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Risk Assessment andRisk Management Plan for

DIR 136

Limited and controlled release of cotton genetically modified for enhanced fibre quality

Applicant: CSIRO

September 2015

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DIR 136 – Risk Assessment and Risk Management Plan (September 2015) Office of the Gene Technology Regulator

Summary of the Risk Assessment and Risk Management Plan

forLicence Application No. DIR 136

DecisionThe Gene Technology Regulator (the Regulator) has decided to issue a licence for this application for a limited and controlled release (field trial) of a genetically modified organism (GMO) into the environment. A Risk Assessment and Risk Management Plan (RARMP) for this application was prepared by the Regulator in accordance with the requirements of the Gene Technology Act 2000 (the Act) and corresponding state and territory legislation, and finalised following consultation with a wide range of experts, agencies and authorities, and the public. The RARMP concludes that this field trial poses negligible risks to human health and safety and the environment and that any risks posed by the dealings can be managed by imposing conditions on the release.

The applicationApplication number DIR 136

Applicant CSIRO

Project title Limited and controlled release of cotton genetically modified for enhanced fibre quality

Parent organism Cotton (Gossypium hirsutum L.)

Introduced genes and modified traits

Partial and full PME3 (pectin methylesterase enzyme) gene from cotton – altered fibre quality

PME4 (pectin methylesterase enzyme) gene from cotton – enhanced fibre quality

PME5 (pectin methylesterase enzyme) gene from cotton – enhanced fibre quality

GAUT1 (galacturonyltransferase) gene from cotton – enhanced fibre quality

nptII gene from Escherichia coli – antibiotic resistance selectable marker

Proposed location One site at the Australian Cotton Research Institute, Narrabri, NSW

Proposed release size Up to 1 hectare (ha) per year

Proposed release dates October 2016 – May 2019†

Primary purpose To assess the agronomic performance and fibre quality of the genetically modified (GM) cotton lines under field conditions

CSIRO proposes to conduct a field trial of GM cotton lines‡ containing introduced cotton genes, or a fragment of a cotton gene, designed to alter pectin profiles in the cotton fibre to enhance fibre quality.

† The proposed release dates have been changed from October 2015 – May 2018 indicated in the consultation RARMP, following a request by the applicant noting that water restrictions will delay the trial.‡ The term ‘line’ is used to denote plants derived from a single plant containing a specific genetic modification resulting from a single transformation event.

Summary I


Risk assessmentThe risk assessment concludes that there are negligible risks to the health and safety of people, or the environment, from the proposed release.

The risk assessment process considers how the genetic modifications and proposed activities conducted with the GMOs might lead to harm to people or the environment. Risks were characterised in relation to both the seriousness and likelihood of harm, taking into account current scientific/technical knowledge, information in the application (including proposed limits and controls), relevant previous approvals and advice received from a wide range of experts, agencies and authorities consulted on the RARMP. Both the short and long term impact were considered.

Credible pathways to potential harm that were considered included exposure to the GM plant material, dispersal of GM seed leading to spread and persistence of the GMOs, and transfer of the introduced genetic material to sexually compatible cotton plants. Potential harms associated with these pathways included toxicity or allergenicity to people, toxicity to other desirable organisms, and environmental harms due to weediness.

The principal reasons for the conclusion of negligible risks are that the proposed limits and controls effectively contain the GMOs and their genetic material and minimise exposure; the introduced genetic modifications are unlikely to cause harm to people or the environment; and the introduced genes are common in the environment.

Risk management planThe risk management plan describes measures to protect the health and safety of people and to protect the environment by controlling or mitigating risk. The risk management plan is given effect through licence conditions.

As the level of risk is considered negligible, specific risk treatment is not required. However, since this is a limited and controlled release, the licence includes limits on the size, location and duration of the release, as well as controls to prohibit the use of GM plant material in human food or animal feed, to minimise dispersal of GM seed or GM pollen from trial sites, to transport GMOs in accordance with the Regulator’s guidelines, to destroy GMOs not required for testing or further planting, and to conduct post-harvest monitoring at the trial site to ensure all GMOs are destroyed.

Summary II


Table of ContentsSUMMARY OF THE RISK ASSESSMENT AND RISK MANAGEMENT PLAN....................................................I

DECISION ..................................................................................................................................................................ITHE APPLICATION............................................................................................................................................................IRISK ASSESSMENT..........................................................................................................................................................IIRISK MANAGEMENT PLAN..............................................................................................................................................II

TABLE OF CONTENTS................................................................................................................................................III

ABBREVIATIONS..........................................................................................................................................................IV

CHAPTER 1 RISK ASSESSMENT CONTEXT........................................................................................................1

SECTION 1 BACKGROUND...........................................................................................................................................1SECTION 2 REGULATORY FRAMEWORK......................................................................................................................1SECTION 3 THE PROPOSED DEALINGS.........................................................................................................................2

3.1 The proposed limits of the dealings (duration, size, location and people)...............................................23.2 The proposed controls to restrict the spread and persistence of the GMOs in the environment..............2

SECTION 4 THE PARENT ORGANISM............................................................................................................................3SECTION 5 THE GMOS, NATURE AND EFFECT OF THE GENETIC MODIFICATION........................................................3

5.1 Introduction to the GMOs.........................................................................................................................35.2 The introduced genes, encoded proteins and their associated effects......................................................45.3 Toxicity/allergenicity of the proteins associated with the introduced genes............................................65.4 Characterisation of the GMO...................................................................................................................7

SECTION 6 THE RECEIVING ENVIRONMENT................................................................................................................86.1 Relevant abiotic factors............................................................................................................................86.2 Relevant agricultural practices.................................................................................................................86.3 Presence of related plants in the receiving environment..........................................................................86.4 Presence of similar genes and encoded proteins in the environment.......................................................9

SECTION 7 RELEVANT AUSTRALIAN AND INTERNATIONAL APPROVALS....................................................................97.1 Australian approvals.................................................................................................................................97.2 International approvals.............................................................................................................................9

CHAPTER 2 RISK ASSESSMENT...........................................................................................................................10

SECTION 1 INTRODUCTION.......................................................................................................................................10SECTION 2 RISK IDENTIFICATION.............................................................................................................................11

2.1 Risk source.............................................................................................................................................112.2 Causal pathway.......................................................................................................................................122.3 Potential harm.........................................................................................................................................132.4 Postulated risk scenarios.........................................................................................................................13

SECTION 3 UNCERTAINTY........................................................................................................................................21SECTION 4 RISK EVALUATION..................................................................................................................................21

CHAPTER 3 RISK MANAGEMENT PLAN...........................................................................................................23

SECTION 1 BACKGROUND.........................................................................................................................................23SECTION 2 RISK TREATMENT MEASURES FOR SUBSTANTIVE RISKS.........................................................................23SECTION 3 GENERAL RISK MANAGEMENT................................................................................................................23

3.1 Licence conditions to limit and control the release................................................................................233.2 Other risk management considerations...................................................................................................26

SECTION 4 ISSUES TO BE ADDRESSED FOR FUTURE RELEASES.................................................................................27SECTION 5 CONCLUSIONS OF THE CONSULTATION RARMP....................................................................................27

REFERENCES ................................................................................................................................................................29

APPENDIX A SUMMARY OF SUBMISSIONS FROM PRESCRIBED EXPERTS, AGENCIES AND AUTHORITIES....................................................................................................................................33

APPENDIX B SUMMARY OF SUBMISSIONS FROM THE PUBLIC.................................................................36

Table of Contents


AbbreviationsAPVMA Australian Pesticides and Veterinary Medicines AuthorityCSIRO Commonwealth Scientific and Industrial Research OrganisationDIR Dealings involving Intentional ReleaseDNA Deoxyribonucleic aciddpa Days post anthesisFSANZ Food Standards Australia New ZealandGalA Galacturonic acidGAUT1 GalacturonyltransferaseGM Genetically modifiedGMO Genetically modified organismha HectareHG HomogalacturonanHGT Horizontal gene transferkm Kilometresm MetresNLRD Notifiable Low Risk DealingNPTII Neomycin phosphotransferase type IINSW New South WalesOGTR Office of the Gene Technology RegulatorPC2 Physical Containment level 2PME Pectin methylesterasePMEI Pectin methylesterase inhibitorRARMP Risk Assessment and Risk Management PlanRegulations Gene Technology Regulations 2001Regulator Gene Technology RegulatorRNA Ribonucleic acidRNAi Ribonucleic acid interferencethe Act The Gene Technology Act 2000

Abbreviations IV

PROPOSED DEALINGSProposed activities involving the GMOProposed limits of the releaseProposed control measures

PARENT ORGANISMOrigin and taxonomyCultivation and useBiological characterisationEcology

PREVIOUS RELEASES

GMOIntroduced or deleted genes (genotype)Novel traits (phenotype)

RISK ASSESSMENT CONTEXT

LEGISLATIVE REQUIREMENTS(including Gene Technology Act and Regulations)

RISK ANALYSIS FRAMEWORK

OGTR OPERATIONAL POLICIES AND GUIDELINES

RECEIVING ENVIRONMENTEnvironmental conditionsAgronomic practicesPresence of related speciesPresence of similar genes


Chapter 1 Risk assessment contextSection 1 Background

1. An application has been made under the Gene Technology Act 2000 (the Act) for Dealings involving the Intentional Release (DIR) of genetically modified organisms (GMOs) into the Australian environment.

2. The Act in conjunction with the Gene Technology Regulations 2001 (the Regulations), an inter-governmental agreement and corresponding legislation that is being enacted in each State and Territory, comprise Australia’s national regulatory system for gene technology. Its objective is to protect the health and safety of people, and to protect the environment, by identifying risks posed by or as a result of gene technology, and by managing those risks through regulating certain dealings with GMOs.

3. This chapter describes the parameters within which potential risks to the health and safety of people or the environment posed by the proposed release are assessed. The risk assessment context is established within the regulatory framework and considers application-specific parameters (Figure 1).

Figure 1. Summary of parameters used to establish the risk assessment context

Section 2 Regulatory framework4. Sections 50, 50A and 51 of the Act outline the matters which the Gene Technology Regulator (the Regulator) must take into account, and the consultation required when preparing the Risk Assessment and Risk Management Plans (RARMPs) that inform the decisions on licence applications. In addition, the Regulations outline further matters the Regulator must consider when preparing a RARMP. In accordance with section 50A of the Act, this application is considered to be a limited and controlled release application, as its principal purpose is to enable the applicant to conduct experiments and the applicant has proposed limits on the size, location and duration of the release, as well as controls to restrict the spread and persistence of the GMOs and their genetic material in the environment. Therefore, the

Chapter 1 – Risk assessment context 1


Regulator was not required to consult with prescribed experts, agencies and authorities before preparation of the Risk Assessment and Risk Management Plan.

