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Office of the Gene Technology Regulator Risk Assessment and Risk Management Plan Application for licence for dealings involving an intentional release into the environment DIR 005/2001 Title: Agronomic assessments and seed increase in eastern Australia of transgenic cotton expressing cry1Ac and cry2Ab genes from Bacillus thuringiensis Applicant: Cotton Seed Distributors Ltd Revised 18 January 2002 Abbreviations aad ANZFA AQIS Bt B.t.k CaMv CMoVb CSD CSIRO DIR DNA DNIR ELISA EMBL EPSPS GM GMAC GMO gox GTTAC GUS IgE IgG IOGTR IPCS JETACAR MAFF MRL mRNA NHMRC NICNAS NOS nptII NLRD NRA OGTR ppm TGA TGAC US EPA US FDA WHO w/v X-gluc μg/g aminoglycoside adenylyltransferase Australia New Zealand Food Authority Australian Quarantine Inspection Service Bacillus thuringiensis Bacillus thuringiensis variety kurstaki cauliflower mosaic virus figwort mosaic virus Cotton Seed Distributors Ltd Commonwealth Scientific and Industrial Research Organisation dealing involving intentional release deoxyribonucleic acid dealing not involving intentional release enzyme linked immunosorbent assay European Molecular Biology Laboratory 5-enolpyruvylshikimate-3-phosphate synthase genetically modified Genetic Manipulation Advisory Committee genetically modified organism glyphosate oxidoreductase Gene Technology Technical Advisory Committee -glucuronidase immunoglobulin E immunoglobulin G Interim Office of the Gene Technology Regulator International Program on Chemical Safety Joint Expert Advisory Committee on Antibiotic Resistance UK Ministry of Agriculture, Fisheries and Food maximum residue limit messenger ribonucleic acid National Health and Medical Research Council National Industrial Chemicals Notification and Assessment Scheme nopaline synthase neomycin phosphotransferase II Notifiable Low Risk Dealing National Registration Authority for Agricultural and Veterinary Chemicals Office of the Gene Technology Regulator parts per million Therapeutic Goods Administrations Technical Grade Active Constituent United States Environmental Protection Agency United States Food and Drug Administration World Health Organisation weight per volume 5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid micrograms per gram TABLE OF CONTENTS Page ABOUT THIS DOCUMENT The regulation of gene technology in Australia ............................................................................ 1 The application ............................................................................................................................ 1 The structure of this document ..................................................................................................... 2 1. 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 EXECUTIVE SUMMARY The licence application ................................................................................................... 3 The new gene technology legislation ............................................................................. 3 Consultation processes.................................................................................................... 4 Background on the GMOs and previous releases ........................................................... 5 The evaluation process ................................................................................................... 5 Conclusions of risk assessment ...................................................................................... 6 Conclusions of the risk management plan ...................................................................... 7 Identification of issues to be addressed for future releases ............................................ 7 Next steps........................................................................................................................ 8 2. ASSESSMENT OF LICENCE APPLICATIONS FOR DEALINGS INVOLVING INTENTIONAL RELEASE INTO THE ENVIRONMENT Australia’s legislative system for regulation of activities involving gene technology....................................................................................................................... 9 Interface with other regulators and government agencies .............................................. 9 The Australia New Zealand Food Authority (ANZFA) ................................................ 10 The National Registration Authority for Agricultural and Veterinary Chemicals (NRA) ............................................................................................................................. 10 Types of dealings with GMOs in Australia today .......................................................... 11 Assessment of the licence application for dealings involving the intentional release of a GMO ........................................................................................................................ 11 What government bodies and experts did the Regulator consult in preparing the risk assessment and risk management plan? .......................................................................... 12 What did the Regulator do after consulting with these government bodies and experts? ........................................................................................................................... 13 Who did the Regulator consult with on the risk assessment and risk management plan? ................................................................................................................................ 13 What issues were raised in the public submissions? ...................................................... 15 What has the Regulator done with the submissions received? ....................................... 16 What information can you obtain on the application and the risk assessment and risk management plan? .......................................................................................................... 16 2.1 2.2 2.2.1 2.2.2 2.3 2.4 2.4.1 2.4.2 2.4.3 2.4.4 2.4.5 2.4.6 3. 3.1 3.2 3.3 3.4 3.5 3.5.1 BACKGROUND ON THE APPLICATION, THE GMOs AND PREVIOUS RELEASES The application ............................................................................................................... 17 The application complied with legislative requirements ................................................ 17 About the organisms to be released ................................................................................ 18 Previous releases of these GMOs in Australia ............................................................... 19 Results from Australian releases of Bollgard II® and Roundup Ready®/ Bollgard II® cotton .......................................................................................................... 20 Agronomic performance ................................................................................................. 20 i 3.5.2 3.5.3 3.6 3.7 3.8 4. 4.1 4.2 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6 4.4 4.5 4.5.1 4.5.2 4.6 4.6.1 4.6.2 4.6.3 4.7 4.7.1 4.7.2 4.7.3 5. 5.1 5.2 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.4 5.5 5.5.1 Insecticidal activity ......................................................................................................... 20 Target range .................................................................................................................... 20 Approvals for general release of INGARD® and Roundup Ready® cotton and issuing of deemed licences by the GMAC ..................................................................... 20 Risk assessment and deemed licence conditions for general release of INGARD® and Roundup Ready® cotton ........................................................................................... 21 Approvals for Bollgard II® cotton in other countries ..................................................... 22 INFORMATION ABOUT THE GMOS AND THE PARENT ORGANISM Summary information about the GMO ........................................................................... 23 The parent organism ....................................................................................................... 24 The introduced genes ...................................................................................................... 25 The cry1Ac gene ............................................................................................................. 25 The cry2Ab gene ............................................................................................................. 26 The CP4 EPSPS gene ..................................................................................................... 26 The uidA reporter gene ................................................................................................... 27 The nptII gene ................................................................................................................. 28 The aad gene................................................................................................................... 28 Bt toxins .......................................................................................................................... 28 Method of gene transfer .................................................................................................. 29 Bollgard II® cotton ........................................................................................................ 29 Bollgard II®/Roundup Ready® cotton ............................................................................. 30 Characterisation of the inserted genetic material and stability of the genetic modification .................................................................................................................... 31 Bollgard II® cotton ........................................................................................................ 31 Roundup Ready® cotton ................................................................................................ 31 Bollgard II®/Roundup Ready® cotton ............................................................................. 31 Expression of the introduced proteins ............................................................................ 32 Bollgard II® cotton .......................................................................................................... 32 Roundup Ready® cotton ................................................................................................ 33 Bollgard II®/Roundup Ready® cotton ........................................................................... 33 RISK ASSESSMENT The Risk Analysis Framework ....................................................................................... 34 The risk assessment process ........................................................................................... 34 Summary of conclusions from risk assessment process ................................................. 35 Hazard identification ...................................................................................................... 35 Hazard and risk characterisation ..................................................................................... 36 Consideration of risks relating to combination of the Roundup Ready® and the Bollgard II® traits ............................................................................................................ 36 Identification of issues to be addressed for future release .............................................. 37 Hazard identification ...................................................................................................... 37 Hazard and risk characterisation ..................................................................................... 38 Toxicity or allergenicity ................................................................................................. 38 A: Nature of the potential toxicity or allergenicity hazard ............................................. 38 Toxicity or allergenicity for humans ........................................................................ 38 Toxicity for other organisms .................................................................................... 39 B: Likelihood of the toxicity or allergenicity hazard occurring ..................................... 39 Toxicity or allergenicity of the introduced proteins................................................... 40 Toxicity or allergenicity of Bollgard II® cotton and Bollgard II®/ ii 5.5.2 5.5.3 5.5.4 5.5.5 5.5.6 Roundup Ready® cotton ........................................................................................... 45 Potential for exposure to Bollgard II® cotton and the introduced proteins ................ 48 C: Conclusions regarding toxicity and allergenicity....................................................... 51 Weediness ....................................................................................................................... 52 A: Nature of the weediness hazard ................................................................................. 52 B: Likelihood of the weediness hazard occurring .......................................................... 52 C: Conclusions regarding weediness ............................................................................ 53 Transfer of introduced genes to other organisms ........................................................... 54 Transfer of introduced genes to other plants .................................................................. 54 A: Nature of the gene transfer hazard ............................................................................. 54 Transfer of genes to other cotton plants ..................................................................... 54 Transfer of genes to other plant species ..................................................................... 54 B: Likelihood of the gene transfer hazard occurring ...................................................... 55 Transfer of genes to other cotton crops or feral cotton populations .......................... 55 Transfer of genes to other plant species ..................................................................... 57 C: Conclusions regarding gene transfer to other plants .................................................. 59 Transfer of introduced genes to other organisms (microorganisms and animals) .......... 60 A: Nature of the gene transfer hazard ............................................................................. 60 B: Likelihood of the gene transfer hazard occurring ...................................................... 61 Transfer of genes to humans or other animals ........................................................... 61 Transfer of genes to bacteria ...................................................................................... 61 Transfer of genes to viruses ....................................................................................... 62 C: Conclusions regarding gene transfer to other organisms ......................................... 62 Insecticide resistance ...................................................................................................... 64 A: Nature of the insecticide resistance hazard ................................................................ 64 B: Likelihood of the insecticide resistance hazard occurring ......................................... 64 C: Conclusions regarding insecticide resistance ............................................................. 65 6. 6.1 6.2 6.2.1 6.2.2 6.2.3 6.2.4 6.2.5 6.3 RISK MANAGEMENT PLAN Summary of risk assessment conclusions ..................................................................... 66 Risk management plan .................................................................................................... 66 Risk of toxicity or allergenicity ...................................................................................... 66 Risks of insecticide resistance ........................................................................................ 67 Risks of weediness or gene transfer................................................................................ 67 General licence conditions .............................................................................................. 67 Monitoring and enforcement of compliance by the OGTR ............................................ 68 Proposed specific risk management licence conditions.................................................. 68 7. 7.1 7.2 7.3 CONSIDERATION OF ISSUES RAISED IN PUBLIC SUBMISSIONS Adequacy of the application and the assessment process ............................................. 73 Compliance and monitoring provisions .......................................................................... 74 Research on biosafety risks ........................................................................................... 75 8. NEXT STEPS .......................................................................................................... 76 9. BIBLIOGRAPHY .................................................................................................... 77 APPENDIX iii ABOUT THIS DOCUMENT The regulation of gene technology in Australia Australia’s first national regulatory system for gene technology was established on 21 June 2001, when the Gene Technology Act 2000 (the Act) took effect. The regulatory system is designed 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 Act establishes a statutory officer, the Gene Technology Regulator (the Regulator), to administer the legislation and make decisions under the legislation. The Regulator is supported by the Office of the Gene Technology Regulator (OGTR), a Commonwealth regulatory body located within the Health and Ageing portfolio. 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. The regulatory system incorporates calls for public input during the assessment of licence applications for dealings involving intentional release of a genetically modified organism (GMO) into the environment (intentional release). The purpose of this document is to seek public comment on the first application of this kind to be assessed under the new regulatory system. The application Cotton Seed Distributors Ltd (CSD) has applied for a licence for the release of a genetically modified insecticidal type of cotton which is registered under the trade name Bollgard II® cotton, and another type, Bollgard II®/Roundup Ready® cotton, which is also resistant to the herbicide Roundup®. Bollgard II®/Roundup Ready® cotton was produced by conventional breeding of Bollgard II® cotton with Roundup Ready® cotton. Roundup Ready® cotton is also genetically modified and was approved for commercial release in Australia in 2000. Both Bollgard II® and Bollgard II®/Roundup Ready® cotton have been trialled previously in Australia. CSD is proposing to carry out a limited release on one site in Queensland, over a total area of 122 hectares. This represents a substantial reduction from the initial proposal in the application, for six sites over a total area of 480 hectares. The release will occur very late in the cotton planting season. Its purpose is to continue large-scale evaluation of the agronomic performance of a number of different cotton varieties and to produce seed for possible future releases. Further releases would be subject to a separate application and assessment process. None of the cotton plants produced in the release, or their by-products, will be used for human or animal feed. Further details about the application can be found in Part 3 of this document. 1 The structure of this document Section 50 of the Act requires the Regulator to prepare a risk assessment and risk management plan in relation to proposed intentional releases. This document presents the risk assessment and risk management plan prepared in relation to the CSD application. The document sets out the various matters that were considered by the Regulator in accordance with Section 51 of the Act in preparing this risk assessment and risk management plan, and also outlines the consultation processes undertaken under Sections 50 and 52 of the Act. Written submissions sought through these consultation processes have been taken into account by the Regulator in finalising the risk assessment and risk management plan and must be considered by the Regulator before making a final decision on the application. The consultation phase is, therefore, an important part of the decision-making process. The structure of the document reflects the matters which the Act and Regulations require the Regulator to consider in preparing the risk assessment and risk management plan. This document: provides an executive summary of the risk assessment and risk management plan. Part 1 refers. provides an overview of the regulatory system for GMOs in Australia and outlines the processes that have been and will be undertaken in relation to this proposal, including a summary of issues raised in public submissions and how these were taken into account. Part 2 refers. summarises the proposed dealings covered by the licence application, and provides background information relating to previous intentional releases of the GMOs and other related GMOs. Part 3 refers. provides detailed information about the GMOs, the parent organisms and the introduced genes. Part 4 refers. details the risk assessment undertaken in accordance with the Risk Analysis Framework developed by the Regulator. Part 5 refers. sets out the conclusions reached as a result of the risk assessment and presents a risk management plan to manage the identified risks. Conditions which will be included in the licence to give effect to the risk management plan are also provided. Part 6 refers. summarises issues raised in public submissions on the risk assessment and risk management plan and how these have been taken into account. Part 7 refers. explains the next steps in the decision-making process. Part 8 refers. 2 1. 1.1 EXECUTIVE SUMMARY The licence application Cotton Seed Distributors Ltd (CSD) has applied for a licence for the release of a genetically modified insecticidal type of cotton which is registered under the trade name Bollgard II® cotton, and another type which is also resistant to the herbicide Roundup®. Bollgard II®/Roundup Ready® cotton was produced by conventional breeding of Bollgard II® cotton with Roundup Ready® cotton. Roundup Ready® cotton is also genetically modified and was approved for commercial release in Australia in 2000. Both Bollgard II® and Bollgard II®/Roundup Ready® cotton have been trialled previously in Australia (refer to Parts 1.4 and 3.4 for details). CSD is proposing to carry out a limited release on one site in Queensland, over a total area of 122 hectares. This represents a substantial reduction from the initial proposal in the application, for six sites over a total area of 480 hectares. The release will occur very late in the cotton planting season. Its purpose is to continue large-scale evaluation of the agronomic performance of a number of different cotton varieties and to produce seed for possible future releases. Further releases would be subject to a separate application and assessment process. None of the cotton plants produced in the release, or their by-products, will be used for human or animal feed. Please note that the application is not available electronically. In the future, the OGTR hopes that electronic submission of applications will be possible, enhancing the accessibility of such information for interested people in the community. 1.2 The new gene technology legislation This is the first application for a licence for an intentional release of a genetically modified organism (GMO) to be assessed under Australia’s new regulatory system for gene technology. The Gene Technology Act 2000 (the Act), which underpins the new regulatory system, took effect on 21 June 2001. The new system is Australia’s first national regulatory system for gene technology and is designed to protect the health and safety of people, and the environment, by identifying risks posed by or as a result of gene technology, and to manage those risks by regulating certain dealings with GMOs. The new regulatory system replaces the former voluntary system overseen by the Genetic Manipulation Advisory Committee (GMAC). The legislation also established a statutory officer, the Gene Technology Regulator (the Regulator) to administer the legislation and make decisions under the legislation. The Regulator is supported by the Office of the Gene Technology Regulator (OGTR), a Commonwealth regulatory body located within the Health and Aged Care portfolio. 3 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 Part 2.2). The requirements under the new legislation for consultation and for considering and assessing licence applications and preparing risk assessment and risk management plans are discussed in detail in Parts 2 and 5 and summarised below. 1.3 Consultation processes In accordance with Section 50 of the Act, the Regulator sought advice in preparing a risk assessment and risk management plan from: (a) the States and Territories; (b) the Gene Technology Technical Advisory Committee (GTTAC); (c) prescribed Commonwealth agencies (Part 2.3 refers); (d) the Environment Minister; and (e) local councils where the release was proposed (the shires of Balonne and Emerald – note that the release is now proposed for Emerald only). As a measure over and above those required under the Act, in order to promote the openness and transparency of the new regulatory system, receipt of the application was also notified to the public by a variety of means outlined in Part 2.3. Advice on the application was received from Queensland, New South Wales, Western Australia, GTTAC, and three prescribed agencies, the National Registration Authority (NRA), the Australia New Zealand Food Authority (ANZFA) and the National Occupational Health and Safety Commission (NOHSC), and the Environment Minister. As required under Section 51 of the Act, the Regulator took this advice into account in the preparation of a risk assessment and risk management plan. In accordance with Section 52 of the Act, the Regulator sought written submissions on the risk assessment and risk management plan from the organisations listed above, and the Environment Minister. In addition, written submissions were sought from the public. Written submissions on the risk assessment and risk management plan were received from New South Wales, the Northern Territory, Queensland, Tasmania, Victoria, and Western Australia, the Emerald Shire Council, GTTAC, and five prescribed agencies, the National Registration Authority (NRA), the Australia New Zealand Food Authority (ANZFA), the National Occupational Health and Safety Commission (NOHSC), the Australian Quarantine and Inspection Service (AQIS) (through Biosecurity Australia), and the Therapeutic Goods Administration (TGA), and the Environment Minister. Fifteen submissions were received from the public (see Part 7). Comments in these submissions were taken into account in finalising the risk assessment and risk management plan presented here. 4 1.4 Background on the GMOs and previous releases The applicant is seeking a licence to release two GMOs: Bollgard II® cotton; and Bollgard II®/Roundup Ready® cotton. Full details of the GMOs and the introduced genes are provided in Part 4. Bollgard II® cotton has been genetically modified by introducing two genes from a soil bacterium to make it resistant to the major pests of cotton in Australia (Part 4.2 refers). The cotton also contains bacterial antibiotic resistance genes and a bacterial ‘reporter’ gene (see Part 4.3). Bollgard II® cotton was derived from INGARD® (Bt) cotton. INGARD® cotton contains an insecticidal gene and was approved for commercial release in Australia in 1996 (see Part 3.3). Bollgard II® was produced by inserting a second insecticidal gene, along with a ‘reporter’ gene, into INGARD® cotton (see Part 4.3). The Bollgard II®/Roundup Ready® cotton was produced through conventional breeding, by crossing Bollgard II® cotton with GM herbicide-tolerant Roundup Ready® cotton (see Part 3.3). Roundup Ready® cotton was approved for commercial release in 2000 (see Part 3.6) There have been 16 previous limited and controlled releases of Bollgard II® cotton in Australia, including four releases of Bollgard II®/Roundup Ready® cotton (see Part 3.4). The previous releases were conducted under the former voluntary system overseen by GMAC. The cotton has also been released in a number of countries overseas (see Part 3.8). More detailed information about the GMO, the parent organism, the genetic modification process, the genes that have been introduced, and the new proteins expressed in the GM cotton are set out in Part 4. 