application for general release of ... for general...non confidential version 1.6 august 2013 page 2...

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Version 1.6 August 2013

APPLICATION FOR GENERAL RELEASE OF GENETICALLY MODIFIED ORGANISMS (GMOs) IN

SOUTH AFRICA: TC1507xNK603 MAIZE

Submitted by:

DuPont Pioneer (Pioneer Hi-Bred International, Inc) 7100 NW 62nd Avenue

P.O. Box 1014 Johnston, IA 50131-1014

USA

As represented by:

DuPont Pioneer (Pioneer Hi - Bred RSA (Pty) Ltd.) P.O Box 8010

Centurion, 0046 Republic of South Africa (RSA)

January 2014

The information presented in this application is limited to the purpose of this application in accordance with the

GMO Act 1997, (Act No. 15 of 1997). The information contained in this application may not be published or used by any third parties for other purposes without the prior consent of Pioneer Hi-Bred

International, Inc.

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APPLICATION FOR GENERAL RELEASE OF GENETICALLY MODIFIED ORGANISMS (GMOs) IN SOUTH AFRICA

This application template is primarily intended for applications dealing with

genetically modified (GM) plants

Applicants are advised to review guidelines available on the Department of Agriculture, Forestry and Fisheries (DAFF) website (www.daff.gov.za)

to assist in the completion of the application

PART I 1. APPLICANT

1.1 Name of applicant The application is submitted by: Pioneer Hi-Bred International, Inc., (DuPont Pioneer) as represented by Pioneer Hi-Bred RSA (Pty) Ltd. 1.2 Address of applicant Pioneer Hi-Bred International, Inc, hereafter referred to as DuPont Pioneer 7100 NW 62nd Avenue P.O. Box 1014 Johnston, IA 50131-1014 USA

DIRECTORATE BIOSAFETY Private Bag X973, Pretoria, 0001

Harvest House Room 167, 30 Hamilton Street, Arcadia, Pretoria, 0002

Tel: 012 319 6382, Fax: 012 319 6298, E-mail: [email protected]

http://www.daff.gov.za/

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2. BRIEF DESCRIPTION OF THE GM PLANT/PRODUCT

Provide a brief description of the plant, the intended function(s) of the genetic

modification(s), and the GM trait(s) of the plant.

TC1507xNK603 maize was obtained by traditional breeding methods between progeny

of the genetically modified maize DAS-Ø15Ø7-1 maize, referred to as TC1507 maize;

and MON-ØØ6Ø3-6 maize, referred to as NK603 maize. As a consequence, the inserts

from these two maize events are not genetically linked and no new genetic modification

has been introduced in the stacked product TC1507xNK603 maize.

TC1507 maize has been genetically modified to express the Cry1F protein and the PAT

protein. Expression of the Cry1F protein confers protection against certain lepidopteran

pests. Expression of the PAT protein, used as a selectable marker in the development

of TC1507 maize, confers tolerance to glufosinate-ammonium herbicide.

NK603 maize has been genetically modified to express the

5-enolpyruvylshikimate-3-phosphate synthase protein from the Agrobacterium sp. strain

CP4 (CP4 EPSPS), conferring tolerance to glyphosate herbicide.

TC1507xNK603 maize therefore has the following agronomic traits:

herbicide tolerance to glufosinate-ammonium and glyphosate herbicides due to

the presence of the PAT and CP4 EPSPS proteins;

insect protection against certain lepidopteran target pests due to the presence of

Cry1F protein.

TC1507 maize and NK603 maize have been deregulated and are authorized for import

and use in food and feed, as well as for general release in South Africa. NK603 was

granted a general release permit in South Africa in 2002 and TC1507 in July 2012. The

event under review, TC1507xNK603, was approved for food and feed use, direct use or

processing, in South Africa in 2011.

In accordance with the OECD guidance for the designation of a unique identifier for

transgenic plants (OECD, 2006), the unique identification code assigned to

TC1507xNK603 maize is DAS-Ø15Ø7-1xMON-ØØ6Ø3-6.

TC1507xNK603 maize is currently available in the food and feed chain due to the

commodity clearance approval granted in South Africa in 2011. This general release

application is intended to allow local seed production for the event. The seed produced

from TC1507xNK603 maize will primarily be used in the production of the three-way

stack TC1507xMON810xNK603 (the principal commercial product). DuPont Pioneer is

therefore applying for a general release of TC1507xNK603, and this will include use in

local hybrid seed production.

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3. CHARACTERISTICS OF THE HOST OR UNMODIFIED RECIPIENT

ORGANISM

3.1 Specific and common names of the recipient or parental organism

or plant

Family name: Poaceae (Gramineae)

Genus: Zea

Species: Zea mays L.

Common name: Maize; corn

3.2 Natural habitat, geographic distribution, geographic origin, and

centres for diversity

Maize originated in Mexico and Guatemala. Maize is grown in South Africa over

a wide range of climatic conditions. However, survival and reproduction of maize

is limited by cool conditions.

The majority of maize is produced between latitudes of 30 and 55 degrees, with

relatively little grown at latitudes higher than 47 degrees anywhere in the world.

The greatest maize production occurs where the warmest month isotherms range

between 21 and 27°C and the freeze-free season lasts 120 to 180 days.

Summer rainfall of 15 cm is the lower limit for maize production without irrigation.

There is no upper limit of rainfall for growing maize, although excess rainfall will

decrease yields.

Maize is a weak competitor outside cultivated fields and does not present weedy

characteristics (CFIA, 1994). In managed ecosystems, maize does not

effectively compete with other cultivated plants or primary colonizers (Alexander,

1988; CFIA, 1994; Del Valle et al., 1983; Raynor et al., 1972; Shaw, 1988).

Maize volunteers are rare and easily controlled by current agronomic practices

including manual or mechanical removal, selective use of herbicides and crop

rotation.

3.3 Reproduction:

3.3.1 Provide detailed information on the mode(s) of reproduction.

Maize (Zea mays L.) is the only cultivated species included in the genus Zea, of

the family Poaceae. It is a highly domesticated agricultural crop with well-

characterised phenotypic and genetic traits. Maize reproduces sexually by wind-

pollination and being a monoecious species, it has separate male staminate

(tassels) and female pistillate (silk) flowers. This gives natural out-crossing

between maize plants but it also enables the control of pollination in the

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production of hybrid seed. Typical of wind-pollinated plants, a large amount of

excess maize pollen is produced for each successful fertilisation of an ovule on

the ear. Wind movements across the maize field cause pollen from the tassel to

fall on the silks of the same or adjoining plants. Studies by various researchers

comparing wind-pollinated species have indicated that maize pollen grains are

relatively large (an average diameter of 90 - 100µm) and heavy (0.25 µg), settle

to the ground rapidly, have a short dispersal distance and therefore, a limited

spatial distribution (Aylor et al., 2003; Jarosz et al., 2005; Luna et al., 2001; Perry

et al., 2012; Pleasants et al., 2001; Sears et al., 2001).

3.3.2 Provide information on specific factors affecting

reproduction.

As a wind-pollinated, monoecious grass species, self-pollination and fertilization,

and cross-pollination and fertilization, are usually possible and frequencies of

each are normally determined by proximity and other physical influences on

pollen dispersal. Tasselling, silking, and pollination are the most critical stages of

maize development, and grain yield is greatly impacted by moisture and

environmental stress.

3.3.3 For pollen spread, identify pollinating agents and the

distances to which pollen is known to spread.

Maize dissemination occurs via pollen or seeds. Maize has been domesticated

for thousands of years, and as a result maize dispersal of individual kernels does

not occur naturally. Wind is the primary pollinating agent for maize. Pollen

shedding from the tassels takes place over a period of 10 to 15 days. Pollen

grains are large, heavy and contain a high percentage of water, characteristics

that limit their dispersal and attachment to plant surfaces, such as leaves.

Generally, viability of shed pollen is 10 to 30 minutes, although it can remain

viable for longer times under favourable conditions (Aylor, 2004; CFIA, 1994;

Luna et al., 2001). However, dispersal of maize pollen tends to be limited as it is

influenced by the large size and limited movement from an originating field. Over

the past decade, significant work has been conducted to characterize the

distance that maize pollen grains can travel from an originating field (e.g.,

Pleasants et al., 2001; Sears et al., 2001). Numerous studies show the majority

(84-92%) of pollen grains travel less than 5 m from the source (e.g., Pleasants et

al., 2001; Sears et al., 2001); father travelling pollen grains are fewer in number

and are therefore less likely to be encountered compared to other pollens or

other food sources (e.g. fungal spores, nectar, etc.).

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3.3.4 Provide information on the generation time.

Maize is an annual crop with a cultural cycle ranging from as short as 10 weeks

to as long as 48 weeks covering the period of seedling emergence to maturity

(OECD, 2003; Shaw, 1988). This variance in maturity allows maize to be grown

over a range of climatic conditions.

3.4 Sexually compatible species:

3.4.1 Provide information on cultivated species, their distribution,

and proximity to general release areas.

The Meso-America region (Mexico, Central American and South America) is the

center of origin for maize and remains the only in situ source of land races (i.e.,

open pollinated varieties that gave rise to modern maize varieties (OECD, 2003).

The closest wild relative of domesticated maize is teosinte. Teosinte varieties

are native to a region extending from northern Mexico to western Nicaragua but

are not present in South Africa. Therefore, there is no risk of gene flow occurring

between cultivated maize and wild relative populations in South Africa.

3.4.2 Give details of wild species and their distribution and

proximity to general release areas.

No wild or weedy relatives of maize are present in South Africa and cross-

pollination with wild type plants will not occur. Maize is not indigenous to South

Africa, but is originally a plant from Central America. Maize depends on man for

its geographical dispersal. Cultivated maize (Zea mays ssp. mays) belongs to

the genus Zea, which includes several other wild species, collectively known as

teosintes. The closest species related to maize is teosinte (Zea mays spp

mexicana), a wild grass found in Mexico and Guatemala. No sexually compatible

wild relatives of maize are found in South Africa.

3.4.3 Identify any plants in the area of general release that may

become cross-pollinated with the host plant.

There is no risk of gene flow occurring between cultivated maize and wild relative

populations in South Africa, because no sexually compatible wild relatives of

maize are found in South Africa. Maize has only one related wild species,

teosinte, which grows only in Mexico and Guatemala.

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3.5 Survivability in the environment:

3.5.1 Provide details on structures produced by the plant for

survival or dormancy.

During the domestication of maize, maize has lost the ability to survive outside

managed agricultural environments. Maize is a non-dormant annual crop and

this lack of dormancy prevents maize seed from readily surviving from one

growing season to the next. Natural regeneration of maize from vegetative tissue

is not known to occur.

3.5.2 Provide information on specific factors affecting survivability.

Maize seed can only survive under favorable climatic conditions. Freezing

temperatures have an adverse effect on maize seed germination and they have

been identified as a major risk in limiting production of maize seed (OECD, 2003;

Shaw, 1988; Wych, 1988). Furthermore, maize is a C4 plant and therefore its

vegetative growth is sensitive to low temperatures. Chlorosis will occur at

temperatures below 15 °C. Temperatures above 45 °C also damage the viability

of maize seed (Craig, 1977).

3.6 Dissemination in the environment:

3.6.1 Provide details on how the plant may disseminate in the

environment

Maize dissemination occurs via kernel (seed/grain) and pollen. Without humans

or animals to aid seed dispersion, kernel movement is limited to within a few feet

of the plant (Abendroth et al., 2011; Fedoroff, 2003). Seed dispersal is generally

limited to cultivated fields. In fact, the inherent properties of maize, the existence

of husks that enclose the ear and the insertion of the individual kernels on the

cob (stiff central spike), reduce the possibility for natural dispersal of seeds.

Survival of maize seed is greatly limited by its sensitivity to diseases and cold.

Because of its highly domesticated nature, maize seed requires the semi-uniform

soil conditions resulting from cultivation in order to germinate and establish in

agricultural habitats (CFIA, 1994). Maize is not an invasive plant because it is a

weak competitor outside the cultivated fields.

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3.6.2 Provide information on specific factors affecting

dissemination.

Mechanical harvesting and transport are ways of disseminating grain and insect

or wind damage may cause mature ears to fall to the ground and prevent their

harvest. Regardless of these routes of dissemination, maize cannot survive

without human assistance.

3.7 Provide information on how the plant is usually utilised in

agriculture, forestry, medicine, etc.

Maize crops are used either as forage or to produce grain. Maize is the most

important grain crop in South Africa, being both the major feed grain and the

staple food for the majority of the South African population. The yellow maize is

mostly used for animal feed production while the white maize is primarily for

human consumption in South Africa. The maize industry is important to the

economy both as an employer and earner of foreign currency because of its

multiplier effects. This is because maize also serves as a raw material for

manufactured products such as paper, paint, textiles, medicine and food (DAFF,

2011).

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4. GENERAL RELEASE

4.1 When will general release be implemented?

The general release of TC1507xNK603 maize may be implemented as soon as

approval has been granted by the South African authorities.

4.2 Where will general release take place?

General release of TC1507xNK603 maize is expected to take place at the same

locations as any other commercial maize in South Africa.

4.3 Detail the type of environment and the geographical areas for which

the plant is suited.

Maize is grown throughout South Africa. However, survival and reproduction in

maize is limited by cool conditions. As mentioned in section 4.2 above,

TC1507xNK603 maize can be grown in the same area as any other commercial

maize. It is of a particular interest in the areas where the target lepidopteran

insect pests are present.

Maize is a weak competitor outside cultivated fields and does not present weedy

characteristics (CFIA, 1994). In addition, the genetic modification in

TC1507xNK603 maize does not confer selective advantage outside managed

agricultural environments.

In managed ecosystems, maize does not effectively compete with other

cultivated plants or primary colonizers (Alexander, 1988; CFIA, 1994; Del Valle,

1983; Raynor et al., 1972; Shaw, 1988).

4.4 Who will undertake the general release?

The general release will be undertaken by DuPont Pioneer, and its affiliated

companies.

4.5 Estimate the amount of production of the GM plant within South

Africa per annum, or the amount of viable plant product to be

imported into South Africa per annum.

Approximately 16 million hectares of biotech maize (white and yellow) was

planted in South Africa for the period 2000 to 2012, producing a grain crop of

over 40 million metric tons including 2012 harvests (James, 2012). For the

2012/13 season only, a total of 2.83 million commercial hectares of maize was

planted in South Africa – white and yellow maize constituted 58% (1.64 million

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hectares) and 42% (1.19 million hectares), respectively. The total maize

plantings included 86% or 2,428 million hectares of biotech maize; of which

49.3% (1.197 million hectares) was stacked Bt and herbicide tolerant genes

(James, 2012).

During 2012, South Africa’s total imports of cereals increased by 22% from R8.6

billion in 2011 to R10.6 billion. Much of the increase was due to maize imports

which increased significantly during this period (DAFF, 2012a).

TC1507xNK603 maize is expected to be part of these imports and local maize

production.

4.6 Give a description of the intended use of the GMO and / or derived

product. Indicate if the derived products are for food / feed or

industrial use.

This application is for general release of TC1507xNK603 maize in South Africa.

TC1507xNK603 maize will be used in the same manner as any other commercial

maize products.

