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I am Anita Bigger, an Expert Regulatory Pharmacologist in the area of
genetic toxicology in the Division of Antiviral Products in the CDER at
the FDA.

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This is a joint presentation with major input from Dr. Kerry Dearfield,
Scientific Advisor for Risk Assessment with the USDA and Dr. Michael
Cimino, Genetic Toxicologist with the USEPA. Both Dr. Dearfield and Dr.
Cimino were unable to attend this Public Hearing but wished to have
their opinions conveyed at the hearing.

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I'll briefly summarize our expertise. Between the three of us, we have
over 80 years experience in the field of genetic toxicology. I spent 18
years performing basic research into mechanisms of mutagenicity and
carcinogenicity and was a study director for mammalian cell mutation
assays. I joined the FDA in 1993. I was CoChair of the CDER Genetic
Toxicology Committee from 1994 to 2006 and participated in the creation
of the current S2 guidances.

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Kerry Dearfield performed basic research on the use of in vitro and in
vivo genetic toxicology assays to study DNA damage by cancer therapeutic
drugs. He was at the EPA for 21 years before joining the USDA in 2005.

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Michael Cimino spent 12 years performing basic research in cytogenetics
and was a study director for in vivo cytogenetics tests. He has been
with the EPA for 24 years. Both Dr. Dearfield and Dr. Cimino were major
contributors to the development of the EPA mutagenicity test battery and
are currently contributors to the EPA Mutagenic Mode of Action
Framework. Both are internationally recognized experts in genetic
toxicology and are often invited as expert peer reviewers for
genotoxicity issues.

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Short term genetic toxicology tests reached prominence in the 1970s.
Throughout the 1980s batteries of tests evolved. By 1991, candidate
assays for a battery had been winnowed down to a small group. An EPA
working group proposed a three test battery consisting of the Ames
assay, an in vitro gene mutation assay that could be the mouse lymphoma
assay or the in vitro chromosomal aberrations assay and, the 3rd
element, an in vivo rodent cytogenetics assay. This working group
consisted of Kerry Dearfield, Michael Cimino, Angela Auletta and Martha
Moore and I'd just like to point that three of this group are
participating in this hearing.

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In 1997, the ICH S2B guidance was finalized. This guidance recommends
the same three test battery.

In 2006, Michael Cimino reported in his overview that the same three
test battery was being used in regulatory agencies worldwide.

In 2008, the focus of the revised ICH S2 guidance is small molecule drug
substances and does not include biologics as defined in the ICH S6
guidance. This is an unreasonable limitation, since most chemicals will
be tested by the same batteries regardless of their ultimate use.

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Option 1 is the same as the current battery with certain exceptions such
as the addition of the in vitro micronucleus assay and lowering of the
limit dose for the in vitro mammalian cell assays and change in the
cytotoxicity level in the mouse lymphoma assay. The last two changes
will lower the sensitivity of the in vitro mammalian cell assays.

Option 2 is different. It consists of an Ames test and two in vivo
tests.

This option eliminates the in vitro mammalian cell assays. These are the
assays that pick up the broadest range of endpoints. Removing these
assays will limit hazard ID

This option is likely to increase animal use and takes the battery in
the opposite direction of the Replacement intention of the 3Rs to find
alternatives to animal testing. This is very important in the EU and
also important in the US.

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In addition, the options for in vivo testing are severely limited. The
in vivo chromosomal aberrations assay and the in vivo micronucleus assay
are the current validated tests. They are similar, but not identical, in
endpoint, as the micronucleus assay can detect aneugens plus clastogens
but may not detect all types of chromosome aberrations (e.g.
translocations).

This slide lists other in vivo tests. Test guidelines exist for tests
listed in the first 6 bullets. Please note the highlighted portions of
the text that indicate that many of these are now considered irrelevant
or insensitive or are not commercially available.

The 7th small bullet in that list addresses Other tests, such as the
Comet assay and transgenics that are without generally-accepted
guidelines and thus are not of immediate value.