1.5 The evaluation process An assessment of the potential hazards and likely risks associated with the proposed release was carried out in accordance with the Act, using a Risk Analysis Framework developed by the Regulator (see Part 5). A risk assessment and risk management plan was then prepared to address these risks. Written submissions were then sought as discussed above, and taken into account in finalising the risk assessment and risk management plan. In preparing the risk assessment and risk management plan, information presented by the applicant, the scientific literature, information from other national and international regulatory agencies, and advice from scientific experts, as well as submissions and advice from the Environment Minister, State and Territory Governments, GTTAC, Commonwealth agencies and the public (see Parts 2.4) was considered and assessed. The legislation requires the Regulator to consider a number of specific issues in preparing the risk assessment and risk management plan (see Part 5.2). These include: the properties of the parent organism; the effect of the genetic modification; the potential for dissemination or persistence of the GMO or its genetic material in the environment and any provisions for 5 limiting this; the extent or scale of the proposed dealings; and any likely impacts of the proposed dealings on the health and safety of people. The legislation also requires the Regulator to consider the potential of the GMO, in the short and long term, to: be harmful to other organisms; adversely affect any ecosystems; transfer genetic material to other organisms; spread or persist in the environment; have a selective advantage in the environment; and be toxic, allergenic or pathogenic to other organisms. 1.6 Conclusions of risk assessment The risk assessment and risk management plan identifies and evaluates a number of possible hazards that could arise as a direct result of the genetic modification of Bollgard II® cotton or Bollgard II®/Roundup Ready® cotton, including: the potential for the genetically modified cotton to be harmful to other organisms, including humans, because it is toxic or allergenic; the potential for the genetically modified cotton to be harmful to the environment because of inherent weediness or increased potential for weediness; and the potential for the new genes introduced into the cotton to transfer to non-GM cotton crops and wild or native cottons, or to other organisms, with adverse consequences; and the potential for resistance to the insecticidal proteins produced by the introduced genes to develop in target insects in the long term. The detailed risk assessment is provided in Part 5, with a summary of the main conclusions presented in Part 5.3. In summary, the risk assessment process identified no substantive additional risks to public health and safety or to the environment arising from the genetic modification of Bollgard II® and Bollgard II®/ Roundup Ready® cotton, compared to those posed by conventional cotton, because: Bollgard II® and Bollgard II®/ Roundup Ready® cotton are not likely to prove more toxic or allergenic to humans or other organisms, other than some lepidopteran insects, than conventional cotton (lepidopteran insects are moths and butterflies); the risk of the Bollgard II® or Bollgard II®/ Roundup Ready® cotton establishing as a weed is low and not likely to be greater than that of conventional cotton; the potential for transfer of the introduced genes to non-GM cotton crops is negligible because it is being planted very late in the cotton growing season and no other cotton crops in the area will be flowering at the same time; the potential for transfer of the introduced genes to wild or native cotton is very low because of the geographical isolation and genetic incompatibility with the native species; the likelihood of transfer of the introduced genes to other organisms is low, but even if such transfer occurred would be unlikely to pose any hazard to human health and safety or the environment; and the risk of development of target insects resistant to the insecticidal proteins is very low, due to the limited scope of the proposed release and the presence of two insecticidal proteins. 6 During the evaluation process, a range of issues were identified that would need to be addressed for future commercial releases. These include further information and data requirements, and the need to consider the use of antibiotic resistance marker genes in the longer term (see Part 5.3.4). 1.7 Conclusions of the risk management plan On the basis of the assessment to date, the risk management plan proposes that the identified risks can be managed to protect human health and safety and the environment by including a number of specific conditions in the licence to minimise the spread and persistence of Bollgard II® and Bollgard II® /Roundup Ready® cotton, or the modified genetic material, in the environment (Parts 6.2 and 6.3 refer). The risk assessment supports the issuing of a licence by the Regulator. The licence would incorporate a number of conditions to achieve effective risk management during and after the release. The licence conditions, and the reasons behind them, are set out in detail in Part 6.3. The conditions include requirements to isolate the cotton crop from other cotton by at least 50 metres, to undertake research to confirm the efficacy of the 50 metre isolation zone, to destroy any viable material not required for subsequent releases (which would require separate licences) after the harvest, and to monitor the release site after harvest and remove any cotton plants (volunteers) that germinate or regrow after the release for a period of 12 months. The licence would also contain a number of general conditions, including conditions required by the Act, that also relate to risk management. For example, there are conditions requiring the applicant to inform people covered by the licence of their obligations, including providing access to inspectors appointed by the Regulator for the purpose of monitoring compliance with the licence conditions, and to inform the Regulator of any additional information about risks to human health or safety or to the environment, unintended effects of the release, or contraventions of the licence conditions. The licence holder must also comply with Guidelines issued by the Regulator, including Guidelines for the Transport of GMOs. The Regulator also has additional options for risk management available. The Regulator has the legislative capacity to enforce compliance with licence conditions and to direct a licence holder to take any steps deemed necessary to protect the health and safety of people or the environment. The Regulator, through the services of the OGTR, will also independently monitor sites where intentional releases are authorised. At least 20 % of all sites will be visited each year. 1.8 Identification of issues to be addressed for future releases During the evaluation process, a range of data and information requirements were identified which would be required before any future commercial release could be contemplated. These include further information and data on: the potential toxicity of Bollgard II cotton, including more information on potential toxicity to non-target pests; the potential for gene flow from cotton to related species; 7 whether growth of Bollgard II cotton is likely to be limited by insect predation, and the potential impact on weediness of the cotton; and the expression levels of the introduced proteins; insect resistance management strategies for Bollgard II cotton; The general issue of the use of antibiotic resistance marker genes may also need to be considered in the longer term (see Part 5.3.4). 1.9 Next steps As required under Division 5 of Part 5 of the Act, the Regulator must make a decision on whether or not to issue a licence for the proposed release, and on the licence conditions, having regard to the risk assessment and risk management plan, written submissions received on the plan, and a number of other matters set out in the legislation and discussed in detail in Part 7. 8 2. ASSESSMENT OF LICENCE APPLICATIONS FOR DEALINGS INVOLVING INTENTIONAL RELEASE INTO THE ENVIRONMENT This part of the document provides details about the new regulatory system for gene technology in Australia and the requirements under this system for assessing licence applications for release of GMOs into the environment. In particular, it outlines the comprehensive consultation processes that are undertaken in relation to such applications. 2.1 Australia’s legislative system for regulation of activities involving gene technology The Office of the Gene Technology Regulator (OGTR) is a Commonwealth regulatory body located within the Health and Aged Care portfolio. The OGTR was established to support the Gene Technology Regulator (the Regulator) in administering the Gene Technology Act 2000 (the Act) which came into effect on 21 June 2001. The Act is supported by the Gene Technology Regulations 2001 (the Regulations). The Act and Regulations underpin a national regulatory system which aims to protect human health and safety, and to protect the environment, by identifying risks posed by or as a result of gene technology, and by managing those risks by regulating certain dealings with genetically modified organisms (GMOs). The new regulatory system established under the Act replaces the former voluntary system which was overseen by the Genetic Manipulation Advisory Committee (GMAC). In summary, the Act prohibits persons from dealing with GMOs (e.g. research, manufacture, production, commercial release and import) unless the dealing is: - exempt; - a Notifiable Low Risk Dealing (NLRD) - that is, contained research work which has been demonstrated to pose minimal risk to workers, the general public or the environment; - on the Register of GMOs ; or - licensed by the Regulator. Detailed information about the national regulatory system and the gene technology legislation is available from the OGTR website at: www.ogtr.gov.au 2.2 Interface with other regulators and government agencies Other government regulatory requirements must also be met in respect of the release of GMOs, and the use of products of GMOs, including those of the Australia New Zealand Food Authority (ANZFA) and the National Registration Authority for Agricultural and Veterinary Chemicals (NRA). 9 2.2.1 The Australia New Zealand Food Authority (ANZFA) ANZFA is responsible for carrying out safety assessments and for the labelling of foods derived from GMOs, under Standard A18 of the Australian Food Standards Code. None of the cotton plants from the proposed release, or any of their by-products, will be used in human food, so no approval is required by ANZFA. Any use of the GM cotton or its by-products in human food would necessitate an application to ANZFA. Further information about food safety assessments and food labelling is available from ANZFA: Australia New Zealand Food Authority PO Box 7186 Canberra Mail Centre ACT 2610 Phone: (02) 6271 2222 Fax: (02) 6271 2278 E-mail: [email protected] http://www.anzfa.gov.au 2.2.2 The National Registration Authority for Agricultural and Veterinary Chemicals (NRA) The NRA undertakes the safety assessment of agricultural and veterinary chemicals. Currently, this includes INGARD® and Bollgard II® cotton which are regarded as plant pesticides. The NRA regulates how agricultural and veterinary chemicals are labelled and also sets the maximum residue levels (MRLs) for agricultural chemicals in agricultural produce, particularly produce entering the food chain. MRLs set by the NRA are referred to ANZFA for incorporation into the Food Standards Code. In the case of commercially released INGARD® cotton, the NRA is also responsible for ensuring implementation of an insecticide resistance management plan (see Part 5.5.4). The management plan was required to satisfy the NRA that the insecticidal protein would remain effective. The NRA is currently considering an application from Cotton Seed Distributors for a permit for the proposed release. Further details on this should be sought from the NRA: National Registration Authority for Agricultural and Veterinary Chemicals PO Box E240 KINGSTON ACT 2604 Phone: (02) 6272 5158 Fax: (02) 6272 4753 Email: [email protected] http://www.affa.gov.au/nra 10 2.3 Types of dealings with GMOs in Australia today As noted above in Part 2.1, dealings with GMOs require a licence, unless they fall into the low risk exempt or notifiable low risk dealing categories or are on the Register of GMOs. For work requiring a licence, there are two major categories: dealings that do not involve the intentional release in the environment (DNIR); and dealings that involve the intentional release of a GMO in the environment (DIR). The DNIR category includes contained work carried out in laboratories and other facilities designed to prevent the release of the GMO into the environment. Examples of this type of work are basic medical or biological research undertaken by research organisations such as universities and other research institutions, or the manufacture of recombinant proteins such as insulin by biotechnology companies. The DIR (intentional release) category covers work ranging from limited releases (field trials) at the initial stages of research and development, through to commercial releases of GMOs. The initial limited releases are carried out in the open environment to obtain information on the agronomic performance of a GMO, its interaction with the environment, and to gain more knowledge of potential hazards and risk management strategies. These releases are initially carried out on a restricted scale and for a limited period, under conditions that minimise the potential for spread of the GMO. As more knowledge is gained about the potential risks, releases may be approved on a larger scale or with less stringent conditions. 2.4 Assessment of the licence application for dealings involving the intentional release of a GMO The application submitted by CSD in respect of Bollgard II® and Bollgard II®/Roundup Ready® cotton seeks a licence for the intentional release of certain GMOs into the Australian environment. The Act is designed to ensure a comprehensive analysis of the hazards and risk posed by the proposed release, and to identify measures necessary to manage those risks so that the health and safety of people and the environment are protected. The Act provides for wide consultation to be undertaken in assessing applications for the intentional release of a GMO into the environment. Significant consultation with parties outside the OGTR is required. For example, in relation to this application, the Regulator must: consult on the application with State and Territory governments, relevant local governments, the Gene Technology Technical Advisory Committee (GTTAC), Commonwealth agencies, the Environment Minister (Section 50 of the Act) and, where the proposed release is considered to pose significant risks to the health and safety of people, or to the environment, the Australian community (Section 49 of the Act); consult on the risk assessment and risk management plan with State and Territory governments, relevant local governments, GTTAC, Commonwealth agencies, the 11 Environment Minister and, for all licence applications, the Australian community (Section 52 of the Act); and consider submissions on the risk assessment and risk management plan (Section 52 of the Act) in deciding whether or not to issue a licence and in setting the licence conditions (Sections 55-58 of the Act). Information on these steps is set out below. 2.4.1 What government bodies and experts did the Regulator consult in the preparing the risk assessment and risk management plan? Extensive consultation on the preparation of the risk assessment and risk management plan is central to the evaluation process. Sections 50 and 52 of the Act provide that the Regulator must seek advice on the preparation of the risk assessment and the risk management plan from the following: (a) the States and Territories; and (b) the Gene Technology Technical Advisory Committee (GTTAC); and (c) each Commonwealth authority or agency prescribed by the Regulations for the purposes of this paragraph (the prescribed agencies are the Australia New Zealand Food Authority (ANZFA); the Australian Quarantine and Inspection Service (AQIS); the National Health and Medical Research Council (NHMRC); the National Industrial Chemicals Notification and Assessment Scheme (NICNAS); the National Registration Authority for Agricultural and Veterinary Chemicals (NRA); and the Therapeutic Goods Administration (TGA); and (d) the Environment Minister; and (e) any local council that the Regulator considers appropriate. In accordance with the Act, the licence application submitted by CSD was provided to: (a) the designated Department in each State and Territory of Australia; (b) the Gene Technology Technical Advisory Committee; (c) the prescribed agencies: - ANZFA; - AQIS; - NHMRC; - NICNAS; - NRA; and - TGA; (d) the Minister for the Environment and Heritage, Senator the Hon Robert Hill; and (e) the Chief Executive Officers of the Balonne and Emerald shires, Queensland (note that the proposed release will now only occur in the latter). 12 Advice on the application was received from the Environment Minister, the Queensland, New South Wales, and Western Australian Governments, the NRA, ANZFA, and NICNAS and GTTAC. In summary, the responses indicated that the following matters should be addressed in the risk assessment and risk management plan: gene transfer to other species including microbes, non-transgenic cotton crops, naturalised cotton populations and native species, and potential ecological impacts; potential for dissemination of GM pollen and seed beyond the release site; persistence of the Cry1Ac and Cry2Ab proteins in soil; mode of action and species specificity of the Cry proteins; impacts of the nptII and GUS proteins on the environment; toxicity of the Cry2Ab protein; the potential for pollen transfer by insects; hazards associated with the Roundup Ready modification; and interaction between the introduced genes/traits Comments were also provided on conditions that might be included in the licence to manage possible risks. All concerns raised by these parties have been addressed in this risk assessment and risk management plan. In line with the principles of openness and transparency that guided development of the regulatory system, the Regulator also publicly notified receipt of the application. A notice advising availability of the application was placed on the OGTR website, and every person and organisation registered on the OGTR mailing list (a total of over 1000 individuals or organisations) was notified by direct mail. 2.4.2 What did the Regulator do after consulting with these government bodies and experts? The Regulator has prepared a risk assessment and risk management plan in accordance with Sections 50 and 51 of the Act. All comments received on application in relation to the preparation of the risk assessment and risk management plan through the consultation process were taken into account. Details of the preparation of risk assessment process and risk management plan are provided in Parts 5 and 6. 2.4.3 plan? Who did the Regulator consult with on the risk assessment and risk management In accordance with Section 52 of the Act, the Regulator sought written submissions on the risk assessment and risk management plan from: (a) the designated Department in each State and Territory of Australia; (b) the Gene Technology Technical Advisory Committee; 13 (c) the prescribed agencies: - ANZFA; - AQIS; - NHMRC; - NICNAS; - NRA; and - TGA; (d) the Minister for the Environment and Heritage, Senator the Hon Robert Hill; and (e) the Chief Executive Officers of Balonne and Emerald shires, Queensland. Section 52 of the Act also required the Regulator to publicly notify the availability of the risk assessment and risk management plan and seek written submissions. The Act specifically requires that: a notice be published in a newspaper circulating generally in all States. An advertisement was placed in the 17 November 2001 edition of The Weekend Australian newspaper. In addition, while not required under the Act, a further advertisement was placed in the 17 November 2001 edition of The Courier-Mail, because the application relates to a proposed release in Queensland. a notice be published in the Gazette. A notice, therefore, appeared in Commonwealth of Australia: Government Notices Gazette of 21 November 2001; and a notice be published on the OGTR website. The following documents, therefore, were made available on the ‘Whats New’ part of the OGTR website as of 17 November 2001: - the early notification on risk assessment; a summary information sheet about the application; a summary information sheet about the risk assessment and risk management plan; and the risk assessment and risk management plan. Copies of these documents were also available from the OGTR. In addition, although not required by the Act, every person and organisation registered on the OGTR mailing list (a total of over 1000 individuals or organisations) received by direct mail a notification of the availability of the risk assessment and risk management plan and an invitation to comment on them. Written submissions on the risk assessment and risk management plan were received from the New South Wales, the Northern Territory, Queensland, Tasmanian, Victorian, and Western Australian governments, the Emerald Shire Council, GTTAC, and five prescribed agencies, the NRA, ANZFA, NOHSC, AQIS and the TGA, and the Environment Minister. Sixteen submissions were received from the public, and a summary of the issues raised in the public submissions is provided in Part 2.4.5 and the Appendix. 14 2.4.4 What issues were raised in the public submissions? In response to the public notifications described in Part 2.4.4, the GTR received 16 written submissions: 5 from private individuals; 2 from agricultural organisations; 2 from environmental interest groups; 2 from health or food interest groups; 1 from an organisation involved in biotechnology; and 4 from consumer or public interest organisations. The analysis of these submissions revealed a range of concerns: 9 raised concerns about potential risks to the environment and 6 raised concerns about potential risks to human health and safety. These were taken into account, together with relevant available scientific knowledge, in finalising the risk assessment and risk management plan (see Parts 5 and 6), except where they related to matters such as food safety and labelling, and pesticide use and safety, which are the responsibility of other regulatory authorities (see Part 2.2); 13 expressed concerns about the release of the GMO, this included concerns about: - the adequacy of the application and the assessment process (see Part 7.1); - concerns about compliance with conditions imposed by the GTR (see Part 7.2); - the need for more research on GMOs (see Part 7.3). 13 raised broader concerns not related to risks to human health and safety or the environment including: - - the need for sustainable development in agriculture; liability for any damage arising as a result of the release; market issues, including the need to consider the potential benefits of a GMO, and socioeconomic issues such as acceptance of GM products by consumers and producers; concerns about multinational corporations or monopolies and patenting of GMOs; and philosophical concerns about gene technology. These issues fall outside the scope of the risk assessment process defined by the Act and Regulations and have therefore not been specifically addressed in this document. 2 of the submissions expressed support for the proposed release. A more detailed summary of the issues raised in each submission, and how these were considered, is provided in the Appendix. 15 In addition, the Regulator will write to each of the organisations or individuals that made a submission on the application, to advise them of the outcome of the application and how their input was considered. 2.4.5 What has the Regulator done with the submissions received? All relevant issues raised in written submissions on the risk assessment and risk management plan have been taken into account in finalising the risk assessment and risk management plan, and will also be taken into account by the Regulator in making a decision as to whether or not to issue a licence for the proposed release. Under Section 56 of the Act, the Regulator must not issue a licence unless satisfied that any risks posed by the proposed release are able to be managed in such a way as to protect the health and safety of people and the environment. The full requirements under the legislation for making decisions on a licence application are discussed in Part 8. 2.4.6 What information can you obtain on the application and the risk assessment and risk management plan? Any interested party may obtain copies of the following documents from the OGTR: the finalised risk assessment and risk management plan as set out in this document; the full licence application submitted by CSD; a summary of the proposed intentional release and the risk assessment and risk management plan, for interested parties who may not wish to consider the detailed assessment, but who want access to some information about the proposal. 