Dr.
Casciano convened the meeting at 1:00 p.m. on June 11. He announced Dr. Daniel Acosta as the new
Chair of the National Center for Toxicological Research (NCTR) Science Advisory
Board (SAB). Dr. Acosta announced the
appointment of three new SAB members.
The new members are: Dr. Jerry Kaplan, University of Utah School of
Medicine; and Dr. Kenneth Tindall, North Carolina Biotechnology Center; the
third newly appointed member, Dr. Elizabeth Barbehenn, from Public Citizen, was
unable to attend today's meeting. He
also announced that Dr. Marcy Rosenkrantz agreed to serve for an additional two
years, and introduced Dr. Leonard Schechtman the new Executive Secretary to the
SAB, and Dr. James MacGregor, the new Deputy Director for Washington
Operations.
Board
members present were Drs. Daniel Acosta, Catherine Donnelly, Nancy Gillett,
Marcy Rosenkrantz, Jerry Kaplan, and Kenneth Tindall. SAB liaisons present
include: Drs. Marilyn Lightfoote (by phone), CDRH; Patricia Hansen, CFSAN;
Linda Youngman, CVM; and Richard Kennedy, UAMS. Other attendees included Dr. Bernard
Schwetz, Acting Principal Deputy Commissioner, and NCTR staff.
Dr.
Casciano, welcomed and introduced Dr. Schwetz who thanked the Board members,
the liaisons from the other centers, ORA, and UAMS. He commented that this SAB has had more of an impact on what
happens at the NCTR than any other Boards of Scientific Counselors or SABs in
other Centers. He said that through
these efforts, the SAB has helped to resolve problem areas and promote better
communication and collaboration. Dr.
Schwetz pointed out that peer review of Agency operating components was
becoming more formalized. He also said
that such peer reviews were conducted at NCTR when he was Director, and have
since been initiated at other FDA centers.
The Center for Devices and Radiological Health (CDRH) is undergoing
review at this time; the Center for Veterinary Medicine (CVM), the Center for
Drug Evaluation and Research (CDER), and the Office of Regulatory Affairs (ORA)
are preparing for reviews; and the Center for Food Safety and Applied Nutrition
(CFSAN) will undergo a review in the near future. He explained this was not a peer review of individuals, but of
the entire science program within a given center. Centers that do not have a formal review program in place, as do
NCTR and the Center for Biologics Evaluation and Research (CBER), will be
reviewed “from the top down to the protocol level” for the first time.
Next, Dr. Acosta requested approval of the June,
2000 minutes. Dr. Hansen (CFSAN) said
that she would like to expand the discussion on dietary supplement work and
priority setting. She agreed to provide
the Executive Secretary with written comments, which he will review, and insert
if appropriate. A motion was made and
seconded to accept the minutes as written, with this one possible modification.
Organization
and Background
Dr.
Casciano provided information on the organization of FDA and NCTR. He noted that FDA has five product centers
and one research center (NCTR). Each
center has a different mandate and mission and it is incumbent on NCTR staff to
understand what those are so they can interface effectively with the other
centers. He stated the mission of each
of the centers and ORA and provided examples of NCTR interaction. He also reviewed:
·
Resource
information over various fiscal years.
·
The full time
employee (FTE) allocation over the last eight years.
·
NCTR's
attempts to maintain a level of productivity through a postdoctoral program and
by using on-site contractors.
·
Additional
resources received from the National Institute of Environmental Health Sciences
(NIEHS). through an interagency agreement (IAG) with the National Toxicology
Program (NTP).
·
How these
additional dollars allow NCTR to maintain a critical mass of scientists.
Dr.
Casciano said FDA requested additional dollars in FY 2002 and that the
appropriated dollars included a percentage of what was asked for and the first
cost of living increase in ten years.
He added that an effort is being made by FDA to enhance its involvement
and visibility in the area of proteomics.
FDA has requested additional $2.5 M dollars in the proteomics/genomics
area and additional dollars for antimicrobial dietary supplements. Food safety has a high visibility in the new
administration, and there is a potential for obtaining additional funding. Areas that impact directly on NCTR’s mandate
to respond to FDA needs are providing highly credible scientific data to the
NTP Bioassay Program, thereby resulting in greater confidence in risk
assessments. NCTR is also a major
player in the food safety initiative. Dr.
Casciano indicated that the Division of Chemistry has a system that allows detection
of seafood decomposition, and it is attempting to adapt this system to detect
decomposition in poultry. In the
proteomics field, the analytical technology is transferable to bioterrorism as
well as the food safety initiative.
NCTR is also doing work in the area of DNA adducts. The Division of Microbiology is working with
an artificial GI tract that can be used to help evaluate the effects of dietary
supplements and perhaps genetically modified food. The Center has done work in
nutrition and on caloric restriction, efforts that have generated a number of
new hypotheses. The Center has also
developed and modified transgenic animals for use in detecting mutagenic and
carcinogenic agents.
Addressing
primarily the new SAB members, Dr. Casciano reviewed some of the Board’s
functions, which include:
·
Advise the
NCTR Director on science, budget and other issues.
·
Review and
approve site visit reports. Dr.
Casciano said that SAB members might be asked to chair or be a member on a site
visit team (SVT) if the program to be reviewed is in their specific area(s) of
expertise. Ad hoc experts are also
included as SVT members. Reviews are
done on a cyclical basis, which averages about once every three years.
·
Help in the
decision making process on various undertakings within the Center. For example, a discussion on the possibility
of moving a new subcommittee under the umbrella of the SAB was on the agenda,
and Dr. MacGregor would present the rationale for the move. (
TAB 1)
Dr.
Casciano summarized the various technical approaches being pursued to assess
toxicity and extrapolate results to the human.
These range from cellular and biochemical mechanistic systems, various
"-omics" technologies (e.g., genomics, proteomics), various other
biomarkers, and transgenic models. The
overriding interest is to develop better predictive models for the human. NCTR's efforts in these areas will result in
a decrease in the use of animals as the dependence on in vitro systems relevant
to the in vivo situation increases.
There will be a tremendous amount of data generated from these various
–omics technologies and computational science will need to be applied to aid in
the understanding of data being developed.
NCTR is building its infrastructure to accommodate development of
proteomic-applicable analytical equipment, microarray chip technology, and the
different toxicogenomic technologies.
Dr.
Casciano then addressed recruiting and retention issues at NCTR, acknowledging
that such efforts are difficult and that probably 20 to 25% of the staff will
turn over in the next five years. He added that NCTR’s location is not
ideal for recruiting the staff that we’re interested in, and we have to compete
in a tight job market. He said that the
individuals we’re interested in generally have a professional spouse who is
also seeking employment. We’re looking
for creative solutions and Dr. Schwetz mentioned some of them today. We’re utilizing creative recruitment,
recruiting individuals for a new group -- the cellular and molecular toxicology
group, and we are in the process of doing some heavy leveraging.
Dr.
Casciano then discussed NCTR's interactions with NIEHS, which has a
toxicogenomics group and with which we are actively communicating to convey our
interest in participating in this area.
It is necessary for FDA to be a player in this area, since industry is
already using these technologies, and will be developing safety data generated
from these technologies in the near future.
FDA needs to be ready to respond and if we’re not, we will take the most
conservative approach and become bottlenecks to the
process and not be catalysts. He said
NCTR needed to develop highly creative interactions with our colleagues in
academia and industry. NCTR has the
opportunity of purchasing academicians through the Intergovernmental Personnel
Act, which allows us to hire academicians for a year or two. Under this program, they can spend their
time here or work at their home institutions on problems that are of interest
to the NCTR and/or the FDA. Any advice that the Board can provide regarding fostering
positive relationships in these areas would be very appreciated.
The
questions from the Board members focused on NCTR financial aspects. Dr. Casciano responded that FDA’s ownership of the NCTR facility allows NCTR
to better manage its funds. He said
leveraging dollars from NIEHS and other FDA centers with which NCTR maintains strong
ties (e.g., CFSAN, CVM), is another funding approach that will be pursued. Projected 2003 funding is expected to be
adequate to sustain current activities and, in addition, a 4% increase has been
requested.
Nonclinical
Studies Subcommittee
Dr.
MacGregor was next on the agenda; he proposed that the Nonclinical Study
Subcommittee (NCSS) of the Advisory Committee for Pharmaceutical Science (ACPS)
be moved under the SAB. He provided the
Board with an overview on the NCSS and addressed the concept and related
activities of this Subcommittee.
Members of the Board asked for additional information and for time to
study the concept. Dr. Tindall agreed
to research the proposal and report back to the Board at it’s next
meeting. (TAB
2)
Endocrine
Disrupter and Knowledge Base Program
The
assembled group then reviewed NCTR's response to the SAB Site Visit Report on
the Endocrine Disrupter and Knowledge Base Program’s (EDKB). Dr. Sheehan provided a handout that
included: (a) the program's fiscal year 2000 research accomplishments (for
comparison with the 2001 plan), (b) a list of 19 publications, and (c) the
response to the SAB report. Dr. Sheehan
then provided an update on the EDKB program. (TAB
3)
Dr. Rosenkrantz noted the absence of a response to
the site visit team's (SVT) suggestion on mainstreaming the knowledge-based
work into the rest of the activities of the Center. Dr. Sheehan said he thought that the Board's visit to ROW should
have given her an idea of its work. Dr.
