OGTR document

Risk Assessment and

Risk Management Plan for

DIR 130

Limited and controlled release of wheat genetically modified for improved grain quality

Applicant: Murdoch University

March 2015PAGE INTENTIONALLY LEFT BLANK

Summary of the Risk Assessment and Risk Management Plan

for

Licence Application DIR 130

Decision

The Gene Technology Regulator (the Regulator) has decided to issue a licence application for the intentional release 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 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 application

Application number

DIR 130

Applicant:

Murdoch University

Project Title:

Limited and controlled release of wheat genetically modified for improved grain quality[footnoteRef:1] [1: The title of the project as supplied by the applicant is ‘Field trial of glutenin transgenic wheat for grain quality improvement’.]

Parent organism:

Bread wheat (Triticum aestivum L.)

Introduced genes and modified traits:

· Dx5 and Dy10 genes from wheat (improved grain quality)

· bar gene from Streptomyces hygroscopicus (selectable marker – herbicide tolerance)

Proposed release dates:

May 2015 – December 2017

Proposed location:

One site in the local government area of Katanning, Western Australia

Proposed release size:

600 square metres each year

Primary purpose

To assess whether the introduction and expression of the genes will increase the strength of dough

Risk assessment

The risk assessment concludes that there are negligible risks to the health and safety of people, or the environment, from the proposed release. No additional controls are required to manage these neglible risks beyond those proposed in the application.

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

Credible pathways to potential harm that were considered included: unintended exposure to the GM plant material; increased spread and persistence of the GM wheat relative to unmodified plants; and transfer of the introduced genetic material to non GM wheat, or other sexually compatible plants. Potential harms associated with these pathways included toxicity to people and other animals, allergic reactions in people and environmental harms associated with weediness.

The principal reasons for the conclusion of negligible risks are that the introduced genes are either identical, or similar, to those already existing in the environment (some of these genes are present in unmodified wheat); and the proposed limits and controls effectively contain the GMOs and their genetic material and minimise exposure.

Risk management plan

The 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, as this is a limited and controlled release, the licence includes limits on the size, location and duration of the release, as well as controls including containment provisions at the trial site; prohibiting the use of GM plant materials in human food or animal feed; destroying GM plant materials not required for further studies; transporting GM plant materials in accordance with the Regulator’s guidelines; and conducting post-harvest monitoring at the trial site to ensure all GMOs are destroyed.

DIR 130 – Risk Assessment and Risk Management PlanOffice of the Gene Technology Regulator

Summary II

Table of Contents

Summary of the Risk Assessment and Risk Management PlanI

DecisionI

The applicationI

Risk assessmentI

Risk management planII

Table of ContentsIII

AbbreviationsIV

Chapter 1Risk assessment context1

Section 1Background1

Section 2Regulatory framework1

Section 3The proposed dealings2

3.1The proposed limits of the dealings (size, location, duration and people)2

3.2The proposed controls to restrict the spread and persistence of the GMOs and their genetic material in the environment3

Section 4The parent organism4

Section 5The GMOs, nature and effect of the genetic modification4

5.1Introduction to the GMOs4

5.2The introduced genes, encoded proteins and their associated effects5

5.3Toxicity/allergenicity or other adverse effects upon health associated with the introduced genes, their encoded proteins and associated products6

5.4Characterisation of the GMOs7

Section 6The receiving environment8

6.1Relevant abiotic factors8

6.2Relevant agricultural practices8

6.3Presence of related plants in the receiving environment8

6.4Presence of similar genes and encoded proteins in the environment8

Section 7Relevant Australian and international approvals9

7.1Australian approvals9

7.2International approvals of GM wheat9

Chapter 2Risk assessment10

Section 1Introduction10

Section 2Risk Identification11

2.1Risk source11

2.2Causal pathway12

2.3Potential harm13

2.4Postulated risk scenarios13

Section 3Uncertainty26

Section 4Risk evaluation27

Chapter 3Risk management29

Section 1Background29

Section 2Risk treatment measures for identified risks29

Section 3General risk management29

3.1Licence conditions to limit and control the release29

3.2Other risk management considerations32

Section 4Issues to be addressed for future releases34

Section 5Conclusions of the RARMP34

References35

Appendix ASummary of submissions from prescribed experts, agencies and authorities44

Appendix BSummary of submissions from the public45

Table of ContentsIII

Abbreviations

APVMA

Australian Pesticides and Veterinary Medicines Authority

CCI

Confidential Commercial Information as declared under section 185 of the Gene Technology Act 2000

DAFWA

Department of Agriculture and Food, Western Australia

DIR

Dealings involving Intentional Release

FSANZ

Food Standards Australia New Zealand

GM

Genetically modified

GMO

Genetically modified organism

ha

Hectare

HMW-GS

High molecular weight glutenin subunit

HMW-GSs

High molecular weight glutenin subunits

LGA

Local government area

LMW-GS

Low molecular weight glutenin subunit

LMW-GSs

Low molecular weight glutenin subunits

m

Metres

NGNE

New Genes for New Environments

NLRD

Notifiable low risk dealings

OGTR

Office of the Gene Technology Regulator

PC2

Physical Containment level 2

ppm

Parts per million

RARMP

Risk Assessment and Risk Management Plan

Regulations

Gene Technology Regulations 2001

Regulator

Gene Technology Regulator

TGA

Therapeutic Goods Administration

the Act

The Gene Technology Act 2000

Abbreviations IV

Risk assessment context

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 genetically modified organisms (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).

PROPOSED DEALINGS

Proposed activities involving the GMO

Proposed limits of the release

Proposed control measures

PARENT ORGANISM

Origin and taxonomy

Cultivation and use

Biological characterisation

Ecology

PREVIOUS RELEASES

GMO

Introduced 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 ENVIRONMENT

Environmental conditions

Agronomic practices

Presence of related species

Presence of similar genes

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

Regulatory framework

Sections 50, 50A and 51 of the Act outline the matters that the Gene Technology Regulator (the Regulator) must take into account, and who must be consulted with, in 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 Gene Technology Act 2000 (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 Gene Technology Regulator (the Regulator) was not required to consult with prescribed experts, agencies and authorities before preparation of the Risk Assessment and Risk Management Plan (RARMP; see section 50 of the Act).

4. 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. The advice from the prescribed experts, agencies and authorities and how it was taken into account is summarised in Appendix A. Five public submissions were received and their considerations are summarised in Appendix B.

5. The Risk Analysis Framework (OGTR 2013) 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.

6. 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), Australian Pesticides and Veterinary Medicines Authority (APVMA), Therapeutic Goods Administration (TGA), 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.

The proposed dealings

7. Murdoch University proposes to release up to 35 lines[footnoteRef:2] of genetically modified (GM) wheat into the environment under limited and controlled conditions. [2: The term ‘line’ is used to denote plants derived from a single plant containing a specific genetic modification resulting from a single transformation event.]

The purpose of the trial is to assess whether the introduction and expression of the genes will increase the strength of bread dough. Published research has shown that increasing the levels of certain proteins by genetic transformation (ie introducing additional copies of the genes in order to increase the quantity of their corresponding proteins) is a useful way of generating novel doughs for characterisation. In addition, the release will allow the applicant to produce sufficient grain for subsequent replicated trials.

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

· conducting experiments with the GMOs

· breeding the GMOs

· propagating the GMOs

· growing or culturing the GMOs

· transporting the GMOs

· disposing of the GMOs

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

These dealings are detailed further below.

The proposed limits of the dealings (size, location, duration and people)

9. The applicant proposes to grow GM wheat plants between May 2015 and December 2017.

10. The GMOs are proposed to be planted in the New Genes for New Environment (NGNE) facility located at Katanning in Western Australia. This facility is operated by the Department of Agriculture and Food, Western Australia (DAFWA).

11. The maximum area of the trial in any year would be up to 0.06 hectares (ha).

12. Only trained and authorised staff are proposed to be permitted to deal with the GM wheat. Any other visitors to the trial site would be accompanied by an authorised Murdoch University representative and would not deal with the GMOs.

