Biotechnology Consultation Note to the File BNF No. 000138
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Biotechnology Consultation - Note to the File
Biotechnology Notification File BNF No. 000138
December 26, 2013
Altered lignin production (through reduction in guaiacyl (G) lignin), event KK179 alfalfa
Alfalfa; Medicago sativa L.; reduction in guaiacyl lignin monomeric subunit; ribonucleic acid (RNA) interference; suppression of endogenous S-adenosyl-L-methionine: trans-caffeoyl CoA 3-O-methyltransferase (CCOMT); KK179 alfalfa; OECD Unique Identifier number MON-ØØ179-5; Monsanto Company and Forage Genetics International
This document summarizes our evaluation of Biotechnology Notification File (BNF) No. 000138. In a submission dated August 24, 2012, Monsanto Company (Monsanto) in collaboration with Forage Genetics International, LLC (FGI) submitted a safety and nutritional assessment of genetically engineered (GE) alfalfa event KK179 (referred to as KK179 alfalfa in this document). Monsanto and FGI provided additional information on March 1, 2013. FDA evaluated the information in Monsanto and FGI's submissions to ensure that regulatory and safety issues regarding human food and animal feed derived from the new plant variety have been resolved prior to commercial distribution. In our evaluation of BNF No. 000138, we considered all of the information provided by Monsanto and FGI as well as publicly available information and information in the agency's files. Here we discuss the outcome of the consultation, but do not intend to restate the information provided in the final consultation in its entirety.
According to Monsanto and FGI, the intended effect of the genetic modification in KK179 alfalfa is to reduce the content of guaiacyl (G) lignin monomeric subunits and total lignin in forage. To accomplish this objective, Monsanto and FGI introduced an inverted repeat DNA sequence, which results in the production of double-stranded RNA (dsRNA) that suppresses endogenous S-adenosyl-L-methionine: trans-caffeoyl CoA 3-O-methyltransferase (CCOMT gene) RNA transcript levels via the RNA interference (RNAi) pathway. CCOMT methylates caffeoyl coenzyme A to form feruloyl coenzyme A, which are precursors of the G and syringyl (S) lignin monomeric subunits. Monsanto and FGI state that reduced levels of CCOMT protein lead to decreased synthesis of G lignin monomeric subunits. However, S lignin monomeric subunits continue to be synthesized via an alternative pathway. Thus, the effect of CCOMT suppression is limited to reduced G lignin synthesis, resulting in an increased S lignin: G lignin ratio.
The purposes of this evaluation are (1) to assess whether the developers have introduced a substance requiring premarket approval as a food additive into food or feed and (2) to determine whether use of the new plant variety in food or feed raises other regulatory issues under the Federal Food, Drug, and Cosmetic Act (FD&C Act).
Genetic Modification and Characterization
Monsanto and FGI state that the recipient alfalfa plant used for transformation was R2336. R2336 is a conventional FGI proprietary plant (propagated vegetatively via stem cuttings).
Transformation Plasmid and Methods
Monsanto and FGI described the transformation plasmid, PV-MSPQ12633. This plasmid contains two transfer DNA (T-DNA) regions, each delineated by left and right border sequences.
The first T-DNA cassette (CCOMT cassette) contains:
- the Pal21 promoter region from the gene for phenylalanine ammonia-lyase 2 from Phaseolus vulgaris (common bean);
- a partial segment (~0.8 kb) of the CCOMT gene from Medicago sativa configured into an inverted repeat sequence; and
- the 3´untranslated region (UTR) of the nopaline synthase (nos) gene from Agrobacterium tumefaciens pTi, which directs polyadenylation of the RNA transcripts.
The second T-DNA cassette (nptII cassette) contains:
- the promoter and leader sequences from the 35S RNA of cauliflower mosaic virus
- the aminoglycoside 3´-phosphotransferase II (nptII) coding sequence from transposon Tn5 of Escherichia coli; and
- the 3´ UTR of the nos gene from A. tumefaciens pTi, which directs polyadenylation of the RNA transcripts.
