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Risk Assessment and Risk Management Plan for DIR 046/2003 Limited and controlled release of GM fowl adenovirus (FAV) Applicant: Imugene Ltd January 2006 EXECUTIVE SUMMARY INTRODUCTION The Gene Technology Regulator (the Regulator) has made a decision to issue a licence for dealings involving the intentional release (DIR) of genetically modified (GM) fowl adenovirus into the environment, on a limited scale and under controlled conditions, in respect of application (DIR 046/2003) from Imugene Limited. The Gene Technology Act 2000 (the Act) and the Gene Technology Regulations 2001 (the Regulations) govern the process undertaken by the Regulator before a decision is made on whether or not to issue a licence. The decision is based upon a risk assessment and risk management plan prepared by the Regulator in consultation with a wide range of expert groups and authorities and the public. More information on the process required for the comprehensive assessment of licence applications to release a genetically modified organism (GMO) into the environment is available from the Office of the Gene Technology Regulator (OGTR) (Freecall 1800 181 030) or at <http://www.ogtr.gov.au/ir/process.htm>. THE APPLICATION Imugene applied for a licence to inoculate up to 1500 chickens with a GM fowl adenovirus. The release is intended to take place over a 49 day period between February and October 2006 and the proposed release site is a CSIRO animal containment facility at Werribee in the Shire of Wyndham, VIC. The GM fowl adenovirus contains the chicken interferon gamma (Ch IFN-γ) gene which produces the Ch IFN-γ protein. Imugene anticipates that expression of Ch IFN-γ protein in inoculated chickens will stimulate their immune system and assist them to use food more efficiently, thereby increasing their rate of weight gain. The purpose of the release is a proof of concept trial to test the safety and effectiveness of the GM fowl adenovirus in reducing microbial load and increasing weight gain in inoculated chickens. This data would be required by the Australian Pesticides and Veterinary Medicines Authority to support an application to register the GMO for use and sale. RISK ASSESSMENT Background The risk assessment first considered what harm to the health and safety of people or the environment could arise as a result of gene technology, and how it could happen, during the proposed, limited and controlled release of the GM fowl adenovirus into the environment (hazard identification). Hence the risks were assessed in comparison to non-GM fowl adenovirus and in the context of the level of containment provided by the animal containment facility in which the trial would take place. Hazards are particular sets of circumstances (events) that might give rise to adverse outcomes (i.e. cause harm). When an event was considered to have some chance of causing harm, it was identified as posing a risk that required further assessment. Each event associated with an identified risk was then assessed to determine the seriousness of harm (consequence - ranging from marginal to major) and the chance of harm (likelihood - ranging from highly unlikely to highly likely). The level of risk (ranging from negligible to Executive summary (November 2005) I high) was then estimated using a Risk Estimate Matrix (refer to Chapter 2 for more information). Hazard identification Of the 40 events characterised during the hazard identification process, two were selected for additional analysis. The potential adverse outcome associated with both events was increased pest potential in birds other than chickens. The remaining 38 events were not assessed further as they were considered not to give rise to an identified risk (refer to Chapter 2 for more information). Risk estimation Two events were analysed that might increase the pest potential of birds other than chickens as a result of the proposed release of the GM fowl adenovirus: Spread to other birds leading to enhanced immunocompetence as a result of insects or non-avian vertebrates transporting the GM fowl adenovirus out of the animal containment facility. Spread to other birds leading to enhanced immunocompetence as a result of inhaling windborne particles containing the GM fowl adenovirus. The risk assessment considered the consequence and likelihood of harm that might result from each of these events. The estimate of risk for each event is negligible (refer to Chapter 3 for more information). RISK MANAGEMENT The Risk Analysis Framework defines negligible risks as insubstantial, with no present need to invoke actions for their mitigation. However, as the small scale and site of the proposed release were important in establishing the context within which the risks were assessed, containment measures have been imposed to limit the size, duration and location of the release to that requested by the applicant. The licence conditions, detailed in Chapter 5 of the RARMP, require the applicant to limit the number of chickens to 1500; conduct the release during a 49 day period between February and October 2006; prevent use of the GMOs, or materials from the GMOs, in human food or animal feed; maintain physical isolation of the release site; undertake research and carry out post-trial monitoring to ensure all GMOs are destroyed. CONCLUSIONS OF THE RARMP The risk assessment concludes that this limited and controlled release of a GM fowl adenovirus at Werribee in Victoria poses negligible risks to the health and safety of people and the environment. The risk management plan concludes that these negligible risks do not require specific risk treatment measures. Licence conditions have been imposed to contain the release to the size, duration and location requested by the applicant. Executive summary (January 2005) II TABLE OF CONTENTS EXECUTIVE SUMMARY .................................................................................................................................. I INTRODUCTION I THE APPLICATION ................................................................................................................................................ I RISK ASSESSMENT................................................................................................................................................. I Background ............................................................................................................................................... I Hazard identification ............................................................................................................................... II Risk estimation ........................................................................................................................................ II RISK MANAGEMENT ............................................................................................................................................. II CONCLUSIONS OF THE RARMP ........................................................................................................................... II TABLE OF CONTENTS .................................................................................................................................. III ABBREVIATIONS .............................................................................................................................................. V TECHNICAL SUMMARY .................................................................................................................................. 1 INTRODUCTION ................................................................................................................................................... 1 SECTION 1 APPLICATION .............................................................................................................................. 1 SECTION 2 RISK ASSESSMENT....................................................................................................................... 2 SECTION 3 RISK MANAGEMENT .................................................................................................................... 4 3.1 Licence conditions to manage this limited and controlled release ............................................. 5 3.2 Other regulatory considerations ................................................................................................. 5 SECTION 4 CONCLUSIONS OF THE RARMP .................................................................................................. 6 CHAPTER 1 RISK ASSESSMENT CONTEXT .......................................................................................... 7 SECTION 1 SECTION 2 2.1 2.2 2.3 2.4 2.5 SECTION 3 SECTION 4 4.1 4.2 BACKGROUND ............................................................................................................................ 7 THE GMOS AND PROPOSED DEALINGS........................................................................................ 8 The proposed dealings ............................................................................................................... 8 The parent organism................................................................................................................. 11 The GMO ................................................................................................................................. 20 The introduced gene and its product ........................................................................................ 21 Method of genetic modification ............................................................................................... 22 THE RECEIVING ENVIRONMENT ................................................................................................. 23 PREVIOUS AUSTRALIAN AND INTERNATIONAL APPROVALS ...................................................... 24 Previous Australian approvals of the same or similar GM Fowl Adenovirus .......................... 24 International approvals ............................................................................................................. 24 CHAPTER 2 RISK ASSESSMENT ............................................................................................................ 25 SECTION 1 SECTION 2 2.1 2.2 2.3 2.5 2.6 2.7 2.8 2.9 2.10 2.11 SECTION 3 INTRODUCTION ......................................................................................................................... 25 HAZARD CHARACTERISATION ................................................................................................... 26 Production of a substance that is toxic or adversely alters the immune response of people .... 31 Production of a substance that is toxic or adversely alters the immune response in chickens . 33 Production of a substance that is toxic or adversely alters the immune response of birds other than chickens ....................................................................................................................... 33 Production of a substance that is toxic or adversely alters the immune response of organisms other than people or birds .................................................................................................... 36 Altered viral characteristics...................................................................................................... 37 Altered persistence within the host .......................................................................................... 41 Increased transmission of GM FAV8....................................................................................... 41 Spread and persistence of the GM FAV8 in the environment .................................................. 42 Gene transfer between the GM FAV8 and other organisms .................................................... 44 Increased rate of mutation of GM FAV8 ................................................................................. 45 Unauthorised activities ............................................................................................................. 46 RISK ESTIMATE PROCESS FOR IDENTIFIED RISKS ....................................................................... 46 CHAPTER 3 RISK ESTIMATES FOR INCREASED PEST POTENTIAL IN OTHER BIRDS ......... 48 SECTION 1 SECTION 2 2.1 BACKGROUND .......................................................................................................................... 48 CONSEQUENCE AND LIKELIHOOD ASSESSMENTS ....................................................................... 49 Characteristics of the parent organism ..................................................................................... 49 2.4 Table of Contents (January 2005) III DIR 046/2003—Risk Assessment and Risk Management Plan 2.2 Office of the Gene Technology Regulator 2.4 SECTION 3 Event 1: Spread to other birds as a result of insects or non-avian vertebrates transporting the GM FAV8 outside the facility ............................................................................................. 50 Event 2: Spread to other birds as a result of inhaling windborne particles containing the GM FAV8 ................................................................................................................................... 55 Uncertainty ............................................................................................................................... 56 RISK ESTIMATES ....................................................................................................................... 56 CHAPTER 4 RISK MANAGEMENT ......................................................................................................... 59 SECTION 1 SECTION 2 SECTION 3 SECTION 4 4.1 4.2 SECTION 5 SECTION 6 BACKGROUND .......................................................................................................................... 59 OTHER AUSTRALIAN REGULATORS ........................................................................................... 59 RISK TREATMENT MEASURES FOR IDENTIFIED RISKS ................................................................. 59 GENERAL RISK MANAGEMENT .................................................................................................. 60 Licence conditions associated with managing limited and controlled releases ........................ 60 Other risk management considerations .................................................................................... 60 MONITORING AND COMPLIANCE............................................................................................... 62 CONCLUSIONS OF THE RARMP ................................................................................................ 62 CHAPTER 5 LICENCE CONDITIONS ..................................................................................................... 63 2.3 SECTION 1 SECTION 3 REFERENCES INTERPRETATIONS AND DEFINITIONS........................................................................................ 63 SPECIFIC CONDITIONS .............................................................................................................. 68 ............................................................................................................................................. 74 APPENDIX A DEFINITIONS OF RISK ANALYSIS TERMS .................................................................. 81 APPENDIX B SUMMARY OF ISSUES RAISED IN SUBMISSIONS FROM PRESCRIBED EXPERTS, AGENCIES AND AUTHORITIES ON APPLICATION .............................. 83 APPENDIX C SUMMARY OF ISSUES RAISED IN SUBMISSIONS RECEIVED FROM PRESCRIBED EXPERTS, AGENCIES AND AUTHORITIES ON THE CONSULTATION RARMP ................................................................................................. 84 APPENDIX D SUMMARY OF PUBLIC SUBMISSION RECEIVED ON THE CONSULTATION RARMP................................................................................................................................... 85 Abbreviations (January 2006) IV ABBREVIATIONS Ad5 APVMA bp cm CAR CCI cDNA CELO CFA3 CFA40 CFA44 Ch IFN-γ CPE DIR DNA DNIR E1 E3 E4 ELISA EPA FAO FAV FAV1 FAV8 FAV8 (VRI-33) FAV9 FSANZ g/L GI GM GMAC GMO GTTAC hCMV hCMV-ie HPS HS-GAGs IBDV IBH IFN- IFN- IFN-γ IL-12 kb km LD50 Human adenovirus serotype 5 Australian Pesticides and Veterinary Medicines Authority (formerly NRA) Basepair of DNA centimetre Coxsackie and adenovirus receptor Confidential Commercial Information Complementary DNA Chick embryo lethal orphan (an isolate of FAV1) A mildly virulent strain of fowl adenovirus, serotype 8 A hypervirulent isolate of fowl adenovirus, serotype 8 An avirulent isolate of fowl adenovirus, serotype 8 Chicken interferon gamma (gene or protein) Cytopathic effect dealing involving intentional release deoxyribonucleic acid dealing not involving intentional release Early region 1 of mammalian adenoviruses Early region 3 of mammalian adenoviruses Early region 4 of mammalian adenoviruses Enzyme-linked immunosorbent assay Environmental Protection Agency of the United States of America Food and Agriculture Organisation (of the United Nations) Fowl Adenovirus Fowl adenovirus serotype 1 Fowl adenovirus serotype 8 A virulent isolate of FAV8 Fowl adenovirus serotype 9 Food Standards Australia New Zealand (formerly ANZFA) Grams per litre Gastro-intestinal genetically modified Genetic Manipulation Advisory Committee genetically modified organism Gene Technology Technical Advisory Committee human cytomegalovirus human cytomegalovirus immediate early promoter Hydropericardium syndrome Heparan sulfate-glycosaminoglycans Infectious bursal disease virus Inclusion body hepatitis Interferon alpha Interferon beta Interferon gamma Interleukin 12 Kilobase pairs of DNA kilometres 50% of the lethal dose Abbreviations (January 2006) V DIR 046/2003—Risk Assessment and Risk Management Plan MDV mRNA NK cells NLRD NRA OGTR ORF PC2 PCR PPE RAF RARMP RCA RNA SV40 TCID50 TNF- WHO w/v Office of the Gene Technology Regulator Marek’s disease virus messenger ribonucleic acid Natural killer cells Notifiable Low Risk Dealing National Registration Authority for Agricultural and Veterinary Chemicals (now APVMA) Office of the Gene Technology Regulator Open reading frame OGTR certified physical containment level 2 polymerase chain reaction Personal protective equipment OGTR Risk Analysis Framework Risk Assessment and Risk Management Plan Replication competent adenoviruses ribonucleic acid Simian virus 40 50% of the tissue culture infectious dose Tumour necrosis factor alpha World health organisation Weight by volume Abbreviations (January 2006) VI DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator TECHNICAL SUMMARY INTRODUCTION The Gene Technology Regulator (the Regulator) has decided to issue a licence (DIR 046/2003) to Imugene Ltd (Imugene) for dealings involving the intentional release of genetically modified (GM) fowl adenovirus type 8a into the environment, on a limited scale and under controlled conditions. The Gene Technology Act 2000 (the Act), the Gene Technology Regulations 2001 (the Regulations) and corresponding state and territory law govern the comprehensive and highly consultative process undertaken by the Regulator before making a decision whether or not to issue a licence to deal with a GMO. The Regulator’s Risk Analysis Framework explains the approach used to evaluate licence applications and to develop the Risk Assessment and Risk Management Plans (RARMPs) that form the basis of her decisions1. The RARMP for DIR 046/2003 has been finalised in accordance with the gene technology legislation. Matters raised in the consultation process regarding risks to the health and safety of people or the environment from the dealings proposed by the applicant were taken into account by the Regulator in deciding to issue a licence and the conditions that have been imposed. SECTION 1 APPLICATION Title: Applicant: Common name of the parent organism: Scientific name of the parent organism: Modified trait(s): Identity of the gene(s) responsible for the modified trait(s): Limited and controlled release of Fowl adenovirus* Imugene Ltd Fowl adenovirus Fowl adenovirus serotype 8, isolate CFA44 Immunomodulatory protein expression Attenuation Interferon gamma gene from chicken Proposed location(s): Werribee, Shire of Wyndham, Victoria Proposed release size: Up to 1500 chickens inoculated with the GMO Proposed time of release: 1 February 2006-30 October 2006 * Imugene’s title for this dealing was Fowl adenovirus vaccine Imugene applied for a licence to release one GM fowl adenovirus (FAV) into the environment. The trial, which would involve the inoculation of up to 1500 chickens with the GM FAV8 between 1 February 2006 and 31 October 2006, is intended to take place in an animal containment facility at a CSIRO Livestock Industries animal containment facility at Werribee in the Shire of Wyndham, Victoria. 1 More information on the assessment of licence applications and copies of the Risk Analysis Framework are available from the Office of the Gene Technology Regulator (OGTR). Free call 1800 181 030 or at <http://www.ogtr.gov.au/ir/process.htm> and <http://www.ogtr.gov.au/pdf/public/ raffinal2.2.pdf > respectively. Technical summary (January 2006) 1 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator The purpose of the proposed release is to test the ability of the GM FAV8 to increase weight gain and feed conversion ratio of chickens when inoculated either by injection in ovo, by eye-drops or by feeding with water containing the GM FAV8. The GM FAV8 has one introduced gene, the chicken interferon gamma (IFN-γ) gene, which will be expressed under the control of regulatory sequences that do not encode any protein. The chicken IFN-γ protein is an immunomodulatory protein that when expressed in chickens, leads to stimulation of the immune system and increased protection from bacteria, viruses and other parasites. The applicant intends to inoculate chickens in a certified physical containment level 2 (PC2) facility. The inoculated chickens will then be transported between the PC2 facility and the animal containment facility. SECTION 2 RISK ASSESSMENT The risk assessment considered information contained in the application, current scientific knowledge, and issues relating to risks to human health and safety and the environment raised in submissions received during consultation with a wide range of prescribed experts, agencies and authorities on the application (summarised in Appendix B). The Regulator notified the public that a RARMP had been prepared and invited written submissions in relation to the RARMP. Advice on the RARMP was also sought from those consulted on the application. Issues relating to the assessment of risk to the health and safety of people or the environment raised during the consultation period are summarised in Appendices C and D. These appendices also indicate where the issues were addressed in the RARMP. The risk assessment first considered what harm to the health and safety of people or the environment could arise due to gene technology, and how it could happen during this release of GMOs into the environment (hazard identification), in comparison to non-GM fowl adenovirus 8 and in the context of the proposed experimental conditions. A hazard (source of potential harm) may be an event, substance or organism (OGTR 2005). The hazard identification process resulted in the compilation of a list of 40 events that describe sets of circumstances (events) by which the proposed release could potentially give rise to adverse outcomes. A risk is identified when a hazard is considered to have some chance of causing harm to people and/or the environment. Those events that do not lead to an adverse outcome, or could not reasonably occur, do not advance in the risk assessment process. The events that are considered to have the potential to lead to adverse outcomes are analysed further to determine the seriousness of harm (consequence) that could result and how likely it is that the harm would occur. The level of risk is then estimated using the Risk Estimate Matrix (see below and Chapter 2). Technical summary (January 2006) 2 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator LIKELIHOOD RISK ESTIMATE Highly Likely Low Moderate High High Likely Negligible Low High High Unlikely Negligible Low Moderate High Highly Unlikely Negligible Negligible Low Moderate Marginal Minor Intermediate Major CONSEQUENCES Risk Estimate Matrix: A negligible risk is considered to be insubstantial with no present need to invoke actions for mitigation. A low risk is considered to be minimal but may invoke actions for mitigation beyond normal practices. A moderate risk is considered to be of marked concern that will necessitate actions for mitigation that need to be demonstrated as effective. A high risk is considered to be unacceptable unless actions for mitigation are highly feasible and effective. Two of the 40 events characterised in the hazard identification process for the proposed release were identified as requiring further analysis. These were: the spread of the GM FAV8 to other birds as a result of contact with insects or non-avian vertebrates transporting the GMO out of the animal containment facility; or as a result of inhaling windborne particles containing the GMO. The potential adverse outcome associated with both events was increased pest potential in other birds as a result of enhanced immunocompetence arising from the expression of the introduced Ch IFN-γ gene. The identified risks were then assessed in further detail in comparison to the parent organism and in the context of the intended trial. The consequence and likelihood assessments used to derive risk estimates for these two events, are summarised in Table 1 (the detailed risk assessment is in Chapter 3). More information on the remaining 38 events that were considered not to give rise to an identified risk is provided in Chapter 2. All risks were estimated to be negligible for this proposed release i.e. insubstantial with no present need to invoke actions for their mitigation. Table 1.1 Summary table for the risk assessment Event that may give rise to an adverse outcome Event 1 Spread of the GM FAV8 to other birds as a result of contact with insects or non-avian vertebrates transporting the GM FAV8 out of the facility. Consequence assessment Likelihood assessment Risk estimate Marginal Low level exposure of other birds is expected to result in a minimal effect or no effect at all The GM virus is not expected to persist in chicken populations and expression of GM FAV8 will be transient. Therefore, any increased growth promotion/immune system stimulation in other bird populations is Highly unlikely Exposure to viable GM FAV8 is expected to be restricted to other birds in the immediate vicinity of the trial site Routine pest management at the facility will minimise rodent numbers and hence the level of exposure to rodents that may be carrying the GM FAV8 Low levels of exposure Negligible Technical summary (January 2006) Does risk require treatment? No 3 DIR 046/2003—Risk Assessment and Risk Management Plan Event that may give rise to an adverse outcome Consequence assessment expected to be short lived. Small growth Event 2 Spread to other birds as a result of inhaling windborne particles containing the GM FAV8 promoting/immune stimulating effect occurring for limited time in a limited number of bird populations is not expected to increase pest potential Ch-IFN-γ is only likely to be active in bird species that have an IFN-γ protein with a high amino acid sequence homology to Ch-IFN-γ Marginal Low level exposure of other birds is expected to result in a marginal effects or no effect at all The GM virus is not expected to persist in chicken populations and expression of Ch-IFN-γ will be transient. Therefore, any increased promotion/immune system stimulation in bird populations is expected to be short lived. Small growth promoting/immune stimulating effect occurring for limited time in a limited number of bird populations is not expected to increase pest potential Ch IFN-γ is only likely to be active in bird species that have an IFN-γ protein with a high amino acid sequence homology to Ch IFN-γ Office of the Gene Technology Regulator Likelihood assessment Risk estimate Does risk require treatment? Negligible No from insects due to very low levels of viable GM FAV8 carried Opportunity for exposure limited by rapid decline in survival of GM FAV8 under desiccating conditions The small size and short duration of the proposed release limits extent and duration of exposure Highly unlikely Exposure to viable GM FAV8 is expected to be restricted to other birds in immediate vicinity of trial site Exposure limited due to low levels of GM FAV8 in airborne particles and rapid decline in survival under desiccating conditions The small size and short duration of the proposed release limits extent and duration of exposure SECTION 3 RISK MANAGEMENT A risk management plan builds upon the risk assessment to consider whether any action is required to mitigate the identified risks. If a risk is estimated to be higher than negligible, risk treatment measures may be required to protect the health and safety of people and the environment. Technical summary (January 2006) 4 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator The risk assessment considered two events that might lead to a risk to the environment. The risk estimates for the adverse outcome of increased pest potential in other birds associated with both events are negligible. However, containment measures have been imposed to restrict the release to the size, timeframe and location requested by the applicant, because the limited area, site and duration of the release were important considerations when assessing the consequence and likelihood of potential adverse outcomes. In addition, research requirements have been included to address uncertainty about the range of birds that are able to be successfully infected by the GM virus. 