Risk Assessment
and
Risk Management Plan
Application for licence for dealings involving an
intentional release into the environment
DIR 055/2004
Title: Field trial of herbicide tolerant
(Roundup Ready Flex cotton MON 88913) and
herbicide tolerant/insect resistant
(Roundup Ready Flex cotton MON 88913/Bollgard II)
cottons
Applicant: Monsanto Australia Ltd
April 2005
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Abbreviations
aad gene encoding AAD
AAD aminoglycoside adenyltransferase
ANZFA Australia New Zealand Food Authority (now FSANZ)
APVMA Australian Pesticides and Veterinary Medicines Authority
bp basepair
Bt Bacillus thuringiensis
Btk Bacillus thuringiensis variety kurstaki
CaMV cauliflower mosaic virus
CP4 EPSPS EPSPS protein from Agrobacterium sp. strain CP4
cry gene encoding Cry
Cry crystal insecticidal proteins of Bt
CSIRO Commonwealth Scientific and Industrial Research Organisation
DIR dealing involving intentional release
DNA deoxyribonucleic acid
EFSA European Food Safety Authority
ELISA enzyme linked immunosorbent assay
EMBL European Molecular Biology Laboratory
EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
FMV figwort mosaic virus
FSANZ Food Standards Australia New Zealand (formerly ANZFA)
g gram
GM genetically modified
GMAC Genetic Manipulation Advisory Committee
GMO genetically modified organism
GTTAC Gene Technology Technical Advisory Committee
GUS -glucuronidase
ha hectare
IgE immunoglobulin E
kDa kiloDalton
km kilometre
m metre
MRL maximum residue limit
mRNA messenger ribonucleic acid
μg microgram
ng nanogram
nos gene encoding nopaline synthase
nptII gene encoding NPTII
NPTII neomycin phosphotransferase type II
OGTR Office of the Gene Technology Regulator
PCR polymerase chain reaction
T-DNA transfer deoxyribonucleic acid
uidA gene encoding GUS
USDA United States Department of Agriculture
US EPA United States Environmental Protection Agency
US FDA United States Food and Drug Administration
WHO World Health Organisation
Abbreviations
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
TABLE OF CONTENTS
EXECUTIVE SUMMARY .................................................................................................................................... I
THE DECISION I
THE APPLICATION ................................................................................................................................................ I
THE EVALUATION PROCESS ................................................................................................................................III
CONCLUSIONS OF THE RISK ASSESSMENT .......................................................................................................... IV
Toxicity or allergenicity to humans .............................................................................................................. IV
Toxicity to non-target organisms ................................................................................................................... V
Weediness ...................................................................................................................................................... V
Transfer of introduced genes to other organisms .......................................................................................... V
Herbicide and insecticide resistance ............................................................................................................ VI
THE RISK MANAGEMENT PLAN (KEY LICENCE CONDITIONS) .............................................................................. VI
Toxicity or allergenicity to humans .............................................................................................................. VI
Toxicity to non-target organisms ................................................................................................................ VII
Weediness ................................................................................................................................................... VII
Transfer of introduced genes to other organisms ....................................................................................... VII
Herbicide and insecticide resistance .......................................................................................................... VII
General conditions ..................................................................................................................................... VII
Research requirements .............................................................................................................................. VIII
Identification of issues to be addressed for future releases ....................................................................... VIII
Monitoring and enforcement of compliance by the OGTR ........................................................................ VIII
FURTHER INFORMATION .................................................................................................................................. VIII
CHAPTER 1 BACKGROUND ....................................................................................................................... 1
SECTION 1 THE APPLICATION........................................................................................................................ 1
Section 1.1 The proposed dealings ........................................................................................................... 2
Section 1.2 Parent organism .................................................................................................................... 3
Section 1.3 Genetic modifications and their effects .................................................................................. 3
Section 1.4 Method of gene transfer ......................................................................................................... 4
SECTION 2 PREVIOUS RELEASES AND INTERNATIONAL APPROVALS .............................................................. 4
Section 2.1 Previous Australian releases of the same or similar GM cottons .......................................... 4
Section 2.2 Approvals by other Australian government agencies ............................................................ 5
Section 2.3 International approvals ......................................................................................................... 7
CHAPTER 2 SUMMARY OF RISK ASSESSMENT AND RISK MANAGEMENT PLAN...................... 8
SECTION 1 FINALISATION OF THE RISK ASSESSMENT AND RISK MANAGEMENT PLAN .................................. 8
SECTION 2 ISSUES RAISED IN SUBMISSIONS ON THE APPLICATION AND RISK ASSESSMENT AND RISK
MANAGEMENT PLAN .................................................................................................................... 8
SECTION 3 RESEARCH REQUIREMENTS ........................................................................................................10
SECTION 4 IDENTIFICATION OF ISSUES TO BE ADDRESSED FOR FUTURE RELEASES .....................................10
SECTION 5 DECISION ON THE APPLICATION .................................................................................................10
SECTION 6 TABULATED SUMMARY OF THE RARMP (INCLUDING LICENCE CONDITIONS) ...............................11
APPENDIX 1 INFORMATION ABOUT THE GMO ..................................................................................16
SECTION 1 SUMMARY INFORMATION ABOUT THE GMO ..............................................................................16
SECTION 2 THE PARENT ORGANISM .............................................................................................................17
SECTION 3 THE INTRODUCED GENES AND THEIR PRODUCTS.........................................................................18
Section 3.1 The cp4 epsps herbicide tolerance gene and encoded protein .............................................18
Section 3.2 The cry1Ac and cry2Ab insecticidal genes and encoded proteins ........................................20
Section 3.3 The uidA reporter gene and encoded protein .......................................................................20
Section 3.4 The nptII and aad antibiotic resistance marker genes and encoded proteins .......................21
SECTION 4 METHOD OF GENETIC MODIFICATION .........................................................................................22
SECTION 5 CHARACTERISATION OF THE INSERTED GENETIC MATERIAL AND STABILITY OF THE GENETIC
MODIFICATION............................................................................................................................23
SECTION 6 EXPRESSION OF THE INTRODUCED PROTEINS ..............................................................................23
SECTION 7 PLEIOTROPIC EFFECTS OF THE GENETIC MODIFICATION ..............................................................25
SECTION 8 CONSIDERATION OF THE RISKS RELATING TO THE COMBINATION OF THE BOLLGARD II® AND THE
®
ROUNDUP READY FLEX TRAITS .................................................................................................25
SECTION 9 RESEARCH REQUIREMENTS ........................................................................................................26
Table of contents
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 2 TOXICITY AND ALLERGENICITY TO HUMANS .........................................................27
SECTION 1 NATURE OF THE POTENTIAL TOXICITY OR ALLERGENICITY HAZARD ..........................................27
SECTION 2 LIKELIHOOD OF THE TOXICITY OR ALLERGENICITY HAZARD OCCURRING...................................28
Section 2.1 Toxicity and allergenicity of conventionally bred non-GM cotton .......................................28
Section 2.2 Exposure of people to GM cottons ........................................................................................29
Section 2.3 Other sources of CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and NPTII in the environment .........31
Section 2.4 Toxicity and allergenicity of the introduced proteins ...........................................................31
SECTION 3 CONCLUSIONS REGARDING TOXICITY OR ALLERGENICITY..........................................................36
APPENDIX 3 TOXICITY TO NON-TARGET ORGANISMS ...................................................................37
SECTION 1 NATURE OF THE POTENTIAL TOXICITY HAZARD..........................................................................37
SECTION 2 LIKELIHOOD OF THE TOXICITY HAZARD OCCURRING ..................................................................38
Section 2.1 Toxicity of conventionally bred cotton ..................................................................................38
Section 2.2 Other sources of CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and NPTII in the environment .........38
Section 2.3 Potential toxicity hazards for stock and wildlife, including mammals, birds and fish ..........40
Section 2.4 Potential toxicity hazard for invertebrates ...........................................................................41
Section 2.5 Potential toxicity hazard for microorganisms ......................................................................44
SECTION 3 CONCLUSIONS REGARDING TOXICITY TO NON-TARGET ORGANISMS ...........................................47
APPENDIX 4 WEEDINESS ...........................................................................................................................48
SECTION 1 NATURE OF THE WEEDINESS HAZARD .........................................................................................48
SECTION 2 LIKELIHOOD OF THE WEEDINESS HAZARD OCCURRING ...............................................................49
Section 2.1 Inherent weediness of conventional non-GM cotton.............................................................49
Section 2.2 Potential selective advantage conferred by the introduced proteins ....................................50
Section 2.3 Potential weediness of Roundup Ready® Flex and
Roundup Ready® Flex/Bollgard II® cottons..........................................................................51
Section 2.4 Spread of GM cottons beyond the release sites ....................................................................53
Section 2.5 Persistence of the GM cotton at the release sites .................................................................53
SECTION 3 CONCLUSIONS REGARDING WEEDINESS ......................................................................................54
SECTION 4 RESEARCH REQUIREMENTS .........................................................................................................54
APPENDIX 5 TRANSFER OF INTRODUCED GENES TO OTHER ORGANISMS .............................55
SECTION 1 GENE TRANSFER FROM THE GM COTTONS TO OTHER PLANTS ....................................................56
Section 1.1 Nature of the gene transfer hazard to other plants ...............................................................56
Section 1.2 Potential hazards from the introduced genes in other plants ...............................................56
Section 1.3 Likelihood of gene transfer from the GM cottons to other plants .........................................57
SECTION 2 GENE TRANSFER FROM THE GM COTTONS TO MICROORGANISMS ................................................59
Section 2.1 Nature of the gene transfer hazard to microorganisms ........................................................59
Section 2.2 Potential hazards from the introduced genes in microorganisms.........................................59
Section 2.3 Other sources of the introduced genes in the environment, and their potential for horizontal
transfer to microorganisms ...................................................................................................60
Section 2.4 Likelihood of gene transfer from Roundup Ready® Flex or Roundup Ready®
Flex/Bollgard II® cotton to microorganisms ........................................................................62
SECTION 3 GENE TRANSFER FROM ROUNDUP READY® FLEX OR ROUNDUP READY® FLEX/BOLLGARD II®
COTTON TO ANIMALS (INCLUDING HUMANS) ..............................................................................65
Section 3.1 Nature of the gene transfer hazard to animals (including humans) .....................................65
Section 3.2 Potential hazards from the introduced genetic materials in animals (including humans) ...65
Section 3.3 Likelihood of gene transfer from Roundup Ready® Flex or
Roundup Ready® Flex/Bollgard II® cotton to animals (including humans) .........................66
SECTION 4 CONCLUSIONS REGARDING GENE TRANSFER TO OTHER ORGANISMS ..........................................67
Section 4.1 Conclusions regarding gene transfer to other plants ...........................................................67
Section 4.2 Conclusions regarding gene transfer to microorganisms .....................................................68
Section 4.3 Conclusions regarding gene transfer to animals, including humans....................................68
SECTION 5 RESEARCH REQUIREMENTS .........................................................................................................68
APPENDIX 6 DEVELOPMENT OF RESISTANCE TO HERBICIDES OR INSECTICIDES ..............69
SECTION 1 REGULATION OF AGRICULTURAL CHEMICALS IN AUSTRALIA ......................................................69
SECTION 2 NATURE OF THE HERBICIDE AND INSECTICIDE RESISITANCE HAZARDS AND LIKELIHOOD OF THE
HAZARDS OCCURRING ................................................................................................................69
Section 2.1 Herbicide resistance .............................................................................................................69
Table of contents
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Section 2.2 Insecticide resistance ............................................................................................................70
SECTION 5 CONCLUSIONS REGARDING HERBICIDE AND INSECTICIDE RESISTANCE .........................................71
Section 5.1 Herbicide resistance .............................................................................................................71
Section 5.2 Insecticide resistance ............................................................................................................71
APPENDIX 7 LICENCE CONDITIONS ......................................................................................................73
SECTION 1 INTERPRETATIONS AND DEFINITIONS .........................................................................................74
SECTION 2 GENERAL CONDITIONS ...............................................................................................................77
SECTION 3 SPECIFIC CONDITIONS ................................................................................................................79
APPENDIX 8 LEGISLATIVE REQUIREMENTS FOR ASSESSING DEALINGS INVOLVING
INTENTIONAL RELEASES .................................................................................................88
SECTION 1 THE REGULATION OF GENE TECHNOLOGY IN AUSTRALIA ...........................................................88
SECTION 2 THE LICENCE APPLICATION ........................................................................................................88
SECTION 3 THE INITIAL CONSULTATION PROCESSES ....................................................................................89
SECTION 4 THE EVALUATION PROCESSES ....................................................................................................90
SECTION 5 FURTHER CONSULTATION...........................................................................................................91
SECTION 6 DECISION ON LICENCE ................................................................................................................91
APPENDIX 9 SUMMARY OF PUBLIC SUBMISSIONS ...........................................................................93
APPENDIX 10 REFERENCES ........................................................................................................................98
Table of contents
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
EXECUTIVE SUMMARY
THE DECISION
The Gene Technology Regulator (the Regulator) has made a decision to issue a licence for
dealings involving intentional release of GMOs into the environment, in respect of application
DIR 055/2004 from Monsanto Australia Limited (Monsanto).
The licence allows Monsanto to conduct a large scale field trial of up to 1815 hectares over
two seasons from 2005 to 2006. Summer plantings will be conducted in the cotton growing
regions of New South Wales and southern Queensland from September 2005 to May 2006 on
up to 1770 hectares and a maximum of 86 sites. A small part of the plantings will take place
in the winter in northern Australia (potentially including northern Western Australia (WA),
northern Queensland and the Northern Territory (NT)) from March to November 2006 on up
to 45 hectares and a maximum of 5 sites.
The Gene Technology Act 2000 (the Act) and the Gene Technology Regulations 2001 (the
Regulations) set out requirements which the Gene Technology Regulator (the Regulator) must
follow when considering an application for a licence to intentionally release a genetically
modified organism (GMO) into the environment.
For a licence to be issued, the Regulator must be satisfied that the release will not pose any
risks to human health and safety or the environment that can not be managed. As part of the
evaluation process, Section 51 of the Act requires the Regulator to prepare a risk assessment
and risk management plan (RARMP) for each licence application, in consultation with a wide
range of expert groups and stakeholders.
Under Section 52 of the Act, the Regulator is required to seek comment on the RARMP from
those consulted in its preparation and to invite submissions from the public. Matters raised
relating to the protection of human health and safety or the environment are taken into
account in finalising the RARMP, which then forms the basis of the Regulator‘s decision on
whether or not to issue a licence, and if so, what conditions to impose.
The Act is designed to operate in a cooperative legislative framework with other regulatory
authorities that have complementary responsibilities and specialist expertise. As well as
enhancing coordinated decision making, this arrangement avoids duplication. The OGTR
liaises closely with other regulators to ensure the identification, evaluation and management
of risks that may be associated with the development and use of gene technology.
THE APPLICATION
Monsanto Australia Ltd (Monsanto) applied for a licence (application number DIR 055/2004)
for the intentional release of genetically modified (GM) herbicide tolerant (Roundup Ready®
Flex MON 88913) and herbicide tolerant/insect resistant (Roundup Ready® Flex MON
88913/Bollgard II®) cottons into the environment, on a limited scale and under controlled
conditions. Monsanto sought to conduct a large scale field trial over two seasons on up to 91
sites covering a total area of up to 1811 hectares from 2005 to 2006 in the southern summer
growing season and the northern winter growing season.
Roundup Ready® Flex cotton MON 88913 (abbreviated to Roundup Ready® Flex cotton)
contains two copies of a gene (cp4 epsps) derived from a common soil bacterium. The
protein encoded by the cp4 epsps gene is an enzyme1 (CP4 EPSPS) that is able to function in
the presence of glyphosate, the active ingredient in Roundup® herbicides, whereas the enzyme
expressed by the equivalent gene in other, non-herbicide tolerant cottons is inhibited by
1
Enzymes are proteins which catalyse specific biochemical reactions.
EXECUTIVE SUMMARY I
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
glyphosate. As this enzyme is involved in an important biochemical pathway for the
synthesis of essential building blocks of cellular proteins, cottons that do not have the cp4
epsps gene are killed by the application of glyphosate. In contrast, the presence of the
CP4 EPSPS enzyme in Roundup Ready® Flex GM cotton allows the application of Roundup
Ready® herbicide for the control of weeds that emerge in the crop, without damaging the crop
itself.
Roundup Ready® Flex cotton MON 88913/Bollgard II® cotton (abbreviated to Roundup
Ready® Flex/Bollgard II® cotton) was produced by crossing of Roundup Ready® Flex cotton
with GM Bollgard II® cotton via conventional breeding. This introduced two genes that
produce insecticidal proteins which provide resistance to the major caterpillar pests of cotton.
Bollgard II® cotton is approved for commercial release south of latitude 22° South in
Australia (DIR 012/2002).
Roundup Ready® Flex cotton differs from GM Roundup Ready® cotton (also approved for
commercial release south of latitude 22° South; DIR 023/2002) in that the former has two
copies of the cp4 epsps gene, under the control of two different novel promoters2, while the
latter only has one. This means that tolerance to Roundup Ready® herbicide is prolonged in
Roundup Ready® Flex cotton due to enhanced expression of the CP4 EPSPS enzyme in both
level and duration. Yield loss occurs if Roundup Ready® herbicide is applied to Roundup
Ready® cotton beyond the four-leaf stage of growth (approximately five weeks after planting).
The applicant anticipates that Roundup Ready® Flex cottons will be able to tolerate
application of Roundup Ready® herbicide at later stages of plant growth without yield loss,
allowing a wider window to apply herbicide during growth of the cotton crop. This is
intended to increase growers‘ flexibility in the timing of herbicide application for integrated
weed management and is not expected to increase the overall amount of herbicide use.
The aims of this field trial are to transfer the Roundup Ready® Flex trait into elite Australian
cotton varieties, to test the agronomic performance of the GM cottons under Australian field
conditions, to produce seed for future releases (which would require separate applications and
approval processes), to set up demonstration sites for industry, government, researchers and
the wider community and to collect data required for future applications.
The applicant proposed a number of containment measures to minimise the spread and
persistence of the GMOs and the introduced genes from the trial sites. These include:
surrounding the trial sites by pollen traps or isolation zones;
destroying all viable GM materials that remains on the trial sites following
harvest;
ensuring that trial sites are not within 50 metres of natural waterways;
destroying any volunteer GM cotton plants that may occur at the release sites after
completion of the plantings; and
implementation of a management plan which will prevent unintended dispersal of
GM cottonseed from demonstration sites by visitors .
None of the cotton plants from the release, or their by-products, will be used in animal feed or
human food. However, the applicant has been given approval to sell lint from the release.
Lint does not contain genetic material or protein. Transport of the GMOs and materials from
the GMOs will be in accordance with the transport guidelines issued by the Regulator.
2
The term ‗promoter‘ refers to a regulatory sequence that controls the expression of the gene that is linked to it.
EXECUTIVE SUMMARY II
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
A limited and controlled release of Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons was previously approved under licence DIR 035/2003. This field
trial is being conducted from 2003 to 2005 in NSW, Queensland, NT and northern WA. The
licence conditions for DIR 035/2003 contain a requirement to conduct a research program and
a range of data on Roundup Ready® Flex cotton relevant to the assessment of this application
have been collected and provided to the Regulator.
THE EVALUATION PROCESS
A risk assessment and risk management plan (RARMP) has been prepared in relation to
licence application DIR 055/2004 from Monsanto in accordance with the Act, the Regulations
and the Risk Analysis Framework. This framework was developed as part of the
establishment of the regulatory arrangements in consultation with the public, State, Territory
and Australian Government agencies, key stakeholders and the Gene Technology Technical
Advisory Committee3.
Details of the process that the Regulator must follow, including the prescribed consultation
process on the application, and the matters that she must consider in preparing a RARMP, are
set out in Appendix 8 of the RARMP. The complete RARMP along with a review document
‗The Biology and Ecology of Cotton (Gossypium hirsutum) in Australia‘ (produced to further
inform the risk analysis) and a set of 'Questions and Answers‘ on the evaluation of the
application can be obtained from the OGTR by calling 1800 181 030 or from the OGTR‘s
website at www.ogtr.gov.au.
The risk assessment considered information contained in the application (comprising:
information required by the Act and the Regulations on the GMOs; the parent organism; the
proposed dealings, including proposed containment conditions; and potential impacts on
human health and safety and the environment), current scientific knowledge and submissions
received during consultation with expert groups, authorities and the public (issues raised in
submissions are summarised in Chapter 2 and Appendix 9 of the RARMP).
Through this process, potential hazards to human health and safety or the environment that
may be posed by the proposed release of the GM cottons were identified. These have been
evaluated to determine the likelihood of each hazard occurring and the likely impact of each
hazard, were it to be realised.
The identified potential hazards relate to:
toxicity and allergenicity to humans: could Roundup Ready® Flex or
Roundup Ready® Flex/Bollgard II® cottons be more toxic or allergenic than non-
GM cotton to humans as a result of the novel gene products or because of
unintended effects?
toxicity to non-target organisms: could Roundup Ready® Flex or
Roundup Ready® Flex/Bollgard II® cottons be harmful to non-target organisms as
a result of the novel gene products or because of unintended effects?
weediness: could the genetic modifications be harmful to the environment by
increasing the potential for the Roundup Ready® Flex or Roundup Ready®
Flex/Bollgard II® cottons to establish as a problematic weed compared to non-GM
cotton?
3
The Risk Analysis Framework has been recently revised (refer ‗What‘s New?‘ at www.ogtr.gov.au) but was not
applied to this RARMP as the consultation version was completed prior to the review‘s finalisation.
EXECUTIVE SUMMARY III
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
transfer of introduced genes to other organisms: could there be adverse
consequences from potential transfer of the introduced genetic materials to other
cultivated cotton crops, feral or native cottons, or to other organisms? and
herbicide and insecticide resistance: could weeds develop resistance to
glyphosate (if the Roundup Ready® crop-herbicide combination were used
inappropriately) and could target insects develop resistance to the insecticidal
proteins produced by the introduced insecticidal genes in Roundup Ready®
Flex/Bollgard II® cotton?
The Australian Pesticides and Veterinary Medicines Authority (APVMA) has a
complementary regulatory role in respect to this application due to its responsibility for
agricultural chemical use in Australia, including insecticides and herbicides, under the
Agricultural and Veterinary Chemicals Code Act 1994.
For commercial products, the normal form of approval is through registration, but the
APVMA may also issue permits allowing restricted use of an insecticide or herbicide, for
example, for a limited period of time or for a limited area. The APVMA can impose
conditions on the use of insecticides and herbicides in registrations and permits. Further
information about the APVMA‘s assessment and approval processes is contained in Chapter 1
and Appendix 6 of the RARMP.
CONCLUSIONS OF THE RISK ASSESSMENT
The Regulator has concluded that the proposed limited and controlled release of Roundup
Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons on up to 91 sites covering a total
area of up to 1811 hectares, over two planting seasons, does not pose any significant risks to
human health and safety or the environment.
The effect of combining the glyphosate tolerance and the insecticidal genes in the GM cotton
plants was considered, in particular whether this could result in new or increased risks over
and above those posed by the individual traits. This was also assessed in the RARMP for
DIR 012/2002 (commercial release of Roundup Ready®/Bollgard II® cotton expressing the
same introduced proteins). It is considered unlikely that the combination of the two unrelated
traits in Roundup Ready® Flex/Bollgard II® cotton will present new or increased risks to
human health and safety, or to the environment.
The risk assessment of each potential hazard identified above is summarised under a separate
heading below.
Toxicity or allergenicity to humans
Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons are unlikely to prove
more toxic or allergenic to humans via occupational exposure than conventional cotton. The
introduced proteins are the same as those expressed by existing commercially released GM
cottons (Roundup Ready®, Roundup Ready®/Bollgard II® and Bollgard II® cottons). None of
the introduced proteins have any known toxicity or allergenicity to humans and the proteins
are naturally widespread in the environment.
Food Standards Australia New Zealand (FSANZ) is responsible for human food safety
assessment, and FSANZ approval will need to be obtained before products from these GM
cottons could be used in human food in Australia. Oil and linters from Roundup Ready®
cotton and from one of the parental GMOs, Bollgard II® cotton, have previously been
approved by FSANZ for use in human food. FSANZ is currently evaluating an application
from Monsanto to approve food products derived from Roundup Ready® Flex cotton.
EXECUTIVE SUMMARY IV
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Accordingly, cottonseed produced during this trial is not permitted to be used in human food
or animal feed.
The sale of lint4 will be allowed for use in fabric, upholstery and other non-food products.
Lint does not contain DNA or protein.
Toxicity to non-target organisms
Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons are unlikely to prove
more toxic to non-target organisms than conventional cotton. The introduced proteins are the
same as those expressed by existing commercially released GM cottons. Potential non-target
effects of the insecticidal proteins expressed in Roundup Ready® Flex/Bollgard II® cotton
have been considered in detail in the risk assessment and risk management plans for Bollgard
II® cotton (DIR 012/2002) and INGARD® cotton (DIR 022/2002), available at
www.ogtr.gov.au. The toxicity of the introduced insecticidal proteins is highly specific to
larvae of lepidopteran insects, including the major caterpillar pests of cotton, and none of the
other introduced proteins have been shown to be toxic to any organism. The introduced
proteins, or similar proteins, are naturally widespread in the environment. However, exposure
of non-target organisms to the introduced proteins will be low and cottonseed from the release
will not be used for stockfeed.
Weediness
The risk of Roundup Ready® Flex or Roundup Ready® Flex/Bollgard II® cotton establishing
as environmental weeds in the cotton growing regions of NSW and Qld and the areas of the
release in northern Australia is very low, and not likely to be greater than that of non-GM
cotton. This is because the major constraints on weediness of both GM and non-GM cotton in
Australia are water availability, nutrient availability, plant competition, herbivory by non-
lepidopteran species, fire and (in southern Australia) frost. It is highly unlikely that the
genetic modifications will affect the response of the GM cottons to these variables and
thereby alter the weediness of the GM cottons.
Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons express the same
introduced proteins as the commercially released Roundup Ready® and Roundup
Ready®/Bollgard II® cottons, which have not become problematic weeds. However, the site
of insertion and level of expression of the introduced herbicide tolerance genes differ.
Therefore, there is a very low possibility that potential unintended effects resulting from the
genetic modification could alter some aspect of the GM cottons‘ biology that may affect
weediness. No unintended effects on agronomic characteristics, including characteristics
indicative of weediness, have been observed in field trials of Roundup Ready® Flex cotton
conducted to date in Australia and the USA.
Licence conditions have been imposed to minimise the spread and persistence of the GM
cottons in the environment, including cleaning of release sites and equipment after harvest,
secure wrapping of GM materials, post harvest inspections and destruction of volunteer cotton
after harvest (refer to key licence conditions, below).
Transfer of introduced genes to other organisms
Some gene transfer from the GM cottons to other cultivated cottons would be likely under
uncontrolled conditions, although the overall frequency of out-crossing would be very low as
4
The long cotton fibres which are separated from the cottonseed during the ginning process are called lint,
whereas the short, fuzzy fibres that remain on the cottonseed after ginning are known as linters. Lint is used to
produce fabric, whereas linters (after being separated from the cottonseed) are used in a variety of products
including food.
EXECUTIVE SUMMARY V
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
cotton is primarily self-pollinating. Transfer of the introduced genes to other cultivated cotton
would pose the same risks as the risks posed by the GM cottons themselves. Licence
conditions have been imposed to minimise the potential transfer of the introduced genetic
materials to other cotton crops (refer to key licence conditions, below).
Gene transfer to naturalised cotton populations is unlikely because of the geographic distances
between these naturalised populations and the cotton growing regions of NSW and Qld.
However, herbarium records of G. hirsutum and G. barbadense suggest that naturalised
populations may occur, or may have occurred in the past, in northern, central and south
eastern Qld, in northern NT and in northern WA. The remnants of some of these populations,
which may be within pollinating distance of cotton crops, has not been confirmed.
Licence conditions have been imposed to limit cross-pollination with compatible plants
outside the release sites (refer to key licence conditions, below)..
The possibility of transfer of the introduced genes to native cotton, other plant species or other
organisms is negligible because of well established genetic incompatibility. Even if such
transfer occurred it would be unlikely to pose any hazard to human health and safety or the
environment.
Herbicide and insecticide resistance
Roundup Ready® herbicide (containing glyphosate as the active ingredient) is not currently
registered for use on cotton beyond the four-leaf stage of growth. Monsanto has submitted an
application to the APVMA for a permit to use Roundup Ready® herbicide on Roundup Ready®
Flex and Roundup Ready® Flex/Bollgard II® cottons beyond the four-leaf stage during the trial.
The risk of development of herbicide-resistant weeds is being assessed in parallel with this
application, and if necessary, will be managed by the APVMA through permit conditions. The
existing registration for the use of the insecticidal proteins produced by the cry1Ac and
cry2Ab genes in Bollgard II® cotton as insecticidal products contains conditions to address the risk
of development of insecticide resistant pests.
THE RISK MANAGEMENT PLAN (KEY LICENCE CONDITIONS)
As part of the evaluation process for this licence application, a risk management plan has been
developed to address the risks identified (refer to Conclusion of the Risk Assessment, above).
The applicant proposed a number of containment measures to minimise the spread and
persistence of the GMOs and introduced genetic materials. In addition to these, the Regulator
has imposed other licence conditions to implement the risk management measures that will
minimise the potential exposure of humans and other organisms, and limit the likelihood of
spread and persistence of the GMOs or the introduced genetic materials in the environment.
The key licence conditions are outlined below.
Chapter 2 of the RARMP provides a tabulated summary of assessment conclusions and
corresponding management conditions. Full details of the licence conditions are provided in
Appendix 7.
Toxicity or allergenicity to humans
Licence conditions have been imposed which require the applicant to:
limit the scale and duration of the release;
prevent GM plant materials and materials from the pollen trap from being used in
human food;
destroy all GM seed not required for testing or future release;
EXECUTIVE SUMMARY VI
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
securely transport and store retained GM plant materials and seeds; and
report any adverse effects on human health and safety.
Toxicity to non-target organisms
Licence conditions have been imposed which require the applicant to:
limit the scale and duration of the release;
prevent GM cottonseed and cottonseed from the pollen trap from being used as
stockfeed;
destroy all GM seed not required for testing or future release; and
securely transport and store the retained GM plant materials and seeds;
Weediness
Licence conditions have been imposed which require the applicant to:
limit the scale and duration of the release;
prevent GM cottonseed and cottonseed from the pollen trap from being used as
stockfeed;
securely transport and store the retained GM plant materials and seeds;
clean the release sites and equipment used at release sites after harvest; and
inspect release sites after harvest and destroy volunteers.
Transfer of introduced genes to other organisms
Licence conditions have been imposed which require the applicant to:
limit the scale and duration of the release;
at sites south of latitude 22º South, surround the GM cottons with a 20 m pollen
trap of non-GM cotton, or Bollgard II®, Roundup Ready® or
Bollgard II®/Roundup Ready® GM cottons which are approved for commercial
release in southern Australia;
at sites north of latitude 22º South, surround the GM cottons with a 50 m isolation
zone (monitored for volunteers) surrounded by a 400 m zone free of any cotton
populations that would be able to cross with the GM cottons;
securely transport and store the retained GM plant materials and seeds;
clean the release sites and equipment used at release sites after harvest; and
inspect release sites after harvest and destroy volunteers.
Herbicide and insecticide resistance
No conditions have been imposed in relation to management of herbicide resistance in weeds
or insecticide resistance in pests, as the APVMA has responsibility for these issues. The
requirement to comply with any conditions imposed by the APVMA has been noted in the
licence.
General conditions
Any licence issued by the Regulator contains a number of general conditions that are also
relevant to risk management. These include, for example:
EXECUTIVE SUMMARY VII
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
identification of the persons or classes of persons covered by the licence;
a requirement that the applicant allows access to the release sites by the Regulator,
or persons authorised by the Regulator, for the purpose of monitoring or auditing;
and
a requirement to inform the Regulator if the applicant becomes aware of any
additional information about risks to human health or safety or to the
environment.
Research requirements
As mentioned above (see under ‗The Application‘), data relevant to the assessment of the
application was supplied from the current trial with the same GM cottons being conducted
under DIR 35/2003. For the current application (DIR 055/2004), no additional research
requirements have been imposed. In addition, a gene flow research program is being
coordinated by the OGTR with GM cotton licensees, including Monsanto, in a range of
current and potential cotton growing areas.
Identification of issues to be addressed for future releases
In addition to the extra data on gene expression and agronomic characteristics that are
expected to be provided from the trial currently being conducted under DIR 35/2003, the
following information would be required if the applicant were to submit an application for the
commercial release of these GM cottons:
the complete sequence of the introduced DNA; and
sequences of the DNA regions flanking the introduced DNA and results of
database homology searches.
Monitoring and enforcement of compliance by the OGTR
As well as the legislative capacity to enforce compliance with licence conditions, the
Regulator has additional options for risk management. The Regulator can direct a licence
holder to take any steps the Regulator deems necessary to protect the health and safety of
people or the environment. The OGTR also independently monitors releases that the
Regulator has authorised. At least 20% of all field trial sites are inspected each year, in
accordance with a monitoring and compliance strategy based on risk profiling (which takes
into account biological, seasonal, geographical and ecological risk factors), to determine
whether licence holders are complying with the licence conditions, or whether there are any
unintended effects.
FURTHER INFORMATION
Detailed information on the evaluation of the application, including the licence conditions, is
available in the risk assessment and risk management plan for this application, which can be
obtained from the website of the Office of the Gene Technology Regulator
(www.ogtr.gov.au), or by calling 1800 181 030 (please quote application number
DIR 055/2004).
EXECUTIVE SUMMARY VIII
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
CHAPTER 1 BACKGROUND
1. This chapter provides background information about the application and previous releases
of relevant genetically modified organisms (GMOs) into the environment.
SECTION 1 THE APPLICATION
2. The OGTR received an application (licence application number DIR 055/2004) from
Monsanto Australia Limited (Monsanto) for the intentional release of genetically modified
(GM) herbicide tolerant (Roundup Ready® Flex MON 88913) and herbicide tolerant/insect
resistant (Roundup Ready® Flex MON 88913/Bollgard II®) cottons into the environment, on a
limited scale and under controlled conditions. Key information relating to the application is
given below:
Project Title: Field trial of herbicide tolerant (Roundup Ready® Flex
MON 88913) and herbicide tolerant/insect resistant
(Roundup Ready® Flex MON 88913/Bollgard II®)
cottons
Applicant: Monsanto Australia Ltd
PO Box 6051
Melbourne VIC 8008
Common name of the parent organism: Cotton
Scientific name of the parent organism: Gossypium hirsutum L.
Modified trait(s): Prolonged herbicide tolerance, insecticidal action,
antibiotic resistance, reporter gene expression
Identity of the gene(s) responsible for the cp4 epsps gene from Agrobacterium sp. strain CP4
modified trait(s): (herbicide tolerance)
cry1Ac and cry2Ab genes from the bacterium
Bacillus thuringiensis (insecticidal)
nptII gene from the bacterium Escherichia coli
(antibiotic resistance)
uidA gene from the bacterium Escherichia coli
(reporter gene)
Proposed Release Location: New South Wales (NSW), Queensland (Qld), northern
Western Australia (WA), the Northern Territory (NT) (see
Appendix 7 for possible release shires)
Proposed Release Sizes and Dates: Season Number of sites Maximum total
area (hectares)
Summer 2005/06 86 1769
Winter 2006 5 42
Total 91 1811
3. It should be noted that some details of the gene construct, including the plasmid map and
some of the regulatory sequences were declared as Confidential Commercial Information
(CCI) under Section 185 of the Act, in connection with licence application DIR 035/2003.
This information was made available to the prescribed expert groups which were consulted in
the preparation of the risk assessment and risk management plan. However, the applicant has
Chapter 1 Background 1
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
recently indicated that it is no longer considered as necessary to protect this information as
CCI and the declaration has been revoked. All previous CCI information can now be made
public and the relevant information is discussed in this document.
4. Roundup Ready® Flex cotton MON 88913 (abbreviated to Roundup Ready® Flex cotton)
contains two copies of a gene (cp4 epsps), derived from the common soil bacterium
Agrobacterium sp. strain CP4 that have been optimised for expression in plants (refer to
Section 1.3 below and Appendix 1 for more detail).
5. Roundup Ready® Flex/Bollgard II® cotton was produced by conventional breeding of
Roundup Ready® Flex cotton with GM Bollgard II® cotton (initiated under licence
DIR 035/2003, which included the same GM cottons). This introduced two genes that
produce insecticidal proteins which provide resistance to the major caterpillar pests of cotton.
Bollgard II® cotton is approved for commercial release south of latitude 22° South in
Australia (DIR 012/2002).
Section 1.1 The proposed dealings
6. Monsanto seeks approval for the limited and controlled release of herbicide tolerant
Roundup Ready® Flex and herbicide tolerant/insect resistant Roundup Ready®
Flex/Bollgard II® cotton into the environment.
7. Monsanto proposes to conduct a large scale field trial on up to 91 sites covering a total
area of up to 1811 hectares over two planting seasons in the southern summer growing season
(September 2005 to May 2006) and the northern winter growing season (March to November
2006). The summer plantings (1769 hectares on 86 sites) would be conducted in the cotton
growing regions of New South Wales (NSW) and southern Queensland (Qld). The winter
plantings (42 hectares on 5 sites) would take place in northern Western Australia (WA), the
Northern Territory (NT) and northern Qld.
8. The aims of the proposed field trial are to:
transfer the Roundup Ready® Flex trait into elite cotton varieties suitable for use
under Australian conditions;
test agronomic performance including disease resistance (bacterial blight and
fusarium and verticillium wilts);
produce seed for future release, which would require separate applications and
approval processes;
set up demonstration sites for industry, government, researchers and the wider
community; and
collect data required for future applications to the OGTR and other regulators for
commercial release such as levels of novel protein expression and seed composition
(required by the OGTR and Food Standards Australia New Zealand (FSANZ)) and
data on the GM cottons‘ tolerance to glyphosate, weed control effectiveness and
glyphosate residue levels (required by the Australian Pesticides and Veterinary
Medicines Authority (APVMA)).
9. None of the cotton plants from the release, nor any of their by-products, will be used for
animal feed or human food. However, the applicant proposes to sell lint from the release.
Lint does not contain genetic material or protein.
Chapter 1 Background 2
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Section 1.2 Parent organism
10. The parent organism is cultivated cotton (Gossypium hirsutum L.), which is exotic to
Australia and is grown as an agricultural crop in NSW, southern and central Qld and on a trial
basis in WA, the NT and northern Qld. More detailed information on cotton can be found in a
review document ‗The Biology and Ecology of Cotton (Gossypium hirsutum) in Australia‘
that was produced in order to inform the risk assessment processes for licence applications
involving GM cottons. This document is available at www.ogtr.gov.au.
Section 1.3 Genetic modifications and their effects
11. Roundup Ready® Flex cotton contains two copies of the epsps gene from Agrobacterium
species strain CP4. The cp4 epsps gene encodes an enzyme (CP4 EPSPS) that provides
tolerance to glyphosate (Padgette et al. 1993), the active ingredient in Roundup® herbicides.
While conventional cotton is killed by glyphosate, Roundup Ready® herbicide can be applied
to Roundup Ready® Flex cotton for the control of weeds that emerge in the crop without
damaging the crop itself.
12. In plants, the native 1 epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene encodes
an enzyme (EPSPS) that is critical for the biosynthesis of aromatic amino acids (tryptophan,
tyrosine and phenylalanine), essential building blocks for proteins. Glyphosate, the active
ingredient in Roundup® herbicides, functions by inhibiting the activity of the naturally
occurring EPSPS enzyme in plants, thus blocking the biosynthesis of aromatic amino acids
and eventually leading to cell death (Steinrucken & Amrhein 1980). The CP4 EPSPS enzyme
naturally produced in Agrobacterium sp. strain CP4 is insensitive to the effects of glyphosate
(Padgette et al. 1993, Monsanto unpublished) and is able to function normally in the
biosynthesis of aromatic amino acids in the presence of the herbicide. Consequently, in GM
plants containing the bacterial cp4 epsps gene, biosynthesis of aromatic amino acids is not
blocked by glyphosate and the plants are not killed by Roundup Ready® herbicide application.
13. Roundup Ready® Flex cotton differs from GM Roundup Ready® cotton (approved for
commercial release south of latitude 22° South; DIR 023/2002) in that the former has two
copies of the cp4 epsps gene, under the control of two different novel promoters2, while the
latter only has one. This means that tolerance to Roundup Ready® herbicide is prolonged in
Roundup Ready® Flex cotton due to enhanced expression, in both level and duration, of the
CP4 EPSPS enzyme. The applicant has indicated that Roundup Ready® herbicide can be
applied to control target weeds in Roundup Ready® Flex cotton up to the 14 node growth
stage (note that the earliest development of first fruit can occur at about 16 nodes or 165 days
after planting). In contrast, if the application of Roundup Ready® herbicide to a Roundup
Ready® cotton crop were delayed until after 4 nodes of plant growth or 35 days after planting,
for instance due to rainfall, it could no longer be applied. Hence Roundup Ready® Flex
cotton is intended to give growers increased flexibility in the timing of herbicide application
for integrated weed management. It is not expected to increase the overall amount of
herbicide used.
14. Roundup Ready® Flex/Bollgard II® cotton was produced by conventional breeding of
Roundup Ready® Flex cotton with Bollgard II® cotton. Roundup Ready® Flex/Bollgard II®
cotton plants contain all of the genes introduced into each of the parental GM varieties.
15. Bollgard II® cotton contains two insecticidal genes, cry1Ac and cry2Ab, both derived from
a common soil bacterium, Bacillus thuringiensis (Bt). These genes encode proteins that are
1
In the context of genes and other genetic material, the term ‗native‘ refers to genes and regulatory sequences
that are naturally present in the parent organism.
2
The term ‗promoter‘ refers to a regulatory sequence that controls the expression of the gene that is linked to it.
Chapter 1 Background 3
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
toxic to lepidopteran caterpillars, including the two key Helicoverpa pests of cotton
(H. armigera and H. punctigera).
16. Bollgard II® cotton plants also contain two bacterial antibiotic resistance genes, nptII
(conferring resistance to kanamycin and neomycin) and aad (conferring resistance to
streptomycin and spectinomycin) and a reporter gene, uidA (enabling visualisation of plant
tissues in which this gene is being expressed). The aad gene is not expressed in the GM
cotton plants because the bacterial promoter that controls its expression is not active in plants.
This gene was used as a marker to select for bacteria containing the modified DNA in the
laboratory prior to the production of the genetically modified plants.
