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WEIGHT OF EVIDENCE GUIDANCE: EVALUATING RESULTS OF EDSP TIER 1 

SCREENING TO IDENTIFY CANDIDATE CHEMICALS FOR TIER 2 TESTING 

DRAFT FOR PUBLIC COMMENT 

1. PURPOSE AND SCOPE OF DOCUMENT 

 The purpose of this document is to set forth some of the general principles, criteria, 

and considerations EPA generally believes to be relevant using a weight-of-evidence 

WoE) approach to evaluate data submitted as part of EPA’s two-tiered paradigm for 

screening and testing chemicals for endocrine activity under the Endocrine Disruptor 

Screening Program (EDSP). This paper was developed by EPA to provide guidance to 

EPA staff and managers who will be reviewing data submitted in response Orders for 

Tier 1 screening that began October 29, 2009 under the Endocrine Disruptor Screening 

Program (EDSP). Additionally, outside parties submitting data may be interested to 

know how the results from Tier 1 screening are being evaluated. This paper provides 

general guidance and is not binding on either EPA or any outside parties. The use of 

language such as "will," "is," "may," "can," or "should" in this paper does not connote 

any requirement for either EPA or any outside parties. As such, EPA may depart from 

the guidance where circumstances warrant and without prior notice. 

 A WoE evaluation is a process where potentially relevant studies are judged in a 

professional manner for quality. Thereafter, a summary statement is developed 

indicating the potential effects of the compound, the mode of action (MOA), and other 

relevant information. It is not a process that simply involves tallying the number of 

positive and negative results within and among studies. Critical assessment of an entire 

body of available data is taken into account for consistency, coherence, and biological 

plausibility (e.g., see USEPA, 2002 & 2005). Principles articulated in this document are 

equally applicable to a WoE evaluation of data from individual assays with multiple 

endpoints, as well as across the suite of assays in the Tier 1 screening battery. In 

addition, these principles would be generally relevant to the review of other scientifically 

relevant information (OSRI) submitted in response to test orders that request OSRI to 

be considered in lieu of designated screening assays in the Tier 1 battery. Most of the 

principles presented in this document are not unique to chemicals with potential 

endocrine activity but are commonly used for WoE evaluations conducted by EPA 

(USEPA 1991; 1992; 1996; 2002; 2005). The criteria discussed in this document are 

based on EPA’s experience in developing and applying risk assessment guidelines 

involving cancer, reproductive and developmental toxicity, and ecological toxicity. 

Important considerations include the use of expert judgment formed through the 

scientific process, current understanding of endocrine mechanisms of toxicity, and 

knowledge of other fields of toxicology (e.g., developmental, reproductive, neurological 

and immunological toxicology, and toxicokinetics). This document provides a 

transparent scientific approach for broadly evaluating Tier 1 screening data to determine 

if additional Tier 2 testing is necessary. 
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 This document also is expected to comply with the provision in the Office of 

Management and Budget Terms of Clearance for the Information Collection Request for 

the first list of chemicals to be screened under the EDSP and direction in the House 

Appropriations Committee for the Interior and Environment FY 2010 report (HR 2996, 

http://thomas.loc.gov/cgi-bin/cpquery/R?cp111:FLD010:@1(hr180)) that directed EPA 

to: 

“develop and publish criteria for evaluating the results of Tier 1 screening and 

determining whether a chemical should undergo Tier 2 analysis within one year of 

enactment. The process should allow for public input.” 

2. HISTORICAL BACKGROUND 

 This section provides an abbreviated overview of EPA’s EDSP. A more detailed 

history of the program can be found at its website (http://www.epa.gov/endo) and in 

other documents or websites referenced herein. 

 In 1996, Congress amended section 408 of the Federal Food, Drug, and Cosmetic 

Act (FFDCA) to require EPA to: 

“develop a screening program, using appropriate validated test systems and other 

scientifically relevant information, to determine whether certain substances may have an 

effect in humans that is similar to an effect produced by a naturally occurring estrogen, 

or other such endocrine effect as the Administrator may designate” [21 U.S.C. 346a(p)].

(http://www.epa.gov/pesticides/regulating/laws/fqpa/)

 Fundamental to the EDSP is a two-tiered approach involving a battery of Tier 1 

screening assays and individual Tier 2 tests designed to indentify and characterize 

chemicals with the potential to interact with the estrogen, androgen, and/or thyroid (E, A 

and/or T) hormonal systems. Tier 1 consists of a battery of complementary in vitro and 

short term in vivo assays designed to maximize sensitivity for detecting interactions with 

E, A, and/or T. Interactions with E, A, and/or T are evaluated using a range of key 

endpoints involving the MOA [e.g., receptor binding and gene transcription, 

steroidogenesis, hypothalamic-pituitary-gonadal (HPG) and –thyroid (HPT) axes] across 

gender and various taxa (e.g., rodents, amphibians, and fish) as indicated in Table 1. 