The problem of available in vivo tests is exacerbated by the fact that
there are a limited number of target organs validated for analysis in
the in vivo tests. (Bone marrow, peripheral blood, testicular cells and
liver are validated.)

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This slide addresses how aneuploidy is handled in the revised guidance.

The revised S2 guidance states that Option 1 does not include a required
independent test designed specifically to test for aneuploidy. This is
true because tests such as the in vivo and in vitro chromosomal
aberrations assays and the mouse lymphoma assay do not directly detect
aneuploidy. Polyploidy, detectable in the in vitro chromosome
aberrations assay, is not aneuploidy. It is important to be able to
detect aneuploidy and a requirement for testing for aneuploidy should be
added to Option 1.

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Why is the same battery used throughout the world?

It works the way it was designed to work. The tests in the battery cover
the full range of genetic damage possible in humans.

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Why is this battery being replaced in the revision of the ICH S2?

The driving force for this revision is the perceived high level of
"false positives" generated by the in vitro mammalian cell assays. In
this case, false positive refers to the lack of a correlation with the
results of rodent carcinogenicity bioassays.

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CDER has always regulated exclusively on a drug's potential to induce
cancer and has used the genetic toxicology tests early in drug
development as a surrogate to predict carcinogenicity. It is often
stated that many drugs have been put on clinical hold because of "false
positive" findings in the in vitro mammalian cell assays. However, it is
important to note that the data to support this statement are
collectible, granted not easily so. However, the data have not yet been
collected, much less analyzed. This should be done before major changes
are made to the current battery.

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How is the reasoning behind the revision of the ICH S2 flawed?

The battery was designed to detect mutagenic activity, not carcinogenic
activity. We have known since the 1980s that the concordance between
battery and rodent bioassay is not impressive. Therefore, it's not a
surprise that Kirkland and coauthors reached a similar conclusion in a
2005 publication.

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The use of the battery to predict only a drug's carcinogenic potential
ignores the potential for mutagenic compounds to cause heritable
mutations and to contribute to other adverse health conditions. The
current battery captures the full range of mutagenic endpoints to
predict all mutagenic adverse effects. This will not be the case if the
ICH S2 revisions are adopted.

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Problems in the details in the revised guidance. I'll summarize these
briefly.

There seems to be little advantage to eliminating the concurrent
positive control without activation when the activated study is
conducted at the same time in the in vitro assays.

The use of only one sex in the in vivo genotoxicity tests should only be
allowed after sex-specific differences in response to the drug have been
thoroughly ruled out.

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Elimination of the concurrent positive control for every in vivo assay
once the lab has proven its competency can be problematic if there are
new issues in the lab (new technician, new source of animals, new batch
of vehicle), plus this violates existing OECD and EPA guidelines.

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For the in vitro micronucleus test, the methods for measuring
cytotoxicity are controversial as the choice may lead to possible
lowering of the upper limit of dosing which will affect the sensitivity
of the assay. US approval is pending

Micronuclei may be induced by changes in temperature without the
administration of a genotoxic agent. However, as long as the environment
is controlled and animal care is appropriate, chemical-induced changes
in body temperature in the in vivo micronucleus assay do not invalidate
a positive result of the test since the chemical has caused both
effects.

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First, we believe that improved interpretation of battery results can
resolve the issue of "false positives" in the current battery. A number
of efforts have provided guidelines for interpretation and follow-up
strategies. These include a CDER guidance and the consensus reports from
two IWGT working groups.

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Other efforts are ongoing, including WHO/IPCS and ILSI/HESI working
groups.

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In addition, a number of CDER reviewers believe that these efforts have
resulted in improved interpretation of battery results and that clinical
holds have been reduced. Tim Robison spoke of the experience in his
division in his earlier talk at this public hearing.

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A recent publication by Francis Collins outlined the National Toxicology
Program for the 21st Century that is studying the possibility of using
high-throughput and computational toxicology approaches. We recommend
retaining the current battery while working to incorporate molecular
tests to detect mutagenicity as suggested by the National Toxicology
Program for the 21st Century.