5. Section 52 of the Act requires the Regulator to seek comment on the RARMP from the States and Territories, the Gene Technology Technical Advisory Committee, Commonwealth authorities or agencies prescribed in the Regulations, the Minister for the Environment, relevant local council(s), and the public.

6. The Risk Analysis Framework (OGTR 2013a) explains the Regulator’s approach to the preparation of RARMPs in accordance with the legislative requirements. Additionally, there are a number of operational policies and guidelines developed by the Office of the Gene Technology Regulator (OGTR) that are relevant to DIR licences. These documents are available from the OGTR website.

7. Any dealings conducted under a licence issued by the Regulator may also be subject to regulation by other Australian government agencies that regulate GMOs or GM products, including Food Standards Australia New Zealand (FSANZ), the Australian Pesticides and Veterinary Medicines Authority (APVMA), the Therapeutic Goods Administration, the National Industrial Chemicals Notification and Assessment Scheme and the Department of Agriculture. These dealings may also be subject to the operation of State legislation declaring areas to be GM, GM free, or both, for marketing purposes.

Section 3 The proposed dealings8. CSIRO proposes to release up to 45 lines of genetically modified (GM) cotton into the environment under limited and controlled conditions. The purpose of the release is to evaluate the agronomic performance of the GM cotton under Australian field conditions, particularly the fibre quality characteristics.

9. The dealings involved in the proposed intentional release include:

conducting experiments with the GMOs

breeding the GMOs

propagating the GMOs

using the GMOs in the course of manufacture of a thing that is not a GMO

growing the GMOs

transporting the GMOs

disposing of the GMOs

possession, supply or use of the GMOs for any of the purposes above.

10. These dealings are detailed further below.

3.1 The proposed limits of the dealings (duration, size, location and people)11. The release is proposed to take place at one site adjacent to the Australian Cotton Research Institute in Narrabri, New South Wales on a maximum area of 1 hectare per year between October 2016 and May 2019.

12. Only trained and authorised staff would be permitted to deal with the GM cotton.

3.2 The proposed controls to restrict the spread and persistence of the GMOs in the environment13. The applicant has proposed a number of controls to restrict the spread and persistence of the GM cotton and the introduced genetic material in the environment. These include:



locating the trial site at a location not prone to flooding

separating GMOs from other cotton crops by a 20 m wide pollen trap of non-GM cotton

locating the outer edge of the pollen trap at least 50 m away from cotton lines used for breeding purposes

inspecting and cleaning all planting and harvest equipment used at the trial site before using for other purposes

cleaning the trial site after harvest

post-harvest monitoring of the trial site for at least 12 months and destroying any volunteer cotton until the site has been free of volunteers for six months

destroying all plant material from the trial not required for analysis or further experimentation

transporting and storing the GMOs in accordance with the current Regulator’s Guidelines for the Transport, Storage and Disposal of GMOs

not allowing plant material from the GMOs to be used in human food or animal feed.

Section 4 The parent organism14. The parent organism is upland cotton (Gossypium hirsutum L.), which is the most commonly cultivated cotton species worldwide. Cotton is exotic to Australia and is grown as an agricultural crop in New South Wales and Queensland, with occasional trial or small-scale cultivation in Victoria, northern Western Australia and in the Northern Territory. In the 2013-14 growing season more than 99% of the Australian cotton crop was GM, with introduced insect resistance and/or herbicide tolerance GM traits (Roth 2014).

15. Cotton is grown as a source of textile and industrial fibre, cottonseed oil for food use, and cottonseed meal for animal feed. Detailed information about the parent organism is contained in a reference document, The Biology of Gossypium hirsutum L. and Gossypium barbadense L. (cotton), which was produced to inform the risk assessment process for licence applications involving GM cotton plants (OGTR 2013b). The document is available from the OGTR website or on request from the OGTR.

16. G. hirsutum cultivar Coker 315 has been used to develop the GM cotton lines proposed for release. This cultivar has been adapted to tissue culture conditions and can be readily modified by standard techniques. However, it is not commercially grown in Australia.

Section 5 The GMOs, nature and effect of the genetic modification

5.1 Introduction to the GMOs17. The applicant proposes to release up to seven categories of cotton genetically modified for altered fibre quality (Table 1). Categories 1-2 and 4-6 have enhanced expression of one of the four introduced cotton genes PME3, PME4, PME5 and GAUT1 for better fibre quality. Category 3 has reduced expression of the PME3 gene and shorter fibre. The last category is generated by crossing of the fibre-specific PME3 enhanced cotton (Category 1) with any of the constitutive PME4, PME5 or GAUT1 enhanced cotton lines (Categories 4, 5 and 6), producing ‘stacked’ cotton lines containing genetic modifications from both GM parents. In addition, all of the GM cotton lines contain an introduced nptII gene, encoding neomycin phosphotransferase type II, as a selectable marker. More detail on the introduced genes is provided in Table 1. Regulatory sequence elements have also been introduced to achieve appropriate expression of the introduced genes; these are described in Table 2.


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Table 1 GM cotton lines proposed for release

Category Description Introduced Gene*

Protein produced

Source Intended trait Max No of lines

1 Fibre- specific PME3 enhanced PME3 Pectin

methylesterase G. hirsutum Longer and finer lint 10

2 Constitutive PME3 enhanced PME3 Pectin

methylesterase G. hirsutum Longer and finer lint 5

3 PME3 Reduced PME3 partial None (RNAi) G. hirsutum Shorter lint 5

4 Constitutive PME4 Enhanced PME4 Pectin

methylesterase G. hirsutum Longer lint 5

5 Constitutive PME5 enhanced PME5 Pectin

methylesterase G. hirsutum Longer lint 5

6 Constitutive GAUT1 enhanced GAUT1 Galacturonyl-

transferase G. hirsutum Longer lint 5

7Category 1 combined with Categories 4, 5 or 6

PME3 +PME4 or PME5 or GAUT1

PME3 +PME4 or PME5 or GAUT1

G. hirsutum Longer and finer lint 10

* Note: all GM cotton plants also contain the nptII gene as a selectable marker.

Table 2 Regulatory sequences introduced into the GM cotton lines proposed for release

Element Description Function Source Used in (Category)

Exp1 α-expansin gene Gh Exp1 promoter Fibre-specific expression G. hirsutum 1, 7

S1 5’ Subclover stunt virus S1 gene promoter Constitutive expression Subclover stunt virus 1, 2, 3, 4, 5, 6, 7

S7 5’ Subclover stunt virus S7 gene promoter Constitutive expression Subclover stunt virus 2, 3, 4, 5, 6, 7

Intron 1 (PPDK)

First intron of pyruvate dikinase gene Enhanced expression Flaveria trinervia 3

Intron 1 (CAT1)

First intron of catalase 1 gene Enhanced expression Ricinus communis 3

ME 3’ NADP-malic enzyme gene terminator Transcription stop signal Flaveria bidentis 1, 2, 3, 4, 5, 6, 7

S3 3’ Subclover stunt virus S3 gene terminator Transcription stop signal Subclover stunt virus 1, 2, 3, 4, 5, 6, 7

18. All GM cotton lines were produced by Agrobacterium tumefaciens-mediated plant transformation. Information about this transformation method can be found in the document Methods of plant genetic modification available from the Risk Assessment References page on the OGTR website. Stacks between the GM cotton lines were produced by conventional cross-breeding.

5.2 The introduced genes, encoded proteins and their associated effects5.2.1 Pectin methylesterase (PME) genes

19. GM cotton plants in Categories 1, 2, 4 and 5 contain one of the three introduced cotton genes PME3, PME4 and PME5. Cotton plants already contain each of these genes. However, introduction of a second copy of a gene is expected to increase the expression level of the protein encoded by that gene. The PME genes encode pectin methylesterases, which catalyse the de-methylesterification of pectins by converting methoxyl groups into carboxyl groups and releasing methanol (Jolie et al. 2010).

20. Pectin is a component of all higher plant cell walls and has diverse functions in plant growth, morphology, development and plant defence (Mohnen 2008; Pelloux et al. 2007). The pectin family is a large group of complex polysaccharides mainly consisting of homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) (Ochoa-Villarreal et al. 2012). HG is the most abundant homopolymer of the pectins,



comprising over 60% of pectins in plant cell walls and is formed by long chains of linear 1,4-linked α-D-galacturonic acid (GalA) (Mohnen 2008). HG is synthesised in the Golgi complex and secreted to the cell wall in a form with a high level of methyl-esterification (Ochoa-Villarreal et al. 2012). The methylesters are subsequently de-esterified by wall-localised PMEs, leading to free carboxyl groups and the release of methanol and protons (Wolf et al. 2009). De-esterification of HGs can lead to both cell wall stiffening as well as to enzymatic degradation of pectin. Thus, the methyl esterification status of HGs is important for the physical properties of the cell wall and for regulating cell expansion in plants (Held et al. 2011).

21. PME enzymes in plants play important roles in cell extension and pollen tube growth (Bosch & Hepler 2005), embryo development (Levesque-Tremblay et al. 2015), seed germination (Müller et al. 2013) and fibre development (Pinzon-Latorre & Deyholos 2014). The level of PME activity has also been shown to be positively correlated to dormancy breakage of yellow cedar (Cupressus nootkatensis) seeds (Ren & Kermode 2000). PME activity is regulated in part by pectin methylesterase inhibitors (Jolie et al. 2010; Senechal et al. 2015; Wang et al. 2013).

22. Cotton fibres are highly elongated single-celled seed trichomes that initiate from the seed coat. The walls of elongating fibre cells are bilayered with the outer layer enriched in de-esterified pectins and the inner layer enriched in xyloglucans and cellulose. Like other dicot plants, cotton has a large PME gene family but only a few are expressed in cotton fibre. PME3, PME4 and PME5 are the major fibre-expressed PMEs in cotton (Liu et al. 2013). It has been suggested that pectin methylesterases play an important role in cotton fibre elongation (Pelloux et al. 2007) and fibre quality (Al-Ghazi et al. 2009).

23. According to Liu et al. (2013), PME3 is normally expressed during the fibre elongation stage between 5 and 15 days post anthesis§ (dpa). PME4 shares 78% identity with PME3 at the amino acid level and is normally expressed at low levels during later fibre elongation stages of around 15-20 dpa. PME5 is phylogenetically more distant from PME3 and PME4, and shares only 42% and 43% identity with PME3 and PME4 at the amino acid level, respectively. It is normally expressed later in fibre development beyond 20 dpa.

