ina Resuspension Buffer prior to size selection.

The libraries were run on the Sage Scie n ce Pippin Prep S ize Selecti o n system using

2% gel cas s ettes acc o rding to t h e m anufacturer’s instr u ctions. Ten m icroliters of loa d ing

solution were added to 30 μl of ea c h of the pu r ified libraries and m ixed thoroughly by

pipetting. F orty m icroliters of Marker B was loaded in the cassette wel l designate d for

the reference sa m ple, and 40 μl of each DNA library was loaded in the re m aining wells

for analysis. After elution of the d e sir e d size range (~445 bp) of DNA frag m ents, the

DN A s a mp l e i n th e elutio n cha m be r o f th e cassett e wa s remove d fr o m th e cassett e by

pipette and transferred i n t o PC R stri p tubes.

After re m o val from the Pippin Prep, the libr a ries were again put through the AMPure XP

bead cleanup procedure and resuspended in 22.5 μl of Illu m ina Resu s pension Buffer.

Twenty μl of the resuspended library was a dded to five microliters of Illu m ina PCR

Pri m e r Cocktai l an d 2 5 μ l o f Illu m i n a PC R Maste r Mi x an d m ixed thoroughly by

pipetting. The DNA frag m ents were enriched through PCR using the following cycling

conditions : 1 cycl e a t 98° C fo r 3 0 seconds ; 1 0 cycle s a t 98° C fo r 1 0 seconds

, 60° C for

3 0 seconds

, 72° C fo r 3 0 seconds ; 1 cycl e at 72°C for 5 m inutes. Following PCR

a mplificati o n, a final AMPure XP bead clea n u p was per f or m ed on the libr a ries w hich

wer e resuspende d i n 32. 5 μ l o f Illu m i n a R e su s p ensio n Buffer

. Finally

, 1 μ l o f eac h DNA

library was diluted 1:10 in Buffer EB for r unning in a DNA High Sensitivity chip on an

Agilent 2100 Bioanalyzer as described above. A ll purified library DNA was stored in a

-

20° C freezer.

B.1.11 Next

- Generatio n Sequencing

The library sa m ples described above were seq u enced by T he Geno m e Analysis Center

(TGAC, Monsanto) using Illu m ina HiSeq tech nolog y tha t produce s shor t sequenc e reads

(~100 bp long). Sufficient nu m bers of the s e sequenc e frag m e nt s wer e obtaine d (>75x

geno m e coverage) to co m prehensively cover the geno m e s of the test event and the

conventional control ( K ovalic et al., 2012). Sequencin g run s perfor m ed b y th e TGAC

were assessed versus standard QC criteria.

B.1.12 Junction Sequence Analysis Bioinformatics

High

- throughput sequence reads w ere e n riched by m apping to the PV

- ZMIR10871

transfor m ation plas m id sequence using the loc a l align m ent software BlastAll (V2.2.21) in

M ons a n t o Co m pany CR 2 4 0

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order to c o llect all reads that were sourc e d from the plas m id as well as reads with

sequences represe n ting inte g ra t ion point. All collected r e ad s wer e furthe r refine d by

re m oving sequencing artefacts of sequencing adapters

, redundan t read s an d lo w quality

read ends. All quality refined reads were then used to identify junction points with

custom developed bioinfo r m atics tools as det a iled below. All sign ifican t junction s are

reported for both the test and the control sa m ples. All software versions were

docu m ented in the archived data package and the software versions which were used in

thi s stud y hav e bee n archived.

B.1.12.1 Sequencin g Rea d Enrichment

The transfor m ation plas m id P V

- ZMIR10871 sequence was used as reference to find all

reads that w ere e ith e r f ully m atched to the insert plas m id frag m ents or partially m atched

with junction sequences. A junction sequence is characterized by a co m b ination of

transfor m ation plas m id sequence and flanking sequence that is likely to be host geno m e

flanking se q uence o r a n y other c o

- inserted se q uence. Local align m ent with Bla s tAll

(V2.2.21) was perfor m e d to collect all sequencing reads with an e

- Score of less than 1e

- 5

and at least 30 bases m atch of greater than 96.7% identity to the transfor m ation plas m id

(Kovalic et al., 2012). Both rea d s o f th e paired

- en d sequence s w ere collected in all cases.

B.1.12.2 R e ad Quality Refinement

In ord e r to identi f y all duplic a te r e ad pairs, a high quality seg m ent (bases 3

- 4 2 ) o f all

collected pairs was c o mpared to all ot h ers with short sequence align m ent software

(Bowtie v.0.12.3) allowing up to 1 mis m atch. If multiple read pairs were m atched at both

paired reads, such read pai r s were dee m ed redundant and only th e bes t qualit y pai r of

reads was kept for further analysis.

Co mputer software Novoalign (v.2.06.09) was used to remove any adapter sequences at

eithe r en d o f th e sequencin g reads

. Lo w qua lity read ends (with p h re d score s o f 1 2 or

lower) were t r im m ed. Only read s o f 3 0 base s o r lon g er after adapter and q u ality

trim m ing were collected. A custom deve l oped P erl script "far m _gen_ s m_bucket.pl" was

used to per f orm read enrich m ent and read quality refine m ent as described above.

B.1.12.3 Junction Detection

Enriched and quality refined read s o f bot h tes t an d contro l sa mp le s wer e aligne d against

the whole PV

- ZMIR10871 transfor m ation plas m i d sequence in order to detect junction

sequences using custom devel o ped Perl script "fa r m _blast _m ap.pl ". Reads with partial

m atch to the transfor m ation plas m id of at l east 30 bases m atch and 96.7% identity were

collected as potential junction sequences (Kovalic et al., 2012). The c o llecte d read s were

also aligned against the geno m ic s equence c o llection of the host geno m e in order to

re m ove junction reads sourced from the plant endogenous ho m ologues. Custom

developed P erl script “junctions_by_bn.pl” was used to identify the junction position on

the transfor m ation plas m id and their suppo r ting junction reads. F o r each junction

position, all supporting junction reads were ali g ned at the 30 plas m id bases proxi m al to

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 220 o f 3 7 2CB I Delete d Copy

the junction position. The re m aining bases o f thes e read s wer e sorte d t o sho w the

align m ent and the consensus of the flanking junction sequences past th e junctio n point.

B.1.12.4 Effective Se q u encing Depth Determination

A single copy locus from the native pla n t geno m e (Pyruvate decarboxylase ( pdc3 ),

GenBank accession.version: AF370006.2) was selected fr o m the Zea mays geno m e and

used to deter m ine the effective seq u ence depth c overage. All rea d s with at l e ast 30 b ases

m atch and 96.7% identity were considered as reads sourced from this locus. A custom

developed P erl script “ f ar m

_ m atch_reads.p l ” w as used to perform such align m ent and

calc u late the actual depth distribution at t h is loc u s.

B.1.12.5 Positive Spike

- in Controls

To produce “spike

- in” p ositi v e control sa m ples for sequencing, plas m id DNA libraries

wer e create d a s describe d abov e ( B .1. 8

– B.1. 1 0 ) an d the n dilute d to 1 and 1/10 maize

geno m e eq u ivalents (representation of the p las m id DN A a t concentra t ions equivalent to

single copy or 1/10 copy per geno m e) b e fore pooling with s a mples produced from the

contr o l m ateri a ls (as describe d above).

B.1.13 PC R an d DN A Sequen c e Analyse s t o Examine the Insert and Flanking

Sequence s i n MO N 87411

Overlappin g PC R products

, denote d a s Produc t A, Product B, Product C, Product D,

Product E, Product F, Product G and Product H were generated that span the insert and

adjace n t 5 ′ an d 3 ′ flanking DNA sequences in MON 87411. For each frag m ent generation

experi m ental conditions were chosen to su c cessfully produce on

- tar g et a mplifications.

These pro d ucts were analyzed to deter m i n e th e nucleotid e sequenc e o f th e inser t in

MON 8741 1

, as well as that of the DNA flanking the 5 ′ and 3 ′ end s o f th e insert.

The PCR analyses for Product A, Product D

, Product E and Product H were each

conducted using 50 ng of gen o m ic DNA t e mplate in a 50 μ l reaction volu m e. The

reaction contained a final concentration of 0.5 μ M of each pri m er and 1x concentration of

Phusion High Fidelity P CR Master Mix w ith H F Buffer (NEB, Ipswich, MA).

The PCR analyses for Product B, Product C and Product F were each conducted using

50 ng of geno m ic DNA te m plate in a 50 μ l reaction volume. The reaction contained a

final concentration of 0.2 μ M of each pri m er and 1. 2 5 units/reaction of Ex Taq

Poly m erase (Takara Bio Inc., Shiga, Japan).

The PCR a nalysis for Product G was each conducted using 50 ng o f geno m ic DNA

te mplate in a 20 μl re ac tion v o lu m e. The reactio n containe d a fina l concentratio n o f 0.5

μM of each pri m er and 1x concentration of Ph u s io n Hig h Fidelit y PC R Maste r M ix with

HF Buffer (NEB, Ipswich, MA).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 221 o f 3 7 2CB I Delete d Copy

The a mplif i cation of Product A, Product D, Product F and Product H were perfor m ed

under the following cycling conditions: 1 cycle at 9 8 ° C fo r 3 0 seconds ; 2 5 cycle s a t 98 ° C

for 10 seconds, 7 2 ° C for 2 m inutes 20 seconds; 1 cycle at 72 ° C for 5 m inutes.

The a mplification of Product B and Product C were perfor m ed under the following

cycling co n dition s : 1 c y cle at 98 ° C fo r 1 0 seconds ; 1 0 cycle s a t 7 0 ° C, decreasing 1 ° C per

cycle for 30 seconds, 7 2 ° C for 2 m i nutes; 25 c y cles at 9 8 ° C for 10 seconds, 6 0 ° C for 30

seconds

, an d 7 2 ° C for 2 m inutes.

The a mplification of Product E was perfor m e d under the following cycling conditions: 1

cycl e a t 9 8 ° C for 30 seconds; 25 cycles at 9 8 ° C for 10 seconds, 65 ° C fo r 2 0 seconds,

72 ° C for 2 m inutes; 1 cycle at 7 2 ° C for 5 m inutes.

The a mplif i cation of Product G was perfor m e d under the following cycling conditions: 1

cycl e a t 9 8 ° C for 30 seconds; 25 cycles at 9 8 ° C for 10 seconds, 71 ° C fo r 2 0 seconds,

72 ° C for 2 m inutes; 1 cycle at 7 2 ° C for 5 m inutes.

Aliquots of each PCR product were separated on a 1.0% (w/ v ) agarose gel and visualized

by ethidium bro m ide staining to verif y tha t th e product s wer e th e expecte d size

. Prio r to

sequencing, each verified PCR pr o duct was either purified usi n g th e QIAquic k PCR

Purification Kit (Qiagen, Inc., Valencia, CA) according to t h e m anufa c turer’s instructio n s

and quantified using the Nanodrop spectrophoto m eter according to the m anufacturer’s

instructions or purified using Exo

- SAP IT (Affy m etrix, San t a Clara, CA). The purified

PCR produ c ts were se q uenced usi n g m ultiple p ri m ers, including pri m ers used for PCR

a mplification. All s e quencing was per f or m e d by Mon s anto TGAC (The Geno m e

Analysi s Center ) usin g BigDy e te r m inator ch e m istry (Applied Bi o syste m s, Foster City,

CA).

A consensus sequence was gen e rated by co mpiling sequenc e s f rom m ultiple seq u encing

reactions perfor m ed on the overlapping PCR products. This consensus sequence was

aligned to the PV

- ZMIR10871 sequence to det e r m ine the integrity and organization of

the integrated DNA and the 5 ′ and 3 ′ insert

- to

- flan k DN A j u n c tion s i n MO N 87411.

B.1.14 PC R an d DN A Sequen c e Analyse s t o Examine the Integrity of the DNA

Insertion Site in MON 87411.

To exa m ine the MON 87411 T

- DNA insertion site in control m aize, PCR and sequence

analyse s wer e perfor m e d o n genomi c DN A fro m the conventional control m aize. The

pri m ers us e d in this an a lysis were d esign e d fro m th e DN A sequence s flankin g th e insert

i n MO N 87411

. A forwar d pri m e r specif ic t o th e DN A sequen ce flanking the 5 ′ end of

the insert was paired with a reverse pri m er specific to the DNA sequence flanking the 3 ′

end of the insert.

Th e PC R reaction s wer e conducte d usin g 5 0 n g o f gen o mi c DN A templat e i n a 5 0 μ l

reaction v o lu m e. The reaction con t aine d a fina l concentratio n o f 0. 5 μ M of each p ri m er

and 1x concentration of Phus i on High Fidelity P CR Master Mix with H F Buffer (NEB )

. The a mplification was perfor m ed under the followin g cyclin g conditions : 1 cycl e a t 98 ° C

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 222 o f 3 7 2CB I Delete d Copy

for 30 seconds; 25 cycles at 9 8 ° C fo r 1 0 seconds

, 72 ° C for 2 m inutes 20 seconds; 1 cycle

at 72 ° C fo r 5 m i nutes.

A s m all aliquot of each PCR product was s e parated on a 1.0% (w/v) agarose gel and

visualized by ethidium bro m ide staining to verify that the PCR products were the

expected size prior to sequencing. Only the verified PCR product from the conventional

control LH244 was either purified using t h e QIAquick PCR Purification Kit (Qiagen,

Inc.) accor d ing to the m anu f acturer’s instructions and q u antified using the Nanodrop

spectroph o to m eter acc o rding to t h e m anufacturer’s i n str u ctions or purified using Exo

SAP IT (Affy m etrix, Santa Clara, C A ). The purified PCR product was sequenced using

multiple p ri m ers, including pri m ers used f o r PCR a m plification. All sequencing was

perfor m ed by TGAC us i ng BigDye ter m i n ator che m istry (Applied Biosyste m s).

A consensus sequence was gen e rated by co mp iling sequenc e s f rom m u ltiple seq u encing

reactions perfor m ed on the verified PCR produ c t. This consensus sequence was aligned

to t h e 5 ′ and 3 ′ sequence s flankin g th e MO N 8741 1 ins e r t t o deter m in e th e integrit y and

organization of the insertion site.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 223 o f 3 7 2CB I Delete d Copy

B. 2 Suppl e m entar y Results

Junction Sequence Class A:

**R 4** 

Plasmi d DN A sequenc e ^Flank

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 224 o f 3 7 2CB I Delete d Copy

A

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 225 o f 3 7 2CB I Delete d Copy

A

A

A

A

A

**R 4**  **F 1** 

Plasmi d DN A sequenc e ^Flank

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 226 o f 3 7 2CB I Delete d Copy

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 227 o f 3 7 2CB I Delete d Copy

A

**R 5** 

Plasmi d DN A sequenc e ^Flank

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 228 o f 3 7 2CB I Delete d Copy

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 229 o f 3 7 2CB I Delete d Copy

**R 5**  **F 1** 

Plasmi d DN A sequenc e ^Flank

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 230 o f 3 7 2CB I Delete d Copy

A

A

A

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 231 o f 3 7 2CB I Delete d Copy

**R 6** 

Plasmi d DN A sequenc e ^Flank

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 232 o f 3 7 2CB I Delete d Copy

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 233 o f 3 7 2CB I Delete d Copy

A

A

A

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 234 o f 3 7 2CB I Delete d Copy

A

A

A

A

Junction Sequence Class B:

**R 4** 

Plasmi d DN A sequenc e ^Flank

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 235 o f 3 7 2CB I Delete d Copy

A

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 236 o f 3 7 2CB I Delete d Copy

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 237 o f 3 7 2CB I Delete d Copy

**R 4**  **F 1** 

Plasmi d DN A sequenc e ^Flank

A

A

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 238 o f 3 7 2CB I Delete d Copy

A

A

A

A

A

**R 5** 

Plasmi d DN A sequenc e ^Flank

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 239 o f 3 7 2CB I Delete d Copy

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 240 o f 3 7 2CB I Delete d Copy

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 241 o f 3 7 2CB I Delete d Copy

A

**R 5**  **F 1** 

Plasmi d DN A sequenc e ^Flank

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 242 o f 3 7 2CB I Delete d Copy

A

A

A

A

**R 6** 

Plasmi d DN A sequenc e ^Flank

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 243 o f 3 7 2CB I Delete d Copy

A

A

A

A

A

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 244 o f 3 7 2CB I Delete d Copy

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 245 o f 3 7 2CB I Delete d Copy

A

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 246 o f 3 7 2CB I Delete d Copy

Figure B

- 1. Junction sequences o f MO N 8741 1 detecte d b y NGS/JSA

Trim m ed nucleotide ali g n m ent of the detected junction sequences. The detected junction

sequences are ordered by junction sequence c l ass (Kovalic et a., 2012) and then by

length. The junction point bet w ee n th e T

- DN A b o rde r an d fl anking sequence is indicated

by the “^” character. Both align m ents are tri mm ed to include only the 30 plas m id bases

proxi m al to the junction point, as wel l a s al l sequenc e o f th e flank. Both align m ents are

show n 5 ′

- > 3 ′ beginning with the de t ected p las m id sequence.

References for Appendix B

Kovalic

, D.

, C

. Garnaat

, L

. Guo

, Y

. Yan

, J

. G roat, A. Silvanovich, L. Ralston, M. Huang,

Q. Tian, A. Christian, N

. Cheikh, J. Hj elle

, S

. Padgett e an d G

. Bannon

. 2012

. Th e us e of

next generation sequencing and junction seq u ence analysis bioinfor m atics to achieve

molecula r characterizatio n o f crops i mproved through m odern biotechnology. The P l ant

Geno m e Journal 5:149

- 163.

M ons a n t o Co m pany CR 2 4 0

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Appendi x C : Materials

, Me t hod s an d Re s ult s fo r Cha r acterizatio n o f Cry3Bb1

an d CP 4 EPSP S Pro t ein s Produce d i n MO N 87411

C.1 Characteri z ation of Cry3Bb 1 Protei n i n MO N 87411

C.1.1 Materials for Cry3Bb1 Characteri z ation

The MON 87411

- produced Cry3 B b1protein (lot 11355394) was purified from the grain

of MON 87411 (lot 11330153). The MON 87411

- produced Cry3 Bb 1 p r otei n wa s stored

in a

- 80 oC freezer in a b uffer soluti o n con t aining 10 m M sodium carbonate/ b icarbo n ate,

0.1 m M EDTA, pH 10.0.

Th e E

. Coli

- produce d Cry3Bb 1 protei n ( lot 11309151) was used a s th e reference

substance. The Cry3Bb1 pro tei n referenc e sub stance was generated from cell paste

produced by large

- scale fe r m entation of E. coli containing the pMON70855 expression

plas m id. The coding sequence for cry3 Bb 1 containe d o n th e expressio n plas m id

(pMON70855) was confir m ed prior to and a f ter fer m en t ation. The E. coli

- produced

Cry3Bb1 protein was chara c terize d previously.

C.1. 2 Cry3Bb 1 Protei n Purification

The plant

- produced Cry3Bb1 protein was purifie d fro m grai n o f MO N 87411

. The

purification procedures were not perfor m ed under a GLP p l an; however, all procedures

were documented on worksheets and, where applicable, SOPs were followed. Cry3Bbl

protein was extracted fr o m seven batches (~ 3 kg each) of MON 87411 grain with 50 mM

sodium carbonate/bicarbonate, pH 10.8, 1 m M EDTA

, 1m M Benza m idin e HCl

, 0. 5 mM

PMSF, 0.1% Triton X

- 100 (1: 5 (w/v) grain to buffer ratio). The extracts were clarified

by centrifugation. The clarified extract was br o ught to 4 0 % saturation b y the ad d iti o n of

ammonium sulfate and clari f ied by centrifugation. The resulting supernatant was brought

to 50% saturation by the addition of a m m onium sulfate

, clarifie d b y centrifugation

, and

the pellet resuspended in on e

- tenth the o r igi n al volu m e i n Capt o Q Buffe r A (5 0 mM

sodium carbonate/bicarbonate, pH 10.8, 1 m M EDTA

, 1m M Benza m idin e HCl

, 0. 5 mM

PMSF). The diss o lved pell e ts were then d i luted 1:1 with CaptoQ buffer A and loaded

onto a Capto Q (GE H e althcare, Piscataway, NJ) anion exchange col u mn that was pre

equilibrated with CaptoQ buffer A. The colu m n was wash e d with 20% buffer B (50 m M

sodium carbonate/bicarbonate, pH 10.8, 1 m M EDTA

, 1 m M Benza m idin e HCl

, 0. 5 m M

PMSF, 500 m M NaCl). The Cry3Bbl protein w a s elute d wit h 40 % buffe r B

. Th e eluted

protei n wa s dilute d 1: 1 wit h 100m M Tris

- HC l p H 8

, 1 M ammoniu m sulfate

, 1 mM

EDTA, 1 mM Benza m idine HCl, 0.5 m M PMS F

. The diluted sa m ple was loaded onto a

Phenyl Se p harose HP (GE Healthcare) colu m n pre

- equilibrated with P henyl Sepharose

buffer A (50 m M Tris

- HCl pH 8, 0.5 M a m monium sulfate, 1 m M EDTA, 1 mM

Benza m idine HCl, 0.5 m M PMSF )

. The column was w a shed with Phenyl Sepharose

buffer A and then eluted with 38% Phenyl Sepharose buffer B (50 mM Tris

- HCl pH8, 1

m M EDTA, 1 m M Benza m idine HCl, 0.5 m M PMSF). As a final purification step, the

Phenyl Sepharose 38% buffer B pool containing the Cry3Bb1 passed at a slow flow r a te

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 248 o f 3 7 2CB I Delete d Copy

through a P B S equili b rated immunoaffinity col u mn. The c o lu m n consisted of Pr o tein L

Agarose cross

- linked to an anti

- Cry3 B b1 m o noclonal antibody. The bound Cry3Bb1

protein was eluted from the column u s ing 100 m M glycine/150 mM NaCl, pH 2.5.

Fractions were collected, immediately neutr a lized to pH 8.0 using 1 M Tris

- HCl, pH 8.0.

Elution fractions were pooled and concent r ated using Spin X spin concentrators

(Corning, Corning NY) with a 10K M W CO pre t reated with 5% PEG (average m olecular

weigh t 3350)

. Thi s MO N 8741 1 Cry3Bb 1 pu rified from the grain of MON 87411 was

aliqu o ted a n d stored in a

- 80 °C freezer.

The final buffer co m position of the s a mple w a s 10 m M sodium carbonate/bicarbonate,

0. 1 m M EDTA

, p H 10.0

. Th e purifie d MO N 87411

- produce d Cry3Bb 1 protei n was

aliqu o ted, assigned l o t 1 1355394, and stored at in a

- 80°C freezer.

C.1.3 N

- T e rmina l Se q uencin g o f Cry3Bb1

C.1.3.1 Methods

N

- ter m inal sequencing by auto m ated Ed m an d egradatio n che m istr y wa s carrie d ou t i n an

atte mpt to confirm the identity of MON 87411

- produced Cry3Bb1

MON 87411

- produced C ry3Bb1 was separated by SDS

- PAGE and transferred to PVDF

m e mbrane. The blot was stained using Coo m assie Blue R

- 250. The major bands at 77

and ~65 kD a containin g MO N 87411

- produce d Cry3Bb 1 wer e excise d fr o m the blot and

used for N

- ter m inal se q uence anal y sis. The analysis was perfor m ed for 15 cycles using

auto m ated Ed m an degradation che m istry (Hu n kapiller et al., 1983) using an Applied

Biosyste m s 494 Procise Sequencing System e quipped with 140C Microgradient system a

Perkin El m er Series 200 UV/VIS A b sorbance Detect o r with Procis e  Control Software

(version 2.1). Chro m atographic data were collected using SequencePro (Applied

Biosyste m s, Foster City CA; version 2.1). A phenylthiohydantoin

- a m i n o aci d (PTH

- AA)

standard solution (Applied Biosyste m s) was u s ed to chr om atographically c a li b rate the

instru m ent for the analysis. A control protein, β

- lactoglobulin, (Applied Biosyste m s) was

analyzed before and after the sequence analysis of the MON 87411

- produced Cry3Bb1

protein to verify that the sequencer m et perfor m ance criteria f o r repetitive yield and

sequence identity.

C.1.3.2 Results of the N

- terminal Sequence Analysis

N

- ter m inal sequencing analysis was p e rfor m ed on the MON 87411

- produced Cry3Bb1

protein. The reaction did not yield any observable sequence for the ~77.0 kDa band,

presu m ably becau s e t h e N

- ter m inus was b l o cked. It is well do c u m ented t h at the N

ter m inal residue of many proteins is b locke d du e t o post

- translationa l m od i ficatio n in

vivo (Polevoda and Sher m an, 2000). Blocked N

- t e r m inal a m ino acids cannot be directly

sequenced by Ed m an degradation.

N

- ter m inal sequencing of the first 15 a mi no acids was perfor m ed on the ~65 kDa band of

MON 87411

- produced Cry3Bb1. A sequence for the Cry3Bb1 protein deduced from the

cry3Bb1 gene present in grain o f MO N 8741 1 wa s observed

. Th e dat a obtained

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 249 o f 3 7 2CB I Delete d Copy

correspond to the deduced Cry3Bb1 protein be g inning at a m ino acid po s itio n

- 50 (Table

C

- 1). Hence, the sequence infor m a tion con f ir m s the identity of the ~65 kDa Cry3Bb1

band isolated from the grain of MON 87411.

Table C

- 1. N

- Terminal Sequence of the MON 87411

- produced Cry3Bb1

MW Expected Sequence Experimental Results Position

77 kDa MANPNNRSEHDTIKV No Sequence Observed

65 kDa TEDSSTEVLDNSTVK TEDSSTEVLDNSTVK 50

- 64

The expected a m ino acid sequence of the N

- ter m i n us of the

~ 6 5 kDa Cr y 3 Bb1 protein was

deduced from the cry3Bb1 gene present in MON 87411. The experimental sequences obtained

from the MON 87411

- pr o duced Cry3Bb1 were c o mpared to the expected se q uence. The single

letter IUPAC

- IUB amino acid code is A, alanine; D, aspatic acid; E, gluta m ic acid; H, histidine;

I, isoleucine; K, l y sine; L, leucine; M, meth i onine; N, asparagine; P, proline; R, arginine;

S, serine; T, threonine; V, valine.

C.1.4 MALDI

- TOF Tryptic Mass Map Analysis of Cry3Bb1

C.1.4.1 Methods

MALDI

- TOF MS m ass fingerpri n t analysis w as used to con f irm the identity of the

MON 87411

- produced Cry3Bb1 protein. A MON 87411

- produced Cry3Bb 1 protein

sa mple was separated by SDS

- PAGE and t h e gel was stained using Brilliant Blue G

Colloid a l s t ain ( S ig m a

- Aldrich, S t. Louis, M O ). Each ~77 kDa band was excised and

destained w ith 40% (v/v) m et h anol / 1 0 % (v/ v ) acetic ac i d. The excised bands were

washed in 100 m M a mmonium bicarbonate a n d treated with 10 m M dithiothreitol (DTT)

at 37°C for 2 h followed by incubation for 20 m i nute s wit h 1 0 m M iodoaceti c aci d i n the

dark. The excised bands were then washed with 25 m M ammonium bicarbonate, dried

using vacuum centrifugation and rehydrated with 20 μl 20 μg/ m l t r ypsin (Pro m eg a

, Madison, W I). After 1 h, excess liquid was re m oved and the excised bands were

incubated at 38°C overnight in 40 μl of 10% (v/v) acetonitrile in 25 m M a mmonium

bicarbo n ate. The excised bands were sonica t ed for 5 m in a nd the resulting extract was

transferred to new m icrocentrifuge tube labeled Extract 1 and dried using vacuum

centrifugation. The excised bands were then e x tracted two more ti m es, each with 30 μl

of a 60% (v/ v ) acetonitrile, 0.1% (v/ v ) trifluoroacetic acid, 0.1% (v/w) β

- octyl

glucopyranosid e solutio n an d sonicate d for 5 m in. These two extracts were pooled

into a new tube labeled Extract 2 a nd dried using vacuum centrifugation. Extract 1 an d 2

wer e the n treate d wit h 0.1 % trifluo r oacetic acid (T F A ) and dried. The extracts were

solubilized in 50% (v/v) acetonitrile, 0.1 % TF A an d sonicate d fo r 5 m i n

. Extracts wer e

spotte d t o well s o n a n analysi s pl ate and m ixed with either 2,5

- dihydroxybenzoic acid

(DHB, The r m o F i sher Scientific Inc. W altha m

, MA), α

- cyano

- 4

- hydroxycinna m ic acid

( α

- Cyano, Ther m o Fisher Sci e ntific Inc.), or 3,5

- di m e thoxy

- 4

- hydroxycinna m ic acid

(Sinapi n ic a cid, Ther m o Fisher Sci e nti f ic Inc

. ). The sa m ples in DHB, α

- Cyano

, and

Sinapinic acid m atrix w ere analyze d i n th e 30 0 t o 500 0 Da

, th e 50 0 t o 500 0 Da

, an d 500

to 7000 Da range, respectively. CalMix 2 T M was used as the external calibrant

(Sequazy m e T M Peptide Mass Standards kit, AB SciEx, Foster City, CA). The analysis

was perfor m ed on a Voyage r T M DE Pro B i ospectro m etry T M work s t atio n (Applied

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 250 o f 3 7 2CB I Delete d Copy

Biosyste m s) using Voyager Instrument Control Panel software (version 5.10.2) and Data

Explorer data analysis software (version 4.0.0.0). Protonated peptide m asses were

isotopically resolved in reflector mode ( A ebersold 199 3 ; Billeci a n d Stults 1 9 93).

GPMAW32 software (Lighthouse Data, Odense M, Den m a r k) was used to generate an in

silico digest of the Cry3Bb1 protein sequence. Masses within 1 Da of the m onoisotopic

m ass were m atched against the in silico digest of the MON 87411

- produced Cry3Bb1

sequence. All m atching m a sses were t a llied and a coverage m ap was generated for the

m ass fingerprint.

C.1.4.2 Results of M A LDI

- TOF Tryptic Mass Map Analysis

The identity of the MON 87411

- produced Cry3Bb1 protein was conf i r m ed by M A LDI

TOF MS analysis of peptide fragments produced by the trypsin digestion of the MON

87411

- produced Cry3Bb1 protein. There were 41 unique peptides identified that

corresponded to the expected m asses (Table C

- 2). The identified m asses were used to

asse m b l e a peptid e m ap o f th e Cry3Bb 1 pro t ein (Fig u r e C

- 1). T he experimentally

deter m ined coverage of the Cry3 B b1 protein wa s 73 % (47 8 ou t o f 652 a m ino acids).

This anal y sis further co n f ir m s the identity of MON 87411

- produced Cry3Bb1 protein.

As part of the m ass fingerprint analysis, a m ass was identified that corresponded to the

predicted mass of an acetylated N

- ter m inal tryptic frag m ent. The N

- ter m inus of the ~77.0

kDa MON 87411

- produced Cry3Bb1 protein beg i ns with alanine at po s iti o n two.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 251 o f 3 7 2CB I Delete d Copy

Table C

- 2. Summary of the Tryptic M asses Id en tified f o r the

MO N 87411

- Produce d Cry3Bb 1 Using MALDI

- TOF MS

O bs erved Ma s s 1 Ex p ected Ma s s Diff. 2 Fr agm e n t S e qu e n c e 3

564.33 564.31 0.02 45

- 48 EFLR

619.35 619.34 0.01 299

- 303 TELTR

686.45 686.42 0.03 155

- 160 TPLSLR

727.37 727.35 0.02 2

- 7 4 Ac

- ANP N N R

794.44 794.40 0.04 394

- 400 LSFDG Q K*

794.48 0.04 292

- 298 LYSKG V K*

877.13 876.47 0.66 161

- 167 SKRSQDR

897.61 897.58 0.03 513

- 520 ITQLPVVK

925.51 925.47 0.04 573

- 580 YASTTNLR

937.56 937.53 0.03 348

- 355 LRPGYFGK

958.58 958.56 0.02 646

- 653 IEFIPVQL

1026.52 1026.49 0.03 258

- 266 GSTYDAWVK

1067.58 1067.52 0.06 181

- 190 NSMP...AVSK

1128.55 1128.56 0.01 377

- 386 TITS...YGDK

1185.67 1185.70 0.03 556

- 566 VTLN...LLQR

1350.67 1350.64 0.03 170

- 180 ELFS...SHFR

1362.72 1362.70 0.02 490

- 500 GTIP... W THR

1385.68 1385.66 0.02 501

- 512 SDVF....DAEK

1457.84 1457.74 0.10 404

- 417 TIAN....PNGK

1460.93 1460.86 0.07 554

- 566 FKVT....LLQR

1496.82 1496.77 0.05 319

- 331 YGPT....NSIR

1590.97 1590.91 0.06 641

- 653 IYID.....PVQL

1619.90 1619.83 0.07 168

- 180 IREL....SHFR

1649.96 1649.84 0.12 626

- 640 NELI....SNEK

1765.05 1764.98 0.07 304

- 318 DIFT....TLQK

2001.13 2001.03 0.10 332

- 347 KPHL....FHTR

2041.22 2041.08 0.14 581

- 597 LFVQ....YINK

2147.03 2146.93 0.10 472

- 488 AYSH....MQDR

2298.38 2298.25 0.13 273

- 291 EMTL....YDIR

2394.23 2394.10 0.13 356

- 376 DSFN....GSSK

2402.48 2402.35 0.13 191

- 211 FEVL....LLLK

2483.41 2483.32 0.09 521

- 545 AYAL...LFLK

2484.26 2484.06 0.20 212

- 232 DAQV....EFYR

2499.18 2499.08 0.10 425

- 445 VDFS....YDSK

2552.36 2552.20 0.16 237

- 257 LTQQ....NGLR

2637.54 2637.29 0.25 131

- 153 ALAE....NSWK

2655.39 2655.18 0.21 425

- 446 VDFS.....DSKR

2734.57 2734.28 0.29 447

- 471 NNGH....PLEK

3444.04 3443.64 0.40 15

- 44 VTPN... ..L NYK*

3443.71 0.33 404

- 434 TIAN...

... DDQK*

3988.68 3988.93 0.25 15

- 48 VTPN.....EFLR

1 The obs e r ved m a ss was collected from a t leas t one of t h re e m atrice s i n clu d in g α

- cya no, D HB and

s i nap i n i c ac i d. The o bse r v e d m a ss sh ow n i s th e m as s closes t t o th e expec t e d m ass.

2 The da t a r e p r e sent t he ca l cu l ate d d i ff e rence be tw e e n t he e xpect e d m a ss and the observ e d m a ss

3 For p e p ti de m atc hes grea t e r t h a n n i ne a m i no ac i ds i n le ng t h t he f i rst 4 r e s id ues a nd la st 4 res id u e s a r e

sh ow n s e p a ra te d by th ree do t s (

... )

4 A C i s t he abbre v iatio n fo r acetylati on

* T h e expecte d peptid e m asse s ar e ne a r l y identica l ( < 1 d a lton). Because this analysis cou l d not det e rmine

wit h certai n t y w h ic h ex p ecte d p e p ti de wa s act u all y obs e r ve d

, t he p e p t i des w it h a n a s te r i sk ( *) were not

i nc l ud e d i n de t e r m i n i ng se q u e nce c ov e r a ge ( F i g u re C

- 1 ).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 252 o f 3 7 2CB I Delete d Copy

001 M ANPNN R SEH DTI K VTPNSE LQTNHNQYPL ADNPNSTLEE LNYKEFL R MT

051 EDSSTEVLDN STVKDAVGTG ISVVGQILGV VGVPFAGALT SFYQSFLNTI

101 WPSDADPWKA FMAQVEVLID KKIEEYAKSK ALAELQGLQN NFEDYVNALN

151 SW KK TPLSLR SKRSQDRIRE LFSQAESHFR

NSMPSFAVSK FEVLFLPTYA

201 QAANTHLLLL KDAQVFGEEW GYSSEDVAEF Y R RQL K LTQQ YTDHCVNWYN

251

VGLNGLRGST YDAWV K FNRF R R EMTLTVLD LIVLFPFYDI

R LYSKGV K TE

301

LTRDIFTDPI FLLTTLQKYG PTFLSIENSI RKPHLFDYLQ GIEFHTRLRP

351

GYFGKDSFNY WSGNYVETRP SIGSSKTITS PFYGD K STEP VQKLSFDGQK

401

VY R TIANTDV AAWPNG K VYL GVT K VDFSQY DDQKNETSTQ TYDSKRNNGH

451 VSAQDSIDQL PPETTDEPLE KAYSHQLNYA ECFLMQD RR G TIPFFTWTHR

501

SVDFFNTIDA EKITQLPVVK AYALSSGASI IEGPGFTGGN

LLFL K ESSNS

551

IA K FKVTLNS AALLQ R YRVR I R YASTTNLR LFVQNSNNDF LVIYIN K TMN

601 KDDDLTYQTF DLATTNSNMG FSGD K NELII GAESFVSNEK IYIDKIEFIP

651 VQL

Figure C

- 1. MALDI

- TOF MS Coverage Map of the MON 87411

- produced

Cry3Bb1

The a m ino acid sequence of the MON 874 1 1

- pr o d u ced Cr y 3Bb1 protein was deduced from t h e

cry3Bb1 gene present in MON 87411. Boxed regions correspond to t r y ptic peptides that we r e

identified fr o m the MON 874 1 1

- pr o d u ced Cr y 3Bb1 protein sample using MALDI

- TOF MS. In

total, 73% ( 4 78 o ut of 652 a m ino acids) of t h e expected protein sequence was i dentified.

C.1.5 Wester n Blo t Analysis

- Immunoreactivit y o f Cry3Bb1

C.1.5.1 Methods

W estern blot analy s is was per f or m ed as f o llows to c on f irm the identity of the

MON 87411

- produced Cry3Bb1 protein and to co mpare the im m unoreactivity of the

MO N 87411

- produce d an d E. col i

- produce d Cry3Bb 1 prote i n

. MO N 87411

- produced

and E. col i

- produced Cry3Bb1 protein were diluted in 1× LB (62 m M Tris

- HCl, 5% (v/v)

2

- m ercaptoethanol, 2% (w/v) sodium dodec y l sulfate (SDS), 0.005% (w/v) bro m ophenol

blue

, 10 % (v/v ) glycerol

, p H 6.8 ) an d heate d t o 100° C fo r 3 m i nutes

. Eac h protein

sa mple was loaded in d u plic a te at

~ 1

, 2, a n d 3 n g purit y correcte d Cry3Bb 1 protei n per

lane onto a pre

- cast Tris

- glycine ( 4

- 20 %) polyacryla m ide gradient m ini

- gel (Invitrogen).

Pre

- stained molecular w eight s tandard s (Precisio n Plu s Prote in Standards, Bio

- Rad) were

loaded on the gel for molecular weight refer e nce and to verify electrotransfer o f the

proteins to the m e m brane. Following electroph o resi s a t a constan t v o ltage

, protein s were

electrotrans f erred to a P VDF m e mb r ane (Invitrogen).

The m e mbrane was blocked with 5% nonfat dry m ilk (NFDM) in 1 × phosphate buffered

salin e containin g 0.05 % (v/v ) Tween

- 2 0 (PBST ) an d incubate d wit h a goa t anti

- Cry3 B b1

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 253 o f 3 7 2CB I Delete d Copy

antibody (lot G839074) at a dilution of 1:2500 i n 1 % NFDM in PBST. After washing

with PBST, the m e m brane was inc u bated with horseradis h peroxidas e (HRP )

- conjugated

horse a n ti

- g oat IgG (H+L) IgG (Ve c tor Labs, Burlinga m e CA ) a t a dilutio n o f 1:500 0 in

1 % NFDM in PBST and washed again, with PBST. Immunoreactive bands were

detected using the ECL TM detection system (GE Healt h care) and A m ersham Hyperfilm

(GE Healthcare). The film was d e velop e d using a Konica SRX

- 101A auto m ated film

processor (Konica Minolta Medical & Graphic, Inc., Tokyo, Japan).

Quantificatio n o f th e band s o n th e blo t wa s perfor m ed on a GS

- 800 densito m eter with the

supplied Q uantity One  software (version 4.6.7, Bio

- Rad) using the using the lane

selection and contour tool. The signal int ensitie s o f th e i mmuno reactive bands m igrating

at the expected position for the Cry3Bb1 protein were quantified as “contour quantity”

values. The immunoreactivity wa s reporte d i n O D × m m 2

.

C.1.5.2 Results of Cry3Bb1 Prote i n Immunoreactivity E q uivalence

Western blot analysis was conducted us in g goa t anti

- Cry3Bb 1 polyclona l antibod y as

additional m eans to confirm the identity of the Cry3Bb1 protein isolated from the grain of

MON 87411 and to assess the equivalenc e o f th e im m unor e a ctivit y o f the

MO N 87411

- produce d an d E. coli

- produce d Cry3Bb 1 proteins.

The results showed that immunoreactive b ands m igrating at the expected appare n t M W

were present in all lanes loaded with the MON 87411

- produced (Figure C

- 2, lanes 10

- 15)

or E

. col i

- produced (Figure C

- 2, lanes 3

- 8) Cry3Bb1 proteins. For each a m ount loaded,

co mparable signal intensity was observed between the MON 87411

- and E. coli

- p ro d uced

Cry3Bb1 protein bands. As expected, the sign a l intensity i n crea s ed with increa s ing load

a m ounts of the MON 87411

- produced and E

. col i

- produced Cry3Bb1 proteins, thus

supportin g identificatio n o f MO N 87411

- produced Cry3Bb1 protein.

To co mpare the im m unoreacti vit y o f th e MO N 87411

- an d th e E. col i

- produce d Cry3Bb1

proteins, densito m etric analysis was conduct e d on bands that m igrated to the expected

apparent MW for Cry3Bb1 p r oteins (~77

- 55 kDa). The s ignal i n ten s ity ( r eport e d in

O D × m m 2 ) of the immunoreacti v e bands identi f ied by anti

- Cry3Bb1 antibo d ies and

m igrating between the f ull

- length protein at 77.0 kDa and the insectici d al tryptic core (at

~55 kDa) were included in the m ean signal intensities (Table C

- 3). B ecause the m ean

signal intensity of the MON 87411

- produced C ry3Bb1 protein band was within 35% of

the m ean signal intensity o f the E. col i

- produce d Cry3Bb 1 protein

, the

MON 87411

- produced and E. coli

- produced Cry3Bb1 proteins were deter m ined to have

equivalent immunoreactivity.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 254 o f 3 7 2CB I Delete d Copy

L a ne 3 4 5 6 7 8 9 10 1 1 12 13 14 15

k D a

250

150

100

75

50

37

25

20

15

10

Figure C

- 2. Western Blot Analysi s o f MO N 87411

- an d E. coli

- pr o duced Cry3Bb1

Proteins

Aliquots of the MON 87411

- p r oduced Cry3Bb1 p rotein and t he E. coli

- p r oduced C r y 3 Bb1

protein were subjected to SDS

- PAGE and electrotransferred to a PVDF m e m b r a ne. Proteins

were detec t ed using anti

- Cr y 3Bb1 ant i bodies as the pri m ary ant i bodies. Immunoreactive bands

were visualized using HRP

- conjugated second a ry antib o dies and an EC L s y ste m

. The

MWs (kDa) of the standards are shown on the left. Lane 1 and 2 were cropped from the image.

