ORD COMPUTATIONAL TOXICOLOGY PROGRAM
FY04 ACTIVITY REPORT
Prepared by:
ORD Computational Toxicology
Implementation and Steering Committee
October 2004
-------
Table of Contents
I. Executive Summary
II. Acronyms and Abbreviations
III. Background
IV. Current Activities
A. EDC Proof-of-Concept Studies
B. Microbial Source Tracking
C. Augmentation Funding
D. New Starts
E. STAR Grants
F. Partnerships
G. Recruitments
H. Related ORD Research
V. Future Directions
3
4
5
7
8
10
11
11
11
12
13
13
15
Table 1. CTISC Rooster
Table 2. Projects Receiving Augmentation Funding
Table 3A and B. STAR RFA Projects
Table 4. Partnerships Established
Table 5. Related Research in ORD
Appendix A. Augmentation RFA
Appendix B. New Start RFA
Appendix C. STAR RFA for the Environmental Bioinformatic Research Center
16
17
20
23
26
27
28
32
-------
I. EXECUTIVE SUMMARY
Fiscal Year 2004 (FY04) was a year of transition for the Office of Research and Development (ORD)
Computational Toxicology (CompTox) Program. In previous years, a small number of proof-of-concept studies
based on endocrine-disrupting modes of action were initiated; and general framework for a larger program was
established. The framework laid out three general objectives for the program: (1) improving the linkages in the
source-outcome paradigm; (2) providing tools for screening and prioritization of chemicals under regulatory
review; and (3) enhancing quantitative risk assessment. The CompTox Program is now moving from primarily
from a planning phase to an implementation phase. In FY04 the endocrine disrupter proof-of-concept projects
advanced through completion of the development of an estrogen reporter cell line to complement the androgen
receptor cell line developed previously, refinement of QSAR models for receptor binding, and development of an
in vitro cell based assay for steroidogenesis. Three cell-based assays are scheduled for scale-up production
against a larger set of chemicals in FY05. Ten existing intramural research projects received funding to apply
genomic, metabonomic, or computational tools in an effort to quickly expand the program beyond the endocrine-
disrupter domain. Through both internal and external RFAs, a significant number of projects were launched that
will provide the scientific foundations upon which future success will be judged. Through the STAR program, four
projects devoted to developing high-throughput screening assays for endocrine-disrupting chemicals (EDCs) were
funded (including a yeast assay, an invertebrate species, and two fsh species), as were three projects conducting
research on systems-biology models of the hypothalamic-pituitary-gonadal axis (two in small fish models, one in
mammals). Two significant activities, the STAR Center for Environmental Bioinformatic Research and the internal
"New Start" projects, will not be completed until FY05, at which time the program will be able to articulate specific
near-term and long-term research objectives. In addition, ORD has, or is, establishing partnerships with both
public and private sector organizations such as Department of Energy (DOE), Department of Defense (DOD),
National Institutes of Environmental Health Sciences (NIEHS), Food and Drug Association (FDA), Chemical
Industry Institute of Toxicology (CUT) Centers for Health Research, IBM, and Affymetrix as a way to access
complementary expertise and leverage available resources. With many activities initiated during FY04, the
CompTox program is currently poised to begin making significant progress in defining how it will approach and
achieve the objectives set forth in the Framework.
-------
II. Acronyms and Abbreviations
APM annual performance measure
ASTER Assessment Tools for the Evaluation of Risk
ATCC American Type Culture Collection
BBDR biologically based dose-response
CCLs Contaminant Chemica! List Chemicals
CEBS , Chemical Effects in Biological Systems
CUT Chemical Industry Institute of Toxicology
CTISC Computational Toxicology Implementation and Steering Committee
DOD Department of Defense
DOE Department of Energy
DSSTox Distributed Structure-Searchable Toxicity
EA expression analysis
EDC endocrine-disrupting chemical
EDSP Endocrine Disrupter Screen ing Program
EDSTAC Endocrine Disrupters Screening and Testing Advisory Committee
ELISA enzyme linked immunosorbent assay
ER estrogen receptor
FDA Food and Drug Association
FQPA Food Quality Protection Act
FTEs full-time employees
FY fiscal year
GnRH gonadotropin releasing hormone
HAP hazardous air pollutant
HHR human health risk
HPG hypothalamic-pituitary-gonadal
IAG interagency agreement
IHCS immunohistochemical staining
LBD ligand-binding domain
LH luteinizing hormone
mCRADA m? Cooperative Research and Development Agreement
MOD Memorandum of Understanding
MST microbial source tracking
MVA multivariate analysis
MYP multi-year plan
NIEHS National Institutes of Environmental Health Sciences
OPP Office of Pesticide Programs
OPPTS Office of Pesticides, Prevention, and Toxic Substances
ORD Office of Research and Development
OSCP Office of Science Coordination and policy
PI Principle investigator
PK/PD pharmacokinetic/pharmacodynamic
PNNL Pacific Northwest National Laboratory
QSAR quantitative structure activity relationships
RBA relative binding affinity
RFA requests for application
RT-PCR reverse transcriptase-polymerase chain reaction
SP2 Safe Products/Safe Pesticides Multi-year Plan
STAR Science to Achieve Results
TCE trichloroethylene
TH thyroid hormone
VFARs virulence factors
WTEC World Technology Evaluation Center
-------
III. BACKGROUND
The ORD Computational Toxicology Report traces its origin to the FY02 House Appropriations Report
107-159, page 54, in which Congress directed EPA to provide $4 million from available funds "for the research,
development and validation of non-animal alternative chemical screening and prioritization methods, such as
rapid, non-animal screens and Quantitative Structure Activity Relationships (QSAR), for potential inclusion in
EPA "s current and future relevant chemical evaluation programs." To fulfill this directive, EPA embarked on
development of a research program that (1) was consistent with the Congressional mandate; (2) complemented
and leveraged related on-going Agency-sponsored efforts to consider alternative test methods; (3) further
advanced research to support the Agency's mission; and (4) would'not duplicate the mission and programs in this
area conducted by other agencies. An innovative program, entitled Computational Toxicology (CompTox), was
thus initiated that would target these goals and that, in the process, would greatly advance toxicology and risk
assessment as currently practiced by the Agency and the broader environmental sciences community. As
recommended by Congress, the proposed approach was developed in consultation with the Office of Pesticides,
Prevention, and Toxic Substances (OPPTS).
The construction of ORD's CompTox program arose out of the Agency's mission to protect human health
and the environment from harmful chemicals. Fulfilling this obligation requires the assessment of hundreds of
possible hazardous effects for tens-of-thousands of chemicals. Numerous Congressionally mandated programs
have reaffirmed this Agency responsibility, including the Food Quality Protection Act {FQPA), the Candidate
Contaminant List under the Safe Drinking Water Act Amendments, the High Production Volume and Pesticide
Inerts Programs, the Endocrine Screening Program, and the Hazardous Air Pollutants Program under the Clean
Air Act. In meeting these mandates, the Agency is confronted with chemical inventories whose size and diversity
have forced EPA to consider a variety of tiered testing approaches. All of these programs have the potential to
require a large use of resources, including animals, in screening assays and would benefit from the development
and application of more advanced ranking and prioritization methods. Although the use of tiered testing
approaches is designed to minimize unnecessary testing requirements, the potential for requiring large
investments in testing resources and animals still exists.
