Overview

1
Overview

• How does a company take a product from concept to
  market?
• How do we assure the safety of food derived from
  these products?
• How to address specific safety issues?
• Unintended Long term Effects
• Allergy
• Antibiotic Resistance Markers
• Feed Safety

2
Plant Biotechnology Product Path
Transgenic Crops Have Many Hurdles
Discovery
Line Generation
Product Advancement
Post Market
Line Selection
Variety Development
Field Trials
Field Production
Seed Sales
Gene Discovery
GH Field Evaluation
Gene Transfer
Regulatory Dossier
NIH Biosafety Audit
FDA, USDA, EPA, EU Novel Foods, EU 90/220
Discovery Program
Gene Transfer
Gene Discovery
GH Field Evaluation
3
3 Phases of Safety Assessment
Discovery
Line Selection
Product Advancement
Post Market
Transfor- mation
Line Selection
Variety Development
Field Production
Gene Discovery
GH Field Evaluation
Product Concept
Market

• Phase II
• Biological / agronomic equivalence
• Stringent agronomic performance and efficacy
  criteria
• Greater than 99 of all events are eliminated
• Key step in product evaluation for conventional
  varieties

• Phase III
• Detailed product safety
• Food
• Feed
• Environmental

• Phase I
• Safety of gene, protein, crop
• Choice of genes / proteins
• mechanism of action
• Source of genes
• history of safe use
• ethics
• Environmental / ecological considerations

4
Safety Assessment - Phase I
Discovery
Line Selection
Product Advancement

Post Market
Transfor- mation
Line Selection
Variety Development
Field Production
Gene Discovery
GH Field Evaluation
Product Concept
Market

• Safety / Registrability Assessment
• Choice of Genes / Proteins
• trait of interest
• selectable marker
• Source of Genes
• safety (history of safe use)
• ethics
• Ecological Hazards/Concerns
• gene
• specific crop

Early Allergology and Toxicology NIH rDNA
Biosafety Audit GO / NO GO decisions

Environmental Assessments
5
History of Safe Consumption-Cry or Bt Proteins

• Derived from naturally occurring Bacillus
  thuringiensis.
• Present in commercial microbial products used
  globally for gt 35 years, organic growers.
• Microbial products contain mixtures of Bt
  proteins.
• Subjected to extensive safety testing
• Acute
• Subchronic
• Chronic
• Human consumption (oral gavage)

6
History of Safe Consumption - Cry or Bt Proteins

• Receptor-mediated mode-of-action.
• Highly specific, selective to target insect(s).
• No adverse effects related to Bt protein during
  production, use or consumption.
• Studies conducted with specific Bt proteins
  produced in genetically modified products have
  confirmed their safety.
• Plant biotechnology products have been in the
  market since 1995 (potato) and 1996 (corn).

7
Safety Assessment - Phase II
Discovery
Line Selection
Product Advancement

Post Market
Transfor- mation
Line Selection
Variety Development
Field Production
Gene Discovery
GH Field Evaluation
Product Concept
Market

• Stand establishment
• Leaf orientation
• Plant height
• Silk date
• Ear height
• Ear tipfill
• Tassel size
• Dropped ears
• Stalk rating
• Above ear intactness

• Early plant vigor
• Leaf color
• Root strength (lodging)
• Silk color
• Ear shape
• Tassel color
• Reaction to fungicides/herbicides
• Late season staygreen/appearance
• Susceptibility to pathogens/pests
• Yield

• Biological/ agronomic equivalence
• Stringent agronomic performance and efficacy
  criteria
• Greater than 99 of all events are eliminated
• Key step in product evaluation for conventional
  varieties

8
Safety Assessment - Phase III
Discovery
Line Selection
Product Advancement

Post Market
Field Production
Line Selection
Variety Development
Transfor- mation
Gene Discovery
GH Field Evaluation
Product Concept
Market

• Safety Assessments
• Food
• Feed
• Environmental

9
International Scientific Organizations -(FAO /
WHO / OECD)

• Products of plant biotechnology are not
  inherently less safe than those developed by
  traditional breeding.
• Food safety considerations are basically of the
  same nature as conventional breeding - so
  traditional approaches to assess food safety are
  appropriate.
• Extensive safety assessments conducted with plant
  biotechnology products provide equal or greater
  assurance of safety of food products.