Transformed plants were generated using Agrobacterium-mediated transformation of R2336 leaf tissue explants with PV-MSPQ12633. Explants were grown on medium containing kanamycin and timentin, which inhibit the growth of untransformed plant cells and excess Agrobacterium, respectively. The kanamycin-resistant calli that developed into somatic embryos were placed in media conducive to shoot and root development. Monsanto and FGI removed the second T-DNA cassette containing the nptII gene from the transformed plants through conventional breeding and meiotic segregation. A combination of analytical techniques was used to identify a subset of transformed plants that contained the CCOMT cassette but lacked the nptII cassette. A single, transformed R2336 alfalfa plant was crossed with Ms208, an elite, male sterile, conventional alfalfa plant, to produce KK179 alfalfa.2 KK179 alfalfa was chosen based on superior phenotypic characteristics and its molecular profile.
Characteristics, Stability, and Inheritance of the Introduced DNA
Monsanto and FGI characterized the introduced DNA using restriction enzyme digestion of leaf tissues obtained from KK179 alfalfa and near isogenic, conventional comparator genomic DNA followed by Southern blot analyses. Monsanto and FGI conclude that the results of these analyses demonstrate that KK179 alfalfa contains a single intact copy of the CCOMT cassette at a single site of insertion in the alfalfa genome. According to Monsanto and FGI, Southern blot analysis was used to confirm that DNA sequences outside the CCOMT cassette border regions from plasmid PV-MSPQ12633, including backbone DNA sequences and the nptII cassette were not detected in the KK179 alfalfa genome.
Monsanto and FGI consider that the conclusions derived from the Southern blot analyses were confirmed by PCR-based DNA sequencing of the insert and flanking genomic sequences and bioinformatics analysis of the obtained sequences. The DNA sequencing analysis confirmed the integrity of the CCOMT cassette within the inserted sequence and identified the 5' and 3' insert-to-genome DNA junctions.
Monsanto and FGI assessed the stability of the inserted DNA across four generations through Southern blot analysis. Southern blots were hybridized with probes that detect restriction fragments that encompass the entire CCOMT cassette insert and portions of the two border segments.3 Monsanto and FGI conclude that the results across four generations of KK179 alfalfa were as expected for an intact, single copy insert whose integration and inheritance is stable across multiple generations.
During the development of KK179 alfalfa, Monsanto and FGI generated and recorded segregation data, obtained using a PCR-based technique, to assess the heritability of the inserted coding sequence in the genome. Monsanto and FGI state that the results of chi-square analysis of the segregation data from multiple generations of breeding show that the CCOMT cassette is inherited according to Mendelian principles of inheritance. Monsanto and FGI conclude that the results of these analyses confirm that KK179 alfalfa contains a single, intact copy of the CCOMT cassette, stably inserted into the alfalfa genome at a single locus.
Bioinformatics Analyses for Putative Polypeptides
Monsanto and FGI state that a FASTA sequence alignment tool was used to assess the structural relatedness between putative polypeptides encoded by the CCOMT cassette and proteins in the allergen and toxin databases.4 Monsanto and FGI note that two criteria were used to assess structural relatedness: 1) 35% or greater amino acid identity in a sliding window of 80 amino acids, and 2) eight contiguous amino acid match between the insert sequence and any protein sequences in the allergen and toxin databases. Monsanto and FGI conclude that analyses of putative polypeptides in all six reading frames of the KK179 alfalfa insert revealed no significant matches with known allergenic or toxic proteins using the first criterion. Two identical short (eight amino acid) polypeptide matches with a protein sequence in the allergen database were identified. Monsanto and FGI report that these matches map to a region in the CCOMT gene segment and further conclude that the inserted CCOMT cassette sequence will not be translated to any polypeptide product because of the lack of a translatable open reading frame. Bioinformatics analyses were also used to demonstrate that open reading frames spanning the 5´ and 3´ flanking sequences of the inserted DNA junctions did not produce any relevant similarities to known allergens or toxins. Monsanto and FGI conclude that there is no evidence for concern regarding allergenicity and toxicity of putative polypeptides resulting from the insertion of the CCOMT cassette into KK179 alfalfa.
