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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)
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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)
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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)
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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)
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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)
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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)
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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
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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.
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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
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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.
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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).
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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.
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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.
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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.
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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.
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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)
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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)
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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)
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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
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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
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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.
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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
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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
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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
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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).
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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.
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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
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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.
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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
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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.
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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).
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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).
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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
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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
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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.
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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
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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
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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.
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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.
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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 .
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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
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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.
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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.
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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
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 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.
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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.
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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.
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‘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.
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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:
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(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.
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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.
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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
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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
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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.
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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
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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
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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.
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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)
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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)
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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)
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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)
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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)
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