4.7 Identify the parts of the plant to be used for the product, the type of

product, and the use of the product as well as the market sector in

which the product may be marketed.

TC1507xNK603 maize has been obtained by traditional breeding methods

between progeny of the genetically modified TC1507 and NK603 maize. No new

genetic modification has been performed to obtain TC1507xNK603 maize and

the genetic modifications in TC1507 and NK603 maize do not impact the

production processes used for maize. TC1507xNK603 maize will be used in a

manner consistent with current uses of commercial maize grain and maize

products.

White maize is being consumed as “pap” for the major part of the population in

South Africa while yellow maize is mainly cultivated for animal consumption

(DAFF, 2012b; Keetch, et al., 2005). Maize is also being processed into food

products such as highly refined starch by the wet-milling process and maize flour

by the dry-milling process (OECD, 2002). The majority of the starch is used for

sweeteners and fermentation including high fructose maize syrup and ethanol. In

addition to milling, the maize germ can be processed to obtain maize oil.

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4.8 Provide information on the proposed labelling of the product for

marketing.

In accordance with the currently approved Consumer Protection Act Regulations

Section 22 (7/5), TC1507xNK603 maize products will be labeled as “Produced

using genetic modification”. DuPont Pioneer will ensure that all TC1507xNK603

maize seed bags will be labeled with the commercial name of the product/GMO,

and the name and address of the entity responsible for placing the product on the

market in line with applicable law.

4.9 State whether the benefits of the product are available in any other

non-GM form. If so, state why the GM form should be approved for

general release when other, non-modified products are available.

There are no other non-genetically modified maize products available with the

same benefits as those provided by TC1507xNK603 maize. When cultivated,

TC1507xNK603 maize provides protection against insect damage by some

lepidopteran pests as well as tolerance to glufosinate-ammonium and glyphosate

herbicides.

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5. BRIEF SUMMARY OF FIELD TRIALS UNDERTAKEN

5.1 Submit a list of previously authorised activities undertaken by the

applicant with the GMO in:

(a) South Africa

In South Africa, Pioneer has conducted studies to evaluate the impact of

cultivation of TC1507xNK603 maize on key non-target arthropod populations.

(b) Other countries.

TC1507xNK603 maize has been approved for food and feed use in more than 10

countries globally and for the environment in Japan, Canada, USA, Argentina

and Brazil.

(c) Include information on the country, year, location and the

authority from which permission was obtained to run the field

trials.

Country: South Africa

Year: 2011 and 2012

Authority: GMO Registrar/Executive Council of GMOs

5.2 Provide a scientific summary on the field performance of the GM

plant, including a scientific explanation of the efficacy of the

introduced trait for each of the previously authorised activities listed

in 5.1.

5.2.1 South African Trials

CBI Deleted.

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6. INSERTED NUCLEIC ACID SEQUENCES AND THE GM ORGANISM OR

PLANT

6.1 Provide a description of the methods used for genetic modification.

TC1507 and NK603 maize, the parental lines used for the breeding of

TC1507xNK603 maize were obtained by the particle acceleration method.


between progeny of the genetically modified TC1507 and NK603 maize. No

other new genetic modification has been introduced in TC1507xNK603 maize.

The TC1507 maize was modified by the insertion of a synthetic truncated cry1F

gene from Bacillus thuringiensis var. aizawai and a gene for phosphinothricin

acetyltransferase (pat) from Streptomyces viridochromogenes.

Cry1F protein produced from the cry1F gene confers protection against certain

lepidopteran corn pests. PAT protein produced from the pat gene confers the

tolerance to glufosinate herbicide to the plant by detoxifying the herbicide into an

inactive acetylated compound, N-acetyl-L-phosphinothricin.

The NK603 maize was modified by the insertion of cp4 epsps gene from

Agrobacterium tumefaciens strain CP4 (2). The CP4 EPSPS protein encoded by

cp4 epsps gene confers tolerance to glyphosate herbicide.

6.2 Describe the nature and source of the vector used. Provide

information on the potential for mobilisation or transfer of the vector

to other organisms.

TC1507xNK603 maize has been obtained by traditional breeding methods by

crossing TC1507 and NK603 parental GM lines. The individual lines, TC1507

and NK603, were developed respectively with the use of a DNA fragment or a

plasmid, as indicated above. No vector was used in the transformation to obtain

TC1507 and NK603 maize.

The genetic stability of the inserted DNA was demonstrated in TC1507 and

NK603 maize as well as during the traditional breeding process as confirmed by

the stable inheritance and expression of proteins in the stacked TC1507xNK603

maize.

On the basis of current scientific evidence the transfer of genetic material

originating from GM plants to bacteria is a negligible concern. There is no known

mechanism for, or definitive demonstration of, DNA transfer from plants to

microbes under natural conditions (DuPont Pioneer, 2011; EFSA, 2004; EFSA,

2006). Even if such gene transfer would occur, i.e. primarily through homologous

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recombination, the eukaryotic promoters used to drive expression of the

transgenes in the maize plants would show a limited, if any, activity in bacteria.

In addition and as discussed throughout this application, the inserted genes

expressed in TC1507xNK603 maize would not pose any risk to human and

animal health or the environment if expressed in bacteria. In fact, all of the

transgenes in TC1507xNK603 maize are derived from bacteria (Hérouet et al.,

2005; Schnepf et al., 1998).

6.3 Provide detailed information on the vector construct and the region

intended for insertion, including the source of donor DNA and the

size and intended function of each constituent fragment of the

region intended for insertion.

As indicated under section 6.1 above, TC1507xNK603 maize was obtained by

traditional breeding and no vector was used in the transformation to obtain

TC1507 and NK603 maize. Instead, the intended insert in TC1507 maize

consisted of linear DNA fragment PHI8999A containing the cry1F and pat coding

sequences, excised from the small recombinant plasmid PHP8999 by digestion

with Pme I. A linear Mlu I DNA fragment, PV-ZMGT32L containing cp4 epsps

gene, isolated from PV-ZMGT32 plasmid, was used in the transformation to

obtain NK603 maize.

As previously mentioned in sections 6.1 and 6.2, no vector was used in the

transformation to obtain TC1507 maize. The purified linear DNA fragment used

in the transformation that resulted in TC1507 maize, termed insert PHI8999A and

containing the cry1F and pat gene coding sequences and the necessary

regulatory components, was obtained from plasmid PHP8999 following digestion

of the plasmid DNA with the restriction enzyme Pme I.

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Classification and taxonomy of the donor organisms and their history of

use:

Agrobacterium tumefaciens

TAXONOMY:

Class: α-Proteobacteria

Family: Rhizobiaceae

Genus: Agrobacterium

Species: A. tumefaciens

Arabidopsis thaliana

TAXONOMY:

Class: Magnoliopsida

Order: Capparales

Family: Brassicaceae

Genus: Arabidopsis

Species: A. thaliana

Bacillus thuringiensis

TAXONOMY:

Class: Bacillus/Clostridium group

Family: Bacillaceae

Genus: Bacillus

Species: B. thuringiensis

B. thuringiensis subsp. aizawai is the donor of the cry1F gene.

Cauliflower mosaic virus

TAXONOMY:

Family: Caulimoviridae

Genus: Caulimovirus

Species: Cauliflower Mosaic Virus

Escherichia coli

TAXONOMY:

Class: γ-Proteobacteria

Family: Enterobacteriaceae

Genus: Escherichia

Species: E. coli

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Oryza sativa

TAXONOMY:

Class: Liliopsida (Monocotyledones)

Order: Cyperales

Family: Poaceae (Gramineae)

Genus: Oryza

Species: O. sativa

Streptomyces viridochromogenes

TAXONOMY:

Class: Actinobacteria

Family: Streptomycetaceae

Genus: Streptomyces

Species: S. viridochromogenes

Strain: Tü494

Zea mays L.

TAXONOMY:

Family: Poaceae (Gramineae)

Genus: Zea

Species: Z. mays L.

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6.4 Provide information on the sequences actually inserted or deleted in

the GM plant:

TC1507 maize

TC1507 maize contains an almost full-length copy of the DNA insert used in the

transformation (i.e., 6186 bp of the 6235 bp fragment PHI8999A) without internal

rearrangements. The insert used in the transformation of TC1507 maize (insert

PHI8999A) contained the plant optimised coding sequences for the cry1F and

pat genes, together with the necessary regulatory components to drive their

expression. The cry1F gene (1.8 kb; origin: Bacillus thuringiensis subsp.

aizawai) was under the control of the ubiquitin promoter ubiZM1(2) (1.9 kb;

origin: Zea mays) and the ORF25PolyA terminator (0.7 kb; origin: Agrobacterium

tumefaciens pTi15995). The intended function of the cry1F gene was to confer

protection against certain lepidopteran insect pests.

The pat gene (0.5 kb; origin: Streptomyces viridochromogenes strain Tü494) was

under the control of the CaMV35S promoter and terminator (0.5 and 0.2 kb,

respectively; origin: cauliflower mosaic virus). The intended function of the pat

gene was to confer tolerance to the application of glufosinate-ammonium

herbicides.

Both cry1F and pat gene cassettes are intact within the transgenic event and the

DNA sequences of the genes are identical to those in the original plasmid as

revealed by the Southern blot data analysis.

NK603 maize

The insert used in the transformation of NK603 maize (insert PV-ZMGT32L)

contained two copies of the cp4 epsps gene (6.7 kb; origin: Agrobacterium sp.

strain CP4). One of the copies of the cp4 epsps gene was under the control of

the rice actin 1 gene intron (1.4 kb; origin: Oryza sativa); the chloroplast transit

peptide of the epsps gene (0.2 kb, origin: Arabidopsis thaliana); and, the

terminator of the nopaline synthase gene (0.4 kb; origin: Agrobacterium

tumefaciens). The second copy of the cp4 epsps gene was under the control of

the e35S promoter with a duplicated enhancer region (0.6 kb; origin: cauliflower

mosaic virus); the hsp70 gene intron (0.8 kb; origin: Zea mays); the chloroplast

transit peptide of the epsps gene (0.2 kb, origin: Arabidopsis thaliana); and, the

terminator of the nopaline synthase gene (0.4 kb; origin: Agrobacterium

tumefaciens). The intended function of the cp4 epsps gene was to confer

tolerance to the application of glyphosate herbicide.

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6.4.1 The copy number of all detectable inserts, both complete and

partial.

In order to assess stability of the individual events stacked in TC1507xNK603

maize, a detailed molecular analysis has been conducted to confirm that the

copy number, structure and organization of the respective individual inserts in

TC1507xNK603 maize are equivalent to those in TC1507 and NK603 maize.

Summary of Southern blot analysis data

Molecular characterization study, using Southern blot analysis, was conducted in

order to characterize and compare the inserted DNA in the stacked maize

TC1507xNK603 to the individual transgenic lines, TC1507 and NK603. The

study also verified the molecular equivalence of the inserted DNA within a single

generation of plants.

Confirmation of TC1507 and NK603 individual Events

The presence of TC1507 maize was confirmed by the expression of Cry1F and

PAT proteins in all germinating TC1507 hybrid plants but not in NK603 hybrid

plants. All but one of the TC1507xNK603 stacked hybrid plants (plant ID: 02-

114C-15) tested positive for Cry1F and all tested positive for PAT expression.

These results were confirmed by Southern analysis. All controls tested negative

for both Cry1F and PAT assays.

The test plants identified to contain the NK603 event were confirmed for

presence of the event with the 35S promoter probe. The plants containing event

NK603, when analyzed by Southern analysis, exhibited the expected

hybridization pattern. An analysis of DNA extracts digested with EcoR V and

hybridized with probe homologous to the cp4 epsps gene showed that the DNA

insertion in the TC1507xNK603 stacked hybrid was consistent with the inserted

cp4 epsps genes in the NK603 hybrid. In addition, the inserted genes in the

stacked hybrid of TC1507xNK603 were equivalent in all plants analyzed of this

single generation.

Comparison of the TC1507xNK603 stacked hybrid to each of the TC1507 and

NK603 test hybrids

Two restriction enzymes, EcoR V and Sac I., were selected for the

characterization studies based on the NK603 plasmid map of Mlu I fragment. On

the fragment map, EcoR V cuts at base pair (bp) positions 20, 3860, and 6678

while Sac I cuts at bp positions 949, 3058, and 6410. These enzymes also are

mapped on the Pme I fragment used to generate the TC1507 event. EcoR V

cuts this fragment at bp positions 62, 4266, 5166, and 5347 and Sac I cuts at bp

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positions 3944, 6051, and 6063. Three probes were used for characterization of

these test substance plants, 35S promoter, cry1F and pat. The 35S promoter

probe is common to both TC1507 and NK603 and the probe location is indicated

schematically on the fragment maps. The cry1F and pat gene probe information

is indicated on the fragment used to generate the TC1507 event, as these probes

uniquely detect the TC1507 event.

Test samples for TC1507 hybrid, NK603 hybrid, and TC1507xNK603 stacked

hybrid, and one control plant were digested with EcoR V or Sac I to generate

Southern blots for analysis.

When hybridized to the 35S promoter probe, the Southern blot results showed

that the combined hybridization patterns exhibited by the individual events

matched the hybridization pattern of the TC1507xNK603 stacked hybrid plants

and no hybridizing bands were present in control lanes. As a positive control and

reference for the probes, the 35S promoter probe hybridized to the expected

PHP8999 bands. When the blots were hybridized with the cry1F and pat probes,

as expected, only test material containing the TC1507 event hybridized to these

probes and the hybridization pattern was consistent among the test plants.

Verification of the genetic equivalence of the TC1507xNK603 stacked hybrid

DNA insertion within a single generation:

A sample set of 44 DNA extracts from the TC1507xNK603 stacked hybrid plants

were digested with EcoR V and analyzed with the 35S promoter, cry1F and pat

probes. As expected, all stacked hybrid plants exhibited the same hybridization

pattern throughout the study.

The Southern blot analysis results presented for the stacked hybrid of

TC1507xNK603 showed an equivalent banding pattern to the individual lines,

TC1507 and NK603 that were used to comprise the stacked hybrid. From these

analyses, the TC1507xNK603 stacked hybrid is confirmed to be a successful

genetic cross of the two events by traditional breeding methods.

Finally, all but one of the TC1507xNK603 stacked hybrid plants analyzed for this

study showed an expected hybridization pattern for the two individual insertions

and that each plant was equivalent to one another within this single generation.

Forty-three of forty-four plants that were analyzed by these Southern blots also

showed an equivalent hybridization pattern to the 4 plants analyzed for

comparison of the stack to the individual events. In conclusion, the confirmation

of individual events, TC1507 and NK603, and the Southern blots analyses results

confirm the stability and the equivalency of the insertions within the

TC1507xNK603 maize line.

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6.4.2 In the case of deletion(s), the size and function of the deleted

region(s).

Not applicable.

6.4.3 Chromosomal location(s) of insert(s) (nucleus, chloroplasts,

mitochondria, or maintained in non-integrated form), and

methods for its determination.

As expected and as a result of developing TC1507xNK603 maize by traditional

breeding methods, the TC1507 and NK603 maize inserts are integrated into the

nuclear genome of TC1507xNK603 maize as confirmed by the detailed Southern

blot analysis reports.

