IWGT Group 4

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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

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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

• Jon Howe
• Peter Kasper
• David Kirkland
• Makoka Nakajima
• Mike ODonovan
• Ulla Plappert-Helbig
• Les Recio
• Maik Schuler
• Yoshifumi Uno

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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 3Rs
• 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 25328-337,
  1995
• IWGT, Hayashi et al, EMM 35234-252, 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• ICH S2(R) draft guidance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 studiesExample of study design
(Covance)
Bone Marrow Blood Micronucleus Comet in 3
tissues
Dose 1 at 0 hrs
Bone Marrow MicronucleusSampled 24 hrsafter 2nd
dose
Dose 2 at 24 hrs
Dose 3 at 45 hrs
Blood
CometSampled 3hrs after 3rd dose
Stomach
Liver
Sample 48 hrs
Bleed3 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
  erythropoiesis-related 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 studyBackground
information Arguments discussed within ICHS2
revision

• Criteria to qualify a repeated dose test ( 2
  weeks) as acceptable for option 2 (no or positive
  in vitro mammalian test)
• 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
• Criteria to disqualify a repeated dose test for
  option 2
• 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