Transcript Document

The International Society of Regulatory Toxicology and
Pharmacology (ISRTP) 2009 Endocrine Workshop
Lister Hill Auditorium, NIH Campus, Bethesda Maryland
Session 1: Strengths and Weaknesses of the EDSP Screen Assays
(September 9, 2009)
Challenges in Conducting and Interpreting Tier 1
EDSP Screening Assays:
CRO Perspective
Rochelle (Shelley) W. Tyl, Ph.D., DABT
Distinguished Fellow, RTI International
Email: [email protected]
www.rti.org
Phone: 919.541.5972
RTI International is a trade name of Research Triangle Institute
Tier 1 Screening to Detect Interactions With the
Endocrine System (EDSTAC, 1998)
The purpose of T1S is to obtain a minimum, yet
sufficient, set of valid and reliable data to detect
whether a chemical substance or mixture may interact
with the endocrine system. Included in T1S is a battery
of assays designed to detect effects that enhance,
mimic, or inhibit estrogen, androgen, and thyroid
hormone-related processes (mechanism of action). In
contrast to the more extensive and detailed tests of Tier
2, the T1S assays should:
(continued)
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• Be inexpensive, quick, and easy to perform
• Be validated and standardized, defining sensitivity and
specificity against a clearly defined standard
• Be more “sensitive” than they are “specific,” with their
primary objective the minimization of false negative (or
Type II) errors, while allowing an acceptable level of false
positive (or Type I) errors
• Capture multiple endpoints and reflect as many
modes/mechanisms of endocrine action as possible;
• Be broadly predictive across species, gender, and age
• Yield data capable of being interpreted as either positive
or negative to determine whether and how to conduct T2T
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Selected Overarching Principles for Development
of the EDSP (EDSTAC, 1998)
The screening and testing (S&T) strategy should:
• Require the minimal number of S&T necessary to make sound
decisions, to reduce the time and cost needed to make these
decisions
• Examine existing S&T data for adverse endpoints in high-dose groups
and also for physiological changes in low-dose groups
• Not detract from assessments of toxicity of chemical
substances/mixtures via mechanisms other than endocrine disruption
• Provide data that can be used for a broad range of management and
regulatory programs to support international harmonization of the
data’s use
• Use a performance-based approach to select better S&T as they are
developed and validated
• Be conducted at a minimal cost necessary to make the decisions
within the EDSTAC Conceptual Framework
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Six Specific Principles (EDSTAC, 1998)
1. To make decisions within the EDSTAC Conceptual Framework, all
S&T should have well-defined endpoints.
2. The use of animals should be reduced to the minimum needed to
obtain scientifically valid results and interpretations.
3. The results of S&T should support further research on effects of
endocrine disruptors on populations, communities, and ecosystems.
4. In interpreting S&T results, a “weight-of-evidence” approach should be
used, consistent with a principle of prudence in protecting human
health and the environment.
5. S&T results should be reported in a format that facilitates database
development and analysis by a broad array of scientific, regulatory,
and management organizations.
6. Decision criteria, such as those for statistical significance (e.g.,
necessary confidence intervals) and biological plausibility, should be
clearly defined.
