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5TH INTERNATIONAL WORKSHOP
ON GENOTOXICITY TESTING
Basel, August 17-19, 2009
Group 6
Follow-up
of in vivo positive results
• Follow-up of 2005 IWGT and subsequent work done by
HESI IVGT on “follow-up of in vitro positive results”.
• Takes into consideration the new data and information
available on dose-response curves and mode of action.
Participants of “In vivo follow-up” workgroup
Name
Region
e-mail
1
Jan Van Bentem
Europe, Netherlands
Regulatory
[email protected]
2
Ricardo Crebelli
Europe, Italy
Regulatory
[email protected]
3
Kerry Dearfield
USA
Regulatory
[email protected]
4
George Douglas
Canada
Regulatory
[email protected]
5
Peter Farmer
Europe, UK
Academic
[email protected]
6
Elmar Gocke
Europe, Switzerland
Industry
[email protected]
7
Baskhar Gollapudi
USA
Industry
[email protected]
8
Makoto Hayashi
Japan
Regulatory
[email protected]
9
David Lovell
Europe, UK
Academic
[email protected]
10
Werner Lutz
Europe, Germany
Academic
[email protected]
11
Takehiko Nohmi
Japan
Academic
[email protected]
12
Jim MacGregor,
Rapporteur
USA
Consultant
[email protected]
13
Daniel Marzin
Europe, France
Regulatory
[email protected]
14
Martha Moore
USA
Regulatory
[email protected]
15
David Phillips
Europe, UK
Academic
[email protected]
16
Lutz Műller,
Co-Chair
Europe, Switzerland
Industry
[email protected]
17
Véronique Thybaud,
Chair
Europe, France
Industry
[email protected]
Not able to attend
Tripartite group: three main regions, Scientists from academic laboratories, regulatory authorities and industry
5TH IWGT in Basel, 2009
Group 6: Follow-up of positives results
Topics:
Topic 1: Use of in vitro results in the design
and interpretation of in vivo assays
Topic 2: Quantitative aspects of the dose
response curve
Topic 3: Evaluation and impact of Mode of Action
Topic 4: Update on in vivo models
C*: agreement both on principle and wording
No C*: agreement on principle, wording to be refined (lack of
time).
5TH IWGT in Basel, 2009
Group 6: Follow-up of positives results
Topic 1
Use of in vitro results in the design and
interpretation of in vivo assays
• 2005 IWGT and IVGT flow charts
Kerry Dearfield
IVGT Review group: Flow chart for follow-up actions
In vitro “clear” positive results
Analyze the existing genotoxicity data.
1
No further testing.
Consider as
potentially
genotoxic.
No
2
Shown to be due to confounding factors?
e.g. culture conditions & interactions, cellspecific metabolism, impurities.
Yes
Conclude (by WOE
and/or MOA) of low
concern for human
risk associated to the
usage.
No follow-up
necessary.
No
Review all available data
for WOE and MOA:
- SAR, physico-chemistry,
ADME, PB/PK, etc.
5
Yes
Available
information
confirms the
concern.
No
6
Conclude (by WOE
and/or MOA
evaluation) of low
concern.
No follow-up
necessary.
3
4
Conduct additional testing
if data not yet available,
consider:
− in vitro genotoxicity tests to
confirm the results, and
assess the types of genetic
damage,
− assays to evaluate
possible interaction with
DNA (DNA reactive),
− assays to evaluate a non
DNA-reactive mechanism.
3
− Conduct in vivo
tests (selection
depending on
available data).
− Interpret in vivo
results in relation
to all available
information.
6
Evaluate the
in vitro
positive
results with
the in vivo
data.
No
Available
information
confirms the
concern.
5
Yes
Yes
Can the in vivo data
help improve WOE
and assessment of
the potential risk for
human associated to
the usage?
Conclude as
genotoxic.
No follow-up
necessary.
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 1: Statements
• The endpoint studied in vivo needs to be appropriate, e.g.,
the same as that found increased in vitro or a surrogate
shown to be appropriate for predicting that same endpoint.
It is recognized that some systems are not specific to a
single endpoint, and this must be taken into account. C*
• The selection of tissue(s) should consider tissue exposure,
ADME information compound metabolism, and other
toxicological considerations. C*
• If in vitro test(s) are positive, and in vivo tests are
conducted, the risk can be considered to be negligible if
follow-up test(s) in appropriate tissues and endpoints in
vivo at appropriate doses show that the in vitro results are
probably not of concern in vivo. C*
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 2: Quantitative aspects of the dose
response curve
• Viracept case study:
Genotoxicity data in animals, dose-response curves and statistical
assessment, and risk assessment for the human exposure.
