Beneficial Organisms in Agriculture, Food and the Environment: Safety Assessment and Regulation

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Beneficial Microorganisms in Agriculture, Food and the Environment

Cry proteins are active against lepidopteran insects and some variants are also active against dipteran insects, coleopteran insects, or nematodes. VIP proteins, which are produced and secreted during the vegetative growth stage, have activity against coleopteran and lepidopteran insect species. Although each of these components contributes to the overall insecticidal activity of microbial Bt products, the Cry proteins are considered the most important component of commercial Bt formulations.

It is for this reason that the majority of commercially available GM crops with insecticidal activity have been developed to express one or more Bt Cry proteins, which are the focus of this review. Unlike most chemical pesticides, which are contact insecticides, Cry proteins are effective only when ingested by the insect. The activated Cry molecules typically bind to specific receptors on mid-GI-tract epithelial cells of target larval insects and then oligomerize, forming pores consisting of four to six Cry molecules each in cellular membranes. These pores enable excess cations to enter the cell, causing osmotic imbalance.

There is also compelling evidence for ABC transporters serving as receptors for Cry proteins in several lepidopteran species Gahan et al. The many permutations of species-specific insect GI-tract proteases, receptors, and tendencies of each Cry protein to oligomerize in a specific situation result in the biological specificity that provides targeted insecticidal activity and safety to NTOs of Bt sprays and GM crops that produce Bt proteins. An essential component of the highly selective insecticidal properties of most Cry proteins is the requirement that the toxin interact with one or more specific receptors.

Many studies have demonstrated that the GI-tract epithelial surface of non-target insects and mammals, including humans, lack specific high-affinity Cry protein receptors Sacchi et al. The absence of high-affinity binding in mammals may be due in part to the absence of BL2, a glycosylating enzyme present in the GI-tract cells of invertebrates. Experiments with surface plasmon resonance assays indicate that binding to the cadherin receptor occurs at nM concentrations Sacchi et al.

Specificity of Cry protein binding was shown with gut cell brush-border membrane vesicle BBMV binding studies. In these experiments the binding of a labeled Cry protein, which is active against Manduca sexta Lepidoptera larvae, could be displaced by an unlabeled excess of the same Cry protein or other Cry proteins active against M. Extensive evidence has been accumulated on the mechanism of action for Bt microbials, and Cry proteins in particular, and resulted in a generally accepted model wherein Cry proteins bind to specific high-affinity receptors in the gut of target larval insects, oligomerize, and form pores in the cellular membranes of the gut.

The result of pore formation is osmotic shock, cell lysis, and eventual death of gut epithelial cells. Cadherin-like glycoproteins were mentioned above as receptors, but aminopeptidase N, ABC transporters, and alkaline phosphatase have also been identified as Cry protein receptors, which enable pore formation in target organisms. BL2 is a glycosylating enzyme found in target insect pests which is responsible for producing the specific sugar residues that facilitate recognition and binding by Cry proteins to the aminopeptidase N and alkaline phosphatase receptors Federici and Siegel, ; Hammond and Koch, The presence of this protein in insect species appears to contribute to the taxonomic specificity of Cry proteins currently in Bt crops.

The absence of this protein is believed to be a reason why Cry proteins fail to exhibit toxic effects in mammals and other non-insect species. There is an extensive history of safety for human consumption of Cry proteins. The strains found were indistinguishable from those in commercial Bt microbial formulations, indicating that Bt microbial applications were the likely source. Vegetables treated with Bt i. Regulatory agencies have acknowledged the history of safe consumption of Cry proteins. For example, the U. However, in the absence of any toxicological concerns, risk from the consumption of treated commodities is not expected for both the general population and infants and children.

Because of their exceptional safety profile, Bt microbials have historically been exempted from the requirement for a numerical tolerance maximum residue level in countries where they have been registered OECD, , meaning that there is no withdrawal time required for consumption of crops sprayed with Bt microbials. The potential human dietary exposure to Cry proteins from application of commercial Bt microbial pesticide formulations was recently estimated Hammond and Koch, ; Box 1.

Although Bt microbial preparations are safe and efficacious, they are limited in their duration of effectiveness because they can be washed off the plant e. These limitations have been addressed through the introduction of GM crops containing one or more genes encoding one or more Bt proteins, which are produced by the plant throughout the growing season. Another advantage of GM crop products expressing Bt proteins is that only the specific Bt protein s of interest, typically Cry proteins, are produced in the crop, possibly decreasing the spectrum of activity compared to Bt microbial formulations Hammond and Koch, Bt Cry proteins in GM crops authorized for cultivation in one or more countries.

