Kevin M. Crofton

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Recommendations for Developing Alternative Test
Methods for DevelopmentalNeurotoxicity
Evaluation

• Kevin M. Crofton
• CAAT DNT
• November 2008

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Global Cortical Search and Replace

• Criteria ? Recommendations
• Guidance ? Recommendations

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Screening for DNT
Methods Development
Funding
Screening for DNT
Policy
Data Collection
Data Interpretation
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Genesis of the Recommendations Document

• Steering committee recognized
• The need for a collaborative approach across
  industrial, academic and government research labs
• The inherent value in a set of recommendations
  that would further the development of test
  methods and models useful for high-throughput
  screening

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Document Evolution

• Writing committee met in June to draft a document
• Sandra Coecke Kevin Crofton
• Pam Lein Leonora Buzanska
• Holgar Knaut Anna Price
• Andrea Seiler
• Current version includes comments from the
  steering committee

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Givens

• Successful integration of alternative test
  methods into will
• require demonstration of their predictive ability
• development and acceptance of a strategy for data
  interpretation.
• These two issues, prediction and interpretation,
  cannot be determined without the generation of
  data.
• Therefore, this document provides guidance for
  methods development and data collection to
  achieve these aims.

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Assumptions

• No one model will be best
• A battery of methods will be needed
• Each component will provide valuable information
• Select the methods/battery based on data
• Use chemicals to test the models and not models
  to test the chemicals (not looking for
  mechanisms)

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The Recommendations

• Are specific to neurodevelopment, but can easily
  b e used for other areas of toxicology
• Are not roadmap to validation
• There are plenty of those already
• Not everyone has validation as an goal
• Provide a path that does not need to be finished,
  
• Hopefully encourage the development of tests,
  from the beginning, that will be eventually
  applicable in a regulatory context

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Definitions

• Endpoint the biological or chemical process,
  response, or effect assessed by a test method
  (OECD Guidance Document 34, 2005)
• Test system any animal, cellular, subcellular,
  chemical or physical system or a combination
  thereof used in a study (modified from OECD, GLP
  principle, directive 87)
• Test method A process or procedure used to
  obtain information on the biological effects of a
  substance or agent. Toxicological test methods
  generate information regarding the ability of a
  substance or agent to produce a specified
  biological effect under specified conditions.
  Used interchangeably with test and assay.
  (OECD Guidance Document 34, 2005). The test
  method should assess one or more of key aspects
  of human neurodevelopment.

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Recommendations

• Methods should measure endpoint(s) that model key
  neurodevelopmental events
• Phenotypic endpoint (e.g., neurite outgrowth)
• Key event in biochemical pathway (e.g.,
  PI3-kinase)
• Assure that method correctly and accurately
  measure the intended endpoint
• Intuitive and obvious, but not always done
• Dynamic Range
• Characterizing this is important for comparison
  between chemicals

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Recommendations (cont)

• Parametric controls
• Manipulation of assay parameters (e.g, pH, NGF)
• Should demonstrate predictable outcomes
• Response characterization
• Define what is a relevant change in the endpoint
  (e.g. a hit)
• Statistical vs biological
• Concentration range
• Allows comparison between labs and between
  endpoints (e.g., cytotoxicity)
• Endpoint selectivity
• The ability of the test method to discriminate
  the endpoint of concern from other outcomes.

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Recommendations (cont)

• Parametric controls
• Manipulation of assay parameters (e.g, pH, NGF)
• Should demonstrate predictable outcomes

Cell Titer Glo Assay Xia et al EHP 2008
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Recommendations (cont)

• Response characterization
• Define what is a relevant change in the endpoint
  (e.g. a hit)
• Statistical vs biological

Variability analyses with detection levels based
on 3 standard deviations (from Tierno et al
Nature Protocols 21134, 2007).
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Recommendations (cont)

• Endpoint selective controls
• Positives - reliably and consistently alter the
  endpoint by known mechanisms
• Negatives reliably causes no effect
• Training set chemicals
• Includes chemicals that reliably affect (or not)
  the endpoint in vitro (or in vivo if available)
• Goals are to
• evaluate the ability of the method to efficiently
  test moderate numbers of chemicals
• confirmation of positive and negative controls
• generation of historical control data to
  characterize the inherent response range for the
  endpoint

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Recommendations (cont)

• Endpoint selective controls
• Positives - reliably and consistently alter the
  endpoint by known mechanisms
• Negatives reliably causes no effect
• Training set chemicals
• Includes chemicals that reliably affect (or not)
  the endpoint in vitro (or in vivo if available)
• Goals are to
• evaluate the ability of the method to efficiently
  test moderate numbers of chemicals
• confirmation of positive and negative controls
• generation of historical control data to
  characterize the inherent response range for the
  endpoint

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Recommendations (cont)

• Testing set chemicals
• Large set of chemicals (100-200) known to affect
  endpoints of developmental neurotoxicity in vivo,
  (and chemicals do not)
• Purposes
• demonstrate the ability of the method to rapidly
  and efficiently test large numbers of chemicals
• provide data that can be used in determining
  future steps in the process of regulatory
  acceptance
• Specificity and Sensitivity
• Sensitivity active substances that are
  correctly
• Specificity inactive substances that are
  correctly identified.

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Chemicals with Substantial Evidence of
Developmental Neurotoxicty (n?100) (Padilla et al.
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Recommendations (cont)

• Testing set chemicals
• Large set of chemicals (100-200) known to affect
  endpoints of developmental neurotoxicity in vivo,
  (and chemicals do not)
• Purposes
• demonstrate the ability of the method to rapidly
  and efficiently test large numbers of chemicals
• provide data that can be used in determining
  future steps in the process of regulatory
  acceptance
• Specificity and Sensitivity
• Sensitivity active substances that are
  correctly
• Specificity inactive substances that are
  correctly identified.

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Recommendations (cont)

• 15. Data sharing
• Open access databases are highly desirable.
• Sharing data will allow inter-laboratory and
  intra-laboratory comparisons of test methods.

• Examples
• NCCT - DSSTOX, TOxCast, ACToR
• Regulatory data mining and new HTP data
• http//www.epa.gov/ncct/
• Poster 12 Broening et al Neurotoxicity
  Database
• Literature data mining
• Poster 15 Padilla et al DNT Chemicals
• Literature data mining

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Purpose of Draft

• Stimulate and focus discussions of alternative
  testing methods and models at this workshop. This
  discussion is critical to the success of the
  meeting.
• Gain feedback from all attendees on the draft
  recommendations.
• We are assuming that attendees will provide
  feedback
• This feedback is critical to the future of both
  the document as
• There will be an open discussion on the draft
• document at the end of the day today
• and a recap at the end of the meeting

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Thanks