Testing the Safety of Genetically Engineered Foods

(Excerpt from: Assessing the safety and nutritional quality of genetically engineered foods by John Fagan)

Note:
1. To download this document including diagrams as a zipped RTF file, click here.
2. To download the diagrams only click here

In the preceding section we have reviewed in brief, how genetic engineering is capable of introducing dangerous allergens and toxins into foods and reducing nutritional quality (see Section C for further details). It is, of course, important to test for these hazards before a genetically engineered food is placed on the market.

Recognizing that a range of safety testing needs will be encountered, Flowchart VII presents three schemes for testing genetically engineered foods for allergens, toxins, and alterations in nutritional quality. These are intended to illustrate the range of possible approaches for assessing the safety and nutritional quality of recombinant foods. By selecting various elements from this range of possibilities, the regulator can adjust factors, such as stringency of safety assurance, timeliness of assessment, and cost of assessment, to design a protocol that meets local needs and expectations.

Known food allergens, known toxins, and nutritional quality can all be evaluated in a straightforward manner, employing well established in vitro analytical methods. All three testing strategies use the same procedures for this purpose. These are summarized in Flowchart VII and presented in detail in Flowchart VIIIA (common food allergens), Flowchart VIIIB (common toxins), and Flowchart IX (assessment of nutritional quality).

Assessing unanticipated allergens and toxins is more challenging. It is in this area that the three testing strategies described below differ. These strategies employ various combinations of the following three approaches to detecting and characterizing allergens and toxins: 1. In vivo testing using small animals and human subjects for the purpose of screening broadly for allergens and toxins, 2. Molecular characterization of the genetic alterations induced through recombinant DNA modifications, 3. Controlled and monitored commercial release of recombinant foods.

Strategies IA and II both emphasize in vivo testing, and monitored marketing, but differ in the extent of in vivo testing, and thus in the degree of certainty with which one can claim that a given genetically engineered food is safe for human consumption. Strategy IA employs a graduated sequence of in vivo tests that will yield a high degree of certainty that a given genetically engineered food will be free of both long term and short term damaging effects for a high proportion of the population. Strategy II employs much smaller subject populations and less extensive in vivo testing, but still provides substantial protection for the consumer when genetically engineered food is cleared for full-scale marketing.

Strategy IB uses the same level of in vivo testing required in Strategy II, but includes extensive molecular characterization of the GEO and the genetically engineered food. This characterization is designed to assess the extent to which genetic engineering has altered the expression of other genes native to the organism, and/or changed the function or regulation of the metabolic and biosynthetic pathways of the GEO. It is felt that this combination of molecular and in vivo studies provides a safety margin similar to that attained with Strategy IA.

Strategy III is modeled after current regulations in the US. In this strategy, the developer is only required to carry out routine nutritional analyses and to test for common allergens and toxins that are suspected to be present on the basis of the identity or the UMO or on the basis of the source of the recombinant gene(s) used in generating the GEO. This testing scheme fails to assess the possibility of unanticipated but potentially dangerous allergens or toxins, because it does not include procedures, such as in vivo feeding tests with animal or human subjects, that are capable of detecting a wide range of toxins or allergens. Only if concrete evidence is available implying that unanticipated allergens or toxins may be present does this strategy require the developer to carry out further characterization.

In addition, Strategy III does not employ monitored marketing. Thus, genetically engineered foods carried through this testing scheme enter the market without any testing for novel allergens and toxins. Since, in this scheme genetically engineered foods need not be labeled, the consumer is even deprived of the choice of avoiding these inadequately tested genetically engineered foods.

Each of these testing strategies is discussed below. The stages of testing common to all three testing strategies are presented first, followed by discussion of the unique features of each strategy.

Flowchart VIIIA-In vitro screening for common food allergens This flow chart presents a plan for testing for known food allergens. Three basic questions will be asked:

1. If food derived from the unmodified organism (the UMO), from which the transgenic organism was generated, is known to contain allergens, are the levels of these allergens in the GEO within the norms expected for the UMO? 2. If the transgene is derived from an organism that expresses allergens, were those allergenic determinants transferred to the GEO via the transgene? 3. Are other common food allergens present in the food derived from the GEO?

These questions will be answered using the standard laboratory test for allergens, which involves assessment of the reactivity of the test substance with immunoglobin E or sera active against the allergen of interest. These procedures are limited by the fact that they cannot provide information on novel allergens.

