ELEMENTS OF PRECAUTION: RECOMMENDATIONS FOR THE REGULATION O

ELEMENTS OF PRECAUTION: RECOMMENDATIONS FOR THE REGULATION O

Postby admin » Sat Jan 09, 2016 11:51 pm

ELEMENTS OF PRECAUTION: RECOMMENDATIONS FOR THE REGULATION OF FOOD BIOTECHNOLOGY IN CANADA
AN EXPERT PANEL REPORT ON THE FUTURE OF FOOD BIOTECHNOLOGY
prepared by The Royal Society of Canada at the request of Health Canada Canadian Food Inspection Agency and Environment Canada
© The Royal Society of Canada
January 2001

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Table of Contents:

• Prefatory Note
• EXECUTIVE SUMMARY
o Summary of the Expert Panel’s Recommendations
o Recommendations Concerning Underlying Policies and Principles Guiding the Regulation of Agricultural Biotechnology
o Recommendations Concerning Regulations and Guidelines
o Recommendations Concerning the Regulatory Process
o Recommendations Concerning Scientific Capacity for the Regulation of Food Biotechnology
• 1. INTRODUCTION
o The Expert Panel
o Mandate and Terms of Reference
o Terms of Reference
o Interpretation of the Terms of Reference
o Health and Environmental Risks
o Socio-Economic Risks
o Philosophical/Metaphysical Risks
o Scientific and Extra-Scientific Issues in Risk Analysis
o Panel Procedures
o A Note on Terminology
o References
o Note
• 2. THE PAST, THE PRESENT AND THE FUTURE
o Introduction
o The Origins of Genetic Engineering
o Our Food Production System Relies on Few Genetically Selected Species
o Direct Gene Transfer Within and Between Species
o Selecting a Transformed Plant
o Current Products and Future Developments
o GM Plants
o GM Microbes
o GM Animals
o Fish
o Means of introducing transgenes into fish
o Development of growth hormone gene constructs for commercial food production
o Future applications
o Shellfish and Aquatic Plants
o Farm Animals
o Need for a Broader Research Agenda
o References
• 3. THE REGULATORY ENVIRONMENT
o Introduction
o Canadian Regulation of Food Biotechnology
o Overview
o Canadian Food Inspection Agency
o Health Canada
o Environment Canada and Protection of the Environment
o References
o Figure 3.1
o Figure 3.2
• 4. POTENTIAL HUMAN HEALTH IMPACTS
o Introduction
• PART 1: TOXICANT ASSESSMENT
o Resistance Factors
o Recommendations
o References
• PART 2: THREATS TO HUMAN HEALTH FROM ALLERGENS IN GM FOODS
o Mechanisms and Allergic Responses in Food Allergy
o The Increasing Problem of Food Allergies
o The Transfer of Allergens by Genetic Modification
o Potential Risks of Allergenic GM Foods
o Food Allergens: How Much Is Too Much?
o What Are the Most Common Food Allergens?
o Can Genetic Modification Increase the Risk of Development of Food Allergy?
o Can We Accurately Assess or Predict the Allergenicity of a Protein?
o Approach to Allergenicity Assessment
o Source of Donor Gene
o Comparison with Known Allergens
o In Vitro and In Vivo Immunologic Analysis
o In Vitro Assays
o In Vivo Studies
o Physicochemical Characteristics
o Prevalence of allergy to the donor protein
o Potential Changes in Host Allergenicity
o Other Considerations in Allergenicity Assessment
o An Example of the Evaluation Process to Assess Allergenicity
o Summary
o Recommendations
o References
• PART 3. NUTRITION ISSUES
o Introduction
o Impacts of Genetic Engineering
o Testing
o Recommendations
o References
• 5. CONSIDERATIONS IN THE USE OF BIOTECHNOLOGY IN ANIMAL PRODUCTION SYSTEMS .
o Introduction
• PART 1. GENETICALLY MODIFIED ANIMALS
o Potential Threats to Animal Health and Welfare
o Fish
o Changes in muscle cellularity, muscle enzyme activity and gene expression
o Changes in gross anatomy
o Changes to swimming ability and foraging behaviour
o Other pleiotropic effects
o Farm Animals
o Changes in muscle cellularity, muscle enzyme activity and gene expression
o Increased incidence of mutations and other pleiotropic effects
o Altered nutritional and welfare needs of transgenic animals
o Creation/Strengthening of Animal Commodification
o Reservoirs of Pathogens or Antibiotic-resistant Microflora
o Loss of Animal Genetic Resources
o Recommendations
o References
• PART 2. GENETICALLY MODIFIED FEEDS, FEED ADDITIVES AND METABOLIC MODIFIERS ADMINISTERED TO FOOD-PRODUCING ANIMALS
o Potential Novel Threats to Food Quality and Safety
o Potential Novel Threats to Animal Health or Welfare
o Metabolic Enhancers
o Vaccines
o Microbially Derived Feed Supplements and Additives
o Potential Threats from Concentration of GM Products in the Animal’s Food Stream
o Recommendations
o References
• 6. ENVIRONMENTAL RISKS
o Introduction
• PART 1: MICROORGANISMS IN BIOTECHNOLOGY AND THE ENVIRONMENT
o The Microbial Species Concept
o The Diversity of Microorganisms in the Natural Environment
o Direct Effects of GMOs on Soil Microflora
o Lateral Gene Transfer
o Transfer of Antibiotic Resistance Genes
o The Importance of Evaluating Selection
o Recommendations
o References
• PART 2: GM PLANTS
o Environmental Risks
o Could GM Plants Become Invasive?
o Gene Flow Between GM Crops and Wild Plants
o Spread of Transgenes in Wild Plants
o GM Crops and Biodiversity
o Regulatory Implications
o Future Research
o Recommendations
o References
• PART 3: ENVIRONMENTAL IMPACT: AN ENTOMOLOGICAL PERSPECTIVE
o Resistance in the Targeted Pest Species
o Impact on Other Herbivores Attacking the Same Host Plant
o Impact on the Natural Enemies of Herbivores
o Impact on Other Non-Target Insects in the Habitat
o General Conclusions
o Other GM Organisms for Insect Control
o Recommendations
o References
• PART 4: POTENTIAL ENVIRONMENTAL RISKS RESULTING FROM INTERACTIONS BETWEEN WILD AND CULTURED FISH
o Salmonid Aquaculture and the Incidence of Escape Events in Canada
o Genetic Interactions Between Wild and Cultured Fish
o Local Adaptation in Fish
o Genetic Differences Between Wild and Cultured Fish
o Hybridization and Outbreeding Depression in Fish
o Ecological Interactions Between Wild and Cultured Fish
o Interactions Between Wild and Non-Transgenic Cultured Fish
o Interactions Between Wild and Transgenic Fish
o Evaluating the Environmental Safety of Genetically Modified Fish
o Experimental Facilities and Evaluation Protocol
o I. Genetic Introgression
o II. Ecological Interactions
o III. Fish Health
o IV. Changes to Environmental Health Effected by Aquaculture Farms
o Density-dependent Effects and Population Viability
o Sterility of Genetically Modified Fish
o Induction of Triploidy
o Sterility as a Mitigative Tool to Minimize Potential Environmental Risks
o Regulatory Implications
o DFO National Code on Introductions and Transfers of Aquatic Organisms
o DFO Draft Policy on Research with, and Rearing of, Transgenic Aquatic Organisms
o Proposed Aquatic Organism Risk Analysis
o Critique of Current Regulatory Framework and Proposed Risk Aquatic Organism Analysis
o CEPA (Canadian Environmental Protection Act)
o Sterility of Transgenic Fish
o Aquatic Organism Risk Analysis
o Future Research
o Public Perception of Environmental Risks Posed by Cultured Fish
o Recommendations
o References
• 7. SUBSTANTIAL EQUIVALENCE AS A REGULATORY CONCEPT
o Introduction
o The Origins of “Substantial Equivalence”
o How Have New Crop Varieties Normally Been Approved?
o How Have Transgenic Crops Been Treated in This Context?
o How Well Has “Substantial Equivalence” Been Accepted?
o The Role of the “Substantial Equivalence” Concept in the Canadian Regulatory Process
o “Novelty” Versus “Equivalence”
o How Do the Products of Genetic Engineering Differ from the Conventionally Derived Products?
o What Are the Anticipated Consequences of “Precise” Single Gene Modifications?
o Is This Simple Linear Model Valid?
o Assessing the Significance of Differences
o Building Better Evaluation Capacity
o Level One - DNA Structure
o Level Two - Gene Expression
o Level Three - Protein Profiling
o Level Four - Metabolic Profiling
o Can “Substantial Equivalence” Become Scientifically Rigorous?
o Recommendations
o References
• 8. THE PRECAUTIONARY PRINCIPLE AND THE REGULATION OF FOOD BIOTECHNOLOGY
o Introduction
o Current Status
o Controversies Surrounding the Precautionary Principle
o Interpreting the Principle
o Recognition of Scientific Uncertainty and Fallibility
o Presumption in Favour of Health and Environmental Values
o Proactive Versus Reactive Approaches to Health and Environmental Values
o Burden of Proof and Standards of Evidence
o Standards of Acceptable Risk (Safety)
o Implications for the Regulation of Food Biotechnology
o Recommendations
o References
o Notes
• 9. ISSUES IN THE SCIENCE-BASED REGULATION OF BIOTECHNOLOGY
• PART 1: MAINTAINING THE INTEGRITY OF RISK ASSESSMENT SCIENCE
o Regulatory Conflict of Interest
o Confidentiality Versus Transparency in Canadian Regulatory Science
o Validation of the Science
o Increasing Commercialization of University Scientific Research in Biotechnology
o Recommendations
o References
• PART 2: LABELLING OF GENETICALLY MODIFIED FOODS
o Current Labelling Policies on GM Foods
o Socio-Political and Ethical-Philosophical Concerns
o Health Basis for Mandatory Labelling
o Conclusions on Mandatory Labelling
o Voluntary Labelling
o References
• GLOSSARY
• ACRONYMS AND ABBREVIATIONS
• MEMBERS OF THE EXPERT PANEL
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Re: ELEMENTS OF PRECAUTION: RECOMMENDATIONS FOR THE REGULATI

Postby admin » Sat Jan 09, 2016 11:53 pm

Prefatory Note

William Leiss, President
The Royal Society of Canada

January 30, 2001

Dear Dr. Leiss:

We are pleased to enclose a copy of the report Elements of Precaution: Recommendations for the Regulation of Food Biotechnology in Canada. The report has been prepared by the Expert Panel on the Future of Food Biotechnology, established by the Committee on Expert Panels of the Royal Society of Canada, in response to a request from Health Canada, the Canadian Food Inspection Agency, and Environment Canada. Our report addresses the questions posed by these agencies in the Terms of Reference negotiated between the government agencies and the Expert Panel, which are outlined in the Executive Summary and the Introduction of the enclosed report. These Terms of Reference asked the Expert Panel to provide advice on the Canadian regulatory system and the scientific capacity the federal government requires into the 21st century to ensure the safety of new food products being developed through biotechnology.

The enclosed report responds to this request by summarizing the scientific developments that have led to the current status of application of the technology and identifying the social and scientific dynamics that foreshadow new applications of biotechnology. It examines in detail the safety implications of these applications for human and animal health and the natural environment. The report also critically examines the current standard principles and practices governing the regulation of food biotechnology both in Canada and internationally, and makes a series of recommendations in three areas: 1) those concerning fundamental policies and principles governing the regulation of biotechnology, 2) those concerning specific Canadian regulations and guidelines, and 3) those concerning the regulatory process itself.

We are happy to report that the enclosed report represents a broad agreement among the members of the Panel. This is not to imply that every member would express all the arguments and conclusions in exactly the same way, or that some members would not favour additional or even stronger recommendations in some areas. Despite the broad agreement among the members, the Expert Panel has agreed that every member should be free to express his or her own individual interpretations and points of difference freely. It is a tribute to the RSC Committee on Expert Panels that a panel of this size and diversity of expertise and opinion was able to work in such a highly collegial and collaborative manner. Many of the issues with which we were charged are complex and controversial. They generated vigorous debate within the Panel. We would like to thank all the members of the Panel for the energy and time they devoted to the completion of this report in such a collaborative spirit.

This is a large and complex topic. The Panel had to carry out its task in a relatively short time span given the enormity of the task. We were able obtain the information we needed and to meet the deadlines we faced largely due to the able support provided to the Panel by Dr. Geoffrey Flynn, Chair of the Committee on Expert Panels, and Ms. Sandy Jackson, the Project Administrator for the Royal Society of Canada. We are deeply indebted to them for their tireless assistance.

We hope that Health Canada, the Canadian Food Inspection Agency, and Environment Canada will find our recommendations useful in the important task they face in the regulation of food biotechnology in Canada.

Yours sincerely,

Conrad Brunk and Brian Ellis Co-Chairs, RSC Expert Panel on the Future of Food Biotechnology

The Royal Society of Canada The Canadian Academy of the Sciences and Humanities La Société royale du Canada L’Académie canadienne des sciences, des arts et des lettres

Prefatory Note

In November, 1999 Health Canada’s Health Products and Foods Branch approached the Royal Society of Canada with a request to commission an Expert Panel to provide advice to ensure the safety of new food products being developed through biotechnology. The Society agreed to do so, and the Committee on Expert Panels undertook the task of screening and selecting the individuals whose names now appear as the authors of this report for panel service.

The report entitled Elements of Precaution: Recommendations for the Regulation of Food Biotechnology in Canada represents a consensus of the views of all of the Panelists whose names appear on the title page. The Committee wishes to thank the Panel Members and Panel Chairs, the Peer Reviewers, and the Panel staff for completing this very important report within a short period of time.

The Society has a formal and published set of procedures, adopted in October 1996, which sets out how Expert Panel processes are conducted, including the process of selecting Panelists. Interested persons may obtain a copy of those procedures from the Society. The Committee on Expert Panels will also respond to specific questions about its procedures and how they were implemented in any particular case.

The Terms of Reference for this Expert Panel are reproduced elsewhere in this report. As set out in our procedures, the terms are first proposed by the study sponsor, in this case Health Canada, the Canadian Food Inspection Agency and Environment Canada, and accepted provisionally by the Committee. After the Panel is appointed, the terms of reference are reviewed jointly by the Panelists and the sponsor; the Panelists must formally indicate their acceptance of a final Terms of Reference before their work can proceed. These are the terms reproduced in this report.

The Panel first submits a draft of its final report in confidence to the Committee, which arranges for another set of experts to do a peer review of the draft. The Peer Reviewer comments are sent to the Panel, and the Committee takes responsibility for ensuring that the Panelists have addressed satisfactorily the Peer Reviewer comments.

The Panel’s report is released to the public without any prior review and comment by the study sponsor. This arm’s-length relationship with the study sponsor is one of the most important aspects of the Society’s Expert Panel process.

Inquiries about the Expert Panel process may be addressed to the Chair, Committee on Expert Panels, Royal Society of Canada.

Dr. Geoffrey Flynn, FRSC Chair, Committee on Expert Panels

on behalf of the Committee Members for this Panel: William Leiss, FRSC, Queen’s University Christopher Garrett, FRS, FRSC, University of Victoria Daniel Krewski, University of Ottawa David Layzell, Queen’s University John Meisel, CC, FRSC, Professor Emeritus, Queen’s Univesity Gilles Paquet, CM, FRSC, FRSA, Université d’Ottawa

January 30, 2001
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Re: ELEMENTS OF PRECAUTION: RECOMMENDATIONS FOR THE REGULATI

Postby admin » Sat Jan 09, 2016 11:56 pm

EXECUTIVE SUMMARY

This Report is a response to a request to the Royal Society of Canada from Health Canada, the Canadian Food Inspection Agency and Environment Canada that an Expert Panel be assembled to provide advice on a series of questions related to the safety of new food products being developed through the use of new genetic engineering technologies. The Terms of Reference asked the Panel “to provide Health Canada, the Canadian Food Inspection Agency and Environment Canada with advice on our regulatory system and the scientific capacity that the federal government requires into the 21st century to ensure the safety of new food products being developed through biotechnology”. We were specifically charged to address the following issues:

# To forecast:

• the types of food products being developed through biotechnology that could be submitted for regulatory safety reviews by Health Canada and/or the Canadian Food Inspection Agency over the next 10 years;
• the science likely to be used to develop these products; and
• any potential short- or long-term risks to human health, animal health and the environment due to the development, production or use of foods derived from biotechnology.

# To assess approaches and methodologies developed in Canada and internationally to evaluate the safety of foods being developed through biotechnology, including those being developed by the World Health Organization, the Food and Agricultural Organization and the Codex Alimentarius Commission.

# To identify:

• the scientific capacity that will be needed to ensure the safety of new foods derived from biotechnology, including human resources for research, laboratory testing, safety evaluation, and monitoring and enforcement; and
• any new policies, guidelines and regulations related to science that may be required for protecting human health, animal health and environmental health.

This Report addresses these issues in the following way.

Chapter 1 clarifies the Panel’s interpretation of its mandate and the Terms of Reference. It attempts to delineate clearly the range of scientific and non-scientific issues that fall within its mandate, those that fall clearly outside it, and those related issues that need to be addressed to provide comprehensive answers to the questions posed by the mandate. Chapter 1 also summarizes the process by which the Panel produced the Report.

Chapter 2 responds to the mandate to forecast the future directions in the development of agricultural biotechnology. It does so by summarizing the scientific developments that have led to the current status of application of the technology. It identifies the social and scientific dynamics driving its current development, and points to technological developments that are likely to bring new applications of biotechnology. Many of the themes summarized in Chapter 2 are developed in greater detail in subsequent chapters dealing with specific health and environmental risks.

Chapter 3 summarizes the system currently in place for the regulation of agricultural biotechnology in Canada. The chapter recommends implementation of an independent process for auditing of the scientific and ethical aspects of regulatory decision making.

Chapter 4 is the first of three chapters that conduct the scientific identification of the short- and long-term risks the Panel found to be most important for regulatory concern in Canada. It focuses on the direct risks to human health posed by genetically modified (GM) food. Part 1 of Chapter 4 considers the specific problems related to the use of the classical risk assessment methodologies for the assessment of toxicological risks from GM foods, especially the assessment of the safety of whole foods. Part 2 focuses on the critical issues related to the identification of potential allergens in GM foods, and makes recommendations for strengthening the scientific capacity for identifying and assessing the allergenicity of new or unexpected proteins in GM foods. Part 3 points to the need to consider the impacts of genetic engineering modifications on the nutritional value of the resulting food.

Chapter 5 considers the potential direct impacts of genetic engineering upon the health and welfare of agricultural animals, as well as the indirect impacts upon wild animals. Part 1 identifies the risks associated with the genetic modification of fish and farm animals themselves, while Part 2 focuses upon the risks associated with GM feeds, feed additives and metabolic modifiers administered to food-producing animals. Chapter 5 makes a variety of recommendations for the more rigorous assessment of the impacts upon animal health and welfare, genetic diversity and sustainability, as well as upon human consumers of GM animals and animal products.

Chapter 6 identifies what the Panel considered to be the most significant potential risks to various aspects of the natural environment posed by agricultural biotechnology. The chapter is divided into four parts, each dealing with the impacts of potential gene flow upon different sectors of the natural environment — microorganisms and soil microflora, wild and non-GM plants, target and non-target insects, and wild fish. Recommendations following each of these sections identify a series of more refined environmental assessments that need to be added to the Canadian regulatory process to protect more adequately important environmental values.

Chapter 7 introduces a series of three final chapters that deal with critical methodological approaches and assumptions underlying current and proposed regulatory practices in the area of agricultural biotechnology. Chapter 7 is an in-depth analysis and critique of one of the most controversial concepts invoked in both national and international regulatory contexts — that of “substantial equivalence”. The Panel finds the use of “substantial equivalence” as a decision threshold tool to exempt GM agricultural products from rigorous scientific assessment to be scientifically unjustifiable and inconsistent with precautionary regulation of the technology. The Panel recommends a four-stage diagnostic assessment of transgenic crops and foods that would replace current regulatory reliance upon “substantial equivalence” as a decision threshold.

Chapter 8 focuses upon the current debate over the validity and relevance of the so-called “precautionary principle” in the regulation of agricultural biotechnologies. Many national and international regulatory bodies (including Canada) have adopted the “precautionary principle” as a regulatory axiom. In this chapter, the Panel lays out an understanding of the principle it considers to have both scientific and regulatory validity, and recommends its use as an axiom of Canadian regulatory policy. The Panel finds the use of “substantial equivalence” as a standard of safety (as opposed to a decision threshold in assessment of risk) to be, in general, a precautionary standard.

Chapter 9 raises a series of issues the Panel identified during its deliberations that it considered to be of critical importance in maintaining the integrity of science upon which the regulation of agricultural biotechnology should be based, and in maintaining public confidence in the regulatory processes. Part 1 of the chapter raises serious concerns about the undermining of the scientific basis for risk regulation in Canada due to the following factors:

• the conflict of interest created by giving to regulatory agencies the mandates both to promote the development of agricultural biotechnologies and to regulate it;
• the barriers of confidentiality that compromise the transparency and openness to scientific peer review of the science upon which regulatory decisions are based; and
• the extensive and growing conflicts of interest within the scientific community due to entrepreneurial interests in resulting technologies and the increasing domination of the research agenda by private corporate interest.

In Part 2 of Chapter 9, the Panel considers the scientific basis for mandatory labelling of genetically engineered food products, and establishes guidelines for mandatory and voluntary labelling on the basis of health risks. The Panel recognizes that there are broader social, political and ethical considerations in the debate about mandatory labelling of GM foods that lie outside the Panel’s specific mandate, so this discussion is not intended to provide a comprehensive answer to the issue of mandatory labelling.

SUMMARY OF THE EXPERT PANEL’S RECOMMENDATIONS

In light of its investigations, the Panel made the following recommendations. The rationale and complete text of these recommendations are found at the end of each major section of the Report.

Recommendations Concerning Underlying Policies and Principles Guiding the Regulation of Agricultural Biotechnology

7.1 The Panel recommends that approval of new transgenic organisms for environmental release, and for use as food or feed, should be based on rigorous scientific assessment of their potential for causing harm to the environment or to human health. Such testing should replace the current regulatory reliance on “substantial equivalence” as a decision threshold.

7.2 The Panel recommends that the design and execution of all testing regimes of new transgenic organisms should be conducted in open consultation with the expert scientific community.

7.3 The Panel recommends that analysis of the outcomes of all tests on new transgenic organisms should be monitored by an appropriately configured panel of “arms-length” experts from all sectors, who report their decisions and rationale in a public forum.

8.1 The Panel recommends the precautionary regulatory assumption that, in general, new technologies should not be presumed safe unless there is a reliable scientific basis for considering them safe. The Panel rejects the use of “substantial equivalence” as a decision threshold to exempt new GM products from rigorous safety assessments on the basis of superficial similarities because such a regulatory procedure is not a precautionary assignment of the burden of proof.

8.2 The Panel recommends that the primary burden of proof be upon those who would deploy food biotechnology products to carry out the full range of tests necessary to demonstrate reliably that they do not pose unacceptable risks.

8.3 The Panel recommends that, where there are scientifically reasonable theoretical or empirical grounds establishing a prima facie case for the possibility of serious harms to human health, animal health or the environment, the fact that the best available test data are unable to establish with high confidence the existence or level of the risk should not be taken as a reason for withholding regulatory restraint on the product.

8.4 As a precautionary measure, the Panel recommends that the prospect of serious risks to human health, of extensive, irremediable disruptions to the natural ecosystems, or of serious diminution of biodiversity, demand that the best scientific methods be employed to reduce the uncertainties with respect to these risks. Approval of products with these potentially serious risks should await the reduction of scientific uncertainty to minimum levels.

8.5 The Panel recommends a precautionary use of “conservative” safety standards with respect to certain kinds of risks (e.g. potentially catastrophic). When “substantial equivalence” is invoked as an unambiguous safety standard (and not as a decision threshold for risk assessment), it stipulates a reasonably conservative standard of safety consistent with a precautionary approach to the regulation of risks associated with GM foods.

9.1 The Panel recommends that Canadian regulatory agencies and officials exercise great care to maintain an objective and neutral stance with respect to the public debate about the risks and benefits of biotechnology in their public statements and interpretations of the regulatory process.

9.2 The Panel recommends that the Canadian regulatory agencies seek ways to increase the public transparency of the scientific data and the scientific rationales upon which their regulatory decisions are based.

9.3 The Panel recommends that the Canadian regulatory agencies implement a system of regular peer review of the risk assessments upon which the approvals of genetically engineered products are based. This peer review should be conducted by an external (non-governmental) and independent panel of experts. The data and the rationales upon which the risk assessment and the regulatory decision are based should be available to public review.

9.4 The Panel recommends that the Canadian Biotechnology Advisory Commission (CBAC) undertake a review of the problems related to the increasing domination of the public research agenda by private, commercial interests, and make recommendations for public policies that promote and protect fully independent research on the health and environmental risks of agricultural biotechnology.

Recommendations Concerning Regulations and Guidelines

4.1 The Panel recommends that federal regulatory officials in Canada establish clear criteria regarding when and what types of toxicological studies are required to support the safety of novel constituents derived from transgenic plants.

4.3 The Panel recommends that, in view of the availability of suitable alternative markers, antibiotic resistance markers should not be used in transgenic plants intended for human consumption.

4.8 The Panel recommends that approvals should not be given for GM products with human food counterparts that carry restrictions on their use for non-food purposes (e.g. crops approved for animal feed but not for human food). Unless there are reliable ways to guarantee the segregation and recall if necessary of these products, they should be approved only if acceptable for human consumption.

5.1 The Panel recommends that the Canadian Food Inspection Agency (CFIA) develop detailed guidelines describing the approval process for transgenic animals intended for (a) food production or (b) other non-food uses, including appropriate scientific criteria for assessment of behavioural or physiological changes in animals resulting from genetic modification.

6.10 The Panel recommends that companies applying for permission to release a GM organism into the environment should be required to provide experimental data (using ecologically meaningful experimental protocols) on all aspects of potential environmental impact.

6.11 The Panel recommends that an independent committee should evaluate both the experimental protocols and the data sets obtained before approvals of new plants with novel traits are granted.

6.12 The Panel recommends that standard guidelines should be drawn up for the longterm monitoring of development of insect resistance when GM organisms containing “insecticidal” products are used, with particular attention to pest species known to migrate over significant distances.

6.13 The Panel recommends that a moratorium be placed on the rearing of GM fish in aquatic netpens.

6.14 The Panel recommends that approval for commercial production of transgenic fish be conditional on the rearing of fish in land-based facilities only.

Recommendations Concerning the Regulatory Process

4.2 The Panel recommends that regulatory authorities establish a scientific rationale that will allow the safety evaluation of whole foods derived from transgenic plants. In view of the international interest in this area, the Panel further recommends that Canadian regulatory officials collaborate with colleagues internationally to establish such a rationale and/or to sponsor the research necessary to support its development.

4.6 The Panel recommends development of mechanisms for after-market surveillance of GM foods incorporating any novel protein.

4.7 The Panel recommends that the appropriate government regulatory agencies have in place a specific, scientifically sound and comprehensive approach for ensuring that adequate allergenicity assessment will be performed on GM foods.

4.9 The Panel recommends that all assessments of GM foods, which compare the test material with an appropriate control, should meet the standards necessary for publication in a peer-reviewed journal, and all information relative to the assessment should be available for public scrutiny. The data should include the full nutrient composition (Health Canada, 1994), an analysis of any anti-nutrient and, where applicable, a protein evaluation such as that approved by the United Nations Food and Agriculture Organization (FAO).

4.10 The Panel recommends that protocols should be developed for the testing of future genetically engineered foods in experimental diets.

4.11 The Panel recommends that the Canadian Nutrient File should be updated to include the composition of genetically engineered foods and be readily available to the public.

5.2 The Panel recommends that the approval process for transgenic animals include a rigorous assessment of potential impacts on three main areas:

1) the impact of the genetic modifications on animal health and welfare;

2) an environmental assessment that incorporates impacts on genetic diversity and sustainability; and

3) the human health implications of producing disease-resistant animals or those with altered metabolism (e.g. immune function).

5.3 The Panel recommends that the tracking of transgenic animals be done in a manner similar to that already in place for pedigree animals, and that their registration be compulsory.

5.4 The Panel recommends that transgenic animals and products from those animals that have been produced for non-food purposes (e.g. the production of pharmaceuticals) not be allowed to enter the food chain unless it has been demonstrated scientifically that they are safe for human consumption.

5.6 The Panel recommends that the use of biotechnology to select superior animals be balanced with appropriate programs to maintain genetic diversity, which could be threatened as a result of intensive selection pressure.

5.8 The Panel recommends that changes in susceptibility of genetically engineered plants to toxin-producing microbes, and the potential transfer of these to the animal and the food supply, be evaluated as part of the approval process.

5.9 The Panel recommends that a data bank listing nutrient profiles of all GM plants that potentially can be used as animal feeds be established and maintained by the federal government.

5.10 The Panel recommends that university laboratories be involved in the validation of the safety and efficacy of GM plants and animals.

5.11 The Panel recommends that Environment Canada and the Canadian Food Inspection Agency establish an assessment process and monitoring system to ensure safe introductions of GM organisms into Canada, according to the intent of the Canadian Environmental Protection Act.

6.1 The Panel recommends that all ecological information on the fate and effects of transgenic biotechnology products on ecosystems required under existing regulations should be generated and made available for peer review.

6.2 The Panel recommends the carrying out of exhaustive, long-term testing for ecological effects of biotechnology products that pose environmental risks, especially with respect to persistence of the organism or a product of the organism, persistent effects on biogeochemical cycles, or harmful effects resulting from horizontal gene transfer and selection.

6.3 The Panel recommends that, in evaluating environmental risks, scientific emphasis should be placed on the potential effects of selection operating on an introduced organism or on genes transferred to natural recipients from that organism.

6.5 The Panel recommends that the history of domestication, and particularly the time period and intensity of artificial selection, of GM plants should be taken into account when assessing potential environmental impacts. Species with a short history of domestication should receive particularly close scrutiny because they are more likely to pose environmental risks.

6.6 The Panel recommends that environmental assessments of GM plants should pay particular attention to reproductive biology, including consideration of mating systems, pollen flow distances, fecundity, seed dispersal and dormancy mechanisms. Information on these lifehistory traits should be obtained from specific experiments on the particular GM cultivar to be assessed, not solely from literature reports for the species in general.

6.7 The Panel recommends that environmental assessments of GM plants should not be restricted to their impacts on agroecosystems but should include an explicit consideration of their potential impacts on natural and disturbed ecosystems in the areas in which they are to be grown

6.8 The Panel recommends that research data from experiments conducted by industry on the potential environmental impacts of GM plants used in Canadian Environmental Protection Agency assessments should be made available for public scrutiny.

6.16 The Panel recommends that potential risks to the environment posed by transgenic fish be assessed not just case-by-case, but also on a population-by-population basis.

Recommendations Concerning Scientific Capacity for the Regulation of Food Biotechnology

4.4 The Panel recommends that the Canadian government support research initiatives to increase the reliability, accuracy and sensitivity of current methodology to assess allergenicity of a food protein, as well as efforts to develop new technologies to assist in these assessments.

4.5 The Panel recommends the strengthening and development of infrastructures to facilitate evaluation of the allergenicity of GM proteins. This could include development of a central bank of serum from properly screened individuals allergic to proteins which might be used for genetic engineering, a pool of standardized food allergens and the novel GM food proteins or the GM food extracts, maintenance and updating of allergen sequence databases, and a registry of food-allergic volunteers.

