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Of Lists and Legends: An Endocrine Disruptor Update
Ellen Mihaich, Ph.D., DABT CIR Expert Panel Meeting December 5, 2016
1
Endocrine Policy Forum • Consortium of List 1 test order recipients and
other interested/involved parties • Self-funded • Represents >95% of List 1 test order recipients • Additional stakeholders include CLA, ACC, ACI,
CSPA, API, and consulting companies • Objective to address regulatory and policy
issues, technical guidance and science advocacy • To undertake collaborations to tackle common areas
of interest
2
What Is An Endocrine Disruptor?
3
• WHO/IPCS 2002 • An endocrine disruptor is an exogenous substance
or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations.
• Resource and regulatory consequences
US EPA’S LEGISLATIVE MANDATES (1996)
Food Quality Protection Act oMust screen pesticides (including inerts) for possible
estrogenic effects that may affect human health oMust use appropriate validated test systems or other
scientifically relevant information oCan include other endocrine effects
Safe Drinking Water Act (SDWA) oCan screen drinking water contaminants to which
substantial numbers of persons are exposed “Substantial numbers” and “may be found in sources
of drinking water “ not defined
4
EDSP HISTORY: 1996 TO TODAY
1996-1998 Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC)
o EPA multi-stakeholder advisory committee evaluates methods for priority setting, screening, testing, and communications as key program elements
1999 to Today: Endocrine Disruptor Screening Program (EDSP) o EPA adopts EDSTAC recommendations, creates EDSP, and in coordination with
OECD, works to standardize/validate screening and test protocols and guidelines
2009 1st Tier 1 List finalized: contained 67 pesticides and pesticide inerts 2013 Second Tier 1 List finalized; 109 chemicals (41 pesticides)
o Office of Management and Budget has not approved the Information Collection Request (ICR) from EPA
o No test orders can be released until OMB approval of the ICR
Work is ongoing to prioritize the list of “10,000” chemicals for screening so current List 2 chemicals may not be chosen ultimately
5
EDSTAC Conceptual Framework
Tier 1 Screening ◦ Eleven In Vitro and In Vivo Assays ◦ Identify chemicals that can potentially interact with the endocrine
system (Estrogen, Androgen, and Thyroid pathways) ◦ Maximize sensitivity to minimize false negatives ◦ A battery of screening assays, with deliberate redundancy
Tier 2 Testing: ◦ Multigen studies in a range of species ◦ Confirm endocrine activity detected in Tier 1 and characterize
adverse effects and ◦ Establish NOAELs and LOAELs for risk assessment for regulation
of the substance
6
7
Tier 1 * Tier 2
In vitro
ER binding
ER transcriptional activation
AR binding
Steroidogenesis, H295R
Aromatase, recombinant
In vivo (toxicology)
Uterotrophic assay 2-generation rat reproduction study
Hershberger assay (or extended 1-generation study)
Pubertal male assay
Pubertal female assay
In vivo (ecotoxicology)
Fish short-term screening assay Medaka extended 1-generation study
Amphibian metamorphosis assay Larval amphibian growth and development test
Avian 2-generation reproductive test
* Tier 1 screens cost $750,000 to $1 million per chemical
Current EDSP Test Guidelines
List 1 Results 52 chemicals (primarily pesticides) were screened in
most, if not all, of the tier 1 screens oEPA evaluated all of the data and wrote weight of
evidence reports All reports and study summaries are publically available on the EPA
website There was no evidence for potential interaction with any of the endocrine
pathways for 20 chemicals For 14 chemicals that showed potential interaction with one or more
pathways, EPA already has enough information to conclude that they do not pose risks.
Of the remaining 18 chemicals, all 18 showed potential interaction with the thyroid pathway, 17 with the androgen pathway, and 14 with the estrogen pathway.