17. Short regulatory sequences controlling expression of the genes are also present in the
genetically modified cottons. These sequences are derived from the cauliflower mosaic virus
(CaMV), figwort mosaic virus (FMV), Agrobacterium tumefaciens, Glycine max (soybean),
Arabidopsis thaliana (thale cress) and Pisum sativum (pea). Although three of these
organisms (CaMV, FMV and A. tumefaciens) are plant pathogens, the regulatory sequences
comprise only a small part of their total genome and are not in themselves capable of causing
disease.
18. Further details on the introduced genes and their encoded proteins are provided in
Appendix 1, Section 3.
Section 1.4 Method of gene transfer
19. Roundup Ready® Flex cotton was generated by introducing the two copies of the cp4
epsps gene into the cotton on a standard plasmid vector carried by Agrobacterium
tumefaciens. The vector is ‗disarmed‘ since it lacks the genes that encode the tumour-
inducing functions of A. tumefaciens (see Appendix 1, Section 4 for details).
20. Bollgard II® cotton was produced by particle bombardment of the cry2Ab and uidA genes
into GM INGARD® cotton (containing the cry1Ac, nptII and aad genes). This technique
involves coating the DNA containing the genes onto very small tungsten or gold particles
which are ‗shot‘ into the cotton tissue, followed by selection of plants that contained single,
functional copies of the genes. INGARD® cotton was produced using a disarmed plasmid
vector (containing the cry1Ac, nptII and aad genes) carried by A. tumefaciens.
21. Roundup Ready® Flex/Bollgard II® cotton was produced by conventional crossing of
Roundup Ready® Flex cotton with Bollgard II® cotton.
SECTION 2 PREVIOUS RELEASES AND INTERNATIONAL APPROVALS
Section 2.1 Previous Australian releases of the same or similar GM cottons
22. Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons were previously
approved for limited and controlled release in Australia under licence DIR 035/2003 (950
hectares permitted, but less than 100 hectares actually planted). This field trial is being
conducted from 2003 to 2005 in NSW, Qld, NT and northern WA. Licence conditions for
DIR 035/2003 contain a requirement to conduct a research program and a range of data on
Roundup Ready® Flex cotton relevant to the assessment of this application have been
collected and provided to the Regulator.
23. First generation Roundup Ready® cotton, containing only one copy of the same cp4 epsps
gene present in the Roundup Ready® Flex cottons, has been approved for commercial release
since 2000 (refer licence DIR 023/2002). Bollgard II® and Bollgard II®/Roundup Ready®
cotton were approved for commercial release by the Regulator in 2002 (licence
DIR 012/2002).
Chapter 1 Background 4
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
24. All of these commercial releases were restricted to the cotton growing regions of NSW
and Qld south of latitude 22° South because of concerns about the potential weediness of the
GM cottons in the northern tropical areas, as well as the potential for out-crossing to feral
cotton in these areas. Therefore, field trials of Bollgard II® cotton and
Roundup Ready®/Bollgard II® cotton north of 22° South are being conducted under limited
and controlled conditions.
25. Prior to obtaining approval for commercial release, numerous field trials of
Roundup Ready®, Bollgard II® and Roundup Ready®/Bollgard II® cottons were conducted
under the voluntary system overseen by the Genetic Manipulation Advisory Committee
(GMAC), and four licences for limited and controlled releases of Bollgard II® and/or
Roundup Ready®/Bollgard II® cotton have been issued by the Regulator (DIR licences):
Roundup Ready® cotton - 23 limited and controlled releases undertaken by:
CSIRO Division of Plant Industry (PR-55, PR-55X, PR-55X2, PR-55X3, PR-55X5
and PR-55X6);
Deltapine Australia Pty Ltd (PR-32, PR-52, PR-52X, PR-52X2, PR-52X3, PR-71,
PR-71X, PR-71X2, PR-83, PR-83X, PR-83X3, PR-140, PR-140X and PR-143);
Monsanto (PR-83X2 and PR-83X4); and
Cotton Seed Distributors Pty Ltd (PR 55-X4).
Bollgard II® and Roundup Ready®/Bollgard II® cotton - 18 limited and controlled
releases undertaken by:
CSIRO Division of Plant Industry (PR-123, PR-123X, PR-123X2, PR-131,
PR-131X, PR-131X2 and PR-131X3);
Deltapine Australia Pty Ltd (PR-51X4, PR-112, PR-112X, PR-112X2, PR-118, PR-
118X and PR-118X2);
Cotton Seed Distributors (Bollgard II® and Roundup Ready®/Bollgard II® in Qld;
DIR 005/2001);
CSIRO (INGARD®, Bollgard II® and Roundup Ready®/Bollgard II® cotton in WA
and NT; DIR 006/2001);
Department of Agriculture (WA) (Bollgard II® cotton in WA; DIR 009/2002); and
Monsanto (Bollgard II® and Roundup Ready®/Bollgard II® cotton in northern WA,
NT and northern Qld; DIR 012/2002).
26. There have been no reports of adverse effects on human health or the environment
resulting from any releases under these approvals.
Section 2.2 Approvals by other Australian government agencies
27. The OGTR is responsible for assessing the biosafety risks to human health and the
environment associated with development and use of GMOs. Other government regulatory
requirements must also be met in respect of the release of GMOs, and the use of products of
GMOs, including the requirements of the Australian Pesticides and Veterinary Medicines
Authority (APVMA) and Food Standard Australia New Zealand (FSANZ).
2.2.1 Australian Pesticides and Veterinary Medicines Authority
28. The APVMA has a complementary regulatory role in respect to this application due to its
responsibility for agricultural chemical use, including insecticides and herbicides in Australia
Chapter 1 Background 5
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
under the Agricultural and Veterinary Chemicals Code (1994). For commercial products, the
normal form of approval is through registration, but the APVMA may also issue permits
allowing restricted use of an insecticide or herbicide, for example for a limited period of time
or for a limited area.
29. In considering applications for registration or permits, the APVMA also considers and, if
necessary, imposes conditions in relation to a number of issues that are outside the scope of
the Gene Technology Regulator‘s assessment, such as the efficacy of an insecticide or
herbicide, and insecticide and herbicide resistance management. The APVMA can impose
conditions on both registrations and permits.
30. Roundup Ready ® herbicide (containing glyphosate as the active ingredient) is not
currently registered for use on cotton beyond the four-leaf stage of growth. A research permit
from the APVMA will be required before any non-registered use of Roundup Ready®
herbicide can occur. Monsanto has submitted an application to the APVMA for the required
permit.
31. In 1996 and 2003 respectively, the APVMA registered the use of the insecticidal proteins
as produced by the cry1Ac gene in INGARD® cotton, and the cry1Ac and cry2Ab genes in
Bollgard II® cotton, as insecticidal products. As part of the registrations, the APVMA imposed
conditions, including an insecticide resistance management plan that limited the proportion of
GM insecticidal cotton that could be planted each year to 30%. In 2003/04, this limit was
40% per valley per farm unit. Following the withdrawal of INGARD® cotton, this cap has
been lifted for the 2004/05 season in recognition of the reduced risk of insecticide resistance
development from the two gene cotton (Bollgard II®). Growers can now choose to grow up to
100% of their cotton crop as Bollgard II®. However, the precise proportion will depend upon
their choice of refuge crop (i.e. whether it contains cotton or another plant species). More
detailed information is available in Appendix 6 of this RARMP. This trial will have to
comply with all conditions imposed by the APVMA.
32. The APVMA and the OGTR work closely together to ensure thorough, coordinated
assessments are undertaken and, wherever possible, that timing of assessments and decisions
by both agencies coincide. Further information about the use of glyphosate on glyphosate-
tolerant crops and about the management of herbicide and insecticide resistance, is available
from the APVMA:
Australian Pesticides and Veterinary Medicines Authority
PO Box E240
KINGSTON ACT 2604
Phone: (02) 6272 5158
Fax: (02) 6272 4753
Email: contact@apvma.gov.au
http://www.apvma.gov.au
2.2.2 Food Standards Australia New Zealand
33. FSANZ is responsible for human food safety assessment. FSANZ is currently evaluating
an application from Monsanto to approve cottonseed oil and linters derived from Roundup
Ready® Flex cotton. FSANZ approval will need to be obtained before materials from the GM
cottons could be used in food in Australia. Oil and linters from Roundup Ready® cotton and
from one of the parental GMOs, Bollgard II® cotton, have previously been approved by
FSANZ for use in human food.
34. Further information about food safety and food labelling are available from FSANZ:
Chapter 1 Background 6
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Food Standards Australia New Zealand
PO Box 7186
Canberra Mail Centre ACT 2610
Phone (02) 6271 2222
Fax (02) 6271 2278
E-mail info@foodstandards.gov.au
http://www.foodstandards.gov.au
Section 2.3 International approvals
35. In the United States of America, Roundup Ready® Flex cotton was approved for
commercial release in December 2004 (USDA-APHIS 2004) and for use in food in March
2005 (US Food and Drug Administration).
36. Roundup Ready® Flex/Bollgard II® cotton is also approved, as under the current US
regulatory system a stacked GMO is automatically approved if it was produced by crossing of
two GMOs, containing unrelated traits, that have already been approved in the USA, and
Bollgard II® was approved for commercial release in the USA in 2002 (see below).
37. Field trials of Roundup Ready® Flex cotton are currently in progress in Mexico.
Applications have been made for field trials in South Africa and Costa Rica (information
supplied by the applicant).
38. Roundup Ready® and Bollgard II® cotton have been approved for commercial release in
other countries:
The United States - the US Department of Agriculture and the Food and Drug
Administration approved the commercial release and use in food (respectively) of
Roundup Ready® cotton in 1995 and Bollgard II® cotton in 2002;
Canada - the Canadian Food Inspection Agency and Health Canada approved the
commercial release and use in food of Roundup Ready® cotton in 1996, and in
June 2003 they authorised the use of Bollgard II® cotton event 15985 for livestock
feed and human food;
Japan - the Japanese Ministries of Agriculture, Forestry and Fisheries, and Health,
Labour and Welfare approved the commercial release of Roundup Ready® cotton
in 1997 and its use in animal feed in 1998. Bollgard II® cotton was approved for
use in food and animal feed in 2002 and 2003, respectively;
Argentina - approved the commercial release of Roundup Ready® cotton in 1999
and its use in human food and stockfeed in 2000 and 2001, respectively;
The Philippines - approved the use of Roundup Ready® cotton in animal feed and
human food in 2003.
39. Other countries where Roundup Ready® cotton varieties have been approved, or are
pending approval, include India, Israel, Mexico and the European Union. Limited and
controlled releases of Bollgard II® cotton have been approved and carried out in Argentina,
Costa Rica, India, Mexico and South Africa.
40. No country is known to have refused an application for the release of Roundup Ready®,
Roundup Ready® Flex or Bollgard II® cottons.
41. There have been no reports of adverse effects on human health or the environment
resulting from any of these international releases.
Chapter 1 Background 7
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
CHAPTER 2 SUMMARY OF RISK ASSESSMENT AND RISK
MANAGEMENT PLAN
42. The Act and the Regulations require that risks associated with dealings with GMOs are
identified and assessed as to whether they can be managed to protect human health and safety
and the environment (see Appendix 8). This chapter provides a summary of the finalised Risk
Assessment and Risk Management Plan (RARMP) produced in response to application
DIR 055/2004 for Monsanto.
SECTION 1 FINALISATION OF THE RISK ASSESSMENT AND RISK MANAGEMENT
PLAN
43. The Regulator has conducted a risk assessment in relation to the proposed dealings and
prepared a risk management plan in accordance with the Act and the Regulations using a Risk
Analysis Framework as detailed in Appendix 8. The RARMP was finalised after consultation
with expert groups and the public (see Section2). The risk assessment process identified a
number of hazards that may arise from the proposed dealings. The risks posed by these
hazards were assessed as being either 'negligible', 'very low', 'low', 'moderate', 'high' or 'very
high'1 by considering:
the likelihood of the hazard occurring; and
the likely consequences (impact) of the hazards, were they to be realised.
44. The following table (Table 1) lists each of the potential hazards that were considered
during the risk assessment process in the Hazard Identification column and summarises the
assessment of each hazard under the column headed Risk. A comprehensive assessment of
each identified hazard is provided in Appendices 2 to 6, as cross-referenced in the column
headed Summary of Risk Assessment.
45. Where it is considered, on the basis of a combination of possible adverse impacts and
likelihood of occurrence, that risk management may be required to protect the health and
safety of humans and/or the environment, the Risk Management column identifies the
methods selected to limit the potential for risk exposure and the reasons they were chosen.
The risk management plan for the proposed dealing is given effect by specific conditions
within the licence. These relevant conditions are summarised in the final column, headed
Licence Conditions, and detailed in Appendix 7.
SECTION 2 ISSUES RAISED IN SUBMISSIONS ON THE APPLICATION AND RISK
ASSESSMENT AND RISK MANAGEMENT PLAN
46. Comments received in response to the consultation on the application DIR 055/2004,
undertaken with expert groups and key stakeholders as required by Section 50 of the Act, and
with the same stakeholders and the public on the consultation version of the RARMP under
Section 52 of the Act (see Appendix 8), were very important in finalising the RARMP, which
then formed the basis of the Regulator‘s decision on the application.
47. Written submissions in relation to DIR 055/2004, received from the agencies and
authorities prescribed by Section 50 of the Act, suggested that the following issues relating to
risks to human health and safety or the environment, should be addressed in the RARMP:
the potential for unintended genetic effects (Appendix 1 refers);
1
This RARMP was prepared and consulted on prior to finalising a review of the Risk Analysis Framework
which is progressively leading to the application of different terminology to characterise the different elements
of risk assessment.
Chapter 2 Summary of the Risk Assessment and Risk Management Plan 8
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
potential for increased herbicide usage (Appendices 1 and 6 refer).
hazards arising from occupational exposure (Appendix 2 refers);
the potential toxicity or allergenicity of the GM cottons through expression of the
introduced proteins or through altered metabolism (Appendices 2 and 3 refer);
ecotoxicity effects on non-target organisms from the Cry proteins (Appendix 3
refers);
the accumulation, persistence and degradation of the introduced proteins in soil
and water (Appendix 3 refers);
the potential for increased weediness of the GM cottons (Appendix 4 refers);
the potential for dissemination of the GM cottons beyond the release sites
(Chapter 2, Appendices 4 and 5 refer);
the potential for adverse impacts arising from gene transfer to other organisms
(Appendix 5 refers);
the potential for, and management of, gene transfer to other cotton crops,
naturalised cotton populations and native cottons (Appendices 5 and 7 refer); and
the potential for, and management of, development of herbicide and insecticide
resistance (Appendix 6 refers).
48. Written submissions also suggested that additional data be sought from the applicant for
the future development of the GMOs (Chapter 2 refers).
49. The Regulator received 8 submissions from members of the public on the consultation
version of the RARMP for this application. A summary of these written submissions and how
they were considered is provided in Appendix 9. The key issues raised by the public that
related to risks to human health and safety or the environment were:
risks to human health and safety due to the genetic modifications (Appendix 2
refers);
potential weediness of the GM cottons (Appendix 4 refers);
containment of the GM cotton plants, material from the GM cotton plants and the
introduced genetic material during the trial (Appendices 4, 5 and 7 refer);
difficulties that might occur relating to cleaning of the release sites (if unexpected
cleaning of all sites was required) (Appendices 4 and 7 refer); and
procedural concerns (Appendix 8 refers).
50. Other issues raised in submissions included: corporate versus public interest,
marketability/trade implications, political issues, and potential legal issues arising from
difficulties in segregating commercialised GM and non-GM crops. However, the focus of the
gene technology legislation is upon the protection of human health and safety and the
environment. These issues can therefore not be considered within the scope of assessments
required to be conducted under the Act.
51. In accordance with Section 56 of the Act, the Regulator has taken into account all issues
raised in written submissions that related to risks to human health and safety or to the
environment from the release of the GM cottons in finalising the RARMP. These issues were
considered carefully and weighed against the body of current scientific information in
reaching the conclusions set out in this document.
Chapter 2 Summary of the Risk Assessment and Risk Management Plan 9
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 3 RESEARCH REQUIREMENTS
52. Licence conditions for a field trial with the same GM cottons (being conducted under
DIR 35/2003) contain a requirement to conduct a research program. As part of this program,
the following data relevant to the assessment of the proposed release have been collected and
were provided in the application for DIR 055/2004:
phenotypic and agronomic characteristics of the Roundup Ready® Flex cotton;
genetic segregation and molecular characterisation of the introduced genetic
material; and
levels of expression of the introduced CP4 EPSPS protein.
53. Therefore, no additional research requirements have been imposed for this release, given
that further data, including expression levels of the introduced CP4 EPSPS protein under
Australian conditions and additional data on agronomic characteristics, will be soon available
from subsequent seasons of the trial being conducted under DIR 35/2003. In addition, a gene
flow research program is being coordinated by the OGTR with GM cotton licensees,
including Monsanto, in a range of current and potential cotton growing areas.
SECTION 4 IDENTIFICATION OF ISSUES TO BE ADDRESSED FOR FUTURE
RELEASES
54. In addition to the data that are expected to be provided as part of the ongoing trial
mentioned above, the following information would be required if the applicant were to submit
an application for the commercial release of these GM cottons:
the complete sequence of the introduced DNA; and
sequences of the DNA regions flanking the introduced DNA and results of
database homology searches.
55. It should be noted that provision of the above data during this release is not part of the
requirement to manage the risks to human health and safety and the environment from the
release. The risk management measures summarised in Table 1, and given effect by the
imposed licence conditions, will achieve this purpose.
56. It should also be noted that the use of these GM cottons, or products derived from them, in
food would require approval from FSANZ.
SECTION 5 DECISION ON THE APPLICATION
57. Details of the matters that the Regulator must consider in making a decision are provided
in Appendix 8. It is important to note that the legislation requires the Regulator to base the
licence decision on whether risks posed by the dealings are able to be managed so as to
protect human health and safety and the environment.
58. The RARMP concludes that the proposed limited and controlled release of the GM
cottons does not pose significant risks to human health and safety or to the environment as a
result of the genetic modification. Detailed risk analyses based on the available scientific
information are provided in Appendices 2 – 6 in support of this conclusion.
59. Therefore, the Regulator has issued licence DIR 055/2004 in respect of this application.
The Regulator has imposed licence conditions to minimise potential exposure of humans and
other organisms, and to limit the spread and persistence of the GMOs and the introduced
genetic materials in the environment.
Chapter 2 Summary of the Risk Assessment and Risk Management Plan 10
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 6 TABULATED SUMMARY OF THE RARMP (INCLUDING LICENCE CONDITIONS)
Table 1 Summary of the risk assessment and the risk management plan (including licence conditions)
GM cottons: the genetically modified cottons proposed for release.
Lepidoptera: the caterpillar insect pests targeted by the GM cottons belong to this order of insects.
Cry1Ac and Cry 2AB: highly specific insecticidal proteins, derived from the common soil bacterium Bacillus thuringiensis (Bt), which are toxic to lepidopteran caterpillar
pests of cotton.
Bt toxins: the Cry1Ac and Cry2Ab proteins may also be referred to as Bt toxins, because they are two of the many protein toxins that are produced by Bt in
nature.
CP4 EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase (enzyme), the gene for which was derived from the bacterium Agrobacterium species strain CP4,
and provides tolerance to the herbicide glyphosate.
GUS: -glucuronidase (enzyme), encoded by the reporter gene uidA, which enables visualisation of plant tissues in which this gene is being expressed.
NPTII: neomycin phosphotransferase type II (enzyme), encoded by the antibiotic resistance gene nptII, which provides resistance to the antibiotics
kanamycin and neomycin.
N/A Not Applicable
Risk Does Risk
Hazard (combines Summary of Risk Assessment Require Risk Management Is Risk Licence conditions
Identification ‘likelihood’ (refer to Appendices for details) Management? Method(s) and Reasons(s) for selection Managed (see Appendix 7 for detailed licence conditions)
& ‘impact’) ?
TOXICITY AND Very Low See Appendix 2 Yes Prevent seed from entering human Yes Prohibit entry into human food supply: no material from the GM
ALLERGENICITY none of the GM cotton material from the release will be used in human food supply: prevents exposure cottons to be used in human food.
FOR HUMANS: food or animal feed; through food. Destroy seed: destroy all seed not required for testing or future trial.
Food humans are commonly exposed to the CP4 EPSPS, Cry1Ac, Cry2Ab, Destroy all seed not required for Secure transport and storage: the GMOs must be transported
GUS and NPTII proteins or very similar proteins, as these are naturally testing or further trial: prevents within a primary, sealed container that is packed in a secondary
widespread in the environment; unintended exposure. unbreakable container; store in sealed container within a locked
toxicity studies indicate that the introduced proteins are not toxic; Ensure secure transport and storage facility that is signed to indicate GM cotton is stored within.
evidence indicates that the introduced proteins are not allergenic, nor do of retained seed: prevents unintended
they have properties of known allergenic proteins; exposure.
there have been no reported toxic or allergic effects from similar GM
cottons expressing the same proteins that have been extensively field
trialled and are commercially released in Australia; and
FSANZ approval will be required before cottonseed oil or linters from the
GM cottons could be used for human food in Australia.
Chapter 2 Summary of the Risk Assessment and Risk Management Plan 11
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Risk Does Risk
Hazard (combines Summary of Risk Assessment Require Risk Management Is Risk Licence conditions
Identification ‘likelihood’ (refer to Appendices for details) Management? Method(s) and Reasons(s) for selection Managed (see Appendix 7 for detailed licence conditions)
& ‘impact’) ?
TOXICITY AND Very Low See Appendix 2 Yes Limit scale of release: decreases Yes Limit scale: restrict area to 1811 hectares over two growing seasons
ALLERGENICITY cotton lint used in clothing and household items contains no proteins or likelihood of exposure. on up to 91 individual sites with a maximum size of any individual
FOR HUMANS: DNA and cotton products from GM cottons cannot be distinguished from Destroy all seed not required for site of not more than 100 ha.
Occupational non-GM products; testing or further trial: prevents Destroy seed: destroy all seed not required for testing or future trial.
exposure; and cotton pollen is not wind-dispersed and therefore unlikely to be an air- unintended exposure. Secure transport and storage: the GMOs must be transported
wearing & using borne allergen; Ensure secure transport and storage within a primary, sealed container that is packed in a secondary
household items exposure to the introduced proteins through working with cotton plants is of retained seed: prevents unintended unbreakable container; store in sealed container within a locked
containing cotton very low; exposure. facility that is signed to indicate GM cotton is stored within.
products humans are commonly exposed to the CP4 EPSPS, Cry1Ac, Cry2Ab, Report any adverse impacts on Report adverse impacts: any adverse impacts on human health
GUS and NPTII proteins, as these or very similar proteins are naturally human health and safety: ensures and safety must be reported to the Regulator.
widespread in the environment; identification of unexpected adverse
toxicity studies indicate that the introduced proteins are not toxic; impacts.
evidence indicates that the introduced proteins are not allergenic, nor do
they have properties of known allergenic proteins;
there have been no reported toxic or allergic effects from similar GM
cottons expressing the same proteins that have been extensively field
trialled and are commercially released in Australia; and
although dust and lint from cotton can be created at processing facilities,
the use of protective equipment prevents respiratory irritation, and fibre
characteristics of the GM cottons are likely to be the same as for non-GM
cotton.
TOXICITY FOR Very Low See Appendix 3 Yes Limit scale of release: decreases Yes Limit scale: restrict area to 1900 hectares over two growing seasons
OTHER exposure of livestock and wildlife to these GM cottons will be low, and likelihood of exposure. on up to 91 individual sites with a maximum size for any individual
ORGANISMS: cottonseed will not be used as stockfeed; Prevent seed from being used as site of 100 ha.
Mammals and compositional analysis has not indicated any significant difference between stockfeed: prevents exposure of Prevent seed from being used as stockfeed: no cottonseed to be
wildlife, including the GM cotton and non-GM cottons, other than the presence of the animals. used as stockfeed.
birds and fish introduced proteins; Destroy all seed not required for Destroy seed: destroy all seed not required for testing or future trial.
the introduced proteins or very similar proteins are already widespread in testing or further trial: prevents Secure transport and storage: the GMOs must be transported
the environment, through the presence of the bacteria from which they are unintended exposure. within a primary, sealed container that is packed in a secondary
derived; Ensure secure transport and storage unbreakable container; store in sealed container within a locked
the toxicity of Cry1Ac and Cry2Ab is highly specific to lepidopteran insect of retained seed: prevents unintended facility that is signed to indicate GM cotton is stored within.
larvae; exposure.
the CP4 EPSPS, GUS and NPTII enzymes are not known to be toxic to
any organism.
TOXICITY FOR Very Low See Appendix 3 Yes Limit scale of release: decreases Yes Limit scale: restrict area to 1900 hectares over two growing seasons
OTHER the CP4 EPSPS, GUS and NPTII enzymes are widespread in the likelihood of exposure. on up to 91 individual sites with a maximum size for any individual
ORGANISMS: environment and are not known to be toxic to any organisms; Destroy all seed not required for site of 100 ha.
Non-target Bt toxins are widespread in the environment and the toxicity of Cry1Ac and testing or further trial: prevents Destroy seed: destroy all seed not required for testing or future trial.
invertebrates, Cry2Ab is highly specific to lepidopteran insect larvae; unintended exposure. Secure transport and storage: the GMOs must be transported
including soil cotton is not the preferred food source for non-target Lepidoptera; and Ensure secure transport and storage within a primary, sealed container that is packed in a secondary
insects laboratory and field studies suggest that populations of key non-target of retained seed: prevents unintended unbreakable container; store in sealed container within a locked
invertebrates are unlikely to be affected by the Bt toxins. Indeed it is likely exposure. facility that is signed to indicate GM cotton is stored within.
that their populations would be favoured by decreases in the use of broad-
spectrum insecticides associated with the use of insecticidal Bt cottons.
Chapter 2 Summary of the Risk Assessment and Risk Management Plan 12
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Risk Does Risk
Hazard (combines Summary of Risk Assessment Require Risk Management Is Risk Licence conditions
Identification ‘likelihood’ (refer to Appendices for details) Management? Method(s) and Reasons(s) for selection Managed (see Appendix 7 for detailed licence conditions)
& ‘impact’) ?
TOXICITY FOR Very Low See Appendix 3 Yes Limit scale of release: decreases Yes Limit scale: restrict area to 1900 hectares over two growing seasons
OTHER most of the introduced proteins are already widespread in the environment, likelihood of exposure. on up to 91 individual sites with a maximum size for any individual
ORGANISMS: through the presence of the bacteria from which they are derived; Destroy all seed not required for site of 100 ha.
Microorganisms the CP4 EPSPS, GUS and NPTII enzymes are not known to be toxic to testing or further trial: prevents Destroy seed: destroy all seed not required for testing or future trial.
any organisms; and unintended exposure. Secure transport and storage: the GMOs must be transported
the Cry proteins are not known to adversely affect microorganisms. This is Ensure secure transport and storage within a primary, sealed container that is packed in a secondary
supported by specific data for Cry1Ac and other Bt toxins. of retained seed: prevents unintended unbreakable container; store in sealed container within a locked
exposure. facility that is signed to indicate GM cotton is stored within.
WEEDINESS Very Low See Appendix 4 Yes Limit scale of release: decreases Yes Limit scale: restrict area to 1900 hectares over two growing seasons
cotton does not possess characteristics commonly associated with likelihood of escape. on up to 91 individual sites with a maximum size for any individual
weediness, and is not known to be a problematic weed in any Ensure secure transport and storage site of 100 ha.
environment; of retained seed: prevents escape of Secure transport and storage: the GMOs must be transported
the introduced genes in the GM cottons are unlikely to affect these GM plant material into the environment within a primary, sealed container that is packed in a secondary
characteristics; outside the release site. unbreakable container; store in sealed container within a locked
the combination of herbicide tolerance and insect resistance in Roundup Clean equipment used at the release facility that is signed to indicate GM cotton is stored within.
Ready® Flex/Bollgard II® cotton is unlikely to increase the weediness site: prevents escape of GM plant Clean equipment used at the release site: equipment used in
potential of this GMO above the potential for either Roundup Ready® Flex material into the environment outside connection with the GM cottons must be cleaned before being used
or Bollgard II® cotton individually; the release site. for any other purpose. If GM cotton is ginned, the gin must be
other GM cottons containing the same proteins, grown commercially in Prevent cottonseed being used as cleaned immediately following its use, before any other cotton is
Australia, have not become problematic weeds; and stockfeed: prevent dispersal of ginned.
major constraints on weediness of GM and non-GM cotton in Australia are cottonseed. Destroy seed: destroy all seed not required for testing or future trial.
water availability, nutrient availability, plant competition, herbivory by non- Destroy all seed not required for Destroy volunteers: after harvest, the release site must be
lepidopteran species, fire and (in southern Australia) frost. testing or further trial: prevents inspected at least once every two months for at least 12 months and
unintended spread. any cotton volunteers destroyed before flowering.
Destroy any volunteers: prevents
persistence of GM cotton plants.
Chapter 2 Summary of the Risk Assessment and Risk Management Plan 13
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Risk Does Risk
Hazard (combines Summary of Risk Assessment Require Risk Management Is Risk Licence conditions
Identification ‘likelihood’ (refer to Appendices for details) Management? Method(s) and Reasons(s) for selection Managed (see Appendix 7 for detailed licence conditions)
& ‘impact’) ?
GENE TRANSFER: Very Low See Appendix 5 Yes Limit scale of release: decreases Yes Limit scale: restrict area to 1900 hectares over two growing seasons
Plants applicant proposes to surround the trial sites by pollen traps or isolation potential gene transfer. on up to 91 individual sites with a maximum size for any individual
Other cotton zones and isolate the sites from naturalised/feral cotton and all other Surround the GM cotton with a pollen site of 100 ha.
crops and feral cotton crops to minimise gene flow and persistence; and trap or isolation zone: minimises Surround the GM cottons with a pollen trap at sites south of
(naturalised) gene transfer to other cottons would not pose any risks additional to the potential for spread of the introduced latitude 22º South: at sites south of latitude 22º South, each release
cotton risks posed by the GM cottons themselves. genes beyond the release site via pollen site must be surrounded by a 20 m pollen trap of non-GM cotton, or
flow. Bollgard II®, Roundup Ready® or Bollgard II®/Roundup Ready® GM
Prevent cottonseed being used as cottons approved for commercial release in southern Australia;
stockfeed: prevents dispersal of Surround the GM cottons with an isolation zone at sites north of
cottonseed. latitude 22º South: each release site must be surrounded with a
Ensure secure transport and storage 50 m isolation zone (that must be monitored for volunteers)
of retained seed: prevents escape of surrounded by a 400 m zone free of any cotton populations that
GM plant material into the environment would be able to cross with the GM cottons;
outside the release site. Secure transport and storage: the GMOs must be transported
Clean equipment used at the release within a primary, sealed container that is packed in a secondary
site: prevents escape of GM plant unbreakable container; store in sealed container within a locked
material into the environment outside facility that is signed to indicate GM cotton is stored within.
the release site. Clean equipment used at the release site: equipment used in
Destroy all seed not required for connection with the GM cottons must be cleaned before being used
testing or further trial: prevents for any other purpose. If GM cotton is ginned, the gin must be
unintended spread. cleaned immediately following its use, before any other cotton is
Destroy any volunteers: prevents ginned.
persistence of GM cotton plants. Destroy seed: destroy all seed not required for testing or future trial.
Destroy volunteers: after harvest, the release site must be
inspected at least once every two months for at least 12 months and
any cotton volunteers destroyed before flowering.
GENE TRANSFER: Negligible See Appendix 5 No N/A N/A None Required
Plants genetic incompatibility prevents gene transfer to native cottons.
Native cottons
and other plant
genera
GENE TRANSFER: Negligible See Appendix 5 No N/A N/A None Required
Microorganisms the likelihood of gene transfer from plants to bacteria is extremely low, and
has not been demonstrated under natural conditions;
all of the genes introduced into Roundup Ready® Flex and Roundup
Ready® Flex/Bollgard II® cottons, and other genes with similar functions,
are widespread in the environment, and readily available for transfer from
these sources via demonstrated natural mechanisms; and
the introduced regulatory sequences are already widespread and function
in the same way as do native regulatory sequences in plants.
Chapter 2 Summary of the Risk Assessment and Risk Management Plan 14
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Risk Does Risk
Hazard (combines Summary of Risk Assessment Require Risk Management Is Risk Licence conditions
‘likelihood’
Identification (refer to Appendices for details) Management? Method(s) and Reasons(s) for selection Managed (see Appendix 7 for detailed licence conditions)
& ‘impact’) ?
GENE TRANSFER: Negligible See Appendix 5 No N/A N/A None Required
Animals the likelihood of gene transfer from plants to animals is extremely low and
including greatly exceeded by the likelihood of transfer from other sources of the
humans introduced genes;
all of the genes and regulatory elements introduced into Roundup Ready®
Flex and Roundup Ready® Flex/Bollgard II® cottons, and other genes with
similar functions, are widespread in the environment, and are therefore
already available for transfer; and
no products from the GM cottons will be used for human food or animal
feed.
HERBICIDE NA See Appendix 6 This risk is APVMA will impose appropriate N/A Not required.
RESISTANCE The risk of herbicide-resistant weeds developing as a result of the managed by conditions
Roundup Ready® Flex crop-herbicide combination will be managed by the the APVMA Licence notes the requirement to comply with conditions imposed by the
APVMA, under conditions of permits or registration for the use of APVMA, including any herbicide resistance management strategy.
agricultural chemicals in Australia. Therefore, no specific conditions have
been imposed in relation to management of herbicide resistance; however,
the requirement to comply with conditions imposed by the APVMA has
been noted.
INSECTICIDE NA See Appendix 6 This risk is APVMA has imposed appropriate N/A Not required.
RESISTANCE The risk of insects developing resistance to the insecticidal proteins managed by conditions
expressed by Roundup Ready® Flex/Bollgard II® cotton is being managed by the the APVMA Licence notes the requirement to comply with conditions imposed by the
APVMA, under conditions of permits or registration for the use of APVMA, including any insecticide resistance management strategy.
insecticidal genes as agricultural chemicals in Australia. Therefore, no
specific conditions have been imposed in relation to management of
insecticide resistance, however, the requirement to comply with conditions
imposed by the APVMA has been noted.
Chapter 2 Summary of the Risk Assessment and Risk Management Plan 15
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 1 INFORMATION ABOUT THE GMO
60. In preparing the risk assessment and risk management plan (RARMP), the Regulator is
required under Section 49 (2) of the Act to consider the properties of the parent organism and
the effects of the genetic modification.
61. This part of the document addresses these matters and provides detailed information about
the GMOs proposed for release, the parent organism, the genetic modification process, the
genes that have been introduced and the new proteins that are expressed in the genetically
modified cotton.
62. It should be noted that some details of the gene construct, including the plasmid map and
some of the regulatory sequences were declared as Confidential Commercial Information
(CCI) under Section 185 of the Act, in connection with licence application DIR 035/2003.
This information was made available to the prescribed expert groups which were consulted in
the preparation of the risk assessment and risk management plan. However, the applicant has
recently indicated that it is no longer considered as necessary to protect this information as
CCI and the declaration has been revoked. All previous CCI information can now be made
public and the relevant information is discussed in detail in this Appendix.
SECTION 1 SUMMARY INFORMATION ABOUT THE GMO
63. Monsanto Australia Limited (Monsanto) proposes to release two GMOs under application
DIR 055/2004: herbicide tolerant Roundup Ready® Flex cotton MON 88913 (abbreviated as
Roundup Ready® Flex cotton) and herbicide tolerant/insect resistant
Roundup Ready® Flex cotton MON 88913/Bollgard II® cotton (abbreviated as Roundup
Ready® Flex/Bollgard II® cotton).
64. Roundup Ready® Flex cotton MON 88913 is derived from an original genetic
modification event referred to as MON 88913 and will be introduced into elite Australian
cotton varieties by conventional breeding. The Roundup Ready® Flex/Bollgard II® cotton
proposed for release was produced by conventional breeding of Roundup Ready® Flex cotton
and GM Bollgard II® cotton (initiated under licence DIR 035/2003, which included the same
GM cottons). Bollgard II® cotton has been licensed in Australia for commercial release south
of latitude 22° South, and for field trials north of latitude 22° South, and has been considered
previously in detail in the RARMP for DIR 012/2002, available at www.ogtr.gov.au.
Bollgard II® cotton is only discussed in this document in so far as it relates to Roundup
Ready® Flex/Bollgard II® cotton.
65. Roundup Ready® Flex cotton and Roundup Ready® Flex/Bollgard II® cotton both contain
two copies of the cp4 epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene, derived
from the bacterium Agrobacterium sp. strain CP4. The native plant epsps gene encodes an
enzyme (EPSPS) that is critical for the synthesis of aromatic amino acids (amino acids are
essential building blocks for proteins). Glyphosate (N-phosphonomethyl glycine), the active
ingredient in Roundup® herbicides, inhibits this enzyme eventually leading to cell and plant
death. The bacterial gene encodes an enzyme (CP4 EPSPS) that is able to function in the
presence of glyphosate. Thus, the use of these GM cottons allows the application of
glyphosate for the control of weeds that emerge in the crop, without damaging the crop itself.
Further details on the cp4 epsps gene and the CP4 EPSPS protein are provided in Section 3 of
this Appendix.
66. Roundup Ready® Flex cotton differs in three ways from the existing Roundup Ready®
cotton that is being grown commercially south of latitude 22º South in Australia. Firstly,
Roundup Ready® Flex cotton contains two copies of the cp4 epsps gene, rather than one copy
Appendix 1 Information about the GMOs 16
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
as in the commercially released Roundup Ready® cotton. Secondly, expression of each copy
of the cp4 epsps gene is under the control of a different, novel regulatory element (see Section
3.1 of this Appendix for details). These regulatory elements were not present in the
commercially released Roundup Ready® cottons. Thirdly, Roundup Ready® Flex cotton does
not contain any antibiotic resistance genes.
67. The novel regulatory elements and the two copies of the cp4 epsps gene result in increased
expression of the cp4 epsps gene in the Roundup Ready® Flex cottons. Roundup Ready®
cotton has little tolerance to glyphosate in reproductive parts of the plant. This means that
glyphosate can only be applied up to the four leaf stage of growth (i.e.prior to flower
initiation) to control weeds, as application of the herbicide after this stage can lead to yield
loss (Monsanto Australia Limited 2001). Roundup Ready® Flex cotton with its prolonged
expression of the cp4 epsps gene, however, can tolerate glyphosate application at later stages
of growth. As a result, the window in which glyphosate can be applied for weed control is
longer, giving growers increased flexibility in timing herbicide applications for integrated
weed management.
68. Roundup Ready® Flex/Bollgard II® cotton in addition contains the insecticidal genes
cry1Ac and cry2Ab, derived from the common soil bacterium Bacillus thuringiensis (Bt).
These genes encode the highly specific insecticidal proteins Cry1Ac and Cry2Ab, which are
toxic to lepidopteran caterpillar pests of cotton, including Helicoverpa armigera (cotton
bollworm) and H. punctigera (native budworm). Further details on the cry1Ac and cry2Ab
genes and the Bt proteins are provided in Section 3 of this Appendix and in the RARMPs for
DIR 012/2002 (Bollgard II® cotton) and DIR 022/2002 (INGARD® cotton), available at
www.ogtr.gov.au.
69. Roundup Ready® Flex/Bollgard II® cotton contains two antibiotic resistance genes. These
genes were used as selectable markers in the laboratory stages of development of the
Bollgard II® plants, to enable selection of bacteria or plant cells containing the desired genetic
modification. The neomycin phosphotransferase type II (nptII) gene confers resistance to the
antibiotics kanamycin and neomycin. The aminoglycoside adenyltransferase (aad) gene
confers resistance to spectinomycin and streptomycin, but is controlled by a bacterial
promoter that does not function in plants. As a result, the AAD protein is not expressed in the
GM cotton. The antibiotic resistance genes are discussed in more detail in Section 3 of this
Appendix. Potential hazards relating to transfer of these genes to bacteria are discussed in
Appendix 5.
70. Roundup Ready® Flex/Bollgard II® cotton also contains the uidA gene from Escherichia
coli which encodes the bacterial enzyme β-glucuronidase (GUS). The GUS enzyme is a
visual marker that allows the detection of genetically modified tissues using a simple
biochemical stain. More information about the uidA gene and the GUS protein is provided in
Section 3 of this Appendix.
71. The methods used to introduce the genes into cotton are discussed in Section 4 of this
Appendix.
SECTION 2 THE PARENT ORGANISM
72. The parent organism is cultivated cotton (Gossypium hirsutum L.), which is exotic to
Australia and is grown as an agricultural crop in New South Wales, southern and central
Queensland (Qld) and on a trial basis in Western Australia, the Northern Territory and
northern Qld. More detailed information on cotton can be found in a review document 'The
Biology and Ecology of Cotton (Gossypium hirsutum) in Australia' (OGTR 2002), that was
Appendix 1 Information about the GMOs 17
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
produced in order to inform the risk assessment processes for licence applications involving
GM cottons. This document is available at www.ogtr.gov.au.
SECTION 3 THE INTRODUCED GENES AND THEIR PRODUCTS
Section 3.1 The cp4 epsps herbicide tolerance gene and encoded protein
73. The cp4 epsps gene, which confers tolerance to glyphosate (N-phosphonomethyl glycine),
the active ingredient of Roundup® herbicides, was isolated from the Agrobacterium species
strain CP4. This cp4 epsps gene encodes a 47.6 kDa EPSPS protein consisting of a single
polypeptide of 455 amino acids (Padgette et al. 1996).
74. In plants the native epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene encodes an
enzyme (EPSPS) critical for the biosynthesis of aromatic amino acids (tryptophan, tyrosine
and phenylalanine), which are essential building blocks for cellular proteins. During this
biosynthetic process the EPSPS enzyme catalyses the addition of the enolpyruvyl moiety of
phosphoenolpyruvate to shikimate-3-phosphate. EPSPS performs this function in plants,
bacteria, algae and fungi but is absent from mammals, which are not able to synthesise these
aromatic amino acids (Padgette et al. 1993, Monsanto unpublished; Bentley 1990).
75. Glyphosate herbicide functions by inhibiting the activity of the naturally occurring EPSPS
enzyme in plants, thus blocking the biosynthesis of aromatic amino acids and eventually
leading to cell death (Steinrucken & Amrhein 1980). The cp4 epsps gene from
Agrobacterium is naturally insensitive to the effects of glyphosate (Padgette et al. 1993,
Monsanto unpublished), as are a number of other microbial EPSPS enzymes (Schulz et al.