Domain swapping has been shown to be an effective way to change the spectrum of activity of a native Cry protein to include a new target pest. Examples of proteins that have undergone domain swapping for this purpose are Cry1A. Such domain exchanges are possible, in part, because the tertiary structures of these Cry domains are highly conserved Li et al. As another example, the Cry1Fa-like protein in the Bt microbial Lepinox construct contains the core Cry1Fa insecticidal protein moiety fused to a Cry1Ac C-terminal protoxin moiety Baum et al.

This modified protein exhibited improved expression in Bt but retained the insecticidal specificity of the native Cry1Fa protein Gilmer and Baum, Recently, the chimeric eCry3. There are several important, well-established examples that demonstrate that small changes in Bt amino acid sequence do not change the safety profile for NTOs. Regulatory authorities have required functional studies with sensitive insect bioassays to demonstrate that these small changes do not impact biological activity.

If these assays indicate biological activity equivalence of the two protein forms, any other properties of the proteins are considered to be equivalent as well. A well-established example of this approach was used to bridge the existing safety assessment performed for the Cry3Bb1 protein produced by MON maize to support the approval of MON maize, which also produces a variant of the Cry3Bb1 protein.

The proteins differ by only 1 amino acid out of To test for functional equivalence, sensitive insect diet incorporation bioassays were performed with appropriate insect models i. These two forms of the Cry2Ab2 protein differ by a few amino acids on the N-terminus of the proteins and reflect the introduction of a chloroplast transit peptide EPA, a.

Despite the addition of these amino acids, there was no impact on biological activity in a sensitive insect bioassay EPA, a. Critics have raised safety concerns about modifications that have been made to Bt Cry proteins introduced into GM crops. There are two problems with this rationale. Rather, the focus should be on whether the modifications fundamentally alter the structure and functional properties of the protein to a degree that it affects the safety of these proteins See History of Safe Use and Bioinformatics. Thus, despite the changes introduced, the extensive history of safe human consumption of native Cry proteins can be applied to the safety assessment of these modified proteins Hammond et al.

Second, Bt Cry proteins in either their microbial or plant-incorporated protectant i. This is an important designation because biopesticides are generally considered be inherently less toxic and have a narrower spectrum of activity i. Several International Life Sciences Institute ILSI task forces have examined and provided science-based recommendations for the safety assessment of crops derived from agricultural biotechnology.

Principles for the assessment of proteins used in agricultural biotechnology were described in detail by Delaney et al. Both describe a two-tiered approach of hazard identification followed by hazard characterization appropriate to the identified hazards, if any. Regulatory guidance on the assessment of insecticidal proteins has been given by both national and international authorities Mendelsohn et al. Although there are seemingly an unlimited number of changes that could be made to improve protein function, one estimate indicates that only 0.

Looking further into the types of changes that a protein can undergo can help explain this estimate. Substitution of one amino acid residue with another one of similar size and properties might have little effect; on the other hand, substitutions that alter the three-dimensional structure will likely have major deleterious consequences on protein functionality. Modifications that alter the ability of the protein to fold properly are often deleterious because they reduce or eliminate the functional activity.

Although radical changes are possible, amino acid sequence modifications introduced in engineered proteins are normally designed to enhance function with minimal disruption of structure. By retaining the three-dimensional structure of the naturally occurring protein, the general functional characteristics are also preserved in the engineered protein Behe et al.

When in vitro engineering introduces minor changes to the structure or function of a protein, there is little reason to suggest that the engineered protein will become toxic. This has been the case for evolutionary changes within protein families such as those used as food processing enzymes. These changes have not resulted in the enzymes becoming toxic to humans Pariza and Cook, For context, this is similar to selecting a specific drop of water in one of five Olympic-sized swimming pools on the first attempt.

As illustrated by the specificity of Cry proteins for their target insects See Receptor-Mediated Selectivity of Bt Cry Proteins , the effects of protein toxins in susceptible organisms generally depend on recognition of specific molecular targets Rappuoli and Montecucco, In addition to considering the HOSU of related proteins, performing bioinformatic analyses of protein sequence and structure can provide valuable information on the safety of a modified Cry protein or other introduced protein. A bioinformatics evaluation is performed as one of the initial steps in the Tier 1 safety assessment Delaney et al.

