EXPLANATORY STATEMENT

APPLICATION A595

FOOD DERIVED FROM INSECT-PROTECTED CORN LINE MON 89034

Executive Summary

Food Standards Australia New Zealand (FSANZ) received a paid Application from Monsanto Australia Ltd (the Applicant) on 19 December 2006.  The Applicant has requested a variation 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, MON 89034.  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.

MON 89034 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 insecticidal proteins derived from Bacillus thuringiensis, a common soil bacterium.

Corn line MON 89034 is intended to be grown in North America.  However, once commercialised, corn products imported into Australia and New Zealand could contain ingredients derived from MON 89034 corn.  Approval is therefore necessary before these products may enter Australian and New Zealand markets.

Safety Assessment

FSANZ has completed a comprehensive safety assessment of food derived from insect-protected corn line MON 89034, 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 MON 89034 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 MON 89034 is considered as safe and wholesome as food derived from other commercial corn varieties.

Labelling

If approved, food derived from insect-protected corn line MON 89034 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 at low levels in the grain.

Labelling addresses the requirement of section 18(1)(b) of the Act, namely 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 MON 89034 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 this application is the preferred option as the potential benefits to all sectors outweigh the costs associated with the approval.

Purpose

The Applicant seeks amendment to Standard 1.5.2 – Food produced using Gene Technology, to include food derived from insect-protected corn line MON 89034 in the Table to clause 2.

Reasons for Decision

A variation to the Code approving food derived from insect-protected corn line MON 89034 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 MON 89034;

• food derived from insect-protected corn line MON 89034 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 food commodities derived from insect-protected corn line MON 89034 will be required if novel DNA and/or protein is present in the final food; and

• 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, an amendment to the Code.

Consultation

The Initial Assessment was advertised for public comment between 21 March and 2 May 2007.  A total of fourteen submissions were received during this period.  The Draft Assessment was advertised for public comment between 12 December 2007 and

6 February 2008.  A total of thirteen submissions were received.  A summary of these is provided in Attachment 3 to this Report.    

CONTENTS

Introduction

1. Background

1.1 Current Standard

1.2 Description and Purpose of the Genetic Modification

1.3 Overseas Approvals

2. The Issue / Problem

3. Objectives

4. Key Assessment Questions

RISK ASSESSMENT

5. Risk Assessment Summary

5.1 Safety Assessment Process

5.2 Outcomes of the Safety Assessment

5.3 Conclusions

risk management

6. Options

6.1 Option 1 – Prohibit food from corn line MON 89034

6.2 Option 2 – Approve food from corn line MON 89034

7. Impact Analysis

7.1 Affected Parties

7.2 Benefit Cost Analysis

7.3 Comparison of Options

communication and Consultation Strategy

8. Communication

9. Consultation

9.2 World Trade Organization (WTO)

Conclusion

10. Conclusion and Decision

10.1 Reasons for Decision

11. Implementation and Review

Attachment 1 - Draft variation to the Australia New Zealand Food Standards Code

Attachment 2 - Safety Assessment

Attachment 3 - Summary of public submissions in response to the Draft Assessment

Attachment 4 - First Review Report

Introduction

An Application was received from Monsanto Australia Limited on 19 December 2006 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 insect-protected corn line MON 89034.

The genetic modification involves the transfer of two cry genes into corn. These genes are from a common soil bacterium called Bacillus thuringiensis and encode insecticidal proteins (Cry proteins) which protect the plant against feeding damage caused by certain insect pest larvae.

A Final Assessment of the Application has been completed, including a comprehensive safety assessment and consideration of issues raised in public consultation.

1. Background

1.1 Current Standard

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.

1.2 Description and Purpose of the Genetic Modification

The genetic modification in insect-protected corn line MON 89034 involves the introduction of the cry1A.105 and cry2Ab2 genes derived from B. thuringiensis subspecies kurstaki and aizawai.  These genes encode the Cry1A.105 and Cry2Ab2 insecticidal proteins which are selectively toxic to a range of lepidopteran insect larvae including European corn borer, Asian corn borer, southwestern corn borer, sugarcane borer, fall armyworm and corn earworm.

Cry proteins exert their effect on the target insects by causing lysis of midgut epithelial cells, which leads to gut paralysis, cessation of feeding and eventual death of the insect.  The lysis of the midgut epithelial cells is mediated by the binding of the activated Cry protein to specialised receptors on these cells.

The purpose of expressing two different Cry proteins in the corn is to provide extended control over a range of insect pest larvae and also to improve the management of insect resistance to the Cry proteins.  Cry1A.105 and Cry2Ab2 have different modes of action, particularly in the way in which they bind to the insect midgut.  Because of the dual effective dose and the distinct modes of action, the Applicant claims the likelihood of resistance being developed by target insects is low.

Hybrid corn lines derived from MON 89034 are intended for cultivation in North America and are not intended to be grown in either Australia or New Zealand.  Food from MON 89034 corn will therefore be entering the Australian and New Zealand food supply as imported, largely processed food products.

1.3 Overseas Approvals

The Applicant made submissions for food and feed use to the United States Food and Drug Administration in late 2006 and also requested a Determination of Nonregulated Status for MON 89034 from the U.S. Department of Agriculture, as well as a tolerance exemption from the Environmental Protection Agency.

Regulatory submissions for import approvals have been or will be made to countries that import significant corn or corn products, including China, Japan, Canada, Korea, the Philippines and Taiwan.  Feed approval was granted in Japan on 2 October 2007.

2. The Issue / Problem

The Applicant has developed corn line MON 89034 that is protected from feeding damage caused by lepidopteran insect pest larvae.  Before food derived from insect-protected corn line MON 89034 can enter the Australian and New Zealand food supply, it must first be assessed for safety and an amendment to the Code must be approved by the FSANZ Board, and subsequently be 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.

Monsanto Australia Limited has applied to have Standard 1.5.2 varied to include food derived from corn line MON 89034. 

3. Objectives

The purpose of this assessment is to determine whether it would be appropriate to vary the Code to approve the sale and use of food derived from corn line MON 89034 under Standard 1.5.2.  Section 18(1) of the Food Standards Australia New Zealand Act 1991 provides that the objectives of FSANZ in developing or reviewing food regulatory measures or variations of food regulatory measures 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.

Section 18(2) requires FSANZ to 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. Key Assessment Questions

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 MON 89034 comparable to food derived from conventional varieties of corn in terms of its safety for human consumption?

Is 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 MON 89034 has been evaluated according to the FSANZ Guidance Document on the Safety Assessment of Genetically Modified Foods[1].  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.

5. Risk Assessment Summary

5.1 Safety Assessment Process

The safety assessment applied to food from corn line MON 89034 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.

In conducting a safety assessment of food derived from insect-protected MON 89034 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.

5.2 Outcomes of the Safety Assessment

Detailed molecular analyses indicate that one copy of each of the cry1A.105 and cry2Ab2 genes have been inserted at a single site in the plant genome and are stably inherited from one generation to the next.  No antibiotic resistance marker genes are present in MON 89034 corn.
MON 89034 corn expresses two novel proteins, Cry1A.105 and Cry2Ab2.  Both proteins are expressed at relatively low levels in the grain, with the mean concentration for Cry1A.105 and Cry2Ab2 being 5.1 μg/g fresh weight and 1.1 μg/g fresh weight, respectively.  Both proteins as expressed in the plant conform in size and amino acid sequence to that expected, do not exhibit any post-translational modification including glycosylation, and also demonstrate the expected insecticidal 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.  It is well established that the Cry proteins from B. thuringiensis are inherently non-toxic to mammals and have exhibited little potential to be allergenic to humans over their long history of use. 

In addition, bioinformatic studies with the Cry1A.105 and Cry2Ab2 proteins have confirmed the absence of any 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 highly unlikely to be toxic or allergenic in humans.

Compositional analyses were done to establish the nutritional adequacy of MON 89034 corn, and to compare it to conventional corn lines under typical cultivation conditions.  No differences of biological significance were observed between MON 89034 corn and its conventional counterpart.  Food from insect-protected MON 89034 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.

5.3 Conclusions

No potential public health and safety concerns have been identified in the assessment of insect-protected MON 89034 corn. On the basis of the data provided in the present application, and other available information, food derived from insect-protected MON 89034 corn is considered as safe and wholesome as food derived from other corn varieties.

risk management

6. Options

There are no non-regulatory options for this Application.  The two regulatory options available for this Application are:

6.1 Option 1 – Prohibit food from corn line MON 89034

Maintain the status quo by rejecting the Application.

6.2 Option 2 – Approve food from corn line MON 89034

Vary Standard 1.5.2 to permit the sale and use of food derived from food derived from insect-protected corn line MON 89034, with or without specified conditions in the Table to clause 2 of the Standard.

7. Impact Analysis

FSANZ is required to consider the impact of various regulatory (and non-regulatory) options on all sectors of the community in Australia and New Zealand.

7.1 Affected Parties

The affected parties may include the following:

• consumers, particularly those who have concerns about biotechnology;

• food importers and distributors of wholesale ingredients;

• the manufacturing and retail sectors of the food industry; and

• government generally, where a regulatory decision may impact on trade or WTO obligations, and enforcement agencies in particular who will need to ensure that any approved products are correctly labelled.

The cultivation of corn line MON 89034 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 Fisheries (MAF) before growing in either country could be permitted.  MON 89034 corn has been developed primarily for agricultural production overseas and, at this stage, the Applicant has no plans for cultivation in either Australia or New Zealand.

7.2 Benefit Cost 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.

7.2.1 Option 1 – prohibit food from corn line MON 89034

Consumers: Possible restriction in the availability of corn products if MON 89034 corn is present in imported foods.

 No impact on consumers wishing to avoid GM foods, as food from MON 89034 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.

 Impact on monitoring resources, particularly at the border, where unapproved GM material derived from MON 89034 may inadvertently be incorporated into imported food products.

Industry:   Possible restriction on corn imports once MON 89034 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 MON 89034

Consumers: No restriction on imported corn products if derived from MON 89034 corn. Increased choice in the marketplace as a result of mandatory labelling of certain products derived from MON 89034.

 Potential impact on consumers wishing to avoid GM corn by a possible restriction of choice of products, or increased prices for non-GM corn. 

Government: Benefit in that there would be no potential for trade disruption as a result of the detection of unapproved MON 89034 in imported products.

 Approval of MON 89034 corn would ensure no conflict with WTO obligations.

 Likely to be increased costs associated with the additional monitoring required to ensure compliance with the labelling provisions of the Code. 

Industry: Broader market access and increased choice in raw materials for food manufacturing.

 Benefit to importers of processed foods containing corn as an ingredient as foods derived from MON 89034 corn would be compliant with the Code.

 Possible cost to food industry as some food ingredients derived from MON 89034 corn would be required to be labelled as genetically modified.

7.3 Comparison of Options

As food from insect-protected corn line MON 89034 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 it could potentially limit the availability of imported corn products in Australia and New Zealand due to the approval of MON 89034 corn by other countries.