3.1 Licence conditions to manage this limited and controlled release A number of licence conditions have been imposed to limit and control the release, including requirements to: contain the chickens in the animal containment facility; wear personal protective equipment (PPE) when carrying out work in the animal containment facility that is removed prior to leaving to prevent transport of GMOs outside the facility; utilise sentinel animals (uninoculated chickens, pigeons and pest birds) and inoculated pigeons and pest birds to assess the ability of the GM virus to spread to and cause infection in birds outside the known host range of the virus; implement pest control measures to minimise the numbers of rodents and insects entering or exiting the facility; following completion of the trial kill and decontaminate prior to disposal all chickens and sentinel animals involved in the trial; following completion of the trial decontaminate all waste and PPE from the trial including equipment used in the trial; following completion of the trial decontaminate all areas in which the trial has taken place; and conduct regular inspections of the release site following completion of the trial until decontamination is successful. The Regulator has issued guidelines and policies for the transport, supply and storage of GMOs (Guidelines for the transport of GMOs, June 2001; Policy on transport and supply of GMOs, July 2005; and Policy on storage of genetically modified organisms, July 2004). Licence conditions based on these guidelines and policies have also been imposed to control possession, use or disposal of the GMOs for the purposes of, or in the course of, the authorised dealings. 3.2 Other regulatory considerations The GM FAV8 proposed for release is intended for use as a veterinary chemical product and, so is also subject to regulation by the Australian Pesticides and Veterinary Medicines Authority (APVMA). Imugene will require a permit from the APVMA to undertake the proposed release. Data collected during the proposed release on the safety and efficacy of the GM FAV8 could be used to support future applications to the APVMA to register the GMO for use and sale. Technical summary (January 2006) 5 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator SECTION 4 CONCLUSIONS OF THE RARMP The risk assessment concludes that this limited and controlled release of GM FAV8 in the Shire of Wyndham, Victoria poses negligible risks to the health and safety of people and the environment as a result of gene technology. The risk management plan concludes that these negligible risks do not require specific risk treatment measures. Licence conditions have been imposed to contain the release to the size, duration and location requested by the applicant. Technical summary (January 2006) 6 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator CHAPTER 1 RISK ASSESSMENT CONTEXT SECTION 1 BACKGROUND 1. This chapter describes the parameters within which risks that may be posed to the health and safety of people and the environment by the proposed release are assessed, based on scientific evidence. These include the scope and boundaries for the evaluation process required by the gene technology legislation2, details of the intended dealings, the GMO(s) and parent organism(s), previous approvals and releases of the GMOs in Australia or overseas, environmental considerations and relevant agricultural practices. The parameters for the risk assessment context are summarised in Figure 1.1. Figure 1.1 Components of the risk context considered during the preparation of the Risk Assessment RISK ASSESSMENT CONTEXT GMO The genetic modification (genotype) The modified traits (phenotype) DEALINGS Activities involving the GMO PARENT ORGANISM RECEIVING ENVIRONMENT Agronomic practices Environmental conditions Previous releases 2. Sections 49 to 51 of the Gene Technology Act (2000) (the Act) outline the matters which the Regulator must take into account, and who she must consult with, in preparing the RARMPs that form the basis of her decision on licence applications. 3. For this application, establishing the risk assessment context includes consideration of: comparisons with the parent organism the nature and effect of the genetic modification facility management and animal husbandry practices the size, duration and location of release requested by the applicant previous approvals for release of these GMOs in Australia and overseas any previous releases of these or other GMOs relevant to this application. 2 The legislative requirements and the approach taken in assessing licence applications them are outlined in more detail at <http://www.ogtr.gov.au/ir/process.htm> and in the Risk Analysis Framework (OGTR 2005) < http://www.ogtr.gov.au/pdf/public/raffinal2.2.pdf>. Chapter 1 Risk assessment context (January 2006) 7 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 4. Initial consideration of the application under section 49 of the Act determined that public consultation was not required for the preparation of the consultation version of the RARMP. 5. In accordance with section 50 of the Act, the Gene Technology Technical Advisory Committee (GTTAC), State and Territory governments, Australian Government agencies, the Minister for Environment and Heritage and the local council where the release may take place were consulted on matters relevant to the preparation of the RARMP. This advice, and where it was taken into account in the RARMP, is summarised in Appendix B. 6. Under Section 52 of the Act the same experts, agencies and authorities and the public were requested to provide comment on the consultation version of the RARMP. The submissions received and where matters relevant to risks to human health and safety and the environment were taken into account are summarised in Appendices C and D respectively. SECTION 2 2.1 THE GMOS AND PROPOSED DEALINGS The proposed dealings 7. Imugene intends to release a genetically modified (GM) fowl adenovirus (GM FAV8) into the environment under limited and controlled conditions. 8. The fowl adenovirus has been modified by insertion of a copy of the chicken interferon gamma gene (Ch IFN-γ) that will be expressed under the control of the human cytomegalovirus immediate early (hCMV-ie) promoter3 and the polyoma virus4 SV40 polyadenylation sequence5. 9. The applicant anticipates that treatment of chickens with GM FAV8 may remove the need for antibiotic treatment in commercial production facilities by stimulating the immune system of the chickens at an early stage of growth. This is expected to lead to a decreased disease burden normally caused by bacteria and viruses which are common in the poultry industry and lead to decreased productivity. 10. The purpose of the proposed release is early stage research to investigate the ability of GM FAV8 to boost the immune system of inoculated chickens and to provide information on the shedding and spread of the GM virus. The use of the GM virus is expected to lead to an increase in the growth rate and feed conversion ratio by the chickens due to the absence of microbial load. The applicant anticipates using the GMO in future larger scale trials to evaluate the efficacy of the GMO in commercial sized chicken flocks. These trials would be the subject of future applications and would require separate assessments and approvals. 11. Approximately 1090 female white Leghorn broiler chickens will be used in this trial. The trial will comprise 10 treatment groups of 100 chickens per group, a sentinel group of 60 chickens to assess the ability of the GM FAV8 to spread and 3 shedding assessment groups of 10 chickens. Promoter – a short regulatory sequence of DNA that usually precedes the coding region of a gene and enables binding of an RNA polymerase to initiate gene expression. 4 Polyoma virus 5 Polyadenylation sequence – a sequence of DNA at the end of an open reading frame that specifies the addition of a stretch of adenine residues onto the messenger RNA. This signal acts as a terminator of transcription. 3 Chapter 1 Risk assessment context (January 2006) 8 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 12. 300 of the chickens will be obtained as fertilised eggs at age 16 days of incubation to generate female hatchlings, and the remaining 790 female chickens will be obtained as one day old chicks. 13. The chickens that receive the GM FAV8 will be inoculated with 103.7 tissue culture infective doses6 (TCID50)7 either in ovo, by inoculation into the egg, or by eyedrop inoculation following hatching. The groups that are treated by eye-drop inoculation at hatching will be reinoculated by ingestion of water containing 103.7 or 104.4 TCID50 GM FAV8 after 21 or 28 days or both (see Table 1.2). One of the groups inoculated in ovo will also be treated by eye-drop inoculation at hatching. 14. Sentinel8 birds comprising chickens, sparrows, starlings and pigeons, which are within the known host range of at least one FAV serotype, will also be used to assess the ability of the virus to infect or spread to feral bird populations. Five sentinel chickens will be included in nine of the ten treatment groups and the three shedding assessment groups (A-C) (see Table 1.2). Other sentinel birds will also be used in 3 groups of four birds of each test species per group. One group will be inoculated with the GMO in a PC2 animal containment facility under the existing DNIR 068/2002. A second group will be used to test the ability of the GM virus to be passively transmitted within the facility. The third group will be in cages outside the facility to test the ability of the virus to be transmitted to and to infect feral bird populations outside the facility. The latter group of sentinel birds will be housed in proximity to the trial groups. 15. The faeces of sentinel birds used in the trial will be monitored for the presence of the GM virus during the trial. At the completion of the trial, tissue samples from euthanased sentinel animals will be tested for infection with the GM virus by polymerase chain reaction (PCR)9 and histopathology of tissues. 16. Chickens used in the trial will be obtained from commercial flocks. They will already have been vaccinated against Marek’s Disease virus (MDV), infectious bursal disease virus (IBDV) and Newcastle disease virus. 17. Imugene proposes to undertake the trial at one location, a CSIRO Livestock Industries animal containment facility at Werribee, in the Shire of Wyndham, Victoria. 18. The animal containment facility is approximately 3 metres in height with mild steel walls topped with a metre of narrow gauge mesh reaching to the roof to prevent the entry of birds to the facility. Although the facility is not insect or rodent proof, pest control measures to minimise the entry and exit of rodents and insects will be imposed. These measures include fly strips and rodent bait. No other modifications to the facility will be made during the trial. 19. During the proposed release, the applicant intends to transport the GM FAV8 from contained laboratory facilities to the release site. Tissue samples from infected chickens will also be transported to certified Physical Containment level 2 (PC2) laboratories for analysis. 6 Tissue culture infective doses (TCID)- the minimum amount of virus required to produce viral activity in a standard culture of stock laboratory cells. 7 TCID50 - the amount of virus required to produce viral activity in 50% of a standard culture of stock laboratory cells 8 Sentinel animals - animals that are used to test the host range or mode of transmission of an infecting organism 9 PCR – a technique of molecular biology that enables amplification of minute amounts of DNA that can be used to detect the presence of viral DNA. Chapter 1 Risk assessment context (January 2006) 9 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Table 1.2 Treatment Groups for proposed trial of GM FAV8 Treatment (GM FAV8 TCID50) Group No of chickens In ovo Day 18 Eye-drop at hatch In water, Day 21 104.4 1 100 + 5 sentinels 103.7 2 100 + 5 sentinels 103.7 3 100 + 5 sentinels 103.7 104.4 4 100 + 5 sentinels 103.7 103.7 5 100 + 5 sentinels 103.7 6 100 + 5 sentinels 103.7 7 100 + 5 sentinels 103.7 8 100 + 5 sentinels 103.7 9 100 10 100 + 5 sentinels A Highest dose (replicate of group 3) B Lowest dose (replicate of group 7) C In water, Day 28 104.4 104.4 103.7 103.7 103.7 FAV8* FAV8* 103.7 104.4 104.4 Likely commercial dose (replicate of group 6) 103.7 103.7 103.7 Pigeons and Pest birds 1 group of 4 starlings, 1 group of 4 sparrows and 1 group of 4 pigeons (conducted in a PC2 animal containment facility). 103.7 103.7 103.7 Sentinel pigeons and pest birds 1 group of 4 starlings, 1 group of 4 sparrows and 1 group of 4 pigeons (in cages inside facility) 103.7 FAV8* 103.7 1 group of 4 starlings, 1 group of 4 sparrows and 1 group of 4 pigeons (held in cages outside the facility) *- FAV8 used in this treatment group will be non-GM FAV8 Chapter 1 Risk assessment context (January 2006) 10 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 20. The applicant proposes to run the trial for a period of 49 days after which all animals involved in the trial will be euthanized and incinerated following the removal of tissue samples for analysis. 21. Initial treatments (in ovo and eye-drop) will be carried out in a certified PC2 facility on-site, prior to removal of the chickens to the animal containment facility. The ‘in water’ treatment will take place in the animal containment facility. 22. Tissue samples from blood, liver, caecal tonsils, heart, kidney and spleen from groups A-C and sentinel group 11 will be retained for PCR analysis and histopathology10 at the conclusion of the dealing. 23. Tissue samples from all sentinel animals involved in the trial will be removed for analysis. Histopathology will be carried out on samples of liver, tonsils, heart, kidney and spleen. 24. PCR, serology and histopathology will take place in a certified PC2 facility. 25. Personnel undertaking work within the animal containment facility will wear personal protective equipment (PPE) to prevent transport of GMOs outside the facility. This will comprise rubber boots and overalls, which will remain in the animal containment facility and which will be autoclaved following completion of the dealing, and disposable latex gloves. 26. All infected animals will be euthanized prior to autoclaving on-site and incineration by a Medi-waste contractor. 27. At the completion of the study, all litter will be sealed into Medi-waste bags within the animal containment facility and placed into sealed, secure Medi-waste wheeled bins. The bins will be surface decontaminated, wheeled through a disinfectant footbath to the secure storage and waste collection shed where they will be collected for incineration by a Medi-waste contractor and incinerated under controlled conditions. 28. Once the litter has been removed, the inside of the shed will be cleaned and surface treated with the viricidal11 agent Virkon TM at a concentration of 20g/L (2% w/v) for a minimum of 10 minutes wet contact time. 29. The shed will be left vacant at the conclusion of the study and swabbed at days 7 and 14 for assessment of persistent virus by PCR and cytopathic effect (CPE)12 assays. 2.2 The parent organism 30. FAV8 is a member of the Aviadenoviridae genus (ICTV). FAVs have been classified into 11 serotypes13 (1-11) on the basis of serum neutralisation studies. The FAVs have also been classified as belonging to subgroups A-E on the basis of restriction endonuclease analysis (Zsak & Kisary 1984). FAV8 is a member of the E subgroup, which also includes serotype 6 and 7 viruses. FAV8 can be further Histopathology – Fixing, staining sectioning and observation of tissues to look for signs of disease, in this case infection by GM FAV8 11 Viricidal – Able to kill viruses. 12 CPE – Cytopathic effect - the effect on cells when infected by a cytopathic virus such as FAV. In the case of cells infected with FAV, cells become round and filled with virus particles. 13 Serotype – The group to which a genus of micro-organisms belongs, based on the recognition of proteins on the surface of the micro-organism that are recognised by reference antibodies. 10 Chapter 1 Risk assessment context (January 2006) 11 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator subdivided into two subtypes a and b. FAV9, a closely related serotype, that was originally thought to be a FAV8 isolate, is classified as a group D FAV (Ojkic et al. 2002a). 31. The parent organism from which the GMO is derived is the CFA44 isolate of serotype 8 sub-type a of fowl adenovirus. This strain was isolated between 1977 and 1979 as a non-pathogenic14 field isolate from Victorian commercial chicken farms (Erny et al. 1991; Pallister et al. 1993). 32. Although virulent subtypes of FAV8, such as the CFA40 isolate, have been implicated in diseases of fowl such as inclusion body hepatitis (IBH) and respiratory disease, CFA44 was described as an isolate that was mildly- to non-pathogenic15, with no associated symptoms of disease (Erny et al. 1991). The minimum dose of CFA44 required for infection of a chick (MID) was between 10000 and 3x105 TCID50 per chick (Erny et al. 1991; Pallister et al. 1993). This strain can therefore be considered as avirulent16. By comparison, the hypervirulent CFA40 isolate, which was implicated as the cause of IBH, had an MID of 10-100 TCID50 (Pallister et al. 1993). No deaths of chickens were recorded at any dose rate with CFA44 (Erny et al. 1991). 33. CFA44 replicates in the upper respiratory tract and gastro-intestinal tract of susceptible animals. 34. Another strain of FAV8, (E. Surient) is currently used as a vaccine in chickens, to protect them from infection by virulent strains of FAV. The E. Surient strain was isolated by NSW Department of Agriculture in 1989 and registered with the Australian Pesticides and Veterinary Medicines Authority (APVMA) in 2000 for use in Australia (NRA No.52390). While there is no information available about the relationship between the CFA44 isolate proposed for use in the current trial and the E. Surient isolate, on the basis of their common serotype, the CFA and E. Surient strains are expected to be closely related. 2.2.1 Host range of the parent organism 35. Based on phylogenetic analysis of adenovirus and host proteins, it is believed that adenoviruses have co-evolved with their hosts over millions of years (Benko & Harrach 2003). This is supported by the finding that members of the Aviadenoviridae genus have only been isolated from avian species (Davison et al. 2003). Members of the Aviadenoviridae genus do not naturally infect mammals, insects or other organisms. 36. While FAV8 has been isolated from chickens, pigeons and budgerigars (McFerran, 1976), other members of the FAV family have been isolated from turkeys, quail and a mallard duck (McFerran, 2003). Closely related aviadenoviruses have also been isolated from geese, turkeys, ostriches, falcons and parrots (Buchen-Osmond, 2003; Gough et al, 1997; Schrenzel et al, 2005; Raue at al, 2005; Wellehan et al, 2005). 14 Non-pathogenic – Non-disease causing. Virulence – Refers to the severity of disease caused by a micro-organism. The virulence of FAV isolates CFA 40 and CFA44 was is determined by the death of 50% of the chickens at a particular dose of FAV (Erny et al. 1991). 16 Avirulent – without virulence. According to Erny et al, 1991, a virus that has a minimum infective dose of greater than 10 4 TCID50 when administered to day old specific pathogen free (SPF) White Leghorn chickens by the nasal/ocular route. 15 Chapter 1 Risk assessment context (January 2006) 12 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 37. Successful infection of a host cell by a virus is dependent on the completion of a complex pathway of events. These can be simplified into five basic processes: viral attachment, viral entry, viral replication and gene expression, viral assembly and escape of virus particles from the host cell. Viral attachment and entry are determined by interactions between viral encoded proteins on the capsid17 of the virus and host proteins on the exterior and interior of the host cell. Viral capsid proteins interact with receptors on the surface of host cells to attach and mediate entry to the host cell. 38. The host-range18 of adenoviruses is determined by both host-encoded and viralencoded factors. Attachment of adenoviruses to host cells is mediated by two viralencoded proteins present on the surface of the virus: the fiber protein, which is responsible for the initial attachment to cellular receptors; and the penton base, which interacts with host-encoded cell surface molecules known as integrins. 39. FAVs have two fibers per penton base and within the FAV family there is a direct relationship between the length of the fiber and the serotype of the virus (Cao et al. 1998b). 40. The tissue tropism19 of adenoviruses is also influenced by the availability of cellular receptors for the fiber and penton proteins and by host-encoded factors that determine whether replication is able to take place (see below). Cells such as airway epithelial cells lack these receptors which prevents them from becoming infected by adenovirus (Shenk 2001). 41. The restriction of host range of adenoviruses can be mediated at key points during the viral life cycle. Mutations in human adenoviruses that have enabled them to replicate in monkey cells have been found in a sequence-specific DNA binding protein, that increases late phase viral protein synthesis (Cheng et al. 1992). However, the restriction also exists at the level of late viral gene expression that includes production of the fiber protein which is decreased 100-1000 fold in monkey cells (Anderson & Klessig 1983). It has also been observed that the restriction may occur at the level of viral assembly due to low levels of expression of a key viral protein (Pieniazek et al. 1990). 42. The fiber protein of human adenoviruses can interact with a wide variety of cellular receptors that are present on the surface of human cells. These receptors include the Coxsackievirus and adenovirus receptor (CAR) as well as other cell surface proteins and non-protein moieties such as heparan-sulfate glycosaminoglycans (HS-GAGs) and sialic-acid (Zhang & Bergelson 2005). All of these molecules may serve as primary receptors for adenoviruses. 43. Avian homologues of the cell surface molecules discussed above exist, and HSGAGs and sialic acid are ubiquitous throughout the animal kingdom. However, while these molecules can act as receptors for adenoviruses that infect mammals, it is unknown whether the same is true for avian adenoviruses such as FAV8. 44. Entry of the virus into a host cell occurs by a process known as receptormediated endocytosis following interaction of the virus with an integrin molecule. The virus particle is transported to the nuclear pore along a microtubule network and the viral capsid is disassembled to enable import of the viral DNA into the nucleus. Capsid – the protein shell that contains the viral DNA. The capsid comprises solely viral proteins as adenovirus does not have a lipid envelope. 18 Host-range – the range of organisms (eg. animals) that another organism (eg. virus or bacterium) is able to infect 19 Tissue tropism – the range of tissue or cells within an organism in which the virus is able to grow. 17 Chapter 1 Risk assessment context (January 2006) 13 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Replication of the viral DNA is dependent on both host- and viral-encoded factors (McConnell & Imperiale 2004). 45. Experiments with one fowl adenovirus, chick embryo lethal orphan (CELO), a highly virulent FAV1 serotype virus, showed that it was able to transduce20 human cell lines and to express a transgene in these cells lines (Michou et al. 1999; Tan et al. 2001). The ability of CELO to replicate in these cell lines could not be tested. 46. Experiments to determine whether the GM FAV8 that will be used in the proposed dealing could produce a cytopathic effect in a variety of mammalian cell lines, including a human cell line, demonstrated that the recombinant virus could not replicate in these cell lines (data provided in application). This result may indicate either a difference between the FAV1 and FAV8 fiber proteins that prevents entry of FAV 8 into human cells or that there is a host restriction that occurs at the level of viral gene expression. There is little information on the mechanism of host range restriction of fowl adenoviruses such as FAV8. However, sequencing of the complete genome of FAV9, a highly related FAV has shown that this adenovirus is missing the early (E)1, E3 and E4 regions that are present in mammalian adenoviruses such as the human Ad5 viruses mentioned above (Ojkic et al. 2002b). The structure of the FAV9 genome is so different from that of the human Ad5 adenovirus that host range restriction could be mediated at many points. In addition, the co-evolution of FAVs with their avian hosts implies that viral DNA sequences to which proteins can bind and initiate gene expression would be specific to avian cells. For these reasons avian adenoviruses such as FAV8 are not thought likely to replicate in cells of mammalian origin. 47. Although no cytopathic effect was observed during the in vitro experiments described above, it is not known whether the cells were able to transduce the cell lines tested. While this is considered to be highly unlikely due to the restricted host range of adenoviruses, if the GM FAV8 was able to transduce the cell lines, the expression of the Ch-IFN-γ transgene would occur independently of viral replication for as long as the virus was maintained within the cell. The effect of IFN-γ expression in mammalian cells is discussed in Section 2.4. 2.2.2 Virulence21 of the parent organism 48. The virulence of FAVs vary even within the same serotype. While the parent organism that is proposed for use in this dealing, CFA44, is an avirulent isolate which was categorised as non-pathogenic to mildly pathogenic with a minimum infectious dose (MID) of 5x103 to 105 TCID50 , another FAV8 isolate, CFA40, which was isolated at the same time as CFA44 during an outbreak of IBH in Victoria, is defined as a hypervirulent strain with an MID of 10-100 TCID50 (Pallister et al. 1996d). 49. The difference in virulence between these two strains and another FAV8 isolate of mild virulence, CFA3, has been shown to be determined by the sequence of the fiber gene (Pallister & Sheppard 1996c; Pallister et al. 1996c). The only significant variation in the sequences of CFA3 and CFA40 occurred in the region that encoded the fiber knob domain, an area that is also implicated in the determination of tissue tropism. Transduce – the process whereby viruses are taken up by target cells but are not able to be propagated due to a host-mediated restriction. 21 Virulence – the level of disease caused by an organism. 20 Chapter 1 Risk assessment context (January 2006) 14 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 50. Other differences between the mildly virulent CFA3 and CFA44 isolates and the CFA40 isolate were detected by restriction enzyme analysis in the genes that encoded the 100K and hexon proteins (Pallister & Sheppard 1996b). These genes are located in a region of late viral gene expression, consistent with them being structural proteins that are required during virus assembly. 51. The hexon protein is important for virus entry into the cell from the endosome22. Mutations in the gene that encodes the hexon protein may affect the amount of virus that escapes from the endosome into the cell and therefore the virulence of the FAV8 isolate (Pallister & Sheppard 1996a). Functions of the 100K protein in human adenovirus 5 include the transport of newly synthesized hexon monomers (the major structural protein of the Ad capsid) from the cytoplasm to the nucleus and trimerization of hexon monomers (Cepko & Sharp 1983). It is not known if the 100K protein is a determinant of FAV8 virulence. 52. Other possible virulence determinants within the FAV8 genome include a triglyceride lipase protein and a homologue of the FAV1 Gam1 protein. Mutations in a gene that encoded triglyceride lipase in Marek’s disease virus (MDV), an avian virus, reduced the virulence of this virus. Homologues of this putative triglyceride lipase have also been found in the CFA40 isolate of FAV8 and in FAV10 (Washietl & Eisenhaber 2003b). 