ILSI Health and 

Environmental Sciences Institute

EPA Difficult 

Substances W

Alternative methods for EcotoxicitTesting 

11 September 20Michelle EmbryHESI 
Agenda for this talk

• Context• The ‘big picture’• Fish Embryo Test (FET)• Overview of several other new methods 

under development

• Eco-TTC (non-test method)• Conclusions
Context for Alternatives Development

Regulatory drivers

Cosmetics DirectiveAnimal Protection DirectiveREAChTox21 (regulatory and scientific)OECD Fish Testing Framework

Scientific drivers

Mechanistic ecotoxicologyAOPsEndocrine disruptionModeling mammalian toxicity

Societal drivers

Animal welfare pressure (3R’s approach)Increased fish use as an alt to higher vertebratesReflected in regulatory and scientific worlds

Regulato

Needs

S

Societal 

Needs
Need for Balance

• Science has pushed the development of these 

methods:

• A call to use new, more approaches in lieu of 

older, less informative test procedures (e.g., AOPs)

• A call to optimize resource use

• Societal pressures have led industry to develop 

alternative methods:

• A call to reduce animal use

• Regulatory acceptance / implementation is key

• Global commerce underscores the need for 

harmonization (e.g., OECD MAD)

• Issues arise when there are legal differences; 

what happens when methods are not accepted in certain jurisdictions? (e.g., issues with Cosmetics Directive, REACh, etc.)

Regu

Societal 
Why Alternatives Work is Important

From: van der Jagt , Munn, Tørsløv & de Bruijn. 2004. Alternative approaches can reduce the use of test animals under REACH. 

0.00% 10.00% 20.00% 30.00% 40.00% 50.00

Two generation reprotox

Developm. tox screening

Long-term fish

Developm. tox study

Carcinogenicity

Skin sensitisation

Short term repeated dose

Further mutagenicity

Short term fish

BioAccumulation (fish)

Long-term repeated tox

Subchronic tox

Acute oral tox

Acute inhalation tox

In vivo eye irritation

Acute dermal tox

In vivo skin irritation

Long-term bird

Estimated animal need in REACH for required end

(% total test animals needed)

Additional potenti

for fish testin

Missing: EDC testing
Movement Towards IATA

• IATA: Integrated Approaches to 

Testing and Assessment

• OECD Fish Testing FW Project 

an example

• General push towards 

consideration of pathwaybased approaches, integration of new technologies as necessary and when appropriate

http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)16&doclanguage=en
Adverse Outcome Pathways (AOPs)

Chemical

Properties

Receptor/Ligand Interaction

DNA Binding

Protein Oxidation

Gene 

Activation

Protein 

Production

Altered 

Signaling

Protein 

Depletion

Altered 

Physiology

Disrupted Homeostasis

Altered Tissue 

Development or Function

Lethality

Impaired DevelopmenImpaired Reproductio

Cancer

Toxicant

MacroMolecular

Interactions

Cellular 

Responses

Organ

Responses

Organism

Responses
Fish Life Stages and Testing

OECD 236 TGFET (Fish Embryo test)

OECD 203 TG 4 

ZF 2d 2d

Compliant with Dir.2010/63/EU definition 

for scientific purposes

Egg 

(unfertilised 

Oocyte)

Embryo

Hatching

Fertilisation

Sperm entry

through the 

micropyle

EMBRYONIC PERIOD LARVA 

Free swimming 

and feeding larva

Metamorphosis (differentiation of the 

dorsal and caudal fins)

Scaled body

ENDOGENEOUS FEEDING

(autotrophic)

EXOGENEOUS FEEDING

(heterotrophic)

resorption of yolk sac 

JUVENILE

Eleutheroembryo

“ Sac fry ” 

Yolk sac

OECD 210 TG ZF 32d 

Endpoints : lethality, growth, malformations

Endpoint : lethality Endpoint : leth
Fish Embryo Test (FET)

Alternative method to the 96h acute fish toxicity test (OECD TG 202005:

• German Federal Environment Agency (UBA) submitted the draft tes

“Fish embryo toxicity (FET) test” to the OECD Test Guideline Progra

• Used in Germany as a replacement for 48h fish effluent testing

2006:

• OECD established the ad hoc Expert Group on the FET. 

• Following expert meetings, OECD decided to perform a validation s

coordinated by EURL ECVAM and steered by a validation managem(VMG)

2009 – 2011:

• The validation study aimed to assess the transferability, intra- and i

reproducibility of the zebrafish embryo toxicity test (ZFET) as well adomain of chemicals tested

2013

• Validation study reviewed and endorsed by ECVAM in addition to th

to acute fish toxicity, approved at EU level for implementation

• FET became OECD TG 236
Fish Embryo Test (FET)

Parallel to the correspondencWide range of database(Currently ~300 cdifferent MOA anchemical propertIn practical term• 0.1 mg/L FE

fish

• 1 mg/L FET • 10 mg/L FET 

Belanger et al. 2013. Environ. Toxicol. Chem. 32: 1768-83
Fish Embryo Test (FET): Summary

• Correlation between FET and acute fish test is better t

fish tests between species

• Little difference between zebrafish and fathead minno

chemicals tested thus-far

• Already being submitted for registrations / dossier

s

• Use of OECD TG 236 data has not translated to accept

regulatory jurisdictions 

• Potential applications beyond acute testing...
Fish Embryo Test (FET) – chronic toxicity

• Work initiated by HESI to explore application / 

optimization of the FET to predict chronic toxicity

• Focus on OECD 210 TG (Fish Early Life Stage Test); m

frequently used bioassay to predict chronic toxicit

y

• Ongoing work to discover, characterize, and annota

AOPs and prioritize their development (CEFIC LRI, o

• Ultimate goal is to identify alternative test method

predict chronic toxicity in fish

Villeneuve et al. 2014. Environ. Toxico
Fish Embryo Test (FET) – other applications

EDC testing• Development of alternatives for in vivo EDC tests – emerging nee• In-line with current pathway-based efforts (AOPs)• Review paper on existing alternative methodologies (for fish and 

amphibians) 

• HESI committee is currently developing criteria for selection of r

chemicals to use in validating / developing new methodologies.

