United States
Environmental Protection
Agency
RESEARCH AND DEVELOPMENT
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EPA 600/R-03/068
March 2005
U.S. Environmental Protection Agency
Office of Research and Development
Washington, DC 20460
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Notice
This report has been reviewed and approved for release by the Office of Research and Development. Approval
does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of
trade names or commercial products constitute endorsement or recommendation for use
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Contents
Biotechnology Initiative Steering Committee
Foreword vi
Evaluating Potential Risks Associated with Biotechnology Products 1
Background I
Goal 2
Research Approach 2
Key Science Issues with Respect to Biotechnology Products 4
1. Potential Allergenicity of Proteins Introduced into the Food Supply by Gene Transfer 4
2. Potential Ecological Effects of Biotechnology Products on Non-Target Species 5
3. Escape of Altered Plants to the Natural Environment and the Likelihood and Effects of
Gene Transfer 6
4. The Development of Pesticide Resistance in the Target Insect Species 7
5. Risk Management 8
Performance Results and Expected Benefits 8
Projected Outputs 8
Appendix A. Project Sheets Al
1. Potential Allergenicity of Proteins Introduced into the Food Supply by Gene Transfer A2
Potential allergenicity of genetically modified organisms A3
Solicitations through the STAR extramural program to support EPA's
Biotechnology Research Program A6
2. Potential Ecological Effects of Biotechnology Products on Non-Target Species A8
Non-target and ecosystem impacts from genetically modified crops containing
plant-incorporated protectants (PIPs) A10
Genetic evaluation of long-term risks of plant-incorporated protectants:
exposures and effects on non-target species A12
3. Escape of Altered Plants to the Natural Environment and Likelihood and
Impact of Gene Transfer A15
Evaluating gene flow from genetically modified crops and its potential
ecological effects A16
4. The Development of Pesticides Resistance in the Insect Target Species A20
Genetic evaluation of long-term risks of plant-incorporated protectants:
evolution of resistance in targeted pests A21
Field assessment of insecticide resistance management (IRM) for
plant-incorporated protectants (PIPs) A23
5. Risk Management A25
Development of strategic monitoring programs for ecological impact from
plant-incorporated protectants (PIPs) A26
Development of management and field-scale tools to manage the risks of
gene transfer and non-target effects from PIP crops to the environment A28
Development of management and field-scale tools to manage and delay
the development of insect resistance to PIP crops by extending crop life A30
Biotechnology Demonstration Project A33
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Acronyms
ARS Agricultural Research Service
BLM Bureau of Land Management
BPPD Biopesticides and Pollution Prevention Division
Bt Bacillus thurigeiensis
CRADAs Cooperative Research And Development Agreement
DOI Department Of the Interior
EPA Environmental Protection Agency
FDA Food and Drag Administration
FIFRA Federal Insecticide, Fungicide and Rodenticide Act
GIS Geographic Information System
GM Genetically Modified
GMO Genetically Modified Organism
GSF National Research Center for Environment and Health
IPA Intergovernmental Personnel Act
IRM Insecticide Resistance Management
NT AID National Institute of Allergy and Infectious Diseases
NAS National Academy of Sciences
NCEA National Center for Environmental Assessment
NCER National Center for Environmental Research
NERL National Exposure Research Laboratory
NHEERL National Health and Environmental Effects Research Laboratory
NIEHS National Institute of Environmental Health Sciences
NIH National Institutes of Health
NPS National Park Service
NRC National Research Council
NRMRL National Risk Management Research Laboratory
OPP Office of Pesticide Programs
OPPT Office of Pollution Prevention and Toxics
OPPTS Office of Prevention, Pesticides, and Toxic Substances
ORD Office of Research and Development
PCR Polymerase chain reaction
PIP Plant Incorporated Protectant
R&D Research and Development
RFA Request for Assistance
STAR Science to Achieve Results
TSCA Toxic Substances Control Act
USDA U.S. Department of Agriculture
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of
Lawrence W. Reiter, and Environmental Laboratory
R. Fowle 111, and Environmental Effects
Mark Bagley, Laboratory
Fairbrother, National and Environmental Laboratory
Z. Francis, Center for
J. Frederick, Center for Environmental
John Glaser, National Laboratory
Health and Environmental Effects Laboratory
Subhas Sikdar, Risk Laboratory
Uwe Stolz, Exposure Laboratory
Lidia Watrad, and Environmental Laboratory
Ward, and Environmental Effects Laboratory
of Prevention, Toxic
L, Andersen, Office of Pesticide Programs
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In accordance with the Environmental Protection Agency's (EPA's) mission to minimize risks to
human health and to safeguard ecological integrity, the EPA Office of Prevention, Pesticides, and Toxic
Substances (OPPTS) is committed to assessing and mitigating any risk posed by biotechnology-derived crops.
Consequently, the Biotechnology Initiative Steering Committee prepared this plan to guide the implementation of
an integrated Biotechnology Research Program. This plan was formulated with the goal of improving the science
needed to inform Agency decisions about the risk and safety of the products of biotechnology.
Many of the scientific concerns about the risk and safety of genetically engineered crops were raised in
three reports1 from the National Research Council (NRC) of the National Academy of Sciences (NAS). These
scientific concerns served as a departure point for the development of this implementation plan. There are three
main areas of concern: (1) risks to human health of the allergenicity of biotechnology products, (2) risks to
natural ecosystems of gene transfer from engineered organisms to natural species in the wild, and (3) mitigating
the development of resistance and of gene transfer. The breadth of scientific issues related to the safety of
bioengineered crops, as well as limited resources, make it imperative to have a focused, integrated, cross-Office
of Research and Development (ORD) research program that coordinates with ongoing research programs in other
federal agencies. We also needed to ensure that the work funded under this research initiative complements work
underway in other sectors.
The Biotechnology Initiative Steering Committee convened a workshop to meet with colleagues from
OPPTS and key senior officials from other federal agencies, the National Academy of Sciences, and the European
Union to discuss the EPA's proposed Biotechnology Research Program. During our discussions, we wanted to
determine the following: (1) Is our proposed program scientifically sound and relevant? (2) Does it complement
related efforts elsewhere? and (3) Is the proposed work appropriate for EPA? The conclusion by non-EPA
attendees was that our proposed research program met these objectives. We also discussed opportunities for
inter-agency collaboration.
The Plan was then peer reviewed by the Board of Scientific Counselors May 13, 2004. This document
reflects their comments.
'NRC, 2000. Genetically Modified Pest-Protected Plants: Science and Regulation, Washington, DC: National Academy Press.
NRC, 2001. Ecological Monitoring of Genetically Modified Crops: A Workshop Summary, Washington, DC. National Academy Press.
NRC, 2002. Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation, Washington, DC: National Academy Press.
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Background
Biotechnology presents a wealth of opportunities
to improve crop productivity, nutritional value, and
resistance to pests and other stresses. However, there
are potential risks to human health, natural ecological
systems, and existing agricultural systems that need
to be evaluated so that the products of biotechnology
can be properly regulated. Currently, EPA regulates
several biotechnology products (e.g., pesticides,
either produced by plants or by microorganisms,
and non-pesticidal substances such as industrial
enzymes, biosensors, and bioremediation agents
produced using microorganisms). While discussions
continue about whether EPA's scope of regulation
should be broadened to include animals (e.g., insects)
that produce pesticidal substances and plants and/or
animals that produce non-pesticidal substances, no
such products are currently under review by EPA.
From a human-health perspective, a major concern
is the potential toxicity and allergenicity associated
with genetically modified foods. Potential adverse
effects can arise from intended modifications
(i.e., from the pesticidal substance) or from
unintended effects resulting from the production
of an unexpected metabolite. To date, the products
approved by EPA for use in human food have all
been proteins that degrade rapidly and from which
no chronic effects would be expected. This approach
has been accepted by the Federal Insecticide,
Fungicide, and Rodenticide Act (FIFRA) Scientific
Advisory Panel. However, some members of the
public have raised the concern that proteins not
previously part of the food supply could be allergens.
It is, however, well accepted that the genetic material
itself will not cause an acute or chronic toxic effect;
thus DNA has been exempted from tolerance.
The regulation of biotechnology products is also
intended to minimize the risks to human-managed
(i.e., agricultural) and natural ecosystems. Such
risks are associated with the consequences of
unintended release of genetically modified plants
or their bioengineered genes. For example, there
are concerns about the ecological impacts resulting
from replication and persistence of transformed
organisms that could out-compete native species
in a given environment. In terms of the risks to
agricultural systems, there are potentially adverse
long-term consequences of evolved resistance to
the biotechnology product. Pest resistance could
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render related conventional products (e.g., the spores
at Bacillus thurigiensis [Bi] used as pesticidal
sprays) ineffective, reducing crop productivity
or necessitating increased usage of conventional
pesticidal applications (which also threatens
ecosystem health).