The CompTox program, by integrating sophisticated, modern computing {e.g., QSAR} with remarkable
advances in molecular biology (e.g., genomics), provides a potentially viable alternative to traditional, animal-
intensive approaches. It is expected that
the development and application of
computational tools will reduce the
probability that testing will be conducted on
"low risk" chemicals, thus reducing the
numbers of animals used. These better
informed decisions may make the greatest
overall contribution to ensuring that the
animals used in toxicity testing will be utilized
in the most efficient and effective manner possible. In some circumstances, it may ultimately be possible to
replace animals used in screening assays with in vitro or in silico tools. A collateral, albeit longer term benefit of
this program, will be the emergence of an approach that consolidates the information required currently on
several toxicological endpoints into a single assessment tool and thus reduces the number of tests that must be
conducted and the number of animals needed for such tests.
To launch the CompTox program, a proof-of-concept effort was developed that was targeted at methods
for the immediate priority-setting issues facing OPPTS in evaluating endocrine disrupters as mandated by
Congress in the FQPA. Successful development of new models and assays to assist in ranking and prioritizing
possibly thousands of chemicals for endocrine-disrupting potential would provide the means to evaluate the
application of these approaches to the additional legislated chemical- evaluation programs noted above. In the
near term, the approach was designed to provide predictive, computer-based structure-activity models and in vitro
assays that could be used to identify those compounds most likely to disrupt endocrine systems. In the longer
Computational Toxicology Background
FY02 Congressional Mandate
Increasing Needs for Prioritization Tools
Advances in Molecular Biology and Chemistry
Endocrine Disrupter Proof-of-Concept
-------
term, the research efforts will refine existing in vivo assays to increase the amount of diagnostic information
gained for the invested resources by eliminating redundancies among these assays and reducing the number of
animals needed.
Specific research was initiated to provide in vitro assays and computer-based structure-activity models to
identify those chemicals most likely to disrupt endocrine activity. In vitro assays for estrogen and androgen
receptor binding, steroid metabolism, and enzyme assays were developed for validation and use in Tier I
screening of EDCs. Researchers have begun to develop approaches to use QSAR models to detect chemicals
likely to bind to nuclear steroid receptors. Work to date supports the potential use of QSAR models in a screening
context, but more work is needed to establish data-quality criteria for the next iteration of predictive QSAR
models, Including obtaining broad chemical spectrum, high quality data sets to validate the model. This work
focuses on refining the QSAR model for regulatory prioritization of untested chemicals by developing model
parameters to identify chemicals having low affinity binding characteristics such as expected for chemicals of
concern to the EPA (i.e., they are not intentionally designed to be potent receptor ligands as are
Pharmaceuticals). Effects research is also focusing on a second generation of in vitro assays (e.g., transcriptional
activation assays) which could either to compliment or replace current competitive binding assays required by the
FQPA.- In FY05, the number of chemicals evaluated in two of these assays (for estrogen and androgen receptor
function) will be considerably increased through a competitively awarded research contract. More sensitive in
vitro assays for other indicators of EDO activity (i.e., steroid metabolism and other enzymes such as aromatase)
are also being developed. Having more sensitive and specific in vitro tests will improve the effectiveness of
screening and ultimately reduce the need to confirm in vitro observations in in vivo systems. Particular promise is
being experienced with a cell line (the M295R) that has capacity to synthesize estradiol from cholesterol, thus
covering the entire biochemical pathway responsible for steroidogenesis.
Similar to the efforts with EDCs, researchers began addressing concerns about the sensitivity and cost of
tests used to evaluate chemicals for developmental neurotoxicity. Alternative approaches include the
identification of molecular biomarkers of key events in the developing nervous system that could be targets of
chemical perturbation. In silico and in vitro approaches could be used to establish linkage between exposure and
endpoints of concern and thus aid in predictions of effect and extrapolation (i.e., high to low dose, acute to
chronic, and cross species) for risk assessment. If successful, alternative approaches would reduce or eliminate
the need to test large groups of animals and could be used to prioritize chemicals for testing to meet EPA's data
requirements for registration and re-registration of pesticides.
Finally, other effects-based research focused on identifying initial biological changes associated with the
cascade of biochemical and physiological events leading to toxicity using environmentally relevant chemicals
such as the chlorotriazine and conazole pesticides. Delineation of critical steps of toxicological significance is
important for biologically based dose-response (BBDR) models that can be used to develop predictive models for
hazard identification. Such models will reduce the need to use whole animal systems to meet EPA's data-testing
requirements.
Within the exposure program, research focused on the development of a metabolic simulator (i.e., in silico
model of liver function) that can be used with other models of biological function (e.g., endocrine system, central
nervous system). A metabolic simulator could then be used with other biological models to predict effects of
chemicals and their potential metabolites prior to screening or testing. Knowing which chemical or metabolite to
evaluate for potential toxicity would reduce the number of tests that are actually needed to meet EPA's testing
requirements.
-------
chemicals. The first phase began by considering the available crystal structures of the LED of the androgen
receptor. In order to create a target for the binding of potential environmental ligands, the ligand was removed by
computational means. As a test of the method, the drug that was the ligand in the original crystal structure
(R1881) was re-docked at the target. The results demonstrated that the structure R1881 was influenced by
interaction with receptor and that the overall concept was feasible. However, the software used for this was
developed for application in pharmaceutical area where high affinity ligands are sought. In contrast, in a toxicity
prescreen, even chemicals with low binding affinity need to be flagged. In comparing the results of five docking
methods, the results tended to be very similar, but did not consistently find the chemicals that are weak binders.
Therefore, another strategy that invojved a series of 14 different crystal structures of steroid family receptors was
conducted. This phase included a mutated androgen receptor from a prostate cancer cell line that is androgen
independent. This mutated androgen receptor binds many ligands in addition to androgens and was used to help
identify androgen receptor specific binding affinity. For a series of 26 chemicals (including drugs and
environmental chemicals that are weak androgens and estrogens) all chemicals were classified correctly although
additional research is still needed to refine this protein-battery approach (Rabinowitz et al.t American Chemical
Society Annual Meeting (2004)).
ORD researchers also completed development of a permanently transfected cell line for use in evaluating
estrogen receptor function (Wilson et al., Toxicol. Sci., 81:69-77 (2004)). This cell line complements previous
efforts on the androgen receptor (Wilson et al., Toxicol Sci., 66:69-8 (2002). Both cell lines were placed in the
American Type Culture Collection (ATCC) where they are available to the general scientific community for use.
Already, several laboratories around the world are employing them to study estrogenic and androgenic modes of
endocrine disruption. During their development phase, their use has been restricted to a relatively small number
of known receptor ligands of various binding affinities. Current plans are to expand the numbers of chemicals
evaluated by these assays to upwards of 100 using a longer list of known receptor ligands as well as other
chemicals that are of potential interest to the Agency as endocrine disrupters. Success in this "semi-high
throughput" effort would facilitate refinement of QSAR models for receptor binding and would build confidence in
their use as a tool for screening and prioritization of endocrine-disrupting chemicals.