Under Option 2, primary producers would benefit from an increased choice of crop lines with potentially lower production costs and higher yields, which could flow on to other sectors including consumers in Australia and New Zealand as lower food prices.   Government will also benefit in that potential disruption to trade will be avoided. 

While there will be costs to government associated with the additional monitoring required to ensure compliance with the Code, similar costs are also likely to be associated with Option 1, therefore the overall impact on monitoring resources is expected to be neutral.

As MON 89034 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 MON 89034, 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.  In addition, FSANZ will issue a media release drawing journalists’ attention to the matter.

As normally applies to all GM food assessments, the Final Assessment Report for this Application will be available to the public on the FSANZ website and distributed to major stakeholders.

9. Consultation

The Initial Assessment was advertised for public comment between 21 March and 2 May 2007.  A total of fourteen submissions were received during this period.  The Draft Assessment was subsequently advertised for public comment between 12 December 2007 and 6 February 2008, with a total of thirteen submissions being received. A summary of second round public submissions is provided in Attachment 3 to this Report.    

FSANZ has taken the submitters’ comments into account in preparing the Draft and Final Assessments of this Application.  Responses to general issues regarding GM food are available from the FSANZ website[2]. Specific issues relating to food derived from corn line MON 89034 have been addressed in the report.  A discussion of the major issues raised follows.

9.1.1 Animal feeding studies

Two submitters have suggested that the expression of two insecticidal proteins in MON 89034 corn should be enough to make the corn sufficiently different from an earlier GM corn variety [MON 810 corn, approved by FSANZ in November 2000] to warrant the undertaking of animal feeding studies.

9.1.1.1 Response

The similarity or otherwise of corn line MON 89034 to a previously approved corn line is not relevant to FSANZ’s decision regarding the need for animal feeding studies.

FSANZ convened an expert group in June 2007 to consider the need for animal feeding studies in the safety assessment of GM foods.  The report from that meeting is available on the FSANZ website at the following web address:

http://www.foodstandards.gov.au/_srcfiles/Workshop%20Report%20FINAL.pdf. 

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 toxicity studies with 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, if considered appropriate by FSANZ.  FSANZ will therefore continue to assess the need for whole food studies on a case-by-case basis, taking into account the nature of the genetic modification and the results of the comparative assessment.

For this Application, the comparative assessment indicates that food derived from corn line MON 89034 is equivalent to food from conventional corn in terms of its composition and nutritional adequacy.  The corn grain contains low levels of two insecticidal proteins; however the evidence indicates these proteins are non-toxic to mammals, including humans and has limited potential to be a food allergen.   FSANZ has therefore not identified any differences that raise potential safety or nutritional concerns.  For this reason, FSANZ does not believe there would be any justification for undertaking additional testing in laboratory animals.

9.1.2 Monitoring and enforcement costs

Queensland Health and the NSW Food Authority have raised the issue of rising monitoring and enforcement costs for governments in terms of available resources, labour and reagent costs.  Both jurisdictions consider that a national enforcement strategy for GM food approvals could be needed to address these issues.

In a further submission to FSANZ in response to the Draft Assessment Report, both jurisdictions have reaffirmed their earlier submissions.  While acknowledging that the issue of costs associated with enforcement is not unique to GM foods, they argue that special consideration should be given to the high level of sophistication needed to detect GM foods, and the associated additional costs.  They state that while a food business’s documentation may be audited in relation to the GM status of individual ingredients, such audits are most likely to be used when investigating suspected labelling breaches by individual food businesses, whereas GM testing is most likely to be used for general surveillance to identify businesses that are failing to identify the presence of GM material in their food products.  In addition, food businesses are not obliged to maintain a paper trail therefore it is not always possible for enforcement agencies to rely on this as a means of reducing enforcement costs associated with product testing.  This is especially the case with imported products, where the paper trail is usually limited or non-existent.

The NSW Food Authority indicated its intention to raise at a national level the general issue of costs associated with the enforcement of new food standards, with specific emphasis on enforcement costs associated with GM food standards.  Given the limited resources available for monitoring and enforcement of the GM food standard, Queensland Health has indicated its intent to support the development of a national enforcement strategy for GM foods.

9.1.2.1 Response

FSANZ agrees that, over time, the successive approval of new GM foods may significantly impact on monitoring resources, however such costs are largely unavoidable because even if no further GM foods were approved in Australia and New Zealand, such foods will still be entering international trade, therefore monitoring for the presence of any unapproved GM foods would continue to be necessary in order for Standard 1.5.2 to be effectively enforced.

FSANZ welcomes the initiative of the NSW Food Authority to raise this issue at a national level, in particular the intention to present a paper to the Implementation Sub-Committee (ISC),[3] proposing the development of a national compliance and monitoring strategy.  

9.1.3 Approval of foods due to risk of accidental presence

Queensland Health has expressed concern about applications of the nature of Application A595, stating the Queensland Government believes that approving GM foods due to the risk of accidental presence in the Australian food supply is not a good reason to apply food regulatory approval since this GM food is a prohibited substance in food.

9.1.3.1 Response

In response to this Application, FSANZ does not propose a variation to the Code for the reason that food derived from MON 89034 corn could be accidentally present in the food supply.  On the contrary, insect-protected MON 89034 corn was developed as a food crop and is intended for deliberate introduction into the food supply. The Applicant therefore applied to FSANZ, as well as a number of other regulatory agencies around the world, for approval of this product prior to its commercial release.

On occasion, FSANZ has accepted applications for foods from GM crops that have been developed primarily for animal feed (e.g. high lysine corn; Application A549).  This is done to avoid what is known as a ‘split use’ approval, where a GM plant receives approval for use as animal feed but not for human food.  The practice of avoiding ‘split use’ is also practiced in the United States and Canada, which are sources of imported GM foods and food ingredients into Australia and New Zealand.

The practice of ‘split-use’ approvals fell out of favour following an incident in the United States where traces of a GM corn (known as StarLink™ corn), which had been approved for animal feed only, were found in human food products.  The incident caused widespread consumer concern and significant disruption to trade and highlighted that adventitious contamination can occur despite well developed identity preservation and segregation systems being in place. 

To prevent similar incidents occurring in the future it is now common practice for GM plants intended primarily for feed use to also undergo food safety assessment and approval for human food use.  This minimises the risk of unassessed and unapproved products entering the food supply as a result of inadvertent co-mingling of grain/seeds during transport and storage, and also ensures that their use as feed will not pose indirect risks to humans.  As indicated above, this consideration is not relevant to this present application.

9.1.4 Claims about adverse effects in pigs fed GM corn

One submitter claims that pig farmers who have been feeding their pigs GM corn have noticed their pigs have become ‘sterile, contracted cancer or died’ and has asked why these findings have not been studied vigorously by the Australian and New Zealand Governments. 

9.1.4.1 Response

FSANZ is aware that in 2002 some farmers in the United States claimed that the feeding of GM corn to their pigs caused infertility in their sows; effects such as cancer and death were not reported.  FSANZ has not however been able to identify any credible scientific evidence which establishes GM corn as the cause of the alleged infertility.

9.1.5 Presence of transgenic DNA in food

Some submitters have expressed concern about the presence of transgenic DNA in food, stating that it is unstable, will not be completely digested in the stomach and may transfer to gut bacteria or human cells, potentially causing a number of adverse effects.

9.1.5.1 Response

This is a general issue that has been the subject of extensive consideration and publication over the last fifteen plus years, including by the Food and Agriculture Organization and the World Health Organization.  Through these considerations it has been concluded that, as DNA from all living organisms is structurally similar, the presence of transgenic DNA in food products, in itself, poses no additional health risk to consumers.

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.  Therefore, in relation to the presence of transgenic DNA in GM food, the key issue for FSANZ is whether its presence poses any greater risk to human health, than that posed by the significantly larger amounts of non-transgenic (i.e. naturally occurring) DNA already present in food. 

In general, the overwhelming scientific consensus is that the risk to human health of transgenic DNA, should it be taken up by bacteria in the gastrointestinal tract or human cells, can be regarded as equivalent to non-transgenic DNA.  Therefore, FSANZ does not regard this as an issue that requires explicit consideration for each and every GM food application.  Rather, consideration of such issues is already implicit in the molecular characterisation component of the safety assessment.  The molecular characterisation considers in detail the transgenic DNA sequences that are transferred to the plant, and therefore will identify any sequences that may remain in the food, and survive digestion. 

The molecular characterisation thus allows the identification of any DNA sequence that may potentially increase the likelihood of transgenic DNA in GM food being taken up and stably incorporated in either gut bacteria or human cells.  In all the assessments done to date, including that for MON 89034 corn, FSANZ has not identified any transgenic DNA sequence that would raise any valid food safety concerns should it remain present in GM food and be ingested by humans.  

9.2 World Trade Organization (WTO)

As members of the World Trade Organization (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 variation to the Code would have a trade enabling effect as it would permit food derived from MON 89034 corn to be imported into Australia and New Zealand and sold, where currently it is prohibited.   For this reason it was determined there is 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

10.1 Reasons for Decision

A variation to the Code to give approval to the sale and use of food derived from corn line MON 89034 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 MON 89034;

• food derived from insect-protected corn line MON 89034 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 food commodities derived from insect-protected corn line MON 89034 will be required if novel DNA and/or protein is present in the final food; and

• 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, an amendment to the Code.

11. Implementation and Review

Following notification, the variation to the Code will come into effect on gazettal, subject to any request from the Ministerial Council for a review of FSANZ’s decision.

ATTACHMENTS

1. Draft variation to the Australia New Zealand Food Standards Code

2. Safety assessment report

3. Summary of second round public submissions

Attachment 1

Draft variation to the Australia New Zealand Food Standards Code

Standards or variations to standards are considered to be legislative instruments for the purposes of the Legislative Instruments Act (2003) and 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 –

Attachment 2

Safety Assessment

Food Derived From Insect-Protected Mon 89034 Corn

SUMMARY AND CONCLUSIONS

Background

Insect-protected MON 89034 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 two cry genes, encoding insecticidal Cry proteins, derived from Bacillus thuringiensis, a common soil bacterium.  The Cry proteins exert their effect on the insect by causing lysis of midgut epithelial cells, which leads to gut paralysis, cessation of feeding and eventual death of the insect.

Insect-protected MON 89034 corn is claimed to provide extended control over a range of insect pest larvae, compared to crops expressing a single cry gene.  MON 89034 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 MON 89034 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 MON 89034 corn may include flour, breakfast cereals, high fructose corn syrup and other starch products.

B. thuringiensis, the microorganism from which the cry genes were 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 Cry proteins it produces are not known to be toxic to any vertebrates, including humans and other mammals. 

Molecular Characterisation

MON 89034 corn was generated through the transfer of the cry1A.105 and cry2Ab2 genes to the conventional inbred corn line LH172. 

