Current
Regulatory and Scientific Issues
in Genetic Toxicology
Invited Speakers and Abstracts
Jiri Aubrecht,
Pharm.D., Ph.D.
Pfizer Global R & D
Dr. Jiri Aubrecht received his Pharm.D from Charles University and PhD in pharmacology from Czechoslovak Academy of Sciences in Prague, Czech Republic. He completed his postdoctoral fellowship in molecular toxicology and cancer biology at the Harvard School of Public Health in Boston MA. After a short stay in the biotech industry, Dr. Aubrecht joined Pfizer Groton Laboratories in 1999. At Pfizer, he developed and implemented approaches for genetic toxicity screening and evaluated the potential of toxicogenomics for studying toxic mechanisms and risk assessment. Currently, Dr Aubrecht leads a safety biomarkers laboratory. His research interests are application of systems biology-based approaches for investigating toxic mechanisms and biomarker development. Dr Aubrecht serves as a chair of the ILSI HESI toxicogenomic committee.
Why Not a Single Test?
The current genotoxicity toxicity
testing paradigm relies on the genotoxicity
testing battery. Although the current paradigm
has prevented introduction of harmful chemicals
to patients and consumers, the limited understanding
of underlying toxic mechanisms results in a
positive findings in the in vitro chromosome
damage assays with questionable human risk and
relevance providing a challenge to industry
and regulatory agencies. Therefore, the development
of broad mechanisms based approaches is essential.
The cellular response to chemicals triggers
a complex web of molecular pathways involved
in cell survival and/or cell death. The emerging
field of systems toxicology utilizes genomic
science and technologies to gain insights into
mechanisms of toxicity. Recently, systems toxicology
approaches has been used for development biomarkers
and/or applied to risk assessment. In addition,
several international consortia in the US (Critical
Path, HESI) and EU (Carcinogenomics, IMI) have
been pursuing the development of biomarkers
of carcinogenic mechanisms. In this presentation,
we will discuss the current status of systems-based
approaches for studying genotoxic mechanisms,
future developments, and their potential role
in risk assessment.
Robert
Daniel Benz, Ph.D.*
Dr. Robert Daniel Benz is the Acting Director and Database Manager, Informatics and Computational Safety Analysis Staff, Science and Research Staff, Office of Pharmaceutical Science, CDER, US FDA. Previously, Dr. Benz served as the Team Leader and Review Genetic Toxicologist, Office of Premarket Approval, CFSAN, US FDA, and, before that, Associate Scientist, Medical Department, Brookhaven National Laboratory. Dr. Benz received his BS in Biology from the Illinois Institute of Technology and his PhD in Biophysics from the University of California. He is a member of several professional societies and Genetic Toxicology Committees. Dr. Benz has organized several symposia on Computational Toxicology and is a recipient of the GTA Excellence in Science Award in 2007, EMS Service Award in 2004, and AGT Outstanding Service Award in 1998.
* Interactive Workshop Session Moderator
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David
Brusick, Ph.D.*
Dr. Brusick received a Ph.D. in
genetics from Illinois State University in 1970.
He worked at the US FDA and Howard University
School of Medicine before moving to Litton Bionetics
in 1974 to establish a commercial genetic toxicology
testing business.
Dr. Brusick was President of the US Environmental
Mutagen Society in 1978 and Chairman of the
International Commission for Protection Against
Environmental Mutagens and Carcinogens from
1989-1995. He has spent over 30 years in the
CRO industry and during that time participated
in a number of Expert Panels for the EPA, National
Academy of Sciences and IARC. He was also a
member of the EPA GeneTox Steering Committee.
Dr. Brusick is the author of two books, approximately
40 book chapters and over 100 publications primarily
directed towards genetic toxicology testing.
He retired in 2005 and has been consulting during
the past three years.
* Expert Panel Member
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David M. DeMarini, Ph.D.