8
EPA Overall Conclusions For several of the chemicals displaying activity in the Tier 1
screening assays, EPA determined they have enough information to conclude that they do not pose risks. EPA recommended the following higher tier studies: o A comparative thyroid assay for 4 chemicals that showed interaction
with the thyroid pathway in mammals (not one of the validated Tier 2 tests)
o A medaka extended one-generation reproductive test (MEOGRT) for 13 chemicals that showed potential interaction with the estrogen or androgen pathways in wildlife
o A larval amphibian growth and development assay (LAGDA) for 5 chemicals that showed potential interaction with the thyroid pathway in wildlife
9
A Tier 2 Quandary
• Tier 2 is for determining adverse effects and to provide data for risk assessment
• In regulatory toxicology we test at levels that achieve some effect
• How will we determine if that effect is specifically endocrine related? • Not as much of a problem in the US where regulations are based
on risk but, • In Europe, deciding if the effect is “endocrine” or not may have
implications for authorization based solely on hazard
10
If Only WoE Was This Easy!
Courtesy of Tim Ward 11
The Dilemma of Many Endpoints
Chance of a Clean Chemical Screening “Clean” (1- p)n
Tier 1 Endocrine Screening Battery:
89 endpoints; if all independent (0.95)89 = 0.01 89 endpoints; if every 4th independent: (0.95)22 = 0.32
Critical Elements in a Weight of Evidence Assessment
• Reliability of information o Quality of the study, transparency of reporting
• Relevance of the information o Appropriate for the question being asked
• Adequacy (or usefulness) of the information o Fit for regulatory decision-making
• Consistent pattern of response o In support of a particular hypothesis
Borgert et al., 2011. Reg Toxicol Pharmacol 61:185-191. Borgert et al., 2014. Birth Defects Res Part B: Develop Reprod Toxicol 101:90-113. de Peyster and Mihaich, 2014. Regul Toxicol Pharmacol 69:348-370.
12
WOE Conceptual Framework
1. Define Explicit Hypotheses • 8 Total for Tier 1 • Potential E+ / E- / A+ / A- / T+ / T- /Steroidogenesis
induction/Steroidogenesis inhibition 2. Systematic Literature Review 3. Evaluate Data Quality 4. Weight Endpoints Quantitatively or Rank-Ordered
based on Explicit Criteria and Data 5. Weight Results within the context of known positives
and negatives 6. Develop Narrative Interpretation listing all assumptions
13 Framework: Borgert et al. 2011 Reg Tox Pharm 61:185-191 Relevance weightings: Borgert et al. 2014, Birth Defects Research, Part B, 101:90-113
Ranking Endpoints
• No hypothesis can be decided on the results of a single assay – Battery Approach
• Rank the relevance (Wrel) of each endpoint for deciding each hypothesis • Rank #1: specific & sensitive for the hypothesis; interpretable
without other endpoints; in vivo endpoints rarely confounded by artifacts or non-specific activity.
• Rank #2: specific & sensitive for the hypothesis; interpretable alone, but less informative than Rank #1, often due to potential confounding; in vitro assays, many in vivo endpoints
• Rank #3: Relevant but only when corroborative of Rank #1 and #2 endpoints; many apical in vivo endpoints.
