Decision Document DD2009-78 - Determination of the Safety of Pioneer Hi-Bred Production Ltd.'s Corn (Zea mays subsp. mays) Event 98140
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This Decision Document has been prepared to explain the regulatory decision reached under Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits", its companion biology document BIO1994-11, "The Biology of Zea mays L. (Corn/Maize)", and Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".
The Canadian Food Inspection Agency (CFIA) has evaluated information submitted by Pioneer Hi-Bred Production Ltd. This information is in regard to the herbicide resistant corn event 98140. The CFIA has determined that this plant with novel traits (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to currently commercialized corn varieties in Canada.
Taking into account these evaluations, unconfined release into the environment and use as livestock feed of corn event 98140 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of August 26, 2009. Any corn lines derived from corn event 98140 may also be released into the environment and used as livestock feed, provided that (i) no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) it is known based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn in Canada, in terms of their potential environmental impact and livestock feed safety, and (iv) the novel genes are expressed at a level similar to that of the authorized line.
Corn event 98140 is subject to the same phytosanitary import requirements as its unmodified counterpart.
Please note, that the livestock feed and environmental safety assessments of novel feeds and PNTs are critical steps in the potential commercialization of these plant types. Other requirements, such as the evaluation of food safety by Health Canada, have been addressed separately from this review.
Table of Contents
- Brief Identification of the Modified Plant
- Background Information
- Description of the Novel Trait
- Criteria for the Environmental Assessment
- Potential of Corn Event 98140 to Become a Weed of Agriculture or be Invasive of Natural Habitats
- Potential for Gene Flow from Corn Event 98140 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
- Altered Plant Pest Potential of Corn Event 98140
- Potential Impact of Corn Event 98140 on Non-Target Organisms
- Potential Impact of Corn Event 98140 on Biodiversity
- Criteria for the Livestock Feed Assessment
- New Information Requirements
- Regulatory Decision
I. Brief Identification of the Modified Plant
|Designation of the Modified Plant:||Corn Event 98140, OECD Unique Identifier DP-09814Ø-6|
|Applicant:||Pioneer Hi-Bred Production Ltd.|
|Plant Species:||Corn (Zea mays subsp. mays)|
|Novel Traits:||Tolerance to glyphosate herbicide and tolerance to ALS-inhibiting herbicides|
|Trait Introduction Method:||Agrobacterium tumefaciens mediated transformation|
|Proposed Use of the Modified Plant:||Commercial production of corn grain for human consumption and for livestock feed. These plants are not intended to be grown outside the normal cultivation area for corn in Canada|
II. Background Information
Corn event 98140 was developed by Pioneer Hi-Bred Production Ltd. using recombinant DNA technology, resulting in the introduction of the gat4621 and the zm-hra genes. The gat4621 gene encodes a glyphosate N-acetyltransferase (GAT) enzyme derived from Bacillus licheniformis enzymes, which metabolises the herbicide glyphosate, conferring resistance to the plant. The zm-hra gene encodes a modified ALS enzyme that confers resistance to ALS-inhibiting herbicides.
Pioneer Hi-Bred Production Ltd. has provided data on the identity of corn event 98140, a detailed description of the transformation method, data and information on the gene insertion site, gene copy number and levels of gene expression in the plant and the role of the inserted genes and regulatory sequences. The novel proteins were identified and characterized. Data was provided for the evaluation of the potential toxicity of the novel proteins to livestock and non-target organisms and potential allergenicity of the novel proteins to humans and to livestock.
Corn event 98140 was field tested in the United States and Canada and the data for trial year 2006 were submitted. The Canadian locations, Thorndale, ON and Branchton, ON were representative of corn growing regions in Canada.
Agronomic characteristics of corn event 98140 such as seed dormancy, vegetative vigour, time to maturity, silking period, susceptibilities to various corn pests and pathogens, and seed production were compared to those of unmodified corn counterparts.
