Food safety issues are as old as mankind and since time immemorial humans have developed strategies to ensure that the food they eat does not harm them. To produce food with any new technology, there must be appropriate safeguards to protect human and animal health. There exist few written records, but it is reasonable to assume that, historically, the safety of new products of food was established by trial and error. The foods consumed today are generally viewed as safe, based on their long history of such safe use. It is worth noting that this general acceptance of historical safety does not necessarily mean that some traditional foods may not cause adverse health effects under some circumstances.

Microbial contaminants and potentially toxic chemicals, both natural and man-made, are considered to be the sources of most of the risks we face when we eat food. A top priority has been to make sure the public is protected from infectious agents such as food-borne viruses and bacteria, considered to be the leading source of food-borne illness. The safety of the chemicals present in foods is also an important consideration. This is because the average diet consists of numerous chemical substances. Some of these are natural plant chemicals that may be toxic because they are natural pesticides that are produced by plants themselves to protect them against insects and other herbivores; others are applied intentionally as additives or occur as unintentional contaminants such as pesticide residues.

Due to the complexity of food and the natural presence of potential hazards, the assurance of food safety is not a simple matter. Almost any single definition of safe food will be overly simplistic, because safe food is a complex, multifaceted concept. According to the World Health Organization/Food and Agriculture Organization (FAO), food is considered safe if there is reasonable certainty that no harm will result from its consumption under anticipated conditions of use (CAC/GL45-2003). The FAO further explains that the goal of any safety assessment is to provide assurance, in the light of the best available scientific knowledge, that the food does not cause harm when prepared, used and/or eaten according to its intended use. The absolute safety of a food or an ingredient can never be guaranteed. However, with appropriate precautions during development, through manufacture into products, and in distribution, the risk from any food can be kept to an absolute minimum that is generally acceptable to consumers.

The safety of food derived from genetically modified crops should be addressed as an integral part of the much bigger issue of food safety. This is because genetically modified foods are not inherently less safe than their traditional counterparts (OECD 1993). Nevertheless, due to lack of past experience with GM foods and concerns about novel technologies, these foods have been subjected to rigorous safety assessment procedures that are not generally applied to traditional foods. The assessment of the safety of whole foods, which are complex and variable mixtures of many chemicals, is challenging compared to assessing the safety of individual chemicals Consequently new approaches have been developed to assist in assessing the safety of GM foods, including the core concept of “substantial equivalence”. This is a relative, rather than absolute, safety approach that relies on a broad comparison of the properties of the GM crop to those in related varieties of the crop that are not genetically modified and that would be considered as safe to consume.

1. Overview

The main human health concerns associated with foods derived from recombinant-DNA plants variously referred to in this text as genetically modified (GM) foods, or foods derived from genetically engineered (GE) plants can be categorized into two: issues associated with the safety of the newly inserted DNA including possibility of horizontal transfer of these genes; and the impact of the product expressed by the inserted gene (typically a protein).

2. Safety of inserted DNA in a GM food

Questions have been raised in the public domain over the fate of the newly inserted DNA after it has been consumed by humans. For instance, the possibility of transfer of the DNA from the food derived from genetically engineered plant into mammalian cells, gastrointestinal bacteria, or soil bacteria.

DNA is chemically identical regardless of its source i.e. the introduced DNA in a genetically modified organism is identical to any other DNA, regardless of which species the introduced DNA may have come from. The Food and Drugs Administration (FDA) considers all DNA from any source including genetically modified crops to be Generally Regarded as Safe (GRAS) (FDA, 2001). DNA is a completely natural and harmless component of most of the foods that we eat and is mostly degraded during industrial processing and in the gastrointestinal tract. Thus, whenever we eat those foods, we are eating the DNA that they contain.

The digestive system digests all DNA in exactly the same way whether the DNA is from GM food or conventional food. According to a report by the European Food Safety Authority (EFSA) on the fate of genes and proteins in food and feed, “After ingestion, a rapid degradation into short DNA or peptide fragments is observed in the gastro-intestinal tract of animals and humans and to date a large number of experimental studies with livestock have shown that rDNA fragments or proteins derived from GM plants have not been detected in tissues, fluids or edible products of farm animals” (EFSA, 2007). Therefore, if one eats DNA in a GM food or a conventional food, it will not change their own DNA or that of their children.

• Since food is derived from living organisms having genes, it follows that all food derived from these organisms contain DNA.

• The daily intake of DNA by humans in the food they consume is estimated to be about 0.1-1.0 g (Flachowsky, 2007), of that even with a diet wholly composed of GM Food the transgenic DNA would < 0.0001% of the total DNA (Society of Toxicology, 2002).

• A highly unlikely series of events would be required for this small fraction of rDNA to transfer to the mammalian or bacterial genome (WHO, 2000). The transgenic DNA would have to:

  • Survive harvest, drying, storage and milling

  • Survive food processing

  • Be present in the fraction of the plant that is consumed

  • Survive acidic pH and digestion by nuclease both in plant and in the mammalian gastrointestinal tract

  • Compete for uptake with a large excess of dietary DNA

  • Stably integrate into the host chromosome

  • Be incorporated into the host DNA and express in the new host

3. Concern about antibiotic resistance genes in GM foods

The other concern directly associated with inserted genes is the issue of genes that confer resistance to antibiotic or what are commonly referred to as antibiotic resistance marker (ARM) genes in GM foods. ARMs have been used in genetic engineering plant transformation processes and in some instances, the ARM genes remains in the finished product. There has been concern about the effect on human health and safety if genes present in GM foods were able to transfer to microorganisms in the human digestive tract. Of particular concern is the possibility that the genes conferring antibiotic resistance could transfer to disease-causing bacteria in the human digestive tract. If this were to happen, the concern is that it could adversely impact antimicrobial therapy. These concerns about ARMs are addressed below but the possibility of such transfer is low, and such furthermore such antibiotic resistance genes are already common in the microbes in the human gastrointestinal tract.

(i) Is the gene itself and the protein product produced harmful to humans or animals?

ARM genes are not any different from other DNA present in plants and animals, and are digested and processed in the gastro intestinal tract just like DNA from any other source. In addition, ARMs are naturally present in the environment including gut bacteria (Jonas et al., 2001).

When expressed in plant cells, the commonly used ARMs produce proteins that are digested in a similar way to other thousands of dietary proteins that humans consume everyday (Goldstein et al., 2005). The NPT II proteins, expressed by the npt IImarker gene for example are non-allergenic and non-toxic when consumed in foods in animal studies and in biotechnology pharmaceutical agents administered to human beings intravenously (Flavell et al., 1992; Fuchs et al., 1993). In addition, ARM proteins are frequently produced by human intestinal bacteria and thus humans have been exposed to these proteins throughout history. Thus it can be reasonably concluded that ARM genes themselves and the proteins they express, as with other genes and proteins in foods and feed do not pose risks to the health of humans or animals.

(ii) Can ARM genes transfer into bacteria and adversely impact antimicrobial therapy?

The possibility of transferring antibiotic resistance from genetically engineered plants and food derived from them to bacteria that are naturally found in the gut of humans and animals and therefore compromising the effectiveness of antibiotics has been raised. Gene flow amongst bacteria is a well established natural process that occurs in nature (Davison, 1999). In the same vein it is theoretically possible for genes to be transferred from plant to bacteria but this would be an extremely rare occurrence. This is because for an ARM to transfer from plants to gut bacteria, the gene would have to be excised from the plant chromosome without being destroyed by cellular enzymes, survive intact in the gut environment, and be acquired in intact form by a transformation-competent bacterium (WHO, 2000; Jonas et al., 2001). Because all whole foods contain DNA, humans have been exposed to DNA from both plant and gut bacteria throughout evolutionary history and in spite of this there has been no evidence of regular incorporation of intact plant or bacterial genes into human cells (Goldstein et al., 2005).

(iii) Are the antibiotics important to human or animal medicine?

It is important to note that antibiotic resistance genes currently present in GM foods code for resistance to antibiotics that are not widely used in human medicine, because resistance to them is already widespread. For example the npt II gene confers resistance to neomycin, kanamycin and other antibiotics that are not in clinical use any more, while aad-3 gene another ARM confers resistance to two-little used antibiotics, streptomycin and spectinomycin (Gilman et al., 1996). In the future, as genetic engineering techniques improve, antibiotic resistance genes will not be present in GM foods because they will either have been removed during development or have been replaced by other types of marker genes.

In a recent publication, the European Food Safety Authority has reaffirmed that two antibiotic resistance marker genes, npt II and aadA, pose no threat to humans or the environment (EFSA, 2009).

4. What about allergens in GM foods?

Introducing a gene into an organism raises the possibility of the introduction of allergens leading to production of foods that can elicit allergenic reactions in humans. An estimated 3 to 4% of adults and up to 8% of children suffer from food allergies in developed countries (Kanny et al., 2000; Sicherer et al., 2004). It is estimated that 90% of the food allergies are associated with products derived from a few groups of foods including cow’s milk, eggs, fish, crustaceans, peanuts, tree nuts, soybeans, wheat and sesame seeds (FAO, 1995). Although allergies to other foods occur, they tend to be far less common.

Most foods do not cause allergenic reactions in most people, but for people who have any kind of food allergy, certain proteins in food can cause an unusual immune reaction. The proteins that provoke this reaction are known as allergens, and people with allergies generally react to just one or a few allergens in one or two specific foods. The exact site of food absorption and allergy induction is still unknown. It is believed that most food allergens are absorbed in the intestines, prior to initiating an immune response. For an immunological reaction to be triggered, allergenic proteins have to move through the stomach in an immunologically intact form. Food proteins can also be absorbed into the circulation system through the oral mucosa (Dirks et al., 2005; Untersmayr et al., 2007). The majority of ingested food proteins whether naturally present in the transgenic product or introduced through genetic engineering are extensively digested as they travel through the gastrointestinal tract and this renders the resulting peptides non-reactive for antigen recognition (York et al., 1999).