1985; Eschenburg et al. 2002), being still able to function normally in the biosynthesis of
aromatic amino acids. Consequently, in GM plant cells expressing the Agrobacterium cp4
epsps gene, biosynthesis of aromatic amino acids is not blocked in the presence of glyphosate
and the plants are not killed by application of glyphosate.
76. The coding sequence of the cp4 epsps gene introduced into the GM cotton plants has been
modified to achieve optimal expression in plants. Although the gene sequence has been
altered, there is only one amino acid difference between the enzyme produced in Roundup
Ready® Flex cottons and the native Agrobacterium enzyme (information provided by the
applicant; (Padgette et al. 1993, Monsanto unpublished), and the protein activity is not
altered.
77. In plants, the EPSPS enzyme and the site of aromatic amino acid synthesis are located in
the chloroplast. Thus, the cp4 epsps gene in the GM cottons was linked to a chloroplast
transit peptide (CTP) coding region, ctp2 from the epsps gene of the plant Arabidopsis
thaliana (Klee et al. 1987), to provide transport to the cotton chloroplast. The ctp2 sequence
present in Roundup Ready® Flex cotton is the same as that used in the development of
Roundup Ready® cotton. The CTP targets the CP4 EPSPS enzyme to the chloroplast. In
plants, EPSPS is synthesised as a preprotein (i.e.containing the CTP) by free cytoplasmic
ribosomes. The precursor is transported into the chloroplast stroma and proteolytically
processed to yield the mature enzyme (della-Cioppa et al. 1986). Once cleaved from the
mature protein, chloroplast transit peptides are rapidly degraded (della-Cioppa et al. 1986;
Bartlett et al. 1982).
78. Two copies of the cp4 epsps gene are present in the GM cottons and each copy is under
the control of a different promoter. A promoter is a region of DNA linked to a gene that
determines whether a gene is expressed, to what extent and in which plant tissues. Details of
the genetic elements composing the introduced two-gene construct are presented in Table 1.
Appendix 1 Information about the GMOs 18
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
79. One copy of the cp4 epsps gene is controlled by the chimeric P-FMV/TSF1 promoter
consisting of enhancer sequences from the figwort mosaic virus (FMV) 35S promoter
(Richins et al. 1987) and the elongation factor EF-1 alpha promoter from the plant
Arabidopsis thaliana (EF-1 alpha A1 gene; Axelos et al. 1989). The other copy of the
cp4 epsps gene is controlled by the chimeric P-35S/ACT8 promoter consisting of enhancer
sequences of the cauliflower mosaic virus (CaMV) 35S promoter (Kay et al. 1987) and the
act8 actin promoter from A. thaliana (An et al. 1996). These promoters direct the cp4 epsps
genes to be expressed in all plant tissues throughout plant growth.
80. The GM cottons also contain additional non-coding sequences from other plant species for
improved gene expression. These include the non-translated leader (exon 1) and intron
sequences (L-TSF1 and I-TSF1, respectively) from the A. thaliana EF-1 alpha A1 gene
(Axelos et al. 1989), and the non-translated leader and intron/exon sequences (L-ACT8 and
I-ACT8, respectively) from the act8 actin gene of A. thaliana (An et al. 1996).
81. Also required for gene expression in plants is a mRNA termination region, including a
polyadenylation signal. The mRNA termination region for both the cp4 epsps genes in the
GM cottons is the 3‘non-translated region derived from the pea ribulose-1,5-biphosphate
carboxylase small subunit E9 gene (Coruzzi et al. 1984).
82. Each cp4 epsps gene in the Roundup Ready® Flex cotton encodes the same protein as is
expressed in Roundup Ready® cotton, which was approved for commercial release in
Australia (DIR 023/2002).
83. Potential hazards relating to the toxicity and allergenicity of the CP4 EPSPS protein are
discussed in Appendices 2 and 3.
Table 1: Arrangement of genetic elements in the two cp4 epsps genes in the DNA construct
®
introduced into Roundup Ready Flex cotton
Genetic element Function Derived from Reference
P-FMV/TSF1 chimeric promoter consisting of:
enhancer sequence from the FMV Figwort mosaic virus Richins et al., 1987
35S promoter; and
promoter of the elongation factor Arabidopsis thaliana Axelos et al., 1989
EF-1 alpha gene
L-TSF1 leader sequence (exon 1) from the Arabidopsis thaliana Axelos et al., 1989
elongation factor EF-1 alpha gene
I-TSF1 intron sequence from the elongation Arabidopsis thaliana Axelos et al., 1989
factor EF-1 alpha gene
ctp2 targeting chloroplast transit peptide sequence Arabidopsis thaliana Klee et al., 1987
sequence of the Arabidopsis epsps gene,
directing the CP4 EPSPS protein
into the chloroplast
cp4 epsps coding sequence for the Agrobacterium sp. Padgette et al., 1996;
CP4 EPSPS protein strain CP4 (Barry et al. 1997)
E9 termination 3‟ nontranslated region of the pea Pisum sativum (pea) Coruzzi et al., 1984
region ribulose-1,5-bisphosphate
carboxylase small subunit E9 gene
P-35S/ACT8 chimeric promoter consisting of:
enhancer sequence from the Cauliflower mosaic Kay et al., 1987
CaMV 35S promoter; and virus
promoter of the act8 actin gene Arabidopsis thaliana An et al., 1996
L-ACT8 leader sequence from the act8 actin Arabidopsis thaliana An et al., 1996
gene
I-ACT8 intron and flanking exon sequence Arabidopsis thaliana An et al., 1996
from the act8 actin gene
Appendix 1 Information about the GMOs 19
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Genetic element Function Derived from Reference
ctp2 targeting chloroplast transit peptide sequence Arabidopsis thaliana Klee et al., 1987
sequence of the Arabidopsis epsps gene,
directing the CP4 EPSPS protein
into the chloroplast
cp4 epsps coding sequence for the Agrobacterium sp. Padgette et al., 1996;
CP4 EPSPS protein strain CP4 Barry et al., 1997
E9 termination 3‟ nontranslated region of the pea Pisum sativum (pea) Coruzzi et al., 1984
region ribulose-1,5-bisphosphate
carboxylase small subunit E9 gene
Section 3.2 The cry1Ac and cry2Ab insecticidal genes and encoded proteins
84. The cry1Ac and cry2Ab genes of Roundup Ready® Flex/Bollgard II® cotton are derived
from Bacillus thuringiensis (Bt), a ubiquitous soil bacterium. The Cry (for crystalline)
proteins (also called Bt proteins or Bt toxins) are a diverse family of insecticidal proteins
produced by various subspecies of Bt. The cry1Ac and cry2Ab genes are derived from B.
thuringiensis variety kurstaki (Btk). The cry1Ac and cry2Ab genes encode Bt toxins that are
highly specific to lepidopteran insects (moths and butterflies) (Widner & Whiteley 1989;
Dankocsik et al. 1990; Macintosh et al. 1990).
85. The toxic effect of Bt proteins requires alkaline conditions (as provided in the larval insect
gut) to dissolve the crystals, partial digestion by specific proteases to release the active core
toxin and binding to specific receptors found on the insect midgut epithelium surface.
Binding leads to formation of pores in the cell membrane which leads to leakage of
intracellular contents into the gut lumen and water into the cell and eventually to cell death,
gut paralysis and starvation. It is these steps that provide the high degree of target specificity
of each Bt toxin (English & Slatin 1992; Hofmann et al. 1988; Knowles & Dow 1993; Van
Rie et al. 1989).
86. Expression of the cry1Ac and cry2Ab genes in Bollgard II® cotton is controlled by the
enhanced CaMV 35S promoter (Kay et al. 1987; Odell et al. 1985). The mRNA termination
region for the cry1Ac gene in Bollgard II® cotton is derived from the alpha subunit of the
beta-conglycinin gene of soybean and for the cry2Ab gene from the nopaline synthase (nos)
gene of A. tumefaciens.
87. More detailed information about the cry1Ac and cry2Ab genes present in Bollgard II®
cotton, and the Bt toxins, can be found in the risk assessment and risk management plans for
DIR 012/2002 and DIR 022/2002, available at www.ogtr.gov.au.
Section 3.3 The uidA reporter gene and encoded protein
88. Roundup Ready® Flex/Bollgard II® cotton plants contain the uidA reporter gene. The
uidA gene is derived from the common gut bacterium Escherichia coli (E. coli) and encodes
the enzyme β-glucuronidase (GUS) (Jefferson et al. 1986).
89. The uidA gene is the most widely used reporter gene in GM plants (Miki & McHugh
2004). Reporter genes encode enzymes that are easily assayed and are therefore used to
‗report‘ on the activity of a promoter to which the reporter gene is linked. They can also be
linked to a gene to report on the cellular location of the encoded protein, or used as a simple
biochemical tag to identify GM tissues.
90. The GUS protein is a monomer with a molecular mass of 68 kDa, and the GUS enzyme is
active in the form of a tetramer. GUS catalyses the hydrolysis of -glucuronides and, less
efficiently, some -galacturonides. A large variety of -glucuronides exist in nature and they
Appendix 1 Information about the GMOs 20
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
have been described as the detoxified excretion forms of xenobiotics (foreign substances e.g.
drugs) and endogenous compounds (e.g. steroids) in vertebrates (Jefferson & Wilson 1991).
E. coli lives in the digestive tract of vertebrates, including humans (Jefferson et al. 1986), and
the GUS enzyme enables it to metabolise -glucuronides as a main source of carbon and
energy.
91. GUS cleaves the chromogenic substrate X-gluc (5-bromo-4-chloro-3-indolyl -D-
glucuronide) to produce an insoluble blue colour (Jefferson et al. 1987). Endogenous GUS
enzyme activity is found in many other bacterial species, and also in vertebrates and
invertebrates, but there is very little background activity in non-GM plants (Gilissen et al.
1998; Jefferson et al. 1987; see also Appendix 2). Therefore, the production of a blue colour
in particular plant cells after staining with X-gluc indicates that these cells express GUS from
the introduced uidA gene, and have been successfully genetically modified.
92. The uidA gene was used as a marker in the laboratory for selecting successfully modified
Bollgard II® cotton cells after the genetic modification. Expression of this gene in Roundup
Ready® Flex/Bollgard II® cotton plants is controlled by the CaMV 35S promoter (Kay et al.
1987; Odell et al. 1985). The mRNA termination region of the gene is the 3‘non-translated
region of the nos gene from A. tumefaciens (Rogers et al. 1985).
Section 3.4 The nptII and aad antibiotic resistance marker genes and encoded proteins
93. Roundup Ready® Flex cotton does not contain any antibiotic resistance genes. Roundup
Ready® Flex/Bollgard II® cotton contains the nptII and aad antibiotic resistance marker genes
from E. coli. These genes were used in the initial laboratory stages of development of
Bollgard II® cotton plants, to enable selection of cells containing the desired genetic
modification. Both genes are in common use as selectable markers in the production of GM
plants.
94. The nptII gene was isolated from the bacterial Tn5 transposon (from E. coli) (Beck et al.
1982). It encodes an enzyme, neomycin phosphotransferase type II (NPTII), which confers
resistance to the aminoglycoside antibiotics kanamycin and neomycin. NPTII uses ATP to
phosphorylate kanamycin and neomycin, thereby inactivating the antibiotic and preventing it
from killing the NPTII-producing cell. The nptII gene functioned as a selectable marker
during the laboratory stages of cotton plant cell selection following genetic modification,
allowing modified cells to grow in the presence of the antibiotic while inhibiting the growth
of non-modified cells.
95. The NPTII enzyme is widespread in the environment and in food chains, in naturally
occurring kanamycin-resistant microorganisms found in soil and in mammalian digestive
systems (Flavell et al. 1992).
96. Expression of the nptII gene in Roundup Ready® Flex/Bollgard II® cotton plants is
controlled by the CaMV 35S promoter (Kay et al. 1987; Odell et al. 1985). The mRNA
termination region of the gene is the 3‘non-translated region of the nos gene from
A. tumefaciens (Rogers et al. 1985).
97. The aad gene was used in the laboratory, prior to production of the genetically modified
plants, to select for bacteria carrying the plasmid with the modified DNA. This gene was
isolated from the bacterial Tn7 transposon (from E. coli) and confers resistance to the
antibiotics spectinomycin and streptomycin (Davies & Benveniste 1974). The aad gene is not
expressed in the GM cotton plants because it is under the control of its native bacterial
promoter, which is not active in plants, and regulatory elements necessary for its expression in
plants have not been added to the gene.
Appendix 1 Information about the GMOs 21
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
98. Potential hazards relating to the toxicity and allergenicity of the NPTII protein are
discussed in Appendices 2 and 3, and those of antibiotic resistance gene transfer in
Appendix 5.
SECTION 4 METHOD OF GENETIC MODIFICATION
99. The Roundup Ready® Flex cotton was produced by Agrobacterium-mediated DNA
transformation (Zambryski 1992) of a Coker variety of cotton (transformation event 88913).
Coker cotton varieties are US cultivars that are widely used in producing GM cottons because
they can be readily cultured and regenerated in the laboratory. The proposed dealing aims to
transfer the Roundup Ready® Flex trait into cotton varieties suitable for Australian conditions
by conventional crossing.
100. Agrobacterium tumefaciens is a common gram-negative soil bacterium that causes
crown gall disease in a wide variety of plants. Plants can be genetically modified by the
transfer of DNA (transfer-DNA or T-DNA, located between specific border sequences on a
resident plasmid) from A. tumefaciens through the mediation of genes from the vir (virulence)
region of Ti plasmids.
101. Disarmed Agrobacterium strains have been constructed specifically for plant
transformation. The disarmed strains do not contain the genes (iaaM, iaaH and ipt)
responsible for the overproduction of auxin and cytokinin, which are required for tumour
induction and rapid callus growth (Klee & Rogers 1989). Agrobacterium plasmid vectors
used to transfer T-DNAs contain well characterised DNA segments required for their
replication and selection in bacteria, and for transfer of T-DNA from Agrobacterium and its
integration into the plant cell genome (Bevan 1984; Wang et al. 1984). Agrobacterium-
mediated transformation has been widely used in Australia and overseas for introducing new
genes into plants without causing any biosafety problems.
102. In this instance, a typical, disarmed, binary plasmid vector (PV-GHGT35) was used to
introduce the gene construct containing two copies of the cp4 epsps gene (see Table 1) into
cotton variety Coker using standard Agrobacterium transformation protocols. Following co-
cultivation with A. tumefaciens containing the gene construct, cotton cells were cultured in the
presence of glyphosate to select for those cells containing the introduced DNA (since the
cp4 epsps gene confers tolerance to glyphosate). Subsequently cotton plants containing the
introduced gene constructs were regenerated from these cells. The resulting genetically
modified plants were further screened for tolerance to glyphosate over several generations in
the laboratory and under field conditions in the USA. Roundup Ready® Flex cotton is derived
from a single genetic modification, or ‗transformation‘, event (event 88913).
103. Bollgard II® cotton was produced by inserting the cry2Ab and uidA genes into the
genomic DNA of INGARD® cotton variety DP50B (containing the cry1Ac and nptII genes).
Genes were delivered into the cotton meristematic cells by microprojectile bombardment
(transformation event 15985) (McCabe & Martinell 1993). This technique is a well-
established method of plant transformation that uses compressed gas to 'shoot' tiny tungsten or
gold particles coated with the genes to be inserted into plant cells. The introduced genes
become incorporated into the genome of the bombarded plant cells. The uidA gene is used as
a marker to identify plant tissue that contains the introduced gene of interest. INGARD®
cotton was itself produced by Agrobacterium-mediated transformation of a non-GM Coker
cotton variety, introducing the cry1Ac, nptII and aad genes.
104. As indicated above, Roundup Ready® Flex/Bollgard II® cotton was produced by
conventional breeding of GM Roundup Ready® Flex cotton with GM Bollgard II® cotton
under DIR 035/2003.
Appendix 1 Information about the GMOs 22
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 5 CHARACTERISATION OF THE INSERTED GENETIC MATERIAL AND
STABILITY OF THE GENETIC MODIFICATION
105. Southern blot analysis of Roundup Ready® Flex DNA shows that all inserted genetic
elements, including coding regions, promoters and non-coding regions, are present as single
inserts in the Roundup Ready® Flex cotton genome and that no unnecessary vector sequences
are present. PCR analysis of Roundup Ready® Flex DNA demonstrates that the arrangement
and linkage of elements in the introduced DNA are the same as those in the plasmid vector
that was used to introduce the cp4 epsps two-gene construct into cotton (see Table 1 above).
106. Phenotypic segregation and Southern blot analysis from five generations of
backcrossing confirmed the stability of the inserted DNA. No loss in the glyphosate tolerant
phenotype (tested for CP4 EPSPS protein expression and tolerance to Roundup Ready®
herbicide) or loss or rearrangement of the inserted genetic elements has been observed. The
analysis of phenotypic segregation over five generations also demonstrated that the inserted
elements are inherited in a normal Mendelian manner for a single, dominant trait.
107. Bollgard II® cotton contains one complete copy of the cry1Ac, cry2Ab, nptII, aad and
uidA genes, stably integrated at one location in the cotton genome. Detailed information on
the characterisation of the inserted genetic material and stability of the genetic modification in
Bollgard II® cotton is provided in the RARMP for DIR 012/2002 (available at
www.ogtr.gov.au).
108. Detailed molecular characterisation of the insertion sites (i.e. sequencing of the regions
flanking the inserted DNA constructs) would be required before any application for a
commercial release of the GM cottons could be assessed.
SECTION 6 EXPRESSION OF THE INTRODUCED PROTEINS
109. The applicant expects that the two different chimeric promoters, containing the viral
enhancer sequences (from the CaMV or FMV 35S promoters), will cause expression of
CP4 EPSPS from the two copies of the cp4 epsps gene throughout most or all parts of the
Roundup Ready® Flex cotton plant. Roundup Ready® Flex cotton has increased and
prolonged expression of the cp4 epsps gene, compared to commercially released
Roundup Ready® cotton, including expression in the reproductive parts of the plant.
Expression of the CP4 EPSPS protein in Roundup Ready® cotton is significantly lower in
reproductive tissues such as stigmas, anthers and floral buds than in vegetative tissue such as
leaves (Pline et al. 2002a).
110. The reproductive structures of cotton plants are very sensitive to the effects of
glyphosate (Pline et al. 2002b; Pline et al. 2002a) and Roundup Ready® cotton crops sprayed
with glyphosate beyond the four true leaf stage of growth exhibit reduced pollination and
increased boll abortion (Monsanto Australia Limited 2001). Tolerance to glyphosate in the
reproductive parts of Roundup Ready® Flex cotton plants is intended to enable glyphosate
application over the top of the cotton crop at later stages of growth. Field trials conducted in
the USA have shown that reproductive parts of Roundup Ready® Flex cotton plants have
greater tolerance to glyphosate as compared to the same plant parts in Roundup Ready cotton
(determined by measurement of pollen viability and the number of pollen grains attached to
the stigmatic lobe after treatment with glyphosate).
111. Data on the level of expression of the CP4 EPSPS protein in different Roundup Ready®
Flex cotton tissues have been collected from field trials conducted in the United States across
four locations during 2002 (Table 2). Samples of young leaves collected over the growing
season, roots, seeds and pollen were analysed using an enzyme-linked immunosorbent assay
Appendix 1 Information about the GMOs 23
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
(ELISA). All of the values presented in Table 2 are well above the level of quantitation,
which means that they can be reliably and reproducibly quantitated. Because of the limited
quantities of pollen available, protein levels by dry weight could not be determined for pollen.
The CP4 EPSPS protein levels detected in all tissues from negative segregants (progeny of
backcrosses between Roundup Ready® Flex cotton and non-GM cotton that did not inherit the
introduced genes) were less than the assay limits of quantitation presented in Table 2.
112. The CP4 EPSPS protein levels detected in Roundup Ready® Flex cotton are
significantly higher than those detected in Roundup Ready® cotton tissues (42 - 53 and 60 -
299 μg per g fresh weight in leaves and seeds, respectively; detailed information is provided
in the RARMP for DIR 023/2002, available from www.ogtr.gov.au.). The applicant is
currently collecting further data on expression of the CP4 EPSPS protein in Roundup Ready®
Flex cotton under Australian conditions as part of a field trial conducted under DIR 035/2003.
®
Table 2: CP4 EPSPS protein levels in Roundup Ready Flex cotton tissues (± standard
deviation)
CP4 EPSPS protein level CP4 EPSPS protein level LOQ/LOD
(in μg per g fresh weight) (in μg per g dry weight) (in μg per g fresh weight)
Tissue type Mean Range Mean Range
Leaf* A 170 ± 64 64 – 260 970 ± 460 270 - 1700 0.23 / 0.069
Leaf B 270 ± 99 77 – 410 1400 ± 540 480 - 2600 0.23 / 0.069
Leaf C 170 ± 44 63 – 260 960 ± 210 290 - 1000 0.23 / 0.069
Leaf D 160 ± 61 66 – 260 630 ± 230 290 – 1100 0.23 / 0.069
Root 31 ± 11 19 – 64 99 ± 40 57 - 200 0.23 / 0.073
Seed 310 ± 110 67 – 550 340 ±120 72 - 580 2.7 / 1.7
Pollen 4 ± 0.22 3.8 – 4.3 ND ND 0.23 / 0.11
* represent the newest fully expanded leaves (leaf A to D) collected at different time points
throughout the growing season between the seedling stage and crop maturity
LOQ: limit of quantitation
LOD: limit of detection
ND: not determined
113. Expression levels of the Cry1Ac, Cry2Ab, GUS and NPTII proteins in Bollgard II®
cotton have been extensively studied. Detailed information on the expression levels of these
proteins in leaves, seeds and pollen of Bollgard II® cotton is presented and discussed in the
RARMP for DIR 012/2002, available from www.ogtr.gov.au. Some of these data are
summarised in Table 3.
®
Table 3: Expression levels* of the proteins introduced into Bollgard II cotton (± standard
deviation)
Tissue type Year tested Cry2Ab Cry1Ac GUS NPTII
leaf 1998 23.8 ± 6.3 2.75 ± 1.32 106 ± 32 16.6 ± 5.2
1999 6.4 ± 1.4 2.07 ± 0.61 120 ± 28.8 4.57 ± 1.02
seed 1998 43.2 ± 5.7 3.35 ± 0.63 58.8 ± 13.0 10.8 ± 1.2
1999 57.4 ± 13.1 2.60 ± 0.66 104 ± 56.9 12.6 ± 2.27
whole plant 1998 8.8 ± 1.2 0.17 ± 0.08 ND ND
1999 20.8 ± 1.8 0.08 ± 0.01 ND ND
pollen 1998 <0.25 0.02 ± 0.01 ND ND
1999 0.32 0.05 ± 0.07 ND ND
* mean protein levels are reported as μg per g fresh weight of plant tissue
ND: not determined
Appendix 1 Information about the GMOs 24
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 7 PLEIOTROPIC EFFECTS OF THE GENETIC MODIFICATION
114. A single plant gene can have an influence on multiple, sometimes unrelated, plant traits.
This phenomenon is known as pleiotropy. Single genes inserted into a plant by genetic
modification can also be pleiotropic and it is necessary to evaluate genetically modified plants
for unintended, pleiotropic effects of the inserted genes, such as changes in agronomic
characteristics.
115. No unintended or secondary effects have been observed in greenhouse or field trials of
Roundup Ready® Flex cotton in the USA. Phenotypic analysis, including lint yield and plant
growth characteristics, of Roundup Ready® Flex cotton from an Australian field trial (four
sites in the cotton growing regions in NSW and Qld) in 2003/04 was conducted under
DIR 035/2003 to evaluate unintended pleiotropic effects. There was no significant difference
in lint yield between Roundup Ready® Flex and the negative segregant control. There was
also no significant difference in the number of reproductive nodes or boll retention at the first
fruiting positions. The only significant difference in plant growth characteristics observed
was an increased height of Roundup Ready® Flex plants at 80 – 90 days after planting. This
difference, however, no longer existed at the time of harvest. Further evaluation of agronomic
performance under Australian conditions will be conducted in the 2004/05 season (under
DIR 035/2003).
116. The agronomic performance of commercially released Roundup Ready® and
Roundup Ready®/Bollgard II® cottons (expressing the same introduced proteins as the
Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons) has been thoroughly
analysed during numerous field trials and commercial releases of these GM cottons in
Australia and elsewhere in the world. Such analysis has been closely monitored for
differences between the GM and parental non-GM and GM cotton varieties (Hamilton &
Reed 1999; Hamilton et al. 2000).
117. The applicant intends to conduct evaluation of agronomic performance, including
disease resistance (bacterial blight, fusarium and verticillium wilt), of Roundup Ready® Flex
and Roundup Ready® Flex/Bollgard II® cottons as part of the proposed trial.
SECTION 8 CONSIDERATION OF THE RISKS RELATING TO THE COMBINATION
® ®
OF THE BOLLGARD II AND THE ROUNDUP READY FLEX TRAITS
118. In preparing the RARMP, the effect of combining glyphosate tolerance and the insect
resistance of Bollgard II® cotton in the same plant, and whether this could result in new or
increased risks over and above those posed by the introduction of the traits singly, were
considered. This has also been assessed in the RARMP for the commercial release of
Roundup Ready®/Bollgard II® cotton (expressing the same introduced proteins) under
DIR 012/2002.
119. It is noted that:
The gene conferring tolerance to glyphosate and the insecticidal genes of
Bollgard II® cotton operate through independent, unrelated biochemical
mechanisms. There is no evidence of any interaction between the two genes, their
products or their metabolic pathways, and no reason to expect that this is likely to
occur.
There is no evidence or reasonable expectation that synergistic effects are likely to
occur from the combination of the two traits, or that they would result in new or
increased risks relating to human health and safety or the environment.
Appendix 1 Information about the GMOs 25
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Each of the genes integrated into the cotton has been integrated stably into the
cotton genome (see Section 5 of this Appendix for detail).
There have been no reports of any unexpected or unintentional adverse effects
from previous releases of Roundup Ready®/Bollgard II® cotton expressing the
same proteins as Roundup Ready® Flex/Bollgard II® cotton (see Section 7 of this
Appendix).
120. It is considered unlikely that Roundup Ready® Flex/Bollgard II® cotton will present
new or increased risks to human health and safety, or to the environment, over and above
those posed by the introduction of the single traits.
SECTION 9 RESEARCH REQUIREMENTS
121. Licence conditions for a field trial with the same GM cottons (being conducted under
DIR 35/2003) contain a requirement to conduct a research program. As part of this program,
the following data relevant to the assessment of the proposed release have been collected and
were provided in the application for DIR 055/2004:
phenotypic and agronomic characteristics of Roundup Ready® Flex cotton;
genetic segregation and molecular characterisation of the introduced genetic
material; and
levels of expression of the introduced CP4 EPSPS protein.
122. Therefore, no additional research requirements have been imposed for this release,
given that further data, including expression levels of the introduced CP4 EPSPS protein
under Australian conditions and additional data on agronomic characteristics, will be soon
available from subsequent seasons of the trial being conducted under DIR 35/2003. In
addition, a gene flow research program is being coordinated by the OGTR with GM cotton
licensees, including Monsanto, in a range of current and potential cotton growing areas.
123. In addition to the above, additional data, the following information would be required if
the applicant were to submit an application for the commercial release of these GM cottons:
the complete sequence of the introduced DNA; and
sequences of the DNA regions flanking the introduced DNA and results of
database homology searches.
Appendix 1 Information about the GMOs 26
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 2 TOXICITY AND ALLERGENICITY TO HUMANS
124. Under Section 51 of the Act, the Regulator is required to consider risks to human health
and safety and the environment in preparing the risk assessment and risk management plan
(RARMP). This Appendix considers potential hazards that may be posed to human health
and safety as a result of any toxicity or allergenicity of the GMOs or their introduced
proteins.
125. It should be noted that since the commercial release of similar GM cottons,
Roundup Ready® and Roundup Ready®/Bollgard II®, producing the same introduced proteins
as Roundup Ready® Flex cotton MON 88913 (Roundup Ready® Flex cotton) and Roundup
Ready® Flex/Bollgard II® cotton, there have been no reported adverse toxic or allergic effects
on human health resulting from occupational exposure, from ingestion of foods derived from
the oil or linters of these GM cottons or from the use of other products containing their oil,
lint or linters. The Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons
proposed for release in this licence application have previously been assessed during
evaluation of the application for DIR 035/2003.
SECTION 1 NATURE OF THE POTENTIAL TOXICITY OR ALLERGENICITY
HAZARD
126. Toxicity is the cascade of reactions resulting from exposure to a dose of chemical
sufficient to cause direct cellular or tissue injury or otherwise inhibit normal physiological
processes (Felsot 2000b). Allergic responses are immune system reactions, resulting from
stimulation of a specific group of antibodies (known as IgE) or sensitisation of specific tissue
bound lymphocytes (FAO & WHO 2000; Taylor & Lehrer 1996). Allergy has a well-defined
etiology (i.e. biochemical cause) that is quite different from toxicity.
127. Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons differ from
conventional cotton through the expression of one and five additional proteins, respectively.
These are the CP4 EPSPS protein (in both GM cottons) and the Cry1Ac, Cry2Ab, GUS and
NPTII proteins (in Roundup Ready® Flex/Bollgard II® cotton only) (see Appendix 1 for
details of protein expression in the GM cottons). The potential for these cottons to be toxic or
allergenic to humans either due to the expression of the introduced gene products or because
of unintended effects of the genetic modification is considered here.
128. Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons express the
same proteins (both structurally and functionally) as the commercially released
Roundup Ready® and Roundup Ready®/Bollgard II® and Roundup Ready®/INGARD®
cottons. The same Cry proteins are also present in the commercially released Bollgard II®
and INGARD® cottons. Risk assessments and risk management plans were produced for
these GM cottons under DIRs 023/2002, 012/2002 and 022/2002, respectively. The only
expected phenotypic difference between the Roundup Ready® Flex cotton and the
commercially released Roundup Ready® cotton is in the level and duration of expression of
the CP4 EPSPS protein and reproductive tolerance to glyphosate. However, although
Roundup Ready® Flex cotton expresses the same introduced protein as commercially released
Roundup Ready® cotton, because they are derived from different transformation events, the
sites of insertion of the introduced gene will be different. Therefore, there is a possibility,
although considered very low, that this could potentially alter some aspect of plant
metabolism that impacts on toxicity or allergenicity.
129. The Australian Pesticides and Veterinary Medicines Authority (APVMA) is responsible
for the use and safety of herbicides in Australia. Glyphosate-based herbicides (e.g. Roundup
Ready®) are not currently registered for use on cotton beyond the four-leaf stage of growth.
Appendix 2 Toxicity and allergenicity to humans 27
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
A research permit from the APVMA for the use of glyphosate after this stage on Roundup
Ready® Flex cotton will be required. Currently, the applicant holds a permit for the use of
glyphosate on Roundup Ready® Flex cotton grown under the DIR 035/2003 licence and has
submitted an application for the permit required for the proposed release. As part of its
assessment of herbicide use, the APVMA considers any potential human health effects, for
example risks arising through occupational exposure or residues in food and the environment.
Thus risks associated with the use of glyphosate are not considered in the risk assessment of
these GM cottons.
SECTION 2 LIKELIHOOD OF THE TOXICITY OR ALLERGENICITY HAZARD
OCCURRING
130. In assessing the likelihood of adverse impacts due to toxicity or allergenicity of
Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons on human health and
safety, the following factors were considered:
the inherent toxicity and allergenicity of conventionally bred non-GM cotton;
the potential exposure to these GM cottons, to their products and to the
introduced proteins (CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and NPTII ) expressed
in these cottons;
the potential exposure to the CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and NPTII
proteins from other sources in the environment; and
the potential toxicity and allergenicity of the new proteins expressed in the GM
cottons.
Section 2.1 Toxicity and allergenicity of conventionally bred non-GM cotton
131. Cotton is a well-established field crop with a long history of safe use. A
comprehensive review of conventional cotton, including information on its toxicity and
allergenicity, is provided in the document ‗The Biology and Ecology of Cotton
(Gossypium hirsutum) in Australia‘ (OGTR 2002) that was produced in order to inform the
risk assessment processes for licence applications involving GM cotton. This document can
be accessed at www.ogtr.gov.au. Information on non-GM cotton is included here to establish
a baseline for comparison with the GM cottons being considered in this risk assessment.
132. Cotton tissue, particularly the seeds, can be toxic if ingested in large quantities because
of the presence of toxic and anti-nutritional factors including gossypol and cyclopropenoid
fatty acids (e.g. dihydrosterculic, sterculic and malvalic acids).
133. Processed cotton fibre contains 99.8% cellulose and is widely used in pharmaceutical
and medical applications because of its very low allergenicity. Cottonseed oil has been in
common use since the middle of the nineteenth century and achieved GRAS (Generally
Recognised As Safe) status under the United States Federal Food Drug and Cosmetic Act
because of its common use prior to 1958 (ANZFA 2002a).
134. Cotton pollen is large, sticky and not transported easily by wind (OGTR 2002),
therefore its potential to act as an airborne allergen is extremely low. However, inhalation of
cotton dust by mill workers can cause byssinosis, an asthma-like condition, in sensitive
individuals. Preventative measures such as the use of facemasks have been successful in
lowering the incidence of this condition.
Appendix 2 Toxicity and allergenicity to humans 28
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Section 2.2 Exposure of people to GM cottons
135. The applicant proposes to retain the cottonseed produced in the field trial. Seed will be
stored or planted in further plantings authorised by the same licence, or under possible future
licences (subject to further application and assessment processes). There will be no
opportunity for human exposure to the GM cottons through food, as oil or meal derived from
cottonseed produced in the proposed field trial will not be used in human food or animal feed.
The applicant does however propose to sell the lint produced during the release for use in
fabric, upholstery and other non-food products. Hence, exposure of people to the GM cottons
will be through:
wearing cotton clothing or using household items made from GM cotton lint;
working with GM cotton (e.g. on cotton farms, in cotton processing facilities); or
living in or near the areas where GM cotton is grown.
136. As there will be no opportunity for humans to consume food products containing cotton
products from this release of GM cottons, hazards to humans through food do not warrant
detailed discussion here. If, in future applications, the applicant intended to use cotton
products (e.g. cotton linters and cottonseed oil) from these GM cottons in human food,
approval will need to be sought from Food Standards Australia New Zealand (FSANZ).
137. However, cottonseed oil and cotton linters are highly refined and processed, with no
detectable DNA or proteins (Leffler & Tubertini 1976; Sims et al. 1996). Oil and linters
derived from the current commercial release of Roundup Ready® and
Roundup Ready®/Bollgard II® cottons cannot be distinguished from those derived from
non-GM cotton. FSANZ considers that food products (oils and linters) for human
consumption that are derived from Roundup Ready® cotton and from Bollgard II® cotton are
as safe as those derived from conventional cotton varieties (ANZFA 2002a; ANZFA 2000;
ANZFA 2001f).
2.2.1 Exposure to GM cotton products through wearing clothing and using
household products made from cotton lint
138. Cotton fabrics, used in clothing, upholstery, towels and other household products, are
made from the cotton lint (long fibres) that surrounds the cottonseed. Household products
that may contain cotton linters include medical dressings, felt, fine quality paper (including
banknotes in many countries), twine and mops. Cellulose derivatives produced from the
linters may be used in pharmaceuticals, cosmetics, toothpaste, lacquers, paints and a variety
of plastics (Gregory et al. 1999). Cotton fibre is widely used in pharmaceutical and medical
applications because of its very low allergenicity.
139. Processed cotton lint and linters contain no detectable DNA or protein (Leffler &
Tubertini 1976; Sims et al. 1996). Fibre characteristics (length, strength, fineness) of
commercially released Roundup Ready® cotton are the same as for non-GM varieties (Cotton
Seed Distributors 2002). The Roundup Ready® Flex cottons are expected to differ from
Roundup Ready® cotton only in the level of expression of the CP4 EPSPS protein (which is
not present in lint) and the applicant has demonstrated that fibre characteristics are equivalent
to non-GM varieties. Therefore the safety of wearing cotton clothing or using other products
made from Roundup Ready® Flex cottons is not likely to be different from that of
commercially released Roundup Ready® or Roundup Ready®/Bollgard II® cotton, or non-GM
cotton.
Appendix 2 Toxicity and allergenicity to humans 29
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
2.2.2 Exposure to GM cotton through working with cotton and living near cotton
plantations
140. Cotton is a well-established field crop with a long history of safe use. The cotton plant
is not known to be capable of causing disease or other ill health in people through contact. A
comprehensive review of conventional non-GM cotton, including information on its toxicity,
allergenicity and pathogenicity, is provided in the document ‗The Biology and Ecology of
Cotton (Gossypium hirsutum) in Australia‘, available at www.ogtr.gov.au.
141. Humans working with cotton plants will be exposed primarily to the outer waxy cuticle
layer at the plant surface, to the seed coat or to the cotton fibres, all of which are essentially
free of protein. Exposure to proteins (including the new proteins expressed in the GM
cottons) or to other cellular components of the cotton plants will only occur if plant cells were
ruptured.
142. Even if exposure to the introduced proteins in Roundup Ready® Flex and Roundup
Ready® Flex/Bollgard II® cottons occurred, these proteins are not toxic or allergenic to
humans (see Section 2.4). The introduced proteins present in the Roundup Ready® Flex
cottons (the CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and NPTII proteins) are the same as those
present in commercially released Roundup Ready® and Bollgard II® cottons. The GM
cottons proposed for release in this application have previously been approved for a limited
and controlled release under licence DIR 035/2003. There have been no reports of adverse
effects on health through occupational exposure from people working with GM cottons
expressing these proteins or conventional Bt sprays containing the Cry proteins.
143. As the level of expression of CP4 EPSPS protein in Roundup Ready® Flex cotton is
higher than the commercially approved Roundup Ready® cotton (see Appendix 1), the
applicant is currently collecting data on protein expression under Australian environmental
conditions as part of the field trial licensed under DIR 035/2003. These data will be provided
to the Regulator as required under the licence for DIR 035/2003.
144. Cotton pollen is large, sticky and not transported easily by wind (OGTR 2002), thereby
limiting possible exposure to cotton pollen as a potential airborne allergen. The introduced
Cry1Ac, Cry2Ab, GUS and NPTII proteins are expected to be expressed at very low levels in
the pollen of the Roundup Ready® Flex/Bollgard II® cottons, based on expression of the
Cry1Ac protein in INGARD® cotton pollen and the Cry2Ab protein in Bollgard II® cotton
pollen (relative to that in other plant tissues) and the similarity of the promoter elements
controlling the expression of the introduced genes (see Appendix 1). The CP4 EPSPS protein
may be expressed at higher levels in pollen of Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons compared with expression in Roundup Ready® cotton. However,
this protein has no known toxicity or allergenicity (see Section 2.4).
145. The primary processing of cotton at cotton gins, and the bulk handling of cottonseed
and cotton fibre, can create and stir up fine dust and lint particles. Use of personal protective
equipment by exposed workers is commonplace in such facilities, to prevent respiratory
irritations. Fibre characteristics (length, strength, fineness) of Roundup Ready® cotton are
the same as for non-GM cotton varieties (Cotton Seed Distributors 2002). The fibre
characteristics of Roundup Ready® Flex cottons are equivalent to non-GM cotton (data
supplied by the applicant). The cotton lint derived from the Roundup Ready® Flex cottons is
no more likely to induce adverse responses in workers than is conventional cotton. However,
the applicant proposes to continue to assess the fibre characteristics of the Roundup Ready®
Flex cottons as part of the proposed release.
Appendix 2 Toxicity and allergenicity to humans 30
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Section 2.3 Other sources of CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and NPTII in the
environment
146. CP4 EPSPS, Cry1Ac, GUS and NPTII proteins, and the genes encoding them, are
widespread in the environment, through the presence of the bacteria from which they are
derived. The same or similar proteins can be found in or on fresh food.
147. EPSPS enzymes are present in all plants, bacteria and fungi. The difference between
the natural plant enzymes and the bacterially derived CP4 EPSPS is in the amino acid
sequence, not in the physiological function of the expressed protein (see Appendix 1 for
details). Other EPSPS enzymes present in food, from both plant and microbial sources, also
vary to a similar degree in amino acid sequence (Felsot 2000a; Padgette et al. 1996). The
CP4 EPSPS enzyme in Roundup Ready® Flex is derived from the common soil bacterium
Agrobacterium species strain CP4 (Padgette et al. 1996), which can also be found on plants
and plant produce.
148. The presence of Bt toxins in food has increased over the past 30 years due to the
commercial use of Btk (B. thuringiensis var kurstaki) microbial sprays to protect food crops,
including organic crops, from insect attack. Residues of Btk proteins, including Cry1Ac and
Cry2Aa proteins (to which the Cry2Ab protein is closely related (Dankocsik et al. 1990;
Widner & Whiteley 1989), are present on a wide variety of foods, including fresh foods such
as lettuce and tomato, with no reported toxic or allergic responses (ANZFA 1999).
149. The cry1Ac gene present in Roundup Ready® Flex/Bollgard II® cotton plants is similar
to the cry1Ac gene originally sequenced by Adang et al (Adang et al. 1985) from
B. thuringiensis var kurstaki. It encodes a protein that is 99.4% identical to that produced by
the B. thuringiensis var kurstaki bacterium. The cry2Ab gene is not naturally expressed in
soil bacteria or Btk sprays due to an ineffective promoter sequence. The cry2Ab gene present
in the Roundup Ready® Flex/Bollgard II® cotton is 88% identical at the sequence level to the
Cry2Aa protein (Dankocsik et al. 1990; Widner & Whiteley 1989). The Cry2Aa protein is
naturally expressed in B. thuringiensis var kurstaki and present in Btk sprays (information
supplied by the applicant for DIR 012/2002). Related Cry proteins are also produced by
other varieties of B. thuringiensis. Bt spores and their toxic crystal (Cry) proteins are found
widely in soils, on plant leaves and in grain stores (Meadows 1993).
150. The uidA gene which produces the GUS protein is derived from the common gut
bacterium Escherichia coli and is therefore already present in the gut of many animals,
including humans. GUS enzyme activity has been detected in numerous microbial, plant and
animal species, including species used as raw food (Gilissen et al. 1998). The GUS protein
used in GM plants is 99.8 % identical to the E. coli GUS protein.
151. Humans continually ingest kanamycin-resistant microorganisms, some containing the
NPTII enzyme. The diet, especially raw salad, is the major source: estimated conservatively,
each human ingests 1.2 x 106 kanamycin-resistant microorganisms daily (Flavell et al. 1992).
Large numbers of kanamycin- or neomycin-resistant bacteria already inhabit the human
digestive system (Levy et al. 1998), with Flavell et al. (1992) estimating about 1012 per
person.
