Comprehensive safety assurance for enabling Comprehensive safety assurance for enabling

transgenic technologies in agriculturetransgenic technologies in agriculture

Biotec India International 2003Hyderabad, 24 - 26 Sep ‘03

Partha R Das GuptaSyngenta Seeds, Asia-Pacific26 Sep ‘03

Global area under transgenic crops -Global area under transgenic crops -increasing at an average annual rate of 10%increasing at an average annual rate of 10%

Transgenic crops in developing countries -Transgenic crops in developing countries -

assessment of latest positionassessment of latest position

Chinese policymakers consider agricultural biotechnology as a strategic tool for improving national food security, raising agricultural productivity, and creating a competitive position in international agricultural markets. Huang Jikun & Wang Qinfang, AgBioForum 5(4), Aug ‘03

“...unless developing countries keep up with advances in biotechnology, they may lose out again. The cost of leaving some countries behind may be higher than the cost of empowering them to become players in mastering and benefiting from biotechnology.” 58th UN General Assembly Report of the Secretary General, May ‘03

Developing countries ought not to lose outDeveloping countries ought not to lose out;; should go for wide adoption of GM technologyshould go for wide adoption of GM technology

In combination with political and economic reforms, biotechnology can increase crop productivity by increasing yields and improving the nutritional content of crops in developing countries. It will also help provide lower-cost food to low-income consumers. J B Penn, Undersecretary, USDA, In Agricultural Biotechnology and the Developing World, Aug ‘03.

"The world has the technology...to feed 10 billion people”. “...some organizations that influence public policy are impeding research due to unfounded fears”. Norman E Borlaug, recent keynote address in Sacramento.

Wide adoption requires enabling regulations;Wide adoption requires enabling regulations;should be science based and address genuine risksshould be science based and address genuine risks

What is lacking? The Indian exampleWhat is lacking? The Indian example Absence of regulatory secretariat/ technical back-

up to assist RCGM; MEC; GEAC Application form does not cover all questions

relevant to regulatory & allied issues, viz., IPRs Limited scope of verification of data submitted Lack of local availability of advanced capacity In the absence of a dedicated regulatory body,

steps/ actions are left to be taken by committees No room for stakeholders’ dialogue - to bring

about modernisation of existing system Overall process is time consuming, costly

Gene(s) Source(s) Molecular characterisation Insert / copy number / gene

integrity Protein(s)

History of safe use and consumption

Function / specificity / mode of action

Levels Toxicology / allergenicity

Crop Characteristics– Morphology

– Yield

Food / Feed Composition

– Proximate analysis

– Key nutrients

– Key anti-nutrients

Gene / Protein Gene / Protein SafetySafety

Food / Food / FeedFeed

SafetySafety

Essentials of science-based safety assessment -Essentials of science-based safety assessment -biological safetybiological safety

Crop SafetyCrop Safety

Food & feed safety assessmentFood & feed safety assessment - - human and animal health (Bt maize example)human and animal health (Bt maize example)

• Digestibility in simulated gastric and intestinal fluids

• Homology comparisons to known toxins and allergens

• Post-translational modifications to CryIAb protein

• Nutrient composition of grain and leaf tissue

• Acute oral gavage(mice)

• Broiler feeding studies

• Laying hen feeding study

• Dairy cattle feeding study

• Beef cattle feeding studies

• Bobwhite quail

Science-based safety assessment -Science-based safety assessment -environmental safety environmental safety Contd..

Honeybees Parasitic wasps Daphnids Total in-field aerial

populations Total on-plant insect

populations Monarch butterfly

Non-target organisms Earthworms Collembola Ladybird beetles Lacewings Pirate bugs Beetles Spiders Mites

List is constantly expandingList is constantly expanding

Science-based safety assessment -Science-based safety assessment -environmental safetyenvironmental safety

Gene flow via pollen Extent and impact Long and short term effects

Release of Cry protein through root exudates Persistence in the soil and its effects on

rhizosphere Survivability in wild conditions

Weediness Germination and seed longevity

Insect resistance management.

Choice of transgenic events for field release -Choice of transgenic events for field release -underlying safety related considerationsunderlying safety related considerations

Molecular characterisation Data on stability, i.e., consistent trait expression

Product performance Biosafety

History of safe use of protein, e.g., Bt Substantial equivalence as food/ feed, to its

conventional counterpart Environmental safety

Effects on biodiversity (initiate long-term research) Exercise caution re field release in centres of origin

Freedom to operate

Transgenic events for field release -Transgenic events for field release -molecular characterisation relating to safetymolecular characterisation relating to safety

New USDA-Industry approach

Number of copies of transgene in plant genome

Intactness of entire insert

Transgene organised in planta as intended

Are functional units still contiguous?

Presence of any extraneous fragments?