Nevertheless, since almost all allergens are proteins (Bush and Hefle, 1996), there exists a possibility that any novel food protein might be an allergen. If a conventional food that contains allergens is genetically engineered, the GM food may contain those allergens, just as the conventional food does. For example, soy naturally contains proteins that cause an allergic reaction in some people. Unless these specific proteins are removed, they will also be found in GM soy varieties. Similarly, unless their level is increased by the genetic procedures, the level of risk is altered. The possibility of introducing new proteins exhibiting allergenic properties is real but low because of the safety assessment procedures that foods derived from recombinant-DNA plants undergo. These safety assessment procedures conducted on the introduced gene and the protein expressed in the GM product are designed to identify potential allergenic effects that may be associated with the commercialized GM crop/food. In assessing the safety of a GM food, tests are carried out to ensure that the levels of naturally occurring allergens in GM foods have not significantly increased above the natural range in the conventional food and to ensure that the new proteins in GM foods are not likely to be allergenic.

Hence, it seems unlikely that an allergenic risk posed by a GM food is greater than that of conventional foods generated by traditional breeding methods that are not subjected to the same kind of stringent safety evaluation procedures. A good example is the common peanut which is generally considered a safe product but has a history of eliciting mild to severe allergenic reaction in a segment of the population that is sensitive, regardless of how it is produced.

5. Possibility of toxins and antinutrients in GM foods?

All substances whether natural or human-made are potentially toxic depending on the dose received. However, substances classified as toxins are those that can be harmful to health at typical levels of exposure. Naturally occurring toxins are found in various foods, but the vast majority of these are present at concentrations well below the level that would harm the consumer. Some foods contain naturally occurring toxins that cause an adverse effect if the food is eaten in excessively high amounts e.g. cyanogenic glycosides in cassava. Other foods contain naturally occurring toxicants that elicit adverse reactions only if the food is prepared in a manner that allows for the retention of a toxicant that is normally destroyed e.g. lectins in kidney beans. Other foods may elicit deleterious effects on particular segments of the population that may be sensitive to a component in the food e.g. allergenic proteins in peanuts and soybeans. On the other hand food may become contaminated with naturally occurring toxicants produced by microorganisms e.g. botulinum toxin and aflatoxin (Taylor and Hefle, 2002). Thus, toxic substances are naturally present in many conventional foods that are subsequently genetically modified. Therefore unless any toxins present in a conventional food are specifically removed, they will remain in the GM version of the food.

For genetic engineered products the concern is the possibility of the genetic modification introducing a hitherto absent toxic substance for example a newly expressed protein or resulting into elevation of naturally occurring toxic substances. This is however precluded by the fact that as part of the safety assessment of GM foods, the levels of the naturally occurring toxins in the GM food are compared to those of the conventional food to ensure that the levels of the toxins are not elevated above their natural levels. Furthermore, the safety evaluation process requires the amino acid-sequence of a novel protein to be demonstrated not to be similar to known protein toxicants and that the protein is rapidly digested under simulated mammalian conditions. Animal bioassays are also conducted on individual proteins to reveal any potential toxicity.

6. What about Unintended Effects?

The potential occurrence of ‘‘unintended effects’’ is another concern being raised regarding the application of recombinant DNA techniques in the production of foods. Unintended effects are defined as those consistent differences between the GM plant and its appropriate control line, which go beyond the primary expected effect(s) of introducing the target gene(s) (EFSA, 2006). It is important to note that unintended effects are not limited to GE crops, traditional breeders spend a considerable time in their breeding programs back crossing in an attempt to eliminate some of these undesirable characteristics (Rischer and Oksman-Caldentey, 2006). Unintended effects in modern biotechnology may arise due to the nature by which current rDNA techniques introduce genes into the plant. The genes may result in disruption of gene functions, causing changes in levels and activities of enzymes, nutrients and metabolites, or the altered production of proteins or toxins (Cellini et al., 2004). An unintended or unexpected effect does not necessarily imply a health hazard, although obviously the expression of a new trait would require thorough scrutiny to ensure that the safety of the new product is reasonably ensured before it is commercialized.

Over the years there have been documented cases where conventional plant breeding procedures have led to crops with unintended effects. For example, conventional breeding of potatoes to produce a variety with superior characteristics resulted in a variety, the Lenape variety, which had unintentionally high levels of glycoalkaloids (Beier, 1990), a class of naturally occurring toxicants typically found in low levels in commercial potato varieties (Zitnak and Johnston, 1970). On the other hand genetically engineered soybeans altered to produce enhanced levels of the amino acid lysine showed an unexpected decrease in oil content (FAO/WHO, 2000).

Recombinant DNA techniques can be considered to be more precise than conventional breeding methods because only known and precisely characterized genes are transferred (IM and NRC, 2004). In contrast conventional breeding involves transferring thousands of unknown genes with unknown function along with the desired genes. Traditional breeders observe off-types due to unintended effects regularly and they methodologically eliminate these plants through selection during the evaluation process and long before commercialization. The same scrutiny, if not more, is also applied to plants generated by rDNA techniques.

It is important to note that all foods whether derived from plants developed through conventional plant breeding or through genetic engineering, carry potential hazardous substances and must be properly and prudently assessed to ensure an acceptable degree of safety. Indeed because GM crops are regulated to a greater extent and subjected to more rigorous risk assessment procedures, than are conventionally bred, non-GM crops, it is more likely that traits with potentially hazardous characteristics will not go through early development phases (IM and NRC, 2004). Moreover, current methods are constantly being improved and new ones are being developed to improve the detection of unintended effects (Rischer and Oksman-Caldentey, 2006).

Predicting and assessing potential adverse health effects posed by foods modified by a number of methods, including genetic engineering, are challenging. Also, because any form of adverse effect developed during this modification is unintentional, they may be unexpected, which complicates matters further. Nonetheless bodies like Codex Alimentrius Commission (CAC), International Life Science Institute (ILSI), and the Organization of Economic Cooperation Development (OECD) have developed guidelines to be followed in the assessment of the safety of GM foods to be discussed in a different segment of the food safety section. Also, to be discussed will be the safety assessment testing procedures and the interpretation of data generated to reasonably determine that the products developed by rDNA technology are at the very minimum as safe as their conventional counterparts.

1. Introduction

At an early stage in the introduction of recombinant DNA (rDNA) technology, efforts began to define internationally harmonized evaluation strategies for the safety of foods derived from genetically modified organisms (GMOs) (Kuiper, 2001). Biotechnologically derived products, be it the food, food ingredients and foods produced by GM microorganisms, undergo more stringent safety assessment procedures than is required by non-GM foods.

The comparative approach described in the initial food safety assessment report (IFBC, 1990), has laid the basis for later safety evaluation strategies. Other organizations such as the Organization of Economic Cooperation and Development (OECD), Food and Agriculture Organization (FAO), World Health Organization (WHO) and the International Science Institute (ILSI) have developed broad consensus documents that provide further guidelines for safety assessment. These documents have largely been used as the basis for development of individual country guidelines on food safety risk assessment procedures for GM foods. There is therefore general consistency in the national approaches to evaluating the food safety of genetically modified plants (Paoletti et al., 2008).

Evaluation of the safety of GM food has required the development of a new risk assessment approach relative to those previously established for chemical additives. Testing single chemical entities is not at all comparable to testing foods that are by their very nature bulky and complex mixtures. Also, animal feeding studies with whole foods can be problematic due to nutritional problems and diet balancing induced adverse effects not related directly to the material itself (Tomlinson, 2000). The difficulties of applying traditional toxicological testing (on single chemicals) and risk assessment procedures to whole foods meant that an alternative approach was required for the safety assessment of genetically modified (GM) foods. This led to the development of the concept of Substantial Equivalence (OECD, 1993).

The concept of Substantial Equivalence (SE) acknowledges that the goal of the safety assessment is not establishing absolute safety but to consider whether the GM food is safe as its traditional counterpart where such counterpart exists or to an earlier approved GM variety (OECD, 1993). Subsequently, any significant intended and unintended differences become the focus of the food assessment that might includes further toxicological, analytical and nutritional investigations before commercialization. The comparator approach should take into account agronomic, morphological, genetic and compositional aspects in order to make an objective assessment. Particular attention should be paid to the choice of comparator, the design of field trials, and statistical analysis of the generated data in order to obtain good comparative data. The comparator should be a non-transgenic, isogenic line to the GM line. The GM crop and the comparator should be grown in the same environmental conditions to avoid genotypic and phenotypic differences not related to the transformation process (Herman et al., 2007).

Substantial Equivalence is the starting point in a safety evaluation rather than an end point of the assessment. Admittedly, the concept of SE has its own inherent limitations; the definition of “substantial” is sometimes not clear, and this fact may leave much scope for individual (and national) interpretations. There is also limited knowledge, for example, of the levels and toxicity of anti-nutritional factors in crop plants especially in crops that are less economically important, which would make the comparative approach a challenge. In addition, crop varieties and analytical methods used to generate early compositional data might now be outdated compared with present crops and methods (Kok, 2003). There is also the notion that relevant unintended side effects may remain undetected when analyzing only specific compounds. However, consensus by expert panel from FAO, WHO and OECD has agreed that Substantial Equivalence is a powerful, robust and flexible paradigm that provides an adequate level of protection. So far, no biotechnology-derived crop that has gone through the regulatory approval process has caused any food safety problem.