These comparisons of the candidate protein to sequence and structure databases are used to determine whether the protein of interest is similar to any known toxic protein. Analyses of phylogenetic relationships between the candidate protein and others are also useful Codex Alimentarius, ; EFSA, e , ; Hammond et al.

The information gathered from the safety assessments described throughout this manuscript identifies if there is a potential hazard associated with the Cry proteins and are necessary for performing dietary risk assessments. However, a determination of exposure is also required because risk is a function of both hazard and exposure Faustman and Omenn, An estimate of the maximum potential exposure to a Cry protein through the diet can be derived by determining the protein expression level in the grain and the amount of grain consumed. Such values are highly conservative estimates of dietary exposure because they do not account for the lack of stability of the protein during digestion, they do not consider its response to heat or food processing conditions, they ignore that commodity crops are a blend of multiple varieties i.

The harsh conditions encountered by dietary proteins in the GI lumen and during food production processes significantly reduce their stability as detailed below. To approximate the effects of protein exposure to conditions in the mammalian GI tract, a validated in vitro assay to assess the potential stability of proteins to pepsin digestion has been developed. This reliable and reproducible assay uses a fixed pepsin: Cry proteins are readily degraded in this assay EPA, ; Okunuki et al. Under conditions of higher pH and lower ratios of pepsin to Cry protein, Cry1Ab protein is more slowly degraded, as is expected since pepsin becomes less active at a greater pH Guimaraes et al.

Although Guimaraes et al. In pigs and calves, Cry protein fragments are detectable but are progressively reduced in size as they travel down the GI tract. None were detected in the liver, spleen, or lymph nodes Chowdhury et al. In addition, maize fed to animals is generally not processed, other than grinding.

Human dietary exposure to Cry proteins is much lower than that of farm animals owing to the lower percentage of maize in the diet and the processing e. Recalling that risk is a function of both hazard and exposure, the lower exposure encountered by humans puts them at lower risk than animals. This is particularly noteworthy when one considers that the animals did not demonstrate reliable signs of toxicity i. Prior to human consumption, Cry proteins in GM crops typically undergo some sort of thermal processing, which must be considered in the toxicological safety assessment because it can significantly reduce the dietary exposure to functionally active Cry protein.

Temperature increases, pH variation, and physical disruption can overcome the forces that keep a protein folded properly Creighton, and cause denaturation, i. Because of the change in three-dimensional structure, denatured polypeptides lose functional activity, including the ability to specifically bind receptors or other compounds. The impact of food processing on the functional activity of introduced proteins is relevant because for cooked food, it results in safety margins that are conservative and an overestimate of actual exposure i.

For Bt maize containing different Cry proteins, the levels of Cry proteins in grain were approximately 0. Even these values may overestimate exposure, as the calculation for exposure to Cry1Ab Bt protein in Hammond and Jez of 0. Clearly, digestion and food processing greatly limit the dietary exposure of any proteins consumed orally whether transgenic or endogenous. Nonetheless, companies seeking registration of biotech crops often use the highly conservative approach of estimating exposure by determining the protein expression in the grain and multiplying it by the amount of grain consumed.

This obviously overestimates exposure for the reasons detailed above. Despite this conservative approach in estimating dietary exposure i. The allergy assessment of a GM crop is designed to identify potential allergenic risks associated with an introduced protein Codex Alimentarius, A weight-of-evidence approach is taken to assess the allergenic potential of the introduced protein.

This assessment includes determining whether the source organism of the introduced protein is allergenic, an extensive bioinformatics assessment to determine if the introduced protein is similar to known allergens, and determining the level of exposure of the introduced protein in the GM crop, including determining the stability of the protein to digestive enzymes Goodman et al. Bacillus thuringiensis microbials are not considered a human allergen, because despite the extensive use as a biopesticide over the last several decades, there has been only one report of allergic reactions in workers who manufacture or apply Bt microbials Siegel, ; Federici and Siegel, However, this single allergic reaction was attributed to bacterial proteins in the Bt microbial formulation other than the Cry proteins Siegel, ; Federici and Siegel, None of the Bt Cry proteins used to develop GM crops have had any sequence similarity to known human allergens using any of the bioinformatics thresholds for amino acid similarity recommended by international guidance Codex Alimentarius, ; Silvanovich et al.

Another assessment of allergenic potential of the inserted protein is to determine the exposure to the protein.