Section 2.4 Toxicity and allergenicity of the introduced proteins
152. There will be no opportunity for people to consume food products (oil and linters) of
the GM Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons, nor will
cottonseed or its by-products be fed to cattle. However, it is worth noting that the introduced
proteins present in the Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons
are the same as those present in commercially released Roundup Ready and Bollgard II®
Appendix 2 Toxicity and allergenicity to humans 31
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
cottons. Studies using the purified forms of the introduced proteins (CP4 EPSPS, Cry1Ac,
Cry2Ab, NPT II and GUS) present in Roundup Ready and Bollgard II® cottons have been
conducted. Detailed descriptions of the results of these studies are available in the risk
assessment and risk management plans for DIR 012/2002, DIR 022/2002 and DIR 023/2002.
The key results are summarised here.
2.4.1 Toxicity
The CP4 EPSPS protein
153. Purified CP4 EPSPS protein, at acute doses of up to 572 mg/kg body weight, produced
no adverse effects in mice (Harrison et al. 1996a). This is more than a thousand times the
anticipated potential consumption of CP4 EPSPS in commercial food derived from all GM
food crops expressing this enzyme under development by Monsanto at that time (soybean,
potato, tomato, corn) (Harrison et al. 1996a). FSANZ has previously concluded that food
products derived from Roundup Ready cotton expressing the same CP4 EPSPS protein are
as safe as those derived from conventional cotton varieties (ANZFA 2000). Likewise, the US
EPA has issued an exemption from residue tolerance requirements for the CP4 EPSPS
protein, and Roundup Ready cotton has been approved for use in food in a number of other
countries (see Chapter 1, Section 2.4).
The Cry1Ac and Cry2Ab proteins
154. Purified Cry1Ac protein, at acute doses of up to 4300 mg/kg body weight, produced no
adverse effects in mice (Naylor 1993b; Naylor 1993a). Likewise, acute oral toxicity studies
in mice with purified Cry2Ab protein at doses of up to 1450 mg/kg have not shown any
adverse effects (Bechtel 1999, Monsanto unpublished).
155. Multiple studies on the acute oral toxicity of Bt microbial preparations, containing
Cry1Ac and Cry2Aa (to which Cry2Ab is 88% identical), in mammals such as rats and
rabbits have revealed no adverse effects at very high doses (Carter & Ligget 1994;
McClintock et al. 1995; Barbera 1995; Spencer et al. 1996). Two separate studies on humans
found no observable health effect of an oral dose of 1000 mg of Bt microbial spores per day
for 3 or 5 days (McClintock et al. 1995; Betz et al. 2000).
156. A study that investigated the effects of the Cry1Ab protein (86% identical to the
Cry1Ac protein) on a bovine hepatocyte culture concluded that there were no significant
changes to cell morphology or the secretion of albumin or the enzyme lactate dehydrogenase.
These results indicate that Cry1Ab has little acute toxicity on mammalian cells even when
applied directly (Shimada et al. 2003).
157. The US Environment Protection Agency (EPA) considers Cry1Ac protein to be non-
toxic to mammals and has established an exemption from residue tolerance requirements
(EPA 2000). In Australia, the APVMA has also determined that a maximum residue limit
(MRL) for human food and animal feed is not necessary for Bt toxins, indicating that they are
of no toxicological significance (see The MRL Standard, Table 5 at:
www.apvma.gov.au/residues/mrl_standard.shtml). Similarly, FSANZ has concluded that oil
and linters derived from Bollgard II® cotton are as safe as those derived from conventional
cotton varieties (ANZFA 2002a). In this assessment, FSANZ reviewed studies provided by
the applicant, which described the effects of acute toxicity studies performed with Cry2Ab on
mice. No toxicity was found.
Appendix 2 Toxicity and allergenicity to humans 32
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
The GUS protein
158. The function of the GUS protein is described in detail in Appendix 1. Enzymes
performing the same function as the GUS protein are present in many bacteria, invertebrates
and vertebrates. Therefore, humans are already exposed to functionally equivalent proteins in
the natural environment without any evidence of toxicity. In addition, the GUS protein
present in the GM cottons is structurally identical to the GUS protein expressed by the
common bacterium E. coli.
159. Acute oral toxicity studies in mice with purified GUS protein at doses of up to
100 mg/kg did not show any adverse effects (Naylor 1992). Studies feeding humans and
animals with 1010 GUS-containing E. coli bacteria per ingestion also did not show any toxic
or pathogenic reactions (Gilissen et al. 1998).
160. FSANZ has concluded that food derived from glyphosate-tolerant sugarbeet and from
Bollgard II® cotton, both expressing the GUS protein, is safe for human consumption
(ANZFA 2001e; ANZFA 2002c). The US EPA does not consider GUS to be toxic for
mammals and has approved its exemption from the requirement to establish tolerance levels
(EPA 2001b).
161. The GUS protein from E. coli is rapidly (<15 seconds) degraded in simulated gastric
fluid and loses its activity by heating/cooking (Fuchs & Astwood 1996).
162. When the sequence of the GUS protein expressed in the GM cottons was compared to
all protein sequences in publicly available databases, it only shared sequence similarities to
homologous E. coli and other glucuronidase proteins, as expected (information provided by
the applicant). These proteins have not been described as toxic to humans. Metabolites of
E. coli GUS activity are non-toxic (Gilissen et al. 1998).
The NPTII protein
163. The NPTII protein is widespread in the environment and in food chains, in naturally
occurring kanamycin-resistant microorganisms that are found in soil and in mammalian
digestive systems (Flavell et al. 1992), without any suggested toxicity for humans. Proteins
conferring resistance to kanamycin are naturally present in human foods, particularly raw
vegetables (Flavell et al. 1992).
164. NPTII was the most commonly used marker gene recorded in the US field trial
database for 2001 and 2002 (Miki & McHugh 2004). The insertion of the nptII gene into a
wide range of GMOs has not resulted in any adverse effects (Flavell et al. 1992). The nptII
gene was introduced into mammalian cell lines with no effects on viability or growth. During
gene therapy experiments, mammalian cells expressing the NPTII protein have been infused
into cancer patients. Again, no adverse effects have been observed (Flavell et al. 1992).
165. The NPTII protein produced in GM tomatoes has been fed to rodents and reported to be
rapidly inactivated and degraded (Calgene 1990). An acute oral toxicity study in mice, in
which the purified NPTII protein was fed at doses of up to 5 000 mg/kg of body weight (2
500 mg/kg administered twice, four hours apart), did not show any adverse effects (Berberich
et al. 1993, Monsanto unpublished). Another similar study in mice also reported no adverse
effects of NPTII at 5 000 mg/kg of body weight (Fuchs et al. 1993c).
166. The US FDA has concluded that NPTII does not possess any properties that distinguish
it toxicologically from other phosphorylating enzymes in the food supply, which are present
in all plants, animals, invertebrates and microorganisms. NPTII is approved as an additive in
food for human consumption in the US (FDA 1994). The US EPA has also established an
Appendix 2 Toxicity and allergenicity to humans 33
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
exemption for NPTII from the requirement for a residue tolerance limit when used as a ―plant
pesticide inert ingredient‖ (EPA 1994).
167. FSANZ has concluded that food derived from GM corn and GM Bollgard II® cotton,
which both express the NPTII protein, is safe for human consumption (ANZFA 2002b;
FSANZ 2003). Regulatory agencies in other countries have also approved the use of the nptII
gene as a selectable marker in food crops and approved the use of the gene and its encoded
protein in human food and animal feed.
168. Currently kanamycin is not in human clinical use, while neomycin has limited and very
specific clinical uses (TGA, MIMS Online database). Even if plant material containing the
NPTII protein were ingested, this enzyme is not likely to be active outside of living cells, as it
requires specific chemical conditions for activity, including the availability of specific co-
factors (essential for the normal catalytic activity of an enzyme). Neomycin is registered
with the APVMA for limited veterinary uses, however resistance to neomycin and kanamycin
are widespread and other antibiotics are preferred (Flavell et al. 1992).
169. Protein and DNA sequence comparisons using sequences from four separate databases
(Genbank, EMBL, PIR29, Swiss-Prot) indicated that NPTII does not have significant
homology to any proteins listed as food toxins in these databases (FDA 1994).
2.4.2 Allergenicity
170. Although there are no predictive assays available to assess the allergenic potential of
proteins, much is known about the biochemical events associated with allergic reactions, as
well as the kinds of proteins that cause problems (Metcalfe et al. 1996; Taylor & Lehrer
1996).
171. Sequence, structural and biochemical comparisons with known allergens have been
used to predict the allergenicity of uncharacterised proteins (Flavell et al. 1992; Fuchs &
Astwood 1996; Astwood et al. 1996; Kimber et al. 1999; Metcalfe et al. 1996; Taylor &
Lehrer 1996).
172. Some general characteristics of allergens are:
molecular weight ranges between 15-70 kDa;
typically glycosylated;
stable in the mammalian digestive system;
stable during high temperatures involved in cooking or processing; and
present as the major protein component in the specific foods.
173. None of the introduced proteins in Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cotton are derived from known allergens. Nevertheless, no material
produced from the GM cottons in the proposed field trial will be used in human food or
animal feed. The UK Royal Society (The Royal Society, 2002) have concluded that there is
at present no evidence that available GM foods cause allergic reactions, and that the risks
posed by GM plants are in principle no greater than those posed by conventional breeding or
by plants introduced from other areas of the world.
The CP4 EPSPS protein
174. The CP4 EPSPS protein is 47.6 kDa, which is in the typical range for allergenic
proteins. However, it is rapidly denatured by heat, enzymatic digestion and acid in simulated
mammalian gastric fluid (Harrison et al. 1996a; Canadian Food Inspection Agency 1997;
Appendix 2 Toxicity and allergenicity to humans 34
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
ANZFA 2001f). The protein shows no significant sequence homology to allergens
assembled in the Genpept, Pir and SwissProt protein databases (Mitsky 1993, Monsanto
unpublished).
The Cry1Ac and Cry2Ab proteins
175. The Cry1Ac protein is approximately 133 kDa in size, which is significantly larger than
typical allergenic proteins. In addition, it is heat labile and rapidly degraded, (under 30
seconds) under simulated mammalian gastrointestinal conditions (Fuchs et al. 1993a). The
Cry2Ab protein is approximately 71 kDa in size, which is at the upper end of the typical size
range for allergenic proteins. Neither the Cry1Ac nor Cry2Ab protein displays characteristics
common to known food allergen proteins. Searches of allergen sequence databases have
shown no significant matches of the Cry1Ac or Cry2Ab proteins to known allergens
(Metcalfe et al. 1996).
176. While there have been reports in the US claiming allergic reactions to Bt microbial
products in topical insecticidal sprays, these are not due to the Cry1Ac or Cry2Aa proteins
present in the Bt sprays. A survey conducted among farm workers who picked vegetables
treated with Bt microbial products indicated that exposure to Bt products may lead to allergic
skin sensitisation, however there was no clinical allergic disease in any of the workers. Most
reactions in these workers were shown to be due to other constituents of the Bt sprays, and
there was no evidence of antibodies specific to the Cry proteins of the Bt sprays (Bernstein et
al. 1999). The US EPA have also determined that reports of reactions to Bt microbial
products have been due to non-Cry proteins produced during fermentation or to other
ingredients added to the insecticidal formulations (EPA 2001a).
The GUS protein
177. The GUS protein is approximately 68 kDa in size, which is within the typical size range
of allergenic proteins. However the widespread occurrence of GUS and the constant
exposure of humans to the protein without ill effect indicates that the likelihood that GUS
being an allergen is extremely low (Gilissen et al. 1998). In addition, the GUS protein does
not possess glycosylation sites and is rapidly denatured in the simulated mammalian digestive
system (Fuchs & Astwood 1996; ANZFA 2002b).
178. Protein sequence comparisons using sequences from four separate databases (EMBL,
Genbank, PIR29 and Swiss-Prot protein databases) indicated that GUS does not have
significant sequence identity to any known protein food allergens or toxins.
The NPTII protein
179. The NPTII protein is approximately 29 kDa in size, which is within the typical size
range of allergenic proteins. However, it does not possess glycosylation sites, is not stable in
the mammalian digestive system and is heat labile, decreasing the probability that it is
allergenic (ANZFA 2001f; FDA 1994; FDA 1998; Fuchs et al. 1993b). Fuchs et al. (1993)
reported that no NPTII was detected 10 seconds after addition of simulated gastric fluid as
measured by both Western blot and enzymatic activity.
180. Protein sequence comparisons using sequences from four separate databases (EMBL,
Genbank, PIR29 and Swiss-Prot protein databases) indicated that NPTII does not have
significant sequence identity to any known protein food allergens or toxins (Fuchs &
Astwood 1996).
Appendix 2 Toxicity and allergenicity to humans 35
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
181. Other regulatory agencies, in Australia and in other countries, have previously assessed
the use of the nptII gene in food crops (e.g. (ANZFA 2001a; ANZFA 2001b; ANZFA 2001c;
ANZFA 2001d; FSANZ 2003). The FDA has evaluated data submitted for deliberate
releases of GMOs expressing the NPTII protein and concluded that NPTII does not have any
of the characteristics associated with allergenic proteins (FDA 1998).
182. A number of genetically modified food crops containing the nptII gene have been
approved for commercial release both in Australia (DIRs 012/2002, 021/2002 and 022/2002)
and overseas. No adverse effects on humans, animals or the environment have been reported
from these releases (FDA 1998; Flavell et al. 1992; EFB 2001).
SECTION 3 CONCLUSIONS REGARDING TOXICITY OR ALLERGENICITY
183. It is considered that the risk of Roundup Ready® Flex or Roundup Ready® Flex/
Bollgard II® cotton being toxic or allergenic for humans is very low because:
cotton lint used in clothing and household items contains no proteins or DNA and
cotton products from GM cottons cannot be distinguished from non-GM
products;
cotton pollen is not wind-dispersed and therefore unlikely to be an air-borne
allergen;
none of the GM cotton materials from the release will be used in human food or
animal feed;
exposure to the introduced proteins through working with cotton plants is very
low;
humans are commonly exposed to the CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and
NPTII proteins or very similar proteins, as these are naturally widespread in the
environment;
toxicity studies indicate that the introduced proteins are not toxic;
evidence indicates that the introduced proteins are not allergenic, nor do they
have properties of known allergenic proteins;
there have been no reported toxic or allergic effects from similar GM cottons
expressing the same proteins that have been extensively field trialled and are
commercially released in Australia; and
although dust and lint from cotton can be created at processing facilities, the use
of protective equipment prevents respiratory irritation, and fibre characteristics of
the GM cottons are likely to be the same as for non-GM cotton.
184. FSANZ approval will be required before any of the GM materials could be used in food
in Australia. Therefore the imposed licence conditions prohibit the use of any GM materials
in food.
185. The licence requires the licence holder to report any adverse effects on human health
and safety (for example allergic reactions as a result of occupational exposure to the cotton)
or to the environment.
Appendix 2 Toxicity and allergenicity to humans 36
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 3 TOXICITY TO NON-TARGET ORGANISMS
186. Under Section 51 of the Act, the Regulator is required to consider risks to human health
and safety and the environment in preparing the risk assessment and risk management plan
(RARMP). This Appendix considers potential hazards that may be posed through any
toxicity of the GMO or its introduced proteins to non-target organisms.
187. It should be noted that since the commercial release of similar GM cottons,
Roundup Ready® and Roundup Ready®/Bollgard II® cotton, expressing the same introduced
proteins as Roundup Ready® Flex cotton MON 88913 (Roundup Ready® Flex cotton) and
Roundup Ready® Flex/Bollgard II® cotton, there have been no adverse effects on non-target
organisms reported. The Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II®
cottons proposed for release in this licence application have previously been assessed during
evaluation of the application for DIR 035/2003. Expression levels of the herbicide resistance
gene (cp4 epsps) in Roundup Ready® Flex cotton have been provided with this application
(see Appendix 1).
SECTION 1 NATURE OF THE POTENTIAL TOXICITY HAZARD
188. Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons differ from
conventional cotton in the expression of one and five additional proteins, respectively. These
are the CP4 EPSPS protein (in both GM cottons) and the Cry1Ac, Cry2Ab, GUS and NPTII
proteins (in Roundup Ready® Flex/Bollgard II® cotton only). The potential for these cottons
to be toxic to organisms, other than the target pests of Roundup Ready® Flex/Bollgard II®
cotton (lepidopteran caterpillars), is considered here. Any adverse effects could result either
from expression of the introduced gene products or because of unintended effects of the
genetic modifications.
189. If Roundup Ready® Flex or Roundup Ready® Flex/Bollgard II® cotton is toxic for non-
target organisms, the potential hazards could include adverse impacts on:
wildlife, including mammals, fish and birds;
invertebrates, including beneficial insects (pollinators, parasitoids or predators of
insect pests); and
microbial organisms, particularly soil microorganisms.
190. Toxicity for lepidopteran insects may also present indirect hazards, with potential to
harm the natural environment (for example, adverse impacts on native biodiversity) through:
effects on populations of specialist parasitoids and predators that feed on
lepidopteran insects affected by the Bt toxin; and
effects on populations of other organisms that interact with lepidopterans affected
by the Bt toxins.
191. The use of Roundup Ready® herbicide (containing glyphosate as the active ingredient)
on GM cotton crops in Australia is registered by the APVMA. As part of their assessment of
this use, the APVMA considers any potential health and environmental effects, such as
toxicity to other organisms and herbicide residues on crops. Thus, risks associated with the
use of glyphosate are not considered in the risk assessment of these GM cottons. It should be
noted, however, that the cultivation of Roundup Ready® cotton in Australia has led to a shift
in herbicide usage (away from residual or environmentally persistent herbicides) rather than
increased use (Australian Cotton Cooperative Research Centre 2002b; data supplied by the
applicant). Glyphosate-based herbicides are thought to pose a lower risk to the environment
than other commonly used herbicides (Crossan & Kennedy 2004).
Appendix 3 Toxicity to non-target organisms 37
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 2 LIKELIHOOD OF THE TOXICITY HAZARD OCCURRING
192. In assessing the likelihood of adverse impacts due to toxicity of Roundup Ready® Flex
and Roundup Ready® Flex/Bollgard II® cottons, a number of factors were considered
including:
the inherent toxicity of conventionally bred cotton;
the potential exposure to the introduced proteins from other sources in the
environment;
information about the likely routes of exposure to Roundup Ready® Flex and
Roundup Ready® Flex/Bollgard II® cottons and to the introduced proteins, for
example through direct contact with the crop or through contact with soil in which
the crop is grown; and
the potential toxicity of the introduced proteins expressed in the cotton for
particular types of organisms, including mammals, fish and birds, non-target
invertebrates and soil microorganisms.
193. Potential non-target effects of the insecticidal proteins expressed in Roundup Ready®
Flex/Bollgard II® cotton have been considered in detail in the risk assessment and risk
management plans for Bollgard II® cotton (DIR 012/2002) and INGARD® cotton
(DIR 022/2002), available at www.ogtr.gov.au, and are only presented here in summary.
Section 2.1 Toxicity of conventionally bred cotton
194. Cotton is a well established field crop with a long history of safe use. A comprehensive
review of conventional cotton, including information on its toxicity and allergenicity, is
provided in the document ‗The Biology and Ecology of Cotton (Gossypium hirsutum) in
Australia‘ (OGTR 2002) that was produced in order to inform the risk assessment processes
for licence applications involving GM cotton. This document can be accessed at
www.ogtr.gov.au. Information on non-GM cotton is included here to establish a baseline for
comparison with the GM cottons being considered in this risk assessment.
195. Cotton tissue, particularly the seeds, can be toxic if ingested in large quantities because
of the presence of toxic and anti-nutritional factors including gossypol and cyclopropenoid
fatty acids (e.g. dihydrosterculic, sterculic and malvalic acids).
196. Mammals generally avoid feeding on cotton plants due to both the gossypol content and
the morphology of the plant. The presence of gossypol and cyclopropenoid fatty acids in
cottonseed limits the use of whole cottonseed as a protein supplement in animal feed, except
for cattle which are less affected by these components. Inactivation or removal of these
components during processing enables the use of some cottonseed meal for farmed fish,
poultry and swine. The meal and hulls of cottonseed can also be used for cattle feed. Its use
as stockfeed is limited, nonetheless, to a relatively small proportion of the diet and it must be
introduced gradually, to avoid potential toxic effects.
197. Best Management Practices developed for the Australian cotton industry prohibit the
use of cotton trash and stubble as a feed for animals, due to residues of pesticides that could
be found in the cotton trash and stubble.
Section 2.2 Other sources of CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and NPTII in the
environment
198. As discussed in Appendix 2, the CP4 EPSPS, Cry1Ac, Cry2Ab, GUS and NPTII
proteins are widespread in the environment. The genes for these proteins have been derived
Appendix 3 Toxicity to non-target organisms 38
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
from common bacteria found in the soil (Agrobacterium species and Bacillus thuringiensis
[Bt]) or mammalian gut (Escherichia coli), and are hence already natural components of the
environment.
199. EPSPS enzymes are present in all plants, bacteria and fungi. The difference between
the natural plant enzymes and the bacterially derived CP4 EPSPS is in the amino acid
sequence, not in the biochemical function. Other EPSPS enzymes from both plant and
microbial sources also vary to a similar degree in amino acid sequence (Felsot 2000a;
Padgette et al. 1996). The CP4 EPSPS enzyme is derived from the common soil bacterium
Agrobacterium species strain CP4 (Barry et al. 1992; Padgette et al. 1996), which is found in
soil and on plants.
200. The native Cry1Ac is naturally produced by the bacterium Bt variety kurstaki (Btk).
Related Cry toxins are also produced by other varieties of Bt. Spores from a range of Bt
varieties and their crystal toxins are found widely in both the agricultural and natural
environment, including in soil, on plant leaves, in grain stores and in dead insects (Meadows
1993).
201. The Cry2Ab protein is not naturally expressed in soil bacteria or Btk sprays due to an
ineffective promoter sequence of the cry2Ab gene. The cry2Ab gene present in the Roundup
Ready® Flex/Bollgard II® cotton is 88% identical at the sequence level to the Cry2Aa protein
(Dankocsik et al. 1990; Widner & Whiteley 1989). The Cry2Aa protein is naturally
expressed in B. thuringiensis var kurstaki and present in Btk sprays (information supplied by
the applicant for DIR 012/2002). Related Cry proteins are also produced by other varieties of
B. thuringiensis. Bt spores and their toxic crystal (Cry) proteins are found widely in soils, on
plant leaves and in grain stores (Meadows 1993). However, as discussed in Section 2.4.2 of
this Appendix, the toxicity of the Cry2Ab protein expressed in GM cottons has been widely
studied. The presence of Bt toxins in agricultural situations has increased over the past 30
years due to the commercial use of Btk microbial sprays to protect food crops, including
organic crops, from insect attack (ANZFA 1999).
202. The GUS and NPTII proteins are widespread in the environment since they are naturally
produced by the common gut bacterium Escherichia coli. E. coli is widespread in human and
animal digestive systems as well as in the environment.
203. E. coli lives in the digestive tract of vertebrates (Jefferson et al. 1986), and utilises the
GUS enzyme in its carbohydrate and energy metabolism. GUS activity, serving the same
function, is also found in a wide range of other bacteria, including other microorganisms of
the digestive tract and many soil bacteria (Gilissen et al. 1998).
204. GUS activity is very common in almost all tissues of vertebrates. It plays a major role
in the degradation of glycosamino-glucuronides and in the release of active hormones from
steroid hormone-glucuronides. Tissues with high GUS activity include the preputial gland,
kidney, liver and spleen (Gilissen et al. 1998).
205. GUS activity is also present in invertebrates such as molluscs, nematodes and insects
(Gilissen et al. 1998). GUS activity has been detected in over 50 different plant species (Hu
et al. 1990). However, when endogenous GUS activity is found in plants, the activity is very
low and its function is unknown (Gilissen et al. 1998).
206. In summary, GUS enzyme activity has been detected in numerous microbial, plant and
animal species (Gilissen et al. 1998) and can be considered widespread in the environment.
Appendix 3 Toxicity to non-target organisms 39
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
207. Humans (and, by implication, other animals) continually ingest kanamycin-resistant
microorganisms, some containing the NPTII protein (Flavell et al. 1992). Kanamycin-
resistant bacteria have been isolated from soil, river water and sewage (Smalla et al. 1993).
Section 2.3 Potential toxicity hazards for stock and wildlife, including mammals, birds
and fish
2.3.1 Exposure of stock and wildlife to Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons
208. None of the cotton plants from the release, nor any of their by-products, will be used as
stockfeed. As mentioned in Section 2.1 of this Appendix, most mammals avoid feeding on
cotton, GM or non-GM, due to the presence of toxic, anti-nutritional substances and the
morphology of the plant (OGTR 2002). In the field, seed cotton is present as large
lint-covered bolls that are unattractive to avian species (OGTR 2002), so birds are not likely
to be exposed to the introduced proteins in the seeds of Roundup Ready® Flex and Roundup
Ready® Flex/Bollgard II® cottons.
209. Cottonseed and pollen from the release are not expected to enter aquatic habitats in any
significant quantities (OGTR 2002); therefore the level of exposure of aquatic organisms to
the GM cottons will be low. Irrigation practices (Good Management Practice of cotton
industry) used by cotton growers in Australia retain irrigation water run-off, as well as the
first 15 mm of storm water run-off, on-farm to minimise the entry of pesticide residues into
natural waterways. In addition, licence conditions imposed require that GM cotton plantings
be separated from natural waterways by at least 50 metres.
2.3.2 Toxicity of Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II®
cottons to stock and wildlife
210. As noted above, none of the cotton plants from the proposed release, nor any of their
by-products, are permitted to be used as stockfeed. However, it is worth noting that the
introduced proteins present in the Roundup Ready® Flex cottons are the same as those present
in commercially released Roundup Ready® and Bollgard II® cottons. Studies using the
purified forms of the introduced proteins present in Roundup Ready® and Bollgard II® cotton
have been conducted to determine the toxicity of the proteins to animals. Detailed
descriptions of the results of these studies are available in the risk assessment and risk
management plans for DIR 012/2002, DIR 022/2002 and DIR 023/2002. The key results are
presented in Appendix 2, Section 2.4 as well as summarised here.
211. Acute oral toxicity studies in mice with each of the CP4 EPSPS, Cry1Ac, Cry2Ab, GUS
and NPTII proteins have not demonstrated any adverse effects (Naylor 1993b; Naylor 1992;
Bechtel 1999, Monsanto unpublished; Harrison et al. 1996b; Fuchs et al. 1993c) see also,
references in Appendix 2). In trials with rats, quail or catfish fed cottonseed meal at 5 to 20%
of their diets, no significant differences were found in weight gain and feed conversion, nor
during gross autopsy, for animals fed Roundup Ready® cottonseed meal (containing the CP4
EPSPS and NPTII proteins) compared to those fed non-GM cottonseed meal (Canadian Food
Inspection Agency 1997).
212. In addition, compositional data of cottonseed derived from Roundup Ready® Flex
cotton was provided in the current application. The assays were performed on samples
collected from five different US states. Gossypol levels in the GM cottonseed, compared to
those in the non-GM cottonseed (derived from the same genetic background), were not
significantly different in any of these samples. Overall, the cyclopropenoid fatty acids
(dihydrosterculic, sterculic and malvalic acids) levels were not significantly different between
the GM and non-GM cottonseed samples either. However, in one case (samples from
Appendix 3 Toxicity to non-target organisms 40
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
California), a small but significant decrease was measured in the malvalic and sterculic acid
levels in the GM versus the non-GM samples. All differences detected were in the range of
expected values for non-GM cottonseed and the GM cottonseed was therefore considered to
be compositionally equivalent to non-GM cottonseed. .
Section 2.4 Potential toxicity hazard for invertebrates
2.4.1 Exposure of invertebrates to Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons
213. Non-target invertebrates may be directly exposed to Roundup Ready® Flex and
Roundup Ready® Flex/Bollgard II® cottons and to the introduced proteins, through feeding on
the plants. Exposure in the soil may occur either when cotton tissues break down following
incorporation into the soil or as a result of exudation of the introduced proteins through the
roots. Exposure may also occur indirectly, through eating other organisms, including the
lepidopteran target pests of Roundup Ready® Flex/Bollgard II® cotton, which have previously
fed on the plants.
214. Relative exposure will be greatest for herbivorous species feeding on the cotton plants.
Sap feeders, such as aphids, will have minimal exposure to the introduced proteins, as sap is
primarily composed of sugars and mineral salts dissolved in water. However, species feeding
on lepidopteran larvae may be exposed to both the full-length Cry1Ac and Cry2Ab proteins
and the activated core toxins, since their lepidopteran prey may have ingested GM cotton
tissues and metabolised the full-length protein, leaving the toxic core element ‗free‘ in the
insect‘s gut. The feeding behaviour of predators and parasitoids of lepidopterans is therefore
likely to affect their potential exposure to the toxin. The exposure of soil invertebrates will
depend mainly upon the amounts of the introduced proteins that are exuded into the soil and
the levels of the proteins present in the plants upon incorporation into the soil at the end of the
growing season. This is discussed further in Section 2.5.1 in relation to exposure of soil
microorganisms.
215. Pollinator species and various insects that feed on pollen will have lower exposure to
the introduced Cry1Ac, Cry2Ab, GUS and NPTII proteins, because of the expected much
lower expression in pollen, relative to that in other plant tissues. This expectation is based on
the known lower expression of the Cry1Ac, Cry2Ab and CP4 EPSPS proteins in INGARD®,
Bollgard II® and Roundup Ready® Flex cotton pollen, respectively, relative to other plant
tissues, and the fact that the expression of all four proteins is controlled by very similar
promoters. (see Appendix 1) (data supplied by the applicant).
216. Non-target lepidopteran species may be exposed to the GM cotton and may be affected
by the Bt toxins. However, cotton is not the preferred food source for non-target lepidopteran
species, and their populations would be maintained on other types of plants found around field
trial locations. A small number of insectivorous lepidopteran species occur in Australia.
Their feeding habits are highly specialised, with the larvae feeding on either sap-sucking
insects (Homoptera) or meat ants (Iridomyrmex purpureus) (Common 1990). In either case,
exposure to the introduced proteins in the GM cottons will be negligible as sap does not
contain protein (Raps et al. 2001) and ants are not considered pests of cotton (Forrester &
Wilson 1988).
2.4.2 Toxicity of Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II®
cottons for invertebrates
217. The direct effects of the CP4 EPSPS, GUS and NPTII proteins have not been tested on
invertebrates. However, EPSPS enzymes are naturally present in all plants, bacteria, algae
and fungi; GUS enzymes are present in bacteria, invertebrates and animals; and NPTII is a
Appendix 3 Toxicity to non-target organisms 41
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
phosphorylating enzyme, which does not possess any properties that distinguish it
toxicologically from other phosphorylating enzymes present in microorganisms, plants and
animals (FDA 1994). In addition, all three proteins are already widespread in the
environment since they are derived from, and naturally produced by, common bacteria. Thus,
the expression of these bacterial enzymes in plants is not likely to have any novel toxic effects
on invertebrates.
218. Most interest in the toxicity of GM cottons for invertebrates has concentrated on the
insecticidal Cry1Ac and Cry2Ab proteins, which are expressed in Roundup Ready®
Flex/Bollgard II® cotton. Potential non-target effects of the Cry1Ac and Cry2Ab proteins
have been considered in detail in the risk assessment for the commercial release of
Bollgard II® cotton (DIR 012/2002) and in the risk assessment for DIR 022/2002 for the
continued commercial release of INGARD® cotton (expressing only the Cry1Ac protein).
These are available at www.ogtr.gov.au. A summary is presented below along with more
recent data from the literature.
Studies conducted under controlled conditions
219. The toxicity of the Cry1A class of Bt toxins, to which Cry1Ac belongs, has been tested
against approximately 45 insect species representing four orders (van Frankenhuyzen &
Nystrom 2002). Bt toxins of this class are toxic to lepidopteran and in some cases, dipteran
and neuropteran insects. The Cry1Ac protein is active against lepidopteran insects but has
also shown toxicity against at least one dipteran insect (adult Tsetse fly) (van Frankenhuyzen
& Nystrom 2002).
220. Another series of studies examined the effects of purified active core Btk Cry1Ac toxin
on 18 agronomically important invertebrate species, representing six orders (Macintosh et al.
1990). Seven insects, all from the Lepidoptera order, were susceptible to the toxin. None of
the 11 non-lepidopteran species were susceptible (this group included one dipteran insect, a
mosquito).
221. Similarly, other studies with the Cry1Ac protein expressed in INGARD® cotton (Sims
1995; Sims 1994) found that of 14 species tested (representing seven orders), only
lepidopteran species were susceptible to Cry1Ac.
222. Detailed studies of the effect of Cry1Ac on specific beneficial non-target insects have
also been carried out, including:
honey bees (both larvae and adults) (Apis mellifera, Hymenoptera), beneficial
insect pollinators (Maggi 1993a; Maggi 1993b);
a beneficial parasitoid wasp (Nasonia vitripennis, Hymenoptera), which targets
the housefly (Musca domestica) (Palmer & Beavers 1993c; Sims 1994);
ladybird beetles (Hippodamia convergens, Colleoptera), beneficial predatory
insects which feed on aphids and other plant pests commonly found on stems and
foliage of weeds and cultivated plants (Palmer & Beavers 1993b; Sims 1994);
green lacewing larvae (Chrysopa earned, Neuroptera), beneficial predatory insect
commonly found on cotton and other cultivated crops (Palmer & Beavers 1993a;
Sims 1994); and
springtails (Folsomia candida and Xenylla grised, Collembola), scavenging soil
insects important in nutrient cycling (Sims & Martin 1996).
Appendix 3 Toxicity to non-target organisms 42
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
223. There were no adverse effects of Cry1Ac observed, even at concentrations well above
the expression levels found in INGARD® and Bollgard II® cotton, for any of the species
tested in these studies.
224. The toxicity of the Cry2A class of Bt toxins, to which Cry2Ab belongs, has been tested
against approximately 50 insect species representing ten orders (van Frankenhuyzen &
Nystrom 2002). Bt toxins of this class are mainly toxic to lepidopteran insects but in some
cases, dipteran insects can also be affected. The Cry2Ab toxin present in Bollgard II® cotton
is generally considered to have specific toxicity for lepidopterans (Dankocsik et al. 1990;
Widner & Whiteley 1990; Widner & Whiteley 1989). However, there is some evidence that
Cry2Ab is active against at least one dipteran insect, Anopheles gambiae (African malarial
mosquito) (Ahmad et al. 1989).
225. Additional data supplied by the applicant indicate that Cry2Ab is non-toxic for larval or
adult honey bees (Apis mellifera, Hymenoptera) (Maggi 2000a; Maggi 2000b) and that
Cry2Ab also has no significant impact on the mortality of green lacewing larvae
(Chrysoperla carnea, Neuroptera), a ladybird beetle (Hippodamia convergens, Colleoptera), a
parasitic wasp (Nasonia vitripennis, Hymenoptera), or an earthworm (Eisenia fetida,
Haplotaxida) (Palmer & Krueger 2000a; Palmer & Krueger 2000b; Palmer & Krueger 2000c;
Palmer & Krueger 2000d).
226. As questions were raised over the potential impact of Cry2Ab expressed in insecticidal
GM cottons on species of Diptera, the OGTR commissioned additional work by CSIRO
Entomology to investigate the toxicity of Bt cotton plants on a range of dipteran insects.
Three dipteran species were tested (Culex quinquefasciatus, mosquito; Musca domestica,
house-fly; and Chironomus tepperi, bloodworm) using a freeze-dried powder of cotton leaves
containing the Cry2Ab protein. The study was recently completed and found no evidence of
toxicity at exposure levels that were equivalent or higher than would occur in fresh GM cotton
leaves (unpublished CSIRO report).
Studies conducted in the field
227. The effects of INGARD® cotton (expressing the Cry1Ac protein) on non-target
arthropod populations were studied over two seasons in cotton fields near Dalby, Qld
(Addison 2001b; Addison 2001a). After classifying the samples to the level of order, the
results for both seasons suggested that, with the exception of Lepidoptera, the total
(cumulative) abundance of arthropods collected from INGARD® cotton fields was
comparable to their abundance in control (unsprayed conventional cotton) plots.
228. A series of large scale field experiments over three growing seasons in Australia
showed no significant difference in non-target invertebrate faunal diversity or abundance
between unsprayed INGARD® cotton and unsprayed conventional cotton, except for a
reduction in parasitoids of Helicoverpa in one season (Fitt & Wilson 2002). A reduction in
specialist parasitoids is expected due to reductions in the abundance of their host species (the
target pests of INGARD® cotton). This is unlikely to threaten their persistence in the
cropping system, since a significant proportion of the Helicoverpa population is always
present on other crops and in uncultivated areas. The overall abundance of invertebrate
species was higher in the unsprayed INGARD® cotton crop than in sprayed conventional
cotton crop.
229. A US study compared the impact on non-target insects of conventional cotton and
cotton varieties expressing Cry1Ac, relative to the impact of insecticides that may be used in
cotton plantations (Naranjo & Ellsworth 2002). The results indicated that the diversity of
arthropods and the abundance of natural enemies were unaffected by the Cry1Ac toxin
Appendix 3 Toxicity to non-target organisms 43
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
present in the cotton, but were affected by the use of insecticides (Naranjo & Ellsworth 2002).
The relative level of parasitism and predation of two key cotton pests was also unaffected by
the insecticidal toxin.
230. Another, more recent US study compared arthropod abundance (classified to the level
of family) and diversity in field plots of Bt, non-Bt and mixed plantings of Bt and non-Bt
cotton (Sisterson et al. 2004). The Bt cotton contained the same gene as Bollgard®/INGARD®
cotton. They found a decrease in the total arthropod diversity of the Bt versus the non-Bt
plots, which was mainly due to decreased numbers of families of the ‗chewing herbivore‘
group (‗sucking herbivores‘ were not affected). This decrease was also found in the mixed
planting plots, although overall numbers of arthropods were higher in these plots relative to
those in the Bt cotton plots. The authors also found that the locations of the sites and the
mean plant height in each of the plots affected the numbers of arthropods found. Plant height
was not linked to any treatment. They concluded that arthropod abundance and diversity
depended on many factors, including the presence of Bt toxins, which they felt was a minor
factor.
231. As indicated above, studies have found that overall numbers of non-target invertebrates
in INGARD® cotton fields are similar to those in unsprayed non-GM cotton fields and higher
than in conventionally sprayed non-GM cotton fields. Increased numbers of non-target
invertebrates are likely to occur as a direct result of reductions in chemical insecticide usage.
Similarly, the use of INGARD® cotton in China, with the concomitant reduction in insecticide
use, resulted in an average increase of 24% in the number of insect predators over
conventional cotton fields (Xia et al. 1999).
232. The potential for insecticidal Cry toxins expressed by GM plants to have an unexpected,
indirect impact on ecological communities in the natural environment, by affecting
interactions between organisms elsewhere in the ‗food web‘, has been considered. While
such indirect impacts have been demonstrated in several studies (see Groot & Dicke 2002),
the impacts relate to altered arthropod community structure in the agricultural field in which
the GM insecticidal plant was cultivated, and it is likely that affected predator species of
lepidopterans simply dispersed to nearby non-GM habitats where prey numbers were higher
(Groot & Dicke 2002). Nearby non-GM habitats will include the refuge crops required as
part of the Integrated Pest Management plan for Bollgard II® cotton.
Section 2.5 Potential toxicity hazard for microorganisms
2.5.1 Exposure of microorganisms to Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons
233. Microorganisms may be exposed to the GM cotton plants during growth or
decomposition of plant material. After harvest of lint and seed, the remaining cotton plant
residues are typically tilled into the soil, so that soil microorganisms are likely to be exposed
to the introduced proteins as the residues are broken down.
234. Exposure of organisms in soil to the introduced proteins may also occur as a result of
root exudations, as has been observed in Bt corn expressing Cry1Ab (Saxena et al. 1999;
Stotzky 2000b). A recent study (Gupta & Watson 2004) showed that roots of INGARD®
cotton express the Cry1Ac protein and release this protein into soil during growth, although
this was not quantified, nor was the mechanism clear. In contrast, Saxena et al (Saxena et al.
2004) have recently reported that no root exudations of Cry proteins were detectable from the
roots of Bt (INGARD®) cotton, canola or tobacco plants, while Cry proteins were exuded
from roots of Bt corn, potato and rice plants. This trait may vary between different cotton
cultivars.
Appendix 3 Toxicity to non-target organisms 44
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
235. The initial level of exposure to the introduced proteins in soil is likely to decrease with
time, as a result of soil biodegradation. The Cry1Ac protein in INGARD® cotton plant
material has been found to degrade in soil with a half-life in the order of 2 to 46 days (Palm et
al. 1996; Ream 1994). However, soil type and other environmental factors are likely to
influence these values (e.g. (Palm et al. 1996; Tapp & Stotzky 1998b). Donegan et al (1995)
could detect Cry1Ac protein in soil samples at the end of 28- and 56-day experiments but they
did not calculate a half-life for the protein. The Cry1Ac protein adsorbs to various soil
components (e.g. humic acids, clay minerals), rendering it resistant to microbial degradation.
Generally there is an initial rapid decline in Cry1Ac levels over several days, followed by a
more gradual rate of decline. In some soils Cry1Ac was still detectable after several months
(Palm et al. 1996).
236. Head et al (Head et al. 2002) tested for the persistence of the Cry1Ac protein in soils
from six US fields in which three to six consecutive seasons of Bollgard® (known as
INGARD® in Australia) cotton crops had been grown, with incorporation of plant material
into the soil by post harvest tillage. Samples were collected three months after the final
season‘s tillage. Neither enzyme-linked immunosorbent assays (ELISAs), nor bioassays (i.e.
feeding to susceptible insect larvae) detected Cry1Ac protein in any of the samples.
237. Generally in Australia cotton is grown in alkaline soil, with a pH range of 7.5 - 8.5
(Australian Cotton Cooperative Research Centre 2002c). Bt endotoxins begin to desorb from
clay soils at alkaline pH and can then be degraded by soil microorganisms (Tapp et al. 1994;
Tapp & Stotzky 1998a). Thus, the Cry1Ac and Cry2Ab proteins are less likely to accumulate
in alkaline Australian agricultural soils, even in the event of successive seasons of cultivation
of GM cottons expressing these proteins.
2.6.2 Toxicity of Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II®
cottons for microorganisms
238. The direct effects of the CP4 EPSPS, GUS and NPTII enzymes have not been tested on
microorganisms. EPSPS enzymes are present in all plants, bacteria, algae and fungi and
perform the same biochemical function in all organisms in which they are expressed. The
CP4 EPSPS enzyme expressed by the Roundup Ready® Flex cottons is derived from a
common soil bacterium. The GUS enzyme is also derived from a common bacterium and is
already present in soil and water ecosystems. Similar enzymes are present in many bacteria,
invertebrates, vertebrates and some plants and perform similar biochemical functions in these
organisms. Thus, the expression of bacterial EPSPS or GUS enzymes in plants will make a
relatively minor contribution to the overall abundance of these proteins in the environment
and is not likely to have any novel toxic effects on microorganisms.