Positive responses lead to more detailed characterization of the level/activity of the antigenic material detected in the food. The results of these studies, along with clinical data regarding this common allergen, obtained from the literature, are then used to decide whether the food is acceptable for human use, and to formulate labeling and use instructions.

Flowchart VIIIB-In vitro screening for known toxins This flow chart presents a plan for testing for known food toxins. Two basic questions will be asked:

1. If food derived from the unmodified organism (the UMO), from which the transgenic organism was generated, is known to contain toxins, are the levels of these toxins in the GEO within the norms expected for the UMO? 2. If the transgene is derived from an organism that expresses a toxin, was that toxin transferred to the GEO via the transgene, or were genes for enzymes critical to the synthesis of that toxin transferred?

These questions will be answered using specific analytical tests for that toxin. If the toxin is detected, further analytical work will be done to quantitate the level/activity of the toxin in the genetically engineered food. In conjunction with clinical data, these analyses will serve as the basis for deciding if the genetically engineered food is appropriate for commercialization, and to formulate labeling and use instructions.

Flowchart IX-Evaluating Nutritional Quality of Transgenic Foods If the transgenic food lacks known allergens and toxins, its potential acceptability for commercialization is high. Thus, it would be justified to invest the time and resources necessary to carry out thorough nutritional analysis.

Alterations in metabolism due to genetic modifications may lead to changes in the nutritional composition of the transgenic food, compared to norms for the corresponding natural food. Some changes may be direct and intended consequences of a given genetic modification. In such cases, specific measurements should be carried out to quantitate the extent to which the developer has succeeded in accomplishing those intended changes. In other cases, secondary, untended alterations in nutritional content, composition, or bioavailability will occur. Before the transgenic food is placed on the market, it is incumbent upon the developer to detect and quantify such changes in common nutrients and vitamins, at least if they are substantial. For this purpose, the following studies are recommended:

1. Standard quantitative methods will be used to assess common nutrients contained in the transgenic food. This analysis will include: quantity, composition, and bioavailability of protein, fats, carbohydrates, major vitamins, and trace elements. 2. The nutrient content of the transgenic food will be compared to that of the corresponding natural food and to norms for that foodstuff. For instance we would want to know if a genetically engineered tomato contained vitamin C levels equivalent to the unmodified variety of tomatoes from which it was derived and contained levels within the range that is typical of other tomatoes. Significant differences in nutrient content between the transgenic and natural food should be stated clearly on the label, and if radical changes are found, the transgenic food should be given a common name that distinguishes it from the corresponding natural food. 3. Some foods are recognized as primary sources for certain nutrients. Transgenic forms of those foods will be tested in detail to assess possible changes in quantity or quality of such nutrients. Significant differences should be highlighted on the label. 4. Nutritional questions of particular relevance to a given GEO will also be explored in more detail. For instance, the FlavrSavr tomato was promoted on the basis of extended shelf life. It is therefore necessary to quantitatively evaluate the persistence of important nutrients in this tomato over the full range of the claimed storage life of this product. That is, because this tomato appears attractive to the consumer for a long period of time, it is necessary to objectively ascertain whether or not nutritional value is preserved, as well, throughout that period of time. If nutritional value is not preserved, this should be stated on the label.

Flowcharts XA and XB-Strategy IA: High stringency in vivo testing to detect possible unanticipated allergens and toxins After the in vitro tests described in flowcharts VIII and IX have been used to assess the presence of common allergens and toxins, the in vivo tests, described in Flowchart XA and XB, can be carried out to establish that a given genetically engineered food is free of novel, unexpected allergens and toxins.

Most governments specify rigorous, standardized protocols for testing the toxicity and allergenicity of novel substances categorized as drugs and food additives. Because genetic engineering introduces new genetic material, and therefore new constituents, into foods, it is reasonable to test all transgenic foods with the same rigor required for these other novel substances. This point is discussed in greater detail in earlier sections.

The testing strategy presented in Flowchart XA and XB is designed to accomplish this aim. It is adapted from typical governmental standards for testing novel drugs and food additives. These standards have been modified to meet specific needs unique to the evaluation of the safety of transgenic foods. This strategy relies on in vivo animal and human studies as a method of testing allergenicity and toxicity more broadly than is possible with in vitro, biochemical or immunological tests. In order to minimize the risk to human test subjects, safety tests progress from animal studies, to small scale human studies, to larger scale trials, and finally to test-marketing of the transgenic food in selected locations with careful monitoring. Successful completion of these studies lead to full-scale commercialization.

