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Biotechnology Consultation
Note to the File
BNF No. 000097
Date: January 5, 2005
Subject: Bacillus thuringiensis Cry3Bb1 corn line MON 88017
Keywords: maize, corn, Zea mays L., Coleopteran-specific insecticidal protein, Cry3Bb1 protein, 5-enolpyruvylshikimate-3-phosphate synthase protein (EPSPS), Agrobacterium, Bacillus thuringiensis (subspecies kumamotoensis), Diabrotica spp., corn rootworm, MON 88017, glyphosate-tolerant, herbicide-tolerant, Roundup™ Ready
In a submission dated March 30, 2004, the Monsanto Company (Monsanto) provided to the Food and Drug Administration (FDA) a summary of the safety and nutritional assessment they conducted on bioengineered corn rootworm-resistant, glyphosate-tolerant corn line MON 88017. Monsanto submitted additional information on May 18, June 22, and October 18, 2004. Monsanto concluded that food and feed products derived from MON 88017 are as safe and nutritious as those derived from conventional corn varieties currently being marketed.
Monsanto introduced a variant of the cry3Bb1 gene isolated from Bacillus thuringiensis (B.t.) subspecies kumamotoensis strain EG4691 and the cp4 epsps gene isolated from Agrobacterium sp. strain CP4 into corn in order to confer resistance to corn rootworm as well as tolerance to the herbicide glyphosate. The cry3Bb1 gene encodes the Cry3Bb1 protein which confers resistance to corn rootworm. The cp4 epsps gene encodes the CP4 5-enolpyruvylshikimate-3-phosphate synthase protein (CP4 EPSPS) which confers tolerance to glyphosate and is used as a selectable marker.
The Environmental Protection Agency (EPA) regulates plant-incorporated protectants under the Federal Food, Drug, and Cosmetic Act and the Federal Insecticide, Fungicide, and Rodenticide Act. Under EPA regulations, the Cry3Bb1 protein in corn line MON 88017 is considered a pesticidal substance and the CP4 EPSPS protein is considered an inert ingredient. Therefore, the safety assessment of these proteins falls under the regulatory purview of EPA.
Monsanto created corn line MON 88017 using the disarmed binary Agrobacterium tumefaciens transformation vector PV-ZMIR39 and callus material from corn line LH198. PV-ZMIR39 carries the transgenes for insertion into the plant genome between consensus T-DNA border sequences. The T-DNA region of PV-ZMIR39 contains the elements listed in Table 1.
| Genetic Element | Description |
|---|---|
| Left border | Left border sequence from octopine Ti plasmid pTi15955, essential for transfer of T-DNA |
| p-ract1 | Rice actin gene promoter to drive cp4 epsps expression |
| ract1 intron | Rice actin gene first intron, to enhance cp4 epsps expression |
| CTP2 | Chloroplast transit peptide from Arabidopsis thaliana, targets protein expression to chloroplast |
| cp4 epsps | Coding sequence for the CP4 EPSPS protein from Agrobacterium strain CP4 |
| NOS 3' | 3' untranslated region of the nopaline synthase (NOS) coding sequence, terminates transcription and directs polyadenylation |
| p-e35S | Promoter with duplicated enhancer region from cauliflower mosaic virus |
| wt CAB leader | 5' untranslated leader from the wheat chlorophyll a/b-binding protein |
| ract1 intron | Rice actin gene first intron, to enhance cry3Bb1 expression |
| cry3Bb1 | Coding sequence for a synthetic variant of the Cry3Bb1 protein from Bacillus thuringiensis subsp. kumamotoensis |
| tahsp17 3' | 3' untranslated region of wheat heat shock protein 17.3, terminates transcription and directs polyadenylation |
| Right border | Right border sequence from the nopaline Ti plasmid pTiT37, essential for transfer of T-DNA |
The plasmid backbone of PV-ZMIR39 contains a bacterial selectable marker gene (aad) that confers resistance to spectinomycin/streptomycin antibiotics and is used to facilitate cloning and maintenance of the transformation vector in bacterial hosts. The plasmid backbone of PV-ZMIR39 is not expected to be integrated into MON 88017.
