Submission on APPLICATION A549 FOOD DERIVED FROM HIGH LYSINE CORN LY038: to permit the use in food of high lysine corn
 
Submitted to Food Standards Australia/New Zealand (FSANZ)
by
Submitter: New Zealand Institute of Gene Ecology
January 22, 2005

http://www.nzige.canterbury.ac.nz/documents/submissionA549.pdf

Overview
 
This submission from the New Zealand Institute of Gene Ecology (NZIGE) is meant to support Food Standards Australia/New Zealand’s preparation of a Draft Assessment on application A549. Our comments and wording are direct, but our spirit is constructive.  The NZIGE is dedicated to the development for the public good of all responsible biotechnologies. We are an assemblage of serious researchers with independent credentials in the area of biotechnology and its social impact. The activities of the NZIGE were supported in part by a grant for the Biosafety Forecast Service of the Biosafety Capacity Building Package under the auspices of the Norwegian Institute of Gene Ecology (GENØK).
 
A549 is an application to amend the Australia New Zealand Food Standards Code to allow foods derived from corn line LY038 to be sold in Australia and New Zealand. “Corn line LY038 has been genetically modified to have higher than usual levels of the amino acid lysine,” particularly in the corn grain [1]. LY038 was modified by the gene cordapA, sourced from the bacterium Corynebacterium glutamicum, inserted into the corn genome using genetic engineering techniques. The gene “encodes the enzyme dihydrodipicolinate synthase (DHDPS). This enzyme is involved in lysine biosynthesis. The bacterial DHDPS enzyme, unlike the plant DHDPS enzyme, is not sensitive to lysine feedback inhibition, so lysine biosynthesis will continue in the presence of high levels of free lysine.” [2]
 
Our submission begins with introductory material describing who we are and why we are involved. We then provide a summary of the major recommendations gathered from the detailed sections of our submission. These sections are organized into three main parts. In Part One, we undertake risk forecasting, an exercise at the leading edge of the research literature that serves to forewarn of risk where the science is not certain. Novel potential hazards of C. glutamicum Dihydordipicolinate Synthase (cDHDPS) protein, its metabolic products expressed in maize, and other side-effects of inserting DNA into the maize genome were identified to the best of our ability on the very tight timeframe available to us for this phase of consultation. Some of these properties, moreover, will be particularly influenced by the protein’s environment and thus are even more important for assessments of food safety.
 
In Part Two, we review the scientific documents submitted by the Applicant in support of A549. We judged this material by two criteria: 1. Was the science at the best possible standard? and 2. Does the science add up to a package that is sufficient to assure the citizens of Australia and New Zealand that they may safely consume food derived from corn line LY038? In most cases we recommend how, why and when the Applicant should supplement their findings with additional data.
 
In Part Three, we comment upon the Impact Analysis contained in the Initial Assessment Report (IAR). We assess the costs and benefits listed and propose further costs and benefits of the options under consideration.
                                                
The Authority (FSANZ) has made plain “the need for standards to be based on risk analysis using the best available scientific evidence” [3]. Above this need is the objective of the “protection of public health and safety” and “the provision of adequate information relating to food to enable consumers to make informed choices” [4], which requires the Authority to determine if the best scientific evidence available is good enough. Our contribution has therefore been to help the Authority identify areas of scientific uncertainty in the application so that these uncertainties can be addressed during the Authority’s development of a complete assessment.
 
We provide compelling new scientific evidence of risk and hazard. We also cannot exclude certain hazards from the information in the studies submitted by the Applicant and made available to the public by FSANZ. 