The 15 second exposure is shown. Lane designations are as follows:

Lane Sample Amount (ng)

1 Precision Plus Protein Standards Dual color

2 Blank

3 E. coli

- Prod u ced Cr y 3B b 1 protein 1

4 E. coli

- Prod u ced Cr y 3B b 1 protein 1

5 E. coli

- Prod u ced Cr y 3B b 1 protein 2

6 E. coli

- Prod u ced Cr y 3B b 1 protein 2

7 E. coli

- Prod u ced Cr y 3B b 1 protein 3

8 E. coli

- Prod u ced Cr y 3B b 1 protein 3

9 Blank

10 MON 87411

- produced C r y3Bb1 p r otein 1

11 MON 87411

- produced C r y3Bb1 p r otein 1

12 MON 87411

- produced C r y3Bb1 p r otein 2

13 MON 87411

- produced C r y3Bb1 p r otein 2

14 MON 87411

- produced C r y3Bb1 p r otein 3

15 MON 87411

- produced C r y3Bb1 p r otein 3

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 255 o f 3 7 2CB I Delete d Copy

Table C

- 3. Comparison of Immunore a ctive Signal Bet w een MON 87411

- and E

coli

- produced Cry3Bb1Proteins

Mean Signal intensi t y from

MON 87411

- produced C r y3Bb 1 1

(O D x m m 2 ) Mean Signal intensi t y from

E. coli

- produce d Cr y 3 Bb1 1

(O D x m m 2 ) Acceptan c e l i mit s 2 for

MON 87411

- produced

Cr y 3 Bb1(O D x m m 2 )

7.35 7.02 4.56

- 9 .48

1 Eac h valu e r e pres e n t s t he mea n o f si x value s (n=6)

2 Th e ac c ept a nc e li m it s ar e fo r th e MO N 8 74 1 1

- p r o d uc e d C r y 3 Bb 1 p ro t e i n a n d a re based on t he i n ter val

be t we e n + 3 5 % ( 7

. 02 × 1 .3 5) a nd

− 3 5% ( 7.0 2 × 0

. 6 5 ) of th e m ean of t he E

. Coli

- p ro du c ed C r y 3 Bb 1 s i gnal

i n te ns it y acr o s s al l l o a d s.

C.1.6 Molecular Weight Estimat i on of Cry3Bb1 using SDS

- PAGE

C.1.6.1 Methods

MON 87411

- produced and E. coli

- produced Cry3Bb1 protein were diluted in 1× LB and

heated to 95

- 105 °C for 3

- 5 m in. The MO N 87411

- produce d Cry3Bb 1 protei n was

loaded in duplicate at 1, 2, and 3 μg onto a pre

- cast Tris

- glycine ( 4

- 2 0 % ) polyacryla m ide

gradient m ini

- gel (Invitrogen). The E. coli

- produced Cry3Bb1 protein was loaded at 1 μg

tot a l pr o tein in a s ingle lan e

. Broa d Rang e Molecula r W e i ght Stan d ards (Bio

- Rad) were

prepared and loaded on the gel in paralle l

. Following electrophoresis at a constant

voltage, proteins were briefly fixed in 40% (v/v) m ethanol, 7% (v/v) acetic acid and

staine d fo r 1 7 hour s wit h Brill i an t Blu e G

- Colloida l stai n (Sig m a

- Aldrich

, St

. Louis,

MO). Gels were briefly destained in 10% ( v / v ) a cetic a c id, 2 5% (v/ v ) m e thanol f oll o w ed

by 6.5 hours in 25% (v/v) m ethanol. Analysis of the gel was perfor m ed using a Bio

- Rad

GS

- 800 densito m eter s u pplied with Quantity One software (4.6.7). Apparent MW was

reported as an average of all six l a nes containing the MON 87411

- produced Cry3Bb1

protein.

C.1.6.2 Results of Cry3Bb1 Prote i n Molecular Weight E quivalence

The intact MON 87411

- produced Cry3Bb1 protein (Figur e C

- 3

, lane s 3

- 8 ) m i grate d to

the sa m e position on t h e gel as the E

. col i

- produced Cry3Bb1 protein ( F igure C

- 3, lane 2)

and the a p parent MW was calc u lated to be 77.0 kDa (Table C

- 4 ). Because the

experi m entally deter m ined apparent M W of the MON 87411

- produced Cry3Bb1 protein

was within the accepta n ce li m its for equivalence, the MON 87411

- and E. coli

- p ro d uced

Cry3Bb1 proteins were deter m ined to have equivalent apparent m olecular weights.

Table C

- 4. Molecular Weig h t Comparison Be t w een the MON 87411

- and

E. coli

- pro d uced Cry3Bb1 Proteins

Apparent MW of

MON 87411

- Produced

Cr y 3Bb1Protein

(kDa) Apparent MW of

E. coli

- Prod u ced Cr y 3B b 1

Protein 1

(kDa) Preset Accep t a nce L i m its

for the MON 874 1 1

-

Produced

Cr y 3Bb1Protein(kDa)

77. 0 74. 5 72. 9

- 81. 7

1 As r e p or te d o n t he Ce r ti f icat e o f An alysi s f or l ot 1 13 0 9151

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 256 o f 3 7 2CB I Delete d Copy

Lane Sample Amount (

1 Broad Range MW Standards 4.5

2 E. coli

- pro du ced Cr y 3B b 1 protein 1

3 MON 87411

- produced C r y3Bb1 p r otein 1

4 MON 87411

- produced C r y3Bb1 p r otein 1

5 MON 87411

- produced C r y3Bb1 p r otein 2

6 MON 87411

- produced C r y3Bb1 p r otein 2

7 MON 87411

- produced C r y3Bb1 p r otein 3

8 MON 87411

- produced C r y3Bb1 p r otein 3

9 Broad Range MW Standards 4.5

10 Blank L a ne 1 2 3 4 5 6 7 8 9 10

k D a

200

1 16.3

97.4

66.2

45

31

21.5

14.4

6.5

Figure C

- 3. Molecular Weight and Purity Analysis of the MON 87411

- produced

Cry3Bb1 Protein

Aliquots of th e MON 874 1 1

- pro d uced Cry3Bb1 and t he E. coli

- pr o duced Cry3 B b1 pr o teins were

subjected to SDS

- PAGE and gel was stained with Brilliant Blue G

- Colloidal stain. The MWs

(kDa) are sh o w n on the left and correspond to t he standards loaded in Lanes 1 and 9. Lane

designations are as follows:

μg)

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 257 o f 3 7 2CB I Delete d Copy

C.1.7 Glycosylation A n alysis of C ry3Bb1

C.1.7.1 Methods

ECL Glycoprotein Detection Kit ( G E Healthca r e) was used for glycoprotein detection.

Th e MO N 87411

- produce d Cry3Bb 1 protein

, th e E. coli

- produce d Cry3Bb 1 protein

, and

a positive c ontrol, tran sf errin (Si gm a

- Aldrich )

, were diluted in 1× LB and heated to

10 0 ° C fo r 4 m i n

. Tw o a m ount s (~10 0 and ~200 ng) of the intact the

MON 87411

- produced Cry3Bb1 protein (purity cor r ected) and the E. coli

- p ro d uced

Cry3Bb 1 protei n (purit y corrected ) wer e loa d ed onto a pre

- cast T r is

- glycin e 4

- 20%

polyacrylamide gradient m ini

- gel (Invit r ogen). Four a m ounts (~50,

~ 100, ~150, and

~200 ng) of the positive control were loa de d o n th e gel

. Protei n M W Standards

(Precision P lus Protein T M Standards Dual color, Bio

- Rad) were also loa d ed for m olecular

weight reference and to verify electrotransfer of the proteins to t h e m e m b rane. Following

electrophoresis at a constant voltage, pro t eins were electrotransferred to a PVDF

m e mbrane (Invitrogen).

Glycosylation analysis was perfor m ed on the PVDF m e mbrane at room te mpe r ature

using the Am ersh a m EC L TM glycoprotein Detection Module (GE Healt h care) as directed

by the m anufacturer. Glycosylated proteins were detected using E CL TM reagents (GE

Healthcare) and A m ersham Hyp e rfilm (GE Healthcare). The film was developed using a

Konica SRX

- 101A auto m ated film processor (Konica Minolta). An identical gel was run

and electrotransferred to a PVDF m e mbrane in parallel. Proteins w ere stained with

Coo m assie Brilliant Blue R

- 25 0 stainin g solutio n (Bio

- Ra d) an d the n destaine d with

1 × Coo m assie Brilliant Blue R

- 250 Destaining Solution (Bio

- R ad). After washing with

water, the blot was scanned using Bio

- Rad GS

- 800 densito m eter.

C.1.7.2 Results of Glycosylation A n alysis

Eukaryoti c protein s ca n b e post

- translationall y m odif ied with carbohydrate m oieties

(Rade m acher et al., 19 8 8). To test whe t her Cry3Bb1 protein was glycosylated w hen

expressed in the grain of MON 87411, the MON 87411

- produced Cry3Bb1 protein was

analyzed u s ing an ECL T M Glycoprotein Detection Module (GE Healthcare). To assess equivalence of the MON 87411

- and E. col i

- produce d Cry3Bb 1 proteins

, the

E. coli

- produced Cry3Bb1 protein was also analyzed.

A clear glycosylation signal was observed at the expected in the lanes contai n ing the

positi v e c o ntrol (tran sf errin) and the b and inte n sity incr e ased with in c re a sing

concentration (Figure C

- 4 panel A, lanes 1

- 4). In contrast, no glycosylation signals were

observed at the expected m olecular weight for Cry3Bb1 in the lanes containing the

MON 87411

- produced Cry3Bb1 protein (Figu r e C

- 4 panel A, lanes 7 and 8) or

E. coli

- produce d Cry3Bb 1 protei n (Figur e C

- 4 panel A, lanes 9 an d 10)

. Th e band

observed in the E. col i

- p roduced Cr y 3Bb1 sa mple was at a lower m olecular weig h t than

the Cry3Bb1 protein. T his band likely represents a naturally biotinylated protein that co

puri f ied with the E. c o li

- produced Cry3Bb1 protein ( C hoi

- Rhe e an d Crona n 2003),

because b i n ds to the s treptavi d in

- H RP conjug a te and therefore is detectable by t h e ECL

syste m

.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 258 o f 3 7 2CB I Delete d Copy

To confi r m that MON 87411

- and E. col i

- produced Cry3Bb1 protei n s wer e appropriately

loaded for glycosylation analy s is, a second m e mbrane with ide n tical loadings and transfer

ti m e was stained with Coo m assie Blue R250 f o r protein detection ( F igur e C

- 4 Pane l B).

Both the MON 87411

- produced Cry3Bb1 (Figure C

- 4 panel B, lanes 7 and 8) and

E. coli

- produced Cry3Bb1 (Figure C

- 4 panel B, lan e s 9

, an d 10 ) protein s wer e detected.

These data indicate that th e glycosylatio n statu s o f MO N 87411

- produce d Cry3Bb1

protei n i s equivalen t to th a t of t h e E. coli

- produce d Cry3 B b 1 protei n and that neither is

glycosylated.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 259 o f 3 7 2CB I Delete d Copy

A. B.

k D a

250 L a ne 1 2 3 4 5 6 7 8 9 10

k D a

2 50 L a ne 1 2 3 4 5 6 7 8 9 1 0

150

100

75

50

37

25

20

15

10 1 50

1 00

75

50

37

25

20

15

10

Figure C

- 4. Glycosylation Analysis of th e MO N 87411

- produce d Cry3Bb 1 Protein

Aliquots of the transferrin (posit i ve control), E. coli

- pr o duced Cry 3 Bb1 and MON

874 1 1

- pr o d u ced Cr y 3B b 1 were subjected to SDS

- PAGE a n d electrotra n sferred to PVDF

m e m b r a nes. The MWs (kDa) corresp o nd to the Pr e c ision Plus Protein T M Dual Color Standards.

The arrows show the ex p ected m igration of t he MON 87411

- a nd E. coli

- p r oduced C r y 3 Bb1

protein. ( A ) Where present, labeled carboh y dr a te m oieties were detected by addition of

streptavidin conjugated to HRP follow e d by a lu m inol

- based the detection using ECL reagents

and exposure to H y perfi l m ®

. (B) An equivalent b l ot was stained with Coomassie Blue R250 t o

confirm the presence of proteins. L a ne designations and the a pproxi m a te mass loaded are as

follows:

Lane Sample Amount (ng)

1 Transferrin (positive control) 50

2 Transferrin (positive control) 100

3 Transferrin (positive control) 150

4 Transferrin (positive control) 200

5 Precision Plus Protei n T M S t andards

6 Blank

7 MON 87411

- produced C r y3Bb1 p r otein 100

8 MON 87411

- produced C r y3Bb1 p r otein 200

9 E. coli

- pro du ced Cr y 3B b 1 protein 100

10 E. coli

- pro du ced Cr y 3B b 1 protein 200

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 260 o f 3 7 2CB I Delete d Copy

C.1.8 Cry3Bb1 Functional Activity Analysis

C.1.8.1 Methods

Insects. C P B eggs were obtained from French Agricultural Research, Inc. (La m berton,

MN). The eggs were incubated at te m peratu re s rangin g fro m 1 5 ° C t o 2 7 °C

, t o achieve

the desired hatch ti m e.

Bioassays. CPB larvae ( ≤ 30 hours old) were used to m easure biological activity of the

MON 87411

- produced and E. coli

- produced Cry3Bb1 protein sa mples in accordance

with the current version of Monsanto S O P BR

- ME

- 1048

. Th e bioassa y wa s replicated

three ti m es on separate days, each with a separate batch of insects. The

MO N 87411

- produce d an d E

. col i

- p roduced su b stances were run in parall e l during each

bioassay

. E ac h bioassa y replicat e consiste d o f a serie s o f si x dilution s yieldin g a dose

serie s wit h a two

- fol d separ ation factor ranging f rom 0.078

– 2.5 μ g Cry3Bb 1 protein/ m l

d i e t fo r the E. col i

- produced and MON 87411

- produced Cry3Bb 1 an d a singl e buffer

control. The Cry3Bb1 protein dosin g solution s wer e prepa red by d il u ting the resp e ctive

protein with purified water and incorporating the dilution into a Bio

- Serv CPB agar

- based

insect diet (pre

- m ix #F9380B; Frenchtown, NJ). Thi s dos e serie s i n die t wa s chose n to

adequately characterize the d o se

- effect relationshi p o n CP B mortalit y fo r the

E. coli

- produce d an d MO N 87411

- produce d C r y3Bb 1 proteins. The diet m ixture was

then dispensed in 0.5 m l al i quots into a 128 well tray (# BAW128, Bio

- Serv, Frenchtown,

NJ). In d ividual insect larvae were placed on t h ese d iets using a fine paintbrush, with a

target nu m ber of 24 insects per treat m ent. The infested wells were covered b y a

ventilated adhesive cover (# BACV16, Bio

- S e rv, Frenchtown, NJ) and the insects were

allowed to feed for a period o f approxi m a tel y 7 day s i n a n environ m enta l chamber

program m ed at 27 ° C, 60% relative hu m idity and a ligh t ing regi m e of 14 light : 10 dark.

The nu m ber of insects infested and the nu m b e r of surviving ins e cts at e ach dose l e vel

was recorded at the end of the 7 day incubation period. For a bioassay to be acce p ted,

control m ortality cannot be > 20%.

C.1.8.2 Results of Functional Activity

The functional activity of th e MO N 87411

- produce d an d E. col i

- produce d Cry3Bb1

protein was deter m ined Color ad o potat o beetl e d i et

- incorporation bioassays. In this

assay, activity is expressed as L C 50, μg/ m l die t

. The MON 87411

- and E. col i

- produced

Cry3Bb 1 protein s wer e considere d functionally equiv a le n t if t h e L C 50

, o f bot h were

within acceptance li m its of 0.37 μg/ m l of diet to 0.97 μg/ m l; the prediction int er val

calc u lated f rom data o b tained f or the E

. col i

- produce d Cry3Bb 1 protei n activity

. The

LC 50 of the MON 87411

- and E. col i

- produced C ry3Bb1 proteins were deter m ined to be

0.77 μg/ m l diet and 0.67 μg/ m l d i et respectively (Table C

- 5). Because the L C 50 of

MON 87411

- produced and E

. coli

- produced Cry3Bb1 proteins were within the

acceptance li m its (Table C

- 5), t h e proteins were deter m ined to have equivalent functional

acti v ity.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 261 o f 3 7 2CB I Delete d Copy

Tabl e C

- 5

. Cry3Bb 1 Fun ctional Activity Assay

MON 87411

- produced

Cry3Bb1 Protein 1 E. coli

- produce d Cry3Bb1

Protei n 1 Preset Ac c eptance Li m its

fo r MO N 87411

- produced

2 (L C 50, μg/ m l diet) (L C 50, μg/ m l diet) Cry3Bb1 Protein

(L C 50, μg/ m l diet )

0.77 0.67 0.37

– 0 .97

1 Value ref e rs t o m ean bas e d on n = 3

2 Da t a ob t a i n e d f o r t he E

. co l i

- produc e d protei n wa s us e d t o gen e rat e th e acceptanc e limits

C.2 Chara c teri z ation of CP4 EPSPS Protein in MON 87411

C.2.1 Materials for CP4 EPSPS Characteri z a tion

The MON 87411

- produced CP4 EPSPS prote i n (lot 11351676) was purified from the

grai n o f MO N 8741 1 (lo t 11330153)

. Th e MO N 87411

- produce d C P 4 EPSP S protein

was stored in a

- 80 oC freezer in a buffer solution containi n g 50 m M Tris

- HCl, pH 7.5,

50 m M KCl, 2 m M dithiothreitol (DTT), 1 m M benza m idine

- HCl, and 25% glycerol.

The E. coli

- produce d CP 4 EPSP S protei n (lo t 10000739 ) was used as the reference

substance. The CP4 EPSPS protein reference substance w a s generated fr o m cell paste

produced by large

- scale fe r m entation of E. coli containing the pMON21104 expression

plas m id. The coding sequence for cp 4 epsp s contained on the expression plas m i d

(pMON21104) was confir m ed prior to and after fer m en t ation. The E. coli

- produced

CP4 EPSPS protein was char acterize d previously.

C.2.2

. CP 4 EPSP S Protei n Purification

The plant

- produced CP4 EPSPS pr o tein was purifie d fro m grai n o f MO N 87411

. The

purification procedures were not perfor m ed under a GLP p l an; however, all procedures

were docu m ented on worksheets and, where applicable

, S OP s wer e followed

. Three

CP4 EPSPS protein puri f ication batches were prepared to generate a sufficient a m ount of

MON 87411 produced CP4 EPSPS protein. All purificatio n step s wer e perfor m ed at

~4 °C. CP4 EPSPS was purified from an ext r act of ground g rain using a co m bination of

purification m ethods. The puri f ication process f or the first two batches used am m o nium

sulfate fractionation, hydrophobic intera c tion chro m atography, anion

- exchange

chro m atography, and a second hydrophobic interaction chro m atography. The

puri f ic a tion process f or the thi r d batch used ammonium sulfa t e fractionation,

hydrophobic interaction chro m atography, anion

- exchange chro m atography, cellulose

phosphate affinity chro m atography, and hydroxyapatite chro m atography.

Chro m a tograph y fraction s conta i nin g CP 4 EPSP S protei n wer e identifie d b y wester n blot

analysi s o r SDS

- PAG E stain e d gels

. Th e fina l purifie d CP 4 EPSP S protei n produc t fr o m

all t h ree batches was co m bined, concent rate d an d buffer

- exchange d t o prepar e the

CP4 EPSPS protein lot used in the equivale nc e study

. E ac h o f th e thre e purification

procedures are briefly described below and a detailed description of the purification

procedures are filed under Lot 11351676 in the Monsanto R egulatory Archives.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 262 o f 3 7 2CB I Delete d Copy

CP 4 EPSP S protei n Batc h 1

. Grain of MON 87411 was ground in the presence of dry ice

and stored at

- 80 °C until use

. A tota l o f 92 0 g o f g r oun d powde r wa s m i xe d wit h EX

buffer (100 m M Tris

- HCl pH 7.5, 2 m M EDTA, 2 m M benza m idine

- HCl, 4 m M DTT,

1 m M phenyl m ethylsulfonyl fluoride, 1 % po lyvinyl

- polypyrrolidon e an d 1 0 % glycerol)

at a sa mple weight (g) to buffer volu m e ( m l) of approxi m a tely 1:10 for ~1.5 hr. The

slurry was centrifuged to clarify, and the s upernatant was collected, filtered

, an d brought

to 40 % ammonium sulfate saturation by additi o n of solid ammonium sul f ate. Following

equilibration, the 40 % saturated solution was c e ntrifuged to pellet precipitated proteins.

The supernatant was brought to 70 % ammon i um sulfate saturation by addition of the

solid s alt. Following e quili b ration, the pr e ci p itated pr o tein contai n ing the m ajority of

CP4 EPSPS protein was collected by cent r ifugation. The 40

- 70 % a m monium sulfate

pellet was re

- suspended in PS

- A buffer ( 5 0 m M Tris

- HCl

, p H 7.5

, 1 m M DTT

, 1 0 %

glycerol (v/v), and 1.5 M am m onium sulfate). The dissolved suspension was centrifuged,

f ilte r ed, and loaded onto a Phenyl S e pharose Fa s t Flow (GE Healthc a re, Piscataway, NJ)

colu m n equilib r ated with PS

- A bu f fer. Protei n s were elut e d with a li n ear salt gr a dient

tha t decrease d fro m 1. 5 M t o 0 M ammoniu m s ulfate over 6 colu m n volu m es. Fractions

containing the CP4 EPSPS protein were pooled and desalted by dialysis against S30Q

- A

buffer (50 m M Tris

- HCl, pH 7.5, 1 m M EDTA, 1 m M benza m idine

- HCl, and 4 m M

DTT). The desalted sample was loaded onto a Source 30Q (GE He a lthcare) colu m n

equilibrated with S30Q

- A buffer. The boun d CP 4 EPSP S wa s elute d wit h a linea r salt

gradient that increased from 0 M to 0.4 M KCl over 15 colu m n vo l u m es. Fractions

containing CP4 EPSPS were pooled and dial y zed against PS

- A buffer. The dialyzed

sa mple was then loaded onto a Source 15PHE (G E Health c are) colu m n equilib r ated with

PS

- A buffer. Proteins w ere eluted w ith a linear salt gradient that decreased from 1.5 M to

0 M ammonium sulfate over 20 column volu m es. Fractions containing the CP4 EPSPS

protein were pooled and buffer exchanged by dialysis against 50 m M Tris

- HCl pH 7.5,

50 m M KC l , 1 m M benza m idine

- HCl, 2 m M DTT, and 10 % glycerol. The buffer

exchanged s a mple was initially co n cent r ated within t h e di a lysis tubing by packing in

Aquacide II (EMD Che m icals, San Di e go, CA), then further concentrated by

centrifugation in an A m icon Ultra spin concentrator (ULTRACEL

- 10K

, 10,00 0 MWCO,

regenerated cellulose m e mbrane). The s a m p le was then buffer exchanged into 1.2X

fo r m ulation buffer (60 m M Tris

- HCl pH 7.5, 60 m M KC l

, 1.2 m M benza m idine

- HCl,

2. 4 m M DTT

, an d 1 0 % glycerol)

, yieldin g a final volu m e of ~500 μl. This Batch 1

purified CP4 EPSPS s a mple was frozen and stored in a

- 80 ° C freezer.

CP 4 EPSP S protei n Batc h 2

. The purification procedure used for the Batch 2 isolation of

CP4 EPSPS from the grain of MON 87411 foll o wed the sa m e sequence of fractionation

steps as used for Batch 1, except that the in i tial amount of grain extracted for Batch 2 was

~3 kg. Be c ause a larger a m ount of grain was extracted for Batch 2, the bed size of

chro m atography colu m ns, the column gradient volu m e s an d th e dialysi s volu m es used

wer e increased

, a s appropriate

, to accommodate the larger amount s o f sa m p l e handle d at

each step. The final volu m e of Batch 2 protein sa m ple was ~350 μl. This Batch 2

purified CP4 EPSPS s a mple was frozen and stored in a

- 80 ° C freezer.

CP4 EPSPS protein Batch 3

. The initial pu r i f i c ation steps u sed f or the Batch 3 isolation

o f CP 4 EPSP S fr o m th e g r ai n o f MO N 8 741 1 followe d th e sa m e sequenc e of

fractionation steps as used for Batch 1 th r ough the Source 30Q step, starting with

~ 2 kg

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 263 o f 3 7 2CB I Delete d Copy

o f groun d grain

. Followin g Sourc e 30 Q fractionation

, fraction s containin g CP 4 EPSPS

were poole d

, brought to 25 % glycerol ( v /v) a n d stored frozen and

- 20 ° C until the next

step. The pooled sa m ple was thawed, con centrate d an d buffe r exchange d int o CPE

buffe r (1 0 m M ME S p H 5.9

, 1 m M benza m idine

- HCl

, 1 m M DTT

, 1 0 μ M Na

- tungstate,

and 10 % g lycer o l) u s ing 15 m l A m icon Ultracell

- 10K centrifugal c o ncentrators. The

buffer exchanged and c o ncentrated s a mple was diluted with CPE buffer and loaded on a

colu m n of P11 Cellulose Phosphate ( W hat m an/GE Healthc a re, Maid s ton e

, UK)

equilibrated in CPE buffer. The colu m n was washed with CPE buffer followed by CPE

buffer with 0.5 m M phosphoenolpyruvate (PEP). CP4 EPSPS was then eluted with CPE

buffer with 0.5 m M PEP and 0.5 m M shiki m ate

- 3

- phosphate ( S

- 3

- P). Fractions

containin g CP 4 EPSP S wer e poole d an d concen trated using a Viv aspi n 15 R centrifugal

concentrator equipped with a 10,000 M W CO m e mbrane (Sartorius S tedim Biotech,

Goettingen, Ger m any). The glycerol concent r ation was increased to 25 % and the sample

f rozen and held at

- 20 °C until the next s t ep. The sa m ple was thawed, diluted with

CHT

- A buffer (50 m M HEPES pH 7.0, 1 m M benza m idine

- HCl, 1 m M DTT, and 10 %

glycerol), and loaded on a cera m ic hydroxyap a tite (CHT Type 1, Bio

- Rad Laboratories,

Hercules, C A ) colu m n. Protein was eluted w ith linear phosphate gradient from 0 to

50 m M sodium phosphate in CHT

- A buffer over 20 colu m n volu m es. Fractions

containing CP4 EPSPS were pooled and concentrated using an A m icon Ultracel

- 10K

centrifugal concentration dev i ce. The pooled sa m ple was buffer exchanged into 1.2X

fo r m ulatio n buffe r (6 0 m M Tris

- HCl

, p H 7.5

, 6 0 m M KCl

, 2. 4 m M DTT

, 1. 2 m M

benza m idine

- HCl, 10 % glycerol), yield i ng a final volu m e of ~0.5 m l of buffer

exchanged protein concentrate.

Preparation of the final pooled protein product. Th e Batc h 1 an d 2 sa m p le s wer e thaw e d

an d poole d wit h purifie d protei n fro m B a tch 3. The pooled protein sa mple w a s

concentrated to ~0.5 m l by centrifugation in a 4 m l A m icon Ultracel

- 10K centrifugal

concentratio n device

, equippe d wit h a 10,00 0 MWC O m e mbrane

. Buffe r exchang e was

conducted in the sa m e unit using 1.2X fo r m ulation buffer (60 m M Tris

- HCl, pH 7.5,

60 m M KCl, 2.4 m M DTT, 1.2 m M benza m idine

- HCl, 10 % glycerol), to a final volu m e

of 300 μl, then brought to 500 μl volu m e wit h a 20 0 μ l buffe r rins e o f th e spin

concentrator. Glycerol was added bringing the final pro t ein volu m e to 600 μl in 1X

f

or m ulation bu ff er and 25 % g ly c erol. Fin a l bu ff er composition of the sa mple was:

50 m M Tris

- HCl pH 7.5, 50 m M KCl, 2 m M DTT, 1 mM benza m idine

- HCl and 25 %

glycerol. T his CP4 EPSPS protein purified fro m th e grai n o f MO N 8 7 41 1 wa s aliquoted,

assigned lot 11351676, and stored in a

- 80 °C freezer.

C.2.3 N

- T e rmina l Se q uencin g o f CP 4 EPSPS

C.2.3.1 Methods

N

- ter m inal sequencing, carried out by auto m a ted Ed m an degradation che m istry, was used

to confirm the identity of t h e MO N 87411

- produce d CP 4 EPSPS.

A MON 87411

- produced CP4 EPSPS protein sa mpl e wa s separate d b y SDS

- PAG E and

transferred to a PVDF me m brane. The blot was stained using Coo m assie Blue R

- 250 and

destained with 1× Coo m assie Brilliant blue R

- 25 0 Destainin g Solutio n (Bio

- Rad)

. The

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 264 o f 3 7 2CB I Delete d Copy

m ajor band at ~44 kDa containing the test protein was excised from the blot and was used

for N

- ter m inal seq u ence anal y sis. The a n alysi s wa s perfor m ed fo r 1 5 cycle s using

auto m ated Ed m an degradation che m istry ( H unkapiller et al., 1983). An Applied

Biosyste m s 494 Procise Sequencing System with a 140C Microgradient system a nd a

Perkin El m er Series 200 UV/VIS Absorbance D etector and Procis e T M Control Soft w are

(version 2.1) were used. Chro m a tographic data were collected using SequencePro

software ( A pplied Biosyste m s, Foster C ity C A ; version 2.1). A P T H

- AA standard

solution (Applied Biosyste m s) was used to chr om atographically c a lib r ate the instr um ent

for the analysis. A control pr o tein, β

- lactoglobulin, (Applied Biosystems) was analyzed

before and after analysis of the MON 87411

- produced CP4 EPSPS protein.

C.2.3.2 Results of the N

- t ermina l Sequenc e Analysis

Fifteen cycles of N

- ter m inal sequencing was perfor m ed on the MON 87411

- produced

CP4 EPSPS protein. The expected sequence for the CP4 EPSPS pro t ein deduced fr o m

the cp 4 epsp s gene present in MON 87411 was obse r ved. The data obtained correspond

t o th e deduce d CP 4 EPSP S protei n beginnin g a t a m in o aci d positio n 2 (Tabl e C

- 6).

Hence, the s equence infor m ation c o nfir m s the identit y o f th e CP 4 EPSP S protei n isolated

from the seed of MON 87411.

Table C

- 6. N

- Terminal Sequence of the MON 87411

- produced CP4EPSPS

A m ino acid

residue # fr o m

the N

- ter m i n us → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Expected

Sequence → M L H G A S S R P A T A R K S S

│ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Experimental

Sequence →

– L H G A S S R P A T A R K X X

The experimental sequences obtained from t h e M ON 87411

- produced CP4 EPSPS protein w e re

c o m pared to the expected sequence. The single letter IUPAC

- I U B a m ino acid code is M,

methionine; L, leucine; H, histidine; G, g l y c ine; A, alanine, S, serine; R, arginine; P, proline; T,

threonine; K, l y sine; (X) indicates that the residue was not identifiable.

C2.4 MALDI

- TOF Tryptic Mass Map Analysis of CP4 EPSPS

C2.4.1 Methods

MALDI

- TOF MS tryptic m ass fingerprint analysis was u s ed to con f irm the ide n tity of t h e

MON 87411

- produced CP4 EPSPS protei n

. A M O N 87411

- produce d CP 4 EPSPS

protei n sa m p l e wa s separate d b y SDS

- PAG E a n d the gel was stained usin g Brillian t Blue

G

- Colloid a l stain (Sig m a

- Aldrich, St. Louis, MO)

. Th e m a jo r ban d a t ~4 4 kD a ban d was

excised, transferred to a m icrocentrifuge tub e

, and destained. The gel band was washed

in 200 μl of 100 m M ammonium bicarbonate, and the n incubate d i n 10 0 μ l o f 1 0 m M

DTT at 37 °C for 2 h to reduce protein disul f ide bonds. The protein w a s then alkylated in

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 265 o f 3 7 2CB I Delete d Copy

the dark for 20 m in with 100 μl of 20 m M iodoacetic acid and washed with 200 μl of

25 m M a mmonium bicarbonate for 3 × 15 m in washes. The gel band was dried with a

Speed

- Vac  concentrator (Ther m o Fisher Scie n tific, W altham, MA) a n d then rehydrated

with 20 μl of trypsin solution (20 μg/ m l). After 1 h, excess liquid was removed and the

gel was incubated overnight at 37 °C in 40 μl of 10 % ac e to n it r ile in 25 m M am m onium

bicarbonate. The gel b a nd was sonicated for 5 m i n to further elute proteolytic frag m ents.

The res u lti n g ext r act w a s tra n s f erred to a new tube lab e led Extra c t 1 a n d dried u si n g a

Speed

- Vac concentrat or

. The gel band was re

- e x tracted twice with 30 μl of a

60 % acetonitrile, 0.1 % tri f luoroacetic acid, 0.1 % β

- octyl

- glucopyranoside solution and

sonicated for 5 m in. These sa mples were po o le d int o a ne w tub e labele d Extrac t 2 and

dried with a Speed

- Vac concentrator. A solution of 0.1 % tri f luoroacetic acid (TFA) was

added to all Extract 1 and 2 tubes and they wer e drie d a s before

. A solutio n of

50 % aceto n itrile, 0.1 % TFA was added to each t ube and all were sonicated for 5 min.

Each extract (0.3 μl) was spott e d to three wells on an analysis plate. For each extract

0.7 5 μ l o f 2

, 5

- dihydroxybenzoi c aci d ( D HB)

, α

- cyano

- 4

- hydroxycinna m ic acid

( α Cyano), or 3,5

- di m e thoxy

- 4

- hydroxycinna m ic acid (sinapinic acid) (Ther m o F i sher

Scientific) was added to one of the spots. The sa mples in DHB m atrix were analyzed in

the 300 to 5000 Da range. S a m ples in α

- Cyano and Sinapinic acid were analyzed in the

500 to 5000 and 500 to 7000 Da range, respectively. C alMix 2 T M was used as the

exter n al calibrant (Seq u azy m e TM P e ptide Mass Standards kit, AB SciEx). The analysis

was perfor m ed on a Voyage r T M DE Pro B i ospectro m etry T M work s t atio n (Applied

Biosyste m s) using Voyager Instrument Control Panel software (version 5.10.2) and Data

Explorer data analysis software (version 4.0.0.0). Protonated peptide m asses were

isotopically resolved in reflector mode ( A ebersold 199 3 ; Billeci a n d Stults 1 9 93).

GPMAW32 software (Lighthouse Dat a ) wa s use d t o generat e a n i n silic o digest of the

CP4 EPSPS protein sequence. Masse s withi n 1 D a o f th e m onoisotopi c m a s s were

m atched against the in silico diges t o f th e MO N 87411

- produce d Cry3Bb 1 sequence

. All

m atching masses were tallied and a coverage m ap was generated for the m ass fingerprint.

C2.4.2 Results of M A LDI

- TOF Tryptic Mass Map Analysis

The identity of the MON 87411

- produced CP4 EPSPS protein was confir m ed by

MALDI

- TOF MS analysis of peptide frag m ents produced by the trypsin digestion of the

MO N 8741 1

- produce d C P 4 EPSP S p r otein

. Ther e w ere 23 unique peptides identified that

corresponded to the masses expected to be produced by trypsin digestion of the

CP4 EPSPS protein ( T able C

- 7). The ide n tified m asses were used to as s e m b le a

coverage map of the entire CP4 EPSPS pro t ein (Figure C

- 5). The experi m entally

deter m ined m ass coverage of the CP4 EPSPS p rotein was 67.3% (306 out of 455 a m ino

acids).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 266 o f 3 7 2CB I Delete d Copy

Table C

- 7. Summary of the Tryptic M asses Id en tified f o r the

MON 87411

- produced CP4 EPSPS Using MALDI

- TOF MS

Obs e r ved Ma s s 1 Expected Mass Diff e r e nc e 2 Fr a g m e nt Sequence 3

3 8 9 .20 3 8 9 .25 0

. 05 2 25

- 227 T IR

4 7 4 .23 4 7 4 .27 0

. 04 2 28

- 231 LE GR

5 9 9 .3 3 5 9 9 .3 3 0

. 00 2 9

- 33 SI S HR

6 1 6 .33 6 1 6 .34 0

. 01 1 28

- 132 RPM GR

6 2 9 .34 6 2 9 .29 0

. 05 2 01

- 205 DH T EK

7 1 1 .45 7 1 1 .45 0

. 00 1 33

- 138 VL NPLR

7 3 3 .44 7 3 3 .38 0

. 06 3 52

- 357 VK E S DR

8 3 5 .3 9 8 3 5 .3 9 0

. 00 6 2

- 69 A MQ A MG AR

8 6 3 .4 7 8 6 3 .4 6 0

. 01 1 5

- 23 SSG L S G TVR

8 7 2 .4 5 8 7 2 .4 5 0

. 00 3 13

- 320 GV T V P ED R *

8 7 2 .4 6 8 7 2 .5 2 0

. 06 3 58

- 366 LSA V AN G LK*

9 3 0 .49 9 3 0 .51 0

. 02 1 69

- 177 VP MASAQVK

9 4 8 .51 9 4 8 .52 0

. 01 1 61

- 168 TPTP ITYR

9 9 1 .5 6 9 9 1 .5 5 0

. 01 1 4

- 23 KS S G

... G T V R

1 11 5

. 57 1 11 5

. 57 0

. 00 2 95

- 305 L A G G

... A DLR

1 35 7

. 72 1 35 7

. 71 0

. 01 1 46

- 157 SED G ...VTLR

1 35 9

. 67 1 35 9

. 72 0

. 05 3 54

- 366 ESDR

. . . NG L K *

1 35 9

. 63 1 35 9

. 64 0

. 01 3 4

- 46 SFMF.

. . GET R *

1 55 8

. 82 1 55 8

. 83 0

. 01 4 7

- 61 IT GL... N TGK

1 64 6

. 85 1 64 6

. 84 0

. 01 3 89

- 405 GL G N ...LDHR

1 76 3

. 83 1 76 3

. 81 0

. 02 3 67

- 382 L N G V ...LVVR

1 99 3

. 97 1 99 3

. 97 0

. 00 2 06

- 224 ML Q G...D G VR

2 18 3

. 19 2 18 3

. 17 0

. 02 2 75

- 294 T G L I ...I NPR

2 36 7

. 33 2 36 7

. 33 0

. 00 1 78

- 200 SA VL

... I MTR

2 45 0

. 24 2 45 0

. 23 0

. 01 2 4

- 46 IP G D

. . . G ETR*

2 45 0

. 22 0

. 02 1 05

- 127 LTMG...S L T K*

3 24 4

. 52 3 24 4

. 52 0

. 00 7 3

- 1 04 E G DT...T G CR

3 24 9

. 59 3 24 9

. 62 0

. 03 3 21

- 351 A PSM...EELR

4 18 8

. 47 4 18 8

. 26 0

. 21 2 34

- 274 LTG Q. ..NPTR

1 The obs e r ved m a ss was o btai ned fr om at leas t on e o f thre e m atrice s includin g α

- cya no, D H B

, a nd

s i nap i n i c ac i d. The o bs e rv e d m a ss sh ow n i s th e m as s closes t t o th e expec t e d m ass.

2 The da t a r e p r e sen t s t he ca l cu l ate d d i ff e rence be tw e e n t he e xpect e d m a ss and the observ e d m a ss.

3 For p e p ti de m atc hes grea t e r t h a n n i ne a m i no ac i ds i n le ng t h t he f i rst 4 r e s id ues a nd la st 4 res id u e s a r e

s h o w se p a r ate d by th r e e d o t s (...)

* T he exp e cted peptide m a ss e s are nearly identical ( < 1 dalton). Because this a nalysi s di d no t determine

wit h certai n t y w h ic h ex p ecte d p e p ti de wa s act u all y obs e r ve d

, t he p e p t i des w it h a n a s te r i sk ( *) were not

i nc l ud e d i n de t e r m i n i ng se q u e nce c ov e r a ge ( F i g u re C

- 5 ).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 2 67 o f 3 7 2CB I Delete d Copy

0 0 1 M L HG A S S R PA TA R K S S GL S G T V R I PG D K S I S H RS F M F G GL AS G E T R IT G L

0 5 1 L E GE D V I N TG KA M Q A M GA R I R K E G DT W I I D G V GN G G L L AP EA P L D F GN A A

1 0 1 T G CR L T M G LV GV Y D F D ST F I G D A S LT K R P M G R VL N P L R EM GV Q V K S ED G D

1 5 1 R L PV T L R G PK TP T P I T YR V P M A S A QV K S A V L L AG L N T P GI TT V I E P IM T R

2 0 1 D H TE K M L Q GF GA N L T V ET D A D G V R TI R L E G R G KL T G Q V ID VP G D P S ST A F

2 5 1 P L VA A L L V PG SD V T I L NV L M N P T R TG L I L T L Q EM G A D I EV IN P R L A GG E D

3 0 1 V A DL R V R S ST LK G V T V PE D R A P S M ID E Y P I L A VA A A F A EG AT V M N G LE E L

3 5 1 R V KE S D R L SA VA N G L K LN G V D C D E GE T S L V V R GR P D G K GL GN A S G A AV A T

4 0 1 H L DH R I A M SF LV M G L V SE N P V T V D DA T M I A T S FP E F M D LM AG L G A K IE L S

4 5 1 D T KAA

Figur e C

- 5

. MALDI

- TO F M S Coverag e Map of the MON 87411

- Produced CP4

EPSP S Pr o t ein

The a m ino acid sequence of the CP4 E P SPS protein was deduced from the cp4 epsps gene

present in MON 87411. Boxed regions correspond to regions covered by t r yptic peptides that

were una m biguous l y iden t ified from the MON 87411

- produced C P 4 EPSPS pro t ein sa m ple using

MALDI

- TOF MS. In t otal, 67.3 % (306 of 455 total a m ino acids) of the expected protein

sequence was covered b y t he identified peptides.

C.2.5 Wester n Blo t Analysis

- Immunoreactivit y o f CP 4 EPSPS

C.2.5.1 Methods

Western blot analysis was perfor m e d to confirm the identity of the MON 87411

- produced

CP4 EPSPS protein and to co m p a re t h e immunoreactivit y o f th e MO N 87411

- and

E. coli

- produce d proteins.

Th e MO N 87411

- an d E. col i

- produced CP4 EPSPS proteins wer e analyze d concurrently

on the sa m e gel using three protein loadings of 1, 2 and 3 ng. Loadings of the three

concentrations were m ade in duplicate on the gel. In preparation for analysis the

E. coli

- produce d CP 4 EPSP S wa s dilute d wit h stor age buffer to the sa m e purity corrected

concentration as the MON 87411

- produced pro t ein (0.263 mg CP4 EPS P S/ m l )

. Aliquots

of each protein were s u bsequently diluted in water and 1 × (LB), and heated at 95 °C for

4 m i n

. Sample s wer e furthe r dilute d i n 5 × L B t o a fina l conce n tratio n o f 0.2 5 ng/μl

, and

applied to a pre

- cast T ris

- glycine 4

- 20 % po l yacryla m ide gradient gel (Invitrogen). P re

stai n ed m o l ecul a r wei g ht m arkers (Pre c is io n Plu s Protei n Standard s Dua l Color; Bio

Rad) were loaded to verify electrotran s fer of the proteins to the m e m brane and to

esti m ate t h e size of the im m unoreactive b a nds observed. Following electrophoresis,

electrotrans f er to a PVDF m e m br a ne (Invitrogen) was perfor m ed.