The proposed Endocrine Disrupters Screening and Testing Advisory Committee (EDSTAC) Tier 1
screening battery includes an assay for steroidogenesis that relies on culture of testicular slices from adult rats.
To date, it has been necessary to rely on tissues from animals for this assay as most cell lines derived from
tissues that are involved in steroidogenesis loose one or more enzymes necessary for the chemical synthesis of
estrogen from cholesterol. The CompTox program has been funding an effort to evaluate the H295R human
adrenocortical carcinoma cell line for this purpose because it retains and expresses all the key enzymes
necessary for steroidogenesis. This effort has focused on developing and standardizing a multi-plexed Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR)-based protocol ("molecular beacons") to evaluate the
expression of 11 steroidogenic enzymes in conjugation with key intermediates and end products. In several
publications (Hilscherova et al., Toxicol. Sci. in press (2004); Zhang et al., Toxicol. Sci. in press (2004)), this
system has been described and evaluated using a set of known pharamcologic agents. Results from the H295
assay have shown sufficient promise that it will be undergoing jnter-laboratory round robin testing by
OSCP/OPPTS as a potential replacement for the sliced testes assay in the proposed EDSP Tier 1 battery.
Within the exposure program, there have been significant advances in research which applies
computational methods, molecular biology, and chemistry tools on the source-to-dose components of the
paradigm and which furthers integration of these components with adverse effects and altered phenotypes
mentioned above. Both prognosis of environmental and organismal metabolism of stressors and diagnosis of
sources of exposure are being advanced by integration of tools across computational chemistry, as well as
genomics, proteomics, and metabonomics. Prognostic metabolic research has focused on the development of a
metabolic simulator (i.e., in siiico model of liver function) that can be used with other models of biological function
(e.g., endocrine system, central nervous system). A metabolic simulator could then be used with other biological
models to predict effects of chemicals and their potential metabolites prior to screening or testing. Knowing which
chemical or metabolite to evaluate for potential toxicity would reduce the number of tests that are needed to meet
EPA's testing requirements. Diagnostic research has focused on the development of molecular indicators of
-------
exposure of aquatic organisms to environmental stressors. A primary challenge in wildlife organism research is
the lack of any large scale effort to sequence genomes. Consequently, the fathead minnow (Pimephales
promelas), the Agency's principal aquatic toxicity test organism and the subject of an EDSP Tier 1 reproductive
screen, has served a role in the discovery of hundreds of gene sequences and expressed sequence tags. These
sequences are being added to hundreds more being provided by a partnership between ORD and the
Department of Energy's Joint Genome Institute which is conducting high-throughput sequencing of fathead
minnow cDNA libraries. Following DMA microarray development, the potential exists for extensive gene profiling
and the development of scores of unique diagnostic exposure indicators to use for aquatic ecosystem monitoring
and to link to phenotypic changes to reveal mechanisms of action. Species extrapolation opportunities (i.e., fish
to mammals) will then present themselves.
A component of the program with a long-term focus is the study of endocrine disruption from the
perspective of systems biology. A number of environmental chemicals have been shown to alter the
neuroendocrine control of gonadal function by targeting the brain and pituitary, specifically the secretion of
luteinizing hormone (LH). These changes in hormone secretion are known to occur in response to a variety of
yet-unidentified molecular changes induced by the test chemical. Thus, a major challenge in these studies has
been the rapid identification of the mechanism of action for such effects. The primary purpose of this project is to
develop a protocol to identify and characterize the target neuronal pathways involved in impaired LH secretion.
ORD investigators are evaluating a protocol that takes advantage of our knowledge of the well-defined series of
neurona! events that lead up to the generation of the ovulatory surge of LH. This hormonal event is crucial for
oocyte maturation, ovulation, and normal reproductive function in the female. The protocol being tested involves
the use of Enzyme Linked Immunosorbent Assay (ELISA) assays for measurement of the neuropeptides that
regulate gonadotropin releasing hormone (GnRH) and GnRH itself. GnRH represents the final brain signal that
regulates LH secretion in both sexes. Using a systems biology approach, it is hypothesized that by identifying
which of the several neuropeptide and neurotransmitter pathways involved in the disruption of GnRH/LH secretion
will serve as the point of departure identifying the cellular mechanisms involved in the effect of the test chemical.
During 2004 this protocol was tested using the chlorotriazine herbicide atrazine, and a dose dependent change in
GnRH/LH release was identified. Related changes in the neuropeptidergic (i.e., neuropeptide Y, p-endorphin,
and neurotensin) and neurotransmitter (i.e., norepinephrine, dopamine, and serotonin) neurons are currently
being examined in an effort to determine the extent to which these neural pathways are affected by atrazine and
how such changes are related to the already identified decrease in LH release. Once developed and
standardized, it is anticipated that this protocol will provide an approach that can be used to rapidly identify the
neuronal pathway and the cellular mechanism involved in altered neuroendocrine function. Such information can
then be employed to further determine the dose response for the test chemical.
B. Microbial Source Tracking: Microbiological impairment of natural water systems is assessed by
monitoring for the presence of culturable fecal indicator bacteria. These microorganisms are associated with fecal
material from humans and other warm-blooded animals. Their presence in water may also signal the presence of
enteric pathogens that could cause illness in exposed persons. Tracking fecal pollution to its source is a topic of
intense interest in view of the current Total Maximum Daily Load requirements. Microbial Source Tracking (MST)
is one approach to determining the sources of fecal pollution and pathogens affecting a water body. The basic
problem of current MST methods is the need to culture microorganisms to assess the primary sources of
pollution. To circumvent such a problem two molecular approaches are being assessed. First, Bacteroides 16S
rDNA sequence analyses are being done to search for culture-independent bacterial source-tracking markers.
Thus far, fecal clone libraries have been generated from cattle, horses, pigs, goats, sheep, chicken, turkey,
possums, rabbits, coyotes, vultures, squirrels, and armadillos. Preliminary results indicate that a considerable
number of novel sequences are associated with these samples and that some of the sequences might be
potential markers for specific fecal sources. Secondly, genomic subtraction techniques are being developed that
will be capable of enriching for microbial host-specific gene sequences. Studies have focused on DMA
sequences of the fecal indicator Entercoccus faecalis. Genomic DN A of E faecium was used as the subtracting
matrix. Genomic information for both organisms has been made publicly available to facilitate the downstream
analysis of genomic clones. Alignment searches of the sequenced clones yielded known E. faecalis DMA
10
-------
sequences. Most of the enriched sequences were unique to £ faecalis while some showed a poor level of
sequence identity to other bacterial sequences. Analysis of over 300 clones indicated that the enrichment method
selected for sequences within the 16S rDNA, the 23S rDNA, and the 16S-23S spacer region. Other enriched
sequences were linked to gene families of putative virulence-related surface-exposed proteins, sugar- and polyol-
utilization pathways, membrane transporter proteins, bacteriophage proteins, and membrane-anchored lipase
proteins. These results suggest that this subtraction method could identify sequences present in different
genomic environments. The next step is to use genomic subtraction of whole fecal microbial communities to
develop novel molecular markers for fecal contamination and fecal source identification. These molecular
markers will be used to better predict the relationship between microbial water quality and public health risks and
to determine the effects of different microbial pollution sources on watershed biology.