Rosenkrantz said there was concern about how further studies would be funded
through the Center and competed against other projects. She asked how that had gone and suggested
that Dr. Casciano or someone else could answer, but she wanted to have this
issue discussed. Dr. Sheehan said, he’d
never seen the total expenditures from NCTR, but the $3,300,000 plus the
$850,000 for the external validation had defrayed a substantial portion of the
expenses associated with developing the models. He indicated that outside funding sources were a primary means of
program support. Dr. Casciano said the
androgen receptor studies and the EPA work were funded by EPA and NCTR’s
budgetary input is relatively small.
Also, they are providing input into the genomics and proteomics
activities in combination with investigators from chemistry, biology and
biometry, and are directing the NCTR knowledge database into development for
the DNA microarray technology. He said
the SVT critique was taken very positively and the Center had responded to it.
Dr. Rosenkrantz asked for comments and there were
none. She did not ask for a vote
because people have not had a chance to read the Center response, and suggested
that the vote be delayed to allow for the possibility of additional questions.
Division of Microbiology
Dr. Khan responded to the SVT Report in Dr.
Cerniglia’s absence and provided an update on the Division of
Microbiology. The report provided
general comments and recommendations:
·
Strategic
planning -
The SVT asked the Division to develop more collaborative research projects with
other FDA centers and ORA. Dr. Khan
said the Division of Microbiology has about 20 different research projects
(CVM-10, CFSAN-3, ORA-6 and CDRH-1).
·
Food
microbiologist
- The SVT recommended that the Division hire a food microbiologist. Dr. Khan said that the position was
advertised in February and an offer was made, but the applicant declined.
·
Philosophical
paradigm –
The SVT recommended they carry out most of the studies based on the mechanistic
approach. Dr. Khan reported that the
Division followed the recommendation to establish the necessary collaborations.
·
Work
related to antibody resistance -- Dr. Khan explained that, based upon mechanistic
studies, they can determine whether or not the risk of transferring antibody
resistance is justified. This approach cannot be used for regulatory work, but
can determine if antibody resistance can be transferred to other
organisms. Division researchers have
shown that poultry isolates can transfer resistance to human isolates and they
now understand the role that CVM can play in controlling the use of antibiotics
in poultry.
·
Leadership
role – The
SVT asked what would happen if Dr. Cerniglia left and whether there is anyone
being groomed for a leadership role?
Dr. Khan said senior staff are given the opportunity to attend training
and to build leadership skills. Also,
the Division directorship was rotated among the senior staff whenever Dr.
Cerniglia was on travel.
·
Building – Dr. Khan added that the
Division appreciates the recommendation that it needed space and reported the
Division will probably move into the chemistry area after modifications are
completed.
·
Public
relations and communications -- The SVT recommended that the Division develop a
website. Dr. Khan reported that the
site has been completed and that they have posted the skills, research
interests, goals and accomplishments of the Division's principal investigators
(PIs).
Next Dr. Khan addressed comments on the following
research focus areas:
·
Food
borne pathogens, food safety, and methods development – The Centers focused in
this research area are CVM and CFSAN.
NCTR has ten projects with CVM, of which, seven or eight are on food
safety. The SVT recommended that the
Division try to quickly publish this research. Dr. Khan said that they want to
publish quickly, but they want to avoid compromising the quality of the
research. Before publishing they send
the data to everyone involved in the collaborative process for review and
comment.
·
Standards
and controls
- Dr. Khan reported that whenever and wherever necessary the Division always
tries to include standardized methods before publication.
·
Determination
of the role of intestinal microflora in activation or detoxification of
xenobiotics
– The SVT recommended that the Division strengthen this area. Dr. Khan responded that they have developed
chemostat models that mimic the human large intestine to study the effect of
low-level antibiotics used in veterinary medicine (e.g. in food-producing
animals) on human microflora.
·
Environmental
biotechnology -- The Division is continuing its work in
this area. Several researchers are
studying the role of fungi and bacteria and how these microorganisms biodegrade
pollutants in the environment.
·
Use
of microorganisms as models to predict how drugs are metabolized - The Division is using a
number of microorganisms to study the metabolic pathways. It has also begun collaborating with USDA
and is trying to isolate bacteria from fish ponds. Division researchers are studying how antibiotics used in the
fish industry are degraded and whether or not antibiotic resistant bacteria
accumulate in the environment.
·
Microbiology
surveillance and diagnostic support of research - The Division continues
to offer surveillance and diagnostic services to NCTR and the other FDA
centers.
Dr. Acosta asked Dr. Donnelly (SVT Chair) and Dr.
Kennedy (SVT member) if they had any additional comments. Neither wished to comment at this
time. Dr. Casciano said NCTR seriously
considered the SVT's comments about public relations and communication. He reported that NCTR completed development
of an “e-journal” that is being made available on its public website to enhance
communications with other FDA centers, other regulatory bodies, and the international
community. Staffers from FDA and other regulatory bodies will serve as its
editorial board and can contribute to the journal as well. He also said that this journal is a
regulatory research interest journal and could become a vehicle for the regulatory
scientist to disseminate thoughts or
ideas that they would like to develop and have others understand. The Division of Microbiology has contributed
the first paper to the journal’s inaugural issue. Dr. Donnelly asked the status of the food microbiologist
position, because of all the recommendations in the report, she said this was
the most critical and one the SVT strongly recommended. She added that this entailed not just hiring
a food microbiologist, but a senior researcher who could direct research with
applied outcomes. The incumbent in the
position should be able to take advantage of all of the resources that appear
to be coming to the Agency to deal with food safety. Dr. Casciano responded that the Division conducted a search and
identified someone, but this candidate did not accept the position. He added that the Division will renew its
search for a strong candidate to fill the position. In response to the SVT’s comment on leadership, he said Dr.
Cerniglia was making an effort to expose his senior scientists to administrative
roles, as well as to representing him in critical meetings. Dr. Acosta asked Dr. Donnelly if she wanted
to make a recommendation on acceptance.
She moved to accept the report as submitted, the motion was seconded,
and the report was accepted.
Division of Microbiology
Dr. Khan provided the following update:
·
The
Division of Microbiology continues to serve a multipurpose function with
specialized expertise to perform fundamental and applied research.
·
The
Division responds to microbial surveillance and diagnostic needs for research
projects within NCTR and FDA.
·
The
surveillance and diagnostic program has three groups that offer services in the
area of bacteriology, parasitology, mycology, virology, serology, and media
preparation.
·
The
virology and serology group offers its services for surveillance and support
for research scientists. The
surveillance branch maintains a breeding colony, quarantine facility, primate
colony, NTP animal studies, non-NTP animal studies, environmental health,
program assurance, and it oversees the animal husbandry and diet preparation
contractors.
·
The
Division provides research support to the other NCTR divisions such as
Biochemical Toxicology, Chemistry, Genetic & Reproductive Toxicology,
Molecular Epidemiology, and Neurotoxicology.
·
Division
staff performs research in the areas of environmental biotechnology, food
safety, microbial models in toxicology, and intestinal microflora.
There are many technical abilities and skills
available from the Division. Its
current work includes:
·
Collaborative
projects with CVM, ORA, CFSAN, and CDRH. Included among the collaborative
research efforts in the food safety area are the in vitro model and molecular analysis of competitive exclusion
products, applicable to pathogens in the poultry industry.
·
Development
of molecular screening methods for the determination of vancomycin resistance
in selective competitive exclusion products, and characterizing gene markers in
competitive exclusion products. Whether
those markers are transferable and whether competitive exclusion is safe for
use in the poultry industry. NCTR has
ongoing studies on fluoroquinolone resistance in camphylobacer sp. and Salmonella
spp. isolated from poultry.
·
The
Division is using molecular techniques to screen animal feeds for proteins
derived from mammalian tissue to detect possible bovine spongiform
encephalopathy (BSE).
·
Developed
and validated an assay for detecting BSE, which won the 2000 FDA Collaborative Science Award.
·
Developed
molecular methods to identify and quantitate human food borne pathogens in the
animal production environment and developed methods to detect the presence of
these pathogens in a variety of food sources such as vegetables, poultry, fish,
and salads.
·
Initiated,
in collaboration with USDA, a project on biodegradation of veterinary drug
residues. This project was undertaken
in response to a suggestion from the SVT.
·
Developed
a guidance document entitled "Assessing the Effects of Antimicrobial
Residues in Food on the Human Intestinal Microflora."
·
Developed
alternative microbial methods for studying the metabolism of drugs.
·
Developed
a bioluminescent assay to determine the tuberculocidal activity of
disinfectants.
·
Developed
multiplex PCR methods for the determination of vancoymycin resistant genes.
·
Characterized
the microbial cytochrome P-450s and glutathione transferase, the aerolysin
toxins genes, and erythromycin resistance genes (erm) in Staphylococcus sp. isolated from chickens; azoreductase and
nitroreductase enzymes in bacteria isolated from human intestinal tract; and
fluoroquinolone resistant Campylobacter
spp. from poultry samples.
·
Developed
a method to isolate and characterize bacteria that are cutting undesirable
antibiotic resistant markers.
·
Completed
and validated a trial method for the detection of BSE.
·
Discovered
an agent in oyster homogenates that is lethal to V. cholerae and V.
Vulnificus.