Nutritional components of corn event 98140, such as proximates, amino acids and fatty acids were compared with those of unmodified corn counterparts.
The Plant Biosafety Office of the Plant Health and Biosecurity Directorate, in conjunction with the Plant and Biotechnology Risk Assessment Unit (PBRA) of the Science Strategies Division, CFIA, have reviewed the above information, in light of the assessment criteria for determining environmental safety of PNTs, as described in the Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants With Novel Traits". The Plant and Biotechnology Risk Assessment Unit has considered the following:
- the potential of corn event 98140 to become a weed of agriculture or be invasive of natural habitats;
- the potential for gene flow from corn event 98140 to wild relatives whose hybrid offspring may become more weedy or more invasive;
- the potential for corn event 98140 to become a plant pest;
- the potential impact of corn event 98140 or its gene products on non-target species, including humans; and
- the potential impact of corn event 98140 on biodiversity.
The Animal Feed Division of the Animal Health Directorate has also reviewed the above information with respect to the assessment criteria for determining the safety and efficacy of livestock feed, as described in Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources". The Animal Feed Division has considered the following:
- the potential impact of corn event 98140 on livestock nutrition; and
- the potential impact of corn event 98140 on livestock and workers/bystanders
Pioneer Hi-Bred Production Ltd. has provided the CFIA with a method for the detection and identification of corn containing the corn event 98140.
III. Description of the Novel Traits
1. Tolerance to Glyphosate and ALS-Inhibiting Herbicides
Glyphosate blocks the shikimic acid pathway required for biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan in plants, leading to growth suppression or death of the plants. The gat4621 gene encodes an enzyme derived from three B. licheniformis N-acetyltransferase enzymes which inactivates glyphosate by N-acetylation. The sequence of this enzyme is 75 to 78% homologous to the sequences that were used to create it. The gat4621 gene introduced into corn during the transformation process to develop corn event 98140 imparts field level tolerance to glyphosate.
Acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS), is a key enzyme in the synthesis of the essential branched-chain amino acids isoleucine, leucine, and valine. ALS-inhibiting herbicides disrupt synthesis of isoleucine, leucine, and valine in plants, leading to growth suppression or death of the plant. The zm-hra gene encodes a corn ALS enzyme that contains two specific amino acid differences from the wild type enzyme, rendering it resistant to all classes of ALS-inhibiting herbicides. The zm-hra gene, which was introduced into corn during the transformation process to develop corn event 98140, imparts field level tolerance to ALS-inhibiting herbicides.
Both GAT4621 expression and ZM-HRA expression in corn event 98140 are driven by constitutive promoters. Samples of tissues were collected from corn plants at various growth stages from six field trial sites across Canada and the United States. Average GAT4621 and ZM-HRA protein expression across all plant stages was evaluated by enzyme-linked immunosorbent assay. The levels of GAT4621, expressed in micro-grams protein per gram dry weight tissue (µg/g dwt), observed were as follows: 4.3 to 44 µg/g dwt in leaves, 2.6 to 13 µg/g dwt in roots, 3.5 to 28 µg/g dwt in whole plants, 7.9 µg/g dwt in grain, 13 µg/g dwt in pollen, 28 µg/g dwt in stalks, and 16 µg/g dwt in forage. The levels of ZM-HRA observed were as follows: 0.05 to 6.7 µg/g dwt in leaves, 0.03 to 0.55 µg/g dwt in roots, 0.23 to 4.0 µg/g dwt in whole plants, 0.34 µg/g dwt in grain, undetectable µg/g dwt in pollen, 1.4 µg/g dwt in stalks, and 2.7 µg/g dwt in forage.
The GAT4621 and ZM-HRA proteins were purified from the grain of corn event 98140 and characterized. The identities of the purified proteins were confirmed by Western immunoblot analysis, tryptic peptide mass mapping, and N-terminal characterization.
