A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 1057 where further information might be needed to assess and food allergens,where applicable.An understanding safety. of the natural variation of the main agronomic and compositional characteristics of the crop in different 4.1.Information on the parent crop geographies and under different cultivation conditions is essential to interpret data comparing the GM crop to The description of the parent crop should include the chosen comparator.Whole food and substance- information on the origin,genotype,phenotype,diver- specific laboratory animal or farm animal studies that sity,and history of safe use of the parent crop and, may already have been conducted on the parent crop where possible,of other related traditionally-bred vari- may also provide valuable additional information rele- eties and species.The characterisation of the parent vant to planning the safety assessment strategy for the crop then guides the choice of test parameters for the GM crop.The Organisation for Economic Cooperation comparison of the GM crop to a close comparator, and Development is compiling consensus documents for which is usually the non-modified parent crop.Test certain crop species that provide the information parameters mostly include indicators of the crop's considered of most relevance of the characterization development,phenotype,and agronomic performance, of the parent crop.Two types of consensus documents as well as its main endogenous nutrients,and potential are available for the world's major food crops:the first anti-nutrients or biologically active substances,toxins, describes a crop's biology,focusing on attributes that Description of parent Characterisation of gene Characterisation of gene Assessment of crop and selection of donor,transformation products and whole food from appropriate parameters event and inserted DNA metabolites GM crop for compositional tests Y ldentify specifie points of comparison with conventional counterpart(s)and establish degree of equivalence Test programme Fig.1.Designing a test programme. Parent Crop Transformation Process Gene Product(s) GM Crop Background knowledge Background knowledge on Safety assessment Safety assessment of history of safe use *source of transferred gene(s) any toxic potential equivalence to parent phenotype "any allergenic potential crop .DNA construct chemical composition consequences of DNA insertion Hazard Identification/ Characterisation Exposure Assessment Safety Assessment Fig.2.The elements in safety assessment of food derived from a GM crop
where further information might be needed to assess safety. 4.1. Information on the parent crop The description of the parent crop should include information on the origin, genotype, phenotype, diversity, and history of safe use of the parent crop and, where possible, of other related traditionally-bred varieties and species. The characterisation of the parent crop then guides the choice of test parameters for the comparison of the GM crop to a close comparator, which is usually the non-modified parent crop. Test parameters mostly include indicators of the crop’s development, phenotype, and agronomic performance, as well as its main endogenous nutrients, and potential anti-nutrients or biologically active substances, toxins, and food allergens, where applicable. An understanding of the natural variation of the main agronomic and compositional characteristics of the crop in different geographies and under different cultivation conditions is essential to interpret data comparing the GM crop to the chosen comparator. Whole food and substancespecific laboratory animal or farm animal studies that may already have been conducted on the parent crop may also provide valuable additional information relevant to planning the safety assessment strategy for the GM crop. The Organisation for Economic Cooperation and Development is compiling consensus documents for certain crop species that provide the information considered of most relevance of the characterization of the parent crop. Two types of consensus documents are available for the world’s major food crops: the first describes a crop’s biology, focusing on attributes that Fig. 2. The elements in safety assessment of food derived from a GM crop. Fig. 1. Designing a test programme. A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088 1057
1058 A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 are relevant to the environmental safety assessment,and parts.Any known nutritional,anti-nutritional,tox- the second describes a crop's compositional character- icological,or allergenic characteristics of the food crops, istics that are of most importance for the food safety or intolerance to foods derived from this crop should be assessment.