239. NPTII is a phosphorylating enzyme, which does not possess any properties to
distinguish it toxicologically from other phosphorylating enzymes present in microorganisms,
plants and animals (FDA 1994). The function of this enzyme is the phosphorylation
(inactivation) of the antibiotic neomycin (and the related kanamycin). In the environment,
this enzyme is not likely to be active outside of living cells, as it requires specific chemical
conditions for activity, including the availability of specific co-factors. Although antibiotic
production by non-pathogenic bacteria has been implicated in suppression of some plant
diseases (Brimecombe et al. 2001), no evidence for the involvement of neomycin or
kanamycin has been found in a search of the scientific literature. Nor are these antibiotics
used in agriculture for controlling soil-borne disease. Thus, the presence of NPTII in soil is
not expected to impact on microbial populations, nor on plant disease susceptibility.
Furthermore, expression of NPTII in a variety of crop plants (e.g. canola, corn, cotton and
Appendix 3 Toxicity to non-target organisms 45
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
tomato), over several years of agronomic performance testing and commercial cultivation, has
not been linked to any increased occurrence of disease.
240. Potential effects of the Cry1Ac protein on microorganisms have been considered in
detail in the risk assessment for the continued commercial release of INGARD® cotton,
DIR 022/2002, available at www.ogtr.gov.au. Further, in their recent study, Gupta and
Watson (2004) examined the decomposition rates of INGARD®, Roundup Ready® and
Roundup Ready®/INGARD® cotton plants and non-GM cotton plant residues. Their results
indicated that there were no significant differences between the decomposition rates of any of
the samples. Fungal colonisation and total microbial respiratory activity appeared greater on
INGARD® cotton residues. However, the authors did not investigate whether these changes
were beneficial, detrimental or neutral.
241. The effects on soil microorganisms of the Cry1Ac toxin, either purified or in GM cotton
leaves, were examined in greater detail by Donegan et al (Donegan et al. 1995; Donegan &
Seidler 1998). Numbers and types of protozoa, bacteria and fungi, as well as substrate
utilisation tests and DNA fingerprinting of eubacterial ribosomal sequences, were used to
analyse the composition of microbial soil communities. The addition of purified Cry1Ac
toxin to soil did not cause any detectable changes to numbers of culturable microorganisms
(bacteria or fungi). In contrast, the addition of either non-GM leaf material or GM leaf
material from one of two GM cotton lines expressing the Cry1Ac toxin caused increases in
the number of bacteria and fungi present in the soil samples. This was expected, due to the
increased organic matter. In comparison with the non-GM leaves, GM leaves generally
caused a more rapid increase in the number of microorganisms, and when individual bacterial
species were identified, it was found that GM and non-GM leaf tissue supported different
species during decomposition. It was speculated that these results may reflect faster
decomposition and nutrient release from the GM cotton leaves compared to the non-GM
leaves, possibly due to some unknown and unintended effect of the genetic modification (or
the tissue culture process involved) on the plant characteristics, separate from expression of
the Cry1Ac protein, because the addition of purified Cry1Ac protein to non-GM leaves did
not reproduce the effect. No significant differences were found in the numbers of protozoa
between any of the soil samples tested (Donegan et al. 1995; Donegan & Seidler 1998).
242. Donegal et al (1995, 1998) also included a different Bt cotton line expressing the
Cry1Ab protein in some of their experiments. This behaved no differently to its non-GM
parental cotton line in terms of stimulating microbial numbers in soil. The authors concluded
that since no effects were seen when either of the purified proteins were added to soil, the
cotton parental line and the transformation event of each GM cotton line could influence the
interaction of the GM cotton with soil microorganisms.
243. A similar study by Saxena and Stotzky (Saxena & Stotzky 2001), using Cry1Ab
expressing Bt corn, found no toxic effects on earthworms or soil microorganisms including,
nematodes, protozoa, bacteria and fungi. The toxin was found in the guts and casts of
earthworms indicating that it was present in their diet, but it did not affect their mortality or
weight during 40 days of exposure. This study did not investigate the composition of the
microbial populations but did determine that there were no changes in absolute numbers of
microorganisms between soil samples exposed to the Bt and non-Bt corn samples.
244. Purified Btk toxins had no effect on in vitro growth of pure or mixed cultures of gram
positive bacteria (Bacillus subtilis, B. cereus, B. thuringiensis (subspecies kurstaki and
israelensis) and Arthrobacter globiformis), gram negative bacteria
(Agrobacterium radiobacter, Pseudomonas aeruginosa, Proteus vulgaris, P. mirabilis,
E. coli, Enterobacter aerogenes, E. cloacae, Oscillatoria sp.), yeast, (Saccharomyces
Appendix 3 Toxicity to non-target organisms 46
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
cerevisiae, Candida albicans), filamentous fungi (Rhizopus nigricans, Cunninghamella
elegans, Aspergillus niger, Fusarium solani, Penicillium sp.) algae (Chlamydomonas sp.,
Oedogonium sp, Euglena sp.) and diatoms (Stotzky 2000a).
245. No studies have been performed to specifically examine the effect of the Cry2Ab
protein on soil microorganisms.
SECTION 3 CONCLUSIONS REGARDING TOXICITY TO NON-TARGET ORGANISMS
246. It is considered that the risk of Roundup Ready® Flex or Roundup Ready®
Flex/Bollgard II® cotton being toxic to, and adversely affecting, non-target organisms is very
low because:
exposure of stock and wildlife to these GM cottons is low;
compositional analysis has not indicated any significant difference between the
GM cotton and non-GM cottons, other than the presence of the introduced
proteins;
the CP4 EPSPS, GUS and NPTII enzymes are widespread in the environment and
have not been shown to be toxic to any organism;
Bt toxins are widespread in the environment and the toxicity of Cry1Ac and
Cry2Ab is highly specific to lepidopteran insect larvae;
cotton is not the preferred food source for non-target Lepidoptera;
laboratory and field studies suggest that populations of key non-target
invertebrates are unlikely to be affected by the Bt toxins. Indeed it is likely that
their populations would be favoured by decreases in the use of broad-spectrum
insecticides associated with the use of insecticidal Bt cottons; and
the Cry proteins are not known to adversely affect microorganisms. This is
supported by specific data for Cry1Ac and other Bt toxins.
247. The licence requires the licence holder to report any adverse effects on the environment
(e.g. any indication of toxicity of the GM cottons for non-target organisms).
Appendix 3 Toxicity to non-target organisms 47
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 4 WEEDINESS
248. Under Section 51 of the Act, the Regulator is required to consider risks to human health
and safety and the environment in preparing the risk assessment and risk management plan
(RARMP). In this Appendix, risks posed by the proposed dealings to the environment are
considered in relation to the potential for the GMOs to become problematic weeds.
SECTION 1 NATURE OF THE WEEDINESS HAZARD
249. There are numerous definitions of weeds including 'a plant growing where it should not
be'. Weeds become a problem to the community when their presence or abundance interferes
with the intended use of the land they occupy. Weeds may also represent a source of food to
various organisms, hence the introduction of weeds to an environment may also bring about
ecological change by altering the structure of food webs.
250. Weeds are thought to share a number of life history characters that enable them to
rapidly colonise and persist in ecosystems, particularly those that are regularly disturbed (Roy
1990; Williamson & Fitter 1996). These characteristics include:
ability to germinate, survive and reproduce under a wide range of environmental
conditions;
long-lived seed with extended dormancy periods;
rapid seedling growth;
rapid growth to reproductive stage;
long continuous seed production;
ability to self-pollinate but not exclusively autogamous;
use of unspecialised pollinators or wind when outcrossing;
high seed output under favourable conditions;
special adaptations for long distance and short distance dispersal; and
being good competitors.
251. However, because environmental conditions have a big influence on these attributes,
and other factors such as plant community composition and availability of key resources (e.g.
space, water, light and nutrients) influence the potential of a plant species to invade, weedy
characteristics alone are not enough to determine if a plant will become a problematic weed.
Therefore, the most successful predictors of weediness remain taxonomic affinity to other
weedy species and the history of a given species‘ weediness elsewhere in the world (Panetta
1993; Pheloung et al. 1999).
252. Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons differ from
conventional non-GM cotton in the expression of one and five additional proteins,
respectively. These are the CP4 EPSPS protein (in both GMOs) and the Cry1Ac, Cry2Ab,
GUS and NPTII proteins (in Roundup Ready® Flex/Bollgard II® cotton only) (see
Appendix 1).
253. The possibility was considered that Roundup Ready® Flex or Roundup Ready®
Flex/Bollgard II® cotton might have the potential to be harmful to the environment, because
of inherent weediness or increased potential for weediness, either due to expression of the
novel gene products or as a result of unintended effects of the genetic modification.
Appendix 4 Weediness 48
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
254. This could occur if the GM cottons displayed altered characteristics such as increased
fitness or increased fecundity. If the GM cottons were to spread in the environment as weeds,
this could result in impacts such as loss of native biodiversity or other adverse environmental
effects.
255. The use of herbicides in Australia is regulated by the APVMA (see Appendix 6). The
use of Roundup Ready® herbicide (containing glyphosate as the active ingredient) on GM
Roundup Ready® cotton is registered by the APVMA. As part of their assessment of this use,
the APVMA consider any potential environmental effects, such as development of
glyphosate-resistant weeds, levels of glyphosate in the environment and drift from spray
applications. Further approval (permit or registration) from the APVMA will be needed to
extend this use. Therefore, risks associated with the use of glyphosate are not considered in
the risk assessment of these GM cottons. The risk of development of glyphosate-resistant
weeds is also addressed briefly in Appendix 6.
SECTION 2 LIKELIHOOD OF THE WEEDINESS HAZARD OCCURRING
256. In assessing the likelihood of adverse impacts due to weediness of Roundup Ready®
Flex and Roundup Ready® Flex/Bollgard II® cottons, a number of factors were considered,
including:
the inherent weediness of conventionally bred non-GM cotton;
the potential selective advantage conferred by the introduced CP4 EPSPS,
Cry1Ac, Cry2Ab, GUS and NPTII proteins;
the potential weediness of Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons; and
the potential for spread and persistence of the GM cottons beyond the release site.
Section 2.1 Inherent weediness of conventional non-GM cotton
257. Attributes of non-GM cotton associated with potential weediness are discussed in the
document 'The Biology and Ecology of Cotton (Gossypium hirsutum) in Australia' (OGTR
2002) that was produced in order to inform the risk assessment processes for licence
applications involving GM cotton. This document can be accessed at www.ogtr.gov.au. In
summary, the document concludes that non-GM cotton is not a problematic weed in Australia,
because factors including soil moisture, nutrient limitation, temperature and roadside
management practices limit the establishment and/or persistence of cotton seedlings.
Information on the weediness of non-GM cotton is included here to establish a baseline for
comparison with the GM cottons being assessed.
258. Cotton is not considered to possess the characteristics commonly associated with
successful weeds, such as seed dormancy, long persistence in the soil, germination under a
broad range of environmental conditions, rapid vegetative growth, short lifecycle, very high
seed output, high seed dispersal and long-distance seed dispersal (Keeler 1989; Keeler 1985).
259. As mentioned above, another important element in predicting weediness is taxonomic
relationship, considering weediness within a taxon, including its history of weediness in any
part of the world (Bergelson et al. 1998; Panetta 1993; Pheloung 1995). Cotton has been
grown for centuries throughout the world without any reports that it is a serious weed pest.
Cotton is not considered to be a problematic weed in Australia (Groves et al. 2000; Groves et
al. 2002). There are about 50 species of Gossypium (Craven et al. 1994; Fryxell 1992) of
which only one (G. tomentosum) is listed as a weed in the USA (Holm et al. 1997).
Appendix 4 Weediness 49
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Section 2.2 Potential selective advantage conferred by the introduced proteins
2.2.1 CP4 EPSPS
260. The CP4 EPSPS protein could only confer a selective advantage in the presence of
glyphosate. In an agricultural setting, Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons will have increased fitness where glyphosate is applied for weed
control. However, glyphosate is not generally used to control established volunteer cotton
plants as it has limited effectiveness on established cotton (i.e.beyond the seedling stage).
Cultivation or alternative herbicides are the main control strategies employed (Australian
Cotton Cooperative Research Centre 2002a).
261. Glyphosate may be used to control weeds on roadsides. In this situation, spraying is
often limited to areas around fixtures such as signs and guide-posts, while slashing is used in
accessible areas. Evidence suggests that cotton is not a significant weed in these situations
and that resistance to glyphosate is not likely to change this (see Section 2.3).
2.2.2 Cry1Ac and Cry2Ab
262. A detailed discussion of the potential of the Cry1Ac protein to influence weediness is
provided in the risk assessment document for application DIR 022/2002 (commercial release
of INGARD® cotton), available at www.ogtr.gov.au. A detailed assessment of the potential of
both the Cry1Ac and Cry2Ab proteins together to influence weediness is provided in the risk
assessment for application DIR 012/2002 (commercial release of Bollgard II® and
Bollgard II®/Roundup Ready® cotton). The Cry proteins could confer a selective advantage
in areas where lepidopteran insect predation limits one or more of the key life stages of
cotton. Information on the behaviour of GM insecticidal cottons in the environment is
presented below, in Section 2.3.
2.2.3 GUS
263. The GUS enzyme, encoded by the uidA gene, enabled visual identification of plant
tissues in which this gene was being expressed during the development of the GM cottons.
GUS activity is naturally found in microorganisms, vertebrates and invertebrates (Gilissen et
al. 1998) but there is very little activity in plants. The GUS protein is an enzyme involved in
bacterial carbohydrate and energy metabolism and is very unlikely to confer any selective
advantage to cotton that might result in weediness (Gilissen et al. 1998).
2.2.4 NPTII
264. The NPTII protein could confer a selective advantage to GM cotton plants in the
presence of a high concentration of neomycin or kanamycin. Since antibiotics are not applied
to cotton crops, and are not likely to be present in any environment where cotton grows, the
expression of NPTII is highly unlikely to confer any selective advantages on the Roundup
Ready® Flex/Bollgard II® cottons. The ability of cotton cells expressing the NPTII protein
was used in the laboratory to select for successfully modified cells and plants.
2.2.5 CP4 EPSPS and Cry1Ac, Cry2Ab in combination
265. The potential for weediness of Roundup Ready® Flex/Bollgard II® cotton is not likely to
be greater than that for the two parental GM varieties, Roundup Ready® Flex cotton and
Bollgard II® cotton. The herbicide tolerance and the insecticidal genes operate through
independent, unrelated biochemical mechanisms. There is no evidence of any interaction
between the two genes, their proteins or their metabolic pathways, and no reason to expect
that this is likely to occur.
Appendix 4 Weediness 50
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
266. Agronomic characteristics of commercially released Roundup Ready®/Bollgard II®
cotton are similar to those of non-GM cotton (Dr G Constable, Program Leader, CSIRO
Cotton Research Unit, personal communication; data supplied by Monsanto). Expression of
the introduced proteins (Cry1Ac and CP4 EPSPS) is also similar in combined (‗stacked‘) trait
GM cotton plants to that in the respective single trait GM cottons. These data suggest that no
unintended effects are likely to occur as a result of the combined traits in Roundup Ready®
Flex/Bollgard II® cotton.
267. There is the possibility of an additive effect in situations where growth or reproduction
of cotton is limited by both lepidopteran insects and glyphosate use, however, neither of these
factors is identified as significant in limiting weediness of cotton.
Section 2.3 Potential weediness of Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons
268. Many of the characteristics associated with weediness are also important agronomic
characteristics. Consequently these are assessed as part of the agronomic evaluations during
the development of new cotton varieties, including GM varieties. Such characteristics have
been assessed for commercially released Roundup Ready® and Roundup Ready®/Bollgard II®
cotton. The Roundup Ready® Flex cottons differ from these Roundup Ready® cottons only in
improved expression of the CP4 EPSPS protein and tolerance of the reproductive tissues to
glyphosate. Therefore, data gathered in the evaluation of the commercially released cottons
can be useful for evaluation of the potential weediness of the Roundup Ready® Flex and
Roundup Ready® Flex/Bollgard II® cottons. Although the Roundup Ready® Flex cottons
express the same introduced proteins as commercially released Roundup Ready® cottons, the
site of insertion and level of expression of the introduced genes is different. Therefore, there
is a low possibility for pleiotropic effects of the genetic modification, which could potentially
alter some aspect of the cotton biology that may affect weediness (for details see Appendix 1).
269. No unintended or secondary effects on agronomic characteristics, including no effects
on fertility, have been observed in greenhouse or field trials of Roundup Ready® Flex cotton
in the USA (information supplied by the applicant). In addition, seed dormancy-related
characteristics and seed germination did not differ significantly between Roundup Ready®
Flex, Roundup Ready® and non-GM cottons. Studies on agronomic characteristics of the GM
cottons proposed for release are currently being conducted in Australia under the licence
DIR 035/2003. These studies show that there is no significant difference between Roundup
Ready® Flex and non-GM cotton regarding growth and plant morphology. The applicant
proposes to continue these studies as part of the proposed release.
270. Seed survival, germination, vigour, yield and disease susceptibility of Roundup Ready®
and Bollgard II® cotton varieties currently being grown have been evaluated in both
controlled environments and field releases and are within the range of current non-GM cotton
varieties (Moser 2000; Allen et al. 2000; Monsanto Australia Limited 2001; Cotton Seed
Distributors 2002). These data suggest that the introduced genes present in Roundup Ready®
Flex and Roundup Ready® Flex/Bollgard II® cottons are not likely to lead to any unintended
effects on characteristics typically associated with weediness.
271. INGARD® cotton (expressing the Cry1Ac and NPTII proteins) has been in commercial
release since 1996, and Roundup Ready® and Roundup Ready®/INGARD® cottons since
2000. Bollgard II® and Roundup Ready®/Bollgard II® cottons have been in commercial
release since 2002. Since their commercial release, cottonseed from these GM cottons has
been used as stockfeed in Australia.
Appendix 4 Weediness 51
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
272. Data gathered from surveys and monitoring conducted in connection with the
commercial release of Roundup Ready® and Roundup Ready®/INGARD® cotton were
reviewed in the risk assessment for application DIR 023/2002. The potential weediness of
GM cottons expressing Bt proteins has also been considered in the risk assessment for
applications DIR 012/2002 and DIR 022/2002. These assessments are available at
www.ogtr.gov.au, and are only presented here in summary.
273. A survey of the transport routes between Emerald (in the cotton growing region in
central Queensland) and the Atherton Tablelands (north of 22º South in Queensland) indicated
that cotton plants had established in the roadside environment only infrequently, despite 12
years of use of these routes for transporting ginned seed (including GM cottons since their
respective commercial releases) for stockfeed (Farrell & Roberts 2002).
274. In both roadside and farm environments, GM and non-GM volunteers tend to establish
in disturbed environments with increased water availability and limited competition from
other vegetation. Cotton volunteers appear to have negligible ability to invade non-disturbed
habitats (e.g. native bush), irrespective of whether or not they are genetically modified.
Survival beyond one year appears to be limited and there is no indication that cotton has
become a problematic weed in either roadside or farm environments. There were no
indications that GM cotton had enhanced survivorship or reproductive potential as compared
to non-GM cotton in any situation. Factors thought to be preventing establishment and
persistence of volunteers in this environment were: a lack of suitable conditions for
germination, competitions from established species for new germinants and low levels of soil
nutrients.
275. Recent surveys conducted over the 2002 - 2003 cotton growing season (in connection
with licence DIR 023/2003 and DIR 022/2002) on the incidence of cotton volunteers in areas
where stock are fed cottonseed, or graze after being fed cottonseed, in northern Queensland
show that very little cottonseed has been used as stockfeed in the preceding year. Where
cottonseed had been used as stockfeed, the incidence of cotton volunteers was never observed
to be problematic, and volunteers never reached flowering or maturity. Data from the
Northern Territory and northern Western Australia could not be collected since cottonseed
had not been used for stockfeed in these areas.
276. An investigation into the weediness potential of GM insecticidal cottons in northern
Australia, conducted over two growing seasons, examined various habitats such as bushland,
roadsides, cattle areas and waterways (Eastick 2002). Data gathered demonstrate that for each
of cotton's life stages, the performance of GM insecticidal cotton is not significantly different
to that of conventional non-GM cotton. There was no evidence that insecticidal genes
enhanced invasiveness or weediness. Due to low germination, growth and survival in most
natural situations, only limited data could be obtained on reproductive capacity. However,
INGARD® cotton (expressing the Cry1Ac protein) produced significantly higher numbers of
bolls at one of the 13 sites studied (an artificial waterway site) in one of the two seasons.
277. Eastick (2002) concluded that cotton is rarely invasive, irrespective of insecticidal
genes, and that lepidopteran herbivores were not a major constraint on the weedy potential of
GM insecticidal cottons in northern Australia. Rather, it was concluded that water and
nutrient availability, herbivory by non-lepidopteran species (vertebrate and invertebrate),
plant competition and fire were the most significant limitations on the establishment and
persistence of cotton populations. Continued monitoring of the sites with the best cotton
survivorship and seed production from this study for a further two years showed consistent
results with those found in the original study, concluding that GM insecticidal cotton is not
more invasive than non-GM cotton (Eastick 2004, draft report summary provided to OGTR;
Appendix 4 Weediness 52
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
the final data and analysis has yet to be submitted). However, this is yet to be determined
conclusively pending consideration of the final report on the potential weediness of GM
insecticidal cottons in northern Australia.
278. Note that only a small part of the release (42 hectares over five sites) will be conducted
in areas north of latitude 22° South. In addition, the same licence conditions have been
imposed as imposed under the current licence for DIR 012/2000 for latitudes north of
22° South to limit spread and persistence of the GM cottons in northern Australia.
Section 2.4 Spread of GM cottons beyond the release sites
279. The proposed dealings include cultivation of Roundup Ready® Flex and Roundup
Ready® Flex/Bollgard II® cottons and retention of all cottonseed for plantings authorised by
the same licence or possible future licences (subject to further applications and assessment
processes). Cottonseed produced in the field trial will not be used for human food or
stockfeed.
280. Cottonseed may be dispersed beyond the limits of the trial sites on equipment during
and after planting and harvesting. Licence conditions have been imposed to require cleaning
of equipment used in connection with the release, so as to prevent the GM cottonseed
escaping into the environment.
281. Visitors may unintentionally disperse cottonseed from trial sites that are used for
demonstration purposes. To prevent this, Monsanto has developed a risk management plan
which ensures e.g. that all visitors must be accompanied by authorised personnel and must
read, understand and sign a Site Visitor Rules and Agreement form before visiting a site.
282. In the area proposed for release in the Northern Territory (Katherine Region),
cottonseed may be dispersed from the trial site to sinkholes nearby during heavy rain.
Sinkholes are depressions in the surface of the land communicating with underground
passages. Sinkholes generally occur in limestone regions (Tindall limestone in the Katherine
Region) and are often formed by the collapse of a cave roof. Specific licence conditions have
been imposed which require post-harvest monitoring for cotton volunteers in the area between
the release site and any sinkholes to which water from the site may flow, as well as the area
surrounding the sinkholes. Any cotton volunteers detected will need to be destroyed.
283. After harvest, seed cotton may be dispersed beyond the limits of the trial sites during
transportation to ginning facilities. The escape of seed cotton during transportation presents
an opportunity for the GM cotton to spread in the environment. Specific licence conditions
have been imposed which require harvested material to be securely wrapped before
transporting away from the release sites so as to prevent seed cotton escaping into the
environment.
Section 2.5 Persistence of the GM cotton at the release sites
284. Some seed may fall to the ground during harvesting and also be incorporated into the
soil. A soil seed bank represents an opportunity for the GMOs to persist in the environment.
Cotton has little dormancy, meaning seed will germinate with the arrival of favourable soil
moisture and temperature conditions. USA field trials demonstrated that seed dormancy
related characteristics are not altered in Roundup Ready® Flex cotton (data provided by the
applicant).
285. Licence conditions have been imposed to require cleaning of sites after harvest, post
harvest monitoring, and destruction of cotton volunteers to ensure that the GM cottons do not
persist in the environment at the release sites.
Appendix 4 Weediness 53
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 3 CONCLUSIONS REGARDING WEEDINESS
286. It is concluded that the risk of Roundup Ready® Flex or Roundup Ready® Flex/
Bollgard II® cotton establishing as environmental weeds in cotton growing regions of
New South Wales and Queensland and the proposed release areas in northern Australia is
very low, and not likely to be greater than that of conventional non-GM cotton, because:
cotton does not possess characteristics commonly associated with weediness, and
is not known to be a problematic weed in any environment;
the introduced genes in the GM cottons are unlikely to affect these characteristics;
the combination of herbicide tolerance and insect resistance in Roundup Ready®
Flex/Bollgard II® cotton is unlikely to increase the weediness potential of this
GMO above the potential for either Roundup Ready® Flex or Bollgard II® cotton
individually;
other GM cottons containing the same proteins, grown commercially in Australia,
have not become problematic weeds; and
major constraints on weediness of GM and non-GM cotton in Australia are water
availability, nutrient availability, plant competition, herbivory by non-
lepidopteran species, fire and (in southern Australia) frost.
287. It is considered that the risks of the GM cottons establishing as a weed can be managed
to an acceptable level by implementing various strategies to minimise the spread and
persistence of the GM cottons in the environment. Licence conditions (including cleaning
release sites and equipment, secure wrapping of GM materials and destruction of volunteer
cotton after harvest) have been imposed to manage this risk. Refer to Chapter 2 and
Appendix 7 for imposed licence conditions.
SECTION 4 RESEARCH REQUIREMENTS
288. The applicant proposes to collect further data on the agronomic characteristics,
including characteristics indicative of potential weediness, of the GM cottons as part of this
release.
289. A number of research requirements, including the collection of information regarding
the agronomic characteristics indicative of potential weediness of the same GM cottons under
Australian conditions, have already been imposed under the licence for application
DIR 035/2003. Some of these data have been provided in the DIR 055/2004 application and
the additional data, which are currently being collected, will be provided to the Regulator as
required under the DIR 035/2003 licence. Therefore, no additional research requirements
have been imposed for the current application (DIR 055/2004).
Appendix 4 Weediness 54
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 5 TRANSFER OF INTRODUCED GENES TO OTHER
ORGANISMS
290. Under Section 51 of the Act, the Regulator is required to consider risks to human health
and safety and the environment in preparing the risk assessment and risk management plan
(RARMP). This Appendix considers potential hazards that may be posed through the transfer
of the introduced genes/genetic materials from Roundup Ready® Flex or Roundup Ready®
Flex/Bollgard II® cotton to other organisms.
291. Gene transfer is the movement of genetic material between individuals. Within a
species, genetic material is routinely transferred between individuals of successive
generations through sexual reproduction (vertical gene transfer). Hybrids can sometimes be
produced between closely related species through sexual reproduction, although this may
require significant human intervention. For example, in plants, cross pollination of wheat and
rye in the laboratory produced triticale. In animals, fertilisation of a mare by a donkey
produces a mule. Hybrid progeny may be fertile or sterile, meaning hybridisation may or may
not lead to the introgression of new genetic material into a population.
292. Without the application of gene technology, gene transfer is not readily observed
between distantly related species, except for between bacteria and between viruses. However,
transfer of genetic material between sexually incompatible organisms (horizontal gene
transfer) can occur. Detailed examination of DNA sequence similarities reveals that ancestral
plants have occasionally exchanged small DNA fragments with distantly related organisms.
However, there seems to have been only very limited transfer of complete genes from plants
to other types of organisms.
293. The likelihood of a hazard arising from gene transfer is dependent on a number of
factors that must form a necessary chain for a hazard to be realised, including:
opportunity for gene transfer to occur such that the recipient organism is exposed
to the genetic material in the form of pollen, plant cells or DNA;
occurrence of the genetic material being incorporated into the genetic material of
the recipient organism at a site and in a configuration that allows the genetic
material to be functional;
persistence of the transferred genetic material such that the recipient organism is
able to survive, reproduce and maintain the genetic modification; and
significance of the transferred genetic material such that its presence and/or
expression in the recipient organism will result in an adverse impact on human
health and safety, or the environment.
294. For ease of reference, the assessment of gene transfer to other organisms is presented in
four sections:
Section 1 details the nature and likelihood of a hazard arising through transfer of
the introduced genetic materials from Roundup Ready® Flex or Roundup Ready®
Flex/Bollgard II® cotton to other plants, including other cotton plants;
Section 2 details the nature and likelihood of a hazard arising through transfer of
the introduced genetic materials from Roundup Ready® Flex or Roundup Ready®
Flex/Bollgard II® cotton to microorganisms; and
Section 3 details the nature and likelihood of a hazard arising through transfer of
the introduced genetic materials from Roundup Ready® Flex or Roundup Ready®
Flex/Bollgard II® cotton to animals, including humans.
Appendix 5 Transfer of introduced genes to other organisms 55
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Section 4 draws together the conclusions from these sections.
295. A detailed assessment of the likelihood of gene transfer from GM cottons to other
organisms is also presented in the RARMPs for other DIR applications, for example DIRs
012/2002, 022/2002, 023/2002, 035/2003 and 049/2004.
SECTION 1 GENE TRANSFER FROM THE GM COTTONS TO OTHER PLANTS
Section 1.1 Nature of the gene transfer hazard to other plants
296. Transfer of the introduced genes (cp4 epsps, cry1Ac, cry2Ab, uidA, nptII and aad) or
regulatory sequences to other cultivated (including volunteer), naturalised (feral) or native
cotton plants would present the same hazards and have the same potential impacts as the
presence of the genes in the GM cottons (see Appendices 2-6). However, if such a gene
transfer occurred, it would increase the possibility that these genetic materials could further
spread in the environment.
297. If gene transfer to other plant species were to occur, any resulting hazards to the
environment could be highly varied, broadly depending upon the nature of the genetic
materials and of the species to which transfer occurred. Transfer of the introduced genes or
regulatory sequences into other plant species may have adverse effects on biodiversity, in
particular to native flora, if the recipient plants and their progeny gained a selective
advantage, such as enhanced survival or reproductive capacity.
Section 1.2 Potential hazards from the introduced genes in other plants
1.2.1 The cp4 epsps (herbicide tolerance) gene
298. Plants expressing this gene could become tolerant to the herbicide glyphosate. This
could confer a selective advantage on the plants in the presence of glyphosate use.
1.2.2 The cry1Ac and cry2Ab (insecticidal) genes
299. Plants expressing these genes could become toxic to lepidopteran insects. This
resistance to lepidopteran insects could confer a selective advantage on plants normally
controlled by these insects and could result in increased weediness. Expression of the cry
genes could also adversely affect survival of lepidopteran insects and consequently other
organisms linked to lepidopteran insects through food webs.
1.2.3 The uidA (reporter) gene
300. Plants expressing this gene could produce the GUS protein. GUS expression is unlikely
to be toxic or allergenic to humans and other organisms (see Appendix 2 for details) and is
unlikely to affect weediness (see Appendix 3 for details), therefore expression of the GUS
protein in other plants would not be expected to lead to any hazard.
1.2.4 The nptII and aad (antibiotic resistance) genes
301. Expression of the nptII marker gene enabled selection of plant cells containing the
genetic modification in the laboratory. The aad gene is not expressed in plant cells. It was
used in the laboratory to allow selection of bacteria containing the desired genes prior to
generation of GM plant cells.
302. Plants expressing these genes could become resistant to the antibiotics kanamycin and
neomycin (if expressing nptII) and/or streptomycin and spectinomycin (if expressing aad).
This would only have an impact on plant survival if these antibiotics were used on the plants,
or otherwise present in the environment of the plants, and were limiting their growth.
Antibiotics are not generally applied to crops and would not limit their growth except at very
high concentrations not found in the natural or agricultural environment.
Appendix 5 Transfer of introduced genes to other organisms 56
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
303. Antibiotic production by non-pathogenic bacteria has been implicated in suppression of
some plant diseases (Brimecombe et al. 2001). If expression of these genes in plants led to
inactivation of antibiotics in the soil, this could potentially impact on microbial populations or
plant disease susceptibility. However, neither the NPTII enzyme (expressed from the nptII
gene) nor the AAD enzyme (expressed from the aad gene) is likely to be active in the
environment outside of the cells in which it is expressed. Expression of the nptII gene in a
variety of crop plants (for example canola, corn and soybean), over several years of
agronomic performance testing and commercial cultivation, has not been linked to increased
susceptibility to disease, as assessed by regulatory authorities of other countries, including the
Animal and Plant Health Inspection Service (APHIS) of the U. S. Department of Agriculture
(eg. USDA-APHIS 1996) (USDA-APHIS 1996) and the Canadian Food Inspection Agency
(Canadian Food Inspection Agency 1998). Thus, expression of the NPTII or AAD proteins
would not be expected to lead to any hazard.
1.2.5 Promoters and other regulatory sequences
304. If these sequences were to be transferred to other plants without the associated
introduced genes of Roundup Ready® Flex or Roundup Ready® Flex/Bollgard II® cotton, the
expression of native plant genes could be altered with unpredictable effects. The impact
could be highly variable and would be dependent on any resulting phenotypic change
induced.
305. Some of the introduced regulatory sequences are derived from plants (Arabidopsis
thaliana and soybean) or bacteria (E. coli), and others are derived from plant pathogens
(cauliflower mosaic virus, figwort mosaic virus, Agrobacterium tumefaciens). However, the
sequences from the plant pathogens are not pathogenic in themselves nor do they cause any
disease symptoms in GM plants.
306. All of the introduced regulatory sequences operate in the same manner as do native
regulatory sequences in plants. The transfer of native regulatory sequences to a new genetic
context occurs naturally in all plant genomes and could also result in unpredictable effects.
Thus, the potential hazards from the introduced sequences are no different from those posed
by sequence transfer from non-GM plants or sequence transfer occurring within the genome
of a plant species.
Section 1.3 Likelihood of gene transfer from the GM cottons to other plants
1.3.1 Gene transfer to cultivated and volunteer cotton
307. For a detailed consideration of the likelihood of gene transfer occurring, including an
overview of the pollination biology of cotton, see the document ‗The Biology and Ecology of
Cotton (Gossypium hirsutum) in Australia‘ (OGTR 2002). This document is available at
www.ogtr.gov.au and was produced in order to inform the risk assessment processes for
licence applications involving GM cottons.
308. Cotton is primarily self pollinating, and as cotton pollen is large and heavy, it is not
easily dispersed by wind (Jenkins 1992). In a cropping situation, however, a low level of
pollen transfer, by insect pollinators, to nearby cotton plants would be likely (OGTR 2002).
Cotton pollen dispersal studies consistently show that when out-crossing occurs, it is localised
around the pollen source and decreases significantly with distance (OGTR 2002 and
references therein).
309. Studies carried out in southern Australia (Narrabri, New South Wales) demonstrated
that out-crossing events are generally rare, with out-crossing rates declining from 0.4% at 1 m
to below 0.03% at 16 m and not detected at 20 m from the GM cotton plants (Llewellyn &
Appendix 5 Transfer of introduced genes to other organisms 57
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Fitt 1996). These out-crossing frequencies are relatively low compared to those seen in other
countries, possibly due to differences in pollinator species, particularly the absence of bumble
bees in Australia (Llewellyn & Fitt 1996). In the out-crossing experiments conducted in
Narrabri, no bees were detected, and although small numbers of wasps and flies were
recorded, it was suggested that hibiscus beetles were likely to be the major cross-pollinators in
these trials.
310. Gossypium hirsutum (Upland cotton) is the most widely planted commercially
cultivated cotton in Australia. Gossypium barbedense (Pima cotton) is also used for
commercial cotton production, but only to a very minor extent in Australia (OGTR 2002).
G. hirsutum and G. barbedense are closely related and hybridisation between the two species
can occur, yielding fertile progeny (Brubaker et al. 1999). Hybrid progeny exhibit
characteristics intermediate to the parents but typically with lower capacity to produce fruit.
After several generations, progeny of the hybrids revert to the characteristics of one or other
of the parents. G. barbedense and hybrids are not more weedy or difficult to control than is
G. hirsutum (personal communication, Warwick Stiller & Greg Constable, CSIRO).
311. For this release, the likelihood of gene transfer, and hence of any hazard arising through
transfer of the introduced genetic materials, to other cultivated cotton is further minimised
because licence conditions have been imposed to limit cross-pollination to plants outside the
release sites (see Chapter 2 and Appendix 7 for details).
312. Note that the OGTR has developed a gene flow research program, involving various
GM cotton licensees (including the applicant), to collect data on pollen-mediated gene flow.
Research will be conducted in a range of current and potential cotton growing areas, therefore
no additional research requirements relating to gene flow have been imposed.
1.3.2 Gene transfer to naturalised (feral) cotton
313. Transfer of the introduced genes or regulatory sequences to naturalised cotton could
also increase the likelihood that these genetic materials could spread and/or persist in the
environment (away from cotton farming systems). Gene transfer to naturalised cotton
populations is thought to be unlikely because of the geographic distances between these
naturalised populations and the cotton growing regions of NSW and QLD. However,
herbarium records of G. hirsutum and G. barbadense suggest that naturalised populations may
occur, or may have occurred in the past, in northern, central and south eastern Queensland, in
northern Northern Territory and in northern Western Australia (OGTR 2002; information
provided by the applicant). The presence of remnants of some of these populations, which
may be within pollinating distance of cotton crops, has not been confirmed.
314. As part of the licence conditions for DIR 022/2002 (INGARD® cotton), Monsanto is
required to conduct a survey of naturalised cotton populations in Queensland, in locations
suggested by herbarium records. Monsanto has provided maps showing the distribution of
naturalised G. hirsutum and G. barbadense populations in Australia, composed from GPS
coordinates given in Australian herbarium records, in the 2004 Annual Report for licences
DIR 022/2002 and DIR 023/2002. A comparison of shires where cotton is cultivated and
shires where feral cotton populations occur (in Queensland) indicates that feral populations
occur in only three cotton production shires, and one shire adjacent to a cotton production
shire. However, it is very difficult to determine the exact distances between feral and
cultivated populations.
315. Licence conditions have been imposed to limit cross-pollination to plants outside the
release sites (see Chapter 2 and Appendix 7 for details).
Appendix 5 Transfer of introduced genes to other organisms 58
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
1.3.3 Gene transfer to native cottons and other plant species
316. Australian flora contains 17 native Gossypium species, all of which are diploids (C, G or
K genomes), while the cultivated cottons are tetraploids (AD genomes). The native species
with highest potential for hybridising with G. hirsutum is G. sturtianum. Hybrids have been
produced without application of plant hormones, when plants were planted in close proximity
of each other (OGTR 2002). These hybrids were sterile, however, effectively eliminating any
potential for introgression of G. hirsutum genes into G. sturtianum populations.
317. The centre of native Gossypium diversity in Australia is in northern Western Australia
and the Northern Territory. Most of the Australian Gossypium species have limited
distributions and occur at considerable geographic distance from cultivated cotton fields.
Thus, gene transfer from Roundup Ready® Flex or Roundup Ready® Flex/Bollgard II® cotton
to native cottons is prevented not only by genetic incompatibility but also by geographic
constraints to cross-pollination (OGTR 2002).
318. The failure of cross-pollination due to well established genetic incompatibility also
prevents gene transfer from Roundup Ready® Flex or Roundup Ready® Flex/Bollgard II®
cotton to other plant species.
SECTION 2 GENE TRANSFER FROM THE GM COTTONS TO MICROORGANISMS
Section 2.1 Nature of the gene transfer hazard to microorganisms
319. Gene transfer from plants to microorganisms cannot occur through cross pollination.
Horizontal gene transfer is defined as the transfer of genetic material from one organism (the
donor) to another organism (the recipient) which is not sexually compatible with the donor
(Conner et al. 2003). There is growing evidence that horizontal gene transfer has been a
principal force in the evolution of bacteria (Nielsen 1998; Stanhope et al. 2001; Ochman et al.
2000; Smalla et al. 2000).
320. The potential hazards associated with the introduced genes of Roundup Ready® Flex or
Roundup Ready® Flex/Bollgard II® cotton transferring to microorganisms could be highly
varied, broadly depending upon the phenotype of the recipient and any changes to its survival,
reproductive capacity and/or pathogenicity. The impact of any hazard arising through gene
transfer would also depend on other sources of the introduced genetic materials in the
environment.
Section 2.2 Potential hazards from the introduced genes in microorganisms
2.2.1 The cp4 epsps (herbicide tolerance) gene
321. Microorganisms expressing this gene could gain the capacity to synthesise aromatic
amino acids and other aromatic compounds in the presence of glyphosate. However, this
would not have a significant effect on microbial communities, since the ability to rapidly
degrade glyphosate is widespread among microorganisms, and soil microorganism
populations are not significantly affected by glyphosate application (Malik et al. 1989).
2.2.2 The cry1Ac and cry2Ab (insecticidal) genes
322. Microorganisms expressing these genes could become toxic to lepidopteran insects.
This could impact on survival of lepidopteran insects if the recipient microorganisms were
ingested at high levels. Microorganism populations could also be affected if toxicity to
lepidopteran insects gave the recipient a survival or reproductive advantage.
Appendix 5 Transfer of introduced genes to other organisms 59
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
2.2.3 The uidA (reporter) gene
323. If microorganisms were to express the GUS protein, this would not have a significant
effect on microbial communities as the enzyme is involved in bacterial carbohydrate and
energy metabolism and is already widespread among bacterial species (Gilissen et al. 1998).
2.2.4 The nptII and aad (antibiotic resistance) genes
324. Microorganisms could become resistant to the antibiotics kanamycin and neomycin (if
expressing nptII) and/or streptomycin and spectinomycin (if expressing aad). The
consequences of this for human health and safety and the environment would depend on other
characteristics of the microorganism (e.g. pathogenicity), the use and significance of the
antibiotics in clinical and/or veterinary practice and whether these antibiotics limit growth or
survival of the microorganism in other circumstances.
325. Some microorganisms may be limited by antibiotics, either due to the use of antibiotic
medicines or in some limited environmental situations where competing microorganisms
produce antibiotics. Viruses are not limited by antibiotics.
326. Neomycin and kanamycin have limited medical, veterinary, or aquacultural use,
because they have severe side effects and many bacteria are already resistant to them (Flavell
et al. 1992), so alternative antibiotics are preferred. Similarly, streptomycin and
spectinomycin have limited medical and veterinary use and many bacteria are already
resistant to them (Shaw et al. 1993; Heym et al. 1994).