After ele c tr o transfer, the m e mbrane was blo c ked for 1 h with 5 % (w/v) NFDM in PBST)

at room temperature. T he m e mbrane w a s then probed with a 1:10,000 dilution of goat

anti

- CP 4 EPSP S antibod y (lo t G

- 848018 ) i n 1 % NFD M i n PBS T fo r 6 0 m in a t ro o m

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 268 o f 3 7 2CB I Delete d Copy

te mperature. Excess antibody was re m oved us in g washe s wit h PBST

. Finally

, the

m e mbrane was probed with HRP

- conjugated r a bbit anti

- goat IgG (Thermo, Rockford, IL)

at a dilution of 1:5,000 in 1 % NFDM in P B ST for 1 h at room te mpe r ature. Excess

HRP

- conjugate was removed using washes with PBST. All washes were perfor m ed at

room t e mp e rature. Immunoreacti v e bands w e re vis u alized using the ECL detection

system (GE, Healthcare) with exposure to A m e r sham Hyperfilm ECL ( G E, Healthcare).

The film was developed using a Konica SRX

- 101A auto m ated film processor.

Quantification of the bands on the blot was perfor m e d using a Bio

- Rad GS

- 800

densito m eter with the s upplied Quantity O n e s oftware (version 4.4.0) using the b and

volu m e tool. The signal intensities of the i mmunoreacti v e bands observed for the

MON 87411

- and E. col i

- produced proteins m igrating at the expected position on the blot

film were quantified as “adjusted volu m e” values (OD × m m 2 ).

C.2.5.2 Results of CP4EPSPS Pro t ein Immunoreactivity Equivalence

Western blot analysis w as conducted using goa t anti

- CP 4 EPSP S polyclona l antibod y as

an additio n a l m eans to con f irm the identity of the CP4 EPSPS protein i s olated f rom the

grain of MON 87411 and to assess the equivalence of the im m unoreactivity of the

MON 87411

- and the E. col i

- produce d CP 4 EPSP S proteins.

The results d e m onstrated that the anti

- CP4 EPSPS antibody recognized the

MO N 8741 1

- produce d CP 4 EPSP S p r otei n tha t m i g rated to an ident i ca l positio n a s the

E. coli

- produce d CP 4 EPSP S protei n (Figur e C

- 6). Further m ore, the immunoreactive

signal inc r e a sed with i n cr easing a m ounts of CP4 E PSP S protei n loaded.

To c o mpare the im m u n oreactivity of the MON 87411

- produced CP4 EPSPS protein and

the E. col i

- produced C P 4 EPSPS proteins, densito m e tri c analysi s wa s conducte d on

bands that m igrated to the expected app aren t M W fo r CP 4 EPSP S protein s (~4 4 kDa).

The signal intensity (reported in OD × m m 2 ) of the band of interest in l a nes loa d ed with

th e MO N 87411

- produce d an d th e E. coli

- produce d CP 4 EPSP S protei n wa s m e asured

(Tabl e C

- 8)

. Becaus e signa l intensit y o f th e MO N 87411

- produce d CP 4 EPSP S p r otein

band was within 35% of the E. col i

- produced CP4 EPSPS protein, the

MON 87411

- produced and E. coli

- produced CP4 EPSPS p r oteins were deter m ined to

have equivalent im m unoreactivity.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 269 o f 3 7 2CB I Delete d Copy

La n e #

MW 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 15

( k D a )

2 5 0

1 5 0

1 0 0

75

50

37

2 5

20

15

10

Figur e C

- 6

. Wester n Blo t A n alysi s o f MO N 87411

- an d E. coli

- Pro d u ced

CP4 EPSPS Proteins

Aliquots of the MON 87411

- produced CP4 EPSPS protein and the E. coli

- p ro d uced

CP 4 EPSP S protei n wer e separate d b y SDS

- PAGE and electrotransferred to a P V DF

m e mbrane

. Th e m e mbran e wa s incubat e d with anti

- C P 4 EPSP S antibodie s a s the

pri m ary antibodies and immunoreactive bands were visualized using HRP

- conjugated

secondary antibodies and an ECL system (G E Healthcare)

. Approxi m at e m o lecular

weights (kDa) are sho w n on the le f t and corr e spond to the m arkers loaded in lane 1.

Lane 1 was cropped from the i m age. The 3 m in exposure is shown. Lane designations

are as foll o w s:

Lane Sample Amount (ng)

1 Precision Plus Protein Standards Dual color (crop p ed

-

from the i m age)

2 E. coli

- produced CP4 EPSPS protein 1

3 E. coli

- produced CP4 EPSPS protein 1

4 E. coli

- produced CP4 EPSPS protein 2

5 E. coli

- produced CP4 EPSPS protein 2

6 E. coli

- produced CP4 EPSPS protein 3

7 E. coli

- produced CP4 EPSPS protein 3

8 Blank

-

9 MON 87411

- produced C P 4 EPSPS pro t ein 1

10 MON 87411

- produced C P 4 EPSPS pro t ein 1

11 MON 87411

- produced C P 4 EPSPS pro t ein 2

12 MON 87411

- produced C P 4 EPSPS pro t ein 2

13 MON 87411

- produced C P 4 EPSPS pro t ein 3

14 MON 87411

- produced C P 4 EPSPS pro t ein 3

15 Blank

-

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 270 o f 3 7 2CB I Delete d Copy

Table C

- 8. Comparison of Immunore a ctive Signal Bet w een MON 87411

- and E

coli

- produce d CP 4 E P SP S Protein

Mean Signal Intensity from Mean Signal Intensity from Acceptan c e Li m it s 2 for

MON 87411

- Produced

CP4 EPSPS 1

(O D × mm 2 ) E. coli

- Produced CP4 EPSP S 1

(O D × mm 2 ) MON 87411

- Produced

CP4 EPSPS

(O D × mm 2 )

6.0 6.0 3.9

– 8.1

1 Each value r e pres e nts t he mean of s i x va lu es ( n= 6 ).

2 The acc e ptan c e li m its are for the MO N 87411

- produc e d CP 4 E PSP S prote i n and a re bas e d on the i nterval

be t we e n + 3 5 % ( 6

. 0 × 1.35 ) a nd

− 3 5% ( 6. 0 × 0

. 6 5 ) of t he m ean o f t he E

. col i

- produced CP4 E PS P S s i gnal

i n te ns it y acr o s s al l l o a d s.

C.2.6 Molecular Weight Estimat i o n o f CP 4 EPSP S usin g SDS

- PA G E

C.2.6.1 Methods

MON 8741 1

- produced C P 4 EPSPS p rotein and E. col i

- produced CP4 EPSPS protein, as

wel l a s B i o

- Ra d Broa d Rang e m o l ecula r weigh t standards

, wer e dilute d i n wate r and

5 × LB. The diluted sa mples were h e ated to 95 °C for 3

- 5 m in. The

MO N 8741 1

- produce d C P 4 EPSP S p r otei n wa s a nalyzed in duplicate at 1, 2, and 3 μg

protein per lane. The E

. col i

- produced CP4 EPSPS reference standard was analyzed at

1 μg total protein in a single lane. The sa mp les were loaded onto a pre

- cast Tris glycine

4

- 20 % polyacryla m ide gradient m ini

- gel ( I nvitrogen) and electroph o resis was perfor m e d

a t a constan t voltage

. P r ote i n s wer e fixe d b y placin g th e ge l in a solution of 40 % (v/v)

m ethanol and 7 % (v/v) acetic acid for 30 m in, and stained for 18 h with Brilliant Blue G

Colloida l stain

. Gel s wer e destaine d fo r 30 to 45 sec with a solutio n containing

10 % (v/ v ) acetic acid and 25 % (v/v) m ethanol, and for 8 h with 25 % (v/v) m ethanol.

Analysis of the gel w as perfor m ed using a Bio

- Rad GS

- 800 densito m eter with the

supplied Quantity One software (version 4 .4.0)

. Apparen t M W wa s reporte d a s an

average of all six lan e s containin g th e MO N 87411

- p roduced Cry3Bb1 protein.

C.2.6.2 Results of CP4EPSPS Pro t ein Molec u lar Weig h t Equivalence

For m olecular weight and purity analysi s

, the MON 87411

- produced CP4 EPSPS protein

was separated using S D S

- PAGE. Following electrophoresis, the g e l was stained with

Brilliant Blue G

- Colloidal stain and ana l yzed by densito m etry (Figure C

- 7). The

MON 8741 1

- produced CP4 EPSPS p rotein (Figure C

- 7, lanes 3

- 8) m igrated to the sa m e

position on the g e l as t h e E. col i

- produced CP4 EPSPS protein ( F igure C

- 7, lane 2) and

the apparent m olecular weight wa s calculate d t o b e 42. 9 kD a (Tabl e C

- 9)

. Becaus e the

experi m entally deter m ined apparent MW o f MON 87411

- produced CP4 EPSPS protein

was within the pre

- set acceptance li m its f o r equivale n ce (Table C

- 9; 42.6

– 45.1 kDa),

the MON 87411

- produced and E

. col i

- produce d CP 4 EPSP S prote i ns were deter m ined to

have equivalent apparent m olecular weights.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 271 o f 3 7 2CB I Delete d Copy

Table C

- 9. Molecular Weight Comparison Bet w e en the MON 87411

- and

E. coli

- produce d CP 4 EPSP S Proteins

Apparent MW

o f MO N 87411

- Produced

CP 4 EPSP S Protein Apparen t M W of

E. coli

- P ro d uced

CP4 EPSPS Protei n 1 Preset Acceptance Li m its

for MON 87411

- Produced

CP 4 EPSPS

(kDa) (kDa) (kDa)

42.9 43.8 42.6

- 45.1

1 As r e p or te d o n t he Ce r ti f icat e o f An alysi s f or l ot 1 00 0 073 9.

La n e #

MW

( k D a )

2 0 0

. 0

1 1 6

. 2 5

97

. 4

66

. 2

45

. 0

3 1

. 0

21

. 5

14

. 4

6

. 5 1 2 3 4 5 6 7 8 9

Figure C

- 7. Molecular Weight and Pur i ty Analysis of the MON 87411

- produced

CP4EPSPS Protein

Aliquots of the MON 87411

- produced and the E. col i

- produce d CP 4 EPSP S pr o t eins

wer e subjecte d t o SDS

- PAG E an d th e ge l wa s st a ined with Brillia n t Blue G

- colloidal

stain. The M W s (kDa) are shown on the left an d correspon d t o th e standards loa d ed in

Lanes 1 and 9. Lane 10 (blank lane) was cropped from the image. Lane designations are

as f ollows:

Lane Sample Amount (μg)

1 Broad Range MW Standards 4.5

2 E. coli

- produced CP4 EPSPS protein 1

3 MON 87411

- produced C P 4 EPSPS pro t ein 1

4 MON 87411

- produced C P 4 EPSPS pro t ein 1

5 MON 87411

- produced C P 4 EPSPS pro t ein 2

6 MON 87411

- produced C P 4 EPSPS pro t ein 2

7 MON 87411

- produced C P 4 EPSPS pro t ein 3

8 MON 87411

- produced C P 4 EPSPS pro t ein 3

9 Broad Range Molecular Weight Markers 4.5

10 Blank (cropped from the i m age)

--

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 272 o f 3 7 2CB I Delete d Copy

C.2.7 Glycosylation A n alysis of C P 4 EPEPS

C.2.7.1 Methods

ECL Glycoprotein Detection Kit ( G E Healthca r e) was used for glycoprotein detection.

Th e MO N 87411

- produce d CP 4 EPSP S pr o t ein

, th e E. coli

- p ro d uced CP4 EPSPS

protein

, an d a positiv e control

, tra n sferrin (Si gm a

- Aldrich), were diluted in 1× LB and

heated to 100 °C for 4 m in. Two a m oun t s (~100 and ~200 ng) of the intact the

MON 87411

- produced CP4 EPSPS pro t ein (purity corrected) and the E. coli

- p ro d uced

CP4 EPSPS protein (p u rity c o rre c t e d) were l o aded onto a pre

- cast Tris

- glycine 4

– 20 %

polyacrylamide gradient m ini

- gel (Invit r ogen). Four a m ounts (~50,

~ 100, ~150, and

~200 ng) of the positive control were loa de d o n th e gel

. Protei n M W Standards

(Precision P lus Protein T M Standards Dual color, Bio

- Rad,) were also loaded for MW

reference and to verify electrotransfer of the proteins to the m e mbrane. Following

electrophoresis at a constant voltage, pro t eins were electrotransferred to a PVDF

m e mbrane (Invitrogen).

Glycosylation analysis was perfor m ed on the PVDF m e mbrane at room te mpe r ature

using the A m ersh a m ECLTM glycoprotein Det e ction Module (GE Healthcare) as directed

by the m anufacturer. Glycosylated proteins were detected using E C LTM reagents (GE

Healthcare) and A m ersham Hyp e rfilm (GE Healthcare). The film was developed using a

Konica SRX 101A auto m ated film processor (Konica Minolta). An identical gel was run

and electrotran sferred to a PVDF m e mbrane in parallel. Proteins w ere stained with

Coo m assie Brilliant Blue R 250 staining soluti o n (Bio

- Rad ) an d the n destaine d wit h 1×

Co o m assie Brilliant Blue R 250 Destaining Solution ( B io

- Rad). After washing with

water, the blot was scanned using Bio Rad GS

- 800 densito m eter.

C.2.7.2 Results of Glycosylation A n alysis

T o tes t whethe r th e CP 4 EPSP S protei n wa s gly cosylated when expressed in the seed of

MO N 8741 1

, th e MO N 87411

- produce d CP 4 EPSP S protei n wa s analyze d usin g an

ECL TM Glycoprotein Detection Module (GE He a lt h care, Piscataway, NJ). To a s se s s equivalence of the MON 87411

- and E

. col i

- produce d CP 4 EPSP S proteins

, the

E. coli

- produce d CP 4 EPSP S protein

, previousl y bee n show n t o b e fre e o f glycosylation

(Harrison et al. 1996) was also analyzed.

A clear glycosylation signal was observed at t h e expected molecular w eight in the lanes

contai n ing the positi v e control ( t r ans f erri n ) a nd the band intensity increa s ed with

increasing concentration (Figure C

- 8, Panel A, lanes 2

- 5). In contr a st, signals were not

observed in the lanes containin g th e MO N 87411

- o r E. col i

- produce d protei n a t the

expected m olecular weight for the C P 4 EPSPS p rotein (Figure C

- 8 panel A, lanes 6

- 9).

T o confir m tha t sufficien t MO N 87411

- an d E. col i

- produce d CP 4 EPSP S protein s were

present for glycosylation analysis, a se c ond me m brane (with identical loadings and

transfer times) was stained with Coo m assie Blue R250 for protein detection (Figure C

- 8

Panel B). B oth the MON 87411

- and E. coli

- produce d CP 4 EPSP S pro teins were clearly

detected (Figure C

- 8, P a ne l B

, Lane s 6

- 9).

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 273 o f 3 7 2CB I Delete d Copy

These data indicate that the glycosylati o n statu s o f MO N 8 7 411

- produce d CP 4 EPSPS

protein is equivalent to that of the E. col i

- produced CP4 EPSPS protein and that neither is

glycosylated.

A. B

.

La n e # 2 3 4 5 6 7 8 9 1 0 La n e # 1 2 3 4 5 6 7 8 9 10

2 5 0

1 5 0

1 0 0

75

50

37

2 5

20

1 5

10 250

150

100

75

50

37

2 5

20

15

10

Figure C

- 8. Glycosylation Analysis o f th e MO N 87411

- produce d CP 4 EPSPS

Protein

Aliquots of transferrin (positive control), E. col i

- produce d CP 4 EPSP S protei n and

MON 87411

- produced CP4 EPSPS protein were subjected to S D S

- PAGE and

electrotrans f erred to PVDF m e mbranes. The M W s (kDa) correspond to the Precision

Plus Protein T M Stand a rds. The arrows show the expected m igration of the

MO N 87411

- produce d an d E

. col i

- produce d CP 4 EPSP S proteins

. (A ) W h er e present,

labele d carbohydrat e moietie s w e r e detecte d b y additio n o f streptavidin conjugated to

HRP followed by a lu m inol

- based detecti o n using ECL reagents and exposure to

Hyperfil m 

. (B) An equivalent blot was stain e d wit h Coomassi e Blu e R25 0 t o confirm

the presence of proteins. Lane designati ons and the approxi m ate m a ss loaded are as

f ollows:

Lane

1 Sample

Precision Plus Protei n T M S t andards Amount (ng)

-

2 Transferrin (positive control) 50

3 Transferrin (positive control) 100

4 Transferrin (positive control) 150

5 Transferrin (positive control) 200

6 E. coli

- produced CP4 EPSPS protein (negative control) 100

7 E. coli

- produced CP4 EPSPS protein (negative control) 200

8 MON 87411

- produced C P 4 EPSPS pro t ein 100

9 MON 87411

- produced C P 4 EPSPS pro t ein 200

10 Blank

-

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 274 o f 3 7 2CB I Delete d Copy

C.2.8 CP 4 EPSP S Functiona l Activit y Analysis

C.2.8.1 Methods

In pre p aration for analysis the E

. coli

- produce d CP 4 EPSP S wa s dilute d wit h storage

buffer to the sa m e purity corrected conce n tration as the MON 87411

- produce d protein

(0.263 m g CP4 EPSPS / m l). Prior to functional activity analysis, both MON 8741 1

- and

E. coli

- produce d protein s wer e dilute d wit h 50 m M HEPES, pH 7.0 buffer to a purity

correcte d concentratio n o f ~5 0 μg / m l

. Assays for both proteins were conducted in

triplicate. T he reac tion s wer e perfor m e d i n 5 0 m M HEPES

, p H 7.0

, 0. 1 m M a mmonium

molybdate, 1 m M PEP and 5 m M potassium f l uoride with or without 2 m M S

- 3

- P for

2 m in at ~25 ° C. The reactions were initiated b y the addition of PEP. After 2 m in, the

reactions were quenched with phosphate assay reagent (0.033% m alachite green,

1.1% am m o nium m olybdate) and then fixed with 33 % (w/v ) sodiu m citrate

. A standard

curve was prepared using 0 to 10 n m oles of inorganic phosphate in w a ter treated with the

phosphate assay reage n t and 3 3 % (w/v) sodium citrate. T h e absor b ance of each reaction

and each standard was m easured in duplicate at 660 nm using a Powe rW ave X i 

m icroplate reader. The a m ount of inorg ani c phosphat e release d fro m PE P i n each

reaction was deter m ined using the standard curve

. Fo r CP 4 EPSPS

, the specific activity

was defined in unit per mg of protein (U/ m g )

, where a unit (U) is defined as 1 μ m o l e of

inorganic phosphate released from PEP per m in at 25 ° C. Calcul a tio n s of the sp e ci f ic

activities were perfor m ed us i ng Microsoft Excel (2007).

C.2.8.2 Results of Functional Activity

The functional activities of the MON 87411

- and E. coli

- produced CP4 EPSPS proteins

were dete r m ined using a colori m etric ass a y that m e a sures for m a tion of inorganic

phosphate (Pi) from the EPSPS

- catalyzed reaction between shiki m ate

- 3

- phosphate (S

3

- P) and PEP. In this a ssay, prot e in

- sp ec i f ic a c ti v ity is ex p res s ed as u n its per m illigram

of protein (U/mg), where a unit is defined as one μ m ole of inorganic phos p hate released

from PEP per m inute at 25 °C. T he MON 87411

- and E. coli

- p ro d uced CP4 EPSPS

proteins were consi de red to have eq u ivale n t f unctional a c ti v ity if the spe c i f ic activities of

both were within the acceptance li m its of 1.96 to 7.90 U / m g.

The experi m entally deter m ined spec i fi c activitie s fo r th e MO N 87411

- and

E. coli

- produce d CP 4 EPSP S protein s ar e presen ted in Table C

- 10. The specific

activities of MON 87411

- and E

. coli

- produced C P 4 EPSPS p roteins were 5.78 U/ m g and

5.0 0 U / m g o f CP 4 EPSP S protein

, respective l y

. Becaus e th e specifi c activit y o f the

MON 87411

- produced CP4 EPSPS protein falls within t h e preset acceptance criterion

(Table C

- 10), the M O N 87411

- produced CP4 EPSPS pr o tein was considered to have

equivalent functional a c tivity to that of the E. coli

- produce d CP 4 EPSP S protein.

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Tabl e C

- 10

. CP 4 EPSP S Fun ctional Activity Assay

MON 87411

- Produced

CP4 EPSPS Protei n 1 E. coli

- P ro d uced

CP4 EPSPS Protei n 1 Preset Acceptance Li m its for

MON 87411

- Produced C P 4 EPSPS

(U/ m g) (U/ m g) (U/ m g)

5.78 ± 0.19 5.00 ± 0.38 1.96

– 7.90

1 Va l ue r e fe r s t o m ean and s t a nd a r d d e v i a tio n ca l cu late d b a sed on n = 6 whic h i nc lu des th ree r e p li ca t e assa y s

spectropho m etricall y rea d i n duplicat e plat e wells.

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References for Appendix C

Aebersold, R. 1993. Mass spect r o m etry of proteins and peptides in biotechnology.

Current Opinion in Biotechnology 4 : 412

- 419.

Billeci, T.M. and J.T. S t ults. 1993. Tryptic m apping of recombinant pr o teins by m atrix

assisted laser desorption/ionization ma ss spectro m etry. Analytical Che m istry 65: 1709

-

1716.

Choi

- Rhee

, E

. an d J.E

. Cronan

. 2003

. Th e b i oti n carboxylase

- b i oti n carboxy l carrier

protein complex of Escheric h ia coli acet y l

- C o A carboxylase. J Biol Chem 278: 30806

-

30812.

Harrison, L.A., M.R. B a iley, M. W

. Naylor, J. E

. Rea m

, B.C

. H a m m ond

, D.L

. Nida

, B.L.

Burnette

, T.E

. Nickson

, T.A

. Mitsky

, M.L

. Taylor

, R.L

. Fuchs

, an d S.R

. Padgette

. 1996.

The expressed protein in glyphosate

- tol e rant soybean, 5

- enolpyruvylshiki m ate

- 3

- phosphat e synthas e fro m Agrobacterium sp. strain CP4, is rapidly digested in vitro and is

not toxic to acutely gavaged m ice. The Journal O f Nutrition 126: 728

- 740.

Hunkapiller, M. W

., R.M. Hewick, L.E. Hood and W .J. Dreyer. 19 8 3. High

- se n siti v ity

sequencing with gas

- phase sequ enator

. Method s i n Enzy m o l og y 91 : 399

- 413.

Polevoda, B. and F. Sher m an. 2000. N α

- ter m inal acet y lation of eukaryotic proteins. J

Biol Chem 275: 36479

- 36482.

Rade m acher, T. W

., R.B. Parekh and R.A. Dwek. 1988. Glycobiology. Annual Review of

Bioche m istry 57 : 785

- 838.

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App e ndi x D : Material s an d M e thod s Us e d fo r th e Analysi s o f th e Level s of

Cry3Bb 1 an d CP 4 EPSP S Protein s i n MO N 87411

D.1. Materials

Over season leaf (OSL1

- 4), over season root (OSR1

- 4), over season whole plant

(OS W P1

- 4), stover, se n escent root, forage root, forage, grain, pollen and silk tissue

sa mples from glyphosate

- treated MON 87411 were harvested from five field sites in

Argentina during the 2011

– 2012 growing season from starting seed lot 11320173.

E coli

- produce d Cry3Bb 1 (lo t 10000775 ) an d CP 4 EPSPS p rotein (lot 10000739) were

used as the analytical reference stan d ards.

D.2. Characteri z ation of the Materials

The ide n tity of MON 87411 was confir m ed b y conductin g MO N 8741 1 event

- specific

poly m erase chain reaction (PCR) analyses on t h e harve s ted grain from each site.

D.3. Field Design and Tissue Collection

Fiel d trial s wer e initi a te d durin g th e 201 1

– 201 2 plantin g seaso n t o generate

MON 87411 at various m aize growing locat i ons in Argentina. OSL1

- 4, OSR1

- 4,

OSWP1

- 4, stover, senescent root, f orage r o ot, forage, grain, poll e n an d sil k tissue

sa mples from the following field sites were analyzed: Perga m ino, Buenos Aires

(Site Code BAFO); Hunter, Buenos Aires ( S ite Code BAHT); Perga m ino, Buenos Aires

(Site Code BAPE); Sarasa, Buenos Aires (Site Code BASS) and Salto, Buenos A i res

(Site Code BATC). At each site, f our replicated plots of plants contai n ing MON 87411

were planted using a rando m ized co m plete

- bloc k fiel d design

. T i ssue sa m ples were

collected fr o m each replicated p lot at all fie l d s ites. See Ta b l es V

- 1 and V

- 2 for detailed

descri p tio n s of when the sa mples were collected.

D.4. Tissue Processing and Protein Extraction

All tiss u e sa mples harvested were shipped to Monsanto’s processing facility, and were

ground, except pollen, by the Monsanto Sa mple Manage m ent Team to facilitate protein

extraction. The ground tissue sa m ples, and pollen, were st o red in a

- 80 °C freezer u ntil

transferred on dry ice to the anal y tical facility.

D.4.1. Cry3Bb1 Protein

The Cry3Bb1 protein was extracted from m aize tiss u es as descri b ed in Table D

- 1. The

protein extracts were ali q uoted and s t ored frozen in a

- 80 °C freezer u n til analysis.

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Table D

- 1. Cry3Bb1 Protein Extraction Method s 1 for Tissue Samples

S a mple Type Tissue

- to

- B uffer Extra c tion B u ff er

Ratio

50 m M sodium

Lea f 2 /Grai n /Pollen/ S ilk/Roo t 3 /Forag e 4 1:100 carbonate

- bicarbonate

+ 0.1% (w/v) BS A 5

1 Cry3Bb1 pro t ein w a s e xtrac t ed from each tissu e b y a dd i n g th e a p propriat e volu m e o f e xtractio n buffe r and

chro m e stee l beads

, a n d s h a k in g i n a Harb i l m ixe r ( H arbi l Industrie s I n c.

, C o m p t o n

, CA)

. The ex t racted

sa m p le s wer e clarifie d us i ng a ser um filter.

2 Ov e r sea s o n l eaf ( O S L 1

, O S L2, O S L 3

, a nd OS L 4 ).

3 Ov e r sea s o n r oot ( OS R1

, O S R 2, OS R 3, a n d OS R 4 )

, fo rage r oot a n d s e ne s cent ro o t

. 4 For a ge, s to ver a n d ov e r se a s o n w h o l e p l ant ( O S W P 1, O S W P2, O S WP 3

, a nd O S WP 4 ).

5 50 m M sod iu m carbona t e

- b i car bo na t e w it h 0

. 1 % b o v i ne serum a l bu mi n ( B S A) [ 0

. 0 1 5 M N a 2 C O 3 , 0.03 5 M N aHCO 3 , a nd 0

. 1% (w / v) B SA ]

.

D.4.2. CP 4 EPSP S Protein

CP4 EPSPS protein was extracted from m aize tissues sa mples as described in Table D

- 2.

The prot e in extr a cts we r e aliqu o ted a nd stored f r o zen in a

- 80 °C f reezer u ntil analy s is.

Table D

- 2. CP4 EPSPS Protein Extraction Method s 1 for T issue Samples

S a mple Type Tissue

- to

- B uffer

Ratio Extra c tion B u ff er

Lea f 2 /Polle n /Sil k /Forag e 3 1:100 1× PBST + 0.1% (w/v) BS A 5

Grain 1:100 1× TBA + 10 m M DC A 6

Roo t 4 1:50 1× PBST + 0.1% (w/v) BSA

1 CP 4 EPSP S protei n wa s ex t racte d fro m eac h tissu e b y a ddin g t he a p propr i at e volu m e o f extractio n buffer

and c h ro m e steel beads, a n d sh a king in a Harbil m i x e r (Ha rb i l In d ust r ie s I n c.

, C o m p t o n

, CA)

. T he

e x t r acte d sam p le s wer e clarifie d us in g a ser um filter.

2 Ov e r sea s o n l eaf ( O S L 1

, O S L2, O S L 3

, a nd OS L 4 ).

3 For a ge, s to ver a n d ov e r se a s o n w h o l e p l ant ( O S W P 1, O S W P2, O S WP 3

, a nd O S WP 4 ).

4 Ov e r sea s o n r oot ( OS R1

, O S R 2, OS R 3, a n d OS R 4 )

, fo rage r oot a n d s e ne s cent ro o t

. 5 Ph o s p ha t e b uf fer e d sa li ne bu ff e r w it h T we e n

- 2 0 a nd 0 .1% b o v i ne se r um a l bu m i n [ 0

. 00 1 M K H 2 PO 4 , 0

. 01 M N a 2 HP O 4 , 0 .1 37 M N aCl

, a nd 0.00 27 M K C l w it h 0 .0 5% (v / v) T w een

- 20 an d 0

. 1% (w / v) BSA].

6 Tr i s

- b o ra t e bu ff e r (p H 7

. 8) wit h L

- as c o r b i c ac i d a n d 1 0 m M d e ox y c ho li c ac i d [ 0 .1 M T ri s,

0

. 1 M Na 2 B 4 O 7 , 0

. 0 0 5 M M g Cl 2 , 0

. 0 5% (v/ v) T we e n

- 2 0, 0

. 2% (w / v) L

- a sco r b i c ac i d a n d 0

. 01 M D C A]

.

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D.5. Cry3Bb 1 an d CP 4 EPSP S Antibodies

D.5.1. Cry3Bb1 Antibodies

Goa t polyclona l antibodie s specifi c fo r the Cry3Bb1 protein were purified using

Protein G affinity chro m atography. The concentration of the purified IgG was

deter m ined to be 4.5 m g / m l by spectrophoto m etric m ethods. The purified antibody was

stored in (1×PBS) (0.001 M K H 2 PO 4

, 0.0 1 M N a 2 HPO 4

, 0.137 M NaCl, and

0.0027 M KCl).

Purifie d Cry3Bb 1 antibodie s wer e couple d w ith biotin (Ther m o Fishe r Scientific,

Waltha m

, MA) accor d ing to the manufacturer’s instructio n s. The detection reage n t was

NeutrAvidin (Ther m o Fisher Scienti f ic) conjugate d t o horseradis h peroxidase.

D.5.2. CP 4 EPSP S Antibodies

Mouse m onoclonal antibody clone 39B6.1 (IgG2a i s otype, kappa light chain) specific for

th e CP 4 EPSP S protei n wa s purifie d fr o m m o u se ascites fluid using Protei n A affinity

chro m atography. The concentration of the purified IgG was deter m ined to be 2.3 m g / m l

by spectrophoto m etric m ethods. Product i on of the 39B6.1 m onoclonal antibody was

perfor m ed by Strategic Biosolutions (Newark, DE). The purified antibody was stored in

a buffer (0.02 M Na H 2 PO 4

, 0.15 M NaCl, and 15 ppm Proc li n 300)

. Th e detection

reagent was goat anti

- CP4 EPSPS a ntibody, otherwise known as anti

- protein 4 (Si g m a

Aldrich, Saint Louis, MO) conjug ate d t o horseradis h peroxidase.

D.6. Cry3Bb 1 an d CP 4 EPSP S ELIS A Methods

D.6.1. Cry3Bb1 Protein

Goat anti

- Cry3Bb1 capture a n tibodie s wer e dilute d i n a coatin g buffe r (0.01 5 M Na 2 CO 3

, 0.03 5 M NaHCO 3 ,) and im m obiliz e d onto 96

- well m icrotit e r plates a t 5 μ g/ m l f ollowed

by incubation in a 4 ° C refr ig era t o r fo r ≥ 12 hours. Prior to each step in the assay, plates

were washed with 1× PBS containing 0.05 % (v/v) Tween 20 (1× PBST)

. Plates were

blocke d wit h th e additio n o f 15 0 μ l pe r wel l o f 1 × PB S wit h 0.25 % (w/v ) casei n for

60 to 70 m inutes at 37 ° C

. Cry3Bb 1 protei n standar d o r s a mple extr a ct was add e d at

100 μ l pe r wel l an d incubate d fo r 6 0 t o 7 0 m i nute s a t 3 7 ° C. Biotin y lat e d goat anti

Cry3Bb1 antibodies wer e adde d a t 10 0 μ l per well and incub a ted f or 60 to

70 m inutes at 37 ° C. NeutraAvidin

- horseradish p eroxidas e conjugat e wa s adde d at 100 μ l

per well and incubated for 30 to 35 m inutes at 37 ° C

. Plate s wer e develope d by adding

100 μ l per well of horseradish peroxidase s ubstrate, 3,3 ' ,5,5 '

- tetra m ethylbenzidine (TMB,

Kir k egaard & P e rry, Gaithersburg, MD). The enzy m atic re a ction was ter m in a ted by the

addition of 100 μ l per well of 6 M H 3 PO 4

. Quantification of the Cry3Bb1 protein was

acco m p lished by i n terp o lation from a Cry3Bb1 protein standard c u rve that ra n ged from

0.35

- 11.2 ng/ m l.

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D.6.2. CP 4 EPSP S Protein

The CP4 E PSPS ELISA was perfor m ed using plates commercially coated at 2 μg/ m l

(Agr i Diagnosti c Manu f acturing

, Rickreall

, OR)

. CP 4 EPSP S protei n standar d o r sample

extract was added at 100 μ l per well and incubated for 60 to 70 m inutes at 37 ° C. Prior to

each step in the assa y

, plates were was h ed with 1× PBST. Goat anti

- CP4 EPSPS

conjugated to horseradish peroxidase was added at 100 μ l per well a n d incubated f o r

60 to 70 m inutes at 37 ° C. Plates were developed by adding 100 μ l per well of

horseradish peroxidase subst r ate, T MB. The enzy m atic reaction was ter m inated b y the

addition of 100 μ l per well of 6 M H 3 PO 4

. Q u antification of the CP4 EPSPS pro t ein was

acco m plished by interpolation from a CP4 EPSPS protein standard curve that ranged

from 0.456

- 14.6 ng/ m l.

D.7. Moistur e Analysis

Tissue m oisture co n tent was deter m i ned usi n g a Mettler Tol e do HR83 Moistu r e Analyzer

System (Mettler

- Toledo, Inc. Colu m bus, OH). A ho m ogeneous tissue

- specific site pool

(TSSP) was prepared consisting of sa mples of a given tissue type grown at a specific site.

The m ean perce n t m oisture for each TSSP was calc u lated from triplicate analyses. A

TSSP dry weight conversion fact o r (DWCF ) wa s calculate d u s ing MoistureDirect version

4. 0 softwar e a s follows:

D W CF 1

- ( Mean% TSSP Moistu r e

100 )

The D W CF was used to convert protein levels assessed on a μg/g fre s h weigh t (fw ) basis

into levels reported on a μg/g dry weight (dw) basis using the f ollowing c alculatio n :

P r o t ein L e v e l in Dry W ei g ht P r o t ein L e v el F r esh W ei g ht

D W CF

The protein levels (ng/ m l) that w e re r epo r ted to be le s s than or equ a l to the limit of

dete c tion ( L OD) or less than the li m it of quantit a tion (LOQ) on a f resh weight basis were

not reported on a dry weight basis.

D.8. Data Analyses

ELISA plates were analyzed on a SP E CTRA m ax Plus 384 (Molecular Devices,

Sunnyvale, CA) m icroplate spectrophoto m ete r

, usin g a dua l wavelengt h detection

m ethod. Protein concentrations were deter m ined by optical absorbance at a wavelength

of 450 nm with a si m u ltaneous reference r eading of 620 nm. Data reduction analyses

were perfor m ed using Molecular Devices SOFT m ax PRO GxP version 5.4 software.

Absorbance readings a nd protein standard c o ncent r atio n s were f itt e d with a four

para m eter logistic c u r v e f it. Foll o w ing the i n terpol a tion f rom the st a ndard c u rv e

, the

a m ount of protein (ng/ml) in the tissue was reported on a “μg/g fw” basis for data that

were greater than or equal to the LOQ. This conversion utilized a sa mple dilution factor,

and tissue

- to

- buffer ratio. The protein val u es in “μg/g fw” were also converted to

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“μg/g dw” by applying the DWCF. Mic r osoft Excel 2007 (Microsoft, Red m ond, WA)

was used to calc u late t h e protein le v e ls in all m aize tissu e s. The sa mple m eans, standard

deviations (SDs), and ranges wer e als o calculate d usin g Microsof t E x ce l 2007

. All

protein expression levels were rounde d t o tw o significan t figures.

A test sub s tance extract that resulted in unexpectedly negative result by ELISA analysis

was re

- e x tracted twice for the pr o tein of i n terest and re

- analyzed by ELISA to confirm

the r esult. The sa m ple with the con f ir m e d unexpected r esult was om itted f rom all

calc u lations.

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App e ndi x E : Materials

, Method s an d Re s ult s fo r Characterizatio n o f the

DvSnf 7 RN A Produc e d i n MO N 87411

E.1 Materials

Th e MO N 8741 1 RN A wa s extracte d fro m ~V 6 m aize MON 87411 leaf tissue. The

MON 87411 RNA was resuspended in water and stored in a

- 80 °C freezer. The i n vitro

produced DvSnf7 RNA (Dvsnf7_968 RNA; lot 11331164) was used as the reference

substance

. Th e DvSnf7_96 8 RN A wa s in vitro tra n scribed fr o m the plas m i d

pMON149601 and puri f ied using acid phenol:chlorofor m

, precipitated with isopropanol

and resuspended in UltraPure W ater (Invitrogen). The in vi tro transcribe d DvSnf7_968

RNA was previously characterized.

E.2. RNA extraction and Poly(A) enrichment

The MON 87411 RNA was extracted and enric h ed from maize MON 87411 leaf tissue.

The extraction and enrich m ent procedure was not perfor m ed under a GLP plan; however,

all procedures were docu m ented on wor k sheets and, where applicable, SOPs were

followed.

MO N 8741 1 RN A wa s extracte d fro m M O N 8741 1 l ea f tissue

. Prio r to extraction, leaf

tissue was processed into a f ine p owder using a Harbil p aint s h aker. Total RNA was

extracted using an RNeasy Plant Mini Kit (Qiagen, Valencia, C A ) following the

m anufacturer’s instructions. All extracted RNA was eluted by water and stored in a

- 80° C freezer.

MON 87411 poly(A) R NA was enriched fro m MO N 8741 1 RN A usin g th e Ambion

MicroPol y (A ) Puris t MA G ki t (Lif e Technolog ies, Carlsbad, CA) as per m anufacturer’s

specifications

. Al l MO N 8741 1 poly(A ) RN A w a s resuspended in nuclease

- free w ater

and stored in a

- 80 °C freezer.

E.3. Sequ e nc e analyses

E.3.1. Methods

DNA sequence analy s is was perfor m ed to confirm the identity of MON 87411 DvSnf7

RNA and to co mpare its sequen c e t o tha t o f DvSnf7_96 8 RNA.

MO N 8741 1 DvSnf 7 RN A was reverse transcribed using a sequence specific pri m er to

produce cDNA (co mpl e m entary DNA) utilizing an O m niscript Reverse Transcription kit

from Qiagen (Valencia, CA). The m anufac turer’ s specification s wer e followe d wit h the

exception of the incubation ti m e, which was run for 30 m in. The resulting cDNA was

stored in a

- 20 °C freezer.

Tw o overlappin g PC R product s (Produc t A an d P r oduct B) were gener a ted that span the

expected pri m ary transcript sequence of MO N 8741 1 DvSnf 7 RN A ( F igur e E

- 1)

. These

products were analyzed to deter m ine t h e nucleotide sequences of MON 87411 DvSnf7

RNA.

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The PCR amplification of Product A was perfo r m ed on 1 μl of diluted MON 87411

DvSnf 7 cDN A i n a 2 0 μ l reaction volu m e. Each reaction contai n ed a final conce n tration

o f 0. 2 μ M of each pri m er, 1 m M MgCl 2

, 0.2 m M dNTP m ix, and 0.5 units of

Pri m eSTAR® GXL Po ly m erase (Takara Bio Inc., Shiga, Japan). The following cycling

conditions were followed: 38 cycles at 98 ° C for 10 seconds; 60 ° C fo r 1 5 seconds;

68 ° C fo r 5 second s with a n initia l denaturatio n ste p a t 9 8 ° C for 2 m in and a final

extension cycle for 5 m in at 68 ° C.

Th e PC R a mplificatio n o f Produc t B wa s pe r fo r m ed o n 1 μ l o f dilute d MO N 87411

DvSnf 7 cDN A i n a 2 0 μ l reaction volu m e. Each reaction contai n ed a final conce n tration

o f 0. 2 μ M of each pri m er, 1 m M MgCl 2

, 0.2 m M dNTP m ix, and 0.5 units of

Pri m eSTAR® GXL Po ly m erase (Takara Bio Inc., Shiga, Japan). The following cycling

conditions were followed: 35 cycles at 98 ° C for 10 seconds; 60 ° C fo r 1 5 seconds;

68 ° C for 30 seconds w ith an ini t ial denaturat i o n ste p a t 9 8 ° C for 2 m in and a final

extension cycle for 5 m in at 68 ° C.

Aliquots of each PCR product were separated on a 1.2% (w/ v ) agarose gel and visualized

by ethidium bro m ide staining to verif y tha t th e product s wer e th e expecte d size

. Prio r to

sequencing, each verified PCR product was pu r ified using the QIAquick Gel Extraction

Kit (Qiage n

, Inc., Valencia, CA) accor d ing to the m anufacturer’s instructions and

quantified using a Nanodrop spectrophotom e ter according to th e m a nufacturer’s

instr u ctions. The purified PCR products were sequenced using multi p le pri m ers,

including pri m ers used for PCR a mplification.

All sequencing was per f or m ed by Monsanto T G AC (The Geno m e Analysis Center) using

BigDyeTM ter m inator che m istry (Applied Biosyste m s, Fo s ter City, C A ). A con s ensus

sequence w as gene r at e d by co mpiling s eque n ces f rom multi p le s eq u encing rea c tions

perfor m ed on the overlapping PCR products. This consensus sequence was obtained to

deter m ine the identity of MON 87411 DvSnf7 RNA. The consensus sequence was also

aligned to the sequence of DvSnf7_968 cDNA which was established during its

characterization (lot 11331164). This align m ent established the equivalence bet w een

MO N 8741 1 DvSnf 7 RN A an d DvSnf7_96 8 RNA.

E.3.2. Results of Sequence Analyses

In order to characterize the MON 87411 DvS n f7 RNA, a nd to de m o nstrate m olecular

equivalence between it and DvSnf7_968 RNA, the sequence of MON 87411 DvSnf7

cDNA (c o m ple m entary DNA) was deter m i ned

. MO N 8741 1 DvSnf 7 RN A wa s reverse

transcribed to cDNA and a m plified by P CR. The a m plified MON 87411 DvSnf7 c D NA

was sequenced to obtain the pri m ary transcript sequence which contains the predicted

transcription start and polyadenylation sites (Coruzzi et al. 1984), and most i mportantly,

the 240 bp dsRNA functional region (inverted repeat) (Bolognesi et al. 2012). The

consensus sequence of MON 87411 DvSnf7 c D N A wa s aligne d t o th e sequenc e o f cDNA

fr o m D v Snf7_96 8 RN A (DvSnf7_ 9 6 8 cDNA ) whic h was previously characterized under

APS characterization plan 11331164. In t h is A PS characteri zation

, DvSnf7_96 8 RNA

was shown to consist of 968 nucleotides enc o m passing the predicted transcription start,

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 284 o f 3 7 2CB I Delete d Copy

the polyadenylation site, and th e 24 0 b p dsRNA

. Thi s anal ysis de m onstr a ted that the

sequence d region s bet w ee n MO N 8741 1 DvSnf 7 cDN A a n d DvSnf7_ 9 6 8 cDN A a re

identical. More i mportantly, it was shown t ha t ther e i s a 100 % seq u enc e identit y i n the

inverted repeat regions between the M ON 87411 DvSnf7 cDNA consensus sequence and

the DvSnf7_968 cDNA reference sequence (Figure E

- 2). This analysis serves as identity

confir m ation of the M ON 87411 DvSnf7 RNA as well as d e m onstrating the m olecular

equivalenc e betwee n MO N 8741 1 D v Snf 7 RN A an d DvSnf7_96 8 RNA.