This is relevant to the allergenicity assessment because it is generally accepted that increased exposure to a protein increases the possibility that the protein could become an allergen. Exposure is assessed by measuring the protein abundance in the grain See Impact of Heat on Stability and the stability of the protein in the presence of pepsin See Impact of Protein Digestibility.

Unlike the toxicological safety assessment, the heat stability assessment of the protein does not provide useful information to the allergy safety assessment because an allergen does not need to be in a functional state to cause an allergic reaction Privalle et al. For this reason, it is important to recognize that these analyses are not predictive of allergenic potential, but instead should be considered as part of the weight-of-evidence approach. Taken together, the weight-of-evidence approach used to assess the Cry proteins present in Bt crops suggests the allergenic potential of these proteins is low and that they present little allergenic risk.

In the paradigms outlined by Delaney et al. Acute toxicology testing was originally warranted because of the insecticidal i.

As described in subsequent sections, neither short- nor long-term toxicology testing has revealed any concerns about the safety of Bt proteins for human or animal consumption. In general, there are fundamental biological properties of proteins that greatly limit their potential to produce chronic toxic effects when ingested Delaney et al. These properties are discussed in detail in Hammond et al. Considering the first two points above, and that Cry proteins are readily digested See Impact of Protein Digestibility and can be denatured and inactivated during normal food processing See Impact of Heat on Stability , human dietary exposure to functionally active dietary proteins, including Cry proteins, is likely to be negligible.

While acute oral toxicity studies with proteins from GM crops have fallen out of favor in some world areas e. Recently, EFSA stated that they may require a repeat-dose day toxicology study when it considers the available safety information on the introduced protein to be insufficient. What this means in practice has not yet been determined for Cry proteins, but given the substantial HOSU of Cry proteins and the existing weight of evidence for approved traits, there should be sufficient scientific evidence on the safety of such proteins to preclude the need for conducting a day study.

A good way to illustrate this disparity is to consider an extreme example. This amount of shelled corn would be the equivalent of 35 semi-truck loads. Thus, these data represent a considerable weight of evidence that Cry proteins are safe for consumption as food and feed; especially when considered with the safety of microbial Bt pesticides See History of Safety Use of Bt Microbial Products in Agriculture ; as well as bioinformatics See History of Safe Use and Bioinformatics , in vitro digestibility assays See Impact of Protein Digestibility , low dietary exposure levels See Human Dietary Exposure Assessment for Bt Microbial Products , and the absence of toxicity following high dose acute studies See Acute and Short-Term Mammalian Toxicology Testing of Bt Cry Proteins with the Cry proteins tested to-date.

Acute toxicity studies in mice with Cry proteins and Bt microbials. This section will discuss in detail the technical deficiencies of some of the papers that report potential health concerns with Bt microbials or Cry proteins. It will also examine the reasons that, despite past practices, long-term animal testing with Bt crops is not scientifically beneficial or justified.

Despite the considerable history of safe consumption, and the large volume of data demonstrating the safety of both Bt formulations and Cry proteins, some parties still doubt the safety of these products. Occasional publications will make startling claims: Yet upon closer examination, these allegations do not appear to have a strong scientific basis.

When one considers the overall demonstrated safety of Cry proteins and the lower dietary exposure to Bt proteins from consumption of a GM crop than microbial Bt formulations used in conventional and organic agriculture, claims of adverse effects exclusively following the consumption of Bt crops simply do not withstand closer scrutiny.

There is a long safety history of Bt microbial pesticides on agricultural crops See History of Safety Use of Bt Microbial Products in Agriculture , and there is similarly a HOSU of Bt crops since when these crops were first introduced into commerce. Following review of relevant data in submitted dossiers from registrants, the U. Rather, they have considered the weight of evidence comprised in part by HOSU, the demonstrated safety of the trait in mammals i.

Examples of repeat-dose toxicity studies with Bt microbial formulations, Cry proteins, and Bt crops. Rats were treated with famotidine to reduce gastric acid secretion and indomethacin to damage the intestinal epithelium and fed diets with or without Cry1Ab protein 10 ppm. Despite the expectation of less Cry1Ab protein digestion and more absorption of Cry1Ab protein into the circulatory system of the GI-impaired animals, there was no evidence of meaningful toxicological effects changes in clinical blood parameters and histologic appearance of organs in the Cry1Ab-dosed animals.

