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Drs. Vicki Dellarco Anna LowitHealth Effects
DivisionOffice of Pesticide Programs

• Mode of Action/ Human Relevance Analysis For
  Incorporating Mechanistic Data in Human Health
  Risk

May 2006
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Uses of Mechanistic Data in Risk Assessment

• Identify
• Key biological (precursor) events leading to
  adverse toxicities (Mode of Action)
• Inform
• Human relevance of animal findings
• Dose response extrapolation
• Life stage susceptibilities
• Understand
• Common pathways of toxicity (cumulative risk
  assessment)
• Promote
• Consistent harmonized approach to risk assessment
  for all health endpoints

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Mechanism of action (more detailed
understanding at biochemical molecular level)
versus Mode of action (identification of
key obligatory steps)
Exposure
Key event
Key event
Key event
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Mode of Action Framework

• EPAs Guidelines for Carcinogen Risk Assessment
• 1996 Proposed Revisions
• put forth the notion of understanding mode of
  action versus mechanism of action
• 1999 Interim Guidance
• introduced mode of action framework
• 2005 Final Guidance
• minor rewording of MOA framework

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Mode of Action Framework

• Postulated mode of action
• Identify sequence of key events on the path to
  cancer
• Experimental support
• Concordance of dose-response for key events with
  that for tumors
• Temporal relationships for key events tumors
• Biological plausibility Coherence
• Strength, consistency specificity
• Other modes of action
• Identify uncertainties
• Conclusion

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Human Relevance Framework

• Risk Sciences Institute-ILSI
• Comparability or concordance analysis of the key
  events relevant biology between the laboratory
  species humans
• Tumor Responses Meek et al., 2003, Critical
  Reviews in Toxicology Vol 33/Issue 6, 581-653
• Reproductive, Developmental, Neurtoxocity
  Responses Seed et al., 2005 Critical Reviews in
  Toxicology Vol 35/Issue 8-9, 63-781
• extended human relevance analysis to include
  mutagenic carcinogens noncancer end points
• WHO/IPCS Human Relevance Framework (in
  preparation)

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Human Relevance Framework

• Based on three analyses
• Is the Weight of Evidence sufficient to establish
  a MOA in animals (MOA Framework)?
• Are the key events in the animal MOA plausible in
  humans?
• Taking into account kinetic/dynamic factors, is
  the animal MOA plausible in humans?

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Continue with the Dose Response Exposure
Assessment
No,
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Assessing an Animal Mode of Action

• General Points
• Applicable to all chemicals, to all endpoints,
  and to all modes of action
• Evaluation of MOA for tumors or (other adverse
  effects) in different organs
• MOA in different organs may or may not be the
  same
• Site concordance between animals humans

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Assessing an Animal Mode of Action

• General Points
• When a substance operates via a novel MOA, the
  analysis is focused on the chemical entails a
  detailed evaluation via the MOA Framework
• When a substance produces an adverse effect
  consistent with an already established peer
  reviewed MOA through which other chemicals have
  been shown to operate, the analysis is focused on
  the established MOA a determination of whether
  the substance operates via the same key events
  established for the pathway

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Assessing an Animal Mode of Action for Human
Relevance

• General Points
• Concordance Analysis of key events is for the MOA
  is not necessarily a chemical specific
  evaluation.
• Chemical specific generic information relevant
  to the toxicity process can be valuable

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MOA Inform Human Relevance
EPAs Cancer Assessment Review Committee (CARC)
classified atrazine as not likely to be
carcinogenic to humans.
Vinclozolin--Since the androgen receptor is
widely conserved across species lines,
antiandrogenic effects would be expected in
humans.
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MOA Inform Dose Response Extrapolation
Alachlor - . . . a margin of exposure (MOE)
approach (indicative of a non-linear dose
response) should be used for the risk
assessment.
Chloroform . . . a nonlinear approach is more
appropriate for low-dose extrapolation.
Cacodylic Acid . . . nonlinear default
approach (i.e., derivation of a reference dose or
margin of exposure) is regarded as the more
appropriate dose response extrapolation approach.
. .
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Case StudyCacodylic Acid (Dimethylarsinic acid)
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DMAV Mode of Action

• Science Issue Paper Mode of Carcinogenic Action
  for Cacodylic Acid (Dimethylarsinic Acid, DMAV)
  and Recommendations for Dose Response
  Extrapolation (July 26, 2005)
• http//www.epa.gov/oppsrrd1/reregistration/cacodyl
  ic_acid/
• Revised issue paper will be publicly available
  this spring.
• EPAs Science Advisory Board (SAB) reviewed the
  special issue paper in September, 2005
• Draft SAB report December 27, 2005

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Metabolism of Arsenic
Pesticide Chemical
Alternate steps of oxidative methylation
reduction
Methylation Reduction
TMAsV
TMAsIII
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DMAV MODE OF ACTION ANALYSIS
Weight of Evidence

• Extensive experimental cellular and laboratory
  animal data

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DMAV Available Cancer Data

• No epidemiology data
• Standard rodent bioassay
• Bladder carcinogen in rats
• via feed -100 ppm (9.4 mg/kg bw per day)
• via drinking water- 50 200 ppm
• females more sensitive than males
• Not carcinogenic in mice
• Up to 500 ppm in B6C3F (Gurr et al., 1989)
• 121 ppm in C57 XC3H/Anf or AKR (NCI 1969)

