ILSI Health and Environmental
Sciences Institute
1156 15th Street, NW
Second Floor
Washington, DC 20005
USA
1.202.659.3306 office
1.202.659.3617 fax
www.hesiglobal.org
Project Committee on the Relevance and Follow-up of Positive Results in In Vitro
Genetic Toxicity (IVGT) Testing
IVGT Pig-a Meeting
Thursday, April 26, 2012
4:30PM – 6:30PM (Eastern Time)
Action Report - DRAFT
Participants
Marilyn Aardema
Chinami Aruga
Jan van Benthem
Zoryana Cammerer
Xuefei Cao
Julie Clements
Laura Custer
Firouz Darroudi
Stephen Dertinger
Vasily Dobrovolsky*
George Douglas
Rosalie Elespuru
Mick Fellows
Bhaskar Gollapudi
Tsuneo Hashizume
Robert Heflich
Lya Hernandez
Masamitsu Honma
Alan Jeffrey
George Johnson
Peter Kasper
Michelle Kenyon
Tim Lawlor
Matt LeBaron
Elisabeth Lorge
David Lovell
Anthony Lynch
James MacGregor
John Nicolette*
Mike O'Donovan
Stefan Pfuhler
BioReliance Corp
Mitsubishi Tanabe Pharma Corporation
National Institute for Public Health and the Environment (RIVM)
Janssen (Johnson & Johnson)
NCTR
Covance Labs
Bristol-Myers Squibb
Leiden University Medical Center
Litron Laboratories
NCTR
Health Canada
FDA
AstraZeneca
The Dow Chemical Co.
Takeda Pharmaceutical Company Limited
US FDA/NCTR
RIVM
National Institute of Health Sciences
New York Medical College
Swansea University
BfArM
Pfizer Inc.
BioReliance
The Dow Chemical Co.
SERVIER
St George's University of London
GSK
Toxicology Consulting Services
Abbott
AstraZeneca R&D
Procter & Gamble
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ILSI Health and Environmental
Sciences Institute
Lynn Pottenger*
Dan Roberts*
Leslie Recio
Maik Schuler
Robert Smith
Willi Suter
Veronique Thybaud
Jan van Benthem
Bas-jan van der Leede*
Freddy Van Goethem
Paul White
James Whitwell
Kristine Witt
Li You
Errol Zeiger
*by teleconference
The Dow Chemical Company
Bristol-Myers Squibb
Integrated Laboratory Systems
Pfizer Global Research and Development
Covance Laboratories
Novartis AG
Sanofi research and development
National Institute for Public Health and the Environment (RIVM)
Janssen Pharmaceuticals
Janssen R&D (pharmaceutical companies of J & J)
Health Canada
Covance Laboratories Ltd
NIEHS/NTP
US FDA
Errol Zeiger Consulting
Summary of the Meeting
I.
a.
b.
c.
d.
Status of Interlab Trial
Study design, since April 2011
New study design element – use of pre-dose blood samples to eliminate outliers
Overview of chemicals studied to date
Going forward

Steve Dertinger presented the slides and led the discussion.

Roche are studying 6 dose groups (low dose range). George Johnson assisting – study
design.
II.
MNU Case Study
a. Lower LOEL using higher throughput scoring approach

Laura Custer presented the slides and led the discussion.

GSK ran low doses for MNU.

Estimate of inter-animal variability? NCTR – Fisher rat.

Phase III LOD was higher than Phase IV (magnetic bead). Steve Dertinger – encourage
development of historical controls.

Stefan Pfuhler – Comet data? Laura will have poster. Data from Aristolochic acid

Vasily Dobrovolsky – Ab or column move data around? Loss of mutant effect from Phase III
to Phase IV.
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ILSI Health and Environmental
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
Japanese – different method for magnetic enriching of RETs.
III.
OECD Guidance as a Goal
a. What should we be doing now to get our studies into better shape?
b. What should we be doing now to get queued into the system?