Information from each step of this evaluation will be used in two ways. First, it will contribute to the decision whether or not to commercialize the transgenic food under study. Second, it will provide information relevant to the labeling and use of the final product, if commercialization is permitted.

Stage I-Animal studies

Short term animal tests are first carried out to eliminate genetically engineered foods containing powerful toxins or allergens before humans subjects are exposed to them. This series of studies will test the novel food in mice for (a) acute effects observed from feeding at maximum feasible doses for 48 hours and, if possible, for up to 2 weeks and (b) sub-acute effects resulting from feeding at levels proportional to maximum dietary levels in humans for up to 90 days. It should be possible to complete these tests within a period of 120 days. In these studies, standardized protocols will be followed which will evaluate the following parameters: • Maximum tolerated dose, based on autonomic signs • Central nervous system effects • Cardiovascular effects • Metabolic effects • Allergy/Inflammatory effects • Gastrointestinal effects

Because there are physical, chemical, and physiological limitations to the amount of a food that can be administered through feeding, it will not be possible in short term experiments such as these to detect toxins and allergens with the same sensitivity as can be done in toxicological experiments, in which extremely large doses can be administered. Thus, it is of paramount importance to carry out longer term experiments to be assured that a given food is free of significant toxins and allergens. Three dosage levels will be used in in vivo studies: (1) normal dietary level (NDL)-the amount of the food typically consumed by humans in a single meal; (2) maximum dietary level (MDL)-the maximum amount that can be consumed daily on a long term basis (limited, among other things, by the need to consume other foods to meet nutritional requirements, which can be calculated on the basis of nutritional data for the genetically engineered food); (3) maximum feasible intake (MFI)-the maximum amount that can be consumed short term (usually limited by the physiological capacity of the subject). For animal studies, NDL and MDL will be calculated from human doses scaled down proportionally to body weight.

Stage II-Short term, high dose human studies

The objective of this work is initial assessment of the safety of the transgenic food in humans. This work will be done using 20-50 normal volunteers. In an in-patient unit, these volunteers will be administered escalating single and multiple doses of the food until toxic effects are observed or the maximum feasible level, described above, is attained. That dose is continued for 48 hours, monitoring vital signs, physiological parameters, blood chemistry, etc. If toxic effects are not observed during that period, dosage will be dropped to the maximum dietary level, defined above, and the experiment continued for up to 90 days. If at any time serious negative effects are observed, the experiment will be terminated.

These studies will not only define toxic or allergenic levels of the food, but will also provide information concerning the nature of any toxic or allergenic effects that occur in humans. This work can be completed within 4 months.

Stage III-Medium term, moderate dose human studies

These studies assess toxic or allergenic responses that manifest within 6 months, and that occur in the general population at a frequency of greater than 1%. In these studies, 100 to 500 subjects will be fed the genetically engineered food at maximum dietary level for up to 6 months. Vital signs, physiological parameters, blood chemistry, etc. will be monitored, and, if necessary, dosage will be manipulated in response to changes in tolerance of the subjects. This phase of safety testing should be completed within a period of 8 months.

Stage IV-Long term human studies

These studies will assess longer-term effects, and because a larger subject population will be used, will be capable of identifying small subpopulations (less than 0.1% of the population) that may have special problems with the transgenic food under study. Depending on the nature of the food and on the outcome of earlier stages of testing, 1000 to 3000 human volunteers will be fed the genetically engineered food at normal dietary levels daily for 1.5 to 2 years. In addition to vital signs, physiological parameters, and blood chemistry, this study may also provide information on the effects of the food on reproduction and cancer incidence.

Stage V-Test-Marketing with Health-Impact

Monitoring If human trials indicate the safety and desirability of a transgenic food, it will next be test-marketed in selected areas, with careful monitoring to detect impacts on the health of consumers. The monitoring system will include two important elements. (1) Hospitals and other medical facilities in the area will be alerted to the trial, and asked to report any health problems that might be related to consumption of the experimental, genetically engineered food. (2) The transgenic food will be labeled. The label will : (a) clearly designate the food as genetically engineered; (b) specify the source species from which genetic material was obtained to assemble the recombinant DNA molecule(s) used in constructing the transgenic organism; (c) describe the unique nutritional or other characteristics of the transgenic food; (d) tell the consumer that the product is experimental and ask the consumer to report any possible health impacts, minor or major, to the developer; (e) provide a mechanism that will allow the consumer to report health impacts to the developer conveniently and without incurring cost (such as a toll-free telephone number, or local contact address). This phase of safety testing will continue for 2 to 3 years.