E.4. Northern Blot Analysis

E.4.1. Methods

Northern blot analysis was carried out to characterize MON 87411 DvSnf7 RNA a nd to

de m onstrate the m olecular equivale nc e betwee n MO N 8741 1 DvSnf 7 RN A and

DvSnf7_96 8 RNA

. Th e dsRN A region s o f M O N 8741 1 DvSnf 7 RN A an d DvSnf7_968

RNA were specifically ta rgete d throug h RNas e I f treat m ent of the RNAs. RNase I f

preferentiall y digest s singl e strande d RN A leaving behind double stranded regions such

as the active region of DvSnf7. It is this 240 bp dsRNA, that is efficiently taken up by

WC R an d i s processe d b y the RNAi m a chinery, u lti m ately le a ding to mRNA

degradation. Therfore, RNase I f digestion facilitates a d i r ect co m p arison of the size

equivalence between the active dsRNA regions of MON 87411 DvSnf7 RNA and

DvSnf7_96 8 RNA.

DvSnf7_968 RNA and poly (A) RNA from M ON 87411 plant tissues were digested with

RNase I f (New England Biolabs, Ipswich, MA) using the m anuf a cturer’s suggested

reaction conditions for 5 m inutes. Following d i gestion, the sa mples were heat inactivated

at 70 °C for 20 m inutes, precipitated, and resuspended in nuclease

- free water.

RNase I f digested MON 87411 poly (A) RNA and DvSnf7_96 8 RN A w e r e resolve d o n a

~6.6% (w/v) fo r m aldehyde/2.5% agarose gel bu f fered by 1x MESA. The gel was run at

100V for ~2.5 hours. An appropriate m olecular size m arker was included on the gel.

After running, the gel w as stained with SYBR Gold (Life Technologies, Carlsbad, C A )

and the RNA visualized and photographed.

The RNA resolved on the fo r m aldehyde/agarose gel was transferred to a neutral nylon

m e mbrane. The ladder and wells w e re m arked b e fo re UV li g ht c ross

- lin k ing t h e RNA to

the m e m br a ne.

Th e norther n blo t wa s hybridize d wit h a [ α

32 P]

- dATP

- labeled probe specific to the 240

bp dsRNA region of DvSnf7. Prior to hybri d ization, the DvSnf7 probe te mplate was

generated by PCR using plas m id PV

- ZMIR10871 as th e templat e an d p ri m ers specific to

th e DvSnf 7 24 0 b p dsRNA

. Th e PC R product s wer e separate d b y agaros e gel

electrophoresis and purified from the gel u s ing QIAquick Gel Extraction Kit (Qiagen,

Valencia, CA) accor d i n g to t h e m a nufacturer ' s specificati o ns. After purific a tion, t h e

probe te m plate was quantified using a Qubit Fluoro m eter (Life Technologies, Carlsbad,

CA ). Approxi m ately 25 ng of probe t e mplate was lab e led w ith [ α

32 P]

- dATP

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 285 o f 3 7 2CB I Delete d Copy

(~6000 Ci/mmol) using the RadPri m e DNA Labeling System (Life Technologies,

Carlsbad, C A ).

The hybridization was carried o u t at 55 °C and the m e m b rane was w ashed at the same

te mperature. Two exp o sures o f th e blo t wer e generate d usin g Koda k Bio m a x M S film

(East m an Kodak, Rochester, N Y ) in conjunctio n wit h tw o Koda k Bio m a x MS

intensifying screen in a

- 80 °C freezer. The banding in the MON 87411 DvSnf7 RNA

and DvSnf7_968 RNA lanes was visually co m p ared, after a one h our exposure, to

deter m ine their size si m ilarity and t h e identit y o f MO N 8741 1 DvSnf 7 RNA.

E.4.2. Results of the N o rthern Blot Analysis

Pol y (A ) RN A fro m MO N 8741 1 plan t tissue s (MO N 8741 1 pol y (A ) RNA ) and

DvSnf7_96 8 RN A wer e digeste d b y RNas e I f w h ic h preferentiall y digest s singl e stranded

RN A leavin g dsRN A region s intact

. Eac h R Nas e I f d i g ested sa mple was loaded in

triplicate and subje c ted to northern blot analyses. T h e norther n blo t wa s hybridize d with

a probe specific to th e 24 0 b p dsRN A regio n o f DvSnf 7 in order to characterize

MON 87411 DvSnf7 RNA by identifying the presence of the functionally active double

stranded re g ion of DvSn f 7 RNA. Additio n ally, a co mparison of the dsRNA regio n s of

MO N 8741 1 DvSnf 7 RN A an d D vSnf7_968 RNA was conducted using this northern blot

analysis to deter m ine equiv a lence. The results are shown in Figure E

- 3. MON 87411

DvSnf7 RNA contains the expected ~240 bp d s RN A regio n o f DvS nf7 as indicated by

the ~240 base band on the blot (Lanes 4

- 6). T his is the expected size of the double

strande d functionall y activ e regio n o f DvSnf 7 (Bolognes i e t al

. 2012)

. Furthermore,

DvSnf7_96 8 RN A (lane s 1

- 3 ) exhibit s a sin g le band at approxi m ately 240 bases, si m ilar

to MON 87411 DvSnf7 RNA (lanes 4

- 6), i ndicatin g tha t MO N 8741 1 DvSnf 7 RN A and

DvSnf7_968 RNA are equivalent with res p ects to their dsRNA functionally active

regions.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 286 o f 3 7 2CB I Delete d CopyDvSnf7pTranscriptionDvSnf7p P r o d u c t A P r odu c t B

1 2 3 4 5 6 7

12

. 0

3

. 0

2

. 0

1

. 65

1

. 0

0

. 85

0

. 65

0

. 5

0

. 4

0

. 3

0

. 2

0

. 1

D v Snf 7 p D v S n f 7 p

P r odu c t A : ~ 42 5 bp P r o du c t B : ~ 60 9 bp

Figur e E

- 1

. PC R Amp l ifica t io n o f MO N 8741 1 DvSnf 7 cDNA

PCR was performed on MON 87411 DvSnf7 cDNA using two pairs of primers to generate

overlapping PCR fra g m ents for sequencing anal y sis

. To verify the PCR products, the PCR

reactions were run on a g e l. The expected p r oduct size for each a m plicon is provided in the

illustration of the MON 8 7411 DvSn f 7 transcript (A region of I

- Hsp70 is spliced out.) The

MON 87411 DvSnf7 suppression casse t te is depicted above the DvSnf7 transcript. This figure is

a representat i ve of the data generated i n the s t udy a nd the illustrations are not to scale. Lane

designations are as follows:

Lane Sample

1 1 Kb DNA Plus Ladder

2 MON 87411 DvSnf7 cDNA

3 Reverse t r ans c riptase react i on m inus reverse trans c riptase

4 No te m plate control

5 MON 87411 DvSnf7 cDNA

6 Reverse t r ans c riptase react i on m inus reverse trans c riptase

7 No te m plate control

Arrows next to the agaro s e gel image denote t h e size of the DNA, in kilobase pairs, obtained

from the 1kb DNA Plus Ladder (Invitrogen) on the ethidium br o m ide stained ge l

.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 287 o f 3 7 2CB I Delete d Copy

DvS n f 7 8 74 1 1 1

DvS n f7_ 9 68 1

DvS n f 7 8 74 1 1 61

DvS n f7_ 9 68 61

DvS n f 7 8 74 1 1 121

DvS n f7_ 9 68 121

DvS n f 7 8 74 1 1 181

DvS n f7_ 9 68 181

DvS n f 7 8 74 1 1 241

DvS n f7_ 9 68 241

DvS n f 7 8 74 1 1 301

DvS n f7_ 9 68 301

DvS n f 7 8 74 1 1 361

DvS n f7_ 9 68 361

DvS n f 7 8 74 1 1 421

DvS n f7_ 9 68 421

DvS n f 7 8 74 1 1 481

DvS n f7_ 9 68 481

DvS n f 7 8 74 1 1 541

DvS n f7_ 9 68 541

DvS n f 7 8 74 1 1 601

DvS n f7_ 9 68 601

DvS n f 7 8 74 1 1 661

DvS n f7_ 9 68 661 | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

DvS n f 7 8 74 1 1 721

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

DvS n f7_ 9 68 721

Figur e E

- 2. Sequenc e Alignmen t bet w ee n MO N 8741 1 DvSnf 7 cDN A and

DvSnf7_96 8 cDNA

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 288 o f 3 7 2CB I Delete d Copy

DvS n f 7 8 74 1 1 781

DvS n f7_ 9 68 781

DvS n f 7 8 74 1 1 841

DvS n f7_ 9 68 841

DvS n f 7 8 74 1 1 901

DvS n f7_ 9 68 901

DvS n f 7 8 74 1 1 961

DvS n f7_ 9 68 961 | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||| | || | | | ||| | ||

| | ||| | ||

Figur e E

- 2 (continued)

. Sequenc e Alignment bet w een MON 87411 DvSnf7 cDNA

an d DvSnf7_96 8 cDNA

The consensus sequence of MON 87411 DvSnf7 c DNA(top se q uence labeled DvSnf7 87411)

aligned to that of the pr e viously characterized Dv S nf7_968 cDNA (bott o m sequence l a beled

DvSnf7_ 9 68) sequence. The inverted r e peat r e gions are underli n ed. The n umbering i n dicates

base position.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 289 o f 3 7 2CB I Delete d Copy

F ig u r e E

- 3. N o r t h er n B lot A n alysis t o C o nf i r m t h e E qu ival e n c e b e t w ee n t h e d s RN A

in D v S n f 7_968 RN A a n d M ON 87411 D v Snf 7 RN A

T he b l o t w as h y b r i d i z ed w it h a 32 P

- l ab l ed, D v S n f 7 p r obe s pe c i f i c t o t h e 240 bp ds RNA

. L an e s

1

- 3 con t a i n 75 pg of D v S n f 7_968 RN A t h a t w as RN ase I f d i g es t ed and l a nes 4

- 6 con t a i n 200 ng

of M O N 87411 p o l y ( A ) R N A a l so su b j e c t ed t o R N ase I f d i g es t i on. L a n e d e s i g na t i ons a r e a s

f o l l o w s :

L a n e S a m p l e

1 D v S n f 7_968 RN A ( RN ase I f d i g es t e d )

2 D v S n f 7_968 RN A ( RN ase I f d i g es t e d )

3 D v S n f 7_968 RN A ( RN ase I f d i g es t e d )

4 M O N 87411 p o l y ( A ) RN A ( RN ase I f d i g es t e d)

5 M O N 87411 p o l y ( A ) RN A ( RN ase I f d i g es t e d)

6 M O N 87411 p o l y ( A ) RN A ( RN ase I f d i g es t e d)

A rr o w s den o t e t he s i z e of t he RNA

, i n b a ses, o b t a i ned f r om t he L ow R an g e R i bo R u l er l add e r o n

t he SYB R G o l d s t a i n ed g e l

.

M o ns a n to C o m p a n y CR 240

- 13 U1 290 o f 3 7 2CB I Delete d Copy

References for Appendix E

Bolognesi, R., P. R a m a seshadri, J. Anderson, P. Bach m an, W. Clinton, R. Flannagan, O.

Ilagan, C. Lawrence, S. Levine, W

. Moar, G. Mueller

, J

. Tan

, J

. Uff m an

, E

. W i ggins

, G.

Heck and G. Segers. 2012. Characterizing t h e m echani s m of action of double

- stranded

RNA activity against Western c o rn rootworm ( Diabrotica virgifera virgifera LeConte).

PLo S ON E 7:e47534.

Coruzzi

, G.

, R

. Broglie

, C

. Edward s an d N.

- H

. Chua

. 1984

. Tissue

- specifi c an d light

regulated expression of a p ea nuclear gene encoding the s m al l subuni t o f ribulose

- 1

, 5

- bisphosphate carboxylase. EMBO Journal 3:1671

- 1679.

M o ns a n to C o m p a n y CR 240

- 13 U1 291 o f 3 7 2CB I Delete d Copy

App e ndi x F : Material s an d Me t hod s Use d fo r th e Analysi s o f Expressio n Levels

o f DvSnf 7 RN A i n M O N 87411

F.1 Materials

Ove r seaso n lea f (OSL1

, OSL2

, OSL3

, and OSL4), over season root (OSR1, OSR2,

OSR3, and OSR4), over season whole p l ant (OS W P1, OSWP2, OSWP3, and OSWP4),

forage root, forage, senescent root, stover, pollen, silk, and grain tissue sa mples fr o m

glyphosate treated plants grown from start i ng seed lot 11320173 were collected at five

field sites in Argentina during 2011

- 2012 growin g season

. M ON 87411 plots were

treated at the 2

- 4 leaf sta g e with glyphosate herbicide at the label rate (0.9 5 k g active

ingredient [a.i.]/ha). An in vitro transcribe d DvSnf 7 RNA

, DvSnf7_96 8 (lot : 11331164),

was used as an analytical refere n ce standard fo r th e assess m e n t o f D v Snf 7 R N A l e vels

from MON 87411.

F.2 Characteri z ation of the Materials

Th e identit y o f MO N 8 7 41 1 wa s confir m ed b y analysis of the har veste d grai n DN A b y an

event

- speci f ic poly m erase c h ain reaction (PCR) m ethod.

F.3. Field Design and Tissue Collection

Field trials were initiated during the 2011 planting season to generate MON 87411 over

season leaf (OSL1 through OSL4), over season root (OSR1 through OSR4), over season

whole plant (OSWP1 through OSWP4), forage root, forage, senescent root, stover,

pollen, silk, and grain at various m aize gro w ing locatio n s in Argentina. The tissue

sa mples from the following field sites were analyzed : Perga m ino

, Bueno s Aire s (site

code BAFO); Hunter, Buenos Aires (site code BAHT); Perga m ino, Buenos Aires (site

cod e BAPE) ; Sarasa

, Bueno s Aire s (BASS) ; an d Salto

, Bueno s Aire s (sit e cod e BATC).

These field sites were representative of m aize producing r e gion s suitabl e fo r commercial

production. At each site, four replicated plots of plants c o ntai n ing MON 87411 were

planted using a rand om ized complete

- block fiel d design

. Ove r seaso n lea f (OSL1

through OSL4), over season root (OSR1 t h rough OSR4), over season whole plant

(OS W P1 through OSWP4), forage root, forage, senescent root, stover, pollen, silk, and

grain sa m ples were collected from each replicated plot at all field sites. See Table VI

1fo r detaile d description s o f wh en the sa mples were collected.

F.4. Tissue Processing and Total RNA Extraction

Tissue sa mples were s h ipped on dry ice to M o nsanto Co mp any (Saint Louis, Missouri)

and stored in

- 80° C fr e ezers. A l l ti ss u e s a m p l e s excep t f o r po ll en

, we r e roughly gr o und

with dry ice by the Monsanto Sa mple Management Tea m

. The roughly ground samples

and pollen sa m ples were f u rt he r p r ocesse d t o fin e powde r usin g liqui d nitrogen

. T he

prepared tis s ue sa m ples were stored in

- 80° C freezers before and after transferred on dry

ice to the a n alytical facility.

M o ns a n to C o m p a n y CR 240

- 13 U1 292 o f 3 7 2CB I Delete d Copy

Total RNA from leaf (OSL1 through OSL4), root (OSR1 through OSR4), whole plant

(OS W P1 through OSWP4), forage root, senes c ent root, and silk tissues was extracted

using a Trizol m ethod (Cho m czynski and Sacchi 1987) and is briefly described below.

On dry ice, approxi m ately 0.1 gra m s of frozen processe d tissu e wer e transferre d int o a

tube and weighed. Trizol reagent (A m b i on Biotechnology, Austin, TX) was added to the

tub e an d mixe d b y vortexing

. F o llowin g a n incubation at RT, chloroform (Fisher

Scientific, Pittsb u rgh, PA) was added and m ixed. Following another i n cubation at RT,

the tube was centrifuged at 12,000 x g an d ~4° C fo r 1 0 m i nutes

. Th e aqueou s phas e was

transferred to a new tube, a n d th e RN A wa s precipitate d with an equal v o l um e of

isopropyl alcohol (Sig m a, Saint Louis, M O ). To pellet the RNA, the tube was

centrifuged at 12,000 x g an d ~4° C fo r 2 0 m i nutes

. Th e RN A pelle t wa s washe d using

70% (v/v) ethanol and resuspended in nuc l ease

- free water (Gibco by Life Technologies,

Grand Isla n d, NY). All extracted R NA s a mp les were stored in a

- 80°C freezer.

Total RNA from forage, stover, pollen, and g r ain tissues was extracted as follows. On

dry ice, approxi m ately 0.1 gra m s of frozen pro c essed tissue were trans f erred to a tube and

weighed. RNA extraction buffer (25 m M Tris

- HCl pH 8.0, 25 m M EDTA, 75 m M NaCl,

1% SDS, and 1% P

- m ercaptolethanol) was added to the tube and m ixed by vortexing.

An equal volu m e of a c id phenol (pH 4.3): chl o roform [2 : 1 (v/v)] (Sig m a, Saint Louis,

MO; Fisher Scientific, Pittsb u rgh, PA, respectively) was add e d to the tube and m ixed.

The tube was centrifuged at 12,000 x g and 2

- 6 °C for 15 minutes to separate the phases.

The aqueous phase was transferred to a new tube and an equal volu m e of acid phenol

(pH 4.3): chloroform [1:1 (v/v)] was added to the tube and m i xed. The tube was

centrifuged at 12,000 x g and 2

- 6 °C for 10 m i nutes to separate the phases. The aqueous

phase was transferred to a new tube and 1⁄2 volu m e of 15 M LiCl was a dded to the tube

while gently m ixing. The tube was incubated on ice for

~ 2 hours to precipitate R NA.

To pellet the RNA, the tube was centrifuged at 20,000 x g and 2

- 6 °C for 30 m inutes.

The RNA pell e t was washed with 75% (v/ v ) ethanol and resuspended in nuclease

- free

water (Gibco by Life T e chnologies). All ext r acted RNA samples were stored in a

- 80°C

freezer.

Al l extracte d RN A s a mple s wer e quantifi e d usin g a NanoDrop

- 800 0 (Ther m o Scientific,

W i l m ington, DE) acc o r d ing to the manufacturer’s in s tructio n s. Prior to t h e ana l ys is

, each

RNA sample was normali z ed in water to an appropriat e concentratio n m a n ua ll y a n d / o r

usin g a Biome k F X P L a borator y Au t o m ati c Wo r kstatio n ( B e ck m a n Coul t e r Inc.

, Bre a

, CA)

accor d ing to the m anufacturer’s instructions. A l l no r m a li ze d R N A s a m p l e s we re s t o r e d in

a

- 8 0 ° C freeze r befor e analysis.

F.5. Analytical Reference Standard DvSnf7_968

The Analytical Reference Standard DvSnf7_968 was produced by in vit r o transcription.

This in vit r o transcribe d DvSnf 7 RN A c orresponds to the expected in planta D vS n f7

RNA transcribed from the DvSnf7 suppressi o n cassett e i n MO N 87411. The sequence of

the DvSnf7_968 was confir m ed by sequencing a n alysis. The DvSnf7_968 concentration

was 2.21 m g / m l as deter m ined by spectrophotometric analysis.

M o ns a n to C o m p a n y CR 240

- 13 U1 293 o f 3 7 2CB I Delete d Copy

F.6. QuantiGene ® Ple x 2. 0 Assay

Th e no r m al i ze d tota l RN A (Sectio n D.2.4 ) w a s analyze d us i n g a valida t e d QuantiGen e ®

Ple x 2. 0 As s a y ( A f f

y m etrix I n c., Sa nta C la r a, CA ). T his b ead

- ba s ed a ssay r e li e s on a

series of oligonucleotide prob e s that recognize and hybridize specifically to a portion of

the DvSnf7 RNA. Capture probes conjugat e d to a m agnetic polyst y re n e m icrobead are

designed to capture the DvSnf7 RNA, while blockin g probe s ac t t o preven t non

- specific

hybridizatio n t o unrelate d RNA s, thereby increasing assay s p ecificity. The assay also

includes oligonucleotide probes whic h hybridiz e t o th e DvSnf 7 RN A an d th e branched

DNA molecules that m ediate the signal a mpli f ication. Detection of the DvSnf7 RNA is

b y fluorescenc e detectio n o f t h e hybridizatio n co mp le x whi le i t passe s throug h a signal

detector. T he fluorescence intensity is prop o rtional to the nu m ber of the DvSn f 7 RNA

molecules p resent in the sa m ple all o wing t h e DvSnf7 RNA to be quantified by including

a reference standard in the assay. The DvSnf7_968 ( S ection F.5) was used as an

analytical reference sta n dard. To e n sure quality, each assay also includes a backgr o und

control, a negative c ontrol (QC

- ), and a po s itive contr o l (QC+).

F.6.1. Target Specific Probe Set Design and Generation

The probe set used in t h e QuantiGen e ® P l e x 2

. 0 Assay (Affy m etrix Inc.) was designed to

detec t transcript s containin g DvSnf 7 RN A (Affy m etrix Inc. 2010). The probe set was

generated and supplied by Affy m etrix and stored i n a

- 2 0 ° C fr eeze r upon receipt.

Three RNA targets, DvSnf7, actin, and EF

- 1 α

, were inclu d ed in this a s say. The DvSn f 7

RNA was to be m easured and reported. Actin and EF

- 1 α were in c luded in the ass a y to

m eet A ff y met r ix’s r equire m ents f or the QuantiG e n e ® Ple x 2. 0 Assa y design

, whic h ha s to

be at lea s t three targets. However, actin and E F

- 1 α will b e neith e r an al yzed nor r e p orted

since they a r e not r e lev a nt to t h e DvSn f 7 RNA quanti f ic a tio n

.

F.6.2. Backgroun d Control

, Negativ e Quali t y Contro l (QC

- )

, an d Positiv e Q u ality

Control (QC+)

Nuclease

- free water was used as a backg r ound control for each assay since all R NA

sa mples, including the DvSnf7_968 and the t o ta l RN A extracte d fro m th e p l an t ti ssues

,

we r e dissolved and normalized in nuclease

- free w ater.

Tota l RN A extracte d fro m conventiona l m aiz e NL616 9 le a f tissu e w a s u s e d a s a QC

- for

eac h assa y

. Th e l eaf ti s sue sa mp le w as coll ec t e d f r o m NL 6 16 9 p lants g r ow n f r o m s eed

lot 11320471. The absence of the transgene containing the DvSnf7 e xpression cassette

wa s confirme d b y analysi s o f th e lea f DN A extracted from individual plant leaf punches

using an endpoint Taq m a n ® PCR analysis m ethod.

The DvSnf7_968 was spiked to an appropriate c oncentration in a separate aliquot of the

sa m e conventional m aize RNA to serve as a QC+ for each assay.

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F.6.3. Reference Standard Curve Generation

The DvSnf7_968 was spiked in separate al iquot s o f th e sa m e conventiona l m a iz e N L 6 1 69

RNA at eight different concentrations to s e rve as refere n ce stand a rds for each ass a y. A

standard curve was generated fo r eac h assa y plate.

F.6.4. Executio n o f th e QuantiGene ® Ple x 2. 0 Assay

Th e Quant i Gen e ® Pl e x 2. 0 Assa y ( A f f

y m etrix Inc.) w as per f or m ed a c cording to the

m anufacturer’s instructions (Affymetrix Inc. 2010 ) wit h a m odif i cation to introduce a

denaturation step. Briefly, each sample, i n cl u d ing the nuclease

- free water back g round

control, QC

- , QC+, DvSnf7_968 reference sta n dards, and the test sa mp les, was assayed

in t r iplicate wells in a v olu m e of 20 m icroli t e r s (μl). For t h e test s a m ples, the t o tal RNA

was nor m alized to a co n centration such that an equal a m ount of RNA for each tissue type

was added to the assay. To ensure the ca p ture of the DvSnf7 RNA during hybridization,

a denatu r ation step was introd u c e d: the sample was m i xed with t h e targ e t

- s p e ci f ic

oligonucleotide probe set in a well of a 96

- well PCR m i croplate (Axygen Scientific,

Union City, CA) and heated for 5 m inutes a t 9 5 ° C usin g a thermocycle r (Applied

Biosyste m s, Foster, CA). After heating, the samples were kept in the ther m ocycler at

46° C unti l use

. Befor e re m ovin g th e pla t e f rom the ther m ocycler, the hybridization

buffer containing the rest of th e component s fo r th e captur e o f th e DvSnf 7 RN A were

added to each sa m ple well. The PCR m i croplate was then removed from the

ther m ocycler and the content of each well (

~ 100 μl) was transferred to the corresponding

well of a Hybridization Pla t e (Affy m e tri x Inc. ) follow ed b y overnigh t hybridization

(Affymetrix Inc. 2010).

After signal a m plification following the overnigh t hybridization

, al l QuantiGene ®

Ple x 2. 0 Assa y plate s wer e rea d b y a Lu m i nex LX200 analyzer (Lu m inex Corp., Austin,

TX) according to the m a nufacture r ’s instruction s

. The net median fluorescence i n te n sity

(MFI) from each assay well, which is the M F I value subtracted by t h e MFI from the

nuclease

- free water background con t rol, was reported digitally.

F.7. Moist u re Analysis

Tissue m oisture content was deter m ined using an IR

- 200 Moisture Analyzer (Denver

Instru m ent Co m pany, Arvada, CO)

. A ho m o geneou s tissu e

- specific site pool ( T SSP)

wa s prepare d consistin g o f sa mp le s o f a giv e n tissue type grown at a give n site

. The

average percent m oisture for each T S SP was calculated from tri p licate a n alyses. A TSSP

Dry W eight Conversion Factor ( DWCF ) wa s calculate d a s follows:

D W CF 1

- ( Mean% TSSP Moistu r e

100 )

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The D W CF was used to convert Dv S nf7 levels assessed on a μg/g fresh weight (fw) basis

into levels reported on a μg/g dry weight (dw) basis using the f ollowing c alculatio n :

DvSnf7 L e v el in Dry W ei g ht DvSnf7 L e v el in F r esh W ei g ht

D W CF

The D W CFs were o n ly applied to t e st sa m pl e s with DvSn f 7 RNA levels g r eater t h an or

equal to the assay’s li m it of quantit a tion (LOQ).

F.8. Data Analyses

Each net MFI value was converted to a log2 v a lue. The sample m ean ( m ean), standard

deviation ( S D), and coefficient of variation ( C V) f rom the three re p li c ate wells of ea c h

sa mple were calc u lated a nd ret a ined in the r a w data. For each analy s is, a standard curve

was generated based on the log2 values of the theoretical concentrations (ng/ m l) of the

eigh t DvSnf7_96 8 referenc e standard s an d th e log 2 value s o f thei r ne t MFI

, which

established the correlation between the DvSnf7_968 concent r ation in the assay and the

log2 value of the net MFI obtained from the a ssay. Using this standard curve, the net

MFI of the test sa m ples was converte d t o a DvSnf 7 concentr ation (ng/ m l). Each test

sa mple with a DvSnf7 RNA level greater than o r equa l t o th e LO Q wa s converte d t o a

μ g/g f w value using the tot a l RNA/tiss u e r a tio a n d dilution fa ctor. All the μ g/g fw values

were converted to μ g/ g d w value s b y applyin g th e D W CF s. The te s t s a mples with the

DvSn f 7 RNA level lower than or e qual to the a ssay’s LOD or less than the assay ’ s LOQ

were rep o rt e d as ≤ LOD or < LOQ. The m ean, SD, and range of the DvSnf7 RNA levels

in MON 87411 were deter m ined on both fw a n d dw basis f or each tissue type acr o ss all

fiv e sites.

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References for Appendix F

Affy m etrix Inc. 2010. QuantiGene® Plex 2.0 Assay: Using a m agnetic plate washer.

Affy m etrix, Inc., Santa Clara, California.

Cho m czynski, P. and N. Sacchi. 1987. Sing l e

- step m ethod of RNA isolation by acid

guanidinium thiocyanate

- phenol

- chloroform extr a ction. Analytical Biochemistry

162:156

- 159.

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App e ndi x G : Materials

, Methods

, an d Individua l Sit e Result s f o r

Compositiona l Analysi s o f MO N 8741 1 Ma i z e Grai n an d Fo r age

Co mpositi o nal co mparisons between MON 87411 and the conventio n al contr o l m aize

hybrid were perfor m ed using the principles an d analyte s outline d i n th e OEC D consensus

docu m ents for m aize c o mposition (OECD, 2002 )

. These princi p les are accepted gl o bally

and have been e mployed previously in a ssessments of m a i ze p r oducts derived through

biotechn o l o gy. The co m positional asse s s m ent was con d ucted on g rain and f o rage

sa mples h a rvested from a single growing season conducted in Argentina during

2011/2012 under nor m al agrono m ic practices.

G.1. Materials

Harvested grain and forage from M ON 87411, a conventional control that has similar

genetic background to that of MON 87411, and conventional, commercial reference

m aize hybrids were c o mpositionally asses s e d

. The reference hy b rids are li s ted in

Table G

- 1.

Table G

- 1. Conventional Comme r cial Re f er e nce Mai z e Hybrids

Material Na m e Seed Lot # Field Site Codes

Dow AgroSciences Mill 527 113 1 97 5 7 BABE, BA T C

Phillips 717 113 0 00 7 3 BABE, BA F O

, BATC

Stewart S518 112 2 69 2 0 BABE

Syngenta NK 880 113 1 97 5 8 BABE, BAGH, BASS

ACA 2002 113 1 97 5 1 BAFO

NC+ 4443 112 2 67 0 0 BAFO, BA L N

Nider a A X 8 78 113 1 97 5 4 BAFO, BAHT

ACA 430 113 1 97 5 3 BAGH

Gate w a y 6 116 112 2 72 1 1 BAGH

Midland Phi ll ips 7B15P 112 2 67 0 2 BAGH

Stewart S602 112 2 69 1 9 BAHT

Stine 9724 112 9 89 5 1 BAHT

La Tijereta L T 625 113 1 97 5 6 BALN, BA T C

Legac y L7671 112 2 65 9 8 BALN, BA T C

Syngenta NK 940 113 1 97 6 0 BALN

H

- 9180 112 2 67 0 4 BAPE

LG2597 112 2 68 6 2 BAPE

Syngenta NK 910 113 1 97 5 9 BAPE

Dekalb DK747 113 1 97 5 5 BASS

M y c o gen 2 M 746 112 2 67 0 5 BASS

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G.2. Characteri z ation of the Materials

The identities of MON 87411, the conventional control, and reference hybrids were

con f ir m ed prior to use in the co m positional assess m ent.

G.3. Field Production of the Sam p les

Grain and forage sa m ples from MON 87411, the conventional control, and the reference

hybrids were collected from eight replica t e d site s i n Argentin a durin g th e 2011/2012

growin g season

. Th e fiel d site s wer e locate d in: Berdier, Buenos Aire s (BABE) ; three

sites in Perga m ino, Buenos Aires (BAFO, BAPE

, an d BATC) ; Gahan

, Bueno s Aires

(BAGH); Hunter, Buenos Aires (BAHT); Los I ndios, Buenos Aires (BALN); and Sarasa,

Buenos Aires (BASS). Starting seeds were planted in a rando m ized co mplete block

design with four plots for each o f MON 87411, the c o nventional contr o l, and the

reference hybrids. The production was conducted under nor m al agrono m ic field

conditions for their respective geographic r e gions that are typical areas for m aize

production in Argentina. MON 87411 plo t s wer e treate d wit h glyphosat e at

approxi m ately 0.95 kg a.i./hectare at approx i m a tel y th e V 2 growt h stag e t o generate

sa mples under conditions of the intended use of the product.

Forage was collected at early dent (R5) and grain was collected at physiological m aturity.

Forage samples were shipped on dry ice and g rain was shipped at a m bient te mperature

f rom the f i e ld sites to Monsanto C o mpany ( S aint Louis, Missouri). Subsa mples were

ground to a powder, stored in a f reezer set to m aintain

- 20°C located at Monsanto

Co mpany (Saint Louis, Miss o uri). Subsa m pl e s were s hippe d o n dr y ic e t o Covance

Laborat o ries Inc. (Ma d i s on, W isconsin) for co mp ositio n al a n alysis.

G.4. Summary of Analytical Met h ods

Nutrients analyzed in this stud y include d m o isture

, ash

, p r otein

, tota l fat

, carbohydrates

b y calculation

, aci d detergen t fibe r (ADF)

, neu t ral detergent fibe r (NDF)

, tota l dietary

fibe r (TDF)

, a m in o acid s (1 8 components)

, fatty acids (22 co m ponents), m inerals

(calciu m

, copper, iron, m agnesium, m ang a nese, phosphorus, potassiu m

, sodium, and

zinc) and vita m ins [ β

- carotene (referred to as vita m in A)

, B1

, B2

, B6

, E ( α

- tocopherol),

niacin, and f olic acid], in the grain, and m oi s ture, ash, protein, total fat, carbohydrates by

calculation, ADF, NDF, calcium and phosphorus in the forage. The anti

- nutrients

assessed in grain included phytic acid and raffinose. Secondary m etabolites assessed i n

grain included furfural, ferulic acid, and p

- coumaric acid.

All co mpositio n al analyses were per f or m ed at Covance Laborat o rie s

, Inc. (Ma d ison,

W isconsin). Methods for analysis were based on internationally

- r ecognize d procedures

an d literatur e publications

. Brie f descript i on s o f th e m e thod s utilize d fo r th e analyse s are

described below.

G.4.1. 2

- Furaldehyde (Furfural)

The ground sa m ples we r e extracted with 4% t r ichloroacetic acid and injected directly on

a high

- perfor m ance liquid chro m atography ( H PLC) system for quantitation of free

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f ur f urals by ult r aviolet d ete c tion (Albala

- H urtado

, e t al.

, 1997)

. The li m it of quantit a tion

was 0.500 pp m

.

Reference Standard:

• ACRO S O r ganics

, 2

- Furaldehyd e, 99.5%, Lot Nu m ber A0296679

G.4.2. Acid Detergent Fiber

The ANKOM2000 Fiber Analyzer auto m ated t h e process of re m o v al of proteins,

carbohydrates, and ash. Fats and pig m ents were re m oved with an ac e ton e was h prio r to

analysis. The fibrous residue th a t was pri m arily c e ll u lose and lignin and insol u ble

prot e in complexes re m ained in the Ankom f ilter bag, and was deter m ined gravi m etrically

(Goering and Van Soest, 1970; Ko m arek, et al., 1993). The li m it of quantitation was

0.100%.

G.4.3. Amino Acid Composition

The following 18 a m ino acids were analyze d :

• Total alanine • Total l y sine

• Total arginine • Total methionine

• Total aspartic acid (including asparagine) • Total phen y l a lanine

• Total c y stine (including c ysteine) • Total proline

• Total gluta m ic acid (including g l uta m ine) • Total serine

• Total g l y c ine • Total threon i ne

• Total histidine • Total t r y ptophan

• Total isoleucine • Total t y rosine

• Total leucine • Total valine

The sa m pl e s were hydrolyzed in 6N hydrochloric acid for approxi m ately 24 hours at

approxi m ately 106

- 118oC. Phenol was added to the 6N hydrochloric acid to prevent

halogenation of tyrosine. Cystine a n d cysteine were converted to S

- 2

- carboxyeth y lthi o cysteine by the addition of dithiodi p ropi o nic acid. T ryptophan was

hydrolyzed from proteins by heating at approxi m ately 110oC in 4.2N sodium hydroxide

for approxi m ately 20 hours.

The sa mples were analyzed by H P LC after pre

- injection derivati z ati o n. The p ri m ary

a m ino acids were derivatized with o

- phth a laldehyde (OPA) and the secondary amino

acids were derivatized with fluorenyl m ethyl chlorofor m ate (FMOC) before injection

(AOAC

, 2 0 12f ; Barkhol t an d Jensen

, 1989 ; Henderso n an d Brooks

, 2010 ; Henderson

, et

al., 2000; Schuster, 198 8 ). The li m it of quantitation for this study was 0.100 m g/g.

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Referenc e Standards:

Co m ponent Manufacturer Lot No. Purity (%)

L

- Alanine Sigma

- Aldrich BCBC5470 99.8

L

- Arginine Mono h ydro c hloride Sigma

- Aldrich 136 1 811 100

L

- Aspartic Acid Sigma

- Aldrich BCBB9274 100

. 6

L

- C y stine Sigma

- Aldrich 145 1 329 100

L

- Glut a m ic Acid Sigma

- Aldrich 142 3 805 100

. 2

Gl y c ine Sigma

- Aldrich 111 9 375 100

L

- Histidine Mono h ydro c hloride Sigma

- Aldrich BCBB1348 99.9

Mono h ydrate

L

- Isoleucine Sigma

- Aldrich 142 3 806 100

L

- Leucine Sigma

- Aldrich BCBC6907 99.9

L

- L y sine Mo no h ydrochlo r ide Sigma

- Aldrich 136 2 380 100

. 2

L

- Methionine Sigma

- Aldrich 142 3 807 99.9

L

- Phen y lalanine Sigma

- Aldrich BCBC5774 100

L

- Proline Sigma

- Aldrich 141 4 414 99.7

L

- Serine Sigma

- Aldrich 133 6 081 99.9

L

- Threonine Sigma

- Aldrich 140 2 329 100

L

- T y r osine Sigma

- Aldrich BCBC2417 100

L

- Valine Sigma

- Aldrich 135 2 709 100

L

- Trypto p han Sigma

- Aldrich BCBB1284 99.8

G.4.4. Ash

All organic m atter was driven off when the samples were ignited at approxi m ately 550oC

in a m u ffle furnace for at least 5 hours. The re m aining inorga n ic m aterial was

deter m ined gravi m etrically and re f erred to as ash (AOAC, 2012p). The li m it of

quantitation was 0.100%.

G.4.5. Bet a Caroten e (Vitami n A)

S a mples were saponified and extracted with h e xane. The samples were then injected on

a rever s e

- p h ase HPLC system with ult r aviol e t light dete c ti o n. Quantitation was achieved

with a linear regression a n alysis (AOAC, 2012q; Quackenbush, 1987). The li m i t of

quantitation was 0.0200 mg/100g.

Reference Standard:

• Sig m a

- Aldrich, Beta

- Carotene Type I, 98.7% (based on E1%

= 2280 f or La m bda

Maxi m u m of 478 nm to 479 nm in Hexane), Lot Nu m ber 021M1304V

G.4.6. Carbohydrate

The total carbohydrate level was calculated by differenc e usin g th e fres h weight

- derived

data and the following equation (USDA, 1973):

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% carbohydrates

= 100 %

- (% protein + % fat + % m o isture + % ash)

The li m it of quantitation was calculated as 0.100%.

G.4.7. Fat by Acid Hydrolysis

The sa mples were hydrolyzed with hydrochlo r ic acid. The fat was extracted using ether

and hexane. The ext r acts were d r ied down and f ilt e red t h ro u gh a sodium sul f ate c o l u mn.

The re m aining extracts were then evapor ated

, dried

, an d weighe d (AOAC

, 2012 t ; u).

The li m it of quantitation was 0.100%.

G.4.8. Fat by Soxhlet Extraction

The sa mples were weig h ed into a c e llulose t h i m ble containing sodium sulfate and dried

to re m ove e x cess m o isture. Pentane was dripped through the sa m p les t o re m ove the fat.

The extract was then evaporated, dried, a nd weighed (AOAC, 2012v; a). The li m it of

quantitation was 0.100%.

G.4.9. Fatty Acids

The following 22 fatty acids were a n alyze d :

• 8:0 Cap r y lic • 18:0 Stearic

• 10:0 Capric • 18:1 Oleic

• 12:0 Lauric • 18:2 L i noleic

• 14:0 M yristic • 18:3 gamma

- Linolenic

• 14:1 M yristo l eic • 18:3 L i nolen i c

• 15:0 Pentad e canoic • 20:0 Arachid i c

• 15:1 Pentad e cenoic • 20:1 Eicose n oic

• 16:0 Pal m itic • 20:2 Eicosad ie noic

• 16:1 Pal m itoleic • 20:3 Eicosatr i enoic

• 17:0 Heptadecanoic • 20:4 Arachi d onic

• 17:1 Heptadecenoic • 22:0 Behenic

The li p id was extra c ted and sapo n i f ied with 0.5N sodium hydroxide in m ethanol. The

saponification m ixture was m ethylated with 14 % boro n trifluorid e in m ethanol. The

resulting m ethyl e s ters were extracted with heptane containing an i n ternal standard. The

m ethyl esters of the fatty acids were analyzed by gas chro m atography (GC) using

exter n al s tandards for quantitation (AOCS, 2009a; b). The li m it of quantitati o n was

0.00400%.

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Met h y l Oc tanoat e JY10

- W

3

. 0 MA7

- W

1

. 25 9 9 .7

Methy l Decanoate 3.25 1.25 99.6

Met h y l Laura t e 3

. 25 1

. 25 9 9 .8

Met h y l My r i sta t e 3

. 25 1

. 25 9 9 .8

Met h y l My r i sto l ea t e 1

. 0 1

. 25 9 9 .5

Met h y l Pen t a d ecanoa t e 1

. 0 1

. 25 9 9 .6

Met h y l Pen t a d ecenoa t e 1

. 0 1

. 25 9 9 .4

Met h y l Pal m i ta t e 1 0 .0 1 5

. 7 5 9 9 .8

Met h y l Pal m i to l ea t e 3

. 0 1

. 25 9 9 .7

Met h y l Hep t a d ecanoa t e 1

. 0 1

. 25 9 9 .6

Met h y l 1 0

- Hept a d ecenoa t e 1

. 0 1

. 25 9 9 .5

Methy l Stearate 7.0 14.00 99.8

J Y 10

- W Met h y l O l ea t e 1 0 .0 1 5

. 7 5 9 9 .8

M A 7

- W Met h y l L i no l e at e 1 0 .0 1 5

. 7 5 9 9 .8

Met h y l G amma

Linol e n ate 1

. 0 1

. 25 9 9 .4

Met h y l L i no l e na t e 3

. 0 1

. 25 9 9 .5

Met h y l Ara c h i da t e 2

. 0 1

. 25 9 9 .8

Met h y l 1 1

- E i c os e noa t e 2

. 0 1

. 25 9 9 .6

Met h y l 1 1

- 1 4

Eicosadi e n oate 1

. 0 1

. 25 9 9 .5

Met h y l 1 1

- 14

- 17

Eicosatrienoa t e 1

. 0 1

. 25 9 9 .5

Met h y l Ara c h i d o na t e 1

. 0 1

. 25 9 9 .4

Met h y l Be hen a t e 1

. 0 1

. 25 9 9 .8 References :

Manufacturer Lot No. Co m ponent Weight

(%) Puri t y

(%)

N u

- C h e k Pr e p

GL C Refere n c e

S t and a r d

C ov a nce 1

C ov a nce 2

G.4.10. Folic Acid

The sa m pl e s were hydrolyzed in a potassium phosphate buffer with the addition of

ascorbi c aci d t o protec t th e foli c aci d during autoclaving. Followin g hydrolysi s by

autoclaving, the sa m ples were t r eated with a chicken

- pancrea s enzyme and incu b ated

approxi m ately 18 hours to liberate the bound folic acid. The a m ount of folic acid was

deter m ined by co mparing the growth respo n se of the sa m ples, using the bacteria

Lactobacillus casei

, with the growth response of a folic acid standard. This response was

m easured turbidi m etrically (AOAC, 2012c; d; Infant Fo r m ula Council, 1985). The li m i t

of quantitation was 0.0600 μ g/g.