24. GM cotton containing the introduced cotton PME genes are expected to express a higher level of cell-wall located pectin methylesterases that will affect the expansion of fibre cell walls and change the final fibre lengths or thickness.5.2.2 The PME3 RNAi

25. All Category 3 GM cotton plants contain an RNAi (ribonucleic acid interference) construct containing fragments of PME3 gene. The construct acts to suppress expression of the endogenous PME3 gene by a natural regulatory mechanism known as RNAi or gene silencing (Baykal & Zhang 2010). The mechanism for silencing of genes by RNAi was discussed in detail in the RARMPs prepared for DIR 112 and DIR 117. The RNAi construct contains a fragment of the PME3 gene repeated in both sense and antisense orientation, separated by two plant gene introns. Following transcription, the self-complementary regions of mRNA anneal and the introns are spliced out, resulting in double-stranded RNA. This is processed by endogenous cellular RNAi machinery into PME3-specific short interfering RNAs (siRNAs). The siRNAs then direct the degradation of messenger RNA (mRNA) molecules with matching sequence, in this case mRNAs transcribed from the endogenous PME3 gene, before they are translated into proteins. The effect of suppressing expression of PME3 is to reduce the total PME3 activity in elongating fibres, leading to higher levels of methyl esterified pectin in fibre cell walls.

26. The efficiency of gene silencing is generally determined by the extent of homology between the silencing construct and the target gene (usually > 95% homology is required) and

§ Anthesis is the period during which a flower is fully open and functional.


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the length of the homologous region. In plants, introduced silencing constructs have been shown to effectively suppress expression of the target genes but can also give rise to silencing of non-target genes with closely matching sequences (Baykal & Zhang 2010).5.2.3 The GAUT1 gene

27. GM cotton plants in Category 6 contain an introduced cotton GAUT1 (GhGAUT1) gene coding for the pectin synthesis enzyme galacturonyltransferase (GalAT). This protein synthesises the most abundant pectic polysaccharide HG (Atmodjo et al. 2011). GalAT catalyses transfer of GalA from uridine-diphosphate-GalA (UDP-GalA) onto the non-reducing end of polysaccharide and oligosaccharide acceptors to produce polymeric HG (Doong & Mohnen 1998).

28. The GAUT1 gene was initially identified in Arabidopsis (Sterling et al. 2006). It belongs to the carbohydrate-active enzyme glycosyltransferase family 8 (GT8) (Yin et al. 2010). In Arabidopsis, GAUT1 is a member of the GAUT1-related gene family consisting of 15 GAUT and 10 GAUT-like genes, which are all involved in the biosynthesis of pectins (Atmodjo et al. 2011). A GAUT1 gene has been isolated from Gossypium barbadense (Chi et al. 2009). This gene is expressed in the fibres with peak levels at 35 to 40 dpa. It is also expressed in hypocotyls and leaves, but not in roots.

29. It has been proposed that there is a positive correlation between pectin synthesis and fibre elongation (Blamey 2003; Rajasundaram et al. 2014). In cotton, preliminary results showed that GM plants with over-expression of the GhGAUT1gene under the control of the constitutive S7 promoter from the subclover stunt virus display higher levels of GAUT protein in leaf tissues and early fibre elongation compared to control plants (information provided by the applicant).5.2.4 The nptII gene

30. The nptII (also denoted aph(3’)-II) gene was isolated from the common gut bacterium Escherichia coli and encodes neomycin (or aminoglycoside) phosphotransferase type II, which inactivates aminoglycoside antibiotics such as kanamycin and neomycin. The nptII gene is used extensively as a selectable marker in the production of GM plants. Further information about this gene can be found in the document Marker genes in GM plants available from the Risk Assessment References page on the OGTR website.

5.3 Toxicity/allergenicity of the proteins associated with the introduced genes31. All of the four genes (PME3, PME4, PME5 and GAUT1) used to enhance fibre quality in the GM cottons are endogenous to cultivated cotton (G. hirsutum). Therefore, humans and other organisms have a long history of exposure to these genes and their encoded proteins.

32. Endogenously present plant PMEs can positively or negatively affect the structural quality of plant-based foods (cloud stability, viscosity, texture, etc). In the food industry, PME (purified from fungi or other sources) is used as a processing aid in fruit and vegetable juices (Jolie et al. 2010). PMEs are not known to be toxic. The GAUT1 protein is an important enzyme in the pectin synthesis pathways with no reported toxicity. The most well-known plant proteins with toxic properties are lectins and ribosome-inactivating peptides (RIPs) (Wu & Sun 2011), and none of the introduced proteins fall into these categories. In addition, a sequence similarity search of the introduced proteins against the Toxin and Toxin Target Database (T3DB) (Wishart et al. 2015) showed that none of the introduced proteins share sequence similarities with known protein toxins.

33. Some plant PMEs are known allergens. PMEs in kiwi fruit (Actinidia deliciosa) and olive tree (Olea europaea) have been identified as allergens (Bublin 2013; Popovic et al. 2013; Salamanca et al. 2010). Potential identities between each introduced PME protein and allergenic proteins listed in the AllergenOnline database from the Food Allergy Research and


http://www.allergenonline.org/

http://www.t3db.ca/

http://www.t3db.ca/

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Resource Program (FARRP), University of Nebraska (Version 15, updated January 2015) were evaluated using the FASTA sequence alignment program (Pearson & Lipman 1988). It revealed that the three cotton PME proteins all showed more than 35% sequence matches over 80 amino acids, which is the threshold for possible cross-reactivity (Codex Alimentarius Commission 2003; FAO 2001; Goodman 2006; Goodman et al. 2005), to allergenic pectin methylesterases from kiwi fruit (gi160419153) and olive tree (isoforms: gi269996495 and gi68270856). This indicates that the introduced PME proteins potentially share immunological properties with protein allergens.

34. A similar search conducted for the GAUT1 protein did not yield any sequence matches with known allergens, indicating a lack of immunological relevant similarities between this protein and known allergens.

35. Insertion of the PME3 gene fragment as part of a gene silencing construct does not result in expression of a novel protein, but in suppression of the expression of endogenous cotton PME3 protein. This is unlikely to lead to increased toxicity or allergenicity (see more discussion in Risk Scenario 2).

36. No studies on the toxicity or allergenicity of the GM cotton plants and their products have been undertaken to date as the proposed trial is at an early stage.

37. The antibiotic selectable marker gene nptII has previously been assessed by regulatory agencies in Australia and in other countries as not posing a risk to human or animal health or to the environment. FSANZ has approved food derived from GM cotton and GM corn expressing the NPTII protein as safe for human consumption (FSANZ 2002; FSANZ 2003; FSANZ 2010). Further information on risk assessment of this gene can be found in the document Marker genes in GM plants available from the Risk Assessment References page on the OGTR website.

5.4 Characterisation of the GMO5.4.1 Phenotypic characterisation

38. The applicant has carried out a preliminary study, under greenhouse conditions, of the GM cotton lines proposed for release. The applicant has provided information on observed phenotypes of the GM cotton plants (below).

39. GM cotton plants in Categories 1 and 2 (enhanced expression of PME3) showed longer and finer fibres when grown under glasshouse conditions. No other effects on the phenotype of Category 1 plants (fibre-specific enhanced PME3) were observed. However, GM cotton plants with constitutively expressed PME3 (Category 2) were observed to have elongated internodes and to be taller than non-GM cotton plants when grown in the glasshouse.

40. GM cotton plants in Category 4 with constitutive expression of PME4 were taller than a non-GM cotton control. They showed longer but not finer fibres when grown under glasshouse conditions. No other obvious changes to phenotype were observed.

41. GM cotton plants in Category 5 (constitutive expression of PME5) and Category 6 (constitutive expression of GAUT1) showed longer but not finer fibres. No other effects on the plant phenotype were observed.

42. GM cotton plants in Category 3 containing the PME3 RNAi construct had a reduced PME3 transcription level, reduced total PME enzyme activity in elongating fibres, higher levels of methyl esterified pectin in the cell walls and shorter fibres. No other obvious differences in phenotype were observed in comparison to the control non-GM cotton.

43. Further phenotypic data would be collected during the proposed field trials.



http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=68270856




Section 6 The receiving environment44. The receiving environment includes: any relevant biotic/abiotic properties of the geographic regions where the field trial would occur; intended agricultural practices, including those that may be altered in relation to normal practices; other relevant GMOs already released; and any particularly vulnerable or susceptible entities that may be specifically affected by the proposed release (OGTR 2013a).

6.1 Relevant abiotic factors45. The abiotic factors relevant to the growth and distribution of commercial cotton in Australia are discussed in The Biology of Gossypium hirsutum L. and Gossypium barbadense L. (cotton) (OGTR 2013b). To summarise, factors restricting where cotton can be grown in Australia are water availability (through rainfall or irrigation), soil suitability and, most importantly, temperature. Cotton seedlings may be killed by frost, growth and development of cotton plants below 12°C is minimal, and a long, hot growing season is crucial for achieving good yields.

46. The release is proposed to take place at one site in Narrabri, NSW (Section 3.1), which has a temperate climate as defined by the Köppen Classification system used by the Australian Bureau of Meteorology.

47. The proposed site is over 2 km away from the nearest natural waterway and is not prone to flooding.

6.2 Relevant agricultural practices48. The limits and controls of the proposed release are outlined in Sections 3.1 and 3.2 of this Chapter. It is anticipated that the agronomic practices for the cultivation of the GM cotton by the applicant will not differ significantly from industry best practices used in Australia. The GMOs proposed for field release would be planted with a standard cone seeder in rows with 1 m spacing, and the pollen trap would be planted with a commercial seed planter. All cotton plants would be grown following standard cotton agricultural management practices and would receive applications of water, fertilisers, herbicides, and insecticides similar to commercially grown non-GM cotton crops. Conventional cultivation practices for cotton are discussed in more detail in The Biology of Gossypium hirsutum L. and Gossypium barbadense L. (cotton) (OGTR 2013b).

49. The GM cotton seed required for experimentation or future trials will be harvested either by hand or with modified commercial harvesting equipment used by CSIRO for small plot breeding experiments. The remaining cotton including plants from the pollen trap will be picked using a standard picker and destroyed at the site.

50. After harvest, a cereal crop may be planted within the trial site. The crop will be chosen from the approved post-harvest crops list on the OGTR website.

6.3 Presence of related plants in the receiving environment51. The proposed release site is in Narrabri, NSW, which is a commercial cotton growing area. Almost 100% of the cotton commercially cultivated in Australia is GM cotton modified for insect resistance and/or herbicide tolerance (Roth 2014). Commercial cotton grown in Australia is either Gossypium hirsutum or Gossypium barbadense, with 99% of cotton planted in 2006 being G. hirsutum (OGTR 2013b). The GM G. hirsutum proposed for release is capable of crossing with both species of commercially grown cotton. It is worth noting that improved fibre quality is one of the main characteristics for which both G. hirsutum and G. barbadense varieties are selected.


http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/policy-postharvest-htm

http://www.bom.gov.au/

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52. Ephemeral populations of cotton volunteers can be found on cotton farms, by roadsides where cotton seed is transported, or in areas where cotton seed is used as livestock feed (Addison et al. 2007; Eastick & Hearnden 2006).