Goal
The goal of the Biotechnology Research Program
is to provide the scientific information needed to
assess and manage the risks of biotechnology. The
research program will accomplish this by providing
the tools needed to generate information about
biotechnology products, by generating the knowledge
needed to understand the nature and magnitude
of potential risks and benefits resulting from the
use of biotechnology products in commerce, and
by providing the means to prevent or control such
risks. At this time the focus is on the risk from plant
incorporated protectants (PIPs).
The Agency Challenge
The Office of Prevention, Pesticides and Toxic
Substances (OPPTS) evaluates the environmental
risks posed by pesticides and chemicals to safeguard
all Americans, including children and other
vulnerable members of the population, as well
as our most threatened species and ecosystems.
Within OPPTS, the Office of Pesticide Programs
(OPP) regulates the use of all pesticides in the
United States and establishes maximum levels
for pesticide residues including genetically
engineered pesticides. While it is not anticipated
that biotechnology products will pose new types
of risks, these new products are often on the
cutting edge of science and regulatory policy; and
research is needed to ensure that their safety can be
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appropriately evaluated. Also, within OPPTS, the
Office of Pollution Prevention and Toxics (OPPT)
regulates the use of industrial chemicals and certain
biotechnology products, such as microorganisms
used in the manufacture of specialty chemicals and
bioremediation agents. OPPT also implements the
Pollution Prevention Act and hence has an interest
in biotechnology product stewardship that would
lead to "green" chemicals. OPPT has an emerging
interest in certain transgenic plants for uses such as
phytoremediation and enhanced wood production
although OPPT does not implement regulatory
oversight in this area at this time. Currently, most
biotechnology risk assessment research concerns in
OPPTS are affiliated with pesticidal products.
In assessing safety, the basic framework for pesticide
regulation provides guidance as to the nature of
any new risks. EPA has recognized that PIPs (or
genetically engineered plants which produce their
own pesticides) represent potentially different risks
from traditional, chemical pesticides. For example,
while there is very low worker exposure and no
spray drift, there are issues regarding gene flow to
wild relatives and pollen movement spreading the
new pesticides to non-altered crops. In addition, the
level of protein produced is very small; but, because
proteins can be allergens and even low levels of a
new protein might lead to sensitization and eventual
allergic reactions, special emphasis on allergenicity is
given to evaluation of these products.
With respect to environmental risk, effective tools
and methods are needed to minimize the likelihood
of negative ecological effects such as the following:
Ecosystems
(a) harm to non-target species, such as soil
organisms, non-pest insects, birds, and other
animals;
(b) disruptive effects on specific biotic
communities;
(c) irreparable loss of changes in species diversity
and genetic diversity within species.
Agri-Systems
(a) creating new or more vigorous pests and
pathogens;
(b) exacerbating the effects of existing pests
through hybridization with related transgenic
plants or microorganisms.
Both
(a) pleiotropic or epistastic effects on plant
physiology due to emerging metabolic
engineering approaches. [These manipulations,
found in current commercialized transgenic
organisms, may result in unintended effects
in host plants or non-target plants that may
inadvertently receive the transgene.];
(b) rapid development of resistance to the
engineered crop by target pests that may result
in greater use of more harmful pesticide
products over the long term.
With respect to protecting human health, EPA
must assess whether pesticides derived through
biotechnology are at least as safe as their conventional
counterparts; and the EPA must ascertain that any
levels of additional or unique risk are clearly defined.
A significant challenge may occur in the future if
transgenic technology results in more substantial
and complex changes in exposures and/or if such
technology results in compounds that are more toxic.
It is important to note that many pesticidal substances
such as phenols and aldehydes occur naturally in
plants.
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Progress also needs to be made in developing
definitive methods for the identification and
characterization of protein allergens. EPA needs
to be able to estimate accurately the levels of
exposure to the genetically engineered products
that are released in the environment, and EPA needs
the means to evaluate whether such exposures are
potentially harmful. Finally, EPA needs to find
and evaluate ways to prevent or mitigate identified
risks.2 Contributing to the effectiveness of this
program are the integration of science activities
across the risk assessment paradigm and strong
interaction with OPPTS. The proposal also includes
the use of workshops involving scientists with
appropriate expertise from academia, industry, and
other government institutes. These workshops are
designed to develop broad consensus with respect
to research needs and strategies and to coordinate
research efforts not just within EPA, but with the
National Institutes of Health (NIH), the Food and
Drug Administration (FDA), the U.S. Department of
Agriculture (USDA), and the scientific community at
large.
(5) strategies for identifying the risks of concern
and effective risk management technologies to
mitigate these risks when monitoring studies indicate
unintended adverse consequences are likely to occur.
A problem-directed research program has been
developed that focuses on five key issues: (1) the
potential allergenicity of proteins introduced into
the food supply by gene transfer; (2) the potential
ecological effects of biotechnology products on
non-target species; (3) the spread of transgenes to the
natural environment via seed dispersal or gene flow-
to sexually compatible relatives; (4) the development
of pesticide resistance in the target species; and
-Under both FIFRA and TSCA. EPA also has an obligation
to consider the potential benefits of biotechnology pesticide
products.
1.
the by
There are no valid animal models for predicting
allergenicity and the long-term effects from
consuming crops containing PIPs and other
biotechnology products. EPA and FDA are currently
using several screening assays designed to compare
new substances to the properties of known dietary
allergens. However, current screening criteria will
not identify some substances using this approach.
Research Need:
Develop an understanding of the basis for
human semitization to dietary allergens,
develop methods to assess dietary
allergenicity, and apply such methods
to monitor human populations exposed
to genetically modified (GM) foods for
increased allergenicity.
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2. of
on
Species
(a) Questions have been raised about the
effects of PIPs on non-target species. Besides
potential direct toxicity to insects, concerns have
been raised that PIPs are so effective at controlling
insect pests that bird populations might be adversely
affected because of a lack of" food. However, recent
data indicate that the number of song birds increase
in areas where Bt cotton use is high. The data
suggest Bt cotton may increase available food supply
as well as reduce avian mortality (Brandt, et al., EPA
Public Interest Finding and Review of the Benefits
for Monsanto Company's Corn Rootworm-Protected
Field Cora Product [Event MON 863], February 19,
2003).
Research Need:
Baseline and monitoring studies to assess
the structure and dynamics of populations
of beneficial organisms and insect
pests in and around crops grown using
conventional, organic, and biotechnology
pest-management tools.
(b) There are few standardized, validated.
affordable field assays for assessing exposure and
effects on non-target species. This is a significant
gap in our understanding of potential unintended
consequences.
Research Needs:
(i) Develop methods to evaluate the
scale over which biologically meaningful
exposures to non-target species may occur
and the mechanisms (e.g., pollen, insect
movement, predation) that mediate this
exposure;
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(ii) Develop an understanding of the
mechanisms that lead to adverse effects in
non-target species (e.g., honeybees) as a
basis to develop standardized, validated,
affordable field, assays for testing for
effects on important species; and
(Hi) Develop an understanding of the
effects of metabolism in genetically
altered plants as a basis to assess risk and
to develop methods for testing genetically
altered plants for such changes.3
(c) Questions have been raised about the
effects of genetically engineered microorganisms
on non-target species. Recombinant bacteria have
been reviewed by OPPTS for use in biosensors
and bioremediation applications as they apply to
hazardous wastes such as polynuclear aromatic
hydrocarbons and poly chlorinated byphenyls.
Research Need:
Genetically engineered microorganisms
have raised issues of non-target effects
and opportunistic pathogenicity for
aquatic species, wildlife, and, humans.
Better methodologies to detect such
effects prior to release andto monitor for
ecological effects in the field are needed.4
'Increasingly, transgeiiics are being developed with altered
metabolic or signaling pathways, and such constructs may have
secondaiy effects on plant physiology due to pleiotropy or
epistasis. For instance, plants are being developed for altered
metabolism (chemical production, altered wood production,
salt tolerance, and phytoremediation), as well as for novel pest
resistance.
4OPP has reviewed genetically engineered bacteria for use as
pesticides. OPP has relied on the existing data requirements for
microbial pesticides although additional research was conducted
by OPd3 in the past. Future research in this area would be of
value. This need is not being addressed in this research plan.