• No Response (NR)
14
Relevance Rankings By Hypothesis: Estrogen Agonist
15
Estrogen Agonist Hypothesis
Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints
FSTRA ERTA ER binding
Vitellogenin: increased in males ER agonism ER competitive binding
Uterotrophic Assay FSTRA FSTRA
Increased uterine weight (wet/blotted) Secondary sexual characteristics: decreased tubercle score: males
Fecundity
Gonad histopathology: males Female behavior
Behavior: males GSI: males, females
Pubertal Female Assay Gonad histopathology: Follicular atresia
Age&weight @vaginal opening: reduced Fertilization success
Ovary weight: reduced Estradiol
Age at first estrous: reduced Testosterone
Pubertal Male Assay Pubertal Female Assay
Testes weight Growth
Testes histopathology: atrophy Estrous cyclicity
Pubertal Male Assay
Growth
Ventral prostate weight
Epididymides histopathology
Steroidogenesis Assay
Estradiol levels
Relevance Rankings By Hypothesis: Androgen Agonist
16
Androgen Agonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints
Hershberger Pubertal Male Aromatase Weights of Cowper's gland, seminal vesicle, LAB, glans penis, ventral prostate: concordance of all endpoints
Age&weight @preputial separation: if accelerated
Aromatase inhibition
FSTRA Seminal vesicle + coagulating gland weight (wet/blotted)
FSTRA
Secondary sexual characteristics: decreased tubercle score: females
Ventral prostate weight Fecundity
Dorsolateral prostate weight Testosterone and estradiol levels LABC muscle complex weight GSI Epididymis weight Behavior Testes weight Fertilization success Testes histopathology Pubertal Female Epididymides histopathology Growth
FSTRA Age&weight @vaginal opening Vitellogenin: reduced in females Uterus weight Gonad histopathology Ovaries weight
AR Binding Adrenals weight Competitive binding - NO Uterus histopathology
Hershberger assay Ovary histopathology Concordance of two or more endpoints (see rank 1)
Pubertal Male
Growth Testosterone levels
Steroidogenesis Testosterone levels
Hershberger assay Only one of five endpoints respond (see Rank 1)
Relevance Rank-FSTRA Endpoints
17
Endpoint E+ E- A+ A- T+ T- Steroid. induct.
Steroid. inhib.
Vitellogenin R1; m R2; f R2; f R2; f NR NR NR R2; f
2o Sex Characteristic
s
R2; m tubercle
score
NR R1: f tubercle
score
R2; m tubercle
score
NR NR NR NR
Fecundity R3 R3 R3 R3 NR NR NR R3
Estradiol Opt.R3 Opt.R3 Opt.R3 Opt.R3; m NR NR NR Opt.R3
Testosterone Opt.R3 Opt.R3 Opt.R3 Opt.R3; f NR NR NR Opt.R3
Gonad Histopatholog
y
R2 m R2; f R2 R2 NR NR NR R2; m
GSI R3; m f
R3 R3 R3 NR NR NR R3
Behavior R2 m R3 f
R3 R3 R3 NR NR NR R3
Fertilization Success
R3 R3 R3 R3 NR NR NR R3
Adult Survival NR NR NR NR NR NR NR NR
Directionality Important in WoE
• Thyroid agonist hypothesis and Triclosan
18
Rank Assay Endpoint(s) Response Reference(s)
1 Amphibian
Metamorphosis Assay
Asynchronous development
No effect [1] [2]
Thyroid histopathology
No effect [1] [2]
2
Amphibian Metamorphosis
Assay
Developmental stage No effect [1] [2]
Hind limb length No effect [1] [2]
Pubertal Male Assay
Thyroid weight
Serum T4 ↓ {3}
Serum TSH No effect [3]
Pubertal Female Assay
Serum T4 ↓ {4}
Serum TSH No effect [4]
3
Amphibian Metamorphosis
Assay
Snout-vent length No effect* [1] [2]
Wet weight No effect* [1] [2]
Pubertal Female Assay
Growth No effect [4] Age & weight @ vaginal opening ↓
{4}
Blood chemistry
Pubertal Male Assay
Growth No effect [3] Age & weight @ preputial separation
No effect [3]
Pituitary weight No effect [3]
*Snout-vent length and wet weight were reported to have increased in Fort et al. (2011), however, a thyroid agonist would be expected to decrease these parameters so they are shown as “no effect” for this hypothesis. [1] Fort et al. (2010) [2] Fort et al. (2011) [3] Zorrilla et al. (2009) [4] Stoker et al. (2010)
Mihaich et al. in press, Crit Rev Toxicol – WoE for Triclosan
Evaluating the Responses
• Positive or negative responses in Rank 1 • Preliminary indication of support for or against hypothesis