U.S. Environmental Protection Agency, Research
Triangle Park, NC
David M. DeMarini was born in Peoria, Illinois, USA. He received the B.S. (1972), M.S. (1974), and Ph.D. (1980) in Biological Sciences (genetics) at Illinois State University, Normal, IL, studying under Dr. Herman E. Brockman. From 1980-1982, he did postdoctoral research at the Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN. He then was a Research Geneticist at the National Toxicology Program, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC from 1983-1984. He began his current position as a Research Genetic Toxicologist at the US Environmental Protection Agency (US EPA), Research Triangle Park, NC in 1985. He is also an Adjunct Full Professor, School of Public Health, University of North Carolina, Chapel Hill, NC (1991-present). He is a member of the Environmental Mutagen Society (EMS) and the Genetics and Environmental Mutagen Society (GEMS), of which he is a Past-President and Board Member. His editorial positions include Editor of Mutation Research--Reviews (1998-present), Co-Editor of EMS Newsletter (1988-1991), and Book Review Editor of Environmental and Molecular Mutagenesis (1989-1993). His Editorial Board memberships include Mutation Research (1985-1997), Environmental and Molecular Mutagenesis (1984-1989, 1993-present), Mutagenesis (1992-1995, 2005-present), Environmental Health Perspectives (1988-1993), Teratogenesis Carcinogenesis and Mutagenesis (1990-1992), EMS Newsletter (1986-1988), Pan-African EMS Newsletter (1994-present), and Genes and Environment (2006-present). He has organized conferences, symposia, and Alexander Hollaender Genetic Toxicology Training Courses internationally, and has given invited lectures at more than 85 conferences worldwide. He has chaired a variety of committees of the EMS, served on Council, and is a Past-President of EMS (2000-2001). He was Program Chair for the 9th International Conference on Environmental Mutagens (9th ICEM) in 2005. He is the President of the International Association of Environmental Mutagen Societies (IAEMS) for 2005-2009. He has served on both (1986 and 2004) Tobacco Smoking and Cancer Monographs of IARC/WHO in Lyon, France, as well as the Drinking Water/Arsenic IARC Monograph (2004) and Indoor Air Monograph (2006). He served on the US National Academy of Science's Steering Committee on Proteomics in 2002. He and his colleagues received the highest scientific achievement award given by the US EPA in 2004 for their work on the genotoxicity of arsenic. He has published 150 articles in mutagenesis (130 journal articles and 20 book chapters). His research interests are molecular mechanisms of mutagenesis, mutation spectra, complex mixtures, and biomarkers of mutation in humans.
Review of 30 Years of Research on the Occurrence, Genotoxicity, and Carcinogenicity of Disinfection By-products in Drinking Water: A Roadmap for Research
The 30-year literature on the occurrence, genotoxicity,
and carcinogenicity of disinfection by-products
(DBPs) has been published recently [Mutat.
Res. 636, 178; 2007]. Some of the findings
for the 11 DBPs regulated by the U.S. EPA are
(a) 2 DBPs (chloroacetic acid and chlorite)
are not carcinogenic-each in 2 species; (b)
chlorite is not carcinogenic in 3 rodent assays
and has never been tested for genotoxicity;
(c) 1 DBP (bromoacetic acid) has never been
tested for carcinogenicity; (d) 2 DBPs, chloroform
and trichloroacetic acid, are carcinogenic via
nongenotoxic mechanisms; (e) 6 DBPs have significant
genotoxicity data gaps; and (f) 5 DBPs have
been assessed as possible or probable human
carcinogens. Among the 74 newly emerging, unregulated
DBPs reviewed, 29 that occur at sub-low ?g/L
levels are genotoxic; and another 14 that occur
at this level have no toxicological data except
for 2, which are carcinogenic. The toxicity
of DBPs is iodo > bromo > chloro, and
50% of the organic carbon and organic halogens
of drinking water are unknown, i.e., not chemically
characterized. Approximately 30% of the municipal
water suppliers in the U.S. have changed from
chlorination to chloramination, which has resulted
in the formation of newly emergin DBPs, such
as the halonitromethanes and brominated forms
of DBPs. Although more toxic than the regulated
DBPs, these newly identified DBPs are generally
present at much lower concentrations than those
that are regulated. Nonetheless, alternative
disinfection practices result in drinking water
in which extracted organic material is less
mutagenic than extracts of chlorinated water.
Recent molecular epidemiology indicates that
an increased risk for bladder cancer is associated
with dermal/inhalation exposure to drinking
water (from bathing/showering and/or swimming)
rather than to drinking the water and that risk
is enhanced in people carrying the GSTT1-1
gene, which is present in 75% of the U.S. population.
Studies more than a decade ago showed that trihalomethanes
other than chloroform are activated to mutagens
via GSTT1-1 in a transgenic strain of
Salmonella. Recent studies in rodents
and humans support the epidemiology above, but
further studies are needed to clarify if dermal
and inhalation exposure are more important than
oral exposure to trihalomethanes for increased
risk for bladder cancer. Although the mutation
spectra of a wide variety of disinfection by-products
have been determined in Salmonella, no
molecular epidemiology studies have been performed
to determine if mutation patterns in human tumors
associated with chlorinated water exposure contain
the spectra of mutations produced by these compounds.
These and other findings provide guidance for
drinking water and public health research.
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Vasily N. Dobrovolsky, Ph.D.
NCTR/FDA
Dr. Dobrovolsky has developed in vivo mutagenesis
models using transgenic and knockout mice and
contributed to improving high throughput methods
of mutation detection.
1988 - MS degree in biotechnology from Moscow
Institute of Physics and Technology, Russia
1994 - Ph.D. degree in molecular biology from
the Shemyakin Institute of Bioorganic Chemistry,
Russian Academy of Sciences, Russia.