The cry1A.105 gene encodes the 133 kDa Cry1A.105 insecticidal protein, which is a chimeric protein consisting of different functional domains derived from three wild-type Cry proteins from B. thuringiensis subspecies kurstaki and aizawai.  The cry2Ab2 gene encodes the 61 kDa Cry2Ab2 insecticidal protein, which is a variant of the wild-type Cry2Ab2 protein from B. thuringiensis subspecies kurstaki.

Detailed molecular analyses indicate that one copy of each of the cry1A.105 and cry2Ab2 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 MON 89034 corn.

Characterisation of Novel Protein

MON 89034 corn expresses two novel proteins, Cry1A.105 and Cry2Ab2.  Both proteins are expressed at relatively low levels in the grain, with the mean concentration for Cry1A.105 and Cry2Ab2 being 5.1 μg/g fresh weight and 1.1 μg/g fresh weight respectively.

A large number of studies have been done to confirm the identity and physicochemical and functional properties of the expressed Cry1A.105 and Cry2Ab2 proteins, as well as to determine their potential toxicity and allergenicity.  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 activity.

In relation to their potential toxicity and allergenicity, it is worth noting that Cry proteins from B. thuringiensis are inherently non-toxic to mammals and have exhibited little potential to be allergenic to humans over their long history of use.  In addition, bioinformatic studies with the Cry1A.105 and Cry2Ab2 proteins have confirmed the absence of any 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

Compositional analyses were done to establish the nutritional adequacy of MON 89034 corn, and to compare it to conventional corn lines under typical cultivation conditions.  The components analysed were protein, fat, carbohydrate, amino acids, fatty acids, vitamins, minerals, and the anti-nutrient phytic acid.

No differences of biological significance were observed between MON 89034 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 commercial corn hybrids and other conventional corn varieties, and therefore they most likely reflect normal biological variability.   Food from insect-protected MON 89034 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 MON 89034 corn. The introduction of MON 89034 corn into the food supply would therefore be expected to have little nutritional impact.  The nutritional adequacy of food derived from MON 89034 corn was also confirmed using a feeding study in rapidly-growing broiler chicks, which demonstrated that MON 89034 corn is equivalent to its conventional counterpart and other commercial corn hybrids 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 MON 89034 corn. On the basis of the data provided in the present application, and other available information, food derived from insect-protected MON 89034 corn is considered as safe and wholesome as food derived from conventional corn varieties.

1. BACKGROUND

A safety assessment has been conducted on food derived from corn that has been genetically modified (GM) to be protected against feeding damage caused by the larvae of a number of insect species.  The GM corn is referred to as MON 89034 corn.

MON 89034 corn is protected against a range of lepidopteran insect larvae including European corn borer, Asian corn borer, southwestern corn borer, sugarcane borer, fall armyworm and corn earworm.

Protection is achieved through expression in the plant of two insecticidal Cry proteins, Cry1A.105 and Cry2Ab2, derived from Bacillus thuringiensis, a common soil bacterium.  Cry1A.105, encoded by the cry1A.105 gene, is a chimeric protein made up of different functional domains derived from three wild-type Cry proteins from B. thuringiensis subspecies kurstaki and aizawai.  The Cry2Ab2 protein is encoded by the cry2Ab2 gene derived from B. thuringiensis subspecies kurstaki.

The Cry proteins exert their effect on the host insect by causing lysis of midgut epithelial cells, which leads to gut paralysis, cessation of feeding and eventual death of the insect.  The lysis of the midgut epithelial cells is mediated by the binding of the activated Cry protein to specialised receptors on these cells.

Hybrid corn lines containing the MON 89034 transformation event are intended for cultivation in North America and are not intended to be grown in either Australia or New Zealand.  Food from MON 89034 corn will therefore be entering the Australian and New Zealand food supply as imported, largely processed food products.

2. HISTORY OF USE

2.1 Host organism

The host organism is corn (Zea mays L), otherwise known as maize.  Corn is the world’s third leading cereal crop, behind wheat and rice, and is grown in over 25 countries (OECD 2002).  In 2005, worldwide production of corn was over 700 million tonnes, with the United States and China being the major producers (FAOSTAT 2005).

Corn is naturally cross-pollinated and until about 1925, mainly open-pollinated varieties were grown, whereas today mainly hybrid varieties are grown.  Hybrid varieties have more vigorous growth and higher yields.

Field corn has been grown in Mexico and Central America for some 8000 years and in Europe for 500 years and can thus be said to have a long history of safe use as food by humans.  The majority of corn that is grown however is destined for use as animal feed.

Corn is processed using both wet and dry milling techniques.  Dry milling is the oldest way of processing corn kernels for food and feed use.  Three dry milling processes are used: (i) stone grinding, which is used widely in Latin America, Africa, Asia, and by small mills in North America; (ii) dry-grind, which is used for producing ethanol for commercial uses; and (iii) degermining, which is the process most widely used by the food industry.

The degermining process produces grits, meal, flour, oil and hominy feed[4].  Wet milling is used primarily to produce starch, which may be used directly in food products or converted to various sweeteners such as high fructose corn syrup.  The starch also serves as a major source of sugar for the fermentation of beverage alcohol.

The corn line used as the recipient for transferred genes is the proprietary inbred line, LH172 (Eggerling 1994).  This inbred line was used because it responds well to transformation with Agrobacterium and subsequent tissue culture steps.  Inbred corn line LH172 is a yellow dent corn and is used as a parental line for producing first generation hybrid corn.

2.2 Donor Organism

The two Cry proteins expressed in MON 89034 corn are derived from B. thuringiensis. 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’.

B. thuringiensis is a facultative anaerobic, gram-positive spore-forming bacterium that produces characteristic insecticidal proteins, as parasporal crystals, during the sporulation phase.  These crystals are predominantly comprised of one or more Crystal (Cry) and Cytolytic (Cyt) toxins, also called δ-endotoxins. 

These toxins are highly specific to their target insect species, are innocuous to humans, vertebrates and plants, and are completely biodegradable (Bravo et al 2007). 

Over 60 subspecies of B. thuringiensis have been described.  B. thuringiensis subspecies can synthesise more than one type of Cry protein, which are specifically toxic to the insect orders Coleoptera, Diptera, Hymenoptera, and Lepidoptera, and also to nematodes.  The Cyt toxins are mostly found in B. thuringiensis strains that are active against Diptera. 

A number of different commercial B. thuringiensis formulations have been registered worldwide for use as an insecticide to be applied to foliage, soil, and water or food storage facilities.  While the B. thuringiensis spores or vegetative cells may persist in the environment for weeks, months or years, the Cry proteins become inactive within hours or days.

Studies on mammals, particularly laboratory animals, demonstrate that B. thuringiensis is mostly non-pathogenic and non-toxic. B. thuringiensis has been demonstrated to be highly specific in its insecticidal activity and has demonstrated little, if any, direct toxicity to non-target insects. 

 The use of B. thuringiensis products in the field can result in considerable aerosol and dermal exposure in humans.  With the exception of case reports on ocular and dermal irritation, no adverse health effects have been documented after occupational exposure to B. thuringiensis products (McClintock et al 1995).

Studies with human volunteers who ingested and inhaled large quantities of a Btk formulation (B. thuringiensis subspecies kurstaki) did not reveal any adverse effects (Fisher & Rosner 1959).  Similarly, B. thuringiensis present in drinking water or food has not been reported to cause adverse effects on human health (WHO 1999).

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.

3.1 Transformation method

MON 89034 was developed through Agrobacterium-mediated transformation of the proprietary inbred corn line, LH172 (Eggerling 1994), using the transformation vector, PV-ZMIR245.  PV-ZMIR245 contains two separate T-DNAs.  The first T-DNA, designated T-DNA I, contains the cry1A.105 and the cry2Ab2 gene expression cassettes.  T-DNA II contains the nptII gene expression cassette that encodes the neomycin phosphotransferase II (NPTII) enzyme which confers tolerance to antibiotics such as neomycin and paromomycin.

The use of two separate T-DNAs enables the generation of marker free plants by allowing insertion of the T-DNA with the traits of interest (T-DNA I) and the T-DNA with the selectable marker (T-DNA II) into two independent loci within the corn genome.  Following selection of the transformants, the inserted T-DNA encoding the selectable marker can be segregated away through subsequent breeding and genetic selection, while the inserted T-DNA containing the trait(s) of interest is maintained.

Freshly isolated immature corn embryos were used in the transformation.  Following inoculation with Agrobacterium containing plasmid PV-ZMIR245, the immature embryos were transferred to a co-culture medium for 1-3 days to ensure transformation of individual cells.  The immature embryos were then transferred to selection medium containing carbenicillin to eliminate Agrobacterium, and paromomycin to eliminate cells that were not transformed so that only cells containing T-DNA II, with or without T-DNA I, survived.  The resulting transformed cells were then subcultured several times on selection medium and regenerated into R0 plants.

During subsequent breeding at the F1 generation, the unlinked insertions of T-DNA I and T-DNA II were segregated.  Plants containing only the cry1A.105 and cry2Ab2 gene cassettes were selected using molecular analysis, while plants containing the nptII gene cassette were discarded.  The absence of the nptII gene and the NPTII protein was further confirmed by both Southern blot and ELISA.  The steps used in the generation of MON 89034 corn are depicted in Figure 1.

Assembly of plasmid vector PV-ZMIR245 in Escherichia coli

and transfer into Agrobacterium tumefaciens strain AB1

Transformation of the immature embryos of LH172 tissue

with A. tumefaciens ABI containing PV-ZMIR245

Initial selection of transformed cells containing either T-DNA II

or T-DNA II plus T-DNA I on a medium containing paromomycin

Regeneration of transformed corn plants

Separation of unlinked DNA inserted of T-DNA I and T-DNA II

through segregation during traditional breeding

Screening of transformed plants for the presence of

T-DNA I and the absence of T-DNA II

Evaluation of selected plants for agronomic

performance and field efficacy

Selection of MON 89034 as the lead commercial

candidate for further evaluation

Figure 1:  Steps used in the generation of MON 89034 corn

3.2 Description of the breeding process

The MON 89034 breeding process is briefly described below and depicted in Figure 2.

The primary transformant (R0) was cross-pollinated with the recurrent parent LH172 to produce the LH172 BC0F1 generation.  During this breeding step, the T-DNA I and T-DNA II insertions were segregated.  Only plants positive for the cry1A.105 gene and negative for the nptII gene were selected for subsequent breeding steps.

The selected LH171 BC0F1 plants were self-pollinated to generate LH172 BC0F2.  The segregation in the LH172 BC0F2 generation was examined using ELISA for Cry2Ab2 on leaf tissue.  The selected LH172 BC0F2 plants were self-pollinated to generate LH172 BC0F3.  The segregation in LH172 BC0F3 generation was determined using ELISA for Cry1A.105 and immunoassay for CryAb2 in leaf tissue.  The F3 generation was fixed for the DNA insert and not expected to segregate further.  The stability of the F4 and F5 generations was confirmed using an immunoassay for Cry2Ab in leaf tissue.

