The simplified Whole Embryo Culture
the throughput increase of a validated tool
for developmental toxicology

Marta Bareny...
Outline
Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Summary
Whole Embryo Culture (WEC)
developmental toxicology

Whole Embryo Culture
Implantation

DB-ALM report

WEC
WEC
Fetal - per...
[1]
Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Summary

[1]:Flick and Klug, 2006. Curr Pharm Des
48 h
Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Summary

Isolation

Culture

Evaluation

Gestational Day 9...
„a demanding procedure which might be unsuitable for routine use as a first step in a
large scale screening program“

Eval...
[2]

Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Summary

1 mm

CERETOX
Developmental toxicity studies:
- a...
European Partnership for Alternative Approaches to Animal Testing (EPAA)
Whole Embryo Culture

DB-ALM report

Simplified W...
DB-ALM report
Alternative methods for reproductive toxicity testing
Whole Embryo Culture

39

21

9

total

DB-ALM report
...
Advantages:
Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Whole organism
Same species than in in vivo reprodu...
Simplified WEC Assay [4]
Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

All compounds
Spinal cord ≥ 4.83?

Aim...
Simplified WEC Assay

Nonteratogens

Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Summary

Results from 3 in...
Teratogens

Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Summary

3 missclassified out of 33

TOTAL: 10 out ...
Simplified WEC Assay
Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Summary

x 100 = 83%

14
RP: Recursive Par...
Summary
Whole Embryo Culture

DB-ALM report

Simplified WEC Assay

Summary

Simplified study design
Simplified data analys...
Whole Embryo Culture

Thank you

DB-ALM report

Simplified WEC Assay

Summary

Gràcies
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The in vitro choice m barenys iuf_2013

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The in vitro choice m barenys iuf_2013

  1. 1. 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
  2. 2. Outline Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary
  3. 3. Whole Embryo Culture (WEC) developmental toxicology Whole Embryo Culture Implantation DB-ALM report WEC WEC Fetal - period Organogenesis Simplified WEC Assay Summary 0 2 4 6 8 10 12 14 16 18 Gestational Day 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 20 21
  4. 4. [1] Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary [1]:Flick and Klug, 2006. Curr Pharm Des
  5. 5. 48 h Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary Isolation Culture Evaluation Gestational Day 9.5 37ºC temperature 25 rpm rotation Endpoints: Tested substances added to the culture medium Oxygenation Viability Growth Differentiation Malformations
  6. 6. „a demanding procedure which might be unsuitable for routine use as a first step in a large scale screening program“ Evaluation Whole Embryo Culture Crown-rump length DB-ALM report Somite number Simplified WEC Assay Summary Total Morphological Score: Malformations ICmax mal ICNOEC TMS ECVAM Prediction Model 2 IC50 3T3 A. B. C. D. E. F. G. H. J. K. L. M. N. P. Q. R. Yolk sac and blood vessels Allantois Flexion Heart Caudal neural tube Hind brain Mid brain Fore brain Otic system Optic system Olfactory system Branchial bars Maxillary process Mandibular process Fore limb Hind limb I > II & III = non embryotoxic II > I & III = weak embryotoxic III > I & II = strong embryotoxic
  7. 7. [2] Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary 1 mm CERETOX Developmental toxicity studies: - among compounds of the same family - among parent compound and metabolites de Jong et al., 2011 Toxicol Appl Pharmacol Barenys et al., 2012 Reprod Toxicol Boix et al., in preparation [2]: Barenys et al., 2012. Reprod Toxicol
  8. 8. European Partnership for Alternative Approaches to Animal Testing (EPAA) Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary 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
  9. 9. DB-ALM report Alternative methods for reproductive toxicity testing Whole Embryo Culture 39 21 9 total DB-ALM report developmental toxicity really used ZFET - Zebrafish Embryo Test EST - Embryonic Stem Cell Test WEC - Whole Embryo Culture 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 [3]:http://ec.europa.eu/enterprise/epaa/2_activities/2_3_comm_and_ dissem/report_alternative_methods_reproductive_toxicity_testing.pdf
  10. 10. Advantages: Whole Embryo Culture DB-ALM report Simplified WEC Assay Whole organism Same species than in in vivo reproductive toxicity assays High predictivity High reproducibility Summary Disadvantages: High expertise required High cost Labor intensive Low throughput Should I stay or should I go?
  11. 11. Simplified WEC Assay [4] Whole Embryo Culture DB-ALM report Simplified WEC Assay All compounds Spinal cord ≥ 4.83? Aims: ↑ throughput ↓ animal use without reducing predictivity Yes No Heart ≥ 4.75? Teratogen Yes No Nonteratogen N° of somites ≥ 22.575? Summary Simplification achieved by: No Statistical prediction model supported by Teratogen one test concentration two structural endpoints (spinal cord – heart) one growth endpoint (n. of somites) Yes Nonteratogen Prediction model built with data from: 59 compounds (33 teratogens and 26 nonteratogens) 70 +11 compounds ( 5t & 6nt) for independent validation [4]: Zhang et al., 2012. Toxicological Sciences
  12. 12. Simplified WEC Assay Nonteratogens Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary Results from 3 independent laboratories Average scores obtained from two or more experiments with six embryos per experiment 7 missclassified out of 26 [4]: Zhang et al., 2012. Toxicological Sciences
  13. 13. Teratogens Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary 3 missclassified out of 33 TOTAL: 10 out of 59
  14. 14. Simplified WEC Assay Whole Embryo Culture DB-ALM report Simplified WEC Assay Summary x 100 = 83% 14 RP: Recursive Partitioning 70 [4]: Zhang et al., 2012. Toxicological Sciences
  15. 15. Summary Whole Embryo Culture DB-ALM report Simplified WEC Assay 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 What do you want from me? Substance related mechanistical studies  ECVAM protocol n.123 Simplified version  Screening or priority testing Zhang et al., 2012. Toxicological Sciences
  16. 16. Whole Embryo Culture Thank you DB-ALM report Simplified WEC Assay Summary Gràcies
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