16 3. BACKGROUND ON THE APPLICATION, THE GMOs AND PREVIOUS RELEASES This part of the document provides information about the proposed release, and summary information about the GMOs, including information about previous releases into the environment of relevant GMOs. 3.1 The application Cotton Seed Distributors Ltd (CSD) has applied for a licence for the release of a genetically modified insecticidal type of cotton which is registered under the trade name Bollgard II® cotton, and another type, Bollgard II®/Roundup Ready® cotton, which is also resistant to the herbicide Roundup®. Bollgard II®/Roundup Ready® cotton was produced by conventional breeding of Bollgard II® cotton with Roundup Ready® cotton. Roundup Ready® cotton is also genetically modified and was approved for commercial release in Australia in 2000. Both Bollgard II® and Bollgard II®/Roundup Ready® cotton have been released previously in Australia. CSD is proposing to carry out a limited release on one site in Queensland, over a total area of 122 hectares. This represents a substantial reduction from the initial proposal in the application, for six sites over a total area of 480 hectares. The release will occur very late in the cotton planting season. Its purpose is to continue large-scale evaluation of the agronomic performance of a number of different cotton varieties and to produce seed for possible future releases. Further releases would be subject to a separate application and assessment process. None of the cotton plants produced in the release, or their by-products, will be used for human or animal feed. Additional supporting information for the application, on the molecular characterisation of Bollgard II® cotton, was provided by Monsanto Australia Ltd (who developed the initial genetically modified cotton varieties that Cotton Seed Distributors then crossed by conventional breeding with Australian cotton cultivars, see Part 4.2). 3.2 The application complied with legislative requirements The proposal was submitted in accordance with the requirements of Section 40 of the Act. As required by Schedule 4, Part 2 of the Regulations, the application included information about: the parent organism; the GMO; the proposed dealing with the GMO; interaction between the GMO and the environment; risks the GMO may pose to the health and safety of people; risk management; previous assessments; and the suitability of the applicant. 17 The application also contained: additional information required for a GMO that is a plant; additional information for a GMO that is intended to be used as food for human or vertebrate animal consumption (noting that material from this release will not be permitted to be used for human consumption); and supporting information from the Institutional Biosafety Committee. A full copy of the application is available on request from the OGTR. 3.3 About the organisms to be released The organisms to be released are Bollgard II® and Bollgard II®/Roundup Ready® cotton. Bollgard II® cotton, previously known as TWINGARD® cotton, has been genetically modified to make it insecticidal. Bollgard II® cotton contains two genes, cry1Ac and cry2Ab, from a soil bacterium Bacillus thuringiensis (abbreviated as Bt). The cry1Ac and cry2Ab genes (also referred to as Bt genes) encode the insecticidal proteins Cry1Ac and Cry2Ab that protect the cotton against the insect pests Helicoverpa armigera (cotton bollworm) and H. punctigera (native budworm). Caterpillars of these insect species are the major pests of cotton in Australia and are normally controlled by spraying with pesticides. Bollgard II® cotton is derived from INGARD® (Bt) cotton which contains a single insecticidal gene (cry1Ac). Bollgard II® was produced by inserting the cry2Ab gene along with a reporter gene (uidA) into INGARD® cotton. INGARD® cotton was approved for commercial release in Australia in 1996 (see Part 3.6). The uidA reporter gene is from Escherichia coli and codes for an enzyme which enables visual identification of plant tissues in which this gene is being expressed. Bollgard II® cotton also contains two bacterial genes conferring resistance to antibiotics (these are present in the parent INGARD® cotton). The npt II gene confers resistance to kanamycin and neomycin and the aad gene confers resistance to streptomycin and spectinomycin. The aad gene is not expressed in the plants. The extra insecticidal gene has been introduced in Bollgard II® cotton with the aim of delaying the emergence of resistant insects. Ecological modelling shows that the use of two genes specifying two different insecticidal proteins in the same plant, as opposed to the use of a single gene, as in INGARD® cotton, is likely to delay the selection of insects resistant to the insecticidal proteins by a factor of 10 (Roush, 1994). Addition of the extra gene has also increased the efficacy of pest control compared to INGARD® cotton (see Part 4.7). The Bollgard II®/Roundup Ready® cotton is also tolerant to the herbicide glyphosate, the active constituent in Roundup® herbicide. The herbicide tolerance is conferred by the CP4 EPSPS gene from a soil bacterium, Agrobacterium strain CP4, and was introduced into Bollgard II® cotton through conventional breeding with herbicide-tolerant Roundup Ready® cotton. Roundup Ready® and Roundup Ready®/INGARD® cotton were approved for commercial release in 2000 (see Part 3.6). 18 The cotton varieties proposed to be released were produced by conventional breeding of Bollgard II® with the Sicala, Sicot and Siokra cultivars developed by CSIRO, or with Roundup Ready® varieties of the same cultivars produced by conventional breeding with Roundup Ready® cotton. Further details about the parent organisms, the genetic modification process and the introduced genes are provided in Part 4. 3.4 Previous releases of these GMOs in Australia A number of previous releases have been carried out to assess the agronomic performance of Bollgard II® and Roundup® Ready/Bollgard II® cotton and their behaviour in the Australian environment. The first release was in 1999. In these releases, Bollgard II® cotton was called TWINGARD® cotton, and the Cry2Ab gene was designated as the ‘Cry X’ gene. The releases were field trials, that is limited releases carried out under conditions to limit spread or persistence of the GMO in the environment. A number of different Bollgard II® cotton varieties have been grown in various Australian locations and conditions, to select the best varieties for further development. A number of organisations have been involved in carrying out the releases, including CSIRO and the Queensland Department of Primary Industries (QDPI) as well as the cotton seed companies CSD and Deltapine Australia Ltd. All releases carried out to date were assessed and conducted under the former voluntary system, under GMAC’s oversight and in accordance with GMAC guidelines. Each proposed release was notified in the Gazette, on the GMAC or IOGTR website, and by direct mail to the GMAC or IOGTR mailing list, to enable public comment for consideration in the assessment process. Relevant local government councils were also advised directly. Reports were provided to the GMAC or the OGTR at the conclusion of each release. No adverse effects on human health and safety or the environment were reported for any of these releases. Before issuing advice to proceed for these releases, GMAC considered the environmental and human health risks of: 12 releases involving Bollgard II® cotton: PR-51X(4), PR-112, PR-112X, PR-112X(2), PR-118, PR-118X, PR-118X(2), PR-123, PR-123X, PR-123X(2), PR-131, and PR-131X; and 4 releases involving Bollgard II®/Roundup Ready® cotton: PR-131X(2), PR-131X(3), PR-140 and PR-140X. The releases have been undertaken in New South Wales, the Northern Territory, Queensland and Western Australia and were carried out by CSIRO Plant Industry, CSD and Deltapine Australia Pty Ltd. In the largest trial, the proposed planting area was 712 hectares. 19 3.5 Results from Australian releases of Bollgard II® and Roundup Ready®/Bollgard II® cotton Factors assessed in the previous releases included the agronomic performance of the cotton, the quality of the cotton fibre, possible effects of the Cry1Ac and Cry2Ab toxins on non-target invertebrates, and the levels of insecticidal activity. 3.5.1 Agronomic performance The varieties selected for large-scale seed increase by CSD in this proposal have been demonstrated in Australian releases to have equivalent yield and fibre quality to conventional varieties. 3.5.2 Insecticidal activity Bollgard II® cotton is expected to have superior levels of insecticidal activity compared to INGARD® cotton and in particular to augment the late season insect control. Initial studies by CSIRO indicate that this is the case and that Bollgard II® cotton gives much higher levels of control of the major Helicoverpa caterpillar species in Australia (Dr G. Fitt, CSIRO Entomology, CEO Australian Cotton Research Institute, personal communication). This data confirms similar observations in the U.S. where Bollgard II® varieties have been tested over the last three years (Jackson et al., 2000; Akin et al., 2001). 3.5.3 Target range Studies on the target range of Bollgard II® cotton for Australian arthropods, carried out by the Queensland Department of Primary Industry and the Australian Quarantine Inspection Service, indicate that the addition of a second insecticidal gene extends the insecticidal spectrum of INGARD® cotton to other minor lepidopteran pests, but does not increase effects on other non-target insects. Full details of this study were recently been provided to the Regulator with a separate licence application and will be made publicly available during consultation on that application. 3.6 Approvals for general release of INGARD® and Roundup Ready® cotton and issuing of deemed licences by the GMAC Bollgard II® and Bollgard II®/Roundup® Ready cotton are derived from two genetically modified cottons, INGARD® and Roundup Ready® cotton. Both have already been released commercially in Australia by Monsanto Australia Ltd. On 5 August 1996, the INGARD® gene present in INGARD® cotton was registered as an agricultural chemical product by the National Registration Authority for Agricultural and Veterinary Chemicals (NRA), on the basis of advice provided by GMAC and other Commonwealth and State Government Agencies. Planting of INGARD cotton was initially limited to 30 000 hectares, but this has been gradually extended. In June 2000, the NRA varied the conditions of registration allowing up to 30 % (165 000 hectares) of the current cotton crop to be planted to INGARD® cotton for the 2000-2001 season. 20 Roundup® Ready and Roundup® Ready/INGARD® cotton were approved for commercial release on 14 September 2000, by the Minister for Health and Aged Care, the Hon Dr Michael Wooldridge MP, under the previous voluntary system. Under transitional arrangements set out in Section 190 of the Act, GMAC’s advices to proceed for the general release of INGARD® and Roundup Ready® cotton, issued to Monsanto Australia Ltd, were taken to be licences for the purposes of the Act. The licences took effect with the commencement of the new legislation on 21 June 2001. 3.7 Risk assessment and deemed licence conditions for general release of INGARD® and Roundup Ready® cotton In assessing and establishing the conditions of the initial approvals for INGARD® and Roundup® Ready cotton, there was extensive co-operation between the NRA, GMAC, the Interim OGTR (IOGTR, the OGTR’s predecessor under the voluntary system), Commonwealth bodies including Environment Australia, the Environment Protection Agency and the Australia New Zealand Food Authority, as well as State agencies. The NRA and the IOGTR also undertook public consultation as part of the assessment process. Responses were received from representatives of consumer, environmental, farming, cotton industry, and scientific and academic interests. The potential risks to human health and the environment were thoroughly assessed by the NRA, GMAC and the IOGTR. They concluded that risks to human health were negligible and that risks to the environment were low and could be managed. The environmental risks identified by GMAC were that there was a very low risk of outcrossing with native Australian cotton species and, for INGARD® cotton, a low risk that it might persist as a weed in certain regions of Australia, if insect resistance might confer a selective advantage. GMAC considered that these risks could be managed by restricting planting of genetically modified cotton to locations where contact with native cotton would be unlikely. GMAC recommended that the release of Roundup Ready® and INGARD® cotton be restricted in location and scale, until further data were available to fully assess the weediness of the GM cotton, and the likelihood and consequences of outcrossing with native cotton. The previously issued licence conditions therefore restrict the general release of the GM cotton to south of latitude 22 degrees South and require environmental monitoring to obtain further information about the potential risks. (Note that the proposed release would take place south of latitude 22 degrees South.) For Roundup Ready® cotton, it was also considered that there was a risk of development of herbicide-tolerant weeds through inappropriate use of the crop/herbicide combination, in particular overuse of glyphosate. The previously issued licence therefore includes a condition that alternative methods of weed control must be used to eliminate weeds exposed to glyphosate that might have developed resistance to the herbicide. Copies of the licence conditions for INGARD® and Roundup Ready® cotton are available on the OGTR website. The licence numbers are GR-3 for INGARD® cotton and GR-9 for Roundup Ready® cotton. A copy of the risk assessment and risk management plan for Roundup Ready® cotton is also available on the website. 21 3.8 Approvals for Bollgard II® cotton in other countries Limited and controlled releases of Bollgard II® cotton have been approved and carried out in Argentina, Costa Rica, India, Japan, Mexico, South Africa and the United States of America. Limited and controlled releases of Bollgard II®/Roundup Ready®cotton have been approved and carried out in the United States of America. An application for limited release of Bollgard II®/Roundup Ready® cotton in Argentina is under consideration. Commercial release of Bollgard II® is currently being assessed by regulators in the United States of America. Applications for approval for use in food products have been lodged with the United States and Japan (and also Australia, see Part 5.5.1). No country has refused an application for the release of Bollgard II®, Roundup Ready® cotton or Bollgard II®/Roundup Ready® cotton and no adverse effects on human health and safety or the environment have been reported. 22 4. INFORMATION ABOUT THE GMOS AND THE PARENT ORGANISM In preparing the risk assessment and risk management plan, 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. 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. 4.1 Summary information about the GMO Bollgard II® cotton contains two insecticidal genes, cry1Ac and cry2Ab, derived from the common soil bacterium Bacillus thuringiensis variety kurstaki. The Cry1Ac and Cry2Ab proteins (Bt toxins) are highly specific insecticidal proteins that are toxic to the major caterpillar pests of cotton (Hofmann et al., 1988; Van Rie et al., 1989; 1990; Widner and Whitely, 1989; Dankocsik et al., 1990), including Helicoverpa armigera (cotton bollworm) and H. punctigera (native budworm). Further details on the Bt toxins and the cry1Ac and cry2Ab genes are provided in Parts 4.3 and 4.4. Bollgard II®/Roundup Ready® cotton is also tolerant to the herbicide glyphosate, the active constituent in Roundup® herbicide. The herbicide tolerance is conferred by the CP4 EPSPS gene from a soil bacterium Agrobacterium strain CP4, and was introduced into Bollgard II® cotton through conventional breeding with herbicide-tolerant Roundup Ready® cotton. Roundup Ready® and Roundup Ready®/INGARD® cotton were approved for commercial release in 2000 (see Part 3.6). The modified cotton plants also contain antibiotic resistance genes. These genes were used as selectable marker genes in the early laboratory stages of development of the plants, to enable selection of plant cells or bacteria containing the desired genetic modification. Potential risks relating to transfer of these genes to other microorganisms are discussed in Part 5.5.3. The antibiotic resistance genes are the bacterial neomycin phosphotransferase II (nptII) gene, conferring resistance to the antibiotics kanamycin and neomycin; and the aminoglycoside adenylyltransferase (aad) gene. The aad gene confers spectinomycin and streptomycin resistance and is linked to a bacterial promoter that does not function in the plants, so the protein is not actually produced in Bollgard II® plants. The antibiotic resistance genes are discussed in more detail in Part 4.3. A gene from Escherichia coli, the uidA gene, which codes for the bacterial enzyme -glucuronidase (GUS), is also present in the plants. It encodes a reporter or marker gene that allows the detection of genetically modified tissues using a simple biochemical stain. More information about the uidA gene and GUS proteins is provided in Part 4.3. The methods used to introduce the genes into cotton are discussed in Part 4.5. 23 4.2 The parent organism The parent organism is Gossypium hirsutum L. (cultivated cotton). This organism is not native to Australia, although it is not clear exactly how or when cotton was introduced to Australia. Fryxell (1966, 1979) proposed that the cotton arrived in northern Australia via ocean currents from Central America. When this may have occurred is unknown, and it has never been substantiated. The primary evidence for this supposition is the presence along riverbanks in northern Australia of naturalised populations of agronomically primitive morphotypes that pre-date intensive cotton cultivation in Australia. Their morphological features suggest that they are not derived from modern elite cultivars, but rather are feral derivatives of primitive cultivated varieties introduced before 1900, or are the descendants of long-distance transoceanic immigrants. Cotton was introduced as a commercially cultivated crop in Queensland in the 1860s when the American Civil War caused shortages in world cotton supplies. Intensive cotton farming in northern New South Wales and northern Western Australia started in the early 1960s (Hearn and Fitt, 1992). Within Australia, G. hirsutum L. occurs nearly exclusively as a managed cultigen. In cotton districts, transient plants may occur along roadsides, but there is no indication that they are sponsoring self-perpetuating feral populations, despite the number of years in which large areas of cotton have been commercially grown. As noted above, naturalised populations occur along riverbanks in northern Australia, but do not appear to be derived from modern cultivars. The parental line for Bollgard II® cotton is the genetically modified Gossypium hirsutum L. cultivar Delta Pine 50B (531 event) developed and registered for commercial use in the United States as Bollgard® cotton (and in Australia as INGARD® cotton). Delta Pine 50B was originally derived from the conventional variety Coker 312 transformed with the cry1Ac, nptII and aad genes using Agrobacterium gene transfer systems (Part 4.5). The Coker 312 cultivar was used because of its positive response to the tissue culture system used to produce genetically modified plants by Agrobacterium-mediated transformation, but is now grown commercially on a very limited basis, if at all. Delta Pine 50B was produced by conventional backcross breeding to move the modified genetic material in INGARD® cotton into the elite Delta Pine 50 background that is adapted for production in the major cotton growing areas of the United States. Bollgard II® cotton line 15985 was developed by further genetic modification of Delta Pine 50B to introduce the cry2Ab and uidA genes, producing the variety Delta Pine 50BG II (15985 event) (see Part 4.5). Since the Delta Pine 50 or 50B parents are not suited to Australian cotton production systems, Australian seed companies have used conventional breeding techniques to transfer the modified genetic material in Bollgard II® into cultivated cotton varieties more suitable for the Australian environment. Bollgard II®/Roundup Ready® cotton was produced by conventional breeding of Bollgard II® cotton with Roundup Ready® cotton. 24 The plants proposed for release are the progeny from crosses between the genetically modified Delta Pine 50BG II line 15985 and Sicala, Sicot and Siokra cotton cultivars developed by CSIRO, or with Roundup Ready® varieties of the same cultivars produced by conventional breeding with Roundup Ready® cotton. These plants have been evaluated in previous releases (see Part 3.5) and demonstrated good agronomic performance and insect control (Dr D. Llewellyn, Senior Principal Research Scientist, CSIRO Plant Industry, personal communication). In assessing the potential risks for Bollgard II® and Bollgard II®/Roundup Ready® cotton, the risks of the parent organism as whole species, i.e. Gossypium hirsutum, were considered, taking into account that there are a range of commercially available cultivars. It is not considered that there are any significantly different risks, for example relating to potential weediness, or the occurrence of natural toxins, for any of the currently available commercially released cultivars of cotton in Australia, and therefore, that no distinction needs to be made between different cultivars carrying the Bollgard II® genes or Bollgard II®/Roundup Ready® genes. Further details relating to the potential of cotton to be a weed and the potential for transfer of genes from cotton to other organisms, including native Australian cottons, are provided in Parts 5.5.2 and 5.5.3. 4.3 The introduced genes 4.3.1 The cry1Ac gene The cry1Ac gene in Bollgard II® cotton is a chimeric gene that combines parts of two genes isolated from Bacillus thuringiensis variety kurstaki (B.t.k.). Part of the B.t.k cry1Ab gene (nucleotides 1 - 1398, corresponding to amino acids 1 - 466; Fischhoff et al., 1987) was linked to a portion of the B.t.k cry1Ac gene (nucleotides 1399-3534, corresponding to amino acids 467 - 1178; Adang et al., 1985). The cry1Ab region is identical to the analogous region of the cry1Ac gene with the exception of 6 amino acid differences. The hypervariable region responsible for insecticidal specificity is from the B.t.k cry1Ac gene and the chimeric gene is therefore referred to as the cry1Ac gene. To ensure the bacterial gene was expressed optimally in plants, a plant-preferred version by of the chimeric cry1Ac gene was synthesised using the strategy described by Perlak et al. (1990, 1991). The amino acid sequence encoded by the synthetic gene is identical to that of the native B.t.k protein, with the exception that a serine is encoded at position 766, rather than leucine. This was the result of an unintentional change that occurred during the synthesis of the plant-preferred version of the gene. However, the altered amino acid is not present in the insecticidally-active trypsin-resistant core and will not change the host range, which is determined by the amino-terminal portion of the protein (see Part 4.4; Bietlot, 1989). The Cry1Ac protein expressed in Bollgard II® cotton is 99.4 % identical to the B.t.k Cry1Ac protein (Adang et al., 1985). The properties of the protein are discussed in Parts 4.4 and 5.5.1. Expression of the cry1Ac gene is driven by an enhanced 35S promoter from cauliflower mosaic virus (CaMV) (Kay et al., 1987; Odell et al., 1985). A promoter is a small piece of DNA that controls the level of expression of genes, acting like a switch. The mRNA 25 polyadenylation signals, which are required for gene expression in plants, are provided by the non-translated region of the soybean alpha subunit of the beta-conglycinin gene (referred to as the 7S 3’ termination sequence) (Schuler et al., 1982). 4.3.2 The cry2Ab gene The cry2Ab gene in Bollgard II ® cotton was also isolated from B.t.k, by cross-hybridisation with the cry2Aa gene from that strain (Donovan et al., 1988; Dankocsik et al., 1990). The sequence of the cry2Ab gene is 89% identical to the cry2Aa gene. B. thuringiensis cells harbouring the cry2Ab gene produce very little Cry2Ab protein, but when the promoter was replaced with that from the cry2Aa gene high levels of expression were achieved in recombinant B. thuringiensis strains (Dankocsik et al., 1990). A plant-preferred version of the cry2Ab gene was also synthesised using the strategy described by Perlak et al. (1990, 1991). Expression of the cry2Ab gene is also controlled by the enhanced CaMV 35S promoter (Kay et al., 1987; Odell et al., 1985). The mRNA polyadenylation signals are provided by the 3’ non-translated region of the nopaline synthase (NOS) gene from A. tumefaciens (Depicker et al., 1982). The properties of the Cry2Ab protein are discussed in Parts 4.4 and 5.5.1. 4.3.3 The CP4 EPSPS gene The gene for CP4 EPSPS, which confers tolerance to glyphosate (N-(phosphonomethyl)glycine), the active ingredient of Roundup herbicide, was isolated from Agrobacterium sp. strain CP4. 5-enolpyruvylshkikimate-3-phosphate synthase (EPSPS) is a critical enzyme in aromatic amino acid biosynthesis, catalysing the addition of the enolpyruvyl moiety of phosphoenolpyruvate to shikimate-3-phosphate. This enzyme is the target of the herbicide glyphosate. Inhibition of EPSPS by glyphosate prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites (Steinrucken and Amrhein, 1980). CP4 EPSPS is naturally resistant to inhibition by glyphosate (Padgette et al., 1993). The native CP4 EPSPS gene contains some sequences with potential polyadenylation sites that are often A+T rich, a higher G+C content that is not frequently found in dicotyledonous plant genes (63% versus ~50%), concentrated stretches of G and C residues, and codons that may not be frequently used in dicotyledonous plant genes. A plant-preferred version of the gene was synthesised by site-directed mutagenesis (Padgette et al., 1993) and used in the vector for transformation of the cotton plants. Although the gene sequence has been altered, the protein produced from the plant-preferred gene has exactly the same sequence as the Agrobacterium protein. The plant-preferred coding sequence was expressed in E. coli from a PRecA-gene 10L vector (Olins et al., 1988) and the proponent states that EPSPS activity was unaltered when compared with the native CP4 EPSPS gene. The gene is driven by the CMoVb promoter (34S promoter of the caulimovirus figwort mosaic virus) (Gowda et al., 1989; Richins et al., 1987; Sanger et al., 1990). In leaf tissue the 34S promoter is 20-fold more active than the promoter from Agrobacterium tumefaciens T-DNA (MAS or mannopine synthase) and it lacks the root-specific and wound-stimulated 26 expression of the MAS promoter (Sanger et al., 1990). The 3’ region of the gene is from the 3’ non-translated region of the NOS gene from Agrobacterium tumefaciens. The gene coding for CP4 EPSPS is fused with the chloroplast transit peptide-coding region from Arabidopsis thaliana EPSPS (Klee et al., 1987) to target the CP4 EPSPS to the chloroplast (the site of aromatic amino acid biosynthesis). In plants, EPSPS is synthesised as a preprotein (containing a transit peptide) 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, chloroplast transit peptides are rapidly degraded (Bartlett et al., 1982; della-Cioppa et al., 1986). 