The LH172 BC0F4 generation was crossed to 12 different inbred corn lines for subsequent commercial development.  Generations LH172 BC0F6 and LH172 BC0F7 were crossed to LH198 (hybrid seed) and were used in all other analyses (composition and protein expression analyses) and additional field trials.

3.3 Description of the gene construct

The transformation vector PV-ZMIR245 contains three gene expression cassettes (Figure 3).  The cry1A.105 and cry2Ab2 gene expression cassettes are contained in T-DNA I and the nptII gene expression cassette is contained in T-DNA II.

T-DNA I in PV-ZMIR245 contains the cry1A.105 and cry2Ab2 genes.  The cry1A.105 gene encodes the 133 kDa Cry1A.105 insecticidal protein, which is a chimeric protein consisting of domains I and II from Cry1Ab or Cry1Ac[5], a substantial portion of domain III from Cry1F and the C-terminal domain from Cry1Ac.  Cry 1Ac and Cry1Ab are derived from B. thuringiensis subsp. kurstaki and Cry1F is derived from B. thuringiensis subsp. aizawai.  The cry1A.105 coding sequence was optimised for expression in monocotyledonous plants.  The cry2Ab2 gene encodes the 61 kDa Cry2Ab2 insecticidal protein, which is a variant of the wild-type Cry2Ab2 protein from B. thuringiensis subsp. kurstaki.  The cry2Ab2 coding sequence has been modified to change codon usage for optimised expression in plants.

T-DNA II in PV-ZMIR245 contains the nptII gene from E. coli transposon Tn5 (Beck et al 1982).  The nptII gene encodes the enzyme neomycin phosphotransferase II (NPTII) and confers resistance to the aminoglycoside antibiotics, including neomycin, paromomycin, kanamycin and geneticin (G418).  The nptII gene in T-DNA II functions as a dominant selectable marker in the initial stages of plant cell selection following transformation but is segregated away in subsequent generations.

R0

     x LH172

LH172 BC0F1

        

LH172 BC0F2

        

LH172 BC0F3

       

LH172 BC0F4

       

LH172 BC0F5

TI: BC1:F1 x RP                                                

LH172 BC0F6

                     x LH198

LH172 BC0F7

       [LH172 BC0F6 x LH198]F1H

                      x LH198                            

LH172 BC0F6 x F2H1

[LH172 BC0F7 x LH198]F1H

Figure 2:  Breeding Tree for MON 89034 corn

The LH172 BC0F6 x F2H1 generation was used for all the molecular analyses.  Generations in bold were used for molecular stability analyses.

Key: R0 = primary transformant; F(#) = filial generation;  = self pollination; BC(#) = backcross generation; RP = recurrent parent; H = hybrid; TI = trait integration.

Figure 3:  Map of transformation vector PV-ZMIR245

A full description of all the genetic elements in the transformation vector is provided in Table 1.

Table 1:  Description of the genetic elements in PV-ZMIR245

3.4 Characterisation of the genes in the plant

A number of molecular analyses were done to characterise the inserted DNA in MON 89034 corn (LH172 BC0 F6 x F2H1 in Figure 2).  A summary of each of the molecular analyses and the findings are given below.

3.4.1 Southern blot analyses

Genomic DNA from MON 89034 corn was analysed using Southern blot analysis to determine the insert number, the copy number, the integrity of the inserted cry1Ab.105 and cry2Ab2 gene cassettes (T-DNA I), and evaluate the presence or absence of plasmid backbone and selectable marker sequences (T-DNA II).  Conventional corn with the same genetic background as the MON 89034 line was used as the control for these analyses, with the transformation vector, PV-ZMIR245, used as a positive hybridisation control.

Isolated genomic DNA from MON 89034 corn and conventional corn was digested with various restriction enzymes, separated on agarose gels and then subjected to Southern blot analysis.  These analyses demonstrated the presence of a single copy of T-DNA I and confirmed a single site of insertion. .

Further Southern blot analyses were done to separately confirm the presence of the cry1A.105 and cry2Ab2 genes as well as each of the associated regulatory elements.  Genomic DNA was digested with selected restriction enzymes and probed with probes specific to each of the different genetic elements within T-DNA I.  Hybridising bands of the expected size were observed for each of the probes used, confirming the presence of intact cry1A.105 and cry2Ab2 genes as well as all associated regulatory elements.  No unexpected bands were detected.

Southern blot analysis was also used to determine whether any vector backbone sequence is present in the inserted DNA, and also to demonstrate the absence of the nptII gene.  Genomic DNA was probed with overlapping probes spanning the vector backbone of PV-ZMIR245 as well as the nptII coding region.  No detectable hybridisation bands were detected with any of the probes used, indicating the absence of any vector backbone sequence as well as the nptII gene.  The absence of other T-DNA II sequences was also confirmed by probing genomic DNA with overlapping probes spanning T-DNA II.  Hybridising bands consistent with that expected for the genetic elements common to both T-DNA I and T-DNA II were detected (e.g., the 35S promoter, nos terminator, border sequences).  No other hybridising bands were detected.

3.4.2 Polymerase chain reaction and DNA sequence analyses

The organisation of the genetic elements within the insert in MON 89034 corn was confirmed using PCR analysis by amplifying seven overlapping regions of DNA spanning the entire length of the insert (Figure 4).  The PCR products generated, following PCR of genomic DNA from MON 89034 corn, were all of the expected size, indicating that the arrangement of the genetic elements within the insert is the same as in the transformation vector, PV-ZMIR245.

Figure 4:  Overlapping PCR products generated across the insert in MON 89034 corn

The PCR products generated from MON 89034 corn genomic DNA were sequenced to further confirm the organisation of genetic elements within the insert, and determine the 5’ and 3’ insert-to-genomic DNA junctions, and the complete DNA sequence of the inserted DNA and adjacent genomic DNA regions.   A consensus sequence of the inserted DNA was generated by compiling the results of numerous sequencing reactions performed on each of the overlapping PCR products.  This consensus sequence was then aligned to the DNA sequence of T-DNA I in the transformation vector, PV-ZMIR245, to determine, what, if any, changes had occurred during the transformation process.

The sequence analysis indicated that the insert DNA in MON 89034 corn is 9317 nucleotides in length.  The sequence of the inserted T-DNA I was almost identical to that in the transformation vector with one exception.  The e35S promoter that regulates the expression of the cry1A.105 gene has been modified and the right border sequence of the T-DNA has been replaced by a left border sequence.   The modified e35S promoter in MON 89034 corn no longer has the duplicated enhancer elements which are present in the original e35S promoter in PV-ZMIR245.  The Applicant considers this molecular rearrangement has most likely occurred through a recombination event between the two 35S promoters in T-DNA I and T-DNA II, either prior to or during the process of T-DNA transfer to the plant cell.

In addition to the entire insert sequence, the sequence of 2060 base pairs of genomic DNA flanking the 5’ end of the insert and 905 base pairs of genomic DNA flanking the 3’ end of the insert were also determined.

3.4.3 Bioinformatic analysis of the 5’ and 3’ junction regions

Bioinformatic analyses were performed to assess the potential for allergenicity, toxicity and bioactivity of putative polypeptides encoded by the 5’ and 3’ insert-genomic DNA junction regions of MON 89034 corn.  These analyses are entirely theoretical, that is, no empirical evidence exists to suggest that the 5’ and 3’ junction regions in MON 89034 corn are transcribed.

The DNA sequence spanning the 5’ and 3’ junction regions of the MON 89034 corn insertion site were analysed for the presence of translational stop codons (TGA, TAG, TAA) and all six reading frames originating or terminating within the MON 89034 corn insertion were translated from stop codon to stop codon.  Following translation, putative polypeptide sequences greater than or equal to eight amino acids in length were selected for further analysis.  At the 5’ junction region, five deduced putative polypeptides were identified, with another five being identified at the 3’ junction region.

Putative polypeptides from each reading frame were compared to three different databases:

1.             AD6 database – contains allergen, gliadin and glutenin sequences and was assembled from sequences found on the FARRP allergen database[6].  Gene identification numbers for the 1537 sequences found on the FARRP database were used to assemble a list which was used to batch query the NCBI protein sequence database[7];

1.          TOXIN5 database – this database was assembled from public sequence databases, including GenBank and EMBL release 124 and SwissProt release 1.  Protein sequences were retrieved using the STRINGSEARCH function (keyword = toxin) of the Wisconsin package (version 10).  This search was used to identify and retrieve 12,771 separate entries which were then used to compile the TOXIN5 database.  The actual number of unique toxin sequences will be less than 12,771 because of the redundancy associated with the public database and because some entries may contain the word ‘toxin’ but may not be relevant protein toxins; and

1.        ALLPEPTIDES – this database was used to represent all currently known publicly available protein sequences and consisted of SwissProt release 40.0 (May 4, 2005) and NRAA release 65.0 (October 24, 2005).

The overall structural similarity of the putative polypeptides to sequences in each database was assessed using the FASTA algorithm.  The extent of structural relatedness was evaluated using visual inspection of the aligned sequences, the calculated percent identity, and E (expectation) score.  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).  The E score is a statistical measure of the likelihood that the observed similarity could have occurred by chance.  A larger E score indicates a lower degree of similarity.  Typically, alignments between two sequences will need to have an E score of less than 1 x 10-5 to be considered to have significant homology.  

In addition to the FASTA comparisons, searches were also done to identify short amino acid sequences that may represent linear IgE binding epitopes.  An algorithm (ALLERGENSEARCH) was developed to identify matches of eight contiguous amino acids or more.  Segments of eight amino acids were chosen by the Applicant because they consider this to be the smallest number of amino acids that will identify immunologically relevant matches.  The Applicant states that searches using small segments (e.g. 6 or 7 amino acids) lead to high rates of false positive matches and therefore have little predictive value.  The ALLERGENSEARCH algorithm compares the query sequence to each sequence in the allergen database using a sliding window of eight amino acids.

No biologically relevant sequence similarity to know allergens, toxins or other proteins was identified for any of the putative polypeptides.  No short peptide matches, representing putative IgE binding epitopes, were shared between any of the putative polypeptides and proteins in the allergen database.

3.4.4 Conclusion

Detailed molecular analyses indicate that one copy of T-DNA I, containing the cry1A.105 and cry2Ab2 gene expression cassettes, has been inserted at a single genomic locus in MON 89034 corn.  Both coding regions are intact, although during the transformation process, a small rearrangement occurred to the e35S promoter which directs expression of the cry1A.105 gene. This minor modification to the e35S promoter has not affected the expression of the gene or the trait (see Section 4).  The molecular analyses also confirmed the absence of the nptII selectable marker in MON 89034 corn, which was introduced in the original transformation on a second T-DNA and subsequently selectively segregated away. 

3.5 Stability of the genetic changes

A number of analyses were done to demonstrate the stability of the genetic changes in MON 89034 corn.  Segregation analysis over multiple generations was done to determine the heritability and stability of the new trait (the cry1A.105 and cry2Ab2 genes, and Cry1A.105 and Cry2Ab2 proteins). Southern blot analysis over multiple generations was done to determine the stability of the inserted DNA.

