APPLICATIONS IN TERMS OF THE GENETICALLY MODIFIED ORGANISMS … for commodity... · 2015-02-13 · APPLICATIONS IN TERMS OF THE GENETICALLY MODIFIED ORGANISMS ACT, 1997 APPLICATION

Syngenta Bt11xMIR162 maize

February 2015

CONFIDENTIAL BUSINESS INFORMATION DELETED

©2015 Syngenta. All rights reserved

Syngenta South Africa (Pty) Ltd

APPLICATIONS IN TERMS OF THE GENETICALLY MODIFIED ORGANISMS ACT, 1997

APPLICATION FOR COMMODITY CLEARANCE OF

GENETICALLY MODIFIED ORGANISMS (GMO) STACKED-EVENT MAIZE TRAIT PRODUCT Bt11 x MIR162

This document is complete as of February 2015. Since it is submitted as part of a regulatory application, which is subject to an on-going regulatory review, it may be subject to later amendment or replacement. The information may also be supplemented with additional material requested by regulatory authorities. As such, it may only be considered properly with reference to those later amendments or supplementary materials and in the context of the dossier as a whole. Property rights This document contains information which is proprietary to Syngenta. Without the prior written consent of Syngenta, it may (i) not be used by any third party including, but not limited to, any regulatory authority for the support of registration approval of this product or any other product, and (ii) not be published or disclosed to any third party including, but not limited to, any regulatory authority for the support of registration approval of any products. Confidentiality claim This document is considered to be confidential for all purposes other than compliance with the relevant registration procedures. Its submission does not constitute a waiver of any right to confidentiality that may exist in any other country.

Syngenta Bt11 x MIR162 maize

February 2015


©2015 Syngenta. All rights reserved


February 2015


©2015 Syngenta. All rights reserved 1 of 103

APPLICATION1 FOR COMMODITY CLEARANCE OF GENETICALLY MODIFIED ORGANISMS (GMO) – STACKED-EVENT MAIZE TRAIT PRODUCT Bt11 x MIR162

1 DAFF Version 1.2 August 2013

REPUBLIC OF SOUTH AFRICA DEPARTMENT OF AGRICULTURE

Genetically Modified Organisms Act, 1997 (Act No. 15 of 1997)

DIRECTORATE BIOSAFETY Private Bag X973, Pretoria, 0001

Harvest House, 30 Hamilton Street, Arcadia, Pretoria, 0002 Fax: 012 319 6329 / 6385


February 2015




February 2015



TABLE OF CONTENTS LIST OF TABLES .............................................................................................................. 5 LIST OF FIGURES ............................................................................................................ 5 LIST OF ACRONYMS AND ABBREVIATIONS ................................................................ 6

INTRODUCTION ............................................................................................................... 9 1. BRIEF DESCRIPTION OF THE GENETICALLY MODIFIED ORGANISM......... 11 1.1 Include specific and common names of the organism, the country of origin of the plant

and a description of the genetically modified trait. ........................................................... 11

2. COMMODITY CLEARANCE .............................................................................. 12 2.1 Please indicate the type of clearance requested. ................................................................ 12 2.2 Detail specific instructions for the storage and handling of the plant or plant parts. ........ 14 2.3 When will commodity clearances take place? ................................................................... 14

2.4 Where will commodity clearance take place? ................................................................... 14 2.5 Detail the type of environment and the geographical areas for which the plant is suited.

14 2.6 Who will undertake the commodity clearance? ................................................................. 14

2.7 Estimate the amount of production of the genetically modified plant within South

Africa per annum, or the amount that will be imported into South Africa per annum. ..... 15

3. DESCRIPTION OF ANY PRODUCT DERIVED FROM THE PLANT ................. 16 3.1 Identify the part of the plant to be used for the product, the type of product, and the

use of the product, the market sector in which the product will be marketed and the

trade name of the product. ................................................................................................. 16 3.2 Specify the exact conditions of use of the product. ........................................................... 20

3.3 Provide information on the proposed labelling of the product for marketing. .................. 20 3.4 State whether the benefits of the product are available in any other non-genetically

modified form. ................................................................................................................... 20 3.5 Detail specific instructions for the storage and handling of GMO's that will avoid

misuse or escape of the genetically modified plant into an environment for which it

was not intended. ............................................................................................................... 20

3.6 Detail the likelihood of the GMO being exported from South Africa, particularly if

such export could result in the introduction of the plant into its centre of origin. ............. 20

4. FOREIGN GENES AND GENE PRODUCTS ..................................................... 22 4.1 Identify all foreign genes in the genetically modified plant. ............................................. 22 4.2 Describe the gene products that are derived from the foreign genes. ................................ 30

4.3 Describe the biological activity associated with the foreign gene products. ..................... 30 4.4 Provide information on the rate and level of expression of the foreign genes and the

sensitivity of the measurement of the rate and level. ......................................................... 32 4.5 Provide protocols for the detection of the foreign genes in the environment including

sensitivity, reliability and specificity of the techniques. .................................................... 40

5. RESISTANCE ..................................................................................................... 42 5.1 Detail whether the genetically engineered plant is able to initiate resistance, in any

biotic component of the environment, to any biologically active foreign gene product. .. 42 5.2 Detail what methods are available to minimise the risk of resistance developing in the

environment. ...................................................................................................................... 42 5.3 Detail how resistance will be managed during release of the genetically modified plant.

42

6. HUMAN AND ANIMAL HEALTH ........................................................................ 43


February 2015



6.1 State whether the genetically modified plant or its products will enter human or

animal food chains. ............................................................................................................ 43 6.2 Detail the results of experiments undertaken to determine the toxicity/ allergenicity of

the foreign gene products (including marker genes) to humans and animals. ................... 43 6.2.1 Possible modes of exposure: whole GM plant or crop 43

6.2.2 Information relating to the genetic modified plant 44 6.2.3 Food Safety Risk Assessment (Codex Alimentarius Commission) 47

6.2.3.1 Toxicology, allergenicity and pathogenicity ................................................... 47 6.2.3.2 Comparative safety assessment ...................................................................... 54 6.2.3.3 Exposure assessment ...................................................................................... 59 6.2.4 Other concerns related to human health 60

6.2.4.1 Genetic stability of insert and phenotypic stability of GM plant ................... 60 6.2.4.2 Gene transfer, antibiotic resistance ................................................................ 60

6.2.4.3 Susceptibility of humans exposed to the GMO .............................................. 65 6.2.4.4 Effect of processing .......................................................................................... 65 6.3 If the foreign gene products are toxic or allergenic in any way, detail how the

Commodity clearance will be managed to prevent contact with animals or humans that

will lead to discomfort or toxicity...................................................................................... 66 6.4 What are the common/major allergens present in the recipient organism before

modification? ..................................................................................................................... 66 6.5 What evidence is there that the genetic modification described in this application did

not result in over-expression of the possible allergens indicated in 6.4? .......................... 67

6.6 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? .................................................................................... 68 6.7 Indicate the proposed health and safety measures that would be applied to safeguard

employees during the proposed activity. ........................................................................... 69

7. ENVIRONMENTAL IMPACT AND PROTECTION ............................................. 70 7.1 Detail any long-term effect the Commodity clearance of the genetically modified

organism is likely to have on the biotic and abiotic components of the environment. ...... 70

7.2 Provide data and information on ecosystems that could be affected by use of the plant

or its products. .................................................................................................................... 72 7.3 Specify what effect the general release of the genetically modified plant will have on

biodiversity. ....................................................................................................................... 72 7.4 Specify the measures to be taken in the event of the plant or product being misused or

escaping into an environment for which it is not intended. ............................................... 73 7.5 If the foreign genes give rise to crops resistant to agrochemicals, provide information

on the registration of the agrochemicals to be used on the crop. ....................................... 73

8. SOCIO-ECONOMIC IMPACTS ........................................................................... 74 8.1 Specify what, if any, positive or negative socio-economic impacts the genetically

modified plant will have on communities in the proposed region of release. ................... 74

9. WASTE DISPOSAL ............................................................................................ 76 9.1 Where only a portion of the genetically modified plant will be used for the product,

how will the unused plant parts be disposed of? ............................................................... 76

10. MONITORING AND ACCIDENTS ...................................................................... 77 10.1 Indicate the methods and plans for monitoring of the GMO ............................................. 77 10.2 Indicate any emergency procedures that will be applied in the event of an accident in a

comprehensive contingency plan ....................................................................................... 77

11. PATHOGENIC AND ECOLOGICAL IMPACTS .................................................. 78


February 2015



11.1 Submit an evaluation of the foreseeable impacts, in particular any pathogenic and

ecologically disruptive impacts. ........................................................................................ 78

12. RISK MANAGEMENT ........................................................................................ 79 12.1 Please indicate any risk management measures that would be required for Commodity

clearance. ........................................................................................................................... 79

13. APPLICATION AFFIDAVIT ................................................................................ 81 14. RISK ASSESSMENT OF Bt11 x MIR162 MAIZE ..................................................... 83 15. RISK ASSESSMENT AFFIDAVIT ...................................................................... 95 16. REFERENCES .................................................................................................... 97 LIST OF TABLES Table 1. Approved Syngenta applications in RSA that included the Bt11 and MIR162 maize events __ 10 Table 2: Authorisation for use of Bt11 x MIR162 maize and single events thereof, in various countries 12 Table 3. Donor genes and regulatory sequences in pZO1502 (Bt11 maize transformation vector). ___ 24 Table 4. Donor genes and regulatory sequences in pNOV1300 (MIR162 maize transformation vector) 27 Table 5a Comparisons of Cry1Ab concentrations in tissues of a Bt11 maize hybrid and the Bt11 x MIR162

maize hybrid on a dry weight (DW) basis _________________________________________ 35 Table 5b Comparisons of PAT concentrations in tissues of a Bt11 maize hybrid and the Bt11 x MIR162

maize hybrid on a DW basis ___________________________________________________ 36 Table 5c Comparisons of Vip3Aa20 concentrations in tissues of a MIR162 maize hybrid and the

Bt11 x MIR162 maize hybrid on a DW basis ______________________________________ 37 Table 5d Comparisons of PMI concentrations in tissues of a MIR162 maize hybrid and the Bt11 x MIR162

maize hybrid on a DW basis ___________________________________________________ 38 Table 6 Anticipated intake of Cry1Ab, PAT, Vip3Aa20, and PMI proteins from consumption of

Bt11 x MIR162 maize in South Africa ____________________________________________ 60 LIST OF FIGURES Figure 1. Map of pZO1502 used in the transformation of Bt11 maize ........................................................... 23 Figure 2. Map of pNOV1300 used in the creation of MIR162 maize .............................................................. 26


February 2015



LIST OF ACRONYMS AND ABBREVIATIONS A. ipsilon : Agrotis ipsilon ASB: African stemborer A. tumefaciens: Agrobacterium tumefaciens B. thuringiensis (Bt): Bacillus thuringiensis B.C.: Before Christ BCW: black cutworm BLAST: Basic Local Alignment Search Tool bp: base pair CaMV: cauliflower mosaic virus CBI: Confidential Business Information CE: corn earworm CFIA: Canadian Food Inspection Agency C. partellus: Chilo partellus DDGS: Dried Distiller’s Grains with Solubles EURL-GMFF: European Union Reference Laboratory for GM Food and Feed DNA: deoxyribonucleic acid dw: dry weight E. coli: Escherichia coli ECB: European corn borer EFSA: European Food Safety Authority ELISA: enzyme-linked immunosorbent assay FAO: Food and Agriculture Organization FARRP: Food Allergy Research and Resource Program FAW: fall army worm FIFRA: Federal Insecticide, Fungicide, and Rodenticide Act fw: fresh weight g: gram GM: genetically modified GMO: genetically modified organism H. zea: Heliothis zea ILSI: International Life Sciences Institute IVS: Intervening intron sequence kg: kilogram LB: left border LOD: limit of detection LOQ: limit of quantification MCB: Mediterranean corn borer mg: milligram NA: not applicable NCBI: National Center for Biotechnology Information NOEL: no-observed-effect-level NOS: nopaline synthase OECD: Organisation for Economic Co-operation and Development O. nubilalis: Ostrinia nubilalis ori: origin of replication P: probability PAT: phosphinothricin N-acetyltransferase


February 2015



PCR: polymerase chain reaction PEP: phosphoenolpyruvate PMI: phosphomannose isomerase PSB: Pink stemborer RB: right border RSA: Republic of South Africa S. albicosta: Striacosta albicosta S. calamistis : Sesamia calamistis se: sugary enhanced types S. frugiperda: Spodoptera frugiperda SGF: simulated mammalian gastric fluid sh2: supersweet types S. nonagrioides: Sesamia nonagrioides SSB: Spotted stemborer su: sugary types S. viridochromogenes: Streptomyces viridochromogenes US EPA: United States Environmental Protection Agency USA: United States of America Vip: vegetative insecticidal protein WBC: western bean cutworm WHO: World Health Organization Z. mays: Zea mays ZmUbiInt: maize ubiquitin μg: microgram


February 2015




February 2015



APPLICATION FOR COMMODITY CLEARANCE OF GENETICALLY MODIFIED ORGANISMS (GMO)

STACKED-EVENT MAIZE TRAIT PRODUCT Bt11 x MIR162

All scientific and technical information that is considered confidential business information, based on Section 36(1) of the Promotion of Access to

Information Act, 2000 (Act No. 2 of 2000) was deleted. INTRODUCTION The stacked-event maize product Bt11 x MIR162 maize (Zea mays L., corn) (hereafter referred to as ‘Bt11 x MIR162 maize’) is a genetically modified (GM) maize that is produced by conventional breeding of the following GM maize events: Bt11 (hereafter referred to as ‘Bt11 maize’) and MIR162 (hereafter referred to as ‘MIR162 maize’). Bt11 x MIR162 maize expresses 1) the insecticidal protein Cry1Ab and enzyme phosphinothricin acetyltransferase (PAT; that confers tolerance to glufosinate-ammonium in herbicide products), present in Bt11 and 2) the insecticidal protein Vip3Aa20 and the enzyme phosphomannose isomerase (PMI), present in MIR162. Bt11 x MIR162 maize allows farmers to effectively control important lepidopteran maize pests such as Ostrinia nubilalis (European corn borer; ECB) and Agrotis ipsilon (black cutworm; BCW), and to use herbicide products containing glufosinate-ammonium. This application under the Genetically Modified Organisms (GMO) Act (Act 15) of 1997 as amended by the GMO Act, 2006 (Act 23 of 2006) includes the import, food and feed use and processing of Bt11 x MIR162 maize. It does not include cultivation. A detailed description of the scope of the authorisation requested can be found in Section 2.1. Commercial seed will be marketed outside South Africa. Where cultivated, the intended use of Bt11 x MIR162 maize is to provide insect and weed control solutions. Various applications under the GMO Act (Act 15) of 1997 for the authorisation of Syngenta maize have previously been assessed and approved by the Executive Council. This application includes data to facilitate the safety assessment of Bt11 x MIR162 maize and derived food and feed products, and to confirm the conclusions reached in previous safety assessments of the above mentioned single events and proteins Cry1Ab, PAT, Vip3Aa20, and PMI. Some of the data presented in this application represents updated information of that previously provided. To facilitate the review, Table 1 indicates where the information provided in this application has been previously assessed and approved by the Executive Council.


February 2015



Table 1. Approved Syngenta applications in RSA that including Bt11 and

MIR162 maize events

Events proteins

Reference number

Permit number Type Date

Bt11

Cry1Ab, PAT

17/3/1-Novartis-00/003

17/3(6/02/017)

Commodity clearance

1/02/2002

Bt11

Cry1Ab, PAT

17/3/1-Syngenta-

02/014 17/3(5/03/067)

General release

26/92003

Bt11xGA21

Cry1Ab, PAT, mEPSPS

17/3/1-Syngenta-

10/109 39.4(5/10/375)

General release

14/12/2010

Bt11xGA21

Cry1Ab, PAT, mEPSPS

17/3/1- Syngenta

06/082 39.4(6/11/260)

Commodity clearance

26/9/2011

Bt11xMIR604xGA21

Cry1Ab, PAT, PMI, Cry3A, mEPSPS

17/3/1- Syngenta

08/097 39.4(6/11/263)

Commodity clearance

26/9/2011

Bt11x MIR162xMIR604x GA21

Cry1Ab, PAT, Vip3Aa20, PMI, Cry3A, mEPSPS

17/3/1- Syngenta

09/100 39.4(6/11/264)

Commodity clearance

26/9/2011

Bt11xMIR162xGA21

Cry1Ab, PAT, Vip3Aa20, PMI, mEPSPS

17/3/1- Syngenta

09/101 39.4(6/11/265)

Commodity clearance

26/9/2011

Bt11x MIR162xTC1507x GA21

Cry1Ab, PAT, Vip3Aa20, PMI, Cry1F,mEPSPS

39.4.1/ Syngenta

10/112 39.4(6/11/266)

Commodity clearance

26/9/2011

Bt11x MIR162xMIR604xGA21

Cry1Ab, PAT, Vip3Aa20, PMI, Cry3A, mEPSPS

17/3/1- Syngenta

09/100 39.4(6/11/264)

Commodity clearance

26/9/2011

MIR162

Vip3Aa20, PMI

39.4.1/ Syngenta –

13/123 39.4(6/14/046)

Commodity clearance

11/3/2014

Bt11xMIR162xMIR604xTC1507x5307xGA21

Cry1Ab, PAT, Vip3Aa20, PMI, Cry3A, Cry1F, eCry3.1Ab, mEPSPS


11/113 39.4(6/14/049)

Commodity clearance

24/3/2014

Bt11xMIR604xTC1507x5307xGA21

Cry1Ab, PAT, PMI, Cry3A, Cry1F, eCry3.1Ab, mEPSPS


11/114 39.4(6/14/050)

Commodity clearance

24/3/2014

Bt11 x DAS591227 x MIR604 x TC1507 x GA21

Cry1Ab, PAT, Cry34Ab1,Cry35Ab1

PMI, Cry3A, Cry1F, mEPSPS


11/115 39.4(6/14/054)

Commodity clearance

24/3/2014


February 2015



1. BRIEF DESCRIPTION OF THE GENETICALLY MODIFIED ORGANISM 1.1 Include specific and common names of the organism, the country of origin of the plant and a description of the genetically modified trait. Syngenta developed Bt11 x MIR162 maize by combining two individual transformation events through conventional breeding. This stacked-event maize provides control of certain lepidopteran insect pests and tolerance to glufosinate-ammonium in herbicide products. Maize plants derived from Bt11 maize contain the transgene cry1Ab, which encodes the insecticidal protein Cry1Ab, and the transgene pat, which encodes the enzyme PAT. The native, full-length Cry1Ab produced by the soil bacterium Bacillus thuringiensis subsp. kurstaki is active against certain lepidopteran pests of maize, including O. nubilalis (ECB) and Sesamia nonagrioides (Mediterranean corn borer; MCB). The Cry1Ab produced by Bt11 maize is a truncated version of native Cry1Ab that retains activity against lepidopteran insects. The gene pat was derived from the soil bacterium Streptomyces viridochromogenes. PAT acetylates glufosinate-ammonium, thus inactivating it and conferring tolerance to glufosinate-ammonium in herbicide products. PAT was used as a selectable marker in the development of Bt11 maize. Maize plants derived from MIR162 maize contain the transgene vip3Aa20, which encodes the insecticidal protein Vip3Aa20, and the transgene pmi, which encodes the enzyme phosphomannose isomerase (PMI). Vip3Aa20 is a variant of the native Vip3Aa1 protein from the soil bacterium B. thuringiensis strain AB88, and is active against certain lepidopteran pests of maize, including Spodoptera frugiperda (fall armyworm; FAW), A. ipsilon (BCW) and Helicoverpa zea (corn earworm; CE). The gene pmi (also known as manA) was derived from Escherichia coli strain K-12. PMI enables transformed plant cells to utilize mannose as a primary carbon source; it was used as a selectable marker in the development of MIR162 maize. Accordingly, Bt11 x MIR162 maize produces the transgenic proteins, Cry1Ab, PAT, Vip3Aa20, and PMI present in the two individual GM maize events.

o family name: Poaceae (formerly Gramineae) o genus: Zea o species: Z. mays L. o subspecies: Z. mays subsp. mays o event/product: Bt11 x MIR162 o common name: Maize, corn o unique identifier: SYN-BTØ11-1 -9 x SYN-IR162-4 o origin: Maize originates from the Meso-American region, i.e. Mexico

and Central America (CFIA, 2003)


February 2015



2. COMMODITY CLEARANCE 2.1 Please indicate the type of clearance requested. This is an application for the commodity clearance, i.e. full food, feed and processing approval, of Bt11 x MIR162 maize in South Africa. This maize is not at present intended to be produced or cultivated in South Africa. Although Bt11 x MIR162 maize is intended for cultivation outside South Africa, derived products (including grain) of this maize may be commingled with derived products from conventional maize and therefore enter South Africa through the trade routes. See below a list of current authorizations for Bt11 x MIR162, Bt11 and MIR162 per country (Table 2). Table 2: Authorisation for use of Bt11 x MIR162 maize and single events thereof, in various countries

Event Country Type of Approval Year of Approval

Bt11 Argentina All uses July 2001

Bt11 Australia/New Zealand

Food August 2001

Bt11 Belarus/ Kazakhstan

Food July 2011

Bt11 Brazil All uses July 2008

Bt11 Canada Food August 1996

Bt11 Canada Feed June 1996

Bt11 Canada Cultivation May 1996

Bt11 China Food, feed, processing April 2004

Bt11 Colombia Feed, food, cultivation February 2008 (feed), April 2009 (food), May 2008 (cultivation)

Bt11 EU Food, feed, processing June 1998

Bt11 Indonesia Food September 2011

Bt11 Japan Food, feed, environment September1996 (Food, Feed), October 1996 (Environment)

Bt11 Korea, Republic Of

Food, feed and environment

December 2003 (food), February 2006 (feed and environment)

Bt11 Malaysia Food, feed, processing March 2012

Bt11 Mexico Food and feed July 2007

Bt11 Paraguay Food, feed, cultivation October 2012

Bt11 Philippines Food, feed, processing, cultivation

July 2003 (food, feed, processing),

April 2005 (cultivation)

Bt11 Russian Federation

Food, feed September 2003 (food), December 2006 (feed)


February 2015



Event Country Type of Approval Year of Approval

Bt11 South Africa Cultivation, food/feed/processing

February 2002 (food, feed, processing),

June 2003 (cultivation)

Bt11 Switzerland Food, feed October 1998

Bt11 Taiwan Food June 2004

Bt11 Turkey Feed December 2011

Bt11 United States Food, feed, processing (FDA), Cultivation (USDA),

Environment (EPA)

January 1996 (Cultivation)

May 2006 (Food, Feed, Processing)

August 2006 (Environment)

Bt11 Uruguay Food, Feed, Cultivation May 2004

Bt11 Vietnam Food, Feed, Cultivation August 2014 (Food, Feed), January 2015 (Cultivation)

MIR162 Argentina food, feed, cultivation May 2011

MIR162 Australia food February 2009

MIR162 Belarus food June 2011

MIR162 Brazil food, feed, cultivation September 2009

MIR162 Canada food, feed, cultivation February 2010 (Cultivation, Feed)

March 2010 (Food)

MIR162 Colombia cultivation December 2010 (feed), June 2012 (food), September 2012 (cultivation)

MIR162 European Union

food, feed, processing October 2012

MIR162 Indonesia food August 2011

MIR162 Japan food, feed, cultivation January 2010 (Food), June 2010 (Cultivation, Feed)

MIR162 Kazakhstan food June 2011

MIR162 Korea, Republic Of

food, feed and environment October 2010 (Food), June 2010 (Feed, Environment)

MIR162 Mexico food, feed, processing January 2010

MIR162 New Zealand food February 2009

MIR162 Philippines food, feed, processing February 2010

MIR162 Russian Federation

food June 2011 (food), March 2012 (feed)

MIR162 Taiwan food April 2009

MIR162 South Africa Food, feed, processing March 2014

MIR162 United States Food, feed, processing (FDA), Cultivation (USDA),

Environment (EPA)

December 2008 (food, feed, processing), April 2010 (cultivation), November 2008 (Environment)

MIR162 Uruguay food, feed, cultivation September 2012

MIR162 Vietnam Food, feed August 2014

Bt11xMIR162 Argentina Cultivation October 2011

Bt11xMIR162 United States Environment (EPA) February 2009

Bt11xMIR162 Philippines food, feed, processing October 2013

Bt11xMIR162 Japan food, cultivation March 2010 (Food), April 2014 (Cultivation)


February 2015



2.2 Detail specific instructions for the storage and handling of the plant or plant parts. There are no specific differences between Bt11 x MIR162 maize compared to conventional maize, except for expression of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins. Bt11 x MIR162 maize can be considered as safe as conventional maize and the same practices used for conventional maize would be used for Bt11 x MIR162 maize. Similarly, grain of Bt11 x MIR162 maize could be stored and handled in the same manner as non-genetically modified maize and other genetically modified maize grain already approved for importation into South Africa. 2.3 When will commodity clearances take place? Commodity clearances are done by various grain traders on the international market, depending on the local need in South Africa. These traders would, as per the requirements in terms of the GMO Act (Act 15) of 1997, obtain the necessary permits prior to importation. 2.4 Where will commodity clearance take place? As indicated in Section 2.3 above, commodity clearances are done by various grain traders on the international market, depending on the local need in South Africa. Commodity clearances mainly take place through the different sea ports and would be used as food or feed in all the areas where commercial maize grain is normally sold for these purposes, e.g. the North West Province, Free State, Limpopo Province, Mpumalanga, KwaZulu-Natal and the Eastern/Western Cape regions. 2.5 Detail the type of environment and the geographical areas for which the plant is suited. This is an application for commodity clearance, i.e. full food, feed and processing approval, of Bt11 x MIR162 maize in South Africa. This application is not for general release of Bt11 x MIR162 maize in South Africa. Grain of Bt11 x MIR162 maize could be used as food or feed in all the areas where commercial maize grain is normally sold for these purposes, e.g. the North West Province, Free State, Limpopo Province, Mpumalanga, KwaZulu-Natal and the Eastern/Western Cape regions. 2.6 Who will undertake the commodity clearance? As indicated in Section 2.3 above, commodity clearances are done by various grain traders on the international market, depending on the local need in South Africa.


February 2015



2.7 Estimate the amount of production of the genetically modified plant within South Africa per annum, or the amount that will be imported into South Africa per annum. This is an application for commodity clearance, i.e. full food, feed and processing approval, of Bt11 x MIR162 maize in South Africa. Bt11 x MIR162 maize is not at present intended to be produced or will not be cultivated in South Africa. The amount of Bt11 x MIR162 maize grain in a consignment imported into South Africa will depend on the extent of cultivation of this maize in the country from where the grain will be imported and the local demand.