5.5 The Panel recommends that federal and provincial governments ensure adequate public investment in university-based genomic research and education so that Canada has the capacity for independent evaluation and development of transgenic technologies.

5.7 The Panel recommends that a national research program be established to monitor the long-term effects of GM organisms on the environment, human health, and animal health and welfare.

6.4 The Panel recommends that a detailed analysis be undertaken of the expertise needed in Canada to evaluate environmental effects of new biotechnology products and, if the appropriate expertise is found to be lacking, resources be allocated to improving this situation.

6.9 The Panel recommends that a federally funded multidisciplinary research initiative be undertaken on the environmental impacts of GM plants. Funds should be made available to scientists from all sectors (industry, government and university) with grant proposals subject to rigorous peer review.

6.15 The Panel recommends the establishment of comprehensive research programs devoted to the study of interactions between wild and cultured fish. Reliable assessment of the potential environmental risks posed by transgenic fish can be undertaken only after extensive research in this area.

6.17 The Panel recommends that identification of pleiotropic, or secondary, effects on the phenotype resulting from the insertion of single gene constructs into GM organisms be a research priority.

7.4 The Panel recommends that Canada develop and maintain comprehensive public baseline data resources that address the biology of both its major agroecosystems and adjacent biosystems.

7.5 The Panel recommends that Canada develop state-of-the-art genomics resources for each of its major crops, farm animals and aquacultured fish, and use these to implement effective methodologies for supporting regulatory decision making.
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1. INTRODUCTION

“The risks in biotechnology are undeniable, and they stem from the unknowable in science and commerce. It is prudent to recognize and address those risks, not compound them by overly optimistic or foolhardy behaviour.”
-- Editors - Nature Biotechnology (October 2000)


THE EXPERT PANEL

This Report is submitted in response to a joint request to the Royal Society of Canada from three agencies of the Government of Canada (Health Canada, Canadian Food Inspection Agency, and Environment Canada) that an independent Expert Panel be convened by the Society to advise on a series of questions related to the safety of new food products being developed through the use of new genetic engineering technologies. The specific questions were laid out in provisional Terms of Reference provided to the Royal Society of Canada Committee on Expert Panels in January 2000. The Committee on Expert Panels then selected a group of 15 people from across Canada who represented a wide range of scientific and policy-related expertise relevant to the questions submitted. The Terms of Reference were then reviewed and interpreted at a meeting of the Expert Panel with representatives of the sponsoring government departments in March 2000. The Royal Society agreed to submit the Report of the Expert Panel to the Government of Canada by December 15, 2000. By mutual agreement this deadline was extended to January 31, 2001.

MANDATE AND TERMS OF REFERENCE

The mandate of the Expert Panel on the Future of Food Biotechnology is to provide Health Canada, the Canadian Food Inspection Agency, and Environment Canada with advice on our regulatory system and the scientific capacity that the federal government requires into the 21st century to ensure the safety of new food products being developed through biotechnology.

Terms of Reference

# To forecast:

• the types of food products being developed through biotechnology that could be submitted for regulatory safety reviews by Health Canada and/or the Canadian Food Inspection Agency over the next 10 years;
• the science likely to be used to develop these products; and
• any potential short- or long-term risks to human health, animal health and the environment due to the development, production or use of foods derived from biotechnology.

# To assess approaches and methodologies developed in Canada and internationally to evaluate the safety of foods being developed through biotechnology, including those being developed by the World Health Organization, the Food and Agricultural Organization and the Codex Alimentarius Commission.

# To identify:

• the scientific capacity that will be needed to ensure the safety of new foods derived from biotechnology, including human resources for research, laboratory testing, safety evaluation, and monitoring and enforcement; and
• any new policies, guidelines and regulations related to science that may be required for protecting human health, animal health and environmental health.

INTERPRETATION OF THE TERMS OF REFERENCE

At its first meeting in March 2000, the Expert Panel met with representatives of the sponsoring government departments to discuss and clarify the Terms of Reference. The Terms of Reference were not revised as a result of this meeting, but the discussions helped to clarify the expectations of the sponsors relative to the scope and limits of this study. The discussion with the sponsors made it evident that, although the focus of the Expert Panel’s enquiry was on the scientific aspects of the new technologies and their effective regulation, the Panel would need to address many peripheral issues that touch on the question of the appropriate use of science in the regulation of risks. For example, controversies over such questions as the advisability of labelling genetically engineered food products, the impacts of international trade agreements and international standards upon Canadian food safety, the complex relationship among biotechnology industries, scientists and regulators, are all related to the question of how science should be used to manage effectively the risks associated with genetically engineered products.

The issues raised in the public debate about biotechnology range across a wide spectrum of concerns. They include concerns about impacts upon human and animal health resulting from undetected toxins or allergenic substances in GM food products, about the environmental impacts of transgenic genes proliferating in wild species of plants, about loss of biodiversity, the impact of expanded reliance upon GM crops upon the agricultural economies of less developed nations, and about impacts upon consumers resulting from monopolization in agribusiness. They also include explicitly ethical concerns about splicing genes across plant and animal “kingdom” barriers, producing “unnatural” animal chimeras, about “playing God” with nature, about the rights of consumers to choose whether to expose themselves to unknown or uncertain risks, or just simply to choose what technologies they will support with their purchasing dollars.

These are among the concerns shaping public attitudes about food biotechnology. They are often expressed as concerns about the “risks” and “safety” of genetically engineered foods, because they are perceived as posing “risks” to a wide variety of social and personal values. The Terms of Reference ask the Expert Panel to focus its attention upon a fairly narrow portion of this wide range of issues — those related to human and animal health and environmental impacts. We have tried to respect the limits of the Terms of Reference as much as possible. Accordingly, it is important at the outset to clarify which questions we have taken as most central to our task under the Terms of Reference, and which we have left outside our consideration. However, it is important to understand that answers to questions not specifically within our mandate are often relevant to, and influence answers to questions that are within it. The health and environmental safety issues posed to the Panel in the Terms of Reference, though largely scientific in nature, often cannot be addressed fully without reference to broader ethical, political and social issues and assumptions.

The different types of concerns at issue in the public debate about biotechnology can be classified helpfully in three categories. These categories distinguish three different kinds of values feared to be placed “at risk” by various biotechnologies. These are concerns about: 1) the potential risks to the health of human beings, animals and the natural environment, 2) the potential risks to social, political and economic relationships and values, and 3) the potential risks to fundamental philosophical, religious or “metaphysical” values held by different individuals and groups. Accordingly, we shall refer to these categories of concern about biotechnology as: 1) Health and Environmental Risks, 2) Socio-Economic Risks, and 3) Philosophical/Metaphysical Risks.

We recognize that the borders between these types of questions are not always clearly demarcated, nor are the questions completely independent. Assumptions about social, economic and philosophical questions often enter into deliberations, and thus conclusions, about the magnitude and acceptability of risks. For example, a strong conviction about the extensive benefits of the widespread adoption of biotechnology crops (or the adverse consequences of failing to adopt them) will tend to colour attitudes toward safety issues (i.e. higher risk levels will be viewed as acceptable). We shall attempt in this Report to be attentive both to the distinctions between the types of issues involved in the food biotechnology debate and to those places where they interpenetrate each other.

Health and Environmental Risks

As noted in the Introduction, the Terms of Reference given to the Expert Panel for this Report ask the panel to focus its attention on this first category of concerns — those having to do with potential risks to the health of human beings, animals and the environment posed by current and projected agricultural products of biotechnology. These Terms of Reference specify that the Panel give its attention to three aspects of this question: 1) the forecasting of the food biotechnologies likely to be submitted for regulatory safety reviews, the science behind them, and the risks to human, animal health and the environment posed by these technologies; 2) the assessment of the methods developed nationally and internationally for assuring the safety of these food biotechnologies; and 3) the identification of the scientific capacities and regulatory policies relating to this science that may be required for protecting human, animal and environmental health.

The subsequent chapters of this Report will attempt to address these issues by laying out the characteristics of current and projected food biotechnologies, identifying the significant hazards potentially associated with the products of these technologies, and assessing the potential magnitudes of the risk these might pose to human, animal or environmental health. They will also evaluate and recommend the methodologies and procedures required in order for industry and government regulators to evaluate reliably the risks posed by specific biotech food products in each of these areas, and to manage these risks within safe (acceptable) levels.

Since these Terms of Reference direct the Panel’s attention narrowly to one category of the issues that have emerged in the public debate about food biotechnology, it therefore goes without saying that the Panel does not intend that this Report represents a comprehensive response to the whole range of concerns involved in the question of whether the development of any particular agricultural biotechnology, or biotechnology in general, is advisable. Such a comprehensive response would have to address all three categories of concern identified above. We do offer an account of the social and economic factors driving this development but, in order to adhere to the terms of our mandate, this account is solely for the purposes of predicting the course of this development and assessing the potential problems it poses for managing health and environmental risks. Concerns of human, animal and environmental health are among the most critical ones raised in the food biotechnology debate, but they are only a small part of the debate. There are also the other categories of important questions having to do with the economic costs and benefits of agricultural biotechnology, the social impacts on societies at different stages of technological and social development, environmental and social ethics, as well as deeply held philosophical and religious convictions about human interventions in nature. While this Report comments on those issues where they are relevant to health and environmental impacts, it does not presume to address them comprehensively.

Socio-Economic Risks

The second category of concerns expressed in the public debate about food biotechnology relate to the potential risks it poses to a variety of socio-economic values: these include concerns about concentration of the seed industry in the hands of a few multinational companies, with potential dislocation of rural farm communities in favour of a few large agribusinesses. They include concern about the potential effects of biotechnology on farmers in lesser developed countries, who may be at risk of increased dependency on multinational corporations from the developed world, leading to decreasing food self-sufficiency in these areas. The recent furor over the so-called “Terminator” seed technology being developed by agbiotech companies and the USDA, which culminated in Monsanto’s announcement that the technology would not be brought into the marketplace, was generated by just this concern.

Proponents of food biotechnology argue that the socio-economic arguments in fact make the strongest case for its development. They argue that the ability to engineer food crops for greater productivity, adaptation to growth in marginal soils and climates, and enhanced nutritional qualities is essential to meeting the food needs for an expanding world population. Proponents also believe that the technology will improve food quality and lower prices for consumers everywhere. These arguments are elaborated elsewhere in this Report as part of the Panel’s discussion of the social forces shaping biotechnology development. The Panel did not, however, consider it within its mandate to assess the extent to which these claims are reliable, or to evaluate quantitatively or qualitatively the magnitude of the benefits of food biotechnology.

Because we have not made these evaluations of the claimed benefits of agricultural biotechnologies, this Report cannot be read as providing any answers to the question of whether these technologies are socially desirable in the broadest sense. Many experts argue that the “safety” (acceptability of the risks) of these technologies depends upon whether the risks, whatever they may be, are outweighed by the overriding benefits they achieve. This “risk-costbenefit” approach to safety is only one among many safety standards that can be invoked by risk regulators. It tends to function as a less restrictive standard of safety, insofar as it permits, in principle, any level of risk as long as there are off-setting benefits. There are many other types of standards commonly advocated as well, including various “threshold” standards, procedural standards, and even “zero-risk”, which are usually more restrictive, or conservative.

This Report does not include a full discussion of this very important risk management issue. It does, however, address the concept of “substantial equivalence”, which is increasingly invoked as a safety standard as well as a risk assessment decision threshold (Chapters 7 and 8). When invoked as a safety standard, “substantial equivalence” establishes a “threshold” of acceptable risk, requiring that the risks of a GM product not exceed those of its non-GM counterpart, regardless of the magnitude of the benefits it may provide. Used in this way, the Panel notes (Chapter 8) that “substantial equivalence” functions as a fairly precautionary safety standard.

It is evident, then, that major factors influencing the social acceptability of food biotechnology are those having to do with perceptions of the socio-economic and political impacts of the growth of the technology rather than only the questions of risk to health and the environment. These involve strongly held political and ethical values — those related to a sense of social justice in the distribution of costs, risks and benefits, individual and community rights to choose, and democratic ideals of participation in decisions concerning the development of biotechnology. While these considerations are significant factors in the overall social question of the merits of food biotechnology, since they are not centrally within the mandate of this Panel, we comment on them only where necessary to address fully the health and environmental questions within our mandate.

Philosophical/Metaphysical Risks

The public debate about food biotechnology has also included a third category of issues. These relate to concerns that genetic engineering technologies give human beings powers over nature that are deeply unethical, either in themselves or in certain of their applications. These concerns are rooted in fundamental philosophical and theological perspectives on human and animal nature, the natural environment and divinity. The concerns about food biotechnology are part of a deeper view of biotechnology in general, which is considered to involve interventions in the natural world that undermine appropriate human relationships to nature or God. It is primarily the process of genetic engineering that is at issue, rather than its impacts upon health, environment or society. It is the fact that genes are altered and transposed between organisms by processes that would not occur “naturally”, crossing species and kingdom barriers and producing life forms (transgenic plants and animals) that would not by produced by the “natural” processes of evolution.

The critical operative concept here, clearly, is that of what is “natural”. This concept is not a scientific one, but a normative one — a view of how human, animal and plant natures should be, or “how God intends them to be”. The fact that a member of the British royal family can, with the support of a large number of British and European citizens, question whether human beings have the “right to play God” with GM organisms, indicates how widespread and deep-seated these metaphysical concerns can be.

These kinds of concerns about biotechnology are often expressed in less metaphysical and abstract language. They are often expressed as considerations of precaution in the face of uncertainty. Many critics of biotechnology base their arguments on the claim that current biotechnologies are based on a reductionist view of nature that is neither scientifically nor philosophically defensible. They challenge the view that the relationships between genes and the traits of organisms are deterministic and one-to-one, arguing instead that these relationships are complex and often unpredictable, since genes act in consort with other genes, the whole organism and the environment (Heinberg, 1999). The underlying force of these claims is that, because genetic technologies are full of high uncertainties, it is morally irresponsible for human beings to “muck around” with nature in this way.

There is also a set of philosophical and metaphysical concerns that are not so much about biotechnology per se as they are about certain implementations of it. Many people object, in principle, to such interventions as the cloning of human beings and/or animals, the engineering of cross-species chimeras (cat-rabbits, pigs used to grow human organs for xeno-transplantation, etc.). They would not argue that all uses of biotechnology are “unnatural”, but would view these kinds of uses as crossing fundamental lines of moral acceptability. Such practices may be viewed as undermining human conceptions of dignity and equality (e.g. in human cloning) or respect for nature as sacred (e.g. chimeras). In effect, these practices pose risks to fundamental moral values — or moral risks.

An even more concrete and immediate concern of this type relates to the transfer of genes from “taboo” foods into other food products. Religious and ethnic groups that observe religious dietary rules prohibiting the eating of certain animals have obvious problems with the consumption of vegetable or other animal foods that may carry genes taken from the prohibited animal. Vegetarians have similar problems with plants engineered with animal genes. These are all concerns rooted in fundamental philosophical/metaphysical beliefs about the world. This does not make them any less significant in the public debate about food biotechnology.

Again, these philosophical and metaphysical issues, while critically important in the public debate about food biotechnology and in the overall assessment of its social merits, are not taken to be within the mandate of the Expert Panel, and this Report takes no stance with respect to them, except where they may impinge directly upon the matters of health risk assessment and management that do fall within its mandate. One place where they do impinge upon these matters, for example, is in the conception of what constitutes full human or animal health. Traditionally, conceptions of health and “well-being” invoke metaphysical notions of what is “natural”, “normal” or “good”. For this reason, the Panel’s discussion of animal health concerns in Chapter 5 requires discussion of animal welfare.

SCIENTIFIC AND EXTRA-SCIENTIFIC ISSUES IN RISK ANALYSIS

We have categorized the different elements of the biotechnology debate as reflecting concerns about different types of values or concerns “at risk” in order to clarify certain issues that are typically confused in the debate, and to clarify our inclusion in this Report of certain matters we consider relevant to the management of health risks. The confusion is engendered by a common, but quite different, way of distinguishing the issues. It is commonly stated that the biotechnology debate falls into the following three kinds of disagreement: 1) scientific disagreements about types and degrees of risk to human, animal and environmental health; 2) political disagreements about the social and economic impacts of agricultural biotechnology (disagreements based upon different political views); and 3) religious, ethical and philosophical disagreements about whether biotechnology is “unnatural”, “immoral”, “playing God”, etc.

Our characterization of the various aspects of the debate may seem at first sight to be no different from the first. But there is a critically important difference. The common classification assumes that the various issues in the debate can be distinguished according to their method of inquiry. It assumes that the issues in the first category involve empirical questions resolvable primarily by means of scientific method. The issues in the second category are about preferred political and social structures, which involve matters, not only of social science and economics, but also of political and social philosophy not resolvable through scientific investigation. The issues in the third category are characterized as deeply religious, moral and metaphysical. They are not matters of physical or social science at all, but value judgments deeply rooted in culture, ethnicity and tradition, which are generally considered to be unresolvable by any rational method.

The Panel does not accept the common classification of the issues in the biotechnology debate because of its implication that the questions put to it in the Terms of Reference — having to do with the identification of potential risks to human, animal and environmental health — are purely questions of science. There is no doubt that questions about the potential hazards inherent in the products of agricultural biotechnology and the mechanisms for assessing the magnitude of the health risks they pose are primarily scientific, requiring the very best scientific methods and expertise for their resolution. But they are not purely scientific. It is now generally recognized in the scholarly literature on the nature of risk analysis that many aspects of the task of assessing the magnitude of technological risks and managing them within the limits of safety involve judgments and decisions that are not themselves strictly scientific (Salter et al., 1988; Mayo et al., 1991; Shrader-Frechette, 1991). They involve value judgments related to such issues as the appropriate way to handle uncertainties in scientific data and results, assignment of the burden of proof among stakeholders in risk issues, standards of proof, definition of the scope of the risk issue (e.g. should human error be considered part of the risk of the technology?), and, of course, the central issue, already noted, of what levels of risk should be considered “acceptable”. Such “extra-scientific” judgments are inherent in any assessment of risk and in the judgments about the technological and social mechanisms for maintaining it within safe limits. Similar judgments are involved in any attempt to predict future scientific and technological developments, which are always at least partially dependent upon human choices and other undetermined variables.

The Panel recognizes that answers to the questions put to it in the Terms of Reference require the very best in scientific investigation. It is for this reason that the Panel was appropriately constituted to represent expertise from the scientific disciplines most relevant to food biotechnology — including biology, biochemistry, genetics, environmental science, ecology, medical science, animal science, food science, plant science, nutrition, toxicology, entomology, etc. But the Panel also recognizes that the Terms of Reference require investigation into the extrascientific issues that establish the framework for the scientific investigations involved in risk analysis. For this reason, it is appropriate that the Panel membership also included specialists in the “normative” disciplines of law, philosophy and ethics.

PANEL PROCEDURES

The members of the Expert Panel were appointed by the Royal Society in February 2000. On February 17, 2000, the Royal Society of Canada issued a press release announcing the establishment of the Expert Panel, the appointment of the Panel members, and an outline of the Terms of Reference. The press release invited written submissions from any interested parties in Canada on issues relevant to its mandate and objectives. The deadline date for these submissions was set at April 30, 2000. However, in view of the fact that many parties did not receive information about the Panel process in time, this date was subsequently extended by the Expert Panel (in announcements on the Royal Society of Canada web site and in press reports) to July 31.

The Expert Panel convened its first meeting in Ottawa on March 15 and 16, 2000. During these two days, the Panel met with representatives of the sponsoring government departments to discuss and clarify the Terms of Reference. During this meeting, it became evident that two of the original appointees to the Expert Panel (François Pothier and James Germida) would not be able to fulfill all the obligations of membership on the panel. They were subsequently replaced by two alternate members (John Kennelly and Jeremy McNeil). At this meeting, the Expert Panel identified the major scientific and other issues that the Report would need to address in order to answer the questions put to it in the Terms of Reference, and a draft structure for the Report was adopted. Research assignments were parsed out to the members of the Panel for reporting at the subsequent meeting.

The Expert Panel convened a second meeting for three days, June 27 to 29, in Ottawa to consider the preliminary research carried out by the members and distributed to them prior to the meeting. In preparation for this meeting, all submissions from interested parties received by that date were read by all members of the Panel, and issues raised in these submissions were discussed as part of an extended discussion in which the members developed an inventory of the major issues to address and moved toward agreement on the position we wished to take with respect to them, given the research findings to that date. Members left this meeting with a revised Report Outline, and research and drafting assignments for a preliminary Report to be considered at the subsequent meeting.

A third meeting was held, again in Ottawa, on August 8 to 10. At this meeting, the Panel reviewed the additional submissions from the public that had been received in response to the extended deadline of July 31. The results of the initial research and the first round of drafts assigned at the June meeting had been circulated to all members of the Panel and were carefully reviewed by the whole Panel. Additional research needs were identified as well as the preliminary overall direction of the findings.

The initial round of research had also identified a series of additional questions the Panel felt could be answered only by further consultation with personnel from the sponsoring agencies. In response to last-minute requests to the agencies, all arranged quickly to provide spokespersons to meet with us. In separate meetings, the Panel interviewed William Yan, Acting Head, Office of Food Biotechnology, Health Canada; Bart Bilmer, Director, Office of Biotechnology, Canadian Food Inspection Agency; Phil McDonald, Biotechnologist, Plant Biotechnology, Canadian Food Inspection Agency, and James Lauter, Evaluation Specialist, Biotechnology Section, Environment Canada. The Panel would like to express gratitude to these persons for agreeing to meet with us on very short notice, and for their forthcoming responses to our questions.

The final meeting of the Expert Panel took place in Vancouver on October 27 to 29. In the interim, the Panel members had been assigned additional research projects and were asked to bring revised drafts of assigned sections for critique and evaluation by the whole Panel. The Panel members reached agreement on the final revisions necessary for the Report at this meeting.

The first draft of this Report was sent to an anonymous group of seven Peer Reviewers, who were selected by the Royal Society of Canada Committee on Expert Panels. The Panel received comments from three of the Peer Reviewers in time for it to incorporate their suggestions into the final version of the Report. The comments and suggestions of the Peer Reviewers were extremely helpful to the Panel and contributed significantly to the quality of the Report.

We regret that two of the persons originally appointed to assist the Expert Panel were unable to do so due to health reasons. Dr. Thérèse Leroux, who was appointed as a member of the Panel, was unable to attend meetings or to participate in the writing of the Report due to health problems. Dr. Michael Smith, who was appointed as a special advisor to the Panel, was also unable to serve in this capacity due to ill health. The fourth meeting of the Panel was held in Vancouver with the expectation that Dr. Smith would be able to join us. We were saddened by his death only a few weeks before this meeting.

A NOTE ON TERMINOLOGY

Terms that have become part of the common currency of debate about food biotechnology do not always have a unequivocal meaning. Sometimes, the resulting ambiguity is used as a subtle tool in favour of one side or other of the debate. For example, a frequently heard argument against those who question the health or environmental safety of biotechnology is that “There is nothing new about biotechnology — human beings have been using it for centuries in the cultivation of yeasts and cultures, and in the selective breeding and hybridization of plants and animals.” The force of this claim depends, of course, upon a definitional stipulation that “biotechnology” refers broadly to any technique for shaping the genetic characteristics of organisms, as well as a further assumption that new recombinant DNA techniques are no different in character or consequence from the traditional techniques. The latter, of course, is not a question that can be decided by an a priori definition, but only by empirical investigation. [1]

The Expert Panel sought to avoid a priori linguistic solutions to substantive issues. A primary substantive issue in the food biotechnology debate, and in the mandate of this Panel, is that of whether the new recombinant DNA technologies pose unique issues and risks requiring special regulatory expertise and techniques. We, therefore, simply state at the outset how we were using some of the key terms in the debate, so as to lend the greatest clarity and consistency to our discussion. In doing so, we do not presume to be prescribing the proper use of these terms or to be describing their most common usage. Since one of the important questions involved in the assessment of the potential hazards of these products and techniques is that of how they differ, if at all, from traditional means of modifying the genetic character of organisms, the Panel found it necessary at points to evaluate the new technologies against the traditional ones. In order to make this project transparent, we needed to adopt clear terms that refer to the different techniques.

For the purposes of this Report, therefore, the Panel uses the terms “Genetic Engineering”, “Genetic Modification” and “Biotechnology” as fully synonymous terms, referring exclusively to the direct transfer or modification of genetic material using recombinant DNA techniques. They do not refer to other traditional breeding and hybridization techniques not involving these techniques. Although “Biotechnology” and “Genetic Modification” are both sometimes used to refer to all techniques of genetic modification, as defined above, we use them in the more narrow sense, assuming that this is the primary concern of both our sponsors and the general public with respect to the regulation of food biotechnology. Accordingly, in this Report, references to “Genetically Modified Organisms (GMOs)”, “Genetically Engineered (GE) Foods”, or “Transgenic Plants or Foods” are always only references to the products of rDNA technologies. References to non-rDNA techniques are referred to in this Report with such terms as “selective breeding”, “artificial selection”, “hybridization” and “traditional animal/plant breeding”.

REFERENCES

Heinberg, R. 1999. Cloning the Buddha: The Moral Impact of Biotechnology. Wheaton, IL: Quest Books.

Mayo, Deborah G., Rachelle D. Hollander (eds.). 1991. Acceptable Evidence: Science and Values in Risk Management. Oxford: Oxford University Press.

Salter, L., E. Levy, W. Leiss. 1988. Mandated Science. Science and Scientists in the Making of Standards. Boston: Kluwer Academic Publishers.

Shrader-Frechette, K.S. 1991. Risk and Rationality. Berkeley: University of California Press.

NOTE

1. The Convention on Biological Diversity (1992), for example, defines “Biotechnology” as “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.”
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Re: ELEMENTS OF PRECAUTION: RECOMMENDATIONS FOR THE REGULATI

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2. THE PAST, THE PRESENT AND THE FUTURE

INTRODUCTION


Genetically modified (GM) plants have already entered the food stream in many parts of the world, and large increases in acreage for a few GM crops have been observed over the last five years. However, the current generation of genetically modified organisms (GMOs) consists mostly of plants modified for a handful of traits. With the expected availability of genomic information for many species in the next few years, the floodgates of genetic modifications could open and release on the market an unprecedented variety of genetically enhanced products. In parallel with this rapid market penetration, there is increasing concern about the use of genetic engineering for food production, particularly about possible deleterious effects on human health and about the possible impacts of the widespread deployment of GMOs in the environment.

The economic stakes of agricultural biotechnology for Canada are high. We are a net exporter of agricultural products, and 26% of Canada’s biotechnology companies focus on the development of agriculture and agri-food products. It is estimated that the global market for biotechnology applications will reach $50 billion annually by 2005 (Sector Competitiveness Frameworks. Bio-Industries: Part I Overview and Prospects, Bio-Industries Branch, Industry Sector, Industry Canada, March 1997), and the strongest growth is projected for the agri-food sector.

In this chapter, we examine the historical roots of GMO technology, survey its present uses in the areas of crop plants, microbes, fish, and farm animals, and make some forecasts concerning the directions this suite of technologies is likely to follow.

THE ORIGINS OF GENETIC ENGINEERING

Various species of microbes (bacteria and fungi) have for decades been modified for increased production of proteins, amino acids and commodity chemicals. Early work in this area relied primarily on discovery of naturally occurring or mutagenesis-induced variant microbial strains. Often these variant genotypes were blocked in specific metabolic pathways, or they expressed higher levels of a key rate-limiting enzyme, with the result that their metabolic output was being channelled into the desired product. Such mutant strains provided valuable biological tools for researchers, and for the fermentation industry they also represented a key commercial asset.

As our understanding of microbial metabolism expanded, the detailed structure of the pathways of interest to the fermentation industry was slowly uncovered. Many of the biosynthetic enzymes involved were identified, and the genes encoding those enzymes were eventually isolated. Seminal to the development of genetic engineering was the discovery in the 1970s that different DNA fragments can be assembled to form new human-made DNA molecules. In 1972, a team led by Paul Berg at Stanford University used restriction enzymes to cut two DNA molecules from two different sources. They then spliced these two foreign pieces together to form a functional hybrid DNA molecule. This new molecule is referred to as recombinant DNA. Genetically modified (or genetically engineered) organisms are made of cells which contain a recombinant DNA (rDNA) molecule.

With the development of DNA manipulation techniques, it became possible to build on the knowledge of microbial biology and to create engineered microbes artificially, through direct insertion of modified genes into a desired strain, or through replacement of an existing gene. While more predictable than screening mutagenized populations, and thus potentially a more rapid path to the desired genotype, this approach had the commercial disadvantage of being accessible to anyone who had the appropriate background knowledge and training. If a competitor could create the same engineered strain within a short time, the initial developer of a new strain would have gained comparatively little commercial advantage.

The situation changed in 1980 with the U.S. Supreme Court decision (Diamond v Chakrabarty), which granted a patent for a GM bacterial strain specifically engineered to break down petroleum residues. This extended the legal definition of intellectual property (IP) as it provided patent protection for the first time to living organisms. In Canada, a recent decision of the Supreme Court (President and Fellows of Harvard College v. Commissioner of Patents (2000), A-334-98, Fd. Court Appeal) also paved the way for the patenting of life forms in this country. Newly engineered microbial strains thus moved from simply being trade secrets to forming part of their “owners” IP portfolio, to be traded, sold or protected by litigation, as necessary. These patented strains now comprise a vast array of “micro-reactors” whose industrial products range from amino acids, antibiotics and insulin to enzymes and alcohols.

OUR FOOD PRODUCTION SYSTEM RELIES ON FEW GENETICALLY SELECTED SPECIES

The limited array of plant and animal species that humans rely upon for most of our food supply have been genetically selected in order to improve their performance and quality. In this case, however, the process has taken place over millennia. In addition, the process, at least in its early stages, was not systematic. Nevertheless, cultivar development is essentially analogous to microbial strain improvement that has been exploited so successfully by the fermentation industry in this century. Natural mutations (and later, induced mutations) were visually identified in the wild or cultivated populations in which they occurred, and selected on the basis of their more desirable properties (better flavour, easier harvest, larger size, etc.). Only in the last hundred years was this process placed on a scientific footing, with the discovery of the genetic basis of heritable traits. An understanding of these underlying genetic mechanisms enabled plant and animal breeders to move and combine desirable traits in a much more systematic fashion, with the result that gains in food production attributable to genetic improvement in agriculture soared in the 20th century. As a result of these efforts, there are virtually no food products on supermarket shelves that have not been improved by plant breeders (fiddleheads and wild blueberries are examples of a few remaining unimproved food plants). Plant and animal breeding have contributed enormously to our current standard of living by ensuring a generally abundant and nutritious food supply, but the plants we consume daily are significantly different from their original wild forms.

To create new plant varieties, breeders have relied on making sexual crosses between individuals that possess desirable characteristics. They then examine the progeny from these parents, looking for individuals that combine as many of the favourable characteristics as possible from each of the parents. Several such selected individuals will typically be crossed with other genotypes, or self-fertilized, to create further progeny generations, each of which will be tested for performance and quality. Nevertheless, conventional plant breeding that relies upon pollen transfer has remained a relatively slow process, and one that depends on chance for the creation of assortments of improved allelic combinations. Each cycle of improvement in a given species usually requires carrying out large numbers of controlled crosses between promising parental types, and years of work to select and evaluate the resulting progeny.