6.4.4 The organisation of the inserted genetic material at the

insertion site.

A detailed description of the organization of the genetic material inserted in

TC1507xNK603 maize shows that both the parental insertions have remained

unchanged during stacking. It was, therefore, concluded that the

TC1507xNK603 maize line is a stable conventional cross between the TC1507

and NK603 maize parental lines. There is no new genetic modification(s)

introduced in TC1507xNK603 maize.

6.5 Describe the trait(s) and characteristics which have been introduced

or modified:

6.5.1 Identify all inserted sequences and genes in the GM plant.

(a) Method used for the genetic modification of TC1507xNK603 maize


between progeny of the genetically modified TC1507 and NK603 maize. No new

genetic modification has been introduced in TC1507xNK603 maize. In

TC1507xNK603 maize, the Cry1F, PAT and CP4 EPSPS proteins are

expressed.

The expression of the Cry1F protein in TC1507xNK603 maize is the result of the

presence of TC1507 maize. The Cry1F protein acts to provide protection against

certain lepidopteran pests. TC1507xNK603 maize also expresses the PAT

protein, used as a selectable marker during transformation, which confers

tolerance to the application of glufosinate-ammonium herbicide.

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The expression of the CP4 EPSPS protein in TC1507xNK603 maize is the result

of the presence of NK603 maize. The CP4 EPSPS protein confers tolerance to

glyphosate which allows the development of these genetically modified plants to

continue despite the presence of glyphosate herbicide.

(b) Molecular characterization of TC1507xNK603 maize

As previously described in section 6.4.1, molecular analyses were performed to

determine the stability and equivalency of the TC1507 and NK603 DNA

insertions in the TC1507xNK603 maize plants. Event-specific polymerase chain

reaction (PCR) analysis was also conducted to confirm the presence of the

TC1507 and NK603 insertions in the TC1507xNK603 plants

6.5.2 Describe the gene products that are derived from the inserted

genes.

In TC1507xNK603 maize, the Cry1F, PAT and CP4 EPSPS proteins are

expressed.

The expression of the Cry1F protein in TC1507xNK603 maize is the result of the

presence of event TC1507. The Cry1F protein, encoded by a synthetic truncated

cry1F gene from Bacillus thuringiensis (Bt) subsp. aizawai, acts to provide

protection against certain lepidopteran pests. TC1507xNK603 maize, like

TC1507 maize, also expresses the PAT protein encoded by the phosphinothricin

acetyltransferase (pat) gene isolated from Streptomyces viridochromogenes,

used as a selectable marker during transformation, which confers tolerance to

the application of glufosinate-ammonium herbicide.

The expression of the CP4 EPSPS protein in TC1507xNK603 maize is the result

of the presence of the event NK603. The CP4 EPSPS protein expressed in

TC1507xNK603 maize confers tolerance to glyphosate and allows these maize

plants to continue to develop normally in the presence of glyphosate.

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6.5.3 Describe the biological activity associated with the inserted

sequences or inserted gene products.

As described in Section 6.5.2 above, TC1507xNK603 maize confers herbicides

tolerance to glufosinate ammonium and glyphosate due to the presence of the

PAT and CP4 EPSPS proteins; and protection against lepidopteran target pests

due to the presence of Cry1F protein.

Biological activity of the Cry1F proteins

Expression of the Cry1F protein confers protection against certain lepidopteran

pests. The Cry1F protein belongs to the 3-domain family of δ-endotoxins

produced by the bacterium Bacillus thuriengiensis (Bt) (Bravo et al., 2007; de

Maagd et al., 2003; Pigott and Ellar, 2007). The mechanism by which Cry

proteins kill target insects (their “mode of action”) has been well characterized

(reviewed in Soberón et al., 2010). Numerous Cry proteins have been identified

allowing systematic classification according to sequence homology, which is

highly correlated with target insect spectrum of activity (Höfte and Whiteley,

1989; Schnepf et al., 1998). The Cry1 class of proteins (which targets

Lepidoptera) are produced by Bt as relatively insoluble parasporal crystalline

inclusions comprised of the proteins in their protoxin forms of approximately 130-

140 kDa in size (Bravo et al., 2007; Schnepf et al., 1998). Upon ingestion by

susceptible insects, the protoxins dissolve in the alkaline conditions of the insect

gut and are processed by proteases to release the active core toxin from the

amino-terminal portion of the molecule. The activated Cry1 toxins are typically

55- 65 kDa in size. Cry toxins bind to specific receptors on the apical microvilli of

insect midgut epithelial cells (Pigott and Ellar, 2007). The identity of the specific

receptor that binds a given Cry toxin determines its specific biological activy and

thereby can differentiate the mode of action of different Cry proteins, e.g. distinct

receptors seem to be involved in binding of Cry1F. Binding of the activated toxin

is followed by oligomerisation of multiple toxin molecules and insertion into the

epithelial cell membrane forming a pore that causes osmotic cell lysis leading to

insect death.

Biological activity of PAT and CP4 EPSPS proteins

The PAT enzyme expressed in TC1507xNK603 maize acetylates glufosinate to

its inactive form, thereby preventing its competition with glutamate, and rendering

the PAT expressing plants to develop in the presence of glufosinate-ammonium.

Glutamine synthetase is a key enzyme in the glutamine biosynthesis pathway

catalysing the glutamate-glutamine transition. Glufosinate-ammonium is a

structural homolog of glutamate and disrupts normal glutamine synthesis by

competing with glutamate in the reaction.

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The CP4 EPSPS protein is a key enzyme in the biosynthesis of aromatic amino

acids in plants and microbes. Glyphosate binds to the plant EPSPS protein,

thereby impairing its normal enzyme activity, resulting in plant cell death. The

EPSPS protein expressed in TC1507xNK603 maize is a bacterial form of the

enzyme with a lower affinity for glyphosate. Therefore the presence of the

glyphosate insensitive EPSPS enzyme in TC1507xNK603 maize cells allows

normal biosynthesis of aromatic amino acids (OECD 1999a).

6.6 Provide information on the expression of the inserted sequences:

DuPont Pioneer conducted the protein expression studies during the 2002-2003

growing season. The study had two objectives (1) to evaluate the substantial

equivalence of the TC1507xNK603 maize treated with glyphosate to a non-

transgenic control by comparing their agronomic characteristics and nutrient

composition results, and (2) to evaluate and compare the level of expression of

Cry1F, PAT and CP4 EPSPS proteins in grain obtained from maize

TC1507xNK603 and control maize line.

Tissue samples were collected from leaf, root, whole plant, pollen, stalk, forage

and grain for the test and control hybrids for protein expression levels. The

tissue concentrations of all three proteins were measured using quantitative

enzyme-linked-immunosorbent-assay (ELISA) systems.

The expression of Cry1F, PAT and CP4 EPSPS proteins was detected in all

tissues assayed for the test hybrid, with the exception of the PAT protein which

was not detected in pollen during the R1 maize growth stage. None of the

proteins (Cry1F, PAT and CP4 EPSPS) were detected in any of the control

tissues.

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6.6.1 Provide information on the rate and level of expression of the

inserted sequences or inserted genes and the sensitivity of

the measurement of the rate and level.

The highest and lowest mean protein expression levels (ng/mg dry weight)

across all tissues for Cry1F, PAT and CP4 EPSPS proteins for each hybrid are

indicated in below table and described underneath:

Highest/Lowest Protein Expression Levels across all Tissues (ng/mg dry weight) Hybrid Cry1F PAT CP4 EPSPS

TC1507xNK603 28.6 / 1.1 4.32 / <LLOQ 351 / 9.8

Control <LLOQ / <LLOQ <LLOQ / <LLOQ 0.03 / <LLOQ

The mean expression levels of Cry1F across tissues of the test hybrid ranged

from 1.12 ng/mg dry weight in R6 leaf to 28.6 ng/mg dry weight in R1 pollen. The

mean expression levels of PAT across tissues of the test hybrid ranged from

below the lower limit of quantitation (<LLOQ) ng/mg dry weight in R1 pollen to

4.32 ng/mg dry weight in V9 leaf.

6.6.2 State whether expression is constitutive or inducible.

In agreement with the constitutive expression patterns predicted for the

promoters used to drive expressions, the field studies with individual TC1507 and

NK603 maize plants confirmed that the proteins, were in general, expressed

throughout the different plant tissues and developmental stages.

6.6.3 Provide information on the parts of the plant where the

inserted sequences or inserted genes are expressed.

As described above under sections 6.6 and 6.6.1, TC1507xNK603 maize

expresses Cry1F, PAT and CP4 EPSPS proteins in all the tissue samples

collected from leaf, root, whole plant, pollen, stalk, forage and grain.

The quantitative range for the Cry1F protein assay was 0.5 ng/ml to 10.0 ng/ml.

The LLOQ in ng/mg dry weight for each tissue was based on extraction volume

(μl) to weight ratios, the limit of quantitation for the ELISA in ng/ml, and the

dilutions used for analysis. In this study, the sample LLOQ on a ng/mg dry

weight basis for Cry1F was 0.135 ng/mg dry weight for root and grain, 0.09 ng/ml

dry weight for stalk and whole plant forage, 0.27 ng/mg dry weight for leaf and

0.54 ng/mg dry weight for pollen tissues.

The mean expression levels of Cry1F in TC1507xNK603 maize was

detected in all tissues assayed for the test hybrid and ranged from 1.12

ng/mg dry weight in R6 leaf to 28.6 ng/mg dry weight in R1 pollen.

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The quantitative range for the PAT protein assay was 0.25 ng/ml to 5.0 ng/ml.

The LLOQ in ng/mg dry weight for each tissue was based on extraction volume

(μl) to weight ratios, the limit of quantitation for the ELISA in ng/ml, and the

dilutions used for analysis. In this study, the sample LLOQ on a ng/mg dry

weight basis for PAT was 0.14 ng/mg dry weight for leaf, 0.27 ng/mg dry weight

for pollen and 0.068 ng/mg dry weight for stalk, root and grain; 0.09 whole plant

R4 growth stage and 0.45 for stalk and the other whole plant tissues.

Expression of the PAT protein across tissues of TC1507xNK603 maize

ranged from <LLOQ ng/mg dry weight in R1 pollen to 4.32 ng/mg dry

weight in V9 leaf.

The quantitative range for the CP4 EPSPS protein assay was 0.5 ng/ml to 10.0

ng/ml. The LLOQ in ng/mg dry weight for each tissue was based on extraction

volume (μl) to weight ratios, the limit of quantitation for the ELISA in ng/ml, and

the dilutions used for analysis. In this study, the sample LLOQ on a ng/mg dry

weight basis for CP4 EPSPS was 0.09 ng/mg dry weight for root and grain, 0.135

ng/mg dry weight for stalk and whole plant forage, 0.27 ng/mg dry weight for leaf

and 0.54ng/mg dry weight for pollen tissues.

Expression of the CP4 EPSPS protein across tissues of TC1507xNK603

maize ranged from 9.79 ng/mg dry weight in R6 leaf to 351 ng/mg dry

weight in R1 pollen. The expression of CP4 EPSPS protein was detected

in all tissues assayed for the test hybrid.

With the exception of one CP4 EPSPS whole plant sample, all control samples

were negative for Cry1F, PAT and CP4 EPSPS proteins. In conclusion, no

statistically different protein expression comparisons were made when

TC1507xNK603 maize was compared with individual maize events, TC1507 and

NK603.

6.7 Provide protocols for the detection of the inserted sequences or

inserted genes in other plants in the environment including

sensitivity, reliability and specificity of the techniques.

DuPont Pioneer conducted an event confirmation by Real-Time Polymerase

Chain Reaction (PCR) analysis of maize tissue containing the combined trait

product TC1507xNK603. The results for the qualitative real-time event-specific

PCR analysis utilizing primer and probe sets for TC1507 and NK603 maize DNA

insertions confirmed that the TC1507xNK603 maize tissue of the seed lot tested

contains both TC1507 and NK603 events.

As described under section 6.4.1, molecular analyses were also performed to

determine the stability and equivalency of the TC1507 and NK603 DNA

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insertions in TC1507xNK603 maize plants. Southern blot analyses performed

confirmed that each of the insertions was equivalent in TC1507xNK603 maize

after crossing by comparison to the individual lines.

6.8 Provide information on the genetic stability of the inserted

sequences.

As described in section 6.4 of this application, molecular analyses were

performed to determine the stability and equivalency of the TC1507 and NK603

DNA insertions in the TC1507xNK603 maize plants. Results of molecular

characterization studies confirmed that inserted sequences are genetically

stable.

6.9 Provide information on the phenotypic stability of the GM plant.

TC1507xNK603 maize has been shown to be phenotypically stable. The results

obtained from detailed molecular analysis, agronomic characterization and

protein expression analysis of TC1507xNK603 maize plants have confirmed the

stable inheritance and expression of the cry1F, pat and cp4 epsps genes as a

result of traditional breeding between progeny of TC1507 and NK603 maize.

6.10 Provide information on any change in the ability of the GM plant to

transfer genetic material to bacteria, plants, or other organisms.

Plant to bacteria gene transfer

On the basis of current scientific evidence the transfer of genetic material

originating from GM plants to bacteria is a negligible concern. There is no known

mechanism for, or definitive demonstration of, DNA transfer from plants to

microbes under natural conditions (EFSA, 2004; EFSA 2006). Even if such gene

transfer would occur, i.e. primarily through homologous recombination, the

eukaryotic promoters used to drive expression of the transgenes in the maize

plants would show limited, if any, activity in bacteria. In addition and as

discussed throughout this application, the inserted genes expressed in

TC1507xNK603 maize would not pose any risk to human and animal health or

the environment if expressed in bacteria. In fact, all of the transgenes in

TC1507xNK603 maize are derived from bacteria (Hérouet et al., 2005; Schnepf

et al., 1998).

Plant to plant gene transfer

There are no other cultivated or wild plant species that are sexually compatible

with maize in South Africa. Maize plants will intra-pollinate and transfer genetic

material between maize lines. The extent of pollination between maize will

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depend upon wind patterns, humidity and temperature. Maize pollen grains are

heavy, with a rapid settling rate, and show limited dispersal and viability

capacities.

6.11 Provide information on how the GM plant differs from the recipient

plant in:

6.11.1 General agronomic traits.

The agronomic characteristics of TC1507xNK603 maize were comparable to

those of conventional maize represented by non-genetically modified (non-GM),

and near-isoline control maize. The results are summarized below.

The agronomic characteristics of TC1507xNK603 maize treated with glyphosate

and control hybrids were evaluated in the field trials, at six separate sites during

the 2003 growing season.

The following agronomic characteristics were evaluated for each maize line in

each block: time to silking, time to pollen shed, plant height, ear height, stalk

lodging, root lodging, stay green, final population, disease incidence, insect

damage, pollen viability (shape) and pollen viability (color). The results of this

study demonstrated that the agronomic characteristics of TC1507xNK603 maize

were comparable to those of conventional maize.

Furthermore, analysis of transgene protein expression in various plant tissues

from the single events and the TC1507xNK603 maize stack demonstrated that

Cry1F and CP4 EPSPS proteins were expressed in all tissues of the

TC1507xNK603 maize. With the exception of pollen, expression of PAT protein

was also detected in all tissues of TC1507xNK603 maize.