3.5.1 Segregation analyses

For the segregation analysis, Chi-square test of inheritance data over four generations of MON 89034 corn was done to determine the heritability and stability of the new traits.  The confirmation of the presence of the gene and stability of the trait was based on: (i) ELISA to detect the Cry2Ab2 and Cry1A.105 proteins; and (ii) PCR assay to detect the presence of the cry1A.105 and cry2Ab2 genes.  The Chi-square test is based on testing the observed segregation ratio of the Cry proteins to the ratio that is expected according to Mendelian principles as shown in Table 2.

The results of the Chi-square test[8] are summarised in Table 3.  All Chi-square values indicate no significant differences between observed and expected genetic ratios across all tested generations of MON 89034 corn.  These results are consistent with the molecular characterisation data indicating a single site of insertion for the cry1A.105 and cry2Ab2 gene expression cassettes.

Table 2:  Expected segregation ratios for MON 89034 corn generations

aSee breeding tree in Figure 2.

bn.a. = not applicable.

cTo confirm segregation, LH172BC0F1 plants were backcrossed to the recurrent parent (LH172) to produce this generation (not shown in Figure 2).

dTo confirm segregation, the LH172BC1F1 plants were self pollinated to produce two different plant populations of this generation (not shown in Figure 2).

Table 3:  Segregation analyses of MON 89034 corn

3.5.2 Stability of the inserted DNA

To determine the stability of the inserted DNA, Southern blot analyses were done using genomic DNA isolated from multiple generations of MON 89034 corn (see Figure 2; the generations used are in bold).  For these analyses, DNA samples were digested with Ssp I which cleaves once within the inserted DNA and in both the 5’ and 3’ genomic flanking regions.  This produces two DNA fragments of ~8.2 and >4.3 kb.  The stability of the inserted DNA was confirmed using overlapping T-DNA I probes spanning the entire inserted DNA sequence.  Genomic DNA isolated from corn with the same genetic background as MON 89034 corn was used as a negative control, and was also spiked with DNA from PV-ZMIR245 to serve as a positive hybridisation control.

Genomic DNA extracted from the different MON 89034 generations produced two bands of ~8.2 kb and ~7.4 kb.  The ~8.2 kb band is the expected size for the 5’ border fragment and the ~7.4 kb band is consistent with the expected band size of >4.3 kb for the 3’ border fragment.  These bands were consistent with bands detected in other Southern analyses and are also identical across multiple generations.

3.5.3 Conclusion

The results of the segregation analysis are consistent with a single site of insertion for the cry1A.105 and cry2Ab2 gene expression cassettes and confirm the results of the molecular characterisation.  Molecular analysis of both self pollinated and cross-fertilised lines, representing a total of seven different generations, indicates that the inserted DNA is stably transformed and inherited from one generation to the next.

3.6 Presence of antibiotic resistance genes

No genes that encode resistance to antibiotics are present in the genome of MON 89034 corn.  The molecular characterisation confirmed the absence of both the aad and nptII genes, which were used in the cloning and transformation process.

4. CHARACTERISATION OF THE NOVEL PROTEIN

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.

4.1 Description and function of novel protein

MON 89034 corn expresses two different Cry proteins, Cry1A.105 and Cry2Ab2.  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 Cry1A.105 and Cry2Ab2 proteins.  The Cry1A.105 and Cry2Ab2 proteins produced in E. coli were engineered so their amino acid sequence matched that of the plant-produced Cry1A.105 and Cry2Ab2 proteins. The equivalence of the bacterial-produced proteins to the plant-produced proteins was determined as part of the protein characterisation.

4.1.1 Mode of action of Cry proteins

The general mechanism of insecticidal activity of Cry proteins is well understood (Gill et al 1992, Schnepf et al 1998, Zhuang & Gill 2003, Bravo et al 2007), with the mode of action being characterised principally in lepidopteran insects.  The Cry proteins belong to a class of bacterial toxins known as pore-forming toxins (PFT) that are secreted as water-soluble proteins, which after undergoing conformational change, are able to insert into, or translocate across, the cell membranes of their host.  There are two main groups of PFT: (i) the α-helical toxins in which the α-helix regions form the trans-membrane pore; and (ii) the β-barrel toxins, that insert into the membrane by forming a β-barrel composed of β-sheet hairpins from each monomer (Parker & Feil 2005).  The Cry proteins belong to the α-helical group of PFT, along with other toxins such as exotoxin A (from Pseudomonas aeruginosa) and diphtheria toxin.

The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming pores in the apical microvilli membrane of the cells, which subsequently leads to ion leakage and cell lysis.  The crystal inclusions ingested by susceptible larvae dissolve in the alkaline environment of the gut, and the solubilised inactive protoxins are cleaved by midgut proteases yielding 60-70 kDa protease resistant proteins (Bravo et al 2005).  Toxin activation involves the proteolytic removal of an N-terminal peptide (25 – 30 amino acids for Cry1 toxins, 58 residues for the Cry3A and 49 for Cry2Aa) and approximately half of the remaining protein from the C-terminus in the case of the long Cry protoxins.  The activated toxin then binds to specific receptors on the brush border membrane of the midgut epithelium columnar cells (de Maagd et al 2001, Bravo et al 2005) before inserting into the membrane.  Toxin insertion leads to formation of lytic pores in microvilli apical membranes (Aronson & Shai 2001, Bravo et al 2005).  Subsequently cell lysis and disruption of the midgut epithelium releases the cell contents providing spores a germinating medium leading to a severe septicemia and insect death.

4.1.2 Cry1A.105

Cry1A.105 is a full-length Cry protein consisting of 1177 amino acids with a molecular weight of 133 kDa.  It is a chimeric protein consisting of domains I and II from Cry1Ab or Cry1Ac[9], substantially domain III from Cry1F, and the C-terminal domain from Cry1Ac (Figure 5).

Cry1A.105 was designed using a domain exchange strategy to achieve high levels of activity against the target insect pests.  Domain exchange is a naturally occurring mechanism that increases protein diversity in B. thuringiensis (De Maagd et al 2001, Masson 2002, De Maagd et al 2003).  A domain exchange strategy has been used to switch the functional domains of Cry1 proteins to develop a commercial biopesticide with improved specificity to lepidopteran insect pests. 

Figure 5:  Schematic representation of the origin of Cry1A.105 protein domains

The overall amino acid sequence identity between Cry1A.105 and Cry1Ac, Cry1Ab, and Cry1F is 93.6%, 90.0% and 76.7%, respectively (see Table 4).  According to this, Cry1A.105 is most closely related to Cry1Ac, but is also has a high degree of amino acid identity with Cry1Ab. 

Table 4:  Amino acid sequence identity between Cry1A.105 and Cry1Ac, Cry1Ab and Cry1F proteins

4.1.3 Cry2Ab2

Cry2Ab2 is an insecticidal protein consisting of 637 amino acids with a predicted molecular weight of 61 kDa and is a variant of the wild-type Cry2Ab2 protein from B. thuringiensis subsp. kurstaki.  The cry2Ab2 gene expressed in MON 89034 has been modified to change the codon usage for optimised expression in the plant. The Cry2Ab2 variant protein expressed in MON 89034 has 88% amino acid sequence identity to Cry2Aa, which is present in a number of registered pest control products in Australia and New Zealand. 

Cry2Ab2 is targeted to the chloroplast using a chloroplast transit peptide (CTP) from the maize ribulose 1,5-bisphosphate carboxylase small subunit which is fused to the N-terminus of Cry2Ab2.  CTPs are typically cleaved from the mature protein upon translocation into the chloroplast, and then rapidly degraded (Bruce 2000).  The CTP used in MON 89034 has a potential cleavage site located three amino acids upstream from the start of the Cry2Ab2 protein sequence (Figure 6); therefore the Cry2Ab2 protein expressed in MON 89034 has an additional three amino acids (MQA) at the N-terminus, compared to the wild-type protein.  An additional amino acid (aspartic acid) was also inserted in the N-terminus of the plant expressed Cry2Ab2 in order to facilitate the cloning of the cry2Ab2 gene.  These additional amino acids were also included in the N-terminus of the E. coli-produced Cry2Ab2 protein.

MON 89034 Cry2Ab2  M-Q-A1-M-D2-N-S-V-L-N

E. coli-produced Cry2Ab2  M-Q-A1-M-D2-N-S-V-L-N

Wild-type Cry2Ab2   -M-    -N-S-V-L-N 

 1 M-Q-A - predicted amino acids from chloroplast transit peptide

 2 D - an additional amino acid included to facilitate cloning

Figure 6:  Comparison of the N-terminus of the MON 89034, E. coli and wild-type Cry2Ab2 proteins.

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 Cry1A.105 and Cry2Ab2 proteins isolated from MON 89034 and to compare them to E. coli-produced proteins.   These techniques included sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry, Western blot analysis, matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry, N-terminal sequencing (Cry2Ab2 only), glycosylation analysis, and insect bioactivity assays.

The E. coli-produced proteins were used as reference standards for determination of protein concentration and immunoblot analysis using N-terminal peptide 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 negative control in glycosylation analyses, and as a reference and a positive control in immunoblot analyses using anti-Cry1A.105 and anti-Cry2Ab2 antibodies.

1.         Cry1A.105

The Cry1A.105 protein was purified at 4ºC from an extract of ground grain from MON 89034 corn using a combination of ammonium sulphate fractionation, anion exchange chromatography, and immunoaffinity chromatography.

Protein identity

The identity of the plant-produced Cry1A.105 was confirmed by Western blot analysis using anti-Cry1A.105 antibody and the N-terminal peptide antibody, and MALDI-TOF MS tryptic mass map analysis:

1.             Western blot using anti-Cry1A.105 antibody – the plant-produced Cry1A.105 protein and the E. coli-produced reference standard were loaded onto the same gel.  Immunoreactive bands migrating between approximately 85 and 130 kDa were observed in both samples.   The major protein band, migrating with an apparent molecular weight of ~130 kDa was present in both the plant-produced and E. coli-produced samples and is consistent with the molecular weight of 133 kDa expected for the full length protein.  The bands of lower molecular weight are partial Cry1A.105 fragments, which the Applicant believes represent proteolytic degradation products;

1.          Western blot using N-terminal peptide antibody – the antibodies were raised against a synthetic peptide consisting of the first 14 amino acids of the Cry1A.105 N-terminus (MDNNPNINECIPYN).  The E. coli-produced Cry1A.105 protein containing the same N-terminal sequence, a Cry1A.105 trypsin-resistant core protein lacking the N-terminus, and the plant-produced Cry1A.105 protein were all loaded onto the same gel.  The E. coli-produced protein served as the positive control and the trypsin-resistant core of Cry1A.105 served as the negative control.  A ~130 kDa immunoreactive band was observed in both the plant- and E. coli-produced Cry1A.105 samples.  An immunoreactive band of ~85 kDa was also observed in both samples, which suggests it represents a C-terminal degradation of the Cry1A.105 protein.  No immunoreactive bands were observed for the trypsin-resistant core Cry1A.105 protein. 