53. There are 17 native species of Gossypium in Australia, most of which can be found in the Northern Territory and the north of Western Australia. Generally, they are found in native vegetation and not in disturbed/modified habitats such as agricultural areas (Groves et al. 2002).

54. Well established genetic incompatibility prevents crossing of native cotton species with cultivated cotton in the natural environment (discussed in OGTR 2013b).

6.4 Presence of similar genes and encoded proteins in the environment55. The PME3, PME4, PME5 and GAUT1 genes were isolated from cotton. Therefore, these genes and their encoded proteins are widespread in the Australian environment.

56. The nptII gene is isolated from the common gut bacteria E. coli (Section 5.2.4). This gene is also present in GM cotton cultivars already commercially planted in Australia, including Bollgard II®, Bollgard III®, Roundup Ready Flex®/ Bollgard II®, and Roundup Ready Flex®/ Bollgard III®.

Section 7 Relevant Australian and international approvals

7.1 Australian approvals7.1.1 Approvals by the Regulator

57. None of the GM cotton lines included in this application has previously been approved by the Regulator for release in Australia.

58. Information on previous DIR licences for GM cotton is available from the GMO Record on the OGTR website. The Regulator has previously approved 35 field trials and 9 commercial releases of GM cotton. There have been no credible reports of adverse effects on human health or the environment resulting from any of these releases.7.1.2 Approvals by other government agencies

59. There are no approvals of these GM cotton lines, including pending approvals, from other Australian authorities.

7.2 International approvals60. None of the GM cotton lines covered in this application has been approved for release in any other countries.



Chapter 2 Risk assessmentSection 1 Introduction

61. The risk assessment identifies and characterises risks to the health and safety of people or to the environment from dealings with GMOs, posed by or as the result of gene technology (Figure 2). Risks are identified within the context established for the risk assessment (see chapter 1), taking into account current scientific and technical knowledge. A consideration of uncertainty, in particular knowledge gaps, occurs throughout the risk assessment process.

Figure 2 The risk assessment process

62. Initially, risk identification considers a wide range of circumstances whereby the GMO, or the introduced genetic material, could come into contact with people or the environment. Consideration of these circumstances leads to postulating plausible causal or exposure pathways that may give rise to harm for people or the environment from dealings with a GMO in the short and long term. These are called risk scenarios.

63. A number of risk identification techniques are used by the Regulator and staff of the OGTR, including checklists, brainstorming, reported international experience and consultation (OGTR 2013a). A weed risk assessment approach is used to identify traits that may contribute to risks from GM plants. In particular, novel traits that may increase the potential of the GMO to spread and persist in the environment or increase the level of potential harm compared with the parental plant(s) are used to postulate risk scenarios (Keese et al. 2013). Risk scenarios postulated in previous RARMPs prepared for licence applications of the same or similar GMOs are also considered.

64. Postulated risk scenarios are screened to identify those that are considered to have some reasonable chance of causing harm. Pathways that do not lead to harm, or could not plausibly occur, do not advance in the risk assessment process.

65. Substantive risks (i.e. those identified for further assessment) are characterised in terms of the potential seriousness of harm (Consequence assessment) and the likelihood of harm (Likelihood

Chapter 2 – Risk assessment 10


assessment). Risk evaluation then combines the Consequence and Likelihood assessments to estimate the level of risk and determine whether risk treatment measures are required. The potential for interactions between risks is also considered.

Section 2 Risk Identification66. Postulated risk scenarios are comprised of three components:

i. The source of potential harm (risk source).

ii. A plausible causal linkage to potential harm (causal pathway).

iii. Potential harm to an object of value, people or the environment.

67. The risk context, including the following factors, is taken into account when postulating relevant risk scenarios:

the proposed dealings, which may be to conduct experiments, develop, produce, breed, propagate, grow, import, transport or dispose of the GMOs, use the GMOs in the course of manufacture of a thing that is not the GMO, and the possession, supply and use of the GMOs in the course of any of these dealings

the proposed limits including the extent and scale of the proposed dealings

the proposed controls to restrict the spread and persistence of the GMO and

characteristics of the parent organism(s).

2.1 Risk source68. The sources of potential harms can be intended novel GM traits associated with one or more introduced genetic elements, or unintended effects/traits arising from the use of gene technology.

69. As discussed in Chapter 1, the GM cotton lines and stacks have been modified by the introduction of one or two of the four cotton genes or a partial cotton gene sequence. These introduced genes or partial gene are considered further as potential sources of risk.

70. In addition, all of the GM cotton lines and stacks contain the nptII antibiotic resistance selectable marker gene. This gene and its product have already been extensively characterised and assessed as posing negligible risk to human or animal health or to the environment by the Regulator as well as by other regulatory agencies in Australia and overseas. Further information about this gene can be found in the document Marker genes in GM plants available from the Risk Assessment References page on the OGTR website. As this gene has not been found to pose a substantive risk to either people or the environment, its potential effects will not be further considered for this application.

71. The introduced genes or partial gene are controlled by introduced regulatory sequences. The regulatory sequences are derived from plants and a plant virus (see Table 2). Regulatory sequences are naturally present in plants, and the introduced elements are expected to operate in similar ways to endogenous elements. There is no evidence that regulatory sequences themselves have toxic or allergenic effects (EPA 1996). Although the viral sequences are derived from a plant pathogen, they only constitute small fractions of the genomes and cannot themselves cause disease. Hence, potential harms from the regulatory elements will not be considered further. However, the introduced regulatory sequences, especially the promoters, control gene expression and hence the distribution and concentration of the derived proteins and siRNA molecules in the GM plants. The effects of protein or siRNA molecules and their levels, especially in relation to toxicity and allergenicity, will be considered below.

72. The genetic modifications have the potential to cause unintended effects in several ways including altered expression of endogenous genes by random insertion of introduced DNA in the



genome, increased metabolic burden due to expression of the introduced proteins, novel traits arising out of interactions with non-target proteins and secondary effects arising from altered substrate or product levels in biochemical pathways. However, these types of effects also occur spontaneously and in plants generated by conventional breeding. Accepted conventional breeding techniques such as hybridisation, mutagenesis and somaclonal variation can have a much larger impact on the plant genome than genetic engineering (Schnell et al. 2015). Plants generated by conventional breeding have a long history of safe use, and there are no documented cases where conventional breeding has resulted in the production of a novel toxin or allergen in a crop (Steiner et al. 2013). Therefore, unintended effects resulting from the process of genetic modification will not be considered further.

2.2 Causal pathway73. The following factors are taken into account when postulating plausible causal pathways to potential harm:

routes of exposure to the GMOs, the introduced gene(s) and gene product(s) potential effects of the introduced gene(s) and gene product(s) on the properties of the

organism potential exposure to the introduced gene(s) and gene product(s) from other sources in the

environment the environment at the site(s) of release agronomic management practices for the GMOs spread and persistence (invasiveness) of the GMOs, including establishment and

reproduction dispersal by natural means and by people tolerance to abiotic conditions (e.g. climate, soil and rainfall patterns) tolerance to biotic stressors (e.g. pest, pathogens and weeds) tolerance to cultivation management practices gene transfer to sexually compatible organisms gene transfer by horizontal gene transfer (HGT) unauthorised activities.

74. The potential for horizontal gene transfer (HGT) from GMOs to other organisms, and any possible adverse outcomes, have been reviewed in the literature (Keese 2008) and assessed in many previous RARMPs. HGT was most recently considered in the RARMP for DIR 108 (available from the GMO Record on the OGTR website). In previous assessments of HGT no substantive risk was identified, due to the rarity of these events and because the wild-type gene sequences are already present in the environment and available for transfer via demonstrated natural mechanisms. Therefore, HGT will not be further considered for this application.

75. The potential for unauthorised activities to lead to an adverse outcome has been considered in many previous RARMPs, most recently in the RARMP for DIR 117 (available from the GMO Record on the OGTR website). In previous assessments of unauthorised activities, no substantive risk was identified. The Act provides for substantial penalties for unauthorised dealings with GMOs or non-compliance with licence conditions, and also requires the Regulator to have regard to the suitability of an applicant to hold a licence prior to the issuing of the licence. These legislative provisions are considered sufficient to minimise risks from unauthorised activities. Therefore, unauthorised activities will not be considered further.

2.3 Potential harm76. Potential harms from GM plants include:


http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/ir-1


harm to the health of people or desirable organisms, particularly toxicity/allergenicity reduced establishment or yield of desirable plants reduced products or services from the land use restricted movement of people, animals, vehicles, machinery and/or water reduced quality of the biotic environment (e.g. providing food or shelter for pests or

pathogens) or abiotic environment (e.g. negative effects on fire regimes, nutrient levels, soil salinity, soil stability or soil water table)

reduced biodiversity for nature conservation.

77. These harms are based on those used to assess risk from weeds (Keese et al. 2013; Standards Australia Ltd et al. 2006). Judgements of what is considered harm depend on the management objectives of the land where the GM plant may spread and persist. A plant species may have different weed risk potential in different land uses such as dryland cropping or nature conservation.

2.4 Postulated risk scenarios78. Four risk scenarios were postulated and screened to identify substantive risk. These scenarios are summarised in Table 3 and more detail of these scenarios is provided later in this Section. Postulation of risk scenarios considers impacts of the GM cotton or its products on people undertaking the dealings, as well as impacts on people and the environment if the GM plants or genetic material were to spread and/or persist.

79. In the context of the activities proposed by the applicant and considering both the short and long term, none of the four risk scenarios gave rise to any substantive risks.

Table 3 Summary of risk scenarios from dealings with GM cotton genetically modified for altered fibre qualityRisk scenario

Risk source Causal pathway Potential harm/s Substantive risk?

Reasons

1 GM cotton expressing introduced genes for enhanced fibre quality

Cultivation of GMOs at the trial site

Exposure of people who deal with the GMOs or of animals at the trial site to introduced proteins

Toxicity or allergenicity in people or toxicity to desirable organisms

No GM plant material will not be used in human food or animal feed.

The introduced proteins are endogenous to cultivated cotton and are not known to be toxic to people or other organisms.

The limited scale and short duration of the trial minimise exposure of people and other organisms to the GM plant material.

2 GM cotton expressing introduced gene silencing construct

Cultivation of GMOs at the trial site

Exposure of people who deal with the GMOs or of animals at the trial site to introduced siRNA


No GM plant material will not be used in human food or animal feed.

The silencing construct does not lead to expression of a protein.

The expressed siRNA is unlikely to affect expression of genes in animals which ingest the GM cotton, and even if it did any effects would be transient.

The limited scale and short duration of the trial minimise exposure of people and other organisms to the GM plant material.



Risk scenario

Risk source Causal pathway Potential harm/s Substantive risk?