Scholz et al. 2013. Crit Rev in Toxicol 43: 45-72

Effluent testing• Effluent testing is the single largest source of use of aquatic verte

globally for a regulatory purpose and is a subject separate from chregulation

• FET has been used in Germany for effluent testing since 2005• How might alternative approached be utilized for effluent testing

FET, etc.
Bioaccumulation

• Increase in regulatory needs for bioaccumulation asse

e.g. UNEP Stockholm Convention on POP chemicals (2

Canadian Domestic Substance List (2006); EU REACH (

• Several strategies* for bioaccumulation testing (or wa

been published taking into account:

• Existing information

• Non-testing methods• Testing methods

*Examples:

ECETOC report (de Wolf et HESI in vitro workshop (WREACH guidance (ECHA 20OSIRIS project (Lombardo 
Bioaccumulation: in vitro metabolism

Loss via 

egestion

(kE

)

Loss via 

gills

(k2

)

Loss via 

metabolism

(kMET

)

Uptake via 

gills

(k1

)

Uptake via 

food

Accumulation of a chemical in an 

organism is the result of absorption, 

distribution, metabolism, and excretion 

(ADME).

• BCF models based on log 

screen chemicals for potebioaccumulative propertieaccount for “M” (i.e., biot

• Some QSAR models predic

rates based on chemical sBCF/BAFTM programme of 

• Only waterborne uptake

• BCF = k1 / (k

2 + kE + k

In v
In vitro Metabolism Ring-Trial

• Further evaluate in vitro methods (trout S9 and hepatocytes) used 

chemical biotransformation in fish in order to support OECD TG de

• As of April 2014, the HESI ring trial is part of the official OECD Test 

Programme Workplan [U.S. and European Commission are leads]

• Aims:

• Compare in vitro assay performance across participating 

• Compare predicted BCF values using the in vitro data to i

measured BCF values

• Provide recommendations for future use of these method

bioaccumulation assessments

• Provide data that can be used to validate modeled bioacc

predictions

• Provide information required to develop an OECD TG for 
Bioaccumulation: in vitro metabolism

REACH endpoint specific guidance – Bioaccumulation (ECHA, 2“In vitro methods have the potential to provide important data 

bioaccumulation assessments...These methods may become an 

part of future test strategies, but their applicability is currently to the lack of standardised protocols, limited validation based o

data sets. Further evaluation work is necessary before they can recommended for use within an ITS.

”
Gill Cell Line Ring Trial

Rationale:• Gill epithelia is the primary target and uptake site of water contamin• Damage to gill epithelia may reflect impaired functioning of the orga

and eventually death

• Cells tolerate exposure to simple buffers 

allows direct analysis of 

environmental samples

• Ongoing round-robin test of the RTgill-W1 cell line assay to predi

acute toxicity – led by Eawag (7 labs and 6 test chemicals)

• Follow-up to the CEFIC-LRI CellSens Project
Non-testing Approaches: eco-TTC Develop

Threshold of Toxicological Concern (TTC)

• An approach for establishing an exposure lev

chemicals, below which no appreciable risk thuman health and/or the environment is exp

• TTC proposes that a de minimis value for tox

can be identified for many chemicals, includ

those of unknown toxicity

• TTC was originally applied to assess chemica

food:

• Flavorings• Food contact materials• Pharmaceutical impurities
Environmental TTC Approaches (eco-TTC)

• De Wolf et al. 2005. ET&• Compiled aquatic toxicity 

• ECETOC EAT databas• USEPA fathead minn• EU Existing substanc• Utrecht guppy datab

• Binned by Mode of Actio• PNECs derived using EU p

Application Factors

• Eco-TTC of 0.1 μg/L for V

See also: Gross et al. (2010); Williams et al. (2011) for additional eco-TTC like approache
Benefits of an eco-TTC

• Maximizes resource use (animals, time, $$• Allows screening-level assessment for 

chemicals with little or no toxicity data• Groups without a QSAR (or no hope for one) 

polymers? UVCBs*?

• New chemistries• Difficult to test substances (?)

• Particularly useful for screening assessme

when production is low

• Supports read-across*Unknown or variable composition, complex reaction products, biological mat
HESI eco-TTC Project

• Led by the HESI Animal Alternatives in Environmenta

Risk Assessment Committee

• Scoping meeting held Feb 2014; multi-sector 

participation

• General strategy to explore this approach in a ste

wise manner developed

• Initial analysis is ongoing, utilizing readily-availab

databases (D. deZwart, RIVM) and industry data when made available

• Strong interest from multiple sectors / groups

• Exploring additional data sources and partne
Overall Conclusions

• Several new, alternative eco test methods available o

development

• Integration into an IATA approach is necessary• Stakeholder involvement early and often is key to 

application and adoption of methods

• Must work together to overcome barriers to develop

and use of alternative methods

See: Braunbeck et al., “Bottlenecks in the development and 

implementation of alternative experimental test methods in ecotoxicology” ALTEX, in press.
Questions?

membry@hesiglobal.org