There were no treatment-related adverse effects seen on reproductive performance in animals fed Bt crops or other transgenic crops Snell et al. According to the review, the majority of these studies showed no adverse effects. As a result, these studies should not be used to inform the risk assessment process for these crops. The issues surrounding whole food testing of GM crops Bt and non- Bt have recently been examined by two groups of authors Bartholomaeus et al.

Considering the limitations of whole food testing, such as low sensitivity and difficulty in defining the test material, both groups concluded that routine whole food testing does not add meaningful information to the risk assessment of GM crops and cannot be scientifically justified. Another recent review article evaluated the reliability of a number of the published toxicology studies carried out with GM crops and proteins in general, and Cry proteins and Bt crops in specific, by adapting the ToxRTool Koch et al.

When its objective criteria were adapted for feeding studies and used to evaluate studies with Bt crops most of the studies were considered to be reliable. Of the studies determined to be reliable, none found evidence of adverse effects from the consumption of GM crops containing Cry proteins or of purified Cry proteins Koch et al.

The biological relevance of these studies to assessing potential health risks from human consumption of foods derived from Bt crops is limited because administration of Cry proteins by IN, IP and IR routes of exposure do not predict risks from IG or dietary intake. IN, IP, and IR routes of exposure bypass the protective barriers of the GI tract and are not particularly relevant when characterizing hazards that might be associated with a protein that will be consumed as food.

However, when an IG route of exposure was used, it was necessary to include a magnesium—aluminum hydroxide suspension to see an effect on these studies. This anti-acid suspension was used to neutralize the pH of the GI tract, thereby preventing digestion of the Cry protein See Impact of Protein Digestibility and eliminating a formidable element of the GI tract barrier.

Additionally aluminum hydroxide is a known antigenic adjuvant Mannhalter et al. While this may not seem like an exceedingly large dose at first glance, this amount of Cry1Ac translates into at least 50,fold greater exposure than estimates of Cry1Ab human intake Hammond and Jez, , which conservatively presumed that Bt maize grain was consumed uncooked. These experimental conditions clearly exceed any reasonable potential human dietary intakes. Furthermore, since maize grain is normally processed e.

Thus the differences in dietary exposure between the aforementioned mouse study and humans would increase from 50, to 5,, Hammond and Jez, One potential explanation for this discrepancy in results is that Cry protein preparations used in the studies where antibody production was observed were contaminated with E. The Cry proteins used in these studies were produced and purified from E. Additionally, the routes of exposure and amounts of protein required to elicit a response were very different from the dietary exposure of Cry proteins in GM crops See Human Dietary Exposure Assessment for Cry Proteins in Bt Crops.

Given the low dietary exposures to Cry proteins from consumption of foods derived from Bt crops, the potential to induce or enhance an immune response in humans is unlikely Guimaraes et al. This concentration is many orders of magnitude higher than the potential dietary exposures a mammalian digestive tract might encounter from consumption of food derived from Bt maize Hammond and Cockburn, ; See Human Dietary Exposure Assessment for Cry Proteins in Bt Crops and may have been directly responsible for the observed binding simply due to the law of mass action i. The Cry1Ac concentration used in these studies was too high to be toxicologically relevant as it greatly exceeded levels toxic to target insect pests, where binding to receptors occurs at much lower concentrations 0.

While one group reported that an insecticidal Cry protein bound to rat BBMV, they also note it did so with low affinity. Additionally, the bound protein could not be displaced by a 10,fold excess of unlabeled Cry protein Hofmann et al. Taken together, the low affinity and inability to displace the labeled protein with unlabeled protein indicates non-specific, or non-receptor-mediated, binding. In another study, Bt microbial spore preparations containing various Cry proteins were reported to cause hematotoxicity in mice when administered by oral gavage Mezzomo et al.

This result could be due to spore components other than Cry proteins as the cause of the hematologic effects because they used distilled water as the negative control and not an appropriate control Bt spores lacking the Cry genes. Because the Bt spore formulation also contained many other bacterial proteins not present in the negative control water , the cause of this observed effect is confounded with other components and the results cannot be specifically attributed to Cry proteins.

Among the claimed findings in these statistical re-analyses were effects on the hematopoietic system. As noted by Bartholomaeus et al. Moreover, the results of some of these studies Velimirov et al. A recent paper reported the detection of Cry1Ab protein in the serum of non-pregnant women, pregnant women, and the cord blood of their fetuses Aris and Leblanc, The authors did not report that they had validated the assay for Cry1Ab protein levels in human serum. Given that diet data were not reported for the test subjects, and that Cry1Ab is commonly used in both conventional and organic agriculture, linking their findings to GM crop consumption is speculative.