·
Discovered
new strains of bacteria and fungi that degrade environmental pollutants and
explained novel biodegradation pathways for xenobiotics.
Plans for fiscal year 2003 include:
·
Initiation
of a new chemostat experiment for testing veterinary fluoroquinolones.
·
Isolation
and molecular characterization of fluoroquinolone resistant Salmonella spp. and E.. coli from chicken and turkey litter.
·
A
study for the plasmid profile, genetic fingerprinting, and strain typing of
vancomycin resistant organisms isolated from competitive exclusion
products.
·
Characterize
fluoroquinolone resistant camphylobacters at the molecular level using PCR-RFLP
and pulse field gel electrophoresis and correlate the data that have been
obtained from poultry litter with the environmental data available for human
subjects.
·
Collaborate
with USDA on biodegradation rates and metabolic fate of antibiotics used in
aquaculture.
·
Working
on diadzein and genistein to try to detect the specific bacteria (from the
human intestinal tract) that convert these phytoestrogens to estrogenic and
non-estrogenic end products.
·
Evaluate
the effect of fluoroquinolones on anaerobic bacteria from the human intestinal
tract. Including development of
resistance, mechanism of resistance development, impact on metabolic
activities, and the dissemination of resistance to bacterial pathogens.
·
Identify
the metabolites produced from norloxacin and sarafloxacin by fungi grown on
poultry litter.
·
Isolation
of new strains of fungi from litter in poultry houses and screen for
biotransformation of fluoroquinolones.
·
Complete
in vitro assay of competitive
exclusion products.
·
Maintain
a world-class research program to solve current issues facing FDA, so the
Agency can make sound science-based regulatory decisions on microbiology. (TAB
4)
Dr. Khan opened the floor for questions. Dr. Tindall asked if any of the researchers
were thinking of pursuing patents. Dr.
Khan said there were none, but that when they felt they had something they
would definitely go in that direction.
Dr. Tindall also asked about the practice for technology transfer. Dr. Casciano said the Center had a
Technology Transfer Office that integrates with a similar group at the Agency
level, and investigators are encouraged to use that office when they felt they had
a useful discovery.
Dr. Donnelly observed that it would seem logical to
extend the current work on Salmonella
DT 104 to Salmonella newport, because
S. newport has now acquired that same
gene cassette. Dr. Khan said that they
isolated several strains of Salmonella
from poultry litter and that he wasn’t sure, but thought S. newport was being studied.
Dr. Acosta asked about external grants or other
resources to do all of these projects.
Dr. Khan said that most of this was money from FDA funds. Dr. Casciano added that there were directed
funds from Congress and the Food Safety Initiative, which comes through CFSAN
and CVM. He also said that when the
dollars collide, we collaborate and leverage within the Agency. Dr. Acosta asked the percentage; Dr. Casciano
said probably ten percent for this Division.
Dr. Kaplan asked if NCTR received grants. Dr. Casciano said that because we are part of the Public Health
Service (PHS) and that due to NIH policy, we cannot directly apply for grants,
but we can serve as co-investigators and co-PIs. In addition, we can get some funds through either equipment or
post docs. He also said that if we
have an expertise that is unavailable in academia, or if there is a need for a
fast track, there are mechanisms for obtaining dollars from other PHS agencies
through an IAG.
Division of Neurotoxicology
Dr. Slikker, provided an update on the Division of
Neurotoxicology. He explained that the
focus of this Division is on the development and validation of quantitative
biomarkers and to use these biomarkers to explain toxic mechanisms and increase
the certainty of underlying assumptions used in risk assessments for
neurotoxicants. Neurotoxicity can be
defined as any adverse effect on the structure or function of the central and/or
peripheral nervous systems. Within FDA,
this includes biological, chemical, or physical agents. Adverse effects include any unwanted effects
that diminish the ability of an organism to survive, reproduce or adapt to its
environment. These effects can be
permanent or reversible. He also
provided the Board with copies of his slide presentation. (TAB
5)
Dr. Slikker identified various approaches used by
the Division, which include:
·
Multidisciplinary
approach, because many of the relevant effects can be measured by
neurochemical, neurophysiological, neuropathological, or behavioral techniques.
·
Discipline-continuum
approach, trying to link together the various kinds of approaches. Everything from psychological testing that
can be done in human and animal models, through behavioral assessments,
physiological assessments, and various kinds of motor function tests. This kind of linkage can be done using
genomics, proteomics, also computational modeling. Neurobiological approaches
including microdialysis allows the collection of information from the nervous
system in a waking animal. Protein
biochemistry is very critical with proteomics and cell culture approaches.
·
Dr.
Slikker discussed the disciplines represented by researchers within the
division. These include a neurochemical
specialist, a neurophysiologist, a developmental neurobehavioral toxicologist,
a pharmacokinetic specialist, and an experimental neurohistologist. Several Division members have been trained
and have experience in the areas of genomics and proteomics.
Dr. Slikker said the Division could provide the
expertise needed to solve integrated problems within the area of neurotoxicology.
Next, Dr. Slikker provided specific examples for
behavioral assessment tools. He said
they had testing laboratories at the Arkansas Children’s Hospital satellite lab
and at the University of Arkansas at Little Rock. The tests are quantitative in nature. Since it is impossible to test every chemical in children, they
have a primate center at NCTR that is capable of providing animals for general
toxicology and pharmacokinetics studies.
They also have a breeding colony, which can be used to study different
stages of development during pregnancy.
Dr. Slikker also said that they have the ability to behaviorally
evaluate up to 90 primates a day. He
said extramural funding had been good, consisting of over 0.5 million dollars
per year over the last 13 years, and productivity has been excellent.
Dr. Slikker discussed ongoing studies within the
Division, and provided examples of study drugs, which range from THC to
chlorpromazine (major tranquilizer), Diazepam (minor tranquilizer), morphine
and atropine, and marijuana. The
Division is also collaborating with CFSAN and CDER. One protocol focuses on the use of clontech arrays to examine for
gene expression alterations. The
hypothesis is that substituted amphetamines may produce long-term
ultrastructural and neurochemical changes and that this could be monitored by
looking at specific DNA expression.
Dr. Slikker said that Division staff publishes in
prominent peer-reviewed scientific journals and has published about 40
articles, reviews and book chapters per year, over the last six years. They also provide leadership on various
kinds of committees, such as those that oversee CFSAN's Redbook II, the
Reproductive and Developmental Toxicity group, EPA, and the WHO working group
on pharmaceutical agents. In the last
year, they have been on four committees dealing with risk assessment in
children. The Division also serves on a
CDER committee that evaluates various pharmaceuticals including anticonvulsant
agents.
Dr. Slikker added that the Division believes in
maintaining a diverse portfolio of leveraging opportunities, which include the
following CRADAs:
·
A
primate study conducted at the NCTR, which is funded by NIDA to the University
of Arkansas at Little Rock. One of our previous postdoctoral fellows is PI on
this award and through a CRADA mechanism, reimburses NCTR at $250,000 a year,
for five years.
·
The
NTP/IAG on Endocrine Disrupters, undertaken with EPA, that is valued at
approximately $200,000 a year. This collaboration addresses quantitative
risk assessment procedures and investigates some of the quantitative methods of
comparing structure and activity.
·
A
CRADA with AstraZeneca, where the Division interacts with industry on broad
questions of how anticonvulsants affect learning and memory. These CRADAs help supplement current
NCTR/FDA funding.
Next, Dr. Slikker discussed projects and protocols
that had either been approved or just begun in the past year.
·
Use
of multiple cDNA arrays to look at temporary changes after exposure to amphetamines. Collaborators include NIOSH, other divisions
within NCTR, and Wake Forrest University.
·
A
long-term look at anticonvulsant treatment with AstraZeneca.
·
Another
protocol under review is on ephedrine-type compounds. This protocol is currently at the second stage of review. Collaborators include Syed Ali and Bill
Girley from UAMS, who is an expert on dietary supplements.
·
Another,
in conjunction with Wright Patterson Air Force Base, looks at
ephedrine-containing dietary supplements.
These concept papers have been approved.
·
Another,
in collaboration with colleagues from Duke University, considers the
developmental effects of nicotine in a rodent model.
·
Another
approved concept paper is on mitochondria energy metabolism to assess the claim
that dietary supplements modulate metabolism
Future directions were also addressed.
·
Thimerosal
is an issue within CBER and CDER. CBER
is pushing this through as an NTP compound.
This is important because it is used as a preservative in many vaccines
that children receive on a routine basis.
NCTR and CDER think that it is very important to be able to evaluate
thimerosal exposure in developing monkeys.
·
Infant
formula is being discussed with CFSAN.
·
There
is ongoing dialogue with CDER to look at the pediatric use of the dissociate
anesthetic, Ketamine, and how to evaluate its safety.
·
Imaging
development with the other centers.
·
Transgenics
- the Division has shown the importance of using primates in certain
studies.
·
Genomics
and proteomics are important areas to amplify for the future.
In closing, Dr. Slikker said that FDA working
groups enhance communication and provide an opportunity to keep up with FDA
needs, and help to anticipate future needs.
One such group is the FDA Intercenter Neurotoxicity Working Group. The working group assists FDA in addressing
neurotoxicity regulatory concerns. He
also said there was an FDA intranet site that allows FDA researchers to communicate
and schedule meetings. This approach of
interacting with other agency researchers interested in safety assessment,
especially neurotoxicity safety assessment, lets all the centers communicate
about issues and try to come up with solutions.