The two key elements in the safety evaluation of GM foods are:

(i) assessing the safety of the genetic material introduced into the plant – this includes the identity of the source of genetic material, the nucleotide sequence of the DNA construct being inserted, the number of inserted sites, and the stability of the insertion in the plant genome;

(ii) safety of the newly introduced trait(s) or expressed product(s) typically a single new protein encoded by the inserted DNA or two new proteins if a marker gene has been used. The assessment of GM crops looks at the following key issues;

• Molecular and phenotypic characterization

• Transformation process

• Safety of new products

• Occurrence and implications of unintended effects

• Pathogenic, toxicity and anti-nutrient effect

• Allergenicity of new products

• Role of the new food in the diet

• Influence of food processing

2. Summary of Food Safety Assessment Guidelines

The summary list below enumerates the kind of information/data that would be contained in a food safety assessment dossier compiled by an applicant for the purpose of safety evaluation by a competent authority, based on CAC/GL45-2003. The list is by no means exhaustive and conversely, not all the information in this list has to be provided as part of the safety dossier. The information in the dossiers will obviously vary from product to product and should be evaluated on a case by case basis.

3. Description of the Recombinant-DNA Plant

• Identification of the crop.

• Name of the transformation event(s).

• Purpose of the modification, sufficient to aid in understanding the nature of the food being submitted for safety assessment.

4. Description of the Host Plant and its Use as Food

• Common or usual name; scientific name and, taxonomic classification.

• History of cultivation and development through breeding, in particular identifying traits that may adversely impact on human health.

• Information on the host plant’s genotype and phenotype relevant to its safety, including any known toxicity or allergenicity.

• History of safe use as a food.

• How plant is typically cultivated, transported and stored.

• Information on special processing required to make the plant safe to eat.

• Part of the plant used as a food source.

• Important macro- or micro-nutrients the food contributes to the diet.

• If the food is important to particular subgroups of the population.

5. Description of the Donor Organisms

• Common and scientific name.

• Taxonomic classification.

• Information about the natural history of the organism as concerns human health.

• Information on naturally occurring toxins, anti-nutrients and allergens.

• In case a microorganism is the donor organism, additional information on human pathogenicity and the relationship to known human pathogens.

• Information on the past and present use, if any, in the food supply and exposure route(s) other than intended food use (e.g. possible presence as contaminants).

6. Description of the Genetic Modification(s)

• Information on the specific method used for the modification.

• Information on the DNA used to modify the plant including the source (e.g., plant, microbial, viral, synthetic), identity and expected function in the plant.

• Details of all genetic components of the vector used to produce or process DNA for transformation of the host organism.

• Information on all the genetic components including marker genes, regulatory and other elements affecting the function of the DNA.

• Location and orientation of the sequence in the final vector/construct and function.

7. Characterization of the Genetic Modification(s)

• Information on the DNA insertions into the plant genome including:

  • characterization and description of the inserted genetic material.

  • number of insertion sites.

  • organization of the inserted genetic material at each insertion site including copy number.

  • sequence data of the inserted material and of the flanking regions bordering the site of insertion, sufficient to identify substance (s) expressed as a consequence of the insertion.

  • identification of any open reading frames within the inserted DNA or created by the insertions with contiguous plant genomic DNA including those that could result in fusion proteins.

• For any expressed substances in the rDNA plant the information to be provided include:

  • gene product(s) (e.g. a protein or an untranslated RNA).

  • gene product(s)’ function.

  • phenotypic description of the new trait(s).

  • level and site of expression of the expressed gene product(s) in the plant, and the levels of its metabolites in the edible portions.

  • amount of the target gene product(s), where possible, if the function of the expressed sequence(s)/gene(s) is to alter the accumulation of a specific endogenous mRNA or protein.

  • information on deliberate modifications made to the amino acid sequence of the expressed protein result in changes in its post-translational modification or affect sites critical for its structure or function.

• Additional information to be provided:

  • to demonstrate whether the arrangement of the genetic material used for insertion has been conserved.

  • to show whether the intended effect of the modification has been achieved and that all expressed traits are expressed and inherited in a manner that is stable through several generations consistent with laws of inheritance.

  • to demonstrate newly expressed trait(s) are expressed as expected in the appropriate tissues in a manner and at levels that are consistent with the associated regulatory sequences driving the expression of the corresponding gene.

  • any evidence to suggest that one or several genes in the host plant has been affected by the transformation process.

  • to confirm the identity and expression pattern of any new fusion proteins.

  • may be necessary to examine the inheritance of the DNA insert itself or the expression of the corresponding RNA if the phenotypic characteristics cannot be measured.

8. Compositional Analyses of Key Components

• Proximate composition including ash, moisture content, crude protein, crude fat, and various carbohydrate.

• Protein amino acid profile.

• Quantitative and qualitative composition of total lipids, i.e., saponifiable and nonsaponifiable components, complete fatty acid profile, phospholipids, sterols, cyclic fatty acids and known toxic fatty acids.

• Composition of the carbohydrate fraction e.g., sugars, starches, chitin, tannins, nonstarch polysaccharides and lignin.

• Qualitative and quantitative composition of micronutrients, i.e., significant vitamin and mineral analysis.

• Presence of naturally occurring or adventitious anti-nutritional factors e.g., phytates, trypsin inhibitors, etc.

• Predictable secondary metabolites, physiologically active (bioactive) substances, other detected substances.

9. Assessment of Possible Toxicity

• Indicate if the donor organism(s) is a known source of toxins.

• Amino acid sequence homology comparison of the newly expressed protein and known protein toxins and anti-nutrients.

• Demonstrate the susceptibility of each newly expressed protein to pepsin digestion.

• Where a host other than the transgenic plant is used to produce sufficient quantities of the newly expressed protein for toxicological analyses, demonstrate the structural, functional and biochemical equivalence of the non-plant expressed protein with the plant expressed protein.

• Oral toxicity study(s) completed for newly expressed proteins.

10. Assessment of Possible Allergenicity (Proteins)

• Indicate if the donor organism(s) is a known source of allergens.

• Amino acid sequence homology comparison of the newly expressed protein and known allergens.

• Demonstrate the susceptibility of each newly expressed protein to pepsin digestion.

• Where a host other than the transgenic plant is used to produce sufficient quantities of the newly expressed protein for toxicological analyses, demonstrate the structural, functional and biochemical equivalence of the non-plant expressed protein with the plant expressed protein.

• For those proteins that originate from a source known to be allergenic, or have sequence homology with a known allergen, additional immunological assays be warranted.

1. Introduction

At an early stage in the introduction of recombinant DNA (rDNA) technology, efforts began to define internationally harmonized evaluation strategies for the safety of foods derived from genetically modified organisms (GMOs) (Kuiper, 2001). Biotechnologically derived products, be it the food, food ingredients and foods produced by GM microorganisms, undergo more stringent safety assessment procedures than is required by non-GM foods.

The comparative approach described in the initial food safety assessment report (IFBC, 1990), has laid the basis for later safety evaluation strategies. Other organizations such as the Organization of Economic Cooperation and Development (OECD), Food and Agriculture Organization (FAO), World Health Organization (WHO) and the International Science Institute (ILSI) have developed broad consensus documents that provide further guidelines for safety assessment. These documents have largely been used as the basis for development of individual country guidelines on food safety risk assessment procedures for GM foods. There is therefore general consistency in the national approaches to evaluating the food safety of genetically modified plants (Paoletti et al., 2008).

Evaluation of the safety of GM food has required the development of a new risk assessment approach relative to those previously established for chemical additives. Testing single chemical entities is not at all comparable to testing foods that are by their very nature bulky and complex mixtures. Also, animal feeding studies with whole foods can be problematic due to nutritional problems and diet balancing induced adverse effects not related directly to the material itself (Tomlinson, 2000). The difficulties of applying traditional toxicological testing (on single chemicals) and risk assessment procedures to whole foods meant that an alternative approach was required for the safety assessment of genetically modified (GM) foods. This led to the development of the concept of Substantial Equivalence (OECD, 1993).

The concept of Substantial Equivalence (SE) acknowledges that the goal of the safety assessment is not establishing absolute safety but to consider whether the GM food is safe as its traditional counterpart where such counterpart exists or to an earlier approved GM variety (OECD, 1993). Subsequently, any significant intended and unintended differences become the focus of the food assessment that might includes further toxicological, analytical and nutritional investigations before commercialization. The comparator approach should take into account agronomic, morphological, genetic and compositional aspects in order to make an objective assessment. Particular attention should be paid to the choice of comparator, the design of field trials, and statistical analysis of the generated data in order to obtain good comparative data. The comparator should be a non-transgenic, isogenic line to the GM line. The GM crop and the comparator should be grown in the same environmental conditions to avoid genotypic and phenotypic differences not related to the transformation process (Herman et al., 2007).

Substantial Equivalence is the starting point in a safety evaluation rather than an end point of the assessment. Admittedly, the concept of SE has its own inherent limitations; the definition of “substantial” is sometimes not clear, and this fact may leave much scope for individual (and national) interpretations. There is also limited knowledge, for example, of the levels and toxicity of anti-nutritional factors in crop plants especially in crops that are less economically important, which would make the comparative approach a challenge. In addition, crop varieties and analytical methods used to generate early compositional data might now be outdated compared with present crops and methods (Kok, 2003). There is also the notion that relevant unintended side effects may remain undetected when analysing only specific compounds. However, consensus by expert panel from FAO, WHO and OECD has agreed that Substantial Equivalence is a powerful, robust and flexible paradigm that provides an adequate level of protection. So far, no biotechnology-derived crop that has gone through the regulatory approval process has caused any food safety problem.