February 2015



3. DESCRIPTION OF ANY PRODUCT DERIVED FROM THE PLANT 3.1 Identify the part of the plant to be used for the product, the type of product, and the use of the product, the market sector in which the product will be marketed and the trade name of the product. South Africa imports maize, primarily as grain, depending on local surpluses and shortages, as well as regional demand. This grain is normally used to produce food and feed products. Grain of Bt11 x MIR162 maize might be imported under the same circumstances and used for the same purposes. Maize has three possible uses: as food, as feed for livestock and as raw material for industry. As a food, the whole grain, either mature or immature, may be used; or the maize may be processed by dry milling techniques to give a relatively large number of intermediary products, which can in turn have a great number of applications in a large variety of foods; wet milling is a process applicable mainly in the industrial use of maize. The various food and feed products that can be derived from maize (OECD, 2002; FAO, 2003), including Bt11 x MIR162 maize, are described in the paragraphs below. a) Primary food uses of field maize

Historically, maize grain has been used by the indigenous people of the Western Hemisphere. Traditional foods include atole and masa of Latin America; arepa of Colombia; and hominy of South-Eastern United States. In South Africa, during the 19th century, whole ground maize meal was provided by small mills scattered throughout the country. Urbanization led to the development of milling systems that provided low fat meal with a prolonged shelf-life. Maize is used as a basic food item by people throughout the world, especially in areas of subsistence agriculture. Traditional hand milling or small scale stone milling is carried out in many areas. The maize meal produced is generally used by local populations, and is used to prepare breads, snacks, porridge and fermented products. Maize constitutes a staple food in many regions of the world. Introduced into Africa by the Portuguese in the 16th century, maize has become Africa's most important staple food crop. Maize meal is made into a thick porridge in many cultures: from the polenta of Italy, the angu of Brazil, the mămăligă of Romania, to cornmeal mush in the USA, pap in South Africa and sadza, nshima and ugali in other parts of Africa. Maize meal is also used as a replacement for wheat flour, to make cornbread and other baked products. Masa is the main ingredient for tortillas, atole and many other dishes of Mexican food. Popcorn consists of kernels of certain varieties that explode when heated, and are eaten as a snack. Roasted dried maize cobs with semi-hardened kernels, coated with a seasoning mixture of fried chopped spring onions with salt added to the oil, is a popular snack food in Vietnam. Cancha, which are roasted maize chulpe kernels, is a very popular snack food in Peru, and also appears in traditional Peruvian ceviche. Unleavened bread, called makki di roti, is a popular bread eaten in the Punjab region of India and Pakistan.


February 2015



Specific to South Africa is the popular use of maize as “green mielies”. These are maize cobs harvested before full maturity (still green) and cooked or roasted for direct consumption. Corn on the cob is also a common dish in USA, Canada, UK, Cyprus and South America. Corn flakes are a common breakfast cereal in many countries all over the world. Maize can also be prepared as hominy, in which the kernels are soaked with lye in a process called nixtamalization; or grits, which are coarsely-ground hominy. This is commonly eaten in the South Eastern United States and is a dish handed down from Native Americans, who called the dish sagamite. The Brazilian dessert canjica is made by boiling maize kernels in sweetened milk.

b) Products from Wet Milling

The maize kernel is composed of the pericarp, germ and endosperm. Processes have been devised to separate these components of the maize kernel, with wet milling as the most important one. Maize kernels prepared for wet milling will be steeped in hot water and sulphur dioxide to soften the pericarp. The softened kernel is cracked by machine and hydrocloned to separate the germ from the endosperm.

The germ portion is pressed to separate the oil that is used in food, i.e. salad or cooking, and for margarine. The chemical composition of maize oil, which has low levels of linolenic acid and adequate levels of tocopherols, contribute to maize oil's good stability during storage and cooking. Maize oil has a high level of polyunsaturated fatty acids that makes it favourable for health and nutritional considerations.

The pressed germ is dried and added to maize gluten feed. Fibrous hulls, removed from the endosperm portion, are also added to maize gluten feed. The gluten portion is dried and used as maize gluten meal. Starch is the primary product of maize wet milling. Starch can be used to produce a variety of foods. Modified starch includes white and yellow dextrin, roasted or dextrinated starch, starch modified by acid or alkali treatment, bleached starch, physically modified starch and starch treated by amylolitic enzymes. About 40% of the starch is consumed as food, or industrial purposes, while 60% is converted to sweeteners. Maize syrups are high in fructose, dextrose and maltodextrins. Examples of food uses of some maize starches include alcoholic beverages, baked goods, cereals, sweets, fats and oils, ice cream and syrups and "Corn flour". Furthermore, dry milling and wet milling processes are used for the production of ethanol or gasohol from maize. Approximately one third of gasohol is produced by the dry milling process and two thirds by the wet milling process. Over 70 percent of the products produced from these processes are in the form of starch that is used for ethanol production. The remaining material, which comprises of about 11 percent of cellulose, hemicelluloses, leftover starch and sugars, are used to make animal feed supplements (FAO, 2003).

c) Products from Dry Milling

Maize dry milling is a mechanical process for separating the embryo and pericarp from the endosperm, followed by dry milling fractionation of the endosperm into


February 2015



coarse particles (grits). Dry milling is also used to produce a wide variety of food and non-food products. Except for the maize being eaten as kernel on the cob and popcorn, all other maize products are based on milled maize.

Food products derived from dry milling includes hominy, grits, meal and flour, which are produced from the hard endosperm and are devoid of embryo and pericarp. Flaking grits are used to produce corn flakes which are one of the most popular ready-to-eat breakfast cereals in the world. Maize flakes are produced by a process that involves high temperature and pressure which gelatinize the starch and denatures the protein of the grit. Coarse grits and medium grits are used to manufacture cereal products and snack foods. Maize grits or hominy grits are mid-sized endosperm particles primarily consumed as a side dish for breakfast. Maize grits are prepared by boiling; commercial "instant" grits are made by steaming, cooking and drying maize grits. Fine grits is used as a brewing adjunct by the brewing industry. Maize grits can also be used for the manufacturing of wallpaper paste and glucose by chemical hydrolysis. Coarse or granulated maize is used in pancake and muffin mixes, corn snacks, cereal products and other baking products. Fine maize meal is used for maize bread, infant’s foods and breakfast cereal. Maize flour is used for bread and pancake mixes, infant’s foods, biscuits, wafers, as filler and carriers in meat products, and in breakfast cereals. Bran is a by-product of this process used as a dietary source of fibre.

d) Products from the Distilling Industry.

Maize grits and whole kernels are used to produce liquor. Whisky and gin are the main "pure" products from maize distilling. Chicha and chicha morada are drinks typically made from particular types of maize. Chicha is fermented and alcoholic, whereas chichi morada is a soft drink commonly consumed in Peru.

e) Feed Processing

Animal feed is produced as a by-product of milling, or the whole plant may be used for animal feed. The material can be fed directly, or preserved. Gluten meal and gluten feed are by-products of the wet processing of maize. Maize grain is usually ground, rolled, pelleted or extruded. Corn Hominy Feed is a by-product of the dry corn milling process. With a typical analysis of 9% protein (minimum), 4% fat (minimum), and 6% fiber (maximum), hominy is an excellent feed for all animals. Hominy looks and handles similar to ground corn, is a great energy source and is very palatable.

f) Uses of Sweet maize

Sweet maize has been bred from field maize. Genetics offer specific adaptations to define preferences for processing and taste. Consumer demands for higher sugar content, improved sweetness and increased flavour has led to the production of new varieties of maize. There are three main categories of sweet maize:

o "sugary types" (su), also called "normal" or "standard"


February 2015



o "sugary enhanced types" (se); o "super sweet types" (sh2), also called "shrunken" or "super sweet".

Almost all the sweet maize varieties developed since the mid 1930's until the seventies were sugary hybrids. This denomination comes from their sweetness, which is a result of the presence of the sugary gene (su gene). Field maize does not have this gene and stores energy for the germinating embryo by depositing starch in the endosperm. Starch in field maize is synthesized from sugars, mainly sucrose, that is produced in the leaves and translocated to the seeds during the grain filling stage. All types of sweet maize disrupt this process of starch synthesis, resulting in the accumulation of sugar in the endosperm that is not rapidly converted to starch. This confers the sweet taste of sweet maize, whereas field maize has a "starchy" taste. Sugary enhanced hybrids carry the se gene, which is different from the su gene. It is a "modifier", which only has an effect when the su gene is also present. It changes the effects of the su gene and results in higher sugar content in the plant (hence the name "sugar enhancer"). The growth of se hybrids does not require isolation from sugary hybrids. Pollination by sugary varieties will produce normal sugary kernels on the ears. Maximum expression of these qualities will be observed when pollen comes from a se variety.

In 1974, the shrunken-2 (sh2) gene was discovered. The presence of this gene in a line confers 50% more sugar compared to the presence of the su gene. Unlike the su gene, the sh2 gene is independent and recessive. Cross-pollination of a sh2 hybrid by a non-sh2 hybrid will produce kernels of the "starch type". The su and se hybrids are mainly used for canning as it is possible to adjust taste before sterilization with additional sugar/salt. Sh2 hybrids are increasingly grown for the fresh market (they represent 75% of this particular market) and for the frozen food industry.

Today, sweet maize is grown mainly in the Americas, Europe, Asia, Eastern Europe, Israel and Africa.

g) Products from dry-grind ethanol processing

During this process, the starch contained in the maize grain is hydrolysed into glucose which is then used to produce ethanol by fermentation. Starch is a major component of maize grain. It consists of a mixture of amylose (linear glucose chains) and amylopectin (branched glucose chains) and is processed using enzymes such as alpha-amylases, glucoamylases, pullulanases, alpha-glucosidases and glucose isomerases, depending on the intended use. Dry-grind ethanol production involves starch enzymatic hydrolysis steps, a fermentation step and a distillation step and produces three final co-products: fuel ethanol, CO2 and Dried Distiller’s Grains with Solubles (DDGS). The co-products obtained after the removal of ethanol by distillation from the yeast fermentation are DDGS, which can be used either separately or combined. DDGS are commonly


February 2015



used in animal feed, with the distillers grains either in a wet or dry form. DDGS is high in protein, fibre and fat content and is sold as an animal feed ingredient, primarily in ruminant diets.

3.2 Specify the exact conditions of use of the product. Bt11 x MIR162 maize grain can be used in the same manner as any other commercial maize grain. 3.3 Provide information on the proposed labelling of the product for marketing. Commodity clearances are done by various grain traders on the international market, depending on the national need in South Africa. Imported grain would be labelled according to the South African labelling requirements. 3.4 State whether the benefits of the product are available in any other non-genetically modified form. If so, state why the genetically modified 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 Bt11 x MIR162 maize. This is not an application for general release. Seed of Bt11 x MIR162 maize will not be sold or commercially grown in South Africa. This is an application for commodity clearance. Grain of this stacked product could be contained in imported grain consignments when entering the South African market. The use of Bt11 x MIR162 maize grain would be the same as with any other grain imported into South Africa. 3.5 Detail specific instructions for the storage and handling of GMO's that will avoid misuse or escape of the genetically modified plant into an environment for which it was not intended. The presence of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins in Bt11 x MIR162 maize does not change any of the typical crop characteristics of this maize. Grain of Bt11 x MIR162 maize can be stored and handled in the same manner as commercial maize. The Bt11 x MIR162 maize will be labelled clearly according to the South African labelling requirements. 3.6 Detail the likelihood of the GMO being exported from South Africa, particularly if such export could result in the introduction of the plant into its centre of origin. Field maize is a grain crop domesticated by indigenous people in Mesoamerica during prehistoric times. Between 1700 and 1250 B.C., the crop spread through large regions of the Americas. After European contact with the Americas in the late 15th and early 16th centuries, explorers and traders carried maize back to Europe and introduced it to other countries. Maize spread to the rest of the world due to its ability to grow in diverse climates.


February 2015



The probability of South Africa exporting Bt11 x MIR162 maize grain, in particular to Central America, the centre of origin for maize, is unlikely since commodity grain is normally not re-exported to Central America.


February 2015



4. FOREIGN GENES AND GENE PRODUCTS 4.1 Identify all foreign genes in the genetically modified plant. Bt11 x MIR162 maize is a GM maize that is produced by conventional breeding of the following GM maize events: Bt11 and MIR162 maize. No further genetic modification to produce this stack has taken place. Maize plants derived from Bt11 maize contain the transgene cry1Ab, which encodes the insecticidal protein Cry1Ab, and the transgene pat, which encodes the enzyme PAT. Maize plants derived from MIR162 maize contain the transgene vip3Aa20, which encodes the insecticidal protein Vip3Aa20, and the transgene pmi, which encodes the enzyme PMI. The foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual Bt11 and MIR162 maize. Accordingly, Bt11 x MIR162 maize produces the transgenic proteins, Cry1Ab, PAT, Vip3Aa20, and PMI that provide control of certain lepidopteran insect pests and tolerance to glufosinate-ammonium in herbicide products. Molecular analyses were performed to characterize the maize inserts. As indicated in Section 2.1, Table 1, information on gene and protein sequences as well as molecular characterisation has already been provided by Syngenta for Bt11 and MIR162 maize for assessment under the South African GMO Act (Act No. 15 of 1997). These dossiers were assessed and approved by the Advisory Committee and Executive Council of South Africa prior to this application. However, for ease of reference, a summary of the information previously provided is provided together with the information on Bt11 x MIR162 maize. Elements expected to be transferred to the plant cell and integrated into the plant genome during T-DNA transfer are categorized by the gene cassette in which they are contained. The elements of the plasmid necessary for its replication and selection in various bacterial hosts are categorized as plasmid backbone. These elements were not expected to be transferred to the plant cell and integrated into the plant genome during T-DNA transfer. The left and right borders are categorized as border regions since only a portion of each border is expected to be integrated into the plant genome (Tzfira et al. 2004).

a) Bt11 maize

Bt11 maize was produced using a protoplast transformation/regeneration system (Negrutiu et al., 1987). The NotI restriction fragment of vector pZO1502, a derivative of plasmid pUC18, was used for the transformation. The genetic elements in plasmid pZO1502, the Bt11 transformation plasmid, are listed in Table 3 and shown in Figure 1. Table 3 contains a description of each constituent of plasmid pZO1502, including the size in base pairs (bp) and the position within the plasmid. A vector map of pZO1502 is shown in Figure 1. The size, function and donor organism of each element of the NotI fragment is summarized in Table 4. The NotI fragment contains a truncated Bt gene which has been derived from the cry1Ab gene of B. thuringiensis; this gene is under the control of the 35S promoter from the CaMV, including the intervening intron sequence IVS6 from maize, and the NOS terminator from Agrobacterium tumefaciens. It contains the pat gene from S. viridochromogenes encoding a phosphinothricin acetyl transferase; this gene is under the control of the 35S promoter from the CaMV, including the intron IVS2


February 2015



pZO1502

7240 bp

bp

cryIAb (1848 bp)

pat (552 bp) amp (861 bp)

IVS6-ADH1 (471 bp)

IVS2-ADH1 (180 bp)

35S promoter (509 bp)

35S promoter (418 bp)

ColE1 ori (674 bp)

NOS terminator (253 bp)

NOS terminator (253 bp)

NdeI (5495)

SphI (2809)

EcoRI (2148)

NotI (35)

NotI (1063)

BglII (6852)

BglII (7119)

from maize and the NOS terminator from A. tumefaciens. It also contains the E. coli origin of replication. The NotI fragment does not contain the E. coli amp gene present on pZO1502 (Figure 1; Table 3) which confers resistance of bacterial cells to ampicillin.

Figure 1. Map of pZO1502 used in the transformation of Bt11 maize


February 2015



Table 3. Donor genes and regulatory sequences in pZO1502 (Bt11 maize transformation vector).

Genetic element Size (bp) Position Description

Active ingredient cassette

35S promoter 509 2153 to 2661

Promoter from the cauliflower mosaic virus (CaMV) 35S gene (Gardner et al., 1981), supplemented with the intron sequence 6 (471 bp) from the maize alcohol dehydrogenase (adh) 1 gene (Freeling and Bennet, 1985) to enhance gene expression in maize (Mascarenhas et al., 1990).

Intervening sequence 22 2662 to 2683 Region used for cloning.

IVS6-ADH1 471 2684 to 3154 Intervening intron sequence 6 from maize adh1 (National Center for Biotechnology Information [NCBI] accession number X04049.1).


cry1Ab 1848 3168 to 5015 Modified cry1Ab, which encodes a Cry1Ab protein that confers resistance to certain lepidopteran insect pests. Originally cloned from B. thuringiensis var. kurstaki HD-1 (Perlak et al., 1991).


NOS terminator 253 5023 to 5275 Terminator sequence from the nopaline synthase (NOS) gene of A.tumefaciens (NCBI accession number V00087.1). Provides a polyadenylation site (Bevan et al., 1983).


Selectable marker cassette

35S promoter 418 5698 to 6115

Promoter from the CaMV 35S gene (Gardner et al., 1981), supplemented with the intron sequence 2 (180 bp) from maize adh1 (Freeling and Bennet, 1985) to enhance gene expression in maize (Mascarenhas et al., 1990).


IVS2-ADH1 180 6122 to 6301 Intervening intron sequence 2 from maize adh1 (NCBI accession number X04049.1).


pat 552 6307 to 6858

S. viridochromogenes strain Tü494 gene encoding the selectable marker PAT. The native coding sequence (Wohlleben et al., 1988) was codon-optimized for enhanced expression in maize. The synthetic gene pat was obtained from Hoechst Schering AgrEvo GmbH, Germany. PAT confers resistance to herbicides containing glufosinate (i.e. phosphinothricin).


NOS terminator 253 6870 to 7122 Terminator sequence from the NOS gene of A. tumefaciens (NCBI accession number V00087.1). Provides a polyadenylation site (Bevan et al., 1983).



February 2015




Vector backbone components: 1. Components present on the 6.2-kb NotI restriction fragment of pZO1502 used for transformation



ColE1 ori 674 1122 to 1795 Origin of replication (ori) that permits replication of plasmids in E. coli. Similar to NCBI accession number V00268.1 (Itoh and Tomizawa, 1979).


2. Components absent from the 6.2-kb NotI restriction fragment of pZO1502 used for transformation, due to its release from the plasmid by the NotI digest



amp 861 97 to 957 Beta-lactamase gene from E. coli that confers resistance to ampicillin (similar to NCBI accession number L08752.1). It functions as a selectable marker for plasmid amplification.


b) MIR162 maize

MIR162 maize was produced by transformation of immature maize embryos derived from a proprietary Z. mays line via A. tumefaciens-mediated transformation (Negrotto et al., 2000; Hoekema et al., 1983). The plasmid pNOV1300 was used for transformation. Information on the Agrobacterium strain LBA4404 used for transformation and its disarmed Ti plasmid can be found in Hoekema et al. (1983) and Ooms et al. (1982). Replication of pNOV1300 in LBA4404 is made possible via homologous recombination with an “acceptor vector” pSB1 (Komari et al., 1996). The genetic elements in plasmid pNOV1300, the MIR162 maize transformation plasmid, are listed in Table 4 and shown in Figure 2. Table 4 contains a description of each constituent of plasmid pNOV1300, including size in base pairs and the position within the plasmid.

The region intended for insertion contains the vip3Aa19 gene, a modified version of the native vip3Aa1 gene from B. thuringiensis. The gene inserted in MIR162 maize differs from the vip3Aa19 gene by two nucleotides. These transformation-induced nucleotide changes in the vip3Aa19 coding sequence resulted in one single amino acid change in the encoded protein. The new gene incorporated into the MIR162 maize genome was designated vip3Aa20 (GenBank Accession Number DQ539888; NCBI2). One of these transformation-induced nucleotide changes resulted in an amino acid change in the encoded protein: methionine at position 129 of Vip3Aa19 has been substituted by isoleucine (M129I). The other nucleotide change resulted in an altered codon; however, it did not result in an amino acid substitution. The gene expressed in MIR162 maize was designated vip3Aa20 and the encoded protein Vip3Aa20. This gene is under the control of the maize polyubiquitin promoter, the intron #9 from the maize phosphoenolpyruvate (PEP) carboxylase gene and the 35S terminator from the

2 NCBI: National Center for Biotechnology Information, US National Library of Medicine,

http://www.ncbi.nlm.nih.gov/sites/entrez?db=Protein



February 2015



pNOV1300

14405 bp

vip3Aa19 (2370 bp)

pmi (1176 bp)

spec (789 bp)

LB (25 bp)

RB (25 bp)

iPEPC9 (108 bp)

ZmUbiInt (1993 bp)

ZmUbiInt (1993 bp) ColE1ori (807 bp)

35S terminator (70 bp)

NOS (253 bp)

cos (432 bp)

KpnI (4786)

HindIII (189)

XmaI (8293)

EcoRV (6965)

SphI (199)

SphI (2586)

SphI (4797)

SphI (14324)

Acc65I (4782)

cauliflower mosaic virus (CaMV). It also contains the pmi gene from E. coli encoding a phosphomannose isomerase; this gene is under the control of the maize polyubiquitin promoter and the nopaline synthase (NOS) terminator from A. tumefaciens.

Figure 2. Map of pNOV1300 used in the creation of MIR162 maize.


February 2015



Table 4. Donor genes and regulatory sequences in pNOV1300 (MIR162 maize transformation vector)

Genetic Element Size (bp) Function

ACTIVE INGREDIENT CASSETTE

Intervening sequence

174 Region used for cloning

ZmUbiInt 1993

Promoter region from Z. mays polyubiquitin gene which contains the first intron (Entrez

Accession Number S94464; NCBI3). Provides constitutive expression in monocots (Christensen

et al., 1992).



vip3Aa19 2370

A modified version of the native vip3Aa1 gene (Estruch et al., 1996) found in the B. thuringiensis

strain AB88 which was isolated from sour milk. The vip3Aa19 gene in vector pNOV1300 was

modified to accommodate the preferred codon usage in maize (Murray et al., 1989). The

vip3Aa19 gene (Entrez Accession Number DQ539887; NCBI3) encodes a Vip3Aa19 protein that

differs from the Vip3Aa1 protein encoded by the vip3Aa1 gene by a single amino acid at

position 284. The vip3Aa1 gene encodes lysine at position 284 and the vip3Aa19 gene

encodes glutamine. Vip3Aa proteins confer resistance to several lepidopteran insect pests.



iPEPC9 108 Intron #9 from the phosphoenolpyruvate carboxylase gene (Entrez Accession Number X15239;

NCBI3) from Z. mays (Hudspeth and Grula, 1989).



35S Terminator 70

Terminator sequence from the 35S DNA from the CaMV genome (Similar to Entrez Accession

Number AF140604; NCBI3). Its function is to provide a polyadenylation sequence (Franck et al.,

1980).

SELECTABLE MARKER CASSETTE



ZmUbiInt 1993

Promoter region from Z. mays polyubiquitin gene which contains the first intron (Entrez

Accession Number S94464 (NCBI3)). Provides constitutive expression in monocots

(Christensen et al., 1992).



pmi 1176

E. coli pmi gene encoding the enzyme phosphomannose isomerase (PMI) (Entrez Accession

Number M15380 (NCBI3)); this gene is also known as manA. Catalyzes the isomerization of

mannose-6-phosphate to fructose-6-phosphate (Negrotto et al., 2000). Used as a selectable

marker during transformation.



NOS Terminator 253

Terminator sequence from the nopaline synthase gene of A. tumefaciens (Entrez Accession

Number V00087 (NCBI3)). Its function is to provide a polyadenylation site (Depicker et al.,

1982).

3

NCBI: National Center for Biotechnology Information, US National Library of Medicine, http://www.ncbi.nlm.nih.gov/sites/entrez?db=Protein



February 2015



Genetic Element Size (bp) Function



VECTOR BACKBONE COMPONENTS

LB (left border)

25

Left border region of T-DNA from A. tumefaciens nopaline Ti-plasmid (Entrez Accession

Number J01825 (NCBI3)). Short direct repeat that flanks the T-DNA and is required for the

transfer of the T-DNA into the plant cell (Zambryski et al., 1982).



spec 789

Spectinomycin adenylyltransferase, aadA gene from E. coli Tn7 (Entrez Accession Number

X03043 (NCBI3)). Confers resistance to erythromycin, streptomycin, and spectinomycin; used

as a bacterial selectable marker (Fling et al., 1985).



ColE1ori 807 Origin of replication that permits replication of plasmid in E. coli. (Similar to Entrez Accession

Number V00268 (NCBI3)) (Itoh and Tomizawa, 1979).



cos 432 The sequence that is cut to produce the cohesive, single-stranded extensions located at the

ends of the linear DNA molecules of certain phages, such as lambda (Sanger et al., 1982).



RB (right border)

25

Right border region of T-DNA from A. tumefaciens nopaline Ti-plasmid (Entrez Accession

Number J01826 (NCBI3)). Short direct repeat that flanks the T-DNA and is required for the

transfer of the T-DNA into the plant cell (Wang et al., 1984).

c) Bt11 x MIR162 maize

The Bt11 x MIR162 maize is produced by conventional breeding of the following GM maize events: Bt11 and MIR162 maize. No further genetic modification to produce this stack has taken place. Bt11 x MIR162 maize produced by conventional breeding combining Bt11 and MIR162 maize has stably inherited the cry1Ab and pat genes from Bt11 and the vip3Aa20 and pmi genes from MIR162 maize, retaining the hybridization patterns as predicted. Accordingly, the foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual Bt11 and MIR162 maize. As described above, data from Southern analyses conducted for individual Bt11 and MIR162 maize events confirmed the presence of the inserts at single loci in each of the individual events. Additional Southern blot analyses were conducted to confirm the integrity of the transgenic inserts in Bt11 x MIR162 maize. Four gene-specific probes, the cry1Ab-, pat-, vip3Aa19-, and pmi-specific probe, were used to compare the hybridization patterns of Bt11 x MIR162 maize with the


February 2015



hybridization patterns observed for the corresponding single events. A vip3Aa19-specific probe was used for the vip3Aa20 Southern analysis. The nucleotide sequences of vip3Aa19 and vip3Aa20 differ by two nucleotides and are 99.9% identical. This does not affect the ability of the vip3Aa19-specific probe to hybridize to the vip3Aa20 sequence present in MIR162 maize. DNA from non-transgenic, near-isogenic NP2222/NP2171 maize was included in each analysis to identify any endogenous DNA sequences that might cross-hybridize with the gene-specific probes. Genomic DNA was analyzed with two restriction digestion strategies. In the first strategy, maize genomic DNA was digested with an enzyme that cut within the corresponding single-event insert; the other recognition sites for this enzyme were located in the maize genome flanking the single-event insert. This first strategy was used twice, with two different enzymes. In the second strategy, maize genomic DNA was digested with two enzymes that cut within the single-event insert such that a DNA fragment of predictable size was released.

Southern blot analyses using the cry1Ab-specific probe and the pat-specific probe were used to assess the genetic integrity of the Bt11 insert in Bt11 x MIR162 maize. Restriction enzymes ApoI, SphI, BspHI, and XhoI were used with the cry1Ab-specific probe, and restriction enzymes HindIII, SphI, BspHI, and XhoI were used with the pat-specific probe for these Southern blot analyses.

Southern blot analyses using the vip3Aa19-specific probe and the pmi-specific probe were used to assess the genetic integrity of the MIR162 insert in Bt11 x MIR162 maize. Restriction enzymes KpnI, EcoRV, HindIII, and XmaI were used with the vip3Aa19-specific probe and restriction enzymes KpnI, BamHI, HindIII, and XmaI were used with the pmi-specific probe for these Southern blot analyses.

Detection of one hybridization band of the expected size demonstrated that Bt11 x MIR162 maize contains a single copy per genome of cry1Ab from Bt11 maize as expected. In analyses with all restriction digestion enzymes, the DNA hybridization patterns for Bt11 maize and Bt11 x MIR162 maize were identical. These results demonstrated that the integrity of the cry1Ab cassette from Bt11 maize was preserved during conventional breeding to produce Bt11 x MIR162 maize.