Since 1994 has been employed at NCTR/FDA first
as a postdoctoral fellow, then as a staff fellow
and currently as a staff scientist.
Pig-A Gene - A New Endogenous Target for Detection of in vivo Mutation Using Flow Cytometry
The product of the X-linked Pig-A gene is involved
in the synthesis of glycosyl phosphatidyl inositol
(GPI) molecule that anchors multiple proteins
to the cell membrane. Mutation of the Pig-A
gene has been associated with disruption of
GPI synthesis and deficiency of appropriate
markers at the surface of the cell. We developed
a method for rapid detection of Pig-A mutants
lacking GPI-anchored markers in rat peripheral
blood cells using high throughput flow cytometry.
The presentation will give historical perspective
for method development, outline the methodology
of mutants detection and discuss the potential
advantages of the new method when compared to
the traditional battery of genotoxic tests.
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Rosalie
K. Elespuru
Food and Drug Administration, CDRH
ILSI and ICH Battery Maintenance: Increased Voltage or Hazard Identification Short Circuit?
ILSI and ICH groups are actively
involved in updating genotoxicity testing standards
by critical examination of the current ICH test
battery and by exploring ways to integrate new
technologies. With regard to in vitro mammalian
assays, the ILSI and ICH approaches seem quite
different. ILSI is evaluating current and potential
future assays in terms of validation, relevance,
and follow-up related to assessment of risk.
There is a potential for increased value of
in vitro mammalian assays if in vitro/in vivo
correlations can be demonstrated (increased
voltage). The ICH group seems to already have
decided that the in vitro mammalian tests are
no longer useful, by eliminating them entirely
in one option of a new test battery (short circuit
for hazard id?). Open discussion and data evaluation
will hopefully lead to the harmonization of
the ILSI and ICH approaches, leading to the
integration of new assays. However, this requires
the hard work of validation, within the context
of the main purpose of short-term genotoxicity
tests, hazard identification.
Patricia Escobar, Ph.D.
Boehringer-Ingelheim Pharmaceuticals, Inc.
Dr. Escobar received her B.S. in Microbiology, and M.S. in genetic toxicology from the Universidad de los Andes in Bogotá, Colombia. Dr. Escobar obtained her Ph.D. in Molecular toxicology from University of Pittsburgh. Then she did her postdoctoral work in the Molecular Epidemiology and Toxicology laboratory at University of California, Berkeley.
Prior to joining Boehringer Ingelheim she was a genetic toxicology study director at BioReliance Corporation and was also involved on new assays development. Dr. Escobar has been invited to deliver numerous talks regarding genetic toxicology and the Comet Assay in the U.S. and abroad.
New Applications of the Comet Assay in Genetic Toxicology Testing
The Comet assay, also known as Single Cell
Gel Electrophoresis (SCGE), is a microgel electrophoretic
technique that has the ability to detect DNA
damage at a single cell level. The alkaline
(pH 13) version of the comet assay enables the
detection of a broad spectrum of DNA damage,
which is measured as single and double strand
breaks, and single strand breaks as a result
of alkali-labile sites or nucleotide excision
repair.
The alkaline Comet assay can be used in in vitro
and in vivo test systems. The in vivo (rodent)
Comet Assay is increasingly being used to evaluate
the genotoxic potential of industrial chemicals
and pharmaceutical compounds. In addition, the
in vivo comet assay is gaining popularity as
part of the genotoxic hazard identification
package, as a follow up or complementary test
after an in vitro genetox positive. This assay
is also being discussed in the regulatory arena
as a possible second in vivo assay in the newly
proposed S2 (R1) ICH guidelines.
The in vitro comet assay is seen as a candidate
for screening in early drug discovery/development
and/or to complement the already existing cytogenetic
methods. The major advantages of the in vitro
comet assay are: 1) that cell proliferation
is not needed for an assessment of genotoxic
potential, 2) almost any mammalian cell type
can be used for testing, 3) small number of
cells are needed so high-throughput methods
may be used, 4) a small amount of test compound
is needed for testing and 5) results are obtained
relatively quickly and in some instances can
be automated.
The versatility of the Comet assay and its potential
applications in genetic toxicology testing will
be discussed.
Sheila M. Galloway, Ph.D.
Merck Research Laboratories, West Point, PA
Rationale for the Proposed Revisions to ICH S2 Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
The ICH S2A and S2B guidances (1996-7) recommended
a battery of two in vitro and one in vivo genotoxicity
tests for pharmaceuticals. Based on the last
10 - 15 years experience with testing pharmaceuticals,
we know we need to reduce our reliance on in vitro assays carried out under somewhat extreme
conditions on the principle of hazard identification,
and consider tests/protocols that identify potential
genotoxic effects under more realistic conditions,
to provide information more useful to human
risk evaluation.
