The Mammalian Safety Assessment of GM Crops - ILSI …ilsi.org/hesi/wp-content/uploads/sites/11/2016/05/6-7-Ladics-Tox... · GMOs for 21st Century –part of the ... • The overall

The Mammalian Safety Assessment of

GM Crops

Gregory S. Ladics, Ph.D., DABT, Fellow ATS

DuPont Pioneer Agricultural Biotechnology

Wilmington, DE USA

[email protected]

Biotechnology

• Genetic manipulation of organisms is notnew - humans have been doing this for thousands of years

• plant & animal breeding

Food crops

Evolution of Zea mays from ancestral teosinte (left) to modern

corn (right). The middle figure shows possible hybrids of teosinte

& early corn varieties

Evolution & breeding of food plants

• “Descendants” of the wild mustard

– Brassica spp.

Global Population Growth and Percent of

Growth by Region 2010-2050

Source: United Nations

Global Harvest Initiative, 2011 GAP Report

GMOs for 21st Century – part of the solution to meet growing food demand

Insect and Disease

Resistance

Nutritional

QualityAbiotic

Stresses

Herbicide

Resistance

Genetic Yield Potential

•Biotech crops have potential advantages to environment

-reduced tillage = reduced erosion

-reduced exposure to certain pesticides

-improved water and nitrogen use efficiency

-allow improved use of sub-optimal lands

-increase in yields to avoid land conversion

• Biotech crops have several potential advantages to

consumers

-reduction in exposure to certain pesticides

-lower mycotoxin levels

-enhanced traits –oil, vitamins, nutrients

Historically we learned to eat “safely” through experience:

Food/Feed Safety AssessmentRELATIVE SAFETY

GM-Crops must be “as safe as” Non-GM

But, you can NOT guarantee absolute safety!

SAFETY FOCUS IS ON THE NEW GENE-PROTEIN

Wheat must be avoided by those with celiac disease

Legumes (beans/peas) must be cooked to inactivate lectins and trypsin

inhibitors

Allergic individuals must avoid specific foods

How do we know GM

products are safe?

• There is a comprehensive

safety assessment program!

Product Safety Assessment

AllergenicityToxicology

(mammalian)

Human Dietary Exposure

Assessment

Environmental

Gene Flow

Environmental Fate/Exposure

Ecotoxicology

Insect Resistance Management

Holistic approach

Inherent toxicity? HOSU of source? Toxic?

Gene Gene product GM PlantHost plant

Allergenic?

Substantial

Equivalence ?

Inherent allergenicity?Insertion

consequences?

Horizontal transfer?

Increase of toxicity?

Increase of allergenicity?

-Comparison of the GM crop to a conventional equivalent with a History

of Safe Use (HOSU) guides the safety assessment

Substantial Equivalence

• The overall safety assessment of GM food begins with the concept known as “substantial equivalence”.

• Takes into account both intended and potential unintended effects that may occur in the GM plant compared to a conventional counterpart that has a history of safe use.

• Involves identifying any changes introduced through the genetic modification: nutrients, antinutrients, and natural toxicants, as well as any phenotypic or agronomic changes.

• Substantial equivalence is not a safety assessment in itself, but is a starting point, which is used to structure the safety assessment of the GM crop.

Substantial Equivalence: Nutrients

Proximates and Fiber (7)Crude Protein

Crude Fat

Ash

Crude Fiber

Acid detergent fiber

Neutral detergent fiber

Carbohydrates

Fatty Acids (25)Caprylic acid (C8:0)

Capric acid (C10:0)

Lauric acid (C12:0)

Myristic acid (C14:0)

Myristoleic acid (C14:1)

Pentadecanoic acid (C15:0)

Pentadecenoic acid (C15:1)

Palmitic acid (C16:0)

Palmitoleic acid (C16:1)

Heptadecanoic acid (C17:0)

Heptadecenoic acid (C17:1)

Stearic acid (C18:0)

Oleic acid (C18:1)

Linoleic acid (C18:2)

(9,15) Isomer of linoleic acid (C18:2)

Linolenic acid (C18:3)

γ-linolenic acid (C18:3)

Arachidic acid (C20:0)

Eicosenoic acid (C20:1)

Eicosadienoic acid (C20:2)

Eicosatrienoic acid (C20:3)

Arachidonic acid (C20:4)

Behenic acid (C22:0)

Erucic acid (C22:1)

Lignoceric acid (C24:0)