327. Antibiotic production by non-pathogenic bacteria has been implicated in suppression of
some plant diseases (Brimecombe et al. 2001). Thus, transfer of these genes to new bacterial
species, including plant pathogens, could potentially impact on microbial populations or plant
disease susceptibility. However, the common occurrence of these and similar genes in
microbial populations (see Section 2.3 below) makes the contribution of GM plants to this
hazard insignificant.
2.2.5 Promoters and other regulatory sequences
328. If these sequences were to be transferred to microorganisms without the associated
introduced genes of Roundup Ready® Flex or Roundup Ready® Flex/Bollgard II® cotton, the
expression of endogenous genes could be altered with unpredictable effects. The impact
could be highly variable and would be dependent on the resulting phenotypic change induced.
329. Some of the introduced regulatory sequences are derived from plants (Arabidopsis
thaliana and soybean) or bacteria (E. coli), and others are derived from plant pathogens
(cauliflower mosaic virus, figwort mosaic virus, Agrobacterium tumefaciens). The sequences
derived from plant pathogens are, however, not pathogenic by themselves nor do they cause
any disease symptoms in GM plants.
330. All of the introduced regulatory sequences operate in the same manner as do native
regulatory elements of plants. The transfer of native regulatory elements from plants to a
microorganism could also result in unpredictable effects. Thus, the likelihood of a hazard
arising due to transfer of the introduced sequences is no different to that of transfer of other
native regulatory elements from non-GM plants.
Section 2.3 Other sources of the introduced genes in the environment, and their
potential for horizontal transfer to microorganisms
331. Information on other sources of the introduced genes in the environment is discussed
here to provide baseline information on the prevalence and transfer of these genes that would
happen naturally, irrespective of the GM cottons. Gene transfer between bacteria is a well
Appendix 5 Transfer of introduced genes to other organisms 60
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
established natural process that is central to their survival and evolution (Nielsen 1998;
Stanhope et al. 2001; Ochman et al. 2000; Smalla et al. 2000; EFSA 2004). Thus, where the
introduced genes already exist in bacterial populations, the likelihood of transfer between
bacterial species would greatly exceed that from GM plants to bacteria. The presence of the
introduced genes in bacterial populations also provides a widespread source of these genes for
any potential transfer to other microorganisms.
332. All of the introduced genes in Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons are already widespread in the environment, being derived from
common soil or gut bacteria. The regulatory sequences are derived from common plant
viruses, bacteria or plants.
2.3.1 The cp4 epsps (herbicide tolerance) gene
333. All plants, bacteria and fungi carry epsps genes. The difference between the cp4 epsps
gene in Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons and other
epsps genes, and the encoded enzymes, is in their DNA and amino acid sequences, not in their
physiological functions (see Appendix 1 for details). The cp4 epsps gene is derived from the
common soil bacterium Agrobacterium sp. strain CP4 (Barry et al. 1992; Padgette et al.
1996), which is found in soil and on plants. The encoded CP4 EPSPS enzyme is naturally
insensitive to glyphosate (Padgette et al. 1993, Monsanto unpublished), as are a number of
other microbial EPSPS enzymes (Schulz et al. 1985; Eschenburg et al. 2002). Thus,
insensitivity to glyphosate is already widespread in microbial populations.
2.3.2 The cry1Ac and cry2Ab (insecticidal) genes
334. The cry1Ac and cry2Ab insecticidal genes expressed in Roundup Ready® Flex/
Bollgard II® cotton occur naturally in the common soil bacterium Bacillus thuringiensis (Bt).
Bt has been isolated from a wide range of sources such as forest, soil, grain dust, bat dung, sea
water and dead insects (Martin & Travers 1989).
335. Many Bt toxin genes are not carried in chromosomal DNA, but are encoded on extra-
chromosomal DNA, known as plasmids. Plasmids are known to be exchanged between
bacterial species in nature by conjugation and transformation. The native cry1Ac gene has
been identified on a plasmid of Bt kurstaki strain HD-73 (Lereclus et al. 1993). It has been
demonstrated in the laboratory that Bt strains can interchange toxin-encoding plasmids with
other Bt strains and with other bacterial species (Glare & O'Callaghan 2000). Horizontal gene
transfer may also occur by transduction mediated by bacteriophages (Glare & O'Callaghan
2000).
2.3.3 The uidA (reporter) gene
336. The uidA gene is derived from the common gut bacterium E. coli, which is widespread
in the environment and is also present in the digestive tracts of vertebrates, including humans
(Gilissen et al. 1998; Jefferson et al. 1986). GUS enzyme activity is found in plants,
vertebrates, invertebrates and many common soil microorganisms (Gilissen et al. 1998).
Genes encoding GUS enzymes are thus widespread in microbial populations.
2.3.4 The nptII and aad (antibiotic resistance) genes
337. The nptII and aad genes were originally isolated from mobile genetic elements
(transposons) found in the plasmids and chromosomes of E. coli. E. coli is widespread in
human and animal digestive systems as well as in the environment. Transposons are readily
transferable between bacterial species in nature. The nptII gene is associated with transposon
Tn5 (Beck et al. 1982) and is observed in gram negative bacteria and Pseudomonas sp. While
it is widely dispersed in the environment, other genes that also confer resistance to neomycin
Appendix 5 Transfer of introduced genes to other organisms 61
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
and kanamycin are more common, and also readily transferable among bacterial species
(Smalla et al. 1994; Belgian Biosafety Server 1999). The aad gene is found in several
transposons (e.g. Tn7 and Tn21) and occurs at high frequency among gram-negative bacteria
(Belgian Biosafety Server 1999).
2.3.5 The promoters and other regulatory sequences
338. All of the gene regulatory sequences introduced into the GM cottons function in the
same way as do native regulatory sequences in plants.
339. The CaMV 35S promoter is already ubiquitous in the environment and in human diets,
through the presence of the cauliflower mosaic virus (CaMV) from which it is derived. In
addition, the CaMV 35S promoter occurs at far higher levels in plants naturally infected with
CaMV than in GM plants (Hull et al. 2000). The FMV 35S promoter is functionally very
similar to the CaMV 35S promoter and is already present in the environment through the
presence of the figwort mosaic virus (FMV) from which it is derived.
340. The nos terminator sequence is derived from A. tumefaciens. This is a common soil
bacterium and the genetic element is therefore widespread in the environment. Expression of
the aad gene is regulated by its own bacterial promoter from the common bacterium E. coli.
341. All other regulatory elements are derived from other plants (Arabidopsis, soybean and
pea) that are widespread in the environment.
Section 2.4 Likelihood of gene transfer from Roundup Ready® Flex or Roundup
Ready® Flex/Bollgard II® cotton to microorganisms
342. Most gene transfers have been identified through analyses of gene sequences (Ochman
et al. 2000; Worobey & Holmes 1999). In general, gene transfers are detected over
evolutionary time scales of millions of years (Lawrence & Ochman 1998). Most gene
transfers have been from virus to virus (Lai 1992) or between bacteria (Ochman et al. 2000).
In contrast, transfers of plant genes to other organisms such as bacteria, fungi or viruses are
exceedingly rare (see Sections 2.4.1, 2.4.2 and 2.4.3 of this Appendix).
2.4.1 Bacteria
343. Natural transformation is a mechanism by which transfer of DNA from plants to
microorganisms could have occurred during evolution (Bertolla & Simonet 1999) and is the
only mechanism likely to contribute to a horizontal gene transfer from GM plants to bacteria
(Smalla et al. 2000; EFSA 2004). Natural transformation is a process by which competent
bacteria can generate genetic variability by taking up and integrating free DNA that is present
in their surroundings. This uptake of DNA does not necessarily depend on DNA sequence,
thus indicating the potential of gene transfer from divergent donor organisms (Nielsen 1998).
344. A number of steps and conditions would need to be fulfilled for functional natural
transformation to occur (Bertolla & Simonet 1999). Many of these are highly unlikely,
making the overall likelihood of gene transfer, and of resulting hazards, extremely low:
release of the DNA molecules from plant cells into the environment;
persistence of the free DNA in the environment;
presence of bacterial genotypes capable of developing competence for natural
transformation;
appropriate biotic and abiotic conditions for the development of the competent
state;
uptake of DNA fragments;
Appendix 5 Transfer of introduced genes to other organisms 62
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
integration of the transforming DNA into the chromosome or resident plasmid,
via recombination, or autonomous replication of the transforming DNA;
expression of the genes by the recipient bacterium; and
selective advantage to fix (maintain) the transferred DNA in the gene pool of the
recipient species.
345. Horizontal gene transfer from plants to bacteria has not been demonstrated under natural
conditions (Syvanen 1999) and deliberate attempts to induce such transfers have so far failed
(e.g. Schlüter et al. 1995; Coghlan 2000). However, the initial steps in the chain occur
readily: DNA is released from plant cells during normal decomposition and free DNA from
GM plants has been shown to persist in the environment for several months (Widmer et al.
1997). Many bacterial species can become naturally competent for transformation under the
right conditions (Lorenz & Wackernagel 1994).
346. Transfer of plant DNA to bacteria has been demonstrated only under highly artificial
laboratory and glasshouse conditions, between homologous sequences and under conditions
of selective pressure (De Vries et al. 2001; Gebhard & Smalla 1998; Nielsen et al. 2000;
Mercer et al. 1999; De Vries & Wackernagel 1998), and even then only at a very low
frequency.
347. Using antibiotic selection to detect extremely rare events, Acinobacter sp. cells
containing a defective copy of the neomycin resistance (nptII) gene (with 10 base pairs (bp) or
317 bp of DNA deleted) were observed to incorporate DNA from GM plants (sugarbeet,
tomato, potato or oilseed rape) carrying the intact nptII gene, leading to restoration of
neomycin resistance. Without the artificially introduced homology in the recipient strain, no
uptake of DNA could be detected in Acinobacter sp. (De Vries et al. 2001; Nielsen et al.
2000) or in Pseudomonas stutzeri (De Vries et al. 2001).
348. A recent study reported evidence of low frequency transfer of a small fragment (180 bp)
of an introduced gene derived from GM soybean to microorganisms within the small intestine
of human ileostomists (i.e. individuals in which the terminal ileum is resected and digested
material is diverted from the body to a colostomy bag) (Netherwood et al. 2004). However,
only very low concentrations (1-3 copies per 106 bacteria) of the small fragment were
detected in samples of microorganisms taken from the small bowels of three of seven
ileostomists. Furthermore, the small fragment was only detected after two steps of
amplification: (i) extensive culturing of the microorganism samples, and (ii) Polymerase
Chain Reaction (PCR) analysis. The introduced gene could not be detected in faeces from
human volunteers with intact digestive tracts following the consumption of a meal containing
GM soya, indicating that the introduced gene is normally completely degraded in the large
intestine.
349. Introduced genetic materials acquired by bacteria are unlikely to be of significance
unless they are expressed or alter the expression of host genes. There are barriers to the
expression of exogenous genes in bacteria. For example:
many plant promoters will not be active in bacteria;
processing of the intermediate RNA may be required for protein expression (e.g.
removal of introns to generate functional mRNA for translation), which can not
occur in bacteria;
coding sequences of plant genes may not be efficiently translated in bacteria due
to differences in codon usage (note that the coding sequences of the bacterially
Appendix 5 Transfer of introduced genes to other organisms 63
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
derived cry1Ac, cry2Ab and cp4 epsps genes were modified to enhance expression
in plants); and
processing of an encoded 'pro-protein' may be required for production of a
functional product (e.g. cleavage of the Chloroplast Transit Peptide from plant
EPSPS pro-proteins, see Appendix 1).
350. Prokaryotes have efficient genomes and generally do not contain extraneous DNA
sequences. If the genes are not useful to the organism then there will be no selective
advantage in maintaining them in the genome, and they are not likely to persist. Thus, the
risk of gene transfer leading to harmful consequences is extremely low and greatly exceeded
by the likelihood of transfer of these genes and regulatory sequences from sources other than
the GM cottons (see Section 2.3).
2.4.2 Viruses
351. There is a possibility that introduced viral sequences could recombine with the genome
of another virus infecting the GM cottons to create a novel virus. This is most likely to occur
via homologous recombination, which relies on a high level of sequence similarity.
Homologous recombination occurs naturally within viral populations and results in hybrids
with essentially the same properties as the parental virus (Candresse 1997; Fraile et al. 1997;
Padidam et al. 1999).
352. Homologous recombination between introduced viral genes and infecting viruses has
been observed in GM plants, although at low frequencies and under conditions of selective
pressure (Greene & Allison 1994; Adair & Kearney 2000; Borja et al. 1999; Schoelz &
Wintermantel 1993; Gal et al. 2002; Frischmuth & Stanley 1998; Gal et al. 1991; Greene &
Allison 1996). These cases involved restoration of an infective virus through
complementation of a defective virus by viral sequences introduced into a GM plant genome.
Interactions between introduced viral sequences in GM plants and infecting viruses, and an
assessment of the likelihood of hazards to human health and the environment arising from
these interactions, are discussed in detail in the RARMP for licence application
DIR 047/2003.
353. The major conclusions from the risk assessment were:
while virus-to-virus gene transfer has an important role in viral evolution, the
incorporation of non-viral genes into viral genomes is very rare (Mayo & Jolly
1991);
strong selective pressure would be required for any gene transfer to persist in the
environment; and
viral sequences are already present within plant genomes (Harper et al. 2002) and,
like introduced viral genes in GM plants, these sequences have the potential to
undergo recombination with infecting viruses.
354. The GM cottons proposed for release only contain pieces of the CaMV and FMV
genomes, which by themselves are not pathogenic and operate in the same manner as do
native promoters in plants. The CaMV and FMV promoters, and the introduced genes linked
to them in the GM cottons, would not offer any selective advantage to a virus if they were
transferred.
355. It is very unlikely that any of the other genes or regulatory elements in the GM cottons
would confer a selective advantage on a virus if recombination were to occur.
Appendix 5 Transfer of introduced genes to other organisms 64
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
356. Thus, the likelihood of gene transfer leading to harmful consequences is extremely low
and greatly exceeded by the likelihood of transfer from other sources of these genes and
regulatory sequences (see Section 2.3 of this Appendix).
2.4.3 Fungi
357. Fungi are known to be transformable, and horizontal gene transfer from plants to plant-
associated fungi has been claimed. Uptake of DNA from the host plant by Plasmodiophora
brassicae (#4107 Bryngelsson et al. 1988; #4108 Buhariwalla & Mithen 1995) and uptake of
the hygromycin gene from a GM plant by Aspergillus niger (Hoffman et al. 1994) have been
reported. However, stable integration and inheritance of the plant DNA in the genome of
these fungi has not been substantiated by experimental evidence (Nielsen 1998).
358. Thus, the likelihood of gene transfer leading to harmful consequences is extremely low
and greatly exceeded by the likelihood of transfer from other sources of these genes and
regulatory sequences (see Section 2.3 of this Appendix).
SECTION 3 GENE TRANSFER FROM ROUNDUP READY® FLEX OR ROUNDUP
® ®
READY FLEX/BOLLGARD II COTTON TO ANIMALS (INCLUDING
HUMANS)
Section 3.1 Nature of the gene transfer hazard to animals (including humans)
359. The potential hazards associated with the introduced genes in Roundup Ready® Flex or
Roundup Ready® Flex/Bollgard II® cotton transferring to animals, including humans, could
be highly varied, broadly depending on the nature of the genetic materials and of the species
to which it transferred, and on any changes to the survival or reproductive capacity of the
recipient or its progeny.
360. Hazards associated with the expression of the introduced genes in animal cells would
only be realised if the genes inserted into the genome near a native promoter or if the
associated regulatory sequences were transferred with the gene and were active in the new
host.
361. Insertion of transferred genetic materials into the genome of an animal cell could result
in the knockout of a native gene in that cell, with varied consequences for the cell and the
animal. However, this hazard is not related to the GM nature of the source of the transferred
DNA. This is because in the GM cottons the inserted DNA behaves in the same manner as
does any native genomic DNA. The insertion of DNA into the genome of an animal cell from
any source, either from elsewhere within the same genome or from another species, could
have the same result. As this possible outcome of gene transfer is not related to the genetic
modification it is not discussed further.
Section 3.2 Potential hazards from the introduced genetic materials in animals
(including humans)
3.2.1 The cp4 epsps (herbicide tolerance) gene
362. The expression of this gene in animals would not be expected to lead to any significant
effects, since this gene encodes an enzyme in a biosynthesis pathway that is not present in
animals, including humans.
3.2.2 The cry1Ac and cry2Ab (insecticidal) genes
363. Animals could express the cry genes and become toxic to lepidopteran insect larvae.
However, there is only a small number of insectivorous lepidopteran species in Australia and
these feed on sap-sucking insects or ants (Common 1990). In either case, exposure to the
Appendix 5 Transfer of introduced genes to other organisms 65
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
introduced proteins in the GM cottons is negligible as sap does not contain protein (Raps et al.
2001) and ants are not considered pests of cotton (Forrester & Wilson 1988). As a result,
expression of the cry genes in animals is not likely to pose any consequences for lepidopteran
insects, nor would it confer a selective advantage to the animal.
3.2.3 The uidA (reporter) gene
364. Animals could express the -glucuronidase (GUS) enzyme. Since GUS enzymes and
their genes naturally occur in animals and in their intestinal fauna (#1142 Gilissen et al 1998),
transfer of the uidA gene to animals is not likely to lead to any hazard.
3.2.4 The nptII and aad (antibiotic resistance) genes
365. Animal cells could gain the ability to inactivate the corresponding antibiotics. If the
transfer occurred to humans or other animals treated with these antibiotics, this may affect the
efficacy of the treatment. If transferred and expressed, the NPTII and AAD enzymes would
generally only be active within the recipient animal cells, where appropriate conditions and
co-factors for activity exist, therefore interference with any antibiotic treatment is unlikely.
366. Neomycin and kanamycin have limited medical, veterinary or aquacultural use, because
they have severe side effects and many bacteria are already resistant to them (#425 Flavell et
al 1992), so alternative antibiotics are preferred. Similarly, streptomycin and spectinomycin
have limited medical and veterinary use and many bacteria are already resistant to them
(#1376 Shaw et al 1993; #7119 Heym et al 1994).
367. Antibiotics are not used to control vertebrate animals, so no selective advantage would
result from expression of the resistance genes in animals.
3.2.5 Promoters and other regulatory sequences
368. If these sequences were to be transferred to animals without the associated genes of
Roundup Ready® Flex or Roundup Ready® Flex/Bollgard II® cotton, the expression of native
genes in the animals could be altered with unpredictable effects. The impact could be highly
variable and would be dependent on the resulting phenotypic change induced. However, the
same is true of any plant gene regulatory sequences, if transferred into a new genetic context.
Thus, the potential hazard is generally not increased relative to that of transfer from non-GM
plants.
369. Some of the introduced regulatory sequences are derived from plant pathogens
(cauliflower mosaic virus, figwort mosaic virus, Agrobacterium tumefaciens). However,
these sequences are not pathogenic in themselves nor do they cause any disease symptoms in
GM plants.
370. All of the introduced regulatory sequences operate in the same manner as do native
regulatory elements in plants. The transfer of native regulatory sequences to a new genetic
context, either within a species or from non-GM plants to animals, could also result in
unpredictable effects. Thus, no new hazard will arise due to the presence of the introduced
regulatory sequences in the GM cottons.
Section 3.3 Likelihood of gene transfer from Roundup Ready® Flex or Roundup
Ready® Flex/Bollgard II® cotton to animals (including humans)
371. The most significant route for entry of foreign DNA into animals, including humans, is
via food as it passes through the gastrointestinal tract. The epithelial lining of the
gastrointestinal tract is exposed to foreign DNA released from food. Microorganisms
colonise the whole length of the gastrointestinal tract, aiding the digestive process. However,
the proportion of DNA derived from the introduced genetic materials of GM plants in the
Appendix 5 Transfer of introduced genes to other organisms 66
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
animal diet is extremely low. For example, it has been calculated that in a diet comprising
40% GM maize, the introduced gene(s) would represent 0.00042% of total dietary DNA
intake (Beever & Kemp 2000). The UK Royal Society have concluded that consumption of
introduced DNA in GM foods, viewed in the context of the normal diet, poses no new or
additional risk. Animals, including humans, consume large amounts of DNA derived not only
from food organisms but also from contaminating microorganisms and viruses (The Royal
Society, 2002).
372. A recent study reported evidence of low frequency transfer of a small fragment of an
introduced gene derived from GM soybean to microorganisms within the small intestine of
human ileostomists. In this study, the potential for gene transfer from intestinal
microorganisms to mammalian intestinal epithelial cells was also investigated in the
laboratory but could not be detected, despite the use of very large numbers of GM bacteria
carrying the introduced gene (Netherwood et al. 2004).
373. The fate of DNA in the digestive tract of various animals has been studied and is
discussed in detail in the risk assessments for other licence applications, including for
DIR 020/2002, DIR 021/2002 and DIR 022/2002. These risk assessments concluded that the
likelihood of transfer via food is extremely low and not greater than the likelihood of transfer
from other sources of the introduced genetic materials in the environment (Section 2.3 of this
Appendix).
374. Even in the rare event of plant DNA uptake by animal cells, a further step of
chromosomal integration has not been demonstrated. Furthermore, any uptake of plant DNA
is likely to occur in non-reproductive (somatic) cells such as immune system or gut
epithelium cells, and the introduced gene would not be transmitted to the cells of the progeny.
375. No products from the GM cottons in the proposed field trial will be used for human
food or animal feed. Thus, the likelihood of gene transfer to animals, including humans, is
negligible.
SECTION 4 CONCLUSIONS REGARDING GENE TRANSFER TO OTHER ORGANISMS
Section 4.1 Conclusions regarding gene transfer to other plants
376. It is considered that risks arising through gene transfer from Roundup Ready® Flex or
Roundup Ready® Flex/Bollgard II® cotton to non-GM cotton (G. hirsutum and G.
barbedense) and other plants are very low to negligible because:
the applicant proposes to surround the trial sites by pollen traps or isolation zones
and isolate the sites from naturalised/feral cotton and all other cotton crops to
minimise gene flow and persistence;
gene transfer to other cottons would not pose any risks additional to the risks
posed by the GM cottons themselves; and
genetic incompatibility prevents gene transfer to native cottons and to other plant
species.
377. Gene transfer to other cultivated, volunteer and naturalised cotton will be limited by the
licence conditions imposed, including the requirement to surround the trial sites with a pollen
trap or isolation zone (see Chapter 2 and Appendix 7).
Appendix 5 Transfer of introduced genes to other organisms 67
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Section 4.2 Conclusions regarding gene transfer to microorganisms
378. It is considered that risks arising through transfer of the introduced genetic materials
from Roundup Ready® Flex or Roundup Ready® Flex/Bollgard II® cotton to microorganisms
is negligible because:
the likelihood of gene transfer from plants to bacteria is extremely low, and has
not been demonstrated under natural conditions;
all of the genes introduced into Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons, and other genes with similar functions, are widespread
in the environment, and readily available for transfer from these sources via
demonstrated natural mechanisms; and
the introduced regulatory sequences are already widespread and function in the
same way as do native regulatory sequences in plants.
Section 4.3 Conclusions regarding gene transfer to animals, including humans
379. It is considered that risks arising through transfer of the introduced genetic materials
from the GM cottons to animals, including humans, are negligible because:
the likelihood of gene transfer from plants to animals is extremely low and greatly
exceeded by the likelihood of transfer from other sources of the introduced genes;
all of the genes and regulatory elements introduced into Roundup Ready® Flex
and Roundup Ready® Flex/Bollgard II® cottons, and other genes with similar
functions, are widespread in the environment, and are therefore already available
for transfer; and
no products from the GM cottons will be used in human food or animal feed.
SECTION 5 RESEARCH REQUIREMENTS
380. The OGTR has developed a gene flow research program, involving various GM cotton
licensees (including the applicant), to collect data on pollen-mediated gene flow. Research
will be conducted in a range of current and potential cotton growing areas, therefore, no
additional research requirements relating to gene flow have been imposed.
Appendix 5 Transfer of introduced genes to other organisms 68
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 6 DEVELOPMENT OF RESISTANCE TO HERBICIDES
OR INSECTICIDES
381. Under Section 51 of the Act, the Regulator is required to consider risks to human health
and safety and the environment in preparing the risk assessment and the risk management
plan. In this Appendix, risks posed by the proposed dealing to the environment are
considered in relation to the potential for the development of herbicide tolerance among
weeds and insecticide resistance among target pests.
SECTION 1 REGULATION OF AGRICULTURAL CHEMICALS IN AUSTRALIA
382. Regulation of agricultural chemicals, including herbicides and insecticides, is
principally the responsibility of the Australian Pesticides and Veterinary Medicines Authority
(APVMA) under the Agricultural and Veterinary Chemicals Code 1994 (the Ag Vet Code
Act). Roundup Ready® Flex/Bollgard II® cotton falls under the Ag Vet Code Act definition
of an agricultural chemical product due to its production of insecticidal substances, and is thus
subject to regulation by the APVMA.
383. The APVMA operates the national system that evaluates, registers and regulates
agricultural and veterinary chemical products. Any changes to the use of a product that is
already on the market must also be referred to the APVMA. For commercial products, the
normal form of approval is through registration, but the APVMA may also issue permits for
experimental work that allow restricted use of an agricultural chemical, for example for a
limited period of time or for a limited area.
384. In considering applications for registration or permits, as well as considering potential
health and environmental impacts, the APVMA also considers a number of issues that are
outside the scope of the Gene Technology Regulator‘s assessment, such as efficacy and the
trade implications of residues. The hazard of development of resistance to agricultural
chemicals is also part of the APVMA‘s assessment of agricultural chemical use. The
APVMA can impose conditions on the use of chemical products in both registrations and
permits. These conditions can include restrictions on use, implementation of a resistance
management plan, and ongoing reporting on compliance.
385. The Gene Technology Act 2000 requires the Regulator to consult the APVMA in
relation to the assessment of licence applications involving intentional release of GMOs to the
environment. The Gene Technology (Consequential Amendments) Act 2000 places a
reciprocal obligation upon the APVMA to consult the Gene Technology Regulator in relation
to certain decisions regarding registrations and permits for an agricultural chemical that is or
contains a genetically modified product.
386. The APVMA and the OGTR work closely together to ensure the thorough, coordinated
assessment of these parallel proposals, and that the decisions by both agencies coincide.
Further information about the APVMA‘s assessment and approval processes can be obtained
from www.apvma.gov.au.
SECTION 2 NATURE OF THE HERBICIDE AND INSECTICIDE RESISITANCE
HAZARDS AND LIKELIHOOD OF THE HAZARDS OCCURRING
Section 2.1 Herbicide resistance
387. There is some potential for development of herbicide resistant weeds if Roundup
Ready® herbicide is used inappropriately on Roundup Ready® Flex and Roundup Ready®
Flex/Bollgard II® cottons. Selection for herbicide resistance has an increased probability of
Appendix 6 Development of insecticide resistant pests and herbicide tolerance 69
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
occurring in the long-term if the GM cottons were grown on a large scale and without taking
any steps to manage the risk.
388. Roundup Ready® herbicide is not currently registered for use on cotton beyond the
four-leaf stage of growth. A research permit from the APVMA for use of glyphosate after
this stage on Roundup Ready® Flex and Roundup Ready® Flex/Bollgard II® cottons will be
required for the proposed trial or any further release. Future trials or commercial release of
the GM cottons would also require further applications and approvals by the Regulator.
389. As part of the Deed of Agreement between the Australian Government and Monsanto for
the commercial release of Roundup Ready® and Roundup Ready®/INGARD® cotton in 2000,
Monsanto were required to develop a crop management plan designed to minimise the
potential for development of weeds resistant to glyphosate (Monsanto Australia Limited
2001) (refer DIR 023/2002). This plan has been endorsed by the herbicide resistance
subcommittee of the Transgenic and Insect Management Strategy (TIMS) committee of the
Australian Cotton Growers Research Association and is enforced by Monsanto under its
Technology User Agreement. Included is a requirement to prevent seed set of weeds that
have survived exposure to Roundup Ready® herbicide. Compliance with the current crop
management plan and undertaking of a weed management audit endorsed by the TIMS
subcommittee is required by the APVMA in connection with the use of Roundup Ready®
herbicide and the continued commercial release of Roundup Ready® cotton, as noted in the
licence DIR 023/2003 issued by the Regulator in June 2003.
390. Data collected by Monsanto from growers of Roundup Ready® cotton varieties as part
of the weed management audit (provided in support of licence application DIR 023/2003)
indicates general compliance with this plan. Over three seasons of cultivation of these GM
cottons, there has been no indication of a shift in weeds resistant to glyphosate and no change
in the level of grower satisfaction with this technology.
391. Monsanto has submitted an application to the APVMA for a research permit for the use
of glyphosate (Roundup Ready® herbicide) after the four-leaf stage on Roundup Ready® Flex
and Roundup Ready® Flex/Bollgard II® cottons for the proposed release. The APVMA would
impose conditions if it considered this necessary to manage any identified risks.
Section 2.2 Insecticide resistance
392. Extensive cultivation of Bollgard II® cotton varieties, including Roundup Ready®
Flex/Bollgard II® cotton, could potentially result in the emergence of resistance to the Cry1Ac
and Cry2Ab proteins in the target species (Helicoverpa armigera and H. puntigera) and other
susceptible lepidopteran species feeding on cotton. This would result in a reduction in the
efficacy of these cottons for the control of insect pests, and could also have impacts on the use
of Bt microbial sprays to control insects in other agricultural systems. Potential adverse
effects include attenuation of the potential benefits of growing Bollgard II® cotton varieties to
the environment and human health.
393. It should also be noted that Bollgard II® cotton was developed with the specific
intention of reducing the risk of insects developing resistance to the Cry1Ac or Cry2Ab
proteins. The expression of two insecticidal proteins (which differ sufficiently in their
mechanisms of action) in Bollgard II® cotton, and the fact that the overall insecticidal activity is
increased relative to INGARD® cotton, is expected to delay the emergence of resistant insects. In
2004, INGARD® cotton has been withdrawn from the market in Australia in favour of
Bollgard II® cotton.
394. Bollgard II® cotton falls under the Ag Vet Code Act definition of an agricultural
chemical product, due to its production of two insecticidal substances, and is thus subject to
Appendix 6 Development of insecticide resistant pests and herbicide tolerance 70
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
regulation by the APVMA. In July 2003, the APVMA registered the use of the insecticidal
proteins as produced by the cry1Ac and cry2Ab genes in GM Bollgard II® cotton as insecticidal
products. An insecticide resistance management plan for Bollgard II® cotton has been developed
by the TIMS Committee of the Australian Cotton Growers' Research Association in
consultation with the APVMA (Monsanto Australia Limited 2004). The APVMA requires
implementation of this plan as a condition of registration. The insecticide resistance
management plan is designed to minimise resistance development and requires growers to
employ a number of measures designed to achieve this objective.
395. A condition within this resistance management plan specified that the amount of
combined INGARD® and Bollgard II® cotton to be grown by one grower (as defined by the
grower‘s trading name) in a valley could not exceed 40% of the total cotton being grown by
that grower in that valley. A grower could choose to plant up to 40% Bollgard II® and no
INGARD®. With the withdrawal of INGARD® cotton from the market, the 40% cap has been
lifted for the 2004/05 season. Growers can now choose to grow up to 100% of their cotton
crop as Bollgard II®, however, as part of the insecticide resistance management strategy,
refuge crops must also be grown, to allow Bollgard II® sensitive insects to survive. There
may be non Bt-cotton or other specific plant species.
e.g. for an irrigated Bollgard II® crop of 50 ha:
Refuge Type Size of refuge % of Bollgard II® in a
required (ha) grower‘s total cotton crop
Irrigated, sprayed non-Bt cotton 50 50 %
Irrigated, unsprayed non-Bt cotton 5 90.9 %
Irrigated pigeon pea 2.5 100 %
Irrigated sorghum 7.5 100 %
Irrigated corn 10 100 %
396. Each refuge area must be at least 2 hectares in size. A guide to the 2004/05 Bollgard II®
resistance management plan is available at www.monsanto.com.au/content/cotton/
bollgard_ii_cotton/rmp.pdf.
397. Where Roundup Ready® Flex/Bollgard II® cotton is grown in the proposed trial, these
and all other conditions imposed by the APVMA will need to be complied with.
SECTION 5 CONCLUSIONS REGARDING HERBICIDE AND INSECTICIDE
RESISTANCE
Section 5.1 Herbicide resistance
398. The potential risk of herbicide-resistant weeds developing if the Roundup Ready® Flex
crop-herbicide combination is used inappropriately will be managed by the APVMA, under
conditions of permits or registration for the use of agricultural chemicals in Australia.
Therefore, no specific licence conditions have been imposed in relation to management of
herbicide resistance; however, the requirement to comply with conditions imposed by the
APVMA has been noted.
Section 5.2 Insecticide resistance
399. The potential risk of insects developing resistance to the insecticidal proteins expressed
by Roundup Ready® Flex/Bollgard II® cotton is being managed by the APVMA, under
conditions of registration for the use of insecticidal genes as agricultural chemicals in
Australia. Therefore, no specific licence conditions have been imposed in relation to
Appendix 6 Development of insecticide resistant pests and herbicide tolerance 71
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
management of insecticide resistance; however, the requirement to comply with conditions
imposed by the APVMA has been noted.
Appendix 6 Development of insecticide resistant pests and herbicide tolerance 72
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 7 LICENCE CONDITIONS
Gene Technology Regulation in Australia
The Gene Technology Act 2000 (Cth) and corresponding state and territory legislation form a
substantial part of a range of integrated regulatory measures relevant to controlling genetically
modified organisms (GMOs) and their use.
The Gene Technology Regulator is required to consult with, and take into account advice
from, a range of stakeholders, including regulatory authorities, on risks to human health and
safety and the environment in assessing applications for dealings involving the intentional
release of GMOs into the Australian environment.
Note in relation to approval of genetically modified foods for human consumption
Food Standards Australia New Zealand (FSANZ, formerly the Australia New Zealand Food
Authority), is responsible for human food safety assessment. Monsanto has applied to
FSANZ for evaluation of material from the GM Cottons for use in human food. FSANZ
approval would need to be obtained before any parts of the GM Cottons such as oil and linters
derived from GM Cotton seed could be used as human food. This licence contains a
condition that prohibits this use.
Note in relation to Herbicide and Insecticide Resistance Management
The Gene Technology (Consequential Amendments) Act (2000) requires the Australian
Pesticides and Veterinary Medicines Authority (APVMA) to consult the Gene Technology
Regulator for the purposes of making certain decisions regarding registration or issuing a
permit for a chemical product that is or contains a genetically modified product.
One of the genetically modified organisms referred to in this licence also falls into the
Agricultural and Veterinary Chemicals Code (1994) definition of an agricultural chemical
product, due to its production of an insecticidal substance, and therefore is subject to
regulation by the APVMA.
The APVMA has imposed conditions in connection with the insecticidal activity of one of the
parent organisms (Bollgard II®) for the purpose of managing the development of insecticide
resistance in the target pest species. Conditions of this licence do not relate to management of
insecticide resistance, and do not replace any conditions set by the APVMA. The licence
holder must comply with any conditions imposed by the APVMA in relation to dealings with
this GMO.
The GMOs referred to in this licence have been modified to be tolerant to a herbicide. The
APVMA has responsibility for setting registration conditions for the use of herbicides in
Australia, including implementation of herbicide resistance management programs.
Conditions of this licence do not relate to use of herbicides, and do not replace any conditions
set by the APVMA. The licence holder must comply with any conditions imposed by the
APVMA in relation to the use of herbicides in connection with these GMOs.
Note about where dealings with GMOs are being undertaken pursuant to this licence
Information about where dealings with GMOs are being undertaken pursuant to this licence
can be found here [insert hyperlink]:
Appendix 7 Licence conditions 73
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 1 INTERPRETATIONS AND DEFINITIONS
This licence does not authorise dealings with GMOs that are otherwise prohibited as a result
of the operation of State legislation declaring areas to be GM, GM free, or both, for marketing
purposes.
In this licence:
(a) words and phrases used in this licence have the same meaning as they do in the Act
and the Regulations;
(b) words importing a gender include any other gender;
(c) words in the singular include the plural and words in the plural include the singular;
(d) words importing persons include a partnership and a body whether corporate or
otherwise;
(e) references to any statute or other legislation (whether primary or subordinate) are a
reference to a statute or other legislation of the Commonwealth of Australia as
amended or replaced from time to time and equivalent provisions, if any, in
corresponding State law, unless the contrary intention appears;
(f) where any word or phrase is given a defined meaning, any other part of speech or
other grammatical form in respect of that word has a corresponding meaning;
(g) specific conditions prevail over standard conditions to the extent of any
inconsistency.
In this licence:
‘Act’ means the Gene Technology Act 2000 (Cth) and equivalent provisions in corresponding
State law.
‘Annual Report’ means a written report provided to the Regulator within 90 days of each
anniversary of this licence containing all the information required by this licence to be
provided in the Annual Report.
‘Burial site’ means a site at which seed from the GMOs or the Pollen Trap plants is destroyed
by burial under at least 1 metre of soil.
‘Clean’ (or ‘Cleaned’), as the case requires, means:
(a) in relation to a Location or other area, the Destruction of the GMOs, Pollen Trap
plants and Plant Material in that Location or area, to the reasonable satisfaction of
the Regulator; or
(b) in relation to Equipment, the removal and Destruction of the GMOs, Pollen Trap
plants and Plant Material from the Equipment, to the reasonable satisfaction of the
Regulator.
Appendix 7 Licence conditions 74
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
‘Cotton’ means plants of the species Gossypium hirsutum L.
‘Destroy’, (or ‘Destroyed’ or ‘Destruction’) means, as the case requires, killed by one or
more of the following methods:
(a) stalk pulling; or
(b) uprooting by ploughing; or
(c) burning; or
(d) treatment with herbicide; or
(e) hand weeding; or
(f) autoclaving; or
(g) in respect of seed only, burial under at least 1 metre of soil.
Note (1): ‘As the case requires’ has the effect that, depending on the circumstances, one or
more of these techniques may not be appropriate. For example, in the case of killing the
remains of harvest of the GMOs, treatment of post harvest remains by herbicide would not be
a sufficient mechanism.
Note (2): Where method (g) is adopted, this licence contains additional conditions relating to
burial as a method of destruction.
‘Equipment’ includes harvesters, seeders, storage equipment, transport equipment (eg bags,
containers, trucks), clothing and tools.
‘GM’ means genetically modified.
‘GMOs’ means the genetically modified organism or organisms authorised for release by this
licence.
‘Insect-proof Glasshouse’ means a facility used for growing plants that is constructed,
maintained and used in such a manner as to prevent insects entering and/or leaving the
facility.
‘Isolation Zone’ means the area of land extending outwards 50 metres in all directions from
the outer edge of a Location.
‘Location’ means an area of land where the GMOs are planted and grown and for purposes of
this licence does not include an insect-proof glasshouse.
‘Natural Waterways’ means waterways other than irrigation channels, holding dams or
storage ponds used to collect water runoff from irrigated areas.
‘OGTR’ means the Office of the Gene Technology Regulator.
Appendix 7 Licence conditions 75
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
‘Plant Material’ means viable parts of the GMOs or Pollen Trap plants, including seed,
stubble and pollen, whether from the plant itself or derived from or produced by the plant.
‘Pollen Trap’ means an area of land, extending at least 20 metres in all directions from the
outside edge of a Location.
‘Pollen Trap plant’ means Cotton from a Pollen Trap.
‘Population of Cotton or Gossypium barbadense’ means a group, or groups, of 5 or more
plants per 10 square meters of land belonging to the species Gossypium hirsutum or
Gossypium barbadense.
‘Regulator’ means the Gene Technology Regulator.
‘Sign-off’ means a notice in writing from the Regulator, in respect of a place or a Location,
whichever applies, that post harvest inspection conditions no longer apply in respect of that
place or Location.
‘Volunteer plants’ means progeny of the GMOs or Pollen Trap plants, or regrowth of
previous GM or non-GM cotton plants.
Appendix 7 Licence conditions 76
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 2 GENERAL CONDITIONS
Duration of Licence
1. This licence remains in force until it is suspended, cancelled or surrendered. No
dealings with GMOs are authorised during any period of suspension.
Holder of Licence
2. The holder of this licence (‗the licence holder‘) is Monsanto Australia Ltd.
Project Supervisor
3. The Project Supervisor in respect of this licence is identified at Attachment A.
4. The licence holder must immediately notify the Regulator in writing if any of the
contact details of the Project Supervisor change.
No dealings with GMOs except as authorised by this licence
5. Persons covered by this licence must not deal with the GMOs except as expressly
permitted by this licence.
Persons covered by this GMO licence
6. The persons covered by this licence are the licence holder and employees, agents or
contractors of the licence holder and other persons who are, or have been, engaged to
undertake any activity in connection with GMOs grown in a Location pursuant to this
licence.
Informing people of their obligations
7. The licence holder must inform any person covered by this licence, to whom a particular
condition of this licence applies, of the following:
(a) the particular condition (including any variations of it);
(b) the cancellation or suspension of the licence;
(c) the surrender of the licence.
8. The licence holder must provide the Regulator, on the Regulator‘s written request,
signed statements from persons covered by this licence that the licence holder has
informed those people of the conditions of this licence that apply to them.
Applicant to notify of circumstances that might affect suitability
9. The licence holder must immediately, by notice in writing, inform the Regulator of:
(a) any relevant conviction of the licence holder occurring after the commencement
of this licence;
(b) any revocation or suspension of a licence or permit held by the licence holder
under a law of the Australian Government, a State or a foreign country, being a
law relating to the health and safety of people or the environment;
(c) any event or circumstances occurring after the commencement of this licence that
would affect the capacity of the holder of his licence to meet the conditions in it.
Appendix 7 Licence conditions 77
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Licence holder must provide information on matters related to suitability
10. The licence holder must provide information related to the licence holder‘s ongoing
suitability to hold a licence when requested to do so in writing by the Regulator and
must provide the information within a time period stipulated by the Regulator.
Additional information to be given to the Regulator
11. It is a condition of a licence that the licence holder inform the Regulator if the licence
holder:
(a) becomes aware of additional information as to any risks to the health and safety of
people, or to the environment, associated with the dealings authorised by the
licence; or
(b) becomes aware of any contraventions of the licence by a person covered by the
licence; or
(c) becomes aware of any unintended effects of the dealings authorised by the
licence.
12. The licence holder must provide the information required by paragraphs (a) (b) and (c)
of the immediately preceding condition to the Regulator as soon as practically and
reasonably possible and must also include the information in the Annual Report.