Dr. Acosta asked for questions/comments. Dr. Rosenkrantz asked about the work of the
computational science group. Dr.
Slikker said that NCTR has been able to obtain funding from EPA, Research
Triangle Park, to collaborate with them to develop a biologically based model. They hired a post doc and bought equipment
to do the modeling. Dr. Slikker said
that a manuscript was submitted and they are hoping for a good return from this
effort. Dr. Rosenkrantz asked if ROW was involved and Dr. Slikker responded it
was. Dr. Rosenkrantz asked if that included
imaging as well. Dr. Slikker said imaging
was more complicated and is being done in conjunction with JIFSAN (a consortium
of CFSAN and the University of Maryland) and CDER (who has relationships at
Duke University and various other universities), so the imaging is not at NCTR. Dr. Slikker added that NCTR does have
imaging capability at UAMS and that they are trying to build on those
relationships so that NCTR can have access to equipment. However, right now they are going through
JIFSAN and the CDER relationship with Duke and other locations. Dr. Casciano said the proteomic and
genomic efforts are interfacing with the bioinformatics group and that they are
developing software to image and identify intensity for protein spots, plus
lend creditability to the development of data sets. Dr. Casciano also said that this is a team effort and these
developmental projects are applicable across various platforms.
National Toxicology Program (NTP)
Dr. Allaben provided an update on the NTP. He provided background information on the
FDA/NIEHS Inter- Agency Agreement (IAG).
He explained that originally this was a five-year concept, but it has
been so effective that it’s now an open-ended agreement.
In 1996, the NCTR entered into an agreement with
NTP to do endocrine disruptor studies.
We looked at five putative endocrine disrupters. These are complex, time
consuming, multigenerational studies, and NCTR was the only facility that could
perform these types of studies. In 1998
resources for developing a phototoxicity research and testing laboratory were
added. This is a state-of-the-art
facility that does not exist anywhere else in government. We are hoping to get appropriate resources
from NIEHS for renovation and expansion of the phototoxicology research
laboratory and for additional animal rooms to support these studies. Negotiations are ongoing.
Dr. Allaben continued by adding that in 2000,
additional high priority FDA compounds, including some of the dietary
supplements, were brought in under the IAG, and most recently we are entering
in to a complex series of studies, the AIDS therapeutic mixtures study. Some of the compounds tested under the IAG
are: chloral hydrate, fumonisin B1, malachite green, urethane/ethanol,
riddelline, glycolic and salicylic acids.
Dr. Allaben added that the endocrine disruptor chemicals, the
phototoxicology nominations, dietary supplements, AIDS therapeutic studies,
thimerosal and other important chemicals are coming in under the umbrella of
the IAG. With regard to endocrine disrupters, we are looking at methoxychlor,
genistein, nonylphenol, vinclozolin, and ethinyl estradiol. Aloe vera is the first dietary supplement
under study. Dr. Allaben also explained
that some of the nominations not included in the IAG include radio frequency,
DNA based safety assessment of selected vaccines, therapeutics, and two
antibiotics that the Agency needed genotoxic information on. He added that the NTP provided an
opportunity to obtain information on P53 studies with Senna, a replacement for
Phenylthalene and TG-AC studies with pilocarpine. These are all nominations that will be coming to NCTR for testing
under the IAG umbrella.
Dr. Allaben said that the science we look at comes
through our Toxicology Study Selection and Review Committee (TSSRC), a
scientific oversight body that comments on the utility and the quality of the
science not only from a pure scientific perspective but also from an Agency
perspective. He added that scientists
from all of the FDA product centers are at the table during the design
phase. We go through a series of
protocol reviews involving NCTR, the product center, and NIEHS. Dr. Allaben explained that the
responsibility of the oversight group is to review research concepts and plans
and the protocols are discussed and commented on. The committee recommends any protocol modifications with regard
to dose level selection, design changes, and it meets twice a year to review
new science projects and to monitor the ongoing. Dr. Allaben described the benefits of the TSSRC, which include
enhancement of the regulatory decision process, support for quantitative risk
assessments, encouragement for new/innovative research approaches, and speeding
the research and testing process. He
said that the TSSRC also provides data to the regulators who are at the table
during the design, and during the monitoring of those studies, and they are the
first to see the results of these efforts.
He added that the process also
utilizes NCTR scientific and contract staff, provides FTEs and post doc
support, facilities renovation, equipment purchase, and provides resources for
travel to scientific meetings. Dr.
Allaben summarized his presentation by saying that this IAG provides a benefit
package that is good for NTP and NCTR and is an example of leveraging to the
best possible extent. (TAB
6)
Phototoxicity Research and Testing Laboratory
Dr. Paul Howard provided an update on the
Phototoxicity Research and Testing Laboratory.
He said there were four centers for research excellence within the NTP,
which are:
·
The
NTP Center for Evaluation of Risk to Human Reproduction, which is composed of a
panel of experts to develop expert opinions on the risk of certain chemicals to
human reproduction.
·
The
Interagency Center for the Evaluation of Alternative Toxicological Methods,
which works in collaboration with ICCVAM (the Interagency Coordinating
Committee on the Validation of Alternative Methods) to evaluate the validation
status of alternative methodologies.
·
The
National Center for Toxicogenomics (this is really not under the NTP; it is
under the NIH).
·
The
Center for Phototoxicology, which conducts toxicological studies that expose
animals to light emitting sources.
Examples of studies:
·
Palmitate O, a sunscreen agent, is the
dimethyl amino ester of PABA, and has been under the NTP study for about 2 1/2
years.
·
Aloe
vera, which was nominated for an NTP study by the National Cancer
Institute. Aloe vera is absolutely everywhere. The question is what are the
phototoxicological properties of this compound.
·
Retinyl
Palmitate - What is the effect on the skin of very high concentrations of
retinol? Photocarcinogenic studies have
not been conducted with retinol or retinyl palmitate and it needs to be needs
to be looked at.
Dr. Howard added that the Aloe vera and retinyl
palmitate studies would start this fiscal year.
Dr. Howard also provided an update on current
studies, which include:
·
Looking
at a transgenic mouse with TP-rats, in collaboration with Lynda Chin at Harvard
University.
·
A
CRADA with Argus Research Laboratories to understand dose response and to
develop a historical database on photocarcinogenesis.
·
Palmitate
O, which is one of the most widely used sunscreen agents.
·
Compounds
related structurally to methoxy-psoralen.
What phototoxicological properties do they have?
·
FD&C
dyes 27 and 28, which are in lipstick and red based facial applications. The NTP asked if these compounds get out of
red-based lipstick and red-based facial applications and into the skin? If they do, we will be studying these
compounds. But only if
pharmacologically valid.
Dr. Howard also reported that the group is working
with NIEHS to look at potential biomarkers of PUVA-induced skin cancer. Very few things we know induce skin cancer,
one is psoralen plus UVA, which induces basal squamous cell carcinomas and
melanoma. He added that we are looking
at developing biomarkers for skin changes.
We are trying to meet the basic science research and testing needs of
the NTP/NIEHS, and FDA. (TAB
7)
Dr.
Kaplan, asked if they ever see cancer in the absence of inflammation from
sunlight, and did they identify any intermediate steps on the way to the cancer
outcome? Dr. Howard responded “yes and
no”. The TSSRC takes an extremely
thorough look at these efforts, and one of the questions that came out of the
review was where was this facility going.
The TSSRC observed that this is satisfactory applied toxicology but
questioned what other things are being done?
One issue that came up was the need to understand the role of
inflammation of the immune system in skin cancer development. Dr. Howard explained that immunosuppression
leads to enhanced skin cancer incidence and that we need to address what is
happening in our model and how we can adjust our model to best mimic the
human. He posed the question of what
would happen if we “immuno-enhanced” an animal with the same light
regimen? Will or will not the skin
cancers, etc. be induced? Dr. Kaplan
said another question is, whether mouse models like the Tg AC mouse, which is
expected to exhibit accelerated reactions to phototoxic carcinogenesis, could
be employed. Dr. Casciano said NCTR was just starting the phototoxicity
facility and such efforts were helping to justify its existence. He also said that the phototoxicity group is
focusing on applied technology using systems that were acceptable for 20 or 30
years. Dr. Casciano explained that as
we learn more about these systems, we have more questions. He added that Dr. Howard is expanding the
areas of study by looking at other possible sources to answer the questions
being asked.
The
meeting adjourned for the day.
The
meeting reconvened on June 12, 2001.
The
first order of business was to revisit the EDKB discussion presented by Dr.
Sheehan on the previous day. Dr.
Rosenkrantz, in response to Dr. Sheehan's statement that the reason this information
was not on the Internet was due to firewall issues, recommended that the SAB go
on record saying this problem needs to be fixed. Dr. Rosenkrantz said that scientific work has to be communicated
and the only way to do that on a broad scale is through the Internet. However, she said that she understood this
was a result of FDA regulations. Dr.
Sheehan agreed, saying that he is frustrated with the firewall issue. Dr. Casciano said recent leadership meetings
have discussed transparency and that use of the Internet was one more way to
enhance that transparency.
