Development of concepts WHO, Comparative assessment Unintended effects, SAFOTEST Allergenicity

1. Development of concepts • WHO, Comparative assessment • Unintended effects, SAFOTEST • Allergenicity • HGT, GMOBILITY • Risks from the environment ? • Conclusions • Alexander Haslberger • 7/04

2. Breeding: Irradiation Irradiator at Institute of Radiation Breeding Ibaraki-ken, JAPAN (http://www.irb.affrc.go.jp/)

3. Uninteded Changes in convent. breeding Published examples of unintended changes in conventional crops that have evaded pre-release evaluation include: • At least two potato varieties withdrawn after commercial release due to abnormally high levels of toxic glycoalkaloids in their tubers (Zitnak & Johnson, 1970, Am. Pot. J. 47: 256-260. Hellenas et al., 1995, J. Sci. Food Agric. 68: 249-255). • A pest-resistant celery variety with abnormally high levels of psoralens which caused light sensitive rashes and burns in pickers (Ames & Gold, 1990; Proc,. Natl. Acad. Sci. USA 87: 7777-7786). • A Spring barley variety, Chariot, which was selected for high malting quality in Cambridge, UK, but when grown in the northern UK shows high levels of grain splitting and the resulted is a reduction in quality (R. Ellis, SCRI pers comm.; comments on the UK recommended lists for cereals, 1999-2002).

4. Unintended Changes in GM plants Nature Biotechnology Haslberger, 2003

5. FAO/WHO expert consultation, 2003 Insertion of a transgene sometimes can affect expression of another gene(s). Expression of the transgene ideally should have no undesired effects on the expression of other host genes or health of the host. Other outcomes, however, have been observed. The transgene can be silenced by methylation or through other mechanisms. Because expression of the transgene often is controlled by novel regulatory elements outside of the host’s normal homeostatic feedback mechanisms, expression of the transgene can have pleiotropic effects, that is, effects upon multiple traits of the host. The use of viral and transposon vectors poses the hazard that the transgene might subsequently move within the genome

6. Existing traditional foods are considered to be safe, through their long history of use, even though they may contain anti-nutritional or toxic substances The concept of Substantial Equivalence embodies the idea that conventional foods can serve as a basis for the safety assessment of GM foods, since most of these foods are obtained from them Concept of Substantial EquivalenceOECD, 1993

7. Criticism on the S.E. Erik Millstone, Eric Brunner and Sue Mayer Nature, October 7, 1999 Showing that a genetically modified food is chemically similar to its natural counterpart is not adequate evidence that it is safe for human consumption

8. Effects on environmental factors on Glucosinolates in PGS- Rape Novak WK, Haslberger AG., Food Chem Toxicol 2000: 38 Range Controls: 7,3- 71,2 meal 16,1-41,1 seed Recommend: 30 DROUGHT-STRESS ?

9. Canada and the S.E; biotechnology-derived products that are considered to be the same as their conventional counterparts should not be exempted from testing.. Canadian Food Inspection AgencyScience BranchOffice of Biotechnology An Overview of the Royal Society Report on the Future of Food Biotechnology

10. CODEX/ WHO/FAO 2001: New Concept of risk assessment and the Substantial Equivalence * No safety assessment * Starting point for safety assessment * Comparison between the GM organism and its closest traditional counterpart * Identification of intended and unintended differences on which further safety assessment should be focused

11. Hazard-, risk- , safety- assessments

12. Risk Analysis, in general Risk Assessment Risk Management Process Initiation  Science based  Policy based Uncertainty ? Risk Communication  Interactive exchange of information and opinions concerning risks

13. Codex guideline principles • The key elements of the Principles are: • there should be a pre-market food safety assessment, on a case-by-case basis, for foods derived from biotechnology. The data and information used in this assessment should be of a quality that would withstand scientific peer review; • the food safety assessment is based on a comparative analysis with a "conventional counterpart" to ensure that the resulting biotech food is no less safe than the foods normally consumed by the population; • risk management measures should be proportional to the risks identified in the safety assessment and may include measures such as labelling, post-market monitoring and product tracing;