Reasons

3 GM cotton expressing introduced genes for enhanced fibre quality or the gene silencing construct

Dispersal of GM seed outside trial limits

GM seed germinates

Establishment of populations of the GM plants


Reduced establishment or yield of desirable plants

No The proposed controls minimise dispersal of GM cotton seeds.

Cotton has limited ability to survive outside agricultural settings and the genetic modifications are not expected to increase its weediness.

The GM cotton is susceptible to standard weed control measures.

4 GM cotton expressing introduced genes for enhanced fibre quality or the gene silencing construct

Pollen from GM plants fertilise other sexually compatible plants

GM hybrid seed germinates

GM hybrids spread and persist



No Cotton has limited ability to outcross.

The proposed limits and controls would minimise pollen flow to cotton plants outside the trial site.

Hybrids between the GMOs and commercial GM cotton lines are not expected to have enhanced toxicity or weediness.

2.4.1 Risk scenario 1

Risk source GM cotton expressing introduced genes for enhanced fibre quality

Causal pathway

Cultivation of GMOs at trial sites

Exposure of people who deal with the GMOs or of animals at the trial site to introduced proteins

Potential harm Toxicity or allergenicity in people or toxicity to desirable organisms

Risk source

80. The source of potential harm for this postulated risk scenario is GM cotton expressing introduced genes for enhanced fibre quality.

Causal pathway

81. Workers who cultivate, harvest, gin, transport, experiment or conduct other dealings with the GM cotton grown would be exposed to cotton plant material. As the applicant proposes that only authorised staff deal with the GM cotton, other people are not expected to be exposed to the GM plants or plant material. Potential pathways of exposure to the introduced proteins are ingestion, inhalation or dermal contact. There is little potential for human ingestion of the introduced proteins, as the applicant proposes that no GM plant material would be used as food. GM plant material that could potentially be airborne and inhaled includes pollen or cotton dust produced during the harvesting or ginning processes. However, cotton pollen is heavy, sticky and not easily dispersed by wind (OGTR 2013b), and people who enter cotton gins typically wear protective face masks (International Fibre Centre website). Workers could come into skin contact with the introduced proteins if they touch damaged plants where cell contents have been released. However, no staff have experienced or observed adverse health effects during development of the GM cotton plant (information provided by the applicant).

82. The applicant proposes to test fibre quality of lint from GM cotton, spun into yarn and woven into fabric at a non-certified facility. Processed cotton lint contains over 99% cellulose and does not contain detectable protein (OGTR 2013b). Therefore, people handling lint from GM cotton would not be exposed to the introduced proteins.


http://www.ifc.net.au/


83. Non-human organisms may be exposed directly to the introduced proteins through ingesting the GM plants, or exposed indirectly through the food chain, or exposed through contact with dead plant material (soil organisms). Livestock would not be expected to ingest the introduced proteins as the GM plant material is not to be used as animal feed. Wild mammals and birds generally avoid feeding on cotton plants, in particular finding the seed unpalatable because of its high gossypol content (OGTR 2013b). A range of invertebrates would be expected to ingest GM cotton plant material. The small scale and short duration of the proposed field trial would restrict the total number of invertebrates exposed to the introduced proteins.

Potential harm

84. Toxicity is the adverse effect(s) of exposure to a dose of a substance as a result of direct cellular or tissue injury, or through the inhibition of normal physiological processes (Felsot 2000). Allergenicity is the potential of a substance to elicit an immunological reaction following its ingestion, dermal contact or inhalation, which may lead to tissue inflammation and organ dysfunction (Arts et al. 2006).

85. Non-GM cotton produces natural toxins for defence against herbivory including gossypol and cycloprenoid fatty acids (OGTR 2013b). The introduced genes are not involved in the metabolic pathways associated with these toxins and are therefore unlikely to affect the levels of endogenous toxins in cotton.

86. The introduced genes for fibre quality (PME3, PME4, PME5 and GAUT1) encode enzymes that are involved in pectin synthesis (GAUT1) and modification (PME3, PME4 and PME5) (Chapter 1, Section 5.2). These genes are endogenous cotton genes but their expression levels and spatial distributions in GM cotton are expected to be different when they are under the control of a non-endogenous promoter. As discussed in Chapter 1 Section 5.3, the proteins encoded by these genes are not known to be toxic to people or to other organisms. The GAUT1 protein has no similarity to known allergens. However, the PME proteins have sequence similarity to known allergens present in kiwifruit and olive pollen. Increased expression levels and expression of these proteins in additional tissues may lead to increased allergenicity towards people who are sensitive to kiwifruit or olive pollen. However, cotton pollen is not usually airborne and the GMOs would not be used in human food.

87. The GAUT1 enzyme is involved in pectin synthesis. The expression level of this enzyme is expected to increase the amount of pectin in the cell wall. Pectin is a food additive approved by FSANZ (code number 440 under Schedule 8 of the Australia New Zealand Food Standards Code).

88. When a fruit (such as tomato) is homogenised for preparing fruit juice, the PME activity can cause rapid pectin breakdown by hydrolysing pectin methylesters to produce polygalacturonic acid and methanol (Anthon & Barrett 2012). Human consumption of high levels of methanol can be toxic to the nervous system, particularly affecting the eyesight. Overexpression of the introduced cotton PME genes may lead to higher levels of PME activities in the GM cotton, which could result in increased methanol levels in the GM cotton tissues if natural mechanisms for removing methanol from cells are overloaded. However, the GMOs would not be used in human food or animal feed.

Conclusion

89. Risk scenario 1 is not identified as a substantive risk because the GM plant material will not be used in human food or animal feed; the introduced proteins are endogenous to cultivated cotton and not known to be toxic; and the limited scale, short duration and the proposed controls of the trial minimise exposure of people and other organisms to the GM plant material. Therefore, this risk could not be greater than negligible and does not warrant further detailed assessment.



2.4.2 Risk Scenario 2

Risk source GM cotton expressing introduced gene silencing construct

Causal pathway

Cultivation of GMOs at trial sites

Exposure of people who deal with the GMOs or of animals at the trial site to introduced siRNA

Potential harm Toxicity or allergenicity in people or toxicity to desirable organisms

Risk source

90. The source of potential harm for this postulated risk scenario is the GM cotton expressing the introduced gene silencing construct (Category 3). The silencing construct containing PME3 gene fragments is designed to produce siRNAs that reduce or suppress expression of the PME3 gene, thus reducing PME3 activity in developing cotton fibre.

Causal pathway

91. People and other organisms may be exposed to this GM cotton in the same way as described in Risk Scenario 1. Exposure is restricted by the same factors discussed in Risk Scenario 1.

Potential harm

92. The potential harms arising from this risk scenario are toxicity or allergenicity in people, or toxicity to desirable organisms, as a result of exposure to the GM cotton. Transcription of the gene fragment in the silencing construct results in hairpin RNA, which enters the RNAi pathway rather than being translated into a protein. Therefore, the introduction of the silencing construct does not lead to expression of a novel protein that could potentially be toxic or allergenic. All known food allergens are proteins, those derived from plants coming chiefly from peanut, tree nuts, wheat and soybean (Delaney et al. 2008; Herman & Ladics 2011).

93. The possibility exists that, rather than direct toxicity, RNAi constructs may give rise indirectly to altered toxicity or allergenicity. The gene silencing activity of RNAi in plants requires a high level of sequence complementarity. However, in the unlikely event that short sequences from the RNAi construct were a sufficiently close match to expressed non-target sequences, RNAi constructs could potentially give rise to off-target silencing effects within the plant. Silencing of non-target genes may lead to changes other than the intended effects, potentially including changes to levels of endogenous toxins or allergens. However, as discussed in Section 2.1, these types of effects also occur spontaneously and in plants generated by conventional breeding. Any off-target effects are not expected to influence the expression of any endogenous compounds that have toxic or allergenic properties to a greater extent than could occur through conventional breeding between different cotton varieties.

94. By targeting the PME3 gene, the silencing construct reduces PME3 activity in elongating fibre cells. As a result, the GM cotton has higher levels of methyl esterified pectin in the cell walls and shorter fibres. No other phenotypic changes were observed when compared to non-GM cotton (Chapter 1, Section 5.4.1). Since the endogenous PME3 gene is expressed specifically in developing fibre, it is expected that the genetic modification will only change the pectin profiles in fibres and not affect any other metabolic pathways.

95. Hairpin RNA transcribed from the silencing construct is processed into siRNAs (short interfering RNAs), which fall under a general category of small RNAs that also includes microRNAs (miRNAs). siRNAs and miRNAs are common in both plants and animals and are believed to play regulatory roles in many biological processes. Animals and plants naturally produce thousands of different siRNA molecules and these are consumed by humans and other organisms whenever they eat plant or animal cells. Zhang et al. (2011) reported that natural plant miRNAs can be absorbed by mammals through food intake, and have the potential to modulate



gene expression in animals. This risk has been further analysed by other researchers but remains controversial (Liang et al. 2014; Petrick et al. 2013). More detailed discussion of this issue can be found in the RARMP for DIR 131.

96. The possibility exists that siRNAs produced in GM cotton lines could, after ingestion, modulate expression of human or animal genes, with unknown physiological effects. The siRNAs would need to be produced at high levels in GM cotton, a large amount of the GM cotton would need to be consumed, the siRNA would need to match a target sequence in a human or animal gene, and be taken up by cells expressing that gene. As noted above, the GM cotton will not be used for human food or animal feed. Mammals do not have genes that are homologous to the cotton PME3 gene targeted by the introduced silencing construct (ExPASy, NCBI). Even if siRNAs were acquired through eating GM cotton and did affect gene expression, it is expected that any effect would be transient as described in Zhang et al (2011). None of these GM cotton lines will be used for human food or animal feed, and the trial is of small scale and short duration.

Conclusion

97. Risk scenario 2 is not identified as a substantive risk due to the introduced gene fragment not coding for any protein; mammals do not have genes homologous to the cotton PME3 gene targeted by the introduced silencing construct; the siRNAs produced are unlikely to affect expression of genes in animals which ingest the GM cotton (and any such effects would be transient); and the proposed limits and controls would minimise exposure of people and other organisms to the GM plant material. Therefore this risk could not be greater than negligible and does not warrant further detailed assessment.


Risk source GM cotton expressing introduced genes for enhanced fibre quality or gene silencing construct

Causal pathway

Dispersal of GM seed outside trial limits

GM seed germinates

Establishment of populations of the GM plants

Potential harms

Toxicity or allergenicity in people or toxicity to desirable organismsor


Risk source

98. The source of potential harm for this postulated risk scenario is GM cotton expressing introduced genes for enhanced fibre quality or the gene silencing construct.

Causal Pathway

99. The first step in the causal pathway for this risk scenario is dispersal of GM seed outside the trial limits. This could occur due to persistence of viable GM seeds at the trial site after the intended duration of the trial, or through physical movement of GM seeds to areas outside the trial site.