Safety Assessment of the CCOMT RNAi
Monsanto and FGI note that it is extremely unlikely that the CCOMT cassette in KK179 alfalfa will encode for a new protein. Transcription of the CCOMT cassette produces a single-stranded RNA containing an inverted repeat, which folds back on itself to form double-stranded RNA (dsRNA), suppressing endogenous CCOMT transcript levels via an RNAi pathway. Monsanto and FGI state that dsRNAs are commonly found in plants and other eukaryotes where they are involved in endogenous gene suppression. Monsanto and FGI state that nucleic acids are present in the cells of living organisms, including plants and animals used as food by humans and animals, and do not raise a safety concern as a component of food or feed. Monsanto and FGI note that RNA-based suppression mechanisms have been used to develop several new plant varieties that have been evaluated by the FDA and approved by several international regulatory authorities. Monsanto and FGI conclude that the expression product from the CCOMT cassette does not raise any safety concerns.
Evidence for Suppression of CCOMT Gene Transcript
Monsanto and FGI examined the transcription of the endogenous CCOMT and actin genes from each of four replicate samples of root tissue and forage tissue from KK179 alfalfa and corresponding near-isogenic conventional control plants. Northern blots showed clear reduction in transcripts of the CCOMT gene in root and forage tissues of KK179 alfalfa compared to those from the conventional control alfalfa, while transcripts of the actin gene from KK179 alfalfa and control alfalfa were similar to each other. Monsanto and FGI conclude that the relative decrease in CCOMT RNA transcript level in KK179 alfalfa confirms that the endogenous CCOMT gene is effectively silenced.
Food and Feed Use
Alfalfa (Medicago sativa L.) is grown as a perennial forage crop that is harvested several times throughout the growing season. It is the principal forage crop cultivated in the United States for animal feed. Alfalfa forage products, including hay, haylage, and silage, are valued for their high protein content and highly digestible fiber for ruminants and horses. Small amounts of alfalfa leaf meal may be used in monogastric animal diets.
Small amounts of alfalfa are used in human food, primarily as sprouts, and in dietary supplements and herbal teas. However, Monsanto and FGI state that the intended use of KK179 alfalfa is in animal feed and that it is not intended for human consumption.
Scope of Analysis
Monsanto and FGI analyzed the composition of forage samples from KK179 alfalfa (Syn1) and an appropriate conventional alfalfa population that has a genetic background similar to that of KK179 alfalfa (Syn1) but lacks the gene construct [hereafter described as the control (Syn1)].5 Monsanto and FGI also assessed the composition of forage from 14 conventional commercial alfalfa varieties (“reference varieties”) grown concurrently with KK179 alfalfa (Syn1) in the field. Monsanto and FGI used data derived from the reference varieties to generate a 99% tolerance interval for each component.6
Study Design - Compositional analyses
Monsanto and FGI state that forage samples were obtained from the first cutting of alfalfa grown at each of six field sites in the United States during the 2011 growing season. Each field site utilized a randomized complete block design with four blocks per site of KK179 alfalfa (Syn1), control (Syn1), and 14 reference varieties. Monsanto and FGI indicate that the alfalfa plants at each of the field sites were grown under normal agronomic field conditions for their respective geographic regions. The above ground portion of plants between 1% and 10% bloom were harvested from plants in the center of each individual plot at each site. Monsanto and FGI measured and evaluated 54 components in forage samples.7 These components included:
- Proximates [moisture, crude protein, crude fat, ash, and carbohydrates (by calculation)]
- Amino acids
- Cell wall components [acid detergent fiber (ADF), neutral detergent fiber (NDF), and acid detergent lignin (ADL)8]
- Intermediates in the lignin biosynthesis pathway (free phenylalanine, ferulic acid, total polyphenols, p-coumaric acid, and sinapic acid)
- Isoflavones and coumestans (daidzein, glycitein, genistein, formononetin, coumestrol, and biochanin A)
- Anti-nutrients [canavanine and saponins (total bayogenin, total hederagenin, total medicagenic acid, total soyasapogenol B, total soyasapogenol E, total zahnic acid, and total saponins)]
Monsanto and FGI excluded seven components (daidzein, glycitein, genistein, coumestrol, formononetin, biochanin A, and sinapic acid) from statistical analyses because over 50% of the observations were below the assay limit of quantitation.