Proposed revisions include more options: In
addition to the traditional battery, an equally
acceptable battery is an Ames test and two in vivo genotoxicity assays, usually a micronucleus
assay in rodent hematopoeitic cells and a second
assay in a different tissue, generally liver.
The reasoning behind this second option without
an in vitro mammalian cell assay includes the
following: When an in vitro mammalian cell assay
(CAbs or MLA) is positive, clearly negative
results in two well conducted in vivo assays,
in appropriate tissues and with demonstrated
adequate exposure, are considered sufficient
evidence for lack of genotoxic potential in vivo. Thus a test strategy in which two in vivo
assays are conducted treats the case where no
in vitro mammalian assay is done as if an in vitro test had been positive.
Integration of in vivo genotoxicity tests into
existing repeat-dose toxicology studies is recommended
wherever possible, and under Option 2, stringent
criteria are given for demonstrating that the
dose in the toxicology study is sufficiently
high, to ensure adequate sensitivity.
For the in vitro mammalian cell assays, the
micronucleus test is added as a third alternative
assay, and there is emphasis on limiting top
concentration and toxicity, and avoiding precipitating
dose levels. A 1 mM limit concentration is recommended,
based on experience with detecting convincing
genotoxins (remembering that many are also detected
in the Ames test), and on the principle that
this far exceeds human clinical exposures, even
to drugs that accumulate in tissues.
For in vivo assays, scoring of micronuclei in
rat blood (reticulocytes) is now acceptable
provided the sample size is sufficiently large
and methods are used to identify the most recently
formed red blood cells. There is now substantial
experience and further development of in vivo
assays that are applicable to a variety of tissues
e.g., DNA strand breakage assays such as the
single cell gel electrophoresis assay or "Comet"
assay, transgenic mutation models, and the micronucleus
assay using tissues other than bone marrow.
It is noted that misleading positive results
can occur in vivo, for example increases in
micronuclei related to regeneration of red blood
cells and not to induced genotoxicity, and the
potential for increases in DNA strand breakage
secondary to other cellular processes including
toxicity.
Because of the limited experience with integrating
the Comet assay into repeat dose toxicology
studies, collaborative trials are under way
to assess the sensitivity, and potential for
false positives or false negatives in such a
study design, in addition to obtaining agreement
on an acceptable protocol for the Comet assay.
While OECD guidelines were developed for genotoxicity
assays in parallel with the ICH guidances in
the early 1990s, and there was a purposeful
attempt to develop methods applicable to genotoxicity
testing for all types of chemicals (industrial,
agricultural, medical) it is clear that certain
attributes of pharmaceutical testing justify
specific modifications for drugs, and differences
from existing OECD guidelines are pointed out
and justified in the revised ICH guidance.
Elmar Gocke, Ph.D.
F. Hoffmann-La Roche Ltd
Risk assessment of genotoxic impurities: the case of EMS
The presence of Ethyl Methansesulfonate (EMS)
in tablets of a HIV medication necessitated
a detailed risk assessment of potential toxic
effects in the exposed patients. Although there
are numerous in vitro and in vivo studies on
the genotoxic activity of EMS, no lifetime carcinogenicity
studies, repeat dose mutation data or exposure
analysis were available to serve as solid basis
for such a risk assessment.
For alkylating agents like EMS it is generally
assumed that the dose response for mutagenicity
(and by default also for carcinogenicity) is
linear indicating that exposure even at very
low levels carries a finite risk. A recent in vitro genotoxicity study (Doak et al 2007; Cancer
Res. 67, 3904ff) provided evidence, however,
that the dose response curve for mutagenic and
clastogenic activity was thresholded, suggesting
that a safe dose range can be defined. In contrast,
Ethylnitrosourea (ENU) did not show a thresholded
dose response. We decided to verify the existence
of a threshold for EMS in the in vivo MNT and
MutaMouse tests. Dose levels ranging from 1.25
to 260 mg/kg/day were applied for up to 28 days.
As reference we included ENU at doses of 1.1
to 22 mg/kg/day. The studies were further supported
by in depth metabolism and exposure analyses
and a general toxicity study in rats.
Our investigations showed that daily doses of
up to 25 mg/kg/day did not induce mutations
in the lacZ gene in the three organs tested
(bone marrow, liver, GI tract). Doses up to
80 mg/kg/day did not induce micronuclei in mouse
bone marrow. Only at higher dose levels the
genotoxic activity of EMS became apparent. The
thresholds were affirmed by statistical analysis.
For ENU no threshold was apparent.
High safety factors were calculated based on
the exposure and Cmax assessment in the treated
animals at NOEL versus the patients at their
respective exposure levels, providing reassurance
that they do not carry a risk for mutagenic
effects. Since genotoxicity is considered to
be at the base for other toxic effects of EMS
(carcinogenicity, teratogenicity), the patients,
consequently, also do not carry a risk for these
adverse events.