4.3.4 The uidA reporter gene The uidA or gusA gene encoding the enzyme ß-glucuronidase (GUS) is the most widely used reporter gene in genetically modified plants (Jefferson et al., 1987; Gilissen et al., 1998). A reporter gene is a gene that encodes an enzyme with an easily assayable activity that is used to report on the expression of a gene or promoter of interest. It allows the study of expression of a gene for which the gene product is not known, or is not easy to identify, or it can be used as a simple biochemical tag to identify transgenic tissues. The GUS enzyme cleaves the chromogenic substrate X-gluc (5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid), resulting in the production of an insoluble blue color in those plant cells expressing GUS activity. Non-GM plant cells themselves do not in general contain any GUS activity, although there are unconfirmed reports of a GUS-like activity in some plant tissues. Therefore, the production of a blue colour in a particular cell after staining with X-gluc indicates that these cells have been successfully genetically modified and contain the uidA gene. Particle bombardment is not particularly efficient at introducing genes into plants, and screening for the uidA reporter gene facilitates identification and recovery of plant material containing the genetic modification. The tight linkage between the uidA gene and the cry2Ab gene has the additional advantage that GUS staining can be used to follow the segregation of the two genes in segregating populations in backcross breeding programs. The uidA gene is from the bacterium Escherichia coli. E. coli has evolved to survive in the mammalian intestine, and the enzyme encoded by the uidA gene enables it to utilize as its sole carbon source -glucuronides excreted in mammalian guts as by-products of the detoxification of compounds including certain antibiotics and hormones. E. coli GUS has a monomer molecular weight of about 68,200 daltons, and the active form is probably a tetramer. GUS is an exo-hydrolase; it will not cleave glucuronides in internal positions within polymers. The enzyme is specific for -D-glucuronides, with some tolerance for -galacturonides. It is inactive against -glucosides, -galactosides, -mannosides, or glycosides in the alpha configuration. Expression of the uidA gene in Bollgard II® cotton is controlled by the CaMV 35S promoter (Kay et al., 1987; Odell et al., 1985). The mRNA polyadenylation signals are provided by the 3’ non-translated region of the NOS gene from A. tumefaciens (Depicker et al., 1982). 27 4.3.5 The nptII gene The nptII gene was isolated from the bacterial Tn5 transposon (Beck et al., 1982). It encodes the enzyme neomycin phosphotransferase type II (nptII) which confers resistance to aminoglycoside antibiotics such as kanamycin and neomycin. The nptII enzyme uses ATP to phosphorylate neomycin, and the related kanamycin, thereby inactivating these antibiotics and preventing them from killing the cells producing nptII. The nptII gene functions as a selectable marker in the initial laboratory stages of cotton plant cell selection following transformation (Horsch et al., 1984; DeBlock et al., 1984) and is expressed in the Bollgard II® and Bollgard II®/Roundup Ready® cotton. The gene is controlled by the CaMV 35S promoter (Kay et al., 1987; Odell et al., 1985). The 3’ region of the gene is from the 3’ non-translated region of the NOS gene from A. tumefaciens (Rogers et al., 1985). 4.3.6 The aad gene The aad gene was isolated from the bacterial Tn7 transposon and is under the control of its own bacterial promoter. This gene codes for an enzyme, 3”(9)-O-aminoglycoside adenylyltransferase (aad), which allows selection of GMOs on medium containing the antibiotics spectinomycin or streptomycin. The aad enzyme adenylates either the 3’-hydroxy on the amino-hexose III ring of streptomycin or the 9-hydroxyl on the actinamine ring of spectinomycin (Davies and Benveniste, 1974). The nucleotide sequence of this gene in the Tn7 transposon has been determined by Fling et al. (1985). The gene is not expressed in the Bollgard II® or Bollgard II®/Roundup Ready® cotton because the bacterial promoter is not active in plants. The gene was used in the laboratory prior to the production of the genetically modified plants to select for bacteria containing the modified DNA. 4.4 Bt toxins Cry1Ac and Cry2Ab are two of a diverse family of insecticidal proteins (Bt proteins or Bt toxins) expressed by the bacterium Bacillus thuringiensis. The Bt proteins are grouped in classes that exhibit different insect specificities. Cry1Ac and Cry2Ab toxins are highly specific for lepidopteran insects (moths and butterflies) (Widner and Whitely, 1989; Macintosh, 1990; Dankocsik et al., 1990). During sporulation, Bt proteins are produced in cytoplasmic crystalline inclusions which are soluble in alkaline aqueous solutions and insoluble in aqueous solutions at neutral or acidic pH (Bulla et al., 1977). When ingested, the Bt protein crystal dissolves in the alkaline environment of the larval insect gut. In many cases, activation of the toxin by cleavage with specific proteases in the gut is required. The proteases cleave the carboxy-terminal domain of the Cry1Ac protein and approximately 28 amino acids from the amino-terminal end of the protein, leaving an active core of approximately 600 amino acids (Chroma and Kaplan, 1990; Bietlot et al., 1989). The Cry2A proteins are smaller (Cry2Aa and Cry2Ab are both 633 28 amino acids) and may not require activation by a protease (Gill et al., 1992; Karim et al., 2000). The active Bt toxins diffuse through the midgut membrane of the target lepidopteran insects and bind to specific high affinity receptors in the midgut epithelium surface (Hofmann et al., 1988; Van Rie et al., 1989; 1990; Karim et al., 2000). Non-target insects, mammals, birds and fish do not possess these receptors and are therefore not susceptible to the toxic effects of these insecticidal proteins. Competition studies indicate that Cry1Ac and Cry2Aa bind to different receptors in target insects (Morse et al., 2001). Binding of Bt toxins to the gut receptors leads to formation of pores in the cell membrane, and leakage of the intracellular contents (for example potassium ions) into the gut lumen and water into the epithelial gut cells (Sacchi, et al., 1986; Knowles et al., 1993; English & Slatin, 1992). The larval gut epithelial cells swell due to osmotic pressure and lyse. The gut becomes paralysed because of changes in the electrolyte and pH balance and the insects stop eating and die (Goldberg and Tjaden, 1990). The pores formed by Cry2Aa, a toxin closely related to the Cry2Ab in Bollgard II® cotton, differ from those formed by Cry1Ac (English et al., 1994), suggesting a mechanistic difference in insecticidal activity between these two types of insecticidal proteins. This is supported by structural analyses of the crystallised Cry2Aa toxin (Morse et al., 2001). Detailed studies on the mode of action of Cry2Ab are not yet available, but high similarity of the Cry2Ab and Cry2Aa protein sequences suggests that they share common biochemical mechanisms. The Cry1Ac protein expressed in INGARD® cotton was compared by Western blot analysis with commercially available microbial pesticides containing Bt toxin (Berberich and Fuchs, 1992). This study showed that the protein expressed by the INGARD® cotton is similar in molecular weight and immunological reactivity to one or more proteins contained in the commercial Bt products Dipel® (Abbott Laboratories) and Thuricide® (Sandoz Inc.). Further, it has been demonstrated that the biological activity and species-specificity of the full-length Cry1Ac protoxin expressed in INGARD® cotton is equivalent to that of the active B.t.k Cry1Ac core toxin (Sims, 1994e). 4.5 4.5.1 Method of gene transfer Bollgard II® cotton The cry2Ab and uidA genes were inserted into the genomic DNA of the genetically modified INGARD® cotton variety Delta Pine 50 B (event 531) as isolated DNA fragments delivered into the cotton cells by projectile bombardment (McCabe and Martinell, 1993). Projectile bombardment is a physical delivery system whereby minute gold or tungsten beads coated with DNA are shot into cells that have the capacity to develop or differentiate into shoots or whole plants. The uidA gene can be used as a marker gene to identify plant tissue that is stably transformed with the introduced genes and from which seed can be selected and transformants recovered (see Part 4.3). 29 The INGARD® cotton contains the cry1Ac, nptII and aad genes originally inserted into the genomic DNA of the Coker 312 cotton variety by Agrobacterium-mediated transformation with plasmid PV-GHBK0. The Agrobacterium-mediated DNA transformation system is well understood (Zambryski, 1992). The plasmid vector, PV-GHBK04, is a binary, single-border transformation vector. The plasmid contains well characterised DNA segments required for selection and replication of the plasmid in bacteria as well as Agrobacterium sequences essential for DNA transfer from Agrobacterium and integration in the plant cell genome (Bevan, 1984, Wang et al., 1984). Agrobacterium tumefaciens is a common gram-negative soil bacterium that causes crown gall disease in a wide variety of plants. The molecular biology of crown gall disease shows that plants can be genetically transformed by the transfer of DNA (T-DNA, located between specific border sequences) from A. tumefaciens through the mediation of the genes (vir region) of Ti plasmids. Disarmed Agrobacterium strains have been constructed specifically for plant transformation. The disarmed strains do not contain the genes (iaaM, iaaH and ipt) for the overproduction of auxin and cytokinin, which are required for tumour induction and rapid callus growth (Klee and Rogers, 1989). A useful feature of the Ti plasmid is the flexibility of the vir (virulence) region to act in either cis or trans configurations to the T-DNA. This has allowed the development of two types of transformation systems: (i) co-integration vectors that join the T-DNA that is to be inserted into the plant and the vir region in a single plasmid (Stachel and Nester, 1986); (ii) binary vectors that have the T-DNA and vir regions segregated on two plasmids (Bevan, 1984). Both provide functionally equivalent transformation systems. 4.5.2 Bollgard II®/Roundup Ready® cotton Roundup Ready® cotton was produced by inserting the CP4 EPSPS, nptII and aad genes into the genomic DNA of Coker 312 line 1445 cotton. The method used to insert the genes was the same as that described above for generating INGARD® cotton, that is via Agrobacterium-mediated transfer, using a binary, single border transformation vector, plasmid vector PV-GHGT07 (Bevan, 1984; Wang et al., 1984). Bollgard II®/ Roundup Ready® cotton was produced through conventional breeding from Bollgard II® and Roundup Ready® parent cultivars. 30 4.6 4.6.1 Characterisation of the inserted genetic material and stability of the genetic modification Bollgard II® cotton Southern blot analysis was used to demonstrate that two T-DNA copies inserted in a head-to-tail arrangement were present in the genome of INGARD® cotton from which Bollgard II® cotton was derived. One T-DNA insert contains full-length copies of the cry1Ac, nptII and aad genes. The second insert is a partial copy, containing only a portion of the cry1Ac gene that does not encode the insecticidally active region of the Bt protein. Southern blot analysis of three generations of backcrossed INGARD® cotton progeny and segregation data indicate that the two inserts are tightly linked. Expression of the Cry1Ac protein, determined by ELISA, was stable through four generations of backcrossing with elite cultivars, with segregation ratios as expected (data supplied by Monsanto Australia Ltd). Southern blot analysis of Bollgard II® DNA shows that one full-length copy of each of the cry2Ab and uidA (GUS) genes is present. The stability of the DNA insert and expression of the Cry2Ab protein in Bollgard II® cotton across five plant breeding generations was confirmed by data from Southern blot, ELISA and Western blot analysis (Doherty et al. 2000a,b; Bookout et al., 2001). Segregation data for the cry2Ab and uidA genes from the CSIRO plant breeding program, indicates that they are inherited in a Mendelian manner, suggesting that they are present at a single locus and are tightly linked (data provided in the application). 4.6.2 Roundup Ready® cotton Southern blot analysis was used to demonstrate that a single copy of the nptII, CP4 EPSPS and aad genes has been inserted in Roundup Ready® cotton. The insert was stably maintained in the cotton genome for three generations (R3 – R5 of line 1445) (data supplied by Monsanto Australia Ltd). The gox gene, from the bacterium Ochromobacterium anthropii (encoding the glyphosate oxidoreductase enzyme (GOX)), although present in the intermediate plasmid vector, was not transferred to the plant genome. The T-DNA region of the insert was truncated at a point before the gox gene would have begun (this is not uncommon, see for example Bakkeren et al., 1989 and De Block et al., 1984). 4.6.3 Bollgard II®/Roundup Ready® cotton No data have been presented for Bollgard II®/Roundup Ready® cotton in relation to the stability of the genetic modifications. However, it should be noted that the proposed release will be a limited field trial in one season only, and therefore involve only one generation of the GMO. The combination of the two traits in the one GMO was achieved by conventional breeding, and the stability of the genetic modifications in Bollgard II®/Roundup Ready® cotton can therefore be inferred from the stability of the Bollgard II® and Roundup Ready® cotton varieties over several generations. There is also evidence for the stability of the genetic modifications in the Bollgard II®/Roundup Ready® cotton from glasshouse and field studies that have demonstrated continued efficacy of the Bollgard II® insecticidal and Roundup Ready® herbicide tolerance traits, in releases carried out since 1999 (Dr D. Llewellyn, Senior Principal Research Scientist, CSIRO Plant Industry, personal communication). 31 4.7 Expression of the introduced proteins 4.7.1 Bollgard II® cotton Penn et al. (2001) measured the mean concentration of Cry1Ac in flower buds and growing tips of both INGARD® and Bollgard II® cotton grown at four sites over two years. They found no statistical difference in Cry1Ac expression (by ELISA assays or quantitative insect bioassays) between INGARD® and Bollgard II® cotton. The average Cry1Ac expression determined by ELISA was 10 g/g (micrograms per gram) tissue. Cry2Ab levels in Bollgard II® cotton, on the other hand, were much higher, about 400 g/g tissue. Quantitative bioassays (Greenplate, 1999) with Heliothis virescens using incorporation of plant tissues in synthetic diets compared to known concentrations of purified Cry1Ac, were used to evaluate the overall lepidopteran insecticidal activity of different tissues and at different times. The mean insecticidal activity was expressed as g Cry1Ac equivalents/g dry weight (Penn et al., 2001). In Bollgard II® plants this represents the combined insecticidal activity of the Cry1Ac and Cry2Ab proteins. In INGARD® cotton insecticidal activity was highest in growing tips (24 g Cry1Ac equivalents/g tissue), slightly lower in flower buds (20 g Cry1Ac equivalents/g tissue) and lower still in large leaves (18 g Cry1Ac equivalents/g tissue). Bollgard II® had consistently higher mean lepidopteran activity with 81, 90 and 50 g Cry1Ac equivalents/g dry weight in growing tips, flower buds and large leaves, respectively. Averaged over all sites, tissues and seasons the Bollgard II® cotton was 3.9 times more effective in controlling H. virescens than the corresponding INGARD® cotton line. The relatively higher level of expression of the Cry2Ab protein compensates for its lower insecticidal activity against many of the target insect pests of cotton. These data support the observed higher insect control of Bollgard II® towards a range of important lepidopteran pests (Akin et al., 2001). The bioassays described above were also used to measure the mean insecticidal activity of small flower buds from Bollgard II® and INGARD® cotton over an eight-week period following planting (Penn et al., 2001). The insecticidal activity of Bollgard II® cotton dropped from 107 g Cry1Ac equivalents/g tissue at 2 weeks to 52 g Cry1Ac equivalents/g tissue by eight weeks, but was still 2.9 fold higher than the single gene INGARD® cotton that had declined from 25 to 18 g Cry1Ac equivalents/g tissue in the same period. This is in line with the drop in efficacy of INGARD® cotton towards the end of the growing season seen for Australian crops, and suggests that Bollgard II® cotton should perform much better late in the growing season than INGARD® cotton. Expression of the GUS protein is present at very low levels, at less than 0.007% dry weight in Bollgard II® cottonseed, equivalent to 70 ppm (parts per million) (data provided by Monsanto). Expression of the nptII protein is likely to be at levels similar to that observed in the parental INGARD® cotton, with less than 4 g/gram of seed or leaf (4 ppm) (data provided by Monsanto). 32 4.7.2 Roundup Ready® cotton The amounts of the introduced proteins were measured in leaf and seed samples of Roundup Ready® cotton line 1445 (Nida et al., 1994, 1995, 1996) by ELISA. CP4 EPSPS was detected at low levels in both leaf (52 g/g tissue) and seed (60-82 g/g tissue) of Roundup Ready® cotton but, as expected, were not detected in the parental Coker 312 line. Similarly, nptII was detected at low levels in leaf (45 g/g tissue) and seed (7 g/g tissue) of Roundup Ready® cotton, but not in the parental line. Treatment of the plants with glyphosate did not alter the levels of CP4 EPSPS or NPT II. The proportion of CP4 EPSPS and nptII protein in cotton seed is very low, representing only 0.02–0.028 % and 0.0022 %, respectively, of the total protein. As expected, the GOX and aad proteins were not detected in Roundup Ready® cotton. The aad gene was not expressed in plants because its promoter does not function in plants, and the gox gene was not inserted into the plant genome (see Part 4.6.2). CP4 EPSPS was also detected by Western blot analysis of protein extracts of Roundup Ready® cotton seed (Barry et al., 1993). An antibody specific for CP4 EPSPS reacted with a protein of 48 kD. This is the expected molecular weight for the protein minus the chloroplast transport peptide, confirming that this peptide is cleaved during transport into the chloroplast. 4.7.3 Bollgard II®/Roundup Ready® cotton Expression of the Cry1Ac, Cry2Ab, CP4 EPSPS and GUS proteins in Bollgard II®/Roundup Ready® cotton has not been measured directly, but is likely to be similar to that of the parent Bollgard II® and Roundup Ready® cotton plants. This is supported by evidence from releases of Bollgard II®/Roundup Ready® cotton that the levels of insect and herbicide tolerance are equivalent to those of the parent varieties (Dr D. Llewellyn, Senior Principal Research Scientist, CSIRO Plant Industry, personal communication). The levels of nptII in Bollgard II®/Roundup Ready® cotton, however, may be higher than for the parent varieties, since two copies of the nptII gene, one each from Roundup Ready® and Bollgard II® cotton, are present. As noted above, nptII levels in Bollgard II® cotton are likely to be less than 4 g/gram of seed or leaf (4 ppm). The levels of expression in Roundup Ready® cotton leaf is around 45 μg/g of tissue so that the maximum level in Bollgard II®/Roundup Ready® cotton would be expected to be around 50 μg/g (50 ppm) (data provided by Monsanto Australia Ltd). 33 5. RISK ASSESSMENT This part of the document explains the risk assessment process, outlines the potential hazards that have been identified and the risks posed by these hazards, and highlights specific areas where the Regulator is seeking input through the consultation processes. 5.1 The Risk Analysis Framework The risk assessment was 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). The Framework was developed in consultation with the States and Territories, Commonwealth government agencies and the public. It takes into account the requirements of the Act and the Gene Technology Regulations 2001, and guidelines and risk assessment strategies in use in related agencies both in Australia and overseas. The purpose of the Risk Analysis Framework 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. 5.2 The risk assessment process In undertaking the risk assessment, the following were considered and analysed: the data presented in the proponent’s application, including additional information supplied by Monsanto (see Part 3.1); data provided previously to GMAC or the IOGTR in respect of previous applications for commercial release of INGARD and Roundup Ready® cotton (see Parts 3.6 and 3.7); submissions or advice from States and Territories, Commonwealth agencies and the Environment Minister; advice from GTTAC; advice from CSIRO experts; information from other national and international regulatory agencies; and current scientific knowledge and the scientific literature. In considering this information and preparing the risk assessment and risk management plan, the following specific matters were taken into account, as required by section 51 of the Act and set out in section 49: 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, or will become, a GMO (see Part 4.2); the effect, or the expected effect, of genetic modification that has occurred, or will occur, on the properties of the organism (see Part 4.4); provisions for limiting the dissemination or persistence of the GMO or its genetic material in the environment (see Parts 6.2 and 6.3); the potential for spread or persistence of the GMO or its genetic material in the environment (see Parts 5.5.2 and 5.5.3) and ; 34 the extent or scale of the proposed dealings (see Part 3.1); any likely impacts of the proposed dealings on the health and safety of people (see Parts 5.5.1 and 5.5.3). In accordance with Regulation 10 of the Regulations, the following were also taken into account: any previous assessment, in Australia or overseas, in relation to allowing or approving dealings with the GMO (see Parts 3.4-3.8); the potential of the GMO concerned to: - be harmful to other organisms (see Part 5.5.1); - adversely affect any ecosystems (see Parts 5.5.2 and 5.5.3); - transfer genetic material to another organism (see Part 5.5.3); - spread, or persist, in the environment (see Part 5.5.2); - have, in comparison to related organisms, a selective advantage in the environment (see Part 5.5.2); and - be toxic, allergenic or pathogenic to other organisms (see Part 5.5.1). Regulation 10 also requires the Regulator to consider both the short and long term when taking these factors into account. Through the risk assessment process, a number of potential hazards were identified. The risks posed by these hazards were evaluated by considering: the likelihood of the hazard occurring; the likely consequences if the hazard were to be realised; and the availability of mechanisms for effectively managing identified risks. The detailed risk assessment is presented below, in two parts: 5.3 hazard identification: Part 5.4 refers. hazard and risk characterisation (in which the likelihood of occurrence of the hazard and any adverse impacts are considered): Part 5.5 refers. Summary of conclusions from risk assessment process 5.3.1 Hazard identification The risk assessment identified number of possible hazards that could arise as a direct result of the genetic modification of Bollgard II® and Bollgard II®/Roundup Ready® cotton, including: the potential for the genetically modified cotton to be harmful to other organisms because it is toxic or allergenic; the potential for the genetically modified cotton to be harmful to the environment because of inherent weediness or increased potential for weediness; 35 the potential for the new genes introduced into the cotton to transfer to non-GM cotton crops and wild or native cottons, or to other organisms, with adverse consequences; and the potential for resistance to the insecticidal proteins produced by the introduced genes to develop in target insects in the long term. 5.3.2 Hazard and risk characterisation In summary, it is concluded that there are no substantive additional risks to public health and safety or to the environment arising from the genetic modification of Bollgard II® and Bollgard II®/Roundup Ready® cotton, compared to those posed by conventional cotton because: Bollgard II® and Bollgard II®/ Roundup Ready® cotton are not likely to prove more toxic or allergenic to humans or other organisms, other than some lepidopteran insects, than conventional cotton (lepidopteran insects are moths and butterflies); the risk of the Bollgard II® or Bollgard II®/ Roundup Ready® cotton establishing as a weed is low and not likely to be greater than that of conventional cotton; the potential for transfer of the introduced genes to non-GM cotton crops is negligible because it is being planted very late in the cotton growing season and no other cotton crops in the area will be flowering at the same time; the potential for transfer of the introduced genes to wild or native cotton is very low because of the geographical isolation and genetic incompatibility with the native species; the likelihood of transfer of the introduced genes to other organisms is low, but even if such transfer occurred would be unlikely to pose any hazard to human health and safety or the environment; and the risk of development of target insects resistant to the insecticidal proteins is very low, due to the limited scope of the proposed release and the presence of two insecticidal proteins. 5.3.3 Consideration of risks relating to combination of the Roundup Ready® and the Bollgard II® traits In preparing the risk assessment, the effect of the combining of the Roundup Ready® glyphosate tolerance and the Bollgard II® insecticidal traits in the same plant, and whether this would result in new or increased risks over and above those posed by the introduction of the single traits, were considered, noting the following: The Roundup Ready® herbicide tolerance and Bollgard II® insecticidal genes 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 arising from the combination of the two traits, are likely to occur, or that they would result in new or increased risks relating to human health and safety or the environment. 36 Each of the genes introduced into the cotton has been stably integrated into the cotton genome (see part 4.6) and there is no evidence or reasonable expectation that recombination between the introduced genes has occurred or will occur in the future. There have been no reports of any unexpected or unintended adverse effects in previous releases of Bollgard II®/Roundup Ready® cotton (see Part 3.5). It was therefore considered unlikely that Bollgard II®/Roundup Ready® would 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. 5.3.4 Identification of issues to be addressed for future releases During the evaluation process, a range of data and information requirements were identified which would be required before any future commercial release could be contemplated. These include further information and data on: the potential toxicity of Bollgard II cotton, including more information on potential toxicity to non-target pests; the potential for cotton to outcross with native cotton species; whether growth of Bollgard II cotton is likely to be limited by insect predation, and the potential impact on weediness of the cotton; and the expression levels of the introduced proteins; insect resistance management strategies for Bollgard II cotton. The general issue of the use of antibiotic resistance marker genes may also need to be considered in the longer term. This issue has recently been addressed by international food standard setting bodies, including the FAO/WHO Expert Consultation on Foods Derived from Biotechnology (29 May-2 June 2000, Geneva Switzerland) and the Codex Ad Hoc Intergovernmental Taskforce on Foods Derived from Biotechnology (November 2000, Tokyo) and the OECD. The international bodies accept that there is no evidence of human health and safety problems with the use of antibiotic resistance marker genes in GM foods (e.g. the nptII gene). However, they have also stated that alternative transformation technologies that do not result in antibiotic resistance marker genes in foods are encouraged in the future development of recombinant DNA plants, where such technologies are available and demonstrated to be safe. While this issue is not directly relevant to the current application, nor to applications made in the near future, the OGTR is currently considering possible options for the gradual phasing out the use of antibiotic resistance marker genes in the longer term. 5.4 Hazard identification This part of the risk analysis presents a summary of the possible hazards that were considered and assessed, and the conclusions that were drawn. It is followed by a detailed description of these matters. 