53. The FAV1 Gam1 protein has been shown to have an anti-apoptotic23 activity when expressed in mammalian cells (Chiocca et al. 1997b). The activity of this protein acts to prevent cell death thereby preventing exposure of infecting adenovirus to the host immune system. Gam1 acts to induce expression of host cellular genes by inhibition of expression of another cellular gene, histone deacetylase and is essential for viral DNA replication (Chiocca et al. 2002). 2.2.3 Pathogenicity24 of the parent organism 54. Adenoviruses enter susceptible hosts either by the mouth, the nasopharynx (nose) or the ocular conjunctiva (eyes). They are able to infect epithelial cells of the respiratory system or the gastrointestinal (GI) tract and can also infect lymphoid tissue in these regions which leads to the type of immune response generated by the host. 55. Diseases caused by group E avian adenoviruses such as FAV8 include IBH, hydropericardium syndrome (HPS) and respiratory diseases (McFerran & Smyth 2000b) and have also been associated with gizzard erosion (Ono et al. 2003). 56. IBH/HPS are thought to be multi-factorial diseases that require the action of more than one virus or prior immunosuppression to enable onset of the diseases (Toro et al. 2000). 57. While hypervirulent strains of FAV8, such as the CFA40 strain, were isolated from an outbreak of IBH in Victoria (Erny et al. 1991), the parent organism, FAV8 CFA44, is not associated with the development of IBH and is asymptomatic when inoculated systemically into day old chickens (Scott Tyack, personal communication). Endosome – an intracellular organelle formed by invagination of the cell wall in response to an extracellular stimulus such as virus binding. 23 Anti-apoptotic – virus infection of cells can lead to cell death by a mechanism known as apoptosis which is characterised by a cessation of DNA replication, DNA fragmentation, and rupture of endogenous cell enzymes which cause the cell to fragment. The anti-apoptotic effect demonstrated by adenoviruses prevents this process occurring. 24 Pathogenicity – the ability of an organism to cause disease. 22 Chapter 1 Risk assessment context (January 2006) 15 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 58. Another isolate of FAV8, E. Surient, has already been approved for use as a vaccine against infection with more virulent strains of FAV8 by the APVMA. The E. Surient isolate is not associated with the development of IBH. 59. Animal models have been used to determine the oncogenic potential of a number of avian adenoviruses (Fadly et al. 1976; Dhillon & Jack 1997a). Although avian adenoviruses are species specific such that natural routes of exposure to animals other than avian species are highly unlikely to result in infection, other routes of exposure such as by subcutaneous or intra-cerebral inoculation can result in infection. Members of FAV8 subtype b have been implicated in the formation of tumours in hamster models of adenovirus infection. However, an isolate of subtype a, of which the CFA44 isolate is a member, was not able to produce tumours in hamsters (Dhillon & Jack 1997b). 60. Determinants of adenovirus pathogenicity are unknown. However, as for virulence, pathogenicity may be related to the ability of the virus to enter and replicate in certain cell types. This is dependent on the cell and tissue tropism of the virus and is determined both by both viral factors such as the fiber protein, the 100k protein and the penton base (Seth et al. 1984) and by host-encoded proteins. 2.2.4 Antibody status of host organisms and immune response to adenovirus infection 61. The chickens that will be used in this dealing will either be obtained as day old female chickens or as fertilised eggs obtained at age 16 days incubation. Both will be obtained from a single commercial source. 62. It is unknown whether either the day old chickens or the hatchlings will possess any maternal antibodies to FAV8 that may affect the efficacy of the treatment. However, the day old chicks are likely to have been vaccinated against common viral diseases of chickens such as MDV, infectious bursal disease virus (IBDV) and Newcastle disease virus. Hatchlings are unlikely to have been vaccinated against these diseases. 63. If there is no prior exposure to FAV8, then it would be expected that the immune response of the chickens, following inoculation with the GM FAV8, would follow the classic antibody response to viral infection. If maternal antibodies are present, then it has been demonstrated that these are only fully effective for 6-8 days so that after this time, the chickens would not have any immunity against FAV infection. 64. The immune response to adenovirus infection occurs following a number of steps including virus uptake and gene expression that leads to viral assembly and viral antigen presentation on the cell surface. 65. Following uptake of adenovirus by susceptible cells, the adenovirus capsid is disassembled by proteolytic degradation within the endosome. DNA is transported through the nuclear pore to the nucleus whereupon expression of early adenovirus genes cause the host cell to enter the DNA replication phase so that viral DNA replication can begin (Shenk 2001). 66. Because the GM FAV8 will be administered in ovo¸ conjunctivally and orally in water, it is likely that lymphoid cells in the oropharynx and gut will be induced to produce a mucosal immune response to the GM FAV8 infection in which high level Chapter 1 Risk assessment context (January 2006) 16 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator of immunoglobulin A (IgA) would be expected to provide immunity to further infection. This form of immunity would be expected to last for between 6-8 weeks (McFerran & Smyth 2000a). 67. The immune response to adenovirus infection involves both cell-mediated (release of cytokines and proliferation of certain cells of the immune system) and humoral (development of antibodies against the infecting adenovirus) arms of the immune system. The cell-mediated response occurs immediately following infection, with antibodies being produced later. 68. One of the initial host responses to infection by adenovirus is the secretion of the cytokines interferon (IFN)- and IFN-, tumour necrosis factor (TNF)- and interleukin (IL)-12 by the infected cell. These cytokines bind to receptors on the infected cell and on neighbouring cells and induce the expression of genes which act to repress viral DNA replication. 69. IFN / also induce expression of cell surface molecules (MHC II) on antigen presenting cells which leads to the efficient recognition of virus, activate natural killer (NK) cells to kill virus-infected cells and promote the release of other cytokines such as IFN-γ from NK cells. 70. The release of IFN-γ provides a potent antiviral, antibacterial and antiparasitic defence mechanism (Johnson et al. 2000c). IFN-γ activates macrophages to produce nitric oxide radicals which help to destroy viruses before the organism has produced antibodies against the virus. In addition to this activity, IFN-γ induces expression of a large number of genes and leads to the activation of cells of the immune system. A receptor for IFN-γ is expressed on virtually all nucleated cells (Biron and Sen 2001). 71. IFN-γ has previously been shown to act as an adjuvant to increase the protective effect of vaccination against influenza in mice (Cao et al. 1992). It is possible that because IFN-γ acts to increase viral clearance, any active infection of cells by adenoviruses that express this gene may be cleared more efficiently than wild type adenoviruses. 72. Human adenoviruses are known to have evolved strategies for evading the host immune system. These include preventing the expression of antigen recognition molecules on the surface of cells of the immune system and by down-regulating the expression of receptors that are required for the recognition of apoptotic signals. Both of these mechanisms of evasion involve the disruption of intracellular protein trafficking (Windheim et al. 2004). 73. The regions of the human adenovirus genome that encode immune evasion functions are the E1A, E1B and E3 regions (Burgert et al. 2002). The homologous regions of FAV9, an adenovirus that is closely related to FAV8, encode none of the proteins that are responsible for immune evasion in human adenoviruses. This indicates that host immune evasion may not play a significant role in the persistence and life cycle of fowl adenoviruses such as FAV8 (Ojkic et al. 2002c). 74. Despite the lack of similarity between the E1 and E3 regions of human and fowl adenoviruses, other regions are highly conserved. In addition, fowl adenoviruses such as FAV8, FAV9 and CELO possess an E5 region which encodes a protein, Gam1 that in CELO is known to have anti-apoptotic activity (Chiocca et al. 1997a). Thus it may be that this protein represents the extent to which fowl adenoviruses have adapted to evade the immune system in avian species. Chapter 1 Risk assessment context (January 2006) 17 DIR 046/2003—Risk Assessment and Risk Management Plan 2.2.5 Office of the Gene Technology Regulator Transmission of FAV8 75. FAVs can either be transmitted both horizontally and, in some cases, vertically (McFerran & Smyth 2000d). 76. Horizontal transmission mainly occurs via the faecal-oral route. The virus is shed in the faeces and in nasal and tracheal mucosa and conjunctival secretions (McFerran & Smyth 2000e). 77. The spread of the virus mainly occurs by direct contact between birds or by indirect contact facilitated by people, or other fomites. Airborne spread of the virus, in contaminated aerosols, is thought to occur only over a short distance (Cook 1974). However, contaminated litter from a poultry house may provide a mechanism for virus spread if transported. 78. Vertical transmission of FAVs has been observed in FAV1 isolates. Chicks hatching from infected eggs may shed virus in faeces from the time of hatching (McFerran & Smyth 2000f). 79. Experiments with a virulent isolate of FAV8 (VRI-33) that was isolated from an outbreak of IBH (Kefford & Borland 1979) and that was used to infect both hens and roosters demonstrated that, by contrast to FAV1, vertical transmission by this isolate of FAV8 was an infrequent event (Reece et al. 1985b). Vertical transmission has not been observed for CFA44 (Scott Tyack, personal communication). 80. Although flies are not known to be vectors of FAV8, experiments conducted by the applicant showed that viable virus could be isolated from the surface of flies that had been exposed to 105 TCID50/ml of CFA44 for a period of 12 hours after exposure (data provided in application and discussed further in Chapter 3, section 2.2.2). 2.2.6 Persistence and latency of FAV8 81. Human adenoviruses such as Ad5 are able to persist and be shed from the tonsils, adenoids and intestines of infected hosts (Horwitz 1996). However, it is unknown whether this is due to viral latency, in which the genome of the virus is maintained within a host cell in an inactive state, or whether it is due to a sub-clinical level of adenovirus infection. No mechanism by which an adenovirus can establish latency has yet been documented. 82. There is no evidence that fowl adenovirus is able to remain latent within cells and persist over long periods. 2.2.7 Environmental stability of FAV8 83. FAVs are resistant to trypsin, alcohol and lipid solvents, but are sensitive to a 1:1000 dilution of formaldehyde and desiccation on fomites, although this can be stabilised by the presence of faecal material (Abad et al. 1994d). 2.2.8 Recombination of the parent organism 84. All viruses share the ability to incorporate new characteristics by the processes of random mutation and recombination that occur during propagation of the genetic Chapter 1 Risk assessment context (January 2006) 18 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator material. While mutations occur randomly and are largely independent of the sequence of the DNA or the RNA of the viral genome, the majority of viral recombination is sequence-dependent, relying on the pairing and exchange of DNA between highly similar sequences, usually found within viruses of the same family or genus. This type of recombination is known as homologous recombination and is a common phenomenon within natural virus populations. Homologous recombination results in hybrids with essentially the same properties as the parental virus (Candresse 1997). 85. The ability of adenoviruses to acquire foreign DNA and replicate efficiently when containing foreign DNA such as a transgene is limited by the capacity of the virus. Adenoviruses are only able to incorporate finite amounts of foreign DNA, so that most recombination events are likely to lead to non-viable viruses that have not gained any selective advantage. 86. Homologous recombination can take place between viruses that infect the cell at the same time (co-infection) (Meinschad & Winnacker 1980c) or that infect the cell at different times (superinfection) (Munz et al. 1983; Young et al. 1984b). It can also take place between the infecting virus and the cellular genome (Duigou & Young 2005c). 87. As discussed below, homologous recombination is responsible for the majority of viral recombination. However, non-homologous recombination, in which the sequences that are transferred are unrelated to those at the site of the recombination event, also occurs at an extremely low frequency. 88. For DNA viruses such as adenoviruses that replicate in the nucleus of the infected cell, recombination occurs at the same time as DNA replication and can even occur late in the replication cycle (Young et al. 1984a). 89. A study of adenovirus recombination, using two human adenoviruses (Ad2 and Ad5) showed that in related adenoviruses there were multiple recombination events within the genome (Meinschad & Winnacker 1980b). These homologous recombination events were mediated by short regions of DNA homology that were between 45 bp and 156 bp in length (Mautner & Boursnell 1983). The frequency of adenovirus recombination events was between 3-6% after one mixed infection, and up to 25% following three mixed infections (Meinschad & Winnacker 1980a). These figures indicate that homologous recombination between highly related adenoviruses is a common occurrence. 90. While the above results were derived from human adenoviruses, it has also been shown that recombination may have taken place between adenoviruses from different genera (Nagy et al. 2002). This event may have occurred during virus evolution prior to the splitting of the adenovirus genera. 91. Recombination can take place between adenoviruses and the cellular genome. This has only been observed in cell lines that are used for the packaging of replication defective adenoviruses25 (Hehir et al. 1996; Murakami et al. 2002; Duigou & Young 2005a). The frequency of recombination events that can lead to the generation of replication competent adenoviruses (RCA)26 was shown to be extremely low (2 x 10-8 replication defective adenoviruses – adenoviruses that have had an essential region such as the E1A or E1B region removed. These adenoviruses are packaged in cell lines that contain the missing regions integrated into the cellular genome. 26 replication competent adenoviruses - adenoviruses that have become replication competent by acquisition by recombination of missing genes. 25 Chapter 1 Risk assessment context (January 2006) 19 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator to 5.6 x 10-10) (Duigou & Young 2005b). These recombination events were due to the presence of short single regions of homology between the adenovirus and the adenovirus genes that had been integrated into the packaging cell line. 92. Non-homologous recombination events leading to RCA generation, were also observed. However, these only occurred at a frequency of 1 in 7.5 x 10-12 viruses (Murakami et al. 2004). 93. Integration of both human and avian adenoviruses into the genome of the infected cell has been observed (Ishibashi et al. 1980b) (Sutter et al. 1978). In avian adenoviruses, this appears to be limited to CELO, a FAV1 virus that is able to transform infected cells due to its oncogenic capacity (Ishibashi et al. 1980a). 94. Although there are no reports of FAV8 integration into the host genome or oncogenic transformation of infected cells, it is possible that integration of FAV8 may occur with an extremely low frequency. However, integration of wild type adenovirus by homologous recombination into the chromosomes of cells would preclude the natural FAV8 lytic infection cycle whereby viruses are released from the cell. (Doerfler 1982). Therefore, in avian cells, FAV8 is highly unlikely to integrate into the host genome. 2.3 The GMO 95. A single GM FAV is proposed for release. The GM FAV contains the wild type chicken IFN-γ gene (Ch IFN-γ). 96. Short regulatory sequences (promoters and polyadenylation sequences) that control expression of the introduced genes are also present in the GM FAV. The expression of Ch IFN-γ will be controlled by the human cytomegalovirus immediate early promoter (hCMV-ie) (Stinski and Roehr, 1985) and the SV40 polyadenylation sequence (Clontech). The regulatory sequences are not infectious or capable of causing disease symptoms. 97. The length of the expression construct is approximately 1400 bp and comprises the hCMV-ie promoter, the Ch IFN-γ gene and the SV40 poly A sequence. 98. The construct containing the promoter, transgene and polyadenylation sequence has been inserted into a region at the right hand end of the FAV8 genome. The sequence of this region, which has been provided by the applicant, indicates that the site of insertion is not within any FAV open reading frames (ORFs)27 that may encode viral proteins. 99. Restriction endonuclease digestion of the site at which the construct has been inserted has resulted in the deletion of a 54 bp sequence. The deletion is not within the published ORFs of the FAV8 genome (Ojkic & Nagy 2000d) and is not within the gene encoding the fiber protein, which has been shown to responsible for variations in virulence between fowl adenoviruses (Pallister et al. 1996b). 100. The insertion site lies less than 10 bp from the beginning of a putative ORF with no homology to any known proteins and no known function, and 900 bp from the start of an ORF that encodes the FAV8 homologue of GAM1, an anti-apoptotic protein that prevents the death of cells infected with this virus (Lehrmann & Cotten 1999). On ORFs – regions of nucleic acid within the genome of an organism that, when expressed, can give rise to significant stretches of amino acids that may form a protein product. 27 Chapter 1 Risk assessment context (January 2006) 20 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator the other strand, the insertion site is approximately 1.2 kb upstream of a gene that encodes a putative triglyceride lipase homologue, a protein that has been implicated as a virulence determinant (Washietl & Eisenhaber 2003a). 101. It is unknown whether the insertion of the construct at this site will have any effect on the expression of these ORFs. However, insertion of the construct into the CFA40, a hypervirulent isolate of FAV8 has an attenuating effect since the GM CFA40 replicates to lower viral titres than the parental non-GM strain of FAV8 (data provided in application). 2.4 The introduced gene and its product 102. The IFN-γ gene encodes a member of the interferon Type II cytokine family. When expressed, IFN-γ has wide ranging biological effects on cells of the immune system and stimulates immune cells to kill bacteria, viruses and both intracellular and extracellular parasites (Samuel 2001; Pestka et al. 2004). 103. The interferon family is conserved between animal species. The γ-interferons are highly species-specific and are naturally produced by T cells and natural killer (NK) cells of the immune system. 104. The presence of a cleavable signal peptide at the N-terminus of the Ch IFN-γ pro-protein28, leads to its secretion from infected cells. Once secreted, it enters the bloodstream where it acts upon its target cells, ie. macrophages and NK cells. 105. Expression of IFN-γ results in anti-viral activity due to the up-regulation of expression of components of the immune system. Specifically, class I and class II molecules of the major histocompatibility complex (MHC), that are involved in the degradation of foreign antigens, are induced on monocytes and many other normal cell types such as endothelial cells (Adolf 1985). In addition, macrophages are activated to release nitrite oxide radicals, and NK cells and T-helper cell activity is also up-regulated. 106. The introduced version of Ch IFN-γ gene was cloned from a complementary DNA (cDNA) expression library (Digby & Lowenthal 1995b). The cDNA sequence (GenBank Accession No. X99774) does not contain any of the intron29 sequences that are present in the copy of the gene that is present in the chicken genome (GenBank Ref:Y07922). The genomic Ch IFN-γ is 6755 bp in length whereas the cDNA that has been cloned into the GMO is 943bp. 107. The Ch IFN-γ protein shows significant amino acid sequence identity to IFN-γ proteins from turkey (97% identity), pheasant (97%), Japanese quail (93%) and guineafowl (87%). IFN-γ from chicken and turkey are cross-reactive in biological assays (Lawson et al. 2001a). In addition, IFN-γ from duck, which shares only 67% identity at the amino acid level with Ch IFN-γ, showed anti-viral activity in chicken fibroblasts indicating that this level of amino acid identity was sufficient to enable cross reactivity. It has been speculated that only IFN-γ proteins that share greater than 60% identity are likely to be cross-reactive (Scheerlinck 1999a). 108. Ch IFN-γ is less than 40% identical to the IFN-γ protein of mammals. It is only 39% identical to the IFN-γ protein of bovine species, and 35% identical to that of Pro-protein – an immature form of a protein that has to be proteolytically cleaved to produce the mature form of the protein. Intron – a sequence of DNA within a gene that does not encode any of the protein product. Intron sequences are removed following transcription of the gene into messenger RNA 28 29 Chapter 1 Risk assessment context (January 2006) 21 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator human IFN-γ. Expression of Ch IFN-γ in bovine fibroblasts failed to protect these cells from virus-induced lysis, indicating that Ch IFN-γ was not active in this species (Digby & Lowenthal 1995a). Similarly, the expression of murine IFN-γ, which is 41% identical to human IFN-γ in human cells failed to inhibit viral growth in these cells (Bukreyev et al. 1999b). These results suggest that if the Ch IFN-γ gene was expressed in organisms other than birds, it is unlikely that the protein would be biologically active. The host specificity of IFN-γ is almost certainly determined at the level of receptor binding of the IFN-γ protein. 109. Human IFN-γ has been thoroughly assessed in conventional safety models used for xenobiotics (Terrel and Green 1993). Acute toxicity studies with human IFN-γ have revealed that an extremely high dose (1.5mg/kg/day), which is 100-1000 times the recommended clinical dose, over a period of 4 weeks, led to mortality in nonhuman primates (http://www.medsafe.govt.nz/profs/Datasheet/i/Imukininj.htm). The level of expression of Ch IFN-γ from the hCMV-ie promoter is unlikely to lead to serum concentrations of IFN-γ that are significantly higher than physiological concentrations. This is because of the possibility that expression from the hCMV-ie promoter in non-human cells does not lead to levels of expression that are as high as those observed in human cells (Addison et al. 1997). 2.4.1 Expression of the Ch IFN-γ protein in the GM FAV 110. In the GM FAV8, expression of the Ch IFN-γ gene is controlled by the hCMVie promoter and by the SV40 polyadenylation signal. 111. The hCMV-ie promoter is regarded as a strong promoter that results in constitutive gene expression in mammalian cells. Although no data is available on the level of gene expression, trials with the GM FAV construct in chickens in contained facilities indicated that the level of IFN-γ expression from this construct led to an increased feed conversion ratio and weight gain. 112. The promoter controlling expression of the Ch IFN-γ gene in the GM FAV is expected to lead to expression of the Ch IFN-γ protein in the chicken until the GM FAV is eradicated by the host immune system. Because FAV8 infects cells of the respiratory and gastro-intestinal (GI) tract, Ch IFN-γ would be expected to be expressed in these cells. 113. An mRNA termination region, including a polyadenylation signal is also required for efficient gene expression in mammalian cells. This signal is required for the addition of a stretch of adenine nucleotides onto the end of the messenger RNA (mRNA) strand following transcription. In GM FAV8, these signals are provided by a sequence from the polyoma virus SV40. The SV40 polyadenylation signal is widely used in molecular biology and is present on a large number of expression vectors. This sequence is 130 bp in length and is untranslated. 2.5 Method of genetic modification 114. The GM FAV8 was constructed by insertion of an expression cassette into the genome of the CFA44 isolate of FAV8. A similar procedure has been described for introduction of genes into the FAV10 genome (Sheppard et al. 1998) and into the CFA40 strain of FAV8 (Johnson et al. 2000b). Chapter 1 Risk assessment context (January 2006) 22 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 115. An expression cassette containing hCMV-ie, Ch IFN-γ and the SV40-polyadenylation sequence was removed from a cloning plasmid by digestion with the NotI restriction enzyme and cloned into a plasmid cloning vector. This vector was then digested with the restriction enzyme NheI and mixed with genomic FAV8 CFA44 DNA that had been cleaved by incubation with the restriction enzyme SpeI. Digestion of FAV8 CFA44 with SpeI resulted in a 54bp deletion in the right hand end of the FAV8 genome. Monolayers of chicken kidney cells were then transfected with the recombinant viral DNA and passaged onto chicken kidney cells until a cytopathic effect, indicative of the production of infectious virus, was observed. The insert was sequenced to confirm the proposed site of insertion of the expression cassette. 116. The GMO has been passaged many times and restriction analysis of the GM FAV has shown that the expression construct is stable. SECTION 3 THE RECEIVING ENVIRONMENT 117. The receiving environment forms part of the context in which the risks associated with dealings involving the GMO are assessed. This includes the size, duration and location of the dealings where the release would occur; intended animal husbandry 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 2005). 118. The size and duration of the proposed release proposed release are outlined in Section 2, along with a description of the CSIRO Livestock Industries animal containment facility at Werribee in the Shire of Wyndham, Victoria where the proposed release is to occur. 119. Because the dealing is being conducted in the containment facility, the biotic interaction between the GMO and the environment is expected to be minimal. 120. The applicant intends to treat all waste water either by heat treatment or by chemical decontamination and maintain a high level of hygiene on the site (eg all equipment will be cleaned on entry and exit to prevent the transfer of the virus). 121. The region in which the trial will be conducted is an agricultural area that produces sheep, cattle and pigs. There are no chicken producers in this area. No changes are contemplated to agronomic practices in this area due to the trial being conducted. 122. There is a zoological park (Victoria Open Range Zoo) approximately 5km from the trial site. 123. The Ch IFN-γ protein is not produced naturally by wild type strains of FAV8, an endemic virus, but is already present in chickens. This forms part of the baseline data for determining the risks from exposure to this protein as a result of the proposed release of the GM FAV8. Chapter 1 Risk assessment context (January 2006) 23 DIR 046/2003—Risk Assessment and Risk Management Plan SECTION 4 4.1 4.1.1 Office of the Gene Technology Regulator PREVIOUS AUSTRALIAN AND INTERNATIONAL APPROVALS Previous Australian approvals of the same or similar GM Fowl Adenovirus Approvals by GMAC or the Regulator 124. No release of GM FAV8 has previously been approved. 125. The Regulator has previously issued a licence (DNIR 068/2003) for dealings with the same or similar GMOs to be conducted in facilities certified by the Regulator to physical containment level 2 (PC2). 4.1.2 Approvals by other Australian government agencies 126. The Regulator is responsible for assessing risks to the health and safety of people and the environment associated with the use of gene technology. As the GM FAV8 is intended for use as a veterinary chemical, it is also subject to the regulatory requirements of the Australian Pesticides and Veterinary Medicines Authority (APVMA). This is discussed further in Chapter 4, Risk Management. 