100. The applicant proposes a number of control measures to prevent persistence of GM seeds in the seed bank at the trial site. These include destroying GMOs that remain in the trial site after harvest, cultivating the site after harvest to encourage decomposition or germination of remaining seed, destroying any volunteers found prior to flowering, and post-harvest monitoring of the trial site for at least twelve months and until the site has been clear of volunteers for six months. It is not expected that expression of the introduced genes for enhanced fibre quality or the gene silencing construct would increase the ability of the GMOs to survive these standard control measures.


http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=

http://enzyme.expasy.org/EC/2.4.1.13



101. Cotton seeds are enclosed in large, heavy bolls that remain attached to the plant. At maturity the bolls split open and the fibres can facilitate seed dispersal by wind over distances less than 100 m (OGTR 2013b). Cotton seed is not normally physically transported by runoff after rainfall or irrigation. Extreme weather conditions such as flooding or high winds may cause dispersal of plant parts. The proposed release site is located at least 2 km from the nearest river and protected by flood levee banks. Although there are irrigation channels in the proximity, these channels do not flow into natural waterways and the applicant proposes to monitor the channels post-harvest for cotton volunteers.

102. Wild mammals and birds generally avoid feeding on cotton plants, in particular finding the seed unpalatable because of its high gossypol content (OGTR 2013b). Therefore, wild animals are unlikely to disperse GM cotton seeds from the trial site. GM cotton seeds would not be used as stock feed, so would not be dispersed by stock.

103. Dispersal of cotton seeds by authorised people entering the trial site would be minimised by cleaning all equipment, including clothing, used with the GM cotton before using it for any other purpose. GM seed cotton would be ginned separately from any other cotton crop in an approved facility to avoid accidentally mixing GM cotton seed with other cotton seed, then dispersing the mixed cotton seed. The applicant proposes to contain GM plant materials during transportation and storage in accordance with the Regulator’s Guidelines for the Transport, Storage and Disposal of GMOs. Only plant material needed for experimentation would be transported outside the site. Spillage of GM seed during transport to and from the release site would be rare and could be readily controlled through cleaning and monitoring of the site of the spill.

104. The spread and persistence of non-GM cotton plants are limited by a number of biotic and abiotic factors, especially cold stress in southern Australia and water stress in non-irrigated environments throughout almost all of Australia. Feral cotton populations are sparse and ephemeral in all current cotton growing regions of Australia (OGTR 2013b). A study found that even when cotton was sown in cleared sites in northern Australian with high water availability, the cotton plants did not establish stable populations (Eastick & Hearnden 2006). Modelling of climactic factors limiting cotton persistence indicate that cotton has naturalisation potential only in the coastal regions of north-east Australia (Rogers et al. 2007), which are distant from the proposed trial site. A few small populations of naturalised cotton are reported in northern Australia, but these are not derived from modern cultivars (OGTR 2013b), and may have a greater ability to survive outside agricultural settings than modern cotton cultivars.

105. The introduced PME and GAUT1 genes are endogenous to cotton and are primarily involved in fibre development. Over-expression of these genes or the gene silencing construct is expected to alter the pectin profiles in cell walls. As pectin has diverse functions in plant growth, morphology, defence and seed dormancy, as discussed in Chapter 1, Section 5.2, these characteristics may be altered in the GM cotton lines. However, preliminary studies by the applicant showed that, under glasshouse conditions, all GM cotton lines in Categories 1, 3, 5 and 6 displayed normal agronomic characteristics apart from expected changes in lint fibres. GM cotton plants in Categories 2 and 4 with over-expression of the PME3 gene and PME4 gene, respectively, showed increased height relative to non-GM cotton (Chapter 1, Section 5.4.1). There is no evidence suggesting that the genetic modifications have altered seed production characteristics or tolerance to abiotic or biotic stresses that could enhance the potential for dispersal or persistence of the GM cotton lines proposed for release. Fibre quality is one of the main characteristics for which cotton is bred. Non-GM cotton varieties with enhanced fibre quality have not been reported to spread more easily or persist longer than other cotton varieties.

Potential harms

106. The potential harms from this risk scenario are toxicity or allergenicity in people or toxicity to desirable organisms, or reduced establishment or yield of desirable plants.



107. Risk Scenarios 1 and 2 considered the potential for the introduced genes or gene silencing construct to lead to toxicity or allergenicity, and did not identify any substantive risks.

108. The GM cotton could reduce the establishment or yield of desirable plants in agricultural settings if GM cotton volunteers grew in other crops. If this happened, the GM cotton volunteers could easily be controlled by standard measures such as application of herbicides or cultivation.

109. The GM cotton could reduce the establishment or yield of desirable plants in the natural environment if the GM cotton spread and persisted as a weed in nature reserves, displacing native vegetation. However, as discussed above, cotton has limited potential to survive outside agricultural settings, and the introduced proteins or the gene silencing construct are not expected to increase its ability to spread and persist. In the unlikely event of GM cotton plants establishing themselves in nature reserves, small and ephemeral feral GM cotton populations would be unlikely to cause harms associated with weediness such as reducing desired plants or restricting physical movement.

Conclusion

110. Risk scenario 3 is not identified as a substantive risk because the proposed controls minimise dispersal of GM cotton seeds, cotton has limited ability to survive outside agricultural settings and the introduced proteins or gene silencing construct are not expected to increase its weediness, and the GM cotton is susceptible to standard weed control measures. Therefore, this risk could not be greater than negligible and does not warrant further detailed assessment.


Risk source GM cotton expressing introduced genes for enhanced fibre quality or gene silencing construct

Causal pathway

Pollen from GM plants fertilising other sexually compatible plants

GM hybrid seed germinates

GM hybrids spread and persist

Potential harms

Toxicity or allergenicity in people or toxicity to desirable organismsor


Risk source

111. The source of potential harm for this postulated risk scenario is GM cotton expressing introduced genes for enhanced fibre quality or a gene silencing construct.

Causal pathway

112. The first step in the causal pathway for this risk scenario is pollen from GM plants fertilising other sexually compatible plants. Cotton is predominantly self-pollinating, with pollen that is large, sticky and heavy and generally not dispersed by wind. Pollen can be transported by insect pollinators, chiefly honeybees, but gene flow studies have shown that outcrossing occurs at low levels and decreases rapidly with distance (OGTR 2013b). The only sexually compatible plants are G. hirsutum or G. barbadense plants, as native Gossypium species are not sexually compatible with cotton (Chapter 1, Section 6.3). It is not expected that the introduced genes for enhanced fibre quality or the gene silencing construct would alter the pollen dispersal characteristics of the GM cotton.

113. The applicant has proposed to restrict pollen flow by surrounding the trial site with a 20 m pollen trap of non-GM cotton. In addition, the applicant has proposed to destroy any post-harvest cotton volunteers on the trial site before flowering. These controls would minimise the potential for pollinators to transfer pollen from GMOs to related plants outside the trial sites.



114. Some outcrossing is expected to occur between the GMOs and other cotton plants grown at the trial site, i.e. non-GM comparator cotton plants and cotton plants in the pollen trap. As non-GM cotton plants grown in the trial site and pollen traps are expected to produce a small proportion of hybrid seeds, the applicant has proposed that non-GM cotton planted in the trial site and/or in the pollen trap will be handled as if they are the GMOs. The limits and controls proposed for the GMOs would minimise dispersal of hybrid seeds (see Risk Scenario 3).

115. Any hybrid seeds that did occur outside of the trial limits would be unlikely to establish a stable population, as discussed in Risk Scenario 3. If the GMOs pollinated a commodity cotton crop, a small amount of hybrid seed containing the introduced proteins could enter human food and animal feed. If the GMOs pollinated a cotton crop intended for seed production, hybrid seed could be purposefully dispersed and grown, leading to wider exposure to the introduced proteins. The applicant has proposed locating the outer edge of the pollen trap at least 50 m away from cotton lines used for breeding purposes, to further minimise gene flow.

Potential harms

116. The potential harms from this risk scenario are toxicity or allergenicity in people or toxicity in desirable organisms, or reduced establishment or yield of desirable plants.

117. Risk Scenarios 1 and 2 considered the potential for the introduced genes or gene silencing construct to lead to toxicity or allergenicity, and did not identify any substantive risks. This is also expected to be the case if the introduced proteins are expressed in hybrids with other cotton.

118. The potential for the GMOs to reduce establishment or yield of desirable plants was discussed in Risk Scenario 3. Cotton plants expressing the introduced proteins or the gene silencing construct are unlikely to spread and persist in nature reserves or to survive standard weed management practices for cotton volunteers in agricultural settings.

Conclusion

119. Risk scenario 4 is not identified as a substantive risk because cotton has limited ability to outcross, the proposed limits and controls would minimise pollen flow to cotton plants outside the trial site, and hybrids between the GMOs and commercial GM cotton lines are not expected to have enhanced toxicity or weediness. Therefore, this risk could not be greater than negligible and does not warrant further detailed assessment.

Section 3 Uncertainty120. Uncertainty is an intrinsic property of risk and is present in all aspects of risk analysis**. Uncertainty in risk assessments arises from sources such as incomplete knowledge and inherent biological variability. Uncertainty is addressed by approaches including balance of evidence, conservative assumptions, and applying risk management measures that reduce the potential for risk scenarios involving uncertainty to lead to harm. If there is residual uncertainty that is important to estimating the level of risk, the Regulator will take this uncertainty into account in making decisions.

121. As field trials of GMOs are designed to gather data, there are generally data gaps when assessing the risks of a field trial application. However, field trial applications are required to be limited and controlled. Even if there is uncertainty about the characteristics of a GMO, limits and controls restrict exposure to the GMO, and thus decrease the likelihood of harm.

122. For DIR 136, uncertainty is noted particularly in relation to:

Potential for increased allergenicity due to higher concentrations of PME proteins

** A more detailed discussion of uncertainty is contained in the Regulator’s Risk Analysis Framework available from the Risk Assessment References page on the OGTR website or via Free call 1800 181 030.




Potential for altered levels of endogenous toxic metabolites

Potential effects of the genetic modifications on weediness characteristics.

123. Additional data, including information to address these uncertainties, may be required to assess possible future applications with reduced limits and controls, such as a larger scale trial or the commercial release of these GMOs.

124. Chapter 3, Section 4, discusses information that may be required for future release.

Section 4 Risk Evaluation125. Risk is evaluated against the objective of protecting the health and safety of people and the environment to determine the level of concern and, subsequently, the need for controls to mitigate or reduce risk. Risk evaluation may also aid consideration of whether the proposed dealings should be authorised, need further assessment, or require collection of additional information.

126. Factors used to determine which risks need treatment may include:

risk criteria level of risk uncertainty associated with risk characterisation interactions between substantive risks.

