The document describes efforts to simplify the whole embryo culture (WEC) assay to increase its throughput for developmental toxicology screening. The traditional WEC assay is time-intensive and requires extensive expertise. A simplified WEC assay was developed using a statistical prediction model based on measurements of just two structural endpoints and one growth endpoint in rat embryos exposed to test compounds. This simplified assay achieved 83% predictivity compared to the traditional WEC assay in classifying compounds as teratogenic or non-teratogenic, while reducing animal use and costs by 50%. The simplified WEC assay shows potential as a higher throughput alternative for developmental toxicity screening.

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. Outline
Whole Embryo Culture
DB-ALM report
Simplified WEC Assay
Summary

3. Whole Embryo Culture (WEC)
developmental toxicology
Whole Embryo Culture
DB-ALM report
Implantation
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. Whole Embryo Culture
[1]
DB-ALM report
Simplified WEC Assay
Summary
[1]:Flick and Klug, 2006. Curr Pharm Des

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. „a demanding procedure which might be unsuitable for routine use as a first step in a
large scale screening program“
Evaluation
Whole Embryo Culture
DB-ALM report
Simplified WEC Assay
Summary
Crown-rump length
Somite number
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. [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. 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. DB-ALM report
Alternative methods for reproductive toxicity testing
DB-ALM report
39
21
9
total
Whole Embryo Culture
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. 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. 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. 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. Teratogens
Whole Embryo Culture
DB-ALM report
Simplified WEC Assay
Summary
3 missclassified out of 33
TOTAL: 10 out of 59

14. Simplified WEC Assay
Whole Embryo Culture
DB-ALM report
Simplified WEC Assay
Summary
14
RP: Recursive Partitioning
70
x 100 = 83%
[4]: Zhang et al., 2012. Toxicological Sciences

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. Whole Embryo Culture
Thank you
DB-ALM report
Simplified WEC Assay
Summary
Gràcies