127. The APVMA has previously approved the use of a wild type FAV8 isolate (E.Surient) (NRA Registration No. 52390) for use as a vaccine for chickens which protects day old chicks from a clinical disease that is induced by infection with more virulent strains of FAV. 4.2 International approvals 128. There have been no international approvals for field trials of the GM FAV8 strain. Chapter 1 Risk assessment context (January 2006) 24 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator CHAPTER 2 RISK ASSESSMENT SECTION 1 INTRODUCTION 129. Risk assessment is the overall process of identifying the sources of potential harm (hazards) and determining both the seriousness and the likelihood of any adverse outcome that may arise. The risk assessment (summarised in Figure 2.1) considers risks from the proposed dealings with the GMO that could result in harm to the health and safety of people or the environment posed by or as a result of gene technology. Figure 2.1 The risk assessment process. RISK CONTEXT GMO Dealings Parent organism Receiving environment HAZARD POTENTIAL ADVERSE OUTCOME Source of potential harm CONSEQUENCES Severity of harm RISK ESTIMATE IDENTIFIED RISK LIKELIHOOD Chance of harm Note: words in bold are defined in Appendix A. 130. Once the risk assessment context has been established (Chapter 1) the next step is hazard identification to examine what harm could arise and how it could happen during this release of GMOs into the environment. 131. It is important to note that the term ‘hazard’ is used in a technical rather than a colloquial sense in this document. The hazard is a source of potential harm. There is no implication that the hazard will necessarily lead to harm. A hazard can be an event, a substance or an organism (OGTR 2005). 132. Hazard identification involves consideration of events (including causal pathways) that may lead to harm. These events are particular sets of circumstances that might occur through interactions between the GMO and the receiving environment as a result of the proposed dealings. 133. A number of hazard identification techniques are used by the Regulator and staff of the OGTR, including the use of checklists, brainstorming, commonsense, reported international experience and consultation (OGTR 2005). In conjunction with these techniques, hazards identified from previous RARMPs prepared for licence applications for the same or similar GMOs are also considered. 134. The hazard identification process resulted in the compilation of a list of events. Some of these events lead to more than one adverse outcome and each adverse outcome can result from more than one event. Chapter 2 Risk assessment (January 2006) 25 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator SECTION 2 HAZARD CHARACTERISATION 135. The list of events compiled during hazard identification are characterised to determine which events constitute a risk to the health and safety of people or the environment posed by or as a result of gene technology. 136. A risk is identified only when there is some chance that harm will occur. Those events that do not lead to an adverse outcome or could not reasonably occur do not constitute an identified risk and will not advance in the risk assessment process. Risks associated with the remaining events are analysed further to determine the seriousness of harm (consequence) and chance of harm (likelihood). The identified risks must be posed by or result from gene technology. 137. The criteria used by the Regulator to determine harm are described in Chapter 3 of the Risk Analysis Framework (OGTR 2005). Harm is assessed in comparison to the parent organism and in the context of the receiving environment. The risk assessment process focuses on measurable criteria for determining harm. 138. The following factors have been taken into account during the analysis of events that may give rise to harm: the proposed dealings, which may include experimentation, development, production, breeding, propagation, use, growth, importation, possession, supply, transport or disposal of the GMOs the characteristics of the non-GM parent routes of exposure to the GMOs, the introduced gene(s) and its product(s) potential effects of the introduced gene(s) and its product(s) expressed in the GMOs potential exposure to the introduced gene(s) and its product(s) from other sources in the environment facility management and animal husbandry practices for the GMOs the size and duration of the release. 139. There have been no previous releases of this GM FAV8. However, the regulator has previously issued DNIR licence 068/2002 for research in contained PC2 laboratory facilities using the same and very similar GMOs. 140. Forty events were characterised in relation to the proposed release. They are summarised in Table 2.1 and discussed in detail later in this section. Events that share a number of common features are grouped together in broader hazard categories as indicated in the table. Two of these events were considered to lead to an identified risk that required further analysis. Chapter 2 Risk assessment (January 2006) 26 DIR 046/2003—Risk Assessment and Risk Management Plan Table 2.1 Hazard category (relevant section) Production of a substance that is toxic or adversely alters the immune response of people (Section 2.1) Summary of events that may give rise to an adverse outcome Event that may give rise to an adverse outcome (relevant sub-section) Ingestion of chicken products (e.g. meat, eggs) infected with the GMO or containing the Ch IFN-γ protein expressed by the introduced gene. (Sub-section 2.1.1) Contact with the GMO during inoculation, in chicken waste Potential adverse outcome Toxicity or adverse immune reactions in people Identified risk? Reason No Chickens from this trial will not be used as food and no eggs will be produced during the trial. Toxic or adverse immune reactions in people No Exposure to the GM FAV8 is expected to be very limited and the expressed Ch IFN-γ is not expected to be toxic or induce adverse immune responses in people. No adverse effects have been observed in workers exposed to a nonGM FAV8 chicken vaccine. Toxicity or adverse immune reactions in people No Exposure to increased levels of viral antigens produced as a result of Ch IFN-γ expression. (Sub-section 2.1.4) Production of a substance that is toxic or adversely alters the immune response in chickens inoculated with the GMO. Toxicity or adverse immune reactions in people Toxicity or adverse immune reactions in chickens No Exposure to the GM FAV8 is expected to be very limited and the expressed Ch IFN-γ is not expected to be toxic or induce adverse immune responses in people. No adverse effects have been observed in workers exposed to a nonGM FAV8 chicken vaccine. No change in expression of viral antigens is expected as a result of the genetic modifications. No Previous trials using chickens inoculated with same or very similar GMOs have not identified any adverse effects in chickens. Ingestion of chicken products infected with the GMO and/or containing the Ch IFN-γ protein expressed from the introduced gene. (Sub-section 2.3.1) Toxicity or adverse immune reactions in other birds No Chickens and chicken products from this trial will not be used in animal feed Contact with the GMO in chicken food or water, waste products or others waste materials or items in contact with the GMO. (Sub-section 2.3.2) Toxicity or adverse immune reactions in other birds No Other birds will be excluded from the facility and will not come into contact with chicken products or items that have been in contact with the GMO. products or other waste materials, when handling items in contact with the GMO or carried by other organisms, including people. (Sub-section 2.1.2) Inhalation of particles containing the GMO. (Sub-section 2.1.3) Production of a substance that is toxic or adversely alters the immune response in chickens (Section 2.2) Production of a substance that is toxic or adversely alters the immune response of birds other than chickens (Section 2.3) Office of the Gene Technology Regulator Chapter 2 Risk assessment (January 2006) 27 DIR 046/2003—Risk Assessment and Risk Management Plan Hazard category (relevant section) Production of a substance that is toxic or adversely alters the immune response of organisms other than people or birds (Section 2.4) Altered viral characteristics (host range, tissue tropism, mode of transmission, latency/chronic infection and pathogenicity or virulence) (Section 2.5) Office of the Gene Technology Regulator Event that may give rise to an adverse outcome (relevant sub-section) Contact with other organisms (including people) carrying the GMO outside the facility. (Subsection 2.3.3) Potential Identified Reason adverse risk? outcome Toxicity or No Exposure of other birds to the GMO adverse immune carried by other organisms is expected reactions in other to be limited and Ch IFN-γ is not birds expected to have an adverse effect on other birds. Inhalation of particles containing the GMO. (Sub-section 2.3.4) Toxicity or adverse immune reactions in other birds No Survival of the GMO in dust is expected to be limited and Ch IFN-γ is not expected to have an adverse effect on other birds. Contact with wash water containing the GMO. (Sub-section 2.3.5) Toxicity or adverse immune reactions in other birds No The opportunity for exposure is very limited and other birds are not expected to receive an infective dose from contact with wash water. Microorganisms or invertebrates come into contact with the GMO or the expressed Ch IFN-γ protein. (Sub-section 2.4.1) Non-avian, non-human vertebrates ingest chicken products infected with the GMO or containing the Ch IFN-γ protein expressed by the introduced gene. (Sub-section 2.4.2) Non-avian, non-human vertebrates ingest or come into contact with the GMO in chicken food or water, chicken waste products or other waste materials, on items in contact with the GMO or carried by other organisms. (Sub-section 2.4.3) Non-avian, non-human vertebrates inhale particles (e.g. dust or water droplets) containing the GMO. (Sub-section 2.4.4) Toxicity or adverse immune reactions No Ch IFN-γ is not expected to be active in or have an adverse effect on microorganisms or invertebrates Toxicity or adverse immune reactions in nonavian vertebrates No Chickens from this trial will not be used in animal feed and no eggs will be produced during trial. Toxicity or adverse immune reactions in nonavian vertebrates No The GM FAV8 is not known to naturally infect organisms other than birds. No evidence of infection or adverse effects has been observed in mice and rabbits inoculated orally or intra-ocularly with the GM FAV8. Toxicity or adverse immune reactions in nonavian vertebrates No Alteration of viral capsid by incorporation of Ch IFN- γ. (Sub-section 2.5.1) Disease in humans or other vertebrates, Disease in chickens or other birds Disease in humans or other vertebrates, Disease in chickens or other birds No The GM FAV8 is not known to infect organisms other than birds. No evidence of infection or adverse effects was observed in mice and rabbits orally administered with the GM FAV8. Ch IFN- γ is not expected to be incorporated into the capsid of the GM FAV8 or to alter viral characteristics. Alteration of viral capsid as a result of deleting 54 base pairs. (Sub-section 2.5.2) Chapter 2 Risk assessment (January 2006) No The 54bp deletion is not expected to alter the viral capsid. 28 DIR 046/2003—Risk Assessment and Risk Management Plan Hazard category (relevant section) Event that may give rise to an adverse outcome (relevant sub-section) Unintended change in amino acid sequence of protein determinants of viral characteristics. (Sub-section 2.5.3) Recombination with another adenovirus leading to acquisition of new viral characteristics and infection of humans or other nonavian vertebrates. (Sub-section 2.5.4) Recombination with another adenovirus leading to acquisition of new viral characteristics and an alteration in the pattern of infection in chickens. (Sub-section 2.5.5) Recombination with another adenovirus leading to acquisition of new viral characteristics and infection of other birds. (Sub-section 2.5.6) Recombination with viruses other than adenoviruses leading to acquisition of new viral characteristics. (Sub-section 2.5.7) Acquisition of new viral characteristics (host range, tissue tropism, pathogenicity or virulence) by transcapsidation. (Sub-section 2.5.8) Expression of Ch IFN-γ suppresses immune system. (Sub-section 2.5.9) Expression of the Ch IFN- γ gene leads to the development of interferon-resistant virus variants. (Sub-section 2.5.10) Chapter 2 Potential adverse outcome Disease in humans or other vertebrates, Disease in chickens or other birds Disease in humans or other vertebrates Office of the Gene Technology Regulator Identified risk? Reason No The genetic modifications are not expected to have any effect on protein determinants of viral characteristics. No Human and other non-avian adenoviruses do not infect chickens. Therefore, the GM FAV8 cannot acquire the ability to infect people and other non-avian vertebrates as a result of recombination with other adenoviruses. Recombination occurs normally in nonGM fowl adenoviruses and the genetic modifications to the GM FAV8 are not expected to increase the frequency of recombination or provide any selective advantage to other adenoviruses. Any recombination events that might occur are unlikely to result in any selective advantage for the GM FAV8 or the recipient virus. Disease in chickens No Disease in other birds No Disease in humans or other vertebrates, Disease in chickens or other birds Disease in humans or other vertebrates, Disease in chickens or other birds Disease in humans or other vertebrates, Disease in chickens or other birds No Recombination with viruses other than adenoviruses is a rare event that occurs on an evolutionary timescale and is not expected to occur during this trial. No Transcapsidation is transient and self limiting and is not expected to result in an adverse effect. No IFN-γ plays a positive, stimulatory role in immune responses and does not suppress the immune system. Disease in chickens, Disease in other birds No The GM virus will be cleared by the immune system within 7-14 days. The low level of transient Ch IFN-γ expression will not cause any selection pressure that will give rise to interferonresistant sub-populations. Risk assessment (January 2006) 29 DIR 046/2003—Risk Assessment and Risk Management Plan Hazard category (relevant section) Altered persistence within host (Section 2.6) Increased transmission of GM FAV8 (Section 2.7) Spread and persistence of the GM FAV8 in the environment (Section 2.8) Gene transfer between GM virus and other organisms (Section 2.9) Office of the Gene Technology Regulator Event that may give rise to an adverse outcome (relevant sub-section) Expression of Ch IFN-γ induces latency/chronic infection. (Sub-section 2.6.1) Potential adverse outcome Disease in chickens Deleting 54 base pairs induces latency/chronic infection. (Sub-section 2.6.2) Expression of Ch IFN-γ increases transmission. (Sub-section 2.7.1) Deleting 54 base pairs increases transmission of the virus. (Sub-section 2.7.2) Disease in chickens No Disease in chickens No Disease in chickens No Increased number of viral particles produced. (Sub-section 2.7.3) Spread by other birds that come into direct contact with inoculated chickens, faeces, or items that have been in contact with the GMO (Sub-section 2.8.1) Spread to other birds as a result of insects or non-avian vertebrates transporting the GM FAV8 out of the facility. (Sub-section 2.8.2) Spread by animals that come into contact with waste material containing the GMO that is washed out of trial location. (Sub-section 2.8.3) Spread to other birds as a result of inhaling windborne particles containing the GM FAV8. (Sub-section 2.8.4) Expression of Ch IFN-γ increases stability of the GM FAV8 contributing to persistence in the environment. (Sub-section 2.8.5) Spillage of material containing the GMO during transport. (Sub-section 2.8.6) Recombination between the GM FAV8 and the chicken genome. (Sub-section 2.9.1) Disease in chickens No Increased pest potential in other birds No Increased pest potential in other birds Yes See Chapter 3 Event 1 Increased pest potential in other birds No The opportunity for exposure is very limited and animals are not expected to come into contact with an infective dose. Increased pest potential in other birds Yes See Chapter 3 Event 2 Increased pest potential in other birds No Ch IFN-γ is not expected to increase the environmental stability of the GMO or contribute to increased persistence. Increased pest potential in other birds No Toxicity, adverse immune reactions or disease in chickens. No All material containing the GMO will be transported and stored in accordance with OGTR guidelines. Exchange of the Ch IFN-γ gene between the GM FAV8 and the chicken genome is not expected to result in any adverse effects in chickens. Chapter 2 Risk assessment (January 2006) Identified risk? Reason No FAV8 is not known to contain genes associated with persistence and the expression of Ch IFN-γ is not expected to induce persistence. The 54bp deletion is not in a coding region and is not expected to affect persistence. Ch IFN-γ does not play any role in virus transmission and is not expected to increase transmission. The 54 bp deletion is not in a coding region or in a region of the virus associated with determinants of transmission. The available evidence indicates that the genetic modification will not increase the number of viral particles produced No direct contact can occur since all other birds are excluded from the facility. 30 DIR 046/2003—Risk Assessment and Risk Management Plan Hazard category (relevant section) Increased rate of mutation in the GMO (Section 2.10) Unauthorised activities (Section 2.11) Event that may give rise to an adverse outcome (relevant sub-section) Recombination between the GM FAV8 and genome of other birds. (Sub-section 2.9.2) Gene transfer between the GM FAV8 and the genome of nonavian, non-viral organisms. (Sub-section 2.9.3) The genetic modifications destabilise the GM FAV8 leading to increased rate of mutation. Use of the GMO outside of the propose licence conditions (noncompliance). 2.1 Potential adverse outcome Toxicity, adverse immune reactions or disease in other birds. Increased pest potential in other birds Disease in nonavian organisms Office of the Gene Technology Regulator Identified risk? Reason No Exchange of the Ch IFN-γ gene between the GM FAV8 and the genome of birds other than chickens is not expected to result in any adverse effects in other birds. No Since the GM FAV8 is not expected to infect any organism other than birds, gene transfer to non-avian organisms is not expected. The GM FAV8 remains stable after repeated cycles of replication in vitro and passage through chickens. Unpredictable effects No Disease in chickens, disease or other birds. Increased pest potential in other birds No The Act provides for substantial penalties for non-compliance and unauthorised dealings with GMOs and also requires a test of the suitability of the applicant to hold a licence prior to the issuing of a licence by the Regulator. Production of a substance that is toxic or adversely alters the immune response of people 141. Toxicity is the cascade of reactions resulting from exposure to a dose of a chemical that is sufficient to cause direct cellular or tissue injury, or otherwise inhibit normal physiological processes (Felsot 2000). Toxic proteins are known to act via acute mechanisms rather than through chronic exposure (Sjoblad et al. 1992). Toxicity may occur through ingestion, contact with the skin or eyes or inhalation. The level of acute toxicity is often expressed as the LD50. This is the amount of a substance given in a single dose that causes death in 50% of a test population of an organism. 142. Immunogenicity is a normal reaction of the immune system to a foreign antigen. The production of antibodies to a foreign protein used as a therapeutic agent may have no effect, may alter the pharmacokinetics/efficacy of the therapeutic protein or may cross react with endogenous proteins with the potential for serious adverse effects. Serious adverse reactions to therapeutic cytokines are rare (Herzyk 2003) 143. Allergic responses are also adverse immune system reactions, resulting from stimulation of a specific group of antibodies (known as IgE) or sensitisation of specific tissue bound lymphocytes (Taylor & Lehrer 1996; FAO/WHO 2001). Allergy has a well-defined aetiology (ie biochemical cause) that is distinct from toxicity. 144. A search of the Allermatch™ database (http://www.allermatch.org/) revealed that the Ch IFN-γ protein sequence shows no significant homology with protein sequences of known allergens. Although Ch IFN-γ is glycosylated and a single contiguous stretch of six amino acids is homologous with a sequence of six amino acids from a peanut allergen, a comparison of contiguous 8 amino stretches shows no homology to any known allergen. Ch IFN-γ is not expected to induce an allergic response in people. Chapter 2 Risk assessment (January 2006) 31 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 145. Ch IFN-γ is the only substance that could be produced as a result of the genetic modifications to FAV8. Ch IFN-γ can only be produced in amounts sufficient to induce a toxic or adverse immune response if cells are infected with the GM FAV8. Avian adenoviruses only infect birds and data supplied by the applicant demonstrates that human cells are not infected with the GM FAV8. Therefore, Ch IFN-γ would not be produced in amounts that would be sufficient to induce a toxic or adverse immune response in people. 2.1.1 Ingestion of chicken products infected with the GMO or containing the Ch IFN-γ protein 146. The applicant has indicated that none of the chickens used in this trial will enter the human food chain. All chickens used in the trial will be destroyed by incineration. Hens will not be of laying age and no eggs will be produced. Therefore, a risk is not identified and the potential for toxicity or adverse immune reactions in people as a result of ingestion of the chicken products containing the GMO or the ChIFN-γ will not be assessed further. 2.1.2 Contact with the GMO during inoculation, in chicken waste products or other waste materials, when handling items in contact with the GMO or carried by other organisms, including people 147. Only people who work with the chickens used in the trial may come into contact with the GMO during handling chickens or waste products, administering the GMO and cleaning the facility. Contact with the GMO during administration would only occur as a result of an accidental splash of GMO added to drinking water or administered via the eye to chicks. Although less likely, accidental self injection during administration to eggs is also possible. No adverse effects have been reported for the widespread use of non-GM FAV8 vaccine approved for use and sale by the APVMA (see Chapter 1, section 2.2). 148. Any dermal contact with the GMO would be minimised by the use of appropriate personal protective equipment (PPE) such as gloves, overalls/laboratory coats as proposed by the applicant to minimise the possibility of the GMO being carried outside the facility by workers. The applicant proposed that all protective equipment be removed prior to exit and held within an ante-room through which workers will enter and exit the facility. 149. Since the proposed release is to be conducted on only one site with a single trial of six weeks, the extent, frequency and duration of human contact with the GM FAV8 is expected to be limited. Therefore, a risk is not identified and the potential for the production of a substance that is toxic or results in an adverse immune response in people as a result of contact with the GMO will not be further assessed. 2.1.3 Inhalation of particles containing the GMO 150. Particles of dust and water droplets in the spray generated during cleaning of the facility may contain the GM FAV8. People involved in the proposed trial may be exposed to these particles during the trial. People other than those directly involved in the trial are less likely to be exposed to particles containing the GMO since they are not expected to be within the vicinity of the trial site and the concentration of particles in the air is expected to decrease rapidly as distance from the trial site increases. 151. Experiments performed by the applicant showed no evidence of infection or any adverse effects in mice and rabbits given an oral dose of the GM FAV8. On the basis Chapter 2 Risk assessment (January 2006) 32 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator of the results from this oral dosing experiment, it is reasonable to expect that inhalation of the GMO is also unlikely to induce any toxic or adverse immune response in people after similar exposures. As noted above no adverse effects have been reported for the use of a non-GM FAV8 vaccine. 152. GM FAV8 does not infect human cells and therefore Ch IFN-γ is not expected to be produced in significant amounts in exposed people. 153. Data supplied by the applicant demonstrates that the GM FAV8 is susceptible to desiccation with up to 99.9% of viral particles being inactivated within 48 hours following evaporation of solution containing GM FAV8 under ambient conditions. Therefore, any dust particles inhaled by the workers are only likely to contain nonviable viral particles that are incapable of expression of Ch IFN-γ. Therefore, a risk is not identified and the potential for the production of a substance that is toxic or adversely alters the immune response in people as a result of inhalation of particles containing the GMO will not be assessed further. 2.1.4 Exposure to increased levels of viral antigens produced as a result of Ch IFN-γ expression 154. Since the inserted Ch IFN-γ gene is expressed under the control of the hCMV-ie promoter its expression is independent of the expression of other viral proteins and is not expected to have any unintended effect on the expression of other viral proteins. In addition, since adenoviruses including FAV are widespread in the environment people, especially those who are in regular contact with birds, would already be exposed to the adenoviral antigens. Therefore, a risk is not identified and the potential for an increase in the levels of expression of viral antigens resulting in toxic or adverse immune responses in people will not be assessed further. 2.2 Production of a substance that is toxic or adversely alters the immune response in chickens 155. The only substance expected to be produced as a result of the genetic modifications to FAV8 is Ch IFN-γ. Ch IFN-γ is produced naturally by chickens as a normal part of immune responses. The applicants have previously administered the same and very similar GMOs to chickens during trials conducted under DNIR licence 068/2002. No adverse reactions, including auto-immunity, were observed during these trials. Therefore, no risk is identified and the potential for production of a substance that is toxic or adversely alters the immune response of chickens will not be assessed further. 2.3 Production of a substance that is toxic or adversely alters the immune response of birds other than chickens 156. Other birds will be excluded from the facility in which the trial will occur. However, other birds may be exposed to the GMO if chicken products are used in feed or they come into contact with materials containing the GMO that may leave the trial site eg airborne particles, waste materials etc. 2.3.1 Ingestion of food products from chicken infected with the GMO and/or containing the Ch IFN-γ protein expressed from the introduced gene. 157. The applicant has indicated that none of the chickens or products from chickens used in this trial will be used as animal food. All chickens used in the trial will be destroyed by incineration. Hens will not be of laying age hence no eggs will be produced. Therefore, no risk is identified and the potential for toxic or adverse Chapter 2 Risk assessment (January 2006) 33 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator immune reactions in other birds resulting from ingestion of chicken products will not be assessed further. 2.3.2 Contact with the GMO in chicken food or water, waste products or others waste materials or items in contact with the GMO 158. The facility in which the proposed trial is to take place has been designed and built in manner that excludes other birds. There is no opportunity for direct exposure to chicken faeces, bedding or items such as food or water containers that have been in contact with the GMO. These materials and items that may contain or have been in contact with the GMO will either be cleaned and disinfected or collected in sealed containers prior to exist from the facility. This will prevent exposure of other birds outside of the facility to the GMO. Therefore, no risk is identified and the potential for toxic or adverse immune reactions in other birds resulting from contact with substances produced by the GMO in chicken waste products, other waste materials or items in contact with the GMO not be assessed further. 