Transcript ECVAM - IHCP

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Analysis of published data for top
concentration of cytotoxicity testing
in mammalian genotoxicity testing
J. Parry, E. Parry, P. Phrakonkham, R. Corvi
In vitro Methods/European Centre for the Validation of Alternative Methods
(ECVAM)
IWGT, Basel, August 2009
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European Centre for the Validation of Alternative Methods
ECVAM
• Validation
• Research
• Communication
EU Policy support
• 7th Amendment to
Cosmetics Directive
• REACH
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ECVAM key area
genotoxicity / carcinogenicity
Taskforce
Validation
Refine
Reduction
Reduce
Replace
Policy support (eg. REACH ITS)
Research support
CARCINOGENOMICS
COMICS
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Genotoxicity testing requirements for REACH
In vitro gene mutation study in bacteria
In vitro study
in mammalian cells (2 tests)
> 10t
if positive
In vivo somatic cell genotoxicity study
if positive classified as mutagen
1t-10t
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ECVAM Workshop
Italy, April 2006
Objectives:
1. Review data from currently available systems
2. Review the performance of new test systems
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ECVAM Workshop on false positives
Follow up activities
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Recommended lists of genotoxic and non-genotoxic chemicals for
assessment of the performance of new or improved genotoxicity tests:
A follow-up to an ECVAM Workshop
D. Kirkland, Mut Res, 653 (2008), 99-108
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Collaboration with COLIPA and NC3Rs on improvement of current in
vitro tests
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Analysis of published data for top concentration and upper limit of
cytotoxicity in mammalian cell genotoxicity testing.
(ECVAM commissioned this analysis to J. & L. Parry)
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Analysis of published data for top concentration of
cytotoxicity in mammalian genotoxicity testing
Objectives
• Review of existing data to determine whether concentration as 10
mM (or 5000 µg/ml) are needed to detect in vivo genotoxins and DNAreactive, mutagenic carcinogens using in vitro mammalian cell tests
or whether a lower level can be justified.
• Identified chemicals positive in mammalian cell tests at > 1mM
• Data from Ames tests were reviewed to see if chemicals would be
detected in other parts of the standard battery if high concentration
was needed to produce positive results in the mammalian cell tests.
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Analysis of top concentration of cytotoxicity testing in
mammalian genotoxicity testing
Screen Carcinogenicity Genotoxicity eXpert (CGX)*
database of rodent carcinogens
Consider chemicals with genotoxicity data
Data for CA, MLA, MNT, SCE, Ames
Focus on CA and MLA
Ames
Establish databases
*Kirkland et al., Mutation Research 584 (2005) 1-256
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Content of the databases
Whenever possible available original data were consulted in the original
publications
CA
• NTP DB
• Ishidate et al., 1988
• Additional publications
MLA
Most of original papers reviewed because of concerns about criteria
used for interpretation of data Mitchell et al., 1997
(for 2/3 chemicals original data reviewed)
• NTP DB
• Additional publications
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General Criteria used for building up the databases
• Follow the OECD and current Guidelines
• In cases were several publications available, most
reliable were taken into consideration, the study
that fulfilled current guideline criteria overruled the
other studies
• For positive results lowest effective concentration
was reported
• For negative and/or equivocal results highest
concentration available was reported
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Criteria used for interpretation of CA data
conservative approach
• Available raw data (i.e. NTP DB and additional
publications)
• Criteria used in the NTP considered
• Ishidate et al., 1988
– Authors conclusions reported
– Ishidate criteria for a positive call were
considered quite conservative, therefore adequate
for this analysis
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Criteria used for interpretation of MLA data
Applied to most of the chemicals reported
• Available raw data
For a positive call:
–
–
–
–
Exclusion of RTG data < 10%