Elmar Gocke and Lutz Müller
• Quantitative aspects of in vivo risk and the potential role of
new flow cytometric genetic toxicity assays
Jim MacGregor
• Analysis of dose-response curves (threshold, NOEL, etc)
Werner Lutz
• Potential modifiers of dose-response curves. Could
additional effects (e.g. induction of cell proliferation) lower
the apparent threshold?
Véronique Thybaud
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 2: Statements
For risk assessment – in vivo follow up of positives (after
review of Viracept data):
• Transgenic animals are acceptable surrogates to internal genes
for risk assessment. C*
• For risk assessment, internal dose is a key element, and may
include:
– Cmax/AUC
– Surrogate endpoints, DNA/protein adducts.
• Nonlinear response curves can be shown in vivo, even with
DNA-reactive agents, but must be demonstrated with appropriate
data.
– Statistical scrutiny must be applied.
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 2: Statements
• Hazard-screening is sufficient for public health protection if current
screening tests (in vitro mutation and chromosomal aberration plus
in vivo chromosomal damage) are negative, but when in vivo
genotoxicity is identified then additional in vivo data are necessary
to define risk in relation to exposure.
• Dose and exposure metrics must be justified in each situation.
• Cross-species extrapolation should consider the same factors used
for other toxicity endpoints:
e.g., relative metabolism, PK differences, surface area scaling, plus
DNA repair differences and relative apoptosis efficiency. C*
• Secondary factors (i.e., potential modifiers of dose-response
curves) such as cell proliferation may modify mutagenic responses,
therefore risk assessment must take this into account. C*
– The impact of the secondary mechanisms) may be tissue specific. C*
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 2: Statements
• Agents documented to induce genetic damage via
interaction with non-DNA targets are expected to exhibit a
“threshold” below which damage does not occur.
– For such “threshold” mechanisms, the NOGEL is appropriate metric
to which additional safety margins may be applied.
• For “DNA-reactive” genotoxicants:
– DNA primary damage can be used for exposure, while stable
mutations should be used for risk assessment.
– Some agents may exhibit a [“practical threshold”]: i.e., a dose
below which exposure does not add significantly to background
rates of DNA damage.
– Additional data are necessary to determine if generalizations can
be made to define “practical thresholds” of concern for
genotoxic/mutagenic damage.
– Consensus is needed about appropriate analytical (statistical)
methods of defining thresholds and risk levels (e.g., margin of
exposure).
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 3: Evaluation and impact of Mode of Action
• Genotoxic versus non-genotoxic mechanisms: feed-back
from Genotoxic and Carcinogenic Thresholds in Tokyo.
Takehiko Nohmi
• Use of in vivo mutation data to inform MOA for cancer.
Martha Moore
• Examples of species, tissue and high dose specific
effect, and mechanistic studies.
Daniel Marzin
• Evaluation (and regulation) of in vivo positives with
adequate negative carcinogenicity data: example a
substance recently evaluated in an European regulatory
body.
Riccardo crebelli
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 3: Statements
• The better the information and data (e.g., MOA)
the more certainty you will have in interpreting
the dose-response curve. This leads to less
uncertainty when determining an acceptable
exposure level. C*
• In case of MOA analysis and extrapolation to
human:
– All data should be used, not only genotoxicity data.
– In vitro studies may be designed to address
mechanistic questions, and aid extrapolation to
human.
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 3: Statements
• Mode of action of individual compounds is decisive
for risk assessment.
• Mechanisms underlying the shape of the doseresponse curve should be investigated more
thoroughly in vitro and in vivo.
• Genotoxicity should be examined in target organs of
chemical carcinogens, using the same species and
strains, when possible.
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 3: Statements (may need more work)
• In vivo mutagenicity can be associated with adverse
effects other than cancer, and this endpoint warrants
consideration in risk assessment.
– Negative carcinogenicity data may not give by default
reassurance on the lack of genotoxicity in vivo for in vitro
genotoxins.
– For in vitro genotoxins the possibility of somatic (and eventually
germ cell) effects in vivo is to be considered, even in presence of
negative carcinogenicity data.
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 4: Update on in vivo Models
•
Current status of F344 gpt delta rats for in vivo
mutagenesis.
Takehiko Nohmi
• Comparison between transgenes and endogenous
genes.
George Douglas
5TH IWGT in Basel, 2009
Follow-up of in vivo positives results
Topic 4: Statements
• TGR assays provide data of comparable quality and
predictivity for carcinogenicity compared to other
standard mutagenicity tests.
• They can be used effectively to follow up results of
other in vivo tests, (as well as in vitro tests).
• TGR assays fill a need in current regulatory
practices (e.g. in vivo follow-up).
• Need to continue development of in vivo assays,
esp. multi-endpoint, multi-species assays (including
Human).
• Promising assays include:
– Pig-a, flow cytometric micronucleus assays
– gptΔ rats and mice.
5TH INTERNATIONAL WORKSHOP
ON GENOTOXICITY TESTING
Basel, August 17-19, 2009
Thank you!