C. Augmentation Funding: In order to facilitate the rapid incorporation of "omic" tools into the ORD
research program, the CTISC set aside nearly $1M for small grants to augment existing efforts so that this
component could be added. In total, 10 projects (Table 2) received an average of S75K in funding that covered
genomics (5 projects), proteomics (1 project), metabonomics (2 projects), and database development (2 projects).
The RFA for this activity is provided in Appendix A. The genomic studies include efforts to study androgenic
exposures in fish, thyroid disruption in frogs, and toxicity pathway identification In rodents exposed to conazole
fungicides. This last project involves a novel approach to microbial source tracking. The proteomic study
involves analysis of effects of estrogenic chemicals in fish, while the metabonomic studies include applying NMR
spectroscopy to rainbow trout exposed to conazoles (one of the first such applications to aquatic species).
Additionally it involves mass spectrometry analysis of lipids in lavage fluid from humans exposed to diesel
exhaust in the search for novel biomarkers of exposure or effect. Finally, the database projects include migrating
the Assessment Tools for the Evaluation of Risk (ASTER) database to a more accessible web-based approach
and expanding the annotated datasets available under the Distributed Structure-Searchable Toxicity (DSSTox)
effort.
D. New Starts: As noted previously, awards for the new start proposals are expected in October 2004
and will be summarized in the next CompTox Accomplishment Report. The RFA for this activity is provided in
Appendix B.
E. The STAR Program: ORDs extramural research program sponsored three RFAs that supported
the CompTox program. The first of these was directed at the development of screening systems for endocrine
systems which may lead to the development of high-throughput screening systems to assist in the prioritization of
chemicals for further screening and in the testing of their potential as endocrine disrupters. In view of the
estimated tens of thousands of chemicals under consideration for screening for endocrine disrupting activity, it is
essential that rapid, sensitive, and reproducible high-throughput screening systems be developed. This request
for applications (http://es.epa.gov/ncer/rfa/archive/Qrants/03/current/2p03high_thrQughput.html) invited research
proposals that explored novel approaches to the development of such systems that can be used to identify
chemicals with estrogen, androgen, or thyroid hormone activities. The projects are listed in Table 3A and include
assays on development of an improved yeast-cell-reporter assay for estrogens and androgens and development
of two fish models (zebra fish and medaka) and an invertebrate model (Daphnia) that could be used as screening
tools for evaluating endocrine disruption. All projects received funding in late September 2003.
11
-------
STAR RFAs
High Throughput EDC Screens
Systems Biology Models
Center for Environmental Bioinformatics
The second RFA was devoted to supporting research developing systems-level biological models of the
hypothalamic-pituitary-thyroid/gonadal axis in small fish or laboratory rodents for assessment of endocrine
disruption. The RFA (http://es.epa.gpy/ncer/rfa/current/20g.gj_comptox.htmi) closed in January 2004, and three
subsequent awards have been made (see Table 3B). Significantly, one of these awards is being made as a
cooperative agreement so that the academic scientists can work more closely with EPA investigators in
Cincinnati, Duluth, and Gulf Breeze to hasten the development of systems biology models of the fathead minnow,
a fish species commonly used in aquatic toxicology tests. EPA scientists will be focusing on the androgen
system, steroidogenesis pathways, and
pituitary function while the academic
laboratory will focus on estrogen sensitive
pathways of endocrine disruption. The
other two projects include one using a set
of model endocrine-disrupting chemicals
in the medaka, and one studying gene
network alterations induced in the rat
uterus by estrogens. These projects
received funding in September 2004.
The final element of activity within the STAR program involved development of an RFA to establish an
EPA Center for Environmental Bioinformatic Research that will be able to apply multi-disciplinary computational
approaches across the source-to-outcome continuum. The RFA for the Center is provided in Appendix C.
Research conducted through the intramural CompTox program will form a significant fraction of the data upon
which novel and innovative bioinformatic approaches will be applied. The funding level will be $1M per year for
up to 5 years. The solicitation will be announced in October 2004, and awards are anticipated to be announced in
early 2005.
F. Partnerships: ORO has reached out to establish a number of partnerships with both public and
private sector organizations, (see Table 4) as a way to leverage resources and to enlist the assistance of those
with complementary expertise. A partnership with the DOE
provides sequencing support for the construction of
microarrays for the fathead minnow, bioinformatics support for
microarray and metabonomic studies, computational support
for metabolic simulation, virulence-factor assessment of water-
borne microorganisms, and preparations for the virtual
metabonomics facility. IBM is assisting EPA with quantum
mechanic/molecular models of metabolic rate constants for
pyrethroid metabolism, with advanced pattern recognition
algorithms for microarray data, and by establishing a
metabonomics node within the NIEHS Chemical Effects in
Biological Systems (CEBS) database. Discussions are ongoing with the DOD regarding the use of genomic
analysis of C. elegans as an alternative animal model for neurotoxicity and the pooling of resources to advance
the science of ecotoxicogenomics. A partnership with FDA will assist in development of regulatory acceptance of
genomic data because both groups have begun to address this issue through a joint effort. Finally, a Material
Cooperative Research and Development Agreement (mCRADA) with Affymetrix is providing material and
bioinformatic tools to compare in vivo and in vitro genomic toxicity pathways for the conazole project.
Established Partnerships
Dept. of Energy
Dept. of Defense
NIEHS
IBM
CUT
Affymetrix ,
G. Recruitments: Over the past year, ORD has hired
new staff with expertise in various aspects of computational
toxicology. Dr. David Mustra joined NCER a toxicologist after
completing two postdoctoral research positions: one with the
Center for Marine Biotechnology and Biomedicine at the Scripps
12
Recruitments
NMR Spectrometrist
Bioinformaticians
Toxicogenomicist
-------
Institution of Oceanography and the other in the Department of Molecular and Experimental Medicine at the
Scripps Research Institute. Dr. Mustra received his Ph.D. in Pharmacology and Toxicology from Dartmouth
College, and will be project officer for the Center for Environmental Bioinformatics when it is awarded in FY05.
Drs. Mitchell Kostich and Ronglin Wang were recruited to provide bioinformatics support centered in the NERL-
Cincinnati laboratory, but serving NERL's greater computational toxicology effort. Mitchell Kostich received his
Ph.D. in Biology from Johns Hopkins University and spent several years as a senior scientist in the
pharmaceutical industry; Ronglin Wang, who received his Ph.D. in plant physiology from Iowa State University,
conducted computational biology research as part of the rice genome project in industry. NERL-Athens is
recruiting a senior level expert in NMR spectroscopy to operate the Metabonomics Facility. NHEERL has an
ongoing recruitment effort to hire a senior level researcher in toxicogenomics. In addition, several postdoctoral
fellows have been recruited to conduct CompTox-related research; and efforts are underway to recruit others
through an ORD-wide competitive process.