Monsanto characterized the integration and number of insertions in MON 88017, using restriction enzyme digestion and Southern blot analysis of MON 88017 genomic DNA. The results indicate integration of a single, intact copy of the T-DNA sequence, including both the cp4 epsps cassette and cry3Bb1 cassette. Monsanto reports that no detectable backbone sequences from the PV-ZMIR39 vector have been integrated into MON 88017.
Monsanto assessed the genetic stability of the cry3Bb1 and cp4 epsps genes across 10 generations using Southern blot and segregation analysis. Monsanto states that the integration is stable and that the segregation of transgenes in MON 88017 shows a Mendelian inheritance pattern.
Corn, Zea mays L., is grown worldwide as a source of food and feed. Corn grain and its processed fractions, an important source of starch and sugar, are consumed in a multitude of food and animal feed products. Corn forage is extensively consumed as an animal feed by ruminants.
Monsanto evaluated the composition of forage and grain from MON 88017 relative to a non-engineered corn line of similar genetic background (conventional control) as well as to twelve commercially available conventional corn hybrid lines.
Compositional analyses were performed on forage and grain. Monsanto analyzed corn grown at three field sites, with three replicates of MON 88017, the conventional control, and four different commercially available conventional hybrids at each site (a total of twelve commercially available conventional hybrid lines) in a randomized complete block design. The MON 88017 plots received an application of glyphosate herbicide according to label directions. Statistical analyses of the compositional data were conducted using a mixed model analysis of variance method. The composition of forage and grain produced by MON 88017 was compared to the conventional control corn with similar genetic background. Four sets of analyses were made based on data from each of the three replicated field sites as well as from a combination of all three field sites. Statistically significant differences were declared at the 5% significance level. Using the data for each component obtained from the twelve different commercial hybrid lines, a "99% tolerance interval" was calculated to contain, with 95% confidence, 99% of the values contained in the population of commercial corn hybrids.
Forage was harvested at the late dough/early dent stages (R4 - R6 growth stage). Monsanto determined the levels of 9 components of corn forage:
No statistically significant differences were found between forage from MON 88017 and forage from the conventional control.
Bulk grain samples were harvested when the grain was mature (R6 growth stage). Monsanto determined the levels of 68 components of corn grain and subsequently conducted a statistical analysis on the following 53 components:1
Monsanto conducted a combined statistical analysis of data from all of the field trials and found no statistically significant differences in the levels of all measured components between the bioengineered MON 88017 line and the conventional control line, except for vitamin B1 and for linolenic and arachidic acid when expressed as a percentage of total fatty acids.2 Linolenic acid was more abundant in MON 88017 relative to the conventional control line, while arachidic acid and vitamin B1 were reduced in MON 88017 relative to the conventional control line. However, the differences in linolenic acid and arachidic acid were not observed when the data were expressed on a dry matter basis. All the reported values were within literature ranges, and within Monsanto's calculated 99% tolerance interval.3
Monsanto has concluded that corn line MON 88017 is not materially different in composition, safety, or any relevant parameter from corn now grown, marketed and consumed. At this time, based on Monsanto's data and information, the agency considers Monsanto's consultation on corn line MON 88017 to be complete.
/s/
Jeremiah Fasano, Ph.D.
(1)The following 15 analytes had greater than 50% of observations below the limit of quantitation of the assay and were excluded from statistical analysis: sodium, caprylic acid (8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), myristoleic acid (14:1), pentadecanoic acid (15:0), pentadecenoic acid (15:1), heptadecanoic acid (17:0), heptadecenoic acid (17:1), gamma-linolenic acid (18:3), eicosadienoic acid (20:2), eicosatrienoic acid (20:3), archidonic acid (20:4), and 2-furaldehyde.
(2) Monsanto also found statistically significant differences in some nutrient levels at specific locations, but except for the nutrients already cited, these differences were not consistent across all locations.
(3) While vitamin B1 analytical values were outside the literature range cited by Monsanto, these values were within the ranges found in the International Life Sciences Institute Crop Composition Database (version 2.0, released April 5, 2004, available at: www.cropcomposition.org) as well as the Organisation for Economic Co-operation and Development document "Consensus Document on Compositional Considerations for New Varieties of Maize (Zea Mays): Key Food and Feed Nutrients, Anti-Nutrients and Secondary Plant Metabolites." (Series on the Safety of Novel Foods and Feeds, No. 6, 2002).