In a few cases, the products of classical breeding methods have generated products with undesirable effects on human health. There are two examples of potato varieties that were conventionally bred, but had to be withdrawn because of unacceptably high levels of glycoalkaloids. The first is the Lenape variety, which was bred from a wide cross between Solanum tuberosum and S. chacoense; it was never released for commercial purposes (Zitnak and Johnston, 1970). The second variety had been released on the market in Sweden, but was later withdrawn (Hellenas et al., 1995). An analogous problem was detected with a celery line that was bred and almost released for commercial purposes. It was found to induce contact dermatitis in field workers and chemical analysis showed that high levels of furanocoumarins were accumulating in this genotype (Trumble et al., 1990)

DIRECT GENE TRANSFER WITHIN AND BETWEEN SPECIES

Because DNA has fundamentally the same gene-coding properties whether it comes from bacteria, salmon or plants, the same molecular biology tools that have enabled extensive gene modification in microbes have been applied to the isolation and manipulation of plant and animal genes. However, moving modified genes efficiently into plant or animal genomes is much more difficult than the corresponding manipulations of microbial genomes. Research into the nature of a common plant disease in the late 1970s led to the discovery of a naturally occurring gene transfer system for plant systems. The bacterial pathogen that causes “crown gall” disease on many plants, Agrobacterium tumefaciens, is able to successfully colonize its host plant because it can transfer a small set of its own genes directly and permanently into the host plant genome (i.e. it transforms part of the plant) (reviewed by Nester et al., 1984). Once established in the plant genome, these bacterial genes take over part of the metabolism of the infected cell and redirect it in a way that provides shelter (the visible gall) and sustenance specifically for the invading bacteria.

The initial report of this natural gene transfer inspired a wave of related research, out of which came the discovery that the Agrobacterium gene transfer process was largely insensitive to the nature of the genes being transferred. As long as a few key portions of the transferred Agrobacterium DNA (T-DNA) were included, the gene transfer process was capable of inserting into the plant cell genome any other “piggybacking” DNA (Chiton et al., 1980). This could include other genes obtained from plants, animals or microbes. In effect, researchers found that it was possible to “hijack” the Agrobacterium system and develop it as a vehicle for transferring new genes into plants.

One limitation of the Agrobacterium gene transfer system is the fact that Agrobacterium is not equally enthusiastic about infecting all species of plants. Large groups of commercially important plants, notably the cereal grains and conifers, are not hosts for Agrobacterium and the gene transfer system therefore does not work well in these plants. It appears, however, that while plant transformation using Agrobacterium is an efficient process in some plants, the actual incorporation of foreign DNA into a plant genome does not absolutely require this bacterial gene transfer system. Simply coating the foreign DNA onto microprojectiles (e.g. tiny gold beads), and blasting these into living plant cells at high velocities, will also work (Paskowski et al., 1984). The DNA coat is presumed to leach off the microprojectile surface once it is inside the recipient cell, and a small fraction of the DNA becomes incorporated into the cell’s genome through a largely unknown process. This gene transfer technique has a much lower efficiency than does Agrobacterium-mediated gene transfer, and the incorporated DNA sequence has often been reorganized by the time it is stably inserted into the plant genome (Kohli et al., 1998). Nevertheless, the “gene gun” method has one advantage – it will, in principle, allow any plant species to be transformed, including those that are not suitable hosts for Agrobacterium.

SELECTING A TRANSFORMED PLANT

Both the Agrobacterium and “gene gun” methods are capable only of transforming a very small percentage of all the cells in the piece of plant tissue being treated. In order to create a transformed plant made only of cells carrying the new gene, two further steps must be successfully completed. First, a plant must be regenerated that is solely derived from one or more of the original transformed cells and, second, in this process all non-transformed cells must be eliminated.

Plant regeneration (i.e. the process of generating a full-size plant from a single cell) often proves to be more difficult than the actual gene transfer process itself. While the latter technologies are now routine, our understanding of the process of plant regeneration remains largely empirical. Even different varieties of a plant species often differ drastically in their ability to be regenerated from small starting tissue pieces (see e.g. Puddephat et al., 1996), so that procedures need to be customized for each new genotype of interest.

Elimination of untransformed cells is normally accomplished by adding a second gene called a selectable marker to the transferred DNA. The selectable marker gene typically encodes an enzyme that will be expressed in every transformed cell, and will confer on that cell the ability to survive in the presence of a selection agent (a chemical capable of killing plant cells). This selection agent can be an antibiotic, herbicide or other anti-metabolite. If the selection agent is an antibiotic, for example, the selectable marker gene might encode an enzyme that is designed to destroy that type of antibiotic and thus allow the cell to avoid being poisoned. As a consequence of the metabolic protection provided by the selectable marker gene, when the treated plant tissue is placed on a growth medium containing the selection agent only those cells that have been transformed will survive, while non-transformed cells will die. This selection process is often combined with the regeneration process, so that the only regenerated plants recovered are those that arose from transformed cells. Since all the cells in the regenerated plant are ultimately derived from that one progenitor cell, and their genes (including the new transgene(s)) are duplicated and shared at each cell division, the transgene(s) will now be present in every cell of the new transgenic plant. In the first generation of commercial GMO crops, the new genes inserted into the plant have always included a selectable marker gene, most commonly either an antibiotic resistance gene or a herbicide resistance gene.

The insertion of single genes into plant genomes using either the Agrobacterium or gene gun procedures is now a routine laboratory procedure, and the earliest commercial products of crop genetic engineering have been derived from insertion of single transgenes into important crop species. However, it should be emphasized that the initial population of transformed plants created in the laboratory by these methods is far from homogeneous. Both of the common gene transfer techniques lead to near-random insertion events (i.e. the location of the new gene within the recipient genome cannot be predicted) (Kohli et al., 1998). Therefore, each transformed individual will carry the transgene at a different location within its genome. In many cases, they will carry multiple copies of the transgene, some of which will be functional while others may not be.

Sorting through the transgenic population and identifying those individuals that appear to express the transgene in an appropriate and useful manner requires considerable time, effort and expertise. Eventually, a limited number of lines that display the desired trait in the laboratory or greenhouse trials in a stable manner will be chosen for more extensive testing and analysis, including field trials for a number of years at multiple locations. The latter program is very similar to the evaluation process by which new crop varieties generated through conventional breeding are assessed for their ability to perform better than existing varieties, but field trials for transgenic varieties in Canada require formal approvals from the relevant regulatory agencies (see Chapter 3).

It is important to note that the utility of this process of gene transfer (genetic engineering) is largely dependent on its integration into conventional breeding programs, where it can provide a source of genetic variation. The agronomic acceptability of a transgenic variety thus derives in large part from the quality of the parental germplasm into which the transgene has been incorporated. Since the success of any crop breeding program in creating highly selected and well-adapted breeding lines is based on having access to a wide range of genetic resources, it is a key priority for plant breeders to ensure that a high level of genetic diversity is maintained in the species of interest, and its relatives.

CURRENT PRODUCTS AND FUTURE DEVELOPMENTS

GM Plants


The three types of GMO crops that first received approval for commercial release in Canada were all designed to address field-level problems faced by growers of large-scale field crops. Herbicide-resistant crops have been promoted as tools to potentially simplify weed control over large monoculture plantings and permit growers to use herbicides less damaging for the environment. Insect damage control through use of Bt gene-containing crop varieties (Bt is derived from a natural insecticide produced by bacteria) have also been promoted as tools to allow farmers (for some crops in some regions) to reduce the number of applications of pesticides. Virus-resistant crops may decrease the need for pesticides for control of insects that transmit the virus from plant to plant. The rapid adoption of GMO crop varieties by growers in Canada can be interpreted as evidence that these first-generation products have provided positive financial and/or management outcomes for the farmer.

The very first GMO varieties approved for commercial release represent the initial output of a development and testing process that usually takes at least five years to complete. The large majority (92%; Ferber, 1999) of GM crops planted in 1999 were modified for only two characteristics: either herbicide resistance or insect resistance. While there are thus relatively few functionally proven genes available for plant genetic engineering, there are dozens of candidate products at various stages in the development “pipeline”. The DNA sequence of the first plant genome to be fully characterized has recently been completed (Arabidopsis Genome Initiative, 2000), and the genome of more agriculturally relevant species are expected in the coming few years. Knowledge of these complete DNA sequences will accelerate the identification of the function of many more genes, and concurrent applications of that knowledge in crop improvement.

Canada is the third largest grower of GM crops in the world (behind the US and Argentina). Canadian food safety approvals have been granted for at least 45 plants with novel traits, including canola, corn, tomato, potato, soybean, cottonseed and squash (CFIA, 2000). The number of different plant-transgene combinations tested in field trials continues to increase: 178 submissions for field trials were made in 2000 versus 40 in 1990. However, many of these products are essentially variants on the initial introductions. Since specific crop varieties are often better adapted to different soil, climate and pest situations, they will usually perform best in specific conditions. A transgene whose addition to the genome can superimpose a useful new trait can therefore be moved relatively easily into an array of agronomically well-adapted genotypes, either by breeding or by transformation of the relevant existing varieties. In this way, genetic engineers can take advantage of the classical breeding efforts that created the well-adapted lines in the first place. A substantial part of the second wave of GMO products thus consists of a wider range of crop varieties carrying herbicide resistance, Bt or anti-viral transgenes, or a combination of these.

However, other GMO traits are also beginning to reach the commercial release stage. Some of these are intended to directly address grower concerns, such as transgenes that confer resistance to fungal or bacterial disease, increased nematode resistance, enhanced frost tolerance or increased photosynthetic efficiency (CFIA, 1999). Other transgenes may modify plant fertility in specific ways that greatly simplify the production of hybrid seed, thus allowing farmers to benefit from the productivity gains associated with hybrid vigour (CFIA, 1999). Future developments will likely include varieties improved for altered flowering time, modified edible oil profiles, increased productivity, enhanced disease resistance, increased resistance to environmental stresses and improved product quality. Plants may be used for the production of compounds with a variety of uses, from pharmaceuticals to precursors of plastics.

Noticeably absent from the first generation of GM crops have been varieties that bring direct consumer benefits. It is thus ironic that the first plant product derived from biotechnology to be put on the supermarket shelves in the US was the Flavr-Savr tomato, marketed in the US in 1985 by the company Calgene. This tomato variety was created to satisfy consumer demand for a flavourful product year-round. By increasing firmness of the fruit, the tomato could be left to ripen on the vine and still be transported to market without the losses associated with a soft ripe tomato. Firmness was increased by genetically reducing the activity of an enzyme (polygalacturonase) involved in fruit softening. However, the Flavr-Savr variety was not a commercial success, and most agbiotech companies focused their initial efforts on the major American field crops, notably corn, cotton and soybeans.

Few transgenic plants currently contain more than two or three genes. A number of transgene combinations are in trials, where traits such as herbicide resistance and fertility management are “stacked” in one variety. However, most scientists agree that many important crop plant characteristics result from the combined action of many genes, sometimes as many as several dozens. Current gene transfer techniques tend to be limited in the size of the new DNA they can efficiently insert into the recipient. These limits are likely to be overcome in the near future, with the advent of new systems for transfer of very large DNA sequences, up to the size of partial or full chromosomes (Hamilton et al., 1996; Wordragen et al., 1997). However, it remains to be established whether rational design of such large gene combinations can create effective and predictable new biological functions in a transgenic plant.

Further back in the GMO pipeline can be found a much wider array of products, some of which are intended to directly address consumer preferences. These include food crops with controlled ripening, altered flower colour, increased protein content, reduced allergenicity, non-bruising and higher vitamin and mineral content. An example is the introduction of genes that produce beta-carotene (the precursor of vitamin A) in rice. The resulting “golden rice” potentially contains sufficient beta-carotene to meet human vitamin A requirements from rice alone (Ye et al., 2000).

There is also great commercial interest in the use of transgenic plants to produce industrial enzymes, pharmaceutical peptides, vaccines and other proteins of pharmaceutical interest (“molecular pharming”). For example, the enzyme lysozyme, which was previously isolated from excess egg whites, can now be produced at a far lower cost as a recombinant protein in transgenic corn.

Finally, the potential now exists to replace many microbially derived animal feed additives in current use with plants that have been GM to directly enhance the animal’s feed supply. For example, a good supply of sulphur-containing amino acids is important for wool-producing ruminant animals. To address this need, it may be possible to express in a transgenic forage crop a novel protein of particularly high cysteine content.

All of these developments are the result of insertion of genes that either express new proteins (and thus new enzymatic properties), or express a “silencing” version of an existing gene in the transgenic plant that is able to reduce the effect of the native gene. Other changes in transgenes under development include the use of selectable markers that are not based on antibiotic resistance genes (e.g. Kunkel et al., 1999), and the use of transgene constructions that allow the selectable marker to be either functionally silenced once it has performed its task during the gene transfer process, or entirely deleted from the transgenic plants (Zubko et al., 2000).

The first generation transgenic crops almost all use a strong viral gene promoter to ensure that high levels of gene effect are created in the plant. However, this promoter typically induces constant gene expression in all parts of the plant. More sophisticated gene control mechanisms are now being tested which allow the transgene to be expressed only in specific tissues of the GMO plant, or at specific times in the plant’s life cycle. This capability will allow the transgene product to be targeted to the tissue where it is maximally effective, and suppresses gene product accumulation in other tissues or at other times. This could reduce internal and external collateral impacts (e.g. Bt toxin production is not needed in pollen and can create negative effects in the biosphere), and could also reduce the metabolic cost to the plant of having to accumulate products unnecessarily. Other gene control systems (inducible promoters) force the transgene to remain silent until the plant is subjected to particular treatments (e.g. sprayed with an inducer chemical) or growth condition (e.g. drought, frost, insect feeding) (e.g. Zuo and Chua, 2000).

Plant genetic engineering is presently used almost exclusively to place new genes into plant genomes with the intent of adding a novel genetic capability to the plant, or increasing or decreasing the activity of a pre-existing gene in the plant. The largely random nature of transgene insertions associated with current methods makes it impractical to consider directed gene replacement (i.e. specifically replacing an existing gene with a modified incoming version of that gene). However, it is clear that the technology is developing rapidly to allow targeted gene insertions, which will allow for more subtle changes in a plant’s genotype. For instance, altering a single amino acid in a protein sequence can have a marked effect on the cellular function of that protein, and thus produce significant changes in metabolism or physiology. To engineer such an alteration of the resident copy of a gene requires changing only one or two bases in the DNA structure of the existing gene, a technically demanding process that will nevertheless probably become feasible within the next 10 years. Replacement of an existing gene with an introduced engineered gene (homologous recombination) has already been achieved on an experimental basis in Arabidopsis while the use of mutagenic oligonucleotides has been shown to create targeted single base changes in plant DNA (Beetham et al., 1999; Zhu et al., 1999), albeit with low efficiencies in both cases.

These approaches allow genetic changes to be made on the resident copy of existing genes, as in many naturally occurring mutations. No new functional genetic elements (e.g. transgenes) are thus introduced into the genome, and since the existing gene control elements remain unaltered, no novel gene promoters have to be incorporated.

GM Microbes

As discussed earlier, genetically engineered microbial strains are already a component of current fermentation technologies. Enrichment, isolation and modification of naturally occurring microorganisms (“bioprospecting”) will likely continue to be a source of biological material for production of enzymes, industrial chemicals and pharmaceuticals, as well as a source of novel genetic material. In agriculture, amino acid supplements (e.g. lysine, threonine and tryptophan) and many of the enzymes used to enhance the nutritive value of animal feeds (e.g. phytase, ßglucanase, arabinoxylanase, proteinase, cellulase) are produced by fermentation, often with GM organisms. Live, GM bacteria and their products can also be used in feed harvest, storage and processing. For example, GM Lactobacillus sp. are used in silage production to control the aerobic and anaerobic phases of fermentation.

In a variation of the bioprospecting approach, methods have been developed for rapid screening of DNA randomly isolated and cloned directly from environmental samples without prior isolation of the organisms. Since only a small fraction of microbial species in natural environments appear to be culturable, this direct sampling can provide an array of genetic material for biotechnological applications.

While individual optimized microbial strains can be extremely useful, defined microbial consortia, containing two or more known species or strains of microorganisms, could offer greater potential. Such consortia are already applied under defined conditions in industrial processes such as dairy product manufacturing, but a wider suite of applications is likely in the future. Increased emphasis on recycling and reusing waste products of various industries and the residential waste stream could result in treatment processes based on the activities of defined, engineered microbial consortia. Examples may include upgrading or modification of wood wastes derived from forestry and pulp and paper manufacturing, petroleum refining byproducts, agricultural wastes and mining wastes.

Undefined microbial consortia are also used extensively, and could be engineered using gene transfer. “Community engineering” of this sort is currently done using donor strains to introduce mobile genetic elements (plasmids and/or transposons) containing specific genes or operons into microbial communities. This approach allows biotechnologists to modify community function in a way that mimics the natural processes of gene exchange that occur when microbial communities are selected for particular functions using traditional enrichment methods.

Plant–microbe interactions have long been exploited for enhanced agricultural production. Plant growth-promoting rhizobacteria and mycorrhizae are presently used as seed or root inoculants to enhance plant growth. Forest tree mycorrhizae and mycorrhizae-bacteria associations are also gaining use. Future developments are likely to include the use of engineered microbial inoculants with improved ability to enhance growth, nutrient and water absorption, and stress tolerance. Since these undefined communities of microorganisms interact intimately with host plant tissues, the process of genetic modification could involve either the plant or the bacteria that are attracted to these root cells during plant growth, or both (O’Connell et al., 1996). For example, engineering the plant to produce specific chemoattractants and/or growth substrates in root exudates, and at the same time engineering bacteria to specifically recognize these signals and/or grow in response to them, would allow researchers to establish a defined plant–microbe interaction in the soil. Many uses of these engineered symbioses can be envisioned:

• enhancing phytoremediation of toxic organic chemicals and metals,
• controlling gene expression in rhizosphere bacteria by engineering plants to release specific effectors,
• bolstering the presence of disease-suppressive microbial populations, or
• enhancing nutrient uptake by encouraging the growth of microorganisms that mobilize and absorb nutrients such as phosphate or nitrate.

Another undefined microbial community important to food production exists in the rumen of some major farm animal species (e.g. cattle). The introduction of the tetracycline-resistant TcR a gene into Prevotella ruminicola was the first transfer of a gene into rumen bacteria (Flint et al., 1988). Since then, gene transfer has been used to introduce cellulase activity into a number of hind-gut bacteria, acid tolerance into cellulolytic rumen bacteria, improved protein (essential amino acid) yield by rumen bacteria and hydrogen scavenging to reduce methanogenesis in rumen bacteria. The current limitation to this technology appears to be ability of the GM organism to become successfully established in the natural rumen or hind-gut environment.

GM Animals

Animal cells generally lack the totipotency of plant cells (i.e. it is not possible to regenerate a fully differentiated animal from a single somatic cell). This precludes the use of low frequency transformation methods that rely upon chemical selection and regeneration. Researchers have therefore relied primarily on direct injection of new DNA into the nuclei of the host organism at a very early stage of development, a procedure that is both technically demanding and limited in its through-put. However, the recent development of methods of somatic cell nuclear transfer, and the production of clones from these somatic cells for livestock species, indicates that the limitations of pronuclear microinjection for the production of GM farm animals may soon be overcome.

The context for technology development in animal production systems has also been different than the situation in plants. Reproduction technologies in large animals are less developed or inefficient compared to plants. Trait manipulation will require a more complete understanding of the genetic basis of animal biology than is presently available. In addition, with the exception of fish and poultry, populations of animals tend to be replaced slowly, and distribution channels of genetic material are more local and diffuse. These characteristics restrict the potential for the rapid and large-scale market penetration by GM genotypes, such as has occurred in crop systems. Industry investment is limited both by this, and by the challenge of maintaining control of any modified germplasm they may create, since there is no equivalent to Plant Breeders Rights in the animal industry. This latter issue may change, however, with advances in genetic marker technologies that will allow precise genotyping and identification, and the recent Supreme Court of Canada decision that allowed patenting of animal life forms.

Fish

Work on transgenic fish has focused on the development of enhanced phenotypes for the aquaculture industry, the study of gene regulation and function, developmental genetics, and the use of animals for production of human hormones such as insulin.

Research on transgenic fish has occurred at a very rapid pace. Beginning with the first report of a transgenic fish in 1985 (Zhu et al., 1985), 13 species had been GM for purposes related to food production and scientific study by the late 1980s (Kapuscinski and Hallerman, 1991), 17 by the mid 1990s (Sin, 1997), and as many as 35 in 2000 (Table 1; Devlin, 2000; Reichhardt, 2000). The first application to be made in North America for the commercial production of a transgenic fish (growth-enhanced Atlantic salmon, Salmo salar) was made in early 2000 in the United States (Niiler, 2000).

Means of introducing transgenes into fish

The method most commonly employed to introduce novel gene constructs into fish is microinjection of the transgene into the cytoplasm of the developing embryo (MacLean and Rahman, 1994). Millions of copies of the transgene are injected as soon as possible after fertilization, usually at the one- or two-cell stage. Because of the large size of their nuclei, unfertilized oocytes of the medaka (Oryzia latipes) have been microinjected directly into the nucleus (Matsumoto et al., 1992). Based on a broad examination of work on genetically engineered salmonids, Devlin (1997) found that retention rates of micro-injected novel DNA by recipient fish can be as high as 50% among individuals that have recently resorbed their yolk sac, declining significantly to rates of 1% to 5% among individuals 6 to 12 months of age. Novel genes can also be introduced into fish via electroporation, a procedure in which fertilized eggs, and occasionally sperm (Sin et al., 1993), are immersed in a solution containing foreign DNA and then subjected to electric pulses (Inoue and Yamashita, 1997). The likelihood of successful transfer of foreign DNA by electroporation is typically very low, although it has been reported to be as high as 7% in surviving embryos (Inoue and Yamashita, 1997). Gene constructs can also be introduced via transfection of novel genes into embryonic stem cells, followed by their reintroduction into the inner cell mass of the developing embryo. Although this method may allow for precise manipulation of host genes, embryonic stem cell research in fish is still in its early stages (Devlin, 1997).

Table 1. Examples of fish that have been successfully genetically engineered.

Species / Reference

rainbow trout (Oncorhynchus mykiss) cutthroat trout (O.clarki) chinook salmon (O. tshawytscha) coho salmon (O. kisutch) Atlantic salmon (Salmo salar) brown trout (S. trutta) Arctic char (Salvelinus alpinus) African catfish (Clarias gariepinus) channel catfish (Ictalurus punctatus) Indian catfish (Heteropneustes fossilis) Japanese medaka (Oryzias latipes) zebrafish (Danio rerio) common carp (Cyprinus carpio) tilapia (Oreochromis niloticus) northern pike (Esox lucius) goldfish (Carasius auratus) silver crucian carp (C. auratus linda) red crucian carp (C. auratus auratus) mud carp (Cirrhinus chinensis) wuchang bream (Megalobrama amblycephala) loach (Misgurnus anguillicaudatus) mud loach (M. mizolepis) gilthead seabream (Sparus auratus) blackhead bream (Acanthopagrus schlegli) largemouth bass (Micropterus salmoides) striped bass (Morone americanus) killifish (Fundulus sp.) walleye (Stizostedion vitreum) / Chourrout et al. (1986) Devlin (1997) Devlin (1997) Devlin et al. (1994a) Fletcher et al. (1988) Sin (1997) Pitkanen et al. (1999) Müller et al. (1992) Dunham et al. (1987) Sheela et al. (1999) Inoue et al. (1990) Stuart et al. (1988) Chen et al. (1993) Brem et al. (1988) Gross et al. (1992) Zhu et al. (1985) MacLean et al. (1987) Sin (1997) MacLean et al. (1987) MacLean et al. (1987) Zhu et al. (1986) Nam et al. (2000) Knibb (1997) Sin (1997) Goldburg (1998) Goldburg (1998) Khoo (1995) Khoo (1995)


Development of growth hormone gene constructs for commercial food production

Initial research on transgenic fish in Canada focused on the transfer of an antifreeze protein gene from marine fish (e.g. winter flounder, Pseudopleuronectes americanus) to a commercially viable fish in the aquaculture industry, Atlantic salmon (Salmo salar) (Fletcher et al., 1988; Shears et al., 1991). Although the expansion of salmon aquaculture to the cold, coastal waters of the Northwest Atlantic remains one goal, most aquaculture-related research on transgenic fish has focused on the use of gene constructs to promote unregulated growth (Devlin, 1997). The commercial motivation for this work lies in the significantly reduced period of time required to rear fish to market size. The increase in growth rates achieved by transgenic fish (typically 200% to 600%, depending on the species) greatly exceeds the 5% to 10%, one-generation increases commonly achieved by artificial selection (Dunham and Devlin, 1999). Despite these large increases in growth rate, these transgenic fish do not attain final sizes greater than those achieved by non-transgenic fish. The transfer and expression of gene constructs to promote unregulated growth has now been reported for at least 15 species of fish (Dunham and Devlin, 1999; Pinkert and Murray, 1999). In Canada, research on growth hormone gene constructs has focused almost entirely on salmonids, notably Atlantic and Pacific salmon.

Future applications

Given the recent application for a GM, growth-enhanced Atlantic salmon in the United States, it is reasonable to expect that a similar application to CFIA for a growth-enhanced salmon will be forthcoming. There are, however, several other genes or gene products that have been, or are likely to be, the focus of research on genetically engineered fish in aquaculture. Novel gene constructs in fish that may form part of an application to CFIA during the next 10 years could include genes that:

1. cause over-expression of hormones such as prolactin that are involved in the transformation of anadromous fish from salt to fresh water, thereby making it theoretically possible to raise marine fish in fresh water;

2. change the pattern of expression of gonadotropin genes to allow for manipulation of the length of reproductive cycles;

3. expand the tolerance of aquaculture fish to wider ranges of environmental conditions;

4. modify the biochemical characteristics of the flesh to enhance nutritional and/or organoleptic qualities;

5. improve host resistance to a variety of pathogens;

6. control sexual maturation to prevent carcass deterioration near the end of the life cycle in Pacific salmon;

7. control sex differentiation and sterility; and

8. enable fish to use plants as a source of protein.

Shellfish and Aquatic Plants

Research on transgenic shellfish (e.g. mussels, abalone, clams) and aquatic plants is less developed than that on transgenic fish. The first successful gene transfer in a bivalve mollusc was the introduction of retroviral vectors into the dwarf surf clam (Mulinia lateralis) (Lu et al., 1996). Another species in which considerable research has been undertaken is the Japanese abalone (Haliotis diversicolor) into which growth hormone (Powers et al., 1997) and other gene constructs (Tsai et al., 1997) have been introduced. Gene constructs have also been introduced into Pacific oyster (Cadoret et al., 1997). In marine plants, there is a report (Kuebler et al., 1994) of transfer of a reporter gene into the protoplasts of Porphyra miniata, a commercially important red algae in southeast Asia.

The temporal lag in research on transgenic shellfish and marine plants will almost certainly translate into a similar lag in the time that will elapse before approval is sought from CFIA for the commercial production of GM shellfish or algae. Despite this lag, it is not improbable that a request will be made to CFIA within the next 10 years.

Farm Animals

Over the next five to ten years, much of the biotechnology research and development will be driven by corporate strategies to capture the potential economic value of transgenic technology for increased growth rate and altered carcass composition in meat-producing animals and compositional modification of milk and eggs.

A critical requirement to realize commercial application of genetic modifications, particularly for traits like fertility and disease resistance that are controlled by many genes, is the development of better genetic tools. Genomic analysis technologies have recently become integrated into research on all livestock species. Once the information (i.e. identity of genomic regions that encode quantitative trait loci of economic importance) and technologies (e.g. cell culture-based transgenesis) are in place, there is little doubt that breeding companies will be in a position to offer animals bred from proprietary germplasm. Such animals will have traits conferring production efficiency, or will in some way meet consumer demand, for example, by offering improved nutritional value.

Another potential application of transgenic technology in livestock production is to increase the safety of animal products for human consumption through strategies that might increase disease resistance. Genetic modifications could reduce product susceptibility to spoilage or bacterial contamination. The recent demonstration in mice, using a gene knockout strategy, of the inactivation of the prion gene involved in transmissible spongiform encephalopathies (TSE) (Flechsig et al., 2000), raises the possibility that similar genetic modifications may be achieved in livestock species to reduce their susceptibility to specific diseases (e.g. to prevent scrapie in sheep).

NEED FOR A BROADER RESEARCH AGENDA

Agricultural biotechnology is an input industry where products are developed and priced to cover the costs of research and development. Many argue that conversion from industrial agriculture to more sustainable systems that depend less on chemicals for their productivity would eliminate the need for some of the currently projected products of biotechnology. There are probably alternatives to some biotechnology products; many of these alternatives are likely not other products, but instead the systems and methods of sustainable agriculture. It seems likely that much more research and discussion will be required to enable society to make informed choices between these alternative approaches to food production. This exploration will need to address both societal concerns about how food is produced, and assessment of “global” (or societal) costs of the choices to be made.

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Re: ELEMENTS OF PRECAUTION: RECOMMENDATIONS FOR THE REGULATI

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3. THE REGULATORY ENVIRONMENT

INTRODUCTION


Immediately following the discovery of recombinant DNA techniques, concern was expressed within the scientific community about the potential of these technologies for creating unpredictable risks to humans or the environment. The initial response, arising from a gathering of scientific experts at Asilomar, California (Berg et al., 1975), was a self-imposed moratorium on extension of the technologies until the associated risks could be better assessed. During the period of this moratorium, extreme caution was employed in creating and handling recombinant microorganisms, and numerous studies examined their potential for presenting unanticipated phenotypes, and for modifying and transmitting the recombinant genomes.

As evidence accumulated that these organisms, while novel, could be managed and controlled using the same well-established procedures used for safely handling naturally occurring microorganisms, restrictions on contained uses of recombinant microbes gradually eased. Governments at various levels developed regulations designed to ensure public safety, based on extensive knowledge of the characteristics and behaviours of recombinant DNA in microbial systems. The resulting local and international regulatory environments have now operated successfully for over two decades, and have allowed commercial exploitation of the power of this technology in the fermentation industry, as described in Chapter 2. It is worth noting, however, that environmental release of GM microbes remains severely restricted.

With the more recent development of transgenic technology for plants and animals, a new set of challenges has faced government regulatory agencies. Compared to most microbes, crop plants, farm animals and fish are much more complex organisms, and they are generally produced and maintained directly in the outdoor environment, rather than in a confined laboratory or fermentor tank. They are also more recognizably part of both the human visual landscape and our food supply system. As governments have struggled to deal with these challenges, themes such as “substantial equivalence” and the “precautionary principle” have come to dominate the debate, and these are explored more fully in Chapters 7 and 8 of the Report. In this chapter, we present a brief overview of the current regulatory environment in Canada, both as a reference point for the Report and to help highlight some of the problems encountered in regulation of GM products.

CANADIAN REGULATION OF FOOD BIOTECHNOLOGY

Overview


In Canada, a GM product may undergo assessment by several agencies, but the Canadian Food Inspection Agency (CFIA) plays the lead role. CFIA has direct responsibility for any necessary field trials for crop plants, and for approval of any GM feed for animals. Health Canada, on the other hand, has responsibility for assessment of food safety. CFIA operates under the powers of the Seeds Act, the Plant Protection Act, the Feeds Act, the Fertilizer Act, and the Health of Animals Act. It also shares some responsibilities with Environment Canada under the Canadian Environmental Protection Act (CEPA), and with Health Canada under the Pest Control Products Act (PCPA) and the Food and Drugs Act. The Canadian Environmental Protection Act is umbrella legislation that is apparently intended to serve as a regulatory “safety net” for any biotechnology products not currently regulated by another federal act.