Tiered testing with representative species to assess potential toxicity to NTOs was originally developed as a relevant and reliable process to make testing more efficient, avoid unnecessary tests for the assessment of conventional pesticides to NTOs, and enable mutual acceptance of data by regulatory authorities globally. Tiered testing is designed to first represent worst-case exposure scenarios in laboratory assays and only progress to more realistic scenarios if the lower-Tiered tests fail to indicate acceptable risk Romeis et al.

This has resulted in the development of a harmonized Tier I NTO battery that has been used in support of Bt crop registrations for approximately two decades Carstens et al. Species tested in the Tier 1 battery are selected to protect valued ecosystem services e. Following this approach could limit the collection of hazard data to those species that are relevant for the ecological risk assessment. Tier I high-dose studies can be broad in scope and allow control over experimental variables and exposure conditions that produce statistically reliable results by testing a sufficiently large number of organisms at a limit dose Romeis et al.

A limit dose is a single treatment level that is a sufficiently high exposure level i. Importantly, lack of adverse effects in limit dose testing provides sufficient confidence to address uncertainties, allowing risk assessors to conclude that there is no unacceptable risk to the environment and that no further data are required Rose, When screening studies conducted in a laboratory setting have suggested potentially unacceptable risk, additional higher-Tier studies can be designed to assess risk under more realistic exposure conditions Romeis et al.

These higher-Tier studies would only be sequentially implemented if unacceptable adverse effects were observed at the Tier I screening level. In addition to invertebrate testing, mammalian and avian studies are performed.

Introduction

Forty-two-day broiler growth studies are performed to assess chronic risk and short-term quail feeding studies are performed to support the avian assessment EPA, a. After publication of Rosi-Marshall et al. However, since the publication of Rosi-Marshall et al. Criticisms of the study included: Consequently, EPA d , stopped uniformly requiring additional aquatic invertebrate studies to assess hazard to aquatic invertebrate species.

This meta-analysis concluded that Tier I laboratory studies have been accurate and conservative in evaluating the environmental safety of Bt crops.

New methodology for evaluation of potential toxicity in microbes

These conclusions are also consistent with findings by Rauschen et al. Over the past several years, a number of meta-analyses have been published examining the safety of Bt crops to NTOs. Recently an update and review of the previous NTO meta-analyses was published by Naranjo This study focused on two environmental elements: The review concluded that hazards identified in the laboratory are commonly an artifact of the experimental laboratory procedures and do not manifest in the field. For example, apparent negative effects can be caused by Bt -intoxicated prey or hosts because of their low quality, rather than as a direct result of Bt toxicity.

Additionally, the analyses clearly demonstrated that potentially minor indirect negative effects of Bt crops demonstrated in the field are insignificant in comparison with alternative pest suppression measures based on use of traditional chemical insecticides. Taken together, this body of work demonstrates the high level of taxonomic specificity for the Bt proteins currently approved for cultivation. This assessment is conducted under diverse geographies to capture the range of environmental conditions.

Safety assessment and regulatory issues

Additionally, environmental interaction data are collected in field experiments for Bt crop and the conventional comparator to evaluate potential adverse effects. These data include measurements of disease and insect susceptibility and crop damage under natural infestation pressure. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a GM crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This aspect of the assessment addresses uncertainty regarding the potential effect the Bt crop may have on plant populations in the wild.

This concern has been considered for each of the Bt crops currently registered by the U. EPA determined in these assessments that there is no significant risk of gene capture and expression of any Bt endotoxin by wild or weedy relatives of soybean and corn in the U. However, for cotton there is a possibility for gene transfer in locations where wild or feral cotton relatives exist. Therefore, EPA requires stringent sales and distribution restrictions on Bt cotton within these geographic areas to prevent outcrossing or hybridization from the crop to sexually compatible relatives.

To improve the efficacy, pest spectrum, and durability of crops producing plant incorporated protectants PIPs , either products producing individual PIPs have been combined through conventional breeding or individual events have been produced that express more than one PIP. These products have been developed with PIPs that have been shown to act independently and do not exhibit cross-resistance EPA, , b. The focus of this section on safety assessment for combined trait PIPs and the impact of combination of PIPs on target pests is outside the scope of this review and has recently been covered in a comprehensive review Head and Greenplate, As the number of transgenic traits has increased, the number of genes potentially introduced into a single GM crop product has also increased and has led to questions whether such traits might interact.