Absence of vector backbone sequence

Locus number to be decided based on above informationLocus number to be decided based on above information

pZM5

VIP3Aa (2370 bp)

PMI (1176 bp)

LB (25 bp)RB (25 bp)

PEPC intron #9 (108 bp)

ZmUbi promoter (1993 bp)

ZmUbi promoter (1993 bp)

35S Term (70 bp)

NOS Term (253 bp)

VIP3 Probe (2370 bp)ZmUbi probe (1993 bp)ZmUbi probe (1993 bp)

PMI probe (1176 bp)

HindIII (293)

KpnI (4890)

SmaI (8399)

XmnI (3015)

Molecular characterisation -Molecular characterisation -confirmation by Southern dataconfirmation by Southern data

Copy number of the elements and absence of extraneous Copy number of the elements and absence of extraneous fragments of the elementsfragments of the elements

KpnI KpnI

Molecular characterisation -Molecular characterisation -confirmation by insert sequence dataconfirmation by insert sequence data

Intactness of entire insert and contiguousness of functional Intactness of entire insert and contiguousness of functional elementselements

Additional information on basepair changes, small Additional information on basepair changes, small rearrangements and/or deletions rearrangements and/or deletions

pZM5

VIP3Aa (2370 bp)

PMI (1176 bp)

LB (25 bp)RB (25 bp)

PEPC intron #9 (108 bp)

ZmUbi promoter (1993 bp)

ZmUbi promoter (1993 bp)

35S Term (70 bp)

NOS Term (253 bp)

Insert sequence (8386 bp)

HindIII (293)

KpnI (4890)

SmaI (8399)

XmnI (3015)

Molecular characterisation -Molecular characterisation -absence of vector backbone sequenceabsence of vector backbone sequence

Southern with a probe that corresponds to the entire region outside Southern with a probe that corresponds to the entire region outside of the borders in the plasmid used for transformationof the borders in the plasmid used for transformation

pZM514405 bp

VIP3Aa (2370 bp)

PMI (1176 bp)

Spec (789 bp)

LB (25 bp)

RB (25 bp)

PEPC intron #9 (108 bp)

Cos (432 bp)

ZmUbi promoter (1993 bp)

ZmUbi promoter (1993 bp)

COLE 1 (807 bp)

35S Term (70 bp)

NOS Term (253 bp)

Vector backbone probe (6019 bp)

Molecular characterisation Molecular characterisation New New USDA-Industry approach, 2003USDA-Industry approach, 2003: : SummarySummary

SequenceN/DSmall rearrangements

SequenceN/DSmall deletions

SequenceN/DBasepair changes

Additional information

SouthernSouthernAbsence of backbone

SequenceSouthernContiguousness of functional elements

SequenceSouthernIntactness of insert

SouthernSouthernCopy number

Proposed approachPrevious approachQuestion addressed

Characteristics of transgenic events/ expressed proteins - Characteristics of transgenic events/ expressed proteins - currently not fit for approval (e.g., in USA)currently not fit for approval (e.g., in USA)

Acute oral mammalian toxicity symptoms of proteins

Significant resistance of proteins to rapid gastric digestion

Homology of insert sequence with known toxins or allergens

Phenotypic or genetic instability Unexpected deviation >20% in compositional

analysis Unexpected ecotoxicity or undesirable

environmental profile

a b a b a b a b a b a b a b a b

z1 z4a z4b z10 z11b z12 WT pZPsC

23.1

9.4

6.6

4.4

2.3

kb

23.1

9.4

6.6

4.4

2.3

kb

Restriction enzymes: a) I-SceI b) KpnI Probe: psy

Restriction enzymes: a) I-SceI b) KpnI Probe: crtI

23.1

a b a b a b a b a b a b a b a b

z1 z4a z4b z10 z11b z12 WT pZCycH

9.4

6.6

4.4

kb

Restriction enzymes: a) I-SceI b) SpeI Probe: cyc

Golden Rice example

„Scientifically correct“ Zvit11b event, e.g.,•High expression•1.6 g/g -carotene

However•Not likely to receive regulatory approval!

Because•No clean integration pattern•Multiple integrations•Recombination events•Hygromycin selection•Beyond Border transfer•Construct „ill“-defined

Zvit11b

Courtesy: Ingo Potrykus

Some commonly raised safety concerns,Some commonly raised safety concerns,and ground realitiesand ground realities (1)

Gene silencing There is indeed a probability

More in multiple copy events than with a single copy; more likely with bioloistic than other techniques

Could be transcriptional, or post-transcriptional Observed during the early days for GM technology Also utilised for intentionally developing a product,

e.g., virus-resistant plants; Flavr Savr Tomato!Therefore, Hundreds of events have to be screened However,

So far there has not been a single case of a regulatory So far there has not been a single case of a regulatory failure on this groundfailure on this ground

Some commonly raised safety concerns,Some commonly raised safety concerns,and ground realitiesand ground realities (2)

Gene flow from transgenics to landraces, wild relatives, etc, e.g., maize in Mexico

Maize and teosinte have coexisted as separate entities in spite of their hybrids naturally occurring in farmers’ fields at a certain frequency.

Gene flow from transgenics is similarly expected At a higher frequency in a cross pollinated crop, viz.,

Maize than a self pollinated crop viz., Rice. While dealing with a transgene in its centre of origin

Risk-benefit analysis will be necessary Relevant information is not readily available

Long-term impact studies need to be undertaken Stakeholders should invest in research

Summing upSumming up

Regulation of GM crops is fast changing Regulatory science is evolving as a separate branch New techniques of safety assessment are emerging Product development should go side by side with

safety assessment Applicant should do “internal audit” before applying

for government approval Safety techniques call for proper capacity building Many critical concerns have no ready answers, yet

Long-term research is necessary Developing countries facing real challenge of food

security, should adopt GM technology early They should modernise their regulatory system Engage in dialogues with stakeholders Invest in regulatory science related research