Vitamins (24)Vitamin B1

Vitamin B2

Pantothenic acid

Vitamin B6

Niacin

Folic acid

α-tocopherol

β-tocopherol

δ-tocopherol

γ-tocopherol

Total tocopherols

Isoflavones

Genistin

Genistein

Malonylgenistin

Acetylgenistin

Daidzin

Daidzein

Malonyldaidzin

Acetyldaidzin

Glycitin

Glycitein

Malonylglycitin

Acetylglycitin

Oligosaccharides (4)Sucrose

Stachyose

Raffinose

Secondary Metabolites & Anti-Nutrients (4)Coumestrol

Lectins

Phytic acid

Trypsin Inhibitor

Amino Acids (18)Methionine

Cystine

Lysine

Tryptophan

Threonine

Isoleucine

Histidine

Valine

Leucine

Arginine

Phenylalanine

Glycine

Alanine

Aspartic acid

Glutamic acid

Proline

Serine

Tyrosine

Minerals (9)Calcium

Phosphorous

Magnesium

Manganese

Copper

Iron

Potassium

Sodium

Zinc

Holistic approach



Allergenic?

Substantial Equivalence ?

Inherent allergenicity? Insertion consequences?




Protein Allergy Assessment

Adverse Reactions To FoodFood hypersensitivity

Non-immune mediated

Food intolerance

Immune-mediated

Food allergy

IgENon-IgE

Celiac Disease (gluten)

inflammatory

disease small intestine

-Metabolic (lactose intolerance)

-Pharmacologic

-idiosyncratic

(asthma and sulfites;

headaches and chocolate,

cheese, or aspartame)nausea, vomiting

urticaria, difficulty

breathing, anaphylactic

shock

IgE Mediated Symptoms

10 to 20 minutes after

eating:

• hives

• angioedema

• asthma

• diarrhea/vomiting

• atopic dermatitis

• anaphylaxis

Protein-specific IgE is the key

mediator in Food Allergy

allergen

Peanut

(Ara h 1)

Sensitized

Antigen

Specific

B cells

Make IgE

(2 IgE epitopes)IgE

FceRIMast cellsrelease

histamine

& leukotrienes

What Are The Potential Protein

Allergenicity Concerns with

Agricultural Biotechnology?

Categories of Potential Health Risks

Relative to Protein Allergenicity

(in order of risk)

1. Transfer an existing allergen or cross-reactive protein into another crop.

2. Creation of food allergens de novo (i.e., potential to become a new allergen)

3. Alteration or quantitative increase of endogenous (existing) allergens (i.e., increasing the hazard of currently allergenic foods)

Codex guideline – Weight of Evidence

Protein Allergenicity Assessment

Weight

Of

Evidence

CODEX, 2009

Glycosylation

Stability to Pepsin

Expression Levels

Heat Stability

Bioinformatics

Gene source

Stability to Trypsin

Immunological

Methods**

**if necessary


Relative to Allergenicity

1. Transfer an existing allergen or cross-reactive protein into another crop

2. Alteration or quantitative increase of endogenous (existing) allergens

3. Creation of food allergens de novo

1. Bioinformatics/Immunological methods

2. Analytical methods

3. Physical properties of protein (e.g., stability in SGF; heat)

Endpoints to reduce

potential risk per

CODEX (2009):

Potential

Risk:

What is Bioinformatics?

Definition:“the comparative analysis of protein sequences intended

to evaluate structural and functional relationships”

Core PrinciplesProtein structure is determined by amino acid sequence

Similar amino acid sequences have similar structure

Similar sequence and structure infers a common ancestor gene and related function.

How does bioinformatics help?

Allows one primary question to be asked:Is the protein an existing allergen?

Allows one secondary question to be

asked:Is the protein likely to cross-react with an existing

allergen?

Bioinformatics is not intended to answer

whether a protein will “become” an

allergen

Search Strategy

• Allergen Search

– Compare amino acid sequence of query protein to database containing sequences of food, dermal and respiratory allergens.

University of Nebraska Allergen Database

• Industry sponsored, peer-reviewed allergen database at

Univ. Nebraska

– Peer-reviewed by clinical and research allergists from around the world: Japan, Europe, and U.S.

– Well-defined criteria; posted on database website.

– Inclusion of protein allergens (food, dermal, respiratory) based on available data in the public literature.

– Updated once a year (Version 14)

– Available free to the general public

– www.allergenonline.org

26

Allergen Search Strategy

– Compare amino acid sequence of query protein to database containing sequences of food, dermal and respiratory allergens.

