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APPLICATION A1001
FOOD DERIVED FROM INSECT-PROTECTED CORN
LINE MIR162
EXPLANATORY STATEMENT
Executive Summary
Purpose
Food Standards Australia New Zealand (FSANZ) received an Application from Syngenta
Seeds Pty Ltd (the Applicant) on 10 October 2007. The Applicant has requested an
amendment to the Australia New Zealand Food Standards Code (the Code), specifically to
Standard 1.5.2 – Food produced using Gene Technology, to permit the sale and use of food
derived from a new genetically modified (GM) variety of corn, MIR162. Standard 1.5.2
prohibits a food produced using gene technology from being sold or used as an ingredient or
component of any food unless it is listed in the Table to clause 2 of that Standard.
MIR162 corn has been genetically modified to be protected against feeding damage caused
by the larvae of certain insect pest species. Protection is achieved through the expression in
the plant of an insecticidal protein derived from Bacillus thuringiensis, a common soil
bacterium.
Corn line MIR162 is intended to be grown in North America. However, once
commercialised, corn products imported into Australia and New Zealand could contain
ingredients derived from MIR162 corn. Approval is therefore necessary before these
products may enter the Australian and New Zealand markets.
The Application is being assessed under the General Procedure and is at the Approval stage.
Safety Assessment
FSANZ has completed a comprehensive safety assessment of food derived from insectprotected corn line MIR162, as required under Standard 1.5.2. The assessment included
consideration of (i) the genetic modification to the plant; (ii) the potential toxicity and
allergenicity of the novel proteins; and (iii) the composition of MIR162 corn compared with
that of conventional corn varieties.
No public health and safety concerns were identified as a result of the safety assessment.
i
On the basis of the available evidence, including detailed studies provided by the Applicant,
food derived from insect-protected corn line MIR162 is considered as safe and wholesome
as food derived from other commercial corn varieties.
Labelling
If approved, food derived from insect-protected corn line MIR162 will be required to be
labelled as genetically modified if novel DNA and/or novel protein is present in the final food.
Studies conducted by the Applicant show that the novel proteins are present in the grain.
Labelling addresses the objective set out in section 18(1)(b) of the Food Standards Australia
New Zealand Act 1991 (FSANZ Act); the provision of adequate information relating to food
to enable consumers to make informed choices.
Impact of regulatory options
Two regulatory options were considered in the assessment: (1) no approval; or (2) approval
of food derived from insect-protected corn line MIR162 based on the conclusions of the
safety assessment.
Following analysis of the potential costs and benefits of each option on affected parties
(consumers, the food industry and government), approval of food derived from insectprotected corn line MIR162 is the preferred option as the potential benefits to all sectors
outweigh the costs associated with the approval.
Assessing the Application
In assessing the Application and the subsequent variation to Standard 1.5.2, FSANZ has
had regard to the following matters as prescribed in section 29 of the FSANZ Act:

Whether costs that would arise from a variation to Standard 1.5.2 approving food
derived from insect-protected corn line MIR162 outweigh the direct and indirect
benefits to the community, Government or industry that would arise from this food
regulatory measure.

There are no other measures that would be more cost-effective than a variation to
Standard 1.5.2 that could achieve the same end.

There are no relevant New Zealand standards.

There are no other relevant matters.
Decision
Approve the variation to Standard 1.5.2 – Food produced using Gene Technology, to
include food derived from insect-protected corn line MIR162 in the Table to clause 2.
Reasons for Decision
A variation to Standard 1.5.2 to permit the sale and use of food derived from insect-protected
corn line MIR162 in Australia and New Zealand is approved on the basis of the available
scientific evidence, for the following reasons:
ii

the safety assessment did not identify any public health and safety concerns
associated with the genetic modification used to produce insect-protected corn line
MIR162;

food derived from insect-protected corn line MIR162 is equivalent to food from the
conventional counterpart and other commercially available corn varieties in terms of its
safety for human consumption and nutritional adequacy;

labelling of certain foods derived from insect-protected corn line MIR162 will be
required if novel DNA and/or protein is present in the final food;

a regulation impact assessment process has been undertaken that also fulfils the
requirement in New Zealand for an assessment of compliance costs. The assessment
concluded that the preferred option is option 2, a variation to Standard 1.5.2;

there are no relevant New Zealand standards; and

there are no other measures that would be more cost-effective than a variation to
Standard 1.5.2 that could achieve the same end.
Consultation
Public submissions were invited on the Assessment Report between 11 April and 23 May 2008.
Comments were specifically requested on the scientific aspects of this Application, in particular,
information relevant to the safety assessment of food from insect-protected corn MIR162. A
total of 24 submissions was received. A summary of these is provided in Attachment 3 to this
Report.
As this Application is being assessed as a general procedure, there was one round of public
comment. Responses to the Assessment Report were used to develop this Approval Report
for the Application. The main issues raised in public comments are discussed in the
Approval Report.
iii
CONTENTS
INTRODUCTION ................................................................................................................... 2
1.
2.
THE ISSUE / PROBLEM .................................................................................................. 2
CURRENT STANDARD ................................................................................................... 2
2.1
Background ........................................................................................................ 2
2.2
Overseas approvals ............................................................................................ 2
3. OBJECTIVES ................................................................................................................ 3
4. QUESTIONS TO BE ANSWERED ...................................................................................... 3
RISK ASSESSMENT............................................................................................................. 3
5.
RISK ASSESSMENT SUMMARY....................................................................................... 4
5.1
Safety Assessment Process ............................................................................... 4
5.2
Outcomes of the Safety Assessment .................................................................. 4
5.3
Conclusions ........................................................................................................ 5
RISK MANAGEMENT ........................................................................................................... 5
6.
OPTIONS ..................................................................................................................... 5
6.1
Option 1 – Continue to prohibit food from corn line MIR162 ................................ 5
6.2
Option 2 – Approve food from corn line MIR162 ................................................. 5
7. IMPACT ANALYSIS ........................................................................................................ 5
7.1
Affected Parties .................................................................................................. 5
7.2
Benefit Cost Analysis .......................................................................................... 6
7.3
Comparison of Options ....................................................................................... 7
COMMUNICATION AND CONSULTATION STRATEGY ...................................................... 7
8.
9.
COMMUNICATION ......................................................................................................... 7
CONSULTATION ............................................................................................................ 7
9.1
Public Consultation ............................................................................................. 7
9.2
World Trade Organization (WTO) ..................................................................... 13
CONCLUSION..................................................................................................................... 13
10.
CONCLUSION AND DECISION ................................................................................... 13
10.1 Reasons for Decision........................................................................................ 13
11.
IMPLEMENTATION AND REVIEW ............................................................................... 14
ATTACHMENT 1 - DRAFT VARIATION TO THE AUSTRALIA NEW ZEALAND FOOD STANDARDS CODE
........................................................................................................................................ 15
ATTACHMENT 2 - SAFETY ASSESSMENT REPORT ................................................................. 16
ATTACHMENT 3 - SUMMARY OF ISSUES RAISED IN PUBLIC SUBMISSIONS............................... 67
ATTACHMENT 4 – 1ST REVIEW REPORT ................................................................................ 72
1
INTRODUCTION
An Application was received from Syngenta Seeds Pty Ltd on 10 October 2007 seeking an
amendment to Standard 1.5.2 – Food produced using Gene Technology, in the Australia
New Zealand Food Standards Code (the Code), to approve food derived from insectprotected corn line MIR162.
The genetic modification involved the transfer of two genes into corn. One gene is from a
common soil bacterium called Bacillus thuringiensis and encodes an insecticidal protein
(Vip3A) which protects the plant against feeding damage caused by certain insect pest
larvae. The second gene, from another common bacterium called Escherichia coli, was used
in the plant transformation process as it encodes an enzyme (PMI) which allows the
transformed corn plants to grow on media containing the sugar mannose.
FSANZ completed a full scientific evaluation of food derived from MIR162 corn according to
FSANZ guidelines1, to assess its safety for human consumption. The Assessment Report
was released in April 2008, and public comment was sought on the safety assessment and
proposed recommendations. Comments received were considered in completion of this
Approval Report.
1.
The Issue / Problem
The Applicant has developed corn line MIR162 that is protected from feeding damage
caused by certain lepidopteran insect pest larvae. Before food derived from insect-protected
corn line MIR162 can enter the Australian and New Zealand food supply, it must first be
assessed for safety and a variation to Standard 1.5.2 must be approved by the FSANZ
Board, and subsequently notified to the Australia and New Zealand Food Regulation
Ministerial Council (Ministerial Council). An amendment to the Code may only be gazetted
once the Ministerial Council process has been finalised.
Syngenta Seeds Pty Ltd has therefore applied to have Standard 1.5.2 amended to include
food derived from corn line MIR162.
2.
Current Standard
2.1
Background
Standard 1.5.2 prohibits a food produced using gene technology from being sold or used as
an ingredient or component of any food unless it is listed in the Table to clause 2 of that
Standard.
2.2
Overseas approvals
Insect-protected corn line MIR162 is intended for commercialisation in the United States and
Canada. Submissions for food, feed and environmental approvals have been made to the
appropriate US agencies (Environmental Protection Agency, Food and Drug Administration,
Department of Agriculture) and Canadian agencies (Health Canada and the Canadian Food
Inspection Agency). Regulatory submissions for food, feed and environmental approvals
have also been made in Colombia, Brazil and Argentina. Regulatory submissions for import
approvals for MIR162 corn have been made in key export markets, for example, Japan,
Taiwan, Korea, Philippines and Mexico.
FSANZ (2007). Safety Assessment of Genetically Modified Foods – Guidance Document.
http://www.foodstandards.gov.au/_srcfiles/GM%20FINAL%20Sept%2007L%20_2_.pdf
1
2
3.
Objectives
In developing or varying a food standard, FSANZ is required by its legislation to meet three
primary objectives which are set out in section 18 of the FSANZ Act. These are:

the protection of public health and safety;

the provision of adequate information relating to food to enable consumers to make
informed choices; and

the prevention of misleading or deceptive conduct.
In developing and varying standards, FSANZ must also have regard to:

the need for standards to be based on risk analysis using the best available scientific
evidence;

the promotion of consistency between domestic and international food standards;

the desirability of an efficient and internationally competitive food industry;

the promotion of fair trading in food; and

any written policy guidelines formulated by the Ministerial Council.
4.
Questions to be answered
Based on information provided by the Applicant on the nature of the genetic modification, the
molecular characterisation, the characterisation of the novel proteins, the compositional
analysis and any nutritional issues, is food derived from corn line MIR162 comparable to food
derived from conventional varieties of corn in terms of its safety for human consumption?
Is there other information available, including from the scientific literature, general technical
information, independent scientists, other regulatory agencies and international bodies, and
the general community, that needs to be considered?
Are there any other considerations that would influence the outcome of this assessment?
RISK ASSESSMENT
Food from insect-protected corn line MIR162 has been evaluated according to FSANZ
guidelines2. The summary and conclusions from the full safety assessment report (at
Attachment 2) are presented below. In addition to information supplied by the Applicant,
other available resource material including published scientific literature and general
technical information was used for the assessment.
FSANZ (2007). Safety Assessment of Genetically Modified Foods – Guidance Document.
http://www.foodstandards.gov.au/_srcfiles/GM%20FINAL%20Sept%2007L%20_2_.pdf
2
3
5.
Risk Assessment Summary
5.1
Safety Assessment Process
In conducting a safety assessment of food derived from insect-protected MIR162 corn, a
number of criteria have been addressed including: a characterisation of the transferred
genes, their origin, function and stability in the corn genome; the changes at the level of
DNA, protein and in the whole food; compositional analyses; evaluation of intended and
unintended changes; and the potential for the newly expressed proteins to be either
allergenic or toxic in humans.
The safety assessment applied to food from corn line MIR162 addresses only food safety
and nutritional issues. It therefore does not address: environmental risks related to the
environmental release of genetically modified (GM) plants used in food production; the safety
of animal feed or animals fed with feed derived from GM plants; or the safety of food derived
from the non-GM (conventional) plant.
5.2
Outcomes of the Safety Assessment
Detailed molecular analyses indicate that one copy of each of the vip3Aa20 and pmi genes
has been inserted at a single site in the plant genome and the genes are stably inherited
from one generation to the next. No antibiotic resistance marker genes are present in
MIR162 corn.
MIR162 expresses two novel proteins, Vip3Aa20 and PMI. The Vip3Aa20 protein is
expressed at moderate levels in the corn grain, with a mean concentration of 43.6 µg/g fresh
weight. The PMI protein is expressed at relatively low levels in the grain, with the mean
concentration of 1.9 μg/g fresh weight.
The Vip3Aa20 protein is 99.7% identical to the native Vip3Aa1 protein, differing by two amino
acids, at positions 129 and 284. The amino acid change at position 129 occurred during the
plant transformation process. Apart from the unexpected amino acid change at position 129,
both proteins conform in size and amino acid sequence to that expected, do not exhibit any
post-translational modification including glycosylation, and also demonstrate the predicted
insecticidal (Vip3A) or enzymatic (PMI) activity.
In relation to potential toxicity and allergenicity, B. thuringiensis has been extensively studied
and has a long history of safe use as the active ingredient in a number of insecticide products
for use in agriculture as well as home gardens.
In addition, bioinformatic studies with the Vip3Aa20 and PMI proteins have confirmed the
absence of any biologically significant amino acid sequence similarity to known protein toxins
or allergens and digestibility studies have demonstrated that both proteins would be rapidly
degraded in the stomach following ingestion, similar to other dietary proteins. Acute oral
toxicity studies in mice with both proteins have also confirmed the absence of toxicity. Taken
together, the evidence indicates that both proteins are unlikely to be toxic or allergenic in
humans.
Compositional analyses were done to establish the nutritional adequacy of MIR162 corn, and
to compare it to conventional corn under typical cultivation conditions. No differences of
biological significance were observed between MIR162 corn and its conventional
counterpart. Food from insect-protected MIR162 corn is therefore considered to be
compositionally equivalent to food from conventional corn varieties and its introduction into
the food supply would therefore be expected to have little nutritional impact.
4
5.3
Conclusions
No potential public health and safety concerns have been identified in the assessment of
insect-protected MIR162 corn. On the basis of the data provided in the present application,
and other available information, food derived from insect-protected MIR162 corn is
considered as safe and wholesome as food derived from conventional corn varieties.
RISK MANAGEMENT
6.
Options
There are no non-regulatory options for this Application. The two regulatory options available
for this Application are:
Option 1 – Continue to prohibit food from corn line MIR162
6.1
Reject the Application, thus maintaining the status quo by not amending Standard 1.5.2 to
approve food derived from insect-protected corn line MIR162.
Option 2 – Approve food from corn line MIR162
6.2
Amend Standard 1.5.2 to permit the sale and use of food derived from insect-protected corn
line MIR162, with or without specified conditions in the Table to clause 2 of the Standard.
7.
Impact Analysis
In the course of developing food regulatory measures suitable for adoption in Australia and
New Zealand, FSANZ is required to consider the impact of all options on all sectors of the
community, including consumers, the food industry and governments in both countries. The
regulatory impact assessment identifies and evaluates, though is not limited to, the costs and
benefits of the regulation, and its health, economic and social impacts.
7.1
Affected Parties
The affected parties may include the following:

Consumers of food products containing corn, particularly those concerned about
biotechnology.

Industry sectors:
-

food importers and distributors of wholesale ingredients;
processors and manufacturers of food products containing corn;
food retailers.
Government:
-
enforcement agencies;
national government, in terms of trade and World Trade Organization (WTO)
obligations.
5
The cultivation of corn line MIR162 in Australia or New Zealand could have an impact on the
environment, which would need to be assessed by the Office of the Gene Technology
Regulator (OGTR) in Australia, and by various New Zealand Government agencies including
the Environmental Risk Management Authority (ERMA) and the Ministry of Agriculture and
Forestry (MAF) before growing in either country could be permitted. MIR162 corn has been
developed primarily for agricultural production overseas and the Applicant has indicated that,
at this stage, there are no plans for cultivation in either Australia or New Zealand.
7.2
Benefit Cost Analysis
7.2.1
Option 1 – continue to prohibit food from corn line MIR162
Consumers:
Possible restriction in the availability of corn products if MIR162 corn is present
in imported foods.
No impact on consumers wishing to avoid GM foods, as food from MIR162
corn is not currently permitted in the food supply.
Government: Potential impact if considered inconsistent with WTO obligations but impact
would be in terms of trade policy rather than in government revenue.
Likely to be increased costs associated with monitoring required for
unapproved GM material derived from MIR162 corn which may inadvertently
be incorporated into imported food products destined for Australia and New
Zealand.
Industry:
Possible restriction on corn imports once MIR162 corn is commercialised
overseas.
Potential longer-term impact - any successful WTO challenge has the potential
to impact adversely on food industry.
7.2.2
Option 2 – approve food from corn line MIR162
Consumers:
Broader availability of imported corn products as there would be no restriction
on imported corn products derived from MIR162 corn.
Potentially a wider range of imported corn products at lower prices.
Appropriate labelling would allow consumers wishing to avoid GM corn to do
so.
Government: Benefit that if MIR162 corn was detected in corn imports, approval would
ensure compliance of those products with the Code. This would ensure no
potential for trade disruption on regulatory grounds.
Approval of MIR162 corn would ensure no conflict with WTO responsibilities.
This option could impact on monitoring resources, as certain foods derived
from MIR162 corn will be required to be labelled as genetically modified.
Industry:
Food manufacturers gain broader market access and increased choice in raw
materials.
6
Importers of processed foods containing corn as an ingredient would benefit as
foods derived from MIR162 corn would be compliant with the Code.
Retailers may be able to offer a broader range of corn products.
Possible cost to food industry as some food ingredients derived from MIR162
corn would be required to be labelled as genetically modified.
7.3
Comparison of Options
As food from insect-protected corn line MIR162 has been found to be as safe as food from
conventional varieties of corn, Option 1 is likely to be inconsistent with Australia and New
Zealand’s WTO obligations. Option 1 would also offer little benefit to consumers wishing to
avoid GM foods, as approval of MIR162 corn by other countries could limit supplementation
of the Australian and New Zealand market with imported corn products.
As MIR162 corn has been found to be safe for human consumption and the potential benefits
outweigh the potential costs, Option 2, an amendment to Standard 1.5.2 giving approval to
insect-protected corn line MIR162, is therefore the preferred option.
COMMUNICATION AND CONSULTATION STRATEGY
8.
Communication
FSANZ has applied a communication strategy to this Application that involves advertising the
availability of assessment reports for public comment in the national press and placing the
reports on the FSANZ website.
Public comment on the Assessment was sought prior to preparation of this Approval Report.
As normally applies to all GM food assessments, the Assessment and Approval Reports will
be available to the public on the FSANZ website. In addition, FSANZ will issue a media
release drawing journalists’ attention to the matter.
The Applicant and individuals and organisations that made submissions on this Application
will be notified at each stage of the Application. This Approval Report and the decision of the
FSANZ Board to approve the draft variation to Standard 1.5.2 will be notified to the
Ministerial Council. If the approval of food derived from insect-protected corn line MIR162 is
not subject to review, the Applicant and stakeholders, including the public, will be notified of
the gazettal of changes to Standard 1.5.2 in the national press and on the website. In
addition, FSANZ provides an information service to the jurisdictions on changes to the Code.
9.
Consultation
9.1
Public Consultation
The Assessment Report was advertised for public comment between 11 April and 23 May 2008.
Comments were specifically requested on the scientific aspects of this Application, in particular,
information relevant to the safety assessment of food from insect-protected corn MIR162. As
this Application is being assessed under a General Procedure, there was one round of public
comment.
A total of 24 submissions was received. A summary of these is provided in Attachment 3 to
this Report. FSANZ has taken the submitters’ comments relevant to food safety into account
in preparing the Approval Report for this Application.
7
Responses to general issues, such as GM food labelling and the use of company generated
data in safety assessments, are available from the FSANZ website3. Specific issues relating
to food derived from MIR162 corn have been addressed in the report. The main issues are
discussed below.
9.1.1
MIR162 corn has not been approved anywhere else in the world
A number of private submissions raised concerns that MIR162 corn has not been approved
anywhere in the world, and Queensland Health requested information on the progress of
international regulatory approvals of MIR162 corn, whether additional information had been
sought from the Applicant, and, if any approval has been declined, the reasons.
9.1.1.1 Response
As MIR162 corn is intended for cultivation in North America, it will not be grown
commercially, nor be present in the food supply prior to receiving appropriate regulatory
approvals in the United States and Canada.
Submissions for regulatory approval of new foods and food ingredients are often made to a
number of countries at similar times, such that the assessment processes are undertaken in
parallel. Submissions have been made to the appropriate agencies in a number of countries
for cultivation and/or feed/food import of MIR162 corn. The Applicant provided the following
update on the status of submissions for regulatory approvals in other countries.
Country
3
GLOBAL REGULATORY TIMELINES FOR MIR162
Submission Type
Status
US EPA
EUP
Submitted May 2007
Anticipated
Approval
Q4 2008
US EPA
US FDA
USDA
Canada
Canada
Canada
Mexico
Colombia
Brazil
Argentina
Japan
Japan
Japan
Korea
Korea
Taiwan
Aus/New Zealand
Philippines
China
EU
Switzerland
Section 3 registration
Food safety
Environmental release
Food safety
Feed safety
Environmental release
Food & feed safety
Food, feed & cultivation
Food, feed & cultivation
Food, feed & cultivation
Food safety
Feed safety
Cultivation
Food safety
Environmental
Food safety
Food import
Food, feed & processing
Food, feed & processing
Food, feed & import
Import
Submitted May 2007
Submitted Aug 2007
Submitted Aug 2007
Submitted Dec 2007
Submitted Dec 2007
Submitted Dec 2007
Submitted Dec 2007
Submitted Dec 2007
Submitted Dec 2007
Submitted Dec 2007
Submitted Oct 2007
Submitted Nov 2007
Submit Mar 2008
Submitted Nov 2007
Submit Apr 2008
Submitted Nov 2007
Submitted Oct 2007
Submitted Nov 2007
Plan to submit Q1 2009
Plan to submit Sep 2008
Plan to submit Nov 2008
Q4 2008
Q4 2008
Q1 2009
Q1 2009
Q1 2009
Q1 2009
Q4 2008
Q2 2009
Q2 2009
Q3 2011
Q2 2009
Q2 2009
Q2 2009
Q1 2009
Q2 2009
Q2 2009
Q4 2008
Q3 2009
Q3 2012
Q3 2011
Q3 2011
http://www.foodstandards.gov.au/foodmatters/gmfoods/frequentlyaskedquest3862.cfm
8
Country
Russia
Russia
South Africa
India
GLOBAL REGULATORY TIMELINES FOR MIR162
Submission Type
Status
Food safety
Feed safety
Cultivation
TBD
Plan to submit July 2008
Plan to submit Q2 2010
Plan to submit July 2008
TBD
Anticipated
Approval
Q2 2010
Q2 2011
Q4 2009
The Applicant has also advised that no requests for additional studies have been made. The
Applicant received requests for information and/or clarification from Health Canada regarding
details of the compositional analysis and from the US FDA also primarily regarding the
compositional analyses. These questions were addressed by the Applicant.
The Applicant has advised that all applications are currently progressing with the various
agencies and there are no outstanding questions. No approvals have been declined.
9.1.2
Enforcement costs
The NSW Food Authority and Queensland Health have reiterated comments that there are
extensive costs incurred in monitoring for the presence of GM food, as detection of GM foods
is more complex and expensive than other food regulatory measures. Queensland
expressed concern that the Benefit Cost Analysis is qualitative in nature, and stated it was
particularly interested in the enforcement costs determined by FSANZ. Queensland also
believes there is a need to consider a national enforcement strategy surrounding GM food
approvals. The NSW Food Authority has indicated its intent to host an Implementation SubCommittee (ISC)4 workshop to consider the cost impacts of monitoring GM food.
9.1.2.1 Response
FSANZ is aware of the concerns raised by jurisdictions in relation to monitoring and
enforcement costs associated with GM foods and agrees that, over time, the successive
approval of new GM foods may significantly impact on monitoring resources. This would also
be the case with the ever-increasing number of new food additives, processing aids and
novel technologies approved in the Code. This issue is therefore not unique to GM foods,
and nor is it specific to any particular GM application.
Following the receipt of submissions, FSANZ contacted a number of jurisdictions to try and
obtain quantitative information regarding the associated costs in order to try and better reflect
this in the benefit cost analysis. The jurisdictions that were contacted indicated they
undertake a range of testing and monitoring activities in relation to GM foods, as well as
other types of foods. Testing for GM foods is significantly more expensive than the cost of
other sorts of testing.
In 2003, a review of labelling of GM foods was undertaken by FSANZ which included an
assessment of compliance and enforcement activities and information from GM food surveys
including a previous ISC Coordinated Survey. The ISC Coordinated Survey included a
business record audit and product testing. A total of 51 samples were tested under the
survey, costing a total of $AUD33,660 or $AUD660 per sample. FSANZ understands that
the current cost of testing (per sample) remains approximately the same as it was in 2003.
4
ISC comprises heads of the appropriate Australian (Commonwealth and State/Territory) and New
Zealand inspection and enforcement agencies and is responsible, among other things, for overseeing
the development and implementation of a consistent approach across jurisdictions to enforcing food
regulation and standards.
9
At this stage however it is not possible to ascertain the total cost of monitoring activities for
GM foods, and obviously this will vary between jurisdictions, depending on the amount of
monitoring that is being undertaken. In the absence of detailed quantitative information, it is
therefore only possible for the benefit cost analysis to be qualitative in nature.
FSANZ believes it is important to recognize that, because GM foods are continually entering
international trade, the costs of monitoring are largely unavoidable and will arise irrespective
of whether or not GM foods are approved in Australia and New Zealand. In the case of
approved GM foods, monitoring is required to ensure compliance with the labelling
requirements, and in the case of GM foods that have not been approved, monitoring is
required to ensure they are not illegally entering the food supply. The costs of monitoring are
thus expected to be comparable, whether a GM food is approved or not. Any regulatory
decision take by FSANZ is therefore unlikely to significantly affect the cost impact on
jurisdictions, in terms of their responsibilities to enforce the Code.
The increased monitoring and enforcement burden being placed on jurisdictions was
discussed by ISC at its meeting in April 2008, where it was agreed that a national compliance
and monitoring strategy for GM foods is required to assist consistent implementation of
Standard 1.5.2. It was also agreed to hold a workshop to develop a draft strategy for
consideration by ISC. FSANZ welcomes the planned discussion of these issues by ISC and
will work with the jurisdictions in developing a national compliance and monitoring strategy
for GM food.
9.1.3
Lack of long-term studies and reliance on 44-day broiler study
A number of submissions questioned the lack of long term studies supporting the safety of
food derived from MIR162 corn, and indicated that a feeding trial with 180 broiler chickens for
44 consecutive days is not adequate to prove its safety as food.
9.1.3.1 Response
FSANZ’s safety assessment of food derived from insect-protected corn line MIR162, included
consideration of (i) the genetic modification to the plant; (ii) the potential toxicity and
allergenicity of the novel proteins; and (iii) the composition of MIR162 corn compared with
that of conventional corn varieties. No public health and safety concerns were identified as a
result of the safety assessment. The safety assessment did not rely on the results of the
feeding study in rapidly-growing broiler chicks.
FSANZ considers that a scientifically-informed comparative assessment of GM foods with
their conventional counterparts can generally identify any potential adverse health effects or
differences requiring further evaluation. In the majority of circumstances, animal toxicity
studies with whole foods are not likely to contribute any further useful information to the
safety assessment and are therefore not warranted. As a consequence, FSANZ does not
require that animal feeding studies with whole GM foods be undertaken on a routine basis.
FSANZ acknowledges there may be future GM applications, particularly for foods with
intentional modifications to composition, where the results of animal toxicity studies may be
informative. FSANZ therefore continues to assess the need for animal studies on a case-bycase basis, taking into account the nature of the genetic modification and the results of the
comparative assessment. While FSANZ does not routinely require animal toxicity studies to
be undertaken, where such studies already exist, Applicants are expected to provide these to
FSANZ to evaluate as additional supporting information.
10
As part of a continual review of FSANZ’s scientific approach to the safety assessment of GM
foods, FSANZ convened an expert panel in June 2007 to develop guidance and
recommendations on the role animal feeding studies can play in the safety assessment of
GM foods. The expert panel recommended that FSANZ should continue with its case-bycase assessment of GM foods on the basis of best available science. The panel noted that
whole-food animal feeding studies may be informative in some limited circumstances, but
concurred with FSANZ that any potential adverse health effects can generally be identified by
a scientifically informed comparative assessment of the GM food against its conventional
counterpart. The report from the expert panel is available from
www.foodstandards.gov.au/_srcfiles/Workshop%20Report%20FINAL.pdf
In the case of MIR162 corn, the Applicant submitted a feeding study comparing the nutritional
performance of MIR162 corn with a non-transgenic near-isogenic control corn and a
commercially available conventional corn. This study was evaluated by FSANZ as additional
supporting information of the nutritional equivalence of MIR162 corn to conventional corn
varieties, but was not considered essential to the safety assessment of food derived from
MIR162 corn. The detailed compositional studies are considered adequate to establish the
nutritional adequacy of food derived from insect-protected MIR162 corn. However,
evaluation of the feeding study in rapidly-growing broiler chicks confirmed that MIR162 corn
is equivalent to its conventional counterpart and commercial corn in its ability to support
typical growth and well-being.
9.1.4
Lack of consideration of the safety of animal feed or animals fed with feed derived
from GM plants
Queensland Health considers there is an obligation to address the safety of animal feed or
animals fed with feed derived from GM plants. A private submitter also expressed concern
FSANZ has not considered the safety of animals fed with GM plants.
9.1.4.1 Response
Many animal feeds are derived from the same GM food crops that are used for human
consumption, and the majority of grain and forage derived from corn is used in animal feed.
Concerns are often expressed that this practice may pose an indirect risk to humans, through
consumption of the meat, milk and eggs derived from such animals. Typically, such GM
crops have already been assessed by FSANZ for their safety as human food. It seems
highly unlikely that a GM crop, found as safe for human food, would pose an indirect risk to
humans via food products derived from animals (e.g. meat, milk, eggs) that had eaten the
GM crop, when no direct risk to humans had been identified in the human food safety
assessment.
Assessment of the safety of GM animal feed is beyond the jurisdiction of FSANZ.
Furthermore, the definition for ‘food produced using gene technology’ in Standard 1.5.2 does
not include food derived from animals that have been fed GM feed. That is, food derived
from animals that have been fed GM feed are not themselves classed as a food produced
using gene technology, or GM animals, and therefore are not subject to pre-market approval
under Standard 1.5.2.
For GM crops intended for cultivation in Australia, the assessment conducted by the OGTR
includes an assessment of the safety of the crop for use as stock feed.
While FSANZ does not have the legislative mandate to assess the safety of food derived
from animals fed GM feed, the scientific evidence published so far indicates that feeding GM
plant material to livestock does not affect the nutritional value or safety of the meat, milk and
eggs derived from those animals.
11
This issue is discussed in the OECD document entitled Considerations for the safety
assessment of animal feedstuffs derived from genetically modified plants5.
9.1.5
There may be unintended effects of the two amino acid changes in the Vip3Aa20
protein
Three submissions raised concerns with the amino acid differences between the Vip3Aa1
protein present in B. thuringiensis and the Vip3Aa20 protein in MIR162, and question the
effect of these changes on toxicity, allergenicity and nutritional value, and whether other
changes could have gone unnoticed.
9.1.5.1 Response
The safety assessment of food derived from insect-protected corn line MIR162, included
consideration of the potential toxicity and allergenicity of the Vip3Aa20 protein, and took into
account the two amino acid differences between Vip3Aa20 and the native Vip3Aa1 protein.
The Vip3Aa20 protein in corn line MIR162 is nearly identical in amino acid sequence to that
encoded by the native vip3Aa1 gene from B. thuringiensis, with the exception of two amino
acids: lysine-284 has been substituted by glutamine, and methionine-129 has been substituted
by isoleucine. The vip3Aa1 gene was modified to the synthetic vip3Aa19 gene to
accommodate the preferred codon usage in corn, resulting in Vip3Aa19 having a single amino
acid change from the native Vip3Aa1 protein at position 284. A transformation induced change
in the vip3Aa19 coding sequence that was incorporated in the MIR162 corn genome resulted
in a second amino acid difference between Vip3Aa1 and Vip3Aa20, at position 129.
Studies meeting FSANZ data requirements were done to confirm the identity and
physicochemical and functional properties of the expressed Vip3Aa20 protein, as well as to
determine its potential toxicity and allergenicity. Apart from the unexpected amino acid
change at position 129, the Vip3Aa20 protein conforms in size and amino acid sequence to
that expected, does not exhibit any post-translational modification including glycosylation,
and also demonstrates the predicted insecticidal activity. Bioinformatic studies with the
Vip3Aa20 protein confirmed the absence of any biologically significant amino acid sequence
similarity to known protein toxins or to known or suspected allergens and digestibility studies
have demonstrated that the Vip3Aa20 protein would be rapidly degraded in the stomach
following ingestion, similar to other dietary proteins. Acute oral toxicity studies in mice also
confirmed the absence of toxicity. The safety of the Vip3Aa protein is further supported by
the long history of safe use of Bt microbial pesticides. Taken together, the evidence indicates
that the Vip3Aa20 protein is unlikely to be toxic or allergenic in humans.
The Vip3Aa20 protein used in some of the studies was prepared by expressing a synthetic
vip3Aa20 gene in an E. coli over-expression system predicted to be identical in amino acid
sequence to Vip3Aa20 expressed in MIR162 corn. A large number of studies have been
done to confirm the equivalence of the MIR162 expressed Vip3Aa20 protein to the E. coliproduced Vip3Aa20 protein (see Attachment 2). The studies support the conclusion that
Vip3Aa20 is expressed as expected in MIR162 corn, and further, that the Vip3Aa20
produced by recombinant E. coli is a suitable surrogate for the plant produced protein.
9.1.6
Food allergies have been reported to have increased following the introduction of
GM crops
Two submissions cited reports of an increase in the prevalence of food allergies following the
introduction of GM foods.
5
http://www.olis.oecd.org/olis/2003doc.nsf/LinkTo/NT0000426A/$FILE/JT00147696.PDF
12
9.1.6.1 Response
FSANZ recognises that food allergies are a serious public health issue. Most people with
food allergy are allergic to one or more of the major allergens (peanuts, tree nuts (e.g.
cashews, almonds, walnuts), shellfish, finned fish, milk, eggs, sesame and soybeans). In
these foods, the major allergenic proteins have been identified. These foods may cause
severe allergic reactions and, under Standard 1.2.3, must be declared on the food label
however small the amount.
While there is some evidence that food allergy is increasing in the population, this is an
increase in the number of people who are allergic to the common allergenic foods, rather
than to new allergens. The reason for this apparent increase in not known.
The potential allergenicity of any new proteins expressed in a GM food is thoroughly
evaluated according the FSANZ Guidance Document: Safety Assessment of Genetically
Modified Foods6. The FSANZ assessment follows internationally recognised best-practise
methods for assessing potential allergenicity, as elaborated in the Codex Guideline for the
Conduct of Food Safety Assessment of Foods Derived from Recombinant DNA Plants. If a
GM food were found to pose a new allergenic risk, that food would not be approved.
In the case of MIR162 corn, the evidence indicates that neither the Vip3Aa20 nor PMI
proteins are likely to be allergenic to humans (see Attachment 2).
9.2
World Trade Organization (WTO)
As members of the WTO, Australia and New Zealand are obligated to notify WTO member
nations where proposed mandatory regulatory measures are inconsistent with any existing or
imminent international standards and the proposed measure may have a significant effect on
trade.
The draft variation to the Code would have a trade-enabling effect as it would permit food
derived from MIR162 corn to be imported into Australia and New Zealand and sold, where
currently it is prohibited. For this reason it was determined there was no need to notify this
Application as a Sanitary and Phytosanitary (SPS) measure in accordance with the WTO
Agreement on the Application of SPS Measures.
CONCLUSION
10.
Conclusion and Decision
Decision
Approve the variation to Standard 1.5.2 – Food produced using Gene Technology, to
include food derived from insect-protected corn line MIR162 in the Table to clause 2.
10.1
Reasons for Decision
A variation to Standard 1.5.2 to permit the sale and use of food derived from insect-protected
corn line MIR162 in Australia and New Zealand is approved on the basis of the available
scientific evidence, for the following reasons:
6
http://www.foodstandards.gov.au/_srcfiles/GM%20FINAL%20Sept%2007L%20_2_.pdf
13