The datasets were assessed using a mixed model of variance. Monsanto and FGI conducted statistical analyses on data obtained from each field site and from a combination of all six field sites. Each individual component for KK179 alfalfa (Syn1) was compared with that of the control (Syn1). Statistical significance was declared at 5% level (p ≤ 0.05). Monsanto and FGI state that when a statistically significant difference in a component was found between KK179 alfalfa (Syn1) and control (Syn1) in the combined-site comparison, an analysis was conducted to assess whether the difference was biologically meaningful from a feed safety or nutritional perspective. This analysis included magnitude of differences, reproducibility across individual sites, and comparison of the mean values for the components in KK179 alfalfa (Syn1) to the 99% tolerance interval for the population of reference varieties and values in published literature.
Results of Analyses
Monsanto and FGI report that there were no statistically significant differences between KK179 alfalfa (Syn1) and control (Syn1) for 44 of 47 components. Statistically significant differences between KK179 alfalfa (Syn1) and control (Syn1) were detected for ash, ferulic acid, and canavanine; they were small in magnitude. Furthermore, the mean levels of ash, ferulic acid, and canavanine were within the 99% tolerance interval of the reference varieties and within the range of values found in the published literature.
Intended Compositional Change - Lignin
Two separate analytical methods, semi-automated and manual methods,9 were used to measure the level of ADL in KK179 alfalfa (Syn1), control (Syn1), and reference varieties. A decrease in mean level of ADL in KK179 alfalfa (Syn1) compared to control (Syn1) was statistically significant when the semi-automated method was used for the analysis (5.39% and 6.93% of dry weight (DW), respectively). Monsanto and FGI point out that the manual method is not as reproducible as the semi-automated method and thus, only large differences would be likely to be discernable using the manual method.
The content of the individual lignin subunits (G lignin, S lignin, H lignin (p-hydroxyphenyl lignin), caffeyl lignin (derived from caffeyl aldehyde), and 5-hydroxyguaiacyl lignin in the cell wall was also measured in the samples of KK179 alfalfa (Syn1), control (Syn1), and reference varieties.10 Because more than 50% of the observations for caffeyl lignin and 5-hydroxyguaiacyl lignin were below the assay limit of quantitation, these two lignin subunits were excluded from the statistical analyses. Monsanto and FGI report a statistically significant decrease in the concentration of G lignin subunits in KK179 alfalfa (Syn1) compared to control (Syn1) (68.1 versus 83.7 μmoles per gram of cell wall residue). The mean values for G lignin subunits for KK179 alfalfa (Syn1) and control (Syn1) fell within the 99% tolerance interval for the population of reference varieties. At the same time, there was an increase (not significant) in H lignin and S lignin subunits. As a result of these changes, the S lignin: G lignin ratio increased from 0.58 in the control (Syn1) to 0.80 in KK179 alfalfa (Syn1).
Summary of Compositional Analyses
Monsanto and FGI conclude that the genetic modification in KK179 alfalfa does not meaningfully impact composition other than the intended reduction in the amount of G lignin monomeric subunits that accumulate in forage. Monsanto and FGI further conclude that the differences in the levels of the components described above are not considered to be biologically meaningful from a feed/food safety and nutritional perspective.
