ADVERSE OUTCOME PATHWAYS:

DATA FOR CHLORPYRIFOS AT VARYING 

LEVELS OF BIOLOGICAL ORGANIZATION 

WILLIAM R. MUNDY, PH. D.

RESEARCH TOXICOLOGIST

USEPA, OFFICE OF RESEARCH AND 

DEVELOPMENT

FIFRA SAP April 10, 2012
Introduction

 Adverse Outcome Pathway

 concept

 example

 evaluating data

2

 Adverse Outcome Pathways for 

Chlorpyrifos

 overview

 example for neurite outgrowth

 levels of biological organization

 relationship to AChE inhibition
Adverse Outcome Pathway

3

An Adverse Outcome Pathway (AOP) is a conceptual framework that 

portrays existing knowledge concerning the linkage between a direct 

molecular initiating event and an adverse outcome, at a level of 

biological organization relevant to risk assessment. 

(Chemical Safety and Sustainability Research Program, 2011; Ankley et al. 2010, Environ. 

Toxicol. Chem., 29(3): 730-741.)

 incorporates the concepts of mechanism of action (detailed 

understanding of the molecular basis of the toxic effect) and 

mode of action (set of likely key events that lead to a toxic 

effect)
Adverse Outcome Pathway

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Chemical

Property

Profile

Receptor/Ligan

d

Interaction

DNA Binding

Protein 

Oxidation

Gene Activation

Protein 

Production

Altered 

Signaling

Protein 

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 

Recruitme

nt

Extinction

Population

Responses

Oxidation

Protein 

Depletion

Development

or Function Cancer

Initiating Event Adverse Outcome

 Propose a set of biologically plausible linkages to apical 

toxicity

 Place data collected at different levels of organization into 

context

 Focus on chemical mechanisms related to adverse outcome
Biologic

inputs

“Normal” 

Biological

Function

Adaptive

Responses

Early cellular

changes

Exposure

Uptake-Delivery to Target Tissues

Perturbation

Cellular response pathway

II. Adverse Outcome Pathways –

definition and example

Adverse

Outcomes

(e.g., mortality, 

Reproductive

Impairment)

Cell inury, 

Inability to 

regulate

Responses

Molecular

initiating event

Perturbed cellular 

response pathway

Adverse outcome

relevant to

risk assessment

Toxicity Pathway

Adverse Outcome Pathway

Modified From NRC 2007
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Toxicant Macro-Molecular

Interactions

Cellular 

Responses

Organ

Responses

Individual

Responses

glutamate (kainate)

receptor activation

hippocampal

cell death 

intracellular Ca2+

dysregulation

domoic acid

(EAA)

memory loss

Adverse Outcome Pathway:

Domoic Acid

Molecular

initiating event

Key events or 

predictive

relationships 

spanning

levels of biological 

organization 

Adverse outcome

relevant to 

risk assessment
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Toxicant Macro-Molecular

Interactions

Cellular 

Responses

Organ

Responses

Individual

Responses

glutamate (kainate)

receptor activation

hippocampal

cell death 

intracellular Ca2+

dysregulation

domoic acid

(EAA)

memory loss

Adverse Outcome Pathway:

evaluation of data

 Plausibility: theoretical basis for biological connections

 Temporal relationship: effects at lower level must precede the 

next

 Dose-response: dose for effect at lower level should be ≤ the 

next
Potential Adverse Outcome Pathways 

for Chlorpyrifos

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Domain Chemical MacroMolecular 

Interactions

Cellular 

Responses

Organ 

Responses

Individual 

Responses

AChE as 

morphogen

In Vitro In Vitro

In Vivo

In Vivo

(zebrafish)

Cholinergic In Vitro In Vitro

chlorpyrifos, 

chlorpyrifos 

oxon, or 

trichloropyridino

l

Cholinergic

receptors

In Vitro In Vitro

Endocannabin

oid system

In Vitro

In Vivo

ROS In Vitro

In Vivo

In Vivo

Serotonergic

system

In Vitro

In Vivo

In Vivo In Vivo

Tubulin, MAPs In Vitro

In Vivo

In Vitro

In Vivo
Potential Adverse Outcome Pathway: 

Morphogenic site

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Toxicant Macro-Molecular

Interactions

Cellular 

Responses

Organ

Responses

Individual

Responses

Binding to AChE

(morph site)

CPF, CPO, TCP Neurite Growth Neural Connectivity Cognition

Molecular

initiating event

Key events or 

predictive

relationships 

spanning

levels of biological 

organization 

Adverse outcome

relevant to 

risk assessment

CPF-esterase 

(morph?)

CPO-esterase 

(morph?) 

CPO-morph

decrease in vitro

(± AChE 

inhibition)

decrease in vivo 

(+ AChE 

inhibition)

CPF (in vitro, in 

vivo)

CPO (in vitro, in 

vivo)

TCP (in vitro)
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Domain Chemical MacroMolecular 

Interactions

Cellular 

Responses

Organ 

Responses

Individual 

Responses

AChE as 

morphogen

chlorpyrifos, 

chlorpyrifos 

oxon, or 

trichloropyridi

morphogenic 

site of AChE

neurite growth axon length

Cholinergic

receptors

M2 receptor cAMP

Endocannabi

noid system

CB1 receptor

FAAH

Potential Adverse Outcome Pathways for 

Chlorpyrifos

trichloropyridi

nol ROS oxidative 

stress

oxidative

stress 

response 

genes

Serotonergic

system

changes in 

level of 

serotonin, 

serotonin 

turnover, 

receptors, etc.

changes in 

level of 

serotonin, 

serotonin 

turnover, 

receptors,

etc., by brain 

region

changes in 

behavior, both 

basal and 

after drug 

challenge

Tubulin, binding to axonal 

Initiating 
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 Assessed AChE activity concurrently

 Concentration/dose-response

 Significant response in absence of significant AChE inhibition

Adverse Outcome Pathways for 

Chlorpyrifos: Relationship to AChE 

Inhibition

Experimental model Response

Primary neuron culture decrease neurite outgrowth

(in vitro)

Rat increase oxidative stress genes in 

CNS

(developmental exposure)

Primary neuron culture inhibition of axonal transport

(in vitro)

Rat* change in serotonergic tone of 

adult (developmental exposure) CNS

* AChE measured in separate experime

13 Questions?