Robert Heflich presented the slides and led the discussion.

Pig-a is no longer an emerging technique – entering a different realm. IWGT developing Piga WG. Give advice to IWGT…for developing OECD guideline.

Maik Schuler – validation - 1) are we ready to initiate process, 2) if not, what do we need,
and 3) what is HESI’s role?

David Kirkland – what is regulatory need? Is this the scope? When and why did we first
start needing complimentary tests? Bone marrow MN was not working. Have to test liverspecific carcinogens, site of contact carcinogens – these are the compounds that you have to
test to support complimentary. If complimentary is not scope – what is it?

Jim MacGregor – Pig-a is first efficient way of measuring mutation. Here is an apical
endpoint that has high sensitivity. Cheaper and quicker. Need to describe what it misses.
Easy integration. Applicability to human.

Bhaskar Gollapudi – new guidance for crop protection materials in Europe – if you have
positive finding in vitro mutation assay, then you have to do in vivo mutation assay. Pig-a
could meet this need once there is a guideline. We are close to being ready for guideline.
Still need to establish – sequencing.

Bob Heflich – need to support mutagenicity of a particular substance – can do other
nucleated cells and sequence Pig-a gene.

George Douglas – You don’t have to be ready to put in a proposal. Can be proposal for
development “of” a proposal. Proposal has to go out through national coordinator
(regulatory channels – need to convince them). DRP – don’t need it and consider not doing
it. But need to have accepted and harmonized protocol for which you do your validation
studies. Comet assay validation not doing DRP. Validation mostly for replacement tests –
this is not a replacement test. High outlier frequency – what are those? Long manifestation
time – longer than bone marrow – why?

A lot of people here have OECD experience – have smaller group discussion. Have Steering
Team that can review recent guidelines and identify problems (in vitro MN, Comet) – can
have IWGT group look at protocol issues. Try to get more data between now and IWGT.
IV.
Summary
a. Conclusions
b. Data gaps that need to be addressed
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ILSI Health and Environmental
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
Lynn Pottenger – include low non-tumorigenic doses in addition to tumorigenic doses.

Is new Pig-a design sufficiently robust and sensitive for dose-response relationships? 28
day, 6/dose. Sample size 5/6 can pick up 2 SD over background with resolving power 80%
(David Lovell). If compare to other assays – has at least as much power as other assays.
Conclusions – not enough info yet.

Additional compounds?

V.
o
Organ-specific carcinogens. Negative genetoxins that are bone marrow toxins – can
strip off Pig-a. Cross linking agents. Large deletions – Topo2 inhibitors.
o
Job for a working group? Many groups asking Steve D for compound lists/ideas.
Formalize this as a WG? 30 chems tested in rat, 13 in mouse. Expected negative
compounds and target limitations.
o
Pfizer – tested etoposide and EMH and tested negative – but need to retest in new
design. Need enough to gain confidence and understand limitations. Need
consideration of negatives.
o
George Douglas – TG assay validation – not enough mutagens/carcinogens tested,
and negatives. If Pig-a is highlighting sensitivity – yes – need negatives. Proving a
negative is a challenge. Positives in literature – probably will be positive (Bob
Heflich). Confounders need to be tested (Azothiaprin – but it is also a mutagen).
Bhaskar Gollapudi – nongenotoxic carcinogens (induce tumors by peroxisome
prolif. for example) – gives confidence on sensitivity of assay. John Nicollette –
effect of stress on bleeding out rats. David Kirkland – look at ECVAM list, review
carcinogenicity data in literature – though this list was for in vitro tests, but list was
derived from what we know about in vivo tests.
Rosie Elespuru – test as many known human carcinogens. Get info on dosimetry. How
increased is sensitivity if you add more animals? ICH – open ended.
Adjourn
Hearing no additional discussion, the meeting was adjourned at 6:30PM.
________________________________
James Kim, Ph.D., DABT
Senior Scientific Program Manager, HESI
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