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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.

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# Outline

• Overview of case study


## • Areas of scientific uncertainty in the AOP


## • Populations of concern
• AOP Development


## • Research results used to construct the AOP

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# Acknowledgements

May 24 -26, 2011 4 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 5

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:May 24 -26, 2011 6

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? May 24 -26, 2011 7May 24 -26, 2011 8



## 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

May 24 -26, 2011 9 • 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.May 24 - 26,2011 10



## 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

May 24 -26, 2011 12 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

• D eveloping 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).

• D eficiencies 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. May 24 -26, 2011 13

## 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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### S

pecies 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 May 24 -26, 2011 18

# 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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