“Systematic approach to investigate outliers of the … · Nils Klüver, PhD 8th EPAA ANNUAL CONFERENCE Brussels, 16.11.2012 “Systematic approach to investigate outliers of the

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!

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Thank you for your attention!

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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

Collaboration with L‘Oréal

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Hypothesis for outliers Priority list of outliers

Chemical analytics

Discussion of results

Feasibility

ZFET decision tree

Dr. Marc Léonard