The diversity in endocrine endpoints within and among the Tier 1 screening assays is 

expected to provide corroborating information and support a WoE evaluation to yield a 

decision as to whether or not the chemical under evaluation requires additional testing 

in Tier 2. 

 Tier 2 testing consists of a group of individual in vivo tests designed to include males 

and females with an intact hypothalamic-pituitary-gonadal axis, multiple pathways of 

exposure and life-stages, and various taxa to further identify and characterize chemicalinduced interactions with E, A, and/or T for risk assessment. Although the endocrine 

system is included, Tier 2 tests are designed to quantify dose-response relationships in 

a larger context of toxicity and potential adversity that may involve other biological 
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systems (e.g., neurological, immunological, hepatic, renal, and cardiovascular) to be 

used for risk assessment. While the Tier 2 mammalian two-generation reproductive 

toxicity test is considered valid, other Tier 2 tests are at various stages in the validation 

process (Table 2). 

Table 1: EDSP Tier 1 battery of screening assays and complementary modes of 

action*. 

Screening 

Assays 

*Modes of Action 

Receptor Binding Steroidogenesis HPG Axis HPT Axis 

E Anti-E A Anti-A E A 

In vitro 

ER Binding ■ ■ 

ER α

Transcriptional 

Activation 

■ 

AR Binding ■ ■ 

Steroidogenesis 

H295R ■ ■ 

Aromatase 

Recombinant ■ 

In vivo 

Uterotrophic ■ 

Hershberger ■ ■ 

Pubertal Male ■ ■ ■ ■ ■

Pubertal Female ■ ■ ■ ■ ■

Fish Short-term 

Reproduction 

(male & female) 

■ ■ ■ ■ ■ ■ ■ 

Amphibian 

Metamorphosis ■

*A mode of action is defined as a sequence of key events and processes, starting with interaction of an 

agent with a cell, proceeding through operational and anatomical changes, and resulting in an adverse 

outcome (USEPA, 2005). These assays encompass certain key events within a mode of action (e.g., 

receptor binding) as well as certain pathways (e.g., steroidogenesis) through which a chemical can 

interact with the E, A, or T hormonal systems. 
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Table 2. EDSP Tier 2 tests. 

Mammalian two-generation reproductive toxicity test 

*Avian two-generation toxicity test - Japanese quail two-generation toxicity test 

*Amphibian growth and development test - Larval Amphibian Growth and Development Assay 

*Fish multigeneration test - Medaka Multigeneration Test 

*Invertebrate two-generation test - Mysid two-generation test 

*Proposed Tier 2 test currently at various stages of the validation process. 

 Most of the proposed Tier 1 screening assays completed the validation process in 

2008 and another in 2009. Subsequent to review [Federal Register Notice of January 

24, 2008 (73 FR 4216)] by the Federal Insecticide Fungicide and Rodenticide Act 

Scientific Advisory Panel (FIFRA SAP) and, based on a final SAP report (SAP, 2008), 

EPA accepted the current EDSP Tier 1 battery (Table 1). Availability of test guidelines 

for each of the 11 screening assays in Tier 1 was published in a Federal Register Notice 

October 21, 2009 (74 FR 54416). 

3. TESTING METHODS AND ENDPOINTS 

3.1. EDSP Tier 1 screening assays 

 The basis for the endpoints in each of the in vitro and in vivo Tier 1 screening assays 

has been described (http://www.epa.gov/scipoly/oscpendo/pubs/assayvalidation/tier1battery.htm) in 

Integrated Summary Reports associated with the validation process (see “peer review 

of individual assays” at website), summarized in a Technical Review Document for SAP 

review of the battery (see “external review” at website) and listed in EPA test guidelines 

for each of the assays (see “Test guidelines for Series 890” at website). The endpoints 

associated with each assay were determined through the validation process to be 

sensitive and specific to detect interaction with the E, A, or T hormonal systems. 

 For chemicals having estrogen- or androgen-like activity, the in vitro receptor binding 

assays provide information on the potential binding characteristics of a compound. 

Comparatively, the in vitro estrogen receptor transcriptional activation or steroidogenic 

assays are cell-based and provide mechanistic as well as some functional information 

on induction/inhibition of gene transcription or degree of steroid hormone production, 

respectively. 