• The transgenic protein cDHDPS may have a different risk spectrum when a component of food.
• cDHDPs and its catabolic products could create novel risks in processed or cooked food.
• The creation of novel RNA molecules by insertion of DNA into the maize genome could create species of RNA that are harmful to humans, possibly through food. 
• The molecular characterization of the DNA inserted into the maize genome, the
  LY038 event, and DNA donated from the transgenic Cre-recombinase line used to create the LY038 maize line, is incomplete. The present data does not exclude, with a high level of confidence, the possibility that corn line LY038 contains additional novel genes, be they derived from the expression of fragments of inserted DNA or novel fusion proteins created at the junctions of inserted DNA and the maize genome. 
•  The molecular characterization of the transgenic protein cDHDPS produced by the genetically modified plant is flawed because the Applicant has not demonstrated that all novel proteins were included in this analysis. 
• The digestibility study of cDHDPS, required as part of an assessment of
  allergenicity, does not meet FAO/WHO standards for concentration of pepsin or standard comparisons to known allergens. Moreover, the digestibility study was fundamentally flawed by not using material from the actual genetically modified organism that the people of Australia and New Zealand would be eating. 
•  An adequate molecular characterization of all novel RNA molecules, that may pose a risk to consumers, is missing along with microarray analysis of the
  transcriptome of the LY038 line. There is published evidence that genetic components of the LY038 event produce novel RNA molecules. There is also evidence in animal studies that some small RNA molecules can be transmitted through food, causing lasting, sometimes heritable, effects on consumers and their children. 
•  The data comparing the composition of the transgenic lines to commercial reference lines of maize may be skewed by selective choice of commercial lines.
  The commercial reference lines chosen may inflate the 99% tolerance interval to more closely match the composition of LY038, thereby reducing the apparent number of significant compositional differences between the LY038 line and conventional corn. 
•  The compositional analysis does not appear to fully support the conclusion of equivalence between LY038 and its closest relative. The comparison found 103 (26% of total comparisons across 5 field studies) statistically significant differences between LY038 and the negative segregant. 
•  The acute toxicity study was fundamentally flawed by not using material from the actual genetically modified organism that the people of Australia and New
  Zealand would be eating. 
•  The broiler performance study may have falsely overestimated the positive effects of LY038 on chickens due to the choice of commercial reference controls. 
•  The broiler performance study indicates some unexplained negative effect on growth over the first 21 days when broilers were fed LY038. 
•  A549 lacks a subchronic toxicity study of adequate duration to conclude that the amino acid levels in LY038 are safe. 
•  A549 lacks a long-term toxicity and carcinogenicity study necessary to conclude that the amino acid levels in LY038 are safe. 

We also provide information and analysis indicating that the Impact Analysis is currently incomplete in some respects and mistaken in others.  Addressing these deficiencies would significantly shift the balance of the analysis.
 
We encourage a precautionary approach when assessing LY038. The scientific community is not uniformly convinced about the adequacy of existing risk assessments (Pusztai, et al. 2003), comfortable with the evidence that genetically modified food organisms are generally safe (Pryme and Lembcke 2003), nor confident that if approval were revoked, a GMO could be removed from the food chain before it caused harm (Heinemann, et al. 2004).
 
FSANZ has stated that the primary data [5] received from Applicants in support of their claims “enables a more rigorous analysis of experimental outcomes than the summary data of the type submitted in support of publication of a scientific article in a peer reviewed journal.” On the contrary, the data we have seen in A549 is not so different from that included in papers we have reviewed for journals. Nevertheless, direct access to the primary data is certainly an important requirement. It is important to note that, just as when peer-reviewing papers for publication, the reviewer cannot ‘tweak’ the experiment or explore all the unwritten parameters. This can lead to mistakes in reviewing. And while the publication of a paper with a flaw generally has very little influence on the daily lives of most citizens, the change in the New Zealand and Australia Food Code has implications for tens of millions of people directly and, because it may be connected to changes in global agriculture, it could have global ramifications. Therefore, the standard of review must both be better and more interrogating than for routine research results submitted for publication. 

We have the view that truly good biotechnologies will be vindicated by not just the best available science, but science adequate to the task of making a sound decision on safety.

Our a priori view is this: it is not a given that the science of the day is adequate for the task. It is possible for an applicant to do state-of-the-art analyses and not meet a standard of risk identification or resolution that may be necessary.
 
Should the best available science be ambiguous on A549, then New Zealand’s precautionary stance (as defined by the Convention on Biodiversity and the Hazardous Substances and New Organisms Act 1996 and amendments) must take priority.
 
Principal authors of this submission: Assoc. Prof. Jack A. Heinemann (NZIGE, Biosafety Forecast Service), Camilo Rodriguez-Beltran (NZIGE, Biosafety Forecast Service), Dr. Joanna Goven (NZIGE, Biosafety Forecast Service), Billie Moore (NZIGE, Biosafety Forecast Service), Leighton Turner (NZIGE), Dr. Thomas Bøhn (GENØK), Assoc. Prof. Juliet A. Gerrard (NZIGE), Marina Cretenet (NZIGE, Biosafety Forecast Service) and Prof. Terje Traavik (GENØK, Biosafety Forecast Service)

[1] FSANZ (2004). Initial Assessment Report: Application A549 Food Derived from High Lysine Corn LY038, p. 6.
[2] Ibid, p. 9.
[3] FSANZ (2004). Initial Assessment Report: Application A549 Food Derived from High Lysine Corn LY038, p. 9.
[4] Ibid, pp. 8-9.
[5] Not ‘raw’ data as indicated in the FSANZ document “FSANZ Response to Article Entitled ‘GE Foods and Human Health Safety Assessments’ By Dr Judy Carman, Spokesperson on GE Food, Public Health Association of Australia”, unless FSANZ receives machine print-outs and traces as well as photographs and tables.