Full-scale marketing

Even after test-marketing is complete, labeling is required, not only for continued monitoring of safety, but also to provide the consumer with sufficient information to make informed purchasing decisions.

Labeling should: (1) specify that the product is genetically engineered; (2) indicate any unique characteristics of the transgenic food relative to the natural counterpart; (3) provide a mechanism for consumer feed-back to the developer; (4) provide information on special handling or preparation requirements.

Flowchart XC-Strategy IB: Safety assessment emphasizing molecular characterization of the genetically engineered organism. Strategy IB, presented in Flowchart XC uses a combination of in vivo tests and molecular characterization of the GEO to eliminate several classes of unanticipated allergens and toxins that might be present in the genetically engineered food. This molecular characterization should reveal if genetic engineering has disrupted the normal gene structure or normal gene expression of the GEO in obvious ways. If such disruptions have not occurred, then unanticipated changes in the quality or safety of the genetically engineered food are less likely. In the light of such evidence, it should be possible to reduce the extent of in vivo testing without compromising the safety of human subjects and consumers.

The first step in this analysis is to consider the nature of the transgene. If it encodes a protein(s) that is known to be non-toxic and non-allergenic, and if it is unlikely to catalyze reactions that modify cellular metabolism in such a way as to generate toxins, then one class of risks is eliminated.

The possibility remains, however, that the transgene or its protein product, might modify cellular gene expression, thereby causing the GEO to produce new toxins or allergens or higher levels of toxins or allergens than the UMO (via mechanisms discussed in detail earlier and in even greater detail in Section C). This possibility is explored by investigating the following three questions:

1. Does the insertion site of the transgene interrupt one or more open reading frames within the DNA of the GEO? If an open reading frame is interrupted, then the expression of at least one gene is blocked. The question then arises as to the identity of that gene and the function of the protein that it encodes, and the actual effect of the loss of that protein on the metabolism and regulation of the GEO. 2. If there are mRNAs actively expressed from the sequences within the 20 kb domains flanking the insertion site of the transgene, are the levels and patterns of expression of those mRNAs unchanged in the GEO, compared to the UMO? The genes whose expression is most likely to be disrupted by the inserted transgene are those nearby. This experiment evaluates the expression of these genes directly. 3. Is the transgene expressed in the parts of the GEO that are normally used for food?

If these questions all yield negative answers, then it is unlikely that the gene expression of the GEO is significantly different from that of the UMO, and there is sufficient confidence that the genetically engineered food is safe to advance to monitored marketing (Stage V).

If any one of these questions yields a positive answer, then it is necessary to assess gene expression more fully in the GEO. For this purpose we propose the use of the differential display technique or another method capable of exhaustively comparing the mRNA profile of the GEO to that of the UMO. If no significant changes in the mRNA profile are observed, it can be concluded that the genetic alterations carried out have not significantly disrupted gene expression in the GEO. These techniques require significant technical expertise, and are somewhat time consuming. However, in some cases the developer will prefer this approach to in vivo testing. If no significant alterations in mRNA expression profile are detected, the developer can proceed directly to monitored marketing. If significant alterations are observed, in vivo testing is required, both Stage I and Stage II, before proceeding to monitored marketing.

Flowchart XI-Strategy II: Safety assessment employing limited in vivo testing of the genetically engineered organism. This strategy is similar to Strategy IA, except only short-term human testing is carried out with a small number of subjects. All of the stages included in this strategy are discussed in detail earlier. This strategy will eliminate those toxins and allergens that are most severe in their effects. Although this approach is less costly and time consuming, its use carries the disadvantage of placing a significantly larger number of people at risk than would be the case with Strategy I. Because fewer than 100 subjects are used, allergens or toxins to which fewer than 1% of the population are susceptible will not be detected. Furthermore, because Strategy II employs only short term studies, serious long term problems would also remain undetected.

Flowchart XII-Strategy III: Safety assessment employing primarily in vitro analysis. As discussed earlier, Strategy III focuses only on common allergens and toxins. It investigates the possibility of other allergens and toxins only in cases where circumstantial evidence happens to arise indicating their presence. As implemented in the US, this approach does not require that genetically engineered foods be labeled. We strongly recommend as a minimal safety measure that all genetically engineered foods be labeled so that any problems that arise can be traced.