Kevin M' Crofton

1
Developmental Neurotoxicity Testing The
Challenge

• Kevin M. Crofton
• Neurotoxicology Division
• National Health and Environmental Effects
  Research Laboratory
• Office of Research and Development
• US EPA, RTP, NC USA
• March 13, 2006

2
Testing Needs

• EPA is charged with protecting public health
• Large numbers of chemicals identified for testing
  (e.g., pesticide inerts, HPVs, CCLs) with no
  risk-based criteria for setting testing
  priorities
• Different regulatory authorities/different
  testing requirements with no scientific basis for
  flexible testing approach
• Current guideline testing is expensive, time
  consuming and requires large numbers of animals

3
Regulatory Drivers for Testing
4
Current DNT Testing Guidelines

• Tools for Testing
• EPA 870.6300 DNT Guideline
• OECD 426 (draft3)
• Basic Requirements in vivo
• Developmental exposure followed by assessments
  of
• Growth and developmental landmarks
• Motor and sensory behaviors
• Cognitive function
• Neurohistopathology and morphometrics

5
Criticism of the Current Approach

• Expensive, time consuming
• Not used as first tier screen (only when
  triggered)
• Current methods are insensitive (e.g., cognition)
• Data is too variable (e.g., motor activity)
• Pathological/morphometrics (wrong ages, not
  sensitive)
• Not testing enough chemicals, therefore not
  protecting public health

Note Some criticisms are real some are
opinions
6
Current Testing Approach versus Reality

• Critical Need - Test 1,000s of chemicals for
  potential to produce developmental neurotoxicity
• Since 1991 we have tested only 80 chemicals
• Cant meet critical need with current in vivo
  test method
• We cannot test our way out of this problem!
• 0.7 1.0 million per chemical
• 1.5 years per study
• We need new approaches that are faster and more
  cost-efficient

7
Solutions

• Overall Goal Move from expensive and time
  consuming in vivo testing batteries to faster and
  cheaper batteries that offer useful data for risk
  assessments
• How do we get there from here?
• Short-term Refine - Better use
• Mid-term Reduce - Develop targeted testing
• Long-term Replace - Develop alternative methods

8
New Approaches

• Develop alternative testing approaches
• Use in vitro high-throughput cell culture models
• Use alternative species (non-mammalian) with
  conserved neurodevelopmental processes
• Develop in silico models
• Such approaches will
• Reduce costs and animal use
• Facilitate screening of large numbers of
  chemicals (high-throughput)
• Provide data for prioritization of chemicals for
  further targeted testing

9
Research Approaches - In Vitro

• In vitro tests based on key events of brain
  development
• Proliferation, migration, differentiation,
  neurite outgrowth, synaptogenesis
• Potential endpoints
• Cell-based
• Biochemical markers
• Conserved molecular (signaling) events

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In Vitro - Example
Method High-Throughput Imaging(automated,
image-analysis software)
Output Automated analyses of 96 wells in 20-30
min
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No NGF
NGF

• Effect of NGF on neurite extension and branching
• 12 point dose-response from one 96 well plate

Courtesy of Cellomics www.cellomics.com/
12
In Vitro - Example
Method Manual tracing of neurites in PC12 cells
Each chemical required manual image acquisition
and tracings taking weeks to months
0.03 uM Methylmercury
Data of Barone and Mundy
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Research ApproachesAlternative Species

• Use non-mammalian species (e.g., fish, worms) for
  development of DNT methods
• Key Allows assessment of intact developing
  nervous system (development analogous to
  mammals)
• Higher throughput testing compared to in vivo
  rodent models

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Alternative Species - Fish

• Medaka
• Neurodevelopment rapid and similar to other
  vertebrates
• Transparent egg and embryo (and fish)
• Assessment of intact CNS structure and function
• Low cost

Ethanol Alters Caspase Activity in Medaka
Morphometric Analysis of Medaka
Data from Oxendine et al
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Alt. Species Zebrafish
Apoptosis in zebrafish embryo brain measured by
image analysis of acridine orange staining
2,4-D
DDT
Atrazine
Dose response curves for apoptosis in the brain
at 96 hr post-fertilization
Dieldrin
Malathion
Nonophenol
Courtesy of Catherine Willett Phylonix
Pharmaceuticals, Inc. www.phylonix.com/
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Science Challenges

• Evaluate different model systems for application
  to DNT
• In vitro cultures (human and animal cell lines)
• Alternative species (e.g., C. elegans, Medaka,
  Zebrafish)
• Demonstration of proof-of-concept
• Predictability
• Gold standards?
• Build open databases
• Develop decision frameworks
• How, exactly will the data be used to prioritize
  further testing?

17
Policy Challenges

• Costs
• Must prioritize scarce resources for
  developmental neurotoxicity research
• Regulatory
• Need parallel integration of new methods into
  policy
• How to use data to prioritize testing
• Can data be used for risk decisions?
• How do we measure success?

18
The Key to Success

• Working Together
• Lots of disparate perspectives
• We are really all working towards the same goal
• We need to synergize, not antagonize

19
Thank You