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.