The proposed controls to restrict the spread and persistence of the GMOs and their genetic material in the environment

The applicant has proposed a number of controls to restrict the spread and persistence of the GM wheat lines and the introduced genetic material in the environment, including:

· locating the NGNE facility at least 50 m from the nearest waterway

· surrounding the facility by a 1.8 m fence to exclude large animals and using a bird proof netting (these are existing features of the NGNE facility)

· implementing a rodent control program

· surrounding the facility by a 10 m monitoring zone and 190 m isolation zone in which no sexually compatible plants will be grown

· cleaning any equipment used with the GM plants before removal from the site, and the disposal of any material collected during cleaning in a manner approved by the Regulator

· separating the GM wheat plants by a buffer of at least 4 m if other GM wheat DIRs are being grown side by side in the same facility

· monitoring the planted locations at least once every fortnight during the flowering of the GM plants

· post-harvest monitoring of the trial site (once every 35 days) and destruction of any volunteer wheat for at least 24 months

· destroying any plant material collected during cleaning by autoclaving, hammer-milling, incineration, burial, or any other method approved by the Regulator

· ploughing back waste material and stubble from harvesting into the soil

· grain/dough testing in the PC2 laboratory at Katanning, all laboratory equipment being subjected to cleaning both before and after use

· transporting and storing GM material in accordance with the Regulator's Guidelines for the Transport, Storage and Disposal of GMOs (2011)

· not allowing GM plant material or products to be used for human food or animal feed.

13. Figure 2 shows the proposed site layout at the Katanning NGNE facility, including some of these controls. These controls, and the limits outlined above, have been taken into account in establishing the risk assessment context (this Chapter), and their suitability for containing the proposed release is evaluated in Chapter 3, Section 3.1.1.

NGNE site

 

 

 

 

 

190 m wide

Isolation Zone, inspected for wheat during the flowering of GMOs

10 m wide

Monitoring

Zone, where the growth of plants is controlled

2 m wide Buffer

Zone where the growth

of plants is controlled

Planting area where GM wheat is planted

Fence

Figure 2 Proposed trial layout, including some of the controls (not drawn to scale)

The parent organism

14. The parent organism is bread wheat (Triticum aestivum L.), which is exotic to Australia. Commercial wheat cultivation occurs in the wheat belt from south eastern Queensland through New South Wales, Victoria, Tasmania, southern South Australia and southern Western Australia.

15. All lines were initially in the cultivar Bobwhite, but were backcrossed into the cultivars Calingiri, Wyalkatchem, Westonia, and IGW2836 (NLRD 2341/2007).

16. Detailed information about the parent organism is contained in the reference document The Biology of Triticum aestivum L. em Thell (bread wheat) (OGTR 2008), which was produced to inform the risk assessment process for licence applications involving GM wheat plants. This document is available from the OGTR website.

The GMOs, nature and effect of the genetic modification

Introduction to the GMOs

The applicant proposes to release up to 35 lines of GM wheat. The lines were originally produced in the wheat cultivar Bobwhite by biolistics 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.

Each GM wheat line has been transformed with a construct containing one of two genes of interest, or a hybrid of these genes. The GM plants also contain the selectable marker gene bar, originating from Streptomyces hygroscopicus.

The expression of the individual Dx5 and Dy10 genes in the GM wheat lines are controlled by their native 5’ and 3’ regulatory sequences. For the Dy10Dx5 hybrid gene, the expression is controlled by the Dy10 promoter and the Dx5 terminator. The bar gene is driven by the promoter consisting of the 5’ untranslated exon and first intron of the maize ubiquitin (ubi-1) gene (Christensen & Quail 1996; Christensen et al. 1992), and followed by the nos terminator from Agrobacterium tumefaciens. A summary of these genes is presented in Table 1.

Table 1 Summary of genes and regulatory elements introduced into the GM wheat plants.

Promoter (origin)

Gene of interest (origin)

Terminator (origin )

Dx5 (wheat)

Dx5 (wheat)

Dx5 (wheat)

Dy10 (wheat)

Dy10 (wheat)

Dy10 (wheat)

Dy10 (wheat)

Dy10-Dx5 hybrid (Sequences of wheat genes Dx5 and Dy10)

Dx5 (wheat)

Ubi-1 (maize)

bar (Streptomyces hygroscopicus)

nos (Agrobacterium tumefaciens)

The introduced genes, encoded proteins and their associated effects

Glutenin genes

The application involves a field trial of GM wheat plants containing either of two wheat glutenin genes, Dx5 and Dy10 (Anderson & Greene 1989; Anderson et al. 1989), or a hybrid gene consisting of a single open reading frame of sequences derived from both Dx5 and Dy10 (Blechl & Anderson 1996). The hybrid gene codes for a mature protein that consists of amino acids 1-124 from Dy10 fused N-terminal to amino acids 130-848 from Dx5 (Blechl & Anderson 1996).

Glutenins are seed storage proteins that act in germinating seeds as a supply of elements such as carbon, nitrogen and sulphur. They are divided into two classes: low molecular weight glutenin subunits (LMW-GSs) and high molecular weight glutenin subunits (HMW-GSs) (Akagawa et al. 2007; Battais et al. 2008; Wieser 2007).

The production of wheat dough is dependent upon the structure and composition of the glutenin and gliadin seed storage proteins, which in the presence of water crosslink to form a matrix, gluten. In particular, the HMW-GSs, such as Dx5 and Dy10, play a critical role in determining the viscoelastic properties that are crucial for the formation of bread dough (Shewry et al. 2003). Bread wheat has three loci, designated Glu-A1, Glu-B1 and Glu-D1, that contain HMW-GS genes, these being located on the long arms of chromosomes 1A, 1B and 1D, respectively (Shewry et al. 2003; Shewry & Tatham 1997). Each locus contains two genes, one gene encoding an x-type and the other a y-type subunit. The amino acid sequences of these subunits are characterised by highly repeated blocks of sequence. Both x -type and y -type subunits contain hexapeptide and nonapeptide motifs, the major difference between these two types of subunits being that the former also contains tripeptide motifs (Shewry et al. 1992; Shewry et al. 2003).

Each gene has alleles. As specfic x-type and y-type alleles usually occur together at a locus, their separation by recombination is difficult; this gene pair hence constitute what can be regarded as a single allele. The Dx5 and Dy10 genes occur together as an ‘allele’ (Glu-D1d) of the locus located on chromosome 1D. Other ‘alleles' at this locus include Dx2+Dy12 (Glu-D1a) and Dx4+Dy12 (Glu-D1b) (Zheng et al. 2011). In many plants, certain HMW-GS genes are expressed at low levels or silent (Forde et al. 1985; Wieser & Zimmermann 2000). As such, a plant commonly produces a smaller number of HMW-GS proteins than may be theoretically predicted (Anjum et al. 2007; Shewry et al. 2003).

The properties of dough that are linked to glutenin have been investigated by a number of studies that have involved the transformation into wheat of glutenin genes (Blechl et al. 2007). Many of these experiments have involved the co-bombardment of immature embryos with high copy number plasmids containing the genes of interest and a plasmid with a herbicide tolerant gene to enable selection of transformants. In one study, aimed at evaluating agronomic performance of bread wheat lines transformed with glutenin genes, the introduction of Dx5 and Dy10 did not show significant changes in traits associated with performance (Bregitzer et al. 2006). Other research with varieties of bread wheat transformed with the Dx5 and Dy10 genes has shown that both these genes affect the mixing properties of dough, but in different ways (Blechl et al. 2007). Increasing the level of the Dy10 protein over five times above that normally found in wheat plants did not prevent dough mixing. However, smaller increases in the Dx5 protein led to greater dough strength, problems with mixing, higher amounts of polymeric protein and lower sedimentation values. It has been suggested that these differences between these two HMW-GSs is possibly due to variations between the sizes and composition of their repetitive domains, and their abilities to form both intermolecular and intramolecular disulphide bonds (Blechl et al. 2007).