10
Assessment of Food/Feed Safety

• Standard - Reasonable certainty that no harm
  will result from intended uses under the
  anticipated conditions of consumption.
• Food is not inherently safe.
• Considered to be safe based on experience.
• Not absolute but relative safety

as safe as ...
11
Substantial Equivalence

• Compare food (or food components) from
  genetically-modified crop to conventional
  counterpart
• Origin of genes
• Agronomic parameters
• Composition (key nutrients / anti-nutrients)
• Consumption

Confirmation of substantial equivalence
equals as safe as
12
Outcomes

• Substantially equivalent to conventional
  counterpart
• Substantially equivalent to conventional
  counterpart except for introduced trait(s)
• Not substantially equivalent to accepted food or
  food component

no further testing
focus assessment on trait(s) / gene product(s)
combined nutritional / toxicological assessment
13
Compositional Analyses to Establish Substantial
Equivalence (Corn Example)
Grain Forage - Protein - Protein - Fat -
Fat - Fiber - Fiber - Starch - Amino acid
composition - Fatty acid composition - Ash -
Sugars - Calcium - Phosphorous
Evaluate Key - Nutrients - Vitamins - Minerals -
Anti-nutrients - Toxicants - Allergens -
Others List depends on crop
14
Product Safety

• Integration of protein, composition, expression
  and feeding studies
• Protein
• make protein standard and fully characterize
  (GLP)
• source, bioinformatics, digestive fate, and acute
  toxicity for traditional dietary proteins
• case-by-case studies for exotic proteins
• Whole-food toxicology (case by case)
• Animal Performance
• (wholesomeness / acceptance)

15
History of Safe Use

• Studies as confirmatory of safety
• Examples Bts
• Extensively used pesticide for 35 years
• No occupational allergy / toxicology
• Extensive database of classical toxicology
• sub-chronic feeding
• chronic feeding
• HUMAN ACUTE ORAL GAVAGE
• Well described biochemical function

16
Bioinformatics Tools

• - Database of all known protein allergens
• - Contains 475 AA sequences
• - Immunologically relevant match
• - Database of all known protein toxins
• - Currently contains 4000 AA sequences
• - Test for biologically relevant match
• - All known genes / proteins
• - Expanding rapidly with genomics
• - Identify homology, helps define function

• Allergens
• Toxins
• All Others

17
Digestive Fate

• Purpose Evaluate in simulated gastric and
  intestinal fluids, the digestibility of proteins.
• Key parameter in allergy assessment
• Stable proteins likely bad actors
• Experimental - GLP
• SGF - 1 hour, SIF - 24 hours
• High resolution SDS-PAGE - resolve 12 AA
• Immunoreactivity assessed
• Bioactivity assessed (e.g. EC50 for insecticides)

18
Heat Stability

• Purpose evaluate immunoreactivity of protein
  after cooking
• Required for Japan
• Test substance is usually grain, from which
  biologically extractable protein is evaluated
• Assesses loss of extractibility,
  immunoreactivity, and possible degradation
• Relevance to safety remains undescribed

19
Mouse Acute Oral Toxicity

• Purpose test for mammalian toxicity (LD50)
• Almost no proteins are orally toxic, but for the
  few which cause problems, most act through acute
  mechanisms
• Single large acute dose, 10-14 days
• End points health, body weight, food
  consumption, necropsy (retain tissues for a rainy
  day)

20
Mouse Oral Acute Toxicity

• Protein Crop Dose mg/kg)
• Cry1Ac Cotton, Tomato 4200
• NPTII Cotton, Potato, Tomato 5000
• Cry3A Potato 5200
• Cry1Ab Corn 4000
• CP4 EPSPS Soybean, Cotton, Canola, Sugarbeet 572
• GUS Soybean, Sugarbeet 100
• GOX Canola, Cotton, Corn, Sugarbeet 100
• ACC Deaminase Tomato 602

21
Will the Consumption of Food From GM Crops That
Contain Antibiotic Resistance Markers Increases
Clinical Antibiotic Resistance?