Detection of one hybridization band of the expected size demonstrated that Bt11 x MIR162 maize contains a single copy per genome of pat from Bt11 maize as expected. In analyses with all restriction digestion enzymes, the DNA hybridization patterns for Bt11 maize and Bt11 x MIR162 maize were identical. These results demonstrated that the integrity of the pat cassette from Bt11 maize was preserved during conventional breeding to produce Bt11 x MIR162 maize. Detection of one hybridization band of the expected size demonstrated that Bt11 x MIR162 maize contains a single copy per genome of vip3Aa20 from MIR162 maize as expected. In analyses with all restriction digestion enzymes, the DNA hybridization patterns for MIR162 maize and Bt11 x MIR162 maize were identical. These results demonstrated that the integrity of the vip3Aa20 cassette in MIR162


February 2015



insert was preserved during conventional breeding to produce Bt11 x MIR162 maize.

Detection of one hybridization band of the expected size demonstrated that Bt11 x MIR162 maize contains a single copy per genome of pmi from MIR162 maize as expected. In analyses with all restriction digestion enzymes, the DNA hybridization patterns for MIR162 maize and Bt11 x MIR162 maize were identical. These results demonstrated that the integrity of the pmi cassette from MIR162 maize was preserved during conventional breeding to produce Bt11 x MIR162 maize.

The expected DNA hybridization patterns were observed in all Southern blot analyses of Bt11 x MIR162 maize, Bt11 maize, MIR162 maize, and non-transgenic, near-isogenic NP2222/NP2171 maize. The DNA hybridization patterns for Bt11 x MIR162 maize corresponded to the hybridization bands observed for Bt11 maize and MIR162 maize, indicating that the integrity of both transgenic inserts were preserved during conventional breeding to produce Bt11 x MIR162 maize.

4.2 Describe the gene products that are derived from the foreign genes. Bt11 x MIR162 maize produced by conventional breeding combining Bt11 and MIR162 maize has stably inherited the cry1Ab and pat genes from Bt11 and the vip3Aa20 and pmi genes from MIR162 maize, retaining the hybridization patterns as predicted. Accordingly, the foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual Bt11 and MIR162 maize. The transgenic proteins expressed in Bt11 x MIR162 maize enable effective insect and weed control:

o a truncated Cry1Ab protein for control of certain lepidopteran pests like the common maize pests: O. nubilalis (ECB), S. nonagrioides (Mediterranean corn borer; MCB), Sesamia calamistis (Pink stemborer; PSB), Busseola fusca (Fuller) (African stemborer; ASB) and Chilo partellus (Swinhoe) (Spotted stemborer; SSB).

o a PAT protein that confers tolerance to herbicide products containing glufosinate ammonium

o a Vip3Aa protein (designated Vip3Aa20) for control of certain lepidopteran pests like Heliothis zea (CE), A. ipsilon (BCW), S. frugiperda (FAW), and Striacosta albicosta (western bean cutworm; WBC).

o a PMI protein, that allows transformed maize cells to utilize mannose as the primary carbon source while maize cells lacking this protein fail to grow.

4.3 Describe the biological activity associated with the foreign gene products. Bt11 x MIR162 maize is a GM maize that is produced by conventional breeding of the following GM maize events: Bt11 and MIR162 maize. No further genetic modification to produce this stack has taken place. Bt11 x MIR162 maize has stably inherited the cry1Ab and pat genes from Bt11 and the vip3Aa20 and pmi genes from MIR162 maize, retaining the hybridization patterns as predicted. The foreign genes in Bt11 x MIR162


February 2015



maize are thus not different from the foreign genes of the individual Bt11 and MIR162 maize. Accordingly, Bt11 x MIR162 maize produces the transgenic proteins, Cry1Ab, PAT, Vip3Aa20, and PMI present in the two individual GM maize events. The traits and characteristics introduced in Bt11 and MIR162 maize are summarized below.

a) Cry1Ab

Protection from feeding damage by pest larvae is provided by expression of a truncated form of a Cry1Ab protein encoded by a modified cry1Ab gene derived from the soil micro-organism B. thuringiensis subsp kurstaki HD-1. The DNA sequence of the gene has been truncated at the 3’ end and modified to increase the level of expression in maize, but the amino acid sequence of the protein has not been altered (Perlak et al., 1991). The change to the plant phenotype involves protection of the Bt11 x MIR162 maize plants from damage by certain lepidopteran insect pests (O. nubilalis (ECB), S. nonagrioides (MCB), S. calamistis (PSB), B. fusca (Fuller) (ASB) and C. partellus (Swinhoe) (SSB)) and shows tolerance to glufosinate-ammonium herbicides.

b) PAT

The tolerance to glufosinate ammonium herbicides is accomplished by expression of a pat gene, derived from the soil micro-organism S. viridochromogenes, strain Tu494, that encodes the enzyme PAT, capable of detoxifying the herbicide (Strauch et al., 1988). The amino acid sequence of the PAT protein encoded by the synthetic pat gene is fully analyzed and the protein is identical to the native protein of S. viridochromogenes (Wohlleben et al, 1988). The PAT protein is responsible for converting L-phosphinothricin in plant cells, the active ingredient in glufosinate ammonium, to an inactive form. In the absence of PAT, application of the herbicide glufosinate leads to reduced production of the amino acid glutamine and increased ammonia levels in plant tissues, resulting in the death of the plant. PAT acetylates phosphinothricin synthesis, thereby inactivating the compound and conferring tolerance to chemically synthesized phosphinothricin compounds, such as the herbicide glufosinate-ammonium. The change to the plant phenotype is that the Bt11 x MIR162 maize plants can survive application of herbicides containing glufosinate-ammonium at normal rates scaling from 450 g to 600 g of active ingredient per hectare.

c) Vip3Aa1

Protection against lepidopteran insect pests is provided through the expression of a vegetative insecticidal protein (Vip) derived from the native Vip3Aa1 protein found in B. thuringiensis strain AB88 (Estruch et al., 1996). Vip proteins are produced during vegetative bacterial growth of B. thuringiensis and are secreted as soluble proteins into the extracellular environment. The activity of Vip3Aa proteins is confined to insects of the order Lepidoptera (Van Frankenhuyzen and Nystrom, 2002). The mechanism by which Vip proteins exert their insecticidal activity has been studied


February 2015



and found to be similar, but not identical, to that which has been previously described for Cry proteins (Lee et al., 2003). Following ingestion, full-length Vip proteins are proteolytically processed into active fragments of approximately 66 kDa which bind to receptors in the mid-gut epithelium of susceptible insects. Competitive binding assays have shown that Vip proteins and Cry proteins bind to different receptors in the insect mid-gut (Lee et al., 2003). Receptor binding is followed by the formation of selective ion channels (pores) in epithelial membranes which leads to cell lysis and death (Yu et al., 1997). Each of these steps plays a role in establishing the insecticidal specificity of a given protein for different insect species. To date, all Vip sequences described fall into three different families: Vip1, Vip2 and Vip3 (Crickmore et al., 2014). While Vip1 and Vip2 are components of a binary toxin that is active against coleopteran species, Vip3 proteins are highly active against several major lepidopteran pests. Vip3A proteins in particular, are very active against lepidopteran species like S. frugiperda (FAW), S. exigua (beet armyworm), H. zea (CE) and A. ipsilon (BCW). The insecticidal activity of Vip3Aa proteins is limited to species within selected families of the order Lepidoptera and no effects on other orders of target or non-target organisms have been recorded (Lee et al., 2003; Estruch et al., 1996; Van Frankenhuyzen and Nystrom, 2002; ILSI, 2012).

d) PMI

Bt11 x MIR162 maize also expresses the PMI enzyme encoded by the pmi gene from E. coli, which serves as a selectable marker. PMI allows transformed maize cells to utilize mannose as the only primary carbon source and therefore to survive on media in which mannose is the sole source of carbon, whereas maize cells lacking the pmi expression will fail to proliferate (Negrotto et al., 2000).

The genetic modification is not intended to change any of the typical crop characteristics of maize (except for the intended resistance against certain lepidopteran pests and tolerance against glufosinate-ammonium herbicides) and the handling and use of Bt11 x MIR162 maize is the same as for non-genetically modified maize.

4.4 Provide information on the rate and level of expression of the foreign genes and the sensitivity of the measurement of the rate and level. Maize plants derived from Bt11 maize produce a Cry1Ab protein that confers resistance to certain lepidopteran pests and a PAT protein that confers tolerance to herbicide products containing glufosinate-ammonium. Maize plants derived from MIR162 maize produce a Vip3Aa20 protein for control of certain lepidopteran pests and a PMI protein, which allows transformed maize cells to utilize mannose as the primary carbon source. The Bt11 x MIR162 maize hybrid was produced by conventional breeding of Bt11 and MIR162 maize. Accordingly, the Bt11 x MIR162 maize hybrid produces the transgenic proteins present in Bt11 and MIR162 maize.

4.4.1 Rate and level of expression of Bt11 x MIR162 maize proteins in different plant tissues

The rate and level of expression of the newly expressed proteins in tissues of Bt11 maize and MIR162 maize during the life cycle of the plant have previously been


February 2015



assessed and approved in South Africa by the Advisory Committee and Executive Council (Table 1).

To characterize the range of expression of the proteins Cry1Ab, PAT, Vip3Aa20, and PMI in various tissues of Bt11 x MIR162 maize plants, the protein concentrations were determined by enzyme-linked immunosorbent assays (ELISA). The maize plants used for this study were grown according to local agronomic practices at a Syngenta Seeds, Inc. field-trial location in Naples, FL, USA in 2008. Plants of two different field corn hybrids were grown in five replicate plots per hybrid, with all plots arranged in a randomized complete block design. Leaf samples were collected at vegetative sixth leaf stage (V6), root samples were collected at reproductive silking stage (R1), and kernel samples were collected at reproductive milk stage (R3). Two replicate samples of each tissue type were collected from each of the five plots per hybrid. The results for the Cry1Ab, PAT, PMI, and Vip3Aa20 concentrations are summarized as ranges on a fresh-weight basis across the two Bt11 x MIR162 maize hybrids. The concentrations of Cry1Ab, PAT, Vip3Aa20, and PMI represent the levels of these proteins in various tissue types of Bt11 x MIR162 maize in two different genotypes at three developmental time points. Concentrations of the proteins were either detected or quantified in all tissue types analysed.

o Concentrations of Cry1Ab ranged from 4.11 to 7.62 μg/g in V6 leaves, 0.76 to 3.90 μg/g in R1 roots, and 0.45 μg/g to 1.01 μg/g in R3 kernels.

o Concentrations of PAT ranged from less than the limit of quantification (LOQ) to 0.05 μg/g in V6 leaves and less than the LOQ to 0.08 μg/g in R1 roots. PAT concentrations in all R3 kernel samples were less than the LOQ.

o Concentrations of Vip3Aa20 ranged from 13.36 to 28.64 μg/g in V6 leaves, 1.12 to 3.65 μg/g in R1 roots and 26.31 to 33.07 μg/g in R3 kernels.

o Concentrations of PMI ranged from 1.02 to 2.31 μg/g in V6 leaves, 0.08 to 0.24 μg/g in R1 roots and 1.61 to 2.61 μg/g in R3 kernels.

4.4.2 Level of expression of Cry1Ab, PAT, Vip3Aa20, and PMI proteins in

different plant tissues in Bt11 x MIR162 maize compared with Bt11 and MIR162 maize

A study has been conducted with the purpose to measure and compare the concentrations of transgenic proteins Cry1Ab, PAT, Vip3Aa20, and PMI in various tissues of a Bt11 x MIR162 maize hybrid with those in maize hybrids derived from Bt11 and MIR162. The Bt11 x MIR162 maize hybrid was produced by conventional breeding of Bt11 and MIR162 maize. Bt11 x MIR162, Bt11 and MIR162 maize hybrids and the non-transgenic, near-isogenic maize hybrid were grown at one field trial location in Uberlandia, Brazil in 2013-2014. The field trials were designed to generate tissue samples from field-grown maize plants cultivated in accordance with common agricultural practices. All hybrids were of the same genetic background and the geographic location selected for the field trial was representative of the agricultural environment where this hybrid maize would typically be grown. Seeds of each hybrid were planted in five replicate plots, and all 20 plots were arranged in a randomized complete block design. From each plot, leaves, roots,


February 2015



whole plants, and kernels were collected from maize plants at two different growth stages. Pollen was collected from maize plants at one growth stage. ELISA was used to quantify the transgenic proteins in each maize tissue sample. Concurrent analysis of tissues from non-transgenic maize confirmed the absence of plant-matrix effects on the analysis method. The concentrations of each transgenic protein in tissues of the Bt11 x MIR162 hybrid were compared to those measured in the corresponding component single event hybrids. The concentrations of Cry1Ab, PAT, Vip3Aa20 and PMI in tissues of the Bt11 x MIR162 maize hybrid were similar to those of the corresponding single-event maize hybrids; Bt11 and MIR162. Although seven significant differences were observed out of 34 statistical comparisons conducted in this study, these differences were not consistently observed across tissue types or developmental stages and are therefore not of biological significance (Table 5a to 5d).


February 2015



Table 5a Comparisons of Cry1Ab concentrations in tissues of a Bt11 maize hybrid and the Bt11 x MIR162 maize hybrid on a dry weight (DW) basis

Concentration (µg/g DW)

Stage Sample Hybrid Mean Range P

V6 leaves Bt11 148.12 141.30 - 158.01 0.937

Bt11 x MIR162 147.66 140.06 - 166.16

roots Bt11 45.72 36.11 - 52.77 0.809

Bt11 x MIR162 44.89 42.14 - 47.02

whole plant Bt11 135.21 114.84 - 148.40 0.278

Bt11 x MIR162 126.48 120.92 - 140.74

R1 leaves Bt11 103.80 92.53 - 114.22 0.005

Bt11 x MIR162 87.18 70.60 - 98.38

roots Bt11 37.24 34.80 - 41.26 0.623

Bt11 x MIR162 36.65 33.85 - 39.69

pollena Bt11 0.120 <LOQ

b - 0.157

0.691 Bt11 x MIR162 0.130 0.0916 - 0.189

whole plant Bt11 52.67 46.58 - 56.51 0.012

Bt11 x MIR162 42.21 31.59 - 47.04

R6 kernels Bt11 7.05 6.50 - 7.77 0.039

Bt11 x MIR162 8.05 7.13 - 8.79

Senescence kernels Bt11 1.93 1.70 - 2.03

0.765 Bt11 x MIR162 1.95 1.73 - 2.04

N = 10 aN= 5 pollen

Results significantly different at P < 0.05 are shown in bold italic type. b LOQ for pollen= 0.05 μg/g

There were not any significant differences in Cry1Ab concentrations in any tissues at the V6 development stage. Cry1Ab concentrations in R1 leaves, whole plants, and R6 kernels of the Bt11 hybrid differed significantly from those of the Bt11 x MIR162 hybrid. However, significant differences were not observed in other growth stages of those same tissue types. Cry1Ab concentrations in R1 pollen, V6 and R1 stages of roots did not differ significantly between the Bt11 x MIR162 and Bt11 hybrids.


February 2015



Table 5b Comparisons of PAT concentrations in tissues of a Bt11 maize hybrid and the Bt11 x MIR162 maize hybrid on a DW basis



V6 leaves Bt11 0.336 0.189 - 0.462 0.178

Bt11 x MIR162 0.417 0.316 - 0.503

roots Bt11 0.866 0.759 - 1.06 0.005

Bt11 x MIR162 1.29 1.07 - 1.42

whole plant Bt11 0.726 0.459 - 0.881 0.850

Bt11 x MIR162 0.709 0.618 - 0.798

R1 leaves Bt11 0.832 0.627 - 1.13 0.446

Bt11 x MIR162 0.739 0.585 - 1.02

roots Bt11 1.08 0.878 - 1.30 0.312

Bt11 x MIR162 1.20 0.913 – 1.50

pollena

Bt11 NA <LODb

NA Bt11 x MIR162 NA <LOD

b

whole plant Bt11 0.804 0.698 - 0.983 0.553

Bt11 x MIR162 0.770 0.643 - 1.00

R6 kernels Bt11 0.0713 0.0555 - 0.0921 0.601

Bt11 x MIR162 0.0757 0.0609 - 0.0862

Senescence kernels Bt11 NA <LODc

NA Bt11 x MIR162 NA <LOD

c

N = 10 aN= 5 pollen

Results significantly different at P < 0.05 are shown in bold italic type.

NA = not applicable b LOD (limit of detection) for pollen= 0.025 μg/g c LOD for kernel = 0.025 μg/g

The concentrations of PAT in leaves and whole plants, R1 roots, and R6 kernels of the Bt11 x MIR162 hybrid did not differ significantly from those of the Bt11 hybrid. A significant difference between the two hybrids was observed from the comparison of PAT concentrations in V6 roots. ELISA analyses of pollen and senescence kernel samples for PAT from both the Bt11 x MIR162 and Bt11 hybrids resulted with measurements less than the limit of detection.


February 2015



Table 5c Comparisons of Vip3Aa20 concentrations in tissues of a MIR162 maize hybrid and the Bt11 x MIR162 maize hybrid on a DW basis



V6 leaves MIR162 188.57 153.36 - 226.60 0.001

Bt11 x MIR162 299.70 286.06 - 313.85

roots MIR162 69.46 62.73 - 75.53 0.623

Bt11 x MIR162 74.36 49.45 - 113.26

whole plant MIR162 152.41 128.16 - 178.68 0.095

Bt11 x MIR162 172.00 157.29 - 188.92

R1 leaves MIR162 188.35 156.16 - 245.13 0.641

Bt11 x MIR162 198.73 157.74 - 238.98

roots MIR162 86.52 40.55 - 145.66 0.190

Bt11 x MIR162 59.98 51.83 - 76.66

pollena

MIR162 61.96 55.12 - 68.60

0.585 Bt11 x MIR162 65.07 53.73 - 82.90

whole plant MIR162 86.31 75.29 - 105.39 0.639

Bt11 x MIR162 90.16 75.95 - 110.55

R6 kernels MIR162 107.90 96.98 - 112.92 0.002

Bt11 x MIR162 128.81 118.27 - 136.76

Senescence kernels MIR162 61.46 38.43 - 76.43

0.416 Bt11 x MIR162 64.43 50.72 - 77.15

N = 10 aN= 5 pollen

Results significantly different at P < 0.05 are shown in bold italic type.

The concentrations of Vip3Aa20 in roots and whole plants, R1 leaves, pollen, and senescence kernels of the Bt11 x MIR162 hybrid did not differ significantly from those of the MIR162 hybrid. A significant difference between the two hybrids was observed from the comparison of Vip3Aa20 concentrations in V6 leaves and R6 kernels.


February 2015



Table 5d Comparisons of PMI concentrations in tissues of a MIR162 maize hybrid and the Bt11 x MIR162 maize hybrid on a DW basis



V6 leaves MIR162 9.15 7.42 - 11.28 0.713

Bt11 x MIR162 9.42 8.63 - 9.82

roots MIR162 3.55 3.17 - 3.73 0.542

Bt11 x MIR162 3.68 3.30 - 4.19

whole plant MIR162 9.77 7.65 - 11.41 0.004

Bt11 x MIR162 6.59 6.05 - 6.94

R1 leaves MIR162 8.32 7.23 - 9.97 0.856

Bt11 x MIR162 8.41 7.30 - 10.34

roots MIR162 3.79 3.11 - 4.38 0.136

Bt11 x MIR162 3.33 2.89 - 3.82

Pollena

MIR162 2.17 1.89 - 2.63

0.432 Bt11 x MIR162 2.29 1.99 - 2.47

whole plant MIR162 6.91 5.54 - 7.92 0.315

Bt11 x MIR162 6.14 4.69 - 8.31

R6 kernels MIR162 3.62 2.92 - 3.95 0.128

Bt11 x MIR162 3.90 3.62 - 4.09

Senescence kernels MIR162 1.27 1.13 - 1.44

0.405 Bt11 x MIR162 1.21 1.09 - 1.32

N = 10 aN= 5 pollen

The concentrations of PMI in leaves and roots, pollen, R1 whole plants, and kernels of the Bt11 x MIR162 hybrid did not differ significantly from those of the MIR162 hybrid. A significant difference between the two hybrids was observed from the comparison of PMI concentrations in V6 whole plants. Although some significant differences were observed out of the 34 statistical comparisons conducted in this study, these differences were not consistently observed across tissue types or developmental stages and are therefore not of biological significance. It can thus be concluded that the concentrations of Cry1Ab, PAT, Vip3Aa20 and PMI in tissues of the Bt11 x MIR162 maize hybrid were not substantially different to those of the corresponding single-event maize hybrids. Therefore, environmental exposure to Cry1Ab, PAT, Vip3Aa20 and PMI from cultivation of Bt11 x MIR162 maize is expected to be no greater than exposure due to the cultivation of the single component events.


February 2015



4.4.3 State whether expression is constitutive or inducible

Expression is constitutive. 4.4.4 Stability of genes

4.4.4.1 Genetic stability As described in Section 4.1 above, data from Southern analyses and DNA sequencing conducted for Bt11 and MIR162 maize confirmed the presence of the inserts at single loci in each of the individual events. This data was assessed and approved by the Advisory Committee and Executive Council prior to this application (Table 1). It demonstrated that single copies of the cry1Ab gene, pat gene and ColE1 origin of replication are present in plants derived from Bt11 maize. As expected the Bt11 maize insert contains two copies of the CaMV 35S promoter, corresponding to the two copies of the promoter present in the transformation plasmid pZO1502. Bt11 maize does not contain the amp gene present on the backbone of pZO1502. Sequence analysis of the entire T-DNA present in Bt11 maize confirmed the overall integrity of the insert and that the contiguousness of the functional elements has been maintained. This information has been previously reviewed by EFSA and has received positive scientific opinions (EFSA, 2005; 2009a). Data from Southern analysis and DNA sequencing demonstrated that MIR162 maize contains (1) a single intact insert, (2) single copies of the vip3Aa20 gene and the pmi gene, (3) two copies of the maize polyubiquitin (ZmUbiInt) promoter (in addition to the endogenous polyubiquitin promoter) corresponding to the two copies of the promoter present in transformation plasmid pNOV1300, (4) one copy of the NOS terminator and (5) none of the backbone sequences from transformation plasmid pNOV1300. The genetic stability of the insert in MIR162 maize has been assessed by Southern blot analysis. Sequence analysis of the entire T-DNA present in MIR162 maize confirmed the intactness of the insert and that the contiguousness of the functional elements within the insert as intended in pNOV1300 has been maintained.

A comparative Southern blot analyses were conducted to confirm the integrity of the transgenic inserts in Bt11 x MIR162 maize by Southern blot analyses. Genomic DNA was analyzed by two restriction digestion strategies. In the first strategy, maize genomic DNA was digested with an enzyme that cut within the corresponding single event insert; the other recognition sites for this enzyme were located in the maize genome flanking the single event insert. This first strategy was used twice with two different enzymes. In the second strategy, maize genomic DNA was digested with two enzymes that cut within the single event insert such that a DNA fragment of predictable size was released. The expected DNA hybridization patterns were observed in all Southern blot analyses of Bt11 x MIR162 maize, Bt11 maize, MIR162 maize, and non-transgenic, near-isogenic maize (NP2222/NP2171). The DNA hybridization patterns for Bt11 x MIR162 maize corresponded to the hybridization bands observed for the single events. This


February 2015



indicates that the integrity of the transgenic inserts from the single events was preserved during conventional breeding to produce Bt11 x MIR162 maize. The results confirmed that the single events are present and that the structure of each insert is retained in the stacked product. 4.4.4.2 Phenotypic stability Previous expression analyses, conducted by Syngenta on each single event, indicated phenotypic stability of the introduced traits in Bt11 and MIR162 maize. These dossiers were assessed and approved by the Advisory Committee and Executive Council prior to this application (Table 1). Bt11 x MIR162 maize F1 seed is produced through conventional breeding of Bt11, and MIR162 single event maize lines which have been stable over multiple generations. Bt11 x MIR162 seed once planted by growers produces grain (F2) which is harvested for food, feed or industrial use. Such grain (F2) or products entering the commodity chain are normally not kept for further sowing. This application is not for general release of Bt11 x MIR162 maize in South Africa.

4.5 Provide protocols for the detection of the foreign genes in the environment including sensitivity, reliability and specificity of the techniques.

The Bt11 x MIR162 maize is a GM maize that is produced by conventional breeding of the following GM maize events: Bt11 and MIR162 maize. No further genetic modification to produce this stack has taken place. Bt11 x MIR162 maize produced by conventional breeding combining Bt11 and MIR162 maize has stably inherited the gene cry1Ab, which encodes the insecticidal protein Cry1Ab, and the gene pat, which encodes the enzyme PAT from Bt11 and gene vip3Aa20, which encodes the insecticidal protein Vip3Aa20, and the gene pmi, which encodes the enzyme PMI from MIR162 maize, retaining the hybridization patterns as predicted. Accordingly, the foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual Bt11 and MIR162 maize. Bt11 x MIR162 maize contains the Cry1Ab, PAT, Vip3Aa20, and PMI genes. The detection methods developed for the single events will also detect the individual events in Bt11 x MIR162 maize. For specific detection of Bt11 maize genomic DNA, a real-time quantitative TaqMan® polymerase chain reaction (PCR) method has been developed using the taxon specific target sequence (Adh1) and the GMO (Bt11) target sequence. One of the oligonucleotide primers is located within the maize specific flanking sequence and the other is located in the insert. This method has been validated for use by the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) and can be found on the EU-RL GMFF website: http://gmo-crl.jrc.ec.europa.eu/summaries/Bt11_CRLVL1007_Validated_Method%20doc.pdf and http://gmo-crl.jrc.ec.europa.eu/summaries/Bt11_CRLVL1007_Val_Report.pdf

http://gmo-crl.jrc.ec.europa.eu/summaries/Bt11_CRLVL1007_Validated_Method%20doc.pdf


http://gmo-crl.jrc.ec.europa.eu/summaries/Bt11_CRLVL1007_Val_Report.pdf


February 2015



For specific detection of MIR162 maize genomic DNA, a real-time quantitative TaqMan® PCR method has been developed using the taxon specific target sequence (Adh1) and the GMO (MIR162) target sequence. This method has been validated for use by theEuropean Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) and can be found on the EU-RL GMFF website: http://gmo-crl.jrc.ec.europa.eu/summaries/MIR162_validated_Method.pdf and http://gmo-crl.jrc.ec.europa.eu/summaries/MIR162_val_report.pdf The Bt11 x MIR162 maize described in this application has been produced by combining the GM maize events: Bt11 and MIR162 through conventional breeding techniques. There was no further genetic modification to produce the stack. As such, the detection methods developed for the single events are appropriate for use on Bt11 x MIR162 maize. Syngenta has confirmed the applicability of these methods on Bt11 x MIR162 maize. Thus, the detection methods provided for Bt11 and MIR162 maize unambiguously detect the single events, as well as the stacked product, in a mixture of seed/grain by using single seed analysis.

http://gmo-crl.jrc.ec.europa.eu/summaries/MIR162_validated_Method.pdf


http://gmo-crl.jrc.ec.europa.eu/summaries/MIR162_val_report.pdf


February 2015



5. RESISTANCE 5.1 Detail whether the genetically engineered plant is able to initiate resistance, in any biotic component of the environment, to any biologically active foreign gene product. This application is for Commodity clearance approval and does not request cultivation approval. Therefore, Bt11 x MIR162 maize described in this application would be imported for food, feed and processing purposes. Under these conditions, no resistance can develop against this product. 5.2 Detail what methods are available to minimise the risk of resistance developing in the environment. Not applicable. This is a Commodity clearance application. 5.3 Detail how resistance will be managed during release of the genetically modified plant. Not applicable. This is a Commodity clearance application.