Commenting on the Microarray data analysis
discussion, Dr. Tindall said that through NCTR’s strong relationship with
NIEHS, the Center should explore interactions with the new National Center for
Toxicogenomics. Dr. Kaplan said he
didn’t understand why this group had to develop their own data analysis tool
when there were so many available and recommended that they evaluate the
available database software. Dr.
Rosenkrantz said this would be an opportunity for the computational science
group. Dr. Tindall said all of the
programs were likely to become more and more involved in various aspects of
genomics and proteomics and should evaluate the available software, servers,
and storage. He also said that no
software solution will be perfect, but it is important to have the
infrastructure in place. Dr.
Rosenkrantz’s motion to accept the response to the EDKB Site Visit report was
approved.
Division
of Biochemical Toxicology
Dr.
Beland provided an update on the Division of Biochemical Toxicology. He explained that this Division is
interested in carcinogenicity and focuses on assessing carcinogenic risk and on
introducing new techniques to assess carcinogenic risk. The Division has approximately 50 staffers,
which include permanent researchers and investigators, several visiting
scientists, and a varying number of postdoctoral students. The distribution of funds drives much of the
Division's scientific research.
Approximately 30% of the division’s discretionary budget comes from FDA
and is used for supplies, travel, and equipment. The other 60-70% comes primarily from NIEHS through NTP. The majority of the research conducted by
the Division is funded externally. Dr.
Beland added that the Division is meeting the Agency's needs, but obtains its
funding from outside sources.
The Biochemical Toxicology Division focuses on four
research areas: efforts in support of NTP nominated chemical compounds, the
neonatal mouse bioassay, dietary folate/methyl deficiency, and analytical
methods development. Within the
Division, funding provided by the NTP IAG supports research efforts for
fumonisin B1, chloral hydrate, urethane and ethanol, malachite green, endocrine
disruptors, phototoxicity studies, dietary supplements, and anti-retroviral
agents. Dr. Beland discussed the
following compounds and focus areas:
·
Fumonisin
B1, which was the first compound investigated under the IAG, and was nominated
by CFSAN.
·
Chloral
hydrate, which is used as a pediatric sedative and was nominated by CDER.
·
Urethane,
which is in a number of food products.
CFSAN needs better dose response data to set regulatory limits. The study is complete and the final report
is being written.
·
Malachite
green is used without approval in the catfish industry as an antifungal
agent. CVM nominated the compound to
determine if enforcement action is necessary.
The protocol information will be available for CVM within the next two
years.
·
Five compounds
were selected for endocrine disrupter studies, these include genistein,
methoxychlor, nonylphenol, ethanol estradiol, and the androgenic
vinclozolin. Mechanistic studies are
underway looking at alteration of hormone levels and the induction of
cytochrome P450.
·
Studies are being done on phototoxicology with alpha and
beta hydroxy acids, aloe vera, and retinyl palmate.
·
Dietary
supplements under study include genistein, riddelliine, and aloe vera.
·
Riddelline is
a compound that is found in some herbal teas.
The NTP asked if we could gather information to determine its genotoxic
potential. It was found that ten DNA
adducts were formed and two have been identified.
·
Aloe vera is
used in skin preparations and is also taken internally. The plant product includes several
fractions, which include aloe vera gel, latex, and a whole leaf
preparation. We know the fractions
contain anthroquinones, which could be genotoxic.
·
In
collaboration with NCI, we developed an immunoassay for AZT and demonstrated
the incorporation of AZT into DNA. The
concern is that these agents are given to pregnant women to prevent development
of HIV in the unborn child. Studies
will be done to determine carcinogenicity, genotoxicity, and metabolism.
·
The neonatal
mouse bioassay responds to very genotoxic compounds, but the assay is also
sensitive to other compounds. The
following chemicals are undergoing tests, benzodiazepines, antihistamines,
lipid peroxidation products, estrogens/antiestrogens, proton pump inhibitors,
mycotoxins, known human carcinogens, and antiretroviral agents.
·
Dietary folate
and methyl deficiency studies to look at nucleotide pool imbalance and
methylation deregulation during hepatocarcinogenesis, folate-dependent
homocysteine metabolism, and methylene tetrahydrofolate reductase polymorphisms
and Down syndrome. These studies have
allowed for additional funding through the CDC and the Arkansas Children’s
Hospital.
·
Two major
laboratories working on analytical methods development, including
immunochemical methods. The development
of antibodies against fumonisin has allowed us to develop a purification system
of ceramide synthase and to develop antibodies against specific DNA
adducts. More recently the Division
began studying catechol estrogens, with funding from the Office of Women’s
Health.
·
The mass
spectrometry group has done work with genistein and daidzein as part of the
endocrine disruptors. We have developed
mass spectrometry methods for oxidative DNA damage from DNA adducts from
tamoxifen and we are expanding these studies to include DNA adducts that come
from components found in hormone replacement therapy. (TAB
8)
Dr.
Acosta asked if there were a decrease in external funding, how would the Division
be effected? Dr. Beland replied he
would go out and solicit funds from other sources. Dr. Acosta asked if the investigators were looking for sources of
external research funds? Dr. Beland
said this has always been encouraged.
Approximately seven years ago a SVT suggested that the Division
concentrate more on FDA funding, but a year or two later the focus shifted once
again to external funding. Dr. Casciano
said with the NIH budget on its way to doubling and the majority of that going
towards extramural funding, NCTR was in a fairly stable situation. He explained that there are very few places
in the world that can do what this group is capable of doing and our uniqueness
allows us to be industrious participants.
Dr. Casciano also said that the excellent mechanistic work that is being
done is applicable to future FDA questions.
He added that good science would continue to be supported. Dr. Beland said that the money they received
from the Office of Women’s Health served as seed money for research that has,
in-turn, brought in additional funding from CDC.
Division
of Molecular Epidemiology
Next,
Dr. Poirier provided an update on the Division of Molecular Epidemiology. The major research areas of the Division
include the identification of genetic polymorphisms that influence carcinogen
metabolism, DNA repair, chemoprevention, and individual cancer
susceptibility. The Division consists
of approximately 25 people at NCTR with a number of collaborators at the
University of Arkansas for Medical Sciences.
Studies
and interests include:
·
Genetic
polymorphisms, DNA adduct detection in humans and molecular epidemiology
studies.
·
Developing a
DNA microchip for large-scale population based studies.
·
Correlate
cytochrome P450s with the onset of puberty in young girls.
·
A case-control
study on colorectal cancer with respect to the H-glutathione transferase A-1
polymorphism.
·
A
retrospective case study on survival of breast cancer after chemotherapy with
respect to GS phenotype, and the recurrence of colorectal polyps with GST
genetic polymorphism, MTHFR polymorphism, and glutathione peroxidase
polymorphism.
·
A study
examining the amount of GSTA-1 protein in the livers of humans compared to the
amount of GSTA-2 protein in the same livers.
·
A study
comparing the overall survival of women with each of the previous phenotypes
with phenotypes of GSTA1.
·
Examination of
hepatic DNA methyl transferase activity in smokers.
·
Determination
of individual methylation profiles, gene expression, and enzyme activity of
CYP1A2 in the human liver.
·
Elevation of
DNA methyl transferase in the livers of smokers compared to nonsmokers.
·
Hypermethylation
in the promoter region of the CYP1A2 gene and that it is associated with
decreased expression.
·
Studies on in vitro toxicity of pancreatic cells in
culture.
·
Research on
biomarkers of pancreatic cancer.
·
Establishing
biomarkers of cancer in high-risk groups such as smokers versus nonsmokers.
·
Develop in vitro predictive bioassays for
chemopreventive agents.
·
Look at the
toxicity of agents such as nicotine, soy, and tea components on pancreatic
cells in culture.
·
Determine the
mechanistic actions of such agents. A
recent result from the study of genistein on the expression of K-ras on
pancreatic cells in culture.
Two
studies are being done in conjunction with an IAG with the National Cancer
Institute:
·
Examining DNA
methylation and cancer risk in humans and experimental animals as well as
abnormal methyl metabolism in non-neoplastic diseases.
·
Examine the
effects of dietary homocysteine on disease.
Future
directions:
·
Breast cancer
response to chemotherapy.
·
Increase the
study populations that will be examined, looking at pharmacokinetics variations
and altered enzyme kinetics.
·
Examine
additional polymorphism and GSTs in the protein and the gene, and of tissue
specific GST expression as potential factors in individual variation in the
disease and chemotherapeutic response.
·
A large-scale
study done in collaboration with the University of Arizona Cancer Center
examining GST.
·
The validation
of DNA snip microassay chip for examination of large-scale population-based
studies.
·
An
investigation of genetic and epi-genetic alterations of specific cells using
laser-capture micro-dissection.
·
Determine
hypermethylation of GST1 promoter as an early marker of prostate cancer in
men. May include additional genes in
studies of hypermethylation.
·
Undertake
mechanistic studies on the biological and pharmacological actions of
chemopreventative agents.
·
Conduct sight
specific methylation studies over the promoter region of the IGF gene and
lymphocytes from a case control study of colon adenomas.
·
Look at the
global hypomethylation studies on H- and K-ras methylation patterns in human
lymphocytes from a case control study.
·
Continue
collaborating with NCI in case-controlled colon adenoma study. Extend collaboration on DNA gene methylation
in rats undergoing hepatocarcinogenesis by methyl deprivation. Complete collaborative clinical studies on
all abnormal methyl metabolism associated with non-neoplastic disease such as
diabetes and arteriosclerosis.