H. Related Research in ORD: Just as the bibliography demonstrated that research related to
CompTox was ongoing throughout ORD, an analysis of the upcoming annual performance measures (APMs)
indicates that research on more predictive tools, either based on fate and transport, metabolic, or dynamic
approaches was also prevalent in other Multi-Year Plans. As shown in Table 5, the 125 APMs identified within
ORDs multi-year plans (MYPs) cover a broad range of media-specific questions and cross human-ecological
boundaries. In general, the projects involve the application of emerging technologies and computational tools to
develop predictive models of pharmacokinetics with a mix of in vitro and in vivo study approaches. There is a
slight emphasis on improving the linkages in the source-to-outcome paradigm as compared with elements
directed at screening, prioritization, and quantitative risk assessment. As expected, this emphasis is not
consistent across MYPs. For example, the screening objective shows higher prominence under Drinking Water,
SP2 and EDCs; whereas the linkage objective is most evident under Air Toxics and Human Health Assessment.
In this analysis, it should be realized that a particular APM could well address multiple objectives of the CompTox
program, but a decision was made to only attribute a particular APM to a particular objective. Summary
information on the APMs from each of the surveyed MYPs is contained in the following paragraphs.
Particulate Matter: The single APM in PM identified as pertinent to CompTox involves characterizing the
mechanism by which inhaled particles affect cardiopulrnonary endpoints in multiple species. A PM study
received augmentation funding from the CompTox program for exploration of the application of
lipidodomics to lung lavage samples from subjects inhaling diesel particles (see Section III.D).
Air Toxics: The thirteen APMs under Air Toxics include pharmacokinetic modeling efforts targeted at
specific chemicals (e.g., hazardous air pollutants, or HAPs), or combinations (TCE and CCU) of chemicals
with attention to extrapolations across exposure duration, route and/or age; mechanism-of-action studies
utilizing in vitro systems to look at the neurotoxicity of VOCs; and genomic and proteomic approaches to
assess carbonyl, HAPs, and chlorine, with a goal of beginning to incorporate computational toxicity tools
in respiratory risk assessment.
Drinking Water: Drinking Water has thirty APMs with relevance to Computational Toxicology
encompassing both toxicological and microbiological research. The toxicology projects include looking at
pharmacokinetic/pharmacodynamic (PK/PD) models of particular Contaminant Chemical List Chemicals
(CCLs), including arsenic, bromodichloromethanes, nitrohalomethanes, and cyanobacteria toxins. There
is a focus on route-to-route, high-to-low, and lab-animal-to-human extrapolation. Additionally, a few
projects will study gene-environment interactions. Other projects are looking at QSAR models for the
CCLs and alternative animal models such as neuronal cultures to study neuronal development. The
microbiological research is using genomic and proteomic tools to identify and characterize pathogens and
to assess virulence factors (VFARs). Such studies include efforts on biofilms, water quality measures,
and on developing tissue-culture models to identify pathogens.
Water Quality: The single APM in Water Quality with relevance to CompTox involves the development of
13
-------
a pharmacokinetic/pharmacodynamic model for predicting individual effects on birds from chronic mercury
exposure.
EDCs: In the EDC area, there is an emphasis within the 27 APMs related to Computational Toxicology
on screening methodologies for. prioritization purposes and on application of molecular biological tools to
the assessment of endocrine disruption in ecologically relevant species. Specific projects are involved
with development of assays, databases, and SAR models related to receptor binding, gene activation,
and steroidogenesis. Within the STAR program, a number of projects are developing other high through-
put assays (see Section III.E}, as well as developing systems level models of the hypothatamic-pituitary-
gonadal axis of small fish and laboratory rodents (see Section III.E). Several intramural projects which
utilize pharmacokinetic or pharmacodynamic modeling approaches also involve with study of the
neuroendocrine axis. Another area of emphasis the development of genomic and proteomic approaches
for studying the effects of estrogens and (anti)-androgens on a variety of aquatic organisms including
fathead minnow, sheepshead minnow, and frogs. At least one project is involve'd with application of
these technologies in the field (the Experimental Lake study in Canada).
Human Health Risk Assessment: The Human Health Risk (HHR) program has the largest number (38) of
APMs relevant to Computational Toxicology, reflecting the core nature of that research area. There is a
general underlying theme of taking advantage of the emerging genomic technologies in terms of
assessing the effects of mixtures (including those with similar and dissimilar modes of action) and
developing principles for the application of predictive pharmacokinetic and pharmacodynamic models.
Another common thread is the study of particular modes of action that may have a common basis in the
induction of cancer versus non-cancer health effects (e.g., signal transduction pathways, oxidative stress
pathways, receptor activation) using both in vitro and in vivo approaches. One area of particular
emphasis in this thread is the application of genomic, proteomic, and metabonomic tools to the study of
chemicals which are hypothesized to induce their toxicities (both cancer and non-cancer) through
modulation of the activity of P450 metabolizing enzymes (a topic receiving augmentation funding from the
CompTox program, see Section III.C). A final thread is the understanding of how modes of action vary as
function of age and how such effects could have long latencies for detection or long latencies for
involvement in toxicity through interaction of aging, exposure, and pre-existing conditions.
Safe Pesticides/Safe Foods: Relevant APMs in SP2 are generally contained within its LTG1, which is to
validate virtual screening methods, a goal very similar to that of Objective 2 of the CompTox Framework.
Predictive models under development include those for generation of activated metabolites of pesticides,
developmental/reproductive toxicity, pyrethroid neurotoxicity, genetic and genomic integrity of sperm,
steroidogenesis, an in vitro system for assessing the age dependent toxicity of cholinesterase-inhibiting
organophosphate pesticides, the effects of pesticidal mixtures on neuronal ion channel function, and for
predicting major neurodevelopmental processes (e.g., apoptosis, neuronal differentiation, axonal
outgrowth, and synaptogenesis) using in vitro systems.
V. FUTURE DIRECTIONS
Building on the momentum established in FY04, the
CompTox program is preparing to articulate specific short-term and
long-term accomplishments in FY05. The program will be increasing
its activity in the endocrine-disrupter proof-of-concept studies by
scaling up the application of bioassays to broader ranges of the
chemicals. It will be awarding a competitive contract to evaluate
predictive toxicogenomic computational models for liver toxicity, and
it will initiate the New Start projects and the STAR Center for
Bioinformatics. The New Start projects will create six or seven
research activities that will form the basis of the intramural research
Projected FY05 Activities
Expand Intramural Program
Begin Scale up Studies
Test Predictive Toxicogenomics
Build Infrastructure
Partner with OPPTS
Establish Bioinformatics Center
Establish Intramural Center
14
-------
program in computational toxicology. The proposals are currently undergoing external scientific review and
internal relevancy review; decisions are expected in late October 2004. Infrastructure building will continue with
the arrival of the ORD Capital Equipment Committee-supported NMR spectrometers in the Athens laboratory.
The'contract was awarded in September 2004, and delivery is expected in late 2004. The facility is expected to
be operational by Spring 2005. A number of research activities are underway to utilize this new capacity to
conduct metabonomic research, including discussions with NIEHS on the use of the CEBS database as a
analytical and storage device for the resulting spectra. Another contract to evaluate commercially available
predictive models of target organ toxicity based upon acute changes in gene expression is expected to be
awarded in November 2004. The contract will be used to test five chemicals in a blinded fashion to evaluate the
utility of the models for EPA relevant chemicals and to potentially prepare for the way for a more extensive use of
that experimental approach.