3. to the
the
(a) The ability of some crops to transfer
introduced genes through hybridization to wild
and/or weedy relatives can make the assessment of
effects on non-target organisms very difficult. There
is one report on Arabidopsis thaliana, which does
not normally outcross, in which a transgenic plant
"... showed a dramatically increased ability to donate
pollen to nearby wildtype mothers compared with
Aribidopsis thaliana mutants expressing the same
mutant allele as the transgenic plants" (Bergelson, et
al.,1998, Promiscuity in Transgenic Plants, Nature
v. 395, p. 25).
Research Need:
Explore the factors influencing gene
transfer rates to provide a basis for better
assessments.
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(b) Some transgenes may have a limited
persistence due to their insert locations.
characteristics of the genetic cassette, or the plants
or microorganisms themselves. This could result in
limiting exposure to the gene product.
Research Need:
Develop methods to evaluate the
persistence/maintenance of transgenes in
plants and microorganisms, the exposure
to those gene products, and, evaluate
whether environmental conditions or
common stressors influence this process.
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Laboratory and small-scale field testing has been
the basis for evaluating the likelihood of resistance
development. Long-term, extensive monitoring has
not been conducted to determine whether the effects
predicted in such tests actually occur in the field.
Research Needs:
(i) Develop models to estimate the
likelihood of the development of insect
resistance that incorporate detailed
biological information for pest species,
including gene flow and, mating patterns
in the wild, geographic and chromosomal
distribution of resistant alleles, and their
additive and non-additive effects on
resistance under selective pressures in the
field; and
(ii) Perform monitoring studies of gene
transfer, the development of resistance to
PIPs by target pests, and. effects on non-
target species (as noted in 2b) to allow
field validation of conclusions regarding
tmnsgenic plants with new pesticide
trails.
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5.
The effectiveness of management strategies to avoid
key risks for extended periods and the effectiveness
of risk management technologies to mitigate risks
associated with unintended adverse consequences
must be evaluated and expanded for new PIP crops.
Research Needs:
(i) Improve strategies to identify the key
risks of concern, develop evaluation
schema to understand the effectiveness
of management strategies, and develop
effective risk management technologies to
mitigate these key risks when monitoring
studies identify the presence of unintended,
adverse consequences; and
(ii) Explore the application of
socioeconomic methods such as benefit-
cost analysis and life-cycle analysis to
better understand issues related to the
public acceptance of genetically altered
products and. to evaluate whether the
genetic alterations produce new organisms
that are not substantially equivalent to
currently existing ones.
By FY 2008, this research program will result in an
improved capability to assess the risks of allergenicity
from GM food, improve the capability to assess the
ecological risks associated with genetically modified
organisms (GMOs), and develop tools to understand
and better manage gene transfer and resistance.
Program performance will be measured in the
following ways:
(a) the use of research products by OPPTS
in the registration and re-registration process,
both in hazard identification and risk
assessment, and in setting risk management
requirements for registration;
(b) general acceptance of these methods by
other regulatory agencies; and
(c) public acceptance of EPA's approach to
regulating GM crops.
The budget for the biotechnology program allows
EPA to begin to address these important issues, but
it is not sufficient to address them all. Consequently,
ORD used the priorities provided by the program
office and a scientific-program view to bring together
the research plan described here. The research will
be limited to PIPs. It will not cover genetically
engineered microorganisms and plants genetically
engineered for non-pesticidal purposes. Those areas
which are not accommodated at this time may be
revisited as a part of the regularly scheduled progress
reviews and incorporated in the program as resources
allow.
The identification of the specific projects that will
be performed by each ORD Laboratory and Center,
as well as those that will be identified for funding
by the Science to Achieve Results (STAR) program,
have been developed by the Biotechnology Initiative
Steering Committee and are included in Appendix A.
The anticipated outputs include the following:
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Endpolnt
&
or
on
'It
Need For
For
NRC (National Council). 2000. Genetically Pest-Protected Plants:
Science Regulation, Washington, DC. National Academy Press.
NRC (National Research Council). 2002A. Environmental Effects of Transgenic Plants:
The Scope and Adequacy of Regulation, Washington, DC. National Academy Press.
NRC (National Council). 2002B. Animal Biotechnology:
Science-Based Concerns, Washington, DC. National Academy Press.
4.
Models to predict dietary allergenicity from
consuming crops containing PIPs and other
biotechnology products will be developed
and verified as a basis to the
potential allergenicity of proteins introduced
into the food supply by gene transfer;
Standardized, verified, and affordable assays
to test for exposure and effects of PIPs on
non-target species as a basis to assess the
potential ecological and other effects from
the use of biotechnology;
Explicit information on pest genetics and
ecology that will validate or improve existing
models to predict development of resistance;
The identification of factors influencing
transfer rates to better understand the
6.
potential for altered plants to escape into the
natural environment and methods to evaluate
the persistence/maintenance of transgenes in
plants as a basis to understand the likelihood
and effect of gene transfer;
The likely safety of biotechnology products
will be evaluated by long-term monitoring
to determine whether the effects predicted in
the laboratory and small-scale tests actually
occur in the field as a basis for evaluating the
likelihood of pesticide resistance
development; and
Risk management strategies to provide
means to mitigate risks when unintended
adverse consequences occur.
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This Appendix contains project which describe the work we to accomplish In to the
three major concerns facing EPA: (1) allergenicity, (2) transfer, (3)
transfer. We have developed five major research As the outpace available resources, It will not be
possible to meet all the needs, nor will it be possible to fully cover Is under plan.
The five major research need(s) are listed followed by project
the related priority work EPA will cover under its Initial biotechnology efforts.
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1
Potential Allergenicity of Proteins Introduced the Food Supply by Gene Transfer
There are no valid models for predicting dietary allergenicity and the long-term effects from consuming
crops containing PIPs and other biotechnology products.
Research Need:
Develop an understanding of the basis for human sensitization to dietary allergens, develop methods
to assess dietary allergenicity, and apply such methods to monitor human populations exposed to
genetically modified foods for increased allergenicity.
There are two projects addressing key research area:
1. Potential allergenicity of genetically modified organisms.
2. Solicitations through the STAR extramural program to support EPA's Biotechnology Research
Program.
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Project Title:
Laboratory/Center:
Program Contact:
Potential AHergenicIty of Genetically Modified Organisms
NHEERL
Mary Jane Belgrade, (919) 541-1821, selgrade.maryjane@epa.gov
Primary Research Objective/Goal: The goal is to develop an animal model suitable for assessing potential
allergenicity relative to other food proteins and for testing hypotheses regarding conditions (e.g., age, genetics)
that contribute to susceptibility. This project focuses on the development of an animal model to predict
allergenicity and will not address the need to monitor human populations.
Background: Biotechnology presents a wealth of opportunities to genetically engineer crops to improve
productivity, enhance resistance to pests and other stressors, and provide nutritional value. However, there is
growing concern that there may be risks to human health that have not been adequately explored. The biggest
concern is that, as a result of the introduction of novel proteins into the food supply, biotechnology may
unwittingly introduce a potent food allergen that could seriously affect the health of susceptible individuals.
Currently, there is no animal model that can be used to test proteins for potential allergenicity following oral
exposure, nor are there other means to readily identify proteins that might be potent allergens. Furthermore,
the mechanisms underlying the development of food allergy and the factors that contribute to individual
susceptibility are poorly understood.
Project Description and Critical Path: EPA sponsored a workshop jointly with FDA and NIEHS entitled
"Assessment of the Allergenic Potential of Genetically Modified Foods" in December 2001 to review the state
of the science and to develop research needs. The development of an animal model to assess the potential
allergenicity of orally administered proteins was determined to be a major research need. Such a model or
models are needed to answer the following questions:
(1) Does dietary exposure to transgenic pesticide
proteins induce immune, inflammatory, and
histopathology responses typical of food allergy?
(2) Is the degree of digestibility inversely related to
risk of allergenicity?
(3) Is early life the most vulnerable time for dietary
allergy sensitization?
(4) Does the food matrix make a difference in
allergic responsiveness?
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(5) How potent is the transgenic pesticide protein in the induction of dietary allergic responses (i.e., what is the
dose-response relationship relative to known food allergens)?
(6) Where there is potential for both respiratory and oral exposure, what are the risks when an individual
sensitized by the respiratory route ingests the protein; and what are the risks of respiratory exposure in an
individual sensitized by the oral route?
The following critical will be followed In order to address questions:
(1) Develop a dietary allergy model in a laboratory rodent using a modification of the respiratory allergy
protocols.
a. Suckling, weanling, and adult rodents (BALB/c mice or Brown Norway rat) will be exposed by
gavage or fed multiple times with various doses of a known food allergen to establish the ability to
induce food-allergy responses. Allergic responsiveness will be judged based on the induction of antigen
specific IgE and IgAin addition to gut mucosal eosinophil influx and respiratory responses. The lung
and skin are the most frequent target organs even when the route of exposure is ingestion. Experimental
conditions that most closely mimic food allergy in humans will be used in subsequent studies.
b. Once the model is developed, rodents will be fed or gavaged multiple times with various doses of a
prototype transgenic pesticide protein. The most likely candidate is the Bt toxin. Various forms of this
toxin with varying degrees of digestibility will be tested. Allergic responsiveness will be assessed based
on results obtained from the above studies. Appropriate positive and negative controls will be
incorporated into the model.