The two key elements in the safety evaluation of GM foods are:

• assessing the safety of the genetic material introduced into the plant – this includes the identity of the source of genetic material, the nucleotide sequence of the DNA construct being inserted, the number of inserted sites, and the stability of the insertion in the plant genome;

• safety of the newly introduced trait(s) or expressed product(s) typically a single new protein encoded by the inserted DNA or two new proteins if a marker gene has been used. The assessment of GM crops looks at the following key issues:

  • Molecular and phenotypic characterization

  • Transformation process

  • Safety of new products

  • Occurrence and implications of unintended effects

  • Pathogenic, toxicity and anti-nutrient effect

  • Allergenicity of new products

  • Role of the new food in the diet

  • Influence of food processing

2. Summary of Food Safety Assessment Guidelines

The summary list below enumerates the kind of information/data that would be contained in a food safety assessment dossier compiled by an applicant for the purpose of safety evaluation by a competent authority, based on CAC/GL45-2003. The list is by no means exhaustive and conversely, not all the information in this list has to be provided as part of the safety dossier. The information in the dossiers will obviously vary from product to product and should be evaluated on a case by case basis.

3. Description of the Recombinant-DNA Plant

• Identification of the crop.

• Name of the transformation event(s).

• Purpose of the modification, sufficient to aid in understanding the nature of the food being submitted for safety assessment.

4. Description of the Host Plant and its Use as Food

• Common or usual name; scientific name and, taxonomic classification.

• History of cultivation and development through breeding, in particular identifying traits that may adversely impact on human health.

• Information on the host plant’s genotype and phenotype relevant to its safety, including any known toxicity or allergenicity.

• History of safe use as a food.

• How plant is typically cultivated, transported, and stored.

• Information on special processing required to make the plant safe to eat.

• Part of the plant used as a food source.

• Important macro- or micro-nutrients the food contributes to the diet.

• If the food is important to subgroups of the population.

5. Description of the Donor Organisms

• Common and scientific name.

• Taxonomic classification.

• Information about the natural history of the organism as concerns human health.

• Information on naturally occurring toxins, anti-nutrients, and allergens.

• In case a microorganism is the donor organism, additional information on human pathogenicity and the relationship to known human pathogens.

• Information on the past and present use, if any, in the food supply and exposure route(s) other than intended food use (e.g. possible presence as contaminants).

6. Description of the Genetic Modification(s)

• Information on the specific method used for the modification.

• Information on the DNA used to modify the plant including the source (e.g., plant, microbial, viral, synthetic), identity and expected function in the plant.

• Details of all genetic components of the vector used to produce or process DNA for transformation of the host organism.

• Information on all the genetic components including marker genes, regulatory and other elements affecting the function of the DNA.

• Location and orientation of the sequence in the final vector/construct and function.

7. Characterization of the Genetic Modification(s)

• Information on the DNA insertions into the plant genome including:

  • characterization and description of the inserted genetic material.

  • number of insertion sites.

  • organization of the inserted genetic material at each insertion site including copy number.

  • sequence data of the inserted material and of the flanking regions bordering the site of insertion, sufficient to identify substance (s) expressed because of the insertion.

  • identification of any open reading frames within the inserted DNA or created by the insertions with contiguous plant genomic DNA including those that could result in fusion proteins.

• For any expressed substances in the rDNA plant the information to be provided include:

  • gene product(s) (e.g. a protein or an untranslated RNA).

  • gene product(s)’ function.

  • phenotypic description of the new trait(s).

  • level and site of expression of the expressed gene product(s) in the plant, and the levels of its metabolites in the edible portions.

  • amount of the target gene product(s), where possible, if the function of the expressed sequence(s)/gene(s) is to alter the accumulation of a specific endogenous mRNA or protein.

  • information on deliberate modifications made to the amino acid sequence of the expressed protein result in changes in its post-translational modification or affect sites critical for its structure or function.

• Additional information to be provided:

  • to demonstrate whether the arrangement of the genetic material used for insertion has been conserved.

  • to show whether the intended effect of the modification has been achieved and that all expressed traits are expressed and inherited in a manner that is stable through several generations consistent with laws of inheritance.

  • to demonstrate newly expressed trait(s) are expressed as expected in the appropriate tissues in a manner and at levels that are consistent with the associated regulatory sequences driving the expression of the corresponding gene.

  • any evidence to suggest that one or several genes in the host plant has been affected by the transformation process.

  • to confirm the identity and expression pattern of any new fusion proteins.

  • may be necessary to examine the inheritance of the DNA insert itself or the expression of the corresponding RNA if the phenotypic characteristics cannot be measured.

8. Compositional Analyses of Key Components

• Proximate composition including ash, moisture content, crude protein, crude fat, and various carbohydrate.

• Protein amino acid profile.

• Quantitative and qualitative composition of total lipids, i.e., saponifiable and nonsaponifiable components, complete fatty acid profile, phospholipids, sterols, cyclic fatty acids and known toxic fatty acids.

• Composition of the carbohydrate fraction e.g., sugars, starches, chitin, tannins, nonstarch polysaccharides and lignin.

• Qualitative and quantitative composition of micronutrients, i.e., significant vitamin and mineral analysis.

• Presence of naturally occurring or adventitious anti-nutritional factors e.g., phytates, trypsin inhibitors, etc.

• Predictable secondary metabolites, physiologically active (bioactive) substances, other detected substances.

9. Assessment of Possible Toxicity

• Indicate if the donor organism(s) is a known source of toxins.

• Amino acid sequence homology comparison of the newly expressed protein and known protein toxins and anti-nutrients.

• Demonstrate the susceptibility of each newly expressed protein to pepsin digestion.

• Where a host other than the transgenic plant is used to produce sufficient quantities of the newly expressed protein for toxicological analyses, demonstrate the structural, functional and biochemical equivalence of the non-plant expressed protein with the plant expressed protein.

• Oral toxicity study(s) completed for newly expressed proteins.

10. Assessment of Possible Allergenicity (Proteins)

• Indicate if the donor organism(s) is a known source of allergens.

• Amino acid sequence homology comparison of the newly expressed protein and known allergens.

• Demonstrate the susceptibility of each newly expressed protein to pepsin digestion.

• Where a host other than the transgenic plant is used to produce sufficient quantities of the newly expressed protein for toxicological analyses, demonstrate the structural, functional and biochemical equivalence of the non-plant expressed protein with the plant expressed protein.

• For those proteins that originate from a source known to be allergenic, or have sequence homology with a known allergen, additional immunological assays be warranted.

1. Introduction

Based on the principles developed by international organizations like the World Health Organization (WHO), the Food and Agriculture Organization (FAO), and the Codex Alimentarius Commission (CAC) jointly established by FAO/WHO, the Organization of Economic and Co-operation and Development (OECD) and the US Food & Drug Administration (FDA), the aim of safety assessment of biotechnologically derived food products is to provide assurance, using the best available scientific knowledge that these foods are as safe to consume as their conventional counterpart and therefore do not cause appreciable harm when prepared, used and/or eaten according to their intended use. In general terms, to determine the safety of genetically modified foods, some of the questions that must be answered in the assessment process include:

• Do the donor and recipient organisms have a history of safe use?

• Are the new substances produced e.g. proteins safe to consume?

• Have potential allergens been introduced into or increased in the food?

• Are there changes in the content of other important substances e.g. toxicants, antinutrients?

• Has the composition and nutritional value changed?

• In what forms will the food or food products derived from it be consumed?

• Do the newly introduced substances survive processing, shipment, storage, and other preparation?

• What is the expected human dietary exposure?

• If an antibiotic resistance or other selectable marker is present, is it safe?

There are also questions to be answered about characterizing the transformation at the molecular genetic level e.g. where is the gene construct incorporated in the plant genome, has the sequence been characterized, are unexpected open reading frames present, number of copies of the gene, stability of expression etc.

Based on the Codex Guidelines for the safety assessment of foods derived from rDNA plants that was covered in a different section, a case study example – Roundup Ready soybean (RR soy), will be used to explain key data that needs to be availed as part of the food safety assessment application dossier and how to interpret the data. The case study information here includes excerpts from an application for food safety assessment dossier submitted by Monsanto company to regulatory authorities in Canada, USA and the UK and made available courtesy of the FAO.

Case Study – Roundup Ready Soybean (RR soy)

Monsanto Company has developed RR soy (event 40-3-2) varieties that confer tolerance to glyphosate, the active ingredient in Roundup herbicide, a herbicide used by farmers to kill weeds. The CP4 enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein is an important enzyme involved in the biosynthesis of aromatic amino acids in plants and microorganisms. (EPSP synthase is not present in animals and therefore glyphosate has a high level of safety for consumers) Inhibition of this enzyme by glyphosate leads to a deficiency in the production of aromatic amino acids and lack of growth in plants. RR soy (event 40-3-2) was produced by introduction of a cp4 epsps gene from a naturally occurring soil bacterium (Agrobacterium) using particle-acceleration transformation into the soybean genome. The cp4 epsps gene encodes for a version of the ESPS enzyme that is tolerant to glyphosate, meaning that Agrobacterium is not killed by the herbicide.

The RR soybean plant therefore produces two EPSPS enzymes: the soybean version already present in the plant, and the bacterial version added in the genetic modification. When the herbicide is applied, the soybean EPSPS enzyme is blocked and cannot function. However, the plant survives because the bacterial EPSPS enzyme that has been transferred into the plant remains active, allowing it to continue to make the necessary amino acids. Thus, the bacterial protein is able to function in the soybean in the same way that it does in the soil bacterium.

2. Description of the Recombinant-DNA Plant

A description of the rDNA plant being presented for safety assessment should be provided to identify the crop, the transformation event(s) to be reviewed and the type and purpose of the modification. The description should be sufficient to aid in the understanding of the nature of the food being submitted for safety assessment.