In conclusion, TC1507xNK603 maize is agronomically equivalent to non-GM

control maize, and similar to the parental single event maize lines assessed.

There is no evidence to expect TC1507xNK603 maize to display different

agronomic characteristics that would raise a safety concern.

6.11.2 Reproduction.

The data collected indicated that the introduced traits in TC1507xNK603 maize

have not changed maize reproductive morphology and persistence or

invasiveness compared to control maize.

In case of unintended release of TC1507xNK603 maize, current agronomic

measures taken to control other commercially available maize can be applied,

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such as selective use of herbicides (with the exception of glufosinate-ammonium

herbicides and glyphosate), and manual or mechanical removal.

Taking into account all the above, there is negligible likelihood for

TC1507xNK603 maize, like any other maize, to become environmentally

persistent or invasive giving rise to any weediness within the context of this

application.

6.11.3 Dissemination, including persistence and invasiveness.

Cultivated maize has been domesticated to the extent that the seeds cannot be

disseminated without human intervention. The TC1507xNK603 maize plants

showed no difference in dissemination compared to non-GM control maize with

comparable genetic background.

6.11.4 Survivability.

Cultivated maize has been domesticated to the extent that it cannot survive

outside managed agricultural environments. Lack of dormancy decreases the

likelihood for maize seeds surviving from one growing season to the next. In

addition, maize does not possess any traits for weediness (OECD, 2003). The

survival characteristics of TC1507xNK603 maize in the environment remain

comparable to those of non-GM maize. Protection against certain lepidopteran

insect pests is not sufficient to allow survival of maize outside the agricultural

habitat, as maize survival in the environment is restricted by a complex

interaction of biotic and abiotic factors. In addition, although expression of the

PAT and CP4 EPSPS proteins in TC1507xNK603 maize confers tolerance to the

herbicides glufosinate-ammonium and glyphosate, respectively, these are broad-

spectrum herbicides that are not routinely used outside agricultural habitats.

Therefore, tolerance to the glufosinate-ammonium and glyphosate do not

enhance the potential for survival of TC1507xNK603 maize in the environment.

6.11.5 Other differences

The results obtained from the field trials carried out in South Africa have

confirmed that TC1507xNK603 maize shows no unexpected changes compared

to non-GM control maize with relation to other agronomic traits, such as stalk

lodging, root lodging, plant height, ear height, final population, stay green,

disease incidence and insect damage.

It is therefore concluded that reproduction, dissemination and survivability

characteristics of TC1507xNK603 maize are comparable to those in conventional

maize.

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7. RESISTANCE DEVELOPMENT

7.1 Detail whether any component of the environment can develop

resistance to any of the foreign gene products in the GM plant.

There is negligible likelihood for TC1507xNK603 maize to become

environmentally persistent or invasive giving rise to any weediness. First,

because maize does not possess any traits for weediness and secondly,

because expression of Cry1F, PAT and CP4 EPSPS proteins in TC1507xNK603

maize does not give rise to traits for weediness.

Maize plants are annuals that generally will not survive in South Africa from one

growing season to the next because of poor dormancy and sensitivity to low

temperature. Despite its non-dormant nature, maize seed can occasionally

persist from one growing season to the next under favourable climatic conditions.

When the temperature and moisture are adequate, the seed will germinate.

These volunteers can be controlled through current agronomic measures taken

to control other commercially available maize, such as selective use of herbicides

(with the exception of glufosinate-ammonium and glyphosate herbicides), and

manual or mechanical removal.

Resistance development in other environmental factors except target insects

There are no other cultivated or wild plant species that are sexually compatible

with maize in South Africa. Maize plants will intra-pollinate and transfer genetic

material between maize lines.

Cultivated maize has been domesticated to the extent that it cannot survive

outside managed agricultural environments. Lack of dormancy prevents maize

seed from readily surviving from one growing season to the next. In addition,

maize does not possess any traits for weediness (OECD, 2003). The survival

characteristics of TC1507xNK603 maize in the environment remain comparable

to those of non-GM maize.

7.2 Highlight the occurrence of resistance in previous field trials /

general releases or in the literature for plants containing the same or

similar genes.

Field-evolved resistance to Bt maize and other Bt crops is well documented in a

review by Tabashnik, et al. (2013). In addition to B. fusca in South Africa that

showed resistance, some of the examples to highlight are those that occurred for

(i) fall armyworm, Spodoptera frugiperda, in Puerto Rico (Storer et al., 2010;

Storer et al., 2012) and(ii) Western corn rootworm, Diabrotica virgifera virgifera

Le Conte, in the United States (Gassmann et al., 2011). The other pest that

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showed resistance were against Bt cotton and are (i) Helicoverpa zea in the USA

and (ii) Pectinophora gossypiella in India (Tabashnik et al., 2013). It should be

highlighted that resistance to insecticides was first documented in 1914 and

today cases of resistance appear between two to twenty years after introduction

of a new insecticide (Roush and Shelton, 1997; Storer et al., 2010).

7.3 Detail what methods are available to minimise the risk of resistance

developing in the environment.

Due to the fact that there is already an Industry IRM plan that was provided to the

Department of Environmental Affairs and the Executive Council of GMOs in

November 2009, DuPont Pioneer’s proposal for TC1507xNK603 maize IRM

strategy will be modelled on the Industry IRM plan. This is because the maize

events that DuPont Pioneer is asking the authorities to deregulate are similar to

other existing Bt maize events that have been commercialized (all the Bt maize

events commercially released in South Africa express cry genes).

Effective management of insect pest populations is fundamental to sustaining

crop production. To date, insect pests are mostly controlled by chemical

insecticides. Decades of experience have taught entomologists that insect

populations adapt, sometimes quickly, to even the best insecticides. Resistance

evolution can be delayed if insecticides are managed appropriately (McCaffery

and Nauen, 2006). Insect resistance is not a new occurrence. Resistance to

insecticides was first documented in 1914 and today cases of resistance may

appear between two to twenty years after introduction of a new insecticide.

Insect resistance has been addressed pro-actively in a number of countries by

the implementation of insect resistance management plans to delay the potential

development of pest resistance and to enable the timely detection of changes in

pest susceptibility. In order to develop and implement an IRM plan, it is

important to consider target pest biology and ecology, product deployment

patterns, local cropping systems; insect susceptibility monitoring,

stakeholder/grower communications, and a remedial action plan should

resistance develop (MacIntosh 2009).

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A proposed plan for addressing insect resistance for TC1507xNK603 maize

would include the following:

(i) Knowledge of pest biology and ecology and local crop landscape

The focal insect species being considered for this IRM plan for TC1507xNK603

are B. fusca and C. partellus.

Busseola fusca is one of the major stem boring insect pests of maize in South

Africa and can be found infesting maize plants in both irrigated and non-irrigated

areas. The naturally occurring variation in populations of B. fusca is exemplified

by lower susceptibility to Bt maize when compared to the sorghum stem borer (C.

partellus) (Van Rensburg, 1999). In South Africa, there are typically two

generations of B. fusca in the east, increasing to three in the west where

generations tend to overlap (Van Rensburg et. al,. 1985). Chilo partellus

generations overlap, and all development stages are present throughout most of

the growing season; November – March (Kfir, 2001).

Maize cultivation in South Africa is unique in the division of the landscape

between irrigated maize fields and non-irrigated maize fields. The continuous,

staggered planting of maize (from September to December) under irrigation

schemes creates an ideal environment where suitable host plants are available

over a long period of time for stem borers to thrive. Later planting dates (mid-

November – early January) also coincide with higher pest infestation levels.

Compliance with insect resistance management refuge requirements is important

to help mitigate the risk of resistance development (Kruger et al., 2009).

Understanding the biology of the target pests as well as the cultivation helps to

adapt an insect resistant management program that fits within an overall

integrated pest management plan, which may be based on various agro-

ecosystems.

(ii) Management to support the development of Bt susceptible insects

A refuge area is an integral component of many IRM strategies (Gould, 1986;

Roush, 1989; Tabashnik, 1994). The purpose of a refuge is to provide a

population of susceptible insects that are readily available to mate with rare

resistant insects that may be emerging from Bt plants. In a sense, the

susceptible alleles provided by the refuge "dilute" any resistance alleles that

remain after selection, making them rare again (Roush, 1994). Refuge is most

effective when the genetics of resistance are recessive and resistance alleles are

rare. With insect protected maize, the presence of a large number of susceptible

individuals arising from a refuge increases the likelihood that a rare resistant

homozygote (RR) will mate with a susceptible homozygote (SS), creating

heterozygous individuals that are subsequently killed by the Bt plants, thus

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diluting resistance in the population. In addition to planting a refuge, insecticide

application within an implemented IPM program may be useful to manage insect

resistance to Bt crops (see Section 3).

Refuge recommendations for TC1507xNK603 are:

Structured Refuge: On-farm fields without the Bt gene

Deliberate planting of non Bt-protected maize or other agronomic crops that are

suitable stem borer hosts will ensure that a refuge exists to support development

of susceptible insects; and ensure that any rare resistant individuals that may

arise from insect protected maize fields are more likely to mate with susceptible

individuals arising from the refuge. This approach is in most cases, especially

where stem borers consistently reduce maize yields, the best approach for

ensuring a refuge.

In view of the above and taking into consideration the biology and reproductive

nature of B. fusca and C. partellus, the presence of alternative hosts, and grower

behaviour, this approach would be suitable for South Africa. This approach is

contained in the current IRM strategy for South Africa and remains applicable for

TC1507xNK603.

Growers may plant the required refuge area by choosing one of the following

options:

Option A:

5% non-Bt maize refuge that is not treated with an insecticide.

Option B:

20% non-Bt maize that may be treated with a non-Bt containing

insecticide/bio-pesticide.

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In addition to planting a refuge according to Option A or B, the grower must also

adhere to certain important requirements when planting the refuge. The non-Bt

maize (i.e. refuge) must:

Be planted under the same growing conditions applicable for the Bt

maize. Thus, if the Bt maize is planted under irrigation, the refuge

maize must also be planted under irrigation,

Every grower planting more than 5 hectares must plant non-Bt maize.

Thus, non-Bt maize fields of neighbouring growers cannot serve as a

refuge,

Refuge “strip” areas must be at least 6 rows wide. Growers are

encouraged to plant refuge maize hybrids with similar maturity and

within a similar timeframe as Bt maize hybrids,

Growers must monitor and scout their fields frequently and

immediately contact their seed representative/agent if defined pest

population thresholds have been reached.

These IRM recommendations are not static and as more information becomes

available during cultivation of Bt products in South Africa, the IRM strategy will be

continuously reviewed and adapted in consultation with the authorities.

(iii) Employment of integrated pest management (IPM) practices for

TC1507xNK603

Insect protected maize is not a stand-alone measure for insect problems in

maize. As indicated previously, insect populations can adapt quickly to even the

best insecticides if those insecticides are not managed correctly. Industry

experiences with chemical insecticides resulted in development of an Integrated

Pest Management (IPM) approach. IPM is regarded as a system of managing

pests designed to be sustainable. IPM involves using the best combination of

cultural, biological and chemical measures for particular circumstances, including

plant biotechnology as appropriate. IPM means the careful consideration of all

available pest control techniques and subsequent integration of appropriate

measures that discourage the development of pest populations and keep

pesticides and other interventions to levels that are economically justified and

reduce or minimise risks to human health and the environment (CropLife

International, 2014). Pertinent components of IPM include: host plant

resistance, biological control, chemical control, and agronomic practices

(Luckmann and Metcalf, 1982; Wiseman, 1994).

Insecticide applications in IPM are utilized judiciously based on field scouting and

associated thresholds. Other factors such as tillage practices, crop growth stage,

presence of biological control agents and weather are also considered for optimal

and appropriate timing of insecticide application when thresholds are reached.

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Agronomic practices are also an important component of an IPM-based IRM

strategy. The major agronomic practice relevant to stem borer resistance

management is planting strategy. The grower will decide which hybrids to plant,

whether they are insect protected or non Bt-protected, and will be advised to

implement the most effective refuge option. Other agronomic practices such as

destruction of the overwintering habitat of the stem borers and other insects by

shredding of the stems and ploughing will also continue to be important.

(iv) Communication and education plan

Maintaining the effectiveness of TC1507xNK603 will depend on good product

stewardship, grower awareness, regular and consistent communication at all

levels of the product chain and grower implementation and support of IRM

recommendations. To be effective, growers must understand more than the

societal and environmental benefits of preserving the effectiveness of insect

protected maize (Kennedy and Whalon, 1995).

Managing insect resistance is not possible unless a workable IRM strategy is

implemented by technology providers and growers. Grower education is the

single most important element of any strategy for promoting compliance to Bt trait

IRM requirements. DuPont Pioneer uses a comprehensive approach to

communicate IRM requirements targeted to both customers and the Pioneer

sales force.

Comprehensive annual training is provided to all dealers. This training includes

trait specific technology information and product management, importance of

IRM and compliance with IRM requirements, expectations for communication of

requirements to customers, and all components of the compliance monitoring

program.

To assist with dealer communications, Technology Guides will be provided for

distribution to all Pioneer Bt trait customers.

The obligation for each grower planting more than 5 hectares to comply with the

refuge requirements will be imposed onto growers through signing of a

contractual agreement during purchase of seed containing the Bt technology.

Prior to signing the agreement as well as during cultivation of Bt crops, the

grower must be made aware of the potential for insect resistance. Regular and

consistent communication through appropriate educational tools at all levels of

the product chain is essential for the future of the technology.

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(v) IRM Compliance

Compliance to the IRM strategy by growers, and in particular compliance with the

refuge requirements, will be monitored through general compliance monitoring

through grower visits.

(vi) Mitigation measures to be implemented if localized insect resistance

occurs after commercial introduction of insect protected maize

Monitoring the potential development of insect resistance will primarily be

conducted through surveillance. The first line of surveillance is the grower, since

the grower, alarmed by decreased product performance or failure in the field,

would be the first to alert the seed supplier to suspected resistant pests.

A critical component of overall IRM strategy is field scouting. The technology

guide that Pioneer supplies to each customer contains information on monitoring

and reporting unexpected damage. Pioneer has protocols in place to ensure

consistent investigation of each report of unexpected levels of pest damage.

Resistance will be suspected if:

a) the maize field in question has been confirmed to be Bt maize

b) the seed planted met purity standards

c) it has been ruled out that a species not susceptible to the protein

(Cry1F) could be responsible for the damage, that no climatic or

cultural reasons could be responsible and that other reasonable

causes for the observed damage have been ruled out.

If insect resistance is suspected, specific actions would be triggered. These

actions are described below.

a) Intensifying field surveillance for Bt maize efficacy in and around the

potential “resistance epicenter” to define the boundaries of the

affected area,

b) Advice growers to incorporate crop residue into the soil or shred

stems following harvest to reduce overwintering larvae survival,

provided this is feasible within the grower’s operation.