Reference Standard:

• USP, Folic acid, 98.9%, Lot Nu m ber Q0G151

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G.4.11. Minerals/ ICP Emission S p ectrometry

The following nine m inerals were analyzed:

• Calci u m • Phospho r us

• Copper • Potassi u m

• Iron • Sodium

• Magnesi u m • Zinc

• Manganese

The sa mp les were dried, precharred, and ash e d overnight in a m uffle furnace set to

m aintain 500oC. The ashed sa mp les were r e

- ashed with nitric acid

, treate d with

hydrochloric acid, taken to dryn ess

, an d pu t int o a solutio n o f 5 % hydrochlori c acid

. The

a m ount of each ele m ent was deter m ined at appropriate w avelen g ths by co m paring the

e m ission of the unknown sa m ple s

, m easured on the inductively coupled plas m a

spectro m eter, with the e m ission of the standard solutions ( A OAC, 2012 e ; g).

Reference Standard:

Inorgani c Venture s Referenc e Stand ards and Li m its of Quantitatio n :

Mi n eral Lot Nu m bers Concentra t ion

(μg/ m L) LOQ (p p m )

Ca l c ium F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 8 4 200, 10 0 0 20

. 0

C o p p e r F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 83 M C A 2.00, 10

. 0 0.5 0 0

Ir on F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 8 5 10.0, 50

. 0 2. 00

Ma g n esi um F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 83 M C A 50.0, 2 5 0 20

. 0

Ma n g a n es e F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 83 M C A 2.00, 10

. 0 0. 3 00

P ho sp ho r us F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 8 4 200, 10 0 0 20

. 0

P ota ssi u m F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 8 4 200, 10 0 0 1 00

S od i u m F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 8 4 200, 10 0 0 1 00

Z inc F 2

- M EB4 1 70 82 MCA

, F 2

- M EB4 1 7 0 83 M C A 10.0, 50

. 0 0. 4 00

G.4.12. Moisture

The sa m ples were dried in a vacuum oven at approxi m ately 100oC. The moisture w e ight

loss was deter m ined and converted to percent m oisture (A O A C, 2012h; i). The li m it of

quantitation was 0.100%.

G.4.13. Neutral Detergent Fiber

The ANKOM2000 Fiber Analyzer auto m ated the proces s o f th e rem oval of protein,

carbohydrate

, an d ash

. Fat s an d pig m ent s wer e re m oved with an a c eton e was h prio r to

analysis. He m icellulose, cellulose, lignin and insoluble protein fraction were left in the

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 30 4 o f 3 7 2CB I Delete d Copy

filter b ag a n d deter m ined gravi m etrically (AACC, 1998; Goering and Van Soest, 1 9 70;

Ko m arek, et al., 1994). The li m it of quantitation was 0.100%.

G.4.14. Niacin

The sa mpl e s were hydrolyzed with sulfuric acid and the pH was adjusted to remove

interferences

. Th e a m oun t o f niaci n wa s deter m ine d b y comparin g th e growt h response

of the sa mp l es, using the bacteria Lactobacillus plantaru m

, with the growth response of a

niacin standard. This response was m easured turbidi m etrically (AOAC, 2012j; c). The

li m it of quantitation was 0.300 μ g/g.

Reference Standard:

• USP, Niacin, 99.8%, Lot Nu m ber J0J235

G.4.15. p

- Coumaric Acid and Ferulic Acid

The ground sa m ples were extracted with m et h anol followed by alkaline hydrolysis and

buffering prior to injection on an analyt i cal HPLC system for quantification of p

cou m aric acid and fer u lic acid by ultra vi o let ( U V) detection (Hager m an and Nicholson,

1982). The li m it of quantitation for the p c ou m aric acid and ferulic acid was 33.3 ppm.

Referenc e Standards:

Co m ponent Ma n ufa c turer Lot No

. Purity (%)

p

- Hy drox y ci n na m ic A cid

(p

- Cou m aric Acid) Si g m a

- A ldrich 0 9 1M 1 1 9 7V 99

. 6

4

- Hy drox y

- 3

-

methox y c i n na m ic A cid

(Ferulic A c id)

4

- Hydroxy

- 3

-

methox y c i n na m ic A cid

(Ferulic A c id) A CR O S Or ganics

A CR O S Or ganics A 02 6 1 3 54

A 02 9 4 7 16 99

. 4

99

. 4

G.4.16. Phytic Acid

The sa mples were extracted using hydrochlor i c aci d an d sonicat i on

, purifie d usin g a

silica based anion exchange c o lu m n, concentrated and injec t ed onto a HPLC syst e m with

a refractive index detector (Lehrfeld

, 1989 ; Lehrfeld

, 1994 ). The li m it of quantitation

was 0.100%.

Reference Standard:

• Sig m a

- Aldrich, Phytic Acid Sodium Salt Hydrate, 97.9%, Lot Nu m b er

BCBH8701V

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G.4.17. Protein

Th e protei n an d othe r organic nitrogen in the sa m ples were converted to am m onia by

digesting the sa m ples with sulfuric acid con t aining a catalyst m ixture. The acid digest

was m ade alkaline. T he ammonia was disti l led and t h e n titrated with a p revi o usly

standardized acid. Instru m entation was used to auto m ate the dige s tio n

, distillation and

titration processes. The per c en t nitroge n wa s calculate d a nd converted to equivalent

protein usi n g the factor 6.25 (AOAC, 2012k; l). The li m it of quantitati o n was 0.100%.

G.4.18. Raffinose

Sugars in the sa mples were extracted with a 50:50 water: m ethanol solution. Aliquots

were taken, dried under i ner t gas

, an d the n recon s titute d wit h a hydroxyla m ine

hydrochloride solution in pyridine containing phenyl

- β

- D

- glucopyranoside as the internal

standard. T he res u lting oxi m es w e re converted to silyl d erivatives b y treat m ent with

hexa m ethyldisilazane and trifluoracetic acid t r eat m ent, and then analyzed by GC using a

fla m e ionization detector (Brobst, 1972; Mason and Slover, 1971). The limit of

quantitation was 0.0500%.

Reference Standard:

• Sig m a

- Aldrich, D

- (+)

- Raffinose pentahydrate, 99.6%, Lot Nu m ber 019K1156

G.4.19. Total Dietary F iber

Duplicate s a mples were gelatini z ed with α

- a m ylase and digested with enzy m es to b r eak

down starch and protei n

. Ethanol was added to each sample to precipitate the s o luble

f iber. The sa mple was f ilte r ed, and the r esid u e was rin s ed with et h anol and ac e to n e to

re m ov e starc h an d protei n degradatio n p roducts and m oisture. Protein content was

deter m ined for one of the duplicates; ash conte n t was deter m ined f or the other. The total

diet a ry f iber in the sa m p le was calc u lated u s ing protein and ash values (AOAC, 2012 m ).

The li m it of quantitation was 1.00%.

G.4.20. Vitami n B 1 (Th iamine Hydrochloride)

The s a mples were autoclaved under weak acid conditions to extract t h e thia m ine. The

resulting solutions were incubated with a buffered enzy m e solution to release any bound

thia m ine. The solutions were purified on a cation

- exchange colu m n

. Aliquots were

reacted with potassium ferricya n ide to convert thia m ine to thiochro m e. The thiochro m e

was extracted into is o butyl alco h ol, m ea s ured on a fluoro m eter, and quantitated by

co mp ariso n t o a know n standar d (AOAC

, 2012n ; o; r). The li m it o f quantitatio n was

1.10 m g/100g.

Reference Standard:

• USP

, Thi a min e Hydrochloride

, 99.7%

, Lo t Numbe r P0K366

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G.4.21. Vitamin B2 (Riboflavi n )

The sa m ples were hydrolyzed with dilute hydroc hlori c aci d an d th e p H wa s adjuste d to

re m ove interferences. The a m ount of riboflavin was deter m ined by co mparing the

growth response of the sa m p les, using the bacteria Lactobacillu s rhamnosus

, with the

growth response of m u l tipoint ribo f lavin st a ndards. The growth response was m easured

turbi d i m etrically (AOAC, 2012s; c )

. The li m it of quantitati o n was 0.200 μ g/g.

Reference Standard:

• USP

, Ribof l a vin

, 99.7%

, Lo t Nu m b e r N1J079

G.4.22. Vitamin B6 (Pyridoxine Hydrochlori d e)

The sa mples were hy d r o lyzed with dilute s u l f uric a cid in t h e aut o clave and the pH was

adjusted to re m ove interferences. The a m ount of pyridoxine was deter m ined by

co mp aring the growth response of th e sa m p les

, usin g th e yeas t Saccharomyces

cerevisiae

, with the growth response of a pyridoxine standard. The response w a s

m easured turbidi m etrically. Results were reported as pyridoxi n e hydrochlorid e (AOAC,

2012b ; Atkins

, e t al.

, 1943)

. Th e li m it of quan t itation was 0.0700 μ g/g.

Reference Standard:

• USP

, Pyridoxin e hydrochloride

, 99.8%

, Lo t Numbe r Q0G409

G.4.23. Vitami n E ( α

- Tocopherol)

The sa mples were saponified to break do w n any fat and release vita m in E. The

saponified m ixtures were extracted with ethyl ether and then quantitated by high

perfor m ance liquid chro m atography using a sili c a colu m n (Cort

, e t al.

, 1983 ; McMurray,

et al., 1980; Speek, et al., 1985). T h e li m it of quantitation was 0.00500 mg/g.

Reference Standard:

• USP, Alpha Tocopherol, 98.9%, Lot Nu m ber N0F068

G.5. Data Processing and Statistical Analysis

A f ter co m p ositio n al a n alyses we r e per f or m ed, data sp r e a dsheets co n t aining in d i v idual

values for each anal y sis were sent to Monsanto Co m pany for re v iew. Data were then

transferred to Certus International, Inc., where they were converted into the appropriate

units and stati s ti c ally a nalyzed. T he f ollowing fo r m ulas were used for re

- expression of

co mposition data for statistical anal y sis (Ta b le G

- 2):

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Table G

- 2. Re

- expression Formulas for S t atistica l Analysi s o f Compositio n Data

C o m ponent From (X) To For m ul a 1

Proxi m a tes (excluding Moisture),

Fiber, Anti

- nutrients % fw t % dw X/d

A m ino Acids (AA) m g/g f w t % dw X/(10d)

Secondary Me tabolites ppm fwt μ g/g dw X/d

Copper, Iron, Manganese, Zinc ppm fwt m g/kg dw X/d

Calci u m

, Magnesi u m

, Phospho r us,

Potassi u m

Folic Acid, Niacin, Vita m in B2,

Vita m in B6 ppm fwt % dw X/(10 4 d)

μ g/g fwt m g/kg dw X/d

Vita m in A, Vita m in B1 m g/100g fwt m g/kg dw 10X/d

Vita m in E m g/g f w t m g/kg dw 10 3 X/d

(100) X j / Σ X, for

Fatty Acids ( FA) % fw t % Total FA each F A j wh ere Σ X

is over all the FA

1 ‘X’ is t he i n dividual sample value; d is the fraction of the sa m ple t hat is dry m a t ter.

In order to co m plete a statistical analysis f or a co m p ositio n al c o nstit u ent in this

co mpositional ass e ss m ent, at lea s t 5 0 % of all t h e values for an anal y te in grain or f o rage

had to b e g r eat e r t h an the assay li m it of quantit a tion (LOQ). Analytes with m ore th a n

50 % o f observation s belo w th e assa y LO Q wer e excluded from sum m aries and a n al y sis.

The following 16 analytes in grain with m ore tha n 50 % o f observation s belo w th e assay

LOQ were excluded from statistical a n aly s i s : 8:0 capr y lic acid, 1 0 :0 capric acid, 12:0

lauric acid, 14:0 m yristic acid, 14:1 m y ristoleic acid, 15:0 pentadecanoic acid, 15:1

pentadecenoic acid, 16:1 pal m itoleic acid, 17: 0 heptadecanoi c ac i d

, 17: 1 heptadecenoic

acid

, 18: 3 gamm a linoleni c ac id, 20:2 eicosadienoic acid, 20 : 3 eicosatrienoic acid, 20:4

arachidonic acid, sodium, and furfural.

Otherwise, individu a l r e sults b elow the LOQ were assigned a value equal to one

- half the

quantitation li m it. Fourteen observations for 22:0 Behenic were assigned a value equal to

one

- hal f o f th e LO Q (0.002 % fwt).

The data were assessed for potential outl i ers using a studentized PRESS residuals

calculation. A PRESS residual is the d i fference between any value and its value

predicted from a statistical m odel that exclu d es the data point. The studentized version

scales these residuals so that t h e val u es t e nd to have a standard nor m al distribution w hen

outlier s ar e absent

. Thus

, m o s t v a lues are ex p ected to b e between ± 3. Extre m e data

points that are also outside of the ± 6 studentize d PRES S res idual ranges are co n si d ered

for exclusion, as outliers, from the fin a l analyses. Seven co mpon e nts had PRESS

residual values outside of the ± 6 range.

Of the seven flagged values, only the vita m i n E value from a conventional reference was

re m oved f rom f urther a nalysis as a n outli e r. The re m aining values were not re m oved

M ons a n t o Co m pany CR 2 4 0

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because they were not extre m e values o r t h ey were dee m ed sufficiently cl o se to

neighboring values to lack sufficient evidence for re m oval.

The outlier test procedure was reapplied to the re m aining vita m in E data to detect

potential o u tliers that were m asked in the first analysis. One vita m in E value from a

commercial re f erence w as ide n ti f ied as an outli e r, but t h e value was not an extre m e value

and was not re m oved as an outlier.

Maize co m positio n al co mponents were stati s tically anal y zed using a m ixed

- model

analy s is of varia n ce with th e SA S MIXE D procedure.

Analyses of the co m bined replicated sites were perfor m ed using m odel (1).

(1) Yijk

= U + Ti + Lj + B(L)jk + LTij + eijk,

where Yijk

= unique i n dividual observation, U

= overall mean

, T i

= substanc e effect,

Lj

= random site effect, B(L)jk

= random block within site effect, LTij

= random site by

substance i n teraction effect, an d eij k

= residua l error.

For each co m ponent analysis, individual m ean co m parison tests of MON 87411 vs.

conventional control were conducted.

A range of observed values from the re ferenc e hybrid s wa s deter m ine d fo r each

analytical co m ponent. Additionally, data from the reference hybri d s were used to

develop 9 9 % t o lera n ce int e rv a ls. A tole r ance i n t e rval is a n int e rv a l t h at one can c lai m

, with a specified degree of c onfidence, contains at least a specifie d proportion

, p

, o f an

entire sa mpled population for the para m eter m easured.

For each c o mpositional co mponent analyzed, two

- sided 9 9 % t o lera n ce intervals were

calc u lated that are ex p ected to c o ntain, w i th 95% confidence, 99% of the qua n tities

expressed in the population of reference hy b rids. Each esti m ate was based upon the

average of all observations per unique refer e nce hybrid. Because n e gative quantities are

not possible, negative calculated lower tolerance bounds were set to zero.

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References for Appendix G

AACC. 1998. Approved m ethods of the A m e r ica n Associatio n o f Cerea l Chemists.

A m erican Association of Cereal Ch e m ists

, Inc.

, St

. Paul

, Minnesota.

Albala

- Hurtado, S., M.T. Veciana

- Nogues, M. Izquierdo

- P ulido and M.C. Vidal

- Carou.

1997. Deter m ination of free and total furfural co m pounds in infant m i lk for m ulas by

high

- perfor m ance liquid chro m atography. Journal of Agricultural and Food Ch e m istry

45 : 2128

- 2133.

AOAC

. 2012a

. AOA C off i cial m e t hod 960.39: Fat (crude) or ether extract in meat.

Association of Analytical Com m unities Internati o nal, Gait h er s burg, Mar y land.

AOAC. 2012b. AOAC official m ethod 961.15: Vita m in B 6 (pyridoxine, pyridoxal,

pyridoxa m ine) in food extracts. A s sociation of Analytical Commu n ities Internati o nal,

Gaithersburg, Maryland.

AOAC

. 2012c

. AOA C off i cial m ethod 960.46: Vita m in assays. Association of Analytical

Communities Internatio n al, Gait h ers b urg, Mar y land.

AOAC

. 2012d

. AOA C officia l m e tho d 992.05 : Tota l folate (Pteroylgluta m ic acid) in

in f ant fo rmula. Association of Analytical C o m m unities Intern a tio n al, Gaith e r s burg,

Maryland.

AOAC. 2012e. AOAC official method 984.27: Calciu m

, copper, iron, m agnesiu m

,

m anganese, phosphorus, potassiu m

, sodiu m

, and zinc in in f ant for m ula. Association of

Analyti c al C o m m unities Inter n ational, Gaithersb u rg, Mar y la n d.

AOAC

. 2012f

. AOA C officia l m e tho d 988.15 : Tryptopha n i n food s an d foo d an d feed

ingre d ients. Associati o n of Analytic a l Communities I ntern a tion a l, Gaithe r s b urg,

Maryland.

AOAC

. 2012g

. AOA C offic ial m e t hod 985.01: Metals and other ele m e nts in plants and

pet foods. Association of Analytical Com m unities Internatio n al, Gait h ers b urg, Mar y land.

AOAC

. 2012h

. AOA C officia l m ethod 925.09: Solids (to t al) and loss on drying

( m oisture) in f lour. Association of Anal y ti c al Communiti e s Int e rnati o nal, Gaith er sburg,

Maryland.

AOAC

. 20 1 2i

. AOA C officia l m ethod 926.08: Loss on drying ( m oisture) in cheese.

Association of Analytical Com m unities Internati o nal, Gait h er s burg, Mar y land.

AOAC

. 20 1 2j

. AOA C officia l m ethod 944.13: Niacin and niac i na m ide (nicotinic acid

and nicotina m ide) in vita m in preparations. Association of Analytical Com m unities

International, Gaithersburg, Maryland.

M ons a n t o Co m pany CR 2 4 0

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AOAC

. 2012k

. AOA C off i cial m e t hod 955.04: Nitrogen (total) in fertilizers Kjeldahl

m ethod. Association of Analytical C o m m unities Inter n ational, Gaithersb u rg, Mar y la n d.

AOAC

. 2012l

. AOA C officia l metho d 979.09 : Protei n i n grain s. Associatio n of

Analyti c al C o m m unities Inter n ational, Gaithersb u rg, Mar y la n d.

AOAC

. 2012 m

. AOA C official m ethod 985.29: Total dieta r y fibe r i n foods

. Association

of Analytical Com m unities Internati o nal, Gait h er s burg, Mar y land.

AOAC

. 2012n

. AOA C off i cial m ethod 942.23: Thia m ine (Vita m in B 1 ) in hu m an a nd pet

foods. Association of Analytical C o mmuniti e s International, Gaithersb u r g, Marylan d

.

AOAC

. 2012o

. AOA C officia l m e t hod 953.17: Thia m ine (Vita m in B 1 ) in g rain p r odu cts.

Association of Analytical Com m unities Internati o nal, Gait h er s burg, Mar y land.

AOAC

. 2012p

. AOA C officia l m e tho d 923.03 : As h o f flour

. Associatio n o f Analytical

Communities Internatio n al, Gait h ers b urg, Mar y land.

AOAC. 2012q. AOAC official method 2005.07 β

· carotene in supple m ents and raw

m aterials reversed

- phase high pressure liquid chro m atographic m ethod. Association of

Analyti c al C o m m unities Inter n ational, Gaithersb u rg, Mar y la n d.

AOAC

. 20 1 2r

. AOA C officia l m ethod 957.17: Thia m ine (Vita m in B 1 ). Associati o n of

Analyti c al C o m m unities Inter n ational, Gaithersb u rg, Mar y la n d.

AOAC

. 2012s

. AOA C officia l metho d 940.33: Riboflavin (Vita m in B 2 ) in v ita m in

prepa r atio ns

. Association of Analytical C o mmunities Inter n ational, Gaithers b urg, Maryland.

AOAC

. 20 1 2t

. AOA C officia l m ethod 922.06: Fat in flour. Association of Analytical

Communities Internatio n al, Gait h ers b urg, Mar y land.

AOAC

. 2012u

. AOA C offic ial m ethod 954.02: Fat (crude) or ether extract in pet food.

Association of Analytical Com m unities Internati o nal, Gait h er s burg, Mar y land.

AOAC. 2012v. AOAC official m ethod 948.22: Fat (crude) in nuts and nut products.

Association of Analytical Com m unities Internati o nal, Gait h er s burg, Mar y land.

AOCS. 2009a. Official Me t hods and Recom m ended Pr a ctices of the A m erican Oil

Ch e m ists' Society. A m erican Oil Che m ists' Soc i ety, Cha m p a ign, Illinois.

AOCS. 2009b. Preparation of m e t hyl esters of fatty acids. A m erican Oil Ch e m i sts'

Society

, Champaign

, Illinois.

M ons a n t o Co m pany CR 2 4 0

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Atkins, L., A.S. Schultz, W .L. W i llia m s an d C.N

. Frey

. 1943

. Y eas t m i crobiological

m ethods for deter m ination of vita m i ns. Indu s trial and Engineering Che m istry, Analytical

Edition 15: 141

- 144.

Barkholt, V. and A.L. Jensen. 1989. A m ino acid analysis deter m ination of cysteine plus

half

- cystine in proteins aft e r hydrochloric acid hydrolysis with a disulfide co mpound as

additive. Analytical Bi oche m i str y 177 : 318

- 322.

Brobst, K.H. 1972. Gas

- liquid chro m atography of tri m ethylsilyl derivatives. Pages 3

- 8 in

Methods in Carbohydrate Che m istry. Volu m e 6. Acade m ic Press, New York, New York.

Cort, W

., T.S. Vicente, E.H. Waysek and B.D. W illia m s. 1983. Vita m in E content of

feedstuffs deter m ined by high

- perfor m ance liquid chro m atographic fluorescence. Journal

of Agriculture and Food Ch e m istry 31: 1330

- 1333.

Goering

, H.K

. an d P.J

. Va n Soest

. 1970

. For a g e fibe r analyse s ( a pparatus

, reagents,

procedures, and so m e applications). Agr icultur e Handboo k No

. 379

. U

. S. Depart m e nt of

Agriculture, Agricultural Research S ervice, W a s hington, D.C.

Hager m an, A.E. and R.L. Nicholson. 1982. High

- perfor m ance liquid chro m atographic

deter m ination of hydroxycinna m ic acids in the m aize m esocotyl. Journal of Agricultural

and Food Che m istry 30: 1098

- 1102.

Henderson, J. W

. and A. Brooks. 2010. I mpro v ed a m ino acid m ethods using Agilent

ZORBAX Eclipse Plus C18 columns for a v a r iety of Agilent LC instru m entation and

separation goals. Agilent Technologies, Inc., W il m ington, D e laware.

Henderson, J. W

., R.D. Ricker, B.A. Bidling m eyer and C. W oodward. 2000. Rapid,

accurate, s ensitive, and reprod u cible HPLC analysis of a m ino acids. Agile n t

Technologies, Inc., W il m ington, D e laware.

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Infant Formula Council, Atlanta, Georgia.

Ko m arek, A.R., J.B. Robertson and P.J. V a n Soest. 1993. A co mparison of m ethods for

deter m ining ADF using the Filter Bag Techn i q u e versus co n ventional filtration. Journal

of An i m al Science 72: 114.

Ko m arek, A.R., J.B. Robertson and P.J. Van Soest. 1994. C o m parison o f the filter b ag

technique to conventional filtration in the V a n Soest NDF analysis of 21 feeds. National

Conference on Forage Quality, Evaluation, and Utilization, A m e rican Society of

Agrono m y, Inc., Crop S cience Society of A m e r ica, I n c., Soil Society of A m erica, Inc.,

Madison

, Wisonsin.

Lehrfeld, J. 1989. High

- perfor m ance liquid chr om atography analysis of phytic acid on a

pH

- stable m acroporous poly m er col u m n. Cereal Ch e m istry 66: 510

- 515.

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- 1 3 U 1 312 o f 3 7 2CB I Delete d Copy

Lehrfeld, J. 1994. HPLC separation and quan t itation of phytic acid and so m e inositol

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. A. Rice. 1980. Influence of extraction

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Develop m ent, Paris, F rance.

Quackenbush, F. W

. 1987. Reverse phase HPLC separation of cis a n d trans

- carotenoids

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- carotenes in food m aterials. J ourna l o f Liqui d Chro m atography

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- 654.

Schuster, R. 1988. Deter m ination of a m ino acids in biological phar m aceutical plant and

food sa m p l es by auto m ated precolu m n d e rivatization and high

- perfo r m ance liquid

chro m atography. Journal of Chro m atography 431: 271

- 284.

Speek, A.J., J. De Schrijver and W

. H.P. Schreurs. 1985. Vita m in E composition of s o me

seed oils as deter m ined by high

- perfor m a nce liquid chromatography with fluoro m etric

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USDA

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. Energ y valu e o f foods : Basi s an d derivation. Agricultural Handbook No.

74. US Depart m ent of Agriculture, Agricult u ral R esearch Service, W ashington, D.C.

M ons a n t o Co m pany CR 2 4 0

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App e ndi x H : Materials

, Methods

, an d Individua l Sit e Result s fo r Seed

Dormanc y an d Germinatio n As s e ssmen t o f MO N 87411

H.1. Materials

Seed ger m ination and dor m ancy characteri s tic s wer e assesse d o n see d fro m MO N 87411,

the conventional control, and reference hybrids produced in replicated field trials during

2012 at the following sites: Jefferson Coun t y, IA (IARL), Warren County, IL (I L MN),

and Pawnee County, KS (KSLA).

H.2. Characteri z ation of the Materials

The identities of the MON 87411 and the conventional control starting seed were verified

by event

- specific poly m erase chain reaction ( P CR) analyses. During th e growin g season,

the field planting order of MON 87411 and t h e conventional control plots was confir m e d

by event

- s p ecific PCR analyses. C h ain

- of

- c ustody docu m entation for all starting seed for

dor m ancy and ger m ination study w as m aintained from ha r vest through ship m ent to the

perfor m ing laboratory with the use of packag i ng labels and plant sa m ple transfer forms.

H.3. Germination Testing Facil it y an d Experimenta l Methods

Ger m ination and dor m ancy evaluations were conducted at BioDiagnostics, Inc. in River

Falls, W I. The prin c i p al inve s ti g ator was qualified to conduct seed ger m ination and

dor m ancy testing consistent with th e standard s establishe d b y th e Associatio n o f Official

Seed Analysts (AOSA), a seed trade associatio n (AOSA / SCS T 2010 ; AOSA

, 2012a;

2012b).

The seed lots (Selfed F 2 grain) of MON 87411, the conventional control, and four

reference h ybrids from each location were tested under seven different te mperature

regi m es. Seven ger m ination chambers were used in the study and each cha m ber was

m aintained dark under one of the following seven temperature regi m es: constant

te mperature of approxi m ately 5, 10, 20, or 30 °C or alternating te mperatures of

approxi m ately 10/20, 10/30, or 20/30° C. T h e alternating te m perature regi m es were

m aintained at the lower te mpe r atur e fo r 1 6 hour s an d th e highe r te m perature for 8 hours.

The te mperature insi d e each ger m ination cha m ber was m onitored and recorded

throughout the duration of the study.

Approxi m ately 100 seeds each o f MON 87 4 11, the c o nventional contr o l, a n d the

reference h ybrids were placed on pre

- m oiste n ed ger m ination towels using a vacu u m

planting syste m

. Additional pre

- m oistened g e r m ination towels were placed on top of the

seed. The ger m ination towels were then rolled up in a wax c over. All rolled ger m ination

towels were labeled and placed i n to an approp r iately labeled bucket. Ea c h bucket within

a te m perat u re re g i m e represented a replicate p e r site. Th e re were 4 re p s per site for a

total of 12 buckets for each te mperature re g ime. Each b u cket cont a i n ed 6 towels or 1

towel per e n try. Buckets were then p laced in the appropriate ger m ination cha m bers.

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Each te mperat u re regi m e constituted a s e p a rate split

- plot experi m ent with four

replications where the w hole

- p lot was t h e l o cati o n where starting seed ( S elfed F 2 grain)

was produced and the sub

- plot was the m aterial type ( i.e

. , MO N 87411

, th e conventional

contr o l or reference m aterial). W hole

- p lots and sub

- plots were rando m ized within each

te mperature regi m e.

A descri p ti o n of each ger m ination characteris t ic evaluated and the ti m ing of evaluations

are pr e sent e d in Table VIII

- 1. The types of data colle c ted depended o n the te mperature

regi m e. Each rolled g e r m ination towel in the AOSA

- recommended temperature regi m e

( i.e

. , alternating 20/30 °C) was a s sessed per i odically during the study for no r m ally

ger m inated, abnor m ally ger m inated, hard (vi abl e an d nonviable)

, dead

, an d fi r m swollen

(viable and nonviable) seed as defined by AOSA guidelines (AOSA, 2012a; 2012b).

AOSA only provides guidelines for testing seed under opti m al te m p e ratures, whereas

additional temperature regi m es were included to test diverse environ m ental conditions.

Therefore, each rolled g er m ination towel in the additional temperature regi m es ( i.e

. , 5,

10, 20, 30, alternating 10/20, and 10/30 °C) was assessed periodically during the study

for ger m inated, hard (viable and nonviabl e ), dead, and firm s w ollen (viable and

nonviable) seed. Because te m perature ext r e m es could affect the develop m ent of

seedlings, AOSA standards were not appli e d and no distinction w as m ade between

nor m al or abnor m al ger m inated seed. Therefo r e

, an y seedlin g wit h a radica l o f 1 m m or

more was classified as g er m inated.

The calculation of percent s e ed in each a s ses sm ent categ o ry was ba se d on the actual

nu m ber of seeds eval u ated ( e.g.

, 99 or 101). Across temperature regi m es, the total

nu m ber of seeds evaluated from each ger m inati o n towel was approxi m ately 100.

W ithin both AOSA and the additio n al te mperature regi m es, hard and fir m

- swollen seeds

re m aining at the final evaluation date were s ubjected to a tetraz o lium (Tz) test for

evaluation of viability according to AOSA standards (AOSA/SCST 2010). The number

o f nonviabl e har d an d nonviabl e firm

- swolle n s e e d wa s adde d t o th e nu m be r o f dea d seed

counted on all collecti o n dates to deter m ine the total per c en t dea d seed

. Tota l count s for

percent viable hard and viable fi r m

- swollen se e d were det er m ined f rom the Tz t e st.

H.4. Statistical Analysis

A n analysi s o f varianc e wa s conducte d usin g SA S  (SAS 2008) accordi n g t o a split

- plot

design (production site as the whole plot and starting seed mate r ial as t h e sub

- pl o t) with

four replications. MON 87411 was co mpared to the conventional control for ger m ination

and dor m ancy characteristics of s e ed produced within each site ( i.e

. , individua l site

analysis) and in a co m b ined

- site analysis in which the data were pooled across all three

sites. The seed ger m ination and d or m ancy characteristics analyzed included perce n t

ger m inated seed, perce n t via b le h ard seed, percen t dea d seed

, an d percent viable fir m

swollen s eed. The percent ger m inated seed w e re categorized as either n or m al ger m i nated

 SA S i s a re g i stere d t r a d e m ar k o f SA S I n stit u te

, I n c.

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or abnor m al ger m inated for the AOSA t e m p e r ature regi m e. The level of statistical

significance was predeter m ined to be 5% ( α =0.05)

. MO N 8741 1 wa s no t statistically

co mpared to the reference hybrids, nor were co mparisons m ade across te m perature

regi m es. T he m in i m u m and m axi m u m m ean v a lues we r e deter m ined f rom the re f erence

m aterials a c ross a ll sites to provid e a rang e o f value s ( i.e

. , referenc e range ) re p rese n t a tive

of commercial m aize hybrids. Results from t h e co m bined

- site analysis are presented in

Table VIII

- 2.

H.5. Individual

- Site Seed Dorma n cy and Germination A n alysis

In the individual site analyse s

, fi ve st a ti s tic a lly s igni f i ca nt di ff eren c es in total were

detected between MON 87411 and the conv e ntional control for the m easured

characteristics ( i.e

. , percent ger m inated, viable hard, d e ad

, o r viabl e fir m

- swolle n seed)

of the seed (Selfed F 2 grain) produced at the IARL, ILMN, and KSLA sites (Table H

- 2).

MON 87411 had fewer dead seed than the conventional control at 10/20° C (0.7 % vs.

2.0%) and more viable firm s w ollen seed than t h e conventional contr o l a t 20° C (0.3 % vs.

0.0% ) fo r th e see d produce d a t th e IAR L site. MON 87411 had m ore ger m inated seed

than the conventional control at 20/30°C (99.3% vs. 97.8%) and fewer dead seed than the

conventional control at 20/30°C (0.3% vs. 1.8%) for the seed produced at the I L MN site.

MON 87411 had m ore ger m inated seed than th e conventiona l contr o l at 5°C (0.5% vs.

0.0%) f or the seed p r oduced at the KSLA site. St a ti s ti c ally sig n i f i c ant di ff ere n ces

betwee n MO N 8741 1 an d th e conventiona l control for ger m ination a m d dor m an c y

characteristics in the individual site analy s es were not consistently detected across

te m perature regi m es or the individual sites. These differe n ces were n o t det e cted in the

co m bined site an a lysis (Section VIII

- 1, Tab l e VIII

- 2 ) and are unlik e ly to be biolo g ic a lly

m eaningful in ter m s of inc r ea s ed pest/weed potential (See F i gure VIII

- 1, Step 2, answer

“no”).

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Table H

- 1. Starting Seed of MON 87411, Conventional Control and Commercial

Mai z e Reference Hybrids Used in Dormancy Assessment

Site Code Material Name Pheno t y pe Monsanto Lot

N u m b er

IARL MON 87411 Gl y p hosate

- Tolerant

and Insect

- Protected 113 5 48 9 9

IARL MPA640 B 1 Conventi o nal Control 113 5 48 9 4

IARL Legacy L7 67 1 Conventi o n al Reference 113 5 48 9 5

IARL Lewis 7007 Conventi o nal Reference 113 5 48 9 6

IARL Gateway 6 11 6 Conventi o nal Reference 113 5 48 9 7

IARL Phillips 717 C onventional Reference 113 5 48 9 8

KSLA MON 87411 Gl y p hosate

- Tolerant

and Insect

- Protected 113 5 49 0 5

KSLA MPA640B Conventi o nal Control 113 5 49 0 0

KSLA Lewis 7007 Conve n ti o nal Reference 113 5 49 0 1

KSLA Midland Phi ll ips 799 Conventi o nal Reference 113 5 49 0 2

KSLA NC+ 5220 Conventi o nal Reference 113 5 49 0 3

KSLA LG2540 Conventi o nal Reference 113 5 49 0 4

ILMN MON 87411 Gl y p hosate

- Tolerant

and Insect

- Protected 113 5 49 1 1

ILMN MPA640B Conventi o nal Control 113 5 49 0 6

ILMN Gateway 4 14 8 Conv e nti o nal Reference 113 5 49 0 7

ILMN H

- 9180 Conventi o nal Reference 113 5 49 0 8

ILMN Stewart S588 Conventi o nal Reference 113 5 49 0 9

ILMN LG2548 Conventi o nal Reference 113 5 49 1 0

1 MP A 64 0B

= LH 2 4 4 × LH2 87

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0

. 0 ( 0

. 0 0) 0

. 3 ( 0

. 2 5) 0

. 5 ( 0

. 2 9) 0

. 3 ( 0

. 2 5) 0

. 5 ( 0

. 2 9) * 0

. 0 ( 0

. 0 0)

0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0)

2

. 0 ( 0

. 9 1) 1

. 0 ( 0

. 4 1) 4

. 3 ( 1

. 3 8) 2

. 3 ( 0

. 4 8) 2

. 8 ( 0

. 7 5) 3

. 3 ( 1

. 1 1)

9 8 .0 ( 0 .9 1) 9 8 .8 ( 0 .2 5) 9 5 .3 ( 1 .4 9) 9 7 .5 ( 0 .6 5) 9 6 .8 ( 0 .8 5) 9 6 .8 ( 1 .1 1)

7 3 .5 ( 2 .6 0) 7 5 .9 ( 1 .3 4) 9 0 .5 ( 1 .5 5) 9 1 .0 ( 1 .2 2) 8 2 .0 ( 2 .4 8) 8 2 .5 ( 4 .0 9)

0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0)

2

. 0 ( 0

. 8 2) 2

. 0 ( 0

. 4 1) 0

. 0 ( 0

. 0 0) 0

. 8 ( 0

. 2 5) 0

. 8 ( 0

. 2 5) 0

. 3 ( 0

. 2 5)

2 4 .5 ( 2 .1 0) 2 2 .1 ( 1 .2 8) 9

. 5 ( 1

. 5 5) 8

. 3 ( 1

. 0 3) 1 7 .3 ( 2 .4 3) 1 7 .3 ( 3 .8 8)

9 8 .0 ( 0 .7 1) 9 9 .0 ( 0 .4 1) 9 9 .3 ( 0 .2 5) 9 9 .3 ( 0 .4 8) 9 8 .5 ( 0 .8 7) 9 8 .8 ( 0 .4 8)

0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0)

1

. 8 ( 0

. 6 3) 1

. 0 ( 0

. 4 1) 0

. 8 ( 0

. 2 5) 0

. 8 ( 0

. 4 8) 1

. 5 ( 0

. 8 7) 1

. 3 ( 0

. 4 8)

0

. 3 ( 0

. 2 5) * 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0 ( 0 .0 0) 0

. 0 ( 0

. 0 0)

9 8 .8 ( 0 .4 8) 9 7 .8 ( 0 .9 5) 9 9 .3 ( 0 .4 8) 9 9 .8 ( 0 .2 5) 9 9 .3 ( 0 .2 5) 9 8 .3 ( 0 .4 8)

0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0)

1

. 3 ( 0

. 4 8) 2

. 3 ( 0

. 9 5) 0

. 8 ( 0

. 4 8) 0

. 3 ( 0

. 2 5) 0

. 8 ( 0

. 2 5) 1

. 8 ( 0

. 4 8)

0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) Tabl e H

- 2

. Germinatio n an d Dormanc y Characteristic s o f MO N 8741 1 an d th e Convent i ona l Contro l See d (Selfe d F 2 Grain)

Produced at each of t h e Three Field Sites

IA R L 1 ILM N 1 KS L A 1

Me a n % ( S .E .) 2 Me a n % ( S .E .) 2 Me a n % ( S.E .) 2

Te m perature 3

Re g i m e A ssess m e nt Cate g o ry M ON 8 7 41 1 C on tr ol M ON 8 7 41 1 C on tr ol M ON 8 7 41 1 C on tr ol

5 °C Ger m inated

Viable Ha r d

Dead

Via b l e F i r m Sw o llen

1 0 ° C Ger m inated

Viable Ha r d

Dead

Via b l e F i r m Sw o llen

20 °C Ger m inated

Viable Ha r d

Dead

Via b l e F i r m Sw o llen

3 0 ° C Ger m inated

Viable Ha r d

Dead

Via b l e F i r m Sw o llen

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9 8 .0 ( 0 .4 1) 9 7 .0 ( 0 .5 8) 9 9 .3 ( 0 .2 5) 1 0 0

. 0 ( 0

. 0 0 ) 9 8 .8 ( 0 .4 8) 9 8 .0 ( 0 .7 1)

0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0)

0

. 7 ( 0

. 4 7) * 2

. 0 ( 0

. 0 0) 0

. 5 ( 0

. 2 9) 0

. 0 ( 0

. 0 0) 0

. 8 ( 0

. 4 8) 0

. 8 ( 0

. 4 8)

1

. 3 ( 0

. 6 3) 1

. 0 ( 0

. 5 8) 0

. 3 ( 0

. 2 5) 0

. 0 ( 0

. 0 0) 0

. 5 ( 0

. 5 0) 1

. 3 ( 0

. 4 8)

9 8 .5 ( 0 .2 9) 9 8 .3 ( 0 .2 5) 9 9 .0 ( 1 .0 0) 9 9 .5 ( 0 .2 9) 9 9 .0 ( 0 .5 8) 1 0 0

. 0 ( 0

. 0 0 )

0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0)

1

. 5 ( 0

. 2 9) 1

. 8 ( 0

. 2 5) 0

. 8 ( 0

. 7 5) 0

. 5 ( 0

. 2 9) 1

. 0 ( 0

. 5 8) 0

. 0 ( 0

. 0 0)

0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 3 ( 0

. 2 5) 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0)

9 7 .5 ( 0 .2 9) 9 8 .5 ( 0 .6 5) 9 9 .3 ( 0 .4 8) * 9 7 .8 ( 0 .6 3) 9 7 .8 ( 0 .7 5) 9 8 .8 ( 0 .4 8)

0

. 8 ( 0

. 4 8) 0

. 0 ( 0

. 0 0) 0

. 5 ( 0

. 2 9) 0

. 5 ( 0

. 2 9) 1

. 3 ( 0

. 4 8) 0

. 5 ( 0

. 2 9)

0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0)

1

. 8 ( 0

. 4 8) 1

. 5 ( 0

. 6 5) 0

. 3 ( 0

. 25 ) * 1

. 8 ( 0

. 8 5) 1

. 0 ( 0

. 4 1) 0

. 8 ( 0

. 2 5)

0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) 0

. 0 ( 0

. 0 0) † 0

. 0 ( 0

. 0 0) Table H

- 2 (continued). Germination and Dormancy Characteristics of MON 87411 and the Conv e n tional Control Seed

(Selfed F 2 Grain) Pr o duced at each of the Three Field S ites

IA R L 1 ILM N 1 KS L A 1

Me a n % ( S .E .) 2 Me a n % ( S .E .) 2 Me a n % ( S.E .) 2

Te m perature 3

Re g i m e A ssess m e nt Cate g o ry M ON 8 7 41 1 C on tr ol M ON 8 7 41 1 C on tr ol M ON 8 7 41 1 C on tr ol

10 / 20 °C Ger m i na t ed

Viable Ha r d

Dead

Via b l e F i r m Sw o llen

10 / 30 °C Ger m i na t ed

Viable Ha r d

Dead

Via b l e F i r m Sw o llen

20 / 30 °C N o r m al Ger mina t ed

( A O S A) A bn or m al Ge r m i na t ed

V i ab l e Ha r d

Dead

V i ab l e F i rm Swo lle n

N o te : T he experi m e n ta l d esi gn wa s a s p lit

- p l ot wit h fo ur re p licati ons.

*Indicate s a s t atisticall y sign i f i cant d i ff e r e n c e be t we e n M O N 8 74 11 a n d t he c o n v e n tio n a l con t rol (α= 0

. 0 5 ) us in g AN O V A.

† N o statistica l c o m p aris on co u l d be m a de d u e t o lac k o f v a ria b ilit y i n t he data.

1 Sit e c od e s ar e a s f o ll o ws : IAR L = Jeffer s o n Co un ty

, I o wa, KS L A = P a w nee C o u n ty

, Ka nsas, a n d I L M N = Wa r r e n C o u n t y I lli no i s

. 2 M ON 8 7 41 1 a nd t he c o nv e n t i onal c on t rol val ues r e p resent m eans w it h s t and a r d e rr or ( S .E.) i n p a ren t he ses. N = 4

. I n s o m e i ns t a n ces

, t he t o ta l p e rce n ta ge of

bo t h MO N 8 7 4 11 an d t he c o n v e n tio n a l co n tr ol d i d not eq u a l 1 0 0% d ue t o nu m e r ica l r oun d in g of t he m ea n s.