In vivo assays integrate effects in a whole organism and provide apical as well as 

mechanistic information from one or multiple endpoints within an assay. Agonistic or 

antagonistic E-, A-, or T-dependent changes can be detected in association with 

reproductive development (e.g., vaginal opening and preputial separation), organ 

weights (e.g., ovaries, uterus, testes, prostate, and thyroid), and corresponding 
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histology of target organs. Mechanistic information such as thyroid hormones 

measured in the pubertal and amphibian assays and vitellogenin in the fish assay can 

be correlated with apical information within the same assays. The use of 

gonadectomized rats in the uterotrophic and Hershberger assays provides mechanistic 

information on specific estrogen- (i.e., uterus) or androgen- (i.e., prostate, seminal 

vesicles, levator ani-bulbocavernosus muscle, Cowper’s glands, and glans penis) 

dependent target organs. As with the in vitro assays, the results of the uterotrophic or 

Hershberger assays are limited to E and A receptor function. On the other hand, results 

involving intact animals (i.e., female and male pubertal, amphibian, and fish assays) 

generally provide more systems information, since more sites of action are involved 

along the HPG and HPT axes that target some of the same E-, A-, or T-dependent 

endpoints. 

3.2. Other Scientifically Relevant Information (OSRI) 

 EPA’s approach to the submission and use of OSRI as part of the EDSP has 

previously been described by EPA and is available in the Federal Docket Management 

System (EPA-HQ-OPPT-2007-1080-0032). In general, OSRI consists of data from 

assays that satisfy the same function as EDSP Tier 1 assays or may include data that 

are indicative of a potential consequence or adverse effect resulting from a chemicalinduced change in the E, A, and/or T hormonal systems. Hence, interference of 

endocrine function may come from effects measured in standard toxicity test guidelines 

or other comparable toxicity studies. Typically, OSRI-types of studies are not designed 

to provide definitive information on the modes or mechanisms of toxicity, but are 

generally focused on measured adverse effects (e.g., ability to become pregnant, 

duration of gestation, signs of difficult or prolonged parturition, sex ratio, or feminization 

or masculinization of offspring, number of pups, stillbirths, gross pathology, and 

histopathology of the vagina, uterus, ovaries, testis, epididymis, seminal vesicles, 

prostate, and thyroid) representing permanent changes with organizational or functional 

consequences. These studies may also encompass a range of life-stages (e.g., twogeneration reproductive study), treatment durations and doses, and provide information 

generated by relevant routes of exposure. 

4. WEIGHT-OF-EVIDENCE APPROACH 

 In evaluating whether additional testing is warranted in Tier 2, EPA anticipates that 

the following key questions would typically be considered as part of EPA’s WoE 

approach: 

• Do existing data provide adequate evidence to conclude whether there is a 

potential for the chemical to interfere with the normal function of the E, A, and/or 

T hormonal systems? 

• If the data indicate a potential to interact with those specific endocrine systems, 

which hormone system is impacted (E, A, and/or T)? 
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 Determination of whether the evidence suggests the substance is or is not a 

candidate for Tier 2 testing is based on evaluation of all relevant data, including any Tier 

1 results. This WoE analysis is conducted on a case-by-case basis by assessing all of 

the individual lines of evidence (Section 4.1) and performing an integrated analysis of 

the data (Section 4.2). 

4.1. Analysis of individual studies 

 In any WoE analysis, a full evaluation of each relevant study is conducted and 

documented. In general, the evaluation of individual studies includes characterization of 

the following: 

• Nature of the effect(s) seen in the study(ies) (e.g., were the effects seen in in 

vitro and/or in vivo assays; were the effects persistent or transient changes; were 

the effects molecular/biochemical changes or adverse outcomes);

• specificity and sensitivity of the effect(s);

• dose- and time- dependent changes, if available; 

• potency of responses and magnitude or severity of changes; and 

• consistency and relationship of the different effects seen within a study. 

 Both statistical and biological significance of the observed effects are relevant in 

evaluating study results. In general, the results of relevant studies are assumed to be 

indicative of interactions with the endocrine system unless data are available that 

demonstrate otherwise (e.g., evidence that the effect is not the consequence of an 

interaction with the endocrine system but a consequence of excessive toxicity to a nonendocrine system). To aid in determining the level of confidence in a study, the 

strengths of the study as well as any attendant limitations and uncertainties shall be 

considered.

4.2. Integrated analysis of data 

Weight-of-evidence assessments must be conducted on a case-by-case basis. 

EPA’s WoE analyses will generally include consideration of the information as follows: 

• Quality of data and the extent to which effects can be replicated within a 

laboratory and across different laboratories; 

• strengths and limitations of in vitro and in vivo results; 

• number and type of effects induced and potency, magnitude, and severity of 

effects; 

• consistency, pattern, range, and interrelationships of effects observed across 

studies, species, strains, and sexes; 

• conditions under which effects occur (e.g., dose, route, duration); and 

• understanding of MOA and biological plausibility of responses.