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Once initial fact-gathering and containment is completed, appropriate follow-up

actions will be implemented. These actions may include one or more of the

following:

a) Collaboratively work with academic researchers to inform customers

and extension agents within the affected area that stem borer

resistance development is suspected and inform them of the region

impacted,

b) Increase field surveillance in and around the “resistance epicenter”,

and collect representative insects for dose-response bioassay

analysis, where appropriate,

c) Recommend to growers and extension agents alternative measures to

reduce or control the stem borer populations in the affected area in

subsequent years, and

Confirmed cases of insect resistance would be communicated to the appointed

Registrar as outlined in the terms of the Genetically Modified Organisms Act,

1997 (Act No. 15 of 1997).

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8. HUMAN AND ANIMAL HEALTH

8.1 State whether the GM plant or its products will enter human or

animal food chains.

TC1507xNK603 maize is already being used for food and feed purposes as the

commodity clearance approval was granted in South Africa in 2011. This

application is for general release of TC1507xNK603 maize in South Africa.

TC1507xNK603 maize will be used in the same manner as any other commercial

maize products.

8.2 Provide information on the anticipated intake or the extent of

exposure to the GM plant.

Maize is consumed in South Africa as pap, green mealies or as high fructose

maize syrup, starches, and oil, which contain negligible amounts of protein, and

as maize flour and popcorn.

According to Steyn (2006), a typical South African adult in rural areas consumes

870g to 1034g of cereals per day; whereas an urban adult consumes about 285g

to 736g per day. This is consistent with the finding of Smale et al. (2011), who

reported that the average consumption is over 100 kg/capita/year in Lesotho,

Malawi, South Africa, Zambia and Zimbabwe; making these countries the highest

consumers of maize in Africa.

The TC1507xNK603 maize, and all food, feed and processed products derived

from TC1507xNK603 maize are expected to form part of similar products from

commercial maize.

Dietary exposure to the insert-related proteins

Using the maximum cereal consumption in South Africa of 1034 g/day, and

assuming all cereal consumed is maize and an average body weight of 55.7 kg

(WHO GEMS, 2008), a consumption of 18.56 g maize/day/kg body weight (bw)

was calculated. This value was used to calculate the theoretical maximum daily

intake (TMDI) for the new proteins expressed in TC1507xNK603 maize, based

on their mean concentrations in maize grain.

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Theoretical Maximum Daily Intake (TMDI) of Insert-Related Proteins Expressed in TC1507xNK603 Maize

1 Based on a maize consumption of 1034 g/person/day and a mean body weight of 55.7 kg

The estimated daily intake of the transgene-related proteins in TC1507xNK603

maize varies from 0.219 to 3.89 x10-4 mg protein per kg body weight per day.

For a person with an average weight of 55.7 kg, this maximally mounts to 14.40

mg protein per day for all transgenic proteins together. These protein

consumption levels are many times below those used in animal toxicology

studies with no reported adverse effects.

This dietary exposure assessment is very conservative as it assumes that 100%

of consumed cereal from maize and that 100% of maize is derived from

TC1507xNK603 maize and it considers that the transgene-related proteins occur

in all maize, maize products and unspecified cereal products.

8.3 Provide information on the comparative assessment of the GM

plant:

8.3.1 The choice of comparator

The comparative assessment of TC1507xNK603 maize and its near-isoline

comparator is based on compositional and agronomic data obtained from field

trials performed during the 2002-2003 growing season.

The study had two objectives (1) to evaluate the substantial equivalence of the

TC1507xNK603 maize treated with glyphosate and a non-transgenic near-isoline

control by comparing their agronomic characteristics and nutrient composition

results, and (2) to evaluate and compare the level of expression of Cry1F, PAT

and CP4 EPSPS proteins in grain obtained from maize TC1507xNK603 and

control maize line.

Protein Concentration in grain [ng/mg dry weight]

TMDI of protein1 [mg/kg bw/day]

Cry1F 2.17 0.040

PAT 0.021 3.89 x10-4

CP4 EPSPS 11.8 0.219

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Test Substance

The test substance (referred to as hybrid) for the TC1507xNK603 maize

consisted of backcross 4 (BC4) generation seed. The test hybrid,

TC1507xNK603, consisted of a cross between TC1507 and NK603 to form the

BC4 hybrid, TC1507xNK603. The test hybrid received two applications of a

herbicide containing the active ingredient glyphosate.

Maize Line: TC1507xNK603

Log Number: T-F-03-109C

Non-GM comparator or Control Substance

The control substance consisted of near-isoline maize seed, which did not

contain event TC1507xNK603

Maize Line: Control

Log Number: C-F-03-82C

8.3.2 The production of material for the comparative assessment,

including locations, replicates and growing seasons.

The two individual maize (Zea mays L.) lines containing events TC1507 and

NK603 were crossed by conventional breeding methods to produce the

combined trait product TC1507xNK603 maize

Field studies were conducted at six separate field sites located in the commercial

maize-growing regions of the United States (4 sites) and Canada (2 sites). The

sites and sites codes were as follows:

Bagley, IA, USA (IA3)

Rochelle, IL, USA (IL1)

Wyoming, IL, USA (IL3)

New Holland, OH, USA, (OH1)

Branchton, Ontario, Canada (ON1)

Thorndale, Ontario, Canada (ON2)

The experimental design at each location was a randomised block design

containing four blocks. Each block contained the TC1507xNK603 maize and a

near-isoline control hybrid for comparative purpose. The plots for

TC1507xNK603 received two applications of a herbicide containing the active

ingredient glyphosate. Three of the four blocks were used to obtain material for

the comparative assessment.

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The nutrient composition samples were collected from Blocks 2, 3 and 4 at each

location. Collections procedures for each tissue type designated for nutrient

composition determinations were as follows:

Forage: Forage samples were collected at approximately the R4 growth stage

from impartially selected, healthy representative plants. Plants used for sampling

contained previously self-pollinated ears. Forage samples did not contain the

roots. Three whole plants from each test and control hybrid were randomly

selected and cut approximately 2.5 cm above the soil surface. Each whole plant

for each respective hybrid was then chopped into sections less than 5 cm in

length and combined to provide a single sample.

Grain: Grain samples were collected once the plant had reached physiological

maturity (R6 growth stage). Each sample consisted of five ears that were

collected from each hybrid (test and control) at each location. A single kernel

from each ear was analyzed for protein expression before it was combined into

one sample.

Compositional analysis of TC1507xNK603 maize

Nutrient composition analysis of maize forage and grain included the

determination of proximates (crude protein, crude fat, ash), crude fiber, acid

detergent fiber (ADF), neutral detergent finder (NDF), carbohydrates and

minerals (calcium and phosphorus).

In addition, nutrient composition analysis of maize grain included the

determination of fatty acids (palmitic, stearic, oleic, linoleic and linolenic acids),

amino acids (methionine, cysteine, lysine, tryptophan, threonine, isoleucine,

histidine, valine, leucine, arginine, phenylalanine, glycine, alanine, aspartic acid,

glutamic acid, proline, serine and tyrosine), minerals (phosphorus, calcium,

copper, iron, magnesium, manganese, potassium, sodium and zinc), vitamins

(beta-carotene, vamin B1, vitamin B2, folic acid and vitamin E), secondary

metabolites (inositol, furfural, p-coumaric acid and ferulic acid), and anti-nutrients

(phytic acid, raffinose and trypsin inhibitor).

The statistical analysis of compositional data was carried out both on an across

location and per individual location basis. For the across location analysis, the

following mixed model was used to describe the data (random effects indicated in

italics):

Response = loc block(loc) entry loc x entry residual

For the individual location analysis that tested for differences between the test

hybrid, TC1507xNK603+glyphosate, and the control hybrid using data from the 3

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replicates at each individual location. The individual analysis used the following

mixed model (random effects indicated in italics):

Response = block entry residual

Based on the data obtained for the nutrient compositional analysis study, it was

concluded that TC1507xNK603 maize is substantially equivalent to conventional

maize of comparable genetic background. Although statistically significant

differences were observed, such differences were not consistent across all six

locations tested.

Comparison of agronomic characteristics between TC1507xNK603 maize and

the parental single events

As described under section 6.11.1 and 8.3.2 above, DuPont Pioneer conducted

the agronomic characteristics of TC1507xNK603 maize during the 2002-2003

growing season.

The following agronomic characteristics were evaluated for each maize line in

each block: time to silking, time to pollen shed, plant height, ear height, stalk

lodging, root lodging, stay green, final population, disease incidence, insect

damage, pollen viability (shape) and pollen viability (color). The results of this

study demonstrated that the agronomic characteristics of TC1507xNK603 maize

were comparable to those of conventional maize represented by non-genetically

modified (non-GM), near-isoline control maize.

There was no statistically significant difference when comparing the agronomic

characteristics of maize TC1507xNK603 and the comparator. Similarly, no

unexpected changes in pollen production, seed production, seed viability and

germination were observed for TC1507xNK603 when compared with the non-GM

maize.

The data collected indicated that the introduced traits in TC1507xNK603 maize

have not changed maize reproductive morphology and persistence or

invasiveness compared to control maize. Taking into consideration all of the

above, there is negligible likelihood for TC1507xNK603 maize, like any other

maize, to become environmentally persistent or invasive giving rise to any

weediness within the context of this application. It was concluded that

TC1507xNK603 maize was agronomically comparable to the non-transgenic

conventional maize.

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8.4 Toxicology:

8.4.1 Detail the results of experiments undertaken to determine the

toxicity to humans and animals of the newly expressed

proteins (including antibiotic markers) or new constituents

other than proteins.

Safety assessment of newly expressed proteins

As described throughout this application, TC1507xNK603 maize has been

obtained by conventional (traditional) breeding between progeny of genetically

modified maize, TC1507 and NK603. Therefore, TC1507xNK603 expresses the

Cry1F, PAT and CP4 EPSPS proteins.

Field studies were conducted to evaluate the substantial equivalence of the

TC1507xNK603 maize to a non-transgenic near-isoline control by comparing

their agronomic characteristics and nutrient compositions. These studies also

evaluated and compared the level of expression of Cry1F, PAT and CP4 EPSPS

proteins in grain obtained from maize TC1507xNK603 and control maize line.

The Cry1F, PAT and CP4 EPSPS proteins were expressed in all tissue samples

assayed.

In addition to the composition, agronomic and protein expression level studies,

DuPont Pioneer conducted a 42 day poultry study and a 13 week rats study to

determine the safety of the newly expressed proteins.

Summary of 42 Day Poultry Feeding Study

The 42 day poultry feeding study with transgenic maize TC1507xNK603 was

conducted during the 2003-2004 growing season. The objective of the study was

to evaluate the nutritional equivalence of the TC1507xNK603 maize line by

comparing the growth performance and carcass yield of broiler chickens fed diets

containing TC1507xNK603 maize with those fed similar diets that were produced

with non-transgenic grain. Three experimental grain lines were produced from

plants that received two sequential treatments containing the active ingredient

glyphosate (TC1507xNK603Gly/Gly), glufosinate (TC1507xNK603Glu/Glu) or

glyphosate followed by glufosinate (TC1507xNK603Gly/Glu). For comparison, a

non-transgenic near isoline of TC1507xNK603 (control maize CH09B/1B5) and

three sources of commercial non-transgenic hybrid maize (reference control

maize lines 33P66, 33J56 and 33R77) were included.

For this study, three diets including starter, grower and finisher were formulated

according to the NRC Nutrient requirements for poultry and were fed in three

phases: starter – days 0 to 21, grower – days 22 to 35, and finisher – days 36 -

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42. The broilers were observed for overall heath, behavioral; changes and/or

evidence of toxicity. Body weights and feed weights were measured for every 7

days. The parameters evaluated at the end of 42 day study included carcass

yield, thighs, breasts, wings, legs, abdominal fat, kidneys and whole liver.

The study indicated that mortality, weight gain, feed efficiency (corrected for

mortalities) and carcass yields were determined to be similar between all

experimental and control groups. It also indicated the safety of the Cry1F, PAT

and CP4 EPSPS proteins expressed in TC1507xNK603 maize. It is therefore

concluded that TC1507xNK603 maize is nutritionally equivalent to near isoline

control and reference conventional maize lines.

Summary of 13 Week Rat Feeding Study

The 13 week (90 days) rat feeding study with genetically modified maize

TC1507xNK603 was also conducted in 2004. The objective of the study was to

evaluate the wholesomeness of rodent diets formulated with the transgenic

maize line TC1507xNK603.

Each experimental test diet contained TC1507xNK603 maize obtained from

plants that received either two sequential applications of a herbicide containing

glyphosate (TC1507xNK603Gly/Gly), glufosinate (TC1507xNK603Glu/Glu) or

glyphosate followed by glufosinate (TC1507xNK603Gly/Glu). For comparison,

one near isogenic non-transgenic hybrid maize line (control substance:

CH09B/1B5) and three non-transgenic commercial hybrid maize lines (reference

substances: 33P66, 33J56, and 33R77) were also evaluated.

For this study, diets were formulated to contain 35 % of maize and rats were

subjected to oral route of exposure in order to assess the wholesomeness of

food. The study parameters and frequency of evaluation were as follows:

Study Parameters Frequency

Body weight Day 0 and weekly thereafter

Food consumption Daily for week1, then weekly

Detailed clinical observations Day 0 and weekly thereafter

Mortality/Moribundity checks Twice daily

Ophthalmology Evaluation Pretest and Week 13

Neurobehavioral Evaluation Pretest and Week 13

Clinical Pathology Evaluation Week 13

Anatomic Pathology Evaluation Week 13

Results:

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No test diet-related differences in body weight, body weight gain, food

consumption, food efficiency, and clinical or ophthalmological observations were

observed following exposure of male or female rats to the TC1507xNK603 maize

diets.

None of the TC1507xNK603 test diets produced any differences in

neurobehavioral parameters in either males or females as per the study

conditions. The following neurobehavioral parameters were evaluated forelimb

grip strength, hindlimb grip strength, behavioral observations and motor activity.

The clinical pathology parameters evaluated included hematology, coagulation,

clinical chemistry and urinalysis. There were no toxicologically significant

differences observed in any of the evaluated parameters in female or male rats

when TC1507xNK603 maize lines were compared to non-transgenic control

maize lines.

The anatomic pathology evaluations included cause of death, organ weight data,

gross observations and microscopic observations. The results indicate no test

diet-related differences were observed in any of these parameters following

exposure of male or female rats to TC1507xNK603 maize diets.

The overall study conclusion was that the exposure of male and female rats to

diets containing TC1507xNK603 maize (irrespective of herbicide treatment),

produced no toxicologically significant differences as compared to rats fed diets

containing non-transgenic maize (near-isogenic or conventional strains). It is

therefore concluded that TC1507xNK603 maize is nutritionally equivalent to near

isoline control and reference conventional maize lines.

Testing of new constituents other than proteins

Detailed compositional analyses of TC1507xNK603 maize, as described in

section 8.3, demonstrated that the composition of TC1507xNK603 maize is

equivalent to that of non-GM maize with comparable genetic background.

Therefore, no testing of any new constituents other than expressed proteins is

necessary.


toxicity of whole GM food or GM feed.

Please refer also to section 8.4.1 above

As described throughout this application, there is no new genetic modification

introduced in TC1507xNK603 maize and molecular characterization of the event

confirmed stability of the introduced inserts. In addition, the nutritional

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assessments of TC1507xNK603 maize have confirmed that whole food and feed

consisting of or derived from TC1507xNK603 maize is equivalent to whole food

and feed consisting of or derived from commercial maize.