(For potatoes see for example OECD,1997, highlighted and become an important element of the 2002b). subsequent safety assessment. The type of information on the parent crop that In addressing the history of use and the importance of should be gathered at the outset of the assessment is the plant-derived foods in the diet,any particular pre- shown in Fig.3. paration,processing,or cooking practices should also be identified.The safe use of plant-derived foods some- 4.1.1.Identity,phenotypic and agronomic performance times depends on special pre-treatment established by Taxonomic identity of the parent crop should be custom and practice'that renders it palatable and safe. established to referenced and internationally acceptable For example,red kidney beans require boiling before principles,including complete name,family name, consumption to render the lectins,protease inhibitors, genus,species,subspecies,cultivar/breeding line,com- and haem-agglutinins inert.In general,the adverse mon name,and sexually compatible wild relatives.Spe- effects to health of most anti-nutrients are caused by the cification of the chemical proximate composition and consumption of raw plant material.Most anti-nutrients key nutrients and anti-nutrients of the host plant is also are not heat stable or are not evenly distributed in the required;the parent crop or conventional counterpart plant;they can hence be inactivated or removed by usually serves as the reference point in the safety measures such a heating,soaking,peeling,or germination. assessment of the GM crop,unless in future,the new trait(s)extensively modify the composition and nutri- 4.1.4.Compositional analysis tional characteristics such that another comparator, Regulators widely agreed that the compositional possibly a combination of non-GM crops and/or assessment should include analyses of the key nutrients, derived foods with a history of human exposure,have to toxins,allergens,anti-nutrients,and biologically active be used as safety standard. substances that are known to be associated with the crop.International consensus continues to develop on 4.1.2.Geographical distributionsource which key components should be analysed in order to Information about the geographical distribution of perform a fully comprehensive compositional analysis cultivation of the parent plant identifying usual climatic of specific food crops.The OECD provides a platform and soil conditions for its growth should be given. for discussions amongst experts representing the Mem- Knowledge of the native distribution centre of origin ber States.The results of these discussions are published and history of domestication provides valuable infor- in the form of a series of consensus documents on com- mation relevant to a crop's cultivation and the occur- positional considerations for new varieties of crops. rence of wild relatives that are potentially sexually Consensus documents on rapeseed (canola),soybean compatible and to which cross-pollination may occur. (OECD,2001a,b),potato,sugar beet,and maize (OECD,2002a,b,c)have been published;consensus 4.1.3.History of use documents for wheat,rice,sunflower,cotton,and barley The novelty,sometimes referred to as'exoticness',of are in preparation.Third party papers exist on a num- food plants is determined by their documented history ber of other key crops such as cotton (Berberich et al., of use in the food supply.It has to be recognised that 1996;Nida et al,.1996). many plant-derived foods that are common in one part The composition of individual plants grown in the of the world may not be part of the food supply in other same field can differ considerably due to natural biolo- Parent Crop Donor,Transgene(s Characterisation of Safety Assessment of Gene Product(s] New GM Crop/Food Delivery Process Identity.Phenotypic Agronomic Performance Geographical Distribution History of Safe Use Compositional Analysis Fig.3.Description of the parent crop
are relevant to the environmental safety assessment, and the second describes a crop’s compositional characteristics that are of most importance for the food safety assessment. (For potatoes see for example OECD, 1997, 2002b). The type of information on the parent crop that should be gathered at the outset of the assessment is shown in Fig. 3. 4.1.1. Identity, phenotypic and agronomic performance Taxonomic identity of the parent crop should be established to referenced and internationally acceptable principles, including complete name, family name, genus, species, subspecies, cultivar/breeding line, common name, and sexually compatible wild relatives. Specification of the chemical proximate composition and key nutrients and anti-nutrients of the host plant is also required; the parent crop or conventional counterpart usually serves as the reference point in the safety assessment of the GM crop, unless in future, the new trait(s) extensively modify the composition and nutritional characteristics such that another comparator, possibly a combination of non-GM crops and/or derived foods with a history of human exposure, have to be used as safety standard. 4.1.2. Geographical distribution/source Information about the geographical distribution of cultivation of the parent plant identifying usual climatic and soil conditions for its growth should be given. Knowledge of the native distribution centre of origin and history of domestication provides valuable information relevant to a crop’s cultivation and the occurrence of wild relatives that are potentially sexually compatible and to which cross-pollination may occur. 