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Integrated Approaches to Testing and 

Assessment Strategy: Use of an Adverse 

Outcome Pathway to Inform Risk Assessment 

Timothy F. McMahon, Ph.D.

Office of Pesticide Programs

U.S. EPA, Washington D.C.

Kevin Crofton, Ph.D.

Tammy Stoker, Ph.D.

Office of Research and Development / 

NHEERL

U.S. EPA, RTP, NC
Purpose

• To illustrate an approach to informing 

risk assessment through use of an 

adverse outcome pathway using 

triclosan as an example. 

• Case study is not a risk assessment for 

triclosan. 

2
Outline

• Overview of case study

• Areas of scientific uncertainty in the 

AOP

• Populations of concern

• AOP Development 

• Research results used to construct the 

AOP

3
Acknowledgements

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Kevin Crofton, Ph.D. (ORD/NHEERL)

Tammy Stoker, Ph.D. (ORD/NHEERL)

R.Woodrow Setzer, Ph.D. (NCCT)

Timothy Leighton (OPP)

Katie Paul, Ph.D. (ORD/NHEERL)

Vicki Dellarco, Ph.D. (OPP)

Jennifer McLain, Ph.D. (OPP)
May 24-26, 2011

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Background on Triclosan as AOP case study

• Antimicrobial pesticide re-assessed by the Agency in 2008 

•Full complement of mammalian toxicity studies that 

investigated general toxicity, as well as studies investigating 

developmental, reproductive, mutagenic, and carcinogenic 

properties.

•Published studies on the effect of triclosan on serum 

thyroid hormones and associated enzymatic pathways: 
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Background, cont.

• Decrease in serum T4 observed in vivo (Crofton, 2007)

•Induction of Cyp2b1 and 3a1 in rat hepatocytes (Jinno et

al., 1997)

•Induction of Cyp2b, Cyp3a, and Cyp4a protein content

and activity in rats (Hanioka et al 1997)

• Accelerated hindlimb development in North American

bullfrog premetamorphic tadpoles (Veldhoen et al., 

2006, 2007)
Background, cont.

Findings raised several questions, including: 

• What are the molecular initiating events for the change in T4 

levels? (can an AOP be identified)

• What is the dose-response relationships for the effect on T4?

• What data are needed to inform interspecies differences in the 

response?

• Is there sensitivity of different life stages to this effect?

• What is the relevance of the change in T4 for adverse outcomes 

in humans?

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Focus on Thyroid

Alterations in thyroid homeostasis is of particular 

concern as disruption of circulating concentrations of 

thyroid hormones have been associated with adverse 

outcomes in humans, including altered 

neurodevelopment. 

Triclosan’s structural similarity with thyroxine, the 

evidence for interaction of triclosan with this hormone 

system in published studies, and evidence of 

widespread environmental occurrence all led to the 

need for further investigation. 
Adverse Outcome Pathway Development

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• The Agency planned and conducted additional research 

on the interaction of triclosan with the thyroid hormone 

system using in vivo and in vitro testing approaches with an 

interest in identifying whether an adverse outcome 

pathway could be identified that would facilitate 

characterization of precursor events for human health risk 

assessment. 

•Studies were also conducted using Tier I EDSP testing 

protocols and other experimental protocols to determine 

whether the developing mammalian organism is more 

susceptible to this effect. 
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Adverse Outcome Pathway 

applications

While the AOP approach is presented as it relates to 

human health, the application is not limited to 

evaluating potential adverse effects in humans. The 

approaches may have utility for informing ecological 

risk assessments across multiple taxa as well. 
Adverse Outcome Pathway-Thyroid Effects

• The additional research conducted on the interaction 

with the thyroid hormone system generated data that 

led to identification of precursor events that the 

Agency proposes as the basis for an adverse 

outcome pathway approach that can be used to 

refine risk assessment. 

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Proposed AOP for Thyroid Effects

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Hepatic

Tissue

Dose 

Actrivate

CAR/PXR

Hepatic

Phase I, II, III

Decreased

T4

Altered 

TR-Regulated

Processes

Adverse

Neuro-Development
Relevance of AOP for Humans

• Developing nervous system is dependent upon 

adequate amounts of thyroid hormones, and 

significant neurological damage can occur when the 

deficiency is present during brain development 

(Obregon, 2007). 

• Deficiencies of thyroid hormone at birth can result in 

deficiencies in mental and neurologic outcome at 2 

and 5 years of age (van Wassenaer, 2002). 

• Identification of this AOP for triclosan represents a 

significant step to refinement of the existing human 

health assessment, where the AOP is relevant to 

potential adverse effects in humans in comparison to 

apical endpoints. 

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Areas of Uncertainty

• Understanding of the physiological 

differences in thyroid hormone regulation and 

pharmacokinetic differences in rats versus 

humans are areas for informing the factors 

applied for interspecies and intraspecies 

extrapolation. 

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Species differences in thyroid regulation 

(inter-species extrapolation) 

• Intended to account for differences in 

response between animals and humans

• Compared to humans, where TBG is the 

major T4 binding protein, most T4 in adult rat 

serum is bound to albumin and transthyretin 

(TTR); rats do not possess TBG after PND 

35. 

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Species differences in thyroid regulation 

(inter-species extrapolation), cont. 

• Binding affinity of T4 for TBG is more than 

100-fold greater than that of albumin or TTR.

• Differences in binding and T4 clearance 

suggests that rats may be more sensitive to 

perturbations of circulating T4 than humans 

and is an important consideration in 

application of the AOP to human populations. 

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Species differences in pharmacokinetics

• Published data show that rats attain higher 

values for area under the curve (AUC) [63.9 

μg*hr/mL] compared to humans [0.2-7.2 

μg*hr/mL] at comparable oral doses

• Rats are also observed to have higher 

plasma levels vs. humans (4,450 ng/mL in 

rats vs 23-574 ng/mL in humans) at 

comparable oral doses

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Populations of Concern

• Infants/children vs. adults

• Results of ORD experimental research 

conducted to investigate whether developing 

rats are more sensitive to hypothyroxinemia 

compared to juvenile and maternal rats 

showed no apparent differences in sensitivity 

to the degree of hypothyroxinemia, and no 

apparent accumulation of triclosan in fetal or 

pup blood or liver compared to maternal 

concentrations

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SAP consultation

• The Agency is seeking the Science 

Advisory Panel’s advice on the scientific 

soundness of use of this AOP and 

knowledge of the qualitative and 

quantitative relationship of the causal 

key events identified in the triclosan 

case study. 

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End of overview

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