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Description of subject
- Food Safety Evaluation of Crops Produced through Biotechnology


What are the Food Safety Issues?
According to guidance provided by government regulators, pre-market food safety evaluation should consider the issues listed below [6, 9].

  1. Safety of the source organism and gene(s)
    1. Safety of the inserted DNA
    2. Safety of DNA Ingestion
    3. Safety of the antibiotic resistance marker (if used)

  2. The food safety issues of the newly introduced product(s)
    1. Potential for Toxicity (protein product)
    2. Potential for Allergenicity (protein product)
    3. Safety of any unintended effects

  3. Equivalence of Composition
  4. Retention of Nutritional Value
  5. The human dietary exposure

The "Safety of the Source Organism" is considered first. One might assume that a gene or genes that are derived from a commonly eaten food crop would not provoke the same degree of scrutiny as would the use of genes from a highly toxic source. In practice, the degree of scrutiny is the same. Risk is minimized if the gene products intended for introduction have been characterized and their function and metabolic fate established. It is a given that they should not encode toxicants, anti-nutrients or other potentially physiologically hazardous activities.

There are few safety issues associated with the ingestion of the newly introduced DNA per se. There exist no reported incidents in which DNA has been shown to be toxic. Despite fears and claims to the contrary, there are also no known instances of plant-derived DNA being taken up and incorporated into the mammalian genome [8]. Dietary DNA is usually degraded when consumed and is quickly hydrolyzed and digested to nucleotides in the human GI tract [8].

Antibiotic resistance markers (ARM) are used to help identify the transformed plants after the transformation (DNA introducing process). Plants that have incorporated the new DNA, including the ARM, will grow in culture that contains the antibiotic. Transformed cells containing the desired newly introduced genes can then be selected for further study. Concerns have been raised about the safety of using antibiotic resistance markers in crops developed through biotechnology. Some fear that ARM genes will transfer to bacteria in the soil and gut and give rise to increased levels of antibiotic resistance. Transfer of plant DNA to bacteria has never been observed in nature, nor has it been possible to demonstrate transfer laboratory experiments. More importantly and unfortunately, antibiotic resistance genes are already widespread in nature. Kanamycin resistance, a marker that is often used in biotechnology, is often present in 10% or more of bacteria randomly isolated from soil. Misuse and poor stewardship of antibiotics in animal agriculture, veterinary and human medicine may have led to the widespread dissemination of ARM genes in nature. There is certainly cause for concern about the indiscriminate use of antibiotics that has led to this situation, but plants containing ARM genes are highly unlikely to contribute to the problem. Banning the use of ARM genes in biotechnology is unlikely to improve the situation [7]. To address these concerns, biotechnologists are developing alternative selection systems that do not utilize ARM genes.

New genes are incorporated into transformed plants in order to confer new desirable traits on the plants. Almost without exception, these traits are the result of the transcription and translation of the genes to synthesize newly acquired proteins in the plant. Toxicological evaluation is routinely performed on purified preparations of the recombinant protein(s) that have been newly introduced. The toxicological evaluation typically answers the following questions:

What Is the Anticipated Human Dietary Exposure?
Data taken from national food consumption surveys can be used to predict the dietary exposure of consumers to the new variety or the proteins that it contains. The data is typically sorted into groups of special nutritional interest such as by age, gender and pregnant or lactating women. Other demographic factors such as ethnicity are considered as well. This evaluation can be complex since ingredients derived from commodities such as corn and soybeans can be found in a large variety of food products. The biotechnology-derived crop is seldom eaten as such; it is usually processed into ingredients and/or incorporated in formulated processed food products. The evaluation of Bt-corn will be used here as an example [10, 11]. The plant-protecting proteins in Bt are referred to as "Cry" proteins. Cry is an abbreviation for the "crystalline" proteins found in Bacillus thuringiensis spores that are toxic to a specific narrow range of insects. Approximately 1% of the whole corn used for food in the US is consumed in products such as tortillas and corn chips, while the balance is milled and fractionated into food ingredients such as starch and corn oil.