On a different level we claim that the presence
of a thresholded dose response allows to revise
the approach to qualification of EMS as genotoxic
impurity according to the PDE principle defined
in ICHQ3C.
B. Bhaskar Gollapudi, Ph.D.
The Dow Chemical Company
The ILSI-HESI Initiative on Relevance and Follow-up of Positive Results in in vitro Genetic Toxicity Testing (IVGT)
A battery of in vitro and in vivo
genetic toxicity tests has been a critical component
of the safety assessment of drugs, pesticides,
and chemicals for many years. It is generally
considered that results from in vitro
studies demonstrate the intrinsic genotoxic
properties of the test compounds. Accumulated
knowledge has shown that the rate of positive
in vitro tests, including the rate of
positive findings not confirmed in vivo
as well as false positives for non-carcinogens,
has been high. There is a sense of urgency on
the need to modify genotoxicity testing paradigms
so as to generate useful information not only
for hazard identification, but also for human
risk assessment. The Health and Environmental
Sciences Institute (HESI) of the International
Life Sciences Institute (ILSI) has launched
an initiative on IVGT with the mission of 1)
improving the scientific basis of the interpretation
of results from in vitro genetic toxicology
tests for purposes of more accurate human risk
assessment, 2) to develop follow-up strategies
for determining the relevance of in vitro
test results to human health, and 3) to provide
a framework for integration of in vitro
testing results into a risk-based assessment
of the effects of chemical exposures on human
health. The IVGT committee held its first workshop
in 2006 (Mutat. Res. 633: 67-79, 2007)
and a second one in 2007 with participation
of international scientists representing various
sectors (academia, industry, and the government).
Based on these workshops, two workgroups were
convened. The first group was charged with creating
a decision tree based on the 2006 IWGT framework
(Mutat. Res. 627: 41-58, 2007) that could
be applied in the case of in vitro positive
results to determine the appropriate follow-up
strategies that takes into consideration quantitative
approaches to data interpretation. A second
workgroup organized a workshop in May of 2008
to examine emerging technologies to improve
the prediction of mutagenic effects in humans;
the proceedings from this workshop will be published
in Environmental and Molecular Mutagenesis.
Jay I. Goodman, Ph.D.
Department of Pharmacology and Toxicology
Michigan State University
Genotoxicity and Carcinogenicity Testing: What Are We Doing and What Should We Be Doing?
There is a need to reconcile the well-established
role that mutagenesis plays in carcinogenesis
with the fact that not all carcinogens are mutagens
and the current view that non-mutagenic events
also underlie carcinogenesis. Progress can be
made towards resolving this apparent paradox
by considering the importance of epigenetic
alterations in the transformation of a normal
cell into a frank malignancy. The term epigenetics
refers to heritable mechanisms, e.g., DNA methylation
(5-methylcytosine content of DNA), histone code
and non-coding RNAs (e.g., miRNA) that are superimposed
on DNA base sequence and regulate transcription.
Thus inheritance should be considered on a dual
level. The transmission of genes (i.e., DNA
base sequence), in both the somatic sense and
from one generation to another, is distinct
from the mechanisms involved in the transmission
of regulated states of gene activity. Epigenetics
is a term used to describe the latter.
A compound is classified as genotoxic if it,
or a metabolite, can bind to DNA (Weisburger
and Williams, 1981). The discipline of genotoxicity
evaluation is focused, to an extent that appears
to be excessive, on employing tests, especially
combinations of different tests, for genotoxicity
as short-term predictors of carcinogenicity.
In my opinion, an inordinate amount of time
and money is being spent on looking for correlations
between carcinogenicity and genotoxicity. Carcinogenesis
involves more than mutagenesis. Yes, there is
a need to develop short-term tests that can
accurately predict the carcinogenic potential
of chemicals, and approximately 70% of the Ames
test positive compounds do turnout to be carcinogens
when subjected to the standard rodent bioassay.
However, the simple carcinogen equals genotoxin
(typically presumed to act as a mutagen) concept
is a failed paradigm. Thus, a continued emphasis
on trying to "make" all carcinogens
fit into the category of mutagens is actually
counterproductive and it tends to obscure current
insight regarding the biology/molecular biology
of carcinogenesis. Furthermore, it is instructive
to reflect on fact that while a compound might
test positive in a particular test, or combination
of tests, for genotoxicity this does not demonstrate
that it will, indeed, act as a mutagen in vivo.
In my opinion, science would be better served
if the discipline of genotoxicity refocused
its major effort away from attempts to use genotoxicity
as a short-term test for carcinogenicity and
towards discerning the true mutagenic potential
of chemicals. Specifically, there is a need
to improve our ability to evaluate the potential
of compounds to cause mutations in vivo, under
realistic conditions, e.g., rational doses and
routs of exposure.
