introduction to alternatives to animal testing in toxicology

Center for Alternatives to Animal Testing (CAAT)

Johns Hopkins University, Baltimore, USA – University of Konstanz, Germany

Francois Busquet, PhD

Introduction to General Toxicology

WHAT IS MY BACKGROUND?

•Toxicologist - 10 years•Alternative test methods•Regulatory affairs•Ecotoxicology

•PhD with Zebrafish egg model for teratogenicity screening test (humans) – Private sector•Post-Doc with Zebrafish egg model for ecotoxicology (environment) – Government

WHO I WORK FOR?

1981 2010

WHAT IS TOXICOLOGY ABOUT?

TESTING PRODUCT SAFETY

PROTECTION FOR

TOXICOLOGY DOGMA

Paracelsus “Everything is poisonous, only the dose makes the poison” – XVIth Century

• How much? e.g. Dose• How long? e.g. Time Exposure• When? e.g. Critical Window• Which effects? e.g. Lethality

TOXICOLOGY PRINCIPLES

WHAT ARE THE COMMON IN VIVO MODELS?

HUMAN ENV

WHAT ARE ALTERNATIVES TEST METHODS?

3Rs

REPLACEMENT

REDUCTION

REFINEMENT

WHY USING IN VITRO MODELS?

ANIMAL WELFARE DILEMMA

PREDICTIVITY

STATISTICS

TG 203 TG 236

7 FISH X 6 GROUPS = 42 20 FISH EGGS X 7 GROUPS = 140

LOW COST & TIME

=X

10 millions US dollars + 5 years time

X =

10,000 US dollars + 2 weeks vs. 6 months

EU REGULATION

+ =+

=++

EU REGULATION

+ =

=

+

++ =

TRENDS IN US

An atmosphere of departure in toxicology 2007

New technologies from biotech and (bio-)informatics revolution

Mapping of pathways of toxicity (PoT)

NAS vision report Tox-21c

Tox-21c Reg. Outputs

EDSP21 =

in vitro HTSin silico

Full replacement

for Tier 1

EDSP20

STATUS OF IN VITRO MODELS ACCEPTANCE IN

EU

EU Status – ACCEPTED

EU Status – ACCEPTED

EU Status – ON GOING

EU Status – ON GOING

EAWAG – INSTITUTE

VIDEO DEMONSTRATION AT EAWAG

- SWITZERLAND

ZebraFish Danio rerio Embryo

as an Alternative Method for Eco and Human toxicology

WHY EVERYBODY WANT TO WORK WITH ZEBRAFISH

EGGS?

•Easy maintenance and mating•High Fecundity rate = laying down transparent eggs all year long •Embryological devt is fast (96h) and well described (Kimmel et al. 1995)

•Low cost model•Genome sequenced•GFP•HTP transposable•A key model in Germany Nagel, 2002•Regulatory RELEVANT•Academic model

ZEBRAFISH HANDLING & CARE IN THE

LABORATORY

ZEBRAFISH ANATOMY

• Male & Female• Age / Life Span / Size

ZEBRAFISH REPRODUCTION

• Approximate generation time: 3-4 months

• Spawning interval: 2-3 days• Up to 400 eggs per week• Eggs: non-adherent and fully

transparent• Rapid embryonic development: 3

days = precursor organs(ideal 28.5° C, tolerated: 23 – 33° C)

•Water quality (Cl, NO32-)

•Number of fish•Size of the fish tank•Food 24/7•Water renewal•Physico-chemical properties •Cleaning

ZEBRAFISH HUSBANDRY

ZEBRAFISH AQUARIUM

• Male/Female ratio• Mass spawning vs. spawning unit• Spawning traps• Stimulus - plant• Light onset• No stones!• Live Food!

ZEBRAFISH EGG PRODUCTION & COLLECTION

Acute toxicity in fish (Replacement)

Testing of WW

Developmental Toxicity (Teratogenicity)

Safety Pharmacology

Mammalian Organotoxicity

General Model for vertebrate developmental biology Genomic

ResearchDrug Discovery

ZF AS A UNIVERSAL MODEL

Acute toxicity in fish (Replacement)

Testing of WW

Developmental Toxicity (Teratogenicity)

Safety Pharmacology

Mammalian Organotoxicity

General Model for vertebrate developmental biology Genomic

ResearchDrug Discovery

ZF AS A UNIVERSAL MODEL

ZF Developmental Stages (26° C)

200 µm

8 cells stage

1 hpf

Yolk sac

200 µm

12 hpf

Somites

Eye

Head

Chorion

ZF Developmental Stages (26° C)

24 hpf

Eye

Sacculi/Otoliths

Taildetached

Cerebellum

Notochord

Brain

Heart cavity

Yolk

ZF Developmental Stages (26° C)

48 hpf Sacculi/Otoliths

Pigmentation

Spinal chord

ZF Developmental Stages (26° C)

Sacculi/Otoliths

Heart

Fin Spinal chord

72 hpf

Intestine

ZF Developmental Stages (26° C)

Beginning coagulation

1 h 24 h

Normal

Lethal effects

48 h

E = eye; S = somites; Ch = chorion; C = chorda; TD = tail detached; TND = tail not detached