Case Study

The RR soy (event 40-3-2) was developed to allow for the use of glyphosate, the active ingredient in the herbicide Roundup, as a weed control option. This genetically engineered soybean line contains a form of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that allows event 40-3-2 to survive the otherwise lethal application of glyphosate. The EPSPS gene put into event 40-3-2 was isolated from a strain of the common soil bacterium Agrobacterium tumefaciens called CP4. The form of EPSPS enzyme produced by this gene is tolerant to glyphosate.

3. Description of the Host Plant and Donor Organism and Use as Food

This section of the data looks at the phenotypic and agronomic description of the plant, history of use of the conventional food including identifying the edible components of the food, food products commonly containing these edible components and processing requirements. May also include information of on how the plant is typically cultivated, transported and stored and whether special processing is required to make the plant safe to eat, and the plants normal role in the diet. As far as the donor organism is concerned, there should be information for its identification and it is particularly important to determine if the donor organism (s) or other closely related members of the family naturally exhibit characteristics of pathogenicity or toxin production, or have other traits that affect human health.

Case Study

RR soy has been tested in field trials in the United States, Central and South America, Europe, Central Europe and Canada since 1991. Data collected from trials conducted in over a 3-year period prior to commercialization in the United States demonstrate that RR soy does not differ significantly from conventional soybeans in morphology, seed production (yield), agronomic characteristics such as time to flowering and pod set, or vigor (germination or persistence). In addition, RR soy was monitored for its susceptibility to diseases and insects, and there were no differences observed in disease severity or insect infestations between RR soy and the control plants.

For RR soy, the host (recipient) organism is the soybean plant and the part of the soy plant that is consumed by humans is the seed — the soybean. Soybeans (soy) can be cooked and consumed whole or processed into many types of foods. Foods made from soy include soy beverages, tofu, soy oil, soy flour and lecithin. Some of the processed products containing soy may include breads, pastries, snack foods, baked products, fried products, edible oil products and special purpose foods such as infant formula.

The donor organism for RR soy is the common soil bacterium Agrobacterium (species strain CP4), from which the EPSPS gene was derived. Agrobacterium is found everywhere in the environment, so it could be expected to be commonly encountered in foods harvested directly from soil, such as cassava, sweet potatoes etc. As Agrobacterium has been ingested by humans over a long period of time, and there is no evidence that it causes toxicity or allergenicity in humans, it is reasonable to assume that the donor organism is safe and acceptable.

4. Description and Characterization of the Genetic Modification

All characteristics of the genetic insert must be known including the identity of the source of genetic material, the nucleotide sequence of the DNA construct being inserted, the number of insertion sites, and the stability of the insertion in the plant genome. The data requirement related to the genetic modifications serve to allow a detailed a detailed understanding of the resulting genetic insertions and their locations in the host plant. A detailed description of the molecular characteristics of the rDNA plant is required in order to demonstrate that the developer has critically analyzed the plant and its products, including all introduced genes and expressed proteins.

• Method used for modification

A detailed description of the method used to insert the new genetic material should be provided.

Case Study

RR soy was produced using a particle-acceleration transformation method. In this process, the plant cells are bombarded with microscopic particles of gold or tungsten coated with DNA that contains the new EPSPS gene from the donor organism (Agrobacterium). The aim is to get the new gene across the plant cell wall, so that it can be incorporated into the plant cell’s genetic material.

• Function

To understand how the new gene(s) function in the plants one would need to know the new gene(s) and their products, in this case the enzyme EPSPS, the genetic material that controls how, where and when the new genes are switched on and the genetic material that targets any new proteins to specific parts of the cell.

Case Study

RR soy contains a single new (cp4 epsps) gene, which codes for the EPSPS enzyme. In front of the bacterial EPSPS gene in RR soy is a regulatory sequence that tells the plant to turn the gene on, known as E35S promoter. At the other end of the EPSPS gene, another regulatory sequence tells the plant where the EPSPS gene ends, known as NOS 3′. A further regulatory sequence is the chloroplast transit peptide sequence whose function is to tell the plant cell to transport the bacterial EPSPS enzyme into the chloroplast of the cell.

• Characterization of the New Gene

This is detailed information on the arrangement of the new genetic material in the genome of the host organism including how many complete or incomplete copies of the new genetic material are present. It is also useful to compare the DNA sequence of the new genetic material in the modified plant’s genome with that of the original DNA to determine if there are any unexpected changes in the DNA sequence in the plant.

• Stability of the Genetic Changes

The genetic changes in a genetically modified plant must be stable. The new genetic material is considered to have become a stable part of the host genome if they remain the same over several generations of plants produced by conventional breeding. The This means that the newly introduced traits should be shown to pass from one generation to the next in a normal predictable way, following the principles of inheritance.

Case Study

A single full copy of the bacterial EPSPS gene plus flanking DNA sequences was present in the genome of RR soy. This gene was intact and of the correct size and sequence.

Case Study

The inserts are inherited in the expected Mendelian pattern and the stability of the inserts has been demonstrated by molecular analysis of the 3rd through the 6th generations of event 40-3-2. In addition, RR soy event has been in commercial production globally since 1996 with consistent product performance.

5. Effects of the new gene on human health

Concerns have been expressed that new genes, particularly antibiotic resistance genes, in GM foods might be able to transfer to disease-causing bacteria in the human digestive tract. Whether new genetic material in GM foods could transfer to gut bacteria and impact on human health has been considered in detail by the World Health Organization (WHO) and several European expert advisory panels, and in numerous scientific papers published in peer reviewed journals. There is general agreement that current genetic materials in GM foods has passed risk assessments and are not likely to present risks for human health. In addition, no effects on human health have been shown as a result of the consumption of such foods by the general population in the countries where they have been approved (WHO, 2009).

Two issues here (a) gene transfer to gut microorganisms (b) transfer into the human host (the gene may not be expressed but could incorporated fragments cause mutations?). The possibility of this happening is very low due to the highly unlikely series of events that would be required for plant DNA to transfer genes horizontally into mammalian or bacterial genomes (Chassy, 2002). However, this possibility cannot be completely discounted (Jonas et al., 2001). Nevertheless, in a recent report the European Food Safety Authority has reaffirmed that two commonly used antibiotic resistance marker genes, npt II and aadA, pose no threat to humans or the environment (EFSA, 2009).

Case Study

As RR soy contains no antibiotic resistance genes, this is not a concern in this case. The only new gene in RR soy that could potentially be transferred to cells in the human digestive tract is the bacterial EPSPS gene. No impact is expected on human health if this gene were to transfer to bacteria in the human gut, because the EPSPS enzyme in RR soy would function in the same way as the EPSPS enzyme naturally present in the gut bacteria. Finally, there is no evidence that DNA sequences from ingested foods have ever been taken up by human cells and incorporated into human DNA (Chassy, 2002).

6. Characterization of New Proteins

• Nature of the new protein

The nature and function of any new proteins in the GM food is also examined as part of the assessment process. This information also includes verification that the size of the new protein is as expected, and to quantify how much new protein is present in particular tissues. The presence and level of new proteins in particular components of GM varieties used as food, or in food preparation, may present safety issues. This should be assessed in the parts of GM plants that are actually eaten. It is possible that the new protein is only expressed in non-edible parts of the plant, inactivated, denatured or removed by heat or processing (cooking) or only present at very low levels in the edible part of the plant.

Case Study

The CP4 EPSPS protein produced in RR soy is functionally similar to a diverse family of EPSPS proteins typically present in food and feed derived from plant and microbial sources. In the safety assessment, the level of the bacterial EPSPS enzyme in fresh or processed edible parts of soy was found to make up less than 0.1% of the total protein. The EPSPS enzyme was shown to have no activity in the edible parts of the soy, because the enzyme is inactivated by heat during processing of the food.

• Potential toxicity of new protein

In this section, the assessment examines the potential toxicity of any new proteins in the GM food. Information should be provided to ensure that genes coding for known toxins or anti-nutrients present in the donor organisms are not transferred to recombinant-DNA plants that do not normally express those toxic or anti-nutritious characteristics.

In the case of proteins, the assessment should focus on amino acid sequence similarity between the protein and known protein toxins and anti-nutrients as well as stability to heat or processing and to degradation in appropriate representative gastric and intestinal model systems. Appropriate oral toxicity studies may need to be carried out in cases where the protein present in the food is not similar to proteins that have previously been consumed safely in food.

Non-protein substances that have not been consumed in food should be assessed on a case-by-case basis depending on the identity and biological function in the plant of the substance and dietary exposure. This may require the isolation of the new substances from the recombinant-DNA plant, or the synthesis or production of the substance from an alternative source, in which case the material should be shown to be biochemically, structurally, and functionally equivalent to that produced in the recombinant-DNA plant.

Case Study

The EPSPS protein does not show meaningful amino acid sequence similarity when compared to known protein toxins. Rapid degradation of the CP4 EPSPS protein correlates with limited exposure to the gastrointestinal tract and little likelihood that the protein can produce pharmacological, toxic, or allergenic effects. There were no treatment-related adverse effects in mice when CP4 EPSPS protein was administered orally at dosages of up to 572 mg/kg of body weight. This dose represents a significant—greater than 1,000-fold—safety margin relative to the highest potential human consumption of CP4 EPSPS protein. Finally, there is no history of EPSPS proteins being toxic. The bacterial EPSPS enzyme in the soybean has a similar structure and function to the EPSPS enzyme naturally present in the soybean and other plants forming part of the human food supply. On the basis of the evidence described above, the bacterial EPSPS enzyme in RR soy is unlikely to be toxic.

• Potential allergenicity of new protein

This component of the assessment looks at whether any new protein present in the RR soy is likely to cause an allergic reaction in some people. The stepwise approach for evaluating the allergenic potential of the genetically modified food examines the following parameters:

• • Source of the protein – Genes derived from known allergenic sources are assumed to encode an allergen unless scientific evidence demonstrates otherwise. Also attention should be paid to the choice of the expression host, because the post-translational modifications allowed by different hosts may impact on the allergenic potential of the protein.