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References for Appendix H

AOSA/SCST. 2010. Tetrazolium testing handbook. A ssociation of Official Seed Analysts

and the Society of Com m ercial Seed Technologists, Ithaca, New York.

SAS

. 2008

. SAS/STA T softwar e versio n 9.2

. SA S Institute, Inc., Cary, North Carolina.

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App e ndi x I : Materials

, Methods

, an d I n div i dua l Sit e Result s fro m Phenotypic,

Agronomi c , an d Environmenta l Interactio n Assessmen t o f MO N 8741 1 under

Fiel d Co nditions

I.1. Materials

Agrono m ic, phenotypic, and environ m ental i n teraction characteri s tics were assessed for

MO N 87411

, th e conventiona l control

, an d 2 2 referenc e hybrid s grow n unde r si m ilar

agrono m ic conditions. Four refere n c e hybrids were pla n ted p er site (Ta b le I

- 1).

I.2. Characteri z ation of the Materials

The presence or absence of the MON 87411 event in the starting seed of MON 87411 and

the conventional control was verified by e v ent

- specific pol ym erase chain reaction (PCR)

analyses. N o m olecular analyses were perfor m ed on the reference starting seed.

I.3. Field S ites and Plot Design

Field trials were established in 2012 at nine sites that provided a range of environmental

and agron om ic conditions re p res e ntative of U .S. m aize growing re g i o ns (Se c tion VIII,

Table VI I I

- 3). The P r incip a l In v e stig a tor at e ach site w a s f a m iliar with the g r owth,

production, and evaluation of m aize characteristics.

At all sites, seed of MON 87411, the conv e ntional control, and four conventional

reference hybrids were planted in a rando m i zed co m pl e te block design with four

replications

. Th e plante d plo t dimensions varied between s it e s, due to va r iability in

available planting equi pm ent and data collec t ion (Table I

- 2 ). At IABG (Iowa), NEYO

(Nebraska), NCBD (No r th Carolina), and PAHM (Pennsylvania) sit e s, each replicated

plo t consiste d o f 1 6 row s o f m a i z e sp aced approxi m ately 0.76

- 0.96 m apart and

approxi m ately 6 m long. Phenotypic and qu a litati v e environ m ental inter a ctio n s data

were collected from rows 2 and 3 (rows 14 an d 1 5 a t NCB D site)

. Row s si x an d eight

were used to collect arthropod sa mples us in g stick y traps

. Row s 9

, 10

, 11

, an d 1 2 were

designated for visual counts of arthropod abundance

. Row s 1 4 an d 1 5 ( r ow s 2 an d 3 at

NCBD site) were used to assess plant da m age caused by corn earworm ( Helicoverpa

zea ) and European corn borer ( O s trinia nubil a li s ).

At IARL (Iowa), IL M N (Illin o is )

, INSH (Indiana), KSLA (Kansas), and NEDC

( Neb ra ska ) s ite s

, eac h re p li ca t e d p l o t co n sisted of s ix rows of m aize spaced

approxi m ately 0.76 m apart and approxi m ately 6 m long

. Th e plot s wer e separate d by

two rows of conventional m aize along t hei r length

. Phenotypi c an d qualitative

environ m ental int e ra c ti o ns data we r e colle c t ed from rows 4 and 5 except one plot at

ILM N an d tw o plot s a t NEDC.

I.4. Planti n g and Field Operations

Planting infor m ation, soil description, and cro p ping history of the t r ial area are listed in

Table I

- 2. Prior to planting, the Principal Investigator at each site prepared the plot area

with a proper seed bed accordi n g to local agrono m ic practice s

, includin g tillage,

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fertilization, and pest manage m ent. During the growing season, all plots were assessed

fo r agronomi c condition s an d pes t populati ons, including pest art hropods

, disease s and

weeds. Fe r tiliz e r, ir r i g ation, ag r i c ultu r al che m icals, and other m anage m ent practices

were applied as necessary. Maintenance op e rations were perfor m ed un i fo r m ly across all

plots.

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Tabl e I

- 1

. Startin g See d fo r Phen otypic, Agronomic, and Environmental

Interaction Assessment

Site Cod e 1 Material Name Monsanto Lot Nu m ber Pheno t y pe

A l l M ON 8 7 41 1 1 13 3 31 76 G lyph o sa t e

- To le r a nt and

Insect

- Protected

Al l MPA64 0B 2 1 13 3 31 70 Con v e n t i on a l C o n t rol

Jaco bs e n See d JS4 4 3 1 1 12 6 70 96 Co v e n tio n a l R e f e r e n c e

IA B G

IARL

IL M N

IN S H

KS L A

NCBD

NEDC

NE YO

PAHM P h illi ps 7 1 3 1 13 0 00 72 Co v e n tio n a l Refere n c e

LG 2 61 5 C L 1 12 2 68 63 Co v e n tio n a l R e f e r e n c e

Pi on ee r 3 2 B8 1 1 12 2 65 78 Co v e n tio n a l R e f e r e n c e

Le g ac y L 7 67 1 1 12 2 65 98 Co v e n tio n a l R e f e r e n c e

Le w i s 7 00 7 1 12 2 65 59 Co v e n tio n a l R e f e r e n c e

G ate w a y 6 11 6 1 12 2 72 11 Co v e n tio n a l R e f e r e n c e

P h illi ps 7 1 7 1 13 0 00 73 Co v e n tio n a l R efere n c e

G ate w a y 4 14 8 1 12 7 30 05 Co v e n tio n a l R e f e r e n c e

H

- 9 18 0 1 12 2 67 04 Co v e n tio n a l R e f e r e n c e

Ste w a rt S58 8 1 12 2 69 18 Co v e n tio n a l R e f e r e n c e

LG 2 54 8 1 12 6 67 31 Co v e n tio n a l R e f e r e n c e

G ate w a y 6 15 8 1 12 7 30 06 Co v e n tio n a l R e f e r e n c e

LG 2 62 0 1 12 2 68 61 Co v e n tio n a l R e f e r e n c e

Ste w a rt S51 8 1 12 2 69 20 Co v e n tio n a l R e f e r e n c e

Le g ac y L 7 67 1 1 12 2 65 98 Co v e n tio n a l R e f e r e n c e

Le w i s 7 00 7 1 12 2 65 59 Co ven tio nal R e fer e nce

Mi d la nd P h ill ips 7 9 9 1 12 2 67 03 Co v e n tio n a l R efere n c e

N C + 522 0 1 12 2 67 01 Co v e n tio n a l R e f e r e n c e

LG 2 54 0 1 12 6 67 30 Co v e n tio n a l R e f e r e n c e

LG 2 54 8 1 12 6 67 31 Co v e n tio n a l R e f e r e n c e

Ste w a rt S60 2 1 12 2 69 19 Co v e n tio n a l R e f e r e n c e

Mi d la nd P h ill ips 7 9 9 1 12 2 67 03 Co v e n tio n a l R efere n c e

G ate w a y 6 15 8 1 12 7 30 06 Co v e n tio n a l R e f e r e n c e

G ate w a y 4 14 8 1 12 7 30 05 Co v e n tio n a l R e f e r e n c e

Mi d la nd P h ill ips 7 9 9 1 12 2 67 03 Co v e n tio n a l R efere n c e

N C + 444 3 1 12 2 67 00 Co v e n tio n a l R e f e r e n c e

LG 2 54 0 1 12 6 67 30 Co v e n tio n a l R e f e r e n c e

LG 2 54 0 1 12 6 67 30 Co v e n tio n a l R e f e r e n c e

N C + 522 0 1 12 6 67 01 Co v e n tio n a l R e f e r e n c e

My cog e n 2 M7 46 1 12 2 67 05 Co ven tio nal R e fer e nce

G ate w a y 6 15 8 1 12 7 30 06 Co v e n tio n a l R e f e r e n c e

P h illi ps 7 1 7 1 13 0 00 73 Co v e n tio n a l R efere n c e

Ste w a rt S58 8 1 12 2 69 18 Co v e n tio n a l R e f e r e n c e

Pi on ee r 3 3 T5 6 1 12 2 65 80 Co v e n tio n a l R e f e r e n c e

G ate w a y 4 14 8 1 12 7 30 05 Co v e n tio n a l R e f e r e n c e

1 Site code: I A BG

= Gre e ne Co u nt y

, I A; I ARL

= J e ff e r s on C o unt y

, IA; IL M N

= W a r r en C ou nt y

, IL; I N S H

= B o o ne C o unty

, IN; KSL A

= Paw n e e Cou n ty

, KS; N C B D

= Pe r q u i m a ns Cou n ty

, NC ; NEDC

= B u tler

Co u nt y

, NE; N EYO

= Y or k C o u nt y

, NE; P A HM = B e rks Co u nt y

, P A.

2 MP A 64 0B

= LH 2 4 4 × LH2 8 7.

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Tabl e I

- 2

. Fiel d an d Planting Information

Planting Harvest Approxi m ate

Plantin g Rate Approxi m ate

Plot Size Rows Previous Crop

Site 1 Dat e 2 Dat e 2 (seed s / m ) (m × m ) per Plot Soil Type % O M 3 2011

IABG 05/09 / 20 1 2 10/05 / 20 1 2 7.2 6.1 × 1 2 .2 16 Loam 4.0 So y b ean

IARL 05/11 / 20 1 2 10/12 / 20 1 2 7.2 6.1 × 4

. 6 6 Sil t y Cl a y Loam 3.4 So y b ean

ILMN 05/10 / 20 1 2 10/01 / 20 1 2 6.9 6.2 × 4

. 6 6 Sil t y Cl a y Loam 4.5 So y b ean

INSH 05/11 / 20 1 2 11/02 / 20 1 2 7.2 6.1 × 4

. 6 6 Silt Loam 2.5 Maize

KSLA 05/11 / 20 1 2 09/21 / 20 1 2 7.2 6.1 × 4

. 6 6 Silt Loam 2.6 Sorghum

NCBD 05/11 / 20 1 2 09/20 / 20 1 2 6.6 6.1 × 1 5 .5 16 Sandy Loam 2.6 Cotton

NEDC 05/05 / 20 1 2 09/11 / 20 1 2 7.2 6.2 × 4

. 6 6 Silt Loam 2.6 So y b ean

NEYO 05/08 / 20 1 2 10/09 / 20 1 2 7.2 6.1 × 1 2 .2 16 Silt Loam 3.0 So y b ean

PAHM 05/19 / 20 1 2 10/19 / 20 1 2 8.2 6.1 × 1 2 .2 16 Loam 1.6 Vegetables 4

Note: All plots were thinned to a uniform density of approxi m a tely 31 plants (IABG, INSH, KSLA, NCBD, NEYO, and PAHM) and 38 p l an t s

(IARL, ILMN, and NEDC) per 6.0 m row.

1 Site code: I A BG = Greene Coun t y, I A ; IARL = Jefferson Coun t y, I A ; ILMN

= Warren Coun t y

, IL; IN S H = Boone Coun t y, IN; KSLA = Pawnee

Coun t y

, KS; NCBD

= Perqui m a ns Co u n t y

, NC; NEDC

= Butler Coun t y

, NE; NEYO

= York Coun t y, N E ; PAHM

= Berks Coun t y

, PA.

2 Planti n g and Harvest Date

= m m /dd/yyyy.

3 % OM

= Pe r cent Organic Matter.

4 Vegetables

= peppers, tomatoes, potatoes, cabbage, maize.

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I.5. Phenotypic Observations

The descri p t ion of the chara c teristics m easured and the d e signated develop m ental stages

where observations o cc u rred a re lis te d in Section V III, Table VIII

- 1.

I.6. Environmental O b servations

Environ m ental i n te r acti o ns ( i.e

. , i n teractions between the c r op plants a n d their rec e iving

environ m ent) were used to character iz e MO N 8741 1 b y evaluatin g plan t respons e to

abiotic stressors, disease da m age, and arthropod

- related d a m age using qualitative

m ethods described in section I.7

. I n addition

, specifi c a r thropo d da m a g e (cor n ear w o r m

da m age and European corn borer) and art h ropo d abundanc e wer e evaluate d usin g the

quantitative m ethods des c ribed in Section I.8.

I.7. Plant Response to Abiotic Stress, Diseas e Damage

, an d Arthropod

- Related

Damage

MON 87411 and the conventional control were e v aluated at all sites for plant response to

abiotic s tre s sors, d isea s e da m age, and art h ropo d da m a ge

. A targe t o f thre e abiotic

stressors, three diseases, and three arthropod pests were evaluated four ti m es during the

following four crop develop m ental stages: V6

– V8 ; V12

- VT ; R1

- R3 ; an d R5

- R6.

Abiotic stressor, disease da m age and arth r opod da m age observations wer e collecte d from

each pl o t using the cate g orical scale of increa s i n g severity li s ted below:

Category Severity of plant da m age

None No sy m pto m s observed

Slight Sy mpto m s not da m aging to plant develop m ent ( e.g

. , m i no r feeding

or m inor lesions); m itigation li k ely n o t req u ired

Moderate Inter m ediate between slight and sev e re; lik e ly re q uires m itig at ion

Sever e Sy mpto m s da m aging to plant develop m ent ( e.g

. , stuntin g o r death);

m itigation u nlik e ly to be e ff ective

Method used for selecting s t ressors at each field site:

1. Prior to each data collection, m aize was surve y e d i n proxi m it y t o th e stud y are a o r the

border rows of the study f or abiotic stre s sors ( e

. g

. , drought), diseases ( e

. g

. , gray leaf

spot), and arthropod da m age ( e.g.

, cor n fle a beetle).

2. The Princi p al Inve s ti g ator chose three a b i o tic stre s sors, three di s eases, and three

arthropod species that are actively causing da m age for s ubsequent evaluation in the

study plots. The Principal Investigators were requested to select additional stressors

if present.

3. If fewer th a n three abiotic stressors, diseases, or arthropod species were present, the

cooperator chose additional abiotic stressor s

, diseases, and arthropod species that are

known to commonly occur in that geograph i cal region and cause da m age at the study

site at t h at ti m e.

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4. All plots at a site were rated for the s a m e abiotic stressors, diseases, and arthropod

pests at a given observation, even if that s e lected stressor w as not present in so m e or

all of the p l o ts.

5. If a selected stressor was not present, the cooperator rec o rded the rating as “none”.

As indicated above, the Principal Investi g ator at each field site chose abiotic stressors,

diseases, and arthropod pests th a t w e r e eithe r activel y caus ing plant injury i n th e study

area o r were likely to occur in m aize during the given observation per i o d

. Therefore

, t h e

type of abiotic stressors, diseases, a nd arthropod pests ass e sse d varie d between

observations at a site and between sites.

In addition, ear and kernel rot disease and stalk rot disease w e re evaluated at harvest (R6

growt h stage ) usin g th e abov e categorica l scale

. Ea r an d k e rne l ro t diseas e dat a were

collected by evaluating five non

- syste m atically selected ears (one per plant) from e ach

plot. The h usks were p ulled b ack and each ear was exa m ined for disease. To evaluate

stalk r o t, five non

- syste m atically selected stalks in each plot were cut lo n gitudi n ally. The

stalks were then exa m ined for disease.

I.8. Arthropod Abundance

Specific arthropod (corn earworm and European corn borer) da m age and arthropod

abundance were assessed quantitatively from observations/collections perfor m ed at

IABG

, NEYO

, NCBD

, a n d PAH M sites.

Corn earw o rm d a m age was evaluated at R5 growth stage by exa m ining ears from t e n

plants (5 c o nsecutive plants per row) in each p l o t. The husks were pull e d back and each

ear was exa m ined for corn earworm da m age u s ing a plastic film grid (size of each grid

0.5 c m 2 ). Da m age (c m 2 ) per plant was cal c ulated as t h e total number of grid cells

m atching the da m age area m ultiplied by 0.5 (each grid cell

= 0.5 c m 2 ).

European c o rn borer da m age was evaluated at R6 gro w th stage by exa m ining ten plants

(5 consecutive plants per row) in each plot. Damage was assessed by splitting each of ten

plants and counting the nu m ber of feeding galleries per plant and length of fe e ding

gallery (c m .) in each stalk.

Arthropods were collected using yellow sti c ky traps five ti m es during the growing season

at the f ollo w ing int e rval s : late veget a tive

– V T , R1, R2, R3, a nd R4 growth stage. Sticky

traps (two per plot) were de p loyed in rows 6 and 8 of each plot at the appro x i m a t e

m idpoint between the ground level and the t op of the pl a nt canopy for all arthropod

collectio n s. At each specifi e d c o llection, traps were de p loyed for appro x i m ately 7 days.

Sticky traps were then sent to the University of Arkansas, Fayetteville, AR for arthropod

identificatio n an d enumeration

. A m a x i m u m of twelve arthropods were enu m erated for

each collection. Seven preselected arthropods ( o r arthropod g roups), na m ely aphid, corn

flea beetle, leafhopper, spider, m icro parasitic hy m enoptera, ladybird beetle, and m inute

pirate bug were enu m erated at all sites f o r each collection ti m e. Additionally, for each

individual collection ( e

. g

. , Collection 1, IABG site), four non

- syste m atically selected

sa mples were exa m ined to deter m ine the m o st abundant arthropods to obtain a total of up

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to twelve arthropods to be enu m erated for t h at particular collection and site. Thus, the

suit e o f arthropod s assesse d ofte n varie d betwee n collection s from a site and between

sites due to differences in te mporal acti v ity and geographical d i stribution of arthropod

taxa.

Five visual counts were conducted during t h e growing season at approxi m ately VT

- R1,

R1, R2, R3, and R4

- R5 from five non

- syste m at i cally s e le c t e d plants per plot to colle c t

abundance data per plot. Visual counts were m a de by ex a m ining the stalk, the leaf blade,

the leaf collar, the ear tip, the silk, and the tassel of each plant.

I.9. Dat a Assessment

Experien c ed scientists f a m iliar with the exp e ri m ental de s ign and evalu a tion criteria were

involved in all co mponents of dat a collection

, sum m ar i z a tion

, an d analysis

. Study

personnel assessed that m easure m e nts were t a ken properly, data were consistent with

expectations based on exper i ence with the crop, and the experi m ent was care f ully

monitored. Prior to analy s is, t h e over a ll d atas e t was evalu a ted f or evide n ce of

biologically relevant changes and for possible evidence of an unexpected plant respo n se.

Any unexpected observations or issues duri n g the t r i a ls that would impact the tri a l

objectives were noted. Data were then sub j ected to data sum m arization o r s tatistical

analy s is as indicated I. 1 0.

I.10. Environmental Interactions Evalua tio n Criteri a fo r Qualitativ e Data

I.10.1 Agronomic and Phenotypic Data

Plant vigor data were summarized but not sub j e cted to an a n alysis of variance (ANOVA).

MON 87411 was considered different from the conventional control in vigor if the ranges

of vigor of MON 87411 did n o t overla p wit h th e rang e o f vigo r o f th e conventional

control across all re p lications

. An y observe d differ e nce s betwee n th e MO N 8741 1 and

conventional control were further assessed in the context of the range of the reference

m aterials, and for consistency at other sites.

An ANOVA was conducted according to a rando m ized co mplete block design using

SAS  to co mpare MON 87411 and the conve n tional control for the 13 phenotypic

characteristics listed in Table VIII

- 1

, wit h th e exceptio n o f plant vigor. The level of

statistical s ignificance was p redeter m i ne d t o b e 5 % ( α = 0.05). Co mparisons of

MON 87411 and the conventional control were conducte d withi n s i t e (individua l site

analy s is) a n d in a combined

- s ite analysis, in which the d ata were p o oled acr o ss sites.

MON 87411 and the conventional control m ateria l s were not statistically co mpared to the

reference materials. The reference range for each m easured phenoty p ic characteristic

was deter m i ned from the m in i m u m and m axi m u m mean values from the 22 conventional

reference hybrids that w ere i n cluded across a ll s i tes.

 SA S i s a re g i stere d t r a d e m ar k o f SA S I n stit u te

, I n c.

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Data exclu d ed from analysis and t h e reasons for exclusion are li s ted in Table I

- 3.

I.10.2 Environmental Interaction Data

The environ m ental interaction data ( i.e

. , pla n t respo n se to abi o tic s tress o rs, d i s ease

da m age, and arthropod d a m age) are categori c al and were not subjected to ANOVA.

MON 87411 and conventional control were con s idered different in susceptibilit y or

tolerance if the range of i n jury s y m pto m s of each did not overlap across all four

replications. Any observed differences were further as s essed in the co n text of the range

of the reference m aterials, and for consistency at other sites.

An ANOVA was conducted according to a rando m ized co mplete block design S AS ®

(SAS 2008; 2012) for corn earworm (CE W ) da m age, European corn borer ( E CB)

da m a ge

, an d arthropo d abundance

. Th e leve l o f statistica l significance was

predeter m ined to be 5 % ( α = 0.05). MON 87411 was compared to the conventional

contr o l at e ach site (i n dividu a l

- s ite a n alysis) for CEW da m age, ECB da m age, and the

arthropod abundance. Addit i onall y

, corn ear w orm d a m age and E u r o pean c o rn b orer

da m age data were pooled across sites (co m bin e d

- site analysis) for statistical co m p arison

betwee n MO N 8741 1 an d th e conventiona l c ontrol. Mini m u m and m ax i m u m m e a n

values were calculated for CEW da m age and ECB d a m age from 15 reference hybrids that

were included at IABG, NEYO, NCBD, and PAHM sites. The reference ran g e for

arthropo d abundanc e evaluate d f r o m a give n collectio n an d sit e wa s deter m ine d fro m the

m in i m u m and m axi m u m m ean values collected from the reference m aize at the site.

For the arthropod abundance data, statistical analyses and significance testing of

differences between MON 87411 and the conv e ntional control m a t erials were only

perfor m ed for the arthropods pre s ent in sufficient nu m bers to esti m ate the m aterial mean

arthropod counts and the variat i on of the m eans. An inclu s io n criterio n wa s established

wher e a give n arthropo d m u s t hav e a n avera g e count per plot per collection ti m e (across

all m aterial s ) of ≥ 1.

Data exclu d ed from analysis and t h e reasons for exclusion are li s ted in Table I

- 3.

I.11. Statistical Analysis

In individual site assess m ents of p l ant v i gor, MON 87411 and the conventiona l control

were considered different if the range of vigor values did no t overla p acros s al l four

replications

. Ther e w e r e n o differ e nce s observe d bet w ee n MO N 8741 1 an d the

conventional control in plant vigor at all sites ( T able I

- 4).

In the in d i v idual

- site a nalysis, a total of 11 statistically significa n t differences were

detecte d ou t o f 10 7 comparison s betwee n M O N 87411 and the conventional control

(Table I

- 4). These differences were distributed a m ong eight of the 13 phenotypic

characteristics

. MO N 8741 1 ha d a lowe r earl y stand count than the conventional control

at the IABG (82.3 vs. 86.3 plants per plot) and PAHM (72.5 vs. 78.8 plants per plot) sites

but a higher early stand count at the NEDC site (95.3 vs. 89.8 plants per plot).

MON 87411 had fewer days to 50% pollen shed than the conventional control at the

NCB D sit e (59. 3 vs

. 60. 5 days ) an d m o r e d a y s t o 50 % silkin g tha n th e conventional

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control at the NEYO site (66.5 vs. 65.5 days). MON 87411 exhibited a higher stay green

rating (less green tissue) than the convent iona l contro l a t th e KS LA site (1.8 vs. 1.0

rating). MON 87411 had a higher final stand count than the conventional control at the

KSLA site (77.3 vs.74.0 plants/plot). Gra i n m oisture percentage was low e r for

MON 87411 than the conventional control at the IARL (16.9 vs. 17.7 %) and PAHM

(21. 1 vs

. 22. 3 % ) sites . The test weight was h ighe r fo r MO N 8741 1 tha n the

conventional control at the PAHM s ite (68.5 vs. 65.8 kg/hl). MON 87411 had a higher

yield than the conventi o na l contro l a t th e INS H sit e (11. 5 vs

. 9. 2 Mg/ha)

. Th e statistical

differences between MON 87411 a nd the convent i onal control detected in the individual

sit e analysi s fo r earl y stan d count

, day s t o 50 % polle n shed

, day s t o 50 % silking

, stay

green, final stand count, grain m o isture percentage, test weight, an d yiel d wer e not

detected in the co m bined

- site a n alysis. Thus

, th e difference s detecte d fo r these

phenotypic characteristics do not i n dicate a co n siste n t resp o nse associated with t h e trait

and are unlikely to be biolog i cally m eaningful, in ter m s of i ncrea s ed pest/weed p o tential

of MON 87411 co mpared to the conventional m aize (See Figure VIII

- 1, Step 2, answer

“no”).

I.12. Individual Field Site Plant Gro w th, Develo p ment, and Environmental

Interactio n s Results a n d Discussi o n

Plan t Respons e t o Abioti c Stressor

, D i seas e Damage

, an d Arthropod

- relate d Damage:

I n th e individual

- sit e assess m ent

, n o diff e rences were observed betwee n MO N 8741 1 and

the conventional control for any of the 100 co m p arisons for the assessed abiotic stressors,

including cold, drought, flood, frost, hail, heat, nutrient deficiency, soil co m paction,

sunscald, and wind (Table I

- 5).

I n th e individual

- sit e assess m ent

, n o diff e rences were observed between MON 87411 and

the conventional control for any of the 119 co mparisons for the assessed diseases,

including anthracnose, bacterial leaf spot, e a r rot

, eyespot

, Fusariu m sp.

, Goss’ s bacterial

wilt, gray leaf spot, leaf b light, m aize rough d w arf virus, Pythium sp., Rhizoctonia sp.,

rust, s e edli n g blight, s m ut, st a lk r o t, and Stewart ’ s bact e ri a l wilt (Table I

- 6 ).

I n th e individual

- sit e assess m ent

, n o diff e rences were observed between MON 87411 and

the conventional control for any of the 102 c o mparisons f or the as s e s sed arthro p ods,

including aphid, ar m ywor m

, billbug, cutwor m

, c o r n earwor m

, cor n fle a beetle

, rootwo r m

beetle, European corn borer, grasshopper, Jap a nese beetle, sap beetle, spider m ite, stink

bug, and wireworm adult (Table I

- 7).

Cor n Earwor m an d Europea n Cor n Bore r Damage:

In the individual

- site analysis, one statistica l ly significant difference was detected out of

12 co mparisons between MON 87411 and the conventional control for CEW and ECB

a m ong all observations at all four sites (Tabl e I

- 8)

. CE W d a m a g e wa s highe r for

MON 87411 than the conventional contr o l a t th e NCB D sit e (3. 3 vs

. 1. 5 c m 2 d a m a ged

area p e r pl o t ). This s tati s tic a l di ff er e nce in t h e individual

- site analysis wa s no t detecte d in

the co m bined

- site anal ys is. Thus, this differ e nce was not indicative of a consistent plant

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response associated with the trait and is unlikely to be biologically m eaningful in ter m s of

increased pest potential of MON 87411 co m pared to the conventional control (See Figure

VIII

- 1, Step 2, answer “ n o”).

Sticky Trap:

A total of 108 statistical co m parisons were m ade between MON 87411 and the

conventional control for arthropod abundance involving the following arthropods: aphid,

corn flea b eetle, delp h acid pla n t h opper, green lacewing, ladybird b eetle, leaf h opper,

m icro

- parasitic hy m enoptera, m acro

- parasit i c hy m enoptera, sap beetle, m inute pirate bug,

and spider (Table I

- 9). L ack of sufficient arthropod ab u ndanc e preclude d statistical

co mparisons between MON 87411 and the conventiona l contro l fo r 7 9 additional

co mparisons; however, descri p ti v e stati s ti c s w e re provided for these comparisons ( T able

I

- 9).

No statistically significant difference s wer e detecte d betwee n MO N 8741 1 an d the

conventional control for 104 out of 108 co m p arisons (Table I

- 9). T he abundance of

aphids was higher in MON 87411 t han the conventional control in Collection 3 (1.8 vs.

0.0 per plot) at the NEYO site. The m ean value for aphid abundance on MON 87411 was

within the range of the reference hybrids. The abundance of de l phacid planthoppers was

lowe r i n MO N 8741 1 tha n th e con ventional control in Collection 1 at the IABG site (0.0

vs. 1.8 per plot). MON 87411 had higher abund a nc e tha n th e conventiona l contro l for

spiders (3.3 vs. 0.3 per plot) in Collection 5 and lower abundance than the conventional

control for m inute pirate bugs (1.0 vs. 3.0 per p l ot) in Collection 1 at the PAHM site.

The m ean values for delphacid planthopper, m inute pirate bug, and spider abundance on

MO N 8741 1 wer e outsid e th e respectiv e range s of reference hybrids. However

, these

di ff erences were not c o nsiste n tly d ete c ted a c r o ss colle c tio n s or s ites ( T able I

- 9). T hus,

these differences in aphid, delphacid p l anthopper, m inute pirate bug, and spider

abundance were not indicative of a consistent response assoc i ated with the trait and are

not considered biologically m e aningful in ter m s of increa s ed pest potenti a l

of MON 87411 co mpa r ed to conve n tion a l m aize (See Secti o n VIII.B.2.).

Visua l counts:

A total of 61 statistical co mparisons wer e m a d e betwee n MO N 8741 1 an d the

conventional control for arth ropo d abundanc e involvin g th e fo llowin g pes t an d beneficial

arthropods: ant

- like flower bee tle

, clic k beetle

, cor n fle a b eetle, ladybird beetle adult,

ladybird beetle larvae, m inute pirate bug, sap beetle, shining flower beetle, and spider

(Table I

- 10). Lack of sufficient arthropod abundance precluded statistical co mparisons

between MON 87411 and the conventional contr o l for 152 additi o nal co m parison s ;

however, the descriptive statistics were provided for these comparisons (Table I

- 10).

No statistically significant difference s wer e detecte d betwee n MO N 8741 1 an d the

conventiona l contro l fo r 6 0 ou t o f 6 1 co mp arison s (Tabl e I

- 10)

. Th e abundanc e o f ant

like flower beetles was lower in MON 87411 t h an the conventional control in Collection

2 at the I A BG site (0.8 vs. 4.3 per plot). The m ean value for ant

- like flower beetle

abundance on MON 87411 was lower than the rang e o f referenc e hybrid s (1. 8

– 3. 3 per

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plot). However, the d ifference for ant

- like fl o w er beetle was not consiste n tly detected

across c o ll e ctions a t the IABG site (T able I

- 1 0 )

. Thus

, thi s differenc e i n ant

- lik e flower

beetle abundance was not indicati v e of a consistent response associated w ith the trait and

is not considered biologically meaningful in ter m s of increased pest pot e nti a l

of MON 87411 co mpa r ed to conve n tional m aize (See Secti o n VIII.B.2.).

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Table I

- 3. Data Missing or Excluded from Analysis

Site Cod e 1 Material Name Material T y pe Plots Character i s ti c s Reason for Exclusion

IALL, ILCY,

ILPH, and

ILWY All All All Pheno t ypic and environ m ental interactions

data Extre m e weather conditions (strong

wind or d rought) that caused

extensive damage across the plots

IAR L MON 87411 Tes t 406 Root lo d ged plants Wind da m a ge

IAR L MPA640B

Legac y L7671 Control

Refere n c e 106

107 Moisture ( % ), shelled plot weight, and t est

weight Incorrect data collection;

Mechanical errors

INSH Legac y L767 1 Refere n c e 205 Da y s to 50% silking and da y s to 50% pollen

she d Identified as an outlier

NEDC

PAHM MON 87411

Midland

Phillips 799

MON 87411

Phillips 717

Pioneer 33T 5 6

MPA640B Test

Refere n c e

Test

Refere n c e

Refere n c e

Control 104

106

106

206

306

406 Final stand count Identified as an outlier

Sticky trap d a ta and visual count Missing data

PAH M MPA640B Control 406 Test weight Missing data

NED C All All All Environ m ental interactions evaluation # 4

(Ani m a l damage)

Environ m ental interaction evaluation # 4 I m proper sel e c tion of stressor

I m proper sel e c tion of stressor

NEY O All All All

NEY O All All All (Black stem disease)

Environ m ental interaction evaluation # 3

(abiotic stressor) Missing data for two abiot i c

stressors

IAR L All All All Environ m ental interaction evaluation # 2 In c o rrec t dat a collection

IAR L All All All Environ m ental interaction evaluation # 3 and 4

(Bean leaf beetle)

Environ m ental interaction evaluation # 2 I m proper sel e c tion of stressor

I m proper sel e c tion of stressor

KSLA All All All (Velvetbean caterpillar)

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Table I

- 3 (continued). Data Miss i ng or Excluded from Analysis

Site Cod e 1 Material Name Material T y pe Plots Character i s ti c s Reason for Exclusion

NCBD All All All Environ m ental interaction evaluation # 1

(Other arthropod) I m proper sel e c tion of stressor

IABG All All All Environ m ental interaction evaluation # 4

(Other arthropod) I m proper sel e c tion of stressor

IABG All All All Visual count

– Collection # 1 Incorrect data collection

INSH LG2620 Refere n c e 101 Environ m ental interaction evaluation # 1

(Bacterial blight

- Pseud o m onas) Incorrect data collection; not rated

for all plots

1 Sit e c od e : IAB G = Gree ne C o u n ty

, IA ; IAL L = St or y

, IA ; IAR L = Jefferso n Cou n ty

, IA ; ILC Y = Cli n to n, IL ; ILP H = C h a m p ai gn, IL ; IL WY = Sta rk, I L; IL MN

= W ar r e n Cou n ty

, IL; I NS H

= B o o ne C o unty

, IN; KS L A

= Paw n e e C o u n ty

, KS ; NC B D

= Per qu i m a ns C o u n ty

, N C ; NED C

= B u tle r Cou n ty

, NE ; N E YO

= Yor k

C o u n ty

, NE ; PAH M = Ber ks C o u n ty

, PA.

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Table I

- 4. Individual Site Phenotypic Comparison of MON 87411 Compared to the Conventional Control

Site Phenotypic Characteri s tics

Plant Vigor (1

- 9 r a tin g ) 2 Early stand count (#/plot) Days to 50% pollen shed

Range Mean (S.E. ) 3 Mean (S.E. ) 3

Cod e 1 MO N 8741 1 Contro l MO N 8741 1 Contro l MO N 8741 1 Control

IABG 2

– 3 1

– 3 82. 3 (1.11)* 86. 3 (1.44) 65. 3 (0.25) 65. 3 (0.48)

IARL 7

– 8 5

– 7 84. 8 (2.25) 82. 3 (3.12) 62. 8 (0.63) 62. 0 (0.41)

ILMN 2

– 3 1

– 3 83. 0 (0.41) 82. 8 (1.18) 62. 0 (0.00)† 62. 0 (0.00)

INSH 4

– 6 3

– 4 76. 5 (3.52) 83. 5 (1.44) 66. 5 (1.66) 64. 8 (0.75)

KSLA 2 2 97. 3 (0.75) 96. 0 (0.82) 56. 0 (0.00) 56. 0 (0.00)

NC BD 3

– 4 3

– 4 71. 3 (3.04) 72. 3 (1.65) 59. 3 (0.25)* 60. 5 (0.50)

NEDC 2 2 95. 3 (1.49)* 89. 8 (1.70) 68. 0 (0.41) 68. 8 (0.25)

NEYO 1 1 76. 0 (0.82) 78. 8 (1.44) 66. 3 (0.48) 65. 5 (0.29)

PAHM 3

– 4 3

– 5 72. 5 (1.89)* 78. 8 (1.55) 63. 5 (0.50) 63. 0 (0.41)

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Table I

- 4 (continued). Indiv i dual Site Phenotypic Comparison of MON 8741 1 Compare d t o th e Conventiona l Control

Site Phenotypic Characteri s tics

Day s t o 50 % silkin g Stay

- gr e en rating (1

- 9 s c ale) Ear height (c m )

Mean (S.E. ) 3 Mean (S.E. ) 3 Mean (S.E. ) 3

Cod e 1 MON 87411 Contro l MO N 8741 1 Contro l MO N 8741 1 Control

IABG 64. 3 (0.25) 65. 3 (0.63) 7. 3 (1.03) 7. 0 (0.00) 96. 8 (2.19) 101.4 (1.22)

IARL 61.0 (0.58) 61. 0 (0.00) 3. 0 (0.00) 3. 5 (0.29) 97. 7 (3.95) 90. 8 (2.40)

ILMN 62. 0 (0.00)† 62. 0 (0.00) 4. 0 (0.00) 4. 0 (0.00) 107.9 (1.03) 109.5 (0.81)

INSH 67. 3 (1.49) 66. 0 (0.82) 4. 5 (0.29) 5. 5 (0.87) 97. 1 (2.21) 93. 6 (2.51)

KSLA 58. 0 (0.00) 58. 0 (0.00) 1. 8 (0.25)* 1. 0 (0.00) 103. 2 (1.45) 102.2 (1.13)

NC BD 58. 8 (0.63) 59. 8 (0.25) 5. 5 (0.50) 6. 0 (0.00) 126. 9 (2.69) 130.1 (1.73)

NEDC 68. 3 (0.25) 68. 3 (0.48) 9. 0 (0.00) 9. 0 (0.00) 113. 5 (0.83) 107.8 (4.33)

NEYO 66. 5 (0.50)* 65. 5 (0.29) 6. 3 (0.25) 6. 0 (0.00) 107. 7 (1.05) 104.3 (1.20)

PAHM 64. 8 (0.63) 64. 3 (0.25) 8. 8 (0.25) 8. 0 (0.41) 85. 9 (3.05) 93.3 (5.68)

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Table I

- 4 (continued). Indiv i dual Site Phenotypic Comparison of MON 8741 1 Compare d t o th e Conventiona l Control

Phenotypic Characteri s tics

Plant height (c m ) Dropped ears (#/plot) Stal k lodge d p lants (#/ p lot)

Mean (S.E. ) 3 Mean (S.E. ) 3 Mean (S.E. ) 3

Site

Cod e 1 MO N 87411 Control MO N 87411 Control MO N 87411 Control

IABG 259. 8 (2.51) 266. 0 (6.25) 0.0 (0.00)† 0. 0 (0.00) 0. 3 (0.25) 0. 0 (0.00)

IARL 200. 6 (9.72) 202. 7 (3.79) 0. 0 (0.00) 0. 0 (0.00) 0. 0 (0.00)† 0. 0 (0.00)

ILMN 257. 5 (1.56) 263. 7 (3.37) 0. 0 (0.00) 0. 0 (0.00) 0. 3 (0.25) 0. 0 (0.00)

INSH 214. 0 (9.56) 204. 2 (7.99) 0. 5 (0.29) 1. 3 (0.48) 3. 3 (0.63) 3. 3 (0.85)

KSLA 223. 7 (3.50) 221. 9 (1.02) 0. 0 (0.00)† 0. 0 (0.00) 0. 0 (0.00)† 0. 0 (0.00)

NC BD 259. 2 (0.67) 262. 2 (1.09) 5. 3 (0.95) 7. 8 (0.95) 0. 3 (0.25) 0. 0 (0.00)

NEDC 252. 4 (4.75) 247. 3 (5.50) 0.0 (0.00) 0. 0 (0.00) 1. 8 (0.85) 1. 0 (0.41)

NEYO 260. 4 (1.63) 257. 1 (2.47) 0. 3 (0.25) 0. 0 (0.00) 1. 0 (0.71) 0. 3 (0.25)

PAHM 217. 2 (3.54) 227. 2 (7.10) 0. 0 (0.00) 0. 3 (0.25) 4. 3 (0.85) 5. 8 (2.06)

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Table I

- 4 (continued). Indiv i dual Site Phenotypic Comparison of MON 8741 1 Compare d t o th e Conventiona l Control

Site Phenotypic Characteri s tics

Roo t lodge d plant s (#/plot ) Final stand count (#/plot) Grain m oisture ( % )

Mean (S.E. ) 3 Mean (S.E. ) 3 Mean (S.E. ) 3

Cod e 1 MO N 8741 1 Contro l MO N 8741 1 Contro l MO N 8741 1 Control

IABG 0. 3 (0.25 ) 1. 0 (1.00 ) 60. 3 (0.25 ) 60. 8 (0.48 ) 17. 0 (0.79 ) 16. 9 (0.59)

IARL 0.0 (0.00 ) 0. 8 (0.48 ) 61. 5 (0.50 ) 61. 8 (0.25 ) 16. 9 (0.15) * 17. 7 (0.33)

ILM N 0. 0 (0.00) † 0. 0 (0.00 ) 62. 0 (0.71 ) 61. 5 (0.50 ) 19. 7 (0.28 ) 19. 5 (0.17)

INSH 0. 5 (0.50 ) 0. 0 (0.00 ) 70. 0 (1.47 ) 69. 3 (2.95 ) 18. 2 (0.26 ) 18. 3 (0.14)

KSL A 0. 0 (0.00) † 0. 0 (0.00 ) 77. 3 (0.48) * 74. 0 (0.00 ) 15. 1 (0.19 ) 15. 4 (0.43)

NC BD 0. 0 (0.00 ) 0. 3 (0.25 ) 67. 8 (1.03 ) 67. 0 (0.58 ) 20. 0 (0.19 ) 20. 0 (0.19)

NED C 0. 0 (0.00 ) 0. 0 (0.00 ) 62. 0 (0.00 ) 61. 3 (0.25 ) 14. 8 (0.33 ) 15. 6 (0.35)

NEY O 0. 0 (0.00) † 0. 0 (0.00 ) 61. 0 (1.35 ) 60. 8 (0.75 ) 15. 4 (0.22 ) 15. 2 (0.20)

PAH M 0. 0 (0.00) † 0. 0 (0.00 ) 62. 3 (0.63 ) 62. 5 (0.29 ) 21. 1 (0.27) * 22. 3 (1.01)

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MO N 87411 Control MO N 87411 Control

76. 9 (0.80) 77. 8 (0.33) 11. 7 (1.18) 9. 5 (0.42)

76. 6 (0.64) 75. 8 (0.64) 8.2 (1.00) 8. 3 (0.43)

69. 6 (0.18) 69. 9 (0.18) 14. 4 (0.29) 14. 9 (0.04)

71. 6 (1.01) 73. 2 (1.18) 11. 5 (0.66)* 9. 2 (0.77)

74. 8 (0.36) 75. 5 (0.40) 14. 1 (0.48) 14. 0 (0.51)

69. 0 (0.41) 70. 1 (0.78) 9.5 (0.28) 9. 3 (0.59)

72. 1 (1.00) 73. 5 (1.07) 7. 5 (1.16) 8. 0 (0.52)

76. 2 (0.28) 76. 3 (0.18) 12. 5 (0.07) 13. 0 (0.17)

68. 5 (0.46)* 65. 8 (0.88) 10. 4 (0.27) 11. 1 (0.48) Table I

- 4 (continued). Indiv i dual Site Phenotypic Comparison of MON 8741 1 Compare d t o th e Conventiona l Control

Site

Cod e 1

IABG

IARL

ILMN

INSH

KSLA

NC BD

NEDC

NEYO

PAHM Phenotypic Characteri s tics

Tes t weigh t (kg/hl ) Y ield (M g / h a)

Mean (S.E. ) 3 Mean (S.E. ) 3

Note: The experi me ntal design was a rando m ized c o m plete block with four rep l icates per site.

*Indicates statistically sig n ificant difference between MON 87411 and the conv e ntional cont r o l ( α =0.05) using ANONA.