The studies described above were conducted in a wheat background containing endogenous Dx5 and Dy10 genes. Other work has involved separately transforming the Dx5 and Dy10 genes into a genetic background that does not contain these genes, thus enabling the individual effects of these two genes on bread quality to be more accurately evaluated (Leon et al. 2009). Expression of Dy10 alone resulted in lines with greater dough strength than those expressing Dx5. Conventional crossing of these lines demonstrated that a combination of Dx5 and Dy10 produced a dough with the greater strength and better dough mixing properties than some other subunit combinations that were evaluated (Leon et al. 2010).

Varieties of durum wheat (Triticum turgidum L. ssp. durum) have also been co-transformed with the wheat genes Dx5 and Dy10 (Gadaleta et al. 2008). The mixing times and peak resistances of dough from the transformed lines were likewise greater than those recorded in the wild-type parents, indicating that expression of the inserted genes was having a positive effect upon strength. With respect to the wild-type parent varieties, yields from the transformed lines were similar.

In another line of research, durum wheat varieties separately expressing the HMW-GS 1Ax1 gene and the Pina gene linked to grain hardness were generated, and then conventional breeding used to combine the two genes (Li et al. 2010). Analysis of these lines suggested that over-expression of both these genes can enhance dough strength. The effects of changing the levels of gliadins on dough properties has also been investigated, the down-regulation of these genes being mediated by RNAi (Gil-Humanes et al. 2012; Gil-Humanes et al. 2008).

Examination of the protein complement of wheat plants that have been transformed via biolistics with HMW-GS genes has revealed that there are frequently HMW-GS proteins that are either larger or smaller than the predicted sizes (Blechl & Vensel 2013). These are likely the result of transformation events that alter the sequence of the genes or result in tandem repeats.

Selectable marker genes

All the GM wheat lines contain the selectable marker gene bar, derived from Streptomyces hygroscopicus. This gene codes for the enzyme phosphinothricin N-acetyltransferase (PAT), which provides tolerance to the broad spectrum herbicide phosphinothricin, also known as glufosinate ammonium. This gene has been used extensively as a selectable marker gene in the production of GM plants. More information on selectable marker genes in general, can be obtained from the OGTR document Marker genes in GM plants, available on the OGTR website.

The plants may also contain the bacterial ampicillin resistance (bla) gene (Sutcliffe 1979), which codes for the enzyme beta-lactamase. The promoter of this gene is specific for prokaryotic organisms; thus the ampicillin protein will not be expressed in plants.

Toxicity/allergenicity or other adverse effects upon health associated with the introduced genes, their encoded proteins and associated products

The introduced genes of interest, Dx5 and Dy10, originate from wheat. This plant is widely consumed by people and animals, and as such people and animals have a long history of exposure to its proteins.

There is no evidence that wheat has any toxic properties. However, a minority of people suffer from allergies to wheat (and other cereals). The two most well characterized allergies to wheat are baker’s asthma (induced by the inhalation of wheat flour during grain processing) and exercise induced anaphylaxis (a reaction that occurs if someone undergoes physical activity soon after consumption of wheat) (Hischenhuber et al. 2006; Tatham & Shewry 2008). These allergies are likely due to a number of compounds in cereals, although the most important triggers are believed to be the -amylase inhibitors and gluten (glutenin and gliadin) seed storage proteins (Battais et al. 2008; Salcedo et al. 2011; Tatham & Shewry 2008). Rashes, wheezing, swelling and abdominal pains are all symptoms of wheat allergy, while pathogenic mechanisms of induction include IgE-mediated and cell-mediated (Hischenhuber et al. 2006). Wheat allergy is often a temporary condition of young children that is outgrown by the age of five (Pietzak 2012).

Both HMW-GSs and LMW-GSs have been implicated in wheat allergy. Experiments have suggested that HMW-GS proteins are allergens in exercise induced anaphylaxis (Matsuo et al. 2005; Morita et al. 2009; Yokooji et al. 2013). Recombinant expressed proteins have been shown to react with sera from patients with wheat allergies (Eriksson et al. 2012; Maruyama et al. 1998). Screening of a wheat cDNA expression library with antisera from wheat allergenic patients identified clones coding for HMW-GS, three likely representing a Dx gene and three Bx7, other experiments identifying the latter as an important allergen (Baar et al. 2014). Three specific sequences in HMW-GSs (which occur in both the Dx5 and Dy10 proteins (Anderson et al. 1989; Sugiyama et al. 1985; Thompson et al. 1985)) have been identified as IgE-binding epitopes (Matsuo et al. 2005).

In the case of LMW-GS proteins, a proteomic study of wheat allergens identified nine members of this class as important allergens, but could not confirm previously reported examples classified as HMW-GS (Akagawa et al. 2007). Techniques such as screening of a wheat cDNA library with sera from patients with wheat allergy have also suggested a connection of LMW-GS proteins to this condition (Baar et al. 2012).

Coeliac (celiac) disease is an autoimmune (genetic) disorder that is triggered by the consumption of gluten (Denham & Hill 2013; Hischenhuber et al. 2006). It is characterised by the immune system attacking the small intestine and inhibiting the absorption of nutrients into the body, likely leading to permanent tissue damage. People can also suffer from gluten intolerance (gluten sensitivity), an adverse reaction to wheat that is neither an allergenic reaction or mediated by the immune system (Pietzak 2012; Sapone et al. 2012).

Distinct varieties of wheat often have different quantities (per grain) of glutenin and gliadin proteins, the quantities in any variety also being influenced by the environment in which the plant is grown (Plessis et al. 2013). From a molecular viewpoint, the synthesis of these proteins is likely regulated primarily at the transcriptional level. Activation and down-regulation of the expression of their respective genes probably involves a number of different transcription factors binding to each other, and either directly or indirectly to DNA promoter elements. Transcription factors that have been associated with gluten protein levels include members of the Opaque2 subfamily of basic leucine zipper (bZIP) factors (storage protein activators) (Albani et al. 1997; Ravel et al. 2009) and DOF (DNA binding with one finger) proteins (Dong et al. 2007; Romeuf et al. 2010). The levels of such transcription factors, and hence seed storage proteins, may be linked to biochemical issues, including the assimilation of nitrogen and the availability of amino acids. Quantitative trait loci linked to the levels of gluten proteins have also been identified and mapped (Zhang et al. 2011).

Characterisation of the GMOs

Stability and molecular characterisation

Based on experiments in the United States, the genes are stably inserted into the genome of the cultivar Bobwhite. There is no reason to expect that conventionally breeding of these genes into other cultivars will affect their stability. However, the sites of insertion and copy numbers of the inserted genes are not known. The proposed field trial will evaluate the expression of the genes in a number of Australian cultivars.

Phenotypic characterisation

Preliminary phenotypic characterisation of the plants in glasshouses has demonstrated that the introduced genes induce no major visible phenotypes or reduce viability.

The receiving environment

The receiving environment includes: any relevant biotic/abiotic properties of the geographic regions where the release 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 2013).

The factors relevant to the growth, distribution and cultivation of commercial wheat can be found in The Biology of Triticum aestivum L. em Thell (Bread Wheat) (OGTR 2008).

It is proposed that the dealings be conducted in the NGNE facility, operated by DAFWA, located at Katanning in Western Australia.

Relevant abiotic factors

The Katanning NGNE facility is purpose built for the trialling of GM plants. Katanning represents the high rainfall environment used for growing wheat in Western Australia. The soil of Katanning is largely alkaline and sodic.

Relevant agricultural practices

It is not anticipated that the agronomic practices for the cultivation of the GM wheat by the applicant will be significantly different from conventional practices for these plants.

GM wheat seeds would be planted in the trial site in winter or early spring. Seed that remain after harvest would be either stored in an approved facility for subsequent use or destroyed. Volunteers would be removed by hand or killed by herbicide application.