The concentrations of GAT4621 and ZM-HRA proteins in corn event 98140 tissues were too low to enable extraction of sufficient amounts for evaluation of environmental and feed safety. To obtain sufficient quantities of the proteins for safety studies, it was necessary to express the gat4621 and zm-hra genes in an E. coli production system. The equivalency of the plant-produced GAT4621 and ZM-HRA proteins to the E. coli - produced proteins was evaluated by comparing their molecular weights, immunological reactivity, tryptic peptide mass map and glycosylation status. Based on the results, the GAT4621 and ZM-HRA proteins produced in corn event 98140 were found to be equivalent to their E. coli -produced counterparts.
The potential mammalian toxicity and allergenicity of the GAT4621 and ZM-HRA proteins was evaluated. These proteins lack sequence similarity to known allergens and protein toxins which have adverse effects to mammals. No adverse effects were observed when the GAT4621 or ZM-HRA proteins were ingested by mice at a dose of approximately 1640 mg/kg body weight and 1236 mg/kg body weight, respectively in acute oral toxicity studies. In vitro digestive fate studies have shown that GAT4621 and ZM-HRA are rapidly degraded in simulated gastric fluid, and heat stability studies have shown that these proteins are heat-labile, unlike protein allergens which are often resistant to digestion with digestive enzymes and heat denaturation. The GAT4621 and ZM-HRA proteins expressed in corn event 98140 are not glycosylated, unlike many known allergens, providing additional evidence that these proteins do not have the properties of known allergens.
2. Development Method
Corn event 98140 was developed through A. tumefaciens-mediated transformation of immature corn embryos with T-DNA plasmid containing both gat4621 and zm-hra genes and their regulatory elements. Corn event 98140 was selected on the basis of tolerance to glyphosate, and the presence of gat4621 and zm-hra genes in the transformant was confirmed by PCR.
3. Stable Integration into the Plant Genome
Molecular characterization by Southern blot analysis demonstrated that corn event 98140 contains one copy of gat4621 and zm-hra gene cassettes inserted at a single site in the corn genome. No additional elements, including intact or partial DNA fragments of the gat4621 and zm-hra gene cassettes or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in corn event 98140.
The stability of the inserted DNA was demonstrated by Southern blot analysis across four generations in the breeding history of corn event 98140. Analysis of the inheritance pattern of the gat4621 and zm-hra genes and the GAT4621 and ZM-HRA proteins across four generations of corn event 98140 confirmed the stability of the inserted DNA. The results of the analysis are consistent with the finding of a single site of insertion that segregates according to the Mendelian laws of genetics.
IV. Criteria for the Environmental Assessment
1. Potential of Corn Event 98140 to Become a Weed of Agriculture or be Invasive of Natural Habitats
The biology of corn (Zea mays) as described in biology document BIO1994-11, "The Biology of Zea mays L. (Corn/Maize)", is such that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Corn does not possess the potential to become weedy due to traits such as lack of seed dormancy, the non-shattering aspect of corn cobs, and poor competitive ability of seedlings. According to the information provided by Pioneer Hi-Bred Production Ltd., corn event 98140 was determined not to be significantly different from their conventional counterparts in this respect.
The PBRA evaluated data submitted by Pioneer Hi-Bred Production Ltd. on the reproductive biology and life history traits of corn hybrids derived from corn event 98140, and determined that early stand establishment, seedling vigor, plant height, ear height, stalk lodging, root lodging, stay green, time to silking, time to pollen shed, yield, pollen viability, insect damage and disease incidence were within the normal range of expression of these traits currently displayed by commercial corn hybrids.
No competitive advantage was conferred to these plants, other than that conferred by tolerance to glyphosate and to ALS-inhibiting herbicides. Tolerance to glyphosate and to ALS-inhibiting herbicides will not, in itself, render corn weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified.
The above considerations, together with the fact that the novel traits have no intended effects on corn weediness or invasiveness, led the PBRA to conclude that the corn event 98140 has no altered weed or invasiveness potential compared to currently commercialized corn.