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Tier 1 In Vitro Assays
EDSTAC in 1998
EDSP in 2009
Alternatives
Estrogen receptor (ER) binding
(cell free) or binding and
transcriptional activation
(transfected cell lines)
Same
None
Androgen receptor (AR)
binding (cell free) or binding
and transcriptional activation
(transfected cell lines)
Same
None
Steroidogenesis assay
with H295R cell line
(human female
adrenocortical carcinoma)
Placental aromatase
(converts
androstenedione or
testosterone to estradiol)
Steroidogenesis assay with
minced testes
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Tier 1 Screening In Vitro Assays (continued)
The in vitro assays, whether done “at the bench” or
through the high throughput prescreening, should:
• Evaluate binding to estrogen, androgen, and perhaps
thyroid nuclear receptors (cell free)
• Evaluate binding to the receptor in the presence and
absence of metabolic capability (e.g., one or more of
the P450 isozymes, CYP1A1, CYP3A4, etc.) in cell
lines
• Distinguish between agonists and antagonists in
functional assays
• Yield dose responses for relative potency of chemical
substances/mixtures with endocrine activity
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Tier 1 Screening In Vitro Assays (EDSTAC, 1998)
Advantages:
• Sensitivity to low concentrations increases detectability
• High specificity of response
• Low cost
• Small amount of chemical substance/mixture required
• In vitro assays can be automated, including use of robotics
• High throughput assays can be developed
• Results can be coupled with QSAR models and for database
screening
• Can be used for complex mixtures (sludge, water contaminants)
• Reduces or replaces animal use
(continued)
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Tier 1 Screening In Vitro Assays
Disadvantages:
• Cell-free ER/AR binding assays cannot differentiate
between agonists and antagonists
• Steroidogenesis assay with H295R cell line (only
recent access is expensive and very restrictive)
• In vitro assays do not include ADME of the whole
animal, so they can (and will) result in false positives
and (worse) false negatives
• They cannot be used as “decision nodes”
• In vivo assays always trump in vitro assays
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Tier 1 In Vivo Assays
EDSTAC in 1998
Rodent 3-day uterotrophic assay
(subcutaneous), adult
ovariectomized
Rodent 20-day pubertal female
assay with thyroid
EDSP in 2009
Modified rodent 3-day
uterotrophic assay
(intraperitoneal or
subcutaneous injection;
OECD TG 440)
None
Alternatives
• Intact weanling rodent 3-day
uterotrophic assay (validated;
OECD TG 440); intact HPG axis,
animal welfare concerns
• Rodent 20-day pubertal male
assay with thyroid
• Intact stimulated weanling rodent
Hershberger assay (less
sensitive); intact HPG axis, animal
welfare concerns
Rodent 5-7 day Hershberger assay
(adult castrate; OECD TG 441)
None
• Rodent 14-day intact adult male
assay with thyroid (EPA SAP: not
acceptable alternative)
• Modified OECD TG 407 (with
endocrine endpoints)
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(continued)
Tier 1 In Vivo Assays (continued)
EDSTAC in 1998
EDSP in 2009
Alternatives
Nonmammalian
Frog metamorphosis assay
(thyroid)
None
None
Fish short-term
reproduction
None
Fish gonadal
recrudescence assay
(phylogenetically farthest
from mammals)
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Tier 1 Screening In Vivo Assays (EDSTAC, 1998)
Advantages:
• Account for absorption, distribution, metabolism, and
excretion (ADME)
• Well-defined, acceptable methods used for decades
• General acceptability in toxicity testing
• Many endpoints are toxicologically relevant and are
used for risk assessment
• Evaluate a broader range of mechanisms
• Provide a comprehensive evaluation of the whole
endocrine system as a unit
• Give comparative perspective to other endpoints of
toxicity
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Tier 1 Screening In Vivo Assays
Disadvantages:
• Phytoestrogens in the feed and bedding can confound
estrogenic results
• Varying levels of “metabolizable energy” in feed can confound
estrogenic results in intact animals
• Incomplete removal of ovaries in the ovx uterotrophic assay can
result in endogenous estrogen production (check at necropsy)
• Castrate/ovariectomized animal assays only detect ER/AR
agonists or antagonists (dependent on receptor binding) and will
not detect EDCs with other mechanisms (e.g., phthalates) or
indirect EDCs
• Intact animal assays detect EDCs with broader range of
mechanisms (direct and indirect), but the intact weanling
Hershberger assay is less sensitive to weak anti-androgens
• There are no T1S assays with in utero or in ovo exposures.
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Interpretation
• In the best of all possible worlds, all of the in vitro and
in vivo screens are negative, so the test chemical
does not go to T2T
or
All the in vitro and in vivo screens are positive, so the
test chemical must go to T2T (at least it is very clear)
• In the real world, it is much more likely that some
screens are positive, some negative, and some
equivocal.
Now What?
“WEIGHT OF EVIDENCE ASSESSMENT”!
