PART III PROCESS SAFETY
5. MON 810, A Case Study on Genetically Modified Maize
The European Corn Borer (Ostrinia nubilalis; ECB) causes massive damage every year to cultivated maize (Essential Biosafety 2001). As reported by some countries (e.g. in Switzerland) the ECB is controlled on approx. 5-20% of maize fields through the release of the parasitic wasp Trichogramma, in other countries (like Germany) are as a countermeasure in addition to the release of Trichogramma applied as well an insecticide (Decis®), to combat the ECB. As permission to use insecticides will in future be limited, it is necessary to find new ways to stop the negative effects of the ECB. One possibility is seen in using insect-resistant maize lines (Essential Biosafety, 2002).
Toxins produced by the bacteria Bacillus thuringiensis, are effective in killing the ECB by provoking death through lysis of the gut lining. The genes from B. thuringiensis have now been cloned and inserted directly into the corn genome for expression in various plant parts, to protect the plant against the ECB. The commercialized transgenic lines vary in their expression of the toxin: NOVARTIS Bt 176 maize expresses the Bt toxin in the kernels, leaves and pollen. NOVAR- TIS Bt 11 expresses toxin in the kernels and MONSANTO’s MON 810 expresses
Integrated approach for safety and nutritional assessment of novel foods
Safety-Nutrition Profile
Biochemistry Toxigenomics
Metabolics/Kinetics Nutrigenomics Toxicology (Clinical/Nutritional)
Dose responses Nutrient-gene interactions Bioavailability Polymorphism
Ranges of Toxicity and functionality
Food Matrix interactions
FIGURE1. Integrated approach for safety evaluation of genetically modified food (Kuiper et al., 2001).
toxin in all parts, except in the pollen (Essential Biosafety 2002). Bt toxins are, depending on the strain of B. thuringiensis from which they originate, very spe- cific in attacking primarily Lepidoptera and Coleoptera.
Under the trade name YieldGard®, a maize line MON 810 was developed through a specific genetic modification securing resistance to attack by the European corn borer (ECB, Ostrinia nubilalis), a major insect pest of maize in agriculture. The novel variety produces a truncated version of the insecticidal pro- tein, Cry1Ab, derived from Bacillus thuringiensis. Delta-endotoxins, such as the Cry1Ab protein expressed in MON 810, act by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry1Ab is insecticidal only to lepidopteran insects, and its specificity of action is directly attributed to the pres- ence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.
This GM line of maize received its first regulatory approval in the USA in 1995 and has since been approved for environmental release and use in livestock feed and/or human food from a number of countries in America, Asia, Australia and Europe (Table 2).
Maize, taken generally, is the third most important crop in the world after rice and wheat; with an annual production over 600 million tones (FAOSTAT Database Records,). It has been grown as a commercial crop in many countries worldwide, starting some 8000 years ago in Mexico and Central America (Mexico, 1994;
OECD, 2002). In Europe it has been known for 500 years.
Maize is naturally cross-pollinated and until 1925 mainly open pollinated vari- eties were cultivated. To produce hybrid seed the tassels are removed from the plants prior to pollen shedding, so that only one sort of pollen will be received by the silks (OECD, 2002). The hybrid plants grown from this seed give more vig- orous growth and higher yields. The two leading maize producers are USA and China.
TABLE2. Regulatory approval status of insect-resistance maize MON 810
Approved for Approved for
Country Environment food/feed
Argentina 1998 1998
Australia 2000
Bulgaria 2000 2000
Canada 1997 1997
European Union 1998 1998
Japan 1996 1997
Russia 2000
South Africa 1999 1999
Switzerland 2000
United Kingdom 1997
United States of America 1995 1996
The behaviour and characteristics of MON 810 have been studied in a range of field experiments in USA and Europe since 1992 (Essential Bisafety, 2001). The assessment of the interaction of this maize line with the environment has included studies on:
● susceptibility to insects and diseases
● survival capacity (volunteers)
● seed multiplication capacity (yields)
● Cry1Ab protein expression in leaves and grain
● seed composition analysis
● safety for birds
● safety for mammals.
Observation and field trials have demonstrated that the mode and reproduction of MON 810 are typical of other maize varieties (Essential Biosafety, 2002).
Expression levels of the Cry1Ab, CP 4 EPSPS, GOX and NPTII proteins are shown in Table 3.
Disease and pest susceptibilities of MON 810 were tested and no differences in agronomic quality, disease, or insect susceptibility other than European corn borer control were detected between MON 810 and non-transgenic plants (Essential Biosafety 2002). Diseases observed included northern leaf blight (Excerohilum turcicum), southern leaf blight (Bipolaris maydis), bacterial leaf blight (Erwinia stewartii), common corn snut (Ustilago maydis), maize stripe virus and common maize rust (Puccinia sorghi).
In 1990, the FAO and WHO agreed that the comparison of a final product with one having an acceptable standard of safety provides an important element of safety assessment (OECD, 2002). The OECD further elaborated this concept and advocated the approach to safety assessment based on substantial equivalence as being the most practical one to address the safety of foods and food components derived through modern biotechnology. It was concluded that the safety assessment of genetically modified foods requires an integrated and stepwise, case-by-case TABLE3. Summary of specific protein levels measured in tissues of MON 810
PROTEIN Protein concentration in µg/g of fresh weight
LEAF GRAIN WHOLE PLANT POLLEN
Cry1Ab 9.35 0.31 4.15 0.09
CP4 EPSPS nd nd nd na
GOX nd nd nd na
NPTII na na na na
Abbreviations:
Cry1Ab – insecticidal protein of the delta-endotoxin group, product of cry1Ab gene
CP4 EPSPS – product of the epsps marker gene (plant selectable marker enabling the identification of corn cells with introduced cry1Ab gene)
GOX – product of glyphosate oxidase encoding gene gox (another selectable marker)
NPTII – enzyme neomycin phosphotransferase II, product of gene nptII, the bacterial selectable marker conferring resistance to kanamycin or neomycin
nd – not detected na – not analyzed
approach, which can be aided by a structured series of questions. A comparative approach focusing on the determination of similarities and differences between the genetically modified food and its conventional counterpart aids in the identification of potential safety and nutritional issues and is considered the most appropriate strategy for the safety and nutritional quality of genetically modified foods. It should be seen as a key step in the safety assessment process although it is not a safety assessment in itself, it does not characterize hazard.
An important element in the determination of substantial equivalence is elabo- ration of compositional comparison, where a comparison of critical components can be carried out at the level of the food source (i.e. species) or the specific food product (FAO, 1996). Critical components are determined by identifying key nutrients and key toxicants and antinutrients for the food source in question. The comparison of critical components should be between the modified variety and non-modified comparators with an appropriate history of safe use. The data for the non-modified comparator can be the natural ranges published in the literature for commercial varieties or those measured levels in parental or other edible vari- eties of the species. The comparator used to detect unintended effects for all crit- ical components should ideally be the near isogenic parental line grown under identical conditions.
Yield comparisons for MON 810 and representative controls were assessed in 9 different locations of USA (Essential Biosafety, 2001; Essential Biosafety, 2002). A comparison of a non-transgenic hybrid with the same hybrid in which one parent was a backcross-derived MON 810 line, showed no significant differ- ence between these hybrids in yield.