the safety assessment did not identify any public health and safety concerns
associated with the genetic modification used to produce insect-protected corn line
MIR162;

food derived from insect-protected corn line MIR162 is equivalent to food from the
conventional counterpart and other commercially available corn varieties in terms of its
safety for human consumption and nutritional adequacy;

labelling of certain foods derived from insect-protected corn line MIR162 will be
required if novel DNA and/or protein is present in the final food;

a regulation impact assessment process has been undertaken that also fulfils the
requirement in New Zealand for an assessment of compliance costs. The assessment
concluded that the preferred option is Option 2, a variation to Standard 1.5.2;

there are no relevant New Zealand standards; and

there are no other measures that would be more cost-effective than a variation to
Standard 1.5.2 that could achieve the same end.
11.
Implementation and Review
The FSANZ Board’s decision will be notified to the Ministerial Council. Following notification,
the proposed draft variation to Standard 1.5.2 is expected to come into effect on gazettal,
subject to any request from the Ministerial Council for a review of FSANZ’s decision.
ATTACHMENTS
1.
2.
3.
Draft variation to the Australia New Zealand Food Standards Code
Safety assessment report
Summary of issues raised in public submissions
14
Attachment 1
Draft variation to the Australia New Zealand Food Standards Code
Subsection 87(8) of the FSANZ Act provides that standards or variations to standards are
legislative instruments, but are not subject to disallowance or sunsetting
To commence: on gazettal
[1]
Standard 1.5.2 of the Australia New Zealand Food Standards Code is varied by
inserting in Column 1 of the Table to clause 2 –
Food derived from insect-protected corn line
MIR162
15
Attachment 2
Safety Assessment Report
APPLICATION A1001 – FOOD DERIVED FROM INSECT-PROTECTED
CORN LINE MIR162
SUMMARY AND CONCLUSIONS
Background
Insect-protected MIR162 corn has been genetically modified (GM) for protection against
feeding damage caused by larvae of a number of insect species. Protection is conferred by
expression in the plant of a modified vip3Aa gene, encoding an insecticidal Vip protein,
derived from Bacillus thuringiensis, a common soil bacterium. The Vip proteins exert their
effect on the insect by forming pores in the gut membranes of sensitive species leading to
disruption of the midgut transmembrane potential and eventual insect death. MIR162 corn
also expresses the pmi gene, encoding the enzyme phosphomannose isomerase (PMI),
which serves as a selectable marker, enabling plants to grow on mannose.
MIR162 corn has been developed for commercial cultivation in North America and may
therefore enter the Australian and New Zealand food supply as imported, largely processed
food products.
In conducting a safety assessment of food derived from insect-protected MIR162 corn, a
number of criteria have been addressed including: a characterisation of the transferred
genes, their origin, function and stability in the corn genome; the changes at the level of
DNA, protein and in the whole food; compositional analyses; evaluation of intended and
unintended changes; and the potential for the newly expressed proteins to be either
allergenic or toxic in humans.
This safety assessment report addresses only food safety and nutritional issues. It therefore
does not address: environmental risks related to the environmental release of GM plants
used in food production; the safety of animal feed or animals fed with feed derived from GM
plants; or the safety of food derived from the non-GM (conventional) plant.
History of Use
Corn is the world’s third leading cereal crop, behind wheat and rice, and is grown in over 25
countries. Corn-derived products are routinely used in a large number and diverse range of
foods and have a long history of safe use. Products derived from MIR162 corn may include
flour, breakfast cereals, high fructose corn syrup and other starch products.
B. thuringiensis, the microorganism from which the vip3A gene was derived, has been
extensively studied and commercially exploited for over 40 years as the active ingredient in a
number of insecticide products used in agriculture as well as home gardens. B. thuringiensis
therefore has had a long history of safe use and the Vip protein it produces is not known to
be toxic to any vertebrates, including humans and other mammals.
Molecular Characterisation
Corn line MIR162 contains two novel genes. The first, vip3Aa20, derived from
B. thuringiensis, encodes the insecticidal protein Vip3Aa20. The vip3Aa20 gene is a
modified version of the native vip3Aa1 gene found in the B. thuringiensis strain AB88.
16
The second gene present in MIR162 corn, pmi, is present as a selectable marker and
encodes the enzyme phosphomannose isomerase derived from Escherichia coli.
Detailed molecular analyses indicate that one copy of each of the vip3Aa20 and pmi genes
has been inserted at a single site in the plant genome and the genes are stably inherited
from one generation to the next. No antibiotic resistance marker genes are present in
MIR162 corn.
Characterisation of Novel Protein
MIR162 expresses two novel proteins, Vip3Aa20 and PMI. The Vip3Aa20 protein is
approximately 89 kDa and 789 amino acids in length. The Vip3Aa20 protein is 99.7%
identical to the native Vip3Aa1 protein and differs by only 2 amino acids, at positions 129 and
284. The amino acid change at position 129 occurred during the plant transformation
process. The phosphomannose isomerase (PMI) protein is approximately 42.8 kDa and 391
amino acids and catalyses the reversible interconversion of mannose 6-phosphate and
fructose 6-phosphate. The Vip3Aa20 protein is expressed at moderate levels in the corn
grain, with a mean concentration of 43.6 µg/g fresh weight. The PMI protein is expressed at
relatively low levels in the grain, with the mean concentration of 1.9 μg/g fresh weight.
A large number of studies have been done to confirm the identity and physicochemical and
functional properties of the expressed Vip3Aa20 and PMI proteins, as well as to determine
their potential toxicity and allergenicity. Apart from the unexpected amino acid change at
position 129, both proteins conform in size and amino acid sequence to that expected, do not
exhibit any post-translational modification including glycosylation, and also demonstrate the
predicted insecticidal (Vip3A) or enzymatic (PMI) activity.
Bioinformatic studies with the Vip3Aa20 and PMI proteins have confirmed the absence of any
biologically significant amino acid sequence similarity to known protein toxins or allergens
and digestibility studies have demonstrated that both proteins would be rapidly degraded in
the stomach following ingestion, similar to other dietary proteins. Acute oral toxicity studies
in mice with both proteins have also confirmed the absence of toxicity. The safety of the
Vip3Aa protein is further supported by the long history of safe use of Bt microbial pesticides.
Taken together, the evidence indicates that both proteins are unlikely to be toxic or allergenic
in humans.
Compositional Analyses
Compositional analyses were done to establish the nutritional adequacy of MIR162 corn, and
to compare it to a non-transgenic conventional corn under typical cultivation conditions. The
components analysed were protein, fat, carbohydrate, amino acids, fatty acids, vitamins,
minerals, the antinutrients phytic acid and raffinose, and the secondary metabolites furfural,
ferulic acid, p-coumaric acid.
No differences of biological significance were observed between MIR162 corn and its
conventional counterpart. Some minor differences in key nutrients were noted, however the
levels observed were within the range of values measured for the comparator corn hybrid
and other conventional corn varieties, and therefore they most likely reflect normal biological
variability. Food from insect-protected MIR162 corn is therefore considered to be
compositionally equivalent to food from conventional corn varieties.
Nutritional Impact
The detailed compositional studies are considered adequate to establish the nutritional
adequacy of food derived from insect-protected MIR162 corn.
17
The introduction of MIR162 corn into the food supply would therefore be expected to have
little nutritional impact. The nutritional adequacy of food derived from MIR162 corn was also
confirmed using a feeding study in rapidly-growing broiler chicks, which demonstrated that
MIR162 corn is equivalent to its conventional counterpart and commercial corn in its ability to
support typical growth and well being.
Conclusion
No potential public health and safety concerns have been identified in the assessment of
insect-protected MIR162 corn. On the basis of the data provided in the present application,
and other available information, food derived from insect-protected MIR162 corn is
considered as safe and wholesome as food derived from conventional corn varieties.
1.
INTRODUCTION
Syngenta Seeds Pty Ltd has submitted an application to Food Standards Australia New
Zealand (FSANZ) to vary Standard 1.5.2 – Food produced using Gene Technology – in the
Australia New Zealand Food Standards Code (the Code) to include food from a new
genetically modified (GM) corn variety. The GM corn variety is known as corn line MIR162.
Corn line MIR162 has been genetically modified to be resistant to a number of lepidopteran
pests of corn, including fall armyworm (Spodoptera frugiperda), corn earworm/cotton
bollworm (Helicoverpa zea), black cutworm (Agropis ipsilon) and western bean cutworm
(Striacosta albicosta). Protection is conferred by the expression in the plant of the
bacterially-derived vip3Aa20 gene, which produces the insecticidal protein Vip3Aa20, a
variant of the native insecticidal Vip3Aa1 protein. A selectable marker gene, pmi, encodes
phosphomannose isomerase and allows transformed cells to utilise carbon from
phosphomannose media.
Unlike Syngenta’s Bt11 corn varieties, MIR162 has no insecticidal activity against European
corn borer (Ostrinia nubilalis). The insect protection of MIR162 will be combined with Bt11 by
conventional breeding. In regions where corn rootworm infestations are problematic for
growers, these two traits will also be combined with Syngenta’s trait, MIR604, which has
been genetically modified to be resistant to Western corn rootworm (Diabrotica vigifera
vigifera), Northern corn rootworm (Diabrotica berberi), and Mexican corn rootworm
(Diabrotica vigifera zeae). Both the Bt11 and MIR604 traits have previously been assessed
by FSANZ and food derived from these lines approved for human consumption.
Bt-based formulations are widely used as biopesticides on a variety of cereal and vegetable
crops grown organically or under conventional agricultural conditions. Several registered
Bt-based microbial pest control products contain Vip3Aa or Vip3Aa-like proteins and it is
likely that small quantities of these proteins are present in the food supply.
Corn is the world’s third leading cereal crop, behind wheat and rice, and is grown in over 25
countries (OECD, 2002). In 2006, worldwide production of corn was nearly 700 million
tonnes, with the United States and China being the major producers (FAOSTAT, 2008). The
majority of grain and forage derived from corn is used in animal feed. Corn grain is also used
in industrial products, such as ethyl alcohol by fermentation and highly refined starch by wet
milling.
Corn is not a major crop in Australia or New Zealand. Domestic production of corn in
Australia and New Zealand is supplemented by the import of a small amount of corn-based
products, largely as high-fructose corn syrup, which is not currently manufactured in either
Australia or New Zealand. Such products are processed into breakfast cereals, baking
products, extruded confectionery and food coatings.
18
Other corn products such as cornstarch are also imported and used by the food industry for
the manufacture of dessert mixes and sauces. Corn may also be imported in finished
products such as corn chips and canned corn, or dry milled goods such as cornflour.
Corn line MIR162 will be grown in North America and is not intended for cultivation in
Australia or New Zealand. Therefore, if approved, food from this line may enter the Australian
and New Zealand food supply as imported food products.
2.
HISTORY OF USE
2.1
Donor Organisms
Bacillus thuringiensis
The source of the vip3Aa gene used in this GM corn is the ubiquitous soil and plant
bacterium Bacillus thuringiensis (Bt) strain AB88. The vip3Aa20 gene in MIR162 corn is a
synthetic version of the native bacterial gene.
The effect of B. thuringiensis products on human health and the environment was the subject
of a critical review by the WHO International Programme on Chemical Safety (WHO, 1999).
The review concluded that ‘B. thuringiensis products are unlikely to pose any hazard to
humans or other vertebrates or the great majority of non-target invertebrates provided that
they are free from non-B. thuringiensis microorganisms and biologically active products other
than the insecticidal proteins’.
Bt proteins are used widely as an insecticide in both conventional and organic agriculture. In
Australia, various Bt insecticidal products are registered with the Australian Pesticides and
Veterinary Medicines Authority (APVMA) for use on cotton, vegetables, fruits, vines, oilseeds,
cereal grains, herbs, tobacco, ornamentals, forestry and turf. Numerous registered
formulations contain Vip3Aa or Vip3Aa-like proteins. The very wide use of formulations
containing the Bt insecticidal proteins indicates that people eating and handling fresh foods
are commonly in contact with this protein.
Insecticidal products using Bt were first commercialised in France in the late 1930s (Nester et
al., 2002) and were first registered for use in the United States by the Environment Protection
Agency (EPA) in 1961 (EPA, 1998).
The EPA thus has a vast historical toxicological database for B. thuringiensis, which indicates
that no adverse health effects have been demonstrated in mammals in any infectivity/
pathogenicity/ toxicity study (McClintock et al., 1995; EPA, 1998; Betz et al., 2000). This
confirms the long history of safe use of Bt formulations in general, and the safety of
B. thuringiensis as a donor organism.
Escherichia coli
The bacterium Escherichia coli is the source of the pmi gene in MIR162. E. coli belongs to
the Enterobacteriaceae, a relatively homogeneous group of rod-shaped, Gram-negative,
facultative aerobic bacteria. Members of the genus Escherichia are ubiquitous in the
environment and are normally found in the digestive tracts of vertebrates, including humans.
The vast majority of E. coli strains are harmless to humans, although some strains can cause
diarrhoea in travellers and E. coli is also the most common cause of urinary tract infections.
More recently, a particularly virulent strain of E. coli, belonging to the enterohaemorrhagic E.
coli group, known as 0157:H7, has come to prominence as a food-borne pathogen
responsible for causing serious illness.
19
This particular group of pathogenic E. coli are distinct from the strains of E. coli (the K-12
strains) that are used routinely in laboratory manipulations. The K-12 strains of E. coli have
a long history of safe use and are commonly used as protein production systems in many
commercial, including pharmaceutical and food ingredient, applications (Bogosian and Kane,
1991).
Agrobacterium tumefaciens
The species Agrobacterium tumefaciens is a Gram-negative, non-spore forming, rod-shaped
bacterium commonly found in the soil. It is closely related to other soil bacteria involved in
nitrogen fixation by certain plants.
Agrobacterium naturally contains a plasmid (the Ti plasmid) with the ability to enter plant cells
and insert a portion of its genome into plant chromosomes. Normally therefore,
Agrobacterium is a plant pathogen causing root deformation mainly with sugar beets, pome
fruit and viniculture crops. However, adaptation of this natural process has now resulted in
the ability to transform a broad range of plant species without causing adverse effects in the
host plant.
2.2
Host Organism
Corn (Zea mays L), otherwise known as maize, is the world’s third leading cereal crop,
behind wheat and rice, and is grown in over 25 countries worldwide (OECD, 2002). In 2006,
worldwide production of corn was nearly 700 million tonnes, with the United States and China
being the major producers (FAOSTAT, 2008).
The majority of grain and forage derived from corn is used as animal feed, however corn also
has a long history of safe use as food for human consumption. The grain can be processed
into industrial products such as ethyl alcohol (by fermentation), and highly refined starch (by
wet-milling) to produce starch and sweetener products. In addition to milling, the corn germ
can be processed to obtain corn oil and numerous other products (White and Pollak, 1995).
Corn plants usually reproduce sexually by wind-pollination. This provides for natural outcrossing between plants, but it also presents an opportunity for plant breeders to produce
hybrid seed by controlling the pollination process. Open pollination of hybrids in the field
leads to the production of grain with properties derived from different lines and, if planted,
would produce lower yields (CFIA, 1994). The commercial production of corn now utilises
controlled cross-pollination of two inbred lines (using conventional techniques) to combine
desired genetic traits and produce hybrid varieties known to be superior to open-pollinated
varieties in terms of their agronomic characteristics. This inbred-hybrid concept and resulting
yield response is the basis of the modern corn seed industry and hybrid corn varieties are
used in most developed countries for consistency of performance and production.
3.
MOLECULAR CHARACTERISATION
Molecular characterisation is necessary to provide an understanding of the genetic material
introduced into the host genome and helps to frame the subsequent parts of the safety
assessment. The molecular characterisation addresses three main aspects: the
transformation method together with a detailed description of the DNA sequences introduced
to the host genome; a characterisation of the inserted DNA including any rearrangements
that may have occurred as a consequence of the transformation; and the genetic stability of
the inserted DNA and any accompanying expressed traits.
20
Study submitted:
Long, N. (2007) Molecular characterization of the transgenic DNA in event MIR162 Maize. Syngenta
Seeds Biotechnology Report No. SSB-119-07.
3.1
Method used in the genetic modification
Corn line MIR162 was produced by Agrobacterium-mediated transformation of Zea mays line
NP2500 x NP2499. The Agrobacterium-mediated DNA transformation system is the basis of
natural plasmid-induced crown-gall formation in many plants and is well understood
(Zambryski, 1992). The genes of interest were inserted into the plasmid between DNA
sequences known as the Left and Right Borders (LB and RB). These border sequences were
isolated from the Ti plasmid of Agrobacterium and normally delimit the DNA sequence
(T-DNA) transferred into the plant.
Immature embryos were co-cultivated with A. tumefaciens strain LB4404 carrying the
transformation plasmid pNOV1300. Between the right and left borders, this plasmid contains
the vip3Aa and pmi genes and regulatory elements as shown in Figure 1 and Table 1. The
vip3Aa19 gene in vector pNOV1300 was modified from the native vip3Aa1 gene in
B. thuringiensis strain AB88 to accommodate the preferred codon usage in corn. The
Vip3Aa19 protein differs from the original Vip3Aa1 protein by a single amino acid: lysine at
position 284 is changed to glutamine.
Embryos producing embryogenic callus were transferred to cell culture medium containing
mannose, with the pmi gene used as a selectable marker. Regenerated plantlets were tested
for the presence of both the pmi and vip3Aa genes, and absence of the spectinomycin (spec)
antibiotic resistance gene by PCR analysis. Plants meeting these criteria were transferred to
the greenhouse for propagation. These steps are summarised in Figure 2.
Table 1: Description of genetic elements in transformation vector pNOV1300
Genetic
Element
Location in
pNOV1300
(bp)
Size
(bp)
Function
Active Ingredient Cassette
ZmUbiInt
200 - 2192
1993
vip3Aa19
2214 - 4583
2370
iPEPC9
4600 - 4707
108
Promoter region from Z. mays polyubiquitin gene which
contains the first intron (EntrezTM Accession Number S94464;
(NCBI, 2006). Provides constitutive expression in monocots.
A modified version of the native vip3Aa1 gene found in the
Bacillus thuringiensis strain AB88. The vip3Aa19 gene in
vector pNOV1300 was modified to accommodate the
preferred codon usage in corn. The vip3A19 gene (EntrezTM
Accession Number DQ539887; (NCBI 2006) encodes a
Vip3Aa19 protein that differs from the Vip3Aa1 protein
encoded by the vip3Aa1 gene by a single amino acid at
position 284. The vip3Aa1 gene encodes lysine at position
284 and the vip3Aa19 gene encodes glutamine. A
transformation induced change in the vip3Aa19 coding
sequence resulted in two single nucleotide differences for the
insert gene, designated vip3Aa20 (Entrez Accession number
DQ539888;(NCBI 2006). The Vip3Aa19 protein differs by a
single amino acid from the Vip3Aa20 protein. Vip3Aa proteins
confer resistance to several lepidopteran insect pests.
Intron #9 from the phosphoenolpyruvate carboxylase gene
(Entrez Accession Number X15239; (NCBI 2006) from Z.
mays.
21
Genetic
Element
35S
Terminator
Location in
pNOV1300
(bp)
4710 - 4779
Size
(bp)
Function
70
Terminator sequence from the 35S RNA from the cauliflower
mosaic virus genome (Similar to EntrezTM Accession Number
AF140604; (NCBI 2006). Its function is to provide a
polyadenylation sequence.
Selectable Marker Cassette
ZmUbilInt
4798 - 6790
199
3
pmi
6803 - 7978
117
6
NOS
Terminator
8039 - 8291
253
Promoter region from Z. mays polyubiquitin gene which
contains the first intron (EntrezTM Accession Number S94464;
(NCBI 2006). Provides constitutive expression in monocots.
E. coli pmi gene encoding the enzyme phosphomannose
isomerase (PMI) (EntrezTM Accession Number M15380;
(NCBI 2006); this gene is also known as manA. Catalyzes the
isomerisation of mannose-6-phosphate to fructose-6phosphate. Used as a selectable marker during
transformation.
Terminator sequence from the nopaline synthase gene of A.
tumefaciens (EntrezTM Accession Number V00087; (NCBI
2006). Its function is to provide a polyadenylation site.
Vector Backbone Components
LB
(left
border)
8362 - 8386
25
spec
9562-10350
789
ColE1ori
1154912355
807
cos
1273613167
432
RB
(right
border)
1 - 25
25
Left border region of T-DNA from A. tumefaciens nopaline Tiplasmid (EntrezTM Accession Number J01825; (NCBI 2006).
Short direct repeat that flanks the TDNA and is required for
the transfer of the T-DNA into the plant cell (Zambryski et al.,
1982).
Spectinomycin adenylyltransferase, aadA gene from E. coli
Tn7 (EntrezTM Accession Number X03043 (NCBI, 2006)).
Confers resistance to erythromycin, streptomycin, and
spectinomycin; used as a bacterial selectable marker (Fling et
al., 1985).
Origin of replication that permits replication of plasmid in E.
coli. (Similar to EntrezTM Accession Number V00268 (NCBI,
2006 (Itoh and Tomizawa, 1979).
The sequence that is cut to produce the cohesive, singlestranded extensions located at the ends of the linear DNA
molecules of certain bacteriophages, such as lambda (Sanger
et al., 1982).
Right border region of T-DNA from A. tumefaciens nopaline
Ti-plasmid (EntrezTM Accession Number J01826 (NCBI,
2006)). Short direct repeat that flanks the T-DNA and is
required for the transfer of the T-DNA into the plant cell (Wang
et al., 1984).
22
35S terminator (70 bp)
ZmUbiInt (1993 bp)
HindIII
(189)
Acc65I/KpnI (4786)
ZmUbiInt (1993 bp)
EcoRV (6965)
pmi (1176 bp)
iPEPC9 (108 bp)
NOS (253 bp)
XmaI (8293)
vip3Aa19 (2370 bp)
RB (25 bp)
LB (25 bp)
vip3Aa19 probe (2370 bp)
Figure 1: Genes and regulatory elements inserted in MIR162
23
vip3Aa1 gene from B.t.
strain AB88 characterized
Optimization and synthesis of vip3Aa19 and pmi
genes and assembly of pNOV1300 vector in E. coli
Transformation of NP2500 x NP2499 embryos with
Agrobacterium tumefaciens containing plasmid vector
pNOV1300
Selection of transformation events
on medium containing mannose
Regeneration of transformed
cells into maize plants
Evaluation of transformants
for insecticidal efficacy
Introgression of transformation events into elite inbred
lines for evaluation of agronomic performance
Selection of MIR162 as lead event
Regulatory studies to
characterize human and
environmental risks
Introgression of transgenes
into commercial breeding
lines and field evaluations
Figure 2: Steps in development of corn line MIR162
24
3.2
Function and regulation of novel genes
vip3Aa20 gene
The Vip3Aa proteins confer resistance to several lepidopteran insect pests. Corn line
MIR162 expresses a modified version of the native Vip3Aa1 protein found in the B.
thuringiensis strain AB88. The Vip3Aa19 protein encoded by the vip3Aa19 gene in vector
pNOV1300 differs from the Vip3Aa1 protein in B. thuringiensis strain AB88 by a single amino
acid: lysine at position 284 is changed to glutamine (K284Q). Transformation induced
changes in the vip3Aa19 coding sequence resulted in two nucleotide differences for the
inserted gene, designated vip3Aa20 (Entrez Accession number DQ539888; NCBI, 2006).
One of these nucleotide changes resulted in a single amino acid change: methionine at
position 129 of Vip3Aa19 has been substituted by isoleucine (M129I). The other change was
a silent mutation, not resulting in an amino acid change.
The regulatory elements are described in Table 1. The vip3Aa20 gene is regulated by the
promoter and first intron from the Zea mays polyubiquitin gene, which confers constitutive
expression, and the 35S terminator from the cauliflower mosaic virus.
pmi gene
The pmi gene represents the manA gene from E. coli and encodes the enzyme
phosphomannose isomerase (PMI). It was used as a selectable marker gene during the
transformation process. Mannose, a hexose sugar, is taken up by plants and converted to
mannose-6-phosphate by hexokinase. This product cannot be further utilised in plants as
they lack the PMI enzyme. The accumulation of mannose-6-phosphate inhibits
phosphoglucose isomerase, causing a block in glycolysis. It also depletes cells of
orthophosphate required for the production of ATP. Therefore, while mannose has no direct
toxicity on plant cells, it causes growth inhibition (Negrotto et al., 2000). This does not occur
in plants transformed with the pmi gene as they can utilise mannose as a source of carbon.
The pmi gene is regulated by the same polyubiquitin promoter (ZmUbilnt) as the vip3Aa20
gene and the NOS terminator from A. tumefaciens.
No other genes were transferred to corn line MIR162.
3.3
Characterisation of the genes in the plant
Insert DNA sequence analysis
To characterise the integrity of the inserted T-DNA, the sequence of the entire DNA insert
was determined. The sequence of the insert was compared to the DNA sequence of the
transforming plasmid (pNOV1300). In total, 8314 bp of T-DNA had become inserted into the
corn genome. During the transformation process the T-DNA insert was truncated at both the
left border (entire LB plus 32 bp of non-coding sequence) and the right border (the entire RB
plus two bp of non-coding sequence).
While T-DNA border sequences are known to play a critical role in T-DNA insertion into the
genome, this result is not unexpected since insertions are often imperfect, particularly at the
Left T-DNA border (Tinland B. and Hohn B., 1995). Two base pair changes were noted in the
MIR162 insert, both within the vip3Aa19 gene. The first change results in a predicted single
amino acid change at position 129 of Vip3Aa19 from methionine to isoleucine (M129I). The
second nucleotide change was silent and did not result in a predicted amino acid change. As
the vip3Aa gene in MIR162 is slightly different from the vip3Aa19 gene encoded on
pNOV1300, it was designated vip3Aa20 (Entrez Accession number DQ539888; NCBI, 2006).
25
The coding sequences of pmi, the ZmUbiInt promoters, the 35S terminator and the NOS
terminator in MIR162 are identical to those in the pNOV1300 transformation plasmid.
Insert and copy number
Southern blot analysis was used to determine the insert and copy number of the vip3Aa20
and pmi genes and to confirm the absence of DNA sequence from outside the T-DNA
borders of the transformation vector.
Southern blot analyses of pooled leaf tissue from plants in MIR162 backcross generation four
(BC4F1) and negative segregants demonstrate that insertion event MIR162 occurred as an
integration of a single intact T-DNA from plasmid pNOV1300. Probes corresponding to
vip3Aa19, pmi, the ZmUbiInt promoter and the NOS terminator produced the expected
hybridisation patterns. Southern analysis using the pNOV1300 backbone sequence as probe
confirmed that plasmid backbone DNA is not present in corn line MIR162.
Flanking regions and putative Open Reading Frame (ORF) analysis
Corn genomic DNA on either side of the MIR162 T-DNA insert was sequenced. The DNA
sequence of 1000 bp of genomic DNA flanking the 5’ end and the 3’ end of the MIR162 insert
was determined.
Analysis of the sequence flanking the 5’ region of the insert revealed significant homology
with Dissociation1 (Ds1)-related transposable elements. Ds1 is a non-autonomous
transposable element that requires an active Activator (Ac) element to become mobile. The
homology between the 5’ flanking sequence and the Ds1-related elements is over 500 bp
from the insert sequence. Previous studies have shown that the excision of Ds elements can
extend beyond the element, but the largest amount of surrounding DNA that has been shown
to be deleted is 36 bp.
The genomic sequence flanking the 3’ region of the T-DNA insert shows homology with a
sequence defined as a cyclophilin gene, but the region of homology lies outside of the
cyclophilin coding region.
The junction regions between the insert and genomic DNA were further analysed for their
potential to be involved in the production of chimeric proteins. The production of unexpected
chimeric proteins as a result of transgene insertion is of particular relevance to food safety.
In cases where there is 100% molecular identity between the plasmid T-DNA and inserted
DNA in the plant, and all regulatory elements including termination and polyadenylation
signals are intact, there is little likelihood of unintended formation of gene fragments that are
transcriptionally active or likely to produce a chimeric protein. In the case of corn line
MIR162, the transformation event has not resulted in any additions, deletions,
rearrangements or partial insertions of the gene of interest, or its regulatory elements, as
determined by the Southern blot and direct DNA sequencing of the entire insert region. In
addition, analysis of all six potential reading frames at the junctions of both the 5’ and 3’ end
of the DNA insert with the corn genome did not detect the presence of any functional novel
open reading frames.
Conclusion
Molecular analyses have been performed on corn line MIR162 to characterise the novel
genes present in the genome. Results indicate that there is one insertion site consisting of
the entire T-DNA from plasmid pNOV1300. Sequence analysis showed that two single
nucleotide changes had occurred within the insert, both within the vip3Aa gene.
26
One of these resulted in a predicted amino acid change from methionine to isoleucine at
position 129. The T-DNA insert in MIR162 has not disrupted any known endogenous corn
genes, nor resulted in the creation of any unexpected open reading frames.
3.4
Development and breeding of corn line MIR162
A number of corn lines were generated following transformation with vector pNOV1300. The
progeny of these primary transformants (T0 plants) were field tested for both resistance to
insect feeding damage and agronomic performance. Following introgression into multiple
elite lines of corn and further evaluation of agronomic performance and insecticidal efficacy,
event MIR162 was selected for further development and regulatory trials. These steps in the
development of MIR162 are shown schematically in Figure 2.
A pedigree chart showing the further breeding of event MIR162 is shown in Figure 3. The
plant test materials used in studies to support the food safety assessment are also indicated.
Finished hybrids, produced from converted MIR162 inbreds, were used as test materials for
compositional analysis and protein quantification. Parental control hybrids with a genetic
background matched to that of the MIR162 hybrids were utilised in all studies so that the
effect of the transformation event could be assessed.
27
Molecular characterisation & Mendelian inheritance
T0
x NPH8431
F1
NP2161 x
F1
BC1F1
bc – backcross
 - self-pollination
BC1F1
bc
bc
BC2F1
BC2F1
bc
x NP2222
bc
BC3F1
A BC1F1
bc
F1
B BC2F1
bc
BC1F1
BC4F1
x NP2153
F1
F1
bc
bc
bc
x NP2276
BC3F1
C BC4F1
x – cross
bc
bc
F1
T0 – original transformant
x NP2010
F1
B9620 x
A
bc
BC1F1
bc
BC1F1
bc (3 times)
bc (3 times)
BC4F1