The Department of Fisheries and Oceans regulates aquatic organisms under the powers of the Fisheries Act, although it has not yet adopted specific regulations that address GM organisms. Since the issue of transgenic fish raises particular concerns, these have been explored in depth in Chapter 6, Part 4 of the Report.

Canadian Food Inspection Agency

CFIA has responsibility for regulating GM plants, assessing their impact on the environment and biodiversity, including the possibility of gene flow and impact on non-target organisms, and is responsible for ensuring livestock feed safety, including feed composition, toxicology, nutrition and dietary exposure (CFIA a). In April 1999, the Canadian Food Safety and Inspection Bill (C-80) was introduced into the House of Commons to “revise and consolidate certain Acts respecting food agricultural commodities, [and] aquatic commodities” and to amend the various acts under which CFIA operates (House of Commons, 2000).

CFIA is the agency that has the first contact with a biotechnology firm wishing to introduce a new GM crop plant. To obtain permission to proceed with confined field trials, the firm first applies to CFIA. The application documents must provide information on the identity and history of the plant, including any known toxins, and on the nature of the novel trait and the transformation method. The engineered DNA fragment (transgene) must be described, as must the pattern of expression of the transgene, any altered plant characteristics and evidence for stability of the novel trait. With respect to the proposed field trial, any related indigenous species must be identified, and a management plan presented that details methods to ensure reproductive isolation, describes spraying regimes, harvesting practices, proposed post-trial land use, contingency plans, and methods of site monitoring, as well as plans for providing public notification of the field trial (CFIA, 2000). The application may combine data from product-specific testing done by the applicant under contained growth conditions (e.g. laboratory or greenhouse trials) with data extracted from the scientific literature. Once confined field trials have been approved (these are normally limited to one hectare per site and to a maximum of five sites per province), CFIA has the authority to inspect them, as well as the records kept on them.

The information that CFIA makes available to the public regarding their approval decisions explains the basis for approval of unconfined release of a GM plant into the environment, such as the criteria to be addressed in deciding whether environmental safety is threatened, but neither the design of the experiments on which the assessment was based, nor their results, are included in the public Decision Document. Similarly, the latter describes the nutritional criteria to be met for livestock feed without presenting analytical data (CFIA b). Although they are not revealed to the public, these data are evidently collected, since the CFIA regulatory directive of July 10, 2000 reminds applicants that “experiments should generate data which can be used to address the five key criteria of environmental safety assessments” (CFIA 2000). In addition, CFIA directives indicate that statistically valid experimental designs are required for testing plants with novel traits, and that all such work is to be of the standard required for peer-reviewed research publications. In the absence of independent peer review, however, the Decision Document is in no sense equivalent to a peer-reviewed scientific paper, and in the Panel’s view, the decision-making process in general lacks transparency, and thus credibility. This issue is examined further in Chapter 9 of the Report.

Despite the existence of an explicit CFIA decision framework (Figure 3.1), the Panel is of the impression that the actual decision process varies greatly from application to application. This is not necessarily an undesirable situation, since a case-by-case analysis allows the flexibility required to respond appropriately to the unique characteristics of each application. However, this degree of discretion can also make it difficult for applicants to know exactly what the approval requirements will be for their product, a problem that CFIA apparently deals with by establishing an ongoing dialogue with each applicant. This enables the Agency to comment on the application and its possible deficiencies, and to request further experimental data or information, as it deems necessary. Again, while this consultation with its advice process clearly has a positive aspect, the Panel is concerned that, without independent review, it also has the potential for allowing inappropriate decisions to be made.

Symptomatic of the lack of clarity in the current process is the ambiguous application of the principle of “substantial equivalence”. Although “substantial equivalence” is explicitly mentioned in CFIA directives (see 1.2.9, Regulatory Directive 2000-07), and appears to operate as a decision threshold in the schematic representation of the decision-making process (Figure 3.1), in Panel interviews CFIA representatives claimed that it is used more as a guiding principle than as an end point (decision threshold). The problems associated with use of “substantial equivalence” as a decision threshold are explored further in Chapter 7 of the Report.

An additional factor potentially affecting the nature of the CFIA decision process is Canada’s recent (July 1998) commitment to harmonization with the US on regulation of agricultural biotechnology (CIFA c). Officials from CFIA, Health Canada and the Animal and Plant Health Inspection Service (APHIS) of the US Department of Agriculture signed an agreement on commonalities in molecular genetic characterization of transgenic products, and on the development of reviewers’ checklists. An international exchange of information and harmonization of procedures is generally commendable, but it does not lessen the responsibility for thorough assessments in Canada.

Health Canada

Many GM crops are destined, as a whole or as specific parts, for the human food supply system. For this reason, they must not only obtain CFIA approval, but must also be assessed by Health Canada. Health Canada gains its jurisdiction to regulate in this area from the Food and Drugs Act and Regulations, within which GM foods come under the Novel Food Regulations. While this regulation establishes important background criteria, such as the defining of novel foods and setting the time frame for a government response, the more instructive document is that entitled Guidelines for the Safety Assessment of Novel Foods (Health Canada, 1994). These guidelines (as opposed to regulations) specify that a guiding principle in the safety assessment is based on a “comparison of molecular, compositional and nutritional data for the modified organism to those of its traditional counterpart”. They suggest that data should be provided on dietary exposure, nutrient composition, anti-nutrients, and nutrient bioavailability. If concerns still remain following this analysis, “toxicity studies would be required as necessary, on the whole food, food constituent or specific component in question”. Finally, using data supplied by the applicant, Environment Canada and Health Canada consult together to decide whether a product is “toxic” to the environment and human health (Health Canada a).

After reviewing the relevant documents and holding discussions with Health Canada personnel, it appears to the Panel that no formal criteria or decision-making framework exists for food safety approvals of GM products by Health Canada. Decisions are largely made on a caseby- case, ad hoc basis. An applicant’s first contact with Health Canada usually involves an informal meeting at which the applicant may be given a sense of the type of studies to be undertaken and the information to be provided in a full application. Following this initial meeting, and perhaps several more meetings with Health Canada personnel, a full application may be submitted. The contents of this application are based loosely on, though not specifically prescribed by, the Guidelines. Health Canada must respond to this “notification” within 45 days, and then has 90 days to issue a decision. Health Canada reviews the material within 45 days and then either asks for more information, or makes a decision to approve or not to approve. As in the CFIA procedures, the applicant is responsible for supplying all of the data to be evaluated, which may be supplemented by any relevant scientific literature. No independent testing of the safety of a GM food by a governmental or other, independent, laboratory is required.

The decisions for approval of a novel food are made public by Health Canada. These documents provide the product name, the name of the proponent, the decision date and further information in a manner similar to the CFIA Decision Documents (Health Canada b). Again, the data on which the decision was based are not revealed. If an approval is issued, it could be accompanied by specific conditions, such as requiring labelling for possible allergens, because Health Canada has jurisdiction over labelling for health and safety issues.

Approvals of GM food additives, such as flavours and enzymes that are derived from GM microorganisms, are handled somewhat differently from foodstuffs themselves. They are essentially evaluated as new food additives, and the application submitted to Health Canada for approval must therefore present the taxonomy of the source microorganism, the history of the microbial strain including any use as a food, details of the novel DNA construct, and evidence for the absence of any pathogenic characteristics. Unlike approvals for transgenic organisms, the decision documents for these additives are not published. Instead, approvals are reflected solely in additions to the list of permitted food additives that appear in the Canada Gazette. Consistent with this approach, those enzymes permitted as food additives are listed in the Food and Drug Regulations (Table V, Division 16), but there is no indication whether they are derived from GM organisms or not. The current regulations thus treat purified products of “living modified organisms” differently from GM organisms themselves. These products also do not fall within a category of concern in the Cartagena Protocol on Biosafety (www.biodiv.org/biosafe/protocol/).

Environment Canada and Protection of the Environment

Current legislation respecting the environment includes CEPA, PCPA, parts of the Seeds Regulations (Part V) and the Feeds Act. With respect to approvals for GM organisms, the regulations call for information to be provided by the proponent about many aspects of the modified organism’s biology and ecological niche, and concerning potential or actual environmental impacts of its unconfined release.

This information may be provided from published sources (historical information) or generated by the proponent through specific testing of the GM organism in question. However, the latter data, by definition, can presumably only reflect the results of studies conducted in confined holding facilities, rather than testing in the open environment. During the consultative process that accompanies application for approval, Environment Canada/CFIA may waive specific information requirements if the proponent can provide persuasive supporting scientific arguments. The information requirements as listed in the CEPA regulations are quite substantial.

Several examples of ecological information requirements derived from sections of the CEPA Regulations are shown in Figure 3.2, along with an excerpt from the CEPA Regulations for field testing of GM microorganisms.

The Seeds Act provides CFIA with the authority to regulate the quality, testing, inspection and sale of seeds in Canada, while the Seeds Regulations (Part V) define regulatory requirements for both confined and unconfined release of plants with novel traits in Canada. According to CFIA (2000), these regulations address five key criteria for assessment of environmental safety: altered weediness potential, potential for outcrossing, altered plant pest potential, impact on non-target organisms and impact on biodiversity. As described above for CFIA, the generation of these data must use statistically valid experimental designs and protocols that meet the standards required for inclusion in peer-reviewed research publications. CFIA provides additional directives that outline conditions for confined field trials (Regulatory Directive Dir95-01) and more recent directives that amend these conditions (http://www.cfia-acia.agr.ca/english/pla ... 1_3e.html; Oct. 27, 2000). The purposes of these directives and amendments are to define methods of reproductive isolation, including isolation distances or buffer zones, to place restrictions on postharvest land use, to restrict the size and number of trials, and to provide improved guidelines on provision of information such as site maps.

The Pest Management Regulatory Agency (PMRA) of Health Canada is charged with the regulation of biological control agents for use in food production in Canada. An example of a recent regulatory decision for a naturally occurring viral biological control agent (for reducing codling moth damage on apple trees) may be found at the following site (http://www.hc-sc.gc.ca/pmra-arla/englis ... w_IE.html; Oct. 27, 2000). Thus far, GM control agents have not been presented to the PMRA for approval, but given the rapid advances in technology it is only a matter of time before this happens. The Panel determined through consultation with PMRA that new regulations and guidelines for GM pest control agents are presently under development.

REFERENCES

Berg, P., D. Baltimore, S. Brenner, R.O.R. Roblin and M.F. Singer. 1975. Asilomar conference on recombinant DNA molecules. Science 188: 991–994.

CFIA (Canadian Food Inspection Agency) a. Regulation of Biotechnology in Canada. At: <www.cfia-acia.agr.ca/english/ppc/biotech/bioteche.shml>

CFIA, b. Plant Biotechnology Decision Documents. At: <www.cfiaacia. agr.ca/english/plaveg/pbo/dde.shtml>

CFIA, c. Canada and United States, Bilateral on Agricultural Biotechnology. At: <www.cfiaacia. agr.ca/english/plaveg/pbo/usda01_e.shtml>

CFIA. 2000. Regulatory Directive 2000-07: Guidelines for the Environmental Release of Plants with Novel Traits Within Confined Field Trials in Canada. At: <www.cfiaacia. agr.ca/english/plaveg/pbo/dir0007e.shtml>

Health Canada 1994, Guidelines for the safety assessment of novel foods. Complete document, PDF<www.hc-sc.gc.ca/food-aliment/english/subjects/novel_foods_and ingredient.html>

Health Canada, a. It’s Your Health. Assessing the Health Risks of Biotechnology Products Under the Canadian Environmental Protection Act (CEPA). At: <www.hcsc. gc.ca/ehp/ehd/catalogue/general//iyh/assesbio.htm>

Health Canada, b. Novel Food – Decisions. At: <www.hc-sc.gc.ca/foodaliment/ english/subjects/novel_foods_and _ingredient/decisions.htm>

House of Commons of Canada. 2000. Bill C-80. At: <parl.gc.ca/36/1/parlbus/house/bills/government/C-80/C-80_1/C-80_cover-E.htm > or as link from <www.cfia-acia.gc.ca>

Figure 3.1: A schematic representation of the safety-based model for the regulation of plants*

STEP 1: FAMILIARITY / __

1.1 SPECIES: Has the plant species been grown or released into the environment in Canada? / IF YES, GO TO 1.2; IF NO/UNKNOWN, GO TO STEP 3

1.2 TRAIT: Is the trait similar to those already introduced into that species? / IF YES, GO TO 1.3; IF NO/UNKNOWN, GO TO STEP 3

1.3 TRAIT INTRODUCTION METHOD: Has the method been used before in that plant species? / IF YES, GO TO 1.4; IF NO/UNKNOWN, GO TO STEP 3

1.4 CULTIVATION: Will cultivation practices be similar to those previously used for this plant species in Canada? / IF YES, GO TO STEP 2; IF NO/UNKNOWN, GO TO STEP 3

STEP 2: SUBSTANTIAL EQUIVALENCE / __

2.1 In considering the following five criteria, and using data or sound scientific rationale, is it known that this plant will not result in altered environmental interaction compared to its counterpart(s)? / IF YES AND NON-TRANSGENIC, EXEMPT FROM SEEDS REGULATIONS, PART V1; IF YES AND TRANSGENIC, GO TO 2.2
; F NO/UNKNOWN, GO TO STEP 3

2.1.1 Altered weediness potential 2.1.2 Gene flow to related species 2.1.3 Altered plant pest potential 2.1.4 Potential impact on non-target organisms 2.1.5 Potential impact on biodiversity / --

2.2 For traits introduced by rDNA methodologies, are the specific genetic elements the same as those previously approved by the CFIA in the same species? / IF YES, EXEMPT FROM SEEDS REGULATIONS, Part V1; IF NO/UNKNOWN, GO TO STEP 3

STEP 3: ENVIRONMENTAL SAFETY ASSESSMENT BY CFIA / __

If acceptable risk, approve to regulate under Seeds Regulations, Part V If unacceptable risk, approval is refused. / --

1 While an environmental safety assessment under the Seeds Regulations, Part V is not required, the plant may still be subject to regulation under other government Acts.


Figure 3.2: CEPA Regulations dealing with the introduction of GM microorganisms into small-scale field trials

(Excerpts from: Canada Gazette Part II, Vol. 131, No. 5, p. 694. Canadian Environmental Protection Act: Regulations amending the new substances notification regulations. Schedule XVII: Information required in respect of microorganisms for introduction in an experimental field study)

Part 1. (f) a description of the biological and ecological characteristics of the micro-organism, including:

(i) the infectivity, pathogenicity to non-human species, toxicity and toxigenicity,

(ii) the conditions required for, and conditions that limit, survival, growth and replication,

(iii) the life cycle, where the micro-organism is not indigenous,

(iv) the resistance to antibiotics and tolerance to metals and pesticides, where the micro-organism is not indigenous,

(v) the involvement in biogeochemical cycling, where the micro-organism is not indigenous, and

(vi) the mechanisms of dispersal of the micro-organism and modes of interaction with any dispersal agents;

and Part 1. (i) where the micro-organism is not indigenous, the dispersal by gene transfer of traits of pathogenicity to non-human species, toxigenicity and resistance to antibiotics, including a description of:

(i) the genetic basis for pathogenicity to non-human species, toxigenicity and resistance to antibiotics,

(ii) the capability to transfer genes, and

(iii) the conditions that might select for dispersal of traits of pathogenicity to non-human species, toxigenicity and resistance to antibiotics, and whether the conditions are likely to exist at the site of the experimental field study or within the range of dispersal of the micro-organism; and

(j) a description of the geographic distribution of the microorganism.

and Part 3. The following information in respect of the site of the experimental field study:

(a) the location and a map;

(b) the size;

(c) the distance to populated areas;

(d) the distance to any protected areas;

(e) a description of the geological landscape at the site and surrounding the site;

(f) a description of the biological diversity found at the site and surrounding the site, including

(i) the identification of the endangered or threatened species, and

(ii) where infectivity, pathogenicity to non-human species, toxicity and toxigenicity have been identified in subparagraph 1(f)(i), the identification of the receptor species;

(g) a comparison of the natural habitat of the micro-organism to the habitat at the site of the experimental field study, and the nature of the selection that may operate on the microorganism at that site; and

(h) where the micro-organism is indigenous, data to demonstrate that it is indigenous.

4. The following information in respect of the experimental field study:

(d) a description of the procedures for transporting the micro-organism to and from the site of the experimental field study;

(e) a description of the procedures and design for the experimental field study, including

(i) the method of application of the micro-organism,

(ii) the quantity, frequency and duration of application of the micro-organism, and

(iii) any activities associated with the experimental field study;

(f) a description of any procedures for monitoring the micro-organism and its ecological effects at the site of the experimental field study, during and after the experimental field study;

(g) a description of the security measures at the site of the experimental field study;

(h) a description of any contingency plans for accidental release;

(i) a description of any recommended procedures for terminating the experimental field study; and

(j) a description of any confinement procedures and biosafety conditions for the micro-organism at the site of the experimental field study, and a description of their effectiveness.

5. The following information in respect of the environmental fate of the micro-organism:

(a) a description of habitats where the micro-organism may persist or proliferate;

(b) the estimated quantities of the micro-organism in the air, water and soil at the points of introduction and the estimated population trends; and

(c) any other information on the environmental fate of the micro-organism.

6. The following information in respect of the ecological effects of the micro-organism:

(a) the involvement of the micro-organism in adverse ecological effects; and

(b) the potential of the micro-organism to have adverse environmental impacts that could affect the conservation and sustainable use of biological diversity.
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Part 1 of 2

4. POTENTIAL HUMAN HEALTH IMPACTS

INTRODUCTION


In this chapter, we examine the question of potential direct risks to human health that might arise from introduction of GM food products into the food supply system. These risks are generally categorized in three ways: possible creation of novel toxicants, possible shifts in the nutrient content of the food, and the possible creation of novel allergens. Each of these categories will be dealt with separately.

PART 1: TOXICANT ASSESSMENT

Assessment of the potential risk associated with a novel product intended for human consumption is routine practice internationally. This practice has given rise to an extensive body of knowledge derived from studies in laboratory animals and from studies of human exposure to chemical residues, microbiological contaminants and pharmaceutical agents, or to modifications in the concentrations of otherwise endogenously present substances. The goal of risk assessment is to inform the decision-making process in order to ensure public protection against unacceptable risks. The therapeutic use of life-saving drugs that may be associated with adverse side effects reflects the careful balance of risk and benefit. This paradigm has been broadly successful in supporting the development of regulations for protection of consumers from adverse health impacts in a very complex and chemically diverse modern society. Of particular interest to the Panel, however, is the suitability of the application of this traditional assessment paradigm for the challenges presented by food biotechnology.

Potential adverse health impacts in humans from exposure to toxicants in the food supply are expressed as a function of the probability, frequency and amount of exposure to the toxicants that are likely to occur (plus the severity of the resulting harm). The toxicological profile of the toxicant, whether endogenously produced or exogenously added to the food, is normally described through a series of well-characterized studies providing important information on the likely behaviour of the toxicant in the human body, and the biological end points most likely to be effected. The toxicological profile that results from these studies, and the dose required to achieve the effects, is then considered in the light of the expected frequency, intensity and duration of exposure to the toxicant under typical use conditions. From this analysis, an expression of anticipated risk can be developed. This model for the expression of risk for food components has been well described by the US National Academy of Sciences (1983) and is widely accepted internationally as the basis for informed decision making for a wide array of chemicals, including pesticides, therapeutic drugs and environmental contaminants.

The implementation of the risk assessment paradigm normally consists of four steps: 1) hazard identification, 2) dose–response evaluation, 3) exposure assessment and 4) risk characterization.

These have been described as follows:

1. Hazard identification is the determination of whether a substance, such as a constituent in food, is or is not causally linked to particular health effects. Hazard is usually determined experimentally in controlled toxicology studies with known doses or exposures to the toxicant under study. In practice, statistical considerations have resulted in the use of a “maximum tolerated dose” (MTD), the highest practical dose that can be administered, in most studies carried out in laboratory animals (Lu and Sielken, 1991). In the specific context of food safety assessment, the World Health Organization (WHO) (2000a) has defined “hazard” as a biological, chemical or physical agent in, or condition of, food with the potential to cause an adverse health effect.

2. Dose–response evaluation is the determination of the relationship between the magnitude of exposure and the probability of occurrence of the adverse effect under study. Dose–response assessment is the mechanism used to assess the potency or severity of the hazard in question. Many substances may lead to adverse effects only at high levels of exposure and may thus be considered to pose less severe hazards. Conversely, some substances may induce significant adverse effects even at very limited exposures and would thus be considered to pose a more severe hazard (e.g. classical anaphylactic responses to very low doses of an allergen).

3. Exposure assessment is the determination of the extent of exposure to a toxicant under a particular set of exposure circumstances. Exposure assessment includes the determination of the magnitude of the exposure, the frequency of the exposure and the duration of the exposure.

4. Risk characterization considers these first three factors and is often reported as a quantitative assessment of the probability of an adverse effect under defined exposure conditions. Hazard identification, dose–response assessment and exposure assessment are all essential elements of this risk assessment. In the specific context of food safety, WHO (2000a) defines “risk” as the function of the probability of an adverse health effect and the severity of that effect, resulting from a hazard in food.

Standard toxicological human health risk assessment as outlined above is science-based, but its accuracy depends on the degree of variability and uncertainty encountered in the assessment studies, which can lead to difficulty in extrapolation (SOT, 2000). Variability arises from the range of differences found within a natural population (e.g. genetic variability in sensitivity to a toxicant) while uncertainty is generated by incomplete knowledge (e.g. inadequate gathering of data), or measurement error. Nevertheless, quantitative risk assessment related to specific chemically defined toxicants is widely used, and can address an array of end points, including cancer and other health risks, microbiological risk and certain ecological/environmental risks (Solomon et al., 1996).

On one level, the assessment of the safety of whole GM foods can be considered simply to require a comparison of the safety of the whole GM food when compared to the food or food constituent from which it is derived. Indeed, some authors have suggested that a useful and practical approach for such comparisons can be based on the concept of the “substantial equivalence” (WHO, 1995) of the whole GM food to the non-modified food already in the diet. The scientific robustness of this approach in the assessment of the risks of novel foods continues to be the subject of considerable scientific debate (WHO, 2000b; 2000c) and is extensively reviewed elsewhere in this Report (see Chapter 7). As indicated in that review, where substantial equivalence can be rigorously substantiated, toxicological assessment of the whole GM food would not be warranted. However, the Panel also concluded that, for the purposes of the safety assessment of GM foods for human consumption, “substantial equivalence” should be considered to have been achieved only if, within scientific certainty, there is equivalence in the genome, proteome and metabolome of the GM food when compared to that of the native food. In the absence of such evidence, the Panel felt that direct assessment of potential health impacts is called for, including toxicological testing. It then becomes necessary to consider whether the traditional toxicological paradigm can be applied in those cases.

Potential adverse health effects from GM food could result from over-expression of an existing protein or other toxicologically active constituent, resulting in much greater exposure to that constituent than previously encountered by humans in their diet. While exposure in this case would be to the same constituent as in the native food, and is thus likely to result in the same toxicological end point, exposure to much greater levels of the constituent in the GM food could lead to adverse health effects which could not be predicted by the absence of these effects at much lower levels of exposure to the constituent in the native food. In other words, the likelihood of a toxicological effect is very much related not only to the nature of the substance to which we are exposed, but also to the amount of exposure as well. In the general case, it would not be unusual to expect that as exposure increases, so might the adverse effects. In this scenario, the protein or metabolite in question can be subjected to the traditional toxicological evaluation, including repeat exposure feeding studies in laboratory animals conducted at a MTD, a dose which is, by design, typically hundreds to thousands of times greater than what might be encountered under actual human exposure conditions.

The Panel recognized that genetic engineering of crop plants may also result in the expression of a constituent which would not otherwise be found in the plant species, but which does occur naturally. This phenomenon is illustrated in transgenic corn which is modified to express the Bt endotoxin (Cry3A), a protein which would never be found in this plant species, but which is normally expressed in the ubiquitous Bacillus thuringiensis microbe. In such cases, human exposure to the protein may already appear to be widespread and hence of little toxicological importance. However, the Panel noted that, because of dietary intake patterns of corn and corn products, human exposure to this protein in Bt-corn is predictably much greater than would otherwise be expected to occur. In the particular case of the Bt endotoxin, the Cry3A protein has been extensively tested for potential impacts on human health without adverse effects being reported, but whether this is true for other novel proteins intended for de novo expression or over-expression in crop plants is uncertain. It is also worth noting that many proteins, such as the Bt endotoxin, are rapidly destroyed when exposed to heat (as may occur in food processing) and are very labile under the acidic conditions of the human intestinal tract. In such cases, it can reasonably be expected that the protein would be readily broken down to toxicologically trivial components, thereby eliminating any potential concern of a classical toxicological response associated with food exposure to the native protein.

The successful application of the traditional toxicological paradigm to assessment of the health hazards that may be associated with dietary exposure to whole GM foods, or modified constituents of foods, depends entirely on our ability to identify the hazards. Where the modified constituent is a single new protein or metabolite, as discussed above, identification and testing of that constituent can be pursued within the framework of the toxicological paradigm. If, however, the hazard results from a pleiotropic response, and involves multiple changes in either protein or metabolic constituents that are not readily predicted from the genetic manipulation, the first step in the risk assessment procedure (hazard identification) seems likely to fail. Thus, while the Panel felt that the traditional toxicological paradigm could adequately assess the safety of individual known hazards, more complex changes in whole foods present a serious methodological challenge. GM whole foods are complex mixtures which, for reasons of nutritional balance, can be administered in feeding trials only at doses that are much more characteristic of typical human exposure. This precludes traditional safety factor considerations, “acceptable daily intake” estimations, and application of the widely accepted principles of the MTD in the design and interpretation of risk assessment studies (WHO, 1999; 2000e; 2000g).

In addition to this limitation, there is considerable uncertainty as to either the appropriate duration of studies, or the most meaningful indicators to monitor. In a consultation paper submitted to WHO (WHO, 2000a), Walker has suggested that a sub-chronic study of 90 days’ duration in rats is the minimum requirement which could address the safety of repeated consumption of a GM food in the diet. A recent WHO expert consultation (WHO, 2000g) also concluded that, where toxicology studies are deemed to be necessary, such studies should be limited to no less than a 90-day repeat exposure study, unless proliferative or other important biological alterations indicated the need for further investigation. In contrast, the position recently adopted by the International Conference on Harmonization (ICH) on technical requirements for the safety assessment of pharmaceuticals (ICH, 1995; 1998) concluded that, where repeatedexposure studies were used for pharmaceutical safety testing, such studies should be of at least 180 days’ duration. In addition, the ICH consultation noted that the determination of the need for carcinogenicity testing should include, among other things, an assessment of any evidence of preneoplastic lesions in the repeated-dose studies. It is noteworthy that, WHO also indicated the need for the assessment of proliferative changes (pre-neoplastic) in short-term repeat-exposure studies in order to determine the need for longer term chronic toxicity/carcinogenicity studies. However, WHO also concluded that such an assessment could be made from 90-day studies, while ICH concluded that a study of at least 180 days’ duration would be required. Similarly, ICH (1997) has also indicated that the safety evaluation of biotechnology-derived pharmaceuticals should include a repeat exposure study of 180 days duration for those products for which human exposure is likely to exceed six months, an exposure scenario which would almost certainly apply for foods. Again, while the ICH position is directed at pharmaceuticals and not foods, the selection of study type and duration would appear to have a similar biological basis for both foods and pharmaceuticals to which long-term human exposure can be reasonably anticipated. The Panel noted that both the US National Research Council Report and the WHO Expert Report (WHO, 2000f) indicated that further toxicology studies, in addition to the 90-day studies described above, could be required to support the safety of transgenic foods. However, the WHO Report indicated only that the appearance of proliferative changes in the 90-day studies might trigger the need for further studies. The Panel was concerned that proliferative changes might not be expected to appear after only 90 days of exposure, and uncertain whether such changes, even if they did appear, provide a useful basis for triggering the need for further studies other than those directed at carcinogenicity or chronic toxicity.

In general, the Panel found that regulatory requirements related to toxicological assessment of GM food appeared to be ad hoc and provided little guidance either as to when specific studies would be required or what types of studies would be most informative. In particular, the Panel was unaware of any validated study protocols currently available to assess the safety of GM foods in their entirety (as opposed to food constituents) in a biologically and statistically meaningful manner. The Panel therefore concurs with the US National Research Council (NRC, 2000) in recommending the immediate initiation of research into the development of practical and scientifically robust approaches for the safety assessment of such foods.

Resistance Factors

A particularly controversial area in the application of gene transfer technology has been the use of marker genes which are co-introduced along with the DNA coding for the desired trait, thereby allowing confirmation that the gene transfer has been successfully completed. Historically, the most common marker genes selected (WHO, 2000c) have been those that code for resistance to herbicides or antibiotics. The concern related to the use of antibiotic resistance genes has focused on the possibility that these genes could find their way into pathogenic microbes, thereby potentially compromising the clinical efficacy of antibiotics used in human medicine or livestock production. Although this concern has been heightened by the rise in drug resistant bacteria and the declining effectiveness of many antibiotics, the Panel agrees with the position of the Royal Society (1998) that the widespread use of antibiotics as feed additives, coupled with the indiscriminate use of antibiotics in human medicine, likely poses a far greater risk for the selection of antibiotic resistant bacteria than transfer of marker genes from plants. However, in view of the availability of alternative technologies that eliminate the need to use antibiotic resistance genes as markers in transgenic plants, the Panel endorses the position already adopted by others (OECD, 2000; WHO, 2000d) on this topic and recommends that antibiotic resistance markers should not be used in any GM food intended for sale in Canada.

RECOMMENDATIONS

4.1 The Panel recommends that federal regulatory officials in Canada establish clear criteria regarding when and what types of toxicological studies are required to support the safety of novel constituents derived from transgenic plants.

4.2 The Panel recommends that regulatory authorities establish a scientific rationale that will allow the safety evaluation of whole foods derived from transgenic plants. In view of the international interest in this area, the Panel further recommends that Canadian regulatory officials collaborate with colleagues internationally to establish such a rationale and/or to sponsor the research necessary to support its development.

4.3 The Panel recommends that, in view of the availability of suitable alternative markers, antibiotic resistance markers should not be used in transgenic plants intended for human consumption.

REFERENCES

ICH. The International Conference on harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline. Guideline on the Need for Carcinogenicity Studies of Pharmaceuticals. Recommendation for Adoption, 29 November 1995.

ICH. The International Conference on harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline. Preclinical Safety Evaluation of Biotechnology - Derived Pharmaceuticals. Recommended for Adoption, 16 July 1997.

ICH. The International Conference on harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline. Duration of Chronic Toxicity Testing in Animals (Rodent and Non Rodent Toxicity Testing). Recommended for Adoption, 2 September 1998.

Lu, F.C., R.L. Sielken Jr., Sielken Inc. Assessment of safety/risk of chemicals: inception and evolution of the ADI and dose-response modeling procedures. Toxicol. Lett. 1991 Dec; 59(1-3): 5–40.