2.3.3 Contact with other organisms (including people) carrying the GMO outside the facility 159. The likelihood of people carrying the GM FAV8 outside the facility will be greatly reduced by the appropriate use and removal of protective equipment that limits dermal exposure to the GMO. 160. Insects and small vertebrates such as rodents will be able to access the facility in which the proposed trial is to take place. As a result of contact with faeces, bedding or other materials in the facility, these organisms could carry the GM FAV8 outside the facility and birds outside the facility may be exposed to the GM FAV8 as a result of contact with these organisms. Since the GMO is primarily transmitted via the fecaloral route, other birds would have to ingest insects or small vertebrates that may have come into contact with the GMO in chicken faeces in order for a possible infection to occur. 161. Data supplied by the applicant suggests that the GMO is shed into chicken faeces at low levels since it took two to three successive 7 day periods or passages before any cytopathic effect was observed in a cell culture assay to detect the GM FAV8 in the faeces of inoculated chickens. In addition, data supplied by the applicant shows that, the GM FAV8 survived for only 48 hours or less in bedding material similar to that to be used when conducting the proposed trial. Furthermore, data supplied by the applicant shows that infectious GM FAV8 could only be detected on flies for between 4 and 12 hours after they were exposed to high levels of the GM FAV8. Given the results of these studies, only low levels of active GM FAV8 particles are expected to be present on any non-avian vertebrate or insect that may come into contact with faeces from the treated chickens and any GM FAV8 on these animals is not expected to survive for extended periods. 162. As outlined in Chapter 1, the level of expression of the Ch IFN-γ under the control of the hCMV-ie promoter in non-human cells may be no higher than endogenous levels of expression. In chickens given doses of very similar GMOs containing the Ch IFN-γ gene, the applicant stated that they were not able to measure Ch IFN-γ levels above the background levels normally present in untreated chickens. To date the only reproduceable assay for the effects of the GM FAV8 has been increased weight gain in chickens. This suggests that Ch IFN-γ levels in chickens as a result of expression of the gene introduced into the GM FAV8 will be low. Chapter 2 Risk assessment (January 2006) 34 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 163. If other birds were to become infected with the GM FAV8, Ch IFN-γ would only have full biological activity in birds closely related to chickens (Huang et al. 2001b; Lawson et al. 2001b). Based on the laboratory work conducted to date in chickens, the levels of expression of Ch IFN-γ are expected to be very low. Therefore, toxic effects of IFN-γ which can occur at high doses (Terrell & Green 1993; Bussiere et al. 1996), are not expected. 164. Other birds may mount an immune response to the Ch IFN-γ. A normal immune response would increase the clearance of the Ch IFN-γ from the systemic circulation of the birds and is unlikely to result in any adverse effects. 165. As discussed in Section 2.1, a search of the Allermatch™ database (http://www.allermatch.org/) revealed that the Ch IFN-γ protein sequence shows no significant homology with protein sequences of known allergens. Therefore, a risk is not identified and the potential for toxicity or adverse immune responses in other birds as a result of exposure to other organisms carrying the GM FAV8 will not be assessed further. 2.3.4 Inhalation of particles containing the GMO 166. As discussed in Section 2.1.3, articles of dust generated during the trial may contain the GM FAV8. Other birds in the area could be exposed to dust particles that escape from the facility during the trial. However, the number of infectious GM FAV8 particles present in dust is expected to be very low because the GM FAV8 is not expected to survive desiccation that would occur in dust particles. Birds outside of the facility are therefore expected to have very limited exposure to viable viral particles that might be present in dust. 167. As discussed in section 2.3.3, the level of expression of the Ch IFN-γ is expected to be low and toxic effects are not expected to occur at low levels. The Ch IFN-γ is not expected to be allergenic and normal immune responses would increase the clearance of Ch IFN-γ from the systemic circulation. Therefore, a risk is not identified and the potential for toxicity or adverse immune reactions in other birds as a result of exposure to particles containing the GM FAV8 will not be assessed further. 2.3.5 Contact with wash water containing the GMO 168. At the end of the proposed trial, the applicant intends to wash the interior surfaces of the facility prior to treating surfaces with a virucidal compound. The wash water enters open drains within the facility prior to collecting in a small pit outside of the facility before being pumped into a holding tank and then to another tank for disinfection by heat treatment at 80°C. 169. Animals such as other birds may come into contact with wash water that may contain the GM FAV8, especially when the wash water is collected in the small pit outside of the facility. However, the concentration of virus particles in the wash water is expected to be very low since few active viral particles are expected to survive desiccation on the interior surfaces of the facility and the washing process is expected to greatly dilute any remaining active virus present. Furthermore, the opportunity for exposure is limited because the wash water is expected to be present in the small pit outside the facility for only a very short time prior to being pumped into a holding tank and subsequently decontaminated by heat treatment. Therefore, no risk is identified and the potential for toxicity or adverse immune reactions in other Chapter 2 Risk assessment (January 2006) 35 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator birds as a result of contact with contaminated wash water will not be assessed further. 2.4 Production of a substance that is toxic or adversely alters the immune response of organisms other than people or birds 170. Exposure of organisms other than people or birds to a substance produced by the GMO may induce a toxic or adverse immune response. Routes of exposure could include consumption of inoculated chickens or eggs or ingestion or contact with chicken food, water, faeces or other materials such as bedding or feathers containing the GMO. Exposure may also occur as a result of contact with other organisms carrying the GMO or contact with items such as food or water containers that have been in contact with the GMO. 2.4.1 Microorganisms or invertebrates come into contact with the GMO or the expressed Ch IFN-γ protein 171. Although microorganisms and invertebrates may be exposed to the GMO or substances produced by the GMO, avian adenoviruses do not infect microorganisms or invertebrates (Büchen-Osmond 2003). Therefore, the GMO is not expected to infect micro-organisms or invertebrates. Although some microorganisms may be exposed to substances produced by the GMO when degrading chickens cells that may be present in faeces, skin or feathers, the only substance expected to be produced as a result of the genetic modification is Ch IFN-γ, to which microorganisms and invertebrates would already be exposed normally since it is expressed naturally in chicken cells. Ch IFN-γ is not expected to be toxic to microorganisms or be toxic or induce any adverse immune reaction in invertebrates. Therefore, no risk is identified and the potential for toxic effects in microorganisms and toxic or adverse immune effects in invertebrates as a result of exposure to the GMO or the Ch IFN-γ will not be further assessed. 2.4.2 Non-avian, non-human vertebrates ingest chicken products infected with the GMO or containing Ch IFN- γ expressed by the introduced gene 172. The applicant has indicated that none of the chickens or products from chickens used in this trial will be used as animal food. All chickens used in the trial will be destroyed by incineration. Hens will not be of laying age hence no eggs will be produced. Therefore, no risk is identified and the potential for toxic or adverse immune reactions in animals other than people or birds resulting from ingestion of chicken products will not be assessed further. 2.4.3 Non-avian, non-human vertebrates ingest or come into contact with the GMO in chicken food or water, chicken waste products or other waste materials, on items in contact with the GMO or carried by other organisms. 173. Small vertebrates such as rodents or lizards may enter the facility within which the trials are proposed to occur and may be exposed to the GMO or materials containing the GMO. The only substance expected to be produced as a result of the genetic modification is Ch IFN-γ and it is only produced when the GMO infects cells. Therefore, non-avian, non-human vertebrates could only be exposed to substances produced by the GMO if they ingest or otherwise come into contact with materials that contain chicken cells infected with the GMO or they become infected with the GMO. Since the GMO only infects birds and any Ch IFN-γ/chicken cells would be rapidly degraded outside of chickens, exposure of non-avian, non-human vertebrates to Ch IFN- γ would be extremely low. Chapter 2 Risk assessment (January 2006) 36 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 174. The applicant submitted data from an experiment in which mice and rabbits were given an oral dose of GM FAV8. There was no evidence of infection and no adverse effects were observed in any animals in this experiment. On the basis of the results of this experiment, the GMO and Ch IFN-γ are not expected to be toxic or to induce any adverse immune reaction in Non-avian, non-human vertebrates. Therefore, no risk is identified and the potential for toxic or adverse immune reactions in animals other than people or birds as a result of exposure to the GMO or Ch IFN-γ produced by the GMO will not be assessed further. 2.4.4 Non-avian, non-human vertebrates inhale particles containing the GMO 175. As discussed in Section 2.1.3 articles of dust including dander and water droplets in the spray generated during cleaning of the facility may contain low numbers of the GM FAV8. Animals such as livestock or rodents in the area may be exposed to these particles during the trial. Animals that are not in the immediate vicinity of the trial site are less likely to be exposed to particles containing the GMO since the concentration of airborne particles is expected to rapidly decrease as distance from the trial site increases. The number of viable present in dust is expected to be very low due to their inability to survive desiccation. 176. As outlined in Section 2.4.3, experiments performed by the applicant showed no evidence of infection or any adverse effects in mice and rabbits given an oral dose of the GM FAV8. On the basis of the results from this oral dosing experiment, it is reasonable to expect that inhalation of the GMO is also unlikely to induce any toxic or adverse immune response in non-avian vertebrates. Therefore, no risk is identified and the potential for toxic or adverse immune reactions in animals other than people or birds as a result of exposure to substances produced by the GMO will not be assessed further. 2.5 Altered viral characteristics The host range, tissue tropism, virulence/pathogenicity and the pattern of viral infection (e.g. acute, latent or chronic infection) depend on the nature of the interaction between the virus and the cells it infects. The mode of transmission of the virus is also determined by its tissue tropism ie. the types of tissues/cells infected by the virus and the shedding of the virus from those tissues/cells. Viral capsid proteins interact with receptors on the surface of host cells to attach to and penetrate into the cell. In the case of the adenoviruses, host range and tissue tropism determinants in the viral capsid include the penton fiber protein and penton base proteins, which interact the CAR receptor and integrins or other receptors on the surface of cells (Russell 2000b; Shenk 2001). The virus can only infect those cells that contain the appropriate receptors and permit replication and exit of infectious virus particles. In FAV8 a single gene that codes for the capsid fiber protein has been shown to be a major determinant of virulence (Pallister et al. 1996a) (see also Chapter 1). 2.5.1 Alteration in viral capsid by incorporation of Ch IFN-γ 177. Ch IFN-γ is unlikely to be incorporated into the viral capsid when capsid assembly takes place in chicken cells. Alterations in structural polypeptides involved in capsid assembly and viral packaging can interfere with the formation and release of mature virus particles (Shenk 2001). Even if Ch IFN-γ were to be incorporated into the GM viral capsid assembly, it is likely that it would destabilise the capsid assembly and prevent the packaging of mature viral particles. Chapter 2 Risk assessment (January 2006) 37 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 178. Ch IFN-γ is not a determinant of host range, tissue tropism or mode of transmission and is unlikely to alter these properties of the GM FAV8. 179. IFN- γ has many biological effects, including antiviral activity as well as stimulation and regulation of the immune response. Ch IFN-γ is not a viral determinant of pathogenicity/virulence. 180. Adenoviruses are not transmitted by vectors such as insects (Büchen-Osmond 2003) and the genetic modification is not expected to confer upon the GM FAV8 the ability to be transmitted by insect vectors. 181. The fiber and penton proteins are major determinants of the mode of transmission of FAV8 since they determine which cells may be infected by the virus and hence the cells from which virus may be shed and therefore the modes by which the virus may be transmitted. The inserted genetic material is distant from the regions of the viral genome that code for these determinants of the mode of transmission and is not expected to alter tissue tropism or mode of transmission of the GM FAV8. Therefore, no risk is identified and the potential for alteration of the characteristics of the GM FAV8 as a result of incorporation of Ch IFN-γ into the viral capsid will not be assessed further. 2.5.2 Alteration of viral capsid as a result of the 54 bp deletion 182. Restriction endonuclease digestion of the site at which the construct has been inserted has resulted in the deletion of a 54 bp sequence. The deletion is not within the published ORFs of the FAV8 genome (Ojkic & Nagy 2000c). The 54 bp deletion is not in a region of the viral genome that encodes caspsid proteins and therefore will not alter the capsid structure. Therefore, no risk is identified and the potential for alteration of the viral characteristics of the GM FAV8 as a result of the 54bp deletion altering the viral capsid will not be assessed further. 2.5.3 Unintended change in amino acid sequence of determinants of viral characteristics 183. The 54bp deletion and the inserted genetic material are not in an ORF or the region of the viral genome that codes for determinants of the viral characteristics under consideration in this section. Therefore, the genetic modification is unlikely to modify the amino acid sequence of these determinants. Therefore, a risk is not identified and the potential for alteration of the characteristics of the GM FAV8 as a result of an unintended change in the amino acid sequence of protein determinants of these properties will not be assessed further. 2.5.4 Recombination with another adenovirus leading to acquisition of new viral characteristics and infection of humans or other non-avian vertebrates 184. Genetic recombination events with other adenoviruses that may be co-infecting chicken cells may occur and may have the potential to alter the characteristics of the GM FAV8 (see also Chapter 1). In order for the GM FAV8 to infect human cells or cells of other non-avian vertebrates it would have to acquire the host range determinants of adenoviruses that normally infect those species. In order for recombination to occur both the DNA from the adenovirus that normally infects people or non-avian vertebrates and the DNA from the GM FAV8 must both be present in the nucleus of the infected chicken cells at the same time. Adenoviruses generally have a restricted host range and human adenoviruses and adenoviruses that infect other non-avian vertebrates are not known to infect birds (Büchen-Osmond 2003). Therefore, the GM FAV8 is not expected to undergo recombination with Chapter 2 Risk assessment (January 2006) 38 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator human adenoviruses or adenoviruses of other non-avian vertebrates. Therefore, a risk is not identified and the potential for recombination with other adenoviruses leading to the acquisition of new viral characteristics and infection of humans or other non-avian vertebrates will not be assessed further. 2.5.5 Recombination with another adenovirus leading to acquisition of new viral characteristics and an alteration in the pattern of infection in chickens 185. Other avian adenoviruses could infect chickens along with the GM FAV8. If another avian adenovirus was present within the nucleus of chicken cells at the same time as the GM FAV8, recombination could occur and may result in the acquisition of new characteristics by the GM FAV8. New characteristics acquired by the GM FAV8 have the potential to lead to changes in tissue tropism, shedding and mode of transmission or the establishment of latent or chronic infections in chickens. 186. The majority of viral recombination is sequence-dependent and occurs by a mechanism known as homologous recombination. However, non-homologous recombination, in which the sequences that are transferred are unrelated to those at the site of the recombination event can also occur at an extremely low frequency. As outlined in Chapter 1, homologous recombination occurs normally in non-GM fowl adenoviruses. The genetic modifications to the GM FAV8 are not expected to increase the frequency of recombination since other fowl adenoviruses do not contain sequences homologous with Ch IFN-γ. Homologous recombination results in hybrids with essentially the same properties as the parental virus (Candresse 1997) and virtually all non-homologous recombination events are likely to lead to non-viable viruses or viruses that have not gained any selective advantage. Therefore, a risk is not identified and the potential for recombination with other adenoviruses leading to the acquisition of new viral characteristics and an alteration of the pattern of infection in chickens will not be assessed further. 2.5.6 Recombination with another adenovirus leading to the acquisition of a new viral characteristics and infection of other birds 187. FAV8 may infect birds other than chickens (McFerran et al. 1976b) and other avian adenoviruses could infect chickens along with the GM FAV8. If DNA from another avian adenovirus was present within the nucleus of chicken cells at the same time as DNA from the GM FAV8, recombination could occur and may result in the acquisition of new characteristics by the GM FAV8. New characteristics acquired by the GM FAV8 and infection of other birds may lead to disease in other birds. 188. However, recombination occurs normally in non-GM fowl adenoviruses and the genetic modifications to the GM FAV8 are not expected to increase the frequency of recombination with other fowl adenoviruses or provide any selective advantage to other adenoviruses. Therefore, a risk is not identified and the potential for recombination with other adenoviruses leading to the acquisition of new viral characteristics and an infection of other birds will not be assessed further. 2.5.7 Recombination with viruses other than adenoviruses leading to the acquisition of new viral characteristics 189. Genetic recombination events with viruses other than adenoviruses that may be co-infecting chicken cells has the potential to alter the characteristics of the GM FAV8. Most reports of recombination involve exchange of genetic information between related viruses. Recombination between unrelated viruses occurs rarely and Chapter 2 Risk assessment (January 2006) 39 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator is evident only on evolutionary timescales.Therefore, it is considered unlikey to take place during the short period for which the proposed trial is to be conducted. 190. In addition, the applicant has stated that chickens to be used in the proposed trial will be vaccinated against the viruses that cause Mareks disease (a herpesvirus), Newcastle disease (a paramyxovirus) and Infectious bursal disease (a birnavirus). Therefore, chickens will not be infected with these viruses and recombination with these viruses will not occur. Therefore, a risk is not identified and the potential for recombination with viruses other that adenoviruses leading to the acquisition of new viral characteristics will not be assessed further. 2.5.8 Acquisition of new viral characteristics by transcapsidation 191. If more than one virus co-infects the same cell there is the possibility of the genome of one virus being enclosed by the capsid proteins of another virus. This phenomenon is known as transcapsidation. Transcapsidation has the potential to alter the characteristics of the GM FAV8. However, transcapsidation is transient and self limiting as it is not maintained in subsequent rounds of viral infection. Therefore, long-term adverse impacts are not expected. Therefore, a risk is not identified and the potential for transcapsidation leading to the acquisition of new viral characteristics will not be assessed further. 2.5.9 Expression of the Ch IFN-γ gene suppresses the immune system 192. Suppression of the immune system may result in altered viral characteristics such as increased virulence. IFN-γ is a potent immunomodulatory compound that has many biological effects, including antiviral activity. Ch IFN-γ enhances antibody responses when co- administered with antigen (Lowenthal et al. 1998) and inhibits the development of adverse effects associated with the infection of chickens with the parasite Eimeria (Lillehoj & Choi 1998). Given the role that IFN-γ plays in immune responses, the expression of ChIFN-γ is unlikely to suppress the immune response. Therefore, a risk is not identified and the potential for expression of Ch IFN-γ to suppress the immune system and alter viral characteristics will not be assessed further. 2.5.10 Expression of the Ch IFN-γ gene leads to the development of interferon-resistant virus variants 193. Interferon-α resistant variants of several viruses such as measles virus and hepatitis B virus (HCV) are known to occur. In the case of measles virus, these are naturally occurring subpopulations (Carrigan & Knox 1990). Resistance to interferonα can also occur during chronic HBV infection, though this occurred following long term (16 week) administration of interferon-α (Terui et al. 2000). There are no reports of any viruses being resistant to interferon-γ. This may be due to the mode of action of IFN-γ, which stimulates both arms of the immune system leading to efficient viral clearance. 194. Both HBV and measles virus are RNA viruses that undergo high levels of spontaneous mutation during replication of the viral RNA genome. Mutation rates in DNA viruses, such as adenoviruses are much lower as they do not involve replication of viral RNA. 195. While the GM FAV8 will express low levels of Ch IFN-γ, which cannot be detected above background levels, the virus will be cleared by the immune system within 7-14 days. It is therefore considered to be highly unlikely that this IFN-γ Chapter 2 Risk assessment (January 2006) 40 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator expression will give rise to interferon-resistant sub-populations. Therefore, a risk is not identified and the potential for expression of Ch IFN-γ to lead to the development of interferon-γ resistant mutants will not be assessed further. 2.6 Altered persistence within the host Persistent infections are those that are not cleared from the host following the primary infection but remain associated with specific cell types within the host. A latent infection can be characterised by a lack of demonstrable infectious virus between episodes of recurrent disease. By contrast, chronic infection is characterised by a continued presence of infectious virus following the primary infection and may include chronic or recurrent disease. During persistent infections the viral genome may be either stably integrated into the DNA of the host cell or maintained outside of the host genome (episomally). Common viral mechanisms that can contribute to persistent infections include immune modulation/evasion and modulation/down regulation of viral gene expression. 2.6.1 Expression of Ch IFN- γ induces latency/chronic infection 196. Human adenoviruses are known to be capable of persistent infection, with proteins encoded within the E1 and E3 regions of the viral genome and the VA RNA (small viral RNAs) genes thought to contribute to persistence (Russell 2000a; Shenk 2001). Based on sequence data for the very closely related FAV9 which does not contain the E1 and E3 regions, FAV8 is not expected to contain sequences that show significant homology to these regions of human adenoviruses (Cao et al. 1998a; Ojkic & Nagy 2000a). Therefore, FAV8 is unlikely to establish persistent infections. There is no evidence that GM FAV8 is able to establish latent or chronic infections. 197. IFN-γ is not a viral determinant involved in evasion of the host immune system. Expression of IFN-γ has been shown to boost the immune response and expedite the viral clearance in vivo for a variety of GM viruses (Kohonen-Corish et al. 1990; Giavedoni et al. 1997; Binder & and Griffin 2001). For these reasons, the expression of Ch IFN-γ is considered unlikely to contribute to viral persistence. Therefore, a risk is not identified and the potential for expression of Ch IFN-γ to alter persistence of GM FAV8 within the host will not be further assessed. 2.6.2 Deleting 54 base pairs induces latency/chronic infection 198. Restriction endonuclease digestion of the site at which the construct has been inserted has resulted in the deletion of a 54 bp sequence. The deletion is not within the published open reading frames (ORF) of the FAV8 genome (Ojkic & Nagy 2000b). The 54 base pairs deleted from the FAV8 genome are not located in a region that encodes proteins known to be associated with persistent infection. Therefore, a risk is not identified and the potential for deleting 54 base pairs to alter persistence of GM FAV8 within the host will not be further assessed. 2.7 2.7.1 Increased transmission of GM FAV8 Expression of Ch IFN-γ increases transmission 199. Avian adenoviruses can be transmitted both horizontally (from infected bird to uninfected bird) and, in some cases, vertically (from the parent into the egg). The main route of horizontal transmission is via the faecal-oral route as a result of direct contact with infected faeces. Over a short distance, transmission via inhalation of airborne particles containing the virus is also possible (McFerran & Smyth 2000c; Toro et al. 2001b). Chapter 2 Risk assessment (January 2006) 41 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 200. Successful horizontal transmission from an infected bird to an uninfected bird requires the entry of an infectious dose of the GM FAV8 into the host animal, attachment and penetration of the virus into the chicken cells, replication of the virus within those cells, exit from the cells, excretion from the bird and transport of an infectious dose to another chicken wherein the cycle is repeated. Viral capsid proteins such as the viral fiber and penton proteins and their interaction with host cell receptors are likely to be major determinants of horizontal transmission of the GM FAV8. 201. Ch IFN- γ is not known to play any role in the transmission of the virus. Given the role of IFN-γ in antiviral defenses of the host, it is more likely to reduce viral transmission. 202. Some fowl adenoviruses may be transmitted vertically (Toro et al. 2001a), but there is experimental evidence that indicates that the vertical transmission of FAV-8 is an infrequent event (Reece et al. 1985a). Vertical transmission of GM FAV8 in other birds that may become infected is also expected to be an infrequent occurrence. Since the hens used for the proposed trial will not be of laying age, vertical transmission of the GM FAV8 will not occur in chickens used in this trial. Therefore, a risk is not identified and the potential for altered transmission of GM FAV8 as a result of the expression of Ch IFN-γ will not be assessed further. 