• Use and misuse of antibiotics in clinical/
  veterinary applications are the major causes of
  antibiotic resistance
• Antibiotic Resistance Markers (ARMs) remain
  critical for production of GM plants with limited
  alternatives
• Plant expressed ARM proteins (NPTII) have been
  shown to be safe
• Potential to impact clinical/ veterinary use is
  remote
• Potential for gene transfer from plant to microbe
  is virtually zero (lt10-14-10-27)
• ARMs are ubiquitous in nature (10 to 40 of gut
  and soil microbes)
• ARMs are specifically selected with limited
  clinical/ veterinary use (NPTII, AAD)

22
Status of Antibiotic Resistance Markers

• Regulatory agencies and international scientific
  reviews have concluded that ARMs pose no
  significant safety concern
• SCP, SCF, SCAN
• Based on public perceived concern, alternatives
  are being evaluated, but will not be available
  for 6 to 7 years

23
Principles of Allergy Assessment

• Avoid transfer of known allergens
• Assume genes from allergenic sources encode an
  allergen until proven otherwise
• Assess the allergenic potential of all introduced
  proteins
• Where allergens are identified, consider
  alternative sources or technical strategies to
  mitigate hazard, or discontinue development

24
Food Allergy Assessment
Source of Gene (Allergenic)
Yes
No
Yes
Sequence Similarity
Solid Phase Immunoassay
Commonly Allergenic
Less Commonly Allergenic
No (lt5 sera)
No
No
Stability to Digestion/ Processing
Yes
Yes
No (gt5 sera)
Skin Prick Test
No
Yes
No
Yes
Consider Options
DBPCFC (IRB)
Consult with Reg. Agency
Yes
No
No Concern
25
Brazil Nut Saga

• Pioneer Hi-Bred International
• tried to correct the methionine deficiency in
  soybeans
• Brazil nuts rich in methionine Brazil nut
  storage protein expressed in transgenic soybean
  successfully increases methionine content
• but, was the Brazil nut storage protein an
  allergen?

26
Brazil Nut Saga

• Testing of Brazil nut storage protein
• University of Nebraska/University of Wisconsin
• 9 Brazil-nut allergic subjects
• 8/9 had antibodies that recognized the Brazil nut
  storage protein as an allergen
• 3 were skin-prick tested with the Brazil nut
  storage protein and the protein elicited an
  allergic response in all 3

27
Brazil Nut Saga

• Product development discontinued
• Still, however, negative publicity for Pioneer
  Hi-Bred International when they did the right
  thing
• Jeremy Rifkin/USA Today editorial

28
Approach for Safety Assessment
Food / Feed Safety
Gene / Protein
Substantial Equivalence

• Gene(s)
• Source(s)
• Molecular characterization
• Insert / copy number / gene integrity
• Protein(s)
• History of safe consumption
• Function / specificity / mode-of-action
• Levels
• Toxicology / allergenicity
• Amino Acid homology
• Digestibility
• Acute oral toxicity
• Clinical

• Crop Characteristics
• Morphology
• Yield
• Food / Feed Composition
• Proximate analysis
• Key nutrients
• Key anti-nutrients
• Feed performance studies
• Wholesomeness

29
Summary

• Plant biotechnology products must meet stringent
  performance standards during development
• agronomics, yield, morphology and line selection
  restores biological equivalence and reduces
  uncertainties relative to risk
• Continuous regulatory oversight occurs throughout
  development and full authorization process
• Candidate genes / proteins are assessed prior to
  transformation

30
Summary

• All products are thoroughly assessed for food,
  feed and environmental assessment prior to
  regulatory approval
• Food / feed safety based on substantial
  equivalence and safety of expressed proteins
• Proteins currently produced in plants have
  history of safe use