BC4F1

BC4F2
BC4F2
BC4F3
BC4F3
BC4F4
BC4F4




x NP2391
x NP2391
D F1

E F2
F F1

G F2
Molecular characterisation & Mendelian inheritance
Molecular characterisation & Mendelian inheritance
Protein characterisation
Composition analysis (Forage and grain)
Protein Quantification – Hybrid 1
Protein Quantification – Hybrid 2
42-day broiler chicken
B
C
C
D&E
D&E
F&G
G
Figure 3: Pedigree diagram for MIR162 and test materials
28
3.5
Stability of the genetic changes
A number of analyses were done to demonstrate the stability of the genetic changes in corn
line MIR162. Southern blot analysis over multiple generations was done to determine the
stability of the inserted DNA. Segregation analysis over multiple generations was done to
determine the heritability and stability of the vip3Aa20 and pmi genes and Vip3Aa20 and PMI
proteins.
Generational stability
To determine whether the insert in corn line MIR162 is stable over a number of generations,
Southern blot analysis was conducted on samples from three backcross generations –
BC1F1, BC2F1 and BC4F1 (see Figure 3). Genomic DNA from MIR162 and a negative control
from the BC4F1 generation was digested with Acc65I, a restriction enzyme that digests once
in the DNA insert but not within the region of the vip3Aa19-specific probe (Figure 1).
Southern analysis identified the same single hybridisation band in each of these generations
when probed with DNA specific to the vip3Aa19 gene, as expected. No hybridisation was
detected in the negative control genomic DNA. The consistent and predicted hybridisation
pattern in several generations of MIR162 indicates that the insert is stably integrated into the
corn genome.
Segregation analysis
Segregation analysis was performed on MIR162 plants over three generations to assess
whether the DNA insert is inherited in a stable and predictable manner. PCR analyses were
conducted on individual plants from three generations of MIR162 to determine the
inheritance ratios of both the vip3Aa20 and the pmi genes. For each generation tested, the
expected inheritance frequency was 1:1 positive to negative for both traits, as a result of
back-crossing a hemizygous transgenic plant to a non-transgenic recurrent parent. The
expected and observed ratios of positive and negative plants were analysed by Chi square
analysis to determine if the trait is segregating in a Mendelian fashion. The expected and
observed segregation frequencies of the vip3Aa20 and pmi genes are shown in Tables 2 and
3.
Table 2: Observed vs. expected genotype for vip3Aa for multiple MIR162 generations
as determined by PCR analysis
Trait
BC1F1
O*
21
20
41
BC2F1
E*
20.5
20.5
41.0
O*
45
52
97
Positive
Negative
Total
X2 value
0.000
0.371
* O = Observed values and E = Expected values
X2= sum [|(Observed-expected)|- 0.5]2/expected
E*
48.5
48.5
97.0
BC4F1
O*
148
139
287
E*
143.5
143.5
287.0
0.223
Table 3: Observed vs. expected genotype for pmi for multiple MIR162 generations as
determined by PCR analysis
Trait
BC1F1
O*
21
20
41
BC2F1
E*
20.5
20.5
41.0
O*
45
52
97
Positive
Negative
Total
X2 value
0.000
0.371
* O = Observed values and E = Expected values
X2= sum [|(Observed-expected)|- 0.5]2/expected
29
E*
48.5
48.5
97.0
BC4F1
O*
148
139
287
E*
143.5
143.5
287.0
0.223
No significant differences between observed and expected segregation frequencies were
found. The critical Chi square value to reject the hypothesis of 1:1 segregation at the 5%
level is 3.84. The Chi square values obtained are less than 3.84 for each gene in all
generations tested. This indicates that the insert in MIR162 is segregating in a predictable
manner according to Mendelian principles.
Stability of protein expression
Study submitted:
Pence, K. (2006) Stability of Vip3Aa20 and phosphomannose isomerise (PMI) protein expression
across multiple generations of maize (corn) derived from transformation event MIR162. Syngenta
Seeds Biotechnology Report No. SSB-002-06.
The stability of Vip3Aa20 and PMI protein expression was evaluated over the same three
generations of corn line MIR162. Individual plants derived from the three backcross
generations BC1F1, BC2F1 and BC4F1 (see Figure 3) were genotyped by PCR as described
in the preceding section. Leaf tissue from five plants hemizygous for the transgenes in each
generation, along with tissue from one near-isogenic, non-transgenic plant, was collected
and analysed by enzyme-linked immuno-sorbent assay (ELISA). The mean Vip3Aa20 and
PMI concentrations in leaves measured in the three backcross generations are given in
Table 4. The concentrations of Vip3Aa20 and PMI are consistent across generations.
Vip3Aa20 and PMI were either not detectable or not quantifiable in the non-transgenic control
samples analysed.
Table 4: Vip3Aa20 and PMI Concentrations in Leaf Tissue from Multiple Generations
of corn line MIR162
Generation
BC1F1
BC2F1
BC4F1
Mean μg
Vip3Aa20/gdw ± SD
(range)
77.94 ± 4.32
(72.83 – 84.18)
74.33 ± 4.07
(68.26 – 78.33)
78.80 ± 5.42
(74.61 – 87.50)
Mean μg
PMI/gdw ± SD
(range)
10.47 ± 0.54
(9.95 – 11.16)
10.59 ± 0.65
(9.86 – 11.16)
10.34 ± 1.23
(8.80 – 11.93)
N = 5 (samples analysed). Values have not been corrected for extraction efficiency.
Stability of insect protection phenotype
Studies submitted:
Huber, S.A., White, J., Mroczkiewicz, S. and Ward, D.P. (2007) Insecticidal Efficacy Field Evaluations
with MIR162 Maize Hybrids in 2005 and 2006. Syngenta Seeds Biotechnology Report No. SSB-52207.
White, J., Sagers, J., Meehan, M. and Meghji, M. (2007) Fall Armyworm Tolerance of a MIR162 Maize
Hybrid: 2006 Field Trial Results. Syngenta Seeds Biotechnology Report No. SSB-503-07.
White, J., Meehan, M. and Meghji, M. (2007) Corn Earworm Tolerance of a MIR162 Maize Hybrid:
2006 Field Trial Results. Syngenta Seeds Biotechnology Report No. SSB-502-07.
Field trials were conducted to assess the insecticidal efficacy of MIR162 corn in comparison
to a conventional corn hybrid control and a conventional corn hybrid treated with a
commercial insecticide standard.
30
The results of trials conducted at multiple locations which represented the major corn
growing regions of the continental United States demonstrate that MIR162 provides
significant crop protection against feeding damage caused by black cutworm (Agropis
ipsilon), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea),
southwestern corn borer (Diatraea grandiosella), beet armyworm (Spodoptera exigua),
common stalk borer (Pepaipema nebris) and western bean cutworm (Striacosta albicosta).
Conclusion
The results of the segregation analysis are consistent with a single site of insertion for both
the vip3Aa20 and the pmi gene expression cassette and confirm the results of the molecular
characterisation. The studies indicate that the T-DNA insert is stably integrated into the corn
genome in corn line MIR162 and is inherited as expected over several generations. The level
of expression of both the Vip3Aa20 and PMI proteins is stable across multiple backcross
generations of corn line MIR162. The Vip3Aa20 protein in MIR162 corn has insecticidal
activity against a range of lepidopteran insects.
3.6
Antibiotic resistance marker genes
No antibiotic marker genes are present in insect-protected corn line MIR162. The molecular
characterisation shows that the region outside the T-DNA of plasmid pNOV1300 was not
integrated into the corn genome during transformation. Consequently, the spectinomycin
resistance gene, which was used as a bacterial selectable marker gene, is not present in
insect-protected corn line MIR162. The absence of the bacterial marker gene in the plant was
confirmed by Southern hybridisation analysis using a probe corresponding to the backbone
of the transformation vector, pNOV1300, as described in section 3.3.
4.
CHARACTERISATION OF NOVEL PROTEINS
In considering the safety of novel proteins it is important to consider that a large and diverse
range of proteins are ingested as part of the normal human diet without any adverse effects,
although a small number have the potential to impair health, e.g., because they are allergens
or anti-nutrients.
As proteins perform a wide variety of functions, different possible effects have to be
considered during the safety assessment including potential toxic, anti-nutritional and
allergenic effects. To effectively identify any potential hazards requires knowledge of the
characteristics, concentration and localisation of all novel proteins expressed in the organism
as well as a detailed understanding of their biochemical function and phenotypic effects. It is
also important to determine if the novel protein is expressed as expected, including whether
any post-translational modifications have occurred.
Corn line MIR162 contains two novel proteins: Vip3Aa20 and PMI. A number of different
analyses were done to determine the identity, physiochemical properties, in planta
expression, bioactivity and potential toxicity and allergenicity of the two proteins. Because
the expression of proteins in planta is usually too low to allow purification of sufficient
quantities for safety assessment studies, a bacterial expression system was used to
generate large quantities of the Vip3Aa20 and PMI proteins. The Vip3Aa20 and PMI proteins
produced in E. coli were engineered so their amino acid sequence matched that of the plantproduced Vip3Aa20 and PMI proteins. The equivalence of the bacterial-produced proteins to
the plant-produced proteins was determined as part of the protein characterisation.
31
4.1
Biochemical function and phenotypic effects
Vip3Aa20
The Vip3Aa20 protein in corn line MIR162 has insecticidal activity against several
lepidopteran pests of corn. The vip3Aa20 gene in corn line MIR162 is a variant of the vip3Aa
gene present in B. thuringiensis (I) strain AB88. The vip3Aa20 gene encodes a Vip3Aa20
protein of approximately 89 kDa and 789 amino acids in length. The Vip3Aa20 protein is
99.7% identical to the native Vip3Aa1 protein and differs by only 2 amino acids, at positions
129 and 284. The Applicant states that both these substitutions are conservative and do not
significantly impact insecticidal activity.
The Vip (vegetative insecticidal protein) proteins in Bt are produced during vegetative
bacterial growth and are secreted as soluble proteins into the extracellular environment. Bt
cultures continue to produce Vip protein during the stationary phase of development and
sporulation. This contrasts with the other insecticidal Bt crystal (Cry) proteins that are
produced during sporulation and spontaneously form small crystals that are contained within
cytoplasmic inclusions.
Following ingestion of Vip3A by target insects, the protein is activated by insect gut proteases
and binds to the membranes of midgut epithelial cells. Subsequent pore formation in the cell
membranes leads to disruption of the midgut transmembrane potential and eventual insect
death.
Both Vip and Cry proteins are proteolytically activated to insecticidal toxic cores in the
lepidopteran gut and form pores in the gut membranes of sensitive species. However,
Vip3Aa has been shown to have significantly different receptor binding properties and pore
forming properties to Cry proteins, indicating that Vip and Cry proteins have different targets
and specific modes of action. The mode of action of Vip3Aa proteins is highly specific to
insects (Lee et al., 2003; Lee et al., 2006).
Phosphomannose isomerase
The phosphomannose isomerase (PMI) protein in MIR162 is a protein of approximately
42.8 kDa and 391 amino acids that catalyses the reversible interconversion of mannose
6-phosphate and fructose 6-phosphate. The pmi gene, which expresses PMI in MIR162, is
derived from E. coli. The lack of PMI in many plant species allows it to be utilised as a
selectable marker for transformation. Plant cells that have been transformed with the E. coli
manA (pmi) gene are able to survive and grow on media containing mannose as the only or
primary energy source (Miles and Guest, 1984). Plants lacking this enzyme are unable to grow
on culture media containing mannose.
Mannose is converted to mannose 6-phosphate by hexokinase in the plant. In the absence of
PMI, this accumulates, inhibiting the enzyme phosphoglucose isomerase and blocking
glycolysis. In addition, depletion of the pyrophosphate required for ATP production and
transcriptional repression of genes involved in photosynthesis and glyoxylate cycle occurs
(Privalle, 2002; Freeze, 2002).
4.2
Protein characterisation
A range of analytical techniques was used to determine the identity as well as the
physicochemical and functional properties of the plant-produced Vip3Aa20 and PMI proteins
isolated from MIR162 corn and to compare them to E. coli-produced proteins. These
techniques included Western blot analysis, peptide mass mapping analysis, insect bioactivity
assays, enzyme activity assays and glycosylation analysis.
32
The E. coli-produced proteins were used as reference standards for determination of protein
concentration and immunoblot analysis using polyclonal antibody. These proteins were also
used as reference standards to evaluate equivalence between plant- and E. coli-produced
proteins for molecular weight and functional activity assays, as a reference and a positive
control in immunoblot analyses using anti-Vip3Aa20 and anti-PMI antibodies, and as a
reference and negative control in glycosylation analysis.
Vip3Aa20
Studies submitted:
Graser, G and Stacy, C. (2006) Characterization of the Vip3A Protein Expressed in Event MIR162Derived Maize (Corn) and Comparison with Microbially Produced and Plant-Derived Vip3A Test
Substances. Syngenta Seeds Biotechnology Report No. SSB-017-06.
Graser, G and Stacy, C. (2006) Characterization of Microbially-Produced Vip3A Test Substance
MIR162VIP3A-0106 and Comparison with Vip3A Expressed in Event MIR162-Derived Maize (Corn).
Syngenta Seeds Biotechnology Report No. SSB-023-06.
Vip3Aa20 expressed in MIR162 is nearly identical in amino acid sequence to that encoded by
the native vip3Aa1 gene from B. thuringiensis, with the exception of two amino acids: lysine284 has been substituted by glutamine, and methionine-129 has been substituted by
isoleucine. The Vip3Aa19 protein encoded by the synthetic vip3Aa19 gene (modified to
accommodate the preferred codon usage in corn) on transformation plasmid pNOV1300 differs
from the native Vip3Aa1 protein encoded by the native vip3Aa1 gene from Bt strain AB88 by a
single amino acid at position 284.
The second amino acid difference between Vip3Aa1 and Vip3Aa20, at position 129, is the
result of a transformation induced change in the vip3Aa19 coding sequence that was
incorporated in the MIR162 corn genome. These differences in the Vip3A proteins are shown
in Table 5.
The Vip3Aa20 protein was purified from a homogenate of leaf tissue from MIR162 corn
hybrids using a combination of ammonium sulphate fractionation and dialysis, and
subsequent immunoaffinity chromatography. A negative control substance was prepared
from leaf material derived from a corresponding negative segregant corn. The Vip3Aa20
protein prepared by expressing a synthetic vip3Aa20 gene in an E. coli over-expression
system is predicted to be identical in amino acid sequence to Vip3Aa20 expressed in MIR162
corn. E. coli produced Vip3Aa20 was purified with anion exchange chromatography and
dialysis.
Table 5: Comparison of the Amino Acid Sequence Differences of Vip3Aa Proteins
Examined in this Study
Protein
Vip3Aa1
Vip3Aa19
native Vip3A in
Bt strain AB88
Codon
optimised
version on
pNOV1300
Amino
acid at
position
129
Amino
acid at
position
284
Source
M
K
B. thuringiensis
M
Q
Recombinant
E. coli
33
Vip3Aa Test
Samples
Protein
Vip3Aa20
MIR162 corn
Vip3Aa20
Amino
acid at
position
129
Amino
acid at
position
284
Source
Vip3Aa Test
Samples
I
Q
MIR162 corn
I
Q
Recombinant E.
coli
LPMIR162-0105 and
0106;
IAPMIR162-0105
and 0106
MIR162VIP3A-0106
Protein identity
The identity of the plant- and microbially-produced Vip3Aa20 was confirmed by Western blot
analysis using anti-Vip3A antibody, and peptide mass mapping analysis. The identity of the
microbially produced Vip3Aa20 was also confirmed by N-terminal amino acid sequence
analysis:
(i)
Western blot using anti-Vip3A antibody – the plant-produced Vip3Aa20 protein and the
E. coli-produced reference standard were subjected to SDS-PAGE on the same gel.
After blotting, goat anti-Vip3Aa immunoaffinity-purified polyclonal antibodies and
donkey anti-goat IgG linked to alkaline phosphatase were used to detect the presence
of immunoreactive Vip3Aa. The major immunoreactive bands corresponded to the
predicted molecular weight of ~89 kDa in both the plant-produced and E. coli-produced
samples. The bands of lower molecular weight are partial Vip3A fragments, which the
Applicant states are likely to represent proteolytic degradation products;
(ii)
Peptide mass mapping analysis – Vip3Aa20 protein isolated from MIR162 and
recombinant E. coli was reduced, alkylated with iodoacetamide and digested with
trypsin. The mass analysis of the Vip3Aa20-derived peptides was performed on a
quadrupole time-of-flight mass spectrometer. The detected peptide masses were
compared to a protein database. The ability to identify a protein using this method is
dependent on matching a sufficient number of observed tryptic mass fragments to
expected (theoretical) mass fragments. In general, a protein identification made by
peptide mapping is considered to be reliable if the measured coverage of the sequence
is 15% or higher with a minimum of five matched peptides (Jensen et al., 1997). The
tryptic mass fragments identified from the plant- and microbially derived Vip3Aa20 that
matched the expected tryptic peptides generated in silico covered ~40% and 45%,
respectively, of the predicted total Vip3Aa20 amino acid sequence. The identified
peptides were representative of regions throughout the sequence of Vip3Aa20,
including peptides close to the N- and C- termini.
(iii)
N-terminal amino acid sequence analysis – Vip3Aa20 protein isolated from
recombinant E. coli was immobilized on a membrane and subjected to N-terminal
amino acid sequence analysis using Edman-based chemistry. The analysis confirmed
the predicted amino acid sequence for the first twelve amino acids. The N-terminal
sequence of the plant derived protein could not be determined due to technical
difficulties. The Applicant states this is most likely due to N-terminal blockage of the
protein.
Insect bioassays
The insecticidal activity of the plant- and E. coli-produced Vip3Aa20 protein was determined
using an insect bioassay with freshly hatched first-instar fall armyworm (S. frugiperda). The
bioassay included a range of concentrations of Vip3Aa20 protein overlayed on insect diet.
34
Each treatment included 24 larvae that were allowed to feed for around nine days, when
mortality was assessed.
The plant- and microbially-derived Vip3Aa20 showed comparable responses with the highest
dose producing up to 100% and 88% mortality respectively, with LC50 values that were not
significantly different by t-test (Table 6). No mortality was observed in negative controls
using insect diet treated with water or buffer. These results indicate that the plant- and
E. coli-produced Vip3Aa20 proteins can be regarded as equivalent in terms of their
insecticidal activity.
Table 6: Comparative Bioactivity of plant and microbially-derived Vip3Aa20 in Diet
Surface Bioassay with First-Instar S. frugiperda
Test Substance
Plant derived Vip3Aa20
MIR162VIP3A-0106
E. coli derived Vip3Aa20
LPMIR162-0106
LC50 [ng Vip3Aa20/cm2]
(95% Confidence Intervals)
225 ng/cm2 (155 - 289)
318 ng/cm2 (232 - 451)
Glycosylation analysis
To assess whether post-translational glycosylation of the plant-produced Vip3Aa20 protein
occurred, the purified protein sample was subjected to glycosylation analysis. Creatinase, a
non-glycosylated protein was included as a negative control. Transferrin, known to be a
glycosylated protein, was used as a positive control. The test and control protein samples
were separated by SDS-PAGE and protein-bound glycan moieties were labelled with
digoxigenin and detected with an anti-digoxigenin antibody coupled to alkaline phosphatase.
The transferrin protein produced a clearly visible band of approximately the expected
molecular weight upon DIG Glycan analysis. No signal was detected for either the
creatinase control or the plant-produced Vip3Aa20 protein. As prokaryotic organisms lack
the capacity for protein glycosylation, the E. coli-produced Vip3Aa20 protein is expected to
act as a negative control. This was confirmed in a similar, subsequent experiment.
These results indicate that the plant-produced Vip3Aa20 protein is not glycosylated and is
equivalent to the E. coli-produced Vip3Aa20 protein in terms of its lack of glycosylation.
Conclusion
A large number of studies have been done on the Vip3Aa20 protein to confirm its identity and
physicochemical and functional properties as well as to determine its equivalence to E. coliproduced Vip3Aa20 protein. Vip3Aa20 produced by MIR162 corn and recombinant E. coli
were both shown to have the predicted molecular weight of ~89 kDa and both
immunologically cross-reacted with the same anti-Vip3A antibody in Western blot analysis.
Peptide mass mapping analysis provided additional evidence of the identity and equivalence
of Vip3Aa20 expressed in MIR162 corn and in recombinant E. coli. Vip3Aa20 protein from
both sources had comparable bioactivity against sensitive lepidopteran larvae. Analysis of
protein glycosylation supports the conclusion that Vip3Aa20 protein produced by MIR162
corn and recombinant E. coli is not glycosylated.
The studies support the conclusion that Vip3Aa20 is expressed as expected in MIR162 corn,
and further, that the Vip3Aa20 produced by recombinant E. coli is a suitable surrogate for the
plant produced protein.
35
PMI
Study submitted:
Stacy, C. and Graser, G. (2006) Characterization of Phosphomannose Isomerase (PMI) Produced in
Event MIR162 Maize and Comparison to PMI as Contained in Test Substance PMI-0198. Syngenta
Seeds Biotechnology Report No. SSB-037-06.
The size, immunoreactivity and specific enzymatic activity of PMI extracted from MIR162
corn leaf tissue was compared to PMI purified from recombinant E. coli. PMI protein was
extracted from a homogenate of leaf tissue from greenhouse- grown MIR162 corn hybrids A
negative control substance was prepared from leaf material from near-isogenic, nontransgenic corn hybrids.
The PMI protein isolated from recombinant E. coli from an over-expression system is
predicted to be identical in amino acid sequence to PMI expressed in MIR162 corn, with the
exception of an additional, non-functional 16 amino acids at the N-terminus (comprising a 13
amino acid T7-Tag® to facilitate purification and 3 amino acids arising from the polylinker).
E. coli produced PMI was purified by extraction and differential ammonium sulphate
concentration, followed by chromatography and ion exchange chromatography.
Protein identity
The molecular weight and immunoreactivity of PMI produced by MIR162 corn was
determined by Western blot analysis. The plant-produced PMI protein and the E. coliproduced reference standard were subjected to SDS-PAGE on the same gel. After blotting,
goat anti-PMI polyclonal antibodies, raised against recombinant E. coli produced PMI, and
donkey anti-goat IgG linked to alkaline phosphatase were used to bind to the primary
antibody and visualized by development with an alkaline phosphatase substrate solution. The
Western blot was examined for the presence of immunoreactive PMI. A single
immunoreactive PMI band corresponding to the predicted molecular weight of ~42.8 kDa was
observed in the MIR162-derived plant extract. The microbially expressed PMI protein
showed one major band with a slightly lower mobility than the plant expressed PMI protein,
which is consistent with its slightly higher predicted molecular weight of ~44.4 kDa, resulting
from the addition of 16 amino acids at the N-terminus. A second minor immunoreactive band
of ~90 kDa was also observed for E. coli derived PMI. The Applicant states that, as this
protein cross-reacts with the anti-PMI antibody and shows the mobility of the molecular
weight of two PMI proteins, it likely represents a dimer of the PMI protein.
Enzymatic Activity
The enzymatic activity of plant- and microbially-derived PMI was measured in triplicate in a
coupled enzyme activity assay, as shown diagrammatically below.
PMI
PGI
G6PDH
Mannose 6-P  Fructose 6-P  Glucose 6-P + NADP  6-Gluconolactone + NADPH
(PMI = Phosphomannose isomerase; PGI = Phosphoglucose isomerase;
G6PDH = Glucose 6-phosphate dehydrogenase)
Briefly, protein extract was added to an assay mixture containing NADP, mannose-6hosphate, PGI and G6PDH. PMI activity was measured by monitoring the formation of
NADPH at 340 nm on a spectrophotometer. The specific activity of PMI was determined
using the known molar extinction coefficient of NADPH. The concentration of PMI added to
the assay was determined, with one unit of PMI defined as 1 µmol of NADP reduced to
NADPH per minute.
36
The mean specific activity for PMI extracted from MIR162 corn was 55.5 ± 4.18 U/mg PMI,
while for microbially-produced PMI it was 33.2 ± 2.37 U/mg PMI. The Applicant concludes
that, considering the expected levels of variability in the measurements, the PMI extracted
from MIR162 corn has similar activity to PMI from recombinant E. coli.
Glycosylation potential
The Applicant states that it is unlikely that PMI expressed in MIR162 corn is posttranslationally glycosylated. The PMI protein contains no consensus amino acid sequences
for N-glycosylation, although O-glycosylation could theoretically occur at the serine or
threonine residue present in the protein (Privalle, 2002). However, PMI is not expected to be
post-translationally glycosylated in MIR162 corn, because its expression is not targeted to a
cellular glycosylation pathway. Mass spectrometric analysis of human PMI indicates that this
protein is also not post-translationally modified (Freeze, 2002). In addition, as the Western
blot analysis shows that the plant-derived PMI has a clear higher mobility (indicating a lower
molecular weight) than microbially-produced PMI (which has 16 additional amino acids at the
N-terminus and therefore a higher molecular weight); a massive glycosylation (e.g. more than
10 gluc units) of the PMI in MIR162 corn can therefore be excluded.
Conclusion
Studies conducted on the PMI protein have demonstrated that the protein expressed in
MIR162 corn conforms in size and immunoreactivity to that expected and also exhibits the
expected enzymatic activity. The E. coli-produced protein was also shown to be equivalent