Expanding beyond the proof-of-concept studies to more real world situations, the FV05 funding initiative
will target the issue of screening and prioritization of evaluation for the non-food use pesticidal inerts. The need
for ORD to help develop approaches for prioritizing chemicals for subsequent screening and testing was
highlighted by the OPPTS/ORD Senior Management Retreat of August 2004. From that retreat, a strategic
direction focusing on development of quantitative structure activity models, high throughput screening assays,
and pollution prevention strategies was identified. An action item from this retreat was to develop a plan for
conducting two proof-of-concept projects building on the earlier EDC projects and the FY05 pesticidal inerts
initiative. Additional planning meetings are being scheduled to develop more specific timeline and events.
Finally, in order to provide a firm foundation for this program to flourish, ORD is establishing a Center for
Computational Toxicology. This Center will provide leadership to the Agency on computational methods that can
be applied to a variety of research activities supported by the Center or that are relevant to its objectives. Details
of the structure, function, and composition of the Center are currently being worked out; preliminary decisions are
expected in October 2004. Establishment of the Center should increase the visibility of the program to the outside
world and provide a hub at which the further development of toxicology as a predictive science can be achieved.
Thus, in conjunction with the awarding of a Center for Environmental Bioinformattc Research by the STAR
program, ORD will have the structure and capabilities to make significant progress in the area of computational
toxicology.
15
-------
D)
C
'§
55
•o
R>
O
1 .
icology Implemen
X
15
a
E
o
o
Q
tt
O
1
•5
o
1ST
Table 1. Compi
Committee (CTI
E
Karen Hammerstro
Ines Pagan
HI
o
Elaine Francis
David Mustra
o:
UJ
o
z
Greg Toth
Eric Weber
Of.
LU
z
*n
1
.'S-J
Gary Ankley
Jack Fowie
Robert Kavlock (Cr
Larry Reiter (Execu
Hugh Tilson
Doug Wolf
I
NHEERl
o
0)
£
o
D
NRMRL
Dennis Pagano
OAQPS
Vickie Dellarco
Tim McMahon
OPPTS
Steve Kueberuwa
Clifton Townsend
O
David Macarus
o
'9
££.
(D
-------
;'I
ifi §
f.Elcl1
m » C O •" o>
itlll^
**?*«§•
s?Ui
3«Slt
1 S 2 §.< •=
sf^iiff
.2 < .« § •- -° e
IS]^^^^,
.itjH!
JttiM
iii?if
•5 £ S15 •
o
Rt
J- Q.£
I
o> 1
£ J» 5
-------
Jill
o JS
fS.
S S
i|*l
III 1
.
12
* 8
P
1
is
J
5
-8 2
2 o
II!
S S o =
-------
TJ
I
i
o
I
8
o
c
a
•5
5f ?s
= * m-o
S1H*
l|i-~l
jj-,S n n c
<5 i £ %,&
i c •- i1»
t: D)^- C As TJ
l|5||l
JS -0 TJ 41 Q._
•s|«4
'3?I*1
f>* O *3
•° 5-9
!«8:
8'Sl'l'I^i^
8ltf*«l*l*
lltllfisl"
flliillilii
-------
O
CM
-------
"IsS
o*
!l-
_ .2 a «
Jllip
^Jlc-nlS
S^^o^mS"
i:M*5a**°
«2«"S'S-« =
itilill!
c ^^o » g E . gs
l^g|§illi
^ | o .9 > S _
5 "
K&
•H M .T
« • .£ « • .g
Illiii
^Isl3-
a.S5
€^5
£ O
H Jl
42
•g.
PS
S
a
5'
£'
«
1 I
^ .. CQ
sts
111
Z 3 O
I
ill
C ffl $
O <0
-------
fc
CN
(N
-------
C TT lw * f\ f\ ^
?
PI-
te
i|H
lilt
to
informatics c
roarray anal
ctral analysis.
Ap
Bioi
.2
a
§ 5
will be
that
toxicity, reaction ki etc.) for chemicals
that are known to p activated
metabolites and wh xicity pathways are
known, signature-b SARs will be
constructed from this d. Using the
QSARS, the next stage of the project
to select signatures (structural motifs)
s, et
roduce
se toxi
d Q
ata.
o '
CO
CM
or
UJ
8.
I 1
o »
a co
-------
Tf
eg
-------
ID £
I *
« a
I 1
a. 03
it!
11!
SS.8-
uo
CN
OC
UJ
2
o
-------
dj O)
d j—
**
0) m
€ 3
"i§
-------
APPENDIX A.
Request for Augmented Funding for
Computational Toxicology Research
February 6,2004
Background: The Computational Toxicology Implementation Steering Committee (CTISC) of ORD has
reserved approximately $750K in FY04 extramural funds to augment existing research projects to
address computational toxicology issues. We anticipate making between 6 and 12 awards through this
request. The CTISC seeks to use these funds to leverage current efforts and is particularly interested in
assisting in the development of near term products that can demonstrate the potentials of a
computational toxicology approach. Therefore, submissions should emphasize the ability of this
funding to produce relatively rapid and relevant products for computational toxicology that would not
occur with existing funding levels. Guidance on the objectives of the Computational Toxicology
program can be found at wwvv. epa.gQV/comptox. ORD staff interested in this funding should submit the
information requested below to Robert Kavlock (CTISC Chair) by March 15,2004. Funding decisions
will be made after review of the submissions by the CTISC for relevancy, scientific merit, and
timeliness of the product and will be announced approximately four weeks later. Requests should not
exceed three single spaced pages.
Project Description:
Principal Investigators (include organizational identity):
Current Funding Source and Level (GPRA Subobiective. FTEs and extramural Hinds available):
Existing APMs, if any, for Project:
Linkase to Computational Toxicology Framework (be specific as to objective):
Amount Requested^
Purpose (describe what the funds will be applied to do. also provide indication of relevant products to
demonstrate onsoins progress):
Intended Funding Vehicle:
Rationale (whatbenefits to computational toxicology will be afforded):
Products (include estimate time of completion):
27
-------
-------
IV. CURRENT ACTIVITIES:
Design Team Role
Develop Framework
Inventory Research
Conduct Workshop
As evidenced by the above discussion, ORD has begun to transition from the EDC-based proof-of-
concept-based program initiated by FY02 Congressional directive to a broader context of predictive models. A
major step in this process was establishment of the cross-ORD Computational Toxicology Design Team in April
2003 that was given three tasks: (1) develop a Framework document that would provide the rational and
foundation for an expanded research program; (2) provide an inventory of existing research being conducted in
ORD that related to the Framework; and (3) have the Framework peer reviewed and presented to the ORD
scientific community. In meeting these goals, the Design Team developed "A Framework for a Computational
Toxicology Program in ORD" (EPA/600/R-03/065) that received a favorable review by an ad hoc panel of the EPA
Science Advisory on September 12, 2003. The Framework defined computational toxicology as "the application
of mathematical and computer models and molecular biological approaches to improve the Agency's prioritization
of data requirements and risk assessments." It identified three main objectives under which to focus activities of
the program: (1) improving the linkages in the source-to-outcome
paradigm; (2) developing screening and prioritization tools; and (3)
enhancing quantitative risk assessment. Subsequently, a public
workshop was held on September 29-30, 2003, at which the Framework
was presented and workgroups addressed how the ORD research
program could address the needs (Kavlock, et al., Reproductive
Toxicology, in press (2004)}. The final goal of constructing a
bibliography was also completed; and the material related to the
Framework, the workshop, and bibliography were posted on a website (www.epa.gov/connptox). This
bibliography, which covers the period of 2000-2004 and contains 158 peer-reviewed publications related to the
three objectives of the Framework, provides a reference base upon which to track progress in the future
development of the CompTox program. The majority of these publications (72%) address the Linkage objectives;
the remaining address Prioritization (18%) and Quantitative Risk Assessment (9%).