127. Four risk scenarios were postulated whereby the proposed dealings might give rise to harm to people or the environment. In the context of the control measures proposed by the applicant, and considering both the short and long term, none of these scenarios were identified as substantive risks. The principal reasons for these conclusions are summarised in Table 3 and include:

none of the GM plant material or products will enter human food or animal feed supply chains

widespread presence of the introduced genes and their encoded proteins in the environment and lack of known toxicity

limited ability of the GM cotton plants to establish populations outside cultivation limited ability of the GM cotton plants to transfer the introduced genetic material to other

cotton plants limits on the size, location and duration of the release proposed by CSIRO suitability of controls proposed by CSIRO to restrict the spread and persistence of the GM

cotton plants and their genetic material.

128. Therefore, risks to the health and safety of people, or the environment, from the proposed release of the GM cotton plants into the environment are considered to be negligible. The Risk Analysis Framework (OGTR 2013a), which guides the risk assessment and risk management process, defines negligible risks as risks of no discernible concern with no present need to invoke actions for mitigation. Therefore, no controls are required to treat these negligible risks. Hence, the Regulator considers that the dealings involved in this proposed release do not pose a significant risk to either people or the environment.



Chapter 3 Risk management planSection 1 Background

129. Risk management is used to protect the health and safety of people and to protect the environment by controlling or mitigating risk. The risk management plan addresses risks evaluated as requiring treatment and considers limits and controls proposed by the applicant, as well as general risk management measures. The risk management plan informs the Regulator’s decision-making process and is given effect through licence conditions.

130. Under section 56 of the Act, the Regulator must not issue a licence unless satisfied that any risks posed by the dealings proposed to be authorised by the licence are able to be managed in a way that protects the health and safety of people and the environment.

131. All licences are subject to three conditions prescribed in the Act. Section 63 of the Act requires that each licence holder inform relevant people of their obligations under the licence. The other statutory conditions allow the Regulator to maintain oversight of licensed dealings: section 64 requires the licence holder to provide access to premises to OGTR inspectors and section 65 requires the licence holder to report any information about risks or unintended effects of the dealing to the Regulator on becoming aware of them. Matters related to the ongoing suitability of the licence holder are also required to be reported to the Regulator.

132. The licence is also subject to any conditions imposed by the Regulator. Examples of the matters to which conditions may relate are listed in section 62 of the Act. Licence conditions can be imposed to limit and control the scope of the dealings. In addition, the Regulator has extensive powers to monitor compliance with licence conditions under section 152 of the Act.

Section 2 Risk treatment measures for substantive risks133. The risk assessment of risk scenarios listed in Chapter 2 concluded that there are negligible risks to people and the environment from the proposed field trial of GM cotton. These risk scenarios were considered in the context of the scale of the proposed release (Chapter 1, Section 3.1), the proposed containment measures (Chapter 1, Section 3.2), and the receiving environment (Chapter 1, Section 6), and considering both the short and the long term. The risk evaluation concluded that no controls are required to treat these negligible risks.

Section 3 General risk management134. The limits and controls proposed in the application were important in establishing the context for the risk assessment and in reaching the conclusion that the risks posed to people and the environment are negligible. Therefore, to maintain the risk context, licence conditions have been imposed to limit the release to the proposed size, location and duration, and to restrict the spread and persistence of the GMOs and their genetic material in the environment. The conditions are discussed and summarised in this Chapter and listed in the licence.

3.1 Licence conditions to limit and control the release3.1.1 Consideration of limits and controls proposed by CSIRO

135. Sections 3.1and 3.2 of Chapter 1 provide details of the limits and controls proposed by CSIRO in their application. These are taken into account in the four risk scenarios postulated for the proposed release in Chapter 2. Many of the proposed control measures are considered standard for GM crop trials and have been imposed by the Regulator in previous DIR licences. The appropriateness of these controls is considered further below.

136. The applicant proposes that the release will take place at one site in Narrabri, NSW. The total planting area each year will not exceed 1 ha and the duration of the proposed release would be

Chapter 3 – Risk management 22


limited to three growing seasons. The small size and short duration of the trial would limit exposure to the GMOs (Risk Scenarios 1 and 2).

137. The applicant proposes that only authorised and trained personnel would be permitted to deal with the GMOs. A standard licence condition requires that all persons dealing with the GMOs must be informed of any applicable licence conditions. These measures would limit the potential exposure of humans to the GMOs (Risk Scenarios 1 and 2).

138. The applicant does not propose using any of the GM plant material for human or animal consumption. Therefore, a condition in the licence prohibits material from the trial from being used for human food or animal feed. This control will restrict exposure of humans and other organisms to the GMOs (Risk Scenarios 1 and 2) and the potential for GM cotton seed to be dispersed outside the trial limits (Risk Scenario 3).

139. The applicant proposes to test the fibre quality of lint from GM cotton grown in the trial at a non-certified facility. At the end of the experimentation, all material derived from the lint will either be destroyed or stored in certified PC2 laboratories. As discussed in Risk Scenario 1, cotton lint is free of DNA and protein so is not a means of exposure to GM material. Therefore, the licence does not impose conditions on experimentation, transport and sale of lint from GM cotton, other than the prohibition of use for food or feed as described above.

140. The applicant proposes to clean all equipment used with the GMOs before using the equipment for other purposes. Equipment used on the trial site would be cleaned on site. The GM cotton would be ginned separately from other cotton crops and the gin would be cleaned after use to prevent GM cotton seed mixing with other seed. These measures are appropriate to restrict potential dispersal of GM cotton seed outside the trial sites (Risk Scenario 3).

141. After the trial site has been harvested, the applicant proposes to destroy all GMOs except for plant material required for testing and cotton seed required for further authorised planting. Cotton seeds have low dormancy levels and do not generally form a viable seed bank, however, dormancy can be induced in cotton seeds by low soil temperature and/or soil moisture (OGTR 2013b). The applicant proposes post-harvest cultivation of the trial sites to promote cotton seed germination or decomposition. A licence condition requires tillage in the spring or summer following the harvest, and provision of adequate soil moisture, so that soil temperature and moisture will be suitable for cotton seed germination. These measures would restrict the persistence of a GM cotton seed bank after the duration of the trial (Risk Scenario 3).

142. The applicant proposes that each trial site will be monitored post-harvest at least every two months for a minimum of twelve months and until the site has been clear of volunteers for at least six months. During this period any cotton volunteers will be destroyed before flowering. However, the proposed frequency of monitoring may not be sufficient. The OGTR’s experience from previous GM cotton trials indicates that if there is vigorous growth of a post-harvest crop, cotton volunteers in the 2-4 leaf stage may be missed during inspections, and also that in particularly favourable weather conditions the period between the 4 leaf stage and the beginning of flowering can be less than 40 days. Therefore, a licence condition requires post-harvest monitoring at least every 35 days, with destruction of any cotton volunteers, for a period of at least 12 months and until no volunteers are detected for a continuous 6 month period. These measures would restrict the persistence of GMOs after completion of the trial (Risk Scenario 3).

143. The applicant proposes that GMOs will be transported and stored according to the Regulator’s current Guidelines for the Transport, Storage and Disposal of GMOs (OGTR website). These protocols would restrict the potential for dispersal of GM seeds outside the trial sites (Risk Scenario 3). This is included as a licence condition.

144. The applicant proposes to locate the trial sites more than two km away from the nearest river. The land is protected by flood levees. The applicant has also proposed to monitor the adjacent irrigation channels after the trial and destroy any volunteers. The licence includes standard DIR


http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/transport-guide-1


licence conditions that require: that the site be at least 50 m from the nearest natural waterway or man-made waterway that flows into a natural waterway; that any area where the GMOs may have dispersed, including irrigation channels, be monitored after harvest; and immediate notification of any extreme weather conditions affecting the site during the proposed release. These measures will minimise likelihood for the GM cotton establishing outside the proposed release site, including as a result of extreme weather events (Risk Scenario 3).

145. The applicant proposes that the trial site will be surrounded by a 20 m wide pollen trap to control pollen flow from the GMOs to cotton plants outside the trial site. The plants within the pollen trap would be non-GM cotton and would be managed so as to flower at the same time as the GMOs. As discussed in Risk Scenario 4, cotton is predominantly self-pollinating and outcrossing rates decrease rapidly with distance. A 20 m pollen trap around GM cotton was found to be an effective buffer under Australian conditions (Llewellyn et al. 2007). Therefore, using a 20 m pollen trap would minimise gene transfer to cotton plants outside the trial sites (Risk Scenario 4). Although the applicant proposes that the plants in the pollen trap will be non-GM, the licence permits pollen trap plants to be either non-GM or GM cotton approved for commercial release in Australia. The applicant proposes to maintain a path of up to 3 m in width through the pollen trap to allow access of vehicles and equipment to the GMO planting area. Since previous cotton licences have allowed the access path to be up to 2.5 m, which has been proven to be adequate for access, the licence specifies a path of up to 2.5 m wide.

146. The applicant proposes to locate the outer edge of the pollen trap at least 50 m away from cotton lines used for breeding purposes, to further minimise gene flow. As discussed above, a 20 m pollen trap is considered adequate to control pollen flow; this is not included as a licence condition.

147. The applicant intends to plant both GM and non-GM cotton in the trial site. The applicant proposes to treat all non-GM cotton from the trial site or cotton from the pollen trap as if it were the GM cotton in this application. This measure would minimise exposure to or dispersal of hybrid seed resulting from outcrossing between the GMOs and other cotton (Risk Scenario 4).

148. The applicant proposes that GMOs may be transported, stored or subjected to experiments in certified physical containment facilities under a Notifiable Low Risk Dealing (NLRD) authorisation in accordance with applicable requirements of the Gene Technology Regulations 2001. This is considered appropriate but is not included in the licence as it would be conducted under a separate valid authorisation.3.1.2 Summary of licence conditions to be implemented to limit and control the release

149. A number of licence conditions have been imposed to limit and control the proposed release, based on the above considerations. These include requirements to:

limit the duration of the field trial to between October 2016 and May 2019

limit the field trial to a maximum area of 1 ha per year at one site in Narrabri, NSW

locate the trial site at least 50 m away from waterways

restrict gene flow via pollen from the field trial site by surrounding the trial site with a 20 m pollen trap of non-GM cotton or GM cotton approved for commercial release

ensure that pollen trap plants flower for the same period of time as the GMOs

treat any non-GM cotton planted in the planting area or pollen trap plants as if they were the GMOs

clean all equipment used with the GMOs before using it for any other purpose

gin the GMOs separately from any other cotton crop

use tillage and irrigation to promote germination of any cotton seeds remaining in the trial site after harvest



monitor the trial site for at least 12 months after harvest and destroy any cotton volunteers until no volunteers are detected for a continuous 6 month period

destroy all GMOs from the trial that are not required for testing or future planting

transport and store the GMOs in accordance with the Regulator’s Guidelines for the Transport, Storage and Disposal of GMOs

not allow GM plant material to be used for human food or animal feed.