In late 2000, FDA recalled foods that contained, or might contain, StarLink corn. StarLink corn had been approved by the EPA for use as animal feed, but had not yet been approved for use in human foods. It was found that StarLink corn had become inadvertently intermingled with corn destined for food processors. DNA from StarLink was detected in taco shells and other whole-corn containing products. Data on human exposure to Cry9c, the Bt protein that was in StarLink corn, provide a useful example. Since StarLink corn was only 0.4% of the US corn crop, only very small amounts entered the human food chain [12]. The dietary exposure to Cry9c by 99th percentile consumers of corn products (Table 1) was predicted to be less than 1 µg/day of Cry9c. The table breaks out the data for Hispanics, since it was thought that their higher consumption of dry-milled corn in the form of tortillas and other whole corn products might lead to higher levels of StarLink consumption. This appears not to have been the case. This author is unaware of any reported toxin that is potent enough to cause a biological effect at these predicted 99th percentile levels of exposure. The policy of zero-tolerance for unapproved ingredients and additives justified the recall by FDA. Nonetheless, numerous reports of adverse effects were attributed to the consumption of Cry9C tainted products. The CDC has been unable to determine if Starlink consumption was the cause any of these incidents or that any of the adverse effects were related to allergic reactions to Cry9C [13].

The dietary exposure to the Cry proteins from human food-approved Bt crops that are in the marketplace today is estimated to be in the range of 1–10 µg/day if all corn were Bt-corn. By comparison, the daily consumption of protein is usually between 100–300 million µg/day [12]. In this context, exposure to new proteins in products developed via biotechnology is very low.

Does the Protein Cause Observable Adverse Effects When Feed at Dietary Levels Is Far in Excess of Those Anticipated to Occur in the Human Diet?
Experimental animals are fed doses in the range of 1 to 5 mg/kg/day [9, 11]. The studies can be evaluations of acute toxicity lasting no more than a few days or weeks, or evaluations of chronic toxicity, with studies lasting for three months to a full life cycle.

None of the Cry proteins thus far evaluated has produced any adverse effect at any level of administration to animals. As will be seen in the next section, this is likely due to their rapid digestibility. Compared to an estimated human exposure of about 1 µg/kg/day, this represents a five millionfold safety factor. In contrast, the toxicological standard routinely used in food safety evaluations is that there should be no observable effect at dietary levels only a hundredfold higher than those that would occur in the diet.

Is the Protein Digested in the Human GI Tract?
In vitro digestibility studies using simulated gastric fluid reveals that the Cry proteins in all of the Bt-corn products thus far approved for human consumption is digested by gastric fluid in approximately 30 seconds [9, 11]. The Cry9C protein present in Starlink corn, however, appears to be more stable to digestion. At low pH values (fasted state), it is digested in 30 minutes, while at higher pH values (feeding) the protein is somewhat more stable [9]. It is thought that proteins that are quickly digested would be unable to exert significant biological activity. If a protein such as Cry9C is not rapidly degraded, it could in principle retain biological activity (see the discussion of Cry9C and allergenicity below).

Is the Protein Destroyed by Food Processing?
Food processing operations such as thermal preservation, dry-milling or wet-milling often degrade and denature proteins giving rise to loss of their biological activity. The Cry proteins found in approved Bt-corn varieties are easily degraded by food-processing operations [9, 11]. In contrast, Cry9C appears somewhat more stable to processing, although it has been established that it is destroyed by wet-milling [13]. The partial destruction by food processing of Cry9C makes it unlike the other Cry proteins that are totally denatured and broken down by thermal processing. From a toxicological point of view, Cry proteins present very little hazard due to their large margin of safety and the fact that they are readily degraded.

Is the Protein Potentially a Food Allergen?
Food allergies affect 6% to 8% of children and 1% to 2% of adults. Ingestion of an allergen against which a person has become sensitized can produce symptoms as mild as oral irritation or minor gastric discomfort, or—albeit infrequently—as severe as anaphylactic shock followed by death. Some of the most common foods in our diet can cause allergy: corn, eggs, soy, rice, wheat, brazil nuts, peanuts, seafood, crustaceans and milk. Most food allergens are proteins, often major protein components of the offending food. Repeated ingestion of large amounts of the proteins is usually necessary for sensitization to occur. Food allergens typically are resistant, or at least partially resistant, to digestion and food processing. Food allergy is a complex and rapidly evolving field that has received increased attention in recent years [14]. More research is needed, however, in at least three general areas: 1) the nature of the human food allergic response, 2) an understanding of the structure(s) and sequences that lead to allergencity that is robust enough to allow predictions of allergencity to be made and 3) the development of a validated animal model with which allergencity can be predicted.