(2) Assess the responses to the transgenic pesticide protein allergen in both a purified form and in a food matrix.
The food matrix is the way in which most human ingestion will occur, and it may provide an adjuvant effect.
Therefore, exposure to the purified protein alone may not be adequate to assess its potential allergenicity.
Using the model protocol, rodents will be gavaged with both the purified protein and an equivalent amount
of the protein in a food matrix. Comparison of the responses should provide insight into the role of the food
matrix in dietary allergy.
(3) Assess the relative potency of transgenic pesticide proteins when compared to known food allergens. Using
the model protocol, responses to transgenic pesticide proteins will be compared to the responses of a range
(strong to weak allergy inducers) of established food allergens.
(4) Use the model to assess effects of respiratory exposure following oral sensitization and oral exposure
following respiratory sensitization.
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Schedule:
Publish Workshop Proceedings:
Begin work to develop animal model:
Hire first post-doc:
Develop animal models
Test Bt toxin & digestibility theory
Demonstrate vulnerability of newborns
Projected Outputs/Impacts:
February 2003
Spring 2003 (Depends entirely on when the animal facility
in the new building opens)
By end of 2003
2005
2006
2008
FY 2008 APG:
FY 2003 APM:
FY 2005 APM:
FY2008 APM:
Improved capability to assess the risks of allergenicity from genetically altered food.
Publish results of jointly sponsored (FDA& NIEHS) workshop.
Develop models and methods for assessing potential allergenicity.
Demonstrate the vulnerability of newborns/ identify windows of vulnerability.
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Project Title: Solicitations through the STAR Extramural Program to Support
EPA's Biotechnology Research Program
Laboratory/Center: NCER
Program Contact: Elaine Francis, (202) 564-6789, francis.elaine@epa.gov
Primary Research Objective/Goal: Engage the external research community to assist EPA's
understanding of the basis for human sensitization to dietary allergenicity and to develop methods to assess
dietary allergenicity.
Background: EPA has developed a problem-driven Biotechnology Research Program that focuses on
five key issues of most importance to the Office of Prevention, Pesticides, and Toxic Substances (OPPTS):
(1) potential allergenicity of proteins introduced into the food supply by gene transfer; (2) potential
ecological effects of biotechnology products on non-target species; (3) escape of altered plants to the natural
environment and likelihood and effects of gene transfer; (4) the development of pesticides resistance in
the target species; and (5) strategies for identifying the key risks of concern and effective risk management
technologies to mitigate these key risks when the monitoring studies indicate unintended adverse
consequences are likely to occur.
The extramural grants program through the Science to Achieve Results (STAR) program will issue a
solicitation(s) for proposals from scientists in academia and not-for-profit organizations for research that
will complement our intramural research program. Those proposals that are of highest quality, of greatest
relevance to the Agency, and that provide a balance to EPA's Biotechnology Research Program's portfolio
will be awarded.
Project Description and Critical Path: The Biotechnology Research Program framework has
identified a number of research needs under each of the five key issues identified above. ORD's intramural
program does not have the capacity nor the capability to address all of these needs. ORD will use its
STAR program to engage the academic and not-for-profit community to help address the critical data gaps.
USDA has been issuing solicitations for extramural proposals for the last several years in four of the five
key areas in which EPA is interested. The issue not covered in the USDA solicitations is the one dealing
with potential allergenicity. This is also an area which has limited intramural capacity and is an area of
extremely high importance to OPPTS. It seems most appropriate, therefore, to consider this issue as the
highest priority for which a solicitation will be developed. NCER will work with USDA, NIATD, and
NIEHS to determine whether they are interested in issuing a joint solicitation. If they are not, NCER will
issue an Request for Assistance (RFA) by itself. The specific topics of interest related to the key issue of
allergenicity will be developed working with the Biotechnology Research Planning Committee to ensure
that the solicitation does not duplicate what can best be done intramurally and that it targets specific topics
of highest priority that would complement the intramural research.
-------
Schedule:
Potential partners from other federal agencies who are interested in leveraging resources and issuing a joint
solicitation will be sought starting in FY03. A solicitation supported by either EPA alone or by EPA in
conjunction with other federal agencies will be issued in FY04. Awards of grants will be made in FY04-05. If
EPA issues a solicitation by itself, then three years' worth of resources will be needed to support a reasonable
extramural program. Therefore, only one solicitation would be issued in the FY04-06 period. If however, other
federal partners are interested in leveraging resources, it may be possible to issue a second solicitation during
this period of time.
Projected Outputs/Impacts:
FY 2004 APM: Issue a solicitation for research proposals.
FY 2008-2009 series of APMs: Reports on individual grant results of extramural research.
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2
Potential Ecological Effects of Biotechnology Products on Non-Target
Questions have been raised about the effects of PIPs on non-target species.
Research Need:
Baseline and monitoring studies to assess the structure and dynamics of populations of beneficial
organisms and insect pests in and around, crops grown using conventional, organic, and biotechnology
pest-management tools.
There is one project addressing key research need:
1. Non-target and ecosystem impacts from genetically modified crops containing plant-incorporated
protectants (PIPs).
There are few standardized, validated, affordable assays for assessing exposure and effects on non-target
species.
Research Needs:
(i) Develop methods to evaluate the scale over which biologically meaningful exposures to non-target
species may occur and the mechanisms (e.g., pollen dispersal, insect movement, predaiion) that mediate
this exposure;
(ii) Develop an understanding of the mechanisms thai lead, to adverse effects in non-target species (e.g.,
honeybees) as a basis to develop a standardized, validated, affordable assay for testing for effects on
important species; and
(Hi) Develop an understanding of the effects of metabolism in genetically altered plants as a basis to
assess risk and to develop methods for testing genetically altered plants for such changes. [Increasingly,
transgenics are being developed with altered metabolic or signaling pathways, and such constructs
may have secondary effects on plant physiology due to pleiotropy or epistasis. These plants are being
developed for altered metabolism (chemical production, altered wood production, salt tolerance, and
phytoremedialion), as well as for novel pest resistance.]
-------
Questions have been raised about the effects of genetically engineered microorganisms on non-target species.
Research Need:
Genetically engineered microorganisms have raised issues of non-target effects and opportunistic
pathogeni city for aquatic species, wildlife, and humans. Better methods to detect such effects prior to
release and to monitor for ecological effects in the field, are needed. [OPP has reviewed genetically
engineered bacteria for use as pesticides. OPP has relied on the existing data requirements for microbial
pesticides although additional research of value was conducted by ORD in the past, and future research
in this area would be of value, ]
One project was developed to meet needs 2bi and 2bii. No discrete projects were developed to meet needs 2biii
and 2c. However, the results from other research areas (e.g., 2a and 3) will likely include or necessitate the
development of the needed assays for the experimental approach.
-------
Project Title:
Non-Target Genetically Crops
(PIPs)
Laboratory/Center:
NCEA
Bob Frederick, (202) 564-3207, frederick.bob@epa.gov
Objective/Goal: The goals are to develop methods to measure direct effects and
secondary trophic level effects on non-target organisms, to characterize assessment endpoint(s), and to develop
predictive strategies to evaluate potential ecosystem-level effects.
Background: The risk of unintended and unexpected adverse effects on non-target organisms and ecosystems
is a key issue in environmental risk assessment of PIP crop plants. While there has been considerable
examination of the effects of Bt crops on certain non-target organisms, particularly using species-specific
laboratory testing, more work is needed to examine effects (or lack of effects) at the field level. Field censuses
documenting species diversity and abundance are important, but they require appropriate baseline studies
against which to compare results from agro- and other-ecosystems containing PIP crop plants. This research
will be structured to answer the questions (1) What are the potential ecological and other effects from the use of
biotechnology products on non-target species? and (2) What are the effective strategies for identifying the key
risks of concern effective risk management technologies to mitigate these key risks when the monitoring
studies indicate unintended adverse consequences?
for
-------
Project Description and Critical Path: NCEA will develop standardized and streamlined methodologies
to conduct baseline assessments of agricultural and near-field ecosystems non-target species diversity and
abundance. In addition to broad field censuses, particular plant and animal species may serve as indicators of
environmental impacts of PIP crop plant releases. Bioindicators may be efficient and sensitive tools to predict
adverse impacts during product evaluation as well as to measure the long-term impacts of environmental
releases. NCEA will identify a suite of ecologically significant indicator species at different trophic levels in,
for example, Bt corn and cotton agro-ecosystems.