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Mode of Action Measurable Key Events in Target
Tissue
DMAIII Metabolite
Urothelial Toxicity
Urinary bladder from a female F344 rat treated
with 100 ppm DMAV
Sustained
Regenerative Proliferation
BrdU Labeling
Hyperplasia
Urinary Bladder Tumors
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Compensatory regeneration in rat bladder at weeks
8 10 following ingestion of DMAV
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Association of Key Precursor Events Bladder
Tumors in F344 Rats
Temporal
Dose Response Concordance
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Cacodylic Acid Key Events Temporal Relationship
DMAIII ? DMAV
Urinary bladder from a female F344 treated with
100 ppm DMAV
Urothelial Cytotoxicity Regenerative
Proliferation Hyperplasia Tumors
6 hours 1 Week 8-10 weeks 104 weeks
BrdU labeling
Urinary bladder tumors
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Cacodylic Acid Key EventsCytotoxicity/Regenerati
ve Proliferation

• Strength, Consistency Specificity
• Consistency of association found in repeated
  experiments within a lab among different labs
• Inhibition of DMAV ?DMAIII reduced cytotoxicity
• Cessation of exposure to DMAV results in recovery
  of tissue (i.e., hyperplasia)
• Biological Plausibility Coherence
• Regenerative proliferation associated with
  persistent toxicity appears to be a risk factor
  for bladder cancer in humans

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Characterization of Cacodylic Acids Genotoxicity

• Neither DMAV or DMAIII are direct acting
  point/gene mutagens
• Both are clastogenic but DMAIII is the more
  potent
• In vitro data only
• DNA damage appears to result from an indirect
  mechanism (ROS/oxidative damage)
• DMAIII ? DMAV

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Chromosomal Aberrations

• For the oxidative DNA damage to be relevant to
  the carcinogenic process (i.e., clonally
  expanded), stable chromosomal mutations must be
  formed
• Formation of chromosomal mutations requires DNA
  replication because chromosomal alterations are
  produced by errors of replication on a damaged
  DNA template.
• frequency of chromosomal mutations will be a
  function of the regenerative proliferative
  response.
• All these events--genetic errors, cytotoxicity,
  stimulation in cell proliferaiton -- must occur
  to result in bladder tumors.

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Other Modes of Action or Key Events

• No other MOA with sufficient scientific support
• Direct DNA reactivity
• Formation of solids
• Changes in urinary chemistry physiology

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Mode of Action Conclusions

• Sequence of key events leading to bladder tumors
  measurable supported by robust data
• Biologically plausible
• Uncertainties do not discount scientific support
• cellular target for cytotoxicity not understood
• unknown cytotoxic metabolites found in urine
  (after drinking water exposure)

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Human Relevance of DMAVs Mode of Action
Metabolism to DMAIII
Urothelial Cytotoxicity
?
Regenerative Proliferation
Hyperplasia Bladder tumors
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Concordance Analysis of Key Events in Rats
Humans Qualitative Quantitative Plausibility
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DMAV Mode of Action (MOA)

• The SAB concurred with EPAs conclusions
• Rat data developed for DMAV most appropriate data
  for quantifying cancer risk
• MOA for the development of bladder tumors in rats
  established
• The rat MOA is expected to be plausible in humans
• The MOA supports nonlinear extrapolation of
  cancer risk to DMAV

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Dose response extrapolation approach

• Dose response extrapolation should be based on
  considerations of MOA which supports nonlinearity
• Must be sufficient DMAIII to produce cell
  killing sufficient cell killing to lead to
  regenerative proliferation
• Cytotoxicity enhanced proliferation need to be
  sustained
• Frequency of chromosomal mutations dependent on
  enhanced proliferation on generation of ROS
  (DMAIII ?DMAV)
• Point of Departure based on cell proliferation
  should be protective of DMAs carcinogenic
  promoting effects

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Dose Response Considerations

• Cancer Guidelines describe a two-step
  dose-response process which separates
• Modeling the observable range of data
• Extrapolation to lower doses
• Nonlinear extrapolation
• Preferred approaches
• PBPK Model--internal dosimetry at the target
  tissue
• e.g. DMAIII
• BBDR Modelpredict biological effect
• e.g., two stage clonal growth
• Interim approach
• Identify a point of departure (POD) based on
  benchmark dose modeling
• Apply uncertainty and safety factors

Key event
POD
Response
Dose
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Quantitative Dose-response Assessment
MOA Established?
No
Yes

• 1. Fit data in observable range
• 2. Linear extrapolation from POD

BBDR model?
Yes
Use model
No
Yes, nonlinear
MOA informs low-dose extrapolation?
No
RfD/RfC or MOE
Yes, linear (including mutagenic MOA)
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Benchmark Dose Modeling Regenerative
Proliferation
Hill Model with 0.95 Confidence Level
Mean Response

BMD10
BMDL10
1
2
3
4
5
6
7
dose
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Cacodylic Acid Summary of benchmark dose
estimates and lower 95 confidence limits for
cytotoxicity, BrdU labeling index, hyperplasia
and tumor data. (Doses in mg/kg/day)
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PBPK Model Application to DMA Risk Assessment
Q What human exposure to DMAV is required to
produce the same target tissue dose of DMA to
bladder that results in tumors in rats exposed to
DMAV?

• Use PBPK model to estimate the environmental
  exposure to DMAV required to achieve the same
  target tissue dose to bladder.

oral exposure
DMAV
metabolism
elimination

• Estimate target tissue dose using various dose
  metrics (e.g., DMAV, DMAIII or TMAO concentration
  in urine or bladder tissue) associated with
  bladder tumor development using DMAV PBPK model.
  
• Current status Agency developing mouse model
  first then scale to rats and humans

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Summary

• Human Relevance Framework
• Identify key events
• Assist in dose response assessment
• Assist in rodent to human extrapolation
• Promote harmonization of risk assessment for all
  endpoints