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“Weight-of-Evidence” Considerations
• At the end of T1S, to determine whether the evidence
warrants a particular conclusion (the substance does
or does not have endocrine activity for EAT) and to
determine progression (or not) to T2T
and
• Consideration on whether results from T1S should
affect the conclusions from T2T (should T1S assay
results be “weighted into” the determination of
whether a substance passes or fails the Tier 2 tests;
is it or is it not an EDC?)
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“Weight-of-Evidence” Considerations (continued)
Information considered in determining “weight-ofevidence” for T1S includes:
• The balance of assays that gave positive/negative
results
• Results of in vitro versus in vivo assays
• The nature of the biological effects induced
• The range of effects observed
• The slope and shape of the dose-response curves
• The level, magnitude, and/or severity of the effects
induced
• The presence/absence of response in multiple taxa
(least important?)
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“Weight-of-Evidence” Approach
• Results of some assays in some taxa, at some level
of severity, are intrinsically worth more than others
and should, therefore, carry more weight in decisions
following T1S and T2T.
• Is there consistency in results of assays with same
mechanism/mode of action (intentional redundancy)?
• The focus of T1S is on sensitivity rather than
specificity. Therefore, the focus is to minimize false
negatives (the risk we miss an EDC) and allow a
certain level of false positives (but problem for
registrants, unnecessary animal usage, costly, etc.)
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Final Comments
• In vitro assays are more useful for determining mechanism than for
determining endocrine activity (I think that they should be done last).
• Most estrogen EDCs to date have been estrogen agonists or estrogenic
(estrogen mimics).
• Most androgen EDCs to date have been androgen antagonists or antiandrogenic (one exception: an androgen agonist, trenbolone, to fatten
cattle found in feed lot effluents).
• The number of animals per group are rather small (typically six), but the
endpoints are almost all organ weights and are therefore objective, not
subjective.
• The 15-day intact adult male assay relies heavily on hormone assays
(organ weights and histopathology are included, but the exposure period
to an intact adult is brief), so the timing of necropsy and blood sampling
is exquisitely important (many/most of the hormones are cyclically
released with different peak timings: hourly, diurnally, etc.).
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Final Comments (continued)
• Analyze the feed and bedding (phytoestrogens can be major
confounders)!
• Make sure the necropsy team is trained, experienced, and
consistent.
• Routes of administration were not considered terribly important
at the time of EDSTAC. Now we know that ADME of
xenobiotics is absolutely dependent on route with both
quantitative and qualitative differences.
• Rodents have enterohepatic circulation, so even if the
xenobiotic is metabolized (detoxified) in the gut, it can lose the
glucuronide or sulfate in the liver and pass via the bile duct back
into the gut (prolonging exposure to the parent compound).
• Metabolism to a toxic metabolite can also occur and can be
route dependent.
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Final Comments (continued)
• EDSTAC never got around to in vitro thyroid receptor
assays, but we know a lot more now about the roles
and regulation of the thyroid gland (TSH, T3, T4, rT3,
etc.).
• Adult surgical models versus intact weanling models:
strengths and weaknesses.
• One outstanding deficit in T1S: no evaluation of
consequences of prenatal/prehatch exposures (not in
EDSTAC or EDSP); T2T does include these.
• The more the CROs know about your chemical and
results of other tests, the more they can assist you in
study design, conclusions, and interpretation.
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Take-away Points
• T1S assays should be interpreted as part of the
screening battery
• EPA should define an optimal T1S battery
• EPA should provide guidance on interpreting T1S
battery results
• There is intentional redundancy in T1S in vivo assays
• Some endpoints have more inherent variability than
others (e.g., body weights, organ weights, age at
vaginal patency and preputial separation, hormone
levels, etc.)
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Take-away Points (continued)
• Should use appropriate statistical tests
• Use “worst case scenarios” to detect endocrine active
substances/mixtures
• If we accept “false positives,” it will be expensive, unnecessary
use of many more animals, and a waste of time
• T1S is not about assessing risk
• T1S is not about endocrine disruption
• T1S is designed to identify those chemicals/mixtures capable of
interacting with the endocrine system to go on to T2T (or not)
THANK YOU!
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