The poultry and rat feeding studies over a period of 42 days and 13 weeks,

respectively, have been carried out with TC1507xNK603 maize; non-GM control

maize with comparable genetics; and grains from three commercial maize

hybrids as summarized in section 8.4.1 above. The results from both studies

reported no adverse effects and have showed that TC1507xNK603 maize is

nutritionally equivalent to non-transgenic and commercial maize. It is therefore

concluded that TC1507xNK603 maize is as safe as non-transgenic maize and

not toxic for human or animal consumption.

In addition, the absence of any similarity between the insert-related protein

sequences and known proteins with toxic characteristics or which could produce

metabolites that may impact the safety or nutritional quality of food and feed

products derived from TC1507xNK603 maize has also been assessed and the

following conclusions were drawn from these analyses:

Cry1F Protein toxicity assessment

The Cry1F protein sequence returned 494 protein accessions with an

expectation (E) score less than 1.0, all of which referred to Cry proteins or Cry-

related proteins. None of the protein sequences returned by the BLASTp search

identified safety concerns that might arise from the expression of Cry1F in

genetically modified plants.

PAT Protein toxicity assessment

The PAT protein sequence returned 1400 protein accessions with an expectation

(E) score less than 1.0. The highest scoring alignments were attributed to PAT

proteins or related sequences from Streptomyces viridochromogenes and

Streptomyces hygroscopicus. Other accessions represented sequences from

related Streptomyces species, synthetic constructs containing portions of the

PAT proteins, or proteins annotated as phosphinothricin acetyltransferases or

putative acetyltransferases, N-acetyltransferases, or GCN5 or GNAT

acetyltransferases, based upon the possession of an acetyltransferase domain

present in the PAT query sequence, or acetyltransferase related sequences

including sortases, acyltransferases, antibiotic or toxin resistance proteins,

ribosomal-protein-alanine acetyltransferases, or histone acetyltransferases, ArsR

family transcriptional regulators, containing both the Arsenical Resistance

Operon Repressor domain as well as the acetyltransferase domain, etc. None of

the protein sequences returned by the BLASTp search identified any safety

concerns from the expression of PAT protein in genetically modified plants.

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CP4 EPSPS Protein toxicity assessment

The amino acid sequences of the two different CP4 EPSPS proteins were

compared with the amino acid sequences of known toxic proteins using a bio-

informatics approach. No relevant similarities between the sequence of the CP4

EPSPS proteins and sequences of toxic proteins were found. EPSPS proteins

are ubiquitously found in plants and have a history of safe consumption. (EFSA,

2009; EFSA, 2012)

Furthermore, numerous studies have shown that Bt maize grain contains lower

mycotoxin concentrations than isogenic conventional grain (Bakan et al., 2002;

Barros et al., 2009; Wu et al., 2006), an observation that may have positive

effects on animal growth performance (Tiemann and Dänicke, 2007).

8.4.3 Provide information on any changes in natural food and feed

constituents, especially toxins.

As described in sections 8.3 and 8.4.1 above, the comparisons carried out

between the natural constituents of TC1507xNK603 maize and non-GM control

maize with comparable genetic background confirm that there are no statistically

significant differences that would fall outside the normal ranges of variation for

commercial maize.

8.5 Allergenicity:

8.5.1 What are the common/major allergens present in the recipient

organism before modification?

In general, maize has been classified as a "less commonly allergenic food" (Hefle

et al., 1996; Moneret-Vautrin, 1998) and few maize kernel proteins (e.g., the 9

kDa lipid transfer protein) have been identified as allergenic (Lee et al., 2005;

Pastorello et al., 2003; Scibilia et al., 2008). Maize allergy can occur due to the

ingestion of maize or maize derivatives, or due to the inhalation of maize flour or

pollen. Although there have been reports of allergenic reactions to maize, maize

is not considered as a major allergenic food and maize allergens have mainly

been caused by pollen.

Recently, Fasoli et al. (2009) identified vicilin, globulin-2, 50 kDa gamma-zein,

endo-chitinase, thioredoxin and trypsin inhibitor as potential allergens using

proteomic tools. However, the clinical relevance of these identified proteins has

yet to be determined. Assessing the allergenicity of maize proteins is difficult

because of the limited availability of sera from maize-allergic individuals.

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Previously, Pasterello et al. (2003) had identified lipid transfer protein (LTP) as

the only major maize allergen.


allergenicity of the newly expressed gene products (including

antibiotic markers) to humans and animals.

The allergenicity of the newly expressed proteins in the parental TC1507 and

NK603 maize lines has been assessed using a weight-of-the-evidence approach

(Codex Alimentarius Commission, 2003; DuPont Pioneer, 2011; EFSA, 2010;

Ladics and Selgrade, 2009). The weight-of-the- evidence assessment is based

on what is known about food allergens, such as history of exposure and safety of

the gene product (i.e., whether the source of the gene for the new protein is

known to induce allergy), protein sequence (in silico amino acid sequence

homology comparisons to known allergens), physicochemical properties (e.g.,

stability to heat and to pepsin or trypsin digestion in vitro; glycosylation status),

and protein abundance in the crop. None of the newly expressed proteins were

considered having allergenic potential.

Evaluation of the allergenicity of the source organisms

There are several factors involved in establishing a history of safe use of a food

source and these include: the length of time the food has been consumed, the

potential hazard associated with consumption, the way it is consumed and used

and at what levels, and observations from animal and human exposures

(Constable et al., 2007).

One important factor to consider in assessing allergenic potential is whether the

source of the gene being introduced into plants is known to be allergenic.

Neither Bacillus thuringiensis (the source of the Cry1F), and Streptomyces

viridochromogenes (the source of the pat gene), or Agrobacterium sp. (the

source of the cp4 epsps gene), have a history of causing allergy and all three are

common soil bacteria.

Amino acid sequence comparison with known allergens

The comparison of the amino acid sequences of the newly expressed proteins in

TC1507xNK603 maize with the sequences of known protein allergens provides

an important basis for the assessment of potential allergenicity (Codex

Alimentarius Commission, 2003). The amino acid sequence comparison

employed two techniques: a search for continuous, identical stretches of eight or

greater amino acid residues in length; and, an identity search for alignments of

eighty or greater residues possessing a sequence identity of at least 35 %.

Studies have indicated that the use of six or seven contiguous identical amino

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acid matches occurs too commonly across proteins resulting in an unacceptably

high number of false positive findings and should not be utilized for predicting

allergenic potential (Codex Alimentarius Commission, 2003; DuPont Pioneer,

2011; EFSA, 2010).

Cry1F Protein allergernicity assessment

The amino acid sequence of the Cry1F protein from Bacillus thuringiesis was

compared to a dataset of known and putative allergens in order to identify any

potential for cross-reactivity using established criteria (Codex Alimentarius

Commission, 2003; EPA 2010). The evaluation employed two techniques; a

search for continuous, identical stretches of amino acids 8 residues or greater in

length, and an identity search using the FASTA34 alignment algorithm (Pearson

and Lipman, 1988) to search for alignments of 80 residues or longer possessing

a sequence identity of 35% or greater. In the case of Cry1F assessment as a

potential allergen, the Cry1F amino acid sequence, consisting of 605 amino

acids, was used as a query against a dataset of known and putative allergens.

The results of the evaluation against the FARRP10 dataset of known and

putative allergens showed no eight or greater contiguous identical amino acid

stretches in common between the Cry1F protein sequence and any of the protein

sequences in the dataset. None of the FASTA34 alignments between the Cry1F

protein sequence and the sequences in the dataset exceeded the 35% threshold

over 80 or greater amino acids. These data indicate that the Cry1F protein does

not show significant sequence identity with known or putative allergens.

PAT Protein allergernicity assessment

The amino acid sequence of the PAT protein from Streptomyces

viridochromogens was compared to a dataset of known and putative allergens in

order to identify any potential for cross-reactivity using established criteria

(Codex Alimentarius Commission, 2003; FAO/WHO 2001). The evaluation

employed two techniques; a search for continuous, identical stretches of amino

acids 8 residues or greater in length, and an identity search using the FASTA34

alignment algorithm (Pearson and Lipman, 1988) to search for alignments of 80

residues or longer possessing a sequence identity of 35% or greater. The PAT

amino acid sequence consisting of 183 amino acids was used as a query against

a dataset of known and putative allergens. The results of the evaluation against

the FARRP10 dataset of known and putative allergens showed no eight or

greater contiguous identical amino acid stretches in common between the Cry1F

protein sequence and any of the protein sequences in the dataset. None of the

FASTA34 alignments between the PAT protein sequence and the sequences in

the dataset exceeded the 35% threshold over 80 or greater amino acids. These

data indicate that the PAT protein does not show significant sequence identity

with known or putative allergens.

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CP4 EPSPS Protein allergernicity assessment

Similar searches have been conducted for the CP4 EPSPS protein. The

searches indicated that CP4 EPSPS protein did not have any eight or greater

contiguous identical amino acid matches or 80 or greater amino acid stretches

with a 35% or more identity with known protein allergens. Therefore, the amino

acid sequence comparison results support the conclusion that none of the newly

expressed proteins in TC1507xNK603 maize shows any significant sequence

identity with known allergens

Therefore, the amino acid sequence comparison results support the conclusion

that none of the newly expressed proteins in TC1507xNK603 maize shows any

significant sequence identity with known allergens.

Assessment of Protein Physiochemical Properties

The biochemical profile of the Cry1F and PAT proteins also provides a basis for

allergenic assessment when compared with known protein allergens. A

comparison of the amino acid sequence of an introduced protein with the amino

acid sequences of known allergens is a useful indicator of allergenic potential

(FAO/WHO, 2001). None of the protein sequences returned by the BLASTP

search identified safety concerns that might arise from the expression of Cry1F

and PAT proteins in genetically modified plants.

According to Apweiler and colleagues, 1999, over half of the proteins in plants

are estimated to be glycosylated. Glycosylation can alter the physiochemical

properties of a protein, such as its tolerance to heat, functional activity, protein

folding, transport and half-life (Raybould et al., 2013; Solá et al., 2007).

Transgenic proteins in plants are not intended to be glycosylated and

recombinant proteins produced in E. coli are not glycosylated (Raybould et al.,

2013); therefore, demonstrating the absence of glycosylation adds to the weight

of evidence that the protein of interest (POI) and the microbial protein are

functionally equivalent.

As indicated under section 6.6, the insert-related proteins are expressed in low

levels in grain of TC1507xNK603 maize. The maximal expression levels

observed for Cry1F, PAT and CP4 EPSPS were 28.6, 4.32 and 351.0 ng/mg dry

weight, respectively, across all tissues.

Using glycoprotein staining following SDS-PAGE, it was shown that none of the

TC1507xNK603 expressed proteins was glycosylated. The Cry1F, PAT and CP4

EPSPS proteins were all rapidly digested in simulated digestive fluid.

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Furthermore, the rapid degradation of the insert-related proteins during

processing at high temperature has been reported (DuPont Pioneer, 2011).


allergenicity of whole GM food or GM feed.

As described throughout this application, there is no new genetic modification

introduced in TC1507xNK603 maize and molecular characterization of the event

confirmed stability of the introduced inserts.

The poultry and rat feeding studies over a period of 42 days and 13 weeks,

respectively, have been carried out with TC1507xNK603 maize; non-GM control

maize with comparable genetics; and grains from three commercial maize

hybrids as summarized in section 8.4.1. The results from these studies reported

no adverse effects and have showed that TC1507xNK603 maize is nutritionally

equivalent to non-transgenic and commercial maize. It is therefore concluded

that TC1507xNK603 maize is as safe as non-transgenic commercial maize.

Furthermore the nutritional assessment of TC1507 and NK603 maize grain and

forage has confirmed that the GM maize and all food, feed and processed

products derived from such maize are nutritionally equivalent to conventional

maize and to all food, feed and processed products derived from conventional

maize. Therefore, consumption of TC1507xNK603 maize or any derived food

and processed products will have no adverse consequences to human health.

8.5.4 What evidence is there that the genetic modification

described in this application did not result in over-expression

of the possible allergens indicated in 8.5.1, i.e. is the

expression of the possible allergens in the non-GM

counterpart substantially equivalent to that in the GM

organism?

Maize is not considered a major allergenic food (Hefle et al., 1996; Moneret-

Vautrin, 1998) and few maize kernel proteins (e.g., 9 kDa lipid transfer protein)

have been identified as allergenic (Lee et al., 2005; Pastorello et al., 2003;

Scibilia et al., 2008). Recently, Fasoli et al. (2009) identified vicilin, globulin-2, 50

kDa gamma-zein, endo-chitinase, thioredoxin and trypsin inhibitor as potential

allergens using proteomic tools. However, the clinical relevance of these

identified proteins has yet to be determined. Assessing the allergenicity of maize

proteins is difficult because of the lack of documentation of maize-induced

allergic reactions as well as the limited availability of sera from documented

maize-allergic individuals.

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Furthermore and as described in Sections 6.6, 8.4 and 8.5 above, the newly

expressed proteins in TC1507xNK603 maize have been assessed for any

similarity to toxins, anti-nutritive proteins or allergens and they have been shown

not to have the characteristics of known allergenic proteins. Therefore, it can be

concluded that expression of the insert related proteins in TC1507xNK603 maize

is unlikely to alter the allergenicity of maize.

In addition, any potential interaction between the products of the genes

expressed in the two events has been evaluated. The intactness and stability of

the TC1507 and NK603 inserts in a combined event product has been assessed.

Furthermore, the expression of these proteins in various tissues from

TC1507xNK603 plants has been studied and was comparable to the expression

of these proteins in the parental events. The potential for the expression of any

novel ORFs present within or flanking the inserted sequences in maize events

TC1507 and NK603 has been assessed and no safety concern has been

identified. The combined presence of events TC1507 and NK603 in maize did

not affect the composition of grain or forage or any agronomic or phenotypic

characteristics, as assessed in field trials, nor did it change the nutritional

qualities of such maize.

Based on the available information, there is no evidence of any concern that the

genetic modification resulted in over-expression of any possible allergens that

would affect the safety of TC1507xNK603 maize.

8.6 If the newly expressed gene products are toxic or allergenic in any

way, detail how the general release will be managed to prevent

contact with animals or humans that will lead to discomfort or

toxicity.

As described in detail in sections 8.4 and 8.5 above, Cry1F, PAT and CP4

EPSPS proteins expressed in TC1507xNK603 maize are not toxic or allergenic

and therefore TC1507xNK603 maize is considered to be safe for animal or

human consumption. Based on the available information, there is no evidence of

any concern that would affect the safety of TC1507xNK603 maize.

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8.7 Nutritional assessment:

8.7.1 Detail the results of the experiments done in the nutritional

assessment of the GM food. Include information on the

baseline used for consideration of natural variations.

Composition analyses of grain from TC1507xNK603 maize have shown that the

contents of protein, fiber, carbohydrates, fat, ash, minerals, fatty acids, amino

acids, vitamins, secondary metabolites and anti-nutrients are all equivalent to

that found in non-GM maize with comparable genetic background and to the

published range of values in the literature.

