EPA 600/R-03/068

March 2005

Biotechnology Research Program

U.S. Environmental Protection Agency

Office of Research and Development

Washington, DC 20460
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

ii Biotechnology Research Program
Contents

Biotechnology Initiative Steering Committee ....................................................................................................... v

Foreword .............................................................................................................................................................. vi

Evaluating Potential Risks Associated with Biotechnology Products .....................................................................1

 Background ..................................................................................................................................................1

 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 .................................................................................................................................A1

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

Biotechnology Research Program iii
iv Biotechnology Research Program

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 Drug 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

NIAID 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
Biotechnology Initiative Steering Committee

Office of Research and Development

Lawrence W. Reiter, National Health and Environmental Effects Research Laboratory

John R. Fowle III, National Health and Environmental Effects Research Laboratory

Mark Bagley, National Exposure Research Laboratory

Anne Fairbrother, National Health and Environmental Effects Research Laboratory

Elaine Z. Francis, National Center for Environmental Research

Robert J. Frederick, National Center for Environmental Assessment

John Glaser, National Risk Management Research Laboratory

MaryJane Selgrade, National Health and Environmental Effects Research Laboratory

Subhas Sikdar, National Risk Management Research Laboratory

Uwe Stolz, National Exposure Research Laboratory

Lidia Watrud, National Health and Environmental Effects Research Laboratory

Marsha Ward, National Health and Environmental Effects Research Laboratory

Office of Prevention, Pesticides and Toxic Substances

Janet L. Andersen, Office of Pesticide Programs
Foreword

 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.

1

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.

vi Biotechnology Research Program
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.

Evaluating Potential Risks Associated with Biotechnology Products

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 

Biotechnology Research Program 1
render related conventional products (e.g., the spores 

of Bacillus thurigiensis [Bt] 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).

Research Approach

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 

2 Biotechnology Research Program
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.

Biotechnology Research Program 3
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.

Key Science Issues with Respect to 

Biotechnology Products

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 

(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.

1. Potential Allergenicity of Proteins

 Introduced into the Food Supply by

 Gene Transfer

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.

2

Under both FIFRA and TSCA, EPA also has an obligation 

to consider the potential benefits of biotechnology pesticide 

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 (GM) foods for 

increased allergenicity.

4 Biotechnology Research Program
2. Potential Ecological Effects of

 Biotechnology Products on Non-Target

 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 Corn Product [Event MON 863], February 19, 

2003).

(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 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.

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; 

Biotechnology Research Program 5
(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 polychlorinated byphenyls.

3. Escape of Altered Plants to the Natural

 Environment and the Likelihood and

 Effects of Gene Transfer

(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).

3

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. 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.

4

OPP 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 ORD in the past. Future research in this area would be of 

value. This need is not being addressed in this research plan.

(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

(iii) 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

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 and to monitor for 

ecological effects in the field are needed.4

Research Need: 

Explore the factors influencing gene 

transfer rates to provide a basis for better 

assessments.

6 Biotechnology Research Program
(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.

4. The Development of Pesticide

 Resistance in the Target Insect Species

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 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.

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 nontarget species (as noted in 2b) to allow 

field validation of conclusions regarding 

transgenic plants with new pesticide 

traits.

Biotechnology Research Program 7
5. Risk 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.

Performance Results and Expected 

Benefits

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.

Projected Outputs

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: 

8 Biotechnology Research Program

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 benefitcost 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.
1. Models to predict dietary allergenicity from 

 consuming crops containing PIPs and other 

 biotechnology products will be developed 

 and verified as a basis to assess the 

 potential allergenicity of proteins introduced 

 into the food supply by gene transfer; 

2. 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; 

3. Explicit information on pest genetics and 

 ecology that will validate or improve existing 

 models to predict development of resistance; 

4. The identification of factors influencing gene 

 transfer rates to better understand the 

Biotechnology Research Program 9

 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; 

5. 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 

6. Risk management strategies to provide 

 means to mitigate risks when unintended 

 adverse consequences occur. 