While species presence and/or abundance could offer valuable indicators of non-target effects, potential effects
of PIP crop plants should also be examined in terms of ecosystem functions. Relevant ecosystem functions
could include nutrient cycling, predator-prey interactions, and the provision of non-target wildlife habitat. We
plan to develop methods and conduct field assessments of these potential ecosystem-level effects in PIP crop
plants but expect the results will be relevant to environmental releases of other modified crop plants as well.
Schedule:
FY06 APM: Report on a conceptual framework for assessing the ecosystem scale effects
genetically modified crops (NCEA).
FY08 APM: Report on effects ofBt crops on agro-ecosystem functions as a risk assessment
tool (NCEA).
Projected Outputs/Impact: The baseline survey methodology and suite of bioindicators will provide a
needed framework for on-going research requirements to registrants and will be valuable in regulatory decision-
making and long-term environmental monitoring of PIP crop plants by the EPA Office of Pesticide Programs
and possibly other offices. Results of the ecosystem function studies will allow more meaningful interpretation
of monitoring results and will support more accurate assessment of ecosystem impacts from PIP crop plants.
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Project Title:
Laboratory/Center:
Program Contact:
Genetic Evaluation of Long-Term of Plant-Incorporated
Protectants: Exposures and Effects on Non-Target Species
NERL
Mark Bagley, (513)569-7455, bagley.mark@epa.gov
Background: EPA has been given the mandate to assess the environmental risks of GM crops based on the
best available information. Presently, however, the information available to make meaningful decisions about
long-term environmental risks of GMOs is limited. As crops with new traits and stacked transgenes head
through the development pipeline, both potential benefits and potential risks may increase. It is important that
EPA continue to evaluate the usefulness of its current data to ascertain long-term ecological risks and to develop
new types of data where necessary. A long-term risk of special concern is unintended collateral effects of PIPs
on non-target species. A thorough assessment of the effects of GM crops on surrounding ecosystems is urgently
needed, including research into the types of baseline data required for effective monitoring of ecosystem health.
Project Description and Critical Path:
Exposure of PIPs to non-target organisms. Based on the phylogenetic relationships between the target pest
species and non-target species, as well as the mode of action of the PIP transgene, we will identify indicator
species for exposure monitoring in and around J5i-corn and ^-cotton agro-ecosystems. Building on our
experience in developing targeted gene expression assays as indicators of endocrine disrupting compounds
and other environmental contaminants, we will
develop quantitative PCR assays for exposure to
Bt compounds. Target loci for gene expression
analysis will be identified by a combination of
differential display, screening subtractive cDNA
libraries, and identification of homologous genes
from well-described insect species, including
Drosophila melanogaster. General stress
response genes and PIP-responsive genes will
be differentiated, and specific assays will be
developed.
Genetic monitoring of non-target organisms. We will evaluate the population genetic structure of indicator
species (identified as part of the gene expression assessment) in and around sets of Bt-corn and j3r-cotton test
plots in order to establish the baseline conditions for monitoring ecosystem changes due to localized Bt toxin
exposures. Gene flow and genetic diversity parameters within and among populations will be evaluated by
microsatellite analysis in relation to planting histories for GM and non-GM crops.
-------
By taking similar measurements over multiple insect generations and plant cropping cycles, we will be able to
monitor changes in genetic diversity, effective population sizes, and effective numbers of migrants through time.
In conjunction with the exposure assays, we will evaluate changes in population sizes over a period of at least
four years as an initial assessment of the utility of this methodology for long-term monitoring.
Schedule:
FY03 1. Review and report on scientific literature related to genetic monitoring.
2. Begin screening for candidate genes for gene expression assays for non-target insects.
3. Put contracts in place and begin pilot-scale assessments of non-target insect populations around
corn agro-ecosystems.
FY04 1. Produce report on development of PCR-based indicators of resistance evolution.
2. Complete development of microsatellite markers for three non-target insect species.
3. Continue development and assessment of gene expression assays for non-target organisms.
4. Continue and expand monitoring near corn agro-ecosystems.
5. Initiate full-scale monitoring of non-target insect populations near cotton agro-ecosystems.
FY05 1. Collect year 2 (cotton) and year 3 (corn) population genetic data for non-target insect populations.
2. Complete development and evaluation of gene expression assay for non-target insects.
3. Coordinate and host ORD/OPP workshop.
FY06 1. Collect year 3 (cotton) and year 4 (corn) population genetic data for non-target insect populations.
2. Initiate planning for expert panel meeting on genetic monitoring.
FY07 1. Collect year 4 (cotton) and year 5 (corn) population genetic data for non-target insect populations.
2. Complete development and evaluation of a long-term monitoring strategy based on collection of
population genetic and gene expression data.
3. Convene expert panel to develop and refine recommendations for implementation of a genetic
monitoring program.
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Outputs/Impacts: This work will provide the Office of Pesticide Program, Biopesticides and Pollution
Prevention Division (OPP/BPPD) with significant new data and tools to evaluate long-term ecological effects
of genetically modified crops. Results of this research will be communicated through a number of annual
products.
APM 2003: Review of the scientific literature on the use of genetic monitoring for long-term ecosystem
surveillance with special attention to its use in agro-ecosystems.
APM: 2005: Joint ORD/OPP workshop on the analysis of population genetics of invertebrates in agro-
ecosystems.
APM 2006: Report on the development and evaluation of a gene expression assay of PIP exposure to non-
target insects.
APM 2007: Development and evaluation of a genetic monitoring program to assess long-term effects of PIPs
on non-target organisms.
APM 2008: Report on expert panel recommendations for a genetic monitoring program to assess long-term
effects of PIPs on non-target organisms.
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3
Escape of Altered Plants to the Natural Environment Likelihood Impact of Gene
Transfer
The ability of some crops to transfer introduced genes through hybridization to wild and/or weedy
relatives can make the assessment of effects on non-target organisms very difficult.
Research Need:
Explore the factors influencing gene transfer rates to provide a basis for better assessments.
Some transgenes may have a limited persistence due to their insert locations, characteristics of the
genetic cassette, or the plants or microorganisms themselves.
Research Need:
Develop methods to evaluate the persistence/maintenance of transgenes in plants and microorganisms,
the exposure to those gene products, and whether environmental conditions or common stressors
influence this process.
No discrete project was developed to address research need 3a. One project was developed to meet
research need 3b:
Evaluating gene flow from genetically modified crops and its potential ecological effects.
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Project Title:
Evaluating Gene Flow from Genetically Modified Crops and Its
Potential Ecological Effects
Laboratory/Center:
NHEERL (WED lead), Lidia Watrud, (541) 754-4567, watrud.lidia@epa.gov
Primary Research Objective/Goal: There are three specific outcomes for these studies: (1) an
understanding of the potential for transfer of novel genetic material from GM crops to non-target plants and the
associated ecological consequences of such exchange; (2) methods for determining and minimizing amounts
and circumstances of gene transfer from proposed GM crops that can be provided by registrants when applying
to OPP/BPPD for registration of new PIPs; and (3) identification of inputs for probabilistic risk assessment
models of gene flow from GM crops.
Background: Currently, engineered crops are planted on tens of millions of acres in the U.S. alone. Pollen
from transgenic crops may hybridize with related crops or weeds, potentially transferring the transgene to crop-
crop or crop-weed hybrids. The resultant F1 hybrids may in turn self- or out-cross to other compatible species
or may backcross to the transgenic or non-transgenic parent. In addition, the transgenic genes may move via
feral plants or seeds; i.e., over-wintering transgenic plants or seeds that escape cultivation or via seeds that
have fallen from planters, combines, trucks, or railroad cars during routine planting, harvesting, and shipping
activities. While it is commonly argued that cultivated crops would not persist well outside of agronomic
situations due to their need for high soil-fertility levels, little information is available on the survival, fertility,
and out-crossing potential of hybrids formed between crops and compatible weedy or native species. Many
species in each of the two latter categories (weedy and native species) commonly thrive in low fertility soils. It
also is not known how exchange of transgenes will affect the overall fitness of non-crop plants, either enhancing
or decreasing their ability to compete within the natural plant community. Methods are needed to allow such
questions about ecological risks to be adequately addressed during development and deployment of crops with
novel PIP transgenes.
--
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Project Description and Critical Path:
Six major scientific questions will be addressed by research:
(1) How far and with what frequency do transgenes move from GM crops into other plants?
(2) Which biological and non-biological factors affect gene flow?