Presence of related plants in the receiving environment

Wheat (Triticum aestivum L.) is sexually compatible with a number of species within the tribe Triticeae that occur in Australia. Of particular importance are durum wheat (Triticum turgidum ssp. Durum), rye (Secale cereale), and Triticale. Hybridisation with durum wheat occurs readily (Wang et al. 2005), whereas that with rye (Dorofeev 1969; Leighty & Sando 1928; Meister 1921) and Triticale (Ammar et al. 2004; Kavanagh et al. 2010) is rarer. Wheat also readily hybridises with Aegilops species, but no Aegilops species are considered to be naturalised in Australia. Any specimens of Aegilops that have been collected in Australia presumably originate from seed accidently introduced amongst wheat seed, or straying from that brought in for breeding programs (AVH 2012).

Australasia possesses four native Triticeae genera - Australopyrum, Stenostachys, Anthosachne (Elymus), and Connorochloa (Barkworth & Jacobs 2011) – as well as a number of introduced species of Triticeae, such as Elytrigia repens (couch grass) and at least four Thinopyrum species (Bell et al. 2010). Thinopyrum ponticum (tall wheatgrass) has been used as a saltland pasture plant in Australia, and in some regions has come to be classified as a weed (Barrett-Lennard 2003; NYNRMP 2011). Although there has been no concerted investigation of natural hybridisation of these native and introduced Triticeae species with wheat, based on experience of hybridising wheat with most other members of the Triticeae, it is unlikely that it occurs.

As the Katanning NGNE facility is designed to be accommodate more than one trial (which may reflect more than one licence holder), it is possible that other GM and non-GM wheat plants will be grown there in close proximity to those plants that are the subject of this application. Currently, GM wheat plants from DIR 128 which are genetically modified for abiotic stress tolerance or micronutrient uptake may be present.

Presence of similar genes and encoded proteins in the environment

The introduced genes and other genetic elements are from a plant (wheat) that is widespread and prevalent in the environment (see Section 5). It is commonly consumed by people who are therefore exposed to the proteins. Other cereals (that are consumed by people) have glutenin proteins (Shewry & Halford 2002).

The bar selectable marker gene comes from Streptomyces hygroscopicus, which is widespread in the environment.

Relevant Australian and international approvals

Australian approvals

Approval by the Regulator

17. Wheat lines possessing the introduced genes have not previously been approved by the Regulator.

18. Information on previous DIR licences for GM wheat can be found on the GMO Record on the OGTR website.

Approval by other government agencies

19. No other approvals are currently required for this GM wheat trial.

International approvals of GM wheat

20. Field trials of the GM wheat lines in the Bobwhite genetic background have been carried out in the USA in 2002 and 2003 (information provided by the applicant).

21. Field trials of different GM wheat plants have been approved internationally, including in the USA, Canada, Germany, Czech Republic, Denmark, Hungary, Iceland, Italy, Spain, Sweden and the United Kingdom. The traits that have been modified include: novel protein production, disease resistance, insect resistance, altered grain properties and herbicide tolerance[footnoteRef:3]. [3: USDA release permit applications, EU GMO Register, accessed November 2014.]

Chapter 1 – Risk assessment context9

Risk assessment

Introduction

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 3). 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.

RISK ASSESSMENT PROCESS *

Risk

scenarios

Substantive Risks

Risk Evaluation

Consequence assessment

Likelihood assessment

Identification of substantive risks

Negligible risks

RISK IDENTIFICATION

RISK CHARACTERISATION

Risk context

Postulation of risk scenarios

* Risk assessment terms are defined in the Risk Analysis Framework 2013

Figure 3 The risk assessment process

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 (risk scenarios) in the short and long term.

Postulated risk scenarios are screened to identify substantive risks that warrant detailed characterisation. A substantive risk is only identified for further assessment when a risk scenario is 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.

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 2013). 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). In addition, risk scenarios postulated in previous RARMPs prepared for licence applications of the same and similar GMOs are also considered.

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

Risk Identification

Postulated risk scenarios are comprised of three components (Figure 4):

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

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

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

source of

potential harm

(a novel GM trait)

potential harm to

an object of value

(people/environment)

plausible causal linkage

Figure 4 Risk scenario

In addition, the following factors are 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 limit the spread and persistence of the GMO

· characteristics of the parent organism(s).

Risk source

The source 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.

As discussed in Chapter 1, each of the GM wheat lines has been modified by the introduction of one of two HMW-GS genes, or a hybrid of both, that are expected to confer improvement in grain quality. These introduced genes are considered further as potential sources of risk.

The GM lines also contain the bar selection marker gene (see Chapter 1, Section5.2.2). This gene and its product has 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 other regulatory agencies in Australia and overseas. Toxicity feeding study expermiments have failed to establish any deleterious effects of the PAT protein upon animals (Herouet et al. 2005; MacKenzie et al. 2007; Merriman 1996). More information on selectable marker genes can be obtained from the OGTR document Marker genes in GM plants, available on the OGTR website. As this gene has not been found to pose substantive risks to either people or the environment, its potential effects will not be further assessed for this application.

In addition, the GM plants may also contain the bla gene (see Chapter 1, Section5.2.2). There is no evidence that the protein encoded by this gene has toxic or allergenic properties. The RARMPs for DIRs 070/2006 and 071/2006 concluded that the risks posed from the expression of a prokaryotic promoter driven bla gene in GM plants were negligible. Even if the bla gene was expressed in the wheat, human exposure to the beta-lactamase protein is routine as ampicillin resistant bacteria occur widely in the environment. For example, E.coli with such a trait is common in the normal human intestine (EFSA 2004; EFSA 2009). Therefore, this gene will not be assessed further for this application.

The HMW-GS genes include endogenous wheat regulatory sequences while the promoter of the bar gene is derived from a maize ubiquitin gene. There is no evidence that regulatory sequences themselves have toxic or allergenic effects (EPA 1996); such effects for these sequences will not be further assessed for this application. However, regulatory sequences, especially the promoters, control the levels of gene expression and hence the levels of the derived proteins in the GM plants. The effects of these protein levels on, in particular, the toxicity and allergenicity of these plants (or at least materials derived from them), will be discussed below.

Causal pathway

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 GM plant, including

establishment

reproduction

dispersal by natural means and by people

tolerance to abiotic conditions (eg climate, soil and rainfall patterns)

tolerance to biotic stressors (eg pest, pathogens and weeds)

tolerance to cultivation management practices

gene transfer to sexually compatible organisms

gene transfer by horizontal gene transfer (HGT)

unauthorised activities.

Although all of these factors are taken into account, some have been considered in previous RARMPs or are not expected to give rise to substantive risks.

The potential for horizontal gene transfer (HGT) and any possible adverse outcomes has been reviewed in the literature (Keese 2008) as well as assessed in many previous RARMPs. HGT was most recently considered in the RARMP for DIR 108. This and other RARMPs are available from the GMO Record on the OGTR website or by contacting the OGTR. No risk greater than negligible 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 assessed further.

The potential for unauthorised activities to lead to an adverse outcome has been considered in previous RARMPs. The Act provides for substantial penalties for non-compliance and unauthorised dealings with GMOs. The Act also requires the Regulator to have regard to the suitability of the applicant to hold a licence prior to the issuing of a licence. These legislative provisions are considered sufficient to minimise risks from unauthorised activities, and no risk greater than negligible was identified in previous RARMPs. Therefore, unauthorised activities will not be considered further.

Potential harm

Potential harms from GM plants include:

harm to the health of people or desirable organisms, including toxicity/allergenicity

reduced establishment of desirable plants, including having an advantage in comparison to related plants

reduced yield of desirable vegetation

reduced products or services from the land use

restricted movement of people, animals, vehicles, machinery and/or water

reduced quality of the biotic environment (eg providing food or shelter for pests or pathogens) or abiotic environment (eg negative effects on fire regimes, nutrient levels, soil salinity, soil stability or soil water table)

reduced biodiversity through harm to other organisms or ecosystems.

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

Postulated risk scenarios

Five risk scenarios were postulated and screened to identifiy substantive risk. These scenarios are summarised in Table 2 and more detail of these scenarios is provided later in this Section. Postulation of risk scenarios considers impacts of the GM wheat or their 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.

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

Table 2 Summary of risk scenarios from dealings with GM wheat genetically modified for improved grain quality

Risk scenario

Risk source

Causal pathway

Potential harm

Substantive risk?