In addition, nutritional equivalence between TC1507xNK603 maize and non-GM

control maize with comparable genetic background has also been shown in a

poultry feeding study over a 42-day period and a rat feeding study over 13

weeks. These studies have been summarized under section 8.4.1 above.

The broiler feeding study indicated that mortality, weight gain, feed efficiency

(corrected for mortalities) and carcass yields were determined to be similar

between all experimental and control groups. The safety of the Cry1F, PAT and

CP4 EPSPS proteins expressed in TC1507xNK603 maize was also proved in

this study.

In addition, the conclusion on the rodent feeding study was that the exposure of

male and female rats to diets containing TC1507xNK603 maize (irrespective of

herbicide treatment), produced no toxicologically significant differences as

compared to rats fed diets containing non-transgenic maize (near-isogenic or

conventional strains).

Furthermore and taking into account the anticipated dietary intake of

TC1507xNK603 maize products (section 8.2 above), consumption of

TC1507xNK603 maize foods will not give rise to any adverse nutritional impact. It

is therefore concluded that TC1507xNK603 maize is nutritionally equivalent to

near isoline control and reference conventional maize lines.

In addition to the description above, a scientific literature search revealed a

number of papers reporting the results of poultry feeding studies with NK603

maize (Taylor et al., 2003), dairy cow feeding studies with TC1507 and NK603

maize (Donkin et al., 2003; Faust et al., 2007; Grant et al., 2003; Ipharraguerre et

al., 2003), a steer feeding study with NK603 maize (Erickson et al., 2003), and a

growing-finishing pig feeding study with NK603 maize (Hyun et al., 2004). The

results of these studies confirmed that grain from maize lines containing the

single events as well as grain from the stacked event (TC1507xNK603) is

nutritionally equivalent to non-GM control maize grain.

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The baseline used for consideration of natural variants

Publicly available data on commercial maize was compiled from the literature

and was used as the baseline for consideration of natural variations in the

comparisons with TC1507xNK603 maize. The comparator maize used during

the animal feeding studies was a non-transgenic near isoline (control maize

CH09B/1B5) developed in the same genetic background as the TC1507xNK603

maize. The commercially available non-transgenic hybrid maize lines (33P66,

33J56 and 33R77) were also used as reference sources. In addition, a

comparative assessment with non-GM maize of comparable genetic background

has been carried out as described under section 8.3.

8.7.2 Detail the results of the experiments done in the nutritional

assessment of the GM feed. Include information on the

baseline used for consideration of natural variations.

As described in section 8.7.1 and many others above, the consumption of

TC1507xNK603 maize as feed is not expected to give rise to any adverse

nutritional impact. The TC1507xNK603 maize and derived feed products are

nutritionally equivalent to any other commercial maize and derived feed products.

Agronomic studies of TC1507xNK603 maize have adequately indicated that the

agronomic characteristics of TC1507xNK603 maize were comparable to those of

conventional maize. Similarly, no unexpected changes in pollen production,

seed production, seed viability and germination were observed for

TC1507xNK603 when compared with the non-GM maize.

The statistical analysis of compositional data was carried out both on an across

location and per individual location basis. Nutrient compositional analysis data

has indicated that TC1507xNK603 maize is substantially equivalent to

conventional maize of comparable genetic background. Although statistically

significant differences were observed, such differences were not consistent

across all six locations tested. The forage samples from conventional, herbicide-

treated TC1507xNK603 maize and near isoline control maize were collected and

analyzed for different analytes, and the results showed no statistically significant

differences for any of the proximates, fiber, or minerals analyzed in the across

sites analysis between TC1507xNK603 treated with glyphosate and the control

maize.

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8.7.3 Provide information on any changes in natural food and feed

constituents, including toxicants, metabolites and anti-

nutritional factors.

As described in above sections, the comparisons carried out between the natural

constituents of TC1507xNK603 maize and non-GM control maize with

comparable genetic background confirm that there are no statistically significant

differences that would fall outside the normal ranges of variation for commercial

maize.

The combined presence of events TC1507 and NK603 in TC1507xNK603 maize

did not affect the composition of grain or forage or any agronomic or phenotypic

characteristics, as assessed in field trials nor did it change the nutritional qualities

of such maize. Furthermore, the insert-encoded proteins in TC1507xNK603

maize have been assessed and demonstrated lack off similarity to toxins, anti-

nutritive proteins or allergens.

8.8 What are the implications of the proposed activity with regard to the

health and safety of the workers, cleaning personnel and any other

person that will be directly or indirectly involved in the activity?

Please take into consideration the provisions of the Occupational

Health and Safety Act, 1993 (Act No. 85 of 1993 as amended by Act

No. 181 of 1993) (and accompanying regulations) and indicate the

proposed health and safety measures that would be applied.

As discussed throughout this application for general release of TC1507xNK603

maize, the agronomy, composition, nutritional value, allergenicity, and mode of

storage, preparation or cooking of TC1507xNK603 maize is not significantly

different from those of commercial maize. Therefore, there are no health or

safety implications specific for the general release of TC1507xNK603 maize.

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9. ENVIRONMENTAL IMPACT AND PROTECTION

9.1 Pollination and reproduction:

9.1.1 Identify any plants in the area of general release that may

become cross-pollinated with the GM pollen (see 3.4.3).

Cultivated maize (Zea mays ssp. mays) belongs to the genus Zea, which

includes several other wild species, collectively known as teosintes. The closest

species related to maize is teosinte (Zea mexicana), a wild grass found in Mexico

and Guatemala. No sexually compatible wild relatives of maize are found in

South Africa.

9.1.2 How do seeds of the GM plant interact in the environment and

what long term effects will the seed likely have on the

environment.

Maize is no longer able to survive in the wild, since dissemination of its seed

needs human intervention. As a result, maize cannot persist as a weed and is

incapable of sustained reproduction outside of domestic cultivation. Volunteers

are common in many agronomic systems, but they are easily controlled and

maize plants are non-invasive in natural habitats (Gould and Shaw, 1983; OECD,

2003). Expression of the Cry1F, PAT and CP4 EPSPS proteins do not confer a

fitness advantage in TC1507xNK603 maize, and therefore are not expected to

affect survival, weediness or invasiveness in the environment.

9.1.3 In the case of vegetative reproduction, describe methods to

be used to limit vegetative spread of the GM plant into the

environment.

Maize does not reproduce vegetatively hence there are no methods that need to

be used to limit vegetative spread of TC1507xNK603 maize into the environment.

9.2 Detail any effects, especially long-term, that the general release of

the GM plant is likely to have on the biotic and abiotic components

of the environment. Information on the impact on non-target

organisms should be provided.

A detailed risk assessment, characterizing the potential for non-target organisms

in the ecosystem to be exposed to the Cry1F protein in TC1507xNK603 maize

and the potential hazards associated with the Cry1F protein on representative

surrogate species has been conducted and is summarized below.

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In the exposure assessment, three routes were identified through which NTOs

could be exposed to Cry1F protein from TC1507xNK603 cultivated maize: via

soil, via prey mediated transfer, and via plant tissue (green tissue and pollen).

The Cry1F protein has been shown to rapidly dissipate in soil ecosystems and

there has been no accumulation in soil after multiple years of successive planting

with TC1507 maize. Litter decomposition rates are also not affected by Cry

proteins. Based on extensive soil fate and persistence data, the exposure risk of

cultivating TC1507xNK603 maize on soil-dwelling organisms is low. Similarly,

Cry proteins have short residency times and do not bio-accumulate within prey

items. Given these short protein residency times, predatory species have

negligible exposure to Cry1F via prey items. Therefore, pollen is the primary

route through which NTOs could be exposed to Cry1F protein from

TC1507xNK603 maize. However, there are many factors that limit environmental

exposure to maize pollen, and only species that reside (for at least part of their

life cycle) in maize fields or within field margins could realistically be exposed.

The specificity of the Cry1F protein has been well established, and there is low

risk of hazard to non lepidopteran orders. Additionally, for non-target

lepidopteran species, several mitigating factors decrease exposure to maize

pollen and results from the early-tier hazard studies suggest negligible risk. This

low hazard has been confirmed with extensive laboratory bioassays using

multiple representative orders tested at 10X environmentally relevant

concentrations. Additionally, numerous field studies have also confirmed that

non-target organism abundance and diversity are not reduced by

TC1507xNK603 maize in reference to isoline maize.

Based on the well-characterized environmental fate, specificity to lepidopteran

pest species, and lack of effects on non-target organisms, the environmental risk

associated with the cultivation of 1507xNK603 maize in South Africa is low.

9.3 Provide data and information on ecosystems that could be affected

by use of the plant or its products.

TC1507xNK603 maize has been shown to be substantially equivalent to

conventional maize, so the environmental risk assessment can be focused only

on the introduced trait and the potential effects of the expressed insecticidal

proteins on the environment. As described above, a thorough environmental risk

assessment has been performed to characterize the risk associated with

cultivating TC1507xNK603 maize in South Africa. Based on the well-

characterized environmental fate, specificity to lepidopteran pest species, and

lack of effects on non-target organisms, the environmental risk associated with

the cultivation of TC1507xNK603 maize in South Africa is low.

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Plant to bacteria gene transfer

Transfer of genetic material originating from TC1507xNK603 maize to bacteria is

a negligible concern. There is no known mechanism for, or definitive

demonstration of, DNA transfer from plants to microbes under natural conditions.

Plant to plant gene transfer

The potential for transfer of genetic material from TC1507xNK603 maize to other

organisms has not been changed and it will be negligible, as there are no

sexually compatible wild or weedy relatives of Zea mays known to exist in South

Africa.

9.4 Specify what effect the general release of the GM plant will have on

biodiversity.

As discussed in sections 9.1 and 9.2, TC1507xNK603 maize is not invasive and

expression of Cry1F, PAT and CP4 EPSPS proteins in TC1507xNK603 maize

does not provide any selective advantage to maize plants outside managed

agricultural habitats. The Cry1F, PAT and CP4 EPSPS proteins expressed in

TC1507xNK603 maize do not correspond to any new toxic or allergenic

substances; moreover, there are no marker genes coding for antibiotic resistance

in TC1507xNK603 maize.

Interactions between the GM plant and target organisms

The genetic modification in TC1507xNK603 maize provides growers with a highly

specific, effective and environmentally beneficial tool for protection against

certain lepidopteran insect pests.

Interactions of the GM plant with non-target organisms


risk of hazard to non lepidopteran orders. Additionally, for non-target



low hazard has been confirmed with extensive laboratory bioassays using



non-target organism abundance and diversity are not reduced by 1507xNK603

maize in reference to isoline maize.

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associated with the cultivation of 1507xNK603 maize in South Africa is low

Effects on biogeochemical processes

TC1507xNK603 maize is substantially equivalent to the conventional counterpart.

Therefore, there is no evidence to suggest that soil biogeochemical processes

would be influenced by the cultivation of TC1507xNK603 maize relative to isoline

maize.

Impact of GM crops on the environment and biodiversity

A thorough environmental risk assessment for the cultivation of event

TC1507xNK603 maize in South Africa has been previously conducted. Based on

this risk assessment, the cultivation of TC1507xNK603 maize is unlikely to have

adverse effects on non-target arthropods, biodiversity or the environment.

9.5 If the foreign genes give rise to crops tolerant to agrochemicals,

provide information on the registration of the agrochemicals to be

used on the crop.

TC1507xNK603 maize is resistant to glufosinate-ammonium and glyphosate

herbicides. As previously mentioned, glufosinate-ammonium tolerance was

introduced as a selectable marker for the selection process and it will not be

used in commercial maize crops in South Africa.

According to the Association of Veterinary and Crop Associations of South Africa

(AVCASA) records (2013), of all herbicides registered for use on herbicide

resistant maize in South Africa, various glyphosate formulations with various

trade names are registered for use on herbicide resistant GM maize and the

registration holders include: Dow Agrosciences, Syngenta, Monsanto, Villa Crop

Protection, Universal Crop Protection and Kombat.

There are no glufosinate ammonium herbicides registered for use on maize in

South Africa. The only permit holder for glufosinate ammonium (also known as

Basta) herbicide is Bayer, and the herbicide is registered for use on deciduous

crops that include mangoes, peaches and nectarines. Several herbicides are

registered in South Africa in the name of various companies, including DuPont,

Monsanto, Syngenta, Dow AgroSciences, etc. These include ALS-inhibiting

herbicides that would be able to eradicate TC1507xNK603 maize volunteer

plants.

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Additional herbicides that would eradicate all maize plants (both conventional

and herbicide tolerant plants) are available on the market, such as paraquat

dichloride (Gramaxone, Paragone), Dimethyl amine/2,4-D/Dicamba and

Boromoxynil (Brominex, Bromotril).

9.6 Submit an evaluation of the foreseeable impacts, in particular any

pathogenic and ecologically disruptive impacts.

The conclusions of absence of any pathogenicity or any ecologically disruptive

impacts obtained from the detailed evaluations of the proteins expressed in

TC1507xNK603 maize confirmed that the genetic modifications in

TC1507xNK603 maize do not introduce any new pathogenic or ecologically

disruptive substances. In particular, there are no marker genes coding for

antibiotic resistance in TC1507xNK603 maize.

This general release application focuses mainly on cultivation of TC1507xNK603

maize in agricultural environments, and on the safety and potential biodiversity

impacts of TC1507xNK603 maize. Any unintentional release such as that arising

from accidental release of TC1507xNK603 maize (for example, spillage of seed

or grain during transport) will be very limited. Maize is not an invasive plant and

it is a weak competitor outside the cultivated fields. In addition, the genetic

modifications in TC1507xNK603 maize do not alter the survival characteristics of

maize. Under such circumstances no significant ecologically disruptive impacts

can be expected from general release of TC1507xNK603 maize.

Bacillus thuringiensis (Bt) is a naturally occurring soil bacterium which expresses

a variety of proteins with a wide spectrum of activity against agronomically

important pest species. Bt insecticidal proteins have a history of safe use as

both microbial-based sprayable foliar pesticides as well as plant-incorporated

protectants. There is a long history of safe use surrounding the cultivation of

transgenic maize containing the event TC1507. Pathogenic or ecologically

disruptive effects are unlikely as a result of cultivation of TC1507xNK603 maize.

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10. SOCIO-ECONOMIC IMPACTS

10.1 Specify what, if any, positive or negative socio-economic impacts

the GM plant will have on communities in the proposed region of

release. The information may include but is not limited to

information on the impact on the following:

(a) Income, competitiveness or economic markets.

(b) Food security.

(c) Access to genetics and other natural resources previously

available.

(d) Cultural traditions, knowledge and practices.

(e) The continued existence and range of diversity of the biological

resources.

Potential impact on biological/natural resources, cultural traditions, knowledge

and practices

TC1507xNK603 maize has no sexual compatibility with other cultivated or wild

plant species in South Africa. Therefore, TC1507xNK603 maize will intra-

pollinate and will not transfer genetic material to other plant species in the South

Africa. It is generally considered that teosinte (Zea mays ssp. mexicana) is an

ancestor of maize. Teosinte is an ancient wild grass found in Mexico and

Guatemala and it is not present in South Africa. The absence of sexually

compatible wild or weedy relatives of Zea mays in South Africa eliminates any

potential for gene transfer to land races or wild relatives.