Abstract
 
In the IAR, FSANZ invited “individuals and organizations to assist FSANZ in preparing the Draft Assessment for” this application A549 (FSANZ 2004, p. 3). Significant new evidence and analysis that is specifically relevant to the evaluation of LY038 is provided to support FSANZ in their preparation of a Draft Assessment. Our submission relates to “the scientific aspects of this Application, in particular, information relevant to the safety assessment of food from corn line LY038” (FSANZ 2004, p. 7). It also addresses the consistency of the IAR with “the objectives of FSANZ as set out in section 10 of the FSANZ Act” and provides “details of potential costs and benefits of the proposed change
to the Code” (FSANZ 2004, p. 3). 
 
In Part One, we describe important structural differences between mDHDPS and cDHDPS that produce novel challenges for studies on allergenicity, developments in food safety science regarding novel aggregates that could be produced by transgenic cDHDPS, the importance of characterizing post-processing and cooking effects on lysine, lysine catabolites and cDHDPS itself, novel regulatory RNAs that may have physiological effects on human consumers and species of DNA that could have biological effects if taken up by human cells through food.
 
In Part Two, we provide the Authority with a detailed examination of the molecular biological data supplied by the Applicant. We find suggestions in the Applicant’s data that secondary insertions into the LY038 genome may have been overlooked, and find deficiencies in protein identification protocols that could lead to false confidence in the low number of novel proteins and post-translational modifications reported in A549. Most importantly, new evidence suggests that the nos terminator sequence used in LY038 is a recombination hotspot, prone to read-through and may contain a cryptic cis-acting splice sequence that could generate novel RNA molecules and proteins at any place it is inserted into the genome. The lack of holistic proteomic and microarray analysis is a serious deficiency of this application.
 
In Part Three, we evaluate the cost/benefit analysis conducted by the Authority. One of our chief concerns with this analysis is that the costs and benefits included are largely concerned with feed while the Authority is considering whether to amend the food Code. Only when the place of feed-related impacts in a food assessment is clarified can one accurately assess the costs and benefits of Options 1 and 2. We also note that significant costs and benefits have been omitted from the analysis, and evidence is lacking to support those that have been included. In addition, the premises underlying some of the listed costs and benefits require further scrutiny. 
 
Our conclusion is that significant additional information should be provided by the
Applicant before the Australia New Zealand Food Code is amended. There have been important advances in biosafety and risk assessment science that are not uniformly reflected in the standard of reporting in A549. The studies submitted in support of A549 no longer uniformly meet what we see as the standard of the science. 
 
We have raised bona fide issues of safety that have not been addressed by the Authority or the Applicant, which also increases the cost of amending the Code. We have not found in the economic analysis any evidence of how amending Standard 1.5.2 will directly reduce food costs. In fact, we see no evidence that the consumer stands to receive any food benefit from amending the Code, while some consumers will certainly bear additional costs from doing so. We therefore must reject most of the Authority’s speculation on benefits in opting for Option
2.
 
Should the Authority come to recommend that the Code be amended to include event LY038, then it is our opinion that a special condition be imposed upon event LY038. In Column 2 of the Table to Clause 2 of Standard 1.5.2, LY038 but not any other members of the LY038 line currently in existence or that may arise through breeding, hybridization or transformation in the future may benefit from a favorable assessment of LY038. In other words, approval of LY038 does not extend to other lines of maize that share parentage or the I-DNA with LY038.

I. Introduction
 
I.1 This submission is the opinion of the submitter on Application A549 – high lysine corn LY038 ‘MAVERA HVC with Lysine’.
 
I.2 The submitter is the New Zealand Institute of Gene Ecology (NZIGE) and its cooperating partners. Responsibility for the content of this submission rests solely with the authors and NZIGE. The NZIGE is a research organisation
(www.nzige.canterbury.ac.nz). The NZIGE has no commercial interest in the product at the focus of this application, no direct or indirect connections with the Applicant, and has no connections to parties that seek to compete with the Applicant by developing a similar novel food. We received substantial support for the research that informs this application through a grant from GENØK for the Biosafety Forecast Service, and additional support from the University of Canterbury. Our submission is further informed by our own extensive experience in the research areas discussed below. If there were to be a public hearing on the application, we would be pleased to present our view. 
 