• Evaluate sequence for amino acid identity using local alignment programs, such as BLAST (or FASTA) • ≥ 35% identity over an 80 or greater amino acid window

and potential (theoretical) IgE epitope matches.– ≥ 8 contiguous identical amino acids (EFSA 2011; Ladics et al.,

2011, Reg. Toxicol. Pharmacol., 60:46-53).









Endpoints to reduce

potential risk per

CODEX (2009):

Potential

Risk:

• For proteins originating from an allergenic source, or

having significant homology with a known allergen,

specific serum screening is conducted.

• An issue of critical importance to sera screening is the

availability of well characterized, quality human sera from

a sufficient number of patients.

• Potential false positives/equivocal results

Specific IgE Sera Screening









Endpoints to reduce

potential risk per

CODEX (2009):

Potential

Risk:









Endpoints to reduce

potential risk per

CODEX (2009):

Potential

Risk:

32

Stability to Pepsin In Vitro

pH 1.2

Pepsin

Provides a loose correlation for

major food allergens (stable).

This test is not meant to “mimic” real

digestion

• Protein resistance to pepsin evaluated in simulated gastric fluid (pH 1.2) containing 0.3% (w/v) pepsin.

• Digestions performed for time intervals 0, 15 and 30 seconds, 1, 2, 5, 10, 15, 20, 30, and 60 minutes at 37°C.

• Samples (each protein at each time point) then analyzed by SDS polyacrylamide gel electrophoresis and/or Western blot analysis.

• A standardized protocol for evaluating the in vitro pepsin resistance of proteins was established (Thomas et al., Regulatory Toxicology Pharmacology, 39:87-98, 2004).

Molecular

Weight

(kDa)

Protein X

Pepsin

LaneLoad Volume

µLSample ID

1 20 Protein X (~2.3 µg) in water ”Time 0”

2 13 SeeBlue molecular weight marker

3 20 Protein X (~2.3 µg) in SGF “Time 0”

4 20 Protein X in SGF for 0.5 minutes

5 20 Protein X in SGF for 1 minute

6 20 Protein X in SGF for 2 minutes






12 20 SGF control ~60 minutes

Heat Stability

Residual Enzyme Activity after Heating for 15 Minutes

0

20

40

60

80

100

35 40 45 50 55 60

Incubation Temperature, (oC)

En

zym

e A

cti

vit

y,

% o

f U

nh

eate

d

Protein X- chlorsulfuron

Protein X + chlorsulfuron

• Very active research area; no consensus

• Definite need for further evaluation

• selectivity

• sensitivity

• testing with a range of proteins

• None (rodent or non-rodent) validated or widely

accepted

Can animal models identify

allergenic food proteins?

Ladics et al., (2010). Reg. Toxicol. Pharmacol., 56:212-

224

Holistic approach



Allergenic?






Toxicology Safety Assessment

Weight of Evidence

Weight

Of

Evidence

History of Safe Use

Expression Level & Dietary Intake

Bioinformatics

In Vitro Digestibility

Mode of Action

& Specificity

Heat Lability

Safety Assessment Considerations for

All Proteins (in silico/in vitro)

• History of Safe Use: The protein has a history of safe use/consumption in food and the source of the inserted DNA does not raise any toxicological concerns.

• Mode of Action and Specificity: The protein acts as intended with a known spectrum of activity. Define biological function, specificity, and mode of action.

• Bioinformatic Analysis: Comparison of the amino acid sequence of the protein in silico to known sequences to determine similarity to known protein toxins, anti-nutrients.

Bioinformatic Screening Tools-

Regulatory

– BLASTP search against the Genpept dataset (millions proteins)

representing submitted protein sequences and translations of all

submitted nucleotide sequences)

– Default parameters (BLOSUM62 matrix; E cutoff of 1.0, low

complexity filtering turned off, maximum number of

alignments returned)

– Alignment results are manually reviewed for similarities to

proteins that might generate safety concerns (including

literature review, background information when necessary)

Safety Assessment Considerations for

All Proteins (in silico/in vitro)

• In Vitro Digestibility and Heat Lability: The protein is readily degraded by pepsin and/or trypsin preparations, pH, and/or temperature.

• Expression Level and Dietary Intake:Determine level of expression of the protein in the crop or crop by-products- compare to existing exposures for the same or related proteins in existing foods; estimate dietary human exposure.

ILSI IFBiC TF6 protein safety

(toxicology) tiered approach

Tier I. Basic Hazard Assessment₋ History of safe use

₋ Bioinformatics

₋ Mode of action and specificity

₋ In vitro digestibility and heat lability

₋ Expression level and dietary intake

Tier II. Supplementary Hazard Assessment

₋Triggered when concerns raised in Tier I

₋ Acute toxicology studies

₋ Repeat dose toxicology studies

₋ Hypothesis-based toxicology studies

Delaney et al., 2008. Food Chem. Tox. 46, S71-S97

Why Acute Toxicology Studies?

