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This is in contrast to requirements introduced for GMOs and GM foods. The concept of risk assessment of GMOs was first discussed at the Asilomar Conference in 1975. The discovery of recombinant DNA had raised concerns among researchers regarding the potential creation of recombinant viruses whose escape would threaten public health.
Fourteen months after a voluntary moratorium on research involving recombinant DNA techniques, guidelines for the physical and biological containment of riskier experiments were drafted and agreed.
These guiding principles were the basis of the USA guidelines for research in modern biotechnology developed in 1976 by the National Institutes of Health Recombinant DNA Advisory Committee. Early regulatory requirements were intended to prevent the accidental release of microorganisms from research facilities.
In continuation of this, regulation for contained use and deliberate release of GMOs was developed, e.g. EU regulations in 1990. These guidelines elaborated a premarket human-health and environmental-safety assessment requirement for all GMOs and GM foods on the basis that they are novel and have no history of safe food or environmental use.
Many countries have since established specific premarket regulatory systems requiring the rigorous assessment of GMOs and GM foods before their release into the environment and/or use in the food supply.
While many national regulatory bodies base their safety assessment of GMOs and GM foods on shared concepts, differences in regulatory systems have led to disagreements and confusion in their deployment. While the terms ‘safety assessment’ and ‘risk assessment’ are often used interchangeably in some literature, these are two clearly different, but interlinked processes.
To provide international consistency in risk analysis of GMOs and GM foods which incorporates risk assessment, management and communication components, a number of international regulatory and standard-setting bodies have introduced uniform standards.
These include standards for human-health and environmental-safety assessment of GMOs and GM foods, and notification of their movement across national borders.
The objective of uniform global standards for risk assessment would be challenging as countries are bound to reach different decisions on the scope of the assessment, particularly the resolution of whether or not to include social or economic aspects. International regulatory systems covering GM food safety (Codex Principles) and environmental safety (Cartagena Protocol on Biosafety) came into force in 2003.
The concept that allows for the comparison of a final product with one having an acceptable standard of safety is an important element of a GM food safety assessment. This principle was elaborated by FAO, WHO and OECD in the early 1990s and referred to as ‘substantial equivalence’.
The principle suggests that GM foods can be considered as safe as conventional foods when key toxicological and nutritional components of the GM food are comparable to the conventional food (within naturally occurring variability), and when the genetic modification itself is considered safe. However, the concept has been criticised by some researchers.
At a Joint FAO/WHO consultation on foods derived from biotechnology held in 2000, it was acknowledged that the concept of substantial equivalence contributes to a robust safety assessment, but it was also clarified that the concept should represent the starting point used to structure the safety assessment of a GM food relative to its conventional counterpart.
The consultation concluded that a consideration of compositional changes should not be the sole basis for determining safety, and that safety can only be determined when the results of all aspects under comparison are integrated.
Influence of GM Foods on Human Health:
The Codex Alimentarius Commission adopted the – following texts in July 2003: Principles for the risk analysis of foods derived from modern biotechnology; Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants; and Guideline for the conduct of food safety assessment of foods produced using recombinant-DNA microorganisms.
The last two texts are based on the Principles and describe methodologies for conducting safety assessments for foods derived from recombinant-DNA plants and microorganisms, respectively.
The premise of the Principles dictates a premarket assessment, performed on a case-by-case basis and including an evaluation of both direct effects (from the inserted gene) and unintended effects (that may arise as a consequence of insertion of the new gene).
The Codex safety assessment principles for GM foods require investigation of:
(a) Direct health effects (toxicity);
(b) Tendency to provoke allergic reactions (allergenicity);
(c) Specific components thought to have nutritional or toxic properties;
(d) Stability of the inserted gene;
(e) Nutritional effects associated with the specific genetic modification; and
(f) Any unintended effects which could result from the gene insertion.
Codex principles do not have a binding effect on national legislation, but are referred to specifically in the Agreement on the Application of Sanitary and Phytosanitary Measures of the World Trade Organisation, and are often used as a reference in the case of trade disputes.
The 2003 Expert consultation on the safety assessment of foods derived ‘from GM animals, including fish formed the opinion that to further develop the risk-assessment process with current scientific knowledge, integrated toxicological and nutritional evaluations should be conducted in order to identify food-safety issues that may need further investigation.
Both evaluations combine data from the hazard identification and characterisation, and food intake assessment steps. It should be noted that such newly suggested further developments of the risk-assessment process have not yet been considered by Codex, and that the international principles and guidelines for risk analysis and safety assessment of foods derived from biotechnology are as accepted by Codex in 2003.