  • Amino acid sequence homology – The purpose is to assess the extent to which a newly expressed protein is similar in structure to known allergens to assess the allergenic potential of the protein.

  • Pepsin resistance – There exists a correlation between resistance to digestion by pepsin and allergenic potential and therefore the resistance of a protein to degradation in the presence of pepsin under appropriate conditions indicates further analysis should be undertaken to determine whether the expressed protein is potentially allergenic.

  • Specific serum screening – Proteins that originate from a source known to be allergenic, or that have sequence homology with a known allergen, immunological assays should be performed where sera are available. There should be additional testing such as the possible use of skin test and ex vivo protocols even if the in vitro immunoassay comes out negative.

Case Study

The amino acid sequence of the CP4 EPSPS protein was compared to protein sequences associated with allergy and coeliac disease and shown to have no meaningful amino acid sequence similarity with any of the known allergens. The protein does not represent a previously described allergen and does not share potentially immunologically relevant amino acid sequence segments or structure with a known allergen. The in vitro assessment of the CP4 EPSPS protein digestibility indicates that the protein, like other food-derived proteins, is very labile to digestion when compared to many clinically important food allergens.

EPSPS protein is present at low levels, approximately 0.08% of the total protein, in whole RR soy seed. Most food allergens are present as major protein components in the specific food, in amounts ranging from 1% up to 80% of total protein. An assessment of the endogenous allergens in conventional and RR soy was made using sera from patients confirmed to be sensitive to soybean protein and the study demonstrated that the introduction of the EPSPS protein did not cause any discernible changes, either qualitatively or quantitatively, in the composition of the allergenic proteins endogenous to soybean. It has been shown that the processing steps used in the production of soybean oil, one of the main sources of soybean in the human diet, reduce the vast majority of protein such that refined soybean oil did not trigger allergenic reactions in humans who were sensitive to soybean. Based on the evidence described above, the bacterial EPSPS enzyme in RR soy is unlikely to be allergenic.

7. Compositional Analyses of Key Components

The GM crop and the conventional variety from which the GM crop was produced (or a variety as close as possible to the GM crop) are grown at different locations that represent areas where the GM crop might be grown in the future. The edible part, in this case the seed, is harvested and tested to determine its composition. The levels of nutrients and other components of the GM crop are compared to the conventional food and also compared to levels reported in the scientific literature for other varieties of the same crop. Any significant differences between the GM and the conventional crop are then assessed for potential adverse health effects.

• Nutrient analysis

Typical nutrient analyses are:

• • Proximate composition – refers to the levels of ash, moisture, protein, fat, and fiber.

  • Amino acid analysis

  • Fatty acid analysis

  • Carbohydrate analysis

  • Vitamin and mineral analysis

Other compounds present in particular foods may also be measured if they are likely to have a significant impact in the overall diet. For example, the assessment would consider isoflavones in soybeans. Soybeans naturally contain isoflavones and their ingestion has been linked to a number of biochemical effects in mammalian specie including estrogenic and hypocholesterolemic activities (Wang et al., 1990) and have also been reported to contribute deleterious effects on livestock animals fed on soybean meal (Setchell et al., 1987)

Levels of antinutrients – This test is carried out to check that the genetic modification has not significantly increased levels of any known naturally occurring antinutrients in the food above the natural range found in the conventional food. Processing of the foods must also be taken into account, because this may inactivate any antinutrients in the unprocessed food. Antinutrients found in conventional foods include trypsin inhibitors and phytic acid.

Rounded Rectangle: Case Study

The only significant antinutrients known to occur naturally in soybeans are soybean lectin, trypsin inhibitor and phytate. Soybean lectin and trypsin inhibitor are destroyed during the heat treatments or processing applied to all soy products before they are eaten. Nevertheless, no differences were found in the levels of soybean lectin, trypsin inhibitor and phytate between RR soy and conventional soy. Two other components were also measured: raffinose and stachyose – while not strictly antinutrients, increased levels of these carbohydrates would be considered undesirable because they cause flatulence. No differences were found in the levels of raffinose or stachyose between RR soy and conventional soy.

8. Nutritional Assessment

The nutritional adequacy and the ability of a GM food to support typical human growth and well-being should be established. This is usually achieved by understanding the genetic modification and its consequences, and analyzing the composition of the food. If the compositional analysis indicates significant differences in a number of important nutrients or other components, or if there is concern that the bioavailability of key nutrients may be compromised by the genetic changes to the food, then feeding studies in animals can determine whether the food is nutritionally adequate.

Rounded Rectangle: Case Study

In the case of RR soy the extent of the compositional and other data were considered sufficient to establish the nutritional adequacy of the food. These studies confirmed that there were no unexpected changes in palatability or wholesomeness of RR soy. In these studies, RR soy and conventional soy were fed to some animals that commonly eat soybeans including groups of rats, chickens and dairy cows for 4–6 weeks. These animal studies showed that RR soy was palatable and able to support typical growth and wellbeing in rats, chickens and dairy cows. They also confirmed the results of studies showing no acute toxicity when the bacterial EPSPS enzyme was given to mice at high doses. Feeding studies with catfish and quail also gave results that were consistent with the studies on rats, chickens and dairy cows.

9. Other safety issues

Other safety issues that are important on a case-by-case basis should also be considered. For example in the case of RR soy, the possibility of indirect pesticide residue accumulation due to the herbicide tolerance trait would be investigated. In this case traditional methods of chemical safety testing should be applied.

Rounded Rectangle: Case Study

In the case of RR soy, the levels of glyphosate used were established to be within allowable safety limits.

10. Local Concerns

Where appropriate, safety risk assessments undertaken by other competent regulatory authorities may be used to avoid duplication. However, genetically modified foods that have already been approved for safety in one country may have to be re-evaluated in a different country to address any significant differences that may arise with respect to consumption patterns, mode of cultivation of crop, form in which the food is consumed and processing methods.

 

1. Are there risks with eating any food?

Virtually every food we eat possess risk. Based on statistics by the World Health Organization, the primary risk associated with eating food is illness due to microbial contaminants, such as food-borne viruses and bacteria. WHO estimates that foodborne and waterborne diarrheal diseases taken together kill about 2.2 million people annually, 1.9 million of them children. The safety of the chemicals in food, both natural and man-made, is also an important consideration. Some are natural plant chemicals that may be toxic because they are produced by the plants to protect them against insects and other herbivores. For example, solanine, a glycoalkoloid in potatoes, is toxic to humans but naturally protects the plant with its fungicidal and pesticidal properties. Others may be unintentional contaminants such as pesticide residues. International bodies such as the Codex Alimentarius Commission, give guidelines on the maximum residue limits for pesticides in food that are considered safe to eat (http://www.codexalimentarius.net/pestres/data/index.html).

2. What is considered to be safe food?

The World Health Organization/Food and Agriculture Organization (FAO) defines food to be safe if there is reasonable certainty that no harm will result from consumption under the anticipated conditions of use (CAC/GL45-2003). The FAO suggests that safety assessments should provide assurance, in the light of the best available scientific knowledge, that the food does not cause harm when prepared, used and/or eaten according to its intended use. The absolute safety of a food or an ingredient can never be guaranteed. However, with appropriate precautions during production, manufacture into products, and distribution, risk can be kept to an absolute minimum that is generally acceptable to consumers.

3. Are there specific food safety concerns associated with GM foods?

Genetically modified foods are not inherently less safe than their traditional counterparts. This conclusion has been reached by WHO, FAO and all National Academies of Science and established scientific authorities from around the world who have investigated this question, including countries from Europe, Asia, North and South America (e.g., International Council for Science; English, French, Italian, U.S., Mexican, Brazilian, Indian and Chinese Academies of Sciences).Nevertheless, due to lack of past experience with GM foods and concerns about novel technologies, these foods have been subjected to rigorous safety assessment procedures that are not generally applied to traditional foods. The determination of the safety of GM foods relies on a broad comparison of the properties of the GM crop to those of its conventional counterpart with a known history of safe use. The most common food safety concerns raised about GM foods include: Is it dangerous to eat foreign DNA? Can genes from GM foods be transferred to people? What is the possibility that GM foods will be allergenic? What is the possibility of novel toxins and anti-nutrients being produced in GM foods? Is there risk associated with resistance genes included in GM foods? These questions are discussed in the following sections.

4. Is it dangerous to eat foreign DNA?

All the food that we eat is derived from living organisms such as plants and animals; all living organisms contain genes which are made up of DNA. The human digestive system degrades all DNA into small fragments, whether it is from GM or conventional food. Numerous experimental studies with livestock have shown that DNA fragments or proteins derived from GM plants have not been detected in tissues, fluids or edible products of farm animals. “Therefore if one eats DNA in a GM food or a conventional food, it will not change their own DNA or that of their children” (European Food Safety Authority (EFSA), 2007; http://www.efsa.europa.eu/EFSA/Statement/EFSA_statement_DNA_proteins_gastroint.

5. What is the possibility that GM foods will be allergenic?

If a new protein is introduced into a potential GM crop, its allergenic properties must be tested to ensure safety. A series of tests are performed on the protein produced by the introduced gene to identify potential allergenic effects prior to product approval. Tests are also done to be sure that the levels of naturally occurring allergens are not increased in the GM food. If a conventional food that already contains allergens is genetically engineered, the GM food will also contain those allergens, unless specific steps are taken to remove the allergens. For example, soy naturally contains proteins that cause an allergic reaction in some people. Unless these specific proteins are removed, they will also be found in GM soy varieties.