These results indicate that the plant-produced Cry1A.105 protein contains an intact N-terminus and are consistent with published literature which shows that proteolytic fragments of Cry1 proteins have intact N-termini (Gao et al 2006);

1.        Tryptic peptide mapping by MALDI-TOF – the plant-produced protein was heat denatured, chemically reduced, alkylated and digested with trypsin, and the masses of the tryptic fragments were measured.  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). There were 52 tryptic mass fragments identified that matched the expected tryptic peptides generated in silico based on the predicted trypsin cleavage sites in the amino acid sequence.  Overall, the confirmed sequence accounts for 43.8 % (516 of 1177 amino acids) of the full length Cry1A.105 protein.

Glycosylation analysis

To assess whether post-translational glycosylation of the plant-produced Cry1A.105 protein occurred, the purified protein sample was subjected to glycosylation analysis.  As prokaryotic organisms lack the capacity for protein glycosylation, the E. coli-produced Cry1A.105 protein was used as the negative control.  Transferrin and horse radish peroxidase (HRP) proteins, which are known to have multiple covalently-linked carbohydrate modifications, were used as positive controls.  The test and control protein samples were separated by SDS-PAGE and protein-bound carbohydrate moieties were detected using a fluorescent stain.  The transferrin and HRP proteins were detected at the expected molecular weights in a concentration dependent manner, with no signal being detected for either the plant-produced or E. coli-produced Cry1A.105 proteins.

These results indicate that the plant-produced Cry1A.105 protein is not glycosylated and is equivalent to the E. coli-produced Cry1A.105 protein in terms of its lack of glycosylation.

Insect bioassays

The insecticidal activity of the plant- and E. coli-produced Cry1A.105 protein was determined using an insect bioassay with corn earworm. The bioassay was repeated three times, in replicate, on separate days with separate batches of larvae.  Each replicate consisted of a series of five protein levels yielding a dose series ranging from 0.00048 – 0.039 μg Cry1A.105 protein/ml of diet with a 3 fold separation factor between dose levels.  Insect larvae (16 larvae per treatment) were allowed to feed on the diet for 6-7 days.  The combined weight of the surviving insects at each dose level for each source of protein was recorded at the end of the 6-7 day incubation period.  The mean EC50 value[10] for the plant-produced Cry1A.105 protein was 0.0074 ± 0.0017 μg protein/ml diet, and for the E. coli-produced Cry1A.105 protein was 0.012 ± 0.0062 μg protein/ml diet.  Both protein sources also produced similar dose response curves.  These results indicate that the plant- and E. coli-produced Cry1A.105 proteins can be regarded as equivalent in terms of their insecticidal activity.

4.2.2 Cry2Ab2

The Cry2Ab2 protein was purified from an extract of ground grain from MON 89034 corn using a combination of ammonium sulphate fractionation, anion exchange chromatography, and immunoaffinity chromatography.

Protein identity

The identity of the plant-produced Cry2Ab2 protein was confirmed by Western blot analysis using anti-Cry2Ab2 antibody, MALDI-TOF mass spectrometry, and N-terminal sequence analysis:

1.             Western blot analysis using anti-Cry2Ab2 antibodies – samples of the plant-produced Cry2Ab2 protein and E. coli-produced Cry2Ab2 reference protein were loaded onto the same gel. Two immunoreactive bands of approximately 61 kDa and 50 kDa were observed in plant-produced Cry2Ab2 sample, with a single band of 61 kDa being observed in the E. coli-produced sample.  The predicted molecular weight of the full length Cry2Ab2 is 61 kDa.  N-terminal sequence analysis of the ~ 50 kDa band demonstrated that its N-terminal sequence begins at amino acid position 145, indicating it is the product of a N-terminal truncation;

1.          Tryptic peptide mapping by MALDI-TOF – the two major immunoreactive bands cross-reacting with anti-Cry2Ab2 antibodies in the Western blot analysis were further assessed using MALDI-TOF mass spectrometry.  For the ~61 kDa band, a total of 32 observed tryptic peptide masses matched the predicted (theoretical) tryptic peptide masses of the Cry2Ab2 protein.  The overall peptide sequence coverage was 44.4 % out of the 637 amino acid residues of the full length Cry2Ab2 protein.  For the lower molecular weight band migrating at ~50 kDa, a total of 24 observed peptide masses matched the expected tryptic peptide masses of the Cry2Ab2 protein, which represents 47.7 % out of the 493 amino acid residues of this truncated protein;

1.        N-terminal sequence analysis – the N-terminus of the ~61 kDa immunoreactive band from MON 89034 corn was blocked therefore it was not possible to obtain the N-terminal sequence of the protein.  N-terminal sequence of this immunoreactive band did however result in N-terminal sequence starting from amino acid position 24, indicating that a minor portion of the protein co-migrating with the full length protein was proteolytically degraded.  As discussed above, N-terminal sequence analysis of the lower immunoreactive band migrating at ~50 kDa revealed this fragment starts from amino acid residue no.143.  N-terminal sequence analysis of the E. coli-produced Cry2Ab2 protein confirmed the N-terminus as MQAMDN, as expected (see Figure 6).  The results of the N-terminal sequence analysis are summarised in Table 5 below.

Table 5:  Summary of N-terminal sequence analysis of Cry2Ab2 protein from MON 89034 corn1,2

1 Undesignated amino acid assignments are shown as ‘X’, tentative assignments are shown in parentheses

2 The single letter amino acid code is A, alanine; D, aspartic acid; F, phenylalanine; H, histidine; I, isoleucine; L, leucine; Q, glutamine; P, proline; S, serine; T, threonine; and V, valine

3 The amino acid sequence deduced from the coding region of the full length cry2Ab2 gene present in MON 89034 corn

Glycosylation analysis

To assess whether post-translational glycosylation of the plant-produced Cry2Ab2 protein occurred, the purified protein sample was subjected to glycosylation analysis.  The E. coli-produced Cry2Ab2 protein was used as the negative control. 

Transferrin, which is known to have multiple covalently-linked carbohydrate modifications, was used as the positive control.  The transferrin protein was detected at the expected molecular weight in a concentration dependent manner, with no signal being detected for the plant-produced Cry2Ab2 protein.   These results confirm that the plant-produced Cry2Ab2 protein is not glycosylated and also that it is equivalent to the E. coli-produced Cry2Ab2 in terms of its lack of glycosylation.

Insect bioassays

The insecticidal activity of the plant- and E. coli-produced Cry2Ab2 proteins was determined using an insect bioassay with corn earworm.   The bioassay was repeated three times, in replicate, on separate days with separate batches of larvae.  Each replicate consisted of a series of seven dilutions yielding a dose series ranging from 0.016 – 1.0 μg Cry2Ab2 protein/ml of diet with a 2-fold separation factor between dose levels.  Insect larvae (16 larvae per treatment) were allowed to feed on the diet for 7 days.  The combined weight of the surviving insects at each does level for each source of protein was recorded at the end of the 7 day incubation period.  The mean EC50 values for the plant-produced Cry2Ab2 protein was 0.16 ± 0.01 μg protein/ml diet and for the E. coli-produced Cry2Ab2 protein was

0.16 ± 0.04 μg protein/ml diet.  Both protein sources also produced similar dose response curves.  These results indicate that the plant- and E. coli-produced Cry2Ab2 proteins can be regarded as equivalent in terms of their insecticidal activity.

4.2.3 Conclusion

A large number of studies have been done on the Cry1A.105 and Cry2Ab2 proteins to confirm their identity and physicochemical and functional properties as well as to determine their equivalence to E. coli-produced Cry1A.105 and Cry2Ab2 proteins.  These studies have demonstrated that the two proteins expressed in MON 89034 corn both conform in size and amino acid sequence to that expected and also both exhibit the expected insecticidal activity.  

The E. coli-produced proteins were also shown to be equivalent to the plant produced proteins in terms of their size, amino acid sequence, physicochemical properties as well as their insecticidal activity.  The E. coli-produced proteins are therefore suitable to act as substitutes for the plant-produced protein for safety assessment purposes.

4.3 Protein expression levels

Validated ELISA methods were used to quantify the levels of the Cry1A.105 and Cry2Ab2 proteins in tissues from MON 89034 corn grown in field trials in the United States in 2005.  Field sites were selected which represent the major corn growing region of the U.S. and which provide a range of environmental conditions that would be encountered during commercial production. At each site, three replicated plots of MON 89034 corn (the [LH172 BC0F7 x LH198]F1H and LH172 BC0F6 x F2H1generations, see Figure 2) were grown along with a conventional hybrid corn variety with a similar genetic background to the test plants.

Over season leaf (OSL 1-4), over season root (OSR 1-4), over season whole plant (OSWP 1-4), forage, stover[11], forage-root, senescent root, pollen, silk and grain were collected from each replicated plot at all field sites. 

The over season leaf and whole plant samples were collected four times at different growth stages: (1) V2 – V4 stage (2-4 leaf stage); (2) V6 – V8 stage (6-8 leaf); (3) V10 – V12 (10-12 leaf) stage; and (4) pre-VT stage (pre-tasseling).  The over season root samples were collected at: (1) V2 – V4 stage; (2) V6 – V8 stage; (3) V10 – V12 stage; (4) pre-VT stage; (5) early dent stage; and (6) after harvest.  Pollen and silks were collected at approximately 100-120 days after planting and grain was harvested at maturity.  Stover was collected following harvest at approximately 130 – 160 days after planting.

Goat polyclonal antibodies were used for the Cry1A.105 ELISA and a mouse monoclonal antibody was used for the Cry2Ab2 ELISA.  The limits of detection (LOD) and limits of quantitation (LOQ) for each of the tissues are provided in Table 6 below.

Table 6:  LOD and LOQ for the Cry1A.105 and Cry2Ab2 proteins in the tissues analysed

1 fresh weight

The Cry1A.105 and Cry2Ab2 protein levels in the various tissues are summarised in Table 7.  The mean concentration of Cry1A.105 in MON 89034 corn was highest in young leaf (V2 – V4 stage; 85 μg/g fwt) with the mean level in grain being 5.1 μg/g fwt.  For Cry2Ab2, the highest levels were also in young leaf (29 μg/g fwt), with the mean level in grain being 1.1 μg/g fwt.  Data on over season protein levels is not presented in this report but in general shows that the levels of both proteins declined over the growing season.