·
Organize a
workshop on diet, DNA methylation processes, and health. NCI has expressed interest in sponsoring a
joint workshop on the different diseases and the overlapping mechanisms that
seem to be impacted upon or involved with methyl deficiency. (TAB 9)
Division
of Genetic and Reproductive Toxicology (DGRT)
Dr.
Martha Moore provided an update on the activities of the DGRT. The
Division has 31.8 FTEs (full time equivalent).
The Division is composed of 14 principal research scientists, 3 staff
fellows, 12.8 support scientists, and 2 administrative support personnel. They have four research focus areas, two
disciplinary areas, (e.g., genetic toxicology and reproductive/developmental
toxicology) and programs which cross-cut the diet and nutrition program (e.g.,
caloric restriction and dietary restriction program). The Division is expanding to move into the area of general
nutrition and dietary supplements.
Division researchers are working in genetic, developmental and
reproductive toxicology, and are
expanding to include genomics and proteomics. Dr. Moore said the Division's biggest challenge is how to use
these tools to answer questions.
Dr.
Moore explained that the Genetic Toxicology group is a Center for Excellence in
the area of in vivo mutagenesis, and
its current activities include:
·
Development of
the rat hprt gene mutation assay.
·
Trying to
understand mutations associated with the in
vitro mouse lymphoma assay.
·
Mitochondrial
mutations.
·
Developing the
TK heterozygous mouse model.
·
Quantitative
mutagenesis, molecular mutagenesis, and most recently the Division used
molecular techniques to quantitate genomic rearrangements.
·
Development of
new techniques for genotypic selection.
·
Work with
human lymphoblastoid cell lines and trying to understand mutation at the TK
locus and also understanding how the p53 phenotype impacts mutation.
·
A study on
genistein in the p53 mouse.
·
Development of
the FIX in vivo gene mutation
assay.
·
Work with
keratinocytes to develop an hprt gene
mutation assay.
Reproductive/Developmental Toxicology is a
smaller focus group with four members.
They are working on:
·
The EDKB
models.
·
Cell cycle
kinetics and apoptosis.
·
Understanding
how folate impacts the development of the neural tube and the normal neural
tube closure process.
The Diet and Nutrition group is a
cross-cutting team involved in:
·
Caloric restriction
and dietary control studies.
·
Studies with
genistein, including one employing p53 mice.
·
Studying
folate as a dietary issue.
·
Developing a
study to understand the impact of severe malnutrition on the induction of
somatic cell mutations.
The
DGRT is moving into the genomics and proteomics area and has done work in the
areas of:
·
2-D gels.
·
Working with
both micro- and filter arrays to further our understanding how filter arrays
work and the problems in using filter array technology.
·
Computerizing
the data generated and normalizing the data.
·
An
international collaboration to try to understand the microarray technology.
The
framework for DGRT is basic applied research, which includes hazard
characterization and dose response assessment, to improve regulatory
decision-making (risk assessment). In
the process the Division:
·
Generates
chemical-specific information.
·
Does research
in support of the various centers and the Office of Women’s Health.
·
Participates
in open dialogues to better understand the needs of the other centers.
In
the area of hazard characterization, the DGRT is:
·
Developing new
methods (the TK+/- mouse model, phiX-174 gene mutation, keratinocyte hprt, MutEx/ACB-PCR genotypic selection,
EDKB, fluorescent markers).
·
Characterizing
and understanding these new methods.
·
Interpreting
data (in vivo lac-i and hprt, in vivo mouse TK assay, human
lymphoblastoid TK assay, filter arrays, mouse lymphoma TK assay) and using
information for regulatory decision making.
·
Studying the
modes of action for toxicants in hazard characterization and selection of dose
response models (DNA sequence analysis, chromosomal mutations, genomic
rearrangements, gene expression and protein production, folate and neural tube
development, impact of dietary restriction on somatic mutation and
physiological parameters).
·
Rodent/human
extrapolation, response of hepatocytes to toxicants, response to dietary
restriction and nutritional changes.
·
Developing the
necessary guidelines to provide information to the centers on how assays are
done and which should be required (mouse lymphoma TK assay, in vivo gene mutation assays).
Dose
response assessment is composed of several areas:
·
Relevant doses
(dose selection, biomarkers, genomics/proteomics), susceptibility/variability
(fetus/newborn/young child), repair deficiency.
·
PMS2-mismatch
mice, diet (antioxidants, dietary restriction, phytoestrogens), the issue of
rodent/human extrapolation (liver toxicity, biomarkers, diet and nutrition,
genomics/ proteomics), cancer/non-cancer risk assessment.
·
The
development of quantitative models (mechanistic commonality,
genomics/proteomics).
DGRT
is also involved in studies for chemical specific information on genistein,
coumestrol, leucomalachite green, AZT (and other combination drugs for the
treatment of AIDS), and UV light/phototoxicity studies. (TAB
10)
In
closing, Dr. Moore said cross-agency collaboration was an important tool to
complete what needs to be accomplished.
The Division currently has active cross-divisional collaboration
activities with the divisions of Biochemical Toxicology, Biometry and Risk
Assessment, Neurotoxicology, and EDKB.
In addition DGRT maintains
cross-agency collaborations with the Office of Women’s Health, CDER,
CFSAN, and is talking with CBER. She
added that DGRT has cross-departmental collaborations with NIEHS and NTP. Dr. Acosta asked if there was a central
organizing group for the genomics/proteomics effort and was told that DGRT is
starting one. Dr. Lightfoot asked if
there was an Agency-wide genomics/proteomics working group and were they
working and participating with them?
Dr. Casciano responded by saying that they are working with the Office
of Science to coordinate the efforts of the different centers and to generate
common goals with minimal repetition.
Division
of Chemistry
Dr.
Robert Turesky provided an update on the Division of Chemistry. The Division has three units: 1) it supports
a strong commitment to the NTP; 2) the Mass Spectrometry Branch, which conducts
fundamental research and supports chemical mass spectrometry service work; and
3) the Analytical/Biomarker Branch. He
continued by adding that the Division supports additional research efforts in
analytical chemistry, toxicology, NMR spectrometry, spectroscopy, computational
chemistry, and biomarker work. The
Division's mission is to utilize chemical research techniques, including
analytical chemistry, mass spectrometry (MS) and NMR spectrometry,
spectroscopic and computational methods to implement intra-divisional,
intra-center, and FDA-relevant research initiatives in toxicology, risk
assessment, and regulatory compliance.
Historically
the Division has been involved with providing essential support in analytical
chemistry and spectrometry to the NTP as well as for other NCTR divisions. Key research projects of the Division of
Chemistry include:
·
Spectrometric
Data Activity Relationship (SDAR) Models for compounds binding to receptors of
toxic responses (predictive toxicology).
·
NMR
spectroscopy of drug purity with public health implications.
·
Rapid
identification of intact whole bacteria based upon spectral patterns using
MALDI-TOF MS (matrix assisted laser deionization – time of flight mass
spectrometry).
·
Fresh Tag
SensorÔ technology for product safety, quality, and
rapid screening of explosives.
·
Rapid
screening and identification of complex mixtures by pyrolysis-mass spectrometry
with pattern recognition methods.
·
Chemical
characterization of critical chemicals, components, constituents, and biologics
for selected botanical products.
·
Impact of
dietary supplements on woman’s health issues.
·
Comparison of
principal components analysis (PCA) and artificial neural networks (ANN) for
the prediction of qualitative and quantitative biological end points from
spectrometric data.
·
Risk
assessments of dietary contaminants.
The
Division of Chemistry has a very strong support relationship for the NTP
research programs. In addition, the
Division has active collaborations with CVM in studying various drug residues
including Amoxicillin, erythromycin, Lincomycin, and sulfa drugs, which require
development of determinative methods that meet testing requirements for
reliability.
Dr.
Turesky said the Division is a strong proponent of using mass spectrometry for
characterization and identification of various bacterial species and problems
associated with microbial contamination.
Working in support of the Food Safety Initiative, the Division uses mass spectrometry to differentiate strains of
food borne pathogens; this is now a standard procedure used by FDA and
others in the United States and around the world. Different milestones in this area include:
·
MALDI can
differentiate bacteria by genus, species, and strain.
·
Specific
Biomarkers for virulence can be detected by MALDI.
·
Biomarker
proteins can sometimes be detected in contaminated media without pre-MS culture
steps.
Dr.
Turesky added that the Division would continue to adapt and use MS methods in
other emerging areas such as proteomics and bioterrorism. Division researchers can detect proteins and
identify biomarkers in intact bacterial cells and may detect protein/biomarkers
in malignant cells or even in vivo. In the future they hope to:
·
Determine
correlation of toxicity and strain types with MALDI spectra.
·
Develop more
powerful MS methods (MALDI/FTMS).
·
Make more
accurate assignment of biomarker (protein) identity.
Benefits
to FDA, as described by Dr. Turesky, include:
·
The ability to
differentiate strains from more Vibrio species.
·
Detection of
biomarkers associated with antibiotic resistance.
·
Application of
MS to FDA programs in bioterrorism, proteomics.
·
Characterization
of various cell types, possibly malignant cells.
Current
goals and objectives using MAB-TOF MS include:
·
Rapid
chemotaxonomic strain-specific bacterial identification.