The potential direct health effects of GM foods are generally comparable to the known risks associated with conventional foods, and include, for example, the potential for allergenicity and toxicity of components present, and the nutritional quality and microbiological safety of the food.
Many of these issues have not traditionally been specifically assessed for conventional food; but in one area—toxicity of food components—there is ample experience related to the use of animal experiments to test potential toxicity of targeted chemical components.
However, the intrinsic difficulty in testing whole foods, as opposed to specific components, in animal feeding experiments have resulted in the development of alternative approaches for the safety assessment of GM foods. The safety assessment of GM food follows a stepwise process aided by a series of structured questions.
Factors taken into account in the safety assessment include:
i. Identity of gene of interest, including sequence analysis of flanking regions and copy number;
ii. Source of gene of interest;
iii. Composition of GMO;
iv. Protein expression product of the novel DNA;
v. Potential toxicity;
vi. Potential allergenicity; and
vii. Possible secondary effects from gene expression or the disruption of the host DNA or metabolic pathways, including composition of critical macronutrients, micronutrients, antinutrients, endogenous toxicants, allergens and physiologically active substances.
Unintended effects, such as elevated levels of antinutritional or toxic constituents in food, have on occasion been characterised in conventional breeding methods, e.g. glycoalkaloid levels in potatoes.
Organisms derived from conventional breeding methods, including tissue cultures, may have a somewhat enhanced possibility for genetic (and epigenetic—environmentally induced changes that affect the expression of a gene without changing the DNA sequence) instabilities, such as the activity of mobile elements and gene-silencing effects.
These effects could increase the probability of unintended pleiotropic effects (affecting more than one phenotypic trait), e.g. increased or decreased expression of constituents or possibly modifications in expressed proteins, as well as epistasis (the interaction of the inserted gene with other genes).
It has been argued that random insertion of genes in GMOs may cause genetic and phenotypic instabilities but, as yet, no clear scientific evidence for such effects is available.
A better understanding of the impact of natural transposable elements on the eukaryotic genome may shed some light on the random insertion of sequences. Gene expression in conventional and GM crops is subject to environmental influences. Environmental conditions such as drought or heat can stimulate some genes; turning the expression up or down.
The assessment of potential synergistic effects is necessary in the risk assessment of organisms derived from gene stacking, i.e. breeding of GMOs containing genetic constructs with multiple traits. Internationally agreed procedures for the assessment of such organisms are desirable.
Unintended effects can be classified as insertional effects, i.e. related to the position of insertion of the gene of interest, or as secondary effects, associated with the interaction between the expressed products of the introduced gene and endogenous proteins and metabolites. There is common agreement that targeted approaches, i.e. the measuring of single compounds, is very useful and adequate to detect such effects, as has been done with conventionally bred products.
To enhance and improve the identification and analyses of these unintended effects, profiling methods have been suggested. This untargeted approach allows detection of unintended effects at the mRNA (microarray), protein (proteomic) and metabolite (metabolomic) level. It still remains to be seen which of these techniques (once validated) would be useful for routine risk-assessment purposes.
Unintended effects were specifically addressed by the FAO/WHO Expert consultation on the safety aspects of genetically modified foods of plant origin and the Codex Principles for the risk analysis of foods derived from modern biotechnology.
These consultations noted that there is a need to establish the consequences of natural baseline variations, the effects of growing conditions and environmental influences, and the ways to interpret safety- relevant data from profiling techniques.
Adequate methods for the assessment of potential, unintended effects need to be evaluated for specific GMOs case by case, where the assessment already aims to consider unintended toxic and antinutritional factors through analysis of proximal constituents and GM characteristics.
As profiling methods are not in use in routine risk assessment, the second step in the comparative safety assessment has been suggested as a measure for identifying and characterising any unintended effects that may be associated with complex foods.
Natural genetic transformation has been found to occur in different environments, e.g. in food. In addition, it has been shown that ingested DNA from food is not completely degraded by digestion, and that small fragments of DNA from GM foods can be found in different parts of the gastrointestinal tract.
As the consequences of horizontal gene transfer (HGT) may be significant in some human-health conditions, the potential for HGT needs to be part of the risk assessment of GM food. FAO/WHO consultations have also discussed the potential risks of gene transfer from GM foods to mammalian cells or gut bacteria.