to the plant produced proteins in terms of its size and immunoreactivity as well as having
comparable enzymatic activity. The E. coli-produced proteins are therefore suitable as
surrogates for the plant-produced protein for safety assessment purposes.
4.3
Protein expression analysis
Study submitted:
Hill, K. (2006) Quantification of Vip3Aa20 and phophomannose isomerise (PMI) in tissues of maize
derived from transformation event MIR162. Syngenta Seeds Biotechnology Report No. SSB-020-06.
The expression levels of the two novel proteins (Vip3Aa20 and PMI) in corn line MIR162
were determined by ELISA. Plants from two MIR162 hybrid lines (referred to as MIR162-A
and MIR162-B) were analysed. The hybrid plants were hemizygous for event MIR162 and
are representative of the corn varieties that would (upon regulatory approval) be grown
commercially. Two corresponding near-isogenic, non-transgenic control hybrids were also
analysed.
Ten plants per transgenic genotype and two plants from each control genotype were
harvested at each of four growth stages: V9-V12 (~8 weeks after planting), anthesis (pollen
shed, 10-11 weeks), seed maturity (18-20 weeks) and senescence (23-24 weeks) from the
two hybrids grown at two field locations (Illinois and Nebraska). Protein was extracted from
samples of leaves, roots, kernels, silk, pollen, whole plant and silage and analysed
quantitatively for Vip3Aa20 and PMI by ELISA using goat and rabbit polyclonal antibodies.
ELISA values were corrected for extraction efficiency (estimated to be >78% and >75% for
Vip3Aa20 and PMI respectively, for the various plant tissues analysed).
Vip3Aa20
The concentrations of Vip3Aa20 were generally similar between the two MIR162 hybrids for
each tissue type at each stage sampled. The mean concentrations across the two locations
for Vip3Aa20 protein, determined on a dry weight basis, are given in Table 7.
37
Vip3Aa20 concentrations in the near-isogenic, non-transgenic negative control samples were
either below the limit of detection or below the limit of quantification.
Table 7: Vip3Aa20 concentrations on a dry-weight basis in MIR162 plants (corrected
for extraction efficiency)
(range)
Tissue
Leaves
Roots
V9-V12
97.26
(76.12 – 119.12)
31.80
(28.10 – 35.65)
Pith
N/A
Kernels
N/A
Silk
N/A
Pollen
N/A
Whole
91.53
Plants
(88.68 – 96.51)
N/A = Not analysed at this stage.
Anthesis
107.74
(97.10 – 118.80)
28.34
(26.30 – 30.20)
31.71
(29.43 – 36.18)
N/A
97.40
(60.54 – 149.00)
47.13
(41.45 – 53.52)
67.61
(61.68 – 72.63)
Seed Maturity
121.79
(77.25 – 159.66)
20.29
(9.87 – 27.48)
58.21
(52.74 – 63.68)
43.56
(40.47 – 50.50)
Senescence
21.31
(12.93 – 30.28)
21.66
(11.58 – 32.13)
N/A
N/A
N/A
N/A
49.04
(34.84 – 63.14)
34.30
(21.12 – 55.17)
N/A
34.24
(30.90 – 37.67)
PMI
The concentrations of PMI were generally similar between the MIR162 hybrids for each
tissue type at each time point. The mean concentrations across the two locations for PMI
protein, determined on a dry weight basis, are given in Table 8. PMI concentrations in the
near-isogenic, non-transgenic negative control samples were below the limit of detection.
Table 8: PMI concentrations on a dry-weight basis in MIR162 plants (corrected for
extraction efficiency)
(range)
Tissue
Leaves
Roots
V9-V12
11.12
(8.26 – 16.76)
4.32
(3.17 – 7.08)
Pith
N/A
Kernels
N/A
Anthesis
9.75
(6.92 – 14.68)
3.49
(2.51 – 5.22)
2.01
(1.53 – 2.40)
N/A
Seed Maturity
5.77
(4.57 – 7.55)
1.99
(1.08 – 3.09)
2.75
(2.36 – 3.19)
1.93
(1.33 – 2.54)
Senescence
<0.26
(<LOD – <LOQ)
1.51
(0.47 – 2.53)
N/A
0.75
(0.54 –0.97)
20.70
N/A
N/A
(12.60 – 27.16)
5.29
Pollen
N/A
N/A
N/A
(3.82 – 7.62)
Whole
8.74
7.10
3.76
2.36
Plants
(7.49 – 9.75)
(6.32 – 7.61)
(2.16 – 5.37)
(1.85 – 3.09)
Means preceded by ‘<‘ (less than) indicate that the estimated LOQ or LOD was used to represent
the samples when calculating the average.
N/A = Not analysed at this stage.
Silk
N/A
38
Conclusion
Kernels from MIR162 corn are the most likely tissue to be used as food, either as grain or
grain by-products. Both novel proteins are expressed in the grain of MIR162 corn. The
average Vip3Aa20 level in mature kernels from MIR162 corn was 43.56 μg/g DW. The
average PMI level in mature MIR162 corn kernels was 1.93 μg/g DW. Assuming corn grain
contains 10% total protein by weight, Vip3Aa20 represents less than 0.004% of total protein,
and PMI represents less than 0.0002% of total protein by weight.
4.4
Potential toxicity of novel proteins
While the vast majority of proteins ingested as part of the diet are not typically associated
with toxic effects, a small number may be harmful to health. Therefore, if a GM food differs
from its conventional counterpart by the presence of one or more novel proteins, these
proteins should be assessed for their potential toxicity. The main purpose of an assessment
of potential toxicity is to establish, using a weight of evidence approach, that the novel protein
will behave like any other dietary protein. The assessment focuses on: whether the novel
protein has a prior history of safe human consumption, or is sufficiently similar to proteins
that have been safely consumed in food; amino acid sequence similarity with known protein
toxins and anti-nutrients; structural properties of the novel protein including whether it is
resistant to heat or processing and/or digestion. Appropriate oral toxicity studies in animals
may also be considered, particularly where results from the biochemical, bioinformatic,
digestibility or stability studies indicate a concern.
4.4.1 History of use
Vip3Aa20
Study submitted:
Graser, G and Song, S. (2006) Analysis of Vip3A or Vip3A-like proteins in six different commercial
microbial Bacillus thuringiensis products. Syngenta Seeds Biotechnology Report No. SSB-036-06.
B. thuringiensis, the microorganism from which the vip3A gene was derived, has been
extensively studied and commercially exploited for over 40 years as the active ingredient in a
number of insecticide products used in agriculture as well as home gardens. B. thuringiensis
therefore has had a long history of safe use. Analysis of six of the commercially available Btbased insecticides demonstrated that Vip3Aa or Vip3Aa-like proteins were present in all six
Bt products, as analysed by ELISA. The products utilizing Bt subsp. kurstaki contained
sufficient immunoreactive material to be detected by Western blot analysis, while levels in
products containing Bt subsp aizawi had lower levels of detectable Vip3Aa-like proteins.
Products based on both Bt subsp. kurstaki and aizawi are available in Australia and New
Zealand, for use both on commercial agricultural crops (including vegetables, fruits, vines
and herbs) and for home garden use. Since Bt products have been used to control
lepidopteran larvae on a wide variety of crops, including fresh market products, it is likely that
small quantities of Vip3Aa of Vip3Aa-like proteins are present in the non-GM food supply.
PMI
As PMI proteins are ubiquitous in nature, including being present in food plants and animals,
it is likely that small amounts of PMI proteins from various sources have always been present
in food. PMI proteins have been found in food plant species such as Brassica species (Chen
et al., 1989), and in seeds of soybeans and other legumes (Lee and Matheson, 1984).
Genes encoding putative PMI proteins have been characterised from a number of other food
organisms, including yeast and pigs (Proudfoot et al., 1994).
39
The presence of PMI proteins in a variety of food sources supports a history of safe use of
PMI proteins.
4.4.2 Similarities with known protein toxins
Vip3Aa20
Study submitted:
Harper, B. (2007) Vip3Aa20: Assessment of amino acid sequence homology with known toxins.
Syngenta Seeds Biotechnology Report No. SSB-116-07.
Bioinformatic analysis was done to assess the Vip3Aa20 protein for any amino acid
sequence similarity with known protein toxins. The Vip3Aa20 sequence (789 amino acids,
Entrez Accession No. ABG20429) was systematically compared with the latest posting of the
National Center for Biotechnology Information (NCBI) Entrez Protein Database (NCBI, 2007)
using the BLASTP program (Altschul et al., 1997). The NCBI Entrez protein database is a
public database containing over 5 million sequences, representing a good source to identify
any potential protein toxin homologies.
The BLASTP program is frequently used for searching protein sequences for sequence
similarities. The BLAST algorithm searches for short stretches or domains of sequence
similarity by performing local alignments. This detects more similarities that would be found
using the entire query sequence length.
The analysis was designed to identify any proteins in the database that showed significant
amino acid sequence identity to Vip3Aa20, indicating they may be closely related to
Vip3Aa20, and whether any proteins with significant sequence identity to Vip3Aa20 are
known to be toxins, indicating possible implications for the toxic potential of Vip3Aa20. All
database sequences with an Expect value (E-value) of 10 or lower were identified by default
by the BLASTP program. The E-value reflects the degree of amino acid similarity between a
pair of sequences and can be used to evaluate the significance of the alignment.
Comparisons between highly homologous proteins yield E-values approaching zero,
indicating the very low probability that such matches would occur by chance. A larger Evalue indicates a lower degree of similarity. Typically, alignments between two sequences
will need to have an E-value of less than 1 x 10-5 for them to be considered to have
significant homology.
A threshold E-value was calculated below which similarity to the query sequence (Vip3Aa20)
is considered to be statistically significant and not a random result. This was done by
randomly shuffling the primary Vip3Aa20 sequence to create five different 789 amino acid
sequences and using these as target sequences. The shuffled sequences all have the same
overall amino acid composition as Vip3Aa20 but are unlikely to have amino acid sequence
similarity with either Vip3Aa20 or proteins in the NCBI Entrez protein database. Analysis of
these target sequences provides an estimate of the background incidence of random hits that
can be expected for any sequence with the same amino acid composition as Vip3Aa20. The
search results using the five shuffled versions of Vip3Aa20 sequence identified hits with Evalues that ranged from 8 x 10-8 to 8.0. Therefore, a score of 8 x 10-8 was set as the
threshold, and scores below this were considered to represent proteins with significant amino
acid sequence similarity and were those proteins were evaluated for biological significance.
The analysis returned 34 entries with E-values below 8 x 10-8. All identified proteins were
identified as vegetative insecticidal proteins, of which 31 were from B. thuringiensis. The
analyses did not demonstrate any significant similarity between the Vip3Aa20 protein and
other proteins that may potentially be toxic to humans or other animals.
40
PMI
Study submitted:
Harper, B. (2007) Phosphomannose isomerise protein (Entrez accession number AA24109):
Assessment of amino acid sequence homology with known toxins. Syngenta Seeds Biotechnology
Report No. SSB-117-07.
To determine whether the PMI protein sequence has any significant homology with known
protein toxins, the PMI sequence (391 amino acids) was systematically compared to the
latest posting of the National Centre for Biotechnology Information (NCBI) Entrez Protein
Database (NCBI, 2007) containing all the publicly available protein sequences, as described
in the preceding section.
Using five randomly shuffled versions of the PMI sequence (as described above for
Vip3Aa20) the appropriate cut-off E-value was determined to be 0.51 and amino acid
sequences with E values lower than this were considered to be significantly similar and were
evaluated for biological significance.
The database search returned 332 entries with E-values below 0.51. Of these entries, 271
were identified as known or putative phosphomannose isomerise enzymes from 178 species
(E-values from 0.0 to 0.39)
The PMI query sequence showed no significant sequence similarity to any proteins identified
as, or known to be, toxins.
4.4.3 Digestibility
See Section 4.5.
4.4.4 Acute oral toxicity study
Proteins which cause toxicity act via acute mechanisms and generally at very low doses
(Sjoblad et al., 1992). Therefore, when a protein demonstrates no acute oral toxicity at a
high dose level using a standard laboratory mammalian test species, this supports the
determination that the protein will be non-toxic to humans and other mammals, and will not
present a hazard under any realistic exposure scenario, including long-term exposures.
Acute oral toxicity studies using mice were conducted to examine the potential toxicity of the
Vip3Aa20 and PMI proteins. As it is very difficult to extract and purify sufficient quantities of
the subject protein from transgenic corn plants for the acute oral toxicity studies, it has
become standard practice to instead use equivalent proteins that have been produced using
bacterial expression systems. For these studies, E. coli-produced Vip3Aa20 and PMI
proteins were used as the test substances. The equivalence of the E. coli- and MIR162 cornproduced proteins was established using a range of methods including Western blot analysis,
peptide mass mapping analysis, bioactivity assays and glycosylation analysis (see Section
4.2).
Vip3Aa20
Study submitted:
Draper, C. (2007) MIR162 VIP3A-0106 single dose oral toxicity study in mice. Central Toxicology
Laboratory Study No. AM7543-REG. Macclesfield, Cheshire, UK.
41
Test material
Vehicle
Test Species
Dose
Control
Vip3Aa20 protein preparation produced in E. coli (MIR162VIP3A0106, 84% purity)
Corn oil
Alpk:APfCD-1 mice (five males and five females per group)
1250 mg Vip3Aa20 protein/kg body weight (1488 mg test
substance/kg bw) dosed orally by gavage
vehicle only
The mice received 1250 mg/kg bw Vip3Aa20 as two fractions (each 2.5 mL) dosed 2 hours
apart on a single day. This was the highest dose that could be physically administered to the
animals, because of limitations of solubility and viscosity. Mice were observed for two
weeks. Clinical observations, body weights and food consumption were measured daily
throughout the study. At the end of the scheduled study period (day 15), the animals were
killed and examined post mortem. Cardiac blood samples were taken for clinical pathology
(haematology and blood clinical chemistry). Selected organs (adrenal glands, brain,
epididymides (males), heart, ovaries (females), kidneys, liver, spleen and testes (males))
were weighed. Specified tissues were examined in situ and processed for subsequent
histopathology examination.
There were no mortalities and no test substance-related clinical signs in either males or
females. Diarrhoea was observed in both the control and treated groups in both sexes on
days 1 and 2 and was considered to be due to the use of corn oil as a vehicle/control
substance.
The body weights of treated females were significantly higher on days 2 and 3, by 3% and
2% respectively compared to controls. Body weights were not statistically significantly higher
on any of the other 12 study days. The slight increases on days 2 and 3 are not considered
to be adverse and are considered to be sporadic differences incidental to treatment.
There were no obvious differences in food consumption between the control and treated
groups in either sex.
There were no statistically significant differences in haematology parameters between
treated and control animals.
There were no statistically significant differences in blood clinical chemistry parameters
between treated and control females, whereas in males the group mean plasma aspartate
aminotransferase level was statistically significantly lower in the treated group compared to
the control (51.0 vs. 77.8). However, the group mean value for the test group was within the
historical control range of means (43.8-59.6) while the group mean value for the control
group was higher than seen in the historical database. Although the difference is statistically
significant, it is considered to be unrelated to treatment. No statistically significant differences
in other blood clinical chemistry parameters or organ pathology were observed.
There were no statistically significant differences in organ weights between the treated and
control animals.
One treated male was observed to have pelvic dilation of the kidney. This is considered to
be spontaneous in nature and unrelated to treatment. This finding has been seen previously
in control animals of this strain. One control and one treated male were observed to have
hydronephrosis of the kidney. This microscopic finding is synonymous with the macroscopic
observation of pelvic dilation of the kidney. Hydronephrosis has previously been seen in
control animals of this strain and is considered to be unrelated to treatment.
42
In summary, the administration of 1250 mg Vip3Aa20/kg bw to mice produced no adverse
effects on clinical condition, body weight, food consumption, clinical pathology, organ
weights, macroscopic or microscopic pathology that were considered to be related to
Vip3Aa20.
PMI
Study submitted:
Kuhn, J.O. (1999) Phosphomannose isomerise (sample PMI-098): Acute oral toxicity study in mice.
Stillmeadow Inc Study No. 4708-98.
Test material
Vehicle
Test Species
Dose
Control
Phosphomannose isomerase preparation from E. coli (60%
phosphomannose isomerase enzyme)
0.5% w/v aqueous carboxymethyl cellulose
HSD:ICR albino mice (seven males and six females)
5050 mg/kg bw (equivalent to 3080 mg/kg bw PMI protein) in
two gavage doses, 1 hour apart.
vehicle only
The mice received a single dose of 3080 mg/kg bw PMI and were observed for two weeks.
Parameters evaluated included body weights and detailed clinical observations. At the end of
the study all animals were killed and examined post mortem. Brain, liver, kidneys and spleen
were weighed.
One male in the control group and two in the test group died shortly after dosing or were in
distress after dosing and subsequently died. Necropsy revealed perforated oesophagi in
these animals, a sign of gavage error and not test-substance related. One replacement
animal was available for each group and dosed in the same manner on day 0. There was no
test article-related mortality during the study.
No clinical signs of toxicity were observed in either group. There were no test-substance
related effects on body weight, organ weights or gross pathology.
Under the conditions of this study, the acute oral LD50 of the PMI protein in mice is greater
than 3080 mg /kg bw.
4.4.5 Conclusion
The data from the bioinformatics analyses and acute oral toxicity studies, together with
evidence of a history of presence of the novel proteins in food, indicate that neither of the
proteins is likely to be toxic to mammals, including humans.
4.5
Potential allergenicity of novel proteins
While the majority of food allergens are proteins, only a small fraction of the many proteins in
foods are allergenic, therefore the likelihood of a novel protein being allergenic is small.
However, as the use of gene technology can result in additional protein diversity being added
to the food supply, the potential allergenicity of any novel protein present in a GM food is
considered as part of the safety assessment.
The potential allergenicity of novel proteins is evaluated using an integrated, step-wise, caseby-case approach relying on various criteria used in combination, since no single criterion is
sufficiently predictive of either allergenicity or non-allergenicity.
43
The assessment focuses on: the source of the novel protein; any significant amino acid
sequence similarity between the novel protein and known allergens; the structural properties
of the novel protein, including susceptibility to digestion, heat stability and/or enzymatic
treatment; and specific serum screening if the novel protein is derived from a source known
to be allergenic or has amino acid sequence similarity with a known allergen. Applying this
approach systematically provides reasonable evidence about the potential of the novel
protein to act as an allergen.
4.5.1 Source of novel proteins
Vip3Aa20
The Vip3Aa20 protein is derived from B. thuringiensis strain AB88. B. thuringiensis has been
used as the active ingredient in insecticidal sprays for the last 40 years and during that period
has not been associated with any reported allergic reactions associated with its use (Nester
et al., 2002).
PMI
The gene encoding PMI was initially isolated from E. coli, which has no history of
allergenicity.
4.5.2 Similarity to known allergens
A comparison of the amino acid sequence of the introduced proteins to known protein
allergens is one of the steps in a multilevel decision tree to assess allergenic potential
(Metcalfe et al., 1996).
Vip3Aa20
Study submitted:
Harper, B. (2007) Vip3Aa20: Assessment of amino acid sequence homology with known allergens.
Syngenta Seeds Biotechnology Report No. SSB-102-07.
To determine whether Vip3Aa20 has significant sequence identity to proteins known or
suspected to be allergens, the amino acid sequence of Vip3Aa20 (Entrez Accession Number
ABG20429) was compared to the Syngenta Biotechnology, Inc. (SBI) Allergen Database
(version 5.0, 2006). The SBI Allergen Database, containing 1735 non-redundant entries,
was compiled from entries in public protein databases identified as allergens or putative
allergens. Additional amino acid sequences of proteins identified as allergens or putative
allergens in the scientific literature were also included in the database.
Two approaches were used to identify potential similarity between Vip3Aa20 and known or
potential allergens. In the first approach, sequential 80-amino acid peptides of the Vip3Aa20
protein sequence were compared to the protein sequences in the SBI Allergen Database using
the FASTA search algorithm to assess structural similarity.
Any matches of greater than 35% amino acid identity were identified as having significant
identity to the allergen sequence. Related protein sequences are considered to be potentially
cross-reactive if linear identity is 35% or greater in a segment of 80 or more amino acids
(FAO/WHO, 2001). No comparisons met this criterion, leading to the conclusion that Vip3Aa20
does not have significant sequence homology with any known allergenic protein.
In the second approach, the Vip3Aa20 protein sequence was also screened for any matches
of eight contiguous amino acids with any allergen sequences in the SBI Allergen Database.
44
This screens for short, local regions of amino acid identity that might indicate the presence of
common T-cell binding epitopes. There were no matches of eight contiguous amino acids,
representing putative IgE binding epitopes, between Vip3Aa20 and any entry in the SBI
Allergen Database.
PMI
Study submitted:
Harper, B. (2007) Phosphomannose isomerise (Entrez accession number AA24109): Assessment of
amino acid sequence homology with known allergens. Syngenta Seeds Biotechnology Report No.
SSB-101-07, including Report by C. Hilger (2004) titled ‘Evaluation of Syngenta Protein Samples POI1
and POI2 for Reactivity with Serum IgE from a Petient Allergic to Frog Alpha-Parvalbumin from Rana
Species’.
To determine whether PMI has any significant homology with allergenic proteins, the PMI
protein sequence was systematically compared to the SBI Allergen database (version 5.0,
2006).
Overall similarity was examined by comparing sequential 80-amino acid sequences covering
the entire PMI protein sequence to the allergen sequences using the FASTA search
algorithm. Any 80-amino acid peptide having greater than 35% amino acid identity was
defined as having significant similarity to the allergen sequence (FAO/WHO, 2001). There
was no significant similarity between any of the sequential PMI 80-amino acid peptides and
any entries in the database, indicating that PMI does not share overall sequence homology
with any known allergenic protein.
The PMI sequence was also screened for matches of eight or more contiguous amino acids
to identify any short local regions of identity that might indicate the presence of common IgE
binding epitopes. There was one region of eight identical amino acids between PMI and the
known allergen α-parvalbumin from Rana species CH2001 (Entrez Accession Number
CAC83047) (Hilger et al., 2002).
One case of severe food-induced anaphylaxis in a single individual who consumed
Indonesian frogs legs has been shown to be due to the protein α-parvalbumin from Rana
species (Hilger et al., 2002). The same protein from Rana esculenta (the common edible
frog) elicited no response in serum from the same individual.
To determine if IgE antibodies present in this patient’s serum recognised PMI, Syngenta sent
a sample of PMI to Dr Hilger’s laboratory for cross-reactivity analysis (Hilger, 2004). The PMI
sample used in the serum screen analysis was the bacterially derived PMI that had been
shown to be a suitable surrogate for plant-derived PMI protein (see Section 4.2).
No cross reactivity between the human serum IgE and PMI occurred. This indicates that the
allergic patient’s serum IgE does not recognise any portion of the PMI protein as an
allergenic epitope. Therefore, the short stretch of sequence similarity between PMI and Rana
species α-parvalbumin is not biologically relevant and has no implications for the potential
allergenicity of PMI.
4.5.3 Digestibility
Resistance to hydrolysis by digestive proteases has been observed in several food allergens
(Astwood and Fuchs, 1996), therefore a correlation exists between resistance to digestion by
pepsin and allergenic potential. As a consequence, one of the criteria for assessing potential
allergenicity is to determine the stability of novel proteins in conditions mimicking human
digestion.
45
Proteins that are rapidly degraded in such conditions are considered less likely to be involved
in eliciting an allergic response. A pepsin digestibility assay was conducted to determine the
digestive stability of the Vip3Aa20 and PMI proteins.
In addition to the pepsin protocol using simulated gastric fluid (SGF), a second digestibility
study was done using simulated intestinal fluid (SIF) containing pancreatin, which is a
mixture of enzymes including amylase, trypsin, lipase, ribonuclease and protease. The
relevance of the SIF study however is limited because ordinarily an ingested protein would
first be exposed to pepsin-mediated hydrolysis in the acidic environment of the stomach
before being subject to further digestion in the small intestine.
