Transcript Folie 1 - IAEMGS

IWGT Group 4
Improving in vivo genotoxicity testing- the link
to standard toxicity testing
Summary of discussion items, conclusions
and recommendations
Hans-Joerg Martus
Novartis Institutes for BioMedical Research
Basel, Switzerland
IWGT Working Group 4
• Andreas Rothfuss (Chair)
• Masa Honma (Co-chair)
• Hans-Joerg Martus
(Rapporteur)
• Marilyn Aardema
• Brian Burlinson
• Andreas Czich
• Sheila Galloway
• Shuichi Hamada
• Bob Heflich
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Jon Howe
Peter Kasper
David Kirkland
Makoka Nakajima
Mike O‘Donovan
Ulla Plappert-Helbig
Les Recio
Maik Schuler
Yoshifumi Uno
Group 4: Integration of genotoxicity into routine studies
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Topics to be addressed
1.
Combination of MN assay and Comet Assay into acute
studies
2.
Integration of the MNT into repeated-dose toxicity (RDT)
studies
3.
Integration of the Comet Assay into RDT studies
4.
Requirements on top dose for „integrated“ RDT studies
5.
Further tests suitable for integration (not covered due to
time constraints)
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Improving in vivo genotoxicity testing
• Animal welfare recommendations to reduce animal usage
– Reduce “false positive” rate in vitro
– Apply smarter testing strategies in vivo
• Options for improvement in vivo: Combination and
Integration
– Combination of acute assays into one stand-alone study.
– Integration of genotoxicity endpoints into repeat dose toxicity (RDT)
studies
• Chances for Improvement beyond 3R’s
– Improved genotoxicity risk assessment: Interpretation of genotoxicity
results in conjunction with toxicity data and toxicokinetics
– More efficient testing using new technologies
– Potential for resource savings (compound, manpower)
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Current Recommendations
• Linking genotoxicity tests to General Toxicity is not a new concept:
Integration of MN assays has been discussed since a while now…
– EMS workshop, MacGregor et al., EMM 25:328-337, 1995
– IWGT, Hayashi et al, EMM 35:234-252, 2000
- More recent guidances:
- REACh ITS (Integrated Testing Strategy)
• Integration of genotoxicity tests (e.g. MN assay, Comet Assay) into
repeat-dose tox studies, if scientifically justified
– ICH S2(R) draft guidance
• Integration of in vivo genotoxicity endpoints into RDT studies preferable,
if scientifically justified
• If more than one genotoxicity endpoint, incorporation into a single study
is preferred.
– ECVAM workshop on Animal reduction (Pfuhler et al., submitted)
• Integration of MN assay into RDT studies “should be standard if RDT
studies are foreseen for the test compound”.
• Combination of acute MN and Comet Assay studies into one study.
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Topic 1: Combination of MN assay and Comet Assay into acute studies
Example of study design (Covance)
Bone Marrow & Blood Micronucleus + Comet in 3 tissues
Dose 1 at 0 hrs
Dose 2 at 24 hrs
Bone Marrow
Micronucleus
Sampled 24 hrs
after 2nd dose
Dose 3 at 45 hrs
Blood
Stomach
Liver
Comet
Sampled 3hrs
after 3rd dose
Sample 48 hrs
Bleed:3 hrs
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Topic 1: Combination of MN assay and Comet
Assay into acute studies
Consensus statements:
– Combination of MNT and comet technically
feasible and scientifically acceptable as an
alternative to the separate assays
• Promising results obtained with (mostly) model
compounds
• 3d or 4d protocols equally acceptable
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Topic 2: Integration of MNT into repeat-dose
toxicology (RDT) studies (I)
Consensus statements:
• Integration of MNT into RDT is scientifically
acceptable
• There may be situations (e.g. severe bone
marrow toxicity) where an acute study is
preferable
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Topic 2: Integration of MNT into repeated-dose
toxicology (RDT) studies (II)
Consensus statements on „Additional early peripheral
blood sampling time point on day 4“
– Early sampling not routinely required but can help in study
evaluation if data show unsuitability of late sampling time point
– If result from terminal sampling is negative and marked
myelotoxicity is evident then the additional early sampling
timepoint may provide useful data
– Early sampling can be advisable to investigate erythropoiesisrelated effects
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Topic 2: Integration of MNT into repeated-dose
toxicology (RDT) studies (III)
Consensus statements on „Effect of Bleeding“
– Animals bled for TK or other purposes can be used for MN
analysis
• For rats above 9 weeks the current data suggest that bleeding might
not affect response to genotoxins as long as MN frequencies in
control animals are unaffected
• One example exists to suggest that for rats aged 5 weeks bleeding
might affect the MN response
• It it advisable to use minimal volumes and low frequencies when
withdrawing blood to minimize disturbance of erythropoiesis
– No data to indicate generation of false-positive results in rats
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Topic 3: integration of comet into RDT studies
Consensus statements
• Integration of comet into RDT studies
– Integration into RDT is considered scientifically acceptable
– Liver comet assay complements MNT in blood or bone marrow
in detecting in vivo genotoxins
– Practical issues need to be considered
• Cytotoxicity
– Data available so far indicate that cytotoxicity does not generate
increases or decreases in DNA migration
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Topic 4: Top dose in RDT study
Background information: Arguments discussed within ICHS2 revision
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Criteria to qualify a repeated dose test (≥ 2 weeks) as acceptable for option 2 (no or
positive in vitro mammalian test):
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Criteria to disqualify a repeated dose test for option 2:
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Maximum feasible dose (formulation)
Exposure plateau
Limit dose: 1000 mg/kg
Accumulation with repeated dosing
At least 50% of acute MTD
For aneugens and certain hematotoxic clastogens:
– 2 -4 day blood sampling from the multiweek study before substantial
hematotoxicity develops
– Acute in vivo test
MTD alone
Reduced exposure to parent drug with time ( 50%) (in that sex)
• Typically seen in rats, especially males, and attributable to enzyme induction; not
necessarily relevant to human; prevents maximal exposure to parent, although it does
provide exposure to metabolite
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Topic 4: Top dose in RDT study
Consensus statements
• MTD considered acceptable for in vitro negatives
• For in vitro positives (or no in vitro data) MTD is
acceptable in many cases such as:
– RDT MTD (or exposure) close (up to two-fold) to acute MTD (or
exposure)
– Estimated human exposure is lower by a large margin
• If deviating therefrom use proper justification
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Group 4: Overall conclusions
• Combination of MNT and comet is scientifically
justified for both acute and RDT studies
• Most recommendations are based on a limited
data set and need to be refined in the future
• Future prospects:
– Evaluate genotoxins with diverse modes-of-action
– Evaluate compounds with extrahepatic target tissues
(comet)
– Consider involving NTP for future experiments
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