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Application of Adverse Outcome Pathways to U.S. EPA's Endocrine Disruptor Screening Program

Browne P, Noyes PD, Casey WM, Dix DJ
Environmental Health Perspectives (2017) DOI: https://doi.org/10.1289/ehp1304 PMID: 28934726


Publication


Abstract

BACKGROUND:
The U.S. EPA's Endocrine Disruptor Screening Program (EDSP) screens and tests environmental chemicals for potential effects in estrogen, androgen, and thyroid hormone pathways, and it is one of the only regulatory programs designed around chemical mode of action.

OBJECTIVES:
This review describes the EDSP's use of adverse outcome pathway (AOP) and toxicity pathway frameworks to organize and integrate diverse biological data for evaluating the endocrine activity of chemicals. Using these frameworks helps to establish biologically plausible links between endocrine mechanisms and apical responses when those end points are not measured in the same assay.

RESULTS:
Pathway frameworks can facilitate a weight of evidence determination of a chemical's potential endocrine activity, identify data gaps, aid study design, direct assay development, and guide testing strategies. Pathway frameworks also can be used to evaluate the performance of computational approaches as alternatives for low-throughput and animal-based assays and predict downstream key events. In cases where computational methods can be validated based on performance, they may be considered as alternatives to specific assays or end points.

CONCLUSIONS:
A variety of biological systems affect apical end points used in regulatory risk assessments, and without mechanistic data, an endocrine mode of action cannot be determined. Because the EDSP was designed to consider mode of action, toxicity pathway and AOP concepts are a natural fit. Pathway frameworks have diverse applications to endocrine screening and testing. An estrogen pathway example is presented, and similar approaches are being used to evaluate alternative methods and develop predictive models for androgen and thyroid pathways.

Figures


Figure 1. U.S. EPA Endocrine Disruptor Screening Program (EDSP) battery.

U.S. EPA Endocrine Disruptor Screening Program (EDSP) battery of 11 Tier 1 screening assays for activity and Tier 2 tests for identifying dose–response relationships and adverse effectsa. Screening and testing data are interpreted for each endocrine pathway, although intact animal in vivo responses may involve multiple end points and pathways. Levels of biological complexity from molecular interactions through to populations are represented by the Tier 1 and Tier 2 screens and tests, consistent with an adverse outcome pathway (AOP) framework.
A+, androgenic; A–, antiandrogenic; E+, estrogenic; E–, antiestrogenic; HPT axis, hypothalamic–pituitary–thyroid axis.
For more detail about specific test methods and protocols, refer to EDSP test guidelines (https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-890-endocrine-disruptor-screening-program).
aEPA test guidelines harmonized through the OECD.

Figure 2. Adverse outcome pathways (AOPs) begin with a molecular interaction.

Adverse outcome pathways (AOPs) begin with a molecular interaction serving as a molecular initiating event (MIE), leading to a series of key events and eventually to an adverse outcome at the organismal level for human health, and at the population level for ecotoxicology assessments. Toxicity pathways can be considered part of an AOP, including plausibly linked molecular interactions and key events, but may not include an adverse outcome.

Figure 3. EDSP Tier 1 and Tier 2 end points relevant to the female mammalian estrogen agonist.

EDSP Tier 1 and Tier 2 end points relevant to the female mammalian estrogen agonist signaling pathway can be organized using an adverse outcome pathway (AOP) framework. Estrogen receptor (ER) binding and activation [i.e., the molecular initiating event (MIE)] can be linked to several related key events from EDSP Tier 1 and Tier 2 assays, leading to an adverse outcome (e.g., altered development). It should be noted that in this example, the AOP only includes regulatory end points included in U.S. EPA test guidelines used to evaluate the potential endocrine activity of environmental chemicals. EOGRTS, extended one-generation reproductive toxicity study; ERTA, estrogen receptor transactivation; VO, vaginal opening.

Figure 4. EDSP Tier 1 and Tier 2 test assays mapped to an ecotoxicological adverse outcome pathway.

EDSP Tier 1 and Tier 2 test assays mapped to an ecotoxicological adverse outcome pathway (AOP) for an estrogen receptor (ER) agonist in male fish including a molecular initiating event (MIE) of receptor binding and related key events measured in Tier 1 and Tier 2 assays and terminating in an adverse outcome represented by declines in population size and by altered sex composition. FSTRA, fish short-term reproductive assay; MEOGRT, Medaka extended one-generation reproductive test; VTG, vitellogenin (egg precursor protein).

Figure 5. Validation of an estrogen receptor (ER) agonism toxicity pathway model.

Validation of an estrogen receptor (ER) agonism toxicity pathway model based on ToxCast™/Tox21 high-throughput screening (HTS) data requires a robust set of reference chemicals for the relevant molecular initiating event (MIE) and key events (Browne et al. 2015).

Figure 6. Adverse outcome pathway (AOP) networks for estrogenicity and antiestrogenicity.

Adverse outcome pathway (AOP) networks for estrogenicity and antiestrogenicity in (A) mammals and (B) fish based on integrated estrogen receptor (ER) agonist and steroidogenesis modeling outputs along with Endocrine Disruptor Screening Program (EDSP) Tier 1 and Tier 2 assays. Additional modeling may be integrated into the AOP network to further refine the model and toxicity pathway/AOP linkages. CYP11a, cytochrome P450 11a (cholesterol side-change cleavage protein); CYP17, cytochrome P450 17 (e.g., 17α-hydroxylase); E2, 17β-estradiol; EOGRTS, extended one-generation reproductive toxicity study; FSTRA, fish short-term reproductive assay; MEOGRT, Medaka extended one-generation reproductive test; StAR, steroidogenic acute regulatory protein; VO, vaginal opening; VTG, vitellogenin.

Tables


Table 1. U.S. EPA Endocrine Disruptor Screening Program (EDSP) tier 1 screening battery assays.

U.S. EPA Endocrine Disruptor Screening Program (EDSP) tier 1 screening battery assays and tier 2 testing assays, high-throughput screening (HTS) assays, and predictive model alternatives