2.7.2 Deleting 54 base pairs increases transmission of the virus 203. Restriction endonuclease digestion of the site at which the construct has been inserted has resulted in the deletion of a 54 bp sequence. The deletion is not within the published open reading frames (ORF) of the FAV8 genome (Ojkic & Nagy 2000e). The 54 base pairs of DNA deleted from the FAV8 genome are not located in an area known to be associated with determinants of viral transmission of the virus. Therefore, a risk is not identified and the potential for deleting 54 base pairs to alter transmission of GM FAV8 will not be further assessed. 2.7.3 Increased number of viral particles produced 204. If the number of GM FAV8 particles shed from inoculated chickens were to increase as a result of the genetic modification, the transmission of the virus may be increased. However, the results of an in vitro study with a very similar GM FAV8 containing Ch IFN-γ showed no increase in number of viral particles produced (Johnson et al. 2000a). Therefore, increased production of GM FAV8 is considered to be unlikely. In addition, mouse IFN-γ expressed by a syncytial virus vector was shown to attenuate viral replication in vivo in mice (Bukreyev et al. 1999a). On the basis of these results, the genetic modifications are not expected increase the number of GM adenoviral particles shed from inoculated chickens. Therefore, a risk is not identified and the potential for an increased production of viral particles to alter transmission of GM FAV8 will not be further assessed. 2.8 Spread and persistence of the GM FAV8 in the environment 205. If the GM FAV8 infects birds other than chickens, the expressed Ch IFN-γ may have the same or a similar effect as the intended effect in chickens. In this case immune system stimulation and increased weight gain may occur in any other birds that are successfully infected with the GM FAV8. These effects could confer enhanced environmental competitiveness that increased the pest potential of infected birds. Chapter 2 Risk assessment (January 2006) 42 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 2.8.1 Spread by other birds that come into contact with inoculated chickens, faeces or items that have been in contact with the GMO 206. Since birds will be excluded from the facility in which the proposed trial is to be conducted, direct contact with inoculated chickens, faeces and items such as food and water containers or bedding is not likely to occur. Therefore, a risk is not identified and the potential for spread of the GM FAV8 in the environment by birds that come into contact with inoculated chickens, faeces or items in contact with the GMO will not be assessed further. 2.8.2 Spread to other birds as a result of contact with insects or non-avian vertebrates transporting the GMO out of the facility 207. As discussed in Section 2.3.3, insects and small vertebrates such as rodents or lizards would not be excluded from the facility in which the proposed trial is to be conducted. Therefore, they may come into contact with inoculated chickens, infected faeces or other items containing or carrying the GMO e.g. food and water containers or bedding materials that have been in contact with inoculated chickens and/or the GMO. As a result of contact occurring within the facility small non-avian vertebrates may physically carry the GMO from the facility and spread it in the environment. Similarly, although insects are not known to be vectors for adenoviruses, insects that have been in contact with the GMO may physically transmit it outside of the location into the surrounding environment. Therefore, a risk is identified and the potential for spread of the GM FAV8 to other birds as a result of insects or non-avian vertebrates transporting the GMO out of the facility will be analysed further in Chapter 3 event 1. 2.8.3 Spread by animals that come into contact with waste material containing the GMO that is washed out of the trial location 208. The potential for animals outside the facility to come into contact with waste material containing the GMO that is washed out of the trial location has already been discussed in Section 2.3.5. No risk is identified and the potential for spread of the GM FAV8 in the environment as a result of animals coming into contact with contaminated wash water will not be assessed further. 2.8.4 Spread to other birds as a result of inhaling windborne particles containing the GM FAV8 209. As discussed in Section 2.1.3, dust particles, dander (skin cells, fluff etc), small feathers, or water droplets may carry the GMO and may be dispersed by wind beyond the trial location. Animals, including other birds may come into contact with these particles carrying the GMO, contributing to the spread of the GMO in the environment. Therefore, a risk is identified and the potential for spread of the GM FAV8 to other birds as a result of wind carrying particles containing the GMO will be analysed further in Chapter 3 event 2. 2.8.5 Expression of Ch IFN-γ increases the stability of the GM FAV8 contributing to persistence in the environment. 210. An increase in the stability of the virus in the environment may have the potential to increase the persistence of the virus. Since adenoviruses are not enveloped viruses, survival or stability outside of the host is largely determined by environmental factors with desiccation of the virus being of prime importance (Abad et al. 1994c). As discussed in Section 2.1.3, data supplied by the applicant demonstrates that the GM FAV8 is susceptible to desiccation. In adenoviruses capsid Chapter 2 Risk assessment (January 2006) 43 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator proteins are likely to contribute to resistance to desiccation. Since the inserted genetic material is not in a region of the viral genome that codes for capsid proteins and is not incorporated into or expected to alter the viral capsid, viral stability is not expected to be changed. Therefore, a risk is not identified and the potential for an increased stability of GM FAV8 contributing to persistence of GM FAV8 in the environment will not be further assessed. 2.8.6 Spillage of material containing the GMO during transport 211. At the end of the trial the applicant has proposed that all solid waste, including chickens carcasses will be collected into bags that will be sealed prior to being placed into large medical waste bins which will be sealed and disinfected on the exterior surfaces prior to exit from the facility. These waste bins will be collected and transported for incineration. It is possible that waste material may be accidentally spilled during transport. However, having sealed primary and secondary containers for transport greatly reduces the potential for exposure to the GMO. All transport of the GMO and any waste material will be conducted in accordance with OGTR guidelines. 212. Chickens and eggs inoculated with the GMO in the certified PC2 facility will be transported to the animal containment facility within a primary container e.g. crate or carton which will be placed inside a plastic bag. The distance between the site of inoculation and the trial site is very short (less than 20m). As a result, unintended environmental exposure to the GMO is unlikely. Therefore, a risk is not identified and the potential for spillage during transport of material containing the GM FAV8 contributing to spread and persistence of GM FAV8 in the environment will not be further assessed. 2.9 Gene transfer between the GM FAV8 and other organisms 213. Upon infection of chicken cells with the GM adenovirus genetic material may be transferred between the GMO and the host cell genome. One possible adverse outcome that may result if gene transfer occurred would be the duplication of the Ch IFN-γ gene in the host cell genome. This may result in at least twice the amount of IFN-γ protein produced in that cell because the introduced copy of the Ch IFN-γ gene may be expressed continuously. This in turn may lead to prolonged immune stimulation which could be detrimental to the host. 214. The transfer of genetic material between the GM FAV8 and the host cell genome could occur by at least three mechanisms: integration, homologous recombination or non-homologous (illegitimate) recombination. 215. Transfer of material between the GM FAV8 and the host cell genome by integration is unlikely to occur as integration is not part of the adenovirus life cycle (discussed in Chapter 1). Therefore, gene transfer via this mechanism is considered unlikely. 216. While non-homologous recombination can also occur, it is a very rare event and is considered to be less likely to occur than homologous recombination and will not be further assessed. 217. The most likely mechanism by which gene transfer between the GM FAV8 and the host cell genome may occur is homologous recombination, in which regions of DNA that have a high degree of sequence identity are able to pair and exchange. Chapter 2 Risk assessment (January 2006) 44 DIR 046/2003—Risk Assessment and Risk Management Plan 2.9.1 Office of the Gene Technology Regulator Recombination between the GM FAV8 and the chicken genome 218. The Ch IFN-γ gene present in the chicken cells contains stretches of non- amino acid coding DNA, known as introns, between the coding sequence of the Ch-IFN gene. By contrast, only DNA that encodes the Ch IFN-γ protein has been inserted into the GM FAV8. This difference in DNA sequence between the two copies of the Ch IFN-γ gene means that while there will still be homologous sequences between the two copies of the Ch IFN-γ gene, the lengths of this homology, which has an effect on the efficiency of homologous recombination, will be variable. However, there may still be sufficient lengths of sequence homology to initiate homologous recombination events. 219. The most likely outcome of any gene transfer occurring as a result of homologous recombination would be the exchange of DNA between the viral and genomic copies of the Ch IFN-γ gene. This may either have no effect on the expression of the Ch IFN-γ genes or by contrast it may disrupt expression of both genes which would lead to no IFN-γ expression in that cell or infecting virus. Because this recombination will only occur at extremely low frequencies in small number of cells, it is unlikely to result in any adverse effects in the chicken or any selective advantage for the GM FAV8. Therefore, a risk is not identified and the potential for recombination resulting in gene transfer between the GM FAV8 and the chicken genome will not be assessed further. 2.9.2 Recombination between the GM FAV8 and the genome of other birds 220. If other birds are infected with the GM FAV8 there may be an exchange of genetic information by recombination. If there is sufficient homology between the inserted Ch IFN-γ gene and the IFN-γ gene present in the infected bird then homologous recombination may occur. This could result in the transfer of the Ch IFN-γ gene from the virus to the infected bird. However, the frequency of recombination events is expected to be extremely low (see Chapter 1) and any gene transfer from the bird genome to the virus is unlikely to result in any selective advantage for the GM FAV8. Therefore, a risk is not identified and the potential for recombination between the GM FAV8 and the genome of other birds will not be assessed further. 2.9.3 Gene transfer between the GM FAV8 and the genome of non-avian, non-viral organisms 221. Fowl adenoviruses are only known to infect birds. Non-vertebrate organisms are not infected with fowl adenoviruses (Büchen-Osmond 2003). In addition, data from experiments conducted by the applicant shows that the GM FAV8 does not infect mice or rabbits and it does not infect human, pig, monkey, cattle, sheep, hamster or duck cells in culture. Since the GM FAV8 is not expected to infect non-avian organisms, gene transfer between the GM FAV8 and non-avian organisms is not expected to occur. Therefore, a risk is not identified and the potential for gene transfer between the GM FAV8 and non-avian organisms will not be assessed further. 2.10 Increased rate of mutation of GM FAV8 Genetic modification destabilises GM FAV8 leading to increased rate of mutation 222. Data provided by the applicant demonstrates that the genetic modification remains stable after repeated passage in cell culture and also after passage through Chapter 2 Risk assessment (January 2006) 45 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator chickens. This implies that the GM FAV8 remains stable and an increased rate of mutation or rearrangement is unlikely. Therefore, a risk is not identified and the potential for the genetic modification to result in an increased rate of mutation in GM FAV8 and the will not be assessed further. 2.11 Unauthorised activities Use of GMOs outside the proposed licence conditions (non-compliance) 223. If a licence were to be issued, non-compliance with the proposed conditions of the licence could lead to spread and persistence of the GM FAV8 outside of the proposed release area. The adverse outcomes that this event could cause are discussed in Section 2.4. The Act provides for substantial penalties for non-compliance and unauthorised dealings with GMOs. The Act also requires that the Regulator has regard for 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. Therefore, no risk is identified and the potential for an adverse outcome as a result of unauthorised activities will not be assessed further. SECTION 3 RISK ESTIMATE PROCESS FOR IDENTIFIED RISKS 224. Two events from the hazard identification process (refer to Events 1 and 2 in Sections 2.8.2 and 2.8.4) are considered to lead to an identified risk for the adverse outcome of increased pest potential in other birds. 225. Chapter 3 provides a detailed analysis of the consequences and likelihood of these two events in order to obtain an estimate of the level of risk. The risks are assessed against the baselines established by reference to characteristics of the parent organism and aspects of the receiving environment (including the facility management and animal husbandry practices and other management conditions proposed by the applicant as discussed in Chapter 1). 226. Information contained in the application (including information required by the Act and the Regulations on the GMOs, the parent organism, the proposed dealings and potential impacts on the health and safety of people and the environment), current scientific knowledge, and submissions received during consultation with expert groups and authorities were also considered. 227. The consequence assessment considers the seriousness of the harm that could potentially result from each event, while the likelihood assessment considers the chance of the event resulting in harm. Consequence and likelihood assessments are then combined to give an overall risk estimate using the Risk Estimate Matrix (Table 2.2). During the consequence and likelihood assessments, consideration is also given to areas of uncertainty that arise from a lack of knowledge. Chapter 2 Risk assessment (January 2006) 46 DIR 046/2003—Risk Assessment and Risk Management Plan Table 2.2 Office of the Gene Technology Regulator The OGTR Risk Estimate Matrix (OGTR 2005) LIKELIHOOD RISK ESTIMATE Highly Likely Low Moderate High High Likely Negligible Low High High Unlikely Negligible Low Moderate High Highly Unlikely Negligible Negligible Low Moderate Marginal Minor Intermediate Major CONSEQUENCES Risk Estimate Matrix: A negligible risk is considered to be insubstantial with no present need to invoke actions for mitigation. A low risk is considered to be minimal but may invoke actions for mitigation beyond normal practices. A moderate risk is considered to be of marked concern that will necessitate actions for mitigation that need to be demonstrated as effective. A high risk is considered to be unacceptable unless actions for mitigation are highly feasible and effective. 228. Definitions of risk analysis terms used by the Regulator can be found in Appendix A. 229. After an estimate is obtained for each identified risk, the risks are evaluated to determine if risk treatment measures are required to mitigate potential harm (see Chapter 4). Chapter 2 Risk assessment (January 2006) 47 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator CHAPTER 3 RISK ESTIMATES FOR INCREASED PEST POTENTIAL IN OTHER BIRDS 230. This chapter estimates the risks associated with two events that could lead to the adverse outcome of increased pest potential in other birds arising from this proposed release. The risk estimates are based on consequence and likelihood assessments for each event. SECTION 1 BACKGROUND 231. Vertebrate pests can be described as animals which people regard as noxious, troublesome or unwanted. 232. They can be directly or indirectly detrimental to human interests. They can also cause environmental damage by reducing biodiversity as a result of reducing native species richness and abundance or by causing undesirable changes in species composition. Vertebrate pests can also cause degradation of abiotic environments or otherwise interfere with land or water use patterns. In addition, vertebrate pests can be agents for diseases that can affect people or other animals. 233. It is possible for an animal to be a pest in one setting but beneficial or domesticated in another (for example, European rabbits introduced to Australia caused ecological damage beyond the scale they inflicted in their natural habitat). For this reason vertebrate pest potential has often been studied in the context of the invasion and establishment of exotic animals. 234. Predicting which animals will become pests is difficult since pest potential is the result of the complex interactions between an animal and its environment. However, there a number of characteristics that are correlated with the establishment, persistence and spread of pest populations (Ehrlich 1989; Sax & Brown 2000; Bomford 2003): Broad ecological requirements or tolerances High population density in original geographic location Broad diet and ability to incorporate new foods into diet High fecundity Association with disturbed habitats or humans Short generation time Extensive genetic/phenotypic variability and behavioural flexibility Large body size relative similar or related species Taxonomic group 235. The intended effect of the inoculation of chickens with the GM FAV8 containing the Ch IFN-γ gene is to increase the rate of weight gain. Although the mechanism by which this growth promotion occurs is not known it has been postulated to be the result of the stimulation of the chicken immune system by Ch IFN-γ and/or a reduction in the existing load of microbial organisms in the chicken allowing more efficient conversion of food energy (Johnson et al. 2000d). Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 48 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 236. Any susceptible birds in the vicinity of the trial location could become infected with the GM FAV8. This could include introduced exotic birds, existing pest birds and native birds. If pest birds were provided with a competitive advantage as a result of enhanced immunocompetence following infection with the GM FAV8 they may become worse pests. 237. In Australia, there are a range of introduced birds that are considered to be of greater pest risk than other introduced birds. These include the European starling (Sturnus vulgaris), the Indian myna (Acridotheres tristus), Blackbird (Turdus merula), European goldfinch (Cardeulis cardeulis), the house sparrow (Passer domesticus), the laughing turtle dove (Streptopelia senegalensis), the spotted turtle dove (Streptopelia chinensis), the rock dove (Columba livia) and the mallard (Anas platyrhynchos) (Bomford 2003). The Indian myna, the house sparrow and the European starling are amongst the worst pest birds in the world (IUCN/SSC Invasive Species Specialist Group (ISSG) 2005). SECTION 2 CONSEQUENCE AND LIKELIHOOD ASSESSMENTS 238. Consideration is given to events 1 and 2 identified in Chapter 2 (Hazard identification) that may give rise to increased pest potential in other bird populations. For each event the level of risk is estimated from assessments of the seriousness of harm (consequence-ranging from marginal to major) and the chance of harm (likelihood-ranging from highly unlikely to highly likely). 239. The Regulator can only consider risks to human health and safety and the environment posed by, or resulting from, gene technology. For this reason, the level of risk from the proposed dealings with the GMOs is considered relative to the baselines of the relevant characteristics of the non-GM FAV8 and its known effects on birds. 2.1 Characteristics of the parent organism 240. Information on non-GM FAV8 is included here to establish a baseline for comparison with the GM FAV8 being considered in this risk assessment (see also Chapter 1). 241. FAV8 has a restricted host range. There are no reports in the published literature on the isolation of the CFA44 strain of FAV8 from birds other than chickens. Other strains of FAV8 have been isolated from chickens, pigeons and budgerigars. In pigeons FAV8 was associated with IBH, respiratory symptoms, diarrhoea and death. In budgerigars, FAV8 was isolated after a bout of enteritis occurring in an aviary (McFerran et al. 1976a). There is also a report of adenoviruses with close antigenic relationship to FAV8 (TR59) being isolated from ostriches with diarrhoea or producing abnormal eggs (Gough & Drury 1997). For the purposes of this risk assessment it is assumed that both the GM and non GM FAV8 can infect the same wide range of birds. 242. The CFA 44 strain of FAV8 is not pathogenic for chickens. Data supplied by the applicant shows that oral doses of up to 104.7 TCID50 reliably produce infection in all inoculated chickens, but no signs of disease have been observed. FAV8 replicates in the upper respiratory tract and gastro-intestinal tract of susceptible animals and is shed from the gastrointestinal mucosa into the faeces, from oral and nasal mucosa and from conjunctival secretions. Horizontal transmission of FAV8 (including CFA44) occurs mainly the fecal-oral route and spread of the virus mainly occurs by direct Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 49 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator contact between birds or by indirect contact facilitated by people, or other fomites. As discussed in Chapter 2, because the GM FAV8 is susceptible to desiccation, , airborne spread of the virus in contaminated aerosols is expected to occur only over a short distance. 2.2 Event 1: Spread to other birds as a result of insects or non-avian vertebrates transporting the GM FAV8 outside the facility 243. Other birds could be exposed to insects or non-avian vertebrates physically transporting the GM FAV8 as a result of coming into contact with inoculated chickens, faeces or items that have been in contact with the GMO. The risk of increased pest potential in exposed birds would depend on a number of factors including the species of birds that are found in the vicinity, whether the birds become infected with the GM FAV8, the level of expression and the effects (if any) of Ch IFN-γ in those birds, the extent of spread and persistence of the GM FAV8 in bird populations, the importance of increased weight gain/immune system stimulation for pest potential of the infected birds (consequence assessment) and the level and extent of exposure to the GM FAV8 during this release (likelihood assessment). The risk is assessed in the context of the limited scale of the proposed release and against the baseline of the effects of the non-GM FAV8 in other birds. 2.2.1 Consequence assessment 244. A wide range of other bird species is likely to be in the area in which the proposed trial is to be conducted. One of the possible means by which exposure of other birds could occur is by consumption of organisms carrying the GMO. Birds that are omnivorous, insectivorous or birds of prey may be exposed to the GM FAV8 if insects or small non-avian vertebrates such as rodents carrying the GMO are consumed. 245. Birds could also acquire a competitive advantage as a result of infection with the GMO and expression of the Ch IFN-γ. The possible secondary ecological effects of this are less easily predicted, but disruptions to local bird communities could occur. 246. The magnitude of the growth promoting/immune system stimulating effect of Ch IFN-γ would depend, in part, upon the dose of GM FAV8 received by birds. If the dose of GM FAV8 received as a result of ingesting insects or rodents is not sufficient to establish an infection, the GM FAV8 would be rapidly cleared from the bird and there would be no effect in those birds. At low doses, the growth promoting/immune system stimulating effect is expected to be diminished or may not occur at all. In chickens, relatively high doses (greater than 104 TCID50 or higher) of a similar GM FAV8 based on a highly virulent parent strain of FAV8 were required in order to demonstrate a growth promoting effect. After a high dose, chickens in this study had a body weight that was up to 17% higher than untreated birds (Johnson et al. 2000e). Lower doses had no growth promoting effect (M. Johnson pers. comm.). 247. As the GM FAV8 that is the subject of this application is based on an avirulent stain of FAV8, it is more likely to require high doses in order to realise a growth promoting/immune system stimulating effect in other birds. If they are exposed at all, other birds are expected to be exposed to only very low levels of the GM FAV8 as a result of ingesting insects or rodents. Therefore, the subsequent growth promoting/immune system stimulating effect in other birds is expected to be small. 248. The growth promoting/immune system stimulating effect of Ch IFN-γ can only occur if it is biologically active in the bird that is infected with the GM FAV8. The Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 50 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator magnitude of any effect would also depend upon the level of activity of the Ch IFN-γ in the infected bird. 249. IFN-γ is very species specific. It is more likely that a cytokine from a particular species will be active in the cells of another species only if the cytokines share amino acid sequence homology of 60% or greater (Scheerlinck 1999b). Even if the homologous cytokines share significant sequence identity, activity in a second related species could be lower than the native cytokine. For example, human IFN-γ is active in human cells, shows diminished activity in other primate cells and little or no activity in mouse or rat cells(Adolf 1985). Similarly, duck IFN-γ has 67 % sequence identity with Ch IFN-γ, but the activity of duck IFN-γ in chicken cells was approximately 16 times lower than the Ch IFN-γ (Huang et al. 2001a). 250. A phylogenetic analysis has shown that the similarity of avian IFN-γ amino acid sequences parallels the presumed evolutionary relationships between the related bird species tested (Kaiser et al. 1998). Ducks are taxonomically categorised in the order Anseriformes which is closely related to Galliformes, the chicken-like birds. As discussed above, even though they are relatively closely related, duck IFN-γ is less active in chicken cells than Ch IFN-γ. Therefore, the Ch IFN-γ is expected to have a higher level of activity in related Galliformes (chicken-like birds) such as turkeys, quails and pheasants and lower activity in other birds such as Passeriformes (perching birds), Falconiformes (birds of prey), Pscittaciformes (parrots) and Columbiformes (doves and pigeons), that are phylogenetically much less closely related to chickens than are ducks (van Tuinen et al. 2000) (Harrison et al. 2004). 251. For these reasons, the growth promoting/immune system stimulating effect of the Ch IFN-γ expression is expected to be largely restricted to birds that are closely related to chickens i.e. Galliformes. Because the area around the trial site is mainly used for cattle and pig farming and there are no commercial chicken producers, few if any other Galliform birds are expected to be in the vicinity of the trial location. The extent of any growth promoting/immune system stimulating effect is therefore expected to be very limited. A growth promoting /immune system stimulating effect may also be possible in other birds, but the magnitude of the effect is expected to be diminished, if it occurs at all. 252. Ch IFN-γ has a short half life (between 10 minutes and several hours depending upon route of administration) in the chicken (Lowenthal et al. 1999). Therefore, the duration of any growth promoting effect in an individual bird is expected to be limited to the duration of the infection with the GM FAV8 and expression of Ch IFN-γ. 253. Since, there is no evidence that non-GM FAV8 or GM FAV8 can establish latent infections or persist within the host cells, the growth promoting/immune system stimulating effect of Ch IFN-γ expression is expected to be short lived. Data supplied by the applicant demonstrates that the GM FAV8 is shed from chickens for 7-14 days after inoculation with no detectable GM FAV8 found in caecal tonsil or kidney tissue at day 14. This data indicates that the GM FAV8 is rapidly cleared from the chicken and the growth promoting effect of the GM FAV8 may only last for between 7 and 14 days. Clearance of the GM FAV8 from any other bird that may become infected is expected to be very similar to that observed in chickens. After the GM FAV8 has been cleared from the bird, no increased weight gain/immune system stimulation will occur in that bird as a result of Ch IFN-γ expression from the initial infection with the GM FAV8. Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 51 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 254. After an initial infection with a virus there is a period of immunity to reinfection with the same virus. The potential for recurrent infection to occur in any infected bird population will depend upon the persistence of the virus in that population. Viral factors that influence persistence in a population include transmissibility (the number of contacts between infected and non-infected individuals, the proportion of contacts susceptible to infection and the probability of transmission per susceptible contact), generation time and duration of infection (acute or chronic). Host determinants that influence persistence in a population include population size, turnover rate or rate at which susceptible individuals are introduced, population density and the proportion of the population susceptible to infection (Nathanson 2001). 255. If some other birds become infected with the GM FAV8 and other members of the same bird population also become infected, it is possible that reinfection with GM FAV8 may occur after immunity has diminished and the growth promoting/immune system effect may occur during subsequent rounds of re-infection. The period for which immunity to FAV8 is effective in preventing reinfection with the same serotype/strain is not known. The applicant proposes to re-administer the GM FAV8 to chickens 21 or 28 days after the initial inoculation. Whether existing immunity prevents or limits the extent of re-infection at this stage is not known. There are reports of immunity to fowl adenovirus lasting for up to 8 weeks (McFerran et al. 2003). Re-infection could be possible after this period. 256. The GM FAV8 is expected to induce the same level and duration of immunity as the non-GM FAV8. Therefore, if other members of the same bird population are infected with the GM FAV8, it is possible that reinfection with GM FAV8 may occur 8 weeks after initial infection. In this case, if a small growth promoting/immune system stimulating effect of Ch IFN–γ did occur in other birds, it would not be continuous but rather it could occur periodically depending upon the level of immunity in the population and potential for re-infection of individuals. 257. The potential for FAV8 to persist in populations of birds other than chickens is not known. The genetic modifications to FAV8 are not expected to alter its persistence in populations of birds. Since direct contact between individuals is important in the spread of FAV8, spread within a population is expected to be largely limited to populations of relatively high density, such as flocks of pest birds. Furthermore, since none of the pest birds in Australia are closely related to chickens, the growth promoting /immune system stimulating effect, if any, of Ch IFN-γ expression is expected to be minimal. Therefore, even if the GM FAV8 is spread and persists in populations of such birds the increased pest potential, if any, in those bird populations is expected to be minimal. 258. In addition, the Ch IFN-γ gene is not expected to provide any selective advantage for the GM FAV8 and it is not expected to persist in the FAV8 population. Given the role of IFN-γ in viral clearance from a host, the Ch IFN-γ is more likely to be selected against in virus populations. If the Ch IFN-γ gene does not persist in the FAV8 population then the biological effects of the expressed protein will cease in any bird populations. 259. If the growth promoting/immune system stimulating effect of Ch IFN-γ did occur, it is expected to be small as a result of the low levels of exposure to the GM FAV8. In addition, the biological effects of Ch IFN-γ are expected to be largely restricted to birds closely related to chickens and few, if any, of such birds are Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 52 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator expected to be present in the areas immediately surrounding the trial site.The extent of any possible increase in pest potential as a result of ingesting insects or non-avian vertebrates carrying the GMO is expected to be very limited. Furthermore, since the Ch IFN-γ gene is not expected to provide the GM FAV8 with any selective advantage, it is not expected to persist in the FAV8 population and any possible increase in pest potential in populations of other birds as a result of the biological effects of Ch IFN-γ expression will be of limited duration. 260. Therefore the consequences of spread to other birds as a result of insects or nonavian vertebrates transporting the GM FAV8 out of the facility are assessed as marginal. 2.2.2 Likelihood assessment 261. Insects and small vertebrates such as rodents will be able to enter the facility in which the proposed trial is to take place. These organisms could carry the GM FAV8 outside of the facility and birds outside the facility may be exposed to the GM FAV8 as a result of contact with these organisms. Since the GMO is primarily transmitted via the faecal-oral route, ingestion of insects or small vertebrates that may have come into contact with the GMO in chicken faeces is a possible route of exposure for other birds. 262. Since the trial is only to be conducted over a six week period, the numbers of non-avian vertebrates that will gain access to the facility during that time is expected to be small with rodents such as rats and mice being the animals most likely to enter the facility in search of food. Pest control mechanisms in place at the location, including rodent bait and traps, which have been imposed as a condition of the licence, will prevent or greatly reduce the number of rodents that will enter the facility. Furthermore, the amount of virus expected to be present in chicken faeces is very low (see discussion below) and the amount of faeces that is likely to adhere to rodent feet/fur is expected to be very small. Since adenoviruses are susceptible to desiccation (Abad et al. 1994b), the amount of viable GM FAV8 on the rodents is expected to rapidly decline as the adherent faeces desiccates. The period for GM FAV8 survival in faeces on rodents is likely to be similar (up to 4 days) or shorter than survival in bedding material (discussed below). 263. Therefore, the number of opportunities for exposure of other birds to doses of GM FAV8 that are sufficient to result in infection as a result of ingesting a small nonavian vertebrates carrying the GMO is expected to be extremely limited. 264. Insects will also be able to enter to the facility during the trial. Although a variety of insects may enter the facility, flies are expected to be greatest in number and will be attracted to the chicken faeces in the facility during the trial. 265. The use of fly strips as a pest control measure will decrease the numbers of insects that have been in contact with the GM FAV8 and that will leave the facility. However, the results of an experiment conducted by the applicant to determine the ability of flies to transmit the non-GM FAV8 showed that following exposure to high titres (105 TCID50/ml) the non-GM fowl adenovirus could only be detected on flies for between 4 and 12 hours after they were exposed. The GM FAV8 is expected to behave in the same manner as the non-GM FAV8. In addition, shedding and viability studies showed that that the GMO is shed into chicken faeces at low levels for 7-14 days post inoculation and that the GM FAV8 survived for only 48 hours or less in Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 53 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator bedding material, though it could occasionally be detected after 4 days in bedding material. 266. Given the results of both of these studies, only very low levels of active GM FAV8 particles are expected to present on insects that could come into contact with faeces from the inoculated chickens and any GM FAV8 on these insects is expected to be rapidly inactivated. 267. Whether the amount of GM FAV8 found on insects is sufficient to establish infection in other birds depends upon the amount of insects consumed, the minimum infectious dose for the GM FAV8 and whether birds are susceptible to infection by the GM FAV8. The minimum infectious dose is poorly understood for most viruses and varies considerably depending on type and strain of the virus and the route of infection. There is no reliable information on the minimum dose of GM or non-GM FAV8 CAF44 required to establish an infection in any birds, including chickens. The applicant has stated that doses of 104.7 TCID50 reliably produce infection in 100% of chickens. They propose to administer doses of 103.7 TCID50 and higher in the proposed trials. Doses lower than this are considered likely to have a diminished growth promoting/immune system stimulating effect or no effect at all (see discussion above). 268. Since very low levels of the GM FAV8 are excreted in faeces, insects are likely to carry only extremely low levels of the virus out of the facility. Although, the intake of insects by other birds is not known, it is likely that large numbers of insects would have to be ingested in order for birds to receive a dose of GM FAV8 that could establish an infection and/or have a biological effect as a result of expression of Ch IFN-γ. Furthermore, since not all viral particles successfully infect cells due to host barriers to infection such as mucus, gastric acids or local immunity, the likelihood of successful infection from exposure to low doses of GM FAV8 is considered to be very low. 269. Exposure to the GM FAV8 as a result of ingesting flies or rodents carrying the virus is likely to be limited to those birds in the immediate vicinity of the trial site and will be further limited by the short time during which the trial is to be conducted. Birds distant from the trial site are much less likely to be directly exposed as a result of ingesting flies or rodents carrying the GMO. The GM FAV8 is unlikely to spread to populations of birds that are distant from the trial site. Therefore, the extent of exposure of bird populations to the GM FAV8 is likely to be limited to the local area. 270. As outlined in Section 1, the pest potential of vertebrates is the result of the complex interactions between the vertebrate animal and its environment. Prediction of pest potential based solely on any one of the many contributing characteristics is likely to be misleading (Newsome & Noble 1986). As outlined in Section 2.2.1, only marginal biological effects, if any, could occur in other birds that may be exposed to low doses of the GM FAV8. Furthermore, any small biological effect occurring in individuals is not expected to persist in bird populations since the Ch IFN-γ gene is not expected to persist in FAV populations. The extent of exposure is likely to be limited to birds in the region immediately surrounding the trial location and limited by both the small scale and short duration of the trial. Existing pest control procedures at the trial location further limit the opportunity for exposure of other bird populations to the GM FAV8 as a result of insects or non-avian vertebrates transporting the GM FAV8 out of the facility. Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 54 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 271. Therefore the likelihood of spread to other birds as a result of insects and nonavian vertebrates transporting the GM FAV8 out of the facility is assessed as highly unlikely. 2.3 2.3.1 Event 2: Spread to other birds as a result of inhaling windborne particles containing the GM FAV8 Consequence assessment 272. Particles of skin, small feathers and dust generated during the trial may contain the GM fowl adenovirus and other birds in the area could be exposed to dust particles that could be carried from the facility by wind. If other birds inhale sufficient numbers of airborne particles containing the GM FAV8 they could become infected and could experience increased weight gain as a result of Ch IFN-γ expression. If other birds do not inhale sufficient viable GM FAV8 particles to establish an infection there would be no growth promoting/immune system stimulating effect in those birds. 273. As discussed in section 2.2.1, the magnitude of any growth promotion or immune system stimulation as a result of infection with the GM FAV8 and the expression of Ch IFN-γ is expected to be small in birds other than those closely related to chickens and the growth promoting effect is also expected to be diminished, or not occur at all, due to the expected low levels of exposure to the GM FAV8. Furthermore, even if a small growth promoting effect does occur in populations of other birds, the small effect is not considered to significantly increase the pest potential of other birds and the effect will not persist in bird populations since the Ch IFN-γ gene is not expected to persist in the FAV population. 274. Therefore the consequences of a spread of GM FAV8 to other birds as a result of inhaling windborne particles containing the GMO are assessed as marginal. 2.3.2 Likelihood assessment 275. Particles generated during trial may be dispersed from the trial site by wind. The number of particles containing the GMO that will be generated during the period in which live chickens will be present at the location is not known. Although windborne particles containing the GMO are expected to be continuously produced, the amount of windborne particles generated during the live animal phase of the trial is expected to be lower than the amount generated during the cleanup of the bedding material after the sacrifice of the animals. Any spread of the GMO from the trial site will be limited to the period during which the trial is to be conducted. After the trial has ended and the facility decontaminated, the spread of the GMO on windborne particles will cease. 276. The spread of the virus mainly occurs by direct contact between birds or by indirect contact facilitated by people or fomites. Because of its susceptibility to desiccation, airborne spread of the GM virus in contaminated aerosols is thought to occur only over a short distance and the concentration of particles containing the GMO is expected to rapidly decline as distance from the trial site increases. Therefore, exposure to significant numbers of airborne particles containing the GMO is likely to be restricted to those birds in the immediate vicinity of the trial location 277. It is unlikely that birds distant from the trial site will be exposed to sufficient numbers of particles containing the GMO to establish an infection and no growth promoting effect would be observed in these birds. Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 55 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 278. The major source of GM FAV8 in the trial facility is considered to be the GM FAV8 shed in faeces from inoculated chickens. As discussed in Section 2.2.2, only very low levels of GM FAV8 are shed in the faeces of inoculated chickens. Dried faeces containing very low amounts of GM FAV8 could become aerosolised during the trial and other potential pathways for GM FAV8 to become attached to airborne particles include the faecal contamination of dust, feedstuff, bedding or feather materials. Adenoviruses are sensitive to desiccating conditions and the viability of GM FAV8 is expected to rapidly decline as faeces dries (Abad et al. 1994a). Therefore, the amount of viable GM FAV8 in dried faeces is expected to be very low and the amount of viable GM FAV8 present in airborne particles is expected to very rapidly decline under the highly desiccating conditions that would occur in most dust particles. 279. Not all windborne particles that spread from the facility will contain the GMO and those windborne particles that do spread from the facility are expected to contain only very low levels of GM FAV8 which is expected to survive for only a limited time. Therefore, if birds in the vicinity are exposed to viable GM FAV8 as a result of inhaling airborne particles containing GM FAV8, the doses received are likely to be very low. 280. As outlined in Section 2.2.2, if any biological effects occur as a result of exposure to low doses of GM FAV8 and expression of Ch IFN-γ, the effects are expected to be small, likely to be limited to susceptible bird populations in the region immediately surrounding the trial location and unlikely to be persistent in bird populations. 281. Therefore the likelihood of spread of the GM FAV8 to other birds as a result of inhaling airborne particles containing the GMO is assessed as highly unlikely 2.4 Uncertainty 282. Although the available data indicates that FAV8 has a relatively restricted host range, the range of birds that are able to be successfully infected by the virus remains uncertain. This uncertainty will be addressed during the trial as research requirements have been imposed as a condition of the licence that test the ability of the virus to spread to and cause infection in other birds. SECTION 3 RISK ESTIMATES 283. The risk estimates (which can range from negligible to high) are based on a combination of the consequence and likelihood assessments, using the Risk Estimate Matrix (see Chapter 2). 284. The risk estimates for the adverse outcome of increased pest potential in other birds as a result of the proposed release of this GM FAV8 are summarised in Table 3.1. They have been made relative to the baseline of the characteristics of the non-GM FAV8, the known effects of non-GM FAV8 in birds and in the context of the proposed management conditions of the release, including animal husbandry and facility management practices and pest control procedures used at the facility. 285. The consequences of spread to other birds as a result of insects or non-avian vertebrates transporting the GM FAV8 out of the facility has been assessed as marginal, and the likelihood of this occurring has been estimated as highly unlikely. Therefore the risk estimate for event 1 is negligible . Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 56 DIR 046/2003—Risk Assessment and Risk Management Plan Table 3.1 Summary of risk assessment Event that may give rise to an adverse outcome Event 1 Spread of the GM FAV8 to other birds as a result of contact with insects or non-avian vertebrates transporting the GM FAV8 out of the facility. Event 2 Spread to other birds as a result of inhaling windborne particles containing the GM FAV8 Chapter 3 Office of the Gene Technology Regulator Consequence assessment Likelihood assessment Risk estimate Marginal Low level exposure of other birds is expected to result in a minimal effect or no effect at all The GM virus is not expected to persist in chicken populations and expression of GM FAV8 will be transient. Therefore, any increased growth promotion/immune system stimulation inother bird populations is expected to be short lived. Small growth promoting/immune stimulating effect occurring for limited time in a limited number of bird populations is not expected to increase pest potential Ch IFN-γ is only likely to be active in bird species that have an IFN-γ protein with a high amino acid sequence homology to Ch IFN-γ Marginal Low level exposure of other birds is expected to result in a marginal effects or no effect at all The GM virus is not expected to persist in chicken populations and expression of GM FAV8 will be transient. Therefore, any increased promotion/immune system stimulation in bird populations is expected to be short lived. Small growth promoting/immune stimulating effect occurring for limited time in a limited number of bird populations is not expected to increase pest potential Ch IFN-γ is only likely to be active in bird species that have an IFN-γ protein Highly unlikely Exposure to viable GM FAV8 is expected to be restricted to other birds in the immediate vicinity of the trial site Routine pest management at the facility will minimise rodent numbers and hence the level of exposure to rodents that may be carrying the GM FAV8 Low levels of exposure from insects due to very low levels of viable GM FAV8 carried Opportunity for exposure limited by rapid decline in survival of GM FAV8 under desiccating conditions The small size and short duration of the proposed release limits extent and duration of exposure Negligible Does risk require treatment? No Highly unlikely Exposure to viable GM FAV8 is expected to be restricted to other birds in immediate vicinity of trial site Exposure limited due to low levels of GM FAV8 in airborne particles and rapid decline in survival under desiccating conditions The small size and short duration of the proposed release limits extent and duration of exposure Negligible No Risk assessment: increased pest potential in other birds (January 2006) 57 DIR 046/2003—Risk Assessment and Risk Management Plan Event that may give rise to an adverse outcome Consequence assessment Office of the Gene Technology Regulator Likelihood assessment Risk estimate Does risk require treatment? with a high amino acid sequence homology to Ch IFN-γ 286. The consequences of spread to other birds as a result of inhaling windborne particles containing the GM FAV8 has been assessed as marginal, and the likelihood of this resulting in increased pest potential as highly unlikely. Therefore the risk estimate for event 2 is negligible. 287. As the risks of all events that may lead to increased pest potential in other birds are estimated to be negligible, there is no present need to invoke actions for mitigation (OGTR 2005). Therefore, no risk treatment measures for increased pest potential in other birds are proposed. Chapter 3 Risk assessment: increased pest potential in other birds (January 2006) 58 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator CHAPTER 4 RISK MANAGEMENT 288. This chapter evaluates the risks assessed in Chapter 4 to determine whether or not specific treatments are required to mitigate harm that may arise during the proposed limited release. Other risk management considerations required under the Act are also addressed in this chapter. SECTION 1 BACKGROUND 289. 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. 290. Under section 62 of the Act, the Regulator can direct a licence holder to take any steps the Regulator deems necessary to protect the health and safety of people or the environment. Licence conditions can be imposed to limit and control the scope of the dealings and the possession, supply, use, transport or disposal of the GMO for the purposes of, or in the course of, a dealing. In addition, the Regulator has extensive powers to monitor compliance with licence conditions under section 152 of the Act. SECTION 2 OTHER AUSTRALIAN REGULATORS 291. Australia’s gene technology regulatory system operates as part of an integrated legislative framework (OGTR 2005). Other agencies that also regulate GMOs or GM products include FSANZ, APVMA, TGA, NICNAS, NHMRC and AQIS. Dealings conducted under any licence issued by the Regulator may also be subject to regulation by one or more of these agencies. 292. The GM FAV8 proposed for release meets the definition of a veterinary medicine under the Agricultural and Veterinary Chemicals Code Act 1994, due to the production of a substance that has a growth promoting effect in animals, and therefore it is subject to regulation by the APVMA. Imugene has made an application to the APVMA for a permit to undertake the proposed release. 293. The Gene Technology (Consequential Amendments) Act 2000 requires the APVMA to consult the Regulator for the purposes of making certain decisions regarding registration or issuing a permit for a veterinary medicine that is, or contains a product from, a GMO. SECTION 3 RISK TREATMENT MEASURES FOR IDENTIFIED RISKS 294. The detailed risk assessment of Events 1 and 2 contained in Chapter 3 concluded that the risk estimates for both events are negligible. These events were considered in the context of the proposed inoculation of up to 1500 chickens with the GMO in an animal containment facility in the shire of Wyndham, Victoria. 295. The Risk Analysis Framework (OGTR 2005), which guides the risk assessment and risk management process, defines negligible risks as insubstantial with no present need to invoke actions for their mitigation. However, containment measures have been imposed to limit the release to the size, duration and location proposed by the applicant. In addition research requirements have been included to collect additional data to address uncertainty identified in the risk assessment about the range of birds that are able to be successfully infected by the GM virus. Chapter 4 Risk management (November 2005) 59 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator SECTION 4 GENERAL RISK MANAGEMENT 4.1 Licence conditions associated with managing limited and controlled releases 4.1.1 Measures to limit and control the proposed release 296. A number of licence conditions have been imposed to limit and control the release, including requirements to: contain the chickens in the animal containment facility; wear personal protective equipment (PPE) when carrying out work in the animal containment facility that is removed prior to leaving to prevent transport of GMOs outside the facility; utilise sentinel animals (uninoculated chickens, pigeons and pest birds) and inoculated pigeons and pest birds to assess the ability of the GM virus to spread and cause infection in birds outside the known host range of the virus; implement pest control measures to minimise the numbers of rodents and insects entering or exiting the facility; following completion of the trial kill and decontaminate prior to disposal all chickens and sentinel animals involved in the trial; following completion of the trial decontaminate all waste and PPE from the trial including equipment used in the trial; following completion of the trial decontaminate all areas in which the trial has taken place; and conduct regular inspections of the release site following completion of the trial until decontamination is successful. 4.1.2 Measures to control other activities associated with the release 297. The Regulator has issued guidelines and policies for the transport, supply and storage of GMOs (Guidelines for the transport of GMOs, June 2001; Policy on transport and supply of GMOs, July 2005 and Policy on storage of genetically modified organisms, July 2004). Licence conditions would also be imposed to control possession, use or disposal of the GMOs for the purposes of, or in the course of, the authorised dealings. 4.2 Other risk management considerations 298. All DIR licences issued by the Regulator contain a number of general conditions that also relate to risk management. These include, for example: identification of the persons or classes of persons covered by the licence applicant suitability contingency and compliance plans reporting structures, including a requirement to inform the Regulator if the applicant becomes aware of any additional information about risks to the health and safety of people or the environment Chapter 4 Risk management (November 2005) 60 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator a requirement that the applicant allows access to the release sites by the Regulator, or persons authorised by the Regulator, for the purpose of monitoring or auditing. 4.2.1 Applicant suitability 299. 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 include: any relevant convictions of the applicant (both individuals and the body corporate) 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 applicant’s history of compliance with previous approved dealings the capacity of the applicant to meet the conditions of the licence. 300. Before making the decision to issue a licence for this application (DIR 046/2003), the Regulator determined that Imugene Ltd was suitable to hold a licence. 301. Conditions in the licence include a requirement for the licence holder to inform the Regulator of any circumstances that would affect their suitability or their capacity to meet the conditions of the licence. 