E ECh

SC TD

C

Ch

Lack of somites Tail bud not detached

ETND

S

phot

os p

rovi

ded

by J

. Bac

hman

n, U

BA

, Ber

lin

Acute toxicity in fish (Replacement)

Testing of WW

Developmental Toxicity (Teratogenicity)

Safety Pharmacology

Mammalian Organotoxicity

General Model for vertebrate developmental biology Genomic

ResearchDrug Discovery

ZF AS A UNIVERSAL MODEL

•Testing of the toxicity of waste water: Replacement of fish test (Goldorfen-Test) by fish egg assay since 2005 (sewage tax law) DIN 38415, part 6(safes 40.000 – 50.000 animals/year)

48 h/26°C

Lethal endpoints:Coagulated egg ?No formation of somites ?No heartbeat ?No detachment of the tail ?

Waste water

TESTING OF WASTE WATER

0- CONTROL1- UNDILUTED WW2- 50% DILUTED WW3- 33% DILUTED WW4- PC

Acute toxicity in fish (Replacement)

Testing of WW

Developmental Toxicity (Teratogenicity)

Safety Pharmacology

Mammalian Organotoxicity

General Model for vertebrate developmental biology Genomic

ResearchDrug Discovery

ZF A UNIVERSAL MODEL

• newly fertilised zebrafish embryos• 20 embryos/concentration/control• 5 test concentrations• 2 ml/well; 26 ± 1° C and light cycle • 96 h exposure with semi-static renewal procedure• 4 endpoints for acute lethality (24, 48, 72 and 96h)• LC50 calculation at 48 and 96h

FET TG236

Acute toxicity in fish (Replacement)

Testing of WW

Developmental Toxicity (Teratogenicity)

Safety Pharmacology

Mammalian Organotoxicity

General Model for vertebrate developmental biology Genomic

ResearchDrug Discovery

ZF A UNIVERSAL MODEL

MAMMALIAN ORGANOTOXICITY

MAMMALIAN ORGANOTOXICITY

Acute toxicity in fish (Replacement)

Testing of WW

Developmental Toxicity (Teratogenicity)

Safety Pharmacology

Mammalian Organotoxicity

General Model for vertebrate developmental biology Genomic

ResearchDrug Discovery

ZF A UNIVERSAL MODEL

Tris-buffer pH 7.4

(0.5% DMSO)

8 hpf

1 dpf

2 dpf3 dpf

2.5 hpf

DEVELOPMENTAL TOXICITY(TERATOGENICITY)

ZF SCORING TERATOGENICITY

CategoryPhysiological/ dysmorphogenic effect 8 hpf 24 hpf 48 hpf 72 hpf

Lethal effect Coagulated egg + + + +

No heart beat +

Teratogenic effects Malformation of head + + +

Malformation of eyes + + +

Modification of the spinal chord + + +

Modification of the chorda + + +

Malformation of tail + + +

Malformation of tail tip (end tail) + + +

Scoliosis + + +

Malformation of sacculi/otoliths + + +

Malformation of heart +

Deformity of yolk + + +

Growth retardation + + +

6 µM AAF, 1 dpf

aYS

aYS: additional yolk sac

2-Acetylaminofluorene (AAF)

125 µM CBZ, 3 dpf

T: Tail

T

control

Carbamazepine (CBZ)

5 mM TEG, 3 dpf

5 mM TEG, 3 dpf

T: Tail; C: Chorda; H: Head

C

H

T

control

Tefagur

S/O: Sacculi/otoliths; H: Head; E: Eye; C: Chorda;

TT: Tail tip; T: Tail

250 µM WRF, 3 dpf500 µM WRF, 3 dpf

S/O

E

S/O

CTT

H

T

control

Warfarin (WFR)

• http://zfin.org/The Zebrafish Model Organism Database

• http://zfatlas.psu.edu/The Zebrafish Atlas

• http://itgmv1.fzk.de/eufishbiomed/eufish_main.phpEUFishBioMed (European Network on Fish Biomedical Models)

ZEBRAFISH LINKS

• Research gate

• http://www.bdpa-detroit.org/portal/index.php/comittees/high-school-computer-competition-hscc/29-education/57-moocs-top-10-sites-for-free-education-with-elite-universities.html• Massive Online Open Course (MOOC)

RESEARCH LINKS

Acknowledgements

Dr. Thomas Broschard

Dr. Nicole Hübler

Dr. Stefan Weigt

Julia PohlAlex Küfner

Andreas Gado

Johann Gally

POSSIBLE FUTURE STEPS FORUVA WELLASSA

WITH ZEBRAFISH EGGS

Short term• Uva x Colombo = national ZF network?

• Research topic on medium to allow fish egg transport by blocking ZF Egg Devt?

• Student project to develop live food from the aquaculture Dpt for ZF?

• ZF unit for student (training)? • Lectures in Zebrafish egg techniques for toxicity?

Medium term• ZF – Research Topics

–Heavy metals & water quality? –Field sampling - pesticides impact & kidney failure disorders?

–Indigenous medicines toxicity?

–Agriculture Dpt? Dvpt of substitutes for pesticides or best practices for farmers?