Evaluating risks with transgenic and gene

edited foods
• Explore implications of intended changes from adding new protein
• Survey unintended changes including accidental genetic alterations
elsewhere from the insert.
Experimental protein toxicology of intended
change
• 18.7 Chemical Risk Evaluation Involves Investigating the Relationship
between Degree of Exposure and Harmful Effects
• Novel proteins can be produced in bacteria, purified, and fed to test
animals to establish their safety for humans. Particular attention is
given to the possible allergenicity of novel proteins.

03/24/2023 2
Experimental safety testing of novel proteins
is possible in animals
• Concepts used to provide empirical evidence of compound safety
from animal tests
• NOEL In animal testing, the “no observed effects dose level.” A high NOEL
value implies low acute toxicity in the test animals (often rodents).
• exposure margin The ratio of the observed NOEL (μg/kg animal body mass
per day) to estimated possible human exposure (μg/kg of human body mass
per day). A high exposure margin means a high safety factor for human
exposure.

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 Tribe 2018

03/24/2023 4
 Flow chart summarising the weight-of-evidence approach for assessment
of allergen risks of newly expressed protein in genetically engineered
organisms. Sources: NAS 2016 , Wal JM 2015.

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Sources of evidence
• Rests of systematic bioinformatic classification of food allergens into
a limited (~12) group of protein families by sequence similarity
• Rapid pepsin digestion (verified by experiment) of candidate protein
under low pH conditions is required as a measure of protection
• Food allergy rests on generation of a particular class of
immunoglobulin (IgE)
 Focusing attention on
evaluating real harms in the
community
Wednesday week 3. 2019
Main points
• Health risks fall on a spectrum from essential strongly verified
causation to uncertain blips in noisy background
• There are challenges in analyzing real world data
• There is a need to consider unavoidable tolerated background genetic
risk (food genetics)
• Misleading inferences are a major distraction using precious scientific
resources badly (vaccines, GMOs)
• Misleading roadblocks can be by-passed by innovation but it is
expensive
Some illustrative data are taken from Kabat
2007 chapter 2 for discussion Thursday
There is a huge spectrum of risks that receive
attention
• Most risks that have been clarified scientifically are those displayed
strong evidence of causation and detectable harm
• The causes of smoking and cervical cancer are the gold-standard
• Many risks discussed widely are ill-defined and at best based on
debatable evidence of harm an miniscule real absolute risk at
background noise level
• Vaccines and GMO food fall into this end of the spectrum
From the weeks required reading
Kabat 2007 11
From the weeks required reading Kabat 2007
 Bradford-Hill considerations for
identification of intrinsic chemical toxicity

• Strength of association between potential risk factor and adverse

outcome
• Consistency of findings from different studies
• Biological gradient shown by a strong dose-response relationship
• Temporal sequence (cause precedes an effect)
• Biological or theoretical plausibility via a characterized mechanism of
action
• Coherence with established knowledge
• Specificity of association, with cause tightly linked to an outcome
After Klaassen 2013 8th ed
03/24/2023 13
A risk issue worth taking about: Prevention of
all cervical cancer
• Strains of human papilloma virus HPV are proven to cause virtually all
cervical cancer
• A triumph of modern biotechnology in identifying and proving HPV as
the essential cause of cervical cancer, enabling superb diagnosis tools,
and the means for elimination of the disease (vaccination)
• Illustration that proof of causation can be scientifically challenging, it
can decisively solved by use of broad search for evidence and
methods of strong inference
• Excellent review Kabat 2016 Chapter 7

14
Kabat 2016 HPV comments in Chapter 7

15
HPV (after Kabat 2016)
• Many different types of HPV
• HPV infects ~10 percent of women world-wide
• Only some HPV types (15/100) cause cancer, most commonly they are HPV-16
and HPV-18
• Proof of this took many years of patient experimentation
• Disease has complex long progression (natural history) from a pre-cancerous
lesion
• Decisive evidence includes (see following slides for details)
• Extremely high relative risk associated with types 16 plus 18
• Strong association between vrus in tissues samples and malignancy
• Convincing evidence of causal mechanism fron vral gene insertion in host DNA
Challenge of interpretation of death rates in a cohort study of 1314 women in a
North England town subjected to 20 years of follow-up (Appleton et al 1996)