For over a decade, combinations of different Cry proteins have been introduced into food and feed crops to expand the number of insect pests that can be controlled and to reduce the potential for the development of insect resistance to the Cry insect-control proteins Bates et al.

Thus, the means by which combined-trait products are evaluated are particularly relevant to the deployment of Bt -containing GM products. As an example, the U. EPA a , b codified their requirements to bridge existing data from a previously registered crop with an insect-control trait to a new combined-trait insect-control product. It is implicit with the approach, provided no interaction synergy is observed with the largest combination of insecticidal products e. The first requirement of the approach is confirmation that the presence and structure of the inserted material has been conserved in the combined-trait product.

The second requirement is that expression of the arthropod-active trait is comparable in the single and combined-trait product, which confirms no biologically meaningful increase in exposure to NTOs. The third requirement is demonstrating a lack of synergism between arthropod-active traits using sensitive insect bioassays. Demonstrating the lack of synergism permits the application of the principle of independent assessment, which has a long history of use in toxicology.

This principle states that if each substance in a mixture acts independently, and the substances are below their NOAELs, their toxicity can be assessed independently EPA, a , b. As applied to Bt crops, satisfying the requirements of this principle enables the use of existing safety studies performed separately for the individual products to assess the safety of the combined-trait product. EPA a has taken the position that for insecticidal traits with a record of proven safety, unless a greater than fold degree of synergism is observed, there is no need to test for human health or non-target effects.

However, in cases where Bt proteins with different, and especially novel, modes of action are being combined, the EPA b has stated that they would be justified in requiring additional testing for human health and NTOs effects for levels of synergism as low as fivefold. Cry proteins that have been shown to be additive or demonstrate potentiation of toxicity in the target insect have not been associated with similar effects in non-target mammalian and avian species Hammond et al. For example, Cry35Ab1 potentiates the activity of Cry34Ab1 against corn rootworm pests, but no evidence of toxicity was observed when the combination was tested in poultry or mice McNaughton et al.

There has been no evidence of toxicity in animal feeding studies of individual Cry proteins or mixtures of Cry proteins in Bt microbial pesticide formulations McClintock et al. As noted by Hammond et al. There was no evidence of synergistic toxicity in crops with combined stacked traits , similar to what was observed years ago when Bt microbial formulations were tested in acute, subchronic, and chronic toxicity studies. Even as new kinds of proteins e. Adoption of Bt crops has been very rapid and attests to their popularity with farmers, who are the primary beneficiaries of their value. For the first time in and continuing in , developing countries planted more hectares of GM crops than industrialized countries James, Herbicide tolerance was the most widely used trait, but insect-resistant GM crops containing Bt proteins were planted on 76 million hectares in , with most of that being maize and cotton.

It is noteworthy that Bt maize MON is grown in five EU countries Spain, Portugal, Romania, the Czech Republic, and Slovakia and in four insect-resistant Bt brinjal eggplant varieties were approved for seed production and initial commercialization in Bangladesh. The start of limited cultivation of Bt brinjal is expected in Brinjal suffers considerable insect damage and as a result the adoption of Bt brinjal in Bangladesh is expected to improve the incomes of thousands of small-holder farmers and consumers in that country, as well as to reduce exposures to chemical insecticides and pesticide poisonings in the areas that adopt the technology.

The most widely used Bt vegetable crop is sweet corn. They demonstrated that non-sprayed Bt varieties produced more clean, marketable ears than conventional isolines, even when the conventional corn was sprayed with chemical insecticides up to eight times. In the most comprehensive report that summarizes global data on GM crops from to Brookes and Barfoot, , the authors reported increased farm income, decreased pesticide use, and decreased greenhouse gasses GHG , which have been attributed to use of GM crops.

The greatest gain, as a result of reduced pesticide use, was for insect-resistant crops. These reductions in insecticide usage rates were for both developed and developing countries, but this benefit was most pronounced for Bt cotton grown in developing countries. Reduced GHG were primarily the result of the adoption of no-till practices.

Although no-till is not necessarily associated with Bt crops, use of Bt crops is expected to lead to reduced fuel consumption due to fewer passes in the field for application of pesticides. As a result of decreased input costs and increases in crop yields, farm income was improved.