Vip3Aa20
Simulated gastric fluid study
Study submitted:
Stacy, C. (2007) In vitro digestibility of Vip3Aa20 under simulated mammalian gastric conditions.
Syngenta Seeds Biotechnology Report No. SSB-038-06.
The in vitro digestibility of Vip3Aa20 protein in SGF containing pepsin was evaluated by
SDS-PAGE and Western blot analysis. Vip3Aa20 protein affinity purified from MIR162 corn
was assessed, as was Vip3Aa20 derived from recombinant E. coli.
Digestibility of the protein in SGF was measured by taking samples at selected time points
(0, 1, 2, 5, 10, 20, 30 and 60 minutes) and subjecting these to SDS-PAGE. Proteins were
visualized by staining the gel or by transferring the protein to a membrane for Western blot
analysis. No intact microbially-derived Vip3Aa20 protein was detectable by Western blot
after one minute exposure to SGF. No intact plant-derived Vip3Aa20 was evident after two
minutes exposure to SGF, as assessed by Western blot.
The plant-derived Vip3Aa20 preparation showed an additional band of ~60kDa after one
minute incubation with SGF, but this band was not visible after 2 minutes digestion. It likely
represents a degradation product of Vip3Aa20. Faint, immunoreactive, low molecular weight
bands (~8 kDa) were observed following one minute incubation in SGF.
These bands, that likely represent proteolytic degradation products of Vip3Aa20, gradually
decreased in intensity following longer incubations with SGF.
Simulated intestinal fluid study
Study submitted:
Stacy, C. (2007) In vitro digestibility of Vip3Aa20 (MIR162VIP3A-0106) under simulated mammalian
intestinal conditions. Syngenta Seeds Biotechnology Report No. SSB-002-07.
The digestibility of Vip3Aa20 protein in SIF containing pancreatin was assessed using
Western blot analysis.
Digestibility of Vip3Aa20 in SIF was measured by incubating microbially-produced Vip3Aa20
with SIF, taking samples at selected time points (0, 5, 15, 30 minutes, 1, 2, 3, 6, 24 and 48
hours), subjecting these to SDS-PAGE and also Western blot analysis using immunoaffinitypurified polyclonal antibodies. The full length Vip3Aa20 was rapidly digested, with no intact
protein detectable after 5 minutes of incubation in SIF. A proteolytic fragment of
approximately 62 kDa was observed at the 5 minute time point. After 15 minutes digestion a
second proteolytic fragment of ~55 kDa was also visible on the Western blot.
46
The intensity of these bands slowly decreased following longer digestion times, but both
bands were still detectable after 48 hours digestion. The intensity of these bands is low
relative to the initial, intact Vip3Aa20 band, indicating that the majority of Vip3Aa20 is rapidly
digested in SIF.
PMI
Study submitted:
Privalle, L. (1999) In vitro digestibility of PMI protein under simulated mammalian gastric and intestinal
conditions. Novartis Seeds Biotechnology Report No. NSB-002-99.
PMI was digested in simulated gastric fluid containing pepsin and in simulated intestinal fluid
containing pancreatin. Samples were examined by SDS-PAGE. PMI was degraded rapidly by
pepsin: no PMI was detected by SDS-PAGE upon immediate sampling of the reaction mix (0
seconds). When the pepsin was diluted to 0.0001X of the standard concentration, no PMI
remained after 10 minutes of incubation. Similarly, no PMI enzymatic activity was detectable
after 10 minutes under these conditions.
PMI was degraded by pancreatin in simulated intestinal fluid after two minutes. In the unlikely
event that PMI survived digestion by pepsin, it would be digested in the mammalian intestinal
environment by pancreatin.
4.5.4 Thermolability
Vip3Aa20
Study submitted:
Stacy, C. (2007) Effect of temperature on the stability of Vip3Aa20 protein. Syngenta Seeds
Biotechnology Report No. SSB-039-06.
The effect of temperature on Vip3Aa20 was determined by incubation for 30 minutes at a
range of temperatures (4ºC, 25ºC, 37ºC, 65ºC, and 95ºC) followed by an insect bioassay
against fall armyworm (S. frugiperda) larvae. Vip3Aa20 exposed to different temperatures
were incorporated at increasing concentrations into insect diet and assessed for insecticidal
activity using freshly hatched larvae. No reduction in Vip3Aa20 bioactivity was observed
following incubation at 25ºC or 37ºC compared to 4ºC. Incubation at 65ºC and 95ºC resulted
in very low mortality, indicating a loss of bioactivity. The data support the conclusion that
Vip3Aa20 is unstable upon heating at temperatures of 65ºC and above.
PMI
Study submitted:
Hill, K. (2003) Effects of temperature on the stability of phosphomannose isomerise. Syngenta Seeds
Biotechnology Report No. SSB-013-03.
The heat stability of PMI was evaluated. Loss of enzyme activity was used to determine the
instability of the protein after exposure to various temperatures (4, 25, 37, 55, 65 and 95ºC)
for 30 minutes. Incubation at ambient temperature (25ºC), or 37ºC for 30 minutes had little
effect on enzyme activity. At 55 ºC, approximately half the enzymatic activity was lost.
Incubation at 65ºC and 95ºC essentially inactivated the protein.
47
4.5.5 Conclusion
A range of information and data have been provided to assess the potential allergenicity of
the two novel proteins in MIR162 corn. Both the Vip3Aa20 and PMI proteins are sourced
from bacteria with a history of human exposure and no association with allergenicity.
Bioinformatic analysis indicates that Vip3Aa20 does not share any significant sequence
similarity with known or suspected allergens. The PMI protein sequence was found to share
a region of sequence identity of eight contiguous identical amino acids with a known allergen,
the protein α-parvalbumin from Rana species. However, further analysis using serum from
the single individual known to have demonstrated IgE-mediated allergy to this specific parvalbumin did not indicate any cross-reactivity with the PMI protein. Both the Vip3Aa20
and PMI proteins are rapidly degraded in simulated mammalian gastric fluid and both are
labile upon heating to temperatures of 65ºC and above. The weight-of-evidence indicates
that the two novel proteins in MIR162 corn are unlikely to allergenic.
4.6
Conclusion
MIR162 corn expresses two novel proteins, Vip3Aa20 and PMI. Vip3Aa20 is expressed at
moderate levels in the grain, with a mean concentration of 44 µg/g dry weight. PMI is
expressed at relatively low levels in the grain, with a mean concentration of 2 μg/g dry
weight.
A large number of studies have been done with the Vip3Aa20 and PMI proteins to confirm
their identity and physicochemical and functional properties as well as to determine their
potential toxicity and allergenicity. These studies have demonstrated that both proteins
conform in size and amino acid sequence to that expected.
Vip3Aa20 does not exhibit any post-translational modification including glycosylation, and
also demonstrate the expected insecticidal activity. PMI exhibited the expected enzymatic
activity.
Bioinformatic studies with Vip3Aa20 have confirmed the absence of any significant amino
acid sequence similarity to known protein toxins or allergens. Although PMI has a short
region of sequence identity with a known allergen, subsequent analysis using human serum
showed this to be of no biological relevance. Digestibility studies have demonstrated that
both proteins would be rapidly degraded in the stomach following ingestion, similar to other
dietary proteins. Acute oral toxicity studies in mice with both proteins have also confirmed
the absence of toxicity in animals. The safety of the Vip3Aa protein is further supported by
the long history of safe use of Bt microbial pesticides. Taken together, the evidence indicates
that both proteins are unlikely to be toxic or allergenic to humans.
5.
COMPOSITIONAL ANALYSES
The main purpose of compositional analysis is to determine if any unexpected changes in
composition have occurred to the food and to establish its nutritional adequacy.
Compositional analysis can also be important for evaluating the intended effect where there
has been a deliberate change to the composition of food.
The classic approach to the compositional analysis of GM food is a targeted one; rather than
analysing every single constituent, which would be impractical, the aim is to analyse only
those constituents most relevant to the safety of the food or that may have an impact on the
whole diet. Important analytes therefore include the key nutrients, toxicants and antinutrients for the food in question (FAO, 1996). The key nutrients and anti-nutrients are those
components in a particular food that may have a substantial impact in the overall diet.
48
They may be major constituents (fats, proteins, carbohydrates or enzyme inhibitors as antinutrients) or minor constituents (minerals, vitamins). Key toxicants are those toxicologically
significant compounds known to be inherently present in an organism, such as compounds
whose toxic potency and level may be significant to health (e.g. solanine in potatoes).
In the case of corn, the key components that should be considered in the comparison include
protein, fat, carbohydrate, amino acids, fatty acids, vitamins, minerals, and the anti-nutrient
phytic acid (OECD, 2002).
Most crops exhibit considerable variability in their nutrient composition. Environmental
factors and the genotype of the plant have an enormous impact on composition. Variation in
these nutrient parameters is a natural phenomenon and is considered to be normal.
Therefore, in addition to a comparison of the composition of the GM food to a closely related
non-GM control, further comparison is made to the range of natural variation found in the
conventional (non-GM) food crop.
5.1
Study design and conduct
To determine whether unexpected changes have occurred in the composition of MIR162
corn as a result of the modification, and to assess its nutritional adequacy, compositional
analyses were done on forage and grain samples collected from MIR162 transgenic hybrid
grown under field conditions with a non-transgenic, near-isogenic hybrid as the conventional
control.
The MIR162 corn hybrid used for this study is a hybrid between the BC4F4 generation
(MIR162 progenitor backcrossed 4x to inbred line NP2276 then self-fertilised 4x) and a
conventional inbred corn line, NP2391 (see Section 3.4). The MIR162 hybrid is designated
NP2276(MIR162)/NP2391. The line used as the comparator for this study is a conventional
NP2276/NP2391 hybrid, which has a genetic background representative of the MIR162
hybrid lines, but without the transferred genes.
Field trials were conducted in the United States in 2005 at six replicated sites. The field sites
were located in regions of the U.S. that represent the agricultural regions where the hybrid
varieties would typically be cultivated7. At each location, one hybrid pair, composed of the
MIR162 hybrid and the corresponding non-transgenic hybrid, was grown in a randomized
complete block design, with three replicates for each genotype. All the corn plants were
grown according to local agronomic practices, including treatment of both hybrids with
conventional pesticides as needed. Plants were self-pollinated by hand and the developing
ears were bagged to avoid cross-pollination.
Selection of analytes for measurement in forage and grain was based on recommendations
of the OECD (OECD, 2002) for comparative assessment of new varieties of corn. Forage
and grain samples were analysed for 65 different components (7 in forage, and 56 in grain).
Compositional analysis of the forage samples included proximates (ash, fat, moisture, protein
and carbohydrates), acid detergent fibre (ADF), neutral detergent fibre (NDF) and minerals
(calcium and phosphorus). For the grain samples, compositional analysis included
proximates, ADF, NDF, total dietary fibre, amino acids, fatty acids, vitamins (A, B1, B2, B6, E,
niacin and folic acid), anti-nutrients (phytic acid, raffinose and trypsin inhibitor), secondary
plant metabolites (furfural, ferulic acid and p-coumaric acid), minerals (calcium, copper, iron,
magnesium, manganese, phosphorus, potassium, selenium, sodium and zinc) and inositol.
All compositional analyses were conducted using methods published and approved by AOAC
International, or other specified industry-standard analytical methods.
7
The sites used were: Stanton, Minnesota; Janesville, Wisconsin; Alleman, Iowa; Seward, Nebraska;
Bloomington, Illinois; Bondville, Illinois.
49
Following compositional analysis, the results were statistically analysed using analysis of
variance across locations for the hybrid pair. For each component analysed for MIR162
corn, the result was compared with that from the conventional counterpart. The statistical
significance of the difference between MIR162 and the control mean values was determined
using a standard F-test. An F-test probability of <5% indicates that the difference between
the genotypes was statistically significant at the 5% level.
An F-test was also used to assess the significance of a potential location x genotype
interaction. A significant outcome (F-test probability <5%) indicates that the effect of
genotype was not consistent across all locations, and undermines the validity of the
comparison of genotypes across locations. In these cases, the average analyte levels
measured at each location were compared to values reported in the literature. For all
analytes measured, the average levels across locations were compared with the range of
natural variation, as reported in the International Life Sciences Institute (ILSI) Crop
Composition Database (ILSI, 2006) and the OECD consensus document (OECD, 2002).
The results of the comparisons of MIR162 and the conventional counterpart are presented in
Tables 9-17.
50
Table 9: Proximate composition of forage from a MIR162 hybrid and a non-transgenic hybrid
Moisture
(% FW)
Protein
(% DW)
Total Fat
(% DW)
Ash
(% DW)
Carbohydrates
(% DW)
ADF
(% DW)
NDF
(% DW)
MIR162
average
range
71.2
66.2-77.2
7.2
3.1-10.1
1.5
0.8-1.9
4.1
3.1-5.8
87.3
82.9-90.7
28.2
23.6-34.2
43.2
35.1-56.1
Non-transgenic
average
range
70.5
64.1-75.8
7.3
3.4-8.9
1.6
0.4-2.3
4.0
2.9-5.7
87.1
83.6-91.7
28.8
23.3-34.8
38.8
32.1-46.9
Percent difference from nontransgenic hybrid1
0.99
-0.91
-5.71
1.11
0.13
-2.10
11.57
F-test Probability for Genotype2
0.184
0.711
0.421
0.796
0.694
0.635
0.010
F-test Probability for Location x
Genotype Interaction
0.730
0.645
0.100
0.948
0.973
0.687
0.286
ILSI (2006)
70.2
7.78
2.039
4.628
85.6
27.00
41.51
49.1 - 81.3
3.14 - 11.57
0.296 - 4.570
1.527 - 9.638
76.4 - 92.1
16.13 - 47.39
20.29 - 63.71
945
945
921
945
945
945
945
25.6 - 34
40 - 48.2
average
range
N
OECD (2002)
range
62 - 78
4.7 - 9.2
1.5 - 3.2
1 ((MIR162-non-transgenic)/non-transgenic)*100.
2 Statistically significant F-test probability (<0.05) indicated in bold and italics type.
2.9 - 5.7
51
Table 10: Calcium and phosphorus composition of forage from a MIR162 hybrid and a non-transgenic hybrid
Calcium
(mg/kg DW)
Phosphorus
(mg/kg DW)
MIR162
average
range
2106
1720-2930
1997
1270-2240
Non-transgenic
average
range
2039
1440-2620
2079
1760-2560
Percent difference from non-transgenic hybrid2
3.24
-3.93
F-test Probability for Genotype3
0.484
0.153
F-test Probability for Location x Genotype
Interaction
0.944
0.066
2028.6
713.9 - 5767.9
481
2066.1
936.2 - 3704.1
481
0.15 - 0.31% DW
0.20 - 0.27%
DW
average
range
N
ILSI (2006)
OECD (2002)
1 For
range
conversion, 1% = 10,000 mg/kg.
2 ((MIR162-non-transgenic)/non-transgenic)*100.
3 Statistically
significant F-test probability (<0.05) indicated in bold and italics type.
52
Table 11: Proximate composition of grain from a MIR162 hybrid and a non-transgenic hybrid
Moisture1
(% FW)
Protein
(% DW)
Fat
(% DW)
Ash
(% DW)
Carbohydrates
(% DW)
ADF
(% DW)
NDF
(% DW)
TDF
(% DW)
Starch
(% DW)
63.1
54.8-68.1
MIR162
average
range
10.3
9.5-11.5
9.8
7.5-11.2
3.8
3.3-4.6
1.4
1.1-1.6
85.0
83.2-87.1
5.0
3.3-7.0
11.7
10.1-13.0
16.8
14.1-19.4
Non-transgenic
average
range
10.5
9.4-12.0
9.6
7.1-11.0
3.8
3.0-4.4
1.3
1.1-1.5
85.3
83.3-88.1
4.6
3.3-6.2
11.1
9.5-12.8
16.3
14.3-17.8
-2.33
1.97
0.29
9.33
-0.36
7.70
5.32
3.18
-2.82
F-test Probability for Genotype3
0.103
0.904
0.012
0.062
0.154
0.016
0.092
0.015
F-test Probability for Location x
Genotype Interaction
0.832
0.538
0.381
0.696
0.309
0.703
0.653
0.455
10.30
6.15 - 17.26
1434
3.555
1.742 - 5.823
1174
1.439
0.616 - 6.282
1410
84.6
77.4 - 89.5
1410
4.05
1.82 - 11.34
1350
11.23
5.59 - 22.64
1349
16.43
8.85 - 35.31
397
57.7
26.5 - 73.8
168
3.0 - 4.3
8.3 - 11.9
11.1
Percent difference from nontransgenic hybrid2
ILSI (2006)
average
range
N
11.3
6.1 - 40.5
1434
OECD (2002)
range
7.0 - 23
6 - 12.7
3.1 - 5.8
1.1 - 3.9
82.2 - 82.9
1 Moisture levels in grain not subject to analysis of variance as grain was mechanically dried after harvest.
2 ((MIR162-non-transgenic)/non-transgenic)*100.
3 Statistically significant F-test probability (<0.05) indicated in bold and italics type.
53
64.9
60.6-69.2
Table 12: Mineral composition of grain from a MIR162 hybrid and a non-transgenic hybrid
Copper
(mg/kg DW)
1.3
0.96-1.95
Iron
(mg/kg DW)
20.2
17.3-22.9
Magnesium
(mg/kg DW)
1252
1090-1480
Manganese
(mg/kg DW)
6.3
4.14-7.97
Phosphorus
(mg/kg DW)
3173
2810-3550
Potassium
(mg/kg DW)
3352
3160-3710
Selenium2,3
(mg/kg DW)
<LOQ - 0.414
N/A
Sodium2,3
(mg/kg DW)
<LOQ - <LOQ
N/A
Zinc
(mg/kg DW)
21.7
18.8-24.3
MIR162
average
range
Calcium
(mg/kg DW)
38.1
29.4-47.2
Non-transgenic
average
range
35.3
25.7-44.0
1.2
1.00-1.58
19.2
15.7-22.5
1218
960-1470
6.1
4.59-8.01
3073
2710-3400
3357
2950-3660
<LOQ - 0.531
N/A
<LOQ - <LOQ
N/A
21.5
19.2-23.8
Percent difference from
non-transgenic hybrid4
7.95
9.63
5.05
2.78
4.33
6.25
-0.15
N/A
N/A
1.01
F-test Probability for
Genotype5
0.002
0.073
0.011
0.102
0.078
0.030
0.916
0.606
F-test Probability for
Location x Genotype
Interaction
0.148
0.360
0.685
0.288
0.697
0.136
0.291
0.326
46.4
1.75
21.81
1193.8
6.18
31.75
21.6
0.73 - 18.5
0.05 - 0.75
0.17 - 731.54
6.5 - 37.2
1344
1249
3842
1810.0 6030.0
1257
0.2
12.7 - 208.4
3273.5
1470.0 5330.0
1349
89
223
1257
234 - 750
mg/100g DW
320 - 720
mg/100g DW
0.001 - 0.1
mg/100g DW
0 - 150
mg/100g DW
1.2 - 3.0
mg/100g DW
average
ILSI6 (2006)
range
N
OECD (2002)
10.42 - 49.07 594.0 - 1940.0
1255
1.69 - 14.30
1257
1256
range
3 -100
0.09 - 1.0
0.1 - 10
82 - 1000
mg/100g DW mg/100g DW mg/100g DW mg/100g DW
1 For conversion, 1 mg/100g = 10 mg/kg.
2 Where some values were <LOQ, statistical comparison was not possible so only the range is shown.
3 LOQ for selenium 0.056-0.057 mg/kg DW, LOQ for sodium 110-114 mg/kg DW.
4 ((MIR162-non-transgenic)/non-transgenic)*100.
5 Statistically significant F-test probabilities (<0.05) indicated in bold and italics type.
6 Below LOQ values are not included.
54
Table 13: Vitamin analysis of grain from a MIR162 hybrid and a non-transgenic hybrid
Vitamin A2
ß-Carotene
Vitamin B1
Thiamine
Vitamin B2
Riboflavin
Vitamin B3
Niacin
Vitamin B6
Pyridoxine
Vitamin B9
Folic Acid
Vitamin E
a-Tocopherol
(mg/100g DW)
(mg/100g DW)
(mg/100g DW)
(mg/100g DW)
(mg/100g DW)
(mg/100g DW)
(mg/g DW)
MIR162
average
range
0.277
0.241-0.316
0.393
0.358-0.433
0.190
0.112-0.238
2.37
2.11-2.83
0.565
0.434-0.694
0.028
0.021-0.034
0.01
0.97-1.54
Non-transgenic
average
range
0.294
0.244-0.358
0.392
0.339-0.443
0.180
0.144-0.226
2.47
2.03-3.15
0.605
0.486-0.738
0.028
0.024-0.033
0.01
1.10-1.54
Percent difference from nontransgenic hybrid3
-5.78
0.03
5.69
-4.12
-6.55
-1.63
-5.46
F-test Probability for Genotype4
0.001
0.983
0.314
0.093
0.002
0.471
0.016
F-test Probability for Location x
Genotype Interaction4
0.048
0.952
0.278
0.639
0.765
0.036
0.917
0.684
0.019 - 4.681
276
0.530
0.126 - 4.000
894
0.125
0.050 - 0.236
704
2.376
1.037 - 4.694
415
0.644
0.368 - 1.132
415
0.0651
0.0147 - 0.1464
895
0.0103
0.0015 - 0.0687
863
2.3 - 8.6
mg/kg DW
0.25 - 5.6
mg/kg DW
9.3 - 70
mg/kg DW
4.6 - 9.6
mg/kg DW
ILSI (2006)
OECD (2002)
average
range
N
range
1 For
conversion, 1 mg/100g = 10 mg/kg.
direct comparison to literature values, original units of RE/g DW were converted to units of mg/100 g DW based on 1 RE = 6 µg beta-carotene.
Institute for Laboratory Animal Research (1995).
3 ((MIR162-non-transgenic)/non-transgenic)*100.
4 Statistically significant F-test probabilities (<0.05) indicated in bold and italics type.
2 For
55
Table 14: Location means of ß-carotene and folic acid in grain from a MIR162 hybrid and a non-transgenic hybrid
Location 1
Location 2
Location 3
Location 5
Location 6
Location 8
Vitamin A1
ß-Carotene
(mg/100g DW)
Vitamin B9
Folic Acid
(mg/100g DW)
MIR162
0.283
0.027
Non-transgenic
0.296
0.028
MIR162
0.304
0.032
Non-transgenic
0.352
0.029
MIR162
0.272
0.027
Non-transgenic
0.280
0.024
MIR162
0.266
0.029
Non-transgenic
0.286
0.032
MIR162
0.292
0.024
Non-transgenic
0.291
0.025
MIR162
0.243
0.025
Non-transgenic
0.260
0.029
average
ILSI (2006)
1
range
0.684
0.0651
0.019 - 4.681
0.0147 - 0.1464
N
276
895
For direct comparison to literature values, original units of RE/g DW were converted to units of mg/100 g DW based on 1 RE = 6 µg beta-carotene.
Institute for Laboratory Animal Research (1995).
56
Table 15: Amino acid composition of grain from a MIR162 hybrid and a non-transgenic hybrid
MIR162
average
range
Asp
(mg/g DW)
6.66
5.29-7.72
Non-transgenic
average
range
6.54
4.85-7.45
3.47
2.64-3.96
5.11
3.68-5.84
19.16
13.2-22.5
8.96
6.51-10.3
3.79
3.13-4.10
7.55
5.24-8.89
2.29
1.96-2.65
4.74
3.52-5.37
Percent difference from nontransgenic hybrid2
1.82
2.31
1.92
2.00
1.80
1.39
1.94
0.92
1.42
F-test Probability for Genotype
0.171
0.059
0.149
0.129
0.119
0.269
0.135
0.545
0.247
F-test Probability for Location x
Genotype Interaction
0.500
0.699
0.307
0.443
0.233
0.265
0.557
0.301
0.329
3.75
2.24 - 6.66
1350
5.12
2.35 - 7.69
1350
20.09
9.65 - 35.36
1350
9.51
4.62 - 16.32
1350
3.85
1.84 - 5.39
1350
7.90
4.39 - 13.93
1350
2.21
1.25 - 5.14
1350
4.90
2.66 - 8.55
1350
0.27 - 0.58
% DW
0.35 - 0.91
% DW
1.25 - 2.58
% DW
0.63 - 1.36
% DW
0.26 - 0.49
% DW
0.56 - 1.04
% DW
0.08 - 0.32
% DW
0.21 - 0.85
%DW
ILSI (2006)
OECD (2002)
1 For
average
6.88
range 3.35 - 12.08
N
1350
range
0.48 - 0.85
% DW
Thr
(mg/g DW)
3.55
2.83-4.06
Ser
(mg/g DW)
5.21
3.95-6.06
Glu
(mg/g DW)
19.54
14.0-23.3
Pro
(mg/g DW)
9.12
6.79-10.8
Gly
(mg/g DW)
3.84
3.26-4.27
Ala
(mg/g DW)
7.70
5.59-9.17
Cys
(mg/g DW)
2.31
1.96-2.62
Val
(mg/g DW)
4.81
3.78-5.61
conversion, 1% = 10 mg/g.
2 ((MIR162-non-transgenic)/non-transgenic)*100.
57
Table 15 continued: Amino acid composition of grain from a MIR162 hybrid and a non-transgenic hybrid
Ile
(mg/g DW)
3.38
2.55-4.00
Leu
(mg/g DW)
12.85
8.86-15.6
Tyr2
(mg/g DW)
3.42
2.58-4.09
Phe
(mg/g DW)
5.09
3.70-6.04
Lys
(mg/g DW)
3.05
2.52-3.44
His
(mg/g DW)
2.87
2.28-3.26
Arg
(mg/g DW)
4.77
3.89-5.30
Trp
(mg/g DW)
0.570
0.453-0.645
MIR162
average
range
Met
(mg/g DW)
2.15
1.76-2.54
Non-transgenic
average
range
2.10
1.71-2.42
3.31
2.35-3.85
12.57
8.28-15.1
3.35
2.35-3.86
4.99
3.43-5.84
2.96
2.47-3.29
2.85
2.20-3.14
4.68
3.64-5.27
0.562
0.479-0.636
Percent difference from nontransgenic hybrid3
2.84
2.23
2.26
2.02
2.05
2.96
0.78
1.96
1.43
F-test Probability for Genotype
0.177
0.113
0.115
0.201
0.128
0.095
0.438
0.276
0.349
F-test Probability for Location x
Genotype Interaction
0.459
0.372
0.561
0.694
0.483
0.256
0.195
0.157
0.090
average
range
N
2.09
1.24 - 4.68
1350
3.68
1.79 - 6.92
1350
13.41
6.42 - 24.92
1350
3.36
1.03 - 6.42
1350
5.25
2.44 - 9.30
1350
3.15
1.72 - 6.68
1350
2.96
1.37 - 4.34
1350
4.33
1.19 - 6.39
1350
0.627
0.271 - 2.150
1350
range
0.1 - 0.46
% DW
0.22 - 0.71
% DW
0.79 - 2.41
% DW
0.12 - 0.79
% DW
0.29 - 0.64
% DW
0.05 - 0.55
% DW
0.15 - 0.38
% DW
0.22 - 0.64
% DW
0.04 - 0.13
%DW
ILSI (2006)
OECD (2002)
1 For
conversion, 1% = 10 mg/g.
non-transgenic value was an outlier and was excluded from analysis.
3 ((MIR162-non-transgenic)/non-transgenic)*100.
2 One
58
Table 16: Fatty acid1 composition of grain from a MIR162 hybrid and a non-transgenic hybrid
16:0 Palmitic
(% of total FA)
18:0 Stearic
(% of total FA)
18:1 Oleic
(% of total FA)
18:2 Linoleic
(% of total FA)
18:3 Linolenic
(% of total FA)
MIR162
average
range
12.78
12.25-13.09
1.84
1.56-1.99
25.49
22.67-26.57
56.99
55.86-59.74
1.81
1.72-1.89
Non-transgenic
average
range
12.69
12.29-13.12
1.88
1.625-2.07
25.22
23.38-26.77
57.36
56.26-59.47
1.75
1.64-1.86
Percent difference from non-transgenic
hybrid2
0.73
-1.91
1.07
-0.65
3.21
F-test Probability for Genotype3
0.275
0.051
0.057
0.026
<0.001
F-test Probability for Location x
Genotype Interaction
0.554
0.934
0.154
0.444
0.163
11.50
7.94 - 20.71
1344
1.82
1.02 - 3.40
1344
25.8
17.4 - 40.2
1344
57.60
36.2 - 66.5
1344
1.20
0.57 - 2.25
1344
0.70 - 1.39% DW
0.67 - 2.81% DW
0.03 - 0.10% DW
ILSI (2006)
average
range
N
OECD (2002)
range
0.29 - 0.79% DW
0.04 - 0.17% DW
1 Five most abundant fatty acids (FA) in maize grain.
2 ((MIR162-non-transgenic)/non-transgenic)*100.
3 Statistically significant F-test probability (<0.05) indicated in bold and italics type.
59
Table 17: Secondary metabolite and anti-nutrient analysis of grain from a MIR162 hybrid and a non-transgenic, near isogenic hybrid
Ferulic Acid
(mg/kg DW)
p-Coumaric Acid
(mg/kg DW)
Inositol
(ppm)
Phytic Acid
(% DW)
Trypsin Inhibitor
(TIU/mg DW)
Furfural2,3
(mg/kg DW)
Raffinose2,3
(% DW)
MIR162
average
range
2682
2490-2980
179
148-202
2957
2410-3530
0.745
0.621-0.871
2.82
2.27-3.72
<LOQ - <LOQ
N/A
<LOQ - 0.116
N/A
Non-transgenic
average
range
2453
2010-2760
157
137-179
2792
2180-3610
0.727
0.593-0.919
2.92
2.38-3.48
<LOQ - <LOQ
N/A
<LOQ - 0.137
N/A
9.31
14.44
5.91
2.38
-3.56
N/A
N/A
F-test Probability for Genotype5
<0.001
<0.001
0.196
0.334
0.416
N/A
N/A
F-test Probability for Location x
Genotype Interaction
0.436
0.073
0.459
0.589
0.671
N/A
N/A
218.4
53.4 - 576.2
1331.5
89.0 - 3765.4
0.745
0.111 - 1.570
2.73
1.09 - 7.18
3.697
3.000 - 6.340
0.132
0.020 - 0.320
817
504
1196
696
14
701
Percent difference from nontransgenic hybrid4
ILSI6 (2006)
average
2201.1
range 291.9 - 3885.8
N
817
OECD (2002)
range 0.02 - 0.3% DW 0.003 - 0.03% DW
0.45 - 1.0
<0.01 ppm
1 For conversion of ferulic acid, 0.02% DW = 200 mg/kg; of p -coumeric acid, 0.003% DW = 30 mg/kg; of furfural, 1 ppm = 1 mg/kg.
2 Where some values were <LOQ, statistical comparison was not possible so only the range is shown.
3 LOQ for furfural 0.55-0.57 mg/kg DW, LOQ for raffinose 0.11-0.15% DW.
4 ((MIR162-non-transgenic)/non-transgenic)*100.
5 Statistically significant F-test probability (<0.05) indicated in bold and italics type.
6 Below LOQ values are not included.
60
0.21 - 0.31
5.2
Forage
Compositional analysis of the forage samples included proximates, ADF, NDF and the
minerals calcium and phosphorus. The forage analyses showed a single statistically
significant difference for NDF between the MIR162 and non-transgenic hybrids. However,
the mean value for NDF from the MIR162 hybrid was within the range of values observed for
