Cell Culture Breakout

1
Cell Culture Breakout

• Sue Marty and Abby Li

2
Endpoint

• Endpoints listed seemed to be accepted
• Need to be left open as science evolves
• no restriction or priority should be imposed
• Glial Function covers many sub-categories

3
Dose Range

• Should there be a set range of doses
• No Restrictions, however some general principles
  will greatly assist interlaboratory comparison
• Include rationalization for dose selection
• Use cytotoxicity as one of the dose limiting
  endpoints (see next slide)
• Relate to plasma level if data available
• Sensitivity of assay can be compared better if
  there is a minimum of 5 doses (need dose
  response)
• When possible, report IC50 levels with Standard
  Deviation
• For highthroughput 100 uM at top end? 1
  standardized dosing regimen for chemicals with
  unknown dose response is needed

4
Specific vs Generalized toxicity

• Cytotoxicity as one anchor since its a widely
  used endpoint that can be used for comparison
• Non-specific effects
• Cytotoxicity could be a specific neurotoxic
  effect (inhibition of proliferation will cause
  cytotoxicity), but then compare with other cell
  types
• Timing is a variable that should also be
  considered
• Details of the parametric controls (e.g. range of
  culture conditions) must be reported

5
Steps to assess system

• Very important to evaluate system in step-wise
  fashion
• Endpoint selective
• Training set chemicals
• We need to select out of Padilla list and other
  sources a training set thats relevant for
  different systems and endpoints
• For example chemicals causing immune-endocrine
  effects may not be relevant to the specific assay
• Important NOT to start with Padilla list before
  going through the other steps
• Limitation is that we dont understand mechanism
  of action

6
Endpoint Specific and Training Set Positive
controls

• Need list of endpoint specific controls for
  different critical endpoints
• Intra and inter laboratory comparison
• Assay development
• Action item everyone send your list
• PC12 ngf dose response
• Transparency in cultural conditions and
  analytical information on chemical purity is
  needed

7
Endpoint Specific and Training Set Negative
controls

• Endpoint Specific Negative controls
• Dose consideration Glucose can be toxic at
  higher dose levels
• Cut-off above 1000 ug/ml
• What characterization is needed?
• Pair positive control with something similar but
  non-neurotoxic to filter out common effects
• Training Set of Negative Controls
• Non-neurotoxic agents with no effects
• Non neurotoxic agents with other toxicity effects
• Need to be able to differentiate specific from
  non-specific effects

8
Questions not discussed

• Is a complete understanding of underlying
  neurobiology necessary for a model/endpoint to be
  useful in a screen?
• Data interpretation of specific vs non-specific
  outcomes
• How divergent should concentration effect curves
  be?

9
Bottom Line Conclusions

• State of science early stage
• Need more data before higher level questions can
  be addressed
• Need to standardize general principles in
  selecting chemicals and setting dose level
• Longer meeting time to discuss principles helpful
  for all labs to follow so that at DNT TestSmart 3
  we can better compare data