4.1.3. History of use The novelty, sometimes referred to as ‘exoticness’, of food plants is determined by their documented history of use in the food supply. It has to be recognised that many plant-derived foods that are common in one part of the world may not be part of the food supply in other parts. Any known nutritional, anti-nutritional, toxicological, or allergenic characteristics of the food crops, or intolerance to foods derived from this crop should be highlighted and become an important element of the subsequent safety assessment. In addressing the history of use and the importance of the plant-derived foods in the diet, any particular preparation, processing, or cooking practices should also be identified. The safe use of plant-derived foods sometimes depends on special pre-treatment established by ‘custom and practice’ that renders it palatable and safe. For example, red kidney beans require boiling before consumption to render the lectins, protease inhibitors, and haem-agglutinins inert. In general, the adverse effects to health of most anti-nutrients are caused by the consumption of raw plant material. Most anti-nutrients are not heat stable or are not evenly distributed in the plant; they can hence be inactivated or removed by measures such a heating, soaking, peeling, or germination. 4.1.4. Compositional analysis Regulators widely agreed that the compositional assessment should include analyses of the key nutrients, toxins, allergens, anti-nutrients, and biologically active substances that are known to be associated with the crop. International consensus continues to develop on which key components should be analysed in order to perform a fully comprehensive compositional analysis of specific food crops. The OECD provides a platform for discussions amongst experts representing the Member States. The results of these discussions are published in the form of a series of consensus documents on compositional considerations for new varieties of crops. Consensus documents on rapeseed (canola), soybean (OECD, 2001a,b), potato, sugar beet, and maize (OECD, 2002a,b,c) have been published; consensus documents for wheat, rice, sunflower, cotton, and barley are in preparation. Third party papers exist on a number of other key crops such as cotton (Berberich et al., 1996; Nida et al., 1996). The composition of individual plants grown in the same field can differ considerably due to natural bioloFig. 3. Description of the parent crop. 1058 A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088
A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 1059 gical variation,since the plant's development and variety of foods and often function as the plant's own metabolism are influenced by a range of biotic and natural pesticides. abiotic factors,including pathogen infection,state of Food allergens are proteins that induce allergic sensi- ripening,growing conditions (location,climate,heat/ tisation in susceptible individuals,such that subsequent drought.soil quality).and storage.The International dietary exposure to the same protein may provoke an Food Biotechnology Council (IFBC;IFBC,1990)has adverse reaction.Exposure of sensitised individuals to noted that there may be significant natural variation in relevant allergens in food may result in serious adverse the composition between samples from individual plants effects. or composite samples from fields of the parent crop line Plants are also a rich source of medicines.which have that are grown in two different locations;the same holds been widely exploited for therapy by the science of true for samples from a GM crop line. pharmacognosy.It follows that many crops consumed A thorough knowledge of the composition of the by humans contain powerful bioactive substances with parent crop should always be established both from the pharmacological activity.Examples include phytoster- literature and from analytical data resulting from field ols,caffeine,and theobromine. trials.Samples should be taken from a range of different It is therefore important to ensure that no new toxins, varieties from the same crop.This represents the refer- anti-nutrients,allergens,or bioactive substances are ence point for subsequent comparison with samples inadvertently introduced,up-or down-regulated as a from the specific GM crop line that is tested.Any sig- consequence of the genetic modification.It is therefore nificant differences in composition between the GM necessary to ensure that transformation does not intro- crop line that fall outside of the range of natural varia- duce new compounds of this type or affect negative tion in levels across a range of varieties of the same crop changes in the levels or characteristics of endogenous that are reproducible then become the focus of further compounds that may impact human health and that are evaluation. already present in the crop plant. There is a great need for continued international standardisation and harmonisation in this area to pro- 4.2.Information on the donor,transgene(s),and vide peer reviewed databases from which the range of delivery process natural variation of the levels of tested parameters can be deduced:such databases may then become the main Molecular characterisation of the recombinant DNA reference or standard to determine the significance of in GM crops is usually done in accordance with broad observed differences in the comparative compositional international guidelines on the safety assessment of analysis of a GM crop line and the comparator.The GM crops.In Europe,there are additional legal OECD and the International Life Sciences Institute requirements for the molecular characterisation of (ILSI)provide