14. The comparative safety assessment The initial step is comprised of a thorough comparison with the closely related conventional counterpart to identify any differences that may have safety implications for the consumer. This comparison includes both phenotypic characteristics as well as a compositional analysis. The phenotypic analysis should also include comparative health parameters. The compositional analysis will focus on key substances in the animal products under scrutiny and will be subject to changes according to latest scientific state-of-the-art. The second step of the CSA comprises the toxicological and nutritional evaluation of the identified differences between the GMO and its comparator. As a result of this second step additional testing may be required and can result in a iterative process in order to obtain all relevant information for the final risk characterisation.

15. Comparative safety assessment, FAO/WHO, 2003, Kok and Kuiper

16. CSA, II Any differences found as a result of the CSA serve as comparable to the hazard identification and hazard characterization steps in a traditional risk assessment paradigm The molecular characterisation should comprise an analysis of the copy number and a sequence analysis of the flanking regions of the place of insertion Food intake assessments will also include an estimate of the extent to which current food products will be replaced by the GM The limitations of standard toxicity testing applied to whole foods Assessment of the replacement factor of important animal-derived sources of micro- and macronutrients by GM animal products in the event of altered composition with relation to these nutrients

17. Molecular characterisation,RR Soya

18. US- EPA: FIFRA consultation 2004 Thus to more fully investigate the possibility that T-DNA inserts are in active genes, Northern hybridization blot hybridization to detect cognate mRNA transcripts should be done using 5’ and 3’ sequences that flank each T-DNA as probes. Comparing the size of transcripts detected by Northern blot in fractionated RNA from non-transformed and transformed lines will assess whether the T-DNA insertion physically disrupted or significantly (2 SD from the norm) affected expression of the cellular gene

20. Control Test Isolate mRNA & label cDNA Isolate mRNA & label cDNA cDNA hybridised to microarray cDNA binds to corresponding gene Expressed only in control Expressed in both conditions Gene induced Not expressed Comparing Gene Expression

21. TOX: Difficulties Animal Feeding Studies Whole Foods Small doses to be fed (bulk, satiety) Nutritional imbalance of the diet Many confounding factors Small safety margins, if any Insufficient sensitivity for specific endpoints     

23. Microarrays/RIKILT

24. Metabolite profiling/ Engel

25. 90 d rat tox for screening of unintended effects

26. GM foods: allergenicity and immune responses FAO/WHO expert consultations, 2001-2003 Distinguish the source of the gene: safe history as food, no history as food, Know allergen: discouraged presently Sequence homology stability, digestibility

27. Continuing discussions: allergenicity • Models for sequence comparison • minimal epitope length, false positive • Additional vitro tests: APCs? • Sera testing • Animal tests? FAO/WHO, 2003: It was recognized that animal models for allergenicity testing, even those that are not yet validated, may be of value to identify potential allergens. It is recommended that additional efforts should be directed to the further development and validation of these models. Post market Monitoring !

28. HGT WP1 Selection, construction and lab bench testing of donor sequences and recipient strains for horizontal gene transfer WP2 Horizontal gene transfer in food systems WP3 Horizontal gene transfer in in vitro model systems WP4 Horizontal gene transfer in vivo WP5 Quantitative risk assessment and evaluation of model systems

29. Our Gut Flora Helps Prevent Colonisation by Pathogens Rapidly colonises gut after birth Comprises more than 1014 organisms More than 400 species An individuals flora is immunologically distinct Symbiotic relationship with host

30. HGT in Food

31. DNA digestion in porcine GI tract material compared to TIM

32. DNA persistence in vivo Gnotobiotic rats (B. subtilis) receiving pDNA Sacrificed after 3 or 5 hr DNA extraction PCR (118-924 bp) • DNA can transiently persist during the passage of the GI tract