TC1507xNK603 stacked maize was developed by traditional breeding to express

the Cry1F, PAT, and CP4 EPSPS proteins. When cultivated, expression of the

Cry1F and Cry1Ab proteins in TC1507xNK603 maize will confer protection

against certain lepidopteran pests, such as the Busseola fusca and Chilo

partellus. The CP4 EPSPS protein expressed in TC1507xNK603 enables plants

at certain growth stages to withstand applications of certain concentrations of

glyphosate herbicide and thus help in improving crop yields for local growers.

Cultural traditions and practices in agronomy are likely to be enhanced, as

growers will have additional options of controlling stem borers, and the cultivation

of TC1507xNK603 maize may require DuPont Pioneer to increase investments in

training growers about insect resistance management, agronomy, and integrated

pest management.


environmentally persistent or invasive or give rise to weediness characteristics.

First, because maize does not possess any traits for weediness and secondly,

expression of Cry1F, PAT and CP4 EPSPS proteins in TC1507xNK603 maize

does not give rise to traits for weediness. Maize plants are annuals that

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generally will not survive in South Africa from one growing season to the next

because of poor dormancy and sensitivity to low temperature. Despite its non-

dormant nature, maize seed can occasionally persist from one growing season to

the next under favourable climatic conditions. When the temperature and

moisture are adequate, the seed will germinate. These volunteers are easily

identified and controlled through current agronomic measures taken to control

other commercially available maize can be applied, such as selective use of

herbicides (with the exception of glufosinate-ammonium and glyphosate), and

manual or mechanical removal.

Furthermore and as discussed throughout this application, the composition,

nutritional value, mode of storage, preparation or cooking, and allergenicity of

TC1507xNK603 maize is not significantly different from those of commercial

maize. As a result, cultivation of TC1507xNK603 maize will not result in any

negative socio-economic impacts on any communities from South Africa.

Income, competitiveness or economic markets and food security

South African maize farmers operate in a global market, and thus need access to

technology to produce competitively in the international arena due to the global

nature of the grain trade. Increased local seed production for maize is expected

following the approval of a general release for TC1507xNK603 maize and this

will enhance skills development in the country as maize seed production is a

specialised field of science.

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11. RISK MANAGEMENT AND POST MARKET MONITORING PLAN

11.1 Please indicate any risk management measures that users of this

trait will have to adhere to with regard to commercial planting and

use.

The environmental risk assessment (ERA) for this application for authorization of

genetically modified TC1507xNK603 maize has been carried out in accordance

with requirements of the GMO Act and based on outcomes of the ERA studies in

South Africa.

The overall conclusion obtained from the ERA confirms that there are no

identified adverse effects to human and animal health or the environment arising

from the product described in this application, which is TC1507xNK603 maize,

for all food and feed uses, and for all food, feed and processed products derived

from TC1507xNK603 maize, including cultivation. Therefore, the risk to human

and animal health or the environment from TC1507xNK603 maize and any

derived products is as negligible as for any commercial maize and any derived

products.

11.2 Please specify an environmental monitoring plan (approach,

strategy, method and analysis) which encompasses but is not

limited to the following:

Further details that informed the inputs of the proposed monitoring plan are listed

below.

(i) Spread, including vegetative spread, of GM plants.

During the domestication of maize, many agronomically significant attributes for

cultivation have been gained whilst losing its ability to survive in the wild.

TC1507xNK603 maize (and any conventional maize) is a non-dormant annual

crop and seeds are the only survival structures. Natural regeneration of maize

(including TC1507xNK603 maize) from vegetative tissue is not known to occur.

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(ii) Environmental impact and protection (focusing on issues such as

weed and insect resistance management; direct and indirect

impacts on non-target organisms).


environmentally persistent or invasive giving rise to any weediness. First,

because maize does not possess any traits for weediness and second,

expression of Cry1F, PAT and CP4EPSPS proteins in TC1507xNK603 maize

does not give rise to traits for weediness.

Maize plants are annuals that generally will not survive in South Africa from one

growing season to the next because of poor dormancy and sensitivity to low

temperature. Despite its non-dormant nature, maize seed can occasionally

persist from one growing season to the next under favourable climatic conditions.

When the temperature and moisture are adequate, the seed will germinate.

These volunteers are easily identified and controlled through current agronomic

measures taken to control other commercially available maize can be applied,

such as selective use of herbicides (with the exception of glufosinate-ammonium

and glyphosate herbicides), and manual or mechanical removal.

Expression of Cry1F, PAT and CP4EPSPS proteins in TC1507xNK603 maize will

not cause any possible immediate and/or delayed effects on biogeochemical

processes resulting from potential direct and indirect interactions of

TC1507xNK603 maize and non-target organisms in the vicinity of

TC1507xNK603 maize.

Insect resistance management

Some of the elements of the plan are listed below.

Refuge Management to support maintaining overall target pest susceptible

to Bt traits

A refuge area is an integral component of many IRM strategies (Gould 1986,

Roush 1989, Tabashnik 1994). The purpose of a refuge is to provide a

population of susceptible insects that are readily available to mate with rare

resistant insects that may be emerging from Bt plants. In a sense, the

susceptible alleles provided by the refuge "dilute" any resistance alleles that

remain after selection, making them rare again (Roush, 1994). Refuge is most

effective when the genetics of resistance are recessive and resistance alleles are

rare. With insect protected maize, the presence of a large number of susceptible

individuals arising from a refuge increases the likelihood that a rare resistant

homozygote (RR) will mate with a susceptible homozygote (SS), creating

heterozygous individuals that are subsequently killed by the Bt plants, thus

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diluting resistance in the population. In addition to planting a refuge, insecticide

application within an implemented IPM program may be useful to manage insect

resistance to Bt crops.

Refuge recommendations for TC1507xNK603 maize:

Structured Refuge: On-farm maize fields without the Bt gene

Deliberate planting of non Bt-protected maize or other agronomic crops that are

suitable stem borer hosts will ensure that a refuge exists to support development

of susceptible insects; and ensure that any rare resistant individuals that may

arise from insect protected maize fields are more likely to mate with susceptible

individuals arising from the refuge. This approach is in most cases, especially

where stem borers consistently reduce maize yields, the best approach for

ensuring a refuge.

In view of the above and taking into consideration the biology and reproductive

nature of B. fusca and C. partellus, the presence of alternative hosts, and grower

behavior, this approach would be suitable for South Africa. This approach is

contained in the current IRM strategy for South Africa and remains applicable for

TC1507xNK603 maize.

Growers may plant the required refuge area by choosing one of the following

options:

Option A:

5% non-Bt maize refuge that is not treated with an insecticide.

In practice this means that for every 95 hectares planted with Bt maize, the

grower must plant 5 hectares of non-Bt maize (thus maize without any Bt genes).

This non-Bt maize may not be treated with any insecticide registered for control

of maize stem borers.

Option B:

20% non-Bt maize that may be treated with a non-Bt containing insecticide/bio-

pesticide.

In addition to planting a refuge according to Option A or B, the grower must also

adhere to certain important requirements when planting the refuge. The non-Bt

maize (i.e. refuge) must:

a) Be planted on the same farm as the Bt maize,

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b) Be planted under the same growing conditions applicable for the Bt

maize. Thus, if the Bt maize is planted under irrigation, the refuge

maize must also be planted under irrigation,

c) Every grower planting more than 5 hectares must plant non-Bt maize.

Thus, non-Bt maize fields of neighboring growers cannot serve as a

refuge,

d) Refuge “strip” areas must be at least 6 rows wide. Growers are

encouraged to plant refuge maize hybrids with similar maturity and

within a similar timeframe as Bt maize hybrids,

e) Growers must monitor and scout their fields frequently and

immediately contact their seed representative/agent if defined pest

population thresholds have been reached.

These IRM recommendations are not static and as more information becomes

available during cultivation of Bt products in South Africa, the IRM strategy may

be reviewed and adapted in consultation with the authorities.

Direct and indirect impacts on non-target organisms (NTO)

A thorough environmental risk assessment characterizing the risk associated

with cultivation of TC1507xNK603 maize in South Africa has been prepared.

In the exposure assessment, three routes were identified through which NTOs

could be exposed to Cry1F protein from TC1507xNK603 cultivated maize: via

soil, via prey-mediated transfer, and via plant tissue (green tissue and pollen).

The Cry1F protein has been shown to rapidly dissipate in soil ecosystems and

there has been no accumulation in soil after multiple years of successive planting

with TC1507 maize. Litter decomposition rates are also not affected by Cry

proteins. Based on extensive soil fate and persistence data, the exposure risk of

cultivating TC1507xNK603 maize on soil-dwelling organisms is low. Similarly,

Cry proteins have short residency times and do not bioaccumulate within prey

items. Given these short protein residency times, predatory species have

negligible exposure to Cry1F via prey items. Therefore, pollen is the primary

route through which NTOs could be exposed to Cry1F protein from

TC1507xNK603 maize. However, there are many factors that limit environmental

exposure to maize pollen, and only species that reside (for at least part of their

life cycle) in maize fields or within field margins could realistically be exposed.


risk of hazard to non-lepidopteran orders. Additionally, for non-target



low-hazard has been confirmed with extensive laboratory bioassays using


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non-target organism abundance and diversity are not reduced by

TC1507xNK603 maize in reference to isoline maize.



associated with the cultivation of TC1507xNK603 maize in South Africa is low.

Weed resistance management

Weed control is a key point in maize production because the absence or the poor

control of weeds considerably reduces quality and quantity of the maize crop.

Weeds are a direct competitor for water, nutritive elements and light with regard

to maize crop.

Glyphosate is the active ingredient of broad-spectrum, non-selective, systemic

herbicides. It controls a broad spectrum of weeds that include grasses,

perennials and woody plants. Glyphosate tolerance provides advantages to the

genetically modified maize plants over the traditional varieties.

Once TC1507xNK603 maize is commercially available, the farmers will be able

to use one broad-spectrum herbicide, glyphosate, for the weeding of their maize

fields, and can spread the herbicide at most of the growth stages and according

to the needs. Another advantage is that this herbicide has a weak remnance in

the soil, which is environmentally preferable.

The feasibility of weeding operations for the farmer will be improved. The

elimination of weeds which are usually in competition with maize crops will be

thus more effective, simplified and less time-consuming for this genetically

modified maize, which will contribute to the quality and yield improvement of the

crop.

Best Management Practices that can be recommended to Minimize Weed

Resistance

a) Use of herbicide tolerant crops does not limit growers to use one

herbicide product. Conventional herbicides can and should still be part of

growers overall management system

b) Limit number of applications of single herbicide or herbicides from the

same mode of action to control target weeds

c) Use mixtures or sequential treatments of an effective alternate mode of

action to control target weeds

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d) Use alternative weed management practices, such as crop rotation,

mechanical cultivation, and delayed planting

The weed resistant management strategy that is currently followed in areas

where NK603 is planted will also be followed: This includes conditions outlined

below:

a) Surveillance plans for volunteer plants

b) Grower educational programs

c) Any problems experienced with the modified crop during the growing

season will be reported to the GMO Registrar

(iii) Pathogenic and ecological impacts

Bacillus thuringiensis (Bt) is a naturally occurring soil bacterium which expresses

a variety of proteins with a wide spectrum of activity against agronomically

important pest species. Bt insecticidal proteins have a history of safe use as

both microbial-based sprayable foliar pesticides as well as plant-incorporated

protectants. There is a long history of safe use surrounding the cultivation of

transgenic maize containing the event TC1507. Pathogenic or ecologically

disruptive effects are unlikely as a result of cultivation of TC1507xNK603 maize.

(iv) Effects on human and animal health.

As mentioned in section 8, and as discussed throughout this application for

general release of TC1507xNK603 maize, the agronomy, composition, nutritional

value, allergenicity, and mode of storage, preparation or cooking of

TC1507xNK603 maize is not significantly different from those of commercial

maize. Therefore, there are no health or safety implications specific for the

general release of TC1507xNK603 maize.

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(v) Impacts of the cultivation, management and harvesting techniques

specific to the GMO.

Communication and education plan

Maintaining the effectiveness of TC1507xNK603 maize will depend on good

product stewardship, grower awareness, regular and consistent communication

at all levels of the product chain and grower implementation and support of IRM

recommendations. To be effective, growers must understand the societal and

environmental benefits of preserving the effectiveness of insect protected maize

(Kennedy and Whalon, 1995).

Managing insect and weed resistance is not possible unless a workable IRM and

WRM strategy is implemented by technology providers and growers. Grower

education is the single most important element of any strategy for promoting

compliance to Bt trait IRM and herbicide trait WRM requirements. DuPont

Pioneer uses a comprehensive, multi-faceted approach to communicate IRM and

WRM requirements targeted to both customers and the sales force.

Comprehensive annual training is provided to all DuPont Pioneer dealers. This

training includes trait specific technology information and product management,

importance of IRM, WRM and compliance with IRM and WRM requirements,

expectations for communication of requirements to customers, and all

components of the compliance monitoring program.

To assist with dealer communications, Technology Guides will be provided for

distribution to all DuPont Pioneer Bt and herbicide trait customers.

The obligation for each grower planting more than 5 hectares to comply with the

refuge requirements will be imposed onto growers through signing of a

contractual agreement during purchase of seed containing the Bt technology.

Prior to signing the agreement as well as during cultivation of Bt and herbicide

tolerant crops, the grower must be made aware of the potential for insect and

weed resistance. Regular and consistent communication through appropriate

educational tools at all levels of the product chain is essential for the future of the

technology.

IRM Compliance

Compliance to the IRM strategy by growers planting more than 5 hectares, and in

particular compliance with the refuge requirements, will be monitored through

general compliance monitoring grower visits.

NON CONFIDENTIAL


Mitigation measures to be implemented if localized insect resistance occurs after

commercial introduction of insect protected maize

Monitoring the potential development of insect resistance will primarily be

conducted through surveillance. The first line of surveillance is necessarily the

grower, since the grower, alarmed by decreased product performance or failure

in the field, would likely be the first to alert the seed supplier to suspected

resistant pests. This surveillance is enforced by the requirement within the

contractual agreement, for growers to regularly scout their farms to enable early

detection.

A critical component of overall IRM strategy is field scouting. With Bt products

currently commercialized, growers are encouraged to scout fields and report

unexpected levels of pest damage to DuPont Pioneer. The technology guide that

Pioneer supplies to each customer contains information on monitoring and

reporting unexpected damage.

This scouting and reporting strategy will continue with the commercialization of

TC1507xNK603 maize.

If insect resistance is suspected, specific actions would be triggered. These

actions are described below.

a) Intensifying field surveillance for Bt maize efficacy in and around the potential

“resistance epicenter” to define the boundaries of the affected area.

b) Advise growers to incorporate crop residue into the soil or shred stalks

following harvest to reduce overwintering larvae survival, provided this is

feasible within the grower’s operation

Also refer to requirements in terms of the Environmental Risk Assessment

Framework for Genetically Modified Organisms.

12. COMPLETE THE AFFIDAVIT. The affidavit is an inseparable part of the application form.

The affidavit has been completed.

NON CONFIDENTIAL


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