The simplified Whole Embryo Culture

the throughput increase of a validated tool for developmental toxicology

Marta Barenys

CERETOX – Centre de Recerca en Toxicologia, Parc Científic de Barcelona IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

Outline

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

Establishment: New, 1978. Biol Rev Camb Philos Soc

REACH - Registration, Evaluation, Authorization and Restriction of Chemicals

ECVAM Validation: Genschow et al., 2002. Altern Lab Anim Piersma et al., 2004. Altern Lab Anim ECVAM DB-ALM Protocol n.123 – Method of Piersma

Gestational Day

Implantation

0 2 4 6 8 10 14 16 18 20 2112

WECOrganogenesis Fetal - period

WEC

Whole Embryo Culture (WEC) developmental toxicology

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

[1]:Flick and Klug, 2006. Curr Pharm Des

[1]

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

48 h

Isolation Culture 37ºC temperature 25 rpm rotation Tested substances added to the culture medium Oxygenation

Evaluation

Endpoints: Viability Growth Differentiation Malformations

Gestational Day 9.5

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

A. Yolk sac and blood vessels B. Allantois C. Flexion D. Heart E. Caudal neural tube F. Hind brain G. Mid brain H. Fore brain J. Otic system K. Optic system L. Olfactory system M. Branchial bars N. Maxillary process P. Mandibular process Q. Fore limb R. Hind limb

ECVAM Prediction Model 2

ICmax mal

ICNOEC TMS

IC50 3T3

I > II & III = non embryotoxic

II > I & III = weak embryotoxic

III > I & II = strong embryotoxic

Evaluation

Crown-rump length

Somite number

Total Morphological Score:

Malformations

„a demanding procedure which might be unsuitable for routine use as a first step in a large scale screening program“

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

[2]: Barenys et al., 2012. Reprod Toxicol

CERETOX Developmental toxicity studies: - among compounds of the same family de Jong et al., 2011 Toxicol Appl Pharmacol

- among parent compound and metabolites Barenys et al., 2012 Reprod Toxicol

Boix et al., in preparation

[2]

1 mm

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

European Partnership for Alternative Approaches to Animal Testing (EPAA) Questionnaire survey - Alternative methods for reproductive toxicity testing Aim: to investigate the real use of each alternative method among industries Participants: 11 large international pharmaceutical and chemical companies ECVAM DataBase service on Alternative Methods to animal experimentation (DB-ALM)

DB-ALM reproductive toxicity report 2011[3]

[3]:http://ec.europa.eu/enterprise/epaa/2_activities/2_3_comm_and_dissem/report_alternative_methods_reproductive_toxicity_testing.pdf

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

WEC: 6 companies substance related mechanistic studies screening or priority testing regulatory purposes studies on species differences (rat/rabbit) part of integrated testing strategy or stand-alone test

DB-ALM report Alternative methods for reproductive toxicity testing

39 total

21 developmental toxicity

9 really used

ZFET - Zebrafish Embryo Test EST - Embryonic Stem Cell Test WEC - Whole Embryo Culture

[3]:http://ec.europa.eu/enterprise/epaa/2_activities/2_3_comm_and_dissem/report_alternative_methods_reproductive_toxicity_testing.pdf

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

Advantages: Whole organism Same species than in in vivo reproductive toxicity assays High predictivity High reproducibility Disadvantages: High expertise required High cost Labor intensive Low throughput

Should I stay or

should I go?

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

[4]: Zhang et al., 2012. Toxicological Sciences

Simplification achieved by: Statistical prediction model supported by one test concentration two structural endpoints (spinal cord – heart) one growth endpoint (n. of somites) Prediction model built with data from: 59 compounds (33 teratogens and 26 nonteratogens) +11 compounds ( 5t & 6nt) for independent validation

Aims: ↑ throughput ↓ animal use without reducing predictivity

Simplified WEC Assay [4]

70

All compounds

Yes No

Teratogen

Spinal cord ≥ 4.83?

Heart ≥ 4.75?

Yes No

Yes No

Nonteratogen N° of somites ≥ 22.575?

Teratogen Nonteratogen

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

Simplified WEC Assay

Results from 3 independent laboratories Average scores obtained from two or more experiments with six embryos per experiment

Nonteratogens

[4]: Zhang et al., 2012. Toxicological Sciences 7 missclassified out of 26

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

Teratogens

3 missclassified out of 33 TOTAL: 10 out of 59

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

Simplified WEC Assay

[4]: Zhang et al., 2012. Toxicological Sciences

70 14

x 100 = 83%

RP: Recursive Partitioning

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

Zhang et al., 2012. Toxicological Sciences

Summary Simplified study design Simplified data analysis Prediction model does not require cytotoxicity data from a cell line No significant reduction in predictivity 50 % reduction of animal use and operational costs

Substance related mechanistical studies ECVAM protocol n.123 Simplified version Screening or priority testing

What do you want from

me?

Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary

Thank you

Gràcies