Reason

1

Introduced genes for improved grain quality

Growing GM plants at the site

Expression of genes in GM plants

Exposure of people who specifically deal with the GM plant material or other organisms that come into contact with the GM plant material in the trial site

· Allergic reactions in people or toxicity in people and other organisms

No

· The limited scale, short duration and other proposed limits and controls minimise exposure of people and other organisms to the GM plant material.

· Plant material from the GMOs would not be used for human food or animal feed.

· The introduced proteins are not known to be toxic. Although they have been associated with allergenicity, there is uncertainty whether they will affect the allergenic threshold in individuals.

2

Introduced genes for improved grain quality

Dispersal of GM seed outside trial limits

Growth of GM plants

Expression of genes in GM plants

Spread and persistence of populations of GM plants outside trial limits

Exposure of people or other organisms to GM plant material

· Allergic reactions in people or toxicity in people and other organisms

· Reduced establishment and yield of desirable plants

· Reduced biodiversity

No

· The limited scale, short duration and other proposed limits and controls minimise the likelihood that GM plant material would leave a trial site.

· The introduced proteins are not known to be toxic. Although they have been associated with allergenicity, there is uncertainty whether they will affect the allergenic threshold in individuals.

· The introduced genes are not expected to increase the ability of the GM plants to spread and persist.

3

Introduced genes for improved grain quality

Dispersal of GM pollen outside trial limits

Vertical transfer of introduced genes to other sexually compatible plants, such as commercial varieties of wheat

Expression of genes in plants

Exposure of people or other organisms to GM plant material

· Allergic reactions in people or toxicity in people and other organisms

· Reduced establishment and yield of desirable plants

· Reduced biodiversity

No

· The limited scale, short duration and other proposed limits and controls minimise the likelihood that GM plant material would leave a trial site.

· The introduced proteins are not known to be toxic. Although they have been associated with allergenicity, there is uncertainty whether they will affect the allergenic threshold in individuals.

· The introduced genes are not expected to increase the ability of the GM plants to spread and persist.

4

Introduced genes for improved grain quality

Dispersal of GM pollen within the NGNE facility

Hybridisation of GM plants of this trial with GM plants (including volunteers) of another trial

Expression of genes in stacked GM plants

Exposure of people or other organisms to GM plant material

· Allergic reactions in people or toxicity in people and other organisms

No

· The limited scale, short duration and other proposed limits and controls minimise exposure of people and other organisms to the GM plant material.

· The introduced proteins are not known to be toxic. Although they have been associated with allergenicity, there is uncertainty whether they will affect the allergenic threshold in individuals.

· The stacking of genes from different GM plants is unlikely to increase the toxicity or allergenicity of the hybrid GM plants.

5

Introduced genes for improved grain quality

Dispersal of GM pollen within NGNE site

Hybridisation of GM plants of this trial with GM plants (including volunteers) of another trial

Dispersal of plants or viable plant material containing stacked genes outside the trial limits

Expression of genes in stacked GM plants

Spread and persistence of populations of GM plants outside a trial site

· Reduced establishment and yield of desirable plants

· Reduced biodiversity

No

· The limited scale, short duration and other proposed limits and controls minimise the likelihood that GM plant material would leave a trial site.

· The introduced genes are not expected to increase the ability of the GM plants to spread and persist.

· The stacking of genes from different GM plants is unlikely to increase the weediness of the hybrid GM plants.

Risk scenario 1

Risk source

Causal pathway

Potential harm

Introduced genes for improved grain quality

Growing GM plants at the site

Expression of genes in GM plants

Exposure of people who specifically deal with the GM plant material or other organisms that come into contact with the GM plant material in the trial site

· Allergic reactions in people or toxicity in people and other organisms

Risk source

The source of potential harm for this postulated risk scenario is the introduced genes for improved grain quality.

Causal pathway

The grain quality improvement genes are expressed in the plant tissues. People who are involved in the breeding, cultivating, harvesting, transporting and experimenting of the GM wheat may be exposed to its products through contact (including inhalation of pollen). This would be expected to mainly occur in the trial site, but could also occur anywhere the GM plant material was transported or used for experimental analysis. Organisms that may be present in the trial site, including birds, rodents and invertebrates, may be exposed to the GM plant material.

22. The proposed limits and controls of the trial would minimise the likelihood that people or other organisms would be exposed to GM plant material. Although people may directly handle the GM plant material, it is not to be used as human food. Further, as the trial is limited to a single site of 0.06 ha, only a small number of people would deal with the GM plant material and a small number of other organisms are likely to be exposed to it. GM plant material is not to be used as animal feed.

23. A fence surrounding the NGNE facility will exclude livestock and other large animals, while rodent control measures will be used to reduce the number of these animals. Further, the facility has bird netting.

24. The applicant has also proposed a series of measures, such as the monitoring and inspecting of the site, together with the cleaning of equipment, the storing of seed and the disposal of waste material that will together help reduce exposure of people to GM plant material in the trial site and the adjacent areas.

Potential harm

People exposed to the proteins expressed from the introduced genes or their associated products may show toxic or allergic reactions, while other organisms may show toxic reactions (Chapter 2, Section 2.3). Proteins are not generally associated with toxic effects. As opposed to small molecular weight chemicals (eg pesticides), proteins have a number of properties that limit their ability to produce toxic effects upon ingestion, these including their likely digestion in the gastrointestinal tract and difficulties they encounter in traversing plasma membranes (Hammond et al. 2013). However, a small number of proteins have been shown to be toxic to humans and mammals, these originating mainly from animals (eg snakes, scorpions) and bacteria (Henkel et al. 2010; Karalliedde 1995). Lectins and protease inhibitors are plant proteins that have toxic properties, but for most people the level of exposure and response to the majority of these compounds is such that they are often classified as anti-nutrients (Delaney et al. 2008). The most well known plant proteins that are definitely toxic to humans are the lectins that consist of a ribosome-inactivating peptide (RIP) linked to a carbohydrate binding peptide, examples being ricin, abrin and modeccin (de Virgilio et al. 2010; Stirpe 2005; Wu & Sun 2011). Other plant proteins, found to be toxic at least to mice, include the urease-like-protein canatoxin from jack beans (Follmer et al. 2001) and an acidic protein and a glycoprotein from soybean (Morais et al. 2010; Vasconcelos et al. 2008; Vasconcelos et al. 1994).

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). The structural and functional properties of plant food allergens can be used to classify them into approximately 30 families, these then being grouped into a small number of superfamilies (Hauser et al. 2008; Radauer & Breiteneder 2007; Salcedo et al. 2008). The major superfamilies are the prolamins, cupins, pathogenesis-related (PR) proteins, profilins and protease inhibitors. Beyond toxicity and allergenicity, specific proteins and chemicals have been associated with autoimmune diseases and food intolerances (ASCIA 2014; Barragan-Martinez et al. 2012; Selmi et al. 2012).

Chapter 1, Section 5.3 presents a review of the potential toxic and allergenic properties of the proteins encoded by the introduced genes. It was concluded that none of the introduced proteins were likely to be toxic to people or other organisms. In respect of the general information on toxicity and plant proteins outlined above, none of the introduced plant proteins can be classified as a lectin or protease inhibitor (ie a likely toxin).

The glutens are prominent members of the prolamin family that have been associated with allergenicity, and, as noted in Chapter 1, Section 5.3, the HMW-GS proteins have been associated with negative effects on the health of people. These effects have been grouped as allergies (eg the consumption of glutenin can induce baker’s asthma or exercise induced anaphylaxis), autoimmune diseases (eg coeliac disease) and ‘gluten sensitivity’ (Sapone et al. 2012). Specific studies that have used the Dx5 and Dy10 proteins, or epitope peptides based on their sequences, have implicated these HMW-GSs in exercise induced anaphylaxis (Matsuo et al. 2005; Morita et al. 2009; Yokooji et al. 2013).