These panels have suggested that it may be prudent in a food- safety assessment to assume that DNA fragments survive in the human gastrointestinal tract and can be absorbed by either the gut microflora or somatic cells lining the intestinal tract.
It was agreed that the assessment needs to take into account a number of factors including, but not limited to, the specific characteristics encoded by the DNA sequences, the characteristics of the receiving organism, and the selective conditions of the local environment of the receiving organisms.
Some scientists have pointed to the present methodological limitations of a comprehensive scientific evaluation of this problem (mainly because of estimations that only approximately 1% of naturally existing bacteria can be cultured, and therefore analysed). Discussion also addresses the consequences of a rare probability of a transfer event against the high numbers of bacteria and genes available for transfer.
The DNA construct used to change the genetic composition of a recipient organism should be considered within an assessment, especially if the gene or its promoter (e.g. cytomegalovirus promoter) has been derived from a viral source.
Sequences unrelated to the target gene could be introduced as part of the construct. Inadvertent introduction of such sequences into the germ-line of a GM animal not only has the potential for creating unintended genetic damage, but can also contribute by recombination to the generation of novel infectious viruses.
A well-known example is the generation of a replication-competent murine leukaemia virus during the development of a vector containing a globin gene. The horizontal transfer of recombinant genetic material to microorganisms has demonstrated an enhanced stability of DNA under certain conditions.
Natural transformation of DNA to bacteria involves the active uptake of extracellular DNA by bacteria in a status of competence or in rare, illegitimate recombination events. The probability of such an event occurring appears to be extremely low, and very much related to the genes, constructs and organisms in question.
Allergies and Immune Responses:
Food allergies or hypersensitivities are adverse reactions to foods triggered by the immune system. Within the different types of reactions involved, non-immunological intolerances to food and reactions involving components of the immune system need to be differentiated. The former may invoke reactions such as bloating or other unpleasant reactions, but are thought not to involve the immune system and called ‘food intolerances’. Allergic reactions to traditional foods are well known.
The major food allergens are proteins in and derived from eggs, fish, milk, peanuts, shellfish, including crustaceans and molluscs (e.g. clams, mussels and oysters), soy, tree nuts (e.g. almonds, Brazil nuts, cashews, hazelnuts/filberts, macadamia nuts, pecans, pine nuts, pistachios and walnuts) and wheat. Whereas the groups of main allergens are well known and advanced testing methods have been elaborated, traditionally developed foods are not generally tested for allergens before market introduction.
The application of modern biotechnology to crops has the potential to make food less safe if the newly added protein proves to cause an allergic reaction once in the food supply.
A well-known case is the transfer of a gene encoding a known allergen, the 2S-Albumin gene from the Brazil nut, to a previously safe soybean variety. When the allergenic properties of the transgenic soybean were tested, sera from patients allergic to Brazil nuts cross-reacted with the transgenic soybean. For this reason, a commercial product was never pursued.
On the other hand, the introduction of an entirely new protein that has not been previously found in the food chain represents a different case. In the first case, guidelines for assessing foods with known allergens are clear.
The second case is more difficult to assess because there is no definitive test to determine the potential allergenicity of a novel protein. Instead, several risk factors provide a rough guide as to the likelihood of allergenicity.
Risk-assessment protocols for food allergy examine four elements:
1. Allergenicity assessment (is the food or elements in the food a potential allergen);
2. Dose response assessment (is there a safe concentration of the allergen);
3. Exposure assessment (how likely is it that people will encounter the allergen); and
4. Susceptible subpopulations (how do those prone to allergy react to this new food).
Elements of an allergenicity assessment include a comparison of the sequence oi the transferred gene (including the flanking regions at the site of insertion) with sequence motifs of allergenic proteins from databanks, an evaluation of the stability of the newly expressed proteins against digestion, and animal and immune tests, as appropriate.
Absence of sequence similarity with allergenic protein epitopes, and low stability under acidic or proteolytic conditions, do not preclude the presence of a potential allergen. There are proven incidents which have contradicted the general rules, e.g. where small modifications in a protein sequence determine allergenicity.
Allergenicity prediction using protein-sequence motifs identified from a new allergen database has been proposed as a new and superior strategy for identifying potential allergens. Some experts consider that the use of sera from polysensitised patients is important for the testing of allergenicity.
Areas of improvement of risk assessment of allergens include mechanistic studies of animal models and genomic techniques. FAO/WHO expert panels have established protocols for evaluating the allergenicity of GM foods on the basis of the weight of evidence. The strategy adopted is applicable to foods containing a gene derived from either a source known to be allergenic or a source not known to be allergenic.