I.3 Our submission relates to “the scientific aspects of this Application, in particular, information relevant to the safety assessment of food from corn line LY038” (FSANZ 2004, p. 7). It also addresses the consistency of the IAR with “the objectives of FSANZ as set out in section 10 of the FSANZ Act” and provides “details of potential costs and benefits of the proposed change to the Code” (FSANZ 2004, p. 3). 
 
I.4 We have done our best to evaluate the scientific documents supplied by the Applicant in support of the application. Some areas of uncertainty may have arisen from the poor reproduction of some material made available by FSANZ for our use. 
 

R. Summary of major recommendations
 
R.1 In order to make an assessment of the changes in protein expression that occur within the plant due to expression of cDHDPS, the Applicant should undertake a routine proteomic analysis (by comparative 2D electrophoresis and mass spectrophotometric analysis of relevant spots) of lysates from whole plant cells and demonstrate that the only change is expression of the inserted gene. Single dimension protein gels of whole plant extracts at various stages of purification should also be supplied, in order to authenticate the purification of cDHDPS from plant tissue. Presentation of only the purified protein is unacceptable.
 
R.2 The Authority should require data from long term (lifetime) animal feeding trials to capture chronic effects, detect carcinogens and co-carcinogens, and proteins that are capable of forming amyloid fibrils.
 
R.3 The Authority should request an analysis of Maillard reaction products or other glycotoxins that could arise from cooking or processing of LY038 corn.
 
R.4 The Applicant should test the potential of in planta-produced cDHDPS to form amyloid fibrils and measure the cytotoxicity of aggregates and intermediate forms compared with native cDHDPS. It would be highly desirable to have the aggregation potential of cDHDPS correlated with changes in pH and other varying physical parameters of the choloroplast.
 
R.5 In order to make an assessment of global changes in the transcriptome, and specific changes cause by the insertion(s) of I-DNA, the Authority should require microarray descriptions capable of detecting novel RNA species in the modified plant, with the RNA source being the plant grown under a variety of relevant field conditions. The microarray should comprehensively represent the genomes of the cultivar of maize modified and unmodified, and any novel RNA species should be tested against the human genome for RNAi activity.
 
R.6 We recommend that I-DNA, especially the Glb1 promoter sequence, be analysed for putative mammalian transcription factor binding motifs.
 
R.7 We recommend that a metabolomic analysis such as NMR combined with
chemometrics and univariant statistics be supplied to the Authority by the Applicant.
 
R.8 The Authority should require the Applicant, at a minimum, to supply data on the digestibility of the cDHDPS protein using a protocol consistent with the FAO/WHO standard (FAO/WHO 2001) and the recommendations of Pusztai et al. (Pusztai, et al. 2003).
 
R.9 We recommend that a compositional analysis that includes the four commercial varieties used in MSL-18883 be requested by the Authority.
 
R.10 We recommend that the Authority dismiss study MSL-18883 for purposes of assessing safety.
 
R.11 The Authority should require the Applicant to submit data on cholesterol concentrations in serum and lipoprotein, and any changes in liver phospholipids, in animal feeding experiments.
 
R.12 We recommend that the Authority request the Applicant to provide a valid
subchronic toxicity study of a minimum of 6 months duration.
 
R.13 We recommend that only after the Applicant demonstrates the safety of LY038 using a higher standard of in vitro and animal in vivo safety tests, then human tests should be completed before another application is lodged with the FSANZ.
 
R.14 A plan for effective post-launch monitoring should be provided by the Applicant and the plan should be subject to a transparent review through the independent scientific community.
 
R.15 The Authority should clarify its proper jurisdiction with regard to this Application; in particular, it should clarify whether and how it is equipped to analyse the impact of the availability or non-availability of LY038 animal feed.
 
R.16 We urge the Authority to disregard the declared intention of the Applicant to segregate LY038 from the human food supply, as it cannot bind the Applicant (or any other party) to this action once the Food Code has been amended. We recommend that the Authority ensure that this declared intention not be permitted to influence the rigour of the application or the analysis of its impacts.
 
R.17 The Authority should clarify the reasoning behind its identification of Affected Parties; in particular, it should clarify why failing to amend the Food Code would prevent animal growers accessing LY038 feed.
 
R.18 The Authority or Applicant should provide evidence for its assertion that the cost to consumers of avoiding LY038, and to government of monitoring for the presence of LY038 in food, will be low.
 
R.19 The Authority should not extend any approval of LY038 to any hybrid line derived from LY038. As in European regulation, all hybrids, whether between LY038 and an unmodified line or another approved modified line, must in this case be treated as a new organism requiring a full safety evaluation.
 
R.20 The Authority should take into account the implications of approving this amendment of the Food Code for New Zealand’s obligations under the Cartagena Protocol.