For the evaluation of protein safety, the ILSI International Food Biotechnology Committee has collaborated with a group of experts to produce a two tiered method to assess the safety of proteins (Delaney et al. 2008; Hammond et al. 2013). The first tier examines five issues: (i) history of safe use of the protein; (ii) bioinformatics analysis; (iii) mode of action; (iv) in vitro digestibility and stability; and (v) expression level and dietary intake. Only if potential safety issues were identified in this evaluation would a second tier assessment be recommended, a prime example of such an assessment being a dose toxicology study.

The proteins Dx5 and Dy10 were examined against the first tier criteria:

(i) History of safe use. The introduced plant proteins come from a plant (wheat) that does not raise any toxicological concerns and has a history of largely safe use in human diets. A minority of people have allergenic reactions to wheat, possess coeliac disease or have ‘gluten sensitivity’.

(ii) Bioinformatic analysis. The introduced proteins do not have any sequence similarity with known toxins. However, they are members of a protein class, the prolamins, that is known to have members with allergenic properties.

(iii) Mode of action. Plants are responsible for the production of a wide range of small molecular weight compounds, usually designated as secondary metabolites, many of which are toxic to humans and herbivore animals (Wink 2009; Wink & Van Wyk 2008). From the perspective of the plant, they help defend the plant against the predatory activities of these groups. The metabolites of greatest concern are the neurotoxins, followed by cytotoxins and compounds that act as poisons in particular organs. The introduced proteins are considered as storage proteins for amino acids that can be used in germination and seedling growth (Shewry & Halford 2002). They do not have enzymatic functions. As such, they are not expected to give rise to any (enzymatic) small molecular weight products that could have toxic properties.

(iv) In vitro digestibility and stability. No data.

(v) Expression level and dietary intake. No data.

The hybrid gene codes for a protein that consists of the 124 N-terminal amino acids from the mature Dy10 protein fused N terminal to the C terminal 719 amino acids from Dx5 (Blechl & Anderson 1996). Such a protein does not have a history of safe use, but the experience of conventional breeding is that the spurious fusing of gene sequences does not lead to proteins that are of health or environmental concern (Steiner et al. 2013; Weber et al. 2012). Further, as discussed above, the sequences of Dx5 and Dy10 themselves have not been associated with toxicity, and it is not expected that a fusion protein of these sequences will have a mode of action that is different from its progenitors.

Although the Dx5 and Dy10 proteins are endogenous to wheat, it is possible that increased quantities of one or both of these proteins may affect the allergenic threshold level of wheat with respect to gluten. The threshold level of a food can be defined as the maximum quantity of a food, containing one or more allergenic proteins, that can be tolerated without producing any adverse (allergenic) reaction. It can be measured by a number of methods, including epidemiological studies, analytical procedures and anecdotal evidence (FDA 2006). In these circumstances, data from points (iv) and (v) may take on greater importance.

The threshold for eliciting an allergic reaction by a single allergen has rarely been determined, varies between individual people, and can vary in any one individual over time due to factors such as stress, exercise and the use of medications (Fernandez et al. 2013). Further, there have been few studies of the concentrations of endogenous food allergens across different cultivars of one species. When such studies have been conducted, allergen concentrations have been discovered to vary widely between cultivars, and a range of environmental factors can affect the level in any individual cultivar (Fernandez et al. 2013; Herman & Ladics 2011). No mechanisms are presently in place to evaluate and segregate ‘low’ and ‘high’ allergenic cultivars of plants that constitute our food supply (Panda et al. 2013). With such a background, it is not possible to accurately answer the question of how much of an increase in an endogenous allergen would lead to a measurable increase in the level of harm.

Nevertheless, it is possible that an elevation in the level of HMW-GSs in GM wheat could increase the spectrum of people who cannot tolerate food derived from this plant. Although people with food allergies largely avoid the offending foods altogether (gluten-free food being defined by standard 1.2.8, clause 16, of the Australia New Zealand Food Standards Code as ‘no detectable gluten’ (http://www.comlaw.gov.au/Details/F2012C00218)), there are individuals with moderate allergies who know, through experience, they can consume limited amounts of the these foods. The consequence to such individuals of an increase in allergen levels in GM food could be an elicitation of a severe allergenic response (Fernandez et al. 2013). However, as the GM wheat will not be used for human food, no harm as a result of humans consuming the GM wheat will occur.

Experiments with the promoter of the Dx5 gene fused to the GUS reporter gene have demonstrated that this promoter drives expression of a gene in the endosperm of seeds, with no expression being detected in leaf, root inflorescence or floret tissues (Lamacchia et al. 2001). Similar results have been obtained when other HMW-GS promoters have been used to drive reporter genes (Furtado et al. 2009; Weibo et al. 2009). As glutenins are classified as seed storage proteins, these patterns of expression are not unexpected. Thus, in relation to the GM wheat of this application, there is unlikely to be a risk of a pollen-induced allergic reaction, but a possible route that people handling plant material may be exposed to the proteins is through contact with seed. Nevertheless, there is uncertainty as to whether the expression of Dx5 and Dy10 would affect the allergenicity of the GM wheat proposed for release.

Gene technology has the potential to cause unintended effects in several ways, including altered expression of an endogenous gene by random insertion of an introduced DNA in the genome, increased metabolic burden due to higher expression of the introduced protein, novel traits arising out of interactions with non-target proteins and secondary effects arising from altered substrate or product levels in biochemical pathways. Such an effect could lead to elevation of the concentration of a normally benign wheat compound to a level where it induces a toxic or allergenic reaction if consumed in an average diet. It is also possible that an entirely novel compound could be produced with such a reaction. In this context, it is important to note that changes of this nature, such as the unexpected increase in the level of an endogenous toxin, can also be induced in plants by conventional methods of plant breeding (Haslberger 2003). However, even though conventional breeding can involve the movement of hundreds and even thousands of genes into a plant, there has never been a report of a completely novel toxin or allergen appearing in a new line of a plant produced by such techniques (Steiner et al. 2013; Weber et al. 2012). The implication is that the movement into wheat of any of the genes that are the subject of this application, none of which belong to any known class of toxin, is unlikely to result in the production (directly or indirectly) of a novel toxin. This includes the production of such a compound via the site of insertion or the production of a fusion protein. In reference to allergenicity, the introduced glutenin proteins are in a protein class containing allergens which is already present in non-GM wheat. However, the GM wheat will not be used for human food.

Conclusion: Risk scenario 1 is not identified as a substantive risk due to the proposed limits and controls designed to minimise exposure of people and other organisms to the GM plant material and the lack of known toxicity of the introduced proteins. Therefore, this risk could not be greater than negligible and does not warrant further detailed assessment.

Risk scenario 2

Risk source

Causal pathway

Potential harm

Introduced genes for improved grain quality

Dispersal of GM seed outside trial limits

Growth of GM plants

Expression of genes in GM plants

Spread and persistence of populations of GM plants outside trial limits

Exposure of people or other organisms to GM plant material

· Allergic reactions in people or toxicity in people and other organisms

· Reduced establishment and yield of desirable plants

· Reduced biodiversity

Risk source

The source of potential harm for this postulated risk scenario is the introduced genes for improved grain quality.

Causal pathway

The grain quality improvement genes are expressed in plant tissues. If seed was dispersed outside the trial site or persisted at the site, this seed could germinate and give rise to plants expressing the introduced genes. These plants could spread and persist in the environment outside the trial limits and people and other organisms may be exposed to GM plant materials.

Dispersal of GM plant material outside the limits of the trial site could occur through the activity of people (including the use of agricultural equipment), the activity of animals such as rodents, herbivores and birds, through extremes of weather such as flooding or high winds, or persistence at the site once the trial has finished.

Wheat lacks seed dispersal characteristics such as stickiness, burrs and hooks, which can contribute to seed dispersal via animal fur (Howe & Smallwood 1982). The intended introduced trait of improved grain quality is not expected to alter these characteristics of seeds.