With the completion of its goals, the Design Team was replaced by the Computational Toxicology
Implementation and Steering Committee (CTISC) in January 2004. Representation on that committee is shown in
Table 1. Membership in the CTISC now includes representatives from the major client offices, as well as the
Regions, in addition to two scientists from each of the Labs and Centers. The CTISC was given the charge of
implementing the broader program while
continuing work with the existing proof-of-
concept activities related to endocrine-
disrupting chemicals. Resources included $2.4
million in the Science to Achieve Results
(STAR) program and $3.5M to initiate new
programs. These are in addition to amounts
within the Labs and Centers. Approximately
23 full-time employees (FTEs) are assigned to the program. The CTISC issued two requests for applications
(RFAs) (Appendix A and B) in February 2004: for the addition of funds to existing programs in order to enhance
the utilization of genomic tools and computational approaches ("Augmentation Projects") and new projects in
computational toxicology ("New Starts"). Following an internal relevancy review of 18 submitted projects totaling
S1.9M, $750K in funds were provided to 10 Augmentation Projects in April 2004. For the "New Start Projects,"
the Committee received 42 Letters of Intent to submit projects, which resulted in receipt of 28 pre-proposals in
April 2004. Following subsequent relevancy review by the Committee, 14 submissions were selected for
development of a full research proposal. These proposals were received on August 20th and are being reviewed
externally for scientific quality and internally for programmatic relevance to ensure that the resulting portfolio
represents the strongest possible foundation for the CompTox program. It is anticipated that approximately 7
awards (with average funding level of $300K per year) will be made in early FY05. The remaining FY04 funds
were devoted to interagency agreements (lAGs) with our federal partners in the Department of Energy for
bioinformatics support and to a competitive contract for a proof-of-concept study on predictive models of organ
toxicity using genomic approaches. All of these elements are ultimately targeted to inform the process of how we
Implementation and Steering Committee
Translate Framework into Research Program
. Ensure Programmatic Relevance
Build Internal and External Partnerships
-------
determine which chemicals need to be tested in which order for which endpoints, thus ultimately making our use
of animal testing procedures more effective and efficient. Details of each activity are provided In the following
sections. Due to the start of the most of these activities during this current year, most of the progress of the
program will be apparent as the initiation of activities rather than the specific research outcomes at this point In
time. That should change dramatically as the new studies come on line and begin to produce new data.
A. EDO Proof-of-Concept Studies: Proof-of-concept studies trying to identify endocrine disrupters
using short-term bioassays or computational approaches formed the early basis of the computational toxicology
program, and progress has been achieved on a number of activities.
Proof-of'Concept Studies
Estrogen Receptor Binding and QSARs
Molecular Docking Models
Transcriptional Activation Assays
Steroidogenesis
Systems Biology Models
Metabolic simulator
In support of the Agency's Endocrine
Disrupter Screening Program (EDSP), QSAR
models are being developed to prioritize
chemicals for endocrine-disrupter screening.
The reliability of the models are being
improved by testing a broader subset of
chemicals more representative of the chemical
structures found on lists of chemicals needing
prioritization. A chemical test system utilizing
in vitro assays along a highly conserved
endocrine-disrupter toxicity pathway has been optimized for development of the high quality test sets needed for
model formulation (Schmieder et al., Environ Sci Tech, in press (2004)).. Algorithms are under development and
evaluation for strategic selection of chemical analogue types within the OPPTS lists of concern (Aladjov et al.,
11th International Workshop on Quantitative Structure-Activity Relationships, 2004). Additionally, models are
being systematically expanded to cover relevant structure space based on chemical selection strategies and
coordinated in vitro testing (Schmieder et ai.. Pure Appl. Chem. 75:2389-2396 (2003)). Another study is being
conducted that will provide a well-defined data set describing the estrogen receptor (ER) binding characteristics of
300 chemicals. These data are necessary to refine and improve current QSAR models which are designed to
detect chemicals likely to bind to the ER and to demonstrate the appropriate methods for evaluating data from
steroid-receptor competitive-binding assays.
During an earlier evaluation of two QSAR computer stimulation models for ER binding, the Agency
determined that neither model was valid for use in chemical prioritization. Each of these models was tested for its
ability to predict the ER relative binding affinity (RBA) of 300 chemicals and compared with the actual RBAs from
laboratory assays for each chemical. Neither of these models preformed adequately with regard to sensitivity,
specificity, and positive predictive probability. Problems with the current ER QSAR models include that they were
developed using competitive binding data from multiple laboratories and that some of the data from chemicals
used to develop the models did not reflect a true competitive inhibition. Thus, using the competitive binding data
from the 300 chemicals used to test the models, additional Kj experiments are being conducted for a subset of 50
chemicals which appeared to exhibit an ability to alter ER binding. These studies will determine whether or not
the chemicals are true competitive inhibitors of ER binding and will provide a more precise estimate of ER binding
affinity. To date, the Kj experiments have been completed on 20 of the test chemicals. The data will be reported
in early FY05. Once these assays are complete, ER binding data from the entire set of 300 chemicals will be
published and used for refining the existing QSAR models. In addition, examples of ER competitive binding
curves that could be observed from a structurally diverse group of chemicals will be included in the EDSP protocol
for the ER competitive binding assay to provide guidelines for data interpretation.
Using a completely in silico method, another was designed to predict the capacity of chemicals to disrupt
the endocrine system through action at the ligand-binding domain (LBD) of steroid hormone receptors. Crystal
structures of steroid receptors were obtained, and computational methods were used to estimate the ability of an
environmental chemical to interact with the target. The computationally intensive procedure for evaluating this
interaction is called "docking" and requires no a priori knowledge of the mode of interaction for unknown
8
-------
Any federal scientist within the USEPA's Office of Research and Development (ORD) is eligible to
submit a pre-proposal, but it is expected that most research supported by the Computational Toxicology
Research Program will be in multi-disciplinary in nature. Thus, pre-proposals that incorporate expertise
from multiple ORD labs and centers, or at minimum, across divisions within a laboratory, will be given
greater consideration while pre-proposals that support an individual scientist will be given low priority
in the review process. Applicants are also encouraged to work with those federal, industrial, and
academic (e.g., STAR grantees) partners, which have begun to be associated with the ORD
Computational Toxicology Program (e.g., Department of Energy laboratories) as well as other
institutions that can complement the efforts of ORD.