platforms for the development of such GM crops in Directive 2001/18/EC (European Com- consensus databases. mission,2001).The Directive requires that the inserted genetic material is well characterised and safe for 4.1.5.Nutrients,anti-nutrients,toxins,and allergens humans and the environment under the conditions of The assessment should focus on those nutrients,tox- the release of the GM crop.Further guidance on data ins,anti-nutrients,allergens,and bioactive constituents to be generated has been given by the European in the host plant or in its close relatives,changes in the Commission's Scientific Steering Committee (European levels of consumption of which might affect human Commission,2003c)and the United Kingdom Advisory health and nutrition.Nutrients are components in a Committee on Environmental Releases (UK ACRE; particular food that may have a substantial nutritional UK ACRE,2001).Both advisory committees empha- impact on the consumer or animal.These may be sise the need to describe the cloning and transfer vectors macro-nutrients (fats,proteins,carbohydrates)or and the recombinant DNA inserted into the GM micro-nutrients(minerals and vitamins). crop.The information and data described in Fig.4 Anti-nutrients are substances that inhibit or block needs to be provided in order to identify and char- important pathways in the human metabolism,or acterise the potential hazards resulting from plant impair digestion.Anti-nutrients may reduce nutrient transformation. utilisation,typically proteins,vitamins,or minerals, thus decreasing the nutritive value of foods (Watzl and 4.2.1.Description of the donor(s) Leitzmann,1995).Toxins are food components that The description of the donor organism(s)should may have a substantial negative impact on human include the classification and taxonomy to international health,including adverse effects other than those related standards (see Section 4.1.1)and should also address to metabolism and digestion.Food toxins such as the any evidence of potential toxicity,allergenicity or neurotoxic solanine,cyanogenic glycosides,erucic acid, pathogenicity.A list of naturally occurring toxins, lectins,and trypsin inhibitors are found in a wide allergens. bioactive substances,and anti-nutrients
gical variation, since the plant’s development and metabolism are influenced by a range of biotic and abiotic factors, including pathogen infection, state of ripening, growing conditions (location, climate, heat/ drought, soil quality), and storage. The International Food Biotechnology Council (IFBC; IFBC, 1990) has noted that there may be significant natural variation in the composition between samples from individual plants or composite samples from fields of the parent crop line that are grown in two different locations; the same holds true for samples from a GM crop line. A thorough knowledge of the composition of the parent crop should always be established both from the literature and from analytical data resulting from field trials. Samples should be taken from a range of different varieties from the same crop. This represents the reference point for subsequent comparison with samples from the specific GM crop line that is tested. Any significant differences in composition between the GM crop line that fall outside of the range of natural variation in levels across a range of varieties of the same crop that are reproducible then become the focus of further evaluation. There is a great need for continued international standardisation and harmonisation in this area to provide peer reviewed databases from which the range of natural variation of the levels of tested parameters can be deduced; such databases may then become the main reference or standard to determine the significance of observed differences in the comparative compositional analysis of a GM crop line and the comparator. The OECD and the International Life Sciences Institute (ILSI) provide platforms for the development of such consensus databases. 4.1.5. Nutrients, anti-nutrients, toxins, and allergens The assessment should focus on those nutrients, toxins, anti-nutrients, allergens, and bioactive constituents in the host plant or in its close relatives, changes in the levels of consumption of which might affect human health and nutrition. Nutrients are components in a particular food that may have a substantial nutritional impact on the consumer or animal. These may be macro-nutrients (fats, proteins, carbohydrates) or micro-nutrients (minerals and vitamins). Anti-nutrients are substances that inhibit or block important pathways in the human metabolism, or impair digestion. Anti-nutrients may reduce nutrient utilisation, typically proteins, vitamins, or minerals, thus decreasing the nutritive value of foods (Watzl and Leitzmann, 1995). Toxins are food components that may have a substantial negative impact on human health, including adverse effects other than those related to metabolism and digestion. Food toxins such as the neurotoxic solanine, cyanogenic glycosides, erucic acid, lectins, and trypsin inhibitors are found in a wide variety of foods and often function as the plant’s own natural pesticides. Food allergens are proteins that induce allergic sensitisation in susceptible individuals, such that subsequent dietary exposure to the same protein may provoke an adverse reaction. Exposure of