NRC (US National Research Council). 2000. Genetically Modified Pest-Protected Plants: Science and Regulation. Committee on Genetically Modified Pest-Protected Plants, Board on Agriculture and Natural Resources, National Research Council. Washington, DC: National Academy Press.

OECD (Organisation for Economic Co-operation and Development). 2000. GM Food Safety: Facts, Uncertainties, and Assessment. The OECD Edinburgh Conference on the Scientific and Health Aspects of Genetically Modified Foods; 28 February – 1 March 2000. Chairman’s Report.

SOT (Society of Toxicology). Communiqué. Spring 2000.

Solomon, K.R., D.B. Baker, P. Richards, K.R. Dixon, S.J. Klaine, T.W. La Point, R.J. Kendall, J.M. Giddings, J.P. Giesy, L.W. Hall, Jr., C.P. Weisskopf, M. Williams. 1996. Ecological risk assessment of atrazine in North American surface waters. Environ. Toxicol. Chem. 15: 31–76.

The Royal Society. 1998. Genetically Modified Plants for Food Use. London.

US National Academy of Sciences. 1983. National Research Council. Risk Assessment in the Federal Government. Washington, DC: National Academy Press.

WHO (World Health Organization). 1995. Application of the Principles of Substantial Equivalence to the Safety Evaluation of Foods or Food Components from Plants Derived by Modern Biotechnology – Report of a WHO Workshop.

WHO. 1999. Principles for the Assessment of Risks to Human Health from Exposure to Chemicals. International Programme on Chemical Safety. Environmental Health Criteria 210. Geneva, Switzerland.

WHO. 2000a. Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology. Food and Agricultural Organization of the United Nations/World Health Organization. Walker, R., Topic 6: Safety Testing of Food Additives and Contaminants and the Long Term Evaluation of Foods Produced by Biotechnology.

WHO. 2000b. Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology. Food and Agricultural Organization of the United Nations/World Health Organization. Tomlinson, N., Topic 1: The Concept of Substantial Equivalence, its Historical Development and Current Use.

WHO. 2000c. Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology. Food and Agricultural Organization of the United Nations/World Health Organization. Pederson, J., Topic 2: Application of Substantial Equivalence Data Collection and Analysis.

WHO. 2000d. Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology. Food and Agricultural Organization of the United Nations/World Health Organization. Ow, D., Topic 12: Marker Genes.

WHO. 2000e. Hazardous Chemicals in Human and Environmental Health. International Programme on Chemical Safety. Geneva.

WHO. 2000f. Safety Aspects of Genetically Modified Foods of Plant Origin. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology. Geneva.

PART 2: THREATS TO HUMAN HEALTH FROM ALLERGENS IN GM FOODS

Food-allergic individuals and their families need to be extremely cautious about the components and ingredients of processed foods they ingest because of the risk that trace amounts of an allergenic food contaminant may cause a severe, potentially life-threatening allergic reaction (Yunginger et al., 1988; Sampson et al., 1992; Canadian Paediatric Society, 1994; Zarkadas et al., 1999). The only current treatment of food allergy is avoidance. A recent report from Montreal indicated that peanut-allergic children and their families experience considerably more impairment in their quality of life and family relations in comparison to children with chronic musculoskeletal disease, attesting to the substantial negative impact of severe food allergies (Primeau et al., 1999).

It is already difficult for food-allergic individuals to understand the different ways crosscontamination of foods can occur, as well as labelling exemptions that allow allergenic foods to remain unlabelled and pose a risk to the allergic consumer (Ham Pong and Zarkadas, 1996; Steinman, 1996; Zarkadas et al., 1999). Bock and Atkins (1989) reported that, in spite of avoidance measures, 75% of peanut-allergic children accidentally ingested peanut over a five-year period. With the widespread penetration of GMO food products in the marketplace, food-allergic people may now need to contend with another variable in deciding what foods are safe to consume (i.e. do any GM food products pose a risk for allergenicity?). The Expert Panel has tried to address this question, and to consider what measures the Canadian government and industry might take to identify the potential risks and to protect the potentially allergic consumer. Allergenicity considerations have been addressed only briefly in reports from some national expert committees on GMOs such as Britain’s Royal Society and the US National Academy of Science, although a more thorough approach has been presented by FAO/WHO and US regulatory agencies, and referred to by the Institute of Food Technologists (Metcalfe et al., 1996; Royal Society, 1998; USEPA, 1999a; IFT, 2000; National Academy of Sciences, 2000; Taylor, 2000). The Canadian Food Inspection Agency (CFIA) has a paucity of published information on its procedure for allergenicity assessment on GMOs (Health Canada, 1994). In the following sections, we discuss issues regarding food allergy that may be relevant to GMOs, potential risks of allergenic GMOs, current technologies available to assess allergenicity and their limitations, and how the technology has been utilized.

Mechanisms and Allergic Responses in Food Allergy

The terms “adverse food reaction” or “sensitivity” are used to mean all types of abnormal reactions to foods, and include food allergy (hypersensitivity) and food intolerance.

Food intolerance is an adverse reaction to a food that does not involve the immune system. Examples of food intolerances include lactose intolerance, the “Chinese restaurant syndrome” caused by monosodium glutamate (MSG) sensitivity, food poisoning, caffeine-induced stimulation and wine-induced migraine. Food allergy or food hypersensitivity, on the other hand, is an adverse immunologic reaction resulting from the ingestion, and in some cases, contact or inhalation of a food or food additive. These two terms are often used interchangeably. The most widely studied mechanism of food allergy is that mediated by immunoglobulin E (IgE), an antibody which, when exposed to an allergen, causes allergy cells in the body (mast cells and basophils) to release a variety of toxic mediators (e.g. histamine and leukotrienes) which then cause an immediate allergic reaction. An allergen is a substance, usually a protein, which causes an adverse reaction by activating immunologic mechanisms. For the remainder of this discussion, reference to an “allergic reaction” will indicate an IgE-mediated reaction (Gell and Coombs classification type 1 hypersensitivity), unless otherwise specified. Other allergic or hypersensitivity reactions to foods which do not involve IgE are usually not well understood and frequently do not have easily measurable or reliable markers to indicate the presence of an immune response. A well-known example of a non-IgE-mediated food hypersensitivity is coeliac disease (gluten-sensitive enteropathy) (Leung, 1998; Zarkadas et al., 1999).

Mediators released during an allergic reaction have a variety of effects on different tissues, and allergic reactions to ingested foods can range in severity from minor itching or skin rash, to anaphylactic shock and death. Allergic reactions to foods frequently occur within minutes of ingestion, but may occasionally be delayed for as long as four hours, and usually last less than 24 hours (Ham Pong, 1990; Zarkadas et al., 1999). Anaphylaxis is a severe, dramatic allergic reaction to a food with potential life-threatening implications. The most frequent causes of anaphylaxisrelated death are upper or lower airway obstruction, and hypotensive shock (Yunginger et al., 1988; Sampson et al., 1992; Leung, 1998; Zarkadas et al., 1999).

The Increasing Problem of Food Allergies

Allergic disorders include allergic rhinitis, asthma, atopic dermatitis (atopic eczema) and food allergies, and these are now among the most common diseases in industrialized countries, with up to 30% prevalence. The incidence of allergic diseases has been estimated to have increased by 30% to 50% in the last 15 years (Kjellman, 1977; Aberg et al., 1995; Moneret- Vautrin, 1998; Habbick et al., 1999). The prevalence of food allergy in the general population varies in different studies, but ranges from 0.3% to 8% in children declining with age to 1% to 2% of adults (Leung, 1998; Zarkadas et al., 1999). Food-related anaphylaxis is felt to be rising in frequency, and one report indicated that food allergy was the cause of 34% of emergency room visits for treatment of anaphylaxis in the US (Kemp et al., 1995). There is some concern that the rising trend in general allergic disorders is also putting a higher proportion of the population at risk for development specifically of food allergies.

The Transfer of Allergens by Genetic Modification

There is no question that allergenic proteins can be transferred by genetic engineering from one organism to another. However, the current generation of GM foods approved for human consumption do not appear to have a significant potential for causing allergic reactions. In fact, there are no validated reports of allergic reactions to currently marketed GM foods as a result of the transgene protein. However, the potential risk for development of toxic or allergic reactions to GM foods will likely increase with advances in the scope and range of genetic modifications, wider acceptance of GM foods, increase in total dietary exposure to novel proteins, introduction of a greater variety of these foods, and more innovative transgenic combinations.

It is useful to review the single confirmed report of recombinant DNA technology transferring an allergenic protein to the host organism. Brazil nut allergy, like other tree nut allergies, can cause anaphylaxis, even when the nut is eaten in small amounts. Brazil nut 2S albumin storage protein was transferred into soybean to increase the content of a sulphur-containing essential amino acid, methionine, as soy is inherently methionine-deficient. The transgenic soy contained higher amounts of methionine, which could then be converted to cysteine by animals fed the soybean meal, reducing the need and cost to supplement the soybean animal feed. However, the transferred brazil nut protein made up a significant fraction, 6%, of the total protein in the soy. Initial evaluation of the brazil nut 2S albumin in a mouse model produced no evidence of allergenic potential. However, evaluation of the allergenic potential in humans by radioallergosorbent test (RAST) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by allergy prick skin test in brazil nut-allergic individuals, all showed that the major brazil nut allergen had been transferred to the soybean (Nordlee et al., 1996). This transgenic soy would therefore have posed a significant risk if ingested by brazil nut-allergic people. As a result, commercial development of this GM soybean ceased and it was never actually consumed by brazil nut-allergic individuals.

It is interesting that this brazil nut gene had been variously transferred to tobacco, bean and canola in the five years prior to discovery of its allergenic potential, without recognition of the potential risks, although none of these transgenic plants has been commercialized (Altenbach et al., 1989, 1992; Aragao et al., 1992; Saalbach et al., 1994). Similarly, a peptide encoding, in part, a portion of the melittin protein (a known allergen from honeybee venom) has been inserted into potatoes to confer bacterial and fungal resistance (Reisman et al., 1988; New Scientist, 1999), although this product has not yet been commercialized.

Transgenic combinations using donor genes from known allergenic sources carry a similar potential for transfer of the allergenic protein, if the transferred gene encodes the allergenic protein.

Potential Risks of Allergenic GM Foods

The clinical risks to consumers eating a GM food to which they are allergic range from minor to severe allergic reactions, including fatal anaphylaxis. A less obvious risk could be that, if the GM food is allergenic, and becomes incorporated as a common dietary staple or supplement, repetitive ingestion by a susceptible atopic population (i.e. genetically predisposed to produce IgE and hence develop allergies) could result in a significant number of people developing a new allergy to such a GM food. Development of an occupational allergy or asthma in food or feed handlers may also occur due to repetitive exposure by contact or inhalation of proteins.

The diagnosis of a food allergy is based, to a great extent, on an accurate history of reproducible allergic reactions resulting from challenge with the suspect food and absence of allergic reactions on avoidance of that food, followed by confirmatory allergy tests or immunologic assays for allergy. However, diagnosis reliant on history alone is confounded by the fact that, even with a suggestive history of food allergy, 60% or fewer of these subjects will have confirmed allergy on evaluation (Bock et al., 1988). A potential risk of allergenic GM foods is that a person allergic to a GM protein may not be able to identify the triggers for his or her allergic reactions if the GM protein is present in several different types of foods. It would therefore be much more difficult to pinpoint the source of the allergic reactions, since there could be several seemingly unrelated sources triggering an allergic reaction. In addition, if the GM allergen is present in a food from one grower but not another, and may be present only seasonally, the identification that a reaction may be due to the GM food is complicated by sporadic and inconsistent reactions to what appear to be the same type of food product.

It is worth noting that allergenic foods can also be GM to become hypoallergenic, as has been achieved for rice by Matsuda et al. (1996). However, in such foods even reduced levels of allergenic protein could still pose a risk for a severely allergic individual.

Food Allergens: How Much Is Too Much?

Genetic engineering to date usually involves insertion of one or a few proteins that constitute a very small fraction (usually less than 0.4%) of the total protein of the transgenic organism. It has been argued that this makes the resultant GM food unlikely to have significant adverse consequences due to the small amount of protein involved (Astwood and Fuchs, 1996b; Metcalfe et al., 1996). However, this argument is not fully valid. Gad c 1 is a parvalbumin protein that constitutes only 0.05% to 0.1% of total cod fish muscle protein, but it is the major cod fish allergen (Bush and Hefle, 1996; Taylor and Lehrer, 1996). Therefore, even a single gene encoding for a highly allergenic protein which constitutes only a small fraction of the host organism, can be sufficient to make that organism allergenic. A food typically causes an allergic reaction on ingestion. The amounts of allergen required to cause allergic reactions can be remarkably small, so that cross-contamination is a major concern when trying to avoid those particular foods. Peanut-allergic individuals have complained of subjective symptoms (e.g. itchy throat) during oral challenge with as little as 0.01 to 0.1 mg of peanut. As a comparison, a peanut kernel can weigh about 700 mg, and a typical serving of peanut butter is about 30 g, implying that 1/70,000 of a peanut kernel can cause minor allergic reactions (Hourihane et al., 1997a; Koetzler and Ferguson, 2000). Anaphylactic death has occurred from 60 mg of casein, the amount found in 2 to 2.5 ml of cow’s milk. Anaphylaxis has been caused by 1 to 2 g of shrimp (one medium-sized shrimp is 4 g) and objective allergic reactions have been provoked by 35 to 100 mg of peanut, 6 to 12 mg of hazelnut, 0.3 ml of cow’s milk, 250 mg soy protein, 1 to 4 g of fish protein, 10 mg ovalbumin (an egg allergen) and 100 to 300 mg of cottonseed in respective allergic individuals (Yman, 1995; Bush and Hefle, 1996; Taylor and Lehler, 1996).

Physical contact with an allergenic food without ingestion can cause contact hives or rash, and if accidentally introduced in the eye, can cause marked eye swelling and even anaphylaxis (Bernstein et al., 1984; Colas de Francs et al, 1991). Patients with severe food allergies have reported allergic reactions to the relevant aerosolized food (e.g. to the smell of cooking seafood, steam from cooking potatoes, and the smell of peanut in an enclosed area such as an airplane) (James et al., 1991; Eng et al., 1996; Ojoda et al., 1997; Sicherer et al., 1999). Typically, these allergic reactions to inhaled food allergens are usually minor although some of the reactions can be more severe, such as respiratory symptoms caused by the smell of peanut. Anaphylaxis is highly unlikely if the inhalant exposure is at low level, but there is at least one reported case of a fatal reaction to the smell of milk proteins (Bosetti et al., 1997). However, for other allergens such as natural rubber latex, there are many reports of anaphylaxis to aerosolized latex particles, and several instances of fatal anaphylaxis from physical contact with mucous membranes, as well as allergic reactions to latex contamination of food products due to gloves used by food handlers (Schwartz, 1995; Landwehr and Boguniewicz, 1996). These allergic reactions to low level allergen exposure highlight the contribution even a small amount of protein can make to the allergenicity of a GM food.

What Are the Most Common Food Allergens?

Nine groups of foods have been identified by an expert committee on food labelling (Agriculture and Agri-Food Canada, and Health Canada Food and Drug Regulations) as being the most likely to cause severe allergic and anaphylactic reactions in Canadians (Zarkadas et al., 1999). These foods are peanuts, tree nuts (almond, brazil nut, cashew, macadamia, hazelnut or filbert, pecan, pine nut, pistachio, walnut), cow’s milk, egg, fish, shellfish (crustaceans and mollusks), soy, wheat and sesame seeds. With the exception of sesame seeds, many of these foods appear on similar lists from the UK, US and the World Health Organization Codex committee (Hide et al., 1994; FAO/WHO, 1998; Zarkadas et al., 1999). These foods account for over 90% of the reported food allergies worldwide. However, a large number of food proteins may cause allergic reactions, and one list has documented 160 such food or food products (Hefle et al., 1996). In addition, allergies to raw fruits and vegetables causing the “Oral Allergy Syndrome”, a usually mild and common type of food allergy affecting the oropharyngeal mucosa, were often not included in epidemiological studies on food allergy. These allergies to raw fruits and vegetables may in fact be the most common single group of food allergies (Pastorello and Ortolani, 1996).

Can Genetic Modification Increase the Risk of Development of Food Allergy?

The development of food and other allergies requires a complex interplay of host and environmental factors. An atopic predisposition, that is an allergic genotype, is crucial to the development of allergies, although some disorders, in particular occupational asthma, can be induced in non-atopic individuals (those who have no genetic predisposition to allergic disorders). Whether the phenotypic expression of an allergic genotype occurs depends on multiple factors. The degree of allergen exposure is important, and in some cases repetitive, prolonged and high level of exposure increases the risk of allergy. However, for allergens such as peanut, sporadic and low level exposure appears to be sufficient to promote sensitization. Total dietary exposure is important and may explain why peanut allergy is more common in North America, rice allergy in Eastern Asia especially Japan, fish allergy in Scandinavia, chickpea allergy in India, wheat allergy in America and Europe (Lehrer et al., 1996), and, on a more local level, edible “bird’s nest” anaphylaxis in Singapore (Goh et al., 1999).

There is a concern that use of a transgene in a staple food, or a common transgene in several types of commonly ingested food, may increase the concentration of such a GM protein in the food stream or occupational environment and thereby increase the risk of development of allergy to that GM protein. Examples of non-GM foods introduced into the North American diet which then began to provoke allergic reactions as consumption increased include kiwi, mango, avocado, and other exotic fruits (Freye, 1989; Gall et al., 1984; Moneret-Vautrin, 1998). The same phenomenon has been occurring in Europe with the increased use of peanut as a food additive. This induction of food allergies by increasing total dietary exposure may be difficult to detect because of an initially low frequency in the population, and because years of ingestion may be required to provoke an allergic response.

The timing of introduction of an allergenic food can determine development of an allergy. Certain food allergies in children (e.g. cow’s milk, wheat, soy and egg allergies) are often self-limited and disappear in early childhood, whereas allergies to peanut, tree nuts, seafood and seeds are usually lifelong (Ham Pong, 1990; Zarkadas et al., 1999). Early introduction of these and other food proteins to the infant’s relatively immature immune system may encourage development of an allergy. Infants and young children therefore appear to be more susceptible to developing food allergies, resulting in a higher incidence. Conversely, delaying introduction of these foods (e.g. by breastfeeding exclusively until age 6 months) can lower the risk of development of an allergy by bypassing the crucial stage of an infant’s life when such a food allergy can be more easily induced by exposure (Host et al., 1999; AAP, 2000).

The potentially widespread use of GM food products as food additives and staple foods, including use in baby foods, may lead to earlier introduction of these novel proteins to susceptible infants either directly or via the presence of the maternally ingested proteins in breast milk. Several maternal dietary food proteins have been detected in breast milk, including bovine milk (betalactoglobulin), egg (ovomucoid and ovalbumin), wheat (gliadin) (Hemmings and Kulangara, 1978; Jakobsson and Lindberg et al., 1983; Cant et al., 1985; Harmatz and Bloch, 1988; Host et al., 1988), and peanut (Vadas 1999). Although controversial, there are sufficient studies to suggest that maternal avoidance of allergenic foods during breast-feeding can reduce the risk of atopic disease, in particular atopic eczema, in the breast-fed infant, and that exposure to these proteins while breast-feeding can promote allergic sensitization and allergic symptoms in the breast-fed infant (Jakobsson, 1983; Cant et al., 1985; Zeiger et al., 1986; Halkens et al., 1992; Zeiger and Heller, 1995; Chandra, 1997; Baumgartner et al., 1998; Ewan, 1998; Host et al., 1988; Vandenplas, 1998; Host et al., 1999; AAP, 2000). There is also the unconfirmed possibility that proteins from the diet of cows can contaminate cow’s milk resulting in indirect exposure especially to infants and young children. Early exposure to inhalant proteins also appears to affect allergy development in susceptible infants (Korsgaard and Dahl, 1983; Businco et al., 1988). One British study reported that 80% of peanut-allergic children had allergic reactions on their first known contact with peanut, indicating that they had previously been exposed inadvertently to peanut in order to become allergic, likely by such means as described above (Hourihane and Kilburn, 1997b).

There is also some evidence to suggest that prenatal sensitization to food and inhalant allergens can occur, and that maternal dietary avoidance during pregnancy may reduce the risk of allergy development in the child. This is an even more controversial area than sensitization via breast-feeding, although there is ample evidence that the human fetus can mount immune responses to in utero allergens from 22 weeks of gestation (Van Asperen et al., 1983; Renz et al., 1991; Piccinni et al., 1993; Jones et al., 1996; Warner et al., 1996; Jones et al., 1998). These issues highlight the susceptibility of children to allergenic dietary proteins, the potential risks to children of allergenic proteins even if consumed mostly by adults, and the risk of inducing food allergy in the population by widespread exposure to allergenic GM proteins.

Some proteins seem to be intrinsically more allergenic than others, and different varieties of the same plant vary in their allergen contents, including peanut, avocado and wheat (Taylor and Lehrer, 1996). Genetic engineering of food plants may have potential pleiotropic effects (collateral changes as a result of the transgene having an effect simultaneously on more than one characteristic) on the host, such as altering the intrinsic allergenicity of the protein itself (e.g. by glycosylation) or the amount of allergenic protein expressed.

The route of exposure of a food allergen can influence development of an allergy. Inhalation or frequent skin contact with certain proteins can provoke an occupational allergy or asthma, and examples of these include psyllium (a laxative derived from the husks of Plantago solidago), natural rubber latex, shellfish (snow crab and prawns), egg, horse dander, and grains (wheat and rye) (Chan-Yeung, 1990; James et al., 1991; Arlian et al., 1992; Anibarro et al., 1993; Kanny and Monteret-Vautrin, 1995; Witteman et al., 1995; Bush and Hefle, 1996; Fanta and Ebner, 1998; Moneret-Vautrin, 1998). Some of these occupationally sensitized workers who react to these proteins by contact or inhalation, may then develop allergic reactions when they ingest the product, as has been reported for egg, psyllium, mare’s milk and natural rubber latex. The transfer of a portion of honeybee venom allergen, melittin, to potatoes (Osusky et al., 2000) raises concern that if the antigenic epitope(s) of melittin happen to be included in the transgene product, commercialization of this GM potato could sensitize consumers to honeybee venom and thus predispose them to a potentially lethal insect sting allergy.

Can We Accurately Assess or Predict the Allergenicity of a Protein?

There are well-recognized specific immunological methods for detecting the presence and quantity of known allergens in a food product. The problem arises where the donor gene and its novel protein are not known to be allergenic, in which case specific immunoreactive diagnostic reagents to assess allergenicity (specifically, IgE from humans allergic to that protein) are not available. In such cases, indirect tests have to be relied on to assess the potential for allergenicity. These indirect tests are fairly non-specific, and therefore must be interpreted with caution. There is currently no single assay or combination that will accurately predict the allergenic potential of proteins from food or non-food sources not previously identified as being allergenic in human subjects.

Nevertheless, these indirect non-immunologic tests are the only techniques currently available to assess the allergenic potential of a novel protein. Full evaluation of a novel protein should include all the steps outlined below, unless allergenicity is confirmed or strongly suspected by initial testing. The National Academy of Sciences (2000) Committee on GM pest-protected plants stated, “The strong likelihood that gene products currently found in commercial transgenic pest-protected plants are not allergens does not remove the need for a minimum of properly planned and executed tests”. A novel protein that has undergone a properly designed and executed series of tests for allergenicity should be considered a low risk for allergenicity if all results are negative (Astwood and Fuchs, 1996b; Lehrer et al., 1996; Metcalfe et al., 1996; Kimber et al., 1999; National Academy of Science, 2000; Taylor, 2000). It would be prudent to monitor for any unanticipated allergic effects following introduction of a GM food where the transgenic protein is novel to the human diet. However, the joint FAO/WHO Expert Consultation Committee (FAO/WHO, 2000) felt that observational studies would be unlikely to identify any long-term adverse effects of GM foods against the background of undesirable effects of conventional foods.

Approach to Allergenicity Assessment

The transgenic protein should be evaluated by:

• Consideration of the source from which the donor gene is derived (i.e. the donor organism)
• Comparison of the donor protein to known allergens
• In vitro and in vivo immunologic analysis to assess allergenic potential
• Assessment of key physicochemical characteristics which are common to allergenic proteins
• Prevalence of known allergy to the donor protein
• Potential changes in endogenous host allergens as a result of gene transfer (pleiotrophic effect)

Source of Donor Gene

If the donor organism has known allergenic proteins, then it must be verified whether the transferred gene has introduced allergenic proteins to the host organism by using standard immunological assays. Donor genes from allergenic non-food sources such as pollen, fungal spores, insect venom, animal dander also need to be considered, if used for gene transfer. There is ample evidence that non-food allergens, if ingested, can provoke allergic reactions. These include royal jelly (secretions of worker honeybees), bee-collected pollen, plant parts (e.g. chamomile, echinacea and psyllium), housedust and storage mites, and mould proteins in flour and lactase enzyme (Subiza et al., 1989; Erban et al., 1993; Tee et al., 1993; Florido-Lopez et al., 1995; Kanny and Moneret-Vautrin, 1995; Binkley, 1996; Thien et al., 1996; Blanco et al., 1997; Moneret-Vautrin, 1998).

Donor genes from common food sources are easiest to evaluate because there is a clear history of previous consumption, presumably with data on presence and frequency of allergic reactions, and thus some availability of specific human IgE for testing. However, if the gene comes from an exotic food source, then previous consumption history by a large portion of the population is unknown, and human IgE to the food may not be available.

The allergenicity of a large number of non-food proteins that could be potentially used in genetic engineering is essentially unknown. Insecticidal crystal protein endotoxin from Bacillis thuringiensis (Bt) is considered safe because of a 30-year history of use as a microbial insecticide with exposure to workers by mostly contact, inhalation and, to a limited extent, ingestion, although a recent report suggests that exposure may cause some immune changes (production of IgG and IgE antibodies) of unclear significance as these were not associated with clinical disease (Bernstein et al., 1999). The common use of substantial levels of this protein (and its congeners) in GM corn and potato, however, is increasing exposure by ingestion, a route not usually encountered.

Comparison with Known Allergens

Many of the known allergenic epitopes (portions of protein responsible for the immune response) are at least 8 to 12 amino acids long. There are databases of known food and non-food allergens which can be used to compare amino acid sequences of novel proteins to known allergens. If a match occurs when comparing the transgenic protein to allergens, then that protein should be considered to be potentially allergenic. The FAO (WHO) expert consultation report (Taylor, 2000) and other authors have suggested a conservative approach of an identity match of eight contiguous amino acids indicating a positive index of allergenicity, although this conservative indexing may overestimate the number of potentially allergenic novel proteins.

Of the approximately tens to hundreds of thousands of proteins that can be found in a plant, only one or two are usually major allergens (Astwood and Fuchs, 1996b; Metcalfe et al., 1996). A major allergen is defined as an allergen to which over 50% of individuals allergic to that food react by in vivo or in vitro testing. Unfortunately, the amino acid sequences for allergenic epitopes are known for only a few allergens, and expanding current databases by identifying and characterizing more food allergens is an area requiring more research. Important foods where the allergenic proteins have been characterized include peanut, cow’s milk, egg, shrimp, codfish, soybean and wheat. Examining the amino acid sequence identifies only epitopes with a common linear amino acid sequence, but some allergens derive their allergenicity by virtue of their tertiary or 3D structure and not their linear structure (Metcalfe et al., 1996). The birch pollen allergen Bet V 3, for instance, contains discontinuous conformational epitopes. These conformational epitopes cannot be detected as being allergenic by linear amino acid sequencing and, indeed, most antibodies produced by an allergic individual to inhalant allergens appear to be toward discontinuous epitopes, but it is unknown whether this applies to antibodies to food allergens (Taylor and Lehrer, 1996).

In Vitro and In Vivo Immunologic Analysis

These are the most sensitive and specific tests of allergenicity. In vitro immunologic analysis assesses the immunochemical reactivity of the transgenic protein and requires specific IgE in sera from individuals known to be allergic to the donor protein in order to determine whether such a protein is allergenic. These assays cannot be performed if no allergic individuals are available to provide IgE for testing. If the donor protein is known to be allergenic, then these are the first tests to be performed and a positive reaction confirms allergenicity. However, occupationally exposed workers can develop IgE antibodies to a protein without clinical allergic disease, and they may then serve as a source of these antibodies for assays. Of interest, therefore, is the recent detection of IgE antibodies to Bt spores in some exposed farm workers since Bt genes have been used extensively in GMOs (Bernstein et al., 1999). Conversely, these assays can also be used to detect the presence of IgE antibodies in an individual to a GM protein in order to assess whether that person may have developed a potential allergy.

In Vitro Assays

In vitro assays can detect the presence and quantity of allergenic proteins in a food, and to a certain degree whether the allergenicity of the protein has been altered. These assays include solid-phase immunoassays such as the radio allergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA), and their respective inhibition assays; immunoblot techniques (e.g. SDS-PAGE); and less commonly used for food allergy evaluation, immunoelectrophoresis and crossed radio immunoelectrophoresis. RAST and ELISA can detect the presence of the test allergen in a GM food and inhibition immunoassays are even more useful to assess both the presence and degree of allergenicity of such a protein. Inhibition assays have been used to detect traces of allergenic proteins contaminating foods, to assess the effects of processing on allergenicity of peanut and soybean-derived products, and to test the allergenicity of a transgenic soybean bio-engineered with a brazil nut gene. Health Canada (Food Research Division, Food Directorate) currently has the ability to test for peanut, egg, soy, milk and hazelnut proteins, using competitive enzyme immunoassays as part of its evaluation process for allergens contaminating processed foods (Health Canada, 2000). Rapid dipstick immunoassay kits for detection of allergenic proteins as food contaminants are being developed (Clare Mills et al., 1997). SDSPAGE with immunoblotting is an excellent method for the separation and detection of allergens, especially where multiple allergens exist in a food.

An accurate immunologic analysis depends on the quality of the material used. Allergenic food proteins have not been standardized, and are thus subject to significant variability in quality, depending on the source. Availability of purified standardized food allergens in sufficient quantities (e.g. derived from recombinant DNA technology) would reduce this variability, and this represents another area requiring research. Reliability of human sera from individuals allergic to the donor protein may be compromised by a) misdiagnosis — the person is not truly allergic, or b) the person may be allergic to only one or some of several possible allergens in the donor food. To overcome these drawbacks, Metcalfe and colleagues (1996) proposed an approach developed by the International Food Biotechnology Council and the Allergy and Immunology Institute of the International Life Sciences Institute (IFT, 2000). They have suggested that serum donors should meet rigid criteria for diagnosis of an allergy (clear-cut, convincing, severe allergic-type reaction to isolated ingestion of that food), and/or positive double-blind, placebo-controlled oral food challenge (DBPCFC) (Bock, 1980; Bock et al., 1988). These authors also suggested that sera from at least 14 such allergic individuals should be used for in vitro assays, for appropriate reliability. If these give negative results, they suggest going on to in vivo assays such as allergy prick skin test, and if necessary, DBPCFC.

One potential drawback of current guidelines for allergenicity assessment of donor proteins from known allergenic sources is the usual presence of multiple allergens in a particular food source, examples of which include peanut, soy, egg and cow’s milk. When 14 test sera are used to assess allergenicity of a GM food, and all are negative, this provides greater than 99.9% assurance that a major allergen from the donor organism has not been transferred, and greater than 95% assurance that a minor allergen affecting at least 20% of the sensitive population has not been transferred (Metcalfe et al., 1996; Taylor, 2000). This would leave a small number of food allergic individuals who are allergic only to minor food allergens at risk if that transgenic food is declared “non-allergenic” on the above statistical basis. However, it is worth emphasizing that a minor food allergen (an allergen to which less than 50% of individuals allergic to that food are allergic) is just as capable of causing severe reactions as a major allergen.

