Presentation at the LIFE EDESIA workshop - 28/02/2018

1. EFSA – IPAM day
3rd LIFE-EDESIA workshop on
Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective
Parma (I) 28.02.2018
In vitro development of alternative testing: from monolayered cells to organoids
COSTANZA ROVIDA
CAAT-Europe – European Centre for Alternatives to Animal Tests
ecopa – European Consensus Platform for Alternatives
LIFE-EDESIA (LIFE12 ENV/IT/000633)
28th February 2018

2. Endocrine Disruptors and the in vitro alternative methods to animal testing
Costanza Rovida
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
1981
Stakeholder
Platform
2012
CAAT EU Policy
Program
2010
Transatlantic
Hub
www.ecopa.eu
www.team-mastery.eu

3. Endocrine Disruptors and the in vitro alternative methods to animal testing
Conceptual Framework for Testing and Assessment of Endocrine Disrupters
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
Level 1
Existing data and non-test Information
Level 2
In vitro assays providing data about selected endocrine mechanism(s) / pathways(s)
(Mammalian and non mammalian methods)
Level 3
In vivo assays providing data about selected endocrine mechanism(s) / pathway(s)
Level 4
In vivo assays providing data on adverse effects on endocrine relevant endpoints
Level 5
In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints
over more extensive parts of the life cycle of the organism
OECD No 150: GUIDANCE DOCUMENT ON STANDARDISED TEST GUIDELINES FOR EVALUATING CHEMICALS FOR ENDOCRINE DISRUPTION

4. Endocrine Disruptors and the in vitro alternative methods to animal testing
Conceptual Framework for Testing and Assessment of Endocrine Disrupters
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
Level 1
Existing data and non-test Information
Level 2
In vitro assays providing data about selected endocrine mechanism(s) / pathways(s)
(Mammalian and non mammalian methods)
Level 3
In vivo assays providing data about selected endocrine mechanism(s) / pathway(s)
Level 4
In vivo assays providing data on adverse effects on endocrine relevant endpoints
Level 5
In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints
over more extensive parts of the life cycle of the organism
OECD No 150: GUIDANCE DOCUMENT ON STANDARDISED TEST GUIDELINES FOR EVALUATING CHEMICALS FOR ENDOCRINE DISRUPTION

5. http://www.oecd.org/env/ehs/testing/OECD_Draft_GIVIMP_in_Human_Safety_Assessment.pdf
Table of content
1. Responsibilities
2. Quality Considerations
3. Facilities
4. Apparatus, material and reagents
5. Test Systems
6. Test and reference items
7. Standard Operating Procedures (SOPs)
8. Performance of the Methods
9. Reporting of Results
10. Storage and retention of records and methodsials

6. ALTEX 34(1), 2017

7. Endocrine Disruptors and the in vitro alternative methods to animal testing
Traditional culture: pan-fried eggs “sunny side up”
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
Cell density ca. 0.1% of tissue,
Dilution of all secreted factors
Cell to cell contact about 2%,
49% plastic, 49% medium
No flow
No steady ingredients,
• Ca. 25% of cell lines misidentified
• 15-25% mycoplasma infected
• Genetic instability
• Culture artifacts

8. http://ec.europa.eu/growth/sectors/chemicals/good-laboratory-practice_en

9. Endocrine Disruptors and the in vitro alternative methods to animal testing
RELIABILITY OF ANIMAL TESTS
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
GLP, standardized protocols, some validated, high doses,
substance effects in healthy animals:

11. Endocrine Disruptors and the in vitro alternative methods to animal testing
Conceptual Framework for Testing and Assessment of Endocrine Disrupters
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
Level 1
Existing data and non-test Information
Level 2
In vitro assays providing data about selected endocrine mechanism(s) / athways(s) (Mammalian and non mammalian
methods)
Level 3
In vivo assays providing data about selected endocrine mechanism(s) / pathway(s)
Level 4
In vivo assays providing data on adverse effects on endocrine relevant endpoints
Level 5
In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints over more extensive
parts of the life cycle of the organism
OECD No 150: GUIDANCE DOCUMENT ON STANDARDISED TEST GUIDELINES FOR EVALUATING CHEMICALS FOR ENDOCRINE DISRUPTION

12. Endocrine Disruptors and the in vitro alternative methods to animal testing
Conceptual Framework for Testing and Assessment of Endocrine Disrupters
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
Level 1
Existing data and non-test Information
Level 2
In vitro assays providing data about selected endocrine mechanism(s) / athways(s) (Mammalian and non mammalian
methods)
Level 3
In vivo assays providing data about selected endocrine mechanism(s) / pathway(s)
Level 4
In vivo assays providing data on adverse effects on endocrine relevant endpoints
Level 5
In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints over more extensive
parts of the life cycle of the organism
We need to move forward!
OECD No 150: GUIDANCE DOCUMENT ON STANDARDISED TEST GUIDELINES FOR EVALUATING CHEMICALS FOR ENDOCRINE DISRUPTION

13. Endocrine Disruptors and the in vitro alternative methods to animal testing
Opportunity from 3D culture
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
• Increased cell survival
• Increased differentiation
• Increased cell – cell interaction
• Reproducing better the complexity
of the organ
• Endpoints need optimization
• More complex – lower
reproducibility
Alépée et al., ALTEX
2014, 31, 441-477.