33. Marker rescue transformation of B. subtilis LTH 5466 in milk and chocolate milk B. subtilis LTH 5466 developed competence during growth () in UHT milk (A) and chocolate milk (B). Marker rescue was observed with E. coli DNA (10 µg/ml) containing nptII gene Transformation frequency -> grey bars Detection limit -> white bars. A B

34. In vivo conjugation in gnotobiotic rats

35. In vivoexperiment 10 ml of inoculum (E. coli pBHR1GFP) overnight at 37 °C in BHI Suspended in 200 ml of physiological solution Administration to mice for 28 days 2 times-week Plating on specific media Decimal dilutions Further analyses PCR Fluorescence Microscope FACS

36. FACS Analysis detected GFP- Donor E. coli but no transkonjugants

37. GFP detection in bacteria from faeces Isolates Co Co GFP protein expression M Co Isolates Co GFP PCR 800bp 99 % similarity with Bacteroides

38. Summary HGT: GMOBILITY Small amounts of plasmid, plant DNA in all parts of the GI tract in all parts of the GI tract development of competence and transformation ( low rates ) possible in vitro No transformation seen from marker gene fragments from plants in vivo models using sensitive models, gnotobiotic rats / marker rescue assays Conjugation in the GI tract

39. FAO/WHO expert consultation, 2003 The DNA construct used to change the genetic make-up of the animal should be considered within an assessment, especially if the gene or its promoter is derived from a viral source There is potential for horizontal transfer of the gene construct: food-ingested foreign DNA may not be completely degraded in the gastrointestinal tract For the food safety assessment, it is prudent to assume that DNA fragments may survive the human gastrointestinal tract and be absorbed by either the gut microflora or somatic cells lining the intestinal tract. In general, the Consultation advocated avoiding the use of any unnecessary DNA sequences including marker genes in the genetic construct.

40. Outcrossing, Herbicides, Pesticides

41. Local factors and agro- ecological base lines • BT- cotton : local factors decide on pesticide reductions and benefits

42. ..analyze technologies, e.g. Organic Farming, Integrative Pest management, precision agriculture, marker directed breeding, GM foods …. for their effects on bio-diversity …as the basis of ecosystem services to humans … ( e.g. sustainable food production …)

43. Wild populations FAO: concern because of crop diversity Genetic diversity Landraces Ex situ collections Modern lines Onset of domestication Modern plant breeding (ca 1900) Before agriculture

44. WHO : GM food aspects need to be seen “holistic” Interaction environment, human health Socio- economic aspects, patenting Globalised trading but regional consequences Ethic aspects ( consultation 2003 )

45. FAO, Food ethics, 2003 While risk assessment is based on science, scientific evidence and analysis cannot always provide immediate answers to questions posed. Much scientific evidence is tentative, as the established processes of science include checking and re checking outcomes in order to obtain the required level of confidence. Decisions usually are defended as based on “science,” and sometimes on economic costs and benefits as well, which offer seemingly objective, verifiable evidence that the policy choice is “correct.” Decisions explicitly based on ethical principles and value preferences can be just as defensible, if the society agrees broadly on the ethical assumptions used to make policy. The emphasis on science and the exclusion of ethical argument as the basis for decisions may polarize the scientific debate.

46. FAO/WHO consultation GM food safety, 2003 includes Ethics Four principles have been established as fundamental in the biomedical field: respect for autonomy, beneficence, non-maleficence and justice (Beauchamp and Childress, 2001). Leiden conference, Sept 2004: Global Code of Ethics for the introduction of Modern Food Biotechnology in global markets Gesche Astrid H., Entsua-Mensah Mamaa and Haslberger G. Alexander

47. QUO VADIS? GM food safety debate has resulted in a high level of safety of present products New products (stacking of different traits, foods with intended changes of nutrients ) will need improved health and environmental assessments

48. Quo vadis IIEPA- FIFRA consultation, 2004: site directed insertion

49. Quo vadis: III