The safety assessment seeks to ensure that new allergens will not be introduced into foods in which consumers would not previously have expected them to occur. Fig. 1 is a flow chart for assessing the potential allergenicity of a product developed through biotechnology [7]. If a gene is isolated from a food that is known to cause food allergies, such as nuts, the path on the left side of the flowchart is followed. IgE-containing sera isolated from allergy sufferers can be used to directly assay allergenicity of the new food. A negative reaction can be followed by a confirmatory skin-prick test. Negative skin-prick reactions can be confirmed through double-blind placebo controlled food challenge protocols. Since sera can be isolated from allergic subjects, a definitive evaluation can be made for proteins isolated from foods containing known allergens. To ensure safety, however, developers of biotech crops avoid taking genes from plants known to contain allergenic proteins.

The pathway described in the right hand portion of Fig. 1 is applied to foods containing novel proteins with no history of consumption and/or no history of food allergencity. If a protein does not resemble known allergens at the level of protein sequence and structure and it is readily digested and/or destroyed by food processing, there is a reasonable likelihood that it will not provoke new food allergies. However, if a protein resists gastric digestion for 30 minutes, as was the case with Cry9c, it is difficult to conclude that it is not allergenic. There is no definitive evidence that Cry9c is an allergen, but since it displays partial resistance to digestion, the possibility that it could be a food allergen can not be eliminated. In the absence of proof that Cry9c was not an allergen, Starlink was not approved by the EPA for food use.

Have Unintended Changes Occurred in the Plant Breeding Process?
The most obvious unintended change that might occur is an alteration of composition or nutritional content. Extensive chemical, biochemical and nutritional analyses are performed on each new variety. Proximate analysis, amino acid analysis, protein profiles, carbohydrate analysis, lipid analysis, fatty acid analysis, vitamin and mineral content are usually determined. In addition, known anti-nutrient or potentially health beneficial compounds which might be important are assayed as well [9, 10, 11]. For example, changes in isoflavone or trypsin inhibitor content in soybeans would be evaluated. In some cases, the concentration of hundreds of cellular metabolites has been determined. A large amount of data has been accumulated on new varieties. The results are easily summarized. No significant differences in composition or nutrient content have been observed in the crop varieties approved to date. Animal feeding studies have been performed in order to determine if the new varieties give similar feed performance to conventional varieties [16]. No differences have been noted between biotechnology-derived and conventional feeds [16]. The animal studies reinforce the conclusion that these varieties have essentially the same nutritional value.

The Substantial Equivalence paradigm focuses attention on any safety issues associated with differences between a novel food and its most appropriate comparator. If no significant compositional differences are observed, SE directs primary attention to the safety of the newly introduced protein as described in the preceding sections. Perhaps it should not be too surprising that plant varieties which have been bred and selected to resemble their conventional counterparts in every detail of growth, hardiness, yield, appearance, size and a host of other agronomic parameters should be identical in composition. It is also reassuring that analysis demonstrates that hundreds of intracellular metabolites are present at identical concentrations as are found in conventional counterparts. Given the highly interconnected and interdependent nature of biochemical pathways in the cell and the tightly coupled regulatory processes that govern the concentration of intermediates and fluxes through metabolic pathways, it is highly unlikely that major changes in specific metabolite concentrations have gone unnoticed. It is impossible to rule out the possibility, however, that the concentration of one or more minor secondary metabolites differs in two varieties of the same plant.

It should also be noted that compositional changes are not per se a safety concern. One can imagine many compositional changes that would enhance safety such as reduction or elimination of an anti-nutrient. Other changes, such as increases in {omega}-3 fatty acids, ß-carotene or Vitamin E, could be health beneficial.

Unintended changes are not new to the food supply. They occur frequently in conventional plant breeding. In fact, the generation of compositional changes, such as enhancing monounsaturated fatty acid content in soybeans or Lysine in corn has sometimes been the objective of plant breeding. There is no reason to suppose that small changes do not occur when biotechnology is applied. No two varieties of the same plant species are identical in composition; representatives of the same variety grown in different locations, soils, climates or years often will have significantly different compositions. It will never be possible to conclude that a product developed via biotechnology, or any other technology, is 100% safe because it is impossible to prove there were no unintended changes in some minor but previously undetected or unappreciated component. The question pursued in the food safety assessment is this: "If any unintended changes did occur, is there any reason to believe these changes would raise more of a safety concern than changes that could and do arise from new crops developed via conventional breeding?"

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