People dealing with GMOs must allow auditing and monitoring of the dealing
13. If a person is authorised by this licence to deal with GMOs and a particular condition of
this licence applies to the dealing by that person, the person must allow the Regulator, or
a person authorised by the Regulator, to enter premises where the dealing is being
undertaken, for the purposes of auditing or monitoring the dealing.
Remaining an Accredited organisation
14. The licence holder must, at all times, remain an accredited organisation in accordance
with the Act and comply with its instrument of accreditation.
Appendix 7 Licence conditions 78
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
SECTION 3 SPECIFIC CONDITIONS
GMOs covered by this licence
1. The GMOs covered by this licence are described at Attachment B.
Permitted dealings
2. The permitted dealings with the GMOs are to plant, grow and conduct experiments with
the GMOs, and the possession, supply, use, transport and disposal of the GMOs for the
purpose of any of the permitted dealings with the GMOs, or in the course of any of these
dealings.
Locations and size of trial
3. The permitted dealings with the GMOs may be undertaken during the cotton growing
seasons in Summer 2005/2006 and Winter 2006 within the Shires set out in the
following table:
Table 1: Shires where permitted dealings with the GMOs may be conducted in Summer
2005/2006 and Winter 2006
NSW QLD NT WA
Balranald Aramac Katherine Broome
Bingara Balonne Wyndham – East Kimberley
Bland Banana
Bogan Burdekin
Bourke Cambooya
Brewarrina Chinchilla
Broken Hill Clifton
Carrathool Dalby
Central Darling Emerald
Coonamble Fitzroy
Deniliquin Flinders
Dubbo Gatton
Forbes Goondiwindi
Griffith Inglewood
Gunnedah Jondaryan
Hay Kingaroy
Jerilderie Milmerran
Lachlan Monto
Moree Plains Murilla
Murrumbidgee Murweh
Narrabri Peak Downs
Narromine Pittsworth
Parry Richmond
Quirindi Rosalie
Tamworth Toowoomba
Walgett Waggamba
Warren Wambo
Yallaroi Warroo
Wondai
4. The GMOs may also be grown in Insect-proof Glasshouses if grown pursuant to the
special conditions contained in conditions 23 to 29.
5. For the cotton growing seasons in Summer 2005/2006 and Winter 2006, the maximum
number of Locations for those growing seasons (where permitted dealings may be
Appendix 7 Licence conditions 79
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
conducted) are set out in Table 2 at Column 2. The maximum combined area of all
Locations where permitted dealings may occur in those growing seasons is limited to the
size set out in Table 2 at Column 3.
Note: This condition does not apply to insect-proof glasshouses. See the definition of
Location.
Table 2: Maximum numbers of Locations and combined areas
Growing season Maximum number Maximum combined
of Locations area of all Locations
Summer 2005/2006 86 1770 ha
Winter 2006 5 45 ha
6. In the Summer 2005/2006 growing season, the maximum size of any individual
Location is 100 hectares. (No individual field trial site can be more than 100 hectares.)
7. The licence holder must be able to access and control a Location to the extent necessary
to comply with this licence, for the duration of the life of the licence.
Notice of planting
8. The licence holder must provide a notice in writing to the Regulator each time the
GMOs are planted at a Location. The notice must set out:
(a) the date on which planting of the GMOs commenced;
(b) details of the Location where the GMOs are planted, including a street address
and GPS coordinates for the Location;
(c) the period during which the licence holder considers the GMOs are likely to
flower; and
(d) the period during which the licence holder considers the GMOs are likely to be
harvested (or Destroyed in lieu of harvest).
9. The notice must be provided to the Regulator within 14 days of the date on which
planting of the GMOs commenced.
Notice of Harvest and Cleaning
10. The licence holder must provide a notice in writing to the Regulator stating when the
Location was harvested and when the Location was Cleaned (following harvest or
Destruction of GMOs at a Location in lieu of harvest). The notice must be provided to
the Regulator within 14 days of the date on which Cleaning of the Location concluded.
Measures to manage Gene Flow
11. Each Location north of latitude 22 degrees South must be surrounded by an Isolation
Zone.
12. Each Location south of latitude 22 degrees South must be surrounded by a Pollen Trap.
Appendix 7 Licence conditions 80
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Restrictions on the use of Isolation Zones
13. A Location cannot be surrounded by an Isolation Zone if there is:
(a) a naturalised Population of Cotton or of Gossypium barbadense within 450m of
the Location; or
(b) a Population of Cotton or Gossypium barbadense within 450m of the Location,
other than a population of Cotton planted pursuant to this licence.
Other conditions about Pollen Traps
14. Each Pollen Trap must be planted out with non-genetically modified Cotton, Bollgard
II Cotton, Roundup Ready Cotton or Bollgard II/Roundup Ready Cotton. These
plants must be grown in such a way as to reasonably promote a dense and vigorous
growth and flowering of the plants at the same time as the GMOs.
15. The edge of every Pollen Trap that is farthest from the GMOs (the ‗outer edge of the
Pollen Trap‘) must not be within 50 metres of a Natural Waterway.
16. Pollen Trap plants must be handled and controlled as if they are the GMOs (ie subject to
other applicable conditions elsewhere in this licence) and Plant Material must be
handled and controlled as if it is the GMOs (ie subject to other applicable conditions
elsewhere in this licence).
17. A Pollen Trap must be able to be accessed and controlled by the licence holder to an
extent that is commensurate with the licence holder‘s rights to access and control the
Location within it.
Other conditions about Isolation Zones
18. No Cotton or Gossypium barbadense of any kind may be grown in an Isolation Zone
while the GMOs are being grown at the Location within it.
19. No flowering Cotton or Gossypium barbadense plants may be present in an Isolation
Zone while the GMOs are being grown at the Location within it.
20. Any vegetative Cotton or Gossypium barbadense plants occurring in an Isolation Zone
must be Destroyed prior to flowering.
21. An Isolation Zone must be able to be accessed and controlled by a licence holder to an
extent that is commensurate with the licence holder‘s rights to access the Location
within it.
22. If a Population of Cotton (other than a Population of Cotton planted pursuant to this
licence) or Gossypium barbadense is planted within 450m of a Location with an
Isolation Zone around it, either this Population or the GMOs in the Location must be
Destroyed prior to flowering. If GMOs in a Location are Destroyed pursuant to this
condition, they are taken to have been harvested for the purposes of this licence.
Special conditions that apply where GMOs are grown in an Insect-proof Glasshouse
23. If the GMOs are grown in an Insect-proof Glasshouse (‗glasshouse‘):
(a) the GMOs must be grown in pots; and
(b) while the GMOs are growing in the glasshouse, sticky pest strips must be
maintained within the glasshouse; and
Appendix 7 Licence conditions 81
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
(c) the glasshouse must be maintained and kept insect-proof at all times; and
(d) the GMOs must be grown only during the period between September 2005 and
November 2006 only in the Shires set out in table 1; and
(e) no more than 10 glasshouses may be used for growing the GMOs at any one time;
and
(f) the licence holder must be able to control and access the glasshouse to the extent
necessary to comply with this licence for the duration of the licence; and
(g) conditions 30 to 43, 61 and 64 apply to GMOs being grown in the glasshouses as
if the glasshouses were Locations; and
(h) all plants growing in the glasshouse must be treated as if they were the GMOs.
24. When any of the GMOs growing in glasshouses are no longer required for purposes of
dealings authorised under this licence:
(a) soil from the pots in which the GMOs were grown, must be relocated, as soon as
practicable, to a Location that is subject to post-harvest monitoring, or, sterilised
using non-pressurised steam sterilisation; and
(b) the GMOs and the Plant Material must be:
(i) relocated to a Location that is subject to post-harvest monitoring, or
(ii) relocated to a facility described in condition 31, or
(iii) Destroyed.
25. When the last GMO in a glasshouse has been Destroyed or relocated, the glasshouse
must be Cleaned.
26. GMOs and Plant Materials must be transported in accordance with the OGTR
Guidelines for the Transport of GMOs (June 2001) issued by the Regulator.
27. Written notices must be provided to the Regulator within 14 days of the first GMO
being planted in each glasshouse. The notice must include the street address and GPS
coordinates of the glasshouse.
28. Written notices must be provided to the Regulator within 14 days of any soil, GMOs or
Plant Material being relocated to a Location under condition 24. The notice must
include the street address and GPS coordinates of the Location to which the soil, GMOs
or Plant Material was relocated and the date of relocation.
29. Written notices must be provided to the Regulator within 14 days of the last GMO in a
glasshouse being Destroyed or relocated. The notice must include the street address and
GPS coordinates of the glasshouse.
Note: The immediately preceding condition is the last of the Special Conditions applying to
glasshouses.
Material from the GMOs may be collected
30. Any material from the GMOs, including Plant Material, may be collected from a
Location for the purpose of conducting experiments on it.
31. Any material from the GMOs, including Plant Material, that is collected may be
transported off the Location to:
(a) a facility certified by the Regulator to physical containment level 2 (PC2); or
Appendix 7 Licence conditions 82
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
(b) a facility approved in writing by the Regulator and signed so as to indicate that
GM Plant Material is stored within the facility,
and may be experimented on and stored in any one or more of these facilities.
32. After any experiments with the material from the GMOs are completed, the Plant
Material must be Destroyed.
Harvest and post-harvest procedures
33. If the GMOs or Pollen Trap plants are harvested, they must be harvested separately from
any other crop.
34. If seed Cotton harvested from the GMOs or from Pollen Trap plants is ginned, it must
be ginned separately from any other crop.
35. Following ginning, seed from the GMOs and Pollen Trap plants must be:
(a) stored in a sealed container, within a locked facility that is signed so as to indicate
that GM Cotton seed is stored within the facility;
(b) exported;
(c) Destroyed by burning; or
(d) Destroyed by burial under at least 1 metre of soil.
36. Any GM Cotton seed obtained from ginning may only be transported to the extent
necessary to store them, export them, Destroy them by burning or burial, or take them to
a facility certified by the Regulator to physical containment level 2 (PC2).
37. Cotton lint obtained from ginning of seed Cotton harvested from the GMOs or Pollen
Trap plants may be sold.
Conditions relating to burial of seed
38. If seed from the GMOs or Pollen Trap plants is buried, the licence holder must:
(a) Within 30 days of burial, provide the Regulator by notice in writing of the precise
location of the Burial site (GPS coordinates and either a street address or other
directions to the Burial site) and the date on which it was buried.
(b) Monitor the burial site at least once every 3 months for a period of 12 months to
identify:
(i) any significant disturbance that may effect the emergence of volunteer
plants and if disturbance is identified, notify the Regulator of appropriate
remedial action taken; and
(ii) any emergence of Volunteer plants. If Volunteer plants are identified, the
Burial site must be Cleaned.
Cleaning – post harvest and generally
39. Equipment, a Location or other area (including a gin) used pursuant to this licence in
respect of GMOs, Pollen Trap plants or Plant Material must be Cleaned.
40. For each Location, either within 14 days of harvest of the GMOs or within 9 months of
planting of the GMOs, whichever occurs first, the Location must be Cleaned.
Appendix 7 Licence conditions 83
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
41. If Equipment is Cleaned, the area in which the Equipment is Cleaned must also be
Cleaned. (For the sake of clarity, it is not necessary for Equipment to be Cleaned only
at a Location.)
42. Cleaning must occur immediately or as soon as practicable after use and before it is used
for any other purpose. (For example, if GM seed is ginned, the gin must be Cleaned
immediately following its use and before any other crop is ginned.)
43. On the request of the Regulator, the Regulator must be provided with written
documentation of the procedures in place to ensure continuing compliance with the
Cleaning conditions in this licence.
Inspection
44. Following Cleaning of a Location or other area, the following places must be inspected
for the existence of Volunteer plants:
(a) the Location;
(b) the Pollen Trap in respect of the Location (if any);
(c) the Isolation Zone in respect of the Location (if any);
(d) irrigation channels and drains through which water flows to and from the Location
and the Pollen Trap;
(e) the area between the Location and any sinkholes into which water from the
Location flows, as well as the immediate area surrounding these sinkholes;
(f) any areas used to Clean Equipment used in connection with the GMOs at the
Location or to Destroy the GMOs, Pollen Trap plants or Plant Material.
45. Inspection must be performed by a person who is able to recognise Volunteer plants.
46. For each Location, all the places required to be inspected must be inspected at least once
every 2 months, commencing on the last day of Cleaning of the Location and continuing
until the Regulator has issued a Sign-off.
47. The results of inspection activities must be recorded in a logbook or paper file. The
logbook or paper file must be available on request for examination or photocopying by
the OGTR. The findings of the inspections as recorded in the logbook or paper file must
be included in the licence holder‘s Annual Report to the Regulator. The logbook or
paper file must contain at least the following:
(a) details of the areas inspected;
(b) details of the date of inspection;
(c) the names of the person or persons who undertook the monitoring and details of the
experience, training or qualification that enabled them to recognise Volunteer plants;
(d) the number of Volunteer plants observed, if any;
(e) details of the development stages reached by the Volunteer plants, if any; and
Appendix 7 Licence conditions 84
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
(f) details of methods used to Destroy Volunteer plants, if any.
48. Any Volunteer plant identified must be Destroyed prior to the plant flowering.
49. For each Location, and all places in respect of that particular Location as listed in
condition 44,
(a) if inspections have been routinely completed in that Location and all those places for
a period of at least 12 months, and,
(b) if inspection records for that Location and all those places show that no Volunteers
have been observed in the most recent 6 month inspection period,
the licence holder may make written application to the Regulator that these inspection
conditions no longer apply to that particular Location and the places in respect of that
particular Location as listed in condition 44.
50. These inspection conditions do not apply in respect of a place if the licence holder has
received a Sign-off.
General conditions on use of Locations post-harvest
51. If the GMOs are grown at a Location, no plants may be planted at the Location, Pollen
Trap or Isolation Zone in respect of the Location until inspection obligations are
completed unless:
(a) the plants are grasses (grass pastures), cereals (cereal crops); or
(b) the plants are plants agreed to in writing by the Regulator.
Transportation of the GMOs, Pollen Trap plants and Plant Material
52. Subject to the conditions immediately below in respect of transportation, the GMOs,
Pollen Trap plants and Plant Material must be transported in accordance with the OGTR
Guidelines for the Transport of GMOs (June 2001) issued by the Regulator.
53. Harvested GMOs, Pollen Trap plants and Plant Material may be transported to a ginning
facility in a cotton module that is:
(a) completely enclosed within 2 layers of tarpaulin (‗double wrapped in tarpaulin‘);
(b) completely enclosed within a layer of tarpaulin inside a layer of shade cloth
(‗double wrapped in tarpaulin and shade cloth‘); or
(c) contained within an enclosed chain-bed truck specifically designed for the
purpose of transporting cotton modules.
54. Fuzzy Cottonseed from the GMOs or Pollen Trap plants may be transported between
Kununurra and Narrabri within calico bags that are tied shut and then placed inside
closed wooden boxes. Wooden boxes used to transport the fuzzy Cottonseed may have
ventilation holes, up to 25mm in diameter, if the ventilation holes are securely covered
with fly-proof wire mesh.
Appendix 7 Licence conditions 85
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
55. Cotton lint derived from GMOs and Pollen Trap plants from ginning is not subject to
transportation conditions.
56. Every container used to transport the GMOs, Pollen Trap plants and Plant Material must
be labelled:
(a) to indicate that it contains genetically modified Cotton; and
(b) with telephone contact numbers for the licence holder and instructions to contact the
licence holder in the event that the container is broken or misdirected.
57. The licence holder must have in place accounting procedures to verify whether the same
quantity of GMOs, Pollen Trap plants or Plant Material sent is delivered and must
document routes, methods and procedures used for transportation of GMOs, Pollen Trap
plants and Plant Material.
Contingency Plans
58. Within 30 days of the date of the commencement of this licence, a written Contingency
Plan must be submitted to the Regulator detailing measures to be taken in the event of
the unintended presence of the GMOs, Pollen Trap plants or Plant Material, outside an
area that must be inspected.
59. The Contingency Plan must include details of procedures to:
(a) ensure the Regulator is notified immediately if the licence holder becomes aware of
the event;
(b) destroy any of the GMOs, Pollen Trap plants or Plant Material; and
(c) inspect for and Destroy any Volunteer plants that may exist as a result of the event.
60. The Contingency Plan must be implemented in the event that the unintended presence of
the GMOs, Pollen Trap plants and Plant Material is discovered outside an area that must
be inspected.
Compliance Management Plan
61. Prior to growing the GMOs, a written Compliance Management Plan must be provided
to the Regulator. The Compliance Management Plan must describe in detail how the
licence holder intends to ensure compliance with these conditions and document that
compliance.
Reporting
62. The licence holder must provide an Annual Report to the Regulator.
Testing methodology
63. The licence holder must provide a written instrument to the Regulator describing an
experimental method that is capable of reliably detecting the presence of the GMOs and
the presence of the genetic modifications described in this licence (at Attachment B) in a
recipient organism. The instrument must be provided within 30 days of the issuing of
this licence.
Appendix 7 Licence conditions 86
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Use of GMOs, Pollen Trap plants and Plant Material
64. The GMOs, Pollen Trap plants and Plant Material must not be used, sold or otherwise
disposed of for any purpose which would involve or result in their use as food for
animals or humans.
Appendix 7 Licence conditions 87
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 8 LEGISLATIVE REQUIREMENTS FOR ASSESSING
DEALINGS INVOLVING INTENTIONAL RELEASES
SECTION 1 THE REGULATION OF GENE TECHNOLOGY IN AUSTRALIA
400. The Gene Technology Act 2000 (the Act) took effect on 21 June 2001. The Act,
supported by the Gene Technology Regulations 2001, an inter-governmental agreement and
corresponding legislation that is being enacted in each State and Territory, underpins
Australia's nationally consistent regulatory system for gene technology. Its objective is to
protect the health and safety of people, and the environment, by identifying risks posed by or
as a result of gene technology, and managing those risks by regulating certain dealings with
genetically modified organisms (GMOs). The regulatory system replaces the former
voluntary system overseen by the Genetic Manipulation Advisory Committee (GMAC).
401. The Act establishes a statutory officer, the Gene Technology Regulator (the Regulator),
to administer the legislation and make decisions under the legislation.
402. The Regulator is supported by the Office of the Gene Technology Regulator (OGTR),
an Australian Government regulatory agency located within the Health and Ageing portfolio.
403. The Act prohibits persons from dealing with GMOs unless the dealing is exempt, a
Notifiable Low Risk Dealing, on the Register of GMOs, or licensed by the Regulator (see
Section 31 of the Act).
404. The requirements under the legislation for consultation and for considering and
assessing licence applications and preparing risk assessment and risk management plans
(RARMPs) are discussed in detail in Division 4, Part 5 of the Act and summarised below.
405. Detailed information about the national regulatory system and the gene technology
legislation is also available from the OGTR website (www.ogtr.gov.au).
SECTION 2 THE LICENCE APPLICATION
406. Licence applications for dealings involving the intentional release (DIR) of a genetically
modified organism into the environment must be submitted in accordance with the
requirements of Section 40 of the Act. As required by Schedule 4, Part 2 of the Regulations,
the application must include information about:
the parent organism;
the GMOs;
the proposed dealing with the GMOs;
interaction between the GMOs and the environment;
risks the GMOs may pose to the health and safety of people;
risk management;
previous assessments of approvals; and
the suitability of the applicant.
407. The application must also contain:
additional information required for a GMO that is:
a plant;
a micro-organism (not living in or on animals and not a live vaccine);
Appendix 8 Legislative requirements for assessing dealings involving intentional releases 88
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
a micro-organism that lives in or on animals;
a live vaccine for use in animals;
a vertebrate animal;
an aquatic organism;
an invertebrate animal;
to be used for biological control;
to be used for bioremediation; and
intended to be used as food for human or vertebrate animal consumption;
supporting information from the Institutional Biosafety Committee.
408. A preliminary screening of an application is undertaken by OGTR staff to determine
whether it complies with the Act and the Regulations, by containing the required information.
If this information is provided in the application, the Regulator may then accept the
application for formal consideration. Section 43 of the Act provides that the Regulator is not
required to consider an application if the application does not contain the required
information.
409. After accepting an application for consideration, the Regulator must decide to issue, or
refuse to issue, a licence. The decision must be taken following an extensive consultation and
evaluation process, as detailed in Sections 3-6 of this Appendix. Regulation 8 of the
Regulations prescribes a period of 170 working days within which this decision must be
taken. This period does not include weekends or public holidays in the Australian Capital
Territory. Also, this period does not include any days in which the Regulator is unable to
progress the application because information sought from the applicant in relation to the
application has not been received.
SECTION 3 THE INITIAL CONSULTATION PROCESSES
410. In accordance with Section 50 of the Act, the Regulator must seek advice in preparing a
RARMP from prescribed agencies:
State and Territory Governments;
the Gene Technology Technical Advisory Committee (GTTAC);
prescribed Australian Government agencies (Regulation 9 of the Gene Technology
Regulations 2001 refers);
the Australian Government Minister for Environment and Heritage; and
relevant local council(s) where the release is proposed.
411. Section 49 of the Act requires that if the Regulator is satisfied that at least one of the
dealings proposed to be authorised by the licence may pose significant risks to the health and
safety of people or to the environment, the Regulator must publish a notice (in national and
regional news papers, in the Gazette and on the OGTR website) in respect of the application,
inviting written submissions on whether the licence should be issued.
412. As a measure over and above those required under the Act, in order to promote the
openness and transparency of the regulatory system, the Regulator may take other steps. For
example, receipt of applications is notified to the public by posting a notice of each
Appendix 8 Legislative requirements for assessing dealings involving intentional releases 89
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
application's receipt on the OGTR website and directly advising those on the OGTR mailing
list. Copies of applications are available on request from the OGTR.
SECTION 4 THE EVALUATION PROCESSES
413. The risk assessment process is carried out in accordance with the Act and Regulations,
using the Risk Analysis Framework (the Framework) developed by the Regulator (available
on the OGTR website). It also takes into account the guidelines and risk assessment strategies
used by related agencies both in Australia and overseas. The Framework was developed in
consultation with the States and Territories, Australian Government agencies, GTTAC and the
public. Its purpose is to provide general guidance to applicants and evaluators and other
stakeholders in identifying and assessing the risks posed by GMOs and in determining the
measures necessary to manage any such risks.
414. In undertaking a risk assessment, the following are considered and analysed:
the data presented in the proponent's application;
data provided previously to GMAC, the interim OGTR or the OGTR in respect of
previous releases of relevant GMOs;
submissions or advice from States and Territories, Australian Government
agencies and the Australian Government Minister for Environment and Heritage
and the public;
advice from GTTAC;
information from other national regulatory agencies; and
current scientific knowledge and the scientific literature.
415. In considering this information and preparing the RARMP, the following specific
matters are taken into account, as set out in Section 49 and required by Section 51 of the Act:
the risks posed to human health and safety or risks to the environment;
the properties of the organism to which the dealings relate before it became a
GMO;
the effect, or the expected effect, of the genetic modification that has occurred on
the properties of the organism;
provisions for limiting the dissemination or persistence of the GMO or its genetic
material in the environment;
the potential for spread or persistence of the GMO or its genetic material in the
environment;
the extent or scale of the proposed dealings; and
any likely impacts of the proposed dealings on the health and safety of people.
416. In accordance with Regulation 10 of the Regulations, the following are also taken into
account:
any previous assessment, in Australia or overseas, in relation to allowing or
approving dealings with the GMO;
the potential of the GMO concerned to:
be harmful to other organisms;
Appendix 8 Legislative requirements for assessing dealings involving intentional releases 90
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
adversely affect any ecosystems;
transfer genetic material to another organism;
spread, or persist, in the environment;
have, in comparison to related organisms, a selective advantage in the
environment; and
be toxic, allergenic or pathogenic to other organisms.
the short and long term when taking these factors into account.
SECTION 5 FURTHER CONSULTATION
417. Having prepared a risk assessment and a risk management plan, the Regulator must,
under Section 52 of the Act, seek comment from stakeholders, including those outlined in
Section 3 and the public.
418. All issues relating to the protection of human health and safety and the environment
raised in written submissions on an application or a risk assessment and a risk management
plan are considered carefully, and weighed against the body of current scientific information,
in reaching the conclusions set out in a final RARMP. Section 56 of the Act requires that
these be taken into account in making a decision on whether or not to issue a licence for the
proposed release.
419. Comments received in written submissions on this RARMP are very important in
shaping the final RARMP and in informing the Regulator's decision on an application. A
summary of public submissions and an indication of where such issues have been taken into
account are provided in an Appendix to the final RARMP.
420. It is important to note that the legislation requires the Regulator to base the licence
decision on whether risks posed by the dealings are able to be managed so as to protect
human health and safety and the environment. Matters in submissions that do not address
these issues and/or concern broader issues outside the objective of the legislation will not be
considered in the assessment process. In most instances, as determined in the extensive
consultation process that led to the development of the legislation, they fall within the
responsibilities of other authorities.
SECTION 6 DECISION ON LICENCE
421. Having taken the required steps for assessment of a licence application, the Regulator
must decide whether to issue or refuse a licence (Section 55 of the Act). The Regulator must
not issue the licence unless satisfied that any risks posed by the dealings proposed to be
authorised by the licence are able to be managed in such a way as to protect the health and
safety of people and the environment.
422. The Regulator must also be satisfied, under Section 57 of the Act, that the applicant is a
suitable person to hold the licence. Section 58 outlines matters the Regulator must consider in
deciding whether a person or company is suitable to hold a licence e.g.:
any relevant convictions;
any relevant revocations or suspensions of a licence or permit; and
the capacity of the person or company to meet the conditions of the licence.
423. The Regulator carefully considers all of this information including information supplied
in a declaration signed by licence applicants.
Appendix 8 Legislative requirements for assessing dealings involving intentional releases 91
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
424. The Monitoring and Compliance Section of the OGTR compiles compliance histories of
applicants, considering all previous approvals to deal with GMOs under the Act and the
previous voluntary system. These histories as well as other information such as follow-up
actions from audits and recent convictions may be taken into account. The ability of an
organisation to provide resources to adequately meet monitoring and compliance
requirements may also be taken into account.
425. If a licence is issued, the Regulator may impose licence conditions (Section 62 of the
Act). For example, conditions may be imposed to:
limit the scope of the dealings;
require documentation and record-keeping;
require a level of containment;
specify waste disposal methods;
manage risks posed to the health and safety of people, or to the environment;
require data collection, including studies to be conducted;
limit the geographic area in which the dealings may occur;
require contingency planning in respect of unintended effects of the dealings; and
limit the dissemination or persistence of the GMO or its genetic material in the
environment.
426. It is also required as a condition of a licence that the licence holder inform any person
covered by the licence of any condition of the licence which applies to them (Section 63 of
the Act). Access to the site of a dealing must also be provided to persons authorised by the
Regulator for the purpose of auditing and monitoring the dealing and compliance with other
licence conditions (Section 64 of the Act). It is a condition of any licence that the licence
holder inform the Regulator of:
any new information as to any risks to the health and safety of people, or to the
environment, associated with the dealings authorised by the licence;
any contraventions of the licence by a person covered by the licence; and
any unintended effects of the dealings authorised by the licence.
427. It should be noted that, as well as imposing licence conditions, the Regulator has
additional options for risk management. The Regulator has the legislative capacity to enforce
compliance with licence conditions, and indeed, to direct a licence holder to take any steps the
Regulator deems necessary to protect the health and safety of people or the environment. The
OGTR also independently monitors trial sites to determine whether the licence holder is
complying with the licence conditions, or whether there are any unforseen problems.
Appendix 8 Legislative requirements for assessing dealings involving intentional releases 92
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 9 SUMMARY OF PUBLIC SUBMISSIONS
428. The Regulator received 8 submissions from members of the public on the RARMP for
this application. A summary of these written submissions and how they were considered is
provided below. The key issues raised by the public that related to risks to human health and
safety or the environment were:
risks to human health and safety due to the genetic modifications (Appendix 2
refers);
potential weediness of the GM cottons (Appendix 4 refers);
containment of the GM cotton plants, material from the GM cotton plants and the
introduced genetic material during the trial (Appendices 4, 5 and 7 refer);
difficulties that might occur relating to cleaning of the release sites (if unexpected
cleaning of all sites was required) (Appendices 4 and 7 refer); and
procedural concerns (Appendix 8 refers).
429. Other issues raised in submissions included: corporate versus public interest,
marketability/trade implications, political issues, and potential legal issues arising from
difficulties in segregating commercialised GM and non-GM crops. However, the focus of the
gene technology legislation is upon the protection of human health and safety and the
environment. These issues can therefore not be considered within the scope of assessments
required to be conducted under the Act.
430. In accordance with Section 56 of the Act, the Regulator has taken into account all issues
raised in written submissions that related to risks to human health and safety or to the
environment from the release of the GM cottons in finalising the RARMP. These issues were
considered carefully and weighed against the body of current scientific information in
reaching the conclusions set out in this document.
Abbreviations: App: Appendix; Ch: Chapter; LC: licence conditions
Issues raised: Av: availability of information; C: containment; EN: environmental risks; FC: food
chain; FS: food safety; G: gene transfer; H: human health and safety; HU: herbicide use;
MA: markets; OSA: outside scope of the assessment; Pc: procedure; RD: resistance development;
RM: risk management; SEG: segregation; T: toxicity; W: weediness
a
Submission from: A: agricultural/industry organisation; E: environmental organisation; I: individual
Sub. a
Type Summary of issues raised Issue Consideration of issue
No.
1 I GM cotton poses a significant threat to the G App 5
genetic integrity of Australian native cotton C. fraseri belongs to a
(Cochlospermum fraseri) different plant family and
cannot cross with cotton
(Gossypium hirsutum). This
is because of well known
genetic differences
Appendix 9 Summary of public submissions 93
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Concerns about biological contamination. The G App 5
rhizomes and bacteria that live in and about Transfer of genetic material
the cotton roots transfer genetic material to between plants and bacteria
other plants through the soil. is known as horizontal gene
transfer. This happens only
very rarely and in for this
application, the risks to
human health and safety and
the environment have been
assessed as negligible.
Is concerned that insects will transfer plant C; RM; FC; G App 5 and LC
debris, seeds, pollen, and so forth, to the rest
of the food chain.
Further pursuits involving the smallest RM; SEG LC
possibility of GM contamination of the Licence conditions prevent
Australian ecosystem should be terminated the use of GM cotton
because of the gross criminal negligence of materials resulting from this
corporations, producers and government with release in feed and food.
regards to previous failure in GM product
containment.
“The pursuit of GM product in the Citizens rights OSA
Commonwealth for the purpose of profit to a under the
minority who are not citizens of the Commonwealth
Commonwealth is in violation of those laws of
the Commonwealth that preserve the right of
every citizen of the Commonwealth to an
equal share of the benefits and responsibilities
of citizenship.”
“Corporate control over the food supply is a Corporate control OSA
threat to the strategic position of the of food chain
Commonwealth and its citizens, and for
government to pursue ends contrary to the
interests of the people of the Commonwealth
is treason and conspiracy to sedition which are
capital crimes requiring the harshest of
penalties to be dealt out to all government
employees, elected officials and corporate
personnel profiting from the introduction of GM
product.”
“Malicious, financial terrorism, pursued Corporate vs OSA
through the courts by corporations involved in Commonwealth
the pursuit of profit to destroy and bankrupt interests
those persons opposed to their purpose reveal
the intention of corporations to be contrary to
the interests of the commonwealth and its
people.”
Is concerned that farmers are incapable of G; MA; RM; SEG App 5 and LC
producing GM cotton in a totally isolated Licence conditions have been
manner that is sustainable and profitable, and imposed to limit gene flow,
yet preserves the requirement of the people of and the spread and
the Commonwealth to 100% containment for persistence of GM cottons
preservation of the genetic integrity of the from the release sites.
Australian ecosystem.
Genetic contamination would result in changes MA; SEG OSA
to the food chain and therefore would create a
risk of contaminating the native food export
industry of Australia. Kangaroo meat, Wild
Boar and Emu would no longer be acceptable
for import by the European Union.
Appendix 9 Summary of public submissions 94
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
2 I “This stuff will poison the environment and WE H, T Apps 2 and 3
do eat it as cotton oil eventually reaches the FS Licence conditions prohibit
poor hapless consumer in the form of cotton the use of GM cotton
seed oil used in the preparation of foods, the materials resulting from this
trash being used as mulch, etc., and surely release in feed and food.
bits get stuck in the clothing made from this FSANZ approval is required
poisoned stuff. Remember DDT/Cane toad? before GM cotton oil can be
etc.” used in human food.
“You can‟t trust them, look at their actions in Suitability of App 8 outlines matters the
the recent bribery case!” applicant Regulator must consider in
deciding whether a company
is suitable to hold a licence.
“Don‟t approve it please.” None Noted
“Pass this on to whoever can help us poor None Noted
defenceless consumers as it seems you
cant/wont/dont! How many have you rejected
so far? I bet it is zero. So much for a job well
done!”
3 I Has the following procedural concerns about the
application:
The summary of the application available on Av GPS-coordinates of the
the OGTR website does not include details on locations will be made
the location of the „threatened‟ crops available on the website after
a licence has been issued
and the GMOs been planted
The following decision is unfortunate: “the Pc Noted
st
Regulator has decided that the proposed (1 stage of consultation with
release does not pose a significant risk” and prescribed expert groups and
“this means that the Regulator is not required authorities)
to seek public comment …until after a RARMP
has been prepared.”
Monsanto and the OGTR should be well Political issue OSA
aware of the repeated, unambiguous
statements made by the NT ALP Government
that there will be no further GM cotton
cropping in the NT
Monsanto‟s application can only be seen as a Political issue OSA
clear provocation of the NT Government‟s
authority, and a disregard for the will of the
people of the NT, who demand an end to the
GM cotton trials
What advice has the office received from the Ch 2 summarises issues
NT Government? raised by all prescribed
agencies and authorities
Notes that “as a measure over and above Pc The RARMP was released for
those required under the Act, in order to public comment on 25/02/05
promote the openness and transparency of for 6 weeks.
the regulatory system, the Regulator may take
other steps” and hopes that at this late stage,
the process can be broadened in scope to
include the unavoidable parameter of public
opinion and local policy.
4 I Objects to this release because:
there is no guarantee of the 1811 hectares RM LC
being de-contaminated if this is found to be Conditions relating to cleaning
desirable or necessary. and monitoring of trial sites
Appendix 9 Summary of public submissions 95
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
5 I “2 years fighting against” None Noted
“You have made your unwanted decision” Licence conditions have been
“I have done all possible to” imposed to ensure that the
“no we are rejecting GM chook food” GM cottons, or products from
the GM cottons, will not be
fed to animals.
6 I Submitted a copy of a chapter of a book on SEG OSA
potential legal issues and GM.
7 I Is 50m sufficient distance from a waterway to G Cotton is insect- rather than
ensure non spread of pollen? Can‟t pollen wind-pollinated therefore its
travel much further than that? pollen is heavy and sticky.
Pollen in a waterway would
be unlikely to come into
contact with cotton flowers
and cause pollination.
Doesn‟t the spraying of insecticides/herbicides RD App 6
merely provoke further insects/pests that are These issues are not unique
resistant to them? Isn‟t that short-sighted? to GMOs. Resistance
When enough plants or insects are resistant to management plans are
roundup, then we will need to create integral to approvals by the
Roundup-2 and breed new crops that are APVMA.
resistant to that.
8 A GM cotton crops have benefited the public and H, EN Noted
the environment through reductions in
pesticide usage and shifts in the types of
pesticides used
Insect resistance to insecticidal cotton is the RD Noted
biggest risk to sustainability. The
sustainability has been improved by the
addition of a second gene.
Herbicide tolerant GM cotton has been widely
adopted across the cotton growing industry
and has encouraged the use of a topically
applied non-residual herbicide.
Decreases the use of pre-emergent products
and topically applied herbicides with greater EN; HU Noted
toxicity than Glyphosate.
Reduced tilling helps maintain soil structure
and decreases erosion.
An enhanced herbicide tolerant trait would be
a great advantage for the industry.
40 years of cotton growing and processing W Noted
throughout the northwestern regions of NSW
has not resulted in the establishment of cotton
as a weed despite spills of seed cotton and
fuzzy cotton seed on farms and along roads
during this time. The introduction of herbicide
tolerant traits has not altered that status to
date.
Company has been involved in a number of RM Noted
GM cotton releases and has been constrained
by and witness to the diligence with which
these releases are conducted and processed.
Appendix 9 Summary of public submissions 96
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Company strongly supports the proposed field RM Noted
®
trial of Roundup Ready Flex and Roundup
® ®
Ready Flex/Bollgard II cottons and is
satisfied that the risk management plan
supporting the limited and controlled release
sufficiently covers that minimal risks attached
to this technology.
Appendix 9 Summary of public submissions 97
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
APPENDIX 10 REFERENCES
Adair, T.L., Kearney, C.M. (2000). Recombination between a 3-kilobase tobacco mosaic
virus transgene and a homologous viral construct in the restoration of viral and nonviral
genes. Archives of Virology 145: 1867-1883.
Adang, M.J., Staver, M.J., Rocheleau, T.A., Leighton, J., Barker, R.F., Thompson, D.V.
(1985). Characterised full-length and truncated plasmid clones of the crystal protein of
Bacillus thuringiensis subsp. kurstaki HD-73 and toxicity to Manduca sexta. Gene 36: 289-
300.
Addison, S. (2001a). Bollgard II - Non-target arthropod East 1999/2000. MAL-200124,
Monsanto Australia, Melbourne. pp 1-21.
Addison, S. (2001b). Bollgard II - Non-target arthropod East 2000/2001. MAL-200126,
Monsanto Australia, Melbourne. pp 1-22.
Ahmad, W., Nicholls, C., Ellar, D.J. (1989). Cloning and expression of an entomocidal
protein gene from Bacillus thuringiensis galleriae toxic to both lepidoptera and diptera. FEMS
Microbiology letters - Federation of European Microbiological Societies, 59: 198-202.
Allen, T. A., Kharboutli, M. S., Capps, C., and Earnest, L. D. (2000). Effectiveness of
Bollgard II® cotton varieties against foliage and fruit feeding caterpillars in Arkansas. In
"Proceedings of the 2000 Cotton Research Meeting", Arkansas Agricultural Experiment
Research Station, Arkansas. pp. 132-135.
An, W.Q., McDowell, J.M., Huang, S., McKinney, E.C., Chambliss, S., Meagher, R.B.
(1996). Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in
vegetative tissues. Plant Journal 10: 107-121.
ANZFA (1999). Full assessment report and regulatory impact statement, A341: Oil and
linters derived from insect resistant cotton. A341, Australia New Zealand Food Authority,
Canberra, Australia. pp 1-59.
ANZFA (2000). Draft risk analysis report, application A355: Food produced from
glyphosate-tolerant cotton line 1445. Australia New Zealand Food Authority, Canberra,
Australia. pp 1-78.
ANZFA (2001a). Final assessment report - Application A379: Oil and linters from
Bromoxynil tolerant cotton transformation events 10211 and 10222. A379, Food Standards
Australia New Zealand, Canberra, Australia. pp 1-85.
ANZFA (2001b). Final assessment report - Application A382: Food derived from insect-
protected potato lines BT-06, ATBT04-06, ATBT04-31, ATBT04-36, and SPBT02-05. A382,
Food Standards Australia New Zealand, Canberra, Australia. pp 1-86.
ANZFA (2001c). Final assessment report - Application A383: Food derived from insect and
potato leafroll virus-protected potato lines RBMT21-129, RBMT21-350, and RBMT22-82.
A383, Food Standards Australia New Zealand, Canberra, Australia. pp 1-75.
Appendix 10 References 98
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
ANZFA (2001d). Final assessment report - Application A384: Food derived from insect and
potato virus Y-protected potato lines RBMT15-101, SEMT15-02 and SEMT15-15. A384,
Food Standards Australia New Zealand, Canberra, Australia. pp 1-78.
ANZFA (2001e). Final assessment report application A378: Food derived from glyphosate-
tolerant sugarbeet line 77 (GTSB77). A378, Australia New Zealand Food Authority,
Canberra, Australia. http://www.foodstandards.gov.au
ANZFA (2001f). Food derived from glyphosate-tolerant cotton line 1445 - A safety
assessment. Australia New Zealand Food Authority, Canberra, Australia.
ANZFA (2002a). Draft assessment report (Full assessment - S.15) Application A436: Oil and
linters derived from insect-protected cotton containing event 15985. Full assessment - S.15
Application A436, Australia New Zealand Food Authority, Canberra, Australia. pp 1-78.
ANZFA (2002b). Draft final risk analysis report application A386: Food derived from insect-
protected, herbicide-tollerant Bt-11 corn. Application A386, Australia New Zealand Food
Authority, Canberra, Australia. pp 1-4.
ANZFA (2002c). Final assessment report (Full assessment - S.15) Application A436: Oil and
linters derived from insect-protected cotton containing event 15985. Full assessment - S.15
Application A436, Australia New Zealand Food Authority, Canberra, Australia. pp 1-80.
http://www.foodstandards.gov.au
Astwood, J.D., Leach, J.N., Fuchs, R.L. (1996). Stability of food allergens to digestion in
vitro. Nature Biotechnology 14: 1269-1273.
Australian Cotton Cooperative Research Centre (2002a). WEEDpak - A guide for integrated
management of weeds in cotton. Australian Cotton Cooperative Research Centre, Narrabri,
NSW.
Australian Cotton Cooperative Research Centre (2002b). Australian dryland cotton:
production guide. 3rd edition. Cotton Research & Development Corporation, Narrabri,
Australia.
Australian Cotton Cooperative Research Centre (2002c). NUTRIpak - A practical guide to
cotton nutrition. Australian Cotton Cooperative Research Centre, Narrabri, NSW.
Axelos, M., Bardet, C., Liboz, T., Le Van, T.A., Curie, C., Lescure, B. (1989). The gene
family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular
cloning, characterization and expression. Molecular and General Genetics 219: 106-112.
Barbera, P.W. (1995). Toxicity/pathogenecity testing of Bacillus thuringiensis strain EG
7826 following acute oral challenge in rats. IITRI Project Number L08574, IIT Research
Institute, Chicago IL.
Barry, G., Kishore, G., Padgette, S., Stallings, W.C. (27-5-1997). Monsanto Company
(St.Louis, MO, USA. United States Patent: Glyphosate-tolerant 5-enolpyruvylshikimate-3-
phosphate synthases. Patent No: 5633435.
Barry, G., Kishore, G., Padgette, S., Taylor, M.L., Kolacz, K., Weldon, M., Re, D., Eichholtz,
D., Fincher, K., Hallas, L.E. (1992). Inhibitors of amino acid biosynthesis: Strategies for
Appendix 10 References 99
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
imparting glyphosate tolerance to crop plants. In "Biosynthesis and Molecular regulation of
Amino acids in Plants", BK Singh, HE Flores, JC Shannon, eds Vol 7. American Society of
Plant Physiologists, Rockville, USA. pp 139-145.