This presentation will focus on genotoxicity
and carcinogenicity testing within the context
discussed above. The importance of rational
dose and route of administration selection for
carcinogenicity testing will be emphasized by
juxtaposing this issue with the fundamental
principle that dose influences mechanism and,
therefore, what happens at high doses does not
necessarily occur at low doses. Thus, an automatic
default to a linear, no threshold assumption
should be questioned. Additionally, the theoretical
and practical implications of the key role that
epigenetic changes play in carcinogenesis shall
be highlighted, especially with regard to enhancing
science-based safety assessment.
David Jacobson-Kram, Ph.D., D.A.B.T.
OND, CDER, FDA
Dr. Jacobson-Kram served as the VP of the Toxicology and Laboratory Animal Health Division at BioReliance Corporation, a contract testing laboratory from 1988 until 2003. Currently, he serves as the Associate Director of Pharmacology and Toxicology in FDA's Office of New Drugs. Over the past twenty years he has served as principal and co-principal investigator on several N.I.H. grants and government contracts and published widely in the areas of genetic and molecular toxicology.
Dr. Jacobson-Kram has served as council member, treasurer and chairman of the Genetic Toxicology Association, executive council member to the Environmental Mutagen Society, Editor of Cell Biology and Toxicology, and as a member of N.I.H. special study sections. In 1996 he became a Diplomate of the American Board of Toxicology (DABT).
Changes to ICH S2: Why They Are Needed, How They Will Help
Genotoxicity is seen only as a predictor of
carcinogenicity prior to drug approval; most
drugs undergo carcinogenicity testing but results
not available until NDA submission. Many people
(hundreds or thousands) including healthy volunteers
will have been exposed to repeated, pharmacologically
active drug doses. Thus, clinical trial participants
are potentially exposed to carcinogens but not
to genotoxic carcinogens. The effects of nongenotoxic
carcinogens are thought to have thresholds and
to be reversible. It has been shown in many
large survey studies that the in vitro mammalian
cell assays (mouse lymphoma and chromosomal
aberrations) have very high sensitivity for
identifying carcinogens but very low specificity
for identifying non carcinogens. When combined
with the Ames test, specificity drops to about
25%. in vitro mammalian cell assays consistently
give positive results in 25 to 30% of studies.
Positive genetox results are often the bases
for clinical holds and additional studies utilizing
resources and animals are generally required
to demonstrate a lack of risk to patients. The
revised guideline gives sponsors a choice of
performing two in vivo endpoints in lieu of
an in vitro mammalian cell assay. The revised
guidance preserves patient safety and will help
expedite development of important new drugs.
Michelle O. Kenyon
Pfizer Global Research and Development
Risk Management Strategies for Ames Mutagens
Due to its high correlation with rodent carcinogenicity, a positive bacterial mutagenicity result often permanently halts the development of pharmaceutical compounds for non-life threatening indications. Continuing the development of a bacterial mutagen requires additional testing in order to order to adequately assess human safety, adding both time and cost to the project. Consequently, screening assays for mutagenicity are often utilized during the early discovery stages of drug development in an attempt to avoid investing in drug candidates which ultimately prove to be mutagens in the GLP Ames assay. Despite the development risks and the high predictive value of mutagenicity screening assays, a small number of compounds are positive in the GLP Ames Assay later in development. We have 3 case examples of drug candidates which showed evidence of mutagenic potential in the GLP Ames assay, for which there was a desire to pursue development to obtain, at minimum, proof of mechanism in the clinic for non-life-threatening indications, assuming that the weight of evidence from further testing demonstrates that the compound is not likely a mammalian genotoxin. The Ames data as well as data from follow-up studies will be discussed. Questions about suitability of the follow-up strategy and perceived regulatory acceptance of that strategy will be posed.
Channa
Keshava, Ph.D.
National Center for Environmental Assessment,
Office of Research and Development
U.S. Environmental Protection Agency
Use of Genotoxicity Data in Mode of Action Analysis for Human Health Risk Assessment
The U.S. Environmental Protection Agency (EPA) released its 2005 Guidelines for Carcinogen Risk Assessment (Cancer Guidelines) and Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (Supplemental Guidance) which indicate increased susceptibility to cancer risks from early life exposure to chemicals with a mutagenic mode of action (MOA). The Supplemental Guidance recommends the application of age-dependent adjustment factors when a mutagenic MOA is determined even in the absence of chemical-specific data for early life exposure. If chemical-specific data are available to derive an adjusted cancer potency value, then those values are used to adjust the risk estimate. The Cancer Guidelines provide a general framework in which genetic toxicity data combined with other information can be used to assess whether a chemical identified as a carcinogen is likely to have a mutagenic MOA. The Guidelines recommend that available data should be evaluated in a consistent and transparent manner by characterizing the weight-of-evidence that a carcinogen is acting by a mutagenic MOA. Due to the increased focus on mutagenic activity in the induction of tumors, EPA is in the process of developing methodology to facilitate analysis of the available data which emphasizes a consistent characterization of weight-of-evidence to determine if mutagenic activity is the likely MOA for tumor induction. This methodology also considers information such as structure-activity relationships and dose-response information. A case study will be discussed. (Disclaimer: The views expressed in this abstract are those of the authors and do not represent the policy of the U.S. Environmental Protection Agency).