A longer term consideration is the potential development of crop volunteers with a combination of herbicide tolerances. Similarly, the use of several crop species in rotation which all rely on tolerance to the same herbicide can lead to the development of significant selection pressure and the potential development of herbicide resistant weeds. This requires the management of this technology as a part of an integrated approach which may include currently available weed control products with alternate modes of action.
Pioneer Hi-Bred Production Ltd. has submitted a herbicide tolerance stewardship plan to the CFIA which was evaluated by PBRA. The stewardship plan contains recommendations to address these concerns, as well as appropriate strategies that will allow for the environmentally safe and sustainable deployment of these traits. In addition, the stewardship plan contains strategies for communication to growers and an efficient mechanism allowing growers to report problems to Pioneer Hi-Bred Production Ltd. Pioneer Hi-Bred Production Ltd. will make this stewardship plan readily available to growers to promote careful management practices for corn event 98140.
2. Potential for Gene Flow From Corn Event 98140 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
Biology document BIO1994-11, "The Biology of Zea mays L. (Corn/Maize)", indicates that there are no wild relatives in Canada that can hybridize with corn.
The PBRA therefore concludes that gene flow from the corn event 98140 to corn relatives is not a concern in Canada.
3. Altered Plant Pest Potential of Corn Event 98140
The intended effect of the novel trait is unrelated to plant pest potential, and corn is not a plant pest in Canada (BIO1994-11). In addition, agronomic characteristics of the modified corn hybrids were shown to be within the range of values displayed by current commercialized corn hybrids, and indicate that the growth habit of corn was not inadvertently altered in such a way as to impact on the plant pest potential of corn. Despite tolerance to glyphosate and ALS-inhibiting herbicides, corn event 98140 volunteers can still be managed by growers using alternative herbicides with different modes of action, or cultivation practices which do not involve the use of these herbicides. Field observations did not indicate modifications of disease or pest susceptibilities.
The PBRA therefore concludes that the corn event 98140 line does not display any altered pest potential in comparison to other commercially available corn lines.
4. Potential Impact of Corn Event 98140 on Non-Target Organisms
The detailed characterizations of the novel genes and resulting enzymes (as briefly summarized in Part III: Description of the Novel Traits) has led to the conclusion that the the novel proteins are not toxic or allergenic and do not result in altered toxic or allergenic properties of corn. The reproductive biology and life history traits of corn event 98140 are not altered as a result of the expression of these two traits and the interaction of corn event 98140 with other organisms is therefore not expected to be altered either.
The PBRA has therefore determined that the unconfined release of corn event 98140 will not result in altered impacts on non-target organisms, including humans, compared to currently available Canadian corn lines.
5. Potential Impact of Corn Event 98140 on Biodiversity
Corn event 98140 expresses no novel phenotypic characteristics which could extend its use beyond the current geographic range of corn production in Canada. Since corn does not out cross to wild relatives in Canada, there will be no transfer of novel traits to species in unmanaged environments. In addition, the novel traits are unlikely to have an impact on plant pest potential or non- target organisms. It is therefore unlikely that corn event 98140 will have any direct effects on biodiversity, in comparison to the effects that would be expected from the cultivation of currently grown corn hybrids.
Corn event 98140 has tolerance to broad spectrum herbicides such as glyphosate and the ALS inhibiting herbicides. The use of these herbicides in cropping systems has the intended effect of reducing local weed populations within agroecosystems. This may result in a reduction in local weed species biodiversity, and may have effects on other trophic levels which utilize these weed species. It must be noted however that the goal of reduction in weed biodiversity in agricultural fields is not unique to the use of PNTs, corn event 98140, or the cultivation of corn. It is therefore unlikely that corn event 98140 will have any indirect effects on biodiversity, in comparison to the effects that would be expected from the cultivation of currently grown corn hybrids.