6. What is the possibility of novel toxins and antinutrients being produced in GM foods?

All substances, whether natural or human-made, are potentially toxic depending on the dose. Substances classified as toxins are those that can be harmful to health at typical levels of exposure. For GM products, there is concern that a new toxic substance will be introduced or the levels of toxic substances already present in the crop might be increased. The products of the new gene are tested to ensure that they are readily digested and are not toxic using simulated mammalian conditions and animal testing as needed. Levels of the naturally occurring toxins are also measured to ensure that they are not elevated above their natural levels. The GM crop is also tested to ensure that nutritional composition has not been significantly altered.

7. What is the risk associated with resistance genes in GM foods?

Antibiotic resistance marker (ARM) genes are commonly used to assist in the process of genetically engineering plants. There has been concern about the effect of these genes on human health and safety, if such genes present in GM foods were able to transfer to microorganisms in the human digestive tract. This question has been studied extensively by many groups (e.g., http://www.efsa.europa.eu/en/news/news/gmo070413.htm).

The DNA in ARM genes are not any different from other DNA present in plants and animals. It is digested and processed in the gastro-intestinal tract just like DNA from any other source. When expressed in plant cells, the commonly used ARM genes have been shown to produce proteins that are digested in a similar way to other thousands of dietary proteins that humans consume every day. In addition, ARM proteins are frequently produced by human intestinal bacteria and thus humans have been exposed to these proteins throughout history.

Therefore, it has been concluded that ARM genes themselves and the proteins they express, as with other genes and proteins in foods and feed do not pose risks to the health of humans or animals. The European Food Safety Authority has recently reaffirmed that the two antibiotic resistance marker genes, npt II and aadA, used for GM plants pose no threat to humans or the environment (EFSA, 2007).It is also important to note that the antibiotic resistance genes currently present in GM foods code for resistance to antibiotics that are not widely used in human medicine, because resistance to them is already widespread. For example, the npt II gene confers resistance to neomycin, kanamycin and other antibiotics that are not in clinical use any more.

It is expected that in the future, as genetic engineering techniques evolve, antibiotic resistance genes will not be present in GM foods because they will either have been removed during development or have been replaced by other types of marker genes.

8. How is the safety of GM foods assessed?

Internationally harmonized evaluation strategies have been developed to test for the safety of foods derived from genetically modified organisms (GMOs). GM- derived products, be they food, food ingredients, or foods produced by GM microorganisms, undergo more stringent safety assessment procedures than is required for non-GM foods.

The approach taken is based on the concept of Substantial Equivalence (SE). SE asks whether the GM food is as safe as its traditional counterpart. (See section below for more details). On a case-by-case basis, toxicological, and nutritional investigations may be required before commercialization.

The comparator approach should consider agronomic, morphological, genetic and compositional aspects in order to make an objective assessment. Particular attention should be paid to the choice of comparator, the design of field trials, and statistical analysis of the generated data in order to obtain good comparative data. The GM crop and the comparator should be grown in the same environmental conditions to avoid genotypic and phenotypic differences not related to the transformation process (Herman et al., 2007).An assessment of GM crops looks at the following key factors:

• Molecular characterization of the new genetic material and transformation process

• Phenotypic characterization of the new product

• Safety of new products

• Occurrence and implications of unintended effects

• Pathogenic, toxicity and anti-nutrient effect

• Allergenicity of new products

• Role of the new food in the diet

• Influence of food processing

9. What kinds of information should be included in a food safety assessment dossier?

1. Description of the Recombinant-DNA Plant

   • Identification of the crop.

   • Name of the transformation event(s).

   • Purpose of the modification, sufficient to aid in understanding the nature of the food being submitted for safety assessment.

2. Description of the Host Plant and its Use as Food

   • Common or usual name; scientific name and, taxonomic classification.

   • History of cultivation and development through breeding, in particular identifying traits that may adversely impact on human health.

   • Information on the host plant’s genotype and phenotype relevant to its safety, including any known toxicity or allergenicity.

   • History of safe use as a food.

   • How plant is typically cultivated, transported and stored.

   • Information on special processing required to make the plant safe to eat.

   • Part of the plant used as a food source.

   • Important macro- or micro-nutrients the food contributes to the diet.

   • If the food is important to particular subgroups of the population.

3. Description of the Donor Organisms

   • Common and scientific name.

   • Taxonomic classification.

   • Information about the natural history of the organism as concerns human health.

   • Information on naturally occurring toxins, anti-nutrients and allergens.

   • In case a microorganism is the donor organism, additional information on human pathogenicity and the relationship to known human pathogens.

   • Information on the past and present use, if any, in the food supply and exposure route(s) other than intended food use (e.g. possible presence as contaminants).

4. Description of the Genetic Modification(s)

   • Information on the specific method used for the modification.

   • Information on the DNA used to modify the plant including the source (e.g., plant, microbial, viral, synthetic), identity and expected function in the plant.

   • Details of all genetic components of the vector used to produce or process DNA for transformation of the host organism.

   • Information on all the genetic components including marker genes, regulatory and other elements affecting the function of the DNA.

   • Location and orientation of the sequence in the final vector/construct and function.

5. Characterization of the Genetic Modification(s)

   • Information on the DNA insertions into the plant genome including:

     • characterization and description of the inserted genetic material.

     • number of insertion sites.

     • organization of the inserted genetic material at each insertion site including copy number.

     • sequence data of the inserted material and of the flanking regions bordering the site of insertion, sufficient to identify substance (s) expressed as a consequence of the insertion.

     • identification of any open reading frames within the inserted DNA or created by the insertions with contiguous plant genomic DNA including those that could result in fusion proteins.

   • For any expressed substances in the rDNA plant the information to be provided include:

     • gene product(s) (e.g. a protein or an untranslated RNA).

     • gene product(s)’ function.

     • phenotypic description of the new trait(s).

     • level and site of expression of the expressed gene product(s) in the plant, and the levels of its metabolites in the edible portions.

     • amount of the target gene product(s), where possible, if the function of the expressed sequence(s)/gene(s) is to alter the accumulation of a specific endogenous mRNA or protein.

     • information on deliberate modifications made to the amino acid sequence of the expressed protein result in changes in its post-translational modification or affect sites critical for its structure or function.

   • Additional information to be provided:

     • demonstrate whether the arrangement of the genetic material used for insertion has been conserved.

     • show whether the intended effect of the modification has been achieved and that all expressed traits are expressed and inherited in a manner that is stable through several generations consistent with laws of inheritance.

     • demonstrate newly expressed trait(s) are expressed as expected in the appropriate tissues in a manner and at levels that are consistent with the associated regulatory sequences driving the expression of the corresponding gene.

     • any evidence to suggest that one or several genes in the host plant has been affected by the transformation process.

     • confirm the identity and expression pattern of any new fusion proteins.

     • may be necessary to examine the inheritance of the DNA insert itself or the expression of the corresponding RNA if the phenotypic characteristics cannot be measured.

6. Compositional Analyses of Key Components

   • Proximate composition including ash, moisture content, crude protein, crude fat, and various carbohydrate.

   • Protein amino acid profile.

   • Quantitative and qualitative composition of total lipids, i.e., saponifiable and nonsaponifiable components, complete fatty acid profile, phospholipids, sterols, cyclic fatty acids and known toxic fatty acids.

   • Composition of the carbohydrate fraction e.g., sugars, starches, chitin, tannins, nonstarch polysaccharides and lignin.

   • Qualitative and quantitative composition of micronutrients, i.e., significant vitamin and mineral analysis.

   • Presence of naturally occurring or adventitious anti-nutritional factors e.g., phytates, trypsin inhibitors, etc.

   • Predictable secondary metabolites, physiologically active (bioactive) substances, other detected substances.

7. Assessment of Possible Toxicity

   • Indicate if the donor organism(s) is a known source of toxins.

   • Amino acid sequence homology comparison of the newly expressed protein and known protein toxins and anti-nutrients.

   • Demonstrate the susceptibility of each newly expressed protein to pepsin digestion.

   • Where a host other than the transgenic plant is used to produce sufficient quantities of the newly expressed protein for toxicological analyses, demonstrate the structural, functional and biochemical equivalence of the non-plant expressed protein with the plant expressed protein.

   • Oral toxicity study(s) completed for newly expressed proteins.

8. Assessment of Possible Allergenicity (Proteins)

   • Indicate if the donor organism(s) is a known source of allergens.

   • Amino acid sequence homology comparison of the newly expressed protein and known allergens.

   • Demonstrate the susceptibility of each newly expressed protein to pepsin digestion.

   • Where a host other than the transgenic plant is used to produce sufficient quantities of the newly expressed protein for toxicological analyses, demonstrate the structural, functional and biochemical equivalence of the non-plant expressed protein with the plant expressed protein.

   • For those proteins that originate from a source known to be allergenic, or have sequence homology with a known allergen, additional immunological assays be warranted.

 

Agrobacterium tumefaciens: The soil bacterium which, when containing the Ti plasmid, is able to form crown galls on a number of dicotyledonous plants.

Allergen: A substance, usually a protein, that causes an allergic reaction.

Allergenicity: The capacity of an allergen to cause an allergic reaction.

Amino acid: The building block components of proteins. Twenty different essential amino acids are used by all living organisms to make proteins.

Antinutrient: A substance within a food that interferes with the uptake of nutrients from food.

Antibiotic resistance marker gene: Genes (usually of bacteria origin) used as selection markers in genetic engineering, because their expression allows cell survival in the presence of normally toxic antibiotic agents. These genes were commonly used in the development and release of first generation genetically engineered crop plants.

Antibody: A protein produced by the immune system in response to an antigen (a molecule that is perceived to be foreign). Antibodies bind specifically to their target antigen to help the immune system render the foreign entity harmless.