Seed dispersal for wheat through endozoochory (the ingestion and excretion of viable seeds) has not been reported. Nevertheless, it cannot entirely be discounted that wheat seeds could be dispersed and germinate after passage through the digestive system of some mammals or birds. For example, viable wheat seeds have been detected in cattle dung (Kaiser 1999). Seeds which survive chewing and digestion by animals are typically small and dormant (Malo & Suárez 1995). Corellas were shown to excrete some viable wheat seeds, although the proportion is extremely low (Woodgate et al. 2011).

Kangaroos, rabbits and rodents are known pests of wheat crops, and cattle or sheep may graze cereals. The site is fenced, limiting the possibility of seed dispersal by any large animals such as cattle, sheep and kangaroos. Rabbits favour soft, green, lush grass (Myers & Poole 1963) and select the most succulent and nutritious plants first (Croft et al. 2002). Although viable seeds from a variety of plant species have been found in rabbit dung, viable wheat seeds were not among them (Malo & Suárez 1995). Other studies have shown that generally very few viable seeds are obtained from rabbit dung (Welch 1985; Wicklow & Zak 1983). Rodents are opportunistic feeders and their diet include seeds and other plant material (Caughley et al. 1998). They may not only eat and destroy seed at the seed source but also hoard seeds (AGRI-FACTS 2002), which increases the possibility of seed dispersal. Only a very limited amount of rodent activity has been observed at trial sites under other GM field trial licences.

Characteristics that influence the spread (dispersal of the plant or its genetic material) and persistence (establishment, survival and reproduction) of a plant species determine the degree of its invasiveness. These characteristics include the ability to establish in competition with other plants, to tolerate standard weed management practices, to reproduce quickly, prolifically and asexually as well as sexually, and to be dispersed over long distances by natural and/or human means.

Baseline information on the weediness of wheat, including factors limiting the spread and persistence of non-GM plants of these species, is given in The Biology of Triticum aestivum L. em Thell (Bread Wheat) (OGTR 2008). In summary, wheat has some characteristics of invasive plants, such as being capable of out-crossing (although it is predominantly self-pollinating) and the ability to germinate or to produce seed in a range of environmental conditions. However, it lacks most of the characteristics that are common to invasive plants, such as the ability to produce a persisting seed bank, rapid growth to flowering, continuous seed production as long as growing conditions permit, high seed output, high seed dispersal and long-distance seed dispersal (Keeler 1989). In addition, wheat has been bred to avoid seed shattering, and white wheat cultivars have little seed dormancy (OGTR 2008).

The expected phenotypic difference between the GM wheat lines and their non-GM progenitors is improved grain quality. This introduced trait is not expected to alter the reproductive or dispersal characteristics of the GM plants. In reference to competitive ability, there is the potential for the GM plants to have an increased distribution in the natural environment and agricultural settings. Although the performance of the GM plants in the field is yet to be determined (which will act as an indication of their performance in natural environments), the trait of improved grain quality cannot readily be interpreted as a cue for the GM plants to increase their invasiveness.

The techniques of conventional breeding (eg selection of plants amongst available germplasm, wide crosses, mutagenesis) have been used to produce varieties of wheat that possess a range of traits. This experience is a useful backdrop against which to view the potential invasiveness of the GM plants with improved grain quality in this application. Crosses with wild relatives have been used to transfer useful genes to crop plants (Goodman et al. 1987; Hajjar & Hodgkin 2007; Maxted & Kell 2009; Prescott-Allen & Prescott-Allen 1998). Wheat is a crop where such transfer has been most fruitful. Wild relatives of wheat that have been exploited as sources of genes include a whole range of Aegilops species (eg Ae. tauschii, Ae. speltoides, Ae. squarrosa), Triticum species (eg T. turgidum subsp. dicoccoides and T. monococcum) and Thinopyrum bessarabicum. Mutagenesis has also been used to generate a number of varieties of cereals (and many other crops) that have a various traits (Ahloowalia et al. 2004; Cheema et al. 1999; Tomlekova 2010)(FAO/IAEA database of mutation enhanced technologies for agriculture).

An increase in the quantity of seed storage proteins in seeds could increase the fitness of the seeds, this in turn increasing the ability of the GM wheat plants to spread and persist. However, no wheat plant that has been generated by any form of conventional breeding has been demonstrated to have increased invasiveness. On the basis of this experience, the genetic modifications are not expected to increase the potential invasiveness of GM plants relative to non-GM plants.

De-domestication, the evolutionary loss of traits gained under domestication, is frequently found amongst the small number of known examples of the evolution of invasiveness amongst existing crop plants, this being most obviously reflected in the acquisition of the ability to more readily disperse seed or fruits (Ellstrand et al. 2010). The only known case of invasiveness in wheat is in Tibet, where ‘semi-wild’ wheat (presumably the result of de-domestication) has shattering heads (brittle rachis) and hulled seeds (toughened glumes preventing free threshing) (Ayal & Levy 2005). Both these traits are absent from domesticated wheat, underlining their importance in marking the boundary between cultivated and weedy forms of this plant; shattering allows easy seed dispersal in the wild, while the hull around grains protects the grain from abiotic and biotic stresses.

If the GM wheat were to lose the trait of non-shattering heads, it would be expected to spread and persist to a greater degree than non-GM commercial varieties, its seed being lost before harvest and contributing greater amounts of seed to a seed bank. As a result, it would be less dependant on human intervention for dispersal. However, there is no reason to believe that the introduction of the genes into wheat is likely to lead to a reversion of either the non-shattering or hull-less traits. The obvious rarity of the natural loss of the non-shattering and hull-less traits in conventionally bred commercial wheat varieties indicate their genetic stability. Further, none of the introduced genes are amongst those known to be associated with shattering or the toughness of the glumes, the latter also being a marker of domestication (Sang 2009).

The proposed limits and controls of the trial would minimise the likelihood of spread and persistence of the GM plants. The small size (up to 0.06 ha per year) will limit the potential for dispersal of GM plant material and exposure to this material. The proposed trial site is surrounded by a fence and only approved staff with appropriate training will have access, this minimising potential for dispersal of seed and exposure to plant material by grazing livestock and people. Dispersal of GM plant material by authorised people entering the proposed trial site would be further minimised by a standard condition of DIR licences which requires the cleaning of all equipment used at the trial site, including clothing. All GM plant material will be transported in accordance with the Regulator’s transport guidelines, which will minimise the opportunity for its dispersal. Furthermore, extra conditions associated with growing the GM plants in the multiuser NGNE facility would also reduce the likelihood of GM plant material being spread. Monitoring of the trial site for volunteers once the GM wheat has been harvested is expected to minimise the likehihood of persistence. Limits and controls are further discussed in Chapter 3.

Potential harm

If GM wheat plants were to establish beyond the trial limits, they could potentially cause one or more of the harms outlined in Section 2.3 of this chapter. As discussed in risk scenario 1, the introduced gene products are not expected to be toxic to humans or other organisms. Although glutenins have been associated with allergenicity, there is uncertainty whether or not their expression in wheat (the organism from which they derive) would affect the endogenous allergenicity of that plant.

With respect to the environment, spread and persistence of the GM plants could reduce the establishment of desired plants. In turn, this could lead to the fragmentation of the habitats of other plants, decreasing the probability of these plants (and the animals that live amongst these plants) maintaining effective breeding populations. As such, there may be a reduction in the biodiversity in regions where the GM plants grew. In reference to native habitats, it must nevertheless be appreciated that there would have to be large numbers of GM plants before the establishment of native plants was affected. In an agricultural setting, a persistent seed bank of GM wheat could reduce the establishment and yield of subsequent crops.

The only expected phenotypic difference between GM wheat and non-GM wheat is altered levels of glutenin. There is no reason to believe that such a trait will have any effect upon the ability of the GM plants to spread and persist, thereby producing any environmental harms. As discussed in Chapter 1, no phenotypic differences were observed between GM wheat and non-GM wheat when grown in glasshouses, and a standard condition of a licence for a field trial would be that the applicant immediately notify the OGTR of any unintended effects.

