Adverse Outcome Pathways (AOPs) focusing on … 6 James Wheeler.pdf · Adverse Outcome Pathways...
Transcript of Adverse Outcome Pathways (AOPs) focusing on … 6 James Wheeler.pdf · Adverse Outcome Pathways...
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What best describes your expertise?
1. Ecotoxicologist
2. Toxicologist
3. High throughput –ologist
4. Still working on it…
5. Other
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Ecoto
xicolo
gist
Toxicolo
gist
High th
rough
put –olo
gist
Still w
orkin
g on it
…Oth
er
12%
57%
16%
6%9%
Understanding the audience
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Outline
● Regulatory background
Potential to increase animal use
● ECETOC task force
● Aromatase inhibition in small fish models
● Case study
● Conclusions
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Regulatory background
● Endocrine disruption has become a topic of increasing public and
regulatory concern
● Policies and legislation in the major regions have been implemented
In the US and Japan a 2-tiered screening and testing programmes are in
place
In Europe hazard based criteria are being implemented which will likely
employ the same screening and testing
● Such testing will require significant additional vertebrate testing
Fish
Amphibians
Birds
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Potential to increase animal use
Low High
Low
H
igh
Animal numbers
Severity
of
the t
est
96 Fish
Short-term
reproduction
480 Fish
Short-term
reproduction
+ Sexual
Development
588 Fish
Medaka
Extended
One
Generation
320 Amphibian
Metamorphosis
480 Amphibian
Larval Growth and
Development
852 birds
Two-
generation
test
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● Examine how AOPs for endocrine active substances might be
constructed and assess the data required to come to sound
conclusions that might have regulatory applications
● Particular utility of AOP for the identification of endocrine disrupting
properties
ECETOC task force
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Molecular Initiating
event
Key event1
Key event2
Key event3
Adverse Outcome
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ECETOC task force
● Avoid the mis-use of AOP to conclude on endocrine disrupting
properties of chemicals
● Build on previous ECETOC work on the identification of endocrine
disrupting properties
● Use recent guidance and examples
ECHA’s mode of action work
US-EPA’s action on integrating mechanistic and apical information
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ECETOC task force
● Acknowledge that the application will influence the required level of
confidence in the AOP
Initiating event
Key events
Individual or population level Adverse Outcome
● Tailor guidance based on proposed application
Priority setting (read across)
Integrated approaches to testing and assessment (IATA)
Hazard identification
Risk assessment
● Trade-off between simplification and completeness
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Aromatase inhibition in small fish models
Aromatase inhibition Initiating event
↓ oestradiol
↓ female vitellogenin
↓ vitellogenin deposition in ovary
↓ fecundity Adverse Outcome
Ankley et al. (2009) Aquat. Toxicol. 92: 168–178
Increased
confidence
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Case study
● ZapazoleTM
● Triazole fungicide
14α-demethylase inhibitor
● Class known to inhibit
cytochrome P450 aromatase
affecting biosynthesis of
oestrogens from androgens
● In vitro steroidogenesis
↓ testosterone
↓ oestradiol
● In vitro aromatase
↓ human recombinant
aromatase
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Case study
● In vivo screening
Fish Short Term Reproduction Test
─ ↔ fecundity and fertility no change
─ ↓ female vitellogenin (mid and high treatments)
─ ↓ female and male gonado-somatic index (GSI)
─ ↑ female histopathologies (high treatment)
● In vivo mid-tier
Fish Sexual Development Test
─ ↔ sex ratio
─ ↓ female yolk accumulation in oocytes (treatment related)
─ ↑ male testicular and sperm development (treatment related)
● In vivo high-tier
Fish Full Life Cycle (no endocrine specific endpoints)
─ ↓ fecundity (high treatment)
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AOP 25: Aromatase inhibition leading to reproductive dysfunction
(in fish)
In vitro
aromatase
inhibition
In vitro
steroidogenesis
In vivo
FSTRT +
FSDT
In vivo
FFLC
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Do you believe ZapazoleTM is an endocrine disrupter in fish?