A review of brazil nut-allergic test subjects used to assess allergenicity of a transgenic soybean containing brazil nut allergens showed that one of the nine test subjects was allergic only to a minor brazil nut allergen (Nordlee et al., 1996). If only a minor allergen from a host source is transferred to a GM food, and the frequency of allergy specifically and solely to the minor allergen is less than 20% of individuals allergic to the host food, then there is some chance, albeit low, that the battery of sera used will not contain IgE to that minor allergen. In such a case, immunological assays may not detect the presence of the minor allergen in the GM food, and other steps in the evaluation process will have to be carried out.
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Re: ELEMENTS OF PRECAUTION: RECOMMENDATIONS FOR THE REGULATI

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Part 2 of 2

In Vivo Studies

Further analysis of allergenicity can be performed using human subjects known to be allergic to the donor protein. This consists of allergy prick skin testing using suitable concentrations of extracts of the host food, the native donor food, and the GM food. These extracts are pricked into the epidermal layers of the allergic individual’s skin, and observed for a localized allergic reaction consisting of a hive at the test site within 15 minutes. This reaction indicates that the test subject’s immune system has identified that particular food as carrying an allergen against which the subject has previously developed IgE antibodies. If desired, these volunteers can be further evaluated by DBPCFC with the GM food to confirm the presence or absence of that particular allergenic protein in the GM food. The confirmation of the presence of an allergen by DBPCFC is the most reliable method, but often is not practical because it requires the physical presence of human volunteers. DBPCFC will most likely be necessary for the final evaluation of a GM food containing a gene from a known allergenic source when all previous evaluations show no indication of allergenicity.

The detection of specific IgE antibodies in an individual does not necessarily mean the presence of a clinical allergy. Other factors may also determine whether an allergic reaction occurs, and those with IgE antibodies but no symptoms on exposure to the relevant allergen have “asymptomatic sensitivity”. In the case of food allergy, only 30% to 40% of individuals with IgE antibodies to foods will have allergic reactions on ingesting the food (Bock et al., 1988; Sampson, 1988). However, high levels of IgE antibodies do increase the probability of a clinical allergy.

The reliability of prick skin testing is clearly affected by the quality of the food allergen extract used. Prick skin tests to some foods, especially fruits and vegetables, must be performed with freshly prepared extracts due to the labile nature of the allergenic protein. Improper extraction of the food proteins may lead to inadequate concentrations of relevant allergens in the skin test extract, which results in false negative tests. Processing, heating or digestion of a protein can destroy protein antigenicity, but it can also enhance the allergenicity by formation of new epitopes or neonantigens. In these cases, allergy testing with the native food may also produce false negative results, which may explain some case reports of allergic reactions to sesame seeds without demonstrable IgE sensitization (Eberlein-Konig et al., 1995). This emphasizes the need in selected cases for food challenges (DBPCFC).

Animal models for in vivo testing may be useful in certain circumstances but there is no animal model currently able to predict accurately human allergic responses and therefore donor protein allergenicity. Examples of current animal models for allergy evaluation include mouse models to evaluate IgE responses to recombinant allergens, and guinea pig and rat models of anaphylaxis. The difficulties in using animal models to assess allergic potential have been documented (Metcalfe et al., 1996; Taylor and Lehrer, 1996; Kimber et al., 1999). An appropriate animal model of food allergy must be such that the test animal is able to mount a human-type IgE antibody response to foods under near natural conditions; that is, it must be able to mount a significant IgE antibody response to the food allergen in question, by the usual provocation route, oral exposure. At the same time, it must be able to tolerate the majority of food proteins. In addition, the animal’s allergic responses should be similar to those seen in humans, be consistent and easily reproducible. Unfortunately, no such model exists. Current animal models mount IgE responses only with difficulty, and under abnormal conditions such as induction by injection of the allergen together with adjuvant to enhance the immune response. Even in these models, the IgE responsiveness can vary at different times under the same conditions.

Use of animal models to screen for potential immunogenicity (ability to mount an immune response by producing IgG antibodies) and using this response as an indicator of potential allergenicity or ability to induce IgE has met with some significant failures with respect to human foods. Guinea pig and rabbit animal models were used to assess the allergenicity of partially hydrolysed cow’s milk whey formulas. These animal models predicted reduced immunogenicity of the whey hydrolysate formulas which were then marketed as “hypoallergenic”, but in fact they remained sufficiently allergenic to cause reactions in most cow’s milk-allergic infants (Palud et al., 1985; Taylor and Lehrer, 1996; Host et al., 1999; AAP, 2000). Assessment of the brazil nut albumin protein in transgenic soybean for potential allergenicity using a mouse model of passive cutaneous anaphylaxis did not elicit an allergic or immune response, leading to the erroneous conclusion that there was no allergenic protein transfer to the soybean (Astwood and Fuchs,1996b; Nordlee et al., 1996).

More reliable animal models mounting human-type IgE responses as described above have the potential to reduce the present dependence on human sera. These could be developed through standard breeding and selection, or perhaps even transgenically. Kleiner et al. (1999) and Li et al. (2000) have recently developed mouse models of cow’s milk allergy and peanut allergy.

Physicochemical Characteristics

Protein allergens tend to have certain characteristics such as molecular weight between 10 and 70 kiloDaltons (kDa), and resistance to acid and proteolytic enzyme digestion (i.e. resistance to gastric digestion). They are usually proteins, often glycosylated (sugar compounds attached to the protein), are relatively stable to heating, have acid isoelectric points, are often water-soluble albumins or salt-soluble globulins, and usually make up a significant percent (1% to 80%) of the protein content of the source material (Matsuda and Nakamura, 1993; Astwood and Fuchs, 1996b; Bush and Heffle, 1996; Metcalfe et al., 1996). GM food proteins showing diagnostic physicochemical characteristics of allergens such as molecular weight, and stability to heat and gastric digestion may then be considered to have a higher potential for allergenicity, if direct immunologic assays are not available.

However, these are not particularly reliable indicators. There are a number of heat-labile or partially heat-labile food proteins which denature and lose conformational epitopes on heating, such as cow’s milk whey beta-lactoglobulin and bovine serum albumin, chicken egg ovomucoid, rice glutenin and globulin, soy glycinin and some peanut proteins (Matsuda and Nakamura, 1993; Bush and Hefle, 1996; Taylor and Lehrer, 1996). Similarly, while food allergens often constitute a significant portion of a food’s proteins, as mentioned previously, the potency of an allergen may compensate for its relative paucity in a food, and some major allergens such as codfish Gad c 1 are present only in small proportions of the food. Likewise, some protein allergens are low molecular weight such as the 9 kDa plant lipid transfer proteins (LTP) which are important allergens of the Prunoideae family which include peaches, plums and cherries (Breiteneder and Ebner, 2000; Rodriguez et al., 2000); LTP from barley used in beer foam formation (Curioni et al., 1999); and the 8 kDa soybean hull protein responsible for asthma outbreaks in Spain (Gonzalez et al., 1991). Interestingly, heating some allergenic proteins may actually increase their allergenicity, in some instances by chemical glycosylation (Maillard reaction). Examples include cow’s milk beta-lactoglobulin, pecan, fish, shrimp, snow crab and limpet (Malanin et al.,, 1995; Berrens, 1996; Taylor and Lehrer, 1996; Moneret-Vautrin, 1998).

It should be noted that some allergenic compounds are not proteins. Known examples are shrimp transfer RNA, inulin (a carbohydrate); and vegetable gums such as carrageenan and tragacanth (Danoff et al., 1978; Yeates, 1991; Tarlo et al., 1995; Bush and Hefle, 1996; Gay- Crosier et al., 2000). In addition, a large number of foods, in particular raw fruits, raw vegetables and spices, have heat-labile proteins (e.g. chitinases and Bet v 1) which cause the Oral Allergy Syndrome. All these exceptions would not be identified as allergens by their physicochemical criteria, or by any other criteria if they were present as a novel protein GM.

Prevalence of Allergy to the Donor Protein

If the prevalence of an allergy to the donor protein is very low, then it may not be recognized or it may be very difficult to obtain sufficient amounts of sera from allergic persons to adequately test for the presence of allergens. Limited testing can lead to missed allergenic characteristics. Metcalfe and colleagues (1996) and Taylor (2000) have suggested that a lower standard of assurance of absence of allergen transfer be used where limited human sera is available for testing. However, this limitation could be overcome by establishing a registry and/or a bank of serum from allergic people.

Potential Changes in Host Allergenicity

When a host organism is being genetically engineered, it must be ensured that the modified host organism has not undergone pleiotropic changes that result in creation of novel allergens. These effects could include the GMO being induced to express higher levels of its own endogenous allergens beyond what might be expected from natural variability; or endogenous or transgenic proteins undergoing post-translational modification including glycosylation or alteration of its 3D structure, perhaps changing allergenicity or creating new allergens. These possibilities can be assessed using the in vitro immunological assays described above. Such changes could increase the severity of an allergy reaction in persons already allergic to the host or donor food, and increase total dietary exposure to a more allergenic food.

Genetic engineering may affect endogenous allergen content in several ways, including altering the host plant metabolic pathways and enhancing allergen production. Storage, and effect of plant hormones such as ethylene, are known to increase the allergenicity of foods such as apple, banana and peach. Stress may also increase the levels of allergenic proteins (e.g. Bet v 1) in some fruits and vegetables (Hsieh et al., 1995; Pastorello and Ortolani, 1996; Breiteneder and Ebner, 2000; Rodriguez et al., 2000; Sanchez-Monge et al., 2000). Different varieties of foods such as peanut, avocado and wheat vary in their allergen content (Bush and Hefle, 1996) and these levels could conceivably be altered further by genetic modification.

Other Considerations in Allergenicity Assessment

Some factors may affect results of allergenicity evaluation, and may need to be considered in assessing or designing studies. The detection of clinically important food allergens may be complicated by the presence of cross-reactive allergens in the food. These cross-reactive allergens may show some similarity to the test allergenic protein and may give positive results, usually weakly, using in vivo and in vitro immunologic assays, but may not cause true allergic reactions. Botanically related plants may have cross-reactive allergens, examples of which are legumes such as peanut, peas, beans and soy. Thus, a peanut-allergic person may have IgE antibodies to peas but can eat peas without allergic reactions. Clusters of allergens also occur when distinct, nonbotanically related plants and foods share similar allergens, as in the birch/celery/spice syndrome, otherwise known as the Oral Allergy Syndrome, where individuals allergic to Bet v 1, the major allergen of birch pollen, also have allergic reactions to homologous pathogenesis-related proteins found in certain fruits, nuts, vegetables and spices (Halmepuro et al., 1984; Breiteneder and Ebner, 2000; Rodriguez et al., 2000). Another important food allergy cluster is the latex-fruit syndrome due to an allergy to Hev b 2, a pathogen-induced endoglucanase enzyme found in natural rubber latex but found also in avocado, banana, chestnut and kiwi (Moller et al., 1998; Breiteneder and Ebner, 2000).

Certain homologous proteins are also found in widely varying species such as tropomyosin in shrimp, chicken, mosquito, cockroach and housedust mites, although the cross-reactivity is probably low (Bush and Hefle, 1996). The degree of sequence homology is important. The major shrimp allergen, tropomyosin, is a protein present in many other foods including beef, pork and chicken with which it shares 60% sequence homology, but beef, pork and chicken tropomyosin are rarely allergenic (Lehrer et al., 1996). In these cases, a definitive answer regarding allergenicity can only be based on specific IgE-based assays.

Some food allergies, such as those involved in the Oral Allergy Syndrome, produce allergenic effects on the oral mucosa during mastication of the food. Oral challenges with these foods by swallowing (e.g. in capsule form as is the preferred method for DBPCFC rather than chewing) may not reproduce the allergic reactions seen in the real-life process of chewing and eating. Foods normally eaten cooked but which may occasionally be handled or eaten raw (e.g. potato) may show a different allergenicity profile depending on how the food is presented for oral challenge. The converse situation also needs to be considered (i.e. foods which increase their allergenicity on heating).

If an allergen is expressed in the host organism, the site of expression such as in the leaves, pollen or edible part of the plant is important in considering risk. There would be different implications if the allergen is not expressed in the edible but rather in the non-edible portion of the plant, or if the allergenic plant proteins might be inhaled during processing of plant parts with the accompanying risk of occupational sensitization.

An Example of the Evaluation Process to Assess Allergenicity

A useful model to examine is the evaluation process used by the US Environmental Protection Agency (USEPA) for potential allergenicity of the Cry9C gene from Bt encoding for an insecticidal crystal protein endotoxin, inserted into GM corn, using the criteria described above (USEPA, 1999a, b). The USEPA used an approach developed during the 1994 Interagency Conference on potential allergenicity in transgenic food crops, which included the USEPA, Food and Drug Administration, and Department of Agriculture (Fox, 1994; USEPA, 1999a). They evaluated the source of the donor gene, which had no known allergenic history despite 30 years of use as a microbial insecticide. However, Bt is not a food product, and this particular BT gene had been modified and therefore had a much shorter exposure history. Comparison with known allergens showed no epitope sequence homology and therefore no resemblance. Immunologic analysis was limited by the absence of material from humans clinically allergic to BT since none has been identified. A brown Norway rat model of IgE immune response was inconclusive although an immune response was provoked. Physicochemical characteristics showed that the Cry9C protein did show some stability to simulated gastric fluid digestion and some heat stability. In addition, its molecular weight of 68.7 kDa fell at the upper end of the range for allergen molecular weight. These physicochemical characteristics suggested a potential for allergenicity, although it was present at a low level (0.17% of total weight), features not usually seen with important allergens. Potential pleiotropic effects were examined by screening the genetically altered Cry9C corn with serum from suspected corn-reactive subjects, which did not detect any alterations in the intrinsic allergenic status of the GM corn.

On the basis of two positive biochemical characteristics found in allergens (relative stability to heat and gastric digestion), the USEPA declined to upgrade Cry9C Bt corn for use as human food in 1999 and left unchanged the approval as an animal feed and for industrial use. Other Bt genes encoding similar endotoxins (e.g. Cry1A and Cry3A) have been approved for human consumption in GM corn and potato as they did not demonstrate the biochemical characteristics shown by the Cry9C gene product, nor did they exhibit any other signs to indicate potential allergenicity (USEPA, 1995).

Unfortunately, the Cry9C corn (termed StarLink (TM)) inadvertently contaminated corn destined for human use resulting in a large recall of corn-derived food products in the US in October 2000. In addition, Cry9C protein was discovered in some non-StarLink seed corn, and although this was felt to be due to physical contamination, cross pollination from StarLink corn could not be ruled out as the source. The accidental introduction of StarLink corn into the human food chain prompted a further review of the potential allergenicity of Cry9C, and of mechanisms for assessing suspected allergenic reactions to StarLink corn. This review was conducted by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP), the primary scientific peer review mechanism of the USEPA (FIFRA, 2000; US EPA, 2000a).

FIFRA-SAP concluded that the Cry9C protein had a medium likelihood of proving to be a potential allergen. They considered that at least 7 of 34 complaints regarding reactions to a corncontaining meal were probably allergic, based on a careful assessment of the history which was compatible with a food allergy, plus opportunity for exposure to the suspected protein, as well as confounding factors (e.g. other allergens). In the final analysis, the determination of whether an allergic reaction occurred to the Cry9C protein in StarLink corn would have to be based on detection of the Cry9C protein in the ingested food, detection of antibodies, especially IgE to Cry9C protein in the subjects’ serum, and if necessary oral food challenge (DBPCFC).

At the strong urging of the USEPA, StarLink corn was voluntarily withdrawn from agricultural use, and all known existing corn contaminated with it has been restricted for nonhuman food use (USEPA, 2000b). Unaccounted StarLink corn could continue to enter the food supply over the next few years, however. This incident underscored the difficulty of restricting a GM food for animal/industrial use when almost indistinguishable non-GM food counterparts are simultaneously available for human consumption. It also highlights the issue of mandatory labelling (discussed in Chapter 9, Part 2). Post-introduction surveillance of a GM food which has medium to high-risk allergenic potential is essential. This medium- to high-risk scenario must be accompanied by appropriate labelling to identify allergic reactions rapidly and accurately. However, where the risk of allergenicity is low, the justification for labelling diminishes from a scientific viewpoint, bearing in mind that the lack of labelling can lead to delayed recognition of the emergence of an allergy, with consequent under-reporting. Therefore, especially when labelling is not required, there should be mechanisms to record, evaluate and fully investigate complaints of suspected allergy, as recommended above for StarLink corn by FIFRA-SAP. In addition, since there may be potential exposure to multiple unlabelled GM proteins from different sources, evaluation of the subjects may have to include a battery of dietary GM proteins.

It could be argued that requiring GM proteins and GM foods to undergo rigorous assessment for allergenicity prior to approval as a food product, with or without labelling, represents a double standard since this process is not required when a novel or exotic non-GM food is introduced. An exotic food may still pose a risk because while there is some history of previous consumption, it may not have been extensive or monitored sufficiently to ensure its safety, and the genetic susceptibility to allergies in its native area may differ. On the other hand, the presence of an exotic or novel food in the diet is more easily identifiable, avoidable, and more easily monitored for the possibility of adverse reactions, compared to a transgenic protein in a food which may not be easily monitored as regards degree and frequency of exposure, or even whether exposure has occurred at all.

Notwithstanding the limits of current technology, a GM food which has undergone a thorough, scientifically valid evaluation process for allergenicity, with negative results, should be considered at low risk to provoke or induce allergic responses and could possibly even be safer than a non-GM novel or exotic food which has not been subjected to the same scrutiny. This evaluation process can significantly minimize allergenicity concerns and perhaps reduce the chances of GM products being used as scapegoats for a variety of real or perceived illnesses. It stands to reason that any GM food with potential or identified allergenicity must either not be approved for human consumption, or if approved, then it must be appropriately labelled.

Summary

The identification of potential allergens in GMOs is accurate and reliable when assessing transgenes from known allergenic sources. It is indirect and non-specific with respect to novel proteins from sources not known to be allergenic and without a history of extensive human exposure. Even for the nine identified major food allergens responsible for most of the severe allergic reactions to foods in Canadians, only some of the allergens have been chemically characterized, and none has been standardized. In vivo and in vitro techniques are available to assess accurately and reliably potential allergenicity when dealing with proteins from known allergenic sources. Where the donor gene comes from an organism not known to be allergenic, or of unknown allergenicity (e.g. an exotic food, or a product not normally ingested as food), assessment becomes more difficult. There is currently no single assay or combination of assays that will accurately predict the allergenic potential of protein from sources not known to be allergenic. Nevertheless, using an array of properly designed and executed assays, and knowledge regarding the characteristics of the transgene, a GM food may then be considered relatively safe for allergic consumers and comparable to its non-GM counterpart, if all tests are negative. Not withstanding negative allergenicity assessments, however, if the transgene is derived from a source of unknown allergenicity, post-introduction surveillance may be prudent to monitor for any unanticipated allergic effects, recognizing that this may be more difficult without corresponding labelling of GM foods.

RECOMMENDATIONS

4.4 The Panel recommends that the Canadian government should support research initiatives to increase the reliability, accuracy and sensitivity of current methodology to assess allergenicity of a food protein, as well as efforts to develop new technologies to assist in these assessments. This would include further research into the identification, purification, characterization and standardization of common food allergens, as well as their respective antibodies (e.g. monoclonal animal antibodies) which can be used in detection systems; development of reliable animal models of human-type IgE antibody responses; identification of specific characteristics which can accurately and specifically identify a novel protein as being allergenic; and development of rapid assays (e.g. dipstick-type assays) for use by food processors and consumers to detect allergenic contaminants.

4.5 The Panel recommends the strengthening of infrastructures, and where none exists, development of these infrastructures to facilitate evaluation of the allergenicity of GM proteins. This could include development of a central bank of serum from properly screened individuals allergic to proteins which might be used for genetic engineering, a pool of standardized food allergens and the novel GM food proteins or the GM food extracts, maintenance and updating of allergen sequence databases, and a registry of food-allergic volunteers. These would enhance the ability of government agencies such as the Canadian Food Inspection Agency to broaden the scope of and its technological ability to detect allergenic proteins.

4.6 The Panel recommends development of mechanisms for after-market surveillance of GM foods incorporating a novel protein, if there are data to indicate its effectiveness, to detect the emergence of consumers developing allergies to such a food either through increase in total dietary exposure over the long term, or occurrence of unanticipated and unpredicted allergic reactions. This could include a central reporting registry and/or epidemiological studies to assess changes in frequency, pattern and clinical presentations of allergy-related complaints. The infrastructure in Recommendation 4.5 could be used to verify scientifically reports of allergic reactions and detect emergence of allergies to GM proteins.

4.7 The Panel recommends that the appropriate government regulatory agencies have in place a specific, scientifically based, comprehensive approach for ensuring that adequate allergenicity assessment will be performed on a GM food, utilizing currently available techniques combined with currently available knowledge of the characteristics of the GM protein relevant to potential allergenicity, and updating testing requirements in keeping with new technologies. Any decision not to complete a full and comprehensive allergenicity assessment should be made only after careful consideration of the scientific rationale to support that omission. The decision to approve or not approve introduction of a GM food and the need for labelling should therefore be based on a rigorous scientific rationale.

4.8 The Panel recommends that approvals should not be given for GM products with human food counterparts that carry restrictions on their use for non-food purposes (e.g. crops approved for animal feed but not for human food). Unless there are reliable ways to guarantee the segregation and recall if necessary of these products, they should be approved only if acceptable for human consumption. If a GM food is found to have acquired additional allergenic properties from gene transfer, then that GM food should either not be marketed, or be properly labelled if marketed

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Sampson, H.A., L. Mendelson, J.P. Rosen. 1992. Fatal and near-fatal anaphylactic reactions to food in children and adolescents. N. Engl. J. Med. 327: 380–84.

Sanchez-Monge, R., C. Blanco, A. Diaz Peralos et al. 2000. Class 1 chitinases, the panallergens responsible for the latex-fruit syndrome, are induced by ethylene treatment and inactivated by heating. J. Allergy Clin. Immunol. 106: 190–95.

Schwartz, H.J. 1995. Latex: a potential hidden “food” allergen in fast food restaurants. J. Allergy Clin. Immunol. 95: 139–40.

Sicherer, S.H., T.J. Furlong, J.D. DeSimone et al. 1999. Peanut allergic reactions on commercial airlines. J. Allergy Clin. Immunol. 103: S237.

Steinman, H.A. 1996. “Hidden” allergens in foods. J. Allergy Clin. Immunol. 98: 241–50.

Subiza, J., J.L. Subiza, M. Hinojosa et al. 1989. Anaphylactic reaction after the ingestion of chamomile tea: a study of cross-reactivity with other composite proteins. J. Allergy Clin. Immunol. 84: 353–58.

Tarlo, S.M., J. Dolovich, C. Listgarter. 1995. Anaphylaxis to carrageenan: a pseudo-latex allergy. J. Allergy Clin. Immunol. 95: 933–36.

Taylor, S.L. 2000. Assessment of the allergenicity of genetically modified foods. Joint FAO/WHO expert consultation on foods derived from biotechnology. Topic 13. Allergenicity 29 May–2 June 2000.

Taylor, S.L., S.B. Lehrer. 1996. Principles and characteristics of food allergens. Crit. Rev. Food Sci. Nutr. 36(S): S91–S118.

Tee, R.D., D.J. Gordon, J.A. Welch et al. 1993. Investigation of possible adverse allergic reactions to mycoprotein (Quorn). Clin. Exp. Allergy 23: 257–70.

Thien, F.C.K., R. Leung, B.A. Baldo et al. 1996. Asthma and anaphylaxis induced by royal jelly. Clin. Exp. Allergy 26: 216–22.

USEPA (US Environmental Protection Agency). 1995. Plant pesticide bacillus thuringiensis CryIII A delta endotoxin and the genetic material necessary for its production: tolerance exemption. Fed. Regist. 60: 21725–28.

USEPA. 1999a. USEPA biopesticides. AgrEvo response to EPA background document. December 1999. At: <www.epa.gov/biopesticides.cry9c/cyy9c-peer_review.htm>

USEPA. 1999b. Biopesticides Cry9C Peer Review. Cry9C food allergenicity background document. 15 Dec 1999. At: <www.epa.gov/biopesticides/cry9c/cyy9c-peer_review.htm>

USEPA. 2000a. Statement by Stephen Johnson, EPA Deputy Assistant Administrator for Pesticides regarding StarLink Corn. 12 Oct, StarLink Corn News Archive. At: <http://www.epa.gov/pesticides/biopesticides/otherdocs/starlink_news.htm>

USEPA. 2000b. Revised Updated Safety Assessment of StarLink (TM) Corn Containing Cry9C Protein. 3 Nov. At: <http://www.epa.gov/scipoly/sap/>

Vadas, P. 1999. Personal communication presented at the 1st Annual Anaphylaxis Symposium, Toronto, Canada.

Van Asperen, P.P., A.S. Kemp, C.M. Mellis. 1983. Immediate food hypersensitivity reactions on the first known exposure to the food. Arch. Dis. Child. 58: 253–56.

Vandenplas, Y. 1998. Myths and facts about breastfeeding: does it prevent later atopic disease? Nutr. Res. 18: 1373–78.

Warner, J.A., A.L. Jones, E.A. Miles et al. 1996. Maternofetal interaction and allergy. Allergy 51: 447–51.

Witteman, A.M., J. van Leeuwen, J. Van der Zee et al. 1995. Food allergens in house dust. Int. Arch. Allergy Immunol. 107: 566–68.

Yeates, H.M. 1991. “Chronic Anaphylaxis Caused by Ingestion of Vegetable Gum Products.” Presented at the 47th annual meeting of the American Academy of Allergy and Immunology, San Francisco.

Yman, I.M. 1995. Food-induced hypersensitivity reactions: a survey over the last 5 years. Allergologie 18: 403.

Yunginger, J.W., G. Kristin, M.D. Sweeny et al. 1988. Fatal food induced anaphylaxis. JAMA 260: 1450–52.

Zarkadas, M, F.W. Scott, J. Salminen et al. 1999. Common allergenic foods and their labelling in Canada – A review. Can. J. Allergy Clin. Immunol. 4: 118–41.

Zeiger, R.S., S. Heller. 1995. The development and prediction of atopy in high risk children: follow-up at age seven years in a prospective randomized study of combined maternal and infant food allergen avoidance. J. Allergy Clin. Immunol. 95: 1179–90.

Zeiger, R.S., S. Heller, M. Mellon et al. 1986. Effectiveness of dietary manipulation in the prevention of food allergy in infants. J. Allergy Clin. Immunol. 78: 224–38.

PART 3. NUTRITION ISSUES

Introduction


A central concern in any modification of traditional food sources must be the impact of such changes on the nutrient content of the food. The components generally considered under this rubric are the content of carbohydrates (simple and complex), proteins and their constituent amino acids, fats and their fatty acid profiles, vitamins, dietary fibre and anti-nutrients. No crop species, in itself, provides an appropriately balanced range of nutrients for human or animal consumers. A diet that effectively meets metabolic needs, therefore, must be derived from multiple sources that complement each other’s nutrient strengths and deficiencies. Nutritionists, dietitians and food specialists work with databases such as the Canadian Nutrient File, which are designed to reflect the average amounts of nutrients in individual foods. Based on such data, which are derived from the history of the commercially grown varieties as a food source, a common crop (e.g. potatoes or corn) would be expected to provide certain nutrients within a known range. If the concentration of a particular nutrient happened to fall at or beyond the extremes of the range, there could be health implications, particularly for humans who rely heavily on that foodstuff in their diet.

Another nutritional parameter that normally attracts regulatory attention is the level of specific anti-nutrients in some foodstuffs. While the definition of an anti-nutrient remains unclear, the term generally refers to plant secondary metabolites that appear to have deleterious effects over time on animal or human consumers. Examples of anti-nutrients whose levels are usually monitored in new Canadian crop varieties include erucic acid and glucosinolates in canola, cyanogenic glycosides in flax, and glycoalkaloids in potatoes.

Impacts of Genetic Engineering

Genetic engineering of common crops in Canada has thus far not focused on nutrient modification, and any impacts on the major nutrients in these first generation GM crops would presumably have to be the result of pleiotropic effects of the transgene(s). To date, GM foods produced from approved GM crops have been judged to be nutritionally equivalent to their non- GM counterparts, presumably on the basis of chemical analyses for the classes of nutrients described above. However, the Panel is unaware of any public data available for confirmation of this assumption. In fact, the only nutritional information related to GM foods available to the public appears in the Decision Documents released by the CFIA for animal feeds (CFIA, Plant Biotechnology Decision Documents, at: www.cfia-acia.ca/english/plaveg/pbo/isda01_.html). This is restricted to a statement about proximate analysis (an unsophisticated procedure that analyzes the material for crude protein, crude fat, ash and moisture levels) and in some cases examines certain groups of amino acids, together with the comment that anti-nutrients did not exceed acceptable levels.

New GM varieties specifically designed to present altered profiles of fatty acids, altered starch qualities, and/or altered protein profiles are all currently under development. Some of the proposed changes have the potential to improve the foodstuff’s nutritional quality, such as GM corn whose storage proteins contain an enhanced level of lysine, the limiting amino acid in that food. Other nutritional modifications being explored include increased vitamin content (e.g. carotenoids, a source of vitamin A – as in “golden rice”), higher iron content, and enhanced concentrations of nutraceuticals such as lignans and bioflavonoids (antioxidants). This ability to fortify traditional foodstuffs is expected to be marketed as a direct consumer benefit of GM foods. While positive impacts can be envisioned, any substantial alteration of food nutrient profiles has potential ramifications that would appear to call for careful monitoring and public reporting.

Testing

Chemical analysis provides the first level of assessment of possible changes in nutrient content in novel foods. Very powerful methodologies are now available for analysis of protein, fatty acid and carbohydrate profiles, as well as scanning for changes in secondary metabolite profiles (see Chapter 7). Nevertheless, food is a complex material with many potential interactions among its components that would be hard to predict simply by scoring the individual chemical classes. Where significant deviations from the profile ranges expected for a food component are detected, it may therefore be desirable to conduct whole organism tests designed to assess nutrient bioavailability. This is analogous to the need, discussed earlier in this chapter, to determine whether novel foods bring with them any new toxicological risks.

The assessment could involve either animal testing, where a suitable animal model has been developed, or testing in human subjects. A foodstuff could be tested as part of a diet fed to experimental animals, which could then be monitored for health and growth over their normal lifetime. Where chemical analysis has detected changes in particular food components, it may be more useful to examine the impacts of those changes by using the specific component as a dietary ingredient. Proteins have been evaluated in this fashion in experimental animals for at least four decades (Campbell and Chapman, 1959). In the early tests done in rats, protein was 10% by weight of the diet and the duration of the feeding was four weeks. A faster method, which gives similar results and involves using an amino acid profile corrected for the digestibility of the protein, took no more than two weeks (Sarwar and McDonough, 1990). This method has been adopted by the FAO/WHO Expert Consultation (1991) on protein quality evaluation. Suitable tests should be available for evaluating specific fat and/or carbohydrate compositions, while testing the impacts of discrete anti-nutrients would follow the well-established protocols developed for toxicological testing.