February 2015



6. HUMAN AND ANIMAL HEALTH 6.1 State whether the genetically modified plant or its products will enter human or animal food chains. As described in Section 3.1, South Africa imports maize, primarily as grain, depending on local surpluses and shortages, as well as regional demand. This grain is normally used to produce food and feed products. Grain of Bt11 x MIR162 maize will be imported under the same circumstances and used for the same purposes. Therefore, Bt11 x MIR162 maize grain and products thereof will enter human and animal food chains. 6.2 Detail the results of experiments undertaken to determine the toxicity/ allergenicity of the foreign gene products (including marker genes) to humans and animals. 6.2.1 Possible modes of exposure: whole GM plant or crop Maize is not considered to be a commonly allergenic food (Iikura et al., 1999) and Bt11 x MIR162 maize plants produce proteins that are very unlikely to be allergenic. Although there have been some reports of occupational allergy to maize dust or pollen allergies in some geographical areas, this is unlikely to be a safety concern unique to the GM crop since maize is not considered to be a major allergenic food and there is no expectation that any of the GM plants have increased allergenic potential compared to their non-GM counterparts. The EFSA GMO Panel recently confirmed that it is unlikely that any interactions between the newly expressed proteins and metabolic pathways of maize would alter the pattern of expression of endogenous proteins/potential allergens and thereby significantly change the overall allergenicity of the whole plant (EFSA, 2010a, 2010b). The recipient organism, maize, has a history of safe use throughout the world and it is not considered as a major allergenic food (EFSA, 2007; Metcalfe et al., 2003). Food allergy to maize is rare (Moneret-Vautrin et al., 1998), although IgE- binding proteins have been identified in maize flour (Pastorello et al., 2000; Pasini et al., 2002). Allergy to maize is detected in a minor fraction of the population of atopic patients. In addition, most individuals with a positive skin prick test or having IgE antibodies against maize were suffering of respiratory allergy and only a few ones displayed a true food allergy upon oral challenge with maize products (Pasini et al., 2002; Jones et al., 1995). Therefore, oral sensitization to maize proteins is very rare (EFSA, 2007). Furthermore, the International Codex Alimentarius Commission has not identified maize among the cereal grains requiring special hypersensitivity labelling (Codex, 1999), nor has the Japanese Ministry of Health and Welfare included maize on its list of foods subject to mandatory or recommended allergen labelling (Ebisawa et al., 2003). Although the allergenicity of the whole crop could theoretically be increased as an unintended effect of the random insertion of the transgene in the genome of the recipient, for example through qualitative or quantitative modifications of the expression pattern of endogenous proteins, this issue is not considered to be relevant since maize is not considered a major allergenic food and possible over-expression of any endogenous


February 2015



protein that is not known to be allergenic would be unlikely to alter the overall allergenicity of the whole plant (EFSA, 2007). Humans and animals may be exposed to the whole Bt11 x MIR162 maize plant either as worker, or orally as consumer, and animals by feed uptake. A food and feed safety risk assessment was conducted to evaluate the possible adverse effects on human and animal health resulting from exposure to Bt11 x MIR162 maize. This has been based on the comparative analysis of the molecular characteristics; expression levels of the novel proteins; well characterised mode of action and biological function of the novel proteins; the agronomic, composition and nutritional properties of the stacked maize event in relation to the single events; dietary human intake, as well as animal feeding studies. Therefore, more detailed information presenting the safety of Bt11 x MIR162 maize and possible modes of exposure for humans can be found in Section 6.2.3, 6.4, 6.5 and 6.6 of this dossier, respectively: 6.2.3.1 (Food Safety), 6.2.3.2 (Comparative Assessment), 6.2.3.3 (Exposure Assessment: Dietary Human Intake), 6.4 and 6.5 (Possible allergens before and after plant modification), 6.6 (Occupational Human Health) and possible mode of exposure for animals in section 6.2.3.2 (Animal feeding study with the broiler). In summary, it can be concluded that none of the components introduced into Bt11 x MIR162 maize are considered to be toxic or allergenic to human or animal health. 6.2.2 Information relating to the genetic modified plant

a) Description of the genetically modified plant

o family name: Poaceae (formerly Gramineae) o genus: Zea o species: Z. mays L. o subspecies: Z. mays subsp. mays o event/product: Bt11 x MIR162 o common name: Maize, corn o unique identifier: SYN-BTØ11-1 -9 x SYN-IR162-4 o origin: Maize originates from the Meso-American region, i.e. Mexico

and Central America (CFIA, 2003)

b) Description of the host plant and its use as foodstuff

Maize has been extensively cultivated and has a history of safe use for food and feed to humans and animals (OECD, 2002; Doebley, 2004; OGTR, 2008). South Africa grows white and yellow maize. White maize represents the majority of the acreage (about two thirds) and is mainly used for human consumption. Yellow maize is mainly grown for grain and largely used to feed domestic animals. Silage maize is also grown, but does not represent a large acreage in South Africa. South Africa also imports and/or exports maize, primarily as grain, depending on local surpluses and shortages, as well as regional demand. Specific for South Africa is also the popular use of maize as “green mielies”. These are maize cobs harvested before full maturity (still green) and cooked or roasted for direct consumption.


February 2015



World-wide, more than 18 million farmers adopted crop biotechnology and cultivate 181 million hectares of GM crops in 2014. Since the first plantings in 1996, cumulative hectares of more than 1.8 billion have been successfully cultivated and thirty eight countries have granted regulatory approvals to biotech crops for use as food, feed or for environmental release since 1994 (James, 2014). South Africa has been involved with biotechnology research and development for over 20 years and has developed a globally competitive biotechnology industry. South Africa planted approximately 15 million hectares of GM maize in the period 2000 to 2013, which included single, as well as stacked traits for insect and herbicide resistance. It was estimated that 2.73 million hectares of maize was planted during the 2013 season, from which 2.36 million hectares were biotech maize. Of this, 28.4% were the single B. thuringiensis (Bt) gene, 18.2% herbicide tolerant, and 53.4% stacked Bt and herbicide tolerant genes (James, 2013). It is estimated that the total farm income benefit for GM maize in South Africa for the period 1996 to 2012 was US$1.105 billion (Brookes and Barfoot, 2014).

South Africa imports maize, primarily as grain, depending on local surpluses and shortages, as well as regional demand. This grain is normally used to produce food and feed products. Grain of Bt11 x MIR162 maize will be imported under the same circumstances and used for the same purposes. The various food and feed products that can be derived from maize (OECD, 2002), including Bt11 x MIR162 maize, are described in detail in Section 3.1 above.

c) Description of genetic modification and donor organisms

i) Description of genetic modification

The Bt11 x MIR162 maize is produced by conventional breeding using different combinations of the GM maize events: Bt11 and MIR162 maize. No further genetic modification to produce this stack has taken place. Bt11 x MIR162 maize therefore contains the cry1Ab and pat genes from Bt11 maize and vip3Aa20 and pmi genes from MIR162 maize. The foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual events. The Bt11 x MIR162 maize plants express the traits present in the single GM maize events through the production of the following proteins:

o a truncated Cry1Ab protein for control of certain lepidopteran pests like the common European maize pests: O. nubilalis (ECB) and S. nonagrioides (MCB).

o a PAT protein that confers tolerance to herbicide products containing glufosinate ammonium

o a Vip3Aa protein (designated Vip3Aa20) for control of certain lepidopteran pests like H. zea (CE), A. ipsilon (BCW), S. frugiperda (FAW), and S. albicosta (WBC).

o a PMI protein, that allows transformed maize cells to utilize mannose as the primary carbon source while maize cells lacking this protein fail to grow.


February 2015



The detailed molecular characterization of Bt11 x MIR162 maize, as explained in Section 4.1, has confirmed that:

o The Bt11 and MIR162 maize inserts are present and their structures have been retained in Bt11 x MIR162 maize

o The expression of the transgenic proteins in Bt11 x MIR162 maize is not substantially different from the expression in the Bt11 x MIR162 single maize events

o The genetic and phenotypic stability of the single events Bt11 and MIR162 maize has been confirmed in the stacked product Bt11 x MIR162 maize

o The molecular characterization raised no safety concerns o Bt11 x MIR162 maize shows no potential for production of new toxins or

allergens o No unintended changes have been identified in Bt11 x MIR162 maize

ii) Donor organisms

(a) Bacillus thuringiensis

The source of native cry1Ab and vip3Aa1 genes is B. thuringiensis. The genus Bacillus is a diverse group of rod-shaped, gram-positive, facultative anaerobic, spore forming bacteria. B. thuringiensis occurs naturally and ubiquitously in the environment. It is a common component of the soil microflora and has been isolated from most terrestrial habitats (Glare and O’Callaghan, 2000). Several subspecies of B. thuringiensis have been described; many of them have been extensively studied and used in commercial insecticide preparations. Insecticidal products using B. thuringiensis have been used for several decades and have a long history of safe use (US EPA, 2001). (b) Streptomyces viridochromogenes

The source of the pat gene is the aerobic bacterium S. viridochromogenes strain Tu494, a gram-positive, sporulating, soil inhabiting bacterium widespread in the environment and with a long history of safe use (OECD, 1999). (c) Escherichia coli

The source of the pmi gene is the common bacterium E. coli, K-12 strain. E. coli belongs to the Enterobacteriaceae, a relatively homogeneous group of rod-shaped, gram-negative, facultative bacteria. Members of the genus Escherichia are ubiquitous in the environment and found in the digestive tract of vertebrates, including humans. The vast majority of E. coli strains are harmless to humans, although some strains can cause diarrhoea and urinary infections. However, this particular group of pathogenic E. coli are distinct from the strains that are routinely used in the laboratory and from which the pmi gene was obtained. The K-12 strains from E. coli have a long history of safe use and are commonly used as protein production systems in many commercial applications.


February 2015



6.2.3 Food Safety Risk Assessment (Codex Alimentarius Commission) A food and feed safety risk assessment was conducted to evaluate the possible adverse effects on human and animal health resulting from exposure to Bt11 x MIR162 maize. This has been based on the comparative analysis of the molecular characteristics; expression levels of the novel proteins; well characterised mode of action and biological function of the novel proteins; the agronomic, composition and nutritional properties of the maize event. Conventional breeding techniques are routinely used to combine individual beneficial traits present in different lines of maize in order to produce new lines that will contain the traits of interest. This is also the case when combining traits introduced in maize by genetic modification. In the case of stacked Bt11 x MIR162 maize, the single events have been extensively assessed for their safety. It was previously advised that when two plants that are substantially equivalent to conventional varieties are crossed by conventional breeding techniques, the combined product can be expected to be substantially equivalent to the single events (WHO, 1995; Crop Life International, 2005). There are no scientific reasons for undertaking additional safety assessments when combining unrelated traits. The safety assessments previously undertaken for individual single trait products should be applicable to the combined trait product. 6.2.3.1 Toxicology, allergenicity and pathogenicity The Bt11 x MIR162 maize described in this application has been produced by combining the GM maize events: Bt11 and MIR162 through conventional breeding techniques. There was no further genetic modification to produce the stack. Maize derived from Bt11 x MIR162 expressed four new proteins:

o a truncated Cry1Ab protein for control of certain lepidopteran pests of maize o a PAT protein that confers tolerance to herbicide products containing glufosinate

ammonium o Vip3Aa20, a variant of the Vip3Aa1 protein produced by B. thuringiensis, which is

active against a number of significant lepidopteran pests of maize o PMI which allows transformed maize cells to utilize mannose as only primary

carbon source.

None of these proteins are considered to be dangerous to human health or the environment. The toxicology, allergenicity and pathogenicity of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins expressed in Bt11 and MIR162 maize have already been provided by Syngenta for assessment under the GMO Act (Act No. 15 of 1997). These dossiers were assessed and approved by the Advisory Committee and Executive Council prior to this application (Table 1). The results of all of the food and feed safety studies concluded that Bt11 x MIR162 maize is not different from conventional maize and it is therefore unlikely to be toxic or allergenic, and that its nutritional effects are no different to those of conventional maize. No new data have been obtained since these approvals and/or applications that would have altered the


February 2015



outcome of such risk assessments. However, for ease of reference, a summary of the information previously provided is provided together with the information on Bt11 x MIR162 maize.

A series of safety studies and existing data on the history of safety of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins were taken into account to support the following conclusions:

o The recipient organism, maize, has a history of safe use throughout the world. o The PAT protein belongs to the class of acetyltransferase enzymes common to

plants and animals. PAT shares very similar two-dimensional structure, immuno-reactivity, molecular weight and functional properties with other acetyltransferase enzymes which occur as a natural component of human and animal diet. There are no reports of toxicity or allergenicity associated with the acetyltransferase enzyme class. The presence of PAT in the food and feed supply does not represent a new exposure.

o Cry1Ab, PAT, Vip3Aa20, and PMI proteins are considered to have a substantial history of safe consumption by humans and animals. It can therefore be considered unlikely to present a health risk to humans or animals

o Neither Cry1Ab, PAT, Vip3Aa20, nor PMI are structurally or functionally related to any protein with the potential to adversely affect human or animal health.

o Exposure to Cry1Ab, PAT, Vip3Aa20, and PMI proteins, based on anticipated levels of intake are likely to be low.

o The Cry1Ab, PAT, Vip3Aa20, and PMI proteins have no significant amino acid homology to known mammalian protein toxins and are readily degraded in in vitro digestibility assays.

o The Cry1Ab, PAT, Vip3Aa20, and PMI proteins show no acute oral toxicity at the highest doses tested in mammalian studies. No test substance related mortalities occurred during the study and no clinical signs attributable to the test substance were observed. There were no effects on clinical condition, body weight, food consumption, clinical pathology, organ weights, macroscopic or microscopic pathology that were considered to be related to the administration of Cry1Ab, PAT, Vip3Aa20, or PMI protein to male and female mice.

o Neither Cry1Ab, PAT, Vip3Aa20, nor PMI has significant amino acid homology to known mammalian protein toxins or to known or putative allergenic proteins.

o The gene sequences of Cry1Ab, PAT, Vip3Aa20, and PMI and their donor organisms are not known to correspond to the sequences of human pathogens, and no sequences of human pathogens have been introduced.

o Although measuring resistance to gastric enzyme degradation is not predictive of exposure or the likelihood of allergic sensitization, it is relevant to note that Cry1Ab, PAT, Vip3Aa20, and PMI proteins were evaluated in simulated mammalian gastric fluid (SGF) containing pepsin. All the proteins were readily degraded in SGF. There is no evidence to suggest that the digestion of Cry1Ab, PAT, Vip3Aa20, and PMI is altered as a result of repeated exposure to the proteins or to expect their accumulation with repeated exposure.

o The transgenic proteins show a lack of acute toxicity and show no significant homology to known protein toxins. They are therefore considered non-toxic and unlikely to present a health risk to humans or animals.


February 2015



It can be concluded that the Cry1Ab, PAT, Vip3Aa20, and PMI proteins produced in Bt11 x MIR162 maize can be considered non-toxic, non-allergenic and unlikely to present a health risk to humans or animals. Based on the known function and mode of action of the newly expressed proteins Cry1Ab, PAT, Vip3Aa20, and PMI, the occurrence of interactions of safety concern between these proteins is unlikely. There is no evidence to suggest that the proteins introduced into Bt11 x MIR162 maize, would alter the characteristics of processed foods and feeds when compared to those derived from conventional maize. Moreover, no evidence of interaction of safety concern between the newly expressed proteins produced has been observed in any studies conducted with Bt11 x MIR162 maize. These conclusions are based on the observations and studies, including those of Bt11 x MIR162 x MIR604 x GA21 summarised later in this section:

o The sources of the transgenes were considered: Cry1Ab, PAT, Vip3Aa20, and PMI came from donors which are not known to be significant causes of food allergies. In addition, no genetic material was obtained from wheat, rye, barley, oats or related cereal grains and the newly expressed proteins are therefore unlikely to have any role in the elicitation of gluten-sensitive enteropathy or other enteropathies which are not IgE mediated.

o No significant alterations in expression levels were observed for each of these proteins in Bt11 x MIR162 maize and the single event maize plants. No significant changes in exposure levels can therefore be expected.

o No biologically relevant differences in composition were detected between the Bt11 x MIR162 x MIR604 x GA21 product (which contains the Bt11 x MIR162 sub combination) and near-isogenic controls.

o No biologically relevant differences in agronomic traits were detected between the Bt11 x MIR162 x MIR604 x GA21 product (which contains the Bt11 x MIR162 sub combination) and near-isogenic controls.

o No adverse effects were observed in broiler chickens fed with diets prepared with Bt11 x MIR162 x MIR604 x GA21 maize grain compared to broiler chickens fed with diets prepared with conventional maize. Since Bt11 x MIR162 is a sub combination of Bt11 x MIR162 x MIR604 x GA21, it is unlikely that there are potential interactions between these proteins that would result in adverse health effects.

o An assessment of amino acid sequence similarity between the transgenic proteins (Cry1Ab, PAT, Vip3Aa20, and PMI) and known or putative toxins has been carried out with updated databases. It showed no biologically relevant amino acid sequence similarity to known or putative toxins.

o Bioinformatic searches using updated databases were performed for sequence homologies and structural similarities between Cry1Ab, PAT, Vip3Aa20, and PMI and known allergens. It showed no biologically relevant amino acid sequence similarity to known or putative allergens.

The conclusions obtained from these analyses have confirmed the results of the previous studies, which showed no similarities between the newly expressed proteins Cry1Ab, PAT, Vip3Aa20, and PMI and known proteins toxic to mammals. It also concludes that the Cry1Ab, PAT, Vip3Aa20, and PMI proteins show no evidence of sharing significant similarities of biological relevance with known allergens and therefore no further testing is required. In particular, no further specific serum screening is considered to be necessary.


February 2015



It can be concluded that the Cry1Ab, PAT, Vip3Aa20, and PMI proteins are unlikely to be toxic, allergenic or pathogenic. In addition, based on the information provided, Syngenta considers it to be unlikely that potential interactions could occur that would change the toxicity, allergenicity or pathogenicity of these proteins in Bt11 x MIR162 maize.

a) Toxicity and pathogenicity

i) Bioinformatic analyses

To determine whether the Cry1Ab, PAT, Vip3Aa20, or PMI proteins had any significant amino acid homology with protein sequences identified as toxins, the sequence of the Cry1Ab, PAT, Vip3Aa20, and PMI amino acids were systematically compared to the latest posting of the National Centre for Biotechnology Information (NCBI 20144) Entrez Protein Database containing all publicly available protein sequences. The BLAST (Basic Local Alignment Search Tool) for Proteins program was used to search the NCBI Entrez® Database to determine whether the Cry1Ab, PAT, Vip3Aa20, or PMI amino acid sequence showed significant similarity to known and putative toxins. The threshold value for determining significance of matches was based on searches conducted with randomly shuffled sequences of the amino acids comprising Cry1Ab, PAT, Vip3Aa20, or PMI. This procedure identified (1) whether any proteins in the database showed significant similarity to the amino acid sequence (i.e. alignments with BLAST for Proteins Expectation values [E -values] below an established threshold), indicating that the amino acid sequence might be closely related to the amino acid sequence, and (2) whether any proteins showing sequence similarity to the amino acid sequence were known or putative toxins.

There were 908 protein sequences identified as having significant sequence similarity to Cry1Ab amino acid sequence; none of these proteins are known or putative human or mammalian toxins. The protein alignments support the conclusion that the Cry1Ab amino acid sequence shows no significant similarity with any known or putative toxins. There were 4211 protein sequences identified as having significant sequence similarity to PAT amino acid sequence. The 1000 most similar sequence alignments are displayed in this report. All 4211 sequences were considered for their potential relevant similarity to PAT and are categorized. None of these proteins were known or putative toxins. The protein alignments from the original report support the conclusion that the PAT amino acid sequence shows no significant similarity with any known or putative toxins. There were 91 protein sequences identified as having significant sequence similarity to Vip3Aa20 amino acid sequence; none of these proteins were known

4 NCBI. 2014. Entrez® Protein database. Bethesda, MD: National Center for Biotechnology Information,

National Library of Medicine, National Institutes of Health.




February 2015



or putative toxins. The protein alignment supports the conclusion that the Vip3Aa20 amino acid sequence shows no significant similarity with any known or putative toxins. There were 3944 protein sequences identified as having significant sequence similarity to PMI amino acid sequence. The first 1000 most similar sequence alignments are displayed in this report. All 3944 sequences were considered for their potential relevant similarity to PMI. None of these proteins were known or putative toxins. The protein alignments support the conclusion that the PMI amino acid sequence shows no significant similarity with any known or putative toxins.

ii) Acute toxicity study in mice

Although the testing of acute exposure of the Cry1Ab, PAT, Vip3Aa20, and PMI protein to the circulatory system were reported and reviewed previously (Table 1), it is summarised for ease of reference below. Single dose acute oral toxicity studies in mice have been performed with Cry1Ab, PAT, Vip3Aa20, and PMI proteins derived from microbial production. Since protein toxins are known to act via acute mechanisms at low doses (Sjoblad et al., 1992), this test is considered appropriate to confirm the safety of the proteins. The acute oral toxicity studies conducted with Cry1Ab, PAT, Vip3Aa20, and PMI proteins confirmed that these proteins are not acutely toxic to mice at the highest dose tested. No test substance-related mortalities occurred during the studies and no clinical signs attributable to the test substance were observed. There were no effects on clinical condition, bodyweight, food consumption, clinical pathology, organ weights, macroscopic or microscopic pathology that were considered to be related to the administration of the proteins to male and female mice. In summary, the data gathered for Cry1Ab, PAT, Vip3Aa20, and PMI proteins expressed in Bt11 maize show no significant homology to known protein toxins, a lack of acute toxicity based on the highest dose tested and that the Cry1Ab and PAT proteins are rapidly digested. These proteins can therefore be considered non-toxic and unlikely to present a health risk to humans or animals (US EPA, 2001). Based on these results, it is concluded that the Cry1Ab, PAT, Vip3Aa20, and PMI protein are very unlikely to be toxic to humans or mammals even under conditions of maximal exposure at a very high dose.

iii) Allergenicity assessment

Because there is no single definitive test to predict food allergenicity in humans, a weight-of-evidence approach was used to assess the potential allergenicity of Cry1Ab, PAT, Vip3Aa20, and PMI. This approach is consistent with the recommendations of the Codex Alimentarius Commission (Codex, 2009).


February 2015



The following types of data were considered in the weight-of-evidence assessment:

o source organism o amino acid sequence similarity to known or putatively allergenic proteins o susceptibility to digestive enzymes o susceptibility to heat inactivation o glycosylation status

(a) Source organism The source of native cry1Ab and vip3Aa1 genes is B. thuringiensis. The genus Bacillus is a diverse group of rod-shaped, gram-positive, facultative anaerobic, spore forming bacteria. The Bt bacteria have no history of allergenicity (Taylor and Hefle, 2001; FAO/WHO, 2001). Bt insecticides have been used for decades and no reports of oral allergies to these preparations have ever been made. A review conducted by the United States Environmental Protection Agency (US EPA) showed that none of the laboratory animal studies submitted to the Agency showed any indication of allergic reactions to Bt or its components (US EPA, 2001). Therefore, cry1Ab and vip3Aa1 genes are not derived from a source known to produce allergenic proteins.

The source of the pat gene is the aerobic bacterium S. viridochromogenes strain Tu494, a gram-positive, sporulating, soil inhabiting bacterium widespread in the environment and with a long history of safe use (OECD, 1999). PAT is not derived from a source known to produce allergenic proteins.

The source of the pmi gene is the common bacterium E. coli, K-12 strain. The K-12 strains from E. coli have a long history of safe use and are commonly used as protein production systems in many commercial applications. PMI is common in nature and found across kingdoms (Reed et al., 2001). PMI is a member of a widely distributed group of enzymes in the cupin protein superfamily; none of the PMIs are known allergens. Very few cupins are allergenic and all allergenic cupins are seed storage proteins, while there are more than 500 PMI proteins in 400 species which are enzymes, not seed storage proteins. Cupins are a large family of proteins with a small portion of their sequences shared at the level of the “cupin fold”. There are many subclasses of cupins and the PMI proteins reside in a subclass that is structurally distinct from the known cupin allergens (Radauer and Brieteneder, 2007). Importantly, the cupin fold is not associated with IgE binding and is not indicative of allergenic potential for the known cupin allergens. (b) Bioinformatic analysis

An extensive updated bioinformatics search for sequence homologies and structural similarities between the expressed proteins (Cry1Ab, PAT, Vip3Aa20, and PMI) and known allergens was performed. To determine whether or not the Cry1Ab, PAT, Vip3Aa20, and PMI amino acid sequence showed biologically relevant similarity to amino acid sequences of


February 2015



known or putative allergens, two different searches were performed against the Food Allergy Research and Resource Program Protein (FARRP) AllergenOnline database, version 14, which contains 1706 amino acid sequences of known and putative allergens. A full-length sequence search using FASTA, and a separate search for exact matches of eight or more contiguous amino acids, was used to compare the amino acid sequence of Cry1Ab, PAT, Vip3Aa20, and PMI to each of the known or putative allergen sequences. In the FASTA search, no sequence similarity greater than 35% shared identity over 80 or more amino acids was observed between the Cry1Ab, PAT, Vip3Aa20, and PMI amino acid sequence and any entry in the FARRP AllergenOnline database. Results from the exact match search show no alignments of eight or more contiguous amino acids between the Cry1Ab, PAT, Vip3Aa20, and PMI amino acid sequence and sequences in the FARRP AllergenOnline database. Together, these results support the conclusion that Cry1Ab, PAT, Vip3Aa20, and PMI shares no biologically relevant amino acid sequence similarity to known or putative protein allergens. In conclusion, assessment of the bioinformatics searches showed that the Cry1Ab, PAT, Vip3Aa20, and PMI proteins have no significant amino acid homology to known or putative allergenic protein sequences that are biologically relevant or have implications for allergenic potential. (c) In vitro digestibility studies

Although measuring resistance to gastric enzyme degradation is not predictive of exposure or the likelihood of allergic sensitization, it is relevant to note that Cry1Ab, PAT, Vip3Aa20, and PMI proteins were evaluated in SGF containing pepsin (Table 1). All the proteins were readily degraded in SGF. There is no evidence to suggest that the digestion of Cry1Ab, PAT, Vip3Aa20, and PMI is altered as a result of repeated exposure to the proteins or to expect their accumulation with repeated exposure. Thus, from data gathered in these studies it can be concluded that Cry1Ab, PAT, Vip3Aa20, and PMI proteins expressed in Bt11 x MIR162 maize are very unlikely to be allergenic.

(d) Testing of the whole GM food/feed on Bt11 x MIR162 maize Bt11 x MIR162 maize is as safe and nutritious as conventional maize since:

o The recipient organism, maize, has a history of safe use throughout the

world. o None of the gene sequences or their donors are known to be pathogenic

to humans and no pathogenic sequences have been introduced. o Exposure to Cry1Ab, PAT, Vip3Aa20, and PMI proteins, based on

anticipated levels of intake are likely to be low. o The Cry1Ab, PAT, Vip3Aa20, and PMI proteins have no significant amino

acid homology to known mammalian protein toxins.


February 2015



o The Cry1Ab, PAT, Vip3Aa20, and PMI proteins have no significant amino acid homology to known or putative allergenic protein sequences that are biologically relevant or have implications for allergenic potential.

o The Cry1Ab, PAT, Vip3Aa20, and PMI proteins show no acute oral toxicity at the highest doses tested in mammalian studies.

o The Cry1Ab, PAT, Vip3Aa20, and PMI proteins are readily degraded in in vitro digestibility assays.

o It is very unlikely that the Cry1Ab, PAT, Vip3Aa20, and PMI protein will be glycosylated in plants.

o Studies comparing the composition of Bt11 x MIR162 x MIR604 x GA21 maize plants (which contain the Bt11 x MIR162 sub combination) and near-isogenic controls have concluded that this maize is substantially equivalent to conventional maize.

o Agronomic performance of Bt11 x MIR162 x MIR604 x GA21 maize plants (which contains the Bt11 x MIR162 sub combination) is similar, and for most parameters, equivalent to their non-transgenic, near-isogenic control counterparts.

o No adverse effects were observed in broiler chickens fed with diets prepared with Bt11 x MIR162 x MIR604 x GA21 maize grain compared to broiler chickens fed with diets prepared with conventional maize. Since Bt11 x MIR162 is a sub combination of Bt11 x MIR162 x MIR604 x GA21, it is unlikely that there are potential interactions between these proteins that would result in adverse health effects.