Sheep Feeding Study
Monsanto and FGI provide summary data from a 28-day lamb feeding study comparing two different diets containing either 100% KK179 alfalfa or 100% near isogenic conventional control hay. Lambs had access to a free choice mineral supplement. The study was conducted as a completely randomized design with a 2 x 2 factorial (diet and sex) arrangement of treatments. Monsanto and FGI state that feed intake, initial and final body weights, average daily gain, gain per unit of feed, and health parameters (body temperature, serum chemistry components, and gross necropsy findings) were similar across dietary treatments, although, as anticipated, there were statistically significant differences in some parameters between the sexes. Monsanto and FGI state that this study confirmed the nutritional wholesomeness of KK179 alfalfa.
FDA evaluated Monsanto and FGI’s submission to determine whether KK179 alfalfa raises any safety or regulatory issues with respect to the intended modification or with respect to the food and feed itself. Based on the information provided by Monsanto and FGI and other information available to the agency, FDA did not identify any issues under the FD&C Act that would require further evaluation at this time.
Monsanto and FGI have concluded that genetically engineered alfalfa variety KK179 alfalfa and the animal feed and food derived from it are as safe as conventional alfalfa varieties and are not materially different in composition or other relevant parameters from other alfalfa varieties now grown, marketed and consumed in the United States. At this time, based on Monsanto and FGI’s data and information, the agency considers Monsanto and FGI’s consultation on KK179 alfalfa to be complete.
1The Pal2 promoter directs transcription of the Pal2 gene within vascular tissue, which results in a pattern of expression that closely mirrors deposition of lignin as the plant matures.
2A single, untransformed R2336 plant was crossed with Ms208 to produce conventional F1 progeny plants. One plant from that cross was selected and used as the near isogenic, conventional comparator to the KK179 alfalfa in the molecular characterization studies. This plant was also used as part of a traditional population breeding program to generate near isogenic conventional plants for other studies described in the submission.
3Alfalfa contains multiple copies of the CCOMT gene that are randomly segregating. Thus, to distinguish the CCOMT cassette from the endogenous CCOMT copies in the KK179 alfalfa genome, Monsanto and FGI included the R2336 x Ms208 cross and the two parental lines – R2336 and Ms208 – as negative controls in the blots probed with CCOMT-containing sequences.
4Bioinformatics searches were conducted using three databases: (1) the allergen, gliadin, and glutenin protein sequence database [(AD_2012) released January 30, 2012] from Food Allergen Research and Resource Program, (2) the toxin protein sequence database [(TOX_2012) released January 30, 2012] from the PRT_2012 database, and (3) the GenBank protein database [(PRT_2012) release 187] from National Center for Biotechnology Information.
5The KK179 alfalfa (Syn1) and near isogenic conventional (Syn1) populations resulted from two generations of modified backcrosses and an inter se (with self) crossing.
6The 99% tolerance interval calculated by Monsanto and FGI represents, with 95% confidence, 99% of the values contained in the population of commercial conventional alfalfa varieties.
7Components for compositional analyses were selected based on the Organisation for Economic Co-operation and Development (OECD) Consensus document on compositional considerations for new varieties of alfalfa and other temperate forage legumes: Key feed nutrients, anti-nutrients and secondary plant metabolites (2005). Monsanto and FGI also included several intermediates in the lignin biosynthetic pathway.
8Acid detergent lignin is an analytical measure of the percentage of the polymerized lignin subunits (primarily G, S, and H lignin monomeric subunits) that fill the spaces in the cell wall of plants.
9The semi-automated method was developed by ANKOM Technology Corporation, Fairport, NY. The manual method is described in T.R. Weston, V. Nayigihugu, and B.W. Hess. 2006. Comparison of techniques for quantitative analysis of acid detergent lignin in roughages. Pages 242-244 in Proceedings, Western Section, American Society of Animal Science, American Society of Animal Science, Champaign, Illinois.
10Lignin composition was measured by gas chromatograph/mass spectrometry after thioacidolysis as described in: Lapierre, C., B. Monties and C. Rolando. 1985. Thioacidolysis of lignin comparison with acidolysis. J. Wood Chem. Technol. 5: 277 and Lapierre, C., B. Pollet and C. Rolando. 1995. New insights into the molecular architecture of hardwood lignins by chemical degradative methods. Res. Chem. Intermediates 21:397.