Table 7:  Summary of Cry1A.105 and Cry2Ab2 protein levels in tissues from MON 89034 corn

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

Cry1A.105

Cry1A.105 is a chimeric protein made up of different functional domains derived from three wild-type Cry proteins from B. thuringiensis subspecies kurstaki and aizawai.  Cry1A.105 consists of domains I and II from Cry1Ab or Cry1Ac[12], substantially domain III from Cry1F, and the C-terminal domain from Cry1Ac.  So while Cry1A.105 does not itself have a history of use, it shares 93.6% amino acid sequence similarity to Cry1Ac, which does have an extensive history of prior human exposure, including consumption in food.  Microbial pesticide products based on B. thuringiensis producing Cry1Ac (e.g. DIPEL®) have been used in both Australia and New Zealand since 1989 and a microbial pesticide that contains a Cry1Ac/Cry1F chimeric protein (Lepinox™) has been approved and used for control of lepidopteran pests in the United States since 1997.  The potential toxicity of both Cry1Ac and Cry1F has been previously assessed by FSANZ and no safety concerns were identified. 

Cry2Ab2

The amino acid sequence of the Cry2Ab2 protein expressed in MON 89034 corn is 88 % identical to the wild type Cry2Ab2 protein produced by B. thuringiensis subsp. kurstaki. 

This protein is present in various microbial pesticide products which have been used for a number of years (e.g. Dipel, Cutlass, Crymax).  In addition, food from insect-protected cotton expressing an identical Cry2Ab2 to that in MON 89034 was approved in Australia and New Zealand in 2002 (Application A436).  In that assessment it was concluded that Cry2Ab2 is unlikely to be toxic to humans.

4.4.2 Specificity

The Cry proteins are a diverse group of proteins which have a defined spectrum of insecticidal activity within particular insect orders (Lepidoptera, Diptera, Coleoptera, and Hymenoptera).  The Cry proteins are not known to be toxic to any vertebrates, including humans and other mammals. 

This high degree of specificity is determined by a number of different factors, including (i) activation of the protein by specific proteolytic enzymes in the insect midgut; (ii) binding of the activated protein to specific midgut receptors; and (iii) changes in protein configuration, which enable the protein to insert into and form pores in the insect midgut membrane.   As a consequence, only insects with specific receptors are affected, and no toxicity is observed in species that lack these receptors, including other insects.  For example, the Cry1Ab, Cry1Ac and Cry1F proteins are active against lepidopteran but not coleopteran insects. 

As the Cry1A.105 protein is a chimeric protein, insect bioassays were done to confirm that it retained its specificity to lepidopteran insects.  Insects used in the bioassay were representative of the Lepidoptera (European corn borer, black cutworm, corn earworm, fall armyworm), Coleoptera (boll weevil, southern corn rootworm) and Hemiptera (western tarnished plant bug, green peach aphid).  These bioassays confirmed that the Cry1A.105 protein is toxic only to lepidopteran insects.

4.4.3 Similarities with known protein toxins

Bioinformatic analyses were done to assess the Cry1A.105 and Cry2Ab2 proteins for any amino acid sequence similarity with known protein toxins.  The TOXIN5 data base, as described in Section 3.4.3 of this report, was assembled for this purpose and the FASTA algorithm was used to assess structural similarity.  Although the FASTA program directly compares amino acid sequences and thus is mainly being used to determine similarity in primary protein structure, the alignment data may also be used to infer both secondary and tertiary structure of proteins.  In addition to the TOXIN5 database, comparisons were also made to the ALLPEPTIDES database (described in Section 3.4.3) which represents all currently known publicly available protein sequences.  Although it may be redundant to search both the TOXIN5 and the ALLPEPTIDES databases for potential similarity to protein toxins, the ALLPEPTIDES database search was used to assess for similarity to other biologically active proteins (e.g. prions), which may not have been annotated with the keyword ‘toxin’.

The extent of similarity was evaluated using visual inspection of the aligned sequences, the calculated percent identity, and E score.  The E score reflects the degree of amino acid similarity between a pair of sequences and can be used to evaluate the significance of the alignment.  A larger E score indicates a lower degree of similarity.  Typically, alignments between two sequences will need to have an E score of less than 1 x 10-5 for them to be considered to have significant homology.

For Cry1A.105, the most significant similarity observed was to Cry1Ac from B. thuringiensis, with 92 % identity over 1182 amino acids and with an E score of zero.  This degree of similarity is expected because the Cry1A.105 protein contains domains I and II as well as the C-terminal domain from Cry1Ac.  For Cry2Ab2, the most significant similarity observed was to Cry2Ab2 from B. thuringiensis (subsp. kurstaki), with 100% identity over 632 amino acids with an E score of zero.  All of the remaining alignments with significant E scores were to other Cry proteins.  The analyses did not demonstrate any significant similarity between the Cry1A.105 and Cry2Ab2 proteins and other proteins that may potentially be toxic to humans or other animals.

4.4.4 Digestibility

See Section 4.5.

4.4.5 Acute oral toxicity study

Acute oral toxicity studies using CD-1 mice were conducted to examine the potential toxicity of the Cry1A.105 and Cry2Ab2 proteins.  For these studies, E. coli-produced Cry1A.105 and Cry2Ab2 were used as the test substances because it was not possible to purify sufficient quantities of the Cry1A.105 and Cry2Ab2 proteins from plant material.  The equivalence of the E. coli- and MON 89034 corn-produced proteins was established using a range of methods including Western blot analysis, MALDI-TOF mass spectrometry, glycosylation analysis and insect bioassay (see Section 4.2).

Cry1A.105

E. coli-produced Cry1A.105 protein was administered by gavage to 10 male and 10 female young adult CD-1 mice as two separate doses administered approximately 4 hours apart on day 0.  The total dose administered was 2072 mg/kg bodyweight.  Separate groups of 10 male and 10 female CD-1 mice were administered vehicle only (carbonate-bicarbonate buffer with reduced glutathione, pH 10 – 11) or bovine serum albumin (1998 mg/kg body weight) as controls.

Following dosing, all mice were observed daily, with body weights and food consumption measured weekly (days 0, 7 and 14).  A gross necropsy examination was performed on all animals at the time of death or at the termination of the study (day 14).

One male animal in the Cry1A.105 treated group was euthanased on day 1 with a suspected gavage injury, which was confirmed on necropsy.  No test article related mortality or clinical signs were observed.  There were no significant differences in body weight, cumulative body weight, or food consumption between the control groups and the Cry1A.105 treated group.  No treatment-related gross pathological findings were observed at necropsy on day 14.

Cry2Ab2

E. coli-produced Cry2Ab2 protein was administered by gavage to 10 male and 10 female young adult CD-1 mice as two separate doses administered approximately 4 hours apart on day 0.  The total dose administered was 2198 mg/kg bodyweight. 

Separate groups of 10 male and 10 female CD-1 mice were administered vehicle only (2mM carbonate-bicarbonate buffer containing 2mM reduced glutathione, pH 10.5) or bovine serum albumin (2442 mg/kg body weight) as controls.

Following dosing, all mice were observed daily, with body weights and food consumption measured weekly (days 0, 7 and 14).  A gross necropsy examination was performed on all animals at the time of death or at the termination of the study (day 14).

There were no unscheduled deaths.  No treatment- related mortality or clinical signs were observed during the study.  There were no significant differences in body weight, cumulative body weight, or food consumption between the control groups and the Cry2Ab2-treated group.  No treatment-related gross pathological findings were observed at necropsy on day 14.

4.5 Potential allergenicity of novel proteins

The potential allergenicity of novel proteins is evaluated using an integrated, step-wise, case-by-case approach relying on various criteria used in combination, since no single criterion is sufficiently predictive of either allergenicity or non-allergenicity.  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

The Cry1A.105 and Cry2Ab2 proteins are both derived from B. thuringiensis subsp. kurstaki and aizawai.  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. Humans using the insecticidal sprays have been shown to develop antibodies to the expressed Cry proteins, but in no case has the presence of these antibodies been linked with any clinical, including allergic, symptoms (Nester et al 2002).

4.5.2 Similarity to known allergens

Bioinformatic analyses were done to assess the Cry1A.105 and Cry2Ab2 proteins for any amino acid sequence similarity with known allergens, gliadins or glutenins.  The AD6 data base, as described in Section 3.4.3 of this report, was assembled for this purpose and the FASTA algorithm was used to assess structural similarity.  In addition to the FASTA comparisons, searches were also done to identify short amino acid sequences that may represent linear IgE binding epitopes.  An algorithm (ALLERGENSEARCH) was developed to identify matches of eight contiguous amino acids or more (see Section 3.4.3).

As with the assessment of similarity to known protein toxins (Section 4.4.3), the extent of structural relatedness was evaluated using visual inspection of the aligned sequences, the calculated percent identity, and E score.  E scores of ~1 or greater are expected to occur for alignments between random, non-homologous sequences (Pearson 2000). 

In terms of the calculated % identity, 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). 

For Cry1A.105, no significant amino acid sequence similarity was observed, with none of the alignments generating an E score of less than 1 x 10-5.  The most significant alignment was to a Kiwi fruit allergen, actinidin.  This alignment demonstrated 24.2 % identity over a 318 amino acid window, with an E score of 2.3.  This alignment does not meet the criteria established by the FAO/WHO (2001) for potential cross-reactivity, nor is the E score reflective of significant homology.  Visual inspection also indicates the alignment is of low significance, with the insertion of many gaps being necessary to optimise the alignment.  When searched using the ALLERGENSEARCH algorithm, no matches, representing putative IgE binding epitopes, were shared between the Cry1A.105 protein and proteins in the AD6 database.

For Cry2Ab2, no significant amino acid sequence similarity was observed, with none of the alignments generating an E score of less than 1 x 10-5.  The most significant alignment was to the Coprinus comatus[13] protein Cop c1, where there was 32.7 % identity over a 52 amino acid window, with an E score of 0.89.    As the E score is close to 1, this alignment is not reflective of significant homology between Cry2Ab2 and the Cop c1 allergen.  In addition, the length of the overlap between the two proteins is relatively short (52 amino acids) when compared to the full length of 637 amino acids of the Cry2Ab2 protein and does not meet the criteria of 35 % identity over 80 amino acids established by the FAO/WHO (2001) for potential cross reactivity.  When searched using the ALLERGENSEARCH algorithm, no matches, representing putative IgE binding epitopes, were shared between the Cry2Ab2 protein and proteins in the AD6 database.

4.5.3 Digestibility

Resistance to hydrolysis by digestive proteases has been observed in several food allergens (Astwood et al 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.  Proteins that are rapidly degraded in such conditions are considered less likely to be involved in eliciting an allergic response.  Recently, a pepsin digestibility assay protocol was standardised in a multi-laboratory evaluation (Thomas et al 2004).  This protocol was followed to determine the digestive stability of the Cry1A.105 and Cry2Ab2 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. 

Because it was not possible to purify sufficient quantities of the Cry1A.105 and Cry2Ab2 proteins from plant material for use in these studies, E. coli-produced Cry1A.105 and Cry2Ab2 were used as the test substances in both the SGF and SIF studies.

Simulated gastric fluid study

Digestibility of the Cry1A.105 and Cry2Ab proteins in SGF was assessed separately using SDS-PAGE and Western blot analysis.  The limit of detection (LOD) of the full length Cry1A.105 and Cry2Ab2 proteins on SDS-PAGE was 0.005 μg, and for Western blot was 1.0 ng for Cry1A.105 and 0.2 ng for Cry2Ab2.