Endpoint NAS 2000 NAS 2002A NAS 2002B

HEALTH EFFECTS

ECOLOGICAL EFFECTS

Allergenicity Detection Methods

Non-target Testing Field

Surveys & Monitoring

Metabolic changes (plieotropic or 

secondary effect) impact on nontargets and/or human health

Impacts of gene flow--beyond

“it occurs”

Resistance management strategies

Monitoring (long-term ecological)

Risk Management

X X

X

X

X

X

X

X

X

X

X

Need For Strategies For

NRC (National Research Council). 2000. Genetically Modified Pest-Protected Plants:

 Science and 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 Research Council). 2002B. Animal Biotechnology:

 Science-Based Concerns, Washington, DC. National Academy Press.
Appendix A. Project Sheets

This Appendix contains project sheets which describe the work we plan to accomplish in order to address the 

three major concerns facing EPA: (1) allergenicity, (2) gene transfer, and (3) mitigating resistance and gene 

transfer. We have developed five major research areas. As the needs outpace available resources, it will not be 

possible to meet all the needs, nor will it be possible to fully cover each need that is addressed under this plan. 

The five major research areas and associated research need(s) are listed followed by project sheets that describe 

the related priority work EPA will cover under its initial biotechnology efforts.
Potential Allergenicity of Proteins Introduced into 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. 

There are two projects addressing this key research area:

 1. Potential allergenicity of genetically modified organisms.

 2. Solicitations through the STAR extramural program to support EPA’s Biotechnology Research 

 Program.

Research Area 1

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.

Biotechnology Research Program A2
Project Title: Potential Allergenicity of Genetically Modified Organisms

Laboratory/Center: NHEERL

Program Contact: MaryJane Selgrade, (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?

A3 Biotechnology Research Program
(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 path will be followed in order to address these 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 IgA in 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.

Biotechnology Research Program A4
Schedule:

Publish Workshop Proceedings: February 2003

Begin work to develop animal model: Spring 2003 (Depends entirely on when the animal facility 

 in the new building opens)

Hire first post-doc: By end of 2003

Develop animal models 2005

Test Bt toxin & digestibility theory 2006

Demonstrate vulnerability of newborns 2008

Projected Outputs/Impacts:

FY 2008 APG: Improved capability to assess the risks of allergenicity from genetically altered food.

FY 2003 APM: Publish results of jointly sponsored (FDA & NIEHS) workshop.

FY 2005 APM: Develop models and methods for assessing potential allergenicity.

FY2008 APM: Demonstrate the vulnerability of newborns/ identify windows of vulnerability.

A5 Biotechnology Research Program
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, NIAID, 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.

Biotechnology Research Program A6
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.

A7 Biotechnology Research Program
Potential Ecological Effects of Biotechnology Products on Non-Target Species

 Questions have been raised about the effects of PIPs on non-target species.

 There is one project addressing this 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 Area 2

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, predation) that mediate 

this exposure;

(ii) Develop an understanding of the mechanisms that 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

(iii) 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 

phytoremediation), as well as for novel pest resistance.]

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.

Biotechnology Research Program A8
 Questions have been raised about the effects of genetically engineered microorganisms on non-target species.

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.

Research Need: 

Genetically engineered microorganisms have raised issues of non-target effects and opportunistic 

pathogenicity 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.]

A9 Biotechnology Research Program
Project Title: Non-Target and Ecosystem Impacts from Genetically Modified Crops

 Containing Plant-Incorporated Protectants (PIPs)

Laboratory/Center: NCEA 

Program Contact: Bob Frederick, (202) 564-3207, frederick.bob@epa.gov

Primary Research 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 and effective risk management technologies to mitigate these key risks when the monitoring 

studies indicate unintended adverse consequences?

Biotechnology Research Program A10

Ecological Risk Assessment

Context for Biotechnology Research

Exposure

Gene Flow/

Invasion

Effects

Target/Non-target

Plants/Animals

Assessment

Ecological Impacts

Risk Management

Mitigation

Pest Adaptation
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 of Bt 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 decisionmaking 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.