Fulfil GEF
Dose-response or trend
Considered also datasets with negative controls with
lower mutant frequency than currently accepted, in case
other criteria were fulfilled
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Considerations related to this retrospective
analysis
• For the purpose of this analysis as many positive compounds as
possible were included
– Included equivocal compounds, when clear positive results were
available for those compounds
– MLA positive results with a negative control with low frequency
mutants
• A subset of data not checked in original papers
• Chemicals classified as carcinogens, classification as in vivo
genotoxins lacking
• In the long term analysis can be improved dependent upon
availability of further data
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Overview of data for CA and MLA
Chemicals with genotoxicity data
404
CA
338
+
197
–
105
MLA
228
i
36
+
148
–
44
i
36
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Distribution of concentrations which produced
positive results in CA and MLA
Positive concentration
(mM)
CA
n° of chemicals (%)
MLA
n° of chemicals (%)
≤1
139 (70.6%)
110 (74.3%)
1 to 10
46 (23.3%)
32 (21.6%)
>10
12 (6.1%)
6 (4.1%)
197
148
Total n° of chemicals
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Ames data for CA positive at 1-10 mM
Tot. n. chemicals = 46
2
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Ames +
Ames e
Ames 17
Chemicals positive at 1-10mM in CA
equivocal/negative Ames
mM
17
mM
Clofibrate
1.03
Methapyrilene hydrochloride
2.51
3-Chloro-2-methylpropene
Technical grade
1.32
N-Methylolacrylamide
2.94
Caffeic acid
1.4
Furfural
3.12
Furan
1.47
Methimazole
3.2
Phenylbutazone
1.63
Hydroquinone
4.1
2-Mercaptobenzothiazole
2.1
Methylphenidate HCl
4.63
Allyl isovalerate
2.11
Furosemide
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Ethionamide
2.4
3-(p-Chlorophenyl)-1-1dimethylurea (AKA monuron)
6.54
Styrene
2.4
alpha-Methylbenzyl alcohol
8.15
Chlorendic acid
2.5
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Ames data for MLA positive at 1-10 mM
Tot. n. chemicals = 32
4
16
Ames +
Ames e
Ames 12
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Chemicals positive at 1-10mM in MLA
equivocal/negative Ames
mM
mM
Methapyrilene hydrochloride
1.01
FD & C red 1 (Ponceau 3R)
3.44
Caffeic acid
1.11
Benzaldehyde
3.77
Chlorobenzene
1.11
Chlorendic acid
3.86
Benzofuran
1.27
Acetaldehyde
3.98
C.I. Direct blue 15
1.51
alpha-Methylbenzyl alcohol
4.1
Furfural
2.1
Furosemide
4.55
Toluene
2.44
Isophorone
5.79
Allyl isovalerate
2.81
Acrylamide
9.8
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Chemicals that require >1mM in mammalian cell tests
(CA or MLA) for a +ve response
Ames -/e
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CA +
16
MLA +
22
CA or MLA +
Ames -/e
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Compounds detected positive at >1mM in one test,
but positive < 1mM in the other
These compounds were removed from final list
•
•
•
•
•
•
Benzaldehyde
Acetaldehyde
Acrylamide
2-chloro-2-methylpropene
2-mercaptobenzothiazole
Hydroquinone
Chemicals that require >1mM in mammalian cell tests
CA
CA +ve (mM)
MLA +ve (mM)
Methapyrilene hydrochloride
2.94
1.01
Caffeic acid
1.4
1.11
Allyl isovalerate
2.11
2.81
Chlorendic acid
2.5
3.86
Furfural
3.12
2.1
Furosemide
6
4.55
alpha-Methylbenzyl alcohol
8.15
4.1
Chlorobenzene
1.11
Benzofuran
1.27
Furan
1.47
Phenylbutazone
1.63
C.I. Direct blue 15
1.51
Ethionamide
2.4
Styrene
2.4
Clofibrate
2.51
N-Methylolacrylamide
1.03
Methimazole
3.2
Isophorone
5.79
Methylphenidate HCl
4.63
3-(p-Chlorophenyl)-1-1-dimethylurea (AKA monuron)
6.54
Toluene
2.44
FD & C red 1 (Ponceau 3R)
3.44
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Initial evaluation on MNT
(not yet checked)
MNT 5
MNT
50
MNT +
45
Ames +
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Ames 19
All MNT +
at < 1mM
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Conclusions
Identified 22 (out of 404) chemicals that are Ames negative/e
and require >1mM in mammalian cell tests (CA or MLA) for a
+ve response
• Are these chemicals all relevant positive?
• Are they supposed to be picked up as genotoxic
carcinogens?
• How many irrelevant chemicals would we avoided if top
concentration reduced to 1mM?
• Work in progress, analysis can be improved in the future,
but let’s start discuss!!
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Aknowledgements
James M. Parry
Elizabeth Parry
Pascal Phrakonkham, ECVAM
David Kirkland, Kirkland Consulting