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Dan
D. Levy, Ph.D.
Food and Drug Administration, CFSAN
What Makes an In Vitro Positive Irrelevant? A Case Study with Eugenol
In a three test battery of short
term genetic toxicity tests, many compounds
are found positive only in the mammalian cell
assays but not in the in vivo micronucleus
or bacterial mutagenesis assays. Because many
of these compounds are not rodent carcinogens,
they are frequently labeled "irrelevant"
positives. Eugenol is one such compound.
Eugenol is an allyl benzene, a class of compounds
found in many plants used for food. Several
allyl benzenes have been tested for carcinogenicity.
Safrole is a moderately potent hepatocarcinogen,
estragole and methyleugenol were less potent
and eugenol was judged non-carcinogenic in mice
and rats. Allyl benzenes cause mutagenic DNA
adducts via a multistep metabolic activation
starting with cytochrome P45 activation followed
by addition of a sulfate group which leaves
to form a DNA-reactive carbonium intermediate.
They are generally Ames negative and positive
or equivocal in mammalian in vitro assays.
The detection of UDS or adducts in liver tends
to correlate with carcinogenicity. Elimination
of these compounds in vivo is relatively
rapid. Addition of sulfate or glucuronate to
the free hydroxyl group on eugenol may allow
for particularly rapid elimination. While there
are no studies that directly compare the kinetics
of eugenol, safrole, methyleugenol and estragole
metabolism the weight of the evidence suggests
that differences in in vivo metabolism
that are unlikely to be detected during in
vitro assays explain the differences in
carcinogenic potency. Detection of genetic damage
by eugenol in vitro rather than being
"irrelevant" is a useful signal of
genetic hazard can be more fully addressed by
the appropriate in vivo assays.
James T. MacGregor, Ph.D., D.A.B.T.
Toxicology Consulting Services
Genetic Toxicology Assessment: Balancing Regulation and Science
Regulatory requirements should employ state-of-the art testing technologies and be supported by the most current scientific information, but the need for validation and consensus-building for regulatory updates often delays adoption of advances in science and technology. When regulatory requirements for genetic toxicology testing were established in the early 1970's, the need for in vivo exposure-response information to support assessment of the risk of genotoxic damage in human exposure situations was clearly delineated. However, the lack of suitable in vivo testing methodologies at that time, coupled with early findings of a strong correlation between mutagenicity in vitro and the outcome of rodent cancer bioassays, led to a regulatory paradigm that relies principally on qualitative interpretation of in vitro tests and very limited in vivo testing. Although recent analyses of accumulated data have shown poor qualitative correlation between the outcomes of in vitro genetic toxicology tests and rodent cancer bioassays (with a particularly high incidence of outcomes of in vitro mammalian cell tests that are categorized as "positive" with agents that are subsequently found not to be carcinogenic in in vivo rodent bioassays), there has been little change in the strategy implemented in the 1970's. These findings support the earlier assumption that in vivo exposure and exposure-response information are needed to derive meaningful estimates of human risk. in vivo technologies that could provide a cost-effective approach to assessment of in vivo dose-response relationships are currently available, but the integration of genetic toxicology endpoints into routine repeat-dose toxicology studies will be necessary to achieve cost-effectiveness. Markers that can be assessed in both humans and laboratory species ("bridging biomarkers") are of particular importance because they can permit direct assessment of human risk via comparative interspecies studies. Potential strategies and cost implications for integrating available and emerging technologies into routine repeat-dose toxicology assessments to achieve multi-tissue analyses will be discussed. Endpoints discussed will include neutral reporter genes in transgenic animals; mutations in endogenous pig-a, hprt, and tk genes; micronucleus incidence, Comet assays, and oncogene mutations.
Martha
Moore, Ph.D.
