Summary of CAATCEFICECVAM Workshop on Developmental Neurotoxicity Testing

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Summary of CAAT-CEFIC-ECVAM Workshop on
Developmental Neurotoxicity Testing
Anna Price and Sandra
CoeckeEuropean Centere for the Validation of
Alternative Methods (ECVAM)Joint Research
CentreEuropean Commission21020 Ispra (VA),
Italy
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ECVAM Primary Mission validation of alternative
methods to reduce, refine and replace animal
tests for safety assessment of chemicals,
cosmetics, pharmaceuticals and biomaterials
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The first workshop on Incorporating In Vitro
Alternative Methods for Developmental
Neurotoxicity Testing (DNT) into International
Hazard and Risk Assessment Strategies Ispra,
Italy, 19 21 April 2005 The Johns Hopkins
Center for Alternatives to Animal Testing (CAAT),
The European Centre for Validation of Alternative
Methods (ECVAM) and The European Chemical
Industry Council (CEFIC)
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Incorporating In Vitro Alternative Methods for
Developmental Neurotoxicity into International
Hazard and Risk Assessment Strategies Sandra
Coecke1, Alan M Goldberg2, Sandra Allen3, Leonora
Buzanska1,4, Gemma Calamandrei5, Kevin Crofton6,
Lars Hareng1, Thomas Hartung1, Holger Knaut 7,
Paul Honegger8, Miriam Jacobs1, Pamela Lein9,
Abby Li10, William Mundy6, David Owen11, Anna
Price1, Steffen Schneider12, Ellen Silbergeld2,
Torsten Reum13, Tomas Trnovec14 and Florianne
Tschudi-Monet8
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Developmental Neurotoxicity Testing (DNT)
Concern about health risks for children has
increased among policy makers, scientists,
representatives of industry, NGOs, the general
public and the media and efforts are being
undertaken to identify DNT-related hazards and
risks.
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• The aim of the DNT workshop
• to develop a roadmap for designing DNT tests
  based
• on specific mechanisms relevant to
  neurodevelopment
• to identify opportunities for reducing,
  refining,
• or replacing (3Rs) the use of animals in DNT
• to develop the process for validating candidate
• alternative methods both scientifically and in
  terms
• of regulatory applications

In vitro
In silico
human
animal
non-mammalian
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• Due to the complexity of the issue two
  breakout groups focussed on two different
  aspects
• 1st Details on the science available
• 2nd Policy and strategy

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Breakout group Details on the science available

• What are the models which are already
  established?
• What endpoints/processes/effects should be
  measured?
• Where to invest?
• How to increase the throughput for DNT
  assessments?
• How to progress towards validation and acceptance
  of alternative DNT approaches?

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Definition of DNT The adverse effects of
substances (xenobiotics) on the nervous system
associated with exposure during
development. The adverse effects may be
expressed at any time during the life span of the
exposed individual.
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• DNT caused by exposure to chemicals refers to the
  adverse effects on any aspect of neurodevelopment
  such as
• neuronal progenitor proliferation
• cell migration
• synaptogenesis
• cell death
• formation of transmitters and receptors
• trimming of connections
• myelinisation
• development of the blood-brain barrier

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• Promising in vitromammalian DNT models
• Human and rodent stem cells
• Immortalised human and rodent
• cell lines
• Primary dissociated neuronal and
• glial cultures
• Re-aggregating brain cell cultures
• Organotypic cultures

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Types of cell and tissue in vitro models
Tissue slices and explants
Increasing complexity
3D models aggregates
Primary cells monolayer cultures
Human cell lines
Increasing information
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Human and rodent stem cells
NF 160

Human embryonic stem cells
Hareng L., Stummann T., KA Reproductive
Toxicology, ECVAM
Mouse Somatic neural stem cells
Conti L. et al., 2005, PLoS Biology, 3, 1-13.
Non-immortilized human cord blood-derived neural
stem cell line
Buzanska L. et al., 2005 Toxicol. in Vitro, 15,
67-74
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• Immortalised human and rodent cell lines

• The major advantages are
• Readily obtainable
• Can be easily expanded in culture
• 3. Can differentiate into a non- dividing cells
  with characteristics of neurons
• (formation of neurites, electrical
  excitability, synthesis of neurotransmitters and
  associated receptors)

Rat PC12 cells

Human CHP-100
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Primary
dissociated neuronal culture

• glial progenitor proliferation
• cell migration
• synaptogenesis
• cell death
• reactive gliosis
• synthesis of transmitters
• expression of various types of
• receptors
• formation of neuronal network
• connections
• long term culture myelinization
• electrical activity