sensitised individuals to relevant allergens in food may result in serious adverse effects. Plants are also a rich source of medicines, which have been widely exploited for therapy by the science of pharmacognosy. It follows that many crops consumed by humans contain powerful bioactive substances with pharmacological activity. Examples include phytosterols, caffeine, and theobromine. It is therefore important to ensure that no new toxins, anti-nutrients, allergens, or bioactive substances are inadvertently introduced, up- or down-regulated as a consequence of the genetic modification. It is therefore necessary to ensure that transformation does not introduce new compounds of this type or affect negative changes in the levels or characteristics of endogenous compounds that may impact human health and that are already present in the crop plant. 4.2. Information on the donor, transgene(s), and delivery process Molecular characterisation of the recombinant DNA in GM crops is usually done in accordance with broad international guidelines on the safety assessment of GM crops. In Europe, there are additional legal requirements for the molecular characterisation of GM crops in Directive 2001/18/EC (European Commission, 2001). The Directive requires that the inserted genetic material is well characterised and safe for humans and the environment under the conditions of the release of the GM crop. Further guidance on data to be generated has been given by the European Commission’s Scientific Steering Committee (European Commission, 2003c) and the United Kingdom Advisory Committee on Environmental Releases (UK ACRE; UK ACRE, 2001). Both advisory committees emphasise the need to describe the cloning and transfer vectors and the recombinant DNA inserted into the GM crop. The information and data described in Fig. 4 needs to be provided in order to identify and characterise the potential hazards resulting from plant transformation. 4.2.1. Description of the donor(s) The description of the donor organism(s) should include the classification and taxonomy to international standards (see Section 4.1.1) and should also address any evidence of potential toxicity, allergenicity or pathogenicity. A list of naturally occurring toxins, allergens, bioactive substances, and anti-nutrients A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088 1059
1060 A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 Parent Crop Donor,Transgene(s) Characterisation of Safety Assessment of and Gene Product(s) New GM Crop/Food Delivery Process Description of Donor Description of Vector DNA Transgene Delivery Process Characterisation of Introduced DNA Characterisation of Insertion Site Fig.4.Description of the donor(s),transgene(s)and delivery process. contained in the donor organism should be drawn up. Agrobacterium donor strain and any plasmids contained Documentation on the history of use of and exposure to in that strain should,however,be described to assess the the donor organism should be cited,where possible. risks of the presence of other sequences that might be This information,together with a detailed under- recognised by the transfer-mediating gene products. standing of the function of any recombinant DNA Using methods of direct transformation of plants, sequences used in the transformation process,facilitates such as particle guns,the preparation of the DNA used the hazard identification of the 'novel elements'that are for plant transformation should be checked for con- transferred to the crop (see also 4.3.2).The introduced taminating sequences of bacterial chromosomal DNA DNA should be shown to be unrelated to any char- or other plasmid DNA.Restriction fragment-prepara- acteristics of the donor organisms that could be harmful tions obtained using gel purification should be checked to human health.If the donor is known to be allergenic, for contaminating vector sequences using,for instance, transferred genes are assumed to encode allergens until Southern blots. evidence to the contrary has been obtained.Assessment of the potential allergenicity of recombinant proteins is 4.2.4.Characterisation of introduced DNA sequences described in more detail in Section 4.3.4. Thorough characterisation of inserted DNA sequen- ces through Southern blotting or polymerase chain 4.2.2.Description of vector DNA reaction (PCR)techniques is standard practice.The A step-wise description of the construction of the number of insertion sites and the copy number of transformation vector should provide details on all introduced DNA sequences have to be determined. organisms used for the amplification of vector DNA.It Characterisation of the inserted DNA merely using should also provide information on the function of all PCR is not sufficient,as it does not unambiguously genetic elements of transformation vectors,including reveal the number of insertion sites and the copy num- coding sequences,promoters and termination signals.A ber of inserted genes.Either Southern blots or a com- vector map with relevant restriction enzyme sites should bination of PCR and Southern blotting yields better also be made available(European Commission,2003c). results.Inserts at one site may be concatemers of the Subsequent proof of absence of vector fragments not same sequence.In particular,the ends of the inserts intended to be transferred is