37 A number of potential hazards arising from the genetic modification of Bollgard II® and Bollgard II®/Roundup Ready® cotton, were identified through: assessment of the application; review of the scientific literature; and review of data from other regulatory bodies and overseas bodies as referenced in Part 2.3. The potential hazards identified were that: 5.5 the genetically modified cotton might be harmful to organisms other than the target lepidopteran pests, because it is toxic or allergenic as a result of the novel gene products expressed in the plants or unforeseen or unintended effects; the genetically modified cotton might be harmful to the environment because of inherent weediness or increased potential for weediness; and the new genes introduced into the cotton to transfer to non-GM cotton crops and wild or native cottons, or to other organisms, with adverse consequences; and; and resistance to the insecticidal proteins produced by the introduced genes may develop in target insects in the long term. Hazard and risk characterisation Each potential hazard identified in Part 5.4 is addressed below, in three steps: A: B: C: 5.5.1 A: Explains the nature of each potential hazard and any adverse impacts these might cause. Examines the likelihood of the potential hazard occurring. Draws conclusions about the risks and their potential impacts. Toxicity or allergenicity Nature of the potential toxicity or allergenicity hazard The possibility was considered that Bollgard II® or Bollgard II®/Roundup Ready® cotton may be harmful to organisms other than the target lepidopteran pests. This could occur if Bollgard II® or Bollgard II®/Roundup Ready® were toxic or allergenic, because of the novel gene products expressed in the plants or unforeseen, unintended effects. Toxicity or allergenicity for humans If the genetically modified cotton is toxic or allergenic, there could be impacts relating to: the safety of human foods containing cottonseed oil (for example blended vegetable oils, margarine, or salad dressings) or cotton linters (which may be used in smallgoods casings, toothpaste, or ice cream). Responsibility for assessment of the safety of food for human consumption lies with the Australia New Zealand Food Authority (ANZFA), not the Gene Technology Regulator (see Part 2.2). However, the Regulator is required to seek advice from ANZFA on the application, and on the risk assessment and risk management plan. It should be noted that none of the cotton from this release, or its by-products will be used for human consumption. 38 the safety of human foods where cotton products are present in the food chain (for example, livestock, poultry or fish that have been fed cotton by-products); occupational health and safety (for example, for farm workers, or factory workers involved in cotton processing); people wearing cotton clothing or using other products containing cotton fibre (for example, medical dressings or tampons) or cottonseed oil (for example, as a pharmaceutical excipient or in cosmetics); and environmental exposure (for example, people breathing cotton pollen). Toxicity for other organisms If Bollgard II® or Bollgard II®/Roundup Ready® cotton is toxic for other non-target organisms, there could be potential impacts relating to: toxicity for beneficial insects (pollinators, parasites or predators of insect pests) or soil biota, with direct impact on growth of crops on farms, as well as secondary ecological effects with potential to harm the natural environment (for example, adverse impacts on biodiversity); and toxicity for grazing animals, including native animals; animal feed safety (for example, animals fed cottonseed meal or hulls); Toxicity for the lepidopteran target organisms, may also present indirect impacts: B: secondary effects on populations of specialist parasites and predators that feed on lepidopteran insects; and secondary effects on populations of organisms that are preyed on by lepidopteran insects. Likelihood of the toxicity or allergenicity hazard occurring In assessing the likelihood of adverse impacts due to toxicity or allergenicity of Bollgard II® and Bollgard II®/Roundup Ready® cotton, a number of factors have been taken into consideration including: the toxicity or allergenicity of the new proteins expressed in the cotton, the Cry1Ac, Cry2ab, CP4 EPSPS, nptII and GUS proteins; other information relating to the toxicity of Bollgard®, Roundup Ready® and Bollgard II®/Roundup Ready® cotton for particular species, including humans and other mammals, non-target invertebrates, soil microorganisms, fish and birds; information about the likely levels and routes of exposure to Bollgard II® and Bollgard II®/Roundup Ready®cotton and the introduced proteins, for example in food or 39 feed, in non-food products containing cottonseed oil or fibre, in residues generated in manufacturing processes, or through direct contact with the crop or contact with soil in which the crop is grown. Toxicity or allergenicity of the introduced proteins Bollgard II® and Bollgard II®/Roundup Ready® cotton differ from conventional cotton in the expression of up to five additional new proteins, the Cry1Ac, Cry2Ab proteins, CP4EPSPS (in Bollgard II®/Roundup Ready® cotton only), nptII and GUS reporter proteins. These have all been considered for their potential toxicity and allergenicity. Cry1Ac protein The Cry1Ac protein present in INGARD® cotton is 99.4 % identical to a naturally occurring Bt toxin, Cry1Ac (Adang et al., 1985, Part 4.3.1 refers). The Cry1Ac protein is expressed in common soil bacteria and therefore already widely present in environment and in food chains. Toxicity for mammals, including humans, and allergenicity The toxic effects of Cry1Ac are highly specific for lepidopteran insects (see Part 4.4). The toxic effects of Bt toxins are mediated through binding to specific receptors on the target insect mid-gut (Hofmann et al., 1988; Van Rie et al., 1989; 1990; Karim et al., 2000) that are not present in organisms other than lepidopterans. In addition, the alkaline conditions required for effective solubility of Bt toxin do not exist in the guts of mammals or most invertebrates. Bt toxins require an alkaline pH of 10 for effective solubility and have extremely limited solubility at the highly acidic pH of human gastrointestinal tract (pH 1.2) (English and Slatin, 1992). Furthermore, the Bt toxin expressed in INGARD® cotton is a full-length protoxin that requires cleavage by a specific protease to convert it to the active core toxin (Part 4.2.5). The Cry1Ac protein is one of a number of insecticidal proteins present in many of the widely used commercial Bt formulations. These are used widely to control insects in many food crops, including fresh produce such as lettuce or tomatoes. Bt protein insecticides, produced by fermentation of the same strain of bacterium from which the cry1Ac gene was derived, have been used traditionally in agriculture over several decades, especially by organic farmers (Cannon, 1993). In fact, the first commercial microbial Bt product (Sporeine) was produced in 1938 in France (Weiser, 1986 cited by Entwistle et al., 1993). The World Health Organisation’s (WHO) International Program on Chemical Safety (IPCS) report on environmental health criteria for Bt concluded that ‘Bt has not been documented to cause any adverse effects on human health when present in drinking water or food’ (IPCS, 2000). There have been no confirmed adverse effects on health either through occupational exposure or ingestion of fresh produce sprayed with Bt insecticides, despite significant oral, dermal and inhalation exposure to the product (Entwistle et al., 1993, US EPA, 2001). While there have also been reports in the US claiming allergic reactions to Bt products in topical sprays, it was determined by the US EPA that these reactions were not due to the bacterium itself or to any of the Cry toxins (US EPA, 2001). A survey conducted in farm workers who picked vegetables treated with Bt microbial products indicates that exposure to Bt products may lead to allergic skin sensitisation and 40 induction of IgE and IgG antibodies. However, there were no reports of clinical allergic disease in any of the workers, or of antibodies to the endotoxin proteins of the Bt sprays (Bernstein et al. 1999). The Cry1Ac protein is unlikely to be a major allergen. It does not display characteristics common to known food allergen proteins, for example: presence as a major component of the food; glycosylation; resistance to degradation by heat, acid and proteases of the digestive system; or derivation from a known allergenic source (Metcalfe et al., 1996, Astwood et al. 1996; Taylor and Lehrer, 1996; Kimber, 1999). The Cry1Ac protein is heat labile and rapidly degraded, in under 30 seconds, under simulated gastrointestinal conditions of the mammalian system (Fuchs, 1993). Searches of allergen sequence databases have shown no significant matches of the Cry1 proteins to known allergens (Metcalfe et al., 1996; Astwood et al., 1996). Acute oral toxicity studies in mice, with purified B.t.k. Cry1Ac proteins at doses of up to 4300 mg/kg, have not shown any adverse effects (Naylor, 1994). Several studies on acute oral toxicity of Bt microbial preparations containing Cry1Ac in rats and rabbits revealed no adverse effects doses of up to thousands of milligrams per kilogram of body weight (Carter and Liggett, 1994; Barbera, 1995; McClintock et al, 1995; Spencer et al., 1996). These studies reported no treatment-related effects on survival, body weight, food consumption, clinical observations, and gross pathology findings at necropsy. A two-year chronic rat feeding study was undertaken with Bt microbial products at doses of up to 8400 mg/kg of body weight/day. A decrease in weight gain was observed at the highest dose, but in the absence of any other adverse findings this was not considered to be related to Cry protein toxicity (McClintock et al, 1995). In two separate studies, human volunteers have been fed 1000 mg of Bt microbial preparations per day for up to 5 days and exhibited no symptoms of toxicity or other ill effects (McClintock et al, 1995). The NRA have issued a Technical Grade Active Constituent (TGAC) exemption for this protein from the requirement to establish a maximum residue limit (MRL) when present in INGARD® cotton or when used as a topical application on food crops (TGAC Exemption 48404, NRA toxicology evaluation and approval 48296, 5 June 2000). The US Environmental Protection Agency considers Cry1Ac protein is non-toxic for mammals and have established an exemption from tolerance requirements (US EPA, 2001). Toxicity for non-target invertebrates A series of studies has been undertaken to demonstrate the effect of Cry1Ac protein on non-target insects. Macintosh et al. (1990) examined the effects of purified active core B.t.k Cry1Ac toxin on 17 agronomically important insect species, representing five orders, and one species of mite. Seven insects, all lepidopterans, were susceptible to the toxin. None of the remaining 11 non-lepidopteran species were susceptible. Another study compared the core B.t.k toxin with recombinant protein equivalent to the full-length Cry1Ac protein expressed in INGARD® cotton (Sims, 1994e; Sims, 1995). Of 14 species tested (representing seven orders), only four lepidopteran species were susceptible to either form of Cry1Ac. The biological activities of the full length and core toxins were very similar. 41 More extensive studies have also been carried out on non-target beneficial insects including: the larval and adult honey bee (Apis mellifera L.), a beneficial insect pollinator (Maggi, 1993a; 1993b); parasitic Hymenoptera (Nasonia vitripennis), a beneficial parasite of the housefly (Musca domestica) (Palmers and Beavers, 1993a; Sims, 1994a); ladybird beetles (Hippodamia convergens), a beneficial predatory insect which feeds on aphids and other plant bugs commonly found on stems and foliage of weeds and cultivated plants (Palmers and Beavers, 1993b; Sims, 1994b) and green lacewing larvae (Chrysopa carnea), a beneficial predatory insect commonly found on cotton and other cultivated crops (Palmers and Beavers, 1993c; Sims, 1994c). There were no adverse effects seen for any of the species tested at the highest dose of full-length recombinant Cry1Ac tested (20 ppm). This was greater than 50 times the maximum Cry1Ac protein expression level in pollen (0.03 ppm) and nectar (0.001 ppm) of INGARD® cotton (see Part 4.2.7 and Table 4). The effects of feeding purified Cry1Ac toxin to collembolans has also been investigated (Sims and Martin, 1996). No adverse effects on the survival or reproduction of Folsomia candida or Xenylla grisea were observed, at doses of up to 200 ppm. There have been reports that pollen from corn containing the closely related Cry1Ab gene was toxic to Monarch butterflies in laboratory feeding studies (Losey et al., 1999). US authorities have concluded, however, that the impact of Bt corn in the field on Monarch butterflies is negligible because of factors that limit environmental exposure (US EPA, 2001). Results from a series of field studies in the US support this conclusion (Sears et al., 2001; Stanley-Horn et al., 2001; Pleasants et al., 2001; Zangerl et al., 2001). A recent study using purified Cry1Ac and Cry1Ab toxins showed that they were toxic for Monarch butterfly larvae. However, it appears that the pollen from corn expressing Cry1Ac is not toxic, as there were no significant differences in the weights of larvae fed pollen from corn expressing Cry1Ac, compared to pollen from non-modified cotton (Hellmich et al., 2001). Toxicity for microorganisms Purified B.t.k 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), Arthrobacter globiformis), gram negative bacteria (Agrobacterium radiobacter, Pseudomonas aeruginosa, Proteus vulgaris, P. mirabilis, Escherichia coli, Enterobacter aerogenes, E. cloacae, Oscillatoria sp.), yeast, (Saccharomyces 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, 2000b). The effect of Cry 1Ac toxin on soil microorganisms was examined by incubating soil with purified Cry1Ac toxin (0.05 g/g) (Donegan et al., 1995). The numbers and types of protozoans, bacteria and fungi were determined at various time points. Substrate utilisation tests and DNA fingerprinting of eubacterial ribosomal sequences were also used to analyse 42 the composition of bacterial soil community. In these experiments, addition of purified Cry1Ac toxin to the soil did not cause any detectable effects on populations of culturable aerobic soil bacteria, fungi or protozoa after exposure for up to 56 days. Cry 2Ab protein The Cry2Ab protein is also a Bt toxin encoded by a gene from Bacillus thuringiensis. The toxic effects of the protein are specific for lepidopteran insects (see Part 4.4). The Cry2Ab protein is closely related (88% identical) to Cry2Aa, which, like Cry1Ac, is one of a number of insecticidal proteins present in many of the widely used commercial Bt formulations. Acute oral toxicity studies in mice, with purified Cry2Ab protein at doses of up to 1450 mg/kg, the highest feasible dose, have not shown any adverse effects (Monsanto Australia Ltd). The Cry2Ab protein is also unlikely to be a major allergen. Data provided in the application show that it does not display characteristics common to known food allergen proteins (discussed for Cry1Ac, above). The Cry2Ab protein is not from a source that is a known allergen, is easily digested, and present at very low levels in the GM cotton (Part 4.7). Searches of sequence databases have shown no significant matches of the Cry2Ab protein to known allergens, toxins or other proteins relevant to animal or human health. CP4 EPSPS protein CP4 EPSPS is derived from a common soil bacterium, Agrobacterium sp. (Zambryski, 1992), that can be found on plant produce (especially raw vegetables), and is functionally and structurally similar to EPSPS proteins present in food and feeds derived from plant and microbial sources. Acute oral toxicity studies in mice, with purified CP4 EPSPS protein at doses of up to 572 mg/kg body weight have not shown any adverse effects. This is more than a thousand times the anticipated consumption level of food products potentially containing CP4 EPSPS protein (Harrison, 1996). CP4 EPSPS is unlikely to be a major allergen. Data provided by Monsanto Australia Ltd show that it does not display characteristics common to known food allergen proteins, discussed for Cry1Ac, above (Canadian Plant Biotechnology Office, Decision Document 97-21, 1997; ANZFA Final Risk Analysis Report Application A355, 2000; Harrison et al., 1996). CP4 EPSPS is not derived from a known allergen, is present at very low levels in the GM cotton (see Part 4.7) and shows no significant protein sequence homology to a database of known toxins or allergens assembled from the Swissprot, Genpept and Pir protein databases. The CP4 EPSPS enzyme is rapidly inactivated by heat and by enzymatic digestion and pH-mediated hydrolysis in simulated mammalian gastric fluid. 43 nptII protein The nptII protein is also ubiquitous 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). The nptII protein does not display characteristics common to known food allergen proteins, discussed for Cry1Ac, above (US FDA, 1998; ANZFA, 1999; Fuchs, 1993). nptII is not derived from a known allergen, is present at very low levels in the GM cotton (see part 4.7) and shows no significant DNA or protein sequence homology to known toxins or allergens in the Genbank, EMBL, Pir and Swiss-Prot databases The nptII enzyme is also heat labile and rapidly inactivated in simulated gastric fluid. Acute oral toxicity studies in mice with the nptII protein have not shown any adverse effects (Berberich et al., 1993). The use of nptII enzyme in tomatoes, canola and cotton has previously been evaluated by the US FDA. The FDA concluded that this enzyme does not have any of the recognised characteristics of food allergens or any attributes that would distinguish it toxicologically from other phosphorylating enzymes in the food supply (FDA 1994, cited in ANZFA, 1999). GUS protein The GUS protein is derived from E. coli and is therefore already present in the gut of many animals, including humans, and in soil and water ecosystems. 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 genetically modified crops is 99.8% homologous to the E. coli GUS protein. The GUS protein does not share any significant homology with known toxins (ANZFA, 2001). In their draft risk analysis report for application A378 ‘Food derived from glyphosate-tolerant sugarbeet line 77 (GTSB77)’ ANZFA concluded that food derived from this plant, which expresses the GUS protein, was safe for human consumption. Acute oral toxicity studies in mice, with purified GUS protein at doses of up to 100 mg/kg, did not show any adverse effects (ANZFA, 2001). The GUS protein is also unlikely to be a major allergen and does not display the characteristics common to known allergen proteins (discussed for Cry1Ac, above) (Fuchs and Astwood, 1996; ANZFA, 2001). Exposure of the GUS protein to simulated mammalian digestive systems resulted in its rapid degradation. The GUS protein does not have chemical or physical characteristics that are typical of known food allergens and does not share significant amino acid sequence similarity with known allergens The US Environmental Protection Agency (US EPA) does not consider GUS to be toxic for mammals and has approved its exemption from the requirement to establish tolerance levels (Federal Register, 2001b). 44 Toxicity or allergenicity of Bollgard II® cotton and Bollgard II®/Roundup Ready® cotton INGARD® cotton Since commercial release in 1996, there have been no reported adverse toxic or allergic effects on health through occupational exposure, ingestion of foods or use of products containing oil or fibre derived from INGARD® cotton, the parent of Bollgard II® cotton. Toxicity for mammals, including humans, and allergenicity A report prepared by ANZFA has concluded that foods derived from INGARD® cotton (oils and linters) are as safe as those derived from conventional cotton (ANZFA Full Assessment Report and Regulatory Impact Assessment A341, 1999). At least 8 overseas countries, including the USA, Canada and Japan, have approved the use of INGARD® cotton products in food (see for example, Canadian Food Inspection Agency, Decision Document 96-14, 1996; Health Canada Novel Food Information Document FD/OFB-096-100-C, 1997). The nutrient composition of INGARD® cotton seed is within the normal range for cottonseed in terms of the concentration of protein, oils, carbohydrate and ash, and amino acid and fatty acid profiles. The levels of known anti-nutritional or toxic factors in INGARD® cotton seed or cottonseed oil, including gossypol, and cyclopropenoid fatty acids (including dihydrosterculic, sterculic and malvalic acids) are also within the range of non-transgenic cotton controls (Keck et al., 1994). Four-week rat feeding studies using raw, ground cottonseed were carried out to compare INGARD® cotton with the parental line (Naylor 1993b, Naylor 1994). There were no significant differences in food consumption and body weight gain in animals fed a diet containing 5 % INGARD® cottonseed, compared to animals fed the same amount of cottonseed from the parental line. At a higher dose of 10%, however, there was some evidence of decreased consumption and weight gain in some groups of animals. This may have been due to reduced palatability as a result of slightly higher levels of sterculic acid in the INGARD® cotton seed compared to the parental line. It should be noted that these levels were still within the range found in normal cottonseed. There was no other evidence of toxicity or other adverse clinical signs during the study or in post mortem analysis of the organs. Toxicity for birds A dietary toxicity study with raw INGARD® cottonseed meal was conducted on the Northern Bobwhite Quail. There was no mortality in birds fed up to 100 000 ppm (10 % w/w, equivalent to 100 seeds/bird/day) for five days. There were no significant differences in feed consumption or body weight between birds INGARD cotton seed meal compared to birds fed cottonseed meal from the parental cotton line (Campbell and Beavers, 1993). In the United States, there have been anecdotal reports of increase in the populations of hummingbirds in Bt cotton fields associated with reductions in the use of various insecticides in these crops (Betz et al., 2000). 45 Toxicity for non-target invertebrates The safety of INGARD® cotton for non-target invertebrates has been demonstrated in studies conducted in Australian field conditions. Since 1994, the CSIRO Cotton Research Unit has carried out a number of studies to investigate the potential impact of INGARD® cotton on non-target invertebrates. Samples of invertebrates were collected from INGARD® cotton crops and sprayed and unsprayed conventional cotton crops. These were sorted and identified to the order level, or to the species level for all commonly recognised cotton pests or beneficial insects. There were no observable negative effects of INGARD® cotton on the abundance or diversity of non-target invertebrates, other than lepidopteran insects, compared to unsprayed fields of conventional cotton. Furthermore, INGARD® cotton fields in Australia required an average of around 50 % fewer insecticide sprays than conventional cotton fields. INGARD® cotton generally harboured significantly higher mean densities of invertebrates than corresponding conventionally sprayed cotton crops, presumably as a direct consequence of the reduction in insecticide treatments (Dr G. Fitt, CSIRO Entomology, CEO Australian Cotton Research Institute, personal communication). 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 what was found in conventional cotton fields (Xia et al., 1999). Other studies have demonstrated no adverse effects of feeding leaves of transgenic cotton containing Cry1Ac to two non-target soil arthropods, a collembolan (Folsomia candida) and an orbatid mite (Oppia nitens), organisms that play key roles as primary feeders or detritivores in soil ecosystems (Yu et al., 1997). Toxicity for microorganisms The effect of GM cotton containing the Cry 1Ac protein on soil microorganisms has also been examined. Soil was incubated with leaves of GM cotton expressing Cry1Ac (1:3 by weight of leaves to soil), or purified Cry1Ac toxin at equivalent levels (0.05 g/g) (Donegan et al., 1995). The numbers and types of protozoans, bacteria and fungi were determined at various time points. Substrate utilisation tests and DNA fingerprinting of eubacterial ribosomal sequences were also used to analyse the composition of bacterial soil community. Donegan et al. reported a short-term stimulatory effect on bacterial and fungal populations, as well as transient changes in the composition of the soil microbial community, for soil containing GM cotton expressing Cry1Ac, relative to soil containing leaves from the parent line. The significance of these changes is unclear; for instance the stimulatory effect may well reflect faster decomposition and release of nutrients from the transgenic leaves compared to the parent plants. 46 Roundup Ready® cotton ANZFA has concluded that foods derived from Roundup Ready cotton (oils and linters) are as safe as those derived from conventional varieties (ANZFA Final Risk Analysis Report Application A355, 2000). At least 5 overseas countries have approved the use in food of products from Roundup Ready cotton (see for example Canadian Plant Biotechnology Office, Decision Document 97-21, 1997; Health Canada Novel Food Information Document FD/OFB-97-08, 1997). The nutrient composition of Roundup Ready cotton is within the normal range for cottonseed in terms of the concentration of protein, oils, carbohydrate and ash, and the amino acid and fatty acid profiles. The levels of known anti-nutritional or toxic factors in Roundup Ready cottonseed or cottonseed oil, including gossypol, and cyclopropenoid fatty acids, are within the range of non-transgenic cotton controls. Treatment of the cotton with glyphosate had no effect on the nutrient composition or the levels of anti-nutritional or toxic factors (Nida et al., 1994; Nida et al., 1995; Canadian Food Inspection Agency, Decision Document 97-21, 1997; Health Canada Novel Food Information Document FD/OFB-97-08, 1997; ANZFA Final Risk Analysis Report Application A355, 2000). It should be noted that the presence of CP4 EPSPS, an enzyme of the aromatic amino acid biosynthetic pathway, does not cause an increase in the levels of aromatic amino acids. In feeding trials of rats, quail, and catfish, with 5 to 20% Roundup Ready cottonseed meal included in the diet, no significant differences in weight gain, feed conversion or gross necroscopy were found for animals fed Roundup Ready cottonseed meal compared to those fed cottonseed meal from control cotton (Canadian Plant Biotechnology Office, Decision Document 97-21, 1997). Bollgard II® cotton For Bollgard II® cotton, there is no evidence or reasonable expectation that it is likely to be harmful for any species other than lepidopteran insects, or specialised prey or parasites that feed on lepidopterans. None of the cotton plants from the proposed release, or their by-products, will be used in human or animal feed. Bollgard II®/Roundup Ready® cotton The risks of allergenicity or toxicity as a result of the genetic modifications in Bollgard II®/Roundup Ready® cotton are likely to be the same as for the parent Roundup Ready® and Bollgard® cotton (discussed above). There is no evidence or reasonable expectation that recombination between the introduced genes, or synergistic effects arising from the combination of the two traits, are likely to occur, or that they would result in new or increased risks relating to toxicity or allergenicity. The Roundup Ready® herbicide tolerance and Bollgard® insecticidal genes operate through independent, unrelated biochemical mechanisms. There is no evidence of any interaction between the two genes or their metabolic pathways and no reason to expect that this is likely to occur. 