In summary, based on the data described above and data described in rest of Section 6.2.3, it can therefore be concluded that the Cry1Ab, PAT, Vip3Aa20, and PMI proteins produced in Bt11 x MIR162 maize can be considered non-toxic, non-allergenic and unlikely to present a health risk to humans or animals.

6.2.3.2 Comparative safety assessment Although there is no reason to indicate that the combination of Bt11 and MIR162 maize by conventional breeding would result in changes in maize composition or in the phenotypic and agronomic characteristics, additional comparative studies were conducted. Syngenta’s position is that, if all of the single events within a stacked event have been through the safety assessment process and have been shown to be of low risk for use as food and feed, then it is not necessary to assess a stacked event consisting of these single events and created through conventional breeding techniques. The exception to this would be if there is a sound hypothesis of a probable interaction between one or more of the events in the stack. Thus, to satisfy the South African regulatory requirements, data of the higher order stack will provide an adequate view of the food and feed safety of any lower order stacks. This approach on stacked events has been adopted in other regions such as European Union e.g. with the guidance from the European Food Safety Authority, EFSA (EFSA, 2007). Compositional analysis, agronomical equivalence and broiler feeding studies, performed with Bt11xMIR162xMIR604xGA21 summarized below, allow us to conclude that Bt11 x MIR162 maize is as safe and nutritious as conventional maize. Molecular analysis and expression studies have also confirmed that the insert in Bt11 x MIR162 maize is


February 2015



stable. Comparative safety assessment of Bt11 x MIR162 maize was evaluated based on the results of the higher level stack Bt11 x MIR162 x MIR604 x GA21 maize. These studies have already been presented in Syngenta’s application for Commodity Clearance of Bt11 x MIR162 x MIR604 x GA21 maize products that was approved by the Advisory Committee and Executive Council of South Africa (39.4(6/11/264)). The use of Bt11 x MIR162 x MIR604 x GA21 maize’s composition, agronomic and broiler data to support safety assessment for Bt11 x MIR162 maize is justified for the following reasons:

o No changes in the reproduction, dissemination or survivability compared to non-transgenic, near-isogenic conventional maize have been observed in field trials conducted with Bt11, MIR162, MIR604 or GA21 maize.

o Thorough safety studies and risk assessments have been conducted for each of the four single events. In particular, compositional analysis of grain and forage derived from Bt11, MIR162, MIR604 and GA21 maize events have been carried out and no consistent patterns emerged to suggest that biologically significant changes in composition or nutritive value of the grain or forage had occurred as an unintended result of transformation or expression of the transgenes.

o There is no reason to anticipate that conventional breeding of Bt11, MIR162, MIR604 and GA21 maize or sub combinations would result in a stacked product that differs from the single events in nutrition, toxicity or allergenic potential to humans or animals.

o None of the proteins expressed by Bt11, MIR162, MIR604 or GA21 maize are known to be toxic or allergenic to humans or animals and there are not known precedents where interactions between non-toxic proteins lead to toxic effects (FIFRA SAP, 2004).

o Therefore combination of the introduced proteins within the quadruple stack product would represent the best chance of finding an adverse effect because any adverse effect caused by the combination of two of the single maize events would also be apparent in Bt11 x MIR162 x MIR604 x GA21 maize.

a) Compositional analysis

In addition to the compositional analysis studies previously reported on Bt11 and MIR162 maize (Table 1), an additional study was performed to support the conclusion that Bt11 x MIR162 maize is no different in composition to conventional maize. Compositional equivalence of Bt11 x MIR162 maize was concluded based on the results of the higher level stack Bt11 x MIR162 x MIR604 x GA21 maize. Levels of key nutritional components in grain and forage from Bt11 x MIR162 x MIR604 x GA21 maize were measured and compared to the levels in grain and forage from non-transgenic, near-isogenic maize, and to the natural variation of these components in other conventional maize varieties. Key nutritional components in forage and grain from Bt11 x MIR162 x MIR604 x GA21 maize were measured and compared with those


February 2015



in forage and grain from non-transgenic, near-isogenic control maize. Bt11 x MIR162 x MIR604 x GA21 maize and the corresponding non-transgenic, near-isogenic control maize were grown at six locations in the USA in 2006. At each location, the hybrids were grown in a randomized complete block design, with three replicates for each genotype. Forage and grain were analyzed for 65 key food and feed nutrients and anti-nutrients; these components were chosen based on the recommendations of the Organisation for Economic Co-operation and Development (OECD) for analysis of new varieties of maize (OECD, 2002). Forage was analyzed for proximates, calcium and phosphorus; grain was analyzed for proximates, starch, minerals, vitamins, amino acids, fatty acids, secondary metabolites, and anti-nutrients. Analysis of variance was used to test for genotype effects and location- by-genotype interactions. In addition, mean levels of nutritional components were compared with the ranges of variation for conventional maize hybrids published in the International Life Sciences Institute (ILSI) Crop Composition Database. In forage, no significant differences were observed for moisture, protein, fat, ash, acid detergent fibre, neutral detergent fiber, calcium or phosphorus. A significant location-by-genotype interaction was observed for carbohydrates. For all components measured in forage, the mean levels (across locations and at each location) were within the ranges of variation for conventional maize hybrids published in the ILSI Crop Composition database5. Grain from the Bt11 x MIR162 x MIR604 x GA21 maize plants, as well as their corresponding non-transgenic, near-isogenic controls, was harvested after physiological maturity was reached and analyzed for the following components:

o proximates (acid detergent fiber, neutral detergent fibre, total dietary fibre, starch, carbohydrates, protein, moisture, fat and ash)

o minerals (calcium, phosphorous, potassium, sodium, iron, copper, magnesium, manganese, selenium and zinc)

o vitamins (Vitamins E, B1, B2, B3, B6, Folic Acid) and β-carotene o amino acids (eighteen amino acids were analysed) o fatty acids (linoleic, oleic, palmitic, stearic and linolenic acids) o secondary metabolites and anti-nutrients (furfural, phytic acid, inositol,

trypsin inhibitor, raffinose, ferulic acid and p-coumaric acid)

In grain, most components analyzed were not significantly different. Statistically significant differences between the two genotypes were noted in levels of 8 nutritional components: neutral detergent fiber, copper, potassium, vitamins B1 and B6, and stearic, oleic and linoleic fatty acids. Statistically significant location-by-genotype interactions were observed for 3 components, and levels below the limit of quantitation precluded statistical comparison of 4 components. For all quantifiable components measured in grain, except vitamin B2 and vitamin E, the mean levels (across locations and at each location) were within the ranges of variation for conventional maize hybrids published in the ILSI Crop Composition database. Mean levels of vitamin B2 were slightly higher than the published ranges at two of the six locations for the transgenic grain and at one location for the non-transgenic grain.

5 International Life Sciences Institute Crop Composition Database: https://www.cropcomposition.org/query/index.html


February 2015



At several locations, vitamin E levels below the limit of quantitation were observed in samples of both the transgenic and non-transgenic grain. For vitamin E, the crop composition database does not show values below the limit of quantitation; however, the limit of quantitation value for vitamin E in this study fell within the range reported in the ILSI database. The results of this study support the conclusion that no biologically significant changes in composition occurred as an unintended result of the transformation process or expression of the transgenes in Bt11 x MIR162 x MIR604 x GA21 maize. Based on these data, it has been demonstrated that the majority of components in Bt11 x MIR162 x MIR604 x GA21 maize did not differ from the control, and when differences did occur, mean levels for the test and control were within ranges considered to be normal for conventional maize. Consistent with approaches of other regions or countries on stacked events, e.g. with the guidance from the European Food Safety Authority, EFSA (EFSA, 2007b), the compositional data of Bt11 x MIR162 x MIR604 x GA21 maize is used to support the risk assessment for Bt11 x MIR162 maize. Therefore, based on the data obtained in the compositional study conducted with Bt11 x MIR162 x MIR604 x GA21 maize and this guidance, it can be concluded that the combination of the single maize events Bt11 and MIR162, using conventional breeding techniques to produce the Bt11 x MIR162 maize product, is unlikely to result in changes in compositional performance with respect to non-transgenic, near-isogenic conventional maize. The conclusion of this study is thus that the composition of Bt11 x MIR162 maize does not materially differ from that of conventional maize (Launis and Kramer 2006; Van Wert, 1994), apart from the intended presence of the introduced proteins Cry1Ab, PAT, Vip3Aa20, and PMI.

b) Agronomic equivalence

In addition to the agronomical equivalence studies previously reported on Bt11 and MIR162 maize (Table 1), an additional study was assessed to confirm that Bt11 x MIR162 maize is no different than conventional maize. Agronomic equivalence of Bt11 x MIR162 maize was evaluated based on the results of the higher level stack Bt11 x MIR162 x MIR604 x GA21 maize. This study has been presented in Syngenta’s application for Commodity Clearance of Bt11 x MIR162 x MIR604 x GA21 maize products that was approved by the Advisory Committee and Executive Council of South Africa (39.4(6/11/264)). Grain yield and agronomic performance of Bt11 x MIR162 x MIR604 x GA21 maize and non-transgenic, near-isogenic maize were evaluated across the US in 2006. Studies with Bt11 x MIR162 x MIR604 x GA21 maize were conducted in a series of replicated trials planted at ten locations in the US, selected to be representative of the range of environmental conditions under which the tested hybrid varieties would typically be grown. Each of the agronomic trials was conducted as a randomized complete block design with five replications per location. In addition to inspections for disease and insect damage, qualitative and quantitative comparisons for a


February 2015



number of morphological and agronomic parameters were made. Up to eighteen separate agronomic parameters and one disease trait were assessed at each location, although not all parameters were assessed at all locations. The characteristics chosen for agronomic comparison were those typically observed by professional maize breeders and agronomists. These agronomic traits cover a broad range of characteristics that encompass the entire life cycle of the maize plant and include data assessing seedling emergence, growth habit, vegetative vigour, days to pollen shed, days to maturity and yield parameters. For each agronomic or disease trait suitable for formal analysis, data were subjected to analysis of variance across locations. The statistical significance of the genotype effect (Bt11 x MIR162 x MIR604 x GA21 vs. the non-transgenic, near-isogenic control) was determined using a standard F-test at the 5% probability. Although there were very small differences in some parameters, these differences were not always consistent across all sites and unlikely to be biologically significant. This data therefore confirms that the agronomic performance of Bt11 x MIR162 x MIR604 x GA21 maize is similar, and for most parameters, equivalent to the non-transgenic, near-isogenic control maize. Bt11 x MIR162 x MIR604 x GA21 maize is therefore unlikely to form feral persistent populations, would not be more invasive or weedy than conventional maize hybrids and would not display higher rates of outcrossing than unmodified maize. Consistent with approaches of other regions or countries on stacked events, e.g. with the guidance from the European Food Safety Authority, EFSA (EFSA, 2007b), the agronomic data of Bt11 x MIR162 x MIR604 x GA21 maize is used to support the risk assessment for Bt11 x MIR162 maize. Therefore, based on the data obtained in the agronomic study conducted with Bt11 x MIR162 x MIR604 x GA21 maize and this guidance, it can be concluded that the combination of the single maize events Bt11 and MIR162, using conventional breeding techniques to produce the Bt11 x MIR162 maize product, is unlikely to result in changes in agronomic performance with respect to non-transgenic, near-isogenic conventional maize.

c) Poultry feeding study Although there is no reason to believe that there will be any adverse effects on broiler chickens when fed with the stacked product, the safety of Bt11 x MIR162 maize has also been evaluated in a 49-day broiler feeding study. The feeding study was conducted with the higher level stack Bt11 x MIR162 x MIR604 x GA21 maize. This study has also been presented in Syngenta’s applications for Commodity Clearance of Bt11 x MIR162 x MIR604 x GA21 as well as Bt11 x MIR162 x GA21 maize products (39.4(6/11/264)). A 49-day broiler feeding study was conducted to evaluate whether standard poultry diets prepared with Bt11 x MIR162 x MIR604 x GA21 maize grain had any adverse effects on male or female broiler chickens as compared to diets prepared with non-transgenic, near-isogenic control grain and other conventional maize lines commercially available. The results of this study showed that the consumption of poultry diets containing Bt11 x MIR162 x MIR604 x GA21 maize grain did not cause


February 2015



any adverse effects on broiler chickens. All diets supported rapid broiler chicken growth at low mortality rates and excellent feed conversion ratios without significant impact on overall carcass yield or quality. The study showed that Bt11 x MIR162 x MIR604 x GA21 maize had no deleterious effects on broiler chickens. These results also allow the conclusion that other stack combinations of the single events Bt11, MIR162, MIR604 and GA21 using conventional breeding techniques, such as Bt11 x MIR162 maize, are unlikely to result in adverse effects on e.g. broiler chickens. Thus, it can be concluded that grain from Bt11 x MIR162 x MIR604 x GA21 and consequently Bt11 x MIR162 maize is safe for food and feed consumption and no differences in wholesomeness are expected with comparable non-GM maize varieties.

6.2.3.3 Exposure assessment It is expected that the introduction of Bt11 x MIR162 maize will replace some of the maize in existing products. However, the genetic modification in Bt11 x MIR162 maize was not intended to change any of the compositional parameters in food and feed, therefore no nutritional changes are expected from the presence of the introduced proteins and no impact on the extent of use can be expected. A dietary assessment for Bt11 x MIR162 maize using the expression data obtained in US studies is included below.

The dietary exposure also takes a worst case assumption that 100% of the maize consumed in South Africa is Bt11 x MIR162 maize. The highest expression value of Cry1Ab, PAT, Vip3Aa20, and PMI in kernels at R6 (the major food tissue of maize) in Bt11 x MIR162 maize has been used. Taking into consideration the level of expression of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins, based on an average maize consumption of 252.6 g/person/day (248.1 g/person/day maize products plus 4.5 g/person/day sweet corn and popcorn kernels) (WHO, 2012), the theoretical daily intake for each of the proteins produced by Bt11 x MIR162 maize (Cry1Ab, PAT, Vip3Aa20, and PMI) was calculated. Conservatively assuming that a 60 kg person consumes 252.6g of maize in one day following maize consumption with 100% Bt11 x MIR162 maize, the acute intake can be estimated as 4.21 g/kg body weight per day. In addition, margins of exposure have been calculated by comparing the no-observed-effect-level (NOEL) from the acute oral toxicity study with each protein to the expected intake level. The results shown in Table 6 indicate that the expected levels of intake of the proteins Cry1Ab, PAT, Vip3Aa20, and PMI through consumption of Bt11 x MIR162 maize in South Africa will be very low.


February 2015



Table 6 Anticipated intake of Cry1Ab, PAT, Vip3Aa20, and PMI proteins from consumption of Bt11 x MIR162 maize in South Africa (assuming that 100% of the maize in the diet is Bt11 x MIR162 maize).

Protein

Maximum expression in kernels

a

(µg/g fresh weight)

Intake (mg/kg body weight/day)

Acute oral NOEL (mg/kg body weight/day)

Margin of exposure

b

Cry1Ab 5.61 0.0236181 1830 7 7483

PAT 0.05 0.0002105 5050 23 990 499

Vip3Aa20 92.32 0.3886672 1250 3 216

PMI 2.6 0.0109460 2072 189 293

a Calculations based on maximum kernel (R6) expression data b Margins of Exposure that exceed 100 are generally considered to support a conclusion that no unacceptable risk is associated with exposure.

Margins of exposure with a minimum factor of 3 216, supporting the conclusion that the risk to consumers is negligible and confirming the results previously obtained. The theoretical consumption of Bt11 x MIR162 maize necessary to reach doses higher than the NOEL is impossible to achieve by normally eating Bt11 x MIR162 maize or maize products. Therefore, the risk of exposure to a toxic dose of PAT from consumption of Bt11 x MIR162 maize is negligible.

6.2.4 Other concerns related to human health

6.2.4.1 Genetic stability of insert and phenotypic stability of GM plant As described in detail in Sections 4.1 and 4.4 above, genetic and phenotypic stability were confirmed in the single maize events as well as the stacked Bt11 x MIR162 maize. 6.2.4.2 Gene transfer, antibiotic resistance This is an application for Commodity clearance, i.e. full food, feed and processing approval, of Bt11 x MIR162 maize in South Africa. Cultivation of Bt11 x MIR162 maize in South Africa is not within the scope of this application. In the rare event that small amounts of maize kernels of Bt11 x MIR162 maize could accidentally find their way into the environment their survival would be very unlikely as maize is highly domesticated and cannot survive without human intervention (Niebur, 1993; Owen, 2005), especially under normal South African climatic conditions. In the rare event that these maize plants were to survive they could be easily controlled using any of the current agronomic measures taken to control other commercially available maize. In the highly unlikely event that cross-pollination did occur, this would not lead to establishment of the transgene in the maize genetic pool as commercially grown hybrids are used mainly for grain production and volunteers can easily be controlled using standard agronomic practices. Maize (Z. mays ssp. mays) hybridizes with a group of taxa collectively called teosinte (OECD, 2003; Owen, 2005). Several types of teosinte are classified as subspecies of Z. mays, whereas others are regarded as separate species of Zea. Teosinte species are natives of Central America and have co-existed with cultivated maize for several thousand


February 2015



years. They have remained genetically distinct from cultivated varieties despite occasional introgression (e.g., US EPA 2010; Baltazar et al. 2005). These species of teosinte do not occur in South Africa and, therefore, gene transfer from Bt11 x MIR162 maize to other sexually compatible plant species is not possible. Therefore, Cry1Ab, PAT, Vip3Aa20, and PMI are unlikely to spread from maize cultivation and persist in the environment as the result of gene flow. There is no evidence from phenotypic or agronomic data from Bt11 x MIR162 x MIR604 x GA21 maize, that the reproductive fitness of Bt11 x MIR162 maize has been unintentionally enhanced as a result of the genetic modification. The traits introduced into Bt11 x MIR162 maize will control some important lepidopteran maize pests and confer a selective advantage only when glufosinate are applied. Maize is not a problematic weed in agriculture. Volunteers can be managed chemically or mechanically. Chemical control of volunteers does not rely solely on herbicides. Maize does not grow outside of cultivation (OECD 2003); therefore, these traits are unlikely to confer a fitness advantage if introduced into other maize varieties though gene flow. The potential for gene flow from Bt11 x MIR162 maize to other cultivated maize varieties therefore is not anticipated to have environmental or agronomic consequences. Based on current scientific knowledge, gene transfer from GM plants to micro-organisms under natural conditions is extremely unlikely, and its establishment would occur primarily through homologous recombination in micro-organisms (EFSA, 2007). The horizontal gene transfer from GM plants to bacteria with subsequent expression of the transgene is regarded as a highly unlikely event under natural conditions, especially in the absence of selective pressure as discussed in detail by EFSA (2006, 2009). In considering the potential impact on human health, it is important to note that humans have always consumed large amounts of maize DNA as a normal component of food and there is no evidence that this consumption has had any adverse effect on human health. Novel DNA sequences in genetically modified foods comprise only a minute fraction of the total DNA in the food (generally less than 0.01%) and are therefore unlikely to pose any special additional risks compared with the large amount of DNA naturally present in all foods. Maize has been consumed for thousands of years by humans and animals and no incident of intact gene transfer has been reported yet. In the very unlikely event that such horizontal gene transfer would take place, no adverse effects on human and animal health or the environment are expected, as no principally new traits would be introduced or expressed in microbial communities. In 1991, the World Health Organization (WHO) issued a report of a Joint FAO/WHO Expert Consultation which looked at strategies for assessing the safety of foods produced by biotechnology (FAO/WHO, 1991). It was concluded by that consultation that as DNA from all living organisms is structurally similar, the presence of transferred DNA in food products, in itself, poses no health risk to consumers. The ability of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins in Bt11 x MIR162 maize to transfer genetic material to other organisms was previously assessed and approved by the Advisory Committee and Executive Council (Table 1).


February 2015



In summary, data gathered to date have shown that:

o The cry1Ab, pat, vip3Aa20 and pmi genes expressed in the Bt11 x MIR162 maize are from bacterial origin and were optimised for expression in plants.

o No changes in the genes were introduced to enhance recombination or gene transfer.

o Both cry1Ab and pat genes are under the control of the 35S promoter which presents limited, if any, activity in prokaryotic organisms. If such genes would be transferred they would not be functional.

o Both vip3Aa20 and pmi genes are under the control of maize promoters which present limited, if any, activity in prokaryotic organisms. If such genes would be transferred they would not be functional.

o Neither the amp gene nor any other antibiotic resistance gene is present in Bt11 x MIR162 maize. Consequently, there is no case of any risk of transfer of such genes.

In conclusion, no change in the ability of Bt11 x MIR162 maize to transfer genetic material to other organisms is observed when compared to conventional maize. In the highly unlikely event that intact gene transfer did occur, the possibility to confer selective advantage or increased fitness to micro-organisms is very limited. It should be noted that if intact gene transfer to bacteria were to occur, no selective advantage is envisaged since the cry1Ab, pat, vip3Aa20, and pmi genes were optimised for expression in plants. Furthermore, no changes in the genes were introduced to enhance recombination or gene transfer and the genes are under the control of maize promoters which present limited, if any, activity in prokaryotic organisms. If such genes would be transferred they would not be functional.

a) Plant to plant gene transfer

This is an application for Commodity clearance, i.e. full food, feed and processing approval, of Bt11 x MIR162 maize in South Africa. Release into the environment of Bt11 x MIR162 maize in South Africa is not within the scope of this application. Since maize is a very common crop in South Africa and it has been cultivated for centuries, its persistence and invasiveness properties are well known and the risks they pose are considered negligible. In the rare event that small amounts of maize kernels of Bt11 x MIR162 maize accidentally find their way into the environment their survival would be very unlikely as maize is highly domesticated and cannot survive without human intervention (Niebur, 1993; Owen, 2005), especially under normal South African climatic conditions. In the rare event that these maize plants were to survive they could be easily controlled using any of the current agronomic measures taken to control other commercially available maize. Survival of maize is dependent upon temperature, seed moisture, genotype, husk protection and stage of development. Maize is not a persistent weed. Maize seed can only survive under a narrow range of climatic conditions. Volunteers are killed by frost or easily controlled by current agronomic practices, including ploughing and the use of selective herbicides.


February 2015



Therefore, the presence of traits that confer tolerance to insect pests or herbicides are unlikely to confer any selective advantage and maize plants containing this type of traits will still be limited mainly by climatic factors. In South African regions with mild and dry winters, maize kernels remaining in the field after harvest can develop into volunteers. However, these volunteer plants develop defectively and tend to have low vigour, rarely producing cobs, which rarely have grain. Therefore, cross-pollination between maize volunteers and other maize crops, although possible, would only occur at very low levels (Palaudelmàs et al., 2009). In the highly unlikely event that cross-pollination did occur, this would not lead to establishment of the transgene in the maize genetic pool as commercially grown hybrids are used mainly for grain production and volunteers can be controlled easily using standard agronomic practices. The only wild taxa known to hybridise spontaneously with maize are species of teosinte (OECD, 2003; Owen, 2005). Populations of sexually compatible wild relatives of maize are not known in South Africa. Therefore, gene transfer from Bt11 x MIR162 maize to other sexually compatible plant species is not possible. Any vertical gene transfer would be limited to other maize plants where cross-pollination between maize varieties under South African cultivation conditions could occur (OECD, 2003; Devos et al., 2005). These properties mean that it can be ruled out that sexually compatible wild relatives of maize could acquire any of the events present in Bt11 x MIR162 maize and represent an agronomic problem in agricultural fields in South Africa or become more invasive in natural habitats.

b) Plant to bacteria gene transfer

Products derived from Bt11 x MIR162 maize can potentially be ingested by humans or animals and some environmental exposure through accidental spillage of grain or through manure or faeces that could lead to gene transfer, is possible. However, based on current scientific knowledge and previous scientific opinions, horizontal gene transfer from GM plants to micro-organisms under natural conditions is extremely unlikely (EFSA, 2009b; Keese, 2008). Transgenic DNA is a component of many food and feed products derived from maize. It is well documented that DNA present in food and feed becomes substantially degraded in the process of digestion in the human or animal gastrointestinal tract. However, a low level of exposure to fragments of ingested DNA, including the recombinant fraction of such DNA, by micro-organisms in the digestive tracts of humans, domesticated animals, and other animals feeding on Bt11 x MIR162 maize or derived products can occur. As no full-length genes from plants have been detected in the large intestine or in faeces (EFSA, 2009b), the concentration and quality of plant DNA accessible to bacteria in receiving environments following the import of Bt11 x MIR162 maize is expected to be very low. Horizontal gene transfer is not an adverse effect as such, but an occurrence that may or may not lead to harm. Therefore, the assessment should focus on the likelihood of harm as a result of gene transfer. The main concern relating to horizontal gene transfer from GM plants to micro-organisms is the transfer of


February 2015



antibiotic resistance genes that could later compromise therapies to combat diseases (Keese, 2008). Current scientific knowledge indicates that horizontal gene transfer of non-mobile DNA fragments between unrelated organisms (such as plants to micro-organisms) is extremely unlikely to occur under natural conditions (EFSA, 2009b; Keese, 2008). In addition to the low concentration of DNA in the gastrointestinal tracts and the lack of competence of most bacteria to take up foreign DNA, the major barrier to such inter-domain transfer is the lack of sufficient DNA sequence similarity for homologous recombination to occur in bacteria. The probability and frequency of horizontal gene transfer of plant DNA (including the recombinant DNA fraction) to exposed micro-organisms is determined by: o the concentration and quality of plant DNA accessible to micro-organisms in

receiving environments, o the presence of micro-organisms with a capacity to develop competence for

natural transformation, i.e. to take up extracellular DNA, o the ability for genetic recombination by which the plant DNA can be

incorporated and thus stabilised in the micro-organism genome (including chromosomes or plasmids),

o the expression and the function of the protein in the recipient, and by o the selective advantage provided by the acquired recombinant gene-encoded

traits. Although the cry1Ab, pat, vip3Aa20, and pmi genes expressed in Bt11 x MIR162 maize are derived from bacteria and in theory, they could provide sufficient DNA similarity for homologous recombination with genes from environmental bacteria, they were optimised for expression in plants. No changes in the genes were introduced to enhance recombination or gene transfer. The genes are under the control of maize promoter or the 35S promoter, which present limited, if any, activity in prokaryotic organisms. If such genes would be transferred they would not be functional. The molecular characterization studies conducted also showed that no antibiotic resistance genes are present in any of the maize events (see Section 6.2.4.2 for more detail). Given the low levels of exposure to micro-organisms that could arise from the import, processing or food and feed use of Bt11 x MIR162 maize in South Africa and the characteristics of the transgenes, it is highly unlikely that horizontal gene transfer will occur (OGTR, 2009). If gene transfer does occur, it is unlikely that the transgenes will become established in the genome of micro-organisms in the environment or human and animal digestive tract. In the very unlikely event that any of the genes were established in the genome of micro-organisms, no adverse effects on human and animal health or the environment are expected and the uncertainty associated with this risk characterization is very low. The conclusion is that the cry1Ab, pat, vip3Aa20, and pmi genes expressed in Bt11 x MIR162 maize are unlikely to be transferred to micro-organisms and, even if they were, this would not lead to human, animal or environmental harm. Thus, the


February 2015



probability that the import, processing or food and feed use of Bt11 x MIR162 maize will result in adverse effects to human or animal or the environment will be negligible. Given the low probability that any of the genes would be transferred to micro-organisms and the fact that even if transfer occurred, harm to humans or animals would be very unlikely, the uncertainties associated with this risk characterisation and the probability of long-term adverse environmental effects is low.