requested as well.The adjacent to plant genomic DNA have to be carefully provision of nucleotide sequence information of the analysed to determine whether any truncated open vector is also considered helpful (UK ACRE,2002). reading frames start within the insert or the plant geno- mic DNA that might yield transcripts that span plant 4.2.3.Transgene delivery genomic DNA and might also produce fusion proteins. Recombinant DNA is in most cases inserted into The UK ACRE guidelines recommend considering to plant cells using Agrobacterium tumefaciens or a particle sequence the recombinant DNA fragments introduced gun.Agrobacterium strains usually contain one vector in to the plant genome,involving the construction of encoding DNA mobilisation and transfer functions and genomic libraries of each transformed plant line that is a separate vector with the recombinant DNA intended to be so characterised (UK ACRE,2002).This recom- for transfer and a recognition site for the transfer-med- mendation is further discussed in Section 5.1 of this iating gene products.Using Agrobacterium,the risk of paper. transfer of random DNA to the plant is relatively small Evidence for the absence of vector backbone not (Gelvin,2000;Hellens and Mullineaux,2000).The intended for transfer is also required using Southern
contained in the donor organism should be drawn up. Documentation on the history of use of and exposure to the donor organism should be cited, where possible. This information, together with a detailed understanding of the function of any recombinant DNA sequences used in the transformation process, facilitates the hazard identification of the ‘novel elements’ that are transferred to the crop (see also 4.3.2). The introduced DNA should be shown to be unrelated to any characteristics of the donor organisms that could be harmful to human health. If the donor is known to be allergenic, transferred genes are assumed to encode allergens until evidence to the contrary has been obtained. Assessment of the potential allergenicity of recombinant proteins is described in more detail in Section 4.3.4. 4.2.2. Description of vector DNA A step-wise description of the construction of the transformation vector should provide details on all organisms used for the amplification of vector DNA. It should also provide information on the function of all genetic elements of transformation vectors, including coding sequences, promoters and termination signals. A vector map with relevant restriction enzyme sites should also be made available (European Commission, 2003c). Subsequent proof of absence of vector fragments not intended to be transferred is requested as well. The provision of nucleotide sequence information of the vector is also considered helpful (UK ACRE, 2002). 4.2.3. Transgene delivery Recombinant DNA is in most cases inserted into plant cells using Agrobacterium tumefaciens or a particle gun. Agrobacterium strains usually contain one vector encoding DNA mobilisation and transfer functions and a separate vector with the recombinant DNA intended for transfer and a recognition site for the transfer-mediating gene products. Using Agrobacterium, the risk of transfer of random DNA to the plant is relatively small (Gelvin, 2000; Hellens and Mullineaux, 2000). The Agrobacterium donor strain and any plasmids contained in that strain should, however, be described to assess the risks of the presence of other sequences that might be recognised by the transfer-mediating gene products. Using methods of direct transformation of plants, such as particle guns, the preparation of the DNA used for plant transformation should be checked for contaminating sequences of bacterial chromosomal DNA or other plasmid DNA. Restriction fragment-preparations obtained using gel purification should be checked for contaminating vector sequences using, for instance, Southern blots. 4.2.4. Characterisation of introduced DNA sequences Thorough characterisation of inserted DNA sequences through Southern blotting or polymerase chain reaction (PCR) techniques is standard practice. The number of insertion sites and the copy number of introduced DNA sequences have to be determined. Characterisation of the inserted DNA merely using PCR is not sufficient, as it does not unambiguously reveal the number of insertion sites and the copy number of inserted genes. Either Southern blots or a combination of PCR and Southern blotting yields better results. Inserts at one site may be concatemers of the same sequence. In particular, the ends of the inserts adjacent to plant genomic DNA have to be carefully analysed to determine whether any truncated open reading frames start within the insert or the plant genomic DNA that might yield transcripts that span plant genomic DNA and might also produce fusion proteins. The UK ACRE guidelines recommend considering to sequence the recombinant DNA fragments introduced in to the plant genome, involving the construction of genomic libraries of each transformed plant line that is to be so characterised (UK ACRE, 2002). This recommendation is further discussed in Section 5.1 of this paper. Evidence for the absence of vector backbone not intended for transfer is also required using Southern Fig. 4. Description of the donor(s), transgene(s) and delivery process. 1060 A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088