(3) How long do transgenes persist in non-target host populations?
(4) What are the unintended ecological consequences to plant communities of gene transfer and/or
dissemination of feral crop plants?
(5) Can unintended ecological effects of genetically modified crops be reduced by designing strategies
to minimize gene flow?
(6) Can probabilistic risk assessment models be developed using parameters identified in our studies?
In order to answer these questions, four lines of research will be conducted.
1. Develop gene tracking methods - qualitative and quantitative molecular methods or other cytological,
biochemical, or morphological markers will be developed to track transgenes (or components thereof)
from GM crops to other crops or non-crop plants. Molecular methods (e.g., PCR) will be developed to
facilitate detection of stress response at the genomic level in support of ecological effects studies.
2. Select compatible crop/non-crop species - compatible plant species will be selected for greenhouse or
field studies of gene transfer. Rates of transfer will be compared between novel and conventional
crop protection genes to provide information on stability and persistence of genetic material.
Differences due to life-history traits (e.g., pollination methods) will be assessed, as well as effects of type
of genetic construct (e.g., nuclear vs. chromosomal inserts; single vs. multiple engineered traits; crop
protection vs. crop nutrient quality traits; protein vs. non-protein metabolically engineered traits, etc.).
3. Evaluate ecological effects - studies will be conducted on the consequences of genetic transfer on
fitness (i.e., survival, yield, biomass production) of recipient plants. The potential for persistence of the
gene through succeeding hybrid and backcross generations of the parental and other compatible species
also will be studied. Differences due to transgene insert locations, characteristics of the genetic cassette,
or the species involved will be evaluated. Fitness consequences of gene expression will be studied in
multispecies communities subjected to various environmental stressors (e.g., herbicide application;
temperature/humidity fluctuations; etc.). Molecular methods such as microarrays will be used to study
genomic level stress responses in relation to fitness parameters.
-------
4. Develop probabilistic risk assessment models - risk assessment methods to evaluate potential adverse
effects of gene transfer from GM crops will be developed using probabilistic methods building on
similar work currently underway for conventional herbicides. This includes estimates of exposure
(e.g., probability of gene transfer for a given crop location, environmental factors, etc.) and effects
(e.g., probability of ecological effects as a consequence of novel genetic material moving into non-crop
plant species).
Projected Outputs/Impacts:
APG (FY08): Understand the factors influencing gene transfer rates from GM crops and the potential
for altered plants to escape into the natural environment and provide methods to evaluate the persistence
maintenance of transgenes in plants as a basis for assessing the likelihood and ecological impact of gene
transfer.
APM (FY04): Quantitative measures for tracking transgenes in crop and non-crop plants.
APM (FY06): Methods for estimating frequency of gene transfer from GM^ crops to non-crop plants.
APM (FYOS): Molecular methods (e.g., microarrays) applied to plant genomes for assessing genetic
change and environmental stress.
APM (FY07): Ecological consequences of movement of transgenes from GM crops to non-crop plants.
APM (FYOS): Probabilistic methods for assessing ecological risk of genetic transfer from GM crops.
Projected Schedule: See table next page.
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Timeline/Outputs Gene Flow
FY 2002
FY 2003
FY 2004
FY2005
FY
Review literature
Identify resources:
plants, traits, people,
and organization
Attend workshop;
identify data
gaps; and select
crops, traits, and
geographies
Draft research plan
Pilot on site
studies of DNA
characteristics,
persistence,
expression, and
transforming ability
Hire NHEERL post-
doc
Identify potential
IPA (academic and
federal agency) and
GSF collaborators
Contribute to
APM on strategy
for updated test
guidelines and
finalize research plan
Approved QA plan
in place
Initiate lab and
chamber studies
with transgenic and
parental plants
Initiate DNA
analyses of plant
and soil materials
from field sites in
US region(s) where
selected crop(s) are
grown
Hire NRC post-doc
Formulate and issue
RFA
Identify US and
international
collaborators
Continue R&D
intramural studies
in laboratory,
chambers, the field,
and model inputs
Initiate extra-mural
R&D via co-ops,
lAGs (USDA-ARS,
DOI-NPS, DOI-
BLM), contracts,
and CRADAs with
the private sector
Identify
collaborators
for international
ecological effects
and molecular
tracking
collaborations in
multi-year field
studies with a
wind and an insect
pollinated crop
Continue intramural
and extramural R&D
Convene meeting
of investigators to
review findings,
methods, problems,
and to define model
parameters
Convene workshop
to develop national
and international
data collection
network
Complete short-term
R&D
Continue long-term
R&D
Produce protocols,
publications, and test
model
Produce Agency
reports: findings,
methods, and white
paper on strategies
to minimize gene
flow effects
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4
The Development of Pesticides in the Insect Target
Laboratory and small-scale field testing has been the basis for evaluating the likelihood of resistance
development. Long-term, extensive monitoring has not been conducted to determine whether the effects
predicted in such tests actually occur in the field.
Research Needs:
(i) Develop models to estimate the likelihood of the development of insect resistance that incorporate
detailed biological information for pest species, including gene flow and mating patterns in the wild,
geographic and chromosomal distribution of resistance alleles, and their additive and non-additive
effects on resistance under selective pressures in the field.
(ii) Perform monitoring studies of gene transfer, the development of resistance to PIP s by target pests,
and effects on non-target species (as noted in 2b) to allow field validation of conclusions regarding
transgenic plants with new pesticide traits.
There are two projects addressing key research need:
1. Genetic evaluation of long-term risks of plant-incorporated protectants: evolution of resistance in
targeted pests.
2. Field assessment of insecticide resistance management (IRM) for PIPs.
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Project Title:
Laboratory/Center:
Program Contact:
Genetic Evaluation of Long-term Risks of Plant-Incorporated
Protectants: Evolution of Resistance in Targeted Pests
NERL
Mark Bagley, (513)569-7455, bagley.mark@epa.gov
Background: The long-term environmental risks posed by GMOs are generally less well understood than
the short-term risks. Thus, it is important that EPA continue to evaluate the usefulness of its current data to
ascertain long-term ecological risks and to develop new types of data where necessary. Adaptation to PIPs by
targeted pests is a long-term risk of special concern. Additional information is needed to evaluate the biological
assumptions of models used to develop EPA's required strategy for resistance management (i.e., high dose,
structured refuge) for different species and traits.
Project Description and Critical Path: Prediction of the likelihood and rate of adaptation by targeted pests
depends on a number of factors, including variation in the toxicity of the plant product over time, the fraction
of each population that is exposed to PIPs, gene flow between exposed and unexposed populations, the number
of genes and amount of genetic variation influencing the trait, dominance and epistatic interactions, and genetic
correlations with other fitness traits. Many of these factors are complex and poorly understood for most species.
We will evaluate genetic parameters for
one model plant-pest system: Bl-com
and western corn rootworm, Diabrotica
virgifera. This system has been identified
by OPP as a concern for development
of Bt resistance. The work will include
laboratory assessments of genetic
variances and covariances between fitness
traits at several levels of exposure to
Bt toxin. In addition, we will examine
the population genetic structure of pest
populations using microsatellite DNA
markers. This analysis of neutral genetic
markers over several generations and
from several populations will provide
information on effective population sizes
and will quantify migration/gene flow.
-------
This information will be used to parameterize models of pest adaptation that will be tested under laboratory-
controlled gene flow scenarios. Using information on target genes of action (e.g., from gene expression studies)
and homologous resistance-conferring genes (e.g., BtR-4 in tobacco budworm and bre-5 in nematodes), we
will design targeted PCR assays for allele frequency changes at candidate resistance loci. Frequencies of these
resistance markers in field populations will be estimated. This approach will be evaluated as an early warning
system for detecting the evolution of resistance by the target pest. If successful, these approaches may also be
applied to other Bt crops and pests such as the j5l-cotton and the tobacco budworm.
Schedule:
FY03 1. Begin development of PCR-based assays for D. virgifera resistance development.
2. Begin development of microsatellite DNA markers for D. virgifera.
3. Begin field collections of D. virgifera for population genetic analysis.
4. Begin in-house cultures of .D. virgifera for laboratory genetic analyses.
FY04 1. Produce report on development of PCR-based indicators of resistance evolution.
2. Complete development of microsatellite markers for D. virgifera and non-target insects.
3. Initiate full-scale laboratory and field analyses of D. virgifera genetics.
FY05 1. Continue laboratory analyses of D. virgifera genetic characteristics, including mapping and QTL
analyses.
2. Complete field analyses of D. virgifera population structure.
FY06 1. Complete laboratory analyses of D. virgifera genetics.
2. Incorporate genetic information on D. virgifera into improved models of resistance evolution and
evaluate their implications for alternative resistance management strategies.