Bartlett, S.G., Grossman, A.R., Chua, N.H. Edelman, M., Hallick, R.B., Chua, N.H., ed.
(1982). Methods in chloroplast molecular biology. Elsevier, Amsterdam.
Bechtel (1999). Acute oral toxicity study of insect protection protein 2 (IPP2) in mice.
Unpublished. Monsanto company, St Louis, MO. Monsanto Report No. MSL:16649.
Beck, E., Ludwig, G., Auerswald, E.A., Reiss, B., Schaller, H. (1982). Nucleotide sequence
and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene
19: 327-336.
Beever, D.E., Kemp, C.F. (2000). Safety issues associated with the DNA in animal feed
derived from genetically modified crops. A review of scientific and regulatory procedures.
Nutrition Abstracts and Reviews, Series B: Livestock Feeds and Feeding 70: 175-182.
Belgian Biosafety Server (1999). Types of antibiotics and related resistance genes. Available
from: http://www.antibioresistance.be/ARmenu.html
Bentley, R. (1990). The shikimate pathway - a metabolic tree with many branches. Critical
Reviews in Biochemistry and Molecular Biology 25: 307-384.
Berberich, Leimgruber, and Regan (1993). Preparation and verification of dose for a mouse
acute oral toxicity study with neomycin phosphotransferase II protein (NPTII), study ML-91-
409. Unpublished. Monsanto Company. Monsanto Report No. MSL:13277.
Bergelson, J., Purrington, C.B., Wichmann, G. (1998). Promiscuity in transgenic plants.
Nature 395: 25.
Bernstein, I.L., Bernstein, J.A., Miller, M., Tierzieva, S., Bernstein, D.I., Lummus, Z.,
Selgrade, M.K., Doerfler, D.L., Seligy, V.L. (1999). Immune responses in Farm workers after
exposure to Bacillus thuringiensis pesticides. Environmental Health Perspectives 107: 575-
582.
Bertolla, F., Simonet, P. (1999). Horizontal gene transfers in the environment: natural
transformation as a putative process for gene transfers between transgenic plants and
microorganisms. Research in Microbiology 150 (6): 375-384.
Betz, F.S., Hammond, B.G., Fuchs, R.L. (2000). Safety and advantages of Bacillus
thuringiensis-protected plants to control insect pests. Regulatory Toxicology and
Pharmacology 32: 156-173.
Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids
Research 12: 8711-8721.
Borja, M., Rubio, T., Scholthof, H.B., Jackson, A.O. (1999). Restoration of wild-type virus by
double recombination of tombusvirus mutants with a host transgene. Molecular Plant-
Microbe Interactions 12 (2): 153-162.
Appendix 10 References 100
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Brimecombe, M.J., De Leij, F.A., Lynch, J.M. (2001). The effect of root exudates on
rhizosphere microbial populations. In "The rhizosphere: biochemistry and organic substances
at the soil-plant interface", R Pinton, Z Varanini, P Nannipieri, eds. Marcel Dekker, Inc., New
York, USA. pp 95-140.
Brubaker, C.L., Brown, A.H.D., Stewart, J.M., Kilby, M.J., Grace, J.P. (1999). Production of
fertile hybrid germplasm with diploid Australian Gossypium species for cotton improvement.
Euphytica 108: 199-213.
Calgene, I. (1990). Request for advisory opinion - kan gene: safety and use in the production
of genetically engineered plants. FDA Docket Number 90A-0416.
Canadian Food Inspection Agency (1997). Decision Document 97-21: Determination of the
safety of cotton lines with Roundup Ready genes (Gossypium hirsutum L.). 97-21, Plant
health and Production Division, Plant Biotechnology Office, pp 1-7.
http://www.inspection.gc.ca/english/plaveg/pbo/dd/dd9721e.shtml
Canadian Food Inspection Agency (1998). Decision Document (DD2003-43):
Determination of the Safety of Monsanto Canada Inc.'s Insect Resistant Corn (Zea
mays L.) Line MON 863. http://www.inspection.gc.ca/english/plaveg/pbo/dd/dd033e.shtml
Candresse, T. (1997). Systematic search for recombination events in plant viruses and viroids.
In "Virus-resistant transgenic plants: potential ecological impact", M Tepfer, E Balázs, eds.
Springer, Berlin, Germany. pp 20-25.
Carter, J.N., Ligget, M.P. (1994). Acute oral toxicity and infectivity/pathogenicity to rats of
EG 7841. HRC Study Report number ECO 6/942538, Huntingdon Research Centre Ltd.,
Huntington Cambridgeshire England.
Coghlan, A. (2000). So far so good: for the moment, the gene genie is staying in its bottle.
New Scientist 2231: 4.
Common, I.F.B. (1990). Moths of Australia. Melbourne University Press, Melbourne.
Conner, A.J., Glare, T.R., Nap, J.P. (2003). The release of genetically modified crops into the
environment. Part II. Overview of ecological risk assessment. Plant Journal 33: 19-46.
Coruzzi, G., Brogue, C., Edwards, C., Chua, N.H. (1984). Tissue-specific and light-regulated
expression of a pea nuclear gene encoding the small subunit of ribulose-1,5-bisphosphate
carboxylase. EMBO Journal 3: 1671-1679.
Cotton Seed Distributors (2002). Wee Waa, NSW, Australia, 2002 -2003 variety guide.
Cotton Seed Distributors Ltd.
Craven, L. A., Stewart, J. M., Brown, A. H. D., and Grace, J. P. (1994). Challenging the
future; the Australian wild species of Gossypium. In "Proceedings of the 1st World Cotton
Research Conference", pp. 278-281.
Crickmore, N., Wheeler, V.C., Ellar, D.J. (1994). Use of an operon fusion to induce
expression and crystallisation of a Bacillus thuringiensis delta-endotoxin encoded by a cryptic
gene. Molecular and General Genetics 242: 365-368.
Appendix 10 References 101
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Crossan, A., Kennedy, I. (2004). A snapshot of Roundup Ready® cotton in Australia: Are
there environmental benefits from the rapid adoption of Roundup Ready® cotton in Australia?
The University of Sydney. http://www.monsanto.com.au/content/links_resources/papers/08-
19-04.pdf
Dankocsik, C., Donovan, W.P., Jany, C.S. (1990). Activation of a cryptic crystal protein gene
of Bacillus thuringiensis subspecies kurstaki by gene fusion and determination of the crystal
protein insecticidal specificity. Molecular Microbiology 4: 2087-2094.
Davies, J.E., Benveniste, R.E. (1974). Enzymes that inactivate antibiotics in transit to their
targets. Annals of the New York Academy of Sciences 235: 130-136.
De Vries, J., Meier, P., Wackernagel, W. (2001). The natural transformation of the soil
bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly
depends on homologous sequences in the recipient cells. FEMS Microbiology Letters 195:
211-215.
De Vries, J., Wackernagel, W. (1998). Detection of nptII (kanamycin resistance) genes in
genomes of transgenic plants by marker-rescue transformation. Molecular and General
Genetics 257: 606-613.
della-Cioppa, G., Bauer, S.C., Klein, B.K., Shah, D.M., Fraley, R.T., Kishore, G.M. (1986).
Translocation of the precursor of 5-enolpyruvylshikimate-3-phiosphate synthase into
chloroplasts of higher plants in vitro. Proceedings of the National Academy of Sciences of the
United States of America 83: 6873-6977.
Donegan, K.K., Palm, C.J., Fieland, V.J., Porteous, L.A., Ganio, L.M., Schaller, D.L., Bucao,
L.Q., Seidler, R.J. (1995). Changes in levels, species and DNA fingerprints of soil
microorganisms associated with cotton expressing the Bacillus thuringiensis var. kurstaki
endotoxin. Applied Soil Ecology 2: 111-124.
Donegan, K.K., Seidler, R.J. (1998). Effect of transgenic cotton expressing the Bacillus
thuringiensis var. kurstaki endotoxin on soil microorganisms- Risk assessment studies. In
"Biotechnology in Australia, Volume 42: Cotton", YPS Bajaj, ed. pp 300-312.
Eastick, R. (2004). Continued monitoring of cotton weediness sites in northern Australia,
Progress Report June 2004.
Eastick, R. (2002). Evaluation of the potential weediness of transgenic cotton in northern
Australia. Technical Bulletin no. 305, Northern Territory Government and Australian Cotton
Cooperative Research Centre, pp 1-177. http://cotton.pi.csiro.au/Assets/PDFFiles/TB3051.pdf
EFB (2001). Antibiotic resistance markers in genetically modified (GM) crops. Briefing
Paper no. 10, European Federation of Biotechnology, pp 1-4. http://efbweb.org
EFSA (2004). Opinion of the scientific panel on genetically modified organisms on the use of
antibiotic resistance genes as marker genes in genetically modified plants. The EFSA Journal
48: 1-18.
English, L., Slatin, S.L. (1992). Mode of action of delta-endotoxins from Bacillus
thuringiensis: a comparison with other bacterial toxins. Insect Biochemistry and Molecular
Biology 22: 1-7.
Appendix 10 References 102
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
EPA (2001a). Biopesticides registration action document: Bacillus thuringiensis plant-
incorporated protectants. US EPA, IIA1-IIC109.
http://www.epa.gov/pesticides/biopesticides/pips/bt_brad.htm
EPA (1994). Neomycin phosphotransferase II; tolerance exemption. Federal Register 59:
49351-49353.
EPA (2000). Biopesticide fact sheet: Bacillus thuringiensis subsp. kurstaki Cry1Ac delta-
endotoxin and its controlling sequences as expressed in cotton (006445) Issued: 4/00. pp 1-12.
http://www.epa.gov/pesticides/biopesticides/factsheets/fs006445t.htm
EPA (2001b). -D Glucuronidase from E. coli and the genetic material necessary for its
production as a plant pesticide inert ingredient: exemption from the requirement of a
tolerance. Federal Register 66: 42957-42962.
Eschenburg, S., Healy, M.L., Priestman, M.A., Lushington, G.H., Schonbrunn, E. (2002).
How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl
shikimate-3-phosphate synthase from Escherichia coli. Planta 216: 129-135.
FAO and WHO (2000). Safety aspects of genetically modified foods of plant origin.
Available from: http://www.who.int/fsf/GMfood/FAO-WHO_Consultation_report_2000.pdf
Farrell, T., Roberts, G. (2002). Survey of cotton volunteers north of latitude 22º south.
Australian Cotton CRC and CSIRO Plant Industry, Narrabri.
FDA (1994). Secondary food additives permitted in food for human consumption; food
additives permitted in feed and drinking water of animals; aminoglycoside 3'-
phosphotransferase II; final rule. 59, United States Food and Drug Administration,
Washington, USA. pp 26700-26711.
FDA (1998). Guidance for Industry: Use of antibiotic resistance marker genes in transgenic
plants. U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition,
Office of Premarket Approval, http://vm.cfsan.fda.gov/~dms/opa-armg.html.
Felsot, A.S. (2000a). Herbicide tolerant genes. Part 1: squaring up Roundup Ready crops.
Agrichemical & Environmental News 173:
Felsot, A.S. (2000b). Insecticidal genes. Part 2: Human health hoopla. Agrichemical &
Environmental News 168: 1-7.
Fitt, G.P., Wilson, L.J. (2002). Non-target effects of Bt-cotton: a case study from Australia. In
"Biotechnology of Bacillus thuringiensis and its environmental impact", RJ Akhurst, CE
Beard, PA Hughes, eds. CSIRO Entomology, Canberra.
Flavell, R.B., Dart, E., Fuchs, R.L., Fraley, R.T. (1992). Selectable marker genes: safe for
plants? Bio/Technology 10: 141-144.
Forrester, N.W., Wilson, A.G.L. (1988). Insect pests of cotton. NSW Agriculture, pp 1-17.
Fraile, A., Alonso-Prados, J.L., Aranda, M.A., Bernal, J.J., Malpica, J.M., García-Arenal, F.
(1997). Genetic exchange by recombination or reassortment is infrequent in natural
populations of a tripartite RNA plant virus. Journal of Virology 71: 934-940.
Appendix 10 References 103
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Frischmuth, T., Stanley, J. (1998). Recombination between viral DNA and the transgenic coat
protein gene of African cassava mosaic geminivirus. Journal of General Virology 79: 1265-
1271.
Fryxell, P.A. (1992). A revised taxonomic interpretation of Gossypium L. (Malvaceae).
Rheedea 2: 108-165.
FSANZ (2003). Final Assessment Report - Application A484: Food from Insect Protected
MON863 Corn.
http://www.foodstandards.gov.au/_srcfiles/A484_Final_Assessment_Report.pdf
Fuchs, R.L., Astwood, J.D. (1996). Allergenicity assessment of foods derived from
genetically modified plants. Food Technology 50: 83-88.
Fuchs, R.L., Berberich, S.A., Serdy, F.S. (1993a). Safety evaluation of genetically engineered
plants and plant products: insect resistant cotton. In "Biotechnology and Safety Assessment",
JA Thomas, LA Myers, eds. Raven Press Ltd, New York, pp 199-212.
Fuchs, R.L., Heeren, R.A., Gustafson, M.E., Rogan, G.J., Bartnicki, D.E., Leimgruber, R.M.,
Finn, R.F., Hershman, A., Berberich, S.A. (1993b). Purification and characterization of
microbially expressed neomycin phosphotransferase II (NPTII) protein and its equivalence to
the plant expressed protein. Biotechnology (NY) 11: 1537-1542.
Fuchs, R.L., Ream, J.E., Hammond, B.G., Naylor, M.W., Leimgruber, R.M., Berberich, S.A.
(1993c). Safety assessment of the neomycin phosphotransferase II (NPTII) protein.
Bio/Technology 11: 1543-1547.
Gal, S., Pisan, B., Hohn, T., Grimsley, N., Hohn, B. (1991). Genomic homologous
recombination in planta. The EMBO Journal 10: 1571-1578.
Gal, S., Pisan, B., Hohn, T., Grimsley, N., Hohn, B. (2002). Agroinfection of transgenic
plants leads to viable cauliflower mosaic virus by intermolecular recombination. Virology 187
(2): 525-533.
Gebhard, F., Smalla, K. (1998). Transformation of Acinetobacter sp. strain BD413 by
transgenic sugar beet DNA. Applied and Environmental Microbiology 64: 1550-1554.
Gilissen, L.J.W., Metz, P.L.J., Stiekema, W.J., Nap, J.P. (1998). Biosafety of E.coli
-glucuronidase (GUS) in plants. Transgenic Research 7: 157-163.
Glare, T.R., O'Callaghan, M. (2000). Bacillus thuringiensis: Biology, Ecology and Safety.
John Wiley & Sons, Chichester, UK.
Greene, A.E., Allison, R.F. (1996). Deletions in the 3' untranslated region of cowpea chlorotic
mottle virus transgene reduce recovery of recombinant viruses in transgenic plants. Virology
225: 231-234.
Greene, A.E., Allison, R.F. (1994). Recombination between viral RNA and transgenic plant
transcripts. Science 263: 1423-1425.
Appendix 10 References 104
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Gregory, S.R., Hernandez, E., Savoy, B.R. (1999). Cottonseed processing. In "Cotton: Origin,
History, Technology and Production", CW Smith, JT Cothren, eds. John Wiley & Sons, New
York. pp 793-819.
Groot, A.T., Dicke, M. (2002). Insect-resistant transgenic plants in a multi-trophic context.
Plant Journal 31: 387-406.
Groves, R.H., Hosking, J.R., Batianoff, D.A., Cooke, D.A., Cowie, I.D., Keighery, B.J.,
Rozefelds, A.C., Walsh, N.G. (2000). The naturalised non-native flora of Australia: its
categorisation and threat to native plant biodiversity. Unpublished report to Environment
Australia by the CRC for Weed Management Systems.
Groves, R.H., Hosking, J.R., Cooke, D.A., Johnson, R.W., Lepschi, B.J., Mitchell, A.A.,
Moerkerk, M., Randall, R.P., Rozefelds, A.C., Waterhouse, B.M. (2002). The naturalised
non-native flora of Australia: its categorisation and threat to agricultural ecosystems.
Unpublished report to Agriculture, Fisheries and Forestry Australia by the CRC for Weed
Management Systems.
Gupta, V., Watson, S. (2004). Ecological impacts of GM cotton on soil biodiversity - below
ground production of Bt by GM cotton and Bt cotton impacts on soil biological processes.
Australian Government Department of Environment and Heritage, pp 1-72.
Hamilton, K.A., Coyler, J., Fabellar, A. (2000). Production of tissue samples from cotton
plants expressing both Bollgard insect protected and Roundup Ready traits grown in 1999
U.S. field trials. MSL-16702, Monsanto Company, USA.
Hamilton, K.A., Reed, A. (1999). Production of tissue samples from cotton plants expressing
both Bollgard insect protected and Roundup Ready traits grown in 1998 U.S. field trials.
MSL-16418, Monsanto Company,
Harper, G., Hull, R., Lockhart, B., Olszewski, N. (2002). Viral sequences integrated into plant
genomes. Annual Review of Phytopathology 40: 119-136.
Harrison, L.A., Bailey, M.R., Naylor, M.W., Ream.J.E., Hammond, B.G., Nida, D.L.,
Burnette, B.L., Nickson, T.E., Mitsky, T.A., Taylor, M.L., Fuchs, R.L., Padgette, S.R.
(1996a). The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikimate-3-
phospate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not
toxic to actutely gavaged mice. Journal of Nutrition 126: 728-740.
Harrison, L.A., Biest, N.A., Leimgruber, R., Padgette, S. (1996b). Preparation,
characterisation, and confirmation of doses for an acute mouse feeding study with B-D-
glucuronidase. Unpublished. Monsanto Report No. MSL:12979, Monsanto Company, St.
Louis.
Head, G., Surber, J.B., Watson, J.A., Martin, J.W., Duan, J.J. (2002). No detection of Cry1Ac
protein in soil after multiple years of transgenic cotton (Bollgard®) use. Environmental
Entomology 31: 30-36.
Heym, B., Honore, N., Truffot-Pernot, C., Banerjee, A., Schurra, C., Jacobs, W.R., Jr., van
Embden, J.D., Grosset, J.H., Cole, S.T. (1994). Implications of multidrug resistance for the
future of short-course chemotherapy of tuberculosis: a molecular study. Lancet 344: 293-298.
Appendix 10 References 105
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Hoffman, T., Golz, C., Schieder, O. (1994). Foreign DNA sequences are received by a wild-
type strain of Aspergillus niger after co-culture with transgenic higher plants. Current
Genetics 27: 70-76.
Hofmann, C., Vanderbruggen, H., Hofte, H., Van Rie, J., Jansens, S., Van Mellaert, H.
(1988). Specificity of Bacillus thuringiensis -endotoxins is correlated with the presence of
high-affinity binding sites in the brush border membrane of target insect midguts.
Proceedings of the National Academy of Sciences of the United States of America 85: 7844-
7848.
Holm, L., Doll, J., Holm, E., Pancho, J., Herberger, J. (1997). World weeds. Natural
histories and distribution. John Wiley and Sons, Inc, USA.
Hu, C.Y., Chee, P.P., Chesney, R.H., Zhou, J.H., Miller, P.D., O'Brien, W.T. (1990). Intrinsic
GUS-like activities in seed plants. Plant Cell Reports 9: 1-5.
Hull, R., Covey, S.N., Dale, P. (2000). Genetically modified plants and the 35S promoter:
assessing the risks and enhancing the debate. Microbial Ecology in Health and Disease 12: 1-
5.
Jefferson, R.A., Burgess, S.M., Hirsh, D. (1986). -Glucuronidase from Escherichia coli as a
gene-fusion marker. Proceedings of the National Academy of Sciences of the United States of
America 83: 8447-8451.
Jefferson, R.A., Kavanagh, T.A., Bevan, M.W. (1987). GUS fusions: -glucuronidase as a
sensitive and versatile gene fusion marker in higher plants. The EMBO Journal 6: 3901-3907.
Jefferson, R.A., Wilson, K.J. (1991). The GUS gene fusion system. Plant Molecular Biology
Manual B-14: 1-33.
Jenkins, J.N. (1992). Cotton. In "OECD Historical Review of Traditional Crop Breeding".
Kay, R., Chan, A., Daly, M., McPherson, J. (1987). Duplication of CaMV 35S promoter
sequences creates a strong enhancer for plant genes. Science 236: 1299-1302.
Keeler, K.H. (1985). Implications of weed genetics and ecology for the deliberate release of
genetically engineered crop plants. Recombinant DNA Technical Bulletin 8: 165-172.
Keeler, K.H. (1989). Can genetically engineered crops become weeds? Bio/Technology 7:
1134-1139.
Kimber, I., Kerkvliet, N.L., Taylor, S.L., Astwood, J.D., Sarlo, K., Dearman, R.J. (1999).
Toxicology of protein allergenicity: prediction and characterization. Toxicological Sciences
48: 157-162.
Klee, H.J., Muskopf, Y.M., Gasser, C.S. (1987). Cloning of an Arabidopsis thaliana gene
encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation
to obtain glyphosate-tolerant plants. Molecular and General Genetics 210: 437-442.
Klee, H.J., Rogers, S.G. (1989). Plant gene vectors and genetic transformation: plant
transformation systems based on the use of Agrobacterium tumefaciens. Cell Culture and
Somatic Cell Genetics of Plants 6: 1-23.
Appendix 10 References 106
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Knowles, B.H., Dow, J.A.T. (1993). The crystal delta-endotoxins of Bacillus thuringiensis:
models for their mechanism of action on the insect gut. Bioassays 15: 469-476.
Lai, M.M.C. (1992). RNA recombination in animal and plant viruses. Microbiological
Reviews 56 (1): 61-79.
Lawrence, J.G., Ochman, H. (1998). Molecular archaeology of the Escherichia coli genome.
Proceedings of the National Academy of Sciences of the United States of America 95: 9413-
9417.
Leffler, H.R., Tubertini, B.S. (1976). Development of cotton fruit: accumulation and
distribution of mineral nutrients. Agronomy Journal 68: 858-861.
Lereclus, D., Delécluse, A., Lecadet, M.M. (1993). Diversity of Bacillus thuringiensis toxins
and genes. In "Bacillus thuringiensis, an environmental biopesticide: theory and practice", PF
Entwistle, JS Cory, MJ Bailey, S Higgs, eds. John Wiley and sons, Chichester, UK. pp 37-69.
Levy, S.B., Marshall, B., Schluederberg, S., Rowse, D., Davis, J. (1998). High frequency of
antimicrobial resistance in human fecal flora. Antimicrobial Agents and Chemotherapy 32:
1801-1806.
Llewellyn, D., Fitt, G. (1996). Pollen dispersal from two field trials of transgenic cotton in the
Namoi valley, Australia. Molecular Breeding 2: 157-166.
Lorenz, M.G., Wackernagel, W. (1994). Bacterial gene transfer by natural genetic
transformation in the environment. Microbiological Reviews 58 (3): 563-602.
Macintosh, S.C., Stone, T.B., Sims, S.R., Hunst, P.L., Greenplate, J.T., Marrone, P.G., Perlak,
F.J., Fischhoff, D.A., Fuchs, R.L. (1990). Specificity and efficacy of purified Bacillus
thuringiensis proteins against agronomically important insects. Journal of Invertebrate
Pathology 56: 258-266.
Maggi, V.L. (1993a). Evaluation of dietary effects of purified B.t.k. endotoxin proteins on
honey bee adults. CAR 181-92, Monsanto Australia Limited.
Maggi, V.L. (1993b). Evaluation of dietary effects of purified B.t.k. endotoxin proteins on
honey bee larvae. CAR 180-92, Monsanto Australia Limited.
Maggi, V.L. (2000a). Evaluation of dietary effects of purified Bacillus thurigiensis Cry2Ab
protein on honey bee larvae. MSL-16961, Monsanto Company, Biotechnology Regulatory
Science Final Report. pp 1-33.
Maggi, V.L. (2000b). Evaluation of the dietary efects of insect protection protein 2 on adult
honey bees (Apis mellifera L.). MSL 16176, California Agricultural Research Inc. pp 1-35.
Malik, J., Barry, G., Kishore, G. (1989). The herbicide glyphosate. Biofactors 2: 17-25.
Martin, P.A.W., Travers, R.S. (1989). Worldwide abundance and distribution of Bacillus
thuringiensis isolates. Applied and Environmental Microbiology 55: 2437-2442.
Appendix 10 References 107
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Mayo, M.A., Jolly, C.A. (1991). The 5'-terminal sequence of potato leafroll virus RNA:
evidence of recombination between virus and host RNA. Journal of General Virology 72:
2591-2595.
McCabe, D.E., Martinell, B.J. (1993). Transformation of elite cotton cultivars via particle
bombardment of meristems. Bio/Technology 11: 596-598.
McClintock, J.T., Schaffer, C.R., Sjoblad, R.D. (1995). A comparative review of the
mammalian toxicity of Bacillus thuringiensis-based pesticides. Pesticide Science 45: 95-105.
Meadows, M.P. (1993). Bacillus thuringiensis in the environment: ecology and risk
assessment. In "Bacillus thuringiensis, an environmental biopesticide: theory and practice",
PF Entwistle, JS Cory, MJ Bailey, S Higgs, eds. John Wiley and sons, Chichester, UK. pp
193-220.
Mercer, D.K., Scott, K.P., Bruce-Johnson, W.A., Glover, L.A., Flint, H.J. (1999). Fate of free
DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA
in human saliva. Applied and Environmental Microbiology 65: 6-10.
Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., Fuchs, R.L.
(1996). Assessment of the allergenic potential of foods derived from genetically engineered
crop plants. Critical Reviews in Food Science and Nutrition 36(S): S165-S186.
Miki, B., McHugh, S. (2004). Selectable marker genes in trangenic plants: applications,
alternatives and biosafety. Journal of Biotechnology 107: 193-232.
Mitsky (1993). Comparative alignment of CP4 EPSPS to known allergenic and toxic proteins
using Fasta algorithm. Unpublished. Monsanto Company, 700 Chesterfield Parkway North, St
Louis, MO, USA 63198. Monsanto Report No. MSL:12820.
Monsanto Australia Limited (2001). Roundup Ready® Cotton Technical Manual. 2.
Monsanto Australia Ltd.
Monsanto Australia Limited (2004). A guide to the 2004/05 Bollgard II resistance
management plan. www.monsanto.com.au/content/cotton/bollgard_ii_cotton/rmp.pdf
Moser, H.S. (2000). Performance of BollgardII® upland cotton strains in Arizona. The 2000
Arizona Cotton Report, The University of Arizona College of Agriculture.
http://ag.arizona.edu/pubs/crops/az1170/
Naranjo, S. E. and Ellsworth, P. C. (2002). Arthropod communities and the transgenic cotton
in the western United States: implications for biological control. In "Proceedings of the First
International Symposium For Biological Control", Van Driesche, R. G. eds, US Forestry
Service, Honolulu, Hawaii.
Naylor, M.W. (1993a). One month feeding study with insect-resistant cottonseed meal in
Sprague-Dawley rats. Monsanto Company The Agricultural Group, St Louise Missouri USA.
Naylor, M.W. (1992). Acute oral toxicity study of beta-glucuronidase (GUS) protein in
albino mice. Unpublished. Monsanto Report No. MSL:12485, Monsanto Company, St. Louis.
Appendix 10 References 108
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Naylor, M.W. (1993b). Acute oral toxicity of Bacillus thuringiensis var. kurstaki [Cry1Ac]
HD-73 protein in albino mice. The Agricultural group Monsanto Company, St. Louis
Missouri USA.
Netherwood, T., Martín-Orúe, S.M., O'Donnell, A.G., Gockling, S., Graham, J., Mathers,
J.C., Gilbert, H.J. (2004). Assessing the survival of transgenic plant DNA in the human
gastrointestinal tract. Nature Biotechnology 22: 204-209.
Nielsen, K.M. (1998). Barriers to horizontal gene transfer by natural transformation in soil
bacteria. APMIS 106: 77-84.
Nielsen, K.M., van Elsas, J.D., Smalla, K. (2000). Transformation of Acinetobacter sp strain
BD413(pFG4 Delta nptII) with transgenic plant DNA in soil microcosms and effects of
kanamycin on selection of transformants. Applied and Environmental Microbiology 66: 1237-
1242.
Ochman, H., Lawrence, J.G., Grolsman, E. (2000). Lateral gene transfer and the nature of
bacterial innovation. Nature 405: 299-304.
Odell, J.T., Nagy, F., Chua, N.H. (1985). Identification of DNA sequences required for
activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810-812.
OGTR (2002). The Biology and Ecology of Cotton (Gossypium hirsutum) in Australia.
http://www.ogtr.gov.au/pdf/ir/biologycotton.pdf
Padgette, Barry, Re, Eichholtz, Weldon, Kolacz, and Kishore (1993). Purification, cloning
and characterisation of a highly glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate
synthase from Agrobacterium sp.strain CP4. Unpublished. Monsanto Company, USA. 1-66.
Monsanto Report No. MSL:12738, 1-66.
Padgette, S.R., Re, D.B., Barry, G.F., Eichholtz, D.E., Delannay, X., Fuchs, R.L., Kishore,
G.M., Fraley, R.T. (1996). New weed control opportunities: development of soybeans with a
Roundup Ready gene. In "Herbicide-resistant crops: agricultural, environmental, economic,
regulatory and technical aspects", SO Duke, ed. CRC Press, Boca Raton. pp 53-84.
Padidam, M., Sawyer, S., Fauquet, C.M. (1999). Possible emergence of new geminiviruses by
frequent recombination. Virology 265: 218-225.
Palm, C.J., Schaller, D.L., Donegan, K.K., Seidler, R.J. (1996). Persistence in soil of
transgenic plant produced Bacillus thuringiensis var. kurstaki -endotoxin. Canadian Journal
of Microbiology 42: 1258-1262.
Palmer, S.J., Beavers, J.B. (1993a). B.t.k. HD-73 protein: Dietary toxicity study with green
lace wing larvae (Chrysopa carnea). WL 93-233, Monsanto Company, St. Louis.
Palmer, S.J., Beavers, J.B. (1993b). B.t.k. HD-73 protein: Dietary toxicity study with ladybird
beetles (Hippodamia convergens). WL 93-232, Monsanto Company, St. Louis.
Palmer, S.J., Beavers, J.B. (1993c). B.t.k. HD-73 protein: dietary toxicity study with parasitic
Hymenoptera (Nasonia vitripennis). WL 93-234, Monsanto Company, St. Louis.
Appendix 10 References 109
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Palmer, S.J., Krueger, H.O. (2000a). Insect protection protein 2: a dietary toxicity study with
green lacewing larvae (Chrysomera carnea). MSL-16171, Monsanto Company St Louis
Misouri. pp 1-33.
Palmer, S.J., Krueger, H.O. (2000b). Insect protection protein 2: a dietary toxicity study with
parasitic hymenoptera (Nasonia vitripennis). MSL-16173, Wildlife International, Ltd. Easton
Maryland, USA. pp 1-33.
Palmer, S.J., Krueger, H.O. (2000c). Insect protection protein 2: a dietary toxicity study with
the ladybird beetle (Hippodamia convergens). MSL- 16172, Wildlife International Ltd. pp 1-
40.
Palmer, S.J., Krueger, H.O. (2000d). Insect protection protein 2: an acute toxicity study with
the earthworm in an artificial soil substrate. MSL - 16177; Study number WL-99--067,
Wildlife International Ltd. pp 1-38.
Panetta, F.D. (1993). A system of assessing proposed plant introductions for weed potential.
Plant Protection Quarterly 8: 10-14.
Pheloung, P.C. (1995). Determining the weed potential of new plant introductions in
Australia. Department of Agriculture, Perth, Australia.
Pheloung, P.C., Williams, P.A., Halloy, S.R. (1999). A weed risk assessment model for use as
a biosecurity tool evaluating plant introductions. Journal of Environmental Management 57:
239-251.
Pline, W.A., Viator, R., Wilcut, J.W., Edmisten, K.L., Thomas, J., Wells, R. (2002a).
Reproductive abnormalities in glyphosate-resistant cotton caused by lower CP4-EPSPS levels
in the male reproductive tissue. Weed Science 50: 438-447.
Pline, W.A., Wilcut, J.W., Duke, S.O., Edmisten, K.L., Wells, R. (2002b). Tolerance and
accumulation of shikimic acid in response to glyphosate applications in glyphosate-resistant
and nonglyphosate-resistant cotton (Gossypium hirsutum L.). Journal of Agricultural and
Food Chemistry 50: 506-512.
Raps, A., Kehr, J., Gugerli, P., Moar, W.J., Bigler, F., Hilbeck, A. (2001). Immunological
analysis of phloem sap of Bacillus thuringiensis corn and of the nontarget herbivore
Rhopalosiphum padi (Homoptera: Aphididae) for the presence of Cry1Ab. Molecular Ecology
10: 525-533.
Ream, J.E. (1994). Aerobic soil degradation of Bacilus thuringiensis var. kurstaki B.t.k. HD-
73 protein bioactivity. MSL 13299, Monsanto Company, St. Louis USA.
Richins, R.D., Scholthof, H.B., Shepherd, R.J. (1987). Sequence of figwort mosaic virus
DNA (caulimovirus group). Nucleic Acids Research 15: 8451-8466.
Rogers, S.G., O'Connell, K., Horsch, R.B., Fraley, R.T. Zaitlin, M., Day, P., Hollaender, A.,
Wilson, C.A., ed. (1985). Biotechnology in plant science. Academic Press Inc, New York.
Roy, J. (1990). In search of the characteristics of plant invaders. In "Biological Invasions in
Europe and the Mediterranean Basin", F di Castri, AJ Hansen, M Debussche, eds. Kluwer
Academic Publishers, Dordrecht, The Netherlands. pp 335-352.
Appendix 10 References 110
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Saxena, D., Flores, S., Stotzky, G. (1999). Insecticidal toxin in root exudates from Bt corn.
Nature 402: 480.
Saxena, D., Stewart, C.N., Altosaar, I., Shu, Q., Stotzky, G. (2004). Larvicidal Cry proteins
from Bacillus thuringiensis are released in root exudates of transgenic B. thuringiensis corn,
potato, and rice but not of B. thuringiensis canola, cotton, and tobacco. Plant Physiology and
Biochemistry 42: 383-387.
Saxena, D., Stotzky, G. (2001). Bacillus thuringiensis (Bt) toxin released from root exudates
and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria,
and fungi in soil. Soil Biology and Biochemistry 33: 1225-1230.
Schlüter, K., Fütterer, J., Potrykus, I. (1995). Horizontal gene transfer from a transgenic
potato line to a bacterial pathogen (Erwinia chrysanthemi) occurs - if at all - at an extremely
low frequency. Bio/Technology 13: 1094-1098.
Schoelz, J.E., Wintermantel, W.M. (1993). Expansion of viral host range through
complementation and recombination in transgenic plants. The Plant Cell 5: 1669-1679.
Schulz, A., Kruper, A., Amrhein, N. (1985). Differential sensitivity of bacterial 5-
enolpyruvyl-shikimate-3-phosphate synthases to the herbicide glyphosate. FEMS
Microbiology Letters 28: 297-301.
Shaw, K.J., Rather, P.N., Hare, R.S., Miller, G.H. (1993). Molecular genetics of
aminoglycoside resistance genes and familial relationships of the aminoglyciside-modifying
enzymes. Microbiological Reviews 57: 138-163.
Shimada, N., Kim, Y.S., Miyamoto, K., Yoshioka, M., Murata, H. (2003). Effects of Bacillus
thuringiensis Cry1Ab toxin on mammalian cells. Journal of Veterinary Medical Science 65:
187-191.
Sims, S., Martin, J. (1996). Effect of the Bacillus thuringiensis insecticidal proteins Cry1Ab,
Cry1Ac,Cry2A and Cry23A on Folsomia candida and Xenylla grisea (Insecta: Collembola).
93-081E1, Monsanto Company, St. Louis.
Sims, S.R. (1994). Sensitivity of insect species to the purified Cry 1Ac insecticidal protein
for Bacillus thuringiensis subsp. kurstaki (B.t.k. HD-73). MSL 13273, Monsanto Company,
St. Louis, USA.
Sims, S.R. (1995). Bacillus thuringiensis subsp. kurstaki (Cry1Ac) protein expressed in
transgenic cotton: effects on beneficial and other non-target insects. Southwestern
Entomologist 20: 493-500.
Sims, S.R., Berberich, S.A., Nida, D.L., Segalini, L.L., Leach, J.N., Ebert, C.C., Fuchs, R.L.
(1996). Analysis of expressed proteins in fibre fractions from insect-protected and glyphosate-
tolerant cotton varieties. Crop Science 36: 1212-1216.
Sisterson, M.S., Biggs, R.W., Olson, C., Carriere, Y., Dennehy, T.J., Tabashnik, B.E. (2004).
Arthropod abundance and diversity in Bt and non-Bt cotton fields. Environmental
Entomology 33: 921-929.
Appendix 10 References 111
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
Smalla, K., Borin, S., Heuer, H., Gebhard, F., van Elsas, J. D., and Nielsen, K. (2000).
Horizontal transfer of antibiotic resistance from transgenic plants to bacteria - are there data to
fuel the debate? Fairbairn, C., Scoles, G., and McHughen, A. eds, University Extension Press,
Saskatchewan, Canada. pp. 146-154.
Smalla, K., Gebhard, F., van Elsas, J. D., Matzk, A., and Schiemann, J. (1994). Bacterial
communities influenced by transgenic plants. Jones, D. D. eds, Division of Agriculture and
Natural Resources, University of California, Oakland, USA. pp. 157-167.
Smalla, K., Van Overbeek, L.S., Pukall, R., van Elsas, J.D. (1993). Prevalence of npt II and
Tn5 in kanamycin-resistant bacteria from different environments. FEMS Microbiology
Ecology, 13: 47-58.
Spencer, T.M., Orozco, E.M., Doyle, R.M. (1996). Petition for determination of non-
regulated status: insect protected corn (Zea mays L.) with Cry1Ac gene from Bacillus
thuringiensis subsp. kurstaki. DEKALB Genetics Corporation,
Stanhope, M.J., Lupas, A., Italia, M.J., Koretke, K.K., Volker, C., Brown, J.R. (2001).
Phylogenetic analyses do not support horizontal gene transfers from bacteria to vertebrates.
Nature 411: 940-944.
Steinrucken, H.C., Amrhein, N. (1980). The herbicide glyphosate is a potent inhibitor of
5-enolpyruvyl-shikimic acid-3-phosphate synthase. Biochemical and Biophysical Research
Communications 94: 1207-1212.
Stotzky, G. (2000a). Monitoring for indirect effects on non-target species, soil microbes,
earthworms, and nematodes. In "Workshop on Ecological Monitoring of Genetically Modified
Crops", National Research Council, Standing Committee on Biotechnology, Food and Fibre
Production, and the Environment, Washington DC USA. pp. 13-14.
Stotzky, G. (2000b). Release, persistence, and biological activity in soil of insecticidal
proteins from Bacillus thuringiensis.
Syvanen, M. (1999). In search of horizontal gene transfer. Nature Biotechnology 17: 833.
Tapp, H., Calamai, L., Stotzky, G. (1994). Adsorption and binding of the insecticidal proteins
from Bacillus thuringiensis subsp.Kurstaki and subsp.tenebrionis on clay minerals. Soil
Biology and Biochemistry 26: 663-679.
Tapp, H., Stotzky, G. (1998a). Insecticidal activity of the toxins from Bacilus thuringiensis
subsp. kurtaski and tenebrionis adsorbed and bound on pure and soil clays. Applied
Environmental Microbiology 61: 1786-1790.
Tapp, H., Stotzky, G. (1998b). Persistence of the insecticidal toxin from Bacillus
thuringiensis subsp. kurstaki in soil. Soil Biology and Biochemistry 30: 471-476.
Taylor, S.L., Lehrer, S.B. (1996). Principles and characteristics of food allergens. Critical
Reviews in Food Science and Nutrition 36: S91-S118.
The Royal Society (2002). Genetically modified plants for food use and human health — an
update. The Royal Society, UK. Policy document 4/02, pp. 1-20.
Appendix 10 References 112
DIR 55/2004 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator
USDA-APHIS (1996). USDA/APHIS Determination on a Petition 95 324 01p of Agritope
Inc., Seeking Nonregulated Status for Delayed-Ripening Cherry Tomato Line 35 1 N.
USDA-APHIS (2004). Monsanto Co.; Availability of Determination of Nonregulated Status
for Cotton Genetically Engineered for Tolerance to the Herbicide Glyphosate. United States
Department of Agriculture, Animal and Plant Health Inspection Service, web site. pp 70-71.
http://www.aphis.usda.gov/brs/fedregister/BRS_20050103a.pdf
van Frankenhuyzen, K. and Nystrom, C. (2002). The Bacillus thuringiensis toxin specificity
database. Available from: http://www.glfc.cfs.nrcan.gc.ca/bacillus
Van Rie, J., Jansens, S., Hofte, H., Degheele, D., Van Mellaert, H. (1989). Specificity of
Bacillus thuringiensis delta -endotoxins: importance of specific receptors on the brush border
membrane of the mid-gut of target insects. European Journal of Biochemistry 186: 239-247.
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: 455-462.
Widmer, F., Seidler, R.J., Donegan, K.K., Reed, G.L. (1997). Quantification of transgenic
plant marker gene persistence in the field. Molecular Ecology 6: 1-7.
Widner, W.R., Whiteley, H.R. (1990). Location of the dipteran specificity region in a
lepidopteran-dipteran crystal protein from Bacillus thuringiensis. Journal of Bacteriology
172: 2826-2832.
Widner, W.R., Whiteley, H.R. (1989). Two highly related insecticidal crystal proteins of
Bacillus thuringiensis subsp. kurstaki possess different host range specificities. Journal of
Bacteriology 171: 965-974.
Williamson, M.H., Fitter, A. (1996). The characters of successful invaders. Biological
conservation 78: 163-170.
Worobey, M., Holmes, E.C. (1999). Evolutionary aspects of recombination in RNA viruses.
Journal of General Virology 80: 2535-2543.
Xia, J.Y., Cui, L.D., Ma, L.H., Dong, S.X., Cui, X.F. (1999). The role of transgenic Bt cotton
in integrated insect pest management. Acta Gossypii Sim 11: 57-64.
Zambryski, P. (1992). Chronicles from the Agrobacterium-plant cell DNA transfer story.
Annual Review Plant Physiology and Plant Molecular Biology 43: 465-490.
Appendix 10 References 113