Adoption of Bt cotton has greatly reduced the abundance of targeted pests in cotton and in other crops with proximity to cotton that are impacted affected by polyphagous target pests Naranjo, Additionally, reductions in insecticide use have facilitated and enabled integrated pest management IPM that, when appropriately managed can play an important part in suppression of other key and periodic pests in cotton. Furthermore, use of Bt cotton has helped to avoid or limit the use of broad-spectrum pesticides against the bollworm complex, which has reduced impacts to beneficial insects that are natural enemies to other cotton pests Naranjo, Consequently, introduction of Bt cotton has enabled the success of biologically based IPM programs and for insecticide use to be nearly eliminated in a system previously dominated by chemical insect control measures.

A major advantage of Bt insect control has been a dramatic reduction in the need for application of conventional small-molecule, chemical insecticides over Bt commodity crops, most particularly corn and cotton.

Adoption of Bt crops has resulted in a net reduction estimated at 50 million kilograms of predominantly organophosphate insecticides from to Brookes and Barfoot, ; this is a considerable advantage in areas where small-holder agriculture is the norm and application is often by hand, as indicated by reductions in pesticide poisoning events Hossain et al. In Burkina Faso in West Africa, the planting of Bt cotton has enabled farmers to reduce the number of chemical insecticide sprays during a growing season from 6 to 2 applications James, India reported pesticide use on Bt cotton has been cut at least in half.

In a survey conducted for the years —, Indian farmers reported Bt cotton use prevented at least 2. Further, there will be continued reductions in the large quantities of soil insecticides that were historically used for control of corn rootworms with the continued adoption and improved efficacy as coleopteran-resistant Bt maize adoption grows in the future.

Another benefit of Bt insect control is the potential for reduced levels of mycotoxin contamination. In general, insect-damaged grain and other plant tissues are more susceptible to infection by fungi than intact tissues. Some of the fungi present in maize-growing areas and able to colonize maize include mycotoxin-producing fungi such as Fusaria Hammond et al. Fusarium species produce the mycotoxin fumonisin, which is known to cause illness in farm animals and possibly in humans Li et al.

Several studies conducted in diverse world areas, and under a wide range of environmental conditions Hammond et al. Reduced fumonisin contamination in Bt maize cannot be guaranteed, but a reduction in fumonisin contamination has generally been observed. This benefit is presumed to be a result of the decrease in insect damage, leading to fewer ports of entry for fungal infection Hammond et al.

Cry Bt proteins, whether in microbial pesticide products or expressed in Bt crops, have been used and consumed safely for decades. The levels of Cry Bt protein in GM crops are very low and are often reduced further by food processing. In addition, extensive testing of Bt proteins, single- Bt trait crops, and stacked trait crops containing Bt proteins has not revealed any harm to non-target insects and other non-target species, including humans.

This environmental safety profile for Bt crops largely reflects the high level of taxonomic specificity that has been achieved with Bt crops currently approved for cultivation. Use of Bt crops provides benefits beyond insect control, such as significantly reducing small-molecule insecticide use for target pests controlled by Bt proteins, reducing applicator exposure to small-molecule insecticides, reducing greenhouse gasses emissions by minimizing field spraying with self-propelled sprayers or other motorized equipment, and by potentially reducing fumonisin levels in maize grain.

Close Find out more. Main Description Microorganisms are widely used in various beneficial applications, including food, pest control, bioremediation, biodegradation, biofuel processes, and plant symbiosis and growth stimulation. Microbes and the law - Safety assessment and regulation of beneficial microorganisms Part I: Food and feed 2: Safety and regulation of microorganisms added to the food and feed chains, including probiotics - Introduction and overview 3: Microbes for human and animal consumption 4: Antibiotic resistance in relation to starter cultures and probiotics 5: Biopreservation of food and feed by postharvest biocontrol with microorganisms Part II: Pest control agents and plant growth promoters 6: Safety and regulation of microbial pest control agents and microbial plant growth promoters - Introduction and overview 7: Microbial control of invertebrate pests 8: Microbial control of plant diseases 9: Safety and regulation of microbial control of weeds Plant growth promotion with microorganisms Part III: Other industrial applications Determining the safety of microorganisms - Introduction and overview Virulence genes in risk assessment of beneficial microorganisms: Occupational safety of microbial agents Part V: Model test systems Model systems for testing microbial pathogenicity, virulence and toxicity - Introduction and overview Nematode and insect models to assay microbial infectivity, virulence and cytotoxicity Assessing potential cytotoxicity of biocontrol microorganisms using invertebrate assays Assessing genotoxic effects of microbial products Assessing the sensitization and irritation properties of microorganisms Part VI:

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