The panels have, however, discouraged the transfer of genes from known allergenic foods unless it can be demonstrated that the protein product of the transferred gene is not allergenic. These principles have been applied by many regulatory agencies assessing the safety of GM foods and have provided the basis for Codex guidelines for the safety assessment of foods derived from biotechnology.
The cellular basis of immune responses is not completely understood, and a better understanding of the interaction of the immune system and foods in general is required in order to decipher whether specific GM foods may have impacts on the immune system apart from allergenicity. The impact of cell-mediated reactions (without involvement of immunoglobulin E antibodies) on hypersensitivity reactions elicited by foods is a matter of current research.
Genetically modified animals have mainly been produced for biomedical research purposes. To date, no GM food animals have been introduced onto international markets. But GM food animals such as fish can be expected in the near future.
In principle, the assessment of food and feed safety for GM animals follows the general principles of the assessment of GMOs outlined above. However, the specificities of the introduction of transgenes into animals, often using viral constructs for introduction into the germ-line, need distinct consideration.
A 2003 report of the Pew Initiative on Food and Biotechnology reviewed techniques for the production, uses and welfare of GM animals, as well as safety aspects. The risk assessment of foods derived from GM animals needs to be undertaken, as for other GM foods, on a case- by-case basis.
This includes an assessment of potential recombination of viral vectors used for transformation with wild-type viruses, especially in poultry, where potential incomplete digestion could lead to intestinal uptake of orally administered proteins, and an assessment of peptide expression that may have hormonal activity (e.g. in fish).
The FAO/WHO expert consultation on the Safety assessment of foods derived from GM animals, including fish held in 2003 addressed the key issues for food safety and evaluated the extent of scientific knowledge with regard to hazard identification and characterisation unique to transgenic animals.
Safety Aspects of Foods produced with GMMs:
The production of food additives or processing aids using GMMs, where the microorganism is not a part of the food, has become an important and generally well-accepted technology, with a significant number of such products on the market. Experience with the purification of proteins in the biomedical field suggests that well-standardised purification protocols are of central importance for the safety of these products.
Where the GMMs are a part of the food matrix (e.g. starter culture containing live or sterilised microbes), certain criteria were established in 2001 by a Joint FAO/ WHO expert consultation on foods derived from biotechnology for assessing the risks that may be associated with the preparation of such foods.
These include the genetic constructs (vectors) used in the GM microorganisms, the pathogenic potential of GMM, and the detrimental effects of a potential gene transfer (considering a higher incidence for gene transfer and the various mechanisms involved).
For GMMs used in foods (e.g. in fermented foods or in functional food preparations), the ensuing risk assessment ought to focus on the effects of a possible interaction between the GMM and endogenous intestinal microflora and the potential immune-stimulatory or immunomodulatory effects of the microorganisms in the event the gastrointestinal tract is colonised.
Small regulatory elements derived from viral DNA are commonly used to drive the expression of transgenes in GMOs. Viral-DNA constructs are sometimes used as transgenes to establish resistance against viral pests, as they express viral proteins that confer viral resistance on plants.
Some scientists suggest that the potential interaction of GM viral constructs with related wild-type viruses needs to be part of the risk assessment, to evaluate the potential of new viral pest strains evolving through mechanisms of recombination.
Insertion of viral vectors into functionally important genes of recipient patients in the field of biomedicine has been reported, and whereas such vectors are not “commonly used in food production, this evidence indicates the limited understanding of mechanisms directing insertion of genetic constructs.
Biopharming:
The potential to produce human proteins in animals has resulted in great interest in new possibilities for human health, but also in efforts to establish appropriate risk- assessment methods. The biosafety aspects of molecular ‘farming’ (or ‘pharming’) can be divided into two major groups: the potential spread of transgenes; and the potential negative effects of the expressed protein on the environment and the consumer.
Practices and guidelines ensuring effective separation of ‘biopharming’ are being investigated. Experts agree that the risk assessment should ensure that proteins designed to produce pharmaceutical products, e.g. in the animal’s milk, cannot find their way to other parts of the animal’s body, possibly causing adverse effects.
Environmental Safety and GMOs:
In many national regulations, the elements of environmental risk assessment (ERA) for GM organisms include the biological and molecular characterisations of the genetic insert, the nature and environmental context of the recipient organism, the significance of new traits of the GMO for the environment, and information on the geographical and ecological characteristics of the environment in which the introduction will take place.