Outputs/Impacts: This work will provide the Office of Pesticide Programs, Biopesticides and Pollution
Prevention Division with significant new data and tools to evaluate long-term ecological impacts of genetically
modified crops. Results of this research will be communicated through a number of annual products.
APM 2004: Report on the development and evaluation of a PCR-based "early warning" assay for adaptation
by target pest populations.
APM 2006: Assessment of pest genetic architecture in order to inform optimized resistance management
plans.
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Project Title:
Laboratory/Center:
Program Contact:
Field Assessment of Insecticide Resistance Management (IRM) for
Plant Incorporated Protectants (PIPs)
NCEA
Bob Frederick, (202) 564-3207, frederick.bob@epa.gov
Primary Research Objective/Goal: The goal is to develop field methods to assess and monitor the effects
of the high-dose/structured refugia IRM strategy on the long-term susceptibility of target pests to Bl endotoxins.
Background: The development of target pest resistance to the Bt transgene[s] used as plant-incorporated
protectants is a serious risk both to the sustainability ofBt crops and to the wider utility of environmentally
"soft" microbial Bl pesticides. Therefore, EPA requires growers of Bt crops to follow the high-dose/structured
refugia strategy to delay or prevent resistance development. This marks the first time EPA has required
resistance management as part of a pesticide registration. Effective management requires sensitive tools for
detecting resistance in field pest populations while the resistant alleles are still sufficiently rare to allow for
corrective action.
Project Description and Critical Path: The research is composed of two parts, each involving the
development and refinement of field-based methodologies to assess and manage Bt resistance in the field.
One component will focus on field testing and validation of the high-dose/structured refugia strategy for
Bt-resistance management. Key assumptions of the models upon which this strategy is based still have not
been tested in field populations of the
target pests. Significant data gaps
exist regarding pest biology, ecology,
and population dynamics, particularly
with respect to dispersal and use of
alternate hosts. Target pests include key
lepidopteran cotton pests, Helicoverpa
zea and Pectinophora gossypiella;
Helicoverpa zea, a pest of both cotton
and corn; and, on corn specifically, the
lepidopteran pest, Oslrinia nubilalis and
two beetle pests, Diabrotica barberi and
D. virgifera. We propose to address these
ecological data gaps in a series of field
and regional-scale studies.
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A second research component will focus on developing appropriate tools to identify and measure Bt resistance
in field populations of the target pests. These tools will include both functional screens or bioassays and
molecular markers. Preliminary markers have been developed for H. virescens and P. gossypiella but have
not yet been tested extensively in field populations. We plan to develop and test additional markers as well as
develop more streamlined bioassay techniques.
Desired Outcomes: The research results will inform regulatory decision-making by OPP and will provide
critical tools to Regional Offices involved in promoting grower compliance with IRM requirements. EPA
will develop tools capable of identifying the evolution ofBt resistance at sufficiently early stages to allow
corrective action to prevent loss of Bt crops as effective and least toxic alternatives to conventional pesticides.
Projected Outputs:
FY06 ARM: Final report outlining appropriate tools for monitoring resistance development in the field and
the use of target pest ecology to refine IRM strategies as they are determined in risk assessment
practice.
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5
Management
The effectiveness of management strategies to avoid key risks for extended periods and the effectiveness
of risk management technologies to mitigate risks associated with unintended adverse consequences must be
evaluated and expanded for new PIP crops.
Research Need:
ft) Improve strategies to identify key risks of concern, develop evaluation schema to understand the
effectiveness of management strategies, and develop of effective new technologies to mitigate these
key risks as when monitoring studies identify the presence of unintended adverse consequences.
There are three projects addressing this key research need.
1. Development of strategic monitoring programs for ecological impact from plant-incorporated
protectants (PIPs).
2. Development of management and field-scale tools to manage the risks of gene transfer and non-target
effects from PIP crops to the environment.
3. Development of management and field-scale tools to manage and delay the development of insect
resistance to PIP crops by extending crop life.
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Project Title:
Laboratory/Center:
Program Contact:
Development of Strategic Monitoring Programs for Ecological Impact
from Plant-Incorporated Protectants (PIPs)
NCEA
Bob Frederick, (202) 564-3207, frederick.bob@epa.gov
Primary Research Objective/Goal: The goal is to determine effective strategies to identify the key risks of
concern and appropriate risk management technologies to mitigate these key risks when the monitoring studies
indicate unintended adverse consequences.
Background: Historically, monitoring programs in association with field releases of genetically modified
organisms have been, explicitly or implicitly, called for as a part of risk assessment/management schemes
or regulatory agenda. However, it is often not clear what should be monitored, why, or for how long.
Recommendations of objectives and methodologies have been made with little understanding of their scientific
basis due to a lack of information. Monitoring for the development of insect resistance to pesticides-identified
as early as 1991-provides the single best example of science-based monitoring program development. This is,
however, only one of many potential ecological concerns associated with GM crops; and often the decision as to
what to monitor for has depended as much on what was possible to monitor as it has on the identified concern.
While wide-ranging, non-specific monitoring programs to detect new or unique effects of genetic engineering
are being suggested, such monitoring may be quite expensive and inefficient. Surely most studies of this nature
will find nothing at great expense even if a previously unknown problem eventually turns up. It will be most
helpful to decision makers and those who will be charged with the design and implementation of monitoring
=. ' ;. programs to
know explicitly
:.: what should be
_ monitored, the
z_ reason behind the
i concern(s) that
generated the need
for monitoring,
the appropriate
methods to conduct
the monitoring
study, and the
purpose for which
"•= the data are to be
collected.
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Project Description and Critical Path: NCEA will convene a meeting of science experts to discuss the
state of the science in environmental monitoring efforts, particularly those related to the determination of
ecological impact from PIP crop plants. The most promising areas for comparison analysis monitoring will
be developed. The comparison approach was described in a recent NRC workshop summary on Ecological
Monitoring of Genetically Modified Crops. The goal is to determine what in-field condition(s) might prove
to be indicative of change (as an early warning indicator) or impact (evidence of a environmental impact, e.g.,
decreasing insect populations). By focusing on the agro-ecosystem condition, it may be possible to ameliorate
the spatio-temporal problems associated with large-scale planting of PIP crop plants and the natural variability
inherent in identifying and tracking ecosystem change. This research will be conducted for a minimum of
three growing seasons. If and where appropriate, the data resulting from the research will be used to test the
power of population change models as predictive tools for ecosystem impact or to assist in development of
cost-effective monitoring efforts.
Projected Outcomes: The research results will be useful both in regulatory decision-making by OPP;
and more generally, they will provide information critical to the risk assessment of PIPs. EPA will have
an evaluation of current and future ecological monitoring methods of potential use for post-registration
surveillance. Results from the field component will lead to constructive evaluation of assumptions made in
risk assessments of GM crops.
Projected Outputs/Impacts:
FY06 APM: Conference report on monitoring strategies for determining ecological impacts as they are
considered in risk assessment from GM crops.
FY07 APM: Report on the field comparison approach to risk-based monitoring for ecological impacts from
genetically modified crops.
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Project Title:
Development of Management and Field-Scale Tools to Manage the
Risks of Gene Transfer Non-Target Effects from PIP Crops to the
Environment
Laboratory/Center:
NRMRL
Background: Biotechnology presents a wealth of opportunities to improve crop productivity, nutritional
value, and resistance to pests and other stresses. However, there are potential risks to human health and
ecological systems that need to be evaluated for the proper regulation of transgenic pesticidal crops. Currently,
EPA regulates biotechnology products that are pesticides produced by plants or by microorganisms. It is
important to continue to develop supporting information to correctly identify the risks of concern associated
with PIP crops and the associated risk management strategies or tactics to mitigate any unintended adverse
consequences connected with these crops. The management portfolio to meet the challenges needs to
be expanded. There are no strategies for identifying the key risks of concern, nor are there effective risk
management technologies to mitigate these key risks when monitoring studies indicate unintended adverse
consequences. It is also important to explore the application of socio-economic methods such as benefit-cost
analysis and life-cycle analysis to better understand issues related to the public acceptance of genetically altered
products and to evaluate whether the genetic alterations produce new organisms that are not substantially
equivalent to currently existing ones.
Project Description Critical Path: Techniques
that have been developed for full field evaluation of
non-target effects in Europe will be evaluated for their
applicability to the U.S. Standardization and testing of the
techniques will be undertaken to understand the directive
force and reliability of data collected through the use of
these techniques. Field testing of selected techniques will
be undertaken to ensure their reliability and the integrity
of information derived from them. The standardized and
tested techniques will be assembled for use by future seed
registrants and public dissemination.