† Indicates p

- values could not be generated due to lack of variabili t y in the data.

1 Site code: I A BG = Greene Coun t y, I A ; IARL = Jefferson Coun t y, I A ; ILMN

= Warren Coun t y

, IL; IN S H = Boone Coun t y, IN; KSLA

= Pawnee

Coun t y

, KS; NCBD

= Perqui m a ns Co u n t y

, NC; NEDC

= Butler Coun t y

, NE; NEYO

= York Coun t y, N E ; PAHM

= Berks Coun t y

, PA.

2 Data we r e n ot subjected to statistical anal y s is. Plant vigor r a ting range ( m inimum

- maxi m um); the range of plant vig o r ratings for the references

is as follows: IABG 1

– 3; I A RL 5

– 8; I LMN 1

– 3; I N SH 3

– 5; K SLA 2; NCBD 3

– 4; NE D C 2; NEYO 1; PAHM 2

– 4

3 MON 87411 and the conventional control values represent means with standard error in parentheses. N

= 4 except where noted in T a ble I

- 3.

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Table I

- 5. Qualit a ti v e Assessmen t : Abiotic Stressor Evaluations Using a Categorical

Scale for MON 87411 and the Conventional C ontrol

Abiotic Str e ssor Nu m ber of

Observatio n s across

Sites 1 N u m ber of Observations where No

Differences were Obser v ed between

MO N 8741 1 and the Conventional

Control

Total 100 100

Cold 1 1

Drought 2 22 22

Flood 3 5 5

Frost 2 2

Hail 8 8

Heat 20 20

Nutrient deficiency 12 12

Soil co m paction 3 3

Sunscald 5 5

W ind 22 22

Note: The experi me ntal design was a rando m ized c o m plete block with four rep l icates per site.

No differences were observed between MON 87411 and the conventional c o ntrol dur i ng a n y

observation for damage caused by any of the assess e d abiotic stressors. Da t a were not subject e d

to statistical anal y s is.

Observational data collected at four cr o p dev e lop m ent stages: V6

– V8; V12

- VT; R1

- R3; a n d R5

-

R6.

1 Site code: I A BG

= Greene Coun t y

, I A ; IARL

= Jefferson Coun t y

, IA; IL M N

= Warren Coun t y

, IL; INSH = Boone Co u n t y, IN; KSLA = Pawnee C o un t y

, KS; NCBD = Perquimans Coun t y

, NC;

NEDC

= Butler Coun t y

, N E; NEYO

= York Coun t y, NE; PAHM

= Berks County, PA.

2 Includes dryness, dr y / heat.

3 Includes wet soil.

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Table I

- 6. Qualit a ti v e Assessment: Disease D a mag e Evaluation s Usin g a Categorical

Scale for MON 87411 and the Conventional C ontrol

Disease Nu m ber of

Observations

across Site s 1 N u m ber of Observations where No

Differences were Observed between

MON 87411 and the Conventional

Control

Total 119 119

Anthracnose 6 6

Bacterial leaf spot 1 1

Ear ro t 2 12 12

Eyespot 5 5

Fusarium sp. 8 8

Goss’s bact e ri a l wilt 10 10

Gray leaf spot 20 20

Leaf blight 3 16 16

Maiz e roug h dwar f virus 2 2

Pythium sp. 2 2

Rhizocto n ia sp. 2 2

Rus t 4 7 7

Seedling blight 2 2

S m ut 8 8

Stalk rot 5 10 10

Stewart’s bacterial wilt 8 8

Note: The experi me ntal design was a rando m ized c o m plete block with four rep l icates per site.

No differences were observed between MON 87411 and the conventional c o ntrol dur i ng a n y

observation for damage c a used by any of the assessed dise a ses. Data we r e not subjected to

statistica l an a l y sis.

Observational data collected at four cr o p dev e lop ment stages: V6

– V8; V12

- VT; R1

- R3; a n d R5

-

R6.

Additional assessments of ear rot disease and stalk rot disease were made o n 5 plant/plots at

harvest.

1 Site code: I A BG

= Greene Coun t y

, I A ; IARL

= Jefferson Coun t y

, IA; IL M N

= Warren Coun t y

, IL; INSH = Boone Co u n t y, IN; KSLA = Pawnee C o un t y

, KS; NCBD = Perquimans Coun t y

, NC;

NEDC

= Butler Coun t y

, N E; NEYO

= York Coun t y, NE; PAHM

= Berks County, PA.

2 Assessed on 5 non

- s y stematically selected plant s

. At ILMN and INSH, s t alk rot data were

collected both on a per pl o t basis and on 5 plant / plots.

3 Includes nor t hern and sou t hern.

4 Includes c o mmon and southern

. 5 Assessed on 5 non

- s y stematically selected plants. At NEDC, s t alk rot data were collected both

on a per pl o t basis and on 5 plant/p l ots.

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Table I

- 7. Qualit a ti v e Assessment: Art h ropod Damage Evaluations Using a

Categorical Scale for MON 87411 and the Conventional Control

Arthropod Nu m ber of

Observatio n s

across Site s 1 N u m b e r o f Observations

where No Differences were

Observed between

MO N 8741 1 and the

Conventional Control

Total 102 102

Aphid (Aphididae) 3 3

A r m y wo r m (Noctuidae ) 2 18 18

Billbugs ( Sphenophoru s parvulu s ) 2 2

Cutworm (Noctuid a e) 3 6 6

Cor n earwor m ( Helicoverpa zea ) 5 5

Corn flea beetle ( Chaetocnema pulicari a ) 5 5

Rootwo r m beetle ( Diabrotica spp. ) 16 16

European corn borer ( O s trinia nu b il a lis ) 9 9

Grasshoppe r ( Melanoplu s spp.) 14 14

Japanese beetle ( Popillia japonic a ) 9 9

Sa p beetl e (Nitidulidae) 3 3

Spider m ites ( Tetranychus spp.) 3 3

Stink bugs (Pentato m idae) 8 8

W irewo r m beetle (Elateridae) 1 1

Note: The experi me ntal design was a rando m ized c o m plete block with four rep l icates per site.

No differences were observed between MON 87411 and the conventional c o ntrol dur i ng a n y

observation for da m a ge caused by any of the assessed arthropods. Data were not subjected to

statistica l an a l y sis.

Observational data collected at four cr o p dev e lop m ent stages: V6

– V8; V12

- VT; R1

- R3; a n d R5

-

R6.

1 Site code: I A BG

= Greene Coun t y

, I A ; IARL

= Jefferson Coun t y

, IA; IL M N

= Warren Coun t y

, IL; INSH = Boone Co u n t y, IN; KSLA = Pawnee C o un t y

, KS; NCBD = Perquimans Coun t y

, NC;

NEDC

= Butler Coun t y

, N E; NEYO

= York Coun t y, NE; PAHM

= Berks County, PA.

2 Includes beet a r m y worm and fall a r m ywo r m

.

3 Includes bl a c k cutwor m

.

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Table I

- 8. Individual

- S ite Analysis: Quant i tative Assessment of Corn Ear w orm and

European Corn Borer Damage to MON 8 7 411 Compared to t h e Conventional

Control

Pest D a mag e a s s e s s ment 1 Sit e 2 MON 87411 Control

Corn earworm

( H

. ze a ) 3 Mean (S.E.) da m a ge area

of 10 plants pe r plot (c m 2 ) IABG 0.7 (0

. 38) 0.5 (0

. 28)

NCBD 3.3 (1

. 25)* 1.5 (0

. 39)

NEYO 3.2 (0

. 13) 3.0 (0

. 22)

PAHM 0.3 (0

. 23) 0.2 (0

. 11)

European corn borer Mean (S.E.) nu m ber of IABG 0.0 (0

. 03) 0.0 (0

. 00)

( O. nubila l i s ) 4 stalk galleries of 10 plants

per plot NCBD 0.1 (0

. 03) 0.1 (0

. 04)

NEYO 0.0 (0

. 00) 0.0 (0

. 00)

PAHM 1.4 (0

. 24) 1.8 (0

. 33)

European corn borer Mean (S.E.) stalk gallery IABG 0.1 (0

. 08) 0.0 (0

. 00)

( O. nubila l i s ) 4 length (c m ) of 10 p lants

per plot NCBD 0.5 (0

. 19) 0.2 (0

. 14)

NEYO 0.0 (0

. 00) 0.0 (0

. 00)

PAHM 5.9 (1

. 46) 7.9 (1

. 46)

Note: The experi me ntal design was a rando m ized c o m plete block with four rep l icates per site.

*Indicates a statistically significant di f ference between MON 87411 a n d t he conventional control

(α=0.05) using ANOVA.

1 MON 87411and the conventional control values represent mea n s with s t andard error in parentheses.

N

= 4.

2 Site code: IABG

= Greene, IA; NC B D

= Perqu i mans, NC; N E YO

= York, NE; PAHM

= Berks, PA.

3 Da m a ge assessment for H. zea was conducted at R5 growth stage.

4 Da m a ge a s sessments for O. nubila l is were conducted at R6 gr ow th stage.

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Table I

- 9. Individual

- S ite Analysis: Art h ropo d Abund a nc e i n Stick y Tra p Sampl es Collecte d fro m MO N 8741 1 Compare d to

th e Conventiona l Control

A p h i d ( A p h i d i d ae) B r o wn Lace wing (He m erobiidae) Corn F l e a Bee t l e (Chryso m elidae)

Pes t A r th r op od Be n e f icia l A r t h ro p od Pes t A r th r op od

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4

1 IA B G 1

. 5 ( 0

. 8 7) 1

. 0 ( 0

. 4 1) 0

. 5

– 3

. 3

— — — 0

. 3 ( 0

. 2 5 ) † 0

. 5 ( 0

. 5 0) 0

. 0

– 0

. 8

N CB D 0

. 8 ( 0

. 7 5 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 1

. 8 0

. 5 ( 0

. 2 9 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 5 0

. 5 ( 0

. 2 9 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 4

. 3

NE Y O 0

. 5 ( 0

. 2 9 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 2

. 0

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

P A HM 0

. 7 ( 0

. 3 3 ) † 0

. 7 ( 0

. 3 3) 0

. 3

– 1

. 0 0

. 7 ( 0

. 3 3 ) † 0

. 3 ( 0

. 3 3) 0

. 0

– 0

. 3 3

. 7 ( 1

. 4 5) 2

. 3 ( 1

. 3 3) 3

. 7

– 1 1 .3

2 IA B G 1

. 3 ( 0

. 7 5) 1

. 0 ( 0

. 4 1) 1

. 3

– 3

. 5 0

. 3 ( 0

. 2 5 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 1

. 0 0

. 5 ( 0

. 5 0 ) † 0

. 5 ( 0

. 5 0) 0

. 0

– 0

. 3

N CB D 0

. 8 ( 0

. 2 5) 0

. 3 ( 0

. 2 5) 0

. 3

– 4

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0) 2

. 0 ( 1

. 0 8) 0

. 5

– 5

. 3

NE Y O 0

. 5 ( 0

. 2 9 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 2

. 3

— — — 0

. 5 ( 0

. 2 9 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 8

P A HM 1

. 0 ( 1

. 0 0 ) † 0

. 7 ( 0

. 6 7) 0

. 0

– 0

. 8

— — — 3

. 0 ( 1

. 5 3) 6

. 7 ( 0

. 8 8) 5

. 7

– 1 2 .5

3 IA B G 1

. 5 ( 0

. 2 9) 0

. 5 ( 0

. 2 9) 1

. 0

– 3

. 8

— — — 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 5

– 1

. 0

N CB D 1

. 5 ( 0

. 6 5) 1

. 0 ( 0

. 7 1) 0

. 0

– 2

. 3

— — — 0

. 8 ( 0

. 4 8) 1

. 3 ( 0

. 9 5) 0

. 3

– 1 6 .0

NE Y O 1

. 8 ( 0

. 4 8 ) * 0

. 0 ( 0

. 0 0) 1

. 0

– 2

. 5

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

P A HM 0

. 3 ( 0

. 3 3 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

— — — 9

. 3 ( 4

. 4 8) 5

. 0 ( 1

. 7 3) 7

. 7

– 2 0 .8

4 IA B G 4

. 0 ( 1

. 7 3) 0

. 8 ( 0

. 2 5) 1

. 5

– 5

. 5 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 3

– 0

. 8 0

. 5 ( 0

. 2 9) 0

. 3 ( 0

. 2 5) 1

. 3

– 4

. 8

N CB D 1

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 1

. 8

— — — 1

. 0 ( 0

. 7 1) 3

. 3 ( 1

. 9 3) 0

. 3

– 1 9 .8

NE Y O 1

. 8 ( 0

. 4 8) 0

. 8 ( 0

. 2 5) 2

. 5

– 7

. 0

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

P A HM 0

. 3 ( 0

. 3 3 ) † 1

. 0 ( 0

. 5 8) 0

. 0

– 1

. 7

— — — 7

. 7 ( 3

. 5 3) 6

. 7 ( 3

. 2 8) 1 2 .3

– 2 3

. 3

5 IA B G 7

. 5 ( 4

. 2 5) 2

. 8 ( 1

. 1 1) 6

. 3

– 1 2 .0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 8 0

. 3 ( 0

. 2 5 ) † 0

. 5 ( 0

. 2 9) 0

. 5

– 1

. 5

N CB D 1

. 0 ( 0

. 7 1) 1

. 0 ( 0

. 4 1) 0

. 5

– 1

. 8

— — — 3

. 3 ( 1

. 1 1) 6

. 8 ( 1

. 0 3) 4

. 3

– 2 7 .8

NE Y O 21.0 ( 5 .5 8) 1 9

. 3 ( 7.0 6) 1 5. 8

– 3 0 .0

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

P A HM 1

. 7 ( 0

. 8 8 ) † 0

. 7 ( 0

. 3 3) 0

. 0

– 1

. 0

— — — 5

. 0 ( 1

. 7 3) 3

. 0 ( 1

. 5 3) 3

. 0

– 1 2 .0

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 343 o f 3 7 2CB I Delete d Copy

Tabl e I

- 9

. (continued)

. Individu a l

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411

Compared to the Conventional C o ntrol

Delphacid Planthopp e r (De l phac i dae) Gras s h opp e r (Acr i didae) Gr ee n Lace w in gs ( Ch r yso p i d ae)

Pes t A r th r op od Pes t A r th r op od Be n e f icia l A r t h ro p od

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4

1 IA B G 0

. 0 ( 0

. 0 0 ) * 1

. 8 ( 0

. 8 5) 1

. 5

– 3

. 5

— — — 3

. 8 ( 1

. 2 5) 2

. 8 ( 1

. 3 1) 1

. 5

– 4

. 8

NCBD 51. 0 ( 5 .58 ) 64. 0 ( 6

. 9 2 ) 63. 8

– 106. 0

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 8 ( 0

. 2 5) 0

. 0

– 0

. 3

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 3

P A HM 6

. 7 ( 1

. 4 5) 3

. 0 ( 1

. 5 3) 3

. 8

– 1 0 .0

— — — — — —

2 IA B G 0

. 8 ( 0

. 4 8) 0

. 5 ( 0

. 2 9) 1

. 3

– 3

. 0

— — — 1

. 8 ( 0

. 8 5) 2

. 5 ( 1

. 1 9) 0

. 0

– 3

. 0

NCBD 10. 0 ( 4 .02 ) 11. 8 ( 2

. 2 9 ) 15. 3

– 23. 0

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

NE Y O 0

. 3 ( 0

. 2 5 ) † 0

. 5 ( 0

. 2 9) 0

. 3

– 0

. 5

— — — 0

. 8 ( 0

. 7 5 ) † 0

. 3 ( 0

. 2 5) 0

. 5

– 1

. 5

P A HM 4

. 7 ( 2

. 7 3) 5

. 0 ( 1

. 7 3) 2

. 3

– 1 0 .5

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5

3 IA B G

— — — — — — — — —

N CB D 8

. 0 ( 1

. 0 8) 5

. 0 ( 2

. 5 2) 9

. 3

– 1 2 .8

— — — — — —

NE Y O 0

. 8 ( 0

. 2 5 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 0

. 5

— — — 4

. 0 ( 1

. 6 8) 2

. 5 ( 0

. 2 9) 1

. 5

– 4

. 8

P A HM 6

. 0 ( 2

. 0 0) 4

. 7 ( 1

. 2 0) 2

. 7

– 8

. 8

— — — — — —

4 IA B G 1

. 3 ( 0

. 7 5 ) † 0

. 3 ( 0

. 2 5) 0

. 5

– 0

. 8

— — — 4

. 3 ( 1

. 1 1) 7

. 0 ( 1

. 2 2) 5

. 8

– 6

. 5

N CB D 4

. 8 ( 1

. 1 1) 6

. 0 ( 0

. 4 1) 7

. 5

– 8

. 3

— — — — — —

NE YO

— — — — — — 0

. 5 ( 0

. 2 9) 0

. 8 ( 0

. 2 5) 0

. 8

– 7

. 3

P A HM 4

. 7 ( 1

. 2 0) 2

. 3 ( 0

. 3 3) 0

. 7

– 1 0 .3

— — — — — —

5 IA B G

— — — — — — 6

. 3 ( 2

. 7 5) 5

. 3 ( 1

. 1 1) 4

. 5

– 9

. 8

N CB D 3

. 3 ( 1

. 2 5) 4

. 3 ( 1

. 4 4) 3

. 8

– 8

. 3

— — — — — —

NE YO

— — — — — — 3

. 0 ( 0

. 7 1) 1

. 5 ( 1

. 1 9) 0

. 3

– 2

. 0

P A HM 1

. 0 ( 0

. 5 8 ) † 0

. 7 ( 0

. 3 3) 0

. 0

– 2

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

— — —

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 344 o f 3 7 2CB I Delete d Copy

Tabl e I

- 9

. (continued)

. Individu a l

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411

Compared to the Conventional C o ntrol

Ladybir d Beet l e (Cocc i nellid a e ) Leafhopp e r (Cicadellidae ) Micro

- Parasiti c Hy m enoptera

Be n e f icia l A r t h ro p o d Pes t A r th r op od Be n e f icia l A r t h ro p od

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 41 1 C on tr ol ra n ge 4 M ON 8 7 41 1 C on tr ol ra n ge 4 M ON 8 7 41 1 C on tr ol ra n ge 4

1 IA B G 1

. 3 ( 0

. 7 5) 0

. 5 ( 0

. 5 0) 1

. 0

– 3

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 3 3 .8 ( 2 .6 6) 4 5 .5 ( 3 .4 0) 3 4 .3

– 6 7

. 3

N CB D 3

. 5 ( 0

. 8 7) 5

. 3 ( 3

. 2 0) 2

. 5

– 3

. 5 3 7

. 8 ( 4

. 5 3 ) 4 0

. 0 ( 5

. 4 3) 3 7 .3

– 5 3

. 0 12 9

. 8 ( 8

. 4 3) 14 1

. 5 ( 2 0 .9 7) 2 1 4

. 5

– 2 7 8 .0

NE Y O 0

. 8 ( 0

. 2 5) 0

. 3 ( 0

. 2 5) 0

. 0

– 7

. 5 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 7

. 5 ( 1

. 9 4) 9

. 0 ( 1

. 4 7) 8

. 8

– 1 3 .0

P A HM 1 5

. 3 (0 8 8) 1 4

. 3 ( 1

. 3 3) 2 1 .5

– 3 2

. 3 4

. 3 ( 1

. 2 0) 4

. 0 ( 2

. 0 8) 3

. 5

– 7

. 3 1 2 8

. 0 ( 1 8

. 0 ) 10 4

. 0 ( 1 8 .3 6) 1 4 5

. 7

– 1 8 9 .3

2 IA B G 0

. 8 ( 0

. 2 5) 1

. 3 ( 0

. 2 5) 1

. 0

– 5

. 8 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 3 6 .3 ( 7 .7 2) 4 4 .8 (1 0

. 4 6 ) 4 8 .0

– 8 8

. 3

N CB D 7

. 5 ( 1

. 8 5) 1 0

. 0 ( 2

. 5 8) 9

. 3

– 1 6 .5 4 0

. 5 ( 8

. 9 1 ) 4 2

. 3 ( 1 5 .1 0) 3 3 .5

– 7 9

. 8 9 2 .3 (1 5

. 3 7 ) 9 7 .8 (1 2

. 2 5 ) 15 3

. 3

– 1 8 7

. 5

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 8 ( 0

. 4 8) 0

. 5

– 1

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 1 4 .3 ( 3 .2 8) 1 7 .3 ( 4 .3 5) 1 4 .3

– 1 7

. 5

P A HM 3 9

. 0 ( 1 1 .1 4) 3 3

. 7 ( 6

. 3 6) 2 6 .3

– 3 6

. 0 3

. 0 ( 1

. 5 3) 5

. 0 ( 1

. 1 5) 3

. 0

– 1 0 .3 10 2

. 7 ( 1 2 .8 1) 1 1 9

. 0 ( 8

. 9 6 ) 11 4

. 0

– 1 4 1

. 8

3 IA B G 0

. 0 ( 0

. 0 0 ) † 0

. 8 ( 0

. 2 5) 0

. 5

– 1

. 5 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 0 3 0 .0 ( 4 .7 8) 2 8 .8 ( 3 .8 8) 3 8 .3

– 10 2

. 3

N CB D 5

. 8 ( 1

. 4 4) 6

. 5 ( 0

. 5 0) 4

. 0

– 7

. 0 4 0

. 8 ( 9

. 0 0 ) 4 3

. 3 ( 1 0 .6 6) 4 8 .0

– 6 9

. 5 6 6 .8 ( 7 .9 6) 6 2 .5 (1 3

. 1 2 ) 6 1 .3

– 8 7

. 3

NE Y O 0

. 3 ( 0

. 2 5 ) † 0

. 8 ( 0

. 4 8) 0

. 3

– 1

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 1 7 .8 ( 1 .0 3) 1 2 .8 ( 1 .6 5) 1 4 .3

– 3 3

. 0

P A HM 2 0

. 7 ( 5

. 9 0) 1 3

. 0 ( 2

. 0 8) 1 0 .3

– 1 4

. 5 2

. 3 ( 0

. 3 3) 3

. 0 ( 1

. 0 0) 1

. 5

– 4

. 5 7 8 .7 (2 2

. 5 1 ) 6 1 .3 ( 2 .9 6) 5 9 .3

– 10 4

. 0

4 IA B G 1

. 3 ( 0

. 4 8) 0

. 5 ( 0

. 5 0) 1

. 3

– 4

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 4 7 .0 ( 5 .1 5) 4 7 .5 ( 3 .3 0) 4 4 .5

– 8 9

. 8

N CB D 8

. 5 ( 1

. 8 5) 7

. 0 ( 1

. 6 8) 5

. 5

– 8

. 0 2 7

. 5 ( 0

. 8 7 ) 3 3

. 5 ( 7

. 6 0) 2 8 .3

– 4 5

. 0 6 5 .8 (1 1

. 5 3 ) 5 6 .5 (1 5

. 5 4 ) 6 1 .5

– 10 4

. 3

NE Y O 0

. 5 ( 0

. 2 9 ) † 0

. 8 ( 0

. 7 5) 0

. 3

– 1

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 1 2 .5 ( 1 .5 0) 9

. 3 ( 2

. 2 9) 1 3 .8

– 6 9

. 0

P A HM 7

. 3 ( 0

. 6 7) 5

. 7 ( 1

. 2 0) 2

. 7

– 5

. 0 3

. 7 ( 0

. 8 8) 4

. 3 ( 1

. 3 3) 4

. 0

– 1 1 .0 1 1 8

. 3 ( 2 4

. 4 4) 8 5 .0 (2 0

. 4 3 ) 10 0

. 0

– 1 7 7

. 0

5 IA B G 2

. 0 ( 0

. 8 2) 2

. 0 ( 2

. 0 0) 1

. 3

– 4

. 5 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 1 7 .5 ( 4 .3 3) 1 3 .8 ( 2 .3 9) 2 0 .0

– 5 8

. 5

N CB D 1

. 5 ( 0

. 2 9) 3

. 8 ( 0

. 4 8) 1

. 8

– 5

. 0 1 7

. 3 ( 2

. 2 5 ) 2 0

. 3 ( 4

. 7 1) 1 9 .5

– 2 7

. 3 9 2 .0 (2 5

. 0 1 ) 6 5 .8 (1 8

. 8 6 ) 8 6 .8

– 10 7

. 5

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 1 1 .3 ( 0 .7 5) 1 3 .5 ( 2 .1 0) 9

. 8

– 2 0 .0

P A HM 5

. 7 ( 0

. 6 7) 4

. 3 ( 2

. 3 3) 2

. 5

– 3

. 7 5

. 0 ( 1

. 0 0) 3

. 3 ( 2

. 0 3) 4

. 0

– 9

. 8 18 2

. 7 ( 2 1 .8 4) 15 2

. 0 ( 3 3 .5 6) 13 6

. 7

– 2 2 9

. 8

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 345 o f 3 7 2CB I Delete d Copy

Tabl e I

- 9

. (continued)

. Individu a l

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411

Compared to the Conventional C o ntrol

Macro

- p a rasitic hy m e noptera Sap Beetle ( N i ti du li d ae ) Mi nu t e P i ra t e B ug ( An t ho c ori dae)

Be n e f icia l A r t h ro p o d Pes t A r th r op od Be n e f icia l A r t h ro p od

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 411 C on tr ol ra n g e 4 M ON 8 7 411 C on tr ol ra n g e 4 M ON 8 7 411 C on tr ol ra n g e 4

1 IA B G

— — — — — — 1

. 0 ( 0

. 7 1) 0

. 8 ( 0

. 4 8) 0

. 8

– 3

. 8

NCBD

— — — — — — 1

. 3 ( 0

. 6 3 ) † 0

. 3 ( 0

. 2 5) 0

. 5

– 0

. 8

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

PAHM

— — — — — — 1

. 0 ( 0

. 5 8 ) * 3

. 0 ( 0

. 5 8) 2

. 0

– 7

. 5

2 IA B G

— — — — — — 1

. 5 ( 0

. 9 6) 3

. 3 ( 0

. 2 5) 3

. 3

– 9

. 5

NCBD

— — — 1

. 3 ( 0

. 4 8 ) † 1

. 0 ( 0

. 7 1) 0

. 0

– 1

. 0 1

. 3 ( 0

. 9 5) 1

. 3 ( 0

. 4 8) 0

. 0

– 1

. 8

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

P A HM 0

. 7 ( 0

. 3 3) 1

. 0 ( 0

. 5 8) 0

. 7

– 2

. 5 0

. 7 ( 0

. 3 3 ) † 1

. 3 ( 0

. 3 3) 0

. 3

– 1

. 8 2

. 7 ( 1

. 2 0) 4

. 7 ( 2

. 1 9) 5

. 0

– 1 0 .0

3 IA B G

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 1

. 3 ( 0

. 4 8) 1

. 0 ( 0

. 4 1) 0

. 8

– 3

. 5

N CB D 0

. 5 ( 0

. 5 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 3 0

. 8 ( 0

. 2 5 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 1

. 0 0

. 3 ( 0

. 2 5) 0

. 5 ( 0

. 2 9) 1

. 0

– 1

. 8

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 3

– 0

. 8

— — — 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

P A HM 0

. 3 ( 0

. 3 3) 1

. 7 ( 0

. 3 3) 0

. 5

– 1

. 7 0

. 3 ( 0

. 3 3 ) † 0

. 0 ( 0

. 0 0) 0

. 7

– 0

. 8 4

. 0 ( 1

. 5 3) 2

. 0 ( 0

. 0 0) 2

. 3

– 1 0 .0

4 IA B G

— — — — — — 0

. 3 ( 0

. 2 5) 1

. 0 ( 0

. 4 1) 2

. 3

– 6

. 3

NCBD

— — — — — — 1

. 0 ( 0

. 5 8) 0

. 8 ( 0

. 2 5) 1

. 5

– 4

. 0

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

PAHM

— — — — — — 0

. 3 ( 0

. 3 3) 0

. 3 ( 0

. 3 3) 2

. 3

– 4

. 0

5 IA B G

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 1

. 0 0

. 5 ( 0

. 2 9) 1

. 8 ( 1

. 4 4) 1

. 3

– 3

. 5

NCBD

— — — 1

. 5 ( 0

. 6 5) 1

. 0 ( 0

. 7 1) 0

. 3

– 1

. 5 1

. 3 ( 0

. 7 5 ) † 0

. 5 ( 0

. 2 9) 0

. 5

– 1

. 5

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 3

— — — 1

. 0 ( 0

. 4 1 ) † 0

. 0 ( 0

. 0 0) 0

. 5

– 0

. 8

P A HM 0

. 3 ( 0

. 3 3 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5

— — — 2

. 0 ( 0

. 5 8) 1

. 0 ( 1

. 0 0) 4

. 0

– 6

. 8

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 346 o f 3 7 2CB I Delete d Copy

Tabl e I

- 9

. (continued)

. Individu a l

- Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87411

Compared to the Conventional C o ntrol

Spid e r ( A r a ne a e )

Beneficia l Arthropod

Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 41 1 C on tr ol Ra ng e 4

1 IA B G 0

. 5 ( 0

. 2 9 ) † 0

. 3 ( 0

. 2 5) 0

. 5

– 0

. 8

N CB D 3

. 3 ( 0

. 4 8) 1

. 0 ( 0

. 4 1) 1

. 5

– 2

. 3

NE Y O 0

. 5 ( 0

. 2 9 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 1

. 3

P A HM 0

. 7 ( 0

. 3 3 ) † 1

. 7 ( 0

. 3 3) 0

. 5

– 1

. 3

2 IA B G 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 1

. 0

N CB D 1

. 5 ( 0

. 2 9) 2

. 5 ( 1

. 5 0) 1

. 5

– 3

. 3

NE Y O 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5

P A HM 1

. 3 ( 0

. 3 3 ) † 0

. 0 ( 0

. 0 0) 1

. 0

– 1

. 5

3 IA B G 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

N CB D 1

. 8 ( 0

. 7 5) 1

. 3 ( 0

. 2 5) 1

. 3

– 3

. 0

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

P A HM 2

. 3 ( 1

. 4 5) 0

. 7 ( 0

. 3 3) 1

. 3

– 2

. 5

4 IA B G 0

. 8 ( 0

. 7 5 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 1

. 0

N CB D 1

. 0 ( 0

. 5 8) 2

. 0 ( 1

. 0 0) 1

. 5

– 4

. 0

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 0

. 3

P A HM 2

. 0 ( 2

. 0 0) 2

. 0 ( 0

. 0 0) 1

. 0

– 1

. 8

5 IA B G 0

. 3 ( 0

. 2 5 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 0

. 5

N CB D 1

. 5 ( 0

. 2 9) 0

. 8 ( 0

. 4 8) 1

. 5

– 2

. 8

NE Y O 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5

P A HM 3

. 3 ( 0

. 8 8 ) * 0

. 3 ( 0

. 3 3) 0

. 3

– 1

. 3

Note: The expe r imental d esign w as a randomized complete bl o ck with four r eplicates per site.

* Indic a tes a st at istic a l l y s ignif i cant d iffe r ence between MON 87 4 11 an d th e con v entiona l co n t ro l (α

= 0

. 05 ) usin g ANO V A.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 347 o f 3 7 2CB I Delete d Copy

† Indicates p

- va lues could n ot be generated where the taxa did not meet inclusi o n criteria (see appendix I .1 0.2).

1 Arthropods were en u m e r ated at five c r op develop ment stages: C ollection 1

= late vegetati v e

– VT growth stage; Collection 2

= R1 growth stage;

Collection 3

= R2 growth stage; Collection 4

= R3 growth stage; Collection 5

= R4 growth stage.

2 Site code: IABG

= Greene, IA; NC B D

= Perqu i mans, NC; N E YO

= York, NE; PAHM

= Berks, PA.

3 MON 87411 and the conventional control values represent means with standard error in par e ntheses. N

= 4 (IABG, N C BD, and N E YO sites); N=

3 (PAHM site).

4 Reference range is calculated from the min i m u m a n d m a x i m um mean values f rom a m ong reference hyb r ids at each si t e

. A dash (

— ) indicates data not available.

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 348 o f 3 7 2CB I Delete d Copy

An t

- like F l ow e r Beetle (An t hicidae) Clic k Beetle Cor n Fle a Bee t le

P o lle n Fee d er Pes t A r th r op od Pes t A r th r op od

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4

1 IA B G

— — — — — — — — —

NCBD

— — — 1

. 0 ( 0

. 7 1) 1

. 8 ( 0

. 8 5) 0

. 8

– 1

. 5 2

. 5 ( 1

. 2 6) 3

. 3 ( 1

. 9 7) 1

. 0

– 5

. 0

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

PAHM

— — — — — — 6

. 3 ( 4

. 3 7) 4

. 7 ( 1

. 4 5) 7

. 8

– 1 0 .3

2 IA B G 0

. 8 ( 0

. 4 8 ) * 4

. 3 ( 2

. 2 9) 1

. 8

– 3

. 3

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

NCBD

— — — 1

. 3 ( 0

. 7 5) 1

. 3 ( 0

. 7 5) 1

. 0

– 1

. 8 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 8

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 Tabl e I

- 10

. Individual

- Sit e Analysis : Art h ropo d Abundanc e i n Visua l Coun t s fro m MO N 8741 1 Compare d t o the

Conventiona l Control

PAHM

— — — — — — 8

. 0 ( 5

. 1 3) 7

. 3 ( 0

. 8 8) 7

. 5

– 1 1 .0

3 IA B G 0

. 5 ( 0

. 5 0) 1

. 0 ( 0

. 7 1) 0

. 8

– 2

. 0

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

NCBD

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 8 ( 0

. 4 8) 0

. 5

– 0

. 8 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

PAHM

— — — — — — 1 2

. 0 ( 3

. 5 1 ) 7

. 7 ( 3

. 8 4) 7

. 8

– 9

. 8

4 IA B G 1

. 3 ( 0

. 9 5) 2

. 8 ( 1

. 4 9) 1

. 5

– 3

. 0

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

NCBD

— — — 0

. 5 ( 0

. 5 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5 0

. 3 ( 0

. 2 5 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 8

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

PAHM

— — — — — — 3

. 7 ( 1

. 7 6) 2

. 0 ( 0

. 5 8) 4

. 0

– 6

. 0

5 IA B G 1

. 8 ( 1

. 1 1) 1

. 8 ( 1

. 4 4) 0

. 5

– 1 .8

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

NCBD

— — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 1

. 3 ( 1

. 2 5) 3

. 3 ( 1

. 8 9) 0

. 8

– 3

. 0

NE YO

— — — — — — 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

PAHM

— — — — — — 2

. 3 ( 0

. 6 7) 7

. 3 ( 3

. 9 3) 5

. 5

– 7

. 3

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 349 o f 3 7 2CB I Delete d Copy

Table I

- 10. (continued). Individual

- Site Analysis: Arthropod Abundance in Visual Count s fro m MO N 8741 1 Compare d t o the

Conventiona l Control

Jap a nese B ee tl e ( S c a r a b ae i d ae) Lacewing Adult (Chry s op i d a e) Lacewin g L arva e (Chrysopid a e )

Pes t A r th r op od Be n e f icia l A r t h ro p o d Be n e f icia l A r t h ro p od

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 411 C on tr ol ra n g e 4 M ON 8 7 411 C on tr ol ra n g e 4 M ON 8 7 411 C on tr ol ra n g e 4

1 IA B G

— — — — — — — — —

N CB D 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 5 ( 0

. 5 0 ) † 0

. 5 ( 0

. 5 0) 0

. 3

– 0

. 5 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

P A HM 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 3 3) 0

. 0

– 0

. 5 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 1

. 0

2 IA B G 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 8 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

N CB D 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 8 ( 0

. 4 8 ) † 0

. 5 ( 0

. 5 0) 0

. 0

– 0

. 5

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

P A HM 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 7 0

. 3 ( 0

. 3 3 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 8

3 IA B G 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

N CB D 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 3 ( 0

. 2 5 ) † 1

. 3 ( 0

. 4 8) 0

. 0

– 0

. 3

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 5 ( 0

. 2 9 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 0

. 5

P A HM 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 3 3) 0

. 0

– 0

. 7 0

. 3 ( 0

. 3 3 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 7

4 IA B G 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5

N CB D 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 0

P A HM 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 7 ( 0

. 6 7 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

5 IA B G 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 3

– 0

. 5 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

N CB D 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5 0

. 0 ( 0

. 0 0 ) † 0

. 5 ( 0

. 5 0) 0

. 0

– 0

. 0

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

P A HM 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 350 o f 3 7 2CB I Delete d Copy

Table I

- 10. (continued). Individual

- Site Analysis: Arthropod Abundance in Visual Count s fro m MO N 8741 1 Compare d t o the

Conventiona l Control

La d y b ir d Beet l e A du l t (C o cc i n elli d ae) La d y b ir d Beet l e Lar v a e (c o cci n elli d ae) Ly gus Bu g ( M iri d ae)

Be n e f icia l A r t h ro p od Be n e f icia l A r t h ro p od Pes t A r th r op od

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4

1 IA B G

— — — — — — — — —

N CB D 0

. 5 ( 0

. 2 9 ) † 0

. 5 ( 0

. 2 9) 0

. 3

– 1

. 0 0

. 5 ( 0

. 5 0) 0

. 5 ( 0

. 5 0) 0

. 3

– 1

. 8

— — —

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

— — —

P A HM 3

. 7 ( 1

. 4 5) 7

. 0 ( 2

. 0 8) 2

. 5

– 3

. 3 0

. 7 ( 0

. 6 7 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5

— — —

2 IA B G 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

— — —

N CB D 0

. 5 ( 0

. 5 0 ) † 0

. 5 ( 0

. 5 0) 0

. 3

– 1

. 0 2

. 8 ( 0

. 9 5) 3

. 5 ( 0

. 5 0) 1

. 5

– 2

. 8

— — —

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

— — —

P A HM 3

. 0 ( 2

. 5 2) 5

. 0 ( 4

. 0 4) 1

. 5

– 2

. 5 0

. 3 ( 0

. 3 3 ) † 0

. 7 ( 0

. 6 7) 0

. 0

– 1

. 3 0

. 0 ( 0

. 0 0 ) † 1

. 0 ( 1

. 0 0) 0

. 0

– 0

. 3

3 IA B G 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

— — —

N CB D 1

. 0 ( 0

. 4 1 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 1

. 5 1

. 3 ( 0

. 6 3 ) † 0

. 3 ( 0

. 2 5) 0

. 8

– 1 .3

— — —

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

— — —

P A HM 1

. 3 ( 0

. 3 3 ) † 0

. 3 ( 0

. 3 3) 0

. 0

– 0

. 8 1

. 3 ( 0

. 6 7 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 1

. 7 0

. 0 ( 0

. 0 0 ) † 0

. 7 ( 0

. 6 7) 0

. 0

– 0

. 3

4 IA B G 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

— — —

N CB D 0

. 8 ( 0

. 4 8 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 8 0

. 5 ( 0

. 5 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

— — —

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

— — —

P A HM 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 3 3) 0

. 0

– 0

. 7 0

. 7 ( 0

. 6 7 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 7 ( 0

. 6 7 ) † 1

. 0 ( 0

. 0 0) 0

. 0

– 1

. 3

5 IA B G 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

— — —

N CB D 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 5 ( 0

. 2 9 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 0

. 5

— — —

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

— — —

P A HM 0

. 0 ( 0

. 0 0 ) † 0

. 7 ( 0

. 3 3) 0

. 0

– 0

. 8 0

. 7 ( 0

. 3 3 ) † 0

. 7 ( 0

. 6 7) 0

. 0

– 1

. 0 0

. 7 ( 0

. 3 3 ) † 0

. 3 ( 0

. 3 3) 0

. 0

– 0

. 5

M ons a n t o Co m pany CR 2 4 0

- 1 3 U 1 351 o f 3 7 2CB I Delete d Copy

Table I

- 10. (continued). Individual

- Site Analysis: Arthropod Abundance in Visual Count s fro m MO N 8741 1 Compare d t o the

Conventiona l Control

Mi nu t e P i ra t e B ug ( An t ho c ori dae) Sap B ee tl e ( N i ti du li d ae ) S h i n i ng Flowe r Beetl e (P h alacri d ae)

Be n e f icia l A r t h ro p o d Pes t A r th r op od P o lle n Fee d er

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4

1 IA B G

N CB D —

1

. 5 ( 0

. 6 5) —

0

. 3 ( 0

. 2 5) —

1

. 0

– 3

. 5 —

4

. 5 ( 2

. 6 3) —

5

. 5 ( 2

. 9 6) —

3

. 8

– 7

. 5 —

6

. 3 ( 1

. 4 9) —

4

. 3 ( 1

. 8 4) —

5

. 8

– 1 0 .5

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

— — —

P A HM 6

. 3 ( 2

. 3 3) 1 0

. 3 ( 5

. 4 9) 8

. 3

- 1 3 .3 6

. 0 ( 2

. 0 8) 4

. 3 ( 1

. 4 5) 5

. 3

– 1 2 .8 2

. 0 ( 0

. 5 8) 5

. 3 ( 3

. 1 8) 1

. 5

– 5

. 3

2 IA B G 5

. 0 ( 0

. 4 1) 4

. 3 ( 1

. 1 1) 1

. 5

- 4

. 5 6

. 3 ( 1

. 4 4) 1 0

. 8 ( 4

. 7 7) 4

. 0

– 7

. 5

— — —

N CB D 2

. 0 ( 0

. 7 1) 2

. 3 ( 0

. 6 3) 0

. 5

- 3

. 3 4

. 8 ( 4

. 0 9) 3

. 5 ( 1

. 5 5) 2

. 8

– 7

. 8 9

. 0 ( 2

. 7 4) 1 5

. 3 ( 3

. 0 9) 7

. 8

– 1 7 .8

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 3

— — —

P A HM 5

. 3 ( 1

. 4 5) 6

. 0 ( 0

. 0 0) 4

. 0

– 1 0 .0 8

. 0 ( 1

. 5 3) 5

. 0 ( 2

. 5 2) 1

. 3

– 7

. 3 1

. 7 ( 0

. 8 8) 4

. 0 ( 1

. 5 3) 2

. 0

– 3

. 5

3 IA B G 1

. 5 ( 0

. 6 5) 0

. 8 ( 0

. 4 8) 0

. 5

– 1

. 8 2

. 0 ( 0

. 9 1) 2

. 0 ( 0

. 5 8) 0

. 3

– 4

. 3

— — —

N CB D 1

. 3 ( 0

. 4 8) 1

. 5 ( 1

. 1 9) 0

. 5

– 1

. 0 2

. 8 ( 1

. 8 0) 1

. 8 ( 1

. 1 8) 1

. 3

– 3

. 8 5

. 3 ( 0

. 8 5) 3

. 8 ( 1

. 1 1) 2

. 0

– 2

. 5

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 4

. 8 ( 0

. 7 5) 4

. 8 ( 0

. 8 5) 1

. 8

– 3

. 0

— — —

P A HM 3

. 3 ( 1

. 2 0) 9

. 3 ( 2

. 7 3) 4

. 0

– 9

. 0 3

. 3 ( 1

. 4 5) 2

. 3 ( 0

. 8 8) 1

. 3

– 5

. 3 1

. 3 ( 0

. 8 8) 1

. 3 ( 0

. 6 7) 0

. 0

– 2

. 8

4 IA B G 0

. 8 ( 0

. 4 8) 1

. 3 ( 0

. 7 5) 1

. 3

– 1

. 8 4

. 0 ( 1

. 4 7) 6

. 8 ( 3

. 6 1) 2

. 3

– 6

. 0

— — —

N CB D 0

. 3 ( 0

. 2 5 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 8 0

. 8 ( 0

. 7 5 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 1

. 3 1

. 0 ( 0

. 5 8 ) † 1

. 3 ( 0

. 6 3) 0

. 0

– 1

. 0

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 1

. 5 ( 0

. 8 7) 2

. 5 ( 0

. 9 6) 0

. 8

– 2

. 8

— — —

P A HM 5

. 0 ( 1

. 5 3) 7

. 3 ( 0

. 6 7) 1

. 7

– 6

. 5 2

. 7 ( 2

. 1 9) 5

. 0 ( 2

. 3 1) 0

. 3

– 2

. 3 1

. 3 ( 0

. 6 7 ) † 0

. 7 ( 0

. 6 7) 0

. 0

– 0

. 7

5 IA B G 1

. 3 ( 0

. 4 8) 1

. 0 ( 0

. 5 8) 0

. 8

– 1

. 8 1 4 .0 ( 7 .3 9) 8

. 8 ( 4

. 8 5) 2

. 5

– 4

. 0

— — —

N CB D 0

. 8 ( 0

. 7 5 ) † 0

. 8 ( 0

. 4 8) 0

. 3

– 1

. 0 3

. 5 ( 0

. 9 6) 4

. 8 ( 1

. 1 1) 1

. 8

– 5

. 0 0

. 5 ( 0

. 2 9 ) † 0

. 5 ( 0

. 2 9) 0

. 3

– 0

. 8

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 1

. 8 ( 0

. 8 5 ) † 1

. 8 ( 1

. 1 1) 0

. 0

– 0

. 5

— — —

P A HM 2

. 0 ( 1

. 0 0) 4

. 7 ( 1

. 4 5) 1

. 3

– 3

. 5 0

. 3 ( 0

. 3 3) 1

. 7 ( 1

. 2 0) 1

. 7

– 2

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

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2 IA B G 1

. 8 ( 0

. 8 5) 1

. 5 ( 0

. 9 6) 0

. 5

– 2

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

NCBD 6

. 8 ( 2

. 6 9) 4

. 8 ( 1

. 2 5) 3

. 5

– 5

. 0 1

. 0 ( 0

. 7 1 ) † 1

. 5 ( 0

. 8 7) 0

. 3

– 1

. 0

NE YO 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

3 IA B G 0

. 3 ( 0

. 2 5 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 5 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 5

NCBD 4

. 5 ( 1

. 3 2) 5

. 0 ( 1

. 4 7) 3

. 3

– 5

. 8 0

. 5 ( 0

. 2 9 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 1

. 8

NE YO 0

. 5 ( 0

. 2 9 ) † 0

. 8 ( 0

. 4 8) 0

. 3

– 1

. 8 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 5

4 IA B G 0

. 3 ( 0

. 2 5 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 8 0

. 5 ( 0

. 2 9 ) † 0

. 5 ( 0

. 5 0) 0

. 3

– 0

. 8

NCBD 2

. 3 ( 1

. 4 4) 3

. 3 ( 2

. 2 9) 3

. 0

– 4

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 5

NE YO 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 0

– 0

. 3 Table I

- 10. (continued). Individual

- Site Analysis: Arthropod Abundance in Visual Count s fro m MO N 8741 1 Compare d t o the

Conventiona l Control

S p i d e r (Ara n eae ) Sti nk b ug (Pentat o m i d ae)

Be n e f icia l A r t h ro p o d Pes t A r th r op od

Mean ( S. E

. ) 3 Refer e nce Mean ( S. E

. ) 3 Refer e nce

C o ll

. 1 Sit e 2 M ON 8 7 41 1 C on tr ol ra n g e 4 M ON 8 7 41 1 C on tr ol ra n g e 4

1 IA B G

— — — — — —

NCB D 7

. 3 ( 1

. 6 0) 4

. 0 ( 1

. 0 8) 3

. 0

- 5

. 8 1

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 2 5) 0

. 3

– 1

. 8

NE Y O 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

PAHM 1

. 0 ( 0

. 5 8) 1

. 7 ( 0

. 8 8) 0

. 8

– 3

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 5

. 7

PAHM 1

. 7 ( 1

. 2 0) 0

. 3 ( 0

. 3 3) 1

. 0

– 2

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

PAHM 1

. 7 ( 0

. 3 3) 1

. 0 ( 0

. 5 8) 1

. 5

– 2

. 7 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 2

. 3

PAHM 2

. 0 ( 1

. 1 5) 0

. 3 ( 0

. 3 3) 1

. 3

– 2

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

5 IA B G 0

. 3 ( 0

. 2 5 ) † 1

. 0 ( 0

. 7 1) 0

. 3

– 0

. 8 0

. 5 ( 0

. 2 9 ) † 0

. 5 ( 0

. 2 9) 0

. 3

– 0

. 5

NCBD 3

. 8 ( 1

. 1 8) 2

. 3 ( 0

. 4 8) 1

. 5

– 5

. 3 0

. 3 ( 0

. 2 5 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 3

NE YO 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0 0

. 0 ( 0

. 0 0 ) † 0

. 0 ( 0

. 0 0) 0

. 0

– 0

. 0

PAHM 1

. 0 ( 0

. 0 0) 2

. 7 ( 1

. 4 5) 1

. 5

- 3

. 3 0

. 0 ( 0

. 0 0 ) † 0

. 3 ( 0

. 3 3) 0

. 0

– 0

. 0

Note: The experi me ntal design was a rando m ized c o m plete block with four rep l icates per site.