47 As noted above, ANZFA have already approved the use in food of products derived from Roundup Ready® cotton, and are currently considering an application for Bollgard II® cotton. If the Bollgard II® cotton application is approved, ANZFA will not require separate approval for Bollgard II®/Roundup Ready® cotton developed through conventional plant breeding. This would also apply to food from any other plant produced by conventional breeding of two genetically modified parents, providing that the parent organism had already been approved. It should be emphasised, that in the case of the release proposed by CSD, none of the cotton plants from the release, or their by-products, will be used in human or animal feed. Potential for exposure to Bollgard II® cotton and the introduced proteins As discussed in Part 4.7.1, the Cry1Ac and Cry2Ab proteins are present at low levels in Bollgard II® cotton, with around 10 and 400 g/g tissue (10 and 400 ppm), respectively, in the buds and growing tips. Expression in pollen and nectar are likely to be even lower, as was the case for Cry1Ac in INGARD® cotton (data supplied by Monsanto), since the same promoter has been used to drive expression of Cry2Ab. The GUS protein is present at around 70 ppm in seed, and nptII is expected to be around 4 ppm (or 50 ppm for Bollgard II®/Roundup Ready® cotton, see Part 4.7.1). The CP4 EPSPS protein is present at around 80 ppm or less in Roundup Ready® cotton. Consequently the level of exposure to the novel proteins in the genetically modified crop is not likely to be significant, and may be further limited depending on possible routes of exposure. The potential for exposure of humans, other animals and soil biota is discussed in detail below. Occupational and environmental exposure for humans The introduced proteins are expressed at very low levels (see Part 4.7.1), as intracellular proteins within the double walled plant cells. Humans working with the plants or the seed would not be exposed to these proteins unless the seeds or tissue ruptured. Even if the plant cells ruptured, the levels of protein expression are very low, as detailed above. Mature lint from cotton is almost pure cellulose and contains little if any protein. So the overall level of exposure for workers handling the cotton, or exposed to soil residues, or residues produced in the ginning process is likely to be negligible. Cotton is largely self-pollinating: the pollen is large and sticky and not easily dispersed by wind. Cotton pollen is therefore not likely to be present in the atmosphere, limiting any possible human exposure to cotton pollen as a potential aeroallergen. Direct occupational or environmental exposure to Cry2Ab, Cry1Ac, GUS and nptII proteins in Bollgard II® cotton plants for farm workers, factory workers, or farming communities will be limited by the scale of the proposed release. 48 Dietary exposure for humans There will be no direct or indirect dietary exposure, since the cotton and its by-products from this release will not be used in food or animal feed. It is possible that limited amounts of Bollgard II® or Bollgard II®/Roundup Ready® cotton pollen might be incorporated in honey, since honey may contain small amounts of pollen. However, hives are generally not placed near cotton fields because of the potential for exposure and loss of foraging bees or contamination of honey with the pesticides normally applied to cotton. The UK Ministry of Agriculture, Fisheries and Food (MAFF) have carried out a study using honey containing pollen from genetically modified canola that expressed the nptII gene. They estimated that a 500 g pot of honey would contain up to 0.005 g of nptII protein (MAFF 1997). This is equivalent to 0.00001 ppm. To put this in perspective, it has been estimated that although amounts as low as 100 g of peanut protein may cause mild reactions in a limited number of individuals, amounts of 2000 to 5000g are required to produce significant reactions in the same patients (cited in MAFF, 1997). The lack of heat stability of the introduced proteins (see above) suggests that cooking or high temperature processing of foods would inactivate the introduced proteins. The rapid gastric digestion of the introduced proteins would further limit any dietary exposure. Exposure in products containing fibre and oil derived from Bollgard cotton Cotton lint contains no detectable nitrogen, and hence no DNA or protein (Leffler and Tubertini, 1976). The refining and processing of cottonseed oil, cotton fibre (lint) or cotton linters, both chemically and thermally, destroys or removes proteins and nucleic acids to below detectable levels (Sims et al., 1996; Sims and Berberich, 1996). Processed cotton fibre contains 99.8% cellulose (AgraFood Biotech, 2000). Given the very low levels of the introduced proteins in Bollgard® and Bollgard II®/Roundup Ready® cotton, exposure to these proteins in products containing cotton fibre, linters or cottonseed oil can be considered negligible. Seed from the proposed release will not in any case be used for oil production. Exposure of livestock and wildlife, including fish and birds Mammals 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 its use as a protein supplement in animal feed except for cattle which are unaffected by these components. Inactivation or removal of these components during processing enables the use of some cottonseed meal for catfish, poultry and swine. Best Management Practices for the Australian cotton industry prohibits the use of cotton trash and stubble as a feed for animals, due to other pesticides that could be found in the cotton trash and stubble. Cotton seed in the field is present as large lint-covered seeds, that are unattractive to avian species, so birds are not likely to be exposed to the insecticidal proteins expressed in the seeds of Bollgard II® and Bollgard II®/Roundup Ready® plants. Cottonseed or pollen is not expected to enter aquatic habitats in any significant quantity, and therefore aquatic species 49 will not be exposed. The proponents propose to ensure that the release site is at least 50 metres from any natural waterways, and because of irrigation practices used by cotton growers in Eastern Australia, water used on the crops remains on the farm, and does not enter natural waterways. Any exposure will be further limited because of the scale of the release. Exposure of non-target invertebrates Non-target invertebrates may be exposed directly, through feeding on the Bollgard II® plants, or indirectly through eating other organisms, including the lepidopteran target organisms, that feed on the plants. Exposure is not expected to be significant, given the low levels of the introduced proteins (see Part 4.7). Exposure will be further limited by the scale of the release. Exposure of soil biota After harvest of lint and seed, the remaining cotton plant residues are typically tilled into the soil, so that soil biota may be exposed to the introduced proteins in the GM cotton. For Cry1Ac in INGARD® cotton, the exposure level as a result of post-harvest tillage is estimated at 1.44 g/acre (Ream, 1994a). This soil exposure will increase in Bollgard II® plants because of the higher levels of expression of the Cry2Ab protein (about 10 fold higher), but will still be relatively small, and further limited because of the scale of the release. Exposure to the other introduced proteins would be even less, since they likely to be expressed at lower levels than the Cry proteins (see Part 4.7.1). It should be noted that Bacillus thuringiensis is a common soil bacterium, and that spores containing Bt toxins including Cry1Ac are already a natural component of soil. Microbial formulations of Bt that contain Cry1Ac, Cry2Ab and similar Bt-toxins are regularly applied as biological pesticides to Australian agricultural soils without adverse effects. Agrobacterium, from which the CP4 EPSPS gene was derived, and E. coli, from which the nptII and GUS gene were derived, are also found in soil. The initial level of exposure is likely to decrease with time, as a result of soil biodegradation. Ream, 1994a have compared the rate of soil biodegradation of Cry1Ac protein in INGARD® cotton plants to that of the purified toxin. The plant-encoded Cry1Ac degraded with a half life of 41 days, compared to 9.3 to 20.2 days for the purified toxin. In another study (Palm, 1996) results were variable but indicated half-lives for Cry1Ac in the order of 2.2 to 46 days. In all cases, there was an initial rapid decline in Cry1Ac levels by day 7 followed by a more gradual rate of decline. However, low levels of Cry1Ac were still detectable at 140 days in some treatments. A soil degradation study conducted with the purified Cry2Aa protein, which is highly similar to Cry2Ab, determined the soil half-life based on biological activity to be 15.5 and 31.7 days for the laboratory and the field, respectively (Sims and Ream, 1997). 50 These results demonstrate that the Cry proteins, as a component of post-harvest plants, are expected to dissipate or degrade when cotton residues are ploughed into the soil after harvest. There is no data on the degradation rates in soil of CP4 EPSPS, nptII and GUS proteins, but the data relating to their stability to digestion in mammalian digestive systems (see part 5.5.1) does not indicate any unusual degree of stability. It has been shown that Bt toxins can bind to clay minerals in soil and that the bound toxin can be protected against microbial degradation and retain insecticidal activity for up to 234 days (Tapp and Stotzky, 1998). However, under most production conditions, cotton is grown in alkaline soil or soil pH ranging from 6.0 – 6.5 (Dr G. Fitt, CSIRO Entomology, CEO Australian Cotton Research Institute, personal communication). At this pH, Bt endotoxins are released from clay and degraded by soil microbes (Crecchio and Stotzky 1998). Many of the experiments examining persistence of Bt proteins reported in the published literature have been conducted in bulk soils or soil components (e.g. Palm, 1996; Koskella and Stotzky, 1997; Stotzky, 2000a). Bulk soil generally does not support populations of microorganisms as high as in the rhizosphere or in cropping situations where plant residues are incorporated into the soil (Griffiths et al., 1999), conditions that are more likely to favour the rapid degradation of Bt toxin. Exposure of organisms in soil to Bt residues may also occur as a result of root exudations, as has been observed for Bt corn expressing Cry1Ab (Saxena et al. 1999; Stotzky, 2000b). However, the mechanism for this is not clear, and it is not known whether a similar process occurs for Bollgard II® cotton. C: Conclusions regarding toxicity and allergenicity It is considered that the likelihood of adverse impacts on humans or other species (other than lepidopteran insects), as a result of toxicity or allergenicity of Bollgard II® or Bollgard II®/Roundup Ready® cotton in the proposed release is very low. There is no evidence that Bollgard II® or Bollgard II®/Roundup Ready® cotton will be more toxic or allergenic to humans or other organisms (other than lepidopteran insects) than conventional cotton. Because of the various factors outlined above, exposure to the genetically modified cotton and the introduced proteins will be minimal. CSD have indicated that no cotton plants from the release or their by-products will be used for human or animal feed. The Regulator may impose conditions on the licence to restrict use of material from the release (see Part 6.3). The scale of the proposed release is relatively small on an agricultural scale, and any environmental impacts due to non-target toxicity are likely to be localised to the specific release site and will therefore be manageable. A secondary impact resulting from the toxicity of Bollgard II® cotton for lepidopteran insects, is that populations of specialist parasites or predators that feed on lepidopterans may be affected, because of the reduction in lepidopteran numbers. This has in fact been observed in CSIRO releases of INGARD® cotton (Dr Gary Fitt, CSIRO Entomology, CEO Australian Cotton Research Institute, personal communication). However, the impacts on these populations, and on any non-target lepidopteran insects, are likely to be far less than the impacts of pesticides used in the cultivation of conventional cotton. 51 There is a theoretical risk that the GM cotton may prove allergenic for a very small number of individuals. However, on the basis of the risk assessment above, the risks are no greater than with any novel food or fibre, including those created by conventional breeding. It should be noted that cotton is widely used in pharmaceutical and medical applications because of its very low allergenicity and purity. 5.5.2 Weediness A: Nature of the weediness hazard The possibility was considered that Bollgard II® or Bollgard II®/Roundup Ready® cotton might have the potential to be harmful to the environment, because of inherent weediness or increased potential for weediness. There is also the possibility that the genetic modification has, either directly or as a result of pleiotropic effects, increased the weediness of the cotton plants. This could result from changes such as increased fitness due to higher levels of insect resistance or increased reproductive capacity. B: Likelihood of the weediness hazard occurring Weediness of unmodified cotton Cotton has been grown for centuries throughout the world without any reports that it is a serious weed pest. Cotton is not listed as a weed in Australia (Tothill et al., 1982) and has no weedy relatives (Keeler et al., 1996). Cotton is not considered to have weedy characteristics, as an annual plant grown in Australia, although a small numbers of plants may occasionally establish on roadsides (Eastick, 2000). Cotton does not possess any of the attributes commonly associated with weeds, such as seed dormancy, long persistence in soil, germination under adverse environmental conditions, rapid vegetative growth, a short life cycle, very high seed output, high seed dispersal and long-distance dispersal of seeds (Keeler, 1985, 1989). In particular, cottonseed does not exhibit dormancy and the seeds cannot persist in the soil for long periods of time. Cotton seeds lose viability quickly under moist conditions. Commercial cotton is always grown from seed, sown when soil temperatures are at least 18C (Duke, 1983). Weediness of Bollgard or Bollgard II®/Roundup Ready® cotton If Bollgard II cotton were to spread in the environment as a weed, this could result in impacts such as loss of environmental biodiversity or adverse effects on agricultural systems. The agronomic characteristics (e.g. germination, seed survival, vigour, yields, disease susceptibility) of Bollgard II or Bollgard II®/Roundup Ready® cotton have been evaluated in glasshouse and releases, and found to be within the range for current commercial conventional cotton varieties and INGARD® cotton varieties (Dr G. Constable, Program Leader, CSIRO Cotton Research Unit, personal communication; Deaton, 1993; Deaton and 52 Beuhler, 1994; Monsanto, 1995; Sheers, 1997; Monsanto, 1998; 1999). The only difference that one would expect between the modified and non-modified cotton is the expression of the five proteins, Cry1Ac, Cry2Ab, CP4 EPSPS, GUS and nptII. There is no evidence, nor any reason to believe, that expression of these proteins in the Bollgard II or Bollgard II®/Roundup Ready® cotton plant would alter any of the characteristic weed attributes listed above. The Cry1Ac and Cry2Ab genes might confer a survival advantage and increase the potential for weediness in regions where insect predation limited its growth, because of an increased ability to tolerate insect feeding or to control pest insects. However, the distribution of wild populations of cotton in Australia is limited by abiotic factors such as water availability and soil type, rather than by insect pressure. Bollgard II/Roundup Ready cotton would have a survival advantage in the presence of glyphosate. However, glyphosate is not used to control cotton plants in agriculture or in natural environments. Its effectiveness on cotton is limited and, because it is not selective, tends to leave bare earth more easily colonised by other weeds. Alternative herbicides are readily available for the control of cotton in the limited cases where this may prove necessary. The nptII protein, encoding resistance to neomycin and kanamycin, will not confer a selective advantage on the cotton, since antibiotics are not used on cotton crops. The GUS protein is also considered very unlikely to confer any selective advantage to cotton that might result in weediness (Gilissen et al., 1998). C: Conclusions regarding weediness It is concluded that the risk of Bollgard II or Bollgard II/Roundup Ready cotton spreading into the environment and causing harm to the environment is low and not likely to be greater than for conventional cotton. In summary, the reasons for these conclusions are that cotton itself is not a weed and the introduced genes are not likely to increase the weediness potential of the plants. It is therefore highly unlikely that Bollgard II or Bollgard II/Roundup Ready cotton will become a weed problem. CSD have proposed various measures to ensure cotton does not spread from the release site, or persist at the site after the harvest, thus reducing the potential for the GM cotton to establish as a weed outside the release site. The Regulator may impose licence conditions to ensure this (see Part 6.3). 5.5.3 Transfer of introduced genes to other organisms In general terms, the types of hazards that might result from transfer of the genes introduced into Bollgard II or Bollgard II®/Roundup Ready® cotton into other organisms could include the production of insecticidal or herbicide-tolerant weeds with potential to compete with native flora populations and subsequent loss of biodiversity, or antibiotic-resistant pathogens with potential to harm human or animal health. 53 The potential hazards are addressed in the following sections, with respect specifically to: other plants (part 5.5.4); and other organisms (Part 5.5.5). 5.5.4 A: Transfer of introduced genes to other plants Nature of the gene transfer hazard Transfer of genes to other cotton plants Transfer of the introduced genes to other cotton plants would present the same hazards and have the same potential impacts as the presence of the genes in Bollgard II or Bollgard II®/Roundup Ready® cotton (see Parts 4.1 and 4.3). However, if transfer occurred to non-GM cotton crops, this would further increase the possibility that the genes could spread in the environment, with flow on impacts depending on the nature of the gene transferred and the species to which it was transferred. Transfer of genes to other plant species Transfer of the introduced genes into other plant species, in particular to native flora, might have adverse effects on biodiversity. Other potential hazards specific to the transferred gene sequences are as follows: i) Insecticidal genes: Plants could become resistant to lepidopteran insects. This could confer a fitness advantage on plants normally controlled by these insects, and could result in increased weediness. There could also be impacts on the lepidopteran insect populations, or specialist predators and parasites that feed on them. ii) Antibiotic resistance marker genes: Plants could become resistant to the antibiotics. This would not in itself have any significant impacts, since antibiotics are not generally used on plants outside of the laboratory. Streptomycin is used in some other countries to control fire blight, a bacterial disease of fruit trees. However, fire blight is not endemic in Australia and is not a disease of cotton. 54 iii) GUS marker gene: Plants would produce the GUS protein. There is no evidence and no reason to believe that this would have any adverse impacts. GUS is not likely to be toxic or allergenic to other organisms, or to increase the weediness of the cotton (Part 5.5.1 and 5.5.2 refer). iv) Herbicide tolerance gene: Plants could become resistant to glyphosate. This would have an impact only if the plant is controlled by glyphosate, on the farm or as a weed in the environment. v) CaMV 35S promoter and other regulatory sequences: If gene transfer did occur, there could be unintended or unexpected effects if the introduced regulatory sequences alter the expression of endogenous plant genes. If such perturbation of normal plant gene expression did occur, the impact would depend on the phenotype. Some of these sequences are derived from plant pathogens (cauliflower mosaic virus, figwort mosaic virus, Agrobacterium tumefaciens). The possibility should be considered that they might have pathogenic properties. B: Likelihood of the gene transfer hazard occurring Transfer of genes to other cotton crops or feral cotton populations Outcrossing rates for cotton The transfer of transgenes from Bollgard II® cotton to other cotton cultivars requires the transfer of Bollgard II® pollen to conventional cotton—and this requires a pollen vector. The reciprocal cross would have no risk implications. Cotton is a facultative self-pollinator, and an opportunistic out-crosser (Oosterhuis and Jernstedt, 1999). Cotton pollen is large and sticky and requires an insect vector for outcrossing—wind dispersal is negligible. Cotton flowers open early in the morning and anther dehiscence and stigma receptivity follows soon after. Tthere is no period of preferential outcrossing. Insect prevalence strongly influences outcrossing rates for cotton (Elfawal et al., 1976; Moresco et al., 1999), and will vary across sites and years (Moffett et al., 1975, 1976; Moresco et al., 1999). Insect visitation rates, however, may overestimate cross-pollination rates because many potential pollinators preferentially target nectaries rather than the pollen (Moffett et al., 1975; Rao et al., 1996). Many field-based assessments estimate outcrossing at 10% or less (Meredith and Bridge, 1973; Gridley, 1974; Theron et al., 1975; Elfawal et al., 1976; Umbeck et al., 1991; Llewellyn and Fitt, 1996). Higher estimates (16.5% to 25%) have been reported in a few cases (Smith 1976; Moresco et al., 1999). Oosterhuis and Jernstedt (1999) suggest that outcrossing rates can reach 80% under some conditions, but provide no substantiating evidence. The level of outcrossing observed in Australian studies of transgenic or conventional cotton is in the order of 1 to 2 % between plants in adjacent rows (Thomson, 1966; Mungomery and Glassop, 1969; Llewellyn and Fitt, 1996). This is relatively low compared to that seen in some other countries . Differences in pollinator species may be responsible for the lower 55 rate, in particular the absence of bumble bees, which are known to be very effective pollinators (Llewellyn and Fitt, 1996). Honeybees were implicated as the chief pollinating agent in a Queensland study (Mungomery and Glassop, 1969), however, since honeybees were not present for a similar study in the Ord River valley (Thomson 1966) it was suggested that native bees might be responsible for the cross pollination. In cotton outcrossing experiments conducted near Narrabri in New South Wales, 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 (Llewellyn and Fitt, 1996). Pollen dispersal distances for cotton Cotton pollen dispersal studies consistently demonstrate that outcrossing is localized around the pollen source and decreases significantly with distance (Thomson (1966); Galal et al., 1972; Theron and Staden, 1975; Elfawal et al., 1976; Chauhan et al., 1983; Umbeck et al., 1991; Llewellyn and Fitt, 1996). This presumably represents the effective foraging range of insect pollinators. The separation distance of 4 metres required in Australia for certified commercial seed production reflects the relatively short distances observed for cotton pollen dispersal in Australian studies. In one CSIRO study carried out in New South Wales, 200 transgenic cotton plants were embedded in an eight-hectare plot of non-transgenic cotton (Llewellyn and Fitt, 1996). Seeds from the non-transgenic cotton were collected and assayed for the nptII protein. Of the 37 000 seeds assayed, only six were found to have been derived from outcrossing of the transgenic pollen and all of these came from within three metres of the transgenic plot. In a second study by Llewellyn and Fitt, at the same location, dispersal of pollen from a block of 3 000 transgenic cotton plants was monitored and 60 000 seeds were assayed. Forty nine cross-pollinated seeds were detected, with the highest level of outcrossing (0.9%) occurring in the first buffer row. Beyond 10 metres, outcrossing events were generally rare, with 0.01% outcrossing detected at distances of 11, 14 and 16 metres, and no outcrossing detected between 16 and 20 metres. Similar findings have been obtained by breeders in previous studies in Australian conditions with non-modified cotton. For example, Thomson (1966) looked at outcrossing from a red leafed (partly dominant) variety of cotton planted within a field of green leafed cotton. This study was carried out in the Ord River valley over two growing seasons. Cross-pollination between adjacent plants, measured as the proportion of red leafed progeny, was in the range of 0 to 5 %, with mean values of 1.63 % and 1.02 %, in the first and second seasons respectively. Very little cross pollination was detected at a distance of more than 3 metres (average less than 0.01%) and none was detected at distances between 3 and 8 metres. Mungomery and Glassop (1969) used a similar experimental design to look at outcrossing during two seasons in Biloela, Queensland. Cross-pollination between adjacent rows of cotton was around 1.7 % in both years, falling to less than 1 % in rows beyond this. No crossing was observed in rows to the north or south of the red leafed cotton, at 32 or 53 metres (the last two distances tested), with the exception of 0.3% outcrossing detected on the northern side at 53 metres, in one of the two growing seasons. 