·
Development of
bacterial databases and search strategies.
·
Applications
to food/seafood borne bacteria, especially Vibrio
spp. (CFSAN & ORA).
·
Development of
patents for new methods.
·
Identification
of bacteria without a prior cell-culture step.
Key
Findings to date include:
·
Demonstrated
that a multiplicity of laboratory variables distort mass spectral fingerprints.
·
Patented a
simple algorithm to correct for method-related spectral changes. (The correction is more practical than using
identical conditions.) US Pat. App. No.
60/239,549 filed 10/10/2000
Future
Experimental and Computational Directions
·
b-test
Py-MAB-TOF- MS (from Dephy, Montreal) at NCTR.
·
Assemble and
validate a 200-sample spectral database using bacteria from CFSAN and ORA
reference collections.
·
License the
patent on using a spectral correction method to mitigate laboratory-based
variations.
·
Develop
algorithms to transform spectra from environmental samples to equivalent
laboratory spectra.
Dr.
Turesky added that Dr. Ang, in collaboration with CFSAN has been establishing
methods to isolate various bioactive components in medicinal or herbal
medications. There has been a great deal
of discussion and controversy because some of these chemicals contain
components that may alter the pharmacological activities of a number of
different drugs and medications. Dr.
Ang has been asked to use her expertise in the chemistry of these compounds to
work with Dr. Leakey who is looking at the activity of herbal medications on
key enzymes, up regulation, down regulation, and metabolic activities. Dr. Ang has been able to isolate hyperforin,
which is found in St. John’s Wort (SJW).
We are now able to conduct in
vitro bioassays to better understand the biochemistry of these
molecules. This work will be extended
to looking at the metabolism of various components of SJW.
Objectives:
·
To develop
human cell-based assays to determine whether a test substance affects key
enzymes involved in the metabolism of pharmaceuticals.
·
To use these
assay systems to investigate potential drug-herbal interactions between
prescribed pharmaceuticals and dietary supplements.
Research
Progress:
·
Extraction and
LC methods developed for four SJW components in tea, fortified drinks, puffs
and snack bars.
·
Methods
developed for five phenolic compounds in echinacea capsules and tablets.
Preliminary
Findings:
·
Developed
methods for isolating hyperforin, the major active ingredient of SJW.
·
Developed or
procured battery of cell lines expressing major isoforms of human drug
metabolizing enzymes; used in inhibition assays.
·
Established
that constituents of Echinacea inhibit enzymes conjugating estrogens.
Future
Work:
·
Develop human hepatocyte-based
assay systems for measuring drug metabolizing enzyme induction.
·
Isolate and
identify the inhibitory constituents of Echinacea and SJW.
·
Investigate
the metabolism of active ingredients of SJW.
·
Apply
inhibition and induction assays to other herbal products.
·
Establish
microarray and proteomic technology for elucidation of mechanisms.
Dr.
Turesky reported that Dr. Beger, in collaboration with Drs. Lay, Miller, and
Wilkes, has been working on the relationship between structure-activity
relationships (SAR) and spectrometric data-activity relationships (SDAR). There are some unique things that 13C
NMR can provide that other spectrometric methods may not, such as providing
information on electron density molecules, configuration and confirmations. An example of some of the success of SDAR
and quantitative spectrometric data-activity relationship (QSDAR) models
include: (a) SDAR model of 108 compounds binding to the estrogen receptor using
NMR and MS data, and (b) QSDAR model of 26 poly- chlorinated dibenzofurans
binding to the aryl receptor using predicted NMR data.
SDAR
Publications and Patents:
·
13C NMR and EI Mass Spectrometric Data to
Produce a Predictive Model of Estrogen Receptor Binding Toxicology and Applied
Pharmacology. 169: 17-25, 2000.
·
Producing 13C
NMR, Infrared Absorption and EI Mass Spectrometric Data Monodechlorination
Models of Chlorobenzenes, Chlorophenols, and Chloroanilines J. Chem. Inf.
Comput. Sci. 40:1449-1455, 2000.
·
Developing 13C
NMR Quantitative Spectrometric Data-activity Relationship (QSDAR) Models to the
Corticosteroid Binding Globulin. J. Comput.-Aided Molec. Design.
·
Models of
Polychlorinated Dibenzodioxins, Dibenzofurans, and Biphenyls Binding Affinity
to the Aryl Hydrocarbon Receptor Developed Using 13C NMR Data. J. Chem. Inf. Comput. Sci.
·
Patent Pending
for “Methods for Predicting the Biological, Chemical, and Physical Properties
of Molecules from Their Spectral Properties.”
Dr. Turesky then discussed the following
future directions of SDAR:
·
Protocol
E0706801: “Continuing to develop SDAR models for the Ames test, neuraltoxicity
(Neurotox), and other toxic endpoints.”
·
Protocol
E0706811: “Developing new strategies for spectrometric models of toxicity”
(ROW).
·
Protocol
E0708301: “Computational predictive system for rodent organ-specific
carcinogenicity” (in collaboration with Biometry, CDER, ROW).
·
Producing
hybrid spectrometric models that incorporate three-dimensional structural
information into the SDAR model.
Dr.
Turesky then said that Dr. Buzatu, in collaboration with Dr. Lay, is taking
complex chemical spectral data from 13C NMR and setting up
artificial neural networks and looking at the different predictive biological
endpoints. One experiment using the
QSDAR-ANN model reflected excellent results for 28 poly-chlorinated biphenyl,
dioxin, and furan toxic equivalence factors (TEFs) using predicted 13C
NMR spectra.
Future
Directions:
·
Currently
developing a quantum mechanical parameter based neural network model for the
prediction of TEFs for dioxins and dioxin-like compounds.
·
Developing an
Internet parallel distributed neural network to handle large data sets as well
as increasing the efficiency of the neural network.
Dr.
Evans has been establishing methods to determine whether NMR can be used as a
rapid screening tool for measuring adulteration and contamination of drugs
using genistein as a model. The
associated protocol “A New Approach to
the NMR Spectroscopy of Drug Purity and the Public Health Implications” has the
following objectives:
·
Determine
properties and develop procedures for use of NMR spectrometer under high
dynamic range conditions.
·
Develop
concepts and methodology for applying spectroscopy to very-low-level impurities
in drugs using results on genistein.
Mass
Spectrometry Applications in FDA Research Initiatives were outlined to include:
·
Allergenicity
·
Bacteria
(taxonomy/speciation)
·
Bioterrorism
·
Drug purity
(chemicals and recombinant proteins)
·
Ion mobility -
MS (protein confirmation determination)
·
Microbial
biotransformation of drugs and antibiotics
·
Proteomics
·
Quality
assurance and compliance programs
·
Rapid
through-put analysis
·
Redox status
(vitamins, lipids, proteins, DNA)
·
Risk
Assessment (biomarkers, DNA- and protein adducts, DNA damage, metabolites)
Dr.
Turesky explained that to have a successful infrastructure for proteomics the
Division recently received funding to obtain access to additional MS
instrumentation. In addition to the
appropriate equipment and tools for proteomics, they need to be able to recruit
and hire people with the appropriate skills.
Dr. Turesky added that Dr. Alex Strasbourg is developing mass
spectrometry methods using model compounds, which will have the potential to
detect BSE.
He
then outlined the following NMR spectroscopy applications in FDA research
initiatives:
·
Computational
chemistry
·
Metabonomics
·
Proteomics -
Drug Interaction
·
Drug Purity
Dr.
Turesky added that Dr. Beger, in collaboration with Dr. James, is looking at
the metabolism modulation of tetrahydrofolate in Down Syndrome, which can only
be done using NMR.
Dr.
Turesky then listed the following research areas supported in FY-2001:
·
Ethinyl
estradiol on bone growth in rats
·
Erythromycin
from farmed animals
·
Malachite
green/leuco malachite green in mice
·
Retinyl
palmitate: isolation and detection
·
DNA adducts of
Tamoxifen
·
Dietary
supplements and herbals: identification of bioactive ingredients
·
Endocrine
disrupters: genistein and daidzein
·
Phytoestrogen
conversion to estrogenic compounds: genistein & daidzein
·
Fluoroquinolone
biotransformation by fungi
·
Microbial
degradation of drugs and feed additives in aquaculture
·
Antihistamine
drugs in neonatal mouse cells (TAB 11)
Dr.
Donnelly noted there was overlap with some of the initiatives in the Division
of Microbiology, and asked if there were attempts to do formal collaboration
and coordinate efforts. Dr. Turesky
responded that there has been strong collaboration with Microbiology on
microbial antibiotic resistance, which requires analytical chemistry and mass
spectrometry. He said that the
characterization of various proteins was initiated by this Division. Dr. Lay said this was larger than just an
NCTR project, as the Vibrio spp. came
from Dolphin Island, and they transported the technology to CFSAN. He added that they are now sequencing the
proteins to differentiate the Vibrios. There are several microbiologists from NCTR
and other FDA facilities involved in this as well as chemists. Dr. Casciano said this is a natural fit
since the two groups have been collaborating for years. The Divisions of Microbiology and Chemistry
have a support and research function and they leverage with each other to
answer some of the questions. Dr.
Donnelly suggested that there was additional work that could be done on
identification of some of the poultry strains by applying mass spectrometry.
Dr.