6.2.4.3 Susceptibility of humans exposed to the GMO Maize is the world’s most widely cultivated cereal with a long history of safe use and the crop itself causes few health problems. The comparative safety assessment conducted with Bt11 x MIR162, Bt11 x MIR162 x MIR604 x GA21 and near-isogenic conventional maize demonstrated that the only differences of biological relevance are the intended introduced genes and the proteins they express: Cry1Ab, PAT, Vip3Aa20, and PMI (See Section 6.2.3 for more information). No biologically significant unintended changes were observed in the GM maize when compared with near-isogenic conventional maize. The release into the environment of Bt11 x MIR162 maize is not within the scope of this application, therefore it is highly unlikely that humans will come into contact with Bt11 x MIR162 maize plants in South Africa. In the unlikely event that small amounts of grain from Bt11 x MIR162 maize could accidentally find their way into the environment this would represent extremely low levels of exposure and the survival of this grain would be very unlikely. Any plants germinating from this grain could be controlled easily using any of the current agronomic measures taken to control other commercially available maize (Owen, 2005). It is expected that the main mode of human exposure to Bt11 x MIR162 maize (and therefore Cry1Ab, PAT, Vip3Aa20, and PMI) will be through ingestion of maize grain or derived products in food. The toxicity and allergenicity assessment conducted for these proteins has shown that Cry1Ab, PAT, Vip3Aa20, and PMI are unlikely to be toxic or allergenic and the expression levels of these proteins in kernels are very low (See Sections 6.2.3 for more information). In summary, it is highly unlikely that the import and use of Bt11 x MIR162 maize in South Africa will result in any adverse effects to human health. 6.2.4.4 Effect of processing A detailed description of the processing technologies used with maize together with detailed information on the composition, level of undesirable substances, nutritional value, metabolism and intended use of maize has been published in the OECD consensus document on compositional considerations for new varieties of maize: key food and feed nutrients, anti-nutrients and secondary plant metabolites (OECD, 2002). In particular, the processing methods used for maize grain include exposure to high temperatures in both the dry- and wet-milling procedures. The initial steeping step of the wet-milling procedure exposes grain to a dilute sulfurous acid solution at temperatures of approximately 49ºC to 54ºC for an extended duration of up to 42 hours. In both the dry-


February 2015



and wet-milling procedures, moistened grain is milled, then dried at temperatures ranging from approximately 54ºC to 71ºC for 30 minutes to several hours. The processing of Bt11 x MIR162 maize will be the same as the processing of non-GM conventional maize (Johnston et al., 2005). Analyses of Bt11 x MIR162 x MIR604 x GA21 maize plants (which contains the Bt11 x MIR162 sub combination) for key nutrients and anti-nutrients have shown that the GM maize grain is compositionally equivalent to conventional maize. Bt11 x MIR162 maize will be produced and processed in the same way as any non-GM maize and there is no evidence to suggest that the expression of the proteins produced in this maize (Cry1Ab, PAT, Vip3Aa20, and PMI) will influence this processing in any way. Based on the information above, there is no reason to believe that the presence of the Cry1Ab, PAT, Vip3Aa20, and PMI in Bt11 x MIR162 maize will alter the processing characteristics in any way. Nor would the presence of the transgenic proteins in the maize grain alter the characteristics of processed foods and feeds when compared to those derived from conventional maize. It is highly unlikely that the processing of Bt11 x MIR162 maize in South Africa will result in any adverse effects to human health. 6.3 If the foreign gene products are toxic or allergenic in any way, detail how the Commodity clearance will be managed to prevent contact with animals or humans that will lead to discomfort or toxicity. The information on the potential toxicity and allergenicity of the foreign gene products expressed in Bt11 x MIR162 maize as described above, allows the conclusion that Bt11 x MIR162 maize is as safe as non-genetically modified maize. Therefore, no special measures for containment are foreseen. 6.4 What are the common/major allergens present in the recipient organism before modification? Maize grain has a history of safe use throughout the world and it is not considered to be a major allergenic food source (Frisner et al., 2000; OECD, 2002; Metcalfe et al., 2003; FARRP, 2014). The prevalence of maize allergy is exceedingly low amongst the human population. Rare cases of occupational allergy to maize dust or maize pollen have been reported and IgE-binding proteins have been identified in maize flour (Pastorello et al., 2000; Pasini et al., 2002). A lipid transfer protein appears to be the major allergen in maize grain (Pastorello et al., 2003). However, in two published studies most individuals with a positive skin prick test or that had IgE antibodies against maize were suffering from respiratory allergy and only a few subjects displayed a true food allergy upon oral challenge with maize products (Pasini et al., 2002; Jones et al., 1995). In summary, oral sensitization to maize grain proteins is very rare (EFSA, 2007). Information on allergy prevalence in South Africa is consistent with information available from other countries. For example, in the USA maize allergy is estimated to affect no more than 0.016% of the general population (Taylor, 2000). In a nationwide survey of food allergy in Japan (Iikura et al., 1999), maize was not identified as a significant cause of food


February 2015



allergy. In addition, on the basis of a retrospective study on patients with histories of food allergy, Moneret-Vautrin et al. (1998) concluded that true food allergy to maize is rare and this has been confirmed by Scibilia et al. (2008). Accordingly, the International Codex Alimentarius Commission has not identified maize among the cereal grains requiring special hypersensitivity labelling (Codex, 2009 ), nor has the Japanese Ministry of Health and Welfare included maize on its list of foods subject to mandatory or recommended allergen labelling (Ebisawa et al., 2003). Although there have been some reports of occupational allergy to maize dust or pollen allergies in some geographical areas, this is unlikely to be a safety concern unique to the GM crop since maize is not considered to be a major allergenic food and there is no expectation that any of the GM plants have increased allergenic potential compared to their non-GM counterparts. The EFSA GMO Panel recently confirmed that it is unlikely that any interactions between the newly expressed proteins and metabolic pathways of maize would alter the pattern of expression of endogenous proteins/potential allergens and thereby significantly change the overall allergenicity of the whole plant (EFSA, 2010a, 2010b).

6.5 What evidence is there that the genetic modification described in this application did not result in over-expression of the possible allergens indicated in 6.4?

Is the expression of the possible allergens in the non-GM counterpart substantially equivalent to that in the GM organism? Although the allergenicity of the whole crop could theoretically be increased as an unintended effect of the random insertion of the transgene in the genome of the recipient, for example through qualitative or quantitative modifications of the expression pattern of endogenous proteins, this matter is not considered to be relevant. Since maize is not considered a major allergenic food and possible over-expression of any endogenous protein that is not known to be allergenic, it would be unlikely to alter the overall allergenicity of the whole plant. The European authority, EFSA (GMO Panel), recently confirmed that it is unlikely that any interactions between the newly expressed proteins and metabolic pathways of maize would alter the pattern of expression of endogenous proteins/potential allergens and thereby significantly change the overall allergenicity of the whole plant (EFSA, 2010c). Bt11 x MIR162 maize produces the proteins Cry1Ab, PAT, Vip3Aa20, and PMI. The potential allergenicity arising from Cry1Ab, PAT, Vip3Aa20, and PMI was discussed in detail under Section 6.2.3. These risk assessments and information supplied concluded that Bt11 x MIR162 maize are not different from conventional maize and are therefore unlikely to be allergenic. To assess the allergenic potential of Cry1Ab, PAT, Vip3Aa20, and PMI a weight of evidence approach (Codex, 2003) was followed by Syngenta, as it is recognized that there are no single definitive tests for allergenicity. A comprehensive assessment was therefore followed, using several characterization studies. The purpose of this approach is to assess protein sequence and other biophysical features of the proteins to identify significant similarities with known allergens. This assessment concluded that the Cry1Ab, PAT,


February 2015



Vip3Aa20, and PMI proteins show no evidence of sharing significant similarities with known allergens, are unlikely to be allergenic and therefore no further testing is required. In addition, based on the information provided, Syngenta considers it to be unlikely that potential interactions could occur that would change the allergenicity of these proteins. It can thus be concluded that the expression of the possible allergens in the non-GM counterpart is substantially equivalent to that in Bt11 x MIR162 maize. Syngenta also closely monitor potential additions to the allergen database and conducts new bioinformatic searches on an annual basis. Bioinformatic searches for sequence homologies and structural similarities between Cry1Ab, PAT, Vip3Aa20, and PMI and known or putative allergens were conducted in 2014. The results of these searches support the conclusion that there are no significant or new similarity matches with known or putative allergens. 6.6 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. 181 of 1993) and accompanied regulations. Maize has a long history of safe use and the crop itself causes few health problems. The information on the potential toxicity and allergenicity of the foreign gene products expressed in Bt11 x MIR162 maize as described above, allows the conclusion that Bt11 x MIR162 maize is as safe as non-genetically modified maize. Therefore, no adverse effects are expected on the health and safety of persons that will be in contact with Bt11 x MIR162 maize. A substantial weight of evidence indicates that there are no harmful unintended changes to Bt11 x MIR162 maize as a result of the transformation process and expression of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins. Hence, the likelihood of immediate or delayed adverse effects on human health either from ingestion or from working with Bt11 x MIR162 maize, or otherwise from coming into contact with the crop in the field, is negligible.

Studies comparing the composition and whole food safety of Bt11 x MIR162 maize plants and non-transgenic maize have been performed. All these studies lead to the conclusion that this maize is substantially equivalent to conventional maize. Finally, expression of the proteins Cry1Ab, PAT, Vip3Aa20, and PMI in food and feed derived from Bt11 x MIR162 maize is unlikely to cause adverse effects through toxicity or allergenicity based on the following information:

o Well-characterized specificity of the biological activity of Cry1Ab, PAT, Vip3Aa20, and PMI.

o No known adverse effects of prior exposure to Cry1Ab, PAT, Vip3Aa20, and PMI proteins in food or feed.

o Cry1Ab, PAT, Vip3Aa20, and PMI have no significant sequence identity to known toxins with known adverse effects in humans or animals.


February 2015



o Cry1Ab, PAT, Vip3Aa20, and PMI have no detectable acute toxicity in mice at high doses.

o Cry1Ab, PAT, Vip3Aa20, and PMI proteins have no significant amino acid homology to known or putative allergenic protein sequences that are biologically relevant or have implications for allergenic potential.

o Cry1Ab, PAT, Vip3Aa20, and PMI are degraded in simulated gastric fluid. o Low dietary exposure to Cry1Ab, PAT, Vip3Aa20, and PMI.

In addition, margins of dietary exposure have been calculated by comparing the NOEL from the acute oral toxicity studies of Cry1Ab, PAT, Vip3Aa20, and PMI protein to the expected intake level. The results (Table 7, Section 6.2.3.3) indicate that expected levels of intake of Cry1Ab, PAT, Vip3Aa20, and PMI through consumption of Bt11 x MIR162 maize in South Africa will be very low. Margins of dietary exposure exceed 3 215 for the Cry1Ab, PAT, Vip3Aa20, and PMI proteins, supporting the conclusion that no unacceptable risk is posed to consumers. 6.7 Indicate the proposed health and safety measures that would be applied to safeguard employees during the proposed activity. As explained above, no adverse effects are expected on the health and safety of persons that will be in contact with Bt11 x MIR162 maize. The same health and safety measure as non-genetically modified maize would be applied.


February 2015



7. ENVIRONMENTAL IMPACT AND PROTECTION 7.1 Detail any long-term effect the Commodity clearance of the genetically modified organism is likely to have on the biotic and abiotic components of the environment. This is an application for Commodity clearance, i.e. full food, feed and processing approval, of Bt11 x MIR162 maize in South Africa. Cultivation of Bt11 x MIR162 maize in South Africa is not within the scope of this application. Bt11 x MIR162 maize is a GM maize that is produced by conventional breeding of the following GM maize events: Bt11 and MIR162 maize. No further genetic modification to produce this stacked-event maize product has taken place. Maize plants derived from Bt11 maize contain the transgene cry1Ab, which encodes the insecticidal protein Cry1Ab, and the transgene pat, which encodes the enzyme PAT. Maize plants derived from MIR162 maize contain the transgene vip3Aa20, which encodes the insecticidal protein Vip3Aa20, and the transgene pmi, which encodes the enzyme PMI. The foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual Bt11 and MIR162 maize. Accordingly, Bt11 x MIR162 maize produces the transgenic proteins, Cry1Ab, PAT, Vip3Aa20, and PMI that provide control of certain lepidopteran insect pests and tolerance to glufosinate-ammonium in herbicide products. Successful establishment and spread of high numbers of plants of Bt11 x MIR162 maize would be necessary to enable any significant interaction with target organisms, which is very unlikely. Any plants germinating from this grain could be easily controlled using any of the current agronomic measures taken to control other commercially available maize (Owen, 2005). a) Effect on biotic environment

(i) Persistence and invasiveness

Maize is a highly domesticated plant and cannot survive without human intervention (OECD, 2003). Maize is an annual crop and seeds are the only survival structures; they cannot be dispersed without mechanical disruption of the cobs and show little or no dormancy. Natural regeneration from vegetative tissue is not known to occur (OECD, 2003). Survival of maize is dependent upon temperature, seed moisture, genotype, husk protection and stage of development. Maize is not a persistent weed. Maize seed can only survive under a narrow range of climatic conditions. Volunteers are killed by frost or easily controlled by current agronomic practices, including cultivation and the use of selective herbicides. In regions with mild and dry winters, maize kernels remaining in the field after harvest can develop volunteers. However, these volunteer plants develop defectively and tend to have low vigour, rarely producing cobs, which rarely have grain. Therefore cross-pollination between maize volunteers and other maize crops, although possible would only occur at very low levels (Palaudelmàs et al., 2009). Populations of sexually compatible wild relatives of maize are not known in South Africa (OECD, 2003).


February 2015



The comparative assessment of phenotypic and agronomic traits conducted for Bt11 x MIR162 maize and near-isogenic conventional maize has shown that the combination of Bt11 and MIR162 maize using conventional breeding has not altered the agronomic and phenotypic characteristics of the stacked-event maize product, apart from the intended modification, which is control of certain lepidopteran insect pests and tolerance to glufosinate-ammonium in herbicide products. The persistence or invasiveness of the stacked product when compared to conventional maize has not increased. Therefore, it is unlikely that Bt11 x MIR162 maize or other maize products containing combinations of fewer of these single events will differ in persistence and invasiveness from conventional maize.

As there are no wild relatives of maize in South Africa, any vertical gene transfer from Bt11 x MIR162 maize would be limited to other maize plants (Devos et al., 2005). Maize is an open-pollinating species and wind often facilitates cross fertilization between plants. Pollen that lands on a silk, will germinate almost immediately after pollination, and within 24 hours will complete fertilization (OECD, 2003). Theoretically, fertilization of other cultivated maize varieties by pollen from Bt11 x MIR162 maize could result in the expression of cry1Ab, pat, vip3Aa20, and/or pmi genes in those varieties, but this will occur only if conditions for cross pollination is favourable in fields adjacent to those where Bt11 x MIR162 maize is grown and only at an extremely low probability. The introduced traits in Bt11 x MIR162 maize are not intended to affect the range or frequency of maize outcrossing, and phenotypic data showed no indication that the genetic modification resulted in enhancement of reproductive characteristics or fertility of Bt11 x MIR162 maize, compared with conventional maize. However, the scope of this application does not include the cultivation of Bt11 x MIR162 maize in South Africa, therefore it is highly unlikely that Bt11 x MIR162 maize would grow in South Africa as a result of import or use in food and feed.

In the unlikely event that small amounts of Bt11 x MIR162 maize grain or grain from the sub-combinations present in the segregating progeny accidentally find their way into the environment, this would represent extremely low levels of exposure and this exposure would be localised and limited in time. The survival of this grain would be very unlikely as successful establishment and spread of high numbers of Bt11 x MIR162 maize plants would be necessary to enable any significant interaction with target organisms. Any plants germinating from this grain could be easily controlled using any of the current agronomic measures taken to control other commercially available maize (Owen, 2005). Therefore, Bt11 x MIR162 maize, or any of the sub-combinations present in the segregating progeny, is extremely unlikely to germinate and survive outside agricultural environments and its potential to interact with target species in South Africa is very low. Thus, the probability of Bt11 x MIR162 maize plants becoming more persistent than the recipient or parental plants in agricultural habitats or more invasive in natural habitats as a result of imported maize kernels of Bt11 x MIR162 maize, can be considered negligible. Thus, Commodity clearance of Bt11 x MIR162 maize is not likely to have significant long-term effects on the environment.


February 2015



(ii) Interactions between the GM plant and target/ non target organisms The comparative safety assessment conducted with Bt11 x MIR162 maize and near-isogenic conventional maize demonstrated that the only expected differences of biological relevance are the introduced genes and the proteins they express: Cry1Ab, PAT, Vip3Aa20, and PMI. The Cry1Ab and Vip3Aa20 proteins expressed by Bt11 x MIR162 maize is intended for protection against certain lepidopteran target pest species. The other proteins expressed are not known to have any effects on target or non-target organisms. In summary, it is highly unlikely that the import and use of Bt11 x MIR162 maize in South Africa will result in interactions with target species and/or non-target species that could result in harmful effects to the environment.

b) Effect on abiotic environment The scope of this application does not include cultivation of Bt11 x MIR162 maize. Therefore, interactions of this maize with the abiotic environment will be highly unlikely. In the unlikely event that small amounts of grain of Bt11 x MIR162 maize accidentally found their way into the South African environment, their survival would be very unlikely, as maize is a highly domesticated plant and cannot survive without human intervention (Niebur, 1993), especially under normal South African climatic conditions. Moreover, these plants can be easily controlled using any of the current agronomic measures taken to control other commercially available maize (Owen, 2005). In the unlikely event that some plants of Bt11 x MIR162 maize survived, the potential effects on the abiotic environment are likely to be the same as those effects resulting from cultivation of non-genetically modified maize. In summary, environmental impacts as a result of interactions between Bt11 x MIR162 maize and the abiotic environment can be considered negligible within the scope of this application. 7.2 Provide data and information on ecosystems that could be affected by use of the plant or its products. Not applicable. This is a Commodity clearance application. 7.3 Specify what effect the general release of the genetically modified plant will have on biodiversity. It is highly unlikely that the import and use of Bt11 x MIR162 maize in South Africa will result in adverse effects on biodiversity. This is an application for Commodity clearance of Bt11 x MIR162 maize in South Africa and release into the environment of Bt11 x MIR162 maize in South Africa is not within the scope of this application.


February 2015



7.4 Specify the measures to be taken in the event of the plant or product being misused or escaping into an environment for which it is not intended. This is an application for the Commodity clearance of Bt11 x MIR162 maize. The Bt11 x MIR162 maize is intended for cultivation outside South Africa and therefore derived products (including grain) of this maize may be commingled with derived products from conventional maize and enters South Africa through the trade routes. Bt11 x MIR162 maize can be considered as safe as conventional maize and the same practices used for conventional maize would be used for Bt11 x MIR162 maize. Commodity clearances are done by various grain traders on the international market, depending on the local need in South Africa, and would only take place under specific commodity clearance permits and conditions such as milling upon entry, which are issued by the Registrar of the GMO Act. Grain of Bt11 x MIR162 maize would therefore be imported, stored and handled in the same manner as non-genetically modified maize grain and other genetically modified maize grain already approved for importation into South Africa. However, in the rare event that small amounts of maize kernels of the stacked product could accidentally find their way into the environment during importation of this product by grain traders, their survival would be very unlikely as maize is highly domesticated and cannot survive without human intervention (Niebur, 1993; Owen, 2005), especially under normal South African climatic conditions. There are no data indicating that Cry1Ab, PAT, Vip3Aa20, and PMI protein expression results in altered seed dormancy, over wintering capacity, or other characteristics that would alter the prevalence of volunteer maize in subsequent growing seasons (ILSI, 2012). In addition, in the rare event that any Bt11 x MIR162 maize seedlings are produced they are unlikely to survive to flowering because they will be killed by frost, or removed by standard agronomic practice, as are seedlings of non-GM maize. The expression of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins does not affect the agronomic characteristics, weediness, persistence or invasiveness potential of Bt11 x MIR162 maize. No unintended effects that could confer a selective advantage or disadvantage have been detected in comparisons of Bt11 x MIR162 maize with non-transgenic, near-isogenic lines. Therefore, no specific measures need to be taken to prevent escape of Bt11 x MIR162 maize into the environment. 7.5 If the foreign genes give rise to crops resistant to agrochemicals, provide information on the registration of the agrochemicals to be used on the crop. Not applicable. This is a Commodity clearance application.


February 2015



8. SOCIO-ECONOMIC IMPACTS

8.1 Specify what, if any, positive or negative socio-economic impacts the genetically modified plant will have on communities in the proposed region of release.

a) The continued existence and range of diversity of the biological resources

This is an application for Commodity clearance of Bt11 x MIR162 maize in South Africa and release into the environment of Bt11 x MIR162 maize in South Africa is not within the scope of this application. No Bt11 x MIR162 maize will be released in the environment thus, as explained under Section 7, the chances of any maize being spilled and as a result negatively affect the existing biodiversity in the environment will be very rare. As there are no wild relatives of maize in South Africa, the potential of genetic transfer and exchange with other organisms is limited to other maize plants. Therefore, no positive or negative socio-economic impacts on the continued existence and range of diversity of the South African biological resources could be expected from the Commodity clearance of Bt11 x MIR162 maize in South Africa.

b) Access to genetics and other natural resources previously available

South Africa is not the centre of origin of maize and there are no wild relatives of maize in South Africa. Furthermore, Bt11 x MIR162 maize will not be released in the South African environment as this is an application for Commodity clearance of Bt11 x MIR162 maize and cultivation of Bt11 x MIR162 maize in South Africa is not within the scope of this application. Therefore, no positive or negative socio-economic impacts on the access to genetics or other natural resources could be expected from the Commodity clearance of Bt11 x MIR162 maize in South Africa.

c) Cultural traditions, knowledge and practices

South Africa imports maize, primarily as grain, depending on national surpluses and shortages, as well as regional demand. This grain is normally used to produce food and feed products. Grain of Bt11 x MIR162 maize will be imported under the same circumstances and used for the same purposes as any other commercial maize. Therefore, no altered socio-economic impacts on the cultural traditions, knowledge and practices of South African communities could be expected from the Commodity clearance of Bt11 x MIR162 maize in South Africa.

d) Income, competitiveness or economic markets

Commodity clearances are done by various grain traders on the international market, depending on the local need in South Africa. Commodity clearance of Bt11 x MIR162 maize would therefore facilitate international grain trade, thereby enabling economical access to the global maize grain trade market by various South African stakeholders (e.g. animal feed manufacturers). Twenty eight countries planted biotech crops on 182 million hectares world-wide in 2013 (James, 2014). The estimated total biotech crop area in South Africa in 2013 was 2.85


February 2015



million hectares. South Africa has been involved with biotechnology research and development for over 20 years and has developed a globally competitive biotechnology industry. South Africa planted approximately 15 million hectares of GM maize in the period 2000 to 2013, which included single, as well as stacked traits for insect and herbicide resistance. It was estimated that 2.73 million hectares maize was planted in 2013 season, from which 2.36 million hectares were biotech maize. Of this, 28.4% were the single Bt gene, 18.2% herbicide tolerant, and 53.4% stacked Bt and herbicide tolerant genes (James, 2013). Biotechnology has had a significant positive impact on farm income derived from a combination of enhanced productivity and efficiency gains. It is estimated that the total farm income benefit for GM maize in South Africa for the period 1996 to 2012 was US$1.105 billion (Brookes and Barfoot, 2014). The estimated GM crop farm income benefit in South Africa from 1996-2010 for herbicide tolerant maize was $3.2 million and for insect resistant maize was $769 million. The total estimated maize planting in 2014/15 is 2.6 million hectares, with an estimated yield of 14.3 million tons (Crop Estimates Committee6).

Therefore, Commodity clearance of Bt11 x MIR162 maize in South Africa can be expected to have a positive impact on the income, competitiveness and economic markets in South African communities.

e) Food security

It is important for Syngenta that the necessary food and feed approvals are obtained in countries where grain or processed products of GM maize events may be traded to, to ensure that the presence of the GM maize in grain consignments would not impede global trade. Syngenta therefore wishes to facilitate access to the global grain trade market by obtaining the necessary Commodity clearance approvals of this stacked product in South Africa. As discussed above, commodity clearances are done by various grain traders on the international market, depending on the local need in South Africa. Commodity clearance of Bt11 x MIR162 maize would therefore facilitate international grain trade in South Africa and can be expected to contribute positively to the maintenance of food security in South African communities.

6 Crop Estimates Committee, www.sagis.org.za/CEC, assessed Jan 2015

http://www.sagis.org.za/CEC


February 2015



9. WASTE DISPOSAL 9.1 Where only a portion of the genetically modified plant will be used for the product, how will the unused plant parts be disposed of? Not applicable. This is an application for Commodity clearance of Bt11 x MIR162 maize in South Africa and release into the environment of Bt11 x MIR162 maize in South Africa is not within the scope of this application. As indicated in Section 2.7, Bt11 x MIR162 maize could enter South Africa as grain or as processed products, which will be used in the manufacturing of food and animal feed products.


February 2015



10. MONITORING AND ACCIDENTS 10.1 Indicate the methods and plans for monitoring of the GMO This is an application for Commodity clearance (full food, feed and processing approval) of Bt11 x MIR162 maize and Bt11 x MIR162 maize is not intended at present for release to the environment in South Africa. The Bt11 x MIR162 maize is intended for cultivation outside South Africa and therefore derived products (including grain) of this maize may be commingled with products derived from conventional maize and enters South Africa through the trade routes. Bt11 x MIR162 maize can be considered as safe as conventional maize and the same practices used for conventional maize would be used for Bt11 x MIR162 maize. Commodity clearances are done by various grain traders on the international market, depending on the local need in South Africa, and would only take place under specific commodity clearance permits and permit conditions, which are issued by the Registrar of the GMO Act. Adherence to these permit conditions would be monitored by the Registrar’s office. Grain of Bt11 x MIR162 maize would therefore be imported, stored and handled in the same manner as non-genetically modified maize grain and other genetically modified maize grain already approved for importation into South Africa. However, in the rare event that small amounts of maize kernels of Bt11 x MIR162 maize could accidentally find their way into the environment during importation of this product by grain traders, their survival would be very unlikely as maize is highly domesticated and cannot survive without human intervention (Niebur, 1993; Owen, 2005), especially under normal South African climatic conditions. In addition, in the rare event that these maize plants were to survive they could be easily controlled using any of the current agronomic measures taken to control other commercially available maize, as the expression of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins does not affect the agronomic characteristics or weediness potential of Bt11 x MIR162 maize. 10.2 Indicate any emergency procedures that will be applied in the event of an accident in a comprehensive contingency plan Importation of grain consignments, which may contain Bt11 x MIR162 maize, are done by grain traders and used as food or feed by different manufacturers in South Africa. If grain of Bt11 x MIR162 maize would accidentally find their way into the environment due to activities by Syngenta South Africa, Syngenta South Africa will inform the Registrar of the GMO Act immediately both verbally and in writing of the accident and provide at the same time, or as soon as possible thereafter, information on (i) the circumstances (ii) the identity and quantity released, (iii) any information that is necessary to assess the impact on the environment and human and animal health and (iv) the measures taken to avoid or mitigate any adverse impact. If unusual observations are reported, more focused in-depth studies can be carried out in order to determine cause of accident. Grain and processed products could be destroyed by milling or burning.


