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

Breeding: IrradiationBreeding: Irradiation

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

Uninteded Changes in convent. breedingUninteded Changes in convent. breeding

Published examples of unintended changes in conventional crops that haveevaded pre-release evaluation include:

• At least two potato varieties withdrawn after commercial releasedue to abnormally high levels of toxic glycoalkaloids in their tubers(Zitnak & Johnson, 1970, Am.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 ofpsoralens 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 highmalting quality in Cambridge, UK, but when grown in the northernUK shows high levels of grain splitting and the resulted is areduction in quality (R. Ellis, SCRI pers comm.; comments on theUK recommended lists for cereals, 1999-2002).

Unintended Changes in GM plantsUnintended Changes in GM plants

Nature Biotechnology

Haslberger, 2003

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

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 EquivalenceConcept of Substantial EquivalenceOECD, 1993OECD, 1993

Criticism on the S.E.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

0

10

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30

40

50

60

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80

Belgium France UK Canada

Comodified

Range Controls: 7,3- 71,2 meal

16,1-41,1 seed

Recommend: 30

Effects on environmental factors on

Glucosinolates in PGS- Rape

DROUGHT-STRESS ?

Novak WK, Haslberger AG., Food Chem Toxicol

2000: 38

Canada and the S.E;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 Agency

Science BranchOffice of Biotechnology

An Overview of the Royal Society Report on the Future of

Food Biotechnology

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 

Hazard-, risk- , safety- assessments

Risk Analysis, in generalRisk Analysis, in general

Risk Assessment Risk Management

Risk Communication

Process Initiation

Science based Policy based

Interactive exchange of information and opinions

concerning risks

Uncertainty ?

Codex guideline principlesCodex 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;

The comparative safety assessmentThe 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.

Comparative safety assessment, FAO/WHO, 2003, Comparative safety assessment, FAO/WHO, 2003, Kok and KuiperKok and Kuiper

CSA, IICSA, 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

Molecular characterisation,Molecular characterisation,RR SoyaRR Soya

US- EPA: FIFRA consultation 2004US- 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

Comparing Gene ExpressionComparing Gene ExpressionControl Test

Isolate mRNA& label cDNA

Isolate mRNA& label cDNA

cDNA hybridised to microarray

Expressed onlyin control

Expressed inboth conditions

Geneinduced

Notexpressed

cDNA binds to corresponding gene

TOX: Difficulties Animal TOX: Difficulties Animal Feeding Studies Whole Feeding Studies Whole

FoodsFoods

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

Microarrays/RIKILT

Metabolite profiling/ Engel

90 d rat tox for screening of unintended effects

GM foods: allergenicity and GM foods: allergenicity and immune responsesimmune 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

• Models for sequence comparison

• minimal epitope length, false positive

• Additional vitro tests: APCs?

• Sera testing

• Animal tests?

Continuing discussions: allergenicity

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 effortsshould be directed to the further development and validation of these models.

Post market Monitoring !

GMOBILITY Safety evaluation of horizontal gene transfer from genetically modified organisms to the microflora of the food chain and human gut

 

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

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

HGT

Our Gut Flora Helps Prevent Our Gut Flora Helps Prevent Colonisation by PathogensColonisation 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

HGT in FoodHGT in Food

DNA digestion in porcine GI tract DNA digestion in porcine GI tract material compared to TIMmaterial compared to TIM

Duodenum Jejunum Ileum

Piglet

TIM1 Piglet TIM1 Piglet

TIM1

527 bp

<30” <30” < 2’ < 30” <1’ 30”- 60’

1617 bp

<30” <30” <30” <30” <30” <30”- 5’

DNA DNA persistence persistence in vivoin vivo

Time Stomach Duodenum Ileum Caecum Colon

3 hr 421 118 924

(+)

421 224

5 hr 421 118 421 924

(+)

924

• 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

Marker rescue transformation of Marker rescue transformation of B. B. subtilissubtilis LTH 5466 in milk and LTH 5466 in milk and

chocolate milkchocolate 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.

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10-5

time (h)

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ells

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FU

/ml)

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B

In vivo In vivo conjugation in conjugation in gnotobiotic ratsgnotobiotic rats

Transfer from L. lactis to E. faecalis

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pCAC4 TC

In vivoIn vivo experimentexperiment

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

Decimal dilutions

Plating on specific media

Further analyses

PCR Fluorescence Microscope FACS

FACS Analysis detected GFP- Donor E. coli

but no transkonjugants

M Co Isolates CoGFP PCR 800bp

Isolates Co Co

GFP protein expression

GFP detection in bacteria from faeces

99 % similarity with Bacteroides

Summary HGT: GMOBILITYSummary 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

FAO/WHO expert consultation, 2003FAO/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.

Outcrossing, Herbicides, Pesticides

• BT- cotton : local factors decide on pesticide reductions and benefits

Local factors Local factors and agro- and agro-

ecological base ecological base lines lines

..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 …)

Before agricultureModern plant breeding (ca 1900)Onset of domestication

Wild populations

Landraces

Modern linesEx situ collections

Gen

etic

div

ersi

tyFAO: concern because of crop diversity

WHO : GM food aspects need to WHO : GM food aspects need to be seen “holistic”be seen “holistic”

• Interaction environment, human health

• Socio- economic aspects, patenting

• Globalised trading but regional consequences

• Ethic aspects ( consultation 2003 )

FAO, Food ethics, 2003FAO, 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.

Four principles have been established as fundamental in the biomedical field:respect for autonomy, beneficence, non-maleficence and justice (Beauchamp and Childress, 2001).

FAO/WHO consultation GM food FAO/WHO consultation GM food safety, 2003 includes Ethicssafety, 2003 includes Ethics

Leiden conference, Sept 2004: Global Code of Ethics for the introduction of Modern Food

Biotechnology in global marketsGesche Astrid H., Entsua-Mensah Mamaa and Haslberger G. Alexander

QUO VADIS?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

Quo vadis IIQuo vadis IIEPA- FIFRA consultation, 2004: EPA- FIFRA consultation, 2004:

site directed insertionsite directed insertion

Quo vadis: III