The risk assessment focuses especially on potential consequences for the stability and diversity of ecosystems, including putative invasiveness, vertical or horizontal gene flow, other ecological impacts, effects on biodiversity and the impact of the presence of GM material in other products.
Different approaches in the ERA regulations of different countries have often resulted in different conclusions on the environmental safety of certain GMOs, especially where the ERA focuses not only on the direct effects of GMOs, but also addresses indirect or long-term effects on ecosystems, e.g. impact of agricultural practices on ecosystems.
Internationally, the concept of ‘familiarity’ was developed also in the concept of environmental safety of transgenic plants. The concept facilitates risk/safety assessments, because to be familiar means having enough information to be able to make a judgement of safety or risk.
Familiarity can also be used to indicate appropriate management practices, including whether standard agricultural practices are adequate or whether other management practices are needed to manage the risk.
Familiarity allows the risk assessor to draw on previous knowledge and experience with the introduction of plants and microorganisms into the environment and informs appropriate management practices. As familiarity depends also on knowledge of the environment and its interaction with introduced organisms, the risk/safety assessment in one country may not be applicable in another country.
Currently, the Cartagena Protocol on Biosafety (CPB) of the Convention on Biological Diversity is the only international regulatory instrument which deals specifically with the potential adverse effects of GMOs (known as living modified organisms (LMOs) under the Protocol) on the environment, taking also into account effects on human health. The Protocol covers transboundary movements of any GM foods that meet the definition of an LMO. Annex III of the Protocol specifies general principles and methodology for risk assessment of LMOs.
The Protocol establishes a harmonised set of international rules and procedures designed to ensure that countries are provided with the relevant information, through the information exchange system called the Biosafety Clearing House.
This Internet-based information system enables countries to make informed decisions before agreeing to the importation of LMOs. It also ensures that LMO shipments are accompanied by appropriate identification documentation.
While the Protocol is the key basis for international regulation of LMOs, it does not deal specifically with GM foods, and its scope does not consider GM foods that do not meet the definition of an LMO. Furthermore, the scope of its consideration of human-health issues is limited, given that its primary focus is biodiversity, in line with the scope of the Convention itself. Consequently, the Protocol alone is not sufficient for the international regulation of GM foods.
In the future, GMOs may gain wider approval for environmental release, either with or without approval to enter them in the human food supply. In such situations, it will be important to consider whether or not to apply post-market monitoring for unexpected environmental spread of the GMOs and their transgenes) that may pose food safety hazards.
Methods for detection of such GMOs and their transgenes in the environment are likely to involve application of two well-established bodies of scientific methodologies:
(1) Diagnostic, DNA-based markers; and
(2) Sampling protocols that are adequate (in terms of statistical power) and cost-effective.
However, there is a need to fully develop appropriate protocols for application of these methods to post-market detection of environmental spread of GMOs and their transgenes.
Monitoring can also be helpful to assure confinement of GMOs during R&D. Post-market monitoring (or surveillance) of GM foods with respect to direct human-health impacts has been raised at international conferences and within the Codex Alimentarius Commission.
Opinions regarding such monitoring vary from neither necessary nor feasible, to being essential to support and improve the results of a risk assessment and enable an early detection of uncharacterised and unintended hazards.
Some have suggested that monitoring of potential long-term effects of GM foods with significantly altered nutritional composition should be mandatory.
The Expert consultation on the safety assessment of foods derived from GM animals held in 2003 identified a need for postmarket surveillance, and therefore a product-tracing system, for:
i. Confirmation of the (nutritional) assessments made during the premarket phase;
ii. Assessment of allergenicity or long-term effects; and
iii. Unexpected effects.
The issue of post-market surveillance is closely related to risk characterisation. In general, potential safety issues should be addressed adequately through premarket studies, as the potential of post-market studies is currently very limited. Post-market surveillance could be useful in certain instances where clear-cut questions require, for instance, a better estimate of dietary exposure and/or the nutritional consequences of GMO-derived food.
Tools to identity or trace GMOs or products derived from GMOs in the environment or food-chain are a prerequisite for any kind of monitoring. Detection techniques (such as polymerase chain reaction; PCR) are in place in a number of countries to monitor the presence of GMOs in food, to enable the enforcement of GM labelling requirements, and to monitor effects on the environment. Attempts to standardise analytical methods for tracing GMOs have been initiated.