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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. Andreas Rothfuss (Chair) Masa Honma (Co-chair)

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iwgt group 4

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

Jon Howe

Peter Kasper

David Kirkland

Makoka Nakajima

Mike O‘Donovan

Ulla Plappert-Helbig

Les Recio

Maik Schuler

Yoshifumi Uno

IWGT Working Group 4

Group 4: Integration of genotoxicity into routine studies

topics to be addressed
Topics to be addressed
  • Combination of MN assay and Comet Assay into acute studies
  • Integration of the MNT into repeated-dose toxicity (RDT) studies
  • Integration of the Comet Assay into RDT studies
  • Requirements on top dose for „integrated“ RDT studies
  • Further tests suitable for integration (not covered due to time constraints)

Group 4: Integration of genotoxicity into routine studies

improving in vivo genotoxicity testing
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)

Group 4: Integration of genotoxicity into routine studies

current recommendations
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 workshopon 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.

Group 4: Integration of genotoxicity into routine studies

slide6
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

Blood

Dose 3 at 45 hrs

CometSampled 3hrs after 3rd dose

Stomach

Liver

Sample 48 hrs

Bleed:3 hrs

Group 4: Integration of genotoxicity into routine studies

topic 1 combination of mn assay and comet assay into acute studies
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

Group 4: Integration of genotoxicity into routine studies

topic 2 integration of mnt into repeat dose toxicology rdt studies i
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

Group 4: Integration of genotoxicity into routine studies

topic 2 integration of mnt into repeated dose toxicology rdt studies ii
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

Group 4: Integration of genotoxicity into routine studies

topic 2 integration of mnt into repeated dose toxicology rdt studies iii
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

Group 4: Integration of genotoxicity into routine studies

topic 3 integration of comet into rdt studies
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

Group 4: Integration of genotoxicity into routine studies

topic 4 top dose in rdt study background information arguments discussed within ichs2 revision
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

Group 4: Integration of genotoxicity into routine studies

topic 4 top dose in rdt study
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

Group 4: Integration of genotoxicity into routine studies

group 4 overall conclusions
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

Group 4: Integration of genotoxicity into routine studies

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