Price A et al., 2001, J. Neuroscience, 21,
6480-6491
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Re-aggregating brain cell cultures

• Reproduces the in vivo-like 3D cytoarchitecture
  of neurons and glial
• cells (microglia, astrocytes and
  oligodendrocytes)
• Within each aggregate, cell migration,
  differentiation and segregation occurs, giving a
  layered structure(neurons in the center, glial
  cells in
• outer part, and stem cells at the periphery)
• Synaptogenesis and myelination take place,
  resulting in spontaneous and evoked electrical
  activity

Zurich M. et al., 2002, J. Neurosci. Res.,1,
108-16
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Extra cellular electrophysiological recordings
for neurotoxicity screening
Rat brain aggregates
Multi electrode array
Evoked field potentials
Map2 staining
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Metabonomic set-up to predict and classify
neurotoxic compounds

liquid handler
Chemical
Mass spectrometry (MS) and statistical analysis
(PCA)
MS
96 well plate
Culture medium cell homogenates
(3D neuronal in vitro model)
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Organotypic cultures

• Derived from explants of undifferentiated
  embryonic brain spinal cord, or sensory organs
• The advantage of this system is the presence of a
  three-dimensional organization
• Suitable for evaluating the electrophysiological
  or pharmacological agents

(Properties of a calcium-activated K1 current on
interneurons in the developing rat Hippocampus.
(Takuya A., et al., 2000, The American Physiol.
Sciety, 22, 77-85)
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• Alternative non-mammalian species
• as model for DNT testing
• Zebrafish
• Medaka fish
• C. Elegans
• Sea Urchin
• Drosophila

(Chaoyong Ma,2004, Modern Drug Discovery 30,
5-9 )
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Non-mammalian species

• Zebrafish and C. Elegans genome is completely
• sequenced and there is a basic understanding
• of gene function and physiological effect
• They offer transparency (the possibility of
• non-invasive imaging)
• Sea urchin suitable to study fertilization
• and early embryonic development
• Drosophila

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Interspecies differences and DNT

• Species differences e.g in synaptogenesis,
  metabolism or in activity of biotransformation
  enzymes are important issues underlying
  mechanisms of toxicity
• Until interspecies differences for DNT are better
  understood both human and rodent cell and tissue
  cultures and alternative species should continue
  to be used

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Metabolism-mediated DNT effects

Inhibition or induction of biotransformation
enzymes can result in the alterations of DNT
processes

• E.g. estrogen formation in the brain is catalyzed
  by cytochrome P450 aromatase isoforms. Estrogen
  is important for developmental processes since it
  regulates
• neuronal proliferation
• survival
• morphology
• synaptogenesis

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Interaction/interplay between endocrine and
immune function

• Chemicals that interfere or mimic endogenous
  hormones and signaling chemicals of the endocrine
  system are endocrine disruptors
• Immune system influences the development of CNS
  e.g macrophages, leucocytes crossing the BBB may
  play an important role in neurodevelopment and in
  the pathophysiology of various diseases

Kinsner A. et al., 2005, J.Neurochem., 95,
1132-1143
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Considerations in evaluating potential models
and endpoints for DNT

• Adaptability for high throughput screening
• Biological complexity of the model in comparison
  to in vivo situation
• Does the model address concerns of regional
  specificity
• Within and between laboratory variability
• Transferability e.g. the technical difficulty
  and expertise needed to set-up the model
• Precision and accuracy of method for quantifying
  dose dependency
• Preliminary indication on the predictive capacity
  
• for a well-defined effect confirmation of
  the applicability domain

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Breakout group Policy and strategy

• How to incorporate data from alternative
  approaches in the regulatory decision process?
• How to complement in vivo approaches?
• Potential of the existing DNT alternative test
  systems?
• Necessary characteristics?
• Identify if possible immediate candidates for
  prevalidation

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It is necessary to develop batteries of tests and
strategies that provide for both mechanisms of
action and the ability to predict human health
consequences
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Stepwise process of introducing alternatives in
DNT 1st step to refine the current in vivo
strategies by integrating information derived
from in vitro, in silico and non-mammalian
alternative test strategies. 2nd step to give
alerts, based on in vitro and in silico testing,
for possible DNT effects would eliminate the
number of chemicals reliant on in vivo mammalian
DNT tests.
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Conclusions

• Detection and characterization of
  chemical-induced DNT effects in the CNS and PNS
  should incorporate in vitro and non-mammalian
  alternative methods as a part of intelligent
  testing strategies for regulatory purposes
• Batteries of models
• Batteries of endpoints