• Consistent positive or negative responses in Ranks 1 and 2 • Sufficient support for or against the hypothesis • Consult Rank 3 for positive, not needed for negative
• Positive responses in Rank 2, but not Rank 1 • Strength of response and corroboration with Rank 3 critical, case-
by-case evaluation needed
19
Hypothesis-Based WoE Framework • Uses explicit relevance rankings of endpoints derived a
priori • hypothesis-based versus hypothesis-generating
• Transparent, objective and consistent interpretation of results for each hypothesis
• Systematic method to resolve inconsistencies in the data and focus on the most definitive information
• Can be applied to Tier 2 and other MoA as well • Can be updated and altered with new information
20
EPA is Shifting Towards Faster Screening Methodologies
Toxicity Testing in the 21st Century o Criticism of EPA for how long the EDSP has taken and an interest in
reducing animal testing has prompted new technologies and processes
o ToxCast™ program at the heart of EPA screening methodology for bioactivity Battery of more than 700 in vitro, high-throughput screens Approximately 15% are directly relevant to EDSP Over 1,800 chemicals already screened and data in the public domain
(actor.epa.gov/dashboard)
oExpoCast™ to be used with the
bioactivity screens in order to consider exposure Currently only for human health
assessment
21
0 .00 1
0 .01
0 .1 1 1 0
A RC e ll a d h e s io n m o le c u le s
C e ll c y c leC y p
C y to k in eD e v e lo p m e n tD N A b in d in g
E RG P C R
G RM R
N u c le a r re c e p to rP R
P ro te a s eP X R
S te ro id h o rm o n eT ra n s p o rte r
E E 2 in th e T o x C a s t B a tte ry
A C 5 0 v a lu e s (µM )
low
er
lim
it o
f c
yto
tox
icit
y
Cy
toto
xic
ity
po
int
Understanding Essentiality of Key Events: Generalized AOP
22
Daniel L. Villeneuve et al. Toxicol. Sci. 2014;142:312-320
Chemical Property Profile
Receptor/Ligand Interaction
DNA Binding
Protein Oxidation
Gene Activation
Protein Production
Altered Signaling
Protein Depletion
Altered Physiology
Disrupted Homeostasis
Altered Tissue Development or Function
Lethality
Impaired Development
Impaired
Reproduction
Cancer
Toxicant Macro-Molecular Interactions
Cellular Responses
Organ Responses
Individual Responses
Structure
Recruitment
Extinction
Population Responses
Adverse Outcome Pathway
CN
NN
Aromatase inhibition
8
0
2
4
6
E2
(ng/
ml)
*
*
0
10
20
Vtg
(mg/
ml)
*
* *Control 2 10 50
Fadrozole (µg/l)
8
0
2
4
6
E2
(ng/
ml)
*
*
0
10
20
Vtg
(mg/
ml)
*
* *Control 2 10 50
Fadrozole (µg/l)
-20 -18 -16-14-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20Exposure (d)
0
2
4
6
8
10
(Tho
usan
ds)
Cum
ulat
ive
Num
ber o
f Egg
s
Control21050
Fadrozole (ug/L)
***
-20 -18 -16-14-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20Exposure (d)
0
2
4
6
8
10
(Tho
usan
ds)
Cum
ulat
ive
Num
ber o
f Egg
s
Control21050
Fadrozole (ug/L)
***
Reduced E2, Vtg synthesis
Impaired vitellogenesis Reduced fecundity
Molecular initiating event
Key events or predictive relationships spanning
levels of biological organization
Adverse outcome relevant to
risk assessment
Adverse Outcome Pathway
Courtesy of G. Ankley 23
Inhibition Aromatase
Reduction E2 levels
Reduction Vitellogenin levels
Reduction Fecundity;
filial ELS survival
Decline Population trajectory
Strong Evidence
Moderate Evidence
Weak Evidence
Comparative Evidence
Direct PPZ evidence in fish
Comparative PPZ evidence in mammals
Comparative PPZ high-throughput evidence
No evidence, potential KE
Key Events
Key Event Relationships
Legend
Possible Adverse Outcome Pathways Leading to a Reduction in Fecundity in Fish Exposed to Propiconazole
Activation CAR/PXR
Increase E2 biotransformation
Increase Cytochrome P450
expression Reduction
E2 synthesis
Decrease Transcription/Translation
Increase Oxidative stress
Dysfunction Mitochondrial
24
A Line of Empirical Evidence: The Acute to Chronic Ratio
• The acute endpoint (e.g. LC50) represents non-specific baseline toxicity.