Rosenkrantz asked if there were other mechanisms for commercializing the
FreshTag™? Dr. Miller said FreshTag™ is
under license to Cox Technologies in Charlotte, North Carolina. He said they worked with Cox from the
initial prototype. In terms of
marketability, Tenneco is looking at building FreshTag™ into a packaging
material. For example, with a zip lock
bag, if you put a fish product or another food that produces ammonia, the bag
will give a clear signal as soon as it decomposes. The process is autolytic and possibly enzymatically generated,
which in turn forms aldehydes from the lipids.
As it turns out, the major compounds of decomposition, such as ammonia,
acids in the case of carbohydrates, sulfurs, and aldehydes can be
detected. Tenneco has expressed
interest in detecting all of these end products in one step.
Division
of Biometry and Risk Assessment
Dr.
Ralph Kodell provided an update on the Division of Biometry and Risk Assessment. The
Division has two Ph.D. researchers, three post doc mathematical statisticians,
two research biologists, two computer specialists, a program support
specialist, and a staff fellow.
He
described the following research highlights:
·
Fumonisin B1
risk modeling.
·
An NTP IAG
study in rats and mice.
·
Risk
assessment for the mechanistic data and traditional bioassay data.
Dr.
Kodell also discussed the Divisions research projects, which include:
·
Cryptosporidium parvum.
·
Cumulative
risk assessment.
·
Computational
toxicology.
·
Looking at the
1,298 chemicals in the Carcinogenic Potency Database (CPDB).
·
Photocarcinogenicity
theory and methods.
·
Analysis of
cDNA microarray data. (TAB
12)
Dr.
Gillett was interested in risk modeling of fumonisin and asked if they were
planning to do this with any of the other NTP products; and if so, would the
Division be involved in the protocol design to help ensure collection of the
appropriate data at the earlier sacrifice time? Dr. Beland said they have wanted to be involved in the design of
the NTP protocols, although they have not yet done so and do not have immediate
plans to do so at this time. Dr.
Casciano said that protocol development is reviewed closely and statisticians
are involved. He also said that Dr.
Kodell’s group is thinking about applying the expertise they have in chemical
dose response, database mining, statistical analysis, and statistical
development to applications in new fields because we at NCTR consider this to
be the future of toxicology. Dr.
Gillett said the purpose of these large, expensive, bioassays is to assess risk
to humans and it is important to get involved early in the study design. Dr. Beland said they would involve Dr.
Kodell’s group. He added that at this
time, they have only completed fumonisin and chloral hydrate. An upcoming study of urethane and alcohol and how these two
compounds interact is expected to be noteworthy.
A
general discussion session followed the presentations made by each of the
Divisions.
Dr.
Gillett asked if there was a provisional committee for genomics and proteomics
at NCTR? Dr. Casciano said that the
initial efforts had focused on identifying individuals who have an interest in
using microarray technology and in building and evaluating the databases. He added that there are committees concentrating
on computational science and another focusing on cellular and molecular
methodologies. Dialog is ongoing with
various centers regarding NCTR's technical expertise and how best to use these
techniques to supply data for regulatory decision-making.
Dr.
Lightfoot had a question regarding antiretroviral agents and a study in
pregnant women. She said one of the few
success stories for antiretroviral agents is that of pregnant women treated
with AZT. What was the motivation for
the research? Is NCTR looking to see if
AZT is more mutagenic than it is therapeutic in the babies of women with
AIDS? Dr. Beland said women and their
babies are currently being treated with multi-drug therapy. The question is whether this is safer than
treatment with AZT by itself, or if such treatment is going to cause future
complications in children who are not HIV positive. He added that NCTR is not advocating discontinuing the treatment,
but rather is questioning whether there are certain other treatments that may
be safer. Dr. Lightfoot asked what
motivated the study? Dr. Beland said
the motivation was the demonstration that AZT is carcinogenic to neonatal mice
when exposed transplacentally. He added
that there have also been some mitochondrial toxicities and death reported in
children who have been treated with AZT and 3TC in Europe.
Dr.
Hansen said CFSAN was interested in pursuing more of the structure-activity
approaches and approaches to constructing tier testing schemes. She said that these approaches could provide
guidance for petitioners and for CFSAN staff.
Dr. Hansen also said that the utility of the work being done in Biometry
was excellent.
Dr.
Youngman said her first visit to NCTR was a real learning experience and that
she previously was unaware of the scope of the work being done here. She said she saw a huge potential for future
collaboration with the Office of Research in Laurel. She said that she spoke to some of the Division Directors after
the presentations and hoped they would consider pursuing new collaborations.
Dr.
Donnelly said the last two days have been incredibly valuable. There is great work being done at NCTR, and
how the Center communicates that work to its constituents is vital. She also said that while listening to some
of the presentations, she got the impression that many of the initiatives and
projects were PI directed, and was not sure they fit within the overall goals
of NCTR. She added that a simple annual
plan is needed to establish strategic goals.
She observed that it seems that it would be very easy for NCTR to draft
an annual plan with strategic goals and have each project fit within a
strategic framework. An outside
reviewer may get the sense that the projects are driven by PI interest or
extramural funding. Dr. Donnelly added
that it would then be easy to see that the project fits not only within the
strategic goals of NCTR, but within the goals of CFSAN, CVM, or one of the
other centers. This structure would
build a constituency base across the centers.
She closed by saying that her frustration is that there is some
fundamentally excellent work going on, but perhaps NCTR staffers are the only
ones that know about it.
Dr.
Casciano responded that the Center has been trying to do that over the last
nine years. The work going on relative
to the NTP is a direct response to FDA needs, and each of the five product
centers would like to have a specifically directed strategic goal. Each product center has different mandates
and methodologies for doing something as simple in concept as risk
assessment. Dr. Casciano continued by
adding that NCTR establishes strategic goals from a concept statement or
prepared paper. This concept statement
can be one page or five pages, whatever it takes to communicate what the
scientist is interested in doing. He
said that if he understands and agrees with the concept, he sends it to the
five product centers for comment. Dr.
Casciano added that NCTR's scientists are very creative and in the last eight
to ten years have developed better communications with the product centers and
now have a greater sense of what their needs are. The rationale is to get early input from the regulatory
centers. If there are three or four
objectives that meet the needs or requirements of the centers, then the PI
develops the protocol. We write
strategic goals broad enough to cover the mandates of all of the centers. Dr. Donnelly responded that she thought that
might be a nice framework for publicity.
Dr. Casciano noted that NCTR could further improve its efforts at
publicity and at conveying its strengths.
Dr.
Rosenkrantz asked if there was a plan for the site visit teams for the coming
year? Dr. Casciano said there would be
a site visit in early fall or winter for the Chemistry Division.
Dr.
Acosta said the Board appreciated the fact that almost all of the Division
heads were present. He added that it is
evident you are very proud of all of the hard work of your staff and that you
have very high standards and operate in a very professional manner.
The
meeting adjourned.
__________________/Signed/_____________________________
___08/09/02________________
Leonard
M. Schechtman, Ph.D., Executive Secretary to the SAB Date
GLOSSARY
of Commonly Used Abbreviations
3TC
- lamivudine, an approved antiretroviral treatment
ACPS
- Advisory Committee for Pharmacological Science
AIDS
- acquired immune deficiency syndrome
ANN
- artificial neural networks
AZT
- zidovudine, an approved antiretroviral treatment
BSE
- Bovine Spongiform Encephalopathy
C13
- Carbon 13
CBER
- Center for Biologics Evaluation and Research
CDC
- Centers for Disease Control and Prevention
CDER
- Center for Drug Evaluation and Research
CDRH
- Center for Devices and Radiological Health
CFSAN
- Center for Food Safety and Applied Nutrition
CRADA-
cooperative research and development agreement
CVM
- Center for Veterinary Medicine
DGRT
- Division of Genetic and Reproductive Toxicology
EDKB
- Endocrine Disrupter and Knowledge Base
ERG
- erythromycin resistance genes
EPA
- Environmental Protection Agency
FTE
- full time equivalent
GS
GST
GSTA
IAG
- Interagency agreement
ICCVAM
- Interagency Coordinating Committee on the Validation of Alternative Methods
LC
- liquid chromatography
MAB
-
MALDI-TOF
MS - matrix assisted laser d-ionization – time of flight mass spectrometry
MTHFR
MS
- mass spectrometry
NCI
- the National Cancer Institute
NCTR
- National Center for Toxicological Research
NCSS
- Non-clinical Studies Subcommittee
NIEHS
- National Institute of Environmental Health Sciences
NIH
- National Institutes of Health
NIOSH
- National Institute of Occupational Safety and Heath
NITA
-
NMR
- Nuclear magnetic resonance
NTP
- National Toxicological Program
ORA
- Office of Regulatory Affairs
PCA
- Principal components analysis
PCR-RFLP
- polymerase chain reaction---
PHS
- Public Health Service
PI
- Principal Investigator
ROW
- company under contract to NCTR to conduct research
SAB
- Science Advisory Board
SDAR
- spectrometric data activity relationship
SAR
- structure activity relationship
SJW
- St. John's Wort
SVT
- site visit team
QSDAR
- quantitative spectrometric data-activity relationship
TEF
- toxic equivalence factors
THC
- tetrahydrocannabinol, a metabolite of marijuana
TSSRC
-- Toxicology Study Selection and Review Committee
UAMS
- University of Arkansas Medical School