Human foods differ from animal feeds in that the emphasis is less on rapid weight gain in a shortened life span, or on enhanced milk production, and more on a long, healthy life as free as possible from disease. Over the many decades of human life, foods are expected to provide all recognized nutritional requirements. Relatively short-term animal tests may yield valuable information, but establishing the impacts of long-term ingestion of a food would involve the systematic monitoring of human populations. This issue is clearly related to the question of labelling of GM foods, and has been explored in Chapter 9 of this Report.

RECOMMENDATIONS

4.9 The Panel recommends that all assessments of GM foods, which compare the test material with an appropriate control, should meet the standards necessary for publication in a peerreviewed journal, and all information relative to the assessment should be available for public scrutiny. The data should include the full nutrient composition (Health Canada, 1994), an analysis of any anti-nutrient, and where applicable, a protein evaluation such as that approved by FAO.

4.10 The Panel recommends that protocols should be developed for the testing of future GE foods in experimental diets.

4.11 The Panel recommends that the Canadian Nutrient File should be updated to include the composition of GE foods and be readily available to the public.

REFERENCES

Campbell, J.A., Chapman, D.G. 1959. Evaluation of protein in foods. Criteria for describing protein value. J. Can. Dietetic Assoc. 21: 51–58.

FAO/WHO Expert Consultation. 1991. Protein Quality Evaluation. Rome.

Health Canada. 1994. Guidelines for the Safety Assessment of Novel Foods. Ottawa.

Sarwar, G., F.E. McDonough. 1990. Evaluation of protein digestibility-corrected amino acid score for evaluating protein quality of foods. J. Assoc. Official Anal. Chem. 73: 347–56.
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5. CONSIDERATIONS IN THE USE OF BIOTECHNOLOGY IN ANIMAL PRODUCTION SYSTEMS

INTRODUCTION


The Canadian regulatory bodies, food producers and processors, and Canadian consumers have experience with food from GM microbes and crops. However, food-producing animals, including fish, differ from bacteria and plants in so many respects that the development and promotion of biotechnology will be substantially different. This chapter will address the animal welfare, food safety and environmental issues related to biotechnology applications in animal production systems, either through the development of a transgenic animal or the use of products derived through biotechnology in animal production systems.

PART 1. GENETICALLY MODIFIED ANIMALS

Modifications in transgenic animals may induce undesirable changes in an animal’s physiology and behaviour. This could lead, for example, to an altered level of production of a natural or genetically altered protein that increases the susceptibility to disease.

Potential Threats to Animal Health and Welfare

Fish


Comparatively little information is available on the effects of transgenesis relative to fish health and welfare. Existing documentation has concentrated, for the most part, on deleterious consequences to fish morphology, respiratory capacity, and locomotion associated with the introduction of growth hormone (GH) gene constructs in some transgenic variants of salmonids, notably Pacific and Atlantic salmon.

Nonetheless, despite the relative paucity of data, it seems clear that pleiotropy (unintended genetically based changes to an organism’s phenotype associated with the introduced gene construct) associated with the introduction of novel gene constructs is the rule rather than the exception in fish. This pleiotropy has been manifested by changes to enzyme activity, gross anatomy, behaviour and, in all likelihood, hormonal activity. The following sections reflect the current status of knowledge relative to impact of transgenesis on fish health and welfare.

Changes in muscle cellularity, muscle enzyme activity and gene expression

Transgenesis has been reported to affect the muscle cellularity and muscle enzyme activity in coho salmon (Oncorhynchus kisutch) containing a GH gene construct (Hill et al., 2000). The levels of activity of two enzymes in the white muscle— phosphofructokinase and cytochrome oxidase — were 275% and 31% higher, respectively, in transgenic fish. This finding is consistent with the hypothesis that the muscle of transgenic fish has greater glycolytic and aerobic requirements than the muscle of non-transgenic fish.

There also is evidence that insertion of single gene constructs can affect the activity of non-targeted genes. Increased gene expression is suggested by elevated levels of transcription (ribosomal proteins and tRNA) and changes in muscle ultrastructure (myosin heavy chain and skeletal "-actin) in transgenic coho salmon relative to their non-transgenic counterparts (Hill et al., 2000). From a human health perspective, the same research documented an increase in the amount of the Ca2+ transport protein, parvalbumin-$, in transgenic coho, a protein that has been identified as a major food allergen in fish (Lindstom et al., 1996).

Changes in gross anatomy

Growth hormone gene constructs can cause significant morphological deformities in fish. For example, Devlin et al. (1995a) have documented morphological abnormalities among transgenic coho salmon in the cranial, jaw and opercular regions. From an animal health perspective, these morphological abnormalities affected the ability of transgenic fish to feed properly and to irrigate their gills at a level that would permit normal rates of respiration. Similar changes to body shape have been observed in transgenic carp (Cyprinus carpio) (Chen et al., 1993; Dunham and Devlin, 1999) and in non-transgenic channel catfish (Ictalurus punctatus) injected with growth hormone (Dunham et al., 1992). Cartilage overgrowth in the cranial and opercular regions has also been associated with increased mortality among the progeny of transgenic coho salmon, again because of their inability to feed normally or to irrigate their gills properly (Devlin et al., 1995b). However, the incidence of such abnormalities can be expected to decrease with selection for transgenic broodstock that produce a reduced range of the phenotypic variability manifested by novel gene constructs.

In addition to these changes to the head region, transgenesis can affect the overall shape of transgenic fish. Ostenfeld et al. (1998) reported that insertion of the pOnMTGH1 gene construct into coho salmon significantly altered the shape and allometry of affected fish. McLean et al. (1997) have suggested that the reduced swimming ability reported for transgenic coho (Farrell et al., 1997) may be attributed in part to changes in skin and pressure drag effected by these changes to body shape.

Changes to swimming ability and foraging behaviour


Transformation with a GH gene construct has been reported to affect the swimming behaviour of salmonids. Farrell et al. (1997) found the critical swimming speeds of growth-enhanced transgenic coho salmon to be significantly lower than those of non-transgenic controls of the same size and same age. These reduced swimming speeds in transgenic coho may be caused by ontogenetic delay or from disruption of the locomotor muscles and [or] their associated respiratory, circulatory and nervous systems (Farrell et al., 1997). Despite this example of reduced swimming speed in GH-enhanced fish, it is not clear that such an effect is a general one. Such reductions, for example, have not been observed between transgenic Atlantic salmon and nontransgenic controls (Abrahams and Sutterlin, 1999).

Increases in overall activity are apparent from simple observation of transgenic salmonids into which a GH gene construct has been introduced. This increased activity appears to be associated with increased feeding rate (Abrahams and Sutterlin, 1999; Devlin et al., 1999) and speed of movement (Abrahams and Sutterlin, 1999). One consequence of this increased activity appears to be reduced vigilance to predators (Abrahams and Sutterlin, 1999), an observation that has also been made in non-transgenic, GH-treated salmonids (Jönsson et al., 1996 a, b).

Other pleiotropic effects

To date, the dominant form of genetic manipulation undertaken for the aquaculture industry has involved growth hormone gene constructs. As suggested by the morphological and enzymatic changes described above, the consequences of increased levels of GH are unlikely to be restricted to increases in growth rate alone.

The growth-promoting effects of GH are achieved in part through the activity of insulinlike growth factor I (IGF-I), a substance produced by the liver and peripheral cells to promote mitosis and/or differentiation of fibroblasts, prechondrocytes and other cells critical to the development of new skeletal and cartilaginous tissue (Goodman, 1993). In addition to the direct effects of GH on the metabolism of target cells in adipose, liver, muscle and pancreatic tissue, GH also can have indirect effects that may affect the health of transgenic fish. For example, Goodman (1993) reported that GH can modify the sensitivity of cells to, as well as production of, other hormones, such as insulin and catecholamines. Indeed, Mori and Devlin (1999) have reported 50% to 83% reductions in the size of the pituitary gland of transgenic coho salmon relative to non-transgenic controls, although it is not known if such changes affect the activity of hormones other than those associated with growth.

Farm Animals

Transgenic research in support of animal agriculture for food production lags behind the progress made with fish, but will undergo a revolution due to the explosive growth of molecularbased technologies being driven by supporting research platforms. Perhaps the most notable is the recent development of methods of somatic cell nuclear transfer and the production of clones from these somatic cells for livestock species (McCreath et al., 2000). This advance overcomes the serious limitations of pronuclear micro-injection for the production of GM livestock species (Polejaeva and Campbell, 2000).

Over the next 5 to 10 years, we should see many of the advances required for development and commercial application of GM germplasm and transgenic animals in dairy cattle, swine, and some poultry. To this end, much of the research and development will be driven by corporate strategies to capture the potential economic value of transgenic technology for improved growth rate and carcass composition in meat-producing animals and compositional modification of milk and eggs. Another critical requirement to realize commercial application, particularly for recalcitrant traits like fertility and disease resistance, is the opening of the genetic “black box”, which is currently taking place as a result of rapid integration of genomics analysis technologies in research on all livestock species (Gellin et al., 2000). Once the information (i.e. identity of genomic regions that encode quantitative trait loci of economic importance) and technologies (e.g. cell culture-based transgenesis) are finally in place, there is little doubt that breeding companies will offer animals bred from proprietary germplasm. Such animals may have traits conferring enhanced production efficiency, or in some way meet consumer demand by, for example, offering improved nutritional value.

Another potential application of transgenic technology in livestock production is to increase the safety of animal products for human consumption through strategies to increase disease resistance and thus reduce reliance on antibiotics. Opportunities exist for genetic modifications that reduce product susceptibility to spoilage or bacterial contamination. The recent demonstration in mice, using a gene knockout strategy, of the inactivation of the prion gene involved in transmissible spongiform encephalopathies (TSE), reveals the possibility that similar genetic modifications may be achieved in livestock species (i.e. to prevent scrapie in sheep and BSE or “mad cow disease” in cattle) to reduce their susceptibility to diseases (Flechsig et al., 2000).

Research efforts in transgenic animals can be categorized into two general areas; the first being production of proteins to modify the normal functioning in the animal (e.g. modification of fat or protein synthesis by the mammary gland, transfer of growth hormone genes into pigs, transfer of cysteine synthesis genes in sheep for enhanced wool production); the second being production of a target protein that is not part of normal animal function (e.g. spider silk production by goats) which may be for food, pharmaceutical or industrial production purposes. The following sections extract information from the published literature relevant to animal health and welfare in order that an understanding of the scope of this issue can be provided.

Changes in muscle cellularity, muscle enzyme activity and gene expression

Production of excess growth hormone in transgenic pigs carrying various growth hormone transgenes (Pursel and Rexroad, 1993) caused multiple physiological effects, including reduced fat carcass, alterations of muscle fibres, thickening of the skin and redistribution of major carcass components, but did not result in “giantism” as was observed in growth hormone-enhanced GM mice (Palmiter et al., 1982). Many of these same effects are not observed in pigs given daily injections of PST.

The carcass fat in transgenic pigs expressing either a bovine, ovine, or human growth hormone gene construct was reduced 84%, 82% and 62 %, respectively, compared to sibling control pigs in a recent study reported by Solomon et al. (1997). Pursel et al. (1996) suggested that the dramatic reductions in carcass fat are related to an interference in insulin’s ability to stimulate lipogenesis, even though insulin was 20-fold higher in the transgenic pig than in the control sibling. As well as reduced carcass fat, major decreases in carcass fatty acid levels have been observed in transgenic pigs, decreases of 70% to 87% in saturated fats, 69% to 89% in monounsaturated fatty acids and 36% to 71% for polyunsaturated fatty acids. The impact of these and other genetic modifications on animal health and welfare, or on food safety, has received little research attention to date.

Pursel et al. (1999) attempted to achieve the same objective by transferring a gene construct that consisted of an avian "-skeletal actin promoter attached to human insulin-like growth factor I into swine. Insulin-like growth factor-I mediates many of the same effects as growth hormone without the dramatic effect on the systemic physiology of the animal. However, the variable response to the transgene in individual animals is apparent. For example, in this study three of 14 transgenic animals died of endocarditis or cardiac hemorrhage, ages ranging from preweaning to just before first parturition. Cause of death may have been associated with the expression of the IGF-I transgene in the cardiac muscle, indicating that control of expression in various tissues will need to be evaluated, not just for individual animals, but also at various physiological stages of life.

Reproductive efficiencies continue to limit progress in development of transgenic animals. Only a small proportion of reconstructed embryos develop to become live offspring. Success with lambs varied from 0.04% with adult cells to 1.7% for fetal-derived cells (Wilmut et al., 1997). Even when considering only the proportion of embryos that became live lambs, the proportion ranged between 3.4% and 7.5 %. There may also be complications at the time of birth. A number of lambs derived by nuclear transfer in the work conducted by Wilmut et al. (1997) died at birth due to congenital abnormalities in the cardiovascular or urinogenital systems. Other problems encountered include large birth weights (perhaps related to culture conditions for the zygote), increased gestation length, immature lung development at birth and slow onset of labour.

These results inevitably trigger major animal welfare concerns and require full consideration prior to release of the technology. While some of the identified problems will be overcome with technology improvements, there will also be situations in which the allowable impact on animal welfare may need careful definition (e.g. the number of times an animal is subjected to Caesarean section in its lifetime).

Increased incidence of mutations and other pleiotropic effects

Current technologies used in the development of transgenic animals have improved control of insertion sites of the construct gene, but examples are accumulating of transgene instability and unexpected patterns of gene expression in transgenic animals. In many cases, the insertional mutation is recessive and is not expressed until subsequent generations. Again, movement of the technology into the commercial arena will require informed debate and decision regarding whether there is an acceptable rate of increase for mutation, and whether any unexpected pattern of gene expression is acceptable.

The biological complexity of animals, the longer generation time and our reduced ability to select for desirable traits in transgenic animals will all delay our ability to quantitatively and qualitatively assess the impact of this technology on the health and welfare of the individual animal or of farm livestock populations as a whole. Assessing the animal welfare advantages (reduced killing of surplus male chicks or castration of males) or disadvantages associated with production systems using GM animals is difficult because there is no consistency of response among transgenic animals at this early stage of technology development. In addition, adverse effects may be identified only when the animals are challenged, or may only be apparent during one stage of the animal’s development. This emphasizes the importance of studying animal welfare and health as an ongoing part of further technological development and monitoring this throughout the life of the transgenic animal.

Altered nutritional and welfare needs of transgenic animals

Genetic manipulations usually have as their target the production of proteins that influence specific metabolic pathways. These alterations can impact on the animal’s inability to synthesize specific enzyme substrates or co-factors. That kind of alteration can change the optimal balance of nutrients required by the animal, and may even alter the requirement for essential nutrients. In traditional animal selection programs, these changes and the resulting dietary or management adjustments are made over an extended period of time and are based on a reasonable working knowledge of the biochemical pathways affected by the selection process.

Suitable facilities and environmental requirements for management of genetically engineered animals will need to be considered prior to release into the commercial agriculture sector, since normal coping mechanisms relative to the animal’s physical and social environment may have been compromised. Similarly, nutritional requirements under normal and stressed conditions need to be determined. The current well-developed science behind modern animal nutrition and management should allow appropriate responses to be devised to meet the novel needs of transgenic animals, once these needs are identified and characterized.

Creation/Strengthening of Animal Commodification

Biotechnology applications in animal populations can occur for both domesticated and non-domestic species. For example, animal sensitivity to the environment could be reduced in order to allow increased nutrient resource allocation to production, or to protect animals from disease. Non-domesticated populations (e.g. Red Deer or Wapiti) could be genetically engineered for increased production of antler velvet or a pharmacological compound in antler velvet. However, as Heap (1995, as reported by Mench, 1999) pointed out in an address to the Royal Society of Agriculture, “Programmes which threaten an animal’s characteristics and form by restricting its ability to reproduce normally, or which may in the future diminish its behaviour or cognition to improve productivity would raise serious intrinsic objections because of their assault on an animal’s essential nature.” Nevertheless, there remains a grey area as to where animal welfare issues begin and ethical issues end, relative to animal management and use, and this uncertainty will be exacerbated by introduction of transgenic technologies. Decisions are, therefore, urgently required regarding the future purpose of the technology. Animal health and welfare (as defined in the glossary) are considered in the process of product approval; however, the mandate of this Panel does not allow it to deal specifically with the ethical issues of technology application in animal production systems.

Reservoirs of Pathogens or Antibiotic-resistant Microflora

Development of animal breeds resistant to a disease would be expected to reduce the short-term requirement for vaccines and medicines. However, creation of resistance to the pathogenic effects of disease agents without blocking infection and continued dissemination of the disease agents (i.e. the animal does not exhibit symptoms but continues to be a carrier) could create additional problems concerning disease epidemiology and control, transmission to other species (including humans) and disease agent mutation (Cunningham, 1999). For example, beef and dairy cattle are currently major reservoirs of enterohemorrhagic Escherichia coli O157.H7 (Shere, 1998). This pathogen is an important cause of food and water contamination, leading to several hundred deaths and thousands of serious illnesses every year in North America. Although this pathogen and related enterohemorrhagic E. coli have co-evolved with humans over the past 4 to 5 million years (Reid, 2000), their incidence has increased over the past two decades as a consequence of changes in farm management practices and increasing encroachment of urban areas and urban water supplies on rural farmland (Shere, 1998; Gagliardi, 2000). Conceivably, changes in farm management practices as a result of biotechnological innovations could increase animal population densities, or alter their ability to act as reservoirs, leading to further increases in the incidences of such pathogens. This risk factor should be investigated during the development of transgenic animal biotechnology products.

Loss of Animal Genetic Resources

Loss of livestock breeds has become an issue in many parts of the developed world where intensive animal agriculture systems require animal uniformity and production efficiencies to maximize economic return (Patterson, 2000). The extent of genetic variation within breeds of livestock influences the rate of genetic progress by selection and the success of genetic resource conservation in the long term. The sequencing of entire animal genomes and identification of single nucleotide polymorphisms in the genomes of agricultural species will provide a better understanding and a more complete characterization of genetic variability at the nucleotide level. However, more accurate selection techniques, allowing production and evaluation of individual animals at an early age, in utero, or even before fertilization in the case of artificial insemination, has the potential to erode existing genetic diversity in our farm animal populations. On the other hand, molecular biology will advance the ability to accurately assess existing genetic variation and could thereby contribute to its preservation of diversity.

Currently, many animal breed associations, and the government, maintain active registries of pedigreed animals in Canada. This has proven to be a useful tool in maintaining the integrity of registered pure breeds or populations of animals. The meat production industry is also engaged in discussions that would allow tracking of individual animals from birth to market as a means of assessing animal management and genetics in terms of final product quality and safety. There is likely to be interest on the part of both the industry and the consumer to maintain similar programs for GM animals, once they enter commercial production systems.

RECOMMENDATIONS

5.1 The Panel recommends that the Canadian Food Inspection Agency (CFIA) develop detailed guidelines describing the approval process for transgenic animals intended for (a) food production or (b) other non-food uses. Furthermore, the Panel recommends that CFIA encourage work with the Canadian Council on Animal Care (CCAC) to engage the scientific community in the development of appropriate scientific criteria for assessment of behavioural or physiological changes in animals resulting from genetic modification. (It is anticipated that applications for GM animals will occur within the next 10 years. It would be advisable to develop the decision process and criteria for each step of the process. The process could then be challenged with a test case.)

5.2 The Panel recommends that the approval process for transgenic animals include a rigorous assessment of potential impacts on three main areas: 1) the impact of the genetic modifications on animal health and welfare; 2) an environmental assessment that incorporates impacts on genetic diversity and sustainability; and 3) the human health implications of producing disease-resistant animals or those with altered metabolism (e.g. immune function). Any negative effects on animal health and welfare and the environment would require justification on the basis of significant benefit to human health or food safety.

5.3 The Panel recommends that the tracking of transgenic animals be done in a manner similar to that already in place for pedigree animals, and that registration be compulsory.

5.4 The Panel recommends that transgenic animals, and products from those animals, that have been produced for non-food purposes (e.g. the production of pharmaceuticals) not be allowed to enter the food chain unless it has been demonstrated scientifically that they are safe for human consumption.

5.5 The Panel recommends that federal and provincial governments ensure adequate public investment in university-based genomic research and education so that Canada has the capacity for independent evaluation and development of transgenic technologies.

5.6 The Panel recommends that the use of biotechnology to select superior animals be balanced with appropriate programs to maintain genetic diversity which could be threatened as a result of intensive selection pressure.

REFERENCES

Abrahams, M.V., A. Sutterlin. 1999. The foraging and antipredator behaviour of growthenhanced transgenic Atlantic salmon. Anim. Behav. 58: 933–42.

Chen, T.T., K. Kight, C.M. Lin, D.A. Powers, M. Hayat, N. Chatakondi, A.C. Ramboux, P.L. Duncan, R.A. Dunham. 1993. Expression and inheritance of RSVLTR-rtGH1 complementary DNA in the transgenic common carp, Cyprinus carpio. Mar. Mol. Biol. Biotechnol. 2: 88–95.

Cunningham, E.P. 1999. Recent Developments in Biotechnology as They Related to Animal Genetic Resources for Food and Agriculture. Document CGRFA-8/99/Inf.9, Commission on Genetic Resources for Food and Agriculture, FAO.

Devlin, R.H., T.Y. Yesaki, E.M. Donaldson, S.J. Du, C.-L. Hew. 1995a. Production of germline transgenic Pacific salmonids with dramatically increased growth performance. Can. J. Fish. Aquat. Sci. 52: 1376–84.

Devlin, R.H., T.Y. Yesaki, E.M. Donaldson, C.-L. Chew. 1995b. Transmission and phenotypic effects of antifreeze/GH gene construct in coho salmon (O. kisutch). Aquaculture 137: 161–69.

Devlin, R.H., J.I. Johnsson, D.E. Smailus, C.A. Biagi, E. Jönsson, B. Th. Björnsson. 1999. Increased ability to compete for food by growth hormone-transgenic coho salmon Oncoryhnchus kisutch (Walbaum). Aqua. Res. 30: 479–82.

Dunham, R.A., A.C. Ramboux, P.L. Duncan, M. Hayat. 1992. Transfer, expression and inheritance of salmonid growth hormone in channel catfish, Ictalurus punctatus, and effects on performance traits. Mol. Mar. Biol. Biotechnol. 1: 380–89.

Dunham, R.A., R.H. Devlin. 1999. Comparison of traditional breeding and transgenesis in farmed fish with implications for growth enhancement and fitness. In J.D. Murray, G.B. Anderson, A.M. Oberbauer, M.M. McGloughlin (eds.), Transgenic Animals in Agriculture. New York: CABI Publishing.

Farrell, A.P., W. Bennet, R.H. Devlin. 1997. Growth-enhanced transgenic salmon can be inferior swimmers. Can. J. Zool. 75: 335–37.

Flechsig, E., D. Shinerling, T. Hegyl, A.J. Rauber, M. Fischer, A. Cozzio, C. von Merling, A. Aguzzi, C. Weissmann. 2000. Prion protein devoid of the octapeptide repeat region restores susceptibility to scrapie in knockout mice. Neuron 27: 399–408.

Gellin, J., S. Brown, J.A. Marshall Graves, M. Rothchild, L. Schook, I. Womack, M. Yerle. 2000. Comparative gene mapping workshop program in agriculturally important animals. Mamm. Genome 11(2): 1400–144.

Gagliardi, J.V., J.S. Karns. 2000. Leaching of Escherichia coli O157:H7 in diverse soils under various agricultural management practices. Appl. Environ. Microbiol. 66: 877–83.

Goodman, H.M. 1993. Growth hormone and metabolism. In M.P. Schreibman, C.G. Scanes, P.K.T. Pang (eds.), The Endocrinology of Growth, Development, and Metabolism in Vertebrates. San Diego: Academic Press.

Hill, J.A., A. Kiessling, R.H. Devlin. 2000. Coho salmon (Oncorhynchus kisutch) transgenic for a growth hormone gene construct exhibit increased rates of muscle hyperplasia and detectable levels of gene expression. Can. J. Fish. Aquat. Sci. 57: 939–50.

Jönsson, E., J.I. Johnsson, B. Th. Björnsson. 1996a. Growth hormone increases predation exposure of rainbow trout. Proc. R. Soc. Lond. B 263: 647–51.

Johnsson, J.I., E. Petersson, E. Jönsson, B. Th. Björnsson, T. Järvi. 1996b. Domestication and growth hormone alter antipredator behaviour and growth patterns in juvenile brown trout, Salmo trutta. Can. J. Fish. Aquat. Sci. 53: 1546–54.

Lindstrom, C.D., T. van Do, I. Hordvik, C. Endresen, S. Elsayed. 1996. Cloning of two distinct cDNAs encoding parvalbumin, the major allergen of Atlantic salmon salmo salar. Scan. J. Immunol. 44: 335–44.

McCreath, K.J., J. Howcroft, K.H.S. Campbell, A. Colman, A.E. Schmeke, A.J. Kind. 2000. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405: 1066–69.

McLean, E., R.H. Devlin, J.C. Byatt, W.C. Clarke, E.M. Donaldson. 1997. Impact of a controlled release formulation of recombinant growth hormone upon growth and seawater adaptation in coho (Oncorhynchus kisutch) and chinook (Oncorhynchus tschawytscha) salmon. Aquacult. 156: 113–28.

Mench, J.A. 1999. Ethics, animal welfare and transgenic animals. In J.D. Murray, G.B. Anderson, A.M. Oberbauer, M.M. McGloughlin (eds.), Transgenic Animals in Agriculture, 251–68. New York: CABI Publishing.

Mori, T., R. Devlin. 1999. Transgene and host growth hormone gene expression in pituitary and nonpituitary tissues of normal and growth hormone transgenic salmon. Mol. Cell. Endocrinol. 149: 129–39.

Ostenfeld, T.H., E. McLean, R.H. Devlin. 1998. Transgenesis changes body and head shape in Pacific salmon. J. Fish Biol. 52: 850–54.

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Pursel, V.G., M.B. Solomon, R.J. Wall. 1996. Genetic engineering of swine. In M.E. Tumbleson, L.B. Schook (eds.), Advances in Swine Biomedical Research, 189–206. Plenum Publishing Corp.

Pursel, V.G., R.J. Wall, A.D. Mitchell, T.H. Elsasser, M.B. Solomon, M.E. Coleman, F. deMayo, R.J. Schwartz. 1999. Expression of insulin-like growth factor-I in skeletal muscle of transgenic swine. In J.D. Murray, G.B. Anderson, A.M. Oberbauer, M.M. McGloughlin (eds.), Transgenic Animals in Agriculture, 1–19. New York: CABI Publishing.

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PART 2. GENETICALLY MODIFIED FEEDS, FEED ADDITIVES AND METABOLIC MODIFIERS ADMINISTERED TO FOOD-PRODUCING ANIMALS

Biotechnology is already widely used in animal production and we can expect an increase in this activity in the future. The primary goals are to influence the nutrition of the animal, improve animal health, modify product characteristics, improve product quality or reduce adverse environmental impacts of animal agriculture. Examples of biotechnology-derived products currently in use include silage inoculants, amino acid supplements, feed enzymes, and pre- and pro-biotics. Information relative to the range of products of biotechnology being studied relative to animal health, nutrition and physiology is found in a review by Bonneau and Laarveld (1998). There continues to be interest in the application of biotechnology for production of metabolic modifiers to improve growth and lactation, feed efficiency and animal product composition. However, there are no such products registered currently for use in Canada. Interest in this area is due to the use of products such as recombinant bovine growth hormone in countries that are trading partners with Canada.

As the acreage of GM crops increases in Canada, a higher portion of the feed ingredients used in livestock production systems will consist of the resulting grains, forages, meals and byproducts. To date, biotechnology applications have focused on improving agronomic characteristics of crops and the quality characteristics required for human food. Improved feeding value of GM crops for animal use is possible but has not been emphasized, mostly because animal feed (grains and oilseed) is often suitable for human consumption or is a by-product of food harvest or processing. This trend is likely to change because movement to targeted crop production for specific types and classes of animal has the potential to reduce animal production costs. The following section identifies some of the potential novel threats associated with the use of GM plants, microorganisms and pharmaceutical products in animal production.

Potential Novel Threats to Food Quality and Safety

Commercial development of feed additives and metabolic modifiers for use in animal production may involve genetic engineering. In many cases, these products (i.e. recombinant GH or IGF-I analogs produced in bacteria) have known benefits on production efficiencies, animal product quality or animal health, but more research is required to determine the potential for negative impacts in commercial settings.

Biotechnology has been responsible for a number of changes in practice relative to the use of vaccines. Issues of biosafety relative to the injection into animal tissues of naked DNA constructs coding for foreign antigen, driven by eukaryotic gene promoters that may be destined for human consumption, are still being explored.

Early technologies rendering GM microbes antibiotic resistant presented the threat of resistance transfer, especially in the animal gut. Although resistance transfer may not be a health threat to the animal, the presence of antibiotic-resistant bacteria in the human food chain is an unnecessary threat to human health.

There are two other areas of potential concern that to date have not yet been addressed to any great extent by the research community. First, there is the potential transmission of toxins from feeds derived from GM plants to the animal, and ultimately into animal products. Plants cohabit with a range of epiphytic micro flora. With any new practice, the epiphytic micro floral populations can change and potentiate toxin production. This is also true for GM plants and, therefore, some monitoring is required. Genetic transformation of plants may have an impact on patterns of gene expression. The resulting changes in the plant’s composition, physiology or morphology will influence the populations and species of micro flora associated with the plant and may thereby lead to the introduction of new, or previously less common, toxins into the animal’s diet. The issue extends to consideration of the behaviour of these altered microbial populations under a range of harvest and storage conditions, and the associated potential for introduction of toxins into animal diets.

A second potential concern focuses on the use of feed additives, digestion enhancers or vaccines against infectious diseases of the gastrointestinal tract. These are designed to improve digestion and gut health, often through the manipulation of gut microflora. Coupled with the diverse range of management conditions to which livestock across Canada are subjected, there may be situations in which such manipulation can cause adverse changes in gut micro floral populations relative to shedding of pathogenic organisms, with the potential contamination of animal products and ground water.

Potential Novel Threats to Animal Health or Welfare

Metabolic Enhancers


Bovine GH (also known as BST, bovine somatotropin) was the first product derived through genetic engineering to be used for modification of animal metabolism. It affects regulation of growth and lactation in cattle. A summary of experiments using genetically engineered BST showed that its administration across a range of doses increases milk production by 10% to 20%, with little effect on milk composition (Bauman, 1999; Etherton and Bauman, 1998). Half the increase in milk yield can be accounted for by an increase in efficiency from the spreading of the maintenance requirement across a larger output. Concerns regarding animal health and safety have focused on the potential for increased incidence of metabolic disease (i.e. ketosis) in the early stages of lactation, compromised immune function (i.e. increased incidence of mammary gland infections), and reduced animal longevity. However, trials conducted to date indicate that the occurrence of these problems is similar to that seen in dairy cows at equal levels of milk production that have not received BST. This suggests that the negative impacts are associated with increased milk production, rather than with the BST itself.

Administration of high doses of recombinant porcine somatotropin to growing pigs has been shown to have adverse effects on animal health, including an increased incidence of stomach ulcers and leg problems associated with osteochondrosis and cartilage soundness (Sejrsen et al., 1996). Radical changes in the composition of tissue fatty acid profiles or shifts in lean tissue to skeletal tissue growth may enhance meat product quality but they also have the potential to increase the animal’s susceptibility to infectious agents or metabolic disease.

