adverse outcome pathways (aops) focusing on … 6 james wheeler.pdf · adverse outcome pathways...

Adverse Outcome Pathways (AOPs) focusing on endocrine active chemicals

James Wheeler

| 2

What best describes your expertise?

1. Ecotoxicologist

2. Toxicologist

3. High throughput –ologist

4. Still working on it…

5. Other

DOW AGROSCIENCES RESTRICTED

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

| 3

Outline

● Regulatory background

Potential to increase animal use

● ECETOC task force

● Aromatase inhibition in small fish models

● Case study

● Conclusions


| 4

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


| 5

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


| 6

● 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


Molecular Initiating

event

Key event1

Key event2

Key event3

Adverse Outcome

| 7

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


| 8

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

| 9

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


| 10

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

| 11

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

| 13

Do you believe ZapazoleTM is an endocrine disrupter in fish?

1. Yes

2. No

3. Unsure


YesNo

Unsure

52%

42%

6%

Case study

| 14

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

| 15

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


| 16

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


| 17

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


| 18

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


| 19

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


| 20

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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adverse outcome pathways (aops) focusing on … 6 james wheeler.pdf · adverse outcome pathways...

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