the non-transgenic hybrid. Also, the mean values for all analytes, including NDF, were
within the range of natural variation reported in the literature (OECD, 2002; ILSI 2006).
5.3
Grain
Nutrients
Proximates
Results of the proximate analysis are shown in Table 11. Statistically significant differences
between MIR162 and the non-transgenic comparator were observed for ash, NDF and
starch. These differences were small (9.33, 5.32 and 2.82% respectively) and the mean
values were within the range of values observed for the non-transgenic hybrid. Additionally,
the mean values for all proximates were within the ranges reported in the literature (OECD,
2002; ILSI 2006). The differences, while statistically significant, are not considered to be
biologically meaningful or nutritionally significant.
Minerals
Results of the mineral analysis are shown in Table 12. Statistically significant differences
were noted for the minerals calcium, iron and phosphorus. These differences were small
(7.95, 5.05 and 6.25% respectively). The mean values were within the ranges observed for
the non-transgenic control line and within the ranges reported in the literature (OECD, 2002;
ILSI 2006). The differences are not considered to be biologically significant.
Vitamins
Vitamin analyses are shown in Table 13. Statistically significant differences between MIR162
and the control line were observed in the levels of Vitamin A (β-carotene), Vitamin B6
(Pyridoxine and Vitamin E (α-tocopherol). The differences observed were small (5.78, 6.55
and 5.46%) with the mean values observed falling within the ranges of values observed for
the non-transgenic control. Additionally, the mean values for all vitamins fell within the
ranges reported in the literature (OECD, 2002; ILSI 2006).
For Vitamin A (β-carotene) and Vitamin B9 (folic acid) a statistically significant genotype-bylocation interaction was noted. This suggests that the effect of genotype was not consistent
across locations and that the comparison of genotypes averaged across locations may not
be valid. Therefore, individual location means for Vitamin A and B9 were assessed, and are
shown in Table 14. The Vitamin A and B9 levels at all locations were within the ranges
reported in the literature (OECD, 2002; ILSI 2006).
Amino acids
Results of the amino acid analyses are shown in Table 15. No significant differences were
noted for any of the 18 amino acids measured and all mean values were within the ranges
reported in the literature (OECD, 2002; ILSI 2006).
61
Fatty Acids
The five most abundant fatty acids in corn grain were analysed. The results are presented in
Table 16. Small but statistically significant differences were noted for linoleic (18:2) and
linolenic (18:3) acid; 0.65 and 3.21% respectively. The mean values observed for these fatty
acids in MIR162 corn were within the ranges of values observed for the non-transgenic
control and literature ranges (ILSI 2006).
Anti-nutrients and secondary plant metabolites
Corn contains a number of anti-nutrients: phytic acid, raffinose, 2,4-dihydroxy-7-methoxy-2H1,4-benzoxazin-3(4H)-one (DIMBOA) and trypsin and chymotrypsin inhibitor. Only phytic
acid and raffinose are considered to be biologically relevant (OECD, 2002). DIMBOA is
present at highly variable levels in corn hybrids and little evidence is available on either its
toxicity or anti-nutritional effects and corn contains only low levels of trypsin and
chymotrypsin inhibitor, neither of which is considered nutritionally significant.
Phytic acid is considered an important anti-nutrient for animals, especially non-ruminants,
since it can significantly reduce the bioavailability of phosphorus in the plant. Feed
formulators add the enzyme phytase to pig and poultry diets to improve the utilisation of
phosphorus. Raffinose is a non-digestible oligosaccharide and is considered an anti-nutrient
because of its gastrointestinal effects (flatulence).
Secondary metabolites are defined as those natural products which do not function directly
in the primary biochemical activities which support the growth, development and
reproduction of the organism in which they occur. Secondary plant metabolites are neither
nutrients nor anti-nutrients but are sometimes analysed as further indicators of the absence
of unintended effects of the genetic modification on metabolism (OECD, 2002).
Characteristic metabolites in corn are furfural and the phenolic acids, ferulic acid and pcoumaric acid.
There are no generally recognised anti-nutrients in corn at levels considered to be harmful,
but for the purposes of comparative assessment, the OECD has recommended considering
analytical data for the content of the anti-nutrients phytic acid and raffinose, and the
secondary metabolites furfural, ferulic acid, p-coumaric acid. These selected secondary
metabolites and anti-nutrients were included in the compositional analysis of MIR162 corn
grain and are shown in Table 17. In addition, data on the levels of the anti-nutrient trypsin
inhibitor and inositol in MIR162 corn grain are provided.
The furfural levels in all transgenic and non-transgenic corn samples were below the limits of
quantitation (LOQ). For raffinose, 16 of 18 MIR162 samples, and 12 of 18 control samples
were <LOQ. Analytes with values <LOQ are not suitable for statistical analysis but
quantifiable levels of raffinose in both the MIR162 and control hybrids were within ranges
reported in the literature.
The levels of the secondary metabolites ferulic acid and p-coumaric acid in MIR162 corn
grain were significantly different from levels in the control grain. However, the mean values
for both metabolities were within the ranges of values observed for the non-transgenic
control, and within the ranges reported in the literature (OECD, 2002; ILSI 2006). Based on
these results, the levels of anti-nutrients and secondary plant metabolites in MIR162 corn
are comparable to those found in conventional corn.
62
5.4
Conclusion
The compositional analyses do not indicate any compositional differences of biological
significance in forage or grain from transgenic corn line MIR162, compared to the non-GM
control. Several minor differences in key nutrients and other constituents were noted,
however, the mean levels observed were within the range of values observed for the nontransgenic comparator and literature ranges. Therefore, all values noted for MIR162 corn
are considered to be within the range of natural variation for corn and suggest than no
unintended changes in composition have occurred in MIR162 corn. On the whole, it can be
concluded that MIR162 corn is equivalent in composition to the non-transgenic comparator
and conventional corn.
6.
NUTRITIONAL IMPACT
In assessing the safety of a GM food, a key factor is the need to establish that the food is
nutritionally adequate and will support typical growth and well being. In most cases, this can
be achieved through an understanding of the genetic modification and its consequences,
together with an extensive compositional analysis of the food.
Where a GM food has been shown to be compositionally equivalent to conventional
varieties, the evidence to date indicates that feeding studies using target livestock species
will add little to the safety assessment and generally are not warranted (OECD, 2003).
If the compositional analysis indicates biologically significant changes to the levels of certain
nutrients in the GM food, additional nutritional assessment should be undertaken to assess
the consequences of the changes and determine whether nutrient intakes are likely to be
altered by the introduction of such foods into the food supply. This assessment should
include consideration of the bioavailability of the modified nutrient.
In this case, MIR162 corn is the result of a simple genetic modification to confer insect
protection with no intention to significantly alter nutritional parameters in the food. In
addition, extensive compositional analyses have been undertaken to demonstrate the
nutritional adequacy of MIR162 corn and these indicate it is equivalent in composition to
grain from conventional corn hybrids.
The Applicant has however submitted a feeding study comparing the nutritional performance
of MIR162 corn with a non-transgenic near-isogenic control corn and a commercially
available conventional corn. This study is evaluated below as additional supporting
information.
6.1
Feeding study in broiler chickens
Study submitted:
Brake, J.T. (2007) Evaluation of Event MIR162 Transgenic maize in broiler chickens. Syngenta Seeds
Biotechnology Report No. SSB-507-07.
The purpose of the study was to evaluate whether standard poultry diets prepared with
MIR162 corn grain had any effect on male of female broiler chicken survival, growth and
feed conversion (an indicator of how efficiently a bird converts feed to live body weight). The
study was conducted using rapidly growing broiler chicks, which are sensitive to changes in
nutrient quality in their diets, and therefore are often used as a model to assess the
wholesomeness of corn.
63
Poultry diets were prepared with three lots of corn grain: grain from MIR162 corn, a nontransgenic, near-isogenic control corn, and a commercially available lot of corn grown in
North Carolina. Diets were formulated on the basis of individual nutrient analyses of grain to
meet standard nutritional recommendations for poultry.
Diets were fed to groups of 90 male and 90 female birds for 44 consecutive days.
Parameters evaluated included survival, body weight, feed conversion and carcass yield.
Broiler chickens fed diets prepared with MIR162 corn grain did not show any adverse effects
compared to chickens fed diets prepared either with the control or commercial grain. There
were no significant differences between the three diets for rates of growth, final body
weights, feed conversion, survival rates or carcass yield.
The results support the conclusion that MIR162 corn is nutritionally comparable to
conventional corn.
7.
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66
Attachment 3
Summary of Issues Raised in Public Submissions
Submitter
Madeleine Love
(Private)
Option
1
Australian Food and
Grocery Council
2
Queensland Health
(whole of
Queensland
government
response)
-
New Zealand Food
Safety Authority
2
Comments
 Has no confidence in the capacity of FSANZ to assess this
Application, due to false statements made in respect of
Application A363 food derived from glyphosate-tolerant canola
GT73.
 The A363 Inquiry Report contains inconsistent statements
about whether canola oil contains negligible or no protein,
however the Executive Summary concludes that all protein has
been removed.
 States that FSANZ has failed to meet the primary obligation to
protect public health and safety and all existing approvals
should be reviewed.
 Claims that epidemiological evidence suggests that
inappropriate assessment and approval of these foods has had
a significant negative impact on the health and well-being of
Australian children.
 Cites a study in the Medical Journal of Australia reporting a
dramatic increase in child food allergies and food anaphylaxis.
 States that there is something going wrong with our food and
that many novel GM proteins have been predicted to be
allergenic but have not been tested by FSANZ nor labelled.
 Requests that the unhealthy population of the USA no longer
be used as the standard for the promotion of consistency
between domestic and international food standards, but rather
that we strive to be internationally consistent with the domestic
food standards of Norway.
 Supports the Application contingent upon a satisfactory safety
assessment by FSANZ.
 Believes GM labelling requirements will provide consumers
with appropriate information on which to base informed choice.
 Neither accepts nor rejects, but will review contingent on the
provision of additional information.
 Requests information on progress of submissions for approval
by overseas regulatory agencies
 Considers there is an obligation to address the safety of animal
feed or animals fed with feed derived from GM plants.
 Remain concerned about the lack of independent, long-term
work on GM issues generally
 Notes an apparent reliance on small studies, such as a
nutritional study with 180 broiler chicks for 44 days.
 Remains concerned that the Benefit Cost Analysis is entirely
qualitative, and is particularly interested in the enforcement
costs determined by FSANZ.
 Acutely aware that approval will impact on monitoring
resources, and suggests that a national enforcement strategy
for GM food be progressed without further delay.
 Has had the Assessment Report reviewed by the Institute of
Environmental Science and Research Limited (ESR).
 ESR and NZFSA agree with the preferred approach to amend
Standard 1.5.2.
67
Submitter
Elise Newnham
(Private)
Option
1
Rachel Carey
(Private)
1
Ivan Jeray (Private)
1
David Rixecker
(Private)
1
Comments
 Feels that FSANZ’s safety assessment procedure is not
rigorous enough.
 Concerned that toxicity tests on mice were only carried out
over a period of two weeks.
 Distressed that FSANZ has not considered the safety of
animals fed with GM plants.
 Concerned that FSANZ has relied on tests carried out and
presented by the company themselves and questions rigour
and objectivity of data. States FSANZ should consider
conducting independent tests.
 Doubts the WTO would have a problem if FSANZ concluded
that GM corn cannot be deemed safe in light of the evidence
presented.
 States that approval is not necessary and we don’t know what
will happen if we do approve it.
 States that the data provided do not exclude public health and
safety concerns.
 Believes the precautionary principle should be applied and the
corn should not be approved until long term, independent
animal feeding studies have been conducted.
 Raises specific concerns –
- Notes the unexpected amino acid change in the Vip3Aa20
protein and questions the effect of this change on toxicity,
allergenicity and nutritional value, and whether other
changes could have gone unnoticed.
- Notes that the ‘safe’ use of Bt in agriculture does not provide
reasonable grounds for concluding that Vip3Aa20 has no
potential toxicity or allergenicity and states that this
conclusion could only be reached through independent
animal feeding studies.
- Questions the conclusion that Vip3Aa20 and PMI would be
rapidly degraded in the stomach and asks whether
independent animal feeding studies prove this conclusively.
- Raises concern about the possibility of transgene transfer to
human gut bacteria and asks whether independent animal
feeding studies demonstrate that the proteins cannot be
taken up by the mammalian gut or other tissues.
 States that GM foods have not been proven safe. Stated
reasons include that GM food will induce antibiotic resistance
and GM food tests on animals are inadequate.
 Notes that some foods are exempt from GM labelling
requirements and states that GM labelling requirements are
not policed
 Protests that FSANZ’s notification did not state that A1001 was
a GM food application.
 Disbelieves this is a safe and profitable decision for Australia.
 States the evidence against the safety of GM crops for human
consumption is undeniable.
 Notes that the Vip3Aa20 protein has two different amino acids
from the normal protein, and that a difference of only one
amino acid in involved in sickle cell anaemia.
 States that the only test involving this crop was a feeding trial
where 180 chickens were fed the corn for 44 days and that this
does not prove that it is safe to eat for our entire lives.
 Believes adopting GM crops will make our crops obsolete on
the global market.
 Believes consumers should be acknowledged before decisions
are made.
68
Submitter
Martina Tschirky
(Private)
Option
1
Michael Byrne
(Private)
1
NSW Food Authority
-
Murray Irwin
(Private)
1
Michael Sartori
(Private)
1
Kylie Richter-Cools
(Private)
1
Comments
 Asks that the approval be refused as it is not possible to rule
out long-term health problems, while existing corn varieties are
proven safe.
 States that old varieties of corn are disappearing in Mexico
because of the aggressive cross-pollination of GE corn.
 Opposes because we do not really know whether GM food is
safe.
 States the introduction of a new gene may have unintended
and initially unobserved side-effects, such as those found in
GM peas.
 Questions whether the GM food has been tested to ensure it is
safe for pregnant women and the developing embryo.
 Concerned that FSANZ does not require animal testing and
relies on ‘paper assessment’ of company data.
 Raises concerns about cross-contamination, effects on wildlife
and the ability of farmers to safe seeds for planting.
 Suggests GM food should be subject to a longitudinal study of
at least 20 years to better ascertain its safety before approval.
 Does not object to the Application.
 Notes that extensive costs are incurred in monitoring for the
presence of GM food.
 States that the NSW Food Authority intends to host an ISC
workshop to consider the cost impacts of monitoring GM food.
 Concerns relate to the lack of adequate testing of this and all
GM organisms.
 Believes that testing of a substance in isolation overlooks the
possibility for effects caused by the combination of other
factors, including other digested proteins.
 Asks why there is not a requirement to prove that a GM food
does not interact negatively with, e.g., commonly available
drugs such as panadol, contraceptive and alcohol or even
existing foods.
 Believes we should take a precautionary approach, which
would dictate that we say no to GM foods until they are proven
to be safe in a complex environment with all variables and
likely combinations having been tested long term.
 States this has not been done with many manufactured
foodstuffs, chemicals or medicines.
 States that the product has not been thoroughly tested.
 Requests that, if approved, all food containing GM foods is
properly labelled.
 Prefers disapproval of all GM foods based on the lack of
adequate testing.
 Appeals to FSANZ to conduct thorough and independent
safety tests before approval.
 Concerned that this GM product has not been approved in the
US nor anywhere else in the world.
 Questions the history of safe use of the soil bacterium Bt and
asks FSANZ to read Genetic Roulette and Seeds of
Deception, by Jeffrey Smith.
 States that the limited feeding trial using 180 broiler chickens
for 44 days is not adequate to prove safety.
 Does not understand why FSANZ is keen to approve, given
the volume of evidence against the safety of GM crops.
69
Submitter
Arthur and
Rosemary
Lathouris
(Private)
Option
1
Ron Dean (Private)
Lillian Lear (Private)
1
1
Tabitha Robortella
(Private)
Robert Vickers
(Private)
1
Food Technology
Association of
Australia
Alison Bleaney
(Private)
2
1
1
Comments
 Oppose as MIR162 is not approved anywhere else in the
world, making Australia and New Zealand guinea pigs for
possible allergies and side effects.
 Believe that tracing any side effects will be virtually impossible
and avoiding product will be difficult as products such as corn
syrup and cornflour go into hundreds of processed food items.
 Oppose due to possible contamination of Australian corn
varieties through cross pollination.
 Object as this GM variety is unnecessary as traditional
varieties already produce good quality corn products.
 Believe the risks outweigh the advantages.
 Opposes as GM issues have not been sufficiently studied.
 Believes GM products should be subject to caution and long
term investigation.
 States that feeding trial with 180 broiler chickens for 44 days is
not a robust study.
 Lists reported evidence of negative effects of GM foods,
including livestock deaths, rise of pests, superweeds and
economic losses.
 Believes there are economic advantages to keeping Australia
GM-free.
 Urges FSANZ to keep Australia GM free.
 Asks FSANZ to not approve GM corn MIR162.
 States that FSANZ has a cavalier attitude to science and
safety testing, more in accord to the used car industry than
controlling the food safety standards of over twenty million
people.
 Cites the FSANZ web site, including ‘the novelty of GM foods
means their long-term effects on human health are unknown.’
 Questions why MIR162 GM corn should be approved in
Australia, making us Syngenta’s laboratory rats, when the corn
has not even been released in the USA.
 Questions the commitment of the Rudd Government to review
the science of GM.
 Believes changes to food labelling laws in 2000 give
consumers no way of selecting non-GM foods, effectively
forcing consumers to consume food containing insecticide.
 Believes FSANZ, OGTR and ABARE have become little more
than conduits for the sales pitch of the biotechnology
corporations.
 Describes the USA appeal to the WTO to force trading
partners to approve GMOs as bullying behaviour
 FTA Australia endorses the comments of its Technical Sub
Committee: The committee agreed with Option 2.
 Opposes GM foods for health reasons
 Attaches submission to the Joint Standing Committee on Gene
Technology in Primary Industries (Tasmania) – the 2002
statement of the Public Health Association of Australia on GM
foods.
70
Submitter
Giles Paul ElwellSutton (Private)
Option
1
L. Sanzaro on behalf
of Croydon
Conservation
Society
1
George Winston
(private)
1
Comments
 States that FSANZ is a body that furthers the interests of the
biotech industry rather than the interests of New Zealand
consumers and does not accept that FSANZ has a valid
jurisdiction over New Zealand.
 Believes FSANZ should renounce from all decisions affecting
the nature of the foods eaten in New Zealand.
 Believes the long-term pleiotropic stability of GM constructs and
their derivatives is poorly researched.
 Cites a ten-fold increase in food allergies and food-borne
diseases in the United States since the introduction of GM
foods.
 Believes the precautionary principle should apply, and a ban on
GM derived foods put in place.
 Believes it is now impossible to find a baseline human
population which has not been exposed to GM foods,
consequently, we have all been subjected to a gigantic and
uncontrolled experiment without our general awareness of
consent.
 Believes this breaches an article of the United Nations Universal
Declaration of Human Rights which recognises the right of every
individual to not be subjected to experimental procedures
without explicit and informed consent.
 Believes there is no robust protocol for GM food labelling, so is
unable to make informed choices in food selections, and
breaches his right to make informed choices.
 Believes BT corn is exceedingly harmful to our natural
environment, including shortening life span of butterflies.
 Believes GM involving insect resistance has a far greater likely
impact on long term human health than short term gain, due to
anticipated problems of pollination resulting from reduced
numbers of bees.
 Cites the apparent 3 to 10% contamination of corn in Mexico as
a potential catastrophe.
 Opposes as the import of GM corn would encourage farming
styles that are not sustainable nor the most beneficial for human
health and survival.
 Urges against approval as this GM product has not been
approved in the US nor anywhere else, it would be the first GM
crop approved by the Rudd government and Australia would be
obliged to notify the WTO if the corn was not approved if it may
have a significant effect on trade.
 Questions the history of safe use of the soil bacterium Bt
 Notes that the Vip3Aa20 protein has two different amino acids
from the normal protein, and that a difference of only one amino
acid in involved in sickle cell anaemia.
 States that a feeding trial with 180 chickens for 44 days is not
adequate to prove that it is safe to eat for our entire lives.
71
Attachment 4
APPLICATION A1001
FOOD DERIVED FROM INSECT-PROTECTED
CORN LINE MIR162
1st REVIEW REPORT
CONTENTS
INTRODUCTION ................................................................................................................ 73
1.
2.
3.
THE ISSUE ................................................................................................................ 73
OBJECTIVES OF REVIEW ............................................................................................ 73
GROUNDS FOR THE REVIEW REQUESTED BY THE MINISTERIAL COUNCIL ................... 73
3.1
Protection of public health and safety .............................................................. 73
4. BACKGROUND ........................................................................................................... 74
5. CONCLUSIONS FROM THE FINAL ASSESSMENT REPORT ............................................... 74
ISSUES ADDRESSED IN FIRST REVIEW ......................................................................... 75
6.
7.
8.
9.
10.
11.
INDEPENDENT LONG TERM ANIMAL FEEDING TRIALS ..................................................... 75
INGESTION OF RECOMBINANT DNA IN FOOD ................................................................ 76
PURITY OF SAMPLES USED FOR COMPOSITIONAL ANALYSES ......................................... 77
THE INCLUSION OF LIVESTOCK FEEDING STUDIES IN NUTRITIONAL ASSESSMENT ............ 77
THE SAFETY OF FOOD DERIVED FROM ANIMALS FED GM FEED .................................. 78
MIR162 CORN HAS NOT BEEN APPROVED ANYWHERE IN THE WORLD ........................ 79
CONCLUSION .................................................................................................................... 79
12.
13.
14.
REVIEW OPTIONS .................................................................................................. 79
DECISION .............................................................................................................. 80
IMPLEMENTATION AND REVIEW ............................................................................... 80
ATTACHMENT 1- DRAFT VARIATION TO THE AUSTRALIA NEW ZEALAND FOOD STANDARDS
CODE......................................................................................................................... 81
ATTACHMENT 2 - EXECUTIVE SUMMARY AND REASONS FOR DECISION FROM THE APPROVAL
REPORT ........................................................................................................................... 82
ATTACHMENT 3 - THE SAFETY OF RECOMBINANT DNA IN FOOD ............................................ 85
72
INTRODUCTION
1.
The Issue
On 3 October 2008, the Australia and New Zealand Food Regulation Ministerial Council
(Ministerial Council) requested a First Review of Application A1001, which seeks approval of
food derived from a genetically modified (GM) corn – namely, insect-protected corn line