302. In addition, any applicant organisation must have access to a properly constituted Institutional Biosafety Committee and be an accredited organisation under the Act. 4.2.2 Compliance and contingency plans 303. The licence requires Imugene to submit a plan detailing how it intends to ensure compliance with the licence conditions and document that compliance. This plan is required before any inoculation with the GM FAV8 occurred. This compliance plan has been submitted by Imugene Ltd. 304. Imugene is also required to submit a contingency plan to the Regulator within 30 days of the issue date of the licence. This plan must measures to be undertaken in the event of any unintended presence of the GM FAV8 outside of the permitted areas. This contingency plan has been submitted by Imugene Ltd. 305. Imugene is also required to provide a method to the Regulator for the reliable detection of the presence of the GMO and the introduced genetic material in a recipient organism. This information is required within 30 days of the issue date of the licence. 4.2.3 Reporting structures 306. The licence obliges 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 release any contraventions of the licence by persons covered by the licence any unintended effects of the release. Chapter 4 Risk management (November 2005) 61 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 307. The licence holder is also obliged to submit an Annual Report within 90 days of the anniversary of the licence containing any information required by the licence, including the results of inspection activities. 308. A number of written notices are also required under the licence that would assist the OGTR in designing and implementing its risk based monitoring program for all licensed dealings. The notices include: expected and actual dates of inoculation expected and actual dates of decontamination SECTION 5 MONITORING AND COMPLIANCE 309. A range of monitoring and compliance activities are undertaken on behalf of the Regulator (OGTR 2005) to check compliance with licence conditions. 310. 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. 311. In cases of non-compliance with licence conditions, the Regulator may also instigate an investigation to determine the nature and extent of non-compliance. The Act provides the Regulator with extensive powers of enforcement to ensure compliance. These include the provision 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 the health and safety of people or the environment could result. SECTION 6 CONCLUSIONS OF THE RARMP 312. The risk assessment concludes that this limited and controlled release of GM FAV8 at a CSIRO Livestock Industries animal containment facility in Werribee, Victoria poses negligible risks to the health and safety of people and the environment as a result of gene technology. 313. The risk management plan concludes that these negligible risks do not require specific risk treatment measures. However, conditions have been imposed in the licence to contain the release to the size, duration and location requested in the application. Chapter 4 Risk management (November 2005) 62 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator CHAPTER 5 LICENCE CONDITIONS SECTION 1 INTERPRETATIONS AND DEFINITIONS This licence does not authorise dealings with GMOs that are otherwise prohibited as a result of the operation of State legislation declaring areas to be GM, GM free, or both, for marketing purposes. In this licence: (a) words and phrases used in this licence have the same meaning as they do in the Act and the Regulations; (b) words importing a gender include any other gender; (c) words in the singular include the plural and words in the plural include the singular; (d) words importing persons include a partnership and a body whether corporate or otherwise; (e) references to any statute or other legislation (whether primary or subordinate) are a reference to a statute or other legislation of the Commonwealth of Australia as amended or replaced from time to time and equivalent provisions, if any, in corresponding State law, unless the contrary intention appears; (f) where any word or phrase is given a defined meaning, any other part of speech or other grammatical form in respect of that word has a corresponding meaning; (g) specific conditions prevail over standard conditions to the extent of any inconsistency. In this licence: ‘Act’ means the Gene Technology Act 2000 (Cth) and equivalent provisions in corresponding State law. ‘Animal Material’ means any organ, tissue or waste from chickens or Sentinel Birds used in the course of this dealing. ‘Annual Report’ means a written report provided to the Regulator within 90 days of each anniversary of this licence containing all the information required by this licence to be provided in the Annual Report. ‘Decontamination’ means, as applicable, the application of the Decontamination Protocol, or decontamination in accordance with the Regulator’s certification conditions, so as to render the GMO non-viable. Chapter 5 Licence conditions (January 2006) 63 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator ‘Destroy’, (or ‘Destroyed’ or ‘Destruction’) means killed by one or more of the following methods: (a) incineration in an EPA approved incinerator; (b) autoclaving; (c) heat treatment; or (d) chemical disinfection in accordance with Appendix E of Australian/New Zealand Standard 2246.3:2002 Safety in laboratories. Part 3: Microbiological aspects and containment facilities (2002). ‘Equipment’ means equipment used in the course of the dealing and includes vehicles, clothing, tools, and cages and also fixtures situated in Location A. ‘GM’ means genetically modified. ‘GMO’ means the genetically modified organism authorised for release by this licence. ‘Location’ means Location A and PC2 animal containment facilties (Certs 507512/2002 and Cert 521/2002). ‘Location A’ means the animal containment facility at the CSIRO Livestock Industries site at Werribee, Victoria. ‘OGTR’ means the Office of the Gene Technology Regulator. ‘Regulator’ means the Gene Technology Regulator. ‘Sentinel Birds’ means any uninoculated chickens and any pigeons, sparrows and starlings used in the dealings to test the ability of the GMO to be transmitted. ‘Shedding’ means the excretion by of recombinant viral particles by birds involved in the dealing. ‘Sign-off’ means a notice in writing from the Regulator that no further testing is required in respect of the Location or other areas used in connection with this licence. ‘Site’ means the Location and the area surrounding the Location within the perimeter fence. ‘Susceptible animal’ means an animal that can be infected by the GMO. Chapter 5 Licence conditions (January 2006) 64 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator SECTION 2 GENERAL CONDITIONS Duration of Licence 1. This licence remains in force until it is suspended, cancelled or surrendered. No dealings with GMOs are authorised during any period of suspension. Holder of Licence 2. The holder of this licence (‘the licence holder’) is Imugene Ltd. Project Supervisor 3. The Project Supervisor in respect of this Licence is identified at Attachment A. 4. The licence holder must immediately notify the Regulator in writing if any of the contact details of the Project Supervisor change. No dealings with the GMO except as authorised by this licence 5. Persons covered by this licence must not deal with the GMO for the purpose of this dealing except as expressly permitted by this licence. Persons covered by this GMO licence 6. The persons covered by this licence are the licence holder and employees, agents or contractors of the licence holder and other persons who are, or have been, engaged to undertake any activity in connection with the GMO authorised by this Licence. Informing people of their obligations 7. The licence holder must inform any person covered by this licence, to whom a particular condition of this licence applies, of the following: (a) the particular condition (including any variations of it); (b) the cancellation or suspension of the licence; (c) the surrender of the licence. 8. The licence holder must provide the Regulator, on the Regulator’s written request, signed statements from persons covered by this licence that the licence holder has informed those people of the conditions of this licence that apply to them. Licence holder to notify of circumstances that might affect suitability 9. The licence holder must immediately, by notice in writing, inform the Regulator of: Chapter 5 Licence conditions (January 2006) 65 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator (a) any relevant conviction of the licence holder occurring after the commencement of this licence; (b) any revocation or suspension of a licence or permit held by the licence holder under a law of the Australian Government, a State or a foreign country, being a law relating to the health and safety of people or the environment; (c) any event or circumstances occurring after the commencement of this licence that would affect the capacity of the holder of this licence to meet the conditions in it. Licence holder must provide requested information on matters related to suitability 10. The licence holder must provide information related to the licence holder’s ongoing suitability to hold a licence when requested to do so in writing by the Regulator and must provide the information within a time period stipulated by the Regulator. Additional information to be given to the Regulator 11. It is a condition of a licence that the licence holder inform the Regulator if the licence holder: (a) becomes aware of additional information as to any risks to the health and safety of people, or to the environment, associated with the dealings authorised by the licence; or (b) becomes aware of any contraventions of the licence by a person covered by the licence; or (c) becomes aware of any unintended effects of the dealings authorised by the licence. 12. The licence holder must provide the information required by paragraphs (a) (b) and (c) of the immediately preceding condition to the Regulator as soon as practically and reasonably possible and must also include the information in the Annual Report. People dealing with the GMO must allow auditing and monitoring of the dealing 13. If a person is authorised by this licence to deal with the GMO and a particular condition of this licence applies to the dealing by that person, the person must allow the Regulator, or a person authorised by the Regulator, to enter premises where the dealing is being undertaken, for the purposes of auditing or monitoring the dealing. Chapter 5 Licence conditions (January 2006) 66 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Remaining an accredited organisation 14. The licence holder must, at all times, remain an accredited organisation in accordance with the Act and comply with its instrument of accreditation. Chapter 5 Licence conditions (January 2006) 67 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator SECTION 3 SPECIFIC CONDITIONS GMO covered by this licence 1. The GMO covered by this licence is described at Attachment B. Permitted dealings 2. This licence authorises the licence holder and persons covered by the licence to conduct all dealings necessary to inoculate a population of chickens and Sentinel Birds with the GMO and to test the safety and efficacy of the GMO. Location, size and timing 3. The Location may contain chickens and Sentinel Birds inoculated with GMO during the period 1 February 2006 until 30 October 2006. 4. The permitted dealings may only be conducted at the Site. 5. Up to 1500 chickens and Sentinel Birds in total may be inoculated with the GMO. 6. Inoculation must occur within the Location. 7. The licence holder must be able to access and control the Site where the GMO is released to the extent necessary to comply with this licence, for the duration of the life of the licence. Notice of permitted dealings 8. The licence holder must provide a notice in writing to the Regulator prior to commencement of the dealings which sets out the dates on which the chickens and Sentinel Birds are to be inoculated with the GMO. 9. The licence holder must provide a notice in writing to the Regulator within 5 days of the commencement of the inoculation of chickens and Sentinel Birds with the GMO which sets out: (a) the date on which the chickens and Sentinel Birds were first inoculated; (b) the number of chickens and Sentinel Birds inoculated; and (c) the number, type and placement of Sentinel Birds used. Persons at Location A must wear protective clothing 10. From the commencement of dealings until the Decontamination of Location A, boots, overalls and gloves must be worn by any person within Location A and must be removed before the person leaves Location A. All persons and wheeled Chapter 5 Licence conditions (January 2006) 68 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Equipment leaving Location A from the commencement of dealings until its Decontamination must pass through a disinfectant footbath which must contain disinfectant capable of inactivating the GMO. 11. At the conclusion of the dealings protective clothing and Equipment potentially contaminated with the GMO must be Decontaminated. 12. Equipment used at Location A which is transported outside Location A prior to Decontamination of Location A must be contained in a manner which prevents dissemination of the GMO (e.g. by double-bagging). Conditions about Susceptible Animals 13. No chickens other than those that are involved in the dealing are to held at the Site. Conditions about Sentinel Birds 14. Sentinel Birds that are used in the dealing must be clearly marked to indicate that they have not been inoculated with the GMO. 15. Sentinel Birds that are being held outside Location A must be caged to prevent their escape from the Site. 16. Faeces from Sentinel Birds must be collected every second day during the course of the dealing and tested for the presence of the GMO. 17. Any Sentinel Birds held outside Location A that are found to be infected with the GMO must be euthanased an soon as practicable and Decontaminated prior to disposal. 18. Faeces from Sentinel Birds that are being held outside Location A must be Decontaminated before disposal. 19. The Regulator must be notified in writing within 7 days if any Sentinel Birds held outside Location A are found to be infected with the GMO. 20. Prior to Decontamination samples of the following tissues must be taken from Sentinel Birds and tested for infection with the GMO, (a) (b) (c) (d) (e) (f) Blood Liver Caecal tonsil Heart Kidney Spleen Chapter 5 Licence conditions (January 2006) 69 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Decontamination of the Location 21. Within 1 month of completion of the trial the Location and any Equipment must be Decontaminated. Note: PC2 animal containment facilities (Certs 507-512/2002 and 521/2002) must be Decontaminated in accordance with the Certification Instrument. General conditions in relation to the Decontamination of Equipment used in connection with this licence 22. If Equipment is used in connection with the GMO or Sentinel Birds held outside Location A then that Equipment must also be Decontaminated. 23. Decontamination must occur immediately or as soon as practicable after the use and before it is used for any other purpose. 24. Any areas in which Equipment is Decontaminated must also be Decontaminated. 25. On the request of the Regulator, the Regulator must be provided with written documentation of the procedures in place to ensure continuing compliance with these Decontamination conditions. Testing for continuing presence of the GMO 26. After Decontamination the licence holder must conduct tests for any continuing presence of viable GMOs at Location A by taking a minimum of 12 swabs from separate points within the facility. Testing must continue until two consecutive negative results have been established. 27. The results of testing activities must be recorded in a logbook. The logbook must be available on request for examination or photocopying by the OGTR. The Regulator must be notified in writing of the findings of the tests as recorded in the logbook within 14 days. These findings must also be included in the licence holder’s annual report to the Regulator. The logbook must contain at least the following: (g) details of the points inspected and, (h) details of the date of decontamination and, (i) details of the dates of testing and, (j) whether the GMO was detected and, (k) if the GMO was detected, details of any subsequent decontamination, including procedures followed, and (l) person(s) doing the inspection. Chapter 5 Licence conditions (January 2006) 70 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 28. Any GMOs detected after 14 days following Decontamination are to be treated as an unintended presence and the licence holder must act in accordance with the Contingency Plan for unintended presence provided to the Regulator pursuant to this licence. 29. If no GMOs are detected by testing following two consecutive weekly tests after Decontamination, the licence holder may make written application to the Regulator for Sign off. Testing methodology 30. The experimental method to be used for detecting the presence of the GMO and the presence of the genetic modifications described in this licence in a recipient organism must be provided to the Regulator within 30 days of issue of the licence. Transportation of the GMO, birds infected with the GMO and Animal Material 31. Live chickens and Sentinel Birds inoculated or infected with the GMO must not be transported off the site. 32. Subject to the conditions immediately below in respect of transportation, the GMOs, Animal Material potentially contaminated with the GMO and swabs used to test for the continuing presence of the GMO must be transported in accordance with the OGTR’s Guidelines for the Transport of GMOs (June 2001) issued by the Regulator. 33. Every container used to transport the GMO, Animal Material potentially contaminated with the GMO and swabs used to test for the continuing presence of the GMO off the Site must be labelled: (a) to indicate that it contains the GMO; and (b) with the telephone contact numbers for the licence holder and instructions to contact the licence holder in the event that the container is broken or misdirected. Disposal of the GMO, chickens and Sentinel Birds 34. Subject to the conditions immediately below, during or after the trial has been conducted, and before the Decontamination of Location A, all Chickens and Sentinel Birds must be euthanased at the Site and Destroyed within 5 days by autoclaving in the PC2 animal facility (Cert 521/2002) and incineration in an EPA approved incinerator. Destruction must occur on the same day as removal from the Location occurs. 35. After the experiments have been conducted and before the Decontamination of Location A, the GMO must be Destroyed, unless stored in accordance with OGTR policy on storage of genetically modified organisms or covered by another Chapter 5 Licence conditions (January 2006) 71 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator appropriate authorisation under the Gene Technology Act 2000. The GMO may be Destroyed during or at the completion of experiments. Pest Control Measures 36. Measures such as rodent baits, rodent traps and fly strips must be put in place in Location A for the duration of the trial. Contingency Plans 37. Prior to the commencement of experiments, a written Contingency Plan must be submitted to the Regulator detailing measures to be taken in the event of the unintended presence of the GMO outside the Location. 38. The Contingency Plan must include details of procedures to ensure the Regulator is notified immediately if the licence holder becomes aware of the event and to decontaminate the area. Compliance Management Plan 39. Prior to dealing with the GMO, a written Compliance Management Plan must be provided to the Regulator. The Compliance Management Plan must describe in detail how the licence holder intends to ensure compliance with each of these conditions and document that compliance and must include: a) a list of the names of all organisations or natural persons who will be persons covered by this licence. Where a name of a person is not known at the time of submitting the Compliance Management Plan the function or position of the person to be covered must be provided; Note: examples of functions or positions are ‘Site manager’, ‘Laboratory worker’ etc; b) an explanation of how the licence holder has informed, or proposes to inform, each person covered by the licence of the conditions of the licence; c) a description of the responsibilities of the licence holder and of each person covered by the licence in relation to the requirements of this licence; d) a description of how any contracts, agreements, or other enforceable arrangements between the licence holder and persons covered by the licence will allow the licence holder to access and control a Site to the extent necessary to comply with this licence, for the duration of the life of the licence; and 40. Where any of the details of the Compliance Management Plan change, the Regulator must be notified of the changes within 14 days of the change occurring. Chapter 5 Licence conditions (January 2006) 72 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Research Requirements 41. During the period of this licence, the licence holder must test for the following in consultation with the OGTR and provide the information to the Regulator either before or during the trial or in the Annual Report: (a) the ability of the GMO to be transmitted to Sentinel Birds inside and outside Location A must be investigated and the results made available to the Regulator. (b) the ability of the GMO to cause an active infection in Sentinel Birds. (c) the shedding, persistence and stability of the GMO. (d) the presence of viral nucleic acid or introduced gene products in tissue samples removed from birds for histopathology. 42. A progress report on the research conducted under the previous condition must be provided to the Regulator in the Annual Report. Reporting 43. The licence holder must provide an Annual Report to the Regulator. GMO must not be consumed 44. The licence holder must ensure that the GMO, birds inoculated with the GMO and Animal Material are not consumed by humans or other animals. Animal ethics approval 45. The dealings authorised by this licence involving the use of animals must not commence unless the Animal Ethics Committee for CSIRO Livestock Industries has first considered and consented in writing to the performance of the dealings. Chapter 5 Licence conditions (January 2006) 73 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator REFERENCES Abad, F.X., Pinto, R.M., Bosch, A. (1994). Survival of enteric viruses on environmental fomites. Applied and Environmental Microbiology 60: 3704-3710. Addison, C.L., Hitt, M., Kunsken, D., Graham, F.L. 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Grouping of fowl adenoviruses based upon the restriction patterns of DNA generated by BamHI and HindIII. Intervirology 22: 110-114. References 80 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator APPENDIX A DEFINITIONS OF RISK ANALYSIS TERMS (* terms defined as in Australia New Zealand Risk Management Standard AS/NZS 4360:2004) Consequence outcome or impact of an adverse event Marginal: there is minimal negative impact Minor: there is some negative impact Major: the negative impact is severe Event* occurrence of a particular set of circumstances Hazard* source of potential harm Hazard identification the process of analysing hazards and the events that may give rise to harm Intermediate the negative impact is substantial Likelihood chance of something happening Highly unlikely: may occur only in very rare circumstances Unlikely: could occur in some circumstances Likely: could occur in many circumstances Highly likely: is expected to occur in most circumstances Quality control to check, audit, review and evaluate the progress of an activity, process or system on an ongoing basis to identify change from the performance level required or expected and opportunities for improvement Risk the chance of something happening that will have an undesired impact Negligible: risk is insubstantial and there is no present need to invoke actions for mitigation Low: risk is minimal but may invoke actions for mitigation beyond normal practices Moderate: risk is of marked concern requiring mitigation actions demonstrated to be effective High: risk is unacceptable unless actions for mitigation are highly feasible and effective Risk analysis the overall process of risk assessment, risk management and risk communication Appendix A Definitions of risk analysis terms (January 2006) 81 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Risk analysis framework systematic application of legislation, policies, procedures and practices to analyse risks Risk assessment the overall process of hazard identification and risk estimation Risk communication the culture, processes and structures to communicate and consult with stakeholders about risks Risk Context parameters within which risk must be managed, including the scope and boundaries for the risk assessment and risk management process Risk estimate a measure of risk in terms of a combination of consequence and likelihood assessments Risk evaluation the process of determining risks that require treatment Risk management the overall process of risk evaluation, risk treatment and decision making to manage potential adverse impacts Risk management plan integrates risk evaluation and risk treatment with the decision making process Risk treatment* the process of selection and implementation of measures to reduce risk Stakeholders* those people and organisations who may affect, be affected by, or perceive themselves to be affected by a decision, activity or risk States includes all State governments, the Australian Capital Territory and the Northern Territory governments Uncertainty imperfect ability to assign a character state to a thing or process; a form or source of doubt Appendix A Definitions of risk analysis terms (January 2006) 82 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator APPENDIX B SUMMARY OF ISSUES RAISED IN SUBMISSIONS FROM PRESCRIBED EXPERTS, AGENCIES AND AUTHORITIES30 ON APPLICATION All issues relating to risks to human health and safety and the environment were considered in the context of currently available scientific evidence that was used in the preparation of the consultation RARMP. Issues raised relating to the Risk Assessment and where they have been considered: risk from a change in viral characteristics (tissue tropism virulence and pathogenicity) (see Chapters 1 and 2) risk of disease in chickens (see Chapters 1 and 2) risk of disease in other avian species (see Chapters 1 and 2) risk of recombination with other viruses (including in people) (see Chapter 2) risk of altering the ecology of native or feral bird populations (see Chapters 2 and 3) risk resulting from any gene flow (both vertical and horizontal) (see Chapter 2) risk resulting from dissemination of the GM virus beyond the intended areas (see Chapters 2 and 3) risk resulting from persistence of the virus (see Chapters 2 and 3 ) risk resulting from occupational exposure (see Chapter 2) the methods to be used for waste disposal, particularly of bird litter (see Chapters 4 and 5) the possible effects of maternal antibody on trial results (see Chapter 1) whether the avian adenovirus uses the same receptors as human adenoviruses (see Chapters 1 and 2) whether the GM virus can enter or replicate in human cells (see Chapter 1) whether the cytokine is functionally active in human cells (see Chapter 1) Issues raised relating to the Risk Management Plan: standard licence conditions for limiting and controlling the release (see Chapters 4 and 5) monitoring any adverse effects (see Chapters 4 and 5) 30 Gene Technology Technical Advisory Committee, State and Territory governments, Australian Government agencies, the Minister for Environment and Heritage and Local councils where the release may occur. Appendix B Summary of agency submissions on the application (January 2006) 83 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator APPENDIX C SUMMARY OF ISSUES RAISED IN SUBMISSIONS RECEIVED FROM PRESCRIBED EXPERTS, AGENCIES AND AUTHORITIES ON THE CONSULTATION RARMP All issues relating to risks to human health and safety 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 and where they have been considered: the ability of the GM virus to infect chickens and other bird species (see Chapters 1, 2, 3 and 5) the ability of insects and non-avian vertebrates to transmit the virus (see Chapters 2, 3 and 5); the activity of the Ch-IFN-γ in other bird species (see Chapters 1, 2 and 3); the level of expression of Ch-IFN-γ (see Chapters 1, 2 and 3); and the possibility and result of recombination between the GMO and other viruses (see Chapters 1 and 2). Appendix C Summary of agency submissions on the Consultation RARMP (January 2006) 84 DIR 046/2003—Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator APPENDIX D SUMMARY OF PUBLIC SUBMISSION RECEIVED ON THE CONSULTATION RARMP Abbreviations used: a Submission from: I: Individual; OSA:. Outside the scope of the assessment Summary of issues raised The vaccine should not be used Appendix D Issue Consideration of issue The Regulator must consider OSA submitted applications Summary of public submissions on the Consultation RARMP (January 2006) 85