Notice of Intent to Submit Pre-Proposal
In order to conduct advance planning for review of the pre-proposals, applicants are requested to notify
the CTISC Chair (see contact information at end of instructions) of the intent to submit an application
under this RFA. The notification should identify the lead investigators and tentative project title and
should be received no later than March 5, 2004.
Review Process and Timeline
The CTISC and selected non-EPA scientists will review the submissions for relevancy and merit of the
application. Review criteria can be found in Section IV of the Framework. Internal EPA reviewers will
s~\ be primarily focusing on the relevancy of the submissions, but will also be evaluating the technical and
4& scientific merit, and potential for impact. External reviewers will be reviewing primarily for technical
and scientific merit. All pre-proposals will receive a summary of the review comments, and those
selected for further work will be given approximately two months for submission of the full, more
detailed, proposal. The preliminary review is expected to be complete by June 1 and subsequent
submission of full proposals on August 1). Those proposals will undergo a second round of review,
with final funding announcement expected in September 2004. In developing the program, CTISC will
be looking to ensure a balanced and integrative portfolio that addresses multiple aspects of the
Framework.
Reporting Requirements
Proposals that are ultimately approved under this RFA will be required to develop a fact sheet on the
project suitable for public distribution and to provide semi-annual updates on research progress to the
CTISC. A meeting of principle investigators to review progress is anticipated during the second year of
the program (i.e., FY06).
Availability of Funds
CTISC anticipates that approximately $1.8M in extramural funding will be available in FY04 and that
an additional S2.5M in FY05 funds will also be available for projects funded by this RFA. We
anticipate funding 6-8 projects, with an average award of $300K per year. Decisions as to which FY
funds will be awarded to a given project will be made taking into consideration the nature of the project,
the use of funds, and the likely time than funds could be committed to approved projects (e.g., if an
application identifies that a competitive cooperative agreement is the funding vehicle of choice, it is
^^^^ most likely that FY05 funds would be used for that project).
29
-------
-------
Any federal scientist within the USEPA's Office of Research and Development (ORD) is eligible to
submit a pre-proposal, but it is expected that most research supported by the Computational Toxicology
Research Program will be in multi-disciplinary in nature. Thus, pre-proposals that incorporate expertise
from multiple ORD labs and centers, or at minimum, across divisions within a laboratory, will be given
greater consideration while pre-proposals that support an individual scientist will be given low priority
in the review process. Applicants are also encouraged to work with those federal, industrial, and
academic (e.g., STAR grantees) partners, which have begun to be associated with the ORD
Computational Toxicology Program (e.g., Department of Energy laboratories) as well as other
institutions that can complement the efforts of ORD.
Notice of Intent to Submit Pre-Proposal
In order to conduct advance planning for review of the pre-proposals, applicants are requested to notify
the CTISC Chair (see contact information at end of instructions) of the intent to submit an application
under this RFA. The notification should identify the lead investigators and tentative project title and
should be received no later than March 5, 2004.
Review Process and Timeline
The CTISC and selected non-EPA scientists will review the submissions for relevancy and merit of the
application. Review criteria can be found in Section IV of the Framework. Internal EPA reviewers will
be primarily focusing on the relevancy of the submissions, but will also be evaluating the technical and
scientific merit, and potential for impact. External reviewers will be reviewing primarily for technical
and scientific merit. All pre-proposals will receive a summary of the review comments, and those
selected for further work will be given approximately two months for submission of the full, more
detailed, proposal. The preliminary review is expected to be complete by June 1 and subsequent
submission of full proposals on August 1). Those proposals will undergo a second round of review,
with final funding announcement expected in September 2004. In developing the program, CTISC will
be looking to ensure a balanced and integrative portfolio that addresses multiple aspects of the
Framework.
Reporting Requirements
Proposals that are ultimately approved under this RFA will be required to develop a fact sheet on the
project suitable for public distribution and to provide semi-annual updates on research progress to the
CTISC. A meeting of principle investigators to review progress is anticipated during the second year of
the program (i,e., FY06).
Availability of Funds
CTISC anticipates that approximately S1.8M in extramural funding will be available in FY04 and that
an additional $2.5M in FY05 funds will also be available for projects funded by this RFA. We
anticipate funding 6-8 projects, with an average award of $300K per year. Decisions as to which FY
funds will be awarded to a given project will be made taking into consideration the nature of the project,
the use of funds, and the likely time than funds could be committed to approved projects (e.g., if an
application identifies that a competitive cooperative agreement is the funding vehicle of choice, it is
most likely that FY05 funds would be used for that project).
29
-------
Other anticipated RFAs
In FY04, the STAR program will be issuing an RFA to fund one or more Centers for Bioinformatics that
will be targeted at providing collaborative efforts to EPA scientists working in the Computational
Toxicology Program. In addition, it is anticipated that an additional internal RFA will be issued by
CTISC later this year to support the proposed FY05 Computational Toxicology Initiative. The intent of
mat initiative is to develop a more efficient and effective process for assessing the risks of pesticidal
inerts and non-food use anti-microbials in support of needs of the Office of Pesticide Programs.
Contact Person:
' Robert Kavlock; Phone: 919-541-2771; email: kavlock.robert@epa. eov
CTISC Committee: Gary Ankley (NHEERL), Jack Fowle (NHEERL), Elaine Francis (NCER), Karen
Hammerstrom (NCEA), Robert Kavlock (NHEERL), hies Pagan (NCEA), Hugh Tilson (NHEERL),
Greg Toth (NERL), Jorge Santo Domingo (NRMRL), Eric Weber (NERL), Doug Wolf (NHEERL),
Doug Young (NRMRL); Executive Lead, Larry Reiter (NHEERL)
Format for Pre-proposals (5 Page limit USUIR 12 point font)
A. Project Title and Introduction
1. Statement of problem
2. Brief Synopsis of Research
B. Which objective (s) of the Computational Toxicology program is (are) addressed with this project and
what is the anticipated impact (be specific).
C. Research Plan
1. Specific Aims/Hypothesis
2. Background and Significance
3. Preliminary Studies (if applicable)
4. Proposed Research Design
5. If there is 'overlap' with research under another goal, provide a brief description of the 'cross-
walk' between the projects and a brief justification of why this work should be considered
for inclusion in Computational Toxicology.
D. Personnel/Research Situation (identify all lead investigators, including any external collaborations)
E. Management Plan (how integration of the research will be achieved across investigators and
organizations)
F. Budget Estimate (at this stage, the CTISC is just seeking budget estimates for planning purposes; a
more detailed budget will be required should the pre-proposal be selected for further development.
Intended vehicle(s) for any external funding actions should be identified.)
G. Projected Milestones (at least annual, tangible products)
30
-------
APPENDIX C. RFA for the STAR Center for Environmental Bioinformatics.
The Request for Applications for the STAT Center can be found at:
http://es.epa.gov/ncer/rf a/2004/2004_comp_tox.html
31
-------
------- |