1. Yes
2. No
3. Unsure
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YesNo
Unsure
52%
42%
6%
Case study
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Case study: What I didn’t tell you
● Environmental exposures are extremely low compared to in vivo effect
levels
● ZapazoleTM is not a potent inhibitor of aromatase
ZapazoleTM IC50 = 68 µM vs fadrozole IC50 = 0.0076 µM
● ZapazoleTM is a known mammalian liver toxicant
Evaluation of fish liver tissues from endocrine studies indicated a
concentration response for
─ Hepatocyte necrosis
─ Decreased hepatocellular vacuolation
─ Female vitellogenin was only reduced in the presence of moderate to severe liver
changes
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Case study
● Fish liver toxicity
Liver site of vitellogenin synthesis
Direct damage / degenerative changes
Enlargement
─ Induction of biotransformation enzymes
─ Increased hormone clearance
● Potential for effects to be
misinterpreted as a
steroidogenic effect
● CEFIC-LRI project: Assessment
of potential endocrine activity in fish – elucidation of the role of liver
toxicity in the vitellogenin response (Profs Braunbeck and Segner)
Mommsen and Walsh 1988
Fish Physiology Vol XIA
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A broader problem: systemic toxicity
Primary
response
Secondary
response
Tertiary
response
Stress
↑ Corticosteroid hormones
↑ Catecholamine hormones
↑↓ Neurotransmitters
↑↓ Gonadotropins
↓ Steroidogenesis
↓ 11-Ketotestosterone ↓ Estradiol → vitellogenin
↓ Ovarian development
(atresia and resorption)
↓ Gonadal Somatic Index
↑↓ Time to sexual maturity
↓ Fecundity
↓ Gamete quality
↑↓ Sex reversal
Fish
Stress as a neuro-endocrine
cascade
– Hypothalamus
– Pituitary
– Cortisol or corticosterone
– Adaptive changes
– Adverse changes
See Wheeler and Coady 2016
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Relevance to AOPs and regulatory decision making
● Specific toxicities (e.g. liver) and systemic stress can lead to
‘endocrine’ responses
Screening assays
Higher tier definitive tests
● These changes should not be mistaken for primary endocrine effects
● AOP applications
Priority setting – less important as false positives acceptable
IATA – could lead to unnecessary testing
Hazard assessment
─ Could lead to misidentification of a chemical as an endocrine disruptor
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Conclusions
● AOPs have great utility for the identification and characterisation of
endocrine disruptors
Increasing confidence in the link between the mechanism and adverse
effect
Potential to significantly reduce animal use
● However, depending on application the level of confidence required
varies
Should be explicitly considered before applying an AOP
Ensure appropriate decisions are made
● Regulatory application needs
Development of better tools for ecotoxicology (wildlife) species
Quality criteria and minimal data requirements
Appropriate exposure assessments (external and at the target site)
See Wheeler and Weltje 2015
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Acknowledgements: ECETOC taskforce
● Richard Green Syngenta
(Chairman)
● Malyka Galay-Burgos
ECETOC
● Lennart Weltje BASF SE
● Sophie Duhayon Total
● Arnd Weyers Bayer
CropScience
● Remi Bars Bayer CropScience
● Ivana Fegert BASF SE
● Uwe Ensenbach Clariant
● Wasma al-Husainy Shell
● Sylvia Jacobi Albemarle
● Rick Becker American
Chemical Council
● Christine Palermo Exxon
Mobil
● Marc Leonard L’Oreal
● Matthew Dent Unilever
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References
● Bars et al. (2011) Science based guidance for the assessment of
endocrine disrupting properties of chemicals. Regulatory Toxicology
and Pharmacology 59, 37-46.
● Bars et al. (2012) Risk assessment of endocrine disrupting chemicals.
Regulatory Toxicology and Pharmacology 64, 143-154.
● Weltje et al. (2013) Refinement of the ECETOC approach to identify
endocrine disrupting properties of chemicals in ecotoxicology.
Toxicology Letters 223, 291-294.
● Wheeler and Weltje (2015) In Response: Adverse outcome
pathways—An industry perspective. ET&C 34(9): 1937-1938.
● Wheeler and Coady (2016) Are all chemicals endocrine disrupters?
IEAM 12(2): 402-403
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