The CFIA has therefore concluded that the potential impact on biodiversity of corn event 98140 is unlikely to be different from that of currently commercialized corn lines.
V. Criteria for the Livestock Feed Assessment
1. Potential Impact of corn event 98140 on Livestock Nutrition
The compositional equivalence of corn event 98140 to its non transgenic, isoline control was determined from replicated trials at four sites in the US and two sites in Canada. Forage and grain samples were collected at each site and analysed for proximate, acid detergent fibre (ADF), neutral detergent fibre (NDF), minerals and amino acids (total, free and acetylated). Grain samples were also analysed for fatty acids, vitamins and secondary metabolites. There were no statistically significant differences between corn event 98140 and the control for protein, fat, ash, crude fibre, ADF, NDF, vitamins, minerals, total and free amino acids in grain and forage samples. All means were within the tolerance interval of the reference commercial varieties and literature ranges. There were no statistically significant differences between transgenic and isoline control grain for fatty acids, inositol, furfural, p-coumaric acid and ferulic acid. All means were within the tolerance interval and/or range of literature values.
The levels of N-acetylglutamate and N-acetylaspartate were statistically significantly elevated in corn event 98140 grain and forage compared to those of the control and were outside the tolerance interval and/or literature values. Additional data also showed statistically significant elevated levels of N-acetylglycine, N-acetylthreonine and N-acetylserine to a minor extent in corn event 98140 grain when compared to that of control corn. The concentration of N-acetylserine was within the statistical tolerance interval of the non-transgenic corn, however the mean values for N-acetylglycine and N-acetylthreonine were outside the tolerance interval. The applicant provided evidence to show that N-acetylated amino acids substitute for the constituent amino acids via metabolic deacetylation. Furthermore, due to the rapid rate of deacetylation of N-acetylglutamate and N-acetylaspartate into free glutamate and aspartate during metabolism as well as the low animal exposure, the increased concentration of N-acetylglutamate and N-acetylaspartate in corn event 98140 poses no risk to livestock. There were also no negative effects of elevated levels of N-acetylglutamate and N-acetylaspartate observed on the performance or health of birds consuming corn event 98140 diets.
Phytic acid, trypsin inhibitor and raffinose were analysed in corn event 98140 grain and compared to the isoline control. No statistically significant differences were observed between corn event 98140 and control and for raffinose, trypsin inhibitor and phytic acid. All means were within the tolerance interval and literature ranges.
To assess the wholesomeness of corn event 98140 for use in animal feed, a 42-day broiler study was conducted. Corn event 98140 corn diets were evaluated by comparing the growth, organ and carcass yield of broiler chickens fed corn event 98140 to those from isoline control (091) and three commercial reference varieties. There were 10 broilers per pen (50% male and 50% female) with 12 pens per treatment in a randomized block design. 120 Ross x Cobb broilers were fed in 3 phases, starter (0-21d), grower (22-35d) and finisher (36-42d) with the corn grain at 58.5%, 64% and 71.5% respectively. Body weight, feed intake, mortality, carcass and organ yield were recorded. No statistically significant differences were observed in mortality, weight gain, feed efficiency, organ and carcass yield variables between broiler consuming diets containing corn event 98140 and the control corn. All means were within the tolerance interval calculated for the study.
The evidence provided by Pioneer Hi-Bred Production Ltd. supports the conclusion that the nutritional composition of corn event 98140 is substantially equivalent to conventional corn varieties with its associated elevated levels of N-acetylglutamate, N-acetylaspartate, N-acetylglycine and N-acetylthreonine. No detrimental effects from these analytes were seen on the health, growth and carcass performance of broilers consuming diets containing corn event 98140.
2. Potential Impact of Corn Event 98140 on Livestock and Workers/Bystanders
The zm-hra gene encodes a modified ALS enzyme that was altered by site-directed mutagenesis to contain two amino acid differences from the wild type ALS enzyme. ALS enzymes are found in a wide variety of plants and micro-organisms. ALS enzymes are not known toxins or allergens and the two amino changes are not expected to change this. The modified ALS enzyme did not have homology with any known allergens or toxins. It was also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. This information suggests that the modified ALS is unlikely to be a novel toxin or allergen.