14. Molecular
properties
Molecular
initiating events
Cellular
responses
Organ
response
Organism
response
ADVERSE OUTCOME PATHWAY

16. Endocrine Disruptors and the in vitro alternative methods to animal testing
Cell Biological Processes necessary for Brain Development
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
With courtesy from William Mundy, U.S. Environmental Protection Agency and John Havel, SRA International, Inc.
Endpoint
Cell Death
Proliferation
Migration
Differentiation
Neurite
Outgrowth
GFAP
Network
Formation
Myelination

17. Endocrine Disruptors and the in vitro alternative methods to animal testing
Neurogenesis in the Mouse and Human Cerebral Cortex
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
Neural stem cells (NSCs)
Ventricular zone (VZ)
Subventricular zone (SVZ)
Inner and Outer SVZ (ISVZ and OSVZ, respectively)
Intermediate zone (IZ)
Cortical plate (CP)
Kelava and Lancaster (2016). Cell Stem Cell
Barenys, M. et al. (2017). Arch Toxicol.
0
50
100
150
200
250
300
350
400
450
0 0.01 0.1 1 5 10 50 100
%respectcontrol
Migration distance
human NPC rat NPC
FGF2 ng/mlFGF2 ng/ml

18. Endocrine Disruptors and the in vitro alternative methods to animal testing
Homogeneity vs Complexity
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
SFEB: Serum free culture of Embryonic bodies
(A)Relationship of the complexity of the cells/tissue produced by individual protocols and the homogeneity
of the cells/tissues generated
(B) Individual regions of cortical tissue from various methods with their relative sizes.

19. Endocrine Disruptors and the in vitro alternative methods to animal testing
Human “mini-brain” developing from iPSC
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
• All cell types but micro-glia
• 350um diameter
• 800 per batch
• Reproducible
• Electrophysiological active
• From patient cells: gene/environment interactions

20. Endocrine Disruptors and the in vitro alternative methods to animal testing
Interference with thyroid serum binding protein transthyretin and subsequent
adverse human neurodevelopmental toxicity
https://aopwiki.org/aops/152#network_view

21. Endocrine Disruptors and the in vitro alternative methods to animal testing
Interference with thyroid serum binding protein transthyretin and subsequent
adverse human neurodevelopmental toxicity
https://aopwiki.org/aops/152#network_view
Type Event ID Title Short name
1MIE 957Binding, Transthyretin in serumBinding, Transthyretin in serum
2KE 958
Displacement, Serum thyroxine
(T4) from transthyretin
Displacement, Serum thyroxine
(T4) from transthyretin
3KE 959
Increased, Free serum
thyroxine (T4)
Increased, Free serum thyroxine
(T4)
4KE 960
Increased, Uptake of thyroxine
into tissue
Increased, Uptake of thyroxine into
tissue
5KE 961
Increased, Clearance of
thyroxine from tissues
Increased, Clearance of thyroxine
from tissues
6KE 281
Thyroxine (T4) in serum,
Decreased
T4 in serum, Decreased
7KE 280
Thyroxine (T4) in neuronal
tissue, Decreased
T4 in neuronal tissue, Decreased
8KE 756
Hippocampal gene expression,
Altered
Hippocampal gene expression,
Altered
9KE 757Hippocampal anatomy, Altered Hippocampal anatomy, Altered
10KE 758
Hippocampal Physiology,
Altered
Hippocampal Physiology, Altered
11AO 402Cognitive Function, Decreased Cognitive Function, Decreased

24. Cell Culture
(one cell type,
few parameters)
Structure / Activity-
Relationships
(Correlations)
Early Alternatives Organo-typic
Cell Culture
(Coculture, Organ function,
often Perfusion)
Today
Automated Cell
Culture (high-
throughput Screening)
Systems Toxicology
(“Virtual Patient”)
Human-on-chip
(Multi-Organ Models
With Microfluidics)
Future
Toxicity Mechanisms
(“Adverse Outcome Pathways”,
“Human Toxome”)
Modeling
(Receptor binding,
Virtual Organs, Kinetics)
Cell Culture
+ Omics or Image
Analysis (high-content)
Integrated
Test Strategies
(combined tests)

25. Endocrine Disruptors and the in vitro alternative methods to animal testing
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
No balloon and no aeroplane will
ever be practically successful (1902)
Heavier-than-air flying
machines are impossible (1895)
William Thomson, known as Lord Kelvin (1824-1907),
President of the Royal Society and inventor of the absolute temperature scale

26. Endocrine Disruptors and the in vitro alternative methods to animal testing
EFSA – IPAM day, 3rd LIFE-EDESIA workshop on Endocrine Disruptors and alternative methods to animal testing - a scientific and regulatory perspective, Parma (I) 28.02.2018
Thank you for the attention!!!