Bacterium: A single-celled life form whose genetic material is not enclosed in a nucleus.

Carbohydrate: Simple sugars and starches present in foods that provide the body with energy and nutrition.

Cauliform mosaic virus CaMV): A good source of strong promoters used in plant cloning vectors.

Codex Alimentarius Commission (CAC): A commission set up by the Food and Agriculture Organization and the World Health Organization of the United Nations in 1962 to develop a code of food standards for all nations.

Composition Analysis: The determination of the concentration of compounds in a plant or animal tissue. Compounds that are commonly quantified are proteins, fats, carbohydrates, minerals, vitamins, amino acids, fatty acids and antinutrients.

Conventional Counterpart: A related organism/variety, its components and/or products for which there is experience of establishing safety based on common use as food.

Cry proteins: A class of crystalline proteins produced by strains of Bacillus thuringiensis and engineered into crop plants to give resistance against insect pests. These proteins are toxic to certain categories of insects (e.g. Lepidopteran etc) but are harmless to mammals and most beneficial insects. Toxin called delta endotoxins.

Dietary exposure: Contact by ingestion between a physical, chemical or biological agent and an organism.

DNA: Stands for deoxyribonucleic acid. DNA is made up of four nucleotides (bases), adenine, guanine, cytosine and thymine on a phosphate backbone. DNA is usually double stranded with base pairing between strands occurring between adenine and thymine, and cytosine and guanine.

Epitope: Also known as allergenic determinant, is the part of a macromolecule that is recognized by the immune system specifically by antibodies, B cells or T cells.

Event – A term used to describe a plant and its offspring that contain a specific insertion of DNA. Events are distinguishable from each other by their unique site of integration of the introduced DNA.

Food additive: Substances added to foods to improve taste, appearance, texture, storage life or other qualities.

Gene: A segment of a DNA strand that contains the instructions to encode for an RNA and/or polypeptide molecule.

Gene expression: The process by which a gene directs the synthesis of mRNA, which in turn directs the synthesis of protein, thereby affecting the phenotype of an organism.

Gene Transfer: The transfer of genes to an organism. Usually used in terms of transfer of a gene to an organism other that the original organism, through the tools of biotechnology.

Genetically modified foods (GM foods): These are foods produced from genetically modified organisms (GMOs) that that have had their genome altered through genetic engineering or foods that contain ingredients from GMOs.

Genetically modified organism (GMO): A life form that has been genetically modified using techniques of modern gene technology.

Gene silencing: Loss of gene expression either through an alteration in the DNA sequence of a structural gene, or its regulatory region; or because of interaction between its transcript and other mRNAs present in the cell.

Genetic engineering: The use of experimental techniques to produce DNA molecules containing new genes or new combinations of genes.

Genome: The total genetic material of a living organism.

Genotype: The genetic make-up of a plant or organism.

Hazard: A biological, chemical, or physical agent, or condition, with the potential to cause an adverse health or environmental effect.

Hazard Characterization: The qualitative and/or quantitative evaluation of the nature of the adverse health effects associated with biological, chemical and physical agents. For chemical agents, a dose-response assessment should be performed if the data are obtainable.

Hazard Identification: The identification of biological, chemical and physical agents capable of causing adverse health or environmental effects.

Herbicide: A substance that is able to kill certain types of plants when applied at specific doses.

Herbicide tolerant: Plants with an increased ability to tolerate commercial applications of herbicide.

Host – An organism that has been genetically modified is sometimes said to be the ‘host’ for genetic material provided by another organism.

Immunoglobulin E (IgE) – A protein antibody that recognizes an allergen. It circulates in the blood and becomes fixed on the surface of specific cells (basophils and mast cells). When IgE on the cell surface binds to allergen, this triggers the release of chemical mediators that provoke the symptoms associated with allergic reactions.

Irradiated food: A food that has been subjected to a measured dose of radiation to reduce the level of bacterial contamination or to control quarantine pests.

Insect protected: Plants that have an inbuilt capacity to protect themselves from damage by particular insect pests.

Isoflavones: Water-soluble chemicals, also known as phytoestrogens,found in many plants and so named because they causeeffects in the mammalian body somewhat similar to those of
estrogen. The most investigated natural isoflavones, genisteinand daidzen, are found in soy products and the herb red clover.

Isogenic line: Genetically identical, used as a basis of comparison in establishing substantial equivalence

Kilodalton (kDa): A kilodalton is a unit used to measure the mass of atoms as well as molecules, such as proteins. One kilodalton is equal to 1000 daltons. One dalton equals the atomic weight of a hydrogen atom (1.66 x 10-24 grams).

Macronutrient: In humans and animals, a substance that is required in relatively large amounts for healthy growth and development, and belongs to one of 3 groups: carbohydrates, fats, and proteins.

Marker gene: A marker gene is a gene attached to the desired gene and used in genetic modification to allow researchers to identify those cells that have successfully taken up the new DNA.

Micronutrient: In humans and animals, a substance, such as a vitamin or trace element, essential for healthy growth and developmentbut required only in minute amounts.

Microorganism: A microscopic life form, usually single-celled, such as bacteria or yeast.

Molecular characterization: This includes DNA sequence data and the mapping of particular functions on the plant chromosome and on the inserted DNA.

Modern biotechnology: The application of In vitro nucleic acid techniques, including recombinant DNA and direct injection of nucleic acid into cells or organelles; or fusion of cells beyond the taxonomic family, that overcome natural physiological reproductive or recombinant barriers and that are not techniques used in traditional breeding and selection.

Novel food: Non-traditional foods for which there is insufficient knowledge in the broad community to enable safe use in the form or context in which the food is presented.

Nutrient: A substance that provides nourishment and promotes growth, health and wellbeing.

Plasmid: Circular extra-chromosomal DNA molecules present in bacteria and yeast. Plasmids replicate autonomously each time the organism divides and are transmitted to the daughter cells.DNA segments are commonly cloned using plasmid vectors.

Pesticide: A substance that is able to kill certain types of pests when applied at specific doses.

Phenotype: An organism(s) expressed physical traits.

Plasmid: A plasmid is a small piece of DNA that can replicate itself within a bacterial cell. Plasmids are used in gene technology as a way to introduce new genes into other cells.

Pleiotropic effects: The simultaneous effect of a given gene on more than one apparently unrelated trait.

Post-translational modification: The addition of specific chemical residues to a protein after it has been translated e.g. sugars (glycosylation), methyl groups (methylation).

Promoter: A DNA sequence that enables a cell to turn a particular gene on.

Recombinant: A transformed cell that contains a recombinant DNA molecule.

Recombinant DNA molecule: A DNA molecule created in the test tube by ligating together pieces of DNA that are not normally contiguous.

Rebonucleic Acid (RNA): RNA stands for ribonucleic acid. RNA is similar in structure to DNA but is generally single stranded. RNA in the cell has many functions, one of these is to act as a template for the synthesis of proteins. RNA that acts as a template for the synthesis of proteins is called messenger RNA or mRNA.

Risk: A function of the probability of an adverse health effect andthe severity of that effect, which is consequential to a hazard(s).

Risk analysis: A process consisting of three components: risk assessment, risk management, and risk communication performed to understand the nature of unwanted, negative consequences to human and animal health, or the environment.

Risk assessment: A scientifically based process consisting of the following steps: (i) hazard identification;(ii) hazard characterization; (iii) exposure assessment; and iv) risk characterization.

Risk management: The process, distinct from risk assessment, of weighing policy alternatives, in consultation with all interested parties, considering risk assessment and other factors relevant for the health protection of consumers and for the promotion of fair trade practices, and, if needed, selecting appropriate prevention and control options.

Secondary metabolism: The production by living organisms of substances not essential for primary metabolic functions or physiology. Their role is associated with interaction with the environment, for example for defence, as elicitors, or as attractants. Some of these have useful pharmacological or nutritional properties, while others are toxic.

Selectable marker: A gene carried by a vector and conferring a recognizable characteristic on cell containing the vector or recombinant DNA molecule derived from the vector.

Sequence Homology: The degree of identity or similarity between 2 selected nucleotide or amino acid sequences.

Sera-binding Tests: Immunological assays that test for the presence of antigen-specific immunoglobulins (for example, IgE) in blood serum, for example serum obtained from individuals allergic to food, pollen, or other environmental antigens. Sera binding tests include assays such as western blotting, Enzyme Linked Immunosorbent Assays (ELISA), ELISA-inhibition, Radio AllergSorbent Test (RAST) and RAST-inhibition techniques.

Substantial equivalent (SE): SE is a concept used in the safety assessment of GM food that states, a food derived from a genetically modified plant or microorganism should be as safe as its traditional counterpart.

Toxin: A substance that can cause adverse health effects under typical circumstances of exposure.

Toxicity: The capacity of a toxin to cause a toxic reaction.

Toxicokinetics: The study of the time-dependent processes related to toxicants as they interact with living organisms. It encompasses absorption, distribution, storage, biotransformation and elimination.

Trait: A distinguishing feature or quality.

Transcription: Transcription is the process of copying information from DNA in the cell to RNA. TheRNA is then used to make proteins by a process called translation.

Transgenic: Adjective describing an organism in which a foreign DNA gene (a transgene) is incorporated into its genome.

Translation: Translation is the process where information contained on an RNA strand is decoded into an amino acid sequence to produce a protein.

Trypsin Inhibitors: Antinutrient proteins present in plants such as soybeans that inhibit the digestive enzyme, trypsin if not inactivated by heating or other processing methods.

Unintended Effect: An effect that was not the purpose of the genetic modification or mutation.

Vector: A DNA molecule, capable of replication in a host organism, into which a gene is inserted to construct a DNA molecule.

Virus: A microscopic particle containing genetic material that is only able to reproduce by infecting a living cell.