 These considerations are part of evaluating the evidence as a whole and 

determining whether or not a chemical has the potential to interact with the endocrine 
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system via E, A, and/or T hormonal pathways. In examining the balance of positive and 

negative results, the relative sensitivity and specificity of the measured endpoints would 

also be considered. Tier 1 in vitro screens can provide some insight into MOA. In 

general, however, Tier 1 in vivo results would carry greater weight than in vitro results 

because in vitro assays inherently lack physiological conditions associated with whole 

tissues or organs and, therefore, have nil or very limited ability to represent metabolic 

processes and pathways leading to endocrine disruption. The relationship between 

endpoints and their impact on normal endocrine function would also typically be 

significant factors in this determination. Concordant effects found in multiple 

interrelated endpoints generally imply a compromise in endocrine function, in contrast to 

isolated or discordant effects. Totality of the evidence is evaluated to determine 

whether such effects can potentially occur across taxa. Generally, consistent positive or 

negative effects across studies and taxa increase confidence in the determination of 

whether or not a chemical has the potential to interact with the endocrine system. 

Additionally, if marginal or weak relationships exist with regard to dose, severity, 

magnitude, and/or incidence, consideration of other available information may also be 

appropriate in determining whether further testing in Tier 2 is warranted. This could 

include consideration of other critical effects (non-endocrine), dose response, what is 

understood about the underlying basis (i.e., toxicity MOA) of these critical effects (i.e.,

non-endocrine or an endocrine MOA not covered by Tier 1) and their human relevance, 

and potential for exposure. Other supportive evidence may also be used in the WoE 

evaluation including pertinent data on related chemicals, metabolisim or toxicokinetics, 

and results of computational models. 

4.3. Summary and conclusions of WoE approach 

 A summary of the WoE analysis is expected to transparently state and explain 

conclusions. It should explain the selection of certain studies or effects as the key basis 

for conclusions. In general, this characterization should be limited to the most 

significant and relevant data, conclusions, and uncertainties. 

 Summary statements for a WoE analysis should generally address key elements as 

follows: 

• Each E, A, and T pathway, including species, gender, and life stages;

• uncertainties and the extent these uncertainties impact the conclusions; 

• relative weight placed on studies and effects (i.e., points at which choices are 

made of critical effects or studies and why); and 

• inconsistent or conflicting results. 

 If Tier 2 testing is indicated, i.e., effects are seen that are mediated through the E, A, 

and/or T hormonal systems, to the extent permitted by the available data, the summary 

should address any potential impact of the results of Tier 2 tests to risk assessment for 

that chemical and provide rationale for any conclusions. This may include, to the extent 

supported by the available data, conclusions regarding the species in which additional 
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testing is warranted and the likelihood that the effect may occur at a lower dose than 

effects seen in existing studies. 
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REFERENCES 

EDSTAC (1998) Endocrine Disruptor Screening and Testing Advisory Committee, Final 

Report, Volume I-II. 

http://www.epa.gov/scipoly/oscpendo/pubs/edspoverview/finalrpt.htm. 

. 

SAP (2008) A set of scientific issues being considered by the Environmental Protection 

Agency regarding: The Endocrine Disruptor Screening Program (EDSP) Proposed Tier 

1 Screening Battery. SAP Minutes No. 2008-03. 

http://www.epa.gov/scipoly/sap/meetings/2008/march/minutes2008-03-25.pdf. 

USEPA (U.S. Environmental Protection Agency) (1991) Guidelines for developmental 

toxicity risk assessment. Federal Register 56(234):63798-63826. Available from: 

http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=23162. 

USEPA (U.S. Environmental Protection Agency) (1996) Guidelines for reproductive 

toxicity risk assessment. Federal Register 61(212):56274-56322. Available from: 

http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=2838. 

USEPA (U.S. Environmental Protection Agency) (1998) Guidelines for neurotoxicity risk 

assessment. Federal Register 63(93):26926-26954. Available from: 

http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=12479. 

USEPA (U.S. Environmental Protection Agency) (2002) A Review of Review of the 

Reference Dose and Reference Concentration Processes. Risk Assessment Forum, 

Washington EPA/630/P-02/002F December 2002 Final Report 

http://www.epa.gov/raf/publications/pdfs/rfd-final.pdf 

USEPA (U.S. Environmental Protection Agency) (2005) Guidelines for carcinogen risk 

assessment Risk Assessment Forum, Washington, DC. EPA/630/P-03/001F. Available 

from: http://www.epa.gov/raf/publications/pdfs/CANCER_GUIDELINES_FINAL_3-25-

05.PDFp://www.epa.gov/