Transcript Group 3

5TH INTERNATIONAL WORKSHOP ON
GENOTOXICITY TESTING
August 17-19, 2009
Topic 3: In Vitro Test Approaches with Better
Predictivity
Stefan Pfuhler (Procter & Gamble, USA)
ICEM Florence Aug 20-25, 2009
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Workshop participants:
Jan van Benthem (RIVM, The Netherlands), Rapporteur
Gladys Ouédraogo (L’Oreal, France)
Raffaella Corvi (ECVAM, Itraly)
Rodger Curren (IIVS, USA)
Kerry Dearfield (USDA FSIS, USA)
Azeddine Elhajouji (Novartis, Switzerland)
Mick Fellows (AstraZeneca, UK), Co-chair
Paul Fowler (Covance, UK)
Roland Frötschl (BfarM, Germany)
Ludovic Le Hegarat (Inserm, France)
Toshio Kasamatsu (KAO, Japan)
Hajime Kojima (JaCVAM, Japan)
Stefan Pfuhler (Procter & Gamble, USA), Chair
Andrew Scott (Unilever, UK)
Gunter Speit (Univ Ulm, Germany)
ICEM Florence Aug 20-25, 2009
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Background:
• High rate of misleading positive results in our current battery of in vitro
tests. As high as 80% when mammalian cell assays are combined (i.e.
Chrom Abs or MLA)
• 7th Amendment to EU Cosmetics Directive (3-test battery): Marketing
and testing ban on ingredients tested in vivo came into force March
2009
-> Valuable compounds may be unnecessarily discarded
• Chemicals: REACH (3-test battery)
-> misleading positive results will lead to unnecessary in vivo
follow-up tests
ICEM Florence Aug 20-25, 2009
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Presentations:
1) Which cell lines should we be using
• Paul Fowler presented results from the Colipa “false positives” project
which compares V79, CHO, CHL, TK6, HepG2, L5178Y, HuLy.
• Mick Fellows presented data generated in conjunction with the
micronucleus OECD guideline finalization
• Azeddine Elhajouji presented data generated at Novartis for comparison
of several cell lines
• Ludowig Le Hegarat presented data generated with HepaRG cells
2) Promising new approaches
• Rodger Curren presented micronucleus data generated in human 3dimensional skin models (Colipa 3D skin project plus additional data from
IIVS, PG, MatTek)
• Gladys Ouédraogo presented Comet assay data in 3-dimensional skin
models (Colipa 3D skin project)
• Hajime Kojima presented 3D Comet assay data generated by JaCVAM
ICEM Florence Aug 20-25, 2009
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Questions that were addressed:
Choice of cell line:
• Are there restrictions for the choice of the cells used for testing?
• Should there be recommendations for the choice of a cell line?
Can that be done now or are more data needed?
New approaches:
• How do we rate the status of the genetox assays performed with
3D human skin equivalents?
• What is the applicability domain of that assay?
• What data would the team like to see before considering this a
valid assay?
