“Systematic approach to investigate outliers of the … · Nils Klüver, PhD 8th EPAA ANNUAL...
Transcript of “Systematic approach to investigate outliers of the … · Nils Klüver, PhD 8th EPAA ANNUAL...
Nils Klüver, PhD
8th EPAA ANNUAL CONFERENCE
Brussels, 16.11.2012
“Systematic approach to investigate outliers
of the fish embryo test to increase its
predictive capacity and applicability domain
for acute fish toxicity and beyond”
EPAA 3Rs Science Award 2012
EPAA industrial partner:
improve the fish embryo toxicity (FET) test to
foster international acceptance as alternative for
the fish acute toxicity test Replacement
Systematic Identification of FET outliers
Reduce uncertainties
Define or extend the applicability domain
Improve predictive capacity
Purpose and aim of the study
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Usage of fish for toxicity and safety evaluation
~200.000 fish per annum in the EU (2008)
most frequently used vertebrates in regulatory
ecotoxicology
REACH (>10t/a)
Plant Protection Products
Biocides
Veterinary Medicinal Products
Effluent Testing
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On a global level, about 3.000.000 fish per year are
used in the USA for effluent testing
Amount of fish test needed within REACH is expected to increase (~1.2 million fish per year)
Acute Fish Toxicity Test – OECD TG 203
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most widely used vertebrate toxicity test in
environmental risk assessment!
control increasing concentrations (≥ 5x)
96 h
7-10 fish per aquarium
takes ≥ 42 animals and overall 5 working days
screen for
mortality
Alternatives?
Fish embryos as alternatives?
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fish embryos up to the stage of independent feeding
are not considered as protected stages (DIRECTIVE
2010/63/EU)
similar as in-vitro
methods and can be
considered as
alternative test system
Fish embryos are already widely used in screening
approaches
FET has emerged as a potential replacement method
for acute fish toxicity testing
The fish embryo toxicity (FET) test
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modified after Lammer et al., 2009
24 h
48 h
72 h
96 h
FET is easy to perform and has a low cost!
Zebrafish embryo toxicity test (ZFET) is a
promising alternative for acute fish test
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Various studies have indicated a high correlation
of fish embryo and acute fish toxicity – similar
sensitivity
The FET is a mandatory component in routine
whole effluent testing in Germany since 2005
OECD draft guideline (2006)
ZFET validation study coordinated
by ECVAM (Phase 2 completed)
Lammer et al., 2009
transferability and very good intra-
and inter-laboratory reproducibility
Limitations?
ZFET outliers
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compounds with higher LC50 in fish embryos
compounds that do not provoke any embryo toxicity
Reasons for ZFET outliers
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Physico-chemical properties (e.g. lipophilicity,
volatilisation, water solubility, molecular size…)
Toxicokinetic (uptake, metabolism)
Toxicodynamic (mode of action)
Project outline
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ZFET Database
update
Data mining for acute fish
toxicity
Outlier
identification / understanding
Implementation and
communication
Embryo toxicity data for >590
compounds is already available
( – UFZ collaboration)
Outlier understanding
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ZFET – outlier testing
• Including potential modifications of exposure scenarios (e.g. closed glass vials, exchange of media, shaking, window, duration)
Chemical analytics and Metabolisation
• Determination of exposure concentration (24h intervals)
• Optional: Internal concentrations (time to reach equilibrium)
• Metabolic activation (include potential metabolites)
MoA related endpoints
• Neurotoxicity (behaviour)
• heart beat measurements
• Others?
Kühnert et al. submitted
3Rs beyond acute fish toxicity testing
Fish early life stage (FELS) – OECD TG 210
Bioaccumulation in fish – OECD TG 305
Endocrine disruption
Developmental Toxicity (human risk)
Acute systemic toxicity (human risk)
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Impact for various industrial sectors including
chemical, cosmetic and pharmaceutical industry
FET has a chance to be accepted in order to replace
the acute fish toxicity test
big impact on the use of fish within REACH!
Summary
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A ZEFT assay OECD draft guideline is available
OECD validation was established
similar sensitivities as acute fish toxicity test
ZFET database
Correlation analysis
Priority list of outliers
Adapting and improving the test system
ZFET decision tree
Suggestion to amend draft test guideline
Advantage of zebrafish embryos
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complexity of all physiological and morphological
interactions (closer to adult compared to cell lines)
Small size, high fecundity, optical transparent, easy
to maintain, low costs…
48 hpf
120 hpf
one cell stage
24 hpf
Generation of ZFET database and outlier
identification
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• Embryo toxicity data for 590 compounds is already available (L‘Oréal – UFZ collaboration)
• include data compilation of S. Belanger (P&G) and colleagues
ZFET
Database
update
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• search in publically available databases (Duluth database, US-EPA ECOTOX, ECHA,EFSA, eChemPortal)
Acute fish toxicity data
Generation of ZFET database and outlier
identification
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• Correlation and statistical analysis
• Definition of an outlier: arbitrary 10-fold difference in zebrafish embryo vs. acute fish toxicity and statistical analysis
• Priority list for outlier characterisation
Outlier identification
Generation of ZFET database and outlier
identification
Database and outlier identification
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Correlation analysis
Critical evaluation of original data for outliers
Hypothesis generation based on existing
knowledge for outliers (e.g. toxicokinetic,
MoA)
Priority list for outlier analysis
(reflecting potential main reasons)
List of ZFET outliers
Implementation and Communication
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Communication of results to national
members of the ZFET validation
management group, ECETOC , ILSI HESI
EUROECOTOX consortium
Defining applicability domain and
suggestions to amend test guideline
Animal Alternatives in Environmental Risk
Assessment Project Committee