US-FDA, NCTR
Overview of the Latest Recommendations for Interpreting Data from the Mouse Lymphoma Assay
The Mouse Lymphoma (MLA) Expert
Workgroup of the International Workshop of Genotoxicity
Testing (IWGT) has conducted a series of workshops
over the past several years. The first meeting
was held in Washington, D.C. in 1999. Subsequently,
meetings have been held in New Orleans, Plymouth,
Aberdeen and San Francisco. The MLA Workgroup
of the IWGT is comprised of experts from Japan,
Europe and the United States. The Workgroup
addressed three main issues of importance to
the assay. These include: (1) the conduct of
a data-based analysis upon which to base a final
recommendation for measuring cytotoxicity; (2)
the criteria for data acceptance and appropriate
approaches for data evaluation; and (3) the
issues related to the International Committee
for Harmonization recommended use of a 24 hr
treatment time (including the ability of the
assay to detect aneugens). This presentation
will provide an overview of these discussions
and include all of the recommendations from
the Workgroup. Recommendations include acceptable
ranges for mutant frequency, cloning efficiency
and suspension growth of the negative/vehicle
controls and on criteria to define an acceptable
positive control response. The recommendation
for the determination of a positive/negative
test chemical response includes both the requirement
that the response exceed a defined value [the
global evaluation factor (GEF)] and that there
also be a positive dose response (evaluated
by an appropriate statistical method).
Paul Nioi, Ph.D.
The Schering-Plough Research Institute
Prior to taking up his position within mechanistic and predictive toxicology, Dr. Nioi was a postdoctoral researcher, again with Schering-Plough. His research was conducted in Dr. Cecil Pickett's laboratory and focused on the molecular biology of the Nrf2 transcription factor and its role in regulating Phase II drug metabolizing enzymes.
Dr. Nioi has a Ph.D in molecular biology from the University of Dundee (UK) and a BSc. in pharmacology from the University of Edinburgh (UK).
Prediction of Non-Genotoxic Carcinogenesis in Rats Using Changes in Gene Expression Following Acute Dosing
Non-genotoxic carcinogenicity of chemicals
is currently routinely evaluated in 2-year rodent
bioassays. Therefore, the development of early
biomarkers for non-genotoxic carcinogenesis
would result in substantial savings in time
and expense. One possible approach to address
this issue is to use changes in gene expression
following acute dosing to predict whether or
not a particular test article is likely to cause
cancer in longer term studies. This talk will
review recent advances in the use of transcriptomics
to predict non-genotoxic carcinogenesis, discuss
the results of a small scale validation study
conducted at Schering-Plough and offer some
suggestions of avenues for future research.
James F. Rusling, Ph.D.
University of Connecticut
Chemistry Department
Electro-optical Toxicity Screening Arrays and Complementary Biocolloid LC-MS Method
While batteries of biological testing protocols exist that can provide good assessment and predictions of toxicity in the general population, new cost-effective procedures based on simpler biochemical systems that are arranged in biosensor formats are emerging that may be very useful for early toxicity screening. This paper describes biosensor arrays employing thin films of DNA and pure metabolic enzymes that show promise in predicting genotoxicity. In these arrays, the enzyme reaction is run in the DNA/enzyme film that acts as a nanoreactor to produce metabolites in close proximity to high concentrations of DNA. The rate of damage to DNA is taken as the genotoxicity endpoint. Formation of nucleobase adducts is detected in the measurement step by catalytic voltammetry, capillary LC-MS after hydrolysis of the DNA, or optically by incorporating an electrochemiluminescent polymer into the biosensor films. The most advanced format of the genotoxicity biosensors feature arrays that can contain many metabolic enzymes, such as cytochrome P450s. Either pure recombinant enzymes or microsomes can be used as enzyme sources. Arrays based on electrochemiluminescence can be read using a simple apparatus featuring a CCD camera. These arrays obtain relative genotoxicity data for a series of enzymes simultaneously. The same film preparation technology provides colloidal nanoreactor particles to obtain DNA-metabolite adducts for structural analysis by LC-MS and CE-LIF arrays.
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Maik
Schuler, Ph.D.*
Pfizer Global Research and Development
* Expert Panel Member
Marie Vasquez
Operations Director
Helix3, Inc.
The Comet Assay: Breaking with Tradition
With the recent S2(R1) modification of ICH
Guidelines for Genotoxicity Testing recommending
the comet assay as a follow up in vivo test
and published guidelines attesting to its "ease
of application",1 laboratories are rushing
to incorporate comet into their regular testing
strategy. However, the testing guidelines, design
and methodology that many are attempting to
apply to comet are based largely on tradition
and on historical data generated from less sensitive
tests with known carcinogens. Meanwhile, misconceptions
about the simplicity of the assay have resulted
in variability issues and the potentially inaccurate
interpretation of results by those less experienced
with using the assay in a true testing environment.
To appropriately and effectively take advantage
of the utility of the comet assay, traditions
must be re-evaluated and the best practice guidelines
may need to be rewritten.
References
1Tice, R.R. et al. Single cell gel/comet
assay: guidelines for in vitro and in
vivo genetic toxicology testing. Environ
Mol Mutagen. 2000; 35(3): 206-21.