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* Indicates a statistically significant dif f erence between MON 87 4 11 and t he conventional c o ntrol ( α = 0.05) using A N OVA.

† Indicates p

- va lues could n ot be generated where the taxa did not meet inclusi o n criteria (see appendix I .1 0.2).

1 Arthropods were en u me r ated at five crop development stages: C ol l ection 1

= VT

- R1 growth stage; Collection 2

= R1 growth stage; Collection 3

= R2 growth stage; Collection 4

= R3 g r owth stage; Collection 5

= R4

- R5 growth stage.

2 Site code: IABG

= Greene, IA; NC B D

= Perqu i mans, NC; N E YO

= York, NE; PAHM

= Berks, PA.

3 MON 87411 and the conventional control values represent mea n s with standard error in parentheses. N

= 4.

4 Reference range is calculated from the min i m u m a n d m a x i m um mean values f rom a m ong reference hyb r ids at each si t e

. A dash (

— ) indicates data not available.

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References for Appendix I

SAS

. 2008

. SAS/STA T softwar e versio n 9.2

. SA S Institute, Inc., Cary, North Carolina.

SAS

. 2012

. SAS/STA T softwar e versio n 9.3

. SA S Institute, Inc., Cary, North Carolina.

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App e ndi x J : Material s an d Me t hod s fo r Polle n Morpholog y an d Viability

Assessment

J.1. Plant Production

MON 87411, the conventional control, and fou r referenc e hybrid s wer e grow n under

si m ilar agrono m ic conditions in a field t r ia l i n Stor y County

, Iowa

. Th e tria l was

arranged in a rando m ized co m pl e te block d e sign with four replications. Each plot

consisted of six rows approxi m ately 6 m in length.

J.2. Flo w er Collection and Pollen Sample Preparation

Tassel bags were placed on three non

- syste m ati c ally selected plants during pollen s h ed.

The following m o rning, pollen was collected from three plants per plot and transferred to

a uniquely labeled tu b e. Pollen collected from each plant in a plot represented a

subsa mple. W ithin approxi m ately 30 m inutes o f collection

, Alexander’ s stai n solution

(Alexander 1980), in a 1:5 dilution with distilled water, was added to each tube (at least

2:1 (v/v) stain to pollen) to fix and stain the poll e n, rendering the poll e n non

- viable

. The

tubes were closed and the contents sha k en until thoroughly m ixed. Subsa mples were

placed on wet ice within 30 m inutes of p o llen collecti o n and m aintained under those

conditions u ntil receipt at t h e perfor m ing laboratory.

J.3. Data Collection

Slides were prepared by aliquoting suspended pollen/stain solution onto a slide. Pollen

characteristics were assessed under an Oly mpu s  BX53 light m icroscope equipped with

an Oly mpu s © DP72 dig i tal color ca m era. The m icroscope and ca m era were connected to

a co mputer running Microsoft W indows X P  and installed with an Olympus © cellSens

(versio n 1.4.1 ) software.

J.3.1. Pollen Viability

When pollen grains were exposed to the stain i ng solution, viable poll e n grain s staine d red

to purple due to the presence of living cytop l as m ic content. Non

- viable pollen grains

stai n ed l i gh t b l u e to g reen o r c o lo rless, and the s h ape appeared ro u nd to collapsed

depending on the degree of hydration. For each pollen sa mple, the nu m ber of viable and

non

- viable pollen grains was counted from a random field of view under the m icroscope.

A m ini m u m of 100 pollen grains were cou n ted for each of the three su b sa m ples per plot.

Mean pollen viability for each replicate was ca l culated from the subsa m ples as sho w n in

Table VIII

- 8.

 O l y m pus Co r p ora tio n.

 W i ndow s X P i s a r e gister e d trad e m ark o f Mi cro s oft C o r p o ra tio n.

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J.3.2. Pollen Diameter

For a single predeter m ined subsa mple per plo t

, p ollen g rain d ia m eter was m easured along

two perpendicular axes for 10 representative pollen grains per replication. Mean pollen

dia m eter for each replicate was calc u lated from t he total of 2 0 dia m eter m easure m ents as

show n i n Tabl e VIII

- 8.

J.3.3. General Pollen Morphology

General pollen m orphology of MON 87411, the c onventiona l control

, an d th e reference

hybrids was observed as s hown in Figure J

- 1.

J.4. Statistical Analysis

An analysis of variance was conducted acc o r d i n g to a rand om ized co mp lete block d esign

SAS  (SAS 2012). The level of statistical s ignificanc e wa s predeter m i ne d t o b e 5%

( α =0.05). MON 87411 was co m p ared to the conventional control for percent viable

pollen and pollen grain dia m e t er

. MO N 8741 1 an d th e conv e ntiona l contro l wer e not

statistically co m pared to the reference hyb r ids. Mini m u m and m ax i m u m m ean values

were calculated for each characteristic from the four reference hybrids. General pollen

morphology was qualitative; the r e f ore, no statistical analy s i s wa s conducte d o n these

observations.

Table J

- 1. Starting Seed for Pollen Morphology and Viability Asses s ment

Material Pheno t y pe Monsanto Lot Nu m ber

MPA640B 1 Conventional Control 11333170

MON 87411 Glyphosate

- Tolerant and Insect

- Protected 11333176

Gateway 6158 Conventional Reference 11273006

Mycoge n 2M746 Conventiona l Reference 11226705

LG2597 Conventiona l Reference 11226862

Phillips 713 Conventiona l Reference 11300072

1 MP A 64 0B

= LH 2 4 4 × LH2 8 7.

 SA S i s a re g i stere d t r a d e m ar k o f SA S I n stit u te

, I n c.

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Figure J

- 1. General Morphology of Pollen from MON 87411, the Conventional

Control, and the Reference Hybrids under 200X Magnification

The m a ize pollen sa m ples were sta i ned with Alexander’s stain diluted 1 : 5 with distilled water.

Viable pollen grains stained red to pur p le, while non

- viable pollen grains stained blue to green

and the shape appeared round to co l lapsed depend i ng on the degree of h ydration.

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References for Appendix J

Alexander

, M.P

. 1980

. A versa t il e stai n fo r polle n fungi

, yeas t an d bacteria

. S t ain

Technology 55:13

- 18.

SAS

. 2012

. SAS/STA T softwar e versio n 9.3

. SA S Institute, Inc., Cary, North Carolina.

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App e ndi x K : Summar y o f Non

- targe t Organis m Stud i es

This appendix provides a summary of the studies conducted to assess the potential effects

of DvSnf7 RNA on non

- tar g et organis m s. All studies were conducted using in vi tr o

produced DvSnf7 RNA, referred to as DvSnf7_968 RNA. W ith the exception of the

earthworm study, all studies utilizing the in vit r o

- produced test substance included a diet

analy s is u s ing a se n sitive insect ( Diabrotica undecimpunctata howardi; Souther n corn

rootwor m ) to confi r m t hat the DvSnf7_968 RNA was biologically active and had the

expected level of biological activity in diet. Additionally, wher e appropriat e base d upon

the diet m atrix, the homogeneity of the t e st m aterial a n d sta b ility o ver t h e p e riod of

storage was also confirmed. A dose confir m ation was not appropriat e f or th e earthwo r m

stud y du e t o th e rapi d degradatio n o f RN A i n the soil m atrix (discussed in Section X.H).

K.1. Evaluatio n o f Potentia l Effect s o f DvSnf7_96 8 RN A t o th e Earth w or m Eisenia

andrei i n a n Acut e Exposur e Stud y i n an Artificial Soil Substrate.

The purpose of this study was to evaluate t h e potential effects of dietary and contact

exposur e o f DvSnf7_96 8 RN A o n adults of the earthworm Eisenia andrei (Oligoc h aeta:

Lu m bricidae) over a 14 day e xposur e period

. Fo r thi s study

, a dilution of DvSnf7_968

RNA was prepared in purified water and i n corporated into a stan d ard artifici a l soil

m edium following the standard m ethodology d e scribed in OECD guideline 207. The test

substanc e wa s evaluate d at a s ingle test co n centration and c o mpared with a wat e r

- tr ea ted

assay contr o l. Treat m ents were m ixed into an a r ti f icial soil in repli c ated 1 L glass jars

(te s t a r ena s /cha m bers). At initi a tion of the te s t, ten adult E

. andre i were p laced on the soil

surfac e o f eac h jar

. Eac h tre at m ent was replicated four ti m e s for a total of 40 earth w orms

exposed per treat m ent. All jars were m ai n tained in an incubator under a 24L: 0D

photoperiod at 20° C. The survival, bio m ass a nd behavior of the wor m s were assessed at

7 and 14 days a f ter c o mmence m ent of the bi o assay. In li e u of a positive control, the

sensitivity o f the E. andrei worm cultu r e used f o r this study was previo u sly con f ir m ed in

a separate testing at t h e T e st Facility, in accor d ance with the guideline, and cond u cted

within 12 months from the start of the s t udy in

- life phase. After 14 d a ys, no effect on

surviv a l (1 0 0% sur v ival in both tr e at m ents), was obser v ed with E

. andre i tre a ted with

DvSnf7_96 8 RN A a t a concentratio n equiv alent to 5000 ng RNA/g soil dry weight.

Additionally, the bio m ass of wo r m s exposed t o DvSnf7_96 8 RN A (

- 8. 4 ± 1.4 % change)

was not si g nificantly reduced co m p ared to the assay control (

- 9.4 ± 2.4 % change ) ( p >

0.05) and there were no adverse observed effec t s on wo r m behavior. Based on the results

of assessments f or m ort a lity, b e havior and change in bio m ass, the NOEC f or

DvSnf7_96 8 RN A wa s conclude d t o be 5000 ng RNA/g soil dry weight.

K.2. Eval u ation of the Potential Diet ar y Effect s o f DvSnf7_96 8 RN A o n Honeybee

Larvae ( Apis mellifera L.).

The purpose of this study was to evaluate t h e p otential d ietary effects of DvSnf7_968

RNA on the survival and develop m ent of honey bee ( Apis mellifera L

. ) l a rvae. Larvae

o f A. mellifera, 2 to 3 days old, were exposed to DvSnf7_968 RNA in a single dose

ad m inistered to the brood cell. A singl e dos e 100 0 ng/ g solution

, i n a 1 0 μ l a liq u ot of

30% (w/v) sucrose/purified water, was added to each larval cell at te s t initiation for a

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total m ass of 11.3 ng DvSnf7/cel l 12

. I n addition

, a n assa y contr ol treate d wit h 1 0 μ L of

30% (w/v) s ucrose/p u rified water was inclu d ed as well as a positi v e co n trol (2000 μg

potassium a r senat e / m l) in a 30% (w/v) sucr o se/ p uri f ied wat e r sol u tion to con f irm that the

test honey b ee larvae were feeding. For each treat m ent there were f our replicate fra m e s

with a target nu m ber of 20 larvae assessed p e r fra m e. Treat m ents were ad m inistered to

the cells and fra m es w e re returned to the hive s fo r incubation

. Treate d broo d cell s were

m apped in each treated fra m e and identified on acetate overlay m aps to in d icate the

study, hive, replicate, and treat m ent group nu m bers. Post

- capping and prior to e m ergence,

al l treate d f r a m e s wer e re m ove d fro m th e hives

, place d int o screene d hiv e boxe s i n a growth

cha m be r an d m aintaine d under a 0 L : 24D photoperiod at a p proxi m ately 27 to 30oC and

47.3 to 72.6% relative hu m idity ( R H). The endpoints m easured were survival at two

different life stages: dosing to cell

- capping (larval stage) and cell

- capping to test

ter m ination (pupal stage). There was 100% survival in both the DvSnf7_968 RNA and

assay co n tr o l tre a t m ents. Additio n ally, e m ergence was initiated in the te s t and contr o l o n

the sa m e day for the DvSnf7_968 RNA trea t m e n t an d th e assa y contro l (da y 14),

approxi m ately 50% e m ergence occurred on t h e sa m e day (day 15) for the DvSnf7_968

RNA treatment and the assay control and 100% e m ergence was achieved on the sa m e day

(da y 17 ) f or th e DvSnf7_96 8 RN A treat m e n t and the assay control. Behavioral

observations at e m ergence indicated no adver s e behavior or m orphological effects.

Survival f or the positi v e contr o l t r eat m ent was 0%, confir m ing the v a lidity of the test

syste m

. Based on no differences in survival and develop m ent between the test and

control treat m ents, t h e NO E C o f t h e D vS n f 7_ 9 68 RN A fo r hon e yb e e l arva e wa s ≥ 11.3

ng/larvae.

K.3. Eval u ation of the Potential Diet ar y Effect s o f DvSnf7_96 8 RN A o n Honeybee

Adults ( Apis mellifera L.).

The purpose of this study was to evaluate t h e p o tential d ietary effects of DvSnf7_968

RNA on the survival of honey bee ( A

pis mellifera L.) adults in 14

- day continuous feeding

study. Honey bee adults, <1 day post

- e m ergence, were exposed to three treat m ents that

included: 1000 ng/g DvSnf7_968 R NA in a 50% (w/v) sucrose/purified water solution,

an assay c o ntrol with 50% (w/v) s ucrose/p u ri fi ed water solution and a positi v e co n tr o l

(200 μ g potassium arsenate/ m l) in a 50% (w/ v ) sucros e /puri f ied water s olution to c o n f irm

that the test bees are feed i ng. For each of the three treat m ents there were four cages

(replicates) containing a target number 20 a dult bees p e r replicate. Each cage was

provided with approxi m ately 10 m l of appropri a te tre a t m ent diet s olution in a vial

inserted on the top of each ca g e. The vial was replaced every two days with fresh

treat m ent diets over the stud y duratio n o f 1 4 days

. Prio r t o test initiation, a dult bees w ere

starved for a m ax i m u m of two hours. Test bees for each treat m ent group were observed

daily for m o rtality, abnor m al behavior, and a ppearance. During the t e st period, adult bees

were m aintained under a 0L: 24D photoperiod, with the exception of the ti m e during

12 A single dose of 10 μ l of 1000 ng/g s olu t i on was a d ded to each larva l cel l fo r a tot a l m as s o f 11. 3 ng

D v S n f7 / ce ll

. T he c onc e n t ra t i on o f 1 00 0 ng / g D v S n f 7_ 9 6 8 R NA i n t he d i et so l u tio n i s c alc u l a t ed b a s e d on

t he d e ns it y o f t he 3 0 % s u cr o s e/wate r (w/v ) s o l u tio n of 1.12 70 g / m l

.

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which m ortality and behavior assess m ents were conducted. Environ m ental conditions

were m aintained at app r oxi m ately 26 to 32oC and 41 to 80% relative hu m idity (RH). The

positi v e co n trol p roduced 100% m o rtality, confi rm ing the vali d ity of the te s t syste m

. Though the study was designed to be a 14

- day exposure, the m or t ality in the assay

contro l exceede d 20 % o n da y 1 0 an d continue d t o inc r ease until t e st te r m ination at day

14. Therefore, day 10 survival data from th e DvSnf7_96 8 RN A treat m en t an d assay

control were analyzed. A 10

- day exposure is considered to be appropriate exposure

duration for a chronic feeding study for a dult honey bees (EFSA, 2013). There w ere no

significant differences (p > 0.05) in m ean survival between the DvSnf7_968 RNA (79%)

and the assay control (76%) treat m ents aft e r 10 days of continuous dietary exposure.

Mean survival on day 14 was not analyzed an d surviva l i n th e DvSnf7_96 8 RN A an d the

assay control was both 30%. The NOEC of the DvSnf7_968 RNA for adult honeybees

was ≥ 1000 ng/g.

K.4. Evaluation of Potential Dietary Ef f

ects of DvSnf7_968 RNA on the Lady B e etle,

Coleomegilla maculata (DeGeer) (Coleoptera: Coccinelli d ae).

The purpose of this study was to evaluate pote n tial dietary effec t s o f th e DvSnf7_968

RNA on t he survival, develop m e nt and growth of the lady beetle, Coleomegilla

maculat a

. The test substance dissolved in wat e r was incorporated i n to an a g ar based

artificial C. maculata diet at a single concentra t io n o f 100 0 n g DvSnf7_96 8 RNA / g of

diet. In addition, appropriate a s sa y contro l (purifie d water ) and a positive c ontrol (120 μg

potassium arsenate/g diet) were included in the study. Each treat m ent was replicated

thre e ti m es an d wit h a targe t o f 2 0 insect s p er die t treat m ent

. Larva e wer e expose d fo r a

m ax i m u m of 18 days. C. maculata larvae originating from the sa m e egg batch were used

for each replicate. All diet treat m ents were ma intained in an incubat o r with a te mperature

o f 27 ° C, a relative humidity ( R H) of 70 % and a 14L: 10D photoperiod. There were no

significant differences (p > 0.05 ) fo r surviva l betwee n th e DvSnf7_96 8 RN A (92% ) and

the assay c o ntrol (90%) tre a t m ents. Addition a lly, there were no significant differences (p

> 0.05) for the m ean percent that deve lope d t o adult s betwee n th e D v Snf7_96 8 RNA

(92%) and the assay control (90%) treat m e n ts. There was no significant difference for

develop m ent days to adult e m ergence between t h e larvae fed the test or control diet (p >

0.05). For the test and the assay control diet treat m ent groups the develop m ent ti m e to

adult e m ergence was 15 days. There was no significant difference with adult bio m ass

between the test and control d i et (p > 0.05). Adults that dev e loped from larvae fed test or

contr o l diet averaged 1 0 .2 m g for both tre a t m ents. Because of significant differences in

body m ass between m a les and females in C. maculata populations, separate analyses

were perfor m ed by gender. There was no sig n ificant difference between the bio m ass of

f

e m ales averaging 10.7 mg and 10.9 m g (p > 0.05) and m ales averaging 9.7 m g and 9.5

mg (p > 0.05) for test and control treat m en t s respectively. T he posi t ive control group had

17 % surviva l an d non e o f th e insect s i n th e positiv e contro l grou p develope d t o th e pupa

stage, confir m ing the validity of the test syste m

. The NOEC of the DvSnf7_968 RNA

for C. maculata wa s conclude d t o b e ≥ 1000 ng/g of diet.

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K.5. Eval u ation of Potential Dieta r y Effect s o f DvSnf7_96 8 RN A o n th e Carabid

ground beetle, Poecilus chalcites ( S ay) (Coleo p tera: Cara b idae)

The purpose of this study was to evaluate potential dietary toxic i t y o f th e DvSnf7_968

RNA against lar v ae of t h e cara b id beetle, Po e cil u s chalcite s

. The test su b stance d issolved

in wat e r w a s inc o rpor at ed i n to an a gar

- ba s ed ar ti f icial d i e t at a sin g le c oncent r ation of

1000 ng DvSnf7_968 RNA/g of di e t. In addition, a ppropriate an assay controls (purified

water) and a positive control (220 μg potassium arsenate/ g diet ) wer e include d i n the

study. Each treat m ent was replicated three t i m es and each replicate was initiated with a

target of 20 insects per diet treat m ent. Larvae were exposed for a m aximum of 35 day s

. All dietary exposures w ere initiated with fir s t instar larvae and lar v ae were individually

housed throughout the study. All diet treat m ents w e re m aintained in an incubator with a

te mperature of 27 ° C

, a R H o f 7 0 % an d a 14L : 10D photoperiod. The endpoints

m easured were survival, develop m e nt to the adult stage, and deve l opment ti m e (days) to

adult e m ergence, and adult bio m ass. There was no significant difference for survival

between the DvSnf7_968 RNA (93%) and the assay control ( 92%) treat m ents and all of

th e subletha l endpoint s ( p > 0.05)

. Fo r th e tes t o r contro l diets

, the percentage that

developed to the adult stage averaged 70% and 75%, respectively, adult bio m ass

averaged 31.9 m g and 32.3 m g, respective l y, and the develop m ent ti m e to adult

e m ergence averaged 33 days for both t r eat m ents. The positive control group had 65%,

survival and none of the insects in the positi v e contr o l gro u p developed to the pupal or

adult stage, confir m ing t he validity of the test s yste m

. T he NO E C of the D v S n f 7_968

RN A fo r P. chalcites wa s ≥ 100 0 ng/ g o f diet.

K.6. Eval u ation of Potential Dietary Effect s o f DvSnf7_96 8 RN A o n th e Insidious

Flo w er Bug, O riu s insidiosu s (Say) (Heteroptera: Anthocoridae)

The purpose of this study was to evaluate potential dietary ef f

ects of DvSnf7_968 RNA

on the survival and develop m ent of O. insidiosus ny m phs over 10 days of continuous

exposure following the m ethodology described in Tan et al., (2011).Three treat m ent diets

were prepared for separ a te feeding exposures to O. insidiosus ny m phs, including an assay

contro l diet

, a DvSnf7_96 8 RN A treat m ent diet at 1000 ng /g diet, and a positive control

diet (100 μ g potassiu m arsenate/ g diet)

. Al l dieta ry exposures were initiated with 5

- day

old ny mphs and each treat m ent con s isted of a t o tal of 40 n y mphs. All test ny mphs were

individually housed in test a r ena s an d supplie d wit h appro p riate treat m ent diet in two

do m es. The do m e diets were replaced every two days. All test ny mphs were allowed to

fee d ad libitum on the treated diet for 10 days. All d i et treat m ents were m aintained in an

incubator with a te m p e rature of 25 ± 5 ° C, a RH of 70 ± 10 % and a 16L: 8D

photoperiod. The test ny mphs w e r e observe d ever y da y to record m ortality and

develop m ent. Survival of the ny mp hs was 93% in both the assay control and DvSnf7_968

RNA treatment diets after 10 days of continuous feeding. Ny mph develop m ent to the

adul t stag e wa s no t significant l y differen t ( p > 0.05 ) a t 95 % and 98% in the assa y control

and DvSnf7_968 RNA treat m ent, respectively. The average develop m ent ti m e of the test

ny mphs was not significantly different (p > 0.05) between the control and test groups,

with m ean develop m ent ti m es of 11.11 ± 0.15 days in the assay control treat m ent and

10.87 ± 0.13 days in DvSnf7_968 RNA treat m ent. In contrast, the O. insidiosus nymphs

fed the positive control treat m ent diet had 100% mortality by day 10, indicating that the

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test system was effective in detecting toxic effects th roug h th e dietar y exposure

. The

NOE C o f th e DvSnf 7 RN A fo r th e insidiou s flowe r bug

, O. insidiosus

, was ≥ 1000 ng/g

diet.

K.7. Evaluation of Potential Dietary Effect s o f DvSnf7_96 8 RN A o n th e Parasitic

Wasp

, Pediobius foveolatus (H y m en optera: Eulophidae)

The purpose of this study was to evaluate potential dietary ef f

ects of DvSnf7_968 RNA

on the survival of adult parasitic wasp, Pediobius foveolatus Crawford over 20 days of

continuous exposure. Three trea t m en t diet s wer e prepare d fo r separate f eeding exposures

to P

. foveolatu s adults. An assay control diet was 30% honey/water ( v /v ) solution

, the

DvSnf7_968 RNA treat m ent diet prepared at 1000 ng/g in a 30% honey/water (v/v)

solution and a positive control (200 μg potassi u m arsenate/ g ) in a 30% honey/water (v/v)

solution. Exposure of P. foveolat u s adults to t h e three t r eat m ent diets was repli c ated f our

ti m es with 10 adult wa s ps per r epli c ate f or a total of 40 wasps per tre a t m ent. All dieta r y

exposures were initiated with newly e m erged adults after approximately 24 hours of

accli m ation. The adult wasps in each replicate w ere housed together in a single are n a and

allowed to f eed ad libitum on the treated diet for 20 days. All diet treat m ents were

m aintained in an incub a tor at a tar g e t te mp erature of 25° C, a t a rget RH of 70 % and a

16L: 8D photoperiod. Mortality was observed every two days at diet replace m ent and the

dead wasps, if any, were re m oved from the tes t arena s a t th e t i m e of observation. T here

was no m ortality of P. foveolat u s adults fed the DvSnf7_968 R NA treat m ent diet or the

assay control diet after 20 days. In contrast, the P. fove o latus ad u lts f ed the positi v e

contr o l treat m ent diet s h owed 100% m ortality at day 12, in d icating t h at the test system

was effective in det e cting toxic effects t h rough the dietary exposure. T he NOEC of the

DvSnf 7 RN A fo r th e parasiti c wasp

, P. foveol a t u s

, was ≥ 100 0 ng/ g diet.

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References for Appendix K

EFSA. 2013. EFSA Guidance Docu m ent on t h e risk as s ess m ent of plant pr o tection

products on bees ( Apis m ellifera

, Bombu s spp. and solitary bees). EFS A Journa l 11:266.

Tan, J., M. S

. Paradise, S .L. Levin, P.M. Bach m a n, J.P. Bach m an, J.P. Uff m an, C. Jiang,

D.B. Carson. 2011. Develop m ent and survival of Orius insidiosus ( S ay) ny mphs on

encapsulated bee polle n

- based diet in a Tier

- I t o xicity assay. Environ m e n tal Ento m o logy

40:1613

- 1621.

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App e ndi x L : Next

- G eneratio n Sequencin g an d Junctio n Sequen c e Analysi s for

th e Cha r acteri z atio n o f DN A Ins e rte d int o Cro p Plants

Background

Safety assess m ents of biotechnology

- der i ved crops include a detailed m olecular

characterization of the inserted DNA sequence and its location within the geno m e (C o dex

Ali m entarius 2009). Typically, molecular cha r acterization has relied on Southern blot

analysis to establish locus and copy nu m b er along with targeted sequencing of

poly m erase chain reaction products spanni n g an y inserte d DN A t o co m p let e the

characterization process. W ith the a dvent of next

- generation sequencing (Shendure and

Ji 2008; Zhang et al. 2011), i mprove m ents in sequencing technologies have enabled

alternative m ethods for m olecular character ization s whic h d o no t r e quir e Souther n blot

analysis. N ext

- Generation Sequencing and Junctio n Sequenc e Analysi s bioinfor m a tics

(NGS/JSA) utilizes sequencing (both nex t

- generation technolo g ie s an d traditional

m ethods) and bioinformatics to produce charact e rizations equivalent to those achieved by

current Southern blot based m ethods.

There are m ulti p le adv a ntages to u s ing next

- g e neration se q uencing and bioin f or m atics,

most notably the robustness, si mplicity and consistency of the m ethod co m pared with

Southern blot studies, which require c u sto m ized experi m ental design for every

transfor m ation event. The new s e quenc i ng

- based m ethod overco m es m any technical

challenges inherent in Souther n blo t analyse s ( e.g.

, f al s e positi v e hy b ridization b ands

resulting from inco m pl e te dig e stion or star activity ( W ei et al. 2008)) and th e nee d for

radioactive 32 P

- labeled probes. This new m e t ho d provide s highe r reproducibility,

because it is less de p e n dent on c o mplex lab b ased p roce d ures. The m ethod described

here is e s s e nti a lly ide n tical f or all trans f or m ation eve n ts and it ro b ustly e s tablishes

molecular characteristics of genetically engineered c rop s (Kovali c e t al

. 2012).

Additionally, si m ilar techniques are being used to character i ze trans g ene integr a tion sites

and insert molecular anato m y in m a mmalian syste m s (DuBos e e t al

. 2013 ; Zhan g e t al.

2012).

Method Synopsis

Molecular characteriz a tio n o f th e inserte d DN A an d assoc i ate d nativ e flankin g sequences

consists of a m u ltistep a pproach to d eter m ine:

1. the nu m ber of insertion sites;

2. the presence/absence plas m id backbone;

3. insert copy nu m ber at eac h insertio n site;

4. DN A sequ e nc e o f eac h inserte d D N A;

5. sequence of the native locus a t eac h insertio n site.

Additionally, current m e thods also establish a description of any genetic rearrange m ents

that m ay have occurred at the insertion site as a consequence of transfor m ation.

Generation a l stability a nalysis, which de m onstrates t h e s t able h erita b ility of i n s e rted

DNA sequ e nces over a n u m ber of b reeding ge nerations

, i s als o routinel y conducted.

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The first step of the m olecular characterization, deter m ination of nu m ber of insert sites, is

conducted using a co m bination of next

- ge n eration sequencing technologies (NGS) and

Junction Sequence Analysis (JSA) bioinfor m a tics (DuBose et al. 2013; Kovalic et al.

2012). A sche m atic representation of the bas i s of the characterizati o n, including the

NGS/JS A methodolog y an d th e dire cted sequencing, is present e d i n Figur e 1 (Kovali c et

al. 2012).

Geno m ic DNA from the transformation eve n t and the co n ventional c o ntrol are u s ed to

generate short (~100 bp) rando m ly distributed sequence frag m ents (s equencin g reads ) in

sufficient nu m bers to ensure co m prehensive coverage of the geno m es (Shendure and Ji

2008) (Figure 1 box 1)

. Sufficient nu m bers of sequence frag m ents are obtained (>75x

effective geno m e coverage) to comprehen s ively cover the geno m es of the sequenced

sa mples (Ajay et al. 2011; Cl ark e an d Carbo n 1976 ; W a n g e t al

. 2008)

. Previou s studies

with a variety of transfor m ation events de m on s trate that 75x coverage o f th e geno m e i s

adequate to provide comprehensive coverage and ensure detection of inserted DNA,

producing results equivalent to Southern blot analysis ( Kovali c e t al

. 2012)

. Th e 75x

coverage used in this method is predicted, based on established and accepted m ethods

(Clarke and Carbon 1976; Lander and W ater m an 1988), to provide geno m e coverage that

would be expected to not m iss a single bas e pair in co mp lex geno m e s (Kovalic et al.

2012). Further m ore, even with know biases in next

- generation s e quencing techniques,

including the Illu m ina sequencing by synth e sis m ethod e m ployed here (Minoche et al.

2011), it has previously been established experi m entally that given deep next

- generation

sequencing, it is possible to achieve co mpreh e nsive coverage of co mp l ex geno m es that

fo r m the foundation for acc u rate whole geno m e stu d ies ( A jay et al. 2 0 11; W ang et al.

2008).

To confirm sufficient sequence coverage in b o th the trans f o r m ation event and the c o ntrol,

the 100 bp sequence reads are analyzed to deter m ine the coverage of a known single

copy endogenous gene, this analysis de m onstrates coverage at >75x m e dian depth in each

sa mple. Further m ore, in order to confirm the m ethod’s ability to detec t an y sequences

derived from the transfo r m ation plas m id, plas m id DN A i s spike d int o conventional

control DNA at a single copy geno m e equival e nt ratio and 1/10 copy geno m e equ i valent

ratio

. Thi s analysi s de m onstrate s tha t an y portio n o f th e plas m id m ay b e detecte d a t a

single copy per geno m e level and 1/10 copy geno m e equivalent level, which is adequate

sensitivity to observe any inserted frag m ent.

Also of note is that although the m ethod pre s ente d her e provide s > 7 5 x coverag e o f the

geno m es under study, accurate asse m bly of co m plete geno m e s e quences for t h e

transfor m ation event and conve n tional control is not technic a lly possible using currently

available sequence assembly tools. This is due to the nature of the sequences generated in

this study, short reads of a single short insert length (Miller et al. 2010), in addition to

li m itation on available sequence asse m bly algo r ith m s (Zhang et al. 2011). The sequences

generated with this m ethod represent datasets sufficient for achieving precise m olecular

characterizatio n o f transfo r m ed DN A i n t ransfor m ation e v ents where reference to a

te mplate sequence (plas m id DNA) i s utilized for co m parison (Kovalic et al. 2012).

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Using bioinfor m atics tools, the sequence reads t h at are derived from the plas m id vector

are selected for further analysis o ut of the co m prehensive geno m ic sequence dataset

produced from the tra n sfo r m ation event. To deter m in e th e inser t number

, th e known

sequence o f the tra ns f o r m ation vector p la s m id is used as an “e

- probe” in the

bioinfor m atics analysis to search for and sel e ct the sequences that contain any portion of

sequence of the plas m id. The DNA sequencing reads with a m atch to the query sequence

havin g a n e

- valu e o f 1 × 1 0 − 5 o r les s an d havin g a m a tc h len gth of at l ea st 30 b ases with

at least 96.7% sequence identity are collect e d. The results of a par a m eter opti m ization

study that syste m atically evaluated m any dif f

erent potential para me ter sets established

these s e le c tion criteria as provi d i n g the best possible c o mbination o f sensiti v ity and

specificity.

Figur e L

- 1

. Sequencin g an d Sequ e nc e Selection

Geno m ic DNA from t he test and control m a terial were sequenced using Illu m ina

HiSeq/TruSeq technology (Illu m ina, Inc.) that produces large nu m bers of short sequence

reads approxi m ately 100 bp in length. Suffic i ent nu m bers of these sequence fragments

were obtai n ed to co m prehensively cover the g e no m es of each sa m ple at >75x average

coverage. U sing these geno m e sequence reads, bioinfor m atics search tools were used to

select all sequence rea d s that are s ignifica n tly si m ilar (as de f ined in the text) to the

transfor m ation plas m id. Only the selected sequence reads were used in further

bioin f or m atics an a lysis to det e r m ine the i n se r t numbe r b y detectin g an d characterizin g all

junction se q uences and the prese n ce or abs e nce of the plas m id backbone sequences by

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lack of detecta b le s e q uences, including the u se of suita b le co n tr o ls for experi m ental

co mprehensiveness and sensitivity.

The nu m ber of DNA in s erts is deter m ined by analyzing the selec t ed sequences for novel

junctions. The junctions of the DNA i n sert and flanking D N A are unique for each

insertion and an exa m ple is shown in Figure 2 below (Kovalic et al. 2012). Therefore,

insertion sites can be recognized by ana l yzing for sequence reads containing such

junctions. Each insertion will prod u ce two unique junction sequence classes

characteristic of the g e n o m ic l o cus, with one at the 5' end of the inse r t, in this ca s e n a m e d

Junction Sequence Class A (JSC

- A), and si m ilar l y one at the 3' end of the insert, J S C

- B

(as illustr a t e d in Figure 3 f rom (Kovalic e t al. 2012)). By evaluating the nu m ber and the

sequence s o f al l uniqu e junctio n classe s dete cted, the nu m ber of i n s er tion s it e s of the

plas m id sequence can be deter m ined. For a single insert, two junction sequence classes

are expecte d

, one each origi n ating from either e n d of the insert, both co n t aining portions

of T

- DNA and flanking sequence.

J u n c t i o n

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C G

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C GT G T

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C GT G TCG

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C GT G TC G GG

**ATC C**  **A T**  **G TA**  **G AT**  **T TC**  **C CG**  **G AC**  **A TG**  **A AG**  **C C**  **A**

A GA A CT A GGA AA C GA C GA C GT G TC G GG A T

P l a s m i d D N A F l a n k in g D N A

Figure L

- 2. Junctions and Junction Sequences

Depicted above are five exa m ple junction s e qu e nces fo r m at t e d and labeled to indicate the

plas m id/flanking DNA portions of the sequences and with the junction point indicated (plas m id

DNA is shown in bold, underlined text and flank DNA is shown in plain t e xt). Junctions are

detected by ex a m ining the NGS d a ta f or seque n ces having port i ons of plasmid sequences that

span less than the full read. Detected junctions are typically characterist i c of plas m id insert i ons

in the geno me. A group of junction sequences which share the same junction point and co mm o n

flanking sequence (as shown above) is c a lled a Junc t ion Sequence Class (or J S C).

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J un c t i o n S e q u e n c e s : C l a s s A I ns e r t J u n c t i o n Re g i on s



## 5 ’ F la n k D N A in s e r t 3 ’ F l a n k

J un c t i o n S e qu e n c e s : Cl a s s B

Figur e L

- 3

. T w o Uniqu e Junctio n Sequen ce Classes are Produced by the Insertion

of a Single Plasmid Region

A sche m at i c representati o n of a single DNA insert i on within the genome showing the inserted

DNA, the 5 ' and 3' flanks (depicted as areas bound e d by dotted lin e s ), and the t wo distinct regions

spanning t he junctions be tw een inserted DNA and flanking DNA (shaded boxes). The group of

~100

- me r sequences in which each read contains sequences fr o m both the DNA insert and the

adjacent fla n king DNA at a given junction is called a Junction Sequence Cl a ss. In this ex a m ple,

two distinct junction sequence cla s ses (in this case: Class A at t h e 5’ end and Class B at the 3’

end) are repr e sented.

The next s t ep in t h e molecular c hara c te r iz a tion is d e te r m ination of the i n se r t copy

nu m ber, integrity of the insert, lack of backbone or oth e r unintende d plas m id sequences,

an d flankin g sequenc e o f th e nat i v e locu s a t th e insertio n site

. Thi s analysi s i s conducted

using directed sequencing, locus

- specific PC R an d DN A s e quencin g analyses

, which

co mple m en t s the NGS/ J SA analyse s

, and is common to both the Sout h ern

- based and the

NGS/JSA c haracterization m ethods. Directed sequencing (locus

- specifi c PC R an d DNA

sequencing analyses) of the t r ans f o r m ation event det e r m ines the co mplete seque n ce of

the i n se r t a nd f lanks. This deter m ines if t h e s equence of the i n se r t is identic a l to the

correspondin g sequenc e i n plas m id vector

, i f eac h genetic ele m ent in t h e inser t i s intact,

if the plasmid vector seque n ce is inserted as a single copy, and es tablishe s n o vector

backbone or other unintended plas m id sequ e nces were inserted in the event. This

co mparison allows a deter m ination of whe t her the T

- DNA ele m ents are present in the

intended order. Furthermore, the geno m ic organ i zation at t h e inse r tion s ite is a sses s e d by

co mparing the insert and flanking sequence to the seque n ce of the insertion site in

conventional control geno m e.

Finally

, th e stabilit y o f t h e T

- DNA across m ultiple generati o ns is evaluated by NGS/JSA

analyses. Geno m ic DNA from multiple gen e rations of the transfor m ation event is

assayed for the nu m ber and sequences of all un i que junction classes, as described above.

This in f ormation is us e d to det e r m ine the nu m ber and id e ntity of ins e rtion s it e s. For a

single insert, two junction sequence classes are expected, both c ontainin g portion s of

T

- DN A an d flankin g sequenc e (Figur e 2)

, wit h one each originating fr o m either end of

the in s ert ( F igure 3). In the ca s e of an eve n t where a s ingle locus is s tably inherited o ver

multiple g e nerations, two identic a l jun c tion s equence c l a sses a re e x pected in al l the

generations tested.

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