56 Umbeck et al. (1991) also investigated pollen dispersal from transgenic cotton embedded in a field or conventional cotton in the United States. They found higher outcrossing rates (up to 5.7% in the first buffer row), but as with the Australian studies, the rate of outcrossing fell rapidly with distance from the transgenic block. The level of outcrossing was generally below 1% at 7 metres, but a low level of sporadic outcrossing was seen at distances of up to 25 metres. Outcrossing at distances greater than 25 metres was not measured. The Australian and US studies cited above measured pollen dispersal through buffer rows of cotton. The outcrossing rate in the absence of buffer rows, between cotton plants separated by bare ground, might be expected to be higher. For instance, Green and Jones (1953) demonstrated that outcrossing through buffer rows decreased outcrossing from 19.5% to 2.6 % at 9.6 metres or 1.0 % at 10.7 metres. By comparison, outcrossing at a distance of 10 metres, in the absence of a buffer, was 4.7 %. Nevertheless, outcrossing in the absence of a buffer did decline with distance, from 6.0% at 5.0 metres, to 4.7% at 10.0 metres, 0.6 % at 25.1 and 50.3 metres. An Egyptian study measured outcrossing from Gossypium barbadense and also demonstrated a rapid decline with distance even in the absence of buffer rows (Galal et al., 1972). The average level of outcrossing varied from 7.8% at 1.1 metres to 0.16% at 35.2 metres. Isolation from feral cotton populations Small feral cotton populations found in northern Australia are confined to beach strands and are geographically isolated from areas of existing or potential cotton cultivation (Hnatuik, 1990). The geographic distances between Bollgard II® and Bollgard II®/Roundup Ready® cotton and naturalised cottons exceed conceivable pollinator foraging ranges, and serves as an effective natural barrier. Isolation from non-GM crops Because the proposed release would occur extremely late in the growing season, there would be no other flowering cotton in the area, so the likelihood of outcrossing to a non-GM cotton crop is extremely remote. It is also proposed to treat the crops with insecticides during the flowering period to limit pollen movement by pollinating insects. Physical isolation and/or buffer rows of non-GM cotton could be used to provide absolute containment of pollen. CSD are proposing to use a minimum isolation distance of 50 metres from other cotton. No buffer rows will be used as the release will be used to produce seed for future trials, and this removes the possibility of contamination by pollen from the buffer. If a licence is issued for the release, CSD will be required to undertake research to confirm the efficacy of the 50 metre isolation zone. Transfer of genes to other plant species The Australian flora contains 17 native Gossypium species that are all members of a distinct group—Gossypium subgenus Sturtia—found exclusively in Australia. They are distant relatives of the cultivated cottons that originated in Americas (Fryxell, 1979; 1992; Fryxell et al., 1992; Seelanan et al., 1999; Brubaker et al., 1999; Liu et al., 2001). The Australian 57 Gossypium species can be apportioned to one of three taxonomic sections within subgenus Sturtia: sect. Sturtia (two species); sect. Hibiscoidea (three species) and sect. Grandicalyx (12 species). Most of the native Australian Gossypium have limited distributions at considerable geographic distances from cultivated cotton fields. None of the native Australian Gossypium species have the properties of invasive agricultural or environmental weeds. Based on the known distributions of the 17 native Gossypium species, G. australe, G. nelsonii and G. sturtianum are the only species whose ranges encompass existing or potential cotton growing regions in eastern Australia. G. australe and G. rotundifolium are the only species whose ranges encompass existing or potential cotton growing areas in northwest Australia. As with conventional cultivated cottons (discussed above), risk accrues only with the deposition of Bollgard II® or Bollgard II®/Roundup Ready® pollen on the stigmas of native Gossypium species and the reciprocal event has no risk implications. Thus, with the exception of the species whose distribution ranges encompass existing or potential cotton growing regions in Australia, the limited distribution and geographic isolation of 13 of 17 native Gossypium effectively insulates them from Bollgard II® or Bollgard II®/Roundup Ready® pollen. Of the remaining species with distributions that encompass existing or potential cotton growing regions (G. australe, G. nelsonii, G. rotundifolium, and G. sturtianum), it is unlikely that their populations will ever occur within pollinator foraging range of Bollgard II® and Bollgard II®/Roundup Ready® cottons. Moreover, the ecological preferences of the native Gossypium species suggest that intimate physical proximity (<1 km) between native Gossypium species and Bollgard II® cotton will be negligible. The wild Gossypium species have no weedy propensity and are only found in native vegetation, not in the human modified environments of the agricultural areas. The wild Gossypium species are particularly intolerant of the heavy clay soils on which most cultivated cotton is grown. In the extremely unlikely event that deposition of Bollgard II® pollen onto a wild Gossypium stigma occurred, the genetic incompatibility between the Bollgard II® cottons and native Gossypium species would preclude transgene escape (reviewed in detail by Brown et al., 1997 and Brubaker et al., 1999). Cultivated cotton is tetraploid (G. hirsutum and G. barbadense, genome aadD) and the native Gossypium species in Australia are diploids (C, G or K genomes) (Stewart, 1994). Consequently hybrids are difficult to effect, even with human manipulation, and are nearly exclusively sterile (Brown et al., 1997; Brubaker et al., 1999). There are two main types of barrier to the spontaneous escape of cotton transgenes into populations of native Gossypium species in eastern Australia and northwestern Australia. These are (i) prezygotic barriers (geographic isolation between endemic species and cotton, disjunct flowering periods, autogamy in isolated plants and competitive disadvantage of foreign pollen in the style); (ii) postzygotic barriers (selective abortion of weak embryos and fruit, hybrid seedling and plant fragility, meiotic sterility of triploids, lack of vigour in hexaploids, poor seed set in hexaploids and sterility of backcross progeny) (Brown et al., 1997). 58 Brubaker et al. (1999) discussed extensive experimental efforts on the hybridisation of tetraploid cotton (G. hirsutum) with pollen from 17 diploid Australian Gossypium species. These experiments were done under artificial (ideal glasshouse) conditions using treatment with gibberellic acid to decrease the frequency of premature capsule abortion. Overall, the average number of seed produced per cotton flower pollinated with wild pollen ranged from 0.05 to 5.9 in contrast with typical intraspecific fecundity of >32 seeds per capsule in cotton (Turner et al., 1977). The species with highest potential for interspecific crossing is G. sturtianum, and it is the only native Gossypium species for which hybrid progeny have been produced as the recipient of cultivated cotton pollen and then only with human intervention. However, hybrids between G. sturtianum and cultivated cotton are sterile regardless of which species served as the pollen recipient, eliminating any potential for transgene exchange (Brown et al., 1997; Brubaker et al., 1999). C: Conclusions regarding gene transfer to plants The likelihood of gene transfer into other plants (including other cotton crops, feral cotton populations or native flora, with potential adverse impacts on biodiversity) is low for transfer to cotton, and negligible for transfer to other plant species. Because the proposed release would occur exceptionally late in the growing season, there would be no other flowering cotton in the area, so the likelihood of outcrossing to a non-GM cotton crop for this particular release is extremely remote. CSD are proposing additional measures, including treatment with pesticides during the flowering period, to limit any possible outcrossing to other cotton, and the Regulator may impose licence conditions to ensure appropriate measure are in place (see Parts 6.2 and 6.3). The conclusions with respect to the specific transferred gene sequences are as follows: i) Insecticidal genes: It is possible that if these genes were transferred to feral, native, or cultivated cotton, the plants might have a survival advantage in regions where insect predation limited their growth. However, cotton and its native relatives are not regarded as weeds in Australia, and their distribution is determined largely by soil type and climatic conditions, rather than insect pressure. ii) Antibiotic resistance genes: There would be no adverse consequences even if outcrossing occurred. Streptomycin is used in some other countries to control fire blight, a bacterial disease of fruit trees. However, fire blight does not occur in Australia, and plants are therefore not treated with streptomycin. iii) GUS marker gene: There would be no adverse consequences even if outcrossing occurred. GUS is not likely to be toxic or allergenic to other organisms, or to increase the weediness of the cotton (Part 5.5.1 and 5.5.2 refer). 59 iv) Herbicide tolerance gene: Outcrossing into plant species other than cotton is extremely unlikely. There would be no adverse consequences even if outcrossing to cotton occurred, since cotton species are not regarded as weeds in Australia and are not controlled by glyphosate on the farm or in the natural environment. v) CaMV 35S promoter and other regulatory sequences: The probability of a hazard arising due to outcrossing of these sequences to other plants is remote, given the low likelihood of gene transfer by outcrossing. Plants are already exposed in nature to the bacteria and viruses from which these sequences are derived. Although some of the regulatory sequences transferred to the plants are derived from plant pathogens, they only represent a very small proportion of the pathogen genome. The sequences are not in themselves infectious or pathogenic. It should be noted that CaMV is already ubiquitous in the environment and in the human diet (Hodgson, 2000a). 5.5.5 A: Transfer of introduced genes to other organisms (microorganisms and animals) Nature of the gene transfer hazard Potential hazards, with respect to the specific gene sequences, are as follows: i) Insecticidal genes: This would not present a hazard to human health or the environment. It should be noted that the insecticidal genes were originally isolated from a common soil bacterium. ii) Antibiotic resistance genes: Transfer of the genes to animals (including humans) or microorganisms other than bacteria (such as viruses) would not present a hazard. However, bacteria that acquired the antibiotic resistance gene(s) could become resistant to those antibiotics. The consequences of this would depend on: the pathogenicity of the microorganism; the use and significance of the antibiotic(s) in clinical and/or veterinary practice; whether resistance to the antibiotic(s) is already widespread in the microbial population. iii) GUS marker gene: Transfer of the genes to animals (including humans) or microorganisms other than bacteria (such as viruses) would not present a hazard. iv) Herbicide tolerance gene: This would not present a hazard to human health or the environment. It should be noted that the herbicide-tolerance gene was originally isolated from a common soil bacterium. 60 v) CaMV 35S promoter and other regulatory sequences: If gene transfer did occur, there could be unintended or unexpected effects if the introduced regulatory sequences alter the expression of endogenous plant genes. If such perturbation of normal plant gene expression did occur, the impact would depend on the phenotype. Some of these sequences are derived from plant pathogens (cauliflower mosaic virus, figwort mosaic virus, Agrobacterium tumefaciens). The possibility should be considered that they might have pathogenic properties. The possibility that the regulatory sequences could recombine with the genome of another virus infecting the plants to create a novel recombinant virus should also be considered. B: Likelihood of the gene transfer hazard occurring Transfer of genes to humans or other animals No evidence has been identified by the Regulator for any mechanism by which the genes could be transferred from Bollgard II or Bollgard II/Roundup Ready cotton plants to humans or animals, nor any evidence that this has occurred during evolutionary history, despite the fact that animals and humans eat large quantities of plant DNA. Transfer of genes to bacteria Transfer of the introduced genes from the Bollgard II or Bollgard II/Roundup Ready cotton to microorganisms is extremely unlikely. Horizontal gene transfer from plants to bacteria has not been experimentally demonstrated under natural conditions (Syvanen, 1999; Nielsen et al. 1997; Nielsen et al. 1998) and deliberate attempts to induce such transfers have so far failed (e.g. Schlüter et al., 1995; Coghlan, 2000). Transfer of plant DNA to bacteria has been demonstrated only under highly artificial laboratory conditions, between homologous sequences and under conditions of selective pressure (Mercer et al. 1999; Gebhard and Smalla, 1998; Nielsen et al., 1998), but even then only at a very low frequency. Phylogenetic comparison of the sequences of plant and bacterial genes suggests that horizontal gene transfer from plants to bacteria during evolutionary history has been extremely rare, if occurring at all (Doolittle, 1999; Nielsen et al. 1998). The cry1Ac and cry2Ab insecticidal genes are already widespread in the environment (they were originally isolated from a common soil bacterium, Bacillus thuringiensis). The nptII, uidA and aad genes are also prevalent in naturally occurring bacteria found in soil and in animal and human digestive systems. The nptII and aad genes occur naturally on transmissible genetic elements (transposons and plasmids) that are readily transferable between bacterial species (Flavell et al., 1992; Pittard, 1997; Langridge, 1997; US FDA Draft Guidance Document on Use of Antibiotic Resistance Marker Genes in Transgenic Plants, 1998). Transfer of the genes from these naturally occurring bacteria, through well documented mechanisms for horizontal transfer between bacteria (Nielsen et al., 1998; 61 Doblhoff-Dier et al. 2000), is far more likely than transfer of the same genes from Bollgard II cotton. The transfer of a gene from a genetically modified plant to bacteria in the human gut would require a series of steps each of which has a very low probability (Pittard, 1997; US FDA Draft Guidance Document on Use of Antibiotic Resistance Marker Genes in Transgenic Plants, 1998). An intact copy of the gene would need to: survive degradation during processing of food in the gut, and by acid and nucleases in the stomach and intestines; be taken up by a bacterium; survive efficient bacterial defence mechanisms for degrading foreign DNA; and become stably integrated into the bacterial genome or on a plasmid, in precise alignment with a bacterial promoter (if this were not co-transferred, intact, from the plant). Finally, for the antibiotic-resistance genes, there would need to be selection pressure with the antibiotic in question for an antibiotic-resistant bacterium to persist and multiply in the gut or the environment. Transfer of genes to other viruses There is a theoretical possibility of recombination between sequences that have been introduced into the Bollgard II® cotton plant genome, and the genome of viruses that might infect the cotton plants (Hodgson, 2000a,b; Ho et al., 2000). Recombination between viral sequences and plant transgenes has only been observed at very low levels, and only between homologous sequences under conditions of selective pressure, e.g. regeneration of infectious virus by complementation of a defective virus, containing a deletion mutation in its coat protein, by sequences transcribed from viral coat gene introduced into a transgenic plant genome (Greene and Allison, 1994, Teycheney and Tepfer, 1999). C: Conclusions regarding gene transfer to other organisms Horizontal gene transfer from plants to animals (including humans) or microorganisms is extremely unlikely. The conclusions, with respect to the specific gene sequences are as follows: i) Insecticidal genes: There would be no adverse consequences even if gene transfer occurred. ii) Antibiotic resistance genes: Transfer of these genes to organisms other than bacteria would not present a hazard, since the antibiotics in question are only used to treat or prevent bacterial infections. Horizontal transfer to bacteria is also extremely unlikely and is considered to pose negligible risks to human health or the environment for the following reasons. Bollgard II cotton contains genes that confer resistance to neomycin, kanamycin, streptomycin and spectinomycin. None of these antibiotics are extensively used in clinical medicine. Streptomycin was formerly used in the treatment of tuberculosis, but is not 62 routinely used today because of its toxicity and the relatively high frequency at which streptomycin-resistant mutants emerge. Only neomycin and kanamycin are used in veterinary practice, and alternative antibiotics are readily available. The use of antibiotic-resistance markers in genetically modified plants and microorganisms to be released into the environment has been researched and reviewed extensively. It has been concluded that the presence of kanamycin-resistance genes in genetically modified plants represents no significant risk to biosafety (Flavell et al., 1992; Pittard, 1997; Langridge, 1997; US FDA Draft Guidance Document on Use of Antibiotic Resistance Marker Genes in Transgenic Plants, 1998; JETACAR, 1999). Flavell et al. (1992) note that the human health analyses need to be viewed against the knowledge that humans continually ingest kanamycin-resistant microorganisms. The diet, especially raw salad, is the major source: at a conservative estimate, each human ingests 1.2 x 106 kanamycin-resistant microorganisms daily. Previous concerns that the nptII protein may itself be toxic or active in human or other animal digestive systems have been effectively eliminated by the work of Fuchs et al. (1993). The existence of the streptomycin/spectinomycin resistance gene on transposons and plasmids found in both gram positive and gram negative bacteria indicates its extensive distribution through the microbial world (Shaw et al., 1993). Although this particular mechanism of resistance does not occur in mycobacteria, resistance to streptomycin and spectinomycin as a result of spontaneous mutations in genes encoding ribosomal RNA occurs at a relatively high frequency because, unlike the enteric microorganisms, mycobacteria contain only a single copy of such genes. In summary, the incidence of naturally occurring bacterial strains resistant to the antibiotics in question is already very high, and the antibiotic resistance genes in these bacteria are often located on transmissible genetic elements that are readily transferable between bacterial species. So, in the unlikely event that the aad or nptII genes were transferred from Bollgard II cotton to a bacterium, this would be unlikely to have any detectable impact on the existing level of resistance in microbial populations. Furthermore, the antibiotics in question are not of major clinical or veterinary significance. iii) GUS marker gene: There would be no adverse consequences even if gene transfer occurred. iv) Herbicide tolerance gene: There would be no adverse consequences even if gene transfer occurred. v) CaMV 35S promoter and other regulatory sequences: As discussed above, horizontal gene transfer from plants to microorganisms or to animals and humans is extremely unlikely. Because recombination between viral sequences and plant genes has only been observed at very low levels, the probability of recombination of the CaMV 35S or CmoVb 34S promoter sequences in the modified cotton with other viruses can be considered to be negligible. While Ho et al. (2000) have postulated that there are risks posed through recombination of the CaMV 35S promoter with the genomes of other viruses infecting the plants to create new viruses, or of integration of the CaMV 35S promoter into other species causing mutations, cancer or reactivation of dormant viruses, these claims have been 63 challenged in the scientific literature (eg Hodgson, 2000 a,b). CaMV is already ubiquitous in the environment and in the human diet and the CaMV 35S promoter is expressed at far higher levels in naturally infected plants than in transgenic plants. 5.5.6 A: Insecticide resistance Nature of the insecticide resistance hazard Extensive cultivation of Bollgard II® cotton could potentially result in the emergence of resistance in target species (Helicoverpa armigera and H. punctigera and other susceptible lepidopteran species feeding on cotton) to the Cry1Ac and Cry2Ab proteins, reducing the efficacy of INGARD® and Bollgard II® cotton for control of insect pests. If this occurred, and resulted in increased pesticide use, there could be adverse effects on the environment and human health. It should be noted that these risks relate to issues of insecticide use in agricultural systems, and as such are not unique to the genetic risk associated with cultivation of INGARD® or Bollgard II® cotton. Although the GTR has given detailed consideration to these risks, any management requirements currently need to be addressed through the NRA or agricultural agencies. B: Likelihood of the insecticide resistance hazard occurring Bollgard II® cotton was developed with the intention of reducing the risk of insecticide resistance developing in the target pests. Ecological modelling shows that the extra gene is likely to delay selection of insects resistant to the insecticidal proteins by a factor of 10 compared to INGARD® cotton (Roush, 1994). The likelihood of insecticide resistance arising as a result of the proposed limited release is very low due to the limited scope of the release, both in area and in time. However, there is a significant likelihood that selection of resistance of Helicoverpa species to the Bt proteins in INGARD® or Bollgard II® cotton crops will eventually occur. Several studies have shown that resistance to Bt can be selected in the laboratory (Tabashnik et al., 1990; Peferoen, 1997). For example, two laboratory strains of Heliothis virescens were selected to become resistant to Cry1Ac and other Bt derived toxins (Gould et al., 1995), and Akhurst et al., (2000) have selected a laboratory strain of the Australian Helicoverpa armigera that is resistant to Cry1Ac. Moar et al. (1995) have selected strains of on Spodoptera exigua resistant to Cry1C and these insects were cross-resistant to Cry1Ab, Cry9C and Cry2A as well as to a recombinant Cry1E-Cry1C fusion protein. In this latter case, no major differences in toxin binding between the susceptible and the resistant insects were observed. Bt resistance in the field has also been demonstrated by studies of the diamondback moth, Plutella xylostella (Tabashnik et al., 1990). The diamondback moth is a major pest of cruciferous vegetables around the world, receives frequent exposure to insecticides, and shows extensive resistance to most insecticides in many growing areas. High levels of resistance to Cry1A toxins have been found in populations of the diamondback moth from the Philippines, Hawaii, Florida and Asia (Tabashnik, et al., 1990; 1994a). 64 Resistance to Bt insecticide appears to be due to one (Tang et al., 1997) or at most a few genes (Tabashnik et al., 1992; 1998; Ferre et al., 1995, Gould et al., 1995,). The mechanisms of insecticidal activity include reduced binding of the toxin to the midgut (Tabashnik et al., 1994b; Gould et al., 1995, Tang et al., 1996), slower interaction of gut proteinases with the protoxin, or the absence of a major gut protein (Oppert et al., 1997). Genetic crosses of the laboratory-selected insecticidal strains of Heliothis virescens demonstrated that a major portion of the resistance in this case was encoded by a single gene (or a set of linked genes) with mostly recessive inheritance (Gould et al., 1995). Studies of resistance in insects from field populations suggest that the common mode of resistance is characterised by a high level of resistance (over 500-fold), reduced toxin binding and a recessive mutation. However there appear to be other modes of resistance which are not recessive and are not associated with reduced toxin binding (Tabashnik et al. 1997; Moar et al., 1995). Reversal of resistance of laboratory strains of diamondback moth derived from resistant field populations has been observed when exposure to Bt insecticide was discontinued over or many generations. Reversal of resistance was associated with restoration of binding of Cry1Ac to brush-border membrane vesicles (Tabashnik et al., 1994b). Gould et al. (1997) estimated the frequency of alleles for resistance in field populations of H. virescens as 1.5 x 10-3. Genetic models indicate that a recessive allele present at this frequency could lead to rapid evolution of resistant populations if Bt toxin producing cotton is grown without adequate refuges for toxin-susceptible larvae (Roush, 1994; Gould et al., 1997). Frequencies of roughly 100 fold higher have been reported for a recessive allele conferring resistance to Cry1Ac toxin in pink bollworm in Arizona cotton fields. However, the frequency of this allele did not increase significantly between 1997 and 1999, even though the Bt cotton was grown in over half the 100 000 hectares planted to cotton, and the efficacy of the Bt cotton remained extremely high (Tabashnik et al., 2000). C: Conclusions regarding insecticide resistance Selection of insects resistant to the Cry1Ac and Cry2Ab protein would ® almost certainly occur if Bollgard cotton were grown widely without ForII Comment taking any stepsontothe deal with this problem. Given particularly the limitedas scope of the Your comments above risk assessment are sought, to whether there arerelease, any additional risks relating the potential resistance in proposed both in scale and to time, and thedevelopment presence ofoftwo target insects. insecticidal proteins, the likelihood of emergence of insects resistant to the Cry1Ac and Cry2Ab proteins, as a result of the trial, is considered minimal. It is therefore not proposed to implement an insecticide resistance management plan for the current release. 65 6. RISK MANAGEMENT PLAN This part of the document recaps the main conclusions from the risk assessment relating to risks to human health and safety or the environment, and details the risk management plan developed by the Regulator to manage these risks. 6.1 Summary of risk assessment conclusions It has been concluded that the proposed release of Bollgard II® cotton in Queensland, south of latitude 22S would not pose any additional risks to human health and safety or to the environment as a result of the genetic modification of the cotton. The main conclusions from the risk assessment are that: Bollgard II® and Bollgard II®/ Roundup Ready® cotton are not likely to prove more toxic or allergenic to humans or other organisms, other than some lepidopteran insects, than conventional cotton (lepidopteran insects are moths and butterflies); the risk of the Bollgard II® or Bollgard II®/ Roundup Ready® cotton establishing as a weed is low and not likely to be greater than that of conventional cotton; the potential for transfer of the introduced genes to non-GM cotton crops is negligible because it is being planted very late in the cotton growing season and no other cotton crops in the area will be flowering at the same time; the potential for transfer of the introduced genes to wild or native cotton is very low because of the geographical isolation and genetic incompatibility with the native species; the likelihood of transfer of the introduced genes to other organisms is low, but even if such transfer occurred would be unlikely to pose any hazard to human health and safety or the environment; and the risk of development of target insects resistant to the insecticidal proteins is very low, due to the limited scope of the proposed release and the presence of two insecticidal proteins. 