The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.
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TestSmart HPV
April 26-27, 1999
Hyatt Fair Lakes
12777 Fair Lakes Circle
Fairfax, VA 22033
A workshop of The Johns Hopkins Center for Alternatives to Animal Testing
TestSmart is a program of the Vision 20/20 forum
This workshop is partially funded through a grant by the Vira I. Heinz Endowment
Abstract for TestSmart--A Humane and Efficient Approach to Screening Information Data Sets (SIDS) Data
Chemical Kinetics in Mammalian Systems -- Research Needs
John M. Frazier
United States Air Force
Introductory Comments
Focus of Attention for HPV Program
- Hazard identification is the process by which we determine whether or not a chemical has the potential to produce a particular toxicological effect.
- Risk characterization is the process by which the quantitative risk of developing a particular toxicological effect will occur given a particular exposure to the chemical. Risk characterization can be inverted to determine the safe level of exposure to a chemical for regulatory purposes.
- The HPV Testing Program is focused on hazard identification. However, we should not lose sight of the beneficial overlap of the two activities.
- Hazard identification is useful as long as it does not produce too many false positives or false negatives.
Toxicological Process
Factors Contributing to the Expression of Selective Toxicity
- Kinetic Factors
- Metabolism (activiation, detoxification)
- Selective transport systems (concentrative uptake, excretion)
- Binding proteins
- Dynamic Factors
- Expression of sensitive molecular targets (enzymes, receptors)
- Protective mechanisms (GSH, Vitamin E, ...)
- Stability of structural components (cytoskeleton, membranes)
- Repair mechanisms
Testing Strategies -- In Vivo Versus In Vitro
| Kinetics | Cellular Dynamics | Systemic Dynamics | Species Extrapolation | |
| In Vivo | + | + | + | - |
| In Vitro | +/- | + | - | + |
Systems Analysis of E-R Relationship
Quantitiative Toxicokinetics
- One of the objectives of quantitative toxicokinetics is to estimate the target tissue dosimetry (target dose metric).
- This objective is accomplished using biologically base kinetic models.
Relation between Site of Action and Target Dosimetry
Exposure-Response Relationships
In Vivo Kinetics Studies
Experimental Design for In Vivo Kinetic Studies
- Basic Design for IV Dosing Studies
- Multiple doses
- Multiple time points
- Replicates
- Collect urine and feces
- Collect exhaled breath
- Collect selected tissues
- Collect blood (separate into plasma and RBC)
- Analyze samples for parent chemical and metabolites
TCA Kinetics in Rat Plasma Following IV Dosing
TCA Excretion Kinetics in Urine
Summary of TCA Kinetics In Vivo Following IV Injection
- Poorly metabolized (<10% 14C-Label exhaled in 24 hours; no metabolites detected in any samples)
- Large fraction of parent chemical excreted in urine (>60% in 24 hours)
- % of dose excreted in urine increases with dose
- Kinetics in the liver and skin differ from those in plasma and other tissues
- Highest concentrations in kidney followed by RBC, liver, ...
Animal Requirements for In Vivo Kinetic Studies
3 Doses
8 Time Points
6 Animals per Time Point
= 144 Rats
What Kinetic Issues are not Answered by Standard Kinetic Experiments?
- How does protein binding affect kinetics?
- What is bioavailability of chemical when exposed via different routes?
- Is biliary excretion/enterohepatic recirculation an important factor?
- Where is the chemical metabolized?
- etc.
Kinetic Requirements for Risk Assessment
Coordinates of BBK Model Domain
- Chemical
- Species
- Gender
- Age
- Physiological State
- Health/Nutrition
- Dose Range
- Route of Exposure
- Dose Schedule
- Time Frame
- Fidelity
- Spatial Resolution
Components of a Model
- Model Architecture
- Mathematical Representation
- Model Parametters
Mammalian Systemic BBK Model
Two Classes of BBK Model Parameters
- Chemical-independent (physiological) parameters
- Chemical-dependent parameters
RBK Model Simulation of In Vivo TCA Kinetics: Plasma Concentration
In Vitro Approaches
Chemical Dependent Model Parameters
Membrane Transport
- Qualititative Issues
- Diffusion
- Mediated Transport
- Transcytosis
- Tissue Specificity
- Quantitative
- Permeability
- TMAX, KT
- Methods
- Membrane Vesicles
- Cells in Culture
- Isolated Perfused Organs
- In Vivo
Binding
- Qualitative Issues
- Reversible
- Irreversible - Adducts
- Quantitative
- BMAX, KB
- Plasma, Tissues
- Methods
- Ultrafiltration
- Dialysis
Metabolism
- Qualitative Issues
- Michaelis-Menton
- Other
- Enzymology
- Quantitative
- VMAX, KM
- Tissue Distribution
- Methods
- Microsomes
- Cytosol
- Cells in Culture
- Isolated Perfused Organs
- In Vivo
Physical Partitioning
- Qualitative Issues
- Volatile/Non-volatile
- Water Soluble/Lipid Soluble
- Quantitative
- Octanol Water Partition Coefficient (QSPR)
- Air/Plasma Partition Coefficient
- Plasma/Tissue Partition Coefficient
- Methods
- Vial Equilibration
- Ultrafiltration
- Osmotic Pump In Vivo
In Vitro Kinetic Studies
TCA Binding to Albumin
TCA Kinetics in IPRL
What Did We Learn from In Vitro Experiments
- TCA is poorly metabolized by the liver → Not sufficient to conclude that TCA is poorly metabolized in vivo
- Binding to albumin is a major factor in determining kinetics of TCA → Non-linear excretion kinetics
- No direct knowledge about other tissues
- TCA is poorly excreted in bile
- Free TCA concentration in liver intracellular water space (mediated transport processes suggested)
Summary of BBK Modeling Strategy
What Data are Required to Estimate the Importance of Kinetic Processes in Target Organ Toxicity?
Recommendations
- Develop a battery of in vitro assays for kinetic parameters that will provide a general idea of expected kinetics
- Develop QSAR methods to predict kinetic parameters
- Establish a database of chemical-independent parameters (mouse, rat, human)
- Conduct research on modeling of fundamental kinetic mechanisms
- Develop a library of "class" models that are acceptable for regulatory risk assessments