ICEM Florence Aug 20-25, 2009
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Resorcinol (3hr + 21hr, +S9)
9
**
***
120
p≥0.01
p≥0.001
***
8
7
% MnBi
6
100
***
V79 3+21 hours -S-9
CHL 3+21 hours -S-9
CHO 3+21 hours -S-9
HuLy 3+21 hours -S-9
HepG2 3+45 hours +S-9
TK6 3+21 hours +S-9
***
5
***
4
***
***
***
***
80
60
40
3
2
20
1
0
0
Solvent
Low
Medium
High
Dose level (toxicity)
Data presented by Paul Fowler
ICEM Florence Aug 20-25, 2009
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% survival (Replication index)
10
3D skin Models in Genotoxicity testing, results from
phase 2: Evaluation of Coded Chemicals
Three chemicals were sent coded from Covance UK to P&G, IIVS, L’Oreal. Each chemical
was tested in at least 2 independent studies in each lab. Results sent to ECVAM for
decoding and analysis
Compound
CAS
number
Vehicle
Results of
3D RSMN assay
P&G, IIVS, L’Oreal
Cyclohexanone
108-94-1
Acetone
-,-,-
Mitomycin C
50-07-7
Acetone
+,+,+
N-Ethyl-Nnitrosourea
759-73-9
Acetone
+,+,+
ICEM Florence Aug 20-25, 2009
Data presented by Rodger Curren
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Data presented by Rodger Curren
50
*
*
*
4
*
2
100
75
4
*
50
* *
*
0
0
5
10
15
0
20
5
10
15
4
**
**
50
**
*
**
2
*
0
0
20
48h
Concentration (mg/ml)
150
5
10
15
20
Concentration (mg/ml)
8
8
*
*
*
125
125
*
75
4
*
50
*
*
*
100
*
75
4
50
*
2
25
25
0
0
0
1
2
3
4
Concentration (mg/ml)
5
2
**
0
% MN
100
Relative % BN cells
6
6
% MN
Relative % BN cells
75
0
0
Concentration (mg/ml)
150
*
25
0
0
6
100
2
25
25
L’Oreal
72h
0
0
2
4
6
Concentration (mg/ml)
8
10
Open symbols are % binucleated cells; Closed symbols are % micronucleated cells
ICEM Florence Aug 20-25, 2009
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% MN
*
Relative % BN cells
*
% MN
100
8
125
6
6
75
*
IIVS
125
*
150
8
Relative % BN cells
P&G
125
Relative % BN cells
150
8
% MN
150
IWGT consensus statements
1.
Data were presented indicating that p53 compromised rodent
cell lines over-estimate genotoxic potential in the
micronucleus test. Therefore, IWGT suggests using p53
competent cells in in vitro MN- or CA-tests.
2.
It has been demonstrated that cell line stability and source
can affect the outcome of genotoxicity assays. Therefore
IWGT recommends to adhere to good cell practice,
characterize all new cells, check regularly for drift and work
from low passage stocks. A common genotoxicity cell bank
with fully characterized stocks of all cells would be useful.
3.
Genotoxicity testing in 3D human reconstructed skin (MN
test and Comet assay ) is a promising new in vitro test for
dermally applied chemicals
ICEM Florence Aug 20-25, 2009
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Statement 1:
Use of p53 competent cells:
• The data presented at the IWGT from the OECD MNvit test
showed that all cell types correctly identify clastogens and
aneugens.
• However, from the data presented on the “misleading
compounds” using the MNvit test there were clear differences
in the response of the various cell types.
• The group agreed that the above would also be applicable to
the CA based on the compatibility of both assays.
• HepaRG is a promising model as the cells appear to have
better phase I and II metabolizing potential than other cell lines.
However, further evaluation is required to confirm the value of
this model for genotoxicity testing
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Statement 3
Use of 3D human reconstructed skin in MN and Comet:
• The advantage of the model is that it resembles the properties of
human skin (barrier function, metabolism) and the route of
exposure is relevant for dermally applied chemicals (e.g.
cosmetics)
• The data presented show that the 3D skin Micronucleus assay is
further advanced and inter and intra-lab reproducibility has been
demonstrated.
• IWGT agreed that the Comet assay should be further evaluated.
The Comet assay is seen as a valuable addition as it is not
dependent on cell proliferation and covers a wider spectrum of
DNA damage.
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Statement 3
Further remarks:
• The metabolic capacity needs to be further evaluated (ongoing)
• It would be valuable to capture the kinetics of penetration and
toxicity in order to establish the ideal sampling time(s) for the
Comet assay
• Recommendations on the use of appropriate vehicles should be
established
• It was agreed that 3D genotoxicity models, once validated, will be
useful (at least) to follow up on positive results from standard in
vitro assays for dermally applied chemicals.
• The applicability domain will be established once the validation is
completed
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