6.2 6.2.1 Risk management plan Risk of toxicity or allergenicity On the basis of the risk assessment, with regard to the first of the risks identified in the risk assessment (Part 5.5.1), the potential toxicity or allergenicity of the cotton, it is not considered necessary to include any management strategies in the risk management plan at this stage. The risks are very low, and the scale of the release is relatively small, limiting any environmental exposure to the GMO. It is noted that the applicant proposes that products from this release will not be used in human food or animal feed, and conditions will be included in the licence, if issued, to restrict use of GM material from the release. 66 6.2.2 Risks of insecticide resistance The risk of development of resistance to either of the Cry1Ac and Cry2Ab proteins in target pests is negligible. Because the proposed release is limited in scope, it is not considered necessary to impose any specific licence conditions to manage this risk. 6.2.3 Risks of weediness or gene transfer It has been concluded that the risks relating to weediness or gene transfer are low and could be managed to an acceptable level by implementing various strategies to minimise the spread and persistence of Bollgard II® and Bollgard II®/Roundup Ready® cotton, or the modified genetic material, in the environment. It is therefore proposed that, if the Regulator decides to issue a licence for the release, the licence would include a number of specific conditions relating to risk management. The proposed conditions include requirements to isolate the cotton crop from other cotton by at least 50 metres, to undertake research to confirm the efficacy of the 50 metre isolation zone, to destroy any viable material not required for subsequent releases (subject to further approvals) after the harvest, and to monitor the release site after the release and remove cotton plants that regrow or sprout from seed remaining on the ground after harvest (volunteers). The proposed licence conditions, and the reason behind them, are set out in detail in Part 6.3. 6.2.4 General licence conditions In addition to the specific risk management conditions proposed in Part 6.3, any licence issued by the Regulator would also contain a number of general conditions including statutory conditions relating to requirements under Sections 61 to 65 of the Act. These conditions apply to all licences issued by the Regulator, and may also be relevant to risk management. For example, there are conditions that will: identify the persons or classes of person covered by the licence; specify the authorised dealings; and require the applicant to: - inform people covered by the licence of their obligations under the licence; - allow access to the release site by the Regulator, or persons authorised by the Regulator for the purposes of monitoring or auditing; - inform the Regulator if the applicant becomes aware of any additional information about risks to human health or safety or to the environment, any unintended effects of the release, or any contraventions of the licence conditions; and - ensure appropriate training for persons covered by the licence. 67 6.2.5 Monitoring and enforcement of compliance by the OGTR 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 releases to determine whether the licence holder is complying with the licence conditions, or whether there are any unforseen problems. 6.3 Proposed specific risk management licence conditions The proposed licence conditions set out below are intended to manage the identified risks, largely through preventing dissemination of the GMOs or their genetic material outside the release site. The proposed conditions also include contingency provisions to cover any unintended release of the GMOs outside the release site, and a requirement to gather data relating to the potential for pollen outcrossing. Qualified Cotton Seed Distributors (CSD) staff would be directly supervising the proposed release if a licence for the release is issued by the Regulator. CSD would be required, under licence conditions, to be proactive in reviewing and assessing any new information that comes to light about the risks and the efficacy of the proposed management strategies during the course of the release. Any licence issued to CSD will be able to be varied at any time to add new conditions, for instance to manage any new risks that are identified, or to improve the existing management strategies. The Regulator will also be proactive in reviewing any new information about risks of the proposed release and may amend licence conditions on the basis of this. Finally, it should be noted that, the Regulator is reviewing all licence conditions for licences carried over from the voluntary system under the transitional arrangements set out in the Act. If as a result of this review, new information becomes available about risks relevant to the proposed release, any licence issued to CSD would be amended if necessary. If the Regulator decides to issue a licence, the following conditions are proposed: Conditions to specify the scope of the trial Location and size of release site 1 2 3 The licence holder and a person covered by this licence may grow the GMOs only at one site, not exceeding 122 hectares in area, in the shire of Emerald in Queensland, the location of which has been notified to the Regulator in accordance with specific condition 2. Prior to commencing to grow the GMOs, the licence holder must notify the Regulator in writing of the location of the site (including the street address or directions, and the GPS coordinates of the site). The notification must also specify the areas of the site where different varieties of the GMOs are to be grown. The licence holder and a person covered by this licence must not grow the GMOs within 50 metres of a natural waterway. 68 Timing of release 4 The licence holder and a person covered by this licence must not plant the GMOs after 28 February 2002. Conditions to minimise gene flow to other cotton crops or native or feral cotton, and to prevent the GM cotton from spreading via waterways Isolation zone 5 6 The licence holder and a person covered by this licence must not grow other cotton within 50 metres of the GMOs. The licence holder must ensure that the isolation zone is able to be accessed in order for the isolation zone to be monitored in accordance with specific conditions 8 to 10, 19 and 20. Monitoring for native Gossypium species 7 8 9 The licence holder must ensure that the release site and the isolation zone are monitored, in accordance with specific condition 9, for wild Gossypium species. The monitoring mentioned in specific condition 8 must: (a) be undertaken by a person able to recognise those species; and (b) must be undertaken at least once every 30 days while the GMOs are flowering. The licence holder must ensure that, during the period when the GMOs are flowering, seed from all native Gossypium species flowering during the same period on the release site and in the isolation zone is harvested and tested for viability and for gene flow as part of the study described in specific conditions 10 to 12. Conditions to carry out research on the efficacy of the 50 metre isolation zone 10 11 12 The licence holder must conduct a study to provide information on the effectiveness of isolation zone in minimising gene flow from the GMOs to conventional cotton and native Gossypium species detected in the isolation zone in accordance with specific condition 8. The licence holder must notify the Regulator, in writing, of the details of the study including the names and qualifications of the researchers. The notification is to be given within 30 days of the commencement of this licence. The licence holder must provide the Regulator with (a) an initial written report of the results of the study on gene flow within 6 months, and a final written report within 12 months, of the date of commencement of this licence. (b) a written report on gene flow to any native Gossypium species detected during monitoring in accordance with specific condition 7 within 6 months of harvesting of the GMOs. 69 Procedures to be taken after the harvest to ensure that the GMOs are segregated from other cotton during ginning, and that harvested material or other GM material from the GMOs remaining on the release site is disposed of appropriately, to prevent continued dissemination of the GMOs in the environment 13 14 15 16 17 The licence holder must ensure that all cotton harvested from the GMOs is ginned separately from other cotton. After ginning, the licence holder or a person covered by this licence must only store seeds harvested from the GMOs in a sealed and locked facility that is marked to indicate that it contains genetically modified cotton seeds. If, after ginning, the seeds harvested from the GMOs are not stored in accordance with specific condition 14, the licence holder and a person covered by this licence must destroy the seeds by burning as soon as reasonably practicable. The licence holder must, within 14 days of harvesting, destroy, or arrange for the destruction of, any parts of the GMOs or whole GMOs remaining on the release site after harvesting, by either stalk pulling, cultivation, burning or herbicide treatment or a combination of these methods. On the request of the Regulator, the licence holder must provide to the Regulator written documentation of the procedures in place for cleaning the gin after ginning of cotton harvested from the GMOs and ensuring that there is no remaining seed or other viable GM material from the GMOs. Procedures to be undertaken after the release for monitoring and destruction of volunteers, to prevent the persistence of the GMO in the environment Post-harvest monitoring 18 19 20 21 The licence holder must ensure that, after harvesting of the GMOs, the release site, the isolation zone and any areas used for cleaning equipment that was used in relation to dealings with the GMOs and GM material from the GMOs are comprehensively and thoroughly monitored for volunteer plants in accordance with specific condition 19. The monitoring mentioned in specific condition 11 must be undertaken: (a) by a person able to recognise volunteer plants; and (b) at least once every 3 months for a period of at least 12 months following the harvest. The licence holder must, within 14 days of the monitoring, provide a written report of the results of the monitoring to the Regulator. The report mentioned in specific condition 20 must include: (a) the names of the a person who undertook the monitoring and details of the experience, training or qualification that enabled them to recognise volunteer plants; (b) the number of volunteer plants observed; (c) details of the development stage reached by any volunteer plants; and (d) details of the methods used to destroy any volunteer plants. Destruction of volunteer plants 22 During the 12 month period of monitoring referred to in specific condition 19, the licence holder must take all reasonable steps to ensure that any volunteer plants in the 70 release site and isolation zone are destroyed, prior to seed set, by cultivation, herbicide treatment, slashing/mowing, burning, or hand weeding. Restrictions to be placed on the use of release site following harvest, to ensure that post-harvest monitoring and destruction of volunteers can be carried out effectively 23 24 25 The licence holder or a person covered by this licence must not grow, or cause or allow to be grown, cotton in the release site within 12 months after the harvest of the GMOs. The grower must ensure that, if any plant is grown in the release site, only the following plants are grown: (a) grass pastures; or (b) cereal crops; or (c) any other plant agreed in writing by the Regulator. If any plants are planted in the release site within 12 months after the harvest of the GMOs, the licence holder must, before planting, notify the Regulator in writing of the date of planting, the details of the type of plant planted and provide the Regulator with a management plan for the detection and destruction of volunteers in accordance with specific conditions 19 and 22. Restrictions to be placed on transport of the GMOs or GM material from the GMOs prevent any escape or dissemination outside the release site 26 27 28 29 30 The licence holder and a person covered by this licence must not transport, or cause or permit the transport of the GMOs or any viable GM material from the GMOs north of latitude 22 degrees south. The licence holder and a person covered by this licence must transport cotton modules containing the GMOs or GM material from the GMOs, only when the cotton modules are covered with a tarpaulin and in a sealed truck. The licence holder and a person covered by this licence must transport any GM material from the GMOs (other than cotton modules) only in a primary sealed container which is packed in a secondary unbreakable container. The cotton modules referred to in specific condition 27, and the primary and secondary containers referred to in special condition 28, must be labelled to indicate that they contain genetically modified cotton. The label must also include the telephone number of the licence holder and instructions to contact the licence holder in the event that a module or container is broken or misdirected. The licence holder must have in place accounting procedures to verify whether the same quantity of GMOs or GM material from the GMOs sent is delivered and must document routes, methods and procedures used for transporting GM material from the GMOs. Requirements for cleaning any equipment used for the release to prevent dissemination of the GMOs or GM material from the GMOs in the environment 31 The licence holder and a person covered by this licence must thoroughly clean any equipment (including harvesters, tents, storage equipment, transport equipment, ginning facility and clothing) used by them at the release site, in the isolation zone, or at the ginning site, in relation to the GMOs and GM material from the GMOs. 71 32 33 34 35 The cleaning must occur as soon as practicable following the use and, except in relation to transport equipment, must occur at the release site, in the isolation zone or at the ginning site. The licence holder and a person covered by this licence must destroy any GMOs or GM material from the GMOs found as a result of the cleaning. If transport equipment is cleaned outside of the release site, the isolation zone or the ginning site, the licence holder must ensure that the area in which the cleaning occurred is monitored for volunteer plants at least once every 3 months for a period of 12 months commencing on the date of the cleaning. On the request of the Regulator, the licence holder or a person covered by this licence must provide to the Regulator written documentation of the procedures in place for complying with specific conditions 31 to 34. Contingency plan to deal with inadvertent release of GM material outside the approved area 36 37 38 Within 30 days of the date of the commencement of this licence, the licence holder must provide a written contingency plan to the Regulator detailing measures to be taken in the event of the unintended presence of the GMOs or GM material from the GMOs outside the release site and the isolation zone. 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) to destroy any of the GMOs or GM material from the GMOs; (c) monitor and destroy any volunteer plants that may exist as a result of the event. The licence holder must implement the contingency plan in the event of the unintended presence of the GMOs of GM material from the GMOs outside the release site and isolation zone. Requirement for a compliance management plan, to ensure compliance with the licence conditions 39 Prior to the commencement of growing of the GMOs, the licence holder must provide a compliance management plan to the Regulator. The plan must describe in detail how the licence holder intends to ensure compliance with these conditions and document that compliance. Annual reporting 40 The licence holder must provide the Regulator with a written report within 90 days of each anniversary of this licence, in accordance with any guidelines issued by the Regulator in relation to annual reporting. 72 7. CONSIDERATION OF ISSUES RAISED IN PUBLIC SUBMISSIONS A summary of the issues raised in the 16 submissions received from the public is provided in Part 2.4.5 and in the Appendix. Most of the public submissions raised issues relating to potential risks to human health and safety or to the environment that have been dealt with in the preceding chapters. In broad terms, these risks related to: toxicity and allergenicity of the GM cotton (see Part 5.5.1); weediness of the GM cotton (see Part 5.5.2); transfer of genes from the GM cotton to other organisms (see Parts 5.5.3-5.5.5); selection for insects resistant to the insecticidal activity of the cotton (see Part 5.5.6). The points raised in submissions were considered carefully, and weighed against the body of current scientific information, in reaching the conclusions set out in this document. Many of the submissions also raised issues that related to matters that are the responsibility of other regulatory authorities, in particular: the use and safety of pesticides; and the labelling and safety of foods derived from GMOs. These are issues that are dealt with by the National Registration Authority for Agricultural and Veterinary Chemicals, and the Australia New Zealand Food Authority, respectively. Contact details for these organisations are provided in Part 2.2. Some of the public submissions also raised a number of broader issues that are outside the direct scope of the gene technology legislation and the risk assessment process (see Part 2.4.5) and so have not been considered here. The remainder of this section discusses some of the more general concerns raised about the application including: the adequacy of the application and the assessment process; compliance and monitoring provisions; and the need for research on biosafety risks. 7.1 Adequacy of the application and the assessment process A number of people expressed views about the assessment process and the adequacy and reliability of information provided by the applicant. In most cases, their concerns focussed on the potential risk of large-scale commercial release rather than on the risks specifically associated with the proposed limited and controlled release. 73 In considering these comments, it should be noted that the risk assessment framework used for the assessment compares favourably with risk assessment processes employed in other countries. The European Community, Canada, the United Kingdom, New Zealand and a range of other countries all adopt a risk-based approach to the assessment of GMOs for release into the environment. The approach adopted in Australia for the assessment of GMOs employs best practice risk assessment, as well as including a consultative process that is considerably more open and transparent than most other countries. All regulatory systems in Australia (including those for therapeutics, imports, agricultural and veterinary chemicals, and for industrial chemicals) involve the submission of data by the applicant. No regulatory system routinely conducts primary research to prepare or validate these data packages. Rather, the data packages are evaluated by relevant experts, and the data are confirmed or disputed through reviews of published data. In keeping with standard regulatory practice, the GTR in its assessment of the biosafety risks of the proposed release: critically evaluated the information provided by the applicant; considered the data against the results from previous releases of the GMO undertaken within Australia and overseen by GMAC; drew on information and concerns expressed in submissions from State and Territory Governments and other government agencies, including Environment Australia, non-government organisations and the general public; undertook a thorough review of current scientific knowledge and the scientific literature; obtained data from other regulatory agencies and international bodies; and sought additional information from the applicant. 7.2 Compliance and monitoring provisions The licence conditions applied to a release can be enforced, and compliance can and will be monitored. The Gene Technology Regulator has enforcement powers that will include the ability to direct a licence holder to take any steps deemed necessary to protect the health and safety of people or the environment, cancel or suspend approvals and seek injunctions. The legislation also provides for fines of up to $1.1 million per day for each breach of regulations by a corporation. In addition to the monitoring that the licence holder would be expected to carry out to meet the licence conditions (see Part 6.3), the Regulator, through the services of the OGTR, will also independently monitor sites where intentional releases are authorised. At least 20 % of all sites will be visited each year. In addition, regular reports which the licence holders are required to provide are assessed to identify any potential problems. 74 7.3 Research on biosafety risks Many submissions were concerned that insufficient research has been done, especially long-term or independent research on potential risks to human health and the environment associated with the release of GMOs. Concern was expressed that the research associated with releases of GMOs was almost exclusively focussed on agronomic issues and did not include the collection and analysis of data to establish the safety of the GMO. In considering these comments, the following points are noted: Considerable research has been done in Australia and overseas that is relevant to identifying the potential risks that Bollgard II® and Bollgard II®/Roundup Ready® cotton present to the environment. This includes limited and controlled releases conducted in Australia over the past eight years (see Parts 2.4, 2.5), and results from monitoring the commercial planting of INGARD® cotton since 1996 (see Part 2.6). Much of this research has been undertaken in collaboration with CSIRO, State agriculture departments and the Australian Cotton Cooperative Research Centre. For example, the OGTR is aware of on-going research on the toxicity of the GM cotton for non-target organisms, insecticide resistance management strategies, weediness, and the potential for outcrossing to native cotton. Many of the submissions also considered that the CSD release should be used to gain information about various risks to human health and safety or the environment. CSD will be required as a condition of the licence to conduct research into the potential for gene flow outside a 50 metre isolation zone. However, it was not considered necessary to require CSD to obtain information on other risks, such as Further research into consequences of the release of Bollgard II® and Bollgard II® /Roundup Ready® cotton on the environment will be required as part of the proposed release and future releases. For the current release, it is proposed that the proponent be required to gather data to confirm the efficacy of the 50 metre isolation zone. Issues that will require further research to provide data for future commercial release applications have been identified during the assessment of the current application (see Part 5.3.4). The safety of GM foods is regulated by ANZFA (see Part 2.7). ANZFA considers that there is no reason to believe that the long-term safety of foods derived from Roundup Ready® or INGARD® cotton will be any less than for foods derived from conventional cotton. However, given the current level of public concern, Australia is participating in several international forums concerned with investigating the feasibility of monitoring GM foods in the marketplace for any long-term effects on human health. There is a significant body of scientific literature and knowledge relating to independent research on the biology of cotton (for example, the potential for weediness or for cross breeding with other plant species). There is also a considerable body of research on the genes and proteins that have been introduced into Bollgard II® cotton and Bollgard II®/Roundup Ready® cotton, and issues such as the potential for gene transfer between species, and the spread of antibiotic resistance. A thorough review of this research was undertaken as part of the risk analysis (see Part 5.2). 75 8. NEXT STEPS The Regulator will make the final decision on whether to issue a licence for the proposed release and on what licence conditions should be included. Before making a decision to issue a licence, the Regulator must: be 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 (Section 56 of the Act); have regard to the risk assessment and risk management plan, and any submissions received under Section 52 of the Act (Section 56 of the Act); and be satisfied that the Cotton Seed Distributors is a suitable person to hold the licence (Sections 57 and 58 of the Act). In deciding whether a person is suitable to hold a licence, the Regulator must have regard to: - any relevant conviction of the body corporate; and - if there is a relevant conviction: whether the offence was committed at a time when any person who is presently a direct of the body corporate was a director; and whether the offence was committed at a time when any officer or shareholder of the body corporate who is presently in a position to influence the management of the body corporate was such an officer or shareholder; and - any revocation or suspension of a licence or permit held under a law relating to the health and safety of people or the environment; and - the capacity of the body corporate to meet the conditions of the licence. The Regulator may set licence conditions to control aspects of the release so as to manage any risks the health and safety of people and the environment. The proposed conditions are detailed in Part 6.3. If the Regulator decides to issue a licence for the release: the Regulator will notify the applicant in writing of the Regulator’s decision as to whether or not to issue a licence, including any conditions imposed; the finalised risk assessment and risk management plan, and summary information will be placed on the OGTR web site; a copy of the licence with details of the GMO, the proposed dealings and the licence conditions will be place on the OGTR web site as part of the Record of GMO and GM product dealings (Section 138 of the Act); and the Regulator will provide written responses to all submissions received during the consultation on the risk assessment and risk management plan. 76 8. BIBLIOGRAPHY 1. Adang, M.J., Staver, M.J., Rocheleau, T. A., Leighton, J., Barker, R. F. and 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. 2. 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