Age Smokers death numbers Non-smokers death

numbers
Overall 139/582 (24%) 230/732 (31%)
ODDS Ratio =0.23/.31
->
0.76
18-24 2/55 (4%) 1/62 (2%)
25-34 3/124 (2%) 5/157 (3%)
45-54 27/130 (21%) 7/121 (15%)
55-64 51/115 (44%) 12/78 (33%)
65-74 29/36 (81%) 101/129 (78%)
75+ 13/13 (100) 64/64 (100)

21
Association between exposures and disease -
> Odds Ratio (a/b)/(c/d)

22
Standard observational approach, null result
for odds ratio is 1

23
Challenge of interpretation of death rates in a cohort study of 1314 women in a
North England town subjected to 20 years of follow-up (Appleton et al 1996)

Age Smokers death numbers Non-smokers death

numbers
Overall 139/582 (24%) 230/732 (31%)
ODDS Ratio =0.23/.31
->
0.76
18-24 2/55 (4%) 1/62 (2%)
25-34 3/124 (2%) 5/157 (3%)
45-54 27/130 (21%) 7/121 (15%)
55-64 51/115 (44%) 12/78 (33%)
65-74 29/36 (81%) 101/129 (78%)
75+ 13/13 (100) 64/64 (100)

24
Conclusion: Population age distribution at
start of study is a confounder

25
Caveat to population studies with polygenic risk
scores (Assignment group 6, genetic profiling)
• Some publications warn of misinterpretation of evidence based on
assuming a homogeneous human population when in practice human
demography is structured (stratified) in subtle and diverse ways
Observational studies are challenging-
confounding factors can interfere
• Age is confounder for observations on smoking and mortality
• Smoking is a confounder for the association between coffee and
cancer
• Other pesticides are confounders for the association between
glyphosate herbicide exposure to farmers and non-Hodgkins
lymphoma

27
28
Speculative false correlations and inferences
interfere with accurate information
• Mercury containing preservatives and autism
• Vaccines and autism
• Pesticides used for mosquito control and microcephaly in Brazil
• GE mosquito release and microcephaly in Brazil

29
Cells in a Petri dish are not people, and experiments with cells
can easily give the wrong answers, GMO Pundit Feb 18 2012
• http://gmopundit.blogspot.com.au/2012/02/cells-in-petri-dish-are-not-people-and.html
• Science-in-reverse can get you into trouble, and put the cart before the horse
Paul Offit uses the expression "science-in-reverse" to explain how simplified biological
experiments can give misleading answers about causes of autism. He's referring to the logical trap
of jumping the gun, and using "in vitro" experiments with isolated component of the human body
exposed to mercury compounds in Petri dishes to formulate a presumptive mechanism for how
the disease occurs, before checking carefully and conclusively that exposure to mercury
compound in a living human actually does cause autism.

NEED TO TRANSLATE IN VIVO SITUATION TO RELEVANT IN VITRO ANALYSIS

30
Where does GE (transgenic) food fit using
these criteria?
• Need to distinguish intended from unintended changes
• It is a potential hazard
• Risk has to be examined partly as a relative risk compared to very low
level background risk already tolerated in existing food with a HOSU.
• The animal feeding studies mentioned for Cry proteins are part of this
assessment as are allergy evaluation of those changes
• The other aspect is whether transgenic food demonstrates higher
unexpected genetic changes than seen in nature and this is discussed
in required reading Custers 2019 to be introduced Thursday.
A few main points as a lead in the Custers
• Massive levels of genetic variability are abundantly documented in
crop genomes and transgene (or gene-edit) changes are relatively
small
• At least one food– sweet potato is a natural transgenic crop with
bacterial Agrobacterium DNA in it.
• About one novel gene in rice evolves de novo every 20000 years of
history (Zhang 2019)