Advancement in genetic engineering, in the case of metabolic modifiers, has resulted in the development of products with pharmacological properties that are incompletely understood. In this situation, thorough study of the new product(s) as well as the technology by which it is produced is required for assessment relative to animal health and welfare and food safety.

Vaccines

Sub-unit vaccines, pathogen attenuation by gene deletion, live vectoring of antigen by insertion of foreign antigen into gene-deleted mutants, and development of “new generation” adjuvants are all processes that have opened the door for new delivery systems for vaccines, for enhanced protection against specific pathogens, and for distinguishing between vaccinated and naturally infected animals. Concerns still being addressed within this technology envelope include consistency of the resulting immune response.

Immunomodulation of growth and lactation can be envisioned as an alternative to direct genetic manipulation of the production or response functions in transgenic animals, and it may be considered more acceptable than exogenous administration of growth or lactation promotants because the need for repeated injection is eliminated. However, this form of permanent modification of the animal’s hormone production pattern is not as well understood and accepted as the promotant approach, and requires further consideration relative to both animal welfare and food quality and safety (Mepham and Forbes, 1995).

Microbially Derived Feed Supplements and Additives

Some of the first GM feeds used by livestock were “single cell” protein products used to replace plant or milk proteins in pre-ruminant and baby pig diets. Crystalline amino acids (e.g. lysine, threonine and tryptophan) are used extensively as supplements in animal diets today. Future developments may include ruminally protected amino acids and use of specific amino acid supplements as stimulants for hormone release (Hurson et al., 1995), or for gut and immune system development in young animals (Gardiner et al., 1995). Microbial enzymes are currently being used to increase the digestibility of nutrients in feeds either in the animal gut (i.e. phytase) or during feed storage and processing to supplement host endogenous enzymes (i.e. protease and amylase), to remove toxins and anti-nutritional factors in feed ingredients (i.e. enzymes to destroy trypsin inhibitors) and to increase digestibility of the non-starch polysaccharides (i.e. ß-glucanase). Manipulation of the gut micro flora to promote growth of beneficial bacteria and/or competitively exclude pathogens can be accomplished through manipulation of the diet composition, or by inclusion of specific microflora, with the goal of improving absorption of nutrients through improved gut health. Many of these amino acids, enzymes, pre- and pro-biotics are produced by fermentation, often with GM organisms. Inclusion of GM-derived proteins in animal diets has not been reported to create novel threats to animal health or welfare. Specific research to investigate potential food or feed safety problems does not appear in the literature.

Live, GM bacteria and their products can be used in feed harvest, storage and processing. For example, GM Lactobacillus sp. is used in silage production to control fermentation. Although these organisms were specifically designed to be competitive in the silo environment, there has been concern that accidental release, either at the time of application or from silo seepage, could create an environmental risk if natural populations are modified. To date, use of GM microbes in the production of animal feeds has not been reported to create novel threats to the environment, although the extent of investigation is very limited.

The introduction of the tetracycline-resistant TcR a gene into Prevotella ruminicola was the first successful transfer of a gene into rumen bacteria (Flint et al., 1988). Since then, gene transfer has been used to introduce cellulase activity into a number of hind-gut bacteria to enhance acid tolerance in cellulolytic rumen bacteria, to improve protein (essential amino acid) yield by rumen bacteria, and to induce hydrogen scavenging in rumen bacteria and thereby reduce methanogenesis. Novel threats to animal health and welfare may result from microbial population shifts that could, for example, cause a reduced capability of gut microflora to adapt to dietary changes.

The current limitation to this technology rests with the GM organism’s ability to compete in the natural rumen or hind-gut environment. Gregg et al. (1993) did report rumen survival for a 50-day period for a GM strain of Bacteriodes fibrisolvens in which the added genetic material did not provide any known competitive advantage.

Transduction and conjugation are well-known mechanisms of transfer of genetic material between microorganisms. The probability of gene transfer in the gastrointestinal tract is dependent on the nature of the GM microbe and the characteristics of the gene construct. A transfer gene that enhances the survival characteristics of the recipient microorganism might provide phage resistance, virulence, adherence, substrate utilization or production of bacterial antibiotics, and could impact animal health and food safety.

No reports of gene transfers from ingested plant or microbial DNA into the epithelial cells of animals have been found, with the exception of genes from infectious agents such as viral DNA. It is assumed that even if such a transfer were to occur, the transformed epithelial cells would not be maintained, because of the continuous replacement of these cell. Further investigation of these assumptions is warranted as the range and source of new gene constructs in the animal gut increase, and as gut cell metabolism is altered due to animal feeding or genetic manipulations.

The potential now exists to replace many of the microbially derived feed additives with plants that are GM to directly enhance the animal’s feed supply. For example, incorporation of phytase in crops would improve phosphorus availability to the animal, as opposed to the current process of supplementing animal diets with recombinant phytase enzyme derived from GM microbes.

To date, no animal health or production problems have been reported to result from to the use of GM grains or oilseeds in feed preparations. Feed industry representatives have reported that introduced gene constructs that reduce the plant’s susceptibility to pests (e.g. Bt corn) also result in a significant reduction of mycotoxins in the plant material (Lobo, 2000), probably due to improved overall plant health. Research is being conducted to test these field observations. Advances in the production of crops that more adequately meet the nutritional needs of the animal may actually reduce the industry demand for dietary additives such as enzymes and amino acids currently derived from GM microbes.

Novel problems related to the production of GM crops for animal consumption would centre around the issue of increased storage and handling capacity requirements at the feed mill. For example, a high-fat grain may be advantageous in poultry diets, but could cause digestive problems if inadvertently added to a ruminant diet. The plant–microbe interactions that lead to mycotoxin production are also still poorly understood. In general, improved plant health will result in less colonization by problem fungi, but certain changes in the plant’s biochemical makeup and morphology may also change the pattern of microbial colonization, thus potentiating conditions for previously undetermined toxin production, or increased mycotoxin production even with similar colonization.

Potential Threats from Concentration of GM Products in the Animal’s Food Stream

Increased production of GM microflora and plants for food production can lead to increased opportunities to use GM-derived byproducts as animal feeds. Byproducts may be unused plant parts (e.g. stem and leaf material following crop harvest), unprocessed plant products that do not meet standards for human use (e.g. immature, high mycotoxin levels), byproducts associated with food or industrial processing, or restaurant waste. The potential for concentration of unidentified anti-nutritive factors or toxins in the byproducts of processing, therefore, needs to be addressed.

With the exception of vegetables, all plants with novel traits approved by the Canadian federal government prior to March 1999 have been approved for animal feed as well as human food (Barrett, 1999). However, upon review of the Supplement to the Decision Document that accompanies the approval of GM plants currently grown in Canada, it was not clear to the Panel that all plant parts are considered in the evaluation process. The potential therefore exists that plant parts destined for feed in animal production systems may not have been specifically tested in that context.

RECOMMENDATIONS

5.7 The Panel recommends that a national research program be established to monitor the long-term effects of GM organisms on the environment, human health, and animal health and welfare. In particular, plant–microbe interactions that could result in increased exposure to toxins in feed or food, and microbial–animal interactions that could increase exposure to human pathogens in food and water need to be studied.

5.8 The Panel recommends that changes in susceptibility of genetically engineered plants to toxin-producing microbes, and the potential transfer of these to the animal and the food supply, be evaluated as part of the approval process.

5.9 The Panel recommends that a data bank listing nutrient profiles of all GM plants that potentially can be used as animal feeds be established and maintained by the federal government.

5.10 The Panel recommends that university laboratories be involved in the validation of the safety and efficacy of GM plants and animals.

5.11 The Panel recommends that Environment Canada and the Canadian Food Inspection Agency establish an assessment process and monitoring system to ensure safe introductions of GM organisms into Canada, according to the intent of the Canadian Environmental Protection Act.

REFERENCES

Barrett, K.J. 1999. Canadian Agricultural Biotechnology: Risk Assessment and the Precautionary Principle. Ph.D. Thesis. University of British Columbia.

Bauman, D.E. 1999. Bovine somatotropin and lactation: from basic science to commercial application. Domest. Anim. Endocrinol. 17(2-3): 101–16.

Bonneau, M., Laarveld, B. 1998. Biotechnology in nutrition, physiology and animal health. In Proceedings, Special Symposium and Plenary Sessions, 312–31. The 8th World Conference on Animal Production. 28 June–4 July, Seoul, Korea.

Etherton, T.D., D.E. Bauman. 1998. Biology of somatotrophin in growth and lactation of domestic animals. Physiol. Res. 78(30): 745–61.

Flint, H.J., A.M. Thomson, J. Bisset. 1988. Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola. Appl. Environ. Microbiol. 54: 555–60.

Gardiner K.R., S.J. Kirk, B.J. Rowlands. 1995. Novel substrates to maintain gut integrity. Nutr. Res. Rev. 8: 43–66.

Gregg, K., G. Allen, T. Baucoup, A. Klieve, M. Lincoln. 1993. Practical genetic engineering of rumen bacteria. In D.J. Farrell (ed.), Recent Advances in Animal Nutrition in Australia, 13–21. Armidale: University of New England.

Hurson, M., M.C. Regan, S.J. Kirk, H.L. Wasserkrug, A. Barbul. 1995. Metabolic effects of arginine in a healthy elderly population. J. Parenter. Enteral Nutr. 19: 227–30.

Lobo, P. 2000. Genetically-enhanced grains’ effect on the feed industry. Feed Manage. 51: 25–26.

Mepham, B.T., J.M. Forbes. 1995. Ethical aspects of the use of immunomodulation in farm animals. Livestock Prod. Sci. 42: 265–72.

Sejrsen, K., N. Oksbjerg, M. Vestergaard, M.T. Sorensen. 1996. Aspects of the use of anabolic steroids in animal production. In Scientific Conference on Growth Promotion in Meat Production, Brussels, Nov. 29 - Dec. 1, 1995, 87–119. Luxembourg: Office for Official Publications of the European Community.
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Part 1 of 4

6. ENVIRONMENTAL RISKS

INTRODUCTION


Many of the concerns surrounding recent developments in agricultural biotechnology centre on the potential ecological effects of these new varieties of organisms, and on their potential for changing agricultural practices that may, in turn, impact on the farm and hinterland environments. The following chapter is divided into four sections that review the science, the developing technologies and the potential environmental concerns for GM microbial, plant and animal (insect and fish) varieties. The Panel has focused its review on those aspects of the biology of GM organisms that are of particular concern with respect to potential environmental risks. For each taxon, where appropriate, the Panel refers to the above regulations or guidelines and we make both research and regulatory recommendations that we believe will strengthen Canada’s environmental protection standards in this area.

PART 1: MICROORGANISMS IN BIOTECHNOLOGY AND THE ENVIRONMENT

No complex life forms exist in isolation from the microbial world. All plants and animals have associated microflora that form commensal, symbiotic, parasitic or pathogenic relationships with their hosts. The vast majority of these relationships benefit both the host species and the microbe, or are selectively neutral in their effects; therefore, we tend to ignore these organisms. Much less common are microbial parasites or pathogens that harm their host, and to which we pay special attention. The communities of microorganisms associated with higher life forms are invariably highly diverse, including representative species of metazoans, protozoans, fungi, algae, bacteria and archaea. The relationships of animals and plants with the microorganisms that surround them or grow within them are the result of millions of years of natural selection, operating on both the host and the associated microflora. These organisms are constantly responding to changes in the physiology and behaviour of one another as variants arise through normal evolutionary processes. Change is continual and multifaceted.

Two important concepts will be described in the next paragraphs that help to place the discussion of potential environmental effects of transgenic organisms into the context of state-of-the- art research in microbial ecology. These concepts are:

• the microbial species concept
• the diversity of microorganisms in the natural environment.

These have been singled out to emphasize the difficulty of making predictions about the effects of transgenic biotechnology products on the microbial environment. Nevertheless, knowledge is growing in this field and we can point out areas of potential concern.

The Microbial Species Concept

This concept is central to discussions of how transgenic organisms might affect microorganisms, microbial communities, the processes they carry out and the ecosystems that depend upon their activity. It is also central to discussions of gene transfer, either from higher plants and animals to their associated microflora, or from microbial biotechnology products to other microorganisms in the surrounding environment. The definition of what constitutes a “species” in bacteria is not well developed. Phenotypic differences (in structure, biochemistry or physiology) between species formed the basis for bacterial identification throughout most of the history of the discipline. In the past 30 years, molecular methods of describing species have been developed. In the past two decades, these molecular methods have been integrated with phenotypic methods to yield a “polyphasic” approach to species identification. There is an ongoing explosion in microbial diversity research that is pushing its way ever further into extreme environments and into the common soil and aquatic habitats of the biosphere (Olsen et al., 1994; Hugenholtz et al., 1998; Whitman et al., 1998). The microbial phylogenetic tree is becoming more and more branched and subdivided and, as a result, the genus and species concept in microbiology is rapidly coming to signify an arbitrarily defined section of the phylogenetic tree. For example, an assumption used by some microbiologists is that a ribosomal RNA (16S rRNA, a commonly used phylogenetic character) sequence difference greater than 2% between two organisms indicates that these are two different species. This definition presents some problems, especially within certain taxonomic groups such as the Proteobacteria where new species are rapidly filling all of the gaps between the branches of the phylogenetic tree. In these highly populated taxonomic groups, a continuum of species variants flows one into another. Because we know most about groups such as the Proteobacteria, they have become a focus of the biotechnology industry. They also represent major colonizers of plant and animal epithelial surfaces, and they are the taxa that contribute many of the harmful bacteria associated with disease.

The Diversity of Microorganisms in the Natural Environment

Microbial diversity is greater, by ecological, phenotypic and genetic measures, than that of any other taxonomic group (Olsen et al., 1994; Tiedje, 1994). Soils are arguably the most complex habitats within the biosphere. They contain a large proportion of the estimated 1030 microbial cells in the biosphere (Whitman et al., 1998). The soil environment is the focus of many concerns associated with the potential environmental effects of transgenic plants and animals. Soils contain enormous numbers of microbial species, although the measured number depends both on how the measurement is carried out and, as explained above, how one defines a microbial species. The number of species in a gram of typical agricultural or forest soils from temperate regions has been estimated to be from thousands to tens of thousands (Torsvik et al., 1990; Ovreas and Torsvik, 1998).

In many natural habitats such as soils, aquatic sediments and marine environments, between 0.01% and 1% of microbial species are currently culturable using standard methods. That means that only a very small sample of microorganisms can actually be studied under laboratory conditions as pure cultures. Limitations of culture methods can be demonstrated by employing culture-independent methods to detect the DNA associated with the uncultured fraction of microorganisms (Hugenholtz et al., 1998). For example, a recent study of grassland soil diversity was conducted by cloning and sequencing of 16S rRNA genes amplified by PCR directly from soil-extracted DNA and comparing these sequences to 16S rRNA genes of over 600 cultured species from the same soil (Felske et al., 1999). The results showed that there was no correlation between the culture collection and the 16SrRNA clone library. This does not mean that the uncultured species can never be cultured in the laboratory and characterized; rather, it reflects limitations in our culture methods and in the resources we have at hand to study the vast diversity of microorganisms that occur in these habitats. As a consequence, we cannot reduce complex microbial communities and their function to a set of known biotic and abiotic interactions. We are beginning to appreciate the fact that we have just scratched the surface in understanding microbial diversity in terms of the numbers of species, their relative abundance, and the differences that exist in diversity between different ecozones (Borneman and Triplett, 1997) or even between different patches in outwardly uniform-looking agricultural soils (Siciliano and Germida, 1999). For the foreseeable future, there must be many unknowns concerning the details of microbial community function in most natural habitats. Despite this limitation, some experimental data exist and certain predictions are possible with regard to the impacts of transgenic organisms on natural microbial environments.

Direct Effects of GMOs on Soil Microflora

First generation transgenic plants and animals, developed through applications of modern biotechnology, usually contain single gene modifications (deletions, insertions, altered regulation). These simple modifications affect the phenotype of the organism, with the objective of adding commercial value to the transgenic crop or animal. The added value may benefit the biotechnology industry, the farmer or the consumer, or some combination of these. Depending on the nature of the modification, an outward change in the normal array of host–microbe associations may or may not occur. Even under the most simplified of conditions, some change is inevitable. For example, transgenic corn cultivar NK4640Bt expressing the Bt toxin gene cryIAb exudes some of the toxin protein from the root into the surrounding rhizosphere and soil, along with other proteins normally present in root exudates (Saxena et al., 1999).

Another obvious route of transgene product exposure in soil is via incorporation of plant material into the soil either during the growing season or post-harvest. Transgenic cotton var. Coker line 81 (cryIAb) and line 249 (cryIAc) release measurable quantities of the truncated Bt toxin during decomposition when incorporated into soil (Palm et al., 1994). Cotton line 81 released 10- to 20-fold more toxin than line 249, commensurate with the level of expression of the Bt toxin in the plant tissues.

These routes of transgene product exposure are novel and will likely elicit a response from the rhizosphere and soil microbial community. For proteolytic microbes in the rhizosphere, novel proteins or peptides represent an additional source of nutrients (peptides, amino acids, carbon and nitrogen) and they will respond, through the action of extracellular proteases, by degrading the novel protein and assimilating the components. This is the underlying cause, along with physical/chemical processes of protein degradation, of the exponential decay of Bt toxin in soil (Tapp and Stotzky, 1998). Plant root and soil microbial proteases can degrade active Bt toxin to inactive peptides within days in artificial soil-free media (Koskella and Stotzky, 1997). In real soils, the protoxin can bind to clay and humus materials and this delays proteolytic degradation, in some cases for months (Saxena et al., 1999). During this phase of novel protein persistence, there may be effects on the range of interacting species from different trophic levels in soils; from bacteria and viruses to protozoans, metazoans and insects.

An important consideration from the ecological perspective is whether release of a single novel protein into the soil microbial community is significant in terms of the effect on soil function. Does the incorporation of novel proteins and peptides into soil have a significant effect on the community structure or biodiversity of the associated microflora and, if it does, is there any reason to be concerned about such a change? Some preliminary studies have addressed this issue (Tomlin, 1994; Donegan et al., 1995; Doyle et al., 1995; Donegan et al., 1997; Heuer and Smalla, 1999; Lottmann et al., 1999; Siciliano and Germida, 1999). Other examples of potential direct effects of transgenic organisms on ecosystem processes have been reviewed recently (Kirk, 2000).

While initial studies of rhizosphere microbial diversity using phenotypic measures indicated differences between the microflora of transgenic versus wild-type canola cultivars (Donegan et al., 1995; Siciliano and Germida, 1999), subsequent studies have shown that these differences can reflect soil microbial community patchiness or heterogeneity within the study area (Germida et al., pers. comm.).

In other words, variation in community structure between patches in different plots can overshadow variation due to the presence/absence of transgene products in the rhizosphere. Similar findings were reported by researchers in Braunschweig, Germany examining the microbial flora associated with transgenic T4-lysozyme-producing potato (Solanum tuberosum) grown under greenhouse and field conditions (Heuer and Smalla, 1999; Lottmann et al., 1999). In these studies, small shifts in species abundance were detected, but the observed effects were minor relative to the natural variability observed in several field samplings.

The potential for GMOs to affect critical soil biogeochemical cycles has been raised. Such an effect would require that steps in specific biogeochemical cycles carried out by microorganisms be inhibited or enhanced, presumably as a result of toxicity to the species involved or a shift in community structure. Arguing against such potential effects is the observed redundancy of functions in microorganisms that are involved in many, if not all, biogeochemical cycles. For an illustration of the redundancy of function in a typical soil biogeochemical function, consider a single step in the nitrogen cycle, the chemoautotrophic oxidation of ammonia to nitrate (Aakra et al., 2000). Despite sampling problems in this study (problems that are associated with most analyses of soil microbial diversity), ammonia-oxidizing bacteria were found to be represented by a complex set of taxonomic clusters (“species”) within the Nitrosospira genus. Unless all of these ammonia-oxidizing species were simultaneously inhibited by the introduction of a GM crop, the function of ammonia oxidation in the soil nitrogen cycle is unlikely to be affected. Of course, it is not inconceivable that the explicit purpose of a biotechnology product (a crop, a microbial inoculant, or an engineered biochemical process) would be to change a step in a biogeochemical cycle. To build on the example above, it may be beneficial to the agronomist, for example, to enhance the oxidation of ammonia to nitrate in the rhizosphere of the crop plant, in order to enhance plant nutrient uptake. In this case, where the biotechnology is in fact designed to modify biogeochemical cycles, risk assessments should be designed to weigh the ecological effects of such a modification. Test systems have been developed to measure such effects (Stotzky, 1993; Jepson et al., 1994).

Lateral Gene Transfer

Gene transfer between closely related and very distantly related microorganisms is an integral part of species evolution in microbial communities. This process can be measured directly (Hoffman et al., 1994; Nakatsu et al., 1995; Dröge et al., 1998; Gebhard and Smalla, 1999; Sengeløv et al., 2000) and it can be inferred from comparative gene or genome analyses (Sundin and Bender, 1996; de Souza et al., 1998; Di Gioia et al., 1998; Ochman et al., 2000; Reid et al., 2000).

Comparative genomics has enabled us to estimate the impact of lateral gene transfer on microbial evolution. Different microbial species vary in the degree to which their genomes are composed of laterally transferred elements. For instance, in the common digestive bacterium Escherichia coli K12, approximately 16% of the genome (or about 700 genes) can be attributed to lateral gene transfer within “recent” evolutionary history (Ochman et al., 2000). To give some perspective on what is meant by “recent”, the same methods of comparison yield an estimate that approximately 16,000 nucleotide base pairs have been successfully introduced into the E. coli genome per million years. This mobile fraction of the genome is composed of genes or other elements either having the hallmarks of lateral gene transfer function (phage-, plasmid- and transposon-related genes) or having DNA of atypical nucleotide sequence composition or patterns of codon usage that distinguishes it from the rest of the genome. Other species of bacteria that occupy less variable and environmentally challenging places may have much less laterally acquired or foreign DNA. For instance, many parasites (Mycoplasma genitalium, Rickettsia prowazekii, Borrelia burgdorferi) have less than 1% laterally transferred DNA. On the other hand, microorganisms inhabiting highly variable habitats that are subject to periodic disturbance, such as soils, sediments or water, are likely to contain greater proportions of laterally transferred genes. Examples include the cyanobacterium Synechocystis PCC6803 and Pseudomonas putida (Ochman et al., 2000, http://www.qiagen.com/sequencing/psputida.html, Oct. 2000).

The contribution of lateral gene transfer to microbial genome evolution can be appreciated by looking at the emergence of beneficial bacteria, such as those that remediate toxic organic pollutants in the environment, and pathogenic bacteria, such as those that cause disease in humans. Recent studies of the emergence of pathogenic E. coli have shown that lateral gene transfer of virulence determinants has occurred repeatedly during the divergence of different pathogenic strains (Reid et al., 2000). For example, the important food- and water-borne pathogen E. coli O157:H7 has acquired numerous pathogenicity determinants over the course of its 4.5-million-year evolution as an animal pathogen (Hacker and Kaper, 2000; Morschhauser et al., 2000; Reid et al., 2000). Since the genome of this pathogen has recently been sequenced, a good perspective on the contribution of gene transfer to the emergence of E.coli O157:H7 as a pathogen will be forthcoming (Perna et al., 2001). Similar hallmarks of horizontal gene transfer mark the Salmonella typhimurium genome (Baumler, 1997).

The examples listed above of gene transfer between different species or genera are very likely gross underestimates of the degree to which lateral gene transfer determines the structure of microbial genomes. This is because the comparative methods used in these studies are less effective at inferring transfer of genes between more closely related species where gene structure is more similar. In addition, lateral gene transfer of this type occurs far more frequently than transfer between distantly related species, thus compounding the underestimate. Therefore, precautions implicit in many regulatory schemes (see Chapter 3) that pertain to microorganisms and that call for information on the capacity of the microorganism to undergo transformation, transduction and conjugation, should take into account the fact that probably all microorganisms take part in these processes of gene exchange, and that in most environments there will be no possibility, and likely no need, to prevent these processes.

In the face of extensive mixing of genes by lateral gene transfer and rapid generation times by simple binary fission, how are bacterial species identities maintained? As indicated in the introductory paragraphs of this section, we base our definition of a microbial species on a suite of structural, physiological and biochemical features and/or arbitrarily defined differences in gene sequences. Lateral gene transfer will erode these differences. On the other hand, the diversity of microbial niches that exist in most natural habitats ensure that unique taxa are selected that are specially adapted to their niche. These taxa will carry a largely invariant set of essential genes, often termed “housekeeping” genes, that are rarely subject to lateral gene transfer or are not selectively advantageous if they are transferred to a new host.

Under this microbial evolutionary paradigm, what is the significance of introducing a foreign gene or set of genes from a crop, animal or other biotechnology product? We cannot know exactly because we cannot know the entirety of interactions and effects that may arise in microbial communities that remain largely uncharacterized. We can discuss some potential risks, from the perspective of our very limited understanding of microbial community structure and function.

Transfer of Antibiotic Resistance Genes

The biotechnology industry has indicated it is no longer developing crops carrying antibiotic resistance markers for commercialization, and a similar trend is likely to occur in the development of transgenic animals for environmental release. There are alternatives to antibiotic selection in the development of transgenic crops, as there are ways to eliminate these genes from the final construct prior to commercialization (Carrer and Maliga, 1995; Iamtham and Day, 2000). These methods were first developed in bacterial systems and have long been available for microbial GMOs (Sanchez-Romero et al., 1998; van Elsas et al., 1998). Many reports and commissions have recommended that the use of genes conferring resistance to human or animal therapeutic antibiotics be avoided in all circumstances where lateral transfer of these genes may occur. Therefore, this potential risk is considered here only in the context of some existing crops, and as background information for understanding the risks of transfer of other genes.

Antibiotic resistance genes are believed to have been derived in many cases from the very microorganisms that produce antibiotics in soil or aquatic habitats. They are found in bacteria isolated from natural environments with no prior, deliberate exposure to antibiotics (Smalla et al., 1993; Dröge et al., 1998) and they can be found in bacteria isolated prior to the era of human discovery and commercialization of antibiotics. The widespread use of antibiotics since the 1940s has resulted in the selection of antibiotic-resistant strains. The latter have acquired resistance genes either by spontaneous mutation of DNA within the strain or by horizontal transfer from another organism (Walsh, 2000). Natural gene mobility contributes an important dimension to the rise of antibiotic resistance in human, animal and plant commensal and pathogenic microorganisms (Health Canada, 1993; Sundin and Bender, 1996; Wireman et al., 1997; Heuer et al, 2000; Lawrence, 2000; Walsh 2000). This history of genetic change within organisms that represents a significant threat to our health and the health of our agricultural systems should be taken as an illustration of the importance of selection.

The Importance of Evaluating Selection

Selection will play a crucial role in determining whether or not a particular gene used for modifications to microorganisms, crops or domestic animals poses a threat to other organisms as a consequence of lateral gene transfer. It is impossible to generalize about the magnitude of this risk, as each gene construct will have a different potential for transfer and, more importantly, for selection in the recipient organism. For instance, the rate of acquisition of foreign DNA of 16 kb (or about 16 genes) per million years for E. coli discussed above is for “successful” integration of foreign genes, under natural selection pressures. Artificial selection accelerates the rate of successful acquisition of foreign genes by orders of magnitude, as determined for both antibiotic resistance genes and pollutant biodegradation genes (Sundin and Bender, 1996; Di Gioia et al., 1998; de Souza et al., 1998). Most bacterial genomes maintain less than 10 Mbp of DNA, and as genes are acquired through lateral transfer, they are also subject to mutation, recombination and deletion. As a result, the genome size remains more or less within the optimal range for that species in its natural habitat, while the genetic makeup of the organism remains in flux. In other words, microorganisms can be viewed as “sampling gene space” rather than accumulating genes (Ochman et al., 2000). The speed with which adaptive mutations can change microbial population structure has been elegantly demonstrated in laboratory evolution studies conducted over 25,000 generations using E. coli (Papadopoulos et al., 1999; Schneider et al., 2000). A large part of the genomic plasticity of this laboratory strain, grown under environmentally stable conditions, has been shown to be due to transposition of insertion sequences (chromosomal mobile genetic elements). This inherent genetic instability therefore contributes substantially to the normal evolutionary change of this species, and by inference all other microbial species. Lateral gene transfer, together with rearrangements and recombination events in recipient organisms, act as driving forces in determining the structures of microbial operons and chromosomes (Lawrence, 2000).

Over the past decade or so, researchers have focused almost all of their efforts on the question of whether or not transgenes and antibiotic resistance markers in plants or animals will transfer to bacteria in the environment. Almost no effort has been expended on the questions of whether or not the genes will be selected in the natural environment and whether or not these genes will pose a risk (Syvanen, 1999). To date, there is no evidence that lateral gene transfer from transgenic crops to the natural microflora of soil has had a significant effect on soil quality or functional ecology. It has proven quite difficult to detect transfer, although there is some evidence that it can occur under somewhat artificial circumstances (Hoffman et al., 1994; de Vries and Wackernagel, 1998; Gebhard and Smalla, 1999). The difficulty here is not so much in detecting a rare event, as in predicting a priori the likely routes of gene transfer, which might be quite complicated. These routes might include uptake of genes from plant residues (Sengeløv et al., 2000) and following animal tissue decay; via ruminant and non-ruminant gut microorganisms and feces (Schubbert et al., 1997, 1998); through plant pollen, root cap cell or root hair cell release; and via a myriad of intermediate vectors including pollinators or their associated microflora (Poppy, 1998; Ramsay et al., 1999); Kaatz study on gut contents of bees (Univ. of Jena); via epiphytic bacteria and insects, or via “rafting” on dispersed particles of plant or soil materials. More importantly, to date there has been no evidence for natural selection acting on any new hosts of genes transferred from transgenic plants or animals. This does not mean that selection cannot operate on these genes. For instance, it is known that mercuric ion released from dental amalgam is at a sufficient concentration in the gut to select for mercury-resistance and genetically linked antibiotic resistance genes in the natural gut bacteria of primates (Wireman et al., 1997). This finding illustrates the subtle nature of selection processes that may come into play.

Where the potential risks of a transgene warrant the cost of the research, case-by-case evaluations of the potential for gene transfer and selection should be done. These studies should place research emphasis on likely means of selection of the transgenes following transfer and how this selection could affect target or non-target microbial communities and ecological processes. Without selection, lateral gene transfer is of little consequence.

RECOMMENDATIONS

6.1 To the extent that the existing regulations, such as those under the Canadian Environmental Protection Agency and the Canadian Food Inspection Agency Acts (Chapter 3), call for ecological information on the fate and effects of transgenic biotechnology products on ecosystems, the Panel recommends that this information should be generated and should be available for peer review.

6.2 If environmental risks are a concern for a particular biotechnology product, especially with respect to persistence of the organism or a product of the organism, persistent effects on biogeochemical cycles, or harmful effects resulting from horizontal gene transfer and selection, then the Panel recommends that exhaustive and long-term testing for these ecological effects be carried out.

6.3 The Panel recommends that, in evaluating environmental risks, scientific emphasis should be placed on the potential effects of selection operating on an introduced organism or on genes transferred to natural recipients from that organism.

6.4 The Panel recommends that a detailed analysis be undertaken of the expertise needed in Canada to evaluate environmental effects of new biotechnology products and, if the appropriate expertise is found to be lacking, resources be allocated to improving this situation.

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