Digestibility of the two proteins in SGF was measured by taking samples at selected time points (0, 0.5, 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 nitrocellulose membrane for Western blot analysis.   The full length Cry1A.105 and Cry2Ab2 proteins were digested below the LOD within 30 seconds, which equates to > 95% of the full length proteins being digested.  Very faint low molecular weight bands (~4.5 kDa for Cry1A.105 and ~5 kDa for Cry2Ab2) were observed on the stained gel after the 30 second time point, but these disappeared fully by the 20 minute time point for Cry1A.105 and 2 minute time point for Cry2Ab2.  The lower molecular weight bands were not visible on Western blot.

Simulated intestinal fluid study

Digestibility of the Cry1A.105 and Cry2Ab2 proteins in SIF was assessed separately using Western blot analysis.  The LOD for the Cry1A.105 protein was 0.1 ng and for Cry2Ab2 was 0.5 ng.

Digestibility of the two proteins in SIF was measured by taking samples at selected time points (0, 5, 15, 30 minutes, 1, 2, 4, 8, 12, and 24 hours) and then subjecting these to SDS-PAGE then transferring to polyvinylidene difluoride membrane for Western blot analysis.  The full length Cry1A.105 was digested below the LOD within 5 minutes of incubation in SIF.  Proteolytic fragments of approximately 60, 32 and 30 kDa were observed at the 5 minute time point.  The 60 kDa band, migrating as a doublet, represents the trypsin-resistant core of the Cry1A.105 protein and was observed for up to 24 hours of digestion.  The 32 kDa fragment was digested below LOD by the 2 hour time point, whereas the 30 kDa band, migrating as a doublet, was observed for up to 24 hours of digestion, however the intensity of the signal decreased over time.  In the case of Cry2Ab2, the full length protein was digested below the LOD within 15 minutes of incubation in SIF.  Several proteolytic fragments, with approximate molecular weights of 60, 55, 50, 40, 12 and 10 kDa, were observed, and displayed varying degrees of stability over the time course although the majority, with the exception of the 50 kDa band, were not detectable by the 24 hour time point.

4.6 Conclusion

MON 89034 corn expresses two novel proteins, Cry1A.105 and Cry2Ab2.  Both proteins are expressed at relatively low levels in the grain, with the mean concentration for Cry1A.105 being 5.1 μg/g fresh weight and for Cry2Ab2, the mean concentration was 1.1 μg/g fresh weight.

A large number of studies have been done with the Cry1A.105 and Cry2Ab2 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, do not exhibit any post-translational modification including glycosylation, and also demonstrate the expected insecticidal activity.

In relation to their potential toxicity and allergenicity, it is worth noting that Cry proteins from B. thuringiensis are inherently non-toxic to mammals and have exhibited little potential to be allergenic to humans over their long history of use.  In addition, bioinformatic studies with the Cry1A.105 and Cry2Ab2 proteins have confirmed the absence of any 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 in animals. 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 anti-nutrients for the food in question.  The key nutrients and anti-nutrients are those components in a particular food that may have a substantial impact in the overall diet.  They may be major constituents (fats, proteins, carbohydrates or enzyme inhibitors as anti-nutrients) 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).

5.1 Study design and conduct

To determine whether unexpected changes have occurred in the composition of MON 89034 corn as a result of the modification, and to assess its nutritional adequacy, compositional analyses were done on forage and grain samples collected from MON 89034, a conventional control line with the same genetic background as MON 89034, and 15 commercial corn hybrids grown under field conditions.

The MON 89034 corn lines used for this study are hybrids between the F6 and F7 generations and a conventional inbred corn line, LH198 (see Section 3.2).  As a consequence, the line used as the comparator for this study is a conventional LH198 x LH172 hybrid, which has a genetic background representative of the MON 89034 hybrid lines, but without the transferred genes.  Commercial corn hybrids were also included in the study as reference lines to provide data for the development of a 99 % tolerance interval for each component analysed.  The commercial hybrids used were all conventional lines[14].

Field trials were conducted in the United States in 2004 at five replicated sites.  The field sites were located in regions of the U.S. that are suitable for the growth of corn and which are representative of commercial corn production[15].  Seed was planted in a randomised complete block design with three replicates per block of each MON 89034, control and reference line.  All the corn lines at each of the field sites were grown under normal agronomic field conditions for their respective geographic regions.

Following compositional analysis, the results were statistically analysed using a mixed model analysis of variance.  Data from the five replicated sites were analysed separately and as a combined data set.  For each component analysed for MON 89034 corn, the result was compared with that from the conventional counterpart, in this case the LH198 x LH172 hybrid line, for the combination of all five sites (i.e., the combined-site) and for each individual site.  A range of measured values from the reference lines was also determined for each component and used as a basis for comparison.  Results were also compared to ranges for individual components in the International Life Sciences Institute (ILSI) Crop Composition Database (ILSI-CCD 2006), as well as to published literature ranges.

A total of 90 ground forage and grain samples were analysed for 77 different components (9 in forage, and 68 in grain).  Compositional analysis of the forage samples included proximates (protein, fat, ash and moisture), acid detergent fibre (ADF), neutral detergent fibre (NDF), minerals (calcium and phosphorus) and carbohydrates by calculation.  For the grain samples, compositional analysis included proximates, ADF, NDF, total dietary fibre, amino acids, fatty acids, vitamins (B1, B2, B6, E, niacin and folic acid), anti-nutrients (phytic acid and raffinose), secondary plant metabolites (furfural, ferulic acid and p-coumaric acid), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium and zinc), and carbohydrates by calculation.  Methods of analysis were based on internationally recognised procedures (e.g., AOAC International methods) or other published methods.

Of the 77 components measured, 16 in grain had greater than 50 % of the analytical values that were below the limit of quantitation, and therefore, were not included in the statistical analyses.  The components not included in the statistical analysis were: sodium, furfural, raffinose, and a number of fatty acids (8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 17:0 heptadecanoic acid, 17;1 heptadecenoic acid, 18:3 gamma linolenic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, and 20:4 arachidonic acid).

Statistically significant differences were determined at the 5 % level of significance (p<0.05).  There were 366 statistical comparisons conducted between MON 89034 and the conventional counterpart (61 comparisons in the combined site and 305 comparisons in the individual sites).  Using the data for each component obtained from the reference lines, a tolerance interval was calculated to contain, with 95 % confidence, 99 % of the values expressed in the population of commercial hybrids.  For those comparisons between MON 89034 and the conventional counterpart for which there was a statistically significant difference (p<0.05), the range of values for MON 89034 was then compared to the 99 % tolerance interval to determine if it was within the range of values for conventional corn hybrids.

The results of the combined site comparisons are presented in Tables 8 – 14.  The results from individual trial sites were also evaluated but are not presented in this report. A summary of the statistically significant differences between MON 89034 and the conventional counterpart, from both combined and individual sites, is presented in Table 15. 

1.               Nutrients

Statistically significant differences between MON 89034 and the control hybrid were observed for three nutrients for the combined-site analyses.  These were: phosphorus in forage (Table 8), and 18:0 stearic and 20:0 arachidic acids in grain (Table 10).  The magnitude of the differences observed were relatively small (3.43 – 19.24 %) compared to the natural variation, with both the mean levels and ranges for MON 89034 corn being well within the 99 % tolerance interval for the commercial corn hybrids.  The levels for these nutrients were also well within the ranges in the ILSI-CCD as well as published literature ranges (See Table 16).

At individual sites, 44 statistically significant differences were observed, although 33 of these were only observed at the one site.  There were no consistent trends observed and, except in two cases, the mean and range of the nutrients measured in MON 89034 corn were well within the tolerance interval for the commercial hybrids.  The mean levels and ranges for calcium and methionine in grain were just outside the tolerance interval but were within the ranges in the ILSI-CCD.  Of the remaining differences observed at more than one site, there were no nutrients that were consistently and statistically different across all sites.  In addition, there were no nutrients that showed statistically significant differences across three sites that had not been found to be different in the combined-site analysis.  Statistically significant differences were observed in as many as two sites for three nutrients (carbohydrate, copper and iron), which had not been found to be different in the combined-site analysis.  The observed differences were all small in magnitude and the mean levels and ranges are all within the tolerance interval for commercial corn hybrids.

Overall, these results show that the nutrient composition of MON 89034 corn is equivalent to that of conventional corn hybrids.  The few statistically significant differences between MON 89034 corn and the conventional counterpart likely reflect the natural variability of the individual components since the mean levels of the specific nutrients in question are well within the tolerance intervals for commercial corn hybrids as well as the ranges in the ILSI-CCD, as well as the scientific literature.

5.3 Anti-nutrients and secondary plant metabolites

Corn contains a number of anti-nutrients: phytic acid, raffinose, 2,4-dihydroxy-7-methoxy-2H-1,4-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).  Raffinose was excluded from the statistical analyses because greater than 50 % of the analytical values were below the limit of quantitation.

Compositional analysis of the grain indicate that phytic acid was present at similar levels in MON 89034 and the conventional counterpart, and no statistically significant differences were observed in any of the comparisons at either the single site or the combined-site analyses (Table 14).

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 p-coumaric acid.  Furfural was excluded from the statistical analyses because greater than 50 % of the analytical values were below the limit of quantitation.

Compositional analysis of the grain demonstrated that ferulic acid was present at similar levels in MON 89034 and the conventional counterpart, and no statistical differences were observed for any of the comparisons.  A statistically significant difference was observed for p-coumaric acid at one site but not at any of the other sites (see Tables 14 and 15).  As there was no consistent trend across sites, this result is not considered biologically meaningful.

Based on these results, the levels of anti-nutrients and secondary plant metabolites in MON 89034 corn are comparable to those found in conventional corn hybrids.

Table 8:  Combined site compositional analysis of forage for MON 89034 corn compared to the conventional counterpart (LH198 x LH172)

1 DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval

2 Values are mean ± S.E.

Table 9:  Combined site amino acid analysis of grain from MON 89034 corn compared to the conventional counterpart (LH198 x LH172)

Table 9 (continued)

1 Values are mean ± standard error (S.E.).

2 CI = confidence interval

Table 10:  Combined site fatty acid analysis of grain from MON 89034 corn compared to the conventional counterpart (LH198 x LH172)

1 Values are mean ± standard error (S.E.).

2 CI = confidence interval

Table 11:  Combined site mineral analysis of grain from MON 89034 corn compared to the conventional counterpart (LH198 x LH172)

1 Values are mean ± standard error (S.E.).

2 CI = confidence interval

Table 12:  Combined site proximate and fibre analysis of grain from MON 89034 corn compared to the conventional counterpart (LH198 x LH172)

1 DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval

2 Values are mean ± S.E.

Table 13:  Combined site vitamin analysis of grain from MON 89034 corn compared to the conventional counterpart (LH198 x LH172)