3.2 Other risk management considerations150. All DIR licences issued by the Regulator contain a number of conditions that relate to general risk management. These include conditions relating to:

applicant suitability

contingency plans

identification of the persons or classes of persons covered by the licence

reporting requirements

access for the purpose of monitoring for compliance.3.2.1 Applicant suitability

151. In making a decision whether or not to issue a licence, the Regulator must have regard to the suitability of the applicant to hold a licence. Under section 58 of the Act, matters that the Regulator must take into account, for either an individual applicant or a body corporate, include:

any relevant convictions of the applicant

any revocation or suspension of a relevant licence or permit held by the applicant under a law of the Commonwealth, a State or a foreign country

the capacity of the applicant to meet the conditions of the licence.

152. On the basis of information submitted by the applicant and records held by the OGTR, the Regulator considers CSIRO suitable to hold a licence. The licence includes a requirement for the licence holder to inform the Regulator of any information that would affect their suitability.

153. In addition, any applicant organisation must have access to a properly constituted Institutional Biosafety Committee and be an accredited organisation under the Act.3.2.2 Contingency plan

154. CSIRO is required to submit a contingency plan to the Regulator before planting the GMOs. This plan will detail measures to be undertaken in the event of any unintended presence of the GM cotton outside permitted areas.

155. CSIRO is also required to provide the Regulator with a method to reliably detect the GMOs or the presence of the genetic modifications in a recipient organism. This methodology is required before planting the GMOs.3.2.3 Identification of the persons or classes of persons covered by the licence

156. The persons covered by the licence are the licence holder and employees, agents or contractors of the licence holder and other persons who are, or have been, engaged or otherwise authorised by the licence holder to undertake any activity in connection with the dealings authorised by the licence. Prior to growing the GMOs, CSIRO is required to provide a list of people and organisations that will be covered by the licence, or the function or position where names are not known at the time.



3.2.4 Reporting requirements

157. The licence requires the licence holder to immediately report any of the following to the Regulator:

any additional information regarding risks to the health and safety of people or the environment associated with the trial

any contraventions of the licence by persons covered by the licence

any unintended effects of the trial.

158. A number of written notices are also required under the licence to assist the Regulator in designing and implementing a monitoring program for all licensed dealings. The notices include:

expected and actual dates of planting

details of areas planted to the GMOs

expected dates of flowering

expected and actual dates of harvest and cleaning after harvest

details of inspection activities.3.2.5 Monitoring for compliance

159. The Act stipulates, as a condition of every licence, that a person who is authorised by the licence to deal with a GMO, and who is required to comply with a condition of the licence, must allow inspectors and other persons authorised by the Regulator to enter premises where a dealing is being undertaken for the purpose of monitoring or auditing the dealing. Post-release monitoring continues until the Regulator is satisfied that all the GMOs resulting from the authorised dealings have been removed from the release site.

160. If monitoring activities identify changes in the risks associated with the authorised dealings, the Regulator may also vary licence conditions, or if necessary, suspend or cancel the licence.

161. In cases of non-compliance with licence conditions, the Regulator may instigate an investigation to determine the nature and extent of non-compliance. The Act provides for criminal sanctions of large fines and/or imprisonment for failing to abide by the legislation, conditions of the licence or directions from the Regulator, especially where significant damage to health and safety of people or the environment could result.

Section 4 Issues to be addressed for future releases162. Additional information has been identified that may be required to assess an application for a commercial release of these GM cotton lines, or to justify a reduction in limits and controls. This includes:

additional molecular and biochemical characterisation of the GM cotton plants including potential for increased toxicity and allergenicity

additional phenotypic characterisation of the GM cotton plants, particularly with respect to traits such as seed dormancy, which may contribute to weediness.

Section 5 Conclusions of the consultation RARMP163. The RARMP concludes that the proposed limited and controlled release of GM cotton poses negligible risks to the health and safety of people or the environment as a result of gene technology, and that these negligible risks do not require specific risk treatment measures.

164. However, conditions have been imposed to limit the release to the proposed size, location and duration, and to restrict the spread and persistence of the GMOs and their genetic material in the



environment, as these were important considerations in establishing the context for assessing the risks.



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Appendix A Summary of submissions from prescribed experts, agencies and authorities††

Advice received by the Regulator from prescribed experts, agencies and authorities on the consultation RARMP is summarised below. All issues raised in submissions that related to risks to the health and safety of people and the environment were considered in the context of the currently available scientific evidence and were used in finalising the RARMP that formed the basis of the Regulator’s decision to issue the licence.

Sub. No. Summary of issues raised Comment1 Agrees with the overall conclusions of the

RARMP.Noted

Suggests considering the effectiveness of proposed use of personal protective equipment (PPE) in limiting exposure to GM material.

The applicant reported no staff experiencing or observing adverse health effects during development of the GM cotton plant. Text has been added to Risk Scenario 1 to note this. The risk of increased toxicity or allergenicity of the GM cotton has been assessed as negligible in the RARMP. Therefore, no prescriptive conditions regarding PPE are imposed in the licence. However, licence conditions require the licence holder to report any adverse effects of the GM cotton on human health.

2 Agrees with the conclusion of the RARMP that the risk of toxicity by expression of the proteins in the GM cotton, or in any other plant they may be transferred to by hybridisation, is negligible. Notes that GM plant material will not be used for animal feed.Conclusion of negligible risk of toxicity can be further supported by indicating that only a small number of plant proteins have ever been shown to be toxic, the most well-known being lectins and ribosome-inactivating peptides (RIPs) (Wu & Sun 2011), and none of the proteins expressed from the introduced genes fall into these categories.Also, mentioned in Paragraph 32 of using a BLAST search against the Entrez Protein dataset needs clarification; it is not obvious why such a search would necessarily identify homology with any known toxin.

Text and references on toxic proteins have been added to Chapter 1, Section 5.3. Text in Paragraph 32 regarding the BLAST search against the Entrez Protein dataset has been removed.

†† Prescribed agencies include GTTAC, State and Territory Governments, relevant local governments, Australian Government agencies and the Minister for the Environment.

Appendix A 32


Sub. No. Summary of issues raised CommentGiven the nature of the genetic modifications and trait, there is no reason to believe that the GM plants, or any other plant to which they may be transferred by hybridisation, will possess an increased ability to spread and persist and induce environmental harms, such as reducing the establishment or yield of desired plants, beyond those current cotton varieties.Although this is a limited and controlled field trial, the RARMP would benefit from considering the environmental harms of the GM plants in the context of available varieties that have been selected to have improved fibre quality. Cumulative knowledge suggests that the environmental management of the GM plants in this application will be no different from currently available varieties that have been selected for the properties of their fibre.

Text to this effect has been added to Chapter 1 Section 6.3 and Risk Scenario 3.

The risk of unintended release into the environment of GM plant material, or gene transfer to non-GM cotton or related species, is appropriately minimised by the proposed limits and controls.

Noted

Generally agrees with Paragraph 161 for issues to be addressed for future releases, but suggests that the risk of toxic effects on native animals is so low that this would unlikely constitute a reason to seek additional molecular and biochemical characterisation of the GM plants.

If the GM cotton were to be commercially released, planting may occur on a very large scale, exposing many animals and people to the GM cotton and its products. Therefore, some additional molecular and biochemical characterisation of the GM plants is considered appropriate.

Although the role of PME in seed dormancy is not recorded in a recent review (Graeber et al. 2012), it is noted that an increase in the activity of PME has been correlated with the cessation of seed dormancy and germination in yellow cedar (Paragraph 21). It is possible that if the expression of the introduced genes affects seed dormancy, then this in-turn may affect the persistence of populations of cotton. This aspect should be addressed if the plants are ever to be commercialised.

Text regarding seed dormancy has been added in Chapter 3, Section 4.

Suggests that if the GM plants ever reach the point of commercialisation, a thorough contrast and comparison of the GM plants with those commercialised varieties which have been bred to have improved fibre quality should be provided.

Noted

Paragraph 76 identifies one of the potential harms as to the ‘health of people or desirable organisms, including toxicity/allergenicity’. As toxicity and allergenicity are referred to in the RARMP, and indeed are likely the most prominent health issues, it is suggested that the word ‘including’ be replaced with ‘particularly’.

Text in paragraph 76 has been modified accordingly.

The RARMP (Paragraphs 31-37 and 84-88) fails to mention that the ingestion of food can also be associated with other adverse health effects, such as food intolerance and autoimmune diseases. Some discussion of these health issues would improve the RARMP.

Since the GM cotton material is not permitted in human food, the other adverse health effects associated with ingestion of food are not considered in this RARMP. FSANZ is responsible for assessing food for human consumption.

Appendix A 33


Sub. No. Summary of issues raised Comment3 Noted that the RARMP was circulated for

comment and no adverse comments were received. Indicated no objection to the issue of a licence for DIR 136.

Noted

4 Notes that the licence will prohibit the use of material from the trial for human or animal consumption. No further comments on the licence application at this stage.

Noted

5 Supported the conclusions of the RARMP that the proposed dealings pose negligible risk of harm to human health and the environment.

Noted

6 No concerns. Cannot see any material risks with this work both in the nature of what is being tested and the control measures that will be in place. Considers this exercise to have negligible biological, human, animal or ecological risks.

Noted

Graeber, K., Nakabayashi, K., Miatton, E., Leubner-Metzger, G., Soppe, W.J. (2012) Molecular mechanisms of seed dormancy. Plant Cell Environ 35: 1769-1786.

Wu, W., Sun, R. (2011) Toxicological studies on plant proteins: a review. Journal of Applied Toxicology 32: 377-386.

Appendix A 34


Appendix B Summary of submissions from the publicThe Regulator received one submission from the public on the consultation RARMP. The issues raised in this submission are summarised in the table below. All issues raised in the submission that related to risks to the health and safety of people and the environment were considered in the context of currently available scientific evidence in finalising the RARMP that formed the basis of the Regulator’s decision to issue the licence.

Issues raised: MT: Marketing and trade.Sub. No. Issue Summary of issues raised Comment

1 None Supports the conclusion of the RARMP that risks to the health and safety of people, or the environment from the proposed release are negligible.

Noted

MT Views maintenance and improvement of cotton fibre quality as critical to the future of the cotton industry. Lint quality is one of the main economic drivers behind the profitability of cotton. The cotton industry is one of Australia’s largest rural export earners. Australia’s international reputation as a reliable supplier of very high quality cotton can be supported and sustained through responsible commercialisation of these fibre quality traits developed by CSIRO.

When deciding whether or not to issue a licence, matters that relate to marketing and trade are outside the legislative responsibility of the Regulator.

Appendix B 35

summary of the risk assessment and risk management web viewdir 136 – risk assessment and risk...

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