It is important to note that no commercially released variety of wheat that is the product of any form of conventional breeding has been recorded to have negatively impacted the environment (or the health of animals and/or animals), beyond that normally associated with these cereals, and subsequently flagged as an environmental weed. These conventionally generated varieties represent a wide range of traits (Goodman et al. 1987; Hajjar & Hodgkin 2007; Maxted & Kell 2009; Prescott-Allen & Prescott-Allen 1998). Therefore, the introduction into wheat of any of the genes that are the subject of this application is unlikely to produce a plant that poses a risk to the environment that is any different from the many wheat plants produced by conventional breeding (National Research Council 1989).

Conclusion: Risk scenario 2 is not identified as a substantive risk due to the proposed limits and controls designed to restrict dispersal of the GM wheat and to minimise exposure of people and other organisms to the GM plant material, and the lack of known toxicity of the introduced proteins. Further, the engineered trait is not associated with weediness. Therefore, this risk could not be greater than negligible and does not warrant further detailed assessment.

Risk scenario 3

Risk source

Causal pathway

Potential harm

Introduced genes for improved grain quality

Dispersal of GM pollen outside trial limits

Vertical transfer of introduced genes to other sexually compatible plants, such as commercial varieties of wheat

Expression of genes in plants

Exposure of people or other organisms to GM plant material

· Allergic reactions in people or toxicity in people and other organisms

· Reduced establishment and yield of desirable plants

· Reduced biodiversity

Risk source

The source of potential harm for this postulated risk scenario is the introduced genes for improved grain quality.

Causal pathway

The grain quality improvement genes are expressed in the plant tissues. Pollen from the GM plants could be transferred outside of the trial site (eg via wind) and fertilise sexually compatible plants, whether they be non-GM wheat, or plants from another sexually compatible species. Alternatively, if seed was dispersed outside the trial site, plants expressing the introduced genes may grow and subsequently disperse pollen. Hybrid plants possessing the introduced genes may form the basis for the spread of these genes in other varieties of wheat, or other sexually compatible plant species. People and other organisms could be exposed to the proteins expressed from the introduced genes through contact with (including inhalation of pollen) or consumption of GM plant material deriving from the plants to which the genes have been transferred.

It should be noted that vertical gene flow per se is not considered an adverse outcome, but may be a link in a chain of events that may lead to an adverse outcome. Baseline information on vertical gene transfer associated with non-GM wheat plants can be found in The Biology of Triticum aestivum L. em Thell (Bread Wheat) (OGTR 2008).

Wheat plants are predominantly self-pollinating and the chances of natural hybridisation occurring with commercial crops or other sexually compatible plants are low and decreases with distance to the GM plants. Outcrossing rates decline significantly over distance, with most pollen falling within the first few metres. Rates are also influenced by the genotype of the variety, and environmental conditions, such as wind direction and humidity.

Wheat is sexually compatible with many species within the genus Triticum, and in closely related genera such as Aegilops, Secale (rye) and Elytrigia (Chapter 1, Section 6.3). Durum wheat (other than bread wheat, the only Triticum species present in Australia) can cross with wheat, although there are no reports of gene flow beyond 40 m (Matus-Cadiz et al. 2004). Hybrids between wheat and Secale cereale are sterile, but treatment with colchicine doubles the chromosome number and results in a fertile plant, the commercialised Triticale (Knupffer 2009). Natural hybridisation between wheat and Triticale rarely occurs (Ammar et al. 2004; Kavanagh et al. 2010), at least partly due to both species being largely self-fertilising (Acquaah 2007). Elytrigia repens does occur as an introduced plant in Australia, but a review of possible means of pollen-mediated gene flow from GM wheat to wild relatives in Europe concluded that there was a minimal possibility of gene flow from wheat to Elytrigia spp. (Eastham & Sweet 2002). Species of Aegilops are not known in Australia. Although specific data is lacking, it is likely that hybridisation of wheat with the four native Australasian Triticeae genera never occurs under natural conditions.

The proposed limits and controls of the trial would minimise the likelihood of the dispersal of pollen and exposure to GM plant material. For example, the applicant proposes to control related species within 200 m of the trial site. Isolation from related species and other wheat cultivation will greatly restrict the potential for pollen flow and gene transfer. In addition, the applicant proposes to perform post-harvest monitoring and to destroy any volunteer plants found at the site to ensure that no GM wheat remain that could then hybridise with sexually compatible plants.

Potential harm

If the vertical transfer of the introduced genes from the GM plants caused the resulting plants to spread and persist in the environment to a degree greater than normally found amongst these species, they may produce one or more harms. These are summarised in Section 2.3 of this chapter. People who are exposed to the proteins expressed from the introduced genes or their associated products through contact or consumption of GM plant material may show toxic or allergenic reactions, while organisms may show toxic reactions from consumption of GM plant material. The GM plants may act to reduce the establishment and yield of desired plants and subsequently reduce biodiversity.

In the rare event of the vertical transfer of the introduced genetic material from the GM plants to non-GM wheat plants or sexually compatible species, it is expected that this material in the recipient will have the properties that it possesses in the GM wheat parent. As discussed in risk scenario 1, the introduced gene products are not expected to be toxic to humans or other organisms. Although they have been associated with allergenicity, there is uncertainty whether their expression in wheat (the organism from which they derive) would affect the endogenous allergenicity of that plant. There is no reason to expect these gene products to have toxic or allergenic properties in a recipient arising from hybridisation with sexually compatible species that are any different from those found in the GM wheat. The uncertainty with respect to allergenicity would apply to any plants that were the product of hybridisation. Risk scenario 2 summarises the reasons that the introduced genes are unlikely to make the GM wheat lines more weedy, these reasons likewise being applicable to any plants to which the genes are transferred via hybridisation.

The traits that have been introduced into the GM plants of this application could become, via vertical gene transfer, combined with traits of other non-GM commercially cultivated wheat plants. However, there is no reason to believe that the resulting plants would possess a level of toxicity or allergenicity greater than that of either parent, or a level of weediness greater than that of either parent.

Conclusion: Risk scenario 3 is not identified as a substantive risk due to the proposed limits and controls designed to restrict dispersal of pollen flow from the GM wheat and to minimise exposure of people and other organisms to the GM plant material, and the lack of known toxicity of the introduced proteins. Further, the introduced trait is not associated with weediness, and it is unlikely that any of the characteristics associated with weeds will occur in hybrids with the GM plants. Therefore, this risk could not be greater than negligible and does not warrant further detailed assessment.

Risk scenario 4

Risk source

Causal pathway

Potential harm

Introduced genes for improved grain quality

Dispersal of GM pollen within the NGNE facility

Hybridisation of GM plants of this trial with GM plants (including volunteers) of another trial

Expression of genes in stacked GM plants

Exposure of people or other organisms to GM plant material

· Allergic reactions in people or toxicity in people and other organisms

Risk source

The source of potential harm for this postulated risk scenario is the introduced genes for improved grain quality.

Causal pathway

The grain quality improvement genes are expressed in the plant tissues. The NGNE facility is a multiuser facility, where at any one time, more than one licence holder may be conducting trials of different GM plants. Pollen from the GM plants of one trial could inadvertently fertilise other sexually compatible GM plants, including volunteers, inside a site. This pollen could be the result of transfer by an agent (eg wind, insects) from one planting area to another, or derived from volunteer plants from either trial. In late 2014, the only licence that is authorised to trial GM plants in the Katanning NGNE facility that are sexually compatible with those of this application is DIR 128, held by The University of Adelaide. This latter licence authorises the growth of GM wheat and barley with either abiotic stress tolerance or micronutrient uptake.

People working in or visiting the NGNE facility could be exposed to the GM hybrid plants or material from the GM hybrid plants, such as pollen. Any animals accessing the site may also be exposed to hybid GM plants.

The applicant has requested permission to grow GM wheat from this licence, and any other licence, next to each other provided they are separated by buffer zones of at least 4 m (2m for this trial and 2m for other trial). The RARMP for DIR 094 considered the possibility of hybridisation between wheat plants occurring over these distances, and concluded that it would be minimal.

The licence for DIR 128 specifies that if the GM plants of that trial are grown at the Katanning NGNE facility at the same time as sexually compatible GM and