MIR162. Approval of this Application involves a variation to Standard 1.5.2 – Food produced
using Gene Technology, of the Australia New Zealand Food Standards Code (the Code).
Following a request for a formal review, FSANZ has three months to complete a response.
In this instance, FSANZ was required to review the decision by 3 January 2009.
2.
Objectives of Review
The objective of this Review is to reconsider the draft variation to Standard 1.5.2 in light of
the Ministerial Council’s grounds for review as outlined in Section 3 below.
3.
Grounds for the Review requested by the Ministerial Council
A First Review of FSANZ’s decision to approve Application A1001 was sought on the
grounds that the proposed amendment to Standard 1.5.2, to permit the sale and use of food
derived from insect-protected corn line MIR162, does not protect public health and safety.
3.1
Protection of public health and safety
A number of reasons has been put forward in asserting that the decision to approve food
derived from corn line MIR162 does not protect public health and safety. Firstly, the First
Review request states that ‘there is no confidence that the construct is safe for human
consumption as there is an absence of independent long term animal feeding trials designed
to measure outcomes relevant to human health.’
Secondly, FSANZ is asked to further clarify what is known about potential health implications
of work establishing proof of principle for persistence and uptake of foreign DNA in and
across the gastrointestinal (GI) tract of mammals. These grounds were used for the First
Review of Application A592 (glyphosate-tolerant soybean line MON89788) and the First
Review Report for A592 included a detailed summary of the scientific evidence relating to GI
uptake of foreign DNA and the potential implications for the health of consumers. However,
the First Review request for A1001 states that FSANZ’s First Review Report for A592 did not
provide that clarity and thus the same concerns stand for A1001.
Thirdly, clarification is requested as to whether MIR162 and control samples used in the
compositional analysis were pure, as contamination of non-GM control samples with GM
material would mask differences and reduce the confidence that can be placed in a
conclusion of equivalence.
The concern arises as a previous safety assessment, for glyphosate-tolerant soybean
MON 89788 (A592) acknowledged contamination of one of the non-GM control samples with
GM material (≤ 3.05%) and the Review request states that such contamination may not be
unusual. It is stated that, for MIR162, FSANZ should determine whether purity was
adequately assessed, the outcome of that assessment, and if contamination occurred, clarify
the policy it applies when evaluating compositional analysis results, including whether a
contamination tolerance has been set.
73
Fourthly, inclusion of the results of a broiler feeding study in the Safety Assessment of
MIR162 is questioned. The First Review request questions whether a study designed to
answer animal production questions can be used to support any conclusion regarding
nutritional impact of MIR162 corn.
Further, FSANZ is requested to outline the scientific evidence utilised to support the
conclusion that ‘feeding GM plant material to livestock does not affect the nutritional value or
safety of the meat, milk and eggs derived from those animals.’
Finally, the First Review request also notes that, to date, no country has given regulatory
approval for insect-protected corn line MIR162.
4.
Background
FSANZ received an Application from Syngenta Seeds Pty Ltd (the Applicant) on
10 October 2007. The Applicant requested an amendment to Standard 1.5.2 to permit the
sale and use of food derived from a new genetically modified variety of corn, MIR162.
Standard 1.5.2 prohibits a food produced using gene technology from being sold or used as
an ingredient or component of any food unless it is listed in the Table to clause 2 of that
Standard.
MIR162 corn has been genetically modified to be protected against feeding damage caused
by the larvae of certain insect pest species. Protection is achieved through the expression in
the plant of an insecticidal protein derived from Bacillus thuringiensis, a common soil
bacterium.
Corn line MIR162 is intended to be grown in North America. However, once
commercialised, corn products imported into Australia and New Zealand could contain
ingredients derived from MIR162 corn. Approval is therefore necessary before these
products may enter the Australian and New Zealand markets.
Prior to approval, FSANZ completed a comprehensive safety assessment of food derived
from insect-protected corn line MIR162. The assessment included consideration of (i) the
genetic modification to the plant; (ii) the potential toxicity and allergenicity of the novel
proteins; and (iii) the composition of MIR162 corn compared with that of conventional corn
varieties.
No public health and safety concerns were identified as a result of the safety assessment.
5.
Conclusions from the Final Assessment Report
The Executive Summary and the reasons for the decision, which were approved by the
FSANZ Board in July 2008, are provided in this Report at Attachment 2.
The Board agreed to the recommendation of the Approval Report to approve food from corn
line MIR162 in view of the findings of the safety assessment that food derived from this line
is as safe and wholesome as food derived from other commercial corn varieties.
74
ISSUES ADDRESSED IN FIRST REVIEW
6.
Independent long term animal feeding trials
The First Review request states that ‘there is no confidence that the construct is safe for
human consumption as there is an absence of independent long term animal feeding trials
designed to measure outcomes relevant to human health.’ The issue of long term animal
feeding studies was addressed in response to submissions received during the public
consultation period for A1001, and has also been addressed in the First Review Report for
Application A595 (food derived from insect-protected corn line MON 89034). FSANZ’s
response to this issue is reiterated below.
FSANZ’s safety assessment of food derived from insect-protected corn line MIR162,
included consideration of (i) the genetic modification to the plant; (ii) the potential
toxicity and allergenicity of the novel proteins; and (iii) the composition of MIR162 corn
compared with that of conventional corn varieties. No public health and safety
concerns were identified as a result of the safety assessment. The safety assessment
did not rely on the results of the feeding study in rapidly-growing broiler chicks.
FSANZ considers that a scientifically-informed comparative assessment of GM foods
with their conventional counterparts can generally identify any potential adverse health
effects or differences requiring further evaluation. In the majority of circumstances,
animal toxicity studies with whole foods are not likely to contribute any further useful
information to the safety assessment and are therefore not warranted. As a
consequence, FSANZ does not require that animal feeding studies with whole GM
foods be undertaken on a routine basis.
FSANZ acknowledges there may be future GM applications, particularly for foods with
intentional modifications to composition, where the results of animal toxicity studies
may be informative. FSANZ therefore continues to assess the need for animal studies
on a case-by-case basis, taking into account the nature of the genetic modification and
the results of the comparative assessment. While FSANZ does not routinely require
animal toxicity studies to be undertaken, where such studies already exist, Applicants
are expected to provide these to FSANZ to evaluate as additional supporting
information.
As part of a continual review of FSANZ’s scientific approach to the safety assessment
of GM foods, FSANZ convened an expert panel in June 2007 to develop guidance and
recommendations on the role animal feeding studies can play in the safety
assessment of GM foods. The expert panel recommended that FSANZ should
continue with its case-by-case assessment of GM foods on the basis of best available
science.
The panel noted that whole-food animal feeding studies may be informative in some
limited circumstances, but concurred with FSANZ that any potential adverse health
effects can generally be identified by a scientifically informed comparative assessment
of the GM food against its conventional counterpart. The report from the expert panel
is available from
www.foodstandards.gov.au/_srcfiles/Workshop%20Report%20FINAL.pdf
75
The GMO Panel of the European Food Safety Authority recently adopted a similar approach
and recommends ‘In cases where molecular, compositional, phenotypic, agronomic and
other analyses have demonstrated equivalence between the GM plant derived food and feed
and their conventional counterpart, except for the inserted trait(s), and results of these
analyses do not indicate the occurrence of unintended effects, the performance of animal
feeding trials with rodents or with target animal species adds little if anything to the overall
safety assessment, and is not recommended’8.
Further information is available on the FSANZ website 9.
7.
Ingestion of recombinant DNA in food
FSANZ is asked to clarify what is known about potential health implications of work
establishing proof of principle for persistence and uptake of foreign DNA in and across the
gastrointestinal (GI) tract of mammals.
The issue of persistence and uptake of recombinant DNA, when ingested, is a general issue
that has been the subject of extensive consideration and publication for more than 15 years.
Based on these deliberations and prolonged scientific discourse, the consensus is that as
DNA from all living organisms is structurally similar, the presence of recombinant DNA in
food products, in itself, poses no additional health risk to consumers (WHO 1991, WHO
1993, Karenlampi 1996, Jonas et al 2001, Gaye & Gillespie 2005, Flachowsky et al 2007,
EFSA 2007)10.
This issue was raised in the First Review requests for Applications A592 (glyphosate-tolerant
soybean line MON89788), A589 (glufosinate ammonium-tolerant rice line LLRICE62) and
A595 (insect-protected corn line MON 89034). In response FSANZ prepared a detailed
review of the scientific evidence relating to GI uptake of foreign DNA and the potential
implications for the health of consumers. This response is presented at Attachment 3 to this
report.
However, the First Review request for Application A1001 states that FSANZ’s First Review
Report for A592 did not provide sufficient clarity and thus the same concerns stand for
A1001.
FSANZ believes that the First Review Report for Application A592 provides a detailed review
of the scientific evidence for persistence and uptake of foreign DNA across the GI tract and
comprehensively discusses the potential health implications of this work. Thus, FSANZ
believes that it has adequately addressed this issue.
In relation to this Application, the transferred DNA in MIR162 corn does not contain any
genetic elements which may significantly increase the likelihood of recombinant DNA in GM
food being taken up and stably incorporated into the genome of either gut micro-organisms
or human cells. Given this, FSANZ does not consider that the issue of persistence and
uptake of recombinant DNA requires specific consideration in the safety assessment of food
derived from insect-protected corn line MIR162; consideration of such issues is already
implicit in the molecular characterisation component of the safety assessment.
8
EFSA (2008) Safety and nutritional assessment of GM plants and derived food
and feed: The role of animal feeding trials Report of the EFSA GMO Panel Working Group on Animal
Feeding Trials. Food and Chemical Toxicology 46 (2008) S2–S70
http://www.efsa.europa.eu/cs/BlobServer/Non_Scientific_Document/gmo_report_feedingtrials.pdf?ssb
inary=true
9 http://www.foodstandards.gov.au/foodmatters/gmfoods/frequentlyaskedquest3862.cfm
10Full citations are listed in Attachment 3.
76
While the issue continues to be an active area of research and publication, FSANZ does not
regard this as an issue that requires specific and explicit consideration for each and every
GM food assessment. FSANZ continues to monitor the scientific literature for studies
relevant to the safety assessment of GM foods and is fully cognisant of the literature relating
to the uptake and persistence of recombinant DNA when ingested as part of GM food.
8.
Purity of samples used for compositional analyses
The First Review request seeks clarification as to whether the samples of GM and control
corn used for compositional analysis were pure.
The Applicant has advised FSANZ that the samples used for the compositional analysis of
MIR162 corn were tested for purity. Testing consisted of one hundred kernel samples from
each lot (MIR162 and control) analyzed by Taqman PCR using a panel of assays to check
for the presence of approved GM lines as well as lines still under development by the
Applicant. No contamination was detected in these samples, which provides 95%
confidence that the lot impurity is 3% or less.
FSANZ is satisfied with the overall conduct of the compositional studies, including
determination of the purity of the samples used in the analyses, and considers that the
conclusions of the study are scientifically valid.
9.
The inclusion of livestock feeding studies in nutritional
assessment
The First Review request questions the validity of including the results of a broiler feeding
study in the safety assessment report for MIR162 corn. The request asserts that such
studies should only be included where they have clear relevance to food safety, and that it is
debatable whether a study designed to answer animal production questions bears such
relevance. The request states that including results of such studies as supporting information
in a safety assessment is misleading.
FSANZ agrees that studies designed to evaluate survival, growth, feed conversion and
carcass characteristics of target livestock species have limited value in the safety
assessment of most GM foods.
In cases where a GM food has been shown to be compositionally equivalent to conventional
varieties, the evidence to date indicates that feeding studies using target livestock species
will add little to the safety assessment and generally are not warranted11.
In the case of MIR162 corn, the Applicant submitted a feeding study comparing the
nutritional performance of MIR162 corn with a non-transgenic near-isogenic control corn and
a commercially available conventional corn. This study was evaluated by FSANZ as
additional supporting information of the nutritional equivalence of MIR162 corn to
conventional corn varieties, but was not considered essential to the safety assessment of
food derived from MIR162 corn. The detailed compositional studies are considered
adequate to establish the nutritional adequacy of food derived from insect-protected MIR162
corn. However, evaluation of the feeding study in rapidly-growing broiler chicks confirmed
that MIR162 corn is equivalent to its conventional counterpart and commercial corn in its
ability to support typical growth and well-being.
11
OECD (2003) Considerations for the safety assessment of animal feedstuffs derived from
genetically modified plants. Series on the Safety of Novel Foods and Feeds, No. 9. Organisation for
Economic Cooperation and Development, Paris.
77
While the broiler feeding study supplied by the Applicant was included in the safety
assessment of MIR162 corn as additional supporting information, the safety assessment
report clearly states that FSANZ considers such studies to be of limited value. However,
FSANZ is also mindful of the concerns of some consumers regarding the perceived need for,
or at least reassurance provided by, such studies, and that exclusion of such studies from
consideration by FSANZ may raise concerns amongst some consumers. Therefore, FSANZ
policy is to include an evaluation of such studies, where they are provided, while bearing in
mind the potential limitations of such studies.
10.
The safety of food derived from animals fed GM feed
The First Review request notes that FSANZ does not have the legislative mandate to assess
the safety of food derived from animals fed GM feed, but seeks elaboration of the scientific
evidence supporting the conclusion stated in the A1001 Approval Report that ‘the scientific
evidence published so far indicates that feeding GM plant material to livestock does not
affect the nutritional value or safety of the meat, milk and eggs derived from those animals.’
The following response to this issue is extracted from the FSANZ Guidance Document on
the Safety Assessment of Genetically Modified Foods12.
Many animal feeds are derived from the same GM commodities that are used for
human consumption and concerns are occasionally expressed that this practice may
pose an indirect risk to humans through consumption of the meat, milk and eggs
derived from such animals.
The OECD has produced a paper as part of their series on the safety of novel foods
and feeds entitled ‘Considerations for the safety assessment of animal feedstuffs
derived from genetically modified plants’13. This paper specifically examined potential
hazards to humans from the presence of novel DNA and protein in animal feedstuffs.
They noted that both DNA and protein are extensively digested when consumed by
animals. It was concluded that while fragments of plant genomic (non-GM) DNA have
been detected in animal food products such as milk, there is no basis to suppose that
recombinant DNA poses hazards any different to other sources of DNA and the
possibility of incorporation of functionally intact DNA or protein derived from GM feeds
into animal products is extremely remote.
In addition to the above report, an overview was conducted of regulatory assessments
of GM foods and to summarise empirical data generated for assessing the safety of
meat, milk and eggs derived from animals fed GM crops that express agronomic input
traits (e.g. herbicide tolerance)14. It was concluded that there are no effects from
feeding GM plant material to livestock and poultry on the nutritional value or safety of
the meat, milk and eggs derived from those animals. Moreover, because most
components of feeds are broken down into smaller components during digestion by
the animal, proteins and DNA derived from the GM plants cannot be detected in milk,
meat or eggs.
12
http://www.foodstandards.gov.au/_srcfiles/GM%20FINAL%20Sept%2007L%20_2_.pdf
OECD (2003) Considerations for the safety assessment of animal feedstuffs derived from
genetically modified plants. Series on the Safety of Novel Foods and Feeds, No. 9. Organisation for
Economic Cooperation and Development, Paris..
14 CAST (2006). Safety of Meat, Milk, and Eggs from Animals Fed Crops Derived from Modern
Biotechnology. Issue Paper 34. Council for Agricultural Science and Technology, Ames, Iowa.
13
78
The European Food Safety Authority has also recently prepared a literature survey on
the fate of recombinant DNA or proteins in meat, milk and eggs from animals fed with
GM feed15. On the basis of this literature survey, EFSA concluded that: (i) DNA and
protein are common constituents of foods and feeds and such substances are rapidly
degraded into small fragments upon digestion by animals and humans; and (ii) a large
number of experimental studies with livestock have shown that recombinant DNA
fragments or proteins derived from GM plants have not been detected in tissues, fluids
or edible products of farm animals or other livestock.
In addition, where GM crops have already been assessed by FSANZ for their safety as
human food, it seems highly unlikely that a GM crop, found as safe for human food, would
pose an indirect risk to humans via food products derived from animals (e.g. meat, milk,
eggs) that had eaten the GM crop, when no direct risk to humans had been identified in the
human food safety assessment.
11.
MIR162 corn has not been approved anywhere in the world
The First Review request notes that, to date, no country has given regulatory approval for
insect-protected corn line MIR162.
As is often the case for new foods and food ingredients, submissions for regulatory approval
of MIR162 corn were made to a number of countries at similar times, such that the
assessment processes are being undertaken in parallel.
Submissions for food, feed and environmental approvals of MIR162 corn have been made to
the appropriate US agencies (Environmental Protection Agency, Food and Drug
Administration, Department of Agriculture) and Canadian agencies (Health Canada and the
Canadian Food Inspection Agency). Regulatory submissions for food, feed and
environmental approvals have also been made in Colombia, Brazil and Argentina.
Regulatory submissions for import approvals for MIR162 corn have been made in key export
markets, for example, Japan, Taiwan, Korea and the Philippines. The Applicant has advised
that MIR162 regulatory approvals are anticipated in 2009.
As MIR162 corn is intended for cultivation in North America, it will not be grown
commercially, nor be present in the food supply, prior to receiving appropriate regulatory
approvals in the United States and Canada.
CONCLUSION
12.
Review Options
There are three options proposed for consideration under this Review:
1.
re-affirm approval of the draft variation to Standard 1.5.2 as notified to the Ministerial
Council; or
2.
re-affirm approval of the draft variation to Standard 1.5.2, subject to any amendments
FSANZ considers necessary; or
15
EFSA (2007). Statement on the fate of recombinant DNA or proteins in meat, milk and eggs from
animals fed with GM feed,
http://www.efsa.europa.eu/cs/BlobServer/Statement/gmo_EFSA_statement_DNA_proteins_gastroint.
pdf?ssbinary=true
79
3.
13.
withdraw approval of the draft variation to Standard 1.5.2 as notified to the Ministerial
Council.
Decision
FSANZ has considered the issues raised by the Ministerial Council in relation to
Application A1001 – Food derived from insect-protected corn line MIR162.
The First Review concludes that the preferred review option is Option 1. FSANZ has
decided to re-affirm the variation to Standard 1.5.2 of the Code to permit the sale of food
derived from insect-protected corn line MIR162, as detailed in Attachment 1.
Decision
FSANZ re-affirms the variation to Standard 1.5.2 of the Code to permit the sale of food derived from insect-protected corn line
MIR162.
14.
Implementation and review
The draft variation to Standard 1.5.2 will come into effect on the date of gazettal.
Attachments
1.
2.
3.
Draft variation to the Australia New Zealand Food Standards Code
Executive Summary and Statement of Reasons from the Final Assessment Report
Safety of recombinant DNA in food
80
Attachment 1
Draft variation to the Australia New Zealand Food Standards Code
Subsection 87(8) of the FSANZ Act provides that standards or variations to standards are
legislative instruments, but are not subject to disallowance or sunsetting
To commence: on gazettal
[1]
Standard 1.5.2 of the Australia New Zealand Food Standards Code is varied by
inserting in the Table to clause 2 –
Food derived from insect-protected corn
line MIR162
81
Attachment 2
Executive Summary and Reasons for Decision from the Approval
Report
Executive Summary
Purpose
Food Standards Australia New Zealand (FSANZ) received an Application from Syngenta
Seeds Pty Ltd (the Applicant) on 10 October 2007. The Applicant has requested an
amendment to the Australia New Zealand Food Standards Code (the Code), specifically to
Standard 1.5.2 – Food produced using Gene Technology, to permit the sale and use of food
derived from a new genetically modified (GM) variety of corn, MIR162. Standard 1.5.2
prohibits a food produced using gene technology from being sold or used as an ingredient or
component of any food unless it is listed in the Table to clause 2 of that Standard.
MIR162 corn has been genetically modified to be protected against feeding damage caused
by the larvae of certain insect pest species. Protection is achieved through the expression in
the plant of an insecticidal protein derived from Bacillus thuringiensis, a common soil
bacterium.
Corn line MIR162 is intended to be grown in North America. However, once
commercialised, corn products imported into Australia and New Zealand could contain
ingredients derived from MIR162 corn. Approval is therefore necessary before these
products may enter the Australian and New Zealand markets.
The Application is being assessed under the General Procedure and is at the Approval stage.
Safety Assessment
FSANZ has completed a comprehensive safety assessment of food derived from insectprotected corn line MIR162, as required under Standard 1.5.2. The assessment included
consideration of (i) the genetic modification to the plant; (ii) the potential toxicity and
allergenicity of the novel proteins; and (iii) the composition of MIR162 corn compared with
that of conventional corn varieties.
No public health and safety concerns were identified as a result of the safety assessment.
On the basis of the available evidence, including detailed studies provided by the Applicant,
food derived from insect-protected corn line MIR162 is considered as safe and wholesome
as food derived from other commercial corn varieties.
Labelling
If approved, food derived from insect-protected corn line MIR162 will be required to be
labelled as genetically modified if novel DNA and/or novel protein is present in the final food.
Studies conducted by the Applicant show that the novel proteins are present in the grain.
Labelling addresses the objective set out in section 18(1)(b) of the Food Standards Australia
New Zealand Act 1991 (FSANZ Act); the provision of adequate information relating to food
to enable consumers to make informed choices.
82
Impact of regulatory options
Two regulatory options were considered in the assessment: (1) no approval; or (2) approval
of food derived from insect-protected corn line MIR162 based on the conclusions of the
safety assessment.
Following analysis of the potential costs and benefits of each option on affected parties
(consumers, the food industry and government), approval of food derived from insectprotected corn line MIR162 is the preferred option as the potential benefits to all sectors
outweigh the costs associated with the approval.
Assessing the Application
In assessing the Application and the subsequent variation to Standard 1.5.2, FSANZ has
had regard to the following matters as prescribed in section 29 of the FSANZ Act:

Whether costs that would arise from a variation to Standard 1.5.2 approving food
derived from insect-protected corn line MIR162 outweigh the direct and indirect
benefits to the community, Government or industry that would arise from this food
regulatory measure.

There are no other measures that would be more cost-effective than a variation to
Standard 1.5.2 that could achieve the same end.

There are no relevant New Zealand standards.

There are no other relevant matters.
Decision
Approve the variation to Standard 1.5.2 – Food produced using Gene Technology, to
include food derived from insect-protected corn line MIR162 in the Table to clause 2.
Reasons for Decision
A variation to Standard 1.5.2 to permit the sale and use of food derived from insect-protected
corn line MIR162 in Australia and New Zealand is approved on the basis of the available
scientific evidence, for the following reasons:

the safety assessment did not identify any public health and safety concerns
associated with the genetic modification used to produce insect-protected corn line
MIR162;

food derived from insect-protected corn line MIR162 is equivalent to food from the
conventional counterpart and other commercially available corn varieties in terms of its
safety for human consumption and nutritional adequacy;

labelling of certain foods derived from insect-protected corn line MIR162 will be
required if novel DNA and/or protein is present in the final food;

a regulation impact assessment process has been undertaken that also fulfils the
requirement in New Zealand for an assessment of compliance costs. The assessment
concluded that the preferred option is option 2, a variation to Standard 1.5.2;
83

there are no relevant New Zealand standards; and

there are no other measures that would be more cost-effective than a variation to
Standard 1.5.2 that could achieve the same end.
Consultation
Public submissions were invited on the Assessment Report between 11 April and 23 May 2008.
Comments were specifically requested on the scientific aspects of this Application, in particular,
information relevant to the safety assessment of food from insect-protected corn MIR162. A
total of 24 submissions was received. A summary of these is provided in Attachment 3 to this
Report.
As this Application is being assessed as a general procedure, there was one round of public
comment. Responses to the Assessment Report were used to develop this Approval Report
for the Application. The main issues raised in public comments are discussed in the
Approval Report.
84
Attachment 3
The safety of recombinant DNA in food
1.
Recombinant DNA is no different to DNA from non-GM sources
All DNA is made up of the same chemical elements; recombinant DNA and DNA from nonGM sources is therefore composed of the same four nucleotides. Genetic modification
results in the re-assortment of sequences of nucleotides but leaves chemical structure
unchanged. Recombinant DNA is therefore chemically identical to non-recombinant DNA.
here is also very little that is unique about the sequences of recombinant DNA, as most gene
constructs that are used for transformation are derived from naturally occurring gene
sequences, the vast majority of which would have been encountered before in food, either
because they are derived from plant genes, or from bacteria or plant viruses that are often
found associated with food (e.g. Bacillus subtilis, a common soil bacterium from which Bt
genes are derived, might often be found on the surface of fresh fruit and vegetables; the
cauliflower mosaic virus from which promoter sequences are often derived is frequently
present in fresh vegetables).
2.
Human beings are exposed to large quantities of foreign DNA and other
nucleic acids (e.g. RNA) from a wide variety of sources on a daily basis as
part of the diet
Nucleic acids are a natural component of food. heir total amount varies according to the
type of food. For example, edible offal and animal muscle tissue comprise a high content of
both DNA and RNA (per gram of tissue), whereas plant storage tissues, such as grains or
potatoes, contain less DNA and RNA because they contain less cell nuclei (Jonas et al
2001). Dietary intake of nucleic acid is therefore influenced heavily by the diet of individuals
and varies widely, but has been estimated to be in the range 0.1-1.0 g/person/day (Doerfler
& Schubbert 1997).
3.
The presence of recombinant DNA in food does not increase the overall
dietary intake of DNA
Genetic modification typically results in the introduction of one or two new genes into an
organism’s genome. Given the large size of plant genomes, the contribution made by
recombinant DNA to the total DNA in the genome will be very small. For example, for corn,
which has an average genome size of 2,292 Mb, transformed with an insert of approximately
5 kb, the inserted recombinant DNA will make up only 2 X 10,000% of the total DNA in the
genome (Jonas et al 2001).
4.
Nucleic acids are broken down during food processing
Food processing may lead to partial or complete degradation or removal of DNA. Physical
and chemical factors, such as shear forces, heat or pH, may cause random cleavage of DNA
strands, thus reducing the average DNA length but not total DNA content (Jonas et al 2001).
Some processes such as the purification of sugar and the production of refined oils will
remove most, if not all, DNA.
A number of studies focussing on various thermal treatments applied to food during
processing (e.g. canning, fermentation), indicate that most DNA (including recombinant
DNA) will be reduced to lengths of approximately 300 base pairs or less (Ebbehoj &
Thomsen 1991, Hupfer et al 1998, Straub et al 1999).
85
DNA fragments of such size are unlikely to encode functional genes, since this would require
not only the full coding region to be present but also the appropriate regulatory sequences
(e.g. promoter, terminator).
5.
Ingested nucleic acids are extensively broken down in the digestive tract
Irrespective of whether GM foods are subject to processing prior to consumption, nucleic
acid will also be broken down during digestion. Ingested DNA is cleaved through acid
hydrolysis and enzymatic digestion (especially by pancreatic and intestinal nucleases) into
small DNA fragments and mixtures of mono-, di-, tri-, oligo- and polynucleotides, which are
then further catabolised into sugar phosphates and purine and pyrimidine bases (Carver &
Walker 1995).
The fate of ingested DNA has been extensively studied and is discussed in a number of
reviews (e.g. Beever & Kemp 2000, Jonas et al 2001). Given the chemical and structural
similarity of all DNA, there is no basis for considering that in vivo hydrolysis and absorption
of recombinant DNA will be different from non-recombinant DNA.
While the vast majority of ingested DNA will be degraded in the GI tract, a number of
studies, including one in humans, have demonstrated that this process may not completely
degrade all ingested DNA, with some incompletely digested DNA fragments being absorbed
and detected transiently in cells of the GI tract as well as blood, liver, spleen and other
organs and tissues. The most quoted of these is the human study reported by Netherwood
et al (2004) as well as the series of studies in mice reported by Schubbert et al (1994, 1997,
and 1998).
In the Netherwood et al study, nineteen human volunteers (twelve with intact digestive tracts,
seven with ileostomies16) were fed GM soy containing the epsps gene. The amount of
recombinant DNA that survived passage through the small bowel varied between the seven
ileostomists, with a maximum of 3.7% recovered from the stoma of one individual. This rate
of recovery was similar to an endogenous soy gene, suggesting the recombinant DNA was
digested similarly to other plant DNA. The epsps gene could not be detected in faeces from
subjects with intact digestive tracts, suggesting that any DNA surviving digestion in the upper
GI tract is readily degraded in the large intestine. The study also found evidence of preexisting transfer of a fragment of the epsps gene between GM soy and a small number of
micro-organisms in the small intestine of the ileostomists. The authors speculated this had
occurred prior to commencement of the study. There was no evidence of the intact epsps
gene being transferred. In subjects with intact digestive tracts, none of the endogenous
bacteria in the faeces were found to contain any epsps gene fragments from GM soy.
In the studies reported by Schubbert et al, M13 bacteriophage DNA was fed to mice at high
doses and transiently detected as fragments in various tissues including foetal tissue. The
vast majority of cells identified as containing M13 DNA fragments appeared to be
macrophages or other differentiated phagocytes of the immune system.
The purpose of such cells is to destroy foreign macromolecules. It has been suggested that
the relatively high frequency of cells that contained M13 DNA is probably related to the
occurrence of unmethylated CpG sequences, which would stimulate macrophages and other
immune cells to phagocytose the fragments (Beever and Kemp, 2000). Unmethylated CpG
sequences are characteristic of bacterial DNA but not DNA in either plants or animals,
therefore M13 DNA is probably not a good model for plant-derived recombinant DNA.
16
An ileostomy involves resection of the terminal ileum and diversion of digesta via a stoma to a
colostomy bag.
86
Other studies undertaken with livestock species ingesting GM plants (e.g. Einspanier et al
2001, Aulrich et al 2002, Reuter & Aulrich 2003, Tony et al 2003, Flachowsky et al 2005,
Broll et al 2005, Mazza et al 2005) have confirmed that plant DNA may be readily detected in
the tissues of animals. In some of these studies, small fragments of recombinant DNA were
also detected in the GI tract or specifically the stomach, and in one case in the blood, liver,
spleen and kidney (Mazza et al 2005), but so far, intact genes of recombinant-DNA origin
have not been detected.
These results clearly indicate that the systemic uptake of ingested foreign DNA is a normal
physiological process, and the demonstration of fragments of DNA in phagocytic cells should
be expected as a natural consequence of that uptake. These cells provide immune
surveillance of the digestive tract and other tissues, and recirculate frequently to the liver as
a normal mechanism of removing debris. The rare appearance of foreign DNA fragments in
a few foetal or neonatal cells should likewise not be of concern as it indicates that a few
macromolecules have crossed the placenta and been engulfed by phagocytes of the foetus.
It should also come as no surprise that, with the improved sensitivity of analytical
techniques, small fragments of recombinant DNA will occasionally be detected. The less
frequent detection of recombinant DNA fragments probably reflects that recombinant DNA
makes up only a very small proportion of the total DNA ingested (see 6.3 above).
6.
Uptake and expression of foreign DNA by micro-organisms inhabiting the
digestive tract is likely to be an extremely rare event
The horizontal DNA transfer of recombinant DNA into gut micro-organisms has been the
subject of intense scientific scrutiny and debate, particularly in relation to the use of antibiotic
resistance genes, and the possibility that such transfer could compromise the therapeutic
use of antibiotics. Some studies are available which demonstrate that, in certain
circumstances, foreign DNA may be taken up and expressed by micro-organisms, at least in
vitro (e.g. Mercer et al 1999). To date, there is no evidence of transfer to and expression of
recombinant DNA in bacteria under natural conditions. Transfer and expression has only
been observed under laboratory conditions and only if homologous recombination is possible
(Nielsen et al 1998).
While such studies provide evidence of the possibility of DNA uptake by bacteria, they do not
provide evidence that recombinant DNA poses any greater risk. The overwhelming scientific
consensus is that, while theoretically possible, the likelihood of transfer and functional
integration of recombinant DNA in gut micro-organisms is extremely low.
The gene transfer mechanisms by which bacteria may acquire new genes (conjugation,
transduction and transformation) are well described and a number of comprehensive reviews
on these processes are available (e.g. Levy & Miller 1989). In food, transfer by all three
mechanisms is believed to be possible, at least from micro-organisms consumed in food,
although studies on gene transfer in the human and animal gut are limited (Jonas et al
2001). The gut and the colon in particular are considered to be a favourable environment for
such transfer because of the high density of micro-organisms; direct cell to cell contact
favours conjugation, and natural transformation is also favoured because of the relatively
high DNA concentration at the recipient cell surface (Paul 1992).
For free DNA however there is only a very low probability per gene and per passage through
the GI tract, of uptake and stable integration into the genome of a bacterial cell. There are
several reasons for this, which are extensively elaborated in Jonas et al (2001), but briefly:

Degradation of DNA through the gastric and ileal passage makes it highly unlikely that
linear DNA molecules of sufficient size will enter the colon;
87

for transformation by linear DNA the bacterial cell must be competent:
-
a bacteria is said to be competent if it is able to naturally take up DNA from the
environment. Competence usually occurs at a particular stage in the bacterial
growth cycle when the bacterium produces a protein called a competence factor.
Only between 1-2% of microbial species are thought to be naturally competent;

DNA transferred through transduction or transformation may be susceptible to
restriction by bacterial restriction endonucleases, which cleave double-stranded DNA;

in the case of linear DNA, homology with sequences in the bacterial genome is
necessary for integration to occur;

to be expressed, the transferred DNA must contain an intact coding region and be
associated with the appropriate bacterial expression signals:
-

most recombinant DNA derived from GM plants will be linked to plant-specific
expression signals which are unlikely to function in bacterial cells;
to be maintained by the bacterial population, acquired DNA must confer a competitive
advantage to the transformed cell.
Therefore, although bacteria possess sophisticated systems for DNA uptake from their
environment, horizontal transfer into and expression of free recombinant DNA present in
food is predicted to be an extremely rare event.
Given the similarity between recombinant DNA and non-recombinant DNA, both in terms of
chemical structure as well as sequence, the likelihood of transfer and functional integration
of recombinant DNA by gut micro-organisms will be theoretically the same as for nonrecombinant DNA present in food.
It might also be argued that, as recombinant DNA would represent only a very small
proportion of the total DNA ingested in food, successful transfer of recombinant DNA to gut
micro-organisms would be far less likely to occur than transfer of non-recombinant DNA.
7.
Should a small proportion of ingested DNA survive digestion in the GI tract,
mammals possess effective mechanisms to avoid incorporation of foreign
DNA into the genome
Mammalian cells have evolved with several mechanisms of defence against the uptake,
integration and continued expression of foreign DNA (Doerfler 1991). In addition to the initial
degradation and/or excretion of foreign DNA that occurs following ingestion and the action of
cells of the immune system e.g. phagocytes, to remove foreign macromolecules, most
mammalian cells produce at least one DNase with exonuclease activity, and these would be
expected to degrade most exogenous DNA, should it actually survive and be taken up by the
cell (Jonas et al 2001).
The nuclear membrane is also a strong barrier against the penetration of nucleic acids. Entry
is tightly regulated by nuclear pores, with nuclear targeting signals required for penetration,
especially in the case of cells that have finished their division and the nuclear envelope is not
disrupted (Gorlick & Mattaj 1996, Guralnick et al 1996, Collas & Aelstrom 1997, Palacios et
al 1997, Popov et al 1998, Zeimienovicz et al 1999, Saphire et al 2000).
88
Should DNA succeed in penetrating the nucleus, and become integrated in the genome, the
evidence indicates that any integrated foreign DNA is likely to be rendered inactive through
targeted methylation (Doerfler 1991, Doerfler et al 1995, Orend et al 1995).
8.
The risk posed by the presence of recombinant DNA in food is no different to
that posed by non-recombinant DNA
While the Review Request raises a number of interesting questions in relation to the
potential impact on human health, should foreign DNA not be inactivated if taken up by cells,
the studies cited (e.g. Palka-Santini et al 2003, Woodhams et al 2007, Rosenberg et al
2007) do not provide any compelling arguments that such health impacts, should they occur,
are likely to be any greater with recombinant DNA compared to non-recombinant DNA.
The study by Malatesta et al (2002) on the ultrastructure of hepatocytes from mice fed GM
soybean17, is interesting in that the authors report that the GM soy-fed mice exhibited some
slight but statistically significant ultrastructural differences in hepatocyte nuclei18 relative to
controls. Cells bearing slightly more irregularly shaped nuclei were postulated to be
indicative of an increased metabolic rate and the slight increase in the number of nuclear
pores was apparently suggestive of increased molecular trafficking between the nucleus and
cytoplasm.
The study itself is quite unusual because it undertakes an investigation at the ultrastructural
level in the absence of any clear evidence of effects in the liver at either the macroscopic or
light microscopic level. Typically, ultrastructural investigations are only undertaken to identify
an underlying mechanism if there is clear evidence of cellular change or clinical signs. In the
Malatesta et al study only 100 cells/mouse were examined. Consequently the relevance of
the subtle ultrastructural morphometrical changes observed are difficult to interpret,
especially in the absence of any corroborating evidence of atypical liver activity (e.g.
classical markers of liver cell damage). In addition, it is not clear that such effects, were they
to be reproduced, would necessarily be attributable to the presence of recombinant DNA
itself. The relevance of this study to the issue of persistence and uptake of recombinant DNA
is therefore questionable.
The main objective of a GM food safety assessment is to identify whether new or altered
hazards are present in the food as a result of the genetic modification, and if present to
determine what risk, if any, they may pose to human health (Codex 2004, FSANZ 2007).
Therefore, the key issue for FSANZ is whether the occurrence of recombinant-DNA in food
poses any greater risk to human health, than that posed by the significantly larger amount of
non-recombinant DNA already present in food.
In general, FSANZ considers the risk to be equivalent between recombinant and nonrecombinant DNA and therefore does not regard this as an issue that requires explicit
consideration for each and every GM food application. Rather, this issue need only be
addressed if the molecular characterisation identifies an element or elements in the gene
construct that may significantly increase the likelihood of recombinant DNA in GM food being
taken up and stably incorporated in either gut micro-organisms or human cells. The
constructs typically used to date contain coding and regulatory sequences that have been
used many times before and are well known not to increase the likelihood of such events
occurring.
17
18
The GM soy line used was glyphosate tolerant soybean line 40-3-2, not MON 89788.
Irregularly shaped nuclei and increased numbers of nuclear pores.
89
9.
Conclusion
The transferred DNA in MIR162 corn does not contain any genetic elements which may
significantly increase the likelihood of recombinant DNA in GM food being taken up and
stably incorporated into the genome of either gut micro-organisms or human cells. Given
this, FSANZ does not consider that the issue of persistence and uptake of recombinant DNA
requires specific consideration in the safety assessment of food derived from insectprotected corn line MIR162; consideration of such issues is already implicit in the molecular
characterisation component of the safety assessment.
References
Aulrich, K., Reuter, T. & Flachowsky, G. (2002). The fate of foreign DNA in farm animals fed with
genetically modified plants. Proc. Soc. Nutr. Physiol. 11: 187 – 188.
Beever, D.E. & Kemp, C.F. (2000). Safety issues associated with the DNA in animal feed derived
from genetically modified crops. A review of scientific and regulatory procedures. Nutrition Abstracts &
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