A11 Biotechnology Research Program
Project Title: Genetic Evaluation of Long-Term Risks of Plant-Incorporated

 Protectants: Exposures and Effects on Non-Target Species

Laboratory/Center: NERL

Program Contact: 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 Bt-corn and Bt-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 Bt-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. 

Biotechnology Research Program A12
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.

A13 Biotechnology Research Program
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.

Biotechnology Research Program A14
Escape of Altered Plants to the Natural Environment and Likelihood and 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. 

 Some transgenes may have a limited persistence due to their insert locations, characteristics of the 

 genetic cassette, or the plants or microorganisms themselves. 

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.

Research Area 3

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.

Research Need: 

Explore the factors influencing gene transfer rates to provide a basis for better assessments.

A15 Biotechnology Research Program
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 cropcrop 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.

Biotechnology Research Program A16
Project Description and Critical Path:

Six major scientific questions will be addressed by this 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.

A17 Biotechnology Research Program
 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 (FY08): Probabilistic methods for assessing ecological risk of genetic transfer from GM crops. 

Projected Schedule: See table next page. 

Biotechnology Research Program A18
FY 2002

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 postdoc

Identify potential 

IPA (academic and 

federal agency) and 

GSF collaborators

Timeline/Outputs Gene Flow Research

FY 2003

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

FY 2004

Continue R&D 

intramural studies 

in laboratory, 

chambers, the field, 

and model inputs

Initiate extra-mural 

R&D via co-ops, 

IAGs (USDA-ARS, 

DOI-NPS, DOIBLM), 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 

FY2005

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 

FY 2006-2007 

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

A19 Biotechnology Research Program
The Development of Pesticides Resistance in the Insect Target Species

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.

There are two projects addressing this 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.

Research Area 4

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 PIPs 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. 

Biotechnology Research Program A20
Project Title: Genetic Evaluation of Long-term Risks of Plant-Incorporated

 Protectants: Evolution of Resistance in Targeted Pests

Laboratory/Center: NERL

Program Contact: 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: Bt-corn 

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.

A21 Biotechnology Research Program
This information will be used to parameterize models of pest adaptation that will be tested under laboratorycontrolled gene flow scenarios. Using information on target genes of action (e.g., from gene expression studies) 

and homologous resistance-conferring genes (e.g., Bt R-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 Bt-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.

Biotechnology Research Program A22
Project Title: Field Assessment of Insecticide Resistance Management (IRM) for

 Plant Incorporated Protectants (PIPs)

Laboratory/Center: NCEA 

Program Contact: 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 Bt 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 of Bt crops and to the wider utility of environmentally 

“soft” microbial Bt 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, Ostrinia 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.

A23 Biotechnology Research Program
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 of Bt 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 APM: 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.

Biotechnology Research Program A24
Research Area 5

Risk 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:

 (i) 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.

25 Biotechnology Research Program
Project Title: Development of Strategic Monitoring Programs for Ecological Impact

 from Plant-Incorporated Protectants (PIPs)

Laboratory/Center: NCEA 

Program Contact: 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 

reason behind the 

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.

Biotechnology Research Program A26
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.

A27 Biotechnology Research Program
Project Title: Development of Management and Field-Scale Tools to Manage the

 Risks of Gene Transfer and 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 and 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.

Biotechnology Research Program A28
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 PIP crops. 

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.

A29 Biotechnology Research Program
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 Glaser, (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, 

and Iowa) where bioengineered corn can 

account for a large percentage of the corn 

crop. The first stage will be to discern the 

utility of remote sensing imagery to identify 

conventional from bioengineered corn. 

Biotechnology Research Program A30
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.

A31 Biotechnology Research Program
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. 

FY 04 APM: 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. 

FY 05 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 APM: 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. 

FY 06 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. 

FY 06 APM: 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. 

FY 06 APM: 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. 

FY 07 APM: 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.

Biotechnology Research Program A32
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.

A33 Biotechnology Research Program