• Protein toxins act through acute

mechanisms after the administration of a

single dose at doses in the nanogram to

milligram per kilogram body weight.

• Therefore, acute oral toxicity studies using

gram per kilogram body weight doses of

the novel protein are appropriate for

assessing the potential toxicity of proteins

Acute Oral Toxicity Test (in vivo)

• Details– Typically conducted in mice (5 male and/or 5

female)

– Recombinant transgene protein (2-5 g)• Need to test protein representative of form expressed in

planta (biochemical equivalence)

– One time oral gavage to “limit” dose:• 2000 mg/kg (OECD)

• Equivalent to 140 g of protein in average human

– Evaluate (14 day observation):• Mortality, body weight, behavioral endpoints, gross

necropsy

– Conclusion:• Transgene protein is or is not acutely toxic

Acute oral toxicity studies conducted with pure transgene

proteins have not demonstrated evidence of adverse

effects

Protein Toxicity

• Proteins have never been shown to be

direct-acting carcinogens, mutagens,

teratogens, or reproductive toxicants.

• Thus, generally not necessary to test

proteins for these toxicological endpoints.

Holistic approach


Gene Gene product GM Plant*Host plant

Allergenic?




- Unintended effects: predictable and unpredictable effects-*Part of plant consumed by humans (typically grain)



Whole Foods

• Subchronic rodent feeding studies

– A general but comprehensive health screen

– Relatively long term exposure that includes development and maturation

• 90 Days in duration (13 weeks); diet fed ad libitum

• Control is closest genetic non-GM comparator; non-GM Reference Standards (acceptable range of normal variation)

• 10-12 animals/sex per test group

– Goal is to determine if unintended differences

occurred during production of a GM resulting in adverse effects

Whole Foods• Subchronic rodent feeding studies

– Nutritional performance (in-life evaluations)• Body weight

• Feed consumption

• Feed efficiency

• Detailed clinical observations

• Neurobehavioral (motor activity; FOB)

• Mortality

– Extensive Clinical Pathology evaluation Following In-Life Phase

• Hematology (16); Coagulation (2)

• Organ Weights; Pathology (~40 organs)

• Clinical Chemistry (18)

• Urinalysis

Results Analyses

Feeding Study

Compare Non-GM vs. GM

Use references to define

acceptable control values

Tolerance

Interval

0

5

10

15

20

25

30

35

40

Non-GM GM

Eff

ect

Substantial Equivalence: Feed Safety and

Performance (Nutritional Equiv.)

Animals are fed GM crops and control conventionally bred crops

in balanced diets: animal production and health is evaluated

No Significant Differences in current GM crops

• Feed Intake

• Body Weight

• Carcass Yield

• Feed Efficiency

• Feed Conversion

• Milk Yield

• Milk Composition

• Digestibility

• Nutrient

Composition

• Acute oral toxicity studies conducted with pure transgene

proteins have not demonstrated evidence of adverse

effects

• GM grains are subjected to analytical evaluations to

establish equivalence of GM grain to non-GM grain

• Long term feeding studies conducted in poultry and

rodents consuming diets formulated with GM grains have

not identified adverse effects

• Large animal feeding studies have demonstrated

nutritional equivalence of GM crops

Conclusions-GM Crops

• Substantial Equivalence

• Agronomic Equivalence (phenology)

• Nutritional Equivalence (crop composition)

Food and Feed Safety Assessment Framework

Nutritional Safety

Rat: 90-day study

Poultry: 42-day study

Allergenicity

Gene origin, bioinformatics, heat and digestion

Protein Safety

Protein characterization and expression

Acute mouse test at a high concentration

Safe

ty A

ssessm

ent

Conclusions

• In contrast to traditional foods, a rigorous safety assessment process exists for GM crops.

• The mammalian safety assessment process of GM food focuses on an evaluation of the allergenic and toxic potential of the introduced novel traits, as well as the wholesomeness of the GM crop.

Conclusions

• To date, no adverse effects, neither allergenic nor toxicological, have been observed with any GM crop that has been approved for human or animal consumption.

The comprehensive safety

assessment program in place for

GM crops works well!

Documents

The Mammalian Safety Assessment of GM Crops - ILSI …ilsi.org/hesi/wp-content/uploads/sites/11/2016/05/6-7-Ladics-Tox... · GMOs for 21st Century –part of the ... • The overall