The GAT4621 protein was derived from three B. licheniformis N-acetyltransferase enzymes, and is 75-78% homologous to these sequences. B. licheniformis is not pathogenic and has been used as a source of industrial enzymes for many years without reports of adverse effects to human health or the environment. The GAT4621 enzyme did not have homology with any known allergens or toxins. It was also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. This information suggests that GAT4621 is unlikely to be a novel toxin or allergen
No adverse effects from the ZM-HRA and GAT4621 proteins were observed in acute oral toxicity studies in mice using approximately 3000 times and 340 times, respectively, the highest predicted livestock dose level per kg body weight. No adverse effects on nutrition or health were observed in a broiler feeding trial comparing corn event 98140 grain to several conventional varieties of corn grain in the diet.
An assessment was also completed on N-acetyl glyphosate and other minor metabolites formed due to the activity of the GAT4621 protein on the pesticide glyphosate. N-acetyl glyphosate was found in all of the tissues of the corn event 98140 that are consumed by livestock (stover, forage, and grain) following application of glyphosate. No adverse effects from N-acetyl glyphosate were observed in a 90-day oral toxicity study in rats using approximately 4800 times the highest predicted livestock dose level per kg body weight. N-acetyl glyphosate and other minor metabolites do not show any evidence of significant toxicological effects in the data considered. N-acetyl glyphosate and other minor metabolites are rapidly excreted and no tissue residues of concern are expected in livestock or products from food animals.
The evidence provided by Pioneer Hi-Bred Production Ltd. indicates there is no potential impact of corn event 98140 on livestock and workers/by-standers when compared to commercialized corn lines.
VI. New Information Requirements
If at any time, Pioneer Hi-Bred Production Ltd. becomes aware of any information regarding risk to the environment, including risk to human or animal health, which could result from release of soybean event 356043 materials in Canada or elsewhere, Pioneer Hi-Bred Production Ltd. will immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of soybean event 356043 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use and environmental release authorizations of soybean event 356043.
VII. Regulatory Decision
Based on the review of the data and information submitted by Pioneer Hi-Bred Production Ltd., and through comparisons of corn event 98140 with unmodified corn counterparts, the Plant and Biotechnology Risk Assessment Unit of the Science Strategies Division, CFIA, has concluded that corn event 98140 is substantially equivalent to commercially cultivated corn lines. The novel genes and their corresponding traits in corn event 98140 do not alter or confer any characteristic that would result in unintended environmental effects following unconfined release. Pioneer Hi-Bred Production Ltd. has provided a stewardship plan to address any concerns associated with the use of glyphosate or ALS-inhibiting herbicides in corn event 98140 production.
Based on the review of submitted data and information by Pioneer Hi-Bred Production Ltd., including comparisons of corn event 98140 with its unmodified corn counterparts, the Animal Feed Division has concluded that the introduced genes and their corresponding traits will not confer to corn event 98140 any characteristic that would raise any concerns regarding the safety or nutritional composition of corn event 98140. Grain corn, its byproducts and corn oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. Corn event 98140 has been assessed and found to be as safe as and as nutritious as traditional corn varieties. Corn event 98140 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.
Unconfined release into the environment and use as livestock feed of corn event 98140 is therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of August 26, 2009. Any corn lines derived from corn event 98140 may also be released into the environment and used as livestock feed, provided that no inter-specific crosses are performed, the intended uses are similar, it is known based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn varieties in Canada, in terms of their potential environmental impact and livestock feed safety, the novel genes are expressed at levels similar to that of the authorized line.
Corn event 98140 is subject to the same phytosanitary import requirements as its unmodified counterpart.
Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of corn event 98140. The Food safety decisions are available on the Health Canada web site.
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