• The chronic endpoint typically reflects a more specific mode of action.
• The more potent an endocrine disrupting agent, the higher will be the acute to chronic ratio (ACR). Ethinyl estradiol ACR = 5,700,000 Most industrial chemical ACR = 10-100
25
Calculating ACR
• Acute and chronic aquatic toxicity studies were assembled from published studies and government reports
• Scientifically valid studies – data quality important • Acute LC50 (mortality) and chronic NOEC for
survival, growth and development, and reproductive endpoints were recorded
• Acute to chronic ratios were determined • Comparisons made between same species • All endpoints, endpoints minus chronic survival, and
reproduction only endpoints • All species combined and then separated by fish,
invertebrate, and aquatic plant/algae
26
ACR Based on Taxa
Fish Invertebrate Algae
27
Now, Does It Make Sense?
• Propiconazole aromatase inhibiting, E and A activity in the same range (or higher) as other non-endocrine bioactivity
0 .00 1
0 .01
0 .1 1 1 0
A RC e ll a d h e s io n m o le c u le s
C e ll c y c leC y p
C y to k in eD e v e lo p m e n tD N A b in d in g
E RG P C R
G RM R
N u c le a r re c e p to rP R
P ro te a s eP X R
S te ro id h o rm o n eT ra n s p o rte r
E E 2 in th e T o x C a s t B a tte ry
A C 5 0 v a lu e s (µM )
low
er
lim
it o
f c
yto
tox
icit
y
Cy
toto
xic
ity
po
int
Propiconazole in the ToxCast Battery • EE2 estrogenic bioactivity much lower than other
bioactivity
28
0 1 2 3 4 5
P P AR
M a tr ix M e ta llo p r o te in a s e
G -P r o te in R e c e p to r
C yto k in e
X e n o b io t ic m e ta b o lis m
E s tr o g e n R e c e p to r
T o x c a s t R e s u lts fo r B P A
A C 5 0 v a lu e s (µ M )
• Bioactivity screen indicates multiple MoA for BPA in the same concentration range
Litany of Lists In 2002, BKH, under contract to DG Environment developed a list of
substances for further evaluation. Other regulatory authorities (e.g., Washington State, Denmark) have used this list to
develop their own lists of endocrine disruptors
UNEP/IPCP “Overview Report I: A Compilation of Lists of Chemicals Recognised as Endocrine Disrupting Chemicals (EDCs) or Suggested as Potential EDCs“ Compilation of lists developed for different purposes Not specific for potentially endocrine active chemicals, Utilizing different methodologies and criteria for designating a chemical as endocrine
active or an endocrine disruptor, No requirement for appropriate data quality or weight of evidence
Endocrine Active Substances Information System (EASIS) Released by the European Commission – DG Environment Searchable database of >500 substances Disclaimer that it has both positive and negative substances No WoE, systematic literature search, or quality criteria apparent https://ec.europa.eu/jrc/en/new/launch-endocrine-active-substances-information-system-easis
29
30
State of the Science of Endocrine Disrupting Chemicals
2002 report
World Health Organization (WHO) and
International Programme on Chemical Safety (IPCS)
2012 report
World Health Organization (WHO) and
United Nations Environment Programme (UNEP)
Critical Review: WHO-UNEP 2012 Report by Lamb et al.