Scrutinizing possible non-target and gene-transfer effects
will continue to be an integral part of the evaluation strategy
used by the Office of Pesticide Programs to register PIP
crops. The information database supporting this inquiry
is very small and largely devoted to specific issues. The
proposed research attempts to close the known information
gaps specifically in the area of management tools.
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Remote sensing and other forms of sensing technology will be tested for their ability to assist the understanding
and modeling of pollen distribution that is the major pathway for gene transfer for the PIP crops. Management
tactics and strategies designed to avoid adverse effects associated with pollen transport will be developed and
evaluated. Certain crops are known for their wide pollen distribution patterns so this detection technique will be
selectively used as a function of crop characteristics.
Projected Outputs/Impacts:
APG (2009): Establish and deliver for future use by Office of Pesticide Programs, EPA regions, and state
and local governmental agencies gene-transfer mitigation and non-target effects tools and
strategies to aid the management of environmental risks associated with PIP crops to help
maintain the biological integrity of the environment.
FY 04 APM: Develop and deliver review of current practice for the management/mitigation of gene-transfer
and non-target effects from PIP crops and tools to assist risk management of adverse effects of
PIP crops.
FY 05 APM: Develop and deliver for future use by EPA regions and state and local agencies preliminary
methods for gene-transfer detection and non-target effects detection to assist the risk
management of the potential adverse effects of PIP crops.
FY 06 APM: Develop and deliver for future use by EPA regions and state and local agencies tools for gene
transfer and non-target effects management to assist the risk management of the potential adverse
effects of PI Per ops.
FY 07 APM^: Conduct and deliver for future use by EPA regions and state and local agencies preliminarily
tested and validated tools for gene transfer and non-target effects management to assist the risk
management of the adverse effects of PIP crops.
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Project Title: Development of Management and Field-Scale Tools to Manage and
Delay the Development of Insect Resistance to PIP Crops by Extending
Crop Life
Laboratory/Center: NRMRL
Program Contact: John Giaser, (513) 569-7568, glaser.john@epa.gov
Background: Pest resistance is the major threat to the technology sustainability of PIP crops. Implicit in the
control of resistance is the ability to monitor the incidence of resistance and to formulate management plans to
intercept any pest resistance at the earliest date possible. Vast crop sizes of > 20 million acres are not amenable
to discrete sampling practices that have the desired statistical power for the prediction of the incidence of
critical resistance levels.
Project Description and Critical Path: Part of this research will focus on the use of remote sensing to
discriminate a reflected light signature for pest infestation. Coincidences such as a signature arising from a
bioengineered crop planting could serve as a tool to direct land-based inspection teams to investigate potential
pest infestations.
There are several laboratory-based techniques that use field-collected insects to determine resistance. These
methods are used in part to support the OPP registration decisions. It is incumbent upon EPA to ensure that
the data derived from these techniques and
methods are of the highest information
content to support OPP decisions. The
methods will be subjected to standardization
and tested for verification and validation
of results. Simulation models have been
used in similar ways by OPP and will be
subjected to standardization followed by
verification and validation scrutiny.
The remote sensing of pest infestation will
begin at the "proof-of-principle" stage
by studying a subset of the corn crop in
the north central part of the Corn Belt
(Minnesota, South Dakota, Nebraska,
U and Iowa) where bioengineered corn can
B account for a large percentage of the corn
• crop. The first stage will be to discern the
• utility of remote sensing imagery to identify
H conventional from bioengineered corn.
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This information will be useful in observing the compliance of growers to the mandated refuge requirements.
Using satellite imagery and field "ground-truthing," the reflected light frequencies indicative of pest infestation
will be selected for lepidopteran insects (European corn borer and others). A composite of several spectral
wavelengths will be explored as indirect information related to pest information. Proof-of-principle evaluation
will follow to evaluate the use of imagery for the earliest identification of field conditions that are indicative of
pest infestations. This scrutiny will push the technology to the level of determining the importance of different
noise contributions to the signal. It may also be possible to initiate the imagery of new corn events that protect
from corn root worm in the first year of large field planting. It is anticipated that the reflected spectral signatures
could be significantly different for these pests. Once the desired signatures are selected they will be tested. The
remote-sensing system will be tested at a "proof-of-practice" level at which time an attempt to image the entire
crop will be undertaken. Similar applications to the transgenic cotton crop will also be pursued. At each stage
of development, reports will show the direction and extent of success that has been accomplished. Operational
manuals and data inspection tools will be developed and published separately.
The scrutiny of resistance detection methodology and simulation modeling will be undertaken initially to
standardize performance and communication related to use. The standardized methods will be tested in a
variety of practitioners' hands to ensure verification and validation of reported results.
The bioengineered crop registration conducted by OPP rests squarely on the reliability of the available data
relating to resistance management. The continued reliance on unstandardized methodology may lead to
assumptions of crop performance that may not be met in the field. While not anticipated, crop failures are
within the realm of potential outcomes. This research strives to develop a firm information basis from which
reliable decisions can be made regarding the resistance management of PIP crops.
Field tools to assess the compliance of resistance management requirements will also be undertaken for use by
EPA regions and states. Toxin detection technology will be evaluated and incorporated in field guidance for its
use in the compliance monitoring.
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Projected Outputs/Impacts:
APG (FY2009):
Establish and deliver for future use by Office of Pesticide Programs, EPA regions, and
state and local governmental agencies basic guidelines and tools to mitigate the
development of resistance in targeted pest populations so as to extend the useful lifetime
of PIP crops to minimize the use of chemical pesticides in agriculture.
FY04APM:
Develop and deliver survey of current practice in management tools for delaying the
development of resistance to PIP crops in targeted insects including modeling, remote
sensing, and laboratory assays to help minimize pesticide use.
FY 05 APM:
Conduct/deliver for future use by Office of Pesticide Programs, EPA regions, and state
and local agencies verified/validated resistance management models for delaying
resistance to PIP crops in target insects minimizing pesticide use.
FY05 APM:
Establish/deliver for future use by Office of Pesticide Programs, EPA regions, and
state and local agencies principles for using remote sensing and GIS to detect pest
infestation to delay insect resistance to PIP minimizing pesticide use.
FY 05
Establish/deliver for future use by Office of Pesticide Programs, EPA regions, and state
and local agencies single laboratory standardization of lab assays to detect pest
insect resistance to PIP crops to minimize pesticide use.
FY06APM:
FY 06 APM::
FY 06 APM::
FY 07 APM::
Develop/deliver for future use by Office of Pesticide Programs, EPA regions, and state
and local agencies data mining tools for models and methods to help manage and delay
insect resistance to PIP crops to minimize pesticide use.
Establish/deliver for future use by Office of Pesticide Programs, EPA, and state and local
agencies proof of application of remote sensing and GIS to detect pest infestation to delay
insect resistance in PIP crops minimizing pesticide use.
Develop/establish/deliver for future use by Office of Pesticide Programs, EPA, and state
and local agencies multi-laboratory standardization of lab assays to detect pest
insect resistance to PIP crops to minimize pesticide use.
Establish/deliver for future use by Office of Pesticide Programs, EPA, and state and local
agencies proof of practice for using remote sensing and GIS to detect pest infestation to
delay insect resistance to PIP minimizing pesticide use.
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Demonstration Project
A demonstration project is being designed to facilitate inter-laboratory participation and cooperation.
Project Title: Biotechnology Demonstration Project
Background: In planning the Biotechnology Initiative Research Program, the Biotechnology Initiative
Steering Committee determined that a demonstration project was needed as a means to foster cross-laboratory
collaboration, to create the opportunity for a productive synergy, and to effectively illustrate the connectedness
of individual laboratory efforts.
Project Description and Critical Path: A demonstration project to assess ecological risk is being
formulated with cross-laboratory cooperation from NCEA, NRMRL, NHEERL, and NERL. This project
provides a means to demonstrate the tools and approaches developed through the larger program. With the
close coordination and involvement of scientists from the Office of Pesticide Programs, the success of this effort
will be measured by the extent to which the tools and approaches are integrated across the risk assessment/risk
management paradigm to inform EPA decisions.
Projected Outputs/Impacts: Conceptually, the project will have two stages. The first will be to prepare a
detailed white paper that (a) reviews the current state of the science in biotechnology risk assessment and how
it is currently used; (b) analyzes expectations from emerging scientific inquiry both within and outside EPA;
and (c) proposes a strategy for the evaluation of new and existing, science-based assessment tools. The second
stage will be to implement the strategy infield experiment(s) designed to incorporate all components of risk
analysis (problem formulation, conceptual model development, risk assessment, risk characterization, and risk
management) and collect data to inform the process.
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