February 2015



11. PATHOGENIC AND ECOLOGICAL IMPACTS 11.1 Submit an evaluation of the foreseeable impacts, in particular any pathogenic and ecologically disruptive impacts. Considering that this is an application for Commodity clearance, that Bt11 x MIR162 maize can be considered as safe as conventional maize and that the same practices used for conventional maize would be used for Bt11 x MIR162 maize, no pathogenic or ecologically disruptive impacts are expected.


February 2015



12. RISK MANAGEMENT 12.1 Please indicate any risk management measures that would be required for Commodity clearance. Bt11 x MIR162 maize is a GM maize that is produced by conventional breeding of the following GM maize events: Bt11 and MIR162 maize. No further genetic modification to produce this stack has taken place. Maize plants derived from Bt11 maize contain the transgene cry1Ab, which encodes the insecticidal protein Cry1Ab, and the transgene pat, which encodes the enzyme PAT. Maize plants derived from MIR162 maize contain the transgene vip3Aa20, which encodes the insecticidal protein Vip3Aa20, and the transgene pmi, which encodes the enzyme PMI. The foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual Bt11 and MIR162 maize. Accordingly, Bt11 x MIR162 maize produces the transgenic proteins, Cry1Ab, PAT, Vip3Aa20, and PMI that provide control of certain lepidopteran insect pests and tolerance to glufosinate-ammonium in herbicide products. This is an application for Commodity clearance of Bt11 x MIR162 maize in South Africa and cultivation of Bt11 x MIR162 maize in South Africa is not within the scope of this application. In the rare event that small amounts of maize kernels of Bt11 x MIR162 maize could accidentally find their way into the environment their survival would be very unlikely. Bt11 x MIR162 maize is therefore unlikely to become more persistent, weedy or invasive than maize varieties currently cultivated in South Africa. No biologically significant unintended changes were observed in Bt11 x MIR162 maize when compared with non-transgenic, near-isogenic lines in agronomic field trials in the United States. Agronomic trials indicate no biologically significant unintended changes in seed dispersal or other traits that might affect the ability of maize to survive without human intervention. Traits measured in field trials include seedling emergence, plant height, failure to produce an ear, dropping of ears before harvest, grain yield and disease incidence. The studies demonstrated that Bt11 x MIR162 maize was equivalent in agronomic parameters to their near-isogenic non-GM counterparts. The absence of unintended changes is not surprising as during domestication of maize, a strong directional selection imposed a genetic bottleneck at loci controlling agronomical valuable traits (Wright et al., 2005). The resulting low genetic variation at these loci, and continued selection during breeding, made it unlikely that unintended effects of transformation of maize could trigger reversion to ancestral weedy traits of teosinte. The comparative safety assessment of relevance to Bt11 x MIR162 maize, as described in detail in Section 6, demonstrated that the only differences of biological relevance are the introduced genes and the proteins they express: Cry1Ab, PAT, Vip3Aa20, and PMI. None of the components introduced into Bt11 x MIR162 maize are considered to be dangerous to human health or the environment. None of the proteins expressed by Bt11 x MIR162 maize are known to be toxic or allergenic to humans or animals. There are no known precedents where interactions between non-toxic proteins lead to toxic effects (FIFRA SAP, 2004). In addition, agronomic, compositional analysis and broiler feeding studies, have confirmed that the Bt11 x MIR162 maize is equivalent to conventional maize and is as safe and nutritious as conventional maize. Throughout all the tests conducted by Syngenta with Bt11 x MIR162 maize no evidence of interaction between the proteins produced by these plants has been observed.


February 2015



Grain of this stacked product could be contained in imported grain consignments and enter the South African market. Commodity clearances into South Africa, which may contain GM grain approved in the country of export, also is milled at the first port of entry in South Africa. The use of Bt11 x MIR162 maize grain would be the same as with any other grain imported into South Africa. As indicated in Section 2.3, commodity clearances are done by various grain traders on the international market, depending on the local need in South Africa. Commodity clearances would take place through the different sea ports and would be used as food or feed in all the areas where non-genetically modified maize grain is normally sold for these purposes, e.g. the North West Province, Free State, Limpopo Province, Mpumalanga, KwaZulu-Natal and the Eastern/Western Cape regions. Bt11 x MIR162 maize can be considered as safe as conventional maize and the same practices used for conventional maize would be used for Bt11 x MIR162 maize. The presence of the Cry1Ab, PAT, Vip3Aa20, and PMI, proteins in Bt11 x MIR162 maize does not change any of the typical crop characteristics of this maize. Labelling of the material will be according to legal requirements. The SABS standard (SANS 910, 2011) on requirements for the receiving, handling, transportation and storage of imported genetically modified commodities not approved for general release, will be followed. The measures pertaining to monitoring of the imported Bt11 x MIR162 maize grain and emergency measures to be taken in an event of accidental spillage would be prescribed under the GMO Act by the Executive Council and will be contained in the permits issued to the importers and the users. Adherence to these permit conditions would be monitored by the GMO Registrar’s office. Exposure to the environment will be limited to unintended release of Bt11 x MIR162 maize, which could occur for example via substantial losses during loading/unloading of the viable commodity including Bt11 x MIR162 maize destined for processing into animal feed or human food products. Exposure can be controlled by clean up measures and the application of current practices used for the control of any adventitious maize plants, such as manual or mechanical removal and the application of herbicides. In order to increase the possibility of detecting any unanticipated adverse effects, a general surveillance system can be implemented that will involve the authorisation of permit holders and operators handling and using of viable Bt11 x MIR162 maize. The operators can be provided with guidance to facilitate reporting of any unanticipated adverse effect from handling and use of viable Bt11 x MIR162 maize. Grain traders, transporters and all handlers in the food supply chain must report to the GMO Registrar’s office any unanticipated adverse effects arising from the handling and use of the GMO commodities.


February 2015



13. APPLICATION AFFIDAVIT The affidavit was competed


February 2015




February 2015



14. RISK ASSESSMENT OF Bt11 x MIR162 MAIZE

Risk assessment: Bt11x MIR162 maize in accordance with Annex III of Cartagena Protocol on Biosafety

Country Taking Decision:

South Africa

Title: Risk Assessment of the stacked-event maize product Bt11 x MIR162 maize (hereafter referred to as ‘Bt11 x MIR162 maize’) in South Africa. This risk assessment is in support of the Syngenta SA (Pty) Ltd. application for Commodity clearance release.

Contact details: Name and Address and contact details of the Applicant Syngenta SA (Pty) Ltd. Thornhill Office Park 94 Bekker Street Midrand, 1685 Tel: +27 11 541 4000 Fax: +27 11 541 4072

LMO information

Name and identity of the living modified organism:

Bt11 x MIR162 maize is a GM that is produced by conventional breeding of the following GM maize events: Bt11 (hereafter referred to as ‘Bt11 maize’) and MIR162 (hereafter referred to as ‘MIR162 maize’). Bt11 maize: Expresses a truncated Cry1Ab protein for control of certain lepidopteran pests and a PAT protein that confers tolerance to herbicide products containing glufosinate ammonium. MIR162 maize: Expresses a Vip3Aa20 protein for control of certain lepidopteran pests and a PMI protein, which acts as a selectable marker enabling transformed plant cells to utilize mannose as the only primary carbon source

Unique identification of the living modified organism:

Bt11 x MIR162: SYN- BTØ11-1 x SYN-IR162-4 Bt11: SYN- BTØ11-1 MIR162:SYN-IR162-4

Transformation event:

Bt11 x MIR162 maize is a GM maize product that is produced by conventional breeding of Bt11 maize and MIR162 maize.

Introduced or Modified Traits:

Altered growth, development and product quality: insect resistance and herbicide tolerance

Techniques used for modification:

Bt11 x MIR162 maize is produced by conventional breeding of Bt11 maize and MIR162 maize. Bt11 maize: Was transformed using a protoplast transformation / regeneration system (Negrutiu et al., 1987).


February 2015




Techniques used for modification:

MIR162 maize: Was produced by transformation of immature maize embryos derived from a proprietary Zea mays line via A. tumefaciens-mediated transformation (Negrotto et al., 2000; Hoekema et al., 1983).

Description of gene modification:

Maize plants derived from Bt11 x MIR162 maize are GM maize that is produced by conventional breeding of Bt11 and MIR162 maize. No further genetic modification to produce this stack has taken place. Accordingly, Bt11 x MIR162 maize produces the transgenic proteins present in Bt11 and MIR162 maize.

o a truncated Cry1Ab protein for control of certain lepidopteran pests like the common maize pests: O. nubilalis (ECB), S. nonagrioides (MCB), S. calamistis (PSB), B. fusca (Fuller) (ASB) and C. partellus (Swinhoe) (SSB).

o a PAT protein that confers tolerance to herbicide products containing glufosinate ammonium.

o a Vip3Aa protein (designated Vip3Aa20) for control of certain lepidopteran pests like H. zea (CE), A. ipsilon (BCW), S. frugiperda (FAW), and S. albicosta (WBC).

o a PMI protein, that acts as a selectable marker trait enabling transformed plant cells to utilize mannose as the only primary carbon source.

Vector characteristics:

No vector was used for the production of Bt11 x MIR162 maize. Bt11 x MIR162 hybrid maize was produced by conventional breeding of Bt11 and MIR162 maize. Bt11: The NotI restriction fragment of vector pZO1502, derivative of commercial available plasmid pUC18, was used for the transformation. MIR162 The plasmid pNOV1300 was used for transformation

Insert or inserts (Annex III.9(d)):

The Bt11 x MIR162 maize is produced by conventional breeding of Bt11 and MIR162 maize. No further genetic modification to produce this stack has taken place. The foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual Bt11 and MIR162 maize. Bt11 The NotI restriction fragment of vector pZO1502, a derivative of plasmid pUC18, was used for the transformation. The NotI fragment contains a truncated Bt gene which has been derived from the cry1Ab gene of B. thuringiensis. This gene is under the control of the 35S promoter from the CaMV, including the intron sequence IVS6 from maize, and the NOS terminator from A. tumefaciens.


February 2015




Insert or inserts It contains the pat gene from S. viridochromogenes encoding a phosphinothricin acetyl transferase; this gene is under the control of the 35S promoter from the CaMV, including the intron IVS2 from maize and the NOS terminator from A. tumefaciens. It also contains the E. coli origin of replication. The NotI fragment does not contain the E. coli amp gene present on pZO1502 which confers resistance of bacterial cells to ampicillin. This information has been previously reviewed by EFSA and has received positive scientific opinions (EFSA, 2009a). Bt11 maize received food, feed and processing approval by the European Commission (EFSA, 2012) and by the Executive Council of South Africa (Table 1). MIR162 The region intended for insertion contains the vip3Aa19 gene7 , a modified version of the native vip3Aa1 gene from B. thuringiensis; this gene is under the control of the maize polyubiquitin promoter, the intron #9 from the maize phosphoenolpyruvate carboxylase gene and the 35S terminator from the cauliflower mosaic virus. It also contains the pmi gene from E. coli encoding a phosphomannose isomerase; this gene is under the control of the maize polyubiquitin promoter and the nopaline synthase terminator from A. tumefaciens.

Recipient organism or parental organisms (Annex III.9(a)):

Taxonomic name/ status of recipient or parental organisms:

Family name: Poaceae Genus: Zea Species: Z. mays L. Subspecies: Z. mays ssp. mays

Common name of recipient or parental organisms:

Maize.

Point of collection or acquisition of recipient or parental organisms:

Maize originates from the Mesoamerican region, i.e. Mexico and Central America (CFIA, 2003).

Characteristics of recipient or parental organisms related to biosafety:

Z. mays reproduces sexually via the production of seed. Although maize is an allogamous species (capable of cross-fertilization), both self-fertilization and cross-fertilization are usually possible. Most maize varieties are protoandrous so pollen shedding precedes silk emergence by up to five days. Pollen dispersal is limited by several factors, including large size (0.1 mm diameter), rapid settling rate and short survivability.

7 The gene conferring protection against lepidopteran insect pests present on the plasmid pNOV1300 is vip3Aa19. The gene inserted in

MIR162 maize differs from the vip3Aa19 gene by two nucleotides. These transformation-induced nucleotide changes in the vip3Aa19 coding sequence resulted in one single amino acid change in the encoded protein. Therefore the gene expressed in MIR162 maize was designated vip3Aa20 and the encoded protein Vip3Aa20.


February 2015





Greater than 98% of the pollen settles to the ground within a maximum distance of 25-50 meters of its source (EEA, 2002; Jarosz et al., 2005). Shed pollen typically remains viable for 10 to 30 minutes, but may remain viable longer under refrigerated and humid conditions (Coe et al., 1988; Herrero and Johnson, 1980; Hoekstra et al., 1989; Jones and Newel, 1948). Fertilization is affected by a number of complicating factors, such as genetic sterility factors and differential growth rates of pollen tubes. 1. Sexual compatibility with other cultivated or wild plant species, including the distribution in South Africa of the compatible species. As there are no wild relatives of maize in South Africa, the potential for genetic transfer and exchange with other organisms is limited to other maize plants. Maize is wind pollinated and pollen distribution and viability depends on prevailing wind patterns, humidity, and temperature. The frequency of cross-pollination and fertilization depends on co-availability of fertile pollen and receptive plants. Wild Zea species have no pronounced weedy tendencies (CFIA, 2003). 2. Survivability (a) Ability to form structures for survival or dormancy Maize is an annual crop. Seeds are the only survival structures; they cannot be dispersed without mechanical disruption of the cobs and show little or no dormancy. Natural regeneration from vegetative tissue is not known to occur. (b) Specific factors affecting survivability Survival of maize is dependent upon temperature, seed moisture, genotype, husk protection and stage of development. Maize seed can only survive under a narrow range of climatic conditions. The biology of maize means that other than deliberate cultivation, the only means by which it can persist in the environment is accidental dispersal of kernels into disturbed ground during harvest or transport; maize cannot reproduce vegetatively (OECD, 2003). Maize kernels spilled in fields during harvest may germinate immediately and seedlings may be killed by frost (Miedema, 1982; OECD, 2003); however, maize can occur as a volunteer weed in areas with mild winters, or when seeds germinate in the spring (OECD, 2003). Volunteers are easily controlled with herbicides or other agronomic practices (Owen, 2005). Maize kernels spilled into disturbed ground outside agriculture can germinate to give occasional feral plants; however, even in areas with mild winters, persistent or invasive populations of feral maize are not observed in South Africa, presumably because of low seed dispersal and seedling survival due to retention of kernels on the ear (Doebley, 2004; Warwick and Stewart, 2005; OECD, 2003).


February 2015





3. Dissemination: (a) ways and extent (e.g. an estimation of how viable pollen and/or seeds declines with distance) of dissemination Maize dissemination may be accomplished through seed dispersal. Seed dispersal does not occur naturally due to the structure of the ear (OECD, 2003). Maize has a large ear with 500 or more kernels attached to its central axis. The kernels are naked and easily digested (cannot survive through the digestive tracts of birds and mammals) and so cannot be dispersed by animals. As ears of maize do not shatter, any ears left on the plant fall to the ground with all the kernels attached; when the hundreds of seeds on the ear germinate, the emerging plants are unable to obtain adequate light and soil to grow and reproduce (Doebley, 2004). Dissemination may also occur via pollen and pollen flow. Pollen dispersal is influenced by wind and weather conditions and is limited by several factors, including large size (0.1 mm diameter), rapid settling rate, short survivability, and physical barriers. Greater than 98% of the pollen settles to the ground within a maximum distance of 25-50 meters of its source (EEA, 2002; Jarosz et al., 2005). (b) specific factors affecting dissemination Maize has a polystichous (arranged in many rows) female inflorescence (group of flowers), called the ear, on a stiff central spike (cob) enclosed in husks (modified leaves). Because of the structure of the ears, seed dispersal of individual kernels does not occur naturally. Maize is non-invasive of natural habitats (OECD, 2003). The rate of dissemination via pollen will be influenced by the size of pollen, wind direction and speed, other weather conditions such as rainfall, the presence of barriers and the degree of synchrony of flowering. Maize pollen is large and heavy and tends to be deposited close to the source plant. In addition, most maize varieties are protoandrous so pollen shedding precedes silk emergence by up to five days. Pollen dispersal is influenced by wind and weather conditions and is limited by several factors, including large size (0.1 mm diameter), rapid settling rate, short survivability, and physical barriers. The pollen grain has a relatively thin outer membrane that gives little environmental protection, consequently shed pollen typically remains viable only for 10 to 30 minutes, but may remain viable longer under refrigerated and humid conditions (Coe et al., 1988; Herrero and Johnson, 1980; Hoekstra et al., 1989; Jones and Newel, 1948). Pollen release can be prevented by detasselling and genetic sterility. 4. Geographical distribution of the plant. Maize is the world’s most widespread cereal with very diverse morphological and physiological traits; it is grown on approximately


February 2015





184 million hectares worldwide (FAOSTAT, 2014 8 ). Maize is distributed over a wide range of conditions: from latitudes 50º North to 50º South, below sea level of the Caspian plains up to 3000m in the Andes Mountains and from semi-arid regions to arid regions (Russell and Hallauer, 1980). The greatest maize production occurs where the warmest month isotherms range between 21º and 27º C and the freeze-free season lasts 120-180 days. 5. Other potential interactions, relevant to the GMO, of the plant with organisms in the ecosystem where it is usually grown, or elsewhere, including information on toxic effects on humans, animals and other organisms. Maize is known to interact with other organisms in the environment including insects, birds, and mammals. It is susceptible to a range of fungal diseases and insect pests, as well as to competition from surrounding weeds (OECD, 2003). Maize is extensively cultivated and has a history of safe use for human food and animal feed. No significant native toxins are reported to be associated with the genus Zea (CFIA, 2003). As there are no wild relatives of maize in South Africa, the potential for genetic transfer and exchange with other organisms is limited to other maize plants. Maize is wind pollinated and pollen distribution and viability depends on prevailing wind patterns, humidity, and temperature. The frequency of cross-pollination and fertilization depends on the co-availability of fertile pollen and receptive plants. 6. Wild plant species Wild Zea species have no pronounced weedy tendencies (CFIA, 2003). The only wild taxa known to hybridise spontaneously with maize are species of teosinte (OECD, 2003; Owen, 2005). Annual teosinte is a wind-pollinated grass. Out-crossing and gene exchange between Z. mays ssp. mexicana and Z. mays ssp. mays do occur, but hybrids have reduced seed dispersal and often reduced viability (OECD, 2003). The natural distribution of Z. mays ssp. mexicana is limited to Mexico and Central America (CFIA, 2003). Although some Tripsacum species (Tripsacum dactyloides, T. floridanum, T. lanceolatum, and T. pilosum) can be crossed with Z. mays ssp mays, hybrids have a high degree of sterility and are genetically unstable. Out-crossing of maize and Tripsacum species is not known to occur in the wild (OECD 2003). No Tripsacum species are present in South Africa. Tripsacum species are geographically restricted to the Americas (CFIA, 2003). Only two species are known to be found north of

8 FAOSTAT. 2014. Statistic Division, Food and Agriculture Organization of United Nations, Rome Italy.

http://faostat.fao.org/site/567/default.aspx#ancor (updated August, 2014)


February 2015





Mexico: T. floridanum which is native to the southern tip of Florida, USA; and T. dactyloides (Eastern gammagrass), which can be found in the northern US. The center of diversity for Tripsacum is the western slopes of Mexico, the same area where teosinte is frequently found (CFIA, 2003). Tripsacum-annual teosinte hybrids have not been produced.

Centre(s) of origin of recipient or parental organisms:


Centres of genetic diversity, if known, of recipient or parental organisms:


Habitats where the recipient or parental organisms may persist or proliferate:

Maize originates from the Mesoamerican region, i.e. Mexico and Central America (CFIA, 2003). Please refer to information provided above regarding geographical distribution of the maize plant. Maize is incapable of sustained reproduction outside domestic cultivation and is non-invasive of natural habitats (OECD, 2003).

Donor organism or organisms (Annex III.9(b)):

Taxonomic name/ status of donor organism(s)

Bacillus thuringiensis The source of native cry1Ab and vip3Aa1 gene is B. thuringiensis. The species is a member of the genus Bacillus, a diverse group of rod-shaped, gram-positive, facultative anaerobic, spore forming bacteria. B. thuringiensis occurs naturally and ubiquitously in the environment. It is a common component of the soil microflora and has been isolated from most terrestrial habitats (Glare and O’Callaghan, 2000). Several subspecies of B. thuringiensis have been described; many of them have been extensively studied and used in commercial insecticide preparations. Insecticidal products using B. thuringiensis have been used for several decades and have a long history of safe use (US EPA, 2001).

Streptomyces viridochromogenes The source of the pat gene is the aerobic bacterium S. viridochromogenes strain Tu494, a gram-positive, sporulating, soil inhabiting bacterium widespread in the environment and with a long history of safe use (OECD, 1999). Escherichia coli The source of the pmi gene is the common bacterium E. coli, K-12 strain. E. coli belongs to the Enterobacteriaceae, a relatively


February 2015




Taxonomic name/ status of donor organism(s)

homogeneous group of rod-shaped, gram-negative, facultative bacteria. Members of the genus Escherichia are ubiquitous in the environment and found in the digestive tract of vertebrates, including humans. The vast majority of E. coli strains are harmless to humans, although some strains can cause diarrhoea and urinary infections. However, this particular group of pathogenic E. coli are distinct from the strains that are routinely used in the laboratory and from which the pmi gene was obtained. The K-12 strain from E. coli has a long history of safe use and is commonly used as a protein production system in many commercial applications.

Common name of donor organism(s):

Bacteria: B. thuringiensis, E. coli , S. viridochromogenes

Point of collection or acquisition of donor organism(s):

B. thuringiensis and E. coli bacteria are widely prevalent in the environment. S. viridochromogenes bacteria are widely prevalent in the environment.

Characteristics of donor organism(s) related to biosafety:

B. thuringiensis and E. coli bacteria are widely prevalent in the environment. S. viridochromogenes bacteria are widely prevalent in the environment.

Intended use and receiving environment

Intended use of the LMO:

Commodity clearance of Bt11 x MIR162 maize in South Africa.

Receiving environment:

No environmental release

Risk assessment summary

Detection/ Identification method of the LMO:

The Bt11 x MIR162 maize is a GM maize that is produced by conventional breeding of Bt11 and MIR162 maize. No further genetic modification to produce this stacked-event maize product has taken place. Bt11 x MIR162 maize produced by conventional breeding combining Bt11 and MIR162 maize has stably inherited the cry1Ab and pat genes from Bt11 and the vip3Aa1 and pmi gene from MIR162 maize, retaining the hybridization patterns as predicted. Accordingly, the foreign genes in Bt11 x MIR162 maize are not different from the foreign genes of the individual Bt11 and MIR162 maize. Bt11 x MIR162 maize contains the cry1Ab, pat, vip3Aa20, and pmi genes. The detection methods developed for the single events will also detect the individual events in Bt11 x MIR162 maize. For specific detection of Bt11 maize genomic DNA, a real-time quantitative TaqMan® PCR method has been developed using the taxon specific target sequence (Adh1) and the GMO (Bt11) target sequence.


February 2015




Detection/ Identification method of the LMO:

This method has been validated for use by the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) and can be found on the EU-RL GMFF website: http://gmo-crl.jrc.ec.europa.eu/summaries/Bt11_CRLVL1007_Validated_Method%20doc.pdf and http://gmo-crl.jrc.ec.europa.eu/summaries/Bt11_CRLVL1007_Val_Report.pdf For specific detection of MIR162 maize genomic DNA, a real-time quantitative TaqMan® PCR method has been developed using the taxon specific target sequence (Adh1) and the GMO (MIR162) target sequence. This method has been validated for use by the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) and can be found on the EU-RL GMFF website: http://gmo-crl.jrc.ec.europa.eu/summaries/MIR162_validated_Method.pdf and http://gmo-crl.jrc.ec.europa.eu/summaries/MIR162_val_report.pdf The Bt11 x MIR162 maize described in this application has been produced by combining Bt11 and MIR162 maize through conventional breeding techniques. There was no further genetic modification to produce the stack. As such, the detection methods developed for the single events should be appropriate for use on Bt11 x MIR162 maize. Syngenta has confirmed the applicability of these methods on Bt11 x MIR162 maize. Thus, the detection methods provided for Bt11 and MIR162 maize unambiguously detect the single events, as well as the stacked product in a mixture of seed/grain by using single seed analysis and the detection methods for each of the single events.

Evaluation of the likelihood of adverse effects:

Maize is planted and harvested as an annual crop. Wild populations with which it could cross-pollinate are uncommon, and not prevalent in South Africa. Maize dissemination can only be accomplished through seed dispersal which does not occur naturally due to the structure of the ear (OECD, 2003). Natural regeneration from vegetative tissue in the field is not known to occur. Maize is predominantly wind pollinated. Plants produce pollen for 10-13 days according to the genotype. Shed pollen typically remains viable only a short time but may remain viable longer under humid conditions. Pollen dispersal is influenced by wind and weather conditions and is limited by several factors, including large size (0.1 mm diameter), rapid settling rate, short survivability, and physical barriers. Greater than 98% of the pollen settles to the ground within a maximum distance of 25-50 meters of its source (EEA, 2002). The pollen grain has a relatively thin outer membrane that gives little








http://gmo-crl.jrc.ec.europa.eu/summaries/MIR162_val_report.pdf


February 2015





environmental protection, consequently shed pollen typically remains viable only for 10 to 30 minutes, but may remain viable longer under refrigerated and humid conditions (Coe et al., 1988; Herrero and Johnson, 1980; Hoekstra et al., 1989; Jones and Newel, 1948). Thus, even in the rare event that small amounts of maize kernels of the stacked product could accidentally find their way into the environment during importation of this product by grain traders, their survival would be very unlikely as maize is highly domesticated and cannot survive without human intervention (Niebur, 1993; Owen, 2005). In addition, in the rare event that these maize plants were to survive they could be easily controlled using any of the current agronomic measures taken to control other commercially available maize. Bt11 x MIR162 maize is unlikely to become more persistent, weedy or invasive than maize varieties currently cultivated in South Africa, as the expression of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins does not affect the overall agronomic characteristics or weediness potential. Maize has a history of safe use for human food and animal feed. No significant native toxins are reported to be associated with the genus Zea (CFIA, 2003) and maize is not considered as a major allergenic food (EFSA, 2007; Metcalfe et al., 2003). The Cry1Ab, PAT, Vip3Aa20, and PMI proteins expressed in Bt11 x MIR162 maize are not derived from a source known to produce allergenic proteins; have no significant amino acid homology to known mammalian protein toxins or to known or putative allergenic protein sequences that are biologically relevant or have implications for allergenic potential; they are readily degraded in in vitro digestibility assays; and, they show no acute oral toxicity in mammalian studies. There is no reason to anticipate that conventional breeding of Bt11 and MIR162 maize would result in stacked products that differ in toxicity to humans or animals. None of the proteins expressed by Bt11 and MIR162 maize are known to be toxic to humans or animals and there are no known precedents where interactions between non-toxic proteins lead to toxic effects (FIFRA SAP, 2004). In addition, compositional analysis and broiler feeding studies, have allow us to conclude that the Bt11 x MIR162 maize is equivalent in composition to conventional maize and is as safe and nutritious as conventional maize. A dietary exposure was assessed taking a worst case assumption that 100% of the maize consumed in South Africa is Bt11 x MIR162 maize. Taking into consideration the level of expression of the Cry1Ab, PAT, Vip3Aa20, and PMI proteins, based on an average maize consumption of 252.6 g/person/day (WHO, 2012), the theoretical daily intake for each of the proteins produced by Bt11 x MIR162 maize (Cry1Ab, PAT, Vip3Aa20, and PMI) was calculated, based on a


February 2015





bodyweight of a 60 kg person. In addition, margins of exposure have been calculated by comparing the NOEL from the acute oral toxicity study with each protein to the expected intake level. The results indicate that the expected levels of intake of the proteins Cry1Ab, PAT, Vip3Aa20, and PMI through consumption of Bt11 x MIR162 maize in South Africa will be very low. Margins of exposure with a minimum factor of 3216, supporting the conclusion that the risk to consumers is negligible and confirming the results previously obtained. Studies comparing the composition and whole food safety of Bt11 x MIR162 maize plants and non-transgenic maize lead to the conclusion that this maize is substantially equivalent to conventional maize. Bt11 x MIR162 maize is highly unlikely to have adverse effects on humans or animals; thus, the effects of Bt11 x MIR162 maize on human or animal health are unlikely to be different from those of non-transgenic maize. Maize is known to interact with other organisms in the environment including insects, birds, and mammals. It is susceptible to a range of fungal diseases and insect pests, as well as to competition from surrounding weeds (OECD, 2003). However, the importation and use as food, feed or for processing of grain from stacked Bt11 x MIR162 maize is highly unlikely to have environmental effects through interactions with non-target organisms.

Evaluation of the consequences:

Cultivation of maize derived from Bt11 x MIR162 maize in South Africa is not within the scope of Syngenta’s Application for Commodity clearance of Bt11 x MIR162 maize. The importation and use as food, feed or for processing of grain from stacked Bt11 x MIR162 maize is not expected to have any adverse consequences on human or animal health or the South African environment. As discussed above, the conclusion reached from the detailed evaluation of the characteristics of Bt11 x MIR162 maize and the likelihood of any adverse effects is that this maize is substantially equivalent to conventional maize and that it is highly unlikely to have any adverse effects on human or animal health or the environment. Therefore, no adverse consequences will result from the importation and use as food, feed or for processing of grain from stacked Bt11 x MIR162 maize in South Africa.

Overall risk:

The overall risk of potential adverse effects from importation of and use as food, feed or for processing of grain from stacked Bt11 x MIR162 maize is negligible. None of the components introduced into Bt11 and MIR162 maize are considered to be dangerous to human health or the environment.


February 2015




Overall risk There is no reason to anticipate that conventional breeding of Bt11 and MIR162 maize would result in stacked products that differ in toxicity to humans or animals. None of the proteins expressed by Bt11 and MIR162 maize are known to be toxic to humans or animals and there are no known precedents where interactions between non-toxic proteins lead to toxic effects (FIFRA SAP, 2004). In addition, compositional analysis and broiler feeding studies, have confirmed that the Bt11 x MIR162 maize is equivalent in composition to conventional maize and is as safe and nutritious as conventional maize. The overall risk for potential adverse effects on human and animal health or the environment as discussed in this document is thus negligible in the context of the intended uses of Bt11 x MIR162 maize.

Recommendation: Full compliance with permit conditions and other risk management conditions imposed by the Competent National Authority.

Actions to address uncertainty regarding the level of risk:

Not applicable.

Additional information

Availability of detailed risk assessment information:

More information on the stacked product and the assessment of risk can be obtained from the Commodity clearance application of Bt11 x MIR162 maize.

Any other relevant information:

No

Attach document: Not applicable

Notes: Not applicable


February 2015



15. RISK ASSESSMENT AFFIDAVIT

The affidavit was competed


February 2015




February 2015



16. REFERENCES

Baltazar, B.M., Sanchez-Gonzales, J.J., de la Cruz-Larios, L., Schoper, J.B. (2005). Pollination between maize and teosinte: an important determinant of gene flow in Mexico. Theor Appl Genet 110:591-526.

Bevan, M., Barnes, W.M., Chilton, M-D. (1983). Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res 11:369–385.

Brookes, G., Barfoot, P. (2014). GM Crops: Global Socio-economic and Environmental Impacts 1996-2012. P.G. Economics Ltd, Dorchester, UK. http://www.pgeconomics.co.uk/page/36/-gm-crop-use-continues-to-benefit-the-environment-and-farmers

CFIA. (2003). The Biology of Zea mays L. (Corn/Maize). Biology Document BIO1994-11 http://www.inspection.gc.ca/english/plaveg/bio/dir/dir9411e.shtml

Codex. (1999). Draft amendment to the General Standard for the Labelling of Prepackaged Foods: Foods that can cause hypersensitivity. Alinorm 99/22, Appendix III, Sec. 4.2.1.4. Adopted at the Twenty-third session of the Codex Alimentarius Commission (Alinorm 99/37).

ftp://ftp.fao.org/codex/ccfics8/fc00_02e.pdf Codex. (2003). Codex principles and guidelines on foods derived from biotechnology.

Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organisation: Rome.

Codex. (2009). Foods Derived from Biotechnology. Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organisation, Rome, Italy. 85 pp.

Coe, E.H.J., Nueffer, M.G., Hoisington, D.A. (1988). The Genetics of Maize. In Corn and Corn Improvement. Agronomy Monographs. Ed. GF Sprague and JW Dudley. American Society of Agronomy, Madison, Wisconsin 18: 81-236.

Crickmore, N., Baum, J., Bravo, A., Lereclus, D., Narva, K., Sampson, K., Schnepf, E., Sun, M., Zeigler, D.R. (2014) "Bacillus thuringiensis toxin nomenclature" http://www.btnomenclature.info/

Crop Life International. (2005). Combined trait plant biotechnology products are those containing more than one biotechnology-derived trait, such as one for insect control and another for herbicide tolerance, or two different insect control traits. https://croplife.org/plant-biotechnology/regulatory-2/combined-events/

Christensen, A. H., Sharrock, R. A., Quail, P. H. (1992). Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Molecular Biology 18: 675-689.

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

Devos, Y., Reheul, D., De Schrijver, A. (2005). The co-existence between transgenic and non-transgenic maize in the European Union: a focus on pollen flow and cross-fertilization. Environmental Biosafety Research 4: 71-87.

Doebley, J. (2004). The genetics of maize evolution. Annual Review of Genetics. 38: 7-59.

Ebisawa, M., Ikematsu, K., Imai, T., Tachimoto, H. (2003). Food Allergy in Japan. Allergy and Clinical Immunology International – Journal of World Allergy Organization 15/5: 214-217.

http://www.pgeconomics.co.uk/page/36/-gm-crop-use-continues-to-benefit-the-environment-and-farmers

http://www.pgeconomics.co.uk/page/36/-gm-crop-use-continues-to-benefit-the-environment-and-farmers

http://www.inspection.gc.ca/english/plaveg/bio/dir/dir9411e.shtml

http://www.btnomenclature.info/

https://croplife.org/plant-biotechnology/regulatory-2/combined-events/


February 2015



EEA. (2002). Maize. Genetically Modified Organisms (GMO's): The significance of gene flow through pollen transfer. A review and interpretation of published literature and recent/current research from the ESF 'Assessing the Impact of GM plants' (AIGM) programme for the ESF and EEA". A. E. K. & S. J. Environmental Issue Report 28 38-42.

EFSA. (2005). Opinion of the Scientific Panel on Genetically Modified Organisms on a request from the Commission related to the notification (Reference C/F/96/05.10) for the placing on the market of insect-tolerant genetically modified maize Bt11, for cultivation, feed and industrial processing, under Part C of Directive 2001/18/EC from Syngenta Seeds. The EFSA Journal 213: 1-33.

EFSA. (2007). Opinion of the Scientific Panel on Genetically Modified Organisms on applications (references EFSA-GMO-UK-2005-19 and EFSA-GMO-RX-Bt11) for the placing on the market of glufosinate-tolerant genetically modified maize Bt11, for food and feed uses, import and processing and for renewal of the authorisation of maize Bt11 as existing product, both under Regulation (EC) No 1829/2003 from Syngenta Seeds S.A.S. on behalf of Syngenta Crop Protection AG. Opinion adopted on 20 April 2005, The EFSA Journal 541: 1-25.

EFSA. (2009a). Scientific opinion of the Scientific Panel on Genetically Modified Organisms on an application (Reference EFSA-GMO-RX-Bt11) for renewal of the authorisation of existing products produced from insect-resistant genetically modified maize Bt11, under Regulation (EC) No 1829/2003 from Syngenta. The EFSA Journal 977: 1-13.

EFSA. (2009b). Statement of EFSA on the consolidated presentation of the joint Scientific Opinion of the GMO and BIOHAZ Panels on the “Use of Antibiotic Resistance Genes as Marker Genes in Genetically Modified Plants” and the Scientific Opinion of the GMO Panel on “Consequences of the Opinion on the Use of Antibiotic Resistance Genes as Marker Genes in Genetically Modified Plants on Previous EFSA Assessments of Individual GM Plants”. The EFSA Journal 1108: 1-8.

EFSA. (2010a). EFSA Panel on Genetically modified Organisms; Scientific Opinion on application (EFSA-GMO-UK-2007-50) for the placing on the market of insect resistant and herbicide tolerant genetically modified maize Bt11 x MIR604 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Syngenta Seeds. The EFSA Journal 8(5) (1614): 1-30.

EFSA. (2010b). Scientific Opinion on application (Reference EFSA-GMO-UK-2008-56) for

the placing on the market of insect resistant and herbicide tolerant genetically

modified maize Bt11 x MIR604 x GA21, for food and feed uses, import and

processing under Regulation (EC) No 1829/2003 from Syngenta Seeds. The EFSA

Journal 8 (5) (1616): 1-30.

EFSA. (2010c). Scientific Opinion on the assessment of allergenicity of GM plants and micro-organisms and derived food and feed. EFSA Panel on Genetically Modified Organisms (GMO Panel). The EFSA Journal 8 (7) (1700): 168 .

EFSA. (2012). Scientific Opinion updating the risk assessment conclusions and risk management recommendations on the genetically modified insect resistant maize Bt11. EFSA Journal 10(12):3018

Estruch, J. J., Warren, G. W., Mullins, M. A., Nye, G. J., Craig, J. A., Koziel, M. G. (1996). Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proceedings of the National Academy of Sciences 91: 5389-5394. (EPA MRID 46880804).


February 2015



FAO. (2003). Maize: Post-harvest Operations. http://www.fao.org/fileadmin/user_upload/inpho/docs/Post_Harvest_Compendium_-_MAIZE.pdf

FAO/WHO. (1991). Strategies for assessing the safety of foods produced by biotechnology. Report of a Joint FAO/WHO Consultation. Geneva, Switzerland, World Health Organization.

FAO/WHO. (2001). “Evaluation of allergenicity of genetically modified foods.” Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology. 22-25 January 2001. Rome, Italy.

FARRP. (2014). FARRP Protein AllergenOnline Database, version 14.0. Lincoln, NB: Food Allergy Research and Resource Program, University of Nebraska in Lincoln. http://www.allergenonline.com

FIFRA SAP. (2004). SAP Report No. 2004-05. Meeting Minutes. FIFRA Scientific Advisory Panel Meeting, June 8-10, 2004, held at the Holiday Inn Arlington, Arlington, Virginia. A Set of Scientific Issues Being Considered by the U.S. Environmental Protection Agency Regarding: Product Characterization, Human Health Risk, Ecological Risk, and Insect Resistance Management for Bacillus thuringiensis (Bt) Cotton Products.

Fling, M. E., Kopf, J., Richards, C. (1985). Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3'(9)-O-nucleotidyltransferase. Nucleic Acids Research 13: 7095-7106.

Franck, A., Guilley, H., Jonard, G., Richards, K., Hirth, L. (1980). Nucleotide sequence of cauliflower mosaic virus DNA (EPA MRID 46880804).Cell. 21: 285-294.

Freeling, M., Bennet, D.C. (1985). Maize Adh1. Annu Rev Genet 19:297–323. Frisner, H., Rosendal, A., Barkholt, V. (2000). Identification of immunogenic maize

proteins in a casein hydrosylate formula. Pediatric Allergy and Immunology. 11, 106-110.

Gardner, RC, Howarth, AJ, Hahn P, Brown-Luedi, M, Sheperd, RJ, Messing, J. (1981). The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing. Nucleic Acids Res 9:2871–2888.

Glare, T. R., O’Callaghan, M. (2000). Bacillus thuringiensis: Biology, Ecology and Safety. Chapter 3 p: 17-19 John Wiley & Sons, Ltd UK.

Herrero, M.P., Johnson, R.R. (1980). High temperature stress and pollen viability of maize. Crop Science 20: 796-800.

Hoekema, A., Hirsch, P., Hooykaas, P., Schilperoort, R. (1983). A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303(12): 179-180.

Hoekstra, F.A., Crow, L.M., Crow, J.H. (1989). Differential desiccation sensitivity of maize and Pennisetum pollen linked to their sucrose content. Plant Cell and Environment 12: 83-91.

Hudspeth, R. L., Grula, J. W. (1989). Structure and expression of the maize gene encoding the phophoenolpyruvate carboxylase isozyme involved in C4 photosynthesis. Plant Molecular Biology 12: 579-589.

Iikura, Y., Imai, Y., Imai, T., Akasawa, A., Fujita, K., Hoshiyama, K., Nakura, H., Kohno, Y., Koike, K., Okudaira, H., Iwasaki, E. (1999). Frequency of immediate-type food allergy in children in Japan. International Archives of Allergy and Immunology 118 (2-4): 251-252.

ILSI. (2012). A review of the environmental safety of Vip3Aa. http://cera-gmc.org/docs/protein_monographs/vip3aa_en.pdf

http://www.fao.org/fileadmin/user_upload/inpho/docs/Post_Harvest_Compendium_-_MAIZE.pdf

http://www.fao.org/fileadmin/user_upload/inpho/docs/Post_Harvest_Compendium_-_MAIZE.pdf

http://www.allergenonline.com/

http://cera-gmc.org/docs/protein_monographs/vip3aa_en.pdf

http://cera-gmc.org/docs/protein_monographs/vip3aa_en.pdf


February 2015



Itoh, Y., Tomizawa, J. (1979). Initiation of replication of plasmid ColE1 DNA by RNA polymerase, ribonuclease H and DNA polymerase I. Cold Spring Harbor Symposium on Quantitative Biology 43: 409-418.

James, C., (2013). Global status of commercialized biotech/GM crops: 2013, ISAAA brief No. 46 http://www.isaaa.org/resources/publications/briefs/default.asp

James, C., (2014). Global status of commercialized biotech/GM crops: 2014, ISAAA brief No. 49 http://www.isaaa.org/resources/publications/briefs/default.asp

Jarosz, N., Loubet, B., Durand, B., Foueillassar, X., Huber, L. (2005). Variation in maize pollen emission and deposition in relation to microclimate. Environmental Science and Technology 39: 4377-4384.

Johnston, D. B., McAloon, A. J., Moreau, R. A., Hicks, K. B., Singh, V., (2005). Composition and economic comparison of germ fractions from modified corn processing technologies. Journal of American Oil Chemical Society 82(8): 603-608.

Jones, M.D., Newel, L.C. (1948). Longevity of pollen and stigmas of grasses: buffalograss Buchloe dactyloides (Nutt.) Engelm. and maize Zea mays L. Journal of American Society of Agriculture 40:195-204.

Jones, S. M., Magnolfi, C. F., Cooke, S. K., Sampson, H. A. (1995). Immunologic cross-reactivity among cereal grains and grasses in children with food hypersensitivity. Journal of Allergy and Clinical Immunology 96(3):341-351.

Keese, P. (2008). Risks from GMOs due to horizontal gene transfer. Environmental

Biosafety Research 7: 123-149.

Komari, T., Hiei, Y., Saito, Y., Murai, N., Kumashiro, T. (1996). Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformation free from selection markers. The Plant Journal 10: 165-174.

Launis, K., Kramer, C. (2006). Compositional Analysis of Grain and Forage Derived from Event Bt11 Hybrid Maize and a Non-transgenic, Near-isogenic Hybrid Maize, Grown During 2005 in the USA: Volume 1 of 2. Report No. SSB-132-06 (unpublished). Research Triangle Park, NC: Syngenta Crop Protection, LLC. Submitted as part of Syngenta’s application for General Release of Bt11 in RSA (permit 39.4(5/10/374)).

Lee, M-K., Walters, F. S., Hart, H., Palekar, N., Chen, J-S. (2003). The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of

Cry1Ab -endotoxin. Applied and Environmental Microbiology 69:4648-4657. Mascarenhas, D., Mettler, I.J., Pierce, D.A., Lowe, H.W. (1990). Intron-mediated

enhancement of heterologous gene expression in maize. Plant Mol Biol 15:913–920.

Metcalfe, D. D., Sampson, H. S., Simon, R. A. (2003). Food allergy: adverse reactions to foods and food additives. 3rd edition. Blackwell Publishing.

Miedema, P. (1982). “The effects of low temperature on Zea mays.” Advances in Agronomy 35: 93-128.

Moneret-Vautrin, D. A., Kanny, G., Beaudouin, E. (1998). L’allergic alimentaire au mais existe-t-elle? Allergy and Immunology 30:230.

Murray, E. E., Lotzer, J., Eberle, M. (1989). Codon usage in plant genes. Nucleic Acids Research 17: 477-498.

NCBI. (2014). National Center for Biotechnology Information, U.S. National Library of Medicine. 8600 Rockville Pike, Bethesda MD, 20894 USA. http://www.ncbi.nlm.nih.gov/nucleotide

http://www.isaaa.org/resources/publications/briefs/default.asp

http://www.isaaa.org/resources/publications/briefs/default.asp

http://www.ncbi.nlm.nih.gov/nucleotide


February 2015



Negrotto, D., Jolley, M., Beer, S., Wenck, A. R., Hansen, G. (2000). The use of

phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Reports 19: 798-803.

Negrutiu, I., Shillito, R., Potrykus, I., Basiani, G., Sala G. (1987). Hybrid genes in the analysis of transformation conditions. Plant Mol. Biol. 8: 363-373.

Niebur, W. S. (1993). "Traditional crop breeding practicies: an historical review to serve as a baseline for assessing the role of modern biotechnology." OECD 113-121.

OECD. (1999). Consensus document on general information concerning the genes and their enzymes that confer tolerance to phosphinothricin herbicide. Series on Harmonisation of Regulatory Oversight in Biotechnology (11).

OECD. (2002). "Consensus Document on Compositional Considerations for New Varieties of Maize (Zea mays): Key Food and Feed Nutrients, Anti-nutrients and Secondary Plant Metabolites." Series on the Safety of Novel Foods and Feeds. No 6. http://www.olis.oecd.org/olis/2002doc.nsf/LinkTo/NT00002F66/$FILE/JT00130429.PDF

OECD. (2003). Consensus Document on the Biology of Zea mays (Maize). Series on Harmonisation of Regulatory Oversight in Biotechnology 27.

OGTR. (2008). The biology of Zea mays L. ssp mays (maize or corn). Australian Government Office of the Gene Technology Regulator. http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/maize-3/$FILE/biologymaize08_2.pdf

OGTR. (2009). Risk Analysis Framework, Australian Government Office of the Gene Technology Regulator.

http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/content/raf-3/$FILE/raffinal4.pdf Ooms, G., Hooykaas, P. J. J., van Veen, R. M., van Beelen, P., Regensburg-Tuink, T.

J.G. and Schilperoort, R. A. (1982). Octopine Ti-Plasmid Deletion Mutants of Agrobacterium tumefaciens with Emphasis on the Right Side of the T-Region. Plasmid 7: 15-29.

Owen, M. D. K. (2005). Maize and soybeans – controllable volunteerism without ferality? In: Gressel, J. (ed.) Crop Ferality and Volunteerism. Boca Raton, FL., CRC Press. pp. 149-165.

Palaudelmàs, M., Peñas, G., Melé, E., Serra, J., Salvia, J., Pla, M., Nadal, A., Messeguer J., (2009). Effect of volunteers on maize gene flow. Transgenic Research 18 (4): 583-594.

Pasini, G., Simonato, B., Curioni, A., Vincenzi, S., Cristaudo, A., Santucci, B., Peruffo, A. D., Giannattasio, M., (2002). IgE-mediated allergy to corn: a 50 kDa protein, belonging to the Reduced Soluble Proteins, is a major allergen. Allergy 57(2): 98-106.

Pastorello, E. A., Farioli, L., Pravettoni, V., Ispano, M., Scibola, E., Trambaiolo, C., Gluffrida, M. G., Ansaloni, R., Godovac-Zimmermann, J., Cont, A., Fortunato, D., Ortoloni, C., (2000). The major maize allergen, which is responsible for food-induced allergic reactions, is a lipid transfer protein. Allergy and Clinical Immunology International – Journal of World Allergy Organization 106(4): 744-751.

http://www.olis.oecd.org/olis/2002doc.nsf/LinkTo/NT00002F66/$FILE/JT00130429.PDF

http://www.olis.oecd.org/olis/2002doc.nsf/LinkTo/NT00002F66/$FILE/JT00130429.PDF

http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/maize-3/$FILE/biologymaize08_2.pdf

http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/maize-3/$FILE/biologymaize08_2.pdf


February 2015



Pastorello, E. A., Pompei, C., Pravettoni, V., Farioli, L., Calamari, A. M., Scibilia, J., Robino, A. M., Conti, A., Iametti, S., Fortunato, D., Bonomi, S., Ortolani, C., (2003). Lipid-transfer protein is the major maize allergen maintaining ige-binding activity after cooking at 100°C, as demonstrated in anaphylactic patients and patients with positive double-blind, placebo-controlled food challenge results. Journal of Allergy Clinical Immunology 112: 775-783.

Perlak, F.J., Fuchs, R.L., Dean, D.A., McPherson, S.L., Fischhoff, D. (1991). Modification of the coding sequence enhances plant expression of insect control protein genes. P Natl Acad Sci USA 88:3324–3328.

Radauer, C. and Breiteneder, H. (2007). Evolutionary biology of plant food allergens. Journal of Allergy and Clinical Immunology 120: 518-525.

Reed, J., Privalle, L., Luann Powell, M., Meghji, M., Dawson, J., Dunder, E., Suttie, J.,

Wenck, A., Launis, K., Kramer, C., Chang, Y., Hansen, G., Wright, M. (2001).

Phosphomannose Isomerase: an efficient selectable marker for plant

transformation. In Vitro Cellular Developmental Biology 37: 127-132.

Russell,W. A., Hallauer, A. R., (1980). "Corn". Hybridization of crop plants. Fehr W.R. and Hadley, H.H. Editors. American Society of Agronomy and Crop Science Society of America, Publishers, Madison, Wisconsin. 299-312.

Sanger, F., Coulson, A. R., Hong, G. F., Hill, D. F., Petersen, G. B. (1982). Nucleotide Sequence of Bacteriophage λ DNA. Journal of Molecular Biology. 162: 729-773.

SANS 910. (2011). Requirements for the receiving, handling, transportation and storage of imported genetically modified commodities not approved for general release. https://www.sabs.co.za/sectors-and-services/sectors/transport/transport_sp.asp; http://www.store.sabs.co.za/sans-910-2011-ed-1-00

Scibilia, J., Pastorello, E. A., Zisa, G., Ottolenghi, A., Ballmer-Weber, B., Pravettoni V., Scovena, E., Robino, A., Ortolani, C. (2008). Maize food allergy: a double-blind placebo-controlled study. Clinical and Experimental Allergy 38: 1943-1949.

Sjoblad, R. D., McClintock, J. T. Y., Engler, R. (1992). Toxicological considerations for protein components of biological pesticide products. Regulatory Toxicol. Pharmacol. 15: 3-9

Strauch, E., Wohlleben, W., Puhler, A. (1988). Cloning of a phosphinothricin N-acetyl transferase gene from Streptomyces viridochromogenes Tü4994 and its expression in Streptomyces lividans and Escherichia coli. Gene. 63: 65-77

Taylor, S. L. (2000). Allergenicity of corn and corn products. Food Allergy Research & Resource Program, University of Nebraska, Lincoln, 7 February.

Taylor, S. L., Hefle, S. L. (2001). “Will genetically modified foods be allergenic?” Journal of Allergenicity and Clinical Immunology 107(5):765-771.

Tzfira, T., Li, J., Lacroix, B., Citovsky, V. (2004). Agrobacterium T-DNA integration: molecules and models. Trends Genet 20:375–383.

US EPA. (2001). Biopesticides Registration Action Document – Bacillus thuringiensis Plant Incorporated Protectants. http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm.

US EPA. (2010). Biopesticides Registration Action Document – Cry1Ab and Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants. http://www.regulations.gov/search/Regs/home.html#documentDetail?R=0900006480b2916e

Van Frankenhuyzen, K., Nystrom C. (2002). "The Bacillus thuringiensis toxin specificity database" http://www.glfc.cfs.nrcan.gc.ca/bacillus (accessed Nov 2014).

https://www.sabs.co.za/sectors-and-services/sectors/transport/transport_sp.asp

http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm

http://www.regulations.gov/search/Regs/home.html#documentDetail?R=0900006480b2916e

http://www.regulations.gov/search/Regs/home.html#documentDetail?R=0900006480b2916e

http://www.glfc.cfs.nrcan.gc.ca/bacillus


February 2015



Van Wert, S. (1994). Petition for Determination of Nonregulated Status: Glufosinate Resistant Corn Transformation Events T14 and MIR162. http://www.aphis.usda.gov/brs/aphisdocs/94_35701p.pdf

Wang, K., Herrera-Estrella, L., Van Montagu, M., Zambryski, P. (1984). Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell 38(2): 455-462.

Warwick, S.I., Stewart, Jr. N. (2005). “Crops Come from Wild Plants—How Doestication Transgenes, and Linkage Together Shape Ferality.” Crop Ferality and Volunteerism 11-30

WHO. (1995). Application of the principles of substantial equivalence to the safety evaluation of foods or food components from plants derived by modern biotechnology. WHO Workshop, pp. 1-80, 1995

WHO. (2012) GEMS/Food consumption cluster diets. http://www.who.int/nutrition/landscape_analysis/nlis_gem_food/en/ (accessed Nov 2014).

Wohlleben, W., Arnold, W., Broer, I., Hilleman, D., Strauch, E., Pühler, A. (1988). Nucleotide sequence of the phosphinothricin-N-acetyltransferase gene from a Streptomyces viridochromogenes Tu494 and its expression in Nicotiana tabacum. Gene 70:25–37.

Wright, S. I., Vroh Bi, I., Schroeder, S. G., Yamasaki, M., Doebley, J. F., McCullen, M. D., Gaut, B. S. (2005). The effect of artificial selection on the maize genome. Science 308: 1310-1314.

Yu C.-G., Mullins M., Warren G. W., Koziel M. G., Estruch J. J. (1997). The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses mid-gut epithelium cells of susceptible insects. Applied Environmental Microbiology. 63(2): 532-536.

Zambryski, P., Depicker, A., Kruger, K., Goodman, H. M. (1982). Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. Journal of Molecular and Applied Genetics 1(4): 361-370.

http://www.aphis.usda.gov/brs/aphisdocs/94_35701p.pdf

Documents

APPLICATIONS IN TERMS OF THE GENETICALLY MODIFIED ORGANISMS … for commodity... · 2015-02-13 · APPLICATIONS IN TERMS OF THE GENETICALLY MODIFIED ORGANISMS ACT, 1997 APPLICATION