• Reviewed described process and methods • Compared to 2002 report • Evaluated all chapters to identify common strengths and
weaknesses • Identified key issues and concerns
• Select examples of unsupported claims • Not a comprehensive assessment or re-evaluation
• Open access manuscript published in peer reviewed journal Reg Tox Pharm: Lamb et al. 2014.Critical comments on the WHO-UNEP State of the Science of Endocrine Disrupting Chemicals – 2012 http://www.ncbi.nlm.nih.gov/pubmed/24530840
31
Overall Concerns Identified by Lamb & colleagues in the WHO-UNEP 2012 Report
• Not an objective, state-of-the-science review • Not an update of WHO-IPCS 2002 report • Causation is often inferred – not established • Controversial topics are poorly addressed • Summary for Decision-Makers
• Not a summary of the main report • New “concerns” raised that were
not in full report
32
Example: Not an Update Semen/Sperm Quality
WHO-IPCS 2002:
WHO-UNEP 2012:
33
Cost of Endocrine Disruptors • In 2015, a series of studies claimed that human
exposure to EDCs is costing the EU hundreds of billions of Euros
• Recently, a similar paper targeted the US, with health costs estimated even higher than in Europe.
• Widely criticized by economists, scientists, and even the European Commission
• Most of the cited chemicals do not meet the WHO-IPCS definition of an ED
• Incomplete and inconsistent correlations between exposure and specific health outcomes
• Lack of transparency in methodology • However, flashy headlines are news!
34
Endocrine Assessments in Japan
35
• Japan is currently developing assays with which to evaluate endocrine properties
• Many are part of the OECD test guideline development program
• The government is also testing selected materials (some based on effects, some based on detection in monitoring programs)
• SPEED 98 and ExTEND Programs
• Considering two-tiered approach that still includes ecological risk assessment
• Considering in vitro assays used to prioritize to enhance efficiency
Endocrine Assessments in China
• Draft of an agro-industrial standard ‘Evaluation Methods for Pesticide Endocrine Disruptors became available in late 2014
• Final version implemented April 2016 • Focus on in vivo mammalian tests
36
Endocrine Assessments in China
• 2 tier system (similar to US-EPA) • Phase 1
• In vitro tests: ER Transcriptional activation and Steroidogenesis assays
• In vivo tests: Uterotrophic, Hershberger and Pubertals • Phase 2
• Extended One-Generation Reproductive Toxicity • Triggered by ‘positives’ at phase 1
37
Elsewhere in the Asia Pacific Region
• Most APAC countries are monitoring developments elsewhere in the world
• Australia publically stated that from a regulatory perspective they consider “endocrine disruption” no different than other modes of action suggesting it will be regulated using a risk-based approach
• Some APAC countries are beginning to request additional OECD endocrine studies if reproductive effects are noted in the standard pesticide submissions
38
Endocrine Assessments in Latin America
• Brazil and Chile are the only Latin American countries where the endocrine issue is under discussion by the regulators
• Although there is gaining interest on the part of NGOs, particularly in Argentina, concerning pesticides and ED
• Brazil appears to be inclined to a hazard-based approach similar to the EU, although they appear open to other possibilities
• Chile, so far, does not appear to be considering a hazard-based approach
39
Quick Quiz Should This Chemical Be Banned?
Assay Result In Vitro ER Transactivation Negative
In Vitro Steroidogenesis Increased Estrogen production at 500 and 1000 µM
Male Rat Pubertal Assay ⬇ body weight. (100 mg/kg/d)
⬇ prostate weight (> 20 mg/kg/d)
⬇ seminal vesicle weight (> 20 mg/kg/d)
⬆ testis weight (> 20 mg/kg/d)
⬇ plasma progesterone (dose-related)
Female Pubertal Assay ⬇ ovarian weight (100 mg/kg/d)
⬇ adrenal gland weight (100 mg/kg/d)
⬆ mRNA levels for FSH (100 mg/kg/d)
⬇ mRNA levels for prolactin (100 mg/kg/d)
⬆ mean length of estrus cycle (100 mg/kg/d)
⬇ (delay) vaginal opening (> 5mg/kg/d)
⬆ age at first estrus by 8 